amanitins and Burkitt-Lymphoma

The carboxyl-terminal domain (CTD) of the large subunit of mammalian RNA polymerase II contains 52 repeats of a heptapeptide that is the target of a variety of kinases. The hyperphosphorylated CTD recruits important factors for mRNA capping, splicing, and 3'-processing. The role of the CTD for the transcription process in vivo, however, is not yet clear. We have conditionally expressed an alpha-amanitin-resistant large subunit with an almost entirely deleted CTD (LS*Delta5) in B-cells. These cells have a defect in global transcription of cellular genes in the presence of alpha-amanitin. Moreover, pol II harboring LS*Delta5 failed to transcribe up to the promoter-proximal pause sites in the hsp70A and c-fos gene promoters. The results indicate that the CTD is already required for steps that occur before promoter-proximal pausing and maturation of mRNA.

Topics: 3' Untranslated Regions; Amanitins; Animals; Burkitt Lymphoma; Cloning, Molecular; Genes, fos; HSP70 Heat-Shock Proteins; Humans; Macromolecular Substances; Mice; Phosphorylation; Promoter Regions, Genetic; Recombinant Proteins; RNA Polymerase II; RNA Splicing; Sequence Deletion; Transcription, Genetic; Tumor Cells, Cultured

The cytosolic chaperonin TRiC is a large protein complex involved in the folding of newly synthesized actin and tubulin. The fertilization of the mouse oocyte is followed by a remodelling of the actin and tubulin filaments. The TRiC subunit TCP1 is expressed only from the 4-cell stage on, even though actin and tubulin are synthesized in the previous stages. We investigated the onset of synthesis of another subunit, TRiC-P5, during early mouse embryogenesis. We report that TRiC-P5 is synthesized at the 2-cell stage in an alpha-amanitin sensitive manner. Thus, it is expressed before TCP1 and is one of the first proteins to be synthesized after zygotic genome activation.

Topics: Amanitins; Animals; Blastocyst; Burkitt Lymphoma; Cell Line; Chaperonin Containing TCP-1; Chaperonins; Cytosol; Embryonic and Fetal Development; Female; Fertilization; Gene Expression Regulation; Humans; Macromolecular Substances; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Protein Biosynthesis; Proteins; Superovulation; Tumor Cells, Cultured; Zygote

The specific silver staining of nucleolar region organizers was applied to Daudi lymphoma control and interferon alpha-treated cells. Isolated nuclei from control and treated samples were used for the kinetic analysis of in vitro RNA and DNA synthesis. Results have shown that interferon treatment induces a reduction of the transcriptional and replicative activities within 90 min. Concomitant to these results is the modification of the organization of nucleoli. Intensity and distribution of silver grains are, in fact, different in treated cells nucleoli as compared to those of controls. Thus, the number and the arrangement of granules could be related to the functional state of the cells suggesting that the transient cascade of interferon-generated signals involves also modulation of nucleolar structure and function in accordance with the hypothesis of a relationship of cell proliferation rate to silver-stained nucleolar protein quantity.

Topics: Amanitins; Burkitt Lymphoma; Cell Nucleolus; Cell Nucleus; DNA Replication; DNA, Neoplasm; Humans; Interferon Type I; Kinetics; Microscopy, Electron; Recombinant Proteins; RNA Polymerase I; RNA, Neoplasm; Silver Staining; Transcription, Genetic; Tumor Cells, Cultured

An in vitro transcription system has been established with a whole-cell extract from the human Burkitt's lymphoma, Daudi, cell line. The B cell extract has been compared with a HeLa cell extract in an effort to study lymphocyte-specific regulatory factors of kappa light chain gene transcription. Both extracts were capable of transcribing Vk genes and other RNA polymerase II dependent genes. Alpha-amanitin at [0.1 micrograms/ml] completely inhibited the accumulation of transcripts in both systems. At low DNA template concentrations the kappa intronic enhancer stimulated Vk promoter transcription 3-7 fold in B cell extracts. The enhancer-dependent transcription was abolished by excising the enhancer from the test plasmid with Eco R1. Both Vk promoter and enhancer-dependent transcription in HeLa extracts was undetectable at low [DNA]. These results demonstrate that kappa enhancer stimulation of Vk transcription in vitro is observed in B cell extracts only under conditions of low DNA template concentration.

Topics: Amanitins; Burkitt Lymphoma; Enhancer Elements, Genetic; Gene Expression Regulation; Genes, Immunoglobulin; HeLa Cells; Humans; Immunoglobulin kappa-Chains; Introns; Promoter Regions, Genetic; Templates, Genetic; Transcription, Genetic; Tumor Cells, Cultured