Drosophila ISWI, a highly conserved member of the SWI2/SNF2 family of ATPases, is the catalytic subunit of three chromatin-remodeling complexes: NURF, CHRAC, and ACF. To clarify the biological functions of ISWI, we generated and characterized null and dominant-negative ISWI mutations. We found that ISWI mutations affect both cell viability and gene expression during Drosophila development. ISWI mutations also cause striking alterations in the structure of the male X chromosome. The ISWI protein does not colocalize with RNA Pol II on salivary gland polytene chromosomes, suggesting a possible role for ISWI in transcriptional repression. These findings reveal novel functions for the ISWI ATPase and underscore its importance in chromatin remodeling in vivo.

Drosophila brahma (brm) encodes the ATPase subunit of a 2 MDa complex that is related to yeast SWI/SNF and other chromatin-remodeling complexes. BRM was identi®ed as a transcriptional activator of Hox genes required for the speci®cation of body segment identities. To clarify the role of the BRM complex in the transcription of other genes, we examined its distribution on larval salivary gland polytene chromosomes. The BRM complex is associated with nearly all transcriptionally active chromatin in a pattern that is generally non-overlapping with that of Polycomb, a repressor of Hox gene transcription. Reduction of BRM function dramatically reduces the association of RNA polymerase II with salivary gland chromosomes. A few genes, such as induced heat shock loci, are not associated with the BRM complex; transcription of these genes is not compromised by loss of BRM function. The distribution of the BRM complex thus correlates with a dependence on BRM for gene activity. These data suggest that the chromatin remodeling activity of the BRM complex plays a general role in facilitating transcription by RNA polymerase II.

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Tetracycline analogs fell into two classes on the basis of their mode of action. Tetracycline, chlortetracycline, minocycline, doxycycline, and 6-demethyl-6-deoxytetracycline inhibited cell-free translation directed by either Escherichia coli or Bacillus subtilis extracts. A second class of analogs tested, including chelocardin, anhydrotetracycline, 6-thiatetracycline, anhydrochlortetracycline, and 4-epi-anhydrochlortetracycline, failed to inhibit protein synthesis in vitro or were very poor inhibitors. Tetracyclines of the second class, however, rapidly inhibited the in vivo incorporation of precursors into DNA and RNA as well as protein. The class 2 compounds therefore have a mode of action that is entirely distinct from the class 1 compounds, such as tetracycline that are used clinically. Although tetracyclines of the second class entered the cytoplasm, the ability of these analogs to inhibit macromolecular synthesis suggests that the cytoplasmic membrane is their primary site of action. The interaction of class 1 and class 2 tetracyclines with ribosomes was studied by examining their effects on the chemical reactivity of bases in 16S rRNA to dimethyl sulfate. Class 1 analogs affected the reactivity of bases to dimethyl sulfate. The response with class 2 tetracyclines varied, with some analogs affecting reactivity and others (chelocardin and 4-epi-anhydrotetracycline) not.The tetracyclines are a group of broad-spectrum antibiotics which are generally considered to prevent bacterial growth by inhibiting protein synthesis. This results from binding of antibiotic to a single site in the 30S ribosomal subunit which prevents attachment of aminoacyl tRNA to the ribosomal acceptor site (3). In order to reach the ribosome, these antibiotics must traverse the hydrophobic lipid bilayer of the bacterial cytoplasmic membrane (3). At physiological pH tetracyclines can exist as an equilibrium mixture of two free base forms: a low-energy, lipophilic nonionized species and a high-energy, hydrophilic zwitterionic structure (7). A solvent-dependent equilibrium between the two forms has been demonstrated with oxytetracycline free base and is supported by X-ray analyses of tetracyclines crystallized from aqueous and nonaqueous solvents (7,15). Both forms are believed to be important for the antibacterial activity of tetracyclines, the low-energy, lipophilic conformational form (Fig. 1A) for uptake across the cytoplasmic membrane and the hydrophilic, zwitterionic structure (Fig. 1B) for binding to the ribosome (7).Chelocardin (14) is a naturally occurring anhydrotetracycline derivative with a modified A ring (Fig. 2). In contrast to the solvent-dependent equilibrium of the two tetracycline species mentioned above, chelocardin apparently exists in the same conformation in both polar and nonpolar solvents, as evidenced by circular dichroism measurements (6). We believe this is related to the planarity of the BCD rings in chelocardin and that a lipophilic form, perhaps related to that of tetracycline, is the preferred species. Thes...