Payne A M, Yue P, Pritchard K, Marston S B
Department of Cardiac Medicine, National Heart and Lung Institute, London, U.K.
Biochem J. 1995 Jan 15;305 ( Pt 2)(Pt 2):445-50. doi: 10.1042/bj3050445.
The recent determination of the genomic sequence of human caldesmon indicates that eight caldesmon mRNA species could be generated by selection of exon 1 or 1', exon 3a or 3ab and/or exon 4. We used reverse transcriptase PCR to determine which transcripts were produced in human, rabbit and sheep artery, vein, lung, intestine, kidney and liver. In all tissues the same three transcripts were present: exons 1'-2-3a-5-6...13, exons 1'-2-3a3b-5-6-...13 and exons 1'-2-3a3b-4-5-6...13. Exon 1 was not present and exon 4 was only present when exon 3b was also present. Three protein isoforms of caldesmon can be distinguished by electrophoresis on high-porosity 6% polyacrylamide gel: 130 kDa, 120 kDa and 70 kDa. The 70 kDa isoform lacks the sequence encoded by exon 3b. We investigated whether the two high-molecular-mass isoforms correspond to the presence and absence of exon 4 using an antiserum specific to the sequence encoded by exon 4. Western-blotting and immunoprecipitation experiments showed that both the 130 kDa and the 120 kDa isoforms were expressed with and without the exon 4 sequence. We therefore propose that the molecular-mass heterogeneity arises from additional first exons, possibly with separate promoter regions, which have not yet been characterized in the genomic sequence.
近期对人钙调蛋白基因组序列的测定表明，通过选择外显子1或1'、外显子3a或3ab以及/或者外显子4，可产生8种钙调蛋白mRNA种类。我们运用逆转录酶PCR来确定在人、兔和羊的动脉、静脉、肺、肠、肾及肝脏中产生了哪些转录本。在所有组织中均存在相同的三种转录本：外显子1'-2-3a-5-6...13、外显子1'-2-3a3b-5-6-...13以及外显子1'-2-3a3b-4-5-6...13。外显子1不存在，且仅当外显子3b也存在时外显子4才会出现。钙调蛋白的三种蛋白质异构体可通过在高孔隙率6%聚丙烯酰胺凝胶上进行电泳加以区分：130 kDa、120 kDa和70 kDa。70 kDa的异构体缺少由外显子3b编码的序列。我们使用针对外显子4编码序列的抗血清，研究了这两种高分子量异构体是否分别对应外显子4的存在与缺失情况。蛋白质免疫印迹和免疫沉淀实验表明，130 kDa和120 kDa的异构体在有和没有外显子4序列的情况下均有表达。因此，我们提出分子量的异质性源于额外的首个外显子，可能具有独立的启动子区域，而这些在基因组序列中尚未得到表征。
