Sheffield V C, Upholt W B
Coll Relat Res. 1985 Jan;5(1):1-8. doi: 10.1016/s0174-173x(85)80042-x.
The copy number of the type II procollagen gene has been determined by quantitative hybridization of cloned DNA to Southern blots of total cellular DNA from chicken. Two different DNA probes have been used. One contains the 289 base pair exon coding for the junction between the triple helical domain and the carboxyl-terminal propeptide and the other contains an 162 base pair exon coding for amino acids 712-765 of the triple helical domain. Both fragments hybridize to single bands present at one copy per haploid genome in genomic DNA from chicken liver or sternal cartilage after these DNAs are cleaved with either HinfI of PstI. When the hybridization stringency and washing conditions were modified sufficiently to stabilize hybridization of heteroduplexes with up to 31% mismatch, no additional hybridizing fragments were detected. These results show that there is not a second copy of this gene or a pseudogene which is more closely related to the type II procollagen gene than the alpha 1 type I procollagen gene. These studies suggest that the cartilage 3 alpha collagen chain, which appears to be very similar to the alpha 1 (II) collagen chain, is encoded by the type II procollagen gene but is differently processed than the major type II gene product, and that the alpha 1 (II) Minor chain identified in bovine type II collagen preparations most likely represents an allelic form of type II collagen rather than a product of a different gene.
通过将克隆的DNA与鸡的总细胞DNA的Southern印迹进行定量杂交,已确定了II型原胶原基因的拷贝数。使用了两种不同的DNA探针。一种包含编码三螺旋结构域与羧基末端前肽之间连接的289个碱基对的外显子,另一种包含编码三螺旋结构域第712 - 765位氨基酸的162个碱基对的外显子。在用HinfI或PstI切割鸡肝或胸骨软骨的基因组DNA后,这两个片段均与单条带杂交,该单条带在单倍体基因组中以每个基因组一份的形式存在。当杂交严谨性和洗涤条件被充分改变以稳定高达31%错配的异源双链体的杂交时,未检测到其他杂交片段。这些结果表明,不存在该基因的第二个拷贝或假基因,其与II型原胶原基因的关系比α1 I型原胶原基因更密切。这些研究表明,软骨3α胶原链似乎与α1(II)胶原链非常相似,由II型原胶原基因编码,但加工方式与主要的II型基因产物不同,并且在牛II型胶原制剂中鉴定出的α1(II)小链很可能代表II型胶原的等位基因形式,而不是不同基因的产物。