Unusually short tandem repeats appear to reach chromosome ends of Rhynchosciara americana (Diptera: Sciaridae)

The characterisation of sequences at chromosome ends of Rhynchosciara americana was continued with the screening of a genomic library using as a probe a short repeat identified in a previous report (M-22, 22 bp) which was found to be specific for noncentromeric termini of this species. Simple repeats, complex tandem and apparently dispersed repeats were present in the genomic clones analysed. Repetitive sequences do not define individual chromosome tips as they were found in all noncentromeric ends. A novel and unusually short tandem repeat type for dipteran chromosome ends (named M-16) composed of 16 nucleotides and frequently associated with M-22 arrays was characterised in this work. Islands of M-16 and M-22 tandem repeats were found in all the genomic clones analysed. Individual probes representative of each repetitive element hybridised not only to all noncentromeric ends of R. americana chromosomes but also to inter-telomeric bridges. This contrasted with the other repeat types which displayed sub-telomeric localisation as seen by double detection of hybridised probe and telomeric reverse transcriptase. Some stretches composed of M-16 and M-22 tandem repeats localised in different regions of the analysed genomic clones were either identical or showed sequence similarity that was unexpectedly higher than the mean sequence similarity observed among repeats within each of their tandem arrays. The occurrence of segmental duplications, as deduced by sequence analyses involving the two repeats that appeared to reach chromosome ends, might indicate the involvement of this type of duplication process in the chromosome end maintenance in this species.

Role of the Mitochondrial Mutations, m. 827A > G and the Novel m. 7462C > T, in the Origin of Hearing Loss

Samples from 30 deaf probands exhibiting features suggestive of syndromic mitochondrial deafness or from families with maternal transmission of deafness were selected for investigation of mutations in the mitochondrial genes MT-RNR1 and MT-TS1. Patients with mutation m. 1555A>G had been previously excluded from this sample. In the MT-RNR1 gene, five probands presented the m. 827A>G sequence variant, of uncertain pathogenicity. This change was also detected in 66 subjects of an unaffected control sample of 306 Brazilian individuals from various ethnic backgrounds. Given its high frequency, we consider it unlikely to have a pathogenic role on hereditary deafness. As to the MT-TS1 gene, one proband presented the previously known pathogenic m. 7472insC mutation and three probands presented a novel variant, m. 7462C>T, which was absent from the same control sample of 306 individuals. Because of its absence in control samples and association with a family history of hearing impairment, we suggest it might be a novel pathogenic mutation.

Curiously composite structures of a retrotransposon and a complex repeat associated with chromosome ends of Rhynchosciara americana (Diptera: Sciaridae)

In Drosophila, telomere retrotransposons counterbalance the loss of telomeric DNA. The exceptional mechanism of telomere recovery characterized in Drosophila has not been found in lower dipterans (Nematocera). However, a retroelement resembling a telomere transposon and termed ""RaTART"" has been described in the nematoceran Rhynchosciara americana. In this work, DNA and protein sequence analyses, DNA cloning, and chromosomal localization of probes obtained either by PCR or by screening a genomic library were carried out in order to examine additional features of this retroelement. The analyses performed raise the possibility that RaTART represents a genomic clone composed of distinct repetitive elements, one of which is likely to be responsible for its apparent enrichment at chromosome ends. RaTART sequence in addition allowed to assess a novel subtelomeric region of R. americana chromosomes that was analyzed in this work after subcloning a DNA fragment from a phage insert. It contains a complex repeat that is located in the vicinity of simple and complex tandem repeats characterized previously. Quantification data suggest that the copy number of the repeat is significantly lower than that observed for the ribosomal DNA in the salivary gland of R. americana. A short insertion of the RaTART was identified in the cloned segment, which hybridized preferentially to subtelomeres. Like RaTART, it displays truncated sequences related to distinct retrotransposons, one of which has a conceptual translation product with significant identity with an endonuclease from a lepidopteran retrotransposon. The composite structure of this DNA stretch probably reflects mobile element activity in the subtelomeric region analyzed in this work.

Chromosome homologies of the highly rearranged karyotypes of four Akodon species (Rodentia, Cricetidae) resolved by reciprocal chromosome painting: the evolution of the lowest diploid number in rodents

Traditionally comparative cytogenetic studies are based mainly on banding patterns. Nevertheless, when dealing with species with highly rearranged genomes, as in Akodon species, or with other highly divergent species, cytogenetic comparisons of banding patterns prove inadequate. Hence, comparative chromosome painting has become the method of choice for genome comparisons at the cytogenetic level since it allows complete chromosome probes of a species to be hybridized in situ onto chromosomes of other species, detecting homologous genomic regions between them. In the present study, we have explored the highly rearranged complements of the Akodon species using reciprocal chromosome painting through species-specific chromosome probes obtained by chromosome sorting. The results revealed complete homology among the complements of Akodon sp. n. (ASP), 2n = 10; Akodon cursor (ACU), 2n = 15; Akodon montensis (AMO), 2n = 24; and Akodon paranaensis (APA), 2n = 44, and extensive chromosome rearrangements have been detected within the species with high precision. Robertsonian and tandem rearrangements, pericentric inversions and/or centromere repositioning, paracentric inversion, translocations, insertions, and breakpoints, where chromosomal rearrangements, seen to be favorable, were observed. Chromosome painting using the APA set of 21 autosomes plus X and Y revealed eight syntenic segments that are shared with A. montensis, A. cursor, and ASP, and one syntenic segment shared by A. montensis and A. cursor plus five exclusive chromosome associations for A. cursor and six for ASP chromosome X, except for the heterochromatin region of ASP X, and even chromosome Y shared complete homology among the species. These data indicate that all those closely related species have experienced a recent extensive process of autosomal rearrangement in which, except for ASP, there is still complete conservation of sex chromosomes homologies.

Characterization of microsatellite loci of Tetragonisca angustula (Hymenoptera, Apidae, Meliponini)

An enriched genomic library was constructed from Tetragonisca angustula, a stingless bee species widely distributed in Brazil. The library was screened using two simple-repeat oligonucleotide probes and 21 microsatellite primer pairs were designed flanking a selection of repeat sequences within positive clones. The polymorphism of the microsatellite loci was analyzed by screening a sample of 19 unrelated T. angustula workers. Fifteen out of 21 loci were shown to be polymorphic, with observed heterozygosity estimates ranging from 0.00 to 0.89. The primers were also successfully used to amplify microsatellite loci from other stingless bee species, Tetragonisca fiebrigi, Tetragonisca weyrauchi, Lestrimelitta maracaia and Schwarziana quadripunctata. The results from variability analyses suggest that the microsatellite loci isolated from T. angustula will be useful in further population studies for the species and also for other Meliponini.

Potential sites of triple-helical nucleic acid formation in chromosomes of Rhynchosciara (Diptera: Sciaridae) and Drosophila melanogaster

Antibodies to specific nucleic acid conformations are amongst the methods that have allowed the study of non-canonical (Watson-Crick) DNA structures in higher organisms. In this work, the structural limitations for the immunological detection of DNA.RNA hybrid duplexes were examined using specific RNA homopolymers as probes for homopolymer polydeoxyadenylic acid (poly(dA)).polydeoxythymidylic acid (poly(dT))-rich regions of Rhynchosciara americana (Diptera: Sciaridae) chromosomes. Anti-DNA.RNA duplexes did not react with the complex formed between chromosomal poly(dA) and exogenous polyuridylic acid (poly(rU)). Additionally, poly(rU) prevented the detection of polyadenylic acid.poly(dT) hybrid duplexes preformed in situ. These results raised the possibility that three-stranded structures rather than duplexes were formed in chromosomal sites. To test this hypothesis, the specificity of antibodies to triple-helical nucleic acids was reassessed employing distinct nucleic acid configurations. These antibodies were raised to the poly(dA).poly(rU).poly(rU) complex and have been used here for the first time in immunocytochemistry. Anti-triplex antibodies recognised the complex poly(dA).poly(rU).poly(rU) assembled with poly(rU) in poly(dA).poly(dT)-rich homopolymer regions of R. americana chromosomes. The antibodies could not detect short triplex stretches, suggesting the existence of constraints for triple-helix detection, probably related to triplex tract length. In addition, anti-poly(dA).poly(rU).poly(rU) antibodies reacted with the pericentric heterochromatin of RNase-treated polytene chromosomes of R. americana and Drosophila melanogaster. In apparent agreement with data obtained in cell types from other organisms, the results of this work suggest that significant triple-helix DNA extensions can be formed in pericentric regions of these species.

Ribosomal RNA gene insertions in the R2 site of Rhynchosciara (Diptera: Sciaridae)

Ribosomal RNA genes of most insects are interrupted by R1/R2 retrotransposons. The occurrence of R2 retrotransposons in sciarid genomes was studied by PCR and Southern blot hybridization in three Rhynchosciara species and in Trichosia pubescens. Amplification products with the expected size for non-truncated R2 elements were only obtained in Rhynchosciara americana. The rDNA in this species is located in the proximal end of the X mitotic chromosome but in the salivary gland is associated with all four polytene chromosomes. Approximately 50% of the salivary gland rDNA of most R. americana larval groups analysed had an insertion in the R2 site, while no evidence for the presence of R1 elements was found. In-situ hybridization results showed that rDNA repeat units containing R2 take part in the structure of the extrachromosomal rDNA. Also, rDNA resistance to Bal 31 digestion could be interpreted as evidence for nonlinear rDNA as part of the rDNA in the salivary gland. Insertions in the rDNA of three other sciarid species were not detected by Southern blot and in-situ hybridization, suggesting that rDNA retrotransposons are significantly under-represented in their genomes in comparison with R. americana. R2 elements apparently restricted to R. americana correlate with an increased amount of repetitive DNA in its genome in contrast to other Rhynchosciara species. The results obtained in this work together with previous results suggest that evolutionary changes in the genus Rhynchosciara occurred by differential genomic occupation not only of satellite DNA but possibly also of rDNA retrotransposons.

Unexpected genetic heterogeneity in a large consanguineous Brazilian pedigree presenting deafness

Nonsyndromic autosomal recessive deafness accounts for 80% of hereditary deafness. To date, 52 loci responsible for autosomal recessive deafness have been mapped and 24 genes identified. Here, we report a large inbred Brazilian pedigree with 26 subjects affected by prelingual deafness. Given the extensive consanguinity found in this pedigree, the most probable pattern of inheritance is autosomal recessive. However, our linkage and mutational analysis revealed, instead of an expected homozygous mutation in a single gene, two different mutant alleles and a possible third undetected mutant allele in the MYO15A gene (DFNB3 locus), as well as evidence for other causes for deafness in the same pedigree. Among the 26 affected subjects, 15 were homozygous for the novel c.10573delA mutation in the MYO15A gene, 5 were compound heterozygous for the mutation c.10573delA and the novel deletion c.9957_9960delTGAC and one inherited only a single c.10573delA mutant allele, while the other one could not be identified. Given the extensive consanguinity of the pedigree, there might be at least one more deafness locus segregating to explain the condition in some of the subjects whose deafness is not clearly associated with MYO15A mutations, although overlooked environmental causes could not be ruled out. Our findings illustrate a high level of etiological heterogeneity for deafness in the family and highlight some of the pitfalls of genetic analysis of large genes in extended pedigrees, when homozygosity for a single mutant allele is expected.

Evolution of Dendrocolaptes platyrostris (Aves: Furnariidae) between the South American open vegetation corridor and the Atlantic forest

The open vegetation corridor of South America is a region dominated by savanna biomes. It contains forests (i.e. riverine forests) that may act as corridors for rainforest specialists between the open vegetation corridor and its neighbouring biomes (i.e. the Amazonian and Atlantic forests). A prediction for this scenario is that populations of rainforest specialists in the open vegetation corridor and in the forested biomes show no significant genetic divergence. We addressed this hypothesis by studying plumage and genetic variation of the Planalto woodcreeper Dendrocolaptes platyrostris Spix (1824) (Aves: Furnariidae), a forest specialist that occurs in both open habitat and in the Atlantic forest. The study questions were: (1) is there any evidence of genetic continuity between populations of the open habitat and the Atlantic forest and (2) is plumage variation congruent with patterns of neutral genetic structure or with ecological factors related to habitat type? We used cytochrome b and mitochondrial DNA control region sequences to show that D. platyrostris is monophyletic and presents substantial intraspecific differentiation. We found two areas of plumage stability: one associated with Cerrado and the other associated with southern Atlantic Forest. Multiple Mantel tests showed that most of the plumage variation followed the transition of habitats but not phylogeographical gaps, suggesting that selection may be related to the evolution of the plumage of the species. The results were not compatible with the idea that forest specialists in the open vegetation corridor and in the Atlantic forest are linked at the population level because birds from each region were not part of the same genetic unit. Divergence in the presence of gene flow across the ecotone between both regions might explain our results. Also, our findings indicate that the southern Atlantic forest may have been significantly affected by Pleistocene climatic alteration, although such events did not cause local extinction of most taxa, as occurred in other regions of the globe where forests were significantly affected by global glaciations. Finally, our results neither support plumage stability areas, nor subspecies as full species. (C) 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103, 801-820.

Alternative promoters in the pst operon of Escherichia coli

The pst operon of Escherichia coli is composed of five genes pstS, pstC, pstA, pstB and phoU, that encode a high-affinity phosphate transport system and a negative regulator of the PHO regulon. Transcription of pst is induced under phosphate shortage and is initiated at the promoter located upstream of the first gene of the operon, pstS. Here, we show by four different technical approaches the existence of additional internal promoters upstream of pstC, pstB and phoU. These promoters are not induced by Pi-limitation and do not possess PHO-box sequences. Plasmids carrying the pst internal genes partially complement chromosomal mutations in their corresponding genes, indicating that they are translated into functional proteins.

The Genetics of Alzheimer`s Disease in Brazil: 10 Years of Analysis in a Unique Population

Alzheimer`s Disease (AD) is the most common type of dementia among the elderly, with devastating consequences for the patient, their relatives, and caregivers. More than 300 genetic polymorphisms have been involved with AD, demonstrating that this condition is polygenic and with a complex pattern of inheritance. This paper aims to report and compare the results of AD genetics studies in case-control and familial analysis performed in Brazil since our first publication, 10 years ago. They include the following genes/markers: Apolipoprotein E (APOE), 5-hidroxytryptamine transporter length polymorphic region (5-HTTLPR), brain-derived neurotrophin factor (BDNF), monoamine oxidase A (MAO-A), and two simple-sequence tandem repeat polymorphisms (DXS1047 and D10S1423). Previously unpublished data of the interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) genes are reported here briefly. Results from others Brazilian studies with AD patients are also reported at this short review. Four local families studied with various markers at the chromosome 21, 19, 14, and 1 are briefly reported for the first time. The importance of studying DNA samples from Brazil is highlighted because of the uniqueness of its population, which presents both intense ethnical miscegenation, mainly at the east coast, but also clusters with high inbreeding rates in rural areas at the countryside. We discuss the current stage of extending these studies using high-throughput methods of large-scale genotyping, such as single nucleotide polymorphism microarrays, associated with bioinformatics tools that allow the analysis of such extensive number of genetics variables, with different levels of penetrance. There is still a long way between the huge amount of data gathered so far and the actual application toward the full understanding of AD, but the final goal is to develop precise tools for diagnosis and prognosis, creating new strategies for better treatments based on genetic profile.