NUCLEOLAR-DERIVED RIBONUCLEIC ACID IN CHROMOSOMES, NUCLEAR SAP, AND CYTOPLASM OF <i>CHIRONOMUS TENTANS</i> SALIVARY GLAND CELLS

Ringborg, Ulrik; Rydlander, L.

doi:10.1083/jcb.51.2.355

Cited by 104 publications

(30 citation statements)

References 23 publications

(19 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1B and 2B). Particularly during in vivo conditions, (pre)ribosomal RNA can also be recorded on the chromosomes (11). In the present in vitro experiments ( Fig.…”

Section: Methodsmentioning

confidence: 49%

See 1 more Smart Citation

Evidence for Transport of 75S RNA from a Discrete Chromosome Region via Nuclear Sap to Cytoplasm in Chironomus tentans

Daneholt

Hosick

1973

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Salivary glands of the dipteran Chironomus tentans were exposed to tritiated pyrimidines for different time periods either in vitro or in vivo. Nonribosomal, high molecular weight RNA molecules of very different sizes (15-100 S) were labeled on chromosomes I-III, while only one main species, 75S RNA, was recorded in Balbiani ring 2, a giant chromosome puff on chromosome IV. 75S RNA was present as a prominent fraction in nuclear sap, but not in cytoplasm, after 90 min of incubation in vitro. During the ensuing 90 min, 75S RNA also appeared in cytoplasm. After 1 week of labeling in vivo, radioactive 75S RNA accumulated heavily in cytoplasm. Absorbance measurements showed that 75S RNA constituted as much as 1.5% of the total salivary-gland RNA. The half-life of 75S RNA was estimated to exceed 35 hr.Available data on the intracellular distribution and metabolic stability of nonribosomal, high molecular weight RNA from Balbiani ring 2 and chromosomes I-III indicate that most of the cytoplasmic 75S RNA is transcribed in the Balbiani ring 2 region of chromosome IV. 75S RNA molecules are, therefore, likely to be transferred from Balbiani ring 2, via nuclear sap, to cytoplasm without being measurably reduced in molecular size.One important step in gene expression of eukaryotic cells is the transfer of genetic information from nuclear DNA to polysomes in cytoplasm. Available data strongly indicate that this information transfer is accomplished by RNA, but the details of the process are far from clear (1). Although different defined messenger RNA species have been demonstrated in cytoplasm (2-5), it has been more difficult to identify the individual corresponding precursors in the nucleus. The main reason for this failure is that the nonribosomal, high molecular weight RNA of the nucleus is a complex mixture of molecules, with concomitant identification problems. In transformed cells, it is feasible to select for one type of RNA, the virusspecific RNA, and thus simplify the analysis (6-8). Another approach is to extensively fractionate the nucleus in order to obtain defined, nonribosomal, high molecular weight RNA species. When such a procedure was applied recently to salivary gland cells of the midge Chironomus tentans, a defined RNA species (75S RNA) was recorded in a restricted chromosome region (9). The present investigation shows that it is possible to follow the migration of that particular RNA species from its site of synthesis via nuclear sap to cytoplasm. Proc. Nat. Acad. Sci. USA 70 (1973) containing labeled, ethanol-soluble substances that would disturb the radioactivity pattern unless extracted), each sample was treated with 100 Al of SDS-Pronase solution.RNA was then precipitated overnight at -20°after 10 Al of 1 M NaCl and 250 ,l of 100% ethanol had been added. Electrophoresis. The technique with agarose gel slabs has been described in detail (13). 1% Agarose gels were prepared in 0.02 M Tris -HCl (pH 8.0)-2 mg/ml of SDS-0.02 M NaCl-2 mM EDTA. After completion of electrophoresis, the gel was cut in...

show abstract

“…1B and 2B). Particularly during in vivo conditions, (pre)ribosomal RNA can also be recorded on the chromosomes (11). In the present in vitro experiments ( Fig.…”

Section: Methodsmentioning

confidence: 49%

“…The appearance in cytoplasm of the finished ribosomal components, 28S and 18S RNA, has been established in vitro (11,15). The small ribosomal RNA component emerged in cytoplasm after about 90 min of incubation.…”

Section: Methodsmentioning

confidence: 99%

Evidence for Transport of 75S RNA from a Discrete Chromosome Region via Nuclear Sap to Cytoplasm in Chironomus tentans

Daneholt

Hosick

1973

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…The synthesis of chromosomal hnRNA in the 16S to 100S range has been found to be sensitive to a-amanitin (9) as well as to 5,6-dichloro-1-,-D-ribofuranosylbenzimidazole (8) and is therefore considered to represent RNA polymerase 1I-based transcription. The nucleolar synthesis of preribosomal 38S RNA and its processing has also been established (24 (Fig. 6).…”

Section: Resultsmentioning

confidence: 99%

“…Injections of cell nuclei were carried out by means of the micromanipulation technique described by De Fonbrune (3). One salivary gland in a volume of modified Cannon medium (24) was placed on the under side of a cover slip and then inverted and placed on a glass plate with a groove (oil chamber). Liquid paraffin was then added in the groove between glass and cover slip.…”

mentioning

confidence: 99%

Microinjection of anti-topoisomerase I immunoglobulin G into nuclei of Chironomus tentans salivary gland cells leads to blockage of transcription elongation.

Egyházi

Durban

1987

Mol. Cell. Biol.

View full text Add to dashboard Cite

other less efficiently transcribing heterogeneous nuclear RNA genes. The pattern of inhibition of growing nascent Balbiani ring chains indicated that the transcriptional process was interrupted at the level of chain elongation. The highly decondensed state of active Balbiani ring chromatin, however, remained unaffected after injection of topoisomerase I antibodies. These data are consistent with the interpretation that topoisomerase I is an essential component in the transcriptional process but not in the maintenance of the decondensed state of active chromatin.Topoisomerases are enzymes that modify the topological structure of DNA by strand breakage and rejoining (for reviews, see references 28 and 29). Type I topoisomerases catalyze unwinding of supercoiled plasmid DNA in steps of single turns and do not require an energy source or divalent cations as cofactors (28,29).The function of eucaryotic topoisomerase I is not yet established, although a role in transcriptional regulation is emerging. A topoisomerase I-like protein was observed near the 5' end and 3' end of the Tetrahymena DNA transcription unit coding for rRNA (16). Furthermore, topoisomerase I has been detected at loci of Drosophila polytene chromosomes that contain transcriptionally active genes; importantly, the sites of heat shock genes immunoreacted with affinity-purified topoisomerase I antibody after, but not before, heat shock induction (13). Photo-cross-linking studies showed that topoisomerase I is concentrated in regions of actively transcribed Drosophila genes and not on nontranscribed flanking regions (15).Despite this suggestive evidence that topoisomerase I might be involved in activation of eucaryotic genes, no direct demonstration exists that topoisomerase I has an essential function in the transcription process.In an attempt to gain information on a possible involvement of topoisomerase I in DNA transcription, we have injected topoisomerase I immunoglobulin G (IgG) into nuclei of living salivary gland cells from Chironomus tentans and analyzed its effect on DNA transcription. Analysis of the transcription of the large tissue-specific puffs, Balbiani rings, enabled us to distinguish between possible effects on RNA synthesis and chromatin structure. We show here that the rate of RNA polymerase I, as well as that of RNA polymerase 1I-promoted transcription, was inhibited after injection of anti-topoisomerase I IgG into the nuclei. The synthesis of * Corresponding author. nucleolar pre-rRNA and of Balbiani ring RNA was lowered by about 80%. The transcription of non-Balbiani ring heterogeneous nuclear RNA (hnRNA) genes was less affected. MATERIALS AND METHODSPolyclonal rabbit anti-topoisomerase I IgG. Rabbits were injected at 1-month intervals with 20 p.g of topoisomerase I purified from Novikoff hepatoma cells (3a, 4). Before injection, topoisomerase I was thoroughly mixed with Freund adjuvant (complete in the first injection and incomplete in subsequent boosters). After the fifth injection, antibody titer could be detected by an enzyme-...

show abstract

“…Sodium dodecyl sulfate buffer (NaDodSO4 buffer) was 0.5% NaDodSO4/0.1 M NaCl/1 mM EDTA/0.01 M Tris, pH 7.4. Dialysis buffer was 0.01 M Tris, pH 7.4/0.1 M NaCl/0.5% NaDodSO4.The incubation mix for RNA synthesis in isolated nuclei contained 6 mM KCI, 1.6 mM MnCl2, 0.1 M (NH4)2SO4, 40,M each of ATP, CTP, and GTP, 4 ,uM UTP, 2 ,uM [3H]UTP (20)(21)(22)(23)(24)(25) ,gCi per 500-Sl sample), and 1 mM dithiothreitol in 50 mM Tris/25% glycerol, pH 7.6. Isolation of Cell Nuclei and Nucleoplasmic and Nucleolar Fractions.…”

mentioning

confidence: 99%

Definition of subclasses of nucleoplasmic RNA

Tamm

1977

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

RNA synthesis in isolated HeLa cell nuclei prepared from cells pretreated with 5,6-dichloro-I-P-D-ribofuranosylbenzimidazole (DRB) is inhibited in a time-dependent manner. After 40-min pretreatment of cells with 60B M DRB in the presence of actinomycin D (0.04 ,g/ml), the rate of RNA synthesis in isolated nuclei, measured by[3HJUTP incorporation, is decreased by 63%. The DRB-resistant one-third of heterogeneous nuclear is distributed over the entire size range of heterogeneous nuclear RNA with some enrichment in the 18S range, as was observed earlier by pulse-labeling whole cells. 5,6-Dichloro-1-f-D-ribofuranosylbenzimidazole (DRB) has a selective and reversible action on nuclear RNA synthesis in mammalian cells by inhibiting two-thirds of heterogeneous nuclear RNA (hnRNA) (1, 2) and >95% of mRNA (2) through an apparent block in chain initiation (3,4 Milwaukee, WI. The phosphate-buffered saline (PBS) was 0.13 M NaCl/2.7 mM KCl/0.82 mM Na2HPO4/1.5 mM KH2PO4/ 0.91 mM CaC12/0.5 mM MgCl2; the pH was 7.3. The lysis buffer was 6 mM KCI/5 mM MgCl2/0.01 M Tris, pH 7.4; to the buffer was added dithiothreitol to a concentration of 1 mM prior to use. High salt buffer was 0.5 M NaCl, 0.05 M MgCl2, and 0.01 M Tris. Sodium dodecyl sulfate buffer (NaDodSO4 buffer) was 0.5% NaDodSO4/0.1 M NaCl/1 mM EDTA/0.01 M Tris, pH 7.4. Dialysis buffer was 0.01 M Tris, pH 7.4/0.1 M NaCl/0.5% NaDodSO4.The incubation mix for RNA synthesis in isolated nuclei contained 6 mM KCI, 1.6 mM MnCl2, 0.1 M (NH4)2SO4, 40,M each of ATP, CTP, and GTP, 4 ,uM UTP, 2 ,uM [3H]UTP (20)(21)(22)(23)(24)(25) ,gCi per 500-Sl sample), and 1 mM dithiothreitol in 50 mM Tris/25% glycerol, pH 7.6. Isolation of Cell Nuclei and Nucleoplasmic and Nucleolar Fractions. The procedures used have been described (1, 6). Pertinent details are given in figure legends.Pulse-Labeling Techniques. Isolated HeLa cell nuclei were labeled with [3H]UTP for 0-30 min at 26°, and 25-,il aliquots were placed on Whatman 3 MM filters that were processed in batches. The acid-precipitable counts were expressed per total sample after subtraction of 0-min counts. RNA was extracted with phenol/chloroform (1). In most experiments, actinomycin D (0.04 ,ug/ml) was used to suppress ribosomal RNA'synthesis.Gel Electrophoresis. The procedure used has been described (1). The marks above the horizontal bar in each panel of Figs. 2, 3 and 4 refer, from left to right, to 28, 18, and 4S marker RNAs from HeLa cell cytoplasm. To obtain size estimates of RNA in the 4-18S range, the logarithms of the molecular weights for marker RNAs were plotted against migration rates, with molecular weights of 1,750,000, 605,000 and 25,000 for 28, 18, and 4S RNA, respectively (7). This gave a linear relaAbbreviations: DRB, 5,6-dichloro-1-#-D-ribofuranosylbenzimidazole; hnRNA, nuclear heterogeneous RNA; [3H]UTP, [3H]uridine 5'-triphosphate; PBS, phosphate-buffered saline; NaDodSO4 buffer, sodium dodecyl sulfate buffer; NP, nucleoplasmic fraction; pre-4S RNA, -4.5S precursor of tRNA.

show abstract

NUCLEOLAR-DERIVED RIBONUCLEIC ACID IN CHROMOSOMES, NUCLEAR SAP, AND CYTOPLASM OF CHIRONOMUS TENTANS SALIVARY GLAND CELLS

Cited by 104 publications

References 23 publications

Evidence for Transport of 75S RNA from a Discrete Chromosome Region via Nuclear Sap to Cytoplasm in Chironomus tentans

Evidence for Transport of 75S RNA from a Discrete Chromosome Region via Nuclear Sap to Cytoplasm in Chironomus tentans

Microinjection of anti-topoisomerase I immunoglobulin G into nuclei of Chironomus tentans salivary gland cells leads to blockage of transcription elongation.

Definition of subclasses of nucleoplasmic RNA

Contact Info

Product

Resources

About