Control of the timing of replication within S-phase of chromosomes or chromosome bands has long been suspected to be a general mechanism of compartmentalizing the genome for differential gene expression. I have developed a method that provides both a direct probe for active and inactive regions of the chromosome and a means to determine the time of replication of these individual sites. By using a retrovirus shuttle vector which contains a selectable marker gene (HSV-tk), to infect tissue culture cells, it is possible to introduce the same single copy sequence in many different chromosomal locations. I have isolated and characterized a series of cell clones which have single or a small number of either active or cryptic copies of the marker gene. To determine the time of replication of the marker sequence, asynchronous or synchronized cell cultures were pulse labeled with bromo-deoxy-uridine. Cells were fractionated according to their position in S-phase by mitotic collection or separately pulse labeled cultures (for synchronized cells). BrdU containing DNA was then degraded selectively by exposure to Hoechst 33257 dye, long wave UV light and S1 nuclease, which makes it possible to analyze each cell fraction with regard to BrdU substitution in the marker sequence. One can thus infer the time of replication of a specific single copy sequence. All clones analyzed, both expressing and cryptic clones, replicate the SW 272 sequence in early S-phase. The findings suggest, that this retrovirus does not integrate randomly into the cellular genome with respect to replication compartments. In one clone the cryptic copy of the viral Tk-gene, could be activated by treatment with 5-aza-cytidine, implying a role of cytosine methylation in regulation of expression in this case. / Source: Dissertation Abstracts International, Volume: 47-07, Section: B, page: 2773. / Thesis (Ph.D.)--The Florida State University, 1986.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_75887 |
| Contributors | STURM, KARIN SIGRID., Florida State University |
| Source Sets | Florida State University |
| Detected Language | English |
| Type | Text |
| Format | 172 p. |
| Rights | On campus use only. |
| Relation | Dissertation Abstracts International |
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