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PubMed2005

The phylogenetic and evolutionary history of a novel alpha-globin-type gene in orangutans (Pongo pygmaeus).

The alpha-globin genes are implicated in human resistance to malaria, a disease caused by Plasmodium parasites. This study is the first to analyze DNA sequences from a novel alpha-globin-type gene in orangutans, a species affected by Plasmodium. Phylogenetic methods show that the gene is a duplication of an alpha-globin gene and is located 5' of alpha-2 globin. The alpha-globin-type gene is notable for having four amino acid replacements relative to the orangutan's alpha-1 and alpha-2 globin genes, with no synonymous differences. Pairwise K(a)/K(s) methods and likelihood ratio tests (LRTs) revealed that the evolutionary history of the alpha-globin-type gene has been marked by either neutral or positive evolution, but not purifying selection. A comparative analysis of the amino acid replacements of the alpha-globin-type gene with human hemoglobinopathies and hemoglobin structure showed that two of the four replaced sites are members of the same molecular bond, one that is crucial to the proper functioning of the hemoglobin molecule. This suggested an adaptive evolutionary change. Functionally, this locus may result in a thalassemia-like phenotype in orangutans, possibly as an adaptation to combat Plasmodium.

Amino Acid Sequence↗

PubMed2004

Evolution of developmental traits.

The evolution of plant development can be studied in many different ways, each of which provides new insights into how plants have been modified over evolutionary time. DNA sequencing shows that most developmental genes are under purifying selection and that obvious adaptive change in proteins is rare. This may indicate that most change occurs in cis-regulatory sequences, that tests for detecting selection lack power, or both. Gene duplications are common and often correlate with divergence of function, as predicted by theory. Studies of gene expression illuminate similarities among structures in disparate plant groups and indicate that the same genes have been deployed repeatedly for similar developmental ends. Comparative functional studies remain uncommon, but promise to illuminate how changing proteins lead to changes in development. Precise characterization of phenotypes by studies of developmental morphology is beginning to occur in some taxonomic groups. The genetic variation necessary for morphological change must originate as allelic polymorphism within populations; such polymorphism has been identified in grasses and in sunflowers, although it is often cryptic.

Arabidopsis↗

PubMed2006

Rh proteins vs Amt proteins: an organismal and phylogenetic perspective on CO2 and NH3 gas channels.

Rh (Rhesus) proteins are homologues of ammonium transport (Amt) proteins. Physiological and structural evidence shows that Amt proteins are gas channels for NH(3), but the substrate of Rh proteins, be it CO2 as shown in green alga, or NH3/NH4+ as shown in mammalian cells, remains disputed. We assembled a large dataset generated of Rh and Amt to explore how Rh originated from and evolved independently of Amt relatives. Analysis of this rich data implies that Rh was split from Amt first to emerge in archaeal species. The Rh ancestor underwent divergence and duplication along speciation, leading to neofunctionalization and subfunctionalization of the Rh family. The characteristic organismal distribution of Rh vs. Amt reflects their early separation and subsequent independent evolution: they coexist in microbes and invertebrates but do not in fungi, vascular plants or vertebrates. Rh gene-duplication was prominent in vertebrates: while epithelial RhBG/RhCG displayed strong purifying selection, erythroid Rh30 and RhAG experienced different episodes of positive selection in each of which adaptive evolution occurred at certain time points and in a few codon sites. Mammalian Rh30 and RhAG were subject to particularly strong positive selection in some codon sites in the lineage from rodents to human. The grounds of this adaptive evolution may be driven by the necessity to increase the surface/volume ratio of biconcave erythrocytes for facilitative gas diffusion. Altogether, these results are consistent with Rh proteins not being the orthologue of Amt proteins but having gained the function for CO2/HCO3- transport, with important roles in systemic pH regulation.

Amino Acid Sequence↗

PubMed2006

The West Nile virus mutant spectrum is host-dependant and a determinant of mortality in mice.

To define the impact of mosquitoes and birds on intrahost WNV population dynamics, the mutant spectra that arose as a result of 20 serial in vivo passages in Culex pipiens and young chickens were examined. Genetically homogeneous WNV was serially passaged 20 times in each host. Genetic diversity was greater in mosquito-passaged WNV compared to chicken-passaged WNV. Changes in the viral consensus sequence occurred in WNV passaged in mosquitoes earlier and more frequently than in chicken-passaged WNV. Analysis of synonymous and nonsynonymous variation suggested that purifying selection was relaxed during passage in mosquitoes. Mortality in mice was significantly negatively correlated with the size of the WNV mutant spectrum. These studies suggest that mosquitoes serve as sources for WNV genetic diversity, that birds are selective sieves, and that both the consensus sequence and the mutant spectrum contribute to WNV phenotype.

Animals↗

PubMed2006

Diversity and evolution of West Nile virus in Illinois and the United States, 2002-2005.

Evolutionary analyses of West Nile virus (WNV) have been limited by uneven sampling across geographic regions and over time. In this study, an expanded data set of 68 WNV envelope gene sequences from the Midwest (Illinois) was created and combined with published sequences to investigate spatial and temporal structuring in the United States viral population. Results indicate an overall lack of geographic structure to WNV in the United States, supporting the notion of WNV as a rapidly expanding pathogen not significantly restricted in its spread by geographic distance. However, analyses of viral genetic diversity show a steady increase in WNV nucleotide-level diversity over time. Additionally, evolutionary rate calculations indicate that WNV has evolved at approximately 0.85 x 10(-3) substitutions/site/year, largely through neutral substitution and purifying selection. Overall, these results show WNV across the United States to be a panmictic viral population that is diversifying and evolving.

Analysis of Variance↗

PubMed2004

Complete genome sequencing of dengue virus type 1 isolated in Buenos Aires, Argentina.

Dengue (DEN) constitutes a major viral arthropod-borne human illness. South America was last considered free of dengue two decades ago when a dramatic increase in the number of dengue fever and hemorrhagic dengue cases had been reported. Five viruses were isolated in Buenos Aires City from the 1999-2000 Paraguay outbreak. RT-PCRs obtained directly from plasma were cloned into pGemT vectors and sequences of the structural genes and NS1 were analyzed. Three viruses were full-length sequenced from RT-PCR obtained from cell-culture isolates. Excess of synonymous over non-synonymous mutations suggested that the structural proteins were under strong functional constraints while a weak purifying selection was operating in the whole polyprotein. Sequence diversity and selective pressures varied among patients but results were significantly above the procedure threshold. One sample showed small-plaque phenotype and impaired growth coupled to 3'untranslated region mutations. Phylogenetic analysis of full-length sequences split Buenos Aires isolates into two clusters within American DEN-1 genotype V: Clade I was phylogenetically linked to Brazilian samples and Clade II with samples from Paraguay and Northeastern Argentina. In Buenos Aires City, only dengue virus serotype 1 imported from Paraguay has been detected, though without evidence of local transmission.

3' Untranslated Regions↗

PubMed2005

In vivo characterization of a vertebrate ultraconserved enhancer.

Genomic sequence comparisons among human, mouse, and pufferfish (Takifugu rubripes (Fugu)) have revealed a set of extremely conserved noncoding sequences. While this high degree of sequence conservation suggests severe evolutionary constraint and predicts a lack of tolerance to change to retain in vivo functionality, such elements have been minimally explored experimentally. In this study, we describe the in-depth characterization of an ancient conserved enhancer, Dc2, located near the dachshund gene, which displays a human-Fugu identity of 84% over 424 basepairs (bp). In addition to this large overall conservation, we find that Dc2 is characterized by the presence of a large block of sequence (144 bp) that is completely identical among human, mouse, chicken, zebrafish, and Fugu. Through the testing of reporter vector constructs in transgenic mice, we observed that the 424-bp Dc2-conserved element is necessary and sufficient for brain tissue enhancer activity. In vivo analyses also revealed that the 144-bp 100% conserved sequence is necessary, but not sufficient, to replicate Dc2 enhancer function. However, the introduction of two separate 16-bp insertions into the highly conserved enhancer core did not cause any detectable modification of its in vivo activity. Our observations indicate that the 144-bp 100% conserved element is tolerant of change at least at the resolution of this transgenic mouse assay and suggest that purifying selection on the Dc2 sequence might not be as strong as we predicted or that some unknown property also constrains this highly conserved enhancer sequence.

Animals↗

PubMed2003

Molecular evolution of the maize sex-determining gene TASSELSEED2 in Bouteloua (Poaceae).

The majority of angiosperms produce hermaphrodite flowers, while a lesser number (20-30%) produce unisexual flowers. Little is known about the molecular biology of sex-determination in angiosperms, however, a few sex-determining genes have been cloned from the model system Zea mays. One of these genes is Tasselseed2 (Ts2) which has been shown to be involved in the arrest of developing pistils in male flowers. In this study, we sequenced a putative homologue of Ts2 in species of Bouteloua, a genus in the grass subfamily Chloridoideae. We found significant genetic variation at Ts2 in Bouteloua relative to other developmental genes characterized in maize and other grass species. We also found that in Bouteluoua, Ts2 is evolving non-neutrally in the hermaphrodite-flowered Bouteloua hirsuta while no difference from neutral expectation was detected at Ts2 in the monoecious/dioecious Bouteloua dimorpha. The putatively neutral gene Alcohol Dehydrogenase1 (Adh1) was also examined for the same species of Bouteloua, and no departure from neutral expectation was detected. Our results suggest that purifying selection may be acting on Ts2 in the hermaphrodite-flowered B. hirsuta while no evidence of selection was detected at Ts2 in the monoecious/dioecious B. dimorpha.

Base Sequence↗

PubMed2005

The mammalian alphaD-globin gene lineage and a new model for the molecular evolution of alpha-globin gene clusters at the stem of the mammalian radiation.

We have explored the evolution of the alpha-globin gene family by comparative sequence and phylogenetic analyses of mammalian alpha-globin genes. Our analyses reveal the existence of a new alpha-globin gene lineage in mammals that is related to the alpha(D)-globin genes of birds, squamates and turtles. The gene is located in the middle of the alpha-globin gene cluster of a marsupial, Sminthopsis macroura and of humans. It exists in a wide variety of additional mammals, including pigs, cows, cats, and dogs, but is a pseudogene in American marsupials. Evolutionary analyses suggest that the gene has generally evolved under purifying selection, indicative of a functional gene. The presence of mRNA products in humans, pigs, and cows also suggest that the gene is expressed and likely to be functional. The analyses support the hypothesis that the alpha(D)-globin gene lineage has an ancient evolutionary origin that predates the divergence of amniotes. The structural similarity of alpha-globin gene clusters of marsupials and humans suggest that an eight gene cluster (5'-zeta2-zeta1-alpha(D)-alpha3-alpha2-alpha1-theta-omega-3'), including seven alpha-like genes and one beta-like globin gene (omega-globin) existed in the common ancestor of all marsupial and eutherian mammals. This basic structure has remained relatively stable in marsupials and in the lineage leading to humans, although omega-globin has been lost from the alpha-globin gene cluster of humans.

Animals↗

PubMed2002

Mutations in the S4-H2 loop of eIF4E which increase the affinity for m7GTP.

Eukaryotic initiation factor 4E (eIF4E) binds the 5'-cap of eukaryotic mRNAs and overexpression of eIF4E in epithelial cell cancers correlates with the metastases/tissue invasion phenotype. Photolabeling of eIF4E with [gamma-32P]8-azidoguanosine 5'-triphosphate (8-N3GTP) demonstrated cross-linking at Lys-119 in the S4-H2 loop which is distant from the m7GTP binding site [Marcotrigiano et al. (1997) Cell 89, 951-961; Friedland et al. (1997) Protein Sci. 6, 125-131]. Modeling studies indicate that 8-N3GTP cross-linked with Lys-119 because it binds a site that is occupied by the second nucleotide of a bound mRNA. Mutagenesis of the S4-H2 loop produced proteins with a 5-10-fold higher affinity for m7GTP than wild-type eIF4E. These mutants of eIF4E may have uses in selectively purifying mRNAs with intact 5'-ends or in determining how the promyelocytic leukemia protein decreases the affinity of eIF4E for mRNA caps.

Animals↗

PubMed1998

Quantitative analysis of methionine enkephalin and beta-endorphin in the pituitary by liquid secondary ion mass spectrometry and tandem mass spectrometry.

This manuscript reviews the use of an off-line combination of liquid chromatography (LC) and mass spectrometry (MS) to quantify endogenous neuropeptides in biological tissues and fluids, and tandem MS (MS/MS) to optimize the molecular specificity of the quantification of native peptides. Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to purify selected endogenous neuropeptides from biological tissues and fluids. Liquid secondary ion MS (LSI-MS), also known as fast atom bombardment (FAB), is used to desorb and to ionize the peptide. The corresponding stable isotope-incorporated synthetic peptide of each peptide is used as the internal standard (I.S.) for quantification. The measurement of methionine enkephalin (ME) and of beta-endorphin1-31 (BE) in the human pituitary is described. This analytical method offers the highest molecular specificity for the measurement of a fully post-translationally modified peptide.

Animals↗

PubMed1998

Development of monoclonal antibodies specific for human glandular kallikrein (hK2): development of a dual antibody immunoassay for hK2 with negligible prostate-specific antigen cross-reactivity.

OBJECTIVES: Human glandular kallikrein (hK2) is a protein that is 80% homologous to prostate-specific antigen (PSA), and, like PSA, is localized to the prostate. We developed a specific immunoassay for hK2 that can be used to evaluate its clinical diagnostic utility. METHODS: We developed monoclonal antibodies (mAbs) specific for hK2 by immunizing with hK2 and screening for clones reactive with hK2 and not PSA. Prototype sandwich assays using these mAbs were tested, and the optimum pair selected. Purified hK2 was used as standard and PSA cross-reactivity was assessed in the assay. Both hK2 and hK2-alpha1-antichymotrypsin (ACT) complexes have been identified in sera of patients with prostate cancer (PCa). Serum samples (n = 671) from healthy volunteers and patients with prostate disease were assayed for hK2 and PSA levels. RESULTS: The assay had a detection limit of less than 0.12 ng/mL and a less than 0.5% cross-reactivity with PSA. The assay preferentially detected free hK2 with a 3.5-fold higher molar response than with hK2-ACT. The mean serum concentration of hK2 in normal control samples was low (0.33 and 0.37 ng/mL for normal healthy men and women, respectively) but was elevated in patients with prostate disease (0.86 and 6.77 ng/mL for patients with benign prostatic hyperplasia and PCa, respectively). Negligible cross-reactivity to hK2 was measured by Tandem PSA assays (Hybritech). CONCLUSIONS: Significant concentrations of hK2, relative to PSA, were detected in human serum, especially in patients with prostate disease. Serum hK2 concentrations were not proportional to PSA concentration. Therefore, hK2 has the potential to be an independent and clinically useful marker for PCa.

Antibodies, Monoclonal↗

PubMed2003

Characterization of beta(2)-microglobulin coding sequence from three non-placental mammals: the duckbill platypus, the short-beaked echidna, and the grey short-tailed opossum.

To further characterize genes of immunological importance from non-placental mammals, cDNAs encoding beta(2)-microglobulin (beta(2)m) were isolated from two prototherians, the platypus and an echidna, and one metatherian, a grey short-tailed opossum. In addition, a second allele of beta(2)m was identified in another metatherian species, the brushtail possum. Analysis of the deduced translations revealed conservation of key residues in these molecules over a long evolutionary history. The types of nucleotide substitutions present among the various taxa are also consistent with purifying selection at this conserved locus. An evolutionary tree of beta(2)m was constructed that supports the classic view of evolution with prototherians as the basal mammalian group.

Amino Acid Sequence↗

PubMed1993

Differential regulation of IL-9-expression after infection with Leishmania major in susceptible and resistant mice.

IL-9 is a pleiotropic lymphokine, one of its activities being the growth stimulation of certain CD4+ T lymphocytes. In murine cutaneous leishmaniasis, depending on the genetic background of the host mouse strain, vigorous proliferation of either mainly Th1 in resistant C57BL/6 mice or Th2-type CD4+ T cells in susceptible BALB/c mice occurs after infection with Leishmania major (L. major). Since little is known about the involvement of IL-9, the possible role of this cytokine with regard to its immunregulatory function was evaluated by comparing its presence in the serum and its expression kinetics in spleen and lymph nodes in resistant and susceptible mice. To this sera of L. major-infected mice were tested functionally for IL-9. In addition the PCR-aided detection of IL-9 mRNA in organs of mice and measurement of the lymphokine in supernatants of restimulated lymph node and spleen cell cultures were used. We show here that although no functionally active IL-9 was detected in sera of both BALB/c and C57BL/6 mice, IL-9 is produced after in vitro antigenic restimulation and its mRNA was found to be expressed in lymph nodes and spleens during an immune response against L. major. Shortly after infection no principal differences in the kinetics of IL-9 expression could be observed, which had its maximum between day 5 and 7 after infection. The rate of production however was higher in the susceptible BALB/c mice. In athymic BALB/c nu/nu mice and in mice depleted of CD4+ T cells no IL-9 production was detectable in vivo at the level of mRNA and no IL-9 was produced after stimulation with L. major antigen in vitro. Treatment of infected mice with cyclosporin A ablates antigen-specific IL-9 production when tested in vitro without affecting its production after polyclonal T cell stimulation. Positively selected, purified CD4+ T cells were fully capable of producing IL-9. From 4 weeks after infection, IL-9 synthesis was observed only in BALB/c mice, correlating with the expansion of antigen-specific Th2 type T helper cells in these mice. Treatment of BALB/c mice with neutralizing anti-IL-4 mAb, a regimen known to lead to subsequent cure of infected BALB/c mice, suppressed late IL-9 synthesis.

Animals↗

PubMed2002

Diversification of olfactory receptor genes in the Japanese medaka fish, Oryzias latipes.

Vertebrate olfactory receptors (OR) exists as the largest multigene family, scattered throughout the genome in clusters. Studies have shown that different animals possess remarkably diverse set of OR genes to recognize diverse odor molecules. In order to examine the evolutionary process of OR diversification, we examined three OR gene subfamilies from Japanese medaka fish (seven lines sampled from four populations). For each subfamily, the sequences of ancestral genes were inferred based on distance method. Examination of d(N)/d(S) ratios for each branch of phylogenetic trees suggested that purifying selection is the major force of evolution in medaka OR genes. However, for the mfOR1 and mfOR2 paralogous gene pairs, a nonrandom distribution of fixed amino acid changes and the d(N)>d(S) in a branch suggested that diversifying selection occurred after gene duplication. The fixed amino acid changes were observed in the third, fifth and sixth transmembrane domains, which has been predicted to serve as a ligand-binding pocket in a structural model. Compatibility test suggested that interlocus recombinations involving the fourth transmembrane domain occurred between the mfOR1 and mfOR2 gene pairs. The pattern of nucleotide substitutions in other OR genes agrees with the hypothesis that a limited number of amino acid residues are involved in odorant binding. Such comparative analyses of paralogous OR genes should provide bases for understanding the evolution, the structure, and the functional specificity of OR genes.

Amino Acid Sequence↗

PubMed2003

A co-opted gypsy-type LTR-retrotransposon is conserved in the genomes of humans, sheep, mice, and rats.

One subset of sequences present within mammalian genomes is the retroelements, which include endogenous retroviruses and retrotransposons. While there are typically thousands of copies of endogenous retroviruses within mammalian hosts, almost no LTR-retrotransposon-like sequences have been identified. Here, we report the presence of a remarkably intact and conserved gypsy-type LTR-retrotransposon sequence within the genomes of several mammals, including humans and mice. Each host probably contains a single orthologous element, indicating that the original, ancestral gypsy LTR-retrotransposon first integrated into mammals over 70 million years ago. It is thus the first described example of a near-intact orthologous retroelement within humans and mice and is one of the most ancient retroelement sequences described to date. Despite their extreme age, the orthologs within each species examined contain a large ORF, between 4.0 and 5.2 kb in length, encoding proteins with sequence similarity to LTR-retrotransposon-derived Capsid (CA), Protease (PR), Reverse Transcriptase (RT), RibonucleaseH (RNaseH), and Integrase (IN). Calculation of nonsynonymous and synonymous nucleotide substitution frequencies indicated that the encoded proteins are under purifying selection, suggesting that these elements have, in fact, been co-opted by their hosts. A possible function for these elements, involving gypsy LTR-retrotransposon restriction in mammals, is discussed.

Amino Acid Sequence↗

PubMed2003

Origin, world-wide dispersion, bio-geographical diversification, radiation and recombination: an evolutionary history of Yam mild mosaic virus (YMMV).

We developed an evolutionary epidemiological approach to understand the regional and world-wide dispersion of Yam mild mosaic virus (YMMV) by retracing its evolutionary history. Analyses of the distribution and the prevalence of YMMV in the Caribbean islands of Guadeloupe and Martinique, and in French Guyana revealed that YMMV has a wide repartition and different prevalence on Dioscorea alata L. (Asian and Oceanic origin), on D. cayenensis Lam.-D. rotundata Poir. (African origin) and on D. trifida L. (Amazon and the Caribbean origin) in this region. Considering the data on the current dispersion of the virus and the evolution and the history of the yams, the phylogenetic analysis of the 3' terminal part of the YMMV genome gave a consistent support of the Asian-Pacific origin of YMMV from D. alata species. The YMMV phylogenetic tree is star-like, suggesting an early split of the genetic lineages. An important part of the clades is constituted by a single lineage arisen by recombination. The largest emerging monophyletic group illustrates well YMMV geographical dispersion. This evolutionary pattern contrasts with the one revealed by the African distinct lineages and by the second significant monophyletic group, for which a host adaptation to D. trifida is suggested. The analysis of the pattern of nucleotide substitutions in the CP gene revealed that purifying selection dominates the evolution of the CP of potyviruses and strongly operates on the YMMV. Switching events, radiation, host and geographical adaptation and recombination events are proposed as major traits of YMMV evolutionary history.

3' Flanking Region↗

PubMed2002

Nucleotide polymorphism at the Atmyb2 locus of the wild plant Arabidopsis thaliana.

DNA variation was studied in a 2.2 kb region of the regulatory gene Atmyb2 using 20 ecotypes of Arabidopsis thaliana and one accession each of Arabis gemmifera and Arabidopsis himalaica. Nucleotide diversity (pi) in the region was 0.0027, which was lower than for other loci in A. thaliana. The MYB domain of the Atmyb2 gene (pi = 0.0036) had a larger variation than the non-MYB region (pi = 0.0013). Tajima's test and Fu and Li's test did not give a significant result. In contrast to the low level of polymorphism, the degree of divergence of the Atmyb2 region was higher between A. thaliana and A. gemmifera (K = 0.0730) than for other loci. The MYB domain (K = 0.0436) had smaller divergence than the non-MYB region (K = 0.0939). The HKA test detected significant discordance in the ratio of polymorphism to divergence in some comparisons. The pattern of low polymorphism and high divergence, which is mainly observed in the non-MYB region of the gene, is inconsistent with the neutral mutation theory. Strong purifying selection after establishment of A. thaliana and a species-specific adaptive process could be invoked to account for this pattern of polymorphism and divergence of Atmyb2.

Arabidopsis↗