A single base mutation in type I procollagen (COL1A1) that converts glycine alpha 1-541 to aspartate in a lethal variant of osteogenesis imperfecta: detection of the mutation with a carbodiimide reaction of DNA heteroduplexes and direct sequencing of products of the PCR
| Autor: | Jp, Zhuang, Constantinos Deltas, Ganguly A, Dj, Prockop |
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| Rok vydání: | 1991 |
| Předmět: |
Adult
Male Aspartic Acid Base Sequence Molecular Sequence Data Glycine Infant Newborn Nucleic Acid Heteroduplexes Nucleic Acid Hybridization Osteogenesis Imperfecta Polymerase Chain Reaction Carbodiimides Mutation Humans Female Genes Lethal Fetal Death Cells Cultured Infant Premature Procollagen Research Article |
| Zdroj: | Europe PubMed Central |
| ISSN: | 0002-9297 |
| Popis: | Skin fibroblasts from a proband with a lethal variant of osteogenesis imperfecta synthesized both apparently normal type I procollagen and a type I procollagen that had slow electrophoretic mobility because of posttranslational overmodifications. The thermal unfolding of the collagen molecules as assayed by protease digestion was about 2 degrees C lower than normal. It is surprising, however, that collagenase A and B fragments showed an essentially normal melting profile. Assay of cDNA heteroduplexes with a new technique involving carbodiimide modification indicated a mutation at about the codon for amino acid 550 of the alpha 1(I) chain. Subsequent amplification of the cDNA by the PCR and nucleotide sequencing revealed a single-base mutation that substituted an aspartate codon for glycine at position alpha 1-541 in the COL1A1 gene. The results here confirm previous indications that the effects of glycine substitutions in type I procollagen are highly position specific. They also demonstrate that a recently described technique for detecting single-base differences by carbodiimide modification of DNA heteroduplexes can be effectively employed to locate mutations in large genes. |
| Databáze: | OpenAIRE |
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