LATE G1/S-PHASE CELL CYCLE-DEPENDENT GENES

腺病毒对半允许和非允许细胞的感染 在培养条件下诱导细胞DNA合成 DNA合成不会发生在未感染的细胞中。DNA合成 腺病毒诱导的几个G_1期缺失 通常伴随血清刺激的DNA的事件 综合。这包括一组细胞周期的表达- 从属基因是下面表达的基因的子集 血清刺激。尽管有例外，但趋势是 结果表明，AD2通常激活G1晚期或S期基因， 而早期和中期的G1期基因不受影响。这些 结果表明：1)腺病毒刺激细胞DNA 通过一种不同的机制进行合成(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查 细胞基因在G1期晚期和S期的高表达 细胞分裂周期。我们的直接目标将是确定一个 晚期G1/S期细胞周期依赖基因转录子的亚群 在腺病毒感染和血清中都有表达- 刺激的细胞。将使用的实验方法如下 1)互补DNA(CDNAs)细菌质粒 细胞Poly(A+)m RNA序列文库的构建 不允许的温度敏感型、G1期细胞周期突变 感染2型腺病毒的叙利亚仓鼠(TsAF8)将被 建造的。2)将使用差异分子杂交 为了从cDNA文库的成员中鉴定那些 与细胞基因转录本相对应的cDNA克隆 在腺病毒感染的细胞中优先表达。3) 这些克隆的cdna插入片段将被用作分子探针。 通过以下方法鉴定和鉴定信使RNA转录本 Northern杂交分析及基因组DNA序列分析 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的细胞中得到证实 在细胞分裂周期的不同阶段，以及在其他ts细胞中 循环突变体在不同阶段阻止生长，当血清- 在有限的温度下受到刺激。一旦这件事发生了 完成后，编码这些序列的细胞基因将 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 它们的表达在与细胞DNA相关时的关键重要性 的复制、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.