Automated identification of conserved synteny after whole-genome duplication

An important objective for inferring the evolutionary history of gene families is the determination of orthologies and paralogies. Lineage-specific paralog loss following whole-genome duplication events can cause anciently related homologs to appear in some assays as orthologs. Conserved synteny—the tendency of neighboring genes to retain their relative positions and orders on chromosomes over evolutionary time—can help resolve such errors. Several previous studies examined genome-wide syntenic conservation to infer the contents of ancestral chromosomes and provided insights into the architecture of ancestral genomes, but did not provide methods or tools applicable to the study of the evolution of individual gene families. We developed an automated system to identify conserved syntenic regions in a primary genome using as outgroup a genome that diverged from the investigated lineage before a whole-genome duplication event. The product of this automated analysis, the Synteny Database, allows a user to examine fully or partially assembled genomes. The Synteny Database is optimized for the investigation of individual gene families in multiple lineages and can detect chromosomal inversions and translocations as well as ohnologs (paralogs derived by whole-genome duplication) gone missing. To demonstrate the utility of the system, we present a case study of gene family evolution, investigating the ARNTL gene family in the genomes of Ciona intestinalis, amphioxus, zebrafish, and human.An important objective for inferring the evolutionary history of gene families and chromosome segments is the determination of orthology and paralogy. A stepwise approach generally uses BLAST (basic local alignment search tool) (Altschul et al. 1997) to define coarse relationships among genes, followed by phylogenetic reconstruction to suggest more detailed hypotheses of descent. Events such as gene duplications or whole genome duplications (WGD), with associated differential gene loss, introduce noise into these analyses. Anomalies, such as lineage-specific paralog loss, can cause anciently related homologs to appear to be orthologs, thereby confusing sequence similarity with functional homology (Postlethwait 2007). Such errors can confound attempts to create nonhuman animal disease models and can obscure recent, species-specific evolutionary change among sister lineages.Orthologs are two genes, one in each of two species, that descended from a single gene in the last common ancestor of those two species. Paralogs are a set of genes derived by duplication within a lineage, and together, a group of paralogs can be co-orthologous to their unduplicated ortholog in a related species. Ohnologs are a special subset of paralogs that result from a whole-genome duplication event (Wolfe 2000). The differential loss of genes that follows a duplication event can create ohnologs gone missing when different ohnologs are lost reciprocally in different lineages.Understanding and distinguishing ohnologs gone missing from orthologs is a pervasive problem in vertebrate genomics due to multiple genome duplication events. Two rounds of whole-genome duplication events, called R1 and R2, likely occurred at the base of the vertebrate lineage after the divergence of non-vertebrate chordates and prior to the appearance of jawed vertebrates (Garcia-Fernàndez and Holland 1994; Spring 1997; Dehal and Boore 2005). A third duplication, called R3, likely occurred in the teleost lineage after the divergence of ray-finned and lobe-finned fishes (Amores et al. 1998; Taylor et al. 2003; Jaillon et al. 2004), but before the radiation of the teleosts. Additional genome duplications punctuated the evolution of other lineages, like salmonids, catastomids, goldfish, Xenopus laevis, and even a rodent (Uyeno and Smith 1972; Allendorf and Thorgaard 1984; Schmid and Steinlein 1991; Risinger and Larhammar 1993; Larhammar and Risinger 1994; Gallardo et al. 1999; David et al. 2003; Mungpakdee et al. 2008a,b). Given the pervasive nature of genome duplication in chordates and the importance of teleost fish and Xenopus laevis as model organisms, it is important to develop automated methods to identify true orthologs among groups of paralogs and to distinguish them from more ancient, nonorthologous homologs.Figure 1 illustrates the problem of distinguishing orthologs following duplication and lineage-specific loss of a gene g and some of its neighboring genes after WGD (R1), speciation (S), and a second WGD event (R2) in one of the descendant lineages. In an idealized case, chromosomes would experience few changes in gene order or gene content, as illustrated by genes of the same color in Figure 1. The most common fate of genes created by a WGD event, however, is pseudogenization and nonfunctionalization (Li 1980; Watterson 1983). Surviving duplicates can develop new functions (Ohno 1970) or partition or lose their existing functions (Force et al. 1999; Lynch and Force 2000; Winkler et al. 2003; Postlethwait et al. 2004; Jovelin et al. 2007; Chain et al. 2008; Conant and Wolfe 2008; Jarinova et al. 2008). From the time of the duplication event to the present, duplicated genes can alter their expression patterns (Force et al. 1999) or their exon structure (Altschmied et al. 2002), or their activities (Zhang et al. 2002; Zhang 2003), and such changes can alter protein–protein interactions or subsequent developmental or physiological functions.Open in a separate windowFigure 1.Differential gene loss following whole-genome duplication creates ohnologs gone missing. This image shows the evolutionary history of a gene g and neighbors undergoing a whole-genome duplication event (R1), a speciation event (S), and a second WGD event (R2) that occurs in only one of the descendant lineages, S2. If no changes to the order or composition of genes on the chromosomes occur over time, most algorithms would find that g1a and g1b are co-orthologous to g1, and that g2a and g2b are co-orthologous to g2. In a more realistic evolutionary history, gene losses and chromosomal rearrangements follow the genome duplication event, including a loss of g1 from the S1 lineage and g2a and g2b from the S2 lineage. Due to these losses, orthology assignment algorithms will usually infer that g1a and g1b are co-orthologous to g2, incorrectly assigning co-orthology where there is none. Extinct genes are shown in gray.In the case of differential gene loss and gene rearrangements in lineages S1 and S2, most reciprocal best-hit BLAST algorithms (Wall et al. 2003) would associate gene g2 with g1a and g1b, and most phylogenetic methods, due to a lack of data, would find that the most likely hypothesis of descent was that genes g2, g1a, and g1b shared their most recent common ancestor; in other words, these methods would incorrectly infer that g1a and g1b were orthologs of g2. The erroneous assignment of orthology presents a problem because it implies that the last common ancestor at time S had a single gene with a set of functions that evolved to g1 (and its subsequent duplicates, g1a and g1b) in S2 and g2 in S1, but in fact, no such gene actually existed.To address this problem and to better infer orthologies and paralogies, we can take advantage of conserved synteny—the tendency of neighboring genes to retain their relative positions and orders on chromosomes over evolutionary time. In a WGD event, duplicated chromosomes (homeologs) initially have gene orders identical to each other and to their immediate ancestor. Between the time of duplication and speciation events, however, genes can be lost from one homeolog or the other (unless preserved by structures such as embedded regulatory elements) (Kikuta et al. 2007), and inversions and other chromosome rearrangements can occur independently on the two duplicated homeologs. These events occurring in the chromosomal vicinity of the gene in question give an identity to all of the genes in the neighborhood. In the example given in Figure 1, we could test the hypothesis that genes g1a and g1b are co-orthologous to gene g2 by first examining the neighbors of g1a and g1b—ensuring that a sufficient number of gene neighbors are also paralogous—and then by checking those neighboring paralogs to ensure that they are orthologous to the neighbors of g2. The conserved syntenic region defined by such genes would confirm (or in this case, reject) the co-orthology of genes g1a and g1b to g2. This approach complements the use of BLAST and phylogenetic reconstruction and provides additional evidence to infer the evolutionary history of gene families independent of sequence identities.Several previous studies examined syntenic conservation at a genomic level to determine the nature of the ancestral chromosomes for that organism''s lineage. Evidence for two rounds of genome duplication in stem vertebrates came from a whole-genome analysis of human, mouse, and fugu pufferfish using the urochordate Ciona intestinalis as an outgroup (Dehal and Boore 2005). Analysis of the Tetraodon nigroviridis (green spotted pufferfish) genome and the construction of a dense meiotic map for medaka supported earlier conclusions (Amores et al. 1998; Postlethwait et al. 1998; Woods et al. 2000; Postlethwait et al. 2002; Taylor et al. 2003; Van de Peer et al. 2003) that a third genome duplication had occurred in the teleost fish. Analysis of Tetraodon and medaka provided evidence for a 12-chromosome ancestral vertebrate genome by calculating conserved syntenic regions between the fish and human genomes (Jaillon et al. 2004; Naruse et al. 2004). Subsequent work reconstructed the ancestral vertebrate genome using data from human, chicken, and medaka genomes (Nakatani et al. 2007) and, in opposition to earlier work (Jaillon et al. 2004; Naruse et al. 2004; Woods et al. 2005), concluded that the osteichthyan ancestor had ∼40 chromosomes. These studies provided insights into the architecture of the ancestral genome, but were not convenient for the study of the evolution of individual gene families, because the methods used did not form individual syntenic clusters (Jaillon et al. 2004; Dehal and Boore 2005; Nakatani et al. 2007); instead, they used hand-curated data (Jaillon et al. 2004; Nakatani et al. 2007) or they downplayed portions of the genome that did not fit into the analysis (Dehal and Boore 2005).We have developed an automated system to identify conserved syntenic regions in a primary genome using an outgroup genome that diverged from the investigated lineage before a whole-genome duplication. Our Synteny Database allows for the analysis of fully or partially assembled genomes (Bridgham et al. 2008) and is optimized for the investigation of individual gene families in multiple lineages. The Synteny Database specializes in comparing genomes that have undergone one or more whole-genome duplications; it is able to detect chromosome inversions and translocations, as well as ohnologs gone missing in the gene families investigated. To demonstrate the utility and use of the system, we present a case study of the evolution of the ARNTL gene family in the amphioxus, Ciona intestinalis, zebrafish, and human genomes.     

Alignathon: a competitive assessment of whole-genome alignment methods

Multiple sequence alignments (MSAs) are a prerequisite for a wide variety of evolutionary analyses. Published assessments and benchmark data sets for protein and, to a lesser extent, global nucleotide MSAs are available, but less effort has been made to establish benchmarks in the more general problem of whole-genome alignment (WGA). Using the same model as the successful Assemblathon competitions, we organized a competitive evaluation in which teams submitted their alignments and then assessments were performed collectively after all the submissions were received. Three data sets were used: Two were simulated and based on primate and mammalian phylogenies, and one was comprised of 20 real fly genomes. In total, 35 submissions were assessed, submitted by 10 teams using 12 different alignment pipelines. We found agreement between independent simulation-based and statistical assessments, indicating that there are substantial accuracy differences between contemporary alignment tools. We saw considerable differences in the alignment quality of differently annotated regions and found that few tools aligned the duplications analyzed. We found that many tools worked well at shorter evolutionary distances, but fewer performed competitively at longer distances. We provide all data sets, submissions, and assessment programs for further study and provide, as a resource for future benchmarking, a convenient repository of code and data for reproducing the simulation assessments.Given a set of sequences, a multiple sequence alignment (MSA) is a partitioning of the residues in the sequences, be they amino acids or nucleotides, into related sets. Here, we are interested in the relationship of evolutionary homology. In other contexts, residues may be aligned with a different aim, as in structural alignments, where residues are aligned if located at the same point in a shared crystal structure (Kolodny et al. 2005). MSA is a fundamental problem in biological sequence analysis because it is a prerequisite for most phylogenetic and evolutionary analyses (Felsenstein 2003; Wallace et al. 2005; Edgar and Batzoglou 2006; Notredame 2007). Most MSAs are termed “global,” made of sequences assumed to be related through the mutational processes of residue substitution, subsequence insertion, and subsequence deletion (collectively, insertions and deletions are termed indels) (for review, see Notredame 2007). The availability of whole-genome sequences has led to an interest in MSAs for complete genomes, including all sequences: genes, promoters, repetitive regions, etc. Termed whole-genome alignment (WGA), this requires the aligner to additionally consider genome rearrangements, such as inversions, translocations, chromosome fusions, chromosome fissions, and reciprocal translocations. Some tools for WGA are also capable of modeling unbalanced rearrangements that lead to copy number change, such as tandem and segmental duplications (Blanchette et al. 2004; Miller et al. 2007; Paten et al. 2008, 2011; Angiuoli and Salzberg 2011). WGA methods have been critical to understanding the selective forces acting across genomes, allowing evolutionary analysis of many potential functional elements (The ENCODE Project Consortium 2012), and in particular, the identification of conserved noncoding functional elements (Drosophila 12 Genomes Consortium 2007; Lindblad-Toh et al. 2011), including cis-regulatory elements (Kellis et al. 2003), enhancers, and noncoding RNAs.The lack of accepted gold standard reference alignments has made it hard to objectively assess the relative merits of WGA methods. Previous evaluations of MSAs can be split into roughly four types: those using simulation, those using expert information, those using direct statistical assessments, and finally those that assess how well an alignment functions for a downstream analysis. We briefly describe and review these approaches (for a more comprehensive review, see Iantorno et al. 2014).In simulation evaluations, a set of sequences and an alignment is generated using a model of evolution. Alignments are created from the simulated sequences and the resulting predictions are compared to the “true” simulated alignment. There are two basic types of simulators for DNA sequence evolution: coalescent simulators and noncoalescent forward-time simulators (Carvajal-Rodríguez 2010). Although useful for modeling populations, coalescent simulators cannot yet efficiently model general sequence evolution, and as a result MSA simulators currently use forward-time approaches. There are numerous forward-time simulators useful for assessing global MSA tools (Stoye et al. 1997; Blanchette et al. 2004; Cartwright 2005; Varadarajan et al. 2008). However, the simulation options for assessing WGA have until recently been absent, essentially because to do so requires modeling both low-level sequence evolution and higher-level genome rearrangements—a formidable challenge given the large and complex parameter space that potentially encompasses all aspects of genome evolution. The sgEvolver simulator (Darling et al. 2004, 2010) is used to generate simulated genome alignments, although it lacks an explicit model for sequence translocation or mobile element evolution. EvolSimulator is a genome simulator, but it has a somewhat simple model of evolution and a focus on ecological parameters (Beiko and Charlebois 2007). Another option, the ALF simulator (Dalquen et al. 2012), models gene and neutral DNA evolution. For this study we used the EVOLVER software, which can simulate full-sized, multichromosome genome evolution in forward time (Edgar et al. 2009). EVOLVER models an explicitly haploid genome and lacks a population model; its framework and expert-curated extensive parameter set are intended to produce “reference-like” genomes, i.e., haploid genomes. EVOLVER models DNA sequence evolution with sequence annotations; a gene model; a base-level evolutionary constraint model; chromosome evolution, including inter- and intrachromosomal rearrangements; tandem and segmental duplications; and mobile element insertions, movements, and evolution.An alternative approach to assessing MSA is to use expert biological information not available to the aligner. Although interpreting the results of simulations is made difficult by the uncertainty to which they approximate reality, the clear advantage of using expert information is that it can be used to assess alignments of actual biological sequences. For protein and RNA alignment there are several popular benchmarks that provide either reference structural alignments or expertly curated alignments (Blackshields et al. 2006; Wilm et al. 2006; Kemena et al. 2013). Nontranscribed DNA alignments are, however, much harder to assess since one lacks an external criterion to assemble objective gold standard references (Kemena and Notredame 2009). This explains why untranslated DNA alignments are usually evaluated using more ad hoc expert information (Margulies et al. 2007; Paten et al. 2008). The main strength of these procedures is that they provide an objective evolutionary context when evaluating the alignment. The difficulty with relying upon such expert information is that it may address only a small fraction of the alignment (e.g., in the referenced papers, coding exons, and ancient repeats), may itself rely on other forms of inference (e.g., ancient repeat analyses have an explicit dependence on the sequence alignment procedures used to determine ancestral repeat relationships), and have unknown variance, generality, and discriminative power.The third approach addresses alignments by statistical measures. For global MSA there are several options, e.g., the T-Coffee CORE/TCS index (Notredame and Abergel 2003; Chang et al. 2014), Heads or Tails (HoT) (Landan and Graur 2008), GUIDANCE (Penn et al. 2010a,b), and StatSigMA-w (Chen and Tompa 2010). For this work, we expand on the probabilistic sampling-based alignment reliability (PSAR) (Kim and Ma 2011) method, which samples pairwise suboptimal alignments to assess the reliability of MSAs. Statistical measures are attractive because they can be used with the complete alignments of real sequences. However, without a gold standard to compare against, they are only a proxy to a true assessment of accuracy.The final category of common assessment methods addresses how well a program generates alignments for a given computational task. This is typically the assessment made by a biologist in choosing an alignment program, i.e., how well does it perform in practice, according to intuition or analysis? Unfortunately, these assessments, often being one-offs, rarely make it into the literature and are difficult if not impossible to generalize from because these assessments are made for the purposes of a given analysis. Notably for WGAs, Bradley et al. (2009) assessed how much alignment methods influenced de novo ncRNA predictions and Margulies et al. (2007) analyzed the effect of different WGAs on the prediction of conserved elements.There have been relatively few independent or community organized assessments of WGA pipelines. Notably, as part of the ENCODE Pilot Project (Margulies et al. 2007), four pipelines were assessed across a substantial number of regions, and Chen and Tompa later compared those alignments using the StatSigMA-w tool (Chen and Tompa 2010). The Alignathon is an attempt to perform a larger and more comprehensive evaluation. It is a natural intellectual successor to the Assemblathon collaborative competitions (Earl et al. 2011; Bradnam et al. 2013). The starting point of the Alignathon is to assume that the problem of genome assembly is largely a solved problem. Although we admit this is currently a dubious assumption, it appears that the problem of genome assembly will shrink in size in the coming years as new sequencing technologies become available and existing assembly software is perfected to take advantage of more numerous, longer, and less error-prone reads (Branton et al. 2008; Schreiber et al. 2013; Laszlo et al. 2014). With this future as a starting point, the question a biologist faces changes from a proximate one of “how do I best assemble the genome of my favorite species?” to a higher level question of “how is my favorite species related to the pantheon of other sequenced species?” Such a question is answered through a WGA. If organized community efforts to sequence large numbers of genomes, such as the Genome 10K Project for vertebrates and 5000 arthropod genomes initiative (i5K) for insects, are to maximally fulfill their promise by revealing and refining the evolutionary history of all of their species, then it is vital that we have the best possible methods for WGA (Genome 10K Community of Scientists 2009; i5K Consortium 2013).     

Optimization and evaluation of single-cell whole-genome multiple displacement amplification

The scarcity of genomic DNA can be a limiting factor in some fields of genetic research. One of the methods developed to overcome this difficulty is whole genome amplification (WGA). Recently, multiple displacement amplification (MDA) has proved very efficient in the WGA of small DNA samples and pools of cells, the reaction being catalyzed by the phi29 or the Bst DNA polymerases. The aim of the present study was to develop a reliable, efficient, and fast protocol for MDA at the single-cell level. We first compared the efficiency of phi29 and Bst polymerases on DNA samples and single cells. The phi29 polymerase generated accurately, in a short time and from a single cell, sufficient DNA for a large set of tests, whereas the Bst enzyme showed a low efficiency and a high error rate. A single-cell protocol was optimized using the phi29 polymerase and was evaluated on 60 single cells; the DNA obtained DNA was assessed by 22 locus-specific PCRs. This new protocol can be useful for many applications involving minute quantities of starting material, such as forensic DNA analysis, prenatal and preimplantation genetic diagnosis, or cancer research.     

Comparative recombination rates in the rat, mouse, and human genomes

Levels of recombination vary among species, among chromosomes within species, and among regions within chromosomes in mammals. This heterogeneity may affect levels of diversity, efficiency of selection, and genome composition, as well as have practical consequences for the genetic mapping of traits. We compared the genetic maps to the genome sequence assemblies of rat, mouse, and human to estimate local recombination rates across these genomes. Humans have greater overall levels of recombination, as well as greater variance. In rat and mouse, the size of the chromosome and proximity to telomere have less effect on local recombination rate than in human. At the chromosome level, rat and mouse X chromosomes have the lowest recombination rates, whereas human chromosome X does not show the same pattern. In all species, local recombination rate is significantly correlated with several sequence variables, including GC%, CpG density, repetitive elements, and the neutral mutation rate, with some pronounced differences between species. Recombination rate in one species is not strongly correlated with the rate in another, when comparing homologous syntenic blocks of the genome. This comparative approach provides additional insight into the causes and consequences of genomic heterogeneity in recombination.     

The first-generation whole-genome radiation hybrid map in the horse identifies conserved segments in human and mouse genomes

A first-generation radiation hybrid (RH) map of the equine (Equus caballus) genome was assembled using 92 horse x hamster hybrid cell lines and 730 equine markers. The map is the first comprehensive framework map of the horse that (1) incorporates type I as well as type II markers, (2) integrates synteny, cytogenetic, and meiotic maps into a consensus map, and (3) provides the most detailed genome-wide information to date on the organization and comparative status of the equine genome. The 730 loci (258 type I and 472 type II) included in the final map are clustered in 101 RH groups distributed over all equine autosomes and the X chromosome. The overall marker retention frequency in the panel is approximately 21%, and the possibility of adding any new marker to the map is approximately 90%. On average, the mapped markers are distributed every 19 cR (4 Mb) of the equine genome--a significant improvement in resolution over previous maps. With 69 new FISH assignments, a total of 253 cytogenetically mapped loci physically anchor the RH map to various chromosomal segments. Synteny assignments of 39 gene loci complemented the RH mapping of 27 genes. The results added 12 new loci to the horse gene map. Lastly, comparison of the assembly of 447 equine genes (256 linearly ordered RH-mapped and additional 191 FISH-mapped) with the location of draft sequences of their human and mouse orthologs provides the most extensive horse-human and horse-mouse comparative map to date. We expect that the foundation established through this map will significantly facilitate rapid targeted expansion of the horse gene map and consequently, mapping and positional cloning of genes governing traits significant to the equine industry.     

Bleomycin sulfate-induced micronuclei in human,rat, and mouse peripheral blood lymphocytes

The sensitivity to micronucleus (MN) induction of human, mouse, and rat peripheral blood lymphocytes (PBLs) exposed to bleomycin sulfate (BLM) in vitro was compared in cytochalasin B-induced binucleated (BN) cells. For the PBLs of each species, either 0, 5, 10, 20, 40, 60, 80, or 160 μg/ml BLM was added to 5 ml aliquots of whole blood for 4 hr at 37°C in a 5% CO₂ atmosphere. Leukocytes were isolated on a density gradient and cultured in the presence of phytohemagglutinin to stimulate blastogenesis, and cytochalasin B was added to each culture at 21 hr postinitiation to prevent cytokinesis. A total of 4,000 BNs/concentration/species was analyzed for MN in two independent experiments. In addition, multiple-MN-BNs were quantitated, and the nucleation index was determined. Significant increases both in total MN-BNs and multiple-MN-BNs were observed at all concentrations in all species. All three species concentration-response curves gave good fits (r² values from 0.87 to 0.95) to either a linear or a square root model (y = mx + b or y = m[x]^0.5 + b, respectively; where y = the percentage of MN-BN, m is the slope, and b is the y-intercept). The MN induction in the human and rat PBLs was not statistically different, but both were significantly less sensitive than the response shown by the BLM-exposed mouse PBLs. This difference in MN susceptibility was observed only at BLM test concentrations ≧ 20 μg/ml. The nucleation index was significantly decreased in all species at either 80 or 160 μg/ml. © 1995 Wiley-Liss, Inc.     

Noninvasive whole-genome sequencing of a human fetus

Analysis of cell-free fetal DNA in maternal plasma holds promise for the development of noninvasive prenatal genetic diagnostics. Previous studies have been restricted to detection of fetal trisomies, to specific paternally inherited mutations, or to genotyping common polymorphisms using material obtained invasively, for example, through chorionic villus sampling. Here, we combine genome sequencing of two parents, genome-wide maternal haplotyping, and deep sequencing of maternal plasma DNA to noninvasively determine the genome sequence of a human fetus at 18.5 weeks of gestation. Inheritance was predicted at 2.8 × 10(6) parental heterozygous sites with 98.1% accuracy. Furthermore, 39 of 44 de novo point mutations in the fetal genome were detected, albeit with limited specificity. Subsampling these data and analyzing a second family trio by the same approach indicate that parental haplotype blocks of ~300 kilo-base pairs combined with shallow sequencing of maternal plasma DNA is sufficient to substantially determine the inherited complement of a fetal genome. However, ultradeep sequencing of maternal plasma DNA is necessary for the practical detection of fetal de novo mutations genome-wide. Although technical and analytical challenges remain, we anticipate that noninvasive analysis of inherited variation and de novo mutations in fetal genomes will facilitate prenatal diagnosis of both recessive and dominant Mendelian disorders.     

D-penicillamine: Mitogenic effect on mouse,rat and human spleen lymphocytes

The effect of D-penicillamine (D-Pen) on the proliferation of cultures of normal mouse, rat, and human spleen lymphocytes and peripheral blood lymphocytes was examined. D-Pen in concentrations of 2×10^–3 M to 8×10^–3 M in serum-free and in serum-containing medium resulted in a highly significant incorporation of³H-TdR by normal mouse and rat spleen cells. Enhanced incorporation of³H-TdR by normal human spleen cells only occurred in serum-containing medium. D-Pen in concentrations of 10^–4 M to 10^–3 M in serum-free and serum-containing medium resulted in significant inhibition of³H-TdR incorporation by normal and mitogen-stimulated mouse and rat spleen cells. Doses of D-Pen greater than 2×10^–2 M strongly inhibited³H-TdR incorporation by both normal and mitogen-stimulated mouse, rat, and human spleen cells and peripheral blood cells. The latter cells were not stimulated or inhibited at lower concentrations of D-Pen.Results from cell depletion and enriching procedures (specific antibody + C' cell killing, employment of athymic, nude spleen cells, adherent and phagocytic cell removal, E rosette cell separation procedures) suggested that target cells in the mouse spleen for D-Pen activation are non-adherent B cells whereas the D-Pen responsive cells in the human spleen probably are T cells.     

Analysis of the fine specificity of rat,mouse and human TAP peptide transporters

Prior to their association with major histocompatibility complex (MHC) class I molecules, peptides generated from cytosolic antigens need to be translocated by the MHC-encoded peptide transporter (TAP) into the lumen of the endoplasmic reticulum (ER). While class I molecules possess well-known binding characteristics for peptides, the fine specificity of TAP for its peptide substrates has not been analyzed in detail. Previously, we have studied the effect of amino acid variations at the N-terminal, the C-terminal, and the penultimate residue on the efficiency of peptide translocation. Using permeabilized cells, we have shown that TAP pre-selects peptides in an allele- and species-specific manner, for which only the C-terminal residue is crucial. This finding is confirmed in the present study by using microsomes containing different TAP. The influence of amino acid substitutions at positions 2 to 7 of 9-residue model peptides on TAP-dependent peptide translocation is systematically examined. Only a few amino acid substitutions at these positions affect the efficiency of peptide translocation significantly, e.g. Pro at position 2 or 3 negatively influences transport whereas Glu at positions 6 and 7 enhances transport. The differences in translocation by the rat TAP alleles a or u, mouse TAP and human TAP are, however, minor for the peptide with internal substitutions used in this study. These results show that the C-terminal residue essentially governs the species-specific substrate specificity of TAP.     

Cytoarchitecture of mouse and rat cingulate cortex with human homologies

A gulf exists between cingulate area designations in human neurocytology and those used in rodent brain atlases with a major underpinning of the former being midcingulate cortex (MCC). The present study used images extracted from the Franklin and Paxinos mouse atlas and Paxinos and Watson rat atlas to demonstrate areas comprising MCC and modifications of anterior cingulate (ACC) and retrosplenial cortices. The laminar architecture not available in the atlases is also provided for each cingulate area. Both mouse and rat have a MCC with neurons in all layers that are larger than in ACC and layer Va has particularly prominent neurons and reduced neuron densities. An undifferentiated ACC area 33 lies along the rostral callosal sulcus in rat but not in mouse and area 32 has dorsal and ventral subdivisions with the former having particularly large pyramidal neurons in layer Vb. Both mouse and rat have anterior and posterior divisions of retrosplenial areas 29c and 30, although their cytology is different in rat and mouse. Maps of the rodent cingulate cortices provide for direct comparisons with each region in the human including MCC and it is significant that rodents do not have a posterior cingulate region composed of areas 23 and 31 like the human. It is concluded that rodents and primates, including humans, possess a MCC and this homology along with those in ACC and retrosplenial cortices permit scientists inspired by human considerations to test hypotheses on rodent models of human diseases.     

Patterns of insertions and their covariation with substitutions in the rat, mouse, and human genomes

The rates at which human genomic DNA changes by neutral substitution and insertion of certain families of transposable elements covary in large, megabase-sized segments. We used the rat, mouse, and human genomic DNA sequences to examine these processes in more detail in comparisons over both shorter (rat-mouse) and longer (rodent-primate) times, and demonstrated the generality of the covariation. Different families of transposable elements show distinctive insertion preferences and patterns of variation with substitution rates. SINEs are more abundant in GC-rich DNA, but the regional GC preference for insertion (monitored in young SINEs) differs between rodents and humans. In contrast, insertions in the rodent genomes are predominantly LINEs, which prefer to insert into AT-rich DNA in all three mammals. The insertion frequency of repeats other than SINEs correlates strongly positively with the frequency of substitutions in all species. However, correlations with SINEs show the opposite effects. The correlations are explained only in part by the GC content, indicating that other factors also contribute to the inherent tendency of DNA segments to change over evolutionary time.     

Construction of comparative cytogenetic maps of the Chinese hamster to mouse, rat and human

We constructed comparative cytogenetic maps of the Chinese hamster to mouse, rat and human by fluorescence in-situ hybridization using 36 cDNA clones of mouse, rat, Syrian hamster, Chinese hamster and human functional genes. In this study, 30 out of the 36 genes were newly mapped to Chinese hamster chromosomes. The chromosomal homology of the Chinese hamster was identified and arranged in 19, 19 and 18 segments of conserved synteny in mouse, rat and human, respectively. Additionally, two of the 19 segments homologous to mouse chromosomes were initially identified in this study. This revised version was published online in July 2006 with corrections to the Cover Date.     

Reconstructing the genomic architecture of ancestral mammals: lessons from human, mouse, and rat genomes

Recent analysis of genome rearrangements in human and mouse genomes revealed evidence for more rearrangements than thought previously and shed light on previously unknown features of mammalian evolution, like breakpoint reuse and numerous microrearrangements. However, two-way analysis cannot reveal the genomic architecture of ancestral mammals or assign rearrangement events to different lineages. Thus, the "original synteny" problem introduced by Nadeau and Sankoff previously, remains unsolved, as at least three mammalian genomes are required to derive the ancestral mammalian karyotype. We show that availability of the rat genome allows one to reconstruct a putative genomic architecture of the ancestral murid rodent genome. This reconstruction suggests that this ancestral genome retained many previously postulated chromosome associations in the placental ancestor and reveals others that were beyond the resolution of cytogenetic, radiation hybrid mapping, and chromosome painting techniques. Three-way analysis of rearrangements leads to a reliable reconstruction of the genomic architecture of specific regions in the murid ancestor, including the X chromosome, and for the first time allows one to assign major rearrangement events to one of human, mouse, and rat lineages. Our analysis implies that the rate of rearrangements is much higher in murid rodents than in the human lineage and confirms the existence of rearrangement hot-spots in all three lineages.     

Preparation and functional properties of monoclonal antibodies to human, mouse and rat OX-2.

We have prepared mouse and rat hybridomas to a 43-kDa molecule expressed in the thymus, on a subpopulation of dendritic cells, and in the brain, in mammalian tissue derived from mouse, rat and human. Using CHO cells transiently transfected with adenovirus vector(s) expressing a cDNA construct for the relevant OX-2 gene, we show these monoclonal antibodies (Mabs) detect a molecule encoded by this construct (rat OX-2 (rOX-2), mouse OX-2 (mOX-2) and human OX-2 (huOX-2), respectively). Furthermore, at least some of the anti-rat Mabs detect determinants expressed on the murine OX-2 molecule, as we predicted in an earlier publication. Previous studies have implied that this molecule might serve an important role in regulation of cell signaling for cytokine production. Using one-way mixed leukocyte reactions we show that when cells are cultured in the presence of the species-specific Mab, cytokine production becomes polarized 'away from' type-2 cytokine production, with preferentially increased expression of type-1 cytokine production.     

Finding the biologically optimal alignment of multiple sequences

OBJECTIVE: Deterministic annealing, which is derived from statistical physics, is a method for obtaining the global optimum in parameter space. During the annealing process, starting from high temperatures which are then lowered, deterministic annealing deterministically find the (global) optimum at each temperature. Thus, deterministic annealing is expected to be more computationally efficient than stochastic sampling strategies to obtain the global optimum. We propose to apply the deterministic annealing technique to the problem of efficiently finding the biologically optimal alignment of multiple sequences. METHODS AND MATERIAL: We take a strategy based on probabilistic models for aligning multiple sequences. That is, we train a probabilistic model using given training sequences and obtain their alignment by parsing, i.e. searching for the most likely parse of each sequence and gaps using the trained parameters of the model. In this scenario, we propose a new stochastic model, which is simple enough to be suited to multiple sequence alignment and, unlike existing stochastic models, say a profile hidden Markov model (HMM), allows us to use similarity scores between symbols (or a symbol and a gap). We further present a learning algorithm for our simple model by combining deterministic annealing with an expectation-maximization (EM) algorithm. We emphasize that our approach is time-efficient, even if the training is done through an annealing process. RESULTS: In our experiments, we used actual protein sequences whose three-dimensional (3D) structures are determined and which are all aligned based on their 3D structures. We compared the results obtained by our approach with those by other existing approaches. Experimental results clearly showed that our approach gave the best performance, in terms of the similarity to the structurally determined alignment, among the approaches tested. Experimental results further indicated that our approach was ten times more efficient in terms of actual computation time than a competing method.     

MAVID: constrained ancestral alignment of multiple sequences

We describe a new global multiple-alignment program capable of aligning a large number of genomic regions. Our progressive-alignment approach incorporates the following ideas: maximum-likelihood inference of ancestral sequences, automatic guide-tree construction, protein-based anchoring of ab-initio gene predictions, and constraints derived from a global homology map of the sequences. We have implemented these ideas in the MAVID program, which is able to accurately align multiple genomic regions up to megabases long. MAVID is able to effectively align divergent sequences, as well as incomplete unfinished sequences. We demonstrate the capabilities of the program on the benchmark CFTR region, which consists of 1.8 Mb of human sequence and 20 orthologous regions in marsupials, birds, fish, and mammals. Finally, we describe two large MAVID alignments, an alignment of all the available HIV genomes and a multiple alignment of the entire human, mouse, and rat genomes.