Identification of the polypeptide encoded by the URF-1 gene of Neurospora crassa mtDNA

Two peptides, potentially representing antigenic determinants of a proposed gene product, were synthesized. The peptide sequences were deduced from the nucleotide sequence of the unidentified reading frame (URF)1 of the Neurospora crassa mitochondrial genome. Specific antisera to the synthetic peptides were produced. The antibodies recognized a single polypeptide species with an apparent relative molecular mass of about 30 000. The mitochondrial origin of this polypeptide was verified by in vivo labelling experiments in the presence of cycloheximide, as well as by in vitro translation using isolated mitochondria. The chemical identification of the protein was performed by partial radiosequencing of the N-terminal portion of the immunoprecipitated URF-1 product. The amount of URF-1 polypeptide present in N. crassa mitochondria is in the range of 1-2%. The protein is a constituent of the inner envelope of the organelle and probably part of a more complex membrane unit.     

Growth of the yeast Hansenula anomala with nitrate as sole source of nitrogen under aerobic and anaerobic conditions

Summary The oxygen requirement ofHansenula anomala growing in batch culture on nitrate as sole source of nitrogen was examined. An aeration rate of 0.03 vvm or a constant oxygen partial pressure of 0.01 bar is sufficient for optimal growth.     

A point mutation in an unusual Sec7 domain is linked to brefeldin A resistance in a Plasmodium falciparum line generated by drug selection

The malaria parasite Plasmodium falciparum has an unusual organization of its secretory compartments. As an approach to a functional identification of auxiliary proteins involved in secretion, a parasite line was generated by drug selection that is resistant to brefeldin A, an inhibitor of the secretory pathway. In the resistant line, neither protein secretion nor parasite viability were affected by the drug. The analysis of a sec7 domain, a conserved structure of guanine nucleotide exchange factors (ARF-GEF) required for the activation of ADP-ribosylation factors, revealed a single methionine-isoleucine substitution in the resistant parasite line. ARF-GEFs are key molecules in the formation of transport vesicles and the main targets of brefeldin A. The methionine residue in this position of sec7 domains is highly conserved and confers brefeldin A sensitivity. Unlike other eukaryotes that have multiple ARF-GEFs, the plasmodial genome encodes a single sec7 domain. This domain shows a distinct structural difference to all sec7 domains analysed so far; two conserved subdomains that are essential for protein function are separated in the plasmodial protein by an insertion of 146 amino acids.     

Unique genetic compartmentalization of the SUF system in cryptophytes and characterization of a SufD mutant in Arabidopsis thaliana

The mobilization of sulfur (SUF) system is one of three systems involved in iron-sulfur cluster biosynthesis and maintenance. In eukaryotes the SUF system is specific for the plastid and therefore of symbiotic origin. Analyses in cryptophytes showed a unique genetic compartmentalization of the SUF system, which evolved by at least two different gene transfer events. We analyzed one of the components, SufD, in the cryptophyte Guillardia theta and in Arabidopsis thaliana. We demonstrated that SufD fulfils house keeping functions during embryogenesis and in adult plants in A. thaliana.     

Subunit composition of the glycyl radical enzyme p-hydroxyphenylacetate decarboxylase. A small subunit, HpdC, is essential for catalytic activity.

p-Hydroxyphenylacetate decarboxylase from Clostridium difficile catalyses the decarboxylation of p-hydroxyphenylacetate to yield the cytotoxic compound p-cresol. The three genes encoding two subunits of the glycyl-radical enzyme and the activating enzyme have been cloned and expressed in Escherichia coli. The recombinant enzymes were used to reconstitute a catalytically functional system in vitro. In contrast with the decarboxylase purified from C. difficile, which was an almost inactive homo-dimeric protein (beta(2)), the recombinant enzyme was a hetero-octameric (beta(4)gamma(4)), catalytically competent complex, which was activated using endogenous activating enzyme from C. difficile or recombinant activating enzyme to a specific activity of 7 U.mg(-1). Preliminary results suggest that phosphorylation of the small subunit is responsible for the change of the oligomeric state. These data point to an essential function of the small subunit of the decarboxylase and may indicate unique regulatory properties of the system.     

Review: origin of complex algae by secondary endosymbiosis: a journey through time

Secondary endosymbiosis—the merging of two eukaryotic cells into one photosynthetic cellular unit—led to the evolution of ecologically and medically very important organisms. We review the biology of these organisms, starting from the first proposal of secondary endosymbiosis up to recent phylogenetic models on the origin of secondarily evolved protists. In addition, we discuss the organelle character of the symbionts based on morphological features, gene transfers from the symbiont into the host and re-import of nucleus-encoded plastid proteins. Finally, we hypothesize that secondary endosymbiosis is more than enslaving a eukaryotic, phototrophic cell, but reflects a complex interplay between host and symbiont, leading to the inseparability of the two symbiotic partners generating a cellular entity.     

Type-3 copper proteins as biocompatible and reusable oxygen sensors

Fluorescently labeled type-3 copper proteins have been proposed previously as solution oxygen sensors by using a FRET mechanism. Herein, we describe how this principle can be adapted to sense O₂ by means of proteins immobilized in optically transparent silica matrices. Specifically, the protein, hemocyanin from Octopus vulgaris N-terminally labeled with Cy5, is immobilized in two different kinds of optically transparent silica matrices, which appear to be a promising platform for enzyme encapsulation. The presented results provide proof of principle that fluorescently labeled proteins immobilized in a silica matrix can be implemented in a reusable, biocompatible and stable oxygen measuring device that might lead to new developments in the field of optical biosensing.     

Synonymous codon usage pattern among the S,M, and L segments in Crimean-congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever (CCHF) virus is one among the major zoonosis viral diseases that use the Hyalomma ticks as their transmission vector to cause viral infection to the human and mammalian community. The fatality of infectious is high across the world especially in Africa, Asia, Middle East, and Europe. This study regarding codon usage bias of S, M, and L segments of the CCHF virus pertaining to the host Homo sapiens, reveals in-depth information about the evolutionary characteristics of CCHFV. Relative Synonymous Codon Usage (RSCU), Effective number of codons (ENC) were calculated, to determine the codon usage pattern in each segment. Correlation analysis between Codon adaptation index (CAI), GRAVY (Hydrophobicity), AROMO (Aromaticity), and nucleotide composition revealed bias in the codon usage pattern. There was no strong codon bias found among any segments of the CCHF virus, indicating both the factors i.e., natural selection and mutational pressure shapes the codon usage bias.     

Rhinovirus-mediated endosomal release of transfection complexes.

Endocytosis is an efficient method for transfer of genes into mammalian cells. Incorporation of adenovirus particles into gene transfer complexes greatly enhances gene delivery, probably by the release of endocytosed DNA into the cytoplasm. We report here that two different serotypes of human rhinovirus (HRV), HRV2 and HRV14, are also able to enhance receptor-mediated gene transfer. The effect of several compounds known to inhibit viral infection on HRV2- and HRV14-enhanced transfection was examined. WIN I(s) and WIN IV, two compounds which inhibit viral uncoating, had different effects on HRV2- and HRV14-enhanced gene transfer to NIH 3T3 cells. While HRV14-enhanced gene transfer was severely reduced in the presence of these compounds, virtually no effects were observed when HRV2 was used. The use of antiviral compounds thus allowed transfection of human cells, which are normally lysed rapidly upon infection with HRV. Viral activity could be mimicked by using a peptide derived from the N terminus of VP1 of HRV2. This peptide possesses pH-dependent membrane-disrupting activity and enhances gene transfer to NIH 3T3 and HeLa cells.