Anthocyanin biosynthesis genes location and expression in wheat–rye hybrids

Studies into gene expression in a foreign background contribute toward understanding of how genes derived from different species or genera manages to co-exist in a common nucleus, on the one hand, and help to estimate possible effectiveness of wide hybridization for cultivated plant improvement, on the other hand. The aim of this study was to investigate conservation of wheat and rye expression networks, using the anthocyanin biosynthesis pathway (ABP) genes as a model system. We isolated and analyzed ABP genes encoding enzymes acting at different steps of the pathway: chalcone-flavanone isomerase (CHI), flavanone 3-hydroxylase (F3H), anthocyanidin synthase (ANS), and anthocyanidin-3-glucoside rhamnosyltransferase (3RT). The rye ABP genes locations we determined (Chi on chromosome 5RL, F3h on 2RL, Ans on 6RL, 3Rt on 5RL, the regulatory Rc—red coleoptile—gene on 4RL) were in agreement with the rearrangements established between rye and wheat chromosomes. Expression of the ABP structural genes was studied in wheat–rye chromosome addition and substitution lines. F3h activation by the Rc gene was found to be critical for the red coleoptile trait formation. It was shown that the rye regulatory Rc gene can activate the wheat target gene F3h and vice versa wheat Rc induces expression of rye F3h. However, lower level of expression of rye F3h in comparison with that of the two wheat orthologues in the wheat–rye chromosome substitution line 2R(2D) was observed. Thus, although work of the wheat and rye ABP gene systems following the formation of wheat–rye hybrids is finely coordinated, some divergence exists between rye and wheat ABP genes, affecting level of gene expression.     

Abscisic acid enhances tolerance to spring freeze stress and regulates the expression of ascorbate–glutathione biosynthesis-related genes and stress-responsive genes in common wheat

Molecular Breeding - Hybrid sterility is a major obstacle to the development of superior inter-subspecific hybrids between indica and japonica subspecies of Asian-cultivated rice. To overcome...     

Variants of CYP46A1 may interact with age and APOE to influence CSF Aβ42 levels in Alzheimer’s disease

Recent studies have suggested that variants of CYP46A1, encoding cholesterol 24-hydroxylase (CYP46), confer risk for Alzheimers disease (AD), a prospect substantiated by evidence of genetic association from several quantitative traits related to AD pathology, including cerebrospinal fluid (CSF) levels of the 42 amino-acid cleavage product of -amyloid (A42) and the tau protein. In the present study, these claims have been explored by the genotyping of previously associated markers in CYP46A1 in three independent northern European case-control series encompassing 1323 individuals and including approximately 400 patients with measurements of CSF A42 and phospho-tau protein levels. Tests of association in case-control models revealed limited evidence that CYP46A1 variants contributed to AD risk across these samples. However, models testing for potential effects upon CSF measures suggested a possible interaction of an intronic marker (rs754203) with age and APOE genotype. In stratified analyses, significant effects were evident that were restricted to elderly APOE 4 carriers for both CSF A42 (P=0.0009) and phospho-tau (P=0.046). Computational analyses indicate that the rs754203 marker probably does not impact the binding of regulatory factors, suggesting that other polymorphic sites underlie the observed associations. Our results provide an important independent replication of previous findings, supporting the existence of CYP46A1 sequence variants that contribute to variability in -amyloid metabolism.     

Estrogen receptor β and its domains interact with casein kinase 2, phosphokinase C, and N-myristoylation sites of mitochondrial and nuclear proteins in mouse brain

The localization of estrogen receptor (ER)β in mitochondria suggests ERβ-dependent regulation of genes, which is poorly understood. Here, we analyzed the ERβ interacting mitochondrial as well as nuclear proteins in mouse brain using pull-down assay and matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS). In the case of mitochondria, ERβ interacted with six proteins of 35-152 kDa, its transactivation domain (TAD) interacted with four proteins of 37-172 kDa, and ligand binding domain (LBD) interacted with six proteins of 37-161 kDa. On the other hand, in nuclei, ERβ interacted with seven proteins of 30-203 kDa, TAD with ten proteins of 31-160 kDa, and LBD with fourteen proteins of 42-179 kDa. For further identification, these proteins were cleaved by trypsin into peptides and analyzed by MALDI-MS using mascot search engine, immunoprecipitation, immunoblotting, and far-Western blotting. To find the consensus binding motifs in interacting proteins, their unique tryptic peptides were analyzed by the motif scan software. All the interacting proteins were found to contain casein kinase (CK) 2, phosphokinase (PK)C phosphorylation, and N-myristoylation sites. These were further confirmed by peptide pull-down assays using specific mutations in the interacting sites. Thus, the present findings provide evidence for the interaction of ERβ with specific mitochondrial and nuclear proteins through consensus CK2, PKC phosphorylation, and N-myristoylation sites, and may represent an essential step toward designing selective ER modulators for regulating estrogen-mediated signaling.     

Branchial morphology and ion–water transport proteins in Antarctic teleosts with different modes of life

In the Antarctic Ocean salt concentration differs from the bottom to the surface owing to the seasonal forming and melting of sea ice. Antarctic teleosts present different lifestyle from benthic to pelagic. While benthic animals face a constant seawater salinity, benthic–pelagic animals have to face different salt concentration. Branchial morphology and ion–water transport proteins were compared in animals with different lifestyle. The ultrastructure of the gills was investigated by scanning electron microscopy (SEM). Na⁺/K⁺/ATPase, Na⁺/K⁺/Cl⁻ cotransport protein NKCC₁ and Aquaporin 3 (AQP3), were investigated by immunohistochemistry. The immunoreactivity for the ion transporter proteins were more intense in the active benthic–pelagic animals and in the icefishes than in the sluggish benthic ones. Conversely, AQP immunoreactivity was stronger in the animals with sedentary lifestyles. The SEM showed the secondary lamellae in the benthic–pelagic animals more densely packed with the exception of the haemoglobin free teleosts.     

Structural and functional comparative study of the complexes formed by viral ø29, Nf and GA-1 SSB proteins with DNA

Single-stranded DNA-binding proteins have in common their crucial roles in DNA metabolism, although they exhibit significant differences in their single-stranded DNA binding properties. To evaluate the correlation between the structure of different nucleoprotein complexes and their function, we have carried out a comparative study of the complexes that the single-stranded DNA-binding proteins of three related bacteriophages, ?29, Nf and GA-1, form with single-stranded DNA. Under the experimental conditions used, ?29 and Nf single-stranded DNA-binding proteins are stable monomers in solution, while GA-1 single-stranded DNA-binding protein presents a hexameric state, as determined in glycerol gradients. The thermodynamic parameters derived from quenching measurements of the intrinsic protein fluorescence upon single-stranded DNA binding revealed (i) that GA-1 single-stranded DNA-binding protein occludes a larger binding site (n=51 nt/oligomer) than ?29 and Nf SSBs (n=3.4 and 4.7 nt/monomer, respectively); and (ii) that it shows a higher global affinity for single-stranded DNA (GA-1 SSB, K(eff)=18.6 x 10(5) M(-1); o29 SSB, K(eff)=2.2 x 10(5) M(-1); Nf SSB, K(eff)=2.9 x 10(5) M(-1)). Altogether, these parameters justify the differences displayed by the GA-1 single-stranded DNA-binding protein and single-stranded DNA complex under the electron microscope, and the requirement of higher amounts of ?29 and Nf single-stranded DNA-binding proteins than of GA-1 SSB in gel mobility shift assays to produce a similar effect. The structural differences of the nucleoprotein complexes formed by the three single-stranded DNA-binding proteins with single-stranded DNA correlate with their different functional stimulatory effects in ?29 DNA amplification.     

Interplay between formation of photosynthetic complexes and expression of genes for iron–sulfur cluster assembly in Rhodobacter sphaeroides?

Photosynthesis Research - Formation of photosynthetic complexes leads to a higher demand for Fe–S clusters. We hypothesized that in the facultative phototrophic alpha-proteobacterium...     

Cloning and characterization of Methoprene-tolerant(Met)and Kruppel homolog 1(Kr-h1)genes in the wheat blossom midge,Sitodiplosis mosellana

Juvenile hormone(JH),a growth regulator,inhibits ecdysteroid-induced meta-morphosis and controls insect development and diapause.Methoprene-tolerant(Met)and Krippel homolog I(Kr-h1)are two proteins involved in JH action.To gain some insight into their function in development of Sitodiplosis mosellana,an insect pest undergoing obligatory larval diapause at the mature 3rd instar stage,we cloned full-length complemen-tary DNAs of Met and Kr-h1 from this specics.SmMet encoded a putative protein,which contained three domains typical of the bHLH-PAS family and eight conserved amino acid residues important for JH binding.SmKr-h1 encoded a protein showing high sequence homology to its counterparts in other specics,and contained all eight highly conserved Zn-finger motifs for DNA-binding.Expression patterns of SmMet and SmKr hl were de-velopmentally regulated and JH III responsive as well.Their mRNA abundance increased as larvae entered carly 3rd instar,pre-diapause and maintenance stages,and peaked during post-diapause quiescence,a pattern correlated with JH titers in this species.Different from reduced expression of SmMer,SmKr-h1 mRNA increased at mid-to-late period of post-diapause development.Topical application of JH II on diapausing larvac also induced the two genes in a dose-dependent manner.Expression of SmuMer and SmKr-h1 clearly declined in the pre-pupal phase,and was significantly higher in female adults than male adults.These results suggest that JH-responsive SmMet and SmKr-h1 might play key roles in diapause induction and maintenance as well as in post-diapause quiescence and adult reproduction,whereas metamorphosis from larvae to pupac might be correlated with their reduced expression.     

Interactome of signaling networks in wheat: the protein–protein interaction between TaRAR1 and TaSGT1

RAR1 and SGT1 are required for development and disease resistance in plants. In many cases, RAR1 and SGT1 regulate the resistance (R)-gene-mediated defense signaling pathways. Lr21 is the first identified NBS-LRR-type R protein in wheat and is required for resistance to the leaf rust pathogen. The Lr21-mediated signaling pathways require the wheat homologs of RAR1, SGT1, and HSP90. However, the molecular mechanisms of the Lr21-mediated signaling networks remain unknown. Here I present the DNA and protein sequences of TaRAR1 and TaSGT1, and demonstrate for the first time a direct protein-protein interaction between them.     

Dynamics of the intrinsically disordered protein NUPR1 in isolation and in its fuzzy complexes with DNA and prothymosin α

Intrinsically disordered proteins (IDPs) explore diverse conformations in their free states and, a few of them, also in their molecular complexes. This functional plasticity is essential for the function of IDPs, although their dynamics in both free and bound states is poorly understood. NUPR1 is a protumoral multifunctional IDP, activated during the acute phases of pancreatitis. It interacts with DNA and other IDPs, such as prothymosin α (ProTα), with dissociation constants of ~0.5 μM, and a 1:1 stoichiometry. We studied the structure and picosecond-to-nanosecond (ps-ns) dynamics by using both NMR and SAXS in: (i) isolated NUPR1; (ii) the NUPR1/ProTα complex; and (iii) the NUPR1/double stranded (ds) GGGCGCGCCC complex. Our SAXS findings show that NUPR1 remained disordered when bound to either partner, adopting a worm-like conformation; the fuzziness of bound NUPR1 was also pinpointed by NMR. Residues with the largest values of the relaxation rates (R₁, R_1ρ, R₂ and η_xy), in the free and bound species, were mainly clustered around the 30s region of the sequence, which agree with one of the protein hot-spots already identified by site-directed mutagenesis. Not only residues in this region had larger relaxation rates, but they also moved slower than the rest of the molecule, as indicated by the reduced spectral density approach (RSDA). Upon binding, the energy landscape of NUPR1 was not funneled down to a specific, well-folded conformation, but rather its backbone flexibility was kept, with distinct motions occurring at the hot-spot region.     

Cell-penetrating fusion peptides OD1 and OD2 interact with Bcr–Abl and influence the growth and apoptosis of K562 cells

The Bcr–Abl oncoprotein is the cause of chronic myelogenous leukemia (CML). Crystal structure analysis suggests that Bcr_30–63 is the core of the Bcr–Abl oligomerization interface for aberrant kinase activity; however, the precise role of other residues of Bcr_1–72 excluding Bcr_30–63 have not been evaluated. In this study, Bcr_30–63 was named OD2 and other residues of Bcr_1–72 were named OD1. Cytoplasmic transduction peptide (CTP) was used to carry molecules into cytoplasm. CTP-OD1 and CTP-OD2 fusion peptides were expressed from a cold-inducible expression system. Our results demonstrated that both fusion peptides could localize into the cytoplasm, specifically interact with the Bcr–Abl protein and further inhibit growth, induce apoptosis, and decrease the phosphorylation of Bcr–Abl in K562 cell lines. However, the viability of THP-1, a Bcr–Abl negative cell line, was unaffected. These results suggested that CTP-OD1 and CTP-OD2 may be an attractive therapeutic option to inhibit the activation of Bcr–Abl kinase in CML.     

Major QTL for carrot color are positionally associated with carotenoid biosynthetic genes and interact epistatically in a domesticated × wild carrot cross

We performed QTL analyses for pigment content on a carotenoid biosynthesis function map based on progeny of a wild white carrot (QAL) which accumulates no pigments × domesticated orange carrot (B493), one of the richest sources of carotenoid pigments—mainly provitamin A α- and β- carotenes. Two major interacting loci, Y and Y ₂ on linkage groups 2 and 5, respectively, control much variation for carotenoid accumulation in carrot roots. They are associated with carotenoid biosynthetic genes zeaxanthin epoxidase and carotene hydroxylase and carotenoid dioxygenase gene family members as positional candidate genes. Dominant Y allele inhibits carotenoid accumulation. When Y is homozygous recessive, carotenoids that accumulate are either only xanthophylls in Y ₂ __ plants, or both carotenes and xanthophylls, in y ₂ y ₂ plants. These two genes played a major role in carrot domestication and account for the significant role that modern carrot plays in vitamin A nutrition.     

Myelin glycosphingolipids,galactosylceramide and sulfatide,participate in carbohydrate–carbohydrate interactions between apposed membranes and may form glycosynapses between oligodendrocyte and/or myelin membranes

Glycosphingolipids (GSLs) can interact with each other by homotypic or heterotypic trans carbohydrate–carbohydrate interactions across apposed membranes, resulting in cell–cell adhesion. This interaction can also provide an extracellular signal which is transmitted to the cytosolic side, thus forming a glycosynapse between two cells. The two major GSLs of myelin, galactosylceramide (GalC) and its sulfated form, galactosylceramide I³-sulfate (SGC), are an example of a pair of GSLs which can participate in these trans carbohydrate–carbohydrate interactions and trigger transmembrane signaling. These GSLs could interact across apposed oligodendrocyte membranes at high cell density or when a membranous process of a cell contacts itself as it wraps around the axon. GalC and SGC also face each other in the apposed extracellular surfaces of the multilayered myelin sheath. Communication between the myelin sheath and the axon regulates both axonal and myelin function and is necessary to prevent neurodegeneration. Participation of transient GalC and SGC interactions in glycosynapses between the apposed extracellular surfaces of mature myelin might allow transmission of signals throughout the myelin sheath and thus facilitate myelin-axonal communication.     

Expression profiles of genes involved in apoptosis and selenium metabolism in articular cartilage of patients with Kashin–Beck osteoarthritis

Kashin–Beck disease (KBD) is a special type of endemic osteoarthritis. It has been suggested that alterations in selenium metabolism and apoptosis play a role in KBD. However, the underlying molecular mechanism remains largely unclear. We performed a microarray analysis using RNA isolated from cartilages of KBD patients and healthy controls, through Significance Analysis of Microarray (SAM) software. Functional gene networks and crucial molecules associated with differentially expressed genes were investigated via Ingenuity Pathway Analysis (IPA) and hub gene analysis. Quantitative real-time PCR was used to check the validation of chip test. We identified 52 up-regulated apoptosis-related genes and 26 down-regulated selenium-related genes between KBD and controls, and these genes associated with the “MYC-mediated apoptosis signaling pathway”. We confirmed the results from array studies with quantitative real-time PCR analysis. Our results suggest that abnormal regulation of selenium metabolism and apoptosis through the MYC mediated signaling pathway contributes to the pathogenesis of KBD, but the relationship between apoptosis gene and selenium gene was not found.     

Hsp90 cochaperones p23 and FKBP4 physically interact with hAgo2 and activate RNA interference–mediated silencing in mammalian cells

Argonaute proteins and small RNAs together form the RNA-induced silencing complex (RISC), the central effector of RNA interference (RNAi). The molecular chaperone Hsp90 is required for the critical step of loading small RNAs onto Argonaute proteins. Here we show that the Hsp90 cochaperones Cdc37, Aha1, FKBP4, and p23 are required for efficient RNAi. Whereas FKBP4 and p23 form a stable complex with hAgo2, the function of Cdc37 in RNAi appears to be indirect and may indicate that two or more Hsp90 complexes are involved. Our data also suggest that p23 and FKBP4 interact with hAgo2 before small RNA loading and that RISC loading takes place in the cytoplasm rather than in association with RNA granules. Given the requirement for p23 and FKBP4 for efficient RNAi and that these cochaperones bind to hAgo2, we predict that loading of hAgo2 is analogous to Hsp90-mediated steroid hormone receptor activation. To this end, we outline a model in which FKBP4, p23, and Aha1 cooperatively regulate the progression of hAgo2 through the chaperone cycle. Finally, we propose that hAgo2 and RNAi can serve as a robust model system for continued investigation into the Hsp90 chaperone cycle.     

G3BP–Caprin1–USP10 complexes mediate stress granule condensation and associate with 40S subunits

Mammalian stress granules (SGs) contain stalled translation preinitiation complexes that are assembled into discrete granules by specific RNA-binding proteins such as G3BP. We now show that cells lacking both G3BP1 and G3BP2 cannot form SGs in response to eukaryotic initiation factor 2α phosphorylation or eIF4A inhibition, but are still SG-competent when challenged with severe heat or osmotic stress. Rescue experiments using G3BP1 mutants show that phosphomimetic G3BP1-S149E fails to rescue SG formation, whereas G3BP1-F33W, a mutant unable to bind G3BP partner proteins Caprin1 or USP10, rescues SG formation. Caprin1/USP10 binding to G3BP is mutually exclusive: Caprin binding promotes, but USP10 binding inhibits, SG formation. G3BP interacts with 40S ribosomal subunits through its RGG motif, which is also required for G3BP-mediated SG formation. We propose that G3BP mediates the condensation of SGs by shifting between two different states that are controlled by the phosphorylation of S149 and by binding to Caprin1 or USP10.