No abstract

Different mutations were generated in the diffused-form early antigen (EA-D) of Epstein-Barr virus (EBV) for study of the effects of these mutations in DNA binding, stimulating the activity of EBV-specific DNA polymerase (EBV-DP), and binding to monoclonal antibody R3. Results revealed that the N-terminal 303 amino acids were essential for DNA binding and were sufficient for the enhancement of the activity of EBV-specific DNA polymerase. Deletion study also showed that the region recognized by the R3 monoclonal antibody was located between aa 315 and aa 377. Our results failed to demonstrate the binding between EA-D and EBV-DP, using the proteins synthesized in vitro, suggesting that direct contact between the two proteins is not required for the EBV-DP activity in vitro. We have generated fusion between EA-D and DNA-binding domain of yeast GAL4 protein; however, this fusion protein was not able to transactivate the promoter containing UAS sequence in P3HR1 cells.

Bacterially expressed Epstein-Barr virus (EBV) DNase was purified to 98% purity and used as the source for characterization of the enzyme activities. Complete digestion of DNA by EBV DNase yielded 5'-monophosphate nucleosides as the final products. During the logarithmic phase of the reaction, EBV DNase acted processively on dsDNA but distributively on ssDNA. Both 5' to 3' and 3' to 5' exonuclease activities were present, although the former was shown to be 10-fold stronger. No significant discrepancy was seen in the liberation of end-labeled nucleotides by DNase when substrates with 5'-protruding, blunt, or 3'-protruding ends were used. EBV DNase was demonstrated also to have an endonuclease activity using supercoiled plasmid DNA as substrate. Two preferential dsDNA cleavage sites were mapped on pBS-TR, a pBlueScript vector containing one copy of the EBV terminal repeat; both are in vector sequences. Finally, an N-terminally truncated EBV major DNA binding protein, but not EA-D, was shown to inhibit EBV DNase activity. This inhibitory effect may due to direct protein-protein interactions between EBV DNase and the major DNA binding protein. The biological significance of these characteristics is discussed.

We demonstrated previously that the 0.4-kilobase DNA fragment from Micromonospora echinospora contains multiple tandem promoters, Pla, Plb, Plc, and P2, which are also functional when cloned into Streptomyces lividans. We now show by in vitro transcription with Streptomyces RNA polymerase that each of these promoters is an authentic initiation site, rather than a processing site for transcripts which initiate further upstream. The DNA sequence requirements for the closely spaced promoters Pla, Plb, and Plc, which are coordinately induced during stationary phase in M. echinospora, were examined by deletional analysis in S. lividans. The Pla and Plb promoters were functional despite deletion of native sequences 5 and 17 base pairs upstream of each initiation site, respectively. Thus, Pla and Plb had greatly reduced upstream DNA sequence requirements compared with typical procaryotic promoters. In contrast, transcription from promoter Plc was significantly decreased when native sequences 34 base pairs upstream were replaced.We have been studying promoter structure in the actinomycete Micromonospora echinospora (NRRL 15839), which grows as multicellular mycelia and has the capacity to form spores after the growing phase. During the stationary phase, M. echinospora produces the calicheamicins (19), a novel family of antitumor antibiotics that causes site-specific double-stranded cleavage of DNA (29).Previously, we defined a set of multiple tandem promoters within a 0.4-kilobase (kb) DNA fragment that has several novel features (1). The P1 promoter region consists of three transcriptional start sites separated by 12 and 17 nucleotides (nt), which are maximally active during stationary phase, concurrent with calicheamicin production. Promoter P2 is located 80 base pairs (bp) downstream from the P1 region and is maximally active during the exponential phase of the life cycle. The P1 and P2 promoters are potentially useful for the investigation of gene activation in actinomycetes during the transition from exponential to stationary phase, when many secondary metabolites are produced (22).The P1 and P2 promoters, and an additional site designated Ptk, are recognized by Streptomyces lividans transformants harboring the 0.4-kb fragment on a plasmid (1). We have therefore utilized the well-characterized genetic system of Streptomyces for more detailed transcriptional analysis. The P1 and P2 promoters belong to the major class of Streptomyces promoters that do not function in Escherichia coli (12,15,16). A consensus sequence has not been identified for this class of promoters. The occurrence of multiple forms of RNA polymerase holoenzyme in Streptomyces spp. which direct transcription from different promoters (6, 28) is consistent with the diversity of promoter sequences found in this organism. Recently, four different genes apparently encoding sigma factors were identified in S. coelicolor by homology to sequences derived from E. coli and Bacillus subtilis (26). In addition, the whiG locus also encodes a putative sigma facto...