Tissue distribution and evolution of fructosamine 3-kinase and fructosamine 3-kinase-related protein

Fructosamine 3-kinase (FN3K) and FN3K-related protein (FN3K-RP) catalyze the phosphorylation of the Amadori products ribulosamines, psicosamines, and, in the case of FN3K, fructosamines. BLAST searches in chordate genomes revealed two genes encoding proteins homologous to FN3K or FN3K-RP in various mammals and in chicken but only one gene, encoding a protein more similar to FN3K-RP than to FN3K, in fishes and the sea squirt Ciona intestinalis. This suggests that a gene duplication event occurred after the fish radiation and that the FN3K gene evolved more rapidly than the FN3K-RP gene. In agreement with this distribution, only one enzyme, phosphorylating ribulosamines and psicosamines but not fructosamines, was found in the tissues from a fish (Clarias gariepinus), whereas two enzymes with specificities similar to either FN3K or FN3K-RP were found in mouse, rat, and chicken tissues. FN3K is particularly active in brain, heart, kidney, and skeletal muscle. Its activity is also relatively elevated in erythrocytes from man, rat, and mouse but barely detectable in erythrocytes from chicken and pig, which correlates well with the low intracellular concentration of glucose in erythrocytes from these species. This is in keeping with the specific role of FN3K to repair protein damage caused by glucose. FN3K-RP was more evenly distributed in tissues, except for skeletal muscle where its activity was particularly low. This may be related to low activity of the pentose phosphate pathway in this tissue, as suggested by assays of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. This finding, together with the high affinity of FN3K-RP for ribulosamines, suggests that this enzyme may serve to repair damage caused by the powerful glycating agent, ribose 5-phosphate.     

Cdc48p interacts with Ufd3p, a WD repeat protein required for ubiquitin-mediated proteolysis in Saccharomyces cerevisiae.

A library of random 10 residue peptides fused to the N-terminus of a reporter protein was screened in the yeast Saccharomyces cerevisiae for sequences that can target the reporter for degradation by the N-end rule pathway, a ubiquitin (Ub)-dependent proteolytic system that recognizes potential substrates through binding to their destabilizing N-terminal residues. One of the N-terminal sequences identified by this screen was used in a second screen for mutants incapable of degrading the corresponding reporter fusion. A mutant thus identified had an abnormally low content of free Ub. This mutant was found to be allelic to a previously isolated mutant in a Ub-dependent proteolytic system distinct from the N-end rule pathway. We isolated the gene involved, termed UFD3, which encodes an 80 kDa protein containing tandem repeats of a motif that is present in many eukaryotic proteins and called the WD repeat. Both co-immunoprecipitation and two-hybrid assays demonstrated that Ufd3p is an in vivo ligand of Cdc48p, an essential ATPase required for the cell cycle progression and the fusion of endoplasmic reticulum membranes. Further, we showed that, similarly to Ufd3p, Cdc48p is also required for the Ub-dependent proteolysis of test substrates. The discovery of the Ufd3p--Cdc48p complex and the finding that this complex is a part of the Ub system open up a new direction for studies of the function of Ub in the cell cycle and membrane dynamics.     

A catalytically inactive gelatinase B/MMP-9 mutant impairs homing of chronic lymphocytic leukemia cells by altering migration regulatory pathways

We previously showed that MMP-9 overexpression impairs migration of primary CLL cells and MEC-1 cells transfected with MMP-9. To determine the contribution of non-proteolytic activities to this effect we generated MEC-1 transfectants stably expressing catalytically inactive MMP-9MutE (MMP-9MutE-cells). In xenograft models in mice, MMP-9MutE-cells showed impaired homing to spleen and bone marrow, compared to cells transfected with empty vector (Mock-cells). In vitro transendothelial and random migration of MMP-9MutE-cells were also reduced. Biochemical analyses indicated that RhoAGTPase and p-Akt were not downregulated by MMP-9MutE, at difference with MMP-9. However, MMP-9MutE-cells or primary cells incubated with MMP-9MutE had significantly reduced p-ERK and increased PTEN, accounting for the impaired migration. Our results emphasize the role of non-proteolytic MMP-9 functions contributing to CLL progression.     

Comparison of GWAS models to identify non-additive genetic control of flowering time in sunflower hybrids

Key message

This study compares five models of GWAS, to show the added value of non-additive modeling of allelic effects to identify genomic regions controlling flowering time of sunflower hybrids.

Abstract

Genome-wide association studies are a powerful and widely used tool to decipher the genetic control of complex traits. One of the main challenges for hybrid crops, such as maize or sunflower, is to model the hybrid vigor in the linear mixed models, considering the relatedness between individuals. Here, we compared two additive and three non-additive association models for their ability to identify genomic regions associated with flowering time in sunflower hybrids. A panel of 452 sunflower hybrids, corresponding to incomplete crossing between 36 male lines and 36 female lines, was phenotyped in five environments and genotyped for 2,204,423 SNPs. Intra-locus effects were estimated in multi-locus models to detect genomic regions associated with flowering time using the different models. Thirteen quantitative trait loci were identified in total, two with both model categories and one with only non-additive models. A quantitative trait loci on LG09, detected by both the additive and non-additive models, is located near a GAI homolog and is presented in detail. Overall, this study shows the added value of non-additive modeling of allelic effects for identifying genomic regions that control traits of interest and that could participate in the heterosis observed in hybrids.

     

Molecular and genetic characterization of the <Emphasis Type="Italic">Ry</Emphasis><Subscript><Emphasis Type="Italic">adg</Emphasis></Subscript> locus on chromosome XI from Andigena potatoes conferring extreme resistance to potato virus Y

Key message

We have elucidated the Andigena origin of the potato Ry_adg gene on chromosome XI of CIP breeding lines and developed two marker assays to facilitate its introgression in potato by marker-assisted selection.

Abstract

Potato virus Y (PVY) is causing yield and quality losses forcing farmers to renew periodically their seeds from clean stocks. Two loci for extreme resistance to PVY, one on chromosome XI and the other on XII, have been identified and used in breeding. The latter corresponds to a well-known source of resistance (Solanum stoloniferum), whereas the one on chromosome XI was reported from S. stoloniferum and S. tuberosum group Andigena as well. To elucidate its taxonomic origin in our breeding lines, we analyzed the nucleotide sequences of tightly linked markers (M45, M6) and screened 251 landraces of S. tuberosum group Andigena for the presence of this gene. Our results indicate that the PVY resistance allele on chromosome XI in our breeding lines originated from S. tuberosum group Andigena. We have developed two marker assays to accelerate the introgression of Ry_adg gene into breeding lines by marker-assisted selection (MAS). First, we have multiplexed RYSC3, M6 and M45 DNA markers flanking the Ry_adg gene and validated it on potato varieties with known presence/absence of the Ry_adg gene and a progeny of 6,521 individuals. Secondly, we developed an allele-dosage assay particularly useful to identify multiplex Ry_adg progenitors. The assay based on high-resolution melting analysis at the M6 marker confirmed Ry_adg plex level as nulliplex, simplex and duplex progenitors and few triplex progenies. These marker assays have been validated and can be used to facilitate MAS in potato breeding.

     

Mutation of a conserved glycine residue modifies the vanadate sensitivity of the plasma membrane H+-ATPase from Schizosaccharomyces pombe

The structural gene pma+1 for the H+-ATPase from the fission yeast Schizosaccharomyces pombe has been isolated and sequenced. The intron-less gene encodes for a protein of Mr = 99,769 which is 75% homologous to those of Saccharomyces cerevisiae and Neurospora crassa. The S. pombe pma+1 gene complements not only S. pombe pma-1-1 but also S. cerevisiae pma-1-4 mutants selected for in vitro vanadate-resistant ATPase activity. The sequence of the S. pombe mutant pma-1-1 allele reveals that the glycine residue 268, which is perfectly conserved in the transduction domain of all animal and fungal transport ATPases sequenced so far, is modified into an aspartate residue by the mutation. Replacement of glycine 268 by aspartate has been monitored by the appearance of a new PvuI restriction site in the mutant DNA. Mitotic cosegregation has been observed between the PvuI site and vanadate-resistant ATPase activity in a growing population of S. pombe transformants.     

A second transport ATPase gene in Saccharomyces cerevisiae

A second transport ATPase gene from Saccharomyces cerevisiae has been identified by hybridization to a PMA1 probe and sequenced. The gene called PMA2 encodes a polypeptide of Mr = 102,157, which, with the exception of the 144 amino-terminal residues, is highly homologous to the structural gene PMA1 for the H+-ATPase. It is localized on the chromosome XVI at 16.7 centimorgan from gal4 and is not essential for haploid growth. Comparison between the upstream, noncoding DNA regions of PMA1 and PMA2 indicates that the two genes are controlled differently. The extensive amino acid sequence homology with the fungal H+-ATPases described so far indicates that the PMA2-encoded protein is also able to function as a H+ pump. This is supported by the observation that in pma1 mutants with reduced plasma membrane ATPase activity, disruption of the PMA2 gene confers the ability to grow under alkaline pH conditions. Slower development of diploids is also observed on normal minimal medium after bilateral disruption of PMA2 in the two parents.     

Increase in the Calcium Content of Cardiac Tissue after Postfixation with Osmium Tetroxide

The concentration of osmium has been measured by destructive chemical analysis in glutaraldehyde fixed heart tissue postfixed with osmium tetroxide and embedded in epoxy resin. After such treatment, the mean atomic number of the specimen (Z) is close to 10, which permits a quantitative analysis of calcium (Ca) by the continuum method, using Z²/A as a correcting factor (A: atomic weight). Wavelength-dispersive X-ray microanalysis has been used to determine the Ca concentration of frog cardiac tissue fixed in glutaraldehyde and embedded in resin. These measurements have been repeated on tissue postfixed in osmium tetroxide; contrary to expectations, the apparent Ca concentration is much higher in osmium-treated than in nontreated tissue. However, this result is observed with OsO₄ solutions prepared in glass, not with solutions prepared in plastic. It is shown by energy dispersive X-ray analysis of droplets that OsO₄ solutions prepared in glass contain large amounts of calcium, potassium and silicon. Care must be taken in preparing OsO₄ fixatives when the fixed tissues are to be subjected to X-ray microanalysis of such elements as Ca or Si.     

Isolation of the structural genes for the alpha and beta subunits of the mitochondrial ATPase from the fission yeast Schizosaccharomyces pombe

The structural genes for the two major subunits of the mitochondrial ATPase were isolated among genomic clones from the yeast Schizosaccharomyces pombe by transformation and complementation of mutants unable to grow on glycerol and lacking either the alpha or the beta subunits. The plasmid pMa1 containing a 2.3-kilobase genomic insert transformed the mutant A23-13 lacking a detectable alpha subunit. The transformant grew on glycerol and contained an alpha subunit of normal electrophoretic mobility. The plasmid pMa2 containing a 5.4-kilobase genomic insert transformed the mutant B59-1 lacking the beta subunit. The transformant grew on glycerol and contained a beta subunit of normal mobility. The structural gene for the beta ATPase subunit for the fission yeast S. pombe was localized within the pMa2 insert by hybridization to a probe containing the beta ATPase gene from the budding yeast Saccharomyces cerevisiae (Saltzgaber, J., Kunapuli, S., and Douglas, M. G. (1983) J. Biol. Chem. 258, 11465-11470). The mRNAs which hybridized to pMa1 and pMa2 were translated by a reticulocyte lysate into polypeptides of Mr = 59,000 and 54,000, respectively. These genes products reacted with an anti-F1-ATPase serum and therefore correspond most probably to precursors of the alpha and beta subunits.     

Use of tool sets by chimpanzees for multiple purposes in Moukalaba-Doudou National Park,Gabon

We report our recent findings on the use of tool sets by chimpanzees in Moukalaba-Doudou National Park, Gabon. Direct observations and evidences left by chimpanzees showed that chimpanzees used sticks as pounders, enlargers, and collectors to extract honey from beehives of stingless bees (Meliponula sp.), which may correspond to those previously found in the same site for fishing termites and to those found in Loango National Park, Gabon. However, we observed chimpanzees using a similar set of tools for hunting a medium-sized mammal (possibly mongoose) that hid inside a log. This is the first report of hunting with tools by a chimpanzee population in Central Africa. Chimpanzees may recognize the multiple functions and applicability of tools (extracting honey and driving prey), although it is still a preliminary speculation. Our findings may provide us a new insight on the chimpanzee’s flexibility of tool use and cognitive abilities of complex food gathering.