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1.
Eukaryot Cell ; 10(9): 1257-63, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21803864

RESUMO

Coordinated regulation of gene expression is a hallmark of the Plasmodium falciparum asexual blood-stage development cycle. We report that carbon catabolite repressor protein 4 (CCR4)-associated factor 1 (CAF1) is critical in regulating more than 1,000 genes during malaria parasites' intraerythrocytic stages, especially egress and invasion proteins. CAF1 knockout results in mistimed expression, aberrant accumulation and localization of proteins involved in parasite egress, and invasion of new host cells, leading to premature release of predominantly half-finished merozoites, drastically reducing the intraerythrocytic growth rate of the parasite. This study demonstrates that CAF1 of the CCR4-Not complex is a significant gene regulatory mechanism needed for Plasmodium development within the human host.


Assuntos
Eritrócitos/parasitologia , Deleção de Genes , Expressão Gênica , Interações Hospedeiro-Parasita/genética , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidade , Fatores de Transcrição/genética , Animais , Proliferação de Células , Eritrócitos/patologia , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Estágios do Ciclo de Vida , Malária Falciparum/parasitologia , Merozoítos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Plasmodium falciparum/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo
2.
BMC Microbiol ; 9: 83, 2009 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-19422698

RESUMO

BACKGROUND: Much of the Plasmodium falciparum genome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of the Plasmodium genome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of the Plasmodium genome. RESULTS: In this study, we investigated the lepidopteran transposon, piggyBac, as a molecular genetic tool for functional characterization of the Plasmodium falciparum genome. Through multiple transfections, we generated 177 unique P. falciparum mutant clones with mostly single piggyBac insertions in their genomes. Analysis of piggyBac insertion sites revealed random insertions into the P. falciparum genome, in regards to gene expression in parasite life cycle stages and functional categories. We further explored the possibility of forward genetic studies in P. falciparum with a phenotypic screen for attenuated growth, which identified several parasite genes and pathways critical for intra-erythrocytic development. CONCLUSION: Our results clearly demonstrate that piggyBac is a novel, indispensable tool for forward functional genomics in P. falciparum that will help better understand parasite biology and accelerate drug and vaccine development.


Assuntos
Elementos de DNA Transponíveis , Genoma de Protozoário , Genômica/métodos , Plasmodium falciparum/genética , Animais , Mutagênese Insercional , Plasmídeos , Plasmodium falciparum/crescimento & desenvolvimento , Transfecção
3.
Neurosci Lett ; 392(1-2): 68-71, 2006 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-16183199

RESUMO

Schizophrenia is a complex multifactorial disorder for which the pathobiology still remains elusive. Dysfunction of the dopamine D2 receptor signaling has been associated with the illness, but numerous studies provide confounding results. This study investigates the association of synonymous polymorphisms (His313 and Pro319) in the dopamine D2 receptor gene with schizophrenia using a case-control approach, with 101 cases and 145 controls. Our results demonstrated that genotype distribution for the His313 polymorphism was significantly different between schizophrenia patients and control subjects (p=0.0012), while the Pro319 polymorphism did not show any association with the disease. The results suggest that the synonymous SNP His313 in DRD2 may be associated with the illness. However, there is a need for further replication studies with larger sample sets.


Assuntos
Predisposição Genética para Doença , Variação Genética , Receptores de Dopamina D2/genética , Esquizofrenia/genética , Adolescente , Adulto , Idade de Início , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Feminino , Frequência do Gene , Genótipo , Histidina/genética , Humanos , Masculino , Polimorfismo Genético , Escalas de Graduação Psiquiátrica , Valina/genética
4.
Biol Psychiatry ; 55(2): 196-9, 2004 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-14732601

RESUMO

BACKGROUND: Chromosome 22q is one of the important regions repeatedly being implicated in schizophrenia. In this region, our group previously reported an association of a CAG repeat marker (22CH3) with schizophrenia in the Indian population. Because Synaptogyrin 1 (SYNGR1), associated with presynaptic vesicles in neuronal cells, lies within 1 million base pairs of this marker, it is a potential candidate gene for schizophrenia. METHODS: We sequenced all six exons and flanking splice junctions of the SYNGR1 gene. We also carried out reverse transcriptase polymerase chain reaction and Northern blot analysis for exon 2 containing transcript of the SYNGR1 gene. RESULTS: We found a novel nonsense mutation (Trp27Ter) in exon 2 of the SYNGR1 gene in a family multiply affected with schizophrenia. Reverse transcriptase polymerase chain reaction and Northern blot analyses revealed that exon 2 containing transcript of this gene is expressed in the brain. CONCLUSIONS: Because the SYNGR1 gene is involved in presynaptic pathways, reduced levels of this protein might play some role in the pathogenesis of schizophrenia.


Assuntos
Códon sem Sentido , Saúde da Família , Proteínas do Tecido Nervoso/genética , Monoéster Fosfórico Hidrolases/genética , Esquizofrenia/genética , Northern Blotting/métodos , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Análise Mutacional de DNA , Éxons , Feminino , Humanos , Masculino , Biologia Molecular/métodos , Linhagem , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Treonina/genética , Triptofano/genética
5.
J Biomol Struct Dyn ; 20(2): 253-63, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12354077

RESUMO

Expansion of GAA repeats in the intron of the frataxin gene is involved in the autosomal recessive Friedreich's ataxia (FRDA). The GAA repeats arise from a stretch of adenine residues of an Alu element. These repeats have a size ranging from 7- 38 in the normal population, and expand to thousands in the affected individuals. The mechanism of origin of GAA repeats, their polymorphism and stability are not well understood. In this study, we have carried out an extensive analysis of GAA repeats at several loci in the humans. This analysis indicates the association of a majority of GAA repeats with the 3' end of an "A" stretch present in the Alu repeats. Further, the prevalence of GAA repeats correlates with the evolutionary age of Alu subfamilies as well as with their relative frequency in the genome. Our study on GAA repeat polymorphism at some loci in the normal population reveals that the length of the GAA repeats is determined by the relative length of the flanking A stretch. Based on these observations, a possible mechanism for origin of GAA repeats and modulatory effects of flanking sequences on repeat instability mediated by DNA triplex is proposed.


Assuntos
Elementos Alu/genética , Repetições de Trinucleotídeos/genética , Região 3'-Flanqueadora , Adenina/química , Algoritmos , Alelos , Cromossomos Humanos Par 22 , Bases de Dados Genéticas , Evolução Molecular , Ataxia de Friedreich/epidemiologia , Ataxia de Friedreich/genética , Frequência do Gene , Genes Recessivos , Genoma Humano , Humanos , Íntrons , Proteínas de Ligação ao Ferro/genética , Modelos Genéticos , Mutação , Polimorfismo Genético , Prevalência , Expansão das Repetições de Trinucleotídeos/genética , Frataxina
6.
PLoS One ; 7(10): e47350, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23077596

RESUMO

BACKGROUND: Aedes aegypti is the primary mosquito vector for dengue virus (DENV) worldwide. Infectivity of dengue virus varies among natural populations of this mosquito. How A. aegypti responds to DENV infection relative to which genes and associated pathways contribute to its differential susceptibility as a vector is not well defined. METHODS/PRINCIPAL FINDINGS: Here, we used custom cDNA microarrays to identify groups of genes that were differentially expressed in midgut tissues between susceptible and refractory strains in a highly time specific manner. While genes involved in protein processing in the endoplasmic reticulum, mRNA surveillance, and the proteasome were significantly up-regulated in the susceptible strain, several metabolic processes including glycolysis, glycan biosynthesis and Wnt pathway were active in the refractory strain. In addition, several key signaling genes were expressed as common responsive genes in both susceptible and refractory mosquitoes that may be necessary for signal transduction to trigger the appropriate host response to the viral infection. These are coordinately expressed in the form of tight gene networks and expression clusters that may be necessary to differentially contribute to the progression of dengue infection between the two strains. CONCLUSIONS: Our data show that highly correlated differential expression of responsive genes throughout the post infection period in A. aegypti midgut tissues is necessary for a coordinated transcriptional response of the mosquito genes to host or defend the viral infection.


Assuntos
Aedes/virologia , Vírus da Dengue/metabolismo , Dengue/genética , Perfilação da Expressão Gênica , Animais , Dengue/transmissão , Dengue/virologia , Vírus da Dengue/genética , Sistema Digestório/metabolismo , Sistema Digestório/virologia , Interações Hospedeiro-Patógeno/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais
7.
Mol Biochem Parasitol ; 176(1): 37-41, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21111761

RESUMO

Previous studies of Brugia malayi promoters have suggested that they are unusual in that they lack the CAAT or TATAA boxes that are often emblematic of eucaryotic core promoter domains. Instead, the region surrounding the spliced leader (SL) addition site appears to function as the core promoter domain in B. malayi. To test the hypothesis that polymorphisms in this SL addition domain are important determinants of promoter activity, a series of domain swap mutants were prepared replacing the SL addition domain of the B. malayi 13kDa large subunit ribosomal protein (BmRPL13) with those of other ribosomal protein (RP) promoters exhibiting a wide range of activities. These constructs were then tested for promoter activity in a homologous transient transfection system. On average, polymorphisms in the SL addition domain were found to be responsible for 80% of the variation in promoter activity exhibited by the RP promoters tested. Essentially all of this effect could be attributable to polymorphisms in the 10nt located directly upstream of the SL addition site. A comparison of the sequence of this domain to the promoter activity exhibited by the domain swap mutants suggested that promoter activity was related to the number of T residues present in the coding strand of the upstream domain. Confirming this, mutation of the upstream domain of the promoter of the BmRPS4 gene to a homogeneous stretch of 10 T residues resulted in a significant increase in promoter activity.


Assuntos
Brugia Malayi/genética , Brugia Malayi/metabolismo , Polimorfismo Genético , Regiões Promotoras Genéticas , RNA Líder para Processamento/genética , RNA Líder para Processamento/metabolismo , Animais , Sequência de Bases , Regulação da Expressão Gênica , Mutagênese Sítio-Dirigida , Proteínas Ribossômicas/genética
8.
Mol Biochem Parasitol ; 169(2): 115-9, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19852985

RESUMO

A 7-nt motif (the trans-splicing motif or TSM) was previously shown to be necessary and sufficient to direct trans-splicing of transgenic mRNAs in transgenic Brugia malayi embryos. Insertion of the TSM into two genes lacking a TSM homologue resulted in trans-splicing of transgenic mRNAs from one transgene but not the other, suggesting that local sequence context might affect TSM function. To test this hypothesis, constructs inserting the TSM into different positions of two B. malayi genes were tested for their ability to support trans-splicing of transgenic mRNAs. Transgenic mRNAs derived from constructs in which the insertion of the TSM did not result in a perturbation of the local predicted secondary structure were trans-spliced, while those in which the TSM perturbed the local secondary structure were not. These data suggest that local secondary structure plays a role in the ability of the TSM to direct trans-splicing.


Assuntos
Brugia Malayi/genética , Conformação de Ácido Nucleico , RNA de Helmintos/química , RNA de Helmintos/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Trans-Splicing , Animais , Animais Geneticamente Modificados , Brugia Malayi/metabolismo , Engenharia Genética/métodos , Modelos Moleculares , Biologia Molecular/métodos , Mutagênese Insercional
9.
Int J Parasitol ; 40(1): 63-71, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19631652

RESUMO

Operons are a common mode of gene organization in Caenorhabditis elegans. Similar gene arrangements suggest that functional operons may exist in Brugia malayi. To definitively test this hypothesis, a bicistronic reporter vector consisting of an upstream firefly luciferase gene and a downstream renilla luciferase gene was constructed. The genome was then surveyed to identify 15 gene pairs that were likely to represent operons. Two of four domains upstream of the 5' gene from these clusters exhibited promoter activity. When constructs replicating the promoter and intergenic arrangement found in the native putative operon were transfected into embryos, both firefly and renilla activities were detected, while constructs with the promoter alone or intergenic region alone produced no activity from the downstream reporter. These data confirm that functional operons exist in B. malayi. Mutation of three U-rich element homologues present in one of the operons resulted in a decrease in downstream renilla reporter activity, suggesting that these were important in mRNA maturation. Hemi-nested reverse transcriptase-PCR assays demonstrated that while the mRNA encoding the native downstream open reading frame of one operon contained an SL1 spliced leader at its 5' end, the renilla gene mRNA produced from the corresponding transgenic construct did not.


Assuntos
Brugia Malayi/genética , Genoma , Família Multigênica , Óperon , Animais , Brugia Malayi/metabolismo , Filariose/parasitologia , Genes Reporter , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Luciferases/genética , Luciferases/metabolismo , Óperon/fisiologia , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trans-Splicing , Transfecção
10.
Mol Biochem Parasitol ; 166(1): 15-21, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19428668

RESUMO

Two promoters from the human filarial parasite Brugia malayi have been mapped in detail. The essential domains of both promoters lacked canonical eukaryotic core promoter motifs. However, the largest contiguous essential domain in both promoters flanked and included the splice leader addition site. These findings suggested that the region flanking the trans-splicing addition site might represent a conserved core domain in B. malayi promoters. To test this hypothesis, the putative promoters of 12 trans-spliced genes encoding ribosomal protein homologues from B. malayi were isolated and tested for activity in a B. malayi transient transfection system. Of the 12 domains examined, 11 produced detectable reporter gene activity. Mutant constructs of the six most active promoters were prepared in which the spliced leader acceptor site and the 10 nt upstream and downstream of the site were deleted. All deletion constructs exhibited >90% reduction in reporter gene activity relative to their respective wild type sequences. A conserved pyrimidine-rich tract was located directly upstream from the spliced leader splice acceptor site which contained a conserved T residue located at position -3. Mutation of the entire polypyrimidine tract or the conserved T individually resulted in the loss of over 90% of reporter gene activity. In contrast, mutation of the splice acceptor site did not significantly reduce promoter activity. These data suggest that the region surrounding the splice acceptor site in the ribosomal promoters represents a conserved essential domain which functions independently of splice leader addition.


Assuntos
Brugia Malayi/genética , Brugia Malayi/metabolismo , Sequência Conservada/genética , Regulação da Expressão Gênica , RNA Líder para Processamento/metabolismo , Motivos de Aminoácidos , Animais , Sequência de Bases/genética , Embrião não Mamífero , Regiões Promotoras Genéticas/genética , Alinhamento de Sequência , Deleção de Sequência
11.
Pharmacogenomics ; 10(2): 277-91, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19207030

RESUMO

AIM: We investigated 16 polymorphisms from three genes, dopamine receptor D2 (DRD2), catechol-O-methyl transferase (COMT) and brain derived neurotrophic factor (BDNF), which are involved in the dopaminergic pathways, and have been reported to be associated with susceptibility to schizophrenia and response to antipsychotic therapy. MATERIALS & METHODS: Single-locus association analyses of these polymorphisms were carried out in 254 patients with schizophrenia and 225 controls, all of southern Indian origin. Additionally, multifactor-dimensionality reduction analysis was performed in 422 samples (243 cases and 179 controls) to examine the gene-gene interactions and to identify combinations of multilocus genotypes associated with either high or low risk for the disease. RESULTS: Our results demonstrated initial significant associations of two SNPs for DRD2 (rs11608185, genotype: chi(2) = 6.29, p-value = 0.043; rs6275, genotype: chi(2) = 8.91, p-value = 0.011), and one SNP in the COMT gene (rs4680, genotype: chi(2) = 6.67, p-value = 0.035 and allele: chi(2) = 4.75, p-value = 0.029; odds ratio: 1.33, 95% confidence interval: 1.02-1.73), but not after correction for multiple comparisons indicating a weak association of individual markers of DRD2 and COMT with schizophrenia. Multifactor-dimensionality reduction analysis suggested a two locus model (rs6275/DRD2 and rs4680/COMT) as the best model for gene-gene interaction with 90% cross-validation consistency and 42.42% prediction error in predicting disease risk among schizophrenia patients. CONCLUSION: The present study thus emphasizes the need for multigene interaction studies in complex disorders such as schizophrenia and to understand response to drug treatment, which could lead to a targeted and more effective treatment.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Catecol O-Metiltransferase/genética , Predisposição Genética para Doença , Variação Genética , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único , Receptores de Dopamina D3/genética , Esquizofrenia/genética , Adulto , Substituição de Aminoácidos , Estudos de Casos e Controles , Éxons , Feminino , Humanos , Índia , Masculino , Valores de Referência , Adulto Jovem
12.
Pharmacogenomics ; 10(3): 385-97, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19290789

RESUMO

AIM: We investigated the catechol-O-methyltrasferase (COMT) gene, which is a strong functional and positional candidate gene for schizophrenia and therapeutic response to antipsychotic medication. MATERIALS & METHODS: Single-locus as well as detailed haplotype-based association analysis of the COMT gene with schizophrenia and antipsychotic treatment response was carried out using seven COMT polymorphisms in 398 schizophrenia patients and 241 healthy individuals from a homogeneous south Indian population. Further responsiveness to risperidone treatment was assessed in 117 schizophrenia patients using Clinical Global Impressions (CGI). A total of 69 patients with a CGI score of 2 or less met the criteria of good responders and 48 were patients who continued to have a score of 3 and above and were classified as poor responders to risperidone treatment. RESULTS: The association of SNP rs4680 with schizophrenia did not remain significant after adjusting for multiple testing. Haplotype analysis showed highly significant association of seven COMT marker haplotypes with schizophrenia (CLUMP T4 p-value = 0.0001). Our results also demonstrated initial significant allelic associations of two SNPs with drug response (rs4633: chi(2) = 4.36, p-value = 0.036, OR: 1.80, 95% CI: 1.03-3.15; and rs4680: chi(2) = 4.02, p-value = 0.044, OR: 1.76, 95% CI: 1.01-3.06) before multiple correction. We employed two-marker sliding window analysis for haplotype association and observed a significant association of markers located between intron 1 and intron 2 (rs737865, rs6269: CLUMP T4 p-value = 0.021); and in exon 4 (rs4818, rs4680: CLUMP T4 p-value = 0.028) with drug response. CONCLUSION: The present study thus indicates that the interacting effects within the COMT gene polymorphisms may influence the disease status and response to risperidone in schizophrenia patients. However, the study needs to be replicated in a larger sample set for confirmation, followed by functional studies.


Assuntos
Antipsicóticos/uso terapêutico , Catecol O-Metiltransferase/genética , Polimorfismo de Nucleotídeo Único , Risperidona/uso terapêutico , Esquizofrenia/tratamento farmacológico , Esquizofrenia/genética , Adulto , Estudos de Casos e Controles , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/genética , Síndrome de DiGeorge/genética , Feminino , Frequência do Gene , Marcadores Genéticos , Genótipo , Humanos , Masculino , Valores de Referência , Esquizofrenia/enzimologia , Deleção de Sequência , Adulto Jovem
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