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Aspergillus fumigatus is a foodborne and airborne human pathogen which produces mycotoxins such as gliotoxin, helvolic acid, fumigallin, and fumigaclavin. The most common disease caused by A. fumigatus is aspergillosis, which is often fatal, especially among AIDS, cancer, and organ-transplant patients. In this study, random cDNA cloning was performed from a cDNA library of A. fumigatus (IMI 385708) constructed on &amp / #955 / ZAP Express. Some of these clones were selected according to their insert sizes and were further subjected to sequence analysis. The sequences were then analyzed by a BLAST search (NCBI Genome Database) to determine the possible functions of these genes. Two of the clones were identified as the primase and 60S ribosomal protein L1-b genes. These genes and a third gene corresponding to the antigenic cell wall galactomannoprotein gene of A. fumigatus were used for the design of three distinct primer pairs. For the primer design, a software was written to differentiate nonconserved regions by multiple sequence alignment. Designed primers were employed in PCR experiments against genomic DNAs of different Aspergillus species. Unique bands were obtained only against A. fumigatus genomic DNA. It was concluded that this PCR-based detection method can be further developed for the rapid detection of A. fumigatus spores from air and food samples.

No description available.

Made available in DSpace on 2016-12-08T16:24:08Z (GMT). No. of bitstreams: 1 PGCA11MA068.pdf: 13593 bytes, checksum: bb2118cc19310d22381d61f71c63aee7 (MD5) Previous issue date: 2011-06-01 / The mitochondrial enzyme Glutamate Dehydrogenase (GDH: EC 1.4.1.2) catalyzes the reversible deamination of the L-glutamate for 2-oxoglutarate (α-ketoglutarate) using NAD+ and NADP+ as coenzymes. It is a complex allosteric enzyme that consists of six identical subunits being its activity influenced by both, the ADP its positive modulator, and by the GTP, its negative modulator.It is one of the most important liver enzymes found in hepatocytes of cattle, sheep and goats. Infections by Fasciola spp or severe acute intoxication by toxins of plants such as Xanthium spp and Senecio spp as well as intoxication by copper result in the release of this enzyme in blood. The increase of the GDH indicates damage or hepatic necrosis in cattle and sheep. This is an enzyme of choice to evaluate the function of the ruminants. In the present study the cDNA, that codifies the GDH enzyme of the hepatocyte of sheep, was synthesized by means of the RT-PCR making use of mRNA extracted from the liver of sheep. Part of the region where the cDNA of the GDH of the ovine is codified was amplified by PCR from primers synthesized through the comparison of the aligned sequences of Ovis aries with those of the Bos taurus,. Homo sapiens, Rattus norvegicus and Mus musculus available in the database, where highly conserved regions, as well as variable regions were identified.The cDNA was cloned in the vector pGEM®-T Easy Vector Systems and inserted in competent cells of the Escherichia coli DH10B by means heat shock. The plasmid DNA was purified and after sequencing, the presence of an insert of 1292 pb was confirmed. The alignment of the sequence deduced of amino acid with other species revealed high homology among the GDH / A enzima mitocondrial Glutamato Desidrogenase (GDH : EC 1.4.1.2) catalisa a desaminação reversível do L- glutamato para 2-oxoglutarato (α-cetoglutarato) usando o NAD+ e NADP+ como coenzimas . É uma enzima alostérica complexa que consiste em seis subunidades idênticas sendo sua atividade influenciada tanto pelo ADP, seu modelador positivo, como pelo GTP, seu modelador negativo. É uma das mais importantes enzimas hepáticas encontradas em hepatócitos de bovinos, ovinos e caprinos. Infecções por Fasciola spp ou intoxicação grave aguda por toxinas de plantas tais como Xanthium spp e Senecio spp e intoxicação por cobre resultam na liberação dessa enzima no sangue. O aumento da GDH indica danos ou necrose hepática em bovinos e ovinos. Esta é a enzima de escolha para avaliar a função hepática dos ruminantes. No presente trabalho o cDNA que codifica a enzima GDH do hepatócito de ovino foi sintetizado por meio de RT-PCR utilizando mRNA extraído do fígado de ovino. Parte da região de codificação do cDNA da GDH de ovino foi amplificada por PCR a partir de oligonucleotídeos iniciadores sintetizados através da comparação das sequências alinhadas de Ovis aries com de Bos taurus, Homo sapiens, Rattus norvegicus e Mus musculus disponíveis em banco de dados, onde foram identificadas regiões bastante conservadas e regiões variáveis. O cDNA foi clonado no vetor pGEM® -T Easy Vector Systems e inserido em células competentes de Escherichia coli DH10B através de choque térmico. O DNA plasmidial foi purificado e após o sequenciamento a presença de um inserto de 1292 pb foi confirmado. O alinhamento da sequência deduzida de aminoácidos com outras espécies revelou alta homologia entre as GDH

In the late 1990s, a series of landmark publications described RNA interference (RNAi) and related RNA silencing phenomena in nematodes, plants and fungi. By manipulating RNA silencing, biologists have been able to create tools for specifically inactivating genes. In organisms from trypanosomes to insects, RNA silencing is now indispensible for studying gene function. RNA silencing has been used in a project aimed at systematically knocking out all genes in the model plant Arabidopsis thaliana. RNA silencing has a natural role in defending eukaryotic cells against virus replication. By assembling virus DNA sequences in a form that triggers RNA silencing, biologists have created plants resistant to specific viruses. In this study, we set out to test if a similar approach would protect plants against infection by the agriculturally important Brassica pathogen, Plasmodiophora brassicae. P. brassicae is an obligate intracellular biotroph, from the little studied eukaryotic supergroup, the Rhizaria. To identify the gene sequences that would be starting material for P. brassicae RNA silencing, new P. brassicae genes were gathered by cDNA cloning or genomic PCR-walking. Using suppression subtractive hybridisation (SSH) and oligo-capping cloning of full-length cDNAs, 76 new gene sequences were identified. A large proportion of the cDNAs were predicted to contain signal peptides for ER translocation. In addition to the new cDNA identified here, partial sequences for the P. brassicae actin and TPS genes were published by other researchers close to the beginning of this study. Using PCR-walking, full-length genomic DNA sequences from both genes were obtained. Later, genomic DNA sequences spanning or flanking a total of 24 P. brassicae genes were obtained. The P. brassicae genes were rich in typical eukaryotic spliceosomal introns. Transcription of P. brassicae genes also appears likely to begin from initiator elements rather than TATA-box-containing promoters. A segment of the P. brassicae actin gene was assembled in hairpin format and transformed into Arabidopsis thaliana. Observation of simultaneous knockdown of the GUS marker gene as well as detection of siRNAs indicated that the hpRNA sequences induced RNA silencing. However, inoculation of these plants with P. brassicae resulted in heavy club root infection. We were unable to detect decreases in actin gene expression in the infecting P. brassicae, at either early or late stages of infection. We conclude that, within the limits of the techniques used here, there is no evidence for induction of RNA silencing in P. brassicae by in planta produced siRNAs.

In the late 1990s, a series of landmark publications described RNA interference (RNAi) and related RNA silencing phenomena in nematodes, plants and fungi. By manipulating RNA silencing, biologists have been able to create tools for specifically inactivating genes. In organisms from trypanosomes to insects, RNA silencing is now indispensible for studying gene function. RNA silencing has been used in a project aimed at systematically knocking out all genes in the model plant Arabidopsis thaliana. RNA silencing has a natural role in defending eukaryotic cells against virus replication. By assembling virus DNA sequences in a form that triggers RNA silencing, biologists have created plants resistant to specific viruses. In this study, we set out to test if a similar approach would protect plants against infection by the agriculturally important Brassica pathogen, Plasmodiophora brassicae. P. brassicae is an obligate intracellular biotroph, from the little studied eukaryotic supergroup, the Rhizaria. To identify the gene sequences that would be starting material for P. brassicae RNA silencing, new P. brassicae genes were gathered by cDNA cloning or genomic PCR-walking. Using suppression subtractive hybridisation (SSH) and oligo-capping cloning of full-length cDNAs, 76 new gene sequences were identified. A large proportion of the cDNAs were predicted to contain signal peptides for ER translocation. In addition to the new cDNA identified here, partial sequences for the P. brassicae actin and TPS genes were published by other researchers close to the beginning of this study. Using PCR-walking, full-length genomic DNA sequences from both genes were obtained. Later, genomic DNA sequences spanning or flanking a total of 24 P. brassicae genes were obtained. The P. brassicae genes were rich in typical eukaryotic spliceosomal introns. Transcription of P. brassicae genes also appears likely to begin from initiator elements rather than TATA-box-containing promoters. A segment of the P. brassicae actin gene was assembled in hairpin format and transformed into Arabidopsis thaliana. Observation of simultaneous knockdown of the GUS marker gene as well as detection of siRNAs indicated that the hpRNA sequences induced RNA silencing. However, inoculation of these plants with P. brassicae resulted in heavy club root infection. We were unable to detect decreases in actin gene expression in the infecting P. brassicae, at either early or late stages of infection. We conclude that, within the limits of the techniques used here, there is no evidence for induction of RNA silencing in P. brassicae by in planta produced siRNAs.

In the late 1990s, a series of landmark publications described RNA interference (RNAi) and related RNA silencing phenomena in nematodes, plants and fungi. By manipulating RNA silencing, biologists have been able to create tools for specifically inactivating genes. In organisms from trypanosomes to insects, RNA silencing is now indispensible for studying gene function. RNA silencing has been used in a project aimed at systematically knocking out all genes in the model plant Arabidopsis thaliana. RNA silencing has a natural role in defending eukaryotic cells against virus replication. By assembling virus DNA sequences in a form that triggers RNA silencing, biologists have created plants resistant to specific viruses. In this study, we set out to test if a similar approach would protect plants against infection by the agriculturally important Brassica pathogen, Plasmodiophora brassicae. P. brassicae is an obligate intracellular biotroph, from the little studied eukaryotic supergroup, the Rhizaria. To identify the gene sequences that would be starting material for P. brassicae RNA silencing, new P. brassicae genes were gathered by cDNA cloning or genomic PCR-walking. Using suppression subtractive hybridisation (SSH) and oligo-capping cloning of full-length cDNAs, 76 new gene sequences were identified. A large proportion of the cDNAs were predicted to contain signal peptides for ER translocation. In addition to the new cDNA identified here, partial sequences for the P. brassicae actin and TPS genes were published by other researchers close to the beginning of this study. Using PCR-walking, full-length genomic DNA sequences from both genes were obtained. Later, genomic DNA sequences spanning or flanking a total of 24 P. brassicae genes were obtained. The P. brassicae genes were rich in typical eukaryotic spliceosomal introns. Transcription of P. brassicae genes also appears likely to begin from initiator elements rather than TATA-box-containing promoters. A segment of the P. brassicae actin gene was assembled in hairpin format and transformed into Arabidopsis thaliana. Observation of simultaneous knockdown of the GUS marker gene as well as detection of siRNAs indicated that the hpRNA sequences induced RNA silencing. However, inoculation of these plants with P. brassicae resulted in heavy club root infection. We were unable to detect decreases in actin gene expression in the infecting P. brassicae, at either early or late stages of infection. We conclude that, within the limits of the techniques used here, there is no evidence for induction of RNA silencing in P. brassicae by in planta produced siRNAs.