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PubMed2002

Genes essential for early events in gonadal development.

The acquisition of a sexually dimorphic phenotype is a key event in mammalian development. The underlying principle of this essential process is that genetic sex, determined by the presence or absence of a Y chromosome at fertilization, directs the embryonic gonads to differentiate into either testes or ovaries. Hormones produced by the testes then trigger the developmental program that leads to male phenotypic sexual differentiation. Without testes and their biochemical products, differentiation proceeds along the female pathway. Recent studies have identified several transcription factors that are required for gonadal development and sexual differentiation, i.e. Wilms' tumor related 1 (WT1), steroidogenic factor 1 (SF-1), SOX9, and GATA4, presumably because they activate the expression of essential target genes. Studies also have identified another transcriptional regulator, designated DAX-1, that inhibits target gene induction by all of these transcriptional activators. This chapter provides an overview of gonadal development and sexual differentiation, reviews the studies that have led to the isolation and characterization of these genes in the gonads, and then discusses how they interact to regulate critical events in sexual differentiation.

DAX-1 Orphan Nuclear Receptor↗

PubMed2001

Essential genes in proximal 3L heterochromatin of Drosophila melanogaster.

We have further characterized essential loci within the centric heterochromatin of the left arm of chromosome 3 (3L) of Drosophila melanogaster, using EMS, radiation and P element mutagenesis. We failed to find any new essential genes, a result that suggests a lower-than-average gene density in this region. Mutations affecting expression of the most proximal gene [lethal 1, l1 or l(3)80Fj] act as dominant suppressors of Polycomb (Pc), behavior which is consistent with a putative trithorax group (trx-G) gene. The third gene to the left of the centromere [lethal 3, l3 or l(3)80Fh] is likely to correspond to verthandi (vtd), a known trx-G gene that plays a role in the regulation of hedgehog (hh) expression and signalling. The intervening gene [lethal 2, l2 or l(3)80Fi] is required throughout development, and mutant alleles have interesting phenotypes; in various allelic combinations that survive, we observe fertility, bristle, wing, eye and cuticle defects.

Animals↗

PubMed2005

Decrypting the biochemical function of an essential gene from Streptococcus pneumoniae using ThermoFluor technology.

The protein product of an essential gene of unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively. ThermoFluor was used to screen the protein against a library of 3000 compounds that were specifically selected to provide information about possible biological functions. The results of this screen identified pyridoxal phosphate and pyridoxamine phosphate as equilibrium binding ligands (K(d) approximately 50 pM, K(d) approximately 2.5 microM, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugars were also identified as ligands of this protein. Sequence comparison with two enzymes of known structure but relatively low overall sequence homology established that several key residues directly involved in pyridoxal phosphate binding were strictly conserved. Screening a collection of generic drugs and natural products identified the antifungal compound canescin A as an irreversible covalent modifier of the enzyme. Our investigation of this protein indicates that its probable biological role is that of a nucleoside diphospho-keto-sugar aminotransferase, although the preferred keto-sugar substrate remains unknown. These experiments demonstrate the utility of a generic affinity-based ligand binding technology in decrypting possible biological functions of a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.

Amino Acid Sequence↗

PubMed2006

[Essential genes as potential targets of antifungal agents in pathogenic yeast Candida].

An important point in the development of an antimicrobial agent is whether its target molecules are essential for growth of the microorganism. From this viewpoint, we focused attention on essential genes as potential targets of antifungal agents in the pathogenic yeast Candida. Here we introduce recent attempts for screening, identification, and characterization of essential genes from a haploid yeast Candida glabrata, using temperature-sensitive mutants. Our experimental results suggesting the essentiality of C. albicans PHO85, the homologue of which is known as a negative regulator of the PHO system and as a non-essential gene in Saccharomyces cerevisiae are also described.

Antifungal Agents↗

PubMed2005

Role of essential genes in mitochondrial morphogenesis in Saccharomyces cerevisiae.

Mitochondria are essential organelles of eukaryotic cells. Inheritance and maintenance of mitochondrial structure depend on cytoskeleton-mediated organelle transport and continuous membrane fusion and fission events. However, in Saccharomyces cerevisiae most of the known components involved in these processes are encoded by genes that are not essential for viability. Here we asked which essential genes are required for mitochondrial distribution and morphology. To address this question, we performed a systematic screen of a yeast strain collection harboring essential genes under control of a regulatable promoter. This library contains 768 yeast mutants and covers approximately two thirds of all essential yeast genes. A total of 119 essential genes were found to be required for maintenance of mitochondrial morphology. Among these, genes were highly enriched that encode proteins involved in ergosterol biosynthesis, mitochondrial protein import, actin-dependent transport processes, vesicular trafficking, and ubiquitin/26S proteasome-dependent protein degradation. We conclude that these cellular pathways play an important role in mitochondrial morphogenesis and inheritance.

Actins↗

PubMed2003

Viable nonsense mutants for the essential gene SUP45 of Saccharomyces cerevisiae.

BACKGROUND: Termination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs) - eRF1 and eRF3. The highly conserved translation termination factor eRF1 in Saccharomyces cerevisiae is encoded by the essential gene SUP45. RESULTS: We have isolated five sup45-n (n from nonsense) mutations that cause nonsense substitutions in the following amino acid positions of eRF1: Y53 --> UAA, E266 --> UAA, L283 --> UAA, L317 --> UGA, E385 --> UAA. We found that full-length eRF1 protein is present in all mutants, although in decreased amounts. All mutations are situated in a weak termination context. All these sup45-n mutations are viable in different genetic backgrounds, however their viability increases after growth in the absence of wild-type allele. Any of sup45-n mutations result in temperature sensitivity (37 degrees C). Most of the sup45-n mutations lead to decreased spore viability and spores bearing sup45-n mutations are characterized by limited budding after germination leading to formation of microcolonies of 4-20 cells. CONCLUSIONS: Nonsense mutations in the essential gene SUP45 can be isolated in the absence of tRNA nonsense suppressors.

Alleles↗

PubMed1976

Method for the isolation of Escherichia coli K-12 mutants deficient in essential genes.

We developed a general procedure for the induction and identification of mutations in chromosomal essential genes that are located in a diploid region of Escherichia coli K-12. The partial diploidy is conferred by an episome that is temperature sensitive for replication so that a mutant strain will form microcolonies at 42 C on complete media if an essential chromosomal gene in the diploid region is defective. Mutations identified by this procedure can be classified into cistrons by a complementation method devised for the purpose. To verify that the procedure works in practice, we fused an episome covering the rif region with an Ftslac+ and used the resulting temperature-sensitive episome to identify chromosomal mutations in essential functions near rif. As expected, a certain proportion of the mutations were in the rif gene, an essential gene that codes for the beta subunit of ribonucleic acid polymerase.

Conjugation, Genetic↗

PubMed2004

Nonsense mutations in the essential gene SUP35 of Saccharomyces cerevisiae are non-lethal.

In the present work we have characterized for the first time non-lethal nonsense mutations in the essential gene SUP35, which codes for the translation termination factor eRF3 in Saccharomyces cerevisiae. The screen used was based on selection for simultaneous suppression of two auxotrophic nonsense mutations. Among 48 mutants obtained, sixteen were distinguished by the production of a reduced amount of eRF3, suggesting the appearance of nonsense mutations. Fifteen of the total mutants were sequenced, and the presence of nonsense mutations was confirmed for nine of them. Thus a substantial fraction of the sup35 mutations recovered are nonsense mutations located in different regions of SUP35, and such mutants are easily identified by the fact that they express reduced amounts of eRF3. Nonsense mutations in the SUP35 gene do not lead to a decrease in levels of SUP35 mRNA and do not influence the steady-state level of eRF1. The ability of these mutations to complement SUP35 gene disruption mutations in different genetic backgrounds and in the absence of any tRNA suppressor mutation was demonstrated. The missense mutations studied, unlike nonsense mutations, do not decrease steady-state amounts of eRF3.

Base Sequence↗

PubMed1999

Disruption of six open reading frames on chromosome X of Saccharomyces cerevisiae reveals a cluster of four essential genes.

In this study we report the construction and basic phenotypic analysis of six Saccharomyces cerevisiae deletion mutants. The open reading frames (ORFs) YJL008C (gene symbol CCT8), YJL010C, YJL011C, YJL012C, YJL017W, and YJL020C from chromosome X have been disrupted by integration of deletion cassettes, comprising the bacterial KanMX4 marker gene and terminal long (LFH) or short (SFH) flanking sequences that are homologous to the 5' and 3' untranslated regions of the respective ORFs. For correct disruption of ORF YJL008C, it was necessary to construct a deletion cassette flanked by 300-350 bp long target guide sequences by LFH-PCR. Transformations using ORF YJL008C gene disruption cassettes synthesized by standard SFH-PCR exclusively resulted in false-positive or multiple integration events, probably because seven additional genes homologous to CCT8 exist in the yeast genome. The other five ORFs have been disrupted using cassettes generated by SFH-PCR, comprising terminal homologous regions of approximately 50 bp to each target site. Correct genomic integration of the reporter modules was verified by analytical PCR and Southern hybridization. Deletion of YJL008C, YJL010C, YJL011C, and YJL012C was found to be lethal, as shown by sporulation and tetrad analysis. This result is in contrast to the finding that only 16-20% of the genes in S. cerevisiae are estimated to be essential. The four essential genes described in this work are clustered, while the two other non-essential ORFs are separated by further ORFs. Although the two viable deletion mutants were tested against 60 different inhibitors, heavy metal ions and salts, no phenotype could be detected that co-segregated with the deletion during meiosis.

Amino Acid Sequence↗

PubMed1999

Disruption and phenotypic analysis of six novel genes from chromosome IV of Saccharomyces cerevisiae reveal YDL060w as an essential gene for vegetative growth.

The disruption of six novel genes (YDL059c, YDL060w, YDL063c, YDL065c, YDL070w and YDL110c), localized on the left arm of chromosome IV in Saccharomyces cerevisiae, is reported. A PCR-based strategy was used to construct disruption cassettes in which the kanMX4 dominant marker was introduced between two long flanking homology regions, homologous to the promoter and terminator sequences of the target gene (Wach et al., 1994). The disruption cassettes were used to generate homologous recombinants in two diploid strains with different genetic backgrounds (FY1679 and CEN. PK2), selecting for geneticin (G418) resistance conferred by the presence of the dominant marker kanMX4. The correctness of the cassette integration was tested by PCR. After sporulation and tetrad analysis of the heterozygous deletant diploids, geneticin-resistant haploids carrying the disrupted allele were isolated. YDL060w was shown to be an essential gene for vegetative growth. A more detailed phenotypic analysis of the non-lethal haploid deletant strains was performed, looking at cell and colony morphology, growth capability on different media at different temperatures, and ability to conjugate. Homozygous deletant diploids were also constructed and tested for sporulation. Only minor differences between parental and mutant strains were found for some deletant haploids.

DNA Primers↗

PubMed2004

Identification of essential and non-essential genes of the guinea pig cytomegalovirus (GPCMV) genome via transposome mutagenesis of an infectious BAC clone.

We report application of a transposition methodology that allows the easy characterization and mutation of genes encoded on an infectious bacterial artificial chromosome (BAC) clone. We characterized mutants generated by transposome (Tn) mutagenesis of a BAC clone of guinea pig cytomegalovirus (GPCMV). A pool of Tn mutant GPCMV BACs were screened initially by restriction profile analysis to verify they were full-length, and subsequently GPCMV BAC DNA from individual mutants was transfected onto guinea pig lung fibroblast cells in order to generate virus. Tn GPCMV BAC mutants were classed as either essential or non-essential gene insertions, depending upon their ability to regenerate viable, replication-competent virus. Representative mutants were more fully characterized. Analysis by sequencing the Tn insertion site on the mutated BACs, and by regeneration of virus using transfection of guinea pig fibroblasts (GPL), demonstrated that a recombinant with a Tn insertion in the UL35 homolog gene (GP35) was a non-essential gene for viral replication in tissue culture. A mutant with an insertion in the UL46 homolog (GP46) was nonviable, a phenotype which could be rescued by homologous recombination of BAC DNA with wild-type UL46 sequences, suggesting an essential role of this putative capsid gene in virus replication.

Amino Acid Sequence↗

PubMed2003

A postgenomic method for predicting essential genes at subsaturation levels of mutagenesis: application to Mycobacterium tuberculosis.

We describe a postgenomic in silico approach for identifying genes that are likely to be essential and estimate their proportion in haploid genomes. With the knowledge of all sites eligible for mutagenesis and an experimentally determined partial list of nonessential genes from genome mutagenesis, a Bayesian statistical method provides reasonable predictions of essential genes with a subsaturation level of random mutagenesis. For mutagenesis, a transposon such as Himar1 is suitable as it inserts randomly into TA sites. All of the possible insertion sites may be determined a priori from the genome sequence and with this information, data on experimentally hit TA sites may be used to predict the proportion of genes that cannot be mutated. As a model, we used the Mycobacterium tuberculosis genome. Using the Himar1 transposon, we created a genetically defined collection of 1,425 insertion mutants. Based on our Bayesian statistical analysis using Markov chain Monte Carlo and the observed frequencies of transposon insertions in all of the genes, we estimated that the M. tuberculosis genome contains 35% (95% confidence interval, 28%-41%) essential genes. This analysis further revealed seven functional groups with high probabilities of being enriched in essential genes. The PE-PGRS (Pro-Glu polymorphic GC-rich repetitive sequence) family of genes, which are unique to mycobacteria, the polyketide/nonribosomal peptide synthase family, and mycolic and fatty acid biosynthesis gene families were disproportionately enriched in essential genes. At subsaturation levels of mutagenesis with a random transposon such as Himar1, this approach permits a statistical prediction of both the proportion and identities of essential genes of sequenced genomes.

Anti-Bacterial Agents↗

PubMed1984

Deletion of an essential gene in Escherichia coli by site-specific recombination with linear DNA fragments.

Deletion of an essential gene in Escherichia coli was accomplished by transformation of linear DNA fragments that have a Kanr gene segment flanked by sequences homologous to closely spaced regions on the E. coli chromosome. Selection for a double crossover within homologous sequences can effectively delete an entire gene. Cell viability is maintained by provision of the essential gene on a plasmid with a temperature-sensitive replicon, resulting in cells which have a temperature-sensitive phenotype.

Base Sequence↗

PubMed2001

Mapping and identification of essential gene functions on the X chromosome of Drosophila.

The Drosophila melanogaster genome consists of four chromosomes that contain 165 Mb of DNA, 120 Mb of which are euchromatic. The two Drosophila Genome Projects, in collaboration with Celera Genomics Systems, have sequenced the genome, complementing the previously established physical and genetic maps. In addition, the Berkeley Drosophila Genome Project has undertaken large-scale functional analysis based on mutagenesis by transposable P element insertions into autosomes. Here, we present a large-scale P element insertion screen for vital gene functions and a BAC tiling map for the X chromosome. A collection of 501 X-chromosomal P element insertion lines was used to map essential genes cytogenetically and to establish short sequence tags (STSs) linking the insertion sites to the genome. The distribution of the P element integration sites, the identified genes and transcription units as well as the expression patterns of the P-element-tagged enhancers is described and discussed.

Animals↗

PubMed2026

Genome-wide identification of conditionally essential genes for growth in the presence of sulfamethoxazole and trimethoprim in sulfamethoxazole- and trimethoprim-resistant Escherichia coli.

UNLABELLED: Resistance to sulfonamides (SULs) and trimethoprim (TMP) in Escherichia coli threatens their clinical relevance. Beyond known resistance mechanisms, little is understood about the cellular responses that enable resistant E. coli to grow under these antibiotic stresses. This study aimed to identify genes that support bacterial growth under SUL and TMP stress. Two saturated transposon mutant libraries were constructed in resistant E. coli MG1655 harboring either dfrA1 or sul2. They were grown with and without 1/2 and 1/4 minimum inhibitory concentration (MIC) of sulfamethoxazole (SMX) or TMP, and mutant depletion was assessed via transposon-directed insertion-site sequencing. At 1/2 MIC, 36 and 89 genes were identified as conditionally essential during SMX and TMP exposure, while 5 and 2 genes were classified as conditionally essential at 1/4 MIC. Genes identified as conditionally essential at 1/4 MIC were also important at 1/2 MIC. Conditionally essential genes belonged to lipopolysaccharide biosynthesis, peptidoglycan metabolism, energy production, membrane integrity, phosphate metabolism, and stress responses, highlighting the role of these factors in maintaining cell stability under SMX and TMP stress. Validation with 10 conditionally essential genes (apaH, mtn, surA, waaO, nlpI, prc, wzxE, fadR, degP, and tpiA) showed that deletion mutants indeed exhibited growth defects and two- to eightfold reductions in MIC under antibiotic stresses compared to their parent strains. This study highlights cellular responses to SMX and TMP under antibiotic stress, and it has identified a list of genes whose products may serve as potential helper drug targets to resensitize resistant E. coli to SMX and TMP treatments. IMPORTANCE: Sulfonamides (SULs) and trimethoprim (TMP) are broad-spectrum antimicrobials. They are commonly used to treat infections in both humans and animals. Resistance against SUL and TMP is widespread in pathogenic bacteria, and there is a need to overcome this problem. One possibility is to target the cellular mechanism by which the resistant bacteria adapt to growth in the presence of the antimicrobials. In this study, we identify the genes, besides the resistance genes, which enable resistant Escherichia coli to grow in the presence of SUL and TMP. We further show that knocking out many of these genes attenuates the resistant E. coli for growth during SUL and/or TMP stress, irrespective of which SUL- or TMP-resistant gene the bacteria carry. The gene products of these genes may serve as potential helper drug targets to resensitize resistant E. coli to sulfamethoxazole and TMP treatments.

Escherichia coli↗

PubMed2004

Screening for novel essential genes of Saccharomyces cerevisiae involved in protein secretion.

We describe here a screening procedure devised for searching new genes involved in protein secretion in Saccharomyces cerevisiae. The screening procedure takes advantage of yeast strains constructed within the EUROFAN project, in which the promoters of the novel essential genes were replaced by the doxycycline-regulated tetO(7)-CYC1 promoter. This promoter is active in normal growth medium but results in downregulation of the gene in the presence of doxycycline. The yeast cells were grown in the presence or absence of doxycycline, and both the growth and secretion of the heat shock protein, Hsp150p, into the culture medium were determined. In seven strains there was a specific effect on protein secretion. In a strain in which the RPN5 gene was downregulated, the level of secreted Hsp150p was increased compared to the control culture. When RER2 was downregulated, cells secreted Hsp150p that was not of the mature size. In five strains, secretion was more severely reduced than cell growth. One of these downregulated genes, YGL098w, was recently reported to encode an ER-located t-SNARE, USE1. Four of the genes detected, NOG2, NOP15, RRP40 and SDA1, encode proteins involved in ribosome assembly, suggesting a possible new signalling pathway between ribosome biogenesis and production of secreted proteins. The results obtained here indicate that the present screen could be successfully used in larger scale to identify novel secretion-related genes.

Blotting, Western↗

PubMed2005

Identification of essential genes in Streptococcus pneumoniae by allelic replacement mutagenesis.

To find potential targets of novel antimicrobial agents, we identified essential genes of Streptococcus pneumoniae using comparative genomics and allelic replacement mutagenesis. We compared the genome of S. pneumoniae R6 with those of Bacillus subtilis, Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus, and selected 693 candidate target genes with > 40% amino acid sequence identity to the corresponding genes in at least two of the other species. The 693 genes were disrupted and 133 were found to be essential for growth. Of these, 32 encoded proteins of unknown function, and we were able to identify orthologues of 22 of these genes by genomic comparisons. The experimental method used in this study is easy to perform, rapid and efficient for identifying essential genes of bacterial pathogens.

Genes, Bacterial↗

PubMed2003

Assessment of the utilization of the antisense RNA strategy to identify essential genes in heterologous bacteria.

We employed an antisense RNA approach to identify essential genes common in both Gram-positive and Gram-negative bacteria by cloning a random library of Streptococcus mutans chromosomal DNA into an expression vector and transforming Escherichia coli. Twelve out of 27 E. coli transformants with growth defective phenotypes contained individual structural genes of S. mutans in the antisense orientation relative to the E. coli promoter. Thirty-three percent of these transformants (4/12) corresponded to the genes (gyrA, ileS, rplE and yihA orthologs) which are essential for bacterial viability.

Escherichia coli↗