Person:

Wartell, Roger M.

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https://hdl.handle.net/1853/71822

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Organizational Unit

School of Biological Sciences

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Now showing 1 - 10 of 28

The formation of adjacent triplex-duplex domains in DNA

(Georgia Institute of Technology, 1999-02) Nam, Kang Hoon ; Abhiraman, Saraswathy ; Wartell, Roger M.

The ability of single-stranded DNA oligomers to form adjacent triplex and duplex domains with two DNA structural motifs was examined. Helix–coil transition curves and a gel mobility shift assay were used to characterize the interaction of single-stranded oligomers 12–20 nt in length with a DNA hairpin and with a DNA duplex that has a dangling end. The 12 nt on the 5'-ends of the oligomers could form a triplex structure with the 12 bp stem of the hairpin or the duplex portion of the DNA with a dangling end. The 3'-ends of the 17–20 nt strands could form Watson–Crick pairs to the five base loop of the hairpin or the dangling end of the duplex. Complexes of the hairpin DNA with the singlestranded oligomers showed two step transitions consistent with unwinding of the triplex strand followed by hairpin denaturation. Melting curve and gel competition results indicated that the complex of the hairpin and the 12 nt oligomer was more stable than the complexes involving the extended single strands. In contrast, results indicated that the extended single-stranded oligomers formed Watson–Crick base pairs with the dangling end of the duplex DNA and enhanced the stability of the adjacent triplex region.
The thermal stability of DNA fragments with tandem mismatches at a d(CXYG)d(CY'X'G) site

(Georgia Institute of Technology, 1996-02-15) Ke, Song-Hua ; Wartell, Roger M.

Temperature-Gradient Gel Electrophoresis (TGGE) was employed to determine the thermal stabilities of 28 DNA fragments, 373 bp long, with two adjacent mismatched base pairs, and eight DNAs with Watson-Crick base pairs at the same positions. Heteroduplex DNAs containing two adjacent mismatches were formed by melting and reannealing pairs of homologous 373 bp DNA fragments differing by two adjacent base pairs. Product DNAs were separated based on their thermal stability by parallel and perpendicular TGGE. The polyacrylamide gel contained 3.36 M urea and 19.2 % formamide to lower the DNA melting temperatures. The order of stability was determined in the sequence context d(CXYG).d(CY'X'G) where X.X' and Y.Y" represent the mismatched or Watson-Crick base pairs. The identity of the mismatched bases and their stacking interactions influence DNA stability. Mobility transition melting temperatures (T u) of the DNAs with adjacent mismatches were 1.0-3.6 degrees C (+/-0.2 degree C) lower than the homoduplex DNA with the d(CCAG).d(CTGG) sequence. Two adjacent G.A pairs, d(CGAG).d(CGAG), created a more stable DNA than DNAs with Watson-Crick A.T pairs at the same sites. The d(GA).d(GA) sequence is estimated to be 0.4 (+/-30%) kcal/mol more stable in free energy than d(AA).d(TT) base pairs. This result confirms the unusual stability of the d(GA).d(GA) sequence previously observed in DNA oligomers. All other DNAs with adjacent mismatched base pairs were less stable than Watson-Crick homoduplex DNAs. Their relative stabilities followed an order expected from previous results on single mismatches. Two homoduplex DNAs with identical nearest neighbor sequences but different next-nearest neighbor sequences had a small but reproducible difference in T u value. This result indicates that sequence dependent next neighbor stacking interactions influence DNA stability.
A program for selecting DNA fragments to detect mutations by denaturing gel electrophoresis methods

(Georgia Institute of Technology, 1994-10-11) Brossette, Stephen ; Wartell, Roger M.

A computer program was developed to automate the selection of DNA fragments for detecting mutations within a long DNA sequence by denaturing gel electrophoresis methods. The program, MELTSCAN, scans through a user specified DNA sequence calculating the melting behavior of overlapping DNA fragments covering the sequence. Melting characteristics of the fragments are analyzed to determine the best fragment for detecting mutations at each base pair position in the sequence. The calculation also determines the optimal fragment for detecting mutations within a user specified mutational hot spot region. The program is built around the statistical mechanical model of the DNA melting transition. The optimal fragment for a given position is selected using the criteria that its melting curve has at least two steps, the base pair position is in the fragment's lowest melting domain, and the melting domain has the smallest number of base pairs among fragments that meet the first two criteria. The program predicted fragments for detecting mutations in the cDNA and genomic DNA of the human p53 gene.
Localization of the intrinsically bent DNA region upstream of the E.cofi rrnB P1 promoter

(Georgia Institute of Technology, 1994-06-25) Gaal, Tamas ; Rao, Lin ; Estrem, Shawn T. ; Yang, Jin ; Wartell, Roger M. ; Gourse, Richard L.

DNA sequences upstream of the rrnB P1 core promoter (-10, -35 region) increase transcription more than 300-fold in vivo and in vitro. This stimulation results from a cis-acting DNA sequence, the UP element, which interacts directly with the alpha subunit of RNA polymerase, increasing transcription about 30-fold, and from a positively acting transcription factor, FIS, which increases expression another 10-fold. A DNA region exhibiting a high degree of intrinsic curvature has been observed upstream of the rrnB P1 core promoter and has thus been often cited as an example of the effect of bending on transcription. However, the precise position of the curvature has not been determined. We address here whether this bend is in fact related to activation of rRNA transcription. Electrophoretic analyses were used to localize the major bend in the rrnB P1 upstream region to position approximately -100 with respect to the transcription initiation site. Since most of the effect of upstream sequences on transcription results from DNA between the -35 hexamer and position -88, i.e. downstream of the bend center, these studies indicate that the curvature leading to the unusual electrophoretic behavior of the upstream region does not play a major role in activation of rRNA transcription. Minor deviations from normal electrophoretic behavior were associated with the region just upstream of the -35 hexamer and could conceivably influence interactions between the UP element and the alpha subunit of RNA polymerase.
Influence of nearest neighbor sequence on the stability of base pair mismatches in long DNA: determination by temperature-gradient gel electrophoresis

(Georgia Institute of Technology, 1993-11-11) Ke, Song-Hua ; Wartell, Roger M.

Temperature-gradient gel electrophoresis (TGGE) was employed to determine the thermal stabilities of 48 DNA fragments that differ by single base pair mismatches. The approach provides a rapid way for studying how specific base mismatches effect the stability of a long DNA fragment. Homologous 373 bp DNA fragments differing by single base pair substitutions in their first melting domain were employed. Heteroduplexes were formed by melting and reannealing pairs of DNAs, one of which was ³²P-labeled on its 5'-end. Product DNAs were separated based on their thermal stability by parallel and perpendicular temperature-gradient gel electrophoresis. The order of stability was determined for all common base pairs and mismatched bases in four different nearest neighbor environments; d(GXT) d(AYC), d(GXG) d(CYC), d(CXA) d(TYG), and d(TXT) * d(AYA) with X,Y = A,T,C, or G. DNA fragments containing a single mismatch were destabilized by 1 to 5°C with respect to homologous DNAs with complete Watson - Crick base pairing. Both the bases at the mismatch site and neighboring stacking interactions influence the destabilization caused by a mismatch. G - T, G - G and G - A mismatches were always among the most stable mismatches for all nearest neighbor environments examined. Purine- purine mismatches were generally more stable than pyrimidine- pyrimidine mispairs. Our results are in very good agreement with data where available from solution studies of short DNA oligomers.
Isolation of proteins involved in cell growth regulation

(Georgia Institute of Technology, 1991) Wartell, Roger M.
Detecting base pair substitutions in DNA fragments by temperature-gradient gel electrophoresis

(Georgia Institute of Technology, 1990-05-11) Wartell, Roger M. ; Hosseini, Seyed Homayoun ; Moran, Charles P., Jr.

A vertical gel electrophoresis apparatus is described which can distinguish DNA fragments differing by single base pair substitutions. The system employs a homogenous polyacrylamide gel containing ureaformamide and a temperature gradient which runs either perpendicular or parallel to the direction of electrophoresis. The temperature-gradient system simplifies several features of the denaturant-gradient system (1) and is relatively inexpensive to construct. Eight homologous 373 bp DNAs differing by one, two, or nine base pair substitutions were examined. DNA electrophoretic mobility changed abruptly with the temperature induced unwinding of DNA domains. GC to AT substitutions at different locations within the first melting domain, as well as an AT to TA transversion were separated with temperature gradients parallel to the electrophoretic direction. The relative stabilities of the DNAs observed in the gels were compared to predictions of DNA melting theory. General agreement was observed however complete correspondence was not obtained.
DNA conformational change in Gal repressor-operator complex: involvement of central G-C base pair(s) of dyad symmetry

(Georgia Institute of Technology, 1988-12-23) Wartell, Roger M. ; Adhya, Sankar Lal

Gal repressor dimer binds to two gal operator sites, O[subscript E] and O[subscript I], which are 16 bp long similar sequences with hyphenated dyed symmetries (11,12). Repressor occupation hinders the reactivity of the N7 atoms in the major groups of guanines, located at positions 1,3 and 8, and the rotational 1′, 3 and 8′ of the symmetries. We have shown that Gal repressor binding to O[subscript E] or O[subscript I] DNA fragments increases the circular dichroism (CD) spectral peak in the 270 to 300 nm range. The CD change is similar to that observed for Lac repressor binding to its operator site (14). It is consistent with a DNA conformational change during complex formation between Gal repressor and O[subscript E] and O[subscript I] DNA. The CD spectral change was not observed when the central 8,8′ C-C base pairs in the DNA-protein complex were replaced by A-T base pairs, whereas substitution of the 1,1′ G-C base pairs do show the accompanying increase in the spectra during repressor binding. The absence of CD change of the Gal repressor complex with DNA mutated at the 8,8′ base pairs suggest that the central G-C base pairs are required for the repressor induced conformational change.
Sequence distributions associated with DNA curvature are found upstream of strong E. coli promoters

(Georgia Institute of Technology, 1987-01-26) Plaskon, R. Richard ; Wartell, Roger M.

The regions upstream from forty-three procaryotic promoters were examined for nucleotide distributions which have been associated with DNA curvature. The analysis procedure assigned a DNA curvature score based on the phasing of the 5' and 3' ends of An and Tn tracts, n greater than or equal to 3. The weighting scheme for the curvature score was based on recent studies which showed that tracts of An and Tn periodically phased with the helix repeat cause DNA curvature. Results show that promoters which have high transcription initiation rates in vivo tend to have high curvature scores in their upstream regions. Regions downstream from the transcription start-point do not have sequences correlated with DNA curvature. Four promoters which have been shown to have upstream activation regions have curvature scores above 1.5 in their -40 to -150 regions. The correlations observed lend support to the hypothesis that DNA curvature is associated with upstream activation of transcription
The catabolite activator protein stabilizes its binding site in the E. coli lactose promoter

(Georgia Institute of Technology, 1985-10-25) DeGrazia, Henry ; Abhiraman, Saraswathy ; Wartell, Roger M.

The effect of catabolite activator protein, CAP, on the thermal stability of DNA was examined. Site specific binding was studied with a 62 bp DNA restriction fragment containing the primary CAP site of the E. coli lactose (lac) promoter. A 144 bp DNA containing the lac promoter region and a 234 bp DNA from the pBR322 plasmid provided other DNA sites. Thermal denaturation of protein-DNA complexes was carried out in a low ionic strength solvent with 40% dimethyl sulfoxide, DMSO. In this solvent free DNA denatured below the denaturation temperature of CAP. The temperature stability of CAP for site specific binding was monitored using an acrylamide gel electrophoresis assay. Results show that both specific and non-specific CAP binding stabilize duplex DNA. Site specific binding to the 62 bp DNA produced a 13.3 degrees C increase in the transition under conditions where non-specific binding stabilized this DNA by 2-3 degrees C.