GTMB 7 - Gene Therapy & Molecular Biology

Buldak and Mirkin: Hypersensitivity of the d(GA) n •d(TC) n dependent on lactose repressorFigure 2. Summary of CAA modifications observed between positions -70 and +5 relative to the TSS. Modified cytosine and/or adenineresidues shown in red occurred only in the absence of IPTG. Modifications shown in green occurred only in the presence of IPTG.Modifications shown in turquoise occurred in both states of promoter induction.These data, therefore, rule out the possibility that themodification of the repeat results from the deformation atthe promoter region.Though modification of the d(GA) n •d(TC) n insert does notseem to be directly linked to the trc promoter activity, it isdowngraded in the presence of IPTG. Since the onlyknown effect of IPTG is to block DNA-binding activity ofthe lactose repressor, we looked at the role of the lactoserepressor in the d(GA) n •d(TC) n insert modification. Tothis end, we first deleted lacI q gene from our test plasmidand transformed the resultant construct into the lacI -background. The data on chemical probing in vivopresented in Figure 5 show that there are no modificationsof the d(GA) n •d(TC) n insert in this setting. At the sametime, the modifications within the promoter region wereidentical to those seen in previous experiments in thepresence of IPTG, showing that trc promoter was in aconstantly induced state. We conclude, therefore, thatfunctional lactose repressor is necessary for a structuralalteration of the d(GA) n •d(TC) n repeat leading to itschemical hyperreactivity in vivo.Lactose repressor is known to bind to at least twooperator sites in our plasmid. The next question istherefore, whether repressor-operator binding is essentialfor the repeat's modification. To answer this question, wedeleted the operator and pseudo-operator, eitherindividually or together. When the primary operator (O1)was deleted, the modification of the d(GA) n •d(TC) n repeatwas similar to that observed in the presence of the normaloperator site (Figure 6). The modification of the promoterregion, as one would expect, became equivalent to that ofthe induced wild-type promoter even in the absence ofIPTG. When the O3 pseudo-operator was deletedindividually, leaving O1 intact, modifications of both theinsert and of the promoter regions (Figure 7A) wereidentical to those seen in the original plasmid assummarized in Figure 2. Furthermore, when both operatorsites were deleted (ΔO1-ΔO3 construct), the results ofmodification were identical to those seen in the case of theΔO1 (Figure 7B). We conclude, therefore, that lactoserepressor per se, rather than its binding to the operatorsites, is necessary for the d(GA) n •d(TC) n hyperreactivity.Figure 3. CAA modification in vivo of the non-transcribedstrand of pTrcCat/Pst. G, C, Maxam-Gilbert sequencing reactionsof plasmid DNA in vitro. IPTG - and + indicate C reactions for invivo CAA-modified plasmid DNAs. Arrows indicate modifiedadenine and cytosine bases.IV. DiscussionWe found that a d(GA) n •d(TC) n repeat does undergostructural changes, accompanied by chemical modification294