CLONING AND CHARACTERIZATION OF THE MURINE FLV GENE

大约有40种鼠基因被证明控制对各种病毒感染的抗性，但迄今为止只有其中之一，Mx基因被克隆和测序。 在小鼠中，对黄病毒诱导的发病率和死亡率的抗性作为常染色体显性性状遗传。 虽然所观察到的Flv基因的抗性和易感等位基因之间的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合成水平上起作用。 来自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。 以前的多点连锁分析映射的黄病毒抗性基因，Flv，在一个0.45 cM的片段在小鼠5号染色体之间的基因座D5Mit408和D5Mit242。 D5Mit159位点与Flv基因紧密连锁。我们建议定位克隆Flv基因。 首先选择并比对来自D5Mit159区域的BAC克隆，然后鉴定这些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。 将对获得的cDNA进行测序，并使用从其末端设计的引物扩增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" cDNA对的序列，并在体内测定中对由其表达的蛋白质进行功能性测试，以确定对黄病毒复制的显性负效应。 在鉴定Flv基因后，将开始研究这种天然病毒抗性的机制。

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.