Molecular cloning of cDNA for proteasomes (multicatalytic proteinase complexes) from rat liver: primary structure of the largest component (C2)

Proteasomes (multicatalytic proteinase complexes) from rat liver are composed of at least 13 nonidentical components [Tanaka, K., Yoshimura, T., Ichihara, A., Ikai, A., Nishigai, M., Morimoto, M., Sato, M., Tanaka, N., Katsube, Y., Kameyama, K., & Takagi, T. (1988) J. Mol. Biol. 203, 985-996]. The nucleotide sequence of one major component (C2) of the proteasomes has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a mixture of synthetic deoxyribonucleotides as a probe. The sequence was composed of 1174 nucleotides including a coding region for the entire protein and noncoding regions of both the 5'- and 3'-sides. The polypeptide deduced from the open reading frame consisted of 263 amino acid residues, and its molecular weight was calculated to be 29,516. The partial amino acid sequences of several fragments (approximately 45% of the total residues), which were obtained by cleavage of C2 with lysyl endopeptidase and cyanogen bromide, were determined by automated Edman degradation and found to be in complete accordance with those deduced from the cDNA sequence. The amino acid composition of C2, determined by chemical analysis, was also consistent with that deduced from the cDNA sequence, indicating that the cloned cDNA actually encoded component C2. Computer analysis revealed little structural similarity of C2 to other proteins reported so far. Northern blot hybridization analyses showed that the mRNA encoding this novel protein C2 was expressed in all the rat tissues examined and in a variety of eukaryotic organisms such as amphibia, birds, and mammals with slight species-specific differences in size.(ABSTRACT TRUNCATED AT 250 WORDS)     

cDNA cloning and sequencing of component C8 of proteasomes from rat hepatoma cells

The primary structure of component C8 of rat proteasomes (multicatalytic proteinase complexes) has been determined by sequencing on isolated cDNA clone. C8 consists of 255 amino acid residues with a calculated molecular weight of 28,417. These values are consistent with those obtained by protein chemical analyses. Computer-assisted homology comparison showed that C8 is a new protein, differing from all proteins reported so far. The overall amino acid sequence of C8 resembles those of most other components of proteasomes reported, such as components C2, C3 and C9 of rat proteasomes and certain components of other eukaryotic proteasomes, such as those of Drosophila and yeast, but shows little similarity with component C5 of rat proteasomes. C8 showed particularly close structural similarity to component YC1 of yeast proteasomes, suggesting that C8 has been highly conserved during evolution and functions ubiquitously in all eukaryotes.     

cDNA cloning and sequencing of component C9 of proteasomes from rat hepatoma cells

The nucleotide sequence of component C9 of rat proteasomes (multicatalytic proteinase complexes) has been determined from a recombinant cDNA clone isolated by screening a Reuber H4TG hepatoma cell cDNA library using synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The predicted sequence of C9 consists of 261 amino acid residues with a calculated molecular weight of 29,496. The C9 component is a novel protein, differing from known proteins, but its primary structure resembles those of other proteasome components, including C2, C3 and C5, although its similarity to C5 is relatively low, suggesting that proteasomes consist of a family of proteins that have evolved from a common ancestor.     

Complete nucleotide sequence of cDNA and predicted amino acid sequence of rat acyl-CoA oxidase

cDNA clones for rat acyl-CoA oxidase were isolated. The 3.8-kilobase mRNA sequence of the enzyme was completely covered by two overlapping clones. The composite cDNA sequence consisted of 3741 bases and contained a 1983-base open reading frame which encodes a polypeptide of 661 amino acid residues. Two species of acyl-CoA oxidase cDNA were identified. They differed in their coding nucleotide sequences, only within a small region. They contained the same number of nucleotides and can be translated in a common reading frame. They are 55% and 50% homologous in the above region at the nucleotide and the amino acid levels, respectively. Both types of cDNA were isolated from a library constructed from mRNA of a single rat, thereby suggesting the occurrence of two species of acyl-CoA oxidase in each rat. The amino terminus of the enzyme was determined to be N-acetylmethionine, which corresponds to the initiator methionine, thus confirming the absence of a terminal presequence. We reported previously that a purified preparation of the enzyme contained three polypeptide components, A, B, and C, and suggested that components B and C are produced by a proteolytic cleavage of component A (Osumi, T., Hashimoto, T., and Ui, N. (1980) J. Biochem. (Tokyo) 87, 1735-1746). We located components B and C on the amino- and the carboxyl-terminal sides of component A. Possible functional significances of several stretches of amino acids of the enzyme are discussed, based on the sequence comparison data between rat and yeast acyl-CoA oxidases.     

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.     

Rat liver glutathione S-transferases. Nucleotide sequence analysis of a Yb1 cDNA clone and prediction of the complete amino acid sequence of the Yb1 subunit

We have constructed a nearly full length cDNA clone, pGTA/C44, complementary to the rat liver glutathione S-transferase Yb1 mRNA. The nucleotide sequence of pGTA/C44 has been determined, and the complete amino acid sequence of the Yb1 subunit has been deduced. The cDNA clone contains an open reading frame of 654 nucleotides encoding a polypeptide comprising 218 amino acids with Mr = 25,919. The NH2-terminal sequence deduced from DNA sequence analysis of pGTA/C44 is in agreement with the first 19 amino acids determined for purified glutathione S-transferase A, a Yb1 homodimer, by Frey et al. (Frey, A. B., Friedberg, T., Oesch, F., and Kreibich, G. (1983) J. Biol. Chem. 258, 11321-11325). The DNA sequence of pGTA/C44 shares significant sequence homology with a cDNA clone, pGT55, which is complementary to a mouse liver glutathione S-transferase (Pearson, W. R., Windle, J. J., Morrow, J. F., Benson, A. M., and Talalay, P. (1983) J. Biol. Chem. 258, 2052-2062). We have also determined 37 nucleotides of the 5'-untranslated region and 348 nucleotides of the 3'-untranslated region of the Yb1 mRNA. The Yb1 mRNA and subunit do not share any sequence homology with the rat liver glutathione S-transferase Ya or Yc mRNAs or their corresponding subunits. These data provide the first direct evidence that the Yb1 subunit is derived from a gene or gene family which is distinct from the Ya-Yc gene family.     

Complete nucleotide sequence of cDNA and deduced amino acid sequence of rat liver arginase

Arginase (EC 3.5.3.1) catalyzes the last step of urea synthesis in the liver of ureotelic animals. The nucleotide sequence of rat liver arginase cDNA, which was isolated previously (Kawamoto, S., Amaya, Y., Oda, T., Kuzumi, T., Saheki, T., Kimura, S., and Mori, M. (1986) Biochem. Biophys. Res. Commun. 136, 955-961) was determined. An open reading frame was identified and was found to encode a polypeptide of 323 amino acid residues with a predicted molecular weight of 34,925. The cDNA included 26 base pairs of 5'-untranslated sequence and 403 base pairs of 3'-untranslated sequence, including 12 base pairs of poly(A) tract. The NH2-terminal amino acid sequence, and the sequences of two internal peptide fragments, determined by amino acid sequencing, were identical to the sequences predicted from the cDNA. Comparison of the deduced amino acid sequence of the rat liver arginase with that of the yeast enzyme revealed a 40% homology.     

Generation of troponin T isoforms by alternative RNA splicing in avian skeletal muscle. Conserved and divergent features in birds and mammals

We describe the isolation and sequence analysis of quail muscle cDNA clones encoding two closely related isoforms of the striated muscle contractile protein, troponin T. The cDNAs represent two troponin T mRNAs that exhibit an unusual sequence relationship. The two mRNAs have identical sequences over hundreds of nucleotides including 3' untranslated regions, but they differ dramatically in a discrete, internally located block of 38 nucleotides. The two alternative sequences of this 38-nucleotide block encode two different but related versions of amino acid residues 230-242, near the C terminus of the protein. These results are consistent with a novel mechanism of troponin T isoform generation by alternative mRNA splicing pathways from a single gene containing two different exons corresponding to amino acids 229-242, as recently proposed by Medford et al. (Medford, R. M., Nguyen, H. T., Destree, A. T., Summers, E., and Nadal-Ginard, B. (1984) Cell 38, 409-421). This proposal was based on analysis of a rat troponin T genomic DNA clone and a cDNA clone corresponding to one of the two alternatively spliced mRNAs. Our analysis of quail troponin T cDNA clones, apparently corresponding to two alternatively spliced mRNA species, provides important new evidence for this novel mechanism of troponin T isoform generation and reveals the differential splicing mechanism to be of great antiquity, antedating the bird-mammal divergence. One of the quail alternative isoform sequences clearly corresponds to one of the rat sequences, but the other quail alternative sequence does not correspond to either of the rat sequences. This result suggests a greater complexity of troponin T gene structure or a greater diversity of troponin T isoform genes than is currently known, and also has implications for the functional significance of the troponin T protein isoform heterogeneity. Comparison of quail and mammal alternative isoform sequences also reveals strongly conserved features which suggest that all the isoform alternative amino acid sequences are variations on a common structural theme.     

Sequence comparison of rat liver phenylalanine hydroxylase and its cDNA clones

Classical phenylketonuria, an inborn error in metabolism, is caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase. The identification of putative cDNA clones coding for rat liver phenylalanine hydroxylase by hybrid-selected translation has previously been reported [Robson, K. J., Chandra, T., MacGillivray, R. T. A., & Woo, S. L. C. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4701-4705]. The authenticity of the clones, however, could not be definitively ascertained at the time because of a lack of amino acid sequence data of the enzyme in the literature. Purified rat liver phenylalanine hydroxylase was subjected to cyanogen bromide treatment, and the resulting fragments were used for N-terminal amino acid sequence analysis. The partial amino acid sequence was then compared to that deduced from an open reading frame in the nucleotide sequence of the cDNA clones. A perfect match of 17 amino acid residues was found between the two sequences following a unique methionine codon present in the nucleotide sequence, thereby providing unambiguous evidence for the identity of the rat liver phenylalanine hydroxylase cDNA clones.     

Rat liver NAD(P)H: quinone reductase nucleotide sequence analysis of a quinone reductase cDNA clone and prediction of the amino acid sequence of the corresponding protein

We have determined the nucleotide sequence of a cDNA clone, pDTD55, complementary to rat liver quinone reductase mRNA (Williams, J.B., Lu, A.Y.H., Cameron, R.G., and Pickett, C.B. (1986) J. Biol. Chem. 261, 5524-5528). The cDNA clone contains an open reading frame of 759 nucleotides encoding a polypeptide comprised of 253 amino acids with a Mr = 28,564. To verify the predicted amino acid sequence of quinone reductase, we have been able to align the amino acid sequences of a cyanogen bromide digest of the purified enzyme to the sequence deduced from the cDNA clone. A comparison of the quinone reductase sequence with other known flavoenzymes did not reveal a significant degree of amino acid sequence homology. These data suggest that the quinone reductase gene has evolved independently from genes encoding other flavoenzymes.     

cDNA encoding type B subunit of rat phosphoglycerate mutase: its isolation and nucleotide sequence.

A cDNA encoding the nonmuscle-specific (type B) subunit of phosphoglycerate mutase (PGAM-B) was isolated and characterized. A cDNA probe, synthesized by the polymerase chain reaction (PCR) from rat liver cell mRNA using mixed primers specific to the amino acid sequence of human PGAM-B, was used to screen a rat liver cell cDNA library. The identity of the cDNA was confirmed by amino acid sequence data for 24 peptides obtained by digesting the purified protein with three different endopeptidases. The coding region encoded a polypeptide composed of 253 amino acid (plus the initiator Met). RNA blot analysis showed a single mRNA species of 1.7 kilobases in rat liver cell. The deduced amino acid sequence of rat PGAM-B was identical to that of human PGAM-B except for only one substitution at position 251 near the carboxyl terminus (valine for the rat and alanine for the human).     

Coding nucleotide sequence of rat liver malic enzyme mRNA

The nucleotide sequence of the mRNA for malic enzyme ((S)-malate NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) from rat liver was determined from three overlapping cDNA clones. Together, these clones contain 2078 nucleotides complementary to rat liver malic enzyme mRNA. The single open reading frame of 1761 nucleotides codes for a 585-amino acid polypeptide with a calculated molecular mass of about 65,460 daltons. The cloned cDNAs contain the complete 3'-noncoding region of 301 nucleotides for the major mRNA species of rat liver and 16 nucleotides of the 5'-noncoding region. Amino acid sequences of seven tryptic peptides (67 amino acids) from the purified protein are distributed through the single open reading frame and show excellent correspondence with the translated nucleotide sequence. The putative NADP-binding site for malic enzyme was identified by amino acid sequence homology with the NADP-binding site of the enoyl reductase domain of fatty acid synthetase.     

Characterization of a genomic and cDNA clone coding for the thioesterase domain and 3' noncoding region of the chicken liver fatty acid synthase gene

The fatty acid synthase (FAS) of animal tissue is a dimer of two identical subunits, each with a Mr of 260,000. The subunit is a single multifunctional protein having seven catalytic activities and a site for binding of the prosthetic group 4'-phosphopantetheine. The mRNA coding for the subunit has an estimated size of 10-16 kb, which is about twice the number of nucleotides needed to code for the estimated 2300 amino acids. We have isolated a positive clone, lambda CFAS, containing FAS gene sequences by screening a chicken genomic library with a segment of a 3' untranslated region of goose fatty acid synthase cDNA clone, pGFAS3, as a hybridization probe. The DNA insert in lambda CFAS hybridizes with synthetic oligonucleotide probes prepared according to the known amino acid sequence of the thioesterase component of the chicken liver fatty acid synthase [Yang, C.-Y., Huang, W.-Y., Chirala, S., & Wakil, S.J. (1988) Biochemistry (preceding paper in this issue)]. Further characterization of the DNA insert shows that the lambda CFAS clone contains about a 4.7-kbp segment from the 3' end of the chicken FAS gene that codes for a portion of the thioesterase domain. Complete sequence analyses of this segment including S1 nuclease mapping, showed that the lambda CFAS clone contains the entire 3' untranslated region of the chicken FAS gene and three exons that code for 162 amino acids of the thioesterase domain from the COOH-terminal end of the fatty acid synthase. Using the exon region of the genomic clone, we were able to isolate a cDNA clone that codes for the entire thioesterase domain of chicken liver fatty acid synthase.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.