Alpha-Amanitin is a well-known specific inhibitor of RNA polymerase II (RNAPII) in vitro and in vivo. It is a cyclic octapeptide which binds with high affinity to the largest subunit of RNAPII, RPB1. We have found that in murine fibroblasts exposure to alpha-amanitin triggered degradation of the RPB1 subunit, while other RNAPII subunits, RPB5 and RPB8, remained almost unaffected. Transcriptional inhibition in alpha-amanitin-treated cells was slow and closely followed the disappearance of RPB1. The degradation rate of RPB1 was alpha-amanitin dose dependent and was not a consequence of transcriptional arrest. Alpha-Amanitin-promoted degradation of RPB1 was prevented in cells exposed to actinomycin D, another transcriptional inhibitor. Epitope-tagged recombinant human RPB1 subunits were expressed in mouse fibroblasts. In cells exposed to alpha-amanitin the wild-type recombinant subunit was degraded like the endogenous protein, but a mutated alpha-amanitin-resistant subunit remained unaffected. Hence, alpha-amanitin did not activate a proteolytic system, but instead its binding to mRPB1 likely represented a signal for degradation. Thus, in contrast to other inhibitors, such as actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, which reversibly act on transcription, inhibition by alpha-amanitin cannot be but an irreversible process because of the destruction of RNAPII.

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A plasmid containing two cloned he atitis B virus genomes in a tandem head-to-tail arrangement gas been introduced into mouse fibroblasts by using cotransformation with the cloned herpes simplex virus thymidine kinase gene. Several copies of the plasmid were integrated into high molecular weight cellular DNA. The original tandem structure of the hepatitis B virus DNA was conserved. Hepatitis B surface antigen was synthesized by all the 15 clones examined. The other viral antigens were not detected. The surface antigen was excreted into the cell culture medium as particles having the same characteristics as those found in human serum. It is estimated that 2-4 X 10' particles were produced per mouse cell per 24 hr in two clones. This value corresponds to approximately24 X 106 surface antigen polypeptides per cell per 24 hr.Hepatitis B virus (HBV) has a limited host range and, in nature, seems to infect only man and perhaps a few additional primates (1). So far, the virus has not been propagated in cell culture, and some viral surface antigen-producing cell lines from human hepatocellular carcinomas constitute the only cell culture systems that synthesize a HBV marker (2,3). For these reasons, study of the virus multiplication at the molecular level has been greatly hampered. Recently, information on the genetic organization of the virus was obtained from the nucleotide sequence of the genome. Genes coding for surface and capsid antigens and perhaps for DNA polymerase are located on the long strand of the genome (4-7). However, nothing is known concerning virus gene expression and its regulation.One approach to the study of HBV gene expression is to examine the functional capacity of the cloned viral DNA after its introduction into a mammalian cell culture. A gene that does not code for a selectable marker can be transferred into a cell by cotransformation of mutant mouse L cells deficient in thymidine kinase (TK) with this gene and the herpes simplex virus (HSV) tk gene (8). It appears that most of the selected.TK+ colonies contain the nonselectable marker. In the present study, mouse LTK-cells were cotransformed with HBV cloned DNA and the HSV cloned tk gene. All the selected TK+ clones synthesized hepatitis B surface antigen (HBsAg) particles which were excreted into the culture medium without apparent damage to the cells.MATERIALS AND METHODS Construction of the Recombinant and Bacterial Transformation. Plasmid pBR322 (200 ng) was digested with EcoRI endonuclease and treated with 2.6 units of alkaline phosphatase (9) in 100 mM Tris'HCI (pH 8.0) at 600C for 60 min. After two phenol extractions and three ether extractions, DNA was precipitated with ethanol. The pellet was dissolved in water and 100 ng of EcoRI-digested HBV DNA was added. The ligation was performed as described (10). Escherichia coli DP50. was grown in L broth medium containing 100.ug of diamninopimelic acid (Sigma) and 20 ug of thymidine (Sigma) per ml. Bacteria were transformed as described (10) with the ligation mixture and were select...

II) 1 (1, 2) and RNAP III (3), with RNAP II being the most sensitive. As a consequence, the incorporation of new ribonucleotides into the nascent RNA chains is blocked (4). Actinomycin D is generally thought to intercalate into DNA thereby preventing the progression of RNA polymerases, with RNAP I being the most sensitive (5, 6). In previous work, we have shown that the average phosphorylation of RNAP II C-terminal domain (CTD) increases in cells exposed to actinomycin D (7, 8). The activity of RNAP II is regulated by multisite phosphorylation on the CTD (9). The underphosphorylated CTD mediates multiple protein-protein interactions involved in the assembly of a preinitiation complex. The subsequent phosphorylation of the CTD occurs along the initiation of transcription and contributes to disrupt some of the interactions that lead to the assembly of the preinitiation complex on promoters. Phosphorylation of RNAP II at this step is required to elongate transcription and mediates the recruitment of various enzymatic complexes involved in processing of the primary transcript (10 -12). In contrast, phosphorylation of the CTD prior to the formation of the preinitiation complex represses the expression of specific genes (13). Hence, the increase in average phosphorylation of the CTD promoted by actinomycin D raises the possibility that different genes may have different susceptibilities to this drug.Several cyclin-dependent kinases (CDK) have been shown to phosphorylate the CTD and regulate transcription. CDK7, and its partner, cyclin H, are subunits of the general transcription factor, TFIIH, a component of the preinitiation complex (14, 15); CDK8 and its partner cyclin C belong to the RNAP II holoenzyme (16, 17); CDK9/PITALRE, and its partners, cyclins T1 and T2, are subunits of the transcription elongation factor P-TEFb (18). 5,6-Dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB) is another widely used transcriptional inhibitor (19) that inhibits CDK7 (20) and CDK9/PITALRE (21). The average CTD phosphorylation is decreased in cells exposed to DRB (7), suggesting that these kinases might contribute to global CTD phosphorylation in vivo.

The monoclonal antibody CC-3 recognizes a phosphodependent epitope on a 255 kDa nuclear matrix protein (p255) recently shown to associate with splicing complexes as part of the [U4/U6.U5] tri-snRNP particle [Chabot et al. (1995) Nucleic Acids Res. 23, 3206-3213]. In mouse and Drosophila cultured cells the electrophoretic mobility of p255, faster in the latter species, was identical to that of the hyperphosphorylated form of RNA polymerase II largest subunit (IIo). The CC-3 immunoreactivity of p255 was abolished by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, which is known to cause the dephosphorylation of the C-terminal domain of subunit IIo by inhibiting the TFIIH-associated kinase. The identity of p255 was confirmed by showing that CC-3-immunoprecipitated p255 was recognized by POL3/3 and 8WG16, two antibodies specific to RNA polymerase II largest subunit. Lastly, the recovery of RNA polymerase II largest subunit from HeLa splicing mixtures was compromised by EDTA, which prevents the interaction of p255 with splicing complexes and inhibits splicing. Our results indicate that p255 represents a highly phosphorylated form of RNA polymerase II largest subunit physically associated with spliceosomes and possibly involved in coupling transcription to RNA processing.

Xenopus laevis oogenesis is characterized by an active transcription which ceases abruptly upon maturation. To survey changes in the characteristics of the transcriptional machinery which might contribute to this transcriptional arrest, the phosphorylation status of the RNA polymerase II largest subunit (RPB1 subunit) was analyzed during oocyte maturation. We found that the RPB1 subunit accumulates in large quantities from previtellogenic early diplotene oocytes up to fully grown oocytes. The C-terminal domain (CTD) of the RPB1 subunit was essentially hypophosphorylated in growing oocytes from Dumont stage IV to stage VI. Upon maturation, the proportion of hyperphosphorylated RPB1 subunits increased dramatically and abruptly. The hyperphosphorylated RPB1 subunits were dephosphorylated within 1 h after fertilization or heat shock of the matured oocytes. Extracts from metaphase II-arrested oocytes showed a much stronger CTD kinase activity than extracts from prophase stage VI oocytes. Most of this kinase activity was attributed to the activated Xp42 mitogen-activated protein (MAP) kinase, a MAP kinase of the ERK type. Making use of artificial maturation of the stage VI oocyte through microinjection of a recombinant stable cyclin B1, we observed a parallel activation of Xp42 MAP kinase and phosphorylation of RPB1. Both events required protein synthesis, which demonstrated that activation of p34 cdc2 kinase was insufficient to phosphorylate RPB1 ex vivo and was consistent with a contribution of the Xp42 MAP kinase to RPB1 subunit phosphorylation. These results further support the possibility that the largest RNA polymerase II subunit is a substrate of the ERK-type MAP kinases during oocyte maturation, as previously proposed during stress or growth factor stimulation of mammalian cells.In Xenopus laevis, as in many species, transcription is arrested in the matured gametes. During oogenesis, proteins and RNAs accumulate to support early embryogenesis. Although the fully grown oocyte contains as much RNA polymerase II (RNAPII) activity as 10 5 individual somatic cells (43), little is known about its properties at maturation, after the germinal vesicle breakdown. From this perspective, we decided to investigate the phosphorylation of the RNAPII largest subunit (RPB1) in the developing and maturing oocytes. The RNAPII core enzyme is an assembly of 12 subunits (29, 45). Extensive studies have assigned an important role to the phosphorylation of the C-terminal domain (CTD) of RPB1 in regulating the initiation of transcription (11, 18). The hypophosphorylated RPB1 (IIa subunit) binds to the TATA box binding protein (TBP) within a preinitiation complex of transcription, this interaction being abolished by phosphorylation of the CTD (28, 52). The steady-state distribution between the hypophosphorylated IIa and the hyperphosphorylated IIo forms of RPB1 results from the antagonistic activity of CTD kinases and CTD phosphatases. A CTD phosphatase has recently been purified and characterized, but few data are available concerning...

The phosphorylation of the C-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II has been investigated in HeLa cells exposed to heat shock. In control cells, the phosphorylated subunit, IIo, and the dephosphorylated subunit, IIa, were found in similar amounts. During heat shock, however, the phosphorylated subunit, IIo, accumulated, whereas the amount of IIa subunit decreased. Since phosphorylation of the CTD had been suggested to play a role in the initiation of transcription and since heat shock was known to perturb gene expression at the level of transcription, the phosphorylation state of RNA polymerase II was examined in cells that had been treated with various inhibitors of transcription. Under normal growth temperature, actinomycin D (over 0.1 microgram/ml) and okadaic acid, a phosphatase inhibitor, were found to inhibit polymerase dephosphorylation. Whereas 5,6-dichlorobenzimidazole riboside (DRB), N-(2-[Methylamino]ethyl)-5-isoquinolinesulfonamide (H-8), and actinomycin D (over 5 micrograms/ml) were found to inhibit polymerase phosphorylation. Actinomycin D concentrations, which inhibited the dephosphorylation process, were lower than those required to inhibit the phosphorylation process. In contrast, during heat shock or exposure to sodium arsenite, a chemical inducer of the heat-shock response, the phosphorylated subunit, IIo, accumulated even in the presence of inhibitors of transcription such as DRB, H-8, and actinomycin D. These experiments demonstrated the existence of a heat-shock-induced CTD-phosphorylation process that might contribute to the regulation of transcription during stress.

The C-terminal domain (CTD) of the RNA polymerase II largest subunit (RPB1) plays a central role in transcription. The CTD is unphosphorylated when the polymerase assembles into a preinitiation complex of transcription and becomes heavily phosphorylated during promoter clearance and entry into elongation of transcription. A kinase associated to the general transcription factor TFIIH, in the preinitiation complex, phosphorylates the CTD. The TFIIH-associated CTD kinase activity was found to decrease in extracts from heat-shocked HeLa cells compared to unstressed cells. This loss of activity correlated with a decreased solubility of the TFIIH factor. The TFIIH-kinase impairment during heat-shock was accompanied by the disappearance of a particular phosphoepitope (CC-3) on the RPB1 subunit. The CC-3 epitope was localized on the C-terminal end of the CTD and generated in vitro when the RPB1 subunit was phosphorylated by the TFIIH-associated kinase but not by another CTD kinase such as MAP kinase. In apparent discrepancy, the overall RPB1 subunit phosphorylation increased during heat-shock. The decreased activity in vivo of the TFIIH kinase might be compensated by a stress-activated CTD kinase such as MAP kinase. These results also suggest that heat-shock gene transcription may have a weak requirement for TFIIH kinase activity.