José Miguel Hermoso

The push-pull mechanism of bacteriophage Ø29 DNA injection

The mechanism of bacteriophage DNA injection is poorly understood, often considered a simple process, driven merely by the packing pressure inside the capsid. In contrast to the well‐established DNA packaging mechanism of Bacillus subtilis phage Ø29, that involves a molecular motor formed by the connector and a viral ATPase, nothing is known about its DNA injection into the cell. We have studied this process measuring DNA binding of p6, a viral genome organization protein. The linear DNA penetrates with a right‐left polarity, in a two‐step process. In the first step ∼65% of the genome is pushed into the cell most probably by the pressure built inside the viral capsid. Thus, synthesis of viral proteins from the right early operon is allowed. This step is controlled, probably by bacterial protein(s) that slow down DNA entry. In the second step at least one of the viral early proteins, p17, participates in the molecular machinery that pulls the remaining DNA inside the cell. Both steps are energy‐dependent, as treatment of cells with azide overrides the whole mechanism, leading to a deregulated, passive entry of DNA.

Signals at the bacteriophage phi 29 DNA replication origins required for protein p6 binding and activity.

Protein p6 of Bacillus subtilis phage phi 29 binds specifically to the ends of the viral DNA that contain the replication origins, giving rise to a nucleoprotein structure. DNA regions recognized by protein p6 have been mapped by deletion analysis and DNase I footprinting. Main protein p6‐recognition signals have been located between nucleotides 62 and 125 at the right phi 29 DNA end and between nucleotides 46 and 68 at the left end. In addition, recognition signals are also present at other sites within 200‐300 bp at each phi 29 DNA end. Protein p6 does not seem to recognize a specific sequence in the DNA, but rather a structural feature, which could be bendability. The formation of the protein p6‐DNA nucleoprotein complex is likely to be the structural basis for the protein p6 activity in the initiation of replication.

Effect of NH4+ ions on phi 29 DNA-protein p3 replication: formation of a complex between the terminal protein and the DNA polymerase.

This work was supported by Community of Madrid (Grupo Estrategico 2000-2003), NIH, grant R01CA77575, and SAF 2001-2245.The transition step from the p3-dAMP initiation complex to the first elongated products, p3-(dAMP)2 and p3-(dAMP)3, requires a dATP concentration higher than that needed for the initiation reaction or for the further elongation of the p3-(dAMP)3 complex. The elongation in phi 29 DNA-protein p3 replication in vitro was strongly inhibited by salt. Under inhibitory salt concentration, the viral protein p6 greatly stimulated phi 29 DNA-protein p3 replication. The effect of protein p6 was not on the rate of elongation but on the amount of elongated product, stimulating the transition from initiation to formation of the first elongation products.Trabajo presentado en 44th Annual Meeting Society for Neuroscience, celebrado en Washington, DC (USA) del 15 al 19 de noviembre de 2014Recent studies have demonstrated that cytochrome c plays an important role in cell death. In the present study, we report that teniposide and various other chemotherapeutic agents induced a dose-dependent increase in the expression of the mitochondrial respiratory chain proteins cytochrome c, subunits I and IV of cytochrome c oxidase, and the free radical scavenging enzyme manganous superoxide dismutase. The teniposide-induced increase of cytochrome c was inhibited by cycloheximide, indicating new protein synthesis. Elevated cytochrome c levels were associated with enhanced cytochrome c oxidase-dependent oxygen uptake using TMPD/ascorbate as the electron donor, suggesting that the newly synthesized proteins were functional. Cytochrome c was released into the cytoplasm only after maximal levels had been reached in the mitochondria, but there was no concomitant decrease in mitochondrial membrane potential or caspase activation. Our results suggest that the increase in mitochondrial protein expression may play a role in the early cellular defense against anticancer drugs.The nucleotide sequence of the 5 end distal region of the human c-K-ras gene promoter was determined. This region, coincident with a variable DNAse I hypersensitive site in native chromatin, contains sequence similarities with known enhancers. A 400 bp MstII DNA fragment of this region stimulated in cis the correctly initiated transcription of the human beta-globin gene in transfected Hela cells. The stimulation of beta-globin transcription (5-6 fold) was dependent on the distance and orientation of the c-K-ras sequences and on the presence of the CCAAT and CACCC elements in the beta-globin promoter. Interaction of nuclear factors with these c-K-ras sequences was analysed by DNAase I footprinting assays using Hela nuclear extracts. A protein binding to these sequences was identified as nuclear factor 1 (NF-1) by DNAase I competition footprinting experiments. However, disruption of the c-K-ras NF-1 binding site by insertion mutagenesis had no effect on the transcriptional activity of the c-K-ras element.Supported by Grant GM-08041 from the National Institutes nof Health, United States Public Health Service.The results presented in this paper indicate that the phi 29 DNA polymerase is the only enzyme required for efficient synthesis of full length phi 29 DNA with the phi 29 terminal protein, the initiation primer, as the only additional protein requirement. Analysis of phi 29 DNA polymerase activity in various in vitro DNA replication systems indicates that two main reasons are responsible for the efficiency of this minimal system: 1) the phi 29 DNA polymerase is highly processive in the absence of any accessory protein; 2) the polymerase itself is able to produce strand displacement coupled to the polymerization process. Using primed M13 DNA as template, the phi 29 DNA polymerase is able to synthesize DNA chains greater than 70 kilobase pairs. Furthermore, conditions that increase the stability of secondary structure in the template do not affect the processivity and strand displacement ability of the enzyme. Thus, the catalytic properties of the phi 29 DNA polymerase are appropriate for a phi 29 DNA replication mechanism involving two replication origins, strand displacement and continuous synthesis of both strands. The enzymology of phi 29 DNA replication would support a symmetrical model of DNA replication.Aided by grants from the National Institutes of Health U.S. Public Health Service, and E. I. Du Pont de Neumours and Company, Inc.This work was supported in part by NRSA, National Institutes of Health Grants NS09463 and NS32501 and from National Science Foundation Grant 9310965.We have recently developed a new method to detect and characterize single base substitutions in transcribed genes which is based on the ability of RNAse A to recognize and cleave single base mismatches in RNA:RNA heteroduplexes. The RNAse A misrnatch cleavage assay was applied to screen human colon carcinoma cell lines and primary tumors for the presence of mutant e-X-ras oncogenes. We have determined that the mutant e-X-ras allele is overexpressed and amplified relative to the normal in the SX-CO-l human colon carcinoma cell lineo The oncogene mutation has been characterized by this method as a glycine to valine substitution at codon 12 of the e-X-ras gene. This result was confirmed by cloning and sequencing. We have previously reported that about 40% of primary human colon tumors contain e-X-ras genes mutant at codon 12 (Forrester et al, Nature 327: 298, 1987). We report here the characterization by molecular cloning and sequencing of the mutation in the e-X-ras oneogene from two of these tumors (tumors 3 and 28). We also describe the histopathologieal eharaeterization of these two tumors and demonstrate, by Southern blot hybridization of NIH3T3 transformants, the simultaneous presenee of mutant e-X-ras and N-ras oncogenes in villous adenoma 28. Our results provide evidence for the frequent assoeiation of ras somatie mutational aetivation in the early stages of tumor development in this common type of human eaneer.The residues forming the 3-->5 exonuclease active site of phi 29 DNA polymerase, located at the N-terminal conserved motifs Exo I, Exo II and Exo III, have been defined by site-directed mutagenesis (Bernad, A., Blanco, L., Lázaro, J. M., Martin, G., and Salas, M. (1989) Cell 59, 219-228; Soengas, M. S., Esteban, J. A., Lázaro, J. M., Bernad, A., Blasco, M. A., Salas, M., and Blanco, L. (1992) EMBO J. 11, 4227-4237). To understand their catalytic role, the residual exonuclease activity of mutants at these active site residues has been kinetically studied. The critical function of residues Asp12, Glu14, Asp66, and Asp169 is supported by a 10(5)-fold reduction in the exonuclease catalytic rate upon single mutation. Residue Tyr165 seems to play a secondary role in the exonuclease reaction based on the 10(2)-10(3)-fold reduced catalytic rate of mutants Y165F and Y165C. Most of the mutants were specially active in the presence of Mn2+ ions, which could be indicative of a direct involvement of these residues in a metal ion-assisted exonucleolytic reaction. The data obtained strongly suggest that the 3-->5 exonuclease active site of phi 29 DNA polymerase is structurally and functionally similar to that of the Escherichia coli DNA polymerase I. In addition, these residues were also very important for the strand displacement ability of phi 29 DNA polymerase, suggesting a structural overlapping of this activity with the 3-->5 exonuclease.phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid similarity. Among them, the conserved region characterized by the amino acid motif Kx3NSxYG has been proposed to form part of the polymerization active site of alpha-like DNA polymerases. Mutants in phi 29 DNA polymerase residue Tyr390 of this conserved motif had been previously described to be affected in DNA-dependent dNTP binding. In this paper, the functional significance of this conserved motif is further studied by the analysis of mutants in conserved residues Asn387, Ser388, and Gly391. Residue Phe393 of phi 29 DNA polymerase has also been selected as target for site-directed mutagenesis because of its conservation within the group of alpha-like DNA polymerases from genomes that replicate by a protein-priming mechanism. Mutant N387Y was shown to be affected both in initiation and polymerization reactions, showing 3-fold higher Km value for dATP and more than 11-fold lower Vmax value than the wild-type enzyme in the initiation reaction; moreover, it was affected in enzyme-DNA translocation. Mutant S388G retained initiation and polymerization activities; interestingly, this mutation significantly increased the efficiency of dNTP incorporation in non-templated reactions. Mutation Gly391 to Asp abolished template-primer binding as shown by gel retardation assays; this mutant was drastically affected in template-dependent dNTP incorporation both in initiation and polymerization reactions, but the efficiency of the non-templated phi 29 terminal protein-deoxynucleotidylation was higher than with the wild-type protein. Mutation Phe393 to Tyr severely decreased initial binding to template-primer DNA molecules, resulting in a reduced activity in DNA primer-dependent polymerization reactions but not in phi 29 terminal protein-dependent ones.Aided by Grants AM-01845, AM-08953, and l-Sol-FR-05099 nfrom the National Institutes o f Health, United States Public nHealth Service, and E. I. Du Pont de Nemours and Company, nInc. A preliminary report o f this work was presented at the nSecond Meeting o f the Federation o f European Biochemical nSocieties (symposium on “Ribonucleic Acid-Structure and nFunction”), Vienna, April 21 to 24, 1965.1 pagina.-- Trabajo presentado al: 4th International Meeting on Apicomplexa in Farm Animals. (Madrid, Spain. 11-14 October ,2017).The hairpin ribozyme catalyzes site-specific cleavage of an RNA substrate using a magnesium-dependent transphosphorylation mechanism. Here, we describe experiments designed to test the importance of ribose 2-hydroxyl groups for ribozyme function. Ribozymes for this work were synthesized in two segments using solid-phase RNA phosphoramidite chemistry. 2-Deoxyribonucleotides were systematically introduced at each of the 50 positions within the ribozyme, and the catalytic activity of the resulting mixed RNA-DNA polymers was measured. Deletion of the 2-hydroxyl group at each of four sites (A10, G11, A24, and C25) was found to result in severe inhibition of cleavage activity (kcat/KM decreased by 100- to 1000-fold), although KM measurements and mobility-shift assays showed that substrate binding was not affected. Identical results were obtained upon substitution of these ribonucleotides with 2-O-methyl derivatives. Inhibition by 2-modified sugars at G11 or A24 was rescued by increased Mg2+ concentrations, suggesting that these 2-hydroxyls may function in magnesium binding. Our results demonstrate that the 2-hydroxyl groups at A10, G11, A24, and C25 provide essential functions for catalysis, possibly forming important tertiary contacts or magnesium coordination sites that are necessary for active site architecture.Supported by Grant GM-08041 from the National Institutes of nHealth, United States Public Health Service.Resumen del trabajo presentado al XXXIII Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular celebrado en Cordoba del 14 al 17 de septiembre de 2010.This article describes the expression pattern and functional analysis of Lazarillo, a novel cell surface glycoprotein expressed in the embryonic grasshopper nervous system, and a member of the lipocalin family. Lazarillo is expressed by a subset of neuroblasts, ganglion mother cells and neurons of the central nervous system, by all sensory neurons of the peripheral nervous system, and by a subset of neurons of the enteric nervous system. It is also present in a few non neuronal cells associated mainly with the excretory system. A monoclonal antibody raised against Lazarillo perturbs the extent and direction of growth of identified commissural pioneer neurons. We propose that Lazarillo is the receptor for a midline morphogen involved in the outgrowth and guidance of these neurons.phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid sequence similarity and sensitivity to inhibitors of eukaryotic DNA polymerase alpha. In this paper, site-directed mutants in the phi 29 DNA polymerase residues Asp249, Ser252, Leu253, and Pro255 of the conserved amino acid motif Dx2SLYP are described. Two mutants, D249E and S252R, were drastically affected in all the synthetic activities, whereas their 3 to 5 exonuclease activity and interaction with the TP primer was normal. Mutant D249E, slightly affected in template-primer binding, was completely inactive in all conditions tested, suggesting that Asp249 could be playing a direct role in catalysis. On the other hand, mutant S252R, strongly affected in template-primer binding, showed some DNA polymerization activity in the presence of Mn2+. Mutants S252G and P255S showed a reduced template-primer binding ability; these mutants, together with mutant L253V, showed metal ion-dependent phenotypes in their synthetic activities and altered sensitivities to the PPi analog phosphonoacetic acid. All these results support the hypothesis that the Dx2SLYP motif forms part of the polymerization active site of the phi 29 DNA polymerase, being the Asp249 residue critical both for protein-primed initiation and DNA polymerization.Poster presentado al Annual Biomedical Research Conference for Minority Students celebrado en California (US) del 7 al 10 de noviembre de 2012.The phage phi 29 regulatory protein p4 activates the late promoter A3 by stabilizing the binding of Bacillus subtilis RNA polymerase (RNAP) as a closed complex. Interaction between the two proteins occurs through amino acid Arg120 in protein p4 and the C-terminal domain of the RNAP alpha subunit (alpha-CTD). In addition to its role as activator of the late transcription, protein p4 represses early transcription from the A2b and A2c promoters, that are divergently transcribed. Binding of p4 to its recognition site at the A3 promoter displaces the RNAP from promoter A2b, both by steric hindrance and by the curvature induced upon p4 binding. At the A2c promoter, the RNAP cooperates with p4 binding in such a way that promoter clearance is prevented. Interestingly, amino acid Arg120 in p4 and the alpha-CTD in B. subtilis RNAP are involved in the interactions that lead to transcription repression at promoter A2c. To investigate how this interaction leads to activation at PA3 and to repression at PA2c, mutant promoters were constructed. In the absence of a -35 consensus box for sigma A-RNAP activation was observed, while in its presence repression occurred. The results support the idea that overstabilization of RNAP at the promoter over a threshold level leads to repression.Resumen del poster presentado al XXXIII Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular celebrado en Cordoba del 14 al 17 de septiembre de 2010.Phi 29 DNA polymerase is able to catalyze two different synthetic reactions: protein-primed initiation and DNA polymerization. We have studied the fidelity of phi 29 DNA polymerase when carrying out these two reactions. Global fidelity was dissected into three steps: insertion discrimination, mismatch elongation, and proofreading. The insertion discrimination of phi 29 DNA polymerase in DNA polymerization ranged from 10(4) to 10(6). The efficiency of mismatch elongation was 10(5)-10(-6)-fold lower than that of a properly paired primer terminus. These factors indicate that DNA polymerization catalyzed by phi 29 DNA polymerase is a highly accurate process. Conversely, the insertion fidelity of protein-primed initiation was quite low, the insertion discrimination factor being about 10(2). Mismatch elongation discrimination was also rather low: mismatched terminal protein (TP).dNMP complexes were elongated from 2- to 6-fold more slowly than the correct TP.dNMP complex. Even more, the 3-->5 exonuclease activity of phi 29 DNA polymerase was unable to act on the TP.dNMP initiation complex, precluding the possibility that a wrong dNMP covalently linked to TP could be excised and corrected. Therefore, protein-primed initiation can be predicted as a quite inaccurate reaction. The problem of maintaining the sequence at the DNA ends is discussed in the context of a recently described model for protein-primed initiation.Formalin-fixed paraffin-embedded tissue specimens obtained by fine needle aspiration of pancreatic masses from 47 patients were examined retrospectively for cytology and the presence of mutant c-K-ras oncogenes. Point mutations of c-K-ras in codon 12 were detected by RNA-DNA RNAse A mismatch cleavage after in vitro DNA amplification of the cellular c-K-ras sequences by the polymerase chain reaction. Of the 36 patients with pancreatic adenocarcinoma, mutant c-K-ras oncogenes were detected in 18 of 25 (72%) with malignant cytologies, 2 of 8 (25%) with atypical cytologies, and 0 of 3 with benign aspiration cytologies. The remaining 11 patients without pancreatic adenocarcinomas did not have mutant c-K-ras genes detectable by the assay. The diagnosis of pancreatic adenocarcinoma was based upon clinical follow-up. The presence of mutant c-K-ras oncogenes did not significantly affect survival in the patients studied. Mutant c-K-ras genes were found at the time of initial clinical presentation in the majority of pancreatic adenocarcinomas, suggesting an important role of the mutation in oncogenesis. In conjunction with cytology, our approach represents an application for cancer diagnosis at the molecular genetic level.Calorie restriction (CR) has been shown to decrease reactive oxygen species (ROS) production and retard aging in a variety of species. It has been proposed that alterations in membrane saturation are central to these actions of CR. As a step towards testing this theory, mice were assigned to 4 dietary groups (control and 3 CR groups) and fed AIN-93G diets at 95 % (control) or 60 % (CR) of ad libitum for 8 months. To manipulate membrane composition, the primary dietary fats for the CR groups were soybean oil (also used in the control diet), fish oil or lard. Skeletal muscle mitochondrial lipid composition, proton leak, and H(2)O(2) production were measured. Phospholipid fatty acid composition in CR mice was altered in a manner that reflected the n-3 and n-6 fatty acid profiles of their respective dietary lipid sources. Dietary lipid composition did not alter proton leak kinetics between the CR groups. However, the capacity of mitochondrial complex III to produce ROS was decreased in the CR lard compared to the other CR groups. The results of this study indicate that dietary lipid composition can influence ROS production in muscle mitochondria of CR mice. It remains to be determined if lard or other dietary oils can maximize the CR-induced decreases in ROS production.To investigate the relationship between RNA folding and ribozyme catalysis, we have carried out a detailed kinetic analysis of four structural derivatives of the hairpin ribozyme. Optimal and suboptimal (wild-type) substrate sequences were studied in conjunction with stabilization of helix 4, which supports formation of the catalytic core. Pre-steady-state and steady-state kinetic studies strongly support a model in which each of the ribozyme variants partitions between two major conformations leading to active and inactive ribozymez substrate complexes. Reaction rates for cleavage, ligation, and substrate binding to both ribozyme conformations were determined. Ligation rates (3 min 21 ) were typically 15-fold greater than cleavage rates (0.2 min 21 ), demonstrating that the hairpin ribozyme is an efficient RNA ligase. On the other hand, substrate binding is very rapid (k on 5 4 3 10 8 M 21 min 21 ), and the ribozymez substrate complex is very stable (K D < 25 pM ;k off < 0.01 min 21 ). Stabilization of helix 4 increases the proportion of RNA molecules folded into the active conformation, and enhances substrate association and ligation rates. These effects can be explained by stabilization of the catalytic core of the ribozyme. Rigorous consideration of conformational isomers and their intrinsic kinetic properties was necessary for development of a kinetic scheme for the ribozyme-catalyzed reaction.The human integrin VLA (very late activation antigens)-4 (CD49d/CD29), the leukocyte receptor for both the CS-1 region of plasma fibronectin (Fn) and the vascular cell surface adhesion molecule-1 (VCAM-1), also mediates homotypic aggregation upon triggering with specific anti-VLA-4 monoclonal antibody (mAb). Epitope mapping of this integrin on the human B-cell line Ramos, performed with a wide panel of anti-VLA-4 mAb by both cross-competitive cell binding and protease sensitivity assays, revealed the existence of three topographically distinct epitopes on the alpha 4 chain, referred to as epitopes A-C. By testing this panel of anti-VLA-4 mAb for inhibition of cell binding to both a 38-kDa Fn fragment containing CS-1 and to VCAM-1, as well as for induction and inhibition of VLA-4 mediated homotypic cell adhesion, we have found overlapping but different functional properties associated with each epitope. Anti-alpha 4 mAb recognizing epitope B inhibited cell attachment to both Fn and VCAM-1, whereas mAb against epitope A did not block VCAM-1 binding and only partially inhibited binding to Fn. In contrast, mAb directed to epitope C did not affect cell adhesion to either of the two VLA-4 ligands. All mAb directed to site A, as well as a subgroup of mAb recognizing epitope B (called B2), were able to induce cell aggregation, but this effect was not exerted by mAb specific to site C and by a subgroup against epitope B (called B1). Moreover, although anti-epitope C and anti-epitope B1 mAb did not trigger aggregation, those mAb blocked aggregation induced by anti-epitope A or B2 mAb. In addition, anti-epitope A mAb blocked B2-induced aggregation, and conversely, anti-epitope B2 mAb blocked A-induced aggregation. Further evidence for multiple VLA-4 functions is that anti-Fn and anti-VCAM-1 antibodies inhibited binding to Fn or to VCAM-1, respectively, but did not affect VLA-4-mediated aggregation. In summary, we have demonstrated that there are at least three different VLA-4-mediated adhesion functions, we have defined three distinct VLA-4 epitopes, and we have correlated these epitopes with the different functions of VLA-4.Phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid similarity. Among them, the two conserved regions characterized by the amino acid motifs D-NSLYP and K--NS(L/V)YG, regions 1 and 2a, respectively, according to Blanco et al. (Blanco, L., Bernad, A., Blasco, M. A. and Salas, M. (1991) Gene (Amst.) 100, 27-38) have been proposed to be part of the polymerization active site of alpha-like DNA polymerases. One phi 29 DNA polymerase mutant in residue Tyr254, located in conserved region 1, and two mutants in residue Tyr390, located in conserved region 2a, have been characterized. The three phi 29 DNA polymerase mutant proteins were affected in polymerization when Mg(2+)-dNTPs were used as substrate. However, when the substrate was Mn(2+)-dNTP, mutants behaved as the wild-type phi 29 DNA polymerase. Mutant Tyr254 to Phe (Y254F) was strongly affected in the protein-primed initiation step of phi 29 DNA replication showing a decreased affinity for Me(2+)-dATP, the initiating nucleotide. Furthermore, the analysis of the template-independent deoxynucleotidylation of the TP by Y254F mutant polymerase is consistent with a change in the relative affinity for dNTPs. On the other hand, mutants Y390F and Y390S were found to be hypersensitive to the dNTP analogs 2-(p-n-butylanilino)dATP and N2-(p-n-butyl-phenyl)dGTP. The results obtained indicate that residues Tyr254 and Tyr390 are involved, directly or indirectly, in Me(2+)-dNTP binding.Lazarillo, a protein recognized by the monoclonal antibody 10E6, is expressed by a subset of neurons in the developing nervous system of the grasshopper. It is a glycoprotein of 45x10(3) M(r) with internal disulfide bonds and linked to the extracellular side of the plasma membrane by a glycosylphosphatidylinositol moiety. Peptide sequences obtained from affinity purified adult protein were used to identify an embryonic cDNA clone, and in situ hybridizations confirmed that the distribution of the Lazarillo mRNA paralleled that of the monoclonal antibody labeling on embryos. Sequence analysis defines Lazarillo as a member of the lipocalin family, extracellular carriers of small hydrophobic ligands, and most related to the porphyrin- and retinol-binding lipocalins. Lazarillo is the first example of a lipocalin anchored to the plasma membrane, highly glycosylated, and restricted to a subset of developing neurons.Trabajo presentado al Annual Biomedical Research Conference for Minority Students celebrada en Nashville (US) del 13 al 16 de noviembre de 2013.In this paper, we show that the phi 29 DNA polymerase, in the absence of DNA, is able to catalyze the formation of a covalent complex between the phi 29 terminal protein (TP) and 5-dAMP. Like the reaction in the presence of phi 29 DNA, TP.dAMP complex formation is strongly dependent on activating Mn2+ ions and on the efficient formation of a TP/DNA polymerase heterodimer. The nature of the TP-dAMP linkage was shown to be identical (a O-5-deoxyadenylyl-L-serine bond) to that found covalently linking TP to the DNA of bacteriophage phi 29, indicating that this DNA-independent reaction actually mimics that occurring as the initiation step of phi 29 DNA replication. Furthermore, as in normal TP-primed initiation on the phi 29 DNA template, this novel reaction showed the same specificity for TP Ser232 as the OH donor and the involvement of the YCDTD amino acid motif, highly conserved in alpha-like DNA polymerases. However, unlike the reaction in the presence of phi 29 DNA, the DNA-independent deoxynucleotidylation of TP by the phi 29 DNA polymerase did not show dATP specificity, being possible to obtain any of the four TP.dNMP complexes with a similar yield. This lack of specificity together with the poor efficiency of this reaction at low deoxynucleoside triphosphate (dNTP) concentration reflect a weak, but similar stability of the four dNTPs at the phi 29 DNA polymerase dNTP-binding site. Thus, the presence of a director DNA would mainly contribute to stabilizing a complementary nucleotide, giving base specificity to the protein-primed initiation reaction. According to all these data, the novel DNA polymerase reaction described in this paper could be considered as a non-DNA-instructed protein-primed deoxynucleotidylation.A cDNA has been isolated from human hippocampus that appears to encode a novel Na(+)-dependent, Cl(-)-independent, neutral amino acid transporter. The putative protein, designated SATT, is 529 amino acids long and exhibits significant amino acid sequence identity (39-44%) with mammalian L-glutamate transporters. Expression of SATT cDNA in HeLa cells induced stereospecific uptake of L-serine, L-alanine, and L-threonine that was not inhibited by excess (3 mM) 2-(methylamino)-isobutyric acid, a specific substrate for the System A amino acid transporter. SATT expression in HeLa cells did not induce the transport of radiolabeled L-cysteine, L-glutamate, or related dicarboxylates. Northern blot hybridization revealed high levels of SATT mRNA in human skeletal muscle, pancreas, and brain, intermediate levels in heart, and low levels in liver, placenta, lung, and kidney. SATT transport characteristics are similar to the Na(+)-dependent neutral amino acid transport activity designated System ASC, but important differences are noted. These include: 1) SATTs apparent low expression in ASC-containing tissues such as liver or placenta; 2) the lack of mutual inhibition between serine and cysteine; and 3) the lack of trans-stimulation. SATT may represent one of multiple activities that exhibit System ASC-like transport characteristics in diverse tissues and cell lines.

Assembly of phage phi 29 genome with viral protein p6 into a compact complex

This work was supported by Community of Madrid (Grupo Estrategico 2000-2003), NIH, grant R01CA77575, and SAF 2001-2245.The transition step from the p3-dAMP initiation complex to the first elongated products, p3-(dAMP)2 and p3-(dAMP)3, requires a dATP concentration higher than that needed for the initiation reaction or for the further elongation of the p3-(dAMP)3 complex. The elongation in phi 29 DNA-protein p3 replication in vitro was strongly inhibited by salt. Under inhibitory salt concentration, the viral protein p6 greatly stimulated phi 29 DNA-protein p3 replication. The effect of protein p6 was not on the rate of elongation but on the amount of elongated product, stimulating the transition from initiation to formation of the first elongation products.Trabajo presentado en 44th Annual Meeting Society for Neuroscience, celebrado en Washington, DC (USA) del 15 al 19 de noviembre de 2014Recent studies have demonstrated that cytochrome c plays an important role in cell death. In the present study, we report that teniposide and various other chemotherapeutic agents induced a dose-dependent increase in the expression of the mitochondrial respiratory chain proteins cytochrome c, subunits I and IV of cytochrome c oxidase, and the free radical scavenging enzyme manganous superoxide dismutase. The teniposide-induced increase of cytochrome c was inhibited by cycloheximide, indicating new protein synthesis. Elevated cytochrome c levels were associated with enhanced cytochrome c oxidase-dependent oxygen uptake using TMPD/ascorbate as the electron donor, suggesting that the newly synthesized proteins were functional. Cytochrome c was released into the cytoplasm only after maximal levels had been reached in the mitochondria, but there was no concomitant decrease in mitochondrial membrane potential or caspase activation. Our results suggest that the increase in mitochondrial protein expression may play a role in the early cellular defense against anticancer drugs.The nucleotide sequence of the 5 end distal region of the human c-K-ras gene promoter was determined. This region, coincident with a variable DNAse I hypersensitive site in native chromatin, contains sequence similarities with known enhancers. A 400 bp MstII DNA fragment of this region stimulated in cis the correctly initiated transcription of the human beta-globin gene in transfected Hela cells. The stimulation of beta-globin transcription (5-6 fold) was dependent on the distance and orientation of the c-K-ras sequences and on the presence of the CCAAT and CACCC elements in the beta-globin promoter. Interaction of nuclear factors with these c-K-ras sequences was analysed by DNAase I footprinting assays using Hela nuclear extracts. A protein binding to these sequences was identified as nuclear factor 1 (NF-1) by DNAase I competition footprinting experiments. However, disruption of the c-K-ras NF-1 binding site by insertion mutagenesis had no effect on the transcriptional activity of the c-K-ras element.Supported by Grant GM-08041 from the National Institutes nof Health, United States Public Health Service.The results presented in this paper indicate that the phi 29 DNA polymerase is the only enzyme required for efficient synthesis of full length phi 29 DNA with the phi 29 terminal protein, the initiation primer, as the only additional protein requirement. Analysis of phi 29 DNA polymerase activity in various in vitro DNA replication systems indicates that two main reasons are responsible for the efficiency of this minimal system: 1) the phi 29 DNA polymerase is highly processive in the absence of any accessory protein; 2) the polymerase itself is able to produce strand displacement coupled to the polymerization process. Using primed M13 DNA as template, the phi 29 DNA polymerase is able to synthesize DNA chains greater than 70 kilobase pairs. Furthermore, conditions that increase the stability of secondary structure in the template do not affect the processivity and strand displacement ability of the enzyme. Thus, the catalytic properties of the phi 29 DNA polymerase are appropriate for a phi 29 DNA replication mechanism involving two replication origins, strand displacement and continuous synthesis of both strands. The enzymology of phi 29 DNA replication would support a symmetrical model of DNA replication.Aided by grants from the National Institutes of Health U.S. Public Health Service, and E. I. Du Pont de Neumours and Company, Inc.This work was supported in part by NRSA, National Institutes of Health Grants NS09463 and NS32501 and from National Science Foundation Grant 9310965.We have recently developed a new method to detect and characterize single base substitutions in transcribed genes which is based on the ability of RNAse A to recognize and cleave single base mismatches in RNA:RNA heteroduplexes. The RNAse A misrnatch cleavage assay was applied to screen human colon carcinoma cell lines and primary tumors for the presence of mutant e-X-ras oncogenes. We have determined that the mutant e-X-ras allele is overexpressed and amplified relative to the normal in the SX-CO-l human colon carcinoma cell lineo The oncogene mutation has been characterized by this method as a glycine to valine substitution at codon 12 of the e-X-ras gene. This result was confirmed by cloning and sequencing. We have previously reported that about 40% of primary human colon tumors contain e-X-ras genes mutant at codon 12 (Forrester et al, Nature 327: 298, 1987). We report here the characterization by molecular cloning and sequencing of the mutation in the e-X-ras oneogene from two of these tumors (tumors 3 and 28). We also describe the histopathologieal eharaeterization of these two tumors and demonstrate, by Southern blot hybridization of NIH3T3 transformants, the simultaneous presenee of mutant e-X-ras and N-ras oncogenes in villous adenoma 28. Our results provide evidence for the frequent assoeiation of ras somatie mutational aetivation in the early stages of tumor development in this common type of human eaneer.The residues forming the 3-->5 exonuclease active site of phi 29 DNA polymerase, located at the N-terminal conserved motifs Exo I, Exo II and Exo III, have been defined by site-directed mutagenesis (Bernad, A., Blanco, L., Lázaro, J. M., Martin, G., and Salas, M. (1989) Cell 59, 219-228; Soengas, M. S., Esteban, J. A., Lázaro, J. M., Bernad, A., Blasco, M. A., Salas, M., and Blanco, L. (1992) EMBO J. 11, 4227-4237). To understand their catalytic role, the residual exonuclease activity of mutants at these active site residues has been kinetically studied. The critical function of residues Asp12, Glu14, Asp66, and Asp169 is supported by a 10(5)-fold reduction in the exonuclease catalytic rate upon single mutation. Residue Tyr165 seems to play a secondary role in the exonuclease reaction based on the 10(2)-10(3)-fold reduced catalytic rate of mutants Y165F and Y165C. Most of the mutants were specially active in the presence of Mn2+ ions, which could be indicative of a direct involvement of these residues in a metal ion-assisted exonucleolytic reaction. The data obtained strongly suggest that the 3-->5 exonuclease active site of phi 29 DNA polymerase is structurally and functionally similar to that of the Escherichia coli DNA polymerase I. In addition, these residues were also very important for the strand displacement ability of phi 29 DNA polymerase, suggesting a structural overlapping of this activity with the 3-->5 exonuclease.phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid similarity. Among them, the conserved region characterized by the amino acid motif Kx3NSxYG has been proposed to form part of the polymerization active site of alpha-like DNA polymerases. Mutants in phi 29 DNA polymerase residue Tyr390 of this conserved motif had been previously described to be affected in DNA-dependent dNTP binding. In this paper, the functional significance of this conserved motif is further studied by the analysis of mutants in conserved residues Asn387, Ser388, and Gly391. Residue Phe393 of phi 29 DNA polymerase has also been selected as target for site-directed mutagenesis because of its conservation within the group of alpha-like DNA polymerases from genomes that replicate by a protein-priming mechanism. Mutant N387Y was shown to be affected both in initiation and polymerization reactions, showing 3-fold higher Km value for dATP and more than 11-fold lower Vmax value than the wild-type enzyme in the initiation reaction; moreover, it was affected in enzyme-DNA translocation. Mutant S388G retained initiation and polymerization activities; interestingly, this mutation significantly increased the efficiency of dNTP incorporation in non-templated reactions. Mutation Gly391 to Asp abolished template-primer binding as shown by gel retardation assays; this mutant was drastically affected in template-dependent dNTP incorporation both in initiation and polymerization reactions, but the efficiency of the non-templated phi 29 terminal protein-deoxynucleotidylation was higher than with the wild-type protein. Mutation Phe393 to Tyr severely decreased initial binding to template-primer DNA molecules, resulting in a reduced activity in DNA primer-dependent polymerization reactions but not in phi 29 terminal protein-dependent ones.Aided by Grants AM-01845, AM-08953, and l-Sol-FR-05099 nfrom the National Institutes o f Health, United States Public nHealth Service, and E. I. Du Pont de Nemours and Company, nInc. A preliminary report o f this work was presented at the nSecond Meeting o f the Federation o f European Biochemical nSocieties (symposium on “Ribonucleic Acid-Structure and nFunction”), Vienna, April 21 to 24, 1965.1 pagina.-- Trabajo presentado al: 4th International Meeting on Apicomplexa in Farm Animals. (Madrid, Spain. 11-14 October ,2017).The hairpin ribozyme catalyzes site-specific cleavage of an RNA substrate using a magnesium-dependent transphosphorylation mechanism. Here, we describe experiments designed to test the importance of ribose 2-hydroxyl groups for ribozyme function. Ribozymes for this work were synthesized in two segments using solid-phase RNA phosphoramidite chemistry. 2-Deoxyribonucleotides were systematically introduced at each of the 50 positions within the ribozyme, and the catalytic activity of the resulting mixed RNA-DNA polymers was measured. Deletion of the 2-hydroxyl group at each of four sites (A10, G11, A24, and C25) was found to result in severe inhibition of cleavage activity (kcat/KM decreased by 100- to 1000-fold), although KM measurements and mobility-shift assays showed that substrate binding was not affected. Identical results were obtained upon substitution of these ribonucleotides with 2-O-methyl derivatives. Inhibition by 2-modified sugars at G11 or A24 was rescued by increased Mg2+ concentrations, suggesting that these 2-hydroxyls may function in magnesium binding. Our results demonstrate that the 2-hydroxyl groups at A10, G11, A24, and C25 provide essential functions for catalysis, possibly forming important tertiary contacts or magnesium coordination sites that are necessary for active site architecture.Supported by Grant GM-08041 from the National Institutes of nHealth, United States Public Health Service.Resumen del trabajo presentado al XXXIII Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular celebrado en Cordoba del 14 al 17 de septiembre de 2010.This article describes the expression pattern and functional analysis of Lazarillo, a novel cell surface glycoprotein expressed in the embryonic grasshopper nervous system, and a member of the lipocalin family. Lazarillo is expressed by a subset of neuroblasts, ganglion mother cells and neurons of the central nervous system, by all sensory neurons of the peripheral nervous system, and by a subset of neurons of the enteric nervous system. It is also present in a few non neuronal cells associated mainly with the excretory system. A monoclonal antibody raised against Lazarillo perturbs the extent and direction of growth of identified commissural pioneer neurons. We propose that Lazarillo is the receptor for a midline morphogen involved in the outgrowth and guidance of these neurons.phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid sequence similarity and sensitivity to inhibitors of eukaryotic DNA polymerase alpha. In this paper, site-directed mutants in the phi 29 DNA polymerase residues Asp249, Ser252, Leu253, and Pro255 of the conserved amino acid motif Dx2SLYP are described. Two mutants, D249E and S252R, were drastically affected in all the synthetic activities, whereas their 3 to 5 exonuclease activity and interaction with the TP primer was normal. Mutant D249E, slightly affected in template-primer binding, was completely inactive in all conditions tested, suggesting that Asp249 could be playing a direct role in catalysis. On the other hand, mutant S252R, strongly affected in template-primer binding, showed some DNA polymerization activity in the presence of Mn2+. Mutants S252G and P255S showed a reduced template-primer binding ability; these mutants, together with mutant L253V, showed metal ion-dependent phenotypes in their synthetic activities and altered sensitivities to the PPi analog phosphonoacetic acid. All these results support the hypothesis that the Dx2SLYP motif forms part of the polymerization active site of the phi 29 DNA polymerase, being the Asp249 residue critical both for protein-primed initiation and DNA polymerization.Poster presentado al Annual Biomedical Research Conference for Minority Students celebrado en California (US) del 7 al 10 de noviembre de 2012.The phage phi 29 regulatory protein p4 activates the late promoter A3 by stabilizing the binding of Bacillus subtilis RNA polymerase (RNAP) as a closed complex. Interaction between the two proteins occurs through amino acid Arg120 in protein p4 and the C-terminal domain of the RNAP alpha subunit (alpha-CTD). In addition to its role as activator of the late transcription, protein p4 represses early transcription from the A2b and A2c promoters, that are divergently transcribed. Binding of p4 to its recognition site at the A3 promoter displaces the RNAP from promoter A2b, both by steric hindrance and by the curvature induced upon p4 binding. At the A2c promoter, the RNAP cooperates with p4 binding in such a way that promoter clearance is prevented. Interestingly, amino acid Arg120 in p4 and the alpha-CTD in B. subtilis RNAP are involved in the interactions that lead to transcription repression at promoter A2c. To investigate how this interaction leads to activation at PA3 and to repression at PA2c, mutant promoters were constructed. In the absence of a -35 consensus box for sigma A-RNAP activation was observed, while in its presence repression occurred. The results support the idea that overstabilization of RNAP at the promoter over a threshold level leads to repression.Resumen del poster presentado al XXXIII Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular celebrado en Cordoba del 14 al 17 de septiembre de 2010.Phi 29 DNA polymerase is able to catalyze two different synthetic reactions: protein-primed initiation and DNA polymerization. We have studied the fidelity of phi 29 DNA polymerase when carrying out these two reactions. Global fidelity was dissected into three steps: insertion discrimination, mismatch elongation, and proofreading. The insertion discrimination of phi 29 DNA polymerase in DNA polymerization ranged from 10(4) to 10(6). The efficiency of mismatch elongation was 10(5)-10(-6)-fold lower than that of a properly paired primer terminus. These factors indicate that DNA polymerization catalyzed by phi 29 DNA polymerase is a highly accurate process. Conversely, the insertion fidelity of protein-primed initiation was quite low, the insertion discrimination factor being about 10(2). Mismatch elongation discrimination was also rather low: mismatched terminal protein (TP).dNMP complexes were elongated from 2- to 6-fold more slowly than the correct TP.dNMP complex. Even more, the 3-->5 exonuclease activity of phi 29 DNA polymerase was unable to act on the TP.dNMP initiation complex, precluding the possibility that a wrong dNMP covalently linked to TP could be excised and corrected. Therefore, protein-primed initiation can be predicted as a quite inaccurate reaction. The problem of maintaining the sequence at the DNA ends is discussed in the context of a recently described model for protein-primed initiation.Formalin-fixed paraffin-embedded tissue specimens obtained by fine needle aspiration of pancreatic masses from 47 patients were examined retrospectively for cytology and the presence of mutant c-K-ras oncogenes. Point mutations of c-K-ras in codon 12 were detected by RNA-DNA RNAse A mismatch cleavage after in vitro DNA amplification of the cellular c-K-ras sequences by the polymerase chain reaction. Of the 36 patients with pancreatic adenocarcinoma, mutant c-K-ras oncogenes were detected in 18 of 25 (72%) with malignant cytologies, 2 of 8 (25%) with atypical cytologies, and 0 of 3 with benign aspiration cytologies. The remaining 11 patients without pancreatic adenocarcinomas did not have mutant c-K-ras genes detectable by the assay. The diagnosis of pancreatic adenocarcinoma was based upon clinical follow-up. The presence of mutant c-K-ras oncogenes did not significantly affect survival in the patients studied. Mutant c-K-ras genes were found at the time of initial clinical presentation in the majority of pancreatic adenocarcinomas, suggesting an important role of the mutation in oncogenesis. In conjunction with cytology, our approach represents an application for cancer diagnosis at the molecular genetic level.Calorie restriction (CR) has been shown to decrease reactive oxygen species (ROS) production and retard aging in a variety of species. It has been proposed that alterations in membrane saturation are central to these actions of CR. As a step towards testing this theory, mice were assigned to 4 dietary groups (control and 3 CR groups) and fed AIN-93G diets at 95 % (control) or 60 % (CR) of ad libitum for 8 months. To manipulate membrane composition, the primary dietary fats for the CR groups were soybean oil (also used in the control diet), fish oil or lard. Skeletal muscle mitochondrial lipid composition, proton leak, and H(2)O(2) production were measured. Phospholipid fatty acid composition in CR mice was altered in a manner that reflected the n-3 and n-6 fatty acid profiles of their respective dietary lipid sources. Dietary lipid composition did not alter proton leak kinetics between the CR groups. However, the capacity of mitochondrial complex III to produce ROS was decreased in the CR lard compared to the other CR groups. The results of this study indicate that dietary lipid composition can influence ROS production in muscle mitochondria of CR mice. It remains to be determined if lard or other dietary oils can maximize the CR-induced decreases in ROS production.To investigate the relationship between RNA folding and ribozyme catalysis, we have carried out a detailed kinetic analysis of four structural derivatives of the hairpin ribozyme. Optimal and suboptimal (wild-type) substrate sequences were studied in conjunction with stabilization of helix 4, which supports formation of the catalytic core. Pre-steady-state and steady-state kinetic studies strongly support a model in which each of the ribozyme variants partitions between two major conformations leading to active and inactive ribozymez substrate complexes. Reaction rates for cleavage, ligation, and substrate binding to both ribozyme conformations were determined. Ligation rates (3 min 21 ) were typically 15-fold greater than cleavage rates (0.2 min 21 ), demonstrating that the hairpin ribozyme is an efficient RNA ligase. On the other hand, substrate binding is very rapid (k on 5 4 3 10 8 M 21 min 21 ), and the ribozymez substrate complex is very stable (K D < 25 pM ;k off < 0.01 min 21 ). Stabilization of helix 4 increases the proportion of RNA molecules folded into the active conformation, and enhances substrate association and ligation rates. These effects can be explained by stabilization of the catalytic core of the ribozyme. Rigorous consideration of conformational isomers and their intrinsic kinetic properties was necessary for development of a kinetic scheme for the ribozyme-catalyzed reaction.The human integrin VLA (very late activation antigens)-4 (CD49d/CD29), the leukocyte receptor for both the CS-1 region of plasma fibronectin (Fn) and the vascular cell surface adhesion molecule-1 (VCAM-1), also mediates homotypic aggregation upon triggering with specific anti-VLA-4 monoclonal antibody (mAb). Epitope mapping of this integrin on the human B-cell line Ramos, performed with a wide panel of anti-VLA-4 mAb by both cross-competitive cell binding and protease sensitivity assays, revealed the existence of three topographically distinct epitopes on the alpha 4 chain, referred to as epitopes A-C. By testing this panel of anti-VLA-4 mAb for inhibition of cell binding to both a 38-kDa Fn fragment containing CS-1 and to VCAM-1, as well as for induction and inhibition of VLA-4 mediated homotypic cell adhesion, we have found overlapping but different functional properties associated with each epitope. Anti-alpha 4 mAb recognizing epitope B inhibited cell attachment to both Fn and VCAM-1, whereas mAb against epitope A did not block VCAM-1 binding and only partially inhibited binding to Fn. In contrast, mAb directed to epitope C did not affect cell adhesion to either of the two VLA-4 ligands. All mAb directed to site A, as well as a subgroup of mAb recognizing epitope B (called B2), were able to induce cell aggregation, but this effect was not exerted by mAb specific to site C and by a subgroup against epitope B (called B1). Moreover, although anti-epitope C and anti-epitope B1 mAb did not trigger aggregation, those mAb blocked aggregation induced by anti-epitope A or B2 mAb. In addition, anti-epitope A mAb blocked B2-induced aggregation, and conversely, anti-epitope B2 mAb blocked A-induced aggregation. Further evidence for multiple VLA-4 functions is that anti-Fn and anti-VCAM-1 antibodies inhibited binding to Fn or to VCAM-1, respectively, but did not affect VLA-4-mediated aggregation. In summary, we have demonstrated that there are at least three different VLA-4-mediated adhesion functions, we have defined three distinct VLA-4 epitopes, and we have correlated these epitopes with the different functions of VLA-4.Phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid similarity. Among them, the two conserved regions characterized by the amino acid motifs D-NSLYP and K--NS(L/V)YG, regions 1 and 2a, respectively, according to Blanco et al. (Blanco, L., Bernad, A., Blasco, M. A. and Salas, M. (1991) Gene (Amst.) 100, 27-38) have been proposed to be part of the polymerization active site of alpha-like DNA polymerases. One phi 29 DNA polymerase mutant in residue Tyr254, located in conserved region 1, and two mutants in residue Tyr390, located in conserved region 2a, have been characterized. The three phi 29 DNA polymerase mutant proteins were affected in polymerization when Mg(2+)-dNTPs were used as substrate. However, when the substrate was Mn(2+)-dNTP, mutants behaved as the wild-type phi 29 DNA polymerase. Mutant Tyr254 to Phe (Y254F) was strongly affected in the protein-primed initiation step of phi 29 DNA replication showing a decreased affinity for Me(2+)-dATP, the initiating nucleotide. Furthermore, the analysis of the template-independent deoxynucleotidylation of the TP by Y254F mutant polymerase is consistent with a change in the relative affinity for dNTPs. On the other hand, mutants Y390F and Y390S were found to be hypersensitive to the dNTP analogs 2-(p-n-butylanilino)dATP and N2-(p-n-butyl-phenyl)dGTP. The results obtained indicate that residues Tyr254 and Tyr390 are involved, directly or indirectly, in Me(2+)-dNTP binding.Lazarillo, a protein recognized by the monoclonal antibody 10E6, is expressed by a subset of neurons in the developing nervous system of the grasshopper. It is a glycoprotein of 45x10(3) M(r) with internal disulfide bonds and linked to the extracellular side of the plasma membrane by a glycosylphosphatidylinositol moiety. Peptide sequences obtained from affinity purified adult protein were used to identify an embryonic cDNA clone, and in situ hybridizations confirmed that the distribution of the Lazarillo mRNA paralleled that of the monoclonal antibody labeling on embryos. Sequence analysis defines Lazarillo as a member of the lipocalin family, extracellular carriers of small hydrophobic ligands, and most related to the porphyrin- and retinol-binding lipocalins. Lazarillo is the first example of a lipocalin anchored to the plasma membrane, highly glycosylated, and restricted to a subset of developing neurons.Trabajo presentado al Annual Biomedical Research Conference for Minority Students celebrada en Nashville (US) del 13 al 16 de noviembre de 2013.In this paper, we show that the phi 29 DNA polymerase, in the absence of DNA, is able to catalyze the formation of a covalent complex between the phi 29 terminal protein (TP) and 5-dAMP. Like the reaction in the presence of phi 29 DNA, TP.dAMP complex formation is strongly dependent on activating Mn2+ ions and on the efficient formation of a TP/DNA polymerase heterodimer. The nature of the TP-dAMP linkage was shown to be identical (a O-5-deoxyadenylyl-L-serine bond) to that found covalently linking TP to the DNA of bacteriophage phi 29, indicating that this DNA-independent reaction actually mimics that occurring as the initiation step of phi 29 DNA replication. Furthermore, as in normal TP-primed initiation on the phi 29 DNA template, this novel reaction showed the same specificity for TP Ser232 as the OH donor and the involvement of the YCDTD amino acid motif, highly conserved in alpha-like DNA polymerases. However, unlike the reaction in the presence of phi 29 DNA, the DNA-independent deoxynucleotidylation of TP by the phi 29 DNA polymerase did not show dATP specificity, being possible to obtain any of the four TP.dNMP complexes with a similar yield. This lack of specificity together with the poor efficiency of this reaction at low deoxynucleoside triphosphate (dNTP) concentration reflect a weak, but similar stability of the four dNTPs at the phi 29 DNA polymerase dNTP-binding site. Thus, the presence of a director DNA would mainly contribute to stabilizing a complementary nucleotide, giving base specificity to the protein-primed initiation reaction. According to all these data, the novel DNA polymerase reaction described in this paper could be considered as a non-DNA-instructed protein-primed deoxynucleotidylation.A cDNA has been isolated from human hippocampus that appears to encode a novel Na(+)-dependent, Cl(-)-independent, neutral amino acid transporter. The putative protein, designated SATT, is 529 amino acids long and exhibits significant amino acid sequence identity (39-44%) with mammalian L-glutamate transporters. Expression of SATT cDNA in HeLa cells induced stereospecific uptake of L-serine, L-alanine, and L-threonine that was not inhibited by excess (3 mM) 2-(methylamino)-isobutyric acid, a specific substrate for the System A amino acid transporter. SATT expression in HeLa cells did not induce the transport of radiolabeled L-cysteine, L-glutamate, or related dicarboxylates. Northern blot hybridization revealed high levels of SATT mRNA in human skeletal muscle, pancreas, and brain, intermediate levels in heart, and low levels in liver, placenta, lung, and kidney. SATT transport characteristics are similar to the Na(+)-dependent neutral amino acid transport activity designated System ASC, but important differences are noted. These include: 1) SATTs apparent low expression in ASC-containing tissues such as liver or placenta; 2) the lack of mutual inhibition between serine and cysteine; and 3) the lack of trans-stimulation. SATT may represent one of multiple activities that exhibit System ASC-like transport characteristics in diverse tissues and cell lines.

Multimeric complexes formed by DNA-binding proteins of low sequence specificity

Some proteins bind to double-stranded DNA with low sequence specificity, forming regular multimeric complexes that extend over large regions of DNA, strongly distorting its conformation. Formation of these complexes at particular DNA sites usually depends on the structural ability of the DNA to follow the path imposed by the protein array. These complexes are found in both prokaryotic and eukaryotic organisms and participate in processes such as DNA replication, transcription and packaging.

Organic amendments and land management affect bacterial community composition, diversity and biomass in avocado crop soils

Background and aimsThe avocado-producing area of southern Spain includes conventional orchards and organic orchards that use different organic amendments. To gain insight into the effects of these amendments, physicochemical properties and microbial communities of the soil were analysed in a representative set of commercial and experimental orchards.MethodsThe population size of several groups of culturable microorganisms was determined by plating on different selective media. Bacterial community structure was studied by denaturing gradient gel electrophoresis (DGGE)ResultsCommercial composts showed the largest effects, especially the animal compost, enhancing the population sizes of some microbial groups and affecting bacterial community structure in superficial and deep soil layers. Moreover, animal and vegetal compost, manure and blood meal addition are related to high bacterial diversity in the superficial soil layer.ConclusionsAll of the organic amendments used in this study affect soil properties in one or more of the characteristics that were analysed. Culturable microbial population data revealed the most evident effects of some of the organic treatments. However, molecular analysis of soil bacterial communities by DGGE allowed the detection of the influence of all of the analysed amendments on bacterial community composition. This effect was stronger in the superficial layer of the avocado soil.

Activation of Replication Origins in ϕ29-related Phages Requires the Recognition of Initiation Proteins to Specific Nucleoprotein Complexes

Protein p6 of Bacillus subtilis phage φ29 activates the initiation of viral DNA replication by forming a multimeric nucleoprotein complex at the origins of replication, located at both ends of the linear genome. This activation requires a precise positioning of the protein p6 array with respect to the initiation site. To investigate this activation mechanism, we have purified the φ29 protein p6 counterparts from the related phages Nf and GA-1 and analyzed the formation of complexes with DNA. In the homologous protein p6-DNA complexes the φ29 and Nf protein arrays showed an identical positioning, different than that of the GA-1 protein array. In contrast, in the heterologous complexes the protein showed a different arrangement except in the case of the Nf protein-φ29 DNA complex. We have also purified the proteins involved in the initiation of replication (terminal protein and DNA polymerase) from phages Nf and GA-1 and measured the ability of the different p6 proteins to activate homologous and heterologous replication origins. The results obtained indicate that the activation requires not only the formation of a specific nucleoprotein complex but also its specific recognition by the proteins involved in the initiation of DNA replication.

Signals in the φ29 DNA-terminal protein template for the initiation of phage φ29 DNA replication

Abstract The protein-free terminal fragments Hind III B and L, from the left and right ends of φ29 DNA, respectively, but not internal fragments of similar size, were active as templates in the formation of the p3-dAMP initiation complex in an in vitro system containing purified φ29 terminal protein p3 and DNA polymerase p2, although the activity was lower than that obtained with the φ29 DNA-p3 complex. These results indicate the existence of specific sequences at the ends of φ29 DNA that allow the initiation of φ29 DNA replication. The template activity of the protein-free terminal fragments was size dependent. The protein-free single strands of the Hind III L fragment were much less active than the corresponding double-stranded fragment. Terminal protein-DNA complexes of phages PZA and φ15, with a terminal protein closely related to the φ29 protein p3, were more active as templates in the initiation reaction with the purified φ29 proteins than the corresponding protein-free DNAs, as it happens in the case of φ29 However, the terminal protein-DNA complexes of phages Nf, B103, and GA-1, with a terminal protein less related or unrelated to the φ29 protein p3, were essentially inactive and became active after removal of the parental terminal protein. These results strongly suggest that the parental terminal protein is the major signal in the template for the initiation of φ29 DNA replication.

Phage Ø29 protein p6: A viral histone-like protein

Abstract Phage O29 protein p6 is one of the most abundant viral proteins in O29-infected B subtilis cells, constituting about 4% of the total cellular proteins (about 3 × 106 copies/cell) at late infection. Electron microscopic studies showed that, in vitro, protein p6 forms heterogeneously-sized complexes all along O29 DNA, suggesting that protein p6 may have a role in genome packaging and organization. The low stability of the protein p6-O29 DNA complexes observed in vitro could reflect the dynamic nature of these complexes, to allow replication, transcription, and encapsidation of the genome. The protein p6-DNA complex consists of a DNA right-handed superhelix wrapped around a multimeric protein core. The DNA in this complex is strongly distorted and compacted. Protein p6 recognition signals have been mapped near the ends of the linear O29 DNA and act as nucleation sites for complex formation. Protein p6 does not recognize a specific sequence, but sequences with specific bendable properties that would favor the formation of the complex. Protein p6 represses transcription from the O29 C2 early promoter, and activates initiation of O29 DNA replication that occurs from both DNA ends. The formation of nucleoprotein complexes at the origins of replication, as well as the specific positioning of protein p6 with respect to the DNA ends are required for the activation of replication. This suggests that the proteins involved in the initiation step of O29 DNA replication, either directly interact with protein p6, or recognize a conformational change at a specific location in the DNA. The mechanism of activation could be the local and transient unpairing of DNA at specific sites, facilitated by the strong distortion of DNA conformation in the nucleoprotein complex.

Formation of a covalent complex between the terminal protein of pneumococcal bacteriophage Cp-1 and 5'-dAMP.

This work was supported by Community of Madrid (Grupo Estrategico 2000-2003), NIH, grant R01CA77575, and SAF 2001-2245.The transition step from the p3-dAMP initiation complex to the first elongated products, p3-(dAMP)2 and p3-(dAMP)3, requires a dATP concentration higher than that needed for the initiation reaction or for the further elongation of the p3-(dAMP)3 complex. The elongation in phi 29 DNA-protein p3 replication in vitro was strongly inhibited by salt. Under inhibitory salt concentration, the viral protein p6 greatly stimulated phi 29 DNA-protein p3 replication. The effect of protein p6 was not on the rate of elongation but on the amount of elongated product, stimulating the transition from initiation to formation of the first elongation products.Trabajo presentado en 44th Annual Meeting Society for Neuroscience, celebrado en Washington, DC (USA) del 15 al 19 de noviembre de 2014Recent studies have demonstrated that cytochrome c plays an important role in cell death. In the present study, we report that teniposide and various other chemotherapeutic agents induced a dose-dependent increase in the expression of the mitochondrial respiratory chain proteins cytochrome c, subunits I and IV of cytochrome c oxidase, and the free radical scavenging enzyme manganous superoxide dismutase. The teniposide-induced increase of cytochrome c was inhibited by cycloheximide, indicating new protein synthesis. Elevated cytochrome c levels were associated with enhanced cytochrome c oxidase-dependent oxygen uptake using TMPD/ascorbate as the electron donor, suggesting that the newly synthesized proteins were functional. Cytochrome c was released into the cytoplasm only after maximal levels had been reached in the mitochondria, but there was no concomitant decrease in mitochondrial membrane potential or caspase activation. Our results suggest that the increase in mitochondrial protein expression may play a role in the early cellular defense against anticancer drugs.The nucleotide sequence of the 5 end distal region of the human c-K-ras gene promoter was determined. This region, coincident with a variable DNAse I hypersensitive site in native chromatin, contains sequence similarities with known enhancers. A 400 bp MstII DNA fragment of this region stimulated in cis the correctly initiated transcription of the human beta-globin gene in transfected Hela cells. The stimulation of beta-globin transcription (5-6 fold) was dependent on the distance and orientation of the c-K-ras sequences and on the presence of the CCAAT and CACCC elements in the beta-globin promoter. Interaction of nuclear factors with these c-K-ras sequences was analysed by DNAase I footprinting assays using Hela nuclear extracts. A protein binding to these sequences was identified as nuclear factor 1 (NF-1) by DNAase I competition footprinting experiments. However, disruption of the c-K-ras NF-1 binding site by insertion mutagenesis had no effect on the transcriptional activity of the c-K-ras element.Supported by Grant GM-08041 from the National Institutes nof Health, United States Public Health Service.The results presented in this paper indicate that the phi 29 DNA polymerase is the only enzyme required for efficient synthesis of full length phi 29 DNA with the phi 29 terminal protein, the initiation primer, as the only additional protein requirement. Analysis of phi 29 DNA polymerase activity in various in vitro DNA replication systems indicates that two main reasons are responsible for the efficiency of this minimal system: 1) the phi 29 DNA polymerase is highly processive in the absence of any accessory protein; 2) the polymerase itself is able to produce strand displacement coupled to the polymerization process. Using primed M13 DNA as template, the phi 29 DNA polymerase is able to synthesize DNA chains greater than 70 kilobase pairs. Furthermore, conditions that increase the stability of secondary structure in the template do not affect the processivity and strand displacement ability of the enzyme. Thus, the catalytic properties of the phi 29 DNA polymerase are appropriate for a phi 29 DNA replication mechanism involving two replication origins, strand displacement and continuous synthesis of both strands. The enzymology of phi 29 DNA replication would support a symmetrical model of DNA replication.Aided by grants from the National Institutes of Health U.S. Public Health Service, and E. I. Du Pont de Neumours and Company, Inc.This work was supported in part by NRSA, National Institutes of Health Grants NS09463 and NS32501 and from National Science Foundation Grant 9310965.We have recently developed a new method to detect and characterize single base substitutions in transcribed genes which is based on the ability of RNAse A to recognize and cleave single base mismatches in RNA:RNA heteroduplexes. The RNAse A misrnatch cleavage assay was applied to screen human colon carcinoma cell lines and primary tumors for the presence of mutant e-X-ras oncogenes. We have determined that the mutant e-X-ras allele is overexpressed and amplified relative to the normal in the SX-CO-l human colon carcinoma cell lineo The oncogene mutation has been characterized by this method as a glycine to valine substitution at codon 12 of the e-X-ras gene. This result was confirmed by cloning and sequencing. We have previously reported that about 40% of primary human colon tumors contain e-X-ras genes mutant at codon 12 (Forrester et al, Nature 327: 298, 1987). We report here the characterization by molecular cloning and sequencing of the mutation in the e-X-ras oneogene from two of these tumors (tumors 3 and 28). We also describe the histopathologieal eharaeterization of these two tumors and demonstrate, by Southern blot hybridization of NIH3T3 transformants, the simultaneous presenee of mutant e-X-ras and N-ras oncogenes in villous adenoma 28. Our results provide evidence for the frequent assoeiation of ras somatie mutational aetivation in the early stages of tumor development in this common type of human eaneer.The residues forming the 3-->5 exonuclease active site of phi 29 DNA polymerase, located at the N-terminal conserved motifs Exo I, Exo II and Exo III, have been defined by site-directed mutagenesis (Bernad, A., Blanco, L., Lázaro, J. M., Martin, G., and Salas, M. (1989) Cell 59, 219-228; Soengas, M. S., Esteban, J. A., Lázaro, J. M., Bernad, A., Blasco, M. A., Salas, M., and Blanco, L. (1992) EMBO J. 11, 4227-4237). To understand their catalytic role, the residual exonuclease activity of mutants at these active site residues has been kinetically studied. The critical function of residues Asp12, Glu14, Asp66, and Asp169 is supported by a 10(5)-fold reduction in the exonuclease catalytic rate upon single mutation. Residue Tyr165 seems to play a secondary role in the exonuclease reaction based on the 10(2)-10(3)-fold reduced catalytic rate of mutants Y165F and Y165C. Most of the mutants were specially active in the presence of Mn2+ ions, which could be indicative of a direct involvement of these residues in a metal ion-assisted exonucleolytic reaction. The data obtained strongly suggest that the 3-->5 exonuclease active site of phi 29 DNA polymerase is structurally and functionally similar to that of the Escherichia coli DNA polymerase I. In addition, these residues were also very important for the strand displacement ability of phi 29 DNA polymerase, suggesting a structural overlapping of this activity with the 3-->5 exonuclease.phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid similarity. Among them, the conserved region characterized by the amino acid motif Kx3NSxYG has been proposed to form part of the polymerization active site of alpha-like DNA polymerases. Mutants in phi 29 DNA polymerase residue Tyr390 of this conserved motif had been previously described to be affected in DNA-dependent dNTP binding. In this paper, the functional significance of this conserved motif is further studied by the analysis of mutants in conserved residues Asn387, Ser388, and Gly391. Residue Phe393 of phi 29 DNA polymerase has also been selected as target for site-directed mutagenesis because of its conservation within the group of alpha-like DNA polymerases from genomes that replicate by a protein-priming mechanism. Mutant N387Y was shown to be affected both in initiation and polymerization reactions, showing 3-fold higher Km value for dATP and more than 11-fold lower Vmax value than the wild-type enzyme in the initiation reaction; moreover, it was affected in enzyme-DNA translocation. Mutant S388G retained initiation and polymerization activities; interestingly, this mutation significantly increased the efficiency of dNTP incorporation in non-templated reactions. Mutation Gly391 to Asp abolished template-primer binding as shown by gel retardation assays; this mutant was drastically affected in template-dependent dNTP incorporation both in initiation and polymerization reactions, but the efficiency of the non-templated phi 29 terminal protein-deoxynucleotidylation was higher than with the wild-type protein. Mutation Phe393 to Tyr severely decreased initial binding to template-primer DNA molecules, resulting in a reduced activity in DNA primer-dependent polymerization reactions but not in phi 29 terminal protein-dependent ones.Aided by Grants AM-01845, AM-08953, and l-Sol-FR-05099 nfrom the National Institutes o f Health, United States Public nHealth Service, and E. I. Du Pont de Nemours and Company, nInc. A preliminary report o f this work was presented at the nSecond Meeting o f the Federation o f European Biochemical nSocieties (symposium on “Ribonucleic Acid-Structure and nFunction”), Vienna, April 21 to 24, 1965.1 pagina.-- Trabajo presentado al: 4th International Meeting on Apicomplexa in Farm Animals. (Madrid, Spain. 11-14 October ,2017).The hairpin ribozyme catalyzes site-specific cleavage of an RNA substrate using a magnesium-dependent transphosphorylation mechanism. Here, we describe experiments designed to test the importance of ribose 2-hydroxyl groups for ribozyme function. Ribozymes for this work were synthesized in two segments using solid-phase RNA phosphoramidite chemistry. 2-Deoxyribonucleotides were systematically introduced at each of the 50 positions within the ribozyme, and the catalytic activity of the resulting mixed RNA-DNA polymers was measured. Deletion of the 2-hydroxyl group at each of four sites (A10, G11, A24, and C25) was found to result in severe inhibition of cleavage activity (kcat/KM decreased by 100- to 1000-fold), although KM measurements and mobility-shift assays showed that substrate binding was not affected. Identical results were obtained upon substitution of these ribonucleotides with 2-O-methyl derivatives. Inhibition by 2-modified sugars at G11 or A24 was rescued by increased Mg2+ concentrations, suggesting that these 2-hydroxyls may function in magnesium binding. Our results demonstrate that the 2-hydroxyl groups at A10, G11, A24, and C25 provide essential functions for catalysis, possibly forming important tertiary contacts or magnesium coordination sites that are necessary for active site architecture.Supported by Grant GM-08041 from the National Institutes of nHealth, United States Public Health Service.Resumen del trabajo presentado al XXXIII Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular celebrado en Cordoba del 14 al 17 de septiembre de 2010.This article describes the expression pattern and functional analysis of Lazarillo, a novel cell surface glycoprotein expressed in the embryonic grasshopper nervous system, and a member of the lipocalin family. Lazarillo is expressed by a subset of neuroblasts, ganglion mother cells and neurons of the central nervous system, by all sensory neurons of the peripheral nervous system, and by a subset of neurons of the enteric nervous system. It is also present in a few non neuronal cells associated mainly with the excretory system. A monoclonal antibody raised against Lazarillo perturbs the extent and direction of growth of identified commissural pioneer neurons. We propose that Lazarillo is the receptor for a midline morphogen involved in the outgrowth and guidance of these neurons.phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid sequence similarity and sensitivity to inhibitors of eukaryotic DNA polymerase alpha. In this paper, site-directed mutants in the phi 29 DNA polymerase residues Asp249, Ser252, Leu253, and Pro255 of the conserved amino acid motif Dx2SLYP are described. Two mutants, D249E and S252R, were drastically affected in all the synthetic activities, whereas their 3 to 5 exonuclease activity and interaction with the TP primer was normal. Mutant D249E, slightly affected in template-primer binding, was completely inactive in all conditions tested, suggesting that Asp249 could be playing a direct role in catalysis. On the other hand, mutant S252R, strongly affected in template-primer binding, showed some DNA polymerization activity in the presence of Mn2+. Mutants S252G and P255S showed a reduced template-primer binding ability; these mutants, together with mutant L253V, showed metal ion-dependent phenotypes in their synthetic activities and altered sensitivities to the PPi analog phosphonoacetic acid. All these results support the hypothesis that the Dx2SLYP motif forms part of the polymerization active site of the phi 29 DNA polymerase, being the Asp249 residue critical both for protein-primed initiation and DNA polymerization.Poster presentado al Annual Biomedical Research Conference for Minority Students celebrado en California (US) del 7 al 10 de noviembre de 2012.The phage phi 29 regulatory protein p4 activates the late promoter A3 by stabilizing the binding of Bacillus subtilis RNA polymerase (RNAP) as a closed complex. Interaction between the two proteins occurs through amino acid Arg120 in protein p4 and the C-terminal domain of the RNAP alpha subunit (alpha-CTD). In addition to its role as activator of the late transcription, protein p4 represses early transcription from the A2b and A2c promoters, that are divergently transcribed. Binding of p4 to its recognition site at the A3 promoter displaces the RNAP from promoter A2b, both by steric hindrance and by the curvature induced upon p4 binding. At the A2c promoter, the RNAP cooperates with p4 binding in such a way that promoter clearance is prevented. Interestingly, amino acid Arg120 in p4 and the alpha-CTD in B. subtilis RNAP are involved in the interactions that lead to transcription repression at promoter A2c. To investigate how this interaction leads to activation at PA3 and to repression at PA2c, mutant promoters were constructed. In the absence of a -35 consensus box for sigma A-RNAP activation was observed, while in its presence repression occurred. The results support the idea that overstabilization of RNAP at the promoter over a threshold level leads to repression.Resumen del poster presentado al XXXIII Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular celebrado en Cordoba del 14 al 17 de septiembre de 2010.Phi 29 DNA polymerase is able to catalyze two different synthetic reactions: protein-primed initiation and DNA polymerization. We have studied the fidelity of phi 29 DNA polymerase when carrying out these two reactions. Global fidelity was dissected into three steps: insertion discrimination, mismatch elongation, and proofreading. The insertion discrimination of phi 29 DNA polymerase in DNA polymerization ranged from 10(4) to 10(6). The efficiency of mismatch elongation was 10(5)-10(-6)-fold lower than that of a properly paired primer terminus. These factors indicate that DNA polymerization catalyzed by phi 29 DNA polymerase is a highly accurate process. Conversely, the insertion fidelity of protein-primed initiation was quite low, the insertion discrimination factor being about 10(2). Mismatch elongation discrimination was also rather low: mismatched terminal protein (TP).dNMP complexes were elongated from 2- to 6-fold more slowly than the correct TP.dNMP complex. Even more, the 3-->5 exonuclease activity of phi 29 DNA polymerase was unable to act on the TP.dNMP initiation complex, precluding the possibility that a wrong dNMP covalently linked to TP could be excised and corrected. Therefore, protein-primed initiation can be predicted as a quite inaccurate reaction. The problem of maintaining the sequence at the DNA ends is discussed in the context of a recently described model for protein-primed initiation.Formalin-fixed paraffin-embedded tissue specimens obtained by fine needle aspiration of pancreatic masses from 47 patients were examined retrospectively for cytology and the presence of mutant c-K-ras oncogenes. Point mutations of c-K-ras in codon 12 were detected by RNA-DNA RNAse A mismatch cleavage after in vitro DNA amplification of the cellular c-K-ras sequences by the polymerase chain reaction. Of the 36 patients with pancreatic adenocarcinoma, mutant c-K-ras oncogenes were detected in 18 of 25 (72%) with malignant cytologies, 2 of 8 (25%) with atypical cytologies, and 0 of 3 with benign aspiration cytologies. The remaining 11 patients without pancreatic adenocarcinomas did not have mutant c-K-ras genes detectable by the assay. The diagnosis of pancreatic adenocarcinoma was based upon clinical follow-up. The presence of mutant c-K-ras oncogenes did not significantly affect survival in the patients studied. Mutant c-K-ras genes were found at the time of initial clinical presentation in the majority of pancreatic adenocarcinomas, suggesting an important role of the mutation in oncogenesis. In conjunction with cytology, our approach represents an application for cancer diagnosis at the molecular genetic level.Calorie restriction (CR) has been shown to decrease reactive oxygen species (ROS) production and retard aging in a variety of species. It has been proposed that alterations in membrane saturation are central to these actions of CR. As a step towards testing this theory, mice were assigned to 4 dietary groups (control and 3 CR groups) and fed AIN-93G diets at 95 % (control) or 60 % (CR) of ad libitum for 8 months. To manipulate membrane composition, the primary dietary fats for the CR groups were soybean oil (also used in the control diet), fish oil or lard. Skeletal muscle mitochondrial lipid composition, proton leak, and H(2)O(2) production were measured. Phospholipid fatty acid composition in CR mice was altered in a manner that reflected the n-3 and n-6 fatty acid profiles of their respective dietary lipid sources. Dietary lipid composition did not alter proton leak kinetics between the CR groups. However, the capacity of mitochondrial complex III to produce ROS was decreased in the CR lard compared to the other CR groups. The results of this study indicate that dietary lipid composition can influence ROS production in muscle mitochondria of CR mice. It remains to be determined if lard or other dietary oils can maximize the CR-induced decreases in ROS production.To investigate the relationship between RNA folding and ribozyme catalysis, we have carried out a detailed kinetic analysis of four structural derivatives of the hairpin ribozyme. Optimal and suboptimal (wild-type) substrate sequences were studied in conjunction with stabilization of helix 4, which supports formation of the catalytic core. Pre-steady-state and steady-state kinetic studies strongly support a model in which each of the ribozyme variants partitions between two major conformations leading to active and inactive ribozymez substrate complexes. Reaction rates for cleavage, ligation, and substrate binding to both ribozyme conformations were determined. Ligation rates (3 min 21 ) were typically 15-fold greater than cleavage rates (0.2 min 21 ), demonstrating that the hairpin ribozyme is an efficient RNA ligase. On the other hand, substrate binding is very rapid (k on 5 4 3 10 8 M 21 min 21 ), and the ribozymez substrate complex is very stable (K D < 25 pM ;k off < 0.01 min 21 ). Stabilization of helix 4 increases the proportion of RNA molecules folded into the active conformation, and enhances substrate association and ligation rates. These effects can be explained by stabilization of the catalytic core of the ribozyme. Rigorous consideration of conformational isomers and their intrinsic kinetic properties was necessary for development of a kinetic scheme for the ribozyme-catalyzed reaction.The human integrin VLA (very late activation antigens)-4 (CD49d/CD29), the leukocyte receptor for both the CS-1 region of plasma fibronectin (Fn) and the vascular cell surface adhesion molecule-1 (VCAM-1), also mediates homotypic aggregation upon triggering with specific anti-VLA-4 monoclonal antibody (mAb). Epitope mapping of this integrin on the human B-cell line Ramos, performed with a wide panel of anti-VLA-4 mAb by both cross-competitive cell binding and protease sensitivity assays, revealed the existence of three topographically distinct epitopes on the alpha 4 chain, referred to as epitopes A-C. By testing this panel of anti-VLA-4 mAb for inhibition of cell binding to both a 38-kDa Fn fragment containing CS-1 and to VCAM-1, as well as for induction and inhibition of VLA-4 mediated homotypic cell adhesion, we have found overlapping but different functional properties associated with each epitope. Anti-alpha 4 mAb recognizing epitope B inhibited cell attachment to both Fn and VCAM-1, whereas mAb against epitope A did not block VCAM-1 binding and only partially inhibited binding to Fn. In contrast, mAb directed to epitope C did not affect cell adhesion to either of the two VLA-4 ligands. All mAb directed to site A, as well as a subgroup of mAb recognizing epitope B (called B2), were able to induce cell aggregation, but this effect was not exerted by mAb specific to site C and by a subgroup against epitope B (called B1). Moreover, although anti-epitope C and anti-epitope B1 mAb did not trigger aggregation, those mAb blocked aggregation induced by anti-epitope A or B2 mAb. In addition, anti-epitope A mAb blocked B2-induced aggregation, and conversely, anti-epitope B2 mAb blocked A-induced aggregation. Further evidence for multiple VLA-4 functions is that anti-Fn and anti-VCAM-1 antibodies inhibited binding to Fn or to VCAM-1, respectively, but did not affect VLA-4-mediated aggregation. In summary, we have demonstrated that there are at least three different VLA-4-mediated adhesion functions, we have defined three distinct VLA-4 epitopes, and we have correlated these epitopes with the different functions of VLA-4.Phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid similarity. Among them, the two conserved regions characterized by the amino acid motifs D-NSLYP and K--NS(L/V)YG, regions 1 and 2a, respectively, according to Blanco et al. (Blanco, L., Bernad, A., Blasco, M. A. and Salas, M. (1991) Gene (Amst.) 100, 27-38) have been proposed to be part of the polymerization active site of alpha-like DNA polymerases. One phi 29 DNA polymerase mutant in residue Tyr254, located in conserved region 1, and two mutants in residue Tyr390, located in conserved region 2a, have been characterized. The three phi 29 DNA polymerase mutant proteins were affected in polymerization when Mg(2+)-dNTPs were used as substrate. However, when the substrate was Mn(2+)-dNTP, mutants behaved as the wild-type phi 29 DNA polymerase. Mutant Tyr254 to Phe (Y254F) was strongly affected in the protein-primed initiation step of phi 29 DNA replication showing a decreased affinity for Me(2+)-dATP, the initiating nucleotide. Furthermore, the analysis of the template-independent deoxynucleotidylation of the TP by Y254F mutant polymerase is consistent with a change in the relative affinity for dNTPs. On the other hand, mutants Y390F and Y390S were found to be hypersensitive to the dNTP analogs 2-(p-n-butylanilino)dATP and N2-(p-n-butyl-phenyl)dGTP. The results obtained indicate that residues Tyr254 and Tyr390 are involved, directly or indirectly, in Me(2+)-dNTP binding.Lazarillo, a protein recognized by the monoclonal antibody 10E6, is expressed by a subset of neurons in the developing nervous system of the grasshopper. It is a glycoprotein of 45x10(3) M(r) with internal disulfide bonds and linked to the extracellular side of the plasma membrane by a glycosylphosphatidylinositol moiety. Peptide sequences obtained from affinity purified adult protein were used to identify an embryonic cDNA clone, and in situ hybridizations confirmed that the distribution of the Lazarillo mRNA paralleled that of the monoclonal antibody labeling on embryos. Sequence analysis defines Lazarillo as a member of the lipocalin family, extracellular carriers of small hydrophobic ligands, and most related to the porphyrin- and retinol-binding lipocalins. Lazarillo is the first example of a lipocalin anchored to the plasma membrane, highly glycosylated, and restricted to a subset of developing neurons.Trabajo presentado al Annual Biomedical Research Conference for Minority Students celebrada en Nashville (US) del 13 al 16 de noviembre de 2013.In this paper, we show that the phi 29 DNA polymerase, in the absence of DNA, is able to catalyze the formation of a covalent complex between the phi 29 terminal protein (TP) and 5-dAMP. Like the reaction in the presence of phi 29 DNA, TP.dAMP complex formation is strongly dependent on activating Mn2+ ions and on the efficient formation of a TP/DNA polymerase heterodimer. The nature of the TP-dAMP linkage was shown to be identical (a O-5-deoxyadenylyl-L-serine bond) to that found covalently linking TP to the DNA of bacteriophage phi 29, indicating that this DNA-independent reaction actually mimics that occurring as the initiation step of phi 29 DNA replication. Furthermore, as in normal TP-primed initiation on the phi 29 DNA template, this novel reaction showed the same specificity for TP Ser232 as the OH donor and the involvement of the YCDTD amino acid motif, highly conserved in alpha-like DNA polymerases. However, unlike the reaction in the presence of phi 29 DNA, the DNA-independent deoxynucleotidylation of TP by the phi 29 DNA polymerase did not show dATP specificity, being possible to obtain any of the four TP.dNMP complexes with a similar yield. This lack of specificity together with the poor efficiency of this reaction at low deoxynucleoside triphosphate (dNTP) concentration reflect a weak, but similar stability of the four dNTPs at the phi 29 DNA polymerase dNTP-binding site. Thus, the presence of a director DNA would mainly contribute to stabilizing a complementary nucleotide, giving base specificity to the protein-primed initiation reaction. According to all these data, the novel DNA polymerase reaction described in this paper could be considered as a non-DNA-instructed protein-primed deoxynucleotidylation.A cDNA has been isolated from human hippocampus that appears to encode a novel Na(+)-dependent, Cl(-)-independent, neutral amino acid transporter. The putative protein, designated SATT, is 529 amino acids long and exhibits significant amino acid sequence identity (39-44%) with mammalian L-glutamate transporters. Expression of SATT cDNA in HeLa cells induced stereospecific uptake of L-serine, L-alanine, and L-threonine that was not inhibited by excess (3 mM) 2-(methylamino)-isobutyric acid, a specific substrate for the System A amino acid transporter. SATT expression in HeLa cells did not induce the transport of radiolabeled L-cysteine, L-glutamate, or related dicarboxylates. Northern blot hybridization revealed high levels of SATT mRNA in human skeletal muscle, pancreas, and brain, intermediate levels in heart, and low levels in liver, placenta, lung, and kidney. SATT transport characteristics are similar to the Na(+)-dependent neutral amino acid transport activity designated System ASC, but important differences are noted. These include: 1) SATTs apparent low expression in ASC-containing tissues such as liver or placenta; 2) the lack of mutual inhibition between serine and cysteine; and 3) the lack of trans-stimulation. SATT may represent one of multiple activities that exhibit System ASC-like transport characteristics in diverse tissues and cell lines.