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

Mol Cell Biol. 1982 Mar; 2(3): 308–319.

Abstract

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.

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Albrecht AM, Biedler JL, Hutchison DJ. Two different species of dihydrofolate reductase in mammalian cells differentially resistant to amethopterin and methasquin. Cancer Res. 1972 Jul;32(7):1539–1546. [PubMed] [Google Scholar]
Alt FW, Kellems RE, Bertino JR, Schimke RT. Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. J Biol Chem. 1978 Mar 10;253(5):1357–1370. [PubMed] [Google Scholar]
Beach LR, Palmiter RD. Amplification of the metallothionein-I gene in cadmium-resistant mouse cells. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2110–2114. [PMC free article] [PubMed] [Google Scholar]
Biedler JL, Spengler BA. Metaphase chromosome anomaly: association with drug resistance and cell-specific products. Science. 1976 Jan 16;191(4223):185–187. [PubMed] [Google Scholar]
Bigger TR, Savage JR. Location of nucleolar organizing regions on the chromosomes of the Syrian hamster (Mesocricetus auratus) and the Djungarian hamster (Phodopus sungorus). Cytogenet Cell Genet. 1976;16(6):495–504. [PubMed] [Google Scholar]
Chandler ME, Yunis JJ. A high resolution in situ hybridization technique for the direct visualization of labeled G-banded early metaphase and prophase chromosomes. Cytogenet Cell Genet. 1978;22(1-6):352–356. [PubMed] [Google Scholar]
Denhardt DT. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966 Jun 13;23(5):641–646. [PubMed] [Google Scholar]
DiPaolo JA, Popescu NC. Cytogenetics of Syrian hamster and its relationships to in vitro neoplastic transformation. Prog Exp Tumor Res. 1979;24:2–16. [PubMed] [Google Scholar]
Dolnick BJ, Berenson RJ, Bertino JR, Kaufman RJ, Nunberg JH, Schimke RT. Correlation of dihydrofolate reductase elevation with gene amplification in a homogeneously staining chromosomal region in L5178Y cells. J Cell Biol. 1979 Nov;83(2 Pt 1):394–402. [PMC free article] [PubMed] [Google Scholar]
Flintoff WF, Davidson SV, Siminovitch L. Isolation and partial characterization of three methotrexate-resistant phenotypes from Chinese hamster ovary cells. Somatic Cell Genet. 1976 May;2(3):245–261. [PubMed] [Google Scholar]
Harper ME, Saunders GF. Localization of single copy DNA sequences of G-banded human chromosomes by in situ hybridization. Chromosoma. 1981;83(3):431–439. [PubMed] [Google Scholar]
Hayashi S, Gillam IC, Delaney AD, Tener GM. Acetylation of chromosome squashes of Drosophila melanogaster decreases the background in autoradiographs from hybridization with [125I]-labeled RNA. J Histochem Cytochem. 1978 Aug;26(8):677–679. [PubMed] [Google Scholar]
Kaufman RJ, Brown PC, Schimke RT. Amplified dihydrofolate reductase genes in unstably methotrexate-resistant cells are associated with double minute chromosomes. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5669–5673. [PMC free article] [PubMed] [Google Scholar]
Kaufman RJ, Brown PC, Schimke RT. Loss and stabilization of amplified dihydrofolate reductase genes in mouse sarcoma S-180 cell lines. Mol Cell Biol. 1981 Dec;1(12):1084–1093. [PMC free article] [PubMed] [Google Scholar]
Kempe TD, Swyryd EA, Bruist M, Stark GR. Stable mutants of mammalian cells that overproduce the first three enzymes of pyrimidine nucleotide biosynthesis. Cell. 1976 Dec;9(4 Pt 1):541–550. [PubMed] [Google Scholar]
LEHMAN JM, MACPHERSON I, MOORHEAD PS. KARYOTYPE OF THE SYRIAN HAMSTER. J Natl Cancer Inst. 1963 Sep;31:639–650. [PubMed] [Google Scholar]
Littlefield JW. Hybridization of hamster cells with high and low folate reductase activity. Proc Natl Acad Sci U S A. 1969 Jan;62(1):88–95. [PMC free article] [PubMed] [Google Scholar]
Miller OJ, Tantravahi R, Miller DA, Yu LC, Szabo P, Prensky W. Marked increase in ribosomal RNA gene multiplicity in a rat hepatoma cell line. Chromosoma. 1979 Feb 21;71(2):183–195. [PubMed] [Google Scholar]
Nunberg JH, Kaufman RJ, Schimke RT, Urlaub G, Chasin LA. Amplified dihydrofolate reductase genes are localized to a homogeneously staining region of a single chromosome in a methotrexate-resistant Chinese hamster ovary cell line. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5553–5556. [PMC free article] [PubMed] [Google Scholar]
Padgett RA, Wahl GM, Stark GR. Structure of the gene for CAD, the multifunctional protein that initiates UMP synthesis in Syrian hamster cells. Mol Cell Biol. 1982 Mar;2(3):293–301. [PMC free article] [PubMed] [Google Scholar]
Padgett RA, Wahl GM, Stark GR. Properties of dispersed, highly repeated DNA within and near the hamster CAD gene. Mol Cell Biol. 1982 Mar;2(3):302–307. [PMC free article] [PubMed] [Google Scholar]
Pall ML. Gene-amplification model of carcinogenesis. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2465–2468. [PMC free article] [PubMed] [Google Scholar]
Popescu NC, DiPaolo JA. Identification of Syrian hamster chromosomes by acetic-saline-Giemsa (ASG) and trypsin techniques. Cytogenetics. 1972;11(6):500–507. [PubMed] [Google Scholar]
Rigby PW, Dieckmann M, Rhodes C, Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. [PubMed] [Google Scholar]
de Saint Vincent BR, Delbrück S, Eckhart W, Meinkoth J, Vitto L, Wahl G. The cloning and reintroduction into animal cells of a functional CAD gene, a dominant amplifiable genetic marker. Cell. 1981 Dec;27(2 Pt 1):267–277. [PubMed] [Google Scholar]
Robins DM, Ripley S, Henderson AS, Axel R. Transforming DNA integrates into the host chromosome. Cell. 1981 Jan;23(1):29–39. [PubMed] [Google Scholar]
Schmid W, Fanconi G. Fragility and spiralization anomalies of the chromosomes in three cases, including fraternal twins, with Fanconi's anemia, type Estren-Dameshek. Cytogenet Cell Genet. 1978;20(1-6):141–149. [PubMed] [Google Scholar]
Seabright M. A rapid banding technique for human chromosomes. Lancet. 1971 Oct 30;2(7731):971–972. [PubMed] [Google Scholar]
Spradling AC, Mahowald AP. Amplification of genes for chorion proteins during oogenesis in Drosophila melanogaster. Proc Natl Acad Sci U S A. 1980 Feb;77(2):1096–1100. [PMC free article] [PubMed] [Google Scholar]
Su TS, Beaudet AL, O'Brien WE. Increased translatable messenger ribonucleic acid for argininosuccinate synthetase in canavanine-resistant human cells. Biochemistry. 1981 May 12;20(10):2956–2960. [PubMed] [Google Scholar]
Swyryd EA, Seaver SS, Stark GR. N-(phosphonacetyl)-L-aspartate, a potent transition state analog inhibitor of aspartate transcarbamylase, blocks proliferation of mammalian cells in culture. J Biol Chem. 1974 Nov 10;249(21):6945–6950. [PubMed] [Google Scholar]
Tantravahi U, Guntaka RV, Erlanger BF, Miller OJ. Amplified ribosomal RNA genes in a rat hepatoma cell line are enriched in 5-methylcytosine. Proc Natl Acad Sci U S A. 1981 Jan;78(1):489–493. [PMC free article] [PubMed] [Google Scholar]
Urlaub G, Chasin LA. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4216–4220. [PMC free article] [PubMed] [Google Scholar]
Wahl GM, Padgett RA, Stark GR. Gene amplification causes overproduction of the first three enzymes of UMP synthesis in N-(phosphonacetyl)-L-aspartate-resistant hamster cells. J Biol Chem. 1979 Sep 10;254(17):8679–8689. [PubMed] [Google Scholar]
Wahl GM, Stern M, Stark GR. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. [PMC free article] [PubMed] [Google Scholar]

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