Control of chromosomal rearrangements in {\it Escherichia coli\/}

A complete physical map of Escherichia coli K-12 strain MG1655 was constructed by digesting chromosomal DNA with the infrequently cutting restriction enzymes NotI, SfiI and XbaI and separating the fragments by pulsed field gel electrophoresis. The map was used to compare six K-12 strains of E. coli. Although several differences were noted and localized, the map of MG1655 was representative of all the K-12 strains tested. The maps were also used to analyze chromosomal rearrangements in the E. coli strain MG1655. The spontaneous and UV induced frequencies of tandem duplication formation were measured at several loci distributed around the chromosome. The spontaneous duplication frequency varied from 10$\sp{-5}$ to 10$\sp{-3}$ and increased at least ten-fold following mild UV irradiation treatment. Duplications of several regions of the chromosome, including the serA region and the metE region, were mapped using pulsed field gel electrophoresis. Duplications of serA were found to be large, ranging in size from 600 kb to 2100 kb. Several of the duplications isolated at serA were caused by ectopic recombination between IS5 elements and between IS186 elements. Duplications of the metE region, however, were almost exclusively the result of ectopic recombination between ribosomal RNA cistrons. Duplication frequencies were determined at both serA and metE in wild type and mismatch repair mutant strains (mutL, mutS, uvrD and recF). Even though all of the mismatch repair mutations increased duplication frequency of metE, the largest increases were observed in the mutL and mutS strains. Duplication frequency of serA was increased less dramatically by mutations in mismatch repair. Several duplications of metE isolated in a wild type and a mismatch repair mutant were mapped. The results showed that the same repeated sequences were used for duplication formation in the mismatch repair mutant as were used in the wild type strain. Several isolates showed evidence of multiple rearrangements indicating that mismatch repair may play a role in stabilizing the genome by controlling chromosomal rearrangement. ^

Genome -wide and locus -specific instability

The discovery of expanded simple repeated sequences causing or associated with human disease has lead to a new area of research involved in the elucidation of how the expanded repeat causes disease and how the repeat becomes unstable. ^ To study the genetic basis of the (CTG)n repeat instability in the DMPK gene in myotonic dystrophy (DM1) patients, somatic cell hybrids were constructed between the lymphocytes of DM1 patients and a variety of Chinese hamster ovary (CHO) cell DNA repair gene deficient mutants. By using small pool PCR (SP-PCR), the instability of the (CTG)n can be quantitated for both the frequency and sizes of length change mutations. ^ Additional SP-PCR analysis on 2/11 subclones generated from this original hybrid showed a marked increase in large repeat deletions, ∼50%. A bimodal distribution of repeats was seen around the progenitor allele and at a large deleted product (within the normal range) with no intermediate products present. ^ To determine if the repair capacity of the CHO cell led to a mutator phenotype in the hamster and hybrid clones, SP-PCR was also done on 3 hamster microsatellites in a variety of hamster cell backgrounds. No variant alleles were seen in over 2500 genome equivalents screened. ^ Human-hamster hybrids have long been shown to be chromosomally unstable, yet information about the stability of repeated sequences was not known. To test if repeat instability was associated with either intact or non-intact human chromosomes, more than 300 microsatellite repeats on 13 human chromosomes (intact and non-intact) were analyzed in eight hybrid cells. No variants were seen between the hybrid and patient alleles in the hybrids. ^ To identify whether DM1 patients have a previously undetected level of genome wide instability or if the instability is truly locus specific, SP-PCR was done on 6 human microsatellites within the patient used to make the hybrid cells. No variants were seen in over 1000 genomes screened. ^ These studies show that the somatic cell hybrid approach is a genetically stable system that allows for the determination of factors that could lead to changes in microsatellite instability. It also shows that there is something inherent about the DM1 expanded (CTG)n repeat that it is solely targeted by, as of yet, and unknown mechanism that causes the repeat to be unstable. (Abstract shortened by UMI.)^

Análisis genómico y funcional de los sistemas de Quorum Sensing en Rhizobium leguminosarum

Rhizobium leguminosarum (Rl) es una alfa-proteobacteria capaz de establecer una simbiosis diazotrófica con distintas leguminosas. A pesar de la importancia de esta simbiosis en el balance global del ciclo del nitrógeno, muy pocos genomas de rhizobios han sido secuenciados, que aporten nuevos conocimientos relacionados con las características genéticas que contribuyen a importantes procesos simbióticos. Únicamente tres secuencias completas de Rl han sido publicadas: Rl bv. viciae 3841 y dos genomas de Rl bv. trifolii (WSM1325 y WSM2304), ambos simbiontes de trébol. La secuencia genómica de Rlv UPM791 se ha determinado por medio de secuenciación 454. Este genoma tiene un tamaño aproximado de 7.8 Mb, organizado en un cromosoma y 5 replicones extracromosómicos, que incluyen un plásmido simbiótico de 405 kb. Este nuevo genoma se ha analizado en relación a las funciones simbióticas y adaptativas en comparación con los genomas completos de Rlv 3841 y Rl bv. trifolii WSM1325 y WSM2304. Mientras que los plásmidos pUPM791a y b se encuentran conservados, el plásmido simbiótico pUPM791c exhibe un grado de conservación muy bajo comparado con aquellos descritos en las otras cepas de Rl. Uno de los factores implicados en el establecimiento de la simbiosis es el sistema de comunicación intercelular conocido como Quorum Sensing (QS). El análisis del genoma de Rlv UPM791 ha permitido la identificación de dos sistemas tipo LuxRI mediados por señales de tipo N-acyl-homoserina lactonas (AHLs). El análisis mediante HPLC-MS ha permitido asociar las señales C6-HSL, C7-HSL y C8-HSL al sistema rhiRI, codificado en el plásmido simbiótico; mientras que el sistema cinRI, localizado en el cromosoma, produce 3OH-C14:1-HSL. Se ha identificado una tercera sintasa (TraI) codificada en el plásmido simbiótico, pero su regulador correspondiente se encuentra truncado debido a un salto de fase. Adicionalmente, se han encontrado tres reguladores de tipo LuxR-orphan que no presentan una sintasa LuxI asociada. El efecto potencial de las señales tipo AHL se ha estudiado mediante una estrategia de quorum quenching, la cual interfiere con los sistemas de QS de la bacteria. Esta estrategia está basada en la introducción del gen aiiA de Bacillus subtilis, que expresa constitutivamente una enzima lactonasa degradadora de AHLs. Para llevar a cabo el análisis en condiciones simbióticas, se ha desarrollado un sistema de doble marcaje que permite la identificación basado en los marcadores gusA y celB, que codifican para una enzima β–glucuronidasa y una β–galactosidasa termoestable, respectivamente. Los resultados obtenidos indican que Rlv UPM791 predomina sobre la cepa Rlv 3841 para la formación de nódulos en plantas de guisante. La baja estabilidad del plásmido que codifica para aiiA, no ha permitido obtener una conclusión definitiva sobre el efecto de la lactonasa AiiA en competitividad. Con el fin de analizar el significado y la regulación de la producción de moléculas señal tipo AHL, se han generado mutantes defectivos en cada uno de los dos sistemas de QS. Se ha llevado a cabo un análisis detallado sobre la producción de AHLs, formación de biofilm y simbiosis con plantas de guisante, veza y lenteja. El efecto de las deleciones de los genes rhiI y rhiR en Rlv UPM791 es más drástico en ausencia del plásmido pUPM791d. Mutaciones en cinI o cinRIS muestran tanto ausencia de señales, como producción exclusivamente de las de bajo peso molecular, respectivamente, producidas por el sistema rhiRI. Estas mutaciones mostraron un efecto importante en simbiosis. El sistema rhiRI se necesita para un comportamiento simbiótico normal. Además, mutantes cinRIS generaron nódulos blancos e ineficientes, mientras que el mutante cinI fue incapaz de producir nódulos en ninguna de las leguminosas utilizadas. Dicha mutación resultó en la inestabilización del plasmido simbiótico por un mecanismo dependiente de cinI que no ha sido aclarado. En general, los resultados obtenidos indican la existencia de un modelo de regulación dependiente de QS significativamente distinto a los que se han descrito previamente en otras cepas de R. leguminosarum, en las cuales no se había observado ningún fenotipo relevante en simbiosis. La regulación de la producción de AHLs Rlv UPM791 es un proceso complejo que implica genes situados en los plásmidos UPM791c y UPM791d, además de la señal 3-OH-C14:1-HSL. Finalmente, se ha identificado un transportador de tipo RND, homologo a mexAB-oprM de P. aeruginosa e implicado en la extrusión de AHLs de cadena larga. La mutación he dicho transportador no tuvo efectos apreciables sobre la simbiosis. ABSTRACT Rhizobium leguminosarum (Rl) is a soil alpha-proteobacterium that establishes a diazotrophic symbiosis with different legumes. Despite the importance of this symbiosis to the global nitrogen cycling balance, very few rhizobial genomes have been sequenced so far which provide new insights into the genetic features contributing to symbiotically relevant processes. Only three complete sequences of Rl strains have been published: Rl bv. viciae 3841, harboring six plasmids (7.75 Mb) and two Rl bv. trifolii (WSM1325 and WSM2304), both clover symbionts, harboring 5 and 4 plasmids, respectively (7.41 and 6.87 Mb). The genomic sequence of Rlv UPM791 was undertaken by means of 454 sequencing. Illumina and Sanger reads were used to improve the assembly, leading to 17 final contigs. This genome has an estimated size of 7.8 Mb organized in one chromosome and five extrachromosomal replicons, including a 405 kb symbiotic plasmid. Four of these plasmids are already closed, whereas there are still gaps in the smallest one (pUPM791d) due to the presence of insertion elements and repeated sequences, which difficult the assembly. The annotation has been carried out thanks to the Manatee pipeline. This new genome has been analyzed as regarding symbiotic and adaptive functions in comparison to the Rlv 3841 complete genome, and to those from Rl bv. trifolii strains WSM1325 and WSM2304. While plasmids pUPM791a and b are conserved, the symbiotic plasmid pUPM791c exhibited the lowest degree of conservation as compared to those from the other Rl strains. One of the factors involved in the symbiotic process is the intercellular communication system known as Quorum Sensing (QS). This mechanism allows bacteria to carry out diverse biological processes in a coordinate way through the production and detection of extracellular signals that regulate the transcription of different target genes. Analysis of the Rlv UPM791 genome allowed the identification of two LuxRI-like systems mediated by N-acyl-homoserine lactones (AHLs). HPLC-MS analysis allowed the adscription of C6-HSL, C7-HSL and C8-HSL signals to the rhiRI system, encoded in the symbiotic plasmid, whereas the cinRI system, located in the chromosome, produces 3OH-C14:1-HSL, previously described as “bacteriocin small”. A third synthase (TraI) is encoded also in the symbiotic plasmid, but its cognate regulator TraR is not functional due to a fameshift mutation. Three additional LuxR orphans were also found which no associated LuxI-type synthase. The potential effect of AHLs has been studied by means of a quorum quenching approach to interfere with the QS systems of the bacteria. This approach is based upon the introduction into the strains Rl UPM791 and Rl 3841 of the Bacillus subtilis gene aiiA expressing constitutively an AHL-degrading lactonase enzyme which led to virtual absence of AHL even when AiiA-expressing cells were a fraction of the total population. No significant effect of AiiA-mediated AHL removal on competitiveness for growth in solid surface was observed. For analysis under symbiotic conditions we have set up a two-label system to identify nodules produced by two different strains in pea roots, based on the markers gusA and celB, encoding a β–glucuronidase and a thermostable β–galactosidase enzymes, respectively. The results obtained show that Rlv UPM791 outcompetes Rlv 3841 for nodule formation in pea plants, and that the presence of the AiiA plasmid does not significantly affect the relative competitiveness of the two Rlv strains. However, the low stability of the pME6863 plasmid, encoding aiiA, did not lead to a clear conclusion about the AiiA lactonase effect on competitiveness. In order to further analyze the significance and regulation of the production of AHL signal molecules, mutants deficient in each of the two QS systems were constructed. A detailed analysis of the effect of these mutations on AHL production, biofilm formation and symbiosis with pea, vetch and lentil plants has been carried out. The effect of deletions on Rlv UPM791 rhiI and rhiR genes is more pronounced in the absence of plasmid pUPM791d, as no signal is detected in UPM791.1, lacking this plasmid. Mutations in cinI or cinRIS show either no signals, or only the small ones produced by the rhiRI system, suggesting that cinR might be regulating the rhiRI system. These mutations had a strong effect on symbiosis. Analysis of rhi mutants revealed that rhiRI system is required for normal symbiotic performance, as a drastic reduction of symbiotic fitness is observed when rhiI is deleted, and rhiR is essential for nitrogen fixation in the absence of plasmid pUPM791d. Furthermore, cinRIS mutants resulted in white and inefficient nodules, whereas cinI mutant was unable to form nodules on any legume tested. The latter mutation is associated to the instabilization of the symbiotic plasmid through a mechanism still uncovered. Overall, the results obtained indicate the existence of a model of QS-dependent regulation significantly different to that previously described in other R. leguminosarum strains, where no relevant symbiotic phenotype had been observed. The regulation of AHL production in Rlv UPM791 is a complex process involving the symbiotic plasmid (pUPM791c) and the smallest plasmid (pUPM791d), with a key role for the 3-OH-C14:1-HSL signal. Finally, we made a search for potential AHL transporters in Rlv UPM791 genome. These signals diffuse freely across membranes, but in the case of the long-chain AHLs an active efflux system might be required, as it has been described for C12-HSL in the case of Pseudomonas aeruginosa. We have identified a putative AHL transporter of the RND family homologous to P. aeruginosa mexAB-oprM. A mutant strain deficient in this transporter has been generated, and TLC analysis shows absence of 3OH-C14:1-HSL in its supernatant. This deficiency was complemented by the reintroduction of an intact copy of the genes via plasmid transfer. The mutation in mexAB genes had no significant effects on the symbiotic performance of R. leguminosarum bv. viciae.

Characteristic enrichment of DNA repeats in different genomes

Using computer programs developed for this purpose, we searched for various repeated sequences including inverted, direct tandem, and homopurine–homopyrimidine mirror repeats in various prokaryotes, eukaryotes, and an archaebacterium. Comparison of observed frequencies with expectations revealed that in bacterial genomes and organelles the frequency of different repeats is either random or enriched for inverted and/or direct tandem repeats. By contrast, in all eukaryotic genomes studied, we observed an overrepresentation of all repeats, especially homopurine–homopyrimidine mirror repeats. Analysis of the genomic distribution of all abundant repeats showed that they are virtually excluded from coding sequences. Unexpectedly, the frequencies of abundant repeats normalized for their expectations were almost perfect exponential functions of their size, and for a given repeat this function was indistinguishable between different genomes.

Mutational inactivation of the proapoptotic gene BAX confers selective advantage during tumor clonal evolution

A remarkable instability at simple repeated sequences characterizes gastrointestinal cancer of the microsatellite mutator phenotype (MMP). Mutations in the DNA mismatch repair gene family underlie the MMP, a landmark for hereditary nonpolyposis colorectal cancer. These tumors define a distinctive pathway for carcinogenesis because they display a particular spectrum of mutated cancer genes containing target repeats for mismatch repair deficiency. One such gene is BAX, a proapoptotic member of the Bcl-2 family of proteins, which plays a key role in programmed cell death. More than half of colon and gastric cancers of the MMP contain BAX frameshifts in a (G)8 mononucleotide tract. However, the functional significance of these mutations in tumor progression has not been established. Here we show that inactivation of the wild-type BAX allele by de novo frameshift mutations confers a strong advantage during tumor clonal evolution. Tumor subclones with only mutant alleles frequently appeared after inoculation into nude mice of single-cell clones of colon tumor cell lines with normal alleles. In contrast, no clones of BAX-expressing cells were found after inoculation of homozygous cell clones without wild-type BAX. These results support the interpretation that BAX inactivation contributes to tumor progression by providing a survival advantage. In this context, survival analyses show that BAX mutations are indicators of poor prognosis for both colon and gastric cancer of the MMP.

RIGS (repeat-induced gene silencing) in Arabidopsis is transcriptional and alters chromatin configuration.

We have previously reported repeat-induced gene silencing (RIGS) in Arabidopsis, in which transgene expression may be silenced epigenetically when repeated sequences are present. Among an allelic series of lines comprising a primary transformant and various recombinant progeny carrying different numbers of drug resistance gene copies at the same locus, silencing was found to depend strictly on repeated sequences and to correlate with an absence of steady-state mRNA. We now report characterization, in nuclei isolated from the same transgenic lines, of gene expression by nuclear run-on assay and of chromatin structure by nuclease protection assay. We find that silencing is correlated with absence of run-on transcripts, indicating that expression is silenced at the level of transcription. We find further that silencing is also correlated with increased resistance to both DNase I and micrococcal nuclease, indicating that the silenced state reflects a change in chromatin configuration. We propose that silencing results when a locally paired region of homologous repeated nucleotide sequences is flanked by unpaired heterologous DNA, which leads chromatin to adopt a local configuration that is difficult to transcribe, and possibly akin to heterochromatin.

The anchorage function of CipA (CelL), a scaffolding protein of the Clostridium thermocellum cellulosome.

Enzymatic cellulose degradation is a heterogeneous reaction requiring binding of soluble cellulase molecules to the solid substrate. Based on our studies of the cellulase complex of Clostridium thermocellum (the cellulosome), we have previously proposed that such binding can be brought about by a special "anchorage subunit." In this "anchor-enzyme" model, CipA (a major subunit of the cellulosome) enhances the activity of CelS (the most abundant catalytic subunit of the cellulosome) by anchoring it to the cellulose surface. We have subsequently reported that CelS contains a conserved duplicated sequence at its C terminus and that CipA contains nine repeated sequences with a cellulose binding domain (CBD) in between the second and third repeats. In this work, we reexamined the anchor-enzyme mechanism by using recombinant CelS (rCelS) and various CipA domains, CBD, R3 (the repeat next to CBD), and CBD/R3, expressed in Escherichia coli. As analyzed by non-denaturing gel electrophoresis, rCelS, through its conserved duplicated sequence, formed a stable complex with R3 or CBD/R3 but not with CBD. Although R3 or CBD alone did not affect the binding of rCelS to cellulose, such binding was dependent on CBD/R3, indicating the anchorage role of CBD/R3. Such anchorage apparently increased the rCelS activity toward crystalline cellulose. These results substantiate the proposed anchor-enzyme model and the expected roles of individual CipA domains and the conserved duplicated sequence of CelS.

The Vi element. A transposon-like repeated DNA sequence interspersed in the vitellogenin locus of Xenopus laevis.

A repeated DNA element in Xenopus laevis is described that is present in about 7500 copies dispersed throughout the genome. It was first identified in the 5' flanking region of one vitellogenin gene and was therefore named the Vi element. Seven copies are present within the vitellogenin gene region, three of them within introns of the genes A1, A2 and B2, and the other four copies in the gene flanking regions. Four of these copies have been sequenced. The Vi element is bounded by a well-conserved 13 base-pair inverted repeat; in addition, it is flanked by a three base-pair direct repeat that appears to be site-specific. The length of these four copies varies from 112 to 469 base-pairs; however, sequence homology between the different copies is very high. Their structural characteristics suggest that length heterogeneity may have arisen by either unequal recombinations, deletions or tandem duplications. Altogether, the characteristics and properties of the Vi element indicate that it might represent a mobile genetic element. One of the four copies sequenced is inserted close (position -535) to the transcription initiation site of the vitellogenin gene B2 in a region otherwise showing considerable homology with the closely related gene B1. Nevertheless, the presence of the Vi element does not seem to influence significantly the estrogen-controlled expression of gene B2. In addition, three alleles of this gene created by length polymorphism in intron 3 and in the Vi element inserted near the transcription initiation site are described.

Modelling Bone Mineral Density in Swiss Breast Cancer Patients Treated with Letrozole, Tamoxifen and Sequences of Letrozole and Tamoxifen in the BIG 1-98 Study (SAKK 21/07).

Background: Bone health is a concern when treating early stage breast cancer patients with adjuvant aromatase inhibitors. Early detection of patients (pts) at risk of osteoporosis and fractures may be helpful for starting preventive therapies and selecting the most appropriate endocrine therapy schedule. We present statistical models describing the evolution of lumbar and hip bone mineral density (BMD) in pts treated with tamoxifen (T), letrozole (L) and sequences of T and L. Methods: Available dual-energy x-ray absorptiometry exams (DXA) of pts treated in trial BIG 1-98 were retrospectively collected from Swiss centers. Treatment arms: A) T for 5 years, B) L for 5 years, C) 2 years of T followed by 3 years of L and, D) 2 years of L followed by 3 years of T. Pts without DXA were used as a control for detecting selection biases. Patients randomized to arm A were subsequently allowed an unplanned switch from T to L. Allowing for variations between DXA machines and centres, two repeated measures models, using a covariance structure that allow for different times between DXA, were used to estimate changes in hip and lumbar BMD (g/cm2) from trial randomization. Prospectively defined covariates, considered as fixed effects in the multivariable models in an intention to treat analysis, at the time of trial randomization were: age, height, weight, hysterectomy, race, known osteoporosis, tobacco use, prior bone fracture, prior hormone replacement therapy (HRT), bisphosphonate use and previous neo-/adjuvant chemotherapy (ChT). Similarly, the T-scores for lumbar and hip BMD measurements were modeled using a per-protocol approach (allowing for treatment switch in arm A), specifically studying the effect of each therapy upon T-score percentage. Results: A total of 247 out of 546 pts had between 1 and 5 DXA; a total of 576 DXA were collected. Number of DXA measurements per arm were; arm A 133, B 137, C 141 and D 135. The median follow-up time was 5.8 years. Significant factors positively correlated with lumbar and hip BMD in the multivariate analysis were weight, previous HRT use, neo-/adjuvant ChT, hysterectomy and height. Significant negatively correlated factors in the models were osteoporosis, treatment arm (B/C/D vs. A), time since endocrine therapy start, age and smoking (current vs. never).Modeling the T-score percentage, differences from T to L were -4.199% (p = 0.036) and -4.907% (p = 0.025) for the hip and lumbar measurements respectively, before any treatment switch occurred. Conclusions: Our statistical models describe the lumbar and hip BMD evolution for pts treated with L and/or T. The results of both localisations confirm that, contrary to expectation, the sequential schedules do not seem less detrimental for the BMD than L monotherapy. The estimated difference in BMD T-score percent is at least 4% from T to L.

Bone mineral density in breast cancer patients treated with adjuvant letrozole, tamoxifen, or sequences of letrozole and tamoxifen in the BIG 1-98 study (SAKK 21/07).

BACKGROUND: The risk of osteoporosis and fracture influences the selection of adjuvant endocrine therapy. We analyzed bone mineral density (BMD) in Swiss patients of the Breast International Group (BIG) 1-98 trial [treatment arms: A, tamoxifen (T) for 5 years; B, letrozole (L) for 5 years; C, 2 years of T followed by 3 years of L; D, 2 years of L followed by 3 years of T]. PATIENTS AND METHODS: Dual-energy X-ray absorptiometry (DXA) results were retrospectively collected. Patients without DXA served as control group. Repeated measures models using covariance structures allowing for different times between DXA were used to estimate changes in BMD. Prospectively defined covariates were considered as fixed effects in the multivariable models. RESULTS: Two hundred and sixty-one of 546 patients had one or more DXA with 577 lumbar and 550 hip measurements. Weight, height, prior hormone replacement therapy, and hysterectomy were positively correlated with BMD; the correlation was negative for letrozole arms (B/C/D versus A), known osteoporosis, time on trial, age, chemotherapy, and smoking. Treatment did not influence the occurrence of osteoporosis (T score < -2.5 standard deviation). CONCLUSIONS: All aromatase inhibitor regimens reduced BMD. The sequential schedules were as detrimental for bone density as L monotherapy.

A test of the master gene hypothesis for interspersed repetitive DNA sequences

Many families of interspersed repetitive DNA elements, including human Alu and LINE (Long Interspersed Element) elements, have been proposed to have accumulated through repeated copying from a single source locus: the "master gene." The extent to which a master gene model is applicable has implications for the origin, evolution, and function of such sequences. One repetitive element family for which a convincing case for a master gene has been made is the rodent ID (identifier) elements. Here we devise a new test of the master gene model and use it to show that mouse ID element sequences are not compatible with a strict master gene model. We suggest that a single master gene is rarely, if ever, likely to be responsible for the accumulation of any repeat family.

Chromosome mapping of repetitive sequences in Anostomidae species: Implications for genomic and sex chromosome evolution

Background: Members of the Anostomidae family provide an interesting model system for the study of the influence of repetitive elements on genome composition, mainly because they possess numerous heterochromatic segments and a peculiar system of female heterogamety that is restricted to a few species of the Leporinus genus. The aim of this study was to isolate and identify important new repetitive DNA elements in Anostomidae through restriction enzyme digestion, followed by cloning, characterisation and chromosome mapping of this fragment. To identify repetitive elements in other Leporinus species and expand on studies of repetitive elements in Anostomidae, hybridisation experiments were also performed using previously described probes of LeSpeI repetitive elements. Results: The 628-base pair (bp) LeSpeII fragment was hybridised to metaphase cells of L. elongatus individuals as well as those of L. macrocephalus, L. obtusidens, L. striatus, L. lacustris, L. friderici, Schizodon borellii and S. isognathus. In L. elongatus, both male and female cells contained small clusters of LeSpeII repetitive elements dispersed on all of the chromosomes, with enrichment near most of the terminal portions of the chromosomes. In the female sex chromosomes of L. elongatus (Z2,Z2/W1W 2), however, this repeated element was absent. In the remaining species, a dispersed pattern of hybridisation was observed on all chromosomes irrespective of whether or not they were sex chromosomes. The repetitive element LeSpeI produced positive hybridisations signals only in L. elongatus, L. macrocephalus and L. obtusidens, i.e., species with differentiated sex chromosomes. In the remaining species, the LeSpeI element did not produce hybridisation signals. Conclusions: Results are discussed in terms of the effects of repetitive sequences on the differentiation of the Anostomidae genome, especially with respect to sex chromosome evolution. LeSpeII showed hybridisation patterns typical of Long Interspersed Elements (LINEs). The differential distribution of this element may be linked to sex chromosome differentiation in L. elongatus species. The relationship between sex chromosome specificity and the LeSpeI element is confirmed in the species L. elongatus, L. macrocephalus and L. obtusidens. © 2012 da Silva et al.; licensee BioMed Central Ltd.

The genomic Echinococcus microsatellite EmsB sequences: from a molecular marker to the epidemiological tool

In the field of molecular and epidemiological parasitology, characterization of fast evolving genetic markers appears as an important challenge to consider the diversity and genetic structure of parasites. The study of respective populations can help us to understand their adaptive strategies to survive and perpetuate the species within different host populations, all trying to resist infection. In the past, the relative monomorphic features of Echinococcus multilocularis, the causative agent of alveolar echinococcosis and a severe human parasitic disease, did not stimulate studies dealing with the genetic variability of Echinococcus species or respective populations. A recently developed, characterized and validated original multilocus microsatellite, named EmsB, tandemly repeated in the genome, offered an additional opportunity for this line of investigation. We have compiled in this review new insights brought by this molecular tracker on the transmission activity of Echinococcus among different hosts and at different geographical scales.

EmsB, a tandem repeated multi-loci microsatellite, new tool to investigate the genetic diversity of Echinococcus multilocularis

In order to explore the genetic diversity within Echinococcus multilocularis (E. multilocularis), the cestode responsible for the alveolar echinococcosis (AE) in humans, a microsatellite, composed of (CA) and (GA) repeats and designated EmsB, was isolated and characterized in view of its nature and potential field application. PCR-amplification with specific primers exhibited a high degree of size polymorphism between E. multilocularis and Echinococcus granulosus sheep (G1) and camel (G6) strains. Fluorescent-PCR was subsequently performed on a panel of E. multilocularis isolates to assess intra-species polymorphism level. EmsB provided a multi-peak profile, characterized by tandemly repeated microsatellite sequences in the E. multilocularis genome. This "repetition of repeats" feature provided to EmsB a high discriminatory power in that eight clusters, supported by bootstrap p-values larger than 95%, could be defined among the tested E. multilocularis samples. We were able to differentiate not only the Alaskan from the European samples, but also to detect different European isolate clusters. In total, 25 genotypes were defined within 37 E. multilocularis samples. Despite its complexity, this tandem repeated multi-loci microsatellite possesses the three important features for a molecular marker, i.e. sensitivity, repetitiveness and discriminatory power. It will permit assessing the genetic polymorphism of E. multilocularis and to investigate its spatial distribution in detail.