Molecular & general genetics : MGG最新文献

We have cloned and sequenced the 5' and 3' ends of the Drosophila homolog of the vertebrate c-ret gene, Ret, and have derived from it the predicted protein sequence of Ret. The extracellular domain of Ret is very widely diverged from that of its vertebrate counterparts but the cadherin motif present in vertebrate c-ret proteins can also be discerned in Ret. As with the vertebrate gene, multiple splice variants were detected at the 5'-end of Ret, one of which inserts an exon with a protein-terminating frameshift into the cDNA. In contrast to human c-ret, which may vary its signalling specificity by using splicing-derived, alternative C-terminal sequences, Ret cDNAs showed no variation at their 3'-ends.

Two oligonucleotide probes derived from conserved motifs in peptide synthetases were hybridized with a cosmid library of Planobispora rosea genomic DNA. Detailed characterization of the physical organization of the positive cosmids indicated the existence of at least eight unlinked contigs containing multiple fragments that hybridized to both probes. Partial sequences of PCR products from the positive cosmids confirmed the existence of peptide synthetase genes. The combined results of hybridizations and physical mapping indicate that, in all likelihood, the isolated P. rosea contigs encode over 40 putative peptide synthetase modules. Similar results were obtained on screening a cosmid library of Actinoplanes teichomyceticus DNA. Furthermore, Southern hybridizations with several actinomycete strains, belonging to different genera, indicate that most strains contain multiple hybridizing bands well in excess of the number expected from the structure of the oligopeptides produced by these strains. Even strains not reported to produce oligopeptides gave clear positive signals when examined with the probes. These results strongly suggest that actinomycetes devote a notable fraction of their genomes to the non-ribosomal synthesis of peptides, and that most strains have the genetic potential to produce more oligopeptides than are currently described.

Using the method for identification of promoters recognized by the sporulation-specific sigma factor sigmaF, we identified a promoter, cspAp, in Streptomyces coelicolor, which showed similarity to the consensus sequence of Bacillus subtilis promoters recognized by the general stress-response sigma factor sigmaB. cspAp directs expression of the cspA gene, which shows sequence similarity to members of the family of major cold shock proteins (CspA) from bacterial species. S1-nuclease mapping using RNA prepared from Escherichia coli containing a two-plasmid system, and from Streptomyces coelicolor at various developmental stages, identified identical transcription start points in both species, corresponding to cspAp. However, the promoter was also active in a S. coelicolor sigF mutant. Transcriptional studies indicated that cspA is transcribed as a monocistronic mRNA. The level of cspA mRNA remains almost constant in all developmental stages, is dramatically increased after cold shock, and decreased after heat shock. Disruption of the S. coelicolor cspA gene did not affect growth or differentiation at 11 degrees C or 30 degrees C.

The kinesin-related Cin8p and cytoplasmic dynein are microtubule-associated motor proteins required for anaphase spindle elongation in the yeast Saccharomyces cerevisiae. Cells deleted for DYN1 (the gene encoding the dynein heavy chain) and carrying the temperature-sensitive allele cin8-3 cannot grow at temperatures above 35 degrees C. Here, we report that the temperature sensitivity of haploid cin8-3 dyn1delta cells is suppressed by the simultaneous presence of the loci MATa and MATalpha, which contain the regulatory genes that determine mating-type and ploidy-dependent phenotypes. The presence of the two MAT loci also rendered haploid cells more resistant to the antimicrotubule drug benomyl. Our results suggest that, in preparation for handling double the amount of DNA in mitosis, properties of microtubules in diploid cells are modified in a pathway controlled by the mating-type regulatory genes.

In the post-genome sequencing era the functional analysis of newly discovered proteins becomes more and more important. In this report we describe a genetic approach to the post-translational regulation of protein function in Saccharomyces cerevisiae by creating conditional lethal mutants. The yeast ORFs YDL139c, YDL147w, ERG3 and ERG11 were tagged with sequences encoding the hormone-binding domains of mammalian steroid receptors by PCR-mediated, targeted integration into the yeast genome. We found that the function of the chimeric proteins is regulated in a hormone-dependent way. This technique provides another important tool for the functional analysis of the yeast proteome.

Most Erwinia amylovora strains form yellow mucoid colonies on solid minimal medium containing asparagine and copper sulfate (MM2Cu). One exception is the strain Ea25/82, which produces white colonies on MM2Cu agar. This strain was transformed with a genomic library of E. amylovora and yellow colonies were recovered. A 1.5-kb fragment was found to complement strain Ea25/82 for color formation, and subsequent sequencing revealed two ORFs. The smaller ORF132(ycfB) overlapped with the end of the larger ORF253(ycfA). The putative protein YcfA shows low homology with K+/Na+ channel transporter ATPases. Resistance genes were inserted in both ORFs, and the E. amylovora strains Ea1/79-YA and Ea1/79-YB were created by site-directed mutagenesis. The mutation in ycfB did not affect color formation, whereas the ycfA mutant formed white colonies on MM2Cu. Sequence analysis of the ycf region in strain Ea25/82 revealed a 1-bp alteration in ycfA and no change in ycfB. Stable complementation of Ea25/82 and Ea1/79-YA, however, required both genes. Carotenoids were not detected in E. amylovora grown in the presence of copper ions. On the other hand, copper-independent secretion of a low-molecular-weight compound with an absorption maximum at 340 nm (CP340) was found for strain Ea1/79, but not for Ea25/82 or the mutant Ea1/79-YA. CP340 formed a complex with copper ions, and complementation with plasmids carrying both ycfA and ycfB restored its release from mutant strains. The compound may be connected with the yellow pigment or function in sensing bacterial population densities.

Net blotch, which is caused by the fungus Pyrenophoral teres Drechs. f. teres Smedeg., presents a serious problem for barley production worldwide, and the identification and deployment of sources of resistance to it are key objectives for many breeders. Here, we report the identification of a major resistance gene, accounting for 65% of the response variation, in a cross between the resistant line C19819 and the susceptible cv. Rolfi. The resistance gene was mapped to chromosome 6H with the aid of two recently developed systems of retrotransposon-based molecular markers, REMAP and IRAP. A total of 239 BARE-1 and Sukkula retrotransposon markers were mapped in the cross, and the 30-cM segment containing the locus with significant resistance effect contained 26 of the markers. The type and local density of the markers should facilitate future map-based cloning of the resistance gene as well as manipulation of the resistance through backcross breeding.

The second intron (bi2) of the cyt b gene from related Saccharomyces species has an extraordinarily conserved sequence and can have different functions in wild-type cells. The protein encoded by the S. cerevisiae intron functions as a maturase to promote intron splicing, while the homologous S. capensis intron encodes a bifunctional protein that acts both as a maturase and as a homing endonuclease (I-ScaI) promoting intron mobility. The protein encoded by intron bi2 belongs to a large gene family characterized by the presence of two conserved LAGLIDADG motifs (P1 and P2). In this study, we analysed a set of splicing-deficient mutants of the S. cerevisiae intron bi2 that carry non-directed mutations affecting the maturase activity, and a set of directed missense mutations introduced into the bifunctional protein encoded by the S. capensis intron. Analysis of these mutations has allowed identification of the residues in the conserved P1 and P2 motifs which are crucial for splicing and homing activities. Moreover, several mutations which are located in the C-terminal part of the protein have been found to affect both functions.

The Rho sub-family of GTPases, comprising Rho, Rac and Cdc42. regulates many biological processes, including morphogenesis, cell polarity, migration, the cell cycle and gene expression. It is important to develop genetic approaches to allow the dissection, in vivo, of the mechanisms of GTPase regulation and signal transmission, and their biological consequences. In this regard, wing development in Drosophila melanogaster is an excellent model system. To investigate the functions of the Drosophila Cdc42 GTPase (Dcdc42), we generated phenotypes during wing development, by expression of the dominant-negative N17 and L89 mutants of Dcdc42. We have identified roles for Dcdc42 in wing growth, and in cell fate choice during the development of the wing veins and the peripheral nervous system. Reduction of Dcdc42 signalling following over-expression of Dcdc42N17 resulted in a broader but more diffuse domain characterised by wing-margin sensory bristles. This was correlated with a broadened stripe of wingless expression along the dorsal-ventral boundary of third-instar wing imaginal discs. Together with genetic interactions with loss- and gain-of-function Notch alleles, these data support a role for wild-type Dcdc42 as a negative regulator of Notch signalling.

The white gene within the transposon A(R)4-24P[white,rosy] inserted at cytological location 24D1-2 in the euchromatic portion of the Drosophila melanogaster genome exhibits a mosaic pattern of expression which is modified by temperature and Y-chromosome number, as in cases of classical position-effect variegation (PEV). The eye colour of the flies in this variegated stock remains mosaic in the presence of the PEV modifier Su(var)3-6, slightly less so with Su(var)3-9 and Su(var)2-5, and full suppression of variegation occurs in the presence of Su(var)3-7. We have induced further transposition of A(R)4-24 and isolated two mosaic stocks with this transgene at new cytological locations. In these stocks, the A(R)4-24 transposon was flanked by the same genomic DNA fragments as in the original location. Spontaneous loss of these fragments leads to reversion of the variegated eye colour to wild-type. We suggest that the flanking DNA fragments from 24D1-2 are capable of inducing position-effect variegation without any association with centromeric heterochromatin. In situ hybridisation and Southern analysis demonstrate that the 5' flanking genomic fragment contains repeated sequences which are abundantly present in heterochromatin.