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The initiation of DNA replication involves a minimum of four factors: a specific DNA sequence (origin), an initiator protein which binds to the origin, a helicase that unwinds the origin and a protein that binds single-stranded DNA that stabilizes the unwound origin. In eukaryotic cells, the origin recognition complex (ORC) is the initiator protein and replication protein A (RPA; ref. 3) is the single-stranded DNA-binding protein. However, the helicase has not been identified and the nature of origins remains elusive, except in the case of Saccharomyces cerevisiae. A unique feature of eukaryotic DNA replication is that it occurs at a few-hundred discrete foci. It has thus been proposed that a real origin must contain a specific DNA sequence and must be attached to replication foci. Using Xenopus laevis egg extracts, we have identified and purified a 170-kD protein, focus-forming activity 1 (FFA-1), which is required for the formation of replication foci. Here we report that FFA-1 has DNA-helicase activity. Moreover, it is a homologue of the human Werner syndrome gene product WRN, a protein associated with premature ageing in humans.

The plasmid virulence genes spvABCD of Salmonella spp. are regulated by SpvR and the stationary-phase sigma factor RpoS. The transcription of spv genes is induced during the post-exponential phase of bacterial growth in vitro. We sought to investigate the relationship between growth phase and RpoS in spv regulation. rpoS insertion mutations were constructed in S. dublin Lane and plasmid-cured LD842 strains, and the mutants were found to be attenuated for virulence and deficient in spv gene expression. We utilized the plasmid pBAD::rpoS to express rpoS independent of the growth phase under the control of the arabinose-inducible araBAD promoter. SpvA expression was induced within 2 h after the addition of 0.1% arabinose, even when bacteria were actively growing. This suggested that the level of RpoS, instead of the growth phase itself, controls induction of the spv genes. However, RpoS did not activate transcription of spvA in the absence of SpvR protein.Using a constitutive tet promoter to express spvR, we found that the spvA gene can be partially expressed in the rpoS mutant, suggesting that RpoS is required for SpvR synthesis. We confirmed that spvR is poorly expressed in the absence of RpoS. With an intact rpoS gene, spvR expression is not dependent on an intact spvR gene but is enhanced by spvR supplied in trans. We propose a model for Salmonella spv gene regulation in which both RpoS and SpvR are required for maximal expression at the spvR and spvA promoters.The virulence of the host-adapted nontyphoid Salmonella strains, including S. dublin, S. choleraesuis, S. gallinarum-pullorum, and S. abortusovis, is associated with the presence of large plasmids of approximately 50 to 100 kb (12, 15). Many isolates of the broad-host range serovars S. typhimurium and S. enteritidis also contain virulence plasmids. Elimination of these plasmids results in the loss of virulence in mouse models of systemic Salmonella infection. It has been shown that the S. dublin plasmid pSDL2 plays an important role in multiplication within the reticuloendothelial system but not in colonization and invasion of the intestine and Peyer's patches in mice (16).While virulence plasmids of different serovars may vary considerably in size and in overall nucleotide sequences, the core virulence genes, designated spv (salmonella plasmid virulence), are highly conserved among all serovars (31). The spv coding region consisting of the regulatory gene spvR and the structural genes spvABCD spans approximately 6 kb (13,23). It has been shown that spvABCD genes form a single operon and are transcribed from the same promoter located upstream of spvA. mRNA transcripts terminate at different sites, resulting in messages of various length containing spvA, spvAB, spvABC, and spvABCD (22). spvR is located directly upstream of the spv-ABCD genes and is transcribed as a distinct message in the same orientation. Mutational analysis of the spv region from S. dublin indicates that spvR and spvB are essential for mouse virulence (41). Studies of S. typhimurium, ho...

Autoinducer-2 (AI-2) is a quorum-sensing signal molecule that controls a variety of cellular activities in response to cell density in both gram-negative and gram-positive bacteria. The production of AI-2 is dependent upon LuxS, the last enzyme in the AI-2 biosynthesis pathway. For this study, we constructed a luxS null mutation (Delta luxS) in Campylobacter jejuni strain 81-176, and showed that it abolished AI-2 production. The Delta luxS mutant had a longer doubling time in Mueller-Hinton (MH) broth and reduced swarming on MH soft agar at 37 degrees C compared to the wild type (wt), whereas growth rate or swarming at 42 degrees C was not affected. The Delta luxS mutant was also more sensitive to hydrogen peroxide (H(2)O(2)) and cumene hydroperoxide than the wt by disc inhibition assays at 42 degrees C, though minimum inhibitory concentration comparisons were inconclusive. Differences in genome-wide gene expression between wt and Delta luxS mutant with and without H(2)O(2) treatments were compared using DNA microarrays. The genes that showed differential expressions (wt/Delta luxS) include operons/pathways involved in AI-2 synthesis and S-adenosylmethionine (SAM) metabolism (metE, metF, and pfs), flagellar assembly/regulation, stress response (ahpC, tpx, and groES), ABC transporters/efflux systems, and two genes of unknown function located downstream of luxS (Cj1199 and Cj1200). The wt/Delta luxS expression ratios of ahpC (encoding alkyl hydroperoxide reductase) and tpx (encoding thiol peroxidase) were increased only with H(2)O(2) treatment, consistent with our finding that the Delta luxS mutant exhibits higher sensitivity to oxidative stress than wt. Our microarray results agreed with the Delta luxS mutant phenotypes, and suggested that LuxS plays a role in central metabolism involving SAM metabolism, but it is uncertain whether AI-2 functions as a true quorum-sensing signal in C. jejuni.

BACKGROUNDLung cancer is the leading cause of cancer death in Taiwan, and the paucity of dependable risk markers has impeded the early management of lung cancer. An association of human papillomavirus (HPV) 16/18 infection with lung cancer among nonsmoking Taiwanese women was revealed in our previous study.METHODSNested PCR was employed to detect HPV 16/18 DNA in the blood circulation of 149 lung cancer patients and 174 noncancer controls. In addition, correlation of prevalence of HPV DNA between the blood circulation and lung tumor tissue was compared from 70 sets of paired tumor tissues and peripheral blood samples available.RESULTSThe results showed that the prevalence rate of HPV 16/18 in the blood circulation of lung cancer cases was significantly higher than that of noncancer controls (47.7% vs. 12.6% for HPV 16, P < 0.0001; 30.9% vs. 5.2% for HPV 18, P < 0.0001). A significantly higher HPV 16 prevalence was detected in female lung cancer patients than that of male (57.6% vs. 41.1%, P = 0.048), as well as in cases with tumor Stages III/IV than those with tumor Stages I/II (54.6% vs. 29.3%, P = 0.006). After adjusting the effects of age, gender, and smoking status, a 6.5‐fold greater risk of lung cancer was demonstrated for those subjects with HPV Type 16 positive (95% CI 3.7–11.3, P < 0.0001), a 9.2‐fold for HPV Type 18 positive (95% CI 4.2–20.2, P < 0.0001), and a 75.7‐fold greatest risk for those with both HPV Type 16 and 18 positive (95% CI 9.8–582.1, P < 0.0001).CONCLUSIONSThese results suggested that the presence of HPV DNA in the blood circulation may serve as a feasible risk marker of lung cancer. Cancer 2003;97:1558–63. © 2003 American Cancer Society.DOI 10.1002/cncr.11191