Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques

ABSTRACT Chromosome-specific probes have been widely used in molecular cytogenetics, being obtained with different methods. In this study, a reproducible protocol for construction of chromosome-specific probes is proposed which associates in situ amplification (PRINS), micromanipulation and degenerate oligonucleotide-primed PCR (DOP-PCR). Human lymphocyte cultures were used to obtain metaphases from male and female individuals. The chromosomes were amplified via PRINS, and subcentromeric fragments of the X chromosome were microdissected using microneedles coupled to a phase contrast microscope. The fragments were amplified by DOP-PCR and labeled with tetramethyl-rhodamine-5-dUTP. The probes were used in fluorescent in situ hybridization (FISH) procedure to highlight these specific regions in the metaphases. The results show one fluorescent red spot in male and two in female X chromosomes and interphase nuclei.

Primer design for complete sequencing of the Avastrovirus ORF2 gene / Desenho de primers para o sequenciamento completo do gene ORF2 em Avastrovirus

Avastrovirus infection is associated with enteric disease, nephritis, and hepatitis in birds. In this study, we present a protocol for the complete sequencing of the ORF2 gene of avian nephritis virus (ANV), chicken astrovirus (CAstV), and turkey astrovirus type 1 (TAstV-1) using a conventional Sanger technique. Previously and newly designed primer pairs targeting both the conserved flanking and internal regions of the ORF2 gene of these three viruses were used. The information derived from the astroviral sequences obtained in this study is fundamental for characterizing this virus and providing data regarding several aspects of disease epidemiology and prevention.

As infecções por avastrovírus estão associados à doença entérica, nefrite e hepatite em aves. Aqui, nos presentamos um protocolo planejado para o sequenciamento completo do gene ORF2 em Avian Nephritis Vírus (ANV), Chicken Astrovirus (CAstV) e Turkey Astrovirus tipo 1 (TAstV-1), usando a técnica de sequenciamento convencional de Sanger. Foram usados primers previamente descritos e desenhados neste estudo, tendo como alvo as regiões conservadas flanqueadoras e internas dentro do gene ORF2 nos três vírus. O conhecimento destas sequencias é um elemento chave para caracterizar o vírus e prover de dados em diversos aspectos da epidemiologia e prevenção da doença.

Primer design for complete sequencing of the Avastrovirus ORF2 gene / Desenho de primers para o sequenciamento completo do gene ORF2 em Avastrovirus

Avastrovirus infection is associated with enteric disease, nephritis, and hepatitis in birds. In this study, we present a protocol for the complete sequencing of the ORF2 gene of avian nephritis virus (ANV), chicken astrovirus (CAstV), and turkey astrovirus type 1 (TAstV-1) using a conventional Sanger technique. Previously and newly designed primer pairs targeting both the conserved flanking and internal regions of the ORF2 gene of these three viruses were used. The information derived from the astroviral sequences obtained in this study is fundamental for characterizing this virus and providing data regarding several aspects of disease epidemiology and prevention.(AU)

As infecções por avastrovírus estão associados à doença entérica, nefrite e hepatite em aves. Aqui, nos presentamos um protocolo planejado para o sequenciamento completo do gene ORF2 em Avian Nephritis Vírus (ANV), Chicken Astrovirus (CAstV) e Turkey Astrovirus tipo 1 (TAstV-1), usando a técnica de sequenciamento convencional de Sanger. Foram usados primers previamente descritos e desenhados neste estudo, tendo como alvo as regiões conservadas flanqueadoras e internas dentro do gene ORF2 nos três vírus. O conhecimento destas sequencias é um elemento chave para caracterizar o vírus e prover de dados em diversos aspectos da epidemiologia e prevenção da doença.(AU)

Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques.

Chromosome-specific probes have been widely used in molecular cytogenetics, being obtained with different methods. In this study, a reproducible protocol for construction of chromosome-specific probes is proposed which associates in situ amplification (PRINS), micromanipulation and degenerate oligonucleotide-primed PCR (DOP-PCR). Human lymphocyte cultures were used to obtain metaphases from male and female individuals. The chromosomes were amplified via PRINS, and subcentromeric fragments of the X chromosome were microdissected using microneedles coupled to a phase contrast microscope. The fragments were amplified by DOP-PCR and labeled with tetramethyl-rhodamine-5-dUTP. The probes were used in fluorescent in situ hybridization (FISH) procedure to highlight these specific regions in the metaphases. The results show one fluorescent red spot in male and two in female X chromosomes and interphase nuclei.

Genetic diversity estimated using inter-simple sequence repeat markers in commercial crops of cupuassu tree / Diversidade genética estimada com marcadores entre sequências simples repetidas em cultivos comerciais de Cupuaçuzeiro

Fifteen ISSR (inter-simple sequence repeat) primers were used to evaluate the genetic diversity among and within commercial crops of T. grandiflorum (Willd. ex Spreng.) K. Schum. For this, 60 specimens were analyzed, distributed in three crops. A total of 102 bands were amplified, with a polymorphism percentage of 52.0% at species level and an average of 6.8 alleles per ISSR primer. The average for polymorphism information content (PIC) index was 0.55. In relation to the genetic diversity index of Nei (H), and Shannon (I), crops analyzed showed the following values: : SAR H = 0,114 e I = 0,177; SSL H = 0,108 e I = 0,162 e SEC H = 0,104 e I = 0,156, considered moderate to low values. AMOVA showed 34.91% of total variance among the crops, and 65.09% within them. The ISSR molecular markers revealed that there is genetic diversity within each commercial crops studied, thus is possible to select superior genotypes that can be used to give more uniform crops. This result has been considered of great relevance, to provide tools for breding implementation programs and design conservation strategies ex situ and in situ.(AU)

Quinze primers ISSR (entre sequências simples repetidas) foram utilizados para avaliar a diversidade genética entre e dentro de pomares comerciais de Theobroma grandiflorum (Willd. ex Spreng.) K. Schum. Para isso, foram analisados sessenta indivíduos, distribuídos nos três cultivos. Um total de 102 bandas foi amplificado, com uma porcentagem de 52,0% de polimorfismo em nível de espécie e média de 6,8 alelos por primer ISSR. A média do Índice de Conteúdo Polimórfico (PIC) foi de 0,55. Em relação aos índices de diversidade gênica de Nei (H) e de Shannon (I), os cultivos analisados apresentaram os valores: SAR H = 0,114 e I = 0,177; SSL H = 0,108 e I = 0,162 e SEC H = 0,104 e I = 0,156, considerados valores de moderados a baixos. A AMOVA revelou 34,91% da variância total entre os cultivos e 65,09% dentro deles. Os marcadores moleculares ISSR revelaram que há diversidade genética dentro de cada cultivo comercial estudado, portanto é possível selecionar genótipos superiores que poderão ser utilizados para originar cultivos mais uniformes. Esse resultado tem sido considerado de grande relevância, por fornecer ferramentas para a implementação de programas de melhoramento e delineamento de estratégias de conservação ex situ e in situ.(AU)

Exequibility of differential gene expression analysis by DDRT-PCR in murine bone marrow cells / Exequibilidade do DDRT-PCR para análise da expressão gênica diferencial em células de medula óssea murina

Model of study: Experimental study. Introduction: Recently, stem cell research has generated great interest due to its applicability in regenerative medicine. Bone marrow is considered the most important source of adult stem cells and the establishment of new methods towards gene expression analysis regarding stem cells has become necessary. Thus Differential Display Reverse Transcription Polymerase Chain Reaction (DDRT-PCR) may be an accessible tool to investigate small differences in the gene expression of different stem cells in distinct situations. Aim: In the present study, we investigated the exequibility of DDRT-PCR to identify differences in global gene expression of mice bone marrow cells under two conditions. Methods: First, bone marrow cells were isolated fresh and a part was cultivated during one week without medium replacement. Afterwards, both bone marrow cells (fresh and cultivated) were submitted to gene expression analyses by DDRT-PCR...

Modelo do estudo: Estudo Experimental. Introdução: Atualmente a pesquisa com células-tronco tem gerado grande interesse devido a sua aplicabilidade no campo na medicina regenerativa. A medula óssea é considerada a maior fonte de células-tronco adultas e o estabelecimento de novos métodos para a análise da expressão gênica torna-se estritamente necessário. Desse modo, o "Differential Display Reverse Transcription Polymerase Chain Reaction (DDRT-PCR)", pode ser uma ferramenta acessível para investigação de pequenas diferenças no nível de expressão gênica em diferentes tipos celulares, sob distintas condições. Objetivo: Neste presente trabalho nós investigamos a exequibilidade do DDRT-PCR na identificação de diferenças no nível de expressão gênica global em células da medula óssea de camundongos sob duas condições. Métodos: Primeiramente, a medula óssea foi isolada frescamente e uma secunda parte foi cultivada por uma semana sem troca de meio. Posteriormente, as células da medula (fresca e cultivada) foram submetidas a análise da expressão gênica, seguindo a metodologia de DDRT-PCR...

Design, Prins-cyclization reaction promoting diastereoselective synthesis of 10 new tetrahydropyran derivatives and in vivo antinociceptive evaluations.

We described in this article the very efficient 2,6-cis ou 2,4,6-cis diastereoselective synthesis (2 or 3 steps, 62-65% global yields) from Prins-cyclization reaction as synthetic key-step to tetrahydropyran rings construction of 10 new congeners compounds (3-12) designed from Naproxen structure. These tetrahydropyran derivatives were in vivo bioevaluated on antinociceptive effect in the acetic acid-induced abdominal writhing test, the tail-flick test, the rota-rod performance and open field tests. All new compounds showed greater antinociceptive activity compared to compound 1a, an analgesic tetrahydropyran derivative previously described by us. We can detach the high activity of tetrahydropyran derivative 10 which presented 87.5% inhibition (14% inhibition was presented by 1a) in the acetic acid-induced abdominal writhing test. Besides that the tail-flick tests indicate compounds 7 and 10 as the most actives. All these new compounds showed no toxicity in mice in all biologically studied models.

Detection of Klinefelter syndrome by G-banding analysis combined with primed in situ labeling technique.

Primed in situ labeling (PRINS) technique is an alternative to in situ hybridization for rapid chromosome screening. We employed triple-color PRINS technique to detect chromosomal abnormalities in Klinefelter syndrome patients diagnosed by G-banding karyotype analysis. Among 1034 infertile male patients, 134 were found to be cytogenetically abnormal, including 70 with chromosomal number abnormalities and 64 with chromosomal structure abnormalities. Among these cytogenetically abnormal patients, 56 were diagnosed as having Klinefelter syndrome. PRINS technique was used on cultured lymphocyte metaphase cells of the Klinefelter syndrome patients; the same result was obtained with G-banding karyotype analysis. PRINS proved to be a rapid and reliable method to detect numerical chromosome abnormalities in peripheral blood lymphocytes in metaphase.

Primed in situ labeling for detecting single-copy genes.

In order to analyze male sterility caused by deletion of SRY and DAZ, we examined the accuracy and cost-effectiveness of a modified primed in situ labeling (PRINS) technique for detection of single-copy genes. Peripheral blood samples were collected from 50 healthy men; medium-term cultured lymphocytes from these samples were suspended in fixative solution and then spread on clean slides. We used four primers homologous to unique regions of the SRY and DAZ regions of the human Y-chromosome and incorporated reagents to increase polymerase specificity and to enhance the hybridization signal. PRINS of SRY and DAZ gave bands at Yp11.3 and Yq11.2, respectively, in all 50 metaphase spreads. The PRINS SRY signals were as distinct as those obtained using traditional fluorescence in situ hybridization (FISH). This new method is ideal for rapid localization of single-copy genes or small DNA segments, making PRINS a cost-effective alternative to FISH. Further enhancement of PRINS to increase its speed of implementation may lead to its wide use in the field of medical genetics.

Physical localization of the retrotransposons Boudicca and Perere 03 in Schistosoma mansoni.

Schistosoma mansoni is 1 of the causative agents of schistosomiasis, an endemic disease in 76 countries of the world. The study of its genome, estimated to be 270 Mb, is very important to understanding schistosome biology, the mechanisms of drug resistance, and immune evasion. Repetitive elements constitute more than 40% of the S. mansoni genome and may play a role in the parasite evolution. The retrotransposons Boudicca, a long terminal repeat (LTR), and Perere 03, a non-LTR, are present in a high number in the S. mansoni genome and were localized with the use of fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS). Bacterial artificial chromosomes (BAC) clones containing the retrotransposons Boudicca and Perere 03 were selected by bioinformatic analysis and used as probes in FISH. Using metaphase chromosomes from sporocysts and the FISH and PRINS techniques, we were able to map these retrotransposons. Perere 03 was localized in the euchromatic regions of the short arm of chromosome 2 and Boudicca in the euchromatic regions of the short arm of chromosomes 2 and Z.

PCR-PRINS-FISH analysis of structurally abnormal sex chromosomes in eight patients with Turner phenotype.

According to cytogenetic analysis, about 50% of Turner individuals are 45,X. The remaining cases have a structurally abnormal X chromosome or are mosaics with a second cell line containing a normal or abnormal sex chromosome. In these mosaics, approximately 20% have a sex marker chromosome whose identity cannot usually be determined by classical cytogenetic methods, requiring the use of molecular techniques. Polymerase chain reaction (PCR), primed in situ labeling (PRINS), and fluorescence in situ hybridization (FISH) analyses were performed in 8 patients with Turner syndrome and 45,X mosaic karyotypes to determine the origin and structure of the marker chromosome in the second cell line. Our data showed that markers were Y-derived in 2 patients and X-derived in the remaining 6 patients. We were also able to determine the breakpoints in the two Y chromosomes. The use of cytogenetic and molecular techniques allowed us to establish unequivocally the origin, X or Y, of the marker chromosomes in the 8 patients with Turner phenotype. This study illustrates the power of resolution and utility of combined cytogenetic and molecular approaches in some clinical cases.

Persistence of DNA from Mycobacterium tuberculosis in superficially normal lung tissue during latent infection.

BACKGROUND: A third of the world's population has latent infection with Mycobacterium tuberculosis, and in areas of low endemicity, most cases of active tuberculosis arise as a result of reactivation of latent bacilli. We sought to establish the cellular location of these latent organisms to facilitate their elimination. METHODS: We applied in-situ PCR to sections of macroscopically normal lung tissue from 13 individuals from Ethiopia and 34 from Mexico who had died from causes other than tuberculosis. Sections of lung tissue from six Norwegian individuals (ie, individuals from a non-endemic population) acted as negative controls, and six Ethiopian tuberculosis cases acted as positive controls. FINDINGS: Control necropsy samples from the Norwegian individuals were all negative by in-situ PCR and conventional PCR, whereas all samples from known Ethiopian tuberculosis cases were positive by both methods. However, in macroscopically normal lung tissue from Ethiopian and Mexican individuals without tuberculous lesions, the in-situ PCR revealed five of 13 and ten of 34 positive individuals, respectively. These results were confirmed by conventional PCR with extracted DNA. Positive cells included alveolar and interstitial macrophages, type II pneumocytes, endothelial cells, and fibroblasts. INTERPRETATION: M. tuberculosis can persist intracellularly in lung tissue without histological evidence of tuberculous lesions. M. tuberculosis DNA is situated not only in macrophages but also in other non-professional phagocytic cells. These findings contradict the dominant view that latent organisms exist in old classic tuberculous lesions, and have important implications for strategies aimed at the elimination of latent and persistent bacilli.

Diagnostico genetico pre-implantacional / Polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH) and primed in situ (PRINS)

O diagnostico genetico pre-implantacional (PGD) e um novo procedimento que pode ser utilizado como diagnostico precoce pre-natal em casais com risco de transmissao de doencas geneticas. Utilizando "polymerase chain reaction" (PCR), "fluorescence in-situ hybridization" (FISH) e "primed in-situ" (PRINS) pode-se determinar o genotipo ou sexo genetico do embriao biopsiado obtido por...

Chromosomal localization of 5S rDNA genes in Leporinus fish (Anostomidae, Characiformes).

The large 45S rDNA chromosome sites have often been analyzed in fish. In contrast, little is known about the 5S genes in this animal group. In the genus Leporinus, the NOR chromosomal location has been shown to be very diverse. In the present work, chromosome mapping of 5S rDNA in three anostomids, Leporinus elongatus, L. obtusidens and L. friderici, is investigated using fluorescence in-situ hybridization (FISH) with PCR-obtained 5S probes and primed in-situ labeling (PRINS). Major 5S rDNA chromosomal sites were found to be subterminally located in a small metacentric pair, while minor ones were detected near the centromeric region of a medium-sized submetacentric pair in all studied species. The 5S rDNA genes were not associated with the NORs or sex chromosomes. A highly conserved chromosomal location of these genes appears to characterize the karyotype evolution of this fish group.

The TATA-box binding protein of Entamoeba histolytica: cloning of the gene and location of the protein by immunofluorescence and confocal microscopy.

A 309 bp DNA fragment from Entamoeba histolytica was amplified by PCR using primers derived from the Acanthamoeba castellanii consensus TATA-box binding protein amino acid sequence. The amplified fragment was used to isolate cDNA and genomic DNA clones containing an ORF encoding the complete E. histolytica TATA-box binding protein (Ehtbp, 702 bp, 234 aa, molecular mass 26 kDa). The EhTBP functional domain showed 55% sequence identity to that of Homo sapiens, 54% to A. castellanii and 37% to Plasmodium falciparum TBPs. In Southern blot experiments we detected a single Ehtbp band, which was transcribed as a 1.3 kb mRNA containing a 420 nt 5' untranslated region. However, the probe hybridized with the 0.8 and 1.5 Mb chromosomes, suggesting that this sequence is diploid. In situ PCR assays showed two signals in 95% of trophozoites, one located in the nucleus and another in EhkO, the novel DNA-containing organelle recently reported. The recombinant E. histolytica TATA-box binding protein was expressed in Escherichia coli. Antibodies against it recognized two proteins of 26 and 29 kDa in E. histolytica nuclear extracts. Confocal microscopy immunofluorescence analysis located the protein in both the nucleus and EhkO.