兔快肌骨骼肌钙ATP酶基因的结构

Structure of the rabbit fast-twitch skeletal muscle Ca2+-ATPase gene.

作者信息

Korczak B, Zarain-Herzberg A, Brandl C J, Ingles C J, Green N M, MacLennan D H

机构信息

Banting and Best Department of Medical Research, Charles H. Best Institute, University of Toronto, Ontario, Canada.

出版信息

J Biol Chem. 1988 Apr 5;263(10):4813-9.

PMID:2965149

Abstract

We have isolated two genomic clones which together encode the Ca2+-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum. One of the two 16.5 kilobase (kb) genomic inserts in the lambda phage vector Charon 4A contains 23 exons extending from the polyadenylation site at the 3' end of the ATPase gene to within 38 nucleotides of the translation initiation codon in the 5' exon. An overlapping genomic insert of 16.5 kb contains the remainder of the 5' exon and a further 8 kb of upstream sequence. S1 nuclease mapping and primer extension analysis of the 5' end of the Ca2+-ATPase mRNA indicate that the transcription initiation site is located 185 base pairs (bp) upstream of the translation initiation codon. A "TATA" box (CA-TAAA) was found at position -30 and the sequence CCAAT was found at position -78 relative to the transcription initiation site. In a previous study (Brandl, C. J., de Leon, S., Martin, D. R., and MacLennan, D. H. (1987) J. Biol. Chem. 262, 3768-3774) cDNAs for neonatal and adult forms of the fast-twitch Ca2+-ATPase were shown to encode different carboxyl-terminal sequences, presumably as a result of alternative splicing. We have now found that these different DNA sequences encoding different carboxyl-terminal sequences are located in different exons. Exon boundaries of the Ca2+-ATPase gene did not correlate well with proposed domain boundaries for the Ca2+-ATPase protein. The locations of exon/intron boundaries were only partially conserved between the Ca2+-ATPase gene and a Na+/K+-ATPase gene (Ovchinnikov, Y. A., Monastyrskaya, G. S., Broude, N. E., Allikmets, R. L., Ushkaryov, Y. A., Melkov, A. M., Smirnov, Y. V., Malyshev, I. V., Dulubova, I. E., Petrukhin, K. E., Gryshin, A. V., Sverdlov, V. E., Kiyatkin, N. I., Kostina, M. B., Modyanov, N. N., and Sverdlov, E. D. (1987) FEBS Lett. 213, 73-80) and they did not follow closely the boundaries of amino acid sequences that are highly conserved among a group of ion transport ATPases.

摘要

我们分离出了两个基因组克隆，它们共同编码兔快肌骨骼肌肌浆网的Ca2 + -ATP酶。λ噬菌体载体Charon 4A中的两个16.5千碱基（kb）基因组插入片段之一包含23个外显子，从ATP酶基因3'端的聚腺苷酸化位点延伸至5'外显子中翻译起始密码子的38个核苷酸内。一个16.5 kb的重叠基因组插入片段包含5'外显子的其余部分和另外8 kb的上游序列。对Ca2 + -ATP酶mRNA 5'端的S1核酸酶图谱分析和引物延伸分析表明，转录起始位点位于翻译起始密码子上游185个碱基对（bp）处。相对于转录起始位点，在-30位置发现了一个“TATA”框（CA-TAAA），在-78位置发现了序列CCAAT。在先前的一项研究中（Brandl，C. J.，de Leon，S.，Martin，D. R.，和MacLennan，D. H.（1987）J. Biol. Chem. 262，3768 - 3774），快肌Ca2 + -ATP酶新生儿和成人形式的cDNA显示编码不同的羧基末端序列，推测是可变剪接的结果。我们现在发现，这些编码不同羧基末端序列的不同DNA序列位于不同的外显子中。Ca2 + -ATP酶基因的外显子边界与Ca2 + -ATP酶蛋白的推测结构域边界相关性不佳。Ca2 + -ATP酶基因与Na + /K + -ATP酶基因（Ovchinnikov，Y. A.，Monastyrskaya，G. S.，Broude，N. E.，Allikmets，R. L.，Ushkaryov，Y. A.，Melkov，A. M.，Smirnov，Y. V.，Malyshev，I. V.，Dulubova，I. E.，Petrukhin，K. E.，Gryshin，A. V.，Sverdlov，V. E.，Kiyatkin，N. I.，Kostina，M. B.，Modyanov，N. N.，和Sverdlov，E. D.（1987）FEBS Lett. 213，73 - 80）之间外显子/内含子边界的位置仅部分保守，并且它们并不紧密遵循一组离子转运ATP酶中高度保守的氨基酸序列边界。