Isolation and identification of a cDNA clone coding for rat uroporphyrinogen decarboxylase.

We have cloned and identified a DNA sequence complementary to the mRNA of uroporphyrinogen decarboxylase ( UroDCase ) from rat. This mRNA is a minor species (0.1%) of the total mRNA from anemic rat spleen. Poly(A)+ mRNA was enriched for UroDCase mRNA to 20% purity by a very efficient procedure involving two successive steps of preparative gel electrophoresis under various denaturing conditions. cDNA prepared from partially purified UroDCase mRNA (1% purity) was cloned in the Pst I site of pBR322 by using the homopolymeric G-C tailing method. Primary screening of 500 clones from this cDNA library was performed with a cDNA probe complementary to highly purified mRNA for UroDCase (20% purity) and UroDCase cDNA clones were finally identified by hybrid-selected translation. The rat cDNA clones obtained hybridize to human UroDCase mRNA. This will permit the isolation of the corresponding human gene and molecular analysis of porphyria cutanea tarda, the commonest type of porphyria.     

Polysome immunoprecipitation of phenylalanine hydroxylase mRNA from rat liver and cloning of its cDNA.

The mRNA for phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1) has been purified from total rat liver mRNAs, of which it constitutes less than 0.25%, to greater than 10% purity in a single step by specific polysome immunoprecipitation. The purified mRNA was used for synthesis and cloning of its cDNA. Recombinant colonies containing phenylalanine hydroxylase DNA sequences were identified by differential hybridization, hybrid-selected translation, and blot hybridization analysis. The rat cDNA clone was capable of hybridizing with human phenylalanine hydroxylase mRNA, which will permit the isolation of the corresponding human gene for analysis of phenylketonuria, a hereditary disorder in phenylalanine metabolism that causes permanent mental retardation in humans.     

Cloning of cDNA sequences of human adenosine deaminase.

Cloned cDNA sequences of human adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) have been isolated from a cDNA library constructed in bacteriophage lambda gt10. The cDNA for the library was prepared from poly(A)+ RNA isolated from a human T-lymphoblast cell line, CCRF-CEM. The library was initially screened by differential plaque hybridization to labeled cDNA prepared from human T- and B-lymphoblast cell lines with a 21-fold difference in levels of translatable ADA mRNA. Two recombinants containing cloned cDNA sequences for ADA were identified by hybridization-selected translation. Both recombinants contained approximately 1,600 base pairs of inserted human DNA. Restriction maps of the two inserts were not identical. One contained approximately 40 base pairs of additional DNA toward the center of the cDNA. The cloned cDNA specifically hybridized to five fragments generated by HindIII digestion of human genomic DNA. It also hybridized to human lymphoblast RNA species 1.6 and 5.8 kilobases in length. The cDNA was used as a probe to estimate ADA mRNA levels in human lymphoblast cell lines. ADA mRNA levels correlate closely with levels of ADA catalytic activity and ADA protein in cell lines containing structurally normal ADA. A leukemic T-lymphoblast line produced 6 to 9 times as much ADA protein and ADA mRNA as transformed B-lymphoblast lines. Two mutant B-lymphoblast lines from patients with hereditary ADA deficiency contained unstable ADA protein but had 3 to 4 times the normal level of ADA mRNA.     

Cloning and expression of human erythropoietin cDNA in Escherichia coli.

Human erythropoietin (Ep) cDNA has been cloned in Escherichia coli by using pBR322 as a vector. Polyadenylylated RNA was isolated from selected human renal carcinomas with elevated Ep titers. The presence of Ep mRNA was detected by immunoprecipitation of in vitro translation products with monoclonal antibody to human Ep. Double-stranded cDNA was synthesized and inserted into the Pst I site of pBR322 by homopolymeric dG . dC tailing. The cDNA library was initially screened by colony hybridization with 32P-labeled cDNA synthesized from size-fractionated mRNA enriched in Ep message. Positive colonies were further screened immunologically by in situ radioimmunoassay with monoclonal antibody to human Ep. Three positive clones were identified that express the Ep gene sequences as a beta-lactamase fusion protein. These clones contain inserts of approximately 1400, 600, and 200 base pairs. Human renal Ep mRNA, of which the translation products immunoreact with anti-Ep on immunoblots, was hybrid-selected by plasmid DNA from these recombinants. Purified human Ep competes with 35S-labeled hybrid-selected translation products for antibody binding.     

Molecular cloning and the nucleotide sequence of cDNA for neuron-specific enolase messenger RNA of rat brain.

The cDNAs to mRNA for rat gamma gamma enolase (neuron-specific enolase; NSE; EC 4.2.1.11) were isolated from a cDNA library by using differential colony hybridization and a hybrid-selected translation assay. By overlapping of the nucleotide sequences of several cDNA inserts, it was found that they spanned 2232 base pairs (bp) which included 1299 bp of the complete coding region, 68 bp of the 5' noncoding region, and 848 bp of the 3' noncoding region, including a polyadenylylation signal. In addition, the poly(A) tail was also found. The amino acid sequence deduced from the nucleotide sequence was composed of 433 amino acids. Southern blot analysis with a cDNA insert detected one hybridizing fragment in rat genomic DNA digested with several different restriction enzymes. Dot-blot and transfer hybridization analyses of poly(A)+ RNA from developing rat brains showed an increase of NSE mRNA 10-30 days after birth.     

Molecular cloning of a vitamin D-dependent calcium-binding protein mRNA sequence from chick intestine.

We have constructed a recombinant cDNA library to facilitate study of the genomic actions of vitamin D3 and its hormonally active metabolite 1,25-dihydroxyvitamin D3 in initiation of the de novo biosynthesis of a 28,000-dalton vitamin D-dependent calcium binding protein (CaBP) present in chick intestine. The recombinant plasmids were prepared by the homopolymeric tailing and hybridization method using as a starting template poly(A)-enriched mRNA obtained from the intestinal mucosa of vitamin D3-replete (+D) chicks. Screening of 9,516 clones in this library was effected by using a comparative in situ colony hybridization technique with two [32P]cDNA probes; these probes were prepared from total poly(A)-RNA from chick intestinal mucosa of vitamin D-deficient (-D) chicks and a poly(A)-RNA specifically enriched for chick intestinal CaBP mRNA by immunoprecipitation of polysomes derived from vitamin D-replete (+D) chicks. We identified 26 clones that consistently displayed a significantly increased hybridization signal when comparing the -D vs. CaBP-enriched probe. Further evaluation of these clones by hybrid-selected translation showed the presence of CaBP-specific sequences. By "RNA gel" analysis of poly(A)-RNA, three independent mRNA species were found to hybridize to a CaBP clone; none of these RNA species were found in -D poly(A)-RNA. With this comparative colony hybridization procedure, we were able to identify CaBP-specific clones corresponding to a mRNA that is 0.1% of the total poly(A)-mRNA. The differential colony hybridization procedure using an enriched vs. a nonenriched probe should be of value in screening for other cDNA clones complementary to rare mRNA species.     

Molecular approach to thermogenesis in brown adipose tissue: cDNA cloning of the mitochondrial uncoupling protein.

The uncoupling protein (UCP) of mammalian brown fat is a specialized and unique component responsible for energy dissipation as heat. Translation and immunoprecipitation from sucrose-fractionated mRNA indicated that the mRNA of UCP sedimented at 14-16 S. A recombinant cDNA library prepared from mRNA of thermoactive brown fat enriched for UCP mRNA has been constructed and cloned in Escherichia coli. Recombinant plasmids were screened by differential colony hybridization to a cDNA probe complementary to poly(A)+ RNA isolated from thermogenic or from weakly thermogenic brown fat. Several differentially hybridizing plasmids were shown to contain UCP cDNA sequences by their ability to select a mRNA coding for an in vitro translation product that was immunoprecipitable with antibodies against UCP. Blot hybridization of brown fat mRNA to a 32P-labeled UCP cDNA probe revealed two major species of mRNA (15S and 18S). As compared to non-thermogenic tissue, a strikingly increased hybridization to the probe was observed with brown fat mRNA from thermoactive tissue. Moreover, hybridization was observed with RNA of brown adipose tissue from rat, hamster, or mouse but not with RNA from rat or mouse liver.     

Structure and expression of rat osteosarcoma (ROS 17/2.8) alkaline phosphatase: product of a single copy gene.

Alkaline phosphatase [ALP; orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] is a ubiquitous enzyme of unknown function expressed at high levels in cells of mineralizing tissues. To study the structure, function, and expression of ALP, a full-length cDNA of rat ALP (2415 bases) was isolated from a ROS 17/2.8 osteosarcoma cell lambda gt10 cDNA library. The predicted amino acid sequence spans 524 residues and includes an N-terminal signal peptide of 17 amino acids, the phosphohydrolase active site, a rather hydrophilic backbone with five potential N-glycosylation sites, and a short hydrophobic C-terminal sequence. ALP negative CHO cells transfected with an expression vector containing the ALP coding sequences express ALP. The rat bone, liver, and kidney ALP shows remarkable 90% homology with the corresponding human enzyme, the most divergent region being the C-terminal hydrophobic domain through which the enzyme may be anchored to the plasma membrane. The rat ALP also shows 50% homology with the human placental and intestinal ALP and 25% homology with the Escherichia coli ALP. The amino acids involved in catalysis show nearly complete homology among all known ALP sequences, suggesting that these enzymes evolved from a common ancestral gene. The rat ALP cDNA pRAP 54, used as a hybridization probe in RNA blot analysis of several tissues that express ALP, revealed the presence of an ALP mRNA of approximately equal to 2500 bases. Furthermore, hybridization patterns derived from Southern blot analysis of rat chromosomal DNA offered molecular evidence that the ALP expressed in ROS 17/2.8 osteosarcoma and various rat tissues, excluding the intestine, is the product of the same single copy gene.     

Molecular cloning and sequencing of a cDNA for olfactory marker protein.

cDNA clones corresponding to mRNA for rat olfactory marker protein (OMP) were isolated from a cDNA library. The library was constructed from olfactory mucosa poly(A)+ RNA enriched for OMP mRNA and cloned into a pBR322-derived plasmid, pMG5. OMP cDNA clones were detected by using a 17-base oligonucleotide probe that contained all 16 possible sequences coding for a known partial amino acid sequence of rat OMP. The identity of these clones was confirmed by hybrid-selected translation and nucleotide sequencing. The sequence of one clone was determined and contained the complete OMP coding region of 486 nucleotides followed by 1630 nucleotides of the 3' untranslated region. The 3' untranslated region included the polyadenylylation signal 16 nucleotides upstream of the poly(A) tail. No other ATG-initiated open reading frame larger than 20 codons was present in register. RNA blot analysis of olfactory mucosa poly(A)+ RNA using this clone as a probe indicated that the level of OMP mRNA, but not its size, declined significantly within a few days following olfactory bulbectomy. OMP mRNA was not detected in 14 nonolfactory rat tissues. Surprisingly, a small amount of OMP mRNA was observed in olfactory bulb. The presence of OMP mRNA in olfactory bulb was confirmed by in vitro translation and immunoprecipitation. These results suggest either that a previously undescribed population of neurons in the olfactory bulb synthesize OMP or that OMP mRNA is transported to the bulb by axonal transport.     

Characterization of cDNA coding for human factor XIIIa.

A cDNA library prepared from human placenta has been screened for sequences coding for factor XIIIa, the enzymatically active subunit of the factor XIII complex that stabilizes blood clots through crosslinking of fibrin molecules. Two oligonucleotides, based on the amino acid sequences of tryptic peptides of factor XIIIa, were used as hybridization probes. Of 0.36 X 10(6) independent recombinants, 1 clone was identified that hybridized to both probes. The insert of 1704 base pairs coded for the amino-terminal 541 amino acid residues of the mature factor XIIIa molecule. Blot-hybridization analysis using this cDNA as a probe showed that the factor XIIIa mRNA from placenta has a size of approximately 4000 bases. The insert was used to rescreen cDNA libraries and to identify further factor XIIIa-specific sequences. The total length of the isolated factor XIIIa cDNA is 3905 bases, and it codes for a protein of 732 amino acids. In spite of the presence of factor XIII in blood plasma, we could not identify a leader sequence typical for secreted proteins.     

Cloning, characterization, and DNA sequence of a human cDNA encoding neuropeptide tyrosine.

In vitro translation of the RNA isolated from a human pheochromocytoma demonstrated that this tumor contained a mRNA encoding a 10.5-kDa protein, which was immunoprecipitated with antiserum raised against porcine neuropeptide Y. Double-stranded cDNA was synthesized from total RNA and inserted into the Pst I site of pUC8. Transformants containing the neuropeptide Y cDNA were identified using the mixed hybridization probe d[A-(A,G)-(A,G)-T-T-(A,G,T)-A-T-(A,G)-T-A-(A,G)-T-G]. The probe sequences were based on the known amino acid sequence, His-Tyr-Ile-Asn-Leu, found in porcine neuropeptide Y. The nucleotide sequence of the cDNA was determined and contained 86 and 174 bases in the 5'- and 3'-untranslated regions, respectively. The coding sequence consisted of 291 bases, suggesting a precursor to neuropeptide Y that was 97 amino acids long (10,839 Da). The deduced amino acid sequence of the precursor suggested that there were at least two sites of proteolytic processing, which would generate three peptides having 28 (signal peptide), 36 (human neuropeptide Y), and 30 (COOH-terminal peptide) amino acid residues. A partial NH2-terminal sequence obtained by Edman degradation of the immunoprecipitated in vitro translation product identified the positions of methionine and leucine in the first 30 residues of the prepropeptide. A highly sensitive single-stranded complementary mRNA hybridization probe specific for neuropeptide Y mRNA was prepared using the bacteriophage SP6 promoter. This probe was used to identify a mRNA corresponding to neuropeptide Y of approximately 800 bases.     

Identification and characterization of an early estrogen-regulated RNA in cultured guinea-pig endometrial cells

A cDNA library was prepared from quiescent guinea-pig endometrial glandular epithelial cells stimulated for 2 h with estradiol-17β (E₂) in the presence of cycloheximide. It was screened by differential hybridization for estrogen-regulated sequences. Six recombinants containing E₂-regulated sequences were identified. One of them, called gecl was then characterized by Northern blot hybridization. The gec1 mRNA was 1800 bases in size. A 2-fold increase in the gec1 mRNA level was achieved at 120 min after E₂ treatment. The E₂ action on gec1 gene required the presence of cycloheximide.The cloned gec1 cDNA was 1 kb in size. The sequence so far determined did not show similarity with well characterized genes. This is the first report on a cloned cDNA probe of early estrogen-induced mRNA in a primary culture of endometrial epithelial cells.     

Molecular cloning of the cDNA coding for rat ornithine transcarbamoylase.

Ornithine transcarbamoylase is a mitochondrial matrix enzyme composed of three identical subunits encoded on the X chromosome. The subunit is synthesized on cytoplasmic polysomes as a precursor that is cleaved during transport into mitochondria. We report here the isolation and characterization of cDNA clones containing sequences corresponding to the mRNA encoding the ornithine transcarbamoylase subunit. cDNA was synthesized using rat liver mRNA enriched by polysome immunoadsorption for the low-abundance messenger species encoding the enzyme subunit. After insertion of cDNA into plasmid pBR322 and cloning in Escherichia coli, identification of the desired plasmids was accomplished by (i) differential colony hybridization using cDNA probes synthesized from mRNA of various tissues; (ii) differential blot hybridization using cDNA probes synthesized from mRNA enriched for or depleted of the ornithine transcarbamoylase message; (iii) hybrid-selected translation assays; and (iv) most definitively, structural analysis, which matched 25 consecutive amino acid residues determined by sequential Edman analysis of the carboxyl-terminal portion of the purified enzyme subunit with coding sequence present in the insert of one of the plasmids.     

Molecular cloning of a cDNA for the chicken progesterone receptor B antigen.

A cDNA for the chicken progesterone receptor B subunit antigen (Mr, 108,000) has been isolated from a cDNA library prepared from size-selected chicken oviduct poly(A)+RNA. A specific monoclonal antibody raised against hen progesterone receptor B subunit (alpha PR-B) was used to screen the library. Recombinant clones reacting with the antibody by virtue of antigen expression were used in hybrid-selected translation. A single clone, pPRB-1, hybridized specifically to a mRNA that yielded a Mr 108,000 protein when translated in vitro and which was immunoprecipitable by the alpha PR-B antibody. This cDNA represents a 470-base-pair portion of the PR-B nucleotide sequence. Additional clones have been subsequently isolated from the recombinant library using the insert from pPRB-1 as a specific probe. A mRNA size of approximately 3000 nucleotides was determined for the chicken progesterone receptor B subunit by formaldehyde/agarose gel electrophoresis and blot hybridization using pPRB-1 as a probe. Preliminary studies show that withdrawal of hormone from chickens treated chronically with estrogen leads to a dramatic decrease in the cellular RNA concentration of receptor B, indicating that target tissue levels of receptor B RNA are under hormonal control.     

Human cholesterol side-chain cleavage enzyme, P450scc: cDNA cloning, assignment of the gene to chromosome 15, and expression in the placenta.

Conversion of cholesterol to pregnenolone is mediated by P450scc [cholesterol, reduced-adrenal-ferrodoxin: oxygen oxidoreductase (side-chain-cleaving), EC 1.14.15.67]. RNA from several human adrenal samples was translated in vitro and immunoprecipitated with anti-bovine P450scc, indicating that P450scc mRNA represents about 0.5% of human adrenal mRNA in normal, hypertrophied, and malignant adrenals. A 1626-base-pair human adrenal P450scc cDNA was cloned in bacteriophage lambda gt10. Primer extension data indicated P450scc mRNA is about 1850 bases long and that all adrenal P450scc mRNA has the same 5' end. A full-length clone containing 1821 bases was obtained from a human testis cDNA library to yield the complete sequence. The encoded human preP450scc contains 521 amino acids with a molecular weight of 60189.65. The testis and adrenal sequences were identical; the human cDNA and amino acid sequences are 82% and 72% homologous, respectively, with the bovine sequences. P450scc cDNA was used to probe DNA from a panel of mouse-human somatic cell hybrids, showing that the single human P450scc gene lies on chromosome 15. The human P450scc gene is expressed in the placenta in early and midgestation; primary cultures of placental tissue indicate P450scc mRNA accumulates in response to cyclic AMP.     

Approach to the isolation of antiproliferative genes

Poly(A) RNAs from normal rat liver and senescent human fibroblasts appear to have more antiproliferative activity than RNAs from regenerating rat liver and early passage human fibroblasts. We have screened two rat liver and one human liver library by differential hybridization and isolated four candidate cDNAs for this antiproliferative activity; one is fibronectin and three others do not match to any sequence in the mammalian portion of the GENBANK database. We are currently testing the antiproliferative nature of these cDNAs by microinjection of hybrid-selected RNA, and we describe an alternative strategy for cloning such genes based on construction of a cDNA library in an RNA expression vector.