LATE G1/S-PHASE CELL CYCLE-DEPENDENT GENES

腺病毒感染半容许和非容许细胞 在培养条件下诱导细胞DNA合成， DNA合成在未感染的细胞中不发生。 dna合成 腺病毒诱导的G1期发生在几个G1期的情况下， 通常伴随血清刺激DNA的事件 合成. 这包括一组细胞周期的表达- 依赖性基因是以下表达的基因的子集 血清刺激 虽然有例外，但趋势 显示Ad2通常激活晚期G1期或S期基因， 而早期和中期G1期基因不受影响。 这些 结果表明：1）腺病毒刺激细胞DNA 通过不同的机制合成（尽管它可以 是血清生长因子使用的机制的一部分。 2)AD2 非允许细胞的感染可用于筛选 在G1/S期晚期表达的细胞基因 细胞分裂周期 我们的近期目标是确定一个 晚期G1/S期细胞周期依赖性基因转录物亚群 它们在腺病毒感染的和血清中表达， 刺激细胞 所使用的实验方法如下： 1）互补DNA（cDNA）细菌质粒 表达的细胞poly（A+）mRNA序列文库， 非允许性温度敏感性G1期细胞周期突变体 感染腺病毒2的叙利亚仓鼠（tsAF 8）将被 构建了 2)将使用差异分子杂交技术 为了从cDNA文库的成员中鉴定那些 对应于细胞基因转录物的cDNA克隆 优先在腺病毒感染的细胞中表达。 3)的 这些克隆的cDNA插入片段将用作分子探针 鉴定和表征信使RNA转录本， 北方印迹分析和基因组DNA测序 印迹分析。 4)细胞周期依赖的RNA水平 这些基因的表达将在同步化的细胞中确定， 在细胞分裂周期的不同阶段，以及在其他TS细胞中， 周期突变体，在不同的阶段，当血清- 在限制性温度下刺激。 一旦这已经 完成后，编码这些序列的细胞基因将被 从叙利亚仓鼠基因组中鉴定和分离 噬菌体文库 该项目的长期目标将是 分析这些基因的结构，并确定 它们的表达至关重要，因为它与细胞DNA有关 复制、形态转化和永生化 原代叙利亚仓鼠真皮成纤维细胞。

Adenovirus infection of semipermissive and nonpermissive cells induces cellular DNA synthesis under culture conditions where DNA synthesis does not occur in uninfected cells. DNA synthesis induced by adenovirus occurs in the absence of several G1-phase events which normally accompany serum-stimulated DNA synthesis. This includes the expression of a set of cell cycle- dependent genes which are a subset of those expressed following serum stimulation. Although there are exceptions, the trend shows that Ad2 generally activates late G1- or S-phase genes, while early and mid G1-phase genes are not affected. These results suggest that: 1) Adenoviruses stimulate cellular DNA synthesis by a mechanism which is not the same (although it could be part of the) mechanism used by serum growth factors. 2) Ad2 infection of nonpermissive cells could be used to screen for cellular genes perferentially expressed in late G1/S-phase of the cell division cycle. Our immediate aim will be to identify a subset of late G1/S-phase cell cycle-dependent gene transcripts which are expressed in both adenovirus-infected and in serum- stimulated cells. The experimental approaches used will be as follows: 1) A complementary DNA (cDNA) bacterial plasmid library of cellular poly(A+) mRNA sequences expressed in a nonpermissive temperature-sensitive, G1-phase cell cycle mutant of Syrian hamster (tsAF8) infected with adenovirus 2 will be constructed. 2) Differential molecular hybridization will be used to identify from among the members of the cDNA library, those cDNA clones which correspond to cellular gene transcripts preferentially expressed in adenovirus-infected cells. 3) The cDNA inserts from these clones will be used as molecular probes to identify and characterize messenger RNA transcripts by Northern blot analysis and genomic DNA sequences by Southern blot analyses. 4) The cell cycle-dependent level of RNA expression of these genes will be ascertained in cells synchronized in different phases of the cell division cycle, and in other ts cell cycle mutants that arrest growth in different phases when serum- stimulated at restrictive temperature. Once this has been accomplished, the cellular genes encoding these sequences will be identified and isolated from a Syrian hamster genomic bacteriophage library. The long range goals of the project will be to analyze the structure of these genes, and to ascertain the critical importance of their expression as it relates to cell DNA replication, morphological transformation and immortalization of primary Syrian hamster dermal fibroblasts.