Molecular & general genetics : MGG最新文献

Glycosylation represents an attractive target for protein engineering of novel antibiotics, because specific attachment of one or more deoxysugars is required for the bioactivity of many antibiotic and antitumour polyketides. However, proper assessment of the potential of these enzymes for such combinatorial biosynthesis requires both more precise information on the enzymology of the pathways and also improved Escherichia coli-actinomycete shuttle vectors. New replicative vectors have been constructed and used to express independently the dnmU gene of Streptomyces peucetius and the eryBVII gene of Saccharopolyspora erythraea in an eryBVII deletion mutant of Sac. erythraea. Production of erythromycin A was obtained in both cases, showing that both proteins serve analogous functions in the biosynthetic pathways to dTDP-L-daunosamine and dTDP-L-mycarose, respectively. Over-expression of both proteins was also obtained in S. lividans, paving the way for protein purification and in vitro monitoring of enzyme activity. In a further set of experiments, the putative desosaminyltransferase of Sac. erythraea, EryCIII, was expressed in the picromycin producer Streptomyces sp. 20032, which also synthesises dTDP-D-desosamine. The substrate 3-alpha-mycarosylerythronolide B used for hybrid biosynthesis was found to be glycosylated to produce erythromycin D only when recombinant EryCIII was present, directly confirming the enzymatic role of EryCIII. This convenient plasmid expression system can be readily adapted to study the directed evolution of recombinant glycosyltransferases.

The chloroplast atpB gene of Chlamydomonas reinhardtii, which encodes the beta subunit of the ATP synthase, contains three in-frame ATGs that are candidate translation initiation codons. An earlier study revealed that the N terminus of the assembled beta subunit maps at the +2 position with respect to the second in-frame methionine codon (Fiedler et al. 1995). Using chloroplast transformation, we have examined the possibility that either of the two additional in-frame ATG codons is competent for translation initiation. We provide evidence that translation of atpB is initiated exclusively at the second ATG codon. We conclude that the beta subunit is not synthesized with an N-terminal leader before its assembly into a functional ATP synthase complex.

Disruption of ipdC, a gene involved in indole-3-acetic acid (IAA) production by the indole pyruvate pathway in Azospirillum brasilense Sp7, resulted in a mutant strain that was not impaired in IAA production with lactate or pyruvate as the carbon source. A tryptophan auxotroph that is unable to convert indole to tryptophan produced IAA if tryptophan was present but did not synthesise IAA from indole. Similar results were obtained for a mutant strain with additional mutations in the genes ipdC and trpD. This suggests the existence of an alternative Trp-dependent route for IAA synthesis. On gluconate as a carbon source, IAA production by the ipdC mutant was inhibited, suggesting that the alternative route is regulated by catabolite repression. Using permeabilised cells we observed the enzymatic conversion of tryptamine and indole-3-acetonitrile to IAA, both in the wild-type and in the ipdC mutant. IAA production from tryptamine was strongly decreased when gluconate was the carbon source.

Schizosaccharomyces pombe expresses a putative transcription factor, named Prr1, which is intriguing in the sense that it contains a bacterial type of phospho-accepting receiver domain, preceded by a mammalian heat shock factor (HSF2)-like DNA-binding domain. The receiver domain is most probably involved in an as yet unidentified histidine-to-aspartate (His-to-Asp) phosphorelay pathway in S. pombe. In this study, the structure, function, and cellular localization of Prr1 were assessed in the context of oxidative stress and His-to-Asp phosphorelay. As the most intriguing result of this study, we found that Prr1 is essential not only for the expression of genes induced by oxidative stress (e.g., ctt1+ and trr1+), but also for the expression of ste11+, which in turn is responsible for the expression of a variety of genes required for sexual development. Accordingly, Prr1-deficient cells are not only hypersensitive to oxidative stress, but also severely defective in conjugation and/or spore formation. These results suggested that the transcription factor Prr1 plays a pivotal role in an as yet unknown signal transduction pathway that is implicated in sexual differentiation. These findings are discussed with special reference to the well-characterized transcription factors Pap1 and Atf1 of S. pombe.

Two ftsZ homologues were isolated from the unicellular primitive red alga Cyanidioschyzon merolae (CmftsZ1 and CmftsZ2). Phylogenetic analysis revealed that CmftsZ1 is most closely related to the ftsZ genes of alpha-Proteobacteria, suggesting that it is a mitochondrial-type ftsZ gene, whereas CmftsZ2 is most closely related to the ftsZ genes of cyanobacteria, suggesting that it is a plastid-type ftsZ gene. Southern analysis indicates that CmftsZ1 and CmftsZ2 are both single-copy genes located on chromosome XIV in the C. merolae genome. Northern analysis revealed that both CmftsZ1 and CmftsZ2 are transcribed, and accumulate specifically before cell and organelle division. The results of Western analysis suggest that CmFtsZ1 is localized in mitochondria.

We have analyzed the sequences of mariner-like element (MLE) transposases, in order to obtain a clearer picture of their phylogenetic relationships. In particular, we have considered their two known structural domains, as well as the nucleic acid sequences of the MLE inverted terminal repeats (ITR). The most consistent tree was obtained using sequences of the catalytic domain of the transposase. The trees obtained with the amino acid sequences of the ITR-binding domain and the ITR sequences themselves were similar to that obtained with the catalytic domain. However, a major difference indicated that the cecropia sub-family is divided into two sub-groups. These new trees were used to examine the evolutionary divergence of mariner-like transposable elements, with particular reference to the possibility that recombination events or gene conversions created mosaic elements during the evolution of transposons.

The alb1 mutant of Lotus japonicus (Ljsym74) forms empty nodules in which most of the bacteria remain in abnormally enlarged infection threads and fail to enter the host plant cells. The alb1 mutant was also found to be defective in differentiation of ramified nodule vascular bundles; only a single vascular bundle differentiates at the proximal end of the alb1 nodules and it fails to differentiate further. Histochemical analysis using fluorescein-conjugated wheat-germ agglutinin (F-WGA) indicated that the mutation in the ALB1 gene specifically affects the differentiation of vascular bundles in nodules. Analysis of nodulin gene expression revealed that the expression of an early nodulin gene, ENOD40, was very low in alb1 nodules. At early developmental stages of alb1 nodules, the pattern of ENOD40 transcription was essentially the same as that in wild-type nodules; transcripts were localized in dividing cortical cells and in the pericycle of the root stele opposite nodule primordia, as in wild-type nodules. However, mature alb1 nodules exhibited very weak or no expression of ENOD40 in the peripheral cells of the undeveloped nodule vascular bundle. The ENOD40 expression pattern in alb1 nodules is distinct from that in another ineffective mutant, fen1 (Ljsym76), in which ENOD40 expression persists prior to premature senescence. These findings lead us to speculate that ENOD40 may play a role in the differentiation of nodule vascular bundles.

We report the cloning of a mouse cDNA encoding the MAK-V protein kinase, with a putative specificity for serine/threonine residues. The mak-v gene is transcribed in adult brain and in the mouse embryo from at least 7.5 dpc. Using the yeast two-hybrid system, we showed that MAK-V interacts with Rabaptin-5, a protein which plays an important role in endocytosis. Functional studies of the MAK-V protein suggest that it regulates endocytosis. We also constructed a human mak-v cDNA and localized the human mak-v gene at 21q22.11. Its chromosomal location suggests that mak-v could be involved in disorders of the nervous system, development or in malignancies.

Replication factor C (RFC) is an essential, multi-subunit ATPase that functions in DNA replication, DNA repair, and DNA metabolism-related checkpoints. In order to investigate how the individual RFC subunits contribute to these functions in vivo, we undertook a genetic analysis of RFC genes from budding yeast. We isolated and characterized mutations in the RFC5 gene that could suppress the cold-sensitive phenotype of rfc1-1 mutants. Analysis of the RFC5 suppressors revealed that they could not suppress the elongated telomere phenotype, the sensitivity to DNA damaging agents, or the mutator phenotype of rfc1-1 mutants. Unlike the checkpoint-defective rfc5-1 mutation, the RFC5 suppressor mutations did not interfere with the methylmethane sulfonate- or hydroxyurea-induced phosphorylation of Rad53p. The Rfc5p suppressor substitutions mapped to amino acid positions in the conserved RFC box motifs IV-VII. Comparisons of the structures of related RFC box-containing proteins suggest that these RFC motifs may function to coordinate interactions between neighboring subunits of multi-subunit ATPases.

Mutations that increase readthrough at a UGA stop codon (informational suppressor mutations) were created in the gene (AS4) that encodes translation elongation factor eEF1A in the filamentous fungus Podospora amserina. The results strongly suggest that the net charge of the eEF1A protein controls the accuracy of translation. Physiological analysis of the mutant strains shows that some of the alleles dominantly increase life span, while only one drastically modifies fertility. This exceptional allele (AS4-56) causes a wide array of phenotypes, including a new growth cessation phenomenon that is different from Senescence or Crippled Growth, previously known degenerative syndromes that are both controlled by AS4. The data emphasise the fact that eEF1A exerts a complex control over cellular physiology.