RDNA transcription

Rhodes D, Klug A (1981) Sequence-dependent helical periodicity of DNA. Nature 292 378-380 Roussel P, Andre C, Comai L, Hernandez-Verdun D (1996) The rDNA transcription machinery is assembled during mitosis in active NORs and absent in inactive NORs. J Cell Biol 133 235-246... 

Santoro and Grummt, using as experimental model the nucleolar remodeling complex NoRC and rDNA transcription in mammalian cells, have shown that... 

G. Bouche, V. Baldin, P. Belenguer, H. Prats, and F. Amalric, Activation of rDNA transcription by FGF-2 key role of protein kinase CKII, Cell Mol Biol Res 40, 547-554 (1994). 

Nucleolar Functions of RecQ Family Members Eukaryotic ribosomal RNA (rRNA) is transcribed from a family of repeated DNA sequences, ribosomal DNA, rDNA. rDNA repeats are G-rich on the non-template strand, not only within the regions that encode ribosomal RNA but also in the spacer regions. rDNA transcription and rRNA biogenesis occur... 

Interspecific cell hybrids between human and murine cells do not express the rDNA genes of both genes. Interspecific crosses in plants and between Xenopus species have also shown a gene-specific dominance effect. This phenomenon is known as nucleolar dominance and, depending on the chromosomal organization of the hybrid, the rDNA of either one of the species is expressed (Reeder, 1985). These findings indicated that there are some aspects of rDNA transcription that are species specific. The incompatibility between different species has then been... 

One of the fractions required to direct rDNA transcription contains the RNA polymerase I enzymatic activity. Purification of mammalian RNA polymerase I indicates that the core enzyme is a multisubunit complex with a molecular mass of >500 kDa. (Hannan et aL, 1998c Song et al.,... 

Actinomycin D inhibits transcription by intercalating into DNA and it is thought to have greater binding affinity for GC-rich regions, which could explain its selectivity for inhibition of Pol I-based rDNA transcription at low doses of 0.001-0.01 /ig/ml (Abelson and Penman, 1975) (Table II). At doses higher than 0.01 /ig/ml. Act D inhibits the expression of both Pol I and Pol II genes. 

Derenzini, M., Sirri, V., Pession, A., Trere, D., Roussel, P Ochs, R. L., and Hernandez-Verdun, D. (1995a). Quantitative changes of the two major AgNOR proteins, nucleolin and protein B23, related to stimulation of rDNA transcription. Exp. Cell Res. 219, 276-282. 

We then tested whether HS-C protein can promote specific and accurate transcription of rat rDNA in an unfractionated extract. Previous studies in our laboratory (2) have shown that unfractionated nuclear extracts from either rat hepatoma or rat liver caimot support rDNA transcription. If the lack of transcription in such extracts is predominantly due to nicks in the template caused by the action of DNase, addition of fraction HS-C should prevent random transcription and yield a distinct band corresponding to the expected size of the transcript. TTiat this is indeed the case was proven by... 

Similar experiments were also performed with unfractionated whole cell extracts. Unlike unfractionated tissue extracts (2), unfractionated whole cell extracts usually yield the correct transcript without addition of exogenous HS-C. However, some preparations of unfractionated cell extracts are unable to support transcription of rDNA. When such preparations were used for rat rDNA transcription, a smeared autoradiogram with no distinct bands was observed. However, addition of HS-C in these preparations prevented random transcription and resulted in the production of the correct transcript (Fig. 2, lanes 3-5). Higher concentrations of HS-C had an inhibitory effect on the transcription, as observed even in reconstituted systems consisting of relatively pure fractions (Fig. 1). 

The present studies have demonstrated that a protein factor which exhibits properties of poly(ADP-ribose) polymerase can prevent nonspecific transcription of rat rDNA in either whole ceU extracts or nuclear extracts derived from tissues. This factor is analogous to that described for RNA polymerase Il-directed transcription (7). Highly purified fractions do not require this factor for accurate transcription of rDNA (Mealey and Jacob, unpublished data) probably due to removal of DNA-nicking enzyme(s) following purification. This observation does not preclude the importance of poly(ADP-ribose) polymerase in vivo in rDNA transcription following DNA damage. Clearly, rDNA can be nicked under these conditions and the requirement for poly(ADP-ribose) polymerase to prevent binding of RNA polymerase I to these nicks will be essential. 

The appearance of a distinct band corresponding to the accurate transcript of rat rDNA in an unfractionated hepatoma nuclear extract in response to exogenous HS-C is of considerable interest. This observation raises the possibility of using such a system to study rDNA transcription in response to a variety of physiological stimuli that are known to alter rRNA synthesis. Unfractionated samples have certain advantages over the fractionated samples since purification procedures could result in differential recovery of the transcription factors from control and test samples. 

In the course of these studies, we observed that RNA polymerase I can be ADP-ribosylated and that this modification can partially inactivate the enzyme (Mealey and Jacob, unpublished data). We are now testing whether ADP-ribosylation of RNA polymerase I can adversely affect rDNA transcription. We have failed to ADP-ribosylate RNA polymerase I completely in the in vitro system and are in the process of developing the optimal conditions for this reaction. Only then can we do meaningful experiments to determine the precise role of ADP-ribosylation of RNA polymerase I or of other essential transcription factor(s) in the transcription of ribosomal RNA genes. 

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