Induction of coiled body-like structures in Xenopus oocytes by U7 snRNA.

The coiled bodies, or "sphere organelles," of amphibian oocytes, first identified by their unique morphology, are large structures of up to 10 microm in diameter. There are 40 to 120 coiled bodies per oocyte nucleus. Most of the coiled bodies are in the nucleoplasm but a few are attached to specific chromosomal loci containing the histone gene repeats. Like the coiled bodies of somatic cells, they contain high concentrations of U7 snRNA, a small nuclear RNA required for 3' end formation of histone transcripts, and of a unique protein called coilin/SPH-1. We show here that increasing the nuclear concentration of U7 snRNA, by injection of either in vitro synthesized U7 snRNA or functional U7 snRNA genes, induces the formation of coiled body-like structures in vivo. In contrast, the formation of these structures is not induced when either a promoterless U7 snRNA gene construct is injected or when functional U7 snRNA genes are co-injected with alpha-amanitin. Increasing the concentration of U1 snRNA or histone mRNA does not induce the formation of these structures, indicating that the formation of these coiled body-like structures is specifically induced by the U7 snRNA. These results suggest that U7 snRNA may be a central nucleating factor of coiled bodies and that the appearance of coiled bodies at histone gene loci results from an increased local concentration of U7 snRNA near the nascent histone pre-mRNAs.

Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators.

The highest sensitivity nucleic acid gel stains developed to date are optimally excited using short-wavelength ultraviolet or visible light. This is a disadvantage for laboratories equipped only with 306- or 312-nm UV transilluminators. We have developed a new unsymmetrical cyanine dye that overcomes this problem. This new dye, SYBR Gold nucleic acid gel stain, has two fluorescence excitation maxima when bound to DNA, one centered at approximately 300 nm and one at approximately 495 nm. We found that when used with 300-nm transillumination and Polaroid black-and-white photography, SYBR Gold stain is more sensitive than ethidium bromide, SYBR Green I stain, and SYBR Green II stain for detecting double-stranded DNA, single-stranded DNA, and RNA. SYBR Gold stain's superior sensitivity is due to the high fluorescence quantum yield of the dye-nucleic acid complexes ( approximately 0.7), the dye's large fluorescence enhancement upon binding to nucleic acids ( approximately 1000-fold), and its capacity to more fully penetrate gels than do the SYBR Green gel stains. We found that SYBR Gold stain is as sensitive as silver staining for detecting DNA-with a single-step staining procedure. Finally, we found that staining nucleic acids with SYBR Gold stain does not interfere with subsequent molecular biology protocols.

Identification and characterization of a sphere organelle protein.

Sphere organelles are nuclear structures in amphibian oocytes that are easily visible by light microscopy. These structures are up to 10 microns in diameter and have been described morphologically for decades, yet their function remains obscure. The present study defines a protein component of the sphere organelle, named SPH-1, which is recognized by a mAb raised against purified Xenopus laevis oocyte nucleoplasm. SPH-1 is an 80-kD protein which is localized specifically to spheres and is undetectable elsewhere on lampbrush chromosomes or in nucleoli. We show using confocal microscopy that SPH-1 is localized to the cortex of sphere organelles. Furthermore, we have isolated a cDNA that can encode SPH-1. When epitope-tagged forms of SPH-1 are expressed in X. laevis oocytes the protein specifically localizes to spheres, demonstrating that the cloned cDNA encodes the sphere antigen. Comparison of the predicted amino acid sequence with sequence databases shows SPH-1 is related to p80-coilin, a protein associated with coiled bodies; coiled bodies are nuclear structures found in plant and animal cells. The sphere-specific mAb stains X. laevis tissue culture cells in a punctate nuclear pattern, showing that spheres or sphere antigens are present in somatic cells as well as germ cells and suggesting a general and essential function for spheres in all nuclei.