Novel quantitative TaqMan real-time PCR assays for detection of Cryptosporidium at the genus level and genotyping of major human and cattle-infecting species.

AIMS: Development of TaqMan MGB real-time PCR assays for quantitative typing of major cattle and human-pathogenic Cryptosporidium species. METHODS AND RESULTS: Three specific TaqMan MGB real-time PCRs, based on the SSU rRNA gene, were directed towards livestock-restricted Cryptosporidium andersoni and Cryptosporidium bovis as well as both human-pathogenic Cryptosporidium parvum and Cryptosporidium hominis. A generic TaqMan assay further identified all known Cryptosporidium species and simultaneously monitored PCR inhibition through an external amplification control. The generic and specific assays were highly reproducible, and all displayed a detection limit of one oocyst per reaction. The specific TaqMan protocols also proved valuable for specifically detecting and quantifying target DNA in the presence of non-target DNA in environmental samples. CONCLUSIONS: All TaqMan MGB real-time PCR assays fulfilled the required specificity and sensitivity criteria, both on laboratory strains and on a surface water matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: No molecular-based method was yet available for the quantitative detection of C. andersoni and the cluster formed by C. bovis, Cryptosporidium ryanae and Cryptosporidium xiaoi. This work provides a novel tool to evaluate the parasite load from domestic ruminants and humans, and to improve assessment and management of microbial risk through better appraisal of the origin and fate of faecal pollutions.

5'-Deoxyribose phosphate lyase activity of human DNA polymerase iota in vitro.

DNA polymerase iota (pol iota) is one of several recently discovered DNA polymerases in mammalian cells whose function is unknown. We report here that human pol iota has an intrinsic 5'-deoxyribose phosphate (dRP) lyase activity. In reactions reconstituted with uracil-DNA glycosylase (UDG), apurinic/apyrimidinic (AP) endonuclease and DNA ligase I, pol iota can use its dRP lyase and polymerase activities to repair G*U and A*U pairs in DNA. These data and three distinct catalytic properties of pol iota implicate it in specialized forms of base excision repair (BER).

An ATM homologue from Arabidopsis thaliana: complete genomic organisation and expression analysis.

ATM is a gene mutated in the human disease ataxia telangiectasia with reported homologues in yeast, Drosophila, Xenopus and mouse. Whenever mutants are available they all indicate a role of this gene family in the cellular response to DNA damage. Here, we present the identification and molecular characterisation of the first plant homologue of ATM. The genomic locus of AtATM ( Arabidopsis thaliana homologue of ATM ) spans over 30 kb and is transcribed into a 12 kb mRNA resulting from the splicing of 79 exons. It is a single copy gene and maps to the long arm of chromosome 3. Transcription of AtATM is ubiquitous and not induced by ionising radiation. The putative protein encoded by AtATM is 3856 amino acids long and contains a phosphatidyl inositol-3 kinase-like (Pi3k-l) domain and a rad3 domain, features shared by other members of the ATM family. The AtAtm protein is highly similar to Atm, with 67 and 45% similarity in the Pi3k-l and rad3 domains respectively. Interestingly, the N-terminal portion of the protein harbours a PWWP domain, which is also present in other proteins involved in DNA metabolism such as human mismatch repair enzyme Msh6 and the mammalian de novo methyl transferases, Dnmt3a/b.

Libraries of Synthetic TALE-Activated Promoters: Methods and Applications.

The discovery of proteins with programmable DNA-binding specificities triggered a whole array of applications in synthetic biology, including genome editing, regulation of transcription, and epigenetic modifications. Among those, transcription activator-like effectors (TALEs) due to their natural function as transcription regulators, are especially well-suited for the development of orthogonal systems for the control of gene expression. We describe here the construction and testing of libraries of synthetic TALE-activated promoters which are under the control of a single TALE with a given DNA-binding specificity. These libraries consist of a fixed DNA-binding element for the TALE, a TATA box, and variable sequences of 19 bases upstream and 43 bases downstream of the DNA-binding element. These libraries were cloned using a Golden Gate cloning strategy making them usable as standard parts in a modular cloning system. The broad range of promoter activities detected and the versatility of these promoter libraries make them valuable tools for applications in the fine-tuning of expression in metabolic engineering projects or in the design and implementation of regulatory circuits.

Ultrasonographic renal volume measurements in early autosomal dominant polycystic disease: comparison with CT-scan renal volume calculations.

PURPOSE: To investigate the correlation and concordance between the ellipsoid volume calculated by ultrasonography measurements (Vol3DUS) and the reference kidney volume measured by CT (VolTDM) in early autosomal dominant polycystic kidney disease (ADPKD). MATERIALS AND METHODS: Prospective study of the correlation and concordance of renal volumes in 24 patients with early ADPKD (48 kidneys analysed separately), with calculation of Vol3DUS using the formula for an ellipsoid in three different manners and VolTDM measurement by manual contouring. Calculations of correlation coefficients (r) and coefficients of intra-class correlation (ICC) with confidence intervals at 95%. RESULTS: The US volume was strongly correlated with the CT volume by using the maximum width in a transverse section (r=0.83) with a mean Vol3DUS=692±348ml [180; 2069]. The most reproducible ultrasonography measurement was the height. When the kidney volume exceeded 800ml, US underestimated the volume. However, the median error was -57.5ml [-1090; 183] and 85% of the Vol3DUS calculated differed by more than 5% from the reference measurement. CONCLUSION: The correlation between the US calculated volumes and the CT volumes was strong. However, the median error with ellipsoid US volume was too high to detect a small renal variation in early ADPKD.

The mouse Kin-17 gene codes for a new protein involved in DNA transactions and is akin to the bacterial RecA protein.

We have sought to characterize the molecular basis of the sensitivity to ionising radiation and to identify the genes involved in the cellular response of mammalian cells to such radiation. Using the Escherichia coli model, we tested the hypothesis that functional domains of RecA protein are represented in proteins of mammalian cells. We review here the results obtained in the detection of nuclear proteins of mammalian cells that are recognized by anti-RecA antibodies. We have called them kin proteins. Kin proteins likely play a role in DNA metabolism. We summarize the cloning of the mouse Kin-17 cDNA and our work on the identification and preliminary characterisation of the biochemical properties of mouse kin17 protein, a new nuclear protein able to recognize bent DNA and suspected to be involved in illegitimate recombination. We briefly describe our latest experiments on the molecular characterisation of the mouse Kin-17 gene. Finally, we discuss the properties of kin17 protein and the possible participation of kin17 protein in DNA transactions like transcription or recombination.

[Immuno-hemolytic transfusion reactions. IV. Analysis, risks and prevention]. / Les accidents immuno-hémolytiques transfusionnels. IV. Analyse, risques et prévention.

The immunological risk of red blood cell transfusions now seems higher than the viral risk. According to studies, severe accidents due to blood incompatibility occur with a frequency estimated at 1/6000 to 1/29000; despite technical progress, the risk does not significantly diminish. The majority of accidents do not originate from laboratory or production stages but from defects in the application of clinical procedures. Preventive measures are based on (i) the elaboration of clinical guidelines, (ii) the compliance to strict rules in carrying out bedside ABO check, and (iii) the realization and interpretation of antibody screening tests. The implementation of quality assurance systems and of the epidemiological surveillance system, which define the basis of a prevention policy, leads to the expectation of an improvement of transfusion safety.

[Immuno-hemolytic transfusion reactions. II Physiopathologic basis and diagnosis]. / Les accidents immuno-hémolytiques transfusionnels. II. Bases physiopathologiques et diagnostic.

Immunological transfusion reactions more than often lead to an activation of the complement proteins and mononuclear cells, inducing a haemolysis from which stem the observed clinical symptoms. In the case of incompatibility, the alloantibodies can lead to an immediate reaction, taking place in the first few minutes or, in the case of a delayed reaction, arising after 24 hours. A standardized clinical and biological evaluation is necessary in order to confirm the diagnosis and to assess the consequences of the antigen-antibody conflict.

[Immuno-hemolytic transfusion reactions. III. Report of 61 cases]. / Les accidents immuno-hémolytiques transfusionnels. III. Etude de 61 cas.

Blood transfusion is mainly bound to immunological and infectious risks. The immunological risk originates from an incompatibility between the blood of the donor and that of the recipient; this risk remains insufficiently assessed. A multicentre study has been carried out by the French Blood Transfusion Society and the National Institute for Blood Transfusion. Sixty-one accidents due to an erythrocyte incompatibility were found: 26 cases with ABO incompatibility, and 35 cases with alloantibodies of other blood group systems. For the former category of accidents, the most frequent cause was due to a failure in the realization of the bedside ABO check. For the latter, the main problem was the achievement and the interpretation of antibody screening. The long term follow-up shows no chronic after-effects of immunological accidents. For each accident, errors have been identified and analysed. It was proven that they all originate from health care establishments.

Ultrasound elastography of the prostate: state of the art.

Prostate cancer is the cancer exhibiting the highest incidence rate and it appears as the second cause of cancer death in men, after lung cancer. Prostate cancer is difficult to detect, and the treatment efficacy remains limited despite the increase use of biological tests (prostate-specific antigen [PSA] dosage), the development of new imaging modalities, and the use of invasive procedures such as biopsy. Ultrasound elastography is a novel imaging technique capable of mapping tissue stiffness of the prostate. It is known that prostatic cancer tissue is often harder than healthy tissue (information used by digital rectal examination [DRE]). Two elastography techniques have been developed based on different principles: first, quasi-static (or strain) technique, and second, shear wave technique. The tissue stiffness information provided by US elastography should improve the detection of prostate cancer and provide guidance for biopsy. Prostate elastography provides high sensitivity for detecting prostate cancer and shows high negative predictive values, ensuring that few cancers will be missed. US elastography should become an additional method of imaging the prostate, complementing the conventional transrectal ultrasound and MRI. This technique requires significant training (especially for quasi-static elastography) to become familiar with acquisition process, acquisition technique, characteristics and limitations, and to achieve correct diagnoses.

A functional OGG1 homologue from Arabidopsis thaliana.

One of the major mutagenic base lesions in DNA caused by exposure to reactive oxygen species is 7,8-dihydro-8-oxoguanine (8-oxoG). Genes coding for DNA repair enzymes that recognise 8-oxoG have been reported in bacteria, yeast, mammals and plants. The prokaryotic and eukaryotic genes are functional homologues but differ in their primary sequence. We have cloned, sequenced, and expressed a new Arabidopsis thaliana cDNA that shows sequence homology to the eukaryotic genes coding for 8-oxoG DNA N-glycosylases (OGG1). The 40.3-kDa enzyme it encodes (AtOGG1) introduces a chain break in a double-stranded oligonucleotide specifically at an 8-oxoG residue. In addition, AtOGG1 can form a Schiff base with 8-oxoG in the presence of NaBH4, suggesting that it is a bifunctional DNA N-glycosylase. Furthermore, expression of AtOGG1 in an Escherichia coli strain that is deficient in the repair of 8-oxoG in DNA suppresses its spontaneous-mutator phenotype. Thus, we have demonstrated that AtOGG1 is not only a structural but also a functional eukaryotic OGG1 homologue.

Purification and characterization of a DNA strand transferase from broccoli.

A protein with DNA binding, renaturation, and strand-transfer activities has been purified to homogeneity from broccoli (Brassica oleracea var italica). The enzyme, broccoli DNA strand transferase, has a native molecular mass of at least 200 kD and an apparent subunit molecular mass of 95 kD and is isolated as a set of isoforms differing only in charge. All three activities are saturated at very low stoichiometry, one monomer per approximately 1000 nucleotides of single-stranded DNA. Strand transfer is not effected by nuclease activity and reannealing, is only slightly dependent on ATP, and is independent of added Mg2+. Transfer requires homologous single- and double-stranded DNA and at higher enzyme concentrations results in very high molecular mass complexes. As with Escherichia coli RecA, transfer by broccoli DNA strand transferase depends strongly on the presence of 3' homologous ends.

Cloning and Characterization of an Arabidopsis thaliana Topoisomerase I Gene.

cDNA and genomic clones encoding DNA topoisomerase I were isolated from Arabidopsis thaliana lambdagt11 and lambdaFix libraries by low stringency hybridization with a Saccharomyces cerevisiae TOP1 probe. The cDNA clones include a 2748-base pair open reading frame predicting an amino acid sequence that is highly homologous to sequences encoded by TOP1 from yeast and human sources. The sequence of the upstream genomic region reveals two putative TATA-like elements and a purine-rich region, but no other obvious controlling elements. Southern blot analysis shows that the gene is present as a single copy in the Arabidopsis genome. When expressed in a S. cerevisiae top1 mutant under the control of the GAL1 promoter, the gene complements the phenotype caused by loss of topoisomerase activity and directs the expression of a protein that cross-reacts with a human anti-topoisomerase I antibody.

Activity of the yeast FLP recombinase in Arabidopsis.

The coding sequence for FLP recombinase, originally from the 2 mu plasmid of Saccharomyces cerevisiae, was introduced into Arabidopsis behind the cauliflower mosaic virus 35S promoter. FLP activity was monitored by the glucuronidase activity resulting from inversion of an antisense-oriented GUS reporter gene flanked by a pair of FRT target sites in inverted repeat. FLP-dependent Gus activity was observed in both transient assays and transgenic plants. The FLP system will be useful for a variety of in planta genetic manipulations.

Misinsertion and bypass of thymine-thymine dimers by human DNA polymerase iota.

Human DNA polymerase iota (pol(iota)) is a recently discovered enzyme that exhibits extremely low fidelity on undamaged DNA templates. Here, we show that poliota is able to facilitate limited translesion replication of a thymine-thymine cyclobutane pyrimidine dimer (CPD). More importantly, however, the bypass event is highly erroneous. Gel kinetic assays reveal that pol(iota) misinserts T or G opposite the 3' T of the CPD approximately 1.5 times more frequently than the correct base, A. While pol(iota) is unable to extend the T.T mispair significantly, the G.T mispair is extended and the lesion completely bypassed, with the same efficiency as that of the correctly paired A. T base pair. By comparison, pol(iota) readily misinserts two bases opposite a 6-4 thymine-thymine pyrimidine-pyrimidone photoproduct (6-4PP), but complete lesion bypass is only a fraction of that observed with the CPD. Our data indicate, therefore, that poliota possesses the ability to insert nucleotides opposite UV photoproducts as well as to perform unassisted translesion replication that is likely to be highly mutagenic.

DNA polymerase iota and related rad30-like enzymes.

Until recently, the molecular mechanisms of translesion DNA synthesis (TLS), a process whereby a damaged base is used as a template for continued replication, was poorly understood. This area of scientific research has, however, been revolutionized by the finding that proteins long implicated in TLS are, in fact, DNA polymerases. Members of this so-called UmuC/DinB/Rev1/Rad30 superfamily of polymerases have been identified in prokaryotes, eukaryotes and archaea. Biochemical studies with the highly purified polymerases reveal that some, but not all, can traverse blocking lesions in template DNA. All of them share a common feature, however, in that they exhibit low fidelity when replicating undamaged DNA. Of particular interest to us is the Rad30 subfamily of polymerases found exclusively in eukaryotes. Humans possess two Rad30 paralogs, Rad30A and Rad30B. The RAD30A gene encodes DNA polymerase eta and defects in the protein lead to the xeroderma pigmentosum variant (XP-V) phenotype in humans. Very recently RAD30B has also been shown to encode a novel DNA polymerase, designated as Pol iota. Based upon in vitro studies, it appears that Pol iota has the lowest fidelity of any eukaryotic polymerase studied to date and we speculate as to the possible cellular functions of such a remarkably error-prone DNA polymerase.

Human DNA polymerase iota promiscuous mismatch extension.

Human DNA polymerase iota is a low-fidelity template copier that preferentially catalyzes the incorporation of the wobble base G, rather than the Watson-Crick base A, opposite template T (Tissier, A., McDonald, J. P., Frank, E. G., and Woodgate, R. (2000) Genes Dev. 14, 1642-1650; Johnson, R. E., Washington, M. T., Haracska, L., Prakash, S., and Prakash, L. (2000) Nature 406, 1015-1019; Zhang, Y., Yuan, F., Wu, X., and Wang, Z. (2000) Mol. Cell. Biol. 20, 7099-7108). Here, we report on its ability to extend all 12 possible mispairs and 4 correct pairs in different sequence contexts. Extension from both matched and mismatched primer termini is generally most efficient and accurate when A is the next template base. In contrast, extension occurs less efficiently and accurately when T is the target template base. A striking exception occurs during extension of a G:T mispair, where the enzyme switches specificity, "preferring" to make a correct A:T base pair immediately downstream from an originally favored G:T mispair. Polymerase iota generates a variety of single and tandem mispairs with high frequency, implying that it may act as a strong mutator when copying undamaged DNA templates in vivo. Even so, its limited ability to catalyze extension from a relatively stable primer/template containing a "buried" mismatch suggests that polymerase iota-catalyzed errors are confined to short template regions.

poliota, a remarkably error-prone human DNA polymerase.

The Saccharomyces cerevisiae RAD30 gene encodes DNA polymerase eta. Humans possess two Rad30 homologs. One (RAD30A/POLH) has previously been characterized and shown to be defective in humans with the Xeroderma pigmentosum variant phenotype. Here, we report experiments demonstrating that the second human homolog (RAD30B), also encodes a novel DNA polymerase that we designate poliota. poliota, is a distributive enzyme that is highly error-prone when replicating undamaged DNA. At template G or C, the average error frequency was approximately 1 x 10(-2). Our studies revealed, however, a striking asymmetry in misincorporation frequency at template A and T. For example, template A was replicated with the greatest accuracy, with misincorporation of G, A, or C occurring with a frequency of approximately 1 x 10(-4) to 2 x 10(-4). In dramatic contrast, most errors occurred at template T, where the misincorporation of G was, in fact, favored approximately 3:1 over the correct nucleotide, A, and misincorporation of T occurred at a frequency of approximately 6.7 x 10(-1). These findings demonstrate that poliota is one of the most error-prone eukaryotic polymerases reported to date and exhibits an unusual misincorporation spectrum in vitro.

Purification and properties of DNA topoisomerase I from broccoli.

We have purified a topoisomerase activity from broccoli (Brassica oleracea var. italica) to near homogeneity. The enzyme is an 80 kDa monomer as judged by gel filtration chromatography and SDS gel electrophoresis, though it may represent a proteolytic fragment of a larger protein. The enzyme is capable of removing both negative and positive supercoils in steps of one, does not absolutely require Mg2+, is only very weakly stimulated by NaCl, is inhibited by camptothecin, and cross-reacts with an antibody directed against human DNA topoisomerase I. These properties identify the enzyme as a eukaryotic type I topoisomerase.