Cloning and expression of a cDNA encoding mouse kidney -amino acid oxidase

A cDNA encoding -amino acid oxidase (DAO;EC 1.4.3.3) has been isolated from a BALB/c mouse kidney cDNA library by hybridization with the cDNA for the porcine enzyme. Analysis of the nucleotide (nt) sequence of the clone revealed that it has a 1647-nt sequence with a 5′-terminal untranslated region of 68 nt that encodes 345 amino acids (aa), and a 3′-terminal untranslated region of 544 nt that contains the polyadenylation signal sequence ATTAAA. The deduced aa sequence showed 77 and 78% aa identity with the porcine and human enzymes, respectively. Two catalytically important aa residues, Tyr²²⁸ and His³⁰⁷, of the porcine enzyme, were both conserved in these three species. RNA blot hybridization analysis indicated that a DAO mRNA, of 2 kb, exists in mouse kidney and brain, but not liver. Synthesis of a functional mouse enzyme in Escherichia coli was achieved through the use of a vector constructed to insert the coding sequence of the mouse DAO cDNA downstream from the tac promoter of plasmid pKK223-3, which was designed so as to contain the lac repressor gene inducible by isopropyl-β- -thiogalactopyranoside. Immunoblot analysis confirmed the synthesis and induction of the mouse DAO protein, and the molecular size of the recombinant mouse DAO was found to be identical to that of the mouse kidney enzyme. Moreover, the maximum activity of the mouse recombinant DAO was estimated to be comparable with that of the porcine DAO synthesized in E. coli cells.     

Cloning and sequence analysis of porcine myoglobin cDNA

Porcine myoglobin cDNA clones have been isolated from a cDNA library prepared from enriched heart-myoglobin mRNA. Sequence analysis revealed 59 nucleotides (nt) in the 5'-untranslated, 462 nt in the amino acid (aa)-coding, and 590 nt in the 3'-untranslated regions. The myoglobin cDNA showed a high G + C content (60%). When the nt sequence of the porcine myoglobin cDNA is compared with those of seal and human myoglobin cDNAs deduced from the corresponding genomic myoglobin genes [Blanchetot et al., Nature 301 (1983) 732-734; Weller et al., EMBO J. 3 (1984) 439-446; Akaboshi, Gene 33 (1985) 241-249], a high degree of homology is observed in the 5'-untranslated region and in parts of the 3'-untranslated region, as well as in the coding region.     

Isolation and sequence determination of cDNA clones for porcine and human lipoamide dehydrogenase. Homology to other disulfide oxidoreductases

A 2.3-kilobase cDNA clone encoding lipoamide dehydrogenase was isolated from a porcine adrenal medulla library in the vector pCD by screening with four synthetic oligonucleotide probes corresponding to amino acid sequence from tryptic peptides of porcine lipoamide dehydrogenase. A 450-bp fragment of the porcine cDNA was used to screen a human small cell lambda gt10 library at reduced stringency. Overlapping human cDNA clones of various lengths were isolated, the largest of which was again 2.3 kilobases in length. Sequencing of both porcine and human cDNAs revealed a short 5'-untranslated region followed by 1530-bp of coding region and 700 bp of 3'-untranslated region preceding a poly(A) tail. The porcine cDNA displayed coding regions corresponding to the known tryptic peptides and a 35-amino acid leader sequence involved in targeting of the protein to the mitochondria. The human lipoamide dehydrogenase cDNA is 96% identical to the porcine at the amino acid level. Alignment of the deduced amino acid sequence of human lipoamide dehydrogenase with human erythrocyte glutathione reductase and mercuric reductase from Tn501 revealed extensive homologies throughout the primary sequence, suggesting that secondary and tertiary structure is also similar among these three enzymes.     

Cloning and sequence analysis of cDNAs encoding mammalian mitochondrial malate dehydrogenase

A cDNA clone, named ppmMDH-1 and covering a part of the porcine mitochondrial malate dehydrogenase (mMDH; L-malate:NAD+ oxidoreductase, EC 1.1.1.37) mRNA, was isolated from a porcine liver cDNA library with a mixture of 24 oligodeoxyribonucleotides as a probe. The sequences of the probe were deduced from the known sequence of porcine mMDH amino acid residues 288-293. ppmMDH-1 covered the coding region for porcine mMDH amino acid residues 17-314 and the 3' untranslated region. Subsequently, mouse mMDH cDNA clones were isolated from a mouse liver cDNA library with the ppmMDH-1 cDNA as a probe. One of the clones, named pmmMDH-1 and containing a cDNA insert of about 1350 base pairs, was selected for sequence analysis, and the primary structure of the mouse precursor form of mMDH (pre-mMDH) was deduced from its cDNA sequence. The sequenced coding regions for the porcine and mouse mMDH mRNAs showed about 85% homology. When the deduced amino acid sequence of the mouse pre-mMDH was compared with that of the porcine mMDH, they shared a 95% homology, and the mouse pre-mMDH yielded a leader sequence consisting of 24 amino acid residues and a mature mMDH, consisting of 314 amino acid residues. The leader sequence contained three basic amino acid residues, no acidic residues, and no hydrophobic amino acid stretch. The mouse mMDH leader sequence was compared with those of three other rodent mitochondrial matrix proteins.     

Gene expression of D-amino acid oxidase in rabbit kidney

Although D-amino acid oxidase (DAO) [EC 1.4.3.3] activity in rabbit kidney extract was undetectable, protein immunoreactive toward rabbit anti-pig kidney DAO antiserum and RNAs that hybridized with fragments of human and pig DAO cDNAs were detected distinctly in the rabbit kidney. A cDNA clone, RD22, was isolated from the rabbit kidney cDNA library by hybridization with a fragment of human DAO cDNA. Analysis of the nucleotide sequence revealed a 2,018 nucleotide sequence encoding a protein consisted of 347 amino acids. The number of amino acid residues was identical with those of human and pig DAOs, and the amino acid sequence showed 80 and 83% identity with pig and human DAOs, respectively. RNAs that hybridized with RD22 DNA fragment also existed in rabbit kidney, and their sizes were the same as those of the RNAs detected with the human and pig DAO cDNA fragments. RD22-derived protein was hardly synthesized by an in vitro expression system. However, a cDNA fragment lacking most of the 5'-untranslated region and its mutants containing base changes around the initiation codon did direct protein synthesis. Moreover, the protein derived from the partial cDNA fragment containing a large part of the coding region sequence showed immunoreactivity toward anti-pig DAO antiserum. The results suggest that one of the causes of the very poor synthesis of DAO protein in rabbit kidney is translational suppression in the synthetic process.     

Molecular cloning and nucleotide sequences of cDNAs specific for rat liver ribosomal proteins S17 and L30

cDNA clones coding for rat liver ribosomal proteins S17 and L30 have been isolated by positive hybridization-translation assay from a cDNA library prepared from 8-9S poly(A)+RNA from free polysomes of regenerating rat liver. The cDNA clone specific for S17 protein (pRS17-2) has a 466-bp insert with the poly(A) tail. The complete amino acid (aa) sequence of S17 protein was deduced from the nucleotide sequence of the cDNA. S17 protein consists of 134 aa residues with an Mr of 15 377. The N-terminal aa sequence of S17 protein determined by automatic Edman degradation is consistent with the sequence data. The aa sequence of S17 shows strong homology (76.9%) to that of yeast ribosomal protein 51 [Teem and Rosbash, Proc. Natl. Acad. Sci. USA 80 (1983) 4403-4407] in the two-thirds N-terminal region. The cDNA clone specific for L30 protein (pRL30) has a 394-bp insert. The aa sequence of L30 protein was deduced from the nucleotide sequence of the cDNA. The protein consists of 114 aa residues with an Mr of 12 652. When compared with the N-terminal aa sequence of rat liver L30 protein [Wool, Annu. Rev. Biochem. 48 (1979) 719-754], pRL30 was found not to contain the initiation codon and 5'-noncoding region. The cDNA showed twelve silent changes in the coding region, one point mutation and one base deletion in the 3'-noncoding region, compared with mouse genomic DNA for L30 protein [Wiedemann and Perry, Mol. Cell Biol. 4 (1984) 2518-2528].     

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.     

Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning.

A lambda gt11 library constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats was screened for rat tropoelastin cDNAs. The first screen, utilizing a human tropoelastin cDNA clone, provided rat tropoelastin cDNAs spanning 2.3 kb of carboxy-terminal coding sequence and extended into the 3'-untranslated region. A subsequent screen using a 5' rat tropoelastin cDNA clone yielded clones extending into the amino-terminal signal sequence coding region. Sequence analysis of these clones has provided the complete derived amino acid sequence of rat tropoelastin and allowed alignment and comparison with published bovine cDNA sequence. While the overall structure of rat tropoelastin is similar to bovine sequence, numerous substitutions, deletions, and insertions demonstrated considerable heterogeneity between species. In particular, the pentapeptide repeat VPGVG, characteristic of all tropoelastins analyzed to date, is replaced in rat tropoelastin by a repeating pentapeptide, IPGVG. The hexapeptide repeat VGVAPG, the bovine elastin receptor binding peptide, is not encoded by rat tropoelastin cDNAs. Variations in coding sequence between rat tropoelastin cDNA clones were also found which may represent mRNA heterogeneity produced by alternative splicing of the rat tropoelastin pre-mRNA.     

Nucleotide sequence of mouse 5-aminolevulinic acid synthase cDNA and expression of its gene in hepatic and erythroid tissues

The cDNA coding for 5-aminolevulinic acid (ALA) synthase (EC 2.3.1.37) in both liver and anemic spleen of the mouse has been cloned. The liver clone was selected by complementation of an Escherichia coli hemA mutant. Erythroid clones were obtained by screening a cDNA library made from mouse anemic spleen RNA, using the liver cDNA as a probe. The sequences of the spleen-derived and liver-derived cDNAs are identical. The nucleotide sequence and predicted amino acid (aa) sequence of a 1.85-kb spleen-derived cDNA is presented. The mouse ALA synthase as sequence displays extensive homology to ALA synthase of chick embryonic liver. The ALA synthase mRNA, detected by Northern blot analysis, was the same size, approx. 2.3 kb, in mouse liver, anemic spleen, and mouse erythroleukemia cells. It is therefore unlikely that different isozymic forms of ALA synthase are present in mouse erythroid and hepatic tissue and this is not the basis for the different effects of heme and porphyrinogenic compounds on the expression of liver and erythroid ALA synthase.     

Structure and expression of cDNA for D-amino acid oxidase active against cephalosporin C from Fusarium solani

D-Amino acid oxidase (DAO) was extracted and purified from cultured mycelia of Fusarium solani M-0718 (FERM P-2688). The enzyme was able to oxidatively deaminate cephalosporin C to 7-beta-(5-carboxy-5-oxopentanamido)cephalosporanic acid. Ninety-eight amino acid residues of the F. solani DAO were determined by sequence analysis of 9 peptides derived from Acromobacter protease I digests of the protein. Complementary DNAs encoding F. solani DAO were isolated from the F. solani cDNA library by hybridization with synthetic oligonucleotide probes corresponding to the partial amino acid sequences. Analysis of the nucleotide sequences of the clones revealed a 1,186-nucleotide sequence with a 5'-terminal untranslated region of 41 nucleotides, an open reading frame of 1,083 nucleotides that encoded 361 amino acids, and a 3'-terminal untranslated region of 62 nucleotides. The amino acid sequence of F. solani DAO had 25% homology to that of porcine kidney DAO [EC 1.4.3.3] and 37% homology to that of Trigonopsis variabilis DAO. The constructed plasmid overproduced F. solani DAO in Escherichia coli. The recombinant DAO had almost the same molecular activity as the native DAO against cephalosporin C.     

The isolation of cDNA clones for human apolipoprotein E and the detection of apoE RNA in hepatic and extra-hepatic tissues.

We have isolated cDNA clones coding for apolipoprotein E (apoE) from a cDNA library prepared from adult human liver mRNA. Mixtures of 128 different oligonucleotides, 17 residues long were synthesised to be complementary to regions of the mRNA corresponding to amino acids 1-6 and 151-156. Five independent apoE clones were selected by direct screening of 5000 recombinants with the two oligonucleotide mixtures. Two overlapping clones contain the 3'-untranslated sequence, the entire coding sequence and an additional 30 bases 5' to the amino terminus of the mature protein. The DNA sequence has been determined spanning the known sites of amino acid substitutions which account for the observed protein polymorphism of apoE. Using the clones as probes in Northern blot analysis of total human liver and kidney RNAs and leucocyte poly(A)+ RNA we have detected a single species of mRNA in liver and kidney of 1.2 kb and two larger species in leucocyte RNA. The level of expression of the mRNA in kidney is approximately 10% of that in liver while the level of apoE RNA sequences in the leucocytes is less than 1% of that in the liver.     

Evidence for transcription and potential translation of the human 1.9 kb HindIII repetitive element.

Recombinant cDNA clones corresponding to the human 1.9kb HindIII repetitive element have been isolated from a cDNA library of liver cytoplasmic polyadenylated RNA. These cDNAs share 95% homology with the reported genomic DNA sequence and a similar amount of homology at the amino acid level with putative coding sequences (see preceding article by Mottez et al). They were isolated as two of four false positives from a human cDNA library in lambda gt11 and were selected with an antibody to an unrelated enzyme. These results provide direct evidence that this repetitive element is transcribed to form poly(A)+ RNA which could be translatable. Also, these observations may add to our understanding of the sources of false positives which are frequently observed in screens of cDNA libraries with antibodies as probes.     

Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae

cDNA clones encoding human lysozyme were isolated from a human histiocytic cell line (U-937) and a human placenta cDNA library. The clones, ranging in size from 0.5 to 0.75 kb, were identified by direct hybridization with synthetic oligodeoxynucleotides. The nucleotide sequence coding for the entire protein was determined. The derived amino acid sequence has 100% homology with the published amino acid (aa) sequence; the leader sequence codes for 18 aa. Expression and secretion of human lysozyme in Saccharomyces cerevisiae was achieved by placing the cloned cDNA under the control of a yeast gene promoter (ADH1) and the alpha-factor peptide leader sequence.     

Isolation of two genomic sequences encoding the Mr = 14,000 subunit of rat prostatein

Complementary DNAs to rat ventral prostate poly(A) RNA were cloned into pBR322 by the "dG-dC tailing" procedure. Clones containing cDNAs to the mRNAs coding for each of the three subunits of a major secretory protein (prostatein) were identified by hybrid-arrested translation. A 457-nucleotide base pair cDNA (E45) and a portion of a 365-base pair cDNA (E85) were analyzed to determine the composite complete DNA coding sequence for the Mr = 14,000 (C3) subunit of prostatein. A sequence of 12-nucleotide bases (TTTGCTGCTATG) in the signal peptide of C3 was noted to be homologous to signal peptide nucleotide sequences reported in cDNAs coding for the other two prostatein subunits, Mr = 6,000 (C1) and 10,000 (C2). Complementary DNA coding for the C3 subunit was used as a hybridization probe to screen an EcoRI rat genomic DNA library. Two unique 12-kilobase genomic clones, each containing mRNA coding sequences within 2.5-3-kilobase fragments, were identified by restriction enzyme mapping and Southern blot analysis. Restriction enzyme sites within the coding regions of both genes were analogous to the cDNA. Differences in restriction enzyme sites in regions of intervening sequences and flanking DNA established the uniqueness of the two genes. It is suggested that both genes may be transcribed in vivo.     

Cloning and functional study of porcine parotid hormone, a novel proline-rich protein

A parotid gland hormone that stimulates intradentinal fluid movement is believed to play a significant role in maintaining the vitality of dentin. This hormone has been purified from porcine parotid glands and partially sequenced in our previous study (Tieche, J. M., Leonora, J., and Steinman, R. R. (1980) Endocrinology 106, 1994-2005). We now report the cloning and functional study of porcine cDNAs that code for this hormone and its complete amino acid sequence. Three cDNA clones were isolated from a porcine parotid cDNA library. The last 30 amino acids encoded by two of the cDNAs agreed with the amino acid sequence of the isolated parotid hormone. In situ hybridization and immunohistochemical staining demonstrated that the acinar cells of the parotid glands were the primary location for both the parotid hormone-related mRNAs and the translation products. A 216-bp fragment of the cDNA that contains the coding sequence for the porcine hormone was subcloned into an expression vector, and the protein expression was detected by immunoblot analysis and quantified by enzyme-linked immunosorbent assay. In addition, the 30-amino acid parotid hormone was synthesized. Both the expressed and the synthetic proteins were biologically active in that they enhanced intradentinal fluid movement as measured by intradentinal dye penetration.     

Isolation and characterization of a cDNA clone for porcine thyroid peroxidase

We undertook the molecular cloning of porcine thyroid peroxidase (TPO). Four oligonucleotide probes were synthesized on the basis of amino acid sequences of 3 tryptic peptides from highly purified porcine TPO. These probes were used to screen a pig thyroid cDNA library. Seven of 16 selected clones (0.45-1.15 kb in size) reacted with all 4 probes. Nucleotide sequencing of the 1.15 kb at the 3'-end of the structural gene revealed the complementary sequence to all 4 probes as well as the nucleotides coding for the entire length of the 3 tryptic peptides. There is an open reading frame of 332 amino acid residues. On Northern blot analysis this gene codes for an mRNA species of 2.85 kb, corresponding to the anticipated size of the mRNA for the intact TPO molecule. We have therefore cloned and characterized a cDNA clone coding for approx. 36% of porcine thyroid peroxidase.     

Characterization of two different genes (cDNA) for cytochrome c oxidase subunit VIa from heart and liver of the rat.

By antibody screening of a rat liver and a rat heart cDNA library in lambda gt11 two clones coding for the liver- and heart-specific subunit VIa of rat cytochrome c oxidase were isolated. In the heart cDNA sequence a TAA stop codon was found in frame 18 bp 5' upstream of the first methionine codon, thus excluding a leader sequence for this protein. The two cDNAs contain the full-length coding region of two subunits. The amino acid sequences of the two subunits show only 50% homology, whereas 74% homology was found between rat heart and bovine heart subunit VIa. By Northern blot analysis it is shown that the gene for subunit VIa from heart is only expressed in heart and skeletal muscle, whereas that from liver is also expressed in kidney, brain, heart and weakly in muscle.     

In photoinhibited photosystem II particles pheophytin photoreduction remains unimpaired

Partial cDNAs coding for human protein S were isolated from a pUC9 human liver cDNA library. Together, the overlapping clones span a (partial) 5'-non-coding region, and the complete protein S coding and 3'-untranslated regions. The derived amino acid sequence deviates at five positions from two previously reported protein S sequences. Two of these differences (Phe instead of Leu at position -16 and Tyr instead of Asp at position 222) are found in regions that are important for the post-translational modification of protein S, the gamma-carboxylation of glutamic acid and the hydroxylation of asparagine, respectively.