Identification of a gene at 16q24.3 that restores cellular senescence in immortal mammary tumor cells.

We have mapped a cellular senescence gene, SEN16, within a genetic distance of 3 - 7 cM, at 16q24.3. Microcell mediated transfer of a normal human chromosome 16, 16q22-qter or 16q23-qter restored cellular senescence in four immortal cell lines, derived from human and rat mammary tumors. The resumption of indefinite cell proliferation, concordant with the segregation of the donor chromosome, confirmed the presence of a senescence gene at 16q23-qter. While microcell hybrids were maintained in selection medium to retain the donor chromosome, sporadic immortal revertant clones arose among senescent cells. Reversion to immortal growth could occur due to inactivation of the senescence gene either by a mutation or a deletion. The analysis for chromosome 16 specific DNA markers, in revertant clones of senescent microcell hybrids, revealed a consensus deletion, spanning a genetic interval of approximately 3 - 7 cM at 16q24.3.

Antisense confirmation of mu- and kappa-opioid receptor mediation of morphine's effects on body temperature in rats.

Previous studies showed that parenterally administered morphine at 4-16 mg/kg markedly increased body temperature in the rat, but higher doses of morphine (> or = 30 mg/kg, subcutaneously, sc) caused a profound decrease in body temperature. Based on the use of selective opioid agonists and antagonists, we postulated that these effects were due to morphine's actions on mu and kappa receptors, respectively. In the present study, we sought to determine whether an antisense (AS) oligodeoxynucleotide (oligo) against cloned mu or kappa opioid receptors could affect morphine-induced body temperature changes. AS oligos were directed against nucleotides 1-18 of the coding region of the mu receptor and 4-21 of the coding region of the kappa receptor. Male SD rats were surgically implanted with intracerebroventricular (icv) cannulae. Rats received icv injections of vehicle or oligo in the animal colony room on days 1, 3 and 5. Either AS oligo or missense (MS) oligo was infused in a volume of 5 microliters over 30 s to freely moving animals. On day 6, the rats were tested. The results showed that icv treatment with an AS oligo against mu opioid receptors, but not an MS oligo against the mu opioid receptor or an AS oligo against the kappa opioid receptor, significantly attenuated the hyperthermia normally produced by a relatively low dose of morphine administered sc. In addition, treatment with an AS oligo against kappa receptors, but not an MS oligo against kappa opioid receptor or an AS oligo against the mu opioid receptor, significantly blocked the hypothermia induced by a high dose of morphine. This study confirms our earlier postulate that morphine at 4 mg/kg, sc, induces an increase in body temperature primarily via mu opioid receptors in the brain and a high dose (30 mg/kg) of morphine administered sc produces a decrease primarily through kappa opioid receptors in the brain.

An antisense oligodeoxynucleotide to mu-opioid receptors inhibits mu-opioid receptor agonist-induced analgesia in rats.

We examined effects of an antisense oligodeoxynucleotide against the mu-opioid receptor on mu-opioid receptor agonist-induced antinociception in the cold water (-3 degrees C) tail-flick test in rats. Rats were injected intracerebroventricularly (i.c.v.) with an antisense, sense or missense oligodeoxynucleotide or artificial cerebrospinal fluid on days 1, 3 and 5. On day 6, antinociceptive effects of opioid agonists were tested. Compared to the artificial cerebrospinal fluid treatment, the cumulative dose-effect curve for subcutaneous (s.c.) morphine was shifted to the right by the antisense oligodeoxynucleotide, but not by the missense oligodeoxynucleotide or the sense oligodeoxynucleotide treatment. Antisense oligodeoxynucleotide treatment reduced the analgesic effect of the mu-opioid receptor agonist PL017 ([N-MePhe3,D-Pro4]morphiceptin), but not the delta-opioid receptor agonist BW373U86 ((+/-)-4-((a-R*)-a-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3- hydroxybenzyl)-N,N-diethyl-benzamide) or the kappa-opioid receptor agonist spiradoline ((+/-)-(5a,7a,8b)-3,4-dichloro-N-methyl-N-[7-(1- pyrrolidinyl)-1-(oxaspiro-[4,5]dec-8-yl]benzeneacetamide monohydrochloride). The drugs were given by i.c.v. injection. These findings indicate that i.c.v. administration of a mu antisense oligodeoxynucleotide specifically blocks mu-, but not delta- or kappa-opioid receptor-mediated analgesia in the rat cold water tail-flick test.

Cloning of a human kappa opioid receptor from the brain.

By using a rat kappa opioid receptor cDNA as a probe to screen a human brain cDNA library, we isolated a 4.0-kb clone (z115) which encompasses a major portion of a human kappa opioid receptor (hkor), extending from the amino acid residue #6 to the 3'-untranslated region. The extreme 5'-region 232-bp fragment of z115 was used as a probe to screen a human genomic DNA library. A 1.6-kb fragment (d2) of one positive clone was found to extend from 5'-untranslated region to beyond the exon/intron junction at residue Arg86. The genomic DNA fragment d2 and the cDNA clone z115 were assembled to generate a clone (d2-z115) containing the entire coding sequence of hkor. Clone d2-z115 has an open reading frame of 1140 bp, which encodes for a 380-amino acid protein. The deduced amino acid sequence has 93.9% and 93.2% identity to rat and mouse kappa receptors, respectively. It also displays approximately 60% identity to both human mu and delta receptors. Northern blot analysis showed that in the human brain there was a single hkor mRNA transcript of 6.0 kb. Among brain regions examined, the amygdala, caudate nucleus, hypothalamus and subthalamic nucleus contained high levels of hkor mRNA. Hkor was cloned into the expression vector pBK-CMV and transiently expressed in COS-1 cells. Hkor had high affinity for [3H] diprenorphine, a nonselective opioid antagonist, and displayed stereospecific binding to naloxone. kappa selective ligands (U50,488H and nor-BNI) had high affinities, whereas mu and delta selective ligands bound with much lower affinities. Dynorphin A (1-17) and alpha-neoendorphin, both endogenous kappa peptides, bound with high affinities. These binding characteristics confirmed that hkor is a kappa receptor, most likely kappa 1 type. Cloning of the human kappa receptor allows investigation of interactions of compounds with the human receptor, instead of rodent receptors, for development of better therapeutic agents.

Differential binding domains of peptide and non-peptide ligands in the cloned rat kappa opioid receptor.

This study was to identify specific regions in kappa opioid receptors that accounted for binding selectivity of kappa ligands. Six chimeric mu/kappa receptors were constructed from cloned rat kappa and mu opioid receptors and transiently expressed in COS-1 cells. All six chimeric mu/kappa receptors bound [3H] diprenorphine with high affinities, indicating that these chimeras retain opioid receptor conformation. Binding affinities of three peptide ligands (dynorphin A, alpha-neo-endorphin, and dynorphin B) and three nonpeptide ligands (norbinaltorphimine, U50,488H, and U69,593) for chimeras were determined and compared to those for mu and kappa opioid receptors. The second extracellular loop and the adjoining C-terminal portion of the fourth transmembrane helix were essential for the high affinity binding of dynorphin A, alpha-neo-endorphin, and dynorphin B to the kappa receptor. The third extracellular loop and the sixth and seventh transmembrane helices played an important role in determining the selectivity of nor-binaltorphimine for the kappa over the mu receptor. U50,488H and U69,593 appeared to require the whole kappa receptor except the second extracellular loop to attain high affinity binding. Thus, the kappa opioid receptor has differential binding domains for peptide and non-peptide ligands.

Intracerebroventricular treatment with an antisense oligodeoxynucleotide to kappa-opioid receptors inhibited kappa-agonist-induced analgesia in rats.

In vivo treatment with an antisense (AS) phosphorothioate oligodeoxynucleotide (oligo) to the rat kappa-opioid receptor selectively inhibited kappa-mediated analgesia in the rat cold-water tail-flick test. Intracerebroventricular (i.c.v.) AS oligo significantly inhibited the analgesic effect of i.c.v. spiradoline, but not that of mu- or delta-opioid agonists. The dose-effect curve for s.c. spiradoline was shifted to the right after AS, but not missense or sense oligo treatment. Thus, AS oligos provide another technique with which to selectively manipulate opioid receptors and further support the role of non-mu opioid receptors in mediating analgesia in rats.

Cloning and expression of a prostaglandin E receptor EP3 subtype from human erythroleukaemia cells.

Prostaglandins inhibit platelet activation by stimulating intracellular cyclic AMP formation. We have postulated that intracellular cyclic AMP levels in platelets are buffered by a distinct prostaglandin receptor that mediates inhibition of cyclic AMP formation. In order to provide evidence for the model, we have cloned the cDNA coding for a prostaglandin receptor EP3 subtype, which is coupled to inhibition of adenylate cyclase, from the megakaryocytic cell line human erythroleukaemia (HEL) cells. A PCR-generated hybridization probe, produced using primers based on the sequence of the mouse prostaglandin EP3 receptor published by Sugimoto, Namba, Honda, Hayashi, Negishi, Ichikawa and Narumiya [(1992) J. Biol. Chem. 267, 6463-6466], was used to screen a lambda gt11 HEL cell cDNA library. The composite full-length cDNA clone HEP3, generated from the two partial clones pHEP3-7 and pHEP3-5, is 1.6 kb long with an open reading frame coding for 390 amino acids. This clone is 83% identical to the alpha subtype of the mouse EP3 receptor. The full-length construct was transfected into COS-1 cells. The cloned receptor exhibited the properties of a prostaglandin EP3 subtype, inhibiting forskolin-stimulated cyclic AMP formation in response to prostaglandin E2 (PGE2) and binding PGE2 with high specificity and a Kd of 3.2 nM. Radiolabelled PGE2 could be displaced by prostaglandins in the order PGE2 = PGE1 > iloprost = PGD2. Northern blot analysis revealed that the receptor is also present in human kidney.

Molecular cloning and expression of a rat kappa opioid receptor.

At least three types of opioid receptors have been identified in the nervous system. In this paper we report molecular cloning and expression of a rat kappa opioid receptor. PCR was performed on double-stranded cDNA derived from poly(A)+ RNA of the rat striatum with primers similar to those of Libert and co-workers [Libert, Parmentier, Lefort, Dinsart, Van Sande, Maenhaut, Simons, Dumont and Vassart (1989) Science 244, 569-572]. One of the PCR products, which had 65% sequence similarity to the mouse delta opioid receptor, was used to screen a rat striatum cDNA library. Two positive clones were isolated and found to be identical. The clone had a 2.1-kb insert, which was termed RKOR-1. RKOR-1 has an open reading frame of 1140 bp and encodes a 380-amino-acid protein. Hydropathy analysis indicates that RKOR-1 has seven putative transmembrane domains with short intra- and extra-cellular loops. Membranes of Cos-7 cells transfected with RKOR-1 exhibited high specific binding for [3H]diprenorphine ([3H]DIP), a non-selective opioid ligand. Naloxone inhibited [3H]DIP binding with stereospecificity. [3H]DIP binding was potently inhibited by selective kappa opioid ligands, with Ki values in the nanomolar or subnanomolar range, but much less potently inhibited by drugs selective for mu or delta receptors. Thus, RKOR-1 represents an opioid receptor with kappa characteristics.

Structure/function analysis of the Ala116-->Lys121 region of endonuclease V by random targeted mutagenesis.

Endonuclease V is the product of the denV gene of bacteriophage T4 and is responsible for the recognition and repair of pyrimidine dimers due to UV irradiation of DNA. This is accomplished by a two-step mechanism involving incision at the site of the lesion followed by cleavage of the phosphate backbone. In order to better understand this molecule, and to validate our new mutagenesis procedure, we have constructed a series of random mutations within the region Ala116-->Lys121 using a random targeted mutagenesis procedure developed for this study. The results presented here suggest an important role for this region in the stabilization of the thymine dimer-containing substrate. These mutants also confirm a direct correlation between survival and both DNA binding and pyrimidine dimer-DNA glycosylase activity. No such correlation exists between survival and AP lyase activity. The results are consistent with the recently published X-ray crystal structure.

Properties of an xeroderma pigmentosum revertant cell line expressing endonuclease V.

We have developed a set of cell lines to help distinguish the sequelae of specific lesions in DNA after UV irradiation. Irradiation results in two primary lesions: cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts. The contributions of each to mutation are considered utilizing a spectrum of cell lines with increasing abilities to repair these lesions. In particular, we focus on a revertant of the XP12Ro(M1) cell line from a patient with Xeroderma pigmentosum, XP129, which is capable of repairing (6-4) photoproducts but not cyclobutane dimers. We have successfully introduced the denV gene into these cells which confers the ability to repair cyclobutane dimers. By comparing the results of a shuttle vector mutation experiment with the vector pZ189, we can correlate specific mutations to specific lesions.

A human nuclear uracil DNA glycosylase is the 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase.

We have isolated and characterized a plasmid (pChug 20.1) that contains the cDNA of a nuclear uracil DNA glycosylase (UDG) gene isolated from normal human placenta. This cDNA directed the synthesis of a fusion protein (Mr 66,000) that exhibited UDG activity. The enzymatic activity was specific for a uracil-containing polynucleotide substrate and was inhibited by a glycosylase antibody or a beta-galactosidase antibody. Sequence analysis demonstrated an open reading frame that encoded a protein of 335 amino acids of calculated Mr 36,050 and pI 8.7, corresponding to the Mr 37,000 and pI 8.1 of purified human placental UDG. No homology was seen between this cDNA and the UDG of herpes simplex virus, Escherichia coli, and yeast; nor was there homology with the putative human mitochondrial UDG cDNA or with a second human nuclear UDG cDNA. Surprisingly, a search of the GenBank data base revealed that the cDNA of UDG was completely homologous with the 37-kDa subunit of human glyceraldehyde-3-phosphate dehydrogenase. Human erythrocyte glyceraldehyde-3-phosphate dehydrogenase was obtained commercially in its tetrameric form. A 37-kDa subunit was isolated from it and shown to possess UDG activity equivalent to that seen for the purified human placental UDG. The multiple functions of this 37-kDa protein as here and previously reported indicate that it possesses a series of activities, depending on its oligomeric state. Accordingly, mutation(s) in the gene of this multifunctional protein may conceivably result in the diverse cellular phenotypes of Bloom syndrome.

A human ADP/ATP translocase gene has seven pseudogenes and localizes to chromosome X.

There are at least three transcriptionally active human ADP/ATP translocase genes. We have isolated seven ADP/ATP translocase pseudogenes from recombinant human genomic libraries. Each pseudogene sequence had more than 85% identity with the sequence of the human ADP/ATP translocase cDNA derived from fibroblast mRNA, but each had mutations that precluded synthesis of a functional protein. Using an intron probe derived from a partial clone of the human fibroblast ADP/ATP translocase gene, we localized the gene to chromosome Xq13----Xq25-26. The gene encoding the skeletal muscle translocase has previously been shown to be on chromosome 4. Therefore, the human ADP/ATP translocase genes are members of a multigene family that includes pseudogenes and has been dispersed to at least two chromosomes.

Structure and regulation of the AMP nucleosidase gene (amn) from Escherichia coli.

The gene for AMP nucleosidase from Escherichia coli (amn) has been sequenced and characterized. The gene codes for a transcript of 1.7 +/- 0.2 kb, and the open reading frame corresponds to a protein of 483 amino acids (Mr = 53848). Amino acid sequences from tryptic peptides of AMP nucleosidase, N-terminal amino acid analysis, and the amino acid composition confirm the gene assignment and the open reading frame of amn. Primer extension studies determined the 5'-end of the amn transcript. The 5'-regulatory region contains overlapping sequences with similarity to the consensus sequences for binding cAMP receptor protein and inorganic phosphate repressor protein. Addition of exogenous cAMP to E. coli deficient in adenylate cyclase resulted in a 3-fold increase in AMP nucleosidase activity. Growth of E. coli on limiting phosphate resulted in an 8-fold increase in the production of AMP nucleosidase. The amn gene was expressed in AMP nucleosidase deficient strains of Azotobacter vinelandii and E. coli. A pUC-amn construct is described that causes approximately 20% of the total protein in E. coli to be produced as AMP nucleosidase. Comparison of the amino acid sequence for AMP nucleosidase with that for yeast AMP deaminase indicates a region in which six of eight amino acids are identical but no other overall homology. The amino acid sequence showed poor agreement with consensus sequences for adenylate binding sites even though the enzyme is known to have a catalytic site for AMP and regulatory sites for MgATP and phosphate.

Inhibition of cellular proliferation by antisense oligodeoxynucleotides to PCNA cyclin.

The proliferating cell nuclear antigen (PCNA or cyclin) is a nuclear protein recently identified as a cofactor of DNA polymerase delta. When exponentially growing Balb/c3T3 cells are exposed to antisense oligodeoxynucleotides to PCNA, both DNA synthesis and mitosis are completely suppressed. A corresponding sense oligodeoxynucleotide has no inhibitory effects. These experiments indicate that PCNA (cyclin) is important in cellular DNA synthesis and in cell cycle progression.

Structural and functional analysis of a growth-regulated gene, the human calcyclin.

Calcyclin was originally defined as a cDNA clone (2A9) whose cognate RNA is growth-regulated and whose sequence shows strong similarities to the sequences of the S-100 protein, a calcium-binding protein, as well as to a subunit of the major cellular substrate for tyrosine kinase. Using the full-length cDNA, we have now isolated from a human genomic library several phages containing calcyclin sequences. One of the phages, ch. 28-10, contains the entire calcyclin gene, plus extensive flanking sequences. The calcyclin gene is a unique copy gene and has 3 exons. The 5' flanking sequence has been characterized, both structurally and functionally. Besides a TATA box, it contains, in the region proximate to the cap site, GC boxes and a sequence with a strong homology to the enhancer core of the SV40 promoter. Other enhancer-like elements are found scattered in both the 5' and 3' flanking regions. The proximate 5' flanking region is very active in driving the transient expression of linked reporters in transfection experiments. Finally, the calcyclin gene has been localized to the long arm of human chromosome 1, near the ski oncogene.

Identification, physical map location and sequence of the denV gene from bacteriophage T4.

The denV gene from bacteriophage T4, which codes for endonuclease V, a small DNA repair enzyme, has been cloned and identified by an approach combining DNA sequencing and genetics, independent of the phenotypic effect of the cloned gene. Appropriate DenV+ and DenV- deletion mutants were mapped physically to define precisely a region encompassing the denV gene. This region was sequenced in order to identify a protein-coding sequence of the correct size for the denV gene (400-500 bp). Finally, identification was confirmed by sequencing the corresponding fragments cloned from four genetically and phenotypically well-characterized denV mutants. The denV gene is located at 64 kb on the T4 genome, adjacent to the ipII gene, and codes for a basic protein of 138 amino acids with a deduced molecular weight of 16,078.

Enzymic detection of uracil in a cloned and sequenced deoxyribonucleic acid segment.

Uracil can occur in DNA either as a result of utilization of dUTP by DNA polymerases or from in situ deamination of cytosine. The latter results in transition mutations following the next round of replication. We describe a technique for the detection of uracil in DNA by a modification of the Maxam-Gilbert sequencing procedure. Reaction of end-labeled DNA with uracil-DNA glycosylase followed by 1 M piperidine results in scission products corresponding to locations of uracils. These are detected by autoradiography following electrophoresis through a sequencing gel. Comparison of these scission products with the DNA sequences elucidates the mechanism of origin of the DNA uracils. The technique was tested with a cloned human DNA sequence grown in a dut-,ung-strain of Escherichia coli, which incorporates uracil in place of thymine in its DNA, and by chemical deamination of cytosines in that sequence. The technique was expanded by use of alkaline and enzymic probes to investigate possible accumulation of uracil, base losses, and other modifications in human liver and brain DNA. No damaged DNA moieties were detected. This method is applicable to the study of any recoverable reiterated sequence by any enzyme preparation that can recognize modifications in DNA.

Cloning and nucleotide sequence analysis of human embryonic zeta-globin cDNA.

Clones of human embryonic alpha-like zeta-globin cDNA were isolated, by detection using cross-hybridization to human alpha-globin cDNA probes, from a cDNA library derived from the mRNA of the human erythroleukemia cell line K562. Nucleotide sequence analysis of these cDNA clones revealed a coding sequence that corresponds perfectly to the independently derived amino acid sequence of the human zeta-globin chain. Comparison of the nucleotide sequence of human zeta-globin cDNA with that of human alpha-globin cDNA confirmed previous estimates of very distant evolutionary divergence between the human zeta- and alpha-globin genes. Nevertheless, the human zeta-globin cDNA sequence shares a remarkable similarity to that of the alpha-globin gene in its codon usage, high G + C base composition, and lack of bias against usage of CG dinucleotides.

Base substitution in an intervening sequence of a beta+-thalassemic human globin gene.

beta globin gene fragments from a patient with homozygous beta+-thalassemia have been cloned and subjected to restriction endonuclease, nucleotide sequence, and in vitro trancription analyses. Restriction endonuclease mapping of the cloned gene fragments revealed no deletions or other rearrangements, and transcription of the thalassemic gene appeared to be normal in vitro. However, nucleotide sequence analysis of the beta+-thalassemic gene fragments permitted identification of a single base change in the body of the small intervening sequence. This nucleotide change creates a sequence much like that of the 3' splice site of the small intervening sequence. The presence of a potential anomalous splicing site as a result of this base change suggests a mechanism for defective posttranscriptional processing of beta globin mRNA precursor molecules in beta+-thalassemia.