John L. Spouge

A DNA barcode for land plants

DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.

Factors underlying spontaneous inactivation and susceptibility to neutralization of human immunodeficiency virus

To determine the factors governing inactivation and neutralization, physical, chemical, and biological assays were performed on a molecular clone of human immunodeficiency type 1 (HIV-1HXB3). This included quantitative electron microscopy, gp120 and p24 enzyme-linked immunosorbent assays, reverse, transcriptase assays, and quantitative infectivity assays. For freshly harvested stocks, the ratio of infectious to noninfectious viral particles ranged from 10(-4) to 10(-7) in viral stocks containing 10(9) to 10(10) physical particles per milliliter. There were relatively few gp120 knobs per HIV particle, mean approximately 10 when averaged over the total particle count. Each HIV particle contained a mean approximately 5 x 10(-17) g of p24 and approximately 2 x 10(-16) g of RNA polymerase, corresponding to about 1200 and 80 molecules, respectively. The spontaneous shedding of gp120 envelope proteins from virions was exponential, with a half-life approximately 30 hr. The loss of RNA polymerase activity in virons was also exponential, with a half-life approximately 40 hr. The physical breakup of virions and the dissolution of p24 core proteins were slow (half-life greater than 100 hr) compared to the gp120 shedding and polymerase loss rates. The decay of HIV-1 infectivity was found to obey superimposed single- and multihit kinetics. At short preincubation times, the loss of infectivity correlated with spontaneous shedding of gp120 from virions. At longer times, an accelerating decay rate indicated that HIV requires a minimal number of gp120 molecules for efficient infection of CD4+ cells. The blocking activity of recombinant soluble CD4 (sCD4) and phosphonoformate (foscarnet) varied with the number of gp120 molecules and number of active RNA polymerase molecules per virion, respectively. These results demonstrate that the physical state of virions greatly influences infectivity and neutralization. The knowledge gained from these findings will improve the reliability of in vitro assays, enhance the study of wild-type strains, and facilitate the evaluation of potential HIV therapeutics and vaccines.

CBOL Protist Working Group: Barcoding Eukaryotic Richness beyond the Animal, Plant, and Fungal Kingdoms

A group of protist experts proposes a two-step DNA barcoding approach, comprising a universal eukaryotic pre-barcode followed by group-specific barcodes, to unveil the hidden biodiversity of microbial eukaryotes.

Protein Antigenic Structures Recognized by T Cells; Potential Applications to Vaccine Design

In summary, our results using the model protein antigen myoglobin indicated, in concordance with others, that helper T lymphocytes recognize a limited number of immunodominant antigenic sites of any given protein. Such immunodominant sites are the focus of a polyclonal response of a number of different T cells specific for distinct but overlapping epitopes. Therefore, the immunodominance does not depend on the fine specificity of any given clone of T cells, but rather on other factors, either intrinsic or extrinsic to the structure of the antigen. A major extrinsic factor is the MHC of the responding individual, probably due to a requirement for the immunodominant peptides to bind to the MHC of presenting cells in that individual. In looking for intrinsic factors, we noted that both immunodominant sites of myoglobin were amphipathic helices, i.e., helices having hydrophilic and hydrophobic residues on opposite sides. Studies with synthetic peptides indicated that residues on the hydrophilic side were necessary for T-cell recognition. However, unfolding of the native protein was shown to be the apparent goal of processing of antigen, presumably to expose something not already exposed on the native molecule, such as the hydrophobic sides of these helices. We propose that such exposure is necessary to interact with something on the presenting cell, such as MHC or membrane, where we have demonstrated the presence of antigenic peptides by blocking of presentation of biotinylated peptide with avidin. The membrane may serve as a short-term memory of peptides from antigens encountered by the presenting cell, for dynamic sampling by MHC molecules to be available for presentation to T cells. These ideas, together with the knowledge that T-cell recognition required only short peptides and therefore had to be based only on primary or secondary structure, not tertiary folding of the native protein, led us to propose that T-cell immunodominant epitopes may tend to be amphipathic structures. An algorithm to search for potential amphipathic helices from sequence information identified 18 of 23 known immunodominant T-cell epitopes from 12 proteins (p less than 0.001). Another statistical approach confirmed the importance of amphipathicity and also supported the importance of helical structure that had been proposed by others. It suggested that peptides able to form a stable secondary structure, especially a helix, more commonly formed immunodominant epitopes. We used this approach to predict potential immunodominant epitopes for induction of T-cell immunity in proteins of clinical relevance, such as the malarial circumsporozoite protein and the AIDS viral envelope.(ABSTRACT TRUNCATED AT 400 WORDS)

Conserved fragments of transposable elements in intergenic regions: evidence for widespread recruitment of MIR- and L2-derived sequences within the mouse and human genomes.

We analysed the distribution of transposable elements (TEs) in 100 aligned pairs of orthologous intergenic regions from the mouse and human genomes. Within these regions, conserved segments of high similarity between the two species alternate with segments of low similarity. Identifiable TEs comprise 40-60% of segments of low similarity. Within such segments, a particular copy of a TE found in one species has no orthologue in the other. Overall, TEs comprise only approximately 20 % of conserved segments. However, TEs from two families, MIR and L2, are rather common within conserved segments. Statistical analysis of the distributions of TEs suggests that a majority of the MIR and L2 elements present in murine intergenic regions have human orthologues. These elements must have been present in the common ancestor of human and mouse and have remained under substantial negative selection that prevented their divergence beyond recognition. If so, recruitment of MIR- and L2-derived sequences to perform a function that increases host fitness is rather common, with at least two such events per host gene. The central part of the MIR consensus sequence is over-represented in conserved segments given its background frequency in the genome, suggesting that it is under the strongest selective constraint.

HIV-1 infection kinetics in tissue cultures

Despite intensive experimental work on HIV-1, very little theoretical work has focused on HIV-1 spread in tissue culture. This article uses two systems of ordinary differential equations to model two modes of viral spread, cell-free virus and cell-to-cell contact. The two models produce remarkably similar qualitative results. Simulations using realistic parameter regimes showed that starting with a small fraction of cells infected, both cell-free viral spread and direct cell-to-cell transmission give an initial exponential phase of viral growth, followed by either a crash or a gradual decline, extinguishing the culture. Under some conditions, an oscillatory phase may precede the extinction. Some previous models of in vivo HIV-1 infection oscillate, but only in unrealistic parameter regimes. Experimental tissue infections sometimes display several sequential cycles of oscillation, however, so our models can at least mimic them qualitatively. Significantly, the models show that infective oscillations can be explained by infection dynamics; biological heterogeneity is not required. The models also display proportionality between infected cells and cell-free virus, which is reassuringly consistent with assumptions about the equivalence of several measures of viral load, except that the proportionality requires a relatively constant total cell concentration. Tissue culture parameter values can be determined from accurate, controlled experiments. Therefore, if verified, our models should make interpreting experimental data and extrapolating it to in vivo conditions sharper and more reliable.

Threshold Average Precision (TAP-k)

Motivation: Since database retrieval is a fundamental operation, the measurement of retrieval efficacy is critical to progress in bioinformatics. This article points out some issues with current methods of measuring retrieval efficacy and suggests some improvements. In particular, many studies have used the pooled receiver operating characteristic for n irrelevant records (ROCn) score, the area under the ROC curve (AUC) of a ‘pooled’ ROC curve, truncated at n irrelevant records. Unfortunately, the pooled ROCn score does not faithfully reflect actual usage of retrieval algorithms. Additionally, a pooled ROCn score can be very sensitive to retrieval results from as little as a single query. Methods: To replace the pooled ROCn score, we propose the Threshold Average Precision (TAP-k), a measure closely related to the well-known average precision in information retrieval, but reflecting the usage of E-values in bioinformatics. Furthermore, in addition to conditions previously given in the literature, we introduce three new criteria that an ideal measure of retrieval efficacy should satisfy. Results: PSI-BLAST, GLOBAL, HMMER and RPS-BLAST provided examples of using the TAP-k and pooled ROCn scores to evaluate sequence retrieval algorithms. In particular, compelling examples using real data highlight the drawbacks of the pooled ROCn score, showing that it can produce evaluations skewing far from intuitive expectations. In contrast, the TAP-k satisfies most of the criteria desired in an ideal measure of retrieval efficacy. Availability and Implementation: The TAP-k web server and downloadable Perl script are freely available at http://www.ncbi.nlm.nih.gov/CBBresearch/Spouge/html.ncbi/tap/ Contact: spouge@ncbi.nlm.nih.gov Supplementary Information: Supplementary data are available at Bioinformatics online.

The Gumbel pre-factor k for gapped local alignment can be estimated from simulations of global alignment

The optimal gapped local alignment score of two random sequences follows a Gumbel distribution. The Gumbel distribution has two parameters, the scale parameter λ and the pre-factor k. Presently, the basic local alignment search tool (BLAST) programs (BLASTP (BLAST for proteins), PSI-BLAST, etc.) use all time-consuming computer simulations to determine the Gumbel parameters. Because the simulations must be done offline, BLAST users are restricted in their choice of alignment scoring schemes. The ultimate aim of this paper is to speed the simulations, to determine the Gumbel parameters online, and to remove the corresponding restrictions on BLAST users. Simulations for the scale parameter λ can be as much as five times faster, if they use global instead of local alignment [R. Bundschuh (2002) J. Comput. Biol., 9, 243–260]. Unfortunately, the acceleration does not extend in determining the Gumbel pre-factor k, because k has no known mathematical relationship to global alignment. This paper relates k to global alignment and exploits the relationship to show that for the BLASTP defaults, 10 000 realizations with sequences of average length 140 suffice to estimate both Gumbel parameters λ and k within the errors required (λ, 0.8%; k, 10%). For the BLASTP defaults, simulations for both Gumbel parameters now take less than 30 s on a 2.8 GHz Pentium 4 processor.

A New Method for Estimating Species Age Supports the Coexistence of Malaria Parasites and Their Mammalian Hosts

Species in the genus Plasmodium cause malaria in humans and infect a variety of mammals and other vertebrates. Currently, estimated ages for several mammalian Plasmodium parasites differ by as much as one order of magnitude, an inaccuracy that frustrates reliable estimation of evolutionary rates of disease-related traits. We developed a novel statistical approach to dating the relative age of evolutionary lineages, based on Total Least Squares regression. We validated this lineage dating approach by applying it to the genus Drosophila. Using data from the Drosophila 12 Genomes project, our approach accurately reconstructs the age of well-established Drosophila clades, including the speciation event that led to the subgenera Drosophila and Sophophora, and age of the melanogaster species subgroup. We applied this approach to hundreds of loci from seven mammalian Plasmodium species. We demonstrate the existence of a molecular clock specific to individual Plasmodium proteins, and estimate the relative age of mammalian-infecting Plasmodium. These analyses indicate that: 1) the split between the human parasite Plasmodium vivax and P. knowlesi, from Old World monkeys, occurred 6.1 times earlier than that between P. falciparum and P. reichenowi, parasites of humans and chimpanzees, respectively; and 2) mammalian Plasmodium parasites originated 22 times earlier than the split between P. falciparum and P. reichenowi. Calibrating the absolute divergence times for Plasmodium with eukaryotic substitution rates, we show that the split between P. falciparum and P. reichenowi occurred 3.0–5.5 Ma, and that mammalian Plasmodium parasites originated over 64 Ma. Our results indicate that mammalian-infecting Plasmodium evolved contemporaneously with their hosts, with little evidence for parasite host-switching on an evolutionary scale, and provide a solid timeframe within which to place the evolution of new Plasmodium species.

FAST OPTIMAL ALIGNMENT

Algorithms often align sequences by minimizing a cost. Such algorithms usually operate by aligning successively longer subsequences until they finish the alignment. Efficient algorithms, such as those of Fickett and Ukkonen, speed the computation by ignoring bad subalignments. A general principle underlies the efficiency of these two algorithms: inequalities can direct computations to promising subalignments. Hence inequalities can be used to suggest alignment algorithms. Inequalities for unweighted end-gaps, affine and concave gap weights, etc., are discussed, and empirical results evaluating new algorithms for single indel costs and weighted end-gaps are presented. Empirical results show the new algorithms are, under certain circumstances, much faster than known algorithms.