Isolation and characterization of human elastin cDNAs, and age-associated variation in elastin gene expression in cultured skin fibroblasts

Abstract

In attempts to isolate human elastin cDNAs, a human placental lambda gt11 cDNA library was screened with a 1.3 kilobase sheep genomic DNA subclone, corresponding to the 3'-end of the elastin mRNA. The four largest clones, the largest being approximately 3 kilobase, were characterized by Northern transfer analyses, restriction endonuclease digestions and dideoxy nucleotide sequencing. Northern transfer analyses of poly(A)+RNA revealed hybridization to mRNA transcripts in the region of 3.5 kilobase. Restriction endonuclease mapping and nucleotide sequencing demonstrated distinct domains characteristic of elastin, and identified areas of variability which apparently reflects alternative splicing of the primary elastin transcripts. To demonstrate the utilization of these cDNAs for studies on elastin gene expression in human cells, elastin mRNA was examined in fibroblast cultures established from the skin of several individuals of varying ages. Northern transfer analyses and slot blot hybridizations demonstrated that elastin gene expression is initiated early during fetal development, and continues at a relatively constant level through several decades. The lowest abundance of elastin mRNA was noted in the cell cultures established for the oldest individual studied (61-year-old female). Demonstration of elastin gene expression in cultured fibroblasts provides a system to study diseases affecting the elastic fibers.