Isolation of a Nuclear Rna Fraction Resembling Dna in Its Base Composition

Sibatani, Atuhiro; Kloet, Siwo R. de; Allfrey, Vincent G.; Mirsky, A. E.

doi:10.1073/pnas.48.3.471

Cited by 141 publications

(25 citation statements)

References 18 publications

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“…The experimental procedures were based on the methods of Sabatani et al (1962), Muramatsu, Smetana and , DiGirolamo, Henshaw and Hiatt (1964), Dahmus and McConnell (1969) and Culp and Brown (1970). The rats were killed by cervical dislocation and the excised tumour tissue, freed from any necrotic material, was collected in medium A (0-25 mol/l sucrose, 3 mmol/l CaCl2, 50 mmol/l Tris-HCl buffer, pH 7-6; DiGirolamo et al., 1964) at 00.…”

Section: Methodsmentioning

confidence: 99%

Intranuclear Distribution of the Inducing Metal in Primary Rhabdomyosarcomata induced in the Rat by Nickel, Cobalt and Cadmium

Webb¹,

Heath²,

Hopkins³

1972

Br J Cancer

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Section: Methodsmentioning

confidence: 99%

Intranuclear Distribution of the Inducing Metal in Primary Rhabdomyosarcomata induced in the Rat by Nickel, Cobalt and Cadmium

Webb¹,

Heath²,

Hopkins³

1972

Br J Cancer

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“…In addition to the ribosomal RNA species and their large precursors, one can see in the sedimentation profile of nuclear RNA small amounts of 48 RNA, and a polydisperse RNA with sedimentation constants between 6S and more than 50S. The latter material presumably represents, at least in part, the heterogeneous nonribosomal type nuclear RNA described in HeLa cells (34) and other animal cells (13,35,36).…”

Section: Sedimentation Profile Of Rna From Isolated Chromosomesmentioning

confidence: 96%

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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“…~[he functional messenger seems to be produced by a single DNA strand (Champe and Benzer, 1962), but it is dubious whether it can be identified with chRNA, since the messenger is expected to display a high U/A ratio (Lanni, 1962). RNA fractions with a DNAlike composition and with a high turnover have been isolated previously from animal nuclei (Sibatani et al, 1962;Georgiev and Mantieva, 1962). Their relation to the present chRNA is unknown.…”

Section: Discussionmentioning

confidence: 98%

THE BASE COMPOSITION OF RIBONUCLEIC ACID IN LAMPBRUSH CHROMOSOMES, NUCLEOLI, NUCLEAR SAP, AND CYTOPLASM OF TRITURUS OOCYTES

Edström

Gall

1963

The Journal of Cell Biology

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The base composition of RNA's extracted from chromosomes, nucleoli, nuclear sap, and cytoplasm of Triturus oocytes has been determined by microelectrophorcsis. The chromosomal RNA has a content of guanine+cytosine equal to that of DNA, but there is no complementarity in the composition as for DNA. Nuclear sap contains a highly variable RNA with a tendency towards high uracil values. Nucleolar and cytoplasmic RNA's are similar in composition and both are of the guanine-cytosine rich type. The chromosomes and nucleoli contain roughly equivalent amounts of RNA, somewhat less than is present in the nuclear sap. The RNA/DNA ratio of the whole chromosomes is about 10. However, the ratio in the synthetically active regions, the loops, is much higher, since the loops contain all the chromosomal RNA hut only a small fraction of the DNA.

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Isolation of a Nuclear Rna Fraction Resembling Dna in Its Base Composition

Cited by 141 publications

References 18 publications

Intranuclear Distribution of the Inducing Metal in Primary Rhabdomyosarcomata induced in the Rat by Nickel, Cobalt and Cadmium

Intranuclear Distribution of the Inducing Metal in Primary Rhabdomyosarcomata induced in the Rat by Nickel, Cobalt and Cadmium

Isolation of Metaphase Chromosomes From Hela Cells

THE BASE COMPOSITION OF RIBONUCLEIC ACID IN LAMPBRUSH CHROMOSOMES, NUCLEOLI, NUCLEAR SAP, AND CYTOPLASM OF TRITURUS OOCYTES

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