Xenopus Drf1, a Regulator of Cdc7, Displays Checkpoint-dependent Accumulation on Chromatin during an S-phase Arrest

We have cloned a Xenopus Dbf4-related factor named Drf1 and characterized this protein by using Xenopus egg extracts. Drf1 forms an active complex with the kinase Cdc7. However, most of the Cdc7 in egg extracts is not associated with Drf1, which raises the possibility that some or all of the remaining Cdc7 is bound to another Dbf4-related protein. Immunodepletion of Drf1 does not prevent DNA replication in egg extracts. Consistent with this observation, Cdc45 can still associate with chromatin in Drf1-depleted extracts, albeit at significantly reduced levels. Nonetheless, Drf1 displays highly regulated binding to replicating chromatin. Treatment of egg extracts with aphidicolin results in a substantial accumulation of Drf1 on chromatin. This accumulation is blocked by addition of caffeine and by immunodepletion of either ATR or Claspin. These observations suggest that the increased binding of Drf1 to aphidicolin-treated chromatin is an active process that is mediated by a caffeine-sensitive checkpoint pathway containing ATR and Claspin. Abrogation of this pathway also leads to a large increase in the binding of Cdc45 to chromatin. This increase is substantially reduced in the absence of Drf1, which suggests that regulation of Drf1 might be involved in the suppression of Cdc45 loading during replication arrest. We also provide evidence that elimination of this checkpoint causes resumed initiation of DNA replication in both Xenopus tissue culture cells and egg extracts. Taken together, these observations argue that Drf1 is regulated by an intra-S-phase checkpoint mechanism that down-regulates the loading of Cdc45 onto chromatin containing DNA replication blocks

Maintenance of replication forks and the S-phase checkpoint by Cdc18p and Orp1p

S-phase and DNA damage checkpoint controls block the onset of mitosis when DNA is damaged or DNA replication is incomplete. It has been proposed that damaged or incompletely replicated DNA generates structures that are sensed by the checkpoint control pathway, although little is known about the structures and mechanisms involved. Here, we show that the DNA replication initiation proteins Orp1p and Cdc18p are required to induce and maintain the S-phase checkpoint in Schizosaccharomyces pombe. The presence of DNA replication structures correlates with activation of the Cds1p checkpoint protein kinase and the S-phase checkpoint pathway. By contrast, induction of the DNA damage pathway is not dependent on Orp1p or Cdc18p. We propose that the presence of unresolved replication forks, together with Orp1p and Cdc18p, are necessary to activate the Cds1p-dependent S-phase checkpoint

A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi

<p>Abstract</p> <p>Background</p> <p>The misdiagnosis of <it>Plasmodium knowlesi </it>by microscopy has prompted a re-evaluation of the geographic distribution, prevalence and pathogenesis of this species using molecular diagnostic tools. In this report, a specific probe for <it>P. knowlesi</it>, that can be used in a previously described TaqMan real-time PCR assay for detection of <it>Plasmodium </it>spp., and <it>Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae </it>and <it>Plasmodium ovale</it>, was designed and validated against clinical samples.</p> <p>Methods</p> <p>A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within the <it>P. knowlesi </it>small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were determined using plasmids containing <it>P. knowlesi </it>DNA and genomic DNA of <it>P. falciparum, P. knowlesi, P. malariae, P. ovale </it>and <it>P. vivax </it>isolated from clinical samples. DNA samples of the simian malaria parasites <it>Plasmodium cynomolgi </it>and <it>Plasmodium inui </it>that can infect humans under experimental conditions were also examined together with human DNA samples.</p> <p>Results</p> <p>Analytical sensitivity of the <it>P. knowlesi</it>-specific assay was 10 copies/μL and quantitation was linear over a range of 10-10⁶copies. The sensitivity of the assay is equivalent to nested PCR and <it>P. knowlesi </it>DNA was detected from all 40 clinical <it>P. knowlesi </it>specimens, including one from a patient with a parasitaemia of three parasites/μL of blood. No cross-reactivity was observed with 67 <it>Plasmodium </it>DNA samples (31 <it>P. falciparum</it>, 23 <it>P. vivax</it>, six <it>P. ovale</it>, three <it>P. malariae</it>, one <it>P. malariae/P. ovale</it>, one <it>P. falciparum/P. malariae, one P. inui and one P. cynomolgi) </it>and four samples of human DNA.</p> <p>Conclusions</p> <p>This test demonstrated excellent sensitivity and specificity, and adds <it>P. knowlesi </it>to the repertoire of <it>Plasmodium </it>targets for the clinical diagnosis of malaria by real-time PCR assays. Furthermore, quantitation of DNA copy number provides a useful advantage over other molecular assays to investigate the correlation between levels of infection and the spectrum of disease.</p

An enclosed in-gel PCR amplification cassette with multi-target, multi-sample detection for platform molecular diagnostics

This work describes a self-contained, simple, disposable, and inexpensive gel capillary cassette for DNA amplification in near point of care settings. The cassette avoids the need for pumps or valves during raw sample delivery or polymerase chain reaction (PCR) amplification steps. The cassette contains capillary reaction units that can be stored at room temperature for up to 3 months. The current cassette configuration format can simultaneously tests up to 16 patients for two or more targets, accommodates different sample types on the same cassette, has integrated positive and negative controls and allows flexibility for multiple geometries. PCR reagents in the cassette are desiccated to allow storage at room temperature with rehydration by raw sample at the time of testing. The sample is introduced to the cassette via a transfer pipette simply by capillary force. DNA amplification was carried out in a portable prototype instrument for PCR thermal cycling with fluorescence detection of amplified products by melt curve analysis. To demonstrate performance, raw genital swabs and urine were introduced to the same cassette to simultaneously detect four sexually transmitted infections. Herpes Simplex Viruses (HSV-1 and HSV-2) were detected from raw genital swabs. Ureaplasma Urealyticum (UU) and Mycoplasma Homonis (MH) were detected from raw urine. Results for multiple patients were obtained in as little as 50'. This platform allows multiparameter clinical testing with a pre-assembled cassette that requires only the introduction of raw sample. Modification of the prototype device to accommodate larger cassettes will ultimately provide high throughput simultaneous testing of even larger numbers of samples for many different targets, as is required for most clinical applications. Combinations of wax and/or polymer cassettes holding capillary reaction units are feasible. The components of the cassette are suited to mass production and robotic assembly to produce a readily manufactured disposable reaction cassette that can be configured for disease-specific testing panels. Rapid testing with a disposable reaction cassette on an inexpensive instrument will permit on the spot evaluation of patients in the clinic for faster medical decision-making and more informed therapeutic choices

Cross-species Malaria Immunity Induced By Chemically Attenuated Parasites

Vaccine development for the blood stages of malaria has focused on the induction of antibodies to parasite surface antigens, most of which are highly polymorphic. An alternate strategy has evolved from observations that low-density infections can induce antibody-independent immunity to different strains. To test this strategy, we treated parasitized red blood cells from the rodent parasite Plasmodium chabaudi with secocyclopropyl pyrrolo indole analogs. These drugs irreversibly alkylate parasite DNA, blocking their ability to replicate. After administration in mice, DNA from the vaccine could be detected in the blood for over 110 days and a single vaccination induced profound immunity to different malaria parasite species. Immunity was mediated by CD4(+) T cells and was dependent on the red blood cell membrane remaining intact. The human parasite, Plasmodium falciparum, could also be attenuated by treatment with seco-cyclopropyl pyrrolo indole analogs. These data demonstrate that vaccination with chemically attenuated parasites induces protective immunity and provide a compelling rationale for testing a blood-stage parasite-based vaccine targeting human Plasmodium species