Proteins Of Known Function Research Articles

Microarray analysis of gene expression profiles in ripening pineapple fruits

BackgroundPineapple (Ananas comosus) is a tropical fruit crop of significant commercial importance. Although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. Understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. In this study we developed a 9277 element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening.ResultsMicroarray analyses identified 271 unique cDNAs differentially expressed at least 1.5-fold between the mature green and mature yellow stages of pineapple fruit ripening. Among these 271 sequences, 184 share significant homology with genes encoding proteins of known function, 53 share homology with genes encoding proteins of unknown function and 34 share no significant homology with any database accession. Of the 237 pineapple sequences with homologs, 160 were up-regulated and 77 were down-regulated during pineapple fruit ripening. DAVID Functional Annotation Cluster (FAC) analysis of all 237 sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin C biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. Quantitative real-time PCR analysis validated the microarray expression results for nine out of ten genes tested.ConclusionsThis is the first report of a microarray based gene expression study undertaken in pineapple. Our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. This study extends our knowledge of the molecular basis of pineapple fruit ripening and non-climacteric fruit ripening in general.

The Accessory Sec Protein Asp2 Modulates GlcNAc Deposition onto the Serine-Rich Repeat Glycoprotein GspB

The accessory Sec system is a specialized transport system that exports serine-rich repeat (SRR) glycoproteins of Gram-positive bacteria. This system contains two homologues of the general secretory (Sec) pathway (SecA2 and SecY2) and several other essential proteins (Asp1 to Asp5) that share no homology to proteins of known function. In Streptococcus gordonii, Asp2 is required for the transport of the SRR adhesin GspB, but its role in export is unknown. Tertiary structure predictions suggest that the carboxyl terminus of Asp2 resembles the catalytic region of numerous enzymes that function through a Ser-Asp-His catalytic triad. Sequence alignment of all Asp2 homologues identified a highly conserved pentapeptide motif (Gly-X-Ser(362)-X-Gly) typical of most Ser-Asp-His catalytic triads, where Ser forms the reactive residue. Site-directed mutagenesis of residues comprising the predicted catalytic triad of Asp2 of S. gordonii had no effect upon GspB transport but did result in a marked change in the electrophoretic mobility of the protein. Lectin-binding studies and monosaccharide content analysis of this altered glycoform revealed an increase in glucosamine deposition. Random mutagenesis of the Asp2 region containing this catalytic domain also disrupted GspB transport. Collectively, our findings suggest that Asp2 is a bifunctional protein that is essential for both GspB transport and correct glycosylation. The catalytic domain may be responsible for controlling the glycosylation of GspB, while other surrounding regions are functionally required for glycoprotein transport.

Multifunctionality and diversity of GDSL esterase/lipase gene family in rice (Oryza sativa L. japonica) genome: new insights from bioinformatics analysis.

BackgroundGDSL esterases/lipases are a newly discovered subclass of lipolytic enzymes that are very important and attractive research subjects because of their multifunctional properties, such as broad substrate specificity and regiospecificity. Compared with the current knowledge regarding these enzymes in bacteria, our understanding of the plant GDSL enzymes is very limited, although the GDSL gene family in plant species include numerous members in many fully sequenced plant genomes. Only two genes from a large rice GDSL esterase/lipase gene family were previously characterised, and the majority of the members remain unknown. In the present study, we describe the rice OsGELP (Oryza sativa GDSL esterase/lipase protein) gene family at the genomic and proteomic levels, and use this knowledge to provide insights into the multifunctionality of the rice OsGELP enzymes.ResultsIn this study, an extensive bioinformatics analysis identified 114 genes in the rice OsGELP gene family. A complete overview of this family in rice is presented, including the chromosome locations, gene structures, phylogeny, and protein motifs. Among the OsGELPs and the plant GDSL esterase/lipase proteins of known functions, 41 motifs were found that represent the core secondary structure elements or appear specifically in different phylogenetic subclades. The specification and distribution of identified putative conserved clade-common and -specific peptide motifs, and their location on the predicted protein three dimensional structure may possibly signify their functional roles. Potentially important regions for substrate specificity are highlighted, in accordance with protein three-dimensional model and location of the phylogenetic specific conserved motifs. The differential expression of some representative genes were confirmed by quantitative real-time PCR. The phylogenetic analysis, together with protein motif architectures, and the expression profiling were analysed to predict the possible biological functions of the rice OsGELP genes.ConclusionsOur current genomic analysis, for the first time, presents fundamental information on the organization of the rice OsGELP gene family. With combination of the genomic, phylogenetic, microarray expression, protein motif distribution, and protein structure analyses, we were able to create supported basis for the functional prediction of many members in the rice GDSL esterase/lipase family. The present study provides a platform for the selection of candidate genes for further detailed functional study.

Affinity‐purification coupled to mass spectrometry: Basic principles and strategies

Identifying the interactions established by a protein of interest can be a critical step in understanding its function. This is especially true when an unknown protein of interest is demonstrated to physically interact with proteins of known function. While many techniques have been developed to characterize protein-protein interactions, one strategy that has gained considerable momentum over the past decade for identification and quantification of protein-protein interactions, is affinity-purification followed by mass spectrometry (AP-MS). Here, we briefly review the basic principles used in affinity-purification coupled to mass spectrometry, with an emphasis on tools (both biochemical and computational), which enable the discovery and reporting of high quality protein-protein interactions.

Ss-Sl2, a novel cell wall protein with PAN modules, is essential for sclerotial development and cellular integrity of Sclerotinia sclerotiorum.

The sclerotium is an important dormant body for many plant fungal pathogens. Here, we reported that a protein, named Ss-Sl2, is involved in sclerotial development of Sclerotinia sclerotiorum. Ss-Sl2 does not show significant homology with any protein of known function. Ss-Sl2 contains two putative PAN modules which were found in other proteins with diverse adhesion functions. Ss-Sl2 is a secreted protein, during the initial stage of sclerotial development, copious amounts of Ss-Sl2 are secreted and accumulated on the cell walls. The ability to maintain the cellular integrity of RNAi-mediated Ss-Sl2 silenced strains was reduced, but the hyphal growth and virulence of Ss-Sl2 silenced strains were not significantly different from the wild strain. Ss-Sl2 silenced strains could form interwoven hyphal masses at the initial stage of sclerotial development, but the interwoven hyphae could not consolidate and melanize. Hyphae in these interwoven bodies were thin-walled, and arranged loosely. Co-immunoprecipitation and yeast two-hybrid experiments showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Woronin body major protein (Hex1) and elongation factor 1-alpha interact with Ss-Sl2. GAPDH-knockdown strains showed a similar phenotype in sclerotial development as Ss-Sl2 silenced strains. Hex1-knockdown strains showed similar impairment in maintenance of hyphal integrity as Ss-Sl2 silenced strains. The results suggested that Ss-Sl2 functions in both sclerotial development and cellular integrity of S. sclerotiorum.

Novel Molluskan Biomineralization Proteins Retrieved from Proteomics: A Case Study with Upsalin

The formation of the molluskan shell is regulated by an array of extracellular proteins secreted by the calcifying epithelial cells of the mantle. These proteins remain occluded within the recently formed biominerals. To date, many shell proteins have been retrieved, but only a few of them, such as nacreins, have clearly identified functions. In this particular case, by combining molecular biology and biochemical approaches, we performed the molecular characterization of a novel protein that we named Upsalin, associated with the nacreous shell of the freshwater mussel Unio pictorum. The full sequence of the upsalin transcript was obtained by RT-PCR and 5'/3' RACE, and the expression pattern of the transcript was studied by PCR and qPCR. Upsalin is a 12 kDa protein with a basic theoretical pI. The presence of Upsalin in the shell was demonstrated by extraction of the acetic-acid-soluble nacre matrix, purification of a shell protein fraction by mono-dimensional preparative SDS-PAGE, and by submitting this fraction, after trypsic digestion, to nano-LC-MS/MS. In vitro experiments with the purified protein showed that it interferes poorly with the precipitation of calcium carbonate. Homology searches also could not affiliate Upsalin to any other protein of known function, leaving open the question of its exact role in shell formation. An antibody raised against an immunogenic peptide of Upsalin was found to be specific to this protein and was subsequently assayed for immunogold localization of the target protein in the shell, revealing the ubiquitous presence of Upsalin in the nacreous and prismatic layers. Recently, with the application of high-throughput proteomic studies to shells, the number of candidate proteins without clear functions has been increasing exponentially. The Upsalin example highlights the crucial need, for the scientific community dealing with biomineralization in general, to dedicate the coming years to designing experimental approaches, such as gene silencing, that focus on the functions of mineral-associated proteins.

Constructing patch-based ligand-binding pocket database for predicting function of proteins

BackgroundMany of solved tertiary structures of unknown functions do not have global sequence and structural similarities to proteins of known function. Often functional clues of unknown proteins can be obtained by predicting small ligand molecules that bind to the proteins.MethodsIn our previous work, we have developed an alignment free local surface-based pocket comparison method, named Patch-Surfer, which predicts ligand molecules that are likely to bind to a protein of interest. Given a query pocket in a protein, Patch-Surfer searches a database of known pockets and finds similar ones to the query. Here, we have extended the database of ligand binding pockets for Patch-Surfer to cover diverse types of binding ligands.Results and conclusionWe selected 9393 representative pockets with 2707 different ligand types from the Protein Data Bank. We tested Patch-Surfer on the extended pocket database to predict binding ligand of 75 non-homologous proteins that bind one of seven different ligands. Patch-Surfer achieved the average enrichment factor at 0.1 percent of over 20.0. The results did not depend on the sequence similarity of the query protein to proteins in the database, indicating that Patch-Surfer can identify correct pockets even in the absence of known homologous structures in the database.

The PduM Protein Is a Structural Component of the Microcompartments Involved in Coenzyme B 12 -Dependent 1,2-Propanediol Degradation by Salmonella enterica

Diverse bacteria use proteinaceous microcompartments (MCPs) to optimize metabolic pathways that have toxic or volatile intermediates. MCPs consist of metabolic enzymes encased within a protein shell that provides a defined environment. In Salmonella enterica, a MCP is involved in B(12)-dependent 1,2-propanediol utilization (Pdu MCP). In this report, we show that the protein PduM is required for the assembly and function of the Pdu MCP. The results of tandem mass spectrometry and Western blot analyses show that PduM is a component of the Pdu MCP. Electron microscopy shows that a pduM deletion mutant forms MCPs with abnormal morphology. Growth tests and metabolite measurements establish that a pduM deletion mutant is unable to form functional MCPs. PduM is unrelated in sequence to proteins of known function and hence may represent a new class of MCP structural proteins. We also report a modified protocol for the purification of Pdu MCP from Salmonella which allows isolation of milligram amounts of MCPs in about 4 h. We believe that this protocol can be extended or modified for the purification of MCPs from diverse bacteria.

Complete genome sequence of the podoviral bacteriophage ΦCP24R, which is virulent for Clostridium perfringens

Bacteriophage ΦCP24R was isolated from raw sewage from a waste treatment plant, and lytic activity was observed against a type A Clostridium perfringens isolate. Electron microscopy revealed a small virion (44-nm-diameter icosahedral capsid) with a short, non-contractile tail, indicative of a member of the family Podoviridae. The phage had a linear, double-stranded DNA genome of 18,919 base pairs (bp) with 41 bp inverted terminal repeats and a type B DNA polymerase, which are characteristics of members of the subfamily Picovirinae. Out of 22 predicted genes in the genome, ten had significant sequence similarity to proteins of known function. Three distinct genes with lytic domains were identified, including a zinc carboxypeptidase domain that has not been previously reported in viruses. The ΦCP24R genome described herein is only the second Clostridium perfringens podovirus genome reported to date.

Topology of 1-Acyl-sn-glycerol-3-phosphate Acyltransferases SLC1 and ALE1 and Related Membrane-bound O-Acyltransferases (MBOATs) of Saccharomyces cerevisiae

In yeast, phosphatidic acid, the biosynthetic precursor for all glycerophospholipids and triacylglycerols, is made de novo by the 1-acyl-sn-glycerol-3-phosphate acyltransferases Ale1p and Slc1p. Ale1p belongs to the membrane-bound O-acyltransferase (MBOAT) family, which contains many enzymes acylating lipids but also others that acylate secretory proteins residing in the lumen of the ER. A histidine present in a very short loop between two predicted transmembrane domains is the only residue that is conserved throughout the MBOAT gene family. The yeast MBOAT proteins of known function comprise Ale1p, the ergosterol acyltransferases Are1p and Are2p, and Gup1p, the last of which acylates lysophosphatidylinositol moieties of GPI anchors on ER lumenal GPI proteins. C-terminal topology reporters added to truncated versions of Gup1p yield a topology predicting a lumenal location of its uniquely conserved histidine 447 residue. The same approach shows that Ale1p and Are2p also have the uniquely conserved histidine residing in the ER lumen. Because these data raised the possibility that phosphatidic acid could be made in the lumen of the ER, we further investigated the topology of the second yeast 1-acyl-sn-glycerol-3-phosphate acyltransferase, Slc1p. The location of C-terminal topology reporters, microsomal assays probing the protease sensitivity of inserted tags, and the accessibility of natural or artificially inserted cysteines to membrane-impermeant alkylating agents all indicate that the most conserved motif containing the presumed active site histidine of Slc1p is oriented toward the ER lumen, whereas other conserved motifs are cytosolic. The implications of these findings are discussed.

A Novel, Noncatalytic Carbohydrate-binding Module Displays Specificity for Galactose-containing Polysaccharides through Calcium-mediated Oligomerization

The enzymic degradation of plant cell walls plays a central role in the carbon cycle and is of increasing environmental and industrial significance. The catalytic modules of enzymes that catalyze this process are generally appended to noncatalytic carbohydrate-binding modules (CBMs). CBMs potentiate the rate of catalysis by bringing their cognate enzymes into intimate contact with the target substrate. A powerful plant cell wall-degrading system is the Clostridium thermocellum multienzyme complex, termed the "cellulosome." Here, we identify a novel CBM (CtCBM62) within the large C. thermocellum cellulosomal protein Cthe_2193 (defined as CtXyl5A), which establishes a new CBM family. Phylogenetic analysis of CBM62 members indicates that a circular permutation occurred within the family. CtCBM62 binds to d-galactose and l-arabinopyranose in either anomeric configuration. The crystal structures of CtCBM62, in complex with oligosaccharides containing α- and β-galactose residues, show that the ligand-binding site in the β-sandwich protein is located in the loops that connect the two β-sheets. Specificity is conferred through numerous interactions with the axial O4 of the target sugars, a feature that distinguishes galactose and arabinose from the other major sugars located in plant cell walls. CtCBM62 displays tighter affinity for multivalent ligands compared with molecules containing single galactose residues, which is associated with precipitation of these complex carbohydrates. These avidity effects, which confer the targeting of polysaccharides, are mediated by calcium-dependent oligomerization of the CBM.

Genome-wide BAC-end sequencing of Musa acuminata DH Pahang reveals further insights into the genome organization of banana

Banana and plantain (Musa spp.) are grown in more than 120 countries in tropical and subtropical regions and constitute an important staple food for millions of people. A Musa acuminata ssp. malaccencis DH Pahang bacterial artificial chromosome (BAC) library (MAMB) was submitted for BAC-end sequencing. MAMB consists of 23,040 clones, with a 140-kbp average insert size, accounting for a five times coverage of the banana genome. A total of 46,080 reads were generated, and 42,750 (92.8%) high-quality sequences were obtained after trimming for vector and quality. Analysis of these data shows a GC content of 41.39%, whereas interspersed repeats comprise 32.3%. The most common repeated sequences found show homology to ribosomal RNA genes, particularly 18S rRNA, while the Ty3/gypsy type monkey retrotransposon is the most common retro element. The sequence data were used to generate a banana-specific repeat library containing 54 new repetitive elements which accounted for 11.86% of the total nucleotides. Simple sequence repeats represent 0.7% of the sequence data and allowed the identification of 2,455 potentially useful marker sites. Functional annotation identified 2,705 sequences that could code for proteins of known function. Microsynteny analysis shows a higher number of co-linear matches to Oryza sativa, in contrast to Arabidopsis thaliana. This database of BAC-end sequences is useful for the assembly of the complete banana genome sequence and is important for identification in functional genomics experiments.

Improvement of the Enediyne Antitumor Antibiotic C-1027 Production by Manipulating Its Biosynthetic Pathway Regulation in Streptomyces globisporus

The production of C-1027 in Streptomyces globisporus was previously increased 2- to 3-fold by manipulating three pathway-specific activators, SgcR1, SgcR2, and SgcR3. In this study, we have further characterized two putative C-1027 regulatory genes, sgcE1 and sgcR, by in vivo inactivation. The HxlR family DNA-binding protein SgcE1 was not essential for C-1027 biosynthesis, since inactivation of sgcE1 showed no effect on C-1027 production. In contrast, the proposed repressive role of the sgcR gene was confirmed by a 3-fold increase in C-1027 production in the ΔsgcR mutant S. globisporus SB1022 strain relative to the wild-type strain. Considering SgcR shows no significant similarity to any protein of known function, it may be representative of a new family of regulatory proteins. Finally, overexpression of the previously characterized activator sgcR1 in S. globisporus SB1022 increased the C-1027 yield to 37.5 ± 7.7 mg/L, which is about 7-fold higher than the wild-type strain.

Proteomic Analysis of Up-accumulated Proteins Associated with Fruit Quality during Autumn Olive (Elaeagnus umbellata) Fruit Ripening

Fruit ripening is a complex phenomenon that makes berries attractive and also determines their nutritional value. Autumn olive ( Elaeagnus umbellata Thunb.) fruit is a rich source of many human health-related nutrients. The changes in pericarp color are initiated at early developmental stages, coinciding with the fast increase in fruit size. Fruit quality traits with special emphasis on soluble sugars, organic acids, lycopene, and total protein contents were assayed during the fruit ripening. In the fully ripe fruit, glucose and fructose were the principal sugars, malic acid was the most abundant organic acid, and lycopene concentration was extremely high. A proteomic analysis was used to identify up-accumulated proteins induced by the ripening. Among 63 up-accumulated protein spots, 43 were successfully identified by MALDI-TOF/TOF-MS. All 43 proteins were novel for autumn olive, and 8 were first reported in the fruit. Twenty-one proteins of known function were involved in sugar metabolism, citric acid cycle, isoprenoid metabolism, fatty acid synthesis, and protein hydrolysis. The possible roles of these 21 accumulated proteins in autumn olive fruit quality are discussed.

Characterization of the Protocatechuate 4,5-Cleavage Pathway Operon in Comamonas sp. Strain E6 and Discovery of a Novel Pathway Gene

The protocatechuate (PCA) 4,5-cleavage (PCA45) pathway is the essential catabolic route for the degradation of various aromatic acids in the genus Comamonas. All of the PCA45 pathway genes, orf1-pmdKEFDABC, as well as another PCA 4,5-dioxygenase gene, pmdA(II)B(II), were isolated from a phthalate-degrading bacterium, Comamonas sp. strain E6. Disruption of pmdB and pmdD in E6, which code for the β subunit of PCA 4,5-dioxygenase and 2-pyrone-4,6-dicarboxylate (PDC) hydrolase, respectively, resulted in a growth defect on PCA, indicating that these genes are essential for the growth of E6 on PCA. On the other hand, inactivation of pmdB(II) did not affect the growth of E6 on PCA. Disruption of pmdK, which is related to a 4-hydroxybenzoate/PCA transporter of Pseudomonas putida, resulted in growth retardation on PCA. The insertional inactivation of orf1 in E6, whose deduced amino acid sequence has no similarity with proteins of known function, led to the complete loss of growth on PCA and the accumulation of PDC and 4-oxalomesaconate (OMA) from PCA. These results indicated the involvement of orf1 in the PCA45 pathway, and this gene, designated pmdU, was suggested to code for OMA tautomerase. Reverse transcription-PCR analysis suggested that the pmdUKEFDABC genes constitute an operon. The transcription start site of the pmd operon was mapped at 167 nucleotides upstream of the initiation codon of pmdU. The pmd promoter activity was enhanced 20-fold when the cells were grown in the presence of PCA. Inducers of the pmd operon were found to be PCA and PDC, but PDC was the more effective inducer.

Gene Expression in Wheat Induced by Inoculation with Puccinia striiformis West

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a destructive disease of wheat (Triticum aestivum L.) worldwide. A suppression subtractive hybridization (SSH) cDNA library was constructed from Pst-infected seedling leaves of an immune resistant germplasm Shaanmai 139. A total of 84 expressed sequence tags with high quality were obtained and alignment of the sequences identified in 56 Unigenes. Blast EST analysis of these Pst-induced genes showed that they were mostly homologous to genes that are induced by cold or heat stress. BlastX searches identified 45 of the Unigenes as similar to those encoding proteins of known function (e value < 1.00E-5), and eight of unknown function. The genes of known function include those with potential biological roles in signal transmission, energy and metabolism, transcription regulation, phenylpropanoid pathway, and defense response. Semi-quantitative reverse transcription-polymerase chain reaction studies confirmed that some of the cDNAs genes were differentially expressed in Pst-infected plants, with 24 and 72 hpi as the two critical time points for the induction of these genes by inoculation with P. striiformis West race CY32 in wheat. Bimodal patterns were presented in Pst-induced genes, such as senescence-associated genes, omega-6 fatty acid desaturase, and acyl-CoA synthetase. This suggests complex patterns of defense-related gene expression in multi-resistance gene cultivars in response to stripe rust infection.

ChemInform Abstract: The Human Genome Project - An Overview

The human genome sequence will underpin human biology and medicine in the next century, providing a single, essential reference to all genetic information. The international program to determine the complete DNA sequence (3,000 million bases) is well underway. As of January 2000, 50% of the sequence is available in the public domain. A comprehensive working draft is expected this year, and the entire sequence is projected to be finished in 2003. DNA sequencing is carried out on mapped, overlapping bacterial clones of 150-200 kb. The working draft comprises assembled unfinished sequence and is released immediately in the public domain. The draft sequence of each clone is then completed, by closing any remaining gaps and resolving any ambiguities, before the entire sequence is checked, annotated, and submitted to the public databases. The sequence of each clone is finished to an accuracy of >99.99%. The availability of a reference sequence of the genome provides the basis for studying the nature of sequence variation, particularly single nucleotide polymorphisms (SNPs), in human populations. SNP typing is a powerful tool for genetic analysis, and will enable us to uncover the association of loci at specific sites in the genome with many disease traits. SNPs occur at a frequency of approximately 1 SNP/kb throughout the genome when the sequence of any two individuals is compared. Programs to detect and map SNPs in the human genome are underway with the aim of establishing a SNP map of the genome during the next two years. The human genome sequence will provide a complete description of all the genes. Annotation of the sequence with the gene structures is achieved by a combination of computational analysis (predictive and homology-based) and experimental confirmation by cDNA sequencing. Detecting homologies between newly defined gene products and proteins of known function helps to postulate biochemical functions for them, which can then be tested. Establishing the association of specific genes with disease phenotypes by mutation screening, particularly for monogenic disorders, provides further assistance in defining the functions of some gene products, as well as helping to establish the cause of the disease. As our knowledge of gene sequences and sequence variation in populations increases, we will pinpoint more and more of the genes and proteins that are important in common, complex diseases. A more detailed understanding of the function of the human genome will be achieved as we identify sequences that control gene expression. Given the availability of gene sequences, the expression status of genes in particular tissues can be monitored in parallel. By comparing corresponding genomic sequences in different species (for example: man, mouse, chicken, and zebrafish), regions that have been highly conserved during evolution can be identified, many of which reflect conserved functions such as gene regulation. These approaches promise to greatly accelerate our interpretation of the human genome sequence.

Rapid screening of membrane topology of secondary transport proteins

Limited experimental data may be very useful to discriminate between membrane topology models of membrane proteins derived from different methods. A membrane topology screening method is proposed by which the cellular disposition of three positions in a membrane protein are determined, the N- and the C-termini and a position in the middle of the protein. The method involves amplification of the encoding genes or gene fragments by PCR, rapid cloning in dedicated vectors by ligation independent cloning, and determination of the cellular disposition of the three sites using conventional techniques. The N-terminus was determined by labeling with a fluorescent probe, the central position and the C-terminus by the reporter fusion technique using alkaline phosphatase (PhoA) and green fluorescence protein (GFP) as reporters. The method was evaluated using 16 transporter proteins of known function from four different structural classes. For 13 proteins a complete set of three localizations was obtained. The experimental data was used to discriminate between membrane topology models predicted by TMHMM, a widely used predictor using the amino acid sequence as input and by MemGen that uses hydropathy profile alignment and known 3D structures or existing models. It follows that in those cases where the models from the two methods were similar, the models were consistent with the experimental data. In those cases where the models differed, the MemGen model agreed with the experimental data. Three more recent predictors, MEMSAT3, OCTOPUS and TOPCONS showed a significantly higher consistency with the experimental data than observed with TMHMM.

From small hosts come big viruses: the complete genome of a second Ostreococcus tauri virus, OtV‐1

Ostreococcus tauri virus (OtV-1) is a large double-stranded DNA virus and a prospective member of the family Phycodnaviridae, genus Prasinovirus. OtV-1 infects the unicellular marine green alga O. tauri, the smallest known free-living eukaryote. Here we present the 191 761 base pair genome sequence of OtV-1, which has 232 putative protein-encoding and 4 tRNA-encoding genes. Approximately 31% of the viral gene products exhibit a similarity to proteins of known functions in public databases. These include a variety of unexpected genes, for example, a PhoH-like protein, a N-myristoyltransferase, a 3-dehydroquinate synthase, a number of glycosyltransferases and methyltransferases, a prolyl 4-hydroxylase, 6-phosphofructokinase and a total of 8 capsid proteins. A total of 11 predicted genes share homology with genes found in the Ostreococcus host genome. In addition, an intein was identified in the DNA polymerase gene of OtV-1. This is the first report of an intein in the genome of a virus that infects O. tauri. Fifteen core genes common to nuclear-cytoplasmic large dsDNA virus (NCLDV) genomes were identified in the OtV-1 genome. This new sequence data may help to redefine the classification of the core genes of these viruses and shed new light on their evolutionary history.

Initial Events Associated with Virus PBCV-1 Infection of Chlorella NC64A.

Chlorella viruses (or chloroviruses) are very large, plaque-forming viruses. The viruses are multilayered structures containing a large double-stranded DNA genome, a lipid bilayered membrane, and an outer icosahedral capsid shell. The viruses replicate in certain isolates of the coccal green alga, Chlorella. Sequence analysis of the 330-kbp genome of Paramecium bursaria Chlorella virus 1 (PBCV-1), the prototype of the virus family Phycodnaviridae, reveals <365 protein-encoding genes and 11 tRNA genes. Products of about 40% of these genes resemble proteins of known function, including many that are unexpected for a virus. Among these is a virus-encoded protein, called Kcv, which forms a functional K(+) channel. This chapter focuses on the initial steps in virus infection and provides a plausible role for the function of the viral K(+) channel in lowering the turgor pressure of the host. This step appears to be a prerequisite for delivery of the viral genome into the host.