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Results for “Genes, Essential”

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PubMed2001

Systematic identification of selective essential genes in Helicobacter pylori by genome prioritization and allelic replacement mutagenesis.

A comparative genomic approach was used to identify Helicobacter pylori 26695 open reading frames (ORFs) which are conserved in H. pylori J99 but highly diverged in other eubacteria. A survey of selected pathways of central intermediary metabolism was also carried out, and genes with a potentially selective role in H. pylori were identified. Forty-five ORFs identified in these two analyses were screened using a rapid vector-free allelic replacement mutagenesis technique, and 33 were shown to be essential in vitro. Notably, 13 ORFs gave essentiality results which are unexpected in view of their known or proposed functions, and phylogenetic analysis was used to investigate the annotation of 7 such ORFs which are highly diverged. We propose that the products of a number of these H. pylori-specific essential genes may be suitable targets for novel anti-H. pylori therapies.

Alleles↗

PubMed1996

Insertional mutagenesis and rapid cloning of essential genes in zebrafish.

Large-scale chemical mutagenesis screens in zebrafish have led to the isolation of thousands of lethal mutations in genes that are essential for embryonic development. However, the cloning of these mutated genes is difficult at present as it requires positional cloning methods. In Drosophila, chemical mutagenesis screens were complemented with P-element insertional mutagenesis which facilitated the cloning of many genes that had been identified by chemical lesions. To facilitate the cloning of vertebrate genes that are important during embryogenesis, we have developed an insertional mutagenesis strategy in zebrafish using a retroviral vector. Here, in a pilot screen of 217 proviral insertions, we obtained three insertional mutants with embryonic lethal phenotypes, and identified two of the disrupted genes. One of these, no arches, is essential for normal pharyngeal arch development, and is homologous to the recently characterized Drosophila zinc-finger gene, clipper, which encodes a novel type of ribonuclease. As it is easy to generate tens to hundreds of thousands of proviral transgenes in zebrafish, it should now be possible to use this screening method to mutate and then rapidly clone a large number of genes affecting vertebrate developmental and cellular processes.

Amino Acid Sequence↗

PubMed2002

Identification of 113 conserved essential genes using a high-throughput gene disruption system in Streptococcus pneumoniae.

The recent availability of bacterial genome sequence information permits the identification of conserved genes that are potential targets for novel antibiotic drug discovery. Using a coupled bioinformatic/experimental approach, a list of candidate conserved genes was generated using a Microbial Concordance bioinformatics tool followed by a targeted disruption campaign. Pneumococcal sequence data allowed for the design of precise PCR primers to clone the desired gene target fragments into the pEVP3 'suicide vector'. An insertion-duplication approach was employed that used the pEVP3 constructs and resulted in the introduction of a selectable chloramphenicol resistance marker into the chromosome. In the case of non-essential genes, cells can survive the disruption and form chloramphenicol-resistant colonies. A total of 347 candidate reading frames were subjected to disruption analysis, with 113 presumed to be essential due to lack of recovery of antibiotic-resistant colonies. In addition to essentiality determination, the same high-throughput methodology was used to overexpress gene products and to examine possible polarity effects for all essential genes.

Cell Division↗

PubMed1994

NAT2, an essential gene encoding methionine N alpha-acetyltransferase in the yeast Saccharomyces cerevisiae.

N alpha-Acetylation is catalyzed by N alpha-acetyltransferases, which transfer acetyl groups from acetyl coenzyme A to the N termini of most eukaryotic proteins co-translationally. NAT1 and ARD1 from the yeast Saccharomyces cerevisiae (Mullen, J. R., Kayne, P. S., Moerschell, R. P., Tsunasawa, S., Gribskov, M., Colavito-Shepanski, M., Grunstein, M., Sherman, F., and Sternglanz, R. (1989) EMBO J. 8, 2067-2075) were previously shown to encode the major N alpha-acetyltransferase, which act on certain proteins having serine, glycine, and alanine but not methionine termini (Sherman, F., Moerschell, R. P., Tsunasawa, S., and Sternglanz, R. (1993) in Methods in Protein Sequence Analysis (Imahori, K., and Sakiyama, F., eds) pp. 173-181, Plenum Publishing Corp., New York). We have identified a second gene, NAT2, that may correspond to the N alpha-acetyltransferase acting on a subset of proteins having methionine termini. Crude extracts of a series of heat-sensitive mutants (Ts-) were screened for acetylation of a 24-amino acid synthetic peptide Met-Asn-Asn- in vitro. One mutant, nat2-1, out of 115 strains examined, lacked acetyltransferase activity, and the mutation co-segregated as a single gene with the heat-sensitive phenotype. The nat2-1 mutants were deficient in the ability to acetylate Met-Asn-Asn- and Met-Glu-Arg-peptides but were able to N alpha-acetylate Ser-Glu-Phe- and Ser-Tyr-Ser- peptides in vitro. The NAT2 wild-type gene was cloned by complementation of the nat2-1 mutant, and the DNA sequence revealed an open reading frame of 288 amino acids. Gene disruption demonstrated that NAT2 is an essential gene, and hybridization analysis indicated that it is located on chromosome VII. Furthermore, there was limited, but significant identities between the yeast N alpha-acetyltransferases Nat1, Ard1, Nat2, and Mak3, although no common motifs could be identified. We propose that NAT2 encodes the major N alpha-acetyl-transferase acting on certain proteins with only methionine termini, and that N alpha-acetylation of some of these proteins is essential for viability.

Acetylation↗

PubMed1998

Identification of linked Legionella pneumophila genes essential for intracellular growth and evasion of the endocytic pathway.

Legionella pneumophila replicates within a specialized phagosome in cultured cells, a function necessary for its pathogenicity. The replicative phagosome lacks membrane marker proteins, such as the glycoprotein LAMP-1, that are indicators of the normal endocytic pathway. We describe the isolation of several Legionella genes essential for intracellular growth and evasion of the endocytic pathway, using a genetic and cell biological approach. We screened 4,960 ethyl methanesulfonate-mutagenized colonies for defects in intracellular growth and trafficking to the replicative phagosome. Six mutant strains of L. pneumophila that had severe intracellular growth defects in mouse bone marrow-derived macrophages were identified. All six mutants were found in phagosomes that colocalized with LAMP-1, indicating defects in intracellular trafficking. The growth defects of two of these strains were complemented by molecular clones from a bank constructed from a wild-type L. pneumophila strain. The inserts from these clones are located in a region of the chromosome contiguous with several other genes essential for intracellular growth. Three mutants could be complemented by single open reading frames placed in trans, one mutant by a gene termed dotH and two additional mutants by a gene termed dotO. A deletion mutation was created in a third gene, dotI, which is located directly upstream of dotH. The delta dotI strain was also defective for intracellular growth in macrophages, and this defect was complemented by a single open reading frame in trans. Based on sequence analysis and structural predictions, possible roles of dotH, dotI, and dotO in intracellular growth are discussed.

Animals↗

PubMed2006

Identification of an essential gene of Listeria monocytogenes involved in teichoic acid biogenesis.

Listeria monocytogenes is a facultative intracellular gram-positive bacterium responsible for severe opportunistic infections in humans and animals. We had previously identified a gene encoding a putative UDP-N-acetylglucosamine 2-epimerase, a precursor of the teichoic acid linkage unit, in the genome of L monocytogenes strain EGD-e. This gene, now designated lmo2537, encodes a protein that shares 62% identity with the cognate epimerase MnaA of Bacillus subtilis and 55% identity with Cap5P of Staphylococcus aureus. Here, we addressed the role of lmo2537 in L. monocytogenes pathogenesis by constructing a conditional knockout mutant. The data presented here demonstrate that lmo2537 is an essential gene of L. monocytogenes that is involved in teichoic acid biogenesis. In vivo, the conditional mutant is very rapidly eliminated from the target organs of infected mice and thus is totally avirulent.

Animals↗

PubMed2006

Identification of an essential gene responsible for D-Asp incorporation in the Lactococcus lactis peptidoglycan crossbridge.

Bacteria such as Lactococcus lactis have D-aspartate (D-Asp) or its amidated derivative D-asparagine (D-Asn), in their peptidoglycan (PG) interpeptide crossbridge. We performed a subtractive genome analysis to identify L. lactis gene yxbA, orthologues of which being present only in bacteria containing D-amino acids in their PG crossbridge, but absent from those that instead insert L-amino acids or glycine. Inactivation of yxbA required a complementing Streptococcus pneumoniae murMN genes, which express enzymes that incorporate L-Ser-L-Ala or L-Ala-L-Ala in the PG crossbridge. Our results show that (i) yxbA encodes D-Asp ligase responsible for incorporation of D-Asp in the PG crossbridge, and we therefore renamed it as aslA, (ii) it is an essential gene, which makes its product a potential target for specific antimicrobials, (iii) the absence of D-Asp may be complemented by L-Ser-L-Ala or L-Ala-L-Ala in the L. lactis PG, indicating that the PG synthesis machinery is not selective for the side-chain residues, and (iv) lactococcal strains having L-amino acids in their PG crossbridge display defects in cell wall integrity, but are able to efficiently anchor cell wall proteins, indicating relative flexibility of lactococcal transpeptidation reactions with respect to changes in PG sidechain composition.

Aspartic Acid↗

PubMed1992

Approaches to the identification of non-essential genes of African swine fever virus.

It is poorly understood why vaccines could not be developed for the control and prevention of African swine fever (ASF) virus infection. The aim of our study was to identify genes non-essential for ASF virus replication because there were indications that certain viral gene products, which apparently are non-essential for viral replication, conferred protection from death due to ASF. A cosmid library representing the genome of ASF virus strain France 64 was established and characterized. Then, in order to inactivate viral genes by insertion, the beta-galactosidase (beta-gal) gene was introduced either randomly or at specific locations of selected cloned DNA fragments. These constructions were transfected into cells which had been previously infected with a cell-culture-adapted viral strain in order to allow the generation of recombinant progeny virus. Viable recombinant progeny was identified by at least one of the following means: (1) expression of beta-gal; (2) detection of beta-gal specific DNA by plaque hybridization, and (3) absence of a functional product of the inactivated gene. Presently, we are characterizing a recombinant virus with an insertionally inactivated thymidine kinase gene.

African Swine Fever Virus↗

PubMed1993

Deletion of the essential gene 24 from the bacteriophage T4 genome.

We deleted the essential gene 24 from the genome of bacteriophage T4. The delta 24 phage is a conditional lethal mutant that can grow only when the host strain supplies the product of gene 24 in trans, or when the phage acquires a functional gene 24 by some type of recombination event. Thus, gene 24 can be used as a selectable marker, for example permitting transposition into the T4 genome and analyses of plasmid-phage recombination [Woodworth and Kreuzer, Mol. Microbiol. 6 (1992) 1289-1296; H.W.E. and K.N.K., manuscript submitted]. We also found that the promoter region of gene 24 allows a low level of autonomous plasmid replication in T4-infected cells, raising the possibility of a previously unrecognized mode of T4 replication initiation.

Bacteriophage T4↗

PubMed1999

Development of a self-cloning system for Actinomadura verrucosospora and identification of polyketide synthase genes essential for production of the angucyclic antibiotic pradimicin.

A self-cloning system for Actinomadura verrucosospora, a producer of the angucyclic antibiotic pradimicin A (PRM A), has been developed. The system is based on reproducible and reliable protoplasting and regeneration conditions for A. verrucosospora and a novel plasmid vector that consists of a replicon from a newly found Actinomadura plasmid and a selectable marker cloned from the Actinomadura strain. The system has an efficiency of more than 10(5) CFU/microgram of DNA. Using this system, we have cloned and identified the polyketide synthase (PKS) genes essential for PRM A biosynthesis from A. verrucosospora. Nucleotide sequence analysis of the 3.5-kb SalI-SphI fragment showed that ketosynthase subunits (open reading frame 1 [ORF1] and ORF2) of the essential PKS genes have strong similarities (59 to 89%) to those for angucyclic antibiotic biosynthesis.

Actinomycetales↗

PubMed2026

Synonymous mutations in essential genes infrequently produce fitness effects in human cell lines.

The assumption that synonymous mutations are fitness-neutral is central to many foundational results in the fields of genetics, genomics, evolutionary biology, and medicine. However, recent results suggest synonymous mutations have pervasive and strong fitness effects. These vigorously debated studies in non-human model systems have even suggested that the proportion of synonymous mutations and their fitness effect sizes are similar to non-synonymous mutations. To probe the fitness effect of synonymous mutations, we utilized recent advances in base editing to test 8558 potential synonymous mutations in 128 highly essential genes in human cell lines. Importantly, our library design excluded splice-proximal sites, ensuring a direct test of codon-level synonymous effects independent of splicing disruption. We find that synonymous mutations rarely have fitness effects on growth, occurring around 37.9-fold (95% CI: 22.16-81.48-fold) less frequently than missense mutations. In this experimental context, these findings demonstrate that synonymous mutations impact cellular fitness far less frequently than missense mutations. These results deviate from earlier reports of widespread synonymous fitness effects in yeast, yet they align with recent prime editing data observed in other human cell lines.

Humans↗

PubMed1997

Disruption of six novel yeast genes reveals three genes essential for vegetative growth and one required for growth at low temperature.

We describe here the construction of six deletion mutants and their basic phenotypic analysis. Six open reading frames (ORFs) from chromosome X, YJR039w, YJR041c, YJR043c, YJR046w, YJR053w and YJR065c, were disrupted by deletion cassettes with long (LFH) or short (SFH) flanking regions homologous to the target locus. The LFH deletion cassette was made by introducing into the kanMX4 marker module two polymerase chain reaction (PCR) fragments several hundred base pairs (bp) in size homologous to the promoter and terminator regions of a given ORF. The SFH gene disruption construct was obtained by PCR amplification of the kanMX4 marker with primers providing homology to the target gene. The region of homology to mediate homologous recombination was about 70 bp. Sporulation and tetrad analysis revealed that ORFs YJR041c, YJR046w and YJR065c are essential genes. Complementation tests by corresponding cognate gene clones confirmed this observation. The non-growing haploid segregants were observed under the microscope. The yjr041c delta haploid cells gave rise to microcolonies comprising about 20 to 50 cells. Most yjr046w delta cells were blocked after one or two cell cycles with heterogeneous bud sizes. The yjr065c delta cells displayed an unbudded spore or were arrested before completion of the first cell division cycle with a bud of variable size. The deduced protein of ORF YJR065c, that we named Act4, belongs to the Arp3 family of actin-related proteins. Three other ORFs, YJR039w, YJR043c and YJR053w are non-essential genes. The yjr043c delta cells hardly grew at 15 degrees C, indicating that this gene is required for growth at low temperature. Complementation tests confirmed that the disruption of YJR043c is responsible for this growth defect. In addition, the mating efficiency of yjr043c delta and yjr053w delta cells appear to be moderately affected.

Base Sequence↗

PubMed2003

Disruption of 12 ORFs located on chromosomes IV, VII and XIV of Saccharomyces cerevisiae reveals two essential genes.

We describe the generation of null-mutants of 12 open reading frames (ORFs), discovered during the systematic sequencing of the Saccharomyces cerevisiae genome. These ORFs are located on chromosome IV (YDL183c), on chromosome VII (YGL139w, YGL140c, YGL141w, YGR280c and YGR284c) or on chromosome XIV (YNL006w, YNR004w, YNR007c, YNR008w, YNR009w and YNR013c). Disruptants were generated using the PCR-based short flanking homology (SFH) strategy in yeast strain FY1679. Tetrad analysis, following sporulation of the heterozygous disruptants, revealed that YGR280c and YNL006w are essential genes for vegetative yeast growth in rich medium. The lethality of the two genes was confirmed by gene complementation analysis. The protein encoded by YNL006w (LST8) is now known to be involved in transport of permeases from the Golgi to the plasma membrane. Basic phenotypic analyses were performed on haploid disruptants from both mating types of 10 non-essential genes. One disruptant (YNR004w) revealed a slow growth rate on glucose-minimal medium at 15 degrees C. For each of the individual ORFs, a disruption cassette and the corresponding cognate gene were cloned into appropriate plasmids.

Base Sequence↗

PubMed2002

Comprehensive essential gene identification as a platform for novel anti-infective drug discovery.

In large part, antimicrobial drug discovery is driven by the breadth and quality of both potential drug targets and available chemical libraries to screen. Traditionally, targets have been few in number and have been limited to those with known function, from which biochemical assays could be implemented into drug screens. Iterations of this same basic approach, applied to a few biochemically-defined targets have identified a limited set of novel antibiotics and even fewer antifungal agents. Indeed, in the last 50 years less than 30 antimicrobial targets have been exploited commercially. Within infectious disease, the industry was driven largely by chemistry-based approaches, simply making new analogs to existing drugs to overcome the growing problem of drug resistance. Elitra Pharmaceutical s approach has been to enable true functional genomics on a genome-wide scale. Elitra s vision has been to identify all of the essential genes directly in the key pathogenic organisms. Having moved rapidly towards the completion of this goal, we are now faced with the enviable challenge of prioritizing enormous target sets and developing novel sensitive screens for those best suited as definitive drug targets. These highly sensitive, cell-based screening paradigms enable re-screening of even well screened chemical libraries to reveal new chemical entities displaying novel modes of action against new targets. In parallel, we have also begun to shift the paradigm from screening targets singly, towards genome-wide approaches to drug screening.

Anti-Infective Agents↗

PubMed1999

Disruption and basic functional analysis of five chromosome X novel ORFs of Saccharomyces cerevisiae reveals YJL125c as an essential gene for vegetative growth.

We describe the disruption and basic functional analysis of five novel open reading frames (ORFs) discovered during the sequencing of the Saccharomyces cerevisiae genome: YJL118w, YJL122w, YJL123c, YJL124c, YJL125c, located on chromosome X. Disruptions have been realized using the long-flanking homology-PCR replacement strategy (LFH-PCR; Wach et al., 1996) in the FY1679 diploid strain. Sporulation and tetrad analysis of these heterozygous deletants were performed, as well as a functional analysis on the haploid deleted strains: different growth conditions (complete glucose and glycerol, minimal media) at three temperatures 15, 30 and 37 degrees C were tested. Analysis revealed YJL125c as an essential gene; the four other ORFs were non-essential and showed no particular phenotype. In addition, the five kanMX4 disruption cassettes were cloned in pUG7 vector. Finally, the five ORFs with their promoter and terminator regions were cloned in the centromeric yeast vector pRS416. The vectors containing the disruption cassettes, the cognate wild-type genes, as well as the deletant strains are available at the EU EUROFAN (EUROSCARF, Frankfurt, DE) genetic and stock centre.

Chromosomes, Fungal↗

PubMed1996

Characterization of the essential gene glmM encoding phosphoglucosamine mutase in Escherichia coli.

Two different approaches to identify the gene encoding the phosphoglucosamine mutase in Escherichia coli were used: (i) the purification to near homogeneity of this enzyme from a wild type strain and the determination of its N-terminal amino acid sequence; (ii) the search in data bases of an E. coli protein of unknown function showing sequence similarities with other hexosephosphate mutase activities. Both investigations revealed the same open reading frame named yhbF located within the leuU-dacB region at 69.5 min on the chromosome (Dallas, W. S., Dev, I. K., and Ray, P. H. (1993) J. Bacteriol. 175, 7743-7744). The predicted 445-residue protein with a calculated mass of 47.5 kDa contained in particular a short region GIVISASHNP with high similarity to the putative active site of hexosephosphate mutases. In vitro assays showed that the overexpression of this gene in E. coli cells led to a significant overproduction (from 15- to 50-fold) of phosphoglucosamine mutase activity. A hexose 1,6-diphosphate-dependent phosphorylation of the enzyme, which probably involves the serine residue at position 102, is apparently required for its catalytic action. As expected, the inactivation of this gene, which is essential for bacterial growth, led to the progressive depletion of the pools of precursors located downstream from glucosamine 1-phosphate in the pathway for peptidoglycan synthesis. This was followed by various alterations of cell shape and finally cells were lysed when their peptidoglycan content decreased to a critical value corresponding to about 60% of its normal level. The gene for this enzyme, which is essential for peptidoglycan and lipopolysaccharide biosyntheses, has been designated glmM.

Amino Acid Sequence↗

PubMed1982

Essential genes and deficiencies in the unc-22 IV region of Caenorhabditis elegans.

Five formaldehyde-induced deficiencies that uncover unc-22 IV, a gene affecting muscle structure in the nematode Caenorhabditis elegans were isolated and positioned. The largest deficiency, sDf2, extends in both directions from unc-22 and is approximately 1.0-2.0 map units in length. The other four deficiencies, sDf7, sDf8, sDf9 and sDf10, are all smaller than sDf2 and are located within the region uncovered by this deficiency. Thirty-seven ethyl methane-sulfonate-induced lethal and sterile mutations linked to unc-22 were isolated and tested for complementation with sDf2. Nineteen lethal mutations failed to complement sDf2. Sixteen of these were further positioned by recombination mapping and also by deficiency mapping with sDf7, sDf8, sDf9 and sDf10. These sixteen mutations define 11 new essential genes in this region. Eight of the genes lie in a 0.9-map unit interval to the left of unc-22, whereas the three remaining genes lie in a region of about 0.2 map units to the right of unc-22. We believe that two of the essential genes identified in this study, let-56 and let-52, are the adjacent genes on either side of unc-22. The lethal mutations exhibit a wide range of terminal phenotypes: from first stage larva to sterile adult.

Animals↗

PubMed2006

A survey of essential gene function in the yeast cell division cycle.

Mutations impacting specific stages of cell growth and division have provided a foundation for dissecting mechanisms that underlie cell cycle progression. We have undertaken an objective examination of the yeast cell cycle through flow cytometric analysis of DNA content in TetO(7) promoter mutant strains representing 75% of all essential yeast genes. More than 65% of the strains displayed specific alterations in DNA content, suggesting that reduced function of an essential gene in most cases impairs progression through a specific stage of the cell cycle. Because of the large number of essential genes required for protein biosynthesis, G1 accumulation was the most common phenotype observed in our analysis. In contrast, relatively few mutants displayed S-phase delay, and most of these were defective in genes required for DNA replication or nucleotide metabolism. G2 accumulation appeared to arise from a variety of defects. In addition to providing a global view of the diversity of essential cellular processes that influence cell cycle progression, these data also provided predictions regarding the functions of individual genes: we identified four new genes involved in protein trafficking (NUS1, PHS1, PGA2, PGA3), and we found that CSE1 and SMC4 are important for DNA replication.

Alleles↗