Polypurine·polypyridine (pur·pyr) tracts are a run of all purines on one strand and all pyrimidines on the complementary DNA strand. Statistical overrepresentation of the tracts in eukarocytes suggests a cellular role or roles. The tracts from triplex DNA <i>in vitro</i> and there is evidence for triplex DNA <i>in vivo</i>. Several cellular roles are possible for triplex DNA. The presence of the tracts in gene 5' flanking regions suggets a regulatory role. This work investigates the role of triplex DNA in the cell, particularly in the regulation of transcription. Proteins mediate DNA looping in the regulation of transcription and in its condensation in chromosomes. Such looping may also be mediated by transmolecular triplexes, formed between separated pur·pyr tracts. Formation of pyr·pur·pyr transmolecular triplexes was investigated using linear and circular plasmid models containing separated pur·pyr tracts able to form a triplex with each other, but not within a tract. Transmolecular triplex loops (T-loops) formed in circular DNA, suggesting a possible regulatory or structural role <i>in vivo</i>. The following model shows a T-loop formed at pH 4. At pH 6, a duplex partially reforms and single-stranded region(s) trap the structure. and single-stranded region(s) trap the structure. T-loops were used as a model to test the Idea that a single-strand extruded by triplex formation in the 5' flanking region of a gene could promote transcription. Transcription was inhibited in T-loops, suggesting such structures could block transcriptional elongation if formed <i>in vivo</i>. The ability of polyamine analogues to promote triplex formation was also tested using T-loops. Pentamines promoted T-loop formation at lower concentrations than tetramines. Spatial distribution of charge was also important. A triplex role in transcriptional regulation was investigated using two examples of human genes with 5' flanking pur·pyr tracts. The effect of triplex-specific antibodies on expression of c-' myc' was investigated using agarose-encapsulated nuclei. Triplex formation between c-'src' promoter pur·pyr tracts was visualized as gel band shift die to dimerization between linear plasmid fragments containing individual tracts. A transmolecular triplex was proposed as one way in which the c-'src' tracts could form a triplex <i>in vivo</i> which might be involved in the regulation of transcription.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-10212004-002131 |
| Date | 01 January 1999 |
| Creators | Ashley, Carolyn |
| Contributors | Lee, Jeremy S. |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-10212004-002131 |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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