Alleviating Telomeric Position Effect Through the Use of AAV Sequences.
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Telomeres, like centromeres, are chromosomal regions made up of heterochromatic DNA. By definition, heterochromatic DNA regions are not transcribed (ie. are silent) while euchromatic DNA regions are transcribed.This phenomenon is known as telomeric position effect, TPE.It has been shown that transcription can be induced in telomeric regions by inserting an inducible promoter next to the telomere. Position effect can overcome and re-silence even some of these inducible genes. We are interested in testing the effects of recombinant adeno-associated virus, rAAV, DNA sequences for insulating genes from TPE. We know rAAV vectors can successfully integrate into the human genome in various locations and remain transcriptionally active. Long-term expression at various locations may be indicative of the recombinant vector having a unique ability to insulate from position effect, even TPE.Yeast is being used as the model system since it has been extensively studied with regard to chromatin effects. Our goal is to construct a yeast telomere containing AAV ITR?s flanking a reporter gene to measure the extent of positional transcriptional effects. Effects will be measured by including a URA3 gene between the ITR sequences which, when transcribed, can produce the amino acid uracil and thus grow on ura- media. We can also test for lack of transcription because, if uracil is present, the indicator 5-FOA is toxic to the cells and kills them.Using differential media we can readily determine whether we have insulation from position effects even if there is an intermediate effect.