Solberg R, Sandberg M, Natarajan V, Torjesen P A, Hansson V, Jahnsen T, Taskén K
Institute of Medical Biochemistry, University of Oslo, Norway.
Endocrinology. 1997 Jan;138(1):169-81. doi: 10.1210/endo.138.1.4864.
The present study reports the exon-intron organization of the human RI alpha gene of cAMP-dependent protein kinase and approximately kilobases (kb) of the 5'-flanking region obtained by isolation and sequencing of several phage clones from human genomic libraries. The RI alpha gene is composed of nine coding exons of varying lengths, separated by introns, giving the gene a total length of at least 21 kb. our recent cloning of a processed RI alpha pseudogene with a 5'-noncoding region different from the previously reported RI alpha complementary RNA indicated that the RI alpha gene may have multiple leader exons giving rise to alternately spliced messenger RNAs (mRNAs). Reverse transcription of human testis RNA followed by PCR identified two different RI alpha mRNA species (RI alpha 1a and RI alpha 1b) containing distinct sequences due to alternately splicing the gene. The previously known RI alpha 1b mRNA revealed low constitutive expression in a human B lymphoid cell line (Reh) and was stimulated only 4- to 6-fold by treatment with cAMP. In contrast, very low levels of the novel RI alpha 1a mRNA were present in untreated Reh cells, but were stimulated 40-to 50-fold by cAMP. The 5'-flanking sequence of the RI alpha gene was G/C rich and did not contain any TATA box. Several putative transcription initiation sites were identified in front of each leader exon (exons 1a and 1b) by the 5'-rapid amplification of complementary DNA ends technique. To determine whether the sequences 5' of both leader exons had promoter activities, the 5'-flanking sequences of exons 1a and 1b were inserted in front of a chloramphenicol acetyltransferase reporter gene, and their ability to direct transcription were examined. Transfection of these constructs into rat GH4C1 cells demonstrated that both constructs had promoter activities, as evidenced by high levels of chloramphenicol acetyltransferase activity.
本研究报告了环磷酸腺苷依赖性蛋白激酶的人RIα基因的外显子-内含子组织以及通过从人基因组文库中分离和测序几个噬菌体克隆获得的约几千碱基（kb）的5'侧翼区域。RIα基因由九个长度各异的编码外显子组成，被内含子隔开，使该基因全长至少21 kb。我们最近克隆了一个加工后的RIα假基因，其5'非编码区与先前报道的RIα互补RNA不同，这表明RIα基因可能有多个前导外显子，产生交替剪接的信使RNA（mRNA）。对人睾丸RNA进行逆转录，然后进行PCR，鉴定出两种不同的RIα mRNA种类（RIα1a和RIα1b），由于该基因的交替剪接而含有不同的序列。先前已知的RIα1b mRNA在人B淋巴细胞系（Reh）中显示出低组成性表达，仅通过用环磷酸腺苷处理可刺激4至6倍。相比之下，未处理的Reh细胞中新型RIα1a mRNA的水平非常低，但通过环磷酸腺苷可刺激40至50倍。RIα基因的5'侧翼序列富含G/C，且不包含任何TATA框。通过5'互补DNA末端快速扩增技术在每个前导外显子（外显子1a和1b）前面鉴定出几个推定的转录起始位点。为了确定两个前导外显子5'端的序列是否具有启动子活性，将外显子1a和1b的5'侧翼序列插入氯霉素乙酰转移酶报告基因的前面，并检测它们指导转录的能力。将这些构建体转染到大鼠GH4C1细胞中表明，两个构建体都具有启动子活性，氯霉素乙酰转移酶活性水平高证明了这一点。
