scholarly journals Gene amplification accompanies low level increases in the activity of dihydrofolate reductase in antifolate-resistant Chinese hamster lung cells containing abnormally banding chromosomes.

1982 ◽  
Vol 94 (2) ◽  
pp. 418-424 ◽  
Author(s):  
J A Lewis ◽  
J L Biedler ◽  
P W Melera
Keyword(s):  
Dihydrofolate Reductase ◽  
Cdna Probe ◽  
Chinese Hamster ◽  
Hybridization Technique ◽  
Chromosome 2 ◽  
Dhfr Gene ◽  
Low Level ◽  
Amplification Process ◽  
Staining Techniques

Three independently-derived, antifolate-resistant Chinese hamster lung cell lines that exhibit low level increases in dihydrofolate reductase (DHFR) activity, i.e., three- to fivefold vs. controls, have been compared with drug-sensitive cells to determine relative DHFR gene content. With a solution hybridization technique that makes use of genomic DNA and a cloned double-stranded Chinese hamster DHFR cDNA probe, it has been found that the enzyme activity increases are associated with an approximately proportionate amplification of DHFR genes. Trypsin-Giemsa staining techniques and hybridizations in situ further show that the amplified DHFR genes are located within abnormally banding regions along chromosome 2q and also suggest that, in each subline, only one chromosome 2 homolog is initially involved in the amplification process.

scholarly journals Assignment of the human dihydrofolate reductase gene to the q11----q22 region of chromosome 5.

1984 ◽  
Vol 4 (10) ◽  
pp. 2010-2016 ◽  
Author(s):  
V L Funanage ◽  
T T Myoda ◽  
P A Moses ◽  
H R Cowell
Keyword(s):  
Dihydrofolate Reductase ◽  
Hybrid Cell ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Chromosome 5 ◽  
Dhfr Gene ◽  
Reductase Gene ◽  
Ovary Cell Line ◽  
Biochemical Analyses ◽  
Human Dihydrofolate Reductase

Cells from a dihydrofolate reductase-deficient Chinese hamster ovary cell line were hybridized to human fetal skin fibroblast cells. Nineteen dihydrofolate reductase-positive hybrid clones were isolated and characterized. Cytogenetic and biochemical analyses of these clones have shown that the human dihydrofolate reductase (DHFR) gene is located on chromosome 5. Three of these hybrid cell lines contained different terminal deletions of chromosome 5. An analysis of the breakpoints of these deletions has demonstrated that the DHFR gene resides in the q11----q22 region.

Matrix attachment regions are positioned near replication initiation sites, genes, and an interamplicon junction in the amplified dihydrofolate reductase domain of Chinese hamster ovary cells

1988 ◽  
Vol 8 (12) ◽  
pp. 5398-5409
Author(s):  
P A Dijkwel ◽  
J L Hamlin
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Replication Initiation ◽  
Matrix Attachment Region ◽  
Dna Fragments ◽  
Dhfr Gene ◽  
Methods Of Analysis ◽  
The Matrix

Genomic DNA in higher eucaryotic cells is organized into a series of loops, each of which may be affixed at its base to the nuclear matrix via a specific matrix attachment region (MAR). In this report, we describe the distribution of MARs within the amplified dihydrofolate reductase (DHFR) domain (amplicon) in the methotrexate-resistant CHO cell line CHOC 400. In one experimental protocol, matrix-attached and loop DNA fractions were prepared from matrix-halo structures by restriction digestion and were analyzed for the distribution of amplicon sequences between the two fractions. A second, in vitro method involved the specific binding to the matrix of cloned DNA fragments from the amplicon. Both methods of analysis detected a MAR in the replication initiation locus that we have previously defined in the DHFR amplicon, as well as in the 5'-flanking region of the DHFR gene. The first of these methods also suggests the presence of a MAR in a region mapping approximately 120 kilobases upstream from the DHFR gene. Each of these MARs was detected regardless of whether the matrix-halo structures were prepared by the high-salt or the lithium 3,5-diiodosalicylate extraction protocols, arguing against their artifactual association with the proteinaceous scaffolding of the nucleus during isolation procedures. However, the in vitro binding assay did not detect the MAR located 120 kilobases upstream from the DHFR gene but did detect specific matrix attachment of a sequence near the junction between amplicons. The results of these experiments suggest that (i) MARs can occur next to different functional elements in the genome, with the result that a DNA loop formed between two MARs can be smaller than a replicon; and (ii) different methods of analysis detect a somewhat different spectrum of matrix-attached DNA fragments.

Moderate-level gene amplification in methotrexate-resistant Chinese hamster ovary cells is accompanied by chromosomal translocations at or near the site of the amplified DHFR gene

1984 ◽  
Vol 4 (1) ◽  
pp. 69-76
Author(s):  
W F Flintoff ◽  
E Livingston ◽  
C Duff ◽  
R G Worton
Keyword(s):  
Gene Amplification ◽  
Structural Gene ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Chromosome 2 ◽  
Dhfr Gene ◽  
Cell Biol ◽  
A Site

In previous studies, we have described several classes of methotrexate-resistant Chinese hamster ovary cell lines. Although the RI class is resistant because of an altered target enzyme, dihydrofolate reductase, the RIII class derived from RI cells is somewhat more resistant because of a moderate amplification of the altered dhfr structural gene (Flintoff et al., Mol. Cell. Biol. 2:275-285, 1982). In one RIII line, a translocation between the short arm (p) of chromosome 2 and the long arm (q) of chromosome 5 was observed, and the amplified RIII gene complex was mapped to the p arm of the 2p-marker chromosome derived from the translocation (Worton et al., Mol. Cell. Biol. 1:330-335, 1981). We tested the hypothesis that chromosomal translocation is a general feature of RIII cells and that such translocation involves a site at or near the dhfr structural gene. Thus, we examined four independently derived RIII-type mutants and found that each had a moderate amplification of the dhfr gene sequences, and karyotype analysis revealed that each carried a translocation involving the 2p arm at or near band 2p25. That this chromosomal rearrangement involves a site near the dhfr locus was demonstrated by mapping the altered but unamplified structural gene coding for the RI phenotype to the short arm of an unaltered chromosome 2. This suggests that a highly specific rearrangement involving an exchange at or near the site of the unamplified gene is a necessary prerequisite for the amplification process. A model for gene amplification involving chromosomal rearrangements and sister chromatid exchange is described.

Direct demonstration of genetic alterations at the dihydrofolate reductase locus after gamma irradiation

1982 ◽  
Vol 2 (1) ◽  
pp. 93-96
Author(s):  
L H Graf ◽  
L A Chasin
Keyword(s):  
Dihydrofolate Reductase ◽  
Gamma Ray ◽  
Reductase Activity ◽  
Chinese Hamster Ovary Cells ◽  
Cdna Probe ◽  
Chinese Hamster ◽  
Genetic Alterations ◽  
Ovary Cells ◽  
Direct Demonstration ◽  
Dna Deletions

Gamma ray-induced mutants of Chinese hamster ovary cells lacking dihydrofolate reductase activity were screened for DNA sequence changes at the locus specifying this activity by using a cloned cDNA probe. Two of nine mutants screened displayed an altered restriction fragment pattern suggesting the occurrence of DNA deletions or rearrangements.

scholarly journals Amplification of the dihydrofolate reductase gene is a mechanism of acquired resistance to methotrexate in patients with acute lymphoblastic leukemia and is correlated with p53 gene mutations

Blood ◽  
1995 ◽  
Vol 86 (2) ◽  
pp. 677-684 ◽  
Author(s):  
E Goker ◽  
M Waltham ◽  
A Kheradpour ◽  
T Trippett ◽  
M Mazumdar ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Gene Amplification ◽  
Dihydrofolate Reductase ◽  
Lymphoblastic Leukemia ◽  
Gene Mutations ◽  
P53 Gene ◽  
Common Mechanism ◽  
Dhfr Gene ◽  
Low Level ◽  
P53 Gene Mutations

Although dihydrofolate reductase (DHFR) gene amplification is a common mechanism of resistance to methotrexate (MTX) in tumor cell lines, with the exception of a few case reports, the incidence of this phenomenon as a mechanism of MTX resistance in the clinic has not been reported. We studied 38 untreated patients and 29 patients in relapse with acute lymphoblastic leukemia (ALL) for gene amplification and p53 gene mutations. Three patients were studied both at diagnosis and at each of two relapses after treatment with MTX. Nine of 29 relapsed patients (31%) had low-level DHFR gene amplification (two to four gene copies) associated with increased levels of DHFR mRNA and enzyme activity. Of significance was a correlation of gene amplification with p53 mutations in seven of nine relapsed patients (P < .001). Low-level DHFR gene amplification may be an important cause of MTX resistance in ALL and strengthens the concept that mutations in the p53 gene may lead to gene amplification as a consequence of defective cell cycle control.

scholarly journals The human interleukin-1 alpha gene is located on the long arm of chromosome 2 at band q13

Blood ◽  
1989 ◽  
Vol 73 (1) ◽  
pp. 104-107 ◽  
Author(s):  
M Lafage ◽  
N Maroc ◽  
P Dubreuil ◽  
R de Waal Malefijt ◽  
MJ Pebusque ◽  
...  
Keyword(s):  
Interleukin 1 ◽  
Cdna Probe ◽  
Proximal Part ◽  
Chromosome 2 ◽  
Interleukin 1 Beta ◽  
Hematopoietic Growth Factors ◽  
Alpha Gene ◽  
Immunologic Reactions ◽  
Beta Gene

Abstract Interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) are two biochemically distinct, but distantly related, polypeptidic cytokines that play a key role in inflammation, immunologic reactions, and tissue repair. Recently, it has been shown that IL-1 alpha is identical to hematopoietin 1, which was described as a hematopoietic growth factor acting on early progenitor cells in synergy with other hematopoietic growth factors. In this report we discuss our use of in situ hybridization on human prometaphase cells with a human IL-1 alpha cDNA probe to localize the human IL-1 alpha gene on the proximal part of the long arm of chromosome 2 at band q13, in the same chromosomal region as the IL-1 beta gene.

scholarly journals High-resolution mapping of the origin of DNA replication in the hamster dihydrofolate reductase gene domain by competitive PCR.

1996 ◽  
Vol 16 (10) ◽  
pp. 5358-5364 ◽  
Author(s):  
C Pelizon ◽  
S Diviacco ◽  
A Falaschi ◽  
M Giacca
Keyword(s):  
Dna Replication ◽  
Dihydrofolate Reductase ◽  
Cultured Cells ◽  
Chinese Hamster ◽  
Single Copy ◽  
Genomic Region ◽  
Competitive Pcr ◽  
Dhfr Gene ◽  
Mapping Procedure ◽  
Dna Replication Origin

By the use of a highly sensitive mapping procedure allowing the identification of the start sites of DNA replication in single-copy genomic regions of untreated, exponentially growing cultured cells (M. Giacca, L. Zentilin, P. Norio, S. Diviacco, D. Dimitrova, G. Contreas, G. Biamonti, G. Perini, F. Weighardt, S. Riva, and A. Falaschi, Proc. Natl. Acad. Sci. USA 91:7119-7123, 1994), the pattern of DNA replication of the Chinese hamster dihydrofolate reductase (DHFR) gene domain was investigated. The method entails the purification of short stretches of nascent DNA issuing from DNA replication origin regions and quantification, within this sample, of the abundance of different adjacent segments by competitive PCR. Distribution of marker abundance peaks around the site from which newly synthesized DNA had emanated. The results obtained by analysis of the genomic region downstream of the DHFR single-copy gene in asynchronous cultures of hamster CHO K1 cells are consistent with the presence of a single start site for DNA replication, located approximately 17 kb downstream of the gene. This site is coincident with the one detected by other studies using different techniques in CHO cell lines containing an amplified DHFR gene domain.

scholarly journals Direct demonstration of genetic alterations at the dihydrofolate reductase locus after gamma irradiation.

1982 ◽  
Vol 2 (1) ◽  
pp. 93-96 ◽  
Author(s):  
L H Graf ◽  
L A Chasin
Keyword(s):  
Dihydrofolate Reductase ◽  
Gamma Ray ◽  
Reductase Activity ◽  
Chinese Hamster Ovary Cells ◽  
Cdna Probe ◽  
Chinese Hamster ◽  
Genetic Alterations ◽  
Ovary Cells ◽  
Direct Demonstration ◽  
Dna Deletions

Gamma ray-induced mutants of Chinese hamster ovary cells lacking dihydrofolate reductase activity were screened for DNA sequence changes at the locus specifying this activity by using a cloned cDNA probe. Two of nine mutants screened displayed an altered restriction fragment pattern suggesting the occurrence of DNA deletions or rearrangements.

Localization of the 68 000-Da human neurofilament gene (NF68) using a murine cDNA probe

Genome ◽  
1988 ◽  
Vol 30 (4) ◽  
pp. 499-500 ◽  
Author(s):  
Martin J. Somerville ◽  
Donald R. McLachlan ◽  
Maire E. Percy
Keyword(s):  
Centromeric Region ◽  
Human Gene ◽  
Cdna Probe ◽  
Chromosome 8 ◽  
Chromosome 7 ◽  
Gene Localization ◽  
Chromosome 2 ◽  
Genomic Probe ◽  
Human Genomic

A recent investigation, using a human genomic probe, has indicated that the 68 000 dalton neurofilament gene (NF68) is on the short arm of chromosome 8. We have used a murine cDNA probe on 65 metaphase spreads in situ to localize the human NF68 gene to 8p21 (20/370 grains; p < 0.0001). In addition, we have found secondary hybridization sites at the centromeric region of chromosome 2 and the long arm of chromosome 7, which are putative loci for other intermediate filaments.Key words: neurofilament, human, gene localization, murine cDNA.

Amplification of a cloned Chinese hamster dihydrofolate reductase gene after transfer into a dihydrofolate reductase-deficient cell line

1983 ◽  
Vol 3 (7) ◽  
pp. 1274-1282
Author(s):  
J D Milbrandt ◽  
J C Azizkhan ◽  
J L Hamlin
Keyword(s):  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Genomic Dna ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Ovary Cell ◽  
Dhfr Gene ◽  
Reductase Gene ◽  
Ovary Cell Line

We have transformed a dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary cell line to the DHFR+ phenotype with a recombinant cosmid (cH1) containing a functional Chinese hamster DHFR gene (J.D. Milbrandt et al., Mol. Cell. Biol. 3:1266-1273, 1983). After exposure of cells to successive increases in methotrexate, we have isolated a resistant cell line (JSH-1) that grows in 1 microM methotrexate. We show here that JSH-1 contains 300 to 500 copies of the integrated cosmid and that these copies are located predominantly at one position on a chromosome identified as Z5a. Hybridization analysis of restriction digests of genomic DNA indicates that the cosmid has been integrated intact into the genome and that upon amplification, the original cosmid/genomic junction fragments are also amplified in JSH-1. Furthermore, the pattern of amplified bands observed in ethidium bromide-stained gels indicates that the unit amplified sequence (amplicon) may be as large as 120 to 135 kilobases and therefore includes considerable amounts of flanking DNA in addition to the 45 kilobases of integrated cosmid. We also show that the protein overproduced by the amplified cosmid in JSH-1 comigrates with the 21,000-dalton polypeptide characteristic of the methotrexate-resistant cell line (CHOC 400) from which cH1 was cloned. However, the DHFR mRNA species overproduced in JSH-1 appear to be larger than those detected in CHOC 400, indicating that not all of the normal transcription and processing signals are preserved in the integrated recombinant cosmid.

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