Uncharacterized Open Reading Frames Research Articles

Mobile elements in a single-filament orange Guaymas Basin Beggiatoa ("Candidatus Maribeggiatoa") sp. draft genome: evidence for genetic exchange with cyanobacteria.

The draft genome sequence of a single orange Beggiatoa ("Candidatus Maribeggiatoa") filament collected from a microbial mat at a hydrothermal site in Guaymas Basin (Gulf of California, Mexico) shows evidence of extensive genetic exchange with cyanobacteria, in particular for sensory and signal transduction genes. A putative homing endonuclease gene and group I intron within the 23S rRNA gene; several group II catalytic introns; GyrB and DnaE inteins, also encoding homing endonucleases; multiple copies of sequences similar to the fdxN excision elements XisH and XisI (required for heterocyst differentiation in some cyanobacteria); and multiple sequences related to an open reading frame (ORF) (00024_0693) of unknown function all have close non-Beggiatoaceae matches with cyanobacterial sequences. Sequences similar to the uncharacterized ORF and Xis elements are found in other Beggiatoaceae genomes, a variety of cyanobacteria, and a few phylogenetically dispersed pleiomorphic or filamentous bacteria. We speculate that elements shared among filamentous bacterial species may have been exchanged in microbial mats and that some of them may be involved in cell differentiation.

Conserved loci of leaf and stem rust fungi of wheat share synteny interrupted by lineage-specific influx of repeat elements

BackgroundWheat leaf rust (Puccinia triticina Eriks; Pt) and stem rust fungi (P. graminis f.sp. tritici; Pgt) are significant economic pathogens having similar host ranges and life cycles, but different alternate hosts. The Pt genome, currently estimated at 135 Mb, is significantly larger than Pgt, at 88 Mb, but the reason for the expansion is unknown. Three genomic loci of Pt conserved proteins were characterized to gain insight into gene content, genome complexity and expansion.ResultsA bacterial artificial chromosome (BAC) library was made from P. triticina race 1, BBBD and probed with Pt homologs of genes encoding two predicted Pgt secreted effectors and a DNA marker mapping to a region of avirulence. Three BACs, 103 Kb, 112 Kb, and 166 Kb, were sequenced, assembled, and open reading frames were identified. Orthologous genes were identified in Pgt and local conservation of gene order (microsynteny) was observed. Pairwise protein identities ranged from 26 to 99%. One Pt BAC, containing a RAD18 ortholog, shares syntenic regions with two Pgt scaffolds, which could represent both haplotypes of Pgt. Gene sequence is diverged between the species as well as within the two haplotypes. In all three BAC clones, gene order is locally conserved, however, gene shuffling has occurred relative to Pgt. These regions are further diverged by differing insertion loci of LTR-retrotransposon, Gypsy, Copia, Mutator, and Harbinger mobile elements. Uncharacterized Pt open reading frames were also found; these proteins are high in lysine and similar to multiple proteins in Pgt.ConclusionsThe three Pt loci are conserved in gene order, with a range of gene sequence divergence. Conservation of predicted haustoria expressed secreted protein genes between Pt and Pgt is extended to the more distant poplar rust, Melampsora larici-populina. The loci also reveal that genome expansion in Pt is in part due to higher occurrence of repeat-elements in this species.

Characterization of the AggR Regulon in Enteroaggregative Escherichia coli

AggR is a transcriptional regulator of enteroaggregative Escherichia coli (EAEC) and has been proposed as the defining factor for typical EAEC strains. Expression of multiple putative virulence factors, including the aggregative adherence fimbriae (AAF), dispersin, the dispersin translocator Aat, and the Aai type VI secretion system, have been found to be regulated by AggR. Here, we confirm the existence of at least 44 AggR-regulated genes using DNA microarray and real-time quantitative reverse transcription-PCR (qRT-PCR); these genes include chromosomal and plasmid-borne loci and 19 previously unsuspected genes. Two previously uncharacterized virulence plasmid-encoded open reading frames (ORFs) (designated ORF3 and ORF4) exhibit significant identity with isoprenoid biosynthesis genes of Bacteria and Archaea. The predicted ORF4 product is closely related to isopentenyl isomerase (IDI) enzymes, whereas the predicted product of the adjacent ORF3 exhibits an aspartate-rich region that is common among trans-isoprenyl phosphate synthases. We show that mutations in these ORFs confer changes in bacterial surface properties. AggR coordinately controls expression of a large number of EAEC genes.

Characterization and crystal structure of the thermophilic ROK hexokinase from Thermus thermophilus

We characterized and determined the crystal structure of a putative glucokinase/hexokinase from Thermus thermophilus that belongs to the ROK (bacterial repressors, uncharacterized open reading frames, and sugar kinases) family. The protein possessed significant enzymatic activity against glucose and mannose, with V(max) values of 260 and 68 μmol·min(-1)·mg(-1) protein, respectively. Therefore, we concluded that the enzyme is a hexokinase. However, the hexokinase showed little catalytic capacity for galactose and fructose. Circular dichroism measurements indicated that the enzyme was structurally stable at 90°C. The crystal structure of the enzyme was determined at a resolution of 2.02 Å, with R(cryst) and R(free) values of 18.1% and 22.6%, respectively. The polypeptide structure was divided into large and small domains. The ROK consensus sequences 1 and 2 were included in the large domain. The cysteine-rich consensus sequence 2 folded into a zinc finger, and the bound zinc was confirmed by both electron density and X-ray absorption fine structure (XAFS) spectrum. The overall structure was a homotetramer that consisted of a dimer of dimers. The accessible surface area buried by the association of the dimers into the tetrameric structures was significantly higher in the T. thermophilus enzyme than in a homologous tetrameric ROK sugar kinase.

Novel E3 Ubiquitin Ligases That Regulate Histone Protein Levels in the Budding Yeast Saccharomyces cerevisiae

Core histone proteins are essential for packaging the genomic DNA into chromatin in all eukaryotes. Since multiple genes encode these histone proteins, there is potential for generating more histones than what is required for chromatin assembly. The positively charged histones have a very high affinity for negatively charged molecules such as DNA, and any excess of histone proteins results in deleterious effects on genomic stability and cell viability. Hence, histone levels are known to be tightly regulated via transcriptional, posttranscriptional and posttranslational mechanisms. We have previously elucidated the posttranslational regulation of histone protein levels by the ubiquitin-proteasome pathway involving the E2 ubiquitin conjugating enzymes Ubc4/5 and the HECT (Homologous to E6-AP C-Terminus) domain containing E3 ligase Tom1 in the budding yeast. Here we report the identification of four additional E3 ligases containing the RING (Really Interesting New Gene) finger domains that are involved in the ubiquitylation and subsequent degradation of excess histones in yeast. These E3 ligases are Pep5, Snt2 as well as two previously uncharacterized Open Reading Frames (ORFs) YKR017C and YDR266C that we have named Hel1 and Hel2 (for Histone E3 Ligases) respectively. Mutants lacking these E3 ligases are sensitive to histone overexpression as they fail to degrade excess histones and accumulate high levels of endogenous histones on histone chaperones. Co-immunoprecipitation assays showed that these E3 ligases interact with the major E2 enzyme Ubc4 that is involved in the degradation related ubiquitylation of histones. Using mutagenesis we further demonstrate that the RING domains of Hel1, Hel2 and Snt2 are required for histone regulation. Lastly, mutants corresponding to Hel1, Hel2 and Pep5 are sensitive to replication inhibitors. Overall, our results highlight the importance of posttranslational histone regulatory mechanisms that employ multiple E3 ubiquitin ligases to ensure excess histone degradation and thus contribute to the maintenance of genomic stability.

“RHEX-26”, a Novel EPO/EPOR-Regulated Mediator of Human Erythroid Progenitor Cell Expansion

Abstract 682Erythropoietin (EPO) and its receptor (EPOR) serve well as an informative paradigm for understanding hematopoietic growth factor effects and receptor action mechanisms. Recently, interest in EPO/EPOR investigations has been heightened based on the clinical emergence of novel EPO mimetics, and by reported EPO/EPOR activities in modulating cellular immunity, diabetic susceptibility, retinopathy and the cytoprotection of select non-hematopoietic tissues. To better understand EPOR action modes, we presently have applied a global phosphoproteomics approach to specifically discover new targets of EPO/EPOR-regulated tyrosine phosphorylation. Using an EPO-dependent erythroid progenitor cell model, our LC-MS/MS approach was first validated for known EPO/EPOR-targets (e.g., JAK2, STATs, SHIPs, PLCgamma, GABs). Second, among certain known targets, new phosphorylation sites were identified. Third, several novel major downstream targets of EPO/EPOR were discovered. One (a previously uncharacterized open reading frame here designated as “RHEX26”) was induced ∼20-fold in tyrosine-phosphorylation by EPO treatment. Quantitative RT-PCR analyses of tissues and primary hematopoietic cells indicate RHEX26 expression to be erythroid- restricted. Antibodies prepared to RHEX26 and tyrosine phosphorylated-RHEX26 confirmed strong EPO-induced phosphorylation; cell fractionation and Western blot studies demonstrated membrane residence of this 26 kDa phosphoprotein; and immunofluorescence microscopy showed sharp RHEX26 sub-localization as an integral plasma membrane protein. In functional analyses, lentiviral shRNA knockdown of RHEX26 proved to markedly inhibit the EPO-dependent expansion of erythroid progenitor cells (clonal colony-forming analyses demonstrated >80% specific inhibition), while effects on cell viability were limited. Mechanistically, RHEX26 is coupled (in part) to GRB2, with co-immunoprecipitation observed specifically upon EPO exposure, but without dependency on RHEX26 tyrosine phosphorylation. Finally, genealogy analyses intriguingly indicate RHEX26 to be well-conserved only in H. sapiens and primates, while no ortholog is present in the rat, mouse, or zebrafish. RHEX26, as a novel EPO/EPOR 26 kDa target, therefore is proposed to comprise an important new Regulator of Human Erythroid eXpansion. Disclosures:Leu:Affymax, Inc.: Employment, own stock in Affymax, Inc. Young:Affymax, Inc.: Employment, own stock in Affymax, Inc. Schatz:Affymax, Inc.: Employment, own stock in Affymax, Inc. Green:Affymax, Inc.: Employment, own stock in Affymax, Inc.

A Novel N,N-8-Amino-8-demethyl-d-riboflavin Dimethyltransferase (RosA) Catalyzing the Two Terminal Steps of Roseoflavin Biosynthesis in Streptomyces davawensis

Streptomyces davawensis synthesizes the antibiotic roseoflavin (RoF) (8-dimethylamino-8-demethyl-D-riboflavin). It was postulated that RoF is synthesized from riboflavin via 8-amino- (AF) and 8-methylamino-8-demethyl-D-riboflavin (MAF). In a cell-free extract of S. davawensis, an S-adenosyl methionine-dependent conversion of AF into MAF and RoF was observed. The corresponding N,N-8-amino-8-demethyl-d-riboflavin dimethyltransferase activity was enriched by column chromatography. The final most active fraction still contained at least five different proteins that were analyzed by enzymatic digestion and concomitant de novo sequencing by MS/MS. One of the sequences matched a hypothetical peptide fragment derived from an as yet uncharacterized open reading frame (sda77220) located in the middle of a (putative) gene cluster within the S. davawensis genome. Expression of ORF sda77220 in Escherichia coli revealed that the corresponding gene product had N,N-8-amino-8-demethyl-d-riboflavin dimethyltransferase activity. Inactivation of ORF sda77220 led to a S. davawensis strain that synthesized AF but not MAF or RoF. Accordingly, as the first identified gene of RoF biosynthesis, ORF sda77220 was named rosA. RosA (347 amino acids; 38 kDa) was purified from a recombinant E. coli strain (as a His(6)-tagged protein) and was biochemically characterized (apparent K(m) for AF = 57.7 ± 9.2 μm; apparent K(D) for AF = 10.0 μm; k(cat) = 0.37 ± 0.02 s(-1)). RosA is a unique enzyme and may be useful for a variety of applications.

ESR1 is co-expressed with closely adjacent uncharacterised genes spanning a breast cancer susceptibility locus at 6q25.1.

Approximately 80% of human breast carcinomas present as oestrogen receptor α-positive (ER+ve) disease, and ER status is a critical factor in treatment decision-making. Recently, single nucleotide polymorphisms (SNPs) in the region immediately upstream of the ER gene (ESR1) on 6q25.1 have been associated with breast cancer risk. Our investigation of factors associated with the level of expression of ESR1 in ER+ve tumours has revealed unexpected associations between genes in this region and ESR1 expression that are important to consider in studies of the genetic causes of breast cancer risk. RNA from tumour biopsies taken from 104 postmenopausal women before and after 2 weeks treatment with an aromatase (oestrogen synthase) inhibitor was analyzed on Illumina 48K microarrays. Multiple-testing corrected Spearman correlation revealed that three previously uncharacterized open reading frames (ORFs) located immediately upstream of ESR1, C6ORF96, C6ORF97, and C6ORF211 were highly correlated with ESR1 (Rs = 0.67, 0.64, and 0.55 respectively, FDR<1×10−7). Publicly available datasets confirmed this relationship in other groups of ER+ve tumours. DNA copy number changes did not account for the correlations. The correlations were maintained in cultured cells. An ERα antagonist did not affect the ORFs' expression or their correlation with ESR1, suggesting their transcriptional co-activation is not directly mediated by ERα. siRNA inhibition of C6ORF211 suppressed proliferation in MCF7 cells, and C6ORF211 positively correlated with a proliferation metagene in tumours. In contrast, C6ORF97 expression correlated negatively with the metagene and predicted for improved disease-free survival in a tamoxifen-treated published dataset, independently of ESR1. Our observations suggest that some of the biological effects previously attributed to ER could be mediated and/or modified by these co-expressed genes. The co-expression and function of these genes may be important influences on the recently identified relationship between SNPs in this region and breast cancer risk.

Evolution and comparative genomics of subcellular specializations: EST sequencing of Torpedo electric organ

Uncharacterized open reading frames (ORFs) in human genomic sequence often show a high degree of evolutionary conservation, yet have little or no tissue EST or protein data suggestive of protein product function. The encoded proteins may have highly restricted expression in specialized cells, subcellular specializations, and/or narrow windows during development. One such highly specialized and minute subcellular compartment is the neuromuscular junction (NMJ), where motorneurons contact muscle fibers. The electric Torpedo ray has evolved to expand the NMJ structure to the size of a large organ (electroplax organ), and we hypothesized that Torpedo electroplax proteins would be candidates for human ESTs expressed at the human NMJ. A total of 9719 primary electroplax cDNA clones were sequenced. We identified 44 human ORFs showing high (> 63%) amino acid identity to Torpedo electroplax transcripts with enrichment for mRNA splicing motifs (SH2 and pre-mRNA splicing domains), an observation potentially important for the strict nuclear domains maintained by myonuclei underlying the NMJ. We generated antibodies against two uncharacterized human genes ( C19orf29 [ Drosophila cactin] and C15orf24) and showed that these were indeed expressed at the murine NMJ. Cactin, a member of the Rel transcription factor family in Drosophila, localized to the postsynaptic cytosol of the NMJ and nuclear membrane. C15orf24 protein localized to the murine postsynaptic sarcolemma. We show a novel approach towards identifying proteins expressed at a subcellular specialization using evolutionary diversity of organ function and cross-species mapping.

Structural Studies of ROK Fructokinase YdhR from Bacillus subtilis: Insights into Substrate Binding and Fructose Specificity

The main pathway of bacterial sugar phosphorylation utilizes specific phosphoenolpyruvate phosphotransferase system (PTS) enzymes. In addition to the classic PTS system, a PTS-independent secondary system has been described in which nucleotide-dependent sugar kinases are used for monosaccharide phosphorylation. Fructokinase (FK), which phosphorylates d-fructose with ATP as a cofactor, has been shown to be a member of this secondary system. Bioinformatic analysis has shown that FK is a member of the “ROK” (bacterial Repressors, uncharacterized Open reading frames, and sugar Kinases) sequence family. In this study, we report the crystal structures of ROK FK from Bacillus subtilis (YdhR) (a) apo and in the presence of (b) ADP and (c) ADP/ d-fructose. All structures show that YdhR is a homodimer with a monomer composed of two similar α/β domains forming a large cleft between domains that bind ADP and d-fructose. Enzymatic activity assays support YdhR function as an ATP-dependent fructose kinase.

Abstract 2916: Novel genes closely adjacent to and coexpressed with ESR1 affect proliferation in ER+ve breast tumors

Abstract Background: Approximately 80% of human breast carcinomas present as estrogen receptor α-positive (ER+ve) disease and ER status is a critical factor in treatment decision-making. However, ER+ve tumors are heterogeneous in both their molecular profiles and response to therapy. Up to 50% of patients derive little or no clinical benefit from treatment and many exhibit de novo or intrinsic resistance to hormonal therapy. Aim: To investigate factors associated with the level of expression of the ER gene that may be potential determinants of the biology of ER+ve tumors. Methods: Pre- and 2wk post-anastrozole tumor biopsies were obtained from 104 postmenopausal women with stage I to IIIB ER+ early breast cancer. RNA extracted from biopsies was analyzed on Illumina 48K microarrays and quantitative trait analysis by Spearman correlation was used to identify genes whose expression in tumors correlated significantly with ESR1. Results: Expression of three previously uncharacterized open reading frames (ORFs) located immediately upstream of ESR1 on chromosome 6q21, RMND1, C6ORF97 and C6ORF211, were highly correlated with ESR1 (Rs=0.672, 0.637 and 0.546 respectively, p&amp;lt;0.0001). Exploration of other publicly available datasets revealed that this relationship was present in other groups of ER+ve tumors. In our tumor set, change in expression of these genes upon estrogen deprivation remained tightly correlated with change in ESR1 expression. Array CGH of microdissected tumor samples on tiling path 32K BAC arrays revealed that DNA copy number changes did not account for the correlation. Analysis of expression of all four genes in cultured MCF7 and BT474 cells by quantitative RT-PCR over a timecourse showed that the correlation was maintained in vitro. Treatment with the ERα antagonist ICI 182,780 did not affect ORF expression or the correlation of the genes with ESR1, suggesting that their transcriptional co-activation is not directly mediated by ERα. siRNA inhibition of C6ORF211 suppressed proliferation in MCF7 cells (1.6-fold, p&amp;lt;0.0001). In pre-treatment tumor biopsies, C6ORF211 correlated with a published proliferation metagene (p=0.04), mirroring the effect of C6ORF211 knockdown on proliferation seen in vitro. C6ORF211 was also found to be expressed more highly in Luminal B versus Luminal A tumors (p=0.0005), despite ESR1 levels not being significantly different between these two molecular subgroups. Summary and conclusions: There is a previously unpublished association between ESR1 and three ORFs immediately adjacent to ESR1 on chromosome 6. Their co-expression is not a result of co-amplification or directly mediated by ERα transcriptional activity. Our data indicate that C6ORF211 influences proliferation in vitro and in tumors hence it remains possible that some of the biological effects previously attributed to ER could be mediated by these co-expressed genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2916.

Distinct Variants at LIN28B Influence Growth in Height from Birth to Adulthood

We have studied the largely unknown genetic underpinnings of height growth by using a unique resource of longitudinal childhood height data available in Finnish population cohorts. After applying GWAS mapping of potential genes influencing pubertal height growth followed by further characterization of the genetic effects on complete postnatal growth trajectories, we have identified strong association between variants near LIN28B and pubertal growth (rs7759938; female p = 4.0 x 10(-9), male p = 1.5 x 10(-4), combined p = 5.0 x 10(-11), n = 5038). Analysis of growth during early puberty confirmed an effect on the timing of the growth spurt. Correlated SNPs have previously been implicated as influencing both adult stature and age at menarche, the same alleles associating with taller height and later age of menarche in other studies as with later pubertal growth here. Additionally, a partially correlated LIN28B SNP, rs314277, has been associated previously with final height. Testing both rs7759938 and rs314277 (pairwise r(2) = 0.29) for independent effects on postnatal growth in 8903 subjects indicated that the pubertal timing-associated marker rs7759938 affects prepubertal growth in females (p = 7 x 10(-5)) and final height in males (p = 5 x 10(-4)), whereas rs314277 has sex-specific effects on growth (p for interaction = 0.005) that were distinct from those observed at rs7759938. In conclusion, partially correlated variants at LIN28B tag distinctive, complex, and sex-specific height-growth-regulating effects, influencing the entire period of postnatal growth. These findings imply a critical role for LIN28B in the regulation of human growth.

Resistance of Saccharomyces cerevisiae to High Concentrations of Furfural Is Based on NADPH-Dependent Reduction by at Least Two Oxireductases

Biofuels derived from lignocellulosic biomass hold promises for a sustainable fuel economy, but several problems hamper their economical feasibility. One important problem is the presence of toxic compounds in processed lignocellulosic hydrolysates, with furfural as a key toxin. While Saccharomyces cerevisiae has some intrinsic ability to reduce furfural to the less-toxic furfuryl alcohol, higher resistance is necessary for process conditions. By comparing an evolved, furfural-resistant strain and its parent in microaerobic, glucose-limited chemostats at increasing furfural challenge, we elucidate key mechanism and the molecular basis of both natural and high-level furfural resistance. At lower concentrations of furfural, NADH-dependent oxireductases are the main defense mechanism. At furfural concentrations above 15 mM, however, (13)C-flux and global array-based transcript analysis demonstrated that the NADPH-generating flux through the pentose phosphate pathway increases and that NADPH-dependent oxireductases become the major resistance mechanism. The transcript analysis further revealed that iron transmembrane transport is upregulated in response to furfural. While these responses occur in both strains, high-level resistance in the evolved strain was based on strong induction of ADH7, the uncharacterized open reading frame (ORF) YKL071W, and four further, likely NADPH-dependent, oxireductases. By overexpressing the ADH7 gene and the ORF YKL071W, we inversely engineered significantly increased furfural resistance in the parent strain, thereby demonstrating that these two enzymes are key elements of the resistance phenotype.

Identification of a Terminal Rhamnopyranosyltransferase (RptA) Involved inCorynebacterium glutamicumCell Wall Biosynthesis

A bioinformatics approach identified a putative integral membrane protein, NCgl0543, in Corynebacterium glutamicum, with 13 predicted transmembrane domains and a glycosyltransferase motif (RXXDE), features that are common to the glycosyltransferase C superfamily of glycosyltransferases. The deletion of C. glutamicum NCgl0543 resulted in a viable mutant. Further glycosyl linkage analyses of the mycolyl-arabinogalactan-peptidoglycan complex revealed a reduction of terminal rhamnopyranosyl-linked residues and, as a result, a corresponding loss of branched 2,5-linked arabinofuranosyl residues, which was fully restored upon the complementation of the deletion mutant by NCgl0543. As a result, we have now termed this previously uncharacterized open reading frame, rhamnopyranosyltransferase A (rptA). Furthermore, an analysis of base-stable extractable lipids from C. glutamicum revealed the presence of decaprenyl-monophosphorylrhamnose, a putative substrate for the cognate cell wall transferase.

TRAPPC2L is a Novel, Highly Conserved TRAPP‐Interacting Protein

Mutations in the trafficking protein particle complex C2 protein (TRAPPC2), a mammalian ortholog of yeast Trs20p and a component of the trafficking protein particle (TRAPP) vesicle tethering complex, have been linked to the skeletal disorder spondyloepiphyseal dysplasia tarda (SEDT). Intriguingly, the X-linked TRAPPC2 is just one of a complement of Trs20-related genes in humans. Here we characterize TRAPPC2L, a novel, highly conserved TRAPP-interacting protein related to TRAPPC2 and the uncharacterized yeast open reading frame YEL048c. TRAPPC2L and TRAPPC2 genes are found in pairs across species and show broad and overlapping expression, suggesting they are functionally distinct, a notion supported by yeast complementation studies and biochemical characterization. RNA interference-mediated knockdown of either TRAPPC2L or TRAPPC2 in HeLa cells leads to fragmentation of the Golgi, implicating both proteins in Golgi dynamics. Gradient fractionation of cellular membranes indicates that TRAPPC2L is found with a portion of cellular TRAPP on very low-density membranes whereas the remainder of TRAPP, but not TRAPPC2L, is found associated with Golgi markers. YEL048c displays genetic interactions with TRAPP II-encoding genes and the gene product co-fractionates with and interacts with yeast TRAPP II. Taken together these results indicate that TRAPPC2L and its yeast ortholog YEL048c are novel TRAPP-interacting proteins that may modulate the function of the TRAPP II complex.

Combination of Several Bioinformatics Approaches for the Identification of New Putative Glycosyltransferases inArabidopsis

Approximately 450 glycosyltransferase (GT) sequences have been already identified in the Arabidopsis genome that organize into 40 sequence-based families, but a vast majority of these gene products remain biochemically uncharacterized open reading frames. Given the complexity of the cell wall carbohydrate network, it can be inferred that some of the biosynthetic genes have not yet been identified by classical bioinformatics approaches. With the objective to identify new plant GT genes, we designed a bioinformatic strategy that is based on the use of several remote homology detection methods that act at the 1D, 2D, and 3D level. Together, these methods led to the identification of more than 150 candidate protein sequences. Among them, 20 are considered as putative glycosyltransferases that should further be investigated since known GT signatures were clearly identified.

Absence of See1p, a widely conserved Saccharomyces cerevisiae protein, confers both deficient heterologous protein production and endocytosis

The uncharacterized Saccharomyces cerevisiae open reading frame (ORF) YIL064w is predicted to encode a cytoplasmic 28 kDa protein, recognized by sequence similarity as a putative S-adenosyl-L-methionine methyltransferase. A micro-scale screening performed in our laboratory with the EUROSCARF S. cerevisiae BY4741 deletion mutant collection identified YIL064w deletion as negatively affecting secretory production of reporter alpha-amylase. The work presented here corroborates the later observations of the yil064w mutant in a larger-scale assay and shows that Yil064p is necessary for the efficient secretory production of two reporter proteins, murine alpha-amylase and fungal polygalacturonase. Further, we analysed endocytosis in the yil064w mutant strain and observed defects at both very early and later stages of endocytic transport in cells in the late logarithmic phase. The defects at very early stages may decisively account for the low transfection (DNA uptake by endocytosis) efficiency that we also observed in the yil064w mutant. These are the first in vivo data reporting a functional role for the protein encoded by ORF YIL064w and identify Yil064p, named here secretion and early endocytosis 1 protein (See1p), as a novel component of intracellular transport.

Chemical-genetic profile analysis in yeast suggests that a previously uncharacterized open reading frame, YBR261C, affects protein synthesis

BackgroundFunctional genomics has received considerable attention in the post-genomic era, as it aims to identify function(s) for different genes. One way to study gene function is to investigate the alterations in the responses of deletion mutants to different stimuli. Here we investigate the genetic profile of yeast non-essential gene deletion array (yGDA, ~4700 strains) for increased sensitivity to paromomycin, which targets the process of protein synthesis.ResultsAs expected, our analysis indicated that the majority of deletion strains (134) with increased sensitivity to paromomycin, are involved in protein biosynthesis. The remaining strains can be divided into smaller functional categories: metabolism (45), cellular component biogenesis and organization (28), DNA maintenance (21), transport (20), others (38) and unknown (39). These may represent minor cellular target sites (side-effects) for paromomycin. They may also represent novel links to protein synthesis. One of these strains carries a deletion for a previously uncharacterized ORF, YBR261C, that we term TAE1 for Translation Associated Element 1. Our focused follow-up experiments indicated that deletion of TAE1 alters the ribosomal profile of the mutant cells. Also, gene deletion strain for TAE1 has defects in both translation efficiency and fidelity. Miniaturized synthetic genetic array analysis further indicates that TAE1 genetically interacts with 16 ribosomal protein genes. Phenotypic suppression analysis using TAE1 overexpression also links TAE1 to protein synthesis.ConclusionWe show that a previously uncharacterized ORF, YBR261C, affects the process of protein synthesis and reaffirm that large-scale genetic profile analysis can be a useful tool to study novel gene function(s).

Molecular isolation and characterization of a novel occlusion body protein gene from Penaeus monodon nucleopolyhedrovirus

The full-length of the occlusion body (OB) protein gene of Penaeus monodon nucleopolyhedrovirus ( PemoNPV) was successfully isolated. The OB gene sequence contained an open reading frame (ORF) of 1359 nucleotides encoding a protein of 452 amino acid residues with a predicted molecular mass of 50.6 kDa. A putative late promoter element, TAAG, was identified 72 nt upstream of the translation start site. The amino acid sequences of tryptic digested peptides of PemoNPV OB protein obtained from LC–MS analysis matched quite well with various regions of deduced amino acid sequences. Recombinant PemoNPV OB proteins specifically reacted with monoclonal antibodies to PemoNPV OB protein. After comparison with nucleotide database, the PemoNPV OB ORF demonstrated 67% identity to an uncharacterized ORF of a baculovirus pathogenic for Penaeus vannamei. However, comparison against protein databases revealed no significant homology to other known proteins. To our knowledge, this PemoNPV OB gene is the first isolated and characterized gene of nucleopolyhedrovirus from shrimp.

Crystal structure of an alkaline serine protease from Nesterenkonia sp. defines a novel family of secreted bacterial proteases

Crystal structure of an alkaline serine protease from Nesterenkonia sp. defines a novel family of secreted bacterial proteases Na Yang, Jie Nan, Erik Brostromer, Rajni Hatti-Kaul, and Xiao-Dong Su* 1 Shenzhen Graduate School of Peking University, Shenzhen 518055, China 2 National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing 100871, China 3 Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, S-221 00 Lund, Sweden