Molecular cloning of cDNA coding for human preprourokinase

A cDNA library was constructed in pBR322 from 18S to 20S mRNA that was extracted from human kidney cells, fractionated on oligo(dT)-cellulose column and sucrose-density gradient, and confirmed for urokinase production in Xenopus laevis oocytes. The Escherichia coli RR1 transformants were hybridized to synthetic oligonucleotide probe prepared according to the known amino acid sequence, Glu 73 to Glu 77 of human urinary urokinase chain B. The entire cloned cDNA covers a 2250-bp region, wherein the 1293-bp sequence codes for preprourokinase consisting of 431 amino acids, with the first 20 residues being a signal peptide. The 5'-untranslated region is at least 80 bp long and the 3'-untranslated region is longer than 850 bp.     

Molecular cloning and sequencing of a cDNA coding for mature human kidney cathepsin H

A cDNA library was established from human kidney RNA and screened with an extended oligonucleotide probe derived from the amino-acid sequence of human cathepsin H. A recombinant clone, pRF15, was isolated and characterized. DNA sequence analysis of its 1106-nucleotide-long insert revealed that pRF15 encodes the complete protein sequence of mature cathepsin H plus 28 amino acids of a propeptide, thus confirming that cathepsin H is synthesized as a larger precursor molecule and posttranslationally processed. Northern blot analysis indicated that cathepsin H is predominantly synthesized in kidney. A high degree of sequence homology was observed with rat cathepsin H, especially within the propeptide. The part of the prosequence coding for the "minichain" is conserved in the prosequence of aleurain, a plant thiol protease.     

Molecular cloning of the cDNA coding for Xenopus laevis prion protein.

Isolation and characterization of the cDNA coding for the 216-residue Xenopus laevis prion protein is reported. Existence of this protein in amphibians was suggested by an EST fragment (accession number BG813008), while a conclusive demonstration is presented here. This protein exhibits a higher identity level to avian and turtle prion (more than 44%) than to mammalian prion (about 28%). Although most of the structural motifs common to known prion proteins are conserved in X. laevis, the lack of repeats represents a substantial difference. Other features worth noting are the presence of not perfectly conserved hydrophobic stretch, which is considered the prion signature, as well as the complete absence of histidine residues.     

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 of cDNA coding for rat plasma glutathione peroxidase

The plasma glutathione peroxidase (PGSH-PO), which is different from erythrocyte glutathione peroxidase (EGSH-PO) in immunochemical property and substrate specificity, was purified from male Wistar rat serum. The amino acid sequence of 5 independent peptides were determined and a cDNA clone for this enzyme was isolated from placental cDNA library. The nucleotide sequence of the cDNA revealed that, similar to EGSH-PO cDNA, the seleno-cysteine was genetically encoded by "TGA" codon. On comparing the nucleotide sequences of EGSH-PO and PGSH-PO, no significant homology was found in the vicinities of "TGA" codons of both enzymes.     

Molecular cloning and sequence analysis of full-length cDNA for rabbit liver NADPH-cytochrome P-450 reductase mRNA

The nucleotide sequence of the mRNA for NADPH-cytochrome P-450 reductase from rabbit liver was determined from a full-length cDNA clone (pFP105). The clone contains 2,269 nucleotides complementary to rabbit liver reductase mRNA. The single open reading frame of 2,037 nucleotides codes for a 679-amino acid polypeptide with a calculated molecular weight of 76,583 daltons. The cloned cDNA contains the complete 3'-noncoding region of 193 nucleotides, including 68 nucleotides of poly(A), and 39 nucleotides of the 5'-noncoding region. The nucleotide sequence in the coding region of cDNA of rabbit reductase (pFP105) showed 85% homology to that of rat reductase (Porter, T.D. & Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U.S. 82, 973-977, and Murakami, H. et al. (1986) DNA 5, 1-10). Rabbit reductase has one more amino acid residue than the rat enzyme, and the amino acid compositions of the two enzymes are similar. The amino acid sequence of the rabbit enzyme showed 91% identity with that of the rat enzyme. The segment related to binding of FMN and FAD was well conserved among rabbit, rat, and pig reductases. The sequence related to AMP moiety-binding was also conserved among these species, and was found in the amino acid sequence of NADH-cytochrome b5 reductase, another flavoenzyme in the microsomal electron transport system.     

Molecular cloning and characterization of double-stranded cDNA coding for bovine chymosin

A full-length cDNA copy of the mRNA encoding calf chymosin (also known as rennin), a proteolytic enzyme with commercial importance in the manufacture of cheese, has been cloned in an f1 bacteriophage vector. The nucleotide sequence of the cDNA was determined, and translation of that sequence into amino acids predicts that the zymogen prochymosin is actually synthesized in vivo as preprochymosin with a 16 amino acid signal peptide. In vitro translation of total poly(A)-enriched RNA from the calf fourth stomach (abomasum) and immunoprecipitation with antichymosin antiserum revealed that a form of chymosin (probably preprochymosin judging from the M_r-value) is the major in vitro translation product of RNA from that tissue. Gel-transfer hybridization of restriction endonuclease-cleaved bovine chromosomal DNA with labeled cDNA probes indicated that the two known forms of chymosin, A and B, must be products of two different alleles of a single chymosin gene.     

Molecular cloning and sequence analysis of cDNA coding for canine gastrin

We have isolated and sequenced cDNA clones encoding for preprogastrin from a canine antral cDNA library. Comparison of this sequence with those of porcine, human, and rat gastrin reveals extensive (83%) homology in the gastrin coding region as well as short regions of conserved nucleotides in the noncoding regions. The canine sequence encodes a preprogastrin of 104 amino acids which consists of a signal peptide, a 37-amino acid prosegment, and a 34-amino acid gastrin sequence, followed by a glycine (the amide donor), and flanked by pairs of arginine residues.     

Molecular cloning of human prostate specific antigen cDNA

A lambda gt11 clone encoding prostate specific antigen has been isolated from a human prostate cDNA library. The cDNA insert of 1415 nucleotides hybridizes specifically to a prostate mRNA species of 1.5 kb. The nucleotide sequence codes for part of a signal peptide, a short propiece and the mature protein of 237 amino acid residues. The Mr for the non-glycosylated protein was 26,089. One potential site for N-linked carbohydrate attachment was identified. The primary structure shows extensive homology with proteases of the kallikrein family.     

Molecular cloning of human haptoglobin cDNA: evidence for a single mRNA coding for alpha 2 and beta chains

Human haptoglobin (Hp) is a plasma glycoprotein composed of alpha and beta polypeptide chains that are covalently associated by disulfide bonds. It had been suggested that alpha and beta polypeptides could be synthesized via a common precursor polypeptide. We report the molecular cloning of DNA complementary to human Hp mRNA. One of the clones, pULB1148, carries a full length copy coding for both alpha 2 and beta polypeptides. In vitro translation of human liver mRNA hybridizing with this cDNA gives a protein mol. wt. of 49000 daltons. The sequence of the alpha 2 beta cDNA shows the presence of a single Arg residue between Gln 142 of the alpha 2 chain and Ileu 1 of the beta chain. With a few minor exceptions, the DNA sequence fits the previously published amino acid sequences. The differences are the presence of an Asp residue at position 52 of alpha 2 instead of Asn, the existence in beta of only one Lys residue between Gly 65 and the following Gln, the presence of Ser and Cys at positions 218-219 instead of Cys-Ser, and of Asp residues at positions 205 and 235 instead of Asn.     

Molecular cloning of cDNA of subunits of ubiquinol-cytochrome c reductase

We present here the preliminary results of the molecular cloning of rat liver ubiquinol-cytochrome c reductase subunits. Using immunoscreening techniques, we have been able to isolate, purify and partially sequence two cDNA clones.     

Molecular cloning and sequence analysis of full-length cDNA coding for mouse contrapsin

Four overlapping cDNA clones encoding contrapsin were isolated from a mouse liver cDNA library constructed in the expression vector, lambda gt11. M13 vector sequence analysis revealed that contrapsin cDNA contained an open reading frame of 1,254 bases encoding 418 amino acids. The N-terminal amino acid sequence of the isolated contrapsin matched residues 30 to 48 of the sequence deduced on nucleotide analysis. One clone, which had the longest 3' untranslated region, contained two sets of tandem polyadenylation signals, AATACA and AATAAA, which were located 497 bases apart, while the remaining three clones terminated at the first signal. The entire reading frame sequence of contrapsin cDNA showed 64% homology with that of human alpha-1-antichymotrypsin.     

Molecular cloning of cDNA of S100 alpha subunit mRNA

The primary structure of the bovine S-100 alpha mRNA on the basis of molecular cloning and sequence analysis of the cDNA are described. The sequence is composed of 532 bp which include the 282 bp of the complete coding region, 89 bp at the 5'-noncoding region, 161 bp at the 3'-noncoding region, polyadenylation signal, ATTAAA and poly(A) tail. Northern blot analysis shows that the size of S-100 alpha mRNA is about 700-800 bases long and a single mRNA occurs in bovine brain. Bovine brain contains both S100 alpha and beta subunits and their mRNAs. In contrast, the rat brain contains only S100 beta subunit and its mRNA.     

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 of cDNA coding for rat proliferating cell nuclear antigen (PCNA)/cyclin.

The 'proliferating cell nuclear antigen' (PCNA), also known as cyclin, appears at the G1/S boundary in the cell cycle. Because of its possible relationship with cell proliferation, PCNA/cyclin has been receiving attention. PCNA/cyclin is a non-histone acidic nuclear protein with an apparent mol. wt of 33000-36000. The amino acid composition and the sequence of the first 25 amino acids of rabbit PCNA/cyclin are known. Using an oligonucleotide probe corresponding to the sequence of the first five amino acids, a cDNA clone for PCNA/cyclin was isolated from rat thymocyte cDNA library. The cDNA (1195 bases) contains an open reading frame of 813 nucleotides coding for 261 amino acids. The 3'-non-coding region is 312 nucleotides long and contains three putative polyadenylation signals. The mol. wt of rat PCNA/cyclin was calculated to be 28 748. The deduced amino acid sequence and composition of rat PCNA/cyclin are in excellent agreement with the published data. Using the cDNA probe, two species of mRNA (1.1 and 0.98 kb) were detected in rat thymocyte RNA. Southern blot analysis of total human genomic DNA suggests that there is a single gene coding for PCNA/cyclin. The deduced amino acid sequence of rat PCNA/cyclin has a similarity with that of herpes simplex virus type-1 DNA binding protein.     

hsp80 of Neurospora crassa: cDNA cloning, gene mapping, and studies of mRNA accumulation under stress.

Using mRNA isolated from Neurospora crassa mycelium, grown for 14 h at normal growth temperature of 28 degrees C, and heat shocked for 1 h at 48 degrees C, a cDNA library was prepared in the expression vector lambda gt11. Following immunoscreening of this library with a polyclonal antiserum raised against a 80-kilodalton heat-shock protein (HSP80), cDNA clones containing 1.1- and 1.4-kilobase inserts were selected. Analysis of the partial nucleotide sequence and the deduced amino acid sequence of the cDNA clones revealed a remarkable extent of homology with other eukaryotic stress-90 family proteins; 85% identity of the amino acid sequence with that of yeast HSP90(82) was seen. The C-terminal end of the sequence contained the MEEVD motif, characteristic of eukaryotic stress proteins with a predominantly cytosolic localization. The gene for N. crassa HSP80 was mapped to the right arm of linkage group V, using restriction fragment length polymorphism mapping. Its expression during heat shock and recovery was monitored by probing Northern blots of RNA isolated from mycelium grown under various stress conditions.     

Molecular cloning of the cDNA coding for regucalcin and its mRNA expression in mouse liver: The expression is stimulated by calcium administration

The molecular cloning of the cDNA coding for a Ca2+-binding proteinregucalcin and its mRNA expression in mouse liver were investigated. ThecDNA clone encoding a regucalcin was isolated from a mouse liver cDNAlibrary and sequenced. Analysis of the sequence of the cloned cDNA showedthat the cDNA encoded the complete amino acid sequence of the mouseregucalcin molecule; the cDNA had an open reading frame of 897 bp. Mouseregucalcin was composed of 299 amino acid residues, and its molecular weightwas estimated to be 33,406 Da. The amino acid sequence of mouse regucalcinhad 94% homology, as compared with that of rat regucalcin. Northernblot analysis with the mouse liver cDNA probe revealed that mouse regucalcinmRNA was mainly present in the liver but only slightly in the kidney with asize of 1.8 kb. Hepatic regucalcin mRNA level of male mouse was higher thanthat of female mouse. A single intraperitoneal administration of calciumchloride (5, 15, and 30 mg Ca2+/100 g body weight) to mice induced aremarkable increase in regucalcin mRNA in the liver; the increase inregucalcin mRNA levels at 30 min after calcium administration wasdose-dependent. The present results demonstrate that regucalcin mRNA in miceis uniquely expressed in the liver, and that its expression is stimulated bycalcium administration.