REGULATION OF MMTV IN T-CELL TUMORS

小鼠乳腺肿瘤病毒（MMTV）是一种B型逆转录病毒， 主要是小鼠的乳腺癌。 MMTV长末端重复序列（LTR） 有一个不寻常的1200 bp的U3区，含有转录调节基因， 该病毒的信号以及长的开放阅读框（ORF）潜在地 编码36 kD蛋白。 该ORF在所有已知的MMTV中是保守的 菌株和观察到的碱基取代给出了很大程度上保守的或 沉默氨基酸的变化;这表明蛋白质功能的保守性。 此外，具有适当的 已在乳腺和肿瘤中鉴定了orf mRNA的结构 来自高乳腺癌小鼠品系。 尽管据报道orf 是一种病毒反式激活因子，这一点尚未得到证实， 尚未确认身份。 最近我们过度表达了未融合的 来自昆虫细胞中的重组杆状病毒的MMTV orf产物。 因为 我们推测，36 kD的ORF产物似乎是一种核蛋白 ORF是转录正或负调节子， 间接或协同参与MMTV诱导的肿瘤。 几 我们将使用各种方法来检验这一假设。 在基因方面，我们 将构建不能合成ORF的MMTV前病毒， 分析其在培养中的复制潜力。 转基因小鼠 将评估过表达ORF产物的组织特异性 ORF的表达、内源性MMTV RNA水平以及 自发性肿瘤 在另一种方法中，我们将使用杆状病毒- 生产ORF蛋白，以开发所需的多克隆抗体， 哺乳动物细胞中ORF产物的鉴定和表征 表达整合的MMTV前病毒。 杆状病毒产生的蛋白质 也将用于生化分析。 细胞内的位置和 将在昆虫细胞中检测ORF的翻译后修饰， 并且部分纯化的ORF将用于评估核酸 蛋白质的结合特性。 最后，杆状病毒ORF蛋白 将用于体外转录反应以确定ORF是否 对MMTV转录有正或负的影响。 因为 MMTV ORF的不寻常性质和病毒的一般保守性， 因此，ORF可能在MMTV中起着独特的作用。 复制或肿瘤发生。

The mouse mammary tumor virus (MMTV) is a B type retrovirus which causes primarily mammary carcinomas in mice. The MMTV long terminal repeat (LTR) has an unusual 1200 bp U3 region containing the transcription regulatory signals of the virus as well as a long open reading frame (orf) potentially encoding a protein of 36 kD. This orf is conserved in all known MMTV strains and the observed base substitutions give largely conservative or silent amino acid changes; this suggests conservation of protein function. In addition, a 1.7 kb spliced transcript which has the appropriate structure for orf mRNA has been identified in mammary glands and tumors from high mammary cancer mouse strains. Although orf has been reported to be a viral transactivator, this has not been confirmed, and the orf product has not been identified. Very recently we have overexpressed the unfused MMTV orf product from a recombinant baculovirus in insect cells. Because the 36 kD orf product appears to be a nuclear protein, we have speculated that orf is a positive or negative regulator of transcription which participates indirectly or cooperatively in MMTV-induced tumors. Several approaches will be used to test this hypothesis. In a genetic approach, we will construct an MMTV provirus which is incapable of orf synthesis for analysis of its replication potential in culture. Transgenic mice overexpressing the orf product will be assessed for the tissue-specific expression of orf, level of endogenous MMTV RNA, and the incidence of spontaneous tumors. In another approach, we will use the baculovirus- produced orf protein to develop polyclonal antibodies needed for identification and characterization of the orf product in mammalian cells expressing integrated MMTV proviruses. The baculovirus-produced protein also will be used for biochemical analysis. The intracellular location and posttranslational modifications of orf will be examined in insect cells, and partially purified orf will be used to evaluate the nucleic acid binding properties of the protein. Finally, the baculovirus orf protein will be employed in in vitro transcription reactions to determine if orf has either positive or negative effects on MMTV transcription. Because of the unusual nature of the MMTV orf and the general conservation of viral genetic information, it is likely that orf plays a unique role in MMTV replication or tumorigenesis.