RESUMEN
RATIONALE: Amplification of distal 3q is the most common genomic aberration in squamous lung cancer (SQC). SQC develops in a multistage progression from normal bronchial epithelium through dysplasia to invasive disease. Identifying the key driver events in the early pathogenesis of SQC will facilitate the search for predictive molecular biomarkers and the identification of novel molecular targets for chemoprevention and therapeutic strategies. For technical reasons, previous attempts to analyze 3q amplification in preinvasive lesions have focused on small numbers of predetermined candidate loci rather than an unbiased survey of copy-number variation. OBJECTIVES: To perform a detailed analysis of the 3q amplicon in bronchial dysplasia of different histological grades. METHODS: We use molecular copy-number counting (MCC) to analyze the structure of chromosome 3 in 19 preinvasive bronchial biopsy specimens from 15 patients and sequential biopsy specimens from 3 individuals. MEASUREMENTS AND MAIN RESULTS: We demonstrate that no low-grade lesions, but all high-grade lesions, have 3q amplification. None of seven low-grade lesions progressed clinically, whereas 8 of 10 patients with high-grade disease progressed to cancer. We identify a minimum commonly amplified region on chromosome 3 consisting of 17 genes, including 2 known oncogenes, SOX2 and PIK3CA. We confirm that both genes are amplified in all high-grade dysplastic lesions tested. We further demonstrate, in three individuals, that the clinical progression of high-grade preinvasive disease is associated with incremental amplification of SOX2, suggesting this promotes malignant progression. CONCLUSIONS: These findings demonstrate progressive 3q amplification in the evolution of preinvasive SQC and implicate SOX2 as a key target of this dynamic process.
Asunto(s)
Cromosomas Humanos Par 3/genética , Amplificación de Genes/fisiología , Neoplasias de Células Escamosas/genética , Lesiones Precancerosas/genética , Factores de Transcripción SOXB1/genética , Anciano , Neoplasias de los Bronquios/genética , Neoplasias de los Bronquios/patología , Neoplasias de los Bronquios/fisiopatología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Invasividad Neoplásica , Neoplasias de Células Escamosas/fisiopatología , Lesiones Precancerosas/clasificación , Lesiones Precancerosas/patologíaRESUMEN
Eimeria tenella is an intracellular protozoan parasite that infects the intestinal tracts of domestic fowl and causes coccidiosis, a serious and sometimes lethal enteritis. Eimeria falls in the same phylum (Apicomplexa) as several human and animal parasites such as Cryptosporidium, Toxoplasma, and the malaria parasite, Plasmodium. Here we report the sequencing and analysis of the first chromosome of E. tenella, a chromosome believed to carry loci associated with drug resistance and known to differ between virulent and attenuated strains of the parasite. The chromosome--which appears to be representative of the genome--is gene-dense and rich in simple-sequence repeats, many of which appear to give rise to repetitive amino acid tracts in the predicted proteins. Most striking is the segmentation of the chromosome into repeat-rich regions peppered with transposon-like elements and telomere-like repeats, alternating with repeat-free regions. Predicted genes differ in character between the two types of segment, and the repeat-rich regions appear to be associated with strain-to-strain variation.
Asunto(s)
Estructuras Cromosómicas/genética , Eimeria tenella/genética , Genes Protozoarios/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Biología Computacional , Repeticiones de Minisatélite/genética , Datos de Secuencia Molecular , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADNRESUMEN
Many methods exist for genotyping--revealing which alleles an individual carries at different genetic loci. A harder problem is haplotyping--determining which alleles lie on each of the two homologous chromosomes in a diploid individual. Conventional approaches to haplotyping require the use of several generations to reconstruct haplotypes within a pedigree, or use statistical methods to estimate the prevalence of different haplotypes in a population. Several molecular haplotyping methods have been proposed, but have been limited to small numbers of loci, usually over short distances. Here we demonstrate a method which allows rapid molecular haplotyping of many loci over long distances. The method requires no more genotypings than pedigree methods, but requires no family material. It relies on a procedure to identify and genotype single DNA molecules, and reconstruction of long haplotypes by a 'tiling' approach. We demonstrate this by resolving haplotypes in two regions of the human genome, harbouring 20 and 105 single-nucleotide polymorphisms, respectively. The method can be extended to reconstruct haplotypes of arbitrary complexity and length, and can make use of a variety of genotyping platforms. We also argue that this method is applicable in situations which are intractable to conventional approaches.
Asunto(s)
Haplotipos , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple , Cromosomas Humanos Par 21 , Cromosomas Humanos X , ADN/química , Interpretación Estadística de Datos , Diploidia , Humanos , Masculino , Análisis de Secuencia de ADNRESUMEN
Cryptosporidium species cause acute gastroenteritis and diarrhoea worldwide. They are members of the Apicomplexa--protozoan pathogens that invade host cells by using a specialized apical complex and are usually transmitted by an invertebrate vector or intermediate host. In contrast to other Apicomplexans, Cryptosporidium is transmitted by ingestion of oocysts and completes its life cycle in a single host. No therapy is available, and control focuses on eliminating oocysts in water supplies. Two species, C. hominis and C. parvum, which differ in host range, genotype and pathogenicity, are most relevant to humans. C. hominis is restricted to humans, whereas C. parvum also infects other mammals. Here we describe the eight-chromosome approximately 9.2-million-base genome of C. hominis. The complement of C. hominis protein-coding genes shows a striking concordance with the requirements imposed by the environmental niches the parasite inhabits. Energy metabolism is largely from glycolysis. Both aerobic and anaerobic metabolisms are available, the former requiring an alternative electron transport system in a simplified mitochondrion. Biosynthesis capabilities are limited, explaining an extensive array of transporters. Evidence of an apicoplast is absent, but genes associated with apical complex organelles are present. C. hominis and C. parvum exhibit very similar gene complements, and phenotypic differences between these parasites must be due to subtle sequence divergence.
Asunto(s)
Cryptosporidium/genética , Genoma de Protozoos , Animales , Cromosomas/genética , Cryptosporidium/clasificación , Cryptosporidium/enzimología , Cryptosporidium/metabolismo , Cryptosporidium parvum/genética , Enzimas/genética , Evolución Molecular , Genes Protozoarios/genética , Genómica , Humanos , Fenotipo , Proteínas Protozoarias/genéticaRESUMEN
The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and animals, and causes an unrelenting infection in immunocompromised individuals such as AIDS patients. We report the complete genome sequence of C. parvum, type II isolate. Genome analysis identifies extremely streamlined metabolic pathways and a reliance on the host for nutrients. In contrast to Plasmodium and Toxoplasma, the parasite lacks an apicoplast and its genome, and possesses a degenerate mitochondrion that has lost its genome. Several novel classes of cell-surface and secreted proteins with a potential role in host interactions and pathogenesis were also detected. Elucidation of the core metabolism, including enzymes with high similarities to bacterial and plant counterparts, opens new avenues for drug development.
Asunto(s)
Cryptosporidium parvum/genética , Cryptosporidium parvum/metabolismo , Enzimas/metabolismo , Genoma de Protozoos , Proteínas Protozoarias/metabolismo , Animales , Antiprotozoarios/farmacología , Metabolismo de los Hidratos de Carbono , Cryptosporidium parvum/patogenicidad , Cryptosporidium parvum/fisiología , ADN Protozoario/genética , Resistencia a Medicamentos/genética , Enzimas/genética , Etanol/metabolismo , Genes Protozoarios , Glucólisis , Intrones , Mitocondrias/genética , Datos de Secuencia Molecular , Familia de Multigenes , Sistemas de Lectura Abierta , Orgánulos/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Purinas/metabolismo , Análisis de Secuencia de ADN , Transcripción GenéticaRESUMEN
The apicomplexan Cryptosporidium parvum is one of the most prevalent protozoan parasites of humans. We report the physical mapping of the genome of the Iowa isolate, sequencing and analysis of chromosome 6, and approximately 0.9 Mbp of sequence sampled from the remainder of the genome. To construct a robust physical map, we devised a novel and general strategy, enabling accurate placement of clones regardless of clone artefacts. Analysis reveals a compact genome, unusually rich in membrane proteins. As in Plasmodium falciparum, the mean size of the predicted proteins is larger than that in other sequenced eukaryotes. We find several predicted proteins of interest as potential therapeutic targets, including one exhibiting similarity to the chloroquine resistance protein of Plasmodium. Coding sequence analysis argues against the conventional phylogenetic position of Cryptosporidium and supports an earlier suggestion that this genus arose from an early branching within the Apicomplexa. In agreement with this, we find no significant synteny and surprisingly little protein similarity with Plasmodium. Finally, we find two unusual and abundant repeats throughout the genome. Among sequenced genomes, one motif is abundant only in C. parvum, whereas the other is shared with (but has previously gone unnoticed in) all known genomes of the Coccidia and Haemosporida. These motifs appear to be unique in their structure, distribution and sequences.
Asunto(s)
Cryptosporidium parvum/genética , Mapeo Físico de Cromosoma/métodos , Análisis de Secuencia de ADN/métodos , Animales , Composición de Base/genética , Centrómero/genética , Criptosporidiosis/diagnóstico , Criptosporidiosis/microbiología , Criptosporidiosis/terapia , Cryptosporidium parvum/aislamiento & purificación , Cryptosporidium parvum/patogenicidad , ADN Protozoario/análisis , Dosificación de Gen , Terapia Genética , Genoma de Protozoos , Datos de Secuencia Molecular , Filogenia , Polimorfismo Genético/genética , Polimorfismo de Nucleótido Simple/genética , Secuencias Repetidas en Tándem/genética , Telómero/genéticaRESUMEN
The genome of the lower eukaryote Dictyostelium discoideum comprises six chromosomes. Here we report the sequence of the largest, chromosome 2, which at 8 megabases (Mb) represents about 25% of the genome. Despite an A + T content of nearly 80%, the chromosome codes for 2,799 predicted protein coding genes and 73 transfer RNA genes. This gene density, about 1 gene per 2.6 kilobases (kb), is surpassed only by Saccharomyces cerevisiae (one per 2 kb) and is similar to that of Schizosaccharomyces pombe (one per 2.5 kb). If we assume that the other chromosomes have a similar gene density, we can expect around 11,000 genes in the D. discoideum genome. A significant number of the genes show higher similarities to genes of vertebrates than to those of other fully sequenced eukaryotes. This analysis strengthens the view that the evolutionary position of D. discoideum is located before the branching of metazoa and fungi but after the divergence of the plant kingdom, placing it close to the base of metazoan evolution.