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J G Gall

Publications and source records attributed to J G Gall.

At least 37 records · Page 2Linked to original sources
PubMed1994

Human p80-coilin is targeted to sphere organelles in the amphibian germinal vesicle.

Cultured vertebrate cells often display one or more coiled bodies in their nuclei. These are spherical structures approximately 0.5-1.0 micron in diameter that contain high concentrations of small nuclear ribonucleoproteins (snRNPs); they are distinct from nuclear speckles and nucleoli, the other major sites of snRNP concentration. Coiled bodies in human cells contain a unique protein, p80-coilin, that has an M(r) = 80 kDa. Cloned p80-coilin cDNA encodes 576 amino acids with a calculated molecular weight of 62.6 kDa. To determine which of several snRNP-containing structures in the amphibian germinal vesicle (GV) might be the homologue of coiled bodies, we injected myc-tagged transcripts of full-length human p80-coilin into the cytoplasm of Xenopus oocytes and followed the fate of the translated proteins with an antibody specific for the tag. Western blots of GV proteins showed rapid appearance of both full-length and truncated p80-coilin in the nucleus. Immunofluorescent staining of spread GV contents demonstrated specific uptake of p80-coilin by the sphere organelle within 1 h after injection. Similar experiments were performed with a series of deletion constructs that lacked progressively longer segments from the carboxy terminus. A construct that contained only the first 102 amino acids (18% of the molecule) was specifically targeted to the sphere organelle. Conversely, a construct lacking the first 92 amino acids failed to localize, although it was imported into the GV. Thus, a relatively short region at the amino terminus of human p80-coilin is both necessary and sufficient for localization in the sphere organelle. Sphere organelles in the GV and coiled bodies in somatic nuclei are clearly related in composition. We suggest that they are homologous organelles with similar functions in preassembly and sorting of RNA processing components. Differences in their composition suggest functional specialization in the two cell types.

Amino Acid Sequence↗
PubMed1994

In vitro assembly of coiled bodies in Xenopus egg extract.

When demembranated sperm nuclei are placed in a Xenopus egg extract, they become surrounded by a nuclear envelope and then swell to form morphologically typical pronuclei. Granules ranging from < 1.0 to approximately 3.0 microns in diameter appear within such nuclei. Bell et al. identified four nucleolar proteins in these "prenucleolar bodies" by immunofluorescent staining (fibrillarin, nucleolin, B23/NO38, 180-kDa nucleolar protein). By in situ hybridization we show that these bodies also contain U3 and U8 small nuclear RNAs (snRNAs), known to be involved in pre-rRNA processing. Moreover, they contain all the snRNAs involved in pre-mRNA splicing (U1, U2, U4, U5, and U6), as well as U7, which is required for histone pre-mRNA 3' end formation. In addition to the nucleolar antigens previously identified, we demonstrated staining with antibodies against the Sm epitope, trimethylguanosine, and coilin. Because the composition of these prenucleolar bodies is closer to that of coiled bodies than to nucleoli, we propose that they be referred to as coiled bodies. The existence of large coiled bodies in transcriptionally inactive pronuclei suggests that they may play a role in the import, assembly, and storage of RNA processing components but are not themselves sites of processing. In transcriptionally active nuclei coiled bodies could serve as sites for initial preassembly and distribution of snRNP complexes for the three major RNA processing pathways: pre-mRNA splicing, pre-rRNA processing, and histone pre-mRNA 3' end formation.

Amino Acid Sequence↗
PubMed1993

U7 small nuclear RNA in C snurposomes of the Xenopus germinal vesicle.

In the amphibian germinal vesicle small nuclear RNAs (snRNAs) occur in morphologically distinct structures called snurposomes. Three types (A, B, and C) have been distinguished on the basis of cytological appearance and snRNA composition. C snurposomes in Xenopus are spherical bodies ranging in diameter from < 1 microns to about 10 microns. They stain intensely with antibodies against trimethylguanosine and the small nuclear ribonucleoprotein-specific Sm antigen but give weak or negative in situ hybridization reactions for the snRNAs involved in pre-mRNA splicing (U1, U2, U4, U5, and U6). We show here that C snurposomes in the Xenopus germinal vesicle contain U7 snRNA, an snRNA of low abundance involved in processing the 3' end of histone pre-mRNA. Xenopus U7 is 58 nucleotides long and is capped at the 5' end with trimethylguanosine. C snurposomes are often associated with B snurposomes, which contain the splicing snRNAs but not U7; B and C snurposomes together constitute a morphologically complex structure known as a sphere or sphere organelle. Although most spheres and C snurposomes are extrachromosomal, a few are attached at the histone gene loci on chromosomes 8, 9, and 16. Because they contain U7 snRNA and occur at the sites of histone pre-mRNA synthesis, C snurposomes presumably play a role in processing histone transcripts.

Animals↗
PubMed1993

A putative zinc-binding protein on lampbrush chromosome loops.

We used mAb A33/22, which recognizes a nuclear protein on the loops of amphibian lampbrush chromosomes, to select cDNA clone PwA33 from an expression library of the newt Pleurodeles waltl. A myc-tagged transcript of clone PwA33 was injected into Pleurodeles oocytes. The translation product localized in the germinal vesicle (GV) and was distributed on the lampbrush loops in a pattern identical to that of the endogenous protein. PwA33 encodes a 71 kDa protein with three distinct domains: a region rich in Cys/His residues that may form zinc fingers, a coiled-coil domain with potential for dimerization and a third 'rfp-like' domain that is shared by several other nuclear proteins. The putative zinc fingers and the coiled-coil domain resemble features in known nucleic acid-binding regulatory proteins. These structures, coupled with a distinctive pattern of expression in embryonic tissues, suggest that A33 may function as a regulatory protein during early development. It is unlikely that the large store of A33 in the GV is bound to DNA. Instead, its association with the nascent transcripts on the lampbrush chromosome loops suggests a role in pre-mRNA synthesis or processing.

Amino Acid Sequence↗
PubMed1993

Snurposomes and coiled bodies.

In the GV of amphibian oocytes, the splicing snRNPs (U1, U2, U4, U5, and U6) occur on most of the lampbrush chromosome loops in association with the nascent transcripts. They also occur in thousands of small extrachromosomal bodies (1-4 microns in diameter) designated B snurposomes. U7 snRNA, which is involved in processing the 3' end of histone pre-mRNAs, occurs in a few dozen extrachromosomal bodies (1-20 microns in diameter) called C snurposomes. C snurposomes often have B snurposomes attached to their surface and B-like inclusions; these compound structures are known as spheres or sphere organelles. One or two sphere organelles are attached to the lampbrush chromosomes at the histone gene loci. Two snRNAs (or snoRNAs) known to be involved in pre-rRNA processing (U3 and U8) occur in the 1000 or so extrachromosomal nucleoli of the GV. We looked for a snurposome that might contain U3 and U8 but not rDNA or rRNA. We were unable to find such a snurposome, but we did identify a hitherto unrecognized population of minute nucleoli in the size range of B snurposomes. Prenucleolar bodies in telophase/early interphase nuclei meet the definition of a pre-rRNA snurposome in that they contain U3 snoRNA and fibrillarin (and probably other processing components) but lack rDNA and do not synthesize rRNA. The structures previously identified as prenucleolar bodies in pronuclei formed in vitro in Xenopus egg extracts share many components with coiled bodies from HeLa nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

A conserved double-stranded RNA-binding domain.

We have identified a double-stranded (ds)RNA-binding domain in each of two proteins: the product of the Drosophila gene staufen, which is required for the localization of maternal mRNAs, and a protein of unknown function, Xlrbpa, from Xenopus. The amino acid sequences of the binding domains are similar to each other and to additional domains in each protein. Database searches identified similar domains in several other proteins known or thought to bind dsRNA, including human dsRNA-activated inhibitor (DAI), human trans-activating region (TAR)-binding protein, and Escherichia coli RNase III. By analyzing in detail one domain in staufen and one in Xlrbpa, we delimited the minimal region that binds dsRNA. On the basis of the binding studies and computer analysis, we have derived a consensus sequence that defines a 65- to 68-amino acid dsRNA-binding domain.

Amino Acid Sequence↗
PubMed1992

5S rRNA-encoding genes of the marsupial frog Gastrotheca riobambae.

The major 5S rRNA gene repeat of the marsupial frog, Gastrotheca riobambae, is 1052 bp in length. It contains a 5S rRNA gene similar to the Xenopus laevis somatic gene, two spacer regions, and a pseudogene. The G. riobambae haploid genome contains about 500 copies of this predominant repeat. This relatively low number of 5S rRNA genes is associated with a limited amplification of the 18S, 5.8S and 28S rRNA genes in oocytes and with a slow rate of early development.

Animals↗
PubMed1992

Localization of the nucleolar protein NO38 in amphibian oocytes.

To examine the role of primary amino acid sequence in the localization of proteins within the nucleus, we studied the nucleolar protein NO38 of amphibian oocytes. We synthesized NO38 transcripts in vitro, injected them into the oocyte cytoplasm, and followed the distribution of the translation products. The injected RNA contained a short sequence encoding an epitope derived from the human c-myc protein. We used an mAb against this epitope to detect translation products from injected RNAs by Western blots and by immunofluoresent staining of cytological preparations. When full-length transcripts of NO38 were injected into oocytes, the translation products accumulated efficiently in the germinal vesicle, and a major fraction was localized in the multiple nucleoli. To identify protein domains involved in this nucleolus-specific accumulation, we prepared a series of carboxy-terminal deletions of the cDNA. Oocytes injected with RNA encoding truncated forms of NO38 were examined for altered patterns of protein accumulation. We defined a domain of about 24 amino acids near the carboxy terminus that was essential for nucleolar localization of NO38. This domain is separated by more than 70 amino acids from two putative nuclear localization signals near the middle of the molecule. Hybrid constructs were made which encoded part of Escherichia coli beta-galactosidase or pyruvate kinase fused to a long segment of NO38 containing the essential domain. Injection of RNA from these constructs showed that the essential domain was not sufficient to target the hybrid proteins to the nucleolus. We suggest that nucleolar accumulation of NO38 requires more than a single linear domain.

Amino Acid Sequence↗
PubMed1992

Assembly and localization of the U1-specific snRNP C protein in the amphibian oocyte.

To study the intranuclear localization of the U1-specific snRNP C protein and its assembly into U1 snRNPs, we injected transcripts encoding a myc-tagged C protein into amphibian oocytes. The distribution of protein translated from the injected RNA was essentially the same in continuous and pulse-label experiments. In both cases the C protein localized within the germinal vesicle in those structures known to contain U1 snRNPs, namely the lampbrush chromosome loops and hundreds of extrachromosomal granules called snurposomes. Oocytes were also injected with an antisense oligodeoxynucleotide that caused truncation of U1 snRNA at the 5' end. In these oocytes, myc-tagged C protein localized normally in the germinal vesicle and could be immunoprecipitated together with truncated U1 snRNA. These experiments suggest that the C protein can enter the germinal vesicle on its own and there associate with previously assembled U1 snRNPs. In transfected tissue culture cells, the myc-tagged C protein localized within the nucleus in a speckled pattern similar to that of endogenous U1 snRNPs.

Amino Acid Sequence↗
PubMed1992

Transcription on lampbrush chromosome loops in the absence of U2 snRNA.

The five small nuclear RNAs (snRNAs) involved in splicing occur on the loops of amphibian lampbrush chromosomes and in hundreds to thousands of extrachromosomal granules called B snurposomes. To assess the role of these snRNAs during transcription and to explore possible relationships between the loops and B snurposomes, we injected single-stranded antisense oligodeoxynucleotides (oligos) against U1 and U2 snRNA into toad and newt oocytes. As shown before, antisense U1 and U2 oligos caused truncation of U1 and complete destruction of U2 snRNAs, respectively. However, injection of any oligo, regardless of sequence, brought on dramatic cytological changes, including shortening of the chromosomes and retraction of the lateral loops, with concomitant shutdown of polymerase II transcription, as well as disappearance of some or all of the B snurposomes. When injected oocytes were incubated for 12 h or longer in physiological saline, these changes were reversible; that is, the chromosomes lengthened, transcription (detected by 3H-UTP incorporation) resumed on newly extended lateral loops, and B snurposomes reappeared. In situ hybridization showed that loops and B snurposomes had negligible amounts of U2 snRNA after recovery from injection of the anti-U2 oligo, whereas these structures had normal levels of U2 snRNA after recovery from a control oligo. Thus, the morphological integrity of B snurposomes and lampbrush chromosome loops is not dependent on the presence of U2 snRNA. Because transcription occurs in the absence of U2 snRNA, we conclude that splicing is not required for transcription on lampbrush chromosome loops.

Animals↗
PubMed1991

Association of RNA with the B and C snurposomes of Xenopus oocyte nuclei.

We studied the time course of [3H]-uridine incorporation into the B and C snurposomes of Xenopus oocyte nuclei. B snurposomes constitute most of the non-nucleolar granules in the 1-4 micron size range; they contain the five splicing small nuclear RNAs (snRNAs; U1, U2, U4, U5 and U6) plus a variety of associated proteins. The organelles referred to as spheres consist of a C snurposome with one or more B snurposomes on its surface. C snurposomes can exist independently of Bs and many are smaller than the structures usually classified as spheres. C snurposomes contain the trimethylguanosine moiety characteristic of snRNAs, as well as the Sm epitope found on several small nuclear ribonucleoproteins (snRNPs), but it is not known which snRNA(s) they contain. When oocytes are incubated with [3H]uridine, all of the nucleoli and chromosome loops label strongly and rapidly. By contrast, labelled RNA appears slowly in the B snurposomes and then only in a fraction of them. After a 24 h incubation, about half of the Bs are labelled, and half are unlabelled or weakly labelled. This observation suggests that there are "mature" and "immature" B snurposomes, and that only the latter acquire newly synthesized RNA. The nature of this RNA is unknown, but it probably includes the splicing snRNAs. B snurposomes on the surface of Cs also constitute a heterogeneous population, some becoming labelled and some remaining unlabelled during a 24 h incubation. An analysis of the label in "doublets" (one B and one C snurposome) suggests that RNA may pass from the Bs to the Cs.

Animals↗
PubMed1991

Histone genes are located at the sphere loci of Xenopus lampbrush chromosomes.

In the anuran Xenopus, as has been demonstrated previously in several species of urodele Amphibia, histone genes lie at the sphere organizer loci of the lampbrush chromosomes. They were located by in situ hybridization of a 3H-labelled histone H4 anti-sense cRNA probe applied to lampbrush preparations in which transcript RNA had been retained, and likewise to preparations in which transcripts were absent but whose DNA had been denatured prior to hybridization. In Xenopus the histone genes lie in intimate association with the spheres that are attached to the lampbrush chromosomes, but they are absent from spheres that lie free in the germinal vesicle. The Anura separated from the Urodela several hundred million years ago, so the sphere organizer/histone gene association is of great antiquity. This suggests that the association has a functional significance, though it is one that has yet to be discovered.

Animals↗
PubMed1991

Small nuclear ribonucleoproteins and heterogeneous nuclear ribonucleoproteins in the amphibian germinal vesicle: loops, spheres, and snurposomes.

We have examined the distribution of snRNPs in the germinal vesicle (GV) of frogs and salamanders by immunofluorescent staining and in situ nucleic acid hybridization. The major snRNAs involved in pre-mRNA splicing (U1, U2, U4, U5, and U6) occur together in nearly all loops of the lampbrush chromosomes, and in hundreds to thousands of small granules (1-4 microns diameter) suspended in the nucleoplasm. The loops and granules also contain several antigens that are regularly associated with snRNAs or spliceosomes (the Sm antigen, U1- and U2-specific antigens, and the splicing factor SC35). A second type of granule, often distinguishable by morphology, contains only U1 snRNA and associated antigens. We propose the term "snurposome" to describe the granules that contain snRNPs ("snurps"). Those that contain only U1 snRNA are A snurposomes, whereas those that contain all the splicing snRNAs are B snurposomes. GVs contain a third type of snRNP granule, which we call the C snurposome. C snurposomes range in size from less than 1 micron to giant structures greater than 20 microns in diameter. Usually, although not invariably, they have B snurposomes on their surface. They may also contain from one to hundreds of inclusions. Because of their remarkably spherical shape, C snurposomes with their associated B snurposomes have long been referred to as spheres or sphere organelles. Most spheres are free in the nucleoplasm, but a few are attached to chromosomes at specific chromosome loci, the sphere organizers (SOs). The relationship of sphere organelles to other snRNP-containing structures in the GV is obscure. We show by immunofluorescent staining that the lampbrush loops and B snurposomes also react with antibodies against heterogeneous nuclear ribonucleoproteins (hnRNPs). Transcription units on the loops are uniformly stained by anti-hnRNP and anti-snRNP antibodies, suggesting that nascent transcripts are associated with hnRNPs and snRNPs along their entire length, perhaps in the form of a unitary hnRNP/snRNP particle. That B snurposomes contain so many components involved in pre-mRNA packaging and processing suggests that they may serve as sites for assembly and storage of hnRNP/snRNP complexes destined for transport to the nascent transcripts on the lampbrush chromosome loops.

Animals↗
PubMed1990

Nucleolin from the multiple nucleoli of amphibian oocyte nuclei.

When fixed preparations of newt germinal vesicle (GV) contents are treated with RNase and are then probed with radiolabeled single-stranded DNA in 0.1-2.0 X SSC, the extrachromosomal nucleoli bind the probe non-specifically. DNA/protein blot analysis of proteins from newt GVs shows that gv95, an acidic protein (pI = 5.0) of Mr = 95,000, is the most prominent non-specific DNA-binding protein. Immunocytochemical analysis with affinity purified antibody directed against gv95 shows that it is located in the multiple nucleoli. We used an antibody directed against rat nucleolin to show that newt gv95 and two similar Xenopus GV proteins are the amphibian versions of nucleolin, a nucleolar ribonucleoprotein originally identified in mammalian cells. We show that mAb 3A10, directed against newt histones H1 and H5, labels gv95 on protein immunoblots and the multiple nucleoli in cytological preparations. These results suggest that histone H1 and nucleolin share a cross-reacting epitope.

Amphibians↗
PubMed1990

A monoclonal antibody that recognizes a phosphorylated epitope stains lampbrush chromosome loops and small granules in the amphibian germinal vesicle.

An mAb library was produced against proteins from the germinal vesicle (GV) of the frog Xenopus laevis; mAb 104 was selected from this library on the basis of its immunofluorescent staining of lampbrush chromosome loops. Chromosomes from several species of frogs and salamanders stained equally well. The antibody also stained the surface of numerous small granules in the GV nucleoplasm. The interior of the same granules was stained by antibodies against small nuclear ribonucleoproteins (snRNPs). mAb 104 also stained somatic nuclei from many vertebrate and invertebrate species, usually in a finely punctate pattern similar to that described for anti-snRNP and other antinuclear antibodies. The staining of somatic nuclei was much stronger during the mitotic stages than during interphase. Immunoblot analysis showed that mAb 104 recognizes a phosphorylated epitope.

Animals↗