The Hus1 homologue of Leishmania major encodes a nuclear protein that participates in DNA damage response

The protozoan parasite Leishmania presents a dynamic and plastic genome in which gene amplification and chromosome translocations are common phenomena. Such plasticity hints at the necessity of dependable genome maintenance pathways. Eukaryotic cells have evolved checkpoint control systems that recognize altered DNA structures and halt cell cycle progression allowing DNA repair to take place. In these cells, the PCNA-related heterotrimeric complex formed by the proteins Hus1, Rad9, and Rad1 is known to participate in the early steps of replicative stress sensing and signaling. Here we show that the Hus1 homolog of Leishmania major is a nuclear protein that improves the cell capability to cope with replicative stress. Overexpression of LmHus1 confers resistance to the genotoxic drugs hydroxyurea (HU) and methyl methanesulfonate (MMS) and resistance to HU correlates to reduced net DNA damage upon LmHus1 expression. (C) 2011 Elsevier B.V. All rights reserved.

Saccharomyces cerevisiae is permissive for replication of bovine papillomavirus type 1

We recently demonstrated that Saccharomyces cerevisiae protoplasts can take up bovine papillomavirus type 1 (BPV1) virions and that viral episomal DNA is replicated after uptake. Here we demonstrate that BPV virus-like particles are assembled in infected S. cerevisiae cultures from newly synthesized capsid proteins and also package newly synthesized DNA, including full-length and truncated viral DNA and S. cerevisiae-derived DNA. Virus particles prepared in S. cerevisiae are able to convey packaged DNA to Cos1 cells and to transform C127 cells. Infectivity was blocked by antisera to BPV1 L1 but not antisera to BPV1 E4. We conclude that S. cerevisiae is permissive for the replication of BPV1 virus.

Kinetics of baculovirus replication and release using real-time quantitative polymerase chain reaction

The study of viral-based processes is hampered by (a) their complex, transient nature, (b) the instability of products, and (c) the lack of accurate diagnostic assays. Here, we describe the use of real-time quantitative polymerase chain reaction to characterize baculoviral infection. Baculovirus DNA content doubles every 1.7 h from 6 h post-infection until replication is halted at the onset of budding. No dynamic equilibrium exists between replication and release, and the kinetics are independent of the cell density at the time of infection. No more than 16% of the intracellular virus copies bud from the cell. (C) 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 476-480, 2002; DOI 10.1002/bit.10126.

Molecular and functional analyses of Kunjin virus infectious cDNA clones demonstrate the essential roles for NS2A in virus assembly and for a nonconservative residue in NS3 in RNA replication

A number of full-length cDNA clones of Kunjin virus (KUN) were previously prepared; it was shown that two of them, pAKUN and FLSDX, differed in specific infectivities of corresponding in vitro transcribed RNAs by similar to100,000-fold (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). In this study, we analyzed a possible genetic determinant(s) of the observed differences in infectivity initially by sequencing the entire cDNAs of both clones and comparing them with the published sequence of the parental KUN strain MRM61C. We found six common amino acid residues in both cDNA clones that were different from those in the published MRM61C sequence but were similar to those in the published sequences of other flaviviruses from the same subgroup. pAKUN clone had four additional codon changes, i.e., Ile59 to Asn and Arg175 to Lys in NS2A and Tyr518 to His and Ser557 to Pro in NS3. Three of these substitutions except the previously shown marker mutation, Arg175 to Lys in NS2A, reverted to the wild-type sequence in the virus eventually recovered from pAKUN RNA-transfected BHK cells, demonstrating the functional importance of these residues in viral replication and/or viral assembly. Exchange of corresponding DNA fragments between pAKUN and FLSDX clones and site-directed mutagenesis revealed that the Tyr518-to-His mutation in NS3 was responsible for an similar to5-fold decrease in specific infectivity of transcribed RNA, while the Ile59-to-Asn mutation in NS2A completely blocked virus production. Correction of the Asn59 in pAKUN NS2A to the wild-type lie residue resulted in complete restoration of RNA infectivity. Replication of KUN replicon RNA with an Ile59-to-Asn substitution in NS2A and with a Ser557-to-Pro substitution in NS3 was not affected, while the Tyr518-to-His substitution in NS3 led to severe inhibition of RNA replication. The impaired function of the mutated NS2A in production of infectious virus was complemented in trans by the helper wild-type NS2A produced from the KUN replicon RNA. However, replicon RNA with mutated NS2A could not be packaged in trans by the KUN structural proteins. The data demonstrated essential roles for the KUN nonstructural protein NS2A in virus assembly and for NS3 in RNA replication and identified specific single-amino-acid residues involved in these functions.

Detection of hepatitis B virus DNA by the polymerase chain reaction in anti-HBE positive chronic hepatitis B patients

Detection of HBV-DNA by PCR was compared with other serological markers (HBsAg, HBeAg and anti-HBe) in a series of49 Chronic Hepatitis B patients, including 12 with a spontaneous clearance of HBsAg. None of these HBsAg negative cases were PCR positive, but 33/37 (89.2%) HBsAg positive cases were PCR positive (p < 0.0001). Among HBsAg positive samples, nine cases were HBeAg positive and anti-HBe negative, all of them PCR positive. Other 3 patients were HBeAg and anti-HBe positive and these cases were also found PCR positive. A third group included 21 patients anti-HBe positive and HBeAg negative: 19 of them were PCR positive and 2 were PCR negative. The last 4 cases were HBeAg and anti-HBe negative, two of them were PCR positive. The detection of anti-HBe viremic cases in the present series suggest that preC variants could occur in our country. In conclusion, the integrated phase o f chronic hepatitis B seems to be less frequent than it was assumed, when only HBeAg or dot blot hybridization techniques were used. The new term "low replication phase" might favorably replace the former "integrated phase".

Active replication of hepatitis B virus (HBV) in HIV type 1 and in HIV type 2 infected patients

To evaluate the effect of concurrent infection by HIV on HBV infection or immunity, we have studied a group of 66 HIV1+ symptomatic Caucasian patients and another of 38 African HIV2+ asymptomatic individuals, concerning their HBV status: serological markers of infection and presence of HBV-DNA in serum, the last taken as sign of hepatitis B virus active replication, were monitored. HIV+ groups were compared with seronegative controls, adequately matched for age, sex and ethnological background. HBV DNA was found in 7.6% of HIV1+ Caucasian patients and 3.2% of seronegative controls; in African HIV2+ individuals 2.6% were also HBV DNA+, a percentage close to that found in HIV2 seronegative controls (2.9%). No correlation was found between HIV infection and HBV active replication. Immunodepression that follows HIV infection over time may be compatible with a degree of T cell function capable of avoiding reinfection with or reactivation of HBV, even in symptomatic stages of acquired immunodeficiency syndrome. Our findings are relevant to the choice of preventive strategies in populations at risk for HIV and HBV infection.

Development of computational tools for the integrated analysis of DNA microarray data with applications in cancer research

The MAP-i Doctoral Program of the Universities of Minho, Aveiro and Porto

Modeling sequencing errors by combining Hidden Markov models.

Among the largest resources for biological sequence data is the large amount of expressed sequence tags (ESTs) available in public and proprietary databases. ESTs provide information on transcripts but for technical reasons they often contain sequencing errors. Therefore, when analyzing EST sequences computationally, such errors must be taken into account. Earlier attempts to model error prone coding regions have shown good performance in detecting and predicting these while correcting sequencing errors using codon usage frequencies. In the research presented here, we improve the detection of translation start and stop sites by integrating a more complex mRNA model with codon usage bias based error correction into one hidden Markov model (HMM), thus generalizing this error correction approach to more complex HMMs. We show that our method maintains the performance in detecting coding sequences.

Results of a collaborative study of the EDNAP group regarding mitochondrial DNA heteroplasmy and segregation in hair shafts.

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the distribution of mitochondrial DNA (mtDNA) heteroplasmy amongst the hairs of an individual who displays point heteroplasmy in blood and buccal cells. A second aim of the exercise was to study reproducibility of mtDNA sequencing of hairs between laboratories using differing chemistries, further to the first mtDNA reproducibility study carried out by the EDNAP group. Laboratories were asked to type 2 sections from each of 10 hairs, such that each hair was typed by at least two laboratories. Ten laboratories participated in the study, and a total of 55 hairs were typed. The results showed that the C/T point heteroplasmy observed in blood and buccal cells at position 16234 segregated differentially between hairs, such that some hairs showed only C, others only T and the remainder, C/T heteroplasmy at varying ratios. Additionally, differential segregation of heteroplasmic variants was confirmed in independent extracts at positions 16093 and the poly(C) tract at 302-309, whilst a complete A-G transition was confirmed at position 16129 in one hair. Heteroplasmy was observed at position 16195 on both strands of a single extract from one hair segment, but was not observed in the extracts from any other segment of the same hair. Similarly, heteroplasmy at position 16304 was observed on both strands of a single extract from one hair. Additional variants at positions 73, 249 and the HVII poly(C) region were reported by one laboratory; as these were not confirmed in independent extracts, the possibility of contamination cannot be excluded. Additionally, the electrophoresis and detection equipment used by this laboratory was different to those of the other laboratories, and the discrepancies at position 249 and the HVII poly(C) region appear to be due to reading errors that may be associated with this technology. The results, and their implications for forensic mtDNA typing, are discussed in the light of the biology of hair formation.

Solid-phase synthesis and NMR studies of modified oligonucleotides forming triplex helix and of oligonucleopeptides mimicking the topoisomerase I-DNA covalent complex

Report for the scientific sojourn carried out at the Institut de Biologia Molecular de Barcelona of the CSIC –state agency – from april until september 2007. Topoisomerase I is an essential nuclear enzyme that modulates the topological status of DNA, facilitating DNA helix unwinding during replication and transcription. We have prepared the oligonucleotide-peptide conjugate Ac-NLeu-Asn-Tyr(p-3’TTCAGAAGC5’)-LeuC-CONH-(CH2)6-OH as model compound for NMR studies of the Topoisomerase I- DNA complex. Special attention was made on the synthetic aspects for the preparation of this challenging compound especially solid supports and protecting groups. The desired peptide was obtained although we did not achieve the amount of the conjugate needed for NMR studies. Most probably the low yield is due to the intrinsic sensitive to hydrolysis of the phosphate bond between oligonucleotide and tyrosine. We have started the synthesis and the structural characterization of oligonucleotides carrying intercalating compounds. At the present state we have obtained model duplex and quadruplex sequences modified with acridine and NMR studies are underway. In addition to this project we have successfully resolved the structure of a fusion peptide derived from hepatitis C virus envelope synthesized by the group of Dr. Haro and we have synthesized and started the characterization of a modified G-quadruplex.

Design and analysis of ChIP-Seq experiments for nuclear factor I DNA-binding proteins

SUMMARY : Eukaryotic DNA interacts with the nuclear proteins using non-covalent ionic interactions. Proteins can recognize specific nucleotide sequences based on the sterical interactions with the DNA and these specific protein-DNA interactions are the basis for many nuclear processes, e.g. gene transcription, chromosomal replication, and recombination. New technology termed ChIP-Seq has been recently developed for the analysis of protein-DNA interactions on a whole genome scale and it is based on immunoprecipitation of chromatin and high-throughput DNA sequencing procedure. ChIP-Seq is a novel technique with a great potential to replace older techniques for mapping of protein-DNA interactions. In this thesis, we bring some new insights into the ChIP-Seq data analysis. First, we point out to some common and so far unknown artifacts of the method. Sequence tag distribution in the genome does not follow uniform distribution and we have found extreme hot-spots of tag accumulation over specific loci in the human and mouse genomes. These artifactual sequence tags accumulations will create false peaks in every ChIP-Seq dataset and we propose different filtering methods to reduce the number of false positives. Next, we propose random sampling as a powerful analytical tool in the ChIP-Seq data analysis that could be used to infer biological knowledge from the massive ChIP-Seq datasets. We created unbiased random sampling algorithm and we used this methodology to reveal some of the important biological properties of Nuclear Factor I DNA binding proteins. Finally, by analyzing the ChIP-Seq data in detail, we revealed that Nuclear Factor I transcription factors mainly act as activators of transcription, and that they are associated with specific chromatin modifications that are markers of open chromatin. We speculate that NFI factors only interact with the DNA wrapped around the nucleosome. We also found multiple loci that indicate possible chromatin barrier activity of NFI proteins, which could suggest the use of NFI binding sequences as chromatin insulators in biotechnology applications. RESUME : L'ADN des eucaryotes interagit avec les protéines nucléaires par des interactions noncovalentes ioniques. Les protéines peuvent reconnaître les séquences nucléotidiques spécifiques basées sur l'interaction stérique avec l'ADN, et des interactions spécifiques contrôlent de nombreux processus nucléaire, p.ex. transcription du gène, la réplication chromosomique, et la recombinaison. Une nouvelle technologie appelée ChIP-Seq a été récemment développée pour l'analyse des interactions protéine-ADN à l'échelle du génome entier et cette approche est basée sur l'immuno-précipitation de la chromatine et sur la procédure de séquençage de l'ADN à haut débit. La nouvelle approche ChIP-Seq a donc un fort potentiel pour remplacer les anciennes techniques de cartographie des interactions protéine-ADN. Dans cette thèse, nous apportons de nouvelles perspectives dans l'analyse des données ChIP-Seq. Tout d'abord, nous avons identifié des artefacts très communs associés à cette méthode qui étaient jusqu'à présent insoupçonnés. La distribution des séquences dans le génome ne suit pas une distribution uniforme et nous avons constaté des positions extrêmes d'accumulation de séquence à des régions spécifiques, des génomes humains et de la souris. Ces accumulations des séquences artéfactuelles créera de faux pics dans toutes les données ChIP-Seq, et nous proposons différentes méthodes de filtrage pour réduire le nombre de faux positifs. Ensuite, nous proposons un nouvel échantillonnage aléatoire comme un outil puissant d'analyse des données ChIP-Seq, ce qui pourraient augmenter l'acquisition de connaissances biologiques à partir des données ChIP-Seq. Nous avons créé un algorithme d'échantillonnage aléatoire et nous avons utilisé cette méthode pour révéler certaines des propriétés biologiques importantes de protéines liant à l'ADN nommés Facteur Nucléaire I (NFI). Enfin, en analysant en détail les données de ChIP-Seq pour la famille de facteurs de transcription nommés Facteur Nucléaire I, nous avons révélé que ces protéines agissent principalement comme des activateurs de transcription, et qu'elles sont associées à des modifications de la chromatine spécifiques qui sont des marqueurs de la chromatine ouverte. Nous pensons que lés facteurs NFI interagir uniquement avec l'ADN enroulé autour du nucléosome. Nous avons également constaté plusieurs régions génomiques qui indiquent une éventuelle activité de barrière chromatinienne des protéines NFI, ce qui pourrait suggérer l'utilisation de séquences de liaison NFI comme séquences isolatrices dans des applications de la biotechnologie.

DNA degradation in avian faecal samples and feasibility of non-invasive genetic studies applied to threatened capercaillie populations

We evaluated the feasibility of using faeces as a non-invasively collected DNA source for the genetic study of an endangered bird population (capercaillie; Tetrao urogallus). We used a multitube approach, and for our panel of 11 microsatellites genotyping reliability was estimated at 98% with five repetitions. Experiments showed that free DNases in faecal material were the major cause of DNA degradation. Our results demonstrate that using avian faeces as a source of DNA, reliable microsatellite genotyping can be obtained with a reasonable number of PCR replicates.

Prostaglandin A1 Inhibits Replication of Classical Swine Fever Virus

Prostaglandins (Pgs) have been shown to inhibit the replication of several DNA and RNA viruses. Here we report the effect of prostaglandin (PgA1) on the multiplication of a positive strand RNA virus, Classical Swine Fever Virus (CSFV) in PK15 cells. PgA1 was found to inhibit the multiplication of CSFV. At a concentration of 5 µg/ml, which was nontoxic to the cells, PgA1 inhibitis virus production in 99%. In PgA1 treated cells the size and number of characteristic Classical Swine Fever focus decreased in amount.

Biological embedding of early life exposures and disease risk in humans: a role for DNA methylation.

BACKGROUND: Following wider acceptance of "the thrifty phenotype" hypothesis and the convincing evidence that early life exposures can influence adult health even decades after the exposure, much interest has been placed on the mechanisms through which early life exposures become biologically embedded. METHODS: In this review, we summarize the current literature regarding biological embedding of early life experiences. To this end we conducted a literature search to identify studies investigating early life exposures in relation to DNA methylation changes. In addition, we summarize the challenges faced in investigations of epigenetic effects, stemming from the peculiarities of this emergent and complex field. A proper systematic review and meta-analyses were not feasible given the nature of the evidence. RESULTS: We identified 7 studies on early life socioeconomic circumstances, 10 studies on childhood obesity, and 6 studies on early life nutrition all relating to DNA methylation changes that met the stipulated inclusion criteria. The pool of evidence gathered, albeit small, favours a role of epigenetics and DNA methylation in biological embedding, but replication of findings, multiple comparison corrections, publication bias, and causality are concerns remaining to be addressed in future investigations. CONCLUSIONS: Based on these results, we hypothesize that epigenetics, in particular DNA methylation, is a plausible mechanism through which early life exposures are biologically embedded. This review describes the current status of the field and acts as a stepping stone for future, better designed investigations on how early life exposures might become biologically embedded through epigenetic effects. This article is protected by copyright. All rights reserved.

The second internal transcribed spacer of nuclear ribosomal DNA as a tool for Latin American anopheline taxonomy: a critical review

Among the molecular markers commonly used for mosquito taxonomy, the internal transcribed spacer 2 (ITS2) of the ribosomal DNA is useful for distinguishing among closely-related species. Here we review 178 GenBank accession numbers matching ITS2 sequences of Latin American anophelines. Among those, we found 105 unique sequences corresponding to 35 species. Overall the ITS2 sequences distinguish anopheline species, however, information on intraspecific and geographic variations is scarce. Intraspecific variations ranged from 0.2% to 19% and our analysis indicates that misidentification and/or sequencing errors could be responsible for some of the high values of divergence. Research in Latin American malaria vector taxonomy profited from molecular data provided by single or few field capture mosquitoes. However we propose that caution should be taken and minimum requirements considered in the design of additional studies. Future studies in this field should consider that: (1) voucher specimens, assigned to the DNA sequences, need to be deposited in collections, (2) intraspecific variations should be thoroughly evaluated, (3) ITS2 and other molecular markers, considered as a group, will provide more reliable information, (4) biological data about vector populations are missing and should be prioritized, (5) the molecular markers are most powerful when coupled with traditional taxonomic tools.