Protein-DNA cross-linking demonstrates stepwise ATP-dependent assembly of T4 DNA polymerase and its accessory proteins on the primer-template.

T4 DNA polymerase, the 44/62 and 45 polymerase accessory proteins, and 32 single-stranded DNA-binding protein catalyze ATP-dependent DNA synthesis. Using DNA primers with cross-linkable residues at specific positions, we obtained structural data that reveal how these proteins assemble on the primer-template. With the nonhydrolyzable ATP analog ATP gamma S, assembly of the 44/62 and 45 proteins on the primer requires 32 protein but not polymerase. ATP hydrolysis changes the position and intensity of cross-linking to each of the accessory proteins and allows cross-linking of polymerase. Our data indicate that the initial binding of the three accessory proteins and ATP to a 32 protein-covered primer-template is followed by ATP hydrolysis, binding of polymerase, and movement of the accessory proteins to yield a complex capable of processive DNA synthesis.     

Hydrosoluble formazan XTT: its application to natural products drug discovery for Leishmania

A colorimetric method for measuring the viability of Leishmania promastigotes is described that is based on the reduction of the tetrazolium salt, XTT, to a water-soluble formazan. Values obtained by the XTT method correlated well with parasite number (r=0.965) and with methods that rely upon the reduction of MTT or MTS (r=0.96 and 0.97, respectively). The IC(50) values obtained by XTT method with amphotericin-B, miltefosine and ketoconazole were similar to those previously reported by other methods. The XTT method proved to be a reliable and convenient method for the screening of methanolic extracts from 1059 plants and was used for the bioassay-guided fractionation of the alkaloid aegeline from Sarcorhachis naranjoana.     

Linking bioprospecting with sustainable development and conservation: the Panama case

The limited international resources for economic aid and conservation can only mitigate poverty and losses of biodiversity. Hence, developing nations must establish the capacity to resolve their problems. Additionally, policy-makers and donors need to obtain scientific input on issues such as global change and ecosystem services. We propose that for nations rich in biodiversity, ecosystem services derived from bioprospecting, or drug discovery, could contribute to economic development. In the case where unstudied samples are shipped abroad for research, the chances of obtaining royalties are infinitesimally small. Therefore developing nations will only realize benefits from bioprospecting through in-country research on their own biodiversity. Policy-makers and donors have failed to appreciate the value of this approach. In order to provide an example of the inherent links between conservation and sustainable economic development, we initiated a drug discovery effort in Panama that emphasizes local benefit. As much of the drug discovery process as possible is conducted in Panamanian laboratories, providing jobs dependent on intact biodiversity and enhancing local research and training. In short, research, plus the spin-offs from research, provide immediate and long-lasting benefits to Panama. The connection between conservation and development has been highlighted in publicity about the project in Panama’s urban media. This provides a constructive alternative to the perception the among the urban populace that economic development inevitably competes with conservation. In summary, our program uses biodiversity to promote human health as well as to support research capacity, economic development and conservation within Panama. The program provides an example of the widely recognized but little developed concept of bioprospecting research as an ecosystem service.     

Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

Background

Protein translocation across the membrane of the Endoplasmic Reticulum (ER) is the first step in the biogenesis of secretory and membrane proteins. Proteins enter the ER by the Sec61 translocon, a proteinaceous channel composed of three subunits, α, β and γ. While it is known that Sec61α forms the actual channel, the function of the other two subunits remains to be characterized.

Results

In the present study we have investigated the function of Sec61β in Drosophila melanogaster. We describe its role in the plasma membrane traffic of Gurken, the ligand for the Epidermal Growth Factor (EGF) receptor in the oocyte. Germline clones of the mutant allele of Sec61β show normal translocation of Gurken into the ER and transport to the Golgi complex, but further traffic to the plasma membrane is impeded. The defect in plasma membrane traffic due to absence of Sec61β is specific for Gurken and is not due to a general trafficking defect.

Conclusion

Based on our study we conclude that Sec61β, which is part of the ER protein translocation channel affects a post-ER step during Gurken trafficking to the plasma membrane. We propose an additional role of Sec61β beyond protein translocation into the ER.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Kinetic characterization of the polymerase and exonuclease activities of the gene 43 protein of bacteriophage T4.

The DNA polymerase from the bacteriophage T4 is part of a multienzyme complex required for the synthesis of DNA. As a first step in understanding the contributions of individual proteins to the dynamic properties of the complex, e.g., turnover, processivity, and fidelity of replication, the minimal kinetic schemes for the polymerase and exonuclease activities of the gene 43 protein have been determined by pre-steady-state kinetic methods and fit by computer simulation. A DNA primer/template (13/20-mer) was used as substrate; duplexes that contained more single-strand DNA resulted in nonproductive binding of the polymerase. The reaction sequence features an ordered addition of 13/20-mer followed by dATP to the T4 enzyme (dissociation constants of 70 nM and 20 microM) followed by rapid conversion (400 s-1) of the T4.13/20-mer.dATP complex to the T4.14/20-mer.PPi product species. A slow step (2 s-1) following PPi release limits a single turnover, although this step is bypassed in multiple incorporations (13/20-mer-->17/20-mer) which occur at rates > 400 s-1. Competition between correct versus incorrect nucleotides relative to the template strand indicates that the dissociation constants for the incorrect nucleotides are at millimolar values, thus providing evidence that the T4 polymerase, like the T7 but unlike the Klenow fragment polymerases, discriminates by factors > 10(3) against misincorporation in the nucleotide binding step. The exonuclease activity of the T4 enzyme requires an activation step, i.e., T4.DNA-->T4.(DNA)*, whose rate constants reflect whether the 3'-terminus of the primer is matched or mismatched; for matched 13/20-mer the constant is 1 s-1, and for mismatched 13T/20-mer, 5 s-1. Evidence is presented from crossover experiments that this step may represent a melting of the terminus of the duplex, which is followed by rapid exonucleolytic cleavage (100s-1). In the presence of the correct dNTP, primer extension is the rate-limiting step rather than a step involving travel of the duplex between separated exonuclease and polymerase sites. Since the rate constant for 13/20-mer or 13T/20-mer dissociation from the enzyme is 6 or 8 s-1 and competes with that for activation, the exonucleolytic editing by the enzyme alone in a single pass is somewhat inefficient (5 s-1/(8 s-1+5 s-1)), ca. 40%. Consequently, a major role for the accessory proteins may be to slow the rate of enzyme.substrate dissociation, thereby increasing overall fidelity and processivity.     

Role of the reticulum in the stability and shape of the isolated human erythrocyte membrane

In order to examine the widely held hypothesis that the reticulum of proteins which covers the cytoplamsic surface of the human erythrocyte membrane controls cell stability and shape, we have assessed some of its properties. The reticulum, freed of the bilayer by extraction with Triton X-100, was found to be mechanically stable at physiological ionic strength but physically unstable at low ionic strength. The reticulum broke down after a characteristic lag period which decreased 500-fold between 0 degrees and 37 degrees C. The release of polypeptide band 4.1 from the reticulum preceded that of spectrin and actin, suggesting that band 4.1 might stabilize the ensemble but is not essential to its integrity. The time-course of breakdown was similar for ghosts, the reticulum inside of ghosts, and the isolated reticulum. However, at very low ionic strength, the reticulum was less stable within the ghost than when free; at higher ionic strength, the reverse was true. Over a wide range of conditions the membrane broke down to vesicles just as the reticulum disintegrated, presumably because the bilayer was mechanically stabilized by this network. The volume of both ghosts and naked reticula varied inversely and reversibly with ionic strength. The volume of the naked reticulum varied far more widely than the ghost, suggesting that its deformation was normally limited by the less extensible bilayer. The contour of the isolated reticulum was discoid and often dimpled or indented, as visualized in the fluorescence microscope after labeling of the ghosts with fluoroscein isothiocyanate. Reticula derived from ghosts which had lost the ability to crenate in isotonic saline were shriveled, even though the bilayer was smooth and expanded. Conversly, ghosts crenated by dinitrophenol yielded smooth, expanded reticula. We conclude that the reticulum is a durable, flexible, and elastic network which assumes and stabilizes the contour of the membrane but is not responsible for its crenation.     

Molecular evolution of a multigene family in group A streptococci

The emm genes are members of a gene family in group A streptococci (GAS) that encode for antiphagocytic cell-surface proteins and/or immunoglobulin-binding proteins. Previously sequenced genes in this family have been named "emm," "fcrA," "enn," "arp," "protH," and "mrp"; herein they will be referred to as the "emm gene family." The genes in the emm family are located in a cluster occupying 3-6 kb between the genes mry and scpA on the chromosome of Streptococcus pyogenes. Most GAS strains contain one to three tandemly arranged copies of emm-family genes in the cluster, but the alleles within the cluster vary among different strains. Phylogenetic analysis of the conserved sequences at the 3' end of these genes differentiates all known members of this family into four evolutionarily distinct emm subfamilies. As a starting point to analyze how the different subfamilies are related evolutionarily, the structure of the emm chromosomal region was mapped in a number of diverse GAS strains by using subfamily-specific primers in the polymerase chain reaction. Nine distinct chromosomal patterns of the genes in the emm gene cluster were found. These nine chromosomal patterns support a model for the evolution of the emm gene family in which gene duplication followed by sequence divergence resulted in the generation of four major-gene subfamilies in this locus.      

Diversity of lactic acid bacteria of the bioethanol process

Background  

Bacteria may compete with yeast for nutrients during bioethanol production process, potentially causing economic losses. This is the first study aiming at the quantification and identification of Lactic Acid Bacteria (LAB) present in the bioethanol industrial processes in different distilleries of Brazil.