Cloning and thyroid hormone regulation of albumin mRNA in Rana catesbeiana tadpole liver.

Thyroid hormones are responsible for the specific biochemical and structural changes that occur during amphibian metamorphosis. In this study we screened a series of cDNAs from a library constructed from T4-treated premetamorphic tadpole liver poly(A)+ RNA in order to identify a clone that could be used to study the influence of T3 on liver-specific gene expression during Rana catesbeiana metamorphosis. The cDNA from one clone exhibited a greater degree of hybridization to liver RNA from thyroid hormone-treated tadpoles than untreated tadpoles and no hybridization to RNA from tail fins of tadpoles of either group. On Northern blots, the mRNA to which the cDNA hybridized was 2.3 kilobases in size. The pattern of hybridization to genomic DNA digested by various restriction enzymes was consistent with the presence of a single gene. Using slot blot analysis we found that the mRNA levels first rose above basal levels only after 5 days of immersion of tadpoles in 12.5 micrograms/liter T3. The mRNA levels increased approximately 10-fold after 7 and 9 days of treatment. Frog livers had mRNA levels that were intermediate between those in untreated tadpoles and tadpoles immersed in T3 for 7 days. Sequence analysis revealed a significant degree of homology to serum albumin and alpha-fetoprotein. While it is known that serum albumin levels rise dramatically during metamorphosis in Rana species, presumably playing a critical role in maintaining water and electrolyte balance during the animals' terrestrial phase, the molecular basis of the induction has not been fully explained.     

In vitro synthesis of L-gulono-gamma-lactone oxidase by rabbit reticulocyte lysate

In vitro synthesis of L-gulono-gamma-lactone oxidase [L-gulono-gamma-lactone:oxygen 2-oxidoreductase, EC 1.1.3.8], one of the microsomal flavin enzymes, was performed with poly(A)+ RNA of rat liver using the rabbit reticulocyte lysate system in order to study the biosynthesis of the enzyme. The apparent molecular weight of the synthesized enzyme protein was almost the same as that of the purified L-gulono-gamma-lactone oxidase from rat liver. It was demonstrated that the enzyme protein was not detectable when guinea pig poly(A)+ RNA was used for the translation, indicating that the mRNA for the enzyme is absent in the guinea pig or, if it exists, is not translatable.     

Isolation of rat liver albumin messenger RNA.

Rat liver albumin messenger RNA has been purified to apparent homogeneity by means of polysome immunoprecipitation and poly(U)-Sepharose affinity chromatography. Specific polysomes synthesizing albumin were separated from total liver polysomes through a double antibody technique which allowed isolation of a specific immunoprecipitate. The albumin-polysome immunoprecipitate was dissolved in detergent and the polysomal RNA was separated from protein by sucrose gradient centrifugation. Albumin mRNA was then separated from ribosomal RNA by affinity chromatography through the binding of poly(U)-Sepharose to the polyadenylate 3' terminus of the mRNA. Pure albumin mRNA migrated as an 18 S peak on 85% formamide-containing linear sucrose gradients and as a 22 S peak on 2.5% polyacrylamide gels in sodium dodecyl sulfate. It coded for the translation of authentic liver albumin when added to a heterologous protein-synthesizing cell-free system derived from either rabbit reticulocyte lysates or wheat germ extracts. Translation analysis in reticulocyte lysates indicated that albumin polysomes were purified approximately 9-fold from total liver polysomes, and that albumin mRNA was purified approximately 74-fold from albumin polysomal RNA. The total translation product in the mRNA-dependent wheat germ system, upon addition of the pure mRNA, was identified as authentic albumin by means of gel electrophoresis and tryptic peptide chromatography.     

Cell-free synthesis of tumor-type poly(A) polymerase

Previous studies in this laboratory suggested that in adult liver, either the gene for the tumor-type poly(A) polymerase is poorly transcribed or the mRNA for this enzyme is largely not expressed. To test these possibilities, total RNA from rat liver and Morris hepatoma 3924A RNA were isolated by using a guanidine thiocyanate method; poly(A+) RNA and poly(A-) RNA were separated by oligo(dT)-cellulose chromatography and used for translation in a rabbit reticulocyte lysate system. After in vitro translation, the products were immunoprecipitated with either purified anti-tumor poly(A) polymerase antibodies or control immunoglobulins. When the polypeptides translated from poly(A+) or poly(A-) hepatoma RNA were precipitated with immune sera, a unique [35S]methionine-labeled 35-kilodalton (kDa) protein was observed. This band was not apparent when control serum was used for the immunoprecipitation. The radiolabeled 35-kDa polypeptide was not evident when the products were incubated with highly purified tumor nuclear poly(A) polymerase prior to immunoprecipitation. Prior incubation of the translation products with bovine serum albumin instead of poly(A) polymerase had no effect on the immunoprecipitation. This 35-kDa protein was not apparent when liver poly(A+) RNA was used to direct translation. These data demonstrate that (a) the tumor enzyme is not synthesized as a precursor, (b) tumor mRNA, but not normal liver mRNA, contains detectable sequences coding for tumor-type poly(A) polymerase, and (c) poly(A) polymerase mRNA also exists as a poly(A-) population.     

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.     

Synthesis of rat liver microsomal cytochrome b5 by free ribosomes

Free and membrane-bound polyribosomes were separated from liver homogenates and characterized by electron microscopy. Using the wheat germ cell-free translation system, total translation products of poly A+RNA extracted from free polyribosomes (poly A+RNAf) showed some correlation to total liver cytosol proteins. In contrast, translation products of poly A+RNA from membrane-bound polyribosomes (poly A+RNAmb) showed some similarity to rat serum. Antibody to purified rat serum albumin immunoprecipitated from only the translation products of poly A+RNAmb a single polypeptide of mol wt 68,000. i.e., 3,000 greater than secreted serum albumin. In contrast, antibody to detergent-extracted cytochrome b5 immunoprecipitated from only the translation products of poly A+RNAf a single polypeptide of mol wt 17,500, identical to that of microsomal cytochrome b5. A consideration of the known properties of cytochrome b5 is consistent with an exclusive site of synthesis on free ribosomes.     

Cloning and expression in Escherichia coli of cDNA for arginase of rat liver

A cDNA expression library constructed in a plasmid pUC8 from poly(A)+ RNA of rat liver was screened immunologically, using an antibody against arginase of rat liver. A cDNA clone was isolated and identified by hybrid-selected translation. The clone contained an insert approximately 1.35 kilobase pairs in length. In the bacterial clone, we detected a specific protein of Mr = about 43,000 that is slightly larger than the purified arginase (Mr = about 40,000) and a high activity of arginase was expressed. The arginase mRNA species of about 1600 bases long was detected in the liver, but not in the small intestine, kidney, spleen and heart of the rats.     

Cell free translation of rat epidermal calcium-binding protein (EP-12) messenger RNA

The primary step in the biosynthesis of 12 KDa rat epidermal calcium binding protein was studied by cell-free protein synthesis. Poly(A)+ rich RNA was extracted and purified from whole newborn rat skin and translated in a lysate system in the presence of labeled methionine. Immunoprecipitation of translation products with a monospecific antibody directed against this protein, which did not react with parvalbumin yielded a product migrating as a single band of molecular weight 12 KDa on polyacrylamide gel electrophoresis. Thus, a mRNA coding for this protein is present in rat skin. The presence of this messenger RNA opens the way for further studies on the regulation of epidermal expression during epidermal cell proliferation and differentiation.     

Effect of post-ischaemic recovery on albumin synthesis and relative amount of translatable albumin messenger RNA in rat liver.

In liver cells recovering from reversible ischaemia, total protein synthesis by postmitochondrial supernatant and membrane-bound and free polyribosomes is not different from that in sham-operated controls. However, the relative proportion of specific proteins is changed, since the incorporation of [3H]leucine in vivo into liver albumin, relative to incorporation into total protein, as determined by precipitation of labelled albumin with the specific antibody, decreases by 40-50% in post-ischaemic livers. Cell-free synthesis by membrane-bound polyribosomes and poly(A)-enriched RNA isolated from unfractionated liver homogenate shows that the decrease in albumin synthesis in liver of rats recovering from ischaemia is due to the relative decrease in translatable albumin mRNA.     

七星瓢虫卵黄原蛋白基因的表达mRNA的体外转译

七星瓢虫成熟雌虫脂肪体总RNA和poly(A)~+RNA中可转译mRNA的水平约为雄虫和不成熟雌虫的两倍,其中所含的卵黄原蛋白mRNA可在体外转译系统中指导卵黄原蛋白多肽的合成。 雌虫取食人工饲料时,其脂肪体RNA中可转译mRNA的水平很低,不能指导卵黄原蛋白多肽的合成。保幼激素类似物能诱导可转译卵黄原蛋白mRNA的出现。     

Hormonal regulation of epidermis-specific protein and messenger RNA synthesis in amphibian metamorphosis

Experiments using a monospecific antibody directed against one type of epidermis-specific keratin from adult skin of the amphibian Xenopus laevis have demonstrated that polysomes synthesizing this protein first appear within larval skin during natural metamorphosis. Further experiments demonstrated that the synthesis of keratin within larval skin could be induced precociously by the thyroid hormone, 3,3′,5-triiodo-l-thyronine, both in vivo and when the isolated larval skin is cultured in vitro. The earliest developmental age responsive to such hormone induction appeared to be Stage $\text{50}\text{52}$ of larval development. This is about 20–24 days before keratin would normally make its appearance within the skin during natural metamorphosis. Hormone treatment of tadpoles at this age will also cause a precocious increase in the amount of keratin messenger RNA present within larval skin. This has been demonstrated directly by the isolation of poly(A)-containing messenger RNA from hormone-treated larvae and its translation in a wheat germ cell-free system to give immunoprecipitable keratin. Peptide analysis of the in vitro translation product indicates that the hormone-induced mRNA probably codes for an initial protein product that is slightly larger than keratin itself.     

Molecular cloning of hepatic mRNAs in Rana catesbeiana responsive to thyroid hormone during induced and spontaneous metamorphosis

Amphibian metamorphosis affords a useful experimental system in which to study thyroid hormone regulation of gene expression during postembryonic vertebrate development. In order to isolate gene-specific cDNA probes which correspond to thyroid hormone-responsive mRNAs, we employed differential colony hybridization of a cDNA library constructed from poly(A)+ RNA of thyroxine-treated premetamorphic tadpole liver. From an initial screening of about 6000 transformants, 32 "potentially positive" colonies were obtained. The recombinant cDNA-plasmids from 13 of these colonies plus two "potentially negative" colonies were purified for further study. Southern blot analysis of the plasmid DNA was employed to determine whether different cDNAs encoded for the same mRNA. The effect of thyroid hormone on the relative levels of specific mRNA species was examined by Northern analysis of liver RNA from premetamorphic tadpoles, thyroxine-treated tadpoles, and adult bullfrogs. Three independent cDNA clones were obtained which encoded thyroid hormone-enhanced mRNAs. We also obtained two independent cDNA clones encoding thyroid hormone-inhibited mRNAs and three independent clones encoding thyroid hormone-unresponsive mRNAs. The levels of two thyroid hormone-enhanced mRNAs and one thyroid hormone-inhibited mRNA were essentially the same in the thyroid hormone-treated tadpole liver and adult liver, suggesting that thyroid hormone induces stable changes in liver gene expression during spontaneous metamorphosis. Using selected cDNAs, RNA dot blot analysis of liver mRNA from tadpoles at different stages of metamorphosis showed that the level of one thyroid hormone-enhanced mRNA increased during late prometamorphosis and metamorphic climax. Similarly, a mRNA which was strongly inhibited by thyroid hormone treatment was observed to decline during prometamorphosis and reach undetectable levels during metamorphic climax. One mRNA was detected which was reproducibly inhibited by thyroid hormone treatment but which remained essentially unchanged during spontaneous metamorphosis. These results provide the first direct evidence for the coordinate and selective pretranslational regulation by thyroid hormone of several liver genes during the developmental process of metamorphosis.     

Regulation of c-erbA-alpha messenger RNA species in tadpole erythrocytes by thyroid hormone.

Putative thyroid hormone (TH) nuclear receptors have been detected in several tissues of Rana catesbeiana tadpoles. T3 receptor number (sites per nucleus) in red blood cells (RBCs) and tail increases substantially just before metamorphic climax or in response to exogenous TH; in contrast, receptor number in liver remains relatively constant. TH receptors in mammals and birds are thought to be encoded by a c-erbA gene. In the present study, two c-erbA cDNAs, one prepared from Xenopus laevis oocytes (XenTR alpha 1) and one prepared from Rana catesbeiana tail (RC12), were used to examine the c-erbA-related mRNA species in Rana catesbeiana tissues and determine their role in the TH induction of tadpole RBC receptor number. XenTR alpha 1 encodes a protein with T3-binding properties typical of TH receptors. RC12 is almost 99% homologous with XenTR alpha 1 at the amino acid level and contains all of the putative T3-binding region and most of the DNA-binding region. Using either cDNA as a probe, it was found that two major species of c-erbA-related mRNA species (2.6 and 4.0 kilobases) were clearly evident in tadpole RBCs, tail, and liver. A third, more diffuse band (approximately 5.0 kilobases) was observed in RBC and tail. In RBCs, but not in liver, the combined level of c-erbA-related mRNA species was increased during spontaneous metamorphosis or after administration of TH. Furthermore, the TH-induced increase in both c-erbA-related mRNA species and receptor number in RBCs was prevented if actinomycin-D was administered with TH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-free translation analysis of messenger RNA in echinoderm and amphibian early development

A wheat germ cell-free translation system has been used to analyze populations of abundant messenger RNA from sea urchin eggs and embryos and from amphibian oocytes and ovaries. We show directly that sea urchin eggs and embryos contain translatable mRNA of three general classes: poly(A)⁺ mRNA, poly(A)^? histone mRNA, and poly(A)^? nonhistone mRNA. Additionally, some histone synthesis appears to be promoted by poly(A)⁺ RNA. Sea urchin eggs seem to contain a higher proportion of prevalent poly(A)^? nonhistone mRNAS than do embryos. Some differences in the proteins encoded by poly(A)⁺ and poly(A)^? RNAs are detectable. Many coding sequences in the egg appear to be represented in both poly(A)⁺ and poly(A)^? RNAs, since the translation products of the two RNA classes exhibit many common bands when run on one-dimensional polyacrylamide gels. However, some of this overlap is probably due to fortuitous comigration of nonidentical proteins. Distinct stage-specific changes in the spectra of prevalent translatable mRNAs of all three classes occur, although many mRNAs are detectable throughout early development. Particularly striking is the presence of an egg poly(A)^? mRNA, encoding a 70,000–80,000 molecular weight protein, which is not detected in morula or later-stage embryos. In amphibian (Xenopus laevis and Triturus viridescens) ovary RNA, the translation assay detects the following three mRNA classes: poly(A)⁺ nonhistone mRNA, poly(A)^? histone mRNA, and poly(A)⁺ histone mRNA. Amphibian ovary RNA appearently lacks an abundant poly(A)^? nonhistone mRNA component of the magnitude detectable in sea urchin eggs. mRNA encoding histone-like proteins is found in the very earliest (small stage 1) oocytes of Xenopus as well as in later stage oocytes. During oogenesis there appear to be no striking qualitative changes in the spectra of prevalent translatable mRNAs which are detected by the cell-free translation assay.     

Sequential up-regulation of thyroid hormone beta receptor, ornithine transcarbamylase, and carbamyl phosphate synthetase mRNAs in the liver of Rana catesbeiana tadpoles during spontaneous and thyroid hormone-induced metamorphosis.

During both spontaneous and thyroid hormone (TH)-induced metamorphosis, the Rana catesbeiana tadpole undergoes postembryonic developmental changes in its liver which are necessary for its transition from an ammonotelic larva to a ureotelic adult. Although this transition ultimately results from marked increases in the activities and/or de novo synthesis of the urea cycle enzymes, the precise molecular means by which TH exerts this tissue-specific response are presently unknown. Recent reports, using RNA from whole Xenopus laevis tadpole homogenates and indirect means of measuring TH receptor (TR) mRNAs, suggest a correlation between the up-regulation of TR beta-mRNAs and the general morphological changes occurring during amphibian metamorphosis. To assess whether or not this same relationship exists in a TH-responsive tissue, such as liver, we isolated and characterized a cDNA clone containing the complete nucleotide sequence for a R. catesbeiana urea cycle enzyme, ornithine transcarbamylase (OTC), as well as a genomic clone containing a portion of the hormone-binding domain of a R. catesbeiana TR beta gene. Through use of these homologous sequences and a heterologous cDNA fragment encoding rat carbamyl phosphate synthetase (CPS), we directly determined the relative levels of the TR beta, OTC, and CPS mRNAs in liver from spontaneous and TH-induced tadpoles. Our results establish that TH affects an up-regulation of mRNAs for its own receptor prior to up-regulating CPS and OTC mRNAs. Moreover, results with cultured tadpole liver demonstrate that TH, in the absence of any other hormonal influence, can affect an up-regulation of both the TR beta and OTC mRNAs.     

Role of type III iodothyronine 5-deiodinase gene expression in temporal regulation of Xenopus metamorphosis

To elucidate the role of type III iodothyronine 5-deiodinase (5-D) in the temporal regulation of amphibian metamorphosis, the regulation of gene expression of 5-D and thyroid hormone receptor beta (TRbeta) in organs of Xenopus laevis was investigated. High levels of TRbeta mRNA in the respective organs were observed at the times of their major morphological changes. Expression of the 5-D gene was highly regulated among the organs during metamorphosis, including up-regulation in the tail and down-regulation in the liver. The tail and liver expressed 5-D gene before their metamorphic changes. These precocious expressions correlated with the lower responsiveness to exogenously added triiodo-L-thyronine (T3) for inducing a high level of TRbeta mRNA expression. However, the same organs responded to lower doses of T3 to regulate 5-D gene expression as seen in spontaneous metamorphosis. The induction of 5-D gene expression was considerably delayed in the intestine, even at an excess dose of T3. Thus, the two genes in a given organ appeared to respond to T3 either with different dose dependencies or with different timetables. The results obtained are also discussed in respect to recent findings in Rana catesbeiana.     

Molecular cloning by hybrid arrest of translation in Xenopus laevis oocytes. Identification of a cDNA encoding the type I iodothyronine 5'-deiodinase from rat liver.

The enzyme(s) catalyzing the 5'-monodeiodination of thyroxine and 3,3',5'-triiodothyronine has not yet been purified, and antibodies of demonstrated specificity are not available. Thus, molecular cloning strategies which rely on the traditional screening techniques of using cDNA probes or monospecific antibodies are problematic. We previously reported that expression of type I 5'-deiodinase can be induced in Xenopus laevis oocytes by the injection of poly (A)+ RNA prepared from rat liver (St. Germain, D.L., and Morganelli, C.M. (1989) J. Biol. Chem. 264, 3054-3056). Using this expression system, we developed a hybrid arrest assay, and with it identified a 241-base pair cDNA which encodes part of this enzyme. The cDNA inhibits translation of 5'-deiodinase activity in oocytes by greater than 99% and 5'-deiodinase mRNA from rat liver poly(A)+ RNA in hybrid selection experiments. The cDNA hybridizes to a 1.9-kilobase RNA species on Northern analysis and demonstrates no significant homology to any previously cloned protein. The application of this hybrid arrest strategy for molecular cloning may prove useful for the isolation of cDNAs for proteins that are low in abundance, difficult to purify, or are subunits of a polymeric functional unit.     

Molecular regulation of ethanol-inducible cytochrome P450-IIEI in hamsters

Liver polysomal poly(A)+ RNA, isolated from hamsters treated with ethanol or pyrazole, was translated in vitro to determine the effect of these compounds on specific mRNA encoding P450-IIEI, an ethanol-inducible P450 isozyme. As assessed by immunoprecipitation of translation products, ethanol and pyrazole increased hepatic P450-IIEI mRNA levels by 160% and 45%, respectively, when compared to controls. In liver microsomes from the same animals, ethanol and pyrazole caused a two-fold increase in microsomal P450-IIEI protein and a two- to three-fold enhancement of microsomal ethanol oxidation and p-nitrophenol hydroxylation. Our results show that the induction of P450-IIEI protein in hamsters by ethanol and pyrazole, an "ethanol-like" inducer, is accompanied by an increase in translatable P450-IIEI mRNA.