Research on the cloning and analyzing of the human collagen gene

1)人α1(XI)胶原基因启动子的分析我们克隆了人COL11A1基因的5‘端，并确定了其主要转录起始点的位置。对直接上游序列的分析表明，缺乏TATA基序，存在几个GC盒。与这一发现一致，引物延伸和核糖核酸酶保护分析发现，在ATG密码子上游318个核苷酸的主要转录起始点周围有几个次要的转录起始点。瞬时转染实验表明，1.6kb的上游区域具有启动子的能力。缺失构建体缩小了其转录活性在核苷酸-541至-199之间的序列。2)一条新的人胶原链的一级结构和基因表达为了确定该链的完整一级结构，我们从人横纹肌肉瘤细胞系(CCL136)的随机扩增的cDNA文库中分离了重叠的克隆，推导出的多肽含有一个1142个氨基酸残基的开放阅读框。预测的多肽由COL1至COL5的五个胶原区组成。在结构上，这种胶原链与Alpha1(XVI)链最相似。用该基因5‘端的Northern杂交分析表明，有11个基因片段的大小在0.7-12kb之间。然而，在多个RNA中，只有两个大小分别为6和12kb的RNA与大多数3‘端的cDNA杂交，这一数据清楚地支持了这一结论，即该cDNA编码了一个不同于已知的α链的胶原α链。我们建议将该胶原链命名为Alpha1(XIX)链。

1) Analysis of the promoter of the human alpha1(XI) collagen geneWe have cloned the 5' portion of the human COL11A1 gene and determined the location of its major start site of transcription. Analysis of the immediate upstream sequence revealed the lack of a TATA motif and the presence of several GC boxes. Consistent with this finding, primer extension and RNase protection assays identified several minor transcriptional start sites around a major one, 318-bp upstream from the ATG codon. Transient transfection experiments has showed the 1.6kb upstream region has the ability of the promoter. Deletion constructs narrowed down the sequence of its transcriptional activity between nucleotides -541 to -199. In this region, there is a single foot printing area containing a sequence with similarity to an AP-3 binding site.2) Primary structure and gene expression of a new human collagen chainTo determine the complete primary structure of the chain, we have isolated overlapping clones from a random-primed cDNA library from human rhabdomyosarcoma cell line (CCL 136) mRNA.The deduced polypeptide contained an open reading frame of 1142 amino acid residues. The predicted polypeptide consists of five collagenous domains of the COL1 to COL5. Structurally, this collagen chain most resembles to the alpha1(XVI) chain. Northern blot analyzes using the 5' end of the cDNA showed 11 mRNA species from 0.7 to 12 kb in size. However, the only two RNAs of 6 and 12kb size out of the multiple RNAs were hybridized to the most 3' end cDNA.The date clearly support the conclusion the cDNAs encode a collagen alpha chain which is different from known alpha chain. We propose to designate this collagen chain as alpha1(XIX) chain.