LARGE EXON SIZE DOES NOT LIMIT SPLICING IN-VIVO
LARGE EXON SIZE DOES NOT LIMIT SPLICING IN-VIVO
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DOI：
10.1128/mcb.14.3.2140
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发表时间：
1994-03-01
影响因子：
5.3
通讯作者：
CHASIN, LA
中科院分区：
文献类型：
--
作者：
CHEN, IT;CHASIN, LA
脊椎动物基因中的外显子大小，除了少数例外，被限制在小于300个碱基。有人提出，这种限制可能来自剪接位点识别的外显子定义模型。在该模型中，下游供体位点增强同一外显子上游受体位点的剪接。这种增强可能需要与外显子两端结合的因子之间的接触;外显子大小限制将促进这种接触。为了验证外显子定义需要邻近性的想法，我们插入了随机DNA。将来自大肠杆菌的片段插入三外显子二氢叶酸还原酶小基因的中央外显子中，并测试扩展的外显子是否被有效拼接。来自扩增的小基因的质粒文库的DNA用于检测缺乏dhfr基因座的CHO细胞缺失突变体。从合并的稳定共转染子群体中分离的DNA的PCR分析显示DNA插入片段大小范围为50至1，500个核苷酸。通过逆转录随后PCR对来自该群体的RNA进行的平行分析显示出相似的大小分布。中心外显子可以拼接成1,400个碱基的mRNA。我们还测试了含有确定大小的外显子插入物的单个质粒克隆。mRNA中包含的最大外显子长度为1,200个碱基，远高于天然存在的外显子的300个碱基的限制。我们的结论是，外显子大小的限制不是外显子定义机制的一部分。
Exon sizes in vertebrate genes are, with a few exceptions, limited to less than 300 bases. It has been proposed that this limitation may derive from the exon definition model of splice site recognition. In this model, a downstream donor site enhances splicing at the upstream acceptor site of the same exon. This enhancement may require contact between factors bound to each end of the exon; an exon size limitation would promote such contact. To test the idea that proximity was required far exon definition, we inserted random DNA. fragments from Escherichia coli into a central exon in a three-exon dihydrofolate reductase minigene and tested whether the expanded exons were efficiently spliced. DNA from a plasmid library of expanded minigenes was used to transfect a CHO cell deletion mutant lacking the dhfr locus. PCR analysis of DNA isolated from the pooled stable cotransfectant populations displayed a range of DNA insert sizes from 50 to 1,500 nucleotides. A parallel analysis of the RNA from this population by reverse transcription followed by PCR showed a similar size distribution. Central exons as large as 1,400 bases could be spliced into mRNA. We also tested individual plasmid clones containing exon inserts of defined sizes. The largest exon included in mRNA was 1,200 bases in length, well above the 300-base limit implied by the sun-ey of naturally occurring exons. We conclude that a limitation in exon size is not part of the exon definition mechanism.