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PubMed1994

Requiem: a novel zinc finger gene essential for apoptosis in myeloid cells.

To identify genes mediating programmed cell death triggered by interleukin 3 (IL-3)-deprivation of myeloid cells, the IL-3-dependent murine myeloid cell line FDCP-1 was used to screen a mammalian cell expression library for cDNAs that would promote survival following withdrawal of IL-3. A unique 892-base pair cDNA was cloned that prevented the programmed cell death response following IL-3 deprivation by causing antisense suppression of an endogenous 2.4-kilobase (kb) mRNA. A 2.3-kb cDNA containing the identical 892-base pair over-lapping sequence was cloned that encoded a deduced 371-amino acid protein containing a single Kruppel-type zinc finger and a cluster of 4 cysteine/histidine-rich repeats resembling atypical zinc fingers. The 2.4-kb mRNA was found to be ubiquitously expressed in murine tissues and its abundance in FDCP-1 cells was not altered in response to IL-3 deprivation. Since expression of this 2.4-kb mRNA was a prerequisite for the apoptosis response following IL-3 deprivation, the gene encoding it was named requiem. Requiem is likely to encode a transcription factor required for the apoptosis response following survival factor withdrawal from myeloid cells.

Amino Acid Sequence↗

PubMed1998

An epimerase gene essential for capsule synthesis in Vibrio vulnificus.

The extracellular capsule polysaccharide (CPS) of Vibrio vulnificus is a primary virulence factor which allows survival of the bacteria in the human host. To study the genes involved in expression of the capsule, we generated mutants that lost the ability to produce CPS following the insertion of a minitransposon into the genome of an encapsulated, clinical strain of V. vulnificus. A genomic region, from one nonencapsulated mutant, containing the transposon and flanking V. vulnificus DNA was cloned, and a probe complementary to the chromosomal DNA immediately adjacent to the transposon was used to locate this fragment in the genome of the encapsulated parent strain. The fragment, which contained a putative capsule gene, was cloned and, when supplied in trans, complemented the mutation in the nonencapsulated mutant to restore capsule production. In addition, virulence studies, using the 50% lethal dose assay, showed that the restoration of capsule production also restored the virulence of the organism. Sequence analysis of the gene disrupted by the transposon revealed that it matched a nucleotide-sugar epimerase of Vibrio cholerae O139, with 75 and 85% identities at the nucleotide and amino acid levels, respectively. In addition, computer analysis recognized epimerases of various organisms as highly similar to the putative epimerase of V. vulnificus. Finally, a combination of PCR amplification and Southern blotting showed that this epimerase is common to at least 10 strains of V. vulnificus that each express a serologically distinct CPS. Our results indicate that the epimerase gene is essential for capsule expression in V. vulnificus.

Amino Acid Sequence↗

PubMed2000

DNA sequence analysis of the tellurite-resistance determinant from clinical strain of Escherichia coli and identification of essential genes.

The tellurite-resistant Escherichia coli strain KL53 was found during testing of the group of clinical isolates for antibiotics and heavy metal ion resistance (Burian et al. 1990). Determinant of the tellurite resistance of the strain was located on the large conjugative plasmid pTE53 and cloned into pACYC184. Three different Ter clones harboring pLK2, pLK18 and pLK20 were isolated (Burian et al. 1998). The smallest functional Ter clone harboring pLK18 was chosen for further analysis. Plasmid pLK18 have been subcloned to obtain convenient DNA fragments for sequencing of tellurite-resistance determinant. Sequencing of this DNA fragments provided complete DNA sequence of the determinant, 5,250 bp in size. The sequence has been compared with nucleotide and protein databank (BLAST programs) and significant homology with the three known operons coding for tellurite resistance has been found (determinat on plasmid pR478 from Serratia marcescens, on plasmid pMER610 from Alcaligenes sp. and chromosomal tellurite resistance genes from Proteus mirabilis). We identified 5 ORFs coding for 5 genes named terB to terF. The clone harboring pLK18 was subjected to the transposition with Tn1737Km to disrupt determinant of the tellurite resistance. Plasmid DNA of several clones containing pLK18 with Tn1737Km was isolated to locate the target site of Tn1737Km. Analyses showed, the genes terB, terC, terD and terE are essential for conservation of the resistance whereas the gene terF is not important in this respect.

DNA, Bacterial↗

PubMed2002

[Pulmonary emphysema in klotho mutant mice (deficient in klotho gene expression): klotho gene essential in postnatal pulmonary integrity].

The homozygous mutant klotho (KL-/-) mouse, which is deficient in klotho gene expression, exhibits multiple phenotypes resembling human aging that include a short life span, arteriosclerosis, osteoporosis, skin atrophy, and ectopic calcifications. Histologic examination of KL-/- mice at 4 weeks of age reveals pulmonary emphysema. The emphysematous changes are not caused by a developmental defect or hypoplasia of the lung. The lungs of heterozygous mutant klotho (KL+/-) mice show emphysematous changes and alveolar calcification which are almost identical to those in KL-/- mice. These observations indicate that the klotho gene expression is essential to maintaining normal alveolar architecture in postnatal life. Pulmonary function tests reveal prolonged expiration time in KL-/- mice. The expression of type IV collagen and surfactant protein-A mRNA is markedly upregulated in KL-/- mice, suggesting that a defect in matrix synthesis or in type II pneumocyte function, or both, may be involved in the pathogenesis of pulmonary emphysema in this mutant. Several animal strains have been reported to develop pulmonary emphysema, but the klotho mouse is unique because it simultaneously exhibits various aging phenotypes. Analysis of pulmonary emphysema in klotho mice will provide a unique insight into the relationship between pulmonary emphysema and aging.

Aging↗

PubMed2003

RAM2: an essential gene in the prenylation pathway of Candida albicans.

Prenylation is a post-translational protein modification process that results in correct protein localization to membranes in the cell. It is mediated by at least three prenyltransferases (PTFs) in eukaryotic cells. The RAM2 gene encodes the common alpha-subunit of two PTFs, farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I). In this study, the RAM2 gene of the prenylation pathway in the opportunistic pathogen Candida albicans was analysed. The heterozygote trisomy test was used to demonstrate that RAM2 is essential to the viability of C. albicans. RAM2 mRNA in the yeast and hyphae growth states was not detected by Northern blot analysis, but was detected by RT-PCR. Drugs that inhibit mammalian PTFs do not alter Candida cell growth, but they do inhibit FTase and GGTase I activities in cell-free enzyme assays. The data from genetic studies and cell-free enzyme assays suggest that the drugs may not have access to the Candida cytoplasm. The regulation of PTF subunits was also examined in a strain in which RAM2 was under the control of a regulable promoter. Overall, this study demonstrated that RAM2 is essential to C. albicans, indicating that protein prenylation is an indispensable cellular process in this yeast.

Alkyl and Aryl Transferases↗

PubMedDate not supplied

Cloning of CDC33: a gene essential for growth and sporulation which does not interfere with cAMP production in Saccharomyces cerevisiae.

The CDC33 gene of Saccharomyces cerevisiae belongs to the class II 'START' genes. Its product is required for the initiation of a new cell division cycle (Hartwell, 1974). Many results suggest that the cAMP signalling pathway is one of the major controlling elements of 'START'. Components of this pathway are encoded by class II 'START' genes. The aim of the present study is to determine whether or not the CDC33 gene interferes with the cAMP signalling pathway. We report here the molecular cloning of the CDC33 gene by complementation of the cdc33-1 thermosensitive mutant. The identity of the cloned gene is confirmed by site-specific reintegration and segregation analysis. This gene is transcribed into a 900-nucleotides mRNA and appears to be relatively abundant in the cell. We also show that the CDC33 gene product is essential for sporulation. cdc33-1 mutant cells are able to enter into the resting state. The cAMP intracellular pool is not modified when the cdc33-1 mutant is shifted to the restrictive temperature. The cdc33-1 mutation is not suppressed by other known elements of the cAMP cascade. All these results suggest that the CDC33 'START' gene does not interfere with the cAMP signalling pathway which controls cell division.

Blotting, Southern↗

PubMed2001

Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for CagA translocation in host cells and induction of interleukin-8.

Helicobacter pylori (Hp) carries a type IV secretion system encoded by the cag pathogenicity island (cag-PAI), which is used to: (i) translocate the bacterial effector protein CagA into different types of eukaryotic cells; and (ii) induce the synthesis and secretion of chemokines, such as interleukin-8 (IL-8). The cag-PAI in Hp 26695 consists of 27 putative genes, six of which were identified as homologues to the basic type IV secretion system represented by the Agrobacterium tumefaciens virB operon. To define the role and contribution of each of the 27 genes, we applied a precise deletion/insertion mutagenesis procedure to knock out each individual gene without causing polar effects on the expression of downstream genes. Seventeen out of 27 genes were found to be absolutely essential for translocation of CagA into host cells and 14 out of 27 for the ability of Hp fully to induce transcription of IL-8. The products of hp0524 (virD4 homologue), hp0526 and hp0540 are absolutely essential for the translocation of CagA, but not for the induction of IL-8. In contrast, the products of hp0520, hp0521, hp0534, hp0535, hp0536 and hp0543 are not necessary for either translocation of CagA or for IL-8 induction. Our data argue against a translocated IL-8-inducing effector protein encoded by the cag-PAI. We isolated a variant of Hp 26695, which spontaneously switched off its capacity for IL-8 induction and translocation of CagA, but retained the complete cag-PAI. We identified a point mutation in gene hp0532, causing a premature translational stop in the corresponding polypeptide chain, providing a putative explanation for the defect in the type IV secretion system of the spontaneous mutant.

Amino Acid Sequence↗

PubMed1994

YBR1012 an essential gene from S. cerevisiae: construction of an RNA antisense conditional allele and isolation of a multicopy suppressor.

The gene YBR1012 was identified during the systematic sequencing of chromosome II of the yeast Saccharomyces cerevisiae. We have inactivated the gene and shown that it is essential for cellular viability. Using antisense RNA technology we have constructed a conditional allele, expression of the antisense RNA strongly inhibits growth. To our knowledge this is the first successful use of antisense RNA technology in S. cerevisiae. Comparison of the deduced ybr1012p sequence with the data banks revealed the presence of a putative phosphatidylinositol kinase domain and a strong homology to the Schizosaccharomyces pombe rad3p. These results suggest that ybr1012p may be involved in signal transduction, possibly related to the control of replication and/or DNA damage repair. The link with DNA damage repair was reinforced by the isolation of the DUN1 gene as a multicopy suppressor of the YBR1012 deletion.

Alleles↗

PubMed1991

Characterization of RPR1, an essential gene encoding the RNA component of Saccharomyces cerevisiae nuclear RNase P.

RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein. We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPR1 (RNase P ribonucleoprotein 1). Gene disruption experiments showed RPR1 to be single copy and essential. Characterization of the gene region located RPR1 600 bp downstream of the URA3 coding region on chromosome V. We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPR1 RNA. The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPR1 transcript is subjected to multiple processing steps to obtain the 369-nucleotide form. Complementation of RPR1-disrupted haploids with one variant of RPR1 gave a slow-growth and temperature-sensitive phenotype. This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPR1 RNA is an essential component of this enzyme.

Base Sequence↗

PubMed1996

Identification and characterization of ispA, a Shigella flexneri chromosomal gene essential for normal in vivo cell division and intracellular spreading.

The virulent phenotype of Shigella requires loci on the chromosome as well as on the large virulence plasmid, and is regulated via a complex web of interactions amongst various chromosomal and large plasmid genes. To further investigate the role of chromosomal loci in virulence, we performed random Tn10 mutagenesis in Shigella flexneri YSH6000T, and isolated an avirulent mutant (V3404) incapable of spreading throughout an epithelial cell monolayer. Although V3404 initially spread intercellularly at the same rate as the wild-type, it gradually slowed down and ceased spreading as a result of increasing defects in cell division, leading to the formation of long filamentous bacteria lacking septa, trapped within cells. In addition, the mutation affected the ability of V3404 to polymerize actin, a prerequisite for intra- and inter-cellular spreading ability. Sequencing of Tn10-flanking DNA revealed that the mutated gene, designated ispA (intracellular septation), was equivalent to a previously sequenced but uncharacterised gene of Escherichia coli located between trp and tonB. Using E. coli sequence data, we cloned the ispA gene from the YSH6000T chromosome and found that it complemented the V3404 mutation. Nucleotide sequencing and in vitro expression experiments revealed that ispA coded for a small (21 kDa), very hydrophobic protein. These results thus show that ispA is an essential virulence gene affecting several functions of the virulence process.

Actins↗

PubMed2003

amfR, an essential gene for aerial mycelium formation, is a member of the AdpA regulon in the A-factor regulatory cascade in Streptomyces griseus.

In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) acts as a chemical signalling molecule that triggers morphological differentiation and secondary metabolism. A transcriptional activator, AdpA, in the A-factor regulatory cascade switches on a number of genes required for both processes, thus forming an AdpA regulon. amfR encoding a regulatory protein similar to response regulators of bacterial two-component regulatory systems and essential for aerial mycelium formation was found to be a member of the AdpA regulon. AdpA bound two sites at nucleotide positions approximately -200 (site 1) and -60 (site 2), with respect to the major transcriptional start point of amfR, and accelerated the transcription of amfR by assisting RNA polymerase in forming an open complex at an appropriate region including the transcriptional start point. Site 2 contributed more to the transcriptional activation of amfR by AdpA than site 1, although AdpA showed a much lower affinity to site 2 than to site 1. The amfR transcription enhanced by AdpA subsequently ceased at day 2 when aerial hyphae began to be formed in the wild-type strain, whereas in an adsA null mutant amfR was continuously transcribed even until day 3. This implied that amfR was repressed growth dependently by a gene product under the control of sigma-AdsA. Transcription of the promoter upstream of amfT depended on amfR, which is consistent with the idea that AmfR serves as an activator for amfTSBA in the amf operon. The observations that the amfR gene contains a TTA codon, a potential target for bldA-mediated regulation, and a conserved Asp-54 residue, which might be phosphorylated by a sensor kinase, suggest that the amf operon is under transcriptional, translational and post-translational control systems.

4-Butyrolactone↗

PubMed1997

ENP1, an essential gene encoding a nuclear protein that is highly conserved from yeast to humans.

A novel gene in Saccharomyces cerevisiae, ENP1, was found to be essential for growth. The ENP1 gene encodes a protein of 483 amino acids (aa). Nucleotide sequence analysis revealed that the deduced aa sequence of this gene exhibited approx. 60% sequence similarity to the deduced aa sequence of proteins of unknown function in Drosophila, Caenorhabditis elegans and humans. No well defined functional motifs were evident upon analysis of the aa sequence. The protein was found to contain 20% acidic aa residues, with most of them being localized to a very negatively charged domain between aa residues 100 and 150. A construct encoding a fusion protein consisting of the Enp1 protein fused to the c-myc epitope that was either under the control of the ENP1 promoter or the GAL1,10 promoter was prepared. The construct was used to express the protein tagged with the c-myc epitope. Despite the presence of a naturally occurring promoter region with homology to the unfolded protein response element, the level of Enp1mycp remained unchanged after growth of the cells in the presence of tunicamycin, an inhibitor of N-linked glycosylation of proteins. Immunohistochemical studies to define the cellular localization of the Enp1myc protein revealed that it was localized to the nucleus. Accession No.: U50779.

Amino Acid Sequence↗

PubMed1985

Bacillus subtilis sigma factor sigma 29 is the product of the sporulation-essential gene spoIIG.

Evidence is presented that the sporulation-essential locus spoIIG codes for both sigma 29 and a structurally related protein, P31. This demonstrates that at least one specific Bacillus subtilis RNA polymerase binding protein provides a critical function in endospore formation. spoIIG-specific RNA is present in B. subtilis cultures that are synthesizing P31 and sigma 29 and is absent in those that are not. A monoclonal antibody specific for an antigenic determinant on P31/sigma 29 detected crossreacting proteins (P25/P21) but not P31 or sigma 29 in a Spo- B. subtilis strain with a mutation at the spoIIG locus (spoIIG41). The appearance of P25 and P21 occurs in this mutant at a time when P31 and sigma 29 would normally appear and suggests that they are homologous proteins. Transformation of the spoIIG41 strain with plasmid DNA carrying the structural gene for spoIIG complements the Spo- phenotype and results in the synthesis of P31, sigma 29, P25, and P21 at the appropriate times during sporulation. In Escherichia coli, the cloned spoIIG sequence encoded a protein that reacted with the anti-P31/sigma 29 monoclonal antibody and had the electrophoretic mobility of authentic P31.

Antibodies, Monoclonal↗

PubMedDate not supplied

Sequence and mutational analysis of ESS1, a gene essential for growth in Saccharomyces cerevisiae.

A newly isolated gene, ESS1, was shown to encode a protein required for vegetative growth in Saccharomyces cerevisiae. The nucleotide sequence of ESS1 revealed a 172 amino acid open reading frame predicting a highly basic, 19.5 kilodalton product. Although the gene was isolated by cross-hybridization with the vertebrate v-sis oncogene, the primary amino acid sequence bears only a slight resemblance to the p28sis protein. ESS1 was shown to be single copy in the yeast genome and transcriptionally active during logarithmic growth. It is located on the right arm of chromosome X, 6 centimorgans distal to ilv3. The genetic map location indicates it is not allelic to any previously characterized mutation in this organism. Both inactivation of ESS1 by gene disruption and overexpression by fusion to a heterologous promoter were detrimental to growth in both haploid and diploid cell types. Under non-permissive conditions, the terminal phenotype of strains containing a suppressible amber mutation within ESS1 was one of aberrant multibudded structures. Examination of this morphology indicates that loss of ESS1 function may lead to a defect in cytokinesis or cell separation.

Amino Acid Sequence↗

PubMed2004

Regulation of tetralin biodegradation and identification of genes essential for expression of thn operons.

The tetralin biodegradation genes of Sphingomonas macrogolitabida strain TFA are clustered in two closely linked and divergent operons. To analyze expression of both operons under different growth conditions, transcriptional and translational gene fusions of the first genes of each operon to lacZ have been constructed in plasmids unable to replicate in Sphingomonas and integrated by recombination into the genome of strain TFA. Expression analysis indicated that the transcription of both genes is induced in similar ways by the presence of tetralin. Gene expression in both operons is also subjected to overimposed catabolic repression. Two additional genes named thnR and thnY have been identified downstream of thnCA3A4 genes. ThnR is similar to LysR-type regulators, and mutational analysis indicated that ThnR is strictly required for expression of the thn operons. Unlike other LysR-type regulators, ThnR does not repress its own synthesis. In fact, ThnR activates its own expression, since thnR is cotranscribed with the thnCA3A4 genes. ThnY is similar to the ferredoxin reductase components of dioxygenase systems and shows the fer2 domain, binding a Cys4[2Fe-2S] iron sulfur center, and the FAD-binding domain, common to those reductases. However, it lacks the NAD-binding domain. Intriguingly, ThnY has a regulatory role, since it is also strictly required for expression of the thn operons. Given the similarity of ThnY to reductases and the possibility of its being present in the two redox states, it is tempting to speculate that ThnY is a regulatory component connecting expression of the thn operons to the physiological status of the cell.

Artificial Gene Fusion↗

PubMed1985

Additional genes essential for replication of the mini-F plasmid from origin I.

We isolated a series of Tn5-insertional mutants from the mini-F plasmid, which has a deletion in the origin II region and replicates exclusively from origin I, and found that the mutants that had Tn5 in either the F4 or the F5 gene were defective in their replication. It is concluded that, in addition to the F3 gene on which we have reported previously, both the F4 and the F5 genes are essential for the replication from origin I.

Bacterial Proteins↗

PubMed2006

Expression profile of RhoGTPases and RhoGEFs during RANKL-stimulated osteoclastogenesis: identification of essential genes in osteoclasts.

UNLABELLED: RhoGTPases regulate actin cytoskeleton dynamics, a key element in osteoclast biology. We identified three novel genes induced during RANKL-stimulated osteoclastogenesis among RhoGTPases and their exchange factors that are essential in osteoclast biology. INTRODUCTION: During the process of differentiation, adhesion to the bone matrix or osteolysis, the actin cytoskeleton of osteoclasts undergoes profound reorganization. RhoGTPases are key regulators of actin dynamics. They control cell adhesion, migration, and morphology through their action on actin cytoskeleton. In mice, there are 18 low molecular weight RhoGTPases. They are activated by guanine nucleotide exchange factors: the RhoGEFs. There are 76 RhoGEFs in mice: 65 belong to the Dbl family and 11 to the CZH family. To identify novel genes among RhoGTPases and RhoGEFs important in osteoclasts, we established the expression profiles of the complete families of RhoGTPases and RhoGEFs during RANKL-stimulated osteoclastogenesis. MATERIALS AND METHODS: The RAW264.7 cell line, mouse bone marrow macrophages, and hematopoietic stem cells were used as precursors for RANKL-induced osteoclastogenesis. Gene arrays and real-time quantitative PCR analyses were performed to establish the transcription profiles of RhoGTPase and RhoGEF genes during differentiation. Small hairpin RNA was used to knock down genes of interest. RESULTS: Of the 18 RhoGTPases and 76 RhoGEFs, the expression of three genes was upregulated by RANKL: the RhoGTPase RhoU/Wrch1, the Dbl family exchange factor Arhgef8/Net1, and the CZH family exchange factor Dock5. The inductions were observed in gene array and real-time quantitative PCR experiments performed in RAW264.7 cells. They were further confirmed in bone marrow macrophages and hematopoietic stem cells. Silencing of Wrch1 and Arhgef8 expression severely inhibited differentiation and affected osteoclast morphology. Dock5 suppression was lethal in osteoclast precursors while having no effect in fibroblasts. CONCLUSIONS: We identified three genes among RhoGTPase signaling pathways that are upregulated during RANKL-induced osteoclastogenesis. These genes are novel essential actors in osteoclasts, most likely through the control of actin cytoskeleton dynamics.

Adaptor Proteins, Signal Transducing↗

PubMed2005

NMR structure of HI0004, a putative essential gene product from Haemophilus influenzae, and comparison with the X-ray structure of an Aquifex aeolicus homolog.

The solution structure of the 154-residue conserved hypothetical protein HI0004 has been determined using multidimensional heteronuclear NMR spectroscopy. HI0004 has sequence homologs in many organisms ranging from bacteria to humans and is believed to be essential in Haemophilus influenzae, although an exact function has yet to be defined. It has a alpha-beta-alpha sandwich architecture consisting of a central four-stranded beta-sheet with the alpha2-helix packed against one side of the beta-sheet and four alpha-helices (alpha1, alpha3, alpha4, alpha5) on the other side. There is structural homology with the eukaryotic matrix metalloproteases (MMPs), but little sequence similarity except for a conserved region containing three histidines that appears in both the MMPs and throughout the HI0004 family of proteins. The solution structure of HI0004 is compared with the X-ray structure of an Aquifex aeolicus homolog, AQ_1354, which has 36% sequence identity over 148 residues. Despite this level of sequence homology, significant differences exist between the two structures. These differences are described along with possible functional implications of the structures.

Amino Acid Sequence↗