scholarly journals Structure of the gene for CAD, the multifunctional protein that initiates UMP synthesis in Syrian hamster cells.

1982 ◽  
Vol 2 (3) ◽  
pp. 293-301 ◽  
Author(s):  
R A Padgett ◽  
G M Wahl ◽  
G R Stark
Keyword(s):  
Electron Microscopy ◽  
Syrian Hamster ◽  
Specific Inhibitor ◽  
Aspartate Transcarbamylase ◽  
Multifunctional Protein ◽  
Intervening Sequences ◽  
Multiple Copies ◽  
Cad Gene ◽  
Polyadenylated Rnas ◽  
Ump Synthesis

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.

Structure of the gene for CAD, the multifunctional protein that initiates UMP synthesis in Syrian hamster cells

1982 ◽  
Vol 2 (3) ◽  
pp. 293-301
Author(s):  
R A Padgett ◽  
G M Wahl ◽  
G R Stark
Keyword(s):  
Electron Microscopy ◽  
Syrian Hamster ◽  
Specific Inhibitor ◽  
Aspartate Transcarbamylase ◽  
Multifunctional Protein ◽  
Intervening Sequences ◽  
Multiple Copies ◽  
Cad Gene ◽  
Polyadenylated Rnas ◽  
Ump Synthesis

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.

Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization

1982 ◽  
Vol 2 (3) ◽  
pp. 308-319
Author(s):  
G M Wahl ◽  
L Vitto ◽  
R A Padgett ◽  
G R Stark
Keyword(s):  
In Situ Hybridization ◽  
Syrian Hamster ◽  
Specific Radioactivity ◽  
Large Chromosome ◽  
Hybridization Technique ◽  
Wild Type ◽  
Aspartate Transcarbamylase ◽  
Metaphase Chromosomes ◽  
Cad Gene

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.

scholarly journals Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization.

1982 ◽  
Vol 2 (3) ◽  
pp. 308-319 ◽  
Author(s):  
G M Wahl ◽  
L Vitto ◽  
R A Padgett ◽  
G R Stark
Keyword(s):  
In Situ Hybridization ◽  
Syrian Hamster ◽  
Specific Radioactivity ◽  
Large Chromosome ◽  
Hybridization Technique ◽  
Wild Type ◽  
Aspartate Transcarbamylase ◽  
Metaphase Chromosomes ◽  
Cad Gene

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.

scholarly journals Identification of an origin of bidirectional DNA replication in the ubiquitously expressed mammalian CAD gene.

1995 ◽  
Vol 15 (8) ◽  
pp. 4136-4148 ◽  
Author(s):  
R E Kelly ◽  
M L DeRose ◽  
B W Draper ◽  
G M Wahl
Keyword(s):  
Dna Replication ◽  
Syrian Hamster ◽  
De Novo ◽  
Chinese Hamster ◽  
Specific Inhibitor ◽  
Housekeeping Gene ◽  
Replication Initiation ◽  
Transcriptional Unit ◽  
Carbamoyl Phosphate ◽  
Cad Gene

Most DNA replication origins in eukaryotes localize to nontranscribed regions, and there are no reports of origins within constitutively expressed genes. This observation has led to the proposal that there may be an incompatibility between origin function and location within a ubiquitously expressed gene. The biochemical and functional evidence presented here demonstrates that an origin of bidirectional replication (OBR) resides within the constitutively expressed housekeeping gene CAD, which encodes the first three reactions of de novo uridine biosynthesis (carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, and dihydroorotase). The OBR was localized to a 5-kb region near the center of the Syrian hamster CAD transcriptional unit. DNA replication initiates within this region in the single-copy CAD gene in Syrian baby hamster kidney cells and in the large chromosomal amplicons that were generated after selection with N-phosphonacetyl-L-aspartate, a specific inhibitor of CAD. DNA synthesis also initiates within this OBR in autonomously replicating extrachromosomal amplicons (CAD episomes) located in an N-phosphonacetyl-L-aspartate-resistant clone (5P20) of CHOK1 cells. CAD episomes consist entirely of a multimer of Syrian hamster CAD cosmid sequences (cCAD1). These data limit the functional unit of replication initiation and timing control to the 42 kb of Syrian hamster sequences contained in cCAD1. In addition, the data indicate that the origin recognition machinery is conserved across species, since the same OBR region functions in both Syrian and Chinese hamster cells. Importantly, while cCAD1 exhibits characteristics of a complete replicon, we have not detected autonomous replication directly following transfection. Since the CAD episome was generated after excision of chromosomally integrated transfected cCAD1 sequences, we propose that prior localization within a chromosome may be necessary to "license" some biochemically defined OBRs to render them functional.

scholarly journals Properties of single-step mutants of Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

1983 ◽  
Vol 3 (11) ◽  
pp. 2089-2098 ◽  
Author(s):  
J Zieg ◽  
C E Clayton ◽  
F Ardeshir ◽  
E Giulotto ◽  
E A Swyryd ◽  
...  
Keyword(s):  
Cell Lines ◽  
Syrian Hamster ◽  
Resistant Cell ◽  
Single Step ◽  
Similar Degree ◽  
Fluctuation Analysis ◽  
Aspartate Transcarbamylase ◽  
Resistant Lines ◽  
Cad Gene

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.

Properties of single-step mutants of Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate

1983 ◽  
Vol 3 (11) ◽  
pp. 2089-2098
Author(s):  
J Zieg ◽  
C E Clayton ◽  
F Ardeshir ◽  
E Giulotto ◽  
E A Swyryd ◽  
...  
Keyword(s):  
Cell Lines ◽  
Syrian Hamster ◽  
Resistant Cell ◽  
Single Step ◽  
Similar Degree ◽  
Fluctuation Analysis ◽  
Aspartate Transcarbamylase ◽  
Resistant Lines ◽  
Cad Gene

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.

Construction of a cDNA to the hamster CAD gene and its application toward defining the domain for aspartate transcarbamylase

1985 ◽  
Vol 5 (7) ◽  
pp. 1735-1742
Author(s):  
K Shigesada ◽  
G R Stark ◽  
J A Maley ◽  
L A Niswander ◽  
J N Davidson
Keyword(s):  
Protein A ◽  
Aspartate Transcarbamylase ◽  
Native Form ◽  
Multifunctional Protein ◽  
E Coli ◽  
Bacterial Enzyme ◽  
Cad Gene ◽  
Mammalian Enzyme ◽  
Quaternary Structures ◽  
Cad Protein

cDNA complementary to hamster mRNA encoding the CAD protein, a multifunctional protein which carries the first three enzymes of pyrimidine biosynthesis, was constructed. The longest of these recombinants (pCAD142) covers 82% of the 7.9-kilobase mRNA. Portions of the cDNA were excised and replaced by a lac promoter-operator-initiation codon segment. The resultant plasmids were transfected into an Escherichia coli mutant defective in aspartate transcarbamylase, the second enzyme of the pathway. Complementation of the bacterial defect was observed with as little as 2.2 kilobases of cDNA sequence, corresponding to the 3' region of the mRNA. DNA sequencing in this region of the hamster cDNA reveals stretches which are highly homologous to the E. coli gene for the catalytic subunit of aspartate transcarbamylase; other stretches show no homology. The highly conserved regions probably reflect areas of protein structure critical to catalysis, while the nonconserved regions may reflect differences between the quaternary structures of E. coli and mammalian aspartate transcarbamylases, one such difference being that the bacterial enzyme in its native form is allosterically regulated and the mammalian enzyme is not.

General method for cloning amplified DNA by differential screening with genomic probes

1982 ◽  
Vol 2 (5) ◽  
pp. 578-587
Author(s):  
O Brison ◽  
F Ardeshir ◽  
G R Stark
Keyword(s):  
Genomic Dna ◽  
Screening Method ◽  
Specific Inhibitor ◽  
Differential Screening ◽  
Gene Copy ◽  
Dna Fragments ◽  
Wild Type ◽  
Aspartate Transcarbamylase ◽  
Gene Coding ◽  
Cad Gene

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.

scholarly journals Construction of a cDNA to the hamster CAD gene and its application toward defining the domain for aspartate transcarbamylase.

1985 ◽  
Vol 5 (7) ◽  
pp. 1735-1742 ◽  
Author(s):  
K Shigesada ◽  
G R Stark ◽  
J A Maley ◽  
L A Niswander ◽  
J N Davidson
Keyword(s):  
Protein A ◽  
Aspartate Transcarbamylase ◽  
Native Form ◽  
Multifunctional Protein ◽  
E Coli ◽  
Bacterial Enzyme ◽  
Cad Gene ◽  
Mammalian Enzyme ◽  
Quaternary Structures ◽  
Cad Protein

cDNA complementary to hamster mRNA encoding the CAD protein, a multifunctional protein which carries the first three enzymes of pyrimidine biosynthesis, was constructed. The longest of these recombinants (pCAD142) covers 82% of the 7.9-kilobase mRNA. Portions of the cDNA were excised and replaced by a lac promoter-operator-initiation codon segment. The resultant plasmids were transfected into an Escherichia coli mutant defective in aspartate transcarbamylase, the second enzyme of the pathway. Complementation of the bacterial defect was observed with as little as 2.2 kilobases of cDNA sequence, corresponding to the 3' region of the mRNA. DNA sequencing in this region of the hamster cDNA reveals stretches which are highly homologous to the E. coli gene for the catalytic subunit of aspartate transcarbamylase; other stretches show no homology. The highly conserved regions probably reflect areas of protein structure critical to catalysis, while the nonconserved regions may reflect differences between the quaternary structures of E. coli and mammalian aspartate transcarbamylases, one such difference being that the bacterial enzyme in its native form is allosterically regulated and the mammalian enzyme is not.

Properties of dispersed, highly repeated DNA within and near the hamster CAD gene

1982 ◽  
Vol 2 (3) ◽  
pp. 302-307
Author(s):  
R A Padgett ◽  
G M Wahl ◽  
G R Stark
Keyword(s):  
Dna Sequences ◽  
Rna Polymerase Iii ◽  
Repeated Sequences ◽  
Repeated Dna ◽  
Multifunctional Protein ◽  
Highly Repeated Dna ◽  
Gene Coding ◽  
Cad Gene ◽  
Polyadenylated Rnas

Dispersed, highly repeated DNA sequences were found within and near the Syrian hamster gene coding for the multifunctional protein CAD. Most of the repeated sequences were homologous to each other and had similar properties. They hybridized to many cytoplasmic polyadenylated RNAs and to 7S and 4.5S cytoplasmic non-polyadenylated RNAs. Cloned DNA fragments containing repeated sequences were transcribed in vitro by RNA polymerase III. The repeated sequences from Syrian hamsters share many properties with the Alu family of repetitive DNA from humans. The hamster sequences were homologous to total repetitive human DNA but only very weakly homologous to two cloned members of the human Alu family.

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