Kimura H, Sogawa K, Sakai Y, Fujii-Kuriyama Y
Department of Biochemistry, Cancer Institute, Tokyo, Japan.
J Biol Chem. 1989 Feb 5;264(4):2338-42.
Two mRNAs for P-450PB-1 and P-450PB-1(ps) are about 2 kilobase pairs long and have identical sequences with each other except for one short region of high variability (Kimura, H., Yoshioka, H., Sogawa, K., Sakai, Y., and Fujii-Kuriyama, Y. (1988) J. Biol. Chem. 263, 701-707). To clarify the origin of the short replacement block between the two mRNAs, we isolated several genomic clones containing relevant gene sequences. Sequence analysis of these genomic clones revealed that the two short segments specific for the two mRNAs are tandemly arranged in a genomic sequence and form exonic sequences equipped with AG and GT sequences on their 5' and 3' ends, respectively, and the putative consensus sequences for the lariat formation. The two short sequences lie between the two exonic sequences coding for the common part of the two mRNAs. Taken together with the structure of the related P-450(M-1) gene (Morishima, N., Yoshioka, H., Higashi, Y., Sogawa, K., and Fujii-Kuriyama, Y. (1987) Biochemistry 26, 8279-8285), all these results clearly demonstrate that the two mRNAs are generated from a single gene by alternative splicing at the eighth exons. The synthesis of the two mRNAs is regulated temporally in livers of male and female rats and brains of the female animals. One of the two mRNAs codes for a monooxygenase of P-450PB-1, and the other (P-450PB-1(ps) mRNA) lacks the sequence coding for the heme-binding site conserved among all species of P-450 molecules, and, therefore, it cannot function as a monooxygenase. The immunoblot analysis using an antibody specific for the 15-mer peptide uniquely encoded by P-450PB-1(ps) mRNA shows that the P-450PB-1(ps) peptide is synthesized at least in rat livers of both sexes in temporally regulated manners and is bound to the microsomal membranes. The function of this peptide remains to be seen.
P-450PB-1和P-450PB-1(ps)的两种信使核糖核酸（mRNA）长度约为2千碱基对，除了一个高变异性的短区域外，它们彼此具有相同的序列（木村浩、吉冈浩、相川克、酒井洋、藤井栗山（1988年）《生物化学杂志》263卷，701 - 707页）。为了阐明这两种mRNA之间短替换片段的起源，我们分离了几个包含相关基因序列的基因组克隆。对这些基因组克隆的序列分析表明，这两种mRNA特有的两个短片段在基因组序列中串联排列，并分别在其5'和3'末端形成带有AG和GT序列以及套索形成假定共有序列的外显子序列。这两个短序列位于编码这两种mRNA共同部分的两个外显子序列之间。结合相关的P-450(M-1)基因结构（森岛直、吉冈浩、东义、相川克、藤井栗山（1987年）《生物化学》26卷，8279 - 8285页），所有这些结果清楚地表明，这两种mRNA是由一个单一基因通过第八外显子的可变剪接产生的。这两种mRNA的合成在雄性和雌性大鼠的肝脏以及雌性动物的大脑中受到时间调控。这两种mRNA中的一种编码P-450PB-1的单加氧酶，另一种（P-450PB-1(ps) mRNA）缺乏在所有P-450分子物种中保守的血红素结合位点编码序列，因此，它不能作为单加氧酶发挥作用。使用对P-450PB-1(ps) mRNA独特编码的15聚体肽具有特异性的抗体进行免疫印迹分析表明，P-450PB-1(ps)肽至少在两性大鼠的肝脏中以时间调控的方式合成，并与微粒体膜结合。该肽的功能尚待观察。
