Amino Acid Sequence Of Domain Research Articles

Identification of novel subgroup A variants with enhanced receptor binding and replicative capacity in primary isolates of anaemogenic strains of feline leukaemia virus.

BackgroundThe development of anaemia in feline leukaemia virus (FeLV)-infected cats is associated with the emergence of a novel viral subgroup, FeLV-C. FeLV-C arises from the subgroup that is transmitted, FeLV-A, through alterations in the amino acid sequence of the receptor binding domain (RBD) of the envelope glycoprotein that result in a shift in the receptor usage and the cell tropism of the virus. The factors that influence the transition from subgroup A to subgroup C remain unclear, one possibility is that a selective pressure in the host drives the acquisition of mutations in the RBD, creating A/C intermediates with enhanced abilities to interact with the FeLV-C receptor, FLVCR. In order to understand further the emergence of FeLV-C in the infected cat, we examined primary isolates of FeLV-C for evidence of FeLV-A variants that bore mutations consistent with a gradual evolution from FeLV-A to FeLV-C.ResultsWithin each isolate of FeLV-C, we identified variants that were ostensibly subgroup A by nucleic acid sequence comparisons, but which bore mutations in the RBD. One such mutation, N91D, was present in multiple isolates and when engineered into a molecular clone of the prototypic FeLV-A (Glasgow-1), enhanced replication was noted in feline cells. Expression of the N91D Env on murine leukaemia virus (MLV) pseudotypes enhanced viral entry mediated by the FeLV-A receptor THTR1 while soluble FeLV-A Env bearing the N91D mutation bound more efficiently to mouse or guinea pig cells bearing the FeLV-A and -C receptors. Long-term in vitro culture of variants bearing the N91D substitution in the presence of anti-FeLV gp70 antibodies did not result in the emergence of FeLV-C variants, suggesting that additional selective pressures in the infected cat may drive the subsequent evolution from subgroup A to subgroup C.ConclusionsOur data support a model in which variants of FeLV-A, bearing subtle differences in the RBD of Env, may be predisposed towards enhanced replication in vivo and subsequent conversion to FeLV-C. The selection pressures in vivo that drive the emergence of FeLV-C in a proportion of infected cats remain to be established.

In Vivo Tagging and Characterization of S‐Glutathionylated Proteins by a Chemoenzymatic Method

In Vivo Tagging and Characterization of S‐Glutathionylated Proteins by a Chemoenzymatic Method

Molecular cloning and characterization of hatching enzyme-like geneII (BmHELII) in the silkworm, Bombyx mori

Hatching enzyme (HE) is an enzyme that digests an egg envelop at the time of embryo hatching. Previously, we have reported a kind of Bombyx mori hatching enzyme-like gene (BmHEL). In this paper, the full length of another BmHEL cDNA sequence (BmHELII, GenBank ID: JN627443) was cloned from bluish-silkworm-eggs. The cDNA was 977bp in length with an open reading frame of 885bp which encodes a polypeptide of 294 amino acids including a putative signal peptide of 16 amino acid residues and a mature protein of 278 amino acids. The deduced BmHELII had a predicted molecular mass of 33.62kDa, isoelectric point of 5.44 and two conserved signature sequences of astacin family. Bioinformatic analysis results showed that the deduced protease domain amino acid sequence of BmHELII had 29.5–87.0% identities to that of HE identified in the other species. The BmHELII gene structure was 6-exon–5-intron, and the promoter region harbored some basal promoter elements and some embryo development related transcription factor binding sites. Semi-quantitative RT-PCR analysis revealed that the relative level of BmHELII transcripts at different stages during egg incubation increased with the development of embryos and reached to a maximum just before hatching, hence declined gradually after hatching. The spatio-temporal expression pattern of BmHELII basically resembled that of hatching enzyme gene. Moreover, the BmHELII transcript was detected in testis of the silkworm, and semi-quantitative RT-PCR analysis showed that it kept at the high level in testis of silkworm from larvae to moth, which suggested that BmHELII might take part in the development of sperm. These results will be helpful to provide a molecular basis for understanding the mechanism underlying silkworm hatching as well as spermatogenesis.

Cloning and expression of ethylene receptor ERS1 at various developmental and ripening stages of mango fruit

Ethylene induces characteristic ripening reactions in climacteric fruits through its binding to histidine-kinase (HK) receptors, activating the expression of ripening genes. Ethylene receptors have been found in Arabidopsis thaliana (Brassicaceae) and some fruits; number and expression patterns differ among species. In mango, only ethylene receptor ETR1 was known. We cloned ERS1 cDNA from mango, and evaluated the expression of Mi-ERS1 and Mi-ETR1 by qPCR in developmental and ripening stages of this fruit. The Mi-ERS1 coding sequence is 1890 bp long and encodes 629 amino acids, similar to ERS1 from other fruits. Also, the amino acid sequence of ERS1 C-terminal HK domain shows the cognate fold after molecular modeling. Mi-ERS1 expression levels increased as mangoes ripened, showing the highest levels at the climacteric stage, while Mi-ETR1 levels did not change during development and ripening. We conclude that the patterns of expression of Mi-ERS1 and Mi-ETR1 differ in mango fruit.

Plant NBR1 is a selective autophagy substrate and a functional hybrid of the mammalian autophagic adapters NBR1 and p62/SQSTM1

(Macro)autophagy encompasses both an unselective, bulk degradation of cytoplasmic contents as well as selective autophagy of damaged organelles, intracellular microbes, protein aggregates, cellular structures and specific soluble proteins. Selective autophagy is mediated by autophagic adapters, like p62/SQSTM1 and NBR1. p62 and NBR1 are themselves selective autophagy substrates, but they also act as cargo receptors for degradation of other substrates. Surprisingly, we found that homologs of NBR1 are distributed throughout the eukaryotic kingdom, while p62 is confined to the metazoans. As a representative of all organisms having only an NBR1 homolog we studied Arabidopsis thaliana NBR1 (AtNBR1) in more detail. AtNBR1 is more similar to mammalian NBR1 than to p62 in domain architecture and amino acid sequence. However, similar to p62, AtNBR1 homo-polymerizes via the PB1 domain. Hence, AtNBR1 has hybrid properties of mammalian NBR1 and p62. AtNBR1 has 2 UBA domains, but only the C-terminal UBA domain bound ubiquitin. AtNBR1 bound AtATG8 through a conserved LIR (LC3-interacting region) motif and required co-expression of AtATG8 or human GABARAPL2 to be recognized as an autophagic substrate in HeLa cells. To monitor the autophagic sequestration of AtNBR1 in Arabidopsis we made transgenic plants expressing AtNBR1 fused to a pH-sensitive fluorescent tag, a tandem fusion of the red, acid-insensitive mCherry and the acid-sensitive yellow fluorescent proteins. This strategy allowed us to show that AtNBR1 is an autophagy substrate degraded in the vacuole dependent on the polymerization property of the PB1 domain and of expression of AtATG7. A functional LIR was required for vacuolar import.

Analysis of the autoproteolytic activity of the recombinant helper component proteinase from zucchini yellow mosaic virus

The multifunctional helper component proteinase (HC-Pro) of potyviruses contains an autoproteolytic function that, together with the protein 1 (P1) and NIa proteinase, processes the polyprotein into mature proteins. In this study, we analysed the autoproteolytic active domain of zucchini yellow mosaic virus (ZYMV) HC-Pro. Several Escherichia coli-expressed MBP:HC-Pro:GFP mutants containing deletions or point mutations at either the N- or C-terminus of the HC-Pro protein were examined. Our results showed that amino acids essential for the proteolytic activity of ZYMV HC-Pro are distinct from those of the tobacco etch virus HC-Pro, although the amino acid sequences in the proteolytic active domain are conserved among potyviruses.

Membrane protein misassembly in disease

Helix–helix interactions play a central role in the folding and assembly of integral α-helical membrane proteins and are fundamentally dictated by the amino acid sequence of the TM domain. It is not surprising then that missense mutations that target these residues are often linked to disease. In this review, we focus on the molecular mechanisms through which missense mutations lead to aberrant folding and/or assembly of these proteins, and then discuss pharmacological approaches that may potentially mitigate or reverse the negative effects of these mutations. Improving our understanding of how missense mutations affect the interactions between TM α-helices will increase our capability to develop effective therapeutic approaches to counter the misassembly of these proteins and, ultimately, disease. This article is part of a Special Issue entitled: Protein Folding in Membranes.

Novel Hemin Binding Domains in the Corynebacterium diphtheriae HtaA Protein Interact with Hemoglobin and Are Critical for Heme Iron Utilization by HtaA

The human pathogen Corynebacterium diphtheriae utilizes hemin and hemoglobin as iron sources for growth in iron-depleted environments. The use of hemin iron in C. diphtheriae involves the dtxR- and iron-regulated hmu hemin uptake locus, which encodes an ABC hemin transporter, and the surface-anchored hemin binding proteins HtaA and HtaB. Sequence analysis of HtaA and HtaB identified a conserved region (CR) of approximately 150 amino acids that is duplicated in HtaA and present in a single copy in HtaB. The two conserved regions in HtaA, designated CR1 and CR2, were used to construct glutathione S-transferase (GST) fusion proteins (GST-CR1 and GST-CR2) to assess hemin binding by UV-visual spectroscopy. These studies showed that both domains were able to bind hemin, suggesting that the conserved sequences are responsible for the hemin binding property previously ascribed to HtaA. HtaA and the CR2 domain were also shown to be able to bind hemoglobin (Hb) by the use of an enzyme-linked immunosorbent assay (ELISA) method in which Hb was immobilized on a microtiter plate. The CR1 domain exhibited a weak interaction with Hb in the ELISA system, while HtaB showed no significant binding to Hb. Competitive binding studies demonstrated that soluble hemin and Hb were able to inhibit the binding of HtaA and the CR domains to immobilized Hb. Moreover, HtaA was unable to bind to Hb from which the hemin had been chemically removed. Alignment of the amino acid sequences of CR domains from various Corynebacterium species revealed several conserved residues, including two highly conserved tyrosine (Y) residues and one histidine (H) residue. Site-directed mutagenesis studies showed that Y361 and H412 were critical for the binding to hemin and Hb by the CR2 domain. Biological assays showed that Y361 was essential for the hemin iron utilization function of HtaA. Hemin transfer experiments demonstrated that HtaA was able to acquire hemin from Hb and that hemin bound to HtaA could be transferred to HtaB. These findings are consistent with a proposed mechanism of hemin uptake in C. diphtheriae in which hemin is initially obtained from Hb by HtaA and then transferred between surface-anchored proteins, with hemin ultimately transported into the cytosol by an ABC transporter.

Characterization of a Central Ca2+/Calmodulin-dependent Protein Kinase IIα/β Binding Domain in Densin That Selectively Modulates Glutamate Receptor Subunit Phosphorylation

The densin C-terminal domain can target Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) in cells. Although the C-terminal domain selectively binds CaMKIIα in vitro, full-length densin associates with CaMKIIα or CaMKIIβ in brain extracts and in transfected HEK293 cells. This interaction requires a second central CaMKII binding site, the densin-IN domain, and an "open" activated CaMKII conformation caused by Ca(2+)/calmodulin binding, autophosphorylation at Thr-286/287, or mutation of Thr-286/287 to Asp. Mutations in the densin-IN domain (L815E) or in the CaMKIIα/β catalytic domain (I205/206K) disrupt the interaction. The amino acid sequence of the densin-IN domain is similar to the CaMKII inhibitor protein, CaMKIIN, and a CaMKIIN peptide competitively blocks CaMKII binding to densin. CaMKII is inhibited by both CaMKIIN and the densin-IN domain, but the inhibition by densin is substrate-selective. Phosphorylation of a model peptide substrate, syntide-2, or of Ser-831 in AMPA receptor GluA1 subunits is fully inhibited by densin. However, CaMKII phosphorylation of Ser-1303 in NMDA receptor GluN2B subunits is not effectively inhibited by densin in vitro or in intact cells. Thus, densin can target multiple CaMKII isoforms to differentially modulate phosphorylation of physiologically relevant downstream targets.

IMGT Collier de Perles for the Variable (V), Constant (C), and Groove (G) Domains of IG, TR, MH, IgSF, and MhSF

INTRODUCTIONThe “IMGT Collier de Perles” (or “IMGT_Collier_de_Perles”) concept is a major concept of numerotation (generated from the NUMEROTATION axiom) of IMGT-ONTOLOGY, the global reference in immunogenetics and immunoinformatics, built by IMGT, the international ImMunoGeneTics information system. The “IMGT Collier de Perles” concept, described here, allows standardized two-dimensional (2D) graphical representations of the domains, based on the IMGT unique numbering. Three leafconcepts (a leafconcept is a concept that corresponds to the finest level of granularity) have been defined: for the variable (V) domain and constant (C) domain of the immunoglobulin superfamily (IgSF) and for the groove (G) domain of the major histocompatibility (MH) superfamily (MhSF). IMGT Colliers de Perles are obtained, starting from V, C, or G domain amino acid sequences, using IMGT/DomainGapAlign and IMGT/Collier de Perles tools. In IMGT/3Dstructure-DB, IMGT Colliers de Perles of V and C domains are provided with hydrogen bonds and those of G domains with IMGT pMH contact analysis. IMGT Colliers de Perles allows one to bridge the gap between sequences and three-dimensional (3D) structures, whatever the species, the IgSF or MhSF protein, or the chain type. They are particularly useful for antibody engineering, sequence-structure analysis, visualization and comparison of positions for mutations, polymorphisms and contact analysis of immunoglobulins (IG), T cell receptors (TR), MH, and related proteins of the immune system (RPI) belonging to the IgSF and MhSF.

IMGT/Collier de Perles: IMGT Standardized Representation of Domains (IG, TR, and IgSF Variable and Constant Domains, MH and MhSF Groove Domains)

INTRODUCTIONIMGT/Collier de Perles is the IMGT online tool that allows one to draw standardized IMGT two-dimensional (2D) graphical representations of protein domains, or IMGT Colliers de Perles, starting from the user’s own domain amino acid (AA) sequences. The use of this tool is described here. IMGT Colliers de Perles can be drawn for three domain types: the variable (V) domain and constant (C) domain of the immunoglobulin (IG) and T cell receptor (TR) and other members of the immunoglobulin superfamily (IgSF), and the groove (G) domain of the major histocompatibility (MH) and other members of the MH superfamily (MhSF). Sequences have to be gapped according to the IMGT unique numbering, using, for example, IMGT/DomainGapAlign. Resulting IMGT Colliers de Perles allow one to quickly visualize amino acids, which are important for the three-dimensional (3D) structural configuration, and to delimit the standardized framework regions (FR-IMGT) and complementarity determining regions (CDR-IMGT) of the IG and TR V-DOMAIN. The length of the strands, loops, and turns and, for the G type, the length of the helix represented in IMGT Colliers de Perles become crucial information in the domain characterization.

DENN Domain Proteins: Regulators of Rab GTPases

The DENN domain is a common, evolutionarily ancient, and conserved protein module, yet it has gone largely unstudied; until recently, little was known regarding its functional roles. New studies reveal that various DENN domains interact directly with members of the Rab family of small GTPases and that DENN domains function enzymatically as Rab-specific guanine nucleotide exchange factors. Thus, DENN domain proteins appear to be generalized regulators of Rab function. Study of these proteins will provide new insights into Rab-mediated membrane trafficking pathways.

Functional Roles of a C-terminal Signaling Complex of CaV1 Channels and A-kinase Anchoring Protein 15 in Brain Neurons

Regulation of CaV1.2 channels in cardiac myocytes by the β-adrenergic pathway requires a signaling complex in which the proteolytically processed distal C-terminal domain acts as an autoinhibitor of channel activity and mediates up-regulation by the β-adrenergic receptor and PKA bound to A-kinase anchoring protein 15 (AKAP15). We examined the significance of this distal C-terminal signaling complex for CaV1.2 and CaV1.3 channels in neurons. AKAP15 co-immunoprecipitates with CaV1.2 and CaV1.3 channels. AKAP15 has overlapping localization with CaV1.2 and CaV1.3 channels in cell bodies and proximal dendrites and is closely co-localized with CaV1.2 channels in punctate clusters. The neuronal AKAP MAP2B, which also interacts with CaV1.2 and CaV1.3 channels, has complementary localization to AKAP15, suggesting different functional roles in calcium channel regulation. Studies with mice that lack the distal C-terminal domain of CaV1.2 channels (CaV1.2ΔDCT) reveal that AKAP15 interacts with neuronal CaV1.2 channels via their C terminus in vivo and is co-localized in punctate clusters of CaV1.2 channels via that interaction. CaV1.2ΔDCT neurons have reduced L-type calcium current, indicating that the distal C-terminal domain is required for normal functional expression in vivo. Deletion of the distal C-terminal domain impairs calcium-dependent signaling from CaV1.2 channels to the nucleus, as shown by reduction in phosphorylation of the cAMP response element-binding protein. Our results define AKAP signaling complexes of CaV1.2 and CaV1.3 channels in brain and reveal three previously unrecognized functional roles for the distal C terminus of neuronal CaV1.2 channels in vivo: increased functional expression, anchoring of AKAP15 and PKA, and initiation of excitation-transcription coupling.

Prediction of 492 human protein kinase substrate specificities.

BackgroundComplex intracellular signaling networks monitor diverse environmental inputs to evoke appropriate and coordinated effector responses. Defective signal transduction underlies many pathologies, including cancer, diabetes, autoimmunity and about 400 other human diseases. Therefore, there is high impetus to define the composition and architecture of cellular communications networks in humans. The major components of intracellular signaling networks are protein kinases and protein phosphatases, which catalyze the reversible phosphorylation of proteins. Here, we have focused on identification of kinase-substrate interactions through prediction of the phosphorylation site specificity from knowledge of the primary amino acid sequence of the catalytic domain of each kinase.ResultsThe presented method predicts 488 different kinase catalytic domain substrate specificity matrices in 478 typical and 4 atypical human kinases that rely on both positive and negative determinants for scoring individual phosphosites for their suitability as kinase substrates. This represents a marked advancement over existing methods such as those used in NetPhorest (179 kinases in 76 groups) and NetworKIN (123 kinases), which consider only positive determinants for kinase substrate prediction. Comparison of our predicted matrices with experimentally-derived matrices from about 9,000 known kinase-phosphosite substrate pairs revealed a high degree of concordance with the established preferences of about 150 well studied protein kinases. Furthermore for many of the better known kinases, the predicted optimal phosphosite sequences were more accurate than the consensus phosphosite sequences inferred by simple alignment of the phosphosites of known kinase substrates.ConclusionsApplication of this improved kinase substrate prediction algorithm to the primary structures of over 23, 000 proteins encoded by the human genome has permitted the identification of about 650, 000 putative phosphosites, which are posted on the open source PhosphoNET website (http://www.phosphonet.ca).

A monoclonal antibody-based enzyme-linked immunosorbent assay for human urinary cotinine to monitor tobacco smoke exposure

Immunoassays for human urinary cotinine [(S)-(−)-cotinine], a major nicotine metabolite, are useful to monitor the degree of tobacco smoke exposure. However, practical monoclonal antibodies for measuring urinary cotinine are not widely available. Here we generated a monoclonal antibody against a newly prepared cotinine–albumin conjugate and developed an enzyme-linked immunosorbent assay (ELISA). Splenocytes from a BALB/c mouse that had been immunized with the conjugate were fused with P3/NS1/1-Ag4-1 myeloma cells, and a hybridoma clone secreting the anti-cotinine antibody, Ab-CT#45 (IgG1κ; amino acid sequences of the variable domains were determined), was established. This antibody was immobilized on microplates to generate an ELISA system in which biotin-labeled cotinine was used as the tracer molecule. After competitive reactions with analyte, the bound biotin residues were monitored colorimetrically with peroxidase-labeled streptavidin. This ELISA produced dose–response curves for cotinine ranging from 0.40–100 ng per assay and was able to discriminate nicotine, cotinine N-oxide, cotinine N-glucuronide (cross-reactivity was each <0.5%, taking cotinine as 100%), (3′R, 5′S)-3′-hydroxycotinine O-glucuronide (0.6%) and (R, S)-norcotinine (1.5%). Another major nicotine metabolite, (3′R, 5′S)-3′-hydroxycotinine, showed 8.4% cross-reactivity, but an analytical recovery test indicated that this metabolite did not result in a significant overestimation of the urinary cotinine levels. The present ELISA has been validated to allow the urine specimens to be “directly” (without pretreatment) measured in order to screen and monitor tobacco smoke exposure, and if necessary, to enable cotinine alone to be more specifically examined after the urine specimens are subjected to a simple chloroform extraction.

Genomic organisation analysis of novel immunoglobulin-like transcripts in Atlantic salmon (Salmo salar) reveals a tightly clustered and multigene family

BackgroundSeveral novel immunoglobulin-like transcripts (NILTs) which have previously been identified in the salmonid species rainbow trout (Oncorhynchus mykiss) contain either one or two extracellular Ig domains of the V-type. NILTs also possess either an immunoreceptor tyrosine-based activating motif (ITAM) or immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic region resulting in different signalling abilities. Here we report for the first time the genomic organisation and structure of the multigene family of NILTs in Atlantic salmon (Salmo salar) using a BAC sequencing approach.ResultsWe have identified six novel Atlantic salmon NILT genes (Ssa-NILT1-6), two pseudogenes (Ssa-NILTp1 and Ssa-NILTp2) and seven genes encoding putative transposable elements in one BAC covering more than 200 kbp. Ssa-NILT1, 2, 4, 5 and 6 contain one Ig domain, all having a CX3C motif, whereas Ssa-NILT3 contains two Ig domains, having a CX6C motif in Ig1 and a CX7C motif in Ig2. Atlantic salmon NILTs possess several ITIMs in the cytoplasmic region and the ITIM-bearing exons are in phase 0. A comparison of identity between the amino acid sequences of the CX3C Ig domains from NILTs varies from 77% to 96%. Ssa-NILT1, 2, 3 and 4 were all confirmed to be expressed either by their presence in EST databases (Ssa-NILT1) or RT-PCR (Ssa-NILT2, 3, and 4) using cDNA as template. A survey of the repertoire of putative NILT genes in a single individual revealed three novel genes (Ssa-NILT7-9) represented by the Ig domain, which together with Ig domains from Ssa-NILT1-6 could be divided into different groups based on specific motifs.ConclusionsThis report reveals a tightly clustered, multigene NILT family in Atlantic salmon. By screening a highly redundant Atlantic salmon BAC library we have identified and characterised the genomic organisation of six genes encoding NILT receptors. The genes show similar characteristics to NILTs previously identified in rainbow trout, having highly conserved cysteines in the Ig domain and several inhibitory signalling motifs in the cytoplasmic region. In a single individual three unique NILT Ig domain sequences were discovered at the genomic DNA level, which were divided into two different groups based on a four residue motif after the third cysteine. Our results from the BAC screening and analysis on the repertoire of NILT genes in a single individual indicates that many genes of this expanding Ig containing NILT family are still to be discovered in fish.

Recombinant DNA-methyltransferase M1.Bst19I from Bacillus stearothermophilus 19: Purification, properties, and amino acid sequence analysis

The M1.Bst19I DNA-methyltransferase gene from restriction-modification system Bst19I (recognition sequence 5'-GCATC-3') in Bacillus stearothermophilus 19 has been cloned in the expressing vector pJW, that carries a tandem of thermo inducible promoters P(R)/P(L) from phage lambda. Highly purified enzyme has been isolated by chromatography on various resins from E. coli cells where it accumulates in a soluble form. Study of M1.Bst19I properties has revealed that enzyme has a temperature optimum 50 degrees C and demonstrates the maximum activity at pH 8.0. M1.Bst19I modifies adenine in sequence 5'-GCATC-3'. Kinetic parameters of M1.Bst19I DNA methylation reaction have been determined as follows: Km for lambda DNA is 0.68 +/- 0.07 microM, Km for S-adenosil-L-methionine is 2.02 +/- 0.31 microM. Catalytical constant (kcat) is 1.8 +/- 0.05 min(-1). Comparative analysis of Target Recognition Domain amino acid sequences for M1.Bst19I and others alpha-N6-DNA methyltransferases has allowed to suppose a presence of two types of the enzymes containing a triplet ATG or ATC in the recognition sequence.

Phosphorylation of Mixed Lineage Leukemia 5 by Cdc2 Affects Its Cellular Distribution and Is Required for Mitotic Entry

The human mixed lineage leukemia-5 (MLL5) gene is frequently deleted in myeloid malignancies. Emerging evidence suggests that MLL5 has important functions in adult hematopoiesis and the chromatin regulatory network, and it participates in regulating the cell cycle machinery. Here, we demonstrate that MLL5 is tightly regulated through phosphorylation on its central domain at the G(2)/M phase of the cell cycle. Upon entry into mitosis, the phosphorylated MLL5 delocalizes from condensed chromosomes, whereas after mitotic exit, MLL5 becomes dephosphorylated and re-associates with the relaxed chromatin. We further identify that the mitotic phosphorylation and subcellular localization of MLL5 are dependent on Cdc2 kinase activity, and Thr-912 is the Cdc2-targeting site. Overexpression of the Cdc2-targeting MLL5 fragment obstructs mitotic entry by competitive inhibition of the phosphorylation of endogenous MLL5. In addition, G(2) phase arrest caused by depletion of endogenous MLL5 can be compensated by exogenously overexpressed full-length MLL5 but not the phosphodomain deletion or MLL5-T912A mutant. Our data provide evidence that MLL5 is a novel cellular target of Cdc2, and the phosphorylation of MLL5 may have an indispensable role in the mitotic progression.

Symptom induction and RNA silencing suppression by the cucumber mosaic virus 2b protein

The 2b protein encoded by Cucumber mosaic virus (CMV) and other cucumoviruses is multifunctional, having roles in local and systemic virus movement, symptom determination, evasion of defense mediated by salicylic acid, and in suppression of antiviral RNA silencing. It also perturbs silencing-mediated regulation of host transcripts, suggesting that another function of 2b protein is to manipulate host gene expression and physiology in a way that may aid the virus. The 2b proteins encoded by the various cucumoviruses (CMV strains, as well as Tomato aspermy virus and Peanut stunt virus) share conserved amino acid sequence domains, suggesting that these might determine specific functions of the protein. We analyzed the effect of mutations in these domains on functions of the 2b protein during viral infection. This revealed that binding of short RNAs, the key determinants of RNA silencing specificity, correlates with RNA silencing suppression activity. Two putative phosphorylation sites were found to be required for virus symptom induction, despite having no influence on RNA silencing suppression. This indicates that the ability to suppress silencing is not the only factor affecting symptom induction by the 2b protein. In accordance with this, our studies also revealed that the 2b protein acts synergistically with some other CMV product(s) to induce symptoms, and that the role of the 2b protein in symptom determination is host species specific.

Discovery and expression profile analysis of AP2/ERF family genes from Triticum aestivum

Throughout its development, common wheat, Triticum aestivum responds to different kinds of adverse abiotic and biotic stress by expressing specific genes that allow it to adapt to these stresses. In this process, genes in the AP2/ERF family encode transcriptional regulators involved in diverse developmental and physiological processes play critical roles. Here, we established an extensive picture of the AP2/ERF family genes in wheat. From 960, 174 ESTs of T. aestivum, 117 putative AP2/ERF family genes were identified by in silico analysis based on the presence of the conserved AP2/ERF domain amino acid sequence of Arabidopsis thaliana. Based on the model species A. thaliana, the AP2/ERF TFs from T. aestivum were classified into five subfamilies with the following number of members: DREB (57), ERF (47), AP2 (9), RAV (3) and Soloist (1). Using the available EST information as a source of expression data, the putative AP2/ERF family genes from T. aestivum were detected in nine kinds of tissues. Transcripts of the genes were shown to be most abundant in leaves, followed by roots and seeds, and the least abundant in stem. Most of the T. aestivum AP2/ERF family genes showed some tissue specificity.