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.     

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.     

Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family.

We have isolated and sequenced a full-length cDNA clone encoding rat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, E.C.1.2.1.12). The entire mRNA is 1269 nucleotides long exclusive of poly(A) and contains respectively 71 and 196 bases of 5' and 3' non-coding regions. Primer extension as well as S1 nuclease protection experiments clearly established that a single (or at least a highly prominent) GAPDH mRNA species is expressed in all rat tissues examined. This sequence allowed the determination of the hitherto unknown primary structure of rat GAPDH which is 333 aminoacids long. Comparison between GAPDH sequences from rat, man and chicken revealed a high degree of sequence conservation at both nucleotide and protein levels.     

Cloning and analysis of cDNA clones for rat kidney alpha-spectrin

We have isolated a 3922-base pair (bp) cDNA clone for rat nonerythroid alpha-spectrin from a rat kidney lambda gt11 cDNA library. Sequence analysis revealed that this cDNA contains an open reading frame of 3090 bp encoding for the C-terminal 1030 amino acid sequence of rat kidney alpha-spectrin. The 3'-untranslated region (including a 38-bp poly(A+) tail) contains an 832-bp sequence. A single mRNA of about 8 kilobase pairs was detected in rat liver, kidney, brain, heart, intestine, lung, testis, stomach, spleen, and muscle with varying abundances, which is consistent with and further confirms the presence of spectrins in nonerythroid tissues as demonstrated previously by immunoblot analysis. Southern blot analysis suggested that there is a single gene for nonerythroid alpha-spectrin. The derived amino acid sequence contains sequence from the spectrin 106-residue internal repeat 12 to the C terminus of rat kidney alpha-spectrin. Sequence comparison with human and chicken nonerythroid alpha-spectrin showed that nonerythroid alpha-spectrin is well conserved during evolution. The rat kidney alpha-spectrin sequence, when compared to rat brain alpha-spectrin, contains an extra 76-amino-acid sequence at the C terminus. Sequence comparison of all the internal repeats available revealed that the internal repeat 3, 4, 5, 6, 7, and 8 has highest sequence similarity with internal repeat 12, 13, 14, 15, 16, and 17, respectively. Therefore, internal repeats 3-8 and 12-17 are most likely derived from an ancestral gene through gene duplication, suggesting that the spectrin gene is derived from a half-spectrin gene by gene duplication and divergence during evolution.     

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.     

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 of cDNA encoding a 55-kDa multifunctional thyroid hormone binding protein of skeletal muscle sarcoplasmic reticulum.

A cDNA clone encoding 55-kDa multifunctional, thyroid hormone binding protein of rabbit skeletal muscle sarcoplasmic reticulum was isolated and sequenced. The cDNA encoded a protein of 509 amino acids, and a comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of the purified protein indicates that an 18-residue NH2-terminal signal sequence was removed during synthesis. The deduced amino acid sequence of the rabbit muscle clone suggested that this protein is related to human liver thyroid hormone binding protein, rat liver protein disulfide isomerase, human hepatoma beta-subunit of prolyl 4-hydroxylase and hen oviduct glycosylation site binding protein. The protein contains two repeated sequences Trp-Cys-Gly-His-Cys-Lys proposed to be in the active sites of protein disulfide isomerase. Northern blot analysis showed that the mRNA encoding rabbit skeletal muscle form of the protein is present in liver, kidney, brain, fast- and slow-twitch skeletal muscle, and in the myocardium. In all tissues the cDNA reacts with mRNA of 2.7 kilobases in length. The 55-kDa multifunctional thyroid hormone binding protein was identified in isolated sarcoplasmic reticulum vesicles using a monoclonal antibody specific to the 55-kDa thyroid hormone binding protein from rat liver endoplasmic reticulum. The mature protein of Mr 56,681 contains 95 acidic and 61 basic amino acids. The COOH-terminal amino acid sequence of the protein is highly enriched in acidic residues with 17 of the last 29 amino acids being negatively charged. Analysis of hydropathy of the mature protein suggests that there are no potential transmembrane segments. The COOH-terminal sequence of the protein, Arg-Asp-Glu-Leu (RDEL), is similar to but different from that proposed to be an endoplasmic reticulum retention signal; Lys-Asp-Glu-Leu (KDEL) (Munro, S., and Pelham, H.R.B. (1987) Cell 48, 899-907). This variant of the retention signal may function in a similar manner to the KDEL sequence, to localize the protein to the sarcoplasmic or endoplasmic reticulum. The positively charged amino acids Lys and Arg may thus interchange in this retention signal.     

Cloning and characterization of a cDNA that encodes a 70-kDa novel human thyroid autoantigen

cDNA clones were isolated by screening a human thyroid carcinoma lambda gt11 library with immunoglobulins purified from serum of a patient with autoimmune Graves' disease. One clone (ML8) containing a 1.25-kilobase (kb) insert hybridized with a single 2.0-kb poly(A+) mRNA in human thyroid and lymphocytes but not in human brain, liver, kidney, or muscle. In addition, this probe also hybridized with a single 2.0-kb poly(A+) mRNA from a rat thyroid cell line (FRTL-5). An apparently full length 2,074-base pair (bp) human cDNA was obtained and sequenced. The nucleotide sequence of the 2,074-bp cDNA includes a 5'-noncoding sequence of 17 bp, a 1827-bp open reading frame, and a 222-bp 3'-noncoding sequence. The canonical polyadenylation signal AATAAA is present 18 bp upstream of the poly(A) tail. This cDNA encodes a 69,812-dalton protein with two potential N-linked glycosylation sites and at least one potential membrane spanning domain. Immunoprecipitation of the in vitro translated protein by sera from several patients with Graves' disease argues that the 69,812-dalton protein is an autoantigen.     

CoA-dependent methylmalonate-semialdehyde dehydrogenase, a unique member of the aldehyde dehydrogenase superfamily. cDNA cloning, evolutionary relationships, and tissue distribution.

Three overlapping cDNA clones encoding methylmalonate-semialdehyde dehydrogenase (MMSDH; 2-methyl-3-oxopropanoate:NAD+ oxidoreductase (CoA-propanoylating); EC 1.2.1.27) have been isolated by screening a rat liver lambda gt 11 library with nondegenerate oligonucleotide probes synthesized according to polymerase chain reaction-amplified portions coding for the N-terminal amino acid sequence of rat liver MMSDH. The three clones cover a total of 1942 base pairs of cDNA, with an open reading frame of 1569 base pairs. The authenticity of the composite cDNA was confirmed by a perfect match of 43 amino acids known from protein sequencing. The composite cDNA predicts a 503 amino acid mature protein with M(r) = 55,330, consistent with previous estimates. Polymerase chain reaction was used to obtain the sequence of the 32 amino acids corresponding to the mitochondrial entry peptide. Northern blot analysis of total RNA from several rat tissues showed a single mRNA band of 3.8 kilobases. Relative mRNA levels were: kidney greater than liver greater than heart greater than muscle greater than brain, which differed somewhat from relative MMSDH protein levels determined by Western blot analysis: liver = kidney greater than heart greater than muscle greater than brain. A 1423-base pair cDNA clone encoding human MMSDH was isolated from a human liver lambda gt 11 library. The human MMSDH cDNA contains an open reading frame of 1293 base pairs that encodes the protein from Leu-74 to the C terminus. Human and rat MMSDH share 89.6 and 97.7% identity in nucleotide and protein sequence, respectively. MMSDH clearly belongs to a superfamily of aldehyde dehydrogenases and is closely related to betaine aldehyde dehydrogenase, 2-hydroxymuconic semialdehyde dehydrogenase, and class 1 and 2 aldehyde dehydrogenases.     

Cloning and characterization of cDNA encoding 3-methylcholanthrene inducible rat mRNA for UDP-glucuronosyltransferase

We have isolated cDNA clones of the mRNA for rat UDP-glucuronosyltransferase that catalyzes the glucuronidation of 4-nitrophenol, by using synthetic oligonucleotides as hybridization probes. The complete nucleotide sequence of the 1,927-base pairs cDNA insert has been determined. With untranslated sequences of 124 and 216 base pairs in the 5'- and 3'-terminal regions, respectively, the cDNA insert contained 1,587 base pairs that encode a complete primary structure of a putative precursor form of 4-nitrophenol UDP-glucuronosyltransferase with a calculated molecular weight of 60,114. The cDNA sequence also indicates the presence of 25 amino acids preceding the sequence determined by microsequence of the isolated protein. This extrapeptide, for the most part, consists of hydrophobic amino acids which are characteristic of the signal peptides as found for secretory proteins and most transmembrane proteins. Furthermore, the deduced amino acid sequence contains a putative halt transfer signal of a hydrophobic segment (residues 487-510), which is flanked on both sides by the peptide segments of highly charged amino acid residues (residues 463-486 and 511-529). These features are consistent with the properties of transmembrane proteins. Specific cDNA probes were used to analyze the induction of the enzyme in rat tissues by treatment with 3-methylcholanthrene. RNA blot analysis showed that 3-methylcholanthrene increased 10- to 15-fold the amount of hybridizable mRNA in liver. The livers and kidneys from 3-methylcholanthrene-treated rats were found to contain almost the same amount of hybridizable mRNA, although the basal level in the kidney was much higher than that of the liver, and the amounts in the lung were much lower than that of the liver and kidney.     

Human gamma-glutamyl transpeptidase cDNA: comparison of hepatoma and kidney mRNA in the human and rat

gamma-Glutamyl transpeptidase (GGT) is a glutathione-metabolizing enzyme that has been extensively studied in relation to hepatocarcinogenesis. Using a cDNA for rat kidney GGT as a probe, we have isolated a full-length cDNA for human GGT from a hepatoma cell-line library. Nucleotide sequence analysis of the clone revealed a 2326-bp insert that includes a 5'-untranslated region of 487 nucleotides (nt), an open reading frame (ORF) of 1707 nt, and a 3'-untranslated region of 132 nt. The ORF encodes a protein with an amino acid sequence that is highly similar to that of the rat GGT precursor peptide, with an overall identity of 79%. The cDNA clone was used to probe Northern blots of hepatoma and kidney RNA from both human and rat. In both species, the GGT mRNA is longer in hepatoma than in kidney. In addition, the human mRNAs were longer than their counterparts in the rat. None of three human hepatocellular carcinomas examined showed a marked elevation in GGT mRNA levels relative to surrounding liver tissue.     

Molecular cloning of the cDNA coding for regucalcin and its mRNA expression in mouse liver: The expression is stimulated by calcium administration

The molecular cloning of the cDNA coding for a Ca2+-binding proteinregucalcin and its mRNA expression in mouse liver were investigated. ThecDNA clone encoding a regucalcin was isolated from a mouse liver cDNAlibrary and sequenced. Analysis of the sequence of the cloned cDNA showedthat the cDNA encoded the complete amino acid sequence of the mouseregucalcin molecule; the cDNA had an open reading frame of 897 bp. Mouseregucalcin was composed of 299 amino acid residues, and its molecular weightwas estimated to be 33,406 Da. The amino acid sequence of mouse regucalcinhad 94% homology, as compared with that of rat regucalcin. Northernblot analysis with the mouse liver cDNA probe revealed that mouse regucalcinmRNA was mainly present in the liver but only slightly in the kidney with asize of 1.8 kb. Hepatic regucalcin mRNA level of male mouse was higher thanthat of female mouse. A single intraperitoneal administration of calciumchloride (5, 15, and 30 mg Ca2+/100 g body weight) to mice induced aremarkable increase in regucalcin mRNA in the liver; the increase inregucalcin mRNA levels at 30 min after calcium administration wasdose-dependent. The present results demonstrate that regucalcin mRNA in miceis uniquely expressed in the liver, and that its expression is stimulated bycalcium administration.     

Rat apolipoprotein E mRNA. Cloning and sequencing of double-stranded cDNA

A 900-base pair clone corresponding to rat liver apolipoprotein E (apo-E) mRNA, and containing a 3'-terminal poly(A) segment, was identified from a library of rat liver cDNA clones in the plasmid pBR322 by specific hybrid selection and translation of mRNA. A restriction endonuclease DNA fragment from this recombinant plasmid was used to clone the 5'-terminal region of the apo-E mRNA by primed synthesis of cDNA. A portion of the double-stranded cDNA corresponding to the 3'-terminal region of apo-E mRNA was subcloned into the bacteriophage M13mp7 and employed as a template for the synthesis of a radioactively labeled, cDNA hybridization probe. This cDNA probe was used in a RNA-blot hybridization assay that showed the length of the apo-E mRNA to be about 1200 nucleotides. The hybridization assay also demonstrated that apo-E mRNA is present in rat intestine, but at about a 100-fold lower level than that of the rat liver. The nucleotide sequence of rat liver apo-E mRNA was determined from the cloned, double-stranded cDNAs. The amino acid sequence of rat liver apo-E was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 311 amino acids. A comparison to the NH2-terminal amino acid sequence of rat plasma apo-E indicated that the first 18 amino acids of the primary translation product are not present in the mature protein and are probably removed during co-translational processing. The coding region was flanked by a 3'-untranslated region of 109 nucleotides, which contained a characteristic AAUAAA sequence that ended 13 nucleotides from a 3'-terminal poly(A) segment. At the 5'-terminal region of the mRNA, 23 nucleotides of an untranslated region were also determined. The inferred amino acid sequence of mature rat apo-E, which contains 293 amino acids, was compared to the amino acid sequence of human apo-E, which contains 299 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall, 69% of the amino acid positions are identical in both proteins. The amino acid identities are clustered in two broad domains separated by a short region of nonhomology, an NH2-terminal domain of 173 residues where 80% are identical, and a COOH-terminal domain of 84 residues where 70% are identical. These two domains may be associated with specific functional roles in the protein.     

Molecular cloning and sequence analysis of the (Na+ + K+)-ATPase beta subunit from dog kidney

cDNA complementary to mRNA coding for the beta subunit of dog renal (Na+ + K+)-ATPase has been cloned into lambda gt11 and the nucleotide sequence of the DNA has been determined. The amino acid sequence of the beta subunit polypeptide has also been deduced from the DNA. The mature form of the dog kidney beta subunit contains 302 amino acids with three potential asparagine-linked attachment sites for carbohydrate. The initiation methionine is removed during processing of the polypeptide to its mature form. Although the beta subunit is an integral membrane protein there is no signal sequence for the polypeptide, and hydropathy analysis predicts that the beta subunit polypeptide spans the cell membrane only once. Secondary structure predictions and a model for the structure of the beta subunit are proposed. DNA sequencing of the 5' non-coding region of the mRNA revealed a 200 bp inverted repeat from the coding region. Blot hybridization of a fragment of the beta subunit cDNA identified a single mRNA species of 2.7 kb in dog kidney and several rat tissues. RNA from rat liver was deficient in mRNA that hybridized to the dog kidney beta subunit cDNA, although mRNA that hybridized to an alpha subunit cDNA was detected. RNA from a human hepatoma cell line, HepG2, however, contained comparable levels of mRNA for both the alpha and the beta subunits.