Expression of human and Chinese hamster hypoxanthine-guanine phosphoribosyltransferase cDNA recombinants in cultured Lesch-Nyhan and Chinese hamster fibroblasts

The results presented in this communication demonstrate that hypoxanthine-guanine phosphoribosyltransferase (HPRT) cDNA can be expressed in both Chinese hamster and human fibroblasts deficient in the endogenous gene product at levels permitting normal growth of the transformants. All the elements necessary for this expression are present in a pBR322-derived plasmid containing HPRT cDNA coding sequence and a retroviral long terminal repeat. These molecules function in both species investigated and, at least in the case of the Chinese hamster transformants, are efficient at the single copy level. Although the effects of the presence of intron sequences and a polyadenylation signal within the plasmids have yet to be evaluated, these studies demonstrate that neither is an absolute requirement for expression of HPRT cDNA sequences in cultured mammalian cells. We describe the construction of recombinant plasmids containing wild type human or Chinese hamster HPRT cDNA sequences in tandem with a retroviral LTR which confer the HPRT+ phenotype in HPRT-deficient V79 and Lesch-Nyhan fibroblasts. Both stable and unstable transformants, that expressed HPRT mRNA and protein, were isolated at high frequency.     

Mouse primase p49 subunit molecular cloning indicates conserved and divergent regions

Primase is a specialized RNA polymerase that synthesizes RNA primers for initiation of DNA synthesis. A full cDNA clone of the p49 subunit of mouse primase, a heterodimeric enzyme, has been isolated using a primase p49-specific polyclonal antibody to screen a lambda gt11 mouse cDNA expression library. The cDNA indicated the subunit is a 417-amino acid polypeptide with a calculated molecular mass of 49,295 daltons. The p49 mRNA is approximately 1500 nucleotides long with a 5'-untranslated region of 74 nucleotides and a 3'-untranslated region of 200 nucleotides. Comparison with a similar sized primase subunit from yeast showed highly conserved amino acid sequences in the N-terminal halves of the polypeptides and included a potential metal-binding domain suggesting the functional importance of this region for DNA binding. In contrast, the 3' portion of the cDNA has rapidly diverged in nucleotide sequence, as primase mRNA can be detected in mouse and rat cells with a 3' probe (including coding and noncoding) but not in RNA from hamster or human cells. A full-length cDNA probe detected mRNA from hamster and human cell lines, indicating a conserved 5' portion and divergent 3' region of the expressed gene. The rapid divergence may be related to the species-specific protein interactions found for the DNA polymerase alpha-primase complex. The mRNA is detected in proliferating but not in quiescent cells consistent with its function in DNA replication.     

The sequence of a cloned cDNA for the beta subunit of bovine thyrotropin predicts a protein containing both NH2-and COOH-terminal extensions

Cloned cDNAs containing sequences coding for the beta subunit of bovine thyrotropin have been identified. The complete nucleotide sequence of the largest of the beta subunit cDNA inserts has been determined. This cDNA contains 35 nucleotides from the 5' untranslated region of thyrotropin beta subunit mRNA and 60 nucleotides coding for an NH2-terminal precursor segment. This is followed by 339 nucleotides which code for the published amino acid sequence of the thyrotropin beta subunit. Following the 339 nucleotide beta subunit coding sequence, no termination codon is encountered for another 15 nucleotides. Thus, the cDNA codes for a thyrotropin beta subunit containing an additional 5 amino acids at the COOH terminus. The cDNA also contains 82 nucleotides of 3' untranslated sequence followed by a short poly(A) segment. Comparison of the bovine cDNA sequence to the recently described mouse thyrotropin beta subunit cDNA sequence reveals considerable homology throughout the coding sequence, including the COOH-terminal extension. These findings suggest the possibility that a thyrotropin beta subunit precursor is processed at both the NH2 and COOH termini.     

Isolation and characterization of mouse MUC18 cDNA gene, and correlation of MUC18 expression in mouse melanoma cell lines with metastatic ability

The cell surface adhesion molecule human MUC18 (huMUC18 or Mel-CAM) has been postulated to play a key pathogenic role in metastatic melanoma progression. To establish an immunocompetent syngeneic mouse model that would greatly facilitate our understanding of the role of MUC18 in the metastatic behavior of melanoma, we cloned and characterized the mouse MUC18 (muMUC18) cDNA gene. The gene was amplified by RT-PCR and RACE of the poly(A)+RNA isolated from the mouse melanoma cell line B16F10/Queens. The cloned muMUC18 cDNA gene contained 28 nucleotides of 5'-UTR, 908 nucleotides of 3'-UTR, and an open reading frame (ORF) of 1947 nucleotides encoding a protein of 648 amino acids, which is two amino acids longer than huMUC18. The size of the muMUC18 mRNA is about 3 kb with a shorter 3'-UTR than the huMUC18 mRNA (about 3.3 kb). Besides, the sequence in the 3' UTR of the two mRNAs is diverse with only 31% identity. The 5'-UTR and coding sequences of the muMUC18 cDNA are 72.4 and 80.6% identical to those of huMUC18, respectively. The deduced amino acid sequence of the muMUC18 cDNA is 76.2% identical to that of huMUC18. The amino acid sequences deduced from MUC18 cDNA sequences from six other mouse melanoma cell lines are identical except one to three residues, suggesting that the muMUC18 cDNA sequence determined in this report is correct. The muMUC18 protein is predicted to be slightly more acidic than the human protein. The levels of muMUC18 mRNA and protein in nine mouse melanoma cell lines were directly proportional to their ability to establish metastatic colonies in lungs of syngeneic mice. Most biological functions of the muMUC18 may be similar to the huMUC18.     

Complete structure of the porcine pro-opiomelanocortin mRNA derived from the nucleotide sequence of cloned cDNA

Polyadenylated RNA isolated from porcine pituitary neurointermediate lobes was used to construct a cDNA library. The library was screened with a rat genomic DNA fragment specific for pro-opiomelanocortin sequences. Two positive clones, pJA-19 and pJA-20, containing respectively 850 bp and 550 bp were characterized. Sequence analysis of the cDNA inserts revealed the complete structure of the porcine pro-opiomelanocortin mRNA. This mRNA would include 129 5'-untranslated nucleotides, 801 nucleotides coding for the 267 amino acids precursor and 162 3'-untranslated nucleotides. Comparison with pro-opiomelanocortin mRNA sequences from other species shows regions of high homology not only in the coding sequences but also in the 5'untranslated region where the first 50 nucleotides are over 80% purines.     

Nucleotide sequence of cloned complementary deoxyribonucleic acid for the alpha subunit of bovine pituitary glycoprotein hormones

Recombinant DNA plasmids containing sequences coding for the alpha subunit of the bovine pituitary glycoprotein hormones have been isolated. The nucleotide sequences of three different cDNA clones have been determined. The largest alpha-subunit cDNA clone was found to contain 713 bases including 77 nucleotides from the 5'-untranslated region, 72 nucleotides coding for a precursor segment, 288 nucleotides coding for the mature alpha subunit, and 276 nucleotides from the 3'-untranslated region of the mRNA followed by a poly(A) segment. This cDNA likely represents most of the bovine alpha-subunit mRNA sequence. Nucleotide sequences were obtained from the cDNA inserts of two other alpha-subunit clones, and several differences among the three cDNA sequences have been detected. These differences in nucleotide sequence may represent either individual variation in genomic sequence or cloning artifacts. Comparison of the bovine alpha-subunit cDNA sequence to the sequences of human, rat, and mouse alpha-subunit cDNAs reveals that the bovine sequence has greater than 70% homology with the other cDNAs. The cloned alpha-subunit cDNA should provide a useful probe for further studies of the structure and expression of this interesting gene.     

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.     

Sequence analysis of the large and small subunits of human ribonucleotide reductase.

We have isolated and sequenced overlapping cDNA clones from a breast carcinoma cDNA library containing the entire coding region of both the R1 and R2 subunits of the human ribonucleotide reductase gene. The coding region of the human R1 subunit comprises 2376 nucleotides and predicts a polypeptide of 792 amino acids (calculated molecular mass 90,081). The sequence of this subunit is almost identical to the equivalent mouse ribonucleotide reductase subunit with 97.7% homology between the mouse and human R1 subunit amino acid sequences. The coding region of the human R2 subunit of ribonucleotide reductase comprises 1170 nucleotides and predicts a polypeptide of 389 amino acids (calculated molecular mass 44,883), which is one amino acid shorter than the equivalent mouse subunit. The human and mouse R2 subunits display considerable homology in their carboxy-terminal amino acid sequences, with 96.3% homology downstream of amino acid 68 of the human and mouse R2 proteins. However, the amino-terminal portions of these two proteins are more divergent in sequence, with only 69.2% homology in the first 68 amino acids.     

Sequence of the cDNA and gene for angiogenin, a human angiogenesis factor

Human cDNAs coding for angiogenin, a human tumor derived angiogenesis factor, were isolated from a cDNA library prepared from human liver poly(A) mRNA employing a synthetic oligonucleotide as a hybridization probe. The largest cDNA insert (697 base pairs) contained a short 5'-noncoding sequence followed by a sequence coding for a signal peptide of 24 (or 22) amino acids, 369 nucleotides coding for the mature protein of 123 amino acids, a stop codon, a 3'-noncoding sequence of 175 nucleotides, and a poly(A) tail. The gene coding for human angiogenin was then isolated from a genomic lambda Charon 4A bacteriophage library employing the cDNA as a probe. The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (4688 base pairs) was then determined. The coding and 3'-noncoding regions of the gene for human angiogenin were found to be free of introns, and the DNA sequence for the gene agreed well with that of the cDNA. The gene contained a potential TATA box in the 5' end in addition to two Alu repetitive sequences immediately flanking the 5' and 3' ends of the gene. The third Alu sequence was also found about 500 nucleotides downstream from the Alu sequence at the 3' end of the gene. The amino acid sequence of human angiogenin as predicted from the gene sequence was in complete agreement with that determined by amino acid sequence analysis. It is about 35% homologous with human pancreatic ribonuclease, and the amino acid residues that are essential for the activity of ribonuclease are also conserved in angiogenin. This provocative finding is thought to have important physiological implications.     

Cloning and sequence analysis of cDNA for mouse prolactin

The present study was undertaken to find out whether or not sexual dimorphism in biological activities and amino acid compositions of mouse prolactin might be due to heterogeneity in mRNA for mouse prolactin Cloned cDNAs for mouse prolactin were first isolated from a mouse pituitary cDNA library by hybridization with a rat prolactin cDNA. Then, one clone of about 140 positive clones obtained from 2000 transformants was subjected to nucleotide sequence analysis and verified to contain a nearly full length of cDNA sequence coding for mouse prolactin precursor. The deduced complete amino acid sequence indicates that the precursor molecule consists of 31 amino acids as the signal peptide and 197 amino acids of prolactin, in which two amino acids were found to be different from the amino acid sequence previously published elsewhere. S1 nuclease mapping analysis using male and female pituitary RNAs indicates that mouse preprolactin is encoded by two mRNAs in both sexes. The two mRNAs differ from each other based upon the deletion of three nucleotides in the coding region for the signal peptide determined by the nucleotide sequence analysis in other cDNA clones. In the present study, no sexual difference was revealed in murine prolactin mRNA.     

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.     

Complete nucleotide and derived amino acid sequences of the fourth component of mouse complement (C4). Evolutionary aspects

The nucleotide sequence coding for the fourth component of mouse complement (C4) has been determined from a cloned genomic DNA fragment and a cloned cDNA fragment. The amino acid sequence of the protein was deduced. The single chain precursor protein (pro-C4) consists of 1719 amino acid residues. The mature beta, alpha, and gamma subunits contain 654, 766, and 291 amino acids, respectively. One potential carbohydrate attachment site is predicted for the beta chain, three for the alpha chain, and none for the gamma chain. From a comparison with human C4 cDNA sequence an extensive overall sequence homology, 79% in nucleotides and 76% in amino acids, is observed. There is conservation in both the position and number of cysteine residues in human and mouse C4. We compared the mouse C4 amino acid sequences with those of mouse C3 and human alpha 2-macroglobulin and the evolutionary relationship among these three proteins is discussed.     

Characterization of the hamster DDT-1 cell aFGF/HGBF-I gene and cDNA and its modulation by steroids.

Syrian hamster DDT-1 cells are derived from smooth muscle of the ductus deferens. DDT-1 cell growth is increased by the addition of testosterone (T). Acidic fibroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF) also known as heparin binding growth factor I and II (HBGF-I and HBGF-II) can replace T in the stimulation of growth in these cells. This phenomenon is correlated with testosterone's ability to elevate aFGF/HBGF-I mRNA. The increase steady-state levels of aFGF/HBGF-I mRNA were documented by northern blots and by in situ hybridization. Using a 520 bp human aFGF/HBGF-I cDNA probe, a genomic clone with a 38 kb DNA insert was isolated from a cosmid library. By restriction enzyme analysis and southern hybridization, it was determined that there are three coding exons. DNA sequence analysis showed all of the coding region and 3' noncoding sequences were on this clone. A 5' noncoding exon not in the 38 kb insert is indicated, based on the cDNA sequences and genomic sequences of aFGF/HBGF-I's from hamster DDT-1 cells and several other species. The cDNA for hamster aFGF/HBGF-I was isolated from a DDT-1 lambda gt11 library and sequenced. Comparison of the coding region of aFGF/HBGF-I from four species shows a greater than 90% conservation of amino acid sequence.     

A universal oligonucleotide probe for acetylcholine receptor genes. Selection and sequencing of cDNA clones for the mouse muscle beta subunit

A region of 25 nucleotides is highly conserved in genes coding for the alpha, beta, gamma, and delta subunits of the nicotinic acetylcholine receptor (AChR) of human, mouse, calf, chicken, and Torpedo. Based on this observation, a 2-fold degenerate oligonucleotide was synthesized and used as a probe to screen a cDNA library made from a mouse myogenic cell line. Clones coding for the beta, gamma, and delta subunits were identified by the probe. The protein sequence deduced from the beta subunit clones codes for a precursor polypeptide of 501 amino acids with a calculated molecular weight of 56,930 daltons, which includes a signal peptide of 23 amino acids. The protein sequence and structural features of the beta subunits of mouse, calf, and Torpedo are conserved. A clone coding for the mouse gamma subunit was isolated, and its identity was confirmed by alignment of its sequence to previously published cDNA sequences for the mouse and calf gamma subunits. The clone contained approximately 200 nucleotides more at its 3' end untranslated region than a mouse gamma clone recently described. Northern blot analysis, utilizing as probes these beta and gamma subunit cDNAs and previously characterized alpha and delta subunit cDNAs, shows that the steady-state levels of the four AChR mRNAs increase coordinately during terminal differentiation of cultured C2 and C2i mouse myoblasts. The increase in mRNA levels can account for the rise of cell surface receptors during myogenesis and suggests that the muscle AChR genes may be regulated during development by a common mechanism. Utilization of this oligonucleotide probe should prove useful for screening a variety of libraries made from different species and tissues which are known to express AChRs.     

Isolation and characterization of cDNA for chicken muscle adenylate kinase

A cDNA clone for muscle adenylate kinase was isolated from a cDNA library of chick skeletal muscle poly(A)+ RNA, and the DNA sequence was determined. The cDNA insert had 854 nucleotides, which consisted of the 5'-untranslated sequence of 57 nucleotides, the sequence of 582 nucleotides coding for 194 amino acids, and the 3'-untranslated sequence of 163 nucleotides and the poly(A) tail of 52 nucleotides. The amino acid sequence predicted from the nucleotide sequence was highly homologous with the reported sequences of human, calf, porcine, and rabbit muscle adenylate kinases. RNA blot analysis of poly(A)+ RNA from various chicken tissues revealed a single species of mRNA of approximately 850 nucleotides and its tissue-specific distribution. The induction of muscle adenylate kinase mRNA synthesis during the chick embryogenesis was also demonstrated by the blot analysis. Southern blot analysis indicated a single gene for muscle adenylate kinase in the chicken genome.