CAD Gene Research Articles

The propensity for gene amplification: A comparison of protocols, cell lines, and selection agents

We have studied cell lines of rodent and human origin for their propensity to become resistant to antifolates (methotrexate, trimetrexate), phosphonacetyl- L-aspartate (PALA), and colcemid, resistances associated with amplification of the DHFR, CAD, and MDR1 genes, respectively. We have employed two different methods: (1) a shallow step-wise selection protocol, where time to attain specified resistance is the quantitative measure, (2) the frequency of resistant colonies at specified drug concentrations. Although there are advantages and disadvantages to both methods, the two methods gave the same relative ranking of cell lines. Striking differences in the propensity for gene amplification (resistance) were found: human cell lines were less prone to amplify genes than Chinese hamster ovary (CHO) cells. This ranking was similar with all of the agents employed. Additionally, we observed that whereas PALA resistance in CHO cells in associated with amplification of the CAD gene, PALA resistance in the two human cell lines studied (HeLaS3 and VA13) was not associated with amplification and/or overexpression of the CAD gene, and thus this resistance to PALA occurs by an unknown mechanism.

Localization of the Chinese Hamster CAD Gene Reveals Homology between Human Chromosome 2p and Chinese Hamster 7q

The trifunctional enzyme CAD catalyzes the first three steps of pyrimidine biosynthesis. By using fluorescence in situ hybridization we have localized the Chinese hamster CAD gene on chromosome 7q11-q13 of diploid fibroblasts. Other genes previously assigned to chromosome 7 include acid phosphatase-1, the M2 subunit of ribonucleotide reductase and ornithine decarboxylase. These genes are also syntenic with CAD on human chromosome 2p. We have then mapped CAD on the pericentromeric region of two different rearranged chromosomes (Z8p and R2q) in a cell line derived from Chinese hamster ovary. The presence of CAD on Z8 and R2 indicates that they derive from rearrangements involving chromosome 7.

Accumulation of anchorage independent cells showing amplified genes (CAD) during the in vitro propagation of CHEF18 Chinese hamster cells

Anchorage independence and gene amplification have frequently been associated with a transformed or tumorigenic phenotype in cultured mammalian cells. However, it is unknown whether these two traits occur as related events during transformation, or are independent features of the transformed phenotype. To clarify this point, immortalized, untransformed CHEF18 Chinese hamster cells were propagated in culture until they became transformed and tumorigenic. The frequencies with which CHEF18 cells formed colonies either in soft agar, in medium containing N-phosphonacetyl-L-aspartate or in the two selective media simultaneously, were determined. The results indicate that anchorage independence and CAD gene amplification spontaneously arose during the propagation of the cells and that their concurrent emergence was not the consequence of independent events. However, the kinetics of their appearance suggests that anchorage independence is the early event whereas gene amplification might represent one of the numerous events which can be dynamically selected in anchorage-independent cells.

Chromosomes bearing amplified genes are a preferential target of chemicals inducing chromosome breakage and aneuploidy

Micronuclei were induced in V79 Chinese hamster cells and in PALA L and MTX M, two derivative cell lines harboring amplified genes, with 1,3-bis(2-chloroethyl)nitrosourea (BCNU) and vinblastine. Spontaneous and induced micronuclei were analyzed for the presence of centromeres by immunofluorescent CREST staining. Micronuclei formed in PALA L cells were also analyzed for the presence of amplified DNA by in situ hybridization with a CAD gene probe. Both cell lines containing amplified genes showed increased micronucleus induction by BCNU and vinblastine. The marker chromosome of PALA L cells was found to be a preferential target for both the clastogenic and the aneugenic action of the two chemicals, DNA amplification seems therefore to be a destabilizing factor of chromosomal structural integrity and mitotic segregation.

CAD gene sequence and the domain structure of the mammalian multifunctional protein CAD

Conference Article| February 01 1993 CAD gene sequence and the domain structure of the mammalian multifunctional protein CAD D. R. Evans; D. R. Evans 1Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar K. Bein; K. Bein 1Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar H. I. Guy; H. I. Guy 1Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar X. Liu; X. Liu 1Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar J. A. Molina; J. A. Molina 1Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar B. H. Zimmermann B. H. Zimmermann 1Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1993) 21 (1): 186–191. https://doi.org/10.1042/bst0210186 Article history Received: September 16 1992 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation D. R. Evans, K. Bein, H. I. Guy, X. Liu, J. A. Molina, B. H. Zimmermann; CAD gene sequence and the domain structure of the mammalian multifunctional protein CAD. Biochem Soc Trans 1 February 1993; 21 (1): 186–191. doi: https://doi.org/10.1042/bst0210186 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: ATCase, apospartate transcarbamylase, CPSase, carbamyl phosphate synthetase, DHOase, dihydro-orotase, GLNase, glutaminase, PRPP, phosphoribosyl pyrophosphate This content is only available as a PDF. © 1993 Biochemical Society1993 Article PDF first page preview Close Modal You do not currently have access to this content.

Structure-function studies in carbamoyl phosphate synthetases.

Tridimensional structure of the ATCase domain On the basis of the amino acid sequence of the ATCase domain, its secondary structure was predicted using the method developed by Levin et al. [15]. The result obtained is strikingly similar to the known secondary structure of the catalytic chain of E. coli ATCase [16]. Simulation of the tridimensional structure was performed by Villoutreix and Ladjimi in this laboratory, using amino acid replacement and energy minimization (to be published elsewhere). Again the result obtained is very close to the known structure of the ATCase catalytic chain of E. coli. A similar result was obtained by Scully and Evans [17], in the case of the CAD protein ATCase domain. Structural comparison of these two proteins should provide information about the evolution of allosteric mechanisms at the molecular level.

Gene amplification and gene deletion are induced at different rates in V79/AP4 Chinese hamster cells.

The effect of mitomycin C (MMC) treatment on gene amplification and gene deletion induction in a V79/AP4 Chinese hamster cell line was investigated. Spontaneous and induced cellular variants resistant to N-phosphonacetyl-L-aspartate (PALA), which selects for carbamyl-P-synthetase, aspartate transcarbamylase and dihydroorotase (CAD) gene amplification events, and to intermediate concentrations of 2,6-diaminopurine (DAP), which selects for adenine phosphoribosyl transferase (aprt) gene deletion events, were isolated. The molecular analysis of spontaneous and induced PALA- and DAP-resistant clones showed that while in the former the CAD gene was amplified, in the latter both mutations and deletions occurred at the aprt gene. These results indicate that MMC favored the rise of gene amplification rather than gene deletion events, and suggest that aberrant DNA replication processes were responsible for the observed gene amplification.

Enzyme Organization in DNA Precursor Biosynthesis

Publisher Summary This chapter discusses the process by which DNA precursors are synthesized and delivered to replication sites and consider the possibility of physical or functional linkage between enzymes of deoxyribonucleoside 5‘-triphosphate (dNTP)’ synthesis and those directly involved in the replication. DNA replication has been shown in both prokaryotic and eukaryotic systems to involve the cooperative function of numerous proteins associated at replication forks. Many metabolic pathways involve multifunctional enzymes or tightly associated multienzyme complexes. Examples of the former are the fattyacyl- CoA synthetase enzyme in vertebrate cells and the eukaryotic “CAD protein” and UMP synthetase, which catalyze the first three and last two reactions, respectively, of the six steps in de novo pyrimidine-nucleotide synthesis. Examples of tightly but non-covalently bound aggregates include the well-known pyruvate and α-ketoglutarate dehydrogenase complexes of mitochondria. The potential biological advantages of enzyme association are fairly obvious and might include: maintenance of locally high metabolite concentrations without the need for physical compartmentation within a membrane; protection of the solvation capacity of cell water, because average concentrations of most metabolites are kept low, even though local concentrations may be much higher; efficiency of regulation of both enzyme synthesis and metabolic fluxes; and relative rapidity with which a metabolic process can respond to changes in the intracellular environment.

Genomic structure of the human caldesmon gene.

The high molecular weight caldesmon (h-CaD) is predominantly expressed in smooth muscles, whereas the low molecular weight caldesmon (l-CaD) is widely distributed in nonmuscle tissues and cells. The changes in CaD isoform expression are closely correlated with the phenotypic modulation of smooth muscle cells. During a search for isoform diversity of human CaDs, l-CaD cDNAs were cloned from HeLa S3 cells. HeLa l-CaD I is composed of 558 amino acids, whereas 26 amino acids (residues 202-227 for HeLa l-CaD I) are deleted in HeLa l-CaD II. The short amino-terminal sequence of HeLa l-CaDs is different from that of fibroblast (WI-38) l-CaD II and human aorta h-CaD. We have also identified WI-38 l-CaD I, which contains a 26-amino acid insertion relative to WI-38 l-CaD II. To reveal the molecular events of the expressional regulation of the CaD isoforms, the genomic structure of the human CaD gene was determined. The human CaD gene is composed of 14 exons and was mapped to a single locus, 7q33-q34. The 26-amino acid insertion is encoded in exon 4 and is specifically spliced in the mRNAs for both h-CaD and l-CaDs I. Exon 3 is the exon that encodes the central repeating domain specific to h-CaD (residues 208-436) together with the common domain in all CaD (residues 73-207 for h-CaD and WI-38 l-CaDs, and residues 68-201 for HeLa l-CaDs). The regulation of h- and l-CaD expression is thought to depend on selection of the two 5' splice sites within exon 3. Thus, the change in expression between l-CaD and h-CaD might be caused by this splicing pathway.

Identification and characterisation of cDNA clones encoding cinnamyl alcohol dehydrogenase from tobacco.

Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) is an enzyme involved in lignin biosynthesis. We have previously isolated pure CAD enzyme as two closely related polypeptides of 44 and 42.5 kDa from tobacco stems. In this paper, we report partial amino acid sequences of these two polypeptides. Based on the peptide sequences mixed oligonucleotides were used to screen a tobacco stem cDNA library and CAD cDNA clones encoding the two polypeptides were identified. DNA sequence comparisons indicate very high sequence identity between these clones both in the coding and in the 5' and 3' untranslated sequences. The close similarity between the two CAD genes leads us to suggest that they do not represent different isoforms but are the same gene from each of the two parental lines of Nicotiana tabacum cv. Samsun. Sequence comparisons with alcohol dehydrogenase 1 (ADH1) from yeast shows sequence similarities of ca. 30%, while comparisons with maize, barley and potato ADH1 sequences show similarities of not more than 23%.

Complete hamster CAD protein and the carbamylphosphate synthetase domain of CAD complement mammalian cell mutants defective in de novo pyrimidine biosynthesis.

The mammalian CAD gene codes for a 240-kDa multifunctional protein that catalyzes the first three steps of de novo pyrimidine biosynthesis. Previously, the longest cDNA construct available was missing approximately 500 bp of coding sequence at the 5' end, thereby lacking the sequence to encode the entire carbamylphosphate synthetase (CPSase) domain. Here, a complete CAD hamster cDNA is constructed, placed into a mammalian expression vector, and transfected into hamster cells deficient in CAD. Transfectants show coordinately restored levels of all three enzyme activities and the presence of full-length CAD protein. A derivative construct of the CAD cDNA was generated that should encode only the CPSase domain. When transfected into mammalian cells, a protein was synthesized that had significant CPSase activity both in vivo and in vitro. The two constructs generated in this study will facilitate the study of CAD structure, function, and allosteric regulation.

Fusions near telomeres occur very early in the amplification of CAD genes in Syrian hamster cells.

Previous analyses by fluorescence in situ hybridization of structures present 20-30 cell generations after the primary events of mammalian gene amplification have shown that tens of megabases of DNA separate each copy of the selected gene in chromosomal arrays that contain up to 15 copies. Since these structures are very unstable, it is necessary to study amplified DNA as soon as possible after it has been formed to relate the structures observed to the primary mechanisms that generated them. Previously, new amplifications of the CAD gene were analyzed in colonies of 10(5) N-(phosphonoacetyl)-L-aspartate-resistant Syrian hamster BHK cells. CAD is on the p arm of chromosome B9 and the amplified genes were usually found in large extensions of B9p, with one copy in its normal position. We now report that dividing drug-resistant cells have been physically separated from static drug-sensitive cells, to allow the amplified structures to be observed only a few cell generations after they have been formed. The most informative results are that about one-third of the newly formed chromosomes carrying amplified CAD genes are dicentric and that about half of these carry two B9q arms. These observations reveal that recombination between the p telomeric regions of two B9 sister chromatids is an important primary event of amplification in this system. The resulting dicentric chromosomes can then enter bridge-breakage-fusion cycles that provide the means to increase the number of CAD genes per cell in successive generations by an asymmetric distribution at each cell division.

Regularities of karyotypic evolution during stepwise amplification of genes determining drug resistance

Analysis of chromosomal alterations during stepwise development of mdr1, dhfr, or CAD gene amplifications in a large number of independently selected Djungarian hamster DM-15 and murine P388 sublines revealed typical patterns of karyotypic evolution, specific for multiplication of each of these genes in each cell type. Some principal similarities of karyotypic evolution were noted in at least two different systems. They include: (i) appearance at the first selection step of a new chromosomal arm bearing the resident gene copy followed at the next selection steps by the formation in these specific chromosomal arms of amplified DNA tandem arrays; (ii) translocations of amplified DNA from its initial site to other, also non-random, chromosomal sites; and (iii) emergence in the cell variants with high degrees of gene amplification of multiple extra-chromosomal elements. The most prominent distinctions among the systems were as follows: (i) different structures, evidently containing amplified DNAs, appeared at the initial steps of amplification of different genes — additional heterogeneously staining regions in specific chromosomal segments in the case of amplification of dhfr or CAD genes in DM-15 cells, and mini-chromosomes in the case of mdr1 gene amplification in both DM-15 and P380 cells; (ii) distinct patterns of location of the amplified mdr1 gene copies are characteristic of Djungarian hamster DM-15 and murine P388 cell derivatives after subsequent steps of selection — at the site of resident gene localization or in some other, also non-random, chromosomal sites in DM-15 sublines, and predominantly extra-chromosomal in P388 sublines. We propose that different mechanisms are responsible for the initial steps of amplification of dhfr and CAD genes on the one hand and the mdr1 gene on the other: non-equal sister-chromatid exchanges and autonomous replication of the extra-chromosomal elements. It seems, however, that both mechanisms may be involved in further rounds of amplification of each of these three genes.

A single base change at a splice acceptor site leads to a truncated CAD protein in Urd-A mutant Chinese hamster ovary cells.

We have previously reported the isolation and characterization of mutant Chinese hamster ovary (CHO-K1) cells of the Urd-A complementation group, which require uridine for growth, are deficient in the activities of the first three enzymes of de novo UMP biosynthesis, and produce markedly reduced amounts of a truncated form of the multifunctional protein CAD, which contains these three enzyme activities. We report here that a single base change of G to A at a highly conserved RNA splice acceptor site is responsible for the phenotype of this mutant. In addition to a small amount of apparently normal CAD mRNA, this mutation causes production of two alternative forms of CAD mRNA in the mutant, one that includes the intron just prior to the mutation and one that excludes the exon just after the mutation. The affected splice site is located at the intron-exon boundary just preceding the exon that encodes the beginning of the aspartate transcarbamylase (ATCase) domain of the CAD protein. Both intron inclusion and exon exclusion during RNA processing introduce a translation stop codon upstream of the region encoding this domain, resulting in the production of the truncated CAD protein seen in the Urd-A mutant. This mutation also results in markedly decreased levels of CAD mRNA and protein in the mutant.

Chromosome rearrangements associated with CAD gene amplification. Experiments with cell hybrids

Resistance to phosphonacetyl- l-aspartate (PALA) is caused by CAD gene amplification. The marker chromosome of a PALA-resistant cell line containing a homogeneously staining region with amplified CAD gene was introduced into PALA-sensitive Chinese hamster cells by microcell-mediated chromosome transfer. Two monochromosomal hybrids containing the marker chromosome in addition to the normal chromosome complement of sensitive cells and 1 tetraploid hybrid containing the complete genomes of donor (resistant) and recipient (sensitive) cells were studied in detail. It was shown that (i) the presence of the marker chromosome was both a necessary and a sufficient condition for the expression of the PALA-resistant phenotype; (ii) the marker chromosome underwent rearrangements in the monochromosomal hybrids, with preferential loss of non-amplified chromosomal regions, while it was not rearranged in the tetraploid hybrid; (iii) unlike the original PALA-resistant cells obtained after long-term selection in the presence of PALA, the PALA-resistant hybrids did not show chromosomal aberrations of other than the marker chromosome. This result indicates that chromosomal aberrations may be due to the selective procedure and is not an inherent property of cells containing amplified genes.

Replication Timing Control Can Be Maintained in Extrachromosomally Amplified Genes

Extrachromosomal elements are common early intermediates of gene amplification in vivo and in cell culture. The time at which several extrachromosomal elements replicate was compared with that of the corresponding amplified or unamplified chromosomal sequences. The replication timing analysis employed a retroactive synchrony method in which fluorescence-activated cell sorting was used to obtain cells at different stages of the cell cycle. Extrachromosomally amplified Syrian hamster CAD genes (CAD is an acronym for the single gene which encodes the trifunctional protein which catalyzes the first three steps of uridine biosynthesis) replicated in a narrow window of early S-phase which was approximately the same as that of chromosomally amplified CAD genes. Similarly, extrachromosomally amplified mouse adenosine deaminase genes replicated at a discrete time in early S-phase which approximated the replication time of the unamplified adenosine deaminase gene. In contrast, the multicopy extrachromosomal Epstein-Barr virus genome replicated within a narrow window in late S-phase in latently infected human Rajii cells. The data indicate that localization within a chromosome is not required for the maintenance of replication timing control.

Replication timing control can be maintained in extrachromosomally amplified genes.

Extrachromosomal elements are common early intermediates of gene amplification in vivo and in cell culture. The time at which several extrachromosomal elements replicate was compared with that of the corresponding amplified or unamplified chromosomal sequences. The replication timing analysis employed a retroactive synchrony method in which fluorescence-activated cell sorting was used to obtain cells at different stages of the cell cycle. Extrachromosomally amplified Syrian hamster CAD genes (CAD is an acronym for the single gene which encodes the trifunctional protein which catalyzes the first three steps of uridine biosynthesis) replicated in a narrow window of early S-phase which was approximately the same as that of chromosomally amplified CAD genes. Similarly, extrachromosomally amplified mouse adenosine deaminase genes replicated at a discrete time in early S-phase which approximated the replication time of the unamplified adenosine deaminase gene. In contrast, the multicopy extrachromosomal Epstein-Barr virus genome replicated within a narrow window in late S-phase in latently infected human Rajii cells. The data indicate that localization within a chromosome is not required for the maintenance of replication timing control.

Synthesis of the nonconserved dihydroorotase domain of the multifunctional hamster CAD protein in Escherichia coli

CAD is the multifunctional protein of higher eukaryotes which catalyzes the first three steps of pyrimidine biosynthesis. Its enzymatic activities exist as independent domains in the order: N terminus-carbamylphosphate synthetase II(CPSase)-dihydroorotase(DHOase)-aspartate transcarbamylase(ATCase)-C terminus. To functionally define the minimum hamster cDNA region required to encode an active DHOase, expression constructs were generated. Many such constructs complement Escherichia coli mutants defective not only in DHOase but also in ATCase. Constructs deleted for most of the sequence encoding the ATCase domain continue to complement E. coli mutants defective in DHOase. All of these smaller constructs also lack the region encoding CPSase. Therefore, a ‘genetic cassette’, containing information for neither the CPSase nor the ATCase domain, can direct the synthesis of a polypeptide with DHOase activity. Interestingly, inclusion of a portion of the DHOase-ATCase interdomain bridge appears to be required for optimum activity.

An unusual Alu repeat sequence within the CAD gene.

There are several hundred thousand members of the Alu repeat family in the human genome. Those Alu elements sequenced to date appear to fit into subfamilies. A novel Alu has been found in an intron of the human CAD gene: it appears to be due to rearrangement between Alu repeats belonging to two different subfamilies. Further sequence data from this intron suggest that the Alu element may have rearranged prior to its entry into the CAD gene. Such findings indicate that, in addition to single nucleotide substitutions and deletions, DNA rearrangements may be a factor in generating the diversity of Alu repeats found in primate genomes.

Molecular cloning of a cDNA encoding the amino end of the mammalian multifunctional protein CAD and analysis of the 5'-flanking region of the CAD gene

CAD is a 243-kDa multidomain polypeptide which catalyzes the first three steps in mammalian de novo pyrimidine biosynthesis. The largest cDNA clone obtained thus far, pCAD142 (Shigesada, K., Stark, G.R., Maley, J. A., Niswander, L. A., and Davidson, J. N. (1985) Mol. Cell. Biol. 5, 1735), lacks the 5' end of the mRNA which encodes the amino terminus of CAD. To clone this missing segment, a synthetic oligonucleotide complementary to pCAD142 and poly(A)+ RNA template, isolated from a Syrian hamster cell line which overproduces the CAD mRNA, were used for cDNA synthesis. The resulting clone pKB11, which has a 1369-base pair (bp) cDNA insert, overlapping pCAD142 by 781 bp, was identified by hybridization methods and sequence analysis and found to contain the entire cDNA sequence for the amino end of the CAD polypeptide. The deduced amino acid sequence is homologous to seven carbamyl phosphate synthetases. Primer extension, oligonucleotide-directed RNase H digestion, and RNA sequencing indicated that pKB11 extends to within 68 bases of the 5' end of the CAD mRNA. This conclusion was confirmed by Northern blotting analysis of the 5'-flanking region of CAD gene. The probable 3' end of an unidentified gene which codes for a 1-kilobase (kb) transcript was identified immediately upstream of the CAD gene. Northern analysis using probes complementary to the region between the CAD and the 1-kb genes detected the presence of a small transcript of less than 300 nucleotides. The sequence revealed three potential translation initiation sites raising the possibility of more than one CAD translation product. The major translation start codon was identified as the first ATG in pKB11 by sequence homology, in vitro transcription and translation, and protein studies. Starting from this ATG within pKB11, the clone encodes a 143-residue domain of unknown function. This study completes the determination of the primary structure of the CAD polypeptide. The CAD mRNA is 7.5 kb in length and has 6675 bp of coding sequence and about 200 bp and 600 bp of untranslated sequence at the 5' and 3' ends, respectively.