Saitta B, Stokes D G, Vissing H, Timpl R, Chu M L
Department of Biochemistry, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Jefferson Medical College, Philadelphia, Pennsylvania 19107.
J Biol Chem. 1990 Apr 15;265(11):6473-80.
We recently reported the isolation and sequencing of two classes of human alpha 2(VI) collagen cDNA clones which share common sequences for the first two-thirds of the molecule but contain a different sequence of either 607 or 887 base pairs at their 3' ends (Chu, M.-L., Pan, T.-C., Conway, D., Kuo, H.-J., Glanville, R. W., Timpl, R., Mann, K., and Deutzmann, R. (1989) EMBO J. 8, 1939-1946). In the present study, we report the sequence of another cDNA clone, which is identical to one class of the previously isolated cDNAs except for a 293-base pair insertion between the common and variable regions. Together, the different classes of cDNAs, referred to as the alpha 2C2, alpha 2C2a, and alpha 2C2a' predict three variant alpha 2 chains of type VI collagen with carboxyl globular domains of 429, 328, and 238 amino acid residues, respectively. In order to explore the mechanisms by which the variations are generated, we isolated and characterized the 3' end of the human alpha 2(VI) collagen gene. The carboxyl globular domain was found to be encoded by six exons which appear to delineate its structural subdomains. The exon/intron arrangement clearly demonstrated that the cDNA variants arose from alternative splicing events by mutually exclusive utilization of the last two exons in conjunction with the selective usage of an internal splice acceptor site in the penultimate exon. The presence of the corresponding mature mRNA transcripts (3.2-3.5 kilobase pairs (kb] in human fibroblasts was shown by Northern blot hybridization, S1 nuclease protection assay, and the polymerase chain reaction. The results indicated that the alpha 2C2 mRNA is the major species, whereas the alpha 2C2a and alpha 2C2a' are the minor forms. Northern blot hybridization also revealed an alpha 2(VI) collagen mRNA of 6.0 kb. This mRNA retained a 2.3-kb intron located between the two alternatively spliced exons and predicted a translational product that is the same as the alpha 2C2a variant.
我们最近报道了两类人α2(VI)胶原蛋白cDNA克隆的分离和测序，这两类克隆在分子的前三分之二具有共同序列，但在其3'端含有607或887个碱基对的不同序列（朱，M.-L.，潘，T.-C.，康威，D.，郭，H.-J.，格兰维尔，R. W.，蒂姆普尔，R.，曼，K.，和多伊茨曼，R.（1989年）《欧洲分子生物学组织杂志》8，1939 - 1946）。在本研究中，我们报道了另一个cDNA克隆的序列，它与先前分离的一类cDNA相同，只是在共同区域和可变区域之间有一个293个碱基对的插入。不同类别的cDNA，即α2C2、α2C2a和α2C2a'，共同预测了VI型胶原蛋白的三种变体α2链，其羧基球状结构域分别含有429、328和238个氨基酸残基。为了探究产生这些变异的机制，我们分离并鉴定了人α2(VI)胶原蛋白基因的3'端。发现羧基球状结构域由六个外显子编码，这些外显子似乎界定了其结构亚结构域。外显子/内含子排列清楚地表明，cDNA变体是通过互斥利用最后两个外显子并结合倒数第二个外显子中一个内部剪接受体位点的选择性使用而由可变剪接事件产生的。通过Northern印迹杂交、S1核酸酶保护分析和聚合酶链反应显示，人成纤维细胞中存在相应的成熟mRNA转录本（3.2 - 3.5千碱基对（kb））。结果表明，α2C2 mRNA是主要种类，而α2C2a和α2C2a'是次要形式。Northern印迹杂交还揭示了一个6.0 kb的α2(VI)胶原蛋白mRNA。该mRNA保留了位于两个可变剪接外显子之间的一个2.3 kb内含子，并预测了一个与α2C2a变体相同的翻译产物。
