The objective of the proposed research is to ultrastructurally characterize basic eukaryotic genetic processes: Chromatin transcription, chromatin replication, nascent transcript processing, and mRNA storage and translation. As our major approach we will use a chromatin spreading technique that allows visualization of dispersed nuclear contents in the electron microscope. Biochemical approaches, such as subcellular fractionation, analytical electrophoresis, and recombinant DNA technology, will supplement our investigation.
Our first aim i s to analyze the genetic activity of specific genes. We are investigating the heat shock transcription units of Drosophila, the late transcription units of Adenovirus 2 in infected Hela cells, the expression and structure of Xenopus 5S gene-contining plasmids when incubated in Xenopus nuclei vs a Xenopus nuclear extract, and the expresion of the Herpes virus thymidine kinase gene when incorporated into a recombinant plasmid and introduced int somatic cells. Secondly, we hope to characterize the ribonucleoprotein structure of pre-messenger and messenger RNA. Specifically, we will investigate the RNP ultrastructure and composition of ascent hnRNA transcripts in Adenovirus infected Hela cells, and we will compare the protein composition of sea urichin egg mRNA to embryo mRNA with the aim of characterizing how these molecules are stored for use after fertilization. Thirdly, using both Drosophila embryos and Adenovirus genomes, we will attempt to visualize chromatin that is engaged simultaneously in replication and transcription and to analyze the result of the physical interaction of these two processes. We hope to gain new information on the transcription unit structure and activity of defined genes, the nuclear packaging and processing of hnRNA, the cytoplasmic storage and utilization of mRNA in early development, and the interaction beween transcription and replication.
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