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.     

Estrogen induces tissue specific changes in the chromatin conformation of the vitellogenin genes in Xenopus.

Nuclei from male Xenopus liver were digested extensively with DNase I and the residual amount of the four vitellogenin genes measured by hybridization with a moderate excess of vitellogenin cDNA. The saturation value was about twofold lower in chromatin isolated from liver cells of estrogen treated than from untreated males or from erythrocytes. Analyzing the disappearance of several defined restriction fragments specific for the A1 and A2 vitellogenin genes, after limited digestion with DNase I, suggested that the entire A1 and A2 vitellogenin genes are about twofold more sensitive to DNase I in chromatin of hepatocytes isolated from estrogen treated than from untreated males. Using the same assay no change in the DNase I sensitivity of the two vitellogenin genes in erythrocyte chromatin was observed. Analysis of the beta 1-globin and an albumin gene demonstrated that the DNase I sensitivity of these genes in both cell types is not altered by estrogen. All these data indicate that estrogen stimulation results in an increased DNase I sensitivity specific for the vitellogenin genes in hepatocytes.     

Placement of small lipovitellin subunits within the vitellogenin precursor in Xenopus laevis

N-terminal amino acid sequence data for the small lipovitellin subunits in Xenopus laevis crystalline yolk platelets indicate that LV2 beta is derived from vitellogenin A2 and that LV2 tau is most likely derived from vitellogenin A1. The small lipovitellin subunits apparently commence within the exon 24 region of the parental vitellogenins, flanking the C-terminal end of phosvitin. As a consequence, we conclude that most of the vitellogenin sequence encoded by exons 30 to 35 is not accounted for by the known yolk proteins.     

Salt concentration-dependency of vitellogenin processing by cathepsin D in Xenopus laevis

The mechanism by which cathepsin D produces only limited proteolysis of vitellogenins (VTG) was studied in Xenopus oocytes. We first examined mature oocytes for the existence of cathepsin D; immunoblot and biochemical analyses revealed the existence of a 43kDa enzyme protein and its proteolytic activity in oocytes during and after the vitellogenesis. By determining the proteolytic activity of the fractions after subcellular fractionation of oocytes, we confirmed that cathepsin D is preserved in the yolk plasma of mature yolk platelets. The reaction of VTG with cathepsin D was examined in vitro at pH 5.6 as a function of NaCl concentrations. Lipovitellins generated from the VTG were preserved for several days at 37°C in the presence of the enzyme if the NaCl concentration was 0.15 mol/L or lower. The amount of lipovitellins decreased with increased molarity of the salt and at 0.5 mol/L NaCl they were rapidly degraded. The precipitates, growing in the reaction tube with 0.15 mol/L NaCl, included all constituents of yolk proteins and were ultrastructurally shown to have crystal structures perforated by empty cavities. No precipitates appeared at 0.5 mol/L NaCl. The results indicate that the limitation on proteolysis of the VTG by cathepsin D is due to the insolubility of yolk proteins at physiological salt concentrations, which explains why yolk can be stored stably in the presence of acid hydrolases over a long period.     

Cloning and characterization of synthetic sequences from the Xenopus laevis vitellogenin structural gene

The mRNA coding for vitellogenin, the yolk protein precursor, has been isolated from the liver of estrogen-stimulated Xenopus laevis. The mRNA has a size of 6.3 kilobases (kb). Optimal conditions were investigated for the synthesis of long complementary DNA (cDNA, referring to DNA synthesized in vitro) copies of the mRNA. Temperature, salt concentration, and enzyme-to-RNA ratio were important factors. Double-stranded cDNA with an average size of 2 to 3 kb was inserted into the vector pMB9 by the poly(dA:dT) method, and the recombinant plasmids were amplified in E. coli. Twenty-one clones with vitellogenin inserts ranging from 1 to 3.7 kb were studied. The regions in the RNA from which these clones had been derived were mapped by R-loop analysis in the electron microscope and by hybridization of the cloned DNAs with specific fractions of mRNA. Slightly more than half of the clones were derived from the 3′-terminal portions of the mRNA while the remaining clones are located internally.     

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.     

Microsomal triglyceride transfer protein promotes the secretion of Xenopus laevis vitellogenin A1

Vitellogenins (Vtg) are ancient lipid transport and storage proteins and members of the large lipid transfer protein (LLTP) gene family, which includes insect apolipophorin II/I, apolipoprotein B (apoB), and the microsomal triglyceride transfer protein (MTP). Lipidation of Vtg occurs at its site of synthesis in vertebrate liver, insect fat body, and nematode intestine; however, the mechanism of Vtg lipid acquisition is unknown. To explore whether Vtg biogenesis requires the apoB cofactor and LLTP family member, MTP, Vtg was expressed in COS cells with and without coexpression of the 97-kDa subunit of human MTP. Expression of Vtg alone gave rise to a approximately 220-kDa apoprotein, which was predominantly confined to an intracellular location. Coexpression of Vtg with human MTP enhanced Vtg secretion by 5-fold, without dramatically affecting its intracellular stability. A comparison of wild type and a triglyceride transfer-defective form of MTP revealed that both were capable of promoting Vtg secretion, whereas only wild type MTP could promote the secretion of apoB41 (amino-terminal 41% of apoB). These studies demonstrate that the biogenesis of Vtg is MTP-dependent and that MTP is the likely ancestral member of the LLTP gene family.