MECHANISTIC APPROACHES TO HCMV MTRIII-INDUCED TRANSFORMATION

本研究的目的是确定转化的机制 人巨细胞病毒（HCMV）mtrII和mtrIII DNA序列。地铁II HCMV株Towne和mtrIII片段可肿瘤性转化 啮齿动物细胞mtrII（980 bp）区域包含三个开放阅读框 (ORF)79、83和34个氨基酸。我们主要关注的是 转化中的mtrII ORF并产生稳定的mtrII转化细胞 通过DNA转染到NIH 3 T3中的具有新霉素抗性标记的细胞系 细胞 这些细胞系在小鼠中产生肿瘤。北方印迹分析 表明在这些转化体中存在mtrII特异性RNA。 在Cos-7细胞中使用氯霉素乙酰转移酶（CAT）测定，或 在CV-1细胞中，我们检测到启动子活性的上游285 bp区域， 当与CAT基因以有义方向连接时， 然而，整个980 bp的mtrII区域没有可检测的启动子 285 bp区域也具有弱的活性， 转录增强子活性仅在有义方向上。没有 通过HIV达特基因检测启动子序列的反式激活，或 这些研究确定了mtrII中的DNA序列可以 顺式激活基因表达，并处于调节 mtrII ORF的表达。使用S1核酸酶分析总RNA 整个mtrII 980 bp序列作为探针鉴定了几种不同的RNA 主要的转录本，被整个980 bp保护 序列，似乎是一个杂交RNA与细胞基因。 另外两 检测到大约490和450个核苷酸的转录物。 进一步的研究是必要的，以映射这些RNA和评估的作用， mtrII介导的转化中的单个ORF。 关于地铁三号线 (7.6 kb），在右手中检测到转化活性 Pst 1/Xbal 3.6 kb片段。 我们对这3.6kb的DNA进行了缺失克隆 使用Stratagene核酸外切酶III系统来确定最小的 转化区。该项目仍在进行中。

The purpose of this study is to identify the mechanism of transformation by human cytomegalovirus (HCMV) mtrII and mtrIII DNA sequences. The mtrII and mtrIII segments of HCMV strain Towne can neoplastically transform rodent cells. The mtrII (980 bp) region contains three open reading frames (ORF) of 79, 83 and 34 amino acids. We primarily focused on the role of mtrII ORFs in transformation and generated stable mtrII transformed cell lines with neomycin resistant marker by DNA transfection into NIH3T3 cells. These cell lines produced tumors in mice. Northern blot analysis indicated the presence of mtrII specific RNAs in these transformants. Using chloramphenicol acetyl transferase (CAT) assays in Cos-7 cells or CV-1 cells, we detected promoter activity in the upstream 285 bp region of mtrII when linked to CAT gene in the sense orientation with respect to ORFs.However, the entire 980 bp mtrII region had no detectable promoter activity regardless of the orientation.The 285 bp region had also weak transcriptional enhancer activity only in the sense orientation. No transactivation of the promoter sequence was detected by HIV TAT gene or CMV IE genes.These studies established that DNA sequences in mtrII can cisactivate gene expression and are in a location to regulate the expression of the mtrII ORFs. S1 nuclease analysis of total RNA using entire mtrII 980 bp sequence as probe identified several distinct RNA species.The major transcript, being protected by the entire 980 bp sequence, seems to be a hybrid RNA with a cellular gene. Two other transcripts of approximately 490 and 450 nucleotides were detected. Further studies are necessary to map these RNAs and to assess the role of the individual ORFs in mtrII mediated transformation. Regarding mtrIII (7.6 kb), the transforming activity was detected in the right hand Pst1/Xbal 3.6 kb fragment. We made deletion clones of this 3.6 kb DNA using a Stratagene exonuclease III system to determine the minimal transforming region. The project is still active.