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J G Gall

Publications and source records attributed to J G Gall.

At least 55 records · Page 3Linked to original sources
PubMed1989

DNA-binding proteins on lampbrush chromosome loops.

When fixed newt lampbrush chromosomes are treated with RNase to remove nascent transcripts and are then probed with radiolabeled single-stranded DNA in 0.1 x SSC, proteins associated with the majority of the lateral loops bind the probe nonspecifically. One or more common hnRNP proteins, several of which are known to bind single-stranded DNA, could be responsible for this generalized binding. In 1.0 x SSC only a relatively small subset of loops continues to bind the probe. In order to characterize this subset of loops, we prepared polyclonal antibodies against DNA-binding proteins initially identified by "Southwestern" analysis. We show by an in situ double labeling experiment that a polyclonal serum raised against gel-eluted histone H1 recognizes the same lateral loops that bind DNA in 1.0 x SSC.

Animals↗
PubMed1989

The sphere organelle contains small nuclear ribonucleoproteins.

We show by immunofluorescence microscopy of amphibian oocyte nuclei that small nuclear ribonucleoproteins (snRNPs) occur in lampbrush chromosome loops, in a few dozen extrachromosomal organelles previously described as "spheres," and in thousands of smaller granules. Spheres are variable in size (up to approximately 20 microns in diameter in the newt Notophthalmus and approximately 10 microns in the frog Xenopus) and are easily distinguishable from nucleoli by morphology and composition. Spheres occur both free in the nucleoplasm and attached to specific chromosome loci, the sphere organizers. Oocyte nuclei of a cricket and a spider contain essentially similar organelles, suggesting that spheres may be common throughout the animal kingdom. We suggest that spheres play a role in the assembly of snRNP complexes for the nucleus comparable to the way that nucleoli assemble ribosomal RNP complexes for the cytoplasm.

Animals↗
PubMed1989

Targeting of a chromosomal protein to the nucleus and to lampbrush chromosome loops.

We have isolated a cDNA clone (SE5A) that encodes a protein on lampbrush chromosome loops of the newt Notophthalmus. In vitro-synthesized transcripts of this clone were injected into Xenopus oocytes, where they were efficiently translated. Most of the translated protein was imported into the oocyte nucleus, and some of it appeared on the chromosome loops. The translation product must contain information that permits its appropriate targeting first to the nucleus and then to the chromosome loops.

Amino Acid Sequence↗
PubMed1989

A novel heterogeneous nuclear RNP protein with a unique distribution on nascent transcripts.

Immediately after the initiation of transcription in eukaryotes, nascent RNA polymerase II transcripts are bound by nuclear proteins resulting in the formation of heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. hnRNP complexes from HeLa cell nuclei contain greater than 20 major proteins in the molecular mass range of 34,000-120,000 D. Among these are the previously described A, B, and C groups of proteins (34,000-43,000 D) and several larger, and as yet uncharacterized, proteins. Here we describe the isolation and characterization of a novel hnRNP protein termed the L protein (64-68 kD by mobility in SDS-polyacrylamide gels). Although L is a bona fide component of hnRNP complexes, it also appears to be a different type of hnRNP protein from those previously characterized. A considerable amount of L is found outside hnRNP complexes, and monoclonal antibodies to the L protein also strongly stain unidentified discrete nonnucleolar structures, in addition to nucleoplasm, in HeLa cell nuclei. Interestingly, the same antibodies stain the majority of nonnucleolar nascent transcripts from the loops of lampbrush chromosomes in the newt, but the most intense staining is localized to the landmark giant loops. The L protein is the first protein of giant loops identified so far, and antibodies to it thus provide a useful tool with which to study these unique RNAs. In addition, isolation and sequencing of cDNA clones for the L protein from human cells predicts a glycine- and proline-rich protein of 60,187 D, which contains two 80 amino acid segments only distantly related to the RNP consensus sequence-type RNA-binding domain. The L protein, therefore, is a new type of hnRNP protein.

Amino Acid Sequence↗
PubMed1987

Self-cleaving transcripts of satellite DNA from the newt.

Satellite 2 of the newt, Notophthalmus viridescens, is a 330 bp tandemly repeated sequence scattered throughout the genome. Cytoplasmic transcripts homologous to satellite 2 are found in a variety of tissues. Most of the transcripts correspond precisely in length to the DNA repeat unit or to whole multiples of that repeat. We show here that dimer-sized satellite 2 transcripts, synthesized with SP6 RNA polymerase from a plasmid clone, undergo site-specific, self-catalyzed cleavage in vitro. The reaction proceeds at neutral pH and requires Mg++ but no other cofactor or energy source. The cleavage products have 5'-hydroxyl and 3'-phosphate groups, at least some of which are in the form of 2',3'-cyclic phosphates. In this respect the reaction resembles the self-cleavage of certain small, infectious RNAs found in plants. Furthermore, the in vitro cleavage of satellite 2 transcripts occurs within a sequence that is homologous to the conserved cleavage site of the infectious RNAs. The existence of monomer and multimer transcripts in the cell suggests that the monomer may arise by site-specific cleavage of long primary transcripts. However, the 5' end of the cellular monomer is 46 or 47 bases upstream of the in vitro cleavage site, suggesting that factors in the cell may modify the cleavage reaction.

Animals↗
PubMed1987

The lampbrush chromosomes of Xenopus laevis: preparation, identification, and distribution of 5S DNA sequences.

Details are given of a technique for making permanent preparations of the lampbrush chromosomes of Xenopus laevis. Stained preparations allow all 18 bivalent chromosomes to be identified, and a working map showing the major features has been constructed. Fifteen of the Xenopus chromosomes have one telomere conspicuously larger than the other; the two smallest chromosomes, and one other, lack large telomeres. Similar preparations, extracted with RNase and denatured, have been hybridized in situ with a 3H-labelled 5S cRNA probe. Chromosomes can be identified in the resulting autoradiographs. 5S DNA sequences are present at all the larger telomeres and at three of the smaller ones, but are absent from the telomeres at both ends of the two smallest chromosomes. There are also five interstitial sites of hybridization. At one of these, label is on the chromosome axis; at the other four, label extends well away from the axis.

Animals↗
PubMed1987

Transcription of the histone loci on lampbrush chromosomes of the newt Notophthalmus viridescens.

We have investigated transcription of the histone gene cluster on lampbrush chromosomes of the newt Notophthalmus viridescens. Clusters of the five histone genes are separated by long tracts of a DNA repeat called satellite 1. Previous in situ hybridization results demonstrated the presence of histone gene coding regions, intergene spacers, and flanking satellite repeats in giant transcription units on lampbrush chromosomes. These results suggested a model in which transcription initiates at histone gene promoters and continues through the rest of the gene cluster into satellite repeats. The readthrough transcription model predicts that spacer regions upstream of the first promoter in the gene cluster should be absent from nascent transcripts on chromosome loops. We have used such upstream spacer probes for in situ hybridization to lampbrush chromosomes. Contrary to our expectation, the results show that upstream spacer regions are transcribed, and indicate that transcription often initiates upstream of the first histone gene promoter. The relationship of giant transcription units in oocyte nuclei to cytoplasmic histone mRNA is discussed.

Animals↗
PubMed1987

Monoclonal antibodies that recognize transcription unit proteins on newt lampbrush chromosomes.

We prepared hybridoma cell lines from mice injected with newt germinal vesicle proteins. We tested culture supernates from these cell lines for antibodies that bound to specific morphological structures in lampbrush chromosome preparations (nucleoli, loops, chromomeres, etc.). Four mAbs that recognize antigens on the lateral transcription loops are described here. We suggest that these antigens are proteins associated with nascent RNA transcripts, although they are not among the 30-40-kD "core" heterogeneous nuclear ribonucleoproteins.

Animals↗
PubMed1986

A transcribed satellite DNA from the bullfrog Rana catesbeiana.

We have cloned and sequenced a 360 bp repeated sequence from genomic DNA of the bullfrog Rana catesbeiana. This sequence, which we call satellite 1, makes up 0.7% of the genome (1.6 X 10(5) repeats) and is scattered throughout the length of all the chromosomes. In situ hybridization with strand-specific probes demonstrated that transcripts from both strands of satellite 1 occur on lamp-brush chromosome loops. We suggest that these transcripts arise by readthrough from upstream structural gene promoters. Sequences that cross-react with satellite 1 were found in genomic DNA of four out of five other Rana species that we tested, but were absent from the DNA of Xenopus, Bombina, and Acris.

Animals↗
PubMed1986

Microinjected Tetrahymena rDNA ends are not recognized as telomeres in Xenopus eggs.

Telomeres are essential structures that stabilize the ends of eukaryotic chromosomes and allow complete replication of linear DNA molecules. We examined the structure and replication of telomeres by observing the fate of the linear extrachromosomal rDNA of Tetrahymena after injection into unfertilized Xenopus eggs. The rDNA replicated efficiently as a linear extrachromosomal molecule, increasing in mass 30-50-fold by 15-20 h after injection. In addition, the molecules increased in length by addition of up to several kilobases of DNA to their termini. Sequence analysis demonstrated that the added DNA bore no resemblance to known telomeres. The junction between the rDNA and added DNA was apparently random, indicating that the addition reaction did not involve a site-specific recombination or integration event. Surprisingly, Southern blot analysis showed that the added DNA did not derive from Xenopus DNA, but rather from co-purifying and therefore co-injected Tetrahymena DNA. The nonspecific ligation of random DNA fragments to the rDNA termini suggests that microinjected Tetrahymena rDNA ends are not recognized as telomeres in Xenopus eggs.

Animals↗
PubMed1986

Transcription of a satellite DNA in the newt.

Satellite 2 is an abundant, 330-bp tandemly repeated sequence in the genome of the newt, Notophthalmus viridescens. This sequence is distributed throughout the genome on each of the 11 chromosomes. Both strands of satellite 2 are transcribed on the lampbrush chromosomes during oogenesis, probably as a result of readthrough from upstream structural gene promoters. In addition to these heterogeneous nuclear transcripts, satellite 2 is homologous to stable, strand-specific cytoplasmic transcripts in a variety of different tissues. The majority of these transcripts correspond in size to the entire satellite 2 repeat unit, or to whole multiples of the repeat. The transcripts present in the ovary have been sequenced by primer extension and were found to be more homogeneous than eight independently cloned satellite 2 DNA repeats. We propose that the stable cytoplasmic transcripts are encoded by a small subset of genomic satellite 2 sequences.

Animals↗
PubMed1985

Giant readthrough transcription units at the histone loci on lampbrush chromosomes of the newt Notophthalmus.

We have studied transcription at the histone loci in oocytes of the newt Notophthalmus viridescens, using in situ hybridization of cloned probes to the nascent RNA on lampbrush chromosome loops. Clusters of the five histone coding sequences are separated by long tracts of a simple sequence DNA, satellite 1. We had previously demonstrated coordinate transcription of histone genes and satellite 1 sequences. We postulated that satellite sequences were transcribed by readthrough from histone gene promoters; that is, transcription initiated at any of the five usual promoters, but did not terminate at the 3' end of the gene. Instead transcription proceeded through downstream sequences in the histone cluster (including spacers and downstream histone genes), and then through the satellite 1 region. Our model led to several specific predictions, in particular that some internal spacer regions between the genes should be well represented in the RNA on loops, that certain sequences should be absent from the loops, and that presence or absence of particular sequences should be correlated with morphological polarity of the transcription unit. We have hybridized ten strand-specific probes to the lamp-brush chromosomes and we find that the patterns of hybridization agree with the readthrough model of transcription.

Animals↗
PubMed1984

Histone RNA in amphibian oocytes visualized by in situ hybridization to methacrylate-embedded tissue sections.

We present an in situ hybridization method for detecting cellular RNAs in tissue sections using methacrylate as the embedding medium. The technique offers the advantage of superior morphological preservation compared with previously published procedures. Since sections can be cut 1 micron or less in thickness, full advantage is taken of the short path length of 3H electrons. Applying this procedure to developing amphibian oocytes, we investigated the accumulation and localization of RNA complementary to the histone genes and their adjacent spacers. Histone RNA begins to accumulate in the cytoplasm of late pachytene-early diplotene oocytes, rapidly reaching a maximum concentration during Dumont stage 1. After this stage the concentration of histone RNA declines. RNA transcribed from histone coding regions is located almost exclusively in the cytoplasm of oocytes. Transcripts of the spacer regions, which are known to be synthesized on oocyte lampbrush chromosomes, do not accumulate in the oocytes. [3H]RNA complementary to U2 small nuclear RNA, used in these experiments as a control, hybridized predominantly to the nucleus of the oocytes.

Animals↗
PubMed1983

Transcription of repetitive sequences on Xenopus lampbrush chromosomes.

We reinvestigated the lampbrush chromosomes of Xenopus laevis and found them well suited for the study or transcription by in situ hybridization to nascent RNA transcripts. Using this technique, we analyzed the transcription of three repetitive sequences that do not show any sequence homology and that differ in their degree of interspersion. We found that they are located on different parts of the chromosomes: two are clustered, one is interspersed. All three of these sequences are transcribed at the lampbrush chromosome stage and transcripts from both strands of each sequence can be detected. The amount of transcription is apparently not proportional to the number of copies of the repetitive sequence at a given chromosomal locus, suggesting that other sequences are involved in the regulation of their transcription.

Animals↗
PubMed1982

The nucleotide sequence at the transcription termination site of the ribosomal RNA gene in Tetrahymena thermophila.

The sequence of 415 nucleotides surrounding the transcription termination site for ribosomal RNA in Tetrahymena thermophila has been determined. The positions of the 3'-ends of mature 26S rRNA, pre-26S rRNA and 35S pre-rRNA were localized within this sequence by hybridization of the purified RNA species to be selected DNA fragments, followed by S1 nuclease treatment of the hybrid and a precise sizing of the RNA-protected DNA fragments on sequencing gels. The 35S pre-rRNA population contained molecules with two distinct 3'-ends, one of which is identical to the end of pre-26S and 26S rRNA, while the other corresponds to a position 15 nucleotides further downstream, which is assumed to be the transcription termination site. The non-coding DNA strand contains a cluster of T's at the putative termination site, and several other T clusters are found further downstream. A short inverted repeat sequence is located near the putative termination site within the transcribed region. The possible role of these structures for transcription termination is discussed.

Animals↗