The Organization of Animal Cell Nuclei

Carnegie Institution Of Washington, D.C., Washington DC

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Abstract

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the functional organization of the animal cell nucleus. Specifically, we concentrate on two molecular processes that take place in the nucleus: the synthesis of RNA by the chromosomes (transcription) and the processing of this RNA into the mature messenger RNA (splicing). We study oocytes of Xenopus and other amphibians because of their exceptional size (~ 1 mm diameter) and the ease with which their giant nucleus (~ 0.4 mm diameter) can be isolated for molecular and structural analysis. We recently discovered that Xenopus oocytes store stable RNA sequences derived from the introns of most transcribed genes. This is an unexpected finding, because intronic sequences are generally destroyed within minutes after being spliced out of new transcripts. This stable intronic sequence (sis) RNA is transmitted to the embryo at fertilization and persists during early development until at least the blastula stage. For this reason we hypothesize the sisRNA may play a regulatory role during gene expression in the oocyte or early embryo. We will extend our studies to include the fly Drosophila, where we can apply sophisticated genetic techniques to better understand the function of sisRNA. We will also continue our studies on the giant lampbrush chromosomes found in oocytes of frogs and salamanders. These chromosomes are so large that one can visualize transcription and splicing on specific genes by conventional light microscopy. We recently developed techniques for imaging transcription units on these chromosomes by superresolution microscopy. We can now examine transcription units with resolution in the range of 50-100 nm, approximately 2-4 X better than achievable by confocal microscopy. We will use immunofluorescent staining and fluorescent in situ hybridization to examine details of RNA transcription and processing at the molecular level. These studies could reveal aspects of RNA transcription and processing that are missed by conventional in vitro molecular studies.