Object-oriented Bayesian networks for evaluating DIP-STR profiling results from unbalanced DNA mixtures.

The genetic characterization of unbalanced mixed stains remains an important area where improvement is imperative. In fact, with current methods for DNA analysis (Polymerase Chain Reaction with the SGM Plus™ multiplex kit), it is generally not possible to obtain a conventional autosomal DNA profile of the minor contributor if the ratio between the two contributors in a mixture is smaller than 1:10. This is a consequence of the fact that the major contributor's profile 'masks' that of the minor contributor. Besides known remedies to this problem, such as Y-STR analysis, a new compound genetic marker that consists of a Deletion/Insertion Polymorphism (DIP), linked to a Short Tandem Repeat (STR) polymorphism, has recently been developed and proposed elsewhere in literature [1]. The present paper reports on the derivation of an approach for the probabilistic evaluation of DIP-STR profiling results obtained from unbalanced DNA mixtures. The procedure is based on object-oriented Bayesian networks (OOBNs) and uses the likelihood ratio as an expression of the probative value. OOBNs are retained in this paper because they allow one to provide a clear description of the genotypic configuration observed for the mixed stain as well as for the various potential contributors (e.g., victim and suspect). These models also allow one to depict the assumed relevance relationships and perform the necessary probabilistic computations.

High-frequency linkage of co-expressing T-DNA in transgenic Arabidopsis thaliana transformed by vacuum-infiltration of Agrobacterium tumefaciens

The efficiency of co-expression and linkage of distinct T-DNAs present in separate Agrobacterium tumefaciens was analysed in Arabidopsis thaliana transformed by the vacuum infiltration method. Co-expression was monitored by the synthesis of three bacterial proteins involved in the production of polyhydroxybutyrate (PHB) in the plastids. Out of 80 kanamycin-resistant transgenic plants analysed, 13 plants were co-transformed with the two distinct T-DNAs and produced PHB. Of those, 7 lines had a kanamycin-resistance segregation ratio consistent with the presence of a single functional insert. Genetic linkage between the distinct T-DNAs was demonstrated for all 13 PHB-producing lines, while physical linkage between the distinct T-DNAs was shown for 12 out of 13 lines. T-DNAs were frequently linked in an inverted orientation about the left borders. Transformation of A. thaliana by the co-infiltration of two A. tumefaciens containing distinct T-DNAs is, thus, an efficient approach for the integration and expression of several transgenes at a single locus. This approach will facilitate the creation and study of novel metabolic pathways requiring the expression of numerous transgenes.

Use of DNA profiles for investigation using a simulated national DNA database: Part II. Statistical and ethical considerations on familial searching.

Familial searching consists of searching for a full profile left at a crime scene in a National DNA Database (NDNAD). In this paper we are interested in the circumstance where no full match is returned, but a partial match is found between a database member's profile and the crime stain. Because close relatives share more of their DNA than unrelated persons, this partial match may indicate that the crime stain was left by a close relative of the person with whom the partial match was found. This approach has successfully solved important crimes in the UK and the USA. In a previous paper, a model, which takes into account substructure and siblings, was used to simulate a NDNAD. In this paper, we have used this model to test the usefulness of familial searching and offer guidelines for pre-assessment of the cases based on the likelihood ratio. Siblings of "persons" present in the simulated Swiss NDNAD were created. These profiles (N=10,000) were used as traces and were then compared to the whole database (N=100,000). The statistical results obtained show that the technique has great potential confirming the findings of previous studies. However, effectiveness of the technique is only one part of the story. Familial searching has juridical and ethical aspects that should not be ignored. In Switzerland for example, there are no specific guidelines to the legality or otherwise of familial searching. This article both presents statistical results, and addresses criminological and civil liberties aspects to take into account risks and benefits of familial searching.

The N-terminal DNA-binding 'zinc finger' of the oestrogen and glucocorticoid receptors determines target gene specificity.

Steroid hormone receptors activate specific gene transcription by binding as hormone-receptor complexes to short DNA enhancer-like elements termed hormone response elements (HREs). We have shown previously that a highly conserved 66 amino acid region of the oestrogen (ER) and glucocorticoid (GR) receptors, which corresponds to part of the receptor DNA binding domain (region C) is responsible for determining the specificity of target gene activation. This region contains two sub-regions (CI and CII) analogous to the 'zinc-fingers' of the transcription factor TFIIIA. We show here that CI and CII appear to be separate domains both involved in DNA binding. Furthermore, using chimaeric ERs in which either the first (N-terminal) (CI) or second (CII) 'zinc finger' region has been exchanged with that of the GR, indicates that it is the first 'zinc finger' which largely determines target gene specificity. We suggest that receptor recognition of the HRE is analogous to that of the helix-turn-helix DNA binding motif in that the receptor binds to DNA as a dimer with the first 'zinc finger' lying in the major groove recognizing one half of the palindromic HRE, and that protein-DNA interaction is stabilized through non-specific DNA binding and dimer interactions contributed by the second 'zinc finger'.

A histone-fold complex and FANCM form a conserved DNA-remodeling complex to maintain genome stability.

FANCM remodels branched DNA structures and plays essential roles in the cellular response to DNA replication stress. Here, we show that FANCM forms a conserved DNA-remodeling complex with a histone-fold heterodimer, MHF. We find that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA-remodeling complex that protects replication forks from yeast to human.

Increased condom use without other major changes in sexual behavior among the general population in Switzerland.

The expression of DNA topoisomerase II alpha and beta genes was studied in murine normal tissues. Northern blot analysis using probes specific for the two genes showed that the patterns of expression were different among 22 tissues of adult mice. Expression levels of topoisomerase II alpha gene were high in proliferating tissues, such as bone marrow and spleen, and undetectable or low in 17 other tissues. In contrast, high or intermediate expression of topoisomerase II beta gene was found in a variety of tissues (15) of adult mice, including those with no proliferating cells. Topoisomerase II gene expression was also studied during murine development. In whole embryos both genes were expressed at higher levels in early than late stages of embryogenesis. Heart, brain and liver of embryos two days before delivery, and these same tissues plus lung and thymus of newborn (1-day-old) mice expressed appreciable levels of the two genes. Interestingly, a post-natal induction of the beta gene expression was observed in the brain but not in the liver; conversely, the expression of the alpha gene was increased 1 day after birth in the liver but not in the brain. However, gene expression of a proliferation-associated enzyme, thymidylate synthase, was similar in these tissues between embryos and newborns. Thus, the two genes were differentially regulated in the post-natal period, and a tissue-specific role may be suggested for the two isoenzymes in the development of differentiated tissues such as the brain and liver. Based on the differential patterns of expression of the two isoforms, this analysis indicates that topoisomerase II alpha may be a specific marker of cell proliferation, whereas topoisomerase II beta may be implicated in functions of DNA metabolism other than replication.

The ATPase activity of RecA is needed to push the DNA strand exchange through heterologous regions.

The role of ATP hydrolysis during the RecA-mediated recombination reaction is addressed in this paper. Recent studies indicated that the RecA-promoted DNA strand exchange between completely homologous double- and single-stranded DNA can be very efficient in the absence of ATP hydrolysis. In this work we demonstrate that the energy derived from the ATP hydrolysis is strictly needed to drive the DNA strand exchange through the regions where the interacting DNA molecules are not in a homologous register. Therefore, in addition to the role of the ATP hydrolysis in promoting the dissociation of RecA from the products of the recombination reaction, as described earlier, ATP hydrolysis also plays a crucial role in the actual process of strand exchange, provided that the lack of homologous register obstructs the process of branch migration.

Effective stiffening of DNA due to nematic ordering causes DNA molecules packed in phage capsids to preferentially form torus knots.

Observation that DNA molecules in bacteriophage capsids preferentially form torus type of knots provided a sensitive gauge to evaluate various models of DNA arrangement in phage heads. Only models resulting in a preponderance of torus knots could be considered as close to reality. Recent studies revealed that experimentally observed enrichment of torus knots can be qualitatively reproduced in numerical simulations that include a potential inducing nematic arrangement of tightly packed DNA molecules within phage capsids. Here, we investigate what aspects of the nematic arrangement are crucial for inducing formation of torus knots. Our results indicate that the effective stiffening of DNA by the nematic arrangement not only promotes knotting in general but is also the decisive factor in promoting formation of DNA torus knots in phage capsids.

The Tightness of the Cotton Swabs Meshing Influences the Chances of Getting Conclusive DNA Profiles

Objective: The chance of obtaining a conclusive DNA profile strongly depends on the quantity of biological material that can be recovered from a crime scene sample. Optimizing the collection strategy is therefore of prime interest. A difference in the level of tightness of the cotton meshed around the shaft has been observed between manufacturers and is hypothesized to affect the collection and subsequent release capacity of cotton swabs. Consequently, we compared the performance of cotton swabs from two different suppliers: Applimed SA and DryswabTM. Methods: These swabs were used to recover 50 ml of blood, either pure or diluted (1:1000 and 1:5000), deposited on both smooth and absorbent surfaces. Performance was compared in terms of ease of use, concentration of extracted DNA, and quality of DNA profiles. DNA quantification was obtained by real-time PCR using the QuantifilerTM Human DNA Quantification Kit. Evaluation of DNA profiles was based on profiles obtained using AmpFlSTR® NGM SElectTM PCR Amplification kit. Results: When considering smooth surfaces, recovered DNA was more concentrated when using the DryswabTM than the Applimed SA cotton swab. More precisely, DNA concentrations ranged from 15.7 to 28.8 ng/ml and 6.7 to 21.2 ng/ml, respectively for samples of pure blood. The same trend was observed for the absorbent surface, with 2.0 to 5.0 ng/ml and 0.9 to 1.4 ng/ml, respectively. Conclusion: Our results illustrate that different cotton swabs produce different results in terms of ease of use and quantity of recovered DNA and this should be taken into consideration when choosing which swab to use at both the crime scene and laboratory. More specifically, results from the present study suggest that looser meshing of the cotton fibres

Affymetrix Whole-Transcript Human Gene 1.0 ST array is highly concordant with standard 3' expression arrays.

The recently released Affymetrix Human Gene 1.0 ST array has two major differences compared with standard 3' based arrays: (i) it interrogates the entire mRNA transcript, and (ii) it uses DNA targets. To assess the impact of these differences on array performance, we performed a series of comparative hybridizations between the Human Gene 1.0 ST and the Affymetrix HG-U133 Plus 2.0 and the Illumina HumanRef-8 BeadChip arrays. Additionally, both RNA and DNA targets were hybridized on HG-U133 Plus 2.0 arrays. The results show that the overall reproducibility of the Gene 1.0 ST array is best. When looking only at the high intensity probes, the reproducibility of the Gene 1.0 ST array and the Illumina BeadChip array is equally good. Concordance of array results was assessed using different inter-platform mappings. Agreements are best between the two labeling protocols using HG-U133 Plus 2.0 array. The Gene 1.0 ST array is most concordant with the HG-U133 array hybridized with cDNA targets. This may reflect the impact of the target type. Overall, the high degree of correspondence provides strong evidence for the reliability of the Gene 1.0 ST array.

Mutation analysis of the ATP7B gene in a new group of Wilson's disease patients: contribution to diagnosis.

Wilson's disease (WD), an autosomal recessive disorder of copper transport with a broad range of genotypic and phenotypic characteristics, results from mutations in the ATP7B gene. Herein we report the results of mutation analysis of the ATP7B gene in a group of 118 Wilson disease families (236 chromosomes) prevalently of Italian origin. Using DNA sequencing we identified 83 disease-causing mutations. Eleven were novel, while twenty one already described mutations were identified in new populations in this study. In particular, mutation analysis of 13 families of Romanian origin showed a high prevalence of the p.H1069Q mutation (50%). Detection of new mutations in the ATP7B gene in new populations increases our capability of molecular analysis that is essential for early diagnosis and treatment of WD.