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Results for “conditionally essential genes”

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PubMed1988

Construction and characterization of the deletion mutant of hupA and hupB genes in Escherichia coli.

Insertion and deletion mutations of the hupB and hupA genes, which encode the HU-1 and HU-2 proteins, respectively, of Escherichia coli, have been constructed in vitro and transferred to the hup loci on the bacterial chromosome. The mutations were constructed by inserting a gene encoding chloramphenicol resistance or kanamycin resistance into the coding region of the hupB or hupA gene, respectively. A complete deletion of the hupA gene was constructed by replacing the entire hupA coding region with the kanamycin resistance gene. Cells in which either the hupB or the hupA gene is defective grow normally, but cells in which both of the hup genes are defective exhibit phenotypes different from the wildtype strain. The hupA-hupB double mutants are cold-sensitive, although their growth rate is normal at 37 degrees C. Furthermore, the viability of the hupA-hupB double mutants is severely reduced when the cells are subjected to either cold shock or heat shock, indicating that the hup genes are essential for cell survival under some conditions of stress. The double mutants also exhibit filamentation when grown in the lower range of permissive growth temperature.

Alleles↗

PubMed2002

Nnf1p, Dsn1p, Mtw1p, and Nsl1p: a new group of proteins important for chromosome segregation in Saccharomyces cerevisiae.

Previously, antibodies were raised against a nuclear envelope-enriched fraction of yeast, and the essential gene NNF1 was cloned by reverse genetics. Here it is shown that the conditional nnf1-17 mutant has decreased stability of a minichromosome in addition to mitotic spindle defects. I have identified the novel essential genes DSN1, DSN3, and NSL1 through genetic interactions with nnf1-17. Dsn3p was found to be equivalent to the kinetochore protein Mtw1p. By indirect immunofluorescence, all four proteins, Nnf1p, Mtw1p, Dsn1p, and Nsl1p, colocalize and are found in the region of the spindle poles. Based on the colocalization of these four proteins, the minichromosome instability and the spindle defects seen in nnf1 mutants, I propose that Nnf1p is part of a new group of proteins necessary for chromosome segregation.

Alleles↗

PubMed2003

Functional analysis in yeast of the Brix protein superfamily involved in the biogenesis of ribosomes.

An extensive homology search based on the sequence of the yeast protein Brx1p (biogenesis of ribosomes in Xenopus, YOL077c) revealed that it is a member of a superfamily of proteins sharing remarkable sequence similarities. Previous work on Brx1p showed that this protein is involved in the process of ribosome biogenesis [Kaser et al., Biol. Chem. 382 (2001) 1637-1647]. Brx1p is the founding member of one of the five existing eukaryotic subfamilies which are all present in yeast. Four of them are represented by one essential gene each and one family is represented by two closely related genes which can functionally replace each other but are essential together for survival. We created conditional alleles of four of the five genes which allowed us to study the effect of depletion of the respective proteins on the ribosome profiles of the strains. In this study we show that not only Brx1p but also three additional superfamily members, namely YHR088w (Rpf1p), YKR081c (Rpf2p) and the homologous proteins Ssf1p (YHR066w)/Ssf2p (YDR312w) are all involved in the multistep process of the assembly of the large ribosomal subunit. This agrees well with the fact that these three proteins, like Brx1p, are located in the nucleolus. Moreover, all four proteins closely interact functionally, because all four mutants are suppressed by the same multicopy suppressor gene.

Cell Nucleolus↗

PubMedDate not supplied

[Characteristics of the effect of implantation of the human ApoA1 gene on the condition of hepatocytes in rats of varying age].

The results obtained show the essential changes in functional state of hepatocyte's plasmatic membrane due to the implantation of human ApoA1 gene to the rat liver. The changes in phospholipid composition, hyperpolarization, increase in activity of membrane bound enzymes, cytochrome P-450 and biosynthesis of liver total proteins have been found. The essential changes characterizing cell effect were more marked in the adult rats, and membrane effect in the old ones.

Aging↗

PubMed1988

Two dominant-acting selectable markers for gene transfer studies in mammalian cells.

We report the development of two dominant-acting genetic markers useful for monitoring gene transfer in mammalian cells that are based on prokaryotic genes encoding key steps in the synthesis of the essential amino acids, tryptophan and histidine. Under appropriate conditions, expression of these genes obviates the nutritional requirements for their respective amino acid products. Expression of the trpB gene of Escherichia coli, which encodes the beta subunit of tryptophan synthase (EC 4.2.1.20), allows mammalian cell survival and multiplication in medium containing indole in place of tryptophan. The hisD gene of Salmonella typhimurium encodes histidinol dehydrogenase (EC 1.1.1.23), which catalyzes the two-step NAD+-dependent oxidation of L-histidinol to L-histidine. In medium lacking histidine and containing histidinol, only mammalian cells expressing the hisD product survive. The selection is a double one in that the provided precursor histidinol is itself toxic to animal cells through its inhibition of histidyl-tRNA synthetase; thus, the dehydrogenase both removes an inhibitor and forms a required end product. Alternatively, the his selection may be carried out under conditions in which the dehydrogenase serves mainly to detoxify histidinol. For either the trp or his selections the substitute nutrient (indole or histidinol) is readily available, inexpensive, stable, permeable to cells, and convertible to the end product in a step controlled by a single gene. Vectors based upon murine retrovirus and papovavirus backbones have been successfully employed for both genes, allowing selection in a range of cell types, including 3T3, CV-1, and HeLa. These dominant selective schemes should provide generally useful and inexpensive alternatives to others currently in use, such as the gpt, neo, hygro, dhfr, and tk selections.

Alcohol Oxidoreductases↗

PubMed1998

The ATP synthase atpHAGDC (F1) operon from Rhodobacter capsulatus.

The atpHAGDC operon of Rhodobacter capsulatus, containing the five genes coding for the F1 sector of the ATP synthase, has been cloned and sequenced. The promoter region has been defined by primer extension analysis. It was not possible to obtain viable cells carrying atp deletions in the R. capsulatus chromosome, indicating that genes coding for ATP synthase are essential, at least under the growth conditions tested. We were able to circumvent this problem by combining gene transfer agent transduction with conjugation. This method represents an easy way to construct strains carrying mutations in indispensable genes.

Amino Acid Sequence↗

PubMed2007

E2f1, E2f2, and E2f3 control E2F target expression and cellular proliferation via a p53-dependent negative feedback loop.

E2F-mediated control of gene expression is believed to have an essential role in the control of cellular proliferation. Using a conditional gene-targeting approach, we show that the targeted disruption of the entire E2F activator subclass composed of E2f1, E2f2, and E2f3 in mouse embryonic fibroblasts leads to the activation of p53 and the induction of p53 target genes, including p21(CIP1). Consequently, cyclin-dependent kinase activity and retinoblastoma (Rb) phosphorylation are dramatically inhibited, leading to Rb/E2F-mediated repression of E2F target gene expression and a severe block in cellular proliferation. Inactivation of p53 in E2f1-, E2f2-, and E2f3-deficient cells, either by spontaneous mutation or by conditional gene ablation, prevented the induction of p21(CIP1) and many other p53 target genes. As a result, cyclin-dependent kinase activity, Rb phosphorylation, and E2F target gene expression were restored to nearly normal levels, rendering cells responsive to normal growth signals. These findings suggest that a critical function of the E2F1, E2F2, and E2F3 activators is in the control of a p53-dependent axis that indirectly regulates E2F-mediated transcriptional repression and cellular proliferation.

Animals↗

PubMed1994

Expressed sequence tags from cultured cells of rice (Oryza sativa L.) under stressed conditions: analysis of transcripts of genes engaged in ATP-generating pathways.

Large-scale sequencing of randomly selected cDNA clones was used to isolate numerous genes in rice (Oryza sativa L.). Total RNA used for cDNA synthesis was prepared from suspension-cultured cells of rice grown under stressed conditions, such as in saline or nitrogen-starvation conditions. A total of 780 cDNA clones were partially sequenced and about 15% could be identified as putative genes. In the library constructed under saline conditions, we identified several genes associated with signal transduction, such as protein kinase and small GTP-binding protein genes. Many stress-related genes were isolated from both the saline and nitrogen-starvation libraries. These results indicate that stress treatment of suspension-cultured cells makes it possible to efficiently isolate various types of plant genes. To examine the usefulness of such tagged cDNAs for the study of gene expression in a specific metabolic pathway, we analyzed mRNA levels of genes engaged in the ATP-generating pathways in cultured cells of rice under different stresses, such as 20% sucrose, salt stress, cold stress and nitrogen-starvation stress. The results suggest that the coordinated induction of several genes in key steps under stressed conditions may be essential for activation of the entire energy-producing pathway to maintain homeostasis in rice cells. Expressed sequence tags identified by random cDNA sequencing provide the opportunity to generate a transcript map of rice genes.

Adaptation, Biological↗

PubMed2003

Characterization of B-type cyclins in the smut fungus Ustilago maydis: roles in morphogenesis and pathogenicity.

Pathogenesis, morphogenesis and cell cycle are connected in the fungal pathogen Ustilago maydis. Here we report the characterization of the catalytic subunit of the cyclin-dependent kinase, encoded by the gene cdk1, and the two B-type cyclins present in this organism, encoded by the genes clb1 and clb2. These cyclins are not redundant and appears to be essential for cell cycle. The analysis of conditional mutants in cyclin genes indicates that Clb1 is required for G1 to S and G2 to M transitions, while Clb2 is specifically required for the onset of mitosis. Both Clb1 and Clb2 carry functional destruction boxes, and expression of derivatives lacking D-boxes arrested cell cycle at a post-replicative stage. High levels of Clb1 generated cells with anomalous DNA content that were hypersensitive to microtubule-destabilizing drugs. In contrast, high levels of Clb2 induce premature entry into mitosis, suggesting that Clb2 is a mitotic inducer in U. maydis. In addition, Clb2 affects morphogenesis, and overexpression of clb2 induces filamentous growth. Furthermore, we have found that appropriate levels of Clb2 cyclin are critical for a successful infection. Mutant strains with half a dose of clb2 or high level of clb2 expression are impaired at distinct stages in the infection process. These data reinforces the connections between cell cycle, morphogenesis and virulence in this smut fungus.

Amino Acid Sequence↗

PubMed2005

Methanol-dependent gene expression demonstrates that methyl-coenzyme M reductase is essential in Methanosarcina acetivorans C2A and allows isolation of mutants with defects in regulation of the methanol utilization pathway.

Methanosarcina acetivorans C2A is able to convert several substrates to methane via at least four distinct methanogenic pathways. A common step in each of these pathways is the reduction of methyl-coenzyme M (CoM) to methane catalyzed by methyl-CoM reductase (MCR). Because this enzyme is used in each of the known pathways, the mcrBDCGA operon, which encodes MCR, is expected to be essential. To validate this prediction, a system for conditional gene inactivation was developed. A heterologous copy of the mcrBDCGA operon was placed under the control of the highly regulated mtaC1 promoter, which directs the expression of genes involved in methanol utilization, and recombined onto the M. acetivorans chromosome. This allowed for disruption of the endogenous mcr operon in the presence of methanol. Because the PmtaC1 promoter is transcribed only during growth on methanol, mcrBDCGA was rendered methanol dependent and the strain was unable to grow in trimethylamine media, strongly suggesting that mcrBDCGA is essential. Upon prolonged incubation, suppressed mutants which expressed mcrBDCGA constitutively could be selected. Expression analysis of PmtaC1::uidA gene fusions in several isolated suppressed mutants suggests that they carry trans-active mutations leading to deregulation of all genes under control of this promoter. Subsequently, proteome analysis of one such suppressed mutant revealed that all known proteins derived from mtaC1 promoter-dependent expression were constitutively expressed in this mutant. This genetic system can therefore be employed for the testing of essential genes and for the identification of genes under a common regulatory mechanism by making regulatory mutations phenotypically selectable.

Gene Deletion↗

PubMed1998

The PY-motif of Bul1 protein is essential for growth of Saccharomyces cerevisiae under various stress conditions.

The previously identified BUL1 gene was found to encode a protein bound to Rsp5-ubiquitin ligase in budding yeast. We have identified the BUL2 gene as a functional homologue of BUL1. The bul1 bul2 double disruptant was sensitive to various stresses, such as high temperature, salts, and a non-fermentable carbon source. Each Bul protein has a putative PY-motif that has been predicted to interact with one of three WW-domains of Rsp5. A mutant Bul1 containing an altered PY-motif was defective in ability to bind to Rsp5 in the two-hybrid system and hardly co-immunoprecipitated with Rsp5. Furthermore, the mutant was not able to overcome all growth defects of the double disruptant. Thus, Bul proteins are essential for growth in various stress conditions, and their functions are mediated through the PY-motif, probably by binding to Rsp5.

Adaptor Proteins, Signal Transducing↗

PubMed1996

A temperature-sensitive splicing mutation in the bimG gene of Aspergillus produces an N-terminal fragment which interferes with type 1 protein phosphatase function.

Progression through anaphase requires high levels of type 1 protein phosphatase (PP1) activity in a variety of eukaryotes, including Aspergillus nidulans. A conditional lethal, temperature-sensitive mutant in one of the Aspergillus PP1 genes, bimG, prevents the normal completion of anaphase when cells are grown at restrictive temperature and this has been shown to be due to a reduction in type 1 phosphatase activity. We show that the bimG11 allele is recessive to the wild-type allele in heterozygous diploids, implying that the mutation is due to loss of function at restrictive temperature, but molecular disruption of the wild-type bimG gene shows that the gene is not essential and has no discernable phenotype under laboratory conditions. Sequence comparison of wild-type and mutant alleles reveals a single base pair difference between the two genes, within the 5' splicing site of the second intron. We demonstrate that the conditional lethal phenotype of bimG11 strains is due to impaired splicing of the mutant mRNA and that this leads to the production of a truncated protein comprising an intact N-subdomain and a modified C-terminus. Over-expression of this truncated form of PP1 in a wild-type haploid produces a lethal phenotype and reduced PP1 activity, supporting the idea that a toxic interfering protein is produced. PP1, therefore, may have at least two spatially separated sites, both of which are required for function. Temperature-sensitive splicing mutations may provide a novel means of engineering conditional versions of other proteins, particularly other phosphatases.

Alleles↗

PubMed2000

Mutational accessibility of essential genes on chromosome I(left) in Caenorhabditis elegans.

We have analyzed a region of approximately 5.4 million base pairs for mutations, which under standard laboratory conditions result in developmental arrest, sterility, or maternal-effect lethality in Caenorhabditis elegans. Lethal mutations were isolated, maintained, and genetically manipulated as homozygotes using sDp2--a duplication of the left half of chromosome I. All of the lethals and rearrangements used in this analysis were balanced by sDp2. Relatively low doses of mutagen, (approximately 15 mM ethylmethane sulfate; EMS), were used so as to limit the occurrence of second-site mutations, thus increasing the probability of recovering single nucleotide substitutions. Treatment of over 32,400 marked chromosomes resulted in 486 analyzed mutations. In this paper, we add 133 previously unidentified let genes, isolated in the EMS screens, and one let gene identified by a gamma-ray induced mutation, to our collection of 103 essential genes. We also recovered lethal alleles of genes for which visible mutants already existed. In total, eight deficiencies and alleles of 237 essential genes were identified. Eighty-nine of the previously unidentified let genes are represented by more than one lethal allele. Statistical analysis indicates a minimum estimate of 400 essential genes in the region of chromosome I balanced by sDp2. This region occupies approximately half of chromosome I, and contains over 1135 protein-coding genes predicted from the genomic sequence data. Thus, approximately one-third of the predicted genes are estimated to be essential. Of these approximately 60% are represented by lethal alleles. Less than 2% of the lethal-bearing strains recovered in our analysis, including the eight genetically definable deficiencies, carried more than one lethal mutation. Several screens were used to recover mutations for this analysis. Because all the mutations were isolated using the same balancer, under similar screening conditions, it was possible to compare intervals within the sDp2 region with each other. The fraction of essential genes that present relatively large targets for EMS was highest within the central cluster (dpy-5 to unc-13).

Animals↗

PubMed2004

Metabolic network analysis of the causes and evolution of enzyme dispensability in yeast.

Under laboratory conditions 80% of yeast genes seem not to be essential for viability. This raises the question of what the mechanistic basis for dispensability is, and whether it is the result of selection for buffering or an incidental side product. Here we analyse these issues using an in silico flux model of the yeast metabolic network. The model correctly predicts the knockout fitness effects in 88% of the genes studied and in vivo fluxes. Dispensable genes might be important, but under conditions not yet examined in the laboratory. Our model indicates that this is the dominant explanation for apparent dispensability, accounting for 37-68% of dispensable genes, whereas 15-28% of them are compensated by a duplicate, and only 4-17% are buffered by metabolic network flux reorganization. For over one-half of those not important under nutrient-rich conditions, we can predict conditions when they will be important. As expected, such condition-specific genes have a more restricted phylogenetic distribution. Gene duplicates catalysing the same reaction are not more common for indispensable reactions, suggesting that the reason for their retention is not to provide compensation. Instead their presence is better explained by selection for high enzymatic flux.

Biomass↗

PubMed2003

A conditionally replicating adenovirus targeted to tumor cells through activated RAS/P-MAPK-selective mRNA stabilization.

The expression of various proteins associated with rapid responses to inflammation and/or proliferation can be controlled at the level of mRNA stability. Because tumor cells continually recapitulate intracellular programs of proliferation, we have used tumor cell-selective stabilization of mRNA as a means to control therapeutic gene expression. We describe an adenoviral vector that is conditionally replication competent in which expression of the essential adenoviral early region 1A (E1A) gene is regulated by ligation to the 3' untranslated region (UTR) of PTGS2 (also known as COX2), the gene encoding prostaglandin-endoperoxide synthase 2, allowing activated RAS/P-MAPK-specific stabilization of its mRNA. Induction of activated RAS supports replication, whereas matched cells in which activated RAS/P-MAPK is not expressed are very poor substrates for viral replication both in vitro and in vivo. Further tumor-targeting strategies will also be required to prevent viral replication at extratumoral sites where PTGS2 is normally induced. Many different genes contain 3' UTRs that control selective mRNA stability under different physiological, pathological and tumor-associated conditions. Therefore, generating tumor selectivity at the level of mRNA stability is a strategy with broad potential applicability in vector design.

Adenoviridae↗

PubMed1997

Multiple modes of activation of the stress-responsive MAP kinase pathway in fission yeast.

The Schizosaccharomyces pombe wis1(+) gene is essential for cell survival under stress conditions. The MAPKK homologue Wis1 is required for activation of the MAPK homologue Spc1, and integrity of the Wis1-Spc1 pathway is required for survival in extreme conditions of heat, osmolarity, oxidation or limited nutrition. We show here that Wis4, a protein kinase of a new MAPKKK class, phosphorylates Wis1 in vitro and activates it in vivo. Win1 is also required for full activation of Wis1, and Win1 rather than Wis4 mediates the osmotic stress signal. Surprisingly, the pathway can still be activated by heat or oxidative stress independently of the phosphorylation of two conserved Wis1 residues. Evidence is presented that the Pyp1 protein tyrosine phosphatase, which dephosphorylates Spc1, is central to this alternative activation mechanism.

Amino Acid Sequence↗

PubMed1999

Pressure response in deep-sea piezophilic bacteria.

Several piezophilic bacteria have been isolated from deep-sea environments under high hydrostatic pressure. Taxonomic studies of the isolates showed that the piezophilic bacteria are not widely distributed in terms of taxonomic positions, and all were assigned to particular branches of the Proteobacteria gamma-subgroup. A pressure-regulated operon from piezophilic bacteria of the genus Shewanella, S. benthica and S. violacea, was cloned and sequenced, and downstream of this operon another pressure regulated operon, cydD-C, was found. The cydD gene was found to be essential for the bacterial growth under high-pressure conditions, and the product of this gene was found to play a role in their respiratory system. Results obtained later indicated that the respiratory system in piezophilic bacteria may be important for survival in a high-pressure environment, and more studies focusing on other components of the respiratory chain have been conducted. These studies suggested that piezophilic bacteria are capable of changing their respiratory system in response to pressure conditions, and a proposed respiratory chain model has been suggested in this regard.

Bacterial Proteins↗

PubMed2005

Conditional depletion of KasA, a key enzyme of mycolic acid biosynthesis, leads to mycobacterial cell lysis.

Inhibition or inactivation of InhA, a fatty acid synthase II (FASII) enzyme, leads to mycobacterial cell lysis. To determine whether inactivation of other enzymes of the mycolic acid-synthesizing FASII complex also leads to lysis, we characterized the essentiality of two beta-ketoacyl-acyl carrier protein synthases, KasA and KasB, in Mycobacterium smegmatis. Using specialized transduction for allelic exchange, null kasB mutants, but not kasA mutants, could be generated in Mycobacterium smegmatis, suggesting that unlike kasB, kasA is essential. To confirm the essentiality of kasA, and to detail the molecular events that occur following depletion of KasA, we developed CESTET (conditional expression specialized transduction essentiality test), a genetic tool that combines conditional gene expression and specialized transduction. Using CESTET, we were able to generate conditional null inhA and kasA mutants. We studied the effects of depletion of KasA in M. smegmatis using the former strain as a reference. Depletion of either InhA or KasA led to cell lysis, but with different biochemical and morphological events prior to lysis. While InhA depletion led to the induction of an 80-kDa complex containing both KasA and AcpM, the mycobacterial acyl carrier protein, KasA depletion did not induce the same complex. Depletion of either InhA or KasA led to inhibition of alpha and epoxy mycolate biosynthesis and to accumulation of alpha'-mycolates. Furthermore, scanning electron micrographs revealed that KasA depletion resulted in the cell surface having a "crumpled" appearance, in contrast to the blebs observed on InhA depletion. Thus, our studies support the further exploration of KasA as a target for mycobacterial-drug development.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗