RESUMO
The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.
Assuntos
Deleção de Genes , Genes Essenciais , Genoma Fúngico , Fases de Leitura Aberta , Saccharomyces cerevisiae/genética , Meios de Cultura , Regulação Fúngica da Expressão Gênica , Marcação de Genes , Genes Fúngicos , Fenótipo , Reação em Cadeia da Polimerase , Recombinação Genética , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
The MEI4 gene product is required for meiotic induction of recombination and viable spore production in the yeast Saccharomyces cerevisiae. DNA sequence analysis shows that the MEI4 gene encodes a 450-amino-acid protein bearing no homology to any previously identified protein. The MEI4 coding region is interrupted by a small intron located near the 5' end of the gene. Efficient splicing of the MEI4 transcript is not dependent on the MER1 protein, which is required for splicing the transcript of another meiotic gene, MER2. Expression of a mei4::lacZ fusion gene is meiosis-specific and depends on both heterozygosity at the mating-type locus and nutrient limitation. Northern (RNA) blot hybridization analysis suggests that MEI4 gene expression is regulated at the level of transcription. A functional MEI4 gene is not required for meiotic induction of transcription of the MER1, MER2, MEK1, RED1, SPO11, or RAD50 gene. Cytological analysis of mei4 mutant strains during meiotic prophase demonstrates that the chromosomes form long axial elements that fail to undergo synapsis. The meiosis II division is delayed in mei4 strains.
Assuntos
Genes Fúngicos , Genes Fúngicos Tipo Acasalamento , Meiose/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Cromossomos Fúngicos/metabolismo , DNA Fúngico , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Cinética , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação , Proteínas Nucleares , Plasmídeos , Reação em Cadeia da Polimerase , Splicing de RNA , Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/ultraestrutura , Esporos Fúngicos/genética , Transcrição GênicaRESUMO
As part of our studies on the fate of the muscle lineage during amphibian limb regeneration, we have isolated genomic and cDNA sequences from a myosin heavy chain in the newt (Notophthalmus viridescens). Notwithstanding the technical problems inherent in analysing the large newt genome, genomic and cDNA sequences have been isolated and subjected to analysis by restriction mapping. Northern hybridization, Southern hybridization and DNA sequencing. We believe these to be the first single copy newt gene sequences to have been subjected to this type of analysis. The newt gene sequences showed a striking difference from mammalian myosins in both the estimated sizes of the gene and its intervening sequences; these being much larger than in the mammalian models, it is speculated that this could contribute to the exceptional size of the newt genome. By contrast, the coding sequences displayed very high levels of sequence homology to mammalian myosins. In particular, the amino acid sequence of the newt myosin was found to have greatest homology with rat and human myosin isotypes having a similar cardio-skeletal muscle expression pattern. Despite a long evolutionary separation, newt and mammalian cardio-skeletal myosins have remained more similar to each other than have the human or rat cardiac forms to skeletal myosins within their own respective species.
Assuntos
Genes , Miosinas/genética , Salamandridae/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , RNARESUMO
The budding yeast MSH4 gene encodes a MutS homolog produced specifically in meiotic cells. Msh4 is not required for meiotic mismatch repair or gene conversion, but it is required for wild-type levels of crossing over. Here, we show that a msh4 null mutation substantially decreases crossover interference. With respect to the defect in interference and the level of crossing over, msh4 is similar to the zip1 mutant, which lacks a structural component of the synaptonemal complex (SC). Furthermore, epistasis tests indicate that msh4 and zip1 affect the same subset of meiotic crossovers. In the msh4 mutant, SC formation is delayed compared to wild type, and full synapsis is achieved in only about half of all nuclei. The simultaneous defects in synapsis and interference observed in msh4 (and also zip1 and ndj1/tam1) suggest a role for the SC in mediating interference. The Msh4 protein localizes to discrete foci on meiotic chromosomes and colocalizes with Zip2, a protein involved in the initiation of chromosome synapsis. Both Zip2 and Zip1 are required for the normal localization of Msh4 to chromosomes, raising the possibility that the zip1 and zip2 defects in crossing over are indirect, resulting from the failure to localize Msh4 properly.
Assuntos
Cromossomos Fúngicos/fisiologia , Troca Genética/fisiologia , Proteínas de Ligação a DNA , Proteínas Fúngicas/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Transporte/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mutação , Proteínas Nucleares , Recombinação Genética/fisiologiaRESUMO
A shuttle mutagenesis system was developed for the dimorphic yeast Yarrowia lipolytica. This system combines transposon insertions generated in Escherichia coli with the transformation of yeast with the Tn-mutagenized DNA. The mini-transposon mTn-3xHA/GFP, used in Saccharomyces cerevisiae for producing stable insertions, was adapted for use in the yeast Y. lipolytica. The mTnYl1 transposon (for mini-Tn of Y. lipolytica) confers resistance to tetracycline in E. coli. It also contains the Y. lipolytica URA3 gene for selection of yeast transformants, and the coding sequence for the S65T mutant form of GFP. The rare cutter endonuclease, I-SceI, restriction site, which enables identification of the chromosomal localization of mutagenized genes, was also incorporated. mTnYl1 was first tested on the ACO1 gene, which encodes an Acyl CoA oxidase isozyme. The mutagenesis system was further validated on a Y. lipolytica genomic DNA library constructed in a pHSS6 derivative vector. Mutants with a particular morphology or defective for alkane, fatty acids and oil degradation were obtained.
Assuntos
Elementos de DNA Transponíveis/genética , Genes Fúngicos , Vetores Genéticos/genética , Mutagênese Insercional , Proteínas de Saccharomyces cerevisiae , Leveduras/genética , Acil-CoA Oxidase , Escherichia coli , Proteínas Fúngicas/genética , Biblioteca Gênica , Genes Reporter , Glucose/farmacologia , Proteínas de Fluorescência Verde , Isoenzimas/genética , Proteínas Luminescentes/genética , Oxirredutases/genética , Resistência a Tetraciclina/genética , Transformação GenéticaRESUMO
Elongation factor 1alpha (EF-1alpha) is an abundant protein in eukaryotic cells, involved chiefly in translation of mRNA on the ribosomes, and is frequently encoded by more than one gene. Here we show the presence of two identical copies of the EF-1alpha gene in the genome of three malaria parasites, Plasmodium knowlesi, P. berghei and P. falciparum. They are organized in a head-to-head orientation and both genes are expressed in a stage specific manner at a high level, indicating that the small intergenic region contains either two strong promoters or a single bidirectional one. Both genes are expressed at the same time during erythrocytic development of the parasite. This expression pattern and the 100% similarity of the two genes excludes the possibility that the duplicated genes developed in accordance to the different types of ribosomes in Plasmodium. It is more likely that the duplication reflects a gene dosage effect. Comparison of codon usage in the Cdc2-related kinase genes (CRK2) of Plasmodium, which are expressed at a very low level, with the EF-1alpha genes indicates the existence of a codon bias for highly expressed genes, as has been shown in other organisms.
Assuntos
Dosagem de Genes , Genes de Protozoários , Fatores de Alongamento de Peptídeos/genética , Plasmodium/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Códon de Terminação , Macaca mulatta , Malária/parasitologia , Dados de Sequência Molecular , Fator 1 de Elongação de Peptídeos , Plasmodium berghei/genética , Alinhamento de SequênciaRESUMO
We report the characterization of BMS-911543, a potent and selective small-molecule inhibitor of the Janus kinase (JAK) family member, JAK2. Functionally, BMS-911543 displayed potent anti-proliferative and pharmacodynamic (PD) effects in cell lines dependent upon JAK2 signaling, and had little activity in cell types dependent upon other pathways, such as JAK1 and JAK3. BMS-911543 also displayed anti-proliferative responses in colony growth assays using primary progenitor cells isolated from patients with JAK2(V617F)-positive myeloproliferative neoplasms (MPNs). Similar to these in vitro observations, BMS-911543 was also highly active in in vivo models of JAK2 signaling, with sustained pathway suppression being observed after a single oral dose. At low dose levels active in JAK2-dependent PD models, no effects were observed in an in vivo model of immunosuppression monitoring antigen-induced IgG and IgM production. Expression profiling of JAK2(V617F)-expressing cells treated with diverse JAK2 inhibitors revealed a shared set of transcriptional changes underlying pharmacological effects of JAK2 inhibition, including many STAT1-regulated genes and STAT1 itself. Collectively, our results highlight BMS-911543 as a functionally selective JAK2 inhibitor and support the therapeutic rationale for its further characterization in patients with MPN or in other disorders characterized by constitutively active JAK2 signaling.
Assuntos
Antineoplásicos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/química , Western Blotting , Proliferação de Células/efeitos dos fármacos , Perfilação da Expressão Gênica , Compostos Heterocíclicos com 3 Anéis/química , Humanos , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/patologia , Inibidores de Proteínas Quinases/químicaAssuntos
Mutagênese Insercional/métodos , Biossíntese de Proteínas , Proteínas/genética , Animais , Anticorpos , Anticorpos Monoclonais , Sequência de Bases , Clonagem Molecular/métodos , Primers do DNA , Elementos de DNA Transponíveis , Escherichia coli/genética , Genes Reporter , Vetores Genéticos , Immunoblotting , Camundongos , Reação em Cadeia da Polimerase/métodos , Proteínas/análise , Coelhos , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
Just as Saccharomyces cerevisiae itself provides a model for so many processes essential to eukaryotic life, we anticipate that the methods and the mindset that have moved yeast biological research "beyond the genome" provide a prototype for making similar progress in other organisms. In this review I describe the experimental processes, results and utility of the current large-scale experimental approaches that use genomic data to provide a functional analysis of the yeast genome.
Assuntos
Genoma Fúngico , Genômica/métodos , Saccharomyces cerevisiae/genética , Pegada de DNA , Bases de Dados Genéticas , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Mutagênese Insercional , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Análise de Sequência de DNA , Deleção de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
MSH4 is a novel meiosis-specific gene required for wild-type levels of spore viability in S. cerevisiae. The predicted product of the MSH4 gene is homologous to the MutS family of proteins; however, msh4-null mutants have no apparent defect in mismatch repair. msh4 mutant strains display wild-type levels of gene conversion and postmeiotic segregation, but they show a reduction in crossing over and a resultant increase in nondisjunction of homologous chromosomes at meiosis I. Immunofluorescence experiments demonstrate that the Msh4 protein is localized to discrete sites on pachytene chromosomes. We propose that Msh4 interacts with a recombination intermediate to influence its resolution.
Assuntos
Adenosina Trifosfatases , Troca Genética/genética , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Proteínas Fúngicas/genética , Meiose/genética , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Esporos Fúngicos/genética , Sequência de Aminoácidos , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Sequência de Bases , Cromossomos Fúngicos/química , Cromossomos Fúngicos/ultraestrutura , Reparo do DNA/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Conversão Gênica/genética , Genes Fúngicos/genética , Imuno-Histoquímica , Dados de Sequência Molecular , Família Multigênica/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento , Mutagênese , Não Disjunção Genética , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homologia de Sequência de Aminoácidos , Esporos Fúngicos/crescimento & desenvolvimentoRESUMO
Analysis of the function of a particular gene product typically involves determining the expression profile of the gene, the subcellular location of the protein, and the phenotype of a null strain lacking the protein. Conditional alleles of the gene are often created as an additional tool. We have developed a multifunctional, transposon-based system that simultaneously generates constructs for all the above analyses and is suitable for mutagenesis of any given Saccharomyces cerevisiae gene. Depending on the transposon used, the yeast gene is fused to a coding region for beta-galactosidase or green fluorescent protein. Gene expression can therefore be monitored by chemical or fluorescence assays. The transposons create insertion mutations in the target gene, allowing phenotypic analysis. The transposon can be reduced by cre-lox site-specific recombination to a smaller element that leaves an epitope tag inserted in the encoded protein. In addition to its utility for a variety of immunodetection purposes, the epitope tag element also has the potential to create conditional alleles of the target gene. We demonstrate these features of the transposons by mutagenesis of the SPA2, ARP100, SER1, and BDF1 genes.
Assuntos
Elementos de DNA Transponíveis , Proteínas Fúngicas/fisiologia , Mutagênese Insercional/métodos , Saccharomyces cerevisiae/genética , Proteínas Virais , Epitopos , Genes Fúngicos , Genes Reporter , Biblioteca Genômica , Proteínas de Fluorescência Verde , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Integrases , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Fenótipo , Proteínas Recombinantes de Fusão , Recombinação Genética , beta-Galactosidase/genéticaRESUMO
A partially redundant oligonucleotide based on conserved protein sequences of cdk and cdc2-like proteins was used to isolate from genomic libraries of Plasmodium falciparum fragments of chromosome XIII carrying a 288-residue open-reading frame encoding a protein kinase sharing 57-58% identity with yeast p34cdc2. Based on sequence data, base composition and the striking similarity with other cdk and related proteins, four intervening sequences were identified. Their removal in vitro allowed expression of the gene, designated PfPK5, in Escherichia coli, the resulting product having kinase activity against casein and histone H1. Western blotting using a polyclonal antibody raised against the expressed protein showed that the kinase was located in the parasite's cytosol and was present in approximately constant amounts throughout the intra-erythrocytic asexual reproductive stage of the life cycle. The PSTAIRE region of the PfPK5 protein differs at three sites from that of p34cdc2, and the gene failed to complement cdc2/28 yeast mutants. However, Western blotting showed that the gene was not expressed in yeast, so this does not eliminate the possibility that it is the malarial version of cdc2.
Assuntos
Proteína Quinase CDC2/genética , Ciclo Celular , Genes de Protozoários , Plasmodium falciparum/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteína Quinase CDC2/metabolismo , Mapeamento Cromossômico , Clonagem Molecular , Teste de Complementação Genética , Íntrons , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/química , RNA Mensageiro/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
We have developed a large-scale screen to identify genes expressed at different times during the life cycle of Saccharomyces cerevisiae and to determine the subcellular locations of many of the encoded gene products. Diploid yeast strains containing random lacZ insertions throughout the genome have been constructed by transformation with a mutagenized genomic library. Twenty-eight hundred transformants containing fusion genes expressed during vegetative growth and 55 transformants containing meiotically induced fusion genes have been identified. Based on the frequency of transformed strains producing beta-galactosidase, we estimate that 80-86% of the yeast genome (excluding the rDNA) contains open reading frames expressed in vegetative cells and that there are 93-135 meiotically induced genes. Indirect immunofluorescence analysis of 2373 strains carrying fusion genes expressed in vegetative cells has identified 245 fusion proteins that localize to discrete locations in the cell, including the nucleus, mitochondria, endoplasmic reticulum, cytoplasmic dots, spindle pole body, and microtubules. The DNA sequence adjacent to the lacZ gene has been determined for 91 vegetative fusion genes whose products have been localized and for 43 meiotically induced fusions. Although most fusions represent genes unidentified previously, many correspond to known genes, including some whose expression has not been studied previously and whose products have not been localized. For example, Sec21-beta-gal fusion proteins yield a Golgi-like staining pattern, Ty1-beta-gal fusion proteins localize to cytoplasmic dots, and the meiosis-specific Mek1/Mre4-beta-gal and Spo11-beta-gal fusion proteins reside in the nucleus. The phenotypes in haploid cells have been analyzed for 59 strains containing chromosomal fusion genes expressed during vegetative growth; 9 strains fail to form colonies indicating that the disrupted genes are essential. Fifteen additional strains display slow growth or are impaired for growth on specific media or in the presence of inhibitors. Of 39 meiotically induced fusion genes examined, 14 disruptions confer defects in spore formation or spore viability in homozygous diploids. Our results will allow researchers who identify a yeast gene to determine immediately whether that gene is expressed at a specific time during the life cycle and whether its gene product localizes to a specific subcellular location.
Assuntos
Proteínas Fúngicas/análise , Expressão Gênica , Genes Fúngicos , Saccharomyces cerevisiae/genética , Sequência de Bases , Citoplasma/química , Imunofluorescência , Proteínas Fúngicas/genética , Meiose , Mitocôndrias/química , Dados de Sequência Molecular , Mutagênese , Fases de Leitura Aberta , Fenótipo , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/crescimento & desenvolvimento , Análise de Sequência de DNA , Esporos Fúngicos/fisiologia , Transformação GenéticaRESUMO
A transposon derived from Escherichia coli Tn3 was introduced into the genome of murine cytomegalovirus (MCMV) to generate a pool of viral mutants. We analyzed three of the constructed recombinant viruses that contained the transposon within the M25, M27, and m155 open reading frames. Our studies provide the first direct evidence to suggest that M25 and M27 are not essential for viral replication in mouse NIH 3T3 cells. Studies in cultured cells and Balb/c mice indicated that the transposon insertion is stable during viral propagation both in vitro and in vivo. Moreover the virus that contained the insertion mutation in M25 exhibited a titer similar to that of the wild-type virus in the salivary glands, lungs, livers, spleens, and kidneys of the Balb/c mice that were intraperitoneally infected with these viruses. These results suggest that M25 is dispensable for viral growth in these organs and the presence of the transposon sequence in the viral genome does not significantly affect viral replication in vivo. The Tn3-based system can be used as a mutagenesis approach for studying the function of MCMV genes in both tissue culture and in animals.
Assuntos
Citomegalovirus/genética , Elementos de DNA Transponíveis/genética , Mutagênese Insercional/métodos , Células 3T3 , Animais , Sequência de Bases , Citomegalovirus/patogenicidade , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/virologia , Primers do DNA/genética , DNA Recombinante/genética , Escherichia coli/genética , Genoma Viral , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Fases de Leitura Aberta , Especificidade de Órgãos , Recombinação Genética , Replicação Viral/genéticaRESUMO
Using a novel multipurpose mini-transposon, we have generated a collection of defined mutant alleles for the analysis of disruption phenotypes, protein localization, and gene expression in Saccharomyces cerevisiae. To catalog this unique data set, we have developed TRIPLES, a Web-accessible database of TRansposon-Insertion Phenotypes, Localization and Expression in Saccharomyces. Encompassing over 250 000 data points, TRIPLES provides convenient access to information from nearly 7800 transposon-mutagenized yeast strains; within TRIPLES, complete data reports of each strain may be viewed in table format, or if desired, downloaded as tab-delimited text files. Each report contains external links to corresponding entries within the Saccharomyces Genome Database and International Nucleic Acid Sequence Data Library (GenBank). Unlike other yeast databases, TRIPLES also provides on-line order forms linked to each clone report; users may immediately request any desired strain free-of-charge by submitting a completed form. In addition to presenting a wealth of information for over 2300 open reading frames, TRIPLES constitutes an important medium for the distribution of useful reagents throughout the yeast scientific community. Maintained by the Yale Genome Analysis Center, TRIPLES may be accessed at http://ycmi.med.yale.edu/ygac/triples.htm
Assuntos
Bases de Dados Factuais , Genes Fúngicos , Saccharomyces cerevisiae/genética , Elementos de DNA Transponíveis , Expressão Gênica , Fases de Leitura Aberta , FenótipoRESUMO
Economical methods by which gene function may be analysed on a genomic scale are relatively scarce. To fill this need, we have developed a transposon-tagging strategy for the genome-wide analysis of disruption phenotypes, gene expression and protein localization, and have applied this method to the large-scale analysis of gene function in the budding yeast Saccharomyces cerevisiae. Here we present the largest collection of defined yeast mutants ever generated within a single genetic background--a collection of over 11,000 strains, each carrying a transposon inserted within a region of the genome expressed during vegetative growth and/or sporulation. These insertions affect nearly 2,000 annotated genes, representing about one-third of the 6,200 predicted genes in the yeast genome. We have used this collection to determine disruption phenotypes for nearly 8,000 strains using 20 different growth conditions; the resulting data sets were clustered to identify groups of functionally related genes. We have also identified over 300 previously non-annotated open reading frames and analysed by indirect immunofluorescence over 1,300 transposon-tagged proteins. In total, our study encompasses over 260,000 data points, constituting the largest functional analysis of the yeast genome ever undertaken.