Characterization of revertants derived from a mouse DNA temperature-sensitive mutant strain, tsFT20, which contains heat-labile DNA polymerase alpha activity

One spontaneous and four N-methyl-N'-nitro-N-nitrosoguanidine-induced revertants of a mouse FM3A mutant, tsTF20, which has heat-labile DNA polymerase alpha activity and cannot grow at 39 degrees C, were isolated and characterized with respect to the thermolability of their DNA polymerase alpha activity, the intracellular level of enzyme activity, growth rate, cell cycle progression, and frequency of initiation of DNA replication at the origin of replicons. DNA polymerase alpha activity in the extracts from the revertant cells showed partial recovery of heat stability. The intracellular level of enzyme activity of the revertant cells was lower than that of wild-type cells even at 33 degrees C. The level of enzyme activity in the revertant cells decreased considerably after a temperature upshift to 39 degrees C, but the DNA synthesizing ability of these cells did not decrease as much as the level of enzyme activity. The growth rates of the wild-type and revertant lines were almost the same at 33 degrees C. At 39 degrees C, the rate for the wild-type increased considerably compared to that at 33 degrees C, while little difference in the growth rates of the revertant lines was observed at the two temperatures. Therefore, the doubling times of the revertant cells were relatively increased compared to those of wild-type cells cultured at the restrictive temperature. Flow microfluorometric analysis and cell cycle analysis to measure labeled mitosis revealed that the increase in the doubling time was due mainly to the increase in the duration of the S phase. Analysis of the center-to-center distance between replicons by DNA fiber autoradiography indicated that the frequency of replicon initiation per unit length DNA at a given time was reduced in the revertant cells growing at 39 degrees C.     

Optimum conditions for electric pulse-mediated gene transfer to mammalian cells in suspension

A pulse-generating machine which delivers exponentially decaying pulses over broad range of pulse lengths was used to determine the optimum pulse conditions for gene transfer to FM3A cells. In the transformation of tk- cells with pTK1, a single pulse of 100-2000 microseconds gave a high transformation frequency at 1.5-6 kV/cm and room temperature, the highest transformation frequency obtained being 3 X 10(-3). As the suspension buffer for cells exposed to the pulse, Saline G was better than PBS(-) for obtaining a large number of transformants because it ensured high cell viability.     

Induction of mutation in mouse FM3A cells by N4-aminocytidine-mediated replicational errors.

To explore the potential use of a nucleoside analog, N4-aminocytidine, in studies of cellular biology, the mechanism of mutation induced by this compound in mouse FM3A cells in culture was studied. On treatment of cells in suspension with N4-aminocytidine, the mutation to ouabain resistance was induced. The major DNA-replicating enzyme in mammalian cells, DNA polymerase alpha, was used to investigate whether the possible cellular metabolite of N4-aminocytidine, N4-aminodeoxycytidine 5'-triphosphate (dCamTP), can be incorporated into the DNA during replication. Using [3H]dCamTP in an in vitro DNA-synthesizing system, we were able to show that this nucleotide analog can be incorporated into newly formed DNA and that it can serve as a substitute for either dCTP or dTTP. dCamTP in the absence of dCTP maintained the activated calf thymus DNA-directed polymerization of deoxynucleoside triphosphates as efficiently as in its presence. Even in the presence of dCTP, dCamTP was incorporated into the polynucleotide. When dCamTP was used as a single substrate in the poly(dA)-oligo(dT)-directed polymerase reaction, it was incorporated into the polynucleotide fraction. The extent of incorporation was 4% of that of dTTP incorporation when dTTP was used as a single substrate. Even in the presence of dTTP, dCamTP incorporation was observed. A copolymer containing N4-aminocytosine residues was shown to incorporate guanine residues opposite the N4-aminocytosines. However, we were unable to observe adenine incorporation opposite N4-aminocytosine in templates. These cell-free experiments show that an AT-to-GC transition can take place in the presence of dCamTP during DNA synthesis, strongly suggesting that the mutation induced in the FM3A cells by N4-aminocytidine is due to replicational errors.     

鲥鱼的驯养与生物学研究Ⅰ.O+龄幼鱼的生长与食性

本文主要描述池塘养殖条件下鲥鱼幼鱼的生长与食性。1)1987-1989年8月至11月幼鱼生长迅速,快于同期珠江天然个体的生长速度;2)体长26.6-94.9mm的幼鱼主要摄食浮游动物,轮虫和浮游植物出现率随个体增大而减少。食物共有18个属种,摄食频度为100%;3)幼鱼的平均饱满指数为75.70‰。各类食物重量百分比的顺序为:桡足类>枝角类>无节幼体>虾类溞状幼体>轮虫>藻类。     

Detection and characterization of a novel factor that stimulates DNA polymerase alpha

A novel factor that stimulates DNA polymerase alpha activity on poly(dA) X oligo(dT) has been identified and partially purified from mouse FM3A cells. The assay system for the factor contained poly(ethylene glycol) 6000. The activities of DNA polymerase alpha on poly(dA) X oligo(dT) in the presence and absence of the stimulating factor were increased greatly by the addition of poly(ethylene glycol). Stimulation by the factor was observed at all the primer to template ratios tested from 0.01 to 0.3. The highest activity was observed at the ratio of 0.05, corresponding to about 3.3 primers on one template in the presence of the factor. The concentration of DNA polymerase alpha used in the assay affected the stimulation by the factor, and the stimulation became more prominent at concentrations of the enzyme lower than 0.04 unit per assay. The stimulating factor lowered the Km value of DNA polymerase alpha for the template-primer, though they had no effect on the Km value for dTTP substrate. The results of product analysis suggested that the stimulation by the factor is mainly due to the increase in the initiation frequency of DNA synthesis from the primers. The stimulating factor specifically stimulated DNA polymerase alpha but not DNA polymerases beta and gamma. Furthermore, the factor formed a complex with DNA polymerase alpha under a certain condition.     

利用单克隆抗体鉴定人甲胎蛋白(HAFP)抗原结构可分性

本文利用两株针对HAFP分子不同抗原决定簇的单克隆抗体,鉴定HAFP酶解片断的抗原抗体反应性质,并同完整HAFP分子进行比较。结果表明,酶解片断上失去了一株单克隆抗体所对应的分子部份,完整保留着另一株单克隆抗体所识别的抗原决定簇,从而证实HAFP分子某些抗原结构之间具有可分割性。     

沙田柚染色体组型及Giemsa带型分析

本文以沙田柚为材料,对其染色体组型及带型进行了观察分析。组型分析:染色体数目2n=18,根据染色体的相对长度分成大小染色体两种类型,前者包括1、2、3、4和5对,后者为6、7、8和9对,根据臂比,9对染色体能够被分成中部着丝点和近中着丝点染色体两种类型。即第5、7,9对为亚中部着丝点,其余为中部着丝点,第6对染色体上有随体;Giemsa带型:除第二对染色体只显中间带外,其余都显着丝点带,并在3、4、8对染色体短臂上和2、3、1对染色体长臂上均显端带,第2、3,6对同源染色体之间的C带显示杂合性。     

Isolation and initial characterization of a temperature-sensitive mutant of mouse FM3A cells defective in cytokinesis

A temperature-sensitive mutant, designated tsFT101, was isolated from a mouse mammary carcinoma cell line, FM3A, and given an initial characterization. In this cell line, cytokinesis was blocked at a non-permissive temperature (39 degrees C), but DNA synthesis and nuclear division proceeded normally for at least 24 h at 39 degrees C as detected respectively by autoradiography and cytofluorometric analysis. As a result, multinucleate cells accumulated at 39 degrees C (more than 95% in 36 h). When the culture was returned to a permissive temperature (33 degrees C) after 24 h of arrest at 39 degrees C, cytokinesis was resumed and there was a rapid decrease in the number of multinucleate cells. At 39 degrees C, tsFT101 cells had less F-actin than cells at 33 degrees C, indicative of the existence of an abnormality in actin polymerization in this mutant.