Boles E, Zimmermann F K
Institut für Mikrobiologie, Technische Hochschule Darmstadt, Germany.
Mol Gen Genet. 1994 May 25;243(4):363-8. doi: 10.1007/BF00280465.
Open reading frames longer than 300 bases were observed in the antisense strands of the genes coding for the glycolytic enzymes phosphoglucose isomerase, phosphoglycerate mutase, pyruvate kinase and alcohol dehydrogenase I. The open reading frames on both strands are in codon register. It has been suggested that proteins coded in codon register by complementary DNA strands can bind to each other. Consequently, it was interesting to investigate whether the open reading frames in the antisense strands of glycolytic enzyme genes are functional. We used oligonucleotide-directed mutagenesis of the PGI1 phosphoglucose isomerase gene to introduce pairs of closely spaced base substitutions that resulted in stop codons in one strand and only silent replacements in the other. Introduction of the two stop codons into the PGI1 sense strand caused the same physiological defects as already observed for pgil deletion mutants. No detectable effects were caused by the two stop codons in the antisense strand. A deletion that removed a section from -31 bp to +109 bp of the PGI1 gene but left 83 bases of the 3' region beyond the antisense open reading frame had the same phenotype as a deletion removing both reading frames. A similar pair of deletions of the PYK1 gene and its antisense reading frame showed identical defects. Our own Northern experiments and those reported by other authors using double-stranded probes detected only one transcript for each gene. These observations indicate that the antisense reading frames are not functional. On the other hand, evidence is provided to show that the rather long reading frames in the antisense strands of these glycolytic enzyme genes could arise from the strongly selective codon usage in highly expressed yeast genes, which reduces the frequency of stop codons in the antisense strand.
在编码糖酵解酶磷酸葡萄糖异构酶、磷酸甘油酸变位酶、丙酮酸激酶和乙醇脱氢酶I的基因的反义链中，观察到了长度超过300个碱基的开放阅读框。两条链上的开放阅读框处于密码子读框内。有人提出，由互补DNA链以密码子读框编码的蛋白质可以相互结合。因此，研究糖酵解酶基因反义链中的开放阅读框是否具有功能就很有意思了。我们使用寡核苷酸定向诱变PGI1磷酸葡萄糖异构酶基因，引入紧密间隔的碱基替换对，导致一条链上出现终止密码子，而另一条链上只有沉默替换。将两个终止密码子引入PGI1有义链会导致与pgil缺失突变体已观察到的相同生理缺陷。反义链中的两个终止密码子未引起可检测到的影响。一个缺失从PGI1基因的-31 bp到+109 bp区域，但在反义开放阅读框之外的3'区域留下83个碱基，其表型与去除两个阅读框的缺失相同。PYK1基因及其反义阅读框的类似一对缺失显示出相同的缺陷。我们自己的Northern实验以及其他作者使用双链探针报道的实验，每个基因仅检测到一种转录本。这些观察结果表明反义阅读框没有功能。另一方面，有证据表明，这些糖酵解酶基因反义链中相当长的阅读框可能源于高表达酵母基因中强烈的选择性密码子使用，这降低了反义链中终止密码子的频率。
