Biosynthesis of Messenger and Ribosomal Ribonucleic Acids in the Nucleolochromosomal Apparatus of Animal Cells

Georgiev, G.P.; Samarina, O. P.; Lerman, M. I.; Смирнов, М. Н.; Severtzov, A. N.

doi:10.1038/2001291a0

Cited by 236 publications

(58 citation statements)

References 9 publications

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“…Some authors describe a block or reduction of transfer of labeled RNA in actinomycinchase experiments (13,16,24,34,38,40, W. Eckert and W. W. Franke, In preparation) . This disagrees with remarks by Perry (27) and Geuskens (14) .…”

Section: Resultsmentioning

confidence: 99%

The Ultrastructure of the Nuclear Envelope of Amphibian Oocytes: A Reinvestigation

Scheer

1970

The Journal of Cell Biology

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Section: Resultsmentioning

confidence: 99%

The Ultrastructure of the Nuclear Envelope of Amphibian Oocytes: A Reinvestigation

Scheer

1970

The Journal of Cell Biology

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“…Chinese hamster cells, kindly provided by Dr L. Siminovitch (Toronto), were grown, in Eagle [2] minimum essential medium (Eurobio) supplemented with 100 U/ml penicillin, 50 pg/ml streptomycin and 10 x calf serum (Flow), in spinner suspension, maintained in the 3 -6 x 1OS/ml concentration range. Whenever autoradiographic studies were planned, [3H]uridine was added at 100 pCi/ml.…”

Section: Cell Culture and Labelingmentioning

confidence: 99%

Ultrastructural Organization and Biochemical Characterization of Chromatin · RNA · Protein Complexes Isolated from Mammalian Cell Nuclei

Bachellerie

Puvion

Zalta

1975

European Journal of Biochemistry

106

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Extranucleolar elements were isolated from mammalian cell nuclei and characterized by correlative biochemical-ultrastructural studies. These complex structures were shown to retain a supra-particle arrangement closely resembling the organizaton in situ of definite nuclear areas. In particular the morphological RNA . protein species, namely perichromatin fibrils and granules, were detected in association with characteristic regions of chromatin. By autoradiographic experiments, we were able to show that perichromatin fibrils represent the morphological state of newly formed heterogeneous nuclear RNA (hnRNA).Chromatin . RNA . protein complexes were further fractionated by means of a treatment dissociating the chromatin component. A minor part of DNA, possibly involved in the biosynthesis and the processing of hnRNA, remains linked to the resulting RNA . protein network.Most of the mechanisms involved in the control of RNA maturation in eukaryotic cells still remain to be elucidated. It has now been clearly established that a class of heterogeneous nuclear RNA (hnRNA) of high molecular weight, rapidly labeled and with a DNA-like composition [l-'51, presents The present study was undertaken in order to establish tentative correlation between biochemical and ultrastructural features of nuclear RNA protein particles and their spatial organization in situ.Abbreviation. hnRNA, heterogeneous nuclear RNA. ~9 1 .We developed an isolation procedure of subnuclear fractions where the ultrastructural organization of definite RNA . protein . chromatin areas appears well preserved. This method could contribute to the characterization of actively transcribed zones in chromatin as well as to a better definition of the events involved in hnRNA metabolism. We here report direct evidence that perichromatin fibrils do indeed represent the morphological state of newly synthesized hnRNA, as previously assumed by autoradiographic studies in situ [18]. MATERIALS AND METHODS Cell Culture and LabelingZadjela Ascitic Hepatoma Cells. Female Wistar rats were inoculated intraperitoneally with approximately 2 x lo7 cells. After 3 days, the cells were collected, washed repeatedly in a phosphate-buffered saline medium (NaC10.12 M, KCl4 mM, Na2HP04 7 mM, KH2P0, 1 mM, NaHCO, 16 mM) and resuspended in Fischer [19] medium (Eurobio), supplemented with 5 % calf serum, 100 U/ml penicillin and 50 pg/ml streptomycin. The cell suspension was then incubated at 37 "C in glass flasks at concentrations varying from 5 x 105/ml to 106/ml according to the experiments planned. Cells were labeled after a 5-h incubation in synthetic medium by addition of radioactive precursors.

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“…One recognizes two peaks, corresponding to the two ribosomal RNA species, and a faster component with a sedimentation constant of about 458. The latter presumably represents the large size ribosomal RNA precursor described in different types of animal cells (31)(32)(33). The presence in the nucleus of 188 RNA in amounts considerably smaller, relative to the major ribosomal RNA component, than found in cytoplasmic ribosomal RNA is in agreement with Penman's observations (29), suggesting that there are no mature ribosomes, but only precursors, in the nucleus: according to this author, the 45S RNA is cleaved into 188 RNA, which is immediately transferred to the cytoplasm, and 358 RNA, which remains in the nucleus to be transformed into 28S RNA.…”

Section: Sedimentation Profile Of Rna From Isolated Chromosomesmentioning

confidence: 99%

Isolation of Metaphase Chromosomes From Hela Cells

Huberman

Attardi

1966

The Journal of Cell Biology

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The authors have developed a method for large-scale isolation of metaphase chromosomes from HeLa cells. The distinguishing feature of this method is the use of a pH sufficiently low (about 3) to stabilize the chromosomes against mechanical damage. Many milligrams of fairly pure, morphologically intact chromosomes can be isolated in 8 hr or less of total working time. The isolated chromosomes contain about 2.0 mg of acid-soluble protein, 2.7 mg of acid-insoluble protein and 0.66 mg of R N A for each milligram of DNA. The R N A bound to the isolated chromosomes consists mainly of ribosomal RNA, but there is also a significant amount of 45S RNA. I N T R O D U C T I O NMany possible biochemical and biophysical approaches to the study of chromosomes in higher organisms have been hindered, until recently, by the lack of suitable procedures for large-scale isolation of chromosomes. Although the methods for isolation of interphase chromosomes, or "chromatin," which have been developed in recent years (1, 2) are satisfactory for certain purposes, a definite need still exists for a procedure which will allow large-scale isolation of morphologically intact metaphase chromosomes. Metaphase chromosomes are an indispensable complement to interphase chromosomes for the general study of chromosome structure. In addition, metaphase chromosomes have the unique advantage of being so condensed that they can be distinguished microscopically both from each other and from contaminating nonchromosomal material. Consequently, one is not limited to studying the average properties of all chromosomes; one can also examine single types of chromosomes.According to our experience, in the isolation of metaphase chromosomes by most previously published methods (3-5), morphological damage to some of the chromosomes cannot be avoided and only partial purification of the chromosomes from cell debris can be achieved. We report here a method for the rapid preparation, in milligram quantities, of fairly pure, morphologically intact metaphase chromosomes from HeLa cells. We also report the results of studies on the chemical composition of isolated chromosomes. M A T E R I A L A N D M E T H O D S Cultivation of CellsHeLa $3 cells (6) were grown in suspension culture in a modified Eagle's medium (7) supplemented with 5% calf serum. For accumulation of metaphase cells, partial synchrony was induced by lowering the culture temperature to 4°C for 1 hr and then returning it to 37°C (8). Ten to 11 hr later, colchicine was added to a final concentration of 0.5 to 1 X 10 -5 M. The cells were harvested by centrifugation 9 to 10 hr after colchicine addition and washed 3 times in 0.137M NaC1, 0.005M KC1, 0.007M NaH2PO4, 0.025 m Tris, pH 7.4. This procedure routinely produced about 30% metaphase cells. 95 on

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Biosynthesis of Messenger and Ribosomal Ribonucleic Acids in the Nucleolochromosomal Apparatus of Animal Cells

Cited by 236 publications

References 9 publications

The Ultrastructure of the Nuclear Envelope of Amphibian Oocytes: A Reinvestigation

The Ultrastructure of the Nuclear Envelope of Amphibian Oocytes: A Reinvestigation

Ultrastructural Organization and Biochemical Characterization of Chromatin · RNA · Protein Complexes Isolated from Mammalian Cell Nuclei

Isolation of Metaphase Chromosomes From Hela Cells

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