Comparative analyses of seven algorithms for copy number variant identification from single nucleotide polymorphism arrays.

Determination of copy number variants (CNVs) inferred in genome wide single nucleotide polymorphism arrays has shown increasing utility in genetic variant disease associations. Several CNV detection methods are available, but differences in CNV call thresholds and characteristics exist. We evaluated the relative performance of seven methods: circular binary segmentation, CNVFinder, cnvPartition, gain and loss of DNA, Nexus algorithms, PennCNV and QuantiSNP. Tested data included real and simulated Illumina HumHap 550 data from the Singapore cohort study of the risk factors for Myopia (SCORM) and simulated data from Affymetrix 6.0 and platform-independent distributions. The normalized singleton ratio (NSR) is proposed as a metric for parameter optimization before enacting full analysis. We used 10 SCORM samples for optimizing parameter settings for each method and then evaluated method performance at optimal parameters using 100 SCORM samples. The statistical power, false positive rates, and receiver operating characteristic (ROC) curve residuals were evaluated by simulation studies. Optimal parameters, as determined by NSR and ROC curve residuals, were consistent across datasets. QuantiSNP outperformed other methods based on ROC curve residuals over most datasets. Nexus Rank and SNPRank have low specificity and high power. Nexus Rank calls oversized CNVs. PennCNV detects one of the fewest numbers of CNVs.

Biogeography in deep time - What do phylogenetics, geology, and paleoclimate tell us about early platyrrhine evolution?

Molecular data have converged on a consensus about the genus-level phylogeny of extant platyrrhine monkeys, but for most extinct taxa and certainly for those older than the Pleistocene we must rely upon morphological evidence from fossils. This raises the question as to how well anatomical data mirror molecular phylogenies and how best to deal with discrepancies between the molecular and morphological data as we seek to extend our phylogenies to the placement of fossil taxa. Here I present parsimony-based phylogenetic analyses of extant and fossil platyrrhines based on an anatomical dataset of 399 dental characters and osteological features of the cranium and postcranium. I sample 16 extant taxa (one from each platyrrhine genus) and 20 extinct taxa of platyrrhines. The tree structure is constrained with a "molecular scaffold" of extant species as implemented in maximum parsimony using PAUP with the molecular-based 'backbone' approach. The data set encompasses most of the known extinct species of platyrrhines, ranging in age from latest Oligocene (∼26 Ma) to the Recent. The tree is rooted with extant catarrhines, and Late Eocene and Early Oligocene African anthropoids. Among the more interesting patterns to emerge are: (1) known early platyrrhines from the Late Oligocene through Early Miocene (26-16.5Ma) represent only stem platyrrhine taxa; (2) representatives of the three living platyrrhine families first occur between 15.7 Ma and 13.5 Ma; and (3) recently extinct primates from the Greater Antilles (Cuba, Jamaica, Hispaniola) are sister to the clade of extant platyrrhines and may have diverged in the Early Miocene. It is probable that the crown platyrrhine clade did not originate before about 20-24 Ma, a conclusion consistent with the phylogenetic analysis of fossil taxa presented here and with recent molecular clock estimates. The following biogeographic scenario is consistent with the phylogenetic findings and climatic and geologic evidence: Tropical South America has been a center for platyrrhine diversification since platyrrhines arrived on the continent in the middle Cenozoic. Platyrrhines dispersed from tropical South America to Patagonia at ∼25-24 Ma via a "Paraná Portal" through eastern South America across a retreating Paranense Sea. Phylogenetic bracketing suggests Antillean primates arrived via a sweepstakes route or island chain from northern South America in the Early Miocene, not via a proposed land bridge or island chain (GAARlandia) in the Early Oligocene (∼34 Ma). Patagonian and Antillean platyrrhines went extinct without leaving living descendants, the former at the end of the Early Miocene and the latter within the past six thousand years. Molecular evidence suggests crown platyrrhines arrived in Central America by crossing an intermittent connection through the Isthmus of Panama at or after 3.5Ma. Any more ancient Central American primates, should they be discovered, are unlikely to have given rise to the extant Central American taxa in situ.

The evolutionary history of ferns inferred from 25 low-copy nuclear genes.

UNLABELLED: • PREMISE OF THE STUDY: Understanding fern (monilophyte) phylogeny and its evolutionary timescale is critical for broad investigations of the evolution of land plants, and for providing the point of comparison necessary for studying the evolution of the fern sister group, seed plants. Molecular phylogenetic investigations have revolutionized our understanding of fern phylogeny, however, to date, these studies have relied almost exclusively on plastid data.• METHODS: Here we take a curated phylogenomics approach to infer the first broad fern phylogeny from multiple nuclear loci, by combining broad taxon sampling (73 ferns and 12 outgroup species) with focused character sampling (25 loci comprising 35877 bp), along with rigorous alignment, orthology inference and model selection.• KEY RESULTS: Our phylogeny corroborates some earlier inferences and provides novel insights; in particular, we find strong support for Equisetales as sister to the rest of ferns, Marattiales as sister to leptosporangiate ferns, and Dennstaedtiaceae as sister to the eupolypods. Our divergence-time analyses reveal that divergences among the extant fern orders all occurred prior to ∼200 MYA. Finally, our species-tree inferences are congruent with analyses of concatenated data, but generally with lower support. Those cases where species-tree support values are higher than expected involve relationships that have been supported by smaller plastid datasets, suggesting that deep coalescence may be reducing support from the concatenated nuclear data.• CONCLUSIONS: Our study demonstrates the utility of a curated phylogenomics approach to inferring fern phylogeny, and highlights the need to consider underlying data characteristics, along with data quantity, in phylogenetic studies.

Comparative analyses of clinical and environmental populations of Cryptococcus neoformans in Botswana.

Cryptococcus neoformans var. grubii (Cng) is the most common cause of fungal meningitis, and its prevalence is highest in sub-Saharan Africa. Patients become infected by inhaling airborne spores or desiccated yeast cells from the environment, where the fungus thrives in avian droppings, trees and soil. To investigate the prevalence and population structure of Cng in southern Africa, we analysed isolates from 77 environmental samples and 64 patients. We detected significant genetic diversity among isolates and strong evidence of geographic structure at the local level. High proportions of isolates with the rare MATa allele were observed in both clinical and environmental isolates; however, the mating-type alleles were unevenly distributed among different subpopulations. Nearly equal proportions of the MATa and MATα mating types were observed among all clinical isolates and in one environmental subpopulation from the eastern part of Botswana. As previously reported, there was evidence of both clonality and recombination in different geographic areas. These results provide a foundation for subsequent genomewide association studies to identify genes and genotypes linked to pathogenicity in humans.

Olfactory Receptor Subgenomes Linked with Broad Ecological Adaptations in Sauropsida.

Olfactory receptors (ORs) govern a prime sensory function. Extant birds have distinct olfactory abilities, but the molecular mechanisms underlining diversification and specialization remain mostly unknown. We explored OR diversity in 48 phylogenetic and ecologically diverse birds and 2 reptiles (alligator and green sea turtle). OR subgenomes showed species- and lineage-specific variation related with ecological requirements. Overall 1,953 OR genes were identified in reptiles and 16,503 in birds. The two reptiles had larger OR gene repertoires (989 and 964 genes, respectively) than birds (182-688 genes). Overall, birds had more pseudogenes (7,855) than intact genes (1,944). The alligator had significantly more functional genes than sea turtle, likely because of distinct foraging habits. We found rapid species-specific expansion and positive selection in OR14 (detects hydrophobic compounds) in birds and in OR51 and OR52 (detect hydrophilic compounds) in sea turtle, suggestive of terrestrial and aquatic adaptations, respectively. Ecological partitioning among birds of prey, water birds, land birds, and vocal learners showed that diverse ecological factors determined olfactory ability and influenced corresponding olfactory-receptor subgenome. OR5/8/9 was expanded in predatory birds and alligator, suggesting adaptive specialization for carnivory. OR families 2/13, 51, and 52 were correlated with aquatic adaptations (water birds), OR families 6 and 10 were more pronounced in vocal-learning birds, whereas most specialized land birds had an expanded OR family 14. Olfactory bulb ratio (OBR) and OR gene repertoire were correlated. Birds that forage for prey (carnivores/piscivores) had relatively complex OBR and OR gene repertoires compared with modern birds, including passerines, perhaps due to highly developed cognitive capacities facilitating foraging innovations.

Three crocodilian genomes reveal ancestral patterns of evolution among archosaurs.

To provide context for the diversification of archosaurs--the group that includes crocodilians, dinosaurs, and birds--we generated draft genomes of three crocodilians: Alligator mississippiensis (the American alligator), Crocodylus porosus (the saltwater crocodile), and Gavialis gangeticus (the Indian gharial). We observed an exceptionally slow rate of genome evolution within crocodilians at all levels, including nucleotide substitutions, indels, transposable element content and movement, gene family evolution, and chromosomal synteny. When placed within the context of related taxa including birds and turtles, this suggests that the common ancestor of all of these taxa also exhibited slow genome evolution and that the comparatively rapid evolution is derived in birds. The data also provided the opportunity to analyze heterozygosity in crocodilians, which indicates a likely reduction in population size for all three taxa through the Pleistocene. Finally, these data combined with newly published bird genomes allowed us to reconstruct the partial genome of the common ancestor of archosaurs, thereby providing a tool to investigate the genetic starting material of crocodilians, birds, and dinosaurs.

Whole genome sequencing identifies circulating Beijing-lineage Mycobacterium tuberculosis strains in Guatemala and an associated urban outbreak.

Limited data are available regarding the molecular epidemiology of Mycobacterium tuberculosis (Mtb) strains circulating in Guatemala. Beijing-lineage Mtb strains have gained prevalence worldwide and are associated with increased virulence and drug resistance, but there have been only a few cases reported in Central America. Here we report the first whole genome sequencing of Central American Beijing-lineage strains of Mtb. We find that multiple Beijing-lineage strains, derived from independent founding events, are currently circulating in Guatemala, but overall still represent a relatively small proportion of disease burden. Finally, we identify a specific Beijing-lineage outbreak centered on a poor neighborhood in Guatemala City.

Interrogation of individual intratumoral B lymphocytes from lung cancer patients for molecular target discovery.

Intratumoral B lymphocytes are an integral part of the lung tumor microenvironment. Interrogation of the antibodies they express may improve our understanding of the host response to cancer and could be useful in elucidating novel molecular targets. We used two strategies to explore the repertoire of intratumoral B cell antibodies. First, we cloned VH and VL genes from single intratumoral B lymphocytes isolated from one lung tumor, expressed the genes as recombinant mAbs, and used the mAbs to identify the cognate tumor antigens. The Igs derived from intratumoral B cells demonstrated class switching, with a mean VH mutation frequency of 4%. Although there was no evidence for clonal expansion, these data are consistent with antigen-driven somatic hypermutation. Individual recombinant antibodies were polyreactive, although one clone demonstrated preferential immunoreactivity with tropomyosin 4 (TPM4). We found that higher levels of TPM4 antibodies were more common in cancer patients, but measurement of TPM4 antibody levels was not a sensitive test for detecting cancer. Second, in an effort to focus our recombinant antibody expression efforts on those B cells that displayed evidence of clonal expansion driven by antigen stimulation, we performed deep sequencing of the Ig genes of B cells collected from seven different tumors. Deep sequencing demonstrated somatic hypermutation but no dominant clones. These strategies may be useful for the study of B cell antibody expression, although identification of a dominant clone and unique therapeutic targets may require extensive investigation.

Bottlenose dolphins exchange signature whistles when meeting at sea.

The bottlenose dolphin, Tursiops truncatus, is one of very few animals that, through vocal learning, can invent novel acoustic signals and copy whistles of conspecifics. Furthermore, receivers can extract identity information from the invented part of whistles. In captivity, dolphins use such signature whistles while separated from the rest of their group. However, little is known about how they use them at sea. If signature whistles are the main vehicle to transmit identity information, then dolphins should exchange these whistles in contexts where groups or individuals join. We used passive acoustic localization during focal boat follows to observe signature whistle use in the wild. We found that stereotypic whistle exchanges occurred primarily when groups of dolphins met and joined at sea. A sequence analysis verified that most of the whistles used during joins were signature whistles. Whistle matching or copying was not observed in any of the joins. The data show that signature whistle exchanges are a significant part of a greeting sequence that allows dolphins to identify conspecifics when encountering them in the wild.

Human E2F6 is alternatively spliced to generate multiple protein isoforms

E2F6 protein belongs to the family of the E2F transcription factors. Here, we showed that the human E2F6 gene contains nine exons distributed along 20.4kbp of genomic DNA on chromosome 2 leading to the transcription of six alternatively spliced E2F6 mRNAs that encode four different E2F6 proteins. Moreover, we identified an E2F6 pseudogene localized on chromosome 22 completely spliced and devoid of exons 2, 3, and 4, and part of exons 1 and 5. Definition of the transcriptional initiation site and sequence analysis show that the gene contains a TATA less, CAAT less, GC-rich promoter with multiple transcription start sites. Regulatory elements necessary for basal transcription reside within a 134bp fragment as determined by transient transfection experiments. © 2004 Elsevier Inc. All rights reserved.

Antibody deficiency due to a missense mutation in CD19 demonstrates the importance of the conserved tryptophan 41 in immunoglobulin superfamily domain formation.

Immunoglobulin superfamily (IgSF) domains are conserved structures present in many proteins in eukaryotes and prokaryotes. These domains are well-capable of facilitating sequence variation, which is most clearly illustrated by the variable regions in immunoglobulins (Igs) and T cell receptors (TRs). We studied an antibody-deficient patient suffering from recurrent respiratory infections and with impaired antibody responses to vaccinations. Patient's B cells showed impaired Ca(2+) influx upon stimulation with anti-IgM and lacked detectable CD19 membrane expression. CD19 sequence analysis revealed a homozygous missense mutation resulting in a tryptophan to cystein (W52C) amino acid change. The affected tryptophan is CONSERVED-TRP 41 located on the C-strand of the first extracellular IgSF domain of CD19 and was found to be highly conserved, not only in mammalian CD19 proteins, but in nearly all characterized IgSF domains. Furthermore, the tryptophan is present in all variable domains in Ig and TR and was not mutated in 117 Ig class-switched transcripts of B cells from controls, despite an overall 10% amino acid change frequency. In vitro complementation studies and CD19 western blotting of patient's B cells demonstrated that the mutated protein remained immaturely glycosylated. This first missense mutation resulting in a CD19 deficiency demonstrates the crucial role of a highly conserved tryptophan in proper folding or stability of IgSF domains.

Study of gene expression in thyrotropin-stimulated thyroid cells by cDNA expression array: ID3 transcription modulating factor as an early response protein and tumor marker in thyroid carcinomas.

Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific induction or repression of a large number of genes. To identify genes differentially regulated by the cAMP-dependent transduction pathway, which is poorly characterized so far, we used the cDNA expression array technology. Hybridizations of Atlas human cDNA expression arrays with (32)P-labeled cDNA probes derived from control or thyrotropin (TSH)-stimulated dog thyrocytes in primary culture generated expression profiles of hundreds of genes simultaneously. Among the genes that displayed modified expression, we selected the transcription factor ID3, whose expression was increased by a cAMP-dependent stimulus. ID3 overexpression after TSH stimulation was first verified by Northern blotting analysis, and its mRNA regulation was then investigated in response to a variety of agents acting on thyrocyte proliferation and/or differentiation. We show that: (1) ID3 mRNA induction was stronger after stimulation of the cAMP cascade, but was not restricted to this signaling pathway, as phorbol myristate ester (TPA) and insulin also stimulated mRNA accumulation; (2) in contrast, powerful mitogens for thyroid cells, epidermal growth factor and hepatocyte growth factor, did not significantly modify ID3 mRNA levels; (3) ID3 protein levels closely parallelled mRNA levels, as revealed by immunofluorescence experiments showing a nuclear signal regulated by TSH; (4) in papillary thyroid carcinomas, ID3 mRNA was downregulated. Our results suggest that ID3 expression might be more related to the differentiating process induced by TSH than to the proliferative action of this hormone.

Characterization of human 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase gene and its expression in mammalian cells

Three β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3β-HSD) catalyze the oxidative conversion of Δ5-3β-hydroxysteroids to the Δ4-3-keto configuration and is therefore essential for the biosynthesis of all classes of hormonal steroids, namely progesterone, glucocorticoids, mineralocorticoids, androgens, and estrogens. Using human 3β-HSD cDNA as probe, a human 3β-HSD gene was isolated from a λ-EMBL3 library of leucocyte genomic DNA. A fragment of 3β-HSD genomic DNA was also obtained by amplification of genomic DNA using the polymerase chain reaction. The 3β-HSD gene contains a 5′-untranslated exon of 53 base pairs (bp) and three successive translated exons of 232, 165, and 1218 bp, respectively, separated by introns of 129, 3883, and 2162 bp. The transcription start site is situated 267 nucleotides upstream from the ATG initiating codon. DNA sequence analysis of the 5′-flanking region reveals the existence of a putative TATA box (ATAAA) situated 28 nucleotides upstream from the transcription start site while a putative CAAT binding sequence is located 57 nucleotides upstream from the TATA box. Expression of a cDNA insert containing the coding region of 3β-HSD in nonsteroidogenic cells shows that the gene encodes a single 42-kDa protein containing both 3β-hydroxysteroid dehydrogenase and Δ5-Δ4-isomerase activities. Moreover, all natural steroid substrates tested are transformed with comparable efficiency by the enzyme. In addition to its importance for studies of the regulation of expression of 3β-HSD in gonadal as well as peripheral tissues, knowledge of the structure of the human 3β-HSD gene should permit investigation of the molecular defects responsible for 3β-HSD deficiency, the second most common cause of adrenal hyperplasia in children.

Genomic organization of the human e1af gene, a member of Ets transcription factors

The E1AF protein belongs to the family of Ets transcription factors and is involved in the regulation of metastasis gene expression. It has recently been reported in an undifferentiated child sarcoma that part of this gene could be fused by translocation to the ews gene. We show here that the human e1af gene, which is located in the q21 region of chromosome 17, is organized in 13 exons distributed along 19 kb of genomic DNA. Its two main functional domains, the acidic domain and the DNA-binding ETS domain, are each encoded by three different exons. The 3'-untranslated region of e1af is 0.7 kb. The 5'-untranslated region is about 0.3 kb and is composed of a first exon upstream from the exon containing the first methionine. These data could possibly accelerate an understanding of the molecular basis of putative inherited diseases linked to E1AF. (C) 1999 Elsevier Science B.V. All rights reserved.

Morphological and molecular characteristics of a new species of Pasteuria parasitic on Meloidogyne ardenensis

A species of the hyper-parasitic bacterium Pasteuria was isolated from the root-knot nematode Meloidogyne ardenensis infecting the roots of ash (Fraxinus excelsior). It is morphologically different from some other Pasteuria pathogens of nematodes in that the spores lack a basal ring on the ventral side of the spore and have a unique clumping nature. Transmission electron microscopy (TEM) showed that the clumps of spores are not random aggregates but result from the disintegration of the suicide cells of the thalli. Sporulation within each vegetative mycelium was shown to be asynchronous. In addition to the novel morphological features 16S rRNA sequence analysis showed this to be a new species of Pasteuria which we have called P. hartismeri. Spores of P. hartismeri attach to juveniles of root-knot nematodes infecting a wide range of plants such as mint (Meloidogyne hapla), rye grass (unidentified Meloidogyne sp.) and potato (Meloidogyne fallax).