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1.
Int J Mol Sci ; 25(10)2024 May 10.
Article in English | MEDLINE | ID: mdl-38791231

ABSTRACT

Ribosomal RNAs (rRNAs) are extensively modified during the transcription and subsequent maturation. Three types of modifications, 2'-O-methylation of ribose moiety, pseudouridylation, and base modifications, are introduced either by a snoRNA-driven mechanism or by stand-alone enzymes. Modified nucleotides are clustered at the functionally important sites, including peptidyl transferase center (PTC). Therefore, it has been hypothesised that the modified nucleotides play an important role in ensuring the functionality of the ribosome. In this study, we demonstrate that seven 25S rRNA modifications, including four evolutionarily conserved modifications, in the proximity of PTC can be simultaneously depleted without loss of cell viability. Yeast mutants lacking three snoRNA genes (snR34, snR52, and snR65) and/or expressing enzymatically inactive variants of spb1(D52A/E679K) and nop2(C424A/C478A) were constructed. The results show that rRNA modifications in PTC contribute collectively to efficient translation in eukaryotic cells. The deficiency of seven modified nucleotides in 25S rRNA resulted in reduced cell growth, cold sensitivity, decreased translation levels, and hyperaccurate translation, as indicated by the reduced missense and nonsense suppression. The modification m5C2870 is crucial in the absence of the other six modified nucleotides. Thus, the pattern of rRNA-modified nucleotides around the PTC is essential for optimal ribosomal translational activity and translational fidelity.


Subject(s)
Peptidyl Transferases , Protein Biosynthesis , RNA, Ribosomal , Saccharomyces cerevisiae , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Peptidyl Transferases/metabolism , Peptidyl Transferases/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomycetales/genetics , Saccharomycetales/metabolism , Ribosomes/metabolism , RNA, Small Nucleolar/genetics , RNA, Small Nucleolar/metabolism , RNA Processing, Post-Transcriptional , RNA, Fungal/genetics , RNA, Fungal/metabolism , Mutation
2.
Heliyon ; 10(6): e27885, 2024 Mar 30.
Article in English | MEDLINE | ID: mdl-38545165

ABSTRACT

Yeasts are single-celled fungi that are widespread around the globe. They are part of a community of microorganisms that use a wide variety of habitats, including fruit surfaces. This study aimed to characterise the culturable epiphytic yeasts associated with apple fruits. The isolated yeast strains were identified by sequencing the 5.8S-ITS region and D1/D2 region of the large subunit ribosomal RNA gene and maintained for long-term storage. A total of 230 yeast isolates belonging to 33 species were recovered. Most of the collected isolates belonged to the phylum Basidiomycota. Members of genera Vishniacozyma, Filobasidium, and Rhodotorula were most frequently isolated. Over half of the species were isolated on only one to three occasions. In seven of the species obtained, the isolates were considerably divergent from their closest relatives and may therefore represent new distinct species. The results of this study demonstrate a high diversity of yeast species associated with apple fruits.

3.
Comput Struct Biotechnol J ; 21: 1249-1261, 2023.
Article in English | MEDLINE | ID: mdl-36817958

ABSTRACT

In three domains of life, proteins are synthesized by large ribonucleoprotein particles called ribosomes. All ribosomes are composed of ribosomal RNAs (rRNA) and numerous ribosomal proteins (r-protein). The three-dimensional shape of ribosomes is mainly defined by a tertiary structure of rRNAs. In addition, rRNAs have a major role in decoding the information carried by messenger RNAs and catalyzing the peptide bond formation. R-proteins are essential for shaping the network of interactions that contribute to a various aspects of the protein synthesis machinery, including assembly of ribosomes and interaction of ribosomal subunits. Structural studies have revealed that many key components of ribosomes are conserved in all life domains. Besides the core structure, ribosomes contain domain-specific structural features that include additional r-proteins and extensions of rRNA and r-proteins. This review focuses specifically on those r-proteins that are found only in archaeal and eukaryotic ribosomes. The role of these archaea/eukaryote specific r-proteins in stabilizing the ribosome structure is discussed. Several examples illustrate their functions in the formation of the internal network of ribosomal subunits and interactions between the ribosomal subunits. In addition, the significance of these r-proteins in ribosome biogenesis and protein synthesis is highlighted.

4.
Nucleic Acids Res ; 49(10): e59, 2021 06 04.
Article in English | MEDLINE | ID: mdl-33684199

ABSTRACT

The elongation step of translation is a key contributor to the abundance, folding and quality of proteins and to the stability of mRNA. However, control over translation elongation has not been thoroughly investigated. In this study, a Renilla-firefly luciferase fusion reporter system was further developed to investigate the in vitro elongation rate and processivity of ribosomes independent of the initiation and termination steps. The reporter mRNA was constructed to contain a single ORF encoding in-frame Renilla luciferase, a specific domain moiety and firefly luciferase. Such a reporter structure enables the quantitative and individual evaluation of the synthesis of a specific domain. As a proof of principle, the synthesis of three protein domains of different lengths and structures was analyzed. Using a cell-free translation assay, both the elongation rate and processivity of ribosomes were shown to vary depending on the domain synthesized. Additionally, a stalling sequence consisting of ten rare arginine codons notably reduced the elongation rate and the processivity of the ribosomes. All these results are consistent with the previously known dynamics of elongation in vivo. Overall, the methodology presented in this report provides a framework for studying aspects that contribute to the elongation step of translation.


Subject(s)
Luciferases, Firefly/metabolism , Peptide Chain Elongation, Translational , Peptide Chain Termination, Translational , RNA, Messenger/metabolism , Ribosomes/metabolism , Genes, Reporter , Saccharomyces cerevisiae
5.
Genetics ; 213(4): 1329-1339, 2019 12.
Article in English | MEDLINE | ID: mdl-31649153

ABSTRACT

Ribosomes of Archaea and Eukarya share higher homology with each other than with bacterial ribosomes. For example, there is a set of 35 r-proteins that are specific only for archaeal and eukaryotic ribosomes. Three of these proteins-eL19, eL24, and eL41-participate in interactions between ribosomal subunits. The eukaryote-specific extensions of r-proteins eL19 and eL24 form two intersubunit bridges eB12 and eB13, which are present only in eukaryotic ribosomes. The third r-protein, eL41, forms bridge eB14. Notably, eL41 is found in all eukaryotes but only in some Archaea. It has been shown that bridges eB12 and eB13 are needed for efficient translation, while r-protein eL41 plays a minor role in ribosome function. Here, the functional interactions between intersubunit bridges were studied using budding yeast strains lacking different combinations of the abovementioned bridges/proteins. The growth phenotypes, levels of in vivo translation, ribosome-polysome profiles, and in vitro association of ribosomal subunits were analyzed. The results show a genetic interaction between r-protein eL41 and the eB12 bridge-forming region of eL19, and between r-proteins eL41 and eL24. It was possible to construct viable yeast strains with Archaea-like ribosomes lacking two or three eukaryote-specific bridges. These strains display slow growth and a poor translation phenotype. In addition, bridges eB12 and eB13 appear to cooperate during ribosome subunit association. These results indicate that nonessential structural elements of r-proteins become highly important in the context of disturbed subunit interactions. Therefore, eukaryote-specific bridges may contribute to the evolutionary success of eukaryotic translation machinery.


Subject(s)
Ribosome Subunits/metabolism , Ribosomes/metabolism , Saccharomyces cerevisiae/metabolism , Models, Molecular , Mutation/genetics , Phenotype , Polyribosomes/metabolism , Protein Binding , Protein Domains , Ribosome Subunits/chemistry , Ribosomes/chemistry , Saccharomyces cerevisiae/growth & development
6.
Nucleic Acids Res ; 47(1): 406-420, 2019 01 10.
Article in English | MEDLINE | ID: mdl-30407570

ABSTRACT

Interactions between subunits in the Saccharomyces cerevisiae ribosome are mediated by universal and eukaryote-specific intersubunit bridges. Universal bridges are positioned close to the ribosomal functional centers, while eukaryote-specific bridges are mainly located on the periphery of the ribosome. Two bridges, eB13 and B6, are formed by the ribosomal protein eL24. The eukaryotic eL24 is composed of an N-terminal domain, a linker region and a C-terminal α-helix. Here, the functions of different domains of eL24 in the S. cerevisiae ribosome were evaluated. The C-terminal domain and the linker region of the eL24 form eukaryote-specific eB13 bridge. Phenotypic characterization of the eL24 deletion mutants indicated that the functional integrity of the eB13 bridge mainly depends on the protein-protein contacts between eL24 and eS6. Further investigation showed importance of the eB13 bridge in the subunit joining in vivo and in vitro. In vitro translation assay demonstrated the role of the eB13 bridge in both initiation and elongation steps of translation. Intriguingly, results of in vitro translation experiment suggest involvement of the N-terminal domain of eL24 in the translation initiation. Therefore, eL24 performs number of tasks required for the optimal ribosome functionality.


Subject(s)
Protein Biosynthesis/genetics , Ribosomal Proteins/genetics , Ribosomes/genetics , Eukaryotic Cells/chemistry , Eukaryotic Cells/metabolism , Models, Molecular , Protein Interaction Maps/genetics , Protein Processing, Post-Translational , Ribosomal Proteins/biosynthesis , Ribosomal Proteins/chemistry , Ribosomes/chemistry , Saccharomyces cerevisiae/genetics , Sequence Deletion/genetics
7.
J Mol Biol ; 428(10 Pt B): 2203-16, 2016 May 22.
Article in English | MEDLINE | ID: mdl-27038511

ABSTRACT

During translation, the two eukaryotic ribosomal subunits remain associated through 17 intersubunit bridges, five of which are eukaryote specific. These are mainly localized to the peripheral regions and are believed to stabilize the structure of the ribosome. The functional importance of these bridges remains largely unknown. Here, the essentiality of the eukaryote-specific bridge eB12 has been investigated. The main component of this bridge is ribosomal protein eL19 that is composed of an N-terminal globular domain, a middle region, and a long C-terminal α-helix. The analysis of deletion mutants demonstrated that the globular domain and middle region of eL19 are essential for cell viability, most likely functioning in ribosome assembly. The eB12 bridge, formed by contacts between the C-terminal α-helix of eL19 and 18S rRNA in concert with additional stabilizing interactions involving either eS7 or uS17, is dispensable for viability. Nevertheless, eL19 mutants impaired in eB12 bridge formation displayed slow growth phenotypes, altered sensitivity/resistance to translational inhibitors, and enhanced hyperosmotic stress tolerance. Biochemical analyses determined that the eB12 bridge contributes to the stability of ribosome subunit interactions in vitro. 60S subunits containing eL19 variants defective in eB12 bridge formation failed to form 80S ribosomes regardless of Mg(2+) concentration. The reassociation of 40S and mutant 60S subunits was markedly improved in the presence of deacetylated tRNA, emphasizing the importance of tRNAs during the subunit association. We propose that the eB12 bridge plays an important role in subunit joining and in optimizing ribosome functionality.


Subject(s)
Eukaryotic Cells/physiology , Ribosome Subunits/physiology , Mutation/genetics , Nucleic Acid Conformation , Protein Biosynthesis/genetics , Protein Biosynthesis/physiology , RNA, Ribosomal/genetics , RNA, Transfer/genetics , Ribosomal Proteins/genetics , Ribosome Subunits/genetics , Saccharomyces cerevisiae/physiology
8.
PLoS One ; 9(7): e101561, 2014.
Article in English | MEDLINE | ID: mdl-24991888

ABSTRACT

Structural studies have revealed that the core of the ribosome structure is conserved among ribosomes of all kingdoms. Kingdom-specific ribosomal proteins (r-proteins) are located in peripheral parts of the ribosome. In this work, the interactions between rRNA and r-proteins of eukaryote Saccharomyces cerevisiae ribosome were investigated applying LiCl induced splitting and quantitative mass spectrometry. R-proteins were divided into four groups according to their binding properties to the rRNA. Most yeast r-proteins are removed from rRNA by 0.5-1 M LiCl. Eukaryote-specific r-proteins are among the first to dissociate. The majority of the strong binders are known to be required for the early ribosome assembly events. As compared to the bacterial ribosome, yeast r-proteins are dissociated from rRNA at lower ionic strength. Our results demonstrate that the nature of protein-RNA interactions in the ribosome is not conserved between different kingdoms.


Subject(s)
Lithium Chloride/toxicity , Ribosomal Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Chromatography, High Pressure Liquid , Isotope Labeling , Lithium Chloride/chemistry , Peptides/analysis , Peptides/isolation & purification , Protein Structure, Quaternary , RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , Ribosomal Proteins/chemistry , Ribosomes/drug effects , Ribosomes/metabolism , Saccharomyces cerevisiae/metabolism , Tandem Mass Spectrometry
9.
Genetics ; 193(2): 467-81, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23172851

ABSTRACT

Mitochondrial metabolism is targeted by conserved signaling pathways that mediate external information to the cell. However, less is known about whether mitochondrial dysfunction interferes with signaling and thereby modulates the cellular response to environmental changes. In this study, we analyzed defective filamentous and invasive growth of the yeast Saccharomyces cerevisiae strains that have a dysfunctional mitochondrial genome (rho mutants). We found that the morphogenetic defect of rho mutants was caused by specific downregulation of FLO11, the adhesin essential for invasive and filamentous growth, and did not result from general metabolic changes brought about by interorganellar retrograde signaling. Transcription of FLO11 is known to be regulated by several signaling pathways, including the filamentous-growth-specific MAPK and cAMP-activated protein kinase A (cAMP-PKA) pathways. Our analysis showed that the filamentous-growth-specific MAPK pathway retained functionality in respiratory-deficient yeast cells. In contrast, the cAMP-PKA pathway was downregulated, explaining also various phenotypic traits observed in rho mutants. Thus, our results indicate that dysfunctional mitochondria modulate the output of the conserved cAMP-PKA signaling pathway.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , Mitochondria/metabolism , Saccharomyces cerevisiae/metabolism , DNA Polymerase I/genetics , DNA-Directed RNA Polymerases/genetics , Down-Regulation , MAP Kinase Signaling System , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mitochondria/genetics , Mitochondrial Proteins/genetics , Mutation , Phosphorylation , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Transcription, Genetic
10.
PLoS One ; 7(3): e33482, 2012.
Article in English | MEDLINE | ID: mdl-22432028

ABSTRACT

Saccharomyces cerevisiae mitochondrial DNA polymerase (Mip1) contains a C-terminal extension (CTE) of 279 amino acid residues. The CTE is required for mitochondrial DNA maintenance in yeast but is absent in higher eukaryotes. Here we use recombinant Mip1 C-terminal deletion mutants to investigate functional importance of the CTE. We show that partial removal of the CTE in Mip1Δ216 results in strong preference for exonucleolytic degradation rather than DNA polymerization. This disbalance in exonuclease and polymerase activities is prominent at suboptimal dNTP concentrations and in the absence of correctly pairing nucleotide. Mip1Δ216 also displays reduced ability to synthesize DNA through double-stranded regions. Full removal of the CTE in Mip1Δ279 results in complete loss of Mip1 polymerase activity, however the mutant retains its exonuclease activity. These results allow us to propose that CTE functions as a part of Mip1 polymerase domain that stabilizes the substrate primer end at the polymerase active site, and is therefore required for efficient mitochondrial DNA replication in vivo.


Subject(s)
DNA Polymerase I/chemistry , DNA Polymerase I/metabolism , DNA Replication , DNA, Fungal/metabolism , DNA, Mitochondrial/metabolism , Mitochondria/enzymology , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Biocatalysis , Exonucleases/metabolism , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/isolation & purification , Mutant Proteins/metabolism , Protein Binding , Sequence Alignment , Sequence Deletion , Structure-Activity Relationship
11.
Biotechnol J ; 7(4): 566-8, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22009669

ABSTRACT

Microbiological production of glutathione using genetically engineered yeast strains has a potential to satisfy the increasing industrial demand of this tripeptide. In the present work accumulation of glutathione in response to YAP1 over-expression in Saccharomyces cerevisiae was studied. The over-expression resulted in intracellular glutathione level over two times higher than in the parent strain. Transcript analyses revealed that, in addition to the genes encoding enzymes in the glutathione biosynthesis pathway (GSH1 and GSH2), the expression levels of the genes in the cysteine biosynthesis pathway (CYS3 and CYS4) were also significantly higher in the YAP1 over-expressed strain. This suggests that YAP1 over-expression affects glutathione accumulation at both its biosynthesis and substrate availability levels.


Subject(s)
Glutathione/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , Gene Expression Regulation, Fungal , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/genetics
12.
Methods Mol Biol ; 824: 417-32, 2012.
Article in English | MEDLINE | ID: mdl-22160912

ABSTRACT

Schizosaccharomyces pombe, the fission yeast, has been a popular and useful model system for investigating the mechanisms of biological processes for a long time. To facilitate purification, localization, and functional analysis of gene products, a wide range of expression vectors have been developed. Several of these vectors utilize the inducible/repressible promoter systems and enable the episomal expression of proteins as fusion proteins with epitope tags attached to their N terminus or C terminus.This chapter provides a detailed protocol for expression of the epitope-tagged proteins from thiamine-regulatable nmt promoter in fission yeast. The yeast culture conditions and procedures for yeast transformation, expression induction, preparation of whole-cell extracts, and analysis of epitope-tagged protein expression by Western blotting are described.


Subject(s)
Epitopes/metabolism , Gene Expression Regulation, Fungal/genetics , Recombinant Proteins/metabolism , Schizosaccharomyces/metabolism , Thiamine/metabolism , Transformation, Genetic/genetics , Blotting, Western/methods , Electrophoresis, Polyacrylamide Gel , Epitopes/genetics , Genetic Vectors/genetics , Promoter Regions, Genetic/genetics , Recombinant Proteins/genetics , Schizosaccharomyces/genetics
13.
J Cell Biol ; 195(3): 467-84, 2011 Oct 31.
Article in English | MEDLINE | ID: mdl-22042620

ABSTRACT

The fission yeast interphase spindle pole body (SPB) is a bipartite structure in which a bulky cytoplasmic domain is separated from a nuclear component by the nuclear envelope. During mitosis, the SPB is incorporated into a fenestra that forms within the envelope during mitotic commitment. Closure of this fenestra during anaphase B/mitotic exit returns the cytoplasmic component to the cytoplasmic face of an intact interphase nuclear envelope. Here we show that Brr6 is transiently recruited to SPBs at both SPB insertion and extrusion. Brr6 is required for both SPB insertion and nuclear envelope integrity during anaphase B/mitotic exit. Genetic interactions with apq12 and defective sterol assimilation suggest that Brr6 may alter envelope composition at SPBs to promote SPB insertion and extrusion. The restriction of the Brr6 domain to eukaryotes that use a polar fenestra in an otherwise closed mitosis suggests a conserved role in fenestration to enable a single microtubule organizing center to nucleate both cytoplasmic and nuclear microtubules on opposing sides of the nuclear envelope.


Subject(s)
Membrane Proteins/metabolism , Nuclear Envelope/metabolism , Nuclear Proteins/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Spindle Apparatus/metabolism , Cell Nucleus/metabolism , Cytoplasm/metabolism , Fluorescent Antibody Technique , Membrane Proteins/genetics , Mitosis , Nuclear Proteins/genetics , Schizosaccharomyces/ultrastructure , Schizosaccharomyces pombe Proteins/genetics
14.
Virus Genes ; 40(3): 423-31, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20155311

ABSTRACT

Cocksfoot mottle virus (CfMV) coat protein (CP) localization was studied in plant and mammalian cells. Fusion of the full-length CP with enhanced green fluorescent protein (EGFP) localized to the cell nucleus whereas similar constructs lacking the first 33 N-terminal amino acids of CP localized to the cytoplasm. CP and EGFP fusions containing mutations in the arginine-rich motif of CP localized to the cytoplasm and to the nucleus in plant cells indicating the involvement of the motif in nuclear localization. In mammalian cells, mutations in the arginine-rich region were sufficient to completely abolish nuclear transport. The analysis of deletions of amino acid residues 1-11, 1-22, and 22-33 of CP demonstrated that there were two separate nuclear localization signals (NLS) within the N-terminus--a strong NLS1 in the arginine-rich region (residues 22-33) and a weaker NLS2 within residues 1-22. Analysis of point mutants revealed that the basic amino acid residues in the region of the two NLSs were individually not sufficient to direct CP to the nucleus. Additional microinjection studies with fluorescently labeled RNA and CP purified from CfMV particles demonstrated that the wild-type CP was capable of transporting the RNA to the nucleus. This feature was not sequence-specific in transient assays since both CfMV and GFP mRNA were transported to the cell nucleus by CfMV CP. Together the results suggest that the nucleus may be involved in CfMV infection.


Subject(s)
Capsid Proteins/genetics , Dactylis/virology , Nuclear Localization Signals , Plant Viruses/genetics , RNA Viruses/genetics , Cell Nucleus/chemistry , Cytoplasm/chemistry , DNA Mutational Analysis , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Point Mutation , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Staining and Labeling
15.
Virus Res ; 146(1-2): 73-80, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19748532

ABSTRACT

The -1 programmed ribosomal frameshifting (-1 PRF) mechanism utilized by many viruses is dependent on a heptanucleotide slippery sequence and a downstream secondary structure element. In the current study, the RNA structure downstream from the slippery site of cocksfoot mottle sobemovirus (CfMV) was proven to be a 12bp stem-loop with a single bulge and a tetranucleotide loop. Several deletion and insertion mutants with altered stem-loop structures were tested in wheat germ extract (WGE) for frameshifting efficiency. The impact of the same mutations on virus infectivity was tested in oat plants. Mutations shortening or destabilizing the stem region reduced significantly but did not abolish -1 PRF in WGE. The same mutations proved to be deleterious for virus infection. However, extending the loop region to seven nucleotides had no significant effect on frameshifting efficiency in WGE and did not hamper virus replication in infected leaves. This is the first report about the experimentally proven RNA secondary structure directing -1 PRF of sobemoviruses.


Subject(s)
Frameshifting, Ribosomal , Nucleic Acid Conformation , Plant Viruses/physiology , Protein Biosynthesis , RNA, Viral/genetics , Amino Acid Sequence , Avena/virology , Base Sequence , Molecular Sequence Data , Mutagenesis, Insertional , Plant Viruses/pathogenicity , RNA, Viral/chemistry , Sequence Deletion , Viral Proteins/biosynthesis
16.
Yeast ; 26(1): 55-66, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19180640

ABSTRACT

A single-step PCR-based epitope tagging enables fast and efficient gene targeting with various epitope tags. This report presents a series of plasmids for the E2 epitope tagging of proteins in Saccharomyces cerevisiae and Schizosaccharomyces pombe. E2Tags are 10-amino acids (epitope E2a: SSTSSDFRDR)- and 12 amino acids (epitope E2b: GVSSTSSDFRDR)-long peptides derived from the E2 protein of bovine papillomavirus type 1. The modules for C-terminal tagging with E2a and E2b epitopes were constructed by the modification of the pYM-series plasmid. The N-terminal E2a and E2b tagging modules were based on pOM-series plasmid. The pOM-series plasmids were selected for this study because of their use of the Cre-loxP recombination system. The latter enables a marker cassette to be removed after integration into the loci of interest and, thereafter, the tagged protein is expressed under its endogenous promoter. Specifically for fission yeast, high copy pREP plasmids containing the E2a epitope tag as an N-terminal or C-terminal tag were constructed. The properties of E2a and E2b epitopes and the sensitivity of two anti-E2 monoclonal antibodies (5E11 and 3F12) were tested using several S. cerevisiae and Sz. pombe E2-tagged strains.


Subject(s)
DNA-Binding Proteins/metabolism , Gene Targeting/methods , Plasmids/genetics , Polymerase Chain Reaction/methods , Saccharomyces cerevisiae/genetics , Schizosaccharomyces/genetics , Viral Proteins/metabolism , Amino Acid Sequence , Base Sequence , DNA-Binding Proteins/genetics , Epitopes/genetics , Epitopes/metabolism , Gene Expression , Genetic Engineering , Genetic Vectors , Molecular Sequence Data , Saccharomyces cerevisiae/metabolism , Schizosaccharomyces/metabolism , Viral Proteins/genetics
17.
Virus Genes ; 32(3): 321-6, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16732485

ABSTRACT

Cocksfoot mottle sobemovirus (CfMV) encodes a non-conserved protein P1 from the 5' ORF1 of genomic RNA. The functions of CfMV P1 are unknown. In the current study we show that P1-deficient CfMV can replicate both in oat leaves and barley suspension culture cells but can not infect oat plants systemically. However, the absence of P1 reduces the efficiency of virus accumulation considerably. The infectivity of the mutant virus restores as a result of the spontaneous transversion. CfMV P1:EGFP shows a very limited cell-to-cell movement in leaf epidermal cells. In Sf9 insect cells CfMV P1 localizes in the fraction of membranes and inclusions but not in soluble cytoplasmic protein fraction.


Subject(s)
Avena/virology , Hordeum/virology , Plant Diseases/virology , RNA Viruses/physiology , RNA Viruses/pathogenicity , Animals , Cells, Cultured , Plant Leaves/virology , Spodoptera , Subcellular Fractions/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Replication
18.
J Gen Virol ; 81(Pt 11): 2783-2789, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11038392

ABSTRACT

The polyprotein of Cocksfoot mottle virus (CfMV; genus SOBEMOVIRUS:) is translated from two overlapping open reading frames (ORFs) 2a and 2b by a -1 ribosomal frameshifting mechanism. In this study, a 12 kDa protein was purified from viral RNA-derived samples that appears to correspond to the CfMV genome-linked protein (VPg). According to the determined N-terminal amino acid sequence, the VPg domain is located between the serine proteinase and replicase motifs and the N terminus of VPg is cleaved from the polyprotein between glutamic acid and asparagine residues. Western blot analysis of infected plant material showed that the polyprotein is processed at several additional sites. An antiserum against the ORF 2a product recognized six distinct proteins, whereas, of these, the VPg antiserum clearly recognized only a 24 kDa protein. This indicates that the fully processed 12 kDa VPg detected in viral RNA-derived samples is a minor product in infected plants. An antiserum against the ORF 2b product recognized a 58 kDa protein, which indicates that the fully processed replicase is entirely or almost entirely encoded by ORF 2b. The origin of the detected cleavage products and a proposed polyprotein processing model are discussed.


Subject(s)
Plant Viruses/metabolism , Viral Proteins/metabolism , Chromosome Mapping , Genome, Viral , Open Reading Frames/genetics , Viral Proteins/genetics
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