Use of the DNA Checkerboard hybridization method for detection and quantitation of Candida species in oral microbiota

The DNA Checkerboard method enables the simultaneous identification of distinct microorganisms in a large number of samples and employs up to 45 whole genomic DNA probes to gram-negative and gram-positive bacterial species present in subgingival biofilms. Collectively, they account for 55%-60% of the bacteria in subgingival biofilms. In this study, we present the DNA Checkerboard hybridization as an alternative method for the detection and quantitation of Candida species in oral cavities. Our results reveal that DNA Checkerboard is sensitive enough and constitutes a powerful and appropriate method for detecting and quantifying Candida species found in the oral cavity.

The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA hybridization method

Molecular methods that permit the simultaneous detection and quantification of a large number of microbial species are currently employed in the evaluation of complex ecosystems. The checkerboard DNA-DNA hybridization technique enables the simultaneous identification of distinct bacterial. species in a large number of dental samples. The original technique employed digoxigenin-labeled whole genomic DNA probes which were detected by chemiluminescence. In this study, we present an alternative protocol for labeling and detecting whole genomic DNA probes in the Checkerboard DNA-DNA hybridization method. Whole genomic DNA was extracted from five bacterial species and labeled with fluorescein. The fluorescein labeled whole genomic DNA probes were hybridized against whole genomic DNA or subgingival plaque samples in a checkerboard hybridization format, followed by chemiluminescent detection. Our results reveal that fluorescein is a viable and adequate alternative labeling reagent to be employed in the checkerboard DNA-DNA hybridization technique. (c) 2007 Elsevier GmbH. All rights reserved.

Hybridization and introgression across different ploidy levels in the Neotropical orchids Epidendrum fulgens and E-puniceoluteum (Orchidaceae)

The hypothesis of gene flow between species with large differences in chromosome numbers has rarely been tested in the wild, mainly because species of different ploidy are commonly assumed to be reproductively isolated from each other because of instantaneous and strong postzygotic barriers. In this study, a broad-scale survey of molecular variation was carried out between two orchid species with different ploidy levels: Epidendrum fulgens (2n = 2x = 24 chromosomes) and Epidendrum puniceoluteum (2n = 4x = 52 chromosomes). To test the strength of their reproductive barriers, we investigated the distribution of genetic variation in sympatric and allopatric populations of these two species and conducted crossing experiments. Nuclear and plastid microsatellite loci were used to genotype 463 individuals from eight populations across the geographical range of both species along the Brazilian coastal plain. All six sympatric populations analysed presented hybrid zones, indicating that hybridization between E. fulgens and E. puniceoluteum is a common phenomenon. Bayesian assignment analysis detected the presence of F(1) and F(2) individuals and also signs of introgression, demonstrating a high potential for interspecific gene flow. Introgression occurs preferentially from E. fulgens to E. puniceoluteum. Pure parental individuals of both species display strong genotype-habitat associations, indicating that environment-dependent selection could be acting in all hybrid zones. This study suggests that hybridization and introgression are evolutionary processes playing a role in the diversification of Epidendrum and indicates the importance of investigations of hybrid zones in understanding reproductive barriers and speciation processes in Neotropical orchid species.

Morphometrical, biochemical and molecular tools for assessing biodiversity. An example in Plebeia remota (Holmberg, 1903) (Apidae, Meliponini)

We see today many efforts to quantify biodiversity in different biomes. It is very important then to develop and to apply other methodologies that allow us to assess biodiversity. Here we present an example of application of three tools with this goal. We analyzed two populations of Plebeia remota from two distinct biomes that already showed several differences in morphology and behavior. Based on these differences, it has been suggested that the populations of Cunha and Prudentopolis do not represent a single species. In order to verify the existence or absence of gene flow between these two groups, we characterized the patterns of mtDNA through RFLP, the patterns of wing venation through geometric morphometry, and the cuticular hydrocarbons through gas chromatography-mass spectrometry. We used bees collected in these two locations and also from colonies which have being kept for around 9 years at Sao Paulo University. We found six different haplotypes in these specimens, of which three of them occurred exclusively in the population of Cunha and three only in the Prudentopolis population. The fact that the populations do not share haplotypes suggests no maternal gene flow between them. The two populations were differentiated by the pattern of the wing veins. They also had different mixtures of cuticle hydrocarbons. Furthermore it was shown that the colonies kept at the university did not hybridize. These two groups may constitute different species. We also show here the importance of using other methodologies than traditional taxonomy to assess and understand biodiversity, especially in bees.

Molecular characterization of a putative heat shock protein cognate gene in Rhynchosciara americana

An hsc70 homologue gene (Rahsc70) of the diptera Rhynchosciara americana was isolated and characterized. We were able to determine the mRNA sequence from an EST of salivary gland cDNA library, and a Rahsc70 cDNA cassette was used as a probe to isolate the genomic region from a genomic library. The mRNA expression of this gene parallels the 2B puff expansion, suggesting its involvement in protein processing, since this larval period corresponds to a high synthetic activity period. During heat shock stress conditions, hsc70 expression decreased. In situ hybridization of polytene chromosomes showed that the Rahsc70 gene is located near the C3 DNA puff. The cellular localization of Hsc70 protein showed this protein in the cytoplasm and in the nucleus.

Molecular characterization of a retrotransposon in the Rhynchosciara americana genome and its association with telomere

Non-LTR retrotransposons, also known as long interspersed nuclear elements (LINEs), are transposable elements that encode a reverse transcriptase and insert into genomic locations via RNA intermediates. The sequence analysis of a cDNA library constructed from mRNA of the salivary glands of R. americana showed the presence of putative class I elements. The cDNA clone with homology to a reverse transcriptase was the starting point for the present study. Genomic phage was isolated and sequenced and the molecular structure of the element was characterized as being a non-LTR retrotransposable element. Southern blot analysis indicated that this transposable element is represented by repeat sequences in the genome of R. americana. Chromosome tips were consistently positive when this element was used as probe in in-situ hybridization. Real-time RT-PCR showed that this retrotransposon is transcribed at different periods of larval development. Most interesting, the silencing of this retrotransposon in R. americana by RNA interference resulted in reduced transcript levels and in accelerated larval development.

Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Classic and molecular cytogenetic analyses reveal chromosomal gains and losses correlated with survival in head and neck cancer patients

Purpose: Genetic biomarkers of head and neck tumors could be useful for distinguishing among patients with similar clinical and histopathologic characteristics but having differential probabilities of survival. The purpose of this study was to investigate chromosomal alterations in head and neck carcinomas and to correlate the results with clinical and epidentiologic variables.Experimental Design: Cytogenetic analysis of short-term cultures from 64 primary untreated head and neck squamous cell carcinomas was used to determine the overall pattern of chromosome aberrations. A representative subset of tumors was analyzed in detail by spectral karyotyping and/or confirmatory fluorescence in situ hybridization analysis.Results: Recurrent losses of chromosomes Y (26 cases) and 19 (14 cases), and gains of chromosomes 22 (23 cases), 8 and 20 (11 cases each) were observed. The most frequent structural aberration was del(22)(q13.1) followed by rearrangements involving 6q and 12p. The presence of specific cytogenetic aberrations was found to correlate significantly with an unfavorable outcome. There was a significant association between survival and gains in chromosomes 10 (P = 0.008) and 20 (P = 0.002) and losses of chromosomes 15 (P = 0.005) and 22 (P = 0.021). Univariate analysis indicated that acquisition of monosomy 17 was a significant (P = 0.0012) factor for patients with a previous family history of cancer.Conclusions: the significant associations found in this study emphasize that alterations of distinct regions of the genome may be genetic biomarkers for a poor prognosis. Losses of chromosomes 17 and 22 can be associated with a family history of cancer.

Classical and molecular cytogenetic analysis in head and neck squamous cell carcinomas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

5S rDNA characterization in twelve Sciaenidae fish species (Teleostei, Perciformes): depicting gene diversity and molecular markers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular cytogenetic analysis of heterochromatin in the chromosomes of tilapia, Oreochromis niloticus (Teleostei : Cichlidae)

The structure of the heterochromatic bands in mitotic chromosomes of the important tropical aquaculture species of tilapia, Oreochromis niloticus, was investigated by the combination of the C-banding technique, chromosomal digestion with two restriction endonucleases and fluorescence in situ hybridization (FISH) of two satellite DNAs (SATA and SATB). The tilapia chromosomes presented heterochromatic bands in the centromeres and in the short arms of almost all chromosomes that were differentially digested by the restriction endonucleases HaeIII and EcoRI. FISH of SATA showed that the satellite sequence is distributed in the centromeric region of all chromosomes of tilapia. FISH also revealed an intense hybridization signal for SATB in only one chromosome pair, but less intense signals were also present in several other pairs. The digestion of tilapia chromosomes by HaeIII and EcoRI was positively correlated with the position of SATA and SATB in chromosomes as revealed by FISH. The results obtained may be useful in future molecular and genetic studies of tilapias.

Chromosome banding and 18S rDNA in situ hybridization analysis of seven species of the family Achiridae (Teleostei : Pleuronectiformes)

Achiridae is an important family of the order Pleuronectiformes widely distributed in North, Central, and South America with freshwater and marine species. In the present study cytogenetic analyses comprising conventional and molecular techniques were carried out in seven species of this family. The following diploid numbers (2n) and fundamental numbers (FN) were obtained: Achirus declivis 2n = 34, FN = 52; Achirus lineatus 2n = 40, FN = 66; Catathyridium jenynsi 2n = 40 and FN = 50; Gymnachirus nudus 2n = 36 and FN = 50; Hypoclinemus mentalis 2n = 38 and FN = 54; Trinectes paulistanus 2n = 42 and FN = 52; and Trinectes sp. 2n = 38 and FN = 54. All species presented a single nucleolar organizer region (NOR) bearing chromosome pair and C-band positive segments mainly distributed at the pericentromeric position. The wide variation observed in chromosome number and FN suggests the occurrence of larger chromosome rearrangements in the family Achiridae if compared with other families of the same order.

Physical mapping of the Nile tilapia (Oreochromis niloticus) genome by fluorescent in situ hybridization of repetitive DNAs to metaphase chromosomes - a review

The Nile tilapia (Oreochromis niloticus) has received increasing scientific interest over the past few decades for two reasons: first, tilapia is an enormously important species in aquaculture worldwide, especially in regions where there is a chronic shortage of animal protein; and second, this teleost fish belongs to the fascinating group of cichlid fishes that have undergone a rapid and extensive radiation of much interest to evolutionary biologists. Currently, studies based on physical and genetic mapping of the Nile tilapia genome offer the best opportunities for applying genomics to such diverse questions and issues as phylogeography, isolation of quantitative trait loci involved in behaviour, morphology, and disease, and overall improvement of aquacultural stocks. In this review, we have integrated molecular cytogenetic data for the Nile tilapia describing the chromosomal location of the repetitive DNA sequences, satellite DNAs, telomeres, 45S and 5S rDNAs, and the short and long interspersed nucleotide elements [short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs)], and provide the beginnings of a physical genome map for this important teleost fish. (C) 2004 Elsevier B.V. All rights reserved.

Molecular identification of hybrids between Neotropical catfish species Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Origin and molecular organization of supernumerary chromosomes of Prochilodus lineatus (Characiformes, Prochilodontidae) obtained by DNA probes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)