Synthesis and processing of high molecular weight RNA by nuclei isolated from embryos of Rana pipiens

Caston, J. Douglas; Jones, Paul H.

doi:10.1016/0022-2836(72)90021-6

Cited by 34 publications

(6 citation statements)

References 26 publications

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“…The methyl-labeled 4.4, 3.7, 2.6, and 2.3 MDal RNA identified in these experiments are similar to pre-rRNA fractions described for other systems (2,5,(23)(24)(25) (Fig. 4).…”

Section: Discussionsupporting

confidence: 65%

“…Based on these data, the relative methyl content of these three fractions, and on literature reports pertaining to other systems (2,(28)(29)(30), a working assumption is that the 18S (0.7 MDal) rRNA arises independently of both the 2.6 and 2.3 MDal intermediates. Accordingly, our observations may be summarized simply as follows: Existence of major and minor processing pathways has been inferred from noninal molecular weight determinations of preribosomal RNA fractions in HeLa cells and Rana pipiens embryonic cells (2,24). Moreover, it has been suggested from electrophoretic patterns that the 4.4 MDal fraction in L-cells may contain at least three distinct components (31) (32,33 …”

Section: Discussionmentioning

confidence: 99%

“…For electrophoresis in 8 M urea, gels were polymerized, removed from the tubes, and then swollen at 250 for 24 The cell suspension was divided into six equal aliquots, which were incubated for the times designated. RNA was extracted and electrophoresed, and total label in each band was quantitated by planometry.…”

Section: Methodsmentioning

confidence: 99%

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Changes in ribosomal RNA processing paths in resting and phytohemagglutinin-stimulated guinea pig lymphocytes.

Purtell¹,

Anthony²

1975

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

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Processing of rRNA was examined in resting and phytohemagglutinin-stimulated guinea pig lymphocytes. Synthesis of 1.7 (28S) and 0.7 (18S) X 106 dalton rRNA was more than 4-fold greater in phytohemagglutinin-stimulated than in resting cells. A 5-to 10-fold increase in flux of molecules through a 2.3 X 106 dalton RNA occurred without a concurrent change in the flux through a 2.6 X 106 dalton fraction in phytohemagglutinin-stimulated cells. In both resting and phytohemagglutinin-stimulated lymphocytes, the 2.3 X 106 dalton intermediate equilibrated with [3Hlmethyl label and pulse-chased prior to the 2.6 X 106 dalton RNA. The data indicate at least two processing paths in guinea pig lymphocytes; one proceeds to rRNA via a 2.3 X 106 dalton intermediate, and another proceeds via a 2.6 X 106 dalton RNA. The increase in rRNA synthesis in phytohemagglutinin-stimulated cells occurs primarily through that path containing the 2.3 X 106 dalton intermediate.In eukaryotes ribosomal RNA (rRNA) is transcribed as a large precursor molecule which is then processed through a series of intermediates into the definitive rRNA products (1, 2). Under certain conditions of metabolic transition within the cell, the rates of synthesis of both the precursors and the definitive rRNA are altered (3-6). Furthermore, data obtained in HeLa cells (7,8), regenerating liver and lens cells (9, 10), hormone-stimulated uterine and mammary gland cells (11,12), and human peripheral lymphocytes (13) indicate that control of rRNA production may not reside exclusively in the rate of transcription of the initial precursor molecule. The processing of this fraction could add a posttranscriptional mechanism for regulating quantitative or qualitative production of rRNA.Phytohemagglutinin (PHA)-induced transformation of lymphocytes provides a system in which gross alterations in the metabolic state of the cell are readily induced. The cells undergo a marked change in morphology and in biochemical metabolism after treatment with PHA, and one of the earliest events is a stimulation of the synthesis and processing of rRNA (14,15).In this paper data are presented which suggest that in the guinea pig lymphocyte the 1.7 X 106 dalton (1.7 MDal) (28S) and 0.7 MDal (18S) rRNA are processed from a 4.4 MDal (45S) precursor through one of at least two processing pathways and that one effect'of PHA is to stimulate processing through only one of these paths. A preliminary report has been communicated (16). METHODSIsolation and Cultures of Lymphocytes. Cervical, axillary, and mesenteric lymph nodes from male guinea pigs were trimmed of fat and teased apart in 10 ml/g wet weight The cells were then suspended at a final concentration of 3.6 or 5.0 X 106 cells per ml in modified medium supplemented with 20 ,og/ml of glutamine, 100 units/ml of penicillin, 100 jig/ml of streptomycin, and 10% heat-inactivated fetal calf serum (complete modified medium). Stationary cultures were maintained at 370 and pH 7.2 in a humidified atmosphere of 5% C02-95% air in 10 X 12 mm round-bott...

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“…The methyl-labeled 4.4, 3.7, 2.6, and 2.3 MDal RNA identified in these experiments are similar to pre-rRNA fractions described for other systems (2,5,(23)(24)(25) (Fig. 4).…”

Section: Discussionsupporting

confidence: 65%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Changes in ribosomal RNA processing paths in resting and phytohemagglutinin-stimulated guinea pig lymphocytes.

Purtell¹,

Anthony²

1975

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

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“…1 and 2 reveals that the effect of blastula PCA-extract differed markedly from that of tubercidin; the former inhibited the labeling of 40S pre-rRNA, whereas the latter enhanced the accumulation of 40S pre-rRNA. For Xenopus, 40S pre-rRNA is accepted to be the primary transcript (2,4,7,8,16) although there have been some reports suggesting the existence of a larger precursor (1,11). Thus, there is the implication that the PCA-extract suppresses formation of the primary transcript.…”

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confidence: 99%

Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos. VII. Inhibition of 40S pre-rRNA synthesis in Xenopus neurula cells.

Shiokawa

1984

Cell Struct. Funct.

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ABSTRACT. A fraction containing acid-soluble materials (PCA-extract) from Xenopus early blastulae preferentially inhibits the incorporation of[3H]uridine into 18S and 28S rRNA in Xenopus neurula cells. Pulse-laneling experiments revealed that whereas tubercidin, a known inhibitor of rRNA processing, produced a marked accumulation of the label in 40S pre-rRNA, Xenopus inhibitor suppressed labeling of the pre-rRNA.When tubercidin was added to cells whose activity for rRNA synthesis had been lowered by the Xenopus inhibitor, there was still an accumulation of label in the pre-rRNA.These results indicate that Xenopus inhibitor suppresses the synthesis of rRNA at transcription rather than at processing.

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“…In the electron microscope, the spacer intercepts between the 40S matrix units of Xenopus and Triturus are often found associated with isolated lateral fibrils or even with dense transcriptional complexes forming the so-called prelude regions (10, 11). In isolated nuclei of Rana, methylated, G+C-rich RNA molecules larger than the 40S pre-rRNA have been observed (12). Moreover, Xenopus cells grown in culture treated with fluorouridine synthesize large RNA molecules that seem to contain sequences of both the 40S pre-rRNA and the external "nontranscribed" spacer (ENTS) of rDNA (13).…”

Section: Introductionmentioning

confidence: 99%

Transcription of spacer sequences in genes coding for ribosomal RNA in Xenopus cells.

Rungger¹,

Achermann²,

Crippa³

1979

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

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Untreated Xenopus cells synthesize RNA molecules (SS-23S) containing sequences complementary to the external "nontranscribed" spacer of the genes coding for rRNA. In cells incubated in the col maturation cleavages of the rRNA precursors are inhibited. Concomitantly, the relative proportion of spacer transcripts detectable in newly synthesized RNA is increased and some sequences complementary to those of the nontranscribed spacer are found in the heavy shoulder of the 40S precursor rRNA peak. In such events transcription initiation seems to take place within the spacer because its middle and right BamHI endonuclease fragments are preferentially transcribed, whereas only few RNA sequences complementary to the left spacer fragment are found. It is concluded that at least some spacer regions contain promoters for transcription and can be transcribed either into a special class of "spacer transcripts" or into molecules covalently linked to rRNA precursor.It is still unclear which sequences of eukaryotic genes coding for pre-rRNA or proteins are actually transcribed and whether transcription initiation starts only from a single defined initiation site. The rRNA genes (rDNA) of Xenopus, which are located in the nucleolus, are among the best-studied eukaryotic genes (for reviews see refs. 1-4). Their first stable transcript is a rRNA molecule (pre-rRNA) of Mr 2.6 X 106 and a sedimentation coefficient of 40 S. After prolonged labeling times no molecules larger than 40S have been, found hybridizing to rDNA (5). However, no 5'-terminal polyphosphates were detected on the 40S pre-rRNA fron cultured Xenopus cells (6). On the other hand, it has recently been shown that the 40S pre-rRNA isolated from oocytes may be capped by an enzyme preparation from vaccinia virus, and thus seems to carry a polyphosphate at its 5' end (7). These results do not, however, exclude other initiation events elsewhere in rDNA, especially if the resulting transcripts are highly unstable.There are various observations in the literature (for reviews see refs. 4, 8, and 9) indicating that the first stable pre-rRNA is not necessarily the only transcript of rDNA. In the electron microscope, the spacer intercepts between the 40S matrix units of Xenopus and Triturus are often found associated with isolated lateral fibrils or even with dense transcriptional complexes forming the so-called prelude regions (10, 11). In isolated nuclei of Rana, methylated, G+C-rich RNA molecules larger than the 40S pre-rRNA have been observed (12). Moreover, Xenopus cells grown in culture treated with fluorouridine synthesize large RNA molecules that seem to contain sequences of both the 40S pre-rRNA and the external "nontranscribed" spacer (ENTS) of rDNA (13). Accordingly, changes in the ultrastructure of rDNA transcription units such as the presence of transcriptional complexes along nearly the entire deoxynucleoprotein axis have been observed after fluorouridine treatment (14).In the present study we ,uCi/ml (1 Ci = 3.7 X 1010 becquerels). Incubation was for eith...

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Synthesis and processing of high molecular weight RNA by nuclei isolated from embryos of Rana pipiens

Cited by 34 publications

References 26 publications

Changes in ribosomal RNA processing paths in resting and phytohemagglutinin-stimulated guinea pig lymphocytes.

Changes in ribosomal RNA processing paths in resting and phytohemagglutinin-stimulated guinea pig lymphocytes.

Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos. VII. Inhibition of 40S pre-rRNA synthesis in Xenopus neurula cells.

Transcription of spacer sequences in genes coding for ribosomal RNA in Xenopus cells.

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