The rabbit C family of short, interspersed repeats. Nucleotide sequence determination and transcriptional analysis

When the entire adeno-associated virus (AAV) genome is inserted into a bacterial plasmid, infectious AAV genomes can be rescued and replicated when the recombinant AAV-plasmid DNA is transfected into human 293 cells together with helper adenovirus particles. We have taken advantage of this experimental system to analyze the effects of several classes of mutations on replication of AAV DNA. We obtained AAV mutants by molecular cloning in bacterial plasmids of naturally occurring AAV variant or defective-interfering genomes. Each of these mutants contains a single internal deletion of AAV coding sequences. Also, some of these mutant-AAV plasmids have additional deletions of one or both AAV terminal palindromes introduced during constructions in vitro. We show here that AAV mutants containing internal deletions were defective for replicative form DNA replication (rep-) but could be complemented by intact wild-type AAV. This indicates that an AAV replication function, Rep, is required for normal AAV replication. Mutants in which both terminal palindromes were deleted (ori-) were also replication defective but were not complementable by wild-type AAV. The cis-dominance of the ori- mutation shows that the replication origin is comprised in part of the terminal palindrome. Deletion of only one terminal palindrome was phenotypically wild-type and allowed rescue and replication of AAV genomes in which the deleted region was regenerated apparently by an intramolecular correction mechanism. One model for this correction mechanism is proposed. An AAV ori- mutant also complemented replication of AAV rep- mutants as efficiently as did wild-type AAV. These studies also revealed an unexpected additional property of the deletion mutants in that monomeric single-stranded single-stranded DNA accumulated very inefficiently even though monomeric single-stranded DNA from the complementing wild-type AAV did accumulate.     

在甜椒花药培养中马来酰胼对小孢子发育的抑制作用

选用甜椒的小孢子单核期花药,用100、300、500、2000ppm的马来酰肼溶液浸泡处理,并设对照,进行无激素MS固体培养基培养。分别取样用各种组化方法对花药内部多糖、蛋白质、核酸及ATP酶进行组化反应和形态学上的观察。与对照组相比,处理组花药外部形态和内部结构出现许多变异。小孢子内多糖,蛋白质含量减少;绒毡层无明显变化;两组中,核酸的含量均无明显变化;ATP酶的活性低于对照组。可能,马来酰肼对于花药中ATP酶等产生抑制作用,致使花药败育。     

海枣曲霉β-半乳糖苷酶的提纯与性质

 通过过聚乙二醇6000-磷酸钾缓冲液双相分离、Sephadex G-100凝胶过滤、DEAE-Sephadex A-50离子交换层析、羟基磷灰石层析及SephadexG-100凝胶过滤等提纯步骤,从海枣曲霉(Aspergillus phoenicis)麦麸培养物抽提液中提纯得到凝胶电泳均一的β-半乳糖苷酶。该酶的最适pH为3.5—4.0,最适温度为60℃(反应15分钟),在pH5.0—8.5之间及60℃以下稳定。在65℃和70℃保温时失活50％的时间分别为27和2分钟。用SDS凝胶电泳法和梯度凝胶电泳法分别测得该酶的分子量为115,000和118,000。薄层凝胶等电聚焦法测得其等电点为pH4.6。     

Metabolism of 9-(1,3-dihydroxy-2-propoxymethyl)guanine, a new anti-herpes virus compound, in herpes simplex virus-infected cells

The metabolism of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), one of the most promising new anti-herpes virus compounds, in HeLa cells infected with herpes simplex virus type 1 was compared with that in the uninfected HeLa cells. In the virus-infected cells, the uptake of DHPG was enhanced and the major metabolites were found to be the mono-, di-, and triphosphate derivatives. The formation of these metabolites was dependent on the extracellular concentration of DHPG (0.5 to 5.0 microM). Virus-induced thymidine kinase was capable of phosphorylating DHPG to its monophosphate which could be further phosphorylated to the di- and triphosphate derivatives by the host cellular enzymes. Incorporation of the DHPG into DNA was observed in virus-infected cells. In contrast with 9-(2-hydroxyethoxymethyl)guanine, DHPG seemed not to serve as a chain terminator, but to be incorporated internally into DNA strands.     

Factors influencing survival and growth of mammalian cells exposed to hypothermia. I. Effects of temperature and membrane lipid perturbers

The Arrhenius plot of the rate of V79 Chinese hamster cell inactivation due to hypothermia has a "break" around 7-10 degrees C with optimum storage temperature for unprotected cells being about 10 degrees C. Addition of the membrane lipid perturber, butylated hydroxytoluene, improves survival of cells when compared to controls at temperatures below this break but not above. Arrhenius plots of growth rates of the cells show breaks at 30 and 40 degrees C. Measurements of membrane fluidity by electron spin resonance or membrane polarization anisotropy by fluorescence spectrophotometry techniques as a function of temperature in these cells also reveal "breaks" centered around 8 and 30 degrees C. Hence, the changes in the rate of cell inactivation and growth as a function of temperature may be related to membrane lipid phase changes.     

Himasthla quissetensis: uptake and utilization of glucose by rediae as determined by autoradiography and respirometry

Daughter rediae of Himasthla quissetensis removed from the digestive gland of Nassarius obsoletus were placed in sterilized seawater fortified with antibiotics. When [³H]-glucose was added to this medium and autoradiographs were made after 3, 9, and 24 hr of exposure, labeling was observed associated with the redial walls and developing germ balls and cercariae within the brood chambers. Respirometric determinations on starved rediae suspended in the seawater medium with and without glucose revealed the rate of oxygen utilization by rediae exposed to exogenous glucose is significantly elevated. These results are interpreted to mean that the daughter rediae of H. quissetensis can take up and utilize glucose.     

Synteny on mouse chromosome 5 of homologs for human DNA loci linked to the Huntington disease gene

Comparative mapping in man and mouse has revealed frequent conservation of chromosomal segments, offering a potential approach to human disease genes via their murine homologs. Using DNA markers near the Huntington disease gene on the short arm of chromosome 4, we defined a conserved linkage group on mouse chromosome 5. Linkage analyses using recombinant inbred strains, a standard outcross, and an interspecific backcross were used to assign homologs for five human loci, D4S43, D4S62, QDPR, D4S76, and D4S80, to chromosome 5 and to determine their relationships with previously mapped markers for this autosome. The relative order of the conserved loci was preserved in a linkage group that spanned 13% recombination in the interspecific backcross analysis. The most proximal of the conserved markers on the mouse map, D4S43h, showed no recombination with Emv-1, an endogenous ecotropic virus, in 84 outcross progeny and 19 recombinant inbred strains. Hx, a dominant mutation that causes deformities in limb development, maps approximately 2 cM proximal to Emv-1. Since the human D4S43 locus is less than 1 cM proximal to HD near the telomere of chromosome 4, the murine counterpart of the HD gene might lie between Hx and Emv-1 or D4S43h. Cloning of the region between these markers could generate new probes for conserved human sequences in the vicinity of the HD gene or possibly candidates for the murine counterpart of this human disease locus.