Laurie-Ahlberg C C, Stam L F
Genetics. 1987 Jan;115(1):129-40. doi: 10.1093/genetics/115.1.129.
The purpose of the work reported here is to identify the molecular basis of the difference in level of expression between the polymorphic Slow and Fast alcohol dehydrogenase (Adh) alleles in Drosophila melanogaster. Previous studies have shown that Fast lines typically have a two- to threefold higher activity level than Slow lines and they also have a substantially higher level of ADH-protein (estimated immunologically). The results of a restriction fragment length polymorphism study in relation to ADH activity variation had previously suggested that the difference in Adh expression between allozymes might not be due entirely to the amino acid replacement substitution, but could be due in part to linkage disequilibrium with a regulatory site polymorphism. Here we describe an approach that makes use of P-element-mediated transformation in order to identify the nucleotide substitution(s) responsible for this difference in ADH level. This approach consists of generating recombinants in vitro between Adh region clones derived from a typical Slow/Fast pair of alleles and then testing for the effects of particular restriction fragments on expression in vivo by transformation. Using this approach, the effect on both ADH activity and ADH-protein level clearly maps to a 2.3-kb restriction fragment that includes all of the Adh coding sequence and some intron and 3' flanking sequence, but excludes all of the 5' flanking sequence of the distal (adult) transcriptional unit. Comparison of Kreitman's DNA sequences for this fragment from several Slow and Fast alleles showing the typical difference in activity level shows that only three nucleotide substitutions distinguish all Fast from all Slow alleles. Thus, it is likely that one or more of these substitutions causes the major difference in Adh expression between allozymic classes. One of these substitutions is, of course, the Slow/Fast amino acid replacement substitution (at 1490) while the other two are nearby third position silent substitutions (at 1443 and 1527). A quantitative analysis of variation among transformant stocks shows that the P-element transformation approach can be used to localize even relatively small effects on gene expression (on the order of 20%).
本文报道的研究工作旨在确定黑腹果蝇中多态性慢型和快型乙醇脱氢酶（Adh）等位基因表达水平差异的分子基础。先前的研究表明，快型品系的活性水平通常比慢型品系高两到三倍，并且它们的ADH蛋白水平（通过免疫估计）也显著更高。先前一项关于与ADH活性变异相关的限制性片段长度多态性研究结果表明，等位酶之间Adh表达的差异可能并非完全归因于氨基酸替换，而是可能部分归因于与调控位点多态性的连锁不平衡。在此，我们描述了一种利用P因子介导的转化来鉴定导致ADH水平这种差异的核苷酸替换的方法。该方法包括在源自典型的慢型/快型等位基因对的Adh区域克隆之间体外产生重组体，然后通过转化测试特定限制性片段对体内表达的影响。使用这种方法，对ADH活性和ADH蛋白水平的影响明确定位于一个2.3kb的限制性片段，该片段包含所有的Adh编码序列以及一些内含子和3'侧翼序列，但不包括远端（成虫）转录单位的所有5'侧翼序列。对来自几个活性水平存在典型差异的慢型和快型等位基因的该片段的Kreitman DNA序列进行比较，结果显示只有三个核苷酸替换区分了所有快型等位基因和所有慢型等位基因。因此，这些替换中的一个或多个可能导致了等位酶类别之间Adh表达的主要差异。其中一个替换当然是慢型/快型氨基酸替换（在1490位），而另外两个是附近的第三位沉默替换（在1443和1527位）。对转化体菌株间变异的定量分析表明，P因子转化方法可用于定位对基因表达相对较小的影响（约20%）。
