Doctoral Dissertations

Date of Award

8-1984

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Salil K. Niyogi

Committee Members

Robert K. Fujimura, K. Bruce Jacobson, Sankar Mitra, Audrey L. Stevens

Abstract

Incubation of supercoiled SV40 DNA with three different HeLa cell free transcription extracts caused the DNA to be converted into ordered nucleoprotein complexes whose physical structure resembled the "beads-on-a-string" morphology of SV40 minichromosomes and eucaryotic chromatin. Nucleoprotein complexes for structural analysis were purified either by velocity sedimentation through sucrose gradients or by gel filtration. The nucleoprotein structures were shown by electron microscopy to be sensitive to protease digestion and to have compaction ratios similar to those for authentic SV40 minichromosomes. Additional studies using micrococcal nuclease and pancreatic DNase 1 digestion indicated that the structures protected nucleosome core-sized DNA fragments which were external to the "beads'. More important, however, were the observations that neither the HeLa cell extracts nor the purified nucleoprotein complexes appeared to contain histones; rather, they seemed to be composed of nonhistone chromosomal proteins. Only purified promoter-containing nucleoprotein complexes were actively transcribed in the presence of additional HeLa cell extract. This suggested that the protein constituents of the complexes may be important in transcription in vitro. It was subsequently shown that both linear and supercoiled Ad2 major late promoter-containing plasmid DNAs incubated with a HeLa whole cell extract were converted to similar ordered nucleoprotein complexes. These complexes, purified by velocity sedimentation onto sucrose cushions, were tested for the ability to serve as template for accurate transcription in vitro. Use of these preformed complexes lessened the stringency of the extract to template ratios and alleviated the bulk DNA dependence of the HeLa whole cell extract. In fact, they eliminated the need for poly[d(I-C)] in reactions containing subthreshold amounts of nucleoprotein template. Nucleoprotein complex, but not DNA templates were accurately transcribed by partially purified RNA polymerase II. These observations indicate that the nucleoprotein complexes enrich the reactions in important transcription components and demonstrate the utility of this method for studying class II gene transcription signals and protein factors.

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