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An enzyme in yeast mitochondria that catalyzes a step in branched-chain amino acid biosynthesis also functions in mitochondrial DNA stability. 总被引:11,自引:0,他引:11
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The yeast mitochondrial high mobility group protein Abf2p is required, under certain growth conditions, for the maintenance of wild-type (rho+) mitochondrial DNA (mtDNA). We have identified a multicopy suppressor of the mtDNA instability phenotype of cells with a null allele of the ABF2 gene (delta abf2). The suppressor is a known gene, ILV5, encoding the mitochondrial protein, acetohydroxy acid reductoisomerase, which catalyzes a step in branched-chain amino acid biosynthesis. Efficient suppression occurs with just a 2- to 3-fold increase in ILV5 copy number. Moreover, in delta abf2 cells with a single copy of ILV5, changes in mtDNA stability correlate directly with changes in conditions that are known to affect ILV5 expression. Wild-type mtDNA is unstable in cells with an ILV5 null mutation (delta ilv5), leading to the production of mostly rho- petite mutants. The instability of rho+ mtDNA in delta ilv5 cells is not simply a consequence of a block in branched-chain amino acid biosynthesis, since mtDNA is stable in cells with a null allele of the ILV2 gene, which encodes another enzyme of that pathway. The most severe instability of rho+ mtDNA is observed in cells with null alleles of both ABF2 and ILV5. We suggest that ILV5 encodes a bifunctional protein required for branched-chain amino acid biosynthesis and for the maintenance of rho+ mtDNA. 相似文献
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The Cpx two-component signal transduction pathway is activated in Escherichia coli mutant strains lacking phosphatidylethanolamine. 总被引:5,自引:0,他引:5
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The CpxA-CpxR two-component signal transduction pathway of Escherichia coli was studied in a mutant (pss-93) lacking phosphatidylethanolamine (PE). Several properties of this mutant are comparable to phenotypes of cpxA point mutants, indicating that this two-component pathway is activated in PE-deficient cells. In contrast to point mutants, cpx operon null mutants have a wild-type phenotype. By use of this information, a cpx operon null allele was introduced into a pss-93 mutant. Certain altered properties of PE-deficient mutants, which were consistent with activation of the Cpx pathway, returned to the wild-type phenotype, namely, active accumulation of proline and thiomethyl-beta-D-galactopyranoside was partially restored to wild-type levels, increased resistance to amikacin returned to wild-type sensitivity, and high levels of degP expression returned to repressed wild-type levels. Elevated levels of acetyl phosphate and nlpE gene product can result in activation of the Cpx pathway. However, inactivation of the nlpE gene or mutations eliminating the ability to make acetyl phosphate did not alter the high level of degP expression in pss-93 mutants. We propose that the lack of PE results in an alteration in cell envelope structure or physical properties, leading to direct activation of the Cpx pathway. 相似文献
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Many promoter-fusion vectors contain an intact beta-lactamase (BLA) gene (bla) to allow measurement of BLA activity as an internal control for plasmid copy number. This approach rests on the assumption that bla is constitutively expressed. To use such vectors for comparison of promoter activity at different growth rates it was necessary to confirm that this is the case under all physiological conditions. The relationship between plasmid copy number and BLA activity at different steady-state growth rates in Escherichia coli HB101 transformed with a ColE1-type plasmid (pBR325) was examined. Both BLA activity and plasmid copy number decreased in a parallel fashion as growth rate increased. This finding was tested further by measuring the growth-rate-regulated expression of the chloramphenicol acetyltransferase (CAT) gene under the control of the rrnB P1 promoter in a plasmid pKK231-1 fusion. The results indicate that BLA activity is a reliable indicator of copy number at a wide range of growth rates and that CAT/BLA ratios can be employed as a valid measure of promoter-specific activity in such plasmid fusions under these different physiological conditions. 相似文献
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Here we report on the construction of a tetracycline inducible expression vector that allows a tightly regulable gene expression in Corynebacterium glutamicum which is used in industry for production of small molecules such as amino acids. Using the green fluorescent protein (GFP) as a reporter protein we show that this vector, named pCLTON1, is characterized by tight repression under non-induced conditions as compared to a conventional IPTG inducible expression vector, and that it allows gradual GFP synthesis upon gradual increase of anhydrotetracycline addition. 相似文献
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A temperature-sensitive splicing mutation in the bimG gene of Aspergillus produces an N-terminal fragment which interferes with type 1 protein phosphatase function. 总被引:3,自引:0,他引:3
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Progression through anaphase requires high levels of type 1 protein phosphatase (PP1) activity in a variety of eukaryotes, including Aspergillus nidulans. A conditional lethal, temperature-sensitive mutant in one of the Aspergillus PP1 genes, bimG, prevents the normal completion of anaphase when cells are grown at restrictive temperature and this has been shown to be due to a reduction in type 1 phosphatase activity. We show that the bimG11 allele is recessive to the wild-type allele in heterozygous diploids, implying that the mutation is due to loss of function at restrictive temperature, but molecular disruption of the wild-type bimG gene shows that the gene is not essential and has no discernable phenotype under laboratory conditions. Sequence comparison of wild-type and mutant alleles reveals a single base pair difference between the two genes, within the 5' splicing site of the second intron. We demonstrate that the conditional lethal phenotype of bimG11 strains is due to impaired splicing of the mutant mRNA and that this leads to the production of a truncated protein comprising an intact N-subdomain and a modified C-terminus. Over-expression of this truncated form of PP1 in a wild-type haploid produces a lethal phenotype and reduced PP1 activity, supporting the idea that a toxic interfering protein is produced. PP1, therefore, may have at least two spatially separated sites, both of which are required for function. Temperature-sensitive splicing mutations may provide a novel means of engineering conditional versions of other proteins, particularly other phosphatases. 相似文献
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Interaction of the Yeast Omnipotent Suppressors Sup1(sup45) and Sup2(sup35) with Non-Mendelian Factors 总被引:6,自引:3,他引:3
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The SUP1 and SUP2 genes code for protein factors intimately involved in the control of translational accuracy. The disrupted alleles of these genes confer a recessive lethal phenotype in both [psi+] and [psi-] genetic backgrounds, indicating an essential function for the corresponding proteins. In [psi+] diploids, heterozygous for the SUP1 null allele, several dominant phenotypes were evident with slow growth and inability to sporulate. These dominant phenotypes disappear after transformation with the multicopy plasmid carrying the wild-type allele of the SUP1 gene. Such dominant phenotypes were not observed for the SUP2 null allele. The incompatibility of multicopy plasmids carrying the SUP2 gene with guanidine hydrochloride-curable cytoplasmic factor(s) was also demonstrated. The possible mechanisms of interaction of the SUP1 and SUP2 genes with the [psi] determinant are discussed. 相似文献
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T Miki T Yasukochi H Nagatani M Furuno T Orita H Yamada T Imoto T Horiuchi 《Protein engineering》1987,1(4):327-332
A novel expression vector pKP1500 for synthesizing unfused protein in Escherichia coli was constructed. pKP1500 perserves the tac promoter, the lacZ SD sequence, unique restriction sites (EcoRI, SmaI, BamHI, SalI, PstI and HindIII) and the rrnB terminators of pKK223-3, but the replication origin is replaced with that of pUC9. Construction of this plasmid is based upon the observation that the copy number control of pUC9 is temperature dependent. At 28 degrees C, the copy number of pKP1500 is less than 25 per chromosome, approximately the same copy number as that of pKK223-3, which contains the replication origin of pBR322, whereas at 42 degrees C, the copy number increases about 10 times and reaches up to 230 copies per chromosome. The main advantage of this system is that the temperature-dependent copy control and regulatable expression of the tac promoter make cells carrying pKP1500 derivatives stable against selective pressure by detrimental overproduction of foreign proteins at a low temperature and permits high expression of cloned DNAs at a high temperature. When chicken lysozyme cDNA carrying the initiation codon (ATG) immediately upstream from the Lys1 codon was inserted downstream from the tac promoter and the SD sequence, the pKP1500 derivative produced lysozyme at about 25% of the total cellular proteins. This value is more than 10 times higher than that obtained with the pKK223-3 derivative carrying the same lysozyme cDNA. By comparison, the expression of eukaryotic genes from the tac promoter reported by others has usually been less than a few % of the total cellular protein.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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The Myxococcus xanthus FprA protein causes increased flavin biosynthesis in Escherichia coli. 总被引:3,自引:2,他引:1
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L J Shimkets 《Journal of bacteriology》1990,172(1):24-30
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Autogenous control of Escherichia coli threonyl-tRNA synthetase expression in vivo 总被引:16,自引:0,他引:16
M Springer J A Plumbridge J S Butler M Graffe J Dondon J F Mayaux G Fayat P Lestienne S Blanquet M Grunberg-Manago 《Journal of molecular biology》1985,185(1):93-104
The regulation of the expression of thrS, the structural gene for threonyl-tRNA synthetase, was studied using several thrS-lac fusions cloned in lambda and integrated as single copies at att lambda. It is first shown that the level of beta-galactosidase synthesized from a thrS-lac protein fusion is increased when the chromosomal copy of thrS is mutated. It is also shown that the level of beta-galactosidase synthesized from the same protein fusion is decreased if wild-type threonyl-tRNA synthetase is overproduced from a thrS-carrying plasmid. These results strongly indicate that threonyl-tRNA synthetase controls the expression of its own gene. Consistent with this hypothesis it is shown that some thrS mutants overproduce a modified form of threonyl-tRNA synthetase. When the thrS-lac protein fusion is replaced by several types of thrS-lac operon fusions no effect of the chromosomal thrS allele on beta-galactosidase synthesis is observed. It is also shown that beta-galactosidase synthesis from a promoter-proximal thrS-lac operon fusion is not repressed by threonyl-tRNA synthetase overproduction. The fact that regulation is seen with a thrS-lac protein fusion and not with operon fusions indicates that thrS expression is autoregulated at the translational level. This is confirmed by hybridization experiments which show that under conditions where beta-galactosidase synthesis from a thrS-lac protein fusion is derepressed three- to fivefold, lac messenger RNA is only slightly increased. 相似文献
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Hypodactyly (Hoxa13Hd) mice have a small deletion within the coding sequence of Hoxa13 and a limb phenotype that is more severe than that of mice with an engineered null allele of Hoxa13. We used whole-mount in situ hybridization, Nile blue sulfate staining and genetic crosses to determine the basis for the phenotypic differences between these two mutants. Expression of Hoxd13 was unaffected in Hoxa13-/- mice, but its domain was reduced at the anterior and posterior margins of the autopod in Hoxa13Hd/Hd limb buds. The maturation of Hoxd11 expression was delayed and expression of Hoxa11 failed to become restricted to the autopod/zeugopod junction in both Hoxa13Hd/Hd and Hoxa13-/- limb buds compared to wild-type mice. Fgf8 expression was normal in both Hoxa13Hd/Hd and Hoxa13-/- mice throughout limb development. A dramatic increase in cell death was observed in limb bud mesenchyme of Hoxa13Hd/Hd mice as early as E11.5 but not in mice homozygous for the null allele. Genetic background was excluded as the basisforthe phenotypic differences. Compound heterozygotes (Hoxa13-/Hd) displayed an intermediate phenotype relative to both homozygotes suggesting that Hoxa13Hd has an effect on the development of the autopod beyond that which may result from a loss of HOXA13 protein. These results showthat Hoxa13Hd has a negative effect on the survival of the mesenchyme in the autopod, unlike the Hoxa13 null mutation, that cannot be explained by a failure of the AER to express Fgfs. In addition, at least one target of HOXA13 may be Hoxa11. 相似文献
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