Molecular cloning and nucleotide sequence of full-length cDNA for ascorbate oxidase from cultured pumpkin cells

A lambda gt11 cDNA expression library was constructed from size-fractionated poly(A)-rich RNA of cultured pumpkin cells. A full-length cDNA clone for ascorbate oxidase mRNA was selected from the library by screening with synthetic oligonucleotides designed from the amino-terminal sequence of ascorbate oxidase protein. The identity of the clone was confirmed by comparing the amino acid sequence deduced by nucleotide sequence analysis with that determined for the amino-terminal sequence of pumpkin ascorbate oxidase. The nucleotide sequence of the cDNA insert was found to contain an open reading frame of 579 codons corresponding to a signal peptide of 30 amino acids and the mature 549-residue ascorbate oxidase. Furthermore, it was found that the amino acid sequence deduced from the nucleotide sequence of the cDNA insert contained four potential N-glycosylation sites and copper-binding amino acid residues located in four regions where the sequence was identical or nearly identical to those of the other known blue multicopper oxidases Neurospora crassa laccase and human ceruloplasmin.     

Molecular cloning and nucleotide sequence of tuna growth hormone cDNA

cDNA for mRNA of tuna growth hormone (GH) was cloned by screening a cDNA library constructed from tuna pituitary gland poly(A)⁺ RNA. The nucleotide sequence of cDNA (911 bases) revealed an open reading frame of 615 nucleotides, including a sequence (51 bases) for a possible secretory protein leader peptide. Noncoding regions were found in the nucleotide sequences up- (5′-terminal: 65 bases) and down- (3′-terminal: 231 bases) stream of the open reading frame. An amino-acid sequence deduced from the nucleotide sequence of the cDNA was identical with that determined in the purified tuna GH. Tuna GH was composed of 187 amino acids, and had a calculated molecular weight of 21275. Amino-acid sequencing showed that there was one possible N-glycosylation site at Asn (Asn-Cys-Thr). Tuna GH showed amino-acid sequence homologies with chum salmon (67%), yellow tail (90%) and with human (32%) growth hormones.     

Molecular cloning and nucleotide sequence of cDNA encoding the human liver S-adenosylmethionine synthetase.

cDNA clone for human liver S-adenosylmethionine synthetase (liver-specific isoenzyme) was isolated from a cDNA library of human liver poly(A)+ RNA. The cDNA sequence encoded a polypeptide consisting of 395 amino acid residues with a calculated molecular mass of 43675 Da. Alignment of the predicted amino acid sequence of this protein with that of rat liver S-adenosylmethionine synthetase showed a high degree of similarity. The coding region of the human liver S-adenosylmethionine synthetase cDNA sequence was 89% identical at the nucleotide level and 95% identical at the amino acid level to the sequence for rat liver S-adenosylmethionine synthetase.     

Molecular cloning and nucleotide sequence of the cDNA for human liver glutamate dehydrogenase precursor

Two cDNA clones (lambda GDHh1 and lambda GDHn61) for glutamate dehydrogenase (GDH) were isolated from a human liver cDNA library in lambda gt11. The clone, lambda GDHh1, was isolated from the library using a synthetic 45mer oligodeoxy-ribonucleotide, the sequence of which was derived from the known amino acid sequence near the NH2-terminus of human liver GDH. Subsequently, lambda GDHn61 was isolated from the same library using lambda GDHh1 as a probe. The inserts of both clones contained an overlapping cDNA sequence for human liver GDH, consisting of a 5'-untranslated region of 70 bp, an open reading frame of 1677 bp, a 3'-untranslated region of 1262 bp and a 15 base poly(A) tract. The predicted amino acid sequence revealed that the human liver GDH precursor consisted of a total of 558 amino acid residues including the NH2-terminal presequence of 53 amino acids. The sequence deduced for the mature enzyme showed 94% homology to the previously reported amino acid sequence of human liver GDH.     

The nucleotide sequence of a complete chicken delta-crystallin cDNA

The nucleotide sequence of a full length cDNA of delta-crystallin mRNA from chicken lens has been determined using a delta-crystallin cDNA clone (pB delta 11), which represents the mRNA sequence of 1530 nucleotides from the poly(A) junction but does not contain the 5'-terminal sequence of 44 nucleotides of the mRNA. The 5'-terminal sequence of the mRNA, absent in the cDNA clone, has been determined with a stretch of cDNA sequence by the primer extension procedure. The amino acid sequence deduced from the nucleotide sequence is consistent with the amino acid sequences of several tryptic peptides, the total amino acid composition, and the mol. wt. of delta-crystallin estimated by SDS-polyacrylamide gel electrophoresis. The computer-assisted analysis predicts high alpha-helical content throughout the polypeptide. Sequence analyses have revealed that gene 1 encodes the mRNA from which the cDNA clone was derived.     

Nucleotide sequence of cloned cDNA specific for rat ribosomal protein L35a

A cDNA clone specific for rat ribosomal protein L35a, which is known to be a tRNA-binding protein, was isolated by hybrid-selected translation from a cDNA library made for 8-9-S poly(A)-rich RNA from regenerating rat liver. The nucleotide sequence of the cDNA was determined. It consists of one base pair from the 5' leading sequence, the entire coding sequence of 333 base pairs and 14 base pairs from the 3' trailing sequence. The primary structure of protein L35a was deduced from the nucleotide sequence. It consists of 109 amino acids with a molecular mass of 12422. The calculated amino acid composition is consistent with that reported for the hydrolysate of L35a. The amino acid sequence showed marked homology with the reported partial sequence of Xenopus leavis ribosomal protein L32, but not significant homology with Escherichia coli ribosomal proteins that bind to tRNA.     

Avocado cellulase: nucleotide sequence of a putative full-length cDNA clone and evidence for a small gene family

A cDNA library was prepared from ripe avocado fruit (Persea americana Mill. cv. Hass) and screened for clones hybridizing to a 600 bp cDNA clone (pAV5) coding for avocado fruit cellulase. This screening led to the isolation of a clone (pAV363) containing a 2021 nucleotide transcribed sequence and an approximately 150 nucleotide poly(A) tail. Hybridization of pAV363 to a northern blot shows that the length of the homologous message is approximately 2.2 kb. The nucleotide sequence of this putative full-length mRNA clone contains an open reading frame of 1482 nucleotides which codes for a polypeptide of 54.1 kD. The deduced amino acid composition compares favorably with the amino acid composition of native avocado cellulase determined by amino acid analysis. Southern blot analysis of Hind III and Eco RI endonuclease digested genomic DNA indicates a small family of cellulase genes.     

Molecular cloning and nucleotide sequence of cDNA coding for rat brain cholecystokinin precursor

A mixture of 14-mer oligodeoxynucleotides was used for the screening of a cDNA clone coding for a cholecystokinin (CCK) precursor from a cDNA library for rat brain microsomal poly(A)RNA. The longest insert is 718 bp long which was verified to contain a nearly full-length cDNA sequence coding for rat CCK precursor, because the size of CCK mRNA was estimated to be about 850 bases long by Northern blotting analysis. Sequence analysis revealed 110 bp in the 5'-untranslated region, 345 bp in the amino acid coding region corresponding to the CCK precursor and 263 bp in the 3'-noncoding region which contains polyadenylation signal AUUAAA and the poly(A) sequence. The precursor may contain a 28 amino acid signal peptide and 12 additional amino acids at the carboxyl terminus.     

Molecular cloning and nucleotide sequence of full-length cDNA for sweet potato catalase mRNA

A nearly full-length cDNA clone for catalase (pCAS01) was obtained through immunological screening of cDNA expression library constructed from size-fractionated poly(A)-rich RNA of wounded sweet potato tuberous roots by Escherichia coli expression vector-primed cDNA synthesis. Two additional catalase cDNA clones (pCAS10 and pCAS13), which contained cDNA inserts slightly longer than that of pCAS01 at their 5'-termini, were identified by colony hybridization of another cDNA library. Those three catalase cDNAs contained primary structures not identical, but closely related, to one another based on their restriction enzyme and RNase cleavage mapping analyses, suggesting that microheterogeneity exists in catalase mRNAs. The cDNA insert of pCAS13 carried the entire catalase coding capacity, since the RNA transcribed in vitro from the cDNA under the SP6 phage promoter directed the synthesis of a catalase polypeptide in the wheat germ in vitro translation assay. The nucleotide sequencing of these catalase cDNAs indicated that 1900-base catalase mRNA contained a coding region of 1476 bases. The amino acid sequence of sweet potato catalase deduced from the nucleotide sequence was 35 amino acids shorter than rat liver catalase [Furuta, S., Hayashi, H., Hijikata, M., Miyazawa, S., Osumi, T. & Hashimoto, T. (1986) Proc. Natl Acad. Sci. USA 83, 313-317]. Although these two sequences showed only 38% homology, the sequences around the amino acid residues implicated in catalytic function, heme ligand or heme contact had been well conserved during evolution.     

Molecular cloning and nucleotide sequence of human pancreatic prechymotrypsinogen cDNA

The cDNA clone encoding human prechymotrypsinogen was isolated from a human pancreas cDNA library and its nucleotide sequence was determined. The sequence consists of a 16 bp 5' non-coding region, a 789 bp amino acid coding region and a 60 bp 3' non-coding region. The predicted product consists of 263 amino acids, including 18 amino acids for a signal peptide and 15 amino acids possible for an activation peptide. Southern blot analyses using the cloned cDNA as a probe revealed that human genomic DNA carries at least two genes that are related to chymotrypsinogen.     

The nucleotide sequence of a full length cDNA clone encoding rat liver urate oxidase

Recently we reported the sequence of a cDNA clone (pUOX-1), isolated from a lambda gt11 cDNA library, which encoded for rat liver urate oxidase (EC 1.7.3.3), but this clone lacked the nucleotide sequences encoding the N-terminal region for this enzyme. Using the cDNA insert from the pUOX-1 clone as a probe, we have now isolated a full length cDNA clone, pUOX-2, from a lambda gt10 library by plaque hybridization. Nucleotide sequence analysis of the pUOX-2 clone showed that it has 1379 base pairs with an open reading frame coding for 303 amino acid residues corresponding to a molecular mass of 34,931 daltons. In addition to the open reading frame the pUOX-2 contains 439 bp of 3'-untranslated and 41 bp of 5'-untranslated sequences. The consensus polyadenylation signal AATAAA precedes a stretch of poly(A)+ residues at the 3' end.     

Molecular cloning and nucleotide sequence of cDNA encoding hydroxyindole O-methyltransferase of bovine pineal glands

Messenger RNA for hydroxyindole O-methyltransferase (EC 2.1.1.4) was partially purified from poly(A)+ RNA isolated from bovine pineal glands by sucrose density gradient centrifugation. The enriched mRNA was used to prepare a cDNA library by use of expression vector lambda gt11. The library was screened with monoclonal antibodies to the enzyme, and three cDNA clones were isolated. These cloned cDNAs cross-hybridized with one another, and their fusion proteins reacted to the monoclonal antibodies with different binding properties. Hydroxyindole O-methyltransferase enzymatic activity was demonstrated in the bacteria lysate infected with lambda HIOMT-A16, the clone that contained the longest insert. An almost full-length cDNA clone was isolated from lambda gt10 cDNA library by use of the lambda HIOMT-A16 cDNA as a probe. The primary structure of hydroxyindole O-methyltransferase was determined by analyzing the nucleotide sequence of the cDNAs. It consisted of 1939 nucleotides including a 1050-nucleotide region coding for 350 amino acids. RNA transfer blot analysis indicated that mRNA encoding hydroxyindole O-methyltransferase was present only in the pineal gland and not in the brain, retina, and liver of cow.     

Primary structure of the beta-subunit of bovine transducin deduced from the cDNA sequence

DNA complementary to the bovine retinal mRNA coding for the beta-subunit of transducin has been cloned by screening a cDNA library with oligodeoxyribonucleotide probes. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 340 amino acid residues (including the initiating methionine). Furthermore, cDNA hybridizable with a transducin beta-subunit cDNA probe has been cloned from a library derived from bovine brain poly(A)+ RNA. Comparison of the cloned cDNAs, in conjunction with blot hybridization analysis and S1 nuclease mapping of poly(A)+ RNA from bovine retina, brain and liver, suggests that the mRNAs coding for the beta-subunits of transducin and other guanine nucleotide binding proteins have the same protein-coding sequence but partly different 5'-noncoding sequences.