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 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.

A mammalian origin of bidirectional DNA replication within the Chinese hamster RPS14 locus

1994 ◽  
Vol 14 (9) ◽  
pp. 5628-5635
Author(s):  
E S Tasheva ◽  
D J Roufa
Keyword(s):  
Dna Replication ◽  
Chinese Hamster ◽  
Housekeeping Gene ◽  
Single Copy ◽  
The Other ◽  
Replication Origins ◽  
Chromosomal Replication ◽  
Dna Binding Sites ◽  
Sequence Encoding ◽  
Copy Dna

Two complementary experimental approaches have been used to identify a chromosomal origin of bidirectional DNA replication within or immediately downstream of the Chinese hamster ribosomal protein S14 gene (RPS14). The replication origin, designated oriS14, maps within a 1.6- to 2.0-kbp region of RPS14 that includes the gene's third and fourth introns, exons IV plus V, and approximately 500 bp of proximal downstream flanking DNA. The nucleic acid sequence encoding oriS14 closely resembles the other mammalian chromosomal replication origins whose primary structures are known. It contains DNA binding sites for a large number of transcription factors, replication proteins, and mammalian oncogenes as well as several dinucleotide repeat motifs, an AT-rich region, and a sequence that is likely to bend the DNA. In contrast to the other well-characterized mammalian replication origins, which are autosomal and therefore carried as two copies per somatic cell, oriS14 is encoded by single-copy DNA within a hemizygous segment of chromosome 2q in CHO-K1 cells. Also, other known mammalian replication origins are situated in nontranscribed, intergenic DNA, whereas the DNA sequence encoding oriS14 substantially overlaps the transcribed portion of a constitutively expressed housekeeping gene.

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.

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.

High-resolution mapping of replication fork movement through the amplified dihydrofolate reductase domain in CHO cells by in-gel renaturation analysis

1989 ◽  
Vol 9 (2) ◽  
pp. 523-531
Author(s):  
T H Leu ◽  
J L Hamlin
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster ◽  
Slight Modification ◽  
Single Copy ◽  
Replication Initiation ◽  
Ovary Cell ◽  
Restriction Fragments ◽  
Labeling Pattern ◽  
Reductase Domain

Utilizing an in vivo labeling method on synchronized cultures, we have previously defined a 28-kilobase (kb) replication initiation locus in the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) (N. H. Heintz and J. L. Hamlin, Proc. Natl. Acad. Sci. USA 79:4083-4087, 1982; N. H. Heintz and J. L. Hamlin, Biochemistry 22:3552-3557, 1983; N. H. Heintz, J. D. Milbrandt, K. S. Greisen, and J. L. Hamlin, Nature [London] 302:439-441, 1983). To locate the origin of replication in this 243-kb amplicon with more precision, we used an in-gel renaturation procedure (I. Roninson, Nucleic Acids Res. 11:5413-5431, 1983) to examine the labeling pattern of restriction fragments from the amplicon in the early S phase. This method eliminates background labeling from single-copy sequences and allows quantitation of the relative radioactivity in individual fragments. We used this procedure to follow the movement of replication forks through the amplicons, to roughly localize the initiation locus, and to estimate the rate of fork travel. We also used a slight modification of this method (termed hybridization enhancement) to illuminate the labeling pattern of smaller restriction fragments derived solely from the initiation locus itself, thereby increasing resolution. Our preliminary results suggest that there are actually two distinct initiation sites in the amplicon that are separated by approximately 22 kb.

Replication in the amplified dihydrofolate reductase domain in CHO cells may initiate at two distinct sites, one of which is a repetitive sequence element

1989 ◽  
Vol 9 (2) ◽  
pp. 532-540
Author(s):  
B Anachkova ◽  
J L Hamlin
Keyword(s):  
Dna Replication ◽  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Replication Initiation ◽  
Ovary Cell ◽  
Sequence Element ◽  
Hybridization Probes ◽  
Ovary Cell Line

To study initiation of DNA replication in mammalian chromosomes, we have established a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) that contains approximately 1,000 copies of the early replicating dihydrofolate reductase (DHFR) domain. We have previously shown that DNA replication in the prevalent 243-kilobase (kb) amplicon type in this cell line initiates somewhere within a 28-kb region located downstream from the DHFR gene. In an attempt to localize the origin of replication with more precision, we blocked the progress of replication forks emanating from origins at the beginning of the S phase by the introduction of trioxsalen cross-links at 1- to 5-kb intervals in the parental double-stranded DNA. The small DNA fragments synthesized under these conditions (which should be centered around replication origins) were then used as hybridization probes on digests of cosmids and plasmids from the DHFR domain. These studies suggested that in cells synchronized by this regimen, DNA replication initiates at two separate sites within the previously defined 28-kb replication initiation locus, in general agreement with results described in the accompanying paper (T.-H. Leu and J. L. Hamlin, Mol. Cell. Biol. 9:523-531, 1989). One of these sites contains a repeated DNA sequence element that is found at or near many other initiation sites in the genome, since it was also highly enriched in the early replicating DNA isolated from cross-linked CHO cells that contain only two copies of the DHFR domain.

Carcinogen-mediated methotrexate resistance and dihydrofolate reductase amplification in Chinese hamster cells

1986 ◽  
Vol 6 (6) ◽  
pp. 1958-1964
Author(s):  
T Kleinberger ◽  
S Etkin ◽  
S Lavi
Keyword(s):  
Gene Amplification ◽  
Dihydrofolate Reductase ◽  
Alkylating Agents ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Dna Amplification ◽  
Simian Virus ◽  
Dhfr Gene ◽  
Cellular Mechanisms ◽  
Methotrexate Resistance

We have investigated different parameters characterizing carcinogen-mediated enhancement of methotrexate resistance in Chinese hamster ovary (CHO) cells and in simian virus 40-transformed Chinese hamster embryo (C060) cells. We show that this enhancement reflects dihydrofolate reductase (dhfr) gene amplification. The carcinogens used in this work are alkylating agents and UV irradiation. Both types of carcinogens induce a transient enhancement of methotrexate resistance which increases gradually from the time of treatment to 72 to 96 h later and decreases thereafter. Increasing doses of carcinogens decrease cell survival and increase the enhancement of methotrexate resistance. Enhancement was observed when cells were treated at different stages in the cell cycle, and it was maximal when cells were treated during the early S phase. These studies of carcinogen-mediated dhfr gene amplification coupled with our earlier studies on viral DNA amplification in simian virus 40-transformed cells demonstrate that the same parameters characterize the amplification of both genes. Possible cellular mechanisms responsible for the carcinogen-mediated gene amplification phenomenon are discussed.

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 High-resolution mapping of replication fork movement through the amplified dihydrofolate reductase domain in CHO cells by in-gel renaturation analysis.

1989 ◽  
Vol 9 (2) ◽  
pp. 523-531 ◽  
Author(s):  
T H Leu ◽  
J L Hamlin
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster ◽  
Slight Modification ◽  
Single Copy ◽  
Replication Initiation ◽  
Ovary Cell ◽  
Restriction Fragments ◽  
Labeling Pattern ◽  
Reductase Domain

Utilizing an in vivo labeling method on synchronized cultures, we have previously defined a 28-kilobase (kb) replication initiation locus in the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) (N. H. Heintz and J. L. Hamlin, Proc. Natl. Acad. Sci. USA 79:4083-4087, 1982; N. H. Heintz and J. L. Hamlin, Biochemistry 22:3552-3557, 1983; N. H. Heintz, J. D. Milbrandt, K. S. Greisen, and J. L. Hamlin, Nature [London] 302:439-441, 1983). To locate the origin of replication in this 243-kb amplicon with more precision, we used an in-gel renaturation procedure (I. Roninson, Nucleic Acids Res. 11:5413-5431, 1983) to examine the labeling pattern of restriction fragments from the amplicon in the early S phase. This method eliminates background labeling from single-copy sequences and allows quantitation of the relative radioactivity in individual fragments. We used this procedure to follow the movement of replication forks through the amplicons, to roughly localize the initiation locus, and to estimate the rate of fork travel. We also used a slight modification of this method (termed hybridization enhancement) to illuminate the labeling pattern of smaller restriction fragments derived solely from the initiation locus itself, thereby increasing resolution. Our preliminary results suggest that there are actually two distinct initiation sites in the amplicon that are separated by approximately 22 kb.

scholarly journals Lagging-Strand, Early-Labelling, and Two-Dimensional Gel Assays Suggest Multiple Potential Initiation Sites in the Chinese Hamster Dihydrofolate Reductase Origin

1998 ◽  
Vol 18 (1) ◽  
pp. 39-50 ◽  
Author(s):  
Shuntai Wang ◽  
Pieter A. Dijkwel ◽  
Joyce L. Hamlin
Keyword(s):  
Dihydrofolate Reductase ◽  
Intergenic Region ◽  
Chinese Hamster ◽  
Intergenic Spacer ◽  
Single Copy ◽  
Matrix Attachment Region ◽  
Two Dimensional ◽  
Template Strand ◽  
Potential Initiation ◽  
Lagging Strand

ABSTRACT There is general agreement that DNA synthesis in the single-copy and amplified dihydrofolate reductase (DHFR) loci of CHO cells initiates somewhere within the 55-kb spacer region between the DHFR and 2BE2121 genes. However, results of lagging-strand, early-labelling fragment hybridization (ELFH), and PCR-based nascent-strand abundance assays have been interpreted to suggest a very narrow zone of initiation centered at a single locus known as ori-β, while two-dimensional (2-D) gel analyses suggest that initiation can occur at any of a large number of potential sites scattered throughout the intergenic region. The results of a leading-strand assay and two intrinsic labelling techniques are compatible with a broad initiation zone in which ori-β and a second locus (ori-γ) are somewhat preferred. To determine how these differing views are shaped by differences in experimental manipulations unrelated to the biology itself, we have applied the lagging-strand, ELFH, neutral-neutral, and/or neutral-alkaline 2-D gel assays to CHOC 400 cell populations synchronized and manipulated in the same way. In our experiments, the lagging-strand assay failed to identify a template strand switch at ori-β; rather, we observed a gradual, undulating change in hybridization bias throughout the intergenic spacer, with hybridization to the two templates being approximately equal near a centered matrix attachment region. In the ELFH assay, all of the fragments in the 55-kb intergenic region were labelled in the first few minutes of the S phase, with the regions encompassing ori-β and ori-γ being somewhat preferred. Under the same conditions, neutral-neutral and neutral-alkaline 2-D gel analyses detected initiation sites at multiple locations in the intergenic spacer. Thus, the results of all existing replicon-mapping methods that have been applied to the amplified DHFR locus in CHOC 400 cells are consistent with a model in which two somewhat preferred subzones reside in a larger zone of multiple potential initiation sites in the intergenic region.

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