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PubMed1994

SPE1 and SPE2: two essential genes in the biosynthesis of polyamines that modulate +1 ribosomal frameshifting in Saccharomyces cerevisiae.

We previously showed that a mutant of Saccharomyces cerevisiae, which cannot make spermidine as a result of a deletion in the SPE2 gene (spe2 delta), exhibits a marked elevation in +1 ribosomal frameshifting efficiency in response to the Ty1 frameshift sequence, CUU AGG C. In the present study, we found that spermidine deprivation alone does not result in increased +1 ribosomal frameshifting efficiency. The high level of +1 ribosomal frameshifting efficiency in spe2 delta cells is the result of the combined effects of both spermidine deprivation and the large increase in the level of intracellular putrescine resulting from the derepression of the gene for ornithine decarboxylase (SPE1) in spermidine-deficient strains.

Enzyme Repression↗
PubMed1996

Polar allele duplication for transcriptional analysis of consecutive essential genes: application to a cluster of Escherichia coli fatty acid biosynthetic genes.

The genes encoding acyl carrier protein and several key fatty acid biosynthetic enzymes are clustered at min 24 of the Escherichia coli chromosome. This cluster of genes is not transcribed as a classical operon, but rather multiple promoters are present and each gene is cotranscribed with at least one other gene. Transcripts specific for single genes ar also present. Transcription of acpP, the gene encoding acyl carrier protein, has been studied in detail. The acpP gene was shown to be transcribed from at least two different promoters by Northern (RNA) blot, primer extension, and deletion analyses, and the major promoter was mapped. We tested whether multiple promoters are necessary to produce acyl carrier protein by use of a new method whereby a transcriptional terminator was integrated into the chromosome upstream of the intact acpP gene. By use of this method (called polar allele duplication), we demonstrate that the promoter located immediately upstream of the coding sequence is sufficient for synthesis of this very abundant protein.

Acyl Carrier Protein↗
PubMed1996

TEL2, an essential gene required for telomere length regulation and telomere position effect in Saccharomyces cerevisiae.

The DNA-protein complexes at the ends of linear eukaryotic chromosomes are called the telomeres. In Saccharomyces cerevisiae, telomeric DNA consists of a variable length of the short repeated sequence C1-3A. The length of yeast telomeres can be altered by mutation, by changing the levels of telomere binding proteins, or by increasing the amount of C1-3A DNA sequences. Cells bearing the tel1-1 or tel2-1 mutations, known previously to have short telomeres, did not respond to perturbations that caused telomere lengthening in wild-type cells. The transcription of genes placed near yeast telomeres is reversibly repressed, a phenomenon called the telomere position effect. The tel2-1 mutation reduced the position effect but did not affect transcriptional repression at the silent mating type cassettes, HMRa and HML alpha. The TEL2 gene was cloned, sequenced, and disrupted. Cells lacking TEL2 function died, with some cells arresting as large cells with three or four small protrusions or "blebs."

Amino Acid Sequence↗
PubMed1987

SSN20 is an essential gene with mutant alleles that suppress defects in SUC2 transcription in Saccharomyces cerevisiae.

Dominant and recessive mutations at the SSN20 locus were previously isolated as extragenic suppressors of mutations in three genes (SNF2, SNF5, and SNF6) that are required in trans to derepress invertase expression. All ssn20 alleles cause recessive, temperature-sensitive lethality. In this study we cloned the SSN20 gene, identified a 4.6-kilobase poly(A)-containing RNA, and showed that disruption of the gene is lethal in a haploid cell. Genetic mapping of SSN20 to a locus on chromosome VII 10 centimorgans distal to cly8 led to the finding that SSN20 is the same gene as SPT6, which affects expression of delta insertions in the 5' noncoding region of HIS4 (F. Winston, D. T. Chaleff, B. Valent, and G. R. Fink, Genetics 107:179-197, 1984). We also showed that an ssn20 mutation restored expression of secreted invertase from deletions of the SUC2 upstream regulatory region; ssn20 restored derepression of SUC2 mRNA in strains with a SUC2 upstream region deletion or a snf2 mutation. Increased or decreased gene dosage of SSN20 also suppressed defects that are suppressed by ssn20 missense mutations. These findings suggest that SSN20 plays a role in general transcriptional processes.

Chromosome Mapping↗
PubMed2005

An essential gene for fruiting body initiation in the basidiomycete Coprinopsis cinerea is homologous to bacterial cyclopropane fatty acid synthase genes.

The self-compatible Coprinopsis cinerea homokaryon AmutBmut produces fruiting bodies without prior mating to another strain. Early stages of fruiting body development include the dark-dependent formation of primary hyphal knots and their light-induced transition to the more compact secondary hyphal knots. The AmutBmut UV mutant 6-031 forms primary hyphal knots, but development arrests at the transition state by a recessive defect in the cfs1 gene, isolated from a cosmid library by mutant complementation. A normal primordia phenotype was achieved when cfs1+ was embedded at both sides in at least 4.0 kb of native flanking DNA. Truncations of the flanking DNA lead to reduction in transformation frequencies and faults in primordia tissue formation, suggesting that the gene is also acting at later stages of development. The cfs1 gene encodes a protein highly similar to cyclopropane fatty acid synthases, a class of enzymes shown in prokaryotes and recently in a plant to convert membrane-bound unsaturated fatty acids into cyclopropane fatty acids. In C. cinerea 6-031, the mutant cfs1 allele carries a T-to-G transversion, leading to an amino acid substitution (Y441D) in a domain suggested to be involved in the catalytic function of the protein and/or membrane interaction.

Agaricales↗
PubMed1998

Study of a region on yeast chromosome XIII that complements pet G199 mutants (COX7) and carries a new non-essential gene.

The mutants of Saccharomyces cerevisiae assigned to complementation group G199 are deficient in mitochondrial respiration and lack a functional cytochrome oxidase complex. Recombinant plasmids capable of restoring respiration were cloned by transformation of mutants of this group with a yeast genomic library. Sequencing indicated that a 2.1-kb subclone encompasses the very end (last 11 amino acids) of the PET111 gene, the COX7 gene and a new gene (YMR255W) of unknown function that potentially codes for a polypeptide of 188 amino acids (about 21.5 kDa) without significant homology to any known protein. We have shown that the respiratory defect corresponding to group G199 is complemented by plasmids carrying only the COX7 gene. The gene YMR255W was inactivated by one-step gene replacement and the disrupted strain was viable and unaffected in its ability to grow in a variety of different test media such as minimal or complete media using eight distinct carbon sources at three pH values and temperatures. Inactivation of this gene also did not affect mating or sporulation.

Amino Acid Sequence↗
PubMed1992

Cloning and expression of the essential gene for guanylate kinase from yeast.

Guanylate kinase catalyzes the reversible transfer of the terminal phosphoryl group of ATP to the acceptor molecule GMP. Detailed analysis of the in vivo function of this enzyme has been limited by the lack of any genetic data. Using oligonucleotides based on amino acid sequence information of the yeast enzyme, the Saccharomyces cerevisiae gene, GUK1, was isolated and characterized. The gene is present in single copy and maps to chromosome IV. Insertional mutagenesis of the GUK1 locus caused recessive lethality, indicating that this enzyme is necessary for vegetative cell growth. Using inducible expression systems, guanylate kinase was produced in large amounts both in S. cerevisiae and in Escherichia coli.

Amino Acid Sequence↗
PubMed1990

Nucleotide sequence and genetic characterization reveal six essential genes for the LIV-I and LS transport systems of Escherichia coli.

The nucleotide sequence of the genes encoding the high affinity, branched-chain amino acid transport systems LIV-I and LS has been determined. Seven genes are present on a 7568-base pair DNA fragment, six of which participate directly in branched-chain amino acid transport. Two periplasmic amino acid-binding proteins are encoded by the livJ (LIV-BP) and livK (LS-BP) genes. These two proteins confer specificity on the LIV-I and LS transport systems. livK is the first gene in a polycistronic message that includes four genes encoding membrane components, livHMGF. The protein products of the livHMGF genes are shared by the two systems. An analysis of the livH and livM DNA sequences suggests that they encode hydrophobic proteins capable of spanning the membrane several times. The LivG and LivF proteins are less hydrophobic, but are also tightly associated with the membrane. Both LivG and LivF contain the consensus sequence for adenine nucleotide binding observed in many other transport proteins. A deletion strain that does not express any of the liv genes was constructed. This strain was used to show that each of the membrane component genes is required for high affinity leucine transport, including two genes, livM and livF, for which no previous genetic evidence had been obtained.

Amino Acid Sequence↗
PubMed2000

Transposon-based approaches to identify essential bacterial genes.

Transposons are a powerful tool for identifying genes essential for bacterial viability. The availability of many bacterial genome sequences and the large number of genes of unknown function therein have inspired the generation of a variety of different approaches. These methods are described and their advantages and disadvantages are discussed.

Bacteria↗
PubMed2003

The trichothecene biosynthesis gene cluster of Fusarium graminearum F15 contains a limited number of essential pathway genes and expressed non-essential genes.

We report for the first time the complete structure and sequence of the trichothecene biosynthesis gene cluster (i.e. Tri5-cluster) from Fusarium graminearum F15, a strain that produces 3-acetyldeoxynivalenol (3-ADON). A putative tyrosinase and polysaccharide deacetylase gene flank the Tri5-cluster: the number of pathway genes between them is less than half the total number of steps necessary for 3-ADON biosynthesis. In comparison with partial Tri5-cluster sequences of strains with 15-acetyldeoxynivalenol and 4-acetylnivalenol chemotypes, the Tri5-cluster from strain F15 contains three genes that are apparently unnecessary for the biosynthesis of 3-ADON (i.e. Tri8 and Tri3, which are expressed, and pseudo-Tri13, which is not expressed). In addition, the Tri7 gene was missing from the cluster. Recombinant TRI3 protein showed limited trichothecene C-15 acetylase activity. In contrast, recombinant TRI8 protein displayed no C-3 deacetylase activity, suggesting that the loss or alteration of function contribute directly to the chemotype difference.

Fusarium↗
PubMed2005

How essential are nonessential genes?

Gene essentiality in bacteria has been identified in silico, focusing on gene persistence, or experimentally, focusing on the growth of knockouts in rich media. Comparing 55 genomes of Firmicutes and Gamma-proteobacteria to identify the genes which, while persistent among genomes, do not lead to a lethal phenotype when inactivated, we show that the characteristics of persistence, conservation, expression, and location are shared between persistent nonessential (PNE) genes and experimentally essential genes. PNE genes show an overrepresentation of genes related to maintenance and stress response. This outlines the limits of current experimental techniques to define gene essentiality and highlights the essential role of genes implicated in maintenance which, although dispensable for growth, are not dispensable from an evolutionary point of view. Firmicutes and Gamma-proteobacteria are mostly differing in the construction of the cell envelope, DNA replication and proofreading, and RNA degradation. In addition to suggesting functions for persistent genes that had until now resisted identification, we show that these genes have many characters in common with experimentally identified essential genes. They should then be regarded as truly essential genes.

Codon↗
PubMed1998

T-DNA mediated disruption of essential gametophytic genes in Arabidopsis is unexpectedly rare and cannot be inferred from segregation distortion alone.

Many genes are thought to be expressed during the haploid phase in plants, however, very few haploid-specific genes have been isolated so far. T-DNA insertion mutagenesis is a powerful tool for generating mutations that affect gametophyte viability and function, as disruption of a gene essential for these processes should lead to a defect in the transmission of the gametes. Mutants can therefore be screened on the basis of segregation distortion for a reporter resistance gene contained in the T-DNA. We have screened the Versailles collection of Arabidopsis transformants for 1:1 KanR:KanS segregation after selfing, focussing on gametophyte mutations which show normal transmission through one gametophyte and cause lethality or dysfunction of the other. Only 1.3% (207) of the 16,000 lines screened were scored as good candidates. Thorough genetic analysis of 38 putative T-DNA transmission defect lines (Ttd) identified 8 defective gametophyte mutants, which all showed 0 to 1% T-DNA transmission through the pollen. During the screen, we observed a high background of low-penetrance mutations, often affecting the function of both gametophytes, and many lines which were likely to carry chromosomal rearrangements. The reasons for the small number of retained lines (all male gametophytic) are discussed, as well as the finding that, for most of them, residual T-DNA transmission is obtained through the affected gametophyte.

Arabidopsis↗
PubMed1984

Inactivation of essential division genes, ftsA, ftsZ, suppresses mutations at sfiB, a locus mediating division inhibition during the SOS response in E. coli.

A dominant sfiB allele has been cloned which renders partial diploids of an sfiB + Escherichia coli host resistant to division inhibition mediated by the SOS response. Transpositional mutagenesis was used to map the position of this sfiB114 allele, carried by a plasmid pLG552 , to an approximately 0.6-kb region overlapping the coding regions for ftsA and ftsZ , two genes essential for normal division. Most Tn 1000 insertions which inactivated sfiB114 also inactivated the ftsA function and caused the disappearance of both a 47-K polypeptide and reduced levels of a 42-K polypeptide in maxi-cells carrying pLG552 . An additional insertion inactivating sfiB114 was mapped to the right of ftsA and resulted in loss of the 42-K but not the 47-K polypeptide in maxi-cells. Moreover, a 2.1-kb BamHI-EcoRI DNA fragment was subcloned which carried ftsA and coded for a 47-K polypeptide but did not carry sfiB114 and did not complement ftsZ . We conclude that sfiB114 is located within ftsZ coding for a 42-K polypeptide. Nevertheless, insertions into ftsZ coding the 47-K polypeptide suppress the sfiB114 allele by substantially reducing the synthesis of the FtsZ ( SfiB114 ) polypeptide. The level of residual FtsZ synthesis was minimal when Tn 1000 was inserted closest to the distal end of ftsA , indicating the presence of a regulatory region essential for maximal expression of ftsZ .

Base Sequence↗