Molecular cloning, cDNA structure, and regulation of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovarian granulosa cells.
Molecular cloning, cDNA structure, and regulation of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovarian granulosa cells.
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DOI：
10.1016/s0021-9258(18)67247-3
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发表时间：
1986-09
期刊：
影响因子：
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通讯作者：
T. Jahnsen;L. Hedin;V. Kidd;W. Beattie;S. Lohmann;U. Walter;J. Durica;T. Schulz;E. Schiltz;M. Browner
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文献类型：
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作者：
T. Jahnsen;L. Hedin;V. Kidd;W. Beattie;S. Lohmann;U. Walter;J. Durica;T. Schulz;E. Schiltz;M. Browner
在大鼠卵巢颗粒细胞中，雌二醇和促卵泡激素（cAMP）选择性诱导II型cAMP依赖性蛋白激酶调节亚基的一种亚型（R-II 51; Mr = 51，000）及其电泳变体（R-II 51.5和R-II 52; Mr分别为51，500和52，000）。为了确定R-II 51的氨基酸序列并深入了解调节卵巢卵泡R-II 51的细胞内含量的分子事件，我们构建了一个λ gt 11 cDNA表达文库，该文库来自于经卵巢刺激的大鼠颗粒细胞的poly（A）+ RNA。从噬菌斑纯化的R-II抗体阳性噬菌体克隆中分离的1.5-丝氨酸蛋白酶（kb）cDNA插入片段选择性地结合R-II 51 mRNA，如通过分析杂交选择的翻译产物所证明的。限制性内切酶图谱和序列分析的1.5-kb的cDNA插入和1.8-和2.2-kb的cDNA插入从两个额外的克隆显示重叠的序列跨越一个区域的3065个核苷酸的大小。1.5和1.8kb的cDNA插入片段分别含有poly（A）加成信号（分别为1508和1761个核苷酸）、末端poly（A）序列和R-II 51的整个编码区（1204个核苷酸），除了5 ′端的少量核苷酸。2.2kb的cDNA插入片段含有394个核苷酸的编码区、一个长的3'非翻译区和两个多聚腺苷酸加成信号（3041和3059个核苷酸）。围绕纯大鼠卵巢R-II 51的自磷酸化位点的氨基酸微序列与从cDNA的核苷酸序列推导的氨基酸序列一致。北方印迹分析表明，两个主要的mRNA物种（1.8和3.2 kb的大小）在ESTA引发大鼠卵巢，大约10倍和50倍以上的R-II mRNA含量在大鼠大脑和大鼠心脏，分别。大鼠肝DNA的Southern印迹分析表明，一个单一的基因编码的R-II 51 mRNA。大鼠卵巢R-II 51、大鼠心脏R-II 54和牛R-II和R-I亚基的已知氨基酸序列之间的结构差异也表明大鼠卵巢R-II 51亚基是不同基因的产物。
One isoform of the regulatory subunit of type II cAMP-dependent protein kinase (R-II51; Mr = 51,000) and its electrophoretic variants (R-II51.5 and R-II52; Mr = 51,500 and 52,000, respectively) are selectively induced by estradiol and follicle-stimulating hormone (cAMP) in rat ovarian granulosa cells. To ascertain the amino acid sequence of R-II51 and to gain insight into the molecular events regulating the intracellular content of ovarian follicular R-II51, we constructed a lambda gt11 cDNA expression library from poly(A)+ RNA of hormone-primed rat granulosa cells. A 1.5-kilobase (kb) cDNA insert, isolated from a plaque-purified R-II antibody positive bacteriophage clone, selectively bound R-II51 mRNA as demonstrated by analysis of the hybrid-selected translation product. Restriction maps and sequence analyses of the 1.5-kb cDNA insert and of the 1.8- and 2.2-kb cDNA inserts from two additional clones showed overlapping sequences which span a region of 3065 nucleotides in size. The 1.5- and 1.8-kb cDNA inserts each contained poly(A) addition signals (1508 and 1761 nucleotides, respectively), terminal poly(A) sequences, and the entire coding region for R-II51 (1204 nucleotides) except for a small number of nucleotides at the 5' end. The 2.2-kb cDNA insert contained 394 nucleotides of the coding region a long 3' untranslated region and two more poly(A) addition signals (3041 and 3059 nucleotides). An amino acid microsequence surrounding the autophosphorylation site of pure rat ovarian R-II51 agreed with the amino acid sequence deduced from the nucleotide sequence of the cDNA. Northern blot analyses demonstrated two major mRNA species (1.8 and 3.2 kb in size) in hormone-primed rat ovaries which were approximately 10- and 50-fold greater than the R-II mRNA content in rat brain and rat heart, respectively. Southern blot analysis of rat liver DNA indicated that a single gene codes for R-II51 mRNA. Structural differences among rat ovarian R-II51, rat heart R-II54, and the known amino acid sequences of bovine R-II and R-I subunits also indicate that the rat ovarian R-II51 subunit is the product of a distinct gene.