Joseph G. Gall - US grants

PROJECT SUMMARY The long-term aim of our research is to understand the functional organization of the animal cell nucleus. Specifically, we will focus on the non-chromosomal components of the nucleus, collectively referred to as nuclear bodies. Our primary experimental material will be Drosophila, which permits unprecedented genetic, molecular, and developmental analysis. We will also study Xenopus oocytes, where the enormous size of the nucleus and its nuclear bodies make it an ideal system for molecular experiments. Finally, we will examine the biogenesis of an unusual nuclear body in oocytes of the mouse. We will concentrate on four nuclear bodies: the Cajal body (CB), the histone locus body (HLB), speckles (interchromatin granule clusters) and the nucleolus. The function of the nucleolus in ribosomal RNA transcription and ribosome biogenesis is well understood, and there is increasing evidence that components of the pre-mRNA and pre-ribosomal RNA processing machinery are assembled or modified in CBs and HLBs. Some or all of the splicing machinery is stored in speckles before transfer to active transcription sites on the chromosomes. In Drosophila we will concentrate on the CB and its signature protein coilin. We recently identified Drosophila coilin and produced null mutants for coil, the gene that encodes coilin. CBs are not detectable in most cells of the mutants, permitting us to study molecular events in cells that lack both coilin and defined CBs. Specifically, we will examine splicing snRNAs, whose post-transcriptional modifications are thought to occur normally in CBs. We will carry out microarray experiments on ovary and testis RNA to determine which genes are up or down regulated in the absence of coilin. We will also examine the composition and behavior of previously unknown nuclear bodies that are inducible in the Drosophila oocyte under experimental conditions. In Xenopus we will follow the biogenesis of CBs and HLBs throughout the entire period of oogenesis to determine the relationship between these two similar nuclear bodies. We will also examine the prominent population of speckles in the oocyte nucleus to determine whether they associate with specific sites on the giant lampbrush chromosomes, and if so, whether the molecular sequences at these sites can be identified using information from the Xenopus tropicalis Genome Project. Finally, we will examine the ultrastructure and molecular composition of the unusual nucleolus-like bodies that occur in late-stage mouse oocytes. These studies on nuclear bodies in three diverse systems will provide insight into the structure and function of the major nuclear organelles and how they interact during transcription and processing of nuclear RNA.

This application is for the purchase of a laser scanning confocal microscope for the Department of Embryology, Carnegie Institution, Baltimore, MD. The confocal microscope is a new form of light optical microscope whose chief advantage is that it permits one to focus on a single plane in a thick object, without interference from out-of-focus parts of the specimen. Used in the epifluorescence mode, the confocal microscope allows one to resolve unprecedented detail in living cells, including single microtubules in the mitotic spindle, elements of the Golgi apparatus and endoplasmic reticulum, and individual bands on the polytene chromosomes of Drosophila. Our group proposes four uses for a confocal microscope. Gall will study the organization of giant chromosomes in the Amphibian oocyte nucleus, including the import and export of specific chromosomal proteins from the nucleus. Pagano will concentrate on the intracellular translocation and metabolism of lipids in living cells. Fire will study myogenesis in the free-living nematode Caenorhabditis using animals transformed with injected genes. Schwartz will examine individual DNA molecules immobilized in agarose gels. Each study involves fluorescent probes, but conventional fluorescence microscopy is difficult with the relatively thick specimens involved (whole cells, whole nuclei, whole animals). The confocal microscope is ideally suited for examining such thick specimens. Used in the epifluorescence mode, the confocal microscope will provide extraordinary spatial localization of molecular probes, permitting novel insight into biological processes.

The proposed studies deal with nuclear proteins and their assembly into macromolecular complexes with RNA on the chromosomes and in other nuclear organelles. Advantage will be taken of the giant nucleus and giant lampbrush chromosomes (LBCs) of the amphibian oocyte. Monoclonal antibodies will be produced against nuclear proteins, whose subnuclear distribution will be determined by immunofluorescence. The extraordinary morphological detail in LBCs allows antigen detection within single transcription units or in defined subnuclear particulates. The antibodies will be used to recover cDNA clones from expression libraries. Protein sequences derived from the cDNA clones will permit deductions about the role(s) of the proteins in nuclear function. In vitro transcriptions made from the cDNA clones will be injected into amphibian oocytes, and the translation products (polypeptides) will be followed biochemically and cytologically from their site of synthesis in the cytoplasm through the nuclear envelope to their final destination on the chromosomes or elsewhere in the nucleus. By appropriate in vitro alteration of the injected transcripts we will determine what amino acid sequences are necessary for transport across the nuclear envelope and for targeting to the final subnuclear destination. Studies will also be conducted on the transcription of the 5S gene sequences on LBCs by in situ nucleic acid hybridization. Finally studies will be carried out on a novel selfcleaving RNA transcribed from a short, repeated sequence of newt DNA, with emphasis on the possible existence of a ribonucleoprotein particle. All of the proposed studies deal with how RNA produced by the chromosomes becomes associated with nuclear proteins in order to carry out basic cellular functions (RNA processing, storage, and transport). Knowledge of such functions is fundamental for understanding cell metabolism in both the normal and diseased state.

The proposed studies deal with the small nuclear ribonucleoproteins (snRNPs) in animal cell nuclei, particularly their organization into macromolecular complexes and larger aggregates visible by conventional light microscopy. snRNPs are an essential part of the nuclear machinery that prepares RNA molecules for export from the nucleus to the cytoplasm - the conversion of pre-messenger (pre-mRNA) molecules to the functional messengers (mRNA). A great deal is known about the biochemical reactions in which snRNPs are involved, primarily the process of splicing, which involves the removal of specific intron sequences from the pre-mRNA and the accurate joining of the remaining pieces. However, the distribution of snRNPs within the nucleus is not well understood. Studies on the giant nuclei of amphibian oocytes show that snRNPs are associated with the pre- mRNA molecules on the chromosomes, as one would expect, but they are also abundant in thousands of nuclear bodies known as snurposomes. The snurposomes may be sites for assembly of snRNPs pinto macromolecular complexes that are then transported to the chromosomes, where they actually function. The proposed studies will characterize the snurposomes of insect oocytes using immunofluorescence to identify the protein components and in situ hybridization to recognize specific snRNA molecules. Based on composition and morphology, several different classes of snurposomes are recognizable in amphibian oocytes, and similar heterogeneity is anticipated in insects. The RNA processing steps in which snRNPs are involved concern the most general and important biochemical reactions within cells. Not only is correct RNA processing essential to normal cell function, but the same biochemical machinery is used by viruses when they infect cells. Thus, knowledge about snRNPs in various cell types has important implications for both normal and diseased cells.