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Biomedical subjects

Paul H Dear

Publications and source records attributed to Paul H Dear.

13 recordsLinked to original sources
PubMed2006

An efficient method for multi-locus molecular haplotyping.

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.

Chromosomes, Human, Pair 21↗
PubMed2006

Use of HAPPY mapping for the higher order assembly of the Tetrahymena genome.

Tetrahymena thermophila is the best studied of the ciliates, a diversified and successful lineage of eukaryotic protists. Mirroring the way in which many metazoans partition their germ line and soma into distinct cell types, ciliates separate germ line and soma into two distinct nuclei in a single cell. The diploid, transcriptionally silent micronucleus undergoes meiosis and fertilization during sexual reproduction and determines the genotype of the progeny; in contrast, the expressed macronucleus contains many copies of hundreds of small chromosomes, determines the cell's phenotype, and is inherited only through vegetative reproduction. Here we demonstrate the power of HAPPY physical mapping to aid the complete assembly of T. thermophila macronuclear chromosomes from shotgun sequence scaffolds. The finished genome, one of only two ciliate genomes shotgun sequenced, will shed valuable additional light upon the biology of this extraordinary, diverse, and, from a genomics standpoint, as yet largely unexplored evolutionary branch of eukaryotes.

Animals↗
PubMed2006

Interrogation of genomes by molecular copy-number counting (MCC).

Human cancers and some congenital traits are characterized by cytogenetic aberrations including translocations, amplifications, duplications or deletions that can involve gain or loss of genetic material. We have developed a simple method to precisely delineate such regions with known or cryptic genomic alterations. Molecular copy-number counting (MCC) uses PCR to interrogate miniscule amounts of genomic DNA and allows progressive delineation of DNA content to within a few hundred base pairs of a genomic alteration. As an example, we have located the junctions of a recurrent nonreciprocal translocation between chromosomes 3 and 5 in human renal cell carcinoma, facilitating cloning of the breakpoint without recourse to genomic libraries. The analysis also revealed additional cryptic chromosomal changes close to the translocation junction. MCC is a fast and flexible method for characterizing a wide range of chromosomal aberrations.

Carcinoma, Renal Cell↗
PubMed2005

Multiplex amplification of the mammoth mitochondrial genome and the evolution of Elephantidae.

In studying the genomes of extinct species, two principal limitations are typically the small quantities of endogenous ancient DNA and its degraded condition, even though products of up to 1,600 base pairs (bp) have been amplified in rare cases. Using small overlapping polymerase chain reaction products, longer stretches of sequences or even whole mitochondrial genomes can be reconstructed, but this approach is limited by the number of amplifications that can be performed from rare samples. Thus, even from well-studied Pleistocene species such as mammoths, ground sloths and cave bears, no DNA sequences of more than about 1,000 bp have been reconstructed. Here we report the complete mitochondrial genome sequence of the Pleistocene woolly mammoth Mammuthus primigenius. We used about 200 mg of bone and a new approach that allows the simultaneous retrieval of multiple sequences from small amounts of degraded DNA. Our phylogenetic analyses show that the mammoth was more closely related to the Asian than to the African elephant. However, the divergence of mammoth, African and Asian elephants occurred over a short time, corresponding to only about 7% of the total length of the phylogenetic tree for the three evolutionary lineages.

Africa↗
PubMed2004

The genome of Cryptosporidium hominis.

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.

Animals↗
PubMed2004

Complete genome sequence of the apicomplexan, Cryptosporidium parvum.

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.

Animals↗
PubMed2003

Integrated mapping, chromosomal sequencing and sequence analysis of Cryptosporidium parvum.

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.

Animals↗
PubMed2003

HAPPY mapping in a plant genome: reconstruction and analysis of a high-resolution physical map of a 1.9 Mbp region of Arabidopsis thaliana chromosome 4.

HAPPY mapping is an in vitro approach for defining the order and spacing of DNA markers directly on native genomic DNA. This cloning-free technique is based on analysing the segregation of markers amplified from high molecular weight genomic DNA which has been broken randomly and 'segregated' by limiting dilution into subhaploid samples. It is a uniquely versatile tool, allowing for the construction of genome maps with flexible ranges and resolutions. Moreover, it is applicable to plant genomes, for which many of the techniques pioneered in animal genomes are inapplicable or inappropriate. We report here its demonstration in a plant genome by reconstructing the physical map of a 1.9 Mbp region around the FCA locus of Arabidopsis thaliana. The resulting map, spanning around 10% of chromosome 4, is in excellent agreement with the DNA sequence and has a mean marker spacing of 16 kbp. We argue that HAPPY maps of any required resolution can be made immediately and with relatively little effort for most plant species and, furthermore, that such maps can greatly aid the construction of regional or genome-wide physical maps.

Journal Article↗
PubMed2003

One by one: Single molecule tools for genomics.

Much of the effort in any genomics programme arises from the need to generate and purify large numbers of identical molecules, since most analytical tools rely on the analysis of bulk DNA. Biological steps such as bacterial cloning--commonly used to prepare bulk samples of defined DNA fragments--are capricious and introduce their own restrictions and distortions. The analysis of single molecules, either directly or by in vitro enzymatic amplification, makes possible the examination of native genomic DNA without the complications and restrictions of biological propagation. Techniques already exist for the in vitro propagation of genomic fragments and for genome mapping, and offer the advantages of speed, flexibility and predictable behaviour. Single molecule sequencing, for which many approaches are being developed, is more challenging, but offers even greater rewards in terms of throughput and read length.

Cloning, Molecular↗
PubMed2003

Detection and genotyping by real-time PCR/RFLP analyses of Giardia duodenalis from human faeces.

A nested PCR assay (TPILC-PCR) was developed to detect and distinguish between Giardia duodenalis assemblages A and B from human faeces by analysis of the triose phosphate isomerase gene (tpi). The assay comprised an initial multiplexed block-based amplification. This was followed by two separate real-time PCR assays specific for assemblages A and B using a LightCycler and SYBR Green I to identify PCR products by melting-point analysis. RFLP analysis was applied to distinguish G. duodenalis assemblage A groups I and II. The real-time nested PCR was evaluated using DNA extracted from purified giardial trophozoites, Cryptosporidium oocysts, whole faeces containing a range of potential pathogens (including G. duodenalis), faecal smears and bacterial suspensions. The assay was specific, sensitive, reproducible and rapid.

Animals↗
PubMed2002

Sequence and analysis of chromosome 2 of Dictyostelium discoideum.

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.

Animals↗