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Single gene disorders are individually rare but collectively common leading causes of neonatal and pediatric morbidity and mortality. Both parents or the mothers of affected individuals with autosomal recessive or X-linked recessive diseases, respectively, are carrier(s). Carrier frequencies of recessive diseases can vary drastically among different ethnicities. This study established a robust pipeline for estimating and ranking carrier frequencies of all known 2699 recessive genes based on genome-wide sequencing data in healthy individuals. The discovery gnomAD cohort contained sequencing data on 76,156 genomes and 125,748 exomes from individuals with seven ethnicity backgrounds. The three validation cohorts composed of the SG10K Project with 4810 genomes on East Asian and South Asian, the ChinaMAP project with 10,588 Chinese genomes, and the WBBC pilot project with 4480 Chinese genomes. Within each cohort, comprehensive selection criteria for various kinds of deleterious variants were instituted, including known pathogenic variants (Type 1), presumably loss-of-function changes (Type 2), predicted deleterious missense variants (Type 3), and potentially harmful in-frame INDELs (Type 4). Subsequently, carrier frequencies of the 2699 genes were calculated and ranked based on ethnicity-specific carrier rates of Type 1 to Type 4 variants. Comparison of results from different cohorts with similar ethnicity background exhibited high degree of correlation, particularly between the ChinaMAP and the WBBC cohorts (Pearson correlation coefficient R = 0.92), confirming the validity of our variant selection criteria and the overall analysis pipeline.
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Alzheimer's disease (AD) is a leading cause of mortality in the elderly. While the coding change of APOE-ε4 is a key risk factor for late-onset AD and has been believed to be the only risk factor in the APOE locus, it does not fully explain the risk effect conferred by the locus. Here, we report the identification of AD causal variants in PVRL2 and APOC1 regions in proximity to APOE and define common risk haplotypes independent of APOE-ε4 coding change. These risk haplotypes are associated with changes of AD-related endophenotypes including cognitive performance, and altered expression of APOE and its nearby genes in the human brain and blood. High-throughput genome-wide chromosome conformation capture analysis further supports the roles of these risk haplotypes in modulating chromatin states and gene expression in the brain. Our findings provide compelling evidence for additional risk factors in the APOE locus that contribute to AD pathogenesis.
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Enfermedad de Alzheimer/genética , Apolipoproteínas E/genética , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/psicología , Apolipoproteínas C/genética , Apolipoproteínas C/metabolismo , Apolipoproteínas E/metabolismo , Encéfalo/metabolismo , Estudios de Casos y Controles , Cognición , Femenino , Expresión Génica , Predisposición Genética a la Enfermedad , Variación Genética , Haplotipos , Humanos , Masculino , Persona de Mediana Edad , Nectinas/genética , Nectinas/metabolismo , Polimorfismo de Nucleótido SimpleRESUMEN
Osteogenesis imperfecta (OI) is an inherited disorder with osteoporosis and recurrent fractures. Children presenting with recurrent fractures and bowing of limbs have severe form of the disorder. Patients carrying homozygous WNT1 mutations have more frequent fractures while heterozygous carriers of the mutation in WNT1 gene are also found to have early onset osteoporosis. We identified a family with novel WNT1 mutation. The index case, a 6 month old child presented with fractures from early infancy. Next generation sequencing (NGS)done for the child didn't show any variations in other OI genes including COL1A1, COL1A2, SERPINH1, CRTAP, LEPRE1, PP1B, 1F1TM5 and BMP1 genes. Sanger sequencing showed 41bp deletion in splice region following exon 1 of WNT1 gene in homozygous state. The mutation was found to be likely pathogenic on bioinformatic analysis. To further characterize the significance of the mutation we studied his mother who is 30 year old with blue sclera and history of backache but no fractures. Her DXA scan of lumber spine showed osteoporosis and she was heterozygous for the mutation. The child's DXA scan showed T-score of -6.4â¯at lumbar spine level. Father also has history of backache and was carrier for the same deletion variant. The child was given 3 doses of zoledronate and did not have any further fractures. Thus, we conclude that this novel variant identified in the child with OI is likely cause for the disease and possibly zoledronate has a role in prevention of fractures in this case.
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Fracturas Óseas/genética , Osteogénesis Imperfecta/genética , Proteína Wnt1/genética , Conservadores de la Densidad Ósea/uso terapéutico , Difosfonatos/uso terapéutico , Variación Genética , Humanos , Imidazoles/uso terapéutico , Lactante , Masculino , Mutación , Osteogénesis Imperfecta/tratamiento farmacológico , Ácido ZoledrónicoRESUMEN
Joubert syndrome and related disorders (JSRD; ORPHA 140874) is a complex set of neurodevelopmental disorders with multiple organ involvement. JSRD is a type of ciliopathy which is caused by the presence of defective primary cilia in an individual. JSRD is commonly inherited in an autosomal recessive pattern, and more than 23 genes are known to be associated with JSRD. We report a novel homozygous mutation identified in the INPP5E gene, c.1303C>T, which leads to a change of an amino acid from arginine to tryptophan at residue 435 in the protein chain. In silico analysis indicates that p.Arg435Trp substitution affects the functionality of the protein product of the gene. Our result adds to the growing body of evidences that underlines the clinical utility of next-generation sequencing in the diagnosis of a genetic disorder when clinical features are inconclusive.
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Aberrant cytosine 5-methylation underlies many deregulated elements of cancer. Among paired non-small cell lung cancers (NSCLC), we sought to profile DNA 5-methyl-cytosine features which may underlie genome-wide deregulation. In one of the more dense interrogations of the methylome, we sampled 1.2 million CpG sites from twenty-four NSCLC tumor (T)-non-tumor (NT) pairs using a methylation-sensitive restriction enzyme- based HELP-microarray assay. We found 225,350 differentially methylated (DM) sites in adenocarcinomas versus adjacent non-tumor tissue that vary in frequency across genomic compartment, particularly notable in gene bodies (GB; p<2.2E-16). Further, when DM was coupled to differential transcriptome (DE) in the same samples, 37,056 differential loci in adenocarcinoma emerged. Approximately 90% of the DM-DE relationships were non-canonical; for example, promoter DM associated with DE in the same direction. Of the canonical changes noted, promoter (PR) DM loci with reciprocal changes in expression in adenocarcinomas included HBEGF, AGER, PTPRM, DPT, CST1, MELK; DM GB loci with concordant changes in expression included FOXM1, FERMT1, SLC7A5, and FAP genes. IPA analyses showed adenocarcinoma-specific promoter DMxDE overlay identified familiar lung cancer nodes [tP53, Akt] as well as less familiar nodes [HBEGF, NQO1, GRK5, VWF, HPGD, CDH5, CTNNAL1, PTPN13, DACH1, SMAD6, LAMA3, AR]. The unique findings from this study include the discovery of numerous candidate The unique findings from this study include the discovery of numerous candidate methylation sites in both PR and GB regions not previously identified in NSCLC, and many non-canonical relationships to gene expression. These DNA methylation features could potentially be developed as risk or diagnostic biomarkers, or as candidate targets for newer methylation locus-targeted preventive or therapeutic agents.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Metilación de ADN , Estudio de Asociación del Genoma Completo/métodos , Neoplasias Pulmonares/genética , Regiones Promotoras Genéticas , Islas de CpG , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodosRESUMEN
There is limited data on the clinical, cellular and molecular changes in relapsed acute promyeloytic leukemia (RAPL) in comparison with newly diagnosed cases (NAPL). We undertook a prospective study to compare NAPL and RAPL patients treated with arsenic trioxide (ATO) based regimens. 98 NAPL and 28 RAPL were enrolled in this study. RAPL patients had a significantly lower WBC count and higher platelet count at diagnosis. IC bleeds was significantly lower in RAPL cases (P=0.022). The ability of malignant promyelocytes to concentrate ATO intracellularly and their in-vitro IC50 to ATO was not significantly different between the two groups. Targeted NGS revealed PML B2 domain mutations in 4 (15.38%) of the RAPL subset and none were associated with secondary resistance to ATO. A microarray GEP revealed 1744 genes were 2 fold and above differentially expressed between the two groups. The most prominent differentially regulated pathways were cell adhesion (n=92), cell survival (n=50), immune regulation (n=74) and stem cell regulation (n=51). Consistent with the GEP data, immunophenotyping revealed significantly increased CD34 expression (P=0.001) in RAPL cases and there was in-vitro evidence of significant microenvironment mediated innate resistance (EM-DR) to ATO. Resistance and relapse following treatment with ATO is probably multi-factorial, mutations in PML B2 domain while seen only in RAPL may not be the major clinically relevant cause of subsequent relapses. In RAPL additional factors such as expansion of the leukemia initiating compartment along with EM-DR may contribute significantly to relapse following treatment with ATO based regimens.
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Antineoplásicos/uso terapéutico , Arsenicales/uso terapéutico , Leucemia Promielocítica Aguda/tratamiento farmacológico , Óxidos/uso terapéutico , Adolescente , Adulto , Antineoplásicos/sangre , Trióxido de Arsénico , Arsenicales/sangre , Línea Celular Tumoral , Niño , Preescolar , Resistencia a Antineoplásicos/genética , Femenino , Perfilación de la Expresión Génica , Células Precursoras de Granulocitos/efectos de los fármacos , Células Precursoras de Granulocitos/patología , Células Precursoras de Granulocitos/fisiología , Humanos , Leucemia Promielocítica Aguda/sangre , Leucemia Promielocítica Aguda/diagnóstico , Masculino , Persona de Mediana Edad , Mutación , Proteínas Nucleares/genética , Óxidos/sangre , Proteína de la Leucemia Promielocítica , Estudios Prospectivos , Recurrencia , Factores de Transcripción/genética , Tretinoina/uso terapéutico , Proteínas Supresoras de Tumor/genética , Adulto JovenRESUMEN
We evaluated the importance of tumor cell selection for generating gene signatures in non-small cell lung cancer. Tumor and nontumor tissue from macroscopically dissected (Macro) surgical specimens (31 pairs from 32 subjects) was homogenized, extracted, amplified, and hybridized to microarrays. Adjacent scout sections were histologically mapped; sets of approximately 1000 tumor cells and nontumor cells (alveolar or bronchial) were procured by laser capture microdissection (LCM). Within histological strata, LCM and Macro specimens exhibited approximately 67% to 80% nonoverlap in differentially expressed (DE) genes. In a representative subset, LCM uniquely identified 300 DE genes in tumor versus nontumor specimens, largely attributable to cell selection; 382 DE genes were common to Macro, Macro with preamplification, and LCM platforms. RT-qPCR validation in a 33-gene subset was confirmatory (ρ = 0.789 to 0.964, P = 0.0013 to 0.0028). Pathway analysis of LCM data suggested alterations in known cancer pathways (cell growth, death, movement, cycle, and signaling components), among others (eg, immune, inflammatory). A unique nine-gene LCM signature had higher tumor-nontumor discriminatory accuracy (100%) than the corresponding Macro signature (87%). Comparison with Cancer Genome Atlas data sets (based on homogenized Macro tissue) revealed both substantial overlap and important differences from LCM specimen results. Thus, cell selection via LCM enhances expression profiling precision, and confirms both known and under-appreciated lung cancer genes and pathways.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Transcriptoma , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Femenino , Humanos , Captura por Microdisección con Láser , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana EdadRESUMEN
Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.
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Cryptococcus neoformans/genética , Genoma Fúngico/genética , ARN de Hongos/genética , Transcriptoma/genética , Virulencia/genética , Cromosomas Fúngicos/genética , ADN de Hongos/genética , Intrones/genéticaRESUMEN
Helminths include both parasitic nematodes (roundworms) and platyhelminths (trematode and cestode flatworms) that are abundant, and are of clinical importance. The genetic characterization of parasitic flatworms using advanced molecular tools is central to the diagnosis and control of infections. Although the nuclear genome houses suitable genetic markers (e.g., in ribosomal (r) DNA) for species identification and molecular characterization, the mitochondrial (mt) genome consistently provides a rich source of novel markers for informative systematics and epidemiological studies. In the last decade, there have been some important advances in mtDNA genomics of helminths, especially lung flukes, liver flukes and intestinal flukes. Fasciolopsis buski, often called the giant intestinal fluke, is one of the largest digenean trematodes infecting humans and found primarily in Asia, in particular the Indian subcontinent. Next-generation sequencing (NGS) technologies now provide opportunities for high throughput sequencing, assembly and annotation within a short span of time. Herein, we describe a high-throughput sequencing and bioinformatics pipeline for mt genomics for F. buski that emphasizes the utility of short read NGS platforms such as Ion Torrent and Illumina in successfully sequencing and assembling the mt genome using innovative approaches for PCR primer design as well as assembly. We took advantage of our NGS whole genome sequence data (unpublished so far) for F. buski and its comparison with available data for the Fasciola hepatica mtDNA as the reference genome for design of precise and specific primers for amplification of mt genome sequences from F. buski. A long-range PCR was carried out to create an NGS library enriched in mt DNA sequences. Two different NGS platforms were employed for complete sequencing, assembly and annotation of the F. buski mt genome. The complete mt genome sequences of the intestinal fluke comprise 14,118 bp and is thus the shortest trematode mitochondrial genome sequenced to date. The noncoding control regions are separated into two parts by the tRNA-Gly gene and don't contain either tandem repeats or secondary structures, which are typical for trematode control regions. The gene content and arrangement are identical to that of F. hepatica. The F. buski mtDNA genome has a close resemblance with F. hepatica and has a similar gene order tallying with that of other trematodes. The mtDNA for the intestinal fluke is reported herein for the first time by our group that would help investigate Fasciolidae taxonomy and systematics with the aid of mtDNA NGS data. More so, it would serve as a resource for comparative mitochondrial genomics and systematic studies of trematode parasites.
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BACKGROUND: Phyto-remedies for diabetic control are popular among patients with Type II Diabetes mellitus (DM), in addition to other diabetic control measures. A number of plant species are known to possess diabetic control properties. Costus pictus D. Don is popularly known as "Insulin Plant" in Southern India whose leaves have been reported to increase insulin pools in blood plasma. Next Generation Sequencing is employed as a powerful tool for identifying molecular signatures in the transcriptome related to physiological functions of plant tissues. We sequenced the leaf transcriptome of C. pictus using Illumina reversible dye terminator sequencing technology and used combination of bioinformatics tools for identifying transcripts related to anti-diabetic properties of C. pictus. RESULTS: A total of 55,006 transcripts were identified, of which 69.15% transcripts could be annotated. We identified transcripts related to pathways of bixin biosynthesis and geraniol and geranial biosynthesis as major transcripts from the class of isoprenoid secondary metabolites and validated the presence of putative norbixin methyltransferase, a precursor of Bixin. The transcripts encoding these terpenoids are known to be Peroxisome Proliferator-Activated Receptor (PPAR) agonists and anti-glycation agents. Sequential extraction and High Performance Liquid Chromatography (HPLC) confirmed the presence of bixin in C. pictus methanolic extracts. Another significant transcript identified in relation to anti-diabetic, anti-obesity and immuno-modulation is of Abscisic Acid biosynthetic pathway. We also report many other transcripts for the biosynthesis of antitumor, anti-oxidant and antimicrobial metabolites of C. pictus leaves. CONCLUSION: Solid molecular signatures (transcripts related to bixin, abscisic acid, and geranial and geraniol biosynthesis) for the anti-diabetic properties of C. pictus leaves and vital clues related to the other phytochemical functions like antitumor, anti-oxidant, immuno-modulatory, anti-microbial and anti-malarial properties through the secondary metabolite pathway annotations are reported. The data provided will be of immense help to researchers working in the treatment of DM using herbal therapies.
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Costus/genética , Genes de Plantas , Hipoglucemiantes/metabolismo , Hojas de la Planta/genética , Transcriptoma , Ácido Abscísico/metabolismo , Monoterpenos Acíclicos , Secuencia de Bases , Carotenoides/metabolismo , Cromatografía Líquida de Alta Presión , Biología Computacional , Costus/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Metiltransferasas/metabolismo , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Monoterpenos/metabolismo , Fitoterapia , Extractos Vegetales/química , Hojas de la Planta/metabolismo , Análisis de Secuencia de ADN , Terpenos/metabolismoRESUMEN
Extracellular signals in development, physiology, homeostasis and disease often act by regulating transcription. Herein we describe a general method and specific resources for determining where and when such signaling occurs in live animals and for systematically comparing the timing and extent of different signals in different cellular contexts. We used recombinase-mediated cassette exchange (RMCE) to test the effect of successively deleting conserved genomic regions of the ubiquitously active Rosa26 promoter and substituting the deleted regions for regulatory sequences that respond to diverse extracellular signals. We thereby created an allelic series of embryonic stem cells and mice, each containing a signal-responsive sentinel with different fluorescent reporters that respond with sensitivity and specificity to retinoic acids, bone morphogenic proteins, activin A, Wnts or Notch, and that can be adapted to any pathway that acts via DNA elements.
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Células Madre Embrionarias/metabolismo , Mutación/genética , Regiones Promotoras Genéticas , Transducción de Señal/genética , Transcripción Genética , Activinas/genética , Activinas/metabolismo , Animales , Secuencia de Bases , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/efectos de los fármacos , Ingeniería Genética , Sitios Genéticos/genética , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas/genética , ARN no Traducido , Ratas , Receptores Notch/genética , Receptores Notch/metabolismo , Recombinación Genética/genética , Elementos de Respuesta/genética , Eliminación de Secuencia/genética , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Tretinoina/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genéticaRESUMEN
PURPOSE OF REVIEW: Chronic obstructive pulmonary disease (COPD) and lung cancer are the leading causes of morbidity and mortality worldwide. The current research is focused on identifying the common and disparate events involved in epigenetic modifications that concurrently occur during the pathogenesis of COPD and lung cancer. The purpose of this review is to describe the current knowledge and understanding of epigenetic modifications in pathogenesis of COPD and lung cancer. RECENT FINDINGS: This review provides an update on advances of how epigenetic modifications are linked to COPD and lung cancer, and their commonalities and disparities. The key epigenetic modification enzymes (e.g. DNA methyltransferases -- CpG methylation, histone acetylases/deacetylases and histone methyltransferases/demethylases) that are identified to play an important role in COPD and lung tumorigenesis and progression are described in this review. SUMMARY: Distinct DNA methyltransferases and histone modification enzymes are differentially involved in pathogenesis of lung cancer and COPD, although some of the modifications are common. Understanding the epigenetic modifications involved in pathogenesis of lung cancer or COPD with respect to common and disparate mechanisms will lead to targeting of epigenetic therapies against these disorders.
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Epigenómica , Neoplasias Pulmonares/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , ADN (Citosina-5-)-Metiltransferasas/genética , ADN-Citosina Metilasas/genética , Histona Acetiltransferasas/genética , Histona Desacetilasas/genética , Histona Demetilasas/genética , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/genética , HumanosRESUMEN
BACKGROUND: Toxoplasma gondii is a member of the phylum Apicomplexa, which consists entirely of parasitic organisms that cause several diseases of veterinary and human importance. Fundamental mechanisms of gene regulation in this group of protistan parasites remain largely uncharacterized. Owing to their medical and veterinary importance, genome sequences are available for several apicomplexan parasites. Their genome sequences reveal an apparent paucity of known transcription factors and the absence of canonical cis-regulatory elements. We have approached the question of gene regulation from a sequence perspective by mining the genomic sequence data to identify putative cis-regulatory elements using a de novo approach. RESULTS: We have identified putative cis-regulatory elements present upstream of functionally related groups of genes and subsequently characterized the function of some of these conserved elements using reporter assays in the parasite. We show a sequence-specific role in gene-expression for seven out of eight identified elements. CONCLUSIONS: This work demonstrates the power of pure sequence analysis in the absence of expression data or a priori knowledge of regulatory elements in eukaryotic organisms with compact genomes.
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Secuencias Reguladoras de Ácidos Nucleicos/genética , Toxoplasma/genética , Algoritmos , Animales , Bases de Datos Genéticas , Regulación de la Expresión Génica , Genoma de Protozoos , Glucólisis/genética , Luciferasas/genética , Luciferasas/metabolismo , Mutación , Regiones Promotoras Genéticas/genética , Proteínas Protozoarias/genética , Proteínas Ribosómicas/genética , TransfecciónRESUMEN
BACKGROUND: Cryptosporidium parvum is a unicellular eukaryote in the phylum Apicomplexa. It is an obligate intracellular parasite that causes diarrhea and is a significant AIDS-related pathogen. Cryptosporidium parvum is not amenable to long-term laboratory cultivation or classical molecular genetic analysis. The parasite exhibits a complex life cycle, a broad host range, and fundamental mechanisms of gene regulation remain unknown. We have used data from the recently sequenced genome of this organism to uncover clues about gene regulation in C. parvum. We have applied two pattern finding algorithms MEME and AlignACE to identify conserved, over-represented motifs in the 5' upstream regions of genes in C. parvum. To support our findings, we have established comparative real-time -PCR expression profiles for the groups of genes examined computationally. RESULTS: We find that groups of genes that share a function or belong to a common pathway share upstream motifs. Different motifs are conserved upstream of different groups of genes. Comparative real-time PCR studies show co-expression of genes within each group (in sub-sets) during the life cycle of the parasite, suggesting co-regulation of these genes may be driven by the use of conserved upstream motifs. CONCLUSION: This is one of the first attempts to characterize cis-regulatory elements in the absence of any previously characterized elements and with very limited expression data (seven genes only). Using de novo pattern finding algorithms, we have identified specific DNA motifs that are conserved upstream of genes belonging to the same metabolic pathway or gene family. We have demonstrated the co-expression of these genes (often in subsets) using comparative real-time-PCR experiments thus establishing evidence for these conserved motifs as putative cis-regulatory elements. Given the lack of prior information concerning expression patterns and organization of promoters in C. parvum we present one of the first investigations of gene regulation in this important human pathogen.
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Cryptosporidium parvum/genética , Perfilación de la Expresión Génica/métodos , Genoma de Protozoos , Genómica/métodos , Elementos Reguladores de la Transcripción/genética , Algoritmos , Animales , Cryptosporidium parvum/metabolismo , Reconocimiento de Normas Patrones Automatizadas , Reacción en Cadena de la PolimerasaRESUMEN
GCN5 is a histone acetyltransferase (HAT) essential for development in mammals and critical to stress responses in yeast. The protozoan parasite Toxoplasma gondii is a serious opportunistic pathogen. The study of epigenetics and gene expression in this ancient eukaryote has pharmacological relevance and may facilitate the understanding of these processes in higher eukaryotes. Here we show that the disruption of T. gondii GCN5 yields viable parasites, which were subsequently employed in a proteomics study to identify gene products affected by its loss. Promoter analysis of these TgGCN5-dependent genes, which were mostly parasite specific, reveals a conserved T-rich element. The loss of TgGCN5 does not attenuate virulence in an in vivo mouse model. We also discovered that T. gondii is the only invertebrate reported to date possessing a second GCN5 (TgGCN5-B). TgGCN5-B harbors a strikingly divergent N-terminal domain required for nuclear localization. Despite high homology between the HAT domains, the two TgGCN5s exhibit differing substrate specificities. In contrast to TgGCN5-A, which exclusively targets lysine 18 of H3, TgGCN5-B acetylates multiple lysines in the H3 tail. We also identify two ADA2 homologues that interact differently with the TgGCN5s. TgGCN5-B has the potential to compensate for TgGCN5-A, which probably arose from a gene duplication unique to T. gondii. Our work reveals an unexpected complexity in the GCN5 machinery of this primitive eukaryote.
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Adenosina Desaminasa/metabolismo , Histona Acetiltransferasas/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/enzimología , Acetilación , Adenosina Desaminasa/química , Adenosina Desaminasa/genética , Secuencia de Aminoácidos , Animales , Núcleo Celular/metabolismo , Exones/genética , Regulación de la Expresión Génica , Histona Acetiltransferasas/química , Histona Acetiltransferasas/genética , Intrones/genética , Modelos Biológicos , Datos de Secuencia Molecular , Mutación/genética , Señales de Localización Nuclear , Regiones Promotoras Genéticas/genética , Unión Proteica , Transporte de Proteínas , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Toxoplasma/citologíaRESUMEN
BACKGROUND: The apicomplexan parasite Cryptosporidium parvum is an emerging pathogen capable of causing illness in humans and other animals and death in immunocompromised individuals. No effective treatment is available and the genome sequence has recently been completed. This parasite differs from other apicomplexans in its lack of a plastid organelle, the apicoplast. Gene transfer, either intracellular from an endosymbiont/donor organelle or horizontal from another organism, can provide evidence of a previous endosymbiotic relationship and/or alter the genetic repertoire of the host organism. Given the importance of gene transfers in eukaryotic evolution and the potential implications for chemotherapy, it is important to identify the complement of transferred genes in Cryptosporidium. RESULTS: We have identified 31 genes of likely plastid/endosymbiont (n = 7) or prokaryotic (n = 24) origin using a phylogenomic approach. The findings support the hypothesis that Cryptosporidium evolved from a plastid-containing lineage and subsequently lost its apicoplast during evolution. Expression analyses of candidate genes of algal and eubacterial origin show that these genes are expressed and developmentally regulated during the life cycle of C. parvum. CONCLUSIONS: Cryptosporidium is the recipient of a large number of transferred genes, many of which are not shared by other apicomplexan parasites. Genes transferred from distant phylogenetic sources, such as eubacteria, may be potential targets for therapeutic drugs owing to their phylogenetic distance or the lack of homologs in the host. The successful integration and expression of the transferred genes in this genome has changed the genetic and metabolic repertoire of the parasite.
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Cryptosporidium parvum/genética , Transferencia de Gen Horizontal/genética , Genoma de Protozoos , Filogenia , Simbiosis/genética , Enzima Ramificadora de 1,4-alfa-Glucano/genética , Animales , Eucariontes/genética , Evolución Molecular , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/genética , Genes/genética , Genes Bacterianos/genéticaRESUMEN
Reports of plant-like and bacterial-like genes for a number of parasitic organisms, most notably those within the Apicomplexa and Kinetoplastida, have appeared in the literature over the last few years. Among the apicomplexan organisms, following discovery of the apicomplexan plastid (apicoplast), the discovery of plant-like genes was less surprising although the extent of transfer and the relationship of transferred genes to the apicoplast remained unclear. We used new genome sequence data to begin a systematic examination of the extent and origin of transferred genes in the Apicomplexa combined with a phylogenomic approach to detect potential gene transfers in four apicomplexan genomes. We have detected genes of algal nuclear, chloroplast (cyanobacterial) and proteobacterial origin. Plant-like genes were detected in species not currently harbouring a plastid (e.g. Cryptosporidium parvum) and putatively transferred genes were detected that appear to be unrelated to the function of the apicoplast. While the mechanism of acquisition for many of the identified genes is not certain, it appears that some were most likely acquired via intracellular gene transfer from an algal endosymbiont while others may have been acquired via horizontal gene transfer.