Regulation of rat growth hormone receptor gene expression

A cDNA encoding the growth hormone (GH) receptor was cloned from rat liver. Both the nucleotide and translated amino acid sequence share greater than 70% similarity with the GH receptors from rabbit and human. An RNA probe was generated from this sequence for use in a solution hybridization assay to quantitate GH receptor mRNA expression in rat tissues. Expression was detected in 9/12 tissues examined, with the highest levels observed in the liver. Expression in liver, kidney, heart and muscle was developmentally regulated, being low at birth and rising to adult levels in 5 weeks. No difference was observed between hepatic expression in males and females, although livers from pregnant rats had elevated levels. Hypophysectomy and GH treatment did not affect hepatic GH receptor mRNA levels.     

Primary structure of rat hepatocyte growth factor and induction of its mRNA during liver regeneration following hepatic injury

Overlapping cDNA clones for rat hepatocyte growth factor (rHGF) were isolated by cross-hybridization with the cloned cDNA for human hepatocyte growth factor (hHGF) and the nucleotide sequence of the cDNA was determined. The entire primary structure of rHGF was deduced from the sequence. Comparison of the amino acid sequences between rat and human HGFs revealed that the two sequences are highly conserved throughout the protein structures, suggesting that rat and human HGFs may be functionally similar. Responses of the rHGF mRNA during liver regeneration in rats were examined by Northern blot hybridization analysis with the aid of the cDNA probe for rHGF. The mRNA levels increased in the liver and spleen but not in the kidney after administration of carbon tetrachloride. At the maximum level of induction, the rHGF mRNA increased in the liver about 4.5-fold over its normal level. The mRNA levels also increased in the liver and spleen after administration of D-galactosamine. On the other hand, no obvious increase of the mRNA was observed in the liver and spleen after partial hepatectomy. These observations suggest that HGF may function as a regulator of liver regeneration following hepatic injury caused by hepatotoxins.     

Phenobarbital-inducible aldehyde dehydrogenase in the rat. cDNA sequence and regulation of the mRNA by phenobarbital in responsive rats

In the rat, a cytosolic isozyme of aldehyde dehydrogenase, designated ALDH-PB, can be induced in the liver by administration of phenobarbital (PB). ALDH-PB activity and mRNA are induced in Long-Evans rats that possess a responsive (R) allele but are not induced in homozygous nonresponsive rats (rr), although the rr genotype is competent to induce other PB-responsive mRNAs. ALDH-PB mRNA is expressed in the basal state (without PB administration) in hepatic tissue in both RR and rr genotypes. We report the complete nucleotide sequence of the rat ALDH-PB mRNA. The protein encoded by the ALDH-PB mRNA is 501 amino acids in length and has a predicted molecular mass of 54,540 daltons. The amino acid sequence predicted from the mRNA demonstrates a strong conservation between the rat ALDH-PB and the human cytosolic aldehyde dehydrogenase hALDH-1. We demonstrate the ALDH-PB, cytochrome P-450b, cytochrome P-450e, and glutathione S-transferase Ya subunit mRNA levels in the liver are altered noncoordinately by administration of PB in RR and rr genotypes. The strikingly different responses to PB administration between the various mRNA species in each of the genotypes suggest that the regulation of specific gene expression by PB may involve multiple pathways.     

Prostatic spermine-binding protein. Cloning and nucleotide sequence of cDNA, amino acid sequence, and androgenic control of mRNA level

cDNA for mRNA of an androgen-dependent spermine-binding protein (SBP) of rat ventral prostate was cloned by inserting cDNA into a dG-tailed expression vector, pUC8, and screening the expression library with anti-SBP antibodies. Hybrid-selected translation using plasmid DNA from positive clones yielded a 34-kDa protein which was immunoprecipitated by affinity-purified anti-SBP antibodies. SBP mRNA is about 1260 bases long as measured by Northern blot hybridization. An amino acid sequence deduced from the nucleotide sequence of the cDNA was identical to an amino acid sequence found in SBP. SBP is extremely rich in acidic residues. Aspartic and glutamic acids, which make up about 33% of the total sequence, comprise 89 of a stretch of 126 amino acids at the carboxyl-terminal end. By dot hybridization analysis, SBP mRNA was not detected in rat liver, kidney, brain, submaxillary gland, or uterus. The prostate levels of SBP mRNA were measured by mRNA translation and dot hybridization. SBP mRNA level decreased to less than 20% of normal 2 days after castration of rats, and this decrease was reversed by 5 alpha-dihydrotestosterone injection into castrated rats.     

Isolation and characterization of cDNAs encoding the rat pituitary gonadotropin-releasing hormone receptor.

Rat pituitary cDNAs encoding the full peptide coding sequence of the rat gonadotropin-releasing hormone receptor were isolated and characterized. The deduced amino acid sequence encodes a protein of 327 residues with seven putative transmembrane domains characteristic of the family of G-protein coupled receptors. It is 95% identical at the amino acid level with the mouse gonadotropin-releasing hormone receptor. An mRNA of 4.5 Kb was identified in the rat pituitary, ovary, and testis, and in murine alpha T3 cells. In addition, a larger mRNA species of 5.0-5.5 Kb was present in these rat tissues, and a smaller mRNA species of 1.8 Kb was present in the rat pituitary and ovary, and in alpha T3 cells. The receptor mRNA levels were increased in the female rat pituitary after ovariectomy compared to levels in intact female rats.     

Tissue kallikrein-binding protein is a serpin. I. Purification, characterization, and distribution in normotensive and spontaneously hypertensive rats

Kallikrein-binding protein was purified to apparent homogeneity from rat serum by Affi-Gel Blue, DEAE-Sepharose CL-6B, Sephacryl S-200 chromatography, and preparative gel electrophoresis or high performance liquid chromatography. The purified protein migrates as a single band of 60 kDa in a sodium dodecyl sulfate-polyacrylamide gel under reducing conditions. It is an acidic protein with isoelectric points ranging from 4.2 to 4.6. The amino terminus of the binding protein is an Asp residue as determined by sequence analysis. It forms a 92-kDa sodium dodecyl sulfatestable complex with kallikrein with a t1/2 of 18 min. Western blot and radioimmunoassay showed a distribution of the kallikrein-binding protein in serum, urine, and various tissues with a 5-10-fold lower amount in spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY). A full length cDNA clone encoding the kallikrein-binding protein was isolated from a rat liver cDNA library by immunoscreening and the translated amino acid sequence matches the amino-terminal 29-amino acid sequence of the binding protein. The cDNA sequence shares 68.8% identity with human alpha 1-antichymotrypsin and is identical to that of a rat hepatic protein. Dot blot analysis shows that kallikrein-binding protein is expressed at high levels in the liver and at low levels in the lung, salivary gland, and kidney. Its mRNA level in the liver decreases by 2-fold after acute phase inflammation and is higher in male than in female rats. Genomic Southern blot analyses reveal restriction fragment length polymorphisms between SHR and WKY rats in the binding protein locus. The results indicate that rat kallikrein-binding protein belongs to the serpin superfamily and its level is significantly reduced in the spontaneously hypertensive rats.     

Identification of rat S14 protein and comparison of its regulation with that of mRNA S14 employing synthetic peptide antisera

The rat S14 gene encodes a protein of unknown function and has an amino acid sequence unrelated to any published sequences. Expression of mRNA S14 and lipogenesis in liver, fat, and mammary gland are regulated coordinately by dietary and hormonal stimuli, suggesting that the S14 protein may be associated with lipogenesis. Antisera to synthetic peptides corresponding to portions of the deduced amino acid sequence of the protein were used to identify the protein and to compare its regulation with that of mRNA S14. Antisera specifically recognized the in vitro translation product of mRNA S14 as defined by its migration on two-dimensional gel electrophoresis. A product of identical Mr was identified on Western blots of liver homogenates from hyperthyroid, carbohydrate-fed rats. Subcellular fractionation showed that S14 protein is primarily cytosolic. The protein was detectable in tissues with abundant S14 gene expression, including hyperthyroid liver and epididymal fat and hypothyroid brown adipose tissue, whereas it was undetectable in hypothyroid liver and euthyroid kidney, testis, and spleen. Diurnal variation in hepatic mRNA S14 correlated with comparable changes in levels of the protein. Surprisingly, no S14 protein was observed in the livers of chronically (3 week) hypothyroid rats treated with triiodothyronine (T3) until 12 h had elapsed, despite attainment of maximal levels of mRNA S14 within 4 h. Rapid appearance of protein after T3 treatment was observed in both euthyroid and short term (4 day) hypothyroid rats, suggesting that long-term hypothyroidism is associated with a defect in the translational efficiency of mRNA S14.     

Isolation and sequence of a cDNA clone which contains the complete coding region of rat phenylalanine hydroxylase. Structural homology with tyrosine hydroxylase, glucocorticoid regulation, and use of alternate polyadenylation sites

Screening of a rat liver cDNA expression library constructed in the vector lambda gt11 with an affinity purified antiserum to rat phenylalanine hydroxylase has resulted in the isolation of two clones which contain the complete coding region (1362 base pairs) of phenylalanine hydroxylase and the entire 3'-untranslated region (562 base pairs). From the nucleotide sequence we deduced the amino acid sequence of the enzyme. The molecular weight is 51,632 (452 amino acids). The rat enzyme is highly homologous to human phenylalanine hydroxylase. The two proteins differ in only 36 amino acids (92% homology), many of which are conservative changes. A dot matrix computer program was used to analyze regions of homology with the amino acid sequence of rat tyrosine hydroxylase. Considerable homology was detected from amino acid 140 in the rat enzyme to the C terminus, but little or no homology was apparent in the N-terminal region. The cDNA clone was used to determine the levels of phenylalanine hydroxylase mRNA in rat tissues using RNA blot hybridization. Two mRNA species were detected, with approximate lengths of 2,000 and 2,400 nucleotides, which appear to derive from use of alternate polyadenylation signals. No difference in mRNA size was found in rats which have different phenylalanine hydroxylase alleles. The kidney was found to contain about 10% of the mRNA found in the liver, and no phenylalanine hydroxylase mRNA was detected in rat brain. Reuber H4 hepatoma cells were also analyzed for phenylalanine hydroxylase mRNA. The parental cells contained mRNA species of the same sizes as in rat liver. Incubation in 10(-6) M hydrocortisone for 24 h resulted in an 18-fold increase in the mRNA level. Mutant hepatoma cells which express very little phenylalanine hydroxylase contained less than 5% of the parental mRNA, but the gene still responded to hydrocortisone.     

Sequence,tissue distribution and developmental changes in rat intestinal oligopeptide transporter

Complementary DNA clones encoding the rat PepT1 small-intestinal oligopeptide transporter were isolated from a jejunal library by cross-hybridization with a rabbit PepT1 cDNA probe. The cDNA sequence indicates that rat PepT1 is composed of 710 amino acids and shows 77% and 83% amino acid sequence identity with rabbit and human PepT1, respectively. Northern blot analysis detected rat PepT1 mRNA in the small intestine and kidney. Intestinal PepT1 mRNA levels were highest in 4-day-old rats, and then decreased reaching the adult level by day 28 after birth. These results indicate that the expressions of PepT1 gene change markedly during development.     

Isolation and characterization of the complete complementary and genomic DNA sequences of human serum amyloid P component

Complementary and genomic DNA clones corresponding to the human serum amyloid P component (SAP) mRNA have been isolated and analyzed. The nucleotide sequences of the cDNA and the corresponding regions of the genomic SAP DNA reported here were identical, and revealed that after coding for a signal peptide of 19 amino acids and the first two amino acids of the mature SAP protein, there is one small intron of 115-base pairs (bp), followed by a nucleotide sequence coding for the remaining 202 amino acid residues. The SAP gene has an ATATAAA sequence 29-bp upstream from the cap site, but there is no CAAT box-like sequence. A possible polyadenylation signal sequence, ATTAAA, was found to be located 28-bp upstream from the polyadenylation site. A comparison of the genomic SAP DNA sequence with that of human C-reactive protein (CRP) revealed a striking overall homology which was not uniform: several highly conserved regions were bounded by non-homologous regions. This comparison provides further support for the hypothesis that SAP and CRP are products of a gene duplication event.     

Characterization of rat transferrin receptor cDNA: the regulation of transferrin receptor mRNA in testes and in Sertoli cells in culture

A 3.4 kilobase cDNA complementary to rat transferrin receptor mRNA has been isolated from an adult rat testis cDNA library. The rat transferrin receptor nucleotide sequence was shown to be 82% similar to the human transferrin receptor sequence over the amino acid coding region and over 90% similar in the sequences known to be responsible for iron regulation in the human mRNA. The mRNA was shown by Northern blot analysis to be regulated by iron levels in Sertoli cells in culture. Iron depletion resulted in at least a 5-fold increase in receptor message in Sertoli cells, as well as in an actively growing testicular cell line (S10-7). The level of transferrin receptor mRNA in cultured Sertoli cells was not influenced by hormones; however, chronic administration of testosterone or FSH to hypophysectomized rats resulted in increased transferrin receptor mRNA levels in the testis. Northern blot analysis of mRNAs from testes of rats synchronized at various stages of the cycle of the seminiferous epithelium showed that transferrin receptor mRNA was differentially regulated throughout the cycle. Northern blots of mRNA from germinal cell populations derived from synchronized tests showed that the message was regulated in the nongerminal cell components of the tubule, most likely the Sertoli cell. The comparison of transferrin receptor mRNA levels in normal testes and testes from hypophysectomized rats, as well as in isolated germinal cells and cultured Sertoli cells, suggested that transferrin receptor mRNA levels were considerably higher in Sertoli cells than in other cell types of the seminiferous tubules.     

Phenobarbital induction of rat liver cytochromes P-450b and P-450e. Quantitation of specific RNAs by hybridization to synthetic oligodeoxyribonucleotide probes

Cytochromes P-450b and P-450e are extremely homologous and immunochemically indistinguishable proteins that are coordinately induced by phenobarbital in rat liver. To assess the effect of phenobarbital on mRNA levels for each of these hemoproteins we performed solution hybridization and Northern blot experiments with synthetic oligodeoxynucleotide probes of defined sequence. Our data demonstrate that phenobarbital administration to rats resulted in marked increases in levels of hepatic mRNA for both cytochrome P-450b and cytochrome P-450e, with a 4- to 5-fold greater accumulation of P-450b mRNA vis à vis P-450e mRNA. The level of hepatic mRNA increased from less than 3 molecules/cell of each mRNA in untreated rats, to 630 and 130 molecules/cell for P-450b and P-450e, respectively, in phenobarbital-treated rats. Data obtained in Northern blot hybridization experiments demonstrated that the size of the mRNAs for each protein were identical, being approximately 1800 bases in length.     

Isolation and characterization of cDNA clones for cytochromes P-450 immunochemically related to rat hepatic P-450 form PB-1

Rat hepatic cytochrome P-450 PB-1 is a prominent constitutive P-450 form whose levels increase approximately 2-3 fold upon phenobarbital administration. Antibodies raised against this protein recognized two major proteins in immunoblots of rat liver microsomal proteins and precipitated comparable amounts of two electrophoretically separable hepatic mRNA translation products. The levels of the two mRNAs encoding these polypeptides were increased substantially upon phenobarbital administration. The anti-PB-1 antibodies were used to screen a cDNA library, and two distinct cDNA clones, pTF-1 and pTF-2, were isolated. These clones contain inserts of 1227 and 410 base pairs, respectively, and show 80% nucleic acid sequence homology in their region of overlap. The DNA sequences of these clones show 54% sequence homology to the corresponding portions of the mRNA encoding P-450 PB-4, a major phenobarbital-inducible form of rat liver P-450, and can be optimally aligned with the PB-4 sequence without introducing insertions or deletions. The level of hepatic mRNA which hybridizes to clone pTF-2 increases approximately 2-4-fold after phenobarbital treatment, whereas mRNA which hybridizes to pTF-1 does not change in concentration after this treatment. mRNA, which hybridizes to pTF-1, is, however, 4-fold more abundant in livers of female rats than in livers of male rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization by cDNA cloning of the mRNA for seminalplasmin, the major basic protein of bull semen

A cDNA library derived from poly(A)+RNA of bull seminal vesicle tissue was screened with synthetic DNA probes specific for seminalplasmin (SAP), the major basic protein of bull semen. From a number of positive clones, pBSV12, containing a 577-bp insert, was identified and sequenced. The derived amino acid sequence comprises the known amino acid sequence of SAP with an amino terminal representing a putative signal sequence; at the carboxyl terminus the sequence contains an additional lysine residue. Present experimental data do not distinguish between two potential SAP precursor molecules, each starting with a methionine residue and differing by 10 amino acid residues in the leader peptide. Comparative Northern analysis reveals a SAP-specific mRNA of 700 bp, which lacks RNA from bovine testis as well as from seminal vesicle tissue of a bull calf; hence, expression of the SAP gene appears to be under androgen and/or developmental control. Southern analysis indicates that one gene appears to specify SAP. SAP-like DNA sequences were detected in ovine and porcine genomic DNA.     

Cloning of rodent cDNA coding the poly(ADP-ribose) polymerase catalytic domain and analysis of mRNA levels during the cell cycle

We have isolated a partial 2.0 kb cDNA (pRATC) encoding the entire 489 amino acids of the NAD binding domain located at the C terminus of the rat poly(ADP-ribose) polymerase. pRATC sequences were analysed and compared with the human mRNA. Our analysis reveals a remarkable homology between the rat and human nucleotide and amino acid sequences. Although a few minor amino acid changes were detected, we have found that the total number of possible phosphorylation sites remained constant in the NAD binding domain of both enzymes. We have also found that a 102 amino acid sequence, containing the putative nucleotide binding site Gly-Lys-Gly (position 378), is perfectly conserved between the rat and human sequences. Strong homology was also detected between pRATC and genomic DNA isolated from various vertebrates. In addition, we have analysed the levels of poly(ADP-ribose) polymerase mRNA throughout the cell cycle. Our results show that the levels of mRNA culminate in the G1 phase. We have also found that the increase in enzymatic activity observed in rats following treatment with phenobarbital did not correspond to an increase in the mRNA levels.     

The rat 78,000 dalton glucose-regulated protein (GRP78) as a precursor for the rat steroidogenesis-activator polypeptide (SAP): the SAP coding sequence is homologous with the terminal end of GRP78

Based on the striking sequence identity between the amino acid sequence of rat steroidogenesis-activator polypeptide (SAP) and the carboxyl terminus of the 78,000 dalton glucose-regulated protein (GRP78), the precursor-product relationship between GRP78 and SAP was investigated in Leydig cells. Immunoblot analysis with peptide antibodies specific for GRP78 and SAP showed that the putative SAP precursor is also immunoreactive with the anti-GRP78 antibody. Genomic blot hybridizations further revealed that GRP78 is neither rearranged nor amplified in the H-540 Leydig cell tumor, the original source for SAP. Further, there appears to be a single copy of the SAP coding sequence within the rat genome. This sequence resides within the last exon of GRP78. Our observations support the hypothesis that, in steroidogenic cells, SAP is likely to be derived from posttranslational processing of a very minor fraction of GRP78.