Showed Amino Acid Sequence Similarity Research Articles

Cell-Penetrating Function of the Poly(ADP-Ribose) (PAR)-Binding Motif Derived from the PAR-Dependent E3 Ubiquitin Ligase Iduna.

Iduna is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that regulates cellular responses such as proteasomal degradation and DNA repair upon interaction with its substrate. We identified a highly cationic region within the PAR-binding motif of Iduna; the region was similar among various species and showed amino acid sequence similarity with that of known cell-penetrating peptides (CPPs). We hypothesized that this Iduna-derived cationic sequence-rich peptide (Iduna) could penetrate the cell membrane and deliver macromolecules into cells. To test this hypothesis, we generated recombinant Iduna-conjugated enhanced green fluorescent protein (Iduna-EGFP) and its tandem-repeat form (d-Iduna-EGFP). Both Iduna-EGFP and d-Iduna-EGFP efficiently penetrated Jurkat cells, with the fluorescence signals increasing dose- and time-dependently. Tandem-repeats of Iduna and other CPPs enhanced intracellular protein delivery efficiency. The delivery mechanism involves lipid-raft-mediated endocytosis following heparan sulfate interaction; d-Iduna-EGFP was localized in the nucleus as well as the cytoplasm, and its residence time was much longer than that of other controls such as TAT and Hph-1. Moreover, following intravenous administration to C57/BL6 mice, d-Iduna-EGFP was efficiently taken up by various tissues, including the liver, spleen, and intestine suggesting that the cell-penetrating function of the human Iduna-derived peptide can be utilized for experimental and therapeutic delivery of macromolecules.

Myotropic activity and immunolocalization of selected neuropeptides of the burying beetle Nicrophorus vespilloides (Coleoptera: Silphidae).

Burying beetles (Nicrophorus sp.) are necrophagous insects with developed parental care. Genome of Nicrophorus vespilloides has been recently sequenced, which makes them interesting model organism in behavioral ecology. However, we know very little about their physiology, including the functioning of their neuroendocrine system. In this study, one of the physiological activities of proctolin, myosuppressin (Nicve-MS), myoinhibitory peptide (Trica-MIP-5) and the short neuropeptide F (Nicve-sNPF) in N. vespilloides have been investigated. The tested neuropeptides were myoactive on N. vespilloides hindgut. After application of the proctolin increased hindgut contraction frequency was observed (EC50 value was 5.47 × 10-8 mol/L). The other tested neuropeptides led to inhibition of N. vespilloides hindgut contractions (Nicve-MS: IC50 = 5.20 × 10-5 mol/L; Trica-MIP-5: IC50 = 5.95 × 10-6 mol/L; Nicve-sNPF: IC50 = 4.08 × 10-5 mol/L). Moreover, the tested neuropeptides were immunolocalized in the nervous system of N. vespilloides. Neurons containing sNPF and MIP in brain and ventral nerve cord (VNC) were identified. Proctolin-immunolabeled neurons only in VNC were observed. Moreover, MIP-immunolabeled varicosities and fibers in retrocerebral complex were observed. In addition, our results have been supplemented with alignments of amino acid sequences of these neuropeptides in beetle species. This alignment analysis clearly showed amino acid sequence similarities between neuropeptides. Moreover, this allowed to deduce amino acid sequence of N. vespilloides proctolin (RYLPTa), Nicve-MS (QDVDHVFLRFa) and six isoforms of Nicve-MIP (Nicve-MIP-1-DWNRNLHSWa; Nicve-MIP-2-AWQNLQGGWa; Nicve-MIP-3-AWQNLQGGWa; Nicve-MIP-4-AWKNLNNAGWa; Nicve-MIP-5-SEWGNFRGSWa; Nicve-MIP-6- DPAWTNLKGIWa; and Nicve-sNPF-SGRSPSLRLRFa).

A Novel β-Glucosidase from Humicola insolens with High Potential for Untreated Waste Paper Conversion to Sugars

Humicola insolens produced a new β-glucosidase (BglHi2) under solid-state fermentation. The purified enzyme showed apparent molecular masses of 116 kDa (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and 404 kDa (gel-filtration), suggesting that it is a homotetramer. Mass spectrometry analysis showed amino acid sequence similarity with a β-glucosidase from Chaetomium thermophilum. Optima of pH and temperature were 5.0 and 65 °C, respectively, and the enzyme was stable for 60 min at 50 °C, maintaining 71 % residual activity after 60 min at 55 °C. BglHi2 hydrolyzed p-nitrophenyl-β-D-glucopyranoside and cellobiose. Cellobiose hydrolysis occurred with high apparent affinity (K M = 0.24 ± 0.01 mmol L(-1)) and catalytic efficiency (k cat/K M = 1,304.92 ± 53.32 L mmol(-1) s(-1)). The activity was insensitive to Fe(+3), Cr(+2), Mn(+2), Co(+2), and Ni(2+), and 50-60 % residual activities were retained in the presence of Pb(2+), Hg(2+), and Cu(2+). Mixtures of pure BglHi2 or H. insolens crude extract (CE) with crude extracts from Trichoderma reesei fully hydrolyzed Whatman no. 1 paper. Mixtures of H. insolens CE with T. reesei CE or Celluclast 1.5 L fully hydrolyzed untreated printed office paper, napkin, and magazine papers after 24-48 h, and untreated cardboard was hydrolyzed by a H. insolens CE/T. reesei CE mixture with 100 % glucose yield. Data revealed the good potential of BglHi2 for the hydrolysis of waste papers, promising feedstocks for cellulosic ethanol production.

Emergence of Sequence Type 779 Methicillin-Resistant Staphylococcus aureus Harboring a Novel Pseudo Staphylococcal Cassette Chromosome mec (SCC mec )-SCC-SCC CRISPR Composite Element in Irish Hospitals

Methicillin-resistant Staphylococcus aureus (MRSA) has been a major cause of nosocomial infection in Irish hospitals for 4 decades, and replacement of predominant MRSA clones has occurred several times. An MRSA isolate recovered in 2006 as part of a larger study of sporadic MRSA exhibited a rare spa (t878) and multilocus sequence (ST779) type and was nontypeable by PCR- and DNA microarray-based staphylococcal cassette chromosome mec (SCCmec) element typing. Whole-genome sequencing revealed the presence of a novel 51-kb composite island (CI) element with three distinct domains, each flanked by direct repeat and inverted repeat sequences, including (i) a pseudo SCCmec element (16.3 kb) carrying mecA with a novel mec class region, a fusidic acid resistance gene (fusC), and two copper resistance genes (copB and copC) but lacking ccr genes; (ii) an SCC element (17.5 kb) carrying a novel ccrAB4 allele; and (iii) an SCC element (17.4 kb) carrying a novel ccrC allele and a clustered regularly interspaced short palindromic repeat (CRISPR) region. The novel CI was subsequently identified by PCR in an additional 13 t878/ST779 MRSA isolates, six from bloodstream infections, recovered between 2006 and 2011 in 11 hospitals. Analysis of open reading frames (ORFs) carried by the CI showed amino acid sequence similarity of 44 to 100% to ORFs from S. aureus and coagulase-negative staphylococci (CoNS). These findings provide further evidence of genetic transfer between S. aureus and CoNS and show how this contributes to the emergence of novel SCCmec elements and MRSA strains. Ongoing surveillance of this MRSA strain is warranted and will require updating of currently used SCCmec typing methods.

The DNA‐binding activity of the Neisseria gonorrhoeae LexA orthologue NG1427 is modulated by oxidation

Neisseria gonorrhoeae is a human-specific organism that is not usually exposed to UV light or chemicals but is likely to encounter reactive oxygen species during infection. Exposure of N. gonorrhoeae to sublethal hydrogen peroxide revealed that the ng1427 gene was upregulated sixfold. N. gonorrhoeae was thought to lack an SOS system, although NG1427 shows amino acid sequence similarity to the SOS response regulator LexA from Escherichia coli. Similar to LexA and other S24 peptidases, NG1427 undergoes autoproteolysis in vitro, which is facilitated by either the gonococcal or E. coli RecA proteins or high pH, and autoproteolysis requires the active and cleavage site residues conserved between LexA and NG1427. NG1427 controls a three gene regulon: itself; ng1428, a Neisseria-specific, putative integral membrane protein; and recN, a DNA repair gene known to be required for oxidative damage survival. Full NG1427 regulon de-repression requires RecA following methyl methanesulphonate or mitomycin C treatment, but is largely RecA-independent following hydrogen peroxide treatment. NG1427 binds specifically to the operator regions of the genes it controls, and DNA binding is abolished by oxidation of the single cysteine residue encoded in NG1427. We propose that NG1427 is inactivated independently of RecA by oxidation.

A novel trypsin Kazal-type inhibitor from Aedes aegypti with thrombin coagulant inhibitory activity

Kazal-type inhibitors play several important roles in invertebrates, such as anticoagulant, vasodilator and antimicrobial activities. Putative Kazal-type inhibitors were described in several insect transcriptomes. In this paper we characterized for the first time a Kazal unique domain trypsin inhibitor from the Aedes aegypti mosquito. Previously, analyses of sialotranscriptome of A. aegypti showed the potential presence of a Kazal-type serine protease inhibitor, in female salivary glands, carcass and also in whole male, which we named AaTI ( A. aegypti trypsin inhibitor). AaTI sequence showed amino acid sequence similarity with insect thrombin inhibitors, serine protease inhibitor from Litopenaeus vannamei hemocytes and tryptase inhibitor from leech Hirudo medicinalis (LDTI). In this work we expressed, purified and characterized the recombinant AaTI (rAaTI). Molecular weight of purified rAaTI was 7 kDa rAaTI presented dissociation constant ( K i) of 0.15 and 3.8 nM toward trypsin and plasmin, respectively, and it weakly inhibited thrombin amidolytic activity. The rAaTI was also able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. AaTI transcription was confirmed in A. aegypti female salivary gland and gut 3 h and 24 h after blood feeding, suggesting that this molecule can act as anticoagulant during the feeding and digestive processes. Its transcription in larvae and pupae suggested that AaTI may also play other functions during the mosquito’s development.

Purification and biochemical characterization of a mycelial glucose- and xylose-stimulated β-glucosidase from the thermophilic fungus Humicola insolens

A mycelial β-glucosidase from the thermophilic mold Humicola insolens was purified and biochemically characterized. The enzyme showed carbohydrate content of 21% and apparent molecular mass of 94 kDa, as estimated by gel filtration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed a single polypeptide band of 55 kDa, suggesting that the native enzyme was a homodimer. Mass spectrometry analysis showed amino acid sequence similarity with a β-glucosidase from Humicola grisea var. thermoidea, with about 22% coverage. Optima of temperature and pH were 60 °C and 6.0–6.5, respectively. The enzyme was stable up to 1 h at 50 °C and showed a half-life of approximately 44 min at 55 °C. The β-glucosidase hydrolyzed cellobiose, lactose, p-nitrophenyl-β- d-glucopyranoside, p-nitrophenyl-β- d-fucopyranoside, p-nitrophenyl-β- d-xylopyranoside, p-nitrophenyl-β- d-galactopyranoside, o-nitrophenyl-β- d-galactopyranoside, and salicin. Kinetic studies showed that p-nitrophenyl-β- d-fucopyranoside and cellobiose were the best enzyme substrates. Enzyme activity was stimulated by glucose or xylose at concentrations up to 400 mM, with maximal stimulatory effect (about 2-fold) around 40 mM. The high catalytic efficiency for the natural substrate, good thermal stability, strong stimulation by glucose or xylose, and tolerance to elevated concentrations of these monosaccharides qualify this enzyme for application in the hydrolysis of cellulosic materials.

NPP-BJ, a nucleotide pyrophosphatase/phosphodiesterase from Bothrops jararaca snake venom, inhibits platelet aggregation

Enzymes of the pyrophosphatase/phosphodiesterase family have multiple roles in extracellular nucleotide metabolism and in the regulation of nucleotide-based intercellular signaling. Snake venoms contain enzymes that hydrolyze nucleic acids and nucleotides, but their function is poorly understood. Here we describe for the first time the isolation and functional characterization of a soluble phosphodiesterase from Bothrops jararaca venom, which shows amino acid sequence similarity to mammalian nucleotide pyrophosphatase/phosphodiesterase 3 (NPP3), and inhibits ADP-induced platelet aggregation. The enzyme, named NPP-BJ, showed an apparent molecular mass of 228 kDa by size exclusion chromatography. NPP-BJ exhibited nuclease activity as well as pyrophosphatase and phosphatase activities, preferentially hydrolyzing nucleoside 5′-triphosphates over nucleoside 5′-diphosphates, but was not active upon nucleoside 5′-monophosphates. Depending on the substrate used, dithiothreitol and EDTA differently inhibited the catalytic activity of NPP-BJ. Platelet aggregation induced by ADP was also abrogated by NPP-BJ, whereas thrombin-induced platelet aggregation was only slightly attenuated. However, polyclonal antibodies raised against NPP-BJ could not abolish the lethal activity of B. jararaca venom. Altogether, these results show that NPP-BJ has a minor contribution to the lethal activity of this venom, but interferes with mechanisms of ADP-induced platelet aggregation.

Purification and characterization of antimicrobial peptides from the skin secretion of Rana dybowskii

Six antimicrobial peptides designated dybowskins were isolated from the skin secretion of Rana dybowskii, an edible frog in Korea. Dybowskin-1 (FLIGMTHGLICLISRKC) and dybowskin-2 (FLIGMTQGLICLITRKC) were isoforms differing in only two amino acid residues at the 7th and 14th positions from the N-terminus, and they showed amino acid sequence similarities with ranalexin peptides. Dybowskin-3 (GLFDVVKGVLKGVGKNVAGSLLEQLKCKLSGGC), dybowskin-4 (VWPLGLVICKALKIC), dybowskin-5 (GLFSVVTGVLKAVGKNVAKNVGGSLLEQLKCKISGGC), and dybowskin-6 (FLPLLLAGLPLKLCFLFKKC) differed in both size and sequence, and they were, in terms of amino acid sequence similarities, related to brevinin-2, japonicin-2, esculentin-2, and brevinin-1 peptides, respectively. All the peptides presented in this paper contained Rana-box, the cyclic heptapeptide domain, which is conserved in other antimicrobial peptides derived from the genus Rana. All the dybowskin peptides showed a broad spectrum of antimicrobial activity against the Gram-positive and Gram-negative bacteria (minimum inhibition concentrations (MIC), 12.5 to >100 μg/ml) and against Candida albicans (MIC, 25 to >100 μg/ml). Especially, dybowskin-4 with valine at its N-terminus was the most abundant and showed the strongest antimicrobial activity among all the dybowskin peptides. This result indicates that the dybowskin peptides from R. dybowskii, whose main habitats are mountains or forests, have evolved differently from antimicrobial peptides isolated from other Korean frogs, whose habitats are plain fields.

Aquabirnaviruses isolated from marine organisms form a distinct genogroup from other aquabirnaviruses

A phylogenetic tree of aquabirnaviruses, including marine birnaviruses (MABV) and infectious pancreatic necrosis virus (IPNV), was developed based on the nucleotide sequences and deduced amino acid sequences of the polyprotein and VP5 genes of genomic segment A. In the polyprotein of MABV strains, the amino acid sequences were very similar, with identities of 98.3-99.7%. Twenty-one unique amino acid residues were found in the deduced amino acid sequences of the polyprotein gene of MABV strains. The phylogenetic tree based on the nucleotide sequence of genomic segment A and polyprotein sequences showed that 31 aquabirnavirus strains were clustered into seven genogroups. All MABV strains isolated in Japan and Korea were clustered into one genogroup which was distinct from other aquabirnaviruses. The seventh genogroup containing all MABV strains showed amino acid sequence similarities of 80.7-90.6% with other genogroups. In VP5, four unique residues were found in MABV strains when compared with IPNV strains. The MABV strains exhibited amino acid sequence similarities of 63.9-86.4% with IPNV strains. The amino acid sequences of VP5 were conserved among MABV strains, but differed from those of IPNV strains. The MABV strains isolated from different host species and different geographical areas were very similar to each other, suggesting that the MABV are distinct from the other genogroups.

Selection of an antibiotic-hypersusceptible mutant of Pseudomonas aeruginosa: identification of the GlmR transcriptional regulator.

Tn501 random mutagenesis was applied to the Pseudomonas aeruginosa wild-type strain PAO1 to select for mutants hypersusceptible to aminoglycoside antimicrobial agents. One such mutant, called 19A, was found to be hypersusceptible to a wide range of antibiotics including aminoglycosides, beta-lactams, fluoroquinolones, colistin, erythromycin, rifampin, and glycopeptides. Light microscopy of the mutant strain revealed abnormal morphology characterized by large, filamentous cells. The drug supersusceptibility of 19A was accompanied by loss of motility, reduced resistance to osmotic and heat shock stress, and impaired growth at low temperatures. The insertion site of the Tn501 transposon in mutant 19A has occurred in an open reading frame (PA5550 according to the PAO1 genome project), whose gene product shows amino acid sequence similarity to the DeoR family of transcriptional repressors. The gene, which we called glmR, is located between the glmS (PA5549) and glmU (PA5552) homologues of E. coli, responsible for the synthesis of UDP-N-acetylglucosamine-1-P, a precursor of both lipopolysaccharide (LPS) and peptidoglycan. We showed that GlmR represses the transcription of the adjacent glmS homologue (PA5549) in P. aeruginosa, possibly affecting the pool of precursors for peptidoglycan and LPS synthesis. To our knowledge GlmR is the first regulator in P. aeruginosa that affects susceptibility to a large variety of antibiotics and is therefore a potential target for novel anti-infective agents.

S1 glycoprotein gene analysis of infectious bronchitis viruses isolated in Korea.

Summary.Fifteen isolates of Infectious bronchitis virus (IBV) were obtained from the kidney, trachea, and cecal tonsil of IB suspected chickens between 2001 and 2002 years in Korea. The S1 glycoprotein gene of IBV isolates were amplified by reverse transcriptase – polymerase chain reaction (RT-PCR) and analyzed by restriction fragment length polymorphism (RFLP) analysis. Fifteen Korean IBV isolates were classified into 4 groups by their RFLP patterns using restriction enzymes, HaeIII, BstYI, and XcmI. The RFLP patterns for 3, 1, and 1 of 15 isolates corresponded to the patterns of IBV Arkansas, Connecticut, and Massachusetts strains, respectively. Ten of 15 isolates generated unique KM91 RFLP pattern that was observed in the IBV KM 91 strain previously isolated in Korea. To confirm genetic diversity in the S1 genes of IBV isolates, viral RNAs of representative 9 of 15 IBV isolates were amplified, cloned, sequenced and compared with published sequences for non-Korean IBV strains. Korean IBV isolates showed amino acid sequence similarity between 61.8% (K446-01 and K161-02) and 96.1% (K281-01 and K210-02) with each other and they showed amino acid sequence similarity between 42.9% (K161-02 and GA980470) and 96.5% (K203-02 and KB8523) compared to non-Korean IBV strains. By phylogenetic tree analysis, Korean IBV field isolates were branched into five clusters in which 3 clusters were differentiated from non-Korean IBV strains. Especially, Korean IBV isolates K069-01, K507-01, K774-01 and K142-02 formed a separate cluster. It seems that IBVs continue to evolve and IBVs showing various genetic differences may cocirculate in Korea.

The ybiT gene of Erwinia chrysanthemi codes for a putative ABC transporter and is involved in competitiveness against endophytic bacteria during infection.

We investigated the role in bacterial infection of a putative ABC transporter, designated ybiT, of Erwinia chrysanthemi AC4150. The deduced sequence of this gene showed amino acid sequence similarity with other putative ABC transporters of gram-negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa, as well as structural similarity with proteins of Streptomyces spp. involved in resistance to macrolide antibiotics. The gene contiguous to ybiT, designated as pab (putative antibiotic biosynthesis) showed sequence similarity with Pseudomonas and Streptomyces genes involved in the biosynthesis of antibiotics. A ybiT mutant (BT117) was constructed by marker exchange. It retained full virulence in potato tubers and chicory leaves, but it showed reduced ability to compete in planta against the wild-type strain or against selected saprophytic bacteria. These results indicate that the ybiT gene plays a role in the in planta fitness of the bacteria.

Platelet-activating Factor (PAF)-dependent Transacetylase and Its Relationship with PAF Acetylhydrolases

Platelet-activating Factor (PAF)-dependent Transacetylase and Its Relationship with PAF Acetylhydrolases

Identification and Functional Characterization of a Na+-independent Neutral Amino Acid Transporter with Broad Substrate Selectivity

We have isolated a cDNA from rat small intestine that encodes a novel Na+-independent neutral amino acid transporter with distinctive characteristics in substrate selectivity and transport property. The encoded protein, designated L-type amino acid transporter-2 (LAT-2), shows amino acid sequence similarity to the system L Na+-independent neutral amino acid transporter LAT-1 (Kanai, Y., Segawa, H., Miyamoto, K., Uchino, H., Takeda, E., and Endou, H. (1998) J. Biol. Chem. 273, 23629-23632) (50% identity) and the system y+L transporters y+LAT-1 (47%) and KIAA0245/y+LAT-2 (45%) (Torrents, D., Estevez, R., Pineda, M., Fernandez, E., Lloberas, J., Shi, Y.-B., Zorzano, A., and Palacin, M. (1998) J. Biol. Chem. 273, 32437-32445). LAT-2 is a nonglycosylated membrane protein. It requires 4F2 heavy chain, a type II membrane glycoprotein, for its functional expression in Xenopus oocytes. LAT-2-mediated transport is not dependent on Na+ or Cl- and is inhibited by a system L-specific inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), indicating that LAT-2 is a second isoform of the system L transporter. Compared with LAT-1, which prefers large neutral amino acids with branched or aromatic side chains, LAT-2 exhibits remarkably broad substrate selectivity. It transports all of the L-isomers of neutral alpha-amino acids. LAT-2 exhibits higher affinity (Km = 30-50 microM) to Tyr, Phe, Trp, Thr, Asn, Ile, Cys, Ser, Leu, Val, and Gln and relatively lower affinity (Km = 180-300 microM) to His, Ala, Met, and Gly. In addition, LAT-2 mediates facilitated diffusion of substrate amino acids, as distinct from LAT-1, which mediates amino acid exchange. LAT-2-mediated transport is increased by lowering the pH level, with peak activity at pH 6.25, because of the decrease in the Km value without changing the Vmax value. Because of these functional properties and a high level of expression of LAT-2 in the small intestine, kidney, placenta, and brain, it is suggested that the heterodimeric complex of LAT-2 and 4F2 heavy chain is involved in the trans-cellular transport of neutral amino acids in epithelia and blood-tissue barriers.

Identification and Molecular Characterization of TM7SF2in the FAUNA Gene Cluster on Human Chromosome 11q13

In this report, the identification and molecular characterization of a novel gene, designated TM7SF2,is reported. This gene was found in the FAUneighboring area (FAUNA) to which other genes have been mapped previously. The FAUNA gene cluster is located at chromosome 11q13 between landmarks H4B and D11S2196E. The TM7SF2gene contains eight coding exons, and their splice site consensus sequences are consistent with the AG/GT rule. Northern blot analysis with a cDNA probe corresponding to TM7SF2revealed varying expression levels of a 1.7-kb transcript in adult human heart, brain, pancreas, lung, liver, skeletal muscle, kidney, ovary, prostate, and testis, but no detectable expression in placenta, spleen, thymus, small intestine, colon (mucosal lining), or peripheral blood leukocytes. The open reading frame in the cDNA sequence codes for a protein of 590 amino acids that is rich in glycine (23%) and arginine (17%) residues in its amino-terminal half and contains seven transmembrane domains in its carboxy-terminal half. The transmembrane region of the putative TM7SF2 protein shows amino acid sequence similarity to those of the lamin B receptor and the C14/C24 sterol reductase.

The euryarchaeotes, a subdomain of Archaea, survive on a single DNA polymerase: fact or farce?

Archaea is now recognized as the third domain of life. Since their discovery, much effort has been directed towards understanding the molecular biology and biochemistry of Archaea. The objective is to comprehend the complete structure and the depth of the phylogenetic tree of life. DNA replication is one of the most important events in living organisms and DNA polymerase is the key enzyme in the molecular machinery which drives the process. All archaeal DNA polymerases were thought to belong to family B. This was because all of the products of pol genes that had been cloned showed amino acid sequence similarities to those of this family, which includes three eukaryal DNA replicases and Escherichia coli DNA polymerase II. Recently, we found a new heterodimeric DNA polymerase from the hyperthermophilic archaeon, Pyrococcus furiosus. The genes coding for the subunits of this DNA polymerase are conserved in the euryarchaeotes whose genomes have been completely sequenced. The biochemical characteristics of the novel DNA polymerase family suggest that its members play an important role in DNA replication within euryarchaeal cells. We review here our current knowledge on DNA polymerases in Archaea with emphasis on the novel DNA polymerase discovered in Euryarchaeota.

Identification and characterization of a gene cluster involved in manganese oxidation by spores of the marine Bacillus sp. strain SG-1

The marine Bacillus sp. strain SG-1 forms spores that oxidize manganese(II) as a result of the activities of uncharacterized components of its spore coat. Nucleotide sequence analysis of chromosomal loci previously identified through insertion mutagenesis as being involved in manganese oxidation identified seven possible genes (designated mnxA to mnxG) in what appears to be an operon. A potential recognition site for the sporulation, mother-cell-specific, RNA polymerase sigma factor, sigmaK, was located just upstream of the cluster, and correspondingly, measurement of beta-galactosidase activity from a Tn917-lacZ insertion in mnxD showed expression at mid-sporulation to late sporulation (approximately stage IV to V of sporulation). Spores of nonoxidizing mutants appeared unaffected with respect to their temperature and chemical resistance properties and germination characteristics. However, transmission electron microscopy revealed alterations in the outermost spore coat. This suggests that products of these genes may be involved in the deposition of the spore coat structure and/or are spore coat proteins themselves. Regions of the deduced protein product of mnxG showed amino acid sequence similarity to the family of multicopper oxidases, a diverse group of proteins that use multiple copper ions to oxidize a variety of substrates. Similar regions included those that are involved in binding of copper, and the addition of copper at a low concentration was found to enhance manganese oxidation by the spores. This suggests that the product of this gene may function like a copper oxidase and that it may be directly responsible for the oxidation of manganese by the spores.

Cloning and Functional Characterization of a System ASC-like Na+-dependent Neutral Amino Acid Transporter

A cDNA was isolated from mouse testis which encodes a Na+-dependent neutral amino acid transporter. The encoded protein, designated ASCT2, showed amino acid sequence similarity to the mammalian glutamate transporters (40-44% identity), Na+-dependent neutral amino acid transporter ASCT1 (57% identity; Arriza, J. L., Kavanaugh, M. P., Fairman, W. A., Wu, Y.-N., Murdoch, G. H., North, R. A., and Amara, S. G.(1993) J. Biol. Chem. 268, 15329-15332; Shafqat, S., Tamarappoo, B. K., Kilberg, M. S., Puranam, R. S., McNamara, J. O., Guadano-Ferraz, A., and Fremeau, T., Jr. (1993) J. Biol. Chem. 268, 15351-15355) and a mouse adipocyte differentiation-associated gene product AAAT (94% identity; Liao, K., and Lane, D.(1995) Biochem. Biophys. Res. Commun. 208, 1008-1015). When expressed in Xenopus laevis oocytes, ASCT2 exhibited Na+-dependent uptakes of neutral amino acids such as L-alanine, L-serine, L-threonine, L-cysteine, and L-glutamine at high affinity with Km values around 20 microM. L-Methionine, L-leucine, L-glycine, and L-valine were also transported by ASCT2 but with lower affinity. The substrate selectivity of ASCT2 was typical of amino acid transport system ASC, which prefers neutral amino acids without bulky or branched side chains. ASCT2 also transported L-glutamate at low affinity (Km = 1.6 mM). L-Glutamate transport was enhanced by lowering extracellular pH, suggesting that L-glutamate was transported as protonated form. In contrast to electrogenic transport of glutamate transporters and the other ASC isoform ASCT1, ASCT2-mediated amino acid transport was electroneutral. Na+ dependence of L-alanine uptake fits to the Michaelis-Menten equation, suggesting a single Na+ cotransported with one amino acid, which was distinct from glutamate transporters coupled to two Na+. Northern blot hybridization revealed that ASCT2 was mainly expressed in kidney, large intestine, lung, skeletal muscle, testis, and adipose tissue. Functional characterization of ASCT2 provided fruitful information on the properties of substrate binding sites and the mechanisms of transport of Na+-dependent neutral and acidic amino acid transporter family, which would facilitate the structure-function analyses based on the comparison of the primary structures of ASCT2 and the other members of the family.

The Transcriptional Activity of the CCAAT-binding Factor CBF Is Mediated by Two Distinct Activation Domains, One in the CBF-B Subunit and the Other in the CBF-C Subunit

CBF-A, CBF-B, and CBF-C together form the heterotrimeric mammalian CCAAT-binding factor, CBF, which binds to DNA to form a CBF-DNA complex. Here we examined the transcription activation function of CBF in an in vitro reconstituted system using the three purified recombinant CBF subunits expressed in Escherichia coli. Two of the subunits, CBF-A and CBF-C, were coexpressed and purified as a CBF-A/CBF-C complex. Addition of the three wild-type recombinant CBF subunits to EL4 cell nuclear extracts depleted of CBF stimulated transcription 5-20-fold from proalpha2(1) collagen promoters and 10-fold from the Rous sarcoma virus long terminal repeat. Two CBF deletion mutants, one containing full-length CBF-A and CBF-C and a CBF-B lacking the NH2-terminal residues 1-224, and the other containing full- length CBF-A and CBF-B and a CBF-C lacking the COOH-terminal residues 114-309, also stimulated transcription from these promoters, but the level of activation was reduced to half that obtained with the full-length CBF subunits. In contrast, a CBF deletion mutant protein containing full-length CBF-A and deleted forms of both CBF-B and CBF-C showed very little transcription activation from these promoters. Hence, this study demonstrates that the heterotrimeric CBF protein consists of two transcription activation domains, one present in CBF-B and the other in CBF-C, and that the two domains act additively in the in vitro assay. The activation domains of both CBF-B and CBF-C, which are rich in glutamine and hydrophobic residues, showed amino acid sequence similarities with each other and with the glutamine-rich activation domain of transcription factor Sp1.