Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis.

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.     

Polymorphism of human red cell glyoxalase I in six ethnic groups of China

Summary A total of 1242 individuals from six Chinese ethnic groups were studied with respect to the glyoxalase I polymorphism using agarose gel electrophoresis. The GLO1^*1 gene frequency and the number of subjects tested in each population are as follows: Uygur 0.2466 (219), Hui 0.1621 (219), Dong 0.1866 (201), Bai 0.1921 (203), Tujia 0.1075 (200), and Maio 0.1600 (200). The differences in the GLO1 gene frequencies between some of these populations are significant.     

北京鸭脑钙调素的分离纯化和性质的研究

钙调素(Calmodulin,简称CaM)是一种多生理功能的调节蛋白,在脑的功能活动中有重要作用。本文采用苯基琼脂糖(phenyl-Sepharose CL 4B)层析和葡聚糖凝胶(Sephadex G-50)过滤法,从北京鸭脑中分离纯化出CaM。纯化的CaM经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和等电聚焦(IEF)电泳鉴定均为一条区带。分子量为19kD,等电点(pI)为4.15,消光系数为1.83。 对纯化的鸭脑CaM的活性和性质进行了研究。它可明显地激活牛环核苷酸磷酸二酯酶活性,在有Ca~(2+)存在的条件下,SDS-PAGE中出现电泳迁移速度的改变,紫外吸收光谱具有已知CaM特有的吸收多峰形,并观察了Ca~(2+)对荧光发射光谱的影响。其氨基酸组成中,1/3是酸性氨基酸,苯丙氨酸和酪氨酸的比例为8:2。与猪CaM和牛CaM的物理化学性质作了比较。     

三乙醇胺基强碱性聚苯乙烯树脂共价固定胰酶的研究

用多孔强碱型三乙醇胺基聚苯乙烯阴离子交换树脂做为载体,用CNBr与载体上的多羟基作用共价偶联了胰酶。红外光谱表明:其共价偶联反应机理与用CNBr活化多糖类载体并接酶的机理相类似。最适偶联条件研究表明:CNBr用量增多,酶蛋白载量增加。但比活下降。偶联pH为10时,固定化酶有适宜的载量和较高的比活。由于胰酶水解蛋白反应释放出H~+质子,这些质子在载体内积累,使微环境内H~+质子浓度增加,进而使得固定化胰酶的pH—活性曲线在pH9～11范围内未出现下降。在变温和60℃恒温下对固定化酶的热稳定性测试表明:固相酶的热稳定性比天然酶的热稳定性有所提高。     

第Ⅷ凝血因子基因内限制性片段长度多态性(RFLP)的研究及其在甲型血友病基因连锁分析中的应用

本文系统地研究了广东地区汉族人群中FⅧ:C基因内BclⅠ,XbaⅠ和BgⅡ位点RFLP的基因频率。多态性位点BclⅠ,XbaⅠ及BglⅠ的切点阳性率分别为63.5%、43.5%和100%。对Bcll和Xbal多态性切点连锁情况研究显示,19.5%的Bcll切点阳性纯合子为Xbal切点杂合子,证明联合应用此两位点RFLP可以把甲型血友病基因连锁分析的有效率提高到65.9%。用RFLP连锁分析对两例甲型血友病家系中的女性进行了致病基因携带者检测,对另一例家系进行了基因产前诊断。     

Microcapillary clonogenic assays for human marrow hematopoietic progenitor cells

The capillary clonogenic cell assay was developed and adapted to culture myeloid and erythroid colonies from human bone marrow cells. The plating efficiencies for femoral bone marrow granulocyte-macrophage progenitors (CFU-gm), erythroid colony-forming units (CFU-e) and erythroid burst-forming units (BFU-e) were 0.143%, 0.229% and 0.141%, respectively. Standard bone marrow progenitor Petri dish assays require a total culture volume of 1 ml per dish, and as such are not suitable for the small numbers of cells often obtained from human bone marrow samples. The microcapillary assay as developed and standardized in our laboratory has the unique advantage of being able to utilize small numbers of cells. This technique is suitable for evaluating the myelotoxicity of investigational new anti-cancer and anti-HIV agents and for further investigation of the mechanisms underlying chemotherapy-induced bone marrow toxicity.