In many bacteria and most eukaryotes, some cytosine residues are modified following DNA replication by enzymatic methylation at the 5' position. This modification does not affect base pairing, but it can cause changes in DNA-protein interactions and helix stability. Examination of a large number of gene sequences indicates a strong, though not absolute, correlation between undermethylation of some sites and gene expression. To determine whether DNA methylation can cause loss of expression potential I and others have initiated studies using genes methylated in vitro at sites likely to be modified in cells, and introduced into suitable test systems. My original goal was to methylate SV4C DNA containing a cloned mammalian gene with the Hpa II methylase, and to analyse transcription of the modified gene during a lytic infection of monkey cells. However, I showed that SV40 DNA loses any in vitro methylation during such an infection. Finally, a plasmid, pTK, containing the thymidine kinase gene from the Herpes simplex type I virus was methylated in vitro and injected into Xenopus laevis oocytes. Unmethylated pTK DNA is transcribed into mRNA and processed into functional thymidine kinase. Using S(,1) nuclease and thymidine kinase activity assays, I have shown that methylated pTK DNA is also efficient in mRNA and protein production when the plasmid is injected at a high concentration. However, preliminary studies indicate that low concentrations of methylated injected pTK DNA may be far less efficiently transcribed. This indicates that methylation of Hpa II sites within the Herpes tk gene probably has some effect on its transcription. Similar results have been reported for the tk gene methylated and assayed in a tk('-) cell line. Much more drastic effects have been reported following investigation of adenovirus early genes and SV40 DNA methylated at Hpa II sites / and tested in the Xenopus system, and of rabbit globin genes methylated at all CG sites. The relatively small depression of synthesis produced in the tk gene may be due to the lack of Hpa II sites in the 5' region of the gene. Whether sites important to transcription are methylated in vivo remains to be confirmed. / Source: Dissertation Abstracts International, Volume: 45-04, Section: B, page: 1090. / Thesis (Ph.D.)--The Florida State University, 1984.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_75316 |
| Contributors | APPLEGATE, MARCIA LYNNE., Florida State University |
| Source Sets | Florida State University |
| Detected Language | English |
| Type | Text |
| Format | 142 p. |
| Rights | On campus use only. |
| Relation | Dissertation Abstracts International |
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