Isolation and partial characterization of genomic clones coding for a human pro-alpha 1 (II) collagen chain and demonstration of restriction fragment length polymorphism at the 3' end of the gene

We have isolated two clones containing 19 kilobases (kb) of the human gene coding for a pro-alpha 1 (II) collagen chain from human lambda genomic DNA libraries. A 3' clone, HC2A, was selected by cross-hybridization with a cDNA clone containing sequences coding for the carboxy propeptide of chick type II procollagen. A second clone, HC2B, was obtained by screening the library with the 5' part of HC2A. The sequence analysis of exon 3 corresponding to the C propeptide reveals the presence of stretches of conserved nucleotides between the human and the chick type II procollagen genes. On Northern blots, the human collagen clone hybridizes strongly to a 5.5-kb RNA for the rat type II procollagen chain. Finally, studies of genomic DNAs from normal individuals reveal the presence of a HindIII and a BamHI polymorphic site at the 3' end of the gene.     

Analysis of cDNA and genomic clones coding for the pro alpha 1 chain of calf type II collagen.

A bovine cDNA library constructed from fetal cartilage RNA was screened with a pro alpha 1(II) collagen specific chicken cDNA. A recombinant clone (Bc 7), with an insert of 1 kb, was identified and shown to contain sequences exhibiting 85% homology with the chicken pro alpha 1(II) collagen C-propeptide. Interspecies comparison strongly suggested that one potential glycosylation site present in the avian C-propeptide is not utilized, since this site is absent in the bovine chain. In addition, two overlapping genomic clones (Pal 3 and Pal 4) were isolated and partially characterized. These clones span 23 kb of DNA and contain approximately 17 kb of the pro alpha 1(II) calf gene. Sequencing of exon 1 has determined the length of the 3' untranslated region and the exact location of the polyadenylation attachment site.     

Isolation and characterization of genomic clones corresponding to the human type II procollagen gene.

A recombinant human DNA library was screened using probes corresponding to the chick alpha 1 (II) procollagen gene. This resulted in the isolation of 2 different genomic clones, LgHCol(II)a and LgHCol(II)b. LgHCol(II)a was identified as corresponding to the alpha 1(II) gene by comparative hybridization and DNA sequence analysis. DNA sequence established that LgHCol(II)a extends at least from amino acid 694 of the triple helix through 54 amino acids of the COOH-propeptide. Hybridization with a probe containing only the exon at the 3' end of the chicken gene suggests that the clone contains the 3' end of the human gene. Thus LgHCol(II)a contains approximately 40% of the coding sequences of the human type II collagen gene.     

Cloning and characterization of an alpha 1-antitrypsin like gene 12 KB downstream of the genuine alpha 1-antitrypsin gene

Cosmid clones containing alpha 1-antitrypsin (alpha 1AT) gene sequences were observed to contain alpha 1AT-like sequences approximately 12 kb downstream of the authentic alpha 1AT gene. Restriction mapping suggested the alpha 1AT-like gene lacks promoter sequences. Cosmid clones from one library contained a truncated alpha 1AT-like gene with a deletion encompassing 1745 bp, including the whole exon IV and part of exon V. Sequencing of exon II of this truncated gene revealed a nucleotide homology of 76% but included critical mutations in the start codon (ATG - greater than ATA) and the 3' exon-intron junction. These results strongly suggest that the truncated alpha 1AT-like gene is a pseudogene, which is present at a frequency of 0.30 in the Dutch population.     

Location of the 11 bp exon in the chicken pro alpha 2(I) collagen gene.

During the fine structural analysis of the 5' end of the 38 kb chicken pro alpha 2(I) collagen gene, we failed to locate an exon, only 11 bp in size, which had been predicted from the DNA sequence analysis of a cDNA clone complementary to the 5' end of the pro alpha 2(I) collagen mRNA (1). We know report the location of this 11 bp exon, exon 2, at the 5' end of a 180 bp Pst I fragment, 1900 bp 3' to exon 1 and 600 bp 5' to exon 3. Its sequence, ATGTGAGTGAG, is highly unusual in that it contains two overlapping consensus donor splice sequences. Moreover, it is flanked by two overlapping donor splice sequences but only one of the four splice sequences is actually spliced (1). The first half of intron 1 also has an unusual sequence: it is 68% GC, contains 88 CpG dinucleotides and 11 Hpa II sites. The second half is more like other intron sequences in the collagen gene with a GC content of 41%, 19 CpG, and no Hpa II sites. However it contains two sequences with 7 and 9 bp homology to the 14 bp SV40 enhancer core sequence. It is suggested that some part of intron 1 may be involved in regulation.     

Complete nucleotide sequence of the high molecular weight human IGF-I mRNA

IGF-I gene expression in mammals typically results in multiple mRNA species ranging in size between 0.7 and 7.6 kb. The smaller mRNA species have largely been characterized by the analysis of nearly full-length cDNAs. This report describes the first complete sequence of the prominent high molecular weight (7.6 kb) IGF-I mRNA species. Isolation and nucleotide sequence analysis of cDNA clones from human adult liver and uterus leiomyoma cDNA libraries resulted in a 7236 bp long sequence followed by a poly(A) tail. The sequence data, in combination with structural analysis of the human IGF-I gene, show that the 7.6 kb human IGF-I mRNA contains 6611 bp of untranslated 3' terminal sequence derived from a single exon. Alternate employment of two polyadenylation signals within the sequence transcribed from this exon generates two mRNAs of 1.1 and 7.6 kb.     

Identification of a mutation that causes exon skipping during collagen pre-mRNA splicing in an Ehlers-Danlos syndrome variant

Recent biochemical studies have shown that the fibroblasts from a patient with Ehlers-Danlos Syndrome Type VIIB produce nearly equal amounts of normal and shortened pro-alpha 2(I) collagen chains (Wirtz, M.K., Glanville, R. W., Steinmann, B., Rao, V. H., and Hollister, D. (1987) J. Biol. Chem. 262, 16376-16385). Compositional and sequencing studies of the abnormal pro-alpha 2(I) chain identified an interstitial deletion of 18 residues corresponding to the N-telopeptide of the collagen molecule. Since this region is encoded by a 54-base pair exon, number 6, the protein defect could have been caused by gene deletion, abnormal pre-mRNA splicing, or both. Here, in order to elucidate the molecular nature of this mutation we have analyzed the sequences of pro-alpha 2(I) collagen cDNA and genomic clones obtained from RNA and DNA of the patient's fibroblasts. Using oligomer-specific cloning we identified a cDNA that contains a 54-base pair deletion corresponding precisely to the sequence of exon 6. Identification of the normal gene was based on the finding of an identical sequence polymorphism in a normal cDNA and in the genomic clone derived from one of the two collagen alleles. The other gene, instead, displayed a base substitution (T to C) in the obligatory GT dinucleotide of the 5' splice-site sequence of intron 6. Analysis of nearly 100 base pairs immediately 5' to exons 5, 6, and 7, and 3' to exons 5 and 7 did not reveal any additional change. Therefore, the data strongly suggest that the observed GT-to-GC transition at the splice donor site of intron 6 generates an abnormally spliced mRNA in which the sequence of exon 5 is joined to the sequence of exon 7. Since skipping of exon 6 does not interfere with the coding frame of the mRNA, the resulting shortened polypeptide, albeit utilized in the assembly of a procollagen trimer, ultimately causes the Ehlers-Danlos Syndrome Type VII phenotype.     

Complete nucleotide sequence of a functional HLA-DP beta gene and the region between the DP beta 1 and DP alpha 1 genes: comparison of the 5'' ends of HLA class II genes.

The complete nucleotide sequence of an HLA-DP beta 1 gene and part of the adjacent DP alpha 1 gene, up to and including the signal sequence exon, were determined. The sequence of the DP beta 1 gene identified it as the DPw4 allele. The six exons of the DP beta 1 gene spanned over 11,000 bp of sequence. The arrangement of the gene was broadly analogous to genes of other class II beta chains. The beta 1 exon was flanked by introns of over 4 kb. Comparisons with published sequences of cDNA clones indicated that an alternative splice junction, at the 3' end of the gene, is used in at least one allele. Variation in choice of splice junction indicates an additional mechanism for allelic variation in class II genes. The sequence also indicated that the DP beta 1 and DP alpha 1 genes are separated by only 2 kb at their 5' ends. Comparison of the 5' ends of the DP alpha 1 and beta 1 genes with other class II sequences, including the DZ alpha gene, showed conservation of several blocks of sequences thought to be involved in control of expression. Some areas of the introns were partially conserved in the DQ beta gene, and several other intron sequences were homologous to sequences found in other unrelated genes.