44 citations found on Xenopus Vg mRNA

J Steroid Biochem Mol Biol 52 (6): 505-515 (1995)

Tissue distribution, hormone regulation and evidence for a human homologue of the estrogen-inducible Xenopus laevis vitellogenin mRNA binding protein.

Dodson RE, Acena MR, Shapiro DJ

Department of Biochemistry, University of Illinois at Urbana-Champaign 61801, USA.

17 beta-estradiol induces the synthesis of massive amounts of the hepatic mRNA encoding the Xenopus laevis egg yolk precursor protein, vitellogenin. Vitellogenin mRNA exhibits a half life of approx. 500 h when 17 beta-estradiol is present, and 16 h after removal of 17 beta-estradiol from the culture medium. We recently reported that Xenopus liver contains a protein, which is induced by 17 beta-estradiol and binds with a high degree of specificity to a binding site in a segment of the 3'-untranslated region (3'-UTR) of vitellogenin mRNA implicated in 17 beta-estradiol stabilization of vitellogenin mRNA. To determine if this mRNA binding protein was specific to this system, or if it was present elsewhere, and regulated by other steroids, we examined the tissue distribution and androgen regulation of this protein. Substantial amounts of the vitellogenin 3'-UTR binding protein were found in several Xenopus tissues including testis, ovary and muscle. In the absence of hormone treatment, lung and intestine contained minimal levels of the mRNA binding protein. Testosterone administration induced the vitellogenin 3'-UTR RNA binding protein in several tissues. Additionally, we found a homologous mRNA binding protein in MCF-7, human breast cancer cells. Although the MCF-7 cell protein was not induced by 17 beta-estradiol, the MCF-7 cell mRNA binding protein appears to be closely related to the Xenopus protein since: (i) the human and Xenopus proteins elicit gel shifted bands with the same electrophoretic mobility using the vitellogenin mRNA 3'-UTR binding site; (ii) The human and Xenopus proteins exhibit similar binding specificity for the vitellogenin 3'-UTR RNA binding site; and (iii) RNA from MCF-7 cells is at least as effective as RNA from control male Xenopus liver in blocking the binding of the Xenopus and human proteins to the vitellogenin mRNA 3'-UTR binding site. Its broad tissue distribution and regulation by both 17 beta-estradiol and testosterone suggests that this mRNA binding protein may play a significant role in steroid hormone regulation of mRNA metabolism in many vertebrate cells. 


Exp Cell Res 217 (2): 227-239 (1995)

Organization of centromeric domains in hepatocyte nuclei: rearrangement associated with de novo activation of the vitellogenin gene family in Xenopus laevis.

Janevski J, Park PC, De Boni U

Department of Physiology, Faculty of Medicine, University of Toronto, Ontario, Canada.

The existence of a function-dependent, nonrandom organization of chromatin domains within interphase nuclei is supported by evidence which suggests that specific chromatin domains undergo spatial rearrangement under conditions which alter gene expression. Exposure to estrogen of male Xenopus laevis hepatocytes in vitro results in de novo activation of vitellogenin mRNA production and vitellogenin protein synthesis and provides an ideal model to study the association between chromatin organization and changes in gene expression. In a test of the hypothesis that the de novo induction of vitellogenesis in male X. laevis is associated with a spatial rearrangement of specific chromatin domains, centromeric regions were localized by immunofluorescent labeling of associated kinetochore proteins in naive and in estrogen-treated, vitellogenic cells. Analyses by confocal scanning laser microscopy of the three-dimensional spatial distribution of kinetochores in estrogen-treated male hepatocytes showed that a significantly greater proportion of signals was associated with the nuclear periphery than in non-estrogen-treated, naive male cells. In hepatocyte nuclei, quantification of kinetochore signal sizes using image analysis showed that these signals were fewer in number and showed greater variation in size than those of cells in metaphase, with larger signals exhibiting total normalized fluorescence intensities of two, three, four, and five times that associated with kinetochore signals of metaphase cells. These observations are taken to reflect the existence of clustering of kinetochores and, by extension, of centromeres in these cells. In summary, the results show that centromeric domains within interphase nuclei of Xenopus hepatocytes occur as clusters and that these domains undergo spatial rearrangement under conditions which alter the transcriptional state of the cell. 


Mol Cell Endocrinol 105 (1): 45-53 (1994)

Thyroid hormone and glucocorticoid independently regulate the expression of estrogen receptor in male Xenopus liver cells.

Ulisse S, Tata JR

Laboratory of Developmental Biochemistry, National Institute for Medical Research, Mill Hill, London, UK.

Earlier studies from our laboratory had shown that triiodothyronine (T3) strongly potentiates the activation by estradiol (E2) of silent vitellogenin (Vit) genes and the autoinduction of estrogen receptor (ER) mRNA in primary cultures of male Xenopus hepatocytes (Rabelo and Tata, 1993). It was, however, not known if T3, or other hormones, could up-regulate ER mRNA in the absence of exogenous E2. We now show that T3 and dexamethasone (Dex), but not progesterone and testosterone, directly induce ER mRNA within 4 h by separate pathways, at doses compatible with the Kd values of their receptors. This induction of ER mRNA is accompanied by a marked enhancement of the activation of the silent Vit B1 gene if E2 is added by 12 h after T3 and Dex, thus suggesting an elevated level of functional ER induced by the two hormones. This conclusion was supported by a higher rate of transcription from an estrogen response element (ERE)-tk-CAT construct transfected into cultured hepatocytes pre-treated with T3 and Dex before incubation with estrogen. Our findings emphasize the importance of hormonal interplay via auto- and cross-regulation of nuclear hormone receptors. 


Mol Cell Biol 14 (5): 3130-3138 (1994)

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

Dodson RE, Shapiro DJ

Department of Biochemistry, University of Illinois, Urbana 61801.

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. 


Mech Dev 45 (1): 49-57 (1994)

Interplay between thyroid hormone and estrogen in modulating expression of their receptor and vitellogenin genes during Xenopus metamorphosis.

Rabelo EM, Baker BS, Tata JR

Laboratory of Developmental Biochemistry, National Institute for Medical Research, Mill Hill, London, UK.

Many postembryonic developmental processes are regulated by an intricate interplay among hormones and growth factors. Thyroid hormone (TH) and estrogen are well known to be individually and obligatorily required for the initiation and progression of amphibian metamorphosis and vitellogenesis. However, whether or not a possible interplay between these two hormones would affect these two developmental processes is not known. Here we report on how triiodothyronine (T3) enhances the precocious activation of vitellogenin (Vit) genes by estradiol (E2) in Xenopus tadpoles during metamorphosis. Using a combination of filter hybridization, RNase protection assay and in situ hybridization, we first show that very low doses (10(-9) M) of exogenous T3 will autoinduce thyroid hormone receptor (TR) mRNA in several tissues of premetamorphic tadpoles. The same treatment enhances and accelerates the precocious activation of the silent vitellogenin genes by E2 at metamorphic climax (stages 60-64) but not before mid-metamorphosis (stages 56-58). This developmental stage dependency may be explained by our finding that, under the same experimental conditions, T3 fails to alter the autoinduction of ER mRNA at mid-metamorphosis but strongly potentiates it at metamorphic climax. Thus a developmental stage specific interplay between thyroid hormone and estrogen determines the kinetics and extent of activation of vitellogenin and estrogen receptor genes during Xenopus postembryonic development. 


Mol Cell Endocrinol 96 (1-2): 37-44 (1993)

Thyroid hormone potentiates estrogen activation of vitellogenin genes and autoinduction of estrogen receptor in adult Xenopus hepatocytes.

Rabelo EM, Tata JR

Laboratory of Developmental Biochemistry, National Institute for Medical Research, Mill Hill, London, UK.

Although the important role of thyroid hormones in regulating metamorphosis of amphibian larvae is well known, it has not been clearly established if thyroid hormones have any function in the activities of adult amphibian tissues. We now describe a strong effect of 3,3',5-triiodothyronine (T3) on adult Xenopus liver cells. Low doses of T3 rapidly (within 6-12 h) potentiate the activation of vitellogenin (Vit) genes by estradiol-17 beta (E2) in primary cultures of adult male and female Xenopus hepatocytes. This effect is developmentally regulated and is first manifested during metamorphic climax. In an attempt to explain this potentiation, we find that T3 also upregulates thyroid hormone receptor beta, but not alpha, transcripts and rapidly enhances the autoinduction of estrogen receptor (ER) mRNA in adult Xenopus hepatocytes. In transient transfection of the Xenopus cell line XTC-2 with an estrogen response element--chloramphenicol transacetylase (ERE-CAT) construct T3 was found to potentiate the transcription by E2 from the transfected ERE, thus suggesting that it enhances the accumulation of functional ER. We conclude that T3 can function in adult amphibian tissues, and discuss the significance of thyroid hormone potentiation of responses to estrogen in reproductive processes. 


Biochem Biophys Res Commun 191 (1): 308-313 (1993)

Attachment of vitellogenin genes to the nucleoskeleton accompanies their activation.

Thorburn A, Knowland J

Department of Biochemistry, University of Oxford, England.

We have investigated the association of an inducible RNA polymerase II gene with the nucleoskeleton using the estrogen-inducible expression of the B2 vitellogenin gene in Xenopus liver as a model system. Using only physiological extraction conditions we find that the promoter region of the gene is strongly associated with the nucleoskeleton when it is transcriptionally active but much less so when it is inactive. We also find that the estrogen receptor protein, which is responsible for activation of this gene, is itself found associated with the nucleoskeleton. Finally, we show that newly synthesized, unspliced vitellogenin mRNA is also found on the nucleoskeleton. Our data suggest that expression of the B2 vitellogenin gene occurs only after it has become attached to the nucleoskeleton. 


J Biol Chem 267 (10): 7053-7059 (1992)

A major estrogen-regulated protein secreted from the liver of Xenopus laevis is a member of the serpin superfamily. Nucleotide sequence of cDNA and hormonal induction of mRNA.

Holland LJ, Suksang C, Wall AA, Roberts LR, Moser DR, Bhattacharya A

Department of Physiology, University of Missouri School of Medicine, Columbia 65212.

Estrogen treatment of Xenopus frogs causes four mRNAs to become highly abundant in the liver. Three of these mRNAs have been previously identified as coding for vitellogenin, ferritin, and serum retinol binding protein. We show here that the fourth abundant liver messenger RNA comprises about 1500 nucleotides and codes for a 45-kDa secreted protein, designated Ep45. A clone complementary to Ep45 mRNA was isolated, and its identity was confirmed by hybridization selection of mRNA that translated in vitro into the Ep45 precursor. Nucleotide sequence analysis of the nearly full length cDNA revealed a total length of 1454 base pairs consisting of: 36 nucleotides of the 5' noncoding region, 1308 base pairs encoding an open reading frame of 436 amino acids, and 110 nucleotides of the 3' untranslated region. Ep45 mRNA may originate from as many as four closely spaced transcription start sites, which are 15 to 21 bases upstream of the first nucleotide of the cDNA clone. The Xenopus laevis genome appears to contain a single Ep45 gene. The deduced amino acid sequence indicates that Ep45 has features typical of a secreted protein, including a signal peptide of 16 amino acids and three potential sites for N-linked glycosylation, and is related to the serine protease inhibitors, a large family of proteins with very diverse physiological functions. Ep45 mRNA was absent in the liver of normal male frogs and increased at least 100-fold in response to estradiol-17 beta. Thus, both Ep45 and vitellogenin mRNAs are switched from undetectable to very high levels, a pattern of expression not found for any other mRNAs in Xenopus liver. 


Mol Endocrinol 5 (2): 159-169 (1991)

A liver protein fraction regulating hormone-dependent in vitro transcription from the vitellogenin genes induces their expression in Xenopus oocytes.

Corthesy B, Corthesy-Theulaz I, Cardinaux JR, Wahli W

Institut de Biologie Animale, Universite de Lausanne, Switzerland.

Xenopus laevis oocytes were used to assay for trans-acting factors shown previously to be involved in the liver-specific regulation of the vitellogenin genes in vitro. To this end, crude liver nuclear extracts obtained from adult estrogen-induced Xenopus females were fractionated by heparin-Sepharose chromatography using successive elutions with 0.1, 0.35, 0.6, and 1.0 M KCl. When these four fractions were injected into oocytes, only the 0.6-M KCl protein fraction significantly stimulated mRNA synthesis from the endogenous B class vitellogenin genes. This same fraction induced estrogen-dependent in vitro transcription from the vitellogenin B1 promoter, suggesting that it contains at least a minimal set of basal transcription factors as well as two positive factors essential for vitellogenin in vitro transcription, i.e. the NF-I-like liver factor B and the estrogen receptor (ER). The presence of these two latter factors was determined by footprinting and gel retardation assays, respectively. In contrast, injection of an expression vector carrying the sequence encoding the ER was unable to activate transcription from the oocyte chromosomal vitellogenin genes. This suggests that the ER alone cannot overcome tissue-specific barriers and that one or several additional liver components participate in mediating tissue-specific expression of the vitellogenin genes. In this respect, we present evidence that the oocyte germinal vesicles contain an NF-I-like activity different from that found in hepatocytes of adult frogs. This observation might explain the lack of vitellogenin gene activation in oocytes injected with the ER cDNA only. 


Mol Cell Biol 10 (12): 6674-6682 (1990)

Activation of chromosomal vitellogenin genes in Xenopus oocytes by pure estrogen receptor and independent activation of albumin genes.

McKenzie EA, Cridland NA, Knowland J

Department of Biochemistry, Oxford, England.

We generate pure estrogen receptor protein in Xenopus oocytes by injecting them with estrogen receptor mRNA synthesized in vitro. A chromosomal vitellogenin gene, which normally responds to estrogen only in liver cells, is activated. Primer extension shows that initiation is accurate, and ribonuclease mapping shows that the first exon is correctly spliced out of the initial transcript. Long transcripts are produced, one being equal in length to poly(A)- vitellogenin mRNA. Immunochemical estimates of receptor levels in the oocyte nuclei suggest that pure receptor, acting alone, cannot activate oocyte vitellogenin genes unless unusually large amounts are present. However, when a receptor-free extract from liver cells is also injected, the amount of receptor required is reduced. Such an extract, but not pure receptor, can also activate albumin genes in oocytes. 


Mol Endocrinol 4 (6): 807-811 (1990)

High concentrations of estrogen stabilize vitellogenin mRNA against cytoplasmic degradation but physiological concentrations do not.

McKenzie EA, Knowland J

Department of Biochemistry, Oxford, United Kingdom.

Using DNA excess filter hybridization to pulse-labeled cellular RNA, we examined the stability of vitellogenin mRNA in Xenopus liver in relation to estrogen concentration. We showed that pharmacological concentrations of estrogen stabilize vitellogenin mRNA against degradation but that physiological concentrations do not. We concluded that there is little foundation for the common belief that estrogen stabilizes vitellogenin mRNA in normal liver cells and that such stabilization contributes to the normal expression of vitellogenin genes. We also discuss the importance of steroid concentration in other contexts, and show that the widespread tendency to use artificially high concentrations may lead to questionable conclusions. 


Nucleic Acids Res 17 (22): 9003-9014 (1989)

Ribosome loading, but not protein synthesis, is required for estrogen stabilization of Xenopus laevis vitellogenin mRNA.

Blume JE, Shapiro DJ

Department of Biochemistry, University of Illinois, Urbana 61801.

We have examined the effect of protein synthesis and of ribosome loading on the estrogen-mediated stabilization of hepatic Xenopus laevis vitellogenin mRNA. Removal of estradiol-17 beta from the culture medium, which destabilizes vitellogenin mRNA, does not alter the density of ribosomes on polysomal vitellogenin mRNA, or change the proportion of vitellogenin mRNA associated with the endoplasmic reticulum. Cycloheximide, which inhibits elongation, without changing the density of ribosomes on vitellogenin mRNA, does not block estrogen-mediated stabilization. In contrast, 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide, (MDMP), which inhibits initiation, greatly reduces the density of ribosomes on vitellogenin mRNA, and completely blocks estrogen-mediated stabilization. Vitellogenin mRNA in MDMP treated cells is degraded at a rate similar to that seen when untreated cells are transferred from medium containing estrogen to estrogen-free medium. This suggests that a ribosome-associated degradative system may not be responsible for vitellogenin mRNA degradation. The failure of estrogen to stabilize vitellogenin mRNA in MDMP-treated cells is not due to the release of vitellogenin mRNA from the endoplasmic reticulum. Vitellogenin mRNA in MDMP-treated cells remains associated with the endoplasmic reticulum in small polysomes containing 3-5 ribosomes. These data demonstrate that maintaining a high density of ribosomes on vitellogenin mRNA, but not continuing protein synthesis, is necessary for estrogen-mediated stabilization of vitellogenin mRNA. 


Mol Endocrinol 3 (10): 1596-1609 (1989)

Identification of two steroid-responsive promoters of different strength controlled by the same estrogen-responsive element in the 5'-end region of the Xenopus laevis vitellogenin gene A1.

Tremea F, Batistuzzo de Medeiros SR, ten Heggeler-Bordier B, Germond JE, Seiler-Tuyns A, Wahli W

Institut de Biologie Animale, Universite de Lausanne.

A structural and functional analysis of the 5'-end region of the Xenopus laevis vitellogenin gene A1 revealed two transcription initiation sites located 1.8 kilobases apart. A RNA polymerase II binding assay indicates that both promoters form initiation complexes efficiently. In vitro, using a transcription assay derived from a HeLa whole-cell extract, the upstream promoter is more than 10-fold stronger than the downstream one. In contrast, both promoters have a similar strength in a HeLa nuclear extract. In vivo, that is in estrogen-stimulated hepatocytes, it is the downstream promoter homologous to the one used by the other members of the vitellogenin gene family, which is 50-fold stronger than the upstream promoter. Thus, if functional vitellogenin mRNA results from this latter activity, it would contribute less than 1% to the synthesis of vitellogenin by fully induced Xenopus hepatocytes expressing the four vitellogenin genes. In contrast, both gene A1 promoters are silent in uninduced hepatocytes. Transfection experiments using the Xenopus cell line B3.2 in which estrogen-responsiveness has been introduced reveal that the strong downstream promoter is controlled by an estrogen responsive element (ERE) located 330 bp upstream of it. The upstream promoter can also be controlled by the same ERE. Since the region comprising the upstream promoter is flanked by a 200 base pair long inverted repeat with stretches of homology to other regions of the X. laevis genome, we speculate that it might have been inserted upstream of the vitellogenin gene A1 by a recombination event and consequently brought under control of the ERE lying 1.5 kilobases downstream. 


J Steroid Biochem 24 (6): 1141-1149 (1986)

Effects of antiestrogens on the induction of vitellogenin and its mRNA in Xenopus laevis.

Riegel AT, Jordan VC, Bain RR, Schoenberg DR

The egg yolk protein precursor vitellogenin is induced by estrogen in the liver of male Xenopus laevis. The large rise in serum vitellogenin is accompanied by a corresponding increase in intracellular levels of vitellogenin and its mRNA. In the present study this model system was used to examine the subcellular sites of action of triphenylethylene antiestrogens (e.g. tamoxifen). Tamoxifen was extensively metabolized to 4-hydroxytamoxifen in Xenopus and both of these antiestrogens were used in this study. Pre-injection with tamoxifen or 4-hydroxytamoxifen suppressed the estrogen-dependent induction of vitellogenin in serum. 4-Hydroxytamoxifen also inhibited the induction of intracellular vitellogenin and its mRNA by estrogen suggesting that this metabolite of tamoxifen is able to inhibit estrogen-induced transcription of the vitellogenin genes. Neither tamoxifen nor 4-hydroxytamoxifen stimulated the production of serum vitellogenin as assayed by a sensitive dot immunoblot assay. However either compound alone induced low amounts of vitellogenin mRNA and stimulated the production of intracellular vitellogenin to levels 10-40% of those produced by similar doses of estradiol. Since 10-40% of the serum levels of vitellogenin produced by estradiol would have been detected by the dot immunoblot assay, these data suggest that antiestrogens may have effects on post-translational processing or secretion of vitellogenin in addition to their effects on vitellogenin transcription. 


Mol Cell Endocrinol 39 (2): 91-98 (1985)

Coordinate estrogen induction of vitellogenin and a small serum protein mRNA in Xenopus laevis liver.

Hayward MA, Barton MC, Shapiro DJ

We have used plus-minus hybridization to identify Xenopus liver cDNA clones of mRNAs whose levels are regulated by estrogen. One clone identified in this way was shown to be a nearly full-length cDNA clone of the mRNA coding for a small 22 000 dalton estrogen-inducible serum protein (EISP). Quantitation of EISP mRNA levels by in vitro translation and by hybridization to the cloned DNA demonstrated a 7-12-fold estrogen induction of EISP mRNA, both in vivo and in primary Xenopus liver cultures. The kinetics of induction of EISP mRNA closely parallel those of the mRNA coding for the abundant estrogen-inducible serum protein, vitellogenin. In contrast, the massive, and toxic, estrogen-mediated accumulation of vitellogenin in serum of male Xenopus laevis is accompanied by a sharp decline in the levels of albumin mRNA and in the levels of the mRNAs coding for several other serum proteins. 


Comp Biochem Physiol [B] 82 (3): 497-505 (1985)

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

Baker BS, Steven J, Tata JR

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. 


Mol Cell Endocrinol 38 (2-3): 151-161 (1984)

Regulation by estrogen receptor of vitellogenin gene transcription in Xenopus hepatocyte cultures.

Perlman AJ, Wolffe AP, Champion J, Tata JR

We have used primary cell cultures of hepatocytes from male or female Xenopus laevis to study the mechanisms by which estrogen induces vitellogenin gene transcription and how primary exposure to estrogen renders cells more responsive to secondary stimulation. We have characterized the estrogen receptor in hormonally naive cells and in hepatocytes treated with estrogen under a variety of conditions. Under all conditions the receptor has a Kd congruent to 4 X 10(-10) M. Hormonally naive male cells contain 300 binding sites whereas female cells or male cells previously exposed to estradiol exhibit 6-7-fold higher levels. In parallel cultures, the absolute rate of vitellogenin gene transcription was determined by hybridization of newly synthesized RNA pulse-labelled with [3H]uridine to cloned Xenopus vitellogenin cDNA. Naive male cells on primary stimulation with estradiol synthesized vitellogenin mRNA at an average rate of approximately 150 moles/cell/h compared to 1200 moles/cell/h for cells previously exposed to estrogen, thus bearing a close correlation with receptor number. Furthermore, we show that the kinetics of the induced up-regulation of receptor exactly parallel those of the increase in the rate of vitellogenin gene transcription upon secondary hormonal stimulation following various periods of primary exposure to estrogen. Addition of cycloheximide to cell cultures during primary estrogen treatment abolishes both receptor up-regulation and increased rate of vitellogenin gene transcription on secondary stimulation. In addition, primary treatment with the antiestrogen tamoxifen prevents both receptor up-regulation and an enhanced rate of transcription or accumulation of vitellogenin mRNA on secondary hormonal exposure. These results demonstrate that estrogen treatment of male Xenopus hepatocytes results in the rapid up-regulation of its own receptor to female levels via new receptor synthesis, and that receptor number is rate-limiting in vitellogenin gene transcription. 


Dev Biol 102 (1): 238-247 (1984)

Unequal activation by estrogen of individual Xenopus vitellogenin genes during development.

Ng WC, Wolffe AP, Tata JR

Using a technique of filter hybridization under very stringent conditions to HindIII fragments of complementary DNA cloned in plasmids, we have measured the accumulation in hepatocytes of mRNA specified by each of the four vitellogenin genes (A1, A2, B1, B2) at different stages of development of Xenopus laevis. The ontogenic competence of embryonic liver to respond to the first exposure to estradiol-17 beta, in terms of activation of transcription of this multigene family, is acquired late in metamorphosis at around Nieuwkoop-Faber stage 58. Upon hormonal induction, the four mRNAs accumulate under non-steady-state conditions at different rates and to different extents at all developmental stages in vivo and in cultured adult hepatocytes. A1 and B1 mRNAs appear more rapidly and accumulate to levels that are five- to eightfold those specified by genes A2 and B2, with higher amounts of B1 than A1 mRNA. A threefold higher absolute rate of synthesis of A1 and B1 mRNAs in hepatocyte cultures, relative to the A2-B2 pair, suggests that hormonal regulation of differential accumulation of vitellogenin mRNA occurs at the transcriptional level. At the early developmental stages (up to stage 61) of acquired competence, there appears to be no fixed pattern of expression, but a pattern of unequal activation of individual genes of the Xenopus vitellogenin multigene family is established thereafter and then retained at all developmental stages of tadpoles, froglets, and in both male and female adults. 



Mol Cell Endocrinol 30 (3): 329-345 (1983)

Rapid estrogen metabolism and vitellogenin gene expression in Xenopus hepatocyte cultures.

Tenniswood MP, Searle PF, Wolffe AP, Tata JR

Male hepatocytes metabolized estradiol-17 beta, 17 alpha-ethinylestradiol and mestranol extremely rapidly (t 1/2 = 40, 60 and 300 min, respectively), whereas these were more stable in cultures of female hepatocytes (t 1/2 = 120, 150 and 640 min, respectively). Vitellogenin mRNA accumulated for only 12 h after a single addition of 10(-6) M estradiol to male hepatocyte cultures; mestranol, but not 17 alpha-ethinylestradiol or diethylstilbestrol, was more potent than the natural hormone. The level and rate of accumulation of vitellogenin mRNA were 5-15 times higher in female than in male hepatocytes, mestranol and estradiol being more potent than 17 alpha-ethinylestradiol and diethylstilbestrol. Ovariectomy, 60 days prior to cell culture, did not alter the metabolism of estradiol or the vitellogenic response of female hepatocytes. On the other hand, a single administration of estradiol in vivo to male Xenopus caused a long-lasting shift (at least 16 weeks) to the female pattern of its metabolism, although the enhanced inducibility of vitellogenin genes was partially reversed between 4 and 16 weeks after hormonal treatment. The addition of fresh estradiol every 4 h to male hepatocyte cultures to compensate for its rapid metabolism resulted in a continuous and sustained accumulation of vitellogenin mRNA at rates comparable to those attained in vivo. Our findings explain the requirement for high levels of estrogen to activate vitellogenin genes and establish Xenopus hepatocyte cultures as a reproducible system for analysing the expression of this multigene family. 


Nucleic Acids Res 11 (10): 2979-2997 (1983)

Vitellogenin B2 gene in Xenopus laevis: isolation, in vitro transcription and relation to other vitellogenin genes.

Germond JE, ten Heggeler B, Schubiger JL, Walker P, Westley B, Wahli W

The isolation of the four Xenopus laevis vitellogenin genes has been completed by the purification from a DNA library of the B2 gene together with its flanking sequences. The overlapping DNA fragments analyzed cover 34 kilobases. The B2 gene which has a length of 17.5 kilobases was characterized by heteroduplex and R-loop mapping in the electron microscope and by in vitro transcription in a HeLa whole-cell extract. Its structural organization is compared with that of the closely related B1 gene. The mRNA-coding sequence of about 6 kilobases is interrupted 34 times in the B1 gene and 33 times in the B2 gene. Sequence homology between the two genes was not only found in exons. In addition, 54% of the intron sequences as well as 63% and 48.5% respectively of the 5' and 3' flanking sequences, show enough homology to form stable duplexes. These findings are compared with earlier results obtained with the two other closely related members of the vitellogenin gene family, the A1 and the A2 genes. 


EMBO J 2 (6): 973-977 (1983)

Partial purification of estradiol receptor from Xenopus laevis liver and levels of receptor in relation to estradiol concentration.

Wright CV, Wright SC, Knowland J

We have used ammonium sulphate precipitation followed by affinity chromatography to partially purify the estrogen receptor from Xenopus laevis liver which may control the genes for vitellogenin, the precursor of the egg yolk proteins. The rate at which receptor binds estradiol explains the kinetics of the induction of vitellogenin synthesis by estradiol, and the dissociation constant (0.5 X 10(-9) M) explains the concentration dependence of the response, which has a threshold of 10(-9) M estradiol, when 67% of the receptor is bound to estradiol. The estradiol concentration in male liver, which does not make vitellogenin, is 0.18 X 10(-9) M, sufficient to saturate 26% of the receptor, while in female liver, which makes vitellogenin continuously, the estradiol concentration is 3.5 X 10(-9) M, giving 88% saturation of receptor, suggesting that the proportion of occupied receptor decides whether or not the vitellogenin genes are active. In the physiological concentration range, estradiol modulates the level of receptor, which varies between 100 binding sites per nucleus in males and 440 in females, but artificially high concentrations of estradiol raise the level to approximately 1000 sites per nucleus. This suggests that the small increase in vitellogenin mRNA induced by physiological concentrations of estradiol is due to pre-existing receptor and that the much larger increases induced by very high concentrations depends on newly-synthesized receptor. 


EMBO J 2 (12): 2271-2279 (1983)

Sequence homologies within the 5' end region of the estrogen-controlled vitellogenin gene in Xenopus and chicken.

Walker P, Brown-Luedi M, Germond JE, Wahli W, Meijlink FC, van het Schip AD, Roelink H, Gruber M, Ab G

In oviparous vertebrates vitellogenin, the precursor of the major yolk proteins, is synthesized in the liver of mature females under the control of estrogen. We have established the organization and primary structure of the 5' end region of the Xenopus laevis vitellogenin A2 gene and of the major chicken vitellogenin gene. The first three homologous exons have exactly the same length in both species, namely 53, 21 and 152 nucleotides, and present an overall sequence homology of 60%. In both species, the 5'-non-coding region of the vitellogenin mRNA measures only 13 nucleotides, nine of which are conserved. In contrast, the corresponding introns of the Xenopus and the chicken vitellogenin gene show no significant sequence homology. Within the 500 nucleotides preceding the 5' end of the genes, at least six blocks with sequence homologies of greater than 70% were detected. It remains to be demonstrated which of these conserved sequences, if any, are involved in the hormone-regulated expression of the vitellogenin genes. 


Nucleic Acids Res 10 (24): 8273-8284 (1982)

Activation of vitellogenin gene transcription is a direct response to estrogen in Xenopus laevis liver.

Hayward MA, Brock ML, Shapiro DJ

Estrogen induces the synthesis of vitellogenin mRNA by activating transcription of the vitellogenin genes. Quantitative inhibition of liver protein synthesis by cycloheximide does not prevent activation of vitellogenin gene transcription. The relative transcription rate of the vitellogenin genes in estrogen stimulated liver is similar in control and cycloheximide treated animals (800-1000 ppm). Selective estrogen activation of vitellogenin gene transcription therefore represents a direct effect of estrogen on vitellogenin gene transcription which can occur without any change in the cells' protein complement. Two other cellular responses to estrogen, the induction of nuclear estrogen receptor, and an increased rate of total nuclear RNA synthesis, are blocked by cycloheximide administration. Since the overall rate of vitellogenin mRNA synthesis is a function of both the selective estrogen activation of vitellogenin gene transcription which is not blocked by cycloheximide and the increased rate of total nuclear RNA synthesis which is blocked by cycloheximide, the total rate of vitellogenin mRNA synthesis is markedly reduced following cycloheximide administration. 


Am J Physiol 243 (1): C1-C6 (1982)

The role of estrogen receptor in Xenopus laevis vitellogenin gene expression.

Hayward MA, Brock ML, Shapiro DJ

Administration of estradiol 17 beta [estra-1,3,5(10)-triene-3,17-beta-diol] to male Xenopus laevis induces the massive synthesis by the liver of the egg yolk precursor phospholipoglycoprotein, vitellogenin, and its cognate mRNAs. Restimulation of male X. laevis that have been previously induced to synthesize vitellogenin mRNA but are inactive in vitellogenin mRNA synthesis at the time of restimulation with estrogen results in more rapid accumulation of vitellogenin mRNA and more efficient transcription of the vitellogenin genes than occurs following primary estrogen stimulation. The estrogen receptor system that mediates estrogen action in this organism exhibits several unusual properties. The cytoplasm of unstimulated liver cells contains high levels of a middle-affinity estrogen-specific binding protein and little if any estrogen receptor. The properties of the estrogen binding protein are consistent with a role in protecting estradiol 17 beta against metabolism, as a fraction of cytoplasmic estradiol 17 beta is not subject to rapid metabolism. In addition, similar binding activities are found in all Xenopus tissues surveyed that respond to steroid hormones. The induction of nuclear estrogen receptor is coincident with the onset of vitellogenin mRNA accumulation. However, an increased level of estrogen receptor is not responsible for the elevated rate of vitellogenin gene transcription observed following restimulation with estrogen. 


Nucleic Acids Res 10 (5): 1515-1533 (1982)

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

Jaggi RB, Wyler T, Ryffel GU

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. 


DNA 1 (4): 345-353 (1982)

Evolutionary conservation of vitellogenin genes.

James TC, Bond UM, Maack CA, Applebaum SW, Tata JR

Homologous and heterologous hybridizations in solution were performed between sheared genomic DNA and DNA complementary to vitellogenin mRNA of Xenopus, chicken, and migratory locust. The kinetics of hybridization and the thermal stability of the hybrids formed suggested a high degree of conservation of coding sequences of insect, amphibian, and avian vitellogenin genes. These cDNA probes hybridized to calf thymus DNA to a slight, but significant, extent, and not at all to Micrococcus lysodektikus DNA. DNA complementary to Xenopus albumin mRNA did not cross-hybridize significantly with locust or chicken DNA. Further evidence for the evolutionary conservation of vitellogenin genes was obtained from Southern blot analysis of restriction endonuclease-digested genomic DNA from a variety of vertebrate and invertebrate oviparous animals (Xenopus, chicken, migratory and desert locusts, yellow meal worm, carab moth, and Mediterranean fruitfly). When probed with cloned vitellogenin cDNAs from Xenopus and migratory locust, the DNA of these organisms showed varying degrees of homology of parts of the vitellogenin coding sequences. Southern blot analysis also showed that a part of the sequence specified in the cloned Xenopus vitellogenin cDNA was represented as repetitive DNA in the locust genome. However, cloned locust vitellogenin cDNA hybridized to discrete fragments of the restricted vertebrate DNA. These studies demonstrate a remarkably high degree of conservation of insect, amphibian, and avian vitellogenin genes. 


Cell 23 (3): 741-746 (1981)

Vitellogenin gene expression in male Xenopus hepatocytes during primary and secondary stimulation with estrogen in cell cultures.

Searle PF, Tata JR

Primary cultures of male Xenopus liver parenchymal cells that retained their competence to respond to estrogen were used to study the hormone-induced activation of the vitellogenin gene in vitro. The accumulation of vitellogenin mRNA in these cells was monitored by a quantitative diazotized paper disc hybridization procedure with a sensitivity of at least 6 pg of sequences complementary to the probe in total RNA samples of 10 micrograms. A short-term time-course analysis showed that vitellogenin mRNA was detectable within 3 hr of exposure to estrogen during primary stimulation, and that the maximum rate of accumulation was reached at 5--6 hr. A long-term time-course analysis of the accumulation of vitellogenin mRNA showed that it is possible to obtain a primary response, a hormone withdrawal effect and an enhanced secondary response in the same batch of cells in a manner analogous to that observed in vivo. Measurement of hormone concentration dependence showed a response at 10(-9) M estradiol, which continued to increase up to at least 10(-6) M estradiol. This requirement for large doses of estradiol for maximal response can be explained by the rapid metabolism of estradiol by the cultured cells. 


J Biol Chem 255 (23): 11308-11312 (1980)

Induction of estrogen receptor and reversal of the nuclear/cytoplasmic receptor ratio during vitellogenin synthesis and withdrawal in Xenopus laevis.

Hayward MA, Mitchell TA, Shapiro DJ

The levels of cytoplasmic and nuclear estrogen receptor have been determined in livers of male Xenopus laevis stimulated by estradiol-17 beta to synthesize vitellogenin mRNA. Estrogen receptor levels were also determined in unstimulated liver and following long term withdrawal of estrogen. In unstimulated liver cells, which do not contain detectable vitellogenin mRNA, more than 80% of the estrogen receptor is located in the nucleus (550 high affinity estrogen binding sites/nucleus), while the cytoplasm contains only 100 high affinity estrogen binding sites/cell. Administration of estradiol-17 beta, which induces massive synthesis and accumulation of vitellogenin mRNA, induces the estrogen receptor as well. The nuclear receptor level rises to approximately 2,000 estrogen binding sites/cell, while the cytosol receptor increases to only 150 sites/cel. Liver cells of male X. laevis which have been withdrawn from estrogen for 70 days exhibit a striking change in receptor levels. The nuclear receptor returns to the level prevailing in unstimulated cells (approximately 500 sites/cell) while the cytosol receptor level rises to more than 1,200 sites/cell (equivalent to 260 fmol/g of tissue). The existence of a pool of cytosol receptor, which is rapidly available for induction of vitellogenin mRNA, may in part explain the shorter lag period and more rapid induction of vitellogenin mRNA observed during secondary estrogen stimulation of withdrawn Xenopus liver cells. 


Eur J Biochem 109 (2): 343-347 (1980)

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

Jaggi RB, Felber BK, Maurhofer S, Weber R, Ryffel GU

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. 


Cell 20 (1): 107-117 (1980)

Comparative analysis of the structural organization of two closely related vitellogenin genes in X. laevis.

Wahli W, Dawid IB, Wyler T, Weber R, Ryffel GU

The structural organization of the two closely related vitellogenin genes A1 and A2 has been determined and compared by electron microscopy. In both genes the mRNA-coding sequence of 6 kb is interrupted 33 times, leading to a total gene length of 21 kb for gene A1 and 16 kb for gene A2. Thus both genes have a mean exon length of 0.175 kb, while the mean intron length is 0.45 kb in gene A1 and 0.31 kb in gene A2. Because the introns interrupt the structural sequence at homologous positions in genes A1 and A2, we suggest that these two genes are the products of a duplication of an ancestral gene which has an intron-exon arrangement similar to that of the extant genes. Since the duplication event, the sequence and length of the analogous introns have changed rapidly, whereas homologous exons have diverged to an extent of only 5% of their sequences. The results suggest different mechanisms of evolution for exons and introns. While the exons evolved primarily by point mutations, such mutations, as well as deletion, insertion and duplication events, were important in the evolution of the introns. 


Proc Natl Acad Sci U S A 77 (3): 1437-1441 (1980)

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

Wahli W, Dawid IB

A gene library of Xenopus laevis was constructed from embryonic DNA partially digested with restriction endonucleases Hae III and Alu I and joined to the phage lambda Charon 4 cloning vector with EcoRI linkers. Nucleotide sequences from three of the four related vitellogenin genes have been isolated. Two of the genes (called A1 and A2) were isolated in their entirety together with long stretches of flanking sequences. These two closely related vitellogenin genes have lengths of about 21 and 16 kilobases, but both produce a vitellogenin mRNA of 6.3 kilobases. 


Cell 19 (1): 53-61 (1980)

Identification, organization and processing intermediates of the putative precursors of Xenopus vitellogenin messenger RNA.

Ryffel GU, Wyler T, Muellener DB, Weber R

To understand the mechanism of estrogen-induced activation of the vitellogenin genes in the liver of Xenopus, it is essential to characterize the transcriptional products of these genes. In this paper we describe large nuclear RNAs containing vitellogenin mRNA sequences as revealed by hybridization of cloned vitellogenin cDNAs to nuclear RNA separated on agarose gels. Putative vitellogenin mRNA precursors, which are recovered as poly(A)-containing RNA, have been identified for the four known vitellogenin mRNAs. From electron microscopic analysis of R loops, prepared between enriched mRNA precursors and cDNA specific for the A1 vitellogenin mRNA, we conclude that the precursor molecules contain sequences complementary to vitellogenin mRNA which are interrupted by additional RNA segments probably representing transcribed introns. Within the 3.7 kb of the 3' end of the A1 vitellogenin mRNA we have discovered seven large and at least five small transcribed introns. Some of the R loops have been found to contain only a few transcribed introns, and we assume that they represent processing intermediates. Comparison of these putative intermediates suggests that the splicing order of different introns does not follow a single pathway. 


Eur J Biochem 99 (1): 23-29 (1979)

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

Widmer HJ, Jaggi RB, Weber R, Ryffel GU

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. 


Cell 16 (3): 535-549 (1979)

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

Wahli W, Dawid IB, Wyler T, Jaggi RB, Weber R, Ryffel GU

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. 


Mol Cell Endocrinol 12 (3): 237-246 (1978)

Synthesis of vitellogenin, an attractive model for investigating hormone-induced gene activation.

Ryffel GU

The estrogen-induced synthesis of vitellogenin in the frog Xenopus and the chicken is an attractive system for investigating the molecular events leading to the activation of a specific gene. In this review article the events occurring at the level of the protein and mRNA in the cytoplasm are discussed. The available data show that the induction of vitellogenin synthesis is due to the accumulation of the corresponding vitellogenin mRNA in the cytoplasm. This suggests that transcriptional or posttranscriptional events in the nucleus are activated by the hormone. The few experiments investigating the processes in the nuclear compartment are reviewed. 


Mol Cell Biochem 21 (3): 145-151 (1978)

Effects of estradiol-17beta in the male Xenopus laevis: isolation and translation of cytoplasmic messenger RNA populations.

Wiggins TL 3d, Tucciarone LM, Lanclos KD

Total Xenopus liver cytoplasmic RNA isolated following long-term estrogen administration (14 days) was fractioned using Sepharose 4B chromatography. One of the Sepharose 4B peaks was shown to contain RNA with a molecular weight reported for vitellogenin mRNA (approximately 34S). The presence of estrogen-induced vitellogenin mRNA in the peak 5 RNA was determined by translation of the RNA in the oocyte and analysis of the oocyte translational products by immunoprecipitation with anti-vitellogenin. Sepharose 4B peaks 2 and 3 were also observed to contain estrogen induced mRNA populations sedimenting between 9--18S. These findings suggest that Sepharose 4B chromatography might prove useful in separating different mRNA populations following estrogen-induced gene activation. 


Mol Cell Endocrinol 12 (2): 151-166 (1978)

Estradiol-induced accumulation of vitellogenin mRNA and secretion of vitellogenin in liver cultures of Xenopus.

Felber BK, Ryffel GU, Weber R

Explants of male Xenopus liver maintained in a serum-free culture medium respond to stimulation by 2 X 10(-8) M 17beta-estradiol with an increasing rate of accumulation of vitellogenin mRNA, as revealed by hybridization of cDNA to the total cytoplasmic RNA extracted from the cultures. A similar response is observed for secretion of 32PO4-labeled vitellogenin into the culture medium. The in vitro response is improved in liver tissue of prestimulated animals, and by adaptation of liver explants to the culture medium prior to hormone treatment, but attains only about 10% of the in vivo response. Since essential features of the in vivo response are maintained in liver explants, organ culture appears suitable for investigating initial events of estradiol action leading to enhanced synthesis of vitellogenin. 


J Biol Chem 253 (13): 4521-4524 (1978)

Rapid accumulation of vitellogenin messenger RNA during secondary estrogen stimulation of Xenopus laevis.

Baker HJ, Shapiro DJ

Accurate quantitation of low concentrations of vitellogenin mRNA by hybridization to vitellogenin cDNA allows analysis of the accumulation of new vitellogenin mRNA sequences throughout secondary estrogen stimulation. Administration of a secondary injection of estradiol-17 beta to male Xenopus laevis which have been withdrawn from estrogen for 60 days results in synthesis of complete vitellogenin mRNA molecules in as little as 1 h after restimulation. Vitellogenin mRNA accumulates at a rate of 13 molecules/cell/min--at least four times the rate observed in primary estrogen stimulation and peaks at a level twice that observed in primary stimulation. Administration of estrogen to male Xenopus laevis evokes stable long lived changes in the pattern of vitellogenin gene expression and constitutes a type of cellular "memory effect." 


Nature 273 (5661): 401-403 (1978)

Translation of Xenopus vitellogenin mRNA during primary and secondary induction.

Farmer SR, Henshaw EC, Berridge MV, Tata JR



Eur J Biochem 86 (1): 225-234 (1978)

Electron-microscopic demonstration of terminal and internal initiation sites for cDNA synthesis on vitellogenin mRNA.

Wahli W, Wyler T, Weber R, Ryffel GU

cDNA synthesized on purified vitellogenin mRNA from Xenopus liver was hybridized to the template in formamide/urea at 22 degrees C to avoid degradation of the RNA. The hybrids formed were visualized by spreading for electron microscopy. Contour length measurements proved that most of the RNA molecules in the hybrids were still intact showing the expected molecular weight of 2.3 x 10(6). The hybridized cDNA corresponded on the average to 12% of the RNA length. In about 80% of the molecules the cDNA was located at one end. Since cDNA synthesis was primed by oligo(dT), the terminal duplex region marks the 3' end of the vitellogenin mRNA molecule. Internal duplex regions were mainly located at a specific position starting about 2800 nucleotides from the 3' end. Since the cDNA hybridizing at the internal position could specifically be synthesized on a vitellogenin RNA fragment isolated on poly(U)-Sepharose as an oligo(A)-containing RNA, we conclude that cDNA synthesis is not only initiated by the poly(A) of the 3' end, but also by a specific internal sequence. 


Proc Natl Acad Sci U S A 74 (6): 2384-2388 (1977)

Regulation by estrogen of the vitellogenin gene.

Skipper JK, Hamilton TH

The vitellogenin gene is inactive in the liver of male Xenopus laevis, unless exogenous estrogen is administered. We have previously shown that conventional doses of estradiol-17beta result in the appearance of new hepatic messenger RNAs, some of which are encoded for vitellogenin. We now report that much higher doses of the hormone (2 mg/frog per day for 4 days) are required to elicit maximal responses. The relative levels of membrane-bound polysomes and vitellogenin mRNA were determined as a function of time and dose of hormone. Translation of total polysomal RNA in a cell-free system derived from wheat germ was used to estimate the relative levels of vitellogenin messenger RNA. Faithful translation of this messenger RNA was indicated by two lines of evidence: labeled cell-free products were immunoprecipitated with antivitellogenin antibody, and the migration of the major labeled product in sodium dodecyl sulfate/acrylamide gels was identical to that of native vitellogenin. Our results establish conditions for maximal estrogen-induced responses in this system, and are compatible with the hypothesis that a major regulatory mechanism of steroid hormones in the control of protein synthesis is that of gene activation and regulation of messenger RNA levels. 


J Biol Chem 251 (10): 3105-3111 (1976)

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

Shapiro DJ, Baker HJ, Stitt DT

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. 


Proc Natl Acad Sci U S A 72 (10): 3934-3938 (1975)

Translation of hormone-induced messenger RNA in amphibian oocytes: I. Induction by estrogen of messenger RNA encoded for vitellogenic protein in the liver of the male African clawed toad (Xenopus laevis).

Lanclos KD, Hamilton TH

Induction of the synthesis of the vitellogenic proteins, lipovitellin and phosvitin, in the liver of the male African clawed toad (Xenopus laevis) was investigated as a function of time after treatment with estradiol-17beta [1,3,5(10)-estratriene-3,17beta-diol]. The appearance of mRNAs encoded for lipovitellin and phosvitin in the cytoplasmic fraction of the liver was assayed by microinjections of hepatic mRNA preparation [either polyribosomes or poly(A)-rich RNA] into oocytes obtained from mature female toads. Oocytes were then incubated in the presence of radioactive amino acid(s) at 19 degrees for periods of time varying from 4 to 18 hr after microinjection. The results show that at 2 hr after hormone treatment more mRNA was present in the cytoplasm, and that from 2 to 72 hr after treatment the level of induced mRNA increased almost linearly to 110% above the control values. Experiments employing specific lipovitellin antiserum indicated no radioactive lipovitellin among the proteins synthesized in oocytes microinjected with hepatic mRNAs isolated from 3 to 9 hr after hormone treatment. However, a marked synthesis of immunoprecipitable, radioactive lipovitellin and an enhanced incorporation of [3H]serine occurred in the oocytes microinjected with hepatic mRNA preparations obtained from toads treated with hormone for 12 or more hr. The identities of the proteins encoded by the mRNAs induced early in estrogen action (2-9 hr) in the male amphibian liver are unknown. It is surmised that some of these proteins may function in the regulation of the subsequent synthesis of the vitellogenic proteins. 


Biochem J 150 (3): 345-355 (1975)

Differential subnuclear distribution of polyadenylate-rich ribonuclei acid during induction of egg-yolk protein synthesis in male Xenopus liver by oestradiol-17 beta.

Tata JR, Baker B

A 4-8-fold increase in the rate of hepatic nuclear RNA synthesis occurred within 11 h after a single injection of oestradiol-17 beta to male Xenopus to induce egg-yolk protein synthesis. 2. By using a gentle procedure for fractionating nuclei into their major structurally different components [J. R. Tata& B. Baker (1974) Exp. Cell Res. 83. 111-124], it was found that the hormone-induced increase in the total amount of newly made RNA was associated with a 2-10-fold increase in the poly(A) content of nuclear RNA. 3. When the poly (A) content of nuclear RNA was determined by hybridization to poly[3H](U) or specific binding to oligo(dT)-cellulose, most of the increase (10-fold) in poly (A) content of newly synthesized RNA was associated with the euchromatin fractions, whereas the increase was less marked in the other subnuclear fractions. 4. Resolution of nuclear RNA into poly (A)-poor and poly(A)-rich RNA species by chromatography on oligo(dT)-cellulose, followed by polyacrylamide-gel electrophoresis with sodium dodecyl sulphate or in the pressence of 99% formamide, revealed that the hormone caused a preferential enhancement of high-molecular-weight (25S-60S) poly (A)-rich HnRNA (heterogeneous nuclear RNA,) much of which was associated with euchromatin and not with the nuclear sap. 5. Induction of vitellogenin in male frogs was in particular characterized by the appearance of a high-molecular-weight polyadenylated component exhibiting a peak at 35-36S, i.e. a molecular weight of approx. 2.05x10(6)+/-0.15x10(6). Although there is no evidence as yet that such a polyadenylated high-molecular-weight nuclear RNA species contains sequences corresponding to vitellogenin mRNA, it is possible that a high proportion of the most stable form of the putative nuclear precursor to vitellogenin mRNA induced by oestrogen in male Xenopus liver may be only marginally bigger than the cytoplasmic mRNA, and may at any one time be predominantly associated with the euchromatin fraction.