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J L Hamlin

Publications and source records attributed to J L Hamlin.

71 records · Page 4Linked to original sources
PubMed1987

Isolation of the amplified dihydrofolate reductase domain from methotrexate-resistant Chinese hamster ovary cells.

We isolated overlapping recombinant cosmids that represent the equivalent of two complete dihydrofolate reductase amplicon types from the methotrexate-resistant CHO cell line CHOC400. The type I amplicons are 260 kilobases long, are arranged in head-to-tail fashion, and represent 10 to 15% of the amplicons in the CHOC400 genome. The type II amplicons are 220 kilobases long, are arranged in head-to-head and tail-to-tail configurations, and constituted the majority of the remaining amplicons in CHOC400 cells. The type II amplicon sequences are represented entirely within the type I unit. These are the first complete amplicons to be cloned from a mammalian cell line.

Animals↗
PubMed1986

Nucleotide sequence and nuclease hypersensitivity of the Chinese hamster dihydrofolate reductase gene promoter region.

We have sequenced the 1240 base pairs (bp) upstream from the translation start site of the hamster dihydrofolate reductase (DHFR) gene. The DNA in the 5' flanking region contains several elements that are homologous in both sequence and relative location to corresponding elements in the human and murine DHFR genes: an 11-bp element adjacent to the ATG codon, a 19-bp element that coincides with the major transcription start site, and two 29-bp upstream elements that are represented 4 times in the murine DHFR gene but only once in the human gene. Two clusters of short, G/C-rich elements conforming to the consensus binding sequence for the transcription factor Spl are located in the upstream region in all three genes. The symmetrical placement of the G/C boxes coincides with a symmetrical DNase I hypersensitive pattern in the chromatin, suggesting that the Spl protein may be involved in maintaining chromatin structure in this region.

Animals↗
PubMed1985

Similar 150-kilobase DNA sequences are amplified in independently derived methotrexate-resistant Chinese hamster cells.

We have isolated overlapping recombinant cosmids that represent 150 kilobases of contiguous DNA sequence from the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). This sequence includes the 25-kilobase dihydrofolate reductase gene and an origin of DNA synthesis. Eight cosmids that span this domain have been utilized as radioactive hybridization probes to analyze the similarities among the dihydrofolate reductase amplicons in four independently derived methotrexate-resistant Chinese hamster cell lines. We have observed no significant differences among the four cell lines within the 150-kilobase DNA sequence that we have examined, except for polymorphisms that result from the amplification of one or the other of two possible alleles of the dihydrofolate reductase domain. We also show that the restriction patterns of the amplicons in these four resistant cell lines are virtually identical to that of the corresponding, unamplified sequence in drug-susceptible parental cells. Furthermore, measurements of the relative copy numbers of fragments from widely separated regions of the amplicon suggest that all fragments in this 150-kilobase region may be amplified in unison. Our data show that in methotrexate-resistant Chinese hamster cells, the amplified unit is large relative to the dihydrofolate reductase gene itself. Furthermore, within the 150-kilobase amplified consensus sequence that we have examined, significant rearrangements do not seem to occur during the amplification process.

Alleles↗
PubMed1983

In vivo effects of cytosine arabinoside on deoxyribonucleic acid replication in Chinese hamster ovary cells. 2. Cytosine arabinoside affects the rate of synthesis but not the pattern of labeling of an amplified chromosomal sequence at the onset of the S period.

The effect of 1-beta-D-arabinofuranosylcytosine (ara-C) on DNA replication in methotrexate-resistant Chinese hamster ovary cells was examined under circumstances in which nuclear DNA synthesis could be distinguished from mitochondrial DNA synthesis. G1-arrested cells were induced to traverse G1 and enter the S phase in the presence of radiolabeled thymidine and various concentrations of the drug. ara-C did not affect the kinetics of G1 traverse and subsequent entry into S after release from isoleucine deprivation, as measured by autoradiography. However, the inhibitor reduced the net rate of thymidine incorporation into nuclear DNA in a dose-dependent fashion. Autoradiography of nuclear matrix-DNA halo structures suggests that the drug inhibits nuclear thymidine incorporation by slowing chain elongation and movement of newly replicated DNA through a matrix-bound replication apparatus. Southern blot analysis of restriction digests of DNA radiolabeled in early S in the presence of ara-C indicates that the synthesis of the early-replicating amplified dihydrofolate reductase domain in these cells begins at sequences identical with those observed in cells synchronized with aphidicolin or hydroxyurea. Progressively lower concentrations of ara-C permit proportionately greater extents of the amplified unit to be replicated. These results suggest that ara-C slows the rate of chain elongation without altering the site at which DNA replication is initiated within individual replicons.

Animals↗
PubMed1983

Organization of a Chinese hamster ovary dihydrofolate reductase gene identified by phenotypic rescue.

We have constructed a genomic DNA library from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) in the cosmid vector pHC79. By utilizing a murine dihydrofolate reductase (DHFR) cDNA clone, we have identified 66 DHFR+ clones among the 11,000 colonies screened by colony hybridization. To isolate a recombinant cosmid containing the entire DHFR gene, we have tested these colonies for their ability to rescue a DHFR- Chinese hamster ovary cell line, using the spheroplast fusion method of gene transfer developed by W. Schaffner (Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980). One clone (cH1) was able to transform DHFR- cells to the DHFR+ phenotype and was shown in hybridization studies to contain all of the gene except a small portion of the 3' untranslated region. We have mapped cosmid cH1 and several overlapping cosmids with a variety of restriction enzymes and have determined the approximate positions of the five (and possibly six) exons within the DHFR gene. Differences between the sizes of homologous genes in hamster cells (24.5 kilobases [kb]) and in mouse cells (31.5 kb) are shown to reside primarily in the length of the 3' intron, which is 8 kb in the hamster gene and 16 kb in length in the mouse gene. Our studies confirm the utility of cosmid libraries for the isolation of large genes, as previously shown by R. de Saint Vincent et al. (Cell 27:267-277, 1981). In addition, a cosmid that contains a functional DHFR gene will be a useful vector for the co-amplification and subsequent overexpression of other cloned genes.

Animals↗
PubMed1983

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

Animals↗
PubMed1982

An amplified chromosomal sequence that includes the gene for dihydrofolate reductase initiates replication within specific restriction fragments.

We have developed a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) containing a 500-fold amplification of a 135-kilobase chromosomal DNA sequence. This sequence includes the gene for dihydrofolate reductase (tetrahydrofolate dehydrogenase, 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3). The high copy number of the amplified sequence permits it to be visualized as a distinct series of restriction fragments in genomic digests separated on ethidium bromide-stained agarose gels. Initiation of DNA replication in the amplified sequence was studied by radiolabeling DNA synthesized during the onset of S phase in synchronized CHOC 400 cells. Autoradiography of Southern blots of labeled genomic digests shows that DNA synthesis initiates in a small subset of the EcoRI fragments derived from the amplified units. These early labeled fragments are not synthesized at later times during S phase, when different subsets of fragments are synthesized. Regardless of the drug used to collect cells at the beginning of S phase, the replication pattern observed remains the same. These data suggest that replication of the amplified sequence initiates at specific sites within each repeated unit and proceeds in nonrandom order throughout the remainder of the sequence--i.e., that initiation of DNA synthesis in the chromosomes of mammalian cells is sequence specific.

Animals↗
PubMed1981

Replication pattern of a large homogenously staining chromosome region in antifolate-resistant Chinese hamster cell lines.

We have investigated the replication pattern of a large, homogenously staining chromosome region (HSR) in two antifolate-resistant Chinese hamster cell lines. This region is believed to be the location of an amplified genetic sequence which includes at least the gene coding for dihydrofolate reductase and which may be present in as many as 200 copies. It is shown that the HSR in both cell lines is among the first chromosome regions to begin DNA synthesis after reversal of an early G1 block. In cells synchronized in the S period with hydroxyurea, it is also clear that the HSR in both cell lines begins replication at many sites within its length in early S. The replicons comprising the HSR therefore may respond to a common initiation signal in early S. In on cell line (A3), replication of the HSR requires, at most, 3 hours of a 7-hour S period; in a second line (MQ19), replication proceeds for approximately 5 hours. In neither line does replication of the HSR occur concomitantly with synthesis of characteristic late replicating regions. These results were confirmed in exponential cultures using a retroactive labeling technique. The significance of these findings is discussed with reference to the possible origin and arrangement of the amplified sequence in these two cell lines.

Animals↗
PubMed1981

Methotrexate-resistant Chinese hamster ovary cells have amplified a 135-kilobase-pair region that includes the dihydrofolate reductase gene.

For the eventual purpose of isolating and studying a single animal cell replicon, we have developed a methotrexate-resistant Chinese hamster ovary cell line that has amplified an early-replicating DNA sequence approximately 500 times; this sequence includes the gene coding for dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). DHFR composes 30% of the cytoplasmic protein in this cell line, and DHFR mRNA represents 25% of the message translatable in vitro. After digestion of genomic DNA from resistant cells with restriction enzymes, a unique set of highly repetitive restriction fragments can be visualized on agarose gels by ethidium bromide staining. These bands are not present in digests of parental DNA. We estimate the total length of the unit repeated sequence to be 135 +/- 15 kilobase pairs. Regardless of the restriction enzyme utilized, a subset of these repetitive fragments hybridizes to radioactive DHFR cDNA. The homogeneously staining regions on mitotic chromosomes in which these amplified sequences are located are shown to be early-replicating, as are the highly repeated restriction fragments themselves. These data suggest that an early replicon can be isolated from this region, and that this entire, normally unique, genomic segment can be cloned and mapped with respect to origins of DNA synthesis and promoters for transcription, as well as other genetic features of interest.

Animals↗
PubMed1978

Control of DNA synthesis in tissue culture cells.

Eukaryotic DNA is functionally divided into thousands of replicons, each of which may be duplicated at a characteristic time within the DNA synthetic (S) period. Our approach toward an understanding of the molecular mechanisms which control orderly eukaryotic DNA synthesis has been: (a) to devise a method of cell synchrony in a suitable tissue culture system wherein all cells in the population enter and traverse the S period with a high degree of synchrony; (b) to determine, utilizing this system, precisely when during the S period critical events and macromolecular syntheses occur; and (c) to examine, by polyacrylamide-gel electrophoresis, the spectrum of proteins which become associated with chromatin during the S period in such a way as to suggest their involvement with DNA synthesis. Possible mechanisms for control are discussed based on the results presented here.

Cell Cycle↗
PubMedDate not supplied

Cloning of the initiation region of a mammalian chromosomal replicon.

In eukaryotic cells, each chromosome is divided into several thousand tandemly arranged replicons, each synthesized at a characteristic time during the S period. Although as yet no eukaryotic DNA sequence known for certain to be a eukaryotic chromosomal replication origin has been isolated, electron microscopic and biochemical evidence suggests that eukaryotic DNA synthesis is initiated at specific sites. We have attempted to establish a system in which functional chromosomal origins could be identified in vivo before their isolation by molecular cloning. For this purpose, we have developed a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) that contains 1,000 copies of a 135-kilobase (kb) early-replicating sequence, and which includes the gene for dihydrofolate reductase (DHFR). We have shown that initiation of DNA synthesis within each repeated unit (amplicon) is restricted to a small subset of restriction fragments at the beginning of the S period, suggesting that these fragments contain or flank origins of DNA synthesis. Here we report molecular cloning and restriction mapping experiments showing that all of these early-labelled fragments (ELFs) are derived from a single locus within each repeated unit. This result implies that synthesis of each amplicon initiates from a single origin of replication at the onset of S, and that an amplicon is formally equivalent to a replicon.

Animals↗