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PubMed2005

A family of fatty acid binding receptors.

The family of G protein-coupled receptors (GPCRs) serves as the target for almost a third of currently marketed drugs, and provides the predominant mechanism through which extracellular factors transmit signals to the cell. The discovery of GPCRs with no known ligand has initiated a frenzy of research, with the aim of elucidating the physiological ligands for these "orphan" receptors and revealing new drug targets. The GPR40 family of receptors, tandemly located on chromosome 19q13.1, exhibit 30-40% homology to one another and diverse tissue distribution, yet all are activated by fatty acids. Since agonists of GPR40 are medium to longchain fatty acids and those for GPR41 and 43 are short-chain fatty acids, the family clearly provides an intriguing example of how the ligand specificity, patterns of expression, and function of GPCRs can diverge through evolution. Here we summarize the identification, structure, and pharmacology of the receptors and speculate on the respective physiological roles that the GPR40 family members may play.

Amino Acid Sequence↗
PubMed2004

Diacylglycerol-dependent binding recruits PKCtheta and RasGRP1 C1 domains to specific subcellular localizations in living T lymphocytes.

Diacylglycerol (DAG) signaling relies on the presence of conserved domain 1 (C1) in its target proteins. Phospholipase C-dependent generation of DAG after T cell receptor (TCR) triggering is essential for the correct immune response onset. Accordingly, two C1-containing proteins expressed in T lymphocytes, Ras guanyl nucleotide-releasing protein1 (RasGRP1) and protein kinase C (PKC), were shown to be fundamental for T-cell activation and proliferation. Although containing the same regulatory domain, they are proposed to relocate to distinct subcellular locations in response to TCR triggering. Here we studied intracellular localization of RasGRP1 and PKC C1 domains in living Jurkat T cells. The results demonstrate that, in the absence of significant primary sequence differences, the C1 domains of these proteins show specific localization within the cell and distinct responses to pharmacological stimulation and TCR triggering. These differences help explain the divergent localization and distinct functional roles of the full-length proteins, which contains them. The properties of these DAG-binding modules allow their characterization as functional markers that discriminate between DAG pools. Finally, we show that by binding to different diacylglycerol forms, overexpression of distinct C1 modules can attenuate DAG-dependent signals originating from the plasma or internal membranes. This is shown by analyzing the contribution of these two lipid pools to PLC-dependent Ras activation in response to TCR triggering.

Amino Acid Motifs↗
PubMed1998

The effect of mismatch repair and heteroduplex formation on sexual isolation in Bacillus.

In Bacillus transformation, sexual isolation is known to be an exponential function of the sequence divergence between donor and recipient. Here, we have investigated the mechanism under which sequence divergence results in sexual isolation. We tested the effect of mismatch repair by comparing a wild-type strain and an isogenic mismatch-repair mutant for the relationship between sexual isolation and sequence divergence. Mismatch repair was shown to contribute to sexual isolation but was responsible for only a small fraction of the sexual isolation observed. Another possible mechanism of sexual isolation is that more divergent recipient and donor DNA strands have greater difficulty forming a heteroduplex because a region of perfect identity between donor and recipient is required for initiation of the heteroduplex. A mathematical model showed that this heteroduplex-resistance mechanism yields an exponential relationship between sexual isolation and sequence divergence. Moreover, this model yields an estimate of the size of the region of perfect identity that is comparable to independent estimates for Escherichia coli. For these reasons, and because all other mechanisms of sexual isolation may be ruled out, we conclude that resistance to heteroduplex formation is predominantly responsible for the exponential relationship between sexual isolation and sequence divergence in Bacillus transformation.

Bacillus↗
PubMed1999

DNA sequence similarity requirements for interspecific recombination in Bacillus.

Gene transfer in bacteria is notoriously promiscuous. Genetic material is known to be transferred between groups as distantly related as the Gram positives and Gram negatives. However, the frequency of homologous recombination decreases sharply with the level of relatedness between the donor and recipient. Several studies show that this sexual isolation is an exponential function of DNA sequence divergence between recombining substrates. The two major factors implicated in producing the recombinational barrier are the mismatch repair system and the requirement for a short region of sequence identity to initiate strand exchange. Here we demonstrate that sexual isolation in Bacillus transformation results almost exclusively from the need for regions of identity at both the 5' and 3' ends of the donor DNA strand. We show that, by providing the essential identity, we can effectively eliminate sexual isolation between highly divergent sequences. We also present evidence that the potential of a donor sequence to act as a recombinogenic, invasive end is determined by the stability (melting point) of the donor-recipient complex. These results explain the exponential relationship between sexual isolation and sequence divergence observed in bacteria. They also suggest a model for rapid spread of novel adaptations, such as antibiotic resistance genes, among related species.

Bacillus↗
PubMed2005

Assessment of protein distance measures and tree-building methods for phylogenetic tree reconstruction.

Distance-based methods are popular for reconstructing evolutionary trees of protein sequences, mainly because of their speed and generality. A number of variants of the classical neighbor-joining (NJ) algorithm have been proposed, as well as a number of methods to estimate protein distances. We here present a large-scale assessment of performance in reconstructing the correct tree topology for the most popular algorithms. The programs BIONJ, FastME, Weighbor, and standard NJ were run using 12 distance estimators, producing 48 tree-building/distance estimation method combinations. These were evaluated on a test set based on real trees taken from 100 Pfam families. Each tree was used to generate multiple sequence alignments with the ROSE program using three evolutionary models. The accuracy of each method was analyzed as a function of both sequence divergence and location in the tree. We found that BIONJ produced the overall best results, although the average accuracy differed little between the tree-building methods (normally less than 1%). A noticeable trend was that FastME performed poorer than the rest on long branches. Weighbor was several orders of magnitude slower than the other programs. Larger differences were observed when using different distance estimators. Protein-adapted Jukes-Cantor and Kimura distance correction produced clearly poorer results than the other methods, even worse than uncorrected distances. We also assessed the recently developed Scoredist measure, which performed equally well as more complex methods.

Base Sequence↗
PubMed2000

Molecular cloning of cDNA encoding two subunits of calcineurin from scallop testis: demonstration of stage-specific expression during maturation of the testis.

Complementary DNAs encoding two subunits of scallop (Patinopecten yessoensis) testis calcineurin were cloned, and the nucleotide sequences of their coding regions were determined. The deduced amino acid sequences of the catalytic subunit, calcineurin A (486 amino acid residues, M(r) 55,005.91), and the regulatory subunit, calcineurin B (170 residues, M(r) 19,237.67), showed high similarity to those of mammalian calcineurins, especially to the brain-type ones rather than to the testis-specific isoforms. Northern blot analysis showed that only a single species for each subunit was expressed in testis and the expression of each subunit increased dramatically from January to March during the maturation stages of the one-year cycle. The period when the maximum amount of mRNAs for calcineurin was expressed corresponds to the one immediately after meiosis, that is, the maturation stage in which 20-80% of the average testis is occupied by spermatozoa. The result is consistent with the one as to the expression of the testis-specific isoform of calcineurin A in mouse, which occurs immediately after meiosis. This is the first report on the stage-specific expression of calcineurin in invertebrate testis and its sequence similarity to the mammalian brain-type isoforms may indicate that the mammalian testis-specific isoforms appeared in evolution after the divergence of mammals from the mollusks and then diverged rapidly for specific functions in testis.

Amino Acid Sequence↗
PubMed2001

Structure-based phylogenetic analysis of short-chain alcohol dehydrogenases and reclassification of the 17beta-hydroxysteroid dehydrogenase family.

Short-chain alcohol dehydrogenases (SCAD) constitute a large and diverse family of ancient origin. Several of its members play an important role in human physiology and disease, especially in the metabolism of steroid substrates (e.g., prostaglandins, estrogens, androgens, and corticosteroids). Their involvement in common human disorders such as endocrine-related cancer, osteoporosis, and Alzheimer disease makes them an important candidate for drug targets. Recent phylogenetic analysis of SCAD is incomplete and does not allow any conclusions on very ancient divergences or on a functional characterization of novel proteins within this complex family. We have developed a 3D structure-based approach to establish the deep-branching pattern within the SCAD family. In this approach, pairwise superpositions of X-ray structures were used to calculate similarity scores as an input for a tree-building algorithm. The resulting phylogeny was validated by comparison with the results of sequence-based algorithms and biochemical data. It was possible to use the 3D data as a template for the reliable determination of the phylogenetic position of novel proteins as a first step toward functional predictions. We were able to discern new patterns in the phylogenetic relationships of the SCAD family, including a basal dichotomy of the 17beta-hydroxysteroid dehydrogenases (17beta-HSDs). These data provide an important contribution toward the development of type-specific inhibitors for 17beta-HSDs for the treatment and prevention of disease. Our structure-based phylogenetic approach can also be applied to increase the reliability of evolutionary reconstructions in other large protein families.

17-Hydroxysteroid Dehydrogenases↗
PubMed2000

A 39-kb sequence around a blackbird Mhc class II gene: ghost of selection past and songbird genome architecture.

To gain an understanding of the evolution and genomic context of avian major histocompatibility complex (Mhc) genes, we sequenced a 38.8-kb Mhc-bearing cosmid insert from a red-winged blackbird (Agelaius phoeniceus). The DNA sequence, the longest yet retrieved from a bird other than a chicken, provides a detailed view of the process of gene duplication, divergence, and degeneration ("birth and death") in the avian Mhc, as well as a glimpse into major noncoding features of a songbird genome. The peptide-binding region (PBR) of the single Mhc class II B gene in this region, Agph-DAB2, is almost devoid of polymorphism, and a still-segregating single-base-pair deletion and other features suggest that it is nonfunctional. Agph-DAB2 is estimated to have diverged about 40 MYA from a previously characterized and highly polymorphic blackbird Mhc gene, Aph-DAB1, and is therefore younger than most mammalian Mhc paralogs and arose relatively late in avian evolution. Despite its nonfunctionality, Agph-DAB2 shows very high levels of nonsynonymous divergence from Agph-DAB1 and from reconstructed ancestral sequences in antigen-binding PBR codons-a strong indication of a period of adaptive divergence preceding loss of function. We also found that the region sequenced contains very few other unambiguous genes, a partial Mhc- class II gene fragment, and a paucity of simple-sequence and other repeats. Thus, this sequence exhibits some of the genomic streamlining expected for avian as compared with mammalian genomes, but is not as densely packed with functional genes as is the chicken Mhc.

Amino Acid Sequence↗
PubMed2004

The light-harvesting complex of photosystem I in Chlamydomonas reinhardtii: protein composition, gene structures and phylogenic implications.

Light-harvesting chlorophyll a/b-binding proteins (LHCI) associated with photosystem I (PSI) and the genes encoding these proteins have been characterized in the unicellular green alga Chlamydomonas reinhardtii, extending previous studies of the PSII-LHCII [Teramoto et al. (2001) Plant Cell Physiol. 42: 849]. In order to assign LHCI proteins in the thylakoid membranes, the PSI-LHCI supercomplex that retains all of the major LHCI proteins was purified. Seven distinct LHCI proteins were resolved from the purified supercomplex by a high-resolution SDS polyacrylamide gel electrophoresis, and their N-terminal amino acid sequences were determined. One LHCI protein (band e) was newly found, although the other six LHCI proteins corresponded to those previously reported. Genomic clones encoding these seven LHCI proteins were newly isolated and the nucleotide sequences were determined. A comprehensive characterization of all members of Lhc gene family in this alga revealed that LHCI proteins are more highly diverged than LHCII, suggesting functional differentiation of the protein components in LHCI. Neighbor joining trees were constructed for LHC proteins from C. reinhardtii and those of Arabidopsis thaliana or Galdieria sulphuraria to assess evolutionary relationships. Phylogenetic analysis revealed that (1). green algal LHCI and LHCII proteins are more closely related to one another than to LHCI proteins in red algae, (2). green algae and higher plants possess seven common lineages of LHC proteins, and (3). Type I and III LHCI proteins are conserved between green algae and higher plants, while Type II and IV are not. These findings are discussed in the context of evolution of multiple diverse antenna complexes.

Algal Proteins↗
PubMed1998

Thermostable glycerol kinase from a hyperthermophilic archaeon: gene cloning and characterization of the recombinant enzyme.

The Pk-glpK gene, which encodes glycerol kinase (GK) from a hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1, was cloned and expressed in Escherichia coli. The amino acid sequence of this enzyme (Pk-GK) deduced from the nucleotide sequence showed 57% identity with that of E. coli GK and 47% identity with that of human GK. Pk-GK, which has a molecular weight of 55902 (497 amino acid residues), was purified from E. coli and characterized. Despite the high sequence similarity, Pk-GK and E. coli GK are greatly divergent in structure and function from each other. Unlike E. coli GK, which exists as a tetramer, Pk-GK exists as a dimer. The preferred divalent cation for Pk-GK is Co2+, instead of Mg2+. The optimum pH and temperature for Pk-GK activity are 8.0 and 80 degrees C, respectively. Pk-GK can utilize other nucleoside triphosphates than ATP as a phosphoryl donor. It is fairly resistant to an allosteric inhibitor of E. coli GK, fructose-1,6-bisphosphate. Determination of the kinetic parameters indicates that the Km value of the enzyme is 15.4 microM for ATP and 111 microM for glycerol and its kcat value is 940 s(-1). The enzyme was shown to be fairly resistant to irreversible heat inactivation and still retained 50% of its enzymatic activity even after heating at 100 degrees C for 30 min. Construction of a model for the three-dimensional structure of the enzyme suggests that the formation of extensive ion-pair networks is responsible for the high stability of this enzyme.

Adenosine Triphosphate↗
PubMed1999

North American and European porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions.

Although North American and European serotypes of porcine reproductive and respiratory syndrome virus (PRRSV) are recognized, only the genome of the European Lelystad strain (LV) has been sequenced completely. Here, the genome of the pathogenic North American PRRSV isolate 16244B has been sequenced and compared with LV. The genomic organization of 16244B was the same as LV but with only 63.4% nucleotide identity. The 189 nucleotide 5' non-coding region (NCR) of 16244B was distinct from the LV NCR, with good conservation (83%) only over a 43 base region immediately upstream of open reading frame (ORF) 1a. Major differences were found in the region encoding the non-structural part of the ORF1a polyprotein, which shared only 47% amino acid identity over 2503 residues of the six non-structural proteins (Nsps) encoded. Nsp2, thought to have a species-specific function, showed the greatest divergence, sharing only 32% amino acid identity with LV and containing 120 additional amino acids in the central region. Nsps encoded by the 5'-proximal and central regions of ORF1b had from 66 to 75% amino acid identity; however, the carboxy-terminal protein CP4 was distinct (42% identity). The ORF 1a-1b frameshift region of 16244B had 98% nucleotide identity with LV. Consistent with previous reports for North American isolates, the six structural proteins encoded were 58 to 79% identical to LV proteins. The 3' NCR (150 nucleotides) was 76% identical between isolates. These genomic differences confirm the presence of distinct North American and European PRRSV genotypes.

5' Untranslated Regions↗
PubMed2000

Codon usage in nucleopolyhedroviruses.

Phylogenetic analyses based on baculovirus polyhedrin nucleotide and amino acid sequences revealed two major nucleopolyhedrovirus (NPV) clades, designated Group I and Group II. Subsequent phylogenetic analyses have revealed three Group II subclades, designated A, B and C. Variations in amino acid frequencies determine the extent of dissimilarity for divergent but structurally and functionally conserved genes and therefore significantly influence the analysis of phylogenetic relationships. Hence, it is important to consider variations in amino acid codon usage. The Genome Hypothesis postulates that genes in any given genome use the same coding pattern with respect to synonymous codons and that genes in phylogenetically related species generally show the same pattern of codon usage. We have examined codon usage in six genes from six NPVs and found that: (1) there is significant variation in codon use by genes within the same virus genome; (2) there is significant variation in the codon usage of homologous genes encoded by different NPVs; (3) there is no correlation between the level of gene expression and codon bias in NPVs; (4) there is no correlation between gene length and codon bias in NPVs; and (5) that while codon use bias appears to be conserved between viruses that are closely related phylogenetically, the patterns of codon usage also appear to be a direct function of the GC-content of the virus-encoded genes.

Animals↗
PubMed2002

Conservation of L and 3C proteinase activities across distantly related aphthoviruses.

The foot-and-mouth disease virus (FMDV) leader (L) proteinase is an important virulence determinant in FMDV infections. It possesses two distinct catalytic activities: (i) C-terminal processing at the L/VP4 junction; and (ii) induction of the cleavage of translation initiation factor eIF4G, an event that inhibits cap-dependent translation in infected cells. The only other member of the Aphthovirus genus, equine rhinitis A virus (ERAV), also encodes an L protein, but this shares only 32% amino acid identity with its FMDV counterpart. Another more distantly related picornavirus, equine rhinitis B virus (ERBV), which is not classified as an aphthovirus, also encodes an L protein. Using in vitro transcription and translation analysis, we have shown that both ERAV and ERBV L proteins have C-terminal processing activity. Furthermore, expression of ERAV L, but not ERBV L, in BHK-21 cells resulted in the efficient inhibition of cap-dependent translation in these cells. We have shown that the ERAV and FMDV L proteinases induce cleavage of eIF4GI at very similar or identical positions. Interestingly, ERAV 3C also induces eIF4GI cleavage and again produces distinct products that co-migrate with those induced by FMDV 3C. The ERBV L proteinase does not induce eIF4GI cleavage, consistent with its inability to shut down cap-dependent translation. We have also shown that another unique feature of FMDV L, the stimulation of enterovirus internal ribosome entry site (IRES) activity, is also shared by the ERAV L proteinase but not by ERBV L. The functional conservation of the divergent ERAV and FMDV proteinases indicates the likelihood of a similar and important role for these enzymes in the pathogenesis of infections caused by these distantly related aphthoviruses.

3C Viral Proteases↗
PubMed2005

Geometrical structure of disordered sphere packings.

The three-dimensional structure of large packings of monosized spheres with volume fractions ranging between 0.58 and 0.64 has been studied with x-ray computed tomography. We search for signatures of organization, classifying local arrangements and exploring the effects of local geometrical constrains on the global packing. This study is the largest and the most accurate empirical analysis of disordered packings at the grain-scale to date, mapping over 380,000 sphere coordinates with precision within 0.1% of the sphere diameters. We discuss topological and geometrical methods to characterize and classify these systems emphasizing the implications that local geometry can have on the mechanisms of formation of these amorphous structures. Some of the main results are (1) the observation that the average number of contacts increases with the volume fraction; (2) the discovery that these systems have a very compact contact network; (3) the finding that disordered packing can be locally more efficient than crystalline packings; (4) the observation that the peaks of the radial distribution function follow power law divergences; (5) the discovery that geometrical frustration plays no role in the formation of such amorphous packings.

Journal Article↗
PubMed2003

Diversity of the superfamily of phloem lectins (phloem protein 2) in angiosperms.

Phloem protein 2 (PP2) is one of the most abundant and enigmatic proteins in the phloem sap. Although thought to be associated with structural P-protein, PP2 is translocated in the assimilate stream where its lectin activity or RNA-binding properties can exert effects over long distances. Analyzing the diversity of these proteins in vascular plants led to the identification of PP2-like genes in species from 17 angiosperm and gymnosperm genera. This wide distribution of PP2 genes in the plant kingdom indicates that they are ancient and common in vascular plants. Their presence in cereals and gymnosperms, both of which lack structural P-protein, also supports a wider role for these proteins. Within this superfamily, PP2 proteins have considerable size polymorphism. This is attributable to variability in the length of the amino terminus that extends from a highly conserved domain. The conserved PP2 domain was identified in the proteins encoded by six genes from several cucurbits, celery (Apium graveolens), and Arabidopsis that are specifically expressed in the sieve element-companion cell complex. The acquisition of additional modular domains in the amino-terminal extensions of other PP2-like proteins could reflect divergence from its phloem function.

Amino Acid Sequence↗
PubMed2004

Effect of priming on activation and localization of phospholipase D-1 in human neutrophils.

Phospholipase D (PLD) plays a major role in the activation of the neutrophil respiratory burst. However, the repertoire of PLD isoforms present in these primary cells, the precise mechanism of activation, and the impact of cell priming on PLD activity and localization remain poorly defined. RT-PCR analysis showed that both PLD1 and PLD2 isoforms are expressed in human neutrophils, with PLD1 expressed at a higher level. Endogenous PLD1 was detected by immunoprecipitation and Western blotting, and was predominantly membrane-associated under control and primed/stimulated conditions. Immunofluorescence showed that PLD had a punctate distribution throughout the cell, which was not altered after stimulation by soluble agonists. In contrast, PLD localized to the phagolysosome membrane after ingestion of nonopsonized zymosan particles. We also demonstrate that tumour necrosis factor alpha greatly potentiates agonist-stimulated PLD activation, myeloperoxidase release, and superoxide anion generation, and that PLD activation occurs via a phosphatidylinositol 3-kinase-sensitive and brefeldin-sensitive ADP-ribosylation factor GTPase-regulated mechanism. Moreover, propranolol, which causes an increase in PLD-derived phosphatidic acid accumulation, caused a selective increase in agonist-stimulated myeloperoxidase release. Our results indicate that priming is a critical regulator of PLD activation, that the PLD-generated lipid products exert divergent effects on neutrophil functional responses, that PLD1 is the major PLD isoform present in human neutrophils, and that PLD1 actively translocates to the phagosomal wall after particle ingestion.

ADP-Ribosylation Factors↗
PubMed1990

Isoform-specific cDNAs for human embryonic, neonatal, and slow skeletal myosin heavy chains.

A series of cDNA fragments encoding immunodetectable portions of the human slow/beta, neonatal, and embryonic isoforms of myosin heavy chain (MHC) were isolated from a human fetal muscle cDNA expression library. A 6 kb fragment isolated on a secondary screen represents the first cloned cDNA encoding a full-length vertebrate MHC (the human embryonic isoform). In the 3'-untranslated regions, 70-80% nucleotide sequence homology exists among orthologous human and rat cDNAs, whereas the homology is less than 65% among the paralogous cDNAs. Furthermore, approximately the same level of untranslated sequence conservation is observed at the 5'-terminus of the embryonic transcript. These results suggest that for both the 3'- and the 5'-untranslated domains, the rate of evolutionary sequence divergence is limited by functional constraints.

Amino Acid Sequence↗
PubMed1992

Therapeutic strategies and outcomes: perspectives from different cultures.

Two forms of psychological therapy, one Japanese the other Euro/American, are described and compared. It is suggested that each identifies comparable goals for individual change. However, each differs in the means by which change is induced. The consensus concerning goals is taken as evidence of universal values in the definition of the fully functioning self. However, the divergence in means by which these goals are attained reflects specific cultural differences. The implications of these differences are related to individualistic versus relational values within the cultures discussed. It is proposed that the comparisons have implications for the practice of any specific form of psychotherapy on members of a culture other than that within which the therapy has developed.

Cross-Cultural Comparison↗