Molecular & general genetics : MGG最新文献

Dehydrins (DHNs; LEA D11) are one of the typical families of plant proteins that accumulate in response to dehydration, low temperature, osmotic stress or treatment with abscisic acid (ABA), or during seed maturation. We previously found that three genes encoding low-molecular-weight DHNs (Dhn1, Dhn2 and Dhn9) map within a 15-cM region of barley chromosome 5H that overlaps a QTL for winterhardiness, while other Dhn genes encoding low- and high-molecular-weight DHNs are located on chromosomes 3H, 4H and 6H. Here we examine the expression of specific Dhn genes under conditions associated with expression of the winterhardiness phenotype. Plants grown at 4 degrees C or in the field in Riverside, California developed similar, modest levels of freezing tolerance, coinciding with little low-MW Dhn gene activity. Dicktoo (the more tolerant cultivar) and Morex (the less tolerant) grown in Saskatoon, Canada expressed higher levels of expression of genes for low-MW DHNs than did the same cultivars in Riverside, with expression being higher in Dicktoo than Morex. Dehydration or freeze-thaw also evoked expression of genes for low MW DHNs, suggesting that the dehydration component of freeze-thaw in the field induces low expression of genes encoding low-MW DHNs. These observations are consistent with the hypothesis that the major chilling-induced DHNs help to prime plant cells for acclimation to more intense cold, which then involves adaptation to dehydration during freeze-thaw cycling. A role for chromosome 5H-encoded DHNs in acclimation to more intense cold seems possible, even though it is not the basis of the major heritable variation in winterhardiness within the Dicktoo x Morex population.

The cry genes that code for the insecticidal crystal proteins of Bacillus thuringiensis (B.t.) have been widely used to develop insect-resistant transgenic plants. The cry3Ca1 gene has been reported to code for a crystal protein which is particularly potent against the Colorado potato beetle (CPB). To explore the biotechnological potential of cry3Ca1, we introduced this gene into transgenic potato plants under the control of the CaMV 35S promoter. In the resulting transformants, the cry3-Ca1 gene was very poorly expressed. In fact, no full-length transcript (2300 nt) could be detected. Instead, only short transcripts of approximately 1100 nt were observed. Analysis of these short transcripts by Northern hybridization, RT-PCR as well as by cloning and sequencing showed that they resulted from premature polyadenylation. These processing events occurred at four sites within the cry3Ca1 coding region (at positions 652, 669, 914 and 981 relative to the translation start site). The sites at which premature polyadenylation took place were not those that showed the highest degree of identity to the canonical AAUAAA motif. Together with other recent data, our findings suggest that premature polyadenylation is an important mechanism which can contribute to the poor expression of transgenes in a foreign host.

The Saccharomyces cerevisiae gene SGS1 encodes a DNA helicase that shows homology to the Escherichia coli protein RecQ and the products of the BLM and WRN genes in humans, which are defective in Bloom's and Werner's syndrome, respectively. Recently, it has been proposed that this helicase is involved in maintaining the integrity of the rDNA and that loss of Sgs1 function leads to accelerated aging. Sgs1 has been isolated on the basis of its genetic interaction with both topoisomerase I and topoisomerase III, as well as in a two-hybrid screen for proteins that interact with the C-terminal portion of topoisomerase II. We have defined the minimal structural elements of Sgs1 required for its interactions with the three topoisomerases, and demonstrate that the complex phenotypes associated with sgs1 mutants are a consequence of a dysfunctional Sgs1-Top3 complex. We also report that the synthetic relationship between mutations in SGS1 and SRS2, which encodes another helicase implicated in recombinational repair, likewise result from a dysfunctional Sgs1-Top3 interaction. Our findings indicate that Sgs1 may act on different DNA structures depending on the activity of topoisomerase I, Srs2 and topoisomerase III.

Uncoupling proteins (UCP) found in the inner mitochondrial membrane of mammals dissipate the proton electrochemical gradient across the inner membrane to produce heat rather than synthesize ATP. Using PCR-based methods, we isolated two novel cDNA clones, WhUCP1a and WhUCP1b, that encode the mitochondrial uncoupling protein of wheat (Triticum aestivum L.). The cDNA clones each contain one ORF which can code for a protein of 286 amino acids with a predicted molecular mass of about 30.5 kDa, although three amino acid substitutions are found between them. The deduced amino acid sequences each possess three typical mitochondrial carrier signature domains and six membrane-spanning domains which are highly conserved in the mitochondrial transporter family. Southern analysis suggested that the WhUCP1 gene may be present in as many as three copies in the wheat genome, and also that WhUCP proteins may be encoded by a small multigene family. Northern analysis revealed that the steady-state level of the WhUCP1 mRNA is quite low. Quantitative RT-PCR clearly showed that expression of the WhUCP1 gene in wheat seedlings is insensitive to low temperature. Our data suggest that WhUCP1 might have functions other than low temperature-induced thermogenesis, although WhUCP1 possesses all the typical features reported for known UCPs.

We have cloned and characterized a gene cluster for anthracycline biosynthesis from Streptomyces galilaeus. This cluster, 15-kb long, includes eight genes involved in the deoxyhexose biosynthesis pathway, a gene for a glycosyltransferase and one for an activator, as well as two genes involved in aglycone biosynthesis. Gene disruption targeted to the activator gene blocked production of aclacinomycins in S. galilaeus. Plasmid pSgs4, containing genes for a glycosyltransferase (aknS), an aminomethylase (aknX), a glucose-1-phosphate thymidylyltransferase (akn Y) and two genes for unidentified glycosylation functions (aknT and aknV), restored the production of aclacinomycins in the S. galilaeus mutants H063, which accumulates aklavinone, and H054, which produces aklavinone with rhodinose and deoxyfucose residues. Furthermore, pSgs4 directed the production of L-rhamnosyl-epsilon-rhodomycinone and L-daunosaminyl-epsilon-rhodomycinone in S. peucetius strains that produce epsilon-rhodomycinone endogenously. Subcloning of the gene cluster was carried out in order to further define the genes that are responsible for complementation and hybrid anthracycline generation.

In the silkworm, Bombyx mori, many eye- and egg-colour mutations affecting the synthesis and accumulation of ommochrome pigments have been described. In order to understand the pigment precursor transporters involved, ABC transporter genes homologous to the Drosophila white gene were isolated from the silkworm. Reverse transcriptase-polymerase chain reactions (RT-PCR) using embryonic mRNA amplified three cDNA fragments, named Bmwh1, Bmwh2 and Bmwh3 that showed homology to the white gene. Since Bmwh3 shows the highest degree of sequence identity and a similar expression pattern to the Drosophila homologue, we characterised this gene further. A 2667-bp Bmwh3 cDNA isolated from an embryonic library has one ORF encoding a polypeptide of 687 amino acids. The predicted protein has one ATP-binding domain, six transmembrane-spanning segments and high similarity to the Drosophila WHITE protein. Southern analysis indicates that Bmwh3 is a single-copy gene. Polyadenylated Bmwh3 transcripts about 2.7 kb long were detected in eggs, Malpighian tubules and pupal heads, but not in testes, posterior silk glands or fat body cells. The level of Bmwh3 mRNA was reduced in w3 and w3ol mutants but normal in other egg- and eye-colour mutants, suggesting that Bmwh3 correspond to the w3 locus. Genetic analysis was used to map the cloned gene to chromosome 10.

The Schizosaccharomyces pombe mutant ehs1-1 mutant was isolated on the basis of its hypersensitivity to Echinocandin and Calcofluor White, which inhibit cell wall synthesis. The mutant shows a thermosensitive growth phenotype that is suppressed in the presence of an osmotic stabiliser. The mutant also showed other cell wall-associated phenotypes, such as enhanced sensitivity to enzymatic cell wall degradation and an imbalance in polysaccharide synthesis. The ehs1 + gene encodes a predicted integral membrane protein that is 30% identical to Saccharomyces cerevisiae Mid1p, a protein that has been proposed to form part of a calcium channel. As expected for such a function, we found that ehs1+ is involved in intracellular Ca2+ accumulation. High external Ca2+ concentrations suppressed all phenotypes associated with the ehs1 null mutation, suggesting that the cell integrity defects of ehs1 mutants result from inadequate levels of calcium in the cell. We observed a genetic relationship between ehs1+ and the protein kinase C homologue pck2+. pck2+ suppressed all phenotypes of ehs1-1 mutant cells. Overproduction of pck2p is deleterious to wild-type cells, increasing 1,3-beta-D-glucan synthase activity and promoting accumulation of extremely high levels of Ca2+. The lethality associated with pck2p, the increase in 1,3-beta-D-glucan synthase production and the strong Ca2+ accumulation are all dependent on the presence of ehs1p. Our results suggest that in fission yeast ehs1p forms part of a calcium channel that is involved in the cell wall integrity pathway that includes the kinase pck2p.

The mechanisms involved in the regulation of mitochondrial DNA (mtDNA) replication, a process that is crucial for mitochondrial biogenesis, are not well understood. In this study, we evaluate the role of DNA polymerase gamma (pol gamma), the key enzyme in mtDNA replication, in both Drosophila cell culture and in developing flies. We report that overexpression of the pol gamma catalytic subunit (pol gamma-alpha) in cultured Schneider cells does not alter either the amount of mtDNA or the growth rate of the culture. The polypeptide is properly targeted to mitochondria, yet the large excess of pol gamma-alpha does not interfere with mtDNA replication under these conditions where the endogenous polypeptide is apparently present in amounts that exceed of the demand for its function in the cell. In striking contrast, overexpression of pol gamma-alpha at the same level in transgenic flies interferes with the mtDNA replication process, presumably by altering the mechanism of DNA synthesis, suggesting differential requirements for, and/or regulation of, mtDNA replication in Drosophila cell culture versus the developing organism. Overexpression of pol gamma-alpha in transgenic flies produces a significant depletion of mtDNA that causes a broad variety of phenotypic effects. These alterations range from pupal lethality to moderate morphological abnormalities in adults. depending on the level and temporal pattern of overexpression. Our results demonstrate that although cells may tolerate a variable amount of the pol gamma catalytic subunit under some conditions, its level may be critical in the context of the whole organism.

FKS1 and FKS2 encode alternative catalytic subunits of the glucan synthases that are responsible for synthesis of beta-1,3-glucan in the Saccharomyces cerevisiae cell wall. Disruption of FKS1 reduces the glucan content of the cell wall, increases chitin content and activates the expression of CWP1, which encodes a glycosylphosphatidylinositol (GPI)-dependent cell wall protein. These cellular responses have been regarded as compensating for cell wall damage in order to maintain cell wall integrity. Here, we report the identification, by genome-wide screening, of 22 genes that are transcriptionally up-regulated in fks1delta cells. Among them, five genes were found to encode GPI-attached proteins, three of which are covalently associated with the cell wall. Deletion and replacement analysis of the promoter regions identified Rlm1-binding sequences as being responsible for the up-regulation following disruption of FKS1. Using the rlm1delta tetOp-FKS1 strain, in which the expression of FKS1 can be repressed by doxycycline, we examined the requirement for Rlm1 for the transcriptional up-regulation of these five genes. Three of the five genes were not up-regulated by doxycycline, indicating that Rlm1 mediates their up-regulation when FKS1 is inactivated. The remaining two genes were up-regulated by doxycycline, suggesting that a transcription factor other than Rlm1 is involved in their response to disruption of FKS1.

Flagellin gene regulation in Legionella pneumophila is modulated by various environmental factors. The expression of the virulent phenotype seems to be linked genetically to flagellum expression. To better understand the mechanisms of flagellin gene expression in L. pneumophila (Lp), we screened a pool of plasmids from a L. pneumophila Corby genomic library for the ability to prevent or reduce luciferase activity in the Escherichia coli strain YK410, which harbours a Lp-pflaA-luxAB fusion. We cloned a DNA fragment encoding the N-terminal part of a protein with significant similarity to members of the LysR family of transcriptional regulators (LTTRs). The entire gene, cloned by inverse PCR, was named flaR. It encodes a protein of 302 amino acids, and computer-assisted analysis of the amino acid sequence revealed a helix-turn-helix motif located near the N-terminus of the protein. The FlaR protein exhibits 21-31% identity to various LTTRs. Furthermore, gel retardation experiments indicate that the FlaR protein is able to bind to its own promoter region and, to a lesser extent, to the flaA promoter of L. pneumophila. The flaR promoter region contains putative LysR binding motifs and two putative Fur boxes. Taken together, these results indicate that FlaR is a DNA-binding protein which belongs to the LTTR family. Southern analysis with a L. pneumophila Corby-specific flaR probe revealed homologous genes in various L. pneumophila strains, but not in the 12 nonpneumophila strains tested so far.