MOLELCULAR BIOLOGY OF PHOTOTRANSDUCTION

我们已经鉴定了人和小鼠的S抗原基因， 来自人的cDNA;来自牛、大鼠和人的Ducin cDNA; Shuzin cDNA 人类和牛的基因和人类的Shuzin基因。的基因序列 测定人和小鼠S抗原。S抗原基因是 长度约50 kbp，包含16个外显子和15个内含子， 由97%的内含子和3%的外显子组成。基因的5 '侧翼区域， 大约1.5 kbp长，没有已知的调控元件， 转录，如TATA，GC或CCAAT盒。有趣的是，5' 人和小鼠基因的侧翼区域表达组织特异性 启动子活性在体外和体内转录测定以及 如转基因小鼠。来自人、大鼠和大鼠的几种表达该蛋白的cDNA， 牛被分离出来，我们确定了它们的DNA序列。每个序列 含有用于通过A-激酶磷酸化的Ser 73。测序结果表明 视网膜和松果体中的β-ducin序列相同 和相同的磷酸化位点。这表明，功能作用 在视网膜和松果体中这种蛋白质的含量是相同的。 视网膜蛋白Shuzin的功能作用是未知的。我们分离 几个cDNAs和测序每个人和牛。整个 测定了人Shuzin基因的序列。这个基因由 两个内含子和三个外显子，并且它具有高度重复的序列， 5 '-非编码区。 我们构建了含有5 '侧翼S抗原基因的融合基因， 细菌基因氯霉素乙酰转移酶上游序列 （CAT）。一种含有小鼠5 ′-侧翼区的杂交基因 将S抗原基因和CAT基因显微注射到转基因小鼠体内。 这些小鼠在视网膜和松果体中表达CAT活性， 这表明1300 bp长的S抗原启动子具有组织特异性 增强子和启动子。 将S抗原cDNA亚克隆到两个表达载体系统中。的 通过凝胶过滤纯化表达的蛋白质，并将其结晶。 这种蛋白质正在研究中。

We have characterized the S-antigen genes from human and mouse, a kinase cDNA from human; phosducin cDNAs from cow, rat, and human; Shuzin cDNAs from human and cow and a Shuzin gene from human. The gene sequences of the human and mouse S-antigens were determined. The S-antigen genes were approximately 50 kbp in length, contained 16 exons and 15 introns, and were comprised of 97% intron and 3% exon. The 5'-flanking regions of the genes, approximately 1.5 kbp long, have no known regulatory elements for transcription such as TATA, GC, or CCAAT boxes. Interestingly, the 5' flanking regions of the human and mouse genes expressed tissue specific promoter activity in both in vitro and in vivo transcription assays as well as in transgenic mice. Several cDNAs of the phosducin from human, rat, and cow were isolated, and we determined their DNA sequences. Each sequence contains a Ser73 for phosphorylation by A-kinase. Sequencing results show that the phosducin in the retina and pineal gland have the same sequences and the same phosphorylation sites. This suggests that the functional role of this protein is the same in the retina and pineal gland. The functional role of the retinal protein Shuzin is unknown. We isolated several cDNAs and sequenced each of these from human and cow. The entire gene sequence of human Shuzin was also determined. This gene is composed of two introns and three exons and it has a highly repetitive sequence in the 5'-noncoding region. We have constructed fusion genes containing a 5'-flanking S-antigen gene sequence upstream of the bacterial gene chloramphenicol acetyl transferase (CAT). A hybrid gene containing the 5'-flanking region of the mouse S-antigen gene and the CAT gene was microinjected into transgenic mice. Those mice expressed CAT activity in the retina and pineal gland, suggesting that the 1300 bp-long S-antigen promoter has a tissue specific enhancer and promoter. S-antigen cDNAs were subcloned into two expression vector systems. The expressed protein was purified by gel filtration, and crystallization of this protein is now in progress.