Developmental regulation of alternative exon usage in the house fly Vssc1 sodium channel gene
Developmental regulation of alternative exon usage in the house fly Vssc1 sodium channel gene
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DOI：
10.1007/s10158-001-0014-1
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发表时间：
2002-04-01
影响因子：
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通讯作者：
Soderlund, David M.
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文献类型：
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作者：
Lee, Si Hyeock;Ingles, Patricia J.;Soderlund, David M.
家蝇cDNA克隆的序列分析vssc 1电压敏感钠通道α亚基转录模板确定了11个假定的选择性剪接外显子。其中9个对应于7个可选外显子（指定为a、B、e、f、h、i和j）和2个互斥外显子（指定为c/d），其先前在黑腹果蝇和绿色果蝇的邻位帕拉钠通道α亚基基因中鉴定，而两个片段代表Vssc 1中新的互斥外显子在单独的Vssc 1 cDNA模板上进行的诊断PCR分析检测了多个部分cDNA中每个推定的选择性外显子的存在或不存在（每个cDNA库42-96个单独的克隆）来自新出现的第一龄幼虫、蛹、第1天成虫头和第1天成虫体。外显子h和i存在于所有发育阶段的所有cDNA克隆中。外显子d也存在于所有发育阶段的编码全长氨基酸序列的所有克隆中;然而，1/42来自成人头部的克隆含有外显子c样片段，其中编码序列由提前终止密码子终止。相比之下，外显子a，B，e，f，j，k和l的频率在发育阶段和成人解剖区域之间不同。作为一个单一的扩增的cDNA片段的Vssc 1区含有替代外显子a，B，c/d，e，f，h和i的分析确定了9个Vssc 1剪接变异体涉及这些外显子。剪接变体包含外显子A，D，H，和I是最丰富的形式在所有的cDNA池检查，但所观察到的模式的剪接变体的表达是特定的每个发育阶段和成人解剖区域。我们的研究结果表明，家蝇Vssc 1基因和果蝇帕拉基因之间的选择性外显子位置和结构具有很强的保守性。黑腹果蝇，但确定显着差异，这些物种之间的外显子使用。
Sequence analysis of cDNA clones amplified by PCR from house fly (Musca domestica L.) Vssc1 voltage-sensitive sodium channel alpha subunit transcript templates identified 11 putative alternatively spliced exons. Nine of these corresponded to the 7 optional exons (designated a, b, e, f, h, i, and j) and 2 mutually exclusive exons (designated c/d) identified previously in the orthologous para sodium channel alpha subunit genes of Drosophila melanogaster and Drosophila virilis, whereas two segments represented new mutually exclusive exons in Vssc1 (designated k/l) located in a region not previously identified as a site of alternative splicing in para. Diagnostic PCR assays on individual Vssc1 cDNA templates detected the presence or absence of each putative alternative exon in multiple partial cDNAs (42-96 individual clones per cDNA pool) from newly emerged first instar larvae, pupae, day 1 adult heads, and day 1 adult bodies. Exons h and i were present in all cDNA clones from all developmental stages. Exon d was also present in all clones from all developmental stages that encoded full-length amino acid sequences; however, 1 of 42 clones from adult head contained an exon c-like segment in which the coding sequence was terminated by a premature stop codon. In contrast, the frequencies of exons a, b, e, f, j, k, and l differed between developmental stages and adult anatomical regions. Analysis of the Vssc1 region containing alternative exons a, b, c/d, e, f, h, and i as a single amplified cDNA segment identified nine Vssc1 splice variants involving these exons. The splice variant containing exons a, d, h, and i was the most abundant form in all cDNA pools examined, but the observed patterns of splice variant expression were specific to each developmental stage and adult anatomical region. Our results document the strong conservation of alternative exon location and structure between the Vssc1 gene of the house fly and the para gene of D. melanogaster but identify marked differences in exon usage between these species.