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

Abstract

Cloning of vitellogenin cDNA of Xenopus laevis revealed that vitellogenin is encoded in a small family of genes representing two distantly related main groups A and B, each comprising two more closely related subgroups A1, Az, and B1, BZ respectively. To characterize the proteins derived from these genes we have isolated the corresponding mRNAs by hybridizing, under stringent conditions, cytoplasmic poly(A)-containing RNA from the liver of estrogen-stimulated Xenopus to filter-bound cDNA clones containing sequences specific for all four vitellogenin genes.Hybridization of the isolated mRNAs with nick-translated cDNA clones revealed that contamination of the mRNAs by those of the other main group was less than 0.1 %. Melting curves of the hybrids prepared with the isolated mRNAs and cDNA clones specific for the four vitellogenin genes showed that the isolated vitellogenin mRNAs are also specific for the four subgroups. Analysis of R loops formed between isolated mRNAs and cDNA clones representing the corresponding subgroup further indicated about 10 % cross-contamination between the more closely related mRNAs. In a reticulocyte lysate each of the four mRNAs coded for a 200000-M, protein immunoprecipitable by monospecific vitellogenin antibody.From these results we conclude that the four different mRNAs A1, A2, B1 and Bz, which all can be isolated efficiently, code for vitellogenin and are expressed simultaneously in response to estrogen stimulation.Estrogen-dependent synthesis of vitellogenin, the precursor of the major egg-yolk proteins in the liver of Xenopus represents an attractive system of gene regulation by hormones, allowing reversible activation of a normally silent gene in male animals to produce a female protein (see review [l -31). Isolation and characterization of the vitellogenin mRNA as well as its accumulation in response to estrogen was reported by various laboratories [4,5]. Recently we demonstrated by restriction analysis and R-loop mapping of various clones of Xenopus vitellogenin cDNA that Xenopus vitellogenin mRNA is composed of four related RNAs [6]. Restriction analysis of genomic DNA demonstrated that all these RNAs are coded for by different genes [6].In our previous experiments we assumed that all these related RNAs are mRNAs and that all code for vitellogenin proteins. In this paper we now report on the isolation of the four vitellogenin mRNAs from poly(A)-containing RNA of estrogen-stimulated Xenopus and it is shown that each of these poly(A)-containing RNAs directs synthesis of a polypeptide in vitro which has about the same size as vitellogenin and is immunoprecipitable with vitellogenin antibody.Abbreviations. cDNA, complementary DNA; Hepes, 4-(2-hydroxyethy1)-1 -piperazineethanesulfonic acid; 1 x NaCl/Cit, 150 mM NaC1/15 mM trisodium citrate; pXlvc, cDNA clone designation : plasmid, Xenopus laevis, vitellogenin, cDNA.Definition. rot or cot product of [RNA] or [DNA] and time of hybridisation (M.s).

Vitellogenesis and choriogenesis in fishes

“…Recently, several Vtg gene transcripts were discovered in the African clawed frog (VtgA1, A2, B1, B2) [74,75] and chicken (Vtg I, II, III) [76,77], along with fish species such as mummichog [78,79], barfin flounder [64], haddock Melanogrammus aeglefinus, a species of cod [80], tilapia Oreochromis aureus [81] and sheepshead minnow Cyprinodon variegatus, an estuarine-species of pupfish [82]. Among fishes, a gene encoding a special form of Vtg lacking the Pv region (phosvitinless Vtg, Pv-less Vtg) was first discovered in zebrafish Danio rerio [83].…”

Quantitation of DNase I sensitivity in Xenopus chromatin containing active and inactive globin, albumin and vitellogenin genes

“…The hybridization solution contained 105 cpm/cm2 of 32P-labelled cloned cDNA nick translated to a specific activity of 1-4 x 108 cpm/lg (17). In all cases, only cloned cDNA which had been isolated from the recombinant plasmid following S1 nuclease or Pst I digestion (17) was used. After hybridization the filters were washed twice at 370 C for 1 h in 50% formamide, 5 x SSC (1 x SSC: 150 mM NaCl, 15 mM trisodium citrate), 0.2% SDS, 0.1% sodium pyrophosphate and 10 pg/ml denatured calf thymus DNA and then twice at 370 C for 30 min in 2 x SSC, 0.2% SDS, 0.1% sodium pyrophosphate and 10 pg/ml denatured calf thymus DNA and then stringently at 37°C for 30 min in 50% formamide, 0.1 x SSC and 0.2% SDS.…”

“…The DNA was dissolved in 2 M urea, 40% formamide, 30 mM Tris-HCl pH 8.5, 1 mM EDTA, 40 Pg/ml of cytochrome C and spread onto distilled water as described (14 (16). The hybridization solution contained 105 cpm/cm2 of 32P-labelled cloned cDNA nick translated to a specific activity of 1-4 x 108 cpm/lg (17). In all cases, only cloned cDNA which had been isolated from the recombinant plasmid following S1 nuclease or Pst I digestion (17) was used.…”

Isolation of two closely related vitellogenin genes, including their flanking regions, from a Xenopus laevis gene library.

“…vidual animals (10,11). Hybridization of cloned cDNA to Southern blots of uncloned genomic DNA digests showed that the different RNAs are transcribed from distinct genes (10).…”

“…The filters were dried for 1 hr at room temperature, treated with alkali, neutralized, and baked for 6 hr at 800C. The filters were preincubated at room temperature for [10][11][12][13][14][15] hr in 50% formamide/0.6 M NaCI/200 mM Tris-HCl, pH 8.0/20 mM EDTA/0.1% sodium pyrophosphate/5 X Denhardt solution (17)/250,gg of E. coli tRNA per ml/100 tg of sheared denatured salmon sperm DNA per ml/0.2% sodium dodecyl sulfate (4 ml per filter). Vitellogenin cDNA clones (9,10) were used as hybridization probes after nick translation to a specific activity of 0.5-1 X 108 cpm/,ug (18).…”