Cloning of the human α2-macroglobulin gene and detection of mutations in two functional domains: the bait region and the thiolester site

摘要: Overlapping genomic clones of the human α2-macroglobulin (α2M) gene were isolated from a cosmid library and used to map 80 kb chromosomal region this gene. Fragments carrying two exons encoding bait exon thiolester site partially sequenced PCR primers designed for amplification both functional domains. By direct sequencing these domains in 30 healthy individuals patients with chronic lung disease three mutations detected. The first was sequence polymorphism occurring near gene, changing Val1000(GTC) Ile1000(ATC), allele frequencies 0.30 (GTC) 0.70 (ATC), respectively. No difference α2M serum levels observed alleles. second mutation occured within one patient, Cys972(TGT) Tyr972(TAT). Since activation internal formed between Cys972 Gln975 each subunits tetrameric is involved covalent cross-linking activating proteinase, predicted interfere function. level normal range patient. In individual we detected an alteration sequence, which usually encoded by different separated intron size 1.6kb. individual, DNA using produced common fragment 1.8 additional variant 0.23kb. This finding, data indicate that he carries alleles gene: regular structure (bait I-intron-bait II), other fused into one. Direct useful tool detection genetic, possibly functional, heterogeneity α2M. This, turn, may provide some insight hitherto unknown physiological role(s) α2M, studying vivo effects naturally ocurring