Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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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

Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a specific inhibitor of the aspartate transcarbamylase activity of the multifunctional protein CAD, overproduce this protein as a result of amplification of the CAD gene. We have used a sensitive in situ hybridization technique to localize CAD genomes in spreads of metaphase chromosomes from several independent PALA-resistant lines and from wild-type PALA-sensitive cells. The amplified genes were always found within chromosomes, usually in an expanded region of the short arm of chromosome B9. In wild-type cells, the CAD gene was also on the short arm of chromosome B9. In one mutant line, 90 to 100 CAD genes were found within an expanded B9 chromosome and 10 to 15 more were near the distal end of one arm of several different chromosomes. Another line contained most the genes in a telomeric chromosome or large chromosome fragment. The amplified genes were in chromosomal regions that were stained in a banded pattern by trypsin-Giemsa. A few double minute chromosomes were observed in a very small fraction of the total spreads examined. The it situ hybridizations were performed in the presence of 10% dextral sulfate 500, which increases the signal by as much as 100-fold. Using recombinant DNA plasmids nick-translated with [125I]dCTP to high specific radioactivity, 10 CAD genes in a single chromosomal region were revealed after 1 week of autoradiographic exposure, and the position of the unique gene could be seen after 1 month.
Mol Cell Biol 1982 Mar
PMID:Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization. 618 Mar 4

The amplified CAD genes in N-(phosphonacetyl)-L-aspartate (PALA)-resistant Syrian hamster cells are located in an expanded chromosomal region emanating from the site of the wild-type gene at the tip of the short arm of chromosome B-9. The terminus of B-9 in PALA-sensitive cells contains a cluster of rRNA genes (i.e., a nucleolus organizer, rDNA). We have used a molecular clone containing sequences complementary to Syrian hamster 28S rRNA to investigate whether rDNA is coamplified with CAD genes in the PALA-resistant mutants. In situ hybridization of this probe to metaphase chromosomes demonstrates that rDNA and CAD genes do coamplify in two independently isolated PALA-resistant mutants. The tight linkage of CAD and rDNA genes was demonstrated by their coordinate translocation from B-9 to the end of the long arm of chromosome C-11 in one mutant. Blot hybridization studies substantiate the in situ hybridization results. Both types of analysis indicate that only one or two rDNA genes, on the average, are coamplified per CAD gene. The data are consistent with the model that unequal exchanges between rDNA genes mediate the amplification of CAD genes in the Syrian hamster mutants that were analyzed.
Mol Cell Biol 1983 Nov
PMID:Co-amplification of rRNA genes with CAD genes in N-(phosphonacetyl)-L-aspartate-resistant Syrian hamster cells. 631 80

Hypertension is a major risk factor for clinically significant atherosclerotic vascular disease in Western Society, although the link between these conditions remains very poorly understood. Recent studies which are reviewed here have demonstrated that major arterial intimal and medial abnormalities occur as a result of hypertension. These include functional changes in endothelial permeability as well as alterations in the endothelial cells themselves with an increase in their turnover and number and distinct changes in morphology. However, endothelial cell loss leading to denudation of the arterial intimal surface appears to be relatively uncommon. Intimal and medial thickening are consistent features of hypertension and result from increases in both cellular and extracellular components. The cells accumulating in the subendothelial space appear to be of both blood-borne and medial origins, although their complete characterization has not been performed as yet. The adherence of blood cells to the endothelial surface appears to be promoted by the presence of hypertension along with their increased entry into the intima through endothelial cell junctions. Medial thickening with hypertension is attributable primarily to increased smooth muscle cell mass, although enhanced deposition of collagen and elastin plays a contributory role. Recent data would indicate that smooth muscle cell hypertrophy rather than hyperplasia is primarily responsible for the greater smooth muscle mass with hypertension. Although elevated DNA content of hypertensive arteries has been demonstrated, such changes may be secondary to a marked increase in cells showing nuclear polyploidy. Prolonged normalization of blood pressure in hypertensive animals can produce considerable regression of arterial changes toward the control state. The changes appear more marked with respect to the cellular rather than the extracellular abnormalities induced by hypertension. In man, little is known about the effects of antihypertensive therapy on the vasculature itself, although clinical complications related to both hemorrhagic or thrombotic strokes are clearly reduced by blood pressure reduction. On the other hand, the influence of treatment on the atherosclerotic process or on the course of coronary artery disease and its complications is not currently understood. The accelerating effect of hypertension on atherosclerosis generally requires a critical level of circulating lipoproteins. Enhanced atherosclerosis is not observed in hypertensive animals without hyperlipoproteinemia or in human subjects with low lipoprotein concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)
Exp Mol Pathol 1984 Aug
PMID:Recent advances in molecular pathology. The effects of hypertension on the arterial wall. 638 Oct 89

Young adult male rhesus monkeys (Macaca mulatta) were fed an atherogenic diet for 38 months. After 38 months of atherosclerosis induction, a baseline group was selected and necropsied to determine the extent and severity of atherosclerosis before regression regimens were begun. The remaining animals were fed diets that varied in cholesterol concentration in order to maintain plasma cholesterol concentrations of approximately 200 or 300 mg/dl for either 24 or 48 months. The progression or regression of atherosclerosis in coronary arteries, abdominal aorta, and carotid arteries was determined by comparing them to the baseline group. Coronary artery atherosclerosis regressed in the majority of animals after 4 years but not after 2 years when plasma cholesterol concentrations were about 200 mg/dl. Among the animals maintained at plasma cholesterol concentrations of about 300 mg/dl, about half the animals progressed in the extent of coronary artery atherosclerosis while about half regressed. The majority of the animals that progressed in lesion extent were genetic hyperresponders to dietary cholesterol whereas those that regressed were predominantly hyporesponders, even though their plasma lipid concentrations were equivalent during the regression phase. The changes seen in atherosclerosis extent in the abdominal aorta were quite similar to the changes seen in coronary arteries. Changes at this site were not pronounced after 2 years, but after 4 years animals with plasma cholesterol concentrations of about 300 mg/dl progressed while the animals at 200 mg/dl were mostly unchanged. No evidence for atherosclerosis regression was found in the common carotid arteries or in the carotid bifurcations.
Exp Mol Pathol 1984 Aug
PMID:A study of atherosclerosis regression in Macaca mulatta. V. Changes in abdominal aorta and carotid and coronary arteries from animals with atherosclerosis induced for 38 months and then regressed for 24 or 48 months at plasma cholesterol concentrations of 300 or 200 mg/dl. 646 38

Syrian hamster cell lines selected in multiple steps for resistance to high levels of N-(phosphonacetyl)-L-aspartate (PALA) contain many copies of the gene coding for the pyrimidine pathway enzyme CAD. Approximately 500 kilobases of additional DNA was coamplified with each copy of the CAD gene in several cell lines. To investigate its structure and organization, we cloned ca. 162 kilobases of coamplified DNA from cell line 165-28 and ca. 68 kilobases from cell line B5-4, using a screening method based solely on the greater abundance of amplified sequences in the resistant cells. Individual cloned fragments were then used to probe Southern transfers of genomic DNA from 12 different PALA-resistant mutants and the wild-type parents. A contiguous region of DNA ca. 44 kilobases long which included the CAD gene was amplified in all 12 mutants. However, the fragments cloned from 165-28 which were external to this region were not amplified in any other mutant, and the external fragments cloned from B5-4 were not amplified in two of the mutants. These results suggest that movement or major rearrangement of DNA may have accompanied some of the amplification events. We also found that different fragments were amplified to different degrees within a single mutant cell line. We conclude that the amplified DNA was not comprised of identical, tandemly arranged units. Its structure was much more complex and was different in different mutants. Several restriction fragments containing amplified sequences were found only in the DNA of the mutant cell line from which they were isolated and were not detected in DNA from wild-type cells or from any other mutant cells. These fragments contained novel joints created by rearrangement of the DNA during amplification. The cloned novel fragments hybridized only to normal fragments in every cell line examined, except for the line from which each novel fragment was isolated or the parental population for that line. This result argues that "hot spots" for forming novel joints are rare or nonexistent.
Mol Cell Biol 1983 Nov
PMID:Structure of amplified DNA in different Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate. 665 63

Eleven independent lines of Syrian hamster cells were selected by using very low levels of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate transcarbamylase. The protocol employed insured that each resistant cell arose during one of the last divisions before selection was applied. Cells of each mutant line contained an amplification of the structural gene for CAD, a trifunctional protein which includes aspartate transcarbamylase and two other enzymes of UMP biosynthesis. Strikingly, despite the minimal selection employed, the degree of amplification of the CAD gene was 6 to 10 times the normal diploid number in all 11 cases. In situ hybridization indicated that the amplified CAD genes were almost always present at a single chromosomal site in each line. Therefore, one of the two alleles was amplified 11- to 19-fold. The rates at which cells became resistant to PALA, determined by fluctuation analysis, were 100 times less dependent on drug concentration than were the frequencies of resistant cells in steady-state populations. The relatively shallow dependence of this rate upon PALA concentration is consistent with our independent observation that most events gave rise to a similar degree of amplification. In six of six cell lines examined, the levels of CAD mRNA and aspartate transcarbamylase activity were elevated two- to fourfold. These lines were resistant to PALA concentrations 20- to 80-fold higher than the ones used for selection. The organization of amplified DNA was examined by hybridizing Southern blots with cloned DNA fragments containing amplified sequences, previously isolated from two cell lines resistant to high levels of PALA. A contiguous region of DNA approximately 44 kilobases long which included the CAD gene was amplified in five of five single-step mutants examined. Outside this region, these mutants shared amplified sequences with only one of the two highly resistant lines.
Mol Cell Biol 1983 Nov
PMID:Properties of single-step mutants of Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate. 665 64

The results of three consecutive experiments are reported in which atherosclerotic lesions of adult male rhesus monkeys produced by a ration relatively rich in calories, cholesterol, and saturated fats and resembling the "average American" table-prepared fare were compared with the atherosclerotic lesions in similar monkeys fed a low-fat, low-cholesterol, and low-calorie, so-called "prudent" table-prepared ration. Each experiment lasted 2 years. The frequency and severity of gross and microscopic aortic atheromatous disease and microscopic coronary artery disease were compared. In addition, the qualitative features of the lesions, several types of analyses of blood lipids, and the reactions of aortic cells to in vivo deposited interstitial aortic deposits of lipoprotein fractions prepared from these animals are described. In general, the animals fed the average American ration had serum cholesterols that were consistently higher (383 +/- 35 mg%) than animals fed the prudent ration (199 +/- 13 mg%). The contrasts in lesion involvement varied from about 6:1 in severity for both aortic and coronary lesions to approximately 3:1 or 4:1 in frequency. The aortic gross surface area involvement at autopsy was 46% for monkeys fed the average American ration compared to 7% for the prudent diet group. In lesions of animals fed the average American ration much of the lipid was extracellular and both cell proliferation and fiber protein deposition were prominent. The small lesions in the animals fed the prudent ration were much more likely to be composed mostly of "foam cell" intimal thickenings. The lesions seen in the animals fed the average American ration resemble those seen in autopsies of many young Americans whose lesions have been studied recently and who demonstrate raised lesions in their coronary arteries and aortas in the third decade.
Exp Mol Pathol 1983 Feb
PMID:Arterial lesions and blood lipids in rhesus monkeys fed human diets. 683 36

Mutant Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, have amplified the gene coding for the multifunctional protein (CAD) that includes this activity. The average amount of DNA amplified is approximately 500 kilobases per gene copy, about 20 times the length of the CAD gene itself. A differential screening method which uses genomic DNAs as probes was developed to isolate recombinant phage containing fragments of amplified DNA. One probe was prepared by reassociating fragments of total genomic DNA from 165-28, a mutant cell line with 190 times the wild-type complement of CAD genes, until all of the sequences repeated about 200 times were annealed and then isolating the double-stranded DNA with hydroxyapatite. This DNA was highly enriched in sequences from the entire amplified region, whereas the same sequences were very rare in DNA prepared similarly from wild-type cells. After both DNAs were labeled by nick translation, highly repeated sequences were removed by hybridization to immobilized total genomic DNA from wild-type cells. A library of cloned DNA fragments from mutant 165-28 was screened with both probes, and nine independent fragments containing about 165 kilobases of amplified DNA, including the CAD gene, have been isolated so far. These cloned DNAs can be used to study the structure of the amplified region, to evaluate the nature of the amplification event, and to investigate gene expression from the amplified DNA. For example, one amplified fragment included a gene coding for a 3.8-kilobase, cytoplasmic, polyadenylated RNA which was overproduced greatly in cells resistant to N-(phosphonacetyl)-L-aspartate. The method for cloning amplified DNA is general and can be used to evaluate the possible involvement of gene amplification in phenomena such as drug resistance, transformation, or differentiation. DNA fragments corresponding to any region amplified about 10-fold or more can be cloned, even if no function for the region is known. The method for removing highly repetitive sequences from genomic DNA probes should also be of general use.
Mol Cell Biol 1982 May
PMID:General method for cloning amplified DNA by differential screening with genomic probes. 689 36

The position of the genetic locus allelic with the cad-2 mutation has been located between units 14 and 15 of the linkage map of S. typhimurium. Fine structure mapping established the gene order as cad flrB nag. The genetic evidence coupled with biochemical evidence indicates that this cad locus is homologous to the ubiF gene of Escherichia coli.
Mol Gen Genet 1980
PMID:A mutation in Salmonella typhimurium imparting conditional resistance to 5-fluorouracil and a bioenergetic defect: mapping of cad. 700 3


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