Brewton R G, Wood B M, Ren Z X, Gong Y, Tiller G E, Warman M L, Lee B, Horton W A, Olsen B R, Baker J R
Department of Cell Biology, University of Alabama at Birmingham 35294, USA.
Genomics. 1995 Nov 20;30(2):329-36. doi: 10.1006/geno.1995.9870.
Type IX collagen is composed of three polypeptides derived from the human genes COL9A1, COL9A2, and COL9A3 that assemble to form a mature collagen molecule with the structure alpha 1(IX)alpha 2(IX)alpha 3(IX). We have identified overlapping cDNA and genomic clones that encode for the entire alpha 3 chain of human type IX collagen. Tryptic peptides from the human alpha 3(IX) collagen chain were subjected to N-terminal amino acid sequencing, and a stretch of 124 contiguous amino acids that included the NC1, COL1, and NC2 domains was obtained. Degenerate oligonucleotide primers were designed based on the amino acid sequences of the human tryptic peptides as well as bovine peptides and sequences from chicken cDNA clones. These primers were used to amplify three overlapping PCR products that covered the majority of the human alpha 3(IX) collagen. PCR products were then used to identify overlapping cDNA clones from a human chondrocyte library. A lambda genomic clone was identified that contained the 5'-most exon that encodes the signal peptide to complete the entire structure of the human alpha 3(IX) collagen chain. Genomic amplification identified a single-strand conformational polymorphism in COL1 that was used to map COL9A3 to chromosome 20q13.3 by linkage analysis. The present study completes the structure of human type IX collagen, and linkage for COL9A3 completes the genomic mapping of cartilage collagen genes. These data will greatly assist the genetic screening of families with degenerative cartilage and eye diseases by allowing investigators to screen for a complete set of candidate collagen gene markers.
IX型胶原蛋白由源自人类基因COL9A1、COL9A2和COL9A3的三条多肽组成，这些多肽组装形成具有α1(IX)α2(IX)α3(IX)结构的成熟胶原蛋白分子。我们已鉴定出编码人类IX型胶原蛋白整个α3链的重叠cDNA和基因组克隆。对来自人类α3(IX)胶原蛋白链的胰蛋白酶肽段进行N端氨基酸测序，获得了一段包含NC1、COL1和NC2结构域的124个连续氨基酸序列。基于人类胰蛋白酶肽段以及牛肽段的氨基酸序列和鸡cDNA克隆的序列设计了简并寡核苷酸引物。这些引物用于扩增覆盖人类α3(IX)胶原蛋白大部分区域的三个重叠PCR产物。然后将PCR产物用于从人类软骨细胞文库中鉴定重叠的cDNA克隆。鉴定出一个λ基因组克隆，其包含编码信号肽的最5'端外显子，从而完成了人类α3(IX)胶原蛋白链的完整结构。基因组扩增在COL1中鉴定出一种单链构象多态性，通过连锁分析将COL9A3定位到20号染色体q13.3区域。本研究完成了人类IX型胶原蛋白的结构，COL9A3的连锁分析完成了软骨胶原蛋白基因的基因组定位。这些数据将极大地有助于对患有退行性软骨和眼部疾病的家族进行基因筛查，使研究人员能够筛查一整套候选胶原蛋白基因标记。
