The overall thrust of the proposed research is the utilization of chromatin spreading methods to ultrastructurally analyze various aspects of transcription, nascent transcript processing and genome replication in order to obtain information about regulation of genetic activity that cannot easily be gained from other approaches. Early emphasis will be placed on improvement of techniques using model systems for in situ hybridization of nucleic acids (amphibian rRNA genes, terminal repeats of Oxytricha) and immuno-detection of proteins (RNA polymerase II, Oxytricha telomin) at the ultrastructural level. Analyses of transcriptional patterns of specific eukaryotic and prokaryotic genes will be made using in situ hybridization (myosin heavy chain genes of Drosophila and quail) and location of transcription units on the E. coli chromosome relative to identifiable rrn operons. The effects of Drosophila and Xenopus rDNA spacer sequences and nascent transcript cleavages on termination of rDNA transcription will be examined using specially constructed termination vectors. The influence of nut site RNA sequences on lambda N gene-mediated antitermination will be studied by visualization of nascent lambda b2 transcript morphology. Sites of snRNP involvement in nascent transcript cleavages during early Drosophila development will be investigated using immunoelectron microscopy. Interactions between the processes of transcription and replication in prokaryotes will be studied using synchronously replicating E. coli cells and plasmids containing inducible replication origins adjacent to derepressible trp operons. Similar interactions will be studied in eukaryotes using synchronously replicating yeast cells, Drosophila embryos and Physarum cultures. Segregation of parental nucleosomes at replication forks during inhibition of protein synthesis will also be analyzed in yeast, Drosophila embryos and Physarum. Finally, overall transcriptional and replicational patterns of polytene chromosomes will be studied with respect to gene organization in band, interband and puff regions. Together, these studies should provide significant new insights into the regulation of genetic activity at the level of individual genes and the physical interactions between the processes of transcription and genome replication.
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