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.     

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.     

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.     

Inhibition of the synthesis of proteins needed for Epstein-Barr virus replication by antisense RNA against the zta gene

Antisense RNA complementary to the Epstein-Barr virus (EBV) Zta gene, an immediate-early gene encoding a transactivator, was applied to inhibit EBV protein synthesis during its lytic cycle. A DNA fragment containing the Zta gene sequence was inserted into an expression vector, pMAMneo, in a sense and antisense direction under a dexamethasone-inducible murine mammary tumor virus LTR promoter, resulting in the construction of plasmids pZ(+) and pZ(–), respectively. Synthesis of Zta protein was reduced in pZ(–)-transfected cells upon dexamethasone induction. Because D-form early antigen and DNA polymerase are essential for viral DNA replication, the contents of these two viral proteins were examined. Amounts of the two lytic proteins were observed to be significantly repressed in pZ(–)-transfected cells. In contrast, both proteins were normally expressed in the sense plasmid pZ(+) or cells transfected with vector alone. Above results demonstrate that Zta antisense RNA can reduce the production of Zta protein and the other lytic proteins, possibly resulting in the inhibition of EBV replication.     

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.     

端粒酶RNA反义基因对肝癌细胞的影响

用RT-PCR的方法钓取端粒酶RNA基因的cDNA,并将其反向插入到逆转录病毒载体pLNCX上,构建hTR基因的反义表达质粒。将质粒经脂质体介导转染人肝癌SMMG-7721细胞中表达。结果表明hTR反义基因的表达有效地封闭或抑制肝癌细胞的端粒酶活性,抑制细胞的生长和增殖,延长细胞的倍增时间并促进细胞凋亡。     

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.