Evolution of chromosomal regions containing transfected and amplified dihydrofolate reductase sequences

A modular dihydrofolate reductase gene has been introduced into Chinese hamster ovary cells lacking dihydrofolate reductase. Clones capable of growth in the absence of added nucleosides contain one to five copies of the plasmid DNA integrated into the host genome. Upon stepwise selection to increasing methotrexate concentrations, cells are obtained which have amplified the transforming DNA over several hundredfold. A detailed analysis of the chromosomes in three clones indicated the appearance of cytologically distinct chromosomal regions containing the amplified plasmid DNA which differ in surrounding sequence composition, structure, and location. Two of the clones examined have extensive, homogeneously staining regions. The DNA in these homogeneously staining regions replicates in the early part of the S phase. The amplified plasmid DNA is found associated at or near the ends of chromosomes or on dicentric chromosomes. We propose that integration of DNA may disrupt telomeric structures and facilitate the formation of dicentric chromosomes, which may then undergo bridge breakage-fusion cycles. These phenomena are discussed in relation to DNA transfer experiments and modes of gene amplification and chromosome rearrangement.

[1]  P. Sharp,et al.  Construction of a modular dihydrofolate reductase cDNA gene: analysis of signals utilized for efficient expression , 1982, Molecular and cellular biology.

[2]  P. Sharp,et al.  Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary dna gene. , 1982, Journal of molecular biology.

[3]  R. Schimke,et al.  Structure of amplified normal and variant dihydrofolate reductase genes in mouse sarcoma S180 cells. , 1982, The Journal of biological chemistry.

[4]  H. Gay,et al.  Variable content of double minute chromosomes is not correlated with degree of phenotype instability in methotrexate-resistant human cell lines. , 1982, Molecular and cellular biology.

[5]  Jack W. Szostak,et al.  Cloning yeast telomeres on linear plasmid vectors , 1982, Cell.

[6]  G. Wahl,et al.  Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization. , 1982, Molecular and cellular biology.

[7]  C. Tyler-Smith,et al.  Gene amplification in methotrexate-resistant mouse cells. III. Interrelationships between chromosome changes and DNA sequence amplification or loss. , 1981 .

[8]  R. Schimke,et al.  Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line , 1981, Molecular and cellular biology.

[9]  R T Schimke,et al.  Relationship of amplified dihydrofolate reductase genes to double minute chromosomes in unstably resistant mouse fibroblast cell lines , 1981, Molecular and cellular biology.

[10]  D. Ledbetter,et al.  In vitro translation of hypoxanthine/guanine phosphoribosyltransferase mRNA: characterization of a mouse neuroblastoma cell line that has elevated levels of hypoxanthine/guanine phosphoribosyltransferase protein. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[11]  J. Haber,et al.  Evidence of Chromosomal Breaks near the Mating-Type Locus of SACCHAROMYCES CEREVISIAE That Accompany MATalpha xMATalpha Matings. , 1981, Genetics.

[12]  W. White,et al.  Methotrexate-resistant Chinese hamster ovary cells have amplified a 135-kilobase-pair region that includes the dihydrofolate reductase gene. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[13]  A. Varshavsky.  Phorbol ester dramatically increases incidence of methotrexate-resistant mouse cells: Possible mechanisms and relevance to tumor promotion , 1981, Cell.

[14]  A. Varshavsky.  On the possibility of metabolic control of replicon "misfiring": relationship to emergence of malignant phenotypes in mammalian cell lineages. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[15]  D. George,et al.  Cloning of DNA from double minutes of Y1 mouse adrenocortical tumor cells: Evidence for gene amplification , 1981, Cell.

[16]  J. Hamlin,et al.  Replication pattern of a large homogenously staining chromosome region in antifolate‐resistant Chinese hamster cell lines , 1981, Journal of cellular physiology.

[17]  F. Ruddle,et al.  Deoxyribonucleic acid-mediated gene transfer in mammalian cells: molecular analysis of unstable transformants and their progression to stability , 1981, Molecular and cellular biology.

[18]  A. Henderson,et al.  Transforming DNA integrates into the host chromosome , 1981, Cell.

[19]  B. Levin,et al.  Double minutes in human large bowel cancer. , 1980, Gastroenterology.

[20]  J. Lewis,et al.  Antifolate-resistant Chinese hamster cells. Evidence for dihydrofolate reductase gene amplification among independently derived sublines overproducing different dihydrofolate reductases. , 1980, The Journal of biological chemistry.

[21]  L. Chasin,et al.  Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[22]  J. Bertino,et al.  Correlation of dihydrofolate reductase elevation with gene amplification in a homogeneously staining chromosomal region in L5178Y cells , 1979, The Journal of cell biology.

[23]  R. Schimke,et al.  Amplified dihydrofolate reductase genes in unstably methotrexate-resistant cells are associated with double minute chromosomes. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[24]  D. Woodcock,et al.  Aberrant double replication of segments of chromosomal DNA following DNA synthesis inhibition by cytosine arabinoside. , 1979, Experimental cell research.

[25]  G R Stark,et al.  Gene amplification causes overproduction of the first three enzymes of UMP synthesis in N-(phosphonacetyl)-L-aspartate-resistant hamster cells. , 1979, The Journal of biological chemistry.

[26]  G. Holmquist,et al.  Telomere replication, kinetochore organizers, and satellite DNA evolution. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[27]  F. Ruddle,et al.  Phenotype stabilisation and integration of transferred material in chromosome-mediated gene transfer , 1979, Nature.

[28]  T. Hsu,et al.  Double minutes in human carcinoma cell lines, with special reference to breast tumors. , 1979, Journal of the National Cancer Institute.

[29]  S. A. Latt,et al.  Enhancement of banding patterns in human metaphase chromosomes by energy transfer. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[30]  L. Chasin,et al.  Amplified dihydrofolate reductase genes are localized to a homogeneously staining region of a single chromosome in a methotrexate-resistant Chinese hamster ovary cell line. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[31]  Stanley N Cohen,et al.  Phenotypic expression in E. coli of a DNA sequence coding for mouse dihydrofolate reductase , 1978, Nature.

[32]  E. Blackburn,et al.  A tandemly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena. , 1978, Journal of molecular biology.

[33]  J. Bertino,et al.  Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. , 1978, The Journal of biological chemistry.

[34]  F. Gilbert,et al.  Double minute chromosomes and the homogeneously staining regions in chromosomes of a human neuroblastoma cell line. , 1977, Science.

[35]  P Berg,et al.  Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. , 1977, Journal of molecular biology.

[36]  B. Spengler,et al.  Metaphase chromosome anomaly: association with drug resistance and cell-specific products. , 1976, Science.

[37]  S. Latt.  Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[38]  A. van der Eb,et al.  A new technique for the assay of infectivity of human adenovirus 5 DNA. , 1973, Virology.

[39]  M. Seabright.  A rapid banding technique for human chromosomes. , 1971, Lancet.

[40]  J. G. Brewen,et al.  Restricted rejoining of chromosomal subunits in aberration formation: a test for subunit dissimilarity. , 1969, Proceedings of the National Academy of Sciences of the United States of America.

[41]  B. Mcclintock,et al.  The Fusion of Broken Ends of Chromosomes Following Nuclear Fusion. , 1942, Proceedings of the National Academy of Sciences of the United States of America.

[42]  B. Mcclintock,et al.  The Stability of Broken Ends of Chromosomes in Zea Mays. , 1941, Genetics.

[43]  A. Henderson,et al.  Chromosome structure and DNA sequence alterations associated with mutation of transformed genes. , 1981, Journal of molecular and applied genetics.

[44]  R. Schimke,et al.  Chromosomal and extrachromosomal localization of amplified dihydrofolate reductase genes in cultured mammalian cells. , 1981, Cold Spring Harbor symposia on quantitative biology.

[45]  R. Worton,et al.  Chromosome stability in CHO cells , 1977, Somatic cell genetics.

[46]  B. Mcclintock,et al.  Chromosome organization and genic expression. , 1951, Cold Spring Harbor symposia on quantitative biology.