Benyajati C, Place A R, Wang N, Pentz E, Sofer W
Nucleic Acids Res. 1982 Nov 25;10(22):7261-72. doi: 10.1093/nar/10.22.7261.
Two formaldehyde-induced, homozygous viable ADH-negative mutants, Adhfn4 and Adhfn6, possess no material that cross-reacts with antibody directed against ADH, no mature mRNA of wild-type size, and greatly reduced amounts of RNA that hybridizes with an Adh probe. We have cloned the genomic DNA sequences from these mutants in bacteriophage lambda Charon 4 and subcloned the Adh region into plasmid vector pBR327. Restriction analyses revealed one small deletion in each of these mutants and DNA sequencing showed that the splice junctions of the 65-base pair (bp) intervening sequence (IVS) were altered. Both cloned mutant Adh genes, as well as the wild-type gene, are capable of promoting correct specific transcription initiation in HeLa cell nuclear extracts in vitro. We conclude that Adhfn4 and Adhfn6 are defective in RNA processing. Our results provide evidence for the importance of the splice junction sequences in normal ADH RNA processing and stabilization in Drosophila. We also speculate that splicing of ADH RNA proceeds in a nonrandom manner: mutations in one of the intervening sequences appear to cause accumulation of a large ADH RNA containing at least one other IVS.
两个由甲醛诱导产生的、纯合存活的乙醇脱氢酶（ADH）阴性突变体Adhfn4和Adhfn6，不含有与抗ADH抗体发生交叉反应的物质，没有野生型大小的成熟mRNA，并且与Adh探针杂交的RNA量大幅减少。我们已将这些突变体的基因组DNA序列克隆到噬菌体λCharon 4中，并将Adh区域亚克隆到质粒载体pBR327中。限制性分析显示这些突变体中的每一个都有一个小缺失，DNA测序表明65个碱基对（bp）的间隔序列（IVS）的剪接连接发生了改变。克隆的突变体Adh基因以及野生型基因，在体外HeLa细胞核提取物中都能够促进正确的特异性转录起始。我们得出结论，Adhfn4和Adhfn6在RNA加工方面存在缺陷。我们的结果为剪接连接序列在果蝇正常ADH RNA加工和稳定中的重要性提供了证据。我们还推测ADH RNA的剪接以非随机方式进行：一个间隔序列中的突变似乎导致了一种包含至少一个其他IVS的大型ADH RNA的积累。
