No presente estudo, foi inicialmente padronizada uma PCR para detecção do DNA viral da semente de Parvovírus Canino utilizado na vacina brasileira Imunovet® (VR-953TM), tendo como alvo o gene VP2. O produto de PCR foi submetido ao seqüenciamento a fim de caracterizar geneticamente a semente vacinal. A seguir, foi padronizada uma reação de PCR em tempo real (RT-PCR) para detecção de um fragmento de 119 pb do gene VP2, a qual foi empregada para avaliar a cinética de replicação da amostra vacinal do CPV em diferentes métodos e tempos de cultivo celular. A correlação entre os resultados do título infeccioso das amostras virais e o número de cópias obtido na RT-PCR foi avaliado pelo Coeficiente de Correlação de Pearson. A PCR padronizada apresentou uma sensibilidade analítica de 457 DICC50/mL. O seqüenciamento do produto de PCR revelou que a amostra vacinal é do tipo CPV-2. A RT-PCR padronizada apresentou uma sensibilidade analítica de 1030 cópias de DNA/mL e uma boa especificidade analítica, pois não detectou o DNA de Adenovírus canino tipos 1 e 2 e Herpesvírus Equino tipo 1. A RT-PCR exibiu Coeficientes de Variação de triplicatas intra-ensaio de 0,43% e inter-ensaio de 0,29%. O Coeficiente de Correlação de Pearson entre o título infeccioso das amostras virais e o número de cópias obtido na RT-PCR foi de 0,55, considerado moderadamente positivo. Considerando que a região alvo da RT-PCR padronizada apresentou 100% de identidade com 93,52% (159/170) das amostras pesquisadas no GenBank pelo BLAST, a RT-PCR padronizada sugere ter um potencial uso no diagnóstico. / In this study, was originally a standard PCR for detection of viral DNA from the canine parvovirus vaccine seed used in Brazilian Imunovet® (VR-953TM), targeting the VP2 gene. The PCR product was subjected to sequencing to genetically characterized the vaccine seed. Then, a reaction was standardized real-time PCR (RT-PCR) to detect a fragment of 119 bp VP2 gene, which was used to evaluate the growth kinetics of the CPV vaccine sample in different methods and cell culture times. The correlation between results of the infectious titre and the number of copies obtained in RT-PCR was evaluated by Pearsons correlation coefficient. The standardized PCR showed an analytical sensitivity of 457 TCID50/mL. The sequencing of the PCR product showed that the vaccine sample is CPV type 2. The standardized RT-PCR showed an analytical sensitivity of 1030 DNA copies/mL and a good analytical specificity, it does not detect the DNA of canine adenovirus type 1 and 2 and equine herpesvirus type 1. The RT-PCR showed coefficients of variation intra-assay triplicates of 0,43% and inter-assay of 0,29%. The Pearsons correlation coefficient between the titre of infectious viral samples and the number of copies obtained in RT-PCR was 0,55, considered moderately positive. Whereas the target region of the standardized RT-PCR showed 100% identity with 93,52% (159/170) of samples surveyed in Genbank by BLAST, the standard RT-PCR suggests a potential diagnostic use.

Análises de diversidade em feijão comum (Phaseolus vulgaris L.), envolvendo caracteres morfológicos e marcadores moleculares, têm mostrado uma estruturação em dois pools gênicos principais, um Mesoamericano e outro Andino, os quais diferem, dentre vários aspectos, quanto à morfologia e fisiologia do grão. Uma nova abordagem que vem sendo usada para estimar a variabilidade genética entre acessos de feijoeiro é a prospecção de polimorfismos de base única (SNPs), tanto em sequências arbitrárias do genoma quanto em sequências gênicas de interesse. Também, o estudo de sequências nucleotídicas que codificam proteínas importantes, tais como a faseolina e a ferro redutase, codificadas, respectivamente, pelos genes Phs e FRO1, deve permitir a geração de novos e informativos marcadores e promover um melhor entendimento da variação existente em P. vulgaris. Assim, os objetivos deste trabalho foram obter as sequências dos genes - Phs e FRO1, acessar a frequência de SNPs em regiões codantes e não-codantes de ambos os genes, e avaliar a utilidade desses polimorfismos para investigar a diversidade genética em 31 genótipos cultivados de feijão comum e um acesso de P. lunatus. Para tanto, primers específicos foram desenhados e as sequências obtidas foram alinhadas, permitindo a identificação de vários sítios polimórficos. Parâmetros moleculares foram estimados pelo software Arlequin e MEGA. As análises de agrupamento foram conduzidas através do método Neighbor-joining. Foram detectadas 361 bases polimórficas, das quais 260 foram do tipo substituição e 101, indel. A frequência de SNPs em regiões não codantes foi duas vezes maior do que em regiões traduzidas, em ambos os genes; também, a substituições do tipo transição foram mais freqüentes do que as do tipo transversão. Os polimorfismos em regiões codantes levaram à substituição de 17 aminoácidos na proteína faseolina, e 14 na enzima ferro redutase. Tais modificações incluem, na maioria das vezes, alterações de aminoácidos com propriedades similares. Vale destacar que sequência predita de aminoácidos da faseolina exibiu uma diversidade elevada entre os acessos investigados, sendo que alguns desses polimorfismos se prestaram para tipificar alguns acessos. Em contrapartida, a enzima ferro redutase exibiu um padrão de aminoácidos mais homogêneo, principalmente para os acessos andinos. As análises de agrupamento revelaram dois clusters bem definidos: um que agrupou os acessos Mesoamericanos e as cultivares brasileiras, e outro que agrupou os Andinos, sugerindo que sequências gênicas são úteis na distinção dos pools gênicos de Phaseolus. Todavia, os polimorfismos do gene -Phs permitiram uma melhor resolução das relações entre os acessos dentro de cada pool gênico, em comparação com os do gene FRO1. / Diversity analysis in common bean (Phaseolus vulgaris L.), based on morphological traits and molecular markers, has revealed the existence of two major gene pools, the Mesoamerican and Andean pools, which differ in several features, including the grain morphology and physiology. A novel approach to estimating genetic variability is in silico mining for single nucleotide polymorphisms (SNP). Arbitrary genome sequences as well as genic sequences were used for this purpose. Additionally, the investigation of nucleotide sequences that code for important proteins, such as phaseolin and iron-reductase, encoded respectively by the Phs and FRO1 genes, should allow new and informative markers to be generated, helping us to gain a better understanding of intraspecific variation in P. vulgaris. Therefore, the aims of this study were to sequence the -Phs and FRO1 genes, to assess SNP frequencies in coding and non-coding regions of both genes, and to assess the possible use of these polymorphisms for investigating the genetic diversity of 31 cultivated genotypes of common bean and one of P. lunatus. Specific primers were designed and the sequences obtained were aligned, allowing us to identify several polymorphic sites. Molecular parameters were estimated using the Arlequin and MEGA software packages. Cluster analyses were conducted using the neighborjoining method. We detected 361 polymorphic sites, consisting of 260 base substitutions and 101 indels. The frequency of SNPs in non-coding regions was twice the frequency in translated regions in both genes. In addition, the occurrence of base transitions was higher than transversions. Polymorphisms in coding regions lead to 17 amino acid substitutions in the phaseolin protein and 14 in the iron reductase enzyme. Most substitutions of this kind include changes that preserve the respective protein functions. The predicted amino acid sequence of phaseolin exhibited high diversity, and some polymorphisms allowed us to typify some accessions. In contrast, the iron reductase enzyme exhibited a more homogeneous pattern of amino acids, especially in the Andean accessions. Cluster analyses revealed two well-defined clusters, one containing the Mesoamerican accessions with the Brazilian cultivars, and another containing the Andean accessions, suggesting that gene sequences are useful for distinguishing the Phaseolus gene pools. Interestingly, the polymorphisms detected in the -Phs gene allowed better visualization of the relationships between accessions in the same pool, in comparison to FRO1.

A Terra Preta Antropogênica (TPA) ocorre somente em sítios arqueológicos na região amazônica. Estes solos de origem antrópica foram enriquecidos em nutrientes, provavelmente pelo manejo de restos orgânicos e do fogo pelas populações pré-colombianas. Em TPA, a presença de material orgânico estável e a grande atividade biológica indicam que este tipo de solo pode ser um local de alta diversidade microbiana, constituindo numa fonte de germoplasma microbiano. Contudo, atualmente não se tem conhecimento da biologia e, sobretudo da estrutura e diversidade das comunidades microbianas deste solo. O conhecimento sobre a diversidade microbiana trará compreensão das funções exercidas pelas comunidades microbianas no solo e o conhecimento das suas interações com outros componentes da biodiversidade, além de benefícios econômicos e estratégicos, como a descoberta de microrganismos potencialmente exploráveis nos processos biotecnológicos. Metabólitos secundários, como antibióticos e toxinas microbianas, podem ser considerados como produtos naturais de importância ecológica. Muitos metabólitos secundários produzidos por fungos e bactérias desenvolvem atividades multifuncionais através da via não-ribossomal, pelas enzimas peptídeo sintetase e policetídeo sintase, que são enzimas multidomínio e podem estar envolvidas na produção de sideróforos e antibióticos. Este estudo teve como objetivo a busca por peptídeos não-ribossômicos produzidos por bactérias isoladas de TPA e suas caracterizações bioquímica e molecular. Através de cultivo, 150 isolados foram selecionados de TPA para análise taxonômica por amplificação e seqüenciamento do gene 16S rRNA, após análise de restrição por enzima de restrição para se conhecer o polimorfismo genético dos isolados. De acordo com o seqüenciamento pôde-se agrupar esses isolados em 17 grupos. Os resultados indicaram a presença dos gêneros Pseudomonas, Bacillus, Arthrobacter, Janthinobacterium, Staphylococcus e Massili. Metabólitos extracelulares foram extraídos dos cultivos bacterianos, usando acetato de etila e clorofórmio, seguidos pela concentração dos extratos, para determinação da capacidade antimicrobiana e a produção de sideróforo foi avaliada usando-se cromoazurol S (CAS). Os resultados mostraram que treze dos 17 isolados apresentaram genes NRPS ou PKS, ou para ambos os genes, associados à produção de peptídeos não-ribossômicos. De todos os isolados estudados, 97% apresentaram produção de sideróforos e algumas espécies dos gêneros Bacillus, Pseudomonas, Arthrobacter e Janthinobacterium mostraram positivas em inibir o crescimento de cinco bactérias testes. A produção de sideróforos do tipo hidroxamato foi positiva para 8 isolados e 7 isolados para o tipo catecol, sendo que 2 isolados não apresentaram reação. Os resultados obtidos em espectrometria de massas Q-TOF indicaram a presença do antibiótico fenazina para o isolado Pseudomonas putida BCM 20, considerado de importância no controle biológico de diversas bactérias patogênicas. / The Anthropogenic Dark Earth (ADE) occurs mainly in archeological sites in the Amazon region. These soils from anthropic origin were enriched with nutrients, probably by the management of organic residues and fire produced by pre-Colombian populations. In ADE, the presence of stable organic matter and high biological activity indicate that this type of soil can be a site of a highly microbial diversity, appointing as a source of microbial germplasm. However, at the present time there is little knowledge about its biology, and especially the structure and diversity of the microbial communities from these soils. The knowledge about microbial diversity will promote a better understanding of the function promoted by the soil microbial community and the knowledge of its interaction with components from biodiversity, likewise the economical and strategic benefits, as for example the discovery of potential microorganisms for exploring biotechnological processes. Secondary metabolites, like antibiotics and microbial toxins, may be considered as natural products of ecological importance. Many of the secondary metabolites produced by fungi and bacteria develop multifunctional activities via non-ribosomal for peptide enzymes and polypeptide synthesis, that are multi-domain enzymes and can be involved in the production of siderophore and antibiotics. This study had the objective of searching for non-ribosomal peptides produced by bacteria isolated from ADE and its biochemical and molecular characterization. Through cultivation in selective media, 150 strains were isolated from ADE for taxonomic analysis by amplification and sequencing of the 16S rRNA gene, after the restriction analysis by restriction enzyme for knowing the genetic strains polymorphism. From the sequencing, it was possible to select these strains in 17 groups, with predominant genera - Pseudomonas, Bacillus, Arthrobacter, Janthinobacterium, Staphylococcus and Massili. Extracellular metabolites were extracted from the cultivated bacteria, using ethylacetate and chloroform, followed by detection of antimicrobial metabolites from bacterial supernatants. Siderophore production was evaluated using ChromoBlue S (CAS). The results from the strains representing the 17 groups showed that at least thirteen strains presented NRPS or PKS gene, or for both genes, associated to the production of non-ribosomal peptides. From all the studied strains, it was found that 97% presented the production of siderophore and that several species from the genera Bacillus, Pseudomonas, Arthrobacter and Janthinobacterium proved to be positive on bacterial growth inhibition. The production of hydroxamate-type siderophore was positive to eight strains and seven strains for the cathecol type, and two strains did not present any reaction. The results obtained from the Q-TOF mass spectrometry confirmed the production of 6-hydroxyphenazine-1-carboxylic acid from the isolated identified as Pseudomonas putida BCM 20. This compound belongs to a fully studied group of antibiotics for biological control against several pathogenic bacteria.

O patógeno Xanthomonas axonopodis pv. passiflorae causa a mancha oleosa ou bacteriose do maracujazeiro, uma doença que acarreta prejuízos à cultura em decorrência da baixa produção de frutos, podendo causar a morte das plantas. Uma coleção de 87 isolados deste patovar, oriundos de 22 cidades dos Estados de São Paulo, Minas Gerais e Paraná e do Distrito Federal, foi usada para estudar a diversidade genética por rep-PCR e AFLP. Nove isolados de outros patovares foram incluídos nas análises genéticas. A técnica rep-PCR revelou pouca diversidade entre os isolados do patovar passiflorae, mas diferenciou, claramente, os diferentes patovares. Todavia, a técnica AFLP revelou considerável diversidade genética entre os isolados do patovar passiflorae. A análise molecular da variância mostrou que a maior parte da diversidade (49,4%) se encontra entre as cidades de coleta. O agrupamento gerado com base nos coeficientes de similaridade e os resultados do teste de atribuição pelo programa Structure revelaram clusters genotípicos homogêneos. Isto evidencia que a variação está mais associada à geografia, ou seja, às cidades de coleta, e que o fluxo desses isolados é pequeno. Cinco conjuntos de primers foram desenhados para a detecção do patógeno em plantas. Desses, um conjunto de primers foi desenhado a partir da seqüência intergênica 16S-23S rRNA e se mostrou específico para o patovar passiflorae. Nenhum amplicon foi detectado nos patovares controles. O restante dos primers foi desenhado a partir do seqüenciamento de locos de AFLP, monomórficos para o patovar passiflorae e ausentes nos demais patovares. Estes primers não foram totalmente específicos; no entanto, todos podem ser recomendados para o diagnóstico da mancha oleosa, uma vez que não há registros de outras Xanthomonas em pomares de maracujá. / The pathogen Xanthomonas axonopodis pv. passiflorae is responsible for the bacterial leaf spot of passion fruits, a disease that provokes commercial losses due to low levels of fruit production and even plant death. A group of 87 isolates of this pathovar, collected from 22 localities of São Paulo, Minas Gerais and Paraná States as in the Federal District was used to evaluate the genetic diversity based on rep-PCR and AFLP. Isolates from other nine pathovars were included in the genetic analyses. Low level of genetic diversity was revealed by the rep- PCR technique, which clearly distinguished the different pathovars. However, considerable diversity between isolates of the pathovar passiflorae was revealed by the AFLP technique. The analysis of molecular variance showed that differences between localities contributed to most part of the variance (49.4%). Groups generated based on similarity coefficients as well as results produced by the software Structure assigning isolates to groups, revealed homogeneous genotypic clusters. This confirms that variance is associated with geographic origin e.g. sampling localities, and that flow of isolates is restricted among localities. Five primer sets were designed for pathogen detection in plants; a primer set was designed for PCR amplification of the intergenic sequence 16-23S rRNA, which was shown to be specific to the pathovar passiflorae. No amplicons were detected in the controls. The remaining primers were designed after sequencing AFLP bands that were monomorphic within the pathovar passiflorae but absent in the other pathovars. These primers were not absolutely specific but all could be recommended for diagnosis of leaf spot as there is no report on the occurrence of other Xanthomonas species in passion fruit orchards.

<p>BACKGROUND: Abetalipoproteinemia (ABL) is a rare autosomal recessive disorder caused by mutations in the gene coding for microsomal triglyceride transfer protein (MTTP).</p><p>AIM: To characterize the genetic basis of ABL in two unrelated patients.</p><p>RESULTS: In the first patient, the substitution c.1911C>T in exon 12 of the <em>MTTP</em> gene, resulting in the protein substitution p.P552L, was discovered using mutation screening. The parents are heterozygous and the proband is a homozygous carrier of this substitution. Using restriction fragment length polymorphism (RFLP), 100 control subjects were analyzed and none carried the substitution indicating that it is a novel <em>MTTP </em>mutation. Sequencing of the other ABL patient showed that the proband carried a homozygous single base insertion, at position  c.2342IVS16+2-3insT, located at the donor splice-site of intron 16 resulting in skipping of exon 16 and truncation of the protein. The proband's mother is heterozygous for the insertion while the father does not carry the insertion. Multiplex ligation-dependent probe amplification (MLPA) did not identify any deletion encompassing exon 16 in the proband, father or mother. Nonpaternity was excluded using polymorphic markers from several chromosomes. Haplotype analysis using markers spanning chromosome 4 revealed  heterodisomy (two homologous chromosomes) of 4p and the distal part of 4q, and isodisomy (duplication of one chromosome) of 4q12-4q26.</p><p>CONCLUSION: These data show that the cause of ABL in one of the patients is a missense mutation, p.P552L, while the cause of ABL in the other patient is due to uniparental disomy, probably resulting from non-disjunstion in meiosis I.</p>

<p>The objective of this study was to develop and validate a six Non-CODIS miniSTR genotyping system and to determine its suitability for forensic casework in South Africa. In Non-CODIS miniSTR genotyping systems, smaller PCR products are amplified and the primers are positioned as close as possible to the repeat region. For this reason, these systems can be valuable in a variety of scenarios including complex paternity cases, missing persons work, and mass fatality disasters.</p>

BACKGROUND: Abetalipoproteinemia (ABL) is a rare autosomal recessive disorder caused by mutations in the gene coding for microsomal triglyceride transfer protein (MTTP). AIM: To characterize the genetic basis of ABL in two unrelated patients. RESULTS: In the first patient, the substitution c.1911C&gt;T in exon 12 of the MTTP gene, resulting in the protein substitution p.P552L, was discovered using mutation screening. The parents are heterozygous and the proband is a homozygous carrier of this substitution. Using restriction fragment length polymorphism (RFLP), 100 control subjects were analyzed and none carried the substitution indicating that it is a novel MTTP mutation. Sequencing of the other ABL patient showed that the proband carried a homozygous single base insertion, at position  c.2342IVS16+2-3insT, located at the donor splice-site of intron 16 resulting in skipping of exon 16 and truncation of the protein. The proband's mother is heterozygous for the insertion while the father does not carry the insertion. Multiplex ligation-dependent probe amplification (MLPA) did not identify any deletion encompassing exon 16 in the proband, father or mother. Nonpaternity was excluded using polymorphic markers from several chromosomes. Haplotype analysis using markers spanning chromosome 4 revealed  heterodisomy (two homologous chromosomes) of 4p and the distal part of 4q, and isodisomy (duplication of one chromosome) of 4q12-4q26. CONCLUSION: These data show that the cause of ABL in one of the patients is a missense mutation, p.P552L, while the cause of ABL in the other patient is due to uniparental disomy, probably resulting from non-disjunstion in meiosis I.

The imprinting status of the SLC22A18AS gene, located in the human chromosome segment 11p15.5, was studied using PCR-SSCP analysis and fetal tissues from a battery of 17 abortuses. This gene showed monoallelic expression (genomic imprinting) in different tissues from two abortuses which were heterozygous for an SNP (c.473G>A) in its coding region. This gene was found to be paternally imprinted (maternally expressed) in five tissues namely lung, liver, brain, kidney and placenta from an abortus. The parental origin of the expressed allele could not be determined in the second abortus as both the mother and the abortus were heterozygous for the SNP. Since paternal blood samples from none of the 17 abortuses could be collected for DNA isolation, the mother's genotype was used to find the origin of the expressed allele. In order to understand the mechanism underlying imprinting of this gene, it was important to understand the nature of the epigenetic marks (imprints) on the two alleles of this gene. Since these epigenetic marks are generally observed in promoters or CpG islands associated with the imprinted genes, the promoters of the SLC22A18AS gene was characterized using transient transfection of putative SLC22A18AS promoter fragments cloned in the pGL3-Basic vector in human cells followed by luciferase reporter assay. Since the promoter of a gene lies upstream to the transcription start site (TSS), TSS of this gene was mapped. In silico approach revealed an EST (CB129046) which had an additional 39 bases upstream to the known mRNA sequence. TSS was then identified by the 5’ primer extension analysis. TSS was found to be 166 bases upstream to the 5’ end of this EST. In order to select cell lines for transient transfection of putative promoter constructs for promoter charaterization, RT-PCR analysis was used to see the expression of this gene in the following available cell lines in the lab: HuH7, HepG2, A549, HeLa, LNCaP and PC3. This gene was found to be maximally expressed in HepG2 cells. Expression of this gene was also observed in A549, HeLa, LNCaP and PC3 cells. HuH7, on the other hand, did not show any detectable expression of this gene. Based on the above data, HepG2 and A549 cells were selected for promoter characterization. Seven putative promoter constructs were transiently transfected in these cells and the promoter activity of different constructs was measured by luciferase assay. The assay identified two promoters for the gene: P1 promoter in a region from -855 to -254 bp and P2 promoter in a region from -1441 to -855. In order to see the presence of putative transcription factor binding sites in the upstream region of the gene, the MatInspector Professional program was used. The gene was found to be devoid of TATA and CCAAT boxes. Most of the putative transcription factor binding sites were present in a region from -855 to -254 bp which spans the P1 promoter, including a binding site for the Sp1 transcription factor. In order to see if Sp1 binds to the promoter of this gene, ChIP assay was performed. Sp1 was shown to bind the region harboring the P1 promoter. In order to see if Sp1 has a role in the regulation of this gene, Sp1 constructs were co-transfected with the SLC22A18AS P1 promoter construct in HepG2 and Sp1-null Drosophila SL2 cells. The results showed that the Sp1 has a positive regulation on the SLC22A18AS promoter activity. As stated earlier, epigenetic marks such as differential methylation of CpG dinucleotides in two alleles are associated with promoters of the genes. Since the promoters for SLC22A18AS were characterized, the presence of allele-specific differentially methylated regions (DMRs) associated with the promoters was investigated. In order to differentiate the two alleles in the promoter regions by SNPs, DNA sequence analysis of the promoter regions was performed in a battery of 17 abortuses to search for SNPs. Abortus no. 3 showed heterozygosity for a C to A change at nucleotide position -445 in the P1 promoter region, while abortus no. 2 showed heterozygosities for G to A and A to G changes at nucleotide positions -919 and -1321 respectively in the P2 promoter region. The alleles in the abortus no. 3 were designated as allele C and allele A. The alleles in abortus no. 2 were designated as allele GG and allele AA. Once the two alleles were differentiated by these SNPs, identification of DMRs was performed using sodium bisulfite genomic DNA sequencing. Genomic DNA from the abortus no. 3 was taken for the identification of DMR in the P1 promoter region, while genomic DNA from abortus no. 2 was taken for the identification of DMR in the P2 promoter region. Sodium bisulfite genomic DNA sequencing of the P1 promoter region showed heavy methylation of both the alleles. No DMR was observed in this region. Sodium bisulfite genomic DNA sequencing of the P2 promoter region using DNA from abortus no. 2 did not show any differential methylation of the two alleles. However, like the P1 promoter region, the P2 promoter region was also heavily methylated. In order to see the methylation status of both the promoter regions in human sperms, sperm DNA from an unrelated healthy volunteer was also subjected to sodium bisulfite genomic sequencing. A dense methylation was observed in both the promoter regions of the gene. Heavy methylation of CpG dinucleotides in these regions corroborates the imprinting result for this gene. Since the methylation epigenetic mark is also known to be associated with CpG islands, CpG Plot/CpG Report analysis was used to identify CpG islands in this gene. The analysis showed the presence of two CpG islands, CpG I and CpG II, in the second intron of the gene. As the CpG I island is known to lack methylated CpGs (Ali et al., unpublished result from our lab), a DMR was sought for the CpG II island region. Heterozygosity was ascertained in this region by sequencing DNA from 17 abortuses. However, none of the abortuses showed heterozygosity. It was reasoned that if there is a differential methylation of the two alleles in this region, half of the clones (alleles) should be unmethylated, and the other half should show methylation. Therefore, DNA from abortus no. 3 was randomly chosen for sodium bisulfite genomic sequence anaylsis to identify DMR. The CpG II island showed heavy methylation. However, a DMR was not identified. In order to see the methylation status of the CpG II island in human sperms, sperm DNA from an unrelated healthy volunteer was also subjected to sodium bisulfite genomic sequencing. Almost all the CpG sites showed methylation. The observation of a dense methylation of both the promoters and CpG II island suggested that methylation has a role in the expression of this gene. In order to confirm this observation, A549 and HuH7 cells were treated with a methyltransferase inhibitor, 5-aza-2’-deoxycytidine. 5-Aza-2’-deoxycytidine treatment in HuH7 cells restored the expression of this gene. Further, the expression of this gene was increased in A549 cells following the drug treatment. These results suggested that DNA methylation has a definite role in the modulation of expression of the SLC22A18AS gene. Histone acetylation is another key epigenetic player which is known to have a role in the expression of genes. In order to study the role of the histone acetylation, HuH7 and A549 cells were treated with TSA, a histone deacetylase inhibitor. Treatment of HuH7 and A549 cells with TSA didn’t have any effect on the expression of this gene. On the other hand, the expression of TPA, a gene shown to be regulated by TSA earlier, increased following the TSA treatment in both cell lines. These results suggested that histone acetylation doesn’t have any effect on the expression of this gene. Based on this observation, it was reasoned that histone acetylation is not associated with the imprinting of this gene. Therefore, we did not look for the allele-specific acetylation of histones in this gene. The SLC22A18AS gene has a weak ORF of 253 amino acids as the translation intiation site does not contain a consensus Kozak sequence for efficient translation. In order to determine if it codes for a protein, Western blot analysis was performed using lysates from A549 cells and human fetal liver tissue, and a polyclonal antibody raised in a rabbit against a bacterially expressed SLC22A18AS protein fragment from amino acids 138 to 245. The Western blot result was negative. It was reasoned that this gene might be expressed at a low level and therefore its expression could not be detected by Western blot analysis. Immunoprecipitation was then performed to enrich the SLC22A18AS protein in the lysates followed by Western blot analysis. SLC22A18AS was shown to be expressed as a 30 kDa band in the immunoprecipiates from A549 cell and human fetal liver tissue lysates. The subcellular localization of this gene was studied by immunofluorescence. The fluorescence immunolocalization was performed on A549 cells with anti-SLC22A18AS antibody. The SLC22A18AS protein was found to be localized in the cytoplasm of A549 cells.

This thesis is about the use of massive DNA sequencing to investigate the transcriptome. During recent decades, several studies have made it clear that the transcriptome comprises a more complex set of biochemical machinery than was previously believed. The majority of the genome can be expressed as transcripts; and overlapping and antisense transcription is widespread. New technologies for the interroga- tion of nucleic acids have made it possible to investigate such cellular phenomena in much greater detail than ever before. For each application, special requirements need to be met. The work presented in this thesis focuses on the transcrip- tome and the development of technology for its analysis. In paper I, we report our development of an automated approach for sample preparation. The procedure was benchmarked against a publicly available reference data set, and we note that our approach outperformed similar manual procedures in terms of reproducibility. In the work reported in papers II-IV, we used different massive sequencing technologies to investigate the transcriptome. In paper II we describe a concatemerization approach that increased throughput by 65% using 454 sequencing,and we identify classes of transcripts not previously described in Populus. Papers III and IV both report studies based on SOLiD sequencing. In the former, we investigated transcripts and proteins for 13% of the human gene and detected a massive overlap for the upper 50% transcriptional levels. In the work described in paper IV, we investigated transcription in non-genic regions of the genome and detected expression from a high number of previ- ously unknown loci. / QC 20100723

Human biotherapy (HBT) or fecal transplants have been shown to be an effective treatment for patients with recurrent Clostridium difficile infection (CDI). This study examines the microbial populations present in CDI patients pre- and post-HBT by extracting bacterial DNA from stool samples and performing pyrosequencing of the 16S rRNA gene. We then compared these microbial populations to those of the donors. We examined 19 pairs of patient samples, of which 14 were clinically cured of CDI, and 5 patients were failures. The successful treatment of CDI was associated with an increase in diversity and richness of the patient's fecal microbiome. The majority of those cured showed an increase in the proportion of Firmicutes and decrease in the proportion of Proteobacteria, although varying antibiotic exposure and innate variability between patients was observed. / MSc thesis / NSERC, CIHR, St. Joseph's Healthcare Hamilton