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Results for “Genes, Essential”

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PubMed1982

Dominant suppressors of a muscle mutant define an essential gene of Caenorhabditis elegans.

The sup-11 1 locus of C. elegans was defined by rare dominant suppressors of unc-93(e1500) III, a mutation that affects muscle structure. All ten of these dominant suppressors have a recessive "scrawny" phenotype. Two additional classes of sup-11 alleles were identified. One class, null alleles, was obtained by reversion of the dominant suppressor activity. These null alleles are recessive embryonic lethals, indicating that sup-11 is an essential gene. Members of the second class, rare semidominant revertants of the "scrawny" phenotype, are partial suppressors of unc-93(e1500). The genetic properties of the dominant suppressor mutations suggest that they are rare missense mutations that confer a novel activity to the sup-11 protein. We consider some of the ways that sup-11 alleles might suppress unc-93(e1500), including the possibilities that the altered sup-11 proteins restore function to a protein complex or are modified products of a gene that is a member of an unc-93 gene family.

Animals↗

PubMed2003

Activity-dependent neuroprotective protein: a novel gene essential for brain formation.

We have recently cloned the novel homeobox-containing activity-dependent neuroprotective protein (ADNP). In the current study, mouse ADNP was shown to be expressed at the time of neural tube closure, detected at E7.5 and increased on E9.5. Expression was augmented in the brain (E12.5), sustained throughout embryogenesis and regulated by VIP. To assess the function of ADNP, knockout mice were established. Detailed analysis revealed cranial neural tube closure failure and death on E8.5-9.0 of the ADNP-knockout embryos. The expression of Oct4, a gene associated with germ-line maintenance was markedly augmented in the knockout embryos. In contrast, the expression of Pax6, a gene crucial for cerebral cortex formation, was abolished in the brain primordial tissue of the knockout embryos. Thus, Pax6 and Oct4 constitute a part of the mechanism of action of ADNP on brain formation, inhibiting germ-line division while activating morphogenesis. In conclusion, ADNP is identified here as a new key gene essential for organogenesis in the developing embryo and may be implicated as a clinical target associated with proper neurodevelopment.

Animals↗

PubMed2006

A simple and effective chromosome modification method for large-scale deletion of genome sequences and identification of essential genes in fission yeast.

The technologies for chromosome modification developed to date are not satisfactorily universal, owing to the typical requirements for special enzymes and sequences. In the present report, we propose a new approach for chromosome modification in Schizosaccharomyces pombe that does not involve any special enzymes or sequences. This method, designated the 'Latour system', has wide applicability with extremely high efficiency, although both the basic principle and the operation are very simple. We demonstrate the ability of the Latour system to discriminate essential genes, with a long chromosomal area of 100 kb containing 33 genes deleted simultaneously and efficiently. Since no foreign sequences are retained after deletion using the Latour system, this system can be repeatedly applied at other sites. Provided that a negative selectable marker is available, the Latour system relies solely upon homologous recombination, which is highly conserved in living organisms. For this reason, it is expected that the system will be applicable to various yeasts.

3-Isopropylmalate Dehydrogenase↗

PubMed1998

Cytoplasmic dynein heavy chain is an essential gene product in Dictyostelium.

We describe here three different approaches to perturb cytoplasmic dynein heavy chain (DHC) gene function in Dictyostelium: integration of a marker into the heavy chain coding sequence by homologous recombination to disrupt transcription, expression of antisense RNA to inhibit translation, and expression of a 158 kDa amino-terminal coding region to perturb the native protein organization. By homologous recombination, we fail to obtain cells that lack an intact DHC gene product. Cells containing antisense orientation plasmids (but not sense) appear to die 4 to 6 days following transformation. Plasmids designed to overexpress an amino-terminal region of the DHC result in substantially reduced transformation efficiency. When expressed at low levels, the truncated amino-terminal product appears capable of dimerizing with an intact heavy chain or with itself, essentially producing a cargo-binding domain lacking mechanochemical activity. This, in turn, likely competes with the native protein's function. These three approaches taken together indicate that the dynein heavy chain is an essential gene in Dictyostelium.

Animals↗

PubMed2003

A screen in Saccharomyces cerevisiae identified CaMCM1, an essential gene in Candida albicans crucial for morphogenesis.

Morphogenesis in Saccharomyces cerevisiae and the pathogenic yeast Candida albicans is governed in part by the same molecular circuits. In S. cerevisiae, FLO11/MUC1 expression has been shown to be modulated by multiple signalling pathways required for pseudohyphal development. We have established a screen in S. cerevisiae to identify regulators of fungal development in C. albicans based on FLO11::lacZ expression as a reporter. This screen identified both known components of the mitogen-activated protein kinase (MAPK) cascade and the cAMP cascade that are important for hyphal development in C. albicans, as well as genes not yet known to be involved in morphogenesis. The Candida homologue of MCM1 is one of the novel factors identified in this screen as being important for morphogenesis. CaMcm1p levels do not vary significantly in different cell types and respond to an autoregulatory feedback mechanism, arguing that CaMcm1p activity is regulated by post-translational modifications. Both overexpression and repression of this essential gene led to the induction of hyphae. Moreover, we found that the expression of HWP1, a hyphae-specific gene, was induced by repression of CaMCM1. The changes in morphology and HWP1 expression were not the result of a change in expression levels of NRG1 or TUP1, known repressors of hyphal development. Thus, CaMcm1p is a component of a hitherto unknown regulatory mechanism of hyphal growth.

Base Sequence↗

PubMed2000

Functional analysis of six novel ORFs on the left arm of chromosome XII in Saccharomyces cerevisiae reveals two essential genes, one of which is under cell-cycle control.

Six novel Open Reading Frames (ORFs) located on the left arm of chromosome XII (YLL044w, YLL042c, YLL040c, YLL038c, YLL035w and YLL034c) have been analysed using short-flanking homology (SFH) gene replacement. Sporulation and tetrad analysis showed that YLL035w and YLL034c are essential for cell growth; yll035w spores arrested after two or three cell divisions, while the majority of yll034c spores stopped growth within two cell cycles after germination. Complementation of the yll035w deletion with its cognate clone, and a promoter-substitution experiment, indicated that the promoter of YLL035w may lie within the adjacent ORF, YLL036c. Transcriptional analysis demonstrated that YLL035w is under cell-cycle regulation. Bioinformatic analyses produced significant matches between YLL034c and mammalian valosin and many other ATPases. The standard EUROFAN growth tests failed to reveal obvious phenotypes resulting from deletion of any of the four non-essential ORFs. Replacement cassettes, comprising the kanMX marker flanked by each ORF's promoter and terminator regions, were cloned into pUG7. All the cognate clones, except for YLL040c, were generated using direct PCR products amplified from genomic DNA or using gap-repair. All clones and strains produced have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt).

Cell Cycle↗

PubMed2001

A Vibrio harveyi insertional mutant in the cgtA (obg, yhbZ) gene, whose homologues are present in diverse organisms ranging from bacteria to humans and are essential genes in many bacterial species.

The cgtA gene product is a member of the subfamily of small GTP-binding proteins that have been identified in diverse organisms ranging from bacteria to humans. In bacteria that sporulate or display another special developmental programme, this gene (referred to as cgtA, obg or yhbZ) appears to be involved in the regulation of these processes. However, this gene has also been found to be essential in all bacterial species investigated to date, although its role in bacteria that do not sporulate and do not undergo a specific development remains unknown. Here the authors characterize a Vibrio harveyi mutant bearing a transposon insertion into the cgtA gene. This mutant reveals a multiple phenotype: it grows more slowly than the wild-type strain in a rich medium; its growth is completely inhibited in minimal media; its survival in 3% NaCl is dramatically reduced; it is very sensitive to UV irradiation; it is more susceptible to mutation upon treatment with different mutagens; its luminescence is decreased; its quorum-sensing regulation is less effective than in the wild-type strain; and the elongated shape of the mutant cells may suggest problems with the regulation of cell division and/or DNA replication. These defects in diverse cellular processes found in the insertional cgtA mutant of V. harveyi indicate that in a bacterium that does not sporulate and does not display other special development programmes, the CgtA protein is involved in the regulation of many crucial biochemical reactions, possibly at the stage of signal transduction.

Amino Acid Sequence↗

PubMed1977

Amber mutations in Escherichia coli essential genes: isolation of mutants affected in the ribosomes.

A method to obtain amber mutations in ribosomal protein genes is described. tit relies on the P1-mediated localized mutagenesis (Hong and Ames, 1971) and on the fact that the recipient strain contains (a) an efficient but genetically unstable suppressor, (b) a particular thermoinducible lambda prophage which kills suppressor hosts at 42 degrees C. Exposure of these bacteria to the high temperature yields frequent suppressor-free derivatives while none will be found if the strain carries an amber mutation in an essential gene. Eleven mutants have been isolated by this method, of which at least six appear to carry amber mutations. All of them map close to, and to the right of spcA, in a region which codes mostly for ribosomal proteins. Three mutants were studied biochemically; all three show defective ribosomal assembly in vivo upon loss of suppression.

Bacteriological Techniques↗

PubMed2004

chk-1 is an essential gene and is required for an S-M checkpoint during early embryogenesis.

The chk1 gene was first discovered in screens for radiation sensitive mutants in S. pombe.(1) Genetic analysis revealed that chk1 is involved in a DNA damage G(2)-M checkpoint. Chk1 becomes activated in response to DNA damage and prevents entry into mitosis by inhibiting the cell cycle machinery. This checkpoint decreases the risk of defective DNA being inherited by daughter cells, therefore reducing the risk of genetic instability. In higher eukaryotes, chk1 homologues have similar checkpoint functions. For example, an avian B-lymphoma cell line that is defective for Chk1 fails to arrest in G(2)-M after DNA damage. Nonetheless, these Chk1 defective cells are viable indicating that Chk1 is not essential for normal somatic cells to divide.(2) In spite of this, mouse and Drosophila homozygous Chk1 mutants die during embryogenesis suggesting that this is an essential gene for embryonic cell cycles.(3,4) What particular role does Chk1 have in directing embryonic cell divisions? Here we used the model organism, C. elegans, to address the role of chk-1 during development. As expected, disruption of chk-1 by RNAi eliminated the DNA damage checkpoint response in C. elegans. In addition, we revealed that chk-1 was predominantly expressed during embryogenesis and in the postembryonic germline. Indeed, we found that chk-1 had an essential role in embryo and germline development. More specifically, disruption of chk-1 expression resulted in embryo lethality, which was attributed to a defect in an intrinsic S-M checkpoint hence causing premature entry into M-phase.

Animals↗

PubMed1985

Cloning of GLN4: an essential gene that encodes glutaminyl-tRNA synthetase in Saccharomyces cerevisiae.

The structural gene for glutaminyl-tRNA synthetase has been isolated from a gene bank of Saccharomyces cerevisiae chromosomal DNA. Cloning was achieved by complementation of a recently described yeast strain that is auxotrophic for glutamine. A multicopy recombinant plasmid with a 5-kilobase-pair genomic insert conferred sixfold elevation in glutaminyl-tRNA synthetase activity and restored a Gln+ phenotype to strains that were Gln- by virtue of a mutant gln4 allele. Subfragments of the 5-kilobase insert directed integration of URA3 to GLN4. Further experiments established that GLN4 is an essential gene that is located on chromosome XV. RNA blots with a GLN4-specific probe detected a single transcript of approximately 2,900 nucleotides.

Amino Acyl-tRNA Synthetases↗

PubMed1985

Characterization of meiosis-deficient mutants by electron microscopy and mapping of four essential genes in the fission yeast Schizosaccharomyces pombe.

Meiosis-deficient mutants of the fission yeast Schizosaccharomyces pombe carrying mei1, mei2, mei3, mei4 and mes1 mutant alleles were characterized by electron microscopy and staining of the nucleus with 4', 6-diamidino-2-phenylindole. Zygotes of either mei1, mei2 or mei3 mutants contained one round nucleus with a single spindle pole body (SPB). These mutants were arrested before premeiotic DNA synthesis. Zygotes of mei4 mutants had one elongated nucleus containing thick electron-dense filaments (linear elements). In the mes1 mutant, the first meiotic division was completed but the SBPs did not duplicate. Modification of the SPB (outer plaque formation) was also blocked and the forespore membrane was not assembled. By haploidization, random spore and tetrad analyses, four essential genes for meiosis (mei2, mei3, mei4 and mes1) were mapped. Gene mei2 was located on chromosome I 14.2 cM distant from ura2. Gene mei3 was linked to ade7 (45.4 cM) on chromosome II. Gene mei4 was linked to cdc2 (0.6 cM) on chromosome II. Gene mes1 was linked to ura3 (25.3 cM) on chromosome I.

Ascomycota↗

PubMed2002

Drosophila nemo is an essential gene involved in the regulation of programmed cell death.

Nemo-like kinases define a novel family of serine/threonine kinases that are involved in integrating multiple signaling pathways. They are conserved regulators of Wnt/Wingless pathways, which may coordinate Wnt with TGFbeta-mediated signaling. Drosophila nemo was identified through its involvement in epithelial planar polarity, a process regulated by a non-canonical Wnt pathway. We have previously found that ectopic expression of Nemo using the Gal4-UAS system resulted in embryonic lethality associated with defects in patterning and head development. In this study we present our analyses of the phenotypes of germline clone-derived embryos. We observe lethality associated with head defects and reduction of programmed cell death and conclude that nmo is an essential gene. We also present data showing that nmo is involved in regulating apoptosis during eye development, based on both loss of function phenotypes and on genetic interactions with the pro-apoptotic gene reaper. Finally, we present genetic data from the adult wing that suggest the activity of ectopically expressed Nemo can be modulated by Jun N-terminal kinase (JNK) signaling. Such an observation supports the model that there is cross-talk between Wnt, TGFbeta and JNK signaling at multiple stages of development.

Animals↗

PubMed2003

Chromosomal deletion formation system based on Tn5 double transposition: use for making minimal genomes and essential gene analysis.

In this communication, we describe the use of specialized transposons (Tn5 derivatives) to create deletions in the Escherichia coli K-12 chromosome. These transposons are essentially rearranged composite transposons that have been assembled to promote the use of the internal transposon ends, resulting in intramolecular transposition events. Two similar transposons were developed. The first deletion transposon was utilized to create a consecutive set of deletions in the E. coli chromosome. The deletion procedure has been repeated 20 serial times to reduce the genome an average of 200 kb (averaging 10 kb per deletion). The second deletion transposon contains a conditional origin of replication that allows deleted chromosomal DNA to be captured as a complementary plasmid. By plating cells on media that do not support plasmid replication, the deleted chromosomal material is lost and if it is essential, the cells do not survive. This methodology was used to analyze 15 chromosomal regions and more than 100 open reading frames (ORFs). This provides a robust technology for identifying essential and dispensable genes.

Chromosome Deletion↗

PubMed1995

Kluyveromyces lactis killer plasmid pGKL2: molecular analysis of an essential gene, ORF5.

The ORF5 of Kluyveromyces lactis killer plasmid pGKL2 (k2) is capable of encoding a small neutral protein of 18 kDa of as yet unassigned function. Although this ORF is located between two larger ORFs, 4 and 6, which it overlaps, RNA analysis showed that it is transcribed monocistronically. One-step gene disruption of ORF5, via in vivo homologous recombination between native plasmid k2 and a transfer vector employing the Saccharomyces cerevisiae LEU2 gene fused to the k2 UCS5 element, yielded Leu+ transformants at high frequencies. The transformants were found to carry a new recombinant form of k2 with ORF5 replaced by the LEU2 marker, termed rk2, in addition to the wild-type plasmids k1 and k2. Northern analysis detected a plasmid-dependent LEU2 transcript distinct in size and regulation from its nuclear counterpart. Recombinant plasmid, rk2, was unable to displace native k2 during Leu+ selective growth; however rk2 was displaced by k2 during non-selective growth. Thus, ORF5 appears to be an essential gene for plasmid integrity and/or maintenance. The ORF5 product was detected by over-expression of an epitope-tagged allele in the baculovirus system. Western analysis using a monoclonal antibody specific for the epitope tag identified a protein band with apparent molecular weight of 20 kDa, corresponding in size to the predicted product.

Amino Acid Sequence↗

PubMed2000

Mouse numb is an essential gene involved in cortical neurogenesis.

During neurogenesis of the mammalian neocortex, neural progenitor cells divide to generate daughter cells that either become neurons or remain as progenitor cells. The mouse numb (m-numb) gene encodes a membrane-associated protein that is asymmetrically localized to the apical cell membrane of dividing cortical progenitor cells and may be segregated to only the apical daughter cell that has been suggested to remain as a progenitor cell. To examine m-numb function during neural development, we generated a loss-of-function mutant allele of m-numb. Mice homozygous for this mutation exhibit severe defects in cranial neural tube closure and precocious neuron production in the forebrain and die around embryonic day 11.5 (E11. 5). These findings suggest that m-numb is an essential gene that plays a role in promoting progenitor cell fate during cortical neurogenesis.

Animals↗

PubMed2001

Genes essential for early events in gonadal development.

The acquisition of a sexually dimorphic phenotype is a critical event in mammalian development. The basic underlying principle of sexual development is that genetic sex--determined at fertilization by the presence or absence of the Y chromosome--directs the embryonic gonads to differentiate into either testes or ovaries. Thereafter, hormones produced by the testes direct the developmental program that leads to male sexual differentiation. In the absence of testicular hormones, the female pathway of sexual differentiation occurs. Recent studies have defined key roles in gonadal development for two transcription factors: Wilms' tumor suppressor 1 (WT1) and steroidogenic factor 1 (SF-1). After presenting a brief overview of gonadal development and sexual differentiation, this chapter reviews the studies that led to the isolation and characterization of WT1 and SF-1, and then discusses how interactions between these two genes may mediate their key roles in a common developmental pathway.

Animals↗

PubMed2001

Extragenic bypass suppressors of mutations in the essential gene BLD2 promote assembly of basal bodies with abnormal microtubules in Chlamydomonas reinhardtii.

bld2-1 mutant Chlamydomonas reinhardtii strains assemble basal bodies with singlet microtubules; bld2-1 cells display flagellar assembly defects as well as positioning defects of the mitotic spindle and cleavage furrow. To further understand the role of the BLD2 gene, we have isolated three new bld2 alleles and three partially dominant extragenic suppressors, rgn1-1, rgn1-2, and rgn1-3. bld2 rgn1-1 strains have phenotypes intermediate between those of bld2 and wild-type strains with respect to flagellar number, microtubule rootlet organization, cleavage furrow positioning, and basal body structural phenotypes. Instead of the triplet microtubules of wild-type cells, bld2 rgn1-1 basal bodies have mixtures of no, singlet, doublet, and triplet microtubules. The bld2-4 allele was made by insertional mutagenesis and identified in a noncomplementation screen in a diploid strain. The bld2-4 allele has a lethal phenotype based on mitotic segregation in diploid strains and in haploid strains generated by meiotic recombination. The lethal phenotype in haploid strains is suppressed by rgn1-1; these suppressed strains have similar phenotypes to other bld2 rgn1-1 double mutants. It is likely that BLD2 is an essential gene that is needed for basal body assembly and function.

Alleles↗

PubMed1997

aarC, an essential gene involved in density-dependent regulation of the 2'-N-acetyltransferase in Providencia stuartii.

The 2'-N-acetyltransferase [AAC(2')-Ia] in Providencia stuartii has a dual function where it is involved in the acetylation of peptidoglycan and certain aminoglycosides. A search for negative regulators of the aac(2')-Ia gene has resulted in the identification of aarC. A missense allele (aarC1) resulted in an 8.9-fold increase in beta-galactosidase accumulation from an aac(2')-lacZ transcriptional fusion. Northern blot analysis demonstrated an increase in aac(2')-Ia mRNA accumulation that was specific to cells at high density. In addition, the aarC1 allele also resulted in a substantial increase in the expression of aarP, a transcriptional activator of the aac(2')-Ia gene. The wild-type aarC gene was isolated by complementation and encodes a predicted protein of 365 amino acids with a molecular mass of 39,815 Da. The predicted AarC protein exhibited 88% amino acid homology to the previously identified GcpE protein of Escherichia coli and 86% homology to a gene product from Haemophilus influenzae. The E. coli gcpE gene was able to functionally complement the aarC1 allele in P. stuartii. The aarC1 allele was identified as a T to G transversion that resulted in a valine to glycine substitution at position 136 in the AarC protein. The aarC gene appears to be essential for cell viability as construction of a disrupted copy (aarC::lacZ) was possible only in cells that carried an episomal copy of aarC or gcpE.

Acetyltransferases↗