Descripción: Xanthan is an industrially important exopolysaccharide produced by the phytopathogenic, gram-negative bacterium Xanthomonas campestris pv. campestris. It is composed of polymerized pentasaccharide repeating malts which are assembled by the sequential addition of glucose-1-phosphate, glucose, mannose, glucuronic acid, and mannose on a polyprenol phosphate carrier (L. Ielpi, R. O. Couso, and M. A. Dankert, J. Bacteriol. 175:2490- 2500, 1993). A cluster of 12 genes in a region designated xpsI or gum has been suggested to encode proteins involved in the synthesis and polymerization of the lipid intermediate. However, no experimental evidence supporting this suggestion has been published. In this work, from the biochemical analysis of a defined set of X. campestris gum mutants, we report experimental data for assigning functions to the products of the gum genes. We also show that the first step in the assembly of the lipid-linked intermediate is severely affected by the combination of certain gum and non- gum mutations. In addition, we provide evidence that the C-terminal domain of the gumD gene product is sufficient for its glucosyl-1-phosphate transferase activity. Finally, we found that alterations in the later stages of xanthan biosynthesis reduce the aggressiveness of X. campestris against the plant.

Descripción: Genes required for xanthan polysaccharide synthesis (xps) are clustered in a DNA region of 13.5 kb in the chromosome of Xanthomonas campestris. Plasmid pCHC3 containing a 12.4-kb insert of xps genes has been suggested to include a gene involved in the pyruvylation of xanthan gum (N. E. Harding, J. M. Cleary, D. K. Cabanas, I. G. Rosen, and K. S. Kang, J. Bacteriol. 169:2854-2861, 1987). An essential step toward understanding the biosynthesis of xanthan gum and to enable genetic manipulation of xanthan structure is the determination of the biochemical function encoded by the xps genes. On the basis of biochemical characterization of an X. campestris mutant which produces pyruvate-free xanthan gum, complementation studies, and heterologous expression, we have identified the gene coding for the ketal pyruvate transferase (kpt) enzyme. This gene was located on a 1.4-kb BamHI fragment of pCHC3 and cloned in the broad-host-range cloning vector pRK404. An X. campestris kpt mutant was constructed by mini-Mu(Tet(r)) mutagenesis of the cloned gene and then by recombination of the mutation into the chromosome of the wild-type strain.

Descripción: Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) are members of the MAPK family and participate in the transduction of stimuli in cellular responses. Their long-term actions are accomplished by promoting the expression of specific genes whereas faster responses are achieved by direct phosphorylation of downstream effectors located throughout the cell. In this study we determined that hERK1 translocates to the mitochondria of HeLa cells upon a proliferative stimulus. In the mitochondrial environment, hERK1 physically associates with (i) at least 5 mitochondrial proteins with functions related to transport (i.e. VDAC1), signalling, and metabolism; (ii) histones H2A and H4; and (iii) other cytosolic proteins. This work indicates for the first time the presence of diverse ERK-complexes in mitochondria and thus provides a new perspective for assessing the functions of ERK1 in the regulation of cellular signalling and trafficking in HeLa cells. © 2009 Galli et al.

Descripción: Background: Functional genetic markers have important implications for genetic analysis by providing direct estimation of functional diversity. Although high throughput sequencing techniques for functional diversity analysis are being developed nowadays, the use of already well established variable markers present in candidate genes is still an interesting alternative for mapping purposes and functional diversity studies. SSR markers are routinely used in most plant and animal breeding programs for many species including Eucalyptus. SSR markers derived from candidate genes (SSR-CG) can be used effectively in co-segregation studies and marker-assisted diversity management. Results: In the present study, eight new non reported SSRs were identified in seven candidate genes for wood properties in Eucalyptus globulus: cinnamoyl CoA reductase (CCR), homocysteine S-methyltransferase (HMT), shikimate kinase (SK), xyloglucan endotransglycosylase 2 (XTH2), cellulose synthase 3 (CesA3), glutathione S-transferase (GST) and the transcription factor LIM1. Microsatellites were located in promoters, introns and exons, being most of them CT dinucleotide repeats. Genetic diversity of these eight CG-derived SSR-markers was explored in 54 unrelated genotypes. Except for XTH2, high levels of polymorphism were detected: 93 alleles (mean of 13.1 sd 1.6 alleles per locus), a mean effective number of alleles (Ne) of 5.4 (sd 1.6), polymorphic information content values (PIC) from 0.617 to 0.855 and probability of Identity (PI) ranging from 0.030 to 0.151. Conclusions: This is the first report on the identification, characterization and diversity analysis of microsatellite markers located inside wood quality candidate genes (CG) from Eucalyptus globulus. This set of markers is then appropriate for characterizing genetic variation, with potential usefulness for quantitative trait loci (QTL) mapping in different eucalypts genetic pedigrees and other applications such as fingerprinting and marker assisted diversity management. © 2012 by Pontificia Universidad Católica de Valparaíso, Chile.

Descripción: Fructokinase (ATP:d-fructose-1-phosphate transferase, EC 2.7.1.3) from rat liver has been purified 400-fold. The purification procedure involves an acid treatment, a heat step at 65°, (NH4)2SO4 fractionation, chromatography on Sephadex G-100 and finally (NH4)2SO4 extraction. The enzyme appears nearly homogenous by density gradient centrifugation but gives a single peak in sedimentation velocity analysis. Purified liver fructokinase has a Km of 0.46-0.80 mM for fructose and 1.56-1.33 mM for MgATP at a K+ concentration of 0.4 and 0.1 M, respectively. The enzyme also phosphorylates l-sorbose and d-tagatose. No difference could be found in the phosphorylation of the pyranose and furanose forms of fructose. The enzyme is inhibited by p-chloromercuribenzoate and is stable up to 50-55°. © 1971.

Descripción: Saccharomyces cerevisiae pyruvate kinase 1 (Pyk1) was demonstrated to be associated to an immunoprecipitate of yeast protein kinase A holoenzyme (HA. Tpk1-Bcy1) and to be phosphorylated in a cAMP-dependent process. Both glutathione S-transferase (GST)-Pyk1 and GST-Pyk2 were phosphorylated in vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GST-Pyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated in vivo, in intact cells overexpressing the protein, or in vitro using crude extracts, as source of protein kinase A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK gene with an attenuated mutation (tpk1w1). The effect of phosphorylation on Pyk activity was assayed in partially purified preparations from three strains, containing different endogenous protein kinase A activity levels. Pyk1 activity was measured at different phosphoenolpyruvate concentrations in the absence or in the presence of the activator fructose 1,6-bisphosphate at 1.5 mM. Preliminary kinetic results derived from the comparison of Pyk1 obtained from extracts with the highest versus those from the lowest protein kinase A activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6-bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity.

Descripción: The effect of chronic exposure to Escherichia coli on morphometrical parameters, different antioxidant defenses, lipid peroxidation and protein oxidation has been studied in digestive gland of the freshwater mussel Diplodon chilensis in the laboratory during 6 weeks. In, a parallel field study, we evaluated morphometrical and oxidative stress parameters in digestive glands of mussels collected from a sewage polluted area (SMA) and from a pristine area (control). Both sites were characterized by analyzing bacteriological and physic-chemical parameters. In the laboratory D. chilensis was able to filter bacteria at high concentrations with a clearing rate of 0.510 ± 0.036. L/h per gram of dry soft tissue mass. The digestive gland mass to shell length ratio (DGM/SL), reduced glutathione (GSH), lipid peroxidation, as concentration of thiobartituric acid reactive substances (TBARS), and glutathione-S-transferase (GST) activity of mussels fed with bacteria were significantly higher than those of control mussels after the fourth week. Fecal bacteria in lake water samples were undetectable in the control, and higher than 24,000. MPN (most probable number)/100. mL in SMA. DGM/SL was higher in SMA. No differences between sites were observed in total lipid and protein content, neither in superoxide dismutase activity. GSH content was higher in SMA, with no difference in the oxidized form. GST activity and MDA were significantly higher in SMA but protein oxidation was not affected. © 2011 Elsevier GmbH.

Descripción: A genetic locus from Acetobacter xylinum involved in acetan polysaccharide synthesis has been characterized. The chromosomal region was identified by screening a genomic library of A. xylinum in a Xanthomonas campestris mutant defective in xanthan polysaccharide synthesis. The A. xylinum cosmid clone can functionally complement a xanthan-negative mutant. The polymer produced by the recombinant strain was found to be indistinguishable from xanthan. Insertion mutagenesis and subcloning of the cosmid clone combined with complementation studies allowed the identification of a 2.3-kb fragment of A. xylinum chromosomal DNA. The nucleotide sequence of this fragment was analyzed and found to contain an open reading frame (aceA) of 1,182 bp encoding a protein of 43.2 kDa. Results from biochemical and genetic analyses strongly suggest that the aceA gene encodes the GDP- mannose:cellobiosyl-diphosphopolyprenol α-mannosyltransferase enzyme, which is responsible for the transfer of an α-mannosyl residue from GDP-Man to cellobiosyl-diphosphopolyprenol. A search for similarities with other known mannosyltransferases revealed that all bacterial α-mannosyltransferases have a short COOH-termina1 amino acid sequence in common.

Descripción: Exposure to sources of UV radiation, such as sunlight, induces a number of cellular alterations that are highly dependent on its ability to affect gene expression. Among them, the rapid activation of genes coding for two subfamilies of proto-oncoproteins, Fos and Jun, which constitute the AP-1 transcription factor, plays a key role in the subsequent regulation of expression of genes involved in DNA repair, cell proliferation, cell cycle arrest, death by apoptosis, and tissue and extracellular matrix remodeling proteases. Besides being regulated at the transcriptional level, Jun and Fos transcriptional activities are also regulated by phosphorylation as a result of the activation of intracellular signaling cascades. In this regard, the phosphorylation of c-Jun by UV-induced JNK has been readily documented, whereas a role for Fos proteins in UV-mediated responses and the identification of Fos-activating kinases has remained elusive. Here we identify p38 MAPKs as proteins that can associate with c-Fos and phosphorylate its transactivation domain both in vitro and in vivo. This phosphorylation is transduced into changes in its transcriptional ability as p38-activated c-Fos enhances AP1-driven gene expression. Our findings indicate that as a consequence of the activation of stress pathways induced by UV light, endogenous c-Fos becomes a substrate of p38 MAPKs and, for the first time, provide evidence that support a critical role for p38 MAPKs in mediating stress-induced c-Fos phosphorylation and gene transcription activation. Using a specific pharmacological inhibitor for p38α and -β, we found that most likely these two isoforms mediate UV-induced c-Fos phosphorylation in vivo. Thus, these newly described pathways act concomitantly with the activation of c-Jun by JNK/MAPKs, thereby contributing to the complexity of AP1-driven gene transcription regulation.