cis-acting mutations that affect rop protein control of plasmid copy number.

A number of pMB1 derivatives provide a trans-acting function that can suppress lethal runaway replication of a temperature-sensitive copy-number mutant of NTP1. Deletion analysis indicates that the region of the pMB1 genome that contains the rop gene is required for this suppression. Mutant derivatives of the temperature-sensitive copy-number mutant plasmid whose conditional lethal phenotype is not suppressed in trans by the region encoding the rop gene have been isolated. These rop-insensitive derivatives contain single nucleotide changes within the RNA I coding region.     

Positive and negative roles of an initiator protein at an origin of replication.

The properties of mutants in the pir gene of plasmid R6K have suggested that the pi protein plays a dual role; it is required for replication to occur and also plays a role in the negative control of the plasmid copy number. In our present study, we have found that the pi level in cell extracts of Escherichia coli strains containing R6K derivatives is surprisingly high (approximately equal to 10(4) dimers per cell) and that this level is not altered in cells carrying high copy number pir mutants. The wild-type and a high copy mutant (Cos405) pir gene were inserted downstream of promoters of different strengths to measure the copy number of an R6K gamma replicon as a function of a 1000-fold range of intracellular pi concentrations. The data demonstrate that reducing the intracellular level of pi to 5% of its normal value can result in a substantial increase in copy number of a gamma origin replicon and that a pi level less than 1% of normal is still permissive for replication. Conversely, increasing the pi level even a few-fold above normal results in a marked inhibition of replication of plasmids containing a single, two, or all three of the R6K origins (alpha, beta, and gamma). We have also shown that the replication inhibition mediated by excess pi is greatly reduced by the pir405 Cos mutation. These results demonstrate that the total level of pi protein is not rate-limiting for a gamma replicon. We have also determined the sensitivity of the pir gene promoter to a wide range of pi concentrations. The activity of this promoter is stimulated by very low pi levels and is almost entirely inhibited when the protein is overproduced 2-fold.     

Temperature-sensitive mutations in the yeast DNA polymerase I gene.

Seven mutations that yield thermolabile DNA polymerases have been isolated in the DNA polymerase I gene, POL1, of Saccharomyces cerevisiae. Strains carrying the mutant genes are temperature sensitive for growth. The pol1 mutants were identified by a method that has general applicability for identification of both temperature-sensitive and null mutations. A plasmid containing a mutagenized pol1 gene was transformed into a strain in which the only functional copy of the POL1 gene was carried on an unstable plasmid. The genes conferring temperature-sensitive growth were detected after elimination of the unstable plasmid containing the wild-type gene. DNA polymerase I isolated from each of the mutants is defective at both 23 degrees C and 36 degrees C. DNA synthesis is deficient in vivo at 36 degrees C in all the mutants, while RNA synthesis is normal in all but one of the mutants. The terminal phenotype of pol1 temperature-sensitive mutants is dumbbell-shaped cells in which the nucleus has migrated to, but apparently not entered, the isthmus separating the mother and the daughter. The POL1 gene is located on chromosome XIV approximately 2 centimorgans away from met4.     

Temperature-sensitive RNA polymerase mutants with altered subunit synthesis and degradation.

A temperature-sensitive mutant having a lethal mutation in the gene for the beta subunit of RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) exhibits an apparent 2- to 3-fold decrease in the rates of both beta and beta' subunit synthesis at the non-permissive temperature, relative to total protein. In contrast, a temperature-sensitive mutant with a lethal mutation in the gene encoding beta' has a 5- to 6-fold increase in the rates of beta and beta' synthesis at 42 degrees. These beta and beta' mutants also exhibit rapid degradation of both subunits at the high temperature.     

母婴传播母子体内乙型肝炎病毒复制与C区启动子变异的关系

目的 了解母婴传播后母子体内 HBV复制程度差异与 C区启动子变异的关系。方法 对 8对复制程度不同的HBV C区启动子基因进行聚合酶链反应（PCR）,应用TA克隆技术构建重组质粒pGEM—CP,每例患者选2个双酶切鉴定正确的克隆测序并分析。结果 HBV复制状态,子高母低组平均变异数于为5.33±1.53,母为9.33±3.06;子低母高组于为8.25±4.27,母为5.00±1.41。母子低复制者C区启动子变异数及变异位点数明显高于高复制者,变异主要集中在BCP和Kunitz类丝氨酸蛋白序列区域,而同复制状态下母子间差异不大。结论母婴传播后母子体内HBV低复制状态与C区启动子变异有关,可能与机体的生长发育状态无关。     

Isolation of temperature-sensitive tyrosine kinase mutants of v-abl oncogene by screening with antibodies for phosphotyrosine.

Temperature-sensitive protein-tyrosine kinase (EC 2.7.1.112) mutants of the oncogene v-abl have been obtained by a direct screening of kinase mutants in bacteria. The v-abl oncogene was expressed in Escherichia coli as a trpE/v-abl fusion protein from the trp promoter. The expression plasmid was mutagenized in vitro and then transfected into E. coli. Bacteria that produced defective tyrosine kinases were distinguished from those producing wild-type v-abl kinases by hybridization with antibodies specific for phosphotyrosine. Two independent mutations that generated temperature-sensitive tyrosine kinases were found to be located in a 12-amino acid region in the tyrosine kinase domain of the v-abl-encoded protein. These mutant v-abl oncogenes displayed temperature-sensitive transforming activity when expressed in NIH 3T3 cells. Cells transformed by these temperature-sensitive tyrosine kinase mutants could be shifted between the transformed and untransformed states by changing their growth temperature. These results confirmed the crucial role of tyrosine kinase activity in the v-abl-mediated oncogenesis.     

Dominant lethal phenotype of a mutation in the -35 recognition region of Escherichia coli sigma 70.

A dominant lethal mutation in the Escherichia coli rpoD gene, which encodes sigma 70, the promoter recognition subunit of RNA polymerase, was isolated after random mutagenesis. The lethal gene was maintained under control of the lac repressor on a low copy plasmid. An amount of lethal sigma 70 that was nearly equimolar with the chromosomally encoded sigma 70 was sufficient to cause cessation of growth. RNA synthesis per unit cell mass was unaffected, but protein synthesis was inhibited by the mutant sigma 70. The amino acid change (Glu-585 to Gln) was in a region of sigma 70 thought to bind the -35 hexamer of the promoter, and the mutant sigma 70 caused increased expression from promoters with nonconsensus bases in the third position of the -35 hexamer. A null mutation of the fis gene could partially suppress the mutant phenotype. These properties are consistent with those expected of a sigma 70 insensitive to growth rate control of rRNA and tRNA promoters.     

Copy-up mutants of the plasmid RK2 replication initiation protein are defective in coupling RK2 replication origins.

The broad host range plasmid RK2 replicates and regulates its copy number in a wide range of Gram-negative bacteria. The plasmid-encoded trans-acting replication protein TrfA and the origin of replication oriV are sufficient for controlled replication of the plasmid in all Gram-negative bacteria tested. The TrfA protein binds specifically to direct repeat sequences (iterons) at the origin of replication. A replication control model, designated handcuffing or coupling, has been proposed whereby the formation of coupled TrfA-oriV complexes between plasmid molecules results in hindrance of origin activity and, consequently, a shut-down of plasmid replication under conditions of higher than normal copy number. Therefore, according to this model, the coupling activity of an initiation protein is essential for copy number control and a copy-up initiation protein mutant should have reduced ability to form coupled complexes. To test this model for plasmid RK2, two previously characterized copy-up TrfA mutations, trfA-254D and trfA-267L, were combined and the resulting copy-up double mutant TFrfA protein TrfA-254D/267L was characterized. Despite initiating runaway (uncontrolled) replication in vivo, the copy-up double-mutant TrfA protein exhibited replication kinetics similar to the wild-type protein in vitro. Purified TrfA-254D, TrfA-267L, and TrfA-254D/267L proteins were then examined for binding to the iterons and for coupling activity using an in vitro ligase-catalyzed multimerization assay. It was found that both single and double TrfA mutant proteins exhibited substantially reduced (single mutants) or barely detectable (double mutant) levels of coupling activity while not being diminished in their capacity to bind to the origin of replication. These observations provide direct evidence in support of the coupling model of replication control.