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PubMed2006

Evolution of ATP synthase subunit c and cytochrome c gene families in selected Metazoan classes.

To investigate the integrated evolution of mitochondrial and nuclear genomes in the eukaryotic cell, we have focused our attention on OXPHOS (oxidative phosphorylation) gene families which encode proteins involved in the main mitochondrial function. The present study reports the phylogenetic analysis of two OXPHOS gene families: ATP synthase subunit c (or lipid binding protein, LBP) and Cytochrome c (Cytc). Both gene families possess a higher expansion trend than the typically low duplication rate of OXPHOS genes in Metazoa, but follow a completely different evolutionary history, especially in mammals. LBP is represented by three well conserved isoforms in all mammals (P1, P2, P3): only P3 possesses a clearly conserved isoform in all Vertebrates, P1 and P2 were already present before the bird-mammal divergence and there are preliminary evidence from the in silico analysis that P1, the most evolutionary divergent isoform, is poorly expressed and not regulated by NRF1. In contrast, Cytc family presents at least two duplicated genes in all the analysed Vertebrates, is subject to a high expansion trend, especially of processed pseudogenes in mammals, and some events of gain and loss of function can be supposed.

Animals↗
PubMed2000

Divergent functional properties of ryanodine receptor types 1 and 3 expressed in a myogenic cell line.

Of the three known ryanodine receptor (RyR) isoforms expressed in muscle, RyR1 and RyR2 have well-defined roles in contraction. However, studies on mammalian RyR3 have been difficult because of low expression levels relative to RyR1 or RyR2. Using the herpes simplex virus 1 (HSV-1) helper-free amplicon system, we expressed either RyR1 or RyR3 in 1B5 RyR-deficient myotubes. Western blot analysis revealed that RyR1- or RyR3-transduced cells expressed the appropriate RyR isoform of the correct molecular mass. Although RyR1 channels exhibited the expected unitary conductance for Cs(+) in bilayer lipid membranes, 74 of 88 RyR3 channels exhibited pronounced subconductance behavior. Western blot analysis with an FKBP12/12.6-selective antibody reveals that differences in gating behavior exhibited by RyR1 and RyR3 may be, in part, the result of lower affinity of RyR3 for FKBP12. In calcium imaging studies, RyR1 restored skeletal-type excitation-contraction coupling, whereas RyR3 did not. Although RyR3-expressing myotubes were more sensitive to caffeine than those expressing RyR1, they were much less sensitive to 4-chloro-m-cresol (CMC). In RyR1-expressing cells, regenerative calcium oscillations were observed in response to caffeine and CMC but were never seen in RyR3-expressing 1B5 cells. In [(3)H]ryanodine binding studies, only RyR1 exhibited sensitivity to CMC, but both RyR isoforms responded to caffeine. These functional differences between RyR1 and RyR3 expressed in a mammalian muscle context may reflect differences in association with accessory proteins, especially FKBP12, as well as structural differences in modulator binding sites.

Animals↗
PubMed2004

Identification of two subfamilies of micropia transposable element in species of the repleta group of Drosophila.

The occurrence, number of insertion sites and antisense RNA expression of micropia transposable element were studied in 26 species that belong to three subgroups (mercatorum, mulleri and hydei) of repleta group of Drosophila. Under high specific PCR, micropia sequences were detected in 11 species, but under less stringent condition, this retrotransposon was detected in all species. The widespread distribution of micropia suggests that this element was already present at the common ancestor of the repleta group of Drosophila. Southern blot analysis showed a variation from 0 to 17 different insertion sites and the occurrence of male-specific sequences. We found that the expression of the 1.0 kb micropia antisense RNA is variable among the species and tissues (soma and testis), which suggests that more than one mechanism regulates transposition in these species. Variation of amplification by PCR and of antisense RNA expression, as well as divergence of nucleotide sequences among the species allow us to suggest that at least two subfamilies of micropia transposable element are harbored by the genome of this species group.

Animals↗
PubMed2006

Evolution on the X chromosome: unusual patterns and processes.

Although the X chromosome is usually similar to the autosomes in size and cytogenetic appearance, theoretical models predict that its hemizygosity in males may cause unusual patterns of evolution. The sequencing of several genomes has indeed revealed differences between the X chromosome and the autosomes in the rates of gene divergence, patterns of gene expression and rates of gene movement between chromosomes. A better understanding of these patterns should provide valuable information on the evolution of genes located on the X chromosome. It could also suggest solutions to more general problems in molecular evolution, such as detecting selection and estimating mutational effects on fitness.

Amino Acid Sequence↗
PubMed2000

Arabidopsis thaliana poly (A) binding protein 2 (PAB2) functions in yeast translational and mRNA decay processes.

The single yeast gene (PAB1) encoding poly (A) binding protein (PABP) has several roles in post-transcriptional processes, including translation initiation and mRNA decay. PABP is encoded by a large gene family in plants. Within Arabidopsis thaliana, the several characterized PABP genes exhibit an extreme degree of sequence divergence and are differentially expressed. Arabidopsis PAB2 is expressed in distinct tissues or during defined developmental windows in most plant organs. In this study we demonstrate that PAB2 restores viability to a yeast pab1 mutant strain. Yeast strains containing wild-type, null (PAB2s) and temperature sensitive (PAB2ts) alleles of PAB2 were used to explore the molecular functions of the plant protein. PAB2 can participate in poly (A) tail shortening, thus demonstrating that it interacts with the yeast poly(A) nuclease complex. PAB2 is required for translation, helping to maintain intact polysome structures. Consistent with its role in translation initiation, poly (A) was found to enhance PAB2 binding to Arabidopsis eIF-iso4G in vitro. In addition, PAB2 can partially restore the linkage between deadenylation, decapping and mRNA decay in yeast. Taken together, our results suggest that Arabidopsis PAB2 participates in many of the same complex post-transcriptional processes identified for yeast PAB1, and is functionally distinct from other characterized Arabidopsis PABPs.

Alleles↗
PubMedDate not supplied

The development and evolution of insect mouthparts as revealed by the expression patterns of gnathocephalic genes.

To understand better both the development and evolution of insect mouthparts, we have compared the expression pattern of several developmentally important genes in insects with either mandibulate or stylate-haustellate mouthparts. Specifically, we examined the expression of the proboscipedia (pb) and Distal-less (Dll) gene products as well as three regulators of pb, Sex combs reduced (Scr), Deformed (Dfd), and cap 'n' collar (cnc). These genes are known to control the identity of cells in the gnathal segments of Drosophila melanogaster and would appear to have similar conserved functions in other insects. Together we have made an atlas of gene expression in the heads of three insects: Thermobia domestica and Acheta domestica, which likely exemplify the mandibulate mouthparts present in the common insect ancestor, and Oncopeltus fasciatus, which has piercing-sucking mouth parts that are typical of the Hemiptera. At the earliest stages of embryogenesis, only the expression of pb was found to differ dramatically between Oncopeltus and the other insects examined, although significant differences were observed later in development. This difference in pb expression reflects an apparent divergence in the specification of gnathal identity between mandibulate and stylate-haustellate mouthparts, which may result from a "phylogenetic homeosis" that occurred during the evolution of the Hemiptera.

Amino Acid Sequence↗
PubMed1990

A pair of tandemly repeated genes code for gp24, a putative adhesion protein of Dictyostelium discoideum.

The glycoprotein gp24 has been implicated in cell-cell adhesion of Dictyostelium discoideum. We have used a cDNA clone that codes for gp24 to screen cloned genomic fragments. Two closely linked genes (GP24A and GP24B) were recognized that generate mRNAs of about 650 base pairs after excision of a small intron and addition of poly(A). They appear to have arisen by tandem duplication of about 800 base pairs, followed by divergence. These genes are expressed within a few hours of the initiation of development; their mRNAs accumulate to a peak at 12 hr and persist until culmination. Both genes have short guanine-rich sequences (G boxes) upstream that have been shown to be involved in transcriptional regulation of other genes expressed during development of Dictyostelium. Their mRNAs code for proteins that are 85% identical. GP24A and GP24B mRNAs code for proteins with a hydrophobic domain followed by a highly charged carboxyl-terminal domain.

Amino Acid Sequence↗
PubMed2001

Two unrelated phosphoenolpyruvate carboxylase polypeptides physically interact in the high molecular mass isoforms of this enzyme in the unicellular green alga Selenastrum minutum.

In the chlorophyte Selenastrum minutum, phosphoenolpyruvate carboxylase (PEPC) exists as two kinetically distinct classes of isoforms sharing the same 102-kDa catalytic subunit (p102). Class 1 PEPC is homotetrameric, whereas Class 2 PEPCs consist of three large protein complexes. The different Class 2 PEPCs contain p102 and 130-, 73-, and 65-kDa polypeptides in different stoichiometric combinations. Immunoblot, immunoprecipitation, and chemical cross-linking studies indicated that p102 physically interacts with the 130-kDa polypeptide (p130) in Class 2 PEPCs. Immunological data and mass spectrometric and sequence analyses revealed that p102 and p130 are not closely related even if a p130 tryptic peptide had significant similarity to a conserved PEPC C-terminal domain from several sources. Evidence supporting the hypothesis that p130 has PEPC activity includes the following. (i) Specific activity expressed relative to the amount of p102 was lower in Class 1 than in Class 2 PEPCs; (ii) reductive pyridoxylation of both p102 and p130 was inhibited by magnesium-phosphoenolpyruvate; and (iii) biphasic phosphoenolpyruvate binding kinetics were observed with Class 2 PEPCs. These data support the view that unicellular green algae uniquely express, regulate, and assemble divergent PEPC polypeptides. This probably serves an adaptive purpose by poising these organisms for survival in different environments varying in nutrient content.

Amino Acid Sequence↗
PubMed2002

Functional properties of the apical Na+-K+-2Cl- cotransporter isoforms.

The bumetanide-sensitive Na(+):K(+):2Cl(-) cotransporter (BSC1) is the major pathway for salt reabsorption in the apical membrane of the mammalian thick ascending limb of Henle. Three isoforms of the cotransporter, known as A, B, and F, exhibit axial expression along the thick ascending limb. We report here a functional comparison of the three isoforms from mouse kidney. When expressed in Xenopus oocytes the mBSC1-A isoform showed higher capacity of transport, with no difference in the amount of surface expression. Kinetic characterization revealed divergent affinities for the three cotransported ions. The observed EC(50) values for Na(+), K(+), and Cl(-) were 5.0 +/- 3.9, 0.96 +/- 0.16, and 22.2 +/- 4.8 mm for mBSC1-A; 3.0 +/- 0.6, 0.76 +/- 0.07, and 11.6 +/- 0.7 mm for mBSC1-B; and 20.6 +/- 7.2, 1.54 +/- 0.16, and 29.2 +/- 2.1 mm for mBSC1-F, respectively. Bumetanide sensitivity was higher in mBSC1-B compared with the mBSC1-A and mBSC1-F isoforms. All three transporters were partially inhibited by hypotonicity but to different extents. The cell swelling-induced inhibition profile was mBSC1-F > mBSC1-B > mBSC1-A. The function of the Na(+):K(+):2Cl(-) cotransporter was not affected by extracellular pH or by the addition of metolazone, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), or R(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1-H-indenyl-5-yl)-oxy]acetic acid (DIOA) to the extracellular medium. In contrast, exposure of oocytes to HgCl(2) before the uptake period reduced the activity of the cotransporter. The effect of HgCl(2) was dose-dependent, and mBSC1-A and mBSC1-B exhibited higher affinity than mBSC1-F. Overall, the functional comparison of the murine apical renal-specific Na(+):K(+):2Cl(-) cotransporter isoforms A, B, and F reveals important functional, pharmacological, and kinetic differences, with both physiological and structural implications.

Amino Acid Sequence↗
PubMed2006

A metabotropic glutamate receptor family gene in Dictyostelium discoideum.

Metabotropic glutamate receptors (mGluRs) are a class of G-protein-coupled receptors that possess a seven transmembrane region involved in the modulation of excitatory synaptic transmission in the nervous system. mGluR orthologs have been identified in Drosophila, Caenorhabditis elegans, and higher organisms. Drosophila possesses two mGluR genes, DmGluRA and DmXR. We screened the Dictyostelium genome data base using the ligand binding domain of rat mGluR1 as bait, and identified a new receptor, DdmGluPR, belonging to the mGluR family. Similar to Drosophila DmXR, the residues of mGluRs involved in the binding of the alpha-carboxylic and alpha-amino groups of glutamate were well conserved in DdmGluPR, but the residues interacting with the gamma-carboxylic group of glutamate were not. The phylogenetic analysis suggests that DdmGluPR diverged after the mGluR family-GABA(B) receptors split but before mGluR family divergence. DdmGluPR mRNA was expressed in vegetative cells and throughout starvation-induced development, but the level of the expression was relatively high until 4 h after starvation. DdmGluPR was localized to the plasma membrane of axenically grown Ax-2 cells expressed as a green fluorescent protein fusion protein. DdmGluPR-null cells grew faster at high cell density and reached higher densities than wild-type cells. DdmGluPR-null cells exhibited delayed aggregates formation upon starvation and impaired chemotaxis toward cAMP. Although expressions of cAR1 and aca, cAMP-signaling components, were rapidly induced and peaked at 2-4 h in wild-type cells, DdmGluPR-null cells displayed sustained and peaked at 8 h of the expressions of these genes. Our findings suggest the involvement of DdmGluPR in the early development of Dictyostelium discoideum.

Amino Acid Sequence↗
PubMed2005

Contributions of heterosis and epistasis to hybrid fitness.

Early-generation hybrid fitness is difficult to interpret because heterosis can obscure the effects of hybrid breakdown. We used controlled reciprocal crosses and common garden experiments to distinguish between effects of heterosis and nuclear and cytonuclear epistasis among morphotypes and advanced-generation hybrid derivative populations in the Piriqueta caroliniana (Turneraceae) plant complex. Seed germination, growth, and sexual reproduction of first-generation hybrids, inbred parental lines, and outbred parental lines were compared under field conditions. Average vegetative performance was greater for hybrids than for inbred lines, and first-season growth was similar for hybrids and outbred parental lines. Hybrid survival surpassed that of inbred lines and was equal to or greater than outbred lines' survival, and more F(1) than parental plants reproduced. Reductions in hybrid fitness due to Dobzhansky-Muller incompatibilities (epistasis among divergent genetic elements) were expressed as differences in vegetative growth, survival, and reproduction between plants from reciprocal crosses for both F(1) and backcross hybrid generations. Comparing performance of hybrids against parental genotypes from intra- and interpopulation crosses allowed a more robust prediction of F(1) hybrids' success and more accurate interpretations of the genetic architecture of F(1) hybrid vigor.

Animals↗
PubMed1993

Divergent fates of P- and E-selectins after their expression on the plasma membrane.

P-selectin and E-selectin are related adhesion receptors for monocytes and neutrophils that are expressed by stimulated endothelial cells. P-selectin is stored in Weibel-Palade bodies, and it reaches the plasma membrane after exocytosis of these granules. E-selectin is not stored, and its synthesis is induced by cytokines. We studied the fate of the two proteins after their surface expression by following the intracellular routing of internalized antibodies to the selectins. By immunofluorescent staining, P-selectin antibody was first seen in endosomes, then in the Golgi region, and finally in Weibel-Palade bodies. In contrast, the E-selectin antibody was detected only in endosomes and lysosomes. Subcellular fractionation of cells after 4 h chase confirmed the localization of P-selectin antibody in storage granules and of the E-selectin antibody in lysosomes. In AtT-20 cells, a mouse pituitary cell line, transfected with P- or E-selectin, only P-selectin was delivered to the endogenous adrenocorticotrophic hormone storage granules after endocytosis. Deletion of the cytoplasmic domain abolished internalization. In summary, after a brief surface exposure, internalized E-selectin is degraded in the lysosomes, whereas P-selectin returns to the storage granules from where it can be reused.

Animals↗
PubMed1979

Sequential Sepharose chromatographic isolation of polysomes and polysomal RNAs depleted in nuclear RNA from Xenopus.

Analyses of sequence complexities, stage specific gene expression, and mRNA sequence divergence require polysomal RNA preparations relatively free of nuclear RNA contamination. Conventional procedures for the isolation of uncontaminated polysomal RNAs which rely on sucrose density centrifugations are laborious and unsuitable for large scale isolations. We describe here a method using sequential Sepharose chromatography for isolating polysomes and polysomal RNAs depleted for nuclear RNA. Polysomes and polysomal RNAs isolated from livers of Xenopus stimulated to produce vitellogenin were capable of directing protein synthesis in vitro and showed little evidence of degradation. The polysomal RNAs contained less than 0.5% of nuclear RNA.

Animals↗
PubMed2003

Recent speciation in the Indo-West Pacific: rapid evolution of gamete recognition and sperm morphology in cryptic species of sea urchin.

The rich species diversity of the marine Indo-West Pacific (IWP) has been explained largely on the basis of historical observation of large-scale diversity gradients. Careful study of divergence among closely related species can reveal important new information about the pace and mechanisms of their formation, and can illuminate the genesis of biogeographic patterns. Young species inhabiting the IWP include urchins of the genus Echinometra, which diverged over the past 1-5 Myr. Here, we report the most recent divergence of two cryptic species of Echinometra inhabiting this region. Mitochondrial cytochrome oxidase 1 (CO1) sequence data show that in Echinometra oblonga, species-level divergence in sperm morphology, gamete recognition proteins and gamete compatibility arose between central and western Pacific populations in the past 250 000 years. Divergence in sperm attachment proteins suggests rapid evolution of the fertilization system. Divergence of sperm morphology may be a common feature of free-spawning animals, and offers opportunities to simultaneously understand genetic divergence, changes in protein expression patterns and morphological evolution in traits directly related to reproductive isolation.

Animals↗
PubMed1994

Sequence of the spike protein of the porcine epidemic diarrhoea virus.

The complete sequence of the spike (S) gene of the Br1/87 isolate of porcine epidemic diarrhoea virus (PEDV) was determined from cDNA clones. The predicted polypeptide was 1383 amino acids long, contained 29 potential N-linked glycosylation sites and showed structural features similar to those of the coronavirus spike protein. The PEDV S protein, like that of the members of the transmissible gastroenteritis virus (TGEV)-related subset, lacks a proteolytic site to yield cleaved amino and carboxy subunits S1 and S2. Viral polypeptide species of the expected M(r), i.e. 170K/190K, were observed in PEDV-infected cells. Sequence comparison confirmed that, within the subset, PEDV was most closely related to the human respiratory coronavirus HCV 229E. However, PEDV S protein has an additional 250 residue N-terminal domain which is absent from HCV 229E and porcine respiratory coronavirus, the respiratory variant of TGEV. Alignment of the S1 regions revealed a second domain of about 90 residues with increased sequence divergence which might possibly express virus-specific determinants.

Amino Acid Sequence↗
PubMed2000

Momentum conservation implies anomalous energy transport in 1D classical lattices

Under quite general conditions, we prove that for classical many-body lattice Hamiltonians in one dimension (1D) total momentum conservation implies anomalous conductivity in the sense of the divergence of the Kubo expression for the coefficient of thermal conductivity, kappa. Our results provide rigorous confirmation and explanation of many of the existing "surprising" numerical studies of anomalous conductivity in 1D classical lattices, including the celebrated Fermi-Pasta-Ulam problem.

Journal Article↗
PubMed2003

Divergent light-, ascorbate-, and oxidative stress-dependent regulation of expression of the peroxiredoxin gene family in Arabidopsis.

Peroxiredoxins (prxs) are peroxidases with broad substrate specificity. The seven prx genes expressed in Arabidopsis shoots were analyzed for their expressional response to changing photon fluence rates, oxidative stress, and ascorbate application. The results reveal a highly variable and gene-specific response to reducing and oxidizing conditions. The steady-state transcript amounts of the chloroplast-targeted prxs, namely the two-cysteine (2-Cys) prxs, prx Q and prx II E, decreased upon application of ascorbate. prx Q also responded to peroxides and diamide treatment. prx II B was induced by tertiary butylhydroperoxide, but rather unaffected by ascorbate. The strongest responses were observed for prx II C, which was induced with all treatments. The two Arabidopsis 2-Cys Prxs and four Prx II proteins were expressed heterologously in Escherichia coli. In an in vitro test system, they all showed peroxidase activity, but could be distinguished by their ability to accept dithiothreitol and thioredoxin as electron donor in the regeneration reaction. The midpoint redox potentials (E(m)') of Prx II B, Prx II C, and Prx II E were around -290 mV and, thus, less negative than E(m)' of Prx II F, 2-Cys Prx A, and 2-Cys Prx B (-307 to -322 mV). The data characterize expression and function of the mitochondrial Prx II F and the chloroplast Prx II E for the first time, to our knowledge. Antibodies directed against 2-Cys Prx and Prx II C showed a slight up-regulation of Prx II protein in strong light and of 2-Cys Prx upon transfer both to high and low light. The results are discussed in context with the subcellular localization of the Prx gene products.

Arabidopsis↗
PubMed2002

Three-beam X-ray diffraction in a semi-infinite perfect crystal.

The approach to multibeam X-ray diffraction by Shen [Acta Cryst. (1986). A42, 525-533] has been carefully re-examined for three-beam Laue-Laue diffraction in a perfect non-absorbing semi-infinite crystal. A modified version of Shen's theory is shown to be equivalent to the Takagi-Taupin formalism. The perturbation of the two-beam integrated power when a third beam is being excited is thus expressed by non-diverging terms. Both Aufhellung and Umweganregung terms are recovered.

Journal Article↗