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We have compared clones of Syrian hamster cells selected for the first amplification of the CAD gene with clones selected for further amplification. The large domain amplified initially was not reamplified as an intact unit. Instead, subregions were reamplified preferentially, and parts of the initial array were often lost. These events reduced the average amount of coamplified DNA accompanying each copy of the selected gene. The degree of amplification was small in the first step (about three extra copies of CAD per cell), but second-step amplifications to a high copy number (up to 60 extra copies per cell) occurred frequently. After several separate steps of amplification, highly condensed arrays that brought many CAD genes close together were formed. In striking contrast to the stability of these highly amplified arrays, the low-copy chromosomal arrays formed early were quite unstable and were often lost completely within 1 or 2 months of growth without selection. The results suggest that different mechanisms may be involved in the first step of amplification and in the later evolution of an already amplified array.
Mol Cell Biol 1989 Jun
PMID:Evolution and stability of chromosomal DNA coamplified with the CAD gene. 256 69

We have determined the 7168 nucleotide DNA sequence corresponding to the messenger RNA of the rudimentary gene of Drosophila melanogaster. By sequence comparison with genes involved in the pyrimidine pathway of prokaryotes and lower eukaryotes, we conclude that the rudimentary gene encodes four enzymically different functions. Each function is restricted to a specific coding domain but in an order different from that previously defined by genetic data. We have found that the corresponding mammalian gene, the CAD gene, exhibits a similar functional organization, and we propose schemes for the evolution of the corresponding coding sequences.
J Mol Biol 1987 Jan 05
PMID:The rudimentary gene of Drosophila melanogaster encodes four enzymic functions. 288 25

In a previous study (G. M. Wahl, B. Robert de Saint Vincent, and M. L. De Rose, Nature (London) 307:516-520, 1984), we used gene transfer of a CAD cosmid to demonstrate that gene position profoundly affects amplification frequency. One transformant, T5, amplified the donated CAD genes at a frequency at least 100-fold higher than did the other transformants analyzed. The CAD genes in T5 and two drug-resistant derivatives were chromosomally located. In this report, we show that a subclone of T5 gives rise to an extrachromosomal molecule (CAD episome) containing the donated CAD genes. Gel electrophoresis indicated that the CAD episome is approximately 250 to 300 kilobase pairs, and a variety of methods showed that it is a covalently closed circle. We show that the CAD episome replicates semiconservatively and approximately once per cell cycle. Since the CAD cosmid, which comprises most of the CAD episome, does not replicate autonomously when transfected into cells, our results indicate that either the process which generated the episome resulted in a cellular origin of DNA replication being linked to the CAD sequences or specific rearrangements within the episome generated a functional origin. The implications of these results for mechanisms of gene amplification and the genesis of minute chromosomes are discussed.
Mol Cell Biol 1987 May
PMID:Characterization of an episome produced in hamster cells that amplify a transfected CAD gene at high frequency: functional evidence for a mammalian replication origin. 288 42

CAD codes for a trifunctional protein involved in the catalysis of the first three enzymatic activities in the de novo pyrimidine biosynthetic pathway, namely, carbamoyl-phosphate synthetase II (EC 6.3.5.5), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3). CAD regulation was studied in the human promyelocyte leukemic line HL-60 as it differentiated into monocytic or granulocytic lineages after induction by 12-O-tetradecanoylphorbol-13-acetate or trans-retinoic acid and dibutyryl cyclic AMP, respectively. Within 12 h of induction of HL-60 cells with either inducer, total cellular levels of CAD RNA essentially disappeared. On the other hand, no apparent decreases in beta-actin RNA levels were seen even 48 h after HL-60 cells were induced, as compared with untreated cells. With nuclear runoff assays, it was clearly shown that the inactivation of CAD gene expression during the induction of HL-60 cells with either inducer was at the transcriptional level. The nuclear runoff experiments also demonstrated that the CAD gene expression was shut down in less than 4 h after induction, well before morphological changes were observed in these cells. At the enzymatic level, the activity of aspartate transcarbamylase, one of the three enzymes encoded by the CAD gene, decreased by about half within 24 h of induction, suggesting a CAD protein half-life of 24 h in differentiating HL-60 cells. Nevertheless, this means that significant levels of aspartate transcarbamylase activity remained even after the cells have stopped proliferating. From the RNA data, it is clear that CAD gene expression is rapidly turned off as promyelocytes begin to terminally differentiate into macrophages and granulocytes. We suspect that the inactivation of the CAD gene in induced HL-60 cells is a consequence of the differentiating cells leaving the cell cycle and becoming nonproliferating.
Mol Cell Biol 1987 May
PMID:Transcriptional regulation of the human CAD gene during myeloid differentiation. 288 43

Although aspartate transcarbamylase (ATCase) is an independent, monofunctional enzyme in Escherichia coli, mammalian ATCase is one of the globular enzymatic domains of the multifunctional CAD protein. We subcloned fragments of the hamster CAD cDNA and assayed polypeptide products expressed in E. coli for ATCase activity in order to isolate a stretch of cDNA which encodes only the ATCase domain. Three such expression constructs contain fragments of hamster CAD cDNA similar in length to the gene encoding the E. coli ATCase catalytic subunit (pyrB). These constructs yield stable proteins with ATCase activity, ascertained by both in vivo and in vitro assays; the clones also possess sequence homology with the pyrB gene at both the 5' and 3' ends. The clone producing the most active ATCase contains cDNA which is analogous to the entire pyrB gene, plus a small amount of CAD sequence upstream of this region. Because these constructs produce independently folded, active ATCase from a piece of cDNA the size of the E. coli pyrB gene, they open the door for the in-depth investigation of the isolated mammalian enzyme domain utilizing recombinant DNA technology. This approach is potentially useful for the analysis of domains of other multifunctional proteins.
Mol Gen Genet 1988 Aug
PMID:The aspartate transcarbamylase domain of a mammalian multifunctional protein expressed as an independent enzyme in Escherichia coli. 290 35

In animals, the first three enzymatic steps of de novo pyrimidine synthesis, carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase, comprise the multifunctional protein known as the CAD protein. Mutants of Chinese hamster ovary cells (CHO-K1, pro-) deficient in CAD protein activities require uridine for growth and are designated Urd-A mutants. To examine further the nature of the genetic alterations in Urd-A mutants and revertants, we have performed a detailed Southern blot hybridization analysis of DNA from wild-type, Urd-A, and revertant cells using as hybridization probes cDNAs complementary to CAD mRNA isolated from Syrian hamster. This has allowed us to identify an apparent alteration in the CAD gene in DNA from Urd-A cells. This alteration is in a region of the gene which appears to correspond to the region of the protein which is hypersensitive to proteases and which seems to be altered in the mutants. Only one of the two CAD alleles present appears to be altered in this way. Study of certain revertants of Urd-A strongly suggests that in some cases reversion has occurred by amplification of the mutant CAD allele.
Somat Cell Mol Genet 1985 Jul
PMID:Identification and localization of DNA alteration in Chinese hamster ovary cell mutants (Urd-) defective in the first three enzymes of de novo pyrimidine synthesis. 299 1

Eucaryotic expression vectors containing the Escherichia coli pyrB gene (pyrB encodes the catalytic subunit of aspartate transcarbamylase [ATCase]) and the Tn5 phosphotransferase gene (G418 resistance module) were transfected into a mutant Chinese hamster ovary cell line possessing a CAD multifunctional protein lacking ATCase activity. G418-resistant transformants were isolated and analyzed for ATCase activity, the ability to complement the CAD ATCase defect, and the ability to resist high concentrations of the ATCase inhibitor N-(phosphonacetyl)-L-aspartate (PALA) by amplifying the donated pyrB gene sequences. We report that bacterial ATCase is expressed in these lines, that it complements the CAD ATCase defect in trans, and that its amplification engenders PALA resistance. In addition, we derived rapid and sensitive assay conditions which enable the determination of bacterial ATCase enzyme activity in the presence of mammalian ATCase.
Mol Cell Biol 1986 Sep
PMID:Escherichia coli aspartate transcarbamylase: a novel marker for studies of gene amplification and expression in mammalian cells. 353 29

We analyzed the amplification of the CAD gene in independently isolated N-(phosphonacetyl)-L-aspartate-resistant clones derived from single parental clones in two mouse cell lines. We report for the first time that the CAD gene is amplified unstably in mouse cells, that the degree of instability varies greatly between clones, and that minute chromosomes and highly unstable chromosomelike structures contain the amplified sequences. These data are most consistent with the idea that the amplified unit in each clone consists of different flanking DNA and that such differences engender amplified sequences with unequal stability. We also introduced the mouse chromosome containing the CAD gene into hamster cells by microcell-mediated chromosome transfer to determine whether the propensity for unstable extrachromosomal amplification of the mouse CAD gene would prevail in the hamster cell nuclear environment. We report that the mouse CAD gene was amplified stably in expanded chromosomal regions in each of seven hybrids that were analyzed. This observation is consistent with the idea that the nuclear environment influences whether mutants containing intra- or extrachromosomally amplified sequences will be isolated.
Mol Cell Biol 1987 Apr
PMID:Unstable and stable CAD gene amplification: importance of flanking sequences and nuclear environment in gene amplification. 360 Jun 32

Transcripts of the CAD gene in Syrian hamster cells are as abundant in the nucleus as in the cytoplasm. This was shown by in situ hybridization of whole cells and by solution and blot hybridization of subcellular fractions. Similar results were obtained both for wild-type cells and for a mutant containing amplified CAD genes in which the level of CAD RNA is 150-fold greater. CAD nuclear RNA is indistinguishable from mature mRNA by gel electrophoresis and blot hybridization. Discrete higher-molecular-weight precursors are undetectable, although the persistence of a short length of intervening sequence in the otherwise fully processed RNA is not excluded. CAD RNA is released from nuclei by sonication in physiological conditions in a ribonucleoprotein form that sediments as a broad peak at about 200S in a sucrose gradient. CAD sequences extracted from nuclei by treatment with EDTA and RNase are found in the 30S particles previously described.
Mol Cell Biol 1985 Mar
PMID:Abundant nuclear ribonucleoprotein form of CAD RNA. 399 Jun 84

Two adjacent fragments of genomic DNA spanning the gene for CAD, which encodes the first three enzymes of UMP biosynthesis, were cloned from a mutant Syrian hamster cell line containing multiple copies of this gene. The mutant was selected for resistance to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, the second enzyme in the pathway. The sizes and positions of about 37 intervening sequences within the 25-kilobase CAD gene were mapped by electron microscopy, and the locations of the 5' and 3' ends of the 7.9-kilobase CAD mRNA were established by electron microscopy and by other hybridization methods. The coding sequences are small (100 to 400 bases), as are most of the intervening sequences (50 to 300 bases). However, there are also several large intervening sequences of up to 5,000 bases each. Two small cytoplasmic polyadenylated RNAs are transcribed from a region just beyond the 5' end of the CAD gene, and their abundance reflects the degree of gene amplification.
Mol Cell Biol 1982 Mar
PMID:Structure of the gene for CAD, the multifunctional protein that initiates UMP synthesis in Syrian hamster cells. 612 80


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