Molecular cloning and nucleotide sequence of a bovine alpha-lactalbumin cDNA

A cDNA clone for the bovine milk protein, alpha-lactalbumin (alpha LA), has been identified using a rat cDNA probe. The bovine cDNA clone is 703 nucleotides (nt) long, contains 8 nt of 5'-untranslated sequence and 269 nt of 3'-untranslated sequence. When compared with previously reported sequences, the bovine alpha LA mRNA sequence has 74% similarity with rat alpha LA mRNA, 79% similarity with human mRNA and 74% similarity with guinea pig mRNA.     

Molecular cloning and nucleotide sequence of a cDNA encoding Euglena gracilis cytochrome c1

The amino acid sequence of the mature protein of Euglena gracilis cytochrome c1 was determined by sequencing of its cDNA. A cDNA expression library was constructed from Euglena poly(A)+ RNA in phage lambda gt11 and screened with an antiserum raised against cytochrome c1 polypeptide isolated from purified E. gracilis complex III. An isolated cDNA clone consisted of 872 base pairs and encoded the mature protein with 243 amino acids. The deduced amino acid sequence contained the unusual heme binding sequence-Phe-Ala-Pro-Cys-His- (Mukai, K. et al. (1989) Eur. J. Biochem. 178, 649-656) instead of the typical sequence,-Cys-X-Y-Cys-His-, commonly found in C-type cytochromes. Comparison of the sequence with those of several other cytochromes c1 revealed that Euglena cytochrome c1 conserved the residues probably ligating heme-iron, those supposed to interact with cytochrome c and regions anchoring the mitochondrial inner membrane.     

Molecular cloning and nucleotide sequence analysis of a cDNA encoding pea cytosolic ascorbate peroxidase

A cDNA clone encoding the cytosolic ascorbate peroxidase of pea (Pisum sativum L.) was isolated and its nucleotide sequence determined. While ascorbate peroxidase shares limited overall homology with other peroxidases, significant homology with all known peroxidases was found in the vicinity of the putative active site.     

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.     

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 cDNA encoding human muscle glycogen debranching enzyme.

cDNA comprising the entire length of the human muscle glycogen debranching enzyme was cloned and its nucleotide sequence determined. The debrancher mRNA includes a 4545-base pair coding region and a 2371-base pair 3'-nontranslated region. The calculated molecular mass of the debrancher protein derived from cDNA sequence is 172,614 daltons, consistent with the estimated size of purified protein (Mr 165,000 +/- 500). A partial amino acid sequence (13 internal tryptic peptides with a total of 213 residues) determined on peptides derived from purified porcine muscle debrancher protein confirmed the identity of the cDNA clone. Comparison of the amino acid sequence predicted from the human glycogen debrancher cDNA with the partial protein sequence of the porcine debrancher revealed a high degree (88%) of interspecies sequence identity. RNA blot analysis showed that debrancher mRNA in human muscle, lymphoblastoid cells, and in porcine muscle are all similar in size (approximately 7 kilobases). Two patients with inherited debrancher deficiency had a reduced level of debrancher mRNA, whereas two other patients had no detectable abnormality in RNA blots. The isolation of the debrancher cDNA and determination of its primary structure is an important step toward defining the structure-function relationship of this multifunctional enzyme and in understanding the molecular basis of the type III glycogen storage disease.     

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.     

Molecular cloning and sequence analysis of cDNA encoding human ferrochelatase

The cDNA encoding human ferrochelatase [EC 4.99.1.1] was isolated from a human placenta cDNA library in bacteriophage lambda gt11 by screening with a radiolabeled fragment of mouse ferrochelatase cDNA. The cDNA had an open reading frame of 1269 base pairs (bp) encoding a protein of 423 amino acid residues (Mr. 47,833) with alternative putative polyadenylation signals in the 3' non-coding regions and poly (A) tails. Amino acid sequencing showed that the mature protein consists of 369 amino acid residues (Mr. 42,158) with a putative leader sequence of 54 amino acid residues. The human enzyme showed an 88% identity to mouse enzyme and 46% to yeast enzyme. Northern blot analysis showed two mRNAs of about 2500 and 1600 bp for ferrochelatase in K562 and HepG2 cells. As full-length cDNA for human ferrochelatase is now available, molecular lesions related to erythropoietic protoporphyria can be characterized.     

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 cDNA encoding nucleoside diphosphate kinase of rice (Oryza sativa L.)

We isolated a rice cDNA encoding nucleoside diphosphate kinase (NDK, EC 2.7.4.6). The deduced amino acid sequence of the rice NDK shows highest homology to spinach NDK-I. The rice NDK gene exhibits a strong codon bias (73.8% GC) in the third position of the codon. DNA blot analysis indicated that at least single NDK gene is present in rice genome.     

Molecular cloning and nucleotide sequence of rat lingual lipase cDNA.

Purified rat lingual lipase (EC3113), a glycoprotein of approximate molecular weight 52,000, was used to generate polyclonal antibodies which were able to recognise the denatured and deglycosylated enzyme. These immunoglobulins were used to screen a cDNA library prepared from mRNA isolated from the serous glands of rat tongue cloned in E. coli expression vectors. An almost full length cDNA clone was isolated and the nucleotide and predicted amino acid sequence obtained. Comparison with the N-terminal amino acid sequence of the purified enzyme confirmed the identity of the cDNA and indicated that there was a hydrophobic signal sequence of 18 residues. The amino acid sequence of mature rat lingual lipase consists of 377 residues and shares little homology with porcine pancreatic lipase apart from a short region containing a serine residue at an analogous position to the ser 152 of the porcine enzyme.     

Molecular cloning and nucleotide sequence of murine 2,3-bisphosphoglycerate mutase cDNA

Cloning and sequencing of a murine cDNA with the entire coding region of 2,3-bisphosphoglycerate mutase is reported, as a prerequisite for further expression studies of this erythroid specific enzyme in Friend mouse erythroleukemia cells. A comparison between species of the deduced amino acid sequences of these proteins shows 20 substitutions between mouse and human and 21 between mouse and rabbit: none of these substitutions are in positions assumed to be in the active site. Amino acid alignment with the other related enzymes, the phosphoglycerate mutases, in combination with crystallographic data from yeast phosphoglycerate mutase, gives some insight into the structure/function correlation for this protein family. Amino acid residues which are most likely critical for either 2,3-bisphosphoglycerate mutase or phosphoglycerate mutase function are pointed out. Concerning the phylogenetic analysis, phosphoglycerate mutases B and M from mammalians appear to have diverged with the yeast enzyme from a common ancestor, before the emergence of the 2,3-bisphosphoglycerate mutases.     

Molecular cloning and nucleotide sequence of a human renin cDNA fragment.

We have studied human renin messenger RNA by hybridization with the mouse submaxillary gland (SMG) renin cDNA probe. The human kidney messenger RNA is about 1.6 kilobase (kb) long, similarly to the mouse SMG renin mRNA. A kidney renin cDNA clone of 1.1 kb length was obtained. A comparison of nucleotide sequences of mouse and human cDNA clones reveals conservation of residues involved in catalytic mechanisms and a potential glycosylation site. The human renin molecular probe allowed us to study renin expression in human chorionic tissue. The chorionic and kidney renin messenger RNAs are similar in length. The Southern blot analysis reveals the presence of a single renin gene in human DNA.     

Molecular cloning and nucleotide sequence of cDNA coding for calf preprochymosin.

DNA complementary to calf stomach mRNA has been synthesised and inserted into the Pst1 site of pAT153 by G-C tailing. Clones containing sequences coding for prochymosin were recognised by colony hybridisation with cDNA extended from a chemically synthesised oligodeoxynucleotide primer, the sequence of which was predicted from the published amino acid sequence of calf prochymosin. Two clones were identified which together contained a complete copy of prochymosin mRNA. The nucleotide sequence is in substantial agreement with the reported amino acid sequence of prochymosin and shows that this protein has a mol.wt. of 40431 and chymosin a mol.wt. of 35612. The sequence also indicates that prochymosin is synthesised as a precursor molecule, preprochymosin, having a 16 amino acid hydrophobic leader sequence analogous to that reported for other secreted proteins.     

Molecular cloning and nucleotide sequence of human alpha 1 acid glycoprotein cDNA

A cDNA clone has been isolated from a pEX expression library that encodes alpha 1 acid glycoprotein. We present the complete nucleotide sequence encoding this protein and compare the derived amino acid sequence with pre-existing data.     

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.     

Molecular cloning and sequence analysis of cDNA encoding human cholesterol 7 alpha-hydroxylase

A complete cDNA clone encoding human cholesterol 7 alpha-hydroxylase has been isolated using a rat P-450ch7 alpha cDNA insert [(1989) FEBS Lett. 257, 97-100] as a probe and totally sequenced. The cDNA contained 1512-base pair open reading frame encoding 504 amino acid residues (Mr 57,630), 39-base pair 5'-untranslated region 1322-base pair 3'-ultranslated region including 20 nucleotides of poly A tail in the total length of 2873 base pairs. The deduced amino acid sequence showed 82% similarity to rat P-450ch7 alpha. Unique amino acid residues were observed in putative binding domains for heme and steroid which are highly conserved in most steroidogenic P-450s.