Kwiatowski J, Skarecky D, Tyler R, Pascual M, Ayala F J
Department of Ecology and Evolutionary Biology, University of California, Irvine 92717.
Free Radic Res Commun. 1991;12-13 Pt 1:429-35. doi: 10.3109/10715769109145813.
The decreased Cu,Zn SOD activity (less than 5%) in a "null" SODCA1 Drosophila melanogaster strain isolated in our laboratory is due to the insertion of a truncated P element into the transcribed region of the Cu,Zn SOD gene. Using a cDNA Cu,Zn SOD probe from a wild type D. melanogaster (F allele) we isolated an EcoRI Cu,Zn SOD clone from an EMBL3 genomic library of the SODCA1 strain, subcloned it, restriction-mapped and partially sequenced it. The 2.5 kb clone consists of a wild-type 1.84 kb EcoRI fragment containing the Cu,Zn SOD gene previously isolated in our laboratory, with an insertion of 0.68 kb derived (by an internal deletion) from an autonomous, 2.9 kb P element. The insertion starts 21 bp upstream from the coding sequence and causes an 8 bp target site duplication characteristic of P elements. A point mutation in the second exon results in a replacement of Asn by Lys at position 96, confirming that the mature protein encoded by the SOCCA1 is the same one encoded by the S allele, commonly found in natural populations. The diminished expression of SODCA1 allele is most possibly due to a reduction of the rate of transcription attributable to the insertion of the P element.
在我们实验室分离得到的“无效”SODCA1黑腹果蝇品系中,铜锌超氧化物歧化酶(Cu,Zn SOD)活性降低(低于5%),这是由于一个截短的P因子插入到了铜锌超氧化物歧化酶基因的转录区域。我们使用来自野生型黑腹果蝇(F等位基因)的cDNA铜锌超氧化物歧化酶探针,从SODCA1品系的EMBL3基因组文库中分离出一个EcoRI铜锌超氧化物歧化酶克隆,将其亚克隆、进行限制酶图谱分析并部分测序。这个2.5 kb的克隆由一个1.84 kb的野生型EcoRI片段组成,该片段包含我们实验室之前分离得到的铜锌超氧化物歧化酶基因,其中插入了一个0.68 kb的片段(通过内部缺失),该片段来源于一个2.9 kb的自主P因子。插入发生在编码序列上游21 bp处,并导致了P因子特有的8 bp靶位点重复。第二个外显子中的一个点突变导致第96位的天冬酰胺被赖氨酸取代,这证实了SOCCA1编码的成熟蛋白与自然种群中常见的S等位基因编码的蛋白相同。SODCA1等位基因表达减少很可能是由于P因子插入导致转录速率降低所致。