Cyclin-dependent kinase 2 (Cdk2) is required for centrosome duplication in mammalian cells. (9/836)

Centrosome duplication is indispensable for the formation of the bipolar mitotic spindle. Surprisingly, even if DNA replication or mitosis is inhibited, centrosome duplication can still occur [1] [2] [3] [4] [5]. Thus, it remains unknown how centrosome duplication is coordinated with the cell cycle. Here, we show that centrosome duplication requires cyclin-dependent kinase 2 (Cdk2) in mammalian cells. We have found that in Chinese hamster ovary (CHO) cells, whereas centrosome duplication is not inhibited by hydroxyurea (HU) treatment, which arrests the cells in S phase, it is inhibited by mimosine treatment, which arrests the cells in late G1 phase. Cdk2 activity was higher in HU-treated cells than in mimosine-treated cells. Remarkably, inhibition of the Cdk2 activity in HU-treated cells with butyrolactone I or roscovitine [6], or by expression of the Cdk inhibitor p21(Waf1/Cip1), blocked the continued centrosome duplication. Moreover, overexpression of Cdk2 reversed the inhibition of centrosome duplication by mimosine treatment. These results indicate a requirement of Cdk2 activity for centrosome duplication and therefore suggest an underlying mechanism for the coordination of centrosome duplication with the cell cycle.  (+info)

Identification and in vivo functional analysis of a virginiamycin S resistance gene (varS) from Streptomyces virginiae. (10/836)

BarA of Streptomyces virginiae is a specific receptor protein for virginiae butanolide (VB), one of the gamma-butyrolactone autoregulators of the Streptomyces species, and acts as a transcriptional regulator controlling both virginiamycin production and VB biosynthesis. The downstream gene barB, the transcription of which is under the tight control of the VB-BarA system, was found to be transcribed as a polycistronic mRNA with its downstream region, and DNA sequencing revealed a 1,554-bp open reading frame (ORF) beginning at 161 bp downstream of the barB termination codon. The ORF product showed high homology (68 to 73%) to drug efflux proteins having 14 transmembrane segments and was named varS (for S. virginiae antibiotic resistance). Heterologous expression of varS with S. lividans as a host resulted in virginiamycin S-specific resistance, suggesting that varS encoded a virginiamycin S-specific transport protein. Northern blot analysis indicated that the bicistronic transcript of barB-varS appeared 1 to 2 h before the onset of virginiamycin M1 and S production, at which time VB was produced, while exogenously added virginiamycin S apparently induced the monocistronic varS transcript.  (+info)

Two-component transcriptional regulation of N-acyl-homoserine lactone production in Pseudomonas aureofaciens. (11/836)

Production of phenazine antibiotics by the biological control bacterium Pseudomonas aureofaciens 30-84 is regulated in part by the PhzI/PhzR N-acyl-homoserine lactone (AHL) response system (L. S. Pierson III, V. D. Keppenne, and D. W. Wood, J. Bacteriol. 176:3966-3974, 1994; D. W. Wood and L. S. Pierson III, Gene 168:49-53, 1996). Two mutants, 30-84W and 30-84.A2, were isolated and were found to be deficient in the production of phenazine, protease, hydrogen cyanide (HCN), and the AHL signal N-hexanoyl-homoserine lactone. These mutants were not complemented by phzI, phzR, or the phenazine biosynthetic genes (phzFABCD) (L. S. Pierson III, T. Gaffney, S. Lam, and F. Gong, FEMS Microbiol. Lett. 134:299-307, 1995). A 2.2-kb region of the 30-84 chromosome which fully restored production of all of these compounds in strain 30-84W was identified. Nucleotide sequence analysis of this region revealed a single open reading frame encoding a predicted 213-amino-acid protein which is very similar to the global response regulator GacA. Strain 30-84.A2 was not complemented by gacA or any cosmid from a genomic library of strain 30-84 but was complemented by gacS (formerly lemA) homologs from Pseudomonas fluorescens Pf-5 (N. Corbel and J. E. Loper, J. Bacteriol. 177:6230-6236, 1995) and Pseudomonas syringae pv. syringae B728a (E. M. Hrabek and D. K. Willis, J. Bacteriol. 174:3011-3020, 1992). Transcription of phzR was not altered in either mutant; however, phzI transcription was eliminated in strains 30-84W and 30-84.A2. These results indicated that the GacS/GacA two-component signal transduction system of P. aureofaciens 30-84 controls the production of AHL required for phenazine production by mediating the transcription of phzI. Addition of exogenous AHL did not complement either mutant for phenazine production, indicating that the GacS/GacA global regulatory system controls phenazine production at multiple levels. Our results reveal for the first time a mechanism by which a two-component regulatory system and an AHL-mediated regulatory system interact.  (+info)

Checkpoint control of the G2/M phase transition during the first mitotic cycle in mammalian eggs. (12/836)

The high incidence of chromosomally abnormal human embryos is frequently assumed to be due to a lack of checkpoint controls operating during early embryogenesis. In our study we have analysed when these mechanisms first become functional. Mouse oocytes treated in late metaphase I with either of two different cyclin-dependent kinase inhibitors [butyrolactone 1 (BL1) or 6-dimethylaminopurine (6-DMAP)] form nuclei in the cytoplasm. BL1-treated eggs enter S-phase at 16-18 h post-treatment and, after completion of DNA synthesis, cleave to 2-cell stage embryos. 6-DMAP treatment results in the rapid initiation of DNA synthesis, its completion by 12 h and then arrest in the G2 phase. Thus, two different cell cycle stages can be obtained at the same time point after the initiation of treatment: G1- after BL1 and G2-staged nuclei after 6-DMAP treatment. That this approach greatly facilitates cell cycle studies has been shown by analysing checkpoint function during the first division. Whilst G2-staged eggs enter M phase within 2-3 h when 6-DMAP is washed out, the onset of M phase is delayed after their fusion to G1 (BL1) cells. Here M phase occurs only after the less advanced nucleus completes DNA replication. Our results indicate that checkpoints in mammalian eggs are functional during the first mitotic cycle.  (+info)

Analysis of quorum-sensing-dependent control of rhizosphere-expressed (rhi) genes in Rhizobium leguminosarum bv. viciae. (13/836)

The rhi genes of Rhizobium leguminosarum biovar viciae are expressed in the rhizosphere and play a role in the interaction with legumes, such as the pea. Previously (K. M. Gray, J. P. Pearson, J. A. Downie, B. E. A. Boboye, and E. P. Greenberg, J. Bacteriol. 178:372-376, 1996) the rhiABC operon had been shown to be regulated by RhiR and to be induced by added N-(3-hydroxy-7-cis-tetradecenoyl)-L-homoserine lactone (3OH, C14:1-HSL). Mutagenesis of a cosmid carrying the rhiABC and rhiR gene region identified a gene (rhiI) that affects the level of rhiA expression. Mutation of rhiI slightly increased the number of nodules formed on the pea. The rhiI gene is (like rhiA) regulated by rhiR in a cell density-dependent manner. RhiI is similar to LuxI and other proteins involved in the synthesis of N-acyl-homoserine lactones (AHLs). Chemical analyses of spent culture supernatants demonstrated that RhiI produces N-(hexanoyl)-L-homoserine lactone (C6-HSL) and N-(octanoyl)-L-homoserine lactone (C8-HSL). Both of these AHLs induced rhiA-lacZ and rhiI-lacZ expression on plasmids introduced into an Agrobacterium strain that produces no AHLs, showing that rhiI is positively regulated by autoinduction. However, in this system no induction of rhiA or rhiI with 3OH,C14:1-HSL was observed. Analysis of the spent culture supernatant of the wild-type R. leguminosarum bv. viciae revealed that at least seven different AHLs are made. Mutation of rhiI decreased the amounts of C6-HSL and C8-HSL but did not block their formation, and in this background the rhiI mutation did not significantly affect the expression levels of the rhiI gene or rhiABC genes or the accumulation of RhiA protein. These observations suggest that there are additional loci involved in AHL production in R. leguminosarum bv. viciae and that they affect rhiI and rhiABC expression. We postulate that the previously observed induction of rhiA by 3OH,C14:1-HSL may be due to an indirect effect caused by induction of other AHL production loci.  (+info)

Possible involvement of cAMP in aerial mycelium formation and secondary metabolism in Streptomyces griseus. (14/836)

In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) triggers secondary metabolism and morphogenesis by binding a repressor protein (ArpA) and dissociating it from DNA. UV-mutagenesis of the A-factor-deficient mutant HH1 generated strain HO2, defective in the synthesis of ArpA and therefore able to form aerial mycelium, spores and streptomycin. Shotgun cloning of chromosomal DNA from wild-type S. griseus in strain HO2 yielded a gene that suppressed aerial mycelium formation and streptomycin production. Nucleotide sequencing and subcloning revealed that the gene encoded a eukaryotic-type adenylate cyclase (CyaA). In mutant HO2 production of cAMP was growth-dependent until the middle of the exponential growth stage; the production profile was the same as in the wild-type strain. However, the amount of cAMP produced was five times larger when mutant HO2 harboured cyaA on the high-copy-number plasmid pIJ486. Consistent with this, supplying cAMP exogenously at a high concentration to mutant HO2 suppressed formation of both aerial mycelium and streptomycin. On the other hand, some lower concentrations of cAMP stimulated or accelerated aerial mycelium formation. No effects of exogenous cAMP on morphogenesis and secondary metabolism were apparent in the wild-type strain. In addition, disruption of the chromosomal cyaA gene in the wild-type strain had almost no effect. Introducing cyaA cloned in either a low- or a high-copy-number plasmid suppressed morphogenesis and secondary metabolism not only in mutant HO2 but also in other arpA mutants, implying that the effects of cAMP became apparent in the arpA-defective background. When mutant HO2 carried cyaA on a plasmid, synthesis of the stringent response factor ppGpp was greatly reduced; this may account for the observed suppression by cAMP of morphogenesis and secondary metabolism. cAMP also affected protein tyrosine phosphorylation, as determined with antiphosphotyrosine antibody.  (+info)

Swarming by Pseudomonas syringae B728a requires gacS (lemA) and gacA but not the acyl-homoserine lactone biosynthetic gene ahlI. (15/836)

Pseudomonas syringae pv. syringae B728a, a causal agent of bacterial brown spot on snap beans, swarms with a characteristic dendritic pattern on semisolid (0.4%) agar plates. Filamentation of swarming cells of B728a was not observed. Mutations in either the gacS (formerly lemA) or gacA gene of B728a eliminate the ability of this P. syringae isolate to swarm without obvious effects on bacterial motility. Three field isolates showed a similar dependence on gacS for swarming. Since gacS and gacA mutants are known to be deficient in N-acyl-L-homoserine lactone (acyl-HSL) production, a mutant was constructed by disruption of the ahlI gene of B728a. This mutant did not make any acyl-HSL detectable by the Agrobacterium traG::lacZ reporter system, yet was unaffected in its ability to swarm. Other phenotypes of gacS and gacA mutations were similarly unaffected in the ahlI mutant.  (+info)

The novel transmembrane Escherichia coli proteins involved in the amino acid efflux. (16/836)

A novel gene of Escherichia coli, rhtB, has been characterized. Amplification of this gene provides resistance to homoserine and homoserine lactone. Another E. coli gene, rhtC, provides resistance to threonine. The homologues of RhtB are widely distributed among various eubacteria and archaea, from one to 12 copies of family members that differ in their primary structure were found in the genomes. Most of them are genes that encode hypothetical transmembrane proteins. Experimental data that indicate participation of the rhtB product in the excretion of homoserine have been obtained.  (+info)