Structure of a Stable G-Hairpin.

In this study, we report the first atomic resolution structure of a stable G-hairpin formed by a natively occurring DNA sequence. An 11-nt long G-rich DNA oligonucleotide, 5'-d(GTGTGGGTGTG)-3', corresponding to the most abundant sequence motif in irregular telomeric DNA from Saccharomyces cerevisiae (yeast), is demonstrated to adopt a novel type of mixed parallel/antiparallel fold-back DNA structure, which is stabilized by dynamic G:G base pairs that transit between N1-carbonyl symmetric and N1-carbonyl, N7-amino base-pairing arrangements. Although the studied sequence first appears to possess a low capacity for base pairing, it forms a thermodynamically stable structure with a rather complex topology that includes a chain reversal arrangement of the backbone in the center of the continuous G-tract and 3'-to-5' stacking of the terminal residues. The structure reveals previously unknown principles of the folding of G-rich oligonucleotides that could be applied to the prediction of natural and/or the design of artificial recognition DNA elements. The structure also demonstrates that the folding landscapes of short DNA single strands is much more complex than previously assumed.

Linear mitochondrial genomes: 30 years down the line.

At variance with the earlier belief that mitochondrial genomes are represented by circular DNA molecules, a large number of organisms have been found to carry linear mitochondrial DNA. Studies of linear mitochondrial genomes might provide a novel view on the evolutionary history of organelle genomes and contribute to delineating mechanisms of maintenance and functioning of telomeres. Because linear mitochondrial DNA is present in a number of human pathogens, its replication mechanisms might become a target for drugs that would not interfere with replication of human circular mitochondrial DNA.

Double-stranded telomeric DNA binding proteins: Diversity matters.

Telomeric sequences constitute only a small fraction of the whole genome yet they are crucial for ensuring genomic stability. This function is in large part mediated by protein complexes recruited to telomeric sequences by specific telomere-binding proteins (TBPs). Although the principal tasks of nuclear telomeres are the same in all eukaryotes, TBPs in various taxa exhibit a surprising diversity indicating their distinct evolutionary origin. This diversity is especially pronounced in ascomycetous yeasts where they must have co-evolved with rapidly diversifying sequences of telomeric repeats. In this article we (i) provide a historical overview of the discoveries leading to the current list of TBPs binding to double-stranded (ds) regions of telomeres, (ii) describe examples of dsTBPs highlighting their diversity in even closely related species, and (iii) speculate about possible evolutionary trajectories leading to a long list of various dsTBPs fulfilling the same general role(s) in their own unique ways.

Extragenomic double-stranded DNA circles in yeast with linear mitochondrial genomes: potential involvement in telomere maintenance.

Although the typical mitochondrial DNA (mtDNA) is portrayed as a circular molecule, a large number of organisms contain linear mitochondrial genomes classified by their telomere structure. The class of mitochondrial telomeres identified in three yeast species, Candida parapsilosis, Pichia philodendra and Candida salmanticensis, is characterized by inverted terminal repeats each consisting of several tandemly repeating units and a 5' single-stranded extension. The molecular mechanisms of the origin, replication and maintenance of this type of mitochondrial telomere remain unknown. While studying the replication of linear mtDNA of C.parapsilosis by 2-D gel electrophoresis distinct DNA fragments composed solely of mitochondrial telomeric sequences were detected and their properties were suggestive of a circular conformation. Electron microscopic analysis of these DNAs revealed the presence of highly supertwisted circular molecules which could be relaxed by DNase I. The minicircles fell into distinct categories based on length, corresponding to n x 0.75 kb (n = 1-7). Similar results were obtained with two other yeast species (P.philodendra and C. salmanticensis) which possess analogous telomeric structure.

Mitochondrial Carriers Link the Catabolism of Hydroxyaromatic Compounds to the Central Metabolism in Candida parapsilosis.

The pathogenic yeast Candida parapsilosis metabolizes hydroxyderivatives of benzene and benzoic acid to compounds channeled into central metabolism, including the mitochondrially localized tricarboxylic acid cycle, via the 3-oxoadipate and gentisate pathways. The orchestration of both catabolic pathways with mitochondrial metabolism as well as their evolutionary origin is not fully understood. Our results show that the enzymes involved in these two pathways operate in the cytoplasm with the exception of the mitochondrially targeted 3-oxoadipate CoA-transferase (Osc1p) and 3-oxoadipyl-CoA thiolase (Oct1p) catalyzing the last two reactions of the 3-oxoadipate pathway. The cellular localization of the enzymes indicates that degradation of hydroxyaromatic compounds requires a shuttling of intermediates, cofactors, and products of the corresponding biochemical reactions between cytosol and mitochondria. Indeed, we found that yeast cells assimilating hydroxybenzoates increase the expression of genes SFC1, LEU5, YHM2, and MPC1 coding for succinate/fumarate carrier, coenzyme A carrier, oxoglutarate/citrate carrier, and the subunit of pyruvate carrier, respectively. A phylogenetic analysis uncovered distinct evolutionary trajectories for sparsely distributed gene clusters coding for enzymes of both pathways. Whereas the 3-oxoadipate pathway appears to have evolved by vertical descent combined with multiple losses, the gentisate pathway shows a striking pattern suggestive of horizontal gene transfer to the evolutionarily distant Mucorales.

Electron microscopic analysis supports a dual role for the mitochondrial telomere-binding protein of Candida parapsilosis.

Linear mitochondrial genomes exist in several yeast species which are closely related to yeast that harbor circular mitochondrial genomes. Several lines of evidence suggest that the conversion from one form to another occurred accidentally through a relatively simple mechanism. Previously, we (L.T. & J.N.) reported the identification of the first mitochondrial telomere-binding protein (mtTBP) that specifically binds a sequence derived from the extreme end of Candida parapsilosis linear mtDNA, and sequence analysis of the corresponding nuclear gene MTP1 revealed that mtTBP shares homology with several bacterial and mitochondrial single-stranded (ss) DNA-binding (SSB) proteins. In this study, the DNA-binding properties of mtTBP in vitro and in vivo were analyzed by electron microscopy (EM). When M13 ssDNA was used as a substrate, mtTBP exhibited similar DNA binding characteristics as human mitochondrial SSB: mtTBP formed protein globules along the DNA substrate, and the bound proteins were randomly distributed, indicating that the binding of mtTBP to M13 ssDNA is not highly cooperative. EM analysis demonstrated that mtTBP is able to recognize the 5' single-stranded telomeric overhangs in their natural context. Using isopycnic centrifugation of mitochondrial lysates of C. papsilosis we show that mtTBP is a structural part of mitochondrial nucleoids of C. parapsilosis and is predominantly bound to the mitochondrial telomeres. These data support a dual role of mtTBP in mitochondria of C. parapsilosis, serving both as a typical mitochondrial SSB and as a specific component of the mitochondrial telomeric chromatin.

Mitochondrial protein phosphorylation: lessons from yeasts.

The genome of Saccharomyces cerevisiae contains as many as 136 protein kinase encoding genes. However, only a limited number of mitochondrial protein kinases have been characterized. A computer-aided analysis revealed that only seven members of this large protein family are potentially localized in mitochondria. The low abundance of mitochondrially targeted protein kinases in yeast reflects the reductive evolution of mitochondrial signaling components and/or the apparent lack of selection pressure for acquiring mitochondrially localized protein kinases encoded by the host genome. This suggests that mitochondria, like obligatory intracellular bacterial parasites, are no longer dependent on signalling mechanisms mediated by protein kinases residing within the mitochondria. Instead, the nucleo-mitochondrial communication system requiring protein phosphorylation may be predominantly regulated by protein kinases, which are cytosolic and/or anchored to the outer mitochondrial membrane.

Mouse serum as a medium supplement for murine immune responses in vitro.

In vitro generation of secondary anti-viral H-2 restricted cytotoxic T cells (Tc cells) alloreactive Tc cells, alloantigen-induced T cell proliferation, bacterial lipopolysaccharide (LPS)-induced B cell proliferation and anti-dinitrophenyl IgM and IgG antibody responses were used to evaluate pools of mouse sera (MS) and other animal sera as medium supplements by comparison with 5-10% fetal calf serum (FCS). Some batches of normal MS were comparable with FCS while others inhibited Tc cell responses when used at 1%, particularly with limiting stimulator cell numbers. In contrast, 2 pools of nude mouse sera and several pools of MS from thioglycollate-injected mice (TMS) used at 1% were consistently comparable with FCS at 5-10% in terms of in vitro generation of effector Tc cells and antibody secreting B cells. TMS at 1% was satisfactory for Tc cell responses either fresh, after heat inactivation at 56 degrees C for 30 min, or after storage for 6 months at -70 degrees C. 0.5% or 2% heat-inactivated rat and rabbit sera (but not guinea pig sera) were also satisfactory for Tc cell generation. Total thymidine incorporation into proliferating B or T cells was 5-15-fold lower in cultures with 1% normal MS, nude MS or TMS than with 5% FCS, but stimulation indices with 1% nude MS and TMS were similar to those with 5% FCS, whereas 1% normal MS apparently completely inhibited stimulation. One percent syngeneic TMS is a convenient, reliable alternative to 5-10% FCS in culture medium, supporting all the murine responses listed above.

Differentiation of the yeasts Williopsis, Zygowilliopsis and Komagataea by karyotypic and PCR analyses.

We used polymerase chain reaction with universal and microsatellite primers, and molecular karyotyping to evaluate the extent of divergence between the genomes of the yeasts currently assigned to the heterogeneous genus Williopsis. Pulsed-field gel electrophoresis of chromosomal DNAs indicates that Zygowilliopsis californica, Komagataea pratensis, Williopsis mucosa, Williopsis salicorniae species and Williopsis sensu stricto complex have clearly different karyotypes. In contrast, the latter six species, Williopsis saturnus, W. beijerinckii, W mrakii, W. suaveolens, W. subsufficiens and W. sargentensis, show similar banding patterns and practically cannot be differentiated on the basis of their karyotypes. The data revealed that a PCR method employing the universal primer N21 is appropriate for the distinction of Williopsis, Zygowilliopsis and Komagataea yeasts. Unique fingerprints were generated with this primer for all 10 species studied while strains of the same species showed nearly identical profiles. The data of UP-PCR are in good agreement with genetic classification and provide support for the species status of the yeasts composing the Williopsis sensu stricto complex. Microsatellite primer (GTG)5 allowing molecular typing of individual strains of the same species may be useful for investigating population structure of the saturn-spored yeasts.

The respiratory complex I in yeast: isolation of a gene NUO51 coding for the nucleotide-binding subunit of NADH:ubiquinone oxidoreductase from the obligately aerobic yeast Yarrowia lipolytica.

We have isolated a gene NUO51 coding for a homologue of the nucleotide-binding subunit of mitochondrial respiratory chain linked NADH:ubiquinone oxidoreductase from the obligately aerobic yeast Yarrowia lipolytica. DNA sequencing revealed a 1464 bp open reading frame encoding a protein with predicted molar mass of about 53.7 kDa. The sequence is highly conserved with its counterparts from filamentous fungi and represents the first yeast homologue of the NADH-binding subunit (51 kDa) of the respiratory complex 1. In addition, PFGE and Southern hybridization analysis indicate that NUO51 is a single copy gene in the genome of Y. lipolytica. The expression of NUO51 by Northern blot analysis was also examined.

Mathematical model of alternative mechanism of telomere length maintenance.

Biopolymer length regulation is a complex process that involves a large number of biological, chemical, and physical subprocesses acting simultaneously across multiple spatial and temporal scales. An illustrative example important for genomic stability is the length regulation of telomeres-nucleoprotein structures at the ends of linear chromosomes consisting of tandemly repeated DNA sequences and a specialized set of proteins. Maintenance of telomeres is often facilitated by the enzyme telomerase but, particularly in telomerase-free systems, the maintenance of chromosomal termini depends on alternative lengthening of telomeres (ALT) mechanisms mediated by recombination. Various linear and circular DNA structures were identified to participate in ALT, however, dynamics of the whole process is still poorly understood. We propose a chemical kinetics model of ALT with kinetic rates systematically derived from the biophysics of DNA diffusion and looping. The reaction system is reduced to a coagulation-fragmentation system by quasi-steady-state approximation. The detailed treatment of kinetic rates yields explicit formulas for expected size distributions of telomeres that demonstrate the key role played by the J factor, a quantitative measure of bending of polymers. The results are in agreement with experimental data and point out interesting phenomena: an appearance of very long telomeric circles if the total telomere density exceeds a critical value (excess mass) and a nonlinear response of the telomere size distributions to the amount of telomeric DNA in the system. The results can be of general importance for understanding dynamics of telomeres in telomerase-independent systems as this mode of telomere maintenance is similar to the situation in tumor cells lacking telomerase activity. Furthermore, due to its universality, the model may also serve as a prototype of an interaction between linear and circular DNA structures in various settings.

Phosphorylation of mitochondrial telomere binding protein of Candida parapsilosis by camp-dependent protein kinase.

Mitochondrial telomere-binding protein (mtTBP) of Candida parapsilosis binds with high affinity to 5' single-stranded overhang of the linear mitochondrial DNA of this yeast (Tomáska, L'., Nosek, J., and Fukuhara, H. (1997) J. Biol. Chem. 272, 3049-3056). Here it is reported that mtTBP is phosphorylated by catalytic subunit of cAMP-dependent protein kinase in vitro. Phosphorylated mtTBP has dramatically reduced ability to bind telomeric oligonucleotide in the gel-mobility retardation assay without affecting the oligomerization of mtTBP in vitro. MtTBP is one of the few mitochondrial proteins and the first mitochondrial single-strand DNA binding proteins that was demonstrated to serve as a substrate for cAMP-dependent protein kinase.

Inhibition of secondary IgG responses by N-acetyl-D-galactosamine.

A range of monosaccharides were tested for their ability to inhibit a variety of in vitro immune response. The most striking specific inhibition was produced by N-acetyl-D-galactosamine (GalNAc). This sugar strongly inhibited the secondary IgG antibody response to two different hapten-carrier systems, but had no effect on primary and secondary IgM responses, generation of cytotoxic T cells to alloantigens and mixed lymphocyte reactions. By exposing secondary antibody cultures to GalNAc for varying periods of time, it was observed that GalNAc only exerted its inhibitory effect on day 4 of the culture, the day when IgG plaque-forming cells first appeared. Furthermore, GalNAc could override the action of T helper factor in T cell-depleted cultures. Collectively, these data indicate that GalNAc inhibits the initiation of IgG synthesis probably by blocking the interaction of a helper factor for IgG synthesis with its target cell.

Identification of a putative mitochondrial telomere-binding protein of the yeast Candida parapsilosis.

Terminal segments (telomeres) of linear mitochondrial DNA (mtDNA) molecules of the yeast Candida parapsilosis consist of large sequence units repeated in tandem. The extreme ends of mtDNA terminate with a 5' single-stranded overhang of about 110 nucleotides. We identified and purified a mitochondrial telomere-binding protein (mtTBP) that specifically recognizes a synthetic oligonucleotide derived from the extreme end of this linear mtDNA. MtTBP is highly resistant to protease and heat treatments, and it protects the telomeric probe from degradation by various DNA-modifying enzymes. Resistance of the complex to bacterial alkaline phosphatase suggests that mtTBP binds the very end of the molecule. We purified mtTBP to near homogeneity using DNA affinity chromatography based on the telomeric oligonucleotide covalently bound to Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified fractions revealed the presence of a protein with an apparent molecular mass of approximately 15 kDa. UV cross-linking and gel filtration chromatography experiments suggested that native mtTBP is probably a homo-oligomer. MtTBP of C. parapsilosis is the first identified protein that specifically binds to telomeres of linear mitochondrial DNA.

Mitochondrial single-stranded DNA-binding proteins: in search for new functions.

During the evolution of the eukaryotic cell, genes encoding proteins involved in the metabolism of mitochondrial DNA (mtDNA) have been transferred from the endosymbiont into the host genome. Mitochondrial single-stranded DNA-binding (mtSSB) proteins serve as an excellent argument supporting this aspect of the endosymbiotic theory. The crystal structure of the human mtSSB, together with an abundance of biochemical and genetic data, revealed several exciting features of mtSSB proteins and enabled a detailed comparison with their prokaryotic counterparts. Moreover, identification of a novel member of the mtSSB family, mitochondrial telomere-binding protein of the yeast Candida parapsilosis, has raised interesting questions regarding mtDNA metabolism and evolution.

Suppression of platelet-derived growth factor receptor tyrosine kinase activity by unsaturated fatty acids.

Treatment of cells with platelet-derived growth factor (PDGF) was previously shown to increase the release of unesterified fatty acids from phospholipids. In view of these observations we examined the effect of various fatty acids on PDGF receptor activity. Unsaturated (oleic, linoleic, linolenic, and arachidonic) but not saturated (stearic and arachidic) fatty acids significantly inhibited the tyrosine kinase activity of the PDGF receptor in intact cells and membrane preparations. Half-maximal inhibition (IC50) was observed between 60 and 100 microM and maximal inhibition between 170 and 200 microM. Lysophospholipids and phospholipids had no effect on receptor activity. Activation of endogenous phospholipase A2 by mellitin in vivo or hydrolysis of phosphatidylcholine by purified phospholipase A2 in vitro inhibited PDGF receptor autophosphorylation similar to that of purified fatty acids. Dimerization of PDGF receptors in vivo was significantly reduced by concentrations of arachidonic acid inhibitory for receptor kinase activity while the binding of PDGF was only marginally affected. These data suggest a possible feedback mechanism resulting in the reduction of PDGF receptor activity by unsaturated fatty acids generated downstream within the PDGF-dependent signal transduction pathway and this effect may be as a direct result of decreased receptor dimerization and/or kinase activity.

Stimulation of yeast adenylyl cyclase activity by lysophospholipids and fatty acids. Implications for the regulation of Ras/effector function by lipids.

A reconstituted system containing membranes prepared from various Saccharomyces cerevisiae strains (CYR1 ras1 ras2) and a recombinant RAS2 protein was used to evaluate the effect of lipids on adenylyl cyclase activity. Incubation of wild-type membranes with lysophosphatidylinositol, lysophosphatidylserine, or lysophosphatidylcholine stimulated adenylyl cyclase activity in the absence and presence of RAS between 2-10-fold depending upon the individual lipid. Unsaturated fatty acids preferentially increased activity 2-3-fold in the presence of RAS. Other phospholipids as well as several detergents had only marginal effects on adenylyl cyclase activity. Lipids had no effect on either the binding or hydrolysis of GTP by RAS. LysoPI decreased both the Km for ATP and the amount of RAS required for enzyme activation. Four catalytically active deletion mutants of the CYR1 protein including one containing only the C-terminal 417 amino acids were similarly responsive to lysoPI when compared to the wild-type enzyme. These data suggest that lysophospholipids and fatty acids, metabolites of the mitogenically responsive enzyme phospholipase A2, may represent a novel mechanism for modulating the activity of downstream effector molecules and their interaction with Ras proteins.

Involvement of a phosphotyrosine protein phosphatase in the suppression of platelet-derived growth factor receptor autophosphorylation in ras-transformed cells.

The nature of the suppression of platelet-derived growth factor (PDGF) receptor autophosphorylation in ras-transformed NIH 3T3 fibroblasts was investigated. The PDGF receptor from ras-transformed cells that had been purified by wheatgerm-lectin affinity chromatography displayed normal PDGF-induced autophosphorylation, indicating that the receptor is not irreversibly modified. Various phosphotyrosine-protein-phosphatase inhibitors did not reverse the inhibition of PDGF-receptor kinase in crude membrane preparations from ras-transformed cells. However, treatment of intact ras-transformed cells both with 2 mM sodium orthovanadate and with 20 microM phenylarsine oxide restored PDGF-receptor tyrosine-kinase activity to a level similar to that observed in normal cells. Direct measurement of the phosphatase activities in crude cellular fractions revealed a 2.5-fold higher membrane-associated phosphotyrosine-protein-phosphatase activity in ras-transformed cells, whereas phosphoserine-protein-phosphatase activity remained unchanged between the cell lines. These data suggest that the suppression of the PDGF-receptor tyrosine-kinase activity in ras-transformed cells is mediated via an inhibitory component, distinct from the receptor, that may be positively regulated by the dephosphorylation of tyrosine residue(s).

Inhibition of secondary IgG responses by monosaccharides: evidence for I-region control.

Certain monosaccharides selectively inhibit secondary IgG responses in vitro. Genetic analyses described in this report revealed that the inhibitory sugars differed between mouse strains and these differences mapped to the I-J and I-C subregions of the murine MHC. These results imply that interaction between T and B lymphocytes can involve the recognition of I-region controlled carbohydrate structures.