CAD Gene Research Articles

Analysis of CAD gene amplification using a combined approach of molecular genetics and cytogenetics.

CAD is a multifunctional protein which catalyzes the first three steps of de novo uridine biosynthesis. Rodent cells resistant to PALA, a specific inhibitor of the ATCase activity of CAD, overproduce the CAD protein and CAD mRNA as a direct result of the amplification of the CAD gene. In order to study the mechanism of CAD gene amplification, a functional Syrian hamster CAD gene was inserted into a cosmid vector using molecular cloning techniques. The cloned genes were assayed for biological function by fusing CAD-deficient Chinese hamster ovary (CHO) cell mutants with protoplasts of E. coli containing the CAD cosmids. Two clones with functional CAD genes were isolated and shown to contain inserts 40 and 45 kb long. The cloned genes could also be introduced into wild type CHO cells by selecting for cells which became resistant to high PALA concentrations in a single step. Transformations of mutant and wild type CHO cells contained multiple active copies of the donated Syrian hamster CAD genes in addition to their endogenous CHO CAD genes. The cloned genes in all transformants analyzed are integrated into host cell chromosomes at single locations defined by in situ hybridization. Independently isolated transformants contain the donated genes in different chromosomes. Co-transformation of CHO cells with two different genes by protoplast fusion is also shown to be possible.

Co-amplification of rRNA genes with CAD genes in N-(phosphonacetyl)-L-aspartate-resistant Syrian hamster cells.

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.

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

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.

Structure of amplified DNA in different Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

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.

Homologous recombination in mammalian cells mediates formation of a functional gene from two overlapping gene fragments.

Chinese hamster cells with a lesion in the CAD gene (cell line Urd-A) require exogenous uridine to survive. Uridine prototrophs could be isolated after introducing two recombinant plasmids containing overlapping fragments of a cloned Syrian hamster CAD gene. In contrast, no uridine prototrophs were obtained after introducing a plasmid containing only one of the two overlapping fragments. DNA restriction analysis showed that the prototrophic transformants contain a functional CAD gene which was formed by a recombination event in the overlapping region of the two clones. Most of the recombination events involved homologous exchanges, and some of them apparently were reciprocal. In situ hybridization analysis revealed that the donated sequences were integrated at a single chromosomal site which was different in each transformant. These results demonstrate the existence of a recombination system(s) in mammalian cells that can catalyze homologous exchanges. Recombination between donated sequences is a means by which this system can be characterized and also utilized for the production of novel gene fusions.

Evidence that the messenger RNA for the multifunctional protein which initiates pyrimidine biosynthesis is encoded by cytogenetic region 15A in Drosphila melanogaster

The protein product of the rudimentary wing locus of Drosophila melanogaster is shown to be a 220,00 daltons polypeptide translated from a 2.5–3.0 × 10 6 daltons messenger RNA. Therefore there are striking similarities between the products of the mammalian cad gene and the Drosophila rudimentary wing locus, which both encode the multifunctional enzyme carbamylphosphate synthetase, aspartate transcarbamylase and dihydroorotase (CPSase-ATCase-DHOase).

General Method for Cloning Amplified DNA by Differential Screening with Genomic Probes

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

A small cadmium resistance plasmid isolated from Staphylococcus aureus

We have isolated from Staphylococcus aureus a plasmid named pIP983, which measures 3.2 kb and specifies resistance to cadmium. The cad gene it carries is of the B type, as indicated by the level of resistance it confers on S. aureus and the sequence homology with the known cadB gene. Sequences homologous to pIP983 were found on several large S. aureus plasmids. They were localized close to the mcr region of pI/258 and pII147, and, at least in the case of the latter plasmid, were not contiguous, but interrupted by nonhomologous DNA.

The cloning and reintroduction into animal cells of a functional CAD gene, a dominant amplifiable genetic marker

Rodent cells resistant to PALA, a specific inhibitor of the aspartate transcarbamylase activity of the multifunctional CAD protein, overproduce CAD as a result of amplification of the CAD gene. We cloned a functional CAD gene from Syrian hamster cells using a cosmid vector. Two independently isolated cosmids containing CAD genes have inserts 40 and 45 kb long. We introduced the cloned genes into CAD-deficient Chinese hamster ovary (CHO) cell mutants by fusing them with protoplasts of Escherichia coli containing the cosmids. We also introduced the cloned genes into wild-type CHO cells by selecting cells that became resistant to high concentrations of PALA following protoplast fusion. The transformants of the mutant and wild-type CHO cells contain multiple active copies of the donated Syrian hamster CAD genes. The cloned genes in three independent transformants are integrated into host-cell chromosomes at single locations identified by in situ hybridization. In two of these transformants, the genes are located in one X chromosome or in a chromosome resembling the X. In the third case, the genes are located in a small metacentric or rearranged chromosome.

Gene amplification causes overproduction of the first three enzymes of UMP synthesis in N-(phosphonacetyl)-L-aspartate-resistant hamster cells.

Mutant Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a transition state analog inhibitor of aspartate transcarbamylase, overproduce CAD, a multifunctional protein which catalyzes the first three reactions of de novo UMP biosynthesis. Increased levels of a single mRNA cause the overproduction of CAD in all PALA-resistant mutants examined thus far. A recombinant plasmid containing a 2,3-kilobase insert complementary to the 3'-proximal region of this 7.9-kilobase mRNA has been prepared and used to show that the CAD gene is amplified in each of the 10 PALA-resistant mutants examined. Rates of association of CAD sequences in DNA isolated from PALA-sensitive and PALA-resistant cells with labeled plasmid DNA indicated that the degree of amplification is approximately equal to the degree of overproduction of protein and mRNA in each mutant. The patterns of digestion of these DNAs with restriction enzymes confirmed this result and showed that the lower limit for the size of the amplified unit is 19 kilobases, much larger than the mRNA. A comparison of restriction endonuclease digests of the cloned cDNA with digests of genomic DNA indicated that part of this difference is attributable to intervening sequences in the CAD gene. A 10.2-kilobase RNA which contains CAD sequences is found in cytoplasmic fractions from some PALA-resistant mutants but not in wild type cells. Restriction patterns were analyzed by a new method in which fragments of DNA are transferred from agarose gels to diazo paper with a high efficiency which is independent of size.