Roughex mediates G(1) arrest through a physical association with cyclin A.

Differentiation in the developing Drosophila eye requires synchronization of cells in the G(1) phase of the cell cycle. The roughex gene product plays a key role in this synchronization by negatively regulating cyclin A protein levels in G(1). We show here that coexpressed Roughex and cyclin A physically interact in vivo. Roughex is a nuclear protein, while cyclin A was previously shown to be exclusively cytoplasmic during interphase in the embryo. In contrast, we demonstrate that in interphase cells in the eye imaginal disk cyclin A is present in both the nucleus and the cytoplasm. In the presence of ectopic Roughex, cyclin A becomes strictly nuclear and is later degraded. Nuclear targeting of both Roughex and cyclin A under these conditions is dependent on a C-terminal nuclear localization signal in Roughex. Disruption of this signal results in cytoplasmic localization of both Roughex and cyclin A, confirming a physical interaction between these molecules. Cyclin A interacts with both Cdc2 and Cdc2c, the Drosophila Cdk2 homolog, and Roughex inhibits the histone H1 kinase activities of both cyclin A-Cdc2 and cyclin A-Cdc2c complexes in whole-cell extracts. Two-hybrid experiments suggested that the inhibition of kinase activity by Roughex results from competition with the cyclin-dependent kinase subunit for binding to cyclin A. These findings suggest that Roughex can influence the intracellular distribution of cyclin A and define Roughex as a distinct and specialized cell cycle inhibitor for cyclin A-dependent kinase activity.

F0F1-ATPase from Vibrio alginolyticus. Subunit composition and proton pumping activity.

An F0F1-ATPase was isolated from the membranes of the marine bacterium Vibrio alginolyticus. Homology between the subunits of the F0-complexes from E. coli and V. alginolyticus was found using antibodies against subunits a, b and c of the E. coli F0F1-ATPase. The F0F1-complex from V. alginolyticus was reconstituted into proteoliposomes, which were competent in ATP-dependent proton uptake. This process was inhibited by triphenyltin, DCCD, and venturicidin. Na+ did not affect proton translocation.

The effect of F0 inhibitors on the Vibrio alginolyticus membrane ATPase.

The inhibition of membrane ATPase from the marine alkalotolerant bacterium Vibrio alginolyticus by DCCD, triphenyltin and venturicidin was studied. DCCD proved to be an irreversible inhibitor, while venturicidin and triphenyltin produced a reversible inhibitory effect. The DCCD-binding proteolipid was identified in the membrane preparations. The effect of the inhibitors on ATPase activity and ATP-dependent Na(+)-transport in V. alginolyticus subcellular vesicles is discussed.

Cyanide-reactive sites in cytochrome bd complex from E. coli.

Cyanide reacts with cytochrome bd from E. coli in an 'aerobically oxidized' state (mainly, an oxygenated complex b558(3+) b595(3+) d(2+)-O2), bringing about (i) decomposition of the heme d2+ oxycomplex (decay of the 648 nm absorption band) and (ii) extensive red shift in the Soret region accompanied by minor changes in the visible range assigned to ferric heme b595. MCD spectra show that the Soret red shift is associated with heme b595(3+) high-to low-spin transition. This is the first unambiguous demonstration that heme b595 can bind exogenous ligands. No reaction of cyanide with b558 is observed. In about 70% of the enzyme which forms the cyano complex, the spin-state transition of b595 decay of heme d oxycomplex match each other kinetically (keff ca. 0.002 s-1 at 50 mM KCN, pH 8.1, 25 degrees C). This points to an interaction between the two hemes. The concerted binding of cyanide to d3+ and b595(3+), perhaps as a bridging ligand, is probably rate-limited by d2+ oxycomplex autoxidation. In the remaining 30% of the isolated bd, there is a rapid phase of cyanide-induced b595 spin-state transition which can be tentatively assigned to that proportion of the enzyme in which heme d is initially in the ferric rather than ferrous-oxy form.

Selection and analysis of rare second-site suppressors of Drosophila RNA polymerase II mutations.

We used a mutagenesis and selection procedure in Drosophila melanogaster to recover rare allele-specific suppressor mutations. More than 11 million flies mutant for one of five recessive-lethal mutations in the two largest subunits of RNA polymerase II were selected for additional mutations that restored viability. Forty-one suppressor mutations were recovered. At least 16 are extragenic, identifying a minimum of three loci, two of which do not map near genes known to encode subunits of RNA polymerase II. At most, 25 are intragenic, 4 reverting the initial altered nucleotide back to wild type. Sequence analysis of interacting mutations in the two largest subunits identified a discrete domain in each subunit. These domains might be contact points for the subunits. Finally, our selections were large enough to allow recovery of multiple independent changes in the same nucleotides yet mutations in other equally likely targets were not recovered. The mutations recovered are not random and might provide insights into possible mechanisms for mutagenesis in eukaryotes.