流行性出血热病毒R22株cDNA克隆及其特异性鉴定

用家鼠型流行性出血热病毒R22株RNA,经polyA接尾,以Oligo-dT做引物,合成cDNA。用pUC18为载体转染E.coli Mc1061,建立cDNA克隆。再经菌落杂交,选择病毒特异性的5个阳性克隆制成缺口翻译探针,与病毒RNA3个片段进行反杂交,确定RNA片段的特异性。结果表明,3个克隆为中(M)片段的cDNA,另两个分别为大(L)和小(S)片段cDNA。核苷酸序列分析证明,克隆的DNA中含病毒特异的核苷酸序列。     

Protein kinase C heterogeneity in GH4C1 rat pituitary cells. Characterization of a Ca2(+)-independent phorbol ester receptor

Clonal GH4C1 rat pituitary cells are heterogeneous with respect to phorbol dibutyrate receptors (PDBu-R) and protein kinase C (PKC) content. GH cell PDBu-Rs can be separated into two categories based on Ca2(+)-modulation of receptor affinity. Approximately 70% of the cytosolic PDBu-Rs demonstrate Ca2(+)-sensitive receptor affinity and redistribute from the soluble to the particulate fraction in the presence of excess Ca2+. The other 30% of the receptors remain in the cytosol in the presence of excess Ca2+. Their receptor affinity is Ca2(+)-independent. Northern blot hybridization and immunoblot analysis showed that GH4C1 cells express Ca2(+)-independent epsilon-PKC as well as Ca2(+)-dependent alpha- and beta-PKCs. Cell lysis in Ca2+ caused the redistribution of greater than 95% of alpha- and beta-PKC to the particulate fraction, whereas approximately 90% of the epsilon-PKC remained in the cytosol. In contrast, brief treatment of GH cell cultures with PDBu or thyrotropin-releasing hormone caused redistribution of all three isozymes. Prolonged treatment with PDBu down-modulated all three isozymes but at different rates and to different extents. In contrast, prolonged thyrotropin-releasing hormone treatment selectively down-modulated epsilon-PKC. These results demonstrate that GH cells have both Ca2(+)-sensitive and -insensitive PKCs and PDBu-Rs and that both populations are regulated by agonists that control prolactin synthesis and secretion by these cells.     

Isolation and characterization of temperature-sensitive mutants of measles virus.

Nine temperature-sensitive (ts) mutants of nonattenuated Edmonston strain measles virus were isolated from wild-type virus which was grown in the presence of 5-fluorouracil. Adsorption, temperature shift, and complementation experiments indicated that all these mutants were restricted at an intracellular stage of infection. However, all the mutants were more rapidly inactivated at 41 C than was wild-type virus, suggesting that the ts product of each mutant either influences or is a structural component of the virus. Three complementation groups were found to be represented among the mutants. Group A contained one mutant and it did not induce synthesis of detectable amounts of viral antigen at the nonpermissive temperature (39 C). Group B consisted of six mutants which did not induce viral antigen synthesis at 39 C and one mutant which did. Group C was represented by one mutant and it induced viral antigen synthesis at 39 C. The two mutants which induced sythesis of viral antigen also induced synthesis of relatively small amounts of virus-specific RNA at 39 C. These mutants, while producing cytoplasmic and nuclear accumulations of viral antigen at 39 C, were restricted in production of syncytia and hemadsorption. All the mutants were less neurovirulent than wild-type virus, as indicated by their inability to produce acute disease in newborn hamsters.     

Physiological and structural analysis of light-harvesting mutants of Rhodobacter sphaeroides.

Two mutants of Rhodobacter sphaeroides defective in formation of light-harvesting spectral complexes were examined in detail. Mutant RS103 lacked the B875 spectral complex despite the fact that substantial levels of the B875-alpha polypeptide (and presumably the beta polypeptide) were present. The B800-850 spectral complex was derepressed in RS103, even at high light intensities, and the growth rate was near normal at high light intensity but decreased relative to the wild type as the light intensity used for growth decreased. Mutant RS104 lacked colored carotenoids and the B800-850 spectral complex, as well as the cognate apoproteins. This strain grew normally at high light intensity and, as with RS103, the growth rate decreased as the light intensity used for growth decreased. At very low light intensities, however, RS104 would grow, whereas RS103 would not. Structural analysis of these mutants as well as others revealed that the morphology of the intracytoplasmic membrane invaginations is associated with the presence or absence of the B800-850 complex as well as of carotenoids. A low-molecular-weight intracytoplasmic membrane polypeptide, which may play a role in B800-850 complex formation, is described, as is a 62,000-dalton polypeptide whose abundance is directly related to light intensity as well as the absence of either of the light-harvesting spectral complexes. These data, obtained from studies of mutant strains and the wild type, are discussed in light of photosynthetic membrane formation and the abundance of spectral complexes per unit area of membrane. Finally, a method for the bulk preparation of the B875 complex from wild-type strain 2.4.1 is reported.     

流行性出血热病毒核酸的提取

本文报告流行性出血热R22和A9株病毒培养,同位素标记及核酸提取的初步研究,並获得了该病毒核酸的3个片段即L、M、S、分子量分别约为:3.8、1.9和0.86×10~5道尔顿,不论用20～70％蔗糖密度梯度离心提纯的病毒,或用30％蔗糖垫层离心的粗制病毒,均获同样结果,但多数情况下,用蔗糖密度梯度离心时,除病毒峰外,还发现主要由细胞组份(即线粒体和核糖体等)组成的另一峰,并经常影响病毒RNA的提取,对如何获得纯净病毒及其核酸进行了讨论。     

Loss of elongation factor P disrupts bacterial outer membrane integrity

Elongation factor P (EF-P) is posttranslationally modified at a conserved lysyl residue by the coordinated action of two enzymes, PoxA and YjeK. We have previously established the importance of this modification in Salmonella stress resistance. Here we report that, like poxA and yjeK mutants, Salmonella strains lacking EF-P display increased susceptibility to hypoosmotic conditions, antibiotics, and detergents and enhanced resistance to the compound S-nitrosoglutathione. The susceptibility phenotypes are largely explained by the enhanced membrane permeability of the efp mutant, which exhibits increased uptake of the hydrophobic dye 1-N-phenylnaphthylamine (NPN). Analysis of the membrane proteomes of wild-type and efp mutant Salmonella strains reveals few changes, including the prominent overexpression of a single porin, KdgM, in the efp mutant outer membrane. Removal of KdgM in the efp mutant background ameliorates the detergent, antibiotic, and osmosensitivity phenotypes and restores wild-type permeability to NPN. Our data support a role for EF-P in the translational regulation of a limited number of proteins that, when perturbed, renders the cell susceptible to stress by the adventitious overexpression of an outer membrane porin.