Orr W C, Orr E C, Legan S K, Sohal R S
Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275, USA.
Arch Biochem Biophys. 1996 Jun 15;330(2):251-8. doi: 10.1006/abbi.1996.0250.
The main objective of this study was to isolate and characterize the catalase gene and accompanying cis-regulatory regions in Drosophila melanogaster. Genomic clones were obtained on the basis of cross-hybridization to catalase cDNA and a 7-kb SalI-KpnI fragment encompassing the catalase gene was introduced into Drosophila by P element-mediated transformation. A single transgene, when placed in a catalase null background, was sufficient to restore resistance to H2O2 as well as reduce susceptibility to early death. DNA sequence of the catalase gene domain was obtained. This included 1365 bp of sequence upstream of the transcription initiation site and 1423 bp downstream of the termination codon. The Drosophila catalase gene is composed of 3 exons, encoding 19, 307, and 180 amino acids, which are separated by 3520- and 96-bp introns. Sequence analysis of the promoter domain is presented, revealing multiple sequence similarities between catalase and Cu,Zn superoxide dismutase promoter domains. Developmental RNA get analysis shows that peaks of catalase mRNA accumulation correspond roughly with major peaks of ecdysone titer during third instar and pupal stages. Candidate ecdysone response element sequences are noted downstream of the catalase polyadenylation site.
本研究的主要目的是分离和鉴定黑腹果蝇中的过氧化氢酶基因及相关的顺式调控区域。基于与过氧化氢酶cDNA的交叉杂交获得了基因组克隆，并通过P因子介导的转化将一个包含过氧化氢酶基因的7kb SalI-KpnI片段导入果蝇。当单个转基因置于过氧化氢酶缺失背景中时，足以恢复对H2O2的抗性并降低对早期死亡的易感性。获得了过氧化氢酶基因结构域的DNA序列。这包括转录起始位点上游1365bp的序列和终止密码子下游1423bp的序列。黑腹果蝇过氧化氢酶基因由3个外显子组成，分别编码19、307和180个氨基酸，它们被3520bp和96bp的内含子隔开。文中展示了启动子结构域的序列分析，揭示了过氧化氢酶和铜锌超氧化物歧化酶启动子结构域之间的多个序列相似性。发育RNA分析表明，过氧化氢酶mRNA积累的峰值大致与三龄幼虫和蛹期蜕皮激素滴度的主要峰值相对应。在过氧化氢酶多聚腺苷酸化位点下游发现了候选蜕皮激素反应元件序列。
