Chromosomal RNA: An artifact of preparation?

Artman, Michael; Roth, Jay S.

doi:10.1016/0022-2836(71)90295-6

Cited by 66 publications

(28 citation statements)

References 9 publications

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“…The results, together with those of Heyden and Zachau [3], obtained on calf thymus chromatin, and the report by Artman and Roth [4] that, depending on the conditions of isolation, the quantity of chromosomal RNA of chicken tissues varied from zero to almost 100% of the total chromatin-associated RNA, provide strong evidence that "chromosomal RNA" is not a distinct and well defined class of RNA.…”

Section: Resultssupporting

confidence: 56%

“…Recently, however, doubts have been raised with regard to the homogeneity and significance of chromosomal RNA. Thus, Heyden and Zachau [3] reported that chromosomal RNA, isolated from calf thymus, consisted to a large extent of degraded tRNA, and Artman and Roth [4] found no evidence for the existence in chicken tissues of a specific low-molecular RNA with the properties described for chromosomal RNA. Moreover, we have recently obtained evidence [5] that only a very small portion of the DNA of rat liver may be present in the form of DNA-RNA hybrids, a finding which does not sup port the hypothesis [ 1, 6,7] that chromosomal RNA functions as a sequence detector for chromosomal proteins.…”

Section: Introductionmentioning

confidence: 99%

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Heterogeneity of chromosomal RNA

Szeszák

Pihl

1972

FEBS Letters

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Section: Resultssupporting

confidence: 56%

Section: Introductionmentioning

confidence: 99%

Heterogeneity of chromosomal RNA

Szeszák

Pihl

1972

FEBS Letters

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“…In so me experiments de signed to study the incorporation of nucleosides in vitro this was followed by three Expt/ Ce ll Res 96 (/975) washes in minimal essential medium (M EM) containing Hanks' salts. Blood enriched in immature red cells (mostly late erythroblasts) was obtained from chicken treated with phenylhydrazine [3,66]. Counting of blood smears from anaemic hens s howed that usually more tha n 70 % cells of the population were erythroblasts, in contrast to the purified erythrocytes from norma l hens which contained less than 2 % not fully mature cells (mostly polychromatic erythroblasts [34]) .…”

Section: Preparation Of Cellsmentioning

confidence: 99%

“…The final preparation contained 218 J)-g of RNA as could be determined colorimetrically, in the presence of only minor quantities of DNA retained, and from the degradation by pancreatic ribonuclease. Having controlled the recoveries of labelled RNA and DNA added to aliquots processed in parallel and that of total DNA after each of the individual preparative steps we have found that the minimal RNA content per hen erythrocyte nucleus, and roughly also per total erythrocyte, is from 0.02 to 0.04 pg, corresponding to about 1-2 % of the DNA present (for chromatin preparations see also [3,40]; published figures for DNA contents per hen or chicken erythrocyte nucleus vary from 1.7 to 3.5 pg [5,17,63,67]). …”

Section: Estimation Of Minimal Rna Content Of Mature Hen Erythrocytesmentioning

confidence: 99%

Characterization and localization of the RNA synthesized in mature avian erythrocytes

Zentgraf

Scheer

Franke

1975

Experimental Cell Research

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Experimental Cell Research 96 (1975) RNA present and synthesized in mature hen erythrocytes in vivo and in vitro has been studied with electron microscopic , autoradiographic , and biochemical methods. The average hen erythrocyte contains a minimum of 0.02-0.04 pg RN A which is predominantly, if not exclusively, located in the nucleus. The incorporation of[3HJuridine into this RNA is very low but is clearly demonstrable in vivo and in vitro using different incubation media. The specific efficiencies of the incorporation assays are compared. The incorporated radioactivity is contained in RN A as shown by its selective sensitivity to hydrolysis in mild alkali and with ribonuclease a nd is not hybridized to DNA as shown by Cs.SO" gradient centrifugation of total hen erythrocyte nucleic acids. LiCI precipitation as well as gel electrophoresis on agarose under denaturing and non-denaturing conditions has revealed that this labelled RNA is of high-molecular weight a nd covers a broad range from ca 0.25 to 5.0 million D, with gel-electrophoretically detectable peaks at positions corresponding to 1.8, 1.0 and 0.5 million D. The pattern obtained is virtually identical after the different incorporation condi tion s and was also sim il ar to that obtained in reticulocyte cells of animals made anaemic with phenylhydrazine. The incorporation is inhibited by actinomycin D with a sensitivity characteristic for mRN A formation a nd is also reduced in the presence of a-amanitin. The labelled RNA remains within th e confinements of the nucleus and does not seem to be transported into the cytoplasm. It appears to be preferentially enriched in the interchromatinic channels . It is concluded that mature hen erythrocytes are capable of transcribing a small part of their genome into pre-mRNA at a very reduced level but that this RNA is not only excluded from functioning in tra nslation but also from nucleocytoplasmic transport. The findings are discussed in relation to the controversial literatu re in this field, a nd possible functions of thi s RNA are hypothesized.Various forms of cell differentiation are characterized by a progressive cessation of the sy nthesis of most, if not all, classes of RNA. In some late stages of such differentiation processes one even notes a general breakdown of RNA-containing structures and the mature cell is almost emptied of RNA . The formation of the red blood cell, in particular in animals with nucleated erythrocytes , has become of special importance in studie s of the programmed switch-off sequence of the transcription of most, and finally perhaps of all, genes. It has been shown that during erythropoiesis the nuclear content and the nuclear envelope is greatly reduced , the chromatin condenses and contains an additional special histone , the nucleolus disappears, concomitant with the cessation of ribosome formation , the number of transcribed genes decreases , leaving the transcription of one class of genes in the Exprl Cell Res 96 (1975)

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“…The possibility that this radioactive material was free poly(ADP-Rib) was investigated by subjecting the non-histone protein fractions to density-gradient centrifugation in a mixture of CsCl and urea, condi-tions which would be expected to break ionic and hydrophobic bonds but not disrupt covalent linkages between proteins and poly(ADP-Rib) [19]. Analysis of the acid-insoluble material from such gradients showed the presence of peaks of radioactivity at the top and bottom of the gradient only (Fig.2).…”

Section: The Occurrence Of Free Poly (Adp-rib) In the Nucleus And Itsmentioning

confidence: 99%

The Modification of Nuclear Proteins by ADP‐ribosylation

Rickwood

MacGillivray

Whish

1977

European Journal of Biochemistry

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When incubated in vitro purified mouse nuclei incorporate NAD into poly(ADP-Rib). Analysis of the product on CsCl/urea gradients showed that a large proportion of the poly(ADP-Rib) was not attached to protein. The free poly(ADP-Rib) did not appear to arise through degradation and its chain length was significantly longer than the poly(ADP-Rib) attached to proteins. Fractionation of the proteins on hydroxyapatite revealed that tissue-specific modification of both the histones and non-histone proteins, had occurred. In the case of one protein species there was evidence for the existence of several forms with different numbers of ADP-Rib residues.It is now more than ten years since the synthesis of poly(ADP-Rib) in isolated nuclei was first demonstrated; it was shown that when nuclei were incubated in vitro with labelled NAD the radioactivity was incorporated exclusively into poly(ADP-Rib) [l]. More recently evidence has been presented to show that this polymer is also synthesized in vivo [2]. Poly(ADP-Rib) is a polynucleotide-like molecule but, in contrast to the other polynucleotides of the cell, its functional role in the cell remains unclear. It has been reported that poly(ADP-Rib) is attached to the nuclear proteins (for a review see [3]) and as such it could modify the characteristics of the proteins to which it is bound, in the same way as phosphorylation and acetylation can also modify the interaction of histones with DNA. Two problems emerge from the evidence presently available. The first concerns which proteins of the nucleus act as acceptors for the poly(ADP-Rib) molecules. So far only the histones have been identified with certainty as being modified by the polymer [4-61, but with the improved techniques now available (see [7]) it is possible to examine the role of most of the nuclear proteins in this process. The second problem relates to the large number of conflicting reports as to the rate of synthesis of poly(ADP-Rib) at different times of the cell cycle [8] and the difficulty of correlating polymer production and its modification of nuclear proteins with particular events in the cell cycle or with changes in gene activity. For some years these laboratories have been involved in developing procedures for the preparation and analysis of nuclear proteins (see [7,9,10]) and in this paper we describe the application of some of our techniques to study the modification of nuclear proteins by poly-(ADP-Rib) in a number of mouse cells.

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Chromosomal RNA: An artifact of preparation?

Cited by 66 publications

References 9 publications

Heterogeneity of chromosomal RNA

Heterogeneity of chromosomal RNA

Characterization and localization of the RNA synthesized in mature avian erythrocytes

The Modification of Nuclear Proteins by ADP‐ribosylation

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