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The Escherichia coli argU10(Ts) phenotype is caused by a reduction in the cellular level of the argU tRNA for the rare codons AGA and AGG
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Sakamoto K Ishimaru S Kobayashi T Walker JR Yokoyama S 《Journal of bacteriology》2004,186(17):5899-5905
The Escherichia coli argU10(Ts) mutation in the argU gene, encoding the minor tRNA(Arg) species for the rare codons AGA and AGG, causes pleiotropic defects, including growth inhibition at high temperatures, as well as the Pin phenotype at 30 degrees C. In the present study, we first showed that the codon selectivity and the arginine-accepting activity of the argU tRNA are both essential for complementing the temperature-sensitive growth, indicating that this defect is caused at the level of translation. An in vitro analysis of the effects of the argU10(Ts) mutation on tRNA functions revealed that the affinity with elongation factor Tu-GTP of the argU10(Ts) mutant tRNA is impaired at 30 and 43 degrees C, and this defect is more serious at the higher temperature. The arginine acceptance is also impaired significantly but to similar extents at the two temperatures. An in vivo analysis of aminoacylation levels showed that 30% of the argU10(Ts) tRNA molecules in the mutant cells are actually deacylated at 30 degrees C, while most of the argU tRNA molecules in the wild-type cells are aminoacylated. Furthermore, the cellular level of this mutant tRNA is one-tenth that of the wild-type argU tRNA. At 43 degrees C, the cellular level of the argU10(Ts) tRNA is further reduced to a trace amount, while neither the cellular abundance nor the aminoacylation level of the wild-type argU tRNA changes. We concluded that the phenotypic properties of the argU10(Ts) mutant result from these reduced intracellular levels of the tRNA, which are probably caused by the defective interactions with elongation factor Tu and arginyl-tRNA synthetase. 相似文献
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Dana L. Swenson Kyoung-Jin Kim Erich W. Six Steven Clegg 《Molecular genetics and genomics : MGG》1994,244(2):216-218
The gene fimU, located on a recombinant plasmid carrying the Salmonella typhimurium type 1 fimbrial gene cluster is closely related to the Escherichia coli tRNA gene argU. The fimU gene complements an E. coli argU mutant that is a P2 lysogen, thereby allowing the phage P4 to grow in this strain but preventing the growth of phage lambda. In addition, fimU was shown to be involved in fimbrial expression since transformants of the E. coli argU mutant could produce fimbriae only in the presence of fimU but not in its absence, whereas in an E. coli argU + strain fimbriation did not require the fimU gene. 相似文献
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Characterization of the cryptic lambdoid prophage DLP12 of Escherichia coli and overlap of the DLP12 integrase gene with the tRNA gene argU. 总被引:12,自引:6,他引:6
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The argU (dnaY) gene of Escherichia coli is located, in clockwise orientation, at 577.5 kilobases (kb) on the chromosome physical map. There was a cryptic prophage spanning the 2 kb immediately downstream of argU that consisted of sequences similar to the phage P22 int gene, a portion of the P22 xis gene, and portions of the exo, P, and ren genes of bacteriophage lambda. This cryptic prophage was designated DLP12, for defective lambdoid prophage at 12 min. Immediately clockwise of DLP12 was the IS3 alpha 4 beta 4 insertion element. The argU and DLP12 int genes overlapped at their 3' ends, and argU contained sequence homologous to a portion of the phage P22 attP site. Additional homologies to lambdoid phages were found in the 25 kb clockwise of argU. These included the cryptic prophage qsr' (P. J. Highton, Y. Chang, W. R. Marcotte, Jr., and C. A. Schnaitman, J. Bacteriol. 162:256-262, 1985), a sequence homologous to a portion of lambda orf-194, and an attR homolog. Inasmuch as the DLP12 att int xis exo P/ren region, the qsr' region, and homologs of orf-194 and attR were arranged in the same order and orientation as the lambdoid prophage counterparts, we propose that the designation DLP12 be applied to all these sequences. This organization of the DLP12 sequences and the presence of the argU/DLP12 int pair in several E. coli strains and closely related species suggest that DLP12 might be an ancestral lambdoid prophage. Moreover, the presence of similar sequences at the junctions of DLP12 segments and their phage counterparts suggests that a common mechanism could have transferred these DLP12 segments to more recent phages. 相似文献
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A gene for expression of horse heart myoglobin in Escherichia coli has been constructed in one step from long synthetic oligonucleotides. The synthetic gene contains an efficient translation initiation signal and used codons that are commonly found in E. coli. Unique restriction sites are placed throughout the gene. It has been inserted in a phagemid vector and is expressed from the lac promoter in E. coli at high efficiency, the soluble heme protein representing approximately 10% of soluble protein. Two versions of horse heart myoglobin were produced with aspartic acid or asparagine at residue 122. Comparison of chromatographic mobilities of these two proteins with authentic horse heart myoglobin identified aspartic acid as the correct residue 122. The availability of this gene, which is designed to facilitate oligonucleotide mutagenesis or cassette mutagenesis, will allow systematic structure-function analysis of horse heart myoglobin. 相似文献
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The Salmonella typhimurium uracil-sensitive mutation use is in argU and encodes a minor arginine tRNA. 总被引:3,自引:1,他引:2
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The use gene of Salmonella typhimurium was previously identified by a mutation conferring sensitivity to uracil in glucose minimal medium. The use gene was cloned and identified as an allele of argU encoding a tRNA for a minor arginine codon (CGG). The uracil-sensitive phenotype was shown to result from a base substitution in the anticodon stem of this tRNA. 相似文献
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This study presents cloning and expression of Actinobacillus pleuropneumoniae Apx I toxin in Escherichia coli expression system to produce fusion protein for the subsequent immunological studies. The gene coding Apx I toxin was amplified from the A. pleuropneumoniae serotype 10 DNA using polymerase chain reaction and cloned to vector under the control of strong, inducible T7 promoter. The presence of insert was confirmed by PCR screening and sequencing after the propagation of recombinant DNA in E. coli cells. The gene coding A. pleuropneumoniae Apx I toxin was extended with a segment to encode a polyhistidine tag linked to its C-terminal sequence allowing a one-step affinity purification of the complex with Ni-NTA resin. Expression of the Apx I coding sequence in E. coli resulted in the formation of insoluble inclusion bodies purified according to a standard purification protocol. The ease of this expression system, the powerful single-step purification and low costs make it possible to produce Apx I in large amounts to further study the role of Apx I in physiological processes. 相似文献
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We report the genetic mapping of pheU , an Escherichia coli gene for phenylalanine tRNA. This gene was located near 94.5 min on the E. coli map. There are no other known tRNA or ribosomal genes in its immediate vicinity. 相似文献
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Cloning and characterization of the Escherichia coli gene coding for alkaline phosphatase. 总被引:11,自引:6,他引:5
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P E Berg 《Journal of bacteriology》1981,146(2):660-667
The Escherichia coli structural gene for alkaline phosphatase, phoA, and a promoter-like mutant of phoA, called pho-1003(Bin) phoA+, were cloned by using plasmid vectors. Initially, these genes were cloned on deoxyribonucleic acid fragments of 28.9 kilobases (kb). Subsequently, they were subcloned on fragments and 4.8 and then 2.7 kilobases. A restriction map was developed, and phoA was localized to a 1.7-kb region. The promoter end of the gene was inferred by its proximity to another gene cloned on the same deoxyribonucleic acid fragment, proC. The stability of the largest plasmid (33.3 kb) was found to be recA dependent, although the subcloned plasmids were stable in a recA+ strain. Synthesis of alkaline phosphatase directed by the phoA+ and pho-1003(Bin) phoA+ plasmids in a phoA deletion strain was assayed under repressing and derepressing levels of phosphate. These data were compared with the copy numbers of the plasmids. It was found that synthesis of alkaline phosphatase was tightly regulated, even under derepressing conditions: a copy number of 17 enabled cells to synthesize only about twofold more enzyme than did cells with 1 chromosomal copy of phoA+. Enzyme levels were also compared for cells containing pho-1003(Bin) phoA+ and phoA+. 相似文献
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Characterization of the sppA gene coding for protease IV, a signal peptide peptidase of Escherichia coli. 总被引:7,自引:5,他引:2
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The sppA gene codes for protease IV, a signal peptide peptidase of Escherichia coli. Using the gene cloned on a plasmid, we constructed an E. coli strain carrying the ampicillin resistance gene near the chromosomal sppA gene and an sppA deletion strain in which the deleted portion was replaced by the kanamycin resistance gene. Using these strains, we mapped the sppA gene at 38.5 min on the chromosome, the gene order being katE-xthA-sppA-pncA. Although digestion of the signal peptide that accumulated in the cell envelope fraction was considerably slower in the deletion mutant than in the sppA+ strain, it was still significant, suggesting the participation of another envelope protease(s) in signal peptide digestion. 相似文献
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Nucleotide sequence of the melA gene, coding for alpha-galactosidase in Escherichia coli K-12. 总被引:7,自引:1,他引:6
Melibiose uptake and hydrolysis in E.coli is performed by the MelB and MelA proteins, respectively. We report the cloning and sequencing of the melA gene. The nucleotide sequence data showed that melA codes for a 450 amino acid long protein with a molecular weight of 50.6 kd. The sequence data also supported the assumption that the mel locus forms an operon with melA in proximal position. A comparison of MelA with alpha-galactosidase proteins from yeast and human origin showed that these proteins have only limited homology, the yeast and human proteins being more related. However, regions common to all three proteins were found indicating sequences that might comprise the active site of alpha-galactosidase. 相似文献
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The nucleotide sequence of the Escherichia coli fus gene, coding for elongation factor G. 总被引:25,自引:4,他引:21
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We have determined the nucleotide sequence of the Escherichia coli fus gene, which codes for elongation factor G. The protein product of the sequenced gene contains 703 amino acids, with a predicted molecular weight of 77,444. The fus gene shows the nonrandom pattern of codon usage typical of ribosomal proteins and other proteins synthesized at a high level. We have identified several potential promoter sequences within the gene. One of these sequences may correspond to the secondary promoter for expression of the downstream tufA gene (encoding elongation factor Tu) whose activity has been described previously (1,2). A comparison of the nucleotide and amino acid sequences of elongation factors G and Tu reveals a limited but significant homology between the two proteins within the 150 amino acid residues at their amino-terminal ends. 相似文献