Detection of a protein encoded by a class II mitochondrial intron of Podospora anserina

Summary In the filamentous fungus Podospora anserina, the amplification as circular DNA molecules of the first intron (intron ) of the CO1 mitochondrial gene, encoding the cytochrome oxidase subunit 1, is known to be strongly associated with aging of strains. In this study we have attempted to detect the protein potentially encoded by the open reading frame (ORF) contained in this intron. This was done by the Western blot technique using specific antisera raised against three polypeptides encoded by three non-overlapping fragments of this ORF adapted to the universal code and overexpressed in Escherichia coli. We examined about thirty independent subclones of Podospora derived from two different geographic races (A, s), using wild-type and mutant strains, young and senescent cultures. A 100 kDa polypeptide, encoded by the class II intron , was detected in five senescent subclones which all showed strong amplification of the intronic sequence (Sen DNA ).     

DNA binding proteins of rat thigh muscle: Purification and characterization of an endonuclease

Two major DNA binding proteins of molecular weights 34,000 and 38,000 have been identified in the 30,000 g supernatant (S-30) fraction of rat thigh muscle extracts. The presence of 38 KD DNA binding protein in the muscle S-30 could be demonstrated only if Triton X-100 treated extracts were used for Afinity chromatography suggesting that this protein may be a membrane associated DNA binding protein. The 38 KD DNA binding protein differed from the 34 KD DNA binding protein also in its chromatographic behaviour in DE-52 columns in which the 38 KD protein was retained, while the 34 KD protein came out in the flow-through in an electrophoretically pure form. The 34 KD DNA binding protein can also be purified by precipitation with MgCl₂. Incubation of 0 15 M NaCl eluates (containing the 38 KD and/or 34 KD DNA binding protein) in the presence of 100 mM Mg²⁺ resulted in the specific precipitation of the 34 KD protein. Prolonged incubation (30 days) of the 0.15 M NaCl eluates containing the two DNA binding proteins at 4°C led to the preferential degradation of the 34 KD DNA binding protein. Nitrocellulose filter binding assays indicated selective binding of purified 34 KD protein to ss DNA. Purified 34 KD DNA binding protein cleaved pBR 322 supercoiled DNA, and electrophoresis of the cleavage products in agarose gels revealed a major DNA band corresponding to the circular form of DNA.     

Localization of a cruciform cutting endonuclease to yeast mitochondria

We have found a cruciform cutting endonuclease in the yeast, Saccharomyces cerevisiae, which localizes to the mitochondria. This activity apparently is associated with the mitochondrial inner membrane since the activity is not released into solution by osmolysis, in contrast to the matrix enzyme, isocitrate dehydrogenase. The cruciform cutting activity appears to be encoded by CCE1. This gene has been shown to encode one of the major cruciform cutting endonucleases present in a yeast cell. In ccel strains, which lack CCE1 endonuclease activity, the mitochondrial cruciform cutting endonucleolytic activity is also absent. Since CCE1 is allelic to MGT1, a gene required for the highly biased transmission of petite mitochondrial DNA in crosses between ⁺ and hypersuppressive ^– cells, it seems likely that the CCE1 endonuclease functions within mitochondria.     

Cyclooxygenases and lipoxygenases are used by the fungus Podospora anserina to repel nematodes

Oxylipins are secondary messengers used universally in the living world for communication and defense. The paradigm is that they are produced enzymatically for the eicosanoids and non-enzymatically for the isoprostanoids. They are supposed to be degraded into volatile organic compounds (VOCs) and to participate in aroma production. Some such chemicals composed of eight carbons are also envisoned as alternatives to fossil fuels. In fungi, oxylipins have been mostly studied in Aspergilli and shown to be involved in signalling asexual versus sexual development, mycotoxin production and interaction with the host for pathogenic species. Through targeted gene deletions of genes encoding oxylipin-producing enzymes and chemical analysis of oxylipins and volatile organic compounds, we show that in the distantly-related ascomycete Podospora anserina, isoprostanoids are likely produced enzymatically. We show the disappearance in the mutants lacking lipoxygenases and cyclooxygenases of the production of 10-hydroxy-octadecadienoic acid and that of 1-octen-3-ol, a common volatile compound. Importantly, this was correlated with the inability of the mutants to repel nematodes as efficiently as the wild type. Overall, our data show that in this fungus, oxylipins are not involved in signalling development but may rather be used directly or as precursors in the production of odors against potential agressors.

Meiotic development initiation in the fungus Podospora anserina requires the peroxisome receptor export machinery

Peroxisomes are versatile organelles essential for diverse developmental processes. One such process is the meiotic development of Podospora anserina. In this fungus, absence of the docking peroxin PEX13, the RING-finger complex peroxins, or the PTS2 co-receptor PEX20 blocks sexual development before meiocyte formation. However, this defect is not seen in the absence of the receptors PEX5 and PEX7, or of the docking peroxins PEX14 and PEX14/17. Here we describe the function of the remaining uncharacterized P. anserina peroxins predictably involved in peroxisome matrix protein import. We show that PEX8, as well as the peroxins potentially mediating receptor monoubiquitination (PEX4 and PEX22) and membrane dislocation (PEX1, PEX6 and PEX26) are indeed implicated in peroxisome matrix protein import in this fungus. However, we observed that elimination of PEX4 and PEX22 affects to different extent the import of distinct PEX5 cargoes, suggesting differential ubiquitination-complex requirements for the import of distinct proteins. In addition, we found that elimination of PEX1, PEX6 or PEX26 results in loss of peroxisomes, suggesting that these peroxins restrain peroxisome removal in specific physiological conditions. Finally, we demonstrate that all analyzed peroxins are required for meiocyte formation, and that PEX20 function in this process depends on its potential monoubiquitination target cysteine. Our results suggest that meiotic induction relies on a peroxisome import pathway, which is not dependent on PEX5 or PEX7 but that is driven by an additional cycling receptor. These findings uncover a collection of peroxins implicated in modulating peroxisome activity to facilitate a critical developmental cell fate decision.     

Identification of calcium binding proteins from brain

A procedure was worked out for purification and identification of calcium-binding proteins from bovine brain using Ca²⁺-dependent, reversible binding to a hydrophobic support, phenyl-Sepharose, as the method of isolation. These proteins could be visualized during and after their separation by running them on non-denaturing polyacrylamide gels, blotting to Zeta-probe paper, and autoradiographing with⁴⁵Ca²⁺. About 24 polypeptides could be seen in this fraction on SDS (Laemmli) gels and about 8–10 native, Ca²⁺-binding proteins could be seen on non-denaturing gels and on blots of their 45Ca²⁺ autoradiographs. Some of these proteins could be purified further by chromatography on DEAE-Sephacel and still retain their⁴⁵Ca²⁺-binding activity.     

Identification of centromeric and telomeric DNA‐binding proteins in rice

Centromeres and telomeres are DNA/protein complexes and essential functional components of eukaryotic chromosomes. Previous studies have shown that rice centromeres and telomeres are occupied by CentO (rice centromere satellite DNA) satellite and G‐rich telomere repeats, respectively. However, the protein components are not fully understood. DNA‐binding proteins associated with centromeric or telomeric DNAs will most likely be important for the understanding of centromere and telomere structure and functions. To capture DNA‐specific binding proteins, affinity pull‐down technique was applied in this study to isolate rice centromeric and telomeric DNA‐binding proteins. Fifty‐five proteins were identified for their binding affinity to rice CentO repeat, and 80 proteins were identified for their binding to telomere repeat. One CentO‐binding protein, Os02g0288200, was demonstrated to bind to CentO specifically by in vitro assay. A conserved domain, DUF573 with unknown functions was identified in this protein, and proven to be responsible for the specific binding to CentO in vitro. Four proteins identified as telomere DNA‐binding proteins in this study were reported by different groups previously. These results demonstrate that DNA affinity pull‐down technique is effective in the isolation of sequence‐specific binding proteins and will be applicable in future studies of centromere and telomere proteins.     

Identification and characterization of the adenosine 3′,5′-cyclic monophosphate binding proteins appearing during the development of Dictyostelium discoideum

A photosensitive, radioactive analogue of cyclic adenosine monophosphate, 8-azido-adenosine 3′,5′-[³²P]monophosphate (8-N₃-cyclic AMP), was used to label the cyclic AMP binding proteins of Dictyostelium discoideum. During development cytosolic proteins appear which are specifically labeled by the photoaffinity agent. The proteins are developmentally regulated since they are only found in starved, developing cells. Unlabeled cyclic AMP competes specifically with the labeled analogue for protein binding sites in contrast to unlabeled 5′-AMP which does not compete. A mutant which develops spores but is deficient in stalk cell production produces a different set of cyclic AMP binding proteins from the parent strain.     

Identification of four proteins from the small subunit of the mammalian mitochondrial ribosome using a proteomics approach

Proteins in the small subunit of the mammalian mitochondrial ribosome were separated by two-dimensional polyacrylamide gel electrophoresis. Four individual proteins were subjected to in-gel Endoprotease Lys-C digestion. The sequences of selected proteolytic peptides were obtained by electrospray tandem mass spectrometry. Peptide sequences obtained from in-gel digestion of individual spots were used to screen human, mouse, and rat expressed sequence tag databases, and complete consensus cDNAs for these species were deduced in silico. The corresponding protein sequences were characterized by comparison to known ribosomal proteins in protein databases. Four different classes of mammalian mitochondrial small subunit ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins are homologs to Escherichia coli S9 and S5 proteins. The presence of these newly identified mitochondrial ribosomal proteins are also investigated in the Drosophila melanogaster, Caenorhabditis elegans, and in the genomes of several fungi.     

Identification and characterization of a novel mitochondrial tricarboxylate carrier

Here we report the identification and functional characterization of a novel mitochondrial tricarboxylate carrier protein, designated BBG-TCC, in rat brain. The cDNA encodes the predicted protein of 342-amino acid residues with five putative membrane-spanning domains. The protein has apparent similarity with a mitochondrial tricarboxylate carrier TCC, but is distinct from the other mitochondria anion transporters. BBG-TCC shows a citrate transport activity. It is specifically expressed in the brain and localizes in the mitochondria of Bergmann glial cells. In contrast, the expression of TCC is rather ubiquitous and strong in neuronal cells in the brain. This new family of proteins may contribute to biosynthesis and bioenergetics in the brain.     

Characterization and purification of a protein binding to the cis-acting element for brain-specific exon 1 of the mouse aromatase gene

The functional differences between male and female brains commit to the existence of androgen that the testis secretes during the perinatal period. Androgen exerts its action on the brain after conversion to estrogen by brain aromatase. The aromatase appears in some neural nuclei such as in the hypothalamus and amygdala, and has been indicated to be involved in the expression of sexuality by the results of neurobehavioral analyses involving aromatase-knockout mice. We analyzed the brain-specific promoter in order to clarify the control mechanism for the expression of brain aromatase, which is deeply concerned in the sexual differentiation of the brain. The 202 bp upstream region of brain-specific exon 1 contains at least three kinds of cis-acting elements, Arom-Aα, -Aβ and -B. In particular, the binding activities as to the Aβ sequence show a tissue-specific pattern. Gel shift analysis revealed that the Aβ binding factor recognizes the TTGGCCCCT sequence. Aβ binding activity is detectable at the perinatal stage, but is undetectable at the adult stage in the brain. Furthermore, a protein which binds to the Aβ sequence was purified from the fetal mouse brain. The molecular mass of the Aβ binding protein was estimated to be 49 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

The crystal structure of the nuclease domain of colicin E7 suggests a mechanism for binding to double-stranded DNA by the H-N-H endonucleases

The bacterial toxin ColE7 contains an H-N-H endonuclease domain (nuclease ColE7) that digests cellular DNA or RNA non-specifically in target cells, leading to cell death. In the host cell, protein Im7 forms a complex with ColE7 to inhibit its nuclease activity. Here, we present the crystal structure of the unbound nuclease ColE7 at a resolution of 2.1A. Structural comparison between the unbound and bound nuclease ColE7 in complex with Im7, suggests that Im7 is not an allosteric inhibitor that induces backbone conformational changes in nuclease ColE7, but rather one that inhibits by blocking the substrate-binding site. There were two nuclease ColE7 molecules in the P1 unit cell in crystals and they appeared as a dimer related to each other by a non-crystallographic dyad symmetry. Gel-filtration and cross-linking experiments confirmed that nuclease ColE7 indeed formed dimers in solution and that the dimeric conformation was more favored in the presence of double-stranded DNA. Structural comparison of nuclease ColE7 with the His-Cys box homing endonuclease I-PpoI further demonstrated that H-N-H motifs in dimeric nuclease ColE7 were oriented in a manner very similar to that of the betabetaalpha-fold of the active sites found in dimeric I-PpoI. A mechanism for the binding of double-stranded DNA by dimeric H-N-H nuclease ColE7 is suggested.     

Structural and biochemical characterization of a new Mg(2+) binding site near Tyr94 in the restriction endonuclease PvuII

We have determined the crystal structure of the PvuII endonuclease in the presence of Mg(2+). According to the structural data, divalent metal ion binding in the PvuII subunits is highly asymmetric. The PvuII-Mg(2+) complex has two distinct metal ion binding sites, one in each monomer. One site is formed by the catalytic residues Asp58 and Glu68, and has extensive similarities to a catalytically important site found in all structurally examined restriction endonucleases. The other binding site is located in the other monomer, in the immediate vicinity of the hydroxyl group of Tyr94; it has no analogy to metal ion binding sites found so far in restriction endonucleases. To assign the number of metal ions involved and to better understand the role of Mg(2+) binding to Tyr94 for the function of PvuII, we have exchanged Tyr94 by Phe and characterized the metal ion dependence of DNA cleavage of wild-type PvuII and the Y94F variant. Wild-type PvuII cleaves both strands of the DNA in a concerted reaction. Mg(2+) binding, as measured by the Mg(2+) dependence of DNA cleavage, occurs with a Hill coefficient of 4, meaning that at least two metal ions are bound to each subunit in a cooperative fashion upon formation of the active complex. Quenched-flow experiments show that DNA cleavage occurs about tenfold faster if Mg(2+) is pre-incubated with enzyme or DNA than if preformed enzyme-DNA complexes are mixed with Mg(2+). These results show that Mg(2+) cannot easily enter the active center of the preformed enzyme-DNA complex, but that for fast cleavage the metal ions must already be bound to the apoenzyme and carried with the enzyme into the enzyme-DNA complex. The Y94F variant, in contrast to wild-type PvuII, does not cleave DNA in a concerted manner and metal ion binding occurs with a Hill coefficient of 1. These results indicate that removal of the Mg(2+) binding site at Tyr94 completely disrupts the cooperativity in DNA cleavage. Moreover, in quenched-flow experiments Y94F cleaves DNA about ten times more slowly than wild-type PvuII, regardless of the order of mixing. From these results we conclude that wild-type PvuII cleaves DNA in a fast and concerted reaction, because the Mg(2+) required for catalysis are already bound at the enzyme, one of them at Tyr94. We suggest that this Mg(2+) is shifted to the active center during binding of a specific DNA substrate. These results, for the first time, shed light on the pathway by which metal ions as essential cofactors enter the catalytic center of restriction endonucleases.     

A nuclear matrix protein related to intermediate filaments proteins is a member of the complex binding alphoid DNA in vitro

A complex of three proteins (of 80, 70, 58 kDa-p80, p70, and p58, respectively) with the ability to bind alphoid DNA (alpha-satDNA) was revealed by gel mobility shift assay (GMSA) in human nuclear matrix. The probes of the alpha-satDNA bound in the GMSA with the greatest specificity, but the complex was capable of binding human satellite 3 fragment. According to ion exchange and affinity chromatography, the complex includes two DNA-binding proteins, p70 and p80, and a non-DNA-binding one, p58, which enhances the specificity of binding to the alpha-satDNA. GMSA, SDS-PAGE and immunoblotting showed that the lamins, as well as constitutive centromeric proteins (CENP-A, CENP-B, CENP-C, CENP-G), were not incorporated into the complex. It was demonstrated by immunoprecipitation assay that p70 and, probably p58, share a common antigen determinant with the rod domain of intermediate filaments (IF) proteins. The results obtained indicate that the nuclear matrix contains at least one IF-related protein that is able to bind specifically to alpha-satDNA in vitro and that this protein is distinct from the lamins.     

Identification and evaluation of universal epitopes in Plasmodium vivax Duffy binding protein

Selected PvDBP-derived synthetic peptides were tested in competition assays with HLA molecules in order to identify and evaluate their binding to a wide range of MHC class II molecules. Binding was evaluated as the peptide’s ability to displace the biotinylated control peptide (HA_306-318) and was detected by a conventional ELISA. Thus, one epitope for the HLA-DR1 molecule, two epitopes for the HLA-DR4 molecule, six epitopes for the HLA-DR7 molecule and three epitopes for the HLA-DR11 molecule displaying a high binding percentage (above 50%) were experimentally obtained. The in vitro results were compared with the epitope prediction results. Two peptides behaved as universal epitopes since they bound to a larger number of HLA-DR molecules. Given that these peptides are located in the conserved PvDBP region II, they could be considered good candidates to be included in the design of a synthetic vaccine against Plasmodium vivax malaria.