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Results for “Genes, Essential”

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PubMed1995

STT3, a novel essential gene related to the PKC1/STT1 protein kinase pathway, is involved in protein glycosylation in yeast.

Mutations of genes involved in the STT1/PKC1 pathway in yeast show staurosporine and temperature sensitivities (stt) which are suppressed by the addition of 1 M sorbitol [Yoshida et al., Mol. Gen. Genet. 242 (1994) 631-640]. Among the stt mutants, stt3-2 shares this phenotype. The STT3 gene encodes a novel 718-amino-acid protein with significant homology to potential transmembrane proteins of Caenorhabditis elegans and mouse mandibular condyle (about 80% homologous and 60% identical). Unlike the STT1/PKC1 gene, STT3 is essential for cell growth irrespective of osmotic support. Pulse-chase experiments show that the sst3 mutants are defective in protein glycosylation. The stt3 mutants are sensitive to hygromycin B and resistant to sodium orthovanadate, whose phenotypes are common to those defective in protein glycosylation.

Alkaloids↗

PubMed1995

A mutation in CSE4, an essential gene encoding a novel chromatin-associated protein in yeast, causes chromosome nondisjunction and cell cycle arrest at mitosis.

The centromere, a differentiated region of the eukaryotic chromosome, mediates the segregation of sister chromatids at mitosis. In this study, a Saccharomyces cerevisiae chromosome mis-segregation mutant, cse4-1, has been isolated and shown to increase the nondisjunction frequency of a chromosome bearing a mutant centromere DNA sequence. In addition, at elevated temperatures the cse4-1 allele causes a mitosis-specific arrest with a predominance of large budded cells containing single G2 nuclei and short bipolar mitotic spindles. The wild-type gene, CSE4, is essential for cell division and encodes a protein containing a domain that is 64% identical to the highly conserved chromatin protein, histone H3. Biochemical experiments demonstrate that CSE4p has similar DNA-binding characteristics as those of histone H3 and might form a specialized nucleosome structure in vivo. Interestingly, the human centromere protein, CENP-A, also contains this H3-like domain. Data presented here indicate that CSE4p is required for proper kinetochore function in yeast and may represent an evolutionarily conserved protein necessary for assembly of the unique chromatin structure associated with the eukaryotic centromere.

Amino Acid Sequence↗

PubMed1998

RPG1: an essential gene of saccharomyces cerevisiae encoding a 110-kDa protein required for passage through the G1 phase.

In Saccharomyces cerevisiae cells a number of genes are required for progression through, or else to pass beyond, the G1 phase. We characterized a novel gene, RPG1, which is also involved in this phase. RPG1 is an essential gene encoding a 110-kDa evolutionarily conserved protein. Elutriated or alpha-factor-synchronized cells of the rpg1-1 temperature-sensitive mutant were arrested in the first cell cycle when shifted to a non-permissive temperature. The cells remained unbudded and neither grew nor duplicated DNA. rpg1-1 cells synchronized in S phase completed mitosis and arrested as unseparated G1 cells after a shift to a non-permissive temperature. Similarly, the asynchronous rpg1-1 cells accumulated in G1 at the non-permissive temperature, but mother and daughter cells did not separate. A bulk of Calcofluor-stained material was localized in the region adjacent to the cell septum. Our data show that Rpg1p is required for passage through the G1 phase and may be involved in growth control. Data published recently indicate that Rpg1p exhibits significant sequence similarity to a subunit of the mammalian translation initiation factor 3.

Amino Acid Sequence↗

PubMed2000

RRS1, a conserved essential gene, encodes a novel regulatory protein required for ribosome biogenesis in Saccharomyces cerevisiae.

A secretory defect causes specific and significant transcriptional repression of both ribosomal protein and rRNA genes (K. Mizuta and J. R. Warner, Mol. Cell. Biol. 14:2493-2502, 1994), suggesting the coupling of plasma membrane and ribosome syntheses. In order to elucidate the molecular mechanism of the signaling pathway, we isolated a cold-sensitive mutant with a mutation in a gene termed RRS1 (regulator of ribosome synthesis), which appeared to be defective in the signaling pathway. The rrs1-1 mutation greatly reduced transcriptional repression of both rRNA and ribosomal protein genes that is caused by a secretory defect. RRS1 is a novel, essential gene encoding a nuclear protein of 203 amino acid residues that is conserved in eukaryotes. A conditional rrs1-null mutant was constructed by placing RRS1 under the control of the GAL1 promoter. Rrs1p depletion caused defects in processing of pre-rRNA and assembly of ribosomal subunits.

Amino Acid Sequence↗

PubMed1992

Identification of acoR, a regulatory gene for the expression of genes essential for acetoin catabolism in Alcaligenes eutrophus H16.

Two hundred thirty-nine base pairs upstream from acoXABC, which encodes the Alcaligenes eutrophus H16 structural genes essential for cleavage of acetoin, the 2,004-bp acoR gene was identified. acoR encodes a protein of 668 amino acids with a molecular mass of 72.9 kDa. The amino acid sequence deduced from acoR exhibited homologies to the primary structures of transcriptional activators such as NifA of Azotobacter vinelandii, NtrC of Klebsiella pneumoniae, and HoxA of A. eutrophus. Striking similarities to the central domain of these proteins and the presence of a typical nucleotide-binding site (GETGSGK) as well as of a C-terminal helix-turn-helix motif as a DNA-binding site were revealed. Between acoR and acoXABC, two different types of sequences with dual rotational symmetry [CAC-(N11 to N18)-GTG and TGT-(N10 to N14)-ACA] were found; these sequences are similar to NtrC and NifA upstream activator sequences, respectively. Determination of the N-terminal amino acid sequence of an acoR'-'lacZ gene fusion identified the translational start of acoR. S1 nuclease protection assay identified the transcriptional start site 109 bp upstream of acoR. The promoter region (TTGCGC-N18-TACATT) resembled the sigma 70 consensus sequence of Escherichia coli. Analysis of an acoR'-'lacZ fusion and primer extension studies revealed that acoR was expressed at a low level under all culture conditions, whereas acoXABC was expressed only in acetoin-grown cells. The insertions of Tn5 in six transposon-induced acetoin-negative mutants of A. eutrophus were mapped within acoR. On the basis of these studies, it is probable that AcoR represents a regulatory protein which is required for sigma 54-dependent transcription of acoXABC.

Acetoin↗

PubMed1992

A new essential gene of Saccharomyces cerevisiae, a defect in it may result in instability of nucleus.

We identified a new essential gene of Saccharomyces cerevisiae, designated NIN1 (nuclear integrity). The nin1-1 mutant cells showed temperature-sensitive growth, and their nuclei underwent deformation and eventually were broken down upon a temperature shift. When the nin1-1 cells growing at 25 degrees C were transferred to a nonpermissive temperature, the nucleus moved to the isthmus and cells with a large bud accumulated. Flow cytometry proved that G2 nuclei accumulated during incubation at a nonpermissive temperature. NIN1 is mapped on chromosome VI, 16 cM centromere-distal to PHO4. Cloning and sequencing of NIN1 revealed that this is a new gene. Disruption of NIN1 resulted in cell death; however, a dead spore clone contained about 30 dead cells, indicating that spores that received the disrupted nin1 allele divided approximately five times before their death. Western blotting using anti-Nin1 antibody reveals that NIN1 produces a doublet consisting of 32 and 34 kDA whose size is close enough to that calculated from the predicted amino acid sequence of Nin1.

Amino Acid Sequence↗

PubMed2002

Marker-fusion PCR for one-step mutagenesis of essential genes in yeast.

We describe a one-step gene replacement method based on fusion PCR that can be used to mutagenize essential genes at their endogenous locus. Marker-fusion PCR can facilitate transfer of alleles between strains as well as PCR-based techniques, such as site-directed and error-prone PCR mutagenesis, all without cloning or strain constructions. With this method, PCR is used to fuse a mutagenized fragment to an overlapping fragment containing a selectable marker flanked by regions of homology to the target. By transforming yeast with these PCR products, specific mutations are introduced at the endogenous locus through homologous recombination. We tested the 'marker-fusion PCR' method using the budding yeast CDC28 gene and were able to efficiently introduce site-directed mutations and integrate genomic or plasmid-borne mutant alleles. As a further application for this method, we used a spiked oligonucleotide to randomize the coding sequence for a single domain of CDC28 and were able to construct highly mutagenized libraries for this region.

Genes, Fungal↗

PubMed1982

Deletion analysis of essential genes of Escherichia coli: investigation of the btuB-rpoBC interval.

A method is described for deletion mapping of essential genes in Escherichia coli. It involves the isolation of secondary-site insertions of lambda cI857plac5 into an F' plasmid. The transposed lacZ gene is useful both for the ready screening of plasmid-phage cointegrates and for rapid analysis of deletions that extend from the site of phage integration into the bacterial genes carried on the substituted plasmid. Such deletions may be used to 'hook-up' bacterial cistrons to the powerful lac promoter. We report the application of this technique to the study of the btuB-rpoBC interval, a cluster of genes encoding components of the transcription-translation apparatus.

Bacteriophage lambda↗

PubMed1990

SAS1 and SAS2, GTP-binding protein genes in Dictyostelium discoideum with sequence similarities to essential genes in Saccharomyces cerevisiae.

We have identified two novel, very closely related genes, SAS1 and SAS2, from Dictyostelium discoideum. These encode small, approximately 20-kilodaton proteins with amino acid sequences thought to be involved in interaction with guanine nucleotides. The protein sizes, spacings of GTP-binding domains, and carboxyl-terminal sequences suggest their relationship to the ubiquitous ras-type proteins. Their sequences, however, are sufficiently different to indicate that they are not true ras proteins. More extensive sequence identity (approximately 55%) is shared with the YPT1 and SEC4 proteins from Saccharomyces cerevisiae. These yeast proteins are essential for growth and are believed to be involved in intracellular signaling associated with membrane function. SAS1 and SAS2 exhibit distinct patterns of genomic organization and developmentally regulated gene expression. SAS1 contains introns and is associated with a developmentally regulated repetitive element. SAS2 is colinear with its mRNA and does not appear to be closely linked with this repetitive element. Both genes are expressed during growth and throughout development. SAS1 is maximally expressed during cytodifferentiation, when two sizes of SAS1 mRNA are detectable. SAS2 mRNA levels are maximal during culmination. On the basis of the expression patterns of the SAS genes and their relationship to the YPT1 and SEC4 genes, we discuss possible functions of the SAS proteins.

Amino Acid Sequence↗

PubMed1994

Amino acid substitution mutations in the herpes simplex virus ICP27 protein define an essential gene regulation function.

ICP27 is an essential herpes simplex virus type 1 (HSV-1) alpha protein that is required for the transition from the beta to the gamma phase of infection. To identify functional regions of ICP27, we constructed 16 plasmids that contain nucleotide substitution mutations in the ICP27 gene. The mutations created XhoI restriction sites, altered one or two codons, and were spaced at semiregular intervals throughout the coding region. Three mutations completely inactivated an essential function of ICP27, as demonstrated by the inability of the transfected plasmids to complement the growth of an HSV-1 ICP27 deletion mutant. These mutations, M11, M15, and M16, mapped in the carboxyl-terminal one-third of ICP27 at residues 340 and 341, 465 and 466, and 488, respectively. In cotransfection assays, all three defective-plasmid mutants retained the transrepression function of ICP27 but were defective at transactivation. To define the lytic functions that are mediated by the transactivation activity of ICP27, we engineered HSV-1 recombinants containing the M11, M15, or M16 mutation. All three viral mutants failed to grow in Vero cells and possessed similar phenotypes. The viral mutants replicated their DNA similarly to the wild-type virus but showed several defects in viral gene expression. These were a failure to down-regulate alpha and beta genes at late times after infection and an inability to induce certain gamma-2 genes. Our results demonstrate that the transactivation function of ICP27 (as it is defined in cotransfection assays) mediates an essential gene regulation function during the HSV-1 infection. This activity is not required for ICP27-dependent enhancement of viral DNA replication. Our work supports and extends previous studies which suggest that ICP27 carries out two distinct regulatory activities during the HSV-1 infection.

Amino Acids↗

PubMed1999

Zimp encodes a homologue of mouse Miz1 and PIAS3 and is an essential gene in Drosophila melanogaster.

The related mouse proteins Miz1 and PIAS3, which have predicted zinc finger domains, interact with the transcription factors Msx2 and STAT3, modulating the ability of Msx2 and STAT3 to regulate transcription. Here, we describe a Drosophila gene, zimp, that encodes a protein with similarity to Miz1 and PIAS3. The zimp gene appears to be post-transcriptionally regulated, as three alternatively spliced forms are detected in a cDNA library screen and on an RNA blot. In addition, all three zimp transcripts are detected in embryonic mRNA, but only two of the transcripts are detected in adult mRNA. The three transcripts have the ability to encode two proteins, of 554 and 522 amino acids. The two Zimp amino acid sequences share an amino-terminal 515-amino-acid region and differ in their carboxy-termini. These proteins and related proteins in other organisms, including mammals, C. elegans, yeast, and plants, share a highly conserved region predicted to form a zinc finger. Deletion of the zimp gene or P-element insertion in zimp is lethal; thus, zimp is an essential gene in Drosophila. These data underscore the potential importance of Zimp-related proteins cross-species, and conservation of the putative zinc finger domain suggests that it is functionally important.

Alternative Splicing↗

PubMed1990

SPT6, an essential gene that affects transcription in Saccharomyces cerevisiae, encodes a nuclear protein with an extremely acidic amino terminus.

SPT6 is an essential gene of Saccharomyces cerevisiae that appears to play a role in transcription. Mutations in the SPT6 (SSN20, CRE2) gene suppress delta insertion mutations in the 5' regions of HIS4 and LYS2 and mutations in cis- and/or trans-acting elements that are required for expression of SUC2 and ADH2. We report here that SPT6 encodes a 170-kilodalton highly charged protein with an extremely acidic amino terminus. By use of an epitope-tagged SPT6 protein, we have determined by indirect immunofluorescence that the SPT6 protein is located in the nucleus.

Amino Acid Sequence↗

PubMed1995

SMC2, a Saccharomyces cerevisiae gene essential for chromosome segregation and condensation, defines a subgroup within the SMC family.

We characterized the SMC2 (structural maintenance of chromosomes) gene that encodes a new Saccharomyces cerevisiae member of the growing family of SMC proteins. This family of evolutionary conserved proteins was introduced with identification of SMC1, a gene essential for chromosome segregation in budding yeast. The analysis of the putative structure of the Smc2 protein (Smc2p) suggests that it defines a distinct subgroup within the SMC family. This subgroup includes the ScII, XCAPE, and cut14 proteins characterized concurrently. Smc2p is a nuclear, 135-kD protein that is essential for vegetative growth. The temperature-sensitive mutation, smc2-6, confers a defect in chromosome segregation and causes partial chromosome decondensation in cells arrested in mitosis. The Smc2p molecules are able to form complexes in vivo both with Smc1p and with themselves, suggesting that they can assemble into a multimeric structure. In this study we present the first evidence that two proteins belonging to two different subgroups within the SMC family carry nonredundant biological functions. Based on genetic, biochemical, and evolutionary data we propose that the SMC family is a group of prokaryotic and eukaryotic chromosomal proteins that are likely to be one of the key components in establishing the ordered structure of chromosomes.

Amino Acid Sequence↗

PubMed1988

Operon structure of dnaT and dnaC genes essential for normal and stable DNA replication of Escherichia coli chromosome.

dnaT and dnaC of Escherichia coli, whose products are components of the primosome, encode genes essential for both normal chromosomal replication and stable DNA replication induced by SOS signals. dnaT and dnaC together with an unknown gene (P-18), which encodes an 18-kDa polypeptide present downstream of dnaC, are cotranscribed from a promoter located 104 base pairs upstream of dnaT. A portion of the transcripts are processed 16 or 27 base pairs upstream of the dnaC coding region and 20% of the transcripts extending through dnaC are terminated at a terminator 103 base pairs downstream of dnaC. The rest of the transcripts pass through this terminator and are terminated downstream of the P-18 gene. The predicted amino acid sequence of the P-18 protein indicate the presence of a 26-amino acid signal peptide at its N terminus, suggesting that this protein may be secreted into the membrane or a periplasmic space. Another gene, which encodes a 14-kDa hydrophobic protein (P-14), was identified upstream of dnaT. The P-14 gene is transcribed from an upstream promoter, and its transcript extends through dnaT and dnaC. These results indicate that dnaT and dnaC constitute an operon with two unknown genes whose products may be associated with a membrane.

Amino Acid Sequence↗

PubMedDate not supplied

A new essential gene of the 'minimal genome' affecting cell division.

The complete sequencing of bacterial genomes has offered new opportunities for the identification of essential genes involved in the control and progression of the cell cycle. For this purpose, we have disrupted ten E. coli genes belonging to the so-called 'minimal genome'. One of these genes, yihA, was necessary for normal cell division. The yihA gene possesses characteristic GTPase motifs and its homologues are present in eukaryotes, archaea and most prokaryotes. Depletion of YihA protein led to a severe reduction in growth rate and to extensive filamentation, with a block beyond the stage of nucleoid segregation. Filamentation was correlated with reduced FtsZ levels and could be specifically suppressed by overexpression of ftsQI, ftsA and ftsZ, and to some extent by ftsZ alone. We hypothesize that YihA, like the Era GTPase, may participate in a checkpoint mechanism that ensures a correct coordination of cell cycle events.

Amino Acid Sequence↗

PubMed2026

Identification of a putative RocS homolog through phenotypic profiling of uncharacterized essential genes in Streptococcus mutans.

Genome-wide viability catalogs produced by transposon sequencing (Tn-seq) and CRISPR interference (CRISPRi) have successfully mapped the essential genome of Streptococcus mutans . In this study, we combined predictive bioinformatics, conditional CRISPRi transcriptional silencing, transmission electron microscopy, transcriptomics, and genetic suppressor screens to investigate nine poorly characterized essential genes in S. mutans . From this screen, phenotypic and genetic analyses identified SMU_393 as a functional homolog of the pneumococcal chromosome segregation factor, RocS. Depletion of SMU_393 resulted in abnormal cell widening, hypersensitivity to DNA damage, and a significant subpopulation of anucleate cells. These phenotypes were bypassed by a spontaneous surface-exposed missense mutation ( dnaA Q197E ) within the AAA+ ATPase domain of the replication initiator. Together, this study refines annotations within the S. mutans essential genome and provides genetic insights into streptococcal chromosome segregation and cell cycle control.

Journal Article↗

PubMed1998

An Escherichia coli gene (yaeO) suppresses temperature-sensitive mutations in essential genes by modulating Rho-dependent transcription termination.

An extragenic multicopy suppressor of the cell division inhibition caused by a MalE-MinE fusion protein in Escherichia coli has been mapped and identified as yaeO, one of the two short open reading frames (ORFs) of an operon located at 4.6 min. Overexpressed yaeO also suppressed some temperature-sensitive mutations in division genes ftsA and ftsQ, in chaperone gene groEL and in co-chaperone gene grpE. Gene yaeO, whose expression is regulated by growth rate, codes for a 9 kDa acidic protein with no obvious resemblance to other proteins. Transcription termination protein Rho co-purified with a histidine-tagged derivative of YaeO protein on Ni2+-NTA agarose columns in a manner that suggested direct YaeO-Rho interaction. In vivo, yaeO expression reduced termination at rho-dependent bacteriophage terminator tL1 and at the terminator of autogenously regulated gene rho. The suppression of temperature-sensitive phenotypes was a consequence of anti-termination, as it could be mimicked by a Prho::Tn10 mutation that reduces the expression and activity of gene rho. Our data indicate that the suppression is not caused by overexpression of the mutated genes, but presumably by indirect stabilization of the mutated proteins.

ATP-Binding Cassette Transporters↗

PubMed2001

Eubacterial diterpene cyclase genes essential for production of the isoprenoid antibiotic terpentecin.

A gene cluster containing the mevalonate pathway genes (open reading frame 2 [ORF2] to ORF7) for the formation of isopentenyl diphosphate and a geranylgeranyl diphosphate (GGDP) synthase gene (ORF1) had previously been cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibiotic, terpentecin (TP) producer (Y. Hamano, T. Dairi, M. Yamamoto, T. Kawasaki, K Kaneda, T. Kuzuyama, N. Itoh, and H. Seto, Biosci. Biotech. Biochem. 65:1627-1635, 2001). Sequence analysis in the upstream region of the cluster revealed seven new ORFs, ORF8 to ORF14, which were suggested to encode TP biosynthetic genes. We constructed two mutants, in which ORF11 and ORF12, which encode a protein showing similarities to eukaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were inactivated by gene disruptions. The mutants produced no TP, confirming that these cyclase genes are essential for the production of TP. The two cyclase genes were also expressed in Streptomyces lividans together with the GGDP synthase gene under the control of the ermE* constitutive promoter. The transformant produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpentetriene), which has the same basic skeleton as TP. The two enzymes, each of which was overproduced in Escherichia coli and purified to homogeneity, converted GGDP into terpentetriene. To the best of our knowledge, this is the first report of a eubacterial DC.

Amino Acid Sequence↗