Localization of the human A1S9 gene complementing the ts A1S9 mouse L-cell defect in DNA replication and cell cycle progression to Xp11.2----p11.4

The temperature-sensitive ts A1S9 mouse L-cell mutant is defective in an X-linked gene essential for progression of cells through the S phase of the cell division cycle. A single copy fragment derived from the complementing human A1S9 gene was used as a probe to localize the gene on the X chromosome. Southern blot analysis of human x rodent hybrids and in situ hybridization to human metaphase chromosomes allowed the regional assignment of the human A1S9 gene to Xp11.2----p11.4.     

Molecular cloning of the cDNA of human X chromosomal gene (CCG1) which complements the temperature-sensitive G1 mutants, tsBN462 and ts13, of the BHK cell line.

The tsBN462 cell line, a temperature-sensitive (ts) mutant isolated from the hamster cell line, BHK21/13 has a ts defect in G1 progression and belongs to the same complementation group as the ts13 cell line. We cloned human cDNA which can complement both tsBN462 and ts13 mutations, from the cDNA library of the secondary ts+ transformant (K-1-1) of tsBN462 cells using, as a probe, the isolated human X chromosomal genomic DNA. The cloned DNA is 5.3 kb long and has an open reading frame of 4662 bp, encoding a protein of 178,768 daltons. The putative protein is hydrophilic with a tandem repeat of 120 amino acids in the C-terminal region. An amino acid sequence (PPKKKRRV), similar to the consensus sequence for the nuclear translocation signal, is located immediately before the tandem repeat of amino acids.     

DNA polymerase-primase complex in wild-type and ts A1S9 mouse L-cells, temperature-sensitive for DNA replication during cell cycle progression

ts A1S9 mutant cells, derived from wild type WT-4 mouse L-cells, are temperature-sensitive (ts) for DNA synthesis and cell division. We try to determine the cause of the arrest of DNA replication in ts A1S9 cells at the nonpermissive temperature by comparing the modifications induced by the shift of temperature on the activity and the synthesis of DNA polymerase-alpha and DNA primase as a function of time. Forty-seven hours after temperature upshift DNA polymerase-alpha activity of ts A1S9 cells was inhibited by 90% while primase activity was barely detectable. By contrast, the activities of both enzymes increased to a plateau level in WT-4 cultured at either temperature and in ts A1S9 cells grown at the low permissive temperature. Study of the synthesis of DNA polymerase-alpha primase and of the structure of the enzyme complex during cell cycle progression was approached by immunoprecipitation of [35S]-labelled cells, with a specific monoclonal antibody directed against DNA polymerase-alpha. We have found that, irrespective of temperature of cultivation of WT-4 or ts A1S9 cells, this antibody precipitated polypeptides of 220, 186, 150, 110, 68-70, 60, and 48 kDa from cell extracts. With ts A1S9 cells cultivated at 38.5 degrees C for 48 hr the polypeptides of 220 and 186 kDa, associated with alpha-polymerase activity, were considerably more abundant than in the control cells, with a concomitant decline in the polypeptides of 60 and 48 kDa, implicated in primase activity. Thus the inhibition of DNA polymerase-alpha cannot be due to a decreased synthesis of the 186 kDa subunit but to its temperature inactivation. Consistent with a recent asymmetric dimeric model where polymerase-alpha complex and polymerase delta complex synthesize co-ordinately at the replication fork lagging and leading DNA strands, the observed alterations of polymerase-alpha and primase content explain the inhibition of DNA synthesis and the cell cycle arrest of the ts A1S9 cells at the nonpermissive temperature.     

Protein synthesis and degradation during expression of the temperature-sensitive defect in ts A1S9 mouse L-cells

The involvement of altered protein metabolism in the expression of the temperature-sensitive (ts) pleiotropic phenotype of ts A1S9 cells was investigated. Cells are ts in growth and DNA replication. They undergo decondensation of their heterochromatin, interruptions of chromatin synthesis, and changes in cell size and morphology at the non-permissive temperature (npt) of 38.5 degrees C. Whereas the rates of incorporation of 3H-leucine, 35S-methionine, and 3H-fucose into proteins were unaffected at 38.5 degrees C, net protein accumulation was greatly reduced. This imbalance resulted from a rapid increase in the rate of protein degradation at the npt. Enhancement of protein degradation was detected within 2-4 hours after temperature upshift and constitutes the earliest metabolic alteration thus far observed during expression of the temperature-sensitive phenotype. The average half-life of proteins performed in ts A1S9 cells at 34 degrees C was decreased four-fold at the npt, and all major cytoplasmic proteins were affected equally. Enhanced protein degradation at the npt was shown to be sensitive to cycloheximide, ammonia, chloroquine, and vinblastine at concentrations that did not affect the basal protein degradation of normally cycling cells. Increased protein degradation at 38.5 degrees C did not involve an equivalent increase in total cellular protease activity. The data obtained are compatible with a model that suggests that temperature inactivation of the ts A1S9 gene product results in activation of a lysosome-mediated mechanism for the rapid degradation of cytoplasmic proteins.     

Molecular cloning and expression of the cDNA for the alpha 1A-adrenergic receptor. The gene for which is located on human chromosome 5.

Pharmacological and molecular cloning studies have demonstrated heterogeneity of alpha 1-adrenergic receptors. We have now cloned two alpha 1-adrenergic receptors from a rat cerebral cortex cDNA library, using the hamster alpha 1B-adrenergic receptor as a probe. The deduced amino acid sequence of clone RA42 encodes a protein of 560 amino acids whose putative topology is similar to that of the family of G-protein-coupled receptors. The primary structure though most closely resembles that of an alpha 1-adrenergic receptor, having approximately 73% amino acid identity in the putative transmembrane domains with the previously isolated hamster alpha 1B receptor. Analysis of the ligand binding properties of RA42 expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 1-adrenergic receptor pharmacology. High affinity for the antagonist WB4101 and agonists phenylephrine and methoxamine suggests that cDNA RA42 encodes the alpha 1A receptor subtype. Northern blot analysis of various rat tissues also shows the distribution expected of the alpha 1A receptor subtype with abundant expression in vas deferens followed by hippocampus, cerebral cortex, aorta, brainstem, heart and spleen. The second alpha 1-adrenergic receptor cloned represents the rat homolog of the hamster alpha 1B subtype. Expression of mRNA for this receptor is strongly detected in liver followed by heart, cerebral cortex, brain stem, kidney, lung, and spleen. This study provides definitive evidence for the existence of three alpha 1-adrenergic receptor subtypes.     

Molecular cloning and primary structure of human chromogranin A (secretory protein I) cDNA

Chromogranin A (CGA), also referred to as secretory protein I, is an acidic protein that has been detected in all neuroendocrine cell types examined and is often present in large amounts relative to other secreted proteins. For example, CGA comprises at least 40% of the soluble protein of the adrenal chromaffin granule, and it appears to be the major secretory protein in the parathyroid secretory granules. CGA complementary DNAs (cDNAs) from bovine adrenal and pituitary have recently been cloned and sequenced and found to be nearly identical. A region of bovine CGA has a high degree of amino acid sequence identity to pancreastatin, a recently isolated porcine peptide that inhibits glucose-induced insulin secretion. This suggests that CGA may be a prohormone. We have cloned and sequenced a human cDNA encoding CGA. This human CGA cDNA has an overall 86% nucleic acid identity to the bovine cDNA. Like the bovine CGA cDNA, the human cDNA has little homology to pancreastatin at the 5' region of this peptide but significant amino acid homology to the carboxyl-terminal portion of pancreastatin where the biologic activity resides. There is an area within the pancreastatin region of human CGA and porcine pancreastatin with a 70% amino acid identity to the calcium-binding moiety of the E-F hand proteins such as parvalbumin and oncomodulin. These data suggest that CGA and pancreastatin may both be members of a larger family of calcium-binding proteins.     

Preliminary characterization of the temperature-sensitive defect in DNA replication in a mutant mouse L cell

Temperature-sensitive ts A1S9 mouse L cells synthesize DNA apparently normally for 6–8 hr upon incubation at 38.5°C. Thereafter, these cells are able to perform limited polydeoxyribonucleotide chain synthesis at the high temperature, but are unable to convert newly replicated small single-strand segments of DNA (of the order of molecular weight 10⁶ daltons) to large molecular weight chromosomal DNA. Data obtained are compatible with a model which suggests that ts A1S9 cells are able to carry out most individual reactions of DNA synthesis at the high temperature, but are temperature-sensitive in a protein which participates in the joining of small DNA segments to make chromosomal DNA strands. When cells are reincubated at a permissive temperature, after the temperature-sensitive lesion has been established, they recover the latter capability several hours before they are able once again to synthesize DNA at normal rates.     

Molecular cloning, structure and expression of the yeast proliferating cell nuclear antigen gene.

The budding yeast Saccharomyces cerevisiae is proving to be an useful and accurate model for eukaryotic DNA replication. It contains both DNA polymerase alpha (I) and delta (III). Recently, proliferating cell nuclear antigen (PCNA), which in mammalian cells is an auxiliary subunit of DNA polymerase delta and is essential for in vitro leading strand SV40 DNA replication, was purified from yeast. We have now cloned the gene for yeast PCNA (POL30). The gene codes for an essential protein of 29 kDa, which shows 35% homology with human PCNA. Cell cycle expression studies, using synchronized cells, show that expression of both the PCNA (POL30) and the DNA polymerase delta (POL3, or CDC2) genes of yeast are regulated in an identical fashion to that of the DNA polymerase alpha (POL1) gene. Thus, steady state mRNA levels increase 10-100-fold in late G1 phase, peak in early S-phase, and decrease to low levels in late S-phase. In addition, in meiosis mRNA levels increase prior to initiation of premeiotic DNA synthesis.     

Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA.

Melanocytes and melanoma cells are known to possess receptors for melanocyte stimulating hormone (MSH). A cDNA clone, designated 11D, has been isolated from human melanoma cells and encodes a MSH receptor. The cloned cDNA encodes a 317 amino acid protein with transmembrane topography characteristics of a G-protein-coupled receptor, but it does not show striking similarity to already published sequences of other G-protein-coupled receptors. When 11D cDNA is expressed in COS-7 cells, it binds an 125I-labelled MSH analogue (NDP-MSH) in a specific manner. The bound ligand could be displaced by melanotropic peptides, alpha-MSH, beta-MSH, gamma-MSH and ACTH (adrenocorticotropic hormone), but not by the non-melanotropic peptide, beta-endorphin. This is the first report of the cloning of the receptor gene of the melanotropin receptor family.     

Molecular cloning of monkey P450 1A1 cDNA and expression in yeast.

Monkey P450 1A1 cDNA (MKah1) was isolated from the lambda gt11 cDNA library of a liver from a 3-methylcholanthrene (3MC)-treated crab-eating monkey using a dog P450 1A1 cDNA fragment as a probe. MKah1 was 2453 bp long and contained an entire coding region for a polypeptide of 512 residues. The nucleotide and deduced amino acid sequences of MKah1 displayed 95% and 94% identity with those of the human P450 1A1 gene, respectively. Even in the 3' noncoding region, MKah1 showed 94% homology with human P450 1A1, whereas it showed less than 69% homology with other mammalian P450 1A1. Monkey P450 1A1 mRNA was not detectable in untreated livers, but was induced by polychlorinated biphenyl and 3MC. The expression plasmid (designated as pMKC-1) was constructed by introduction of the coding region of MKah1 into a yeast expression vector (pAM82) containing the promoter of acid phosphatase (APase). Northern blot analysis revealed that monkey P450 1A1 mRNA was expressed in yeast under the control of the APase promoter. Microsomes from yeast transformed by pMKC-1 catalyzed 7-ethoxycoumarin O-deethylation, benzo(a)pyrene hydroxylation and the mutagenic activation of 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), 3-amino-1-methyl-5H-pyrido(4,3-b)-indole acetate (Trp-P-2) and 2-amino-6-methyldipyrido(1,2-a:3',2'-d)imidazole acetate (Glu-P-1).     

Molecular cloning of AMP deaminase isoform L. Sequence and bacterial expression of human AMPD2 cDNA.

Human AMPD2 cDNA clones have been isolated from T-lymphoblast and placental lambda gt11 libraries utilizing a previously cloned rat partial AMPD2 cDNA as the probe. Alignment analysis of all cDNA clones indicates the presence of intervening sequences in several placental isolates. This has been confirmed by sequencing human AMPD2 genomic clones. Intervening sequences can be removed from the cDNA clones by restriction with endonucleases at unique sites within the proposed open reading frame. This results in a 3292-base pair cDNA proposed to contain the entire AMPD2 open reading frame, which would encode a 760-amino acid polypeptide with a predicted subunit molecular mass of 88.1 kDa. Nucleotide and predicted amino acid comparisons with the 264 base pairs of proposed coding sequences in the rat AMPD2 cDNA demonstrate 91% similarity and identity, respectively. A comparison of the predicted human AMPD1 and AMPD2 polypeptides demonstrates homology in their C-terminal domains. Included in this region is the conserved motif, SLSTDDP, proposed to be part of the catalytic site of all AMP deaminases. In contrast, the predicted N-terminal domains of the human AMPD1 and AMPD2 polypeptides are unique. When placed in a prokaryotic expression vector, the human AMPD2 cDNA expresses AMP deaminase activity which can be precipitated with polyclonal antisera specific for isoform L.     

Molecular cloning and expression of a G25K cDNA, the human homolog of the yeast cell cycle gene CDC42.

G25K is a low-molecular-mass GTP-binding protein with a broad distribution in mammalian tissues. A cDNA clone was isolated by using oligonucleotides corresponding to the partial amino acid sequence of purified human G25K. The cDNA encodes an 191-amino-acid polypeptide containing GTP-binding consensus sequences and a putative farnesylation site at the C terminus. The sequence exhibits 50 and 70% identities to the mammalian rho and rac proteins, respectively, and an 80% identity to the Saccharomyces cerevisiae CDC42 gene product. Insect Sf9 cells infected with recombinant baculovirus vectors expressing the G25K cDNA produced a 25-kDa protein that bound GTP and was recognized by antibodies specifically reactive to G25K. G25K appears to be the human homolog of the CDC42 gene product, since expression of the G25K cDNA in S. cerevisiae suppressed both cdc42-1 and cdc24-4 temperature-sensitive lethal mutations.     

Molecular cloning and expression of the human and mouse homologues of the Drosophila dachshund gene

Recent genetic analysis of the Drosophila dachshund (dac) gene has established that dac encodes a novel nuclear protein that is involved in both eye and leg development. In the Drosophila eye, dac expression appears to be controlled by the product of the eyeless/Pax6 gene. In order to analyze the Pax6 pathway in vertebrates we have isolated and characterized the cDNA and genomic clones corresponding to the human and mouse homologues of Drosophila dac. A full-length human cDNA encoding dachshund (DACH) encodes the 706 amino acids protein with predicted molecular weight of 73 kDa. A 109 amino acid domain located at the N-terminus of the DACH showed significant sequence and secondary structure homologies to the ski/sno oncogene products. Northern blot analysis found human DACH predominantly in adult kidney, heart, and placenta, with less expression detected in the brain, lung, skeletal muscle and pancreas. A panel of human cell lines was studied and most notably a large proportion of neuroblastomas expressed DACH mRNA. Mouse Dach encodes a protein of 751 amino acids with predicted molecular weight of 78 kDa that is 95% identical to the human DACH. RNase protection analysis showed the highest Dach mRNA expression in the adult mouse kidney and lung, whereas lower expression was detected in the brain and testis. RT/PCR analysis readily detected Dach mRNA in the adult mouse cornea and retina. Dach mRNA expression in the mouse E11.5 embryo was observed primarily in the fore and hind limbs, as well as in the somites. Received: 9 February 1999 / Accepted: 19 April 1999     

Molecular cloning of the cDNA for the catalytic subunit of human DNA polymerase delta.

The cDNA of human DNA polymerase delta was cloned. The cDNA had a length of 3.5 kb and encoded a protein of 1107 amino acid residues with a calculated molecular mass of 124 kDa. Northern blot analysis showed that the cDNA hybridized to a mRNA of 3.4 kb. Monoclonal and polyclonal antibodies to the C-terminal 20 residues specifically immunoblotted the human pol delta catalytic polypeptide. A multiple sequence alignment was constructed. This showed that human pol delta is closely related to yeast pol delta and the herpes virus DNA polymerases. The levels of pol delta message were found to be induced concomitantly with DNA pol delta activity and DNA synthesis in serum restimulated proliferating IMR90 cultured cells. The human pol delta gene was localized to chromosome 19 by Southern blotting of EcoRI digested DNA from a panel of rodent/human cell hybrids.