Molecular & general genetics : MGG最新文献

The sfiW locus of Salmonella enterica, previously identified by mutations that suppress the cell division defect of His-constitutive (His(c)) strains, corresponds to serC, the bifunctional gene for phosphoserine-oxoglutarate aminotransferase (SerC) and 2-ketoerythroic acid 4-phosphate transaminase (PdxF). SerC- mutants form small, nearly spherical cells in a wild-type (His+) background, suggesting that the SerC/PdxF product acts as a septation antagonist. Suppression of His(c) filamentation by serC mutations may be explained by loss of the anti-septation activity of SerC/PdxF. The isolation of serC alleles that have lost their biosynthetic activities but are still able to inhibit septum formation suggests that the anti-septation activity of the SerC/PdxF product is unrelated to its known roles in serine and pyridoxine biosynthesis.

The yeast Kluyveromyces lactis is can utilise a wide range of non-fermentable carbon compounds as sole sources of carbon and energy, and differs from Saccharomyces cerevisiae in being able to carry out oxidative and fermentative metabolism simultaneously. In S. cerevisiae, growth on all non-fermentable carbon sources requires Cat8p, a transcriptional activator that controls the expression of gluconeogenic and glyoxylate cycle genes via CSREs (Carbon Source Responsive Elements). The down-regulation of Cat8p by fermentable carbon sources is the primary factor responsible for the tight repression of gluconeogenesis by glucose in S. cerevisiae. To analyse the regulation of gluconeogenesis in K. lactis, we have cloned and characterised the K. lactis homologue of CAT8 (KlCAT8). The gene was isolated by multicopy suppression of a fog2/klsnf1 mutation, indicating a similar epistatic relationship between KlSNF1 and KlCAT8 as in the case of the S. cerevisiae homologues. KlCAT8 encodes a protein of 1445 amino acids that is 40% identical to ScCat8p. The most highly conserved block is the putative Zn(II)2Cys6 DNA-binding domain, but additional conserved regions shared with members of the zinc-cluster family from Aspergillus define a subfamily of Cat8p-related proteins. KlCAT8 complements the growth defect of a Sccat8 mutant on non-fermentable carbon sources. In K. lactis, deletion of KlCAT8 severely impairs growth on ethanol, acetate and lactate, but not on glycerol. Derepression of enzymes of the glyoxylate cycle--malate synthase and particularly isocitrate lyase--was impaired in a Klcat8 mutant, whereas Northern analysis revealed that derepression of KlFBP1 and KlPCK1 does not require KlCat8p. Taken together, our results indicate that in K. lactis gluconeogenesis is not co-regulated with the glyoxylate cycle, and only the latter is controlled by KlCat8p.

Rearrangements of T-DNAs during genetic transformation of plants can result in the insertion of transgenes in the form of repeats into the host genome and frequently lead to loss of transgene expression. To obtain insight into the mechanism of repeat formation we screened 45 transgenic lines of aspen and hybrid aspen transformed with six different gene constructs. The frequency of T-DNA repeat formation among randomly screened transgenic lines was found to be about 21%. In ten transgenic lines direct repeats were detected. An inverted repeat was found in one other transgenic line. Sequencing of the junctions between the T-DNA inserts revealed identical residual right-border repeat sequences at the repeat junctions in all ten transgenic lines that had direct repeats. Formation of "precise" junctions based on short regions of sequence similarity between recombining strands was observed in three transgenic lines transformed with the same plasmid. Additional DNA sequences termed filler DNAs were found to be inserted between the T-DNA repeats at eight junctions where there was no similarity between recombining ends. The length of the filler DNAs varied from 4 to almost 300 bp. Small filler DNAs--a few base pairs long--were in most cases copied from T-DNA near the break points. The large filler sequences of about 300 bp in two transgenic lines were found to be of host plant origin, suggesting that transgene repeat formation occurred as a result of the simultaneous invasion of a receptive site in the host genome by two independent T-DNA strands. On the basis of the results obtained, and in the light of previous reports on T-DNA/plant DNA junctions in aspen and other crop plants, a mechanistic model for transgene rearrangement and filler formation is suggested.

An ATM-like gene was identified in the genome of Caenorhabditis elegans. The putative product of the gene, termed Ce-atl-1 (C. elegans ATM-like 1) consists of 2514 amino acid residues. The C-terminal sequence, which contains a PI-3 kinase-like domain, showed good homology with the products of the gene MEC1/ESR1 from budding yeast, the rad3+ gene of fission yeast and mammalian ATM (ataxia-telangiectasia and rad3+ related) genes. The results of RNA-mediated interference indicated that the major phenotype associated with repression of Ce-atl-1 was lethality (approximately 50-80%) during early embryogenesis. Among the surviving progeny, males (XO animals) arose at a high frequency (2-30%). In addition, 5% of oocyte chromosomes demonstrated aneuploidy due to a defect in pre-meiotic chromosomal segregation. Gene expression analyses indicated that Ce-atl-1 mRNA was expressed in all larval stages and that its level increased about fivefold in the adult stage. The adult expression level was decreased in the glp-4 mutant, which is defective in germ line proliferation. Ce-atl-1 was strongly expressed in both the mitotic and meiotic cells of adult gonads. In summary, Ce-atl-1 appears to be important for early embryogenesis, and loss of its function results in a defect in chromosome segregation, similar to what has been observed for AT-related proteins.

The Ku heterodimer binds to the ends of double-stranded breaks (DSBs) in DNA, and is involved in nonhomologous end joining. HDF1 and HDF2, which have been identified in Saccharomyces cerevisiae as homologues of the Ku70 and Ku80 proteins of mammals, reduce radiosensitivity only when homologous recombination repair is impaired and, therefore, affect DSB repair via nonhomologous recombination. Although it has been reported that homologous recombination is defective in the hdf1 null mutant, the roles of HDF1 and HDF2 in this process are not completely clear. We investigated the effect of HDF1 and HDF2 on intrachromosomal recombination by measuring rates of deletion between direct repeats caused by spontaneous and DNA damage-induced events (DEL recombination). We found a decrease in spontaneous DEL recombination in both TCY5 (hdf1delta) and TCY6 (hdf2delta) strains, suggesting that HDF1 and HDF2 play a role in homologous recombination. As DEL recombination events may occur by sister chromatid conversion and/or single-strand annealing, which is initiated by DSBs, HDF1 and HDF2 may be required to recruit proteins to the damaged ends so as to promote single-strand annealing. The strains TCY5 and TCY6 are also defective in methylmethane sulfonate (MMS)- and X-ray-induced, but not in UV-induced DEL recombination. This confirms that HDF1 and HDF2 are required for the completion of DEL recombination by single strand annealing.

Characterization of the Neurospora crassa mus-25 mutant suggests that it is defective in recombination repair and belongs to the uvs-6 epistasis group. It shows a high sensitivity to the alkylating agents methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), but not to UV radiation. It is barren (i.e. does not produce ascospores) in homozygous crosses. The frequency of MMS-induced mutations at the ad-3 loci is approximately three times higher than in the wild type. The ratio of homologous to nonhomologous integration of the pMTR::HYG plasmid is much lower than in wild type. The mus-25 mutant is epistatic to the mei-3 mutant for MMS sensitivity. mei-3, which is a homololog of the Saccharomyces cerevisiae gene RAD51, is a member of the uvs-6 epistasis group which contains several genes that are homologous to recombination repair genes in other organisms. The mus-25 gene was cloned by identifying a genomic DNA fragment which complements the MMS sensitivity of the mutant. The amino acid sequence deduced from the cloned DNA showed a high degree of homology to the Rad54 protein, which is involved in recombinational repair in S. cerevisiae. Comparison of the nucleotide sequences of the genomic and cDNAs of the mus-25 gene revealed an ORF of 2505 bp with a single 118-bp intron beginning immediately after the second nucleotide of the AUG start codon. The molecular weight of the deduced gene product was 93.5 kDa. The transcript level was raised within 60 min after UV irradiation or MMS treatment, as also observed for the expression of the other N. crassa recombinational repair genes, suggesting the existence of a common mechanism which induces expression of the recombinational repair genes in response to DNA damage.

The rhizobial nodulation gene nodC encodes an N-acetylglucosaminyltransferase that is responsible for the synthesis of chitin oligosaccharides. These oligosaccharides are precursors for the synthesis of the lipo-chitin oligosaccharides that induce cell division and differentiation during the development of nitrogen-fixing root nodules in leguminous plants. The NodC proteins of Mesorhizobium loti and Sinorizobium meliloti yield chitinpentaose and chitintetraose as their main products, respectively. In order to localize regions in these enzymes that are responsible for this difference in product chain length, a set of six chimeric enzymes, comprising different combinations of regions of the NodC proteins from these two bacteria, was expressed in Escherichia coli. The oligosaccharides produced were analyzed using thin-layer chromatography. The major conclusion from this work is that a central 91-amino acid segment does not play any obvious role in determining the difference in the chain length of the major product. Furthermore, the characteristically predominant synthesis of chitintetraose by S. meliloti NodC is mainly dependent on a C-terminal region of maximally 164 amino acids; exchange of only this C-terminal region is sufficient to completely convert the M. loti chitinpentaose synthase into an S. meliloti-like chitintetraose synthase. The N-terminal region of 170 amino acids also plays a role in restricting the length of the major product to a tetramer. However, the role of the C-terminal region is clearly dominant, since exchanging the N-terminal region has no effect on the relative amounts of chitintetraose and -pentaose produced when the C-terminal region of S. meliloti NodC is present. The length of a predicted beta-strand around residue 300 in the C-terminal region of various NodC proteins is the only structural element that seems to be related to the length of the chitin oligosaccharides produced by these enzymes; the higher the amount of chitintetraose relative to chitinpentaose, the shorter the predicted beta-strand. This element may therefore be important for the effect of the C-terminal 164 amino acids on chitin oligosaccharide chain length.

A rice transcript, Rim2, was identified that accumulated in both incompatible and compatible interactions between rice and Magnaporthe grisea. The Rim2 transcript also accumulated in response to treatment with a cell wall elicitor derived from M. grisea. A 3.3-kb RIM2 cDNA clone was isolated and is predicted to encode a protein of 653 amino acids, which shares 32 55% identity with TNP2-like proteins encoded by CACTA transposons of other plants. A 1.05-kb segment of the Rim2 sequence shows 82% nucleotide sequence identity with sequences flanking the A1 and C members of the rice Xa21 disease resistance gene family. The 5'-upstream region of Rim2 was cloned and the transcriptional start sites were identified. The 5' and 3' noncoding termini of Rim2 are AT-rich. A cis-element showing similarity to a sequence that mediates defense-associated transcriptional activation of the tobacco retrotransposon Tnt1, and four motifs that fit the consensus sequence of the elicitor-responsive elements in the promoters of the parsley PR-1 genes were found in the 5'-upstream region. Four imperfect tandem repeats were identified in the 3' noncoding terminus. Southern analysis with genomic DNA from different rice species indicated that Rim2 is present in 3-4 copies per genome. These results suggest that Rim2 may be one component of a large CACTA-like element, whose transcript accumulates in response to attack by pathogens.

The construction of the first balancer chromosome, FiM1, for the medfly Ceratitis capitata is described. This chromosome has three overlapping pericentric inversions and is marked with dominant and recessive mutations. The inversion breakpoints of FiM1 suppress recombination throughout the length of the fifth chromosome, allowing lethal mutations to be recovered and maintained. This chromosome will provide a powerful tool for the manipulation of laboratory stocks, in particular, the recovery of new mutant and transgenic strains. We demonstrate the use of FiM1 for the recovery and maintenance of chromosomes carrying lethal mutations.

We screened for mutant strains of Saccharomyces cerevisiae that are sensitive to overexpression of specific cyclins, and identified mutations in two genes that caused growth inhibition in response to mild overexpression of Clb3. One was the ANP1 gene, which encodes a glycosyltransferase previously identified by a similar strategy using Clb2 instead of Clb3. This paper describes the second strain of S. cerevisiae that is hypersensitive to Clb3 expression. The gene mutated in this strain was identified as PMR1, which encodes a Ca2+-ATPase located in the Golgi membrane. The protein product of pmr1-1 was truncated at residue 409 and thus lacked the C-terminal ATPase domain. The pmr1-1 strain was hypersensitive to over-expression of Clb3, but not Cln2, Clb5 or Clb2. The lethality due to Clb3 expression in pmr1-1 could be suppressed by adding Ca2+ ions to the medium. The pmr1-1 strain proved to be defective in glycosylation, and the defects in glycosylation were exacerbated by high levels of Clb3. On induction of Clb3 expression in the pmr1-1 strain, the cells arrested at anaphase with an elongated daughter bud. We discuss possible interpretations of this synthetic lethal phenotype.