INTRON-EXON STRUCTURE, ALTERNATIVE USE OF PROMOTER AND EXPRESSION OF THE MOUSE COLLAGEN X-GENE, COL10A-1
INTRON-EXON STRUCTURE, ALTERNATIVE USE OF PROMOTER AND EXPRESSION OF THE MOUSE COLLAGEN X-GENE, COL10A-1
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DOI：
10.1111/j.1432-1033.1993.tb17739.x
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发表时间：
1993-04-01
期刊：
影响因子：
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通讯作者：
CHEAH, KSE
中科院分区：
文献类型：
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作者：
KONG, RYC;KWAN, KM;CHEAH, KSE
整个小鼠胶原X基因（Col10a-1）已被分离。该基因由3个外显子和2个内含子组成，全长7.0 kb。外显子2和3共同编码15 bp的5‘未翻译序列、2040 bp的开放阅读框和895个核苷酸的3’非编码区。在该基因的5'侧区，发现了两个一致的TATA-box序列。通过核糖核酸酶保护试验鉴定第一个外显子，并通过引物延伸分析确定Col10a-1 mRNA转录本的5‘端，结果表明，更多的3’ TATA box可能被主要使用，并且在外显子1序列3‘上至少有三个转录起始位点，导致5’非翻译区分别为78、77和55个核苷酸。通过聚合酶链反应对cDNA末端的快速扩增，检测到一个额外的mRNA物种与其他Col10a-1转录本重叠，包括3‘ TATA box序列，得到一个约235个碱基的5’未翻译序列。后一个转录本大约从20 bp 3‘到5’ TATA盒开始。这些数据表明Col10a-1基因的启动子和转录开始有不同的用途。与鸡、牛和人胶原X基因外显子2和3的组合核苷酸和推断氨基酸序列的比较表明，该基因高度相似，表明该基因在整个进化过程中具有保守性。小鼠Col10a-1 mRNA约为3.0 kb，经SDS/PAGE检测，蛋白酶化蛋白几乎等于45 kDa。mRNA和蛋白质的大小与开放阅读框预测的大小相关。逆转录聚合酶链反应实验表明，小鼠胶原X基因在性交后13.5天首次表达，暂时早于软骨内成骨的发生。与普遍接受的x型胶原与软骨内成骨的关联一致，原位杂交分析表明Col10a-1 mRNA仅限于生长软骨的肥大区。
The entire mouse collagen X gene (Col10a-1) has been isolated. The gene is composed of three exons and two introns spanning 7.0 kb of the DNA sequence. Exons 2 and 3 together encode 15-bp of 5' untranslated sequence, a 2040-bp open reading frame and an 895-nucleotide 3' non-coding region. In the 5' flanking region of the gene, two consensus TATA-box sequences were found. Identification of the first exon by ribonuclease-protection assays and the determination of the 5' end of Col10a-1 mRNA transcripts by primer-extension analyses show that the more 3' TATA box is probably predominantly used and that there are at least three transcription start sites in the exon 1 sequence 3' to this, resulting in 5' untranslated regions of 78, 77 and 55 nucleotides. By means of rapid amplification of cDNA ends by polymerase chain reaction, an additional mRNA species was detected which overlapped the other Col10a-1 transcripts, including the 3' TATA box sequence, giving a 5' untranslated sequence of approximately 235 bases. This latter transcript starts approximately 20 bp 3' to the more 5' TATA box. The data suggest alternative use of promoters and transcription starts for the Col10a-1 gene. Comparison of the combined nucleotide and deduced amino acid sequences of exons 2 and 3 with chicken, bovine and human collagen X genes, showed a high degree of similarity indicating conservation of this gene throughout evolution. Mouse Col10a-1 mRNA was shown to be approximately 3.0 kb and the pepsinized protein, as detected by SDS/PAGE, was almost-equal-to 45 kDa. The mRNA and protein sizes correlate with that predicted by the open reading frame. Reverse-transcription polymerase chain reaction assays indicate that the mouse collagen X gene is first expressed at 13.5 days post coitum, temporally preceding the onset of endochondral ossification. In agreement with the generally accepted association of type-X collagen with endochondral ossification, in situ hybridization analyses indicate that Col10a-1 mRNA are restricted to the hypertrophic regions of growth cartilage.