Molecular cloning and nucleotide sequence analysis of rat PCNA/cyclin cDNA

The 'proliferating cell nuclear antigen' (PCNA), also known as cyclin, accumulates in the nuclei of dividing and transformed cells and reacts with autoantibodies from certain lupus patients. A full-length cDNA (1195 bp) clone encoding PCNA/cyclin was isolated from rat thymocyte cDNA library. The nucleotide sequence reveals an open reading frame of 783 nucleotides coding for 28.7 kD protein. The predicted amino acid sequence and composition are in excellent agreement with the published protein data of rabbit PCNA. We report the entire nucleotide sequence of the cDNA and complete amino acid sequence for rat PCNA/cyclin.     

Isolation and expression in Escherichia coli of a cDNA clone encoding porcine pancreatic elastase

We have cloned a DNA that is complementary to the messenger RNA that encodes porcine pancreatic elastase 1 from pancreas using rat pancreatic elastase 1 cDNA as a probe. This complementary DNA contains the entire protein coding region of 798 nucleotides which encodes an elastase of 266 amino acids, and 22 and 136 nucleotides of the 5' and 3'-untranslated sequences. When this deduced amino acid sequence was compared with known amino acid sequences, a carboxy-terminal 240 amino acids long peptide was found to be identical with a mature form of porcine pancreatic elastase 1, except for two amino acids. The porcine enzyme contains the same number of amino acid residues as the rat enzyme, and their amino acid sequences are 85% homologous. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 240 amino acids including a leader and activation peptide of 26 amino acids. We expressed the cloned porcine pancreatic elastase 1 cDNA in E. coli as a lac-fused protein. The resulting fused protein showed enzymatic activity and immunoreactivity toward anti-elastase serum.     

The primary structure of rat ribosomal protein L26

The amino acid sequence of rat ribosomal protein L26 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein. Rat L26 contains 145 amino acids and has a molecular mass of 17,266 Da. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 8-16 copies of the L26 gene. The mRNA for the protein is about 650 nucleotides in length. Protein L26 has a sequence of 9 residues that may be repeated in three places.     

Molecular cloning and expression in Escherichia coli of a cDNA encoding human pancreatic elastase 2

We have cloned a DNA that is complementary to the messenger RNA that encodes human pancreatic elastase 2 from a human pancreatic cDNA library using a cloned cDNA for rat pancreatic elastase 2 messenger RNA. This complementary DNA contains the entire protein coding region of 807 nucleotides which encodes preproelastase of 269 amino acids, and 4 and 82 nucleotides of the 5'- and 3'-untranslated sequences, respectively. When this deduced amino acid sequence was compared with known amino acid sequences it showed 82% homology with rat pancreatic elastase 2. This deduced sequence also contains a 16-amino-acid peptide identical with the N-terminal sequence determined for native human pancreatic proelastase 2. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 241 amino acids including 16 and 12 amino acids for a signal peptide and an activation peptide, respectively. Moreover, the predicted key amino acid residues involved in determining the substrate specificity of mammalian pancreatic elastase 2 are retained in the human enzyme. Cloned human pancreatic elastase 2 cDNA was expressed in E. coli as a mature and pro-form protein. Both resulting proteins showed immunoreactivity toward anti-elastase serum and enzymatic activity. We have also cloned and sequenced a porcine pancreatic elastase 2 cDNA.     

The primary structure of rat ribosomal protein S10

The amino acid sequence of rat ribosomal protein S10 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein. Ribosomal protein S10 contains 165 amino acids and has a molecular mass of 18917 Da. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 17-20 copies of the S10 gene. The mRNA for the protein is about 750 nucleotides in length. Ribosomal protein S10 has several possible internal duplications; one is a tandem repeat of ten residues that is basic and contains two or three prolines.     

The primary structure of rat ribosomal protein L18a

The amino acid sequence of rat ribosomal protein L18a was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L18a contains 175 amino acids and has a molecular mass of 20,047 Da. Hybridization of the cDNA to digests of rat nuclear DNA and to a preparation of poly(A)+ mRNA suggests that there are 8-11 copies of the L18a gene and that the mRNA for the protein is about 700 nucleotides in length. Rat L18a is related to Schizosaccharomyces pombe L17 and perhaps to Halobacterium marismortui L19.     

Identification by molecular cloning of two forms of the alpha-subunit of the human liver stimulatory (GS) regulatory component of adenylyl cyclase

Two DNA molecules complementary to human liver mRNA coding for the alpha-subunit of the stimulatory regulatory component Gs of adenylyl cyclase were cloned. One of the two forms is a full-length cDNA of 1614 nucleotides plus a poly(A) tail of 59 nucleotides. The deduced sequence of 394 amino acids encoded by its open reading frame is essentially identical to that of the alpha-subunits of Gs identified by molecular cloning from bovine adrenals, bovine brain and rat brain. Two independent clones of the other type of cDNA were isolated. Both were incomplete, beginning within the open reading frame coding for the alpha s polypeptide. One codes for amino acids 5 through 394 and the other for amino acids 48 through 394 of the above described cDNA of 1614 nucleotides, and both have the identical 3'-untranslated sequence. They differ from the first cDNA, however, in that they lack a stretch of 42 nucleotides (numbers 214 through 255) and have nucleotides 213 (G) and 256 (G) replaced with C and A, respectively. This results in a predicted amino acid composition of another alpha-subunit of Gs that is shorter by 14 amino acids and contains two substitutions (Asp for Glu and Ser for Gly) at the interface between the deletion and the unchanged sequence. We call the smaller subunit alpha s1 and the larger alpha s2. This is the first demonstration of a structural heterogeneity in alpha s subunits that is due to a difference in amino acid sequence.     

The primary structure of rat ribosomal protein L21

The covalent structure of rat ribosomal protein L21 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein. Ribosomal protein L21 contains 159 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 18,322. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 16-23 copies of the L21 gene. The mRNA for the protein is about 680 nucleotides in length.     

Molecular cloning of the human casein kinase II alpha subunit

A human cDNA encoding the alpha subunit of casein kinase II and a partial cDNA encoding the rat homologue were isolated by using a Drosophila casein kinase II cDNA probe. The 2.2-kb human cDNA contains a 1.2-kb open reading frame, 150 nucleotides of 5' leader, and 850 nucleotides of 3' noncoding region. Except for the first 7 deduced amino acids that are missing in the rat cDNA, the 328 amino acids beginning with the amino terminus are identical between human and rat. The Drosophila enzyme sequence is 90% identical with the human casein kinase II sequence, and there is only a single amino acid difference between the published partial bovine sequence and the human sequence. In addition, the C-terminus of the human cDNA has an extra 53 amino acids not present in Drosophila. Northern analysis of rat and human RNA showed predominant bands of 5.5, 3.1, and 1.8 kb. In rat tissues, brain and spleen had the highest levels of casein kinase II alpha subunit specific RNA, while skeletal muscle showed the lowest. Southern analysis of human cultured cell and tissue genomic DNA using the full-length cDNA probe revealed two bands with restriction enzymes that have no recognition sites within the cDNA and three to six bands with enzymes having single internal sites. These results are consistent with the possibility that two genes encode the alpha subunits.     

Molecular cloning of complementary DNA encoding one of the human pancreatic protease E isozymes

We have cloned a DNA from a human pancreatic cDNA library using a cloned rat pancreatic elastase 1 cDNA as a probe, and determined its nucleotide sequence. This cDNA contains a coding region of 810 nucleotides which encodes a 270-amino-acid protein. The deduced amino acid sequence shows less than 60% homologies with rat and porcine pancreatic elastase 1, although its substrate binding region is homologous with those of the above elastases 1. When this deduced amino acid sequence was compared with known amino acid sequences of pancreatic proteases other than elastases, it was found to contain an amino acid sequence which was highly homologous with the N-terminal amino acid sequence of porcine pancreatic protease E. We also purified human pancreatic protease E isozymes from human pancreatic juice, and determined their N-terminal amino acid sequences. One of the isozymes does not hydrolyze elastin but does hydrolyze a synthetic substrate. Endoglycosidase F digests glycoside bonds of the isozyme. These results suggest that the cDNA cloned by us corresponded to one of the human protease E isozymes.     

The primary structure of rat ribosomal protein S8.

The amino acid sequence of rat ribosomal protein S8 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2- and carboxyl-terminal amino acid sequences of the protein. Ribosomal protein S8 contains 207 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 23,928. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 7-9 copies of the S8 gene. Ribosomal protein S8 contains a possible internal repeat that has 12 or 13 residues, is basic, and occurs 5 times in the protein.     

The primary structure of rat ribosomal protein L12

The covalent structure of the rat 60S subunit protein L12 which is a component of the ribosomal elongation factor binding domain was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein. L12 has 165 amino acids and a molecular weight of 17,834. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 11-13 copies of the L12 gene. The mRNA for the protein is about 800 nucleotides in length. Rat L12 is homologous to Saccharomyces cerevisiae L15. The cDNA contains the highly repetitive DNA sequence, R.dre.1, in the 3' noncoding region.     

Nucleotide sequence of cloned cDNA specific for rat ribosomal protein L27

We constructed cDNA libraries from 8-9-S poly(A)-rich RNA from regenerating rat liver, isolated clones specific for ribosomal protein L27 and determined the nucleotide sequences of the cDNA clones. The longest cDNA consists of 15 base pairs from the 5' leading sequence, the entire coding sequence of 411 base pairs, and the 3' trailing sequence of 59 base pairs including the poly(A) tail. The primary structure of protein L27 was deduced from the sequence of nucleotides. Protein L27 contains 135 amino acids and has a molecular mass of 15,666 Da. The amino-terminal sequence agreed well with the published partial amino acid sequence and the calculated amino acid composition is also consistent with the reported composition determined for the hydrolyzate of L27.     

The primary structure of rat ribosomal protein S7

The amino acid sequence of the rat 40S ribosomal subunit protein S7 was deduced from the sequence of nucleotides in two recombinant cDNAs and confirmed from the amino acid sequence of a cyanogen bromide peptide obtained from the protein. Ribosomal protein S7 has 194 amino acids and has a molecular mass of 22,113. Hybridization of the cDNA to digest of nuclear DNA suggests that there are 14-16 copies of the S7 gene. The mRNA for the protein is about 725 nucleotides in length. Rat S7 is homologous with Xenopus laevis S8. The protein contains a possible internal duplication of 10 residues.     

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.     

Cloning and sequence analysis of a cDNA for a rat liver glutathione S-transferase Yb subunit.

We have isolated a Yb-subunit cDNA clone from a GSH S-transferase (GST) cDNA library made from rat liver polysomal poly(A) RNAs. Sequence analysis of one of these cDNA, pGTR200, revealed an open reading frame of 218 amino acids of Mr = 25,915. The deduced sequence is in agreement with the 19 NH2-terminal residues for GST-A. The sequence of pGTR200 differs from another Yb cDNA, pGTA/C44 by four nucleotides and two amino acids in the coding region, thus revealing sequence microheterogeneity. The cDNA insert in pGTR200 also contains 36 nucleotides in the 5' noncoding region and a complete 3' noncoding region. The Yb subunit cDNA shares very limited homology with those of the Ya or Yc cDNAs, but has relatively higher sequence homology to the placental subunit Yp clone pGP5. The mRNA of pGTR200 is not expressed abundantly in rat hearts and seminal vesicles. Therefore, the GST subunit sequence of pGTR200 probably represents a basic Yb subunit. Genomic DNA hybridization patterns showed a complexity consistent with having a multigene family for Yb subunits. Comparison of the amino acid sequences of the Ya, Yb, Yc, and Yp subunits revealed significant conservation of amino acids (approximately 29%) throughout the coding sequences. These results indicate that the rat GSTs are products of at least four different genes that may constitute a supergene family.     

The primary structure of rat ribosomal protein S19

The covalent structure of rat ribosomal protein S19 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein. Ribosomal protein S19 contains 144 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 15,944. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 15-18 copies of the S19 gene. The mRNA for the protein is about 640 nucleotides in length. Rat S19 is related to Saccharomyces cerevisiae S16A and to Halobacterium marismortui S12.     

cDNA cloning and sequencing for the import precursor of subunit B in H(+)-ATP synthase from rat mitochondria

The nucleotide sequence of the import precursor of subunit b of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 1,124 nucleotides including a coding region for the import precursor of subunit b and noncoding regions of both the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame consisted of 256 and 214 amino acid residues with a molecular weight of 28,867 and 24,628, respectively. The presequence of 42 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.     

Structure of the rat prolactin gene

The organization and sequence of the rat preprolactin gene has been investigated. Analysis of two different plasmids containing pituitary cDNA inserts has provided the complete 681-nucleotide coding sequence of preprolactin as well as 17 nucleotides preceding the initiation codon and 90 nucleotides following the termination codon. Digestion of rat chromosomal DNA with the restriction endonuclease Eco RI followed by size fractionation and hybridization to a labeled prolactin cDNA probe has demonstrated that prolactin genomic sequences are located on 6.0-, 3.9-, and 2.9-kilobase fragments. The 6.0- and 3.9-kilobase fragments were isolated from a library of cloned rat DNA fragments. The sequence of more than 1800 nucleotides of the cloned DNA has been determined. The sequenced region contains coding regions of 180 and 189 nucleotides which specify the COOH-terminal 123 amino acids of the 227-amino-acid sequence of rat preprolactin. These coding regions are separated by an intervening sequence of 597 nucleotides. At least one other large intervening sequence separates this region from the region coding for the NH2-terminal portion of preprolactin. Hybridization experiments suggested that the intervening sequences of the rat prolactin gene contain DNA sequences which are repeated elsewhere in the rat genome.