Sequence of normal canine COL1A1 cDNA and identification of a heterozygous alpha1(I) collagen Gly208Ala mutation in a severe case of canine osteogenesis imperfecta
| Autor: | Joyce A.M. Wootton, Ronald R. Minor, Bonnie G. Campbell, James N. MacLeod |
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| Rok vydání: | 2001 |
| Předmět: |
DNA
Complementary Genotype Dentinogenesis imperfecta Sequence analysis Molecular Sequence Data Biophysics Glycine Biology Biochemistry Collagen Type I Dogs Complementary DNA Sequence Homology Nucleic Acid medicine T7 RNA polymerase Animals Humans Point Mutation Amino Acid Sequence skin and connective tissue diseases Molecular Biology Peptide sequence Polymerase Messenger RNA Alanine Base Sequence RNA Osteogenesis Imperfecta medicine.disease Molecular biology Collagen Type I alpha 1 Chain Disease Models Animal Phenotype biology.protein Collagen medicine.drug |
| Zdroj: | Archives of biochemistry and biophysics. 384(1) |
| ISSN: | 0003-9861 |
| Popis: | The sequence of canine COL1A1 cDNA was determined from four overlapping COL1A1 RT-PCR products generated from canine fibroblast RNA. In the translated region, nucleotide identity between canine and human COL1A1 cDNA was 93.2%, although the canine sequence lacked nucleotides 204 to 215 in the region coding for the N-propeptide. Amino acid identity was 97.7%. Total RNA and type I collagen were collected from cultured skin fibroblasts of a 12-week-old male golden retriever with pathologic fractures suggestive of osteogenesis imperfecta (OI) and dentinogenesis imperfecta. Sequential, overlapping approximately 1,000-bp fragments of COL1A1 and COL1A2 cDNA were each amplified by RT-PCR using primers containing 5' T7 polymerase sites. These PCR products were transcribed with T7 RNA polymerase, hybridized into RNA duplexes, and cleaved at mismatch sites with RNase. The proband had an unique cleavage pattern for the fragment of COL1A1 mRNA spanning nucleotides 709 to 1,531. Sequence analysis identified a G to C point mutation for nucleotide 1,276, predicting a codon change from glycine (GGA) to alanine (GCA) for amino acid 208. This change disrupts the normal Gly-X-Y pattern of the collagen triple helix. Restriction enzyme digestion of the RT-PCR product was consistent with a heterozygous COL1A1 mutation. Type I collagen was labeled with 3H-proline, salt precipitated, and analyzed by SDS-PAGE. Pepsin digested alpha chains were over-hydroxylated, and procollagen processing was delayed. Thus, canine and human OI appear homologous in terms of clinical presentation, etiology, and pathogenesis. |
| Databáze: | OpenAIRE |
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