Characterization of cloned complementary DNA covering more than 6000 nucleotides (97%) of avian vitellogenin mRNA

The messenger RNA coding for chicken vitellogenin, a precursor of the egg-yolk proteins lipovitellin and phosvitin, is synthesized in the liver following estrogen injection. This mRNA is 6600 nucleotides long. We have previously reported the cloning and preliminary characterization of some cDNA fragments representing portions of the vitellogenin mRNA [Biochem. Biophys. Acta, 606, 34--46 (1980)]. In this paper we report the full characterization of a larger series of such clones, representing almost the entire length of the mRNA, by restriction endonuclease mapping, R-loop mapping, RNA-DNA hybridization and by translation in vitro of the RNA which hybridizes to the cloned DNA. From the results we conclude that the chicken vitellogenin mRNA, unlike that of Xenopus laevis, does not vary in sequence over most of its length, although some variations in the cDNA sequences were detected particularly in clones derived from the 3' terminus of the RNA. All sequence variants appear to be present in RNA prepared from single animals. The possible origins of these minor species are discussed. Furthermore, we describe a cDNA clone complementary to an mRNA which is about the same size as vitellogenin mRNA and which codes for an egg yolk protein antigenically related to lipovitellin. This mRNA is synthesized constitutively.     

Vitellogenin genes and their products in closely and distantly related species of Xenopus

Plasma vitellogenins from two closely related species of Xenopus, X. laevis and X. borealis, and a more ancient species, X. tropicalis, exhibited the same size on gel electrophoresis and were immunologically related. Partial peptide maps of 125I-labelled plasma vitellogenins, however, revealed marked differences in th structure and organisation of vitellogenin in the three Xenopus species. Northern blot hybridisation of liver RNA from oestrogen-treated males and females, probed with cloned vitellogenin cDNA, revealed the presence of mRNA of the same size in the three species of Xenopus, which was absent in untreated male liver. Cell-free translation of total liver RNA showed the presence of functional mRNA coding for vitellogenin subunit of the same size (Mr congruent to 210,000). Restriction endonuclease digestion patterns of genomic DNA from the three Xenopus species, using cloned X. laevis vitellogenin cDNA as the hybridisation probe, revealed significant differences in the organisation of these genes, which occur at a higher multiplicity in X. laevis and X. borealis than in X. tropicalis. Thus, despite a high degree of conservation of size, overall sequence and immunological identity of vitellogenin genes and their products in the three species of Xenopus, there is a substantial structural rearrangement during evolution of Xenopus within this multigene family.     

Characterisation of bacterial clones containing DNA sequences derived from Xenopus laevis vitellogenin mRNA.

A 1700 nucleotide DNA sequence derived from Xenopus vitellogenin mRNA has been cloned in the bacterial plasmid pBR322. The identity of the cloned sequence was verified in two ways. Firstly, the plasmid DNA was shown to hybridise to an RNA of the correct size (6,700 nucleotides). This was shown by in situ hybridisation to electrophoretically separated RNA and also by the formation of "R-loops" with purified vitellogenin mRNA. Then, using a novel procedure in which plasmid DNA covalently bound to diazotised paper is used to select complementary mRNA sequences, the cloned sequence was shown to hybridise to an mRNA which directed the synthesis of vitellogenin when translated in a reticulocyte lysate cell-free system.     

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.     

In vitro translation and estradiol-17beta induction of Xenopus laevis vitellogenin messenger RNA.

Administration of estradiol-17beta to male Xenopus laevis induces synthesis and secretion by the liver of the egg yolk precursor protein vitellogenin. RNA extracted from livers of estradiol-17beta-treated Xenopus laevis directs the synthesis of the entire 200,000-dalton vitellogenin monomer in a cell-free protein synthesizing system derived from rabbit reticulocytes. Vitellogenin synthesized in vitro was isolated and quantitated by indirect immunoprecipitation and identified by comparison to authentic [14C]vitellogenin. The in vitro product and [14C]vitellogenin co-migrate on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and they exhibit identical immunoprecipitation curves. Xenopus laevis vitellogenin messenger RNA has a sedimentation coefficient of approximately 30 S in sucrose density gradients. It can be purified approximately 60-fold from cell RNA by poly(U)-Sepharose chromatography and therefore appears to contain a polyadenylate sequence. Vitellogenin mRNA and vitellogenin synthesis in vivo could not be detected in unstimulated male Xenopus laevis. The relative rate of vitellogenin synthesis and the level of vitellogenin mRNA were determined at various times following the administration of estradiol-17beta. Vitellogenin synthesis is maximal 12 days after estradiol-17beta administration when it comprises approximately 70% of cell protein synthesis. The level of vitellogenin mRNA and the intracellular rate of vitellogenin synthesis exhibit a close correspondence from 4 to 16 days after administration of estradiol-17beta.     

Quantitation of vitellogenin messenger RNA in the liver of male xenopus toads during primary and secondary stimulation by estrogen

From livers of estrogen-stimulated female Xenopus toads, large quantities of estrogen-induced, poly(A)-containing RNA could be isolated, showing the same characteristics as vitellogenin mRNA obtained from hormone-treated males.Using cDNA hybridization, vitellogenin mRNA was monitored in the cytoplasmic poly(A)-containing RNA of the liver of male toads during 13 days of primary and the initial phase of secondary stimulation with estrogen.During primary stimulation, low amounts of vitellogenin mRNA, not exceeding 0.18% of the cytoplasmic poly(A)-containing RNA, were first detected after 12 hr of hormone treatment, and vitellogenin mRNA was found to increase on the average to 34% of the cytoplasmic poly(A)-containing RNA on the seventh day of hormone treatment. After 3 days of primary stimulation, accumulation of vitellogenin mRNA leveled off, showing no significant increase in the cytoplasm up to 13 days of hormone treatment. As judged from incorporation of ³²PO₄ into blood plasma proteins of males during primary stimulation, vitellogenin was first detected after 1 day, and its synthesis was found to increase dramatically until the thirteenth day of hormone treatment. This implies that there is a coincidence between appearance and extent of synthesis of vitellogenin and the abundance of vitellogenin mRNA in the cytoplasm, but there is evidence that during later phase of primary stimulation (day 3–13), the increase in synthesis of vitellogenin cannot be attributed anymore to a significant accumulation of vitellogenin mRNA.In male Xenopus, estrogen-induced synthesis of vitellogenin is no more detectable 41 days after hormone injection, and the concentration of vitellogenin mRNA was found to be <0.03% of the cytoplasmic poly(A)-containing RNA. Secondary stimulation by estrogen of these animals results in an at least 30 fold faster accumulation of vitellogenin mRNA in the cytoplasm within the initial 12 hr of hormone treatment. This may explain the faster appearance of vitellogenin in the blood plasma.     

Estradiol and estrogen receptor-dependent stabilization of a minivitellogenin mRNA lacking 5,100 nucleotides of coding sequence.

We have developed a transfection assay to investigate the estrogen-mediated stabilization of cytoplasmic vitellogenin mRNA. A minivitellogenin (MV5) gene containing the 5' and 3' untranslated and coding regions but lacking 5,075 nucleotides of internal coding sequence was constructed. Cotransfection of the MV5 plasmid and a Xenopus estrogen receptor expression plasmid into Xenopus liver tissue culture cells yielded a 529-nucleotide MV5 mRNA, which was specifically stabilized by estrogen. MV5 mRNA exhibited the increased stability indicative of positive regulation when the estradiol-estrogen receptor complex was present and was not destabilized by unliganded estrogen receptor. Transfected estrogen receptor, estradiol, and 529 nucleotides of the 5,604-nucleotide vitellogenin B1 mRNA were sufficient for stabilization.     

Construction and partial characterization of a recombinant DNA probe for locust vitellogenin messenger RNA.

Double-stranded DNA complementary to poly(A)-containing RNA from the fat body of adult female locusts, Locusta migratoria, was synthesized. Hybrid molecules containing this cDNA was constructed in the PstI site of the plasmid pAT 153 by the technique of dC . dG tailing and amplified in Escherichia coli K-12 strain HB 101. Ten colonies of bacteria were identified as carrying recombinant plasmids containing DNA complementary to locust vitellogenin mRNA by (a) 'Northern' blot hybridization analysis and (b) hybrid selection of vitellogenin mRNA and immunological detection of the products of translation of the mRNA. Of the ten recombinant plasmids, one, termed plasmid 4E, containing a cDNA insert of about 650 nucleotides, was characterized in greater detail and a partial restriction map obtained. Using this hybrid plasmid it was possible to derive a value for the average content of vitellogenin mRNA in the adult female locust fat body as 1.5 x 10(5) molecules/cell, and to establish that the haploid genome of L. migratoria contains only one or two genes coding for vitellogenin.     

Isolation and characterization of genomic clones covering the chicken vitellogenin gene

A series of overlapping recombinant clones, which cover the vitellogenin gene, has been isolated from a phage-lambda linked chicken gene library. The DNA of the overlapping clones spans 28 kb of contiguous DNA sequences in the chicken genome. Electron microscopic analysis of hybrids between vitellogenin mRNA and the genomic clones indicates that the chicken vitellogenin gene has a length of approximately 22 kb, about 3.8 times the size of the mRNA. The mRNA sequence is interrupted by at least 33 intervening sequences (introns). Comparison with the vitellogenin gene A2 from Xenopus laevis (Wahli et al., 1980, Cell 20: 107-117) indicates conservation of the number and length of the exons during evolution. Heteroduplex analysis reveals a short stretch of sequence homology between the genes from chicken and frog.     

An estrogen-inducible protein binds specifically to a sequence in the 3'' untranslated region of estrogen-stabilized vitellogenin mRNA.

The 3' untranslated region (3'-UTR) has been implicated in the estrogen stabilization of hepatic Xenopus laevis vitellogenin mRNA. We used RNA gel mobility shift assays to demonstrate that Xenopus liver contains a factor which binds with very high specificity to a segment of the 3'-UTR of vitellogenin B1 and B2 mRNAs. We detected a single high-affinity binding site in the vitellogenin mRNA 3'-UTR and localized the binding site to a 27-nucleotide region. Since binding was abolished by proteinase K digestion, at least a component of the factor is a protein. Following estrogen administration, binding was induced approximately four- to fivefold in extracts from liver polysomes. The hepatic vitellogenin mRNA-binding protein was found in both polysomes and cytosol. Since the protein was also estrogen inducible in cytosol, this represents a genuine induction, not simply recruitment of the cytosolic protein into polysomes. UV cross-linking studies with the 27-nucleotide recognition sequence revealed bands corresponding to bound proteins with apparent molecular weights of 71,000 and 141,000. This appears to be the first example of steroid hormone-inducible proteins binding to an mRNA 3'-UTR. Its induction by estrogen and its sequence-specific binding to a region of vitellogenin mRNA important in estrogen-mediated stabilization suggest that the protein may play a role in the regulation of mRNA stability.     

Isolation of globin messenger RNA of Xenopus laevis

The polypeptide chains of Xenopus laevis hemoglobin have been analyzed by sodium dodecyl sulfate (SDS) and acid-urea gel electrophoresis. Four components can be distinguished, each having an approximate molecular weight of 13,000 daltons. Messenger RNA coding for the globin chains has been isolated and characterized. In a denaturing acrylamide gel the mRNA has an approximate molecular weight of 250,000 daltons. The complexity of the RNA is consistent with the presence of four different mRNA molecules, each of this molecular weight. When the mRNA is assayed in a wheat germ in vitro translation system, four polypeptides are synthesized corresponding to the four globin subunits. The relative proportion of the four synthesized polypeptides appears to vary according to the developmental stage of the red blood cells used for mRNA isolation. Hybridization of a complementary DNA (cDNA) copy of the globin mRNA to Xenopus laevis DNA in DNA excess indicates that each of the globin genes is present in one to three copies per haploid genome.     

The Relationship of the Estrogen Receptor to the Induction of Vitellogenin in Chicken and Xenopus Liver

The mechanisms involved in the regulation of gene expression in eukaryote cells, although an area of active research, are still largely unknown. This is at least partly due to the lack of good experimental model systems. One type of system which is being exploited with some considerable success is the induction of proteins by steroid hormones. Studies on the effects of estrogen and progesterone on the synthesis of the egg white proteins in the chick oviduct, for instance, have yielded substantial insight into both the regulation of protein synthesis by steroid hormones [1] and the arrangement of the DNA sequences coding for these proteins [2, 3].
The need for other good inducible systems clearly exists and the induction of vitellogenin, the precursor of the major egg yolk proteins, by estrogen in the livers of the chicken and frog ( Xenopus laevis ) is one that is attracting increasing interest. In common with the chick oviduct, large amounts of a specific protein are synthesised in response to a well defined hormonal stimulus. However, the induction of vitellogenin also has the advantage that the response is not complicated by the extensive hyperplasia that follows estrogen treatment in the chick oviduct [4, 5] and that vitellogenin may be induced in vitro [6–11].
The aims of this review are first to discuss recent data on the induction of vitellogenin and vitellogenin mRNA both in vivo and in vitro and then to relate this data to the properties of the estrogen receptor, present in chicken and Xenopus liver, which is thought to mediate the induction of vitellogenin by estrogen.     

Quantitation of the accumulation of histone messenger RNA during oogenesis in Xenopus leavis

The accumulation of messenger RNA coding for histone H3 in oogenesis of Xenopus laevis was studied by quantitative hybridization techniques, using a cloned genomic DNA fragment as a probe. This probe was isolated from cloned Xenopus histone DNA and contains most of the H3 coding sequences. Histone H3 mRNA accumulation was found to be completed before the maximum lampbrush stage. Hybridization of RNA blots with DNA probes containing genes for histones H2A, H2B, and H4 suggests the same accumulation pattern for the mRNAs coding for these histones as for histone H3 mRNA. The amount of H3 mRNA in the mature oocyte was established to be 130 ± 68 pg, i.e., about 5 × 10⁸ copies.