Abstract
(Englisch)
|
In collaboration with R. van Driels group, an extensive analysis of intranuclear distribution of premRNA transcription sites and of newly synthesized RNA has been carried out following microinjection of Br-UTP into the cytoplasm of cultured cells. The use of gridded coverslips as cell supports allowed us to follow individual cells during a given period of labeling time. We have been able to confirm that perichromatin regions represent the major transcriptionally active domain in the nucleus and that perichromatin fibrils are the in situ forms of nascent hnRNA transcripts. Although some Br-labeled RNA migrates through nuclear pores towards the cytoplasm, most label accumulates as perichromatin fibrils within the transcription regions. This is in agreement with earlier data reporting an alteration of pre-mRNA processing dependent on the frequency of Br-labeled nucleotides in RNA molecules. Immunoelectron microscopic analysis of several pre-mRNA transcription and processing factors has revealed a strong association of RNA polymerase II, hnRNP core proteins, transcription factor TFIIH, poly(A) polymerase, splicing factor SC-35 and Sm complex of snRNP with perichromatin fibrils. Since nascent transcripts, hnRNPs, RNA polymerase II and Poly(A) polymerase are absent within interchromatin granule clusters, our results strongly support the idea that perichromatin fibrils are also sites of major pre-mRNA processing events (Cmarko et al., Mol.Biol.Cell 10, 211, 1999).
In collaboration with J. Aten's group, we have developed a method allowing one to specifically reveal fractions of DNA labeled in vivo, either with iodinated or chlorinated deoxyuridine (Jaunin et al., J.Histochem.Cytochem., 46, 1203, 1998). In the following experiments, our attention was concentrated in two directions. In one series of assays, we have examined the fine structural localization of DNA replication sites after short Br-dUTP labeling of cells and then investigated the ultrastructural distribution of newly synthesized DNA after different Br-dUTP pulse durations or after pulse-chase assays. In a second series of assays, we have carried out Br-dUTP labeling of cells covering one S phase, followed by chase period of several cell cycles. This approach allows us to label chromosomal domains consisting of one or very few chromosomes.
Finally, we have developed a collaboration with T. Cremer's group, aiming at defining, at an ultrastructural level, chromosome territories within the interphase nucleus. This is realized by means of specific chromosome molecular probes labeled either with biotin or digoxigenin and ultrastructural in situ hybridization.
|
