Search

Background In rice, the cortex and outer tissues play a key role in submergence tolerance. The cortex differentiates into aerenchyma, which are air-containing cavities that allow the flow of oxygen from shoots to roots, whereas exodermis suberification and sclerenchyma lignification limit oxygen loss from the mature parts of roots by forming a barrier to root oxygen loss (ROL). The genes and their networks involved in the cellular identity and differentiation of these tissues remain poorly understood. Identification and characterization of key regulators of aerenchyma and ROL barrier formation require determination of the specific expression profiles of these tissues. Results We optimized an approach combining laser microdissection (LM) and droplet digital RT-PCR (ddRT-PCR) for high-throughput identification of tissue-specific expression profiles. The developed protocol enables rapid (within 3 days) extraction of high-quality RNA from root tissues with a low contamination rate. We also demonstrated the possibility of extracting RNAs from paraffin blocks stored at 4 °C without any loss of quality. We included a detailed troubleshooting guide that should allow future users to adapt the proposed protocol to other tissues and/or species. We demonstrated that our protocol, which combines LM with ddRT-PCR, can be used as a complementary tool to in situ hybridization for tissue-specific characterization of gene expression even with a low RNA concentration input. We illustrated the efficiency of the proposed approach by validating three of four potential tissue-specific candidate genes detailed in the RiceXpro database. Conclusion The detailed protocol and the critical steps required to optimize its use for other species will democratize tissue-specific transcriptome approaches combining LM with ddRT-PCR for analyses of plants.

Parkia pendula (Willd.) Walp. (Fabaceae) is a neotropical species of the genus Parkia more abundantly distributed in Central to South America. From the seeds of P. pendula a glucose/mannose specific lectin (PpeL) was isolated that has been characterised and used as a biotechnological tool but until now this is the first manuscript to analyse P. pendula mRNA expression in seedlings. For this porpoise a Differential display reverse transcription polimerase chain reaction (DDRT-PCR) was used to evaluate the expression of P. pendula lectin mRNAs in non-rooted seedlings. No bands were observed in the agarose gel, indicating the absence of mRNA of PpeL seedlings. our findings confirm that lectins mRNAs are differently regulated among species even if they are grouped in the same class.

The only active compounds able to control phytoplasmas are the antibiotic oxytetracyclines and the direct and fast screening of new antimicrobials compounds on media is very complicated due to the difficulty to culture phytoplasmas. A simple and reliable screening method to evaluate the effects of antimicrobial compounds on phytoplasmas by an ex-vivo approach was developed. By the use of scanning electron microscopy in parallel with molecular tools the direct activity of tetracyclines on phytoplasmas was verified.

Even though the major benefit of arbuscular mycorrhizae is the increased uptake of phosphate from the soil solution and translocation to the plant, changes in the activity of enzymes involved in nitrogen (N) metabolism have been detected in mycorrhizal roots. Using differential display of reverse-transcripts of tobacco roots not-inoculated or inoculated with Glomus intraradices (Gi), we have cloned two partial cDNAs (NtGi2 and NtGi3). The presence of a conserved CobW/HypB/UreG domain and phylogenetic analyses suggest that NtGi2 and NtGi3 encode isoforms of urease accessory protein G (ureG) highly similar to ureG from fungi. The steady state levels of the putative ureG transcripts were shown to be higher in roots colonized by Gi, as compared to non-mycorrhizal controls. Urease activities were also determined in tobacco roots inoculated with Glomus clarum (Gc) or Gi and grown in substrate containing 50, 100 or 150 mg N kg-1 in the form of ammonium sulfate (N-AMS) or urea (N-URE). Urease activities were shown to be induced in mycorrhizal roots fertilized with 100 mg N-AMS kg-1. In Gc-colonized roots fertilized with N-URE, induction of urease activities was observed at the lowest N concentration. In contrast, at the highest N-URE concentration, suppression of urease activities was observed in Gc and Gi-colonized roots, as compared to non-mycorrhizal controls. Urease activities in roots were modulated by soil N availability and source, and arbuscular mycorrhizal fungal inoculation.Muito embora o maior benefício de micorrizas arbusculares seja o incremento na absorção de fosfato da solução do solo e translocação para a planta, alterações nas atividades de enzimas envolvidas no metabolismo de nitrogênio (N) têm sido detectadas em raízes micorrizadas. Usando "differential display of reverse-transcripts" de raízes de tabaco não-inoculadas ou inoculadas com Glomus intraradices (Gi), dois cDNAs parciais (NtGi2 e NtGi3) foram clonados. A presença de um domínio CobW/HypB/UreG conservado e a análise filogenética sugerem que NtGi2 e NtGi3 codificam isoformas de proteínas acessórias da urease G (ureG) altamente similares a ureG de fungos. Os níveis de transcritos dos genes ureG putativos foram mais elevados em raízes colonizadas por Gi, em relação ao controle não-micorrizado. As atividades de urease foram determinadas em raízes de tabaco inoculadas com Glomus clarum (Gc) ou Gi e cultivadas em substrato contendo 50, 100 ou 150 mg N kg-¹, na forma de sulfato de amônio (N-AMS) ou uréia (N-URE). As atividades de ureases foram induzidas em raízes micorrizadas cultivadas com 100 mg N-AMS kg-¹. Em raízes colonizadas por Gc e cultivadas com N-URE, a indução das atividades de urease foi observada na concentração mais baixa de N. Em contraste, na concentração mais elevada de N-URE, supressão das atividades de urease em raízes colonizadas por Gc e Gi, em relação aos controles não-micorrizados, foi observada. As atividades de urease nas raízes foram moduladas pela disponibilidade e fonte de N no solo, e pela inoculação com fungos micorrízicos arbusculares.

We performed annealing control primer (ACP)-based differential-display reverse transcription-polymerase chain reaction (DDRT-PCR) to isolate differentially expressed genes (DEGs) from the stage IV ovary and ovotestis of the rice field eel, Monopterus albus. Using 20 arbitrary ACP primers, 14 DEG expressed-sequence tags were identified and sequenced. The transcriptional expression of one DEG, G2, was significantly greater in the ovotestis than the stage IV ovary. To understand the role of G2 in sex inversion, G2 cDNA was cloned and semi-RT-PCR, real time PCR were performed during gonad development. The full-length G2 cDNA was 650 base pairs (bp) and it comprised a 5′-untranslated region (UTR) of 82 bp, a 3′-UTR of 121 bp and an open reading frame of 444 bp that encoded a 148-amino acid protein. The expression of G2 was weak during early ovarian development until the stage IV ovary, but expression increased significantly with gonad development. We speculate that G2 may play an important function during sex inversion and testis development in the rice field eel, but the full details of the function of this gene requires further research.

Differential gene expression profiling in broiler and Thai native chicken muscle was analysed by using mRNA differential display reverse transcription-PCR (DDRT-PCR) technique. Total RNA was isolated from the pectoralis (breast) muscles at 1, 2, 4 and 6 weeks-age and 8 RNA pools were prepared based on 5 individual intra-breeds with the same age. Differential display banding patterns were produced using two anchor primers dT12VC (V:A, C, G) or dT12VG and 26 arbitrary primers. Comparing the banding patterns of the broiler and native chicken muscles revealed 7 differential markers from 5 primer combinations. In this investigation, the profiling of differentially expression cDNA bands could be divided into 4 categories: (i) bands only detected in broilers (all weeks-age); (ii) bands only detected in the native chickens (all weeks-age); (iii) bands only absent at 1 week-age in both chickens and (iv) bands only absent at 4 and 6 weeks-age of broilers. In category i and ii represented the gene expression profiling in muscle associated with the specific chicken breeds. In category iii and iv represented the transcriptional change upregulated and downregulated genes which associated with aging of chickens, respectively. We have successfully applied DDRT-PCR to identify the differential gene expression pro filing in broiler and Thai native chicken muscles. Further characterization of these differentially expression cDNA bands (consist of reamplification, cloning and sequencing as well as quantitative PCR analysis) provides the important candidate genes for meat quality in chicken.

This study was designed to investigate changes in gene expression associated with stage-specific programmed cell death (PCD) in intersegmental muscles (ISMs) of the moth, Manduca sexta. The technique of differential display reverse transcription PCR was applied to compare mRNA levels before and after the onset of PCD in ISMs. Expression of E75B transcription factor was repressed while another factor, βFTZ-F1, stayed at a very low level. However, gene coding for a translation-initiation factor (eIF1A) was upregulated. Expression of these genes had not been previously reported to be altered in dying ISMs. An ecdysteroid agonist, RH-5849, that prevented PCD in ISMs also blocked these changes.

Parkia pendula (Willd.) Walp. (Fabaceae) is a neotropical species of the genus Parkia more abundantly distributed in Central to South America. From the seeds of P. pendula a glucose/mannose specific lectin (PpeL) was isolated that has been characterised and used as a biotechnological tool but until now this is the first manuscript to analyse P. pendula mRNA expression in seedlings. For this porpoise a Differential display reverse transcription polimerase chain reaction (DDRT-PCR) was used to evaluate the expression of P. pendula lectin mRNAs in non-rooted seedlings. No bands were observed in the agarose gel, indicating the absence of mRNA of PpeL seedlings. our findings confirm that lectins mRNAs are differently regulated among species even if they are grouped in the same class. Parkia pendula (Willd.) Walp. (Fabaceae) é a espécie neotropical do gênero Parkia mais abundantemente distribuída na América Central a do Sul. Das sementes de P. pendula foi isolada uma lectina glicose/manose específica (Ppel) que foi caracterizada e usada como ferramenta biotecnológica, porém até o momento esse é o primeiro artigo a analisar a expressão do mRNA nas plântulas de P. pendula. Para esse propósito uma reação de PCR diferencial de transcriptase reversa (DDRT-PCR) foi utilizada para avaliar a expressão do mRNA da lectina de P. pendula em plântulas não enraizadas. Nenhuma banda foi observada no gel de agarose, indicando a ausência de mRNA das plântulas de PpeL. Nossos achados confirmam que os mRNAs de lectinas são regulados de forma diferentes entre as espécies, mesmo que sejam agrupadas na mesma classe

Parkia pendula (Willd.) Walp. (Fabaceae) is a neotropical species of the genus Parkia more abundantly distributed in Central to South America. From the seeds of P. pendula a glucose/mannose specific lectin (PpeL) was isolated that has been characterised and used as a biotechnological tool but until now this is the first manuscript to analyse P. pendula mRNA expression in seedlings. For this porpoise a Differential display reverse transcription polimerase chain reaction (DDRT-PCR) was used to evaluate the expression of P. pendula lectin mRNAs in non-rooted seedlings. No bands were observed in the agarose gel, indicating the absence of mRNA of PpeL seedlings. our findings confirm that lectins mRNAs are differently regulated among species even if they are grouped in the same class.

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 a 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. Resultados: Inicialmente, foi possível identificar em células da medula óssea com uma semana de cultivo, pequenas alterações morfológicas (células ovais para fibroblastóides) e no perfil de proteínas, por meio da visualização de bandas em SDS-Page gel. Finalmente, a análise da expressão gênica por DDRT-PCR, mostrou uma expressão diferencial com a presença de três bandas (1300, 1000 and 225 pb) exclusivamente expressas na medula óssea fresca e mais duas bandas (400 and 300 pb) presentes somente nas células de medula cultivadas. Conclusões: Em suma, a metodologia de DDRT-PCR mostrou-se eficiente para a identificação de pequenas diferenças no nível de expressão gênica em células da medula óssea sob duas definidas condições. Portanto, nós acreditamos que o DDRT-PCR possa ser designado de forma rápida e eficiente para a análise diferencial de expressão gênica em diferentes tipos de células-tronco, sob diferentes condições.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:27:05Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:27:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-10-01 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. Results: Initially, it was possible to observe in one week-cultured bone marrow cells, changes in morphology (oval cells to fibroblastic-like cells) and protein profile, which was seen through differences in band distribution in SDS-Page gels. Finally through gene expression analysis, we detected three bands (1300, 1000 and 225 bp) exclusively expressed in the fresh bone marrow group and two bands (400 and 300 bp) expressed specifically in the cultivated bone marrow cell group. Conclusions: In summary, the DDRT-PCR method was proved efficient towards the identification of small differences in gene expression of bone marrow cells in two defined conditions. Thus, we expect that DDRT-PCR can be fast and efficiently designed to analyze differential gene expression in several stem cell types under distinct conditions. Mestre em Ciěncias Médicas Universidade Federal de São Paulo Departamento de Medicina (UNIFESP/EPM), São Paulo Mestre em Biologia Celular e Molecular Universidade de São Paulo Departamento de Genética (USP/FMRP), Ribeirão Preto-SP Universidade Estadual Paulista Júlio de Mesquita Filho Departamento de Ciěncias Biológicas (UNESP), Assis-SP LICE Laboratory/UNIFESP,Vila Clementino, Rua Pedro de Toledo, 669, São Paulo 04039-003 Universidade Estadual Paulista Júlio de Mesquita Filho Departamento de Ciěncias Biológicas (UNESP), Assis-SP

Ziel der vorliegenden Dissertation war Teilaspekte der Zusammenhänge der Karzinogenese und den Stoffwechsel maligne transformierter Zellen am Beispiel von Plattenepithelkarzinomen des oberen Aerodigestivtraktes dazustellen. Aus diesem Grunde wurde mittels der Differential Display Methode das aktive mRNS Expressionsprofil von etablierten Plattenepithelkarzinomzellinien des Larynx und benignen Keratinozytenzellinien zur vergleichenden Genanalyse untersucht. Die isolierte mRNS wurde revers transkribiert, die entstandene cDNS Population in einer DDRT-PCR Reaktion radioaktiv markiert und mit einer Primerkombination von einem der 26 dekamer Upstream-Primer und einem der 12 Oligo-T Downstream-Primer amplifiziert. Die elektrophoretische Auftrennung der PCR-Amplikons zeigte im Mittel 80-120 differenzierbare cDNS-Fragmente pro Primerkombination und Zellinie. Bei der vergleichenden Auswertung der Bandenmuster der autoradiographisch nachgewiesenen cDNS Fragmente ließ sich neben der weitgehenden Übereinstimmung zwischen den Zellinien ein Fragment identifizieren, dessen Expression in den Plattenepithelkarzinomzellinien deutlich reduziert war. Nach erfolgter Isolierung, Reamplifikation, Klonierung und Sequenzierung konnte das 148 bp große Fragment bei 99,3 %-iger Homologie einem kodierenden Abschnitt des GALC-Gens zugeordnet werden. Die Konstanz der differentiellen Expression der GALC-mRNS in Plattenepithelkarzinomen des Larynx ließ sich mittels vergleichender Northern-Hybridisierung in Zellinien und Biopsaten bestätigen. In dieser Arbeit zeigt sich erstmalig eine mögliche Beteiligung der GALC in der Karzinogenese von Larynxkarzinomen auf Transkriptionsebene. Eine Verschiebung des Stoffwechselgleichgewichtes zwischen GalCer und Ceramiden durch reduzierte GALC-Expression könnte über eine Akkumulation von Synthesevorstufen und abweichende Expression von GSL sowie eine gesteigerte Umsatz von Ceramiden zur Inhibition der Apoptose, Verlust einer adhäsionsvermittelten Wachstumskontrolle und gesteigerter Motilität führen.

Even though the major benefit of arbuscular mycorrhizae is the increased uptake of phosphate from the soil solution and translocation to the plant, changes in the activity of enzymes involved in nitrogen (N) metabolism have been detected in mycorrhizal roots. Using differential display of reverse-transcripts of tobacco roots not-inoculated or inoculated with Glomus intraradices (Gi), we have cloned two partial cDNAs (NtGi2 and NtGi3). The presence of a conserved CobW/HypB/UreG domain and phylogenetic analyses suggest that NtGi2 and NtGi3 encode isoforms of urease accessory protein G (ureG) highly similar to ureG from fungi. The steady state levels of the putative ureG transcripts were shown to be higher in roots colonized by Gi, as compared to non-mycorrhizal controls. Urease activities were also determined in tobacco roots inoculated with Glomus clarum (Gc) or Gi and grown in substrate containing 50, 100 or 150 mg N kg-1 in the form of ammonium sulfate (N-AMS) or urea (N-URE). Urease activities were shown to be induced in mycorrhizal roots fertilized with 100 mg N-AMS kg-1. In Gc-colonized roots fertilized with N-URE, induction of urease activities was observed at the lowest N concentration. In contrast, at the highest N-URE concentration, suppression of urease activities was observed in Gc and Gi-colonized roots, as compared to non-mycorrhizal controls. Urease activities in roots were modulated by soil N availability and source, and arbuscular mycorrhizal fungal inoculation. Muito embora o maior benefício de micorrizas arbusculares seja o incremento na absorção de fosfato da solução do solo e translocação para a planta, alterações nas atividades de enzimas envolvidas no metabolismo de nitrogênio (N) têm sido detectadas em raízes micorrizadas. Usando "differential display of reverse-transcripts" de raízes de tabaco não-inoculadas ou inoculadas com Glomus intraradices (Gi), dois cDNAs parciais (NtGi2 e NtGi3) foram clonados. A presença de um domínio CobW/HypB/UreG conservado e a análise filogenética sugerem que NtGi2 e NtGi3 codificam isoformas de proteínas acessórias da urease G (ureG) altamente similares a ureG de fungos. Os níveis de transcritos dos genes ureG putativos foram mais elevados em raízes colonizadas por Gi, em relação ao controle não-micorrizado. As atividades de urease foram determinadas em raízes de tabaco inoculadas com Glomus clarum (Gc) ou Gi e cultivadas em substrato contendo 50, 100 ou 150 mg N kg-¹, na forma de sulfato de amônio (N-AMS) ou uréia (N-URE). As atividades de ureases foram induzidas em raízes micorrizadas cultivadas com 100 mg N-AMS kg-¹. Em raízes colonizadas por Gc e cultivadas com N-URE, a indução das atividades de urease foi observada na concentração mais baixa de N. Em contraste, na concentração mais elevada de N-URE, supressão das atividades de urease em raízes colonizadas por Gc e Gi, em relação aos controles não-micorrizados, foi observada. As atividades de urease nas raízes foram moduladas pela disponibilidade e fonte de N no solo, e pela inoculação com fungos micorrízicos arbusculares.

This study is focused on the identification of early response genes involved in the resistance mechanism of one of the most important diseases of wheat, yellow rust. The strategy undertaken was to use differential display reverse transcriptase-PCR method (DDRT-PCR) on two of the yellow rust differential lines of wheat, were infected with the virulent and the avirulent Puccinia striiformis f. sp. tritici races together with appropriate control inoculations. Upon infection of the plant materials (resistant and/or susceptible), different time points were evaluated on DDRT-PCR banding profiles. The expression level differences of 33 genes in DDRT-PCR were further analyzed using quantitative real-time PCR (qRT-PCR). Among which only those (14 gene fragments) resulting reproducible induction or repression levels were reported with measured fold changes. Based on the sequence similarity analysis, all but one were absent in NCBI databank, thus they are newly found wheat sequences. Among those, two genes were identified as full ORF, including 5′ and 3′ end untranslated regions (UTR); namely cyclophilin like protein (putative antifungal activity) and ubiquitin-conjugating enzyme (E2) or Rad6. The sequence homology analysis of the cloned gene fragments revealed that the genes detected may have roles in ubiquitinylation, programmed cell death (apoptosis), putative antifungal activities, disease resistance responses, pathogenesis related responses, and including a few with no known function.

The objective of the present study was to determine by differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) the effects of juvenile hormone (JH) III applied during the late larval 3 (L3) phase on gene expression in Melipona scutellaris. A temporal window of expression of feminizing genes exists during the late L3 and pre-defecating larval phases when these genes can be turned on or off by the action of JH, which is able to mediate the differentiation of female larvae into queens. Combination of the HT11A-AP4 primers revealed differential expression in L3 individuals treated with JH III for 1 h, with weak expression of the transcript, while intense expression was observed for controls and individuals treated for 4 h. Combination of the HT11G-AP4 and HT11G-AP5 primers showed suppression of the gene products for each primer combination in 1-h treated larvae compared to untreated control individuals of the same age and individuals treated for 4 h. Differential gene expression was also observed during development. These results demonstrate that the JH III may suppress or alter gene expression profiles during phase L3 of M. scutellaris.

A técnica Differential Display (DD), descrita primeiramente em 1992, promoveu um grande avanço nos estudos de expressão gênica em eucariotos, permitindo análises comparativas, isolamento e caracterização de novos genes relacionados aos mais variados processos biológicos. A larga aplicação do DD se deve, principalmente, ao fato de ser uma técnica rápida, fácil e necessitar de pequena quantidade de RNA total. Entretanto, o uso de radioisótopos em uma determinada etapa do DD restringe esse método a laboratórios que tenham áreas específicas para manipular esses reagentes e o torna inviável à grande maioria dos centros de pesquisa existentes no país. Assim, esse trabalho busca adaptar a coloração por nitrato de prata, não radioativa, à técnica do DD; utilizando como modelo biológico a espécie Apis mellifera (abelha africanizada). Os resultados têm sido promissores e mostram que a coloração por nitrato de prata é eficiente no DD e pode tornar a técnica mais difundida nos laboratórios nacionais, inclusive aplicada aos estudos de câncer. UNITERMOS: DDRT-PCR, Expressão gênica, Differential Display, método não radioativo.

We have compared gene expression, using the Differential Display Reverse Transcriptase - Polymerase Chain Reaction (DDRT-PCR) technique, by means of mRNA profile in Melipona scutellaris during ontogenetic postembryonic development, in adult worker, and in both Natural and Juvenile Hormone III-induced adult queen. Six, out of the nine ESTs described here, presented differentially expressed in the phases L1 or L2, or even in both of them, suggesting that key mechanisms to the development of Melipona scutellaris are regulated in these stages. The combination HT11G-AP05 revealed in L1 and L2 a product which matches to thioredoxin reductase protein domain in the Clostridium sporogenes, an important protein during cellular oxidoreduction processes. This study represents the first molecular evidence of differential gene expression profiles toward a description of the genetic developmental traits in the genus Melipona.Nesse estudo nós usamos a técnica de Differential Display Reverse Transcriptase - Polymerase Chain Reaction (DDRT-PCR) para comparamos o perfil de mRNA em Melipona scutellaris durante o desenvolvimento ontogenético pós-embrionário e em operárias adultas, rainha natural e induzida pelo Hormônio Juvenil III. Fragmentos diferencialmente expressos foram detectados usando as seguintes combinações de primers: HT11G-AP05; HT11C-AP05; HT11G-OPF12; HT11G-OPA16. Dos 9 ESTs descrito nesse trabalho, 6 tiveram expressão diferencial nas fases de larva L1 e L2, sugerindo serem mecanismos chave no regulação do desenvolvimento larval em Melipona. A combinação HT11G-AP05 revelou em L1 e L2 um produto com similaridade à proteína tioredoxina redutase de Clostridium sporogenes, uma proteína importante durante os processos de oxidoredução. Esse estudo representa as primeiras evidências moleculares do perfil de expressão durante o desenvolvimento ontogenético em abelhas do gênero Melipona.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Social insects inspire great curiosity, interest and a wish to know the social life molecular basis. Differentially expressed genes identification has been used with the objective to understand either the gene function and the molecular mechanisms of different biological processes. In this study, two protocols for the total RNA extraction from Camponotus vittatus were tested and the Differential Display Reverse Transcriptase Polymerase Chain Reaction (DDRT-PCR) technique was used with the aim to identify differentially expressed genes in caste, sex and development stages of this ant. The RNA extraction method, based in Chomczynski e Sacchi (1987), was the most efficacious. The DDRT-PCR technique was efficient for the detection of differentially expressed genes from C. vittatus. Three expressed sequence tags (ESTs) were sequenced and the analysis with bioinformatics tools failed in detect similarity with those sequences from data banks. These sequences may be new and, perhaps, specific of C. vittatus. They were deposited in dbEST database. A differentially expressed fragment, specific of works, was obtained with the primers HT11G and AP03. The results of its sequence and the analysis by bioinformatics revealed that this EST was, in fact, been isolated from the endosymbiotic bacteria of C. vittatus because a grol domain was detected and was observed high similarity with many proteins of the chaperonin family, a kind of protein present in the endosymbiotic bacterias of this genus. Insetos sociais despertam grande curiosidade, interesse e uma vontade de se compreender as bases moleculares da vida social. A identificação de genes diferencialmente expressos tem sido usada para o entendimento não só da função gênica, mas também dos mecanismos moleculares de diferentes processos biológicos. Nesse estudo, foram testados dois protocolos para extração de RNA total de Camponotus vittatus e a técnica de Differential Display Reverse Transcriptase Polymerase Chain Reaction (DDRT-PCR) foi utilizada para a identificação de genes diferencialmente expressos em casta, sexo e nos estágios de desenvolvimento dessa formiga. O método de extração de RNA baseado em Chomczynski e Sacchi (1987) demonstrou ser o mais eficaz. A técnica DDRTPCR foi eficiente para a detecção de genes diferencialmente expressos de C. vittatus. Três expressed sequence tags (ESTs) foram seqüenciadas e análises por bioinformática não detectaram similaridade com seqüências depositadas em banco de dados, podendo ser novas e, talvez, específicas de C. vittatus. Essas seqüências foram depositadas no banco de dados dbEST. Um fragmento diferencialmente expresso em operária foi obtido com a combinação dos primers: HT11G e AP03. Os resultados do seqüenciamento e das análises por bioinformática revelaram que tal EST foi isolada da bactéria endossimbionte de C. vittatus, pois foi detectada a presença de um domínio grol e alta similaridade com diversas proteínas da família das chaperoninas, proteínas presentes nas bactérias endossimbiontes do gênero. Mestre em Genética e Bioquímica

Non radioactive Differential Display Reverse Transcription- PCR (DDRT-PCR) was used to screen molecular variants at expression level in Pyrenophora graminea and Pyrenophora teres, grown in vitro with glucose or plant cell walls as carbon sources. Several differential transcripts from both species were identified, either constitutive or induced by barley leaf cell walls. Three cloned species-specific transcripts were sequenced: one was constitutively expressed in P. graminea and absent in P. teres, two were specifically induced by host leaf cell walls either in P. graminea or in P. teres. For all three, sequence analysis showed significant homology with known pathogenicity-related genes available in databases, coding for toxic metabolites, cell wall degrading enzymes and regulative factors as kinases. These data are discussed in relation to the biology of the two pathogens under study.

Studies comparing in vivo and in vitro functional capacities of leukocytes from non-parturient and periparturient dairy cows have provided substantial evidence that systemic and local mammary immune defenses are deficient around parturition. This evidence has lead to the reasonable hypothesis that immune deficiency underlies the heightened mastitis susceptibility of periparturient cows. Nutrition and vaccine studies substantiate this hypothesis, showing that dietary antioxidant supplementation and rigorous immunization regimes can bolster innate and humoral immunity to the point that mastitis severity and time for return to normal milk production are reduced. However, completely effective resolution of this significant production disease has not been achieved because so little is understood about its complex etiology. In particular, we possess almost no knowledge of how or why immune cells responding to parturient physiology end up with deficient functional capacities. Fluctuations in reproductive steroid hormones and chronic shifts in neuroendocrine hormones with roles in nutrient partitioning and appetite control may affect the expression of critical leukocyte genes in periparturient dairy cows. A thorough understanding of leukocyte biology during periparturition would seem a critical goal for future development of effective mastitis prevention strategies. Recently, our group has begun to use cDNA microarray technology to explore bovine leukocyte RNA for global gene expression changes occurring around parturition. We are working within the context of a hypothesis that the physiology of parturition negatively affects expression of critical genes in blood leukocytes. In the current study we initiated hypothesis testing using leukocyte RNA from a high producing Holstein cow collected at 14 days prepartum and 6 hours postpartum to interrogate a cDNA microarray spotted with >700 cDNAs representing unique bovine leukocyte genes. This analysis revealed 18 genes with ≥1.2-fold higher expression 14 days prepartum than 6 hours postpartum. BLASTN analysis of these genes revealed only one that can be considered a classical immune response gene. All other repressed genes were either unknown or putatively identified as encoding key proteins involved in normal growth and metabolism of cells. Given the critical roles of these repressed genes in normal cell functions, we may have identified good candidates to pursue with respect to periparturient immunosuppression and mastitis susceptibility.

The inflammatory response elicited by various stimuli such as microbial products or cytokines is determined by differences in the pattern of cellular gene expression. We have used the differential display RT-PCR (DDRT-PCR) strategy to identify mRNAs that are differentially expressed in various murine cell types stimulated with pro-inflammatory cytokines, microbial products or anti-inflammatory drugs. Mouse embryonic fibroblasts (MEFs) were treated with IFNs, TNF, or sodium salicylate. Also, peritoneal macrophages from C3H/Hej mice were stimulated with T. cruzi-derived GPI-mucin and/or IFN-g. After DDRT-PCR, various cDNA fragments that were differentially represented on the sequencing gel were recovered, cloned and sequenced. Here, we describe a summary of several experiments and show that, when 16 of a total of 28 recovered fragments were tested for differential expression, 5 (31%) were found to represent mRNAs whose steady-state levels are indeed modulated by the original stimuli. Some of the identified cDNAs encode for known proteins that were not previously associated with the inflammatory process triggered by the original stimuli. Other cDNA fragments (8 of 21 sequences, or 38%) showed no significant homology with known sequences and represent new mouse genes whose characterization might contribute to our understanding of inflammation. In conclusion, DDRT-PCR has proven to be a potent technology that will allow us to identify genes that are differentially expressed when cells are subjected to changes in culture conditions or isolated from different organs.

The development of arbuscular mycorrhiza between obligate symbiotic fungi and host roots must involve activation of symbiosis-related genes. Using a new powerful method, called differential display reverse transcription polymerase chain reaction (DDRT-PCR) developed for eukaryotic cells, we have started to study early molecular events during mycorrhiza formation by pea roots and Glomus mosseae. (DDRT-PCR)-cDNA fragments corresponding to genes that are specifically enhanced or repressed during the first steps of the symbiosis are being isolated.

Cloning of a novel gene in the human kidney homologous to rat munc13s: Its potential role in diabetic nephropathy. Glomerular mesangial cells (MC) are believed to play a pivotal role in development of diabetic nephropathy. We employed differential display reverse transcription polymerase chain reaction (DDRT-PCR) comparing human MC grown under 25 mM and 5.5 mM D-glucose and osmolarity control as a first step to identify possible candidate genes regulated by hyperglycemia. This strategy resulted in cloning of a novel gene in human MC, human munc13 (hmunc13), a human homologue of rat munc13s with the N-terminal segment similar to munc13-1 and the C-terminal segment more similar to munc13-2. Hmunc13 is also expressed in human kidney cortical epithelial cells. By using relative RT-PCR and Northern blot, we have confirmed that expression of hmunc13 in MC is up-regulated by high D-glucose treatment. Together with previous reports that munc13s binds to diacylglycerol (DAG) and that hyperglycemia increases DAG levels, these findings point to a potential role of hmunc13 in mediating some of the acute and chronic changes in MC produced by exposure to hyperglycemia.

In the northeastern region of Brazil, saline soils are constraints to banana production, becoming necessary to understand the mechanisms of salt tolerance. Two bananas genotypes, Tap Maeo, tolerant, and Berlin, sensitive, were subjected to treatment with 50 mol m-3 NaCl or without salt. This study evaluated the effects of salt on the following physiological aspects: leaf area, content and distribution of Na+, membrane integrity, proton AT Pase activity. Besides, a search for differentially expressed genes was performed using the Differential Display technique. Tap Maeo genotype showed the smallest reduction in leaf area, smaller accumulation of Na+ and malondialdehyde (MDA), and higher activity of proton AT Pase activity. Two sequences differentially expressed in the tolerant genotype, (Musa 07, Musa 23), shared a high degree of identity with the amino acid sequences of the genes SOS1 and SOS2, respectively. The clone Musa 10 was highly similar to amino acid sequence of the ascorbate peroxidase gene, and Musa 26, encodes the enzyme betaine aldehyde dehydrogenase. These significant biological markers indicate that salinity tolerance in banana involves at least two simultaneous mechanisms: the activation of the SOS pathway, increasing the extrusion of Na+, and the activation of antioxidative system, increasing the synthesis of APX and betaine aldehyde dehydrogenase enzyme. RESUMO Na região Nordeste do Brasil, solos salinos são restritivos à produção de banana, tornandose necessário compreender os mecanismos de tolerância de sal. Dois genótipos de banana, Tap Maeo, tolerante, e Berlim, sensível, foram submetidos ao tratamento com 50 mol m-3 NaCl ou sem sal. Este estudo avaliou os efeitos do sal sobre os seguintes aspectos fisiológicos: área foliar, conteúdo e distribuição de Na+, integridade da membrana, atividade da AT Pase. Além disso, uma busca por genes diferencialmente expressos foi realizada usando a técnica Differential Display. O genótipo Tap Maeo apresentou a menor redução na área foliar, menor acúmulo de Na+ e malondialdeído (MDA) bem como maior atividade da H+AT Pase. Duas sequências diferencialmente expressas no genótipo tolerante (Musa 07, Musa 23) compartilham alto grau de identidade com as sequências de aminoácidos dos genes SOS1 e SOS2, respectivamente. O clone Musa 10 é muito semelhante à sequência de aminoácidos do gene da peroxidase do ascorbato, e o Musa 26 codifica a enzima aldeído betaína desidrogenase. Estes marcadores biológicos significativos indicam que a tolerância à salinidade em banana envolve pelo menos dois mecanismos simultâneos: a ativação da via SOS, aumentando a extrusão do Na+, e a ativação do sistema antioxidante, aumentando a síntese de APX e da enzima aldeído betaína desidrogenase.