Use of cloned htpR gene of Escherichia coli to introduce htpR mutation into the chromosome

A deletion htpR mutant of Escherichia coli has been constructed on the basis of site-directed mutagenesis. To this end, the chromosomal allele of htpR gene was substituted by a mutant allele introduced into the cell with a recombinant plasmid. The htpR mutant is characterized by a reduced level of proteolysis and therefore by a decreased rate of proteolytic degradation of RNA polymerase of bacteriophage T7. The mutation in htpR is linked with chloramphenicol resistance.     

Molecular cloning and expression of a gene that controls the high-temperature regulon of Escherichia coli.

The high-temperature production (HTP) regulon of Escherichia coli consists of a set of operons that are induced coordinately by a shift to a high temperature under the control of a single chromosomal gene called htpR or hin. To identify more components of this regulon, the rates of synthesis of many polypeptides resolved on two-dimensional polyacrylamide gels were measured in various strains by pulse-labeling after a temperature shift-up. A total of 13 polypeptides were found to be heat inducible only in cells bearing a normal htpR gene on the chromosome or on a plasmid; on this basis these polypeptides were designated products of the HTP regulon. Several hybrid plasmids that contain segments of the E. coli chromosome in the 75-min region were found to carry the htpR gene. A restriction map of this region was constructed, and selected fragments were subcloned and tested for the ability to complement an htpR mutant. The polypeptides encoded by these fragments were detected by permitting expression in maxicells, minicells, and chloramphenicol-treated cells. Complementation was accompanied by production of a polypeptide having a molecular weight of approximately 33,000. This polypeptide, designated F33.4, was markedly reduced in amount in an htpR mutant expected to contain very little htpR gene product. Polypeptide F33.4 is postulated to be the product of htpR and to be an effector that controls heat induction of the HTP regulon.     

htpR-like gene controls cell division and proteolysis in Pseudomonas aeruginosa

As shown in hybridization experiments, the genome of Pseudomonas aeruginosa cells contains a htpR-like gene which controls the expression of heat shock genes in cells of Escherichia coli. By means of specially constructed plasmids, the synthesis of htpR antisense RNA has been found to disturb cell division and proteolytic processes in P. aeruginosa, suggesting the functional relationship of htpR genes in E. coli and Pseudomonas bacteria.     

Thermo-regulated expression of the htpR gene under the control of the PR-promotor of bacteriophage lambda induces supersynthesis of heat shock proteins

The mechanisms of induction of heat shock protein synthesis in E. coli have been studied. For this purpose plasmids in which htpR gene expression is controlled by the PR-promoter of bacteriophage lambda and by the Trp-promoter have been constructed. An effective induction of heat shock proteins requires both an increased content of htpR protein and additional cofactors formed in the cell under heat shock conditions.     

Regulation of proteolytic processes using antisense RNA genes htpR and lon of Escherichia coli in hetero- and homologous genetic systems

Possibility of correction of proteolytic processes in cells of Escherichia coli and Pseudomonas aeruginosa has been studied. For this purpose recombinant plasmids directing the synthesis of antisense RNAs were constructed. In Ps. aeruginosa the synthesis of htpR antisense RNA resulted in 2.5-fold reduction of the intensity of degradation of 3H-puromycin polypeptides under heat shock conditions. An antisense RNA complementary to the 5'- end of E. coli lon gene decreased the same index to the level observed in lon- mutants. Genes homologous to htpR and lon genes of E. coli were found in Pseudomonas: bacteria in hybridisation experiments. This finding suggests that the genetic system of heat shock in these microorganisms is organized in a similar manner.     

Optimizing the expression in E. coli of a synthetic gene encoding somatomedin-C (IGF-I).

Double-stranded DNA encoding the human hormone somatomedin-C (SMC) has been synthesized. This synthetic gene has been inserted into a plasmid bearing the strong leftward promoter (PL) of bacteriophage lambda and expressed in E. coli. Codons for the N-terminal region of SMC which maximized the hormone's synthesis were chosen in an SMC-lac z fusion assay. The amounts of SMC accumulated in E. coli were influenced by mutations at two chromosomal loci, lon and htpR.     

sn-glycerol-3-phosphate acyltransferase tubule formation is dependent upon heat shock proteins (htpR)

Overexpression of the Escherichia coli sn-glycerol-3-phosphate (glycerol-P) acyltransferase, an integral membrane protein, causes formation of ordered arrays of the enzyme in vitro. The formation of these tubular structures did not occur in an E. coli strain bearing a mutation in the htpR gene, the regulatory gene for the heat shock response. The htpR165 mutation was shown by genetic analysis to be the lesion responsible for blockage of tubule formation. Similar amounts of glycerol-P acyltransferase were produced in isogenic htpR+ and htpR165 strains, ruling out an effect of htpR165 on expression of glycerol-P acyltransferase. Further, phospholipid metabolism was not altered in either strain after induction of glycerol-P acyltransferase synthesis. Increased glycerol-P acyltransferase synthesis caused a partial induction of the heat shock response which was dependent upon a wild type htpR gene. The heat shock proteins induced were identified as the groEL and dnaK gene products on two-dimensional gels. These two proteins have been implicated in the assembly of bacteriophage coats. These heat shock proteins appear essential for tubule formation.     

Mutants of Escherichia coli K-12 with increased resistance to ionizing radiation. VI. Increased radioresistance and heat shock proteins

By means of one-dimensional electrophoresis, it is shown that in radiation-resistant Gamr444 and Gamr445 mutants of Escherichia coli K-12 high-molecular weight heat shock proteins are hyperproduced at 32-37 degrees C and are induced more intensively during heat shock (in comparison to the parental wild-type strain AB1157). When the missense htpR15 mutation of the positive regulatory htpR gene for heat shock proteins was introduced by transduction into the genome of the Gamr444 mutant, its enhanced radiation-resistance disappeared but could be restored upon introduction of pKV3 plasmid bearing the htpR+ gene. These data show that heat shock proteins are participating in the enhanced radioresistance of Gamr mutants.     

Mutations in the rpoH (htpR) gene of Escherichia coli K-12 phenotypically suppress a temperature-sensitive mutant defective in the sigma 70 subunit of RNA polymerase.

Escherichia coli K-12 strain 285c contains a mutation in rpoD, the gene encoding the sigma subunit of RNA polymerase. The 70-kilodalton sigma polypeptide encoded by this allele is unstable, and this instability leads to temperature-sensitive growth. We describe the isolation and characterization of four temperature-resistant pseudorevertants of 285c that can grow at high temperature. Each of these revertants increased the stability of the sigma 70 mutant protein. The map position of the suppressor mutations was close to that of the rpoH (htpR) gene. A multicopy plasmid containing the intact rpoH gene restored the temperature-sensitive phenotype. Marker rescue experiments established the positions of three of the alleles within the rpoH gene. One mutation has been sequenced and causes a leucine-to-tryptophan change 7 amino acids from the carboxyl terminus of the rpoH gene product.     

Chemical synthesis and cloning of a gene for human beta-urogastrone

A DNA duplex coding for the 53 amino acids of human beta-urogastrone has been synthesised. Computer assisted design of the gene included restriction endonuclease sites for plasmid insertion, a termination codon and two triplets coding for lysine at the 5'-end of the structural gene. The synthesis involved preparation of 23 oligodeoxyribonucleotides by phosphotriester procedures coupled to rapid HPLC techniques. The gene was constructed in two halves by enzymatic ligation of the oligonucleotides and cloned into a specially constructed chimeric plasmid vector. Escherichia coli K12 MRC8 was transformed by the plasmid and clones containing the full gene sequence were isolated and characterised.     

Expression of a gene for hybrid protein containing the [Val8] amino acid sequence of calcitonin in yeast cells

A recombinant plasmid has been constructed, which directs the synthesis of a hybrid protein, yeast repressible acid phosphatase [Val8]calcitonin, in yeast. The plasmid contains a truncated gene (pho5) acid phosphatase lacking 96 C-terminal amino acids replaced by the synthetic gene for human calcitonin and sequences required for the plasmid propagation in transformed yeast cells. A modified RIA method using immobilisation of protein extracts on solid supports was developed to monitor the expression of the hybrid protein. By use of this method, as well as by standard RIA of CNBr-cleaved protein extracts, synthesis of a calcitonin-related protein was detected in extracts of transformed strains grown under conditions inducing pho5 promoter.     

多靶点复合抗原抗肿瘤基因疫苗的构建及真核表达

目的：构建含有人存活蛋白（survivin）-2B主要T细胞表位区域、人和猴绒毛膜促性腺激素β链的核心片段CTP37区域融合基因的真核表达质粒,并在人胚肾293T细胞中进行表达。方法：通过基因合成和搭桥PCR技术构建含有Survivin2B主要T细胞表位区域、人和猴CTP37区域基因的融合基因2PAG,将其插入含有人IgK链前导信号肽（sig）、人IgG-Fc和糖基磷脂酰肌醇（GPI）锚定信号肽融合基因序列的细胞膜锚定修饰真核表达载体pCI—Fc—GPI中,继而又将酶切后的sig2PAG-FC-GPI融合基因导入含有细小病毒内部核糖体结合位点（IRES）基因且可以共表达人GM-CSF和B7．1融合基因的真核表达载体pVAX1-IRES-GM／B7中;将构建的重组质粒pVAX1-sig-2PAG-FC-GPI-GM／B7（简称pVAX1-2PFcGB）转染293T细胞,利用流式细胞仪和免疫荧光检测其表达情况。结果：2PAG融合基因经测序正确,PCR和酶切鉴定证明已成功连入真核表达载体pVAX-IRES-GM／B7中;流式细胞仪和免疫荧光的检测结果显示,重组质粒pVAX1-2PFcGB在293T细胞中得到很好的表达。结论：成功构建了重组质粒pVAX1-2PFcGB,且在293T细胞中可以有效表达,为对该基因疫苗的后续功能研究奠定了基础。     

Isolation of the R'his plasmids of Vibrio cholerae

V. cholerae strain VT5104 capable of donor activity in conjugation has been constructed by the genetic technique based on plasmid RP4::Mucts62 integration into V. cholerae chromosome due to plasmid homology with Mucts62 inserted into the chromosome. The gene for histidine synthesis has been mobilized and transferred into the recipient cells from VT5104 donor. The conjugants obtained are able to efficiently transfer his+ gene included into the plasmid structure in conjugation with eltor recipient. Thus, the constructed strain VT5104 generates R' plasmids carrying V. cholerae chromosomal genes.     

Gene X of bacteriophage f1 is required for phage DNA synthesis. Mutagenesis of in-frame overlapping genes

The gene II protein of bacteriophage f1 is a site-specific endonuclease required for initiation of phage viral strand DNA synthesis. Within gene II is another gene, X, encoding a protein of unknown function identical to the C-terminal 27% of the gene II protein, and separately translated from codon 300 (AUG) of gene II. By oligonucleotide mutagenesis, we constructed phage mutants in which this codon has been changed to UAG (amber) or UUG (leucine), and propagated them on cells carrying a cloned copy of gene X on a plasmid. The amber mutant makes no gene X protein, and cannot grow in the absence of the complementing plasmid; the leucine-inserting mutant can make gene X protein, and grows normally without the plasmid. Without gene X protein, phage DNA synthesis (particularly viral strand synthesis) is impaired. We discuss this finding in the context of other known in-frame overlapping genes (particularly genes A and A* of phage phi X174), many of which are also involved in the specific initiation of DNA synthesis, and suggest applications for the mutagenic strategy we employed.     

Localization of the DNA segment coding for the synthesis of fraction I on the pYT plague pathogen plasmid]

Fragmentation of the pYT plasmid of the plague pathogen by ten restriction endonucleases has been studied. The evidence has been obtained in support of the possible presence of the movable genetic element containing a HindIII site within the plasmid pYT. The gene encoding the I fraction of the plague pathogen has been cloned. The physical map of the pYT plasmid has been constructed with the use of restriction endonucleases BamHI, XhoI, BstEII, SmaI, EcoRI, and HindIII. The fragment of the plasmid DNA coding for the synthesis of the plague pathogen fraction I has been mapped.