Lee Si Hyeock, Ingles Patricia J, Knipple Douglas C, Soderlund David M
Department of Entomology, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456, USA.
Invert Neurosci. 2002 Apr;4(3):125-33. doi: 10.1007/s10158-001-0014-1. Epub 2001 Nov 10.
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.
通过PCR从家蝇（Musca domestica L.）Vssc1电压门控钠通道α亚基转录模板扩增的cDNA克隆的序列分析鉴定出11个推定的可变剪接外显子。其中9个对应于先前在黑腹果蝇和果蝇直系同源para钠通道α亚基基因中鉴定出的7个选择性外显子（命名为a、b、e、f、h、i和j）和2个互斥外显子（命名为c/d），而两个片段代表Vssc1中位于先前未被鉴定为para可变剪接位点区域的新互斥外显子（命名为k/l）。对单个Vssc1 cDNA模板进行的诊断性PCR检测，在新羽化的一龄幼虫、蛹、1日龄成虫头部和1日龄成虫身体的多个部分cDNA（每个cDNA库42 - 96个个体克隆）中检测每个推定可变外显子的存在与否。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也存在于所有编码全长氨基酸序列的所有发育阶段的克隆中；然而，来自成虫头部的42个克隆中有1个包含一个外显子c样片段，其中编码序列被一个提前的终止密码子终止。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虫解剖区域之间存在差异。将包含可变外显子a、b、c/d、e、f、h和i的Vssc1区域作为单个扩增的cDNA片段进行分析，鉴定出涉及这些外显子的9种Vssc1剪接变体。包含外显子a、d、h和i的剪接变体是所有检测的cDNA库中最丰富的形式，但观察到的剪接变体表达模式在每个发育阶段和成虫解剖区域都是特定的。我们的结果证明了家蝇Vssc1基因和黑腹果蝇para基因之间可变外显子位置和结构的高度保守性，但也确定了这些物种在外显子使用上的显著差异。
