Sarkar F H, Ball D E, Tsang W, Li Y W, Kuo T H
Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201.
DNA Cell Biol. 1993 Jun;12(5):435-40. doi: 10.1089/dna.1993.12.435.
Polymerase chain reaction [PCR, reverse transcriptase-PCR (RT-PCR)] has been used to amplify the mRNA subspecies of the plasma membrane calcium pump isoform 1 (PMCA1) in total RNA extracted from hamster tissues. Two primers were synthesized that encompass the site at which a 154-bp exon is included totally (PMCA1a), partially (PMCA1c and d), or completely excluded (PMCA1b) in the carboxy-terminal regulatory region. PCR amplification revealed two bands (PMCA1b and 1c) that are more abundant in various tissues, while Southern hybridization of the samples after PCR amplification has detected two additional mRNA variants corresponding to PMCA1a and 1d. The distribution of these mRNA variants are tissue specific and correlate well with the pump protein distribution patterns on immunoblot. Since these multiple bands on the immunoblot are not derived from proteolysis, it is suggested that they represent the PMCA1 isozymes encoded by these alternatively spliced mRNAs. To our knowledge, this is the first report to show all four alternatively spliced mRNAs that are simultaneously detected in one single RNA sample using PCR technique. Since these isozymes are different in their regulatory domain, their tissue-specific expression may be physiologically important.
聚合酶链反应[PCR，逆转录聚合酶链反应（RT-PCR）]已用于扩增从仓鼠组织提取的总RNA中质膜钙泵同工型1（PMCA1）的mRNA亚型。合成了两条引物，其涵盖了在羧基末端调节区域中154bp外显子被完全包含（PMCA1a）、部分包含（PMCA1c和d）或完全排除（PMCA1b）的位点。PCR扩增显示出两条条带（PMCA1b和1c），它们在各种组织中更为丰富，而PCR扩增后样品的Southern杂交检测到另外两种对应于PMCA1a和1d的mRNA变体。这些mRNA变体的分布具有组织特异性，并且与免疫印迹上的泵蛋白分布模式良好相关。由于免疫印迹上的这些多条条带并非源自蛋白水解，因此表明它们代表了由这些可变剪接的mRNA编码的PMCA1同工酶。据我们所知，这是第一份使用PCR技术在单个RNA样品中同时检测到所有四种可变剪接mRNA的报告。由于这些同工酶在其调节域中不同，它们的组织特异性表达可能具有重要的生理意义。
