Molecular & general genetics : MGG最新文献

In this paper, we examine the effects of mutations in the 5'UTR of the chloroplast rps7 transcript of Chlamydomonas reinhardtii that reduce the stability of the mRNA. Five point mutants in the rps7 5'UTR were selected on the basis of their failure to accumulate reporter mRNA in Escherichia coli. Each of these mutations produces alterations in the predicted higher-order structures of the rps7 5'UTR that destabilize the mRNA. Cis-acting suppressors of these mutations have been selected in E. coli and in the C. reinhardtii chloroplast that restore message stability and function. No differences in RNA melting and reannealing profiles have been observed between wild type, original mutant, and suppressor 5'UTRs transcribed in vitro. Proteins of 32 kDa and 47 kDa that bind to the wild-type rps7 5'UTR are not detected by UV cross-linking assays performed with any of the mutant rps7 5'UTRs. However, binding of the 32-kDa protein is restored in the six suppressor mutants examined. This suggests that the 32-kDa protein may be involved in protecting the rps7 5'UTR and the attached coding region from digestion by ribonucleases. Alternatively, the binding site for the 32-kDa protein may be independently lost in the rearranged tertiary structure of the mutant 5'UTR that exposes the RNA to degradation and is restored in the suppressor mutants.

Streptomyces reticuli produces a mycelium-associated enzyme (CpeB) which exhibits heme-dependent catalase and peroxidase activity, as well as heme-independent manganese-peroxidase activity. The cpeB gene does not have a promoter of its own. It is co-transcribed together with the adjacent furS gene from at least one promoter, the position of which was deduced on the basis of high-resolution S1 mapping of transcriptional start sites. Physiological and transcriptional studies suggested that FurS acts as a transcriptional repressor in the presence of Mn2+ and Fe2+ ions. A FurS fusion protein was purified, after cloning of the corresponding gene, either from Escherichia coli or Streptomyces lividans transformants. The fusion protein from each host strain can be converted into a form that exhibits reduced electrophoretic mobility following treatment with thiol-reducing agents; in the presence of diamide, in contrast, the mobility of the protein is enhanced. Additional immunological studies have shown that the native S. reticuli FurS also shows these properties, which are due to the presence of redox-sensitive cysteine residues. As revealed by gel-shift and in vitro footprinting studies, only the reduced form of the FurS fusion protein and the reduced FurS protein (partially purified from S. reticuli) is able to bind to a motif upstream of the furS gene. In the absence of first-row divalent ions, the binding site encompasses 22 bp. In the presence of Mn2+, Fe2+, Co2+, Cu2+ or Zn2+, however, the region bound is extended by 18 bp. It is noteworthy that the region upstream of the furA gene in several mycobacteria contains a very similar motif. The predicted mycobacterial FurA shares a high degree of sequence identity with FurS, and the furA gene is linked to one that encodes a catalase-peroxidase (KatG). The implications of these findings are discussed.

LIM-domain proteins participate in important cellular processes in eukaryotes, including gene transcription and actin cytoskeleton organization. They are predominantly found in animals, but have also been identified in yeast and plants. Following the characterization ofa LIM-domain protein in sunflower pollen, we carried out an extensive search for these proteins in flowering plants. We have isolated and studied cDNAs and/or genomic sequences for two novel LIM-domain proteins from sunflower, three from tobacco, and one from Arabidopsis. The plant proteins are structurally related to the cytoskeleton-associated CRP class of LIM proteins in animals, but show several distinctive features, including a second, atypical, LIM domain. We have performed comparative expression studies of these genes, as well as of one other gene from tobacco and two additional Arabidopsis genes whose sequences are available from databases. These studies, carried out by RT-PCR in the presence of gene-specific primers, showed that, in sunflower and tobacco, pollen grains and sporophytic tissues express different sets of LIM proteins. With the exception of one Arabidopsis gene--which has two introns--all the genes analyzed contain four introns at conserved positions, indicating that the ancestral gene from which the various copies evolved in higher plants allready had this split structure.

Neisseria gonorrhoeae lacks several common DNA repair pathways found in other organisms. As recent evidence had indicated that gonococci use recombinational repair to repair UV-induced DNA lesions, this study examined whether the gonococcal RecJ homologue contributes in this repair capacity. The recJ gene from strain MS11 was cloned and sequenced and was found to show a considerable degree of identity to its Escherichia coli homologue. A N. gonorrhoeae delta recJ mutant was constructed and tested for recombinational proficiency as well as for defects in DNA repair. In the absence of the RecJ exonuclease, DNA transformation and pilin switching occurred at wild type levels, indicating that the efficiency of recombination remained unimpaired. In contrast, N. gonorrhoeae delta recJ mutants showed extreme sensitivity to low levels of UV irradiation and to exposure to DNA-alkylating reagents [e.g. ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS)]. Complementation of the gonococcal recJ mutant in cis restored resistance to low-level UV, indicating that the gonococcal RecJ protein is involved in recombinational repair, and can act independently of other single-strand-specific exonucleases. Furthermore, transformation competence was not required for RecJ-dependent DNA repair. Overall, the data show that N. gonorrhoeae recJ mutants present a unique phenotype when compared to their E. coli recJ counterparts, and further support the contention that RecORJ-dependent recombinational repair is a major DNA repair pathway in the genus Neisseria.

The barley stem rust resistance gene rpg4 was physically and genetically localized on two overlapping BAC clones covering an estimated 300-kb region of the long arm of barley chromosome 7(5H). Initially, our target was mapped within a 6.0-cM region between the previously described flanking markers MWG740 and ABG391. This region was then saturated by integrating new markers from several existing barley and rice maps and by using BAC libraries of barley cv. Morex and rice cv. Nipponbare. Physical/genetic distances in the vicinity of rpg4 were found to be 1.0 Mb/cM, which is lower than the average for barley (4 Mb/cM) and lower than that determined by translocation breakpoint mapping (1.8 Mb/cM). Synteny at high resolution levels has been established between the region of barley chromosome 7(5H) containing the rpg4 locus and the subtelomeric region of rice chromosome 3 between markers S16474 and E10757. This 1.7-cM segment of the rice genome was covered by two overlapping BAC clones, about 250 kb of total length. In barley the markers S16474 and E10757 genetically delimit rpg4, lying 0.6 cM distal and 0.4 cM proximal to the locus, respectively.

Full-length transcript sequences were isolated from broad bean root nodules, which encode a novel nodulin designated VfENOD18. The corresponding transcripts were detected in early and in late stages of nodule development and were localized exclusively in the nitrogen-fixing zone III. The VfENOD18 sequence is not only homologous to a number of ESTs from various mono- and dicotyledonous plants, but also to the ATP-binding protein MJ0577 from Methanococcus jannaschii and to a range of bacterial proteins that belong to the MJ0577 superfamily. Hence, VfENOD18 is a member of a ubiquitous family of plant proteins that might function as ATP-binding proteins or ATPases. On the genomic level, VfENOD18 genes can be divided into two groups on the basis of differences in their 5' UTRs. One group lacks the 5' UTR region including the ATG initiation codon, whereas the second group contained the complete 5' UTR region. Further upstream of this VfENOD18 gene, a retrotransposon sequence was identified. The -14/-964 VfENOD18 promoter fragment was devoid of complete organ-specific elements known from other nodulin gene promoters. Nevertheless, this region was able to mediate full promoter activity in the central region of transgenic Vicia hirsuta root nodules.

Hyperactivation of Cdc2 in fission yeast causes cells to undergo a lethal premature mitosis, a phenomenon called mitotic catastrophe. This phenotype is observed in cdc2-3w wee1-50 cells at high temperature and is suppressed by a single recessive mutant, mcs3-12. Mcs3 acts independently of the Wee1 kinase and Cdc25 phosphatase, two major regulators of Cdc2. We have isolated multicopy suppressors of the cell cycle arrest phenotype of mcs3-12 wee1-50 cdc25-22 cells, but did not identify the mcs3 gene itself. Instead several known mitotic regulators were isolated, including the Cdc25 phosphatase, Wis2 cyclophilin, Cek1 kinase, and an Hsp90 homologue, Swo1. We also isolated clones encoding non-functional, truncated forms of the Wee1 kinase and Dis2 type 1 phosphatase. In addition we identified a multicopy suppressor that encodes a structural homologue of the budding yeast SPO12 gene. We find that overexpression of fission yeast spo12 not only suppresses the phenotype of the mcs3-12 wee1-50 cdc25-22 strain, but also that of a win1-1 wee1-50 cdc25-22 strain at high temperature, indicating that the function of spo12 is not directly related to mcs3. We show that spo12 mRNA is periodically expressed during the fission yeast cell cycle, peaking at the G2/M transition coincidently with cdc15. Deletion of spo12, however, has no overt effect on either the mitotic or meiotic cell cycles, except when the function of the major B type cyclin, Cdc13, is compromised.

The phenomenon of transvection has been well characterized for the yellow locus in Drosophila. Enhancers of a promoterless yellow locus in one homologous chromosome can activate the yellow promoter in the other when its own enhancers are blocked by the su(Hw) insulator introduced by the gypsy retrotransposon. Insertion of another gypsy into the neighboring scute locus hinders transvection presumably owing to disruption of chromosomal synapsis between the yellow alleles. We determined the sequences of gypsy required for inhibition of transvection. Two partial revertants of the scD1 mutation were obtained in which transvection between the yellow alleles was restored. Both sc revertants were generated by deletion of nine of the twelve su(Hw)-binding sites of gypsy inserted into the scute locus. This result suggests that the su(Hw) region is required for an interaction between two gypsy elements that disrupts trans activation of the yellow promoter by enhancers located on the homologous chromosome.

Although previously reported attempts to construct recA null mutants in Streptomyces spp. have been unsuccessful, we have used the suicide plasmid pErmdeltaRecA to inactivate the recA gene in Streptomyces rimosus by gene disruption. pErmdeltaRecA carries the erythromycin resistance gene ermE and a 451-bp fragment of the S. rimosus recA gene (encoding amino acids 2-151). An erythromycin-resistant clone with single plasmid integration into the recA gene on the chromosome was analyzed in detail. This clone possesses one inactive copy of recA which lacks the entire promoter region and the ATG start codon, and a second, truncated gene that encodes only first 151 amino acids of the RecA protein. This S. rimiosus rec A mutant can therefore be considered a completely RecA-deficient strain. The mutant strain is highly sensitive to UV light. Introduction of a plasmid carrying the wild type S. rimosus recA gene completely restored the UV resistance of the recA mutant to wild-type levels. recA genes encoding RecA proteins with short deletions at the C-terminus (21 and 51 amino acids) could not fully rescue the UV sensitivity of the S. rimosus recA strain, when introduced in the same way.

sconC, which encodes a negative regulator of sulphur metabolism in Aspergillus nidulans was cloned, sequenced, and found to belong to the highly conserved family of SKP1 genes essential for many cell functions, including cell cycle regulation. The ORF of 722 bp, encoding a protein of 161 amino acids, is interrupted by four introns. There is a fifth intron (135 bp long) in the upstream untranslated sequence. Two point mutations in conserved regions were identified in the mutant alleles sconC3 and sconC1, which result in relief of sulphur metabolite repression. The SCONC protein contains the PEST sequence common for proteins that are subject to rapid turnover. Transformation of the sconC3 mutant with sconB+ restores the wild-type phenotype. The sconB gene encodes a protein containing the F-box, a domain known to interact with Skp1 proteins. By analogy with other systems, it seems likely that the SCONC protein interacts with SCONB. sconC mRNA is present in the sconC3 and sconB2 mutants and the level of the sconC transcript seems not to be significantly regulated by supplementation of the medium with sulphur.