Isolation and Characterization of Ribosomal Ribonucleic Acid

Kirby, K.S.

doi:10.1042/bj0960266

Cited by 541 publications

(116 citation statements)

References 8 publications

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“…In some experiments, the oocytes were separated according to size as described (2). The oocytes were washed and the RNA was extracted by the method of Kirby as described (2,6 (10).…”

Section: Methodsmentioning

confidence: 99%

Presence of tadpole and adult globin RNA sequences in oocytes of Xenopus laevis

Perlman

Ford

Rosbash

1977

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

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The swimming tadpole, which possesses an active circulatory system, was also assayed for the tadpole and adult globin sequences. Whereas the adult sequences are present at approximately the same concentration as in the mature oocyte, the concentration of the tadpole sequences increases at least 300-fold in the first 3 days following fertilization. The lampbrush chromosomes of amphibian oocytes are the sites of intense transcriptional activity (1). Although the RNA sequences present in oocytes have been partially characterized, many questions remain to be answered (2-4). One important question is the extent to which the oocyte sequences are specific for oogenesis and early development.One approach to this problem is to assay ovary RNA for mRNA sequences that code for proteins normally associated with terminally differentiated cells. In this report, a successful search for the globin mRNA sequence of Xenopus laevis in the oocyte is described. In addition, the number of copies of this sequence per oocyte was found to be not markedly different from the number of copies of the&majority of other poly(A)-containing sequences per oocyte.MATERIALS AND METHODS Preparation of RNA. Ovary RNA. Ovaries were removed from mature X. laevis females, washed with Barth X medium (5), and resuspended in Barth X medium containing 1% Pronase and 25 mM EDTA. After 10-15 min, the individual oocytes were essentially free of follicle cells. In some experiments, the oocytes were separated according to size as described (2). The oocytes were washed and the RNA was extracted by the method of Kirby as described (2, 6).Egg RNA. Unfertilized ovulated eggs were dejellied with Barth X containing 2% cysteine (7 (12). were synthesized and purified as described except that [32P]dCTP was used at a final concentration of 32 IiM (13).RNA-DNA Hybridization and Assay. RNA-DNA hybridization was performed as described (14) except that the reactions were at 70°in 1.0 M NaCI/5 mM EDTA/10 mM Tris-HCI, pH 7.4. Crot (product of RNA concentration and time in mol-sec/liter) values were determined in one of two ways. For total RNA, Crot was determined from the optical density by assuming 1 mg = 25 A260 units. For poly(A)-containing RNA, the amount of poly(A) was determined by hybridization with radioactive poly(U) (15). Poly(A)-containing RNA from the tadpole, oocyte, and egg was assumed to have a length of 1500 nucleotides and a 5% poly(A) content. The Crot values were corrected to 0.18 M Na+ at 600 (Crote) as described (13,16). Hybrid formation was monitored with nuclease SI as described (13).Abbreviations: cDNA, complementary DNA; Crot, product of RNA concentration and time in mol-sec/liter; Crote, Crot corrected to 0.18 M Na+ at 600.

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“…In some experiments, the oocytes were separated according to size as described (2). The oocytes were washed and the RNA was extracted by the method of Kirby as described (2,6 (10).…”

Section: Methodsmentioning

confidence: 99%

Presence of tadpole and adult globin RNA sequences in oocytes of Xenopus laevis

Perlman

Ford

Rosbash

1977

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

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“…At this stage the medlium was withdrawn, the aletirone layers were rinsed and further inculbated for 12 hours in a meditum containing GA (0.5 Nim) or GA and various inhibitors (10 FM inhibit a-amylase synthesis completely within 2 and one-half to 4 hours depending on the concentration used. The incorporation of "'C-uridine by the aleurone layers is inhibiited by 1 mm 6-methylpurine in the following way: 30 to 35 % within 2 and one-half hours of its addition and 65 to 70 % after 4 hoturs of its addition.…”

mentioning

confidence: 99%

Hormonal Control of Enzyme Synthesis: On the Mode of Action of Gibberellic Acid and Abscisin in Aleurone Layers of Barley

1967

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Summary. Gibberellic acid (GA) enhances the synthesis otf a-amylase andl ribonutclease in isolated aleurone layers andl this process is inhibited by abscisiin. Removal of gibberellic acid in mid-course of a-amylase productioni results in a slowing down of a-amylase synthesis, suggesting a continued requirement of GA for eiizyme synthesis. This is paralleled by a continuous requirement for RNA synthesis. Addition of 6-methylpurine or 8-azaguanine in mid-course results in ani inhibition of a-amylase synthesis within 3 to 4 hours. However, adtinomycin D added in inid-course is almost without effect. 'rhis is not due to its failuire to enter the cells, because it does inhibit 1'C-uridinie incorporation at this stage. Addition of abscisin to aleuroine layers which are synthesizing a-amylase results in an inhibition o'f this synthesis within 2 to 3 hours. Cycloheximide oIn the other hand inhibits enzyme synthesis immediately upon its addition. These data are consistent with the hyp.othesis that the expression of the GA effect requires the synthesis of enzyme-specific RNA molecules. The similarity in the kinetics of inhilbition between abscisin on the one haind and 8-azaguanine or 6-meth'yl;purine exert its action by inhibiting the synthesis or by preventiing their incorporation into G(ibberellic acid gre,atly enhllanices the syiithesis of atmylolytic enzymes (and several other hydrolytic enzyinmes) in aleuiroine cells of barley (20,23, 27) oni the other suggests that abscisin ma) of these enzyme-specific RNA molectules an active enzyme-svnthesisiilg unit.synthesis b))y abs'cisin cani be partially) o\vercomeyl)

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“…After freeze-thawing the mu particle and whole cell preparation, the sample was ground in liquid nitrogen, and treated with 2'5 per cent, sodium lauryl sulphate at 600 C. RNA was extracted using p-amino salicylate and phenol-m-cresol (Kirby, 1965). The RNA was analysed by electrophoresis on 25 per cent.…”

Section: Methodsmentioning

confidence: 99%

Studies on the RNA of the mate-killer particles of paramecium

Baker

1970

Heredity

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Isolation and Characterization of Ribosomal Ribonucleic Acid

Cited by 541 publications

References 8 publications

Presence of tadpole and adult globin RNA sequences in oocytes of Xenopus laevis

Presence of tadpole and adult globin RNA sequences in oocytes of Xenopus laevis

Hormonal Control of Enzyme Synthesis: On the Mode of Action of Gibberellic Acid and Abscisin in Aleurone Layers of Barley

Studies on the RNA of the mate-killer particles of paramecium

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