Development of 18 Polymorphic Microsatellite Markers for Vinca minor (Apocynaceae) via 454 Pyrosequencing

• Premise of the study: Polymorphic microsatellite markers were developed in Vinca minor (Apocynaceae) to evaluate the level of clonality, population structure, and genetic diversity of the species within its native and introduced range. • Methods and Results: A total of 1371 microsatellites were found in 43,565 reads from 454 pyrosequencing of genomic V. minor DNA. Additional microsatellite loci were mined from publicly available cDNA sequences. After several rounds of screening, 18 primer pairs flanking di-, tri-, or tetranucleotide repeats were identified that revealed high levels of genetic diversity in two native Italian populations, with two to 11 alleles per locus. Clonal growth predominated in two populations from the introduced range in Germany. Five loci successfully cross-amplified in three additional Vinca species. • Conclusions: The novel polymorphic microsatellite markers are promising tools for studying clonality and population genetics of V. minor and for assessing the historical origin of Central European populations.

Polimorfismos del gen Butirilcolinesterasa responsables de reacciones adversas en pacientes consumidores de “cocaína”

La butirilcolinesterasa humana (BChE; EC 3.1.1.8) es una enzima polimórfica sintetizada en el hígado y en el tejido adiposo, ampliamente distribuida en el organismo y encargada de hidrolizar algunos ésteres de colina como la procaína, ésteres alifáticos como el ácido acetilsalicílico, fármacos como la metilprednisolona, el mivacurium y la succinilcolina y drogas de uso y/o abuso como la heroína y la cocaína. Es codificada por el gen BCHE (OMIM 177400), habiéndose identificado más de 100 variantes, algunas no estudiadas plenamente, además de la forma más frecuente, llamada usual o silvestre. Diferentes polimorfismos del gen BCHE se han relacionado con la síntesis de enzimas con niveles variados de actividad catalítica. Las bases moleculares de algunas de esas variantes genéticas han sido reportadas, entre las que se encuentra las variantes Atípica (A), fluoruro-resistente del tipo 1 y 2 (F-1 y F-2), silente (S), Kalow (K), James (J) y Hammersmith (H). En este estudio, en un grupo de pacientes se aplicó el instrumento validado Lifetime Severity Index for Cocaine Use Disorder (LSI-C) para evaluar la gravedad del consumo de “cocaína” a lo largo de la vida. Además, se determinaron Polimorfismos de Nucleótido Simple (SNPs) en el gen BCHE conocidos como responsables de reacciones adversas en pacientes consumidores de “cocaína” mediante secuenciación del gen y se predijo el efecto delos SNPs sobre la función y la estructura de la proteína, mediante el uso de herramientas bio-informáticas. El instrumento LSI-C ofreció resultados en cuatro dimensiones: consumo a lo largo de la vida, consumo reciente, dependencia psicológica e intento de abandono del consumo. Los estudios de análisis molecular permitieron observar dos SNPs codificantes (cSNPs) no sinónimos en el 27.3% de la muestra, c.293A>G (p.Asp98Gly) y c.1699G>A (p.Ala567Thr), localizados en los exones 2 y 4, que corresponden, desde el punto de vista funcional, a la variante Atípica (A) [dbSNP: rs1799807] y a la variante Kalow (K) [dbSNP: rs1803274] de la enzima BChE, respectivamente. Los estudios de predicción In silico establecieron para el SNP p.Asp98Gly un carácter patogénico, mientras que para el SNP p.Ala567Thr, mostraron un comportamiento neutro. El análisis de los resultados permite proponer la existencia de una relación entre polimorfismos o variantes genéticas responsables de una baja actividad catalítica y/o baja concentración plasmática de la enzima BChE y algunas de las reacciones adversas ocurridas en pacientes consumidores de cocaína.

Integration of retrotransposons-based markers in a linkage map of barley

A deeper understanding of random markers is important if they are to be employed for a range of objectives. The sequence specific amplified polymorphism (S-SAP) technique is a powerful genetic analysis tool which exploits the high copy number of retrotransposon long terminal repeats (LTRs) in the plant genome. The distribution and inheritance of S-SAP bands in the barley genome was studied using the Steptoe × Morex (S × M) double haploid (DH) population. Six S-SAP primer combinations generated 98 polymorphic bands, and map positions were assigned to all but one band. Eight putative co-dominant loci were detected, representing 16 of the mapped markers. Thus at least 81 of the mapped S-SAP loci were dominant. The markers were distributed along all of the seven chromosomes and a tendency to cluster was observed. The distribution of S-SAP markers over the barley genome concurred with the knowledge of the high copy number of retrotransposons in plants. This experiment has demonstrated the potential for the S-SAP technique to be applied in a range of analyses such as genetic fingerprinting, marker assisted breeding, biodiversity assessment and phylogenetic analyses.

Use of the checkerboard DNA-DNA hybridisation technique for in vivo detection of cariogenic microorganisms on metallic brackets, with or without use of an antimicrobial agent

Objective: Using checkerboard DNA-DNA hybridisation (CDDH) assay, this randomised clinical study evaluated the contamination of metallic brackets by four cariogenic bacterial strains (Streptococcus mutans, Streptococcus sobrinus, Lactobacillus casei and Lactobacillus acidophilus) and the efficacy of 0.12% chlorhexidine gluconate (CHX) mouthwashes in reducing bacterial contamination. Methods: Thirty-nine 11-33-year-old patients under treatment with fixed orthodontic appliances were enrolled in the study and had 2 new metallic brackets bonded to premolars. Nineteen patients used a 0.12% CHX mouthwash (Periogard (R)) and 20 patients used a placebo mouthwash (control) twice a week. After 30 days, the brackets were removed and samples were obtained for analysis by CDDH. Data were analysed statistically by the Kruskal-Wallis test (alpha = 0.05) using the SAS software. Results: S. mutans, S. sobrinus, L. casei and L. acidophilus were detected in 100% of the samples from both groups. However, brackets of the control group were more heavily contaminated by S. mutans and S. sobrinus (P < 0.01). In the experimental group, although all counts decreased after rinsing with the chlorhexidine solution, there was significant difference only for S. mutans (P = 0.03). Conclusions: The use of 0.12% chlorhexidine gluconate mouthwashes can be useful in clinical practice to reduce the levels of cariogenic microorganisms in patients under treatment with fixed orthodontic appliances. (C) 2011 Elsevier Ltd. All rights reserved.

Estudo da detecção do DNA do papiloma vírus humano (HPV) e da expressão imuno-histoquímica de proteína do ciclo celular no carcinoma epidermóide oral

Oral squamous cell carcinoma (OSCC) is the most common malignancy in oral cavity and human papillomavirus (HPV) may have an important role in its development. The aim of this experiment was to investigate the HPV DNA and viral types in 90 cases of OSCC. Moreover, a comparative analysis between the cases of OSSC with and without HPV DNA was performed by using cell cycle markers p21 and pRb in order to detect a possible correlation of these proteins and HPV infection. DNA was extracted from paraffin embedded tissue and amplified by PCR (polymerase chain reaction) with primers PCO3+ e PCO4+ for a fragment of human β-globin gene. After this procedure, PCR for HPV DNA detection was realized using a pair of generic primers GP5+ e GP6+. Immunohistochemical study was performed by streptoavidin-biotin technique and antibodies against p21 and pRb proteins were employed. Eighty-eight cases were positive for human β-globin gene and HPV DNA was found in 26 (29.5%) of then. It could not be detected significant correlation between HPV and age, sex and anatomical sites of the lesion. The most prevalent viral type was HPV 18 (80.8%). Regarding the immunohistochemical analysis, it was detected significant association between HPV presence and pRb immunoexpression (p=0,044), nevertheless, the same was not observed in relation to p21 protein (p =0,416). It can be concluded that the low detection of HPV DNA in OSCC by the present experiment suggests a possible role of the virus in the development and progression in just a subset of this disease

Isolation and characterization of microsatellite DNA markers for spinner dolphin (Stenella longirostris)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

THE USE OF AMPPD AS AN ALTERNATIVE SUBSTRATE FOR AP-MEDIATED DETECTION OF NONRADIOLABELED DNA PROBES IN EUCALYPTUS-SALIGNA

We present a non-radioactive alternative to Southern's (J. Mol. Biol. 98: 503-517, 1975) DNA-DNA hybridization technique. The use of AMPPD - Disodium 3-(4-Methoxyspiro {1,2-dioxetane-3,2'tricyclo[3.3.1.1(3,7)]decan}-4-yl)phyenyl phosphate as an alternative substrate for AP-mediated detection of digoxigenin-11 dUTP-labeled probes made possible the simple and nonhazardous reuse of blots. We used 0.8 % agarose gels containing 30 mug per lane of Eucalyptus saligna DNA, digested with Eco RI, electrophoresed and blotted on to nylon membranes (Hybond-N, Amersham, UK), using the Southern blotting procedure, and UV irradiated for one minute for DNA fixation. The hybridizations were carried out overnight with digoxigenin labeled random inserts of E. saligna DNA by using the Genius Kit (Boehringer Mannheim). Detection of the DNA-DNA hybrids was performed in the presence of 0.5% blocking agent and the substrates NBT/BCIP were replaced by 0.26 mM AMPPD in the final alkaline assay buffer (50 mul/cm2). After membrane incubation for five minutes at room temperature in a sealed plastic bag, the AMPPD solution was retrieved and stored at 4-degrees-C for reuse. A Kodak X-BRAF QA-S film was pressed firmly onto the bag containing the wet membrane, exposed for two to six hours and then developed. After use, the probes were stripped off and the blots reutilized, three times so far, with the same results.

A SIMPLE TECHNIQUE FOR ISOLATION OF DNA SUITABLE FOR PCR AMPLIFICATION FROM CYTOGENETIC PREPARATIONS

In order to rescue molecular information from chromosome preparations, we describe a rapid procedure to obtain DNA from cytogenetic preparations in microscope slides, stored for one to live years at room temperature. This technique was modified from previously described procedures and the DNA obtained was shown to be suitable for PCR amplification.

Properties of BaBi2Ta2O9 thin films prepared by chemical solution deposition technique for dynamic random-access memory applications

We report on the properties of BaBi2Ta2O9 (BBT) thin films for dynamic random-access memory (DRAM) and integrated capacitor applications. Crystalline BBT thin films were successfully fabricated by the chemical solution deposition technique on Pt-coated Si substrates at a low annealing temperature of 650°C. The films were characterized in terms of structural, dielectric, and insulating properties. The electrical measurements were conducted on Pt/BBT/Pt capacitors. The typical measured small signal dielectric constant and dissipation factor, at 100 kHz, were 282 and 0.023, respectively, for films annealed at 700°C for 60 min. The leakage current density of the films was lower than 10-9 A/cm2 at an applied electric field of 300 kV/cm. A large storage density of 38.4 fC/μm2 was obtained at an applied electric field of 200 kV/cm. The high dielectric constant, low dielectric loss and low leakage current density suggest the suitability of BBT thin films as dielectric layer for DRAM and integrated capacitor applications.

Variabilidade genética intrapopulacional em Myracrodruon urundeuva Fr. All. por marcador AFLP

Levels of genetic variability for in situ and ex situ genetic conservation were estimated in a population of Myracrodruon urundeuva using the PCR (polymerase chain reaction) technique with the AFLP (Amplified fragment-length polymorphism) genetic marker. Seeds for progeny tests were collected from 30 open-pollination trees (matrices) at Paulo de Faria Ecological Station - SP. From this genetic material, three progeny tests were installed on the Teaching and Research Farm of Ilha Solteira Faculty of Engineering - University of São Paulo State (UNESP), which is located in Selvlria - MS, Brazil. The analysis by genetic marker was conducted with three combinations of different starters EcoRl-Msel, resulting in a total number of 137 polymorphic bands, thus forming a table of binary data. These data were used for the analysis of genetic divergence and distance between progenies. High levels of genetic divergence were observed among families. Based on the Analysis of Molecular Variance (AMOVA), it was shown that 16.2% of genetic diversity is found among progenies and 83.8% within progenies, which suggests deviances of random matings. The grouping of progenies, based on genetic distances, suggests that progenies deriving from trees which are close to each other tend to be more similar. This, in turn, indicates that the population originating the seeds may be genetically structured.

Fluorescent amplified fragment length polymorphism (fAFLP) analyses and genetic diversity in Litopenaeus vannamei (Penaeidae)

The Pacific white shrimp, Litopenaeus vannamei (Penaeidae), represents about 95% of all Brazilian shrimp production. The Brazilian L. vannamei foundation broodstock was made up of specimens collected from different American Pacific sites, but little information was collected on the genetic structure of the broodstock. We used the fluorescence amplified fragment length polymorphism (fAFLP) method to study the genetic diversity of L. vannamei broodstock lines 03CMF1 and 03CBF1 originally produced by breeder-shrimps imported mainly from Panama and Ecuador, although wild individuals from other localities may also have been used in producing these two lines. Our results showed a total of 93 polymorphic bands ranging from 50 to 500 bp, the mean Nei's genetic diversity calculated for the total sample was 13.4% and identity and genetic distance analyses indicated high genetic homogeneity within and between both the broodstock lineages studied which suggests that they had similar genetic structure. These results may represent an important tool for the appropriate management of L. vannamei broodstocks. Copyright by the Brazilian Society of Genetics.

Análise de polimorfismos da região controle do dna mitocondrial em indivíduos nascidos e residentes no estado do espírito santo para utilização na identificação humana

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

Variabilidade genética de Fusarium spp. isolados de solos e de bananeiras com sintomas do Mal-do-Panamá

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