Cloning and nucleotide sequence of cDNA encoding human liver serine-pyruvate aminotransferase

Cloned cDNAs for human liver serine-pyruvate aminotransferase (Ser-PyrAT) were obtained by screening of a human liver cDNA library with a fragment of cDNA for rat mitochondrial Ser-PyrAT as a probe. Two clones were isolated from 50,000 transformants. Both clones contained approximately 1.5 kb cDNA inserts and were shown to almost completely overlap each other on restriction enzyme mapping and DNA sequencing. The nucleotide sequence of the mRNA coding for human liver Ser-PyrAT was determined from those of the cDNA clones. The mRNA comprises at least 1487 nucleotides, and encodes a polypeptide consisting of 392 amino acid residues with a molecular mass of 43,039 Da. The amino acid composition determined on acid hydrolysis of the purified enzyme showed good agreement with that deduced from the nucleotide sequence of the cDNA. In vitro translation of the mRNA derived from one of the isolated clones, pHspt12, as well as that of mRNA extracted from human liver, yielded a product of 43 kDa which reacted with rabbit anti-(rat mitochondrial Ser-PyrAT) serum. Comparison of the deduced amino acid sequences of human Ser-PyrAT and the mature form of rat mitochondrial Ser-PyrAT revealed 79.3% identity. Although human Ser-PyrAT appears to be synthesized as the mature size, the 5'-noncoding region of human Ser-PyrAT mRNA contains a nucleotide sequence which would encode, if translated, an amino acid sequence similar to that of the N-terminal extension peptide of the precursor for rat mitochondrial Ser-PyrAT.     

Human apolipoprotein B: identification of cDNA clones and characterization of mRNA.

Apolipoprotein B (apoB) is a major protein component of low density and very low density lipoproteins. Because of its large size and heterogeneity, molecular studies of apoB have been difficult, and its structure and regulation remain poorly understood. We now report the identification of human apoB cDNA clones by antibody screening of hepatoma libraries in the expression vector lambda gt11. Both oligo(dT) primed and random primed libraries were constructed and screened with polyclonal antibodies to intact apoB, as well as with antibodies raised against a synthetic peptide based on the limited amino acid sequence available for apoB. The identity of the clones was unambiguously established by comparisons of the cloned cDNA sequences with apoB amino acid sequences. The clones hybridize to an exceptionally large 20 kb mRNA that is present in liver and intestine but not other tissues examined, consistent with the distribution expected from protein biosynthetic studies. The properties of the mRNA have implications for the biogenesis of the multiple apoB molecular weight forms secreted by liver and intestine.     

Characterization of the cDNA encoding human nucleophosmin and studies of its role in normal and abnormal growth

A cDNA encoding human nucleophosmin (protein B23) was obtained by screening a human placental cDNA library in lambda gtll first with monoclonal antibody to rat nucleophosmin and then with confirmed partial cDNA of human nucleophosmin as probes. The cDNA had 1311 bp with a coding sequence encoding a protein of 294 amino acids. The identity of the cDNA was confirmed by the presence of encoded amino acid sequences identical with those determined by sequencing pure rat nucleophosmin (a total of 138 amino acids). The most striking feature of the sequence is an acidic cluster located in the middle of the molecule. The cluster consists of 26 Asp/Glu and 1 Phe and Ala. Comparison of human nucleophosmin and Xenopus nucleolar protein NO38 shows 64.3% sequence identity. The N-terminal 130 amino acids of human nucleophosmin also bear 50% identity with that of Xenopus nucleoplasmin. Northern blot analysis of rat liver total RNA with a partial nucleophosmin cDNA as probe demonstrated a homogeneous mRNA band of about 1.6 kb. Similar observations were made in hypertrophic rat liver and Novikoff hepatoma. However, the quantity of nucleophosmin mRNA is 50- and 5-fold higher in Novikoff hepatoma and hypertrophic rat liver, respectively, when compared with normal rat liver. Dot blot analysis also showed a nucleophosmin mRNA ratio of 64:5:1 in the three types of rat liver. When the protein levels were compared with Western blot immunoassays, Novikoff hepatoma showed 20 times more nucleophosmin, while only about 5 times more nucleophosmin was observed in hypertrophic rat liver than in unstimulated normal liver.     

Molecular cloning of cDNA for the catalytic subunit of rat liver type 2A protein phosphatase, and detection of high levels of expression of the gene in normal and cancer cells

A cloned cDNA encoding a catalytic subunit of type 2A protein phosphatase from a rat liver cDNA library was obtained by use of a synthetic oligonucleotide corresponding to the tryptic peptide sequence of the purified enzyme. There was only a single amino acid difference between the deduced amino acid sequence of the clone obtained and those of the catalytic subunits, 2A alpha, of the rabbit skeletal muscle, porcine kidney and human liver enzymes, suggesting that this clone was a rat 2A alpha cDNA. On Northern blot analysis using a cDNA fragment as a probe, three mRNA species were detected in rat liver: a major mRNA of 2.0 kb and a minor one of 2.7 kb under high stringency conditions, and also a 1.1 kb mRNA under low stringency conditions. The 2A alpha gene was found to be highly expressed in various tissues of rat, especially the brain. High levels of expression of the gene were also detected in mouse NIH3T3 cells and their transformants, and in human cancer cell lines as well as a human immortalized cell line.     

Molecular cloning, cDNA structure and tissue-specific expression of the human regulatory subunit RI beta of cAMP-dependent protein kinases.

Complementary DNA clones for the regulatory subunit RI beta of cAMP-dependent protein kinases were isolated from a human testis cDNA library using a mouse RI beta cDNA probe. One clone 2.4 kilobases (kb) in length contained an open reading frame of 1137 bases, and encoded a protein of 379 amino acids (excluding the initiator methionine). The human RI beta protein was one amino acid shorter than the corresponding protein in mouse and rat. The nucleotide similarity to mouse and rat sequences was 85.6% and 84.8%, respectively, while the amino acid similarity was 97.6% and 97.3%, respectively. Northern blot analyses revealed a 2.7 kb mRNA in human tissues and a 2.8 kb mRNA in mouse tissues. Both mouse and human RI beta mRNA were found to be expressed in most tissues, and not restricted to brain and testis as reported by others.     

Molecular cloning and nucleotide sequence of cDNA encoding the rat kidney S-adenosylmethionine synthetase.

We previously reported the isolation of a cDNA encoding the liver-specific isozyme of rat S-adenosylmethionine synthetase from a lambda gt11 rat liver cDNA library. Using this cDNA as a probe, we have isolated and sequenced cDNA clones for the rat kidney S-adenosylmethionine synthetase (extrahepatic isoenzyme) from a lambda gt11 rat kidney cDNA library. The complete coding sequence of this enzyme mRNA was obtained from two overlapping cDNA clones. The amino acid sequence deduced from the cDNAs indicates that this enzyme contains 395 amino acids and has a molecular mass of 43,715 Da. The predicted amino acid sequence of this protein shares 85% similarity with that of rat liver S-adenosylmethionine synthetase. This result suggests that kidney and liver isoenzymes may have originated from a common ancestral gene. In addition, comparison of known S-adenosylmethionine synthetase sequences among different species also shows that these proteins have a high degree of similarity. The distribution of kidney- and liver-type S-adenosylmethionine synthetase mRNAs in kidney, liver, brain, and testis were examined by RNA blot hybridization analysis with probes specific for the respective mRNAs. A 3.4-kilobase (kb) mRNA species hybridizable with a probe for kidney S-adenosylmethionine synthetase was found in all tissues examined except for liver, while a 3.4-kb mRNA species hybridizable with a probe for liver S-adenosylmethionine synthetase was only present in the liver. The 3.4-kb kidney-type isozyme mRNA showed the same molecular size as the liver-type isozyme mRNA. Thus, kidney- and liver-type S-adenosylmethionine synthetase isozyme mRNAs were expressed in various tissues with different tissue specificities.     

Thyroid hormone modulates the introduction of a stop codon in rat liver apolipoprotein B messenger RNA

Apolipoprotein B (apoB) biosynthesis by rat liver was studied following thyroid hormone (3,5,3'-triiodo-L-thyronine) administration to hypothyroid rats. Pharmacologic doses of 3,5,3'-triiodo-L-thyronine caused suppression of apoB100 synthesis but did not affect apoB48 levels. There was no detectable apoB100 synthesis in hyperthyroid rats. To examine whether these results were mediated by the previously demonstrated mechanism of RNA modification (Powell, L. M., Wallis, S. C., Pease, R. J., Edwards, Y. H., Knott, T. J., and Scott, J. (1987) Cell 50, 831-840), the DNA sequence corresponding to the C-terminal end of rat apoB48 was determined from rat liver cDNA clones. Rat cDNAs contained a stop codon at an identical position to that found in human and rabbit apoB48 intestinal cDNA. To quantitate the relative amounts of apoB100 and apoB48 message, cDNA was synthesized from hepatic and intestinal apoB RNA and a 207-base pair fragment amplified using the polymerase chain reaction. The products were then differentially hybridized with oligonucleotides specific for apoB100 (containing CAA) or apoB48 (TAA). Control and hypothyroid liver contained approximately equal amounts of CAA and TAA, while hyperthyroid liver contained greater than 90% TAA. All gut samples contained 94-98% TAA. Genomic DNA from rat liver contained only CAA. The results demonstrate that apoB mRNA modification can be hormonally modulated in the adult rat by induction of a mechanism involving substitution of a stop codon into hepatic apoB100 mRNA.     

Cloning and nucleotide sequence of a full-length cDNA for human liver gamma-glutamylcysteine synthetase.

We have cloned and sequenced a full-length cDNA for human liver gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in glutathione biosynthesis. The cDNA consists of 2634 bp containing an open reading frame encoding a protein of 367 amino acids and having a calculated M(r) = 72,773. The nucleotide sequence of the cDNA for human liver GCS shares an 84% overall similarity with the composite rat GCS sequence deduced from three overlapping partial cDNAs (Yan and Meister, JBC 265: 1588-1593, 1990). The deduced amino acid sequences are 94% similar. Comparison of Northern blots of total RNA isolated from rat kidney or liver with that from human kidney revealed the GCS mRNA to be larger in the human tissue (approximately 4.0 kb vs. approximately 3.7 kb). (The sequence for the human liver GCS cDNA has been assigned accession number M90656 in GenBank/EMBL databases.     

Cloning and sequence analysis of cDNA clones for bovine aortic-endothelial-cell transglutaminase.

A cDNA clone encoding transglutaminase was isolated from a bovine-endothelial-cell cDNA library using oligonucleotide probes designed based on partial amino acid sequences of the purified protein. Sequencing of the cDNA insert revealed an open reading frame of 2061 bp coding for a protein of 687 amino acids. The sequence of bovine endothelial-cell transglutaminase was 88, 82, 80, 37, 37 and 37% identical with that of human endothelial, rat macrophage, guinea-pig liver, human and rat keratinocyte transglutaminases, and the human blood-coagulation factor XIIIa subunit, respectively. The cDNA clone was hybridized to a single mRNA species of 3.9 kb in the liver, lung, spleen and heart but not hybridized to RNA from the brain. Northern-blot analysis of mRNA from retinoid-treated cultured vascular endothelial cells revealed that retinoids were able to induce a large increase in the transglutaminase mRNA levels.     

Construction of DNA sequences complementary to rat alpha 1 and alpha 2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D

Type I collagen mRNA from fetal rat calvaria was used as a template for the synthesis of a cDNA that was subsequently inserted in the PstI site of the plasmic vector pBR322 and cloned. Three recombinant plasmids containing type I collagen specific sequences were characterized: p alpha 1R1 is 1600 bp and spans approximately 500 amino acid residues within the triple helical region of alpha 1(I) and p alpha 1R2 is 900 bp in size and covers the entire 3' noncoding and about half of the C-terminal propeptide region of alpha 1(I) collagen mRNA. The third recombinant p alpha 2R2 is 1500 bp and contains alpha 2(I) sequences specific for the entire 3' noncoding and C-terminal propeptide region. Partial nucleic acid sequence data revealed that the decreasing order of amino acid and nucleotide homology to similar regions of the rat cDNA was mouse greater than human greater than chick. Northern hybridization of mRNA after electrophoresis in 0.8% agarose revealed two distinctly different molecular weight patterns characteristic of alpha 1(I) (4.7 and 5.7 kb) and alpha 2(I) (4.2 and 4.5 kb) collagen mRNA when hybridized with the corresponding cDNA probe. Despite the high degree of sequence homology, DNA probes from rat or human produced a significantly reduced hybridization signal when used as an interspecies hybridization probe than to its corresponding mRNA. The rat cDNA probes were used in a dot hybridization assay to measure the type I collagen mRNA content in the fetal rat calvaria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human augmenter of liver regeneration: molecular cloning, biological activity and roles in liver regeneration

The complete amino acid sequence of human augmenter of liver regeneration (hALR) was reported by deduction from nucleotide sequence of its complementary DNA . The cDNA for hALR was isolated by screening a human fetal liver cDNA library and the sequencing of this insert revealed an open reading frame encoding a protein with 125aa and highly homologous (87% ) with rat ALR encoding sequence. The recombinant hALR expressed from its cDNA in transient expression experiments in cos-7 cells could stimulate DNA synthesis of HTC hepatoma cell in the dose-dependent and heat-resistant way. Northern blot analysis with rat ALR cDNA as probe confirmed that ALR mRNA was expressed in the normal rat liver at low level and that dramatically increased in the regenerating liver after partial hepatectomied rat. This size of hALR mRNA is 1.4 kb long and expressed in human fetal liver, kidney and testis. These findings indicated that liver itself may be the resource of ALR and suggested that ALR seems to be an im-portant parac     

Molecular cloning and sequence analysis of cDNA for the catalytic subunit 1 alpha of rat kidney type 1 protein phosphatase, and detection of the gene expression at high levels in hepatoma cells and regenerating livers as compared to rat livers.

A cDNA clone containing the full coding sequence of a type 1 protein phosphatase catalytic subunit 1 alpha has been isolated from a rat kidney lambda gt 10 library. The protein sequence deduced from the cDNA contains 330 amino acid residues with a molecular mass of 38 kDa. The cDNA clone from rat kidney was 89% identical at the nucleotide level in the coding region to type 1 protein phosphatase 1 alpha from rabbit skeletal muscle. However, the two protein sequences were completely identical. The type 1 alpha protein phosphatase from rat kidney shows 49% homology of amino acid sequence to the rat type 2A alpha protein phosphatase. Thus, the protein sequence of type 1 alpha protein phosphatase was completely conserved between rat and rabbit. The mRNA levels of type 1 protein phosphatase were determined in rat liver, AH13, a strain of rat hepatoma, and regenerating rat liver by Northern blot analysis using the cDNA fragment as a probe, under which conditions a single mRNA of 1.5 kb was detected. The mRNA levels of AH13 were remarkably increased when compared to those of normal ivers, whereas the mRNA levels of regenerating livers were slightly but significantly increased. These results demonstrate a marked increase in gene expression of type 1 protein phosphatase in hepatoma cells, suggesting an important role of the type 1 protein phosphatase in hepatocarcinogenesis.     

Hormonal regulation of estradiol biosynthesis, aromatase activity, and aromatase mRNA in rat ovarian follicles and corpora lutea

To determine the molecular basis for changes in aromatase (P450arom) activity in rat ovarian follicles and corpora lutea, seven clones for rat P450arom cDNA have been identified and isolated from a rat granulosa cell λgtll cDNA expression library using a 62 mer deoxyoligonucleotide probe (derived from an amino acid sequence of purified human placental aromatase) and a human placental P450arom cDNA probe. One of the rat P450arom cDNA clones contained an insert 1.2 kb in size. Both the human 1.8 kb cDNA and the rat 1.2 kb cDNA probes hybridized to a single species of P450arom mRNA that was 2.6 kb in size. Northern blot analysis revealed that corpora lutea isolated on day 15 of pregnancy contained high amounts of P450arom mRNA, whereas granulosa cells of antral follicles of hormonally primed, hypophysectomized rats (i.e., those from which mRNA was isolated to construct the cDNA library) contained only low amounts of P450arom mRNA. The lower amounts of P450arom in granulosa cells of preovulatory follicles in the estradiol-follicle-stimulating hormone primed hypophysectomized rats were unexpected because follicles incubated in medium containing testosterone substrate produce more estradiol than do corpora lutea isolated on day 15 of pregnancy and incubated under similar conditions. Additional studies will determine the hormonal events responsible for the elevated amounts and constitutive maintenance of P450arom mRNA and aromatase activity in luteal cells in vivo and in vitro.