Intron‐exon structure, alternative use of promoter and expression of the mouse collagen X gene,Col10a‐1

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Abstract
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 readin 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 ofCol10a‐1mRNA 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 otherCol10a‐1transcripts, 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 theCol10a‐1gene. 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. MouseCol10a‐1mRNA was shown to be approximately 3.0 kb and the pepsinized protein, as detected by SDS/PAGE, was ∼ 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 dayspost coitum, temporally preceding the onset of endochondral ossification. In agreement with the generally accepted association of type‐X collagen with endochondral ossification,in situhybridization analyses indicate thatCol10a‐1mRNA are restricted to the hypertrophic regions of growth cartilage.

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