Comparative analysis of Xenopus tropicalis and Xenopus laevis vitellogenin gene sequences.

Analysis of cDNA clones synthesized from vitellogenin mRNA of X. tropicalis revealed three different types of cDNA clones, i.e. A, A* and B. A and A* clones have a sequence divergence of about 6% and are both related to X. laevis vitellogenin cDNAs of subgroup A1 as well as A2 with a sequence divergence of 6-9%. B clones however, are related to X. laevis cDNA clones of subgroup B1 and B2 with a sequence divergence of about 7%. While the A and B clones correspond to vitellogenin mRNAs of similar abundance, A* clone is complementary to a vitellogenin mRNA about 100 fold less abundant than A and B mRNAs although all three vitellogenin mRNAs are encoded by single copy genes. Furthermore, two forms of A* mRNA were found. One of the two is lacking an internal fragment of about 900 bp. Since this DNA fragment is highly repeated in the genome, we suggest that this A* clone was synthesized from a processing intermediate of the A* precursor vitellogenin mRNA.

Four different vitellogenin proteins of Xenopus identified by translation in vitro.

Kinetic analysis of vitellogenin mRNA translation in a cell-free reticulocyte lysate translation system revealed that a serine-rich sequence, most probably containing the phosvitin molecule, is located toward the end of the translational product and therefore resides near to the carboxy terminus of the vitellogenin molecule. Translation of the four different vitellogenin mRNAs in vitro and cleavage of the translational products with cyanogen bromide revealed that vitellogenin consists of four different polypeptides, each containing a serine-rich sequence toward its carboxy terminus.

Isolation and translation in vitro of four related vitellogenin mRNAs of estrogen-stimulated Xenopus laevis.

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, A2, and B1, B2 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 200 000-Mr protein immunoprecipitable by monospecific vitellogenin antibody. From these results we conclude that the four different mRNAs A1, A2, B1 and B2, which all can be isolated efficiently, code for vitellogenin and are expressed simultaneously in response to estrogen stimulation.

Enrichment and characterization of the DNA coding for vitellogenin in Xenopus laevis.

Purified vitellogenin mRNA of Xenopus laevis was incubated with mechanically sheared DNA in high concentrations of formamide and the resulting R-loops (i.e. RNA . DNA hybrid fragments) separated from the bulk DNA by caesium chloride buoyant density centrifugation. Hybridization with 125I-labeled vitellogenin mRNA revealed a 15--30-fold enrichment of the DNA coding for vitellogenin. Restriction analysis of the R-loop-enriched DNA demonstrated that all known endonuclease HindIII fragments coding for vitellogenin of unfractionated Xenopus DNA were also present in the enriched material, including the specific fragments for the oligo(A)-containing segment of the RNA. Comparison of these restriction data with the structure found in cloned vitellogenin cDNA, indicates the presence of at least one intervening sequence in the genomic DNA coding for vitellogenin.

Vitellogenin in Xenopus laevis is encoded in a small family of genes.

Vitellogenin, the yolk protein precursor, is produced in X. laevis liver from a 6.3 kilobase (kb) mRNA. Sequences of this mRNA have been transcribed into cDNA and cloned in E. coli. Some properties of 21 of these cloned DNAs, ranging in size from 1 to 3.7 kb, have been reported by Wahli et al. (1978b). This paper reports restriction endonuclease mapping, cross hybridization, heteroduplex mapping in the electron microscope and heteroduplex melting experiments with these DNAs. We conclude that the cloned DNAs fall into two main groups of sequences which differ from each other in approximately 20% of their nucleotides. Each main group contains two subgroups which differ from each other by about 5% sequence divergence. By hybridizing cloned DNAs with restricted genomic DNA, we showed that sequences corresponding to all four sequence groups are present in a single animal. Furthermore, we have obtained tentative evidence for the presence of large intervening sequences in genomic vitellogenin DNA. Analysis of R loop molecules demonstrated that all four sequences are present in the vitellogenin mRNA population purified from individual animals. While some alternate explanations are not entirely excluded, we suggest that vitellogenin is encoded by a small family of related genes in Xenopus.