Isolation and Translation in vitro of Four Related Vitellogenin mRNAs of Estrogen-Stimulated Xenopus laevis

Abstract

Cloning of vitellogenin c[complementary]DNA of X. laevis revealed that vitellogenin is encoded in a small family of genes represetnting 2 distantly related main groups A and B, each comprising 2 more closely related subgroups A1, A2, and B1, B2, respectively. To characterize the proteins derived from these genes the corresponding mRNA were isolated by hybridizing, under stringent conditions, cytoplasmic poly(A)-containing RNA from the liver of estrogen-stimulated X. laevis to filter-bound cDNA clones containing sequences specific for all 4 vitellogenin genes. Hybridization of the isolated mRNA with nick-translated cDNA clones revealed that contamination of the mRNA by those of the other main group was < 0.1%. Melting curves of the hybrids prepared with the isolated mRNA and cDNA clones specific for the 4 vitellogenin genes showed that the isolated vitellogenin mRNA are also specific for the 4 subgroups. Analysis of R loops formed between isolated mRNA and cDNA clones representing the corresponding subgroup further indicated about 10% cross-contamination between the more closely related mRNA. In a reticulocyte lysate each of the 4 mRNA coded for a 200,000 Mr (molecular ratio) protein immunoprecipitable by monospecific vitellogenin antibody. The 4 different mRNA (A1, A2, B1 and B2) which all can be isolated efficiently, code for vitellogenin and are expressed simultaneously in response to estrogen stimulation.