Person:
Wartell, Roger M.

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https://hdl.handle.net/1853/71822
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School of Biological Sciences
School established in 2016 with the merger of the Schools of Applied Physiology and Biology
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End date: 1994 × search.filters.applied.f.isOrgUnitOfPublicationTitle: School of Biological Sciences × Start date: 1990 ×

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A program for selecting DNA fragments to detect mutations by denaturing gel electrophoresis methods

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.

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Isolation of proteins involved in cell growth regulation

1991 , Wartell, Roger M.

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Localization of the intrinsically bent DNA region upstream of the E.cofi rrnB P1 promoter

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.

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Detecting base pair substitutions in DNA fragments by temperature-gradient gel electrophoresis

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.

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Influence of nearest neighbor sequence on the stability of base pair mismatches in long DNA: determination by temperature-gradient gel electrophoresis

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.

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