Xenopus Liver Research Articles

Structural and functional characterization and cloning of Xenopus FOSP-1 (frog oviduct-specific protein-1) genes.

As a model for tissue-specific gene expression, our laboratory has been studying the expression of vitellogenin and FOSP-1 (frog oviduct-specific protein-1) genes in Xenopus laevis which are expressed exclusively in the liver and oviduct, respectively, both strictly regulated by estrogen. Whereas the structure and function of Xenopus vitellogenin mRNAs and the upstream regulatory sequences (URS) of their genes are well established, little or no similar information is available for FOSP-1 genes. In this study, using a combination of 5' rapid amplification of cDNA ends (RACE) and reverse-transcriptase PCR, we have identified two gene copies of FOSP-1, termed FOSP-1A and FOSP-1B. Comparison of the sequences of full-length FOSP-1A and partial FOSP-1B cDNAs revealed a high degree of similarity at the 5' end. We next isolated FOSP-1A and FOSP-1B genomic clones. Dot-plot comparison of their URS showed both similarities and differences. Two estrogen-responsive elements (EREs), termed proximal (pERE) and distal (dERE), were identified at -1070/-1082 and -1167/-1179, respectively, in FOSP-1B, but not FOSP-1A, URS. Quantitative electrophoretic mobility shift assay (EMSA) and DNA footprinting with recombinant Xenopus estrogen receptor (xER) expressed in insect Sf9 cells, showed that xER interacted with a higher affinity with dERE than pERE in a hormone-independent manner, and that the two EREs do not act cooperatively. Functional studies involving transient transfection of human MCF-7 cells with a FOSP-1B URS-tkCAT construct confirmed that both EREs act as hormone-inducible cis-acting elements. These studies now pave the way for analysis of tissue specificity of estrogen-inducible gene expression in Xenopus liver and oviduct.

Purification and Characterization of an Estrogen-regulated Xenopus Liver Polysomal Nuclease Involved in the Selective Destabilization of Albumin mRNA

A previous report from this laboratory described an estrogen-regulated endoribonuclease activity on Xenopus liver polysomes which had properties one might expect for a messenger ribonuclease involved in the regulated destabilization of albumin mRNA (Pastori, R. L., Moskaitis, J. E., and Schoenberg, D. R. (1991) Biochemistry 30, 10490-10498). This report describes the purification and properties of this ribonuclease. The purified nuclease fraction contained a doublet of 62 and 64 kDa and a small amount of a 40-kDa peptide. In situ analysis on both denaturing and nondenaturing gels using an albumin transcript as substrate showed all three proteins possess nuclease activity. Peptide mapping and Western blot with a polyclonal antiserum showed the 62- and 64-kDa peptides to be isoforms, and the 40-kDa peptide to be a degradation product of the larger species. Two-dimensional gel electrophoresis further separated the 62- and 64-kDa species into three pairs of proteins, with isoelectric points of 9.6, 9.8, and 9.8. The purified ribonuclease rapidly degraded a full-length albumin transcript, yet had no effect on either a full-length albumin antisense transcript or full-length ferritin transcript. A number of properties of the purified nuclease were characterized, including the effects of salt, divalent cations, EDTA, sulfhydryl reagents, and temperature. Treatment of the polysomal nuclease with micrococcal nuclease had no effect, indicating that this enzyme does not require an RNA cofactor for activity. Finally, primer extension mapped the major cleavage site to an overlapping repeated sequence APyrUGA, with cleavage between and adjacent to the two pyrimidine residues generating fragments with 5'-hydroxyls.

The androgen receptor mRNA is up-regulated by testosterone in both the harderian gland and thumb pad of the frog, Rana esculenta

α32P-labelled cDNA probe from plasmid containing rat androgen receptor (rAR) has been tested in hybridization experiments using RNAs from the Harderian gland and thumb pad of the edible frog, Rana esculenta. Northern blot analysis has shown a high degree of homology between the rAR cDNA and the frog androgen receptor mRNA (fAR mRNA); this has been supported by both the hybridization conditions (high stringency) and the molecular size of fAR mRNA which is quite similar to those described in mammals (9.4 kb). The role of androgens has been further investigated with respect to the kinetics of expression of fAR mRNA in in vivo experiments. In both the Harderian gland and thumb pad, testosterone has increased the levels of fAR mRNA as compared with the untreated groups. The use of cyproterone acetate (CPA) in combination with testosterone has resulted in a loss of the increase in fAR mRNA as compared to testosterone-treated groups, while CPA alone has resembled the control group. In primary cultures of frog Harderian gland and thumb pad cells, the steady-state levels of fAR mRNA have been increased in the cells exposed to testosterone as compared to those not exposed. These findings confirm that, in these androgen target tissues, testosterone exerts an up-regulation on its own receptors, increasing the accumulation of fAR mRNA in the same way as oestrogens up-regulate the expression of their own receptors in Xenopus liver and oviduct cells.

Isolation of the Xenopus complement factor B complementary DNA and linkage of the gene to the frog MHC.

C factor B (Bf) is the key component of the C3 convertase of the alternative C pathway, and its gene resides in the class III region of the mammalian MHC. To elucidate the evolution of both the C system and the MHC, we isolated Bf cDNA clones from Xenopus laevis, an ectothermic vertebrate in which the MHC has been well defined at both the biochemical and functional levels. A part of the serine protease domain of the Xenopus Bf mRNA was amplified by reverse transcriptase-PCR, using degenerate primers corresponding to regions encoding the perfectly conserved amino acid sequences found in both the mouse Bf and C2 proteins. A full length Xenopus Bf cDNA clone was isolated from a Xenopus liver cDNA library. The deduced amino acid sequence of 747 residues showed the same domain structure as mammalian Bf and C2: three short consensus repeat domains, a von Willebrand domain and a serine protease domain. Xenopus Bf has 40% and 30% overall amino acid identity to mouse Bf and mouse C2, respectively. Because the amino acid identity between mouse Bf and mouse C2 is 38%, the gene duplication of Bf/C2 probably occurred before the divergence of amphibians and mammals. Southern blotting analysis of the Xenopus Bf gene showed a close linkage to the MHC, indicating that the Bf gene was linked to the class I and class II genes at the time Xenopus shared a common ancestor with mouse and man, 350 x 10(6) yr ago.

Cloning and Expression of a Xenopus Liver cDNA Encoding a Fructose‐Phosphate‐Insensitive Regulatory Protein of Glucokinase

Xenopus liver contains a protein inhibitor of glucokinase that, in contrast to the mammalian regulatory protein of glucokinase, is insensitive to fructose 6-phosphate and fructose 1-phosphate [Vandercammen A. & Van Schaftingen, E. (1993) Biochem. J. 294, 551-556]. The purpose of this work was to compare the primary structure and other properties of this Xenopus protein with those of its rat liver counterpart. A Xenopus laevis liver cDNA library was screened using the cDNA encoding the rat liver regulatory protein as a probe. The cloned cDNA was 2534 bp long and encoded a 619-amino-acid protein with a molecular mass of 68695 Da and 57% identity with the rat liver regulatory protein. This identity was only about 30% in an internal region (amino acids 349-381) and in the 70 carboxy terminal-residues. The Xenopus cDNA was expressed in Escherichia coli and the recombinant regulatory protein was purified to near homogeneity and found to have the same size, reactivity to antibodies and effects on the kinetics of glucokinase as the protein purified from Xenopus liver. In contrast to the rat liver regulatory protein, both recombinant and native Xenopus regulatory proteins were insensitive to fructose 6-phosphate, fructose 1-phosphate and to physiological concentrations of Pi, and they inhibited Xenopus glucokinase with greater affinity than rat glucokinase. These results allow one to conclude that the fructose-phosphate-insensitive protein of lower vertebrates is homologous to the fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive protein found in mammals.

Thyroid hormone-dependent differential regulation of multiple arginase genes during amphibian metamorphosis.

We have cloned three nonhepatic arginase genes in Xenopus laevis. The deduced amino acid sequences of the three arginases are almost identical and share about 60% identity with mammalian as well as Xenopus liver arginase. Both the liver and nonhepatic arginase genes are activated early during embryogenesis. The liver arginase gene is strongly expressed in tadpole liver, but weakly in other tissues. In contrast, the nonhepatic arginase genes have the strongest expression in the tadpole tail. During metamorphosis, the liver and nonhepatic arginase genes show distinct regulation patterns. In the intestine, both types of arginase genes are activated during the remodeling period. In the tail, the liver arginase gene is activated during tail resorption, whereas the nonhepatic ones are highly expressed throughout all stages examined. Finally, in the hindlimb, the liver arginase is up-regulated slightly during development, whereas the nonhepatic ones have low levels of expression until the end of metamorphosis. During 3,5,3'-L-triiodothyronine (T3)-induced metamorphosis, the nonhepatic arginase genes are activated very quickly, whereas the liver arginase gene is a late T3 response gene. These differential regulation patterns during normal and T3-induced metamorphosis suggest potential functions for the arginases during tissue remodeling.

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

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.

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.

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.

Identification of a novel endothelin receptor in Xenopus laevis liver

Membranes prepared from Xenopus liver displayed high density of high affinity endothelin (ET) binding sites. These sites have the same affinity for [ 125I] ET-1 and [ 125I] ET-3. Scatchard analysis of the specific binding from saturation binding experiments revealed an apparent dissociation constant (K d) of 93.1 and 70.9 pM and maximum binding (B max) of 602 and 651 fmol/mg protein for [ 125I] ET-1 and [ 125I] ET-3, respectively. Competition binding experiments using [ 125I] ET-1 and unlabelled ET-1, ET-3, S6c, and BQ123 indicated that ET-1 and ET-3 were the most potent in displacing [ 125I] ET-1 binding from these membranes (IC 501 and 0.3 nM, respectively), whereas S6c BQ123, selective for ET B and ET A receptors, respectively, did not have any inhibitory effect up to 1 μM. These data clearly indicate that the ET receptors present in Xenopus liver membranes belong to a new subtype of ET receptor. Because it resembled mammalian ET B receptors in its affinities for ET-1 and ET-3, we propose that this receptor be called the ET BX receptor.

Cloning of a new member of the peroxisome proliferator-activated receptor gene family from mouse liver.

Peroxisome proliferators are postulated to elicit predictable pleiotropic responses in the liver by activating a peroxisome proliferator-activated receptor (PPAR). PPARs from mouse liver (mPPAR), rat liver (rPPAR), and Xenopus liver (xPPAR gamma) have been cloned recently. We now report the cloning of a new member from mouse liver which we designate mPPAR gamma. mPPAR gamma cDNA contained an open reading frame encoding a 475-amino acid protein exhibiting 75% amino acid similarity to xPPAR gamma, while it showed only 55% identity with mPPAR. The ligand-binding and DNA-binding domains are best conserved between mPPAR gamma, mPPAR, and xPPAR gamma. Like rPPAR, mPPAR gamma is able to impart peroxisome proliferator responsiveness to the promoter of peroxisomal bifunctional gene, which encodes the second enzyme of the peroxisomal fatty acid beta-oxidation system. Northern blot analysis revealed high expression of mPPAR gamma gene in mouse liver, kidney, and heart and low expression in the lung, testis, brain, skeletal muscle, and spleen. In mice treated with ciprofibrate, a peroxisome proliferator, a 2-fold increase in mPPAR gamma mRNA was observed in the liver and kidney. The presence of two PPARs in the mouse liver suggests the possibility of multiple signaling pathways for the peroxisome proliferator-induced pleiotropic responses.

Thyroid hormone potentiates estrogen activation of vitellogenin genes

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 (T 3) on adult Xenopus liver cells. Low doses of T 3 rapidly (within 6–12 h) potentiate the activation of vitellogenin (Vit) genes by estradiol-17β (E 2) 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 T 3 also upregulates thyroid hormone receptor β, but not α, 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 T 3 was found to potentiate the transcription by E 2 from the transfected ERE, thus suggesting that it enhances the accumulation of functional ER. We conclude that T 3 can function in adult amphibian tissues, and discuss the significance of thyroid hormone potentiation of responses to estrogen in reproductive processes.

The Nuclease That Selectively Degrades Albumin mRNA in Vitro Associates with Xenopus Liver Polysomes through the 80S Ribosome Complex

A ribonuclease activity that has characteristics expected for an enzyme that catalyzes the regulated destabilization of serum protein-coding mRNAs following estrogen administration was previously identified on Xenopus liver polysomes. This enzyme activity is estrogen inducible and selectively degrades mRNAs (e.g., albumin, γ-fibrinogen) that are unstable following estrogen administration to male frogs. This paper reports on the relationship between this enzyme activity and the association of 40S and 60S ribosomal subunits. Ribonuclease activity (as defined by the generation of a specific cleavage fragment from albumin RNA) is found in polysome fractions that contain the majority of the liver mRNA. This activity sediments on sucrose gradients with the large polysome complexes observed in liver of vitellogenic animals. EDTA treatment generates 40S and 60S ribosome subunits and a significant amount of 80S ribosome monomers. Under these conditions, polysomal ribonuclease activity is found both free in solution and with the 80S material. Puromycin treatment generates predominantly 40S and 60S ribosomal subunits. Polysomal ribonuclease activity is found only in solution following puromycin treatment. These data indicate that the Xenopus liver polysomal nuclease requires the association of both ribosomal subunits for complex formation with polysomes. The polysomal nuclease behaves as a basic protein on Mono Q chromatography, with the fractionated material retaining the same differential activity toward albumin versus ferritin mRNA.

Molecular cloning and expression of a metallothionein mRNA in Xenopus laevis.

Metallothioneins (MT) are expressed during early development in species of several groups. To understand MT function in developmental processes, we studied the MT system of Xenopus laevis, a model vertebrate species in experimental embryology. We first purified and sequenced the liver MT from copper-treated animals. This 62-amino-acid protein shares the main structural properties of known vertebrate MT, and is more closely related to avian than to fish or mammalian MT. Using this sequence, we designed oligonucleotide primers to amplify and isolate a MT clone from a XL2 cell line cDNA library. This 752-bp cDNA encodes a putative 62-amino-acid-long protein that is 100% identical with the sequenced MT. Zinc, cadmium, and copper ions are very efficient inducers of MT mRNA accumulation in Xenopus liver and cell lines.

Attachment of Vitellogenin Genes to the Nucleoskeleton Accompanies Their Activation

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.

Developmental regulation and tissue distribution of the liver transcription factor LFB1 (HNF1) in Xenopus laevis.

The transcription factor LFB1 (HNF1) was initially identified as a regulator of liver-specific gene expression in mammals. It interacts with the promoter element HP1, which is functionally conserved between mammals and amphibians, suggesting that a homologous factor, XLFB1, also exists in Xenopus laevis. To study the role of LFB1 in early development, we isolated two groups of cDNAs coding for this factor from a Xenopus liver cDNA library by using a rat LFB1 cDNA probe. A comparison of the primary structures of the Xenopus and mammalian proteins shows that the myosin-like dimerization helix, the POU-A-related domain, the homeo-domain-related region, and the serine/threonine-rich activation domain are conserved between X. laevis and mammals, suggesting that all these features typical for LFB1 are essential for function. Using monoclonal antibodies, we demonstrate that XLFB1 is present not only in the liver but also in the stomach, intestine, colon, and kidney. In an analysis of the expression of XLFB1 in the developing Xenopus embryo, XLFB1 transcripts appear at the gastrula stage. The XLFB1 protein can be identified in regions of the embryo in which the liver diverticulum, stomach, gut, and pronephros are localized. The early appearance of XLFB1 expression during embryogenesis suggests that the tissue-specific transcription factor XLFB1 is involved in the determination and/or differentiation of specific cell types during organogenesis.

Differential regulation and polyadenylation of transferrin mRNA in Xenopus liver and oviduct

Estrogen destabilizes transferrin mRNA in male Xenopus liver in the same manner as observed for albumin and γ-fibrinogen. The present study examined estrogen regulation of transferrin gene expression in female Xenopus liver and oviduct. In female Xenopus liver estrogen causes the same enhanced degradation of transferrin mRNA from the cytoplasm as seen in males. In contrast, transferrin is induced 3- to 4-fold in both oviduct nuclear and cytoplasmic RNA. The similar increase in transferrin RNA in both preparations suggests a transcriptional mechanism is responsible for this stimulation. Therefore, transferrin expression is differentially regulated in these tissues by the same hormone. Previous experiments showed that Xenopus serum albumin mRNA has a very short (17 residue) poly(A) tail that may play a role in its hormone-regulated instability. Transferrin mRNA has a similarly short poly(A) tail in liver of both male and female Xenopus. Estrogen has no effect on transferrin polyadenylation in liver. Similarly short poly(A) is found on transferrin mRNA from estrogen-deprived oviducts in explant culture. However, addition of estradiol to the medium results in the appearance of a 50–200 nucleotide poly(A) concurrent with induction. Therefore, transferrin mRNA is differentially polyadenylated in Xenopus liver and oviduct. In the latter tissue polyadenylation is under hormonal control.

Characterization and developmental expression of Xenopus C/EBP gene

The Xenopus homolog of the transcription factor C / EBP (CCAAT / enhancer core binding protein), cloned from an adult Xenopus liver cDNA library, encodes a protein whose sequence is 67% homologous to that of rat C / EBP at the amino acid level, with virtually identical sequence of the basic-zipper region at the carboxyl terminus. As determined by gel electrophoretic mobility shift assays, the protein synthesized from XC / EBP cDNA bound specifically to the consensus binding site for C / EBP-like proteins. Northern blotting and RNase protection revealed a single species of xC / EBP mRNA of 2.7 kb which was most abundant in adult Xenopus liver, with smaller amounts in spleen, kidney, oviduct and brain and undetectable in heart and skeletal muscle. Although a small amount of this transcript could be detected in unfertilized eggs and early embryos, its accumulation rose sharply at the onset of metamorphosis (stage 55 / 56), and continued to increase through metamorphic climax to reach its highest level in stage 66 froglet liver, but thereafter declining in adult liver. In situ hybridization revealed a uniform pattern of distribution of xC / EBP mRNA in the liver and fat body throughout metamorphosis. Towards the end of metamorphosis, high levels of xC / EBP mRNA were detected in epithelial cells of the digestive tract. However, the spatial pattern of cells expressing the transcript changed markedly in the developing kidney. Our results suggest that xC / EBP may be involved as a transcription factor in the establishment of the adult phenotype during post-embryonic development of Xenopus.

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.

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.

Detection of fibrillarin in nucleolar remnants and the nucleolar matrix

In order to gain further insights into the fundamental structure of the nucleolus, nucleolar remnants of Xenopus and chickens were examined for the presence of fibrillarin and nucleolus organizer region (NOR) silver staining. Nucleolar remnants of Xenopus nucleated red blood cells were found to contain easily detectable amounts of fibrillarin and NOR silver staining. Upon examination of various tissues, fibrillarin and NOR silver staining were detected in nucleoli of Xenopus liver hepatocytes and within nucleoli of oocytes and follicle cells from ovaries of mature female toads. By comparison, nucleolar remnants of adult chicken nucleated red blood cells contained only trace amounts of fibrillarin and NOR silver staining, whereas red blood cell nucleolar remnants of immature chicks had easily detectable amounts of fibrillarin and NOR silver staining. Nucleoli from hepatocytes of both adult and immature chickens demonstrated comparable levels of fibrillarin and NOR silver staining. Since fibrillarin was found in nucleolar remnant structures, we tested for (and detected) its presence in residual nucleoli of in situ nuclear matrix derived from HeLa cells. These findings are discussed in terms of the basic structural and functional organization of the nucleolus.