2,277 research outputs found
Olitsky, Peter K. -- 1935-64 -- Correspondence, Individual -- letter, 1953-05-06
No full textLetter from Gasser, Herbert S. to Schlesinger, R. Walter dated 1953-05-06.Sabin Collection Fair Use Policy</a
Use of a molecular approach for the definitive diagnosis of proliferative larval mesocestodiasis in a cat
No full textPractical PCR tools for the delineation of Contracaecum rudolphii A and Contracaecum rudolphii B (Ascaridoidea : Anisakidae) using genetic markers in nuclear ribosomal DNA
No full textUsing genetic markers defined previously in the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA), PCR-coupled restriction fragment length polymorphism (PCR-RFLP) and specific PCR assays were established for the specific detection of each of two morphologically indistinguishable operational taxonomic units (Contracaecum rudolphii A and Contracaecum rudolphii B) within Contracaecum rudolphii (s.l.) and their differentiation from Contracaecum septentrionale, a closely related congener. Application of these tools to C rudolphii (s.l.) adults from Phalaerocorax carbo sinensis (the Eurasian subspecies of the great cormorant) from Qinghai Lake in China, revealed C. rudolphii B to infect this host. This is the first report of C rudolphii B in P. carbo sinensis outside of Europe (where it was originally detected), supporting the proposal that this species has a broad geographical distribution. Together with other methods, each of these molecular tools will be useful for investigating the ecology of C rudolphii A and C rudolphii B as well as C septentrionale. (c) 2006 Elsevier Ltd. All rights reserved
Regulation of Mec1 (ATR) signaling in budding yeast
No full textCells are continuously challenged by various sources of DNA damage that can contribute to cancer formation if not appropriately repaired. To cope with this threat, cells have conserved mechanisms called the DNA damage checkpoints that sense damaged DNA, stop the cell cycle, and upregulate DNA repair. Central players in these checkpoints are the PI3K-like kinases ATM and ATR (S.c. Tel1 and Mec1). Mec1 senses single stranded DNA (ssDNA) that is exposed at stalled replication forks and activates the S phase checkpoint. However, ssDNA, which is generated at the lagging strand during normal replication, does not cause detectable checkpoint activation. It is unknown how Mec1 is regulated in S phase. To study this, we took advantage of a mutant allele of MEC1, mec1-100, which is proficient for the G2 DNA damage checkpoint, but is compromised in G1-S and intra-S-phase checkpoints.
In the first part of this thesis we aimed at identifying regulatory factors. We screened for spontaneous survivors on a lethal dose of the replication fork-stalling agent hydroxyurea (HU) for mec1-100 cells. We mapped additional mutations in mec1-100 or mutations in either PPH3 or PSY2, which form a highly conserved phosphatase (PP4) complex. In a second, more unbiased, high-throughput screen we combined mec1-100 with a collection of 1525 gene deletions involved in chromatin processes and scored double mutants for HU sensitivity. pph3Δ and psy2Δ were among the top mec1-100 suppressor hits, confirming a strong genetic interaction. Suppression by pph3Δ was correlated with the phosphorylation of the downstream kinase Rad53. However, it did not depend exclusively on Rad53, because residual suppression of mec1-100 by pph3Δ could also be observed in rad53Δ cells. We tested whether Psy2-Pph3 might regulate Mec1 directly, and found a physical interaction between Psy2 and Ddc2-Mec1. Moreover, we found that a phosphorylation site within Mec1 (S1991) is downregulated in mec1-100 cells and restored when Pph3 is also lost. However, we were unable to demonstrate that Pph3 dephosphorylates Mec1 directly in vitro. Phosphorylation required both Mec1 kinase activity and Rad53. Thus, we speculate that Mec1 phosphorylation is achieved through Rad53, which is in turn regulated by Pph3, indicating the existence of a feedback loop from Rad53 back to Mec1. Mutation of the phosphorylation site renders cells sensitive to the radiomimetic drug Zeocin, indicating an important role in the survival of DNA damage. Finally, we applied quantitative phosphoproteomics to identify Mec1 and Pph3 targets. We found that the levels of the majority of the phosphopeptides that are affected by a tel1Δ mec1-100 mutation but not by rad53Δ can be rescued due to additional deletion of PPH3. The data presented here support a model in which Pph3 is a major regulator of Mec1 signaling.
In a second part mec1-100 was further characterized in order to understand the mechanism by which its two point mutations outside of the catalytic domain (F1179S, N1700S) cause defects in the replication checkpoint. We find that the mutations leave kinase activity in vitro, oligomerization and Ddc2-Mec1 interaction intact. Genetic analysis shows that mec1-100 is additive, rather than epistatic with mutation or deletion of any of the canonical checkpoint activating proteins Ddc1, Dna2, Dpb11, Rad24, Mrc1, Rad9, Tel1 or Chk1. Thus, we conclude that mec1-100 does not impair function of any of these proteins. We hypothesized that the mutated region might constitute a regulatory domain that is bound by a yet unknown factor. IP experiments followed by mass spectrometry analysis did not show reproducibly decreased interaction of any protein. Additional detailed biochemical analysis is needed to fully understand the mechanism of the two mec1-100 mutations.
We further characterize intragenic mec1-100 suppressor mutations by mapping them to a homology model. While some mutations reside within the kinase domain, and could influence catalytic activity, others might as well be involved protein-protein interactions. We asked whether suppression would involve Rad24 dependent Mec1 activation. Interestingly, we find that suppression by mutations in residues that might make protein-protein contacts completely requires Rad24. Other suppressor mutations relied less on Rad24. Thus, we conclude that intragenic suppression of mec1-100 HU sensitivity employs at least two different mechanisms: one that is Rad24-dependent and a second that is Rad24–independent. These unpublished results will help in understanding Mec1 function and regulation once structural data is available.
The third experimental part resolves the role of the RecQ helicase Sgs1 in replication checkpoint signaling. It was shown before that Sgs1 and Mec1 synergistically contribute to replication fork stabilization under replication stress. Both interact with the ssDNA binding protein RPA. Here, we created a mutant, sgs1-r1, which lacks the RPA interaction domain. While sgs1-r1 is proficient to stabilize stalled forks under replication stress, it is synthetic lethal with mus81Δ, slx4Δ, slx5Δ and slx8Δ. These could provide alternative means to recover stalled forks by resolving crossover structures, DNA repair or break induced replication. . Sgs1 was previously shown to promote Rad53 activation in a manner independent of its helicase activity. We show here that Sgs1 checkpoint function requires the R1 domain. Mec1 phosphorylates Sgs1 in this domain and Sgs1 phosphorylation allows its binding to Rad53 in vitro and in vivo. We thus propose that Sgs1 serves as a mediator in checkpoint signaling by recruiting Rad53 to stalled replication forks for activation.
This work provides new insights into Mec1 signaling by elucidating the checkpoint function of Sgs1 and defining Psy2-Pph3 as a major regulator of this pathway
Semi-nested PCR for the specific detection of Habronema microstoma or Habronema muscae DNA in horse faeces
No full textHabronema microstoma and Habronema muscae (Spirurida: Habronematidae) are parasitic nematodes which infect the stomach and/or skin of equids. The accurate diagnosis of gastric habronemosis is central to studying its epidemiology, but data on its distribution and prevalence are lacking, mainly due to the limitations of clinical and coprological diagnosis in live horses. To overcome this constraint, a two-step, semi-nested PCR-based assay was validated (utilizing genetic markers in the nuclear ribosomal DNA) for the specific amplification of H. microstoma or H. muscae DNA from the faeces from horses (n = 46) whose gastrointestinal parasite status had been determined at autopsy and whose faeces were examined previously using a conventional parasitological approach. Of these horses examined at autopsy, some harboured adults of either H. microstoma (n= 19) or H. muscae (n =4), and others (n = 7) harboured both species. Most of them were also infected with other parasites, including strongylid nematodes (subfamilies Cyathostominae and Strongylinae), bots and/or cestodes; there was no evidence of metazoan parasites in 2 horses. Larvated spirurid eggs were detected in the faeces of 1 of the 30 horses (3.3 %) shown to be infected with Habronema at autopsy. For this set of 46 samples, the PCR assay achieved a diagnostic specificity of 100 % and a sensitivity of approximately 97 % (being able to specifically detect as little as approximately 0.02 fg of Habronema DNA). The specificity of the assay was also tested using a panel of control DNA samples representing horse, the gastric spirurid Draschia megastoma and 26 other species of parasites from the alimentary tract of the horse. H. microstoma, H. muscae and D. megastoma could be readily differentiated from one another based on the sizes of their specific amplicons in the PCR. The results of this study showed that the performance of the PCR for the diagnosis of gastric habronemosis was similar to that of autopsy but substantially better than the traditional coprological examination procedure used. The ability to specifically diagnose gastric habronemosis in equids should have important implications for investigating the epidemiology and ecology of H. microstoma and H. muscae.[...
Specific identification of Habronema microstoma and Habronema muscae (Spirurida, Habronematidae) by PCR using markers in ribosomal DNA
No full textGastric or cutaneous habronemosis caused by Habronema microstoma Creplin, 1849 and Habronema muscae Carter, 1865 is a parasitic disease of equids transmitted by muscid flies. There is a paucity of information on the epidemiology of this disease, which is mainly due to limitations with diagnosis in the live animal and with the identification of the parasites in the intermediate hosts. To overcome such limitations, a molecular approach, based on the use of genetic markers in the second internal transcribed spacer (ITS-2) of ribosomal DNA, was established for the two species of Habronema. Characterisation of the ITS-2 revealed sequence lengths and G+C contents of 296 bp and 29.5% for H. microstoma, and of 334 bp and 35.9% for H. muscae, respectively. Exploiting the sequence difference (approximately 40%) between the two species of nematode, primers were designed and tested by the polymerase chain reaction (PCR) for their specificity using a panel of control DNA samples from common equid endoparasites, and from host tissues, faeces or muscid flies. Effective amplification from each of the two species of Habronema was achieved from as little as 10 pg of genomic DNA. Hence, this molecular approach allows the specific identification and differentiation of the DNA from H. microstoma and H. muscae, and could thus provide a molecular tool for the specific detection of Habronema DNA (irrespective of developmental stage) from faeces, skin and muscid fly samples. The establishment of this tool has important implications for the specific diagnosis of clinical cases of gastric and cutaneous habronemosis in equids, and for studying the ecology and epidemiology of the two species of Habronema
Semi-nested PCR for the specific detection of Habronema microstoma or Habronema muscae DNA in horse feaces
No full text
Single-strand conformation polymorphism (SSCP) for the analysis of genetic variation
No full textThe accurate analysis of genetic variation has major implications in many areas of biomedical research, including the identification of infectious agents (such as parasites), the diagnosis of infections, and the detection of unknown or known disease-causing mutations. Mutation scanning methods, including PCR-coupled single-strand conformation polymorphism (SSCP), have significant advantages over many other nucleic acid techniques for the accurate analysis of allelic and mutational sequence variation. The present protocol describes the SSCP method of analysis, including all steps from the small-scale isolation of genomic DNA and PCR amplification of target sequences, through to the gel-based separation of amplicons and scanning for mutations by SSCP (either by the analysis of radiolabeled amplicons in mutation detection enhancement (MDE) gels or by non-isotopic SSCP using precast GMA gels). The subsequent sequence analysis of polymorphic bands isolated from gels is also detailed. The SSCP protocol can readily detect point mutations for amplicon sizes of up to 450-500 bp, and usually takes 1-2 days to carry out. This user-friendly, low-cost, potentially high-throughput platform has demonstrated the utility to study a wide range of pathogens and diseases, and has the potential to be applied to any gene of any organis
How Agents do it in Stream Logic Programming
No full textThe key factor that will determine the speed and depth to which multi-agent systems penetrate the commercial marketplace is the ease with which applications can be developed. One approach is to use general purpose languages to construct layers of agent level constructs. Object-oriented languages have been advocated as appropriate for the complexity of distributed systems. According to Gasser and Briot [1992], the key problem with the common forms of object based concurrent programming is the fixed boundaries they give to agents are too inflexible. They do not reflect either the theoretical positions emerging in Multi-agent systems, MAS, nor the reality of multilevel aggregations of actions and knowledge. This paper advocates the use of a rather different type of object based concurrent language, stream logic programming, SLP, that does not have this drawback
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