CLONING AND CHARACTERIZATION OF THE MURINE FLV GENE

鼠 FLV 基因的克隆和表征

• 批准号: 6632084
• 负责人: Margo A Brinton
• 金额: $ 25.03万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6632084
• 关键词: Flaviviridae; animal genetic material tag; artificial chromosomes; cell line; complementary DNA; genetic mapping; genetic susceptibility; immunity; messenger RNA; molecular biology information system; molecular cloning; nucleic acid sequence

Approximately 40 murine genes have been shown to control resistance to various virus infections, but only one of these, the Mx gene has so far been cloned and sequenced. In mice, resistance to flavivirus-induced morbidity and mortality is inherited as an autosomal dominant trait. Although the molecular basis for the observed functional differences between the resistant and susceptible alleles of the Flv gene is not known, our recent data suggest that the product of the Flv gene functions at the level of flavivirus RNA synthesis. Data from both yellow fever virus and dengue virus outbreaks have suggested the possible existence of a human Flv homolog. A previous multipoint linkage analysis mapped the flavivirus resistance gene, Flv, within a 0.45 cM segment on mouse chromosome 5 between loci D5Mit408 and D5Mit242. Tight linkage between the D5Mit159 locus and the Flv gene was observed. We propose to positionally clone the Flv gene. BAC clones from the D5Mit159 region will first be selected and aligned and then the transcriptional units within these BAC clones will be identified. Complete cDNA sequences of candidate genes will then be obtained by direct selection and by exon trapping. The cDNAs obtained will be sequenced and primers designed from their termini will be used to amplify cDNAs for the alleles from congenic resistant and susceptible mouse cells. The sequences of pairs of "resistant" and "susceptible" cDNAs will be determined and compared and the proteins expressed from them will be functionally tested in an in vivo assay for a dominant, negative effect on flavivirus replication. Studies of the mechanism of this natural viral resistance will be initiated after the identification of the Flv gene.

大约 40 个小鼠基因已被证明可以控制对各种病毒感染的抵抗力，但迄今为止只有其中一种 Mx 基因被克隆和测序。 在小鼠中，对黄病毒引起的发病率和死亡率的抵抗力作为常染色体显性性状遗传。 尽管观察到的 Flv 基因抗性和易感等位基因之间功能差异的分子基础尚不清楚，但我们最近的数据表明 Flv 基因的产物在黄病毒 RNA 合成水平上发挥作用。 黄热病病毒和登革热病毒爆发的数据表明可能存在人类 Flv 同源物。 先前的多点连锁分析将黄病毒抗性基因 Flv 定位在小鼠 5 号染色体 D5Mit408 和 D5Mit242 位点之间 0.45 cM 的片段内。 观察到 D5Mit159 基因座和 Flv 基因之间的紧密连锁。我们建议定位克隆 Flv 基因。 首先将选择并比对来自 D5Mit159 区域的 BAC 克隆，然后将鉴定这些 BAC 克隆内的转录单位。 然后通过直接选择和外显子捕获获得候选基因的完整cDNA序列。 获得的 cDNA 将被测序，从其末端设计的引物将用于扩增来自同源抗性和易感小鼠细胞的等位基因的 cDNA。 将确定并比较“抗性”和“易感性”cDNA对的序列，并在体内测定中对由它们表达的蛋白质进行功能测试，以确定对黄病毒复制的显性负面影响。 在鉴定出 Flv 基因后，将启动对这种自然病毒抵抗机制的研究。