检测脱氨基速率的超灵敏方法及应用

脱氨基被认为是引起细胞突变的主要因素,如果这些脱氨基的产物不被修复,将引起转换(transition)突变.为了理解DNA结构和其化学活性的关系,介绍一种新的灵敏的遗传学方法,它应用在DNA特定点的脱氨基速率的测定.这种方法基于M13mp2噬菌体内的1acZα基因中的CCC脯氨酸密码子的反转突变,即每个脱氨基事件表现为在白色菌斑背景中的一个蓝色菌斑,其灵敏度可达10⁵ M13mp2 DNA分子中检验出一个脱氨基事件.此外,该法可以计算脱氨基动力学速率常数和反应活化能.     

A nuclear genetic lesion affecting Saccharomyces cerevisiae mitochondrial translation is complemented by a homologous Bacillus gene.

A novel Bacillus gene was isolated and characterized. It encodes a homolog of Saccharomyces cerevisiae Pet112p, a protein that has no characterized relative and is dispensable for cell viability but required for mitochondrial translation. Expression of the Bacillus protein in yeast, modified to ensure mitochondrial targeting, partially complemented the phenotype of the pet112-1 mutation, demonstrating a high degree of evolutionary conservation for this as yet unidentified component of translation.     

The Mammalian Protein (rbet1) Homologous to Yeast Bet1p Is Primarily Associated with the Pre-Golgi Intermediate Compartment and Is Involved in Vesicular Transport from the Endoplasmic Reticulum to the Golgi Apparatus

Yeast Bet1p participates in vesicular transport from the endoplasmic reticulum to the Golgi apparatus and functions as a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) associated with ER-derived vesicles. A mammalian protein (rbet1) homologous to Bet1p was recently identified, and it was concluded that rbet1 is associated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1. In the present study using rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi apparatus as marked by the Golgi mannosidase II in normal rat kidney cells. Rather, rbet1 is predominantly associated with vesicular spotty structures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm. These structures colocalize with the KDEL receptor and ERGIC-53, which are known to be enriched in the intermediate compartment. When the Golgi apparatus is fragmented by nocodazole treatment, a significant portion of rbet1 is not colocalized with structures marked by Golgi mannosidase II or the KDEL receptor. Association of rbet1 in cytoplasmic spotty structures is apparently not altered by preincubation of cells at 15°C. However, upon warming up from 15 to 37°C, rbet1 concentrates into the peri-Golgi region. Furthermore, rbet1 colocalizes with vesicular stomatitis virus G-protein en route from the ER to the Golgi. Antibodies against rbet1 inhibit in vitro transport of G-protein from the ER to the Golgi apparatus in a dose-dependent manner. This inhibition can be neutralized by preincubation of antibodies with recombinant rbet1. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle docking but before the actual fusion event. Antibodies against rbet1 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. These results suggest that rbet1 may be involved in the docking process of ER- derived vesicles with the cis-Golgi membrane.     

A Role for Cdk2 Kinase in Negatively Regulating DNA Replication during S Phase of the Cell Cycle

Using cell-free extracts made from Xenopus eggs, we show that cdk2-cyclin E and A kinases play an important role in negatively regulating DNA replication. Specifically, we demonstrate that the cdk2 kinase concentration surrounding chromatin in extracts increases 200-fold once the chromatin is assembled into nuclei. Further, we find that if the cdk2–cyclin E or A concentration in egg cytosol is increased 16-fold before the addition of sperm chromatin, the chromatin fails to initiate DNA replication once assembled into nuclei. This demonstrates that cdk2–cyclin E or A can negatively regulate DNA replication. With respect to how this negative regulation occurs, we show that high levels of cdk2–cyclin E do not block the association of the protein complex ORC with sperm chromatin but do prevent association of MCM3, a protein essential for replication. Importantly, we find that MCM3 that is prebound to chromatin does not dissociate when cdk2– cyclin E levels are increased. Taken together our results strongly suggest that during the embryonic cell cycle, the low concentrations of cdk2–cyclin E present in the cytosol after mitosis and before nuclear formation allow proteins essential for potentiating DNA replication to bind to chromatin, and that the high concentration of cdk2–cyclin E within nuclei prevents MCM from reassociating with chromatin after replication. This situation could serve, in part, to limit DNA replication to a single round per cell cycle.     

Structural and functional determinants in adenovirus type 2 penton base recombinant protein.

Discrete domains involved in structural and functional properties of adenovirus type 2 (Ad2) penton base were investigated with site-directed mutagenesis of the recombinant protein expressed in baculovirus-infected cells. Seventeen substitution mutants were generated and phenotyped for various functions in insect and human cells as follows. (i) Pentamerization of the penton base protein was found to be dependent on three amino acid side chains, the indole ring of Trp119, the hydroxylic group of Tyr553, and the basic group of Lys556. (ii) Arg254, Cys432, and Trp439, the stretch of basic residues at positions 547 to 556, and Arg340 of the RGD motif played a critical role in stable fiber-penton base interactions in vivo. (iii) Nuclear localization of penton base in Sf9 cells was negatively affected in mutants W119H or W165H, and, to a lesser extent, by substitutions in the consensus polybasic signal at positions 547 to 549. (iv) Penton base mutants were also assayed for HeLa cell binding, cell detachment, plasmid DNA internalization, and Ad-mediated gene delivery. The results obtained suggested that the previously identified integrin-binding motifs RGD340 and LDV287 were functionally and/or topologically related to other discrete regions which include Trp119, Trp165, Cys246, Cys432, and Trp439, all of which were involved in penton base-cell surface recognition, endocytosis, and postendocytotic steps of the virus life cycle.     

Experimental study and mechanism analysis on bioeffects by nanosecond electromagnetic pulses

The athermal bioeffects caused by nanosecond electromagnetic pulses with body cells was studied by using a broad band transverse EM-wave cell (BTEM CELL). The experimental system and preliminary mechanism analysis were presented.     

布氏田鼠肥满度分析和小型兽类肥满度指标K与KWL（重长指标）的比较

通过对两个肥满度指标的理论和生物学意义分析,以及对布氏田鼠肥满度的研究和实际应用的讨论,认为描述动物的肥满度时,重长指标ＫＷＬ优于指标Ｋ。两指标的最大差别是成体的ＫＷＬ值大于幼体,而成体的Ｋ值小于幼体。布氏田鼠肥满度没有性别差异;有异著的年龄差异,成体鼠的肥满度高于幼鼠;有显著的季节变化,鼠种群春季肥满度最高,夏季降低,秋季回升;有显著的年际变化,高数量年的肥满度高于低数量年。     

不同光学异构体组成的氯氰菊酯对敏感及抗性家蝇的神经电生理学研究

采用敏感家蝇(Musca domestica vicina L.)及由5种不同光学异构体组成的氯氰菊酯选育的抗性家蝇中胸足离体标本,观测五种药剂对足感觉神经纤维冲动发放的影响。各种异构体组成的氯氰菊酯均可引起感觉神经纤维发放的增加,然后逐渐降低,直至完全阻断。以阻断时间和加药剂量为参数,求出神经敏感度。结果表明：五种药剂作用于敏感家蝇的神经敏感度与室内生物测定的LD₅₀值(μg/头)无相关性,而与供试药剂中反α体与顺α体的比例有关,反α体所占比例越多的药物,神经敏感度越高。抗性家蝇的神经敏感度与敏感 家蝇相比大幅度下降,可以认为神经敏感度降低是家蝇对氯氰菊酯产生抗性的主要机制。抗性家蝇中,氯氰菊酯品系(RC₁)的抗生水平最高,但它的神经敏感度较其它抗性品系也高,由此推测,RC₁,的抗性机制与其它晶系有所不同。     

异源八倍体小冰麦体细胞无性系的建立及其染色体变异

从５个异源八倍体小冰麦（Ｔｒｉｔｉｃｕｍ －Ａｇｒｏｐｙｒｏｎ）的叶片、幼穗及成熟胚诱导愈伤组织,建立体细胞无性系,获得大量再生植株。附加一个冰草染色体组的异源八倍体小冰麦杂种无性系中３７．５％ 表现变异,其中非整倍体植株变异较多,很多变异的再生植株形态与小麦近似,同时出现一定数量染色体重排、交换、易位、断裂、融合等变异。结果表明,通过杂种无性系变异进行染色体基因转化及遗传修饰是一条可行的途径。实验还观察了小冰麦愈伤组织分化过程中绿点的形成过程,首次提出两种类型绿点,即芽绿点和根绿点,并描述了两者的差异     

中枢ACTH受体研究进展

除垂体以外,中枢神经系统也含有促肾上腺皮质激素（ＡＣＴＨ）能神经元,其神经纤维在中枢具有较广泛的投射。ＡＣＴＨ相关肽类在中枢发挥着多种生理功能。近年来对于中枢ＡＣＴＨ受体的研究取得很大的进展,现已确认ＡＣＴＨ结合位点在中枢具有广泛的分布。新近克隆出的四种ＡＣＴＨ受体中,有两种是中枢神经系统占优势的受体亚型。