María L. Salas

Terminal protein-primed DNA amplification

By using appropriate amounts of four bacteriophage phi 29 DNA replication proteins--terminal protein, DNA polymerase, protein p6 (double-stranded DNA-binding protein), and protein p5 (single-stranded DNA-binding protein)--it has been possible to amplify limited amounts of the 19,285-bp-long phi 29 DNA molecule by three orders of magnitude after 1 hr of incubation at 30 degrees C. Moreover, the quality of the amplified material was demonstrated by transfection experiments, in which infectivity of the synthetic (amplified) phi 29 DNA, measured as the ability to produce phage particles, was identical to that of the natural phi 29 DNA obtained from virions. The results presented in this paper establish some of the requisites for the development of isothermal DNA amplification strategies based on the bacteriophage phi 29 DNA replication machinery that are suitable for the amplification of very large (> 70 kb) segments of DNA.

Citrate inhibition of phosphofructokinase and the Pasteur effect

This investigation was supported by United States Public Health Service Research Grant GM-08041.

Characterization of an African Swine Fever Virus 20-kDa DNA Polymerase Involved in DNA Repair

African swine fever virus (ASFV) encodes a novel DNA polymerase, constituted of only 174 amino acids, belonging to the polymerase (pol) X family of DNA polymerases. Biochemical analyses of the purified enzyme indicate that ASFV pol X is a monomeric DNA-directed DNA polymerase, highly distributive, lacking a proofreading 3′-5′-exonuclease, and with a poor discrimination against dideoxynucleotides. A multiple alignment of family X DNA polymerases, together with the extrapolation to the crystal structure of mammalian DNA polymerase β (pol β), showed the conservation in ASFV pol X of the most critical residues involved in DNA binding, nucleotide binding, and catalysis of the polymerization reaction. Therefore, the 20-kDa ASFV pol X most likely represents the minimal functional version of an evolutionarily conserved pol β-type DNA polymerase core, constituted by only the “palm” and “thumb” subdomains. It is worth noting that such an “unfingered” DNA polymerase is able to handle templated DNA polymerization with a considerable high fidelity at the base discrimination level. Base excision repair is considered to be a cellular defense mechanism repairing modified bases in DNA. Interestingly, the fact that ASFV pol X is able to conduct filling of a single nucleotide gap points to a putative role in base excision repair during the ASFV life cycle.

African Swine Fever Virus IAP Homologue Inhibits Caspase Activation and Promotes Cell Survival in Mammalian Cells

ABSTRACT African swine fever virus (ASFV) A224L is a member of the inhibitor of apoptosis protein (IAP) family. We have investigated the antiapoptotic function of the viral IAP both in stably transfected cells and in ASFV-infected cells. A224L was able to substantially inhibit caspase activity and cell death induced by treatment with tumor necrosis factor alpha and cycloheximide or staurosporine when overexpressed in Vero cells by gene transfection. We have also observed that ASFV infection induces caspase activation and apoptosis in Vero cells. Furthermore, using a deletion mutant of ASFV lacking the A224L gene, we have shown that the viral IAP modulates the proteolytic processing of the effector cell death protease caspase-3 and the apoptosis which are induced in the infected cells. Our findings indicate that A224L interacts with the proteolytic fragment of caspase-3 and inhibits the activity of this protease during ASFV infection. These observations could indicate a conserved mechanism of action for ASFV IAP and other IAP family members to suppress apoptosis.

Role of the host cell nucleus in the replication of African swine fever virus DNA.

An examination by autoradiography of African swine fever virus-infected alveolar macrophages pulse labeled with [3H]thymidine showed that, at early times of viral DNA replication, the grains were localized exclusively in the nucleus in 20% of the cells, while in 45% the label was found in the cytoplasm. In the remaining 35%, newly synthesized DNA was detected in both the nucleus and the cytoplasm. At later times, the percentage of cells with grains in the nucleus decreased considerably. Pulse-chase experiments indicated that the DNA synthesized in the nucleus is then transported to the cytoplasm. The presence of virus-specific DNA sequences in the nucleus was confirmed by in situ hybridization of infected macrophages. Similar hybridization experiments with African swine fever virus-infected VERO cells followed by confocal microscopy also indicated the existence of a nuclear stage in the localization of the viral DNA. These results suggest a mechanism for African swine fever virus DNA replication with an initial stage in the nucleus followed by a cytoplasmic phase. Specific nuclear forms associated with the hybridization signal have been observed in African swine fever virus-infected macrophages and VERO cells. The nuclear forms seen in macrophages are consistent with a mechanism for the egress of the viral DNA from the nucleus that involves initial budding at the nuclear membrane.

African Swine Fever Virus Protease, a New Viral Member of the SUMO-1-specific Protease Family

African swine fever virus (ASFV) is a complex DNA virus that employs polyprotein processing at Gly-Gly-Xaa sites as a strategy to produce several major core components of the viral particle. The virus gene S273R encodes a 31-kDa protein that contains a “core domain” with the conserved catalytic residues characteristic of SUMO-1-specific proteases and the adenovirus protease. Using a COS cell expression system, it was found that protein pS273R is capable of cleaving the viral polyproteins pp62 and pp220 in a specific way giving rise to the same intermediates and mature products as those produced in ASFV-infected cells. Furthermore, protein pS273R, like adenovirus protease and SUMO-1-specific enzymes, is a cysteine protease, because its activity is abolished by mutation of the predicted catalytic histidine and cysteine residues and is inhibited by sulfhydryl-blocking reagents. Protein pS273R is expressed late after infection and is localized in the cytoplasmic viral factories, where it is found associated with virus precursors and mature virions. In the virions, the protein is present in the core shell, a domain where the products of the viral polyproteins are also located. The identification of the ASFV protease will allow a better understanding of the role of polyprotein processing in virus assembly and may contribute to our knowledge of the emerging family of SUMO-1-specific proteases.

The bacteriophage phi 29 DNA polymerase, a proofreading enzyme.

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.Supported by Grant GM-08041 from the National Institutes of 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.Aided by Grants AM-01845, AM-08953, and l-Sol-FR-05099 from the National Institutes o f Health, United States Public Health Service, and E. I. Du Pont de Nemours and Company, Inc. A preliminary report o f this work was presented at the Second Meeting o f the Federation o f European Biochemical Societies (symposium on “Ribonucleic Acid-Structure and Function”), 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).Supported by Grant GM-08041 from the National Institutes of Health, 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.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.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.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.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.

Two interconvertible forms of yeast phosphofructokinase with different sensitivity to endproduct inhibition

This investigation was supported by United States Public Health Service Research Grant GM-08041.

DNA-dependent RNA polymerase in African swine fever virus

Abstract African swine fever (ASF) virus has a DNA-dependent RNA polymerase activity which does not require the addition of exogenous DNA. The polymerase activity copurifies with ASF virus and reaches a maximum specific activity value of 5–15 pmol UMP/μg of virus protein, when the virus particles are pretreated with NP-40. In vitro RNA synthesis by ASF virus requires higher concentrations of ATP than of GTP or CTP. The RNA product has a sedimentation rate of 6–14 S and anneals specifically with virus DNA.

Polyadenylation, methylation, and capping of the RNA synthesized in vitro by African swine fever virus

Abstract African swine fever (ASF) virus synthesizes in vitro four classes of capped and polyadenylated RNAs with sedimentation coefficients of about 16,12, 10, and 7 S, free poly(A) with a sedimentation coefficient of about 4 S and a material, smaller than 4 S, labeled with S -adenosyl[ methyl - 3 H]methionine and partially polyadenylated. The average chain length of free and RNA-bound poly(A) is about 14 and 33 nucleotides, respectively. The following CAP structures were present in the RNA: m 7 GpppA m (79%), m 7 GpppG m (13%), GpppA m (5.8%), and GpppG m (2.3%). Neither CAP 2 structure nor internal methylations were detected in the RNA.