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.

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.

Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3-4A complex.

Hepatitis C virus (HCV) NS3-4A is a membrane-associated multifunctional protein harboring serine protease and RNA helicase activities. It is an essential component of the HCV replication complex and a prime target for antiviral intervention. Here, we show that membrane association and structural organization of HCV NS3-4A are ensured in a cooperative manner by two membrane-binding determinants. We demonstrate that the N-terminal 21 amino acids of NS4A form a transmembrane alpha-helix that may be involved in intramembrane protein-protein interactions important for the assembly of a functional replication complex. In addition, we demonstrate that amphipathic helix alpha(0), formed by NS3 residues 12-23, serves as a second essential determinant for membrane association of NS3-4A, allowing proper positioning of the serine protease active site on the membrane. These results allowed us to propose a dynamic model for the membrane association, processing, and structural organization of NS3-4A on the membrane. This model has implications for the functional architecture of the HCV replication complex, proteolytic targeting of host factors, and drug design.

Essential role for Pbx1 in corneal morphogenesis.

PURPOSE: The Pbx TALE (three-amino-acid loop extension) homeodomain proteins interact with class 1 Hox proteins, which are master regulators of cell fate decisions. This study was performed to elucidate the role of the Pbx1 TALE protein in the corneal epithelium of mice. METHODS: Pbx1(f/f) mice were crossed with mice containing Cre recombinase under the control of the K14 promoter. Subsequently, the eyes of these mice were dissected and prepared for histologic or molecular analysis. RESULTS: Tissue-specific deletion of Pbx1 in the corneal epithelium of mice resulted in corneal dystrophy and clouding that was apparent in newborns and progressively worsened with age. Thickening of the cornea epithelium was accompanied by stromal infiltration with atypical basal cells, severe disorganization of stromal collagen matrix, and loss of corneal barrier function. High epithelial cell turnover was associated with perturbed expression of developmental regulators and aberrant differentiation, suggesting an important function for Pbx1 in determining corneal identity. CONCLUSIONS: These studies establish an essential role of the Pbx1 proto-oncogene in corneal morphogenesis.

Complex genetics in idiopathic hypogonadotropic hypogonadism.

Idiopathic hypogonadotropic hypogonadism (IHH) is an important human disease model. Investigations of the genetics of IHH have facilitated insights into critical pathways regulating sexual maturation and fertility. IHH has been traditionally considered a monogenic disorder. This model holds that a single gene defect is responsible for the disease in each patient. In the case of IHH, 30% of cases are explained by mutations in one of eleven genes. In recent years, several lines of evidence have challenged the monogenic paradigm in IHH. First, disease-associated mutations display striking incomplete penetrance and variable expressivity within and across IHH families. Second, each locus is responsible for only a small percentage of cases. Third, more than one disease-associated mutation seems to be segregating in some families with IHH, and their combined or separate presence in individuals accounts for the variability in disease severity. Finally, IHH is not strictly a congenital and life-long disorder; occasionally it manifests itself during adulthood (adult-onset IHH); in other cases, the disease is not permanent, as evidenced by normal activity of the hypothalamic-pituitary-gonadal axis after discontinuation of treatment in adulthood (IHH reversal). Together, these observations suggest that IHH is not strictly a monogenic mendelian disease, as previously thought. Rather, it is emerging as a digenic, and potentially oligogenic disease, in which hormonal and/or environmental factors may critically influence genetic predisposition and clinical course. Future investigations of IHH should characterize the extent of the involvement of multiple genes in disease pathogenesis, and elucidate the contributions of epigenetic factors.

Development of a retrospective job exposure matrix for PCB-exposed workers in capacitor manufacturing

Objectives: Polychlorinated biphenyls (PCBs) are considered probable human carcinogens by the International Agency for Research on Cancer and one congener, PCB126, has been rated as a known human carcinogen. A period-specific job exposure matrix (JEM) was developed for former PCB-exposed capacitor manufacturing workers (n=12,605) (1938-1977). Methods: A detailed exposure assessment for this plant was based on a number of exposure determinants (proximity, degree of contact with PCBs, temperature, ventilation, process control, job mobility). The intensity and frequency of PCB exposures by job for both inhalation and dermal exposures, and additional chemical exposures were reviewed. The JEM was developed in nine steps: (1) all unique jobs (n=1,684) were assessed using (2) defined PCB exposure determinants; (3) the exposure determinants were used to develop exposure profiles; (4) similar exposure profiles were combined into categories having similar PCB exposures; (5) qualitative intensity (high-medium-low-baseline) and frequency (continuous-intermittent) ratings were developed, and (6) used to qualitatively rate inhalation and dermal exposure separately for each category; (7) quantitative intensity ratings based on available air concentrations were developed for inhalation and dermal exposures based on equal importance of both routes of exposure; (8) adjustments were made for overall exposure, and (9) for each category the product of intensity and frequency was calculated, and exposure in the earlier era was weighted. Results: A period-specific JEM modified for two eras of stable PCB exposure conditions. Conclusions: These exposure estimates, derived from a systematic and rigorous use of the exposure determinant data, lead to cumulative PCB exposure-response relationships in the epidemiological cancer mortality and incidence studies of this cohort. [Authors]

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

The polymorphic nature of HIV type 1 env V4 affects the patterns of potential N-glycosylation sites in proviral DNA at the intrahost level.

We have previously shown that env V4 from HIV-1 plasma RNA is highly heterogeneous within a single patient, due to indel-associated polymorphism. In this study, we have analyzed the variability of V4 in proviral DNA from unfractionated PBMC and sorted T and non-T cell populations within individual patients. Our data show that the degree of sequence variability and length polymorphism in V4 from HIV provirus is even higher than we previously reported in plasma. The data also show that the sequence of V4 depends largely on the experimental approach chosen. We could observe no clear trend for compartmentalization of V4 variants in specific cell types. Of interest is the fact that some variants that had been found to be predominant in plasma were not detected in any of the cell subsets analyzed. Consistently with our observations in plasma, V3 was found to be relatively conserved at both interpatient and intrapatient level. Our data show that V4 polymorphism involving insertions and deletions in addition to point mutations results in changes in the patterns of sequons in HIV-1 proviral DNA as well as in plasma RNA. These rearrangements may result in the coexistence, within the same individual, of a swarm of different V4 regions, each characterized by a different carbohydrate surface shield. Further studies are needed to investigate the mechanism responsible for the variability observed in V4 and its role in HIV pathogenesis.

Identification and antifungal susceptibility of a large collection of yeast strains isolated in Tunisian hospitals.

Abstract In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used as a rapid method to identify yeasts isolated from patients in Tunisian hospitals. When identification could not be exstablished with this procedure, sequencing of the internal transcribed spacer with 5.8S ribosomal DNA (rDNA) (ITS1-5.8S-ITS2) and D1/D2 domain of large-subunit (LSU rDNA) were employed as a molecular approach for species differentiation. Candida albicans was the dominant species (43.37% of all cases), followed by C. glabrata (16.55%), C. parapsilosis (13.23%), C. tropicalis (11.34%), C. dubliniensis (4.96%), and other species more rarely encountered in human diseases such as C. krusei, C. metapsilosis, C. lusitaniae, C. kefyr, C. palmioleophila, C. guilliermondii, C. intermedia, C. orthopsilosis, and C. utilis. In addition, other yeast species were obtained including Saccharomyces cerevisiae, Debaryomyces hansenii (anamorph known as C. famata), Hanseniaspora opuntiae, Kodamaea ohmeri, Pichia caribbica (anamorph known as C. fermentati), Trichosporon spp. and finally a novel yeast species, C. tunisiensis. The in vitro antifungal activities of fluconazole and voriconazole were determined by the agar disk diffusion test and Etest, while the susceptibility to additional antifungal agents was determined with the Sensititre YeastOne system. Our results showed low incidence of azole resistance in C. albicans (0.54%), C. tropicalis (2.08%) and C. glabrata (4.28%). In addition, caspofungin was active against most isolates of the collection with the exception of two K. ohmeri isolates. This is the first report to describe caspofungin resistant isolates of this yeast.

The region of mouse mammary tumor virus DNA containing the long terminal repeat includes a long coding sequence and signals for hormonally regulated transcription.

Starting from a biologically active recombinant DNA clone of exogenous unintegrated GR mouse mammary tumor virus, we have generated three subclones of PstI fragments of 1.45, 1.1, and 2.0 kb in the plasmid vector PBR322. The nucleotide sequence has been determined for the clone of 1.45 kb which includes almost the complete region of the long terminal repeat (LTR) plus an adjacent stretch of unique sequence DNA. A short region of the 2.0 kb clone, containing the beginning of the LTR, has also been sequenced. Starting with the A of an initiation codon outside the LTR, we detected an open reading frame of 960 nucleotides, potentially coding for a protein of 320 amino acids (36K). Two hundred nucleotides downstream from the termination codon, and approximately 25 nucleotides upstream from the presumptive initiation site of viral RNA synthesis, we found a promoter-like sequence. The sequence AGTAAA was detected approximately 15-20 nucleotides upstream from the 3' end of virion RNA and probably serves as a polyadenylation signal. The 1.45 kb PstI fragment has been transfected into Ltk- cells together with a plasmid containing the thymidine kinase gene of herpes simplex virus. The virus-specific RNA synthesis detected in a Tk+ cell clone was strongly stimulated by the addition of dexamethasone.

The apical localization of transcribing RNA polymerases on supercoiled DNA prevents their rotation around the template.

The interaction of Escherichia coli RNA polymerase with supercoiled DNA was visualized by cryo-electron microscopy of vitrified samples and by classical electron microscopy methods. We observed that when E. coli RNA polymerase binds to a promoter on supercoiled DNA, this promoter becomes located at an apical loop of the interwound DNA molecule. During transcription RNA polymerase shifts the apical loop along the DNA, always remaining at the top of the moving loop. This relationship between RNA polymerase and the supercoiled template precludes circling of the RNA polymerase around the DNA and prevents the growing RNA transcript from becoming entangled with the template DNA.