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Results for “Genes, Essential”

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PubMed2006

Characterization of lptA and lptB, two essential genes implicated in lipopolysaccharide transport to the outer membrane of Escherichia coli.

The outer membrane (OM) of gram-negative bacteria is an asymmetric lipid bilayer that protects the cell from toxic molecules. Lipopolysaccharide (LPS) is an essential component of the OM in most gram-negative bacteria, and its structure and biosynthesis are well known. Nevertheless, the mechanisms of transport and assembly of this molecule in the OM are poorly understood. To date, the only proteins implicated in LPS transport are MsbA, responsible for LPS flipping across the inner membrane, and the Imp/RlpB complex, involved in LPS targeting to the OM. Here, we present evidence that two Escherichia coli essential genes, yhbN and yhbG, now renamed lptA and lptB, respectively, participate in LPS biogenesis. We show that mutants depleted of LptA and/or LptB not only produce an anomalous LPS form, but also are defective in LPS transport to the OM and accumulate de novo-synthesized LPS in a novel membrane fraction of intermediate density between the inner membrane (IM) and the OM. In addition, we show that LptA is located in the periplasm and that expression of the lptA-lptB operon is controlled by the extracytoplasmic sigma factor RpoE. Based on these data, we propose that LptA and LptB are implicated in the transport of LPS from the IM to the OM of E. coli.

Bacterial Outer Membrane Proteins↗

PubMed2002

Regulation of capsule synthesis and cell motility in Salmonella enterica by the essential gene igaA.

Mutants of Salmonella enterica carrying the igaA1 allele, selected as able to overgrow within fibroblast cells in culture, are mucoid and show reduced motility. Mucoidy is caused by derepression of wca genes (necessary for capsule synthesis); these genes are regulated by the RcsC/YojN/RcsB phosphorelay system and by the RcsA coregulator. The induction of wca expression in an igaA1 mutant is suppressed by mutations in rcsA and rcsC. Reduced motility is caused by lowered expression of the flagellar master operon, flhDC, and is suppressed by mutations in rcsB or rcsC, suggesting that mutations in the igaA gene reduce motility by activating the RcsB/C system. A null igaA allele can be maintained only in an igaA(+)/igaA merodiploid, indicating that igaA is an essential gene. Lethality is suppressed by mutations in rcsB, rcsC, and yojN, but not in rcsA, suggesting that the viability defect of an igaA null mutant is mediated by the RcsB/RcsC system, independently of RcsA (and therefore of the wca genes). Because all the defects associated with igaA mutations are suppressed by mutations that block the RcsB/RcsC system, we propose a functional interaction between the igaA gene product and either the Rcs regulatory network or one of its regulated products.

Animals↗

PubMed1988

Structure and expression of yeast DPR1, a gene essential for the processing and intracellular localization of ras proteins.

The ras proteins represent a unique example of membrane proteins which apparently do not utilize the secretory pathway for their membrane localization. Instead, it is believed that palmitic acid, covalently attached to the protein, acts as an anchor to the membranes. Recent identification of yeast mutants defective in the processing of the ras proteins has provided a novel approach for defining these biosynthetic processes. We report here the characterization of yeast DPR1, a gene essential for the processing of the ras proteins. The sequence of the gene indicates that it encodes a protein of 431 amino acids which contains no significant homology with any known proteins. It is a relatively hydrophilic protein with no apparent hydrophobic stretches. The C-terminal half of the encoded protein has an unusually high content of cysteine. The DPR1 gene product has been identified in a cell-free translation system as a protein having an apparent molecular weight of 43 kd. This represents the first step in the investigation of a novel protein-processing pathway, one that is distinct from the secretory pathway.

Amino Acid Sequence↗

PubMed1990

Molecular cloning of the DAC2/FUS3 gene essential for pheromone-induced G1-arrest of the cell cycle in Saccharomyces cerevisiae.

Mating pheromones, known as a and alpha-factors, arrest the division of cells of opposite mating types, alpha and a respectively, in Saccharomyces cerevisiae. I have cloned the DAC2 gene, which is required for both pheromone-induced division-arrest and cell-fusion during conjugation. The constructed dac2::LEU2 null mutation leads to defects in both pheromone-induced division-arrest and cell-fusion during conjugation; it also suppresses the growth defect caused by the gpa1 mutation (a mutation in the alpha subunit of the S. cerevisiae G protein). These results indicate that DAC2 may be the same gene as FUS3, which was recently isolated by Elion et al. (1990) as a gene essential for cell-fusion during conjugation. The dac2::LEU2 null mutant also showed morphological alterations in response to mating pheromones. I show here that the DAC2 product plays an essential role in both the division-arrest signalling pathway of the yeast pheromone response and in cell-fusion during conjugation.

Cloning, Molecular↗

PubMed2006

Rapid identification of non-essential genes for in vitro replication of Marek's disease virus by random transposon mutagenesis.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphomas in poultry. The MDV genome encodes more than 100 genes. However, the role of many of these genes in virus replication is not known. The construction of an infectious bacterial artificial chromosome (BAC) clone of the highly oncogenic RB-1B strain of MDV has been described previously. Virus reconstituted from the BAC clone induced rapid-onset lymphomas in chickens very similar to the wildtype viruses. In this paper, the construction of a high-density random transposon-insertion mutant library of the RB-1B BAC clone using a high throughput in vitro transposon mutagenesis technique is described. Furthermore a PCR screening method, using primers specific for the transposon sequence and the MDV gene(s) of interest, was developed for the rapid identification of specific insertion mutants. The application of the screening method to identify some of the non-essential genes for MDV replication in vitro is described.

Chromosomes, Artificial, Bacterial↗

PubMed2005

Genome-wide survey of non-essential genes required for slowed DNA synthesis-induced filamentous growth in yeast.

We recently discovered that slowed DNA synthesis induces filamentous differentiation in S. cerevisiae. We screened the BY yeast deletion strains and identified four classes of non-essential genes that are required for both slowed DNA-induced filamentous growth and classic forms of filamentous growth: (a) genes encoding regulators of the actin cytoskeleton and cell polarity, ABP1, CAP2 and HUF1 (=YOR300W), in addition to the previously known BNI1, BUD2, PEA2, SPA2 and TPM1; (b) genes that are likely involved in cell wall biosynthesis, ECM25, GAS1 and PRS3; (c) genes encoding possible regulators of protein secretion, SEC66, RPL21A and RPL34B; (d) genes encoding factors for normal mitochondrial function, IML1 and UGO1. These results showed that pseudohyphal formation involves not the only previously known regulation of the actin cytoskeleton/cell polarity but also regulation of cell wall synthesis, protein secretion and mitochondrial function. Identification of multiple classes of genes that are required for both slowed DNA synthesis-induced and classic forms of filamentous growth confirms that slowed DNA synthesis-induced filamentous growth is bone fide filamentous differentiation.

Actins↗

PubMed1999

Saccharomyces cerevisiae GNA1, an essential gene encoding a novel acetyltransferase involved in UDP-N-acetylglucosamine synthesis.

The Saccharomyces cerevisiae gene, YFL017C, for a putative acetyltransferase was characterized. Disruption of YFL017C was lethal, leading to a morphology similar to those caused by the depletion of AGM1 or UAP1, the genes encoding phospho-N-acetylglucosamine mutase and UDP-N-acetylglucosamine pyrophosphorylase, respectively. This implies the involvement of YFL017C in UDP-N-acetylglucosamine synthesis. The recombinant protein for YFL017C displayed phosphoglucosamine acetyltransferase activities in vitro and utilized glucosamine 6-phosphate as the substrate. When incubated with Agm1p and Uap1p, the Yfl017c protein produced UDP-N-acetylglucosamine from glucosamine 6-phosphate. These results indicate that YFL017C specifies glucosamine-6-phosphate acetyltransferase; therefore, the gene was designated GNA1 (glucosamine-6-phosphate acetyltransferase). In addition, whereas bacterial phosphoglucosamine acetyltransferase and UDP-N-acetylglucosamine pyrophosphorylase activities are intrinsic in a single polypeptide, they are encoded by distinct essential genes in yeast. When the sequence of ScGna1p was compared with those of other acetyltransferases, Ile97, Glu98, Val102, Gly112, Leu115, Ile116, Phe142, Tyr143, and Gly147 were found to be highly conserved. When alanine was substituted for these amino acids, the enzyme activity for the substituted Phe142 or Tyr143 enzymes was severely diminished. Although the activity of Y143A was too low to perform kinetics, F142A displayed a significantly increased Km value for acetyl-CoA, suggesting that the Phe142 and Tyr143 residues are essential for the catalysis.

Acetyltransferases↗

PubMed2003

A Kluyveromyces lactis mutant in the essential gene KlLSM4 shows phenotypic markers of apoptosis.

We report the study of Kluyveromyces lactis cells expressing a truncated form of KlLSM4, a gene ortholog to LSM4 of Saccharomyces cerevisiae which encodes an essential protein involved in both pre-mRNA splicing and mRNA decapping. We had previously demonstrated that the first 72 amino acids of the K. lactis Lsm4p (KlLsm4Deltap) can restore cell growth in both K. lactis and S. cerevisiae cells not expressing the endogenous protein. However, cells showed a remarkable loss of viability in stationary phase. Here we report that cells expressing KlLsm4Deltap presented clear apoptotic markers such as chromatin condensation, DNA fragmentation, accumulation of reactive oxygen species, and showed increased sensitivity to different drugs. RNA analysis revealed that pre-mRNA splicing was almost normal while mRNA degradation was significantly delayed, pointing to this as the possible step responsible for the observed phenotypes.

Apoptosis↗

PubMed1997

AUT1, a gene essential for autophagocytosis in the yeast Saccharomyces cerevisiae.

Autophagocytosis is a starvation-induced process responsible for transport of cytoplasmic proteins to the vacuole. In Saccharomyces cerevisiae, autophagy is characterized by the phenotypic appearance of autophagic vesicles inside the vacuole of strains deficient in proteinase yscB. The AUT1 gene, essential for autophagy, was isolated by complementation of the sporulation deficiency of a diploid aut1-1 mutant strain by a yeast genomic library and characterized. AUT1 is located on the right arm of chromosome XIV, 10 kb from the centromere, and encodes a protein of 310 amino acids, with an estimated molecular weight of 36 kDa. Cells carrying a chromosomal deletion of AUT1 are defective in the starvation-induced bulk flow transport of cytoplasmic proteins to the vacuole. aut1 null mutant strains are completely viable but show decreased survival rates during starvation. Homozygous delta aut1 diploid cells fail to sporulate. The selective cytoplasm-to-vacuole transport of aminopeptidase I is blocked in logarithmically growing and in starved delta autl cells. Deletion of the AUT1 gene had no obvious influence on secretion, fluid phase endocytosis, or vacuolar protein sorting. This supports the idea of autophagocytosis as being a novel route transporting proteins from the cytoplasm to the vacuole.

Amino Acid Sequence↗

PubMed1994

The mating-type region of Schizosaccharomyces pombe contains an essential gene encoding a protein homologous to human modulators of HIV transactivation.

In Schizosaccharomyces pombe, an intrachromosomal crossover between the mating type (MT) expression locus and one of the silent donor cassettes is lethal due to the loss of the intervening L region. The region contains one essential gene, let1. This gene was cloned and sequenced. The deduced amino acid (aa) sequence of let1 shows extensive homologies with SUG1 from Saccharomyces cerevisiae. Significant homologies were also found with the human HIV transactivation modulators, MSS1 and TBP-1, as well as with subunit 4 of the mammalian 26 S protease. The data indicate that let1 is a member of a recently defined multigene family of ATPases.

Adenosine Triphosphatases↗

PubMed2000

Generation of recombinant parapoxviruses: non-essential genes suitable for insertion and expression of foreign genes.

Orf virus (OV) is an epitheliotropic poxvirus and belongs to the genus Parapoxvirus (PPV). PPV, especially OV, is regarded as a promising candidate for an expression vector. Among available live vaccines only strain D1701 represents a highly attenuated OV strain with clearly reduced pathogenicity. Therefore, we started to identify potentially non-essential genes or regions of D1701, which might be suitable for insertion and expression of foreign genes. The present contribution reviews some of the progress using the vegf-e (homologue of the mammalian vascular endothelial growth factor) gene locus for the generation of recombinant D1701. The vegf-e gene of D1701 is dispensable for virus growth in vitro and in vivo, and represents a major virulence determinant of OV. It is shown that foreign genes can be inserted and functionally expressed in the vegf-e locus, also leading to the induction of a specific immune response in the non-permissive host. Furthermore, it is reported that adaptation to VERO cells led to the deletion of three further regions of the OV D1701 genome, which seems to be combined with additional virus attenuation in sheep. Molecular analysis of this OV D1701 variant allows the identification of new, potentially non-essential sites in the viral genome.

Animals↗

PubMed1987

Revertants of v-fos-transformed fibroblasts have mutations in cellular genes essential for transformation by other oncogenes.

Morphologic revertants of FBJ murine sarcoma virus (v-fos)-transformed rat-1 fibroblasts were isolated using a novel selection procedure based on prolonged retention of rhodamine 123 within mitochondria of v-fos-transformed versus normal fibroblasts. Two classes of revertants were isolated: class I revertants have sustained mutations in cellular genes, and a class II revertant has a nonfunctional v-fos provirus. Somatic-cell hybridization studies suggested that the revertant phenotype was recessive to the transformed phenotype. Class I revertants were also resistant to retransformation by v-gag-fos-fox, v-Ha-ras, v-abl, and v-mos, but could be retransformed by the trk oncogene and polyoma virus middle T antigen. These results suggest that the class I revertants sustained mutations in one or more cellular genes essential for transformation by some, but not all, oncogenes. Our data suggest the existence of common biochemical pathways for transformation.

Animals↗

PubMed1990

Analysis of the fowlpox virus genome region corresponding to the vaccinia virus D6 to A1 region: location of, and variation in, non-essential genes in poxviruses.

The DNA sequence of the fowlpox virus genome corresponding to the vaccinia virus D6 to A1 region has been determined. Translation of this sequence reveals fowlpoxvirus gene homologues corresponding to the D6, D7, D9, D10, D11, D12, D13 and A1 genes of vaccinia virus. In contrast, no gene homologue for the non-essential vaccinia virus D8 gene was present in fowlpox virus. Instead, a gene transcribed from the opposite strand to the vaccinia virus D8 gene showing no homology to any previously sequenced poxvirus gene was present. The amino terminus of the fowlpox virus D9 homologue had undergone substantial changes, including frameshifts which would be predicted to inactivate the gene. Insertion of a gene cartridge composed of the vaccinia virus p7.5 promoter and the lacZ gene into the fowlpox virus D8, D9 and D10 genes in vitro, followed by recombination into fowlpox virus, was carried out. Stable insertion mutants with the correct genotype were obtained for D8 and D9 which, when tested in chickens did not appear to have been attenuated. No stable insertion mutants were obtained for D10, indicating that this gene probably encodes a function which is essential for virus replication. The D8 and D9 genes of fowlpox virus represent useful insertion sites for the construction of recombinant fowlpox virus vaccines.

Amino Acid Sequence↗

PubMed1998

Genes essential for nod factor production and nodulation are located on a symbiotic amplicon (AMPRtrCFN299pc60) in Rhizobium tropici.

Amplifiable DNA regions (amplicons) have been identified in the genome of Rhizobium etli. Here we report the isolation and molecular characterization of a symbiotic amplicon of Rhizobium tropici. To search for symbiotic amplicons, a cartridge containing a kanamycin resistance marker that responds to gene dosage and conditional origins of replication and transfer was inserted in the nodulation region of the symbiotic plasmid (pSym) of R. tropici CFN299. Derivatives harboring amplifications were selected by increasing the concentration of kanamycin in the cell culture. The amplified DNA region was mobilized into Escherichia coli and then into Agrobacterium tumefaciens. The 60-kb symbiotic amplicon, which we termed AMPRtrCFN299pc60, contains several nodulation and nitrogen fixation genes and is flanked by a novel insertion sequence ISRtr1. Amplification of AMPRtrCFN299pc60 through homologous recombination between ISRtr1 repeats increased the amount of Nod factors. Strikingly, the conjugal transfer of the amplicon into a plasmidless A. tumefaciens strain confers on the transconjugant the ability to produce R. tropici Nod factors and to nodulate Phaseolus vulgaris, indicating that R. tropici genes essential for the nodulation process are confined to an ampliable DNA region of the pSym.

Agrobacterium tumefaciens↗

PubMed1995

Molecular cloning of Rab-related genes in the yeast Yarrowia lipolytica. Analysis of RYL1, an essential gene encoding a SEC4 homologue.

Small GTP-binding proteins of the Rab family are involved in the vesicular traffic inside eukaryotic cells. A gene library from the yeast Yarrowia lipolytica was screened with an oligonucleotide deduced from a highly conserved sequence in the Rab family. Four different genes were isolated. One of them, RYL1, was shown to be essential for cell viability. RYL1p displayed a high similarity with and tight phylogenetic relationships to SEC4p. When placed under the control of the GAL10 promoter, RYL1 was able to specifically relieve the thermosensitivity of a sec4-8 mutant of Saccharomyces cerevisiae. Therefore, it is proposed that RYL1 is a functional homologue of the S. cerevisiae SEC4 gene and is involved in the fusion of secretory vesicles with the plasma membrane in the general protein secretion pathway.

Amino Acid Sequence↗

PubMed1999

The cardiac homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple genes essential for heart development.

Csx/Nkx2.5 is a vertebrate homeobox gene with a sequence homology to the Drosophila tinman, which is required for the dorsal mesoderm specification. Recently, heterozygous mutations of this gene were found to cause human congenital heart disease (Schott, J.-J., Benson, D. W., Basson, C. T., Pease, W., Silberbach, G. M., Moak, J. P., Maron, B. J., Seidman, C. E. and Seidman, J. G. (1998) Science 281, 108-111). To investigate the functions of Csx/Nkx2.5 in cardiac and extracardiac development in the vertebrate, we have generated and analyzed mutant mice completely null for Csx/Nkx2.5. Homozygous null embryos showed arrest of cardiac development after looping and poor development of blood vessels. Moreover, there were severe defects in vascular formation and hematopoiesis in the mutant yolk sac. Interestingly, TUNEL staining and PCNA staining showed neither enhanced apoptosis nor reduced cell proliferation in the mutant myocardium. In situ hybridization studies demonstrated that, among 20 candidate genes examined, expression of ANF, BNP, MLC2V, N-myc, MEF2C, HAND1 and Msx2 was disturbed in the mutant heart. Moreover, in the heart of adult chimeric mice generated from Csx/Nkx2.5 null ES cells, there were almost no ES cell-derived cardiac myocytes, while there were substantial contributions of Csx /Nkx2.5-deficient cells in other organs. Whole-mount &bgr;-gal staining of chimeric embryos showed that more than 20% contribution of Csx/Nkx2. 5-deficient cells in the heart arrested cardiac development. These results indicate that (1) the complete null mutation of Csx/Nkx2.5 did not abolish initial heart looping, (2) there was no enhanced apoptosis or defective cell cycle entry in Csx/Nkx2.5 null cardiac myocytes, (3) Csx/Nkx2.5 regulates expression of several essential transcription factors in the developing heart, (4) Csx/Nkx2.5 is required for later differentiation of cardiac myocytes, (5) Csx/Nkx2. 5 null cells exert dominant interfering effects on cardiac development, and (6) there were severe defects in yolk sac angiogenesis and hematopoiesis in the Csx/Nkx2.5 null embryos.

Animals↗

PubMed2002

Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondria-dependent apoptosis.

Thioredoxin-2 (Trx-2) is a mitochondria-specific member of the thioredoxin superfamily. Mitochondria have a crucial role in the signal transduction for apoptosis. To investigate the biological significance of Trx-2, we cloned chicken TRX-2 cDNA and generated clones of the conditional Trx-2-deficient cells using chicken B-cell line, DT40. Here we show that TRX-2 is an essential gene and that Trx-2-deficient cells undergo apoptosis upon repression of the TRX-2 transgene, showing an accumulation of intracellular reactive oxygen species (ROS). Cytochrome c is released from mitochondria, while caspase-9 and caspase-3, but not caspase-8, are activated upon inhibition of the TRX-2 transgene. In addition, Trx-2 and cytochrome c are co-immunoprecipitated in an in vitro assay. These results suggest that mitochondrial Trx-2 is essential for cell viability, playing a crucial role in the scavenging ROS in mitochondria and regulating the mitochondrial apoptosis signaling pathway.

Amino Acid Sequence↗

PubMed1987

GPA1, a haploid-specific essential gene, encodes a yeast homolog of mammalian G protein which may be involved in mating factor signal transduction.

GPA1 protein of Saccharomyces cerevisiae is homologous to the alpha subunit of mammalian G protein. GPA1 transcript was found in haploid cells but was not detected in diploid cells. Disruption of GPA1 resulted in a haploid-specific lethal phenotype, indicating that GPA1 is a haploid-specific essential gene for cell growth. Upon regulation of expression of GPA1 by the galactose-inducible GAL1 promoter, the loss of GPA1 function was found to lead to cell-cycle arrest at the late G1 phase. Mutants that suppress the lethality of the gpa1::HIS3 mutation showed a sterile phenotype that was not cell-type-specific. These results suggest that GPA1 protein may control the signal for mating-factor-mediated cell-cycle arrest.

Cell Cycle↗