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Genetic analysis of essential genes in the ftsE region of the Escherichia coli genetic map and identification of a new cell division gene, ftsS.

Several conditional lethal mutants of Escherichia coli have been analysed genetically using generalized transduction and lambda transducing vectors. Three temperature-sensitive ftsE mutants were found as was a cold-sensitive ftsE mutant. A new gene was found which mapped close to ftsE, namely ftsS. Both cell division genes map close to the gene which controls the heat-shock regulon (htpR).

Cell Division↗

Caffeine-resistance in fission yeast is caused by mutations in a single essential gene, crm1+.

Caffeine is a base analogue and is known to affect a wide variety of cellular processes. In order to dissect genetically molecules which mediate the biological effects of caffeine, temperature-sensitive (ts) and caffeine-resistant mutants were isolated from fission yeast, Schizosaccharomyces pombe. Surprisingly, all twelve ts isolates contained a mutation in the same locus, crm1. Cells of the ts crm1 mutant showed an abnormal chromosome structure at the restrictive temperature, an elevated expression of Pap1-dependent transcription, and cross-resistance to an unrelated drug such as staurosporine. Overproduction of pap1+ also conferred caffeine resistance, whilst the resistance of the crm1 mutant is abolished in the pap1- background. These results show that the crm1+ gene is a major locus for caffeine resistance, which arises from Pap1-dependent transcriptional activation.

Alkaloids↗

Renal expression of the essential genes associated with sodium transport following a total proctocolectomy in rats.

The water and electrolyte balance is regulated by the renin-angiotensin-aldosterone system in the kidney. We previously reported that the levels of circulating aldosterone dramatically increased following a total proctocolectomy in rats. However, there is no direct evidence regarding whether renal adaptation is accelerated by the induction of aldosterone-associated molecules. To explore this question, Sprague-Dawley rats underwent total proctocolectomies and then were killed 8 weeks later. We investigated the renal expression of 11beta-hydroxysteroid dehydrogenase type 2, the alpha-, beta-, and gamma-subunits of the epithelial sodium channel, and the alpha1- and beta1-subunits of Na+,K+-ATPase mRNAs because those molecules are responsible for the aldosterone specificity for mineralocorticoid receptor, amiloride-sensitive sodium absorption, and sodium extrusion from distal tubules, respectively. A Northern blot analysis demonstrated the kidney to exhibit mRNA induction for all of these molecules, thus supporting the idea that renal adaptation following a total proctocolectomy depends, at least in part, upon the molecular induction which is principally regulated by circulating aldosterone.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

Genetic and phenotypic characterization of mutants in four essential genes that map to the left half of HSV-1 UL DNA.

Several HSV-1 proteins including the major capsid protein (VP5), two minor capsid proteins (VP11-12 and VP18.8), the alkaline nuclease and glycoprotein gH have been reported to be encoded by the left-most one-third of HSV-1 UL DNA. In this paper, we present physical mapping data and phenotypic analysis of six ts mutants whose mutations lie within this region and which collectively represent four functional complementation groups (1-6, 1-7, 1-10, and 1-26). In this study, mutants in complementation group 1-10 were found to be defective in the synthesis of viral DNA, late viral polypeptides, and the formation of mature capsid-like structures--properties characteristic of other ts mutants defective in functions required for viral DNA synthesis. Two DNA-positive mutants in complementation group 1-7 fail to induce capsid formation and probably possess mutations in coding sequences for VP5. Mutants in two other complementation groups (1-6 and 1-26) synthesize significant levels of viral DNA, late polypeptides, and capsids. The functions of the gene products represented by these mutants remain to be determined.

Animals↗

Stability of a host-vector system based on complementation of an essential gene in Escherichia coli.

Antibiotic selection is the most common selection system for plasmid-containing bacteria. This technique, nevertheless, can be a source of problems during the expression of heterologous genes in Escherichia coli. We have developed an alternative selection system based on the complementation of a chromosomal auxotrophic (dapD2) mutation by the corresponding wild type gene carried on a plasmid. We show that the system effectively selects for the presence of plasmid on solid and liquid medium. In addition, we have observed a loss of viability associated with high levels of gene expression and accumulation of a heterologous protein, but the selective power and improved intrinsic stability of the dap+ plasmid, compared to a beta-lactamase (bla) based vector, excludes overgrowth of the culture by plasmidless cells.

Cloning, Molecular↗

Essential genes that regulate apoptosis.

The expression of several genes has been associated with the induction of apoptosis in a wide variety of vertebrate and invertebrate organisms. However, relatively few gene products have been demonstrated to be required for cell death. This review highlights genes that are required for apoptosis and proposes mechanisms by which the proteins encoded by these genes might function.

Journal Article↗

Towards establishing a method to screen for inhibitors of essential genes in mycobacteria: evaluation of the acetamidase promoter.

As a successful pathogen, Mycobacterium tuberculosis has effectively infected one-third of the world's population. Despite the existence of compound libraries developed by recent advances in combinatorial chemistry, few compounds have been screened against M. tuberculosis. The use of a regulable promoter to control the level of expression of a drug target in living organisms has been shown to be advantageous compared with targetless whole-cell-based or in vitro biochemical screening approaches towards antibiotic discovery. In this study, we demonstrate that the acetamidase promoter from Mycobacterium smegmatis responds in a dose-dependent manner to different concentrations of its inducer acetamide. Using this promoter to regulate expression of a zeocin resistance gene in M. smegmatis, we show that the test strain exhibits increased sensitivity to zeocin at a low concentration of acetamide compared with a fully resistant phenotype at high doses of the inducer. This model system has indicated the feasibility of using a regulable promoter in designing a whole-cell-based high throughput screen for specific inhibitors against potential drug targets of M. tuberculosis.

Amidohydrolases↗

mraW, an essential gene at the dcw cluster of Escherichia coli codes for a cytoplasmic protein with methyltransferase activity.

Three new open reading frames, mraZ, mraW and mraR (also called ftsL), were revealed by DNA sequencing immediately upstream of gene pbpB in the dcw cluster of Escherichia coli. We have found that mraW and mraZ are active genes, coding for two proteins with relative molecular masses of 34 800 and 17 300, respectively. MraW is a cytoplasmic protein that under overproduction condition is also loosely bound to the membrane. Soluble MraW was purified up to 90% by a single high performance electrophoresis (HPEC) step from an extract of an overproducing strain. The protein exhibits a S-adenosyl-dependent methyltransferase activity on membrane-located substrates.

Amino Acid Sequence↗

Null mutations in the essential gene yrfF (mucM) are not lethal in rcsB, yojN or rcsC strains of Salmonella enterica serovar Typhimurium.

Insertion of factor MudJ in the intergenic region between divergent genes yrfF and yrfE, at centisome 76 in the genome of Salmonella enterica serovar Typhimurium LT2, confers the characteristics recently described for mucM mutants, i.e. mucoidy and resistance to mecillinam. Cloning of the intergenic region plus either the yrfF or the yrfE gene in a multicopy plasmid showed that only the plasmid carrying the yrfF gene complemented mucM mutants, thus suggesting that mucM mutations are in fact yrfF mutations. A null yrfF mutation obtained by insertion of a kanamycin cassette into the yrfF open reading frame (yrfF28::Kan) produced abortive colonies when transduced to a wild-type strain but was normally accepted by rcsB, rcsC or yojN strains. Neither mutations preventing synthesis of the capsular exopolysaccharide colanic acid (cps, galE) nor rcsA mutations, which reduce expression of cps genes, conferred tolerance to the lethal yrfF28::Kan mutation. Spontaneous suppressor mutations arose very frequently in abortive yrfF28::Kan colonies, and all of them affected either rcsC, yojN, or rcsB genes. Thus, the lethal effect caused by inactivation of gene yrfF appears to be mediated by a function that is dependent on the rcsC-yojN-rcsB phosphorelay system but does not involve synthesis of colanic acid.

Amdinocillin↗

A P-insertion screen identifying novel X-linked essential genes in Drosophila.

The recent determination and annotation of the entire euchromatic sequence of the Drosophila melanogaster genome predicted the existence of about 13600 different genes (Science 287 (2000) 2185; http://www.fruitfly.org/annot/index.html). In parallel, the Berkeley Drosophila Genome Project (BDGP) has undertaken systematic P-insertion screens, to isolate new lethals and misexpressing lines. To date, however, the genes of the X chromosome have been under-represented in the screens performed. In order both to characterize several X-linked genes of prime interest to our laboratories and contribute to the collection of lethal P-insertions available to the community, we performed a P-insertion mutagenesis of the X chromosome. Using the PlacW and PGawB P-elements as mutagens, we generated two complementary sets of enhancer-trap lines, l(1)(T)PL and l(1)(T)PG, respectively, which both contain a reporter gene whose developmental expression can be monitored when driven by nearby enhancer sequences. We report here the characterization of 260 new insertions, mapping to 133 different genes or predicted CGs. Of these, 83 correspond to genes for which no lethal mutation had yet been reported. For 64 of those, we could confirm that lethality was solely due to the P-element insertion. The primary molecular data, reporter gene expression patterns (observed in embryos, third instar larvae and adult ovaries) and proposed CG assignment for each strain can be accessed and updated on our website at the following address: http://www-cbd.ups-tlse.fr:8080/screen.

Animals↗

VACUOLELESS1 is an essential gene required for vacuole formation and morphogenesis in Arabidopsis.

Most plant cells are characterized by the presence of a large central vacuole that in differentiated cells accounts for more than 90% of the total volume. We have undertaken a genetic screen to look for mutants that are affected in the formation of vacuoles in plants. In this study, we report that inactivation of the Arabidopsis gene VACUOLELESS1 (VCL1) blocks vacuole formation and alters the pattern of cell division orientation and cell elongation in the embryo. Consistent with a role in vacuole biogenesis, we show that VCL1 encodes the Arabidopsis ortholog of yeast Vps16p. In contrast to yeast mutants that lack a vacuolar compartment but are viable and morphologically normal, loss of the plant vacuole leads to aberrant morphogenesis and embryonic lethality.

Alleles↗

U2AF35 is encoded by an essential gene clustered in an operon with RRM/cyclophilin in Caenorhabditis elegans.

In most species the 3' splice site is recognized initially by an interaction between the two-subunit splicing factor U2AF with the polypyrimidine (poly(Y)) tract that results in recruitment of the U2 snRNP to the branch-point consensus just upstream. In contrast, in Caenorhabditis elegans, both the poly(Y) tract and the branch-point consensus sequences are missing, apparently replaced by the highly conserved U4CAG/R 3' splice site consensus. Nevertheless C. elegans U2AF65 is very similar to its mammalian and fly counterparts and may recognize the 3' splice site consensus. Here we report the cloning of the C. elegans U2AF35 gene, uaf-2. We show that it lacks an identifiable RS domain, which, in flies, has been shown to play a role in RNA binding, but it contains an extended glycine-rich stretch at its C-terminus. uaf-2 is in an operon with cyp-13, a gene that encodes a cyclophilin with an RRM domain at its N-terminus. We demonstrate by RNA interference that both U2AF genes, uaf-1 (which encodes U2AF65) and uaf-2, are required for viability, whereas cyp-13 is apparently not.

Amino Acid Sequence↗

The meiotic drive system on maize abnormal chromosome 10 contains few essential genes.

In maize, a distal portion of abnormal chromosome 10 (Ab10) causes the meiotic drive of itself as well as many unlinked heterochromatic regions known as knobs. The Ab10 drive system, which encodes trans- as well as cis-acting components, occupies a large region of chromosome 10L equivalent to approximately 3% of the genome. Here we describe five new structural mutations of Ab10 (five deletions and a duplication) that arose from a screen for meiotic drive mutants. The high frequency of breakage events, detected both genetically and cytologically, suggest that the chromosome may be especially unstable. Very large deletions within the drive system are female-transmissible and plants homozygous for deficiencies lacking much of this interval can be grown to maturity. The data suggest that few genes required for normal growth and development lie within the portion of Ab10 responsible for meiotic drive. These and other published data suggest that meiotic drive systems tend to evolve in gene-sparse or otherwise information-poor regions of the genome where they are less likely to negatively affect individual fitness.

Chromosome Aberrations↗

The yeast inositol monophosphatase is a lithium- and sodium-sensitive enzyme encoded by a non-essential gene pair.

Inositol monophosphatases (IMPases) are lithium-sensitive enzymes that participate in the inositol cycle of calcium signalling and in inositol biosynthesis. Two open reading frames (YHR046c and YDR287w) with homology to animal and plant IMPases are present in the yeast genome. The two recombinant purified proteins were shown to catalyse inositol-1-phosphate hydrolysis sensitive to lithium and sodium. A double gene disruption had no apparent growth defect and was not auxotroph for inositol. Therefore, lithium effects in yeast cannot be explained by inhibition of IMPases and inositol depletion, as suggested for animal systems. Overexpression of yeast IMPases increased lithium and sodium tolerance and reduced the intracellular accumulation of lithium. This phenotype was blocked by a null mutation in the cation-extrusion ATPase encoded by the ENA1/PMR2A gene, but it was not affected by inositol supplementation. As overexpression of IMPases increased intracellular free Ca2+, it is suggested that yeast IMPases are limiting for the optimal operation of the inositol cycle of calcium signalling, which modulates the Ena1 cation-extrusion ATPase.

Amino Acid Sequence↗