It is proposed to build a laser Raman microscope which is capable of observing chromosomal material with about one micron resolution. Using this apparatus Raman spectra will be taken of chromosomes from a variety of easily available sources. By focusing a laser beam of about 1 micron or less on various portions of the chromosome and associated cellular apparatus and capturing the Raman light which is back scattered through the microscope objective, it is proposed to obtain both the classical and the resonance Raman spectra of the various chromosomal components including both protein and DNA. In particular the Raman marker band characteristic of the A, B, C, and Z forms of DNA as well as Raman bands sensitive to protein secondary structure will be sought. The presence of the Z marker bands in a portions of a gene would give evidence for the Z form. Polytene chromosomes will be examined in fixed and non-fixed states. The Raman spectra of polytene chromosomes containing bound antibodies to Z-DNA will also be taken to see if evidence can be found for presence of the Z-conformation in the DNA of polytene chromosomes upon antibody binding. This approach could settle the questions of whether or not Z-DNA exists in polytene chromosomes or whether or not antibodies are capable of pulling the DNA into the Z form upon binding. By collecting instanteous Raman spectra of the cellular components of living cells, it is proposed to determine the type, extent of and duration of conformational changes in the DNA and associated proteins during cell division. Thus, it is hoped to answer the questions as to whether Z-DNA is a naturallly occurring structure in genetic material. It is also hoped to improve the understanding of the molecular basis of the hereditary process. Such understanding can only help to formulate a more rational approach to the development of drug therapy for treatment of diseases which involve malfunction of the genetic material.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033825-02
Application #
3283894
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1984-08-01
Project End
1987-07-31
Budget Start
1985-08-01
Budget End
1986-07-31
Support Year
2
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Oregon
Department
Type
Schools of Arts and Sciences
DUNS #
948117312
City
Eugene
State
OR
Country
United States
Zip Code
97403
Whiting, A K; Peticolas, W L (1994) Details of the acyl-enzyme intermediate and the oxyanion hole in serine protease catalysis. Biochemistry 33:552-61
Otto, C; Thomas, G A; Rippe, K et al. (1991) The hydrogen-bonding structure in parallel-stranded duplex DNA is reverse Watson-Crick. Biochemistry 30:3062-9
Evertsz, E M; Thomas, G A; Peticolas, W L (1991) Raman spectroscopic studies of the DNA cro binding site conformation, free and bound to cro protein. Biochemistry 30:1149-55
Thomas, G A; Peticolas, W L (1989) A temperature-dependent Z to B to single-strand transition in d(CGCG). Biopolymers 28:1625-36
Dai, Z Y; Thomas, G A; Evertsz, E et al. (1989) The length of a junction between the B and Z conformations in DNA is three base pairs or less. Biochemistry 28:6991-6
Thomas, G A; Kubasek, W L; Peticolas, W L et al. (1989) Environmentally induced conformational changes in B-type DNA: comparison of the conformation of the oligonucleotide d(TCGCGAATTCGCG) in solution and in its crystalline complex with the restriction nuclease EcoRI. Biochemistry 28:2001-9
Peticolas, W L; Wang, Y; Thomas, G A (1988) Some rules for predicting the base-sequence dependence of DNA conformation. Proc Natl Acad Sci U S A 85:2579-83
Patapoff, T W; Thomas, G A; Wang, Y et al. (1988) Polarized Raman scattering from oriented single microcrystals of d(A5T5)2 and d(pTpT). Biopolymers 27:493-507
Wang, Y; Thomas, G A; Peticolas, W L (1987) Sequence dependence of the B to Z transition in crystals and aqueous NaCl solutions for deoxyoligonucleotides containing all four canonical DNA bases. Biochemistry 26:5178-86
Taillandier, E; Ridoux, J P; Liquier, J et al. (1987) Infrared and Raman studies show that poly(dA).poly(dT) and d(AAAAATTTTT)2 exhibit a heteronomous conformation in films at 75% relative humidity and a B-type conformation at high humidities and in solution. Biochemistry 26:3361-8

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