RecA-mediated annealing of single-stranded DNA and its relation to the mechanism of homologous recombination.

We demonstrate that RecA protein can mediate annealing of complementary DNA strands in vitro by at least two different mechanisms. The first annealing mechanism predominates under conditions where RecA protein causes coaggregation of single-stranded DNA (ssDNA) molecules and where RecA-free ssDNA stretches are present on both reaction partners. Under these conditions annealing can take place between locally concentrated protein-free complementary sequences. Other DNA aggregating agents like histone H1 or ethanol stimulate annealing by the same mechanism. The second mechanism of RecA-mediated annealing of complementary DNA strands is best manifested when preformed saturated RecA-ssDNA complexes interact with protein-free ssDNA. In this case, annealing can occur between the ssDNA strand resident in the complex and the ssDNA strand that interacts with the preformed RecA-ssDNA complex. Here, the action of RecA protein reflects its specific recombination promoting mechanism. This mechanism enables DNA molecules resident in the presynaptic RecA-DNA complexes to be exposed for hydrogen bond formation with DNA molecules contacting the presynaptic RecA-DNA filament.

Molecular detection and identification of zygomycetes species from paraffin-embedded tissues in a murine model of disseminated zygomycosis: a collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) evaluation.

The present study was performed to assess the interlaboratory reproducibility of the molecular detection and identification of species of Zygomycetes from formalin-fixed paraffin-embedded kidney and brain tissues obtained from experimentally infected mice. Animals were infected with one of five species (Rhizopus oryzae, Rhizopus microsporus, Lichtheimia corymbifera, Rhizomucor pusillus, and Mucor circinelloides). Samples with 1, 10, or 30 slide cuts of the tissues were prepared from each paraffin block, the sample identities were blinded for analysis, and the samples were mailed to each of seven laboratories for the assessment of sensitivity. A protocol describing the extraction method and the PCR amplification procedure was provided. The internal transcribed spacer 1 (ITS1) region was amplified by PCR with the fungal universal primers ITS1 and ITS2 and sequenced. As negative results were obtained for 93% of the tissue specimens infected by M. circinelloides, the data for this species were excluded from the analysis. Positive PCR results were obtained for 93% (52/56), 89% (50/56), and 27% (15/56) of the samples with 30, 10, and 1 slide cuts, respectively. There were minor differences, depending on the organ tissue, fungal species, and laboratory. Correct species identification was possible for 100% (30 cuts), 98% (10 cuts), and 93% (1 cut) of the cases. With the protocol used in the present study, the interlaboratory reproducibility of ITS sequencing for the identification of major Zygomycetes species from formalin-fixed paraffin-embedded tissues can reach 100%, when enough material is available.

Expression of human estrogen receptor mutants in Xenopus oocytes: correlation between transcriptional activity and ability to form protein-DNA complexes.

The human estrogen receptor (hER) is a trans-acting regulatory protein composed of a series of discrete functional domains. We have microinjected an hER expression vector (HEO) into Xenopus oocyte nuclei and demonstrate, using Western blot assay, that the hER is synthesized. When nuclear extracts from oocytes were prepared and incubated in the presence of a 2.7 kb DNA fragment comprising the 5' end of the vitellogenin gene B2, formation of estrogen-dependent complexes could be visualized by electron microscopy over the estrogen responsive element (ERE). Of crucial importance is the observation that the complex formation is inhibited by the estrogen antagonist tamoxifen, is restored by the addition of the hormone and does not take place with extracts from control oocytes injected with the expression vector lacking the sequences encoding the receptor. The presence of the biologically active hER is confirmed in co-injection experiments, in which HEO is co-introduced with a CAT reporter gene under the control of a vitellogenin promoter containing or lacking the ERE. CAT assays and primer extensions analyses reveal that both the receptor and the ERE are essential for estrogen induced stimulation of transcription. The same approach was used to analyze selective hER mutants. We find that the DNA binding domain (region C) is essential for protein--DNA complex formation at the ERE but is not sufficient by itself to activate transcription from the reporter gene. In addition to region C, both the hormone binding (region E) and amino terminal (region A/B) domains are needed for an efficient transcription activation.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA evidence, probabilistic evaluation and collaborative tests.

Forensic scientists working in 12 state or private laboratories participated in collaborative tests to improve the reliability of the presentation of DNA data at trial. These tests were motivated in response to the growing criticism of the power of DNA evidence. The experts' conclusions in the tests are presented and discussed in the context of the Bayesian approach to interpretation. The use of a Bayesian approach and subjective probabilities in trace evaluation permits, in an easy and intuitive manner, the integration into the decision procedure of any revision of the measure of uncertainty in the light of new information. Such an integration is especially useful with forensic evidence. Furthermore, we believe that this probabilistic model is a useful tool (a) to assist scientists in the assessment of the value of scientific evidence, (b) to help jurists in the interpretation of judicial facts and (c) to clarify the respective roles of scientists and of members of the court. Respondents to the survey were reluctant to apply this methodology in the assessment of DNA evidence.

Tailed Duplex DNA is the preferred substrate for Rad51 protein-mediated homologous pairing.

Purpose of the Study: To elucidate the mechanism of homologous recombination and double-strand break repair mediated by the eukaryotic recombination pin, Rad51.

Early DNA vaccination of puppies against canine distemper in the presence of maternally derived immunity.

Canine distemper (CD) is a disease in carnivores caused by CD virus (CDV), a member of the morbillivirus genus. It still is a threat to the carnivore and ferret population. The currently used modified attenuated live vaccines have several drawbacks of which lack of appropriate protection from severe infection is the most outstanding one. In addition, puppies up to the age of 6-8 weeks cannot be immunized efficiently due to the presence of maternal antibodies. In this study, a DNA prime modified live vaccine boost strategy was investigated in puppies in order to determine if vaccinated neonatal dogs induce a neutralizing immune response which is supposed to protect animals from a CDV challenge. Furthermore, a single DNA vaccination of puppies, 14 days after birth and in the presence of high titers of CDV neutralizing maternal antibodies, induced a clear and significant priming effect observed as early as 3 days after the subsequent booster with a conventional CDV vaccine. It was shown that the priming effect develops faster and to higher titers in puppies preimmunized with DNA 14 days after birth than in those vaccinated 28 days after birth. Our results demonstrate that despite the presence of maternal antibodies puppies can be vaccinated using the CDV DNA vaccine, and that this vaccination has a clear priming effect leading to a solid immune response after a booster with a conventional CDV vaccine.

A protective cocktail vaccine against murine cutaneous leishmaniasis with DNA encoding cysteine proteinases of Leishmania major.

The protection elicited by the intramuscular injection of two plasmid DNAs encoding Leishmania major cysteine proteinase type I (CPb) and type II (CPa) was evaluated in a murine model of experimental cutaneous leishmaniasis. BALB/c mice were immunized either separately or with a cocktail of the two plasmids expressing CPa or CPb. It was only when the cpa and cpb genes were co-injected that long lasting protection against parasite challenge was achieved. Similar protection was also observed when animals were first immunized with cpa/cpb DNA followed by recombinant CPa/CPb boost. Analysis of the immune response showed that protected animals developed a specific Th1 immune response, which was associated with an increase of IFN-gamma production. This is the first report demonstrating that co-injection of two genes expressing different antigens induces a long lasting protective response, whereas the separate injection of cysteine proteases genes is not protective.

Questioning the utility of RNA-DNA chimera in gene repair.

In principle, we should be glad that Eric Kmiec and his colleagues published in Science's STKE (1) a detailed experimental protocol of their gene repair method (2, 3). However, a careful reading of their contribution raises more doubts about the method. The research published in Science five years ago by Kmiec and his colleagues was said to demonstrate that chimeric RNA-DNA oligonucleotides could correct the mutation responsible for sickle cell anemia with 50% efficiency (4). Such a remarkable result prompted many laboratories to attempt to replicate the research or utilize the method on their own systems. However, if the method worked at all, which it rarely did, the achieved efficiency was usually lower by several orders of magnitude. Now, in the Science's STKE protocol, we are given crucial information about the method and why it is so important to utilize these expensive chimeric RNA-DNA constructs. In the introduction we are told that the RNA-DNA duplex is more stable than a DNA-DNA duplex and so extends the half-life of the complexes formed between the targeted DNA and the chimeric RNA-DNA oligonucleotides. This logical explanation, however, conflicts with the statement in the section entitled "Transfection with Oligonucleotides and Plasmid DNA" that Kmiec and colleagues have recently demonstrated that classical single-stranded DNA oligonucleotides with a few protective phosphothioate linkages have a "gene repair conversion frequency rivaling that of the RNA/DNA chimera". Indeed, the research cited for that result actually states that single-stranded DNA oligonucleotides are in fact several-fold more efficient (3.7-fold) than the RNA-DNA chimeric constructs (5). If that is the case, it raises the question of why Kmiec and colleagues emphasize the importance of the RNA in their original chimeric constructs. Their own new results show that modified single-stranded DNA oligonucleotides are more effective than the expensive RNA-DNA hybrids. Moreover, the current efficiency of the gene repair by RNA-DNA hybrids, according to Kmiec and colleagues in their recent paper is only 4×10-4 even after several hours of pre-selection permitting multiplification of bacterial cells with the corrected plasmid (5). This efficiency is much lower than the 50% value reported five years ago, but is assuredly much closer to the reality.

Genetic architecture of ambulatory blood pressure in the general population: insights from cardiovascular gene-centric array.

Genetic determinants of blood pressure are poorly defined. We undertook a large-scale, gene-centric analysis to identify loci and pathways associated with ambulatory systolic and diastolic blood pressure. We measured 24-hour ambulatory blood pressure in 2020 individuals from 520 white European nuclear families (the Genetic Regulation of Arterial Pressure of Humans in the Community Study) and genotyped their DNA using the Illumina HumanCVD BeadChip array, which contains ≈50 000 single nucleotide polymorphisms in >2000 cardiovascular candidate loci. We found a strong association between rs13306560 polymorphism in the promoter region of MTHFR and CLCN6 and mean 24-hour diastolic blood pressure; each minor allele copy of rs13306560 was associated with 2.6 mm Hg lower mean 24-hour diastolic blood pressure (P=1.2×10(-8)). rs13306560 was also associated with clinic diastolic blood pressure in a combined analysis of 8129 subjects from the Genetic Regulation of Arterial Pressure of Humans in the Community Study, the CoLaus Study, and the Silesian Cardiovascular Study (P=5.4×10(-6)). Additional analysis of associations between variants in gene ontology-defined pathways and mean 24-hour blood pressure in the Genetic Regulation of Arterial Pressure of Humans in the Community Study showed that cell survival control signaling cascades could play a role in blood pressure regulation. There was also a significant overrepresentation of rare variants (minor allele frequency: <0.05) among polymorphisms showing at least nominal association with mean 24-hour blood pressure indicating that a considerable proportion of its heritability may be explained by uncommon alleles. Through a large-scale gene-centric analysis of ambulatory blood pressure, we identified an association of a novel variant at the MTHFR/CLNC6 locus with diastolic blood pressure and provided new insights into the genetic architecture of blood pressure.

DNA binding specificity of different STAT proteins. Comparison of in vitro specificity with natural target sites.

STAT transcription factors are expressed in many cell types and bind to similar sequences. However, different STAT gene knock-outs show very distinct phenotypes. To determine whether differences between the binding specificities of STAT proteins account for these effects, we compared the sequences bound by STAT1, STAT5A, STAT5B, and STAT6. One sequence set was selected from random oligonucleotides by recombinant STAT1, STAT5A, or STAT6. For another set including many weak binding sites, we quantified the relative affinities to STAT1, STAT5A, STAT5B, and STAT6. We compared the results to the binding sites in natural STAT target genes identified by others. The experiments confirmed the similar specificity of different STAT proteins. Detailed analysis indicated that STAT5A specificity is more similar to that of STAT6 than that of STAT1, as expected from the evolutionary relationships. The preference of STAT6 for sites in which the half-palindromes (TTC) are separated by four nucleotides (N(4)) was confirmed, but analysis of weak binding sites showed that STAT6 binds fairly well to N(3) sites. As previously reported, STAT1 and STAT5 prefer N(3) sites; however, STAT5A, but not STAT1, weakly binds N(4) sites. None of the STATs bound to half-palindromes. There were no specificity differences between STAT5A and STAT5B.

IRF6 Screening of Syndromic and a priori Non-Syndromic Cleft Lip and Palate Patients: Identification of a New Type of Minor VWS Sign.

Van der Woude syndrome (VWS), caused by dominant IRF6 mutation, is the most common cleft syndrome. In 15% of the patients, lip pits are absent and the phenotype mimics isolated clefts. Therefore, we hypothesized that some of the families classified as having non-syndromic inherited cleft lip and palate could have an IRF6 mutation. We screened in total 170 patients with cleft lip with or without cleft palate (CL/P): 75 were syndromic and 95 were a priori part of multiplex non-syndromic families. A mutation was identified in 62.7 and 3.3% of the patients, respectively. In one of the 95 a priori non-syndromic families with an autosomal dominant inheritance (family B), new insights into the family history revealed the presence, at birth, of lower lip pits in two members and the diagnosis was revised as VWS. A novel lower lip sign was observed in one individual in this family. Interestingly, a similar lower lip sign was also observed in one individual from a 2nd family (family A). This consists of 2 nodules below the lower lip on the external side. In a 3rd multiplex family (family C), a de novo mutation was identified in an a priori non-syndromic CL/P patient. Re-examination after mutation screening revealed the presence of a tiny pit-looking lesion on the inner side of the lower lip leading to a revised diagnosis of VWS. On the basis of this data, we conclude that IRF6 should be screened when any doubt rises about the normality of the lower lip and also if a non-syndromic cleft lip patient (with or without cleft palate) has a family history suggestive of autosomal dominant inheritance.