Xenopus liver ferritin H subunit: cDNA sequence and mRNA production in the liver following estrogen treatment

In vitro translation of liver mRNA from estrogen-treated Xenopus frogs yields two abundant polypeptides in the range of 20 kDa. DNA clones for one of these translation products were isolated and shown to be complementary to mRNA for the heavy subunit of ferritin. The predicted Xenopus amino acid sequence shares about 86% identity with the ferritin heavy chain from bullfrogs and about 70% identity with the comparable mammalian and avian proteins. Clone identity was confirmed by hybridization selection followed by in vitro translation into translation products of 19.5-20 kDa. The nearly full-length cDNA clone, termed XlferH1, comprises 868 nucleotides plus 22 adenosines of the poly(A) tail, including 134 nucleotides of the 5'-untranslated region, a 528-base coding region for 176 amino acids, and a 206-nucleotide 3'-untranslated region. The clone lacks 22 nucleotides from the 5' end of the mRNA. The level of ferritin mRNA in the liver of estrogen-treated frogs was determined over time. The amount of this mRNA relative to total RNA decreased about 3-fold 14 days after estradiol-17 beta was administered. However, the hormone also elevated total RNA in the liver about 24-fold. Hence, the total ferritin mRNA content of the liver increased to about 8 times its initial amount. This pattern of gene expression was very similar to that for serum retinol binding protein. The estrogen induction of these two mRNAs appeared to parallel the overall stimulation of hepatic RNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat liver glutathione S-transferases. Complete nucleotide sequence of a glutathione S-transferase mRNA and the regulation of the Ya, Yb, and Yc mRNAs by 3-methylcholanthrene and phenobarbital

With the use of cDNA probes reverse transcribed from purified glutathione S-transferase mRNA templates, four cDNA clones complementary to transferase mRNAs have been identified and characterized. Two clones, pGTB38 and pGTB34, have cDNA inserts of approximately 950 and 900 base pairs, respectively, and hybridize to a mRNA(s) whose size is approximately 980 nucleotides. In hybrid-select translation experiments, pGTB38 and pGTB34 select mRNAs specific for the Ya and Yc subunits of rat liver glutathione S-transferases. Clone pGTB33, which harbors a truncated cDNA insert, hybrid-selects only the Ya mRNA. All of the clones, pGTB38, pGTB34, and pGTB33, hybrid-select another mRNA which is specific for a polypeptide with an electrophoretic mobility slightly greater than the Ya subunit. The entire nucleotide sequence of the full length clone, pGTB38, has been determined and the complete amino acid sequence of the corresponding polypeptide has been deduced. The mRNA codes for a protein comprising 222 amino acids with Mr = 25,547. We have also identified a cDNA clone complementary to a Yb mRNA of the rat liver glutathione S-transferases. This clone, pGTA/C36, hybrid-selects only Yb mRNA(s) and hybridizes to a mRNA(s) whose size is approximately 1200 nucleotides. Although the Ya, Yb, and Yc mRNAs are elevated coordinately by phenobarbital and 3-methylcholanthrene, the Ya-Yc mRNAs are induced to a much greater extent compared to the Yb mRNA(s). These data suggest that the mRNAs for each transferase isozyme are regulated independently.     

Isolation and characterization of bone morphogenetic protein-binding proteins from the early Xenopus embryo.

Using a surface plasmon resonance biosensor as a sensitive and specific monitor, we have isolated two distinct bone morphogenetic protein (BMP)-binding proteins, and identified them as lipovitellin 1 and Ep45, respectively. Lipovitellin 1 is an egg yolk protein that is processed from vitellogenin. Both vitellogenin and Ep45 are synthesized under estrogen control in the liver, secreted, and taken up by developing oocytes. In this paper, we have shown that of the TGF-beta family members tested, Ep45 can bind only to BMP-4, whereas lipovitellin 1 can bind to both BMP-4 and activin A. Because of this difference in specificity, we have focused on and further studied Ep45. Kinetic parameters were determined by surface plasmon resonance studies and showed that Ep45 associated rapidly with BMP-4 (k(a) = 1.06 x 10(4) M(-1)s(-1)) and dissociated slowly (k(d) = 1.6 x 10(-4) s(-1)). In Xenopus embryos microinjected with Ep45 mRNA, Ep45 blocked the ability of follistatin to inhibit BMP activity and to induce a secondary body axis in a dose-dependent manner, whereas it had no effect on other BMP antagonists, chordin and noggin. These results support the possibility that Ep45 interacts with BMP to modulate its activities in vivo.     

Structure of Xenopus laevis ribosomal protein L32 and its expression during development.

cDNA clones for Xenopus laevis ribosomal protein L32 have been isolated and sequenced. The deduced amino acid sequence indicates that L32 is a basic protein of 110 amino acids, has a molecular weight of 12,603 and is homologous to the rat ribosomal protein L35. Using the cDNA clone as a probe to follow the expression of this gene during Xenopus development, it has been shown that the pattern of accumulation of this mRNA follows the one previously described for other ribosomal protein mRNAs during oogenesis and embryogenesis. The analysis of the utilization of L32 mRNA during embryogenesis shows that this is controlled by the translational regulation typical of other ribosomal protein mRNAs.     

Comparative nucleotide sequence analysis of two types of larval beta-globin mRNAs of Xenopus laevis.

The complete nucleotide sequence of the cDNA insert of the clone pXGL25 derived from the larval beta II-globin mRNA of Xenopus laevis has been determined. The sequence of 593 nucleotides represents part of the 5'nontranslated region, the coding region for 146 amino acids and the entire 3'nontranslated region. It diverges from the related larval beta I-sequence by 24.9% in the coding region. Alignment of the 5' and 3'nontranslated regions of the two related larval beta-sequences to maximum matching resulted in 31.2% and 46.7% divergence, respectively. Divergence between the corresponding adult and larval sequences considerably exceeds that of related larval sequences, suggesting that larval genes may have arisen by gene duplication prior to genome duplication. In contrast to mammalian beta-globin mRNAs, replacement and silent base substitutions are equally abundant, thus indicating less functional constraint on the larval Xenopus laevis beta-globin chains. The larval beta I- and beta II-globins diverge by 30.8% and show most variation in the alpha 1/beta 2-chain interaction sites.     

Pregnenolone 16 alpha-carbonitrile-inducible P-450 gene family: gene conversion and differential regulation.

A cytochrome P-450 cDNA clone, designated pP450PCN2, homologous to the previously characterized pregnenolone 16 alpha-carbonitrile (PCN)-induced P-450 cDNA (pP450PCN1; F. J. Gonzalez, D. W. Nebert, J. P. Hardwick, and C. B. Kasper, J. Biol. Chem. 260:7435-7441), was isolated from a rat liver cDNA expression library by use of a polyclonal anti-P450PCN1 antibody. This P-450 cDNA contains 2,014 base pairs and yields an open reading frame of a protein consisting of 504 amino acids (Mr = 57,760). P450PCN2 cDNA and protein shared 90% nucleotide and 89% amino acid similarity with P450PCN1 cDNA and protein, respectively. The 5' untranslated, coding, and 3' untranslated regions between the two cDNAs share 94, 93, and 79% similarities, respectively. Nucleotide differences in the coding regions, however, are not evenly distributed. Complete homology exists between the two mRNAs for 425 nucleotides (positions 346 through 771). Other regions of 93 nucleotides containing only one difference and 147 nucleotides containing two differences exist toward the 3' end of the coding regions. These data suggest the possibility that a gene conversion event(s) have occurred subsequent to duplication of the ancestral P450PCN gene. Oligonucleotide probes unique for P450PCN1 and P450PCN2 cDNAs were used to examine the levels of their respective mRNAs in noninduced and PCN-induced liver cells and in male and female rats of various ages. P450PCN1 mRNA was not detectable in either male or female rats at any ages. In contrast, P450PCN2 mRNA was present at a low level in newborn rats and became elevated in both males and females at 1 week of age. Levels of p450PCN2 mRNA continued to increase in males until 12 weeks, whereas the mRNA in females reached peak levels at 2 weeks of age but declined continuously at the onset of puberty (between 4 and 12 weeks). These levels of P45PCN2 mRNA closely parallel the increases in testosterone 6 beta-hydroxylase activity and P450PCN2 protein level, as analyzed by Western blots. P450PCN1 mRNA was induced by PCN, dexamethasone, and phenobarbital in both male and female rats. P450PCN2 mRNA was not significantly induced by PCN or dexamethasone but was readily induced by phenobarbital. Testosterone 6 beta-hydroxylase activity was also induced severalfold by PCN, dexamethasone, and phenobarbital. These data demonstrate that P450PCN1 and P450PCN2 genes are differentially regulated during development and after administration of inducing compounds and furthermore suggest that both enzymes possess testosterone 6 beta-hydroxylase activity.     

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

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

Molecular cloning of the cDNA for androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis

cDNA clones coding for two closely related androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis were selected by screening an epididymal cDNA library constructed in lambda gt 11 with affinity-purified antibody directed against the glycoproteins. The largest clone of 956 nucleotides provided coding information for a protein of 246 amino acids of which the first 19 residues comprise a putative signal peptide sequence which when cleaved would produce a mature protein of 227 residues and a molecular mass of 26 kDa. Confirmation of the identity of the clone was provided by a match between the amino acid sequence predicted from the cDNA sequence and the actual amino acid sequence determined for a tryptic peptide fragment of one of the pure glycoproteins. It is probable that the primary amino acid sequence of the two glycoproteins is identical. Northern blot and slot-blot analysis revealed that the mRNA for the glycoproteins is approximately 1250 nucleotides long and that the concentration of the mRNA in the epididymis is androgen-dependent. The glycoproteins and their mRNAs were unique to the epididymis as determined by Western and Northern blots, respectively, since signals were absent from skin, brain, liver, kidney, heart, skeletal muscle and testis. Cross-reacting proteins of slightly smaller apparent molecular mass were detected in extracts of mouse and guinea-pig epididymis, but not rabbit or bull epididymis. Comparison with existing protein data bases revealed that the epididymal glycoproteins display significant sequence homology with yeast carboxypeptidase Y.     

Molecular characterization of the interferon-induced 15-kDa protein. Molecular cloning and nucleotide and amino acid sequence

We have isolated a cDNA clone for an interferon-induced 15-kDa protein. The cDNA clone was prepared from mRNA isolated from interferon-beta-treated human Daudi cells. The clone of 635 base pairs contains an open reading frame coding for a protein of 145 amino acids, and suggests for the mRNA a 75-base pair 5' untranslated and a 125-base pair 3' untranslated region. Approximately 85% of the amino acid sequence of the 15-kDa protein has been independently obtained from 2 nmol of material using microsequencing technology on the N terminus of the intact protein and on tryptic and chymotryptic peptides. The amino acid sequence of the isolated protein is identical to the amino acid sequence deduced from the cDNA. Northern blot analysis confirmed that the mRNA for the 15-kDa protein is undetectable in untreated cells, but is greatly induced following interferon treatment.     

Rat liver glutathione S-transferases. Construction of a cDNA clone complementary to a Yc mRNA and prediction of the complete amino acid sequence of a Yc subunit

Using polysomal immunoselected rat liver glutathione S-transferase mRNAs, we have constructed cDNA clones using DNA polymerase I, RNase H, and Escherichia coli ligase (NAD+)-mediated second strand cDNA synthesis as described by Gubler and Hoffman (Gubler, U., and Hoffman, B. S. (1983) Gene 25, 263-269). Recombinant clone, pGTB42, contained a cDNA insert of 900 base pairs whose 3' end showed specificity for the Yc mRNA in hybrid-select translation experiments. The nucleotide sequence of pGTB42 has been determined, and the complete amino acid sequence of a Yc subunit has been deduced. The cDNA clone contains an open reading frame of 663 nucleotides encoding a polypeptide comprising 221 amino acids with a molecular weight of 25,322. The NH2-terminal sequence deduced from pGTB42 is in agreement with the first 39 amino acids determined for a Ya-Yc heterodimer by conventional protein-sequencing techniques. A comparison of the nucleotide sequence of pGTB42 with the sequence of a Ya clone, pGTB38, described previously by our laboratory (Pickett, C. B., Telakowski-Hopkins, C. A., Ding, G. J.-F., Argenbright, L., and Lu, A.Y.H. (1984) J. Biol. Chem. 259, 5182-5188) reveals a sequence homology of 66% over the same regions of both clones; however, the 5'- and 3'-untranslated regions of the Ya and Yc mRNAs are totally divergent in their sequences. The overall amino acid sequence homology between the Ya and Yc subunits is 68%, however, the NH2-terminal domain is more highly conserved than the middle or carboxyl-terminal domains. Our data suggest that the Ya and Yc subunits of the rat liver glutathione S-transferases are products of two different mRNAs which are derived from two related yet different genes.     

Characterization of a complementary deoxyribonucleic acid coding for human and bovine plasminogen

A cDNA library was constructed in pBR322 from bovine liver mRNA that was enriched for plasminogen mRNA by polysome immunoprecipitation. A 32P-labeled single-stranded cDNA was then prepared from the enriched bovine mRNA and employed as a probe to screen the cDNA library. The screening was carried out by testing for clones that protect the hybridized 32P-labeled cDNA from S1 nuclease digestion. The longest clone that was found was 581 base pairs in length and coded for the C-terminal 107 amino acids of bovine plasminogen, a 3' noncoding region of 246 nucleotides and a poly(A) tail. The bovine cDNA clone was then used as a probe to screen a human liver cDNA library of 18 000 recombinants. Six isolates were found to contain human plasminogen sequences. The longest clone consisted of 1851 base pairs corresponding to amino acid residues 272-790, followed by a 3' noncoding region of 227 base pairs and a poly(A) tail. Restriction fragments of the human cDNA were then used as probes to screen a human genomic DNA library present in a Charon 4A lambda phage library. Approximately 50 isolates from 10(6) recombinants were identified that hybridized to varying degrees with the cDNA probe. Among these, 10 corresponding to the gene for human plasminogen have been analyzed, and 3 that overlap have been shown to extend from kringle 3 through the 3' noncoding region of the gene. A 160 base pair exon with flanking splice junctions was then characterized and shown to encode for the first half of plasminogen kringle 4, including amino acid residues 346-399.     

The primary structure of human hemopexin deduced from cDNA sequence: evidence for internal, repeating homology.

We have cloned and analyzed a cDNA containing the coding sequence for human hemopexin. We have first identified, by immunological screening of 30.000 colonies of a liver cDNA library in the expression vector pEX1, a clone carrying an insert 1170 base pairs long that shows 100% homology with a known human hemopexin peptide. The complete sequence coding for hemopexin was isolated from a liver cDNA library in the vector pAT218. The DNA insert of 1523 base pairs shows an open reading frame coding for 439 amino acids, a 3' noncoding region of 159 nucleotides long, followed by a poly(A) tail. The insert spans the entire coding region and from which the primary structure of the protein was deduced. By computer assisted analysis of the amino acid sequence, it was possible to recognize a core unit, of about 45 amino acids, which is repeated 8 or possibly even 10 fold along the polypeptide chain. This feature suggests that the gene might have evolved through a series of duplications. This characteristic, together with prediction of secondary structure, suggest a rough model for the tridimensional folding that allows some speculations on the function of hemopexin. Blot hybridization of total RNA from human liver with nick translated hemopexin cDNA detected a message of about 1600 nucleotides. Southern blot experiments to identify the hemopexin gene (s) suggest that it is not a large multi-gene family, but that there is only one or at most a few genes in the human genome.     

Xenopus laevis serum albumins are encoded in two closely related genes.

cDNA clones containing sequences complementary to Xenopus laevis albumin mRNA have been identified in a collection of cDNA clones made from poly(A)+ RNA prepared from male Xenopus laevis liver. Although all the albumin cDNA clones crosshybridise, restriction enzyme and heteroduplex analysis show that there are 2 closely related albumin mRNA sequences. The 2 albumin mRNAs are only mismatched by 8% but could be isolated by positive selection using stringent hybridization conditions. Oocytes injected with the 2 purified mRNAs, secreted either the 68,000 or 74,000 dalton albumin into the culture medium showing that the 2 albumins of X. laevis serum are encoded in the 2 closely related mRNAs. Measurements of the abundance of albumin mRNA show that the 2 albumin mRNAs together account for about 9% of total poly(A)+ RNA in male Xenopus laevis liver but the mRNA coding for the 74,000 dalton mRNA is about twice as abundant as that coding for the 68,000 dalton mRNA.     

Coordinate estrogen-regulated instability of serum protein-coding messenger RNAs in Xenopus laevis

Estrogen causes the cytoplasmic destabilization of albumin and gamma-fibrinogen mRNA in Xenopus laevis liver. The purpose of the present study was to determine whether mRNA destabilization is a generalized phenomenon in response to estrogen, or whether this process is restricted to a particular class of mRNAs. To address this, we have expanded our bank of serum protein-coding cDNA clones to include transferrin, the second protein of inter-alpha-trypsin inhibitor and clone 12B, for which there is no mammalian homolog. Together with albumin and gamma-fibrinogen, these represent more than 85% of the mRNAs encoding liver secreted proteins. Estrogen administration to male Xenopus or to liver explant cultures causes the generalized disappearance of all of these mRNAs. In contrast, estrogen has no effect on actin, ferritin, or poly(A)-binding protein mRNA, all of which encode intracellular proteins. We have previously demonstrated that albumin mRNA is degraded in both messenger ribonucleoprotein and polysome fractions. Sucrose gradient analysis demonstrates the same pattern for degradation of all other serum protein-coding mRNAs. Estrogen has no effect on the amounts or gradient distribution of actin, ferritin, or poly(A)-binding protein mRNA. We conclude that regulated destabilization of mRNAs encoding secreted proteins is a generalized phenomenon in response to estrogen stimulation of Xenopus liver.     

Nucleotide sequence of cloned cDNA specific for rat ribosomal protein L35a

A cDNA clone specific for rat ribosomal protein L35a, which is known to be a tRNA-binding protein, was isolated by hybrid-selected translation from a cDNA library made for 8-9-S poly(A)-rich RNA from regenerating rat liver. The nucleotide sequence of the cDNA was determined. It consists of one base pair from the 5' leading sequence, the entire coding sequence of 333 base pairs and 14 base pairs from the 3' trailing sequence. The primary structure of protein L35a was deduced from the nucleotide sequence. It consists of 109 amino acids with a molecular mass of 12422. The calculated amino acid composition is consistent with that reported for the hydrolysate of L35a. The amino acid sequence showed marked homology with the reported partial sequence of Xenopus leavis ribosomal protein L32, but not significant homology with Escherichia coli ribosomal proteins that bind to tRNA.