LASER RAMAN PROBE OF MOLECULAR STRUCTURES IN LIVING CELLS. This proposal begins with the premise that double helical DNA is polymorphic in nature and may exist in different conformations. The purpose of this research is to determine the role played by DNA conformation in regulating gene expression. We propose two approaches to this problem. First, it is proposed to examine directly the conformation of DNA in certain living cells as it occurs in different regions in a chromosome or chromosomal apparatus. We propose to obtain these measurements as a function of cell division. We seek to determine if there is a difference between the conformation of DNA that is actively being transcribed and DNA in which transcription is repressed. The occurrence of transcription has already been determined as a function of position in certain chromosomal regions such as in the puffs of the chromosomes from the salivary gland Drosphilia. Zygotic transcription is known to be suppressed until after several cell divisions in sea urchin embryos. we will be able to use our recently developed laser Raman microscope with these materials to examine the conformation of DNA in regions that are actively being transcribed and regions where transcription is apparently being repressed. The second approach begins with the premise that if DNA exists in different conformations (such as the A or the Z) in living cells these must occur in specific base sequences. We propose to continue our design, synthesis, purification, and microcrystallization of oligonucleotides in order to determine what we will call the conformational code of DNA. To any oligonucleotide double helical duplex there exists a certain set of conformational states to which it will adapt depending upon its base sequence and the environmental conditions. This correspondence between conformation and base sequence represents a code which one can use to predict the conformational probability of a given base sequence in a living cell. We have found it is very easy to obtain well formed, prismatic, optically perfect microcrystals of oligonucleotides of the order of 10 to 100 microns on an edge. These microcrystals are too small for X-ray diffraction or conventional Raman Spectroscopy, but they produce sharp, beautiful Raman spectra using our laser Raman microscope. From these spectra the DNA conformation can be unambiguously identified.
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