Amplified dihydrofolate reductase genes are located in chromosome regions containing DNA that replicates during the first half of S-phase.

Abstract

To obtain a better understanding of the relationship between metaphase chromosome banding patterns and genome organization, attention was focused on regions of metaphase chromosomes that were found to contain the genes for a specific cellular enzyme, dihydrofolate reductase (DHFR) . These studies involved the use of highly methotrexateresistant mouse lymphoblastoid cells (L5178YR), which contain approximately 300 times the number of DHFR genes present in parental cells (L5178YS) . Karyotypic analysis revealed the presence of two very large, nonhomologous, marker chromosomes that were absent in the parental line . In situ hybridization of 3H-labeled cloned DHFR cDNA to metaphase chromosomes of L5178YR cells was used to localize the DHFR genes to a very large Giemsa (G)-negative region on each of the two large marker chromosomes. Regional patterns of DNA replication in metaphase chromosomes were studied by autoradiographic visualization of [3H]thymidine incorporation and by fluorescent microscopic visualization of bromodeoxyuridine incorporation . Because the amplified DHFR genes were present within two prominent cytogenetic regions on two easily identifiable chromosomes, it was possible to observe the following. The amplified DHFR genes were located in chromosome regions that replicated at the same time during the first half of a 9-h S-phase . DNA replication began simultaneously and terminated simultaneously at many locations throughout each amplified region . We conclude that transcriptionally active DHFR genes are located within large G-negative regions of metaphase chromosomes and that the DNA within these regions replicates during the first half of S-phase .The genome of animal cells is subdivided into 30,000 replication units, or replicons, ranging in size from 50 to 330 kilobasepairs (kb) (13,22,23) . DNA replication proceeds bidirectionally within each replicon from an origin until reaching replication forks proceeding outward from adjacent origins of replication (24, 28) . Groups of replicons are activated in a defined temporal order that is maintained from one S-phase to the next (1,11,39,40,45,47) . Clusters of replicons initiate replication in synchrony and are similar in size and rate of fork movement (21,27) . This clustering of replicon activity may be responsible for the regional patterns of DNA replication observed in metaphase chromosomes (9,12,15,30,35,38,46) . A number of reports have indicated that the DNA of specific cytogenetic loci replicate at characteristic times within S-phase . THE JOURNAL OF CELL BIOLOGY " VOLUME 92 FEBRUARY 1982 531-539 © The Rockefeller University Press " 0021-9525/82/02/0531/09 $1 .00In particular, it has been observed that heterochromatic chromosomal regions are usually the last to complete replication. These include the inactive X chromosome in females (facultative heterochromatin [8,18,36]) and centromeric regions (constitutive heterochromatin [11,16]). The darkly staining Gpositive bands produced by Giemsa staining procedures contain DNA that replicate...

Replication timing control can be maintained in extrachromosomally amplified genes.

“…The apparent lack of timing control in these extrachromosomal amplicons is intriguing, since the single-copy DHFR gene has been observed to replicate in a narrow window either early (19,24,25) or late (19) in S-phase. There are several possible reasons for these differences.…”

“…The methods for labeling cells with 5-bromo-2'-deoxyuridine (BrdU), fractionating cells on the cell sorter, and quantitating the incorporated label were previously described by Gilbert and Cohen (16). Cultures at 90% confluence were trypsinized and plated in fresh medium within 24 to 48 h of analysis to maximize the number of cells in S-phase. This procedure occasionally resulted in populations depleted in G1-phase cells at the time of analysis (e.g., see Fig.…”

“…These elements contain 100 to >1,000 kb of DNA flanking the amplification target gene (37) timing control in such structures is only beginning to be explored. Interestingly, the extrachromosomal elements containing dihydrofolate reductase (DHFR) genes in the mouse cell line 3T6R50 appear to replicate throughout S-phase (34), while the chromosomally located single-copy and amplified DHFR genes in a variety of mouse, human, and hamster cell lines generally replicate in a discrete interval confined to the first half of S-phase (10,19,24).The difference in replication timing of extrachromosomal and chromosomal DHFR amplicons raises the possibility that either the recombination events which generate extrachromosomal amplicons sever the connection between replication origins and adjacent timing control sites or that timing control is an intrinsic property of sequences confined within chromosomes. To …”

DOUBLE MINUTES AND HOMOGENEOUSLY STAINING REGIONS: Gene Amplification in Mammalian Cells

“…Homogeneously staining regions complete replication before the middle of S-phase at a time when the other chromosome regions are still actively synthesizing DNA (25,26,93,129). Replication begins synchronously at many sites along the entire length of the HSR at the beginning of S-phase; termination also appears to be fa irly synchronous (79, 93).…”

The changes in the levels of dihydrofolate reductase mRNA and its gene dosage in 5-fluorouracil-resistant L1210 cells.

“…MTX-resistant cells are well known to have chromosomal abnormalities like HSR or double minute chromosomes (DMs) Kaufman et al 1979;Cowell 1982;Kellems et al 1982), which are associated with DHFR gene amplification. HSR of MTX-resistant cells are common in chromosome 2, although other sites have been also reported.…”