The following alphabetical list represents papers published in 2004 with at least one Whitehead author (in red). Not all of this work was done at the Whitehead Institute. Some of these papers are collaborations with scientists elsewhere. The papers are gathered from PubMed and from Science Citation Index (also known as the Web of Science) Preceding the bibliography is an alphabetical list of the titles of the papers followed by the first author :

-Acrp30/adiponectin is reduced in the serum of obese and diabetic individuals, and the genetic locus of adiponectin is linked to the metabolic syndrome. Hug
-Acyl CoA synthetase 2 (ACS2) over-expression enhances fatty acid internalization and neurite outgrowth. Marszalek
-Analysis of Dscam diversity in regulating axon guidance in Drosophila mushroom bodies. Zhan
-Arabidopsis ALF4 encodes a nuclear-localized protein required for lateral root formation. DiDonato
-Are Sequence Family Variants Useful for Identifying Deletions in the Human Y Chromosome? Repping
-At most three ES cells contribute to the somatic lineages of chimeric mice and of mice produced by ES-tetraploid complementation. Wang
-Basecalling using hidden Markov models. Boufounos
-Bending stiffness of a crystalline actin bundle. Shin
-Bidirectional promoters regulate the monoallelically expressed Ly49 NK receptors. Ensminger
-Biological magic behind the bullets. Stockwell
-Breakpoint region of the most common isochromosome, i(17q), in human neoplasia is characterized by a complex genomic architecture with large, palindromic, low-copy repeats. Barbouti
-Caveolin-1 gene disruption promotes mammary tumorigenesis and dramatically enhances lung metastasis in vivo - Role of Cav-1 in cell invasiveness and matrix metalloproteinase (MMP-2/9) secretion.Williams
-Chromosomes 6 and 13 Harbor Genes that Regulate Pubertal Timing in Mouse Chromosome Substitution Strains .Krewson.
-Colloid surface chemistry critically affects multiple particle tracking measurements of biomaterials.Valentine
-Combined haploid and insertional mutation screen in the zebrafish.Wiellette
-Combining Gene Expression Profiles and Clinical Parameters for Risk Stratification in Medulloblastomas. Fernandez-Teijeiro
-Comparative genomic analysis of the clade B serpin cluster at human chromosome 18q21: amplification within the mouse squamous cell carcinoma antigen gene locus.Askew
-A complex interaction of imprinted and maternal-effect genes modifies sex determination in odd sex (Ods) mice. Poirier
-Computational Identification of Plant MicroRNAs and Their Targets, Including a Stress-Induced miRNA Jones-Rhoades
-Confirmation of a dyslexia susceptibility locus on chromosome 1p34-p36 in a set of 100 Canadian families.Tzenova
-Constitutive activation of the MEK/ERK pathway mediates all effects of oncogenic H-ras expression in primary erythroid progenitors.Zhang
-Control of Centromere Localization of the MEI-S332 Cohesion Protection Protein. Lee
-Control of pancreas and liver gene expression by HNF transcription factors. Odom
-Coordinated replication timing of monoallelically expressed genes along human autosomes. Ensminger
-Corticosteroid pharmacogenetics: association of sequence variants in CRHR1 with improved lung function in asthmatics treated with inhaled corticosteroids.Tantisira
-Cre-lox-regulated conditional RNA interference from transgenes..Ventura
-A cytoskeleton-based functional genetic screen identifies Bcl-xL as an enhancer of metastasis, but not primary tumor growth.Martin
-Defects Arising From Whole-Genome Duplications in Saccharomyces cerevisiae. Andalis
-Defects in adaptive energy metabolism with CNS-Linked hyperactivity in PGC-1 alpha null mice.Lin
-Derepression by depolymerization: Structural insights into the regulation of Yan by Mae.Qiao
-Derivation of embryonic germ cells and male gametes from embryonic stem cells. Geijsen
-Developmentally regulated alterations in polycomb repressive complex 1 proteins on the inactive X chromosome.Plath
-The differential roles of budding yeast Tem1p, Cdc15p, and Bub2p protein dynamics in mitotic exit. Molk
-Direct visualization of transcription factor-induced chromatin remodeling and cofactor recruitment in vivo. Carpenter
-Dnmt1 Expression in Pre- and Postimplantation Embryogenesis and the Maintenance of IAP Silencing. Gaudet
-Dppa3 / Pgc7 / stella is a maternal factor and is not required for germ cell specification in mice. Bortvin
-Drosophila MOS Ortholog Is Not Essential for Meiosis. Ivanovska
-A dyslexia susceptibility locus (DYX7) linked to dopamine D4 receptor (DRD4) region on chromosome 11p15.5 Hsiung.
-Early requirement for fgf8 function during hindbrain pattern formation in zebrafish. Wiellette
-Effects of Q/N-rich, polyQ, and non-polyQ amyloids on the de novo formation of the [PSI+] prion in yeast and aggregation of Sup35 in vitro.Derkatch
-Elastic behavior of cross-linked and bundled actin networks.Gardel
-Elongation of yeast prion fibers involves separable steps of association and conversion. Scheibel
-Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs.Vazquez
-Enhanced detection sensitivity using a novel solid-phase incorporated affinity fluorescent protein biosensor. Zhong
-Epigenetic regulation of translation reveals hidden genetic variation to produce complex traits. True
-Erralpha and Gabpa/b specify PGC-1alpha-dependent oxidative phosphorylation gene expression that is altered in diabetic muscle. Mootha
-Expression of MeCP2 in postmitotic neurons rescues Rett syndrome in mice.Luikenhuis
-Expression of GST-fused kinase domain of human Csk homologous kinase from Pichia pastoris facilitates easy purification. Murthy
-A family of Acrp30/adiponectin structural and functional paralogs. Wong
-A family of human Y chromosomes has dispersed throughout northern Eurasia despite a 1.8-Mb deletion in the azoospermia factor c region. Repping
-Follistatin operates downstream of Wnt4 in mammalian ovary organogenesis. Yao
-Gene amplification as a developmental strategy. Isolation of two developmental amplicons in Drosophila. Claycomb
-Gene expression-based high-throughput screening (GE-HTS) and application to leukemia differentiation Stegmeier
-Genetic and epigenetic regulation of the FLO gene family generates cell-surface variation in yeast. Halme
-Genetic complementation of cytokine signaling identifies central role of kinases in hematopoietic cell proliferation. Koh
-Genetic Dissection of Complex Traits with Chromosome Substitution Strains of Mice. Singer.
-General conditions for predictivity in learning theory. Poggio
-Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Jaillon
-Genome scan analyses and positional cloning strategy in IBD: successes and limitations Wild
-Genome-wide linkage analysis of a composite index of neuroticism and mood-related scales in extreme selected sibships.Nash
-Genomewide linkage analysis of bipolar disorder by use of a high-density single-nucleotide-polymorphism (SNP) genotyping assay. Middleton
-Genome-wide scan in Portuguese Island families implicates multiple loci in bipolar disorder: Fine mapping adds support on chromosomes 6 and 11.Pato
-GermOnline, a cross-species community knowledgebase on germ cell differentiation. Wiederkehr
-Global Position and Recruitment of HATs and HDACs in the Yeast Genome Robert
-Glucose and sucrose: hazardous fast-food for industrial yeast? Verstrepen
-Guest, a transposable element belonging to the Tcl/mariner superfamily is an ancient invader of Neurospora genomes. Ramussen
-Haploview: analysis and visualization of LD and haplotype maps.Barrett
-Hepatic temporal gene expression profiling in Helicobacter hepaticus-infected A/JCr mice. Boutin
-hIan5: the human ortholog to the rat Ian4/Iddm1/lyp is a new member of the Ian family that is overexpressed in B-cell lymphoid malignancies. Zenz
-High efficiency creation of human monoclonal antibody-producing hybridomas. Dessain
-A high-performance multilane microdevice system designed for the DNA forensics laboratory Goedecke
-Hsp104 Catalyzes Formation and Elimination of Self-Replicating Sup35 Prion Conformers Shorter
-Human cloning - the science and ethics of nuclear transplantation. Jaenisch
-Huntingtin aggregates ask to be eaten. Thoreen
-Identification and characterisation of the posteriorly-expressed Xenopus neurotrophin receptor homolog genes fullback and fullback-like. Bromley
-Identification of the linker-SH2 domain of STAT as the origin of the SH2 domain using two-dimensional structural alignment. Gao
-Identification of the proteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation factor 2. Liu
-Inactivation of fatty acid transport protein 1 prevents fat-induced insulin resistance in skeletal muscle. Kim
-Inadvertent cancer research Weinberg
-Increased postischemic brain injury in mice deficient in uracil-DNA glycosylase. Endres
-Inflammatory bowel disease susceptibility loci defined by genome scan meta-analysis of 1952 affected relative pairs. van Heel
-Insulin-like growth factor 2 expressed in a novel fetal liver cell population is a growth factor for hematopoietic stem cells. Zhang
-Interactions among alpha-synuclein, dopamine, and biomembranes - Some clues for understanding neurodegeneration in Parkinson's disease Rochet
-Points of View: lectures: can't learn with them, can't learn without them Lodish
-Lessons from the Genome Sequence of Neurospora crassa: Tracing the Path from Genomic Blueprint to Multicellular Organism. Borkovich
-LIF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells.Daheron
-Lnk Inhibits Tpo-mpl Signaling and Tpo-mediated Megakaryocytopoiesis. Tong
-LIX: a chemokine with a role in hematopoietic stem cells maintenance. Choong
-Local and global cooperativity in the human alpha-lactalbumin molten globule. Quesada
-Loss of heterozygosity and its correlation with expression profiles in subclasses of invasive breast cancers. Wang
-Low temperature or GroEL/ES overproduction permits growth of Escherichia coli cells lacking trigger factor and DnaK Vorderwulbecke
-MAE, a dual regulator of the EGFR signaling pathway, is a target of the Ets transcription factors PNT and YAN.Vivekanand
-Mapping multiple sclerosis susceptibility to the HLA-DR locus in African Americans. Oksenberg
-Mechanisms and implications of imatinib resistance mutations in BCR-ABL.Nardi
-The Meis3 protein and retinoid signaling interact to pattern the Xenopus hindbrain. Dibner
-Metabolic and hormonal interactions between muscle and adipose tissue. Tomas
-Methods for high-density admixture mapping of disease genes. Patterson
-Methods in comparative genomics: genome correspondence, gene identification and regulatory motif discovery.Kellis
-Mice cloned from olfactory sensory neurons. Eggan
-Microarrays for an integrative genomics. Ramaswarwy.
-Microarrays of small molecules embedded in biodegradable polymers for use in mammalian cell-based screens. Bailey
-Microenvironment-driven changes in the expression profile of hematopoietic cobblestone area-forming cells. Choong
-MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5' region. Mallory
-MicroRNA-directed cleavage of HOXB8 mRNA Yekta
-MicroRNA Regulation of NAC-Domain Targets Is Required for Proper Formation and Separation of Adjacent Embryonic, Vegetative, and Floral Organs Mallory
-MicroRNAs. Genomics, Biogenesis, Mechanism, and Function. Bartel
-MicroRNAs modulate hematopoietic lineage differentiation. Chen
-MicroRNAs: something important between the genes. Mallory
-Molecular genetics approaches in yeast to study amyloid diseases. Outeiro
-Monoallelic expression and asynchronous replication of p120 catenin in mouse and human cells.Gimelbrant
-Multiple levels of telomerase regulation. Stewart
-Multivariate proteomic analysis of murine embryonic stem cell self-renewal versus differentiation signaling. Prudhomme
-Mutations in the Drosophila Condensin Subunit dCAP-G: Defining the Role of Condensin for Chromosome Condensation in Mitosis and Gene Expression in Interphase Dej
-New approaches to gene hunting in IBD Daly
-New class of Son-of-sevenless (Sos) alleles highlights the complexities of Sos function Silver.
-nlz Gene family is required for hindbrain patterning in the zebrafish.Hoyle
-No bias in linkage analysis. Abecasis
-Nuclear cloning of embryonal carcinoma cells. Blelloch
-Opinion: Micromanagers of gene expression: the potentially widespread influence of metazoan microRNAs Bartel.
-Origins of mammalian hematopoiesis: In vivo paradigms and in vitro models Lensch
-Origins of variation in the fungal cell surface Verstrepen
-Papillomavirus-mediated neoplastic progression is associated with reciprocal changes in Jagged1 and manic fringe expression linked to notch activation. Veeraraghavalu
-Parental Phenotypes in Family-Based Association Analysis Purcell
-PathBLAST: a tool for alignment of protein interaction networks. Kelley.
-Patterns of flanking sequence conservation and a characteristic upstream motif for microRNA gene identification Ohler
-Plant hormone indoleacetic acid induces invasive growth in Saccharomyces cerevisiae Prusty
-Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody.Colby
-The potential of a chemical graph transformation system.
-Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae. Kellis
-Properties of structured association approaches to detecting population stratification. Purcell
-A proposed role for the Polycomb group protein dRING in meiotic sister-chromatid cohesion.Balicky
-Rapid analysis of the DNA-binding specificities of transcription factors with DNA microarrays. Mukherjee.
-Reconstruction of functionally normal and malignant human breast tissues in mice. Kuperwasser
-Recovering frequencies of known haplotype blocks from single-nucleotide polymorphism allele frequencies Pe'er.
-Regulation of insulin sensitivity adipose tissue-derived hormones and inflammatory cytokines Ruan.
-Reprogramming of a melanoma genome by nuclear transplantation. Hochedlinger
-Relating microstructure to rheology of a bundled and cross-linked F-actin network in vitro Shin.
-Rictor, a Novel Binding Partner of mTOR, Defines a Rapamycin-Insensitive and Raptor-Independent Pathway that Regulates the Cytoskeleton.Sarbassov
-Role for toll-like receptor 4 in dendritic cell activation and cytolytic CD8(+) T cell differentiation in response to a recombinant heat shock fusion protein. Palliser
-Role of proneuregulin 1 cleavage and human epidermal growth factor receptor activation in hypertonic aquaporin induction. Herrlich
-Saccharomyces cerevisiae SSD1-V confers longevity by a Sir2p-independent mechanism Kaeberlein
-SANT domain: a unique histone-tail-binding module? Boyer
-Scaling of F-actin network rheology to probe single filament elasticity and dynamics. Gardel
-Segmental phylogenetic relationships of inbred mouse strains revealed by fine-scale analysis of sequence variation across 4.6 Mb of mouse genome Frazer
-Selective pressures on the olfactory receptor repertoire since the human-chimpanzee divergence Gimelbrant
-Signal integration during development: Insights from the Drosophila eye. Voas
-siRNA Selection Server: an automated siRNA oligonucleotide prediction server. Yuan
-The Six1 homeoprotein stimulates tumorigenesis by reactivation of cyclin A1. Coletta
-Small interfering RNA production by enzymatic engineering of DNA (SPEED). Luo
-Specification of the enveloping layer and lack of autoneuralization in zebrafish embryonic explants.Sagerstrom
-Spinocerebellar ataxia 8 noncoding RNA causes neurodegeneration and associates with staufen in Drosophila. Mutsuddi
-Stochastic yet biased expression of multiple Dscam splice variants by individual cells. Neves
-Strategies of vertebrate neurulation and a re-evaluation of teleost neural tube formation. Lowery
-Structural test of the parameterized-backbone method for protein design.Plecs
-Structure of the acrosomal bundle.Schmid
-Systematic genome-wide screens of gene function. Carpenter
-A systems approach to discovering signaling and regulatory pathways--or, how to digest large interaction networks into relevant pieces.Ideker
-Telomerase: Promise and challenge Ince
-Three-dimensional architecture of the class I ligase ribozyme. Bergman
-Three-dimensional network photonic crystals via cyclic size reduction/infiltration of sea urchin exoskeleton. Ha
-Total serum protein N-glycome profiling on a capillary electrophoresis-microfluidics platform.Callewaert
-Towards a treatment for spinal muscular atrophy: looking for drugs that increase SMN protein levels. Man
-TRA-1/GLI controls development of somatic gonadal precursors in C-elegans Mathies
-A transcriptional profiling study of CCAAT/enhancer binding protein targets identifies hepatocyte nuclear factor 3 beta as a novel tumor suppressor in lung cancer. Halmos
-Transcriptional regulatory code of a eukaryotic genome.Harbison
-Transcriptional response of Candida albicans upon internalization by macrophages. Lorenz
-Transdifferentiation and adult stem cells: Myth or reality? Jaenisch
-Transgenic rescue demonstrates involvement of the Ian5 gene in T cell development in the rat .Michalkiewicz
-Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Yang
-2003 Curt Stern Award address - On low expectations exceeded; or, the genomic salvation of the Y chromosome. Page
-Tyrosol is a quorum-sensing molecule in Candida albicans. Chen
-Under cover: causes, effects and implications of Hsp90-mediated genetic capacitance. Sangster
-Uracil DNA glycosylase activity is dispensable for immunloglobulin class switch .Begum
-Visfatin: A New Adipokine Hug
-When cells get stressed: an integrative view of cellular senescence. Ben-Porath
-Wtl functions in the development of germ cells in addition to somatic cell lineages of the testis.Natoli
-X chromosome in population genetics. Schaffner
-An X-to-autosome retrogene is required for spermatogenesis in mice Bradley
-Zebrafish unplugged reveals a role for muscle-specific kinase homologs in axonal pathway choice.Zhang

Abecasis, G., Cox, N., Daly, M. J., Kruglyak, L., Laird, N., Markianos, K. and Patterson, N. (2004) No bias in linkage analysis. American Journal of Human Genetics. 75 722-723. Full Text.

Andalis, A. A., Storchova, Z., Styles, C., Galitski, T., Pellman, D. and Fink, G. R. (2004) Defects Arising From Whole-Genome Duplications in Saccharomyces cerevisiae. Genetics 167 1109-21. Comparisons among closely related species have led to the proposal that the duplications found in many extant genomes are the remnants of an ancient polyploidization event, rather than a result of successive duplications of individual chromosomal segments. If this interpretation is correct, it would support Ohno's proposal that polyploidization drives evolution by generating the genetic material necessary for the creation of new genes. Paradoxically, analysis of contemporary polyploids suggests that increased ploidy is an inherently unstable state. To shed light on this apparent contradiction and to determine the effects of nascent duplications of the entire genome, we generated isogenic polyploid strains of the budding yeast Saccharomyces cerevisiae. Our data show that an increase in ploidy results in a marked decrease in a cell's ability to survive during stationary phase in growth medium. Tetraploid cells die rapidly, whereas isogenic haploids remain viable for weeks. Unlike haploid cells, which arrest growth as unbudded cells, tetraploid cells continue to bud and form mitotic spindles in stationary phase. The stationary-phase death of tetraploids can be prevented by mutations or conditions that result in growth arrest. These data show that whole-genome duplications are accompanied by defects that affect viability and subsequent survival of the new organism. Full Text

Askew, D. J., Askew, Y. S., Kato, Y., Turner, R. F., Dewar, K., Lehoczky, J. and Silverman, G. A. (2004) Comparative genomic analysis of the clade B serpin cluster at human chromosome 18q21: amplification within the mouse squamous cell carcinoma antigen gene locus. Genomics 84 176-184. The human clade B serpins neutralize serine or cysteine proteinases and reside predominantly within the intracellular compartment. Genomic analysis shows that the 13 human clade B serpins map to either 6p25 (n = 3) or 18q21 (n = 10). Similarly, the mouse clade B serpins map to syntenic loci at 13A3.2 and 1D, respectively. The mouse clade B cluster at 13A3.2 shows a marked expansion in the number of serpin genes (n = 15). The purpose of this study was to determine whether a similar expansion occurred at 1D. Using STS-content mapping, comparative genomic DNA sequence analysis, and cDNA cloning, we found that the mouse clade B cluster at 1D showed nearly complete conservation of gene number, order, and orientation relative to those of 18q21. The only exception was the squamous cell carcinoma antigen (SCCA) locus. The human SCCA locus contains two genes, SERPINB3 (SCCA1) and SERPINB4 (SCCA2), whereas the mouse locus contains four serpins and three pseudogenes. Based on phylogenetic analysis and predicted amino acid sequences, amplification of the mouse SCCA locus occurred after rodents and primates diverged and was associated with some diversification of proteinase inhibitory activity relative to that of humans.Full Text.

Bailey SN, Sabatini DM, Stockwell BR. (2004) Microarrays of small molecules embedded in biodegradable polymers for use in mammalian cell-based screens. PNAS 101 16144-16149.We developed a microarray-based system for screening small molecules in mammalian cells. This system is compatible with image-based screens and requires fewer than 100 cells per compound. Each compound is impregnated in a 200-mum-diameter disc composed of biodegradable poly-(D),(L)-lactide/glycolide copolymer. Cells are seeded on top of these discs, and compounds slowly diffuse out, affecting proximal cells. In contrast with microtiter-based screening, this system does not involve the use of wells or walls between each compound-treated group of cells. We demonstrate detection of the effects of a single compound in a large microarray, that diverse compounds can be released in this format, and that extended release over several days is feasible. We performed a small synthetic lethal screen and identified a compound (macbecin II) that has reduced activity in cells with RNA interference-mediated decrease in the expression of tuberous sclerosis 2. Thus, we have developed a microarray-based screening system for testing the effects of small molecules on mammalian cells by using an imaging-based readout. This method will be useful to those performing small-molecule screens to discover new chemical tools and potential therapeutic agents. PDF

Balicky, E. M., Young, L., Orr-Weaver, T. L., and Bickel, S. E. (2004). A proposed role for the Polycomb group protein dRING in meiotic sister-chromatid cohesion. Chromosoma 112, 231-239. ORD protein is required for accurate chromosome segregation during male and female meiosis in Drosophila melanogaster. Null ord mutations result in random segregation of sister chromatids during both meiotic divisions because cohesion is completely abolished prior to kinetochore capture of microtubules during meiosis I. Previous analyses of mutant ord alleles have led us to propose that the C-terminal half of the ORD protein mediates protein-protein interactions that are essential for sister-chromatid cohesion. To identify proteins that interact with ORD, we conducted a yeast two-hybrid screen using an ORD bait and isolated dRING, a core subunit of the Drosophila Polycomb repressive complex 1. We show that a missense mutation in ORD completely ablates the two-hybrid interaction with dRING and prevents nuclear retention of the mutant ORD protein in male meiotic cells. Using affinity-purified antibodies generated against full-length recombinant dRING, we demonstrate that dRING protein is expressed in the male and female gonads and colocalizes extensively with ORD on the chromatin of primary spermatocytes during G2 of meiosis. Our results suggest a novel role for the Polycomb group protein dRING and are consistent with the model that interaction of dRING and ORD is required to promote the proper segregation of meiotic chromosomes.Full Text.

Barbouti, A., Stankiewicz, P., Nusbaum, C., Cuomo, C., Cook, A., Hoglund, M., Johansson, B., Hagemeijer, A., Park, S. S., Mitelman, F., et al. (2004). The breakpoint region of the most common isochromosome, i(17q), in human neoplasia is characterized by a complex genomic architecture with large, palindromic, low-copy repeats. American Journal of Human Genetics 74, 1-10. Although a great deal of information has accumulated regarding the mechanisms underlying constitutional DNA rearrangements associated with inherited disorders, virtually nothing is known about the molecular processes involved in acquired neoplasia-associated chromosomal rearrangements. Isochromosome 17q, or "i( 17q)," is one of the most common structural abnormalities observed in human neoplasms. We previously identified a breakpoint cluster region for i(17q) formation in 17p11.2 and hypothesized that genome architectural features could be responsible for this clustering. To address this hypothesis, we precisely mapped the i( 17q) breakpoints in 11 patients with different hematologic malignancies and determined the genomic structure of the involved region. Our results reveal a complex genomic architecture in the i( 17q) breakpoint cluster region, characterized by large (similar to 38 - 49-kb), palindromic, low-copy repeats, strongly suggesting that somatic rearrangements are not random events but rather reflect susceptibilities due to the genomic structure. Full Text.

Barrett, J. C., Fry, B., Maller, J. and Daly, M. J. (2004) Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2004 Aug 5 [Epub ahead of print]. Research over the last few years has revealed significant haplotype structure in the human genome. The characterization of these patterns, particularly in the context of medical genetic association studies, is becoming a routine research activity. Haploview is a software package that provides computation of linkage disequilibrium statistics and population haplotype patterns from primary genotype data in a visually appealing and interactive interface. SUMMARY: A program for visualizing and analyzing LD and haplotype patterns in genotype data. AVAILABILITY: http://www.broad.mit.edu/personal/jcbarret/haploview/. PDF

Bartel, D. P., and Chen, C. Z. (2004). Opinion: Micromanagers of gene expression: the potentially widespread influence of metazoan microRNAs. Nat Rev Genet 5, 396-400. We propose that the microRNA milieu, unique to each cell type, productively dampens the expression of thousands of mRNAs and provides important context for the evolution of all metazoan mRNA sequences. For genes that should not be expressed in a particular cell type, protein output is lowered to inconsequential levels. For other genes, dosage is adjusted in a manner that allows for customized expression in different cell types while achieving a more uniform level within each cell type. In these ways, the microRNAs add an extensive layer of gene control that integrates with transcriptional and other regulatory processes to expand the complexity of metazoan gene expression. Full Text.

Bartel, D. P. (2004). MicroRNAs. Genomics, Biogenesis, Mechanism, and Function. Cell 116, 281-297. MicroRNAs (miRNAs) are endogenous approximately 22 nt RNAs that can play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression. Although they escaped notice until relatively recently, miRNAs comprise one of the more abundant classes of gene regulatory molecules in multicellular organisms and likely influence the output of many protein-coding genes. Full Text.

Begum, N. A., Kinoshita, K., Kakazu, N., Muramatsu, M., Nagaoka, H., Shinkura, R., Biniszkiewicz, D., Boyer, L. A., Jaenisch, R. and Honjo, T. (2004) Uracil DNA glycosylase activity is dispensable for immunloglobulin class switch. Science. 305 1160-1163. Activation-induced cytidine deaminase (AID) is required for the DNA cleavage step in immunoglobulin class switch recombination (CSR). AID is proposed to deaminate cytosine to generate uracil (U) in either mRNA or DNA. In the second instance, DNA cleavage depends on uracil DNA glycosylase (UNG) for removal of U. Using phosphorylated histone gamma-H2AX focus formation as a marker of DNA cleavage, we found that the UNG inhibitor Ugi did not inhibit DNA cleavage in immunoglobulin heavy chain (IgH) locus during CSR, even though Ugi blocked UNG binding to DNA and strongly inhibited CSR. Strikingly, UNG mutants that had lost the capability of removing U rescued CSR in UNG(-/-) B cells. These results indicate that UNG is involved in the repair step of CSR yet by an unknown mechanism. The dispensability of U removal in the DNA cleavage step of CSR requires a reconsideration of the model of DNA deamination by AID. Full Text.

Ben-Porath, I., and Weinberg, R. A. (2004). When cells get stressed: an integrative view of cellular senescence. J Clin Invest 113, 8-13. Cells entering a state of senescence undergo a permanent cell cycle arrest, accompanied by a set of functional and morphological changes. Senescence of cells occurs following an extended period of proliferation in culture or in response to various physiologic stresses, yet little is known about the role this phenomenon plays in vivo. The study of senescence has focused largely on its hypothesized role as a barrier to extended cell division, governed by a division-counting mechanism in the form of telomere length. Here, we discuss the biological functions of cellular senescence and suggest that it should be viewed in terms of its role as a general cellular stress response program, rather than strictly as a barrier to unlimited cycles of cell growth and division. We also discuss the relative roles played by telomere shortening and telomere uncapping in the induction of senescence.Full Text.

Bergman, N. H., Lau, N. C., Lehnert, V., Westhof, E., and Bartel, D. P. (2004). The three-dimensional architecture of the class I ligase ribozyme. Rna 10, 176-184. The class I ligase ribozyme catalyzes a Mg(++)-dependent RNA-ligation reaction that is chemically analogous to a single step of RNA polymerization. Indeed, this ribozyme constitutes the catalytic domain of an accurate and general RNA polymerase ribozyme. The ligation reaction is also very rapid in both single- and multiple-turnover contexts and thus is informative for the study of RNA catalysis as well as RNA self-replication. Here we report the initial characterization of the three-dimensional architecture of the ligase. When the ligase folds, several segments become protected from hydroxyl-radical cleavage, indicating that the RNA adopts a compact tertiary structure. Ribozyme folding was largely, though not completely, Mg(++) dependent, with a K(1/2[Mg]) < 1 mM, and was observed over a broad temperature range (20 degrees C -50 degrees C). The hydroxyl-radical mapping, together with comparative sequence analyses and analogy to a region within 23S ribosomal RNA, were used to generate a three-dimensional model of the ribozyme. The predictive value of the model was tested and supported by a photo-cross-linking experiment. Full Text.

Blelloch, R. H., Hochedlinger, K., Yamada, Y., Brennan, C., Kim, M., Mintz, B., Chin, L. and Jaenisch, R. (2004) Nuclear cloning of embryonal carcinoma cells Proc Natl Acad Sci U S A. 2004 Aug 11 [Epub ahead of print]. This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 29, 2003. Embryonal carcinoma (EC) cells have served as a model to study the relationship between cancer and cellular differentiation given their potential to produce tumors and, to varying degrees, participate in embryonic development. Here, nuclear transplantation was used to assess the extent to which the tumorigenic and developmental potential of EC cells is governed by epigenetic as opposed to genetic alterations. Nuclei from three independent mouse EC cell lines (F9, P19, and METT-1) with differing developmental and tumorigenic potentials all were able to direct early embryo development, producing morphologically normal blastocysts that gave rise to nuclear transfer (NT)-derived embryonic stem (ES) cell lines at a high efficiency. However, when tested for tumor or chimera formation, the resulting NT ES cells displayed an identical potential as their respective donor EC cells, in stark contrast to previously reported NT ES cells derived from transfer of untransformed cells. Consistent with this finding, comparative genomic hybridization identified previously undescribed genetic lesions in the EC cell lines. Therefore, nonreprogrammable genetic modifications within EC nuclei define the developmental and tumorigenic potential of resulting NT ES cells. Our findings support the notion that cancer results from the deregulation of stem cells and further suggest that the genetics of ECs will reveal genes involved in stem cell self-renewal and pluripotency. PDF

Borkovich, K. A., Alex, L. A., Yarden, O., Freitag, M., Turner, G. E., Read, N. D., Seiler, S., Bell-Pedersen, D., Paietta, J., Plesofsky, N.,Galagan, J.E,.et al. (2004). Lessons from the Genome Sequence of Neurospora crassa: Tracing the Path from Genomic Blueprint to Multicellular Organism. Microbiol Mol Biol Rev 68, 1-108. We present an analysis of over 1,100 of the approximately 10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.Full Text.

Bortvin, A., Goodheart, M., Liao, M., and Page, D. C. (2004). Dppa3 / Pgc7 / stella is a maternal factor and is not required for germ cell specification in mice. BMC Dev Biol 4, 2. BACKGROUND: In mice, germ cells are specified through signalling between layers of cells comprising the primitive embryo. The function of Dppa3 (also known as Pgc7 or stella), a gene expressed in primordial germ cells at the time of their emergence in gastrulating embryos, is unknown, but a recent study has claimed that it plays a central role in germ cell specification. RESULTS: To test Dppa3's role in germ cell development, we disrupted the gene in mouse embryonic stem cells and generated mutant animals. We were able to obtain viable and fertile Dppa3-deficient animals of both sexes. Examination of embryonic and adult germ cells and gonads in Dppa3-deficient animals did not reveal any defects. However, most embryos derived from Dppa3-deficient oocytes failed to develop normally beyond the four-cell stage. CONCLUSION: We found that Dppa3 is an important maternal factor in the cleavage stages of mouse embryogenesis. However, it is not required for germ cell specification.Full Text.

Boufounos, P., El-Difrawy, S., and Ehrlich, D. (2004). Basecalling using hidden Markov models. Journal of the Franklin Institute-Engineering and Applied Mathematics 341, 23-36. In this paper we propose hidden Markov models to model electropherograms from DNA sequencing equipment and perform basecalling. We model the state emission densities using artificial neural networks, and modify the Baum-Welch reestimation procedure to perform training. Moreover, we develop a method that exploits consensus sequences to label training data, thus minimizing the need for hand labeling. We propose the same method for locating an electropherogram in a longer DNA sequence. We also perform a careful study of the basecalling errors and propose alternative HMM topologies that might further improve performance. Our results demonstrate the potential of these models. Based on these results, we conclude by suggesting further research directions.Full text.

Boutin, S. R., Rogers, A. B., Shen, Z., Fry, R. C., Love, J. A., Nambiar, P. R., Suerbaum, S. and Fox, J. G. (2004) Hepatic temporal gene expression profiling in Helicobacter hepaticus-infected A/JCr mice. Toxicologic Pathology. 32 678-693. Helicobacter hepaticus infection of A/JCr mice is a model of infectious liver cancer. We monitored hepatic global gene expression profiles in H. hepaticus infected and control male A/JCr mice at 3 months, 6 months, and 1 year of age using an Affymetrix-based oligonucleotide microarray platform on the premise that a specific genetic expression signature at isolated time points would be indicative of disease status. Model based expression index comparisons generated by dChip yielded consistent profiles of differential gene expression for H. hepaticus infected male mice with progressive liver disease versus uninfected control mice within each age group. Linear discriminant analysis and principal component analysis allowed segregation of mice based on combined age and lesion status, or age alone. Up-regulation of putative tumor markers correlated with advancing hepatocellular dysplasia. Transcriptionally down-regulated genes in mice with liver lesions included those related to peroxisome proliferator, fatty acid, and steroid metabolism pathways. In conclusion, transcriptional profiling of hepatic genes documented gene expression signatures in the livers of H. hepaticus infected male A/JCr mice with chronic progressive hepatitis and preneoplastic liver lesions, complemented the histopathological diagnosis, and suggested molecular targets for the monitoring and intervention of disease progression prior to the onset of hepatocellular neoplasia.

Boyer, L. A., Latek, R. R., and Peterson, C. L. (2004). The SANT domain: a unique histone-tail-binding module? Nature Reviews Molecular Cell Biology 5, 158-163. Chromatin-remodelling complexes have an important role in all DNA-mediated processes and, although much is known about how these enzymes regulate chromosomal DNA accessibility, how they interact with their histone substrates has remained unclear. However, recent studies have indicated that the SANT domain has a central role in chromatin remodelling by functioning as a unique histone-interaction module that couples histone binding to enzyme catalysis.Full Text.

Bradley, J., Baltus, A., Skaletsky, H., Royce-Tolland, M., Dewar, K. and Page, D. C. (2004) An X-to-autosome retrogene is required for spermatogenesis in mice. Nat Genet 36, 872 - 876 We identified the gene carrying the juvenile spermatogonial depletion mutation (jsd), a recessive spermatogenic defect mapped to mouse chromosome 1 (refs. 1,2). We localized jsd to a 272-kb region and resequenced this area to identify the underlying mutation: a frameshift that severely truncates the predicted protein product of a 2.3-kb genomic open reading frame. This gene, Utp14b, evidently arose through reverse transcription of an mRNA from an X-linked gene and integration of the resulting cDNA into an intron of an autosomal gene, whose promoter and 5' untranslated exons are shared with Utp14b. To our knowledge, Utp14b is the first protein-coding retrogene to be linked to a recessive mammalian phenotype. The X-linked progenitor of Utp14b is the mammalian ortholog of yeast Utp14, which encodes a protein required for processing of pre-rRNA and hence for ribosome assembly. Our findings substantiate the hypothesis that mammalian spermatogenesis is supported by autosomal retrogenes that evolved from X-linked housekeeping genes to compensate for silencing of the X chromosome during male meiosis. We find that Utp14b-like retrogenes arose independently and were conserved during evolution in at least four mammalian lineages. This recurrence implies a strong selective pressure, perhaps to enable ribosome assembly in male meiotic cells.Full Text.

Bromley E, Knapp D, Wardle FC, Sun BI, Collins-Racie L, Lavallie E, Smith JC, Sive HL. Identification and characterisation of the posteriorly-expressed Xenopus neurotrophin receptor homolog genes fullback and fullback-like. Gene Expr Patterns 2004;5(1):135-140. Full Text

Callewaert, N., Contreras, R., Mitnik-Gankin, L., Carey, L., Matsudaira, P. and Ehrlich, D. (2004) Total serum protein N-glycome profiling on a capillary electrophoresis-microfluidics platform. Electrophoresis. 25 3128-3131. We implemented 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled asparagine-linked glycan (N-glycan) profiling on a microfluidic electrophoresis platform. Using 11.5 cm effective length etched channels and 4% linear polyacrylamide as the separation matrix, the major N-glycans in human serum were profiled in 12 min with a resolution comparable to what is achieved for these analytes on gel-based DNA sequencers. This demonstration suggests a practical clinical application for highspeed compact analyzers which might be uniquely based on microfluidic devices. PDF.

Carpenter, A. E., and Belmont, A. S. (2004). Direct visualization of transcription factor-induced chromatin remodeling and cofactor recruitment in vivo. Methods Enzymol 375, 366-381.

Carpenter, A. E., and Sabatini, D. M. (2004). Systematic genome-wide screens of gene function. Nat Rev Genet 5, 11-22. By using genome information to create tools for perturbing gene function, it is now possible to undertake systematic genome-wide functional screens that examine the contribution of every gene to a biological process. The directed nature of these experiments contrasts with traditional methods, in which random mutations are induced and the resulting mutants are screened for various phenotypes. The first genome-wide functional screens in Caenorhabditis elegans and Drosophila melanogaster have recently been published, and screens in human cells will soon follow. These high-throughput techniques promise the rapid annotation of genomes with high-quality information about the biological function of each gene. Full Text.

Chen, H., Fujita, M., Feng, Q., Clardy, J., and Fink, G. R. (2004). Tyrosol is a quorum-sensing molecule in Candida albicans. Proc Natl Acad Sci U S A. The human fungal pathogen Candida albicans shows a significant lag in growth when diluted into fresh minimal medium. This lag is abolished by the addition of conditioned medium from a high-density culture. The active component of conditioned medium is tyrosol, which is released into the medium continuously during growth. Under conditions permissive for germ-tube formation, tyrosol stimulates the formation of these filamentous protrusions. Because germ-tube formation is inhibited by farnesol, another quorum-sensing molecule, this process must be under complex positive and negative control by environmental conditions. The identification of tyrosol as an autoregulatory molecule has important implications on the dynamics of growth and morphogenesis in Candida. PDF.

Chen, C. Z., Li, L., Lodish, H. F., and Bartel, D. P. (2004). MicroRNAs modulate hematopoietic lineage differentiation. Science 303, 83-86. MicroRNAs (miRNAs) are an abundant class of similar to22-nucleotide regulatory RNAs found in plants and animals. Some miRNAs of plants, Caenorhabditis elegans, and Drosophila play important gene-regulatory roles during development by pairing to target mRNAs to specify posttranscriptional repression of these messages. We identify three miRNAs that are specifically expressed in hematopoietic cells and show that their expression is dynamically regulated during early hematopoiesis and lineage commitment. One of these miRNAs, miR-181, was preferentially expressed in the B-lymphoid cells of mouse bone marrow, and its ectopic expression in hematopoietic stem/progenitor cells led to an increased fraction of B-lineage cells in both tissue-culture differentiation assays and adult mice. Our results indicate that microRNAs are components of the molecular circuitry that controls mouse hematopoiesis and suggest that other microRNAs have similar regulatory roles during other facets of vertebrate development. Full Text.

Choong, M. L., Luo, B., and Lodish, H. F. (2004). Microenvironment-driven changes in the expression profile of hematopoietic cobblestone area-forming cells. Annals of Hematology 83, 160-169. Studies with ex vivo cultures of bone marrow have indicated the importance of the adherent layer as a primary reservoir of the most primitive hematopoietic stem cells, from which derivative stem cells and more differentiated progenitors are continuously generated. We used the Affymetrix GeneChip to analyze the mRNA expressions between bone marrow-derived hematopoietic progenitor cells in the cobblestone areas (CA) and the free-floating cells released from the CA formations. Mouse bone marrow hematopoietic progenitor cell line FDCP-Mix and S17 stromal cells were used in this study. Of the 12,000 genes on the chip, only 29 showed more than fivefold higher in CAFC; and for cells in the supernatant, only 55 showed fivefold higher expressions than in the cobblestone area-forming cells (CAFC). The hematopoietic cells in CAFC expressed genes associated with homing, adhesion, and suppression of differentiation, while the free-floating hematopoietic cells showed mature lineage markers and differentiation-specific genes. This confirmed the more primitive nature of the hematopoietic cells in the adherent layer. Of interest in the findings were the discoveries of many secreted and surface protein expressions in CA hematopoietic cells. This may imply interactions among the hematopoietic cells, stromal cells, and the extracellular matrix in CA, which drive the growth, maturation, and differentiation of the hematopoietic cells.

Choong, M. L., Yong, Y. P., Tan, A. C. L., Luo, B., and Lodish, H. F. (2004). LIX: a chemokine with a role in hematopoietic stem cells maintenance. Cytokine 25, 239-245. LIX is a chemokine usually associated with cell migration and activation in neutrophil. While using a microarray approach to dissect the hematopoietic microenvironment, we have discovered that LIX is also expressed in the hematopoietic stromal cells and its expression is associated with hematopoietic supportive phenotypes. LIX microarray profiles were verified using reverse transcription and semi-quantitative polymerase chain reaction. We subsequently tested the effects of LIX on the primary bone marrow derived lineage depleted (Lin(-)) cells. LIX was found to increase the number of long-term culture-initiating cells by 34% (from 1 in 342 to 1 in 255 of Lin(-) cells). LIX also increased the clonogenicity of the long-term culture Lin(-) cells by 2-fold (p = 0.029). When compared to untreated EML hematopoietic progenitor cell line, LIX was found to increase the level of DNA synthesis in EML cells significantly (1.63-fold; p = 0.010), with a corresponding increase in viable cell number by 1.11-fold 96 h after seeding. Similar effect was not observed with the M I mature myeloid leukemia cell line or with the MS5.1 and AFT024 stromal cell lines. This suggested that the proliferative effect of LIX is specific towards the primitive hematopoietic cells. Full Text

Claycomb, J. M., Benasutti, M., Bosco, G., Fenger, D. D., and Orr-Weaver, T. L. (2004). Gene amplification as a developmental strategy. Isolation of two developmental amplicons in Drosophila. Dev Cell 6, 145-155. Gene amplification is known to be critical for upregulating gene expression in a few cases, but the extent to which amplification is utilized in the development of diverse organisms remains unknown. By quantifying genomic DNA hybridization to microarrays to assay gene copy number, we identified two additional developmental amplicons in the follicle cells of the Drosophila ovary. Both amplicons contain genes which, following their amplification, are expressed in the follicle cells, and the expression of three of these genes becomes restricted to specialized follicle cells late in differentiation. Genetic analysis establishes that at least one of these genes, yellow-g, is critical for follicle cell function, because mutations in yellow-g disrupt eggshell integrity. Thus, during follicle cell differentiation the entire genome is overreplicated as the cells become polyploid, and subsequently specific genomic intervals are overreplicated to facilitate gene expression. Full Text.

Colby, D. W., Chu, Y., Cassady, J. P., Duennwald, M., Zazulak, H., Webster, J. M., Messer, A., Lindquist, S., Ingram, V. M. and Wittrup, K. D. (2004) Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody. Proc Natl Acad Sci U S A. 101 17616-17621 Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion in the number of polyglutamine-encoding CAG repeats in the gene that encodes the huntingtin (htt) protein. A property of the mutant protein that is intimately involved in the development of the disease is the propensity of the glutamine-expanded protein to misfold and generate an N-terminal proteolytic htt fragment that is toxic and prone to aggregation. Intracellular antibodies (intrabodies) against htt have been shown to reduce htt aggregation by binding to the toxic fragment and inactivating it or preventing its misfolding. Intrabodies may therefore be a useful gene-therapy approach to treatment of the disease. However, high levels of intrabody expression have been required to obtain even limited reductions in aggregation. We have engineered a single-domain intracellular antibody against htt for robust aggregation inhibition at low expression levels by increasing its affinity in the absence of a disulfide bond. Furthermore, the engineered intrabody variable light-chain (VL)12.3, rescued toxicity in a neuronal model of HD. We also found that VL12.3 inhibited aggregation and toxicity in a Saccharomyces cerevisiae model of HD. VL12.3 is significantly more potent than earlier anti-htt intrabodies and is a potential candidate for gene therapy treatment for HD. To our knowledge, this is the first attempt to improve affinity in the absence of a disulfide bond to improve intrabody function. The demonstrated importance of disulfide bond-independent binding for intrabody potency suggests a generally applicable approach to the development of effective intrabodies against other intracellular targets.PDF

Coletta, R. D., Christensen, K., Reichenberger, K. J., Lamb, J., Micomonaco, D., Huang, L. L., Wolf, D. M., Muller-Tidow, C., Golub, T. R., Kawakami, K., and Ford, H. L. (2004). The Six1 homeoprotein stimulates tumorigenesis by reactivation of cyclin A1. Proceedings of the National Academy of Sciences of the United States of America 101, 6478-6483. Homeobox genes constitute a large family of transcription factors that are essential during normal development and are often dysregulated in cancer. However, the molecular mechanisms by which homeobox genes influence cancer remain largely unknown. Here we show that the tissue-restricted cyclin A1 is a transcriptional target of the Six1 homeoprotein. Both genes are expressed in the embryonic but not the terminally differentiated mammary gland, and Six1-knockout mice show a dramatic reduction of cyclin A1 in the embryonic mammary gland. In addition, both genes are reexpressed in breast cancers. Six1 overexpression increases cyclin Al mRNA levels and activity, cell proliferation, and tumor volume, whereas Six1 down-regulation decreases cyclin A1 mRNA levels and proliferation. Overexpression of Six1 in wild-type mouse embryonic fibroblasts, but not in knockout variants lacking the cyclin A1 gene, induces cell proliferation. Furthermore, inhibition of cyclin A1 in Six1-overexpressing mammary carcinoma cells decreases proliferation. Together these results demonstrate that cyclin A1 is required for the proliferative effect of Six1. We conclude that Six1 overexpression reinstates an embryonic pathway of proliferation in breast cancer by up-regulating cyclin A1.Full Text.

Daheron, L., Opitz, S. L., Zaehres, H., Lensch, W. M., Andrews, P. W., Itskovitz-Eldor, J. and Daley, G. Q. (2004) LIF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells. Stem Cells. 22 770-8. Murine embryonic stem (mES) cells remain undifferentiated in the presence of leukemia inhibitory factor (LIF), and activation of signal transducer and activator of transcription 3 (STAT3) via LIF receptor (LIFR) signaling appears sufficient for maintenance of mES cell pluripotency. Anecdotal and contradictory accounts exist for the action of LIF in the culture of human embryonic stem cells, and the nature of LIF signaling and whether the LIF-STAT3 pathway is conserved in human embryonic stem cells (hESCs) has not been systematically explored. In this study, we show that the LIFRbeta and the signaling subunit gp130 are expressed in hESCs and that human LIF can induce STAT3 phosphorylation and nuclear translocation in hESCs. Nevertheless, despite the functional activation of the LIF-STAT3 signaling pathway, human LIF is unable to maintain the pluripotent state of hESCs. Feeder-free culture conditions that maintain hESCs in an undifferentiated state do not show activation of STAT3, suggesting that distinct signaling mechanisms govern the self-renewal of hESCs. Full Text.

Daly, M. J., and Rioux, J. D. (2004). New approaches to gene hunting in IBD. Inflammatory Bowel Diseases 10, 312-317. Without a doubt, one of the greatest promises of the human genome project has been the expectation that its completion will rapidly accelerate the discovery of the heritable components of complex disease. Such discoveries will educate us as to the underlying causes of disease and hold out great promise for medicine in the future by potentially enabling the prediction of individual risk, the selection of therapy based on genetic profile, and a more rational approach to designing drugs. Unfortunately, discovering these genetic components of complex disease has been much more difficult than initially hoped and, to date, very few positive results have been conclusively confirmed. We review here approaches to tackling the problem of gene finding in complex disease with an emphasis on new approaches that may complement and supplement existing methods that have been making only slow progress toward the elucidation of the genetic architecture of disease. Along the way we highlight the successful work by many groups in studying the genetics of IBD, where significant successes have arrived earlier than in most other complex human diseases.

Dej, K. J., Ahn, C. and Orr-Weaver, T. L. (2004) Mutations in the Drosophila Condensin Subunit dCAP-G: Defining the Role of Condensin for Chromosome Condensation in Mitosis and Gene Expression in Interphase. Genetics. 168 895-906. Chromosomes are dynamic structures that are reorganized during the cell cycle to optimize them for distinct functions. SMC and non-SMC condensin proteins associate into complexes that have been implicated in the process of chromosome condensation. The roles of the individual non-SMC subunits of the complex are poorly understood, and mutations in the CAP-G subunit have not been described in metazoans. Here we elucidate a role for dCAP-G in chromosome condensation and cohesion in Drosophila. We illustrate the requirement of dCAP-G for condensation during prophase and prometaphase; however, we find that alternate mechanisms ensure that replicated chromosomes are condensed prior to metaphase. In contrast, dCAP-G is essential for chromosome condensation in metaphase of single, unreplicated sister chromatids, suggesting that there is an interplay between replicated chromatids and the condensin complex. In the dcap-g mutants, defects in sister-chromatid separation are also observed. Chromatid arms fail to resolve in prophase and are unable to separate at anaphase, whereas sister centromeres show aberrant separation in metaphase and successfully move to spindle poles at anaphase. We also identified a role for dCAP-G during interphase in regulating heterochromatic gene expression. Full Text

Derkatch, I. L., Uptain, S. M., Outeiro, T. F., Krishnan, R., Lindquist, S. L. and Liebman, S. W. (2004) Effects of Q/N-rich, polyQ, and non-polyQ amyloids on the de novo formation of the [PSI+] prion in yeast and aggregation of Sup35 in vitro. Proceedings of the National Academy of Sciences of the United States of America. 101 12934-12939. Prions are infectious protein conformations that are generally ordered protein aggregates. In the absence of prions, newly synthesized molecules of these same proteins usually maintain a conventional soluble conformation. However, prions occasionally arise even without a homologous prion template. The conformational switch that results in the de novo appearance of yeast prions with glutamine/aspargine (Q/N)-rich prion domains (e.g., [PSI+]), is promoted by heterologous prions with a similar domain (e.g., [RNQ(+)], also known as [PIN+]), or by overexpression of proteins with prion-like Q-, N-, or Q/N-rich domains. This finding led to the hypothesis that aggregates of heterologous proteins provide an imperfect template on which the new prion is seeded. Indeed, we show that newly forming Sup35 and preexisting Rnq1 aggregates always colocalize when [PSI+] appearance is facilitated by the [RNQ(+)] prion, and that Rnq1 fibers enhance the in vitro formation of fibers by the prion domain of Sup35 (NM). The proteins do not however form mixed, interdigitated aggregates. We also demonstrate that aggregating variants of the polyQ-containing domain of huntingtin promote the de novo conversion of Sup35 into [PSI+]; whereas nonaggregating variants of huntingtin and aggregates of non-polyQ amyloidogenic proteins, transthyretin, a-synuclein, and synphilin do not. Furthermore, transthyretin and a-synuclein amyloids do not facilitate NM aggregation in vitro, even though in [PSI+] cells NM and transthyretin aggregates also occasionally colocalize. Our data, especially the in vitro reproduction of the highly specific heterologous seeding effect, provide strong support for the hypothesis of cross-seeding in the spontaneous initiation of prion states. Full Text

Dessain, S. K., Adekar, S. P., Stevens, J. B., Carpenter, K. A., Skorski, M. L., Barnoski, B. L., Goldsby, R. A. and Weinberg, R. A. (2004) High efficiency creation of human monoclonal antibody-producing hybridomas. J Immunol Methods. 291 109-22. The native human antibody repertoire holds unexplored potential for the development of novel monoclonal antibody therapeutics. Current techniques that fuse immortal cells and primary B-lymphocytes are sub-optimal for the routine production of hybridomas that secrete human monoclonal antibodies. We have found that a murine cell line that ectopically expresses murine interleukin-6 (mIL-6) and human telomerase (hTERT) efficiently forms stable human antibody-secreting heterohybridomas through cell fusion with primary human B-lymphocytes. The hybrid cells maintain secretion of human antibodies derived from the primary B-lymphocytes through multiple rounds of cloning. Using splenic B-lymphocytes from a patient immunized with a Streptococcus pneumoniae capsular polysaccharide vaccine, we have succeeded in creating hybridomas that secrete human monoclonal antibodies specific for S. pneumoniae antigens. Using peripheral blood lymphocytes, we have similarly cloned a human antibody that binds a viral antigen. These experiments establish that SP2/0-derived cell lines ectopically expressing mIL-6 and hTERT will enable the rapid cloning of native human monoclonal antibodies.Full Text

Dibner, C., Elias, S., Ofir, R., Souopgui, J., Kolm, E. J., Sive, H., Pieler, T. and Frank, D. (2004) The Meis3 protein and retinoid signaling interact to pattern the Xenopus hindbrain. Developmental Biology 271 75-86. In Xenopus embryos, proper hindbrain formation requires activities of both XMeis3 protein and retinoic acid (RA) signaling. In this study, we show that XMeis3 protein and RA signaling differentially interact to regulate hindbrain patterning. The knockdown of XMeis3 protein prevented RA-caudalizing activity from inducing hindbrain marker expression in both explants and embryos. In contrast, inhibition of RA signaling differentially modulated XMeis3 activity. Target genes that are jointly activated by either RA or XMeis3 activities could not be efficiently induced by XMeis3 when RA signaling was inhibited. However, transcription of an XMeis3 target gene that is not an RA target gene was hyper-induced in the absence of retinoid signaling. Target genes jointly induced by RA or XMeis3 protein were synergistically activated in the presence of both activities, while RA treatment inhibits the ability of XMeis3 to activate transcription of neural genes that are not RA targets. HoxD1, an RA direct-target gene was also identified as an XMeis3 direct-target gene. HoxD1 protein acts downstream of XMeis3 to induce hindbrain marker gene transcription. To pattern the hindbrain, RA requires functional XMeis3 protein activity. XMeis3 protein appears crucial for initial hindbrain induction, whereas RA signaling defines the spatial limits of hindbrain gene expression by modifying XMeis3 protein activity Full Text

DiDonato, R. J., Arbuckle, E., Buker, S., Sheets, J., Tobar, J., Totong, R., Grisafi, P., Fink, G. R., and Celenza, J. L. (2004). Arabidopsis ALF4 encodes a nuclear-localized protein required for lateral root formation. Plant J 37, 340-353. Lateral root formation, the primary way plants increase their root mass, displays developmental plasticity in response to environmental changes. The aberrant lateral root formation (alf)4-1 mutation blocks the initiation of lateral roots, thus greatly altering root system architecture. We have positionally cloned the ALF4 gene and have further characterized its phenotype. The encoded ALF4 protein is conserved among plants and has no similarities to proteins from other kingdoms. The gene is present in a single copy in Arabidopsis. Using translational reporters for ALF4 gene expression, we have determined that the ALF4 protein is nuclear localized and that the gene is expressed in most plant tissues; however, ALF4 expression and ALF4's subcellular location are not regulated by auxin. These findings taken together with further genetic and phenotypic characterization of the alf4-1 mutant suggest that ALF4 functions independent from auxin signaling and instead functions in maintaining the pericycle in the mitotically competent state needed for lateral root formation. Our results provide genetic evidence that the pericycle shares properties with meristems and that this tissue plays a central role in creating the developmental plasticity needed for root system development. Full Text.

Eggan, Kevin, Kristin Baldwin, Michael Tackett*, Joseph Osborne, Joseph Gogos, Andrew Chess*, Richard Axel, Rudolf Jaenisch* Mice cloned from olfactory sensory neurons. Nature, published online 15 February 2004.Cloning by nuclear transplantation has been successfully carried out in various mammals, including mice. Until now mice have not been cloned from post-mitotic cells such as neurons. Here, we have generated fertile mouse clones derived by transferring the nuclei of post-mitotic, olfactory sensory neurons into oocytes. These results indicate that the genome of a post-mitotic, terminally differentiated neuron can re-enter the cell cycle and be reprogrammed to a state of totipotency after nuclear transfer. Moreover, the pattern of odorant receptor gene expression and the organization of odorant receptor genes in cloned mice was indistinguishable from wild-type animals, indicating that irreversible changes to the DNA of olfactory neurons do not accompany receptor gene choice.Full Text.

Endres, M., Biniszkiewicz, D., Sobol, R. W., Harms, C., Ahmadi, M., Lipski, A., Katchanov, J., Mergenthaler, P., Dirnagl, U., Wilson, S. H., Meisel, A. and Jaenisch, R. (2004) Increased postischemic brain injury in mice deficient in uracil-DNA glycosylase. Journal of Clinical Investigation 113 1711-1721. Uracil-DNA glycosylase (UNG) is involved in base excision repair of aberrant uracil residues in nuclear and mitochondrial DNA. Ung knockout mice generated by gene targeting are viable, fertile, and phenotypically normal and have regular mutation rates. However, when exposed to a nitric oxide donor, Ung(-/-) fibroblasts show an increase in the uracil/cytosine ratio in the genome and augmented cell death. After combined oxygen-glucose deprivation, Ung(-/-) primary cortical neurons have increased vulnerability to cell death, which is associated with early mitochondrial dysfunction. In vivo, LING expression and activity are low in brains of naive WT mice but increase significantly after reversible middle cerebral artery occlusion and reperfusion. Moreover, major increases in infarct size are observed in Ung(-/-) mice compared with littermate control mice. In conclusion, our results provide compelling evidence that LING is of major importance for tissue repair after brain ischemia. Full Text.

Ensminger, A. W. and Chess, A. (2004) Bidirectional promoters regulate the monoallelically expressed Ly49 NK receptors. Immunity. 21 2-3. Members of the Ly49 gene family of natural killer (NK) cell receptors in mice are expressed in seemingly stochastic combinations such that each NK cell expresses a handful of family members. A transcriptional switch appears to establish this interesting pattern of expression. Full Text

Ensminger, A. W., and Chess, A. (2004). Coordinated replication timing of monoallelically expressed genes along human autosomes. Hum Mol Genet. 2004 13(6) : 651-658. A number of genes in the mammalian genome are expressed from only one of two alleles in either an imprinted or random manner. Those belonging to the random class include X-linked genes subject to X inactivation, as well as a number of autosomal genes, including odorant receptors, immunoglobulins, T-cell receptors, interleukins, natural killer-cell receptors, and pheromone receptors. Monoallelically expressed genes display the unusual property of asynchronous replication and for those genes whose transcription is randomly monoallelic, the asynchronous replication is also random. In mice, recent work has shown that the random asynchronous replication of distributed autosomal genes is coordinated at the whole chromosome level, indicative of chromosome-pair nonequivalence. Here, we show that autosome-pair nonequivalence is present in human cells, and demonstrate its ability to cross the centromere. Additionally, by examining the replication of these genes in a number of human trisomies, we consistently find one allele replicating early and the other two alleles replicating late, similar to previous observations in X trisomies. PDF

Fernandez-Teijeiro, A., Betensky, R. A., Sturla, L. M., Kim, J. Y., Tamayo, P., and Pomeroy, S. L. (2004). Combining Gene Expression Profiles and Clinical Parameters for Risk Stratification in Medulloblastomas. J Clin Oncol. Feb 17 [Epub ahead of print] PURPOSE: Stratification of risk in patients with medulloblastoma remains a challenge. As clinical parameters have been proven insufficient for accurately defining disease risk, molecular markers have become the focus of interest. Outcome predictions on the basis of microarray gene expression profiles have been the most accurate to date. We ask in a multivariate model whether clinical parameters enhance survival predictions of gene expression profiles. PATIENTS AND METHODS: In a cohort of 55 young patients (whose medulloblastoma samples have been analyzed previously for gene expression profile), associations between clinical and gene expression variables and survival were assessed using Cox proportional hazards models. Available clinical variables included age, stage (ie, the presence of disseminated disease at diagnosis), sex, histologic subtype, treatment, and status. RESULTS: Univariate analysis demonstrated expression profiles to be the only significant clinical prognostic factor (P =.03). In multivariate analysis, gene expression profiles predicted outcome independent of other criteria. Clinical criteria did not significantly contribute additional information for outcome predictions, although an exploratory analysis noted a trend for decreased survival of patients with metastases at diagnosis but favorable gene expression profile. CONCLUSION: Gene expression profiling predicts medulloblastoma outcome independent of clinical variables. These results need to be validated in a larger prospective study.

Frazer, K. A., Wade, C. M., Hinds, D. A., Patil, N., Cox, D. R. and Daly, M. J. (2004) Segmental phylogenetic relationships of inbred mouse strains revealed by fine-scale analysis of sequence variation across 4.6 Mb of mouse genome. Genome Research. 14 1493-1500. High-density SNP screening of panels of inbred mouse strains has been proposed as a method to accelerate the identification of genes associated with complex biomedical phenotypes. To evaluate the potential of these studies, a more detailed understanding of the fine structure of sequence variation across inbred mouse strains is needed. Here, we use high-density oligonucleotide arrays to discover all extremely dense set of SNPs in 13 classical and two wild-derived inbred strains in five genomic intervals totaling 4.6 Mb of DNA sequence, and then analyze the segmental haplotype structure defined by these high-density SNPs. This analysis reveals segments ranging from 12 to 608 kb in length within which the inbred strains have a simple and distinct phylogenetic relationship with typically two or three clades accounting for the 13 classical strains examined. The phylogenetic relationships among strains change abruptly and unpredictably from segment to segment, and are distinct in each of the five genomic regions examined. The data suggest that at least 12 strains would need to be resequenced for exhaustive SNP discovery in every region of the mouse genome, that similar to97% of the variation among inbred strains is ancestral (between clades) and similar to3% private (within clades), and provides critical insights into the proposed use of panels of inbred strains to identify genes underlying quantitative trait loci. Full Text

Gao, Q., Hua, J., Kimura, R., Headd, J. J., Fu, X. Y. and Chin, Y. E. (2004) Identification of the linker-SH2 domain of STAT as the origin of the SH2 domain using two-dimensional structural alignment. Molecular & Cellular Proteomics. 3 704-714. The availability of large volumes of genomic sequences presents an unprecedented proteomic challenge to characterize the structure and function of various protein motifs. Primary structural alignment is often unable to accurately identify a given motif due to sequence divergence; however, with the aid of secondary structural prediction for analysis, it becomes feasible to explore protein motifs on a proteome-wide scale. Here we report the use of secondary structural alignment to characterize the Src homology 2 (SH2) domains of both conventional and divergent sequences and divide them into two groups, Src-type and STAT-type. In addition to the basic "alphabetabetabetaalpha" structure (betaB), the Src-type SH2 domain contains an extra beta-strand (betaE or betaE-betaF motif). Alternatively, the linker domain-conjugated SH2 domain in STAT contains the alphaB' motif. Combining BLAST data from betaB core motif sequences with predicted secondary structural alignment, we have screened for SH2 domains in various eukaryotic model systems including Arabidopsis, Dictyostelium, and Saccharomyces. Two novel genes carrying the linker-SH2 domain of STAT were discovered and subsequently cloned from Arabidopsis. These genes, designated as STAT-type linker-SH2 domain factors (STATL), are found in a wide array of vascular and nonvascular plants, suggesting that the linker-SH2 domain evolved prior to the divergence of plants and animals. Using this approach, we expanded the number of putative SH2 domain-bearing genes in Dictyostelium and comparatively studied the secondary structural profiles of both typical and atypical SH2 domains. Our results indicate that the linker-SH2 domain of the transcription factor STAT is one of the most ancient and fully developed functional domains, serving as a template for the continuing evolution of the SH2 domain essential for phosphotyrosine signal transduction. Full Text.

Gardel, M. L., Shin, J. H., MacKintosh, F. C., Mahadevan, L., Matsudaira, P. A. and Weitz, D. A. (2004) Scaling of F-actin network rheology to probe single filament elasticity and dynamics. PHYSICAL REVIEW LETTERS 93 (18): Art. No. 188102 The linear and nonlinear viscoelastic response of networks of cross-linked and bundled cytoskeletal filaments demonstrates remarkable scaling with both frequency and applied prestress, which helps elucidate the origins of the viscoelasticity. The frequency dependence of the shear modulus reflects the underlying single-filament relaxation dynamics for 0.1-10 rad/sec. Moreover, the nonlinear strain stiffening of such networks exhibits a universal form as a function of prestress; this is quantitatively explained by the full force-extension relation of single semiflexible filaments.PDF

Gardel, M. L., Shin, J. H., MacKintosh, F. C., Mahadevan, L., Matsudaira, P., and Weitz, D. A. (2004). Elastic behavior of cross-linked and bundled actin networks. Science 304, 1301-1305. Networks of cross-linked and bundled actin filaments are ubiquitous in the cellular cytoskeleton, but their elasticity remains poorly understood. We show that these networks exhibit exceptional elastic behavior that reflects the mechanical properties of individual filaments. There are two distinct regimes of elasticity, one reflectingbendingof single filaments and a second reflectingstretchingof entropic fluctuations of filament length. The mechanical stiffness can vary by several decades with small changes in cross-link concentration, and can increase markedly upon application of external stress. We parameterize the full range of behavior in a state diagram and elucidate its origin with a robust model. Full Text.

Gaudet, F., Rideout, W. M., 3rd, Meissner, A., Dausman, J., Leonhardt, H., and Jaenisch, R. (2004). Dnmt1 Expression in Pre- and Postimplantation Embryogenesis and the Maintenance of IAP Silencing. Mol Cell Biol 24, 1640-1648. The methylation of intracisternal A-type particle (IAP) sequences is maintained during mouse embryogenesis. Methylation suppresses IAP expression and the potential for mutagenesis by retrotransposition, but it is not clear how methylation of these elements is maintained during the embryonic stages when the bulk of the genome is being demethylated. It has been suggested that the high levels of DNA methyltransferase-1 (Dnmt1) present during cleavage could be important for keeping IAPs methylated. To test this hypothesis, we combined mutant alleles of Dnmt1 with an agouti allele (A(iapy)), which provided a coat color readout for the methylation status of the IAP insertion in the agouti locus. We found that reduction in Dnmt1 levels directly impacted methylation at this locus, leading to stable transcriptional activation of the agouti gene in the adult. Specifically, the short maternal Dnmt1 protein was important in maintaining methylation at the A(iapy) locus in cleavage embryos, whereas the longer Dnmt1 isoform found in somatic cells was important in maintaining IAP methylation during the postimplantation stage. These results underscore the importance of maintaining proper maintenance of methylation patterns during gestation and suggest that interference with this process may stably affect gene expression patterns in the adult and may have profound phenotypic consequences. Full Text.

Geijsen, N., Horoschak, M., Kim, K., Gribnau, J., Eggan, K., and Daley, G. Q. (2004). Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 427, 148-154. Egg and sperm cells ( gametes) of the mouse are derived from a founder population of primordial germ cells that are set aside early in embryogenesis. Primordial germ cells arise from the proximal epiblast, a region of the early mouse embryo that also contributes to the first blood lineages of the embryonic yolk sac(1). Embryonic stem cells differentiate in vitro into cystic structures called embryoid bodies consisting of tissue lineages typical of the early mouse embryo(2,3). Because embryoid bodies sustain blood development, we reasoned that they might also support primordial germ cell formation. Here we isolate primordial germ cells from embryoid bodies, and derive continuously growing lines of embryonic germ cells. Embryonic germ cells show erasure of the methylation markers ( imprints) of the Igf2r and H19 genes, a property characteristic of the germlineage. We show that embryoid bodies support maturation of the primordial germ cells into haploid male gametes, which when injected into oocytes restore the somatic diploid chromosome complement and develop into blastocysts. Our ability to derive germ cells from embryonic stem cells provides an accessible in vitro model system for studies of germline epigenetic modification and mammalian gametogenesis. Full Text.

Gimelbrant, A. A., Ensminger, A. W., Qi, P., Zucker, J. and Chess, A. (2004) Monoallelic expression and asynchronous replication of p120 catenin in mouse and human cells. J Biol Chem. Papers in Press 1 November The number of autosomal mammalian genes subject to random monoallelic expression has been limited to genes highly specific to the function of chemosensory neurons or lymphocytes, making this phenomenon difficult to address systematically. Here we demonstrate that asynchronous DNA replication can be used as a marker for the identification of novel genes with monoallelic expression, and identify p120 catenin, a gene involved in cell adhesion, as belonging to this class. p120 is widely expressed; its presence in available cell lines allowed us to address quantitative aspects of monoallelic expression. We show that the epigenetic choice of active allele is clonally stable, and that biallelic clones express p120 at twice the level of monoallelic clones. Unlike previous reports about genes of this type, we find that expression of p120 can be monoallelic in one cell type and strictly biallelic in another. We show that in human lymphoblasts, the silencing of one allele is incomplete. These unexpected properties are likely to be widespread, as we show that the Tlr4 gene shares them. Identification of monoallelic expression of a nearly ubiquitous gene indicates that this type of gene regulation is more common than previously thought. This has important implications for carcinogenesis and definition of cell identity. PDF

Gimelbrant, A. A., Skaletsky, H., and Chess, A. (2004). Selective pressures on the olfactory receptor repertoire since the human-chimpanzee divergence.PNAS 101 9019-9022. PDF.

Goedecke, N., McKenna, B., El-Difrawy, S., Carey, L., Matsudaira, P., and Ehrlich, D. (2004). A high-performance multilane microdevice system designed for the DNA forensics laboratory. Electrophoresis 25, 1678-1686. Full Text.

Ha, Y. H., Vaia, R. A., Lynn, W. F., Costantino, J. P., Shin, J., Smith, A. B., Matsudaira, P. T. and Thomas, E. L. (2004) Three-dimensional network photonic crystals via cyclic size reduction/infiltration of sea urchin exoskeleton. Advanced Materials. 16 1091-+. Many naturally occurring solids possess periodic structures that give rise to visible photonic crystal properties,([1]) commonly termed structural colors. Some stunning examples are butterfly wings (one-dimensional, 1D), ([2]) abalone shells (1D),([3]) sea-mouse spines (two-dimensional, 2D),([4]) and natural opals (three-dimensional, 3D).([5]) Exploitation of other periodic natural structures, is however limited by the inherently large size scale and the low dielectric contrast of the materials. Furthermore, these generally more complex geometries are a challenge to model correctly in order to obtain correct band diagrams. Here we report the development of a high fidelity cyclic size reduction and infiltration scheme, and apply it to a sea urchin exoskeleton to successfully fabricate a high dielectric contrast 3D photonic crystal exhibiting a stop band in the mid-IR range. The band structure of the exoskeleton is modeled using level set mathematics and agrees well with the experimental reflectivity exhibited by the 3D bicontinuous tellurium network of the replicated urchin Full Text.

Halme, A., Bumgarner, S., Styles, C., and Fink, G. R. (2004). Genetic and epigenetic regulation of the FLO gene family generates cell-surface variation in yeast. Cell 116, 405-415. The FLO gene family of Saccharomyces cerevisiae includes an expressed gene, FLO11, and a set of silent, telomere-adjacent FLO genes. This gene family encodes cell-wall glycoproteins that regulate cell-cell and cell-surface adhesion. Epigenetic silencing of FLO11 regulates a key developmental switch: when FLO11 is expressed, diploid cells form pseudohyphal filaments; when FLO11 is silent, the cells grow in yeast form. The epigenetic state of FLO11 is heritable for many generations and regulated by the histone deacetylase (HDAC) Hda1p. The silent FLO10 gene is activated by high-frequency loss-of-function mutations at either IRA1 or IRA2. FLO10 is regulated by the same transcription factors that control FLO11: Sfl1p and Flo8p, but is silenced by a distinct set of HDACs: Hst1p and Hst2p. These sources of epigenetic and genetic variation explain the observed heterogeneity of cell-surface protein expression within a population of cells derived from a single clone.Full Text.

Halmos, B., Basseres, D. S., Monti, S., D'Alo, F., Dayaram, T., Ferenczi, K., Wouters, B. J., Huettner, C. S., Golub, T. R. and Tenen, D. G. (2004) A transcriptional profiling study of CCAAT/enhancer binding protein targets identifies hepatocyte nuclear factor 3 beta as a novel tumor suppressor in lung cancer. Cancer Research 64 4137-4147. We showed previously that CCAAT/enhancer binding protein alpha(C/EBPalpha), a tissue-specific transcription factor, is a candidate tumor suppressor in lung cancer. In the present study, we have performed a transcriptional profiling study of C/EBPalpha target genes using an inducible cell line system. This study led to the identification of hepatocyte nuclear factor 3beta (HNF3beta), a transcription factor known to play a role in airway differentiation, as a downstream target of C/EBPalpha. We found down-regulation of HNF3beta expression in a large proportion of lung cancer cell lines examined and identified two novel mutants of HNF3beta, as well as hypermethylation of the HNF3beta promoter. We also developed a tetracycline-inducible cell line model to study the cellular consequences of HNF3beta expression. Conditional expression of HNF3beta led to significant growth reduction, proliferation arrest, apoptosis, and loss of clonogenic ability, suggesting additionally that HNF3beta is a novel tumor suppressor in lung cancer. This is the first study to show genetic abnormalities of lung-specific differentiation pathways in the development of lung cancer. Full Text.

Harbison, C. T., Gordon, D. B., Lee, T. I., Rinaldi, N. J., Macisaac, K. D., Danford, T. W., Hannett, N. M., Tagne, J. B., Reynolds, D. B., Yoo, J., Jennings, E. G., Zeitlinger, J., Pokholok, D. K., Kellis, M., Rolfe, P. A., Takusagawa, K. T., Lander, E. S., Gifford, D. K., Fraenkel, E. and Young, R. A. (2004) Transcriptional regulatory code of a eukaryotic genome. Nature. 431 99-104. DNA-binding transcriptional regulators interpret the genome's regulatory code by binding to specific sequences to induce or repress gene expression. Comparative genomics has recently been used to identify potential cis-regulatory sequences within the yeast genome on the basis of phylogenetic conservation, but this information alone does not reveal if or when transcriptional regulators occupy these binding sites. We have constructed an initial map of yeast's transcriptional regulatory code by identifying the sequence elements that are bound by regulators under various conditions and that are conserved among Saccharomyces species. The organization of regulatory elements in promoters and the environment-dependent use of these elements by regulators are discussed. We find that environment-specific use of regulatory elements predicts mechanistic models for the function of a large population of yeast's transcriptional regulators. Full Text.

Herrlich, A., Leitch, V. and King, L. S. (2004) Role of proneuregulin 1 cleavage and human epidermal growth factor receptor activation in hypertonic aquaporin induction. Proc Natl Acad Sci U S A.Early Edition 21 October. Mammalian cells are confronted with changes in extracellular osmolality at various sites, including the aqueous layer above the lung epithelium. Hypertonic shock induces the activation of mitogen-activated protein kinases and the expression of a defined set of genes, including aquaporins. We investigated upstream components of the response to hypertonicity in lung epithelial cells and found that before extracellular signal-regulated kinase activation and aquaporin synthesis, the membrane-bound prohormone neuregulin 1-beta is cleaved and binds to human epidermal growth factor receptor 3 (HER3). The signaling is prevented by matrix metalloproteinase inhibition, inhibition of neuregulin 1-beta binding to HER3, and inhibition of HER tyrosine kinase activity. Inhibition of HER activation interferes with the hypertonic induction of two different aquaporins in three distinct cell lines of mouse and human origin. We propose that ligand-dependent HER activation constitutes a generalized signaling principle in the mammalian hypertonic stress response relevant to aquaporin expression. PDF

Hochedlinger, K., Blelloch, R., Brennan, C., Yamada, Y., Kim, M., Chin, L. and Jaenisch, R. (2004) Reprogramming of a melanoma genome by nuclear transplantation. Genes Dev 18 1875-85. We have used nuclear transplantation to test whether the reprogramming activity of oocytes can reestablish developmental pluripotency of malignant cancer cells. We show here that the nuclei of leukemia, lymphoma, and breast cancer cells could support normal preimplantation development to the blastocyst stage but failed to produce embryonic stem (ES) cells. However, a blastocyst cloned from a RAS-inducible melanoma nucleus gave rise to ES cells with the potential to differentiate into multiple cell types in vivo including melanocytes, lymphocytes, and fibroblasts. Chimeras produced from these ES cells developed cancer with higher penetrance, shorter latency, and an expanded tumor spectrum when compared with the donor mouse model. These results demonstrate that the secondary changes of a melanoma nucleus are compatible with a broad developmental potential but predispose mice to melanomas and other malignant tumors on reactivation of RAS. Our findings serve as a paradigm for studying the tumorigenic effect of a given cancer genome in the context of a whole animal. Full Text.

Hoyle, J., Tang, Y. P., Wiellette, E. L., Wardle, F. C., and Sive, H. (2004). nlz Gene family is required for hindbrain patterning in the zebrafish. Dev Dyn 229, 835-846. This study describes the conserved nlz gene family whose members encode unusual zinc finger proteins. In the zebrafish neurectoderm, both nlz1 and the newly isolated nlz2 are expressed in the presumptive hindbrain and midbrain/hindbrain boundary, where expression of nlz1 is dependent on pax2a. In addition, nlz2 is uniquely expressed more anteriorly, in the presumptive midbrain and diencephalon. Overexpression of Nlz proteins during gastrula stages inhibits hindbrain development. In particular, ectopically expressed Nlz1 inhibits formation of future rhombomeres 2 and 3 (r2, r3), whereas neighboring r1 and r4 are not affected. Conversely, simultaneous reduction of Nlz1 and Nlz2 protein function by expression of antisense morpholino-modified oligomers leads to expansion of future r3 and r5, with associated loss of r4. These data indicate that one function of the nlz gene family is to specify or maintain r4 identity, and to limit r3 and r5 during hindbrain formation. Full Text.

Hsiung, G. Y. R., Kaplan, B. J., Petryshen, T. L., Lu, S., and Field, L. L. (2004). A dyslexia susceptibility locus (DYX7) linked to dopamine D4 receptor (DRD4) region on chromosome 11p15.5. American Journal of Medical Genetics Part B-Neuropsychiatric Genetics 125B, 112-119. Dyslexia is a disability in acquiring reading and spelling skills that is independent of general intelligence and educational opportunity, and is highly heritable. It is known that dyslexia often co-occurs with attention deficit hyperactivity disorder (ADHD), and the 7-repeat allele of the 48-bp tandem repeat in exon 3 of the dopamine D4 receptor (DRD4) has been implicated in ADHD. We, therefore, investigated DRD4 as a candidate gene for dyslexia by testing for linkage and association with 14 markers at and around the DRD4 locus on chromosome 11p15.5. Using 100 families having at least two siblings affected with dyslexia, model-free linkage analysis revealed evidence for linkage to the DRD4-exon 3 repeat (two-point MFLOD = 2.27, P = 0.001) and to HRAS located just proximal to DRD4 (two-point MFLOD = 2.68, P = 0.0004). Evidence for linkage was maximal between DRD4 and HRAS (three-point MFLOD = 3.57, P = 0.00005). However, linkage disequilibrium analysis showed no significant evidence for association between dyslexia and DRD4 or HRAS. In particular, dyslexic subjects showed no significant increase of the DRD4 7-repeat allele associated with ADHD. It is possible that other DRD4 variants, not in strong linkage disequilibrium. with the exon 3 repeat polymorphism, or alternatively, another gene very closely linked to DRD4, may influence susceptibility to dyslexia. Full Text.

Hug, C. and Lodish, H. F. (2004) Visfatin: A New Adipokine. Science (Scienceexpress 16 December). Too much abdominal (visceral) fat increases an individual's risk of developing insulin resistance and other metabolic disorders. In a Perspective, Hug and Lodish discuss the unexpected finding that blood levels of a hormone produced by visceral fat, called visfatin, correlate with obesity (Fukuhara et al.). Surprisingly, visfatin has beneficial, insulin-like activity and is able to bind to the insulin receptor, lowering blood glucose levels. The implications of this finding for diabetes research and therapy are discussed by the Perspective authors.PDF

Hug, C., Wang, J., Ahmad, N. S., Bogan, J. S., Tsao, T. S. and Lodish, H. F. (2004) T-cadherin is a receptor for hexameric and high-molecular-weight forms of Acrp30/adiponectin Proc Natl Acad Sci U S A Epub ahead of print 21 June. Acrp30/adiponectin is reduced in the serum of obese and diabetic individuals, and the genetic locus of adiponectin is linked to the metabolic syndrome. Recombinant adiponectin, administered to diet-induced obese mice, induced weight loss and improved insulin sensitivity. In muscle and liver, adiponectin stimulates AMP-activated protein kinase activation and fatty acid oxidation. To expression-clone molecules capable of binding adiponectin, we transduced a C2C12 myoblast cDNA retroviral expression library into Ba/F3 cells and panned infected cells on recombinant adiponectin linked to magnetic beads. We identified T-cadherin as a receptor for the hexameric and high-molecular-weight species of adiponectin but not for the trimeric or globular species. Only eukaryotically expressed adiponectin bound to T-cadherin, implying that posttranslational modifications of adiponectin are critical for binding. An adiponectin mutant lacking a conserved N-terminal cysteine residue required for formation of hexamer and high-molecular-weight species did not bind T-cadherin in coimmunoprecipitation studies. Although lacking known cellular functions, T-cadherin is expressed in endothelial and smooth muscle cells, where it is positioned to interact with adiponectin. Because T-cadherin is a glycosylphosphatidylinositol-anchored extracellular protein, it may act as a coreceptor for an as-yet-unidentified signaling receptor through which adiponectin transmits metabolic signals.PDF.

Ideker, T. (2004) A systems approach to discovering signaling and regulatory pathways--or, how to digest large interaction networks into relevant pieces Adv Exp Med Biol 547 21-30. In the post-genomic era, the first step in any study of protein function is a homology search against the complete genome sequence of the organism of interest. By analogy, if we also wish to elucidate the cadre of signaling and regulatory pathways in the cell, an extremely powerful first step is to construct a complete network of protein-protein and transcriptional interactions and then search through this network to identify critical pathways in a top-down fashion. Like genomic sequence, the molecular interaction network provides a broad foundation for more directed studies to follow. We illustrate this strategy using a large network of 12,232 interactions in yeast. A variety of applications are discussed, including screening the network to identify pathways responsible for gene expression changes observed in galactose-induced cells, and identifying groups of interacting proteins that are essential (by phenotypic assay) for the cellular response to DNA damage.

Ince, T. A., and Crum, C. P. (2004). Telomerase: Promise and challenge. Human Pathology 35, 393-395.

Ivanovska, I., Lee, E., Kwan, K. M., Fenger, D. D., and Orr-Weaver, T. L. (2004). The Drosophila MOS Ortholog Is Not Essential for Meiosis. Curr Biol 14, 75-80. In metazoan oocytes, a metaphase arrest coordinates the completion of meiosis with fertilization. Vertebrate mos maintains the metaphase II arrest of mature oocytes and prevents DNA replication between the meiotic divisions. We identified a Drosophila homolog of mos and showed it to be the mos ortholog by two additional criteria. The dmos transcripts are present in Drosophila oocytes but not embryos, and injection of dmos into Xenopus embryos blocks mitosis and elevates active MAPK levels. In Drosophila, MAPK is activated in oocytes, consistent with a role in meiosis. We generated deletions of dmos and found that, as in vertebrates, dmos is responsible for the majority of MAPK activation. Unexpectedly, the oocytes that do mature complete meiosis normally and produce fertilized embryos that develop, although there is a reduction in female fertility and loss of some oocytes by apoptosis. Therefore, Drosophila contains a mos ortholog that activates a MAPK cascade during oogenesis and is nonessential for meiosis. This could be because there are redundant pathways regulating meiosis, because residual, low levels of active MAPK are sufficient, or because active MAPK is dispensable for meiosis in Drosophila. These results highlight the complexity of meiotic regulation that evolved to ensure accurate control over the reproductive process. Full Text

Jaenisch, R. (2004) Human cloning - the science and ethics of nuclear transplantation. N Engl J Med. 351: 2787-2791.

Jaenisch, R. (2004) Transdifferentiation and adult stem cells: Myth or reality? Applied Physics a-Materials Science & Processing. 79 1625-1626. The paper by Kruse et al. published in this issue of Applied Physics A claims that cells isolated from pancreas of adult rats or humans can generate cells of multiple lineages. To convincingly show "transdifferentiation" of adult stem cells across lineage boundaries requires stringent experimental criteria such as clonal analyses. The data provided by Kruse et al. fall short of such standards and the conclusion that multipotential cells were isolated is unconvinving.Full Text

Jaillon, O., Aury, J. M., Brunet, F., Petit, J. L., Stange-Thomann, N., Mauceli, E., Bouneau, L., Fischer, C., Ozouf-Costaz, C., Bernot, A., Nicaud, S., Jaffe, D., Fisher, S., Lutfalla, G., Dossat, C., Segurens, B., Dasilva, C., Salanoubat, M., Levy, M., Boudet, N., Castellano, S., Anthouard, R., Jubin, C., Castelli, V., Katinka, M., Vacherie, B., Biemont, C., Skalli, Z., Cattolico, L., Poulain, J., de Berardinis, V., Cruaud, C., Duprat, S., Brottier, P., Coutanceau, J. P., Gouzy, J., Parra, G., Lardier, G., Chapple, C., McKernan, K. J., McEwan, P., Bosak, S., Kellis, M., Volff, J. N., Guigo, R., Zody, M. C., Mesirov, J., Lindblad-Toh, K., Birren, B., Nusbaum, C., Kahn, D., Robinson-Rechavi, M., Laudet, V., Schachter, V., Quetier, F., Saurin, W., Scarpelli, C., Wincker, P., Lander, E. S., Weissenbach, J. and Crollius, H. R. (2004) Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature. 431 946-957. Tetraodon nigroviridis is a freshwater puffer fish with the smallest known vertebrate genome. Here, we report a draft genome sequence with long-range linkage and substantial anchoring to the 21 Tetraodon chromosomes. Genome analysis provides a greatly improved fish gene catalogue, including identifying key genes previously thought to be absent in fish. Comparison with other vertebrates and a urochordate indicates that fish proteins have diverged markedly faster than their mammalian homologues. Comparison with the human genome suggests similar to900 previously unannotated human genes. Analysis of the Tetraodon and human genomes shows that whole-genome duplication occurred in the teleost fish lineage, subsequent to its divergence from mammals. The analysis also makes it possible to infer the basic structure of the ancestral bony vertebrate genome, which was composed of 12 chromosomes, and to reconstruct much of the evolutionary history of ancient and recent chromosome rearrangements leading to the modern human karyotype. Full Text

Jones-Rhoades, M. W. and Bartel, D. P. (2004) Computational Identification of Plant MicroRNAs and Their Targets, Including a Stress-Induced miRNA Mol Cell 14 787-99. MicroRNAs (miRNAs) are approximately 21-nucleotide RNAs, some of which have been shown to play important gene-regulatory roles during plant development. We developed comparative genomic approaches to systematically identify both miRNAs and their targets that are conserved in Arabidopsis thaliana and rice (Oryza sativa). Twenty-three miRNA candidates, representing seven newly identified gene families, were experimentally validated in Arabidopsis, bringing the total number of reported miRNA genes to 92, representing 22 families. Nineteen newly identified target candidates were confirmed by detecting mRNA fragments diagnostic of miRNA-directed cleavage in plants. Overall, plant miRNAs have a strong propensity to target genes controlling development, particularly those of transcription factors and F-box proteins. However, plant miRNAs have conserved regulatory functions extending beyond development, in that they also target superoxide dismutases, laccases, and ATP sulfurylases. The expression of miR395, the sulfurylase-targeting miRNA, increases upon sulfate starvation, showing that miRNAs can be induced by environmental stress.Full Text.

Kaeberlein, M., Andalis, A. A., Liszt, G. B., Fink, G. R. and Guarente, L. (2004) Saccharomyces cerevisiae SSD1-V confers longevity by a Sir2p-independent mechanism Genetics 166 1661-1672.The SSD1 gene of Saccharomyces cerevisiae is a polymorphic locus that affects diverse cellular processes including cell integrity, cell cycle progression, and growth at high temperature. We show here that the SSD1-V allele is necessary for cells to achieve extremely long life span. Furthermore, addition of SSD1-V to cells call increase longevity independently of SIR2, although SIR2 is necessary for SSD1-V cells to attain maximal life span. Past studies of yeast aging have been performed in short-lived ssd1-d strain backgrounds. We propose that SSD1-V defines a previously undescribed pathway affecting cellular longevity and suggest that future studies on longevity-promoting genes should be carried out in long-lived SSD1-V strains. Full Text.

Kelley, B. P., Yuan, B., Lewitter, F., Sharan, R., Stockwell, B. R. and Ideker, T. (2004) PathBLAST: a tool for alignment of protein interaction networks Nucleic Acids Res 32 W83-W88. PathBLAST is a network alignment and search tool for comparing protein interaction networks across species to identify protein pathways and complexes that have been conserved by evolution. The basic method searches for high-scoring alignments between pairs of protein interaction paths, for which proteins of the first path are paired with putative orthologs occurring in the same order in the second path. This technique discriminates between true- and false-positive interactions and allows for functional annotation of protein interaction pathways based on similarity to the network of another, well-characterized species. PathBLAST is now available at http://www.pathblast.org/ as a web-based query. In this implementation, the user specifies a short protein interaction path for query against a target protein-protein interaction network selected from a network database. PathBLAST returns a ranked list of matching paths from the target network along with a graphical view of these paths and the overlap among them. Target protein-protein interaction networks are currently available for Helicobacter pylori, Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster. Just as BLAST enables rapid comparison of protein sequences between genomes, tools such as PathBLAST are enabling comparative genomics at the network level. Full Text.

Kellis, M., Patterson, N., Birren, B., Berger, B. and Lander, E. S. (2004) Methods in comparative genomics: genome correspondence, gene identification and regulatory motif discovery. J Comput Biol 11 319-55. In Kellis et al. (2003), we reported the genome sequences of S. paradoxus, S. mikatae, and S. bayanus and compared these three yeast species to their close relative, S. cerevisiae. Genomewide comparative analysis allowed the identification of functionally important sequences, both coding and noncoding. In this companion paper we describe the mathematical and algorithmic results underpinning the analysis of these genomes. (1) We present methods for the automatic determination of genome correspondence. The algorithms enabled the automatic identification of orthologs for more than 90% of genes and intergenic regions across the four species despite the large number of duplicated genes in the yeast genome. The remaining ambiguities in the gene correspondence revealed recent gene family expansions in regions of rapid genomic change. (2) We present methods for the identification of protein-coding genes based on their patterns of nucleotide conservation across related species. We observed the pressure to conserve the reading frame of functional proteins and developed a test for gene identification with high sensitivity and specificity. We used this test to revisit the genome of S. cerevisiae, reducing the overall gene count by 500 genes (10% of previously annotated genes) and refining the gene structure of hundreds of genes. (3) We present novel methods for the systematic de novo identification of regulatory motifs. The methods do not rely on previous knowledge of gene function and in that way differ from the current literature on computational motif discovery. Based on genomewide conservation patterns of known motifs, we developed three conservation criteria that we used to discover novel motifs. We used an enumeration approach to select strongly conserved motif cores, which we extended and collapsed into a small number of candidate regulatory motifs. These include most previously known regulatory motifs as well as several noteworthy novel motifs. The majority of discovered motifs are enriched in functionally related genes, allowing us to infer a candidate function for novel motifs. Our results demonstrate the power of comparative genomics to further our understanding of any species. Our methods are validated by the extensive experimental knowledge in yeast and will be invaluable in the study of complex genomes like that of the human. PDF

Kellis, M., Birren, B. W., and Lander, E. S. (2004). Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae. Nature 428, 617-624. Whole-genome duplication followed by massive gene loss and specialization has long been postulated as a powerful mechanism of evolutionary innovation. Recently, it has become possible to test this notion by searching complete genome sequence for signs of ancient duplication. Here, we show that the yeast Saccharomyces cerevisiae arose from ancient whole-genome duplication, by sequencing and analysing Kluyveromyces waltii, a related yeast species that diverged before the duplication. The two genomes are related by a 1:2 mapping, with each region of K. waltii corresponding to two regions of S. cerevisiae, as expected for whole-genome duplication. This resolves the long-standing controversy on the ancestry of the yeast genome, and makes it possible to study the fate of duplicated genes directly. Strikingly, 95% of cases of accelerated evolution involve only one member of a gene pair, providing strong support for a specific model of evolution, and allowing us to distinguish ancestral and derived functions. Full Text.

Kim, J. K., Gimeno, R. E., Higashimori, T., Kim, H. J., Choi, H. J., Punreddy, S., Mozell, R. L., Tan, G., Stricker-Krongrad, A., Hirsch, D. J., Lodish, H.F.et al. (2004). Inactivation of fatty acid transport protein 1 prevents fat-induced insulin resistance in skeletal muscle. Journal of Clinical Investigation 113, 756-763. Insulin resistance in skeletal muscle plays a major role in the development of type 2 diabetes and may be causally associated with increases in intramuscular fatty acid metabolites. Fatty acid transport protein 1 (FATP1) is an acyl-CoA synthetase highly expressed in skeletal muscle and modulates fatty acid uptake and metabolism by converting fatty acids into fatty acyl-CoA. To investigate the role of FATP1 in glucose homeostasis and in the pathogenesis of insulin resistance, we examined the effect of acute lipid infusion or chronic high-fat feeding on insulin action in FATP1 KO mice. Whole-body adiposity, adipose tissue expression of adiponectin, intramuscular fatty acid metabolites, and insulin sensitivity were not altered in FATP1 KO mice fed a regular chow diet. In contrast, FATP1 deletion protected the KO mice from fat-induced insulin resistance and intramuscular accumulation of fatty acyl-CoA without alteration in whole-body adiposity. These findings demonstrate an important role of intramuscular fatty acid metabolites in causing insulin resistance and suggest that FATP1 may be a novel therapeutic target for the treatment of insulin resistance and type 2 diabetes. Full Text.

Koh, E. Y., Chen, T., and Daley, G. Q. (2004). Genetic complementation of cytokine signaling identifies central role of kinases in hematopoietic cell proliferation. Oncogene 23, 1214-1220. Molecular evidence suggests a multistep process in the development of acute leukemia. Since inappropriate activation of cytokine signaling cascades is a recurring theme in human leukemia, we performed expression screens to identify genes that transform cytokine-dependent cells. Using retroviral cDNA libraries derived from peripheral blood mononuclear cells of patients with myeloproliferative disorders, we isolated numerous genes that genetically complement cytokine requirements for proliferation of BaF/3 and TF-1 cells. The majority of recovered genes represent members of the kinase family, including several previously linked to leukemogenesis. Our unbiased screen highlights the central role of kinase activation in hematopoietic cell proliferation and identifies a number of potential leukemic oncoproteins.Full Text.

Krewson, T. D., Supelak, P. J., Hill, A. E., Singer, J. B., Lander, E. S., Nadeau, J. H. and Palmert, M. R. (2004) Chromosomes 6 and 13 Harbor Genes that Regulate Pubertal Timing in Mouse Chromosome Substitution Strains. Endocrinology 145 (10): 4447-4451 Variation in the onset of puberty among inbred strains of mice suggests that quantitative trait loci (QTLs) affect neurological and hormonal aspects of sexual maturation. Taking a novel approach toward identifying factors that regulate the hypothalamic-pituitary-gonadal (HPG) axis, we evaluated pubertal timing (as assessed by vaginal opening) in two inbred strains of mice, A/J and C57BL/6J (B6), and in a panel of chromosome substitution strains (CSSs) generated from A/J and B6 mice. In each CSS, a single chromosome from A/J has been substituted in a homozygous fashion for the corresponding chromosome in B6, partitioning the A/J genome into 22 strains with a common host (B6) background. Vaginal opening (VO) occurred significantly earlier in A/J compared with B6 mice. While the majority of the CSSs assessed had a timing of VO that was similar to the progenitor B6 strain, CSSs for chromosomes 6 and 13 each displayed significantly earlier time of VO than B6 mice. F1 (B6 x CSS) mice for chromosomes 6 and 13 displayed phenotypes that were intermediate between the CSS and B6 strains suggesting that the trait was inherited in a codominant manner. These findings demonstrate that chromosomes 6 and 13 harbor QTLs that control the timing of vaginal opening. Identification of the responsible genes may reveal factors that regulate the maturation of the HPG axis and determine the timing of puberty.PDF

Kuperwasser, C., Chavarria, T., Wu, M., Magrane, G., Gray, J. W., Carey, L., Richardson, A., and Weinberg, R. A. (2004). Reconstruction of functionally normal and malignant human breast tissues in mice. Proc Natl Acad Sci U S A. The study of normal breast epithelial morphogenesis and carcinogenesis in vivo has largely used rodent models. Efforts at studying mammary morphogenesis and cancer with xenotransplanted human epithelial cells have failed to recapitulate the full extent of development seen in the human breast. We have developed an orthotopic xenograft model in which both the stromal and epithelial components of the reconstructed mammary gland are of human origin. Genetic modification of human stromal cells before the implantation of ostensibly normal human mammary epithelial cells resulted in the outgrowth of benign and malignant lesions. This experimental model allows for studies of human epithelial morphogenesis and differentiation in vivo and underscores the critical role of heterotypic interactions in human breast development and carcinogenesis.PDF.

Lee, J. Y., Dej, K. J., Lopez, J. M. and Orr-Weaver, T. L. (2004) Control of Centromere Localization of the MEI-S332 Cohesion Protection Protein. Curr Biol 14 1277-83. In mitosis and meiosis, cohesion is maintained at the centromere until sister-chromatid separation. Drosophila MEI-S332 is essential for centromeric cohesion in meiosis and contributes to, though is not absolutely required for, cohesion in mitosis. It localizes specifically to centromeres in prometaphase and delocalizes at the metaphase-anaphase transition. In mei-S332 mutants, centromeric sister-chromatid cohesion is lost at anaphase I, giving meiosis II missegregation. MEI-S332 is the founding member of a family of proteins important for chromosome segregation. One likely activity of these proteins is to protect the cohesin subunit Rec8 from cleavage at the metaphase I-anaphase I transition. Although the family members do not show high sequence identity, there are two short stretches of homology, and mutations in conserved residues affect protein function. Here we analyze the cis- and trans-acting factors required for MEI-S332 localization. We find a striking correlation between domains necessary for MEI-S332 centromere localization and conserved regions within the protein family. Drosophila MEI-S332 expressed in human cells localizes to mitotic centromeres, further highlighting this functional conservation. MEI-S332 can localize independently of cohesin, assembling even onto unreplicated chromatids. However, the separase pathway that regulates cohesin dissociation is needed for MEI-S332 delocalization at anaphase Full Text

Lensch, M. W. and Daley, G. Q. 2004. Origins of mammalian hematopoiesis: In vivo paradigms and in vitro models. Stem Cells in Development and Disease. Current Topics in Developmental Biology. 60.127-196.

Lin, J. D., Wu, P. H., Tarr, P. T., Lindenberg, K. S., St-Pierre, J., Zhang, C. Y., Mootha, V. K., Jager, S., Vianna, C. R., Reznick, R. M., Cui, L. B., Manieri, M., Donovan, M. X., Wu, Z. D., Cooper, M. P., Fan, M. C., Rohas, L. M., Zavacki, A. M., Cinti, S., Shulman, G. I., Lowell, B. B., Krainc, D. and Spiegelman, B. M. (2004) Defects in adaptive energy metabolism with CNS-Linked hyperactivity in PGC-1 alpha null mice. Cell. 119(1) :121- +. PGC-1alpha is a coactivator of nuclear receptors and other transcription factors that regulates several metabolic processes, including mitochondrial biogenesis and respiration, hepatic gluconeogenesis, and muscle fiber-type switching. We show here that, while hepatocytes lacking PGC-1alpha are defective in the program of hormone-stimulated gluconeogenesis, the mice have constitutively activated gluconeogenic gene expression that is completely insensitive to normal feeding controls. C/EBPbeta is elevated in the livers of these mice and activates the gluconeogenic genes in a PGC-1alpha-independent manner. Despite having reduced mitochondrial function, PGC-1alpha null mice are paradoxically lean and resistant to diet-induced obesity. This is largely due to a profound hyperactivity displayed by the null animals and is associated with lesions in the striatal region of the brain that controls movement. These data illustrate a central role for PGC-1alpha in the control of energy metabolism but also reveal novel systemic compensatory mechanisms and pathogenic effects of impaired energy homeostasis. Full Text

Liu, S. H., Milne, G. T., Kuremsky, J. G., Fink, G. R. and Leppla, S. H. (2004) Identification of the proteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation factor 2. Molecular and Cellular Biology. 24 9487-9497. Diphthamide, a posttranslational modification of translation elongation factor 2 that is conserved in all eukaryotes and archaebacteria and is the target of diphtheria toxin, is formed in yeast by the actions of five proteins, Dph1 to -5, and a still unidentified amidating enzyme. Dph2 and Dph5 were previously identified. Here, we report the identification of the remaining three yeast proteins (Dph1, -3, and -4) and show that all five Dph proteins have either functional (Dph1, -2, -3, and -5) or sequence (Dph4) homologs in mammals. We propose a unified nomenclature for these proteins (e.g., HsDph1 to -5 for the human proteins) and their genes based on the yeast nomenclature. We show that Dph1 and Dph2 are homologous in sequence but functionally independent. The human tumor suppressor gene OVCA1, previously identified as homologous to yeast DPH2, is shown to actually be HsDPH1. We show that HsDPH3 is the previously described human diphtheria toxin and Pseudomonas exotoxin A sensitivity required gene 1 and that DPH4 encodes a CSL zinc finger-containing Dnaj-like protein. Other features of these genes are also discussed. The physiological function of diphthamide and the basis of its ubiquity remain a mystery, but evidence is presented that Dph1 to -3 function in vivo as a protein complex in multiple cellular processes Full Text

Lodish, H. F., Rodriguez, R. K. and Klionsky, D. J. (2004) Points of View: lectures: can't learn with them, can't learn without them. Cell Biol Educ. 3 202-11. Full Text.

Lorenz, M. C., Bender, J. A. and Fink, G. R. (2004) Transcriptional response of Candida albicans upon internalization by macrophages. Eukaryotic Cell. 3 1076-1087. The opportunistic fungal pathogen Candida albicans is both a benign gut commensal and a frequently fatal systemic pathogen. The interaction of C. albicans with the host's innate immune system is the primary factor in this balance; defects in innate immunity predispose the patient to disseminated candidiasis. Because of the central importance of phagocytic cells in defense against fungal infections, we have investigated the response of C. albicans to phagocytosis by mammalian macrophages using genomic transcript profiling. This analysis reveals a dramatic reprogramming of transcription in C. albicans that occurs in two successive steps. In the early phase cells shift to a starvation mode, including gluconeogenic growth, activation of fatty acid degradation, and downregulation of translation. In a later phase, as hyphal growth enables C. albicans to escape from the macrophage, cells quickly resume glycolytic growth. In addition, there is a substantial nonmetabolic response imbedded in the early phase, including machinery for DNA damage repair, oxidative stress responses, peptide uptake systems, and arginine biosynthesis. Further, a surprising percentage of the genes that respond specifically to macrophage contact have no known homologs, suggesting that the organism has undergone substantial evolutionary adaptations to the commensal or pathogen lifestyle. This transcriptional reprogramming is almost wholly absent in the related, but nonpathogenic, yeast Saccharomyces cerevisiae, suggesting that these large-scale and coordinated changes contribute significantly to the ability of this organism to survive and cause disease in vivo. Full Text

Lowery, L. A. and Sive, H. (2004) Strategies of vertebrate neurulation and a re-evaluation of teleost neural tube formation. Mech Dev. 121 1189-97. The vertebrate neural tube develops by two distinct mechanisms. Anteriorly, in the brain and future trunk (cervicothoracic) region, 'primary neurulation' occurs, where an epithelial sheet rolls or bends into a tube. Posteriorly, in the future lumbar and tail region, the neural tube forms by 'secondary neurulation', where a mesenchymal cell population condenses to form a solid rod that undergoes transformation to an epithelial tube. Teleost neurulation has been described as different from that of other vertebrates. This is principally because the teleost trunk neural tube initially forms a solid rod (the neural keel) that later develops a lumen. This process has also been termed secondary neurulation. However, this description is not accurate since the teleost neural tube derives from an epithelial sheet that folds. This best fits the description of primary neurulation. It has also been suggested that teleost neurulation is primitive, however, both primary and secondary neurulation are found in groups with a more ancient origin than the teleosts. The similarity between neurulation in teleosts and other vertebrates indicates that this group includes viable models (such as the zebrafish) for understanding human neural tube development. Full Text.

Luikenhuis, S., Giacometti, E., Beard, C. F. and Jaenisch, R. (2004) Expression of MeCP2 in postmitotic neurons rescues Rett syndrome in mice. Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):6033-8. Epub 2004 Apr 06. Mutations in MECP2 are the cause of Rett syndrome (RTT) in humans, a neurodevelopmental disorder that affects mainly girls. MeCP2 is a protein that binds CpG dinucleotides and is thought to act as a global transcriptional repressor. It is highly expressed in neurons, but not in glia, of the postnatal brain. The timing of MeCP2 activation correlates with the maturation of the central nervous system, and recent reports suggest that MeCP2 may be involved in the formation of synaptic contacts and may function in activity-dependent neuronal gene expression. Deletion or targeted mutation of Mecp2 in mice leads to a Rett-like phenotype. Selective mutation of Mecp2 in postnatal neurons leads to a similar, although delayed, phenotype, suggesting that MeCP2 plays a role in postmitotic neurons. Here we test the hypothesis that the symptoms of RTT are exclusively caused by a neuronal MeCP2 deficiency by placing Mecp2 expression under the control of a neuron-specific promoter. Expression of the Mecp2 transgene in postmitotic neurons resulted in symptoms of severe motor dysfunction. Transgene expression in Mecp2 mutant mice, however, rescued the RTT phenotype. Full Text.

Luo, B., Heard, A. D. and Lodish, H. F. (2004) Small interfering RNA production by enzymatic engineering of DNA (SPEED). Proc Natl Acad Sci U S A. 2004 Apr 13;101(15):5494-9. Epub 2004 Mar 15.. Small interfering RNAs (siRNAs) potently silence expression of target genes. In principle siRNA libraries can be used to perform effective genome-scale functional genetic screens in mammalian cells, but their development has been hampered by the need to chemically synthesize thousands of oligonucleotides and to incorporate them into expression vectors. We have developed a technology to efficiently convert a double-stranded cDNA library into a retroviral siRNA library in which 21-base siRNAs are produced in infected cells at high levels and efficiently block expression of their target genes. The key steps are the generation of random cDNA fragments that are fused to a hairpin linker, cleavage with the MmeI endonuclease that creates 20- to 21-bp cDNA fragments, conversion to a double-stranded DNA that contains two copies of the cDNA insert in a head-to-head palindrome, and insertion of the construct downstream of a polymerase III promoter. We constructed a siRNA library with 3 x 10(6) clones from a mouse embryo cDNA library; siRNAs were found against many different genes; and multiple siRNAs can be generated from a single mRNA. We further showed that specific siRNAs were efficiently produced in stably infected mammalian cells and resulted in significant and specific reduction of their target mRNAs. Because no prior knowledge about target transcripts is needed, a cDNA-derived siRNA library will generate siRNAs against unknown transcripts and genes. Finally, cDNA-derived siRNA libraries can be readily generated from any cell type or species, enabling genome-wide functional screens in many biological systems. Full Text.

Mallory, A. C., Dugas, D. V., Bartel, D. P. and Bartel, B. (2004) MicroRNA Regulation of NAC-Domain Targets Is Required for Proper Formation and Separation of Adjacent Embryonic, Vegetative, and Floral Organs. Curr Biol. 14 1035-1046. Background: MicroRNAs (miRNAs) are approximately 21 nucleotide (nt) RNAs that regulate gene expression in plants and animals. Most known plant miRNAs target transcription factors that influence cell fate determination, and biological functions of miRNA-directed regulation have been reported for four of 15 known microRNA gene families: miR172, miR159, miR165, and miR168. Here, we identify a developmental role for miR164-directed regulation of NAC-domain genes, which encode a family of transcription factors that includes CUP-SHAPED COTYLEDON1 (CUC1) and CUC2. Results: Expression of a miR164-resistant version of CUC1 mRNA from the CUC1 promoter causes alterations in Arabidopsis embryonic, vegetative, and floral development, including cotyledon orientation defects, reduction of rosette leaf petioles, dramatically misshapen rosette leaves, one to four extra petals, and one or two missing sepals. Reciprocally, constitutive overexpression of miR164 recapitulates cuc1 cuc2 double mutant phenotypes, including cotyledon and floral organ fusions. miR164 overexpression also leads to phenotypes not previously observed in cuc1 cuc2 mutants, including leaf and stem fusions. These likely reflect the misregulation of other NAC-domain mRNAs, including NAC1, At5g07680, and At5g61430, for which miR164-directed cleavage products were detected. Conclusions: These results demonstrate that miR164-directed regulation of CUC1 is necessary for normal embryonic, vegetative, and floral development. They also show that proper miR164 dosage or localization is required for separation of adjacent embryonic, vegetative, and floral organs, thus implicating miR164 as a common regulatory component of the molecular circuitry that controls the separation of different developing organs and thereby exposes a posttranscriptional layer of NAC-domain gene regulation during plant development.Full Text.

Mallory, A. C., Reinhart, B. J., Jones-Rhoades, M. W., Tang, G., Zamore, P. D., Barton, M. K. and Bartel, D. P. (2004) MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5' region. EMBO J. 2004 Jul 29 [Epub ahead of print].MicroRNAs (miRNAs) are approximately 22-nucleotide noncoding RNAs that can regulate gene expression by directing mRNA degradation or inhibiting productive translation. Dominant mutations in PHABULOSA (PHB) and PHAVOLUTA (PHV) map to a miR165/166 complementary site and impair miRNA-guided cleavage of these mRNAs in vitro. Here, we confirm that disrupted miRNA pairing, not changes in PHB protein sequence, causes the developmental defects in phb-d mutants. In planta, disrupting miRNA pairing near the center of the miRNA complementary site had far milder developmental consequences than more distal mismatches. These differences correlated with differences in miRNA-directed cleavage efficiency in vitro, where mismatch scanning revealed more tolerance for mismatches at the center and 3' end of the miRNA compared to mismatches to the miRNA 5' region. In this respect, miR165/166 resembles animal miRNAs in its pairing requirements. Pairing to the 5' portion of the small silencing RNA appears crucial regardless of the mode of post-transcriptional repression or whether it occurs in plants or animals, supporting a model in which this region of the silencing RNA nucleates pairing to its target. Full Text

Mallory, A. C. and Vaucheret, H. (2004) MicroRNAs: something important between the genes. Current Opinion in Plant Biology. 7 120-125. Non-coding small endogenous RNAs, of 21-24 nucleotides in length, have recently emerged as important regulators of gene expression in both plants and animals. At least three categories of small RNAs exist in plants: short interfering RNAs (siRNAs) deriving from viruses or transgenes and mediating virus resistance or transgene silencing via RNA degradation; siRNAs deriving from transposons or transgene promoters and controlling transposon and transgene silencing probably via chromatin changes; and microRNAs (miRNAs) deriving from intergenic regions of the genome and regulating the expression of endogenous genes either by mRNA cleavage or translational repression. The disruption of miRNA-mediated regulation causes developmental abnormalities in plants, demonstrating that miRNAs play an important role in the regulation of developmental decisions. Full Text.

Man, M. N. T., Lam, L. T., Stockwell, B. R., McKenzie, A. E., Morris, G. E. and Sewry, C. A. (2004) Towards a treatment for spinal muscular atrophy: looking for drugs that increase SMN protein levels. Neuromuscular Disorders. 14 595-596.

Marszalek, J. R., Kitidis, C., Dararutana, A. and Lodish, H. F. (2004) Acyl CoA synthetase 2 (ACS2) over-expression enhances fatty acid internalization and neurite outgrowth. J Biol Chem. 2004 Jun 4;279(23):23882-91. Epub 2004 Mar 29 During neurodevelopment neurons increase phospholipid synthesis to generate additional plasma membrane that makes up the growing neurites. Compared to most cell types, neurons contain a high percentage of the polyunsaturated fatty acids (PUFAs) arachidonic acid (AA) and docosahexaenoic acid (DHA). Utilizing PC12 cell lines as a model neuronal cell line, we examined the internalization rate of AA, DHA and non-essential oleic acid (OA), as well as their effects on neurite outgrowth. When wild type cells were differentiated, the rate of AA and DHA internalization increased 50% more than the rate of OA internalization. When media was supplemented with AA or DHA, the average neurite length was increased by ~40%, but supplementation with the same amount of OA had no effect. We also increased the levels of acyl CoA synthetase-1 (ACS1) and ACS2 proteins to determine whether they contribute to PUFA internalization or neurite outgrowth. Over-expression of ACS1 increased the rate of OA internalization by 55%, and AA and DHA uptake was increased by 25%, but there was no significant change in neurite outgrowth. In ACS2 over-expressing cells, in contrast, the rate of OA internalization increased by 90%, AA by 115% and DHA by 70%. The average aggregate neurite length in ACS2 over-expressing cells was increased by ~40% when the media was supplemented with PUFAs, but there was no change with OA supplementation. Taken together, these results support the hypotheses that ACSs are rate limiting for FA internalization and that ACS2 enhances neurite outgrowth by promoting PUFA internalization. Full Text.

Martin, S. S., Ridgeway, A. G., Pinkas, J., Lu, Y., Reginato, M. J., Koh, E. Y., Michelman, M., Daley, G. Q., Brugge, J. S. and Leder, P. (2004) A cytoskeleton-based functional genetic screen identifies Bcl-xL as an enhancer of metastasis, but not primary tumor growth. Oncogene. 23 4641-4645. Many mouse models of breast cancer form large primary tumors that rarely metastasize. Models with aggressive metastasis express oncoproteins that simultaneously affect growth and apoptosis pathways. To define the role of apoptotic resistance and to model a challenge faced by tumor cells during metastatic dissemination, we focused on apoptosis induced by cell shape change. Inhibiting actin polymerization with Latrunculin-A causes cell rounding and death within hours in nontumorigenic human 10A-Ras mammary epithelial cells. In contrast, MDA-MB-231 metastatic breast tumor cells resist LA-induced death, and survive for days despite cell rounding. Infecting 10A-Ras cells with a MDA-MB-231 retroviral expression library, and selecting with Latrunculin-A repeatedly identified Bcl-xL as a suppressor of cytoskeleton-dependent death. Although Bcl-xL enhances the spread of metastatic breast tumor cell lines, the distinct effects of apoptotic resistance on tumor growth in the mammary gland and during metastasis have not been compared directly. We find that Bcl-xL overexpression in mouse mammary epithelial cells does not induce primary tumor formation or enhance MEK-induced tumorigenesis within the mammary gland environment. However, it strongly enhances metastatic potential. These results with Bcl-xL provide novel evidence that isolated apoptotic resistance can increase metastatic potential, but remain overlooked by assays based on breast tumor growth. Full Text.

Mathies, L. D., Schvarzstein, M., Morphy, K. M., Blelloch, R., Spence, A. M. and Kimble, J. (2004) TRA-1/GLI controls development of somatic gonadal precursors in C-elegans. Development. 131 4333-4343. TRA-1/GLI is best known as a master regulator of sex determination in the nematode C elegans, but its fly and vertebrate homologs (e.g. Ci, GLI) regulate embryonic patterning and cell proliferation. In this paper, we show that TRA-1/GLI controls development of the two somatic gonadal precursors (SGPs) in both XX and XO animals, in addition to its role in sex determination. Normally, SGPs reside at the poles of the gonadal primordium and divide according to intrinsic gonadal axes. In tra-1-null mutants, however, SGPs assume non-polar positions and the polarity of one SGP is reversed. Consistent with its SGP function, TRA-1 protein is present in SGPs; during embryogenesis and early larval development. Previous studies have shown that the ehn-3 gene also affects SGP positions, and we report here that tra-1 and ehn-3 interact genetically. Whereas SGPs in tra-1 and ehn-3 single mutants are largely normal and generate many descendants, those in ehn-3 double mutants do not mature or divide. Furthermore, tra-1 is a dominant enhancer of the ehn-3 gonadal defect, which includes the enhancement of a weak sexual transformation in the gonad. We cloned ehn-3, and found that it encodes a C2H2 zinc-finger protein. A rescuing EHN-3::GFP reporter is predominantly nuclear and expressed specifically in SGPs. The EHN-3 protein is therefore likely to regulate gene expression. We propose that TRA-1/GLI and EHN-3 have overlapping roles in regulation of multiple steps of SGP development. We speculate that regulation of SGP development may be an evolutionarily ancient role of TRA-1/GLI in nematode development. Full Text.

Michalkiewicz, M., Michalkiewicz, T., Ettinger, R. A., Rutledge, E. A., Fuller, J. M., Moralejo, D. H., Van Yserloo, B., MacMurray, A. J., Kwitek, A. E., Jacob, H. J., Lander, E. S. and Lernmark, A. (2004) Transgenic rescue demonstrates involvement of the Ian5 gene in T cell development in the rat. Physiological Genomics. 19 228-232. A single point mutation in a novel immune-associated nucleotide gene 5 (Ian5) coincides with severe T cell lymphopenia in BB rats. We used a transgenic rescue approach in lymphopenic BB-derived congenic F344.lyp/lyp rats to determine whether this mutation is responsible for lymphopenia and to establish the functional importance of this novel gene. A 150-kb P1 artificial chromosome (PAC) transgene harboring a wild-type allele of the rat Ian5 gene restored Ian5 transcript and protein levels, completely rescuing the T cell lymphopenia in the F344.lyp/lyp rats. This successful complementation provides direct functional evidence that the Ian5 gene product is essential for maintaining normal T cell levels. It also demonstrates that transgenic rescue in the rat is a practical and definitive method for revealing the function of a novel gene.

Middleton, F. A., Pato, M. T., Gentile, K. L., Morley, C. P., Zhao, X., Eisener, A. F., Brown, A., Petryshen, T. L., Kirby, A. N., Medeiros, H., Carvalho, C., Macedo, A., Dourado, A., Coelho, I., Valente, J., Soares, M. J., Ferreira, C. P., Lei, M., Azevedo, M. H., Kennedy, J. L., Daly, M. J., Sklar, P. and Pato, C. N. (2004) Genomewide linkage analysis of bipolar disorder by use of a high-density single-nucleotide-polymorphism (SNP) genotyping assay: A comparison with microsatellite marker assays and finding of significant linkage to chromosome 6q22. American Journal of Human Genetics. 74 886-897. We performed a linkage analysis on 25 extended multiplex Portuguese families segregating for bipolar disorder, by use of a high-density single-nucleotide-polymorphism (SNP) genotyping assay, the GeneChip Human Mapping 10K Array (HMA10K). Of these families, 12 were used for a direct comparison of the HMA10K with the traditional 10-cM microsatellite marker set and the more dense 4-cM marker set. This comparative analysis indicated the presence of significant linkage peaks in the SNP assay in chromosomal regions characterized by poor coverage and low information content on the microsatellite assays. The HMA10K provided consistently high information and enhanced coverage throughout these regions. Across the entire genome, the HMA10K had an average information content of 0.842 with 0.21-Mb intermarker spacing. In the 12-family set, the HMA10K-based analysis detected two chromosomal regions with genomewide significant linkage on chromosomes 6q22 and 11p11; both regions had failed to meet this strict threshold with the microsatellite assays. The full 25-family collection further strengthened the findings on chromosome 6q22, achieving genomewide significance with a maximum nonparametric linkage (NPL) score of 4.20 and a maximum LOD score of 3.56 at position 125.8 Mb. In addition to this highly significant finding, several other regions of suggestive linkage have also been identified in the 25-family data set, including two regions on chromosome 2 (57 Mb, NPL = 2.98; 145 Mb, NPL = 3.09), as well as regions on chromosomes 4 (91 Mb, NPL = 2.97), 16 (20 Mb, NPL = 2.89), and 20 (60 Mb, NPL = 2.99). We conclude that at least some of the linkage peaks we have identified may have been largely undetected in previous whole-genome scans for bipolar disorder because of insufficient coverage or information content, particularly on chromosomes 6q22 and 11p11. Full Text.

Molk, J. N., Schuyler, S. C., Liu, J. Y., Evans, J. G., Salmon, E. D., Pellman, D. and Bloom, K. (2004) The differential roles of budding yeast Tem1p, Cdc15p, and Bub2p protein dynamics in mitotic exit. Molecular Biology of the Cell. 15 1519-1532. In the budding yeast Saccharomyces cerevisiae the mitotic spindle must be positioned along the mother-bud axis to activate the mitotic exit network (MEN) in anaphase. To examine MEN proteins during mitotic exit, we imaged the MEN activators Tem1p and Cdc15p and the MEN regulator Bub2p in vivo. Quantitative live cell fluorescence microscopy demonstrated the spindle pole body that segregated into the daughter cell (dSPB) signaled mitotic exit upon penetration into the bud. Activation of mitotic exit was associated with an increased abundance of Tem1p-GFP and the localization of Cdc15p-GFP on the dSPB. In contrast, Bub2p-GFP fluorescence intensity decreased in mid-to-late anaphase on the dSPB. Therefore, MEN protein localization fluctuates to switch from Bub2p inhibition of mitotic exit to Cdc15p activation of mitotic exit. The mechanism that elevates Temlp-GFP abundance in anaphase is specific to dSPB penetration into the bud and Dhc1p and Lte1p promote Tem1p-GFP localization. Finally, fluorescence recovery after photobleaching (FRAP) measurements revealed Tem1p-GFP is dynamic at the dSPB in late anaphase. These data suggest spindle pole penetration into the bud activates mitotic exit, resulting in Tem1p and Cdc15p persistence at the dSPB to initiate the MEN signal cascade.Full Text.

Mootha, V. K., Handschin, C., Arlow, D., Xie, X., St Pierre, J., Sihag, S., Yang, W., Altshuler, D., Puigserver, P., Patterson, N., Willy, P. J., Schulman, I. G., Heyman, R. A., Lander, E. S. and Spiegelman, B. M. (2004) Erralpha and Gabpa/b specify PGC-1alpha-dependent oxidative phosphorylation gene expression that is altered in diabetic muscle. Proc Natl Acad Sci U S A. 101 6570-5. Recent studies have shown that genes involved in oxidative phosphorylation (OXPHOS) exhibit reduced expression in skeletal muscle of diabetic and prediabetic humans. Moreover, these changes may be mediated by the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha). By combining PGC-1alpha-induced genome-wide transcriptional profiles with a computational strategy to detect cis-regulatory motifs, we identified estrogen-related receptor alpha (Erralpha) and GA repeat-binding protein alpha as key transcription factors regulating the OXPHOS pathway. Interestingly, the genes encoding these two transcription factors are themselves PGC-1alpha-inducible and contain variants of both motifs near their promoters. Cellular assays confirmed that Erralpha and GA-binding protein a partner with PGC-1alpha in muscle to form a double-positive-feedback loop that drives the expression of many OXPHOS genes. By using a synthetic inhibitor of Erralpha, we demonstrated its key role in PGC-1alpha-mediated effects on gene regulation and cellular respiration. These results illustrate the dissection of gene regulatory networks in a complex mammalian system, elucidate the mechanism of PGC-1alpha action in the OXPHOS pathway, and suggest that Erralpha agonists may the mechanism of PGC-1alpha action in the OXPHOS pathway, and suggest that Erralpha agonists may ameliorate insulin-resistance in individuals with type 2 diabetes mellitus. Full Text.

Mukherjee, S., Berger, M. F., Jona, G., Wang, X. S., Muzzey, D., Snyder, M., Young, R. A. and Bulyk, M. L. (2004) Rapid analysis of the DNA-binding specificities of transcription factors with DNA microarrays. Nature Genetics. 36 1331-1339. We developed a new DNA microarray-based technology, called protein binding microarrays (PBMs), that allows rapid, high-throughput characterization of the in vitro DNA binding site sequence specificities of transcription factors in a single day. Using PBMs, we identified the DNA binding-site sequence specificities of the yeast transcription factors Abf1, Rap1 and Mig1. Comparison of these proteins' in vitro binding sites with their in vivo binding sites indicates that PBM-derived sequence specificities can accurately reflect in vivo DNA sequence specificities. In addition to previously identified targets, Abf1, Rap1 and Mig1 bound to 107, 90 and 75 putative new target intergenic regions, respectively, many of which were upstream of previously uncharacterized open reading frames. Comparative sequence analysis indicated that many of these newly identified sites are highly conserved across five sequenced sensu stricto yeast species and, therefore, are probably functional in vivo binding sites that may be used in a condition-specific manner. Similar PBM experiments should be useful in identifying new cis regulatory elements and transcriptional regulatory networks in various genomes. Full Text

Murthy, T. V. S. (2004) Expression of GST-fused kinase domain of human Csk homologous kinase from Pichia pastoris facilitates easy purification. Biotechnology Letters. 26 443-449. Human Csk Homologous Kinase (CHK), a protein of 527 amino acid residues, is involved in suppression of breast tumors. The kinase domain of CHK ( amino acid residues 228 to 485) expressed with C-terminal 6HIS fusion in Pichia pastoris is heavily glycosylated. Expression of the C-terminal 6HIS fused kinase domain of CHK, with an N-terminal glutathione S-transferase fusion, in Pichia pastoris alleviated the hyperglycosylation. The expressed protein was purified by affinity chromatography to 1 mg l(-1) culture and remained active. A simple plate assay to identify colonies of P. pastoris expressing the recombinant protein is also presented. Full Text.

Mutsuddi, M., Marshall, C. M., Benzow, K. A., Koob, M. D. and Rebay, I. (2004) The spinocerebellar ataxia 8 noncoding RNA causes neurodegeneration and associates with staufen in Drosophila. Curr Biol. 14 302-8. Spinocerebellar Ataxia 8 (SCA8) appears unique among triplet repeat expansion-induced neurodegenerative diseases because the predicted gene product is a noncoding RNA. Little is currently known about the normal function of SCA8 in neuronal survival or how repeat expansion contributes to neurodegeneration. To investigate the molecular context in which SCA8 operates, we have expressed the human SCA8 noncoding RNA in Drosophila. SCA8 induces late-onset, progressive neurodegeneration in the Drosophila retina. Using this neurodegenerative phenotype as a sensitized background for a genetic modifier screen, we have identified mutations in four genes: staufen, muscleblind, split ends, and CG3249. All four encode neuronally expressed RNA binding proteins conserved in Drosophila and humans. Although expression of both wild-type and repeat-expanded SCA8 induce neurodegeneration, the strength of interaction with certain modifiers differs between the two SCA8 backgrounds, suggesting that CUG expansions alter associations with specific RNA binding proteins. Our demonstration that SCA8 can recruit Staufen and that the interaction domain maps to the portion of the SCA8 RNA that undergoes repeat expansion in the human disease suggests a specific mechanism for SCA8 function and disease. Genetic modifiers identified in our SCA8-based screens may provide candidates for designing therapeutic interventions to treat this disease. Full Text.

Nardi, V., Azam, M. and Daley, G. Q. (2004) Mechanisms and implications of imatinib resistance mutations in BCR-ABL. Curr Opin Hematol. 11 35-43. SUMMARY: PURPOSE OF REVIEW Aside from bone marrow transplantation, a definitive cure for Philadelphia (Ph) chromosome-positive chronic myeloid leukemia (CML) has yet to be developed. Although Imatinib, the first molecularly targeted drug developed for CML has achieved a remarkable success, the emergence of resistance to this agent mitigates the prospect of a cure for this leukemia. Though a variety of resistance mechanisms can arise, in the majority of patients resistance coincides with reactivation of the tyrosine kinase activity of the BCR-ABL fusion oncoprotein. This can result from gene amplification and, more importantly, point mutations that disrupt the bind of imatinib to BCR-ABL itself. In this review, we aim to define and illuminate mechanisms of resistance and describe how drug resistance is shedding new light on kinase domain regulation.RECENT FINDINGS In light of recent studies and publications, it is now clear that Imatinib exerts its inhibitory action by stabilizing the inactive non ATP-binding conformation of BCR-ABL and that mutations even outside the kinase domain can lead to enhanced autophosphorylation of the kinase, thereby stabilizing the active conformation that resists imatinib binding. So far, 25 different substitutions of 21 amino acid residues of BCR-ABL have been detected in CML patients. In addition, it has been recently illustrated that mutations preexist the onset of treatment and that some confer a more aggressive disease phenotype. Finally it has been shown that molecular remission is almost never reached through Imatinib therapy.SUMMARY The most common mechanism of relapse for CML patients treated with Imatinib is the appearance of point mutations in the BCR-ABL oncogene that confer resistance to this drug. Insights into the emerging problem of resistance should promote the rational development of alternative, synergistic, and potentially curative treatment strategies .Full Text.

Nash, M. W., Huezo-Diaz, P., Sterne, A., Purcell, S., Hoda, F., Cherny, S. S., Abecasis, G. R., Prince, M., Gray, J. A., Ball, D., Asherson, P., Mann, A., Goldberg, D., McGuffin, P., Farmer, A., Plomin, R., Craig, I. W. and Sham, P. C. (2004) Genome-wide linkage analysis of a composite index of neuroticism and mood-related scales in extreme selected sibships. Human Molecular Genetics. 13 2173-2182. There is considerable evidence to suggest that the genetic vulnerabilities to depression and anxiety substantially overlap and quantitatively act to alter risk to both disorders. Continuous scales can be used to index this shared liability and are a complementary approach to the use of clinical phenotypes in the genetic analysis of depression and anxiety. The aim of this study (Genetic and Environmental Nature of Emotional States in Siblings) was to identify genetic variants for the liability to depression and anxiety after the application of quantitative genetic methodology to a large community-based sample (n=34 371), using four well-validated questionnaires of depression and anxiety. Genetic model fitting was performed on 2658 unselected sibships, which provided evidence for a single common familial factor that accounted for a substantial proportion of the genetic variances and covariances of the four scales. Using the parameter estimates from this model, a composite index of liability (G) was constructed. This index was then used to select a smaller-but statistically powerful-sample for DNA collection (757 individuals, 297 sibships). These individuals were genotyped with more than 400 microsatellite markers. After the data were checked and cleaned, linkage analysis was performed on G and the personality scale of neuroticism using theregression-based linkage program MERLIN-REGRESS. The results indicated two potential quantitative trait loci (QTL): one on chromosome 1p (LOD 2.2) around 64 cM (43-70 cM) near marker D1S2892 and another on chromosome 6p (LOD 2.7) around 47 cM (34-63 cM) near marker D6S1610. Further exploratory sex-specific analyses suggested that these QTLs might have sex-limited effects. Full Text.

Natoli, T. A., Alberta, J. A., Bortvin, A., Taglienti, M. E., Menke, D. B., Loring, J., Jaenisch, R., Page, D. C., Housman, D. E. and Kreidberg, J. A. (2004) Wtl functions in the development of germ cells in addition to somatic cell lineages of the testis. Developmental Biology. 268 429-440. The Wilms' tumor suppressor gene, Wt1, encodes a transcription factor critical for development of the urogenital system. To identify lineages within the developing urogenital system that have a cell-autonomous requirement for Wt1, chimeric mice were generated from Wt1-null ES cells. Males with large contributions of Wt1-/- cells showed hypoplastic and dysgenic testes, with seminiferous tubules lacking spermatogonia. Wt1-null cells contributed poorly to both somatic and germ cell lineages within the developing gonad, suggesting an unexpected role for Wt1 in germ cell development in addition to a role in the development of the somatic lineages of the gonad. Wt1 expression was detected in embryonic germ cells beginning at embryonic day 11.5 after migrating primordial germ cells (PGCs) have entered the gonad. Germ cells isolated from Wt1-null embryos showed impaired growth in culture, further demonstrating a role for Wt1 in germ cell proliferation or survival. Therefore, Wt1 plays important, and in some cases previously unrecognized, roles in multiple lineages during urogenital development. Full Text.

Neves, G., Zucker, J., Daly, M., and Chess, A. (2004). Stochastic yet biased expression of multiple Dscam splice variants by individual cells. Nature Genetics.Published online 1 Feb. The Drosophila melanogaster gene Dscam is essential for axon guidance and has 38,016 possible alternative splice forms. This diversity can potentially be used to distinguish cells. We analyzed the Dscam mRNA isoforms expressed by different cell types and individual cells. The choice of splice variants expressed is regulated both spatially and temporally. Different subtypes of photoreceptors express broad yet distinctive spectra of Dscam isoforms. Single-cell RT-PCR documented that individual cells express several different Dscam isoforms and allowed an estimation of the diversity that is present. For example, we estimate that each R3/R4 photoreceptor cell expresses 14-50 distinct mRNAs chosen from the spectrum of thousands of splice variants distinctive of its cell type. Thus, the Dscam repertoire of each cell is different from those of its neighbors, providing a potential mechanism for generating unique cell identity in the nervous system and elsewhere. Full Text.

Odom, D. T., Zizlsperger, N., Gordon, D. B., Bell, G. W., Rinaldi, N. J., Murray, H. L., Volkert, T. L., Schreiber, J., Rolfe, P. A., Gifford, D. K., Fraenkel, E.,Young, RA, et al. (2004). Control of pancreas and liver gene expression by HNF transcription factors. Science 303, 1378-1381. The transcriptional regulatory networks that specify and maintain human tissue diversity are largely uncharted. To gain insight into this circuitry, we used chromatin immunoprecipitation combined with promoter microarrays to identify systematically the genes occupied by the transcriptional regulators HNF1alpha, HNF4alpha, and HNF6, together with RNA polymerase II, in human liver and pancreatic islets. We identified tissue-specific regulatory circuits formed by HNF1alpha, HNF4alpha, and HNF6 with other transcription factors, revealing how these factors function as master regulators of hepatocyte and islet transcription. Our results suggest how misregulation of HNF4alpha can contribute to type 2 diabetes. Full Text.

Ohler, U., Yekta, S., Lim, L. P., Bartel, D. P. and Burge, C. B. (2004) Patterns of flanking sequence conservation and a characteristic upstream motif for microRNA gene identification. Rna. 10 1309-1322. MicroRNAs are ~22-nucleotide (nt) RNAs processed from foldback segments of endogenous transcripts. Some are known to play important gene regulatory roles during animal and plant development by pairing to the messages of protein-coding genes to direct the post-transcriptional repression of these messages. Previously, we developed a computational method called MiRscan, which scores features related to the foldbacks, and used this algorithm to identify new miRNA genes in the nematode Caenorhabditis elegans. In the present study, to identify sequences that might be involved in processing or transcriptional regulation of miRNAs, we aligned sequences upstream and downstream of orthologous nematode miRNA foldbacks. These alignments showed a pronounced peak in sequence conservation about 200 bp upstream of the miRNA foldback and revealed a highly significant sequence motif, with consensus CTCCGCCC, that is present upstream of almost all independently transcribed nematode miRNA genes. Scoring the pattern of upstream/downstream conservation, the occurrence of this sequence motif, and orthology of host genes for intronic miRNA candidates, yielded substantial improvements in the accuracy of MiRscan. Nine new C. elegans miRNA gene candidates were validated using a PCR-sequencing protocol. As previously seen for bacterial RNA genes, sequence features outside of the RNA secondary structure can therefore be very useful for the computational identification of eukaryotic noncoding RNA genes. The total number of confidently identified nematode miRNAs now approaches 100. The improved analysis supports our previous assertion that miRNA gene identification is nearing completion in C. elegans with apparently no more than 20 miRNA genes now remaining to be identified. Full Text.

Oksenberg, J. R., Barcellos, L. F., Cree, B. A. C., Baranzini, S. E., Bugawan, T. L., Khan, O., Lincoln, R. R., Swerdlin, A., Mignot, E., Lin, L, .Reich, D.E., et al. (2004). Mapping multiple sclerosis susceptibility to the HLA-DR locus in African Americans. American Journal of Human Genetics 74, 160-167. An underlying complex genetic susceptibility exists in multiple sclerosis ( MS), and an association with the HLA-DRB1* 1501-DQB1* 0602 haplotype has been repeatedly demonstrated in high-risk ( northern European) populations. It is unknown whether the effect is explained by the HLA-DRB1 or the HLA-DQB1 gene within the susceptibility haplotype, which are in strong linkage disequilibrium (LD). African populations are characterized by greater haplotypic diversity and distinct patterns of LD compared with northern Europeans. To better localize the HLA gene responsible for MS susceptibility, case-control and family-based association studies were performed for DRB1 and DQB1 loci in a large and well-characterized African American data set. A selective association with HLA-DRB1* 15 was revealed, indicating a primary role for the DRB1 locus in MS independent of DQB1*0602. This finding is unlikely to be solely explained by admixture, since a substantial proportion of the susceptibility chromosomes from African American patients with MS displayed haplotypes consistent with an African origin. Full Text.

Outeiro, T. F., and Muchowski, P. J. (2004). Molecular genetics approaches in yeast to study amyloid diseases. J Mol Neurosci 23, 49-60. The occurrence of protein aggregates in ordered fibrillar structures known as amyloid, found inside and outside of brain cells, is a feature shared by many neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases. Although the molecular mechanisms that underlie neurodegeneration will ultimately have to be tested in neuronal and animal models, there are several distinct advantages in using model organisms to elucidate fundamental aspects of protein aggregation, amyloid formation, and toxicity. Here, we review recent studies indicating that amyloid formation by disease-causing proteins can be faithfully recapitulated in simple yeast-based models in Saccharomyces cerevisiae. These studies have already contributed to our basic understanding of molecular chaperone function/dysfunction in Huntington's disease, and functional genomics approaches being undertaken currently will likely bear novel insights into the genes and pathways that modulate neuronal cell dysfunction and death in these devastating diseases. A final advantage of using yeast to study amyloid formation and toxicity is the ease and rapidity with which large-scale drug-screening efforts can be conducted in this model organism.

Page, D. C. (2004). 2003 Curt Stern Award address - On low expectations exceeded; or, the genomic salvation of the Y chromosome. American Journal of Human Genetics 74, 399-402. Full Text.

Palliser, D., Huang, Q., Hacohen, N., Lamontagne, S. P., Guillen, E., Young, R. A., and Eisen, H. N. (2004). A role for toll-like receptor 4 in dendritic cell activation and cytolytic CD8(+) T cell differentiation in response to a recombinant heat shock fusion protein. J Immunol 172, 2885-2893. Recombinant heat shock fusion proteins (Hsfp) injected into mice without added adjuvants can stimulate production of CD8 cytolytic T cells. Because initiation of productive immune responses generally requires dendritic cell (DC) activation, the question arises as to whether the Hsfp can activate DC independently of contaminating LPS. Using microarray analyses of DC from LPS-insensitive mice having a point mutation in Toll-like receptor 4 (Tlr4) (C3H/HeJ), or lacking Tlr4 (B10/ScNCr), we show here that unlike a LPS standard, Hsfp activated DC from HeJ mice almost as well as DC from wild-type mice. Consistent with the microarray analysis, the Hsfp's ability to activate DC was not eliminated by polymyxin B but was destroyed by proteinase K. The Hsfp did not, however, stimulate DC from mice lacking Tlr4. In vivo the CD8 T cell response to the Hsfp in mice lacking Tlr4 was impaired: the responding CD8 cells initially proliferated vigorously but their development into cytolytic effector cells was diminished. Overall, the results indicate that this Hsfp can activate DC independently of LPS but still requires Tlr4 for an optimal CD8 T cell response.Full Text.

Pato, C. N., Pato, M. T., Kirby, A., Petryshen, T. L., Medeiros, H., Carvalho, C., Macedo, A., Dourado, A., Coelho, I., Valente, J., Daly, MJ, et al (2004). Genome-wide scan in Portuguese Island families implicates multiple loci in bipolar disorder: Fine mapping adds support on chromosomes 6 and 11. American Journal of Medical Genetics Part B-Neuropsychiatric Genetics 127B, 30-34. As part of an extensive study in the Portuguese Island population of families with multiple patients suffering from bipolar disorder and schizophrenia, we performed an initial genome-wide scan of 16 extended families with bipolar disorder that identified three regions on chromosomes 2, 11, and 19 with genome-wide suggestive linkage and several other regions, including chromosome 6q, also approached suggestive levels of significance. Dick et al. [2003: Am J Hum Genet 73:107114] recently reported in a study of 250 families with bipolar disorder a maxLOD score of 3.61 near marker D6S1021 on chromosome 6q. This study replicates this finding having detected a peak NPL = 2.02 (P = 0.025) with the same marker D6S1021(104.7 Mb). Higher-density mapping provided additional support for loci on chromosome 6 including marker D6S1021 with an NPL = 2.59 (P = 0.0068) and peaking at marker D6S1639 (125 Mb) with an NPL = 3.06 (P = 0.0019). A similar pattern was detected with higher-density mapping of chromosome 11 with an NPL = 3.15 (P = 0.0014) at marker D11S1883 (63.1 Mb). Simulations at the density of our fine mapping data indicate that less than 1 scan out of 10 would find two such scores genome-wide in the same scan by chance. Our findings provide additional support for a susceptibility locus for bipolar disorder on 6q, as well as, suggesting the importance of denser scans. Full Text.

Patterson, N., Hattangadi, N., Lane, B., Lohmueller, K. E., Hafler, D. A., Oksenberg, J. R., Hauser, S. L., Smith, M. W., O'Brien, S. J., Altshuler, D., Daly, M.J.et al. (2004). Methods for high-density admixture mapping of disease genes. American Journal of Human Genetics 74, 979-1000. Admixture mapping (also known as "mapping by admixture linkage disequilibrium," or MALD) has been proposed as an efficient approach to localizing disease-causing variants that differ in frequency ( because of either drift or selection) between two historically separated populations. Near a disease gene, patient populations descended from the recent mixing of two or more ethnic groups should have an increased probability of inheriting the alleles derived from the ethnic group that carries more disease-susceptibility alleles. The central attraction of admixture mapping is that, since gene flow has occurred recently in modern populations ( e. g., in African and Hispanic Americans in the past 20 generations), it is expected that admixture-generated linkage disequilibrium should extend for many centimorgans. High-resolution marker sets are now becoming available to test this approach, but progress will require ( a) computational methods to infer ancestral origin at each point in the genome and (b) empirical characterization of the general properties of linkage disequilibrium due to admixture. Here we describe statistical methods to estimate the ancestral origin of a locus on the basis of the composite genotypes of linked markers, and we show that this approach accurately estimates states of ancestral origin along the genome. We apply this approach to show that strong admixture linkage disequilibrium extends, on average, for 17 cM in African Americans. Finally, we present power calculations under varying models of disease risk, sample size, and proportions of ancestry. Studying similar to2,500 markers in similar to2,500 patients should provide power to detect many regions contributing to common disease. A particularly important result is that the power of an admixture mapping study to detect a locus will be nearly the same for a wide range of mixture scenarios: the mixture proportion should be 10%-90% from both ancestral populations.Full Text.

Pe'er, I. and Beckmann, J. S. (2004) Recovering frequencies of known haplotype blocks from single-nucleotide polymorphism allele frequencies Genetics 166 2001-2006.Prospects for large-scale association studies rely on economical methods and powerful analysis. Representing available SNPs by small subsets and measuring allele frequencies on pooled DNA samples each improve genotyping cost effectiveness, while haplotype analysis may highlight associations in otherwise underpowered studies. This manuscript provides the mathematical framework to integrate these methodologies.Full Text.

Plath, K., Talbot, D., Hamer, K. M., Otte, A. P., Yang, T. P., Jaenisch, R. and Panning, B. (2004) Developmentally regulated alterations in polycomb repressive complex 1 proteins on the inactive X chromosome. Journal of Cell Biology. 167 1025-1035. Polycomb group (PcG) proteins belonging to the polycomb (Pc) repressive complexes 1 and 2 (PRC1 and PRC2) maintain homeotic gene silencing. In Drosophila, PRC2 methylates histone H3 on lysine 27, and this epigenetic mark facilitates recruitment of PRC1. Mouse PRC2 (mPRC2) has been implicated in X inactivation, as mPRC2 proteins transiently accumulate on the inactive X chromosome (Xi) at the onset of X inactivation to methylate histone H3 lysine 27 (H3-K27). In this study, we demonstrate that mPRC1 proteins localize to the Xi, and that different mill proteins accumulate on the Xi during initiation and maintenance of X inactivation in embryonic cells. The Xi accumulation of mPRC1 proteins requires Xist RNA and is not solely regulated by the presence of H3-K27 methylation, as not all cells that exhibit this epigenetic mark on the Xi show Xi enrichment of mPRC1 proteins. Our results implicate mPRC1 in X inactivation and suggest that the regulated assembly of PcG protein complexes on the Xi contributes to this multistep process.Full Text.

Plecs, J. J., Harbury, P. B., Kim, P. S. and Alber, T. (2004) Structural test of the parameterized-backbone method for protein design. Journal of Molecular Biology. 342 289-297. Designing new protein folds requires a method for simultaneously optimizing the conformation of the backbone and the side-chains. One approach to this problem is the use of a parameterized backbone, which allows the systematic exploration of families of structures. We report the crystal structure of RH3, a right-handed, three-helix coiled coil that was designed using a parameterized backbone and detailed modeling of core packing. This crystal structure was determined using another rationally designed feature, a metal-binding site that permitted experimental phasing of the X-ray data. RH3 adopted the intended fold, which has not been observed previously in biological proteins. Unanticipated structural asymmetry in the trimer was a principal source of variation within the RH3 structure. The sequence of RH3 differs from that of a previously characterized right-handed tetramer, RH4, at only one position in each 11 amino acid sequence repeat. This close similarity indicates that the design method is sensitive to the core packing interactions that specify the protein structure. Comparison of the structures of RH3 and RH4 indicates that both steric overlap and cavity formation provide strong driving forces for oligomer specificity. Full Text.

Poggio, T., Rifkin, R., Mukherjee, S., and Niyogi, P. (2004). General conditions for predictivity in learning theory. Nature 428, 419-422. Developing theoretical foundations for learning is a key step towards understanding intelligence. 'Learning from examples' is a paradigm in which systems ( natural or artificial) learn a functional relationship from a training set of examples. Within this paradigm, a learning algorithm is a map from the space of training sets to the hypothesis space of possible functional solutions. A central question for the theory is to determine conditions under which a learning algorithm will generalize from its finite training set to novel examples. A milestone in learning theory(1-5) was a characterization of conditions on the hypothesis space that ensure generalization for the natural class of empirical risk minimization (ERM) learning algorithms that are based on minimizing the error on the training set. Here we provide conditions for generalization in terms of a precise stability property of the learning process: when the training set is perturbed by deleting one example, the learned hypothesis does not change much. This stability property stipulates conditions on the learning map rather than on the hypothesis space, subsumes the classical theory for ERM algorithms, and is applicable to more general algorithms. The surprising connection between stability and predictivity has implications for the foundations of learning theory and for the design of novel algorithms, and provides insights into problems as diverse as language learning and inverse problems in physics and engineering.Full Text.

Poirier, C., Qin, Y. J., Adams, C. P., Anaya, Y., Singer, J. B., Hill, A. E., Lander, E. S., Nadeau, J. H. and Bishop, C. E. (2004) A complex interaction of imprinted and maternal-effect genes modifies sex determination in odd sex (Ods) mice. Genetics. 168 1557-1562. The transgenic insertional mouse mutation Odd Sex (Ods) represents a model for the long-range regulation of Sox9. The mutation causes complete female-to-male sex reversal by inducing a male-specific expression pattern of Sox9 in XX Ods/+ embryonic gonads. We previously described an A/J strain-specific Suppressor of Ods termed Odsm1(A). Here we show that phenotypic sex depends on a complex interaction between the Suppressor and the transgene. Suppression can be achieved only if the transgene is transmitted paternally. In addition, the suppressor itself exhibits a maternal effect, Suggesting that it may act on chromatin in the early embryo .Full Text

Prudhomme, W., Daley, G. Q., Zandstra, P., and Lauffenburger, D. A. (2004). Multivariate proteomic analysis of murine embryonic stem cell self-renewal versus differentiation signaling. PNAS 101(9) : 2900-2905. A number of extracellular stimuli, including soluble cytokines and insoluble matrix factors, are known to influence murine embryonic stem cell self-renewal and differentiation behavioral responses via intracellular signaling pathways, but their net effects in combination are difficult to understand. To gain insight concerning key intracellular signals governing these behavioral responses, we employ a multivariate systems analysis of proteomic data generated from combinatorial stimulation of mouse embryonic stem cells by fibronectin, laminin, leukemia-inhibitory factor, and fibroblast growth factor 4. Phosphorylation states of 31 intracellular signaling network components were obtained across 16 different stimulus conditions at three time points by quantitative Western blotting, and partial-least-squares modeling was used to determine which components were most strongly correlated with cell proliferation and differentiation rate constants obtained from flow cytometry measurements of Oct-4 expression levels. This data-driven, multivariate (16 conditions x 31 components x 3 time points = approximately 1,500 values) proteomic approach identified a set of signaling network components most critically associated (positively or negatively) with differentiation (Stat3, Raf1, MEK, and ERK), proliferation of undifferentiated cells (MEK and ERK), and proliferation of differentiated cells (PKBalpha, Stat3, Src, and PKCepsilon). These predictions were found to be consistent with previous in vivo literature, along with direct in vitro test here by a peptide inhibitor of PKCepsilon. Our results demonstrate how a computational systems biology approach can elucidate key sets of intracellular signaling protein activities that combine to govern cell phenotypic responses to extracellular cues.PDF.

Prusty, R., Grisafi, P., and Fink, G. R. (2004). The plant hormone indoleacetic acid induces invasive growth in Saccharomyces cerevisiae. PNAS Early Edition 9 March 2004.Fungi must recognize plant-specific signals to initiate subsequent morphogenetic events such as filamentation that lead to infection. Here we show that the plant hormone indoleacetic acid (IAA) induces adhesion and filamentation of Saccharomyces cerevisiae. Genome expression profiling of cells treated with IAA identified Yap1, a fungal specific transcription factor, as a key mediator of this response. Strains lacking YAP1 (yap1-1) are hypersensitive to growth on IAA because they accumulate more IAA than can wild type. Members of a family of transporters the amino acid/auxin:proton symport permeases with homology to AUX1, a putative IAA transporter from plants, are up-regulated in the yap1-1 mutant. Deletion of any one of these transporters makes yap1-1 mutants more resistant to IAA by decreasing its uptake. The permease mutants are defective in IAA perception and filamentation. The ability of a fungus to perceive a plant hormone that causes it to differentiate into an invasive form has important implications for plant-pathogen interactions. PDF.

Purcell, S. and Sham, P. (2004) Properties of structured association approaches to detecting population stratification. Hum Hered. 58 93-107. Objective: To examine the properties of the structured association approach for the detection and correction of population stratification. Method: A method is developed, within a latent class analysis framework, similar to the methods proposed by Satten et al. (2001) and Pritchard et al. (2000). A series of simulations illustrate the relative impact of number and type of loci, sample size and population structure. Results: The ability to detect stratification and assign individuals to population strata is determined for a number of different scenarios. Conclusion: The results underline the importance of careful marker selection.

Purcell, S., Sham, P. and Daly, M. J. (2004) Parental Phenotypes in Family-Based Association Analysis. Am J Hum Genet. Dec 21;76(2) [Epub ahead of print] Family-based association designs are popular, because they offer inherent control of population stratification based on age, sex, ethnicity, and environmental exposure. However, the efficiency of these designs is hampered by current analytic strategies that consider only offspring phenotypes. Here, we describe the incorporation of parental phenotypes and, specifically, the inclusion of parental genotype-phenotype correlation terms in association tests, providing a series of tests that effectively span an efficiency-robustness spectrum. The model is based on the between-within-sibship association model presented in 1999 by Fulker and colleagues for quantitative traits and extended here to nuclear families. By use of a liability-threshold-model approach, standard dichotomous and/or qualitative disease phenotypes can be analyzed (and can include appropriate corrections for phenotypically ascertained samples), which allows for the application of this model to analysis of the commonly used affected-proband trio design. We show that the incorporation of parental phenotypes can considerably increase power, as compared with the standard transmission/disequilibrium test and equivalent quantitative tests, while providing both significant protection against stratification and a means of evaluating the contribution of stratification to positive results. This methodology enables the extraction of more information from existing family-based collections that are currently being genotyped and analyzed by use of standard approaches.Full Text

Qiao, F., Song, H. Y., Kim, C. A., Sawaya, M. R., Hunter, J. B., Gingery, M., Rebay, I., Courey, A. J. and Bowie, J. U. (2004) Derepression by depolymerization: Structural insights into the regulation of Yan by Mae. Cell. 118 163-173. Yan, an ETS family transcriptional repressor, is regulated by receptor tyrosine kinase signaling via the Ras/ MAPK pathway. Phosphorylation and downregulation of Yan is facilitated by a protein called Mae. Yan and Mae interact through their SAM domains. We find that repression by Yan requires the formation of a higher order structure mediated by Yan-SAM polymerization. Moreover, a crystal structure of the Yan-SAM/Mae-SAM complex shows that Mae-SAM specifically recognizes a surface on Yan-SAM that is also required for Yan-SAM polymerization. Mae-SAM binds to Yan-SAM with similar to1000-fold higher affinity than Yan-SAM binds to itself and can effectively depolymerize Yan-SAM. Mutations on Mae that specifically disrupt its SAM domain-dependent interactions with Yan disable the derepression function of Mae in vivo. Depolymerization of Yan by Mae represents a novel mechanism of transcriptional control that sensitizes Yan for regulation by receptor tyrosine kinases. Full Text.

Quezada, C. M., Schulman, B. A., Froggatt, J. J., Dobson, C. M., and Redfield, C. (2004). Local and global cooperativity in the human alpha-lactalbumin molten globule. Journal of Molecular Biology 338, 149-158. NMR spectroscopy has been used to follow the urea-induced unfolding of the low pH molten globule states of a single-disulfide variant of human alpha-lactalbumin ([28-111] alpha-LA) and of two mutants, each with a single proline substitution in a helix. [28-111] alpha-LA forms a molten globule very similar to that formed by the wild-type four-disulfide protein, and this variant has been used as a model for the alpha-lactalbumin (alpha-LA) molten globule in a number of studies. The urea-induced unfolding behavior of [28-111] alpha-LA is similar to that of the four-disulfide form of the protein, except that [28-111] alpha-LA is less stable and has greater cooperativity in the loss of different elements of structure. For one mutant, L11P, the helix containing the mutation is highly destabilized such that it is completely unfolded even in the absence of urea. By contrast, for the other mutant, Q117P the helix containing the mutation retains its compact structure. Both mutations, however, show significant long-range destabilization of the overall fold showing that the molten globule state has a degree of global cooperativity. The results reveal that different permutations of three of the four major alpha-helices of the protein can form a stable, locally cooperative, compact structural core. Taken together, these findings demonstrate that the molten globule state of a-LA is an ensemble of conformations, with different subsets of structures linked by a range of long-range interactions. Full Text

Ramaswawy, S. (2004). Microarrays for an integrative genomics. American Journal of Human Biology 16, 174-175.

Ramussen, J. P., Taylor, A. H., Ma, L. J., Purcell, S., Kempken, F., and Catcheside, D. E. A. (2004). Guest, a transposable element belonging to the Tcl/mariner superfamily is an ancient invader of Neurospora genomes. Fungal Genetics and Biology 41, 52-61. Guest is a transposable element of the Tc1/mariner superfamily with 30-40 bp terminal inverted repeats and a TA dinucleotide target site duplication. Guest was originally discovered in the St. Lawrence 74A laboratory strain of the filamentous fungus Neurospora crassa. In this report, Guest iterations subcloned from a cosmid library of the Oakridge 74A strain were used to design PCR primers that permitted the detection of Guest in wild isolates of N. crassa. Guest is present in N. crassa as multiple copies ranging between 100 bp and 2.4kb and is present in the mating type locus of several Neurospora species. Bioinformatic analysis of the entire N. crassa genome (Oakridge 74A strain) detected 48 Guest iterations. All iterations appeared to have been inactivated either by repeat-induced point mutation or sequence deletion, with the majority being remnants less than 400 bp in length. The possible involvement of Guest in the evolution of the variabl region that flanks the mating type idiomorphs in several Neurospora species is discussed. Full Text.

Rangarajan, A., Hong, S. J., Gifford, A. and Weinberg, R. A. (2004) Species- and cell type-specific requirements for cellular transformation. Cancer Cell. 6 171-83. Recent evidence suggests that human cells require more genetic changes for neoplastic transformation than do their murine counterparts. However, a precise enumeration of these differences has never been undertaken. We have determined that perturbation of two signaling pathways-involving p53 and Raf-suffices for the tumorigenic conversion of normal murine fibroblasts, while perturbation of six pathways-involving p53, pRb, PP2A, telomerase, Raf, and Ral-GEFs-is needed for human fibroblasts. Cell type-specific differences also exist in the requirements for tumorigenic transformation: immortalized human fibroblasts require the activation of Raf and Ral-GEFs, human embryonic kidney cells require the activation of PI3K and Ral-GEFs, and human mammary epithelial cells require the activation of Raf, PI3K, and Ral-GEF. Full Text

Repping, S., Korver, C. M., Oates, R. D., Silber, S., Van Der Veen, F., Page, D. C. and Rozen, S. (2004) Are Sequence Family Variants Useful for Identifying Deletions in the Human Y Chromosome? Am J Hum Genet. 75 514-517. Full Text.

Repping, S., van Daalen, S. K. M., Korver, C. M., Brown, L. G., Marszalek, J. D., Gianotten, J., Oates, R. D., Silber, S., van der Veen, F., Page, D. C. and Rozen, S. (2004) A family of human Y chromosomes has dispersed throughout northern Eurasia despite a 1.8-Mb deletion in the azoospermia factor c region. Genomics. 83 1046-1052. The human Y chromosome is replete with amplicons-very large, nearly identical repeats-which render it susceptible to interstitial deletions that often cause spermatogenic failure. Here we describe a recurrent, 1.8-Mb deletion that removes half of the azoospermia factor c (AZFc) region, including 12 members of eight testis-specific gene families. We show that this "b2/b3" deletion arose at least four times in human history-likely on inverted variants of the AZFc region that we find exist as common polymorphisms. We observed the b2/b3 deletion primarily in one family of closely related Y chromosomes-branch N in the Y-chromosome genealogy-in which all chromosomes carried the deletion. This branch is known to be widely distributed in northern Eurasia, accounts for the majority of Y chromosomes in some populations, and appears to be several thousand years old. The population-genetic success of the b2/b3 deletion is surprising, (i) because it removes half of AZFc and (ii) because the gr/gr deletion, which removes a similar set of testis-specific genes, predisposes to spermatogenic failure. Our present findings suggest either that the b2/b3 deletion has at most a modest effect on fitness or that, within branch N, its effect has been counterbalanced by another genetic, possibly Y-linked, factor. Full Text

Robert, F., Pokholok, D. K., Hannett, N. M., Rinaldi, N. J., Chandy, M., Rolfe, A., Workman, J. L., Gifford, D. K. and Young, R. A. (2004) Global Position and Recruitment of HATs and HDACs in the Yeast Genome. Mol Cell. 16 199-209. Chromatin regulators play fundamental roles in the regulation of gene expression and chromosome maintenance, but the regions of the genome where most of these regulators function has not been established. We explored the genome-wide occupancy of four different chromatin regulators encoded in Saccharomyces cerevisiae. The results reveal that the histone acetyltransferases Gcn5 and Esa1 are both generally recruited to the promoters of active protein-coding genes. In contrast, the histone deacetylases Hst1 and Rpd3 are recruited to specific sets of genes associated with distinct cellular functions. Our results provide new insights into the association of histone acetyltransferases and histone deacetylases with the yeast genome, and together with previous studies, suggest how these chromatin regulators are recruited to specific regions of the genome. Full Text .

Rochet, J. C., Outeiro, T. F., Conway, K. A., Ding, T. T., Volles, M. J., Lashuel, H. A., Bieganski, R. M., Lindquist, S. L. and Lansbury, P. T. (2004) Interactions among alpha-synuclein, dopamine, and biomembranes - Some clues for understanding neurodegeneration in Parkinson's disease. Journal of Molecular Neuroscience. 23 23-33. Parkinson's disease (PD) is a neurologic disorder resulting from the loss of dopaminergic neurons in the brain. Two lines of evidence suggest that the protein alpha-synuclein plays a role in the pathogenesis of PD: Fibrillar alpha-synuclein is a major component of Lewy bodies in diseased neurons, and two mutations in alpha-synuclein are linked to early-onset disease. Accordingly, the fibrillization of alpha-synuclein is proposed to contribute to neurodegeneration in PD. In this report, we provide evidence that oligomeric intermediates of the alpha-synuclein fibrillization pathway, termed protofibrils, might be neurotoxic. Analyses of protofibrillar alpha-synuclein by atomic force microscopy and electron microscopy indicate that the oligomers consist of spheres, chains, and rings. alpha-Synuclein protofibrils permeabilize synthetic vesicles and form pore-like assemblies on the surface of brain-derived vesicles. Dopamine reacts with alpha-synuclein to form a covalent adduct that slows the conversion of protofibrils to fibrils. This finding suggests that cytosolic dopamine in dopaminergic neurons promotes the accumulation of toxic alpha-synuclein protofibrils, which might explain why these neurons are most vulnerable to degeneration in PD. Finally, we note that aggregation of alpha-synuclein likely occurs via different mechanisms in the cell versus the test tube. For example, the binding of alpha-synuclein to cellular membranes might influence its self-assembly To address this point, we have developed a yeast model that might enable the selection of random alpha-synuclein mutants with different membrane-binding affinities. These variants might be useful to test whether membrane binding by alpha-synuclein is necessary for neurodegeneration in transgenic animal models of PD.

Ruan, H., and Lodish, H. F. (2004). Regulation of insulin sensitivity by adipose tissue-derived hormones and inflammatory cytokines. Curr Opin Lipidol 15, 297-302. PURPOSE OF REVIEW: The aim of this review is to assess the role of adipose tissue-derived hormones and inflammatory cytokines in the pathogenesis of obesity-linked type II diabetes, with a special focus on articles published between December 2002 and December 2003. RECENT FINDINGS: Insulin resistance is widely recognized as a fundamental defect seen in obesity and type II diabetes. Although the molecular mechanisms triggering the development of insulin resistance remain elusive, recent studies have suggested that adipose tissue and adipose tissue-derived hormones and inflammatory cytokines play essential roles in the overall insulin sensitivity in vivo. Dysfunctions of adipose tissue can lead to systemic insulin resistance. SUMMARY: Understanding the regulation of the metabolic and secretory functions of adipose tissue, as well as its subsequent impact on overall insulin sensitivity, is becoming increasingly important given the therapeutic potential of targeting the root causes of insulin resistance in the treatment of type 2 diabetes and its associated complications, such as cardiovascular and cerebrovascular diseases

Sagerstrom, C. G., Gammill, L. S., Veale, R. and Sive, H. (2004) Specification of the enveloping layer and lack of autoneuralization in zebrafish embryonic explants. Developmental Dynamics Online 12 November. We have analyzed the roles of cell contact during determination of the outermost enveloping layer (EVL) and deeper neurectoderm in zebrafish embryos. Outer cells, but not deeper cells, are specified to express the EVL-specific marker, cyt1 by late blastula. EVL specification requires cell contact or close cell proximity, because cyt1 is not expressed after explant dissociation. The EVL may be homologous to the Xenopus epithelial layer, including the ventral larval epidermis. While Xenopus epidermal cytokeratin gene expression is activated by bone morphogenetic protein (BMP) signaling, zebrafish cyt1 is not responsive to BMPs. Zebrafish early gastrula ectodermal explants are specified to express the neural markers opl (zic1) and otx2, and this expression is prevented by BMP4. Dissociation of zebrafish explants prevents otx2 and opl expression, suggesting that neural specification in zebrafish requires cell contact or close cell proximity. This finding is in contrast to the case in Xenopus, where ectodermal dissociation leads to activation of neural gene expression, or autoneuralization. Our data suggest that distinct mechanisms direct development of homologous lineages in different vertebrates.Full Text.

Sangster, T. A., Lindquist, S., and Queitsch, C. (2004). Under cover: causes, effects and implications of Hsp90-mediated genetic capacitance. Bioessays 26, 348-362. The environmentally responsive molecular chaperone Hsp90 assists the maturation of many key regulatory proteins. An unexpected consequence of this essential biochemical function is that genetic variation can accumulate in genomes and can remain phenotypically silent until Hsp90 function is challenged. Notably, this variation can be revealed by modest environmental change, establishing an environmentally responsive exposure mechanism. The existence of diverse cryptic polymorphisms with a plausible exposure mechanism in evolutionarily distant lineages has implications for the pace and nature of evolutionary change. Chaperone-mediated storage and release of genetic variation is undoubtedly rooted in protein-folding phenomena. As we discuss, proper protein folding crucially affects the trajectory from genotype to phenotype. Indeed, the impact of protein quality-control mechanisms and other fundamental cellular processes on evolution has heretofore been overlooked. A true understanding of evolutionary processes will require an integration of current evolutionary paradigms with the many new insights accruing in protein science. PDF.

Sarbassov,Dos, D. S., Ali, S. M., Kim, D. H., Guertin, D. A., Latek, R. R., Erdjument-Bromage, H., Tempst, P. and Sabatini, D. M. (2004) Rictor, a Novel Binding Partner of mTOR, Defines a Rapamycin-Insensitive and Raptor-Independent Pathway that Regulates the Cytoskeleton. Curr Biol 14 1296-302. The mammalian TOR (mTOR) pathway integrates nutrient- and growth factor-derived signals to regulate growth, the process whereby cells accumulate mass and increase in size. mTOR is a large protein kinase and the target of rapamycin, an immunosuppressant that also blocks vessel restenosis and has potential anticancer applications. mTOR interacts with the raptor and GbetaL proteins to form a complex that is the target of rapamycin. Here, we demonstrate that mTOR is also part of a distinct complex defined by the novel protein rictor (rapamycin-insensitive companion of mTOR). Rictor shares homology with the previously described pianissimo from D. discoidieum, STE20p from S. pombe, and AVO3p from S. cerevisiae. Interestingly, AVO3p is part of a rapamycin-insensitive TOR complex that does not contain the yeast homolog of raptor and signals to the actin cytoskeleton through PKC1. Consistent with this finding, the rictor-containing mTOR complex contains GbetaL but not raptor and it neither regulates the mTOR effector S6K1 nor is it bound by FKBP12-rapamycin. We find that the rictor-mTOR complex modulates the phosphorylation of Protein Kinase C alpha (PKCalpha) and the actin cytoskeleton, suggesting that this aspect of TOR signaling is conserved between yeast and mammals.Full Text.

Schaffner, S. F. (2004). The X chromosome in population genetics. Nat Rev Genet 5, 43-51. Genetic variation records a large amount of information about the history of a species and about the processes that create and shape that variation. Owing to the way in which it is inherited, the X chromosome is a rich resource of easily accessible genetic data, and therefore provides a unique tool for population-genetic studies. The potential of the human X chromosome, which rivals that of the more traditional mtDNA and Y chromosome, has only just begun to be tapped. Full Text.

Scheibel,Thomas , Jesse Bloom, and Susan L. Lindquist* The elongation of yeast prion fibers involves separable steps of association and conversion. Proc. Natl. Acad. Sci. February 24, 2004, vol. 101, no. 8, 2287–2292. A self-perpetuating change in the conformation of the translation termination factor Sup35p is the basis for the prion [PSI+], a protein-based genetic element of Saccharomyces cerevisiae. In a process closely allied to in vivo conversion, the purified soluble, prion-determining region of Sup35p (NM) converts to amyloid fibers by means of nucleated conformational conversion. First, oligomeric species convert to nuclei, and these nuclei then promote polymerization of soluble protein into amyloid fibers. To elucidate the nature of the polymerization step, we created single-cysteine substitution mutants at different positions in NM to provide unique attachment sites for various probes. In vivo, the mutants behaved like wild-type protein in both the [psi-] and [PSI+] states. In vitro, they assembled with wild-type kinetics and formed fibers with the same morphologies. When labeled with fluorescent probes, two mutants, NMT158C and NME167C, exhibited a change in fluorescence coincident with amyloid assembly. These mutants provided a sensitive measure for the kinetics of fiber elongation, and the lag phase in conversion. The cysteine in the mutant NMK184C remained exposed after assembly. When labeled with biotin and bound to streptavidin beads, it was used to capture radiolabeled soluble NM in the process of conversion. This process established the existence of a detergent-susceptible intermediate in fiber elongation. Thus, the second stage of nucleated conformational conversion, fiber elongation, itself contains at least two steps: the association of soluble protein with preformed fibers to form an assembly intermediate, followed by conformational conversion into amyloid. PDF.

Schmid, M. F., Sherman, M. B., Matsudaira, P. and Chiu, W. (2004) Structure of the acrosomal bundle. Nature. 431 104-7. In the unactivated Limulus sperm, a 60- micro m-long bundle of actin filaments crosslinked by the protein scruin is bent and twisted into a coil around the base of the nucleus. At fertilization, the bundle uncoils and fully extends in five seconds to support a finger of membrane known as the acrosomal process. This biological spring is powered by stored elastic energy and does not require the action of motor proteins or actin polymerization. In a 9.5-A electron cryomicroscopic structure of the extended bundle, we show that twist, tilt and rotation of actin-scruin subunits deviate widely from a 'standard' F-actin filament. This variability in structural organization allows filaments to pack into a highly ordered and rigid bundle in the extended state and suggests a mechanism for storing and releasing energy between coiled and extended states without disassembly. Full Text.

Shin, J. H., Gardel, M. L., Mahadevan, L., Matsudaira, P. and Weitz, D. A. (2004) Relating microstructure to rheology of a bundled and cross-linked F-actin network in vitro. Proceedings of the National Academy of Sciences of the United States of America 101 9636-9641. The organization of individual actin filaments into higher-order structures is controlled by actin-binding proteins (ABPs). Although the biological significance of the ABPs is well documented, little is known about how bundling and cross-linking quantitatively affect the microstructure and mechanical properties of actin networks. Here we quantify the effect of the ABP scruin on actin networks by using imaging techniques, cosedimentation assays, multiparticle tracking, and bulk rheology. We show how the structure of the actin network is modified as the scruin concentration is varied, and we correlate these structural changes to variations in the resultant network elasticity.Full Text.

Shin, J. H., Mahadevan, L., So, P. T., and Matsudaira, P. (2004). Bending stiffness of a crystalline actin bundle. Journal of Molecular Biology 337, 255-261. The acrosomal process of the sperm of the horseshoe crab (Limulus polyplienius) is a unique crystalline actin bundle, consisting of multiple actin filaments cross-linked by the actin-bundling protein, scruin. For successful fertilization, the acrosomal bundle must penetrate through a 30 mum thick jelly coat surrounding the egg and thus it must be sufficiently stiff. Here, we present two measurements of the bending stiffness of a single crystalline bundle of actin. Results from these measurements indicate that the actin:scruin composite bundle has an average elastic modulus of 2 GPa, which is similar to that of a single actin filament, and a bending stiffness that is more than two orders of magnitude larger than that of a bundle of uncross-linked actin filaments due to stiffening by the scruin matrix. Full Text.

Shorter, J., Lindquist, S. Hsp104 Catalyzes Formation and Elimination of Self-Replicating Sup35 Prion Conformers. Science 304 1793-1797. The protein-remodeling factor Hsp104 governs inheritance of [PSI+], a yeast prion formed by self-perpetuating amyloid conformers of Sup35. Perplexingly, either excess or insufficient Hsp104 eliminates [PSI+]. In vitro, at low concentrations, Hsp104 catalyzed formation of oligomeric intermediates that proved critical for nucleating Sup35 fibrillization de novo and displayed a conformation common amongst amyloidogenic polypeptides. At higher Hsp104 concentrations, amyloidogenic oligomerization and contingent fibrillization were abolished. Hsp104 also disassembled mature fibers in a manner that initially exposed new surfaces for conformational replication, but eventually exterminated prion conformers. These Hsp104 activities differed in their reaction mechanism and can explain [PSI+] inheritance patterns.Full Text.

Silver, S. J., Chen, F., Doyon, L., Zink, A. W. and Rebay, I. (2004) New class of Son-of-sevenless (Sos) alleles highlights the complexities of Sos function. Genesis 39 263-72. The guanine nucleotide exchange factor (GEF) Son-of-sevenless (Sos) encodes a complex multidomain protein best known for its role in activating the small GTPase RAS in response to receptor tyrosine kinase (RTK) stimulation. Much less well understood is SOS's role in modulating RAC activity via a separate GEF domain. In the course of a genetic modifier screen designed to investigate the complexities of RTK/RAS signal transduction, a complementation group of 11 alleles was isolated and mapped to the Sos locus. Molecular characterization of these alleles indicates that they specifically affect individual domains of the protein. One of these alleles, Sos(M98), which contains a single amino acid substitution in the RacGEF motif, functions as a dominant negative in vivo to downregulate RTK signaling. These alleles provide new tools for future investigations of SOS-mediated activation of both RAS and RAC and how these dual roles are coordinated and coregulated during development.PDF

Singer, J. B., Hill, A. E., Burrage, L. C., Olszens, K. R., Song, J., Justice, M., O'Brien, W. E., Conti, D. V., Witte, J. S., Lander, E. S., and Nadeau, J. H. (2004). Genetic Dissection of Complex Traits with Chromosome Substitution Strains of Mice. Science.18 March Chromosome Substitution Strains (CSSs) have been proposed as a simple and powerful way to identify quantitative trait loci (QTLs) affecting developmental, physiological and behavioral processes. Here, we report the construction of a complete CSS panel for a vertebrate species. The CSS panel consists of 22 mouse strains, each of which carries a single chromosome substituted from a donor strain (A/J) onto a common host background (C57BL/6J). A survey of 53 traits revealed evidence for 150 QTLs affecting serum levels of sterols and amino acids, diet-induced obesity, and anxiety. These results demonstrate that CSSs greatly facilitate detection and identification of genes that control the extraordinary diversity of naturally occurring phenotypic variation in the A/J and C57BL/6J inbred strains. PDF.

Stegmaier, K., Ross, K. N., Colavito, S. A., O'Malley, S., Stockwell, B. R., and Golub, T. R. (2004). Gene expression-based high-throughput screening (GE-HTS) and application to leukemia differentiation. Nature Genetics 36, 257-263. Chemical genomics involves generating large collections of small molecules and using them to modulate cellular states. Despite recent progress in the systematic synthesis of structurally diverse compounds, their use in screens of cellular circuitry is still an ad hoc process(1-4). Here, we outline a general, efficient approach called gene expression based high-throughput screening (GE-HTS) in which a gene expression signature is used as a surrogate for cellular states, and we describe its application in a particular setting: the identification of compounds that induce the differentiation of acute myeloid leukemia cells. In screening 1,739 compounds, we identified 8 that reliably induced the differentiation signature and, furthermore, yielded functional evidence of bona fide differentiation. The results indicate that GE-HTS may be a powerful, general approach for chemical screening. Full Text.

Stockwell, B. R. (2004). The biological magic behind the bullets. Nat Biotechnol 22, 37-38. Full Text.

Tantisira, K. G., Lake, S., Silverman, E. S., Palmer, L. J., Lazarus, R., Silverman, E. K., Liggett, S. B., Gelfand, E. W., Rosenwasser, L. J., Richter, B., Israel, E., Wechsler, M., Gabriel, S., Altshuler, D., Lander, E., Drazen, J. and Weiss, S. T. (2004) Corticosteroid pharmacogenetics: association of sequence variants in CRHR1 with improved lung function in asthmatics treated with inhaled corticosteroids. Human Molecular Genetics 13 1353-1359. Corticosteroids mediate a variety of immunological actions and are commonly utilized in the treatment of a wide range of diseases. Unfortunately, therapy with this class of medications is associated with a large proportion of non-responders and significant side effects. Inhaled corticosteroids are the most commonly used asthma controller therapy. However, asthmatic response to corticosteroids also varies widely between individuals. We investigated the genetic contribution to the variation in response to inhaled corticosteroid therapy in asthma. The association of longitudinal change in lung function and single nucleotide polymorphisms from candidate genes crucial to the biologic actions of corticosteroids were evaluated in three independent asthmatic clinical trial populations utilizing inhaled corticosteroids as the primary therapy in at least one treatment arm. Variation in one gene, corticotropin-releasing hormone receptor 1 (CRHR1) was consistently associated with enhanced response to therapy in each of our three populations. Individuals homozygous for the variants of interest manifested a doubling to quadrupling of the lung function response to corticosteroids compared with lack of the variants (P-values ranging from 0.006 to 0.025 for our three asthmatic populations). As the primary receptor mediating the release of adrenocorticotropic hormone, which regulates endogenous cortisol levels, CRHR1 plays a pivotal, pleiotropic role in steroid biology. These data indicate that genetic variants in CRHR1 have pharmacogenetic effects influencing asthmatic response to corticosteroids, provide a rationale for predicting therapeutic response in asthma and other corticosteroid-treated diseases, and suggests this gene pathway as a potential novel therapeutic target.Full Text.

Thoreen, C. C., and Sabatini, D. M. (2004). Huntingtin aggregates ask to be eaten. Nat Genet 36, 553-554. A new study identifies a protective role for cellular aggregates in Huntington disease by showing that aggregates promote the clearance of mutant protein by activating autophagy through the inhibition of mTOR. This challenges the common view that they are possibly innocuous but probably harmful to the host cell. Full Text.

Tomas, E., Kelly, M., Xiang, X. Q., Tsao, T. S., Keller, C., Keller, P., Luo, Z. J., Lodish, H., Saha, A. K., Unger, R. and Ruderman, N. B. (2004) Metabolic and hormonal interactions between muscle and adipose tissue. Proceedings of the Nutrition Society. 63 381-385. From the perspective of a muscle physiologist, adipose tissue has long been perceived predominantly as a fuel reservoir that provides muscle and other tissues with NEFA when exogenous nutrients are insufficient for their energy needs. Recently, studies have established that adipose tissue is also an endocrine organ. Among the hormones it releases are adiponectin and leptin, both of which can activate AMP-activated protein kinase and increase fatty acid oxidation in skeletal muscle and probably other tissues. Deficiencies of leptin or leptin receptor, adiponectin and IL-6 are associated with obesity, insulin resistance and a propensity to type 2 diabetes. In addition, a lack of adiponectin has been linked to atherosclerosis. Whether this pathology reflects a deficient activation of AMP-activated protein kinase in peripheral tissues remains to be determined. Finally, recent studies have suggested that skeletal muscle may also function as an endocrine organ when it releases the cytokine IL-6 into the circulation during sustained exercise. Interestingly, one of the apparent effects of IL-6 is to stimulate lipolysis, causing the release of NEFA from the adipocyte. Thus, hormonal communications exist between the adipocyte and muscle that could enable them to talk to each other. The physiological relevance of this cross talk clearly warrants further study.

Tong, W. and Lodish, H. F. (2004) Lnk Inhibits Tpo-mpl Signaling and Tpo-mediated Megakaryocytopoiesis..J Exp Med. 2004 200 (5): 569-580 Thrombopoietin (Tpo) is the primary cytokine regulating megakaryocyte development and platelet production. Tpo signaling through its receptor, c-mpl, activates multiple pathways including signal transducer and activator of transcription (STAT)3, STAT5, phosphoinositide 3-kinase-Akt, and p42/44 mitogen-activated protein kinase (MAPK). The adaptor protein Lnk is implicated in cytokine receptor and immunoreceptor signaling. Here, we show that Lnk overexpression negatively regulates Tpo-mediated cell proliferation and endomitosis in hematopoietic cell lines and primary hematopoietic cells. Lnk attenuates Tpo-induced S-phase progression in 32D cells expressing mpl, and Lnk decreases Tpo-dependent megakaryocyte growth in bone marrow (BM)-derived megakaryocyte culture. Consistent with this result, we found that in both BM and spleen, Lnk-deficient mice exhibited increased numbers of megakaryocytes with increased ploidy compared with wild-type mice. In addition, Lnk-deficient megakaryocytes derived from BM and spleen showed enhanced sensitivity to Tpo during culture. The absence of Lnk caused enhanced and prolonged Tpo induction of STAT3, STAT5, Akt, and MAPK signaling pathways in CD41(+) megakaryocytes. Furthermore, the Src homology 2 domain of Lnk is essential for Lnk's inhibitory function. In contrast, the conserved tyrosine near the COOH terminus is dispensable and the pleckstrin homology domain of Lnk contributes to, but is not essential for, inhibiting Tpo-dependent 32D cell growth or megakaryocyte development. Thus, Lnk negatively modulates mpl signaling pathways and is important for Tpo-mediated megakaryocytopoiesis in vivo.Full Text.

True, Heather L. , Ilana Berlin & Susan L. Lindquist (2004) Epigenetic regulation of translation reveals hidden genetic variation to produce complex traits. Nature. 431 184-7. Phenotypic plasticity and the exposure of hidden genetic variation both affect the survival and evolution of new traits1-3, but their contributing molecular mechanisms are largely unknown. A single factor, the yeast prion [PSI+], may exert a profound effect on both4. [PSI+] is a conserved, protein-based genetic element that is formed by a change in the conformation and function of the translation termination factor Sup35p5, and is transmitted from mother to progeny. Curing cells of [PSI+] alters their survival in different growth conditions and produces a spectrum of phenotypes in different genetic backgrounds4. Here we show, by examining three plausible explanations for this phenotypic diversity, that all traits tested involved [PSI+]-mediated read-through of nonsense codons. Notably, the phenotypes analysed were genetically complex, and genetic re-assortment frequently converted [PSI+]-dependent phenotypes to stable traits that persisted in the absence of [PSI+]. Thus, [PSI+] provides a temporary survival advantage under diverse conditions, increasing the likelihood that new traits will become fixed by subsequent genetic change. As an epigenetic mechanism that globally affects the relationship between genotype and phenotype, [PSI+] expands the conceptual framework for phenotypic plasticity, provides a one-step mechanism for the acquisition of complex traits and affords a route to the genetic assimilation of initially transient epigenetic traits.Full Text.

Tzenova, J., Kaplan, B. J., Petryshen, T. L., and Field, L. L. (2004). Confirmation of a dyslexia susceptibility locus on chromosome 1p34-p36 in a set of 100 Canadian families. American Journal of Medical Genetics Part B-Neuropsychiatric Genetics 127B, 117-124. Dyslexia is a common and genetically complex trait that manifests primarily as a reading disability independent of general intelligence and educational opportunity. Strong evidence for a dyslexia susceptibility locus on chromosome 1p34-p36 (near marker D1S199) was recently reported, and an earlier study found suggestive evidence for linkage to the same region. We tested for the presence of a dyslexia gene in this region in a sample of 100 Canadian families using both qualitative and quantitative definitions of the phenotype. Using a qualitative definition of dyslexia (affected, unaffected, or uncertain), the largest multipoint Genehunter Maximum LOD-Score (MLS) in 100 core nuclear families was 3.65 at D1S507, distal to D1S199. Quantitative trait locus (QTL) linkage analysis was performed for four measures of dyslexia (phonological awareness, phonological coding, spelling, and rapid automatized naming speed) employing the variance components approach implemented in Genehunter. Using a model with QTL additive and dominance variance and polygenic additive variance, the multi-point LOD scores maximized proximal to D1S199 (between D1S552 and D1S1622), with peaks of 4.01 for spelling and 1.65 for phonological coding (corresponding LOD scores under 1 degree of freedom were 3.30 and 1.13, respectively). In conclusion, our study confirms and strengthens recent findings of a dyslexia susceptibility gene on chromosome 1p34-p36 (now designated DYX8) Full Text.

Valentine, M. T., Perlman, Z. E., Gardel, M. L., Shin, J. H., Matsudaira, P., Mitchison, T. J. and Weitz, D. A. (2004) Colloid surface chemistry critically affects multiple particle tracking measurements of biomaterials. Biophysical Journal 86 4004-4014. Characterization of the properties of complex biomaterials using microrheological techniques has the promise of providing fundamental insights into their biomechanical functions; however, precise interpretations of such measurements are hindered by inadequate characterization of the interactions between tracers and the networks they probe. We here show that colloid surface chemistry can profoundly affect multiple particle tracking measurements of networks of fibrin, entangled F-actin solutions, and networks of cross-linked F-actin. We present a simple protocol to render the surface of colloidal probe particles protein-resistant by grafting short amine-terminated methoxy-poly(ethylene glycol) to the surface of carboxylated microspheres. We demonstrate that these poly(ethylene glycol)-coated tracers adsorb significantly less protein than particles coated with bovine serum albumin or unmodified probe particles. We establish that varying particle surface chemistry selectively tunes the sensitivity of the particles to different physical properties of their microenvironments. Specifically, particles that are weakly bound to a heterogeneous network are sensitive to changes in network stiffness, whereas protein-resistant tracers measure changes in the viscosity of the fluid and in the network microstructure. We demonstrate experimentally that two-particle microrheology analysis significantly reduces differences arising from tracer surface chemistry, indicating that modifications of network properties near the particle do not introduce large-scale heterogeneities. Our results establish that controlling colloid-protein interactions is crucial to the successful application of multiple particle tracking techniques to reconstituted protein networks, cytoplasm, and cells. Full Text.

van Heel, D. A., Fisher, S. A., Kirby, A., Daly, M. J., Rioux, J. D., and Lewis, C. M. (2004). Inflammatory bowel disease susceptibility loci defined by genome scan meta-analysis of 1952 affected relative pairs. Human Molecular Genetics 13, 763-770. Crohn's disease and ulcerative colitis (the inflammatory bowel diseases) have a strong genetic component. Although over 20 putative susceptibility loci have been identified by individual genome scans, the majority of these loci have not been replicated. Many individual studies are at the lower limit of acceptable power for complex disease linkage analysis. Genome scan meta-analysis (GSMA), by use of sample sizes an order of magnitude greater than individual linkage studies, has increased power to detect novel loci, may confirm or refute regions detected in smaller individual studies, and enables regions to be prioritized for further gene identification efforts. Genome scan data (markers, significance scores) were obtained from 10 separate studies and meta-analysis was performed using the GSMA method. These studies comprised 1952 inflammatory bowel disease, 1068 Crohn's disease and 457 ulcerative colitis affected relative pairs. Study results were divided into 34 cM chromosomal bins, ranked, weighted by study size, summed across studies and bin-by-bin significance obtained by simulation. A region on chromosome 6p (containing the HLA) met genome wide significance for inflammatory bowel disease. Loci meeting suggestive significance for inflammatory bowel disease were 2q, 3q, 5q, 7q and 16 (NOD2/CARD15 region); Crohn's disease, 2q, 3q, 6p, 16 (NOD2/CARD15 region), 17q, 19p; and ulcerative colitis, 2q. Clustering of adjacent bins was observed for chromosomes 6p, 16, 19p. The meta-analysis has identified novel loci and prioritized genomic regions for further gene identification studies.Full Text.

Vazquez, F., Vaucheret, H., Rajagopalan, R., Lepers, C., Gasciolli, V., Mallory, A. C., Hilbert, J. L., Bartel, D. P. and Crete, P. (2004) Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Molecular Cell. 16 69-79. Here we describe a set of endogenous short interfering RNAs (siRNAs) in Arabidopsis, some of which direct the cleavage of endogenous mRNAs. These siRNAs correspond to both sense and antisense strands of a noncoding RNA (At2g27400) that apparently is converted to double-stranded RNA and then processed in 21 nt increments. These siRNAs differ from previously described regulatory small RNAs in two respects. First, they require components of the cosuppression pathway (RDR6 and SGS3) and also components of the microRNA (miRNA) pathway (AGO1, DCL1, HEN1, and HYL1) but not components needed for heterochromatic siRNAs (DCL3 and RDR2), another class of endogenous plant siRNAs. Second, these siRNAs; repress the expression of genes that have little overall resemblance to the genes from which they originate, a characteristic previously reported only for miRNAs. The identification of this silencing pathway provides yet another dimension to posttranscriptional mRNA regulation in plants. Full Text.

Veeraraghavalu, K., Pett, M., Kumar, R. V., Nair, P., Rangarajan, A., Stanley, M. A. and Krishna, S. (2004) Papillomavirus-mediated neoplastic progression is associated with reciprocal changes in Jagged1 and manic fringe expression linked to notch activation. Journal of Virology. 78 8687-8700. Infection by high-risk human papillomaviruses (HPV) and persistent expression of viral oncogenes E6 and E7 are causally linked to the development of cervical cancer. These oncogenes are necessary but insufficient for complete transformation of human epithelial cells in vivo. Intracellular Notch1, protein is detected in invasive cervical carcinomas (ICC), and truncated Notch1 alleles complement the function of E6/E7 in the transformation of human epithelial cells. Here we investigate potential mechanisms of Notch activation in a human cervical neoplasia. We have analyzed human cervical lesions and serial passages of an HPV type 16-positive human cervical low-grade lesion-derived cell line, W12, that shows abnormalities resembling those seen in cervical neoplastic progression in vivo. Late-passage, but not early-passage, W12 and progression of the majority of human high-grade cervical lesions to ICC showed upregulation of Notch ligand and Jagged1 and downregulation of Manic Fringe, a negative regulator of Jagged1-Notch1 signaling. Concomitantly, an increase in Notch/CSL (CBF1, Suppressor of Hairless, Lag1)-driven reporter activity and a decrease in Manic Fringe upstream regulatory region (MFng-URR) -driven reporter activity was observed in late-passage versus early passage W12. Analysis of the MFng-URR revealed that Notch signaling represses this gene through Hairy Enhancer of Split 1, a transcriptional target of the Notch pathway. Expression of Manic Fringe by a recombinant adenovirus, dominant-negative Jagged1, or small interfering RNA against Jagged1 inhibits the tumorigenicity of CaSki, an ICC-derived cell line that was previously shown to be susceptible to growth inhibition induced by antisense Notch1. We suggest that activation of Notch in cervical neoplasia is Jagged1 dependent and that its susceptibility to the influence of Manic Fringe is of therapeutic value. Full Text.

Ventura, A., Meissner, A., Dillon, C. P., McManus, M., Sharp, P. A., Van Parijs, L., Jaenisch, R. and Jacks, T. (2004) Cre-lox-regulated conditional RNA interference from transgenes. Proceedings of the National Academy of Sciences of the United States of America. 101 10380-10385. We have generated two lentiviral vectors for conditional, Cre-loxregulated, RNA interference. One vector allows for conditional activation, whereas the other permits conditional inactivation of short hairpin RNA (shRNA) expression. The former is based on a strategy in which the mouse U6 promoter has been modified by including a hybrid between a LoxP site and a TATA box. The ability to efficiently control shRNA expression by using these vectors was shown in cell-based experiments by knocking down p53, nucleophosmin and DNA methyltransferase 1. We also demonstrate the usefulness of this approach to achieve conditional, tissue-specific RNA interference in Cre-expressing transgenic mice. Combined with the growing array of Cre expression strategies, these vectors allow spatial and temporal control of shRNA expression in vivo and should facilitate functional genetic analysis in mammals. Full Text.

Verstrepen, K. J., Iserentant, D., Malcorps, P., Derdelinckx, G., Van Dijck, P., Winderickx, J., Pretorius, I. S., Thevelein, J. M. and Delvaux, F. R. (2004) Glucose and sucrose: hazardous fast-food for industrial yeast? Trends Biotechnol. 22 531-537. Yeast cells often encounter a mixture of different carbohydrates in industrial processes. However, glucose and sucrose are always consumed first. The presence of these sugars causes repression of gluconeogenesis, the glyoxylate cycle, respiration and the uptake of less-preferred carbohydrates. Glucose and sucrose also trigger unexpected, hormone-like effects, including the activation of cellular growth, the mobilization of storage compounds and the diminution of cellular stress resistance. In an industrial context, these effects lead to several yeast-related problems, such as slow or incomplete fermentation, 'off flavors' and poor maintenance of yeast vitality. Recent studies indicate that the use of mutants with altered responses to carbohydrates can significantly increase productivity. Alternatively, avoiding unnecessary exposure to glucose and sucrose could also improve the performance of industrial yeasts .Full Text.

Verstrepen, K. J., Reynolds, T. B. and Fink, G. R. (2004) Origins of variation in the fungal cell surface Nat Rev Microbiol 2 533-40.The increase in hospital-acquired fungal infections has been attributed to the ability of fungi to adhere not only to human tissues, but also to the plastic prostheses and invasive devices that are used to treat disease. These properties are conferred by a family of fungal cell-surface proteins, called adhesins. Adhesins might also have a central role in the formation of fungal biofilms, which are resistant to antimicrobial drugs. The structure of the genes that encode adhesin-family members, and the sequence homology between them, enables genetic reshuffling of domains to form new genes. Coupled with epigenetic changes in gene expression, these genetic rearrangements provide a reservoir of cell-surface molecules with new functions..

Vivekanand, P., Tootle, T. L. and Rebay, I. (2004) MAE, a dual regulator of the EGFR signaling pathway, is a target of the Ets transcription factors PNT and YAN. Mech Dev. 121 1469-79. Ets transcription factors play crucial roles in regulating diverse cellular processes including cell proliferation, differentiation and survival. Coordinated regulation of the Drosophila Ets transcription factors YAN and POINTED is required for eliciting appropriate responses to Receptor Tyrosine Kinase (RTK) signaling. YAN, a transcriptional repressor, and POINTED, a transcriptional activator, compete for regulatory regions of common target genes, with the ultimate outcome likely influenced by context-specific interactions with binding partners such as MAE. Previous work in cultured cells has led us to propose that MAE attenuates the transcriptional activity of both YAN and POINTED, although its effects on POINTED remain controversial. Here we describe a new layer of complexity to this regulatory hierarchy whereby mae expression is itself directly regulated by the opposing action of YAN and POINTED. In addition, we report that MAE can antagonize POINTED function during eye development; a finding that suggests MAE operates as a dual positive and negative regulator of RTK-mediated signaling in vivo. Together our results lead us to propose that a combination of protein-protein and transcriptional interactions between MAE, YAN and POINTED establishes a complex regulatory circuit that ensures that both down-regulation and activation of the RTK pathway occur appropriately according to specific developmental context.Full Text.

Voas, M. G., and Rebay, I. (2004). Signal integration during development: Insights from the Drosophila eye. Dev Dyn 229, 162-175. The Drosophila eye is a highly ordered epithelial tissue composed of approximately 750 subunits called ommatidia arranged in a reiterated hexagonal pattern. At higher resolution, observation of the constituent photoreceptors, cone cells, and pigment cells of the eye reveals a highly ordered mosaic of amazing regularity. This relatively simple organization belies the repeated requirement for spatially and temporally coordinated inputs from the Hedgehog (Hh), Wingless (Wg), Decapentaplegic (Dpp), JAK-STAT, Notch, and receptor tyrosine kinase (RTK) signaling pathways. This review will discuss how signaling inputs from the Notch and RTK pathways, superimposed on the developmental history of a cell, facilitate context-specific and appropriate cell fate specification decisions in the developing fly eye. Lessons learned from investigating the combinatorial signal integration strategies underlying Drosophila eye development will likely reveal cell-cell communication paradigms relevant to many aspects of invertebrate and mammalian development. Full Text

Vorderwulbecke, S., Kramer, G., Merz, F., Kurz, T. A., Rauch, T., Zachmann-Brand, B., Bukau, B., and Deuerling, E. (2004). Low temperature or GroEL/ES overproduction permits growth of Escherichia coli cells lacking trigger factor and DnaK. Febs Letters 559, 181-187. Escherichia coli trigger factor (TF) and DnaK cooperate in the folding of newly synthesized proteins. The combined deletion of the TF-encoding tig gene and the dnaK gene causes protein aggregation and synthetic lethality at 30degreesC. Here we show that the synthetic lethality of DeltatigDeltadnaK52 cells is abrogated either by growth below 30degreesC or by overproduction of GroEL/GroES. At 23degreesC DeltatigDeltadnaK52 cells were viable and showed only minor protein aggregation. Overproduction of GroEL/GroES, but not of other chaperones, restored growth of DeltatigDeltadnaK52 cells at 30degreesC and suppressed protein aggregation including proteins greater than or equal to 60 kDa, which normally require TF and DnaK for folding. GroEL/GroES thus influences the folding of proteins previously identified as DnaK/TF substrates. Full Text

Wang, Z. and Jaenisch, R. (2004) At most three ES cells contribute to the somatic lineages of chimeric mice and of mice produced by ES-tetraploid complementation. Dev Biol. 275 192-201. Chimeric or entirely embryonic stem (ES) cell-derived mice ("ES mice") can be produced by injecting ES cells into diploid (2n) or tetraploid (4n) host blastocysts, respectively. Usually, between 10 and 15 ES cells are injected into the host blastocyst, but it is not clear how many of the injected cells contribute to the somatic lineages, thus serve as "founder cells" of the embryo proper. We have used genetically labeled ES cells to retrospectively determine the number of founder ES cells that generate the somatic lineages of chimeric and of ES mice. ES cell clones individually labeled with provirus were mixed in equal numbers and injected into 2n or 4n blastocysts to generate chimeric or ES mice. Southern analysis of DNA from the resulting animals indicated that the somatic lineages were most often derived from one or two and sometimes from up to three founder ES cells. The number of founder cells was independent of the total number of cells injected into the host blastocysts. Our results are consistent with the notion that constraints of the host embryo restrict the number of ES cells that can contribute to a chimeric or an ES mouse. Full Text

Wang, Z. G. C., Lin, M., Wei, L. J., Li, C., Miron, A., Lodeiro, G., Harris, L., Ramaswamy, S., Tanenbaum, D. M., Meyerson, M., et al. (2004). Loss of heterozygosity and its correlation with expression profiles in subclasses of invasive breast cancers. Cancer Research 64, 64-71. Gene expression array profiles identify subclasses of breast cancers with different clinical outcomes and different molecular features. The present study attempted to correlate genomic alterations (loss of heterozygosity; LOH) with subclasses of breast cancers having distinct gene expression signatures. Hierarchical clustering of expression array data from 89 invasive breast cancers identified four major expression subclasses. Thirty-four of these cases representative of the four subclasses were microdissected and allelotyped using genome-wide single nucleotide polymorphism detection arrays (Affymetrix, Inc.). LOH was determined by comparing tumor and normal single nucleotide polymorphism allelotypes. A newly developed statistical tool was used to determine the chromosomal regions of frequent LOH. We found that breast cancers were highly heterogeneous, with the proportion of LOH ranging widely from 0.3% to > 60% of heterozygous markers. The most common sites of LOH were on 17p, 17q, 16q, 11q, and 14q, sites reported in previous LOH studies. Signature LOH events were discovered in certain expression subclasses. Unique regions of LOH on 5q and 4p marked a subclass of breast cancers with "basal-like" expression profiles, distinct from other subclasses. LOH on 1p and 16q occurred preferentially in a subclass of estrogen receptor-positive breast cancers. Finding unique LOH patterns in different groups of breast cancer, in part defined by expression signatures, adds confidence to newer schemes of molecular classification. Furthermore, exclusive association between biological subclasses and restricted LOH events provides rationale to search for targeted genes. Full Text.

Weinberg, R. A. (2004) Inadvertent cancer research Cancer Biology & Therapy 3 238-239.

Wiederkehr, C., Basavaraj, R., de Menthiere, C. S., Hermida, L., Koch, R., Schlecht, U., Amon, A., Brachat, S., Breitenbach, M., Briza, P., Orr-Weaver, T et al. (2004). GermOnline, a cross-species community knowledgebase on germ cell differentiation. Nucleic Acids Research 32, D560-D567. GermOnline provides information and microarray expression data for genes involved in mitosis and meiosis, gamete formation and germ line development across species. The database has been developed, and is being curated and updated, by life scientists in cooperation with bioinformaticists. Information is contributed through an online form using free text, images and the controlled vocabulary developed by the GeneOntology Consortium. Authors provide up to three references in support of their contribution. The database is governed by an international board of scientists to ensure a standardized data format and the highest quality of GermOnline's information content. Release 2.0 provides exclusive access to microarray expression data from Saccharomyces cerevisiae and Rattus norvegicus, as well as curated information on similar to700 genes from various organisms. The focus report pages include links to external databases that contain relevant annotation, microarray expression and proteome data. Conversely, the Saccharomyces Genome Database (SGD), S.cerevisiae GeneDB and Swiss-Prot link to the budding yeast section of GermOnline from their respective locus pages. GermOnline, a fully operational prototype subject-oriented knowledgebase designed for community annotation and array data visualization, is accessible at http://www.germonline.org. The target audience includes researchers who work on mitotic cell division, meiosis, gametogenesis, germ line development, human reproductive health and comparative genomics. Full text.

Wiellette, E., Grinblat, Y., Austen, M., Hirsinger, E., Amsterdam, A., Walker, C., Westerfield, M. and Sive, H. (2004) Combined haploid and insertional mutation screen in the zebrafish. Genesis. 40 231-240. To identify genes required for development of the brain and somites, we performed a pilot screen of gynogenetic haploid zebrafish embryos produced from mothers mutagenized by viral insertion. We describe an efficient method to identify new mutations and the affected gene. In addition, we report the results of a small-scale screen that identified five genes required for brain development, including novel alleles of nagie oko, pou5f1, ribosomal protein L36, and n-cadherin, as well as a novel allele of the laminin g1 gene that is required for normal skeletal muscle fiber organization and somite patterning .PDF

Wiellette, E. L., and Sive, H. (2004). Early requirement for fgf8 function during hindbrain pattern formation in zebrafish. Dev Dyn 229, 393-399. Fibroblast growth factor (FGF) signaling is required for normal development of the vertebrate brain, including the isthmus and caudal regions of the hindbrain. Recent work in zebrafish has identified a requirement for the combination of fgf3 and fgf8 functions in specification of rhombomeres 5 and 6 (r5, r6), when evaluated at mid- and late somitogenesis stages. However, when examined earlier in development, during early somitogenesis stages, FGF8 alone is required to initiate r5 and r6 development. Both a mutation in fgf8 and injection of fgf8-targeted antisense morpholino-modified oligonucleotides result in suppression of genes normally expressed in r5 and r6 by the one- to two-somite stage. This expression recovers by the six-somite stage, and we propose that this recovery is a response to activation of fgf3 and to delayed accumulation of fgf8. These data demonstrate an early, nonredundant requirement for fgf8 function in hindbrain patterning.Full Text.

Wild, G. E., and Rioux, J. D. (2004). Genome scan analyses and positional cloning strategy in IBD: successes and limitations. Best Pract Res Clin Gastroenterol 18, 541-553. The past decade has witnessed a tremendous expansion of our knowledge-base of genetics of inflammatory bowel disease. To a large extent, this progress reflects the scientific innovation and impact of the human genome project, which has fueled many laboratory-based studies focusing on the molecular genetics of Crohn's disease and ulcerative colitis. The complementary strategies of genome-wide linkage scanning and candidate gene analysis uncovered a number of genetic loci associated with IBD susceptibility. Notably, the identification of the IBD1 and IBD5 loci is a major scientific discovery. Although many issues related to the function and expression of these genes await elucidation, there is a shared optimism that pivotal clinical applications will emerge from these investigations.

Williams, T. M., Medina, F., Badano, I., Hazan, R. B., Hutchinson, J., Muller, W. J., Chopra, N. G., Scherer, P. E., Pestell, R. G. and Lisanti, M. P. (2004) Caveolin-1 gene disruption promotes mammary tumorigenesis and dramatically enhances lung metastasis in vivo - Role of Cav-1 in cell invasiveness and matrix metalloproteinase (MMP-2/9) secretion. Journal of Biological Chemistry. 279 51630-51646. Caveolin-1 (Cav-1) is the principal structural component of caveolae membrane domains in non-muscle cells, including mammary epithelia. There is now clear evidence that caveolin-1 influences the development of human cancers. For example, a dominant-negative mutation (P132L) in the Cav-1 gene has been detected in up to 16% of human breast cancer samples. However, the exact functional role of caveolin-1 remains controversial. Mechanistically, in cultured cell models, Cav-1 is known to function as a negative regulator of the Rasp-42/ 44 MAP kinase cascade and as a transcriptional repressor of cyclin D1 gene expression, possibly explaining its in vitro transformation suppressor activity. Genetic validation of this hypothesis at the in vivo and whole organismal level has been prevented by the lack of a Cav-1 (-/-)-null mouse model. Here, we examined the role of caveolin-1 in mammary tumorigenesis and lung metastasis using a molecular genetic approach. We interbred a well characterized transgenic mouse model of breast cancer, MMTV-PyMT ( mouse mammary tumor virus-polyoma middle T antigen), with Cav-1(-/-)-null mice. Then, we followed the onset and progression of mammary tumors and lung metastases in female mice over a 14-week period. Interestingly, PyMT/Cav-1 (-/-) mice showed an accelerated onset of mammary tumors, with increased multiplicity and tumor burden ( similar to 2-fold). No significant differences were detected between PyMT/ Cav-1 (-/-) and PyMT/ Cav-1 (+/-) mice, indicating that complete loss of caveolin-1 is required to accelerate both tumorigenesis and metastasis. Molecularly, mammary tumor samples derived from PyMT/Cav-1 (-/-) mice showed ERK-1/2 hyperactivation, cyclin D1 up-regulation, and Rb hyperphosphorylation, consistent with dysregulated cell proliferation. PyMT/Cav-1 (-/-) mice also developed markedly advanced metastatic lung disease. Conversely, recombinant expression of Cav-1 in a highly metastatic PyMT mammary carcinoma-derived cell line, namely Met-1 cells, suppressed lung metastasis by similar to4.5-fold. In vitro, these Cav-1-expressing Met-1 cells (Met-1/ Cav-1) demonstrated a similar to 4.8-fold reduction in invasion through Matrigel-coated membranes. Interestingly, delivery of a cell permeable peptide encoding the caveolin-1 scaffolding domain ( residues 82 - 101) into Met-1 cells was sufficient to inhibit invasion. Coincident with this decreased invasive index, Met-1/ Cav-1 cells exhibited marked reductions in MMP-9 and MMP-2 secretion and associated gelatinolytic activity, as well as diminished ERK-1/2 signaling in response to growth factor stimulation. These results demonstrate, for the first time, that caveolin-1 is a potent suppressor of mammary tumor growth and metastasis using novel in vivo animal model approaches. Full Text.

Wong, G. W., Wang, J., Hug, C., Tsao, T. S. and Lodish, H. F. (2004) A family of Acrp30/adiponectin structural and functional paralogs. Proc Natl Acad Sci U S A. 2004 Jul 1 [Epub ahead of print] Biochemical, genetic, and animal studies in recent years have established a critical role for the adipokine Acrp30/adiponectin in controlling whole-body metabolism, particularly by enhancing insulin sensitivity in muscle and liver, and by increasing fatty acid oxidation in muscle. We describe a widely expressed and highly conserved family of adiponectin paralogs designated as C1q/tumor necrosis factor-alpha-related proteins (CTRPs) 1-7. In the present study, we focus on mCTRP2, the mouse paralog most similar to adiponectin. At nanomolar concentrations, bacterially produced mCTRP2 rapidly induced phosphorylation of AMP-activated protein kinase, acetyl-CoA carboxylase, and mitogen-activated protein kinase in C2C12 myotubes, which resulted in increased glycogen accumulation and fatty acid oxidation. The discovery of a family of adiponectin paralogs has implications for understanding the control of energy homeostasis and could provide new targets for pharmacologic intervention in metabolic diseases such as diabetes and obesity.PDF

Yadav, M. K., Kelley, B. P. and Silverman, S. M. 2004. The potential of a chemical graph transformation system. Graph Transformations, Proceedings. Lecture Notes in Computer Science. 3256. 83-95. Full Text.

Yang, J., Mani, S. A., Donaher, J. L., Ramaswamy, S., Itzykson, R. A., Come, C., Savagner, P., Gitelman, I., Richardson, A. and Weinberg, R. A. (2004) Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis Cell 117 927-39. Metastasis is a multistep process during which cancer cells disseminate from the site of primary tumors and establish secondary tumors in distant organs. In a search for key regulators of metastasis in a murine breast tumor model, we have found that the transcription factor Twist, a master regulator of embryonic morphogenesis, plays an essential role in metastasis. Suppression of Twist expression in highly metastatic mammary carcinoma cells specifically inhibits their ability to metastasize from the mammary gland to the lung. Ectopic expression of Twist results in loss of E-cadherin-mediated cell-cell adhesion, activation of mesenchymal markers, and induction of cell motility, suggesting that Twist contributes to metastasis by promoting an epithelial-mesenchymal transition (EMT). In human breast cancers, high level of Twist expression is correlated with invasive lobular carcinoma, a highly infiltrating tumor type associated with loss of E-cadherin expression. These results establish a mechanistic link between Twist, EMT, and tumor metastasis. Full Text.

Yao, H. H. C., Matzuk, M. M., Jorgez, C. J., Menke, D. B., Page, D. C., Swain, A. and Capel, B. (2004) Follistatin operates downstream of Wnt4 in mammalian ovary organogenesis Developmental Dynamics 230 210-215. Wnt4(-/-) XX gonads display features normally associated with testis differentiation, suggesting that WNT4 actively represses elements of the male pathway during ovarian development. Here, we show that follistatin (Fst) which encodes a TGFbeta superfamily binding protein, is a downstream component of Wnt signaling. Fst inhibits formation of the XY-specific coelomic vessel in XX gonads. In addition, germ cells in the ovarian cortex are almost completely lost in both Wnt4 and Fst null gonads before birth. Thus, we propose that WNT4 acts through FST to regulate vascular boundaries and maintain germ cell survival in the ovary.Full Text.

Yekta, S., Shih, I. H., and Bartel, D. P. (2004). MicroRNA-directed cleavage of HOXB8 mRNA. Science 304, 594-596. MicroRNAs (miRNAs) are endogenous approximately 22-nucleotide RNAs, some of which are known to play important regulatory roles in animals by targeting the messages of protein-coding genes for translational repression. We find that miR-196, a miRNA encoded at three paralogous locations in the A, B, and C mammalian HOX clusters, has extensive, evolutionarily conserved complementarity to messages of HOXB8, HOXC8, and HOXD8. RNA fragments diagnostic of miR-196-directed cleavage of HOXB8 were detected in mouse embryos. Cell culture experiments demonstrated down-regulation of HOXB8, HOXC8, HOXD8, and HOXA7 and supported the cleavage mechanism for miR-196-directed repression of HOXB8. These results point to a miRNA-mediated mechanism for the posttranscriptional restriction of HOX gene expression during vertebrate development and demonstrate that metazoan miRNAs can repress expression of their natural targets through mRNA cleavage in addition to inhibiting productive translation..Full Text.

Yuan, B., Latek, R., Hossbach, M., Tuschl, T. and Lewitter, F. (2004) siRNA Selection Server: an automated siRNA oligonucleotide prediction server Nucleic Acids Res 32 W130-W134. The Whitehead siRNA (short interfering RNA) Selection Web Server (http://jura.wi.mit.edu/bioc/siRNA) automates the design of short oligonucleotides that can specifically 'knock down' expression of target genes. These short sequences are about 21 nt in length, and when synthesized as double stranded RNA and introduced into cell culture, can reduce or eliminate the function of the target gene. Depending on the length of a gene, there are potentially numerous combinations of possible 21mers. Some experimental evidence has already shown that not all 21mers in a gene have the same effectiveness at silencing gene function. Our tool incorporates published design rules and presents the scientist with information about uniqueness of the 21mers within the genome, thermodynamic stability of the double stranded RNA duplex, GC content, presence of SNPs and other features that may contribute to the effectiveness of a siRNA.Full Text.

Zenz, T., Roessner, A., Thomas, A., Frohling, S., Dohner, H., Calabretta, B., and Daheron, L. (2004). hIan5: the human ortholog to the rat Ian4/Iddm1/lyp is a new member of the Ian family that is overexpressed in B-cell lymphoid malignancies. Genes and Immunity 5, 109-116. The family of immune associated nucleotide binding proteins (Ian) is a distinct family of GTP- binding proteins conserved in plants, mice, rats and humans that are associated with immune functions, suggesting involvement in conserved defense mechanisms. Recently, the rat Ian4 (rIan4) was cloned and it appears to be identical to the gene Iddm1/lyp responsible for severe lymphopenia and the development of insulin-dependent diabetes in the BB-DP rat. Here we describe the characterization of a new human member of the Ian family: hIan5. hIan5 is highly homologous to rIan4, has a predicted molecular weight of 35 kDa and contains distinct G motifs of GTP- binding proteins (G-1 to G-4) in the N-terminus. Human Ian5 is anchored to the mitochondria by the hydrophobic COOH-terminal domain. Human Ian5 is highly expressed in lymph node and spleen. Different blood fractions show high hIan5 expression in CD4- and CD8-positive T cells and monocytes, but not in B lymphocytes. In contrast, in B-CLL (chronic lymphocytic leukemia) and mantle cell lymphoma samples, hIan5 mRNA was upregulated. The current data underline the role of hIan5 in T-lymphocyte development and function, and for the first time suggest that upregulation of Ian proteins is associated with B-cell malignancy, possibly by inhibiting apoptosis.Full Text.

Zhan, X. L., Clemens, J. C., Neves, G., Hattori, D., Flanagan, J. J., Hummel, T., Vasconcelos, M. L., Chess, A. and Zipursky, S. L. (2004) Analysis of Dscam diversity in regulating axon guidance in Drosophila mushroom bodies. Neuron. 43 673-686. Dscam is an immunoglobulin (Ig) superfamily member that regulates axon guidance and targeting in Drosophila. Alternative splicing potentially generates 38,016 isoforms differing in their extracellular Ig and transmembrane domains. We demonstrate that Dscam mediates the sorting of axons in the developing mushroom body (MB). This correlates with the precise spatiotemporal pattern of Dscam protein expression. We demonstrate that MB neurons express different arrays of Dscam isoforms and that single MB neurons express multiple isoforms. Two different Dscam isoforms differing in their extracellular domains introduced as transgenes into single mutant cells partially rescued the mutant phenotype. Expression of one isoform of Dscam in a cohort of MB neurons induced dominant phenotypes, while expression of a single isoform in a single cell did not. We propose that different extracellular domains of Dscam share a common function and that differences in isoforms expressed on the surface of neighboring axons influence interactions between them. Full Text

Zhang, C. C. and Lodish, H. F. (2004) Insulin-like growth factor 2 expressed in a novel fetal liver cell population is a growth factor for hematopoietic stem cells. Blood 103 2513-2521. Hematopoietic stem cells (HSCs) undergo dramatic expansion during fetal liver development, but attempts to expand their numbers ex vivo have failed. We hypothesized that unidentified fetal liver cells produce growth factors that support HSC proliferation. Here we describe a novel population of CD3(+) and Ter119(-) day-15 fetal liver cells that support HSC expansion in culture, as determined by limiting dilution mouse reconstitution analyses. DNA array experiments showed that, among other proteins, insulin-like growth factor 2 (IGF-2) is specifically expressed in fetal liver CD3(+) cells but not in several cells that do not support HSCs. Treatment of fetal liver CD3(+)Ter119(-) cells with anti-IGF-2 abrogated their HSC supportive activity, suggesting that IGF-2 is the key molecule produced by these cells that stimulates HSC expansion. All mouse fetal liver and adult bone marrow HSCs express receptors for IGF-2. Indeed, when combined with other growth factors, IGF-2 supports a 2-fold expansion of day-15 fetal liver Lin(-)Sca-1(+)c-Kit(+) long-term (LT)-HSC numbers. Thus, fetal liver CD3(+)Ter119(-) cells are a novel stromal population that is capable of supporting HSC expansion, and IGF-2, produced by these cells, is an important growth factor for fetal liver and, as we show, adult bone marrow HSCs. Full Text.

Zhang, J., Lefebvre, J. L., Zhao, S. and Granato, M. (2004) Zebrafish unplugged reveals a role for muscle-specific kinase homologs in axonal pathway choice. Nature Neuroscience 14 November. En route to their target, pioneering motor growth cones repeatedly encounter choice points at which they make pathway decisions. In the zebrafish mutant unplugged, two of the three segmental motor axons make incorrect decisions at a somitic choice point. Using positional cloning, we show here that unplugged encodes a homolog of muscle-specific kinase (MuSK) and that, unlike mammalian MuSK, unplugged has only a limited role in neuromuscular synaptogenesis. We demonstrate that unplugged is transiently expressed in cells adjacent to the choice point and that unplugged signaling before the arrival of growth cones induces changes in the extracellular environment. In addition, we find that the unplugged locus generates three different transcripts. The splice variant 1 (SV1) isoform lacks the extracellular modules essential for agrin responsiveness, and signaling through this isoform mediates axonal pathfinding, independent of the MuSK downstream component rapsyn. Our results demonstrate a new role for MuSK homologs in axonal pathway selection. Full Text.

Zhang, J., and Lodish, H. F. (2004). Constitutive activation of the MEK/ERK pathway mediates all effects of oncogenic H-ras expression in primary erythroid progenitors. Blood 104 (6): 1679-1687 Oncogenic mutations in ras genes frequently occur in patients with myeloid disorders and in these patients erythropoiesis is often affected. Previously we showed that expression of oncogenic H-ras in purified mouse primary fetal liver erythroid progenitors blocks terminal erythroid differentiation and supports Epo-independent proliferation. As a first step in understanding the underlying molecular mechanisms we examined the signaling pathways downstream of Ras in primary erythroid cells. We found that three major pathways are abnormally activated by oncogenic H-ras: Raf/ERK, PI3-kinase/Akt and RalGEF/RalA. However, only constitutive activation of the MEK/ERK pathway alone could recapitulate all of the effects of oncogenic H-ras expression in blocking erythroid differentiation and inducing Epo-independent proliferation. While expression of a constitutively active Akt kinase (ca.Akt) in erythroid progenitors does not significantly affect erythroid differentiation in the presence of erythropoietin (Epo), coexpression of ca.Akt together with a constitutively active MEK causes prolonged Epo-independent proliferation of erythroid progenitors in addition to a block in differentiation. Moreover, the effects of oncogenic H-ras expression on primary erythroid cells are blocked by the addition of U0126, a specific inhibitor of MEK1/2, allowing normal terminal erythroid proliferation and differentiation. Our data suggest that the interruption of constitutive MEK/ERK signaling is a potential therapeutic strategy to correct impaired erythroid differentiation in patients with myeloid disorders. PDF

Zhong,Joan Q., Yelena Freyzon, Daniel J. Ehrlich and Paul Matsudaira. (2004) Enhanced detection sensitivity using a novel solid-phase incorporated affinity fluorescent protein biosensor - Biomol Eng 2004 Apr;21(2):67-72. We engineered green fluorescent protein (GFP) into affinity fluorescent proteins (aFPs) biosensors. The aFPs detect protein-protein interactions by enhanced fluorescence intensity. In a proof of principle demonstration, aFPs containing haemagglutinin (HA) tag bind specifically to the anti-HA antibody. The sensitivity and specificity is enhanced 28-fold by incorporation of aFPs into solid-phase surface.Full Text.

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