Heat-Sensitive Early Function in Induced λ Nsus Lysogens

Mutations in gene N of lambda prevent killing of the host bacterium after infection. However, derepression of Nsus prophages in nonpermissive (pm(-)) bacteria results in death of the lysogens. When prophages in pm(-)(lambdaCItsA-Nsus) lysogens are derepressed by raising the temperature to 45 C, the cells remain viable as long as they are at 45 C. However, they cannot form colonies at 33 C unless they have been superinfected, at the high temperature, by lambdaCI(+)-Nsus phage which produces repressor at 45 C. A large fraction of these "rescued," heat-inducible lysogens are lysogenized by the superinfecting phage, but lysogenization is not required for rescue. In pm(-)(lambdaCItsA-Nsus) lysogens growing at 45 C, the rate of deoxyribonucleic acid (DNA) synthesis shows a characteristic increase after the temperature is lowered. This increased DNA synthesis, which is correlated with loss of rescue potential, does not occur as long as the cultures are maintained at 45 C.     

DAF1, a mutant gene affecting size control, pheromone arrest, and cell cycle kinetics of Saccharomyces cerevisiae.

The mating pheromone alpha-factor arrests Saccharomyces cerevisiae MATa cells in the G1 phase of the cell cycle. Size control is also exerted in G1, since cells do not exit G1 until they have attained a critical size. A dominant mutation (DAF1-1) which causes both alpha-factor resistance and small cell size (volume about 0.6-fold that of the wild type) has been isolated and characterized genetically and by molecular cloning. Several alpha-factor-induced mRNAs were induced equivalently in daf1+ and DAF1-1 cells. The DAF1-1 mutation consisted of a termination codon two-thirds of the way through the daf1+ coding sequence. A chromosomal deletion of DAF1 produced by gene transplacement increased cell volume about 1.5-fold; thus, DAF1-1 may be a hyperactive or deregulated allele of a nonessential gene involved in G1 size control. Multiple copies of DAF1-1 also greatly reduced the duration of the G1 phase of the cell cycle.     

Hierarchical analysis of variation in the mitochondrial 16S rRNA gene among Hymenoptera

Nucleotide sequences from a 434-bp region of the 16S rRNA gene were analyzed for 65 taxa of Hymenoptera (ants, bees, wasps, parasitoid wasps, sawflies) to examine the patterns of variation within the gene fragment and the taxonomic levels for which it shows maximum utility in phylogeny estimation. A hierarchical approach was adopted in the study through comparison of levels of sequence variation among taxa at different taxonomic levels. As previously reported for many holometabolous insects, the 16S data reported here for Hymenoptera are highly AT-rich and exhibit strong site-to-site variation in substitution rate. More precise estimates of the shape parameter (alpha) of the gamma distribution and the proportion of invariant sites were obtained in this study by employing a reference phylogeny and utilizing maximum-likelihood estimation. The effectiveness of this approach to recovering expected phylogenies of selected hymenopteran taxa has been tested against the use of maximum parsimony. This study finds that the 16S gene is most informative for phylogenetic analysis at two different levels: among closely related species or populations, and among tribes, subfamilies, and families. Maximization of the phylogenetic signal extracted from the 16S gene at higher taxonomic levels may require consideration of the base composition bias and the site-to-site rate variation in a maximum-likelihood framework.      

Killing of Bacillus Spores by Aqueous Dissolved Oxygen, Ascorbic Acid, and Copper Ions

An approach to decontamination of biological endospores is discussed. Specifically, the performance of an aqueous modified Fenton reagent is examined. A modified Fenton reagent formulation of cupric chloride, ascorbic acid, and sodium chloride is shown to be an effective sporicide under aerobic conditions. The traditional Fenton reaction involves the conversion of hydrogen peroxide to hydroxyl radical by aqueous ionic catalysts such as the transition metal ions. Our modified Fenton reaction involves the conversion of aqueous dissolved oxygen to hydrogen peroxide by an ionic catalyst (Cu²⁺) and then subsequent conversion to hydroxyl radicals. Results are given for the modified Fenton reagent deactivating spores of Bacillus globigii. A biocidal mechanism is proposed that is consistent with our experimental results and independently derived information found in the literature. This mechanism requires diffusion of relatively benign species into the interior of the spore, where dissolved O₂ is then converted through a series of reactions which ultimately produce hydroxyl radicals that perform the killing action.     

Isolation and Characterization of New Alleles of the Cyclin-Dependent Kinase Gene CDC28 with Cyclin-Specific Functional and Biochemical Defects

The G₁ cyclin Cln2 negatively regulates the mating-factor pathway. In a genetic screen to identify factors required for this regulation, we identified an allele of CDC28 (cdc28-csr1) that blocked this function of Cln2. Cln2 immunoprecipitated from cdc28-csr1 cells was completely defective in histone H1 kinase activity, due to defects in Cdc28 binding and activation by Cln2. In contrast, Clb2-associated H1 kinase and Cdc28 binding was normal in immunoprecipitates from these cells. cdc28-csr1 was significantly deficient in other aspects of genetic interaction with Cln2. The cdc28-csr1 mutation was determined to be Q188P, in the T loop distal to most of the probable Cdk-cyclin interaction regions. We performed random mutagenesis of CDC28 to identify additional alleles incapable of causing CLN2-dependent mating-factor resistance but capable of complementing cdc28 temperature-sensitive and null alleles. Two such mutants had highly defective Cln2-associated kinase, but, surprisingly, two other mutants had levels of Cln2-associated kinase near to wild-type levels. We performed a complementary screen for CDC28 mutants that could cause efficient Cln2-dependent mating-factor resistance but not complement a cdc28 null allele. Most such mutants were found to alter residues essential for kinase activity; the proteins had little or no associated kinase activity in bulk or in association with Cln2. Several of these mutants also functioned in another assay for CLN2-dependent function not involving the mating-factor pathway, complementing the temperature sensitivity of a cln1 cln3 cdc28-csr1 strain. These results could indicate that Cln2-Cdc28 kinase activity is not directly relevant to some CLN2-mediated functions. Mutants of this sort should be useful in differentiating the function of Cdc28 complexed with different cyclin regulatory subunits.     

A cosmid vector for systematic chromosome walking

We describe the construction of a cosmid, LoristB, that contains SP6 and T7 phage-encoded RNA polymerase promoter sequences that are oriented towards and immediately adjacent to HindIII and BamHI cloning sites. We describe techniques for rapidly generating RNA probes from these promoters that must be complementary to the extreme left or right ends of the cloned DNA and can be used for library screening. Probe preparation requires neither prior knowledge of restriction sites nor fragment isolation. We also make extensive use of cos mapping restriction-mapping protocols that we have devised for our cosmid vectors for generation and alignment of steps in a cosmid walk.     

Deletion of the Cul1 gene in mice causes arrest in early embryogenesis and accumulation of cyclin E.

The stability of many proteins is controlled by the ubiquitin proteolytic system, which recognizes specific substrates through the action of E3 ubiquitin ligases [1]. The SCFs are a recently described class of ubiquitin ligase that target a number of cell cycle regulators and other proteins for degradation in both yeast and mammalian cells [2] [3] [4] [5] [6]. Each SCF complex is composed of the core protein subunits Skp1, Rbx1 and Cul1 (known as Cdc53 in yeast), and substrate-specific adaptor subunits called F-box proteins [2] [3] [4]. To understand the physiological role of SCF complexes in mammalian cells, we generated mice carrying a deletion in the Cul1 gene. Cul1(-/-) embryos arrested around embryonic day 6.5 (E6.5) before the onset of gastrulation. In all cells of the mutant embryos, cyclin E protein, but not mRNA, was highly elevated. Outgrowths of Cul1(-/-) blastocysts had limited proliferative capacity in vitro and accumulated cyclin E in all cells. Within Cul1(-/-) blastocyst cultures, trophoblast giant cells continued to endocycle despite the elevated cyclin E levels. These results suggest that cyclin E abundance is controlled by SCF activity, possibly through SCF-dependent degradation of cyclin E.     

动态分析沙鼠非酒精性脂肪肝病形成及生化影响

目的分析高脂饮食导致的沙鼠NAFLD疾病进展中脂质代谢、肝功能、抗氧化等方面的变化,探讨沙鼠NAFLD的形成机理。方法雄性沙鼠120只,随机分为对照组和模型组。对照组给予普通饲料,模型组高脂饮食建立NAFLD模型。分别于1、2、4、6、8、16周每组各处死10只动物,观察肝脏病理变化,计算肝指数,检测血清CHO、TG、LDL-C、HDL-C、GOP、GPT及肝组织的SOD、GSH-PX、CAT活性和FFA含量。结果模型组病理观察2周形成单纯性脂肪肝,6周动物门管区有轻度炎症,8周出现腺泡Ⅲ带局灶性窦周/细胞周纤维化,16周肝脏中度纤维化;模型组第1、2、4、6、8、16周时CHO、HDL-C、LDL-C及FAA波动性升高,但均高于同期对照组（P〈0.05,P〈0.01）,TG在1、2、4周高于同期对照组（P〈0.05,P〈0.01）;16周末GOT、GPT出现了显著性升高（P〈0.01）。抗氧化酶GSH-PX、CAT、SOD活性模型组在第8～16周显著性降低（P〈0.05,P〈0.01）。结论高脂饮食使沙鼠2周形成单纯性脂肪肝模型后,随饲喂时间延长,16周末可发展为中度肝纤维化。脂质代谢紊乱与氧化应激在沙鼠NAFLD进展中的发挥了不同的作用。     

The adaptor protein shb binds to tyrosine 1175 in vascular endothelial growth factor (VEGF) receptor-2 and regulates VEGF-dependent cellular migration

Previous studies have shown that the adaptor protein Shb is involved in receptor tyrosine kinase signaling. In this study, we demonstrate that Shb is phosphorylated in an Src-dependent manner upon vascular endothelial growth factor (VEGF) stimulation using porcine aortic endothelial cells expressing the human VEGF receptor 2 (VEGFR-2) (KDR). In co-immunoprecipitation experiments, we could detect an interaction between Shb and the VEGFR-2 in human telomerase-immortalized microvascular endothelial cells. Furthermore, in a glutathione S-transferase pull-down assay, the Src homology 2 domain of Shb was shown to interact with phosphorylated tyrosine 1175 in the C-terminal tail of VEGFR-2. VEGF-induced Shb phosphorylation was lost in porcine aortic endothelial cells expressing a chimeric murine VEGFR-2 (Flk-1) with a mutation at the corresponding position. Shb expression was specifically decreased by 80%, in a transient manner, by using the short interfering RNA technique. Reduced Shb expression led to a loss of stimulation of phosphatidylinositol 3-kinase, phosphorylation of focal adhesion kinase at tyrosine 576, the generation of focal adhesions, and stress fiber formation in response to VEGF. Furthermore, we show that VEGF-induced migration is inhibited in Shb short interfering RNA-treated cells. Our data demonstrate that Shb is important for VEGF signaling in endothelial cells. This is achieved by Shb binding to tyrosine 1175 in the VEGFR-2, which regulates VEGF-induced formation of focal adhesions and cell migration, of which the latter occurs in a phosphatidylinositol 3-kinase-dependent manner.