We have investigated the expression of the bacteriorhodopsin gene (bop) and the gas vesicle protein gene (gvpA) in the extremely halophilic archaebacterium Halobacterium halobium, using primer-directed reverse transcription of RNA to quantify message levels. The level of gvpA gene transcript was found to increase about 5-fold from early to mid-logarithmic growth phase, while the level of bop gene transcript increased about 20-fold from mid-logarithmic to stationary phase. Transcriptional induction of both the gvpA and bop genes was significantly reduced by aeration and almost completely blocked by the DNA gyrase inhibitor novobiocin.

The bop gene of wild-type Halobacterium halobium NRC-1 is transcriptionally induced more than 20-fold under microaerobic conditions. bop transcription is inhibited by novobiocin, a DNA gyrase inhibitor, at concentrations subinhibitory for growth. The exposure of NRC-1 cultures to novobiocin concentrations inhibiting bop transcription was found to partially relax plasmid DNA supercoiling, indicating the requirement of high DNA supercoiling for bop transcription. Next, the bop promoter region was cloned on an H. halobium plasmid vector and introduced into NRC-1 and S9, a bop overproducer strain. The cloned promoter was active in both H. halobium strains, but at a higher level in the overproducer than in the wild type. Transcription from the bop promoter on the plasmid was found to be inhibited by novobiocin to a similar extent as was transcription from the chromosome. When the cloned promoter was introduced into S9 mutant strains with insertions in either of two putative regulatory genes, brp and bat, no transcription was detectable, indicating that these genes serve to activate transcription from the bop promoter in trans. Deletion analysis of the cloned bop promoter from a site ϳ480 bp upstream of bop showed that a 53-bp region 5 to the transcription start site is sufficient for transcription, but a 28-bp region is not. An 11-bp alternating purine-pyrimidine sequence within the functional promoter region, centered 23 bp 5 to the transcription start point, was found to display DNA supercoiling-dependent sensitivity to S1 nuclease and OsO 4 , which is consistent with a non-B-DNA conformation similar to that of left-handed Z-DNA and suggests the involvement of unusual DNA structure in supercoiling-stimulated bop gene transcription.The bop gene, encoding bacterio-opsin, the purple membrane protein of Halobacterium halobium, was one of the first archaeal genes to be cloned and sequenced (11). After cloning, several studies focused on the transcription of the bop gene and its regulation by oxygen and light. The start site for transcription was shown to map just 2 nucleotides upstream of the coding region by purification of the message, capping of its 5Ј end, and sequencing (6). Although this established bop mRNA as a primary transcript, no easily identifiable promoter could be found. Only a weak similarity to the TATA-like (box A) element was present (17). Interestingly, an 11-bp-long alternating purine-pyrimidine sequence centered 23 bp 5Ј to the transcription start site was observed and hypothesized to influence transcription by adopting left-handed Z-DNA conformation, which would be stabilized by high salt concentration and DNA supercoiling in H. halobium.The bop gene was shown to be transcriptionally induced at least 20-fold under microaerobic conditions in wild-type H. halobium strains (36, 43). Interestingly, bop gene transcription was inhibited by novobiocin, suggesting the involvement of DNA supercoiling in the modulation of promoter activity (42). Moreover, the involvement of two upstream genes in bop gene tr...

cDNAs corresponding to RNA from the autonomous parvovirus minute virus of mice were cloned into constitutive and inducible expression vectors. These clones generate viral NS2, VP1, and VP2 proteins individually. Initial examination of these clones by transient expression analysis and analysis of stably transformed murine cell lines inducibly expressing these constructs indicated that they will be useful tools for characterizing the function of individual minute virus of mice gene products.

To evaluate the relative importance of alternating d(CG) sequence length, DNA supercoiling, and salt in left-handed Z-DNA formation, plasmids containing short d(CG)n sequences (n = 3-17) with the capability of replicating in either Escherichia coli or the halophilic archaeum Halobacterium halobium were constructed. Z-DNA conformation in the d(CG)n sequences was assayed by (i) a band shift assay using the Z-DNA-specific Z22 monoclonal antibody (ZIBS assay); (ii) an S1 nuclease cleavage-primer extension assay to map B-Z junctions; and (iii) a BssHII restriction inhibition assay. Using the ZIBS assay on plasmids purified from E. coli, the transition from B-DNA to Z-DNA occurred from d(CG)4, to d(CG)5, with 20% of d(CG)4, and 90% of d(CG)5 in Z-DNA conformation. These findings were consistent with the results of S1 nuclease cleavage observed at B-Z junctions flanking d(CG)4 and d(CG)5 sequences. Resistance to BssHII restriction endonuclease digestion was observed only in supercoiled plasmids containing d(CG)8 or longer sequences, indicating that shorter d(CG)n sequences are in dynamic equilibrium between B- and Z-DNA conformations. When a plasmid containing d(CG)4, was isolated from a topA mutant of E. coli, it contained 25% greater linking deficiency and 40% greater Z-DNA conformation in the alternating d(CG) region. In plasmids purified from H. halobium, which showed 30% greater linking deficiency than from E. coli, 20-40% greater Z-DNA formation was found in d(CG)4-6 sequences. Surprisingly, no significant difference in Z-DNA formation could be detected in d(CG)3-17 sequences in plasmids from either E. coli or H. halobium in the NaCl concentration range of 0.1-4 M.