Abstract

The mechanism of gene regulation at the molecular level will be studied with special emphasis on the role of DNA properties and conformations as determinants of specificity. The properties of left-handed DNA will be investigated with restriction fragments of known sequence and recombinant plasmids. The influence of base sequence on the capacity of a region to form a left-handed helix will be studied with synthetic DNA polymers, plasmids containing simple repeating sequences (both of natural and synthetic origin) and with mutants of defined sequence in the structurally interesting segments. Conditions that promote the R to L transition (such as ionic environment, supercoiling, AAF modification of guanosine moieties, and methylation) will be investigated. Attempts will be made to determine the structure and chemical behavior of the junction between a left-handed Z-region and a right-handed B-segment. The biological significance of left-handed DNA will be evaluated in vitro and in vivo. Conformational properties will be determined by circular dichroism, proton and phosphorus NMR, and laser Raman spectroscopy as well as superhelix density determinations on topoisomers of the recombinant molecules. X-ray diffraction studies will be pursued on a collaboratilve basis. Gene cloning will be employed extensively to prepare chemically defined molecules with inserts with the desired sequences. Milligram quantities of the restriction fragment inserts will be purified by high pressure liquid chromatography on RPC-5 An understanding of the details of gene expression is fundamental to our ultimate comprehension of normal cellular growth and divison as well as disease syndromes characterized by unregulated growth such as cancer.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030822-04
Application #
3278709
Study Section
Biophysics and Biophysical Chemistry A Study Section (BBCA)
Project Start
1983-01-01
Project End
1987-12-31
Budget Start
1986-01-01
Budget End
1986-12-31
Support Year
4
Fiscal Year
1986
Total Cost
Indirect Cost

  Institution

Name
University of Alabama Birmingham
Department
Type
School of Medicine & Dentistry
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294

  Publications

Amirhaeri, S; Wohlrab, F; Wells, R D (1995) Differential effects of simple repeating DNA sequences on gene expression from the SV40 early promoter. J Biol Chem 270:3313-9
Klysik, J (1995) An intramolecular triplex structure from non-mirror repeated sequence containing both Py:Pu.Py and Pu:Pu.Py triads. J Mol Biol 245:499-507
Kang, S; Jaworski, A; Ohshima, K et al. (1995) Expansion and deletion of CTG repeats from human disease genes are determined by the direction of replication in E. coli. Nat Genet 10:213-8
Kang, S; Wells, R D (1994) Zinc destabilizes DNA Watson-Crick pairs at AGCT. J Biol Chem 269:9528-32
Wang, Y H; Amirhaeri, S; Kang, S et al. (1994) Preferential nucleosome assembly at DNA triplet repeats from the myotonic dystrophy gene. Science 265:669-71
Lukomski, S; Wells, R D (1994) Left-handed Z-DNA and in vivo supercoil density in the Escherichia coli chromosome. Proc Natl Acad Sci U S A 91:9980-4
Klysik, J; Shimizu, M (1993) Escherichia coli single-stranded DNA-binding protein alters the structure of intramolecular triplexes in plasmids. FEBS Lett 333:261-7
Hartman, D A; Kuo, S R; Broker, T R et al. (1992) Intermolecular triplex formation distorts the DNA duplex in the regulatory region of human papillomavirus type-11. J Biol Chem 267:5488-94
Kang, S; Wohlrab, F; Wells, R D (1992) GC-rich flanking tracts decrease the kinetics of intramolecular DNA triplex formation. J Biol Chem 267:19435-42
Rahmouni, A R; Wells, R D (1992) Direct evidence for the effect of transcription on local DNA supercoiling in vivo. J Mol Biol 223:131-44

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