Neste trabalho realizamos a análise das variações na expressão gênica global do fungo aquático Blastocladiella emersonii submetido ao estresse de carência de oxigênio (hipóxia), utilizando a técnica de microarranjos de cDNA em lâminas contendo 3773 genes distintos. Nos experimentos de hipóxia gradual (diminuição gradual da concentração de oxigênio dissolvido, seguido de reoxigenação) e hipóxia direta (diminuição direta da concentração de oxigênio dissolvido, seguido de reoxigenação) observamos que 650 genes foram diferencialmente expressos em pelo menos uma das condições de estresse e que 534 deles mostraram-se afetados (direta ou indiretamente) pela disponibilidade de oxigênio, uma vez que apresentaram recuperação (ou tendência à recuperação) da sua expressão aos níveis normais, quando as células foram reoxigenadas. Além de modular a expressão de diversos genes sem função conhecida, B. emersonii responde à hipóxia reajustando a expressão de genes responsáveis pela produção e consumo de energia. Pelo menos transcricionalmente, este fungo favorece o metabolismo anaeróbico, através da indução de genes que codificam enzimas da via glicolítica e lactato desidrogenase, ao passo que no ciclo do ácido cítrico, a maioria dos genes encontram-se reprimidos ou não sofrem alteração na expressão. Processos dispendiosos em energia como síntese protéica, metabolismo de aminoácidos, enovelamento de proteínas e transporte por membrana apresentaram perfis predominantemente de repressão gênica quando em carência de oxigênio. Ainda utilizando a técnica de microarranjos, mostramos semelhanças entre os perfis transcricionais nos experimentos hipóxia e de carência de Fe2+ (tratamento com quelante de Fe2+ 2,2´-dipyridyl) sugerem que estes estresses estão de alguma forma relacionados, fornecendo bons indícios de que o íon Fe2+ possa ter um papel importante no mecanismo sensor de oxigênio e/ou de resposta a hipóxia em B. emersonii. Além disso, o tratamento prévio de células submetidas à hipóxia com o antibiótico geldanamicina, um conhecido inibidor da proteína de choque térmico HSP90, levou à diminuição da indução de certos genes de hipóxia, indicando que este fungo pode possuir algum mecanismo semelhante ao do fator de transcrição de hipóxia HIF1-α de mamíferos, uma vez que este fator também é afetado por geldanamicina. Adicionalmente, desenvolvemos um protocolo para transformação de B. emersonii mediada por Agrobacterium tumefasciens que se mostrou promissor. A transferência do T-DNA contendo um gene de resistência a higromicina B, presente no vetor binário pBINPLUS-Hph, foi evidenciada pelo crescimento normal e esporulação das células transformadas, na presença do antibiótico e pela amplificação do gene de resistência no DNA genômico de células transformadas. / In this work we analyzed global gene expression changes in the aquatic fungus Blastocladiella emersonii submitted to oxygen deprivation (hypoxia), using cDNA microarrays containing 3,773 distinct genes. In gradual hypoxia (gradual decrease in dissolved oxygen concentration, followed by reoxygenation) and direct hypoxia (direct decrease of dissolved oxygen concentration, followed by reoxygenation) we observed 650 differentially expressed genes in at least one of the stress conditions tested, 534 of them being affected (directly or indirectly) by oxygen availability, since they showed recovery of normal expression levels or a tendency to recover, when cells were reoxygenated. Besides modulating many genes with no previously assigned function, B. emersonii responds to hypoxia by readjusting the expression levels of genes responsible for energy production and consumption. At least transcriptionally, this fungus seems to favour anaerobic metabolism through the induction of genes encoding glycolytic enzymes and lactate dehydrogenase, while in the TCA-cycle, most genes were repressed or unchanged. Energy-costly processes like protein synthesis, amino acid metabolism, protein folding and transport had their gene expression profiles predominantly repressed during oxygen deprivation. Microarray experiments also showed similarities between the transcriptional profile of genes in hypoxia and iron (II) deprivation (treatment with the iron (II) chelator 2,2\'-dipyridyl), suggesting that these stresses are somehow related, giving good evidence that Fe2+ ion could have a role in the mechanism of oxygen sensing and/or response to hypoxia in B. emersonii. Furthermore, pretreatment of cells subjected to hypoxia with the antibiotic geldanamycin, a known inhibitor of the heat shock protein HSP90, caused a significant decrease in the induction of certain hypoxic genes, indicating that this fungus could have a mechanism similar to that of the mammalian hypoxia transcription factor HIF-1α, which is also affected by geldanamycin. Additionally, we developed an Agrobacterium tumefasciens-mediated protocol for transformation of B. emersonii that has shown to be promising. The capacity to transfer the T-DNA containing a hygromycin B resistance gene, present in the pBINPLUSHph binary vector, was evidenced by the normal growth and sporulation of the transformed cells in the presence of antibiotic and by amplification of the resistance gene from the genomic DNA of transformed cells

Este trabalho mostrou a purificação de uma apirase em extratos de tegumento de vermes adultos de S. mansoni com razão de hidrólise de ADP/ATP aproximadamente 2. Análise da amostra por MALDI-TOF evidenciou a presença de helicase e carboxilesterase, sendo que a atividade de hidrólise de ADP ainda não foi descrita para estas proteínas. Através de \"Western blot\" mostrou-se que a proteína purificada não tem epítopos reconhecíveis por anticorpo anti-apirase de batata, e que a imunoreatividade cruzada com apirase da família CD39 está presente na fração insolúvel que não foi utilizada no processo de purificação. Esta tese caracteriza também a utilização de \"primers\" consenso degenerados na construção de bibliotecas de cDNA com PCR de baixa estringência. A introdução do \"touch-down\" PCR aumentou o número médio de sequências não redundantes por biblioteca de 20 para 40. O \"touch-down\" associado ao uso de primers híbridos consenso-degenerados (40 pb) elevou a média de sequências não redundantes para 300. Este número significa uma diminuição de cerca de 10 vezes na quantidade de trabalho de clonagem e sequenciamento necessário para se gerar o mesmo montante de dados (Fietto et al., 2000, addendum patent application Nº196,716). A diminuição da concentração de sal na reação e abaixamento da velocidade de rampa entre os passos de anelamento e extensão gerou os melhores perfis de amplificação de cDNA. Bibliotecas feitas com primers híbridos mais curtos (25 pb) mostraram-se mais redundantes, refletindo uma maior especificidade por certas sequências alvo. Com estes primers, usando-se RNAm de tegumento, clonamos um fragmento parcial de cDNA altamente homólogo à CD39 de camundongo. Esta sequência serviu como base para o isolamento de grande parte do gene de Schistosoma (2,9 Kb) através de RACE-PCR (Rapid Amplification of cDNA Ends). / This work shows purification of an apyrase from the tegument membrane of S. mansoni adult worms that exhibited an ADP/ATP hydrolysis ratio near 2. MALDI-TOF analyses showed that helicase and carboxylesterase were present in the sample, however in the literature there is no ADPase activity described for either enzyme. Western blot analyses showed that the purified apyrase ha no epitopes common to members of the CD39 apyrase family. On the other hand, a CD39-like apyrase remained in the insoluble fraction that was not used in the purification procedures. This work studied the construction of cDNA libraries with Hybrid Consensus-degenerate Oligonucleotide Primers (40bp) and low stringency RT-PCR. Introduction of touch-down PCR in the standard ORESTES technique improved the number of non-redundant sequences per library twofold. Association of touch-down with hybrid consensus-degenerated oligonucleotide primers increased this number to 300 sequences per library. The modifications resulted in a 10-fold decrease in the amount of work required for libraries construction and high-throughput sequencing (Fietto et al., 2000; addendum to patent application #196,716 - Ludwig Institute for Cancer Research). The best amplification profiles were obtained in RT-PCR reactions with low salt and a slow temperature ramp. Libraries made with shorter hybrid primers (25bp) showed more redundant sequences, reflecting a higher specificity for few templates. These shorter primers were used in combination with tegument mRNA to clone a gene fragment homologous to CD39 of mouse. This sequence was used in RACE-PCR reactions (rapid amplification of cDNA ends) to clone a 2.9 Kb cDNA corresponding to most of the predicted coding sequence of the Schistosoma gene.

No description available.

The Crimson-spotted rainbowfish (Melanotaenia fluviatilis) from the Murray-Darling basin of Australia is a common indicator species in Australian ecotoxicology. Biochemical changes have been investigated in this species, but not molecular markers of ecotoxicological interest. In this study genes of M. fluviatilis were isolated using a cDNA library and sequences analysed. Of 345 randomly selected clones, 94 shared similarity with 26 different genes in other organisms in public databases. Amongst these, reproductive genes coding for vitellogenin, retinol binding protein, sialyltransferase and zona pellucida protein were considered of interest in ecotoxicology. The vitellogenin gene was selected for study as it has been widely used as a molecular marker of exposure to 17â-estradiol (E2) in teleosts. Gene expression was examined via northern blot, RT-PCR and Real-Time PCR relative to the housekeeping gene (18S rRNA). The expression of vitellogenin mRNA was observed a t 12 hours post-exposure, peaked at 48 hours according to northern blot analysis; and cleared within 4 days, partly consistent with RT-PCR. However, Real-time PCR yielded an inconclusive result, probably due to differences between pooled and individual samples. Vitellogenin in blood plasma was confirmed by western blot, found to be significantly increased and retained in the plasma in fish treated with E2 compared to controls. It was concluded that vitellogenin mRNA is a molecular marker of exposure to 17â-estradiol in the rainbowfish, and could potentially be used as a marker of exposure to environmental estrogenic chemicals. Further investigations of the expression of genes in the cDNA library, could establish other molecular markers of ecotoxicological interest in M. fluviatilis.

<p>Autism is a pervasive developmental disorder (PDD) that&rsquo / s incidence is approximately 1 in 158. It is four times more prevalent in males than females and is believed to be caused by both genetic and environmental factors. Research indicates that several genes are involved in autism and it is believed that these genes act together to produce autism. Many genes implicated in this disorder are involved with brain structure formation and brain functioning. Studies have identified the reelin (RELN) gene as necessary for proper formation of brain, which indicates that RELN abnormalities could contribute to the aetiology of several neurogenetic diseases such as schizophrenia, bipolar and autism. The aims of the study were (i) to genotype two SNPs (exonic rs3622691 and intronic rs736707) in the RELN gene using Taqman&reg / SNP Genotyping assays to detect association with autism in three distinct South African (SA) ethnic groups (Black, Caucasian and Mixed), and (ii) to detect candidate genes that are over and under-expressed in the samples taken from a SA Caucasian autistic group and compare those with samples taken from a healthy Caucasian group using cDNA microarray. The Taqman&reg / study indicated significant association for the intronic SNP, rs736707, with a p-value of 0.0009 in the total SA group. More so, the Mixed group displayed the highest significance amongst the ethnic groups, with a p-value of 0.00014. The microarray study yielded 21 genes with 95% significance in the Caucasian sample group. Most genes were hypothetical proteins and formed part of the FAM90A family. The LOC83459 showed the highest level of expression in the autistic samples, while the BTNL8 gene was shown to be highly suppressed in the control samples.</p>

In most mammals, sexual fate is determining genetically by the presence of the SRY gene which encoded the testis-determining factors on the Y chromosome. Likewise, avian sex is determined genetically. At day 3.5 (stage 22; HH) in chicken embryogenesis, the gonadal primordium begins forming. Thus, to identify the novel sex-determinating genes in early chicken embryos, subtractive cDNA libraries from male-minus-female (M-F) and female-minus-male (F-M) of 3 Dpc. embryos were established. Both collected male and female chicken total mRNAs were purified using Dynabeads. After a blund-end restriction endonuclease Rsa I digestion of cDNA, adaptor ligation for tester cDNA was performed. When first and second cDNA hybridization was finished, those nonredundant cDNA between tester and driver will be amplified by two rounds of PCR. Subsequently, TA-cloning was performed and the cDNA fragments were PCR-amplified using M13 primers. PCR products of Clones were first screened by differential screening hybridization to decrease false positive inserts. Then, gene annotation was carried out by data-mining in public databases, GeneBank (NCBI, USA). Finally, 40 known and 71 novel transcripts of M-F cDNA library, 88 known and 128 novel transcripts of F-M cDNA library were identified. In M-F subtracted library, 4 identified known genes were located on Z sex chromosome such as WD repeat domain 36 (WDR36), PC4 and SFRS1 interacting protein 1 (PSIP1), serum response factor binding protein 1 (SRFBP1) and glycine dehydrogenase (decarboxylating) (GLDC). Another two identified known genes, laminin alpha 1 (LAMA1) and leukocyte cell derived chemotaxin 1 (LECT1) were reported be relate to cell differentiation and development. In F-M subtracted library, only Wpkic-8 was located on W sex chromosome. Other identified genes like slowmo homolog 2 (Drosophila) (SLMO2), collagen, type IV, alpha 1 (COL4A1), anterior gradient 2 homolog (Xenopus laevis), transcript variant 2 (AGR2), solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 6 (SLC25A6) and prolyl endopeptidase (PREP) were also found expressed higer in human ovary then testis. PREP was proposed that it may play a role in mediating sperm death by regulating the levels of thyrotropin-releasing hormone analogs and in mediating sperm death associated with necrozoospermia. These transcripts located on W or Z sex chromosome identified from subtracted libraries may play an important role in sex determination mechanism.

Expansion of common bean (<i>Phaseolus vulgaris</i> L.) crops in the northern Great Planes has been hampered due to the lack of cultivars demonstrating sufficient vitality under low temperature conditions. <i>Phaseolus angustissimus</i> L., a wild bean species, has been previously shown to possess the ability to survive low temperatures in field trials. Freezing tolerance experiments under controlled conditions resulted in P. angustissimus demonstrating a greater capacity for freezing tolerance than <i>P. vulgaris</i>, as all P. vulgaris plants studied were dead at -2.5oC while most P. angustissimus plants treated to the same conditions survived. Exposure to chilling temperatures over five days resulted in stunted growth in both species, but the cultivated bean suffered more compared to the wild bean, as noted by a marked loss in tissue water content over the first three days of chilling. Interspecific macroarray hybridizations of a cDNA library from cold acclimated Medicago sativa L. using cDNAs derived from non-chilled and three-day chilled <i>P. vulgaris</i> and <i>P. angustissimus</i> plants showed that <i>P. vulgaris</i> showed more changes in gene expression after three days of chilling. Also, <i>P. vulgaris</i> showed a general trend towards down-regulation of the transcripts sampled on the third day of chilling compared to <i>P. angustissimus</i>. RT-PCR experiments were conducted using cDNAs from plant tissues exposed to various durations of chilling to confirm the results from the macroarray experiment. These time-course RT-PCR experiments revealed expression patterns across various chilling durations in genes identified from the macroarray. Data from these experiments suggest that <i>P. vulgaris</i> and <i>P. angustissimus </i> seedlings respond differently to low temperature exposure, and that some of the changes in <i>P. angustissimus</i> transcripts monitored here may be useful for researchers in better understanding how Phaseolus species can respond better to chilling temperatures.

Expansion of common bean (<i>Phaseolus vulgaris</i> L.) crops in the northern Great Planes has been hampered due to the lack of cultivars demonstrating sufficient vitality under low temperature conditions. <i>Phaseolus angustissimus</i> L., a wild bean species, has been previously shown to possess the ability to survive low temperatures in field trials. Freezing tolerance experiments under controlled conditions resulted in P. angustissimus demonstrating a greater capacity for freezing tolerance than <i>P. vulgaris</i>, as all P. vulgaris plants studied were dead at -2.5oC while most P. angustissimus plants treated to the same conditions survived. Exposure to chilling temperatures over five days resulted in stunted growth in both species, but the cultivated bean suffered more compared to the wild bean, as noted by a marked loss in tissue water content over the first three days of chilling. Interspecific macroarray hybridizations of a cDNA library from cold acclimated Medicago sativa L. using cDNAs derived from non-chilled and three-day chilled <i>P. vulgaris</i> and <i>P. angustissimus</i> plants showed that <i>P. vulgaris</i> showed more changes in gene expression after three days of chilling. Also, <i>P. vulgaris</i> showed a general trend towards down-regulation of the transcripts sampled on the third day of chilling compared to <i>P. angustissimus</i>. RT-PCR experiments were conducted using cDNAs from plant tissues exposed to various durations of chilling to confirm the results from the macroarray experiment. These time-course RT-PCR experiments revealed expression patterns across various chilling durations in genes identified from the macroarray. Data from these experiments suggest that <i>P. vulgaris</i> and <i>P. angustissimus </i> seedlings respond differently to low temperature exposure, and that some of the changes in <i>P. angustissimus</i> transcripts monitored here may be useful for researchers in better understanding how Phaseolus species can respond better to chilling temperatures.

Natural diseases caused by keratin mutations are rare and have only been reported in humans. We have recently identified a heritable skin disorder in Norfolk terriers caused by a mutation in KRT10. Affected dogs have a tendency to form shallow erosions or blisters following mild trauma, which is first noted after the birthing process. As the dogs age, they display generalized hyperpigmentation and scaling that is most severe in the axillary and inguinal regions. The main histologic and ultrastructural features include: marked hyperkeratosis, epidermal hyperplasia, prominent vacuolation of the upper suprabasal layers, eosinophilic intracytoplasmic aggregates (keratin bundles), numerous and frequently enlarged keratohyaline granules, and epidermal hyperplasia. Analysis of an extended pedigree through seven generations confirmed an autosomal recessive mode of inheritance. The keratin 10 mutation was defined as a G-T point mutation in intron 5 that affected splicing at the boundary of exon 4 and intron 5. The primary outcome of the mutation was a 35 bp deletion in exon 4 caused by use of a cryptic splice site. Real-time PCR quantitation of KRT10 confirmed that this mutation led to premature mRNA decay and an average 35-fold decrease in KRT10 message. Organotypic cell culture techniques were used to establish in vitro models for normal and affected Norfolk terriers. After 21 days of culture, normal epidermis was cornified with a compact and multifocally parakeratotic stratum corneum. Affected epidermis largely reproduced the expected morphologic alterations. Immunoblotting and immunohistochemistry for keratin 10 protein and real-time PCR quantitation of KRT10 message showed significantly less keratin expression in vitro than in vivo suggesting that the differentiation program in vitro underwent significant alterations. A diagnostic PCR assay was established for detection of the carrier state. Global analysis of gene expression between normal, carrier and affected dogs was performed with DermArray cDNA microarrays. Affected and carrier dogs showed differential regulation of 320 and 298 genes, respectively, between normal dogs. In affected dogs, 217 were upregulated and 103 were downregulated. In carrier dogs, 222 were upregulated and 76 were downregulated. 72 genes (65 upregulated, 7 downregulated) were altered in both affected and heterozygous dogs.

Two hypotheses currently exist regarding to the determining factors for sexual development and differentiation in birds. One is based on the unbalanced sex chromosome, meaning that avian sex determination is dominated by ¡§Z-chromosome dosage¡¨. The other brings up (reconsider this) the key factor of ¡§W chromosome¡¨ which is a particular sex chromosome in female birds (ZW). In the previous studies, we constructed a female-subtract-male cDNA library before morphological gonad differentiation. After sequencing and annotation, a total of 279 expression sequence taqs (ESTs) were identified, with potentially higher expression levels in females. By utilizing quantitative RT-PCR, 16 potential ESTs and three marker transcripts (HINT1, FET1 and WDR36), which identified to be involved in sexual development at 3, 5, 7, 9 days post-coitum (dpc) was analyzed in chicken embryos. Results indicated that AGR2, CPT2, DUSP19, HINTW, LOC771368 and EY53070791 had higher expression levels in female than in male embryos at 3 and 5 dpc; FET1 expression level in female embryos gradually increased from 3 to 9 dpc. Moreover, both HINT1 and WDR36 were higher expressed in male than in female embryos across 3 to 9 dpc. However, HINT1 exhibited higher expression levels starting at early stage, whereas WDR36 at later stage. Next, we constructed HINT1-GFP fusion protein and overexpressed this protein in chicken B-cell line (DT40), resulting in upregulation of WDR36 expression. On the contrary, overexpressed HINTW-GFP fusion protein in DT40 cells had decreased WDR36 expression level. Moreover, we designed a small hairpin RNA by utilizing RNA interference technique to knockdown expression of HINTW, which resulted in WDR36 upregulation. Finally, we then estimated the regulation of WDR36 promoter activity through analyzing HINT1-GFP overexpression. Results had shown that HINT1-GFP can improve WDR36 promoter activity. Therefore, we suppose that HINT1 can regulate WDR36 transcription via WDR36 proximal promoter region. Ongoing HINT1 responsive element(s) must be identified to characterize whether HINT1 or HINTW regulates WDR36.