In this study we show, by immuno¯uorescence and electron microscopy immuno-gold labelling, that the major transforming protein of Human Papillomavirus type 16 E7 is associated with the nucleolus of cells derived from the HPV16-positive cervical carcinoma line CaSki. The E7 nucleolar staining appeared to be cell cycle dependent, being considerably reduced in the G 2 phase. The total level of the protein in the cell, however, remained constant during all phases. We also show that the cellular protein Rb1, which is targeted by E7, is localised in the nucleus and nucleolus in CaSki cells. Thus, it is possible that the presence of E7 in the nucleolus correlates with a hypothetical function(s) of Rb1 in this particular intranuclear compartment. The nucleolar localisation of HPV16 E7 protein was also observed in the ®ssion yeast Schizosaccharomyces pombe, suggesting that a targeting mechanism of HPV16 E7 protein into the nucleolus is common to both mammalian and yeast systems. Nucleolar localisation of HPV16 E7 protein may be independent from Rb1 since no Rb1 related proteins have been identi®ed in ®ssion yeast.
Deletion mutagenesis analysis of a duplicated gene necessary for Haemophilus influenzae serotype b capsule expression showed that only one functional copy of this gene is required for capsule production and for virulence in infant rats. Mutant strains generated in this study differed from each other and from the parental strain in their ability to maintain the large tandem duplication which contains the genes involved in serotype b capsule expression.
Copy number (Cop) mutants of miniR6-5 and miniF plasmid derivatives containing a beta-lactamase gene were isolated by selection for increased ampicillin-resistance. Mutants which exhibited an increased copy number and a reduced incompatibility response as compared to the respective parent mini-plasmid were obtained from both miniR6-5 and miniF. Characterization of these mutant plasmids has provided the first description of the replicative properties of miniF Cop mutants, and has also facilitated a comparison of plasmids representing the IncFI and IncFII incompatibility groups. Cop mutants from these groups differed in several respects: (i) MiniF Cop mutants were considerably more difficult to obtain and showed a markedly lower transforming efficiency than the corresponding miniR6-5 mutants. (ii) MiniR6-5 Cop mutants were stably maintained in a polA1 strain without selective pressure, whereas miniF Cop mutants severely reduced the viability of this host. (iii) MiniR6-5 replication stopped within a few minutes after inhibition of protein synthesis, whereas miniF replication continued at a declining rate for about one hour in the presence of chloramphenicol. (iv) In contrast to miniR6-5 replication, miniF DNA synthesis was blocked faster by rifampicin than by chloramphenicol. (v) The copy number of miniR6-5 plasmids (but not of miniF) was reduced by about 50% in an rnc strain deficient in RNAase III.
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