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1.  A genetic polymorphism evolving in parallel in two cell compartments and in two clades 
The enzyme phosphoenolpyruvate carboxykinase, PEPCK, occurs in its guanosine-nucleotide-using form in animals and a few prokaryotes. We study its natural genetic variation in Colias (Lepidoptera, Pieridae). PEPCK offers a route, alternative to pyruvate kinase, for carbon skeletons to move between cytosolic glycolysis and mitochondrial Krebs cycle reactions.
PEPCK is expressed in both cytosol and mitochondrion, but differently in diverse animal clades. In vertebrates and independently in Drosophila, compartment-specific paralogous genes occur. In a contrasting expression strategy, compartment-specific PEPCKs of Colias and of the silkmoth, Bombyx, differ only in their first, 5′, exons; these are alternatively spliced onto a common series of following exons. In two Colias species from distinct clades, PEPCK sequence is highly variable at nonsynonymous and synonymous sites, mainly in its common exons. Three major amino acid polymorphisms, Gly 335 ↔ Ser, Asp 503 ↔ Glu, and Ile 629 ↔ Val occur in both species, and in the first two cases are similar in frequency between species. Homology-based structural modelling shows that the variants can alter hydrogen bonding, salt bridging, or van der Waals interactions of amino acid side chains, locally or at one another′s sites which are distant in PEPCK′s structure, and thus may affect its enzyme function. We ask, using coalescent simulations, if these polymorphisms′ cross-species similarities are compatible with neutral evolution by genetic drift, but find the probability of this null hypothesis is 0.001 ≤ P ≤ 0.006 under differing scenarios.
Our results make the null hypothesis of neutrality of these PEPCK polymorphisms quite unlikely, but support an alternative hypothesis that they are maintained by natural selection in parallel in the two species. This alternative can now be justifiably tested further via studies of PEPCK genotypes′ effects on function, organismal performance, and fitness. This case emphasizes the importance, for evolutionary insight, of studying gene-specific mechanisms affected by natural genetic variation as an essential complement to surveys of such variation.
PMCID: PMC3556304  PMID: 23311980
Amino acid polymorphism; Coalescent simulation; Glycolysis; Intramolecular bond variation; Neutral null hypothesis; Parallel evolution; Phosphoenolpyruvate carboxykinase; Selection hypothesis; Splice variation
2.  Bioinformatics Analysis of the FREM1 Gene—Evolutionary Development of the IL-1R1 Co-Receptor, TILRR 
Biology  2012;1(3):484-494.
The TLRs and IL-1 receptors have evolved to coordinate the innate immune response following pathogen invasion. Receptors and signalling intermediates of these systems are generally characterised by a high level of evolutionary conservation. The recently described IL-1R1 co-receptor TILRR is a transcriptional variant of the FREM1 gene. Here we investigate whether innate co-receptor differences between teleosts and mammals extend to the expression of the TILRR isoform of FREM1. Bioinformatic and phylogenetic approaches were used to analyse the genome sequences of FREM1 from eukaryotic organisms including 37 tetrapods and five teleost fish. The TILRR consensus peptide sequence was present in the FREM1 gene of the tetrapods, but not in fish orthologs of FREM1, and neither FREM1 nor TILRR were present in invertebrates. The TILRR gene appears to have arisen via incorporation of adjacent non-coding DNA with a contiguous exonic sequence after the teleost divergence. Comparing co-receptors in other systems, points to their origin during the same stages of evolution. Our results show that modern teleost fish do not possess the IL-1RI co-receptor TILRR, but that this is maintained in tetrapods as early as amphibians. Further, they are consistent with data showing that co-receptors are recent additions to these regulatory systems and suggest this may underlie differences in innate immune responses between mammals and fish.
PMCID: PMC4009816  PMID: 24832504
TILRR; IL-1RI; co-receptor; FREM1; bioinformatics; evolutionary development; signal transduction; TIR activation; IL-1
3.  Evaluating the Evidence for Transmission Distortion in Human Pedigrees 
Genetics  2012;191(1):215-232.
Children of a heterozygous parent are expected to carry either allele with equal probability. Exceptions can occur, however, due to meiotic drive, competition among gametes, or viability selection, which we collectively term “transmission distortion” (TD). Although there are several well-characterized examples of these phenomena, their existence in humans remains unknown. We therefore performed a genome-wide scan for TD by applying the transmission disequilibrium test (TDT) genome-wide to three large sets of human pedigrees of European descent: the Framingham Heart Study (FHS), a founder population of European origin (HUTT), and a subset of the Autism Genetic Resource Exchange (AGRE). Genotyping error is an important confounder in this type of analysis. In FHS and HUTT, despite extensive quality control, we did not find sufficient evidence to exclude genotyping error in the strongest signals. In AGRE, however, many signals extended across multiple SNPs, a pattern highly unlikely to arise from genotyping error. We identified several candidate regions in this data set, notably a locus in 10q26.13 displaying a genome-wide significant TDT in combined female and male transmissions and a signature of recent positive selection, as well as a paternal TD signal in 6p21.1, the same region in which a significant TD signal was previously observed in 30 European males. Neither region replicated in FHS, however, and the paternal signal was not visible in sperm competition assays or as allelic imbalance in sperm. In maternal transmissions, we detected no strong signals near centromeres or telomeres, the regions predicted to be most susceptible to female-specific meiotic drive, but we found a significant enrichment of top signals among genes involved in cell junctions. These results illustrate both the potential benefits and the challenges of using the TDT to study transmission distortion and provide candidates for investigation in future studies.
PMCID: PMC3338262  PMID: 22377632
4.  Two Evolutionary Histories in the Genome of Rice: the Roles of Domestication Genes 
PLoS Genetics  2011;7(6):e1002100.
Genealogical patterns in different genomic regions may be different due to the joint influence of gene flow and selection. The existence of two subspecies of cultivated rice provides a unique opportunity for analyzing these effects during domestication. We chose 66 accessions from the three rice taxa (about 22 each from Oryza sativa indica, O. sativa japonica, and O. rufipogon) for whole-genome sequencing. In the search for the signature of selection, we focus on low diversity regions (LDRs) shared by both cultivars. We found that the genealogical histories of these overlapping LDRs are distinct from the genomic background. While indica and japonica genomes generally appear to be of independent origin, many overlapping LDRs may have originated only once, as a result of selection and subsequent introgression. Interestingly, many such LDRs contain only one candidate gene of rice domestication, and several known domestication genes have indeed been “rediscovered” by this approach. In summary, we identified 13 additional candidate genes of domestication.
Author Summary
The origin of two cultivated rice Oryza sativa indica and O. sativa japonica has been an interesting topic in evolutionary biology. Through whole-genome sequencing, we show that the rice genome embodies two different evolutionary trajectories. Overall genome-wide pattern supports a history of independent origin of two cultivars from their wild population. However, genomic segments bearing important agronomic traits originated only once in one population and spread across all cultivars through introgression and human selection. Population genetic analysis allows us to pinpoint 13 additional candidate domestication genes.
PMCID: PMC3111475  PMID: 21695282
5.  Human Endogenous Retrovirus K106 (HERV-K106) Was Infectious after the Emergence of Anatomically Modern Humans 
PLoS ONE  2011;6(5):e20234.
HERV-K113 and HERV-K115 have been considered to be among the youngest HERVs because they are the only known full-length proviruses that are insertionally polymorphic and maintain the open reading frames of their coding genes. However, recent data suggest that HERV-K113 is at least 800,000 years old, and HERV-K115 even older. A systematic study of HERV-K HML2 members to identify HERVs that may have infected the human genome in the more recent evolutionary past is lacking. Therefore, we sought to determine how recently HERVs were exogenous and infectious by examining sequence variation in the long terminal repeat (LTR) regions of all full-length HERV-K loci. We used the traditional method of inter-LTR comparison to analyze all full length HERV-Ks and determined that two insertions, HERV-K106 and HERV-K116 have no differences between their 5′ and 3′ LTR sequences, suggesting that these insertions were endogenized in the recent evolutionary past. Among these insertions with no sequence differences between their LTR regions, HERV-K106 had the most intact viral sequence structure. Coalescent analysis of HERV-K106 3′ LTR sequences representing 51 ethnically diverse individuals suggests that HERV-K106 integrated into the human germ line approximately 150,000 years ago, after the emergence of anatomically modern humans.
PMCID: PMC3102101  PMID: 21633511
6.  Total Synthesis and Selective Activity of a NewClass of Conformationally Restrained Epothilones 
Stereoselective total syntheses of two novel conformationally restrained epothilone analogues are described. Evans asymmetric alkylation, Brown allylation, and a diastereoselective aldol reaction served as the key steps in the stereoselective synthesis of one of the two key fragments of the convergent synthetic approach.Enzyme resolution was employed to obtain the second fragment as a single enantiomer. The molecules were assembled by esterification, followed by ring-closing metathesis. In preliminary cytotoxicity studies, one of the analogues showed strong and selective growth inhibitory activity against two leukemia cell lines over solid human tumor cell lines. The precise biological mechanism of action and high degree of selectivity of this analogue remain to be examined.
PMCID: PMC2712887  PMID: 17955508
antitumor agents; epothilone analogues; macrolide; natural products; total synthesis
7.  Inhibition of Choline Transport by Redox-active Cholinomimetic Bis-catechol Reagents 
Bioorganic & medicinal chemistry  2007;15(22):7042-7047.
Both N,N′-(2,3-dihydroxybenzyl)-N,N,N′,N′-tetramethyl-1,6-hexanediamine dibromide (DTH, 6) and N,N′-(2,3-dihydroxybenzyl)-N,N,N′,N′-tetramethyl-1,10-decanediamine dibromide (DTD, 7), which are symmetrical bis-catechol substituted hexamethonium and decamethonium analogues, respectively, were found to inhibit high affinity choline transport in mouse brain synaptosomes. Inhibitory properties were evaluated using an extraordinarily sensitive capillary electrophoresis method employing electrochemical detection at an enzyme-modified microelectrode. Dose-response curves were generated for each inhibitor and IC50 values were determined to be 76 μM for 6 and 21 μM for 7. Lineweaver-Burk analysis revealed that both molecules inhibit high affinity choline uptake by a mixed inhibition mechanism. The KI values for 6 and 7 were determined to be 73 ± 1 and 31 ± 2 μM, respectively. The inhibition properties were further compared to a series of mono-catechol analogues, 3-[(trimethylammonio)methyl]catechol (1), N,N-dimethylepinephrine (4) and 6-hydroxy-N,N-dimethylepinephrine (5), as well as the well-characterized hemicholinium inhibitors, hemicholinium-15 (HC-15, 8) and hemicholinum-3 (HC-3, 9).
PMCID: PMC2094012  PMID: 17827016
high-affinity choline transport; cholinomimetic inhibitors; neuronal degradation; capillary electrophoresis with electrochemical detection; enzyme microelectrode
8.  Shiga Toxin–producing Escherichia coli, Idaho 
Emerging Infectious Diseases  2007;13(8):1262-1264.
PMCID: PMC2828089  PMID: 17953111
Escherichia coli; O157; immunoassay; hemolytic uremic syndrome; Idaho; Shiga toxin; letter
9.  Transcription increases multiple spontaneous point mutations in Salmonella enterica 
Nucleic Acids Research  2003;31(15):4517-4522.
The spontaneous rate of G·C→A·T mutations and a hotspot T·A→G·C transversion are known to increase with the frequency of transcription—increases that have been ascribed primarily to processes that affect only these specific mutations. To investigate how transcription induces other spontaneous point mutations, we tested for its effects in repair-proficient Salmonella enterica using reversion assays of chromosomally inserted alleles. Our results indicate that transcription increases rates of all tested point mutations in the induced gene: induction significantly increased the individual rates of an A·T→T·A transversion, an A·T→G·C transition and the pooled rates of the three other point mutations assayed. Although the S.enterica genome is thought to have a mutational bias towards G·C base pairs, transitions creating A·T pairs were approximately 10 times more frequent than the reverse mutation, resulting in an overall mutation pressure to lower G+C contents. Transitions occurred at roughly twice the rate of transversions, similar to results from sequence comparisons; however, several individual transversions are more frequent than the least common transition.
PMCID: PMC169952  PMID: 12888512
10.  Identification of a Subset of Human Immunodeficiency Virus Type 1 (HIV-1), HIV-2, and Simian Immunodeficiency Virus Strains Able To Exploit an Alternative Coreceptor on Untransformed Human Brain and Lymphoid Cells 
Journal of Virology  2003;77(11):6138-6152.
The chemokine receptors CCR5 and CXCR4 are the major coreceptors for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). At least 12 other chemokine receptors or close relatives support infection by particular HIV and SIV strains on CD4+ transformed indicator cell lines in vitro. However, the role of these alternative coreceptors in vivo is presently thought to be insignificant. Infection of cell lines expressing high levels of recombinant CD4 and coreceptors thus does not provide a true indication of coreceptor use in vivo. We therefore tested primary untransformed cell cultures that lack CCR5 and CXCR4, including astrocytes and brain microvascular endothelial cells (BMVECs), for naturally expressed alternative coreceptors functional for HIV and SIV infection. An adenovirus vector (Ad-CD4) was used to express CD4 in CD4− astrocytes and thus confer efficient infection if a functional coreceptor is present. Using a large panel of viruses with well-defined coreceptor usage, we identified a subset of HIV and SIV strains able to infect two astrocyte cultures derived from adult brain tissue. Astrocyte infection was partially inhibited by several chemokines, indicating a role for the chemokine receptor family in the observed infection. BMVECs were weakly positive for CD4 but negative for CCR5 and CXCR4 and were susceptible to infection by the same subset of isolates that infected astrocytes. BMVEC infection was efficiently inhibited by the chemokine vMIP-I, implicating one of its receptors as an alternative coreceptor for HIV and SIV infection. Furthermore, we tested whether the HIV type 1 and type 2 strains identified were able to infect peripheral blood mononuclear cells (PBMCs) via an alternative coreceptor. Several strains replicated in Δ32/Δ32 CCR5 PBMCs with CXCR4 blocked by AMD3100. This AMD3100-resistant replication was also sensitive to vMIP-I inhibition. The nature and potential role of this alternative coreceptor(s) in HIV infection in vivo is discussed.
PMCID: PMC155019  PMID: 12743271
11.  Coordinated Response to Reports of Possible Anthrax Contamination, Idaho, 2001 
Emerging Infectious Diseases  2002;8(10):1093-1095.
In 2001, the intentional release of anthrax spores in the eastern United States increased concern about exposure to anthrax nationwide, and residents of Idaho sought assistance. Response from state and local agencies was required, increasing the strain on epidemiologists, laboratorians, and communications personnel. In late 2001, Idaho’s public health communications system handled 133 calls about suspicious powders. For each call, a multiagency bridge call was established, and participants (public health officials, epidemiologists, police, Federal Bureau of Investigation personnel, hazardous materials officials, and others) determined which samples would be tested by the state public health laboratory. A triage system for calls helped relieve the burden on public safety and health systems.
PMCID: PMC2730284  PMID: 12396922
anthrax; bioterrorism; Idaho
12.  Sodium, an Obligate Growth Requirement for Predominant Rumen Bacteria1 
Applied Microbiology  1974;27(3):549-552.
Sodium is an obligate growth requirement for most currently recognized predominant species of rumen bacteria. The isoosmotic deletion of Na+ from a nutritionally adequate defined medium completely eliminated growth of most species. Growth yields and rates were both a function of Na+ concentration for Na+-requiring species, and Na+ could not be replaced by Rb+, Li+, or Cs+ when these ions were substituted for Na+ at a concentration equivalent to an Na+ concentration that supported abundant growth. Li+, Cs+, or Rb+ was toxic at an Na+-replacing concentration (15 mM) but not at a K+-replacing concentration (0.65 mM). K+ was also an obligate growth requirement for rumen bacteria in media containing Na+ and K+ as major monovalent cations, but K+ could be replaced, for most species, by Rb+. The quantities of Na+ that support rapid and abundant growth of Na+-requiring rumen bacteria show that these organisms are slight halophiles. A growth requirement for Na+ appears more frequent among nonmarine bacteria than has been previously believed.
PMCID: PMC380082  PMID: 4856854

Results 1-12 (12)