Molecular & general genetics : MGG最新文献

SpCCE1 (YDC2) from Schizosaccharomyces pombe is a DNA structure-specific endonuclease that resolves Holliday junctions in vitro. To investigate the in vivo function of SpCCE1 we made an Spcce1:ura4+ insertion mutant strain. This strain is viable and, despite being devoid of the Holliday junction resolvase activity that is readily detected in fractionated extracts from wild-type cells, exhibits normal levels of UV sensitivity and spontaneous or UV-induced mitotic recombination. In accordance with the absence of a nuclear phenotype, we show by fluorescence microscopy that a SpCCE1-GFP fusion localises exclusively to the mitochondria of S. pombe. In Saccharomyces cerevisiae the homologue of SpCCE1, CCE1, is known to function in the mitochondria where its role appears to be to remove recombination junctions and thus facilitate mitochondrial DNA segregation. A similar function can probably be attributed to SpCCE1 in S. pombe, since the majority of mitochondrial DNA from the Spcce1::ura4- strain is in an aggregated form apparently due to extensive interlinking of DNA molecules by recombination junctions. Surprisingly, this marked effect on the conformation of mitochondrial DNA results in little or no effect on proliferation or viability of the Spcce1::ura4+ strain. Possible explanations are discussed.

In eukaryotes, the levels of ribosomal proteins are coordinately regulated under varying nutritional conditions and at different developmental stages. Little is known about how ribosomal protein levels are coupled to the levels of rRNA. The formation of a ribonucleoprotein particle composed of 5S rRNA and a ribosomal protein is an early step in ribosome assembly. To investigate how these two ribosomal components are regulated in Neurospora crassa, we cloned the gene encoding the 5S rRNA-binding ribosomal protein (crp-4) and developed a novel system for measuring relative 5S rRNA transcriptional rates in vivo, using a reporter RNA derived from the 40S precursor RNA. The reporter RNA is cleaved from the 5S rRNA in vivo and therefore allows us to distinguish between changes in the 5S rRNA transcription rate and 5S rRNA stability. Using this system, we found that transcription of 5S rRNA is constitutive and is not coordinated with the levels of crp-4 mRNA or with 40S rRNA levels during a carbon upshift or a carbon downshift.

The gene MUS81 p6ethyl methansulfonate, UV sensitive) was identified as clone 81 in a two-hybrid screen using the Saccharomyces cerevisiae Rad54 protein as a bait. It encodes a novel protein with a predicted molecular mass of 72,316 (632 amino acids) and contains two helix-hairpin-helix motifs, which are found in many proteins involved in DNA metabolism in bacteria, yeast, and mammals. Mus81p also shares homology with motifs found in the XPF endonuclease superfamily. Deletion of MUS81 caused a recessive methyl methansulfonate- and UV-sensitive phenotype. However, mus81delta cells were not significantly more sensitive than wild-type to gamma-radiation or double-strand breaks induced by HO endonuclease. Double mutant analysis suggests that Rad54p and Mus81p act in one pathway for the repair of, or tolerance to, UV-induced DNA damage. A complex containing Mus81p and Rad54p was identified in immunoprecipitation experiments. Deletion of MUS81 virtually eliminated sporulation in one strain background and reduced sporulation and spore viability in another. Potential homologs of Mus81p have been identified in Schizosaccharomyces pombe, Caenorhabditis elegans and Arabidopsis thaliana. We hypothesize that Mus81p plays a role in the recognition and/or processing of certain types of DNA damage (caused by UV and MMS) during repair or tolerance processes involving the recombinational repair pathway.

The ascomycete Fusarium fujikuroi could be transformed stably to hygromycin resistance only when the transforming plasmid contained a fragment of DNA from the fungus. The transformation frequencies were roughly independent of the sequence of the particular fungal DNA fragment used, of its size (1.8 or 6 kb), and of whether this DNA was present only once in the fungal genome or about forty times (the genes for ribosomal RNA). The plasmid was integrated into the fungal genome by homologous recombination in the eighteen transformants tested; ectopic integration was never observed. The carB gene of F. fujikuroi was cloned and shown to complement unpigmented mutants deficient in phytoene dehydrogenase. A mutant carB allele was prepared in vitro and used to transform wild-type protoplasts; the transformants contained a genomic duplication and were heterozygous for carB; the mutant allele replaced the original wild-type allele when this was spontaneously lost in the transformants. This loss was due to gene conversion in some cases and to recombination between repeated sequences in others.

In order to better understand the involvement of the DNA molecule in the replication initiation process we have characterized the structure of the DNA at Autonomously Replicating Sequences (ARSs) in Saccharomyces cerevisiae. Using a new method for anti-bent DNA analysis, which allowed us to take into account the bending contribution of each successive base plate, we have investigated the higher-order structural organization of the DNA in the region which immediately surrounds the ARS consensus sequence (ACS). We have identified left- and right-handed anti-bent DNAs which flank this consensus sequence. The data show that this organization correlates with an active ACS. Analysis of the minimum nucleotide sequence providing ARS function to plasmids reveals an example where the critical nucleotides are restricted to the ACS and the right-handed anti-bent DNA domain, although most of the origins considered contained both left- and right-handed anti-bent DNAs. Moreover, mutational analysis shows that the right-handed form is necessary in order to sustain a specific DNA conformation which is correlated with the level of plasmid maintenance. A model for the role of these individual structural components of the yeast replication origin is presented. We discuss the possible role of the right-handed anti-bent DNA domain, in conjunction with the ACS, in the process of replication initiation, and potentialities offered by the combination of left- and right-handed structural components in origin function.

The proper folding of tubulins prior to their incorporation into microtubules requires a group of conserved proteins called cofactors A to E. In fission yeast, homologues of these cofactors (at least B, D and E) are necessary for the biogenesis of microtubules and for cell viability. Here we show that the temperature-sensitive alp11-924 mutant, which is defective in the cofactor B homologue, contains an opal nonsense mutation, which results in the production of a truncated Alp11B protein (Alp11(1-118). We isolated a tRNA(Trp) gene as a multicopy suppressor of this mutation, which rescues alp11-924 by read-through of the nonsense codon. The truncated Alp1-118 protein lacks the C-terminal half of Alp11B, consisting of a central coiled-coil region and the distal CLIP-170 domain found in a number of proteins involved in microtubule functions. Both of these domains are required for the maintenance of microtubule architecture in vivo. Detailed functional analyses lead us to propose that Alp11B comprises three functional domains: the N-terminal half executes the essential function, the central coiled-coil region is necessary for satisfactory maintenance of cellular alpha-tubulin levels, and the C-terminal CLIP-170 domain is required for efficient binding to alpha-tubulin.

Mutations at several loci affect the sensitivity of the yeast Saccharomyces cerevisiae to staurosporine. We report here the characterization of novel staurosporine- and temperature-sensitive mutants (stt). Cloning and integration mapping showed that the genes STT2/ STT6, STT5, STT7, STT8 and STT9 are allelic to VPS18, ERG10, GPI1, VPS34 and VPS11, respectively. The products of ERG10 and GPI1, respectively, catalyze mevalonate and glycosyl phosphatidylinositol anchor synthesis, while VPS18 and VPS11 genes belong to the class C VPS (Vacuolar Protein Sorting) genes, and the VPS34 gene is classified as a class D VPS. Therefore, staurosporine sensitivity is affected by ergosterol and glycolipid biosynthesis and by vacuolar functions. We found that other vps mutants belonging to classes C and D exhibit staurosporine sensitivity, and that they show calcium sensitivity and fail to grow on glycerol as the sole carbon source; both of the last two characteristics are shared by vacuolar H+-ATPase mutants (vma). As vma mutants were also found to show staurosporine-sensitive growth, staurosporine sensitivity is likely to be affected by acidification of the vacuole. Moreover, wild type yeast cells are more sensitive to staurosporine in alkaline media than in acidic media, suggesting that staurosporine is exported from the cytosol by H+/drug antiporters. Pleiotropic drug resistance (PDR) genes also provide some resistance to staurosporine, because deltapdr5, deltasnq2 and deltayor1 strains are more sensitive to staurosporine than the wild-type strain. This suggests that staurosporine is also exported by the ATP-binding cassette (ABC) transporters on the plasma membrane. vma mutants and vps mutants of classes C and D vps are sensitive to hygromycin B and vanadate, while ABC transporter-depleted mutants do not show such sensitivity, indicating that two systems differ in their ability to protect the cell against different types of drug.

The tryptophan synthase-encoding gene, trpB, of Aspergillus nidulans was cloned and characterized. It was mapped to chromosome I, between the gene medA, which is required for sexual and asexual development, and an ORF encoding a protein with significant similarity to subunit B of vacuolar ATP synthases. The 5' untranslated region was found to be at least 142 nucleotides (nt) long, the poly(A) addition site was localized at position + 216 relative to the stop codon by sequencing of several independent cDNA clones. The trpB gene contains two exons separated by an intron of 105 nt, which is located close to the 5' end of the ORF. Directly upstream of the transcriptional start site, one well conserved potential binding site for the cross-pathway control transcriptional activator CPCA was found. The level of trpB transcript was shown to be regulated by cross-pathway control. A knockout mutant for trpB displays tryptophan auxotrophy, no trpB transcript is detectable, and development is perturbed to an extent that is dependent on the amount of tryptophan added to the medium. The trpB gene encodes a protein of 723 amino acids, with a calculated molecular weight of 77.6 kDa. The deduced amino acid sequence shows 72.6% similarity to the tryptophan synthase of Neurospora crassa. Most amino acid residues essential for catalytic activity in the tryptophan synthase of Salmonella typhimurium are conserved. The linker region joining the two domains of the enzyme is 13 residues longer than the longest connector found so far in tryptophan synthases from fungi.

Ligation of the ubiquitin-like protein SUMO (Smt3p) to other proteins is essential for viability of the yeast Saccharomyces cerevisiae. Like ubiquitin (Ub), SUMO undergoes ATP-dependent activation by a specific activating enzyme. SUMO-activating enzyme is a heterodimer composed of Uba2p and Aos1p, polypeptides with sequence similarities, respectively, to the C- and N-terminal parts of Ub-activating enzyme. To study the function of SUMO conjugation, we isolated uba2 mutants that were temperature-sensitive for growth. In these mutants conjugation of SUMO to other proteins was drastically reduced, even at the temperature permissive for growth. In a screen for spontaneous suppressors of the temperature-sensitive growth phenotype of the mutant uha2-ts9, we isolated a strain with a null mutation (sut9) in a gene of hitherto unknown function (SUT9/YIL031W/SMT4). This gene encodes a protein with similarities to Ulp1p, a dual-function protease that processes the SUMO precursor and deconjugates SUMO from its substrates. The novel protein was therefore termed Ulp2p. Inactivation of ULP2 in a strain expressing wild-type SUMO-activating enzyme resulted in slow and temperature-sensitive growth, and accumulation of SUMO conjugates. Thus, mutations in SUMO-activating enzyme and mutations in Ulp2p suppress each other, indicating that SUMO conjugation and deconjugation must be in balance for cells to grow normally. Other phenotypes of ulp2 mutants include a defect in cell cycle progression, hypersensitivity to DNA damage, and chromosome mis-segregation. Ulp2p is predominantly located within the nucleus, whereas Ulp1p colocalizes with nuclear pore complex proteins, indicating that the apparently distinct functions of the two SUMO deconjugating enzymes are spatially separated.

Vitellogenins (Vtg) are the major yolk proteins in most oviparous organisms. They are encoded by a small number of genes--between one and four depending on the species. Characterization of the Vtg region in the genome of the rainbow trout reveals unusual features, however, in that this locus contains twenty complete genes and ten pseudogenes per haploid genome. The Vtg genes differ from each other by insertion, deletion and rearrangement events, although, at the sequence level, they show a high degree of similarity. Fluorescent in situ hybridization (FISH), pulsed-field gel electrophoresis (PFGE) and Southern analysis indicate that all gene copies are contained in a single 1,500-kb region, and that most of the genes form tandem arrays separated by a conserved 4.5-kb intergenic region. The presence of large reiterated fragments indicates that this region has been subjected to several amplification events. The presence of a retroposon element (called 19) in Vtg intron 9 appears to be responsible for the silencing of at least nine of the ten pseudogenes. Two other incomplete retrotransposons (one LTR- and one LINE-type) and sequences derived from a HIV-like retrovirus are inserted into the conserved intergenic region, very close to the transcription start site. Their presence in all Vtg 5'-flanking regions suggests a possible role in gene amplification at this locus.