CLONING AND CHARACTERIZATION OF THE MURINE FLV GENE

大约有40种小鼠基因被证明可以控制对各种病毒感染的抵抗力，但到目前为止，只有其中一种，Mx基因被克隆并测序。在小鼠中，对黄病毒引起的发病率和死亡率的抗性是作为常染色体显性性状遗传的。虽然观察到的Flv基因抗性和易感等位基因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暴发的数据表明，可能存在人类流感病毒同源物。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位点之间的0.45 cM片段中。观察到D5Mit159位点与Flv基因之间的紧密连锁。我们建议对Flv基因进行定位克隆。首先选择D5Mit159区域的BAC克隆进行比对，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。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。将确定和比较“抗性”和“易感”cdna对的序列，并在体内试验中对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。对这种自然病毒抗性机制的研究将在Flv基因鉴定后开始。

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.