Wei Sheng Wu Xue Bao, 2002 Jun, 42(3), 321 - 5 {Screening strains producing inulinase and cloning of inulinase gene}; Zhang L et al.; A wild-type strain AF10 producing inulinase was screened from soil samples, the strain is identified to be a A . niger . A endoinulinase gene inuA1 from A . niger AF10 was wequenced and analyzed, after PCR amplification . The results shows inuA1 is 1551 bp in length without any intron and contains 4 potential N-glycosylation sites . The conservative sequence is WMNEPN from the N-terminus . The inuA1 was cloned to pUC118 and the recombinant vector pinuA1 was obtained and transformed into E . coli JM109 . The recombinant JM109/inuA1 included inuA1 was obtained. Wei Sheng Wu Xue Bao, 2002 Jun, 42(3), 305 - 10 {Cloning and expression of Vitreoscilla hemoglobin in Streptomyces aureofaciens}; Meng C et al.; Vitreoscilla hemoglobin Gene was cloned in Streptomyces aureofaciens through E . coli-Streptomyces shuttle plasmids constructed by both pIJ699-pUC19(vhb) and pIJ702-pBR322(vhb) . Under low dissolved oxygen conditions, expression of hemoglobin enhanced CTC yield of engineering strain more about 40%-60% that that of control by improving oxygen transmission in cells . Hemoglobin expression by Promoter of tetracycline resistance gene was more effective for oxygen transmission than that by its native promoter regulated by dissolved oxygen concentrations in environments of locally low concentrations of dissolved oxygen. Wei Sheng Wu Xue Bao, 2002 Jun, 42(3), 298 - 304 {Selection and characterization of peptides that specifically binding to hepatitis C virus serine protease}; Du G et al.; The HCV NS3 serine protease that plays important role in the processing of polyprotein and the replication of virus is a prime target for antiviral drugs and therapy research . Based on the crystallographic structure of HCV sreine protease, a single-chain protease was contstructed in which the central sequence of NS4A was fused to the N-terminus of NS3 serine protease domain via a flexible linker and it was expressed at high level in soluble form in E . coli . The purified protease could cleave the recombinant protein NS5ab into two parts . The purified protease was used as target to screen binding peptides from phage displayed peptide library . After three rounds of affinity screening, 37 out of 44 randomly selected phages could bind specifically with the single-chain serine protease and their specificity were verified by competitive ELISA . The 13 sequenced clones represents 6 kinds of sequences of which the amino acids composition is in bias and there is a consensus sequence: {H/F/W}-H-W-X-X-W. Wei Sheng Wu Xue Bao, 2002 Feb, 42(1), 110 - 3 {Cloning of flocculent gene and expression in industrial yeast strain}; Guo W et al.; The partial genomic library of Saccharomyce cerevisiae FL189 possessing strong flocculation ability was constructed using Yeast-E . coli shuttle plasmid YCp50 as vector . Recombinant plasmid containing flocculation gene was obtained by screening the growth of transformants on the selective medium and measurement flocculation, designated as pCF1.pCF1 was introduced into industrial yeast strain PJ208-5-15 . Six transformants PJ208-5-15-1(pCF1)-PJ208-5-15-6(pCF1) possessing strong flocculation ability were obtained . The results of Southern blot and restriction endonuclease analysis showed that the insert is about 4.3 kb and could hybridize with the probe (2.6 kb Eco RV fragment of FLO1) . Flocculation ability assay indicated that the transformants possess strong flocculation ability . Hence, the gene controlling flocculation phenotype exists in the cloned DNA fragment . The restriction endonuclease analysis and the sequence analysis of the insert DNA fragment are in progress. Wei Sheng Wu Xue Bao, 2002 Feb, 42(1), 99 - 104 {Antigen analysis of envelope gene products of avian leukosis virus subgroup J}; Qin A et al.; Envelope glycoprotein of avian leukosis virus Subgroup J (ALV-J) determines the host range of virus infection and cross-neutralization patterns . The truncated envelope genes of avian lecukosis virus subgroup J (ALV-J) were amplified by PCR and cloned them into pGEX-5X-3 vector for expressing envGST-fusion protein . Western blot analysis results showed that the products of truncated env gene expressed in Escherichia coli could reacted with G2, JE9 and I45 monoclonal antibodies (Mabs) specific to envelope protein of ALV-J . Using different Mabs to map the epitopes in the expressed truncated gp85 GST fusion protein, the results showed that Mab G2 and JE9 antibodies recognizing epitope in gp85 was localized between amino acid 65-155 . Mab I45 reacted with the epitope at the location of amino acid 156-233 . It indicated that the specificity of subgroup J virus is determined by the gp85 peptide since GST-gp85 protein expressed in Escherichia coli is not glycosylated. Wei Sheng Wu Xue Bao, 2002 Feb, 42(1), 40 - 4 {The study on cloning and expression of Bt cry1Ab13 gene}; Tan J et al.; B . thuringiensis strain C005 with high insecticidal activity to several kinds of pests, screened from China, was identified that it contained cry1Ab gene by PCR-RFLP . Southern blotting showed that a 8.5 kb positive band of plasmid DNA digested with PstI contained cry1Ab gene . The gene was cloned from Bt C005 and the results of sequence analysis showed that cry1Ab13 gene contained a 3468 bp open reading frame, encoding a 130.6 kD protein composing 1155 amino acids . The IE point of Cry1Ab13 protein was pH 4.845 . The cry1Ab gene has been registered in GenBank (Accession number is AF254640) and named as cry1Ab13 as a novel gene by International Nomenclature Committee of Bt delta-endotoxin genes . SDS-PAGE analysis indicated that 130 kD protein of Cry1Ab13 was expressed in a Bt acrystalliferous mutant cryB- and bioassay results proved that the transformant BiotI81 containing cry1Ab13 gene had high toxicity to Plutella xylostella. Biotechnol Bioeng, 2003 Mar 30, 81(7), 783 - 9 Simultaneous synthesis of enantiomerically pure (S)-amino acids and (R)-amines using coupled transaminase reactions; Cho BK et al.; For the simultaneous synthesis of enatiomerically pure (S)-amino acids and (R)-amines from corresponding alpha-keto acids and racemic amines, an alpha/omega-transaminase coupled reaction system was designed using favorable reaction equilibrium shift led by omega-transaminase reaction . Cloned tyrB, aspC and avtA, and omegataA were co-expressed in E . coli BL21(DE3) using pET23b(+) and pET24ma, respectively . The coupled reaction produced the (S)-amino acids with 73-90% (> 99% ee(S)) of conversion yield and resolved the racemic amines with 83-99% ee(R) for 5 to 10 hours . In designing the coupled reactions in the cell, alanine and pyruvate were efficiently used in the cell as an amine donor for the alanine transaminase and an amino acceptor for the omega-transaminase, respectively, resulting in an alanine-pyruvate shuttling system . The common problem of the low equilibrium constant of the alpha-transaminase can be efficiently overcome by the coupling with the omega-transaminase . However, overcoming the product inhibition of omega-transaminase by the ketone by-product and increasing the decarboxylation rate of the oxaloacetate produced during the transaminase reaction become barriers to further improving the overall reaction rate and the yield of the coupled reactions . Proteins, 2003 Feb 15, 50(3), 474 - 85 Structural studies on MtRecA-nucleotide complexes: insights into DNA and nucleotide binding and the structural signature of NTP recognition; Datta S et al.; RecA protein plays a crucial role in homologous recombination and repair of DNA . Central to all activities of RecA is its binding to Mg(+2)-ATP . The active form of the protein is a helical nucleoprotein filament containing the nucleotide cofactor and single-stranded DNA . The stability and structure of the helical nucleoprotein filament formed by RecA are modulated by nucleotide cofactors . Here we report crystal structures of a MtRecA-ADP complex, complexes with ATPgammaS in the presence and absence of magnesium as well as a complex with dATP and Mg+2 . Comparison with the recently solved crystal structures of the apo form as well as a complex with ADP-AlF4 confirms an expansion of the P-loop region in MtRecA, compared to its homologue in Escherichia coli, correlating with the reduced affinity of MtRecA for ATP . The ligand bound structures reveal subtle variations in nucleotide conformations among different nucleotides that serve in maintaining the network of interactions crucial for nucleotide binding . The nucleotide binding site itself, however, remains relatively unchanged . The analysis also reveals that ATPgammaS rather than ADP-AlF4 is structurally a better mimic of ATP . From among the complexed structures, a definition for the two DNA-binding loops L1 and L2 has clearly emerged for the first time and provides a basis to understand DNA binding by RecA . The structural information obtained from these complexes correlates well with the extensive biochemical data on mutants available in the literature, contributing to an understanding of the role of individual residues in the nucleotide binding pocket, at the molecular level . Modeling studies on the mutants again point to the relative rigidity of the nucleotide binding site . Comparison with other NTP binding proteins reveals many commonalties in modes of binding by diverse members in the structural family, contributing to our understanding of the structural signature of NTP recognition . Arch Virol, 2003 Feb, 148(2), 345 - 55 Involvement of the terminus of grouper betanodavirus capsid protein in virus-like particle assembly; Lu MW et al.; Dragon grouper, Epinephelus lanceolatus, nervous necrosis virus (DGNNV) consists of 180 copies of capsid protein that encapsulates a bipartite genome of single-stranded (+)RNAs . Expressing the open reading frame (ORF) of RNA2 in Escherichia coli forms virus-like particles (VLPs) that resembles native virus . Deleting N- and C-termini revealed different impacts on VLP formation . Deletion of 35 or 52 residues at the N-terminus completely ruined the VLP assembly, presumably due to removal of positively charged residues for binding RNAs . When deletions were restricted to 4, 16, or 25 N-terminal residues, the assembly of VLPs remained . The ability of VLP formation diminished when 4 to 11 C-terminal residues were deleted . The termini that can be deleted without seriously destructing the VLPs are 25 and 3 residues at N- and C-termini, respectively. Proc Natl Acad Sci U S A, 2003 Feb 18, 100(4), 1547 - 51 Epub 2003 Jan 29. Expression and mechanistic analysis of a germacradienol synthase from Streptomyces coelicolor implicated in geosmin biosynthesis; Cane DE et al.; The PCR has been used to amplify a 2,181-bp ORF from Streptomyces coelicolor A3(2), designated SC9B1.20 (= SCO6073), encoding a protein of 726 amino acids and showing significant sequence similarity at the deduced amino acid level in both the N-terminal and C-terminal halves to the known sesquiterpene synthase pentalenene synthase . The full-length recombinant protein was expressed at high levels in Escherichia coli and shown to catalyze the Mg(2+)-dependent conversion of farnesyl diphosphate to the sesquiterpene alcohol (4S, 7R)-germacra-1 (10)E, 5E-diene-11-ol . The enzymatic cyclization had a k(cat) of 6.2 +/- 0.5 x 10(-3) s(-1) and a K(m) for farnesyl diphosphate of 62 +/- 8 nM . Expression of the N-terminal (366 amino acids) domain of the SC9B1.20 protein also gave a fully functional cyclase which converted farnesyl diphosphate to the identical sesquiterpene alcohol with a slightly lower k(cat) of 3.2 +/- 0.4 x 10(-3) s(-1) and a twofold greater k(m) of 115 +/- 14 nM . By contrast, the expressed C-terminal domain of SC9B1.20 had no farnesyl diphosphate cyclase activity . The formation of the germacradienol seems to be the committed step in the formation of geosmin, the characteristic odoriferous constituent of Streptomyces species. J Biol Chem, 2003 Apr 4, 278(14), 12013 - 21 Epub 2003 Jan 28. Regulation of the interleukin-1-induced signaling pathways by a novel member of the protein phosphatase 2C family (PP2Cepsilon); Li MG et al.; Although TAK1 signaling plays essential roles in eliciting cellular responses to interleukin-1 (IL-1), a proinflammatory cytokine, how the IL-1-TAK1 signaling pathway is positively and negatively regulated remains poorly understood . In this study, we investigated the possible role of a novel protein phosphatase 2C (PP2C) family member, PP2Cepsilon, in the regulation of the IL-1-TAK1 signaling pathway . PP2Cepsilon was composed of 303 amino acids, and the overall similarity of amino acid sequence between PP2Cepsilon and PP2Calpha was found to be 26% . Ectopic expression of PP2Cepsilon inhibited the IL-1- and TAK1-induced activation of mitogen-activated protein kinase kinase 4 (MKK4)-c-Jun N-terminal kinase or MKK3-p38 signaling pathway . PP2Cepsilon dephosphorylated TAK1 in vitro . Co-immunoprecipitation experiments indicated that PP2Cepsilon associates stably with TAK1 and attenuates the binding of TAK1 to MKK4 or MKK6 . Ectopic expression of a phosphatase-negative mutant of PP2Cepsilon, PP2Cepsilon(D/A), which acted as a dominant negative form, enhanced both the association between TAK1 and MKK4 or MKK6 and the TAK1-induced activation of an AP-1 reporter gene . The association between PP2Cepsilon and TAK1 was transiently suppressed by IL-1 treatment of the cells . Taken together, these results suggest that, in the absence of IL-1-induced signal, PP2Cepsilon contributes to keeping the TAK1 signaling pathway in an inactive state by associating with and dephosphorylating TAK1. J Biol Chem, 2003 Apr 11, 278(15), 13227 - 34 Epub 2003 Jan 29. Molecular dissection of the contribution of negatively and positively charged residues in S2, S3, and S4 to the final membrane topology of the voltage sensor in the K+ channel, KAT1; Sato Y et al.; Voltage-dependent ion channels control changes in ion permeability in response to membrane potential changes . The voltage sensor in channel proteins consists of the highly positively charged segment, S4, and the negatively charged segments, S2 and S3 . The process involved in the integration of the protein into the membrane remains to be elucidated . In this study, we used in vitro translation and translocation experiments to evaluate interactions between residues in the voltage sensor of a hyperpolarization-activated potassium channel, KAT1, and their effect on the final topology in the endoplasmic reticulum (ER) membrane . A D95V mutation in S2 showed less S3-S4 integration into the membrane, whereas a D105V mutation allowed S4 to be released into the ER lumen . These results indicate that Asp(95) assists in the membrane insertion of S3-S4 and that Asp(105) helps in preventing S4 from being releasing into the ER lumen . The charge reversal mutation, R171D, in S4 rescued the D105R mutation and prevented S4 release into the ER lumen . A series of constructs containing different C-terminal truncations of S4 showed that Arg(174) was required for correct integration of S3 and S4 into the membrane . Interactions between Asp(105) and Arg(171) and between negative residues in S2 or S3 and Arg(174) may be formed transiently during membrane integration . These data clarify the role of charged residues in S2, S3, and S4 and identify posttranslational electrostatic interactions between charged residues that are required to achieve the correct voltage sensor topology in the ER membrane. Faraday Discuss, 2003, 122, 131 - 44; discussion 171-90 Time-resolved and static-ensemble structural chemistry of hydroxymethylbilane synthase; Helliwell JR et al.; The enzyme hydroxymethylbilane synthase (HMBS, EC 4.3.1.8), 313 amino acid residues and MW 34 kDa, also known as porphobilinogen deaminase (PBGD), catalyses the stepwise polymerization of four molecules of porphobilinogen (PBG) to the linear tetrapyrrole 1-hydroxymethylbilane . Several crystallographic structures of HMBS have been previously determined, most recently including by time-resolved Laue protein crystallography of the Lys59Gln mutant form with reaction initiation undertaken by use of a flow cell carrying the substrate PBG . In this paper we review these structures and add new molecular graphics representations and analyses . Moreover we present a new structure refined at 1.66 A resolution using diffraction data recorded at cryo-temperature (100 K) in an attempt at trapping the polypeptide loop (residues 47 to 58) in the vicinity of the enzyme active site, missing in all previous structure determinations . This loop still has not appeared in the electron density maps, in spite of the advantage of cryo-temperature, but nevertheless the 1.66 A cryo-structure extends the ensemble of known HMBS structures . The cryomodel of protein, cofactor and 320 bound water molecules has been refined to a final R-factor and R-free of 0.198 and 0.247 respectively; the PDB deposition codes, coordinates and structure factors are 1GTK and R1GTKSF respectively . Finally a protein comparison study is presented of the Mycobacterium tuberculosis (MTb) HMBS, with the E . coli HMBS . This has been done as preparation for future structural studies on the MTb HMBS from this important disease bearing organism . The overall amino acid sequence identity is 41% . Most interestingly there is a two-residue reduction in length of the loop referred to above (Asp 50 and Gly 58 being missing in the MTb form) . This gives the hope that this loop will be less flexible and thus might become visible to crystallographic analysis. Wei Sheng Wu Xue Bao, 1999 Aug, 39(4), 305 - 14 {Molecular cloning and sequencing of outer capsid protein gene of rice dwarf virus and its expression in Escherichia coli}; Lu R et al.; The S2 full-length cDNA of rice dwarf phytoreovirus which enocodes the viral outer capsid protein was cloned and its complete nucleotide sequence was determined . The results showed that S2 is 3512 bp long with a large open reading frame which encodes a protein of 1116 amino acids . It shares 94.6% and 95.4% identity with RDV of Japanese H isolate in terms of nucleotide and amino acid sequences, respectively, and it also shows some homology with VP2 of rotavirus at the level of amino acid sequence . The search of deduced RDV S2 amino acid sequence in Blast network found that there were 4 leucine-rich motifs in P2 protein, and ten amino acids within the hydrophibic region at amino-terminus could form an alpha-helix . Predicted secondary structure of S2 cDNA indicated that a hairpin and a stem loop are present in the 5'-end within 50 bp, and a stem loop in the 3'-end within 50 bp . RDV S2 partial and full-length sequences were then cloned into expression vector pET-11d & pTrcHisC . SDS-PAGE and Western blot proved that amino- and carborn-termini of P2 were successfully expressed in E . coli. Wei Sheng Wu Xue Bao, 1999 Dec, 39(6), 521 - 6 {The isolation and characterization of type 1 pili from pathogenic Escherichia coli of chicken origin}; Gao S et al.; The pili from pathogenic Escherichia coli isolates 566, 1794 and TK3 of chicken and turkey origin were purified . After mechanic detachment from the bacterial cells, the pili were concentrated by precipitation with ammonium sulfate, dialyzed, and solubilized in buffer containing deoxycholate . The fraction containing the pilus was purified further by ultracentrifugation in a sucrose gradient . After ultracentrifugation, the pili at the density of 1.10 to 1.15 g.cm-3 (between 10%-20% of sucrose gradients) were collected, and the purified pili from strain 566, 1794 and TK3 had an apparent molecular weight of 17,500, 17,000 and 17,000 respectively, which retained their ability to bind the erythrocyte in a mannose-inhibitable fashion . Hyperimmunesera raised in BALB/C mice against the purified pili from strain 1794 reacted positively with type 1 pili from both isolates 566 and TK3 by immuno blot . These results revealed that the three strains either Chinese or north american isolates expressed type 1 pili which had molecular weights from 17,000 to 17,500, and they have common antigenic epitopes. Wei Sheng Wu Xue Bao, 1999 Dec, 39(6), 489 - 94 {Cloning of DNA fragment related to salt tolerance in Sinorhizobium meliloti 042B}; Chen X et al.; Total DNA partially digested by EcoR I was prepared for S . meliloti 042B, in which 15-25 kb DNA fragments were collected . Vector pLAFR I was purified and digested by EcoR I, and then the various DNA fragments of 042B were ligated with pLAFR I by T4DNA ligase . Gene library of S . meliloti 042B was constructed with pLAFR I using E . coli S17-1 as recipient . The number of bacterial recombinants obtained was about 8,000 and 95% of them contained foreign DNA fragments . Using NTG, 042B was mutated on FY plates and 12 sensitive strains were screened at 0.5 mol/L NaCl from 2,000 colonies . One of them was named GZ17 and selected as a recipient strain . By biparental mating the foreign DNA fragments were introduced from gene library of strain 042B into recipient strain GZ17 which is sensitive to 0.5 mol/L NaCl . Then the transconjugants were grown on FY plates containing tetracycline (20 micrograms/mL) and 0.5 mol/L NaCl . A 7 kb inserted DNA fragment related to salt tolerance was obtained . In subcloning experiment, a 4 kb DNA fragment related to salt tolerance was obtained. Wei Sheng Wu Xue Bao, 1999 Jun, 39(3), 226 - 33 {The outer membrane protein (OMP) patterns of Escherichia coli isolates of predominant O serogroups originated from chichens in different regions in China}; Gao S et al.; The purpose of this study is to determine the outer membrane protein (OMP) patterns of avian Escherichia coli isolates with predominant serogroups originated from 18 provinces, autonomous regions and municipal cities in China . Total of 204 isolates belonging to O18, O78, O2, O88, O11 and O26 serogroups respectively, were tested . The outer membrane proteins of these isolates were isolated with the improved N-lauroylsarcosine method and analyzed by SDS-PAGE . 4 different OMP patterns were identified with these isolates of predominant serogroups . 3 different OMP patterns with 56 isolates of O18, 4 with 54 isolates of O78, 2 with 28 isolates O2, 1 with 26 isolates of O88, 3 with 22 isolates of O11 and 1 with 18 isolates of O26 respectively, were found . Isolates with OMP pattern 1 were discovered in 6 different serogroups, and OMP-3 pattern was also shared by O18, O78 and O11 serogroups isolates . These results indicated that the OMP patterns of avian pathogenic Escherichia coli isolates of O18, O78, O2, O11 serogroups which were isolated from different regions in China were heterogeneous, and all of O88 and O26 serogroups isolates just belonged to OMP pattern 1 . Moreover, the OMP pattern 1 was presented in isolates of six different predominant O serogroups. Wei Sheng Wu Xue Bao, 1999 Jun, 39(3), 215 - 9 {Molecular cloning of rat OB gene and its expression in Escherichia coli}; Zhang T et al.; In order to provide rat OB gene product for studying the relationship between obesity and noninfectious diseases, rat OB cDNA was amplified by RT-PCR technique . 460 bp fragment of OB cDNA was subcloned into EcoRI/BamHI site of plasmid pUC 19 . Sequence analysis of OB cDNA revealed that the translation reading frame was identical with that reported in the literature . Thereby plasmid pBV220-rOB was constructed and the specific expression of OB gene in E . coli identified by SDS-PAGE electrophoresis was obtained. Wei Sheng Wu Xue Bao, 1999 Jun, 39(3), 209 - 14 {Expression detection and location analysis of BstNI isoschizomer restriction-modification system gene}; Liu J et al.; Some genetic markers of E . coli HB101 and JM110 were identified, two bacterial strains were used as recipients respectively to detect the expression of a restriction endonuclease(R) gene and a methylase(M) gene of BstNI isoschizomer restriction-modification system . DNA fragment containing the R-M genes was deleted unilaterally with exoIII and 23 deletion subclones were obtained . According to the Enzyme activity of each subclone, R and M gene were located respectively at the regions of 0.2-->1.4 kb and 1.5-->3.3 kb from cloning site PstI . Analysis showed that the R . M system belongs to type II, two genes are controlled by the different promoters; the recognition sequence of this system is the same as that of DNA-cytosine methyltransferase(Dcm), the latter's methylation function can resist the R enzyme . It was interesting that the recombinant plasmid with an R+ M- genotype appeared to be lethal to dcm+ hosts yet . This indicated that the M gene closely linking to R gene is of critical importance for the existence of the R-M system in process of evolution. Wei Sheng Wu Xue Bao, 1999 Apr, 39(2), 178 - 80 {Preparation, purification and identification of sialic acid from Escherichia coli C-8}; Qian S et al.; Colominic acid produced from Escherichia coli C-8 was purified by ammonia sulfate precipitation, dialysis and concentration . After hydrolysis of colominic acid at a pH of 2.5, at 70 degrees C for 4 h, sialic acid was obtained, then purified by chromatography on Dowex1-x8 . Analysis of thin-layer chromatography and absorption spectrum of sialic acid in the orcinol/Fe3+/HCl and the periodic acid/thiobarbituric acid confirmed that its purity was identical with that of standard sample. Wei Sheng Wu Xue Bao, 1999 Apr, 39(2), 100 - 7 {Study of HSP70 gene upstream regulation element on expression efficiency of GST gene in M . smegmatis}; Cheng J et al.; Four different expression vectors were constructed by cloning foreign gene which encode Schistosoma japonicum 26 kD antigen (Sj26GST) into Escherichia coli-Mycobacteria shuttle plasmid pBCG-2000 and investigated their expression efficiency in mycobacteria smegmatis . The plasmid which contains promoter of human mycobacterial tuberculosis heat shock protein 70 was firstly digested with Nco I and modified with two different ways to lead to two kinds of SD sequences, and ligated with Sj26GST encoding gene . Then, the DNA fragment contained HSP70 promoter and Sj26GST gene was obtained and cloned into E . coil-mycobacteria shuttle plasmid pBCG-2000, and finally four recombinant mycobacterial expression vectors that differenciated in SD sequence, orientation and copy number were selected . The expressed native recombinant Sj26GST(rSj26GST) was soluble and could be observed on SDS-PAGE about at the molecular weight of 26 kD obviously . Analysis with protein density scanning indicated: the expression efficiency that containing double-copy promoter-foreign gene vector was the highest and the expressed protein which was about 1.6 times than others was 28% of total protein of Mycobacteria smegmatis . The cloned direction and SD sequence had no significant effect on expression efficiency. Acta Crystallogr D Biol Crystallogr, 2003 Feb, 59(Pt 2), 400 - 3 Epub 2003 Jan 23. De novo purification scheme and crystallization conditions yield high-resolution structures of chitinase A and its complex with the inhibitor allosamidin; Papanikolau Y et al.; The purification scheme of chitinase A (ChiA) from S . marcescens has been extensively revised . The pure enzyme crystallizes readily under new crystallization conditions . The ChiA crystal structure has been refined to 1.55 A resolution and the crystal structure of ChiA co-crystallized with the inhibitor allosamidin has been refined to 1.9 A resolution . Allosamidin is located in the deep active-site tunnel of ChiA and interacts with three important residues: Glu315, the proton donor of the catalysis, Asp313, which adopts two conformations in the native structure but is oriented towards Glu315 in the inhibitor complex, and Tyr390, which lies opposite Glu315 in the active-site tunnel. Acta Crystallogr D Biol Crystallogr, 2003 Feb, 59(Pt 2), 385 - 8 Epub 2003 Jan 23. Improved three-dimensional growth of manganese superoxide dismutase crystals on the International Space Station; Vahedi-Faridi A et al.; Manganese superoxide dismutase was crystallized in microgravity with 35 PCAM experiments (Protein Crystallization Apparatus for Microgravity) on the ISS (International Space Station) from 5 December 2001 to 19 April 2002 . Crystals were very large in size and could easily be seen by eye . Crystals with 0.45 x 0.45 mm cross-sections and of up to 3 mm in length were obtained in several drops: an 80-fold increase in crystal Volume compared with the largest earth-grown crystal . A smaller crystal (0.15 x 0.30 mm in cross-section and 1.6 mm in length) was soaked in cryoprotectant and placed in a cryoloop . Diffraction data were collected at 100 K at the BioCARS bending-magnet beamline . The space group was C222(1), with unit-cell parameters a = 100.64, b = 107.78, c = 179.82 A . Diffraction spots to 1.26 A resolution were observed . Unfortunately, the high-resolution diffraction degraded owing to radiation damage and the resolution limit for the complete data set was 1.35 A . It is anticipated that increasing the crystal Volume and diffraction limit through microgravity crystal growth will enable several types of technically challenging structure determinations. Acta Crystallogr D Biol Crystallogr, 2003 Feb, 59(Pt 2), 381 - 4 Epub 2003 Jan 23. Crystallization of hamster dihydroorotase: involvement of a disulfide-linked tetrameric form; Maher MJ et al.; Dihydroorotase (DHOase) catalyses the formation of L-dihydroorotate (DHO) in the de novo pyrimidine biosynthetic pathway . The type I DHOase domain from hamster forms part of the trifunctional enzyme CAD . The hamster DHOase domain has been cloned and expressed in Escherichia coli . Solutions of the homodimeric protein convert to a homotetrameric species when incubated at ambient temperature . Formation of the tetrameric species is mediated via disulfide linkages between single free cysteine residues on the surface of each monomer . This process is also observed under conditions used for crystallization of the hamster DHOase domain; crystals composed exclusively of the tetrameric species grow from solutions containing as little as 10% tetramer . The crystallization of pure tetrameric DHOase results in two crystal forms: form I, with space group C222(1) and unit-cell parameters a = 127.1, b = 603.5, c = 144.7 A, and form II, with space group P2(1) and unit-cell parameters a = 260.5, b = 148.2, c = 308.0 A, beta = 102.2 degrees . Data have been recorded to 4.3 and 4.0 A resolution, respectively. Acta Crystallogr D Biol Crystallogr, 2003 Feb, 59(Pt 2), 372 - 4 Epub 2003 Jan 23. Crystallization and preliminary X-ray analysis of TBP-interacting protein from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1; Yamamoto T et al.; The 26 kDa TBP (TATA-binding protein) interacting protein from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-TIP26) is a possible transcriptional regulatory protein in Thermococcales . Here, the crystallization of both the native and selenomethionine-substituted proteins and data collection are described . The native crystals belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 73.83, c = 86.41 A, and diffract to 2.2 A using synchrotron radiation . MAD data was collected and a Bijvoet difference Patterson map showed strong peaks sufficient to determine the positions of the Se atoms. Acta Crystallogr D Biol Crystallogr, 2003 Feb, 59(Pt 2), 369 - 71 Epub 2003 Jan 23. Purification, crystallization and preliminary X-ray analysis of a mu-like calpain; Pal GP et al.; The X-ray structure of m-calpain in the absence of Ca(2+) has been described, but it has not been possible to obtain sufficient mu-calpain for structure determination . Comparison of the two structures is of interest in attempting to understand their different Ca(2+) requirements . Here, the crystallization in the absence of Ca(2+) of an inactive mutant hybrid calpain (MW approximately 100 kDa), which contains 85% of the rat mu-calpain sequence and is well expressed in Escherichia coli, is described . The properties of this calpain in its active form and particularly its Ca(2+) requirement are close to those expected for wild-type mu-calpain . Clusters of plate-shaped crystals were obtained by vapour diffusion with polyethylene glycol (M(r) approximately 6000) as precipitating agent in the presence of detergent . The crystals diffract to a resolution of 2.7 A at a synchrotron source . The space group is P2(1), with unit-cell parameters a = 72.7, b = 184.6, c = 86.3 A, beta = 100.7 degrees . There are two molecules in the asymmetric unit, corresponding to a solvent content of 57.1%. Acta Crystallogr D Biol Crystallogr, 2003 Feb, 59(Pt 2), 350 - 2 Epub 2003 Jan 23. Preliminary X-ray characterization of the ribonuclease P (C5 protein) from Escherichia coli: expression, crystallization and cryoconditions; Choe HW et al.; The gene for Escherichia coli ribonuclease P (RNase P) protein (also known as C5 protein) and its mutant C5-C113A have been expressed as GST fusion proteins in E . coli at a high level . After cleavage of the fusion protein, highly purified functional C5 protein is obtained that can be crystallized with 2.5-2.6 M (NH(4))(2)HPO(4)/(NH(4))H(2)PO(4) pH 7.0 at room temperature . These crystals are suitable for X-ray analysis, belong to the space group P3(1)21 or P3(2)21 (unit-cell parameters a = b = 66.67, c = 142.09 A) and diffract to 2.9 A at 100 K using sorbitol and glycerol as cryoprotectants . For three molecules in the asymmetric unit a V(M) of 2.17 A(3) Da(-1) was calculated. Acta Crystallogr D Biol Crystallogr, 2003 Feb, 59(Pt 2), 214 - 24 Epub 2003 Jan 23. Ping-pong cross-validation in real space: a method for increasing the phasing power of a partial model without risk of model bias; Hunt JF et al.; Experimental phases could only be obtained to 4.4 A resolution for crystals of the SecA translocation ATPase . Density modification of these phases exploiting the 65% solvent content of the crystal produced a map from which an approximate backbone model could be built for 80% of the structure . Combining the phases inferred from this partial model with the MIR phases and repeating the density modification produced an improved map from which a more complete backbone model could be built . However, this procedure converged before yielding a map, that allowed unambiguous sequence assignment for the majority of the protein molecule . In order to avoid the likely model bias associated with a speculative attempt at sequence assignment, a real-space cross-validation procedure was employed to facilitate completion of the crystal structure based on partial model phasing . The protein was partitioned into two disjoint sets of residues . Models in which the side chains were built for residues in one of the two sets were used for phase combination and density modification in order to produce improved electron density for interpretation of residues in the other set that had not been included in the model . Residues in the two sets were therefore omitted from the model in alternation except at sites where the side chain could be identified definitively based on phasing with the other set . This ping-pong cross-validation procedure allowed partial model phasing to be used to complete the crystal structure of SecA without being impeded by model bias . These results show that the structure of a large protein molecule can be solved with exclusively low-resolution experimental phase information based on intensive use of partial model phasing and density modification . Real-space cross-validation can be applied to reduce the risk of model bias associated with partial model phasing, streamlining this approach and expanding its range of applicability. RNA, 2003 Jan, 9(1), 33 - 43 Identification of the Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure; Brescia CC et al.; DsrA RNA regulates the translation of two global regulatory proteins in Escherichia coli . DsrA activates the translation of RpoS while repressing the translation of H-NS . The RNA-binding protein Hfq is necessary for DsrA to function in vivo . Although Hfq binds to DsrA in vitro, the role of Hfq in DsrA-mediated regulation is not known . One hypothesis was that Hfq acts as an RNA chaperone by unfolding DsrA, thereby facilitating interactions with target RNAs . To test this hypothesis, we have examined the structure of DsrA bound to Hfq in vitro . Comparison of free DsrA to DsrA bound to Hfq by RNase footprinting, circular dichroism, and thermal melt profiles shows that Hfq does not alter DsrA secondary structures, but might affect its tertiary conformation . We identify the site on DsrA where Hfq binds, which is a structural element in the middle of DsrA . In addition, we show that although long poly(U) RNAs compete with DsrA for binding to Hfq, a short poly(U) stretch present in DsrA is not necessary for Hfq binding . Finally, unlike other RNAs, DsrA binding to Hfq is not competed with by poly(A) RNA . In fact, DsrA:poly(A):Hfq may form a stable ternary complex, raising the possibility that Hfq has multiple RNA-binding sites. RNA, 2003 Feb, 9(2), 231 - 8 Analysis of recombinant yeast decapping enzyme; Steiger M et al.; A critical step in the turnover of yeast mRNAs is decapping . Two yeast proteins, Dcp1p and Dcp2p, are absolutely required for decapping, although their precise roles in the decapping reaction have not been established . To determine the function of both Dcp1p and Dcp2p in decapping, we purified recombinant versions of these proteins from Escherichia coli and examined their properties . These experiments demonstrate that copurification of Dcp1p and Dcp2p yields active decapping enzyme under a variety of conditions . Moreover, Dcp2p alone can have decapping activity under some biochemical conditions . This suggests that Dcp2p can be a catalytic subunit of the decapping complex, and Dcp1p may function to enhance Dcp2p activity, or as an additional active subunit . In addition, recombinant Dcp1p/Dcp2p prefers long mRNA substrates and is sensitive to inhibition by sequestration of the 5' end but not the 3' end of the substrate . This suggests that Dcp1p/Dcp2p contains an additional RNA-binding site spatially distinct from the active site . Finally, using two RNA-binding proteins that enhance decapping in vivo (Edc1p and Edc2p), we can reconstitute the activation of decapping with recombinant proteins . This indicates that the Edc1 and Edc2 proteins act directly on the decapping enzyme. J Biol Chem, 2003 Apr 25, 278(17), 15142 - 52 Epub 2003 Jan 28. Deamidations in recombinant human phenylalanine hydroxylase . Identification of labile asparagine residues and functional characterization of Asn --> Asp mutant forms; Carvalho RN et al.; Recombinant human phenylalanine hydroxylase (hPAH) expressed in Escherichia coli for 24 h at 28 degrees C has been found by two-dimensional electrophoresis to exist as a mixture of four to five molecular forms as a result of nonenzymatic deamidation of labile Asn residues . The multiple deamidations alter the functional properties of the enzyme including its affinity for l-phenylalanine and tetrahydrobiopterin, catalytic efficiency, and substrate inhibition and also result in enzyme forms more susceptible to limited tryptic proteolysis . Asn(32) in the regulatory domain deamidates very rapidly because of its nearest neighbor amino acid Gly(33) (Solstad, T., Carvalho, R . N., Andersen, O . A., Waidelich, D., and Flatmark, T . (2003) Eur . J . Biochem., in press) . Matrix-assisted laser desorption/ionization time of flight-mass spectrometry of the tryptic peptides in the catalytic domain of a 24-h (28 degrees C) expressed enzyme has shown Asn(376) and Asn(133) to be labile residues . Site-directed mutagenesis of nine Asn residues revealed that the deamidations of Asn(32) and Asn(376) are the main determinants for the functional and regulatory differences observed between the 2- and 24-h-induced wild-type (wt) enzyme . The Asn(32) --> Asp, Asn(376) --> Asp, and the double mutant forms expressed for 2 h at 28 degrees C revealed qualitatively similar regulatory properties as the highly deamidated 24-h expressed wt-hPAH . Moreover, deamidation of Asn(32) in the wt-hPAH (24 h expression at 28 degrees C) and the Asn(32) --> Asp mutation both increase the initial rate of phosphorylation of Ser(16) by cAMP-dependent protein kinase (p < 0.005) . By contrast, the substitution of Gly(33) with Ala or Val, both preventing the deamidation of Asn(32), resulted in enzyme forms that were phosphorylated at a similar rate as nondeamidated wt-hPAH, even on 24-h expression . The other Asn --> Asp substitutions (in the catalytic domain) revealed that Asn(207) and Asn(223) have an important stabilizing structural function . Finally, two recently reported phenylketonuria mutations at Asn residues in the catalytic domain were studied, i.e . Asn(167) --> Ile and Asn(207) --> Asp, and their phenotypes were characterized. J Biol Chem, 2003 Apr 18, 278(16), 13728 - 39 Epub 2003 Jan 28. Analysis of human flap endonuclease 1 mutants reveals a mechanism to prevent triplet repeat expansion; Liu Y et al.; Flap endonuclease 1 (FEN1), involved in the joining of Okazaki fragments, has been proposed to restrain DNA repeat sequence expansion, a process associated with aging and disease . Here we analyze properties of human FEN1 having mutations at two conserved glycines (G66S and G242D) causing defects in nuclease activity . Introduction of these mutants into yeast led to sequence expansions . Reconstituting triplet repeat expansion in vitro, we previously found that DNA ligase I promotes expansion, but FEN1 prevents the ligation that forms expanded products . Here we show that among the intermediates that could generate sequence expansion, a bubble is necessary for ligation to produce the expansion product . Severe exonuclease defects in the mutant FEN1 suggested that the inability to degrade bubbles exonucleolytically leads to expansion . However, even wild type FEN1 exonuclease cannot compete with DNA ligase I to degrade a bubble structure before it can be ligated . Instead, we propose that FEN1 suppresses sequence expansion by degrading flaps that equilibrate with bubbles, thereby reducing bubble concentration . In this way FEN1 employs endonuclease rather than exonuclease to prevent expansions . A model is presented describing the roles of DNA structure, DNA ligase I, and FEN1 in sequence expansion. EMBO J, 2003 Feb 3, 22(3), 746 - 56 The alternating ATPase domains of MutS control DNA mismatch repair; Lamers MH et al.; DNA mismatch repair is an essential safeguard of genomic integrity by removing base mispairings that may arise from DNA polymerase errors or from homologous recombination between DNA strands . In Escherichia coli, the MutS enzyme recognizes mismatches and initiates repair . MutS has an intrinsic ATPase activity crucial for its function, but which is poorly understood . We show here that within the MutS homodimer, the two chemically identical ATPase sites have different affinities for ADP, and the two sites alternate in ATP hydrolysis . A single residue, Arg697, located at the interface of the two ATPase domains, controls the asymmetry . When mutated, the asymmetry is lost and mismatch repair in vivo is impaired . We propose that asymmetry of the ATPase domains is an essential feature of mismatch repair that controls the timing of the different steps in the repair cascade. EMBO J, 2003 Feb 3, 22(3), 735 - 45 The RdgC protein of Escherichia coli binds DNA and counters a toxic effect of RecFOR in strains lacking the replication restart protein PriA; Moore T et al.; PriA protein provides a means to load the DnaB replicative helicase at DNA replication fork and D loop structures, and is therefore a key factor in the rescue of stalled or broken forks and subsequent replication restart . We show that the nucleoid-associated RdgC protein binds non-specifically to single-stranded (ss) DNA and double-stranded DNA . It is also essential for growth of a strain lacking PriA, indicating that it might affect replication fork progression or fork rescue . dnaC suppressors of priA overcome this inviability, especially when RecF, RecO or RecR is inactivated, indicating that RdgC avoids or counters a toxic effect of these proteins . Mutations modifying ssDNA-binding (SSB) protein also negate this toxic effect, suggesting that the toxicity reflects inappropriate loading of RecA on SSB-coated ssDNA, leading to excessive or untimely RecA activity . We suggest that binding of RdgC to DNA limits RecA loading, avoiding problems at replication forks that would otherwise require PriA to promote replication restart . Mutations in RNA polymerase also reduce the toxic effect of RecFOR, providing a further link between DNA replication, transcription and repair. EMBO J, 2003 Feb 3, 22(3), 724 - 34 A model for dsDNA translocation revealed by a structural motif common to RecG and Mfd proteins; Mahdi AA et al.; RecG protein differs from other helicases analysed to atomic resolution in that it mediates strand separation via translocation on double-stranded (ds) rather than single-stranded (ss) DNA . We describe a highly conserved helical hairpin motif in RecG and show it to be important for helicase activity . It places two arginines (R609 and R630) in opposing positions within the component helices where they are stabilized by a network of hydrogen bonds involving a glutamate from helicase motif VI . We suggest that disruption of this feature, triggered by ATP hydrolysis, moves an adjacent loop structure in the dsDNA-binding channel and that a swinging arm motion of this loop drives translocation . Substitutions that reverse the charge at R609 or R630 reduce DNA unwinding and ATPase activities, and increase dsDNA binding, but do not affect branched DNA binding . Sequences forming the helical hairpin and loop structures are highly conserved in Mfd protein, a transcription-coupled DNA repair factor that also translocates on dsDNA . The possibility of type I restriction enzymes and chromatin-remodelling factors using similar structures to drive translocation on dsDNA is discussed. Drug Deliv, 2003 Jan-Mar, 10(1), 35 - 40 A kinetic model for predicting the release rate of sparingly-water-soluble drugs from a hydrogel-coated polymeric matrix; Fan YL; Medical devices used for on-target drug delivery are often coated with a hydrogel coating for friction-reduction purpose . Thus, the delivery of a sparingly-water-soluble drug by such a device must diffuse through a nonerodable hydrogel layer . An empirical rate equation has been derived for such a kinetic model and predicts that the rate of drug release from such a device is directly proportional to the loading of the drug in the polymeric matrix . The validity of this kinetic model was examined by measuring the rate of release of 2,4,4'-trichloro-2'-hydroxydiphenyl ether from different hydrogel-coated (ethylene-vinyl acetate) copolymer stents containing a wide range of the drug . The experimentally determined release rates are in reasonably good agreement with those calculated from the empirical rate equation . Bioefficacy test results based on zone-of-inhibition test against Escherichia coli are also in good agreement with the release rate and drug-loading data predicted according to the empirical rate equation. Vet Microbiol, 2003 Apr 29, 92(4), 363 - 77 An RTX operon in hemolytic Moraxella bovis is absent from nonhemolytic strains; Angelos JA et al.; Pathogenic isolates of Moraxella bovis express a calcium-dependent transmembrane pore forming cytotoxin that is an RTX toxin encoded by mbxA . The DNA flanking mbxA was cloned and sequenced to determine if M . bovis contained a classical RTX operon . Open reading frames (ORFs) with deduced amino acid sequence homology to putative activation (RTX C) and transport (RTX B and D) proteins were identified and have been designated MbxC, MbxB, and MbxD, respectively . Thus, hemolytic M . bovis contains a typical RTX operon comprised of four genes arranged (5'-3') mbxCABD . In addition, the deduced amino acid sequences of DNA flanking mbxCABD revealed ORFs with amino acid sequence similarity to transposases (5') . At the 3' end of the mbx gene cluster, an ORF with homology to bacterial tolC genes was identified . Thus, as with the cya RTX operon of Bordetella pertussis, M . bovis appears to have a secretion accessory protein linked to RTX genes . Analysis of genomic DNA isolated from 5 nonhemolytic M . bovis strains by PCR and Southern blotting revealed the absence of mbxCABD . These strains did, however, amplify with primers specific for the 5' region flanking mbxC . M . bovis harbors a classical RTX operon that is absent in nonhemolytic strains. Mol Biochem Parasitol, 2003 Jan, 126(1), 15 - 23 Cloning, heterologous expression in Escherichia coli and characterization of a protein disulfide isomerase from Fasciola hepatica; Salazar-Calderon M et al.; A Fasciola hepatica cDNA clone of 1752 bp was isolated from an adult worm cDNA expression library by immunological screening using a rabbit serum against the excretory-secretory antigens . The nucleotide sequence of the cDNA revealed the presence of an open reading frame of 489 codons which encoded a 55 kDa polypeptide, showing a high degree of homology to protein disulfide isomerases . This putative antioxidant protein cDNA was expressed in Escherichia coli as a GST fusion protein . The cleaved recombinant protein was shown to be biologically active in vitro by mediating the oxidative refolding of reduced RNase . Immunoblotting studies using a specific antiserum raised against the recombinant protein showed the presence of a polypeptide of similar molecular mass in the excretory-secretory extract of the adult parasite . The extracellular location of this protein was also supported by the specific immune responses found against this protein in F . hepatica experimentally infected rabbits. Clin Biochem, 2003 Feb, 36(1), 41 - 9 Antigenicity of a recombinant NS3 protein representative of ATPase/helicase domain from hepatitis C virus; Penton N et al.; It has been shown that the Hepatitis C virus nonstructural NS3 protein possesses at least two enzymatic domains: a serine-protease domain and an adenosine triphosphatase (ATPase)/helicase domain . In this report, a truncated fragment of NS3 (26 kDa), representing main epitopes from the (ATPase)/helicase domain, has been expressed in Escherichia coli . The recombinant protein was purified by Ion Metal Affinity Chromatography (IMAC) with more than 90% purity . The recognition of B-cell linear epitopes in the NS3 protein was evaluated by immunoblot . The recombinant NS3 protein was reduced and carboxymethylated, and the recognition of either conformational and/or linear B-cell determinants was evaluated by ELISA . The inclusion of the recombinant NS3 protein in a third-generation diagnostic system UltraMicroELISA (UMELISA) allowed an increase in the sensitivity, due to the detection of a new variety of false-negative sera in blood donor test samples. J Endocrinol, 2003 Feb, 176(2), R1 - 7 The heparin-binding 10 kDa fragment of connective tissue growth factor (CTGF) containing module 4 alone stimulates cell adhesion; Ball DK et al.; Connective tissue growth factor (CTGF) is a 349-residue mosaic protein that contains four structural modules implicated in protein-protein interactions . To address the functionality of residues 247-349 (containing module 4 alone), this region of CTGF was produced as a maltose binding protein (MBP) fusion protein in E . coli . After removal of MBP, recombinant CTGF commenced at Glu(247), was of M(r) 10 000, was immunoreactive with anti-CTGF{247-260}, bound strongly to heparin, and promoted dose-dependent adhesion of fibroblasts, myofibroblasts, endothelial cells, and epithelial cells . An 8 kDa presumptive C-terminally truncated form of CTGF commencing at Glu(247) also promoted cell adhesion . CTGF-mediated cell adhesion was abolished by heparin or EDTA . These data demonstrate the presence of heparin-binding and cell-adhesion motifs within the C-terminal 103 residues of CTGF and show that CTGF-mediated cell adhesion is heparin-and divalent cation-dependent . Thus, CTGF isoforms comprising essentially module 4 are intrinsically functional in the absence of the other constituent modules of CTGF. Protein Pept Lett, 2002 Dec, 9(6), 553 - 6 Purification, crystallization and preliminary X-ray studies of human augmenter of liver regeneration; Ji CN et al.; Human augmenter of liver regeneration has been expressed in Escherichia coli, purified and crystallized . The crystals belong to space group C222, with unit-cell parameters a=51.7 A, b=78.8 A, c=63.7 A . Diffraction data were collected to 2.80 A with a completeness of 99.9% (99.9% for the last shell), a R(sym) value of 0.092(0.236) and an I/sigma(I) value of 6.2(2.7). Biol Chem, 2002 Dec, 383(12), 1865 - 73 Evaluation of similarities in the cis/trans isomerase function of trigger factor and DnaK; Schiene-Fischer C et al.; Two functionally redundant enzymes, trigger factor and the hsp70 chaperone DnaK, have been found to assist de novo protein folding in E coli . Trigger factor is a peripheral peptidyl prolyl cis/trans isomerase (PPIase) of the large subunit of the ribosome . In contrast, DnaK displays two catalytic features: the secondary amide peptide bond cis/trans isomerase (APIase) function supplemented by the ATPase site . APIases accelerate the cis/trans isomerization of nonprolyl peptide bonds . Both enzymes have affinity for an unfolded polypeptide chain . The diminished low temperature cell viability in the presence of trigger factor variants with impaired PPlase activity indicates that the enhancement of folding rates plays a crucial role in protein folding in vivo . For the DnaK-mediated increase in the folding yield in vitro, the minimal model for APlase catalysis involves the catalyzed partitioning of a rapidly formed folding intermediate as could be inferred from the DnaK/DnaJ/GrpE/ATP-assisted refolding of GdmCl-denatured luciferase . Using three different peptide bond cis/trans isomerization assays in vitro, we could show that there is no overlapping substrate specificity of trigger factor and DnaK . We propose that only if trigger factor recruits supplementing molecules is it capable of exhibiting functional complementarity with DnaK in protein folding. Wei Sheng Wu Xue Bao, 2001 Aug, 41(4), 415 - 20 {Cloning and expression of two garlic virus coat protein genes}; Ma Y et al.; The coat protein(CP) genes of garlic mosaic virus(GMVc) and garlic latent virus(GLVc) isolated from garlic(Allium) plants in Tianjin, China, were amplified from an established cDNA library by PCR method and subsequently expressed in E . coli . using the pET-30a expression system . The determined sequences of GMVc and GLVc CP genes show that the complete GMVc CP gene has 867 nucleotides encoding 289 amino acids . It has 88.5% and 97.2% homology, at the levels of nucleotide and amino acid, respectively, to a reported GMV, indicating that it belongs to Potyvirus . The complete GLVc CP gene has 885 nucleotides coding for 294 amino acids . It has 73.6% and 90.9% homologous percents, in nucleotide and amino acid, respectively, compared to a previously reported GLV, suggesting that it is a member of Carlavirus . The expressed products presented in inclusion body and were analyzed by SDS-PAGE . The molecular weights of GMVc and GLVc CPs appear in 32 kD and 34 kD size, respectively, which are consistent with the deduced sizes of these two CPs . These data will be virtually significant to the further investigation of viruses infecting parlic plant, the control of garlic virus diseases and the production of virus-freed garlic plants. Wei Sheng Wu Xue Bao, 2001 Aug, 41(4), 402 - 7 {Cloning and sequencing of the gene encoding esterase estA from Ralstonia eutropha CH34}; Song S et al.; An esterase-positive clone was isolated by screening a genomic library of Ralstonia eutropha CH34 constructed in Escherichia coli S17-1 with top agar containing alpha-naphtyl acetate and Fast Blue RR . A gne encoding esterase activity, estA was subcloned from this clone . Nucleotide sequencing of estA showed that it was a 825 bp open reading frame, encoding an esterase EstA, composed of 275 amino acids with a predicted molecular mass of 30,785 D . Homology analysis revealed that EstA exhibited significant amino acid similarity with the enzymes involved in the meta-cleavage pathway in the metaboleism of aromatic acid compounds. Wei Sheng Wu Xue Bao, 2001 Dec, 41(6), 693 - 8 {Cloning and sequencing of 16S rRNA gene of Phytoplasma CWB1 strain associated with cactus witches' broom}; Cai H et al.; A 1.5 kb DNA fragment was amplified in DNA samples extracted from Opuntia salmiana porm showed witches'-broom symptom . The result indicates the existence of phytoplasma associated with this disease and this phytoplasma was designated as CWB1 . The amplified fragment was ligated to pGEM-T easy vector and then transformed into JM109 strain of E . coli . Cloned DNA fragments were verified by PCR, restriction endonuclease (EcoRI) digestion and sequence analysis . The result revealed that the 16S rRNA gene of CWB1 consists of 1489 bp and shared 99.7% homology with Faba bean phyllody which belongs to phytoplasma 16S rII-C subgroup . So we can classify this strain into phytoplasma 16S rII-C subgroup. Wei Sheng Wu Xue Bao, 2001 Oct, 41(5), 625 - 9 {Construction of mucosal vaccine derived from HBV surface antigen epitope A and the assay of its immunogenicity}; Liu X et al.; The fusion gene of HBV(adr) surface antigen epitope and B subunit of Cholera toxin was constructed and expressed successfully in E . coli at high yield . After denaturation-renaturation process, SDS-PAGE analysis showed that most of the renatured product reassociated in a pentameric form which was the same as natural CTB . Western blot analysis indicated that the immunogenicity of HBsAg antigen epitope was conserved . Moreover, ELISA analysis of the sera of orally, intranasally and intraperitoneally immunized mice showed that the circulating IgG antibodies to HBsAg were developed . The results may be helpful for constructing the novel mucosal vaccine with high efficacy. Wei Sheng Wu Xue Bao, 2001 Oct, 41(5), 559 - 66 {Homologous recombination in Streptomyces lincolnensis B48}; Tang Y et al.; To study frequency and mechanism of homologous recombination in Streptomyces, an E . coli plasmid which cannot replicate in Streptomyces was transformed into Streptomyces lincolnensis B48 . After homologous recombination between delta lincomycin biosynthetic genes inactivated by thiostrepton resistant gene (tsr) carried on pYYE04al and homologous sequences on the chromosome, S . lincolnensis YY1 and S . lincolnensis, YY2 were obtained on SMA with low thiostrepton concentration . Hybridization of chromosomal DNA samples of S . lincolnensis YY1, S . lincolnensis YY2, standard S . lincolnensis and S . lincolnensis YYc digested with SmaI with the probe of tsr gene gave signal corresponding to a fragment of 1.5 kb in the former two; Nevertheless, hybridization of chromosomal DNA digested with Hind III and Sma I using the probe of delta lacZ' gene resulted in positive fragment of 4.4 kb only in S . lincolnensis YY2 . Southern hybridizations indicate that S . lincolnensis YY1 is the result of homologous exchange while S . lincolnensis YY2 comes from bomologous recombination . To prove the existence of E . coli replicon and ampicillin resistant gene on the chromosome of S . lincolnensis YY2, its DNA digested with SphI was ligated and then transformed into E . coli JM83 competent cell . Two transformants named pSLE1 grew on the plate containing ampicillin . It's confirmed that pSLE1 is a part of pYYE04a1 from its digestion with different enzymes. Wei Sheng Wu Xue Bao, 2001 Oct, 41(5), 523 - 9 {The different functions of glnB and glnZ from Azospirillum brasilense YU62 in the control of nitrogen fixation}; Chen S et al.; The glnB and glnZ genes of A . brasilense have 70% homology at nucleotide sequence . glnB is located in a 3.7 kb Eco RI+ PstI fragment and glnZ is located in a 3.7 kb SalI fragment . Both glnB and glnZ genes were mutagenized by Kmr cassette insertions and glnB- and glnZ- mutants were obtained . glnB- mutant did not have any nitrogenase activity, while glnZ- mutant still has nitrogenase activity . The coding regions of glnB and glnZ were cloned into pVK100 vectors and recombinant plasmids pVK-II and pVK-Z were obtained, respectively . The recombinant plasmids pVK-II and pVK-Z were introduced into glnB- and glnZ- to produce C-glnB and C-glnZ, respectively . C-glnB can restore nitrogenase activity and C-glnZ does not have effect on nitrogenase activity . When pVK-II and pVK-Z were introduced into A . brasilense Yu62 and draT-, respectively, the Yu62-II (containing pVK-II) and draT-II (containing pVK-II) have higher nitrogenase activity than that of wild type Yu62 . In contrast, Yu62-Z (containing pVK-Z) and draT-Z (containing pVK-Z) has no effect on nitrogenase activity . The nifA(-)-II (containg pVK-II) and nifA(-)-Z (containing pVK-Z) still have no nitrogenase activity. J Infect Dis, 2003 Feb 1, 187(3), 418 - 23 Epub 2003 Jan 24. Inheritance of susceptibility to induced Escherichia coli bladder and kidney infections in female C3H/HeJ mice; Hopkins WJ et al.; In the present study, the inheritance of resistance and susceptibility to bladder and kidney infections in BALB/c, C3H/HeJ, F(1), and backcross mice was investigated, and the number of genes contributing to the phenotypes was estimated . Infections were induced in female mice by intravesical inoculation with Escherichia coli, and the number of bacteria in bladder and kidneys was quantified at 10 days . The (BALB/c x C3H/HeJ) F(1) mice had bladder and kidney infection intensities equivalent to those observed in the resistant BALB/c parents . Twelve percent of the (F(1) x C3H/HeJ) backcross mice had severe bladder infections, similar to the susceptible C3H/HeJ parents . Kidney infections ranging in intensity between those observed in BALB/c and C3H/HeJ parents were present in one-half of the backcross mice . Statistical analyses indicated that >/=1 gene is responsible for the increased susceptibility of C3H/HeJ mice and that the trait appears to be recessive. Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 952 - 7 Epub 2003 Jan 27. Determination of the energetics governing the regulatory step in growth hormone-induced receptor homodimerization; Bernat B et al.; Signaling in the human growth hormone (hGH)-human GH receptor system is initiated by a controlled sequential two-step hormone-induced dimerization of two hGH receptors via their extracellular domains (ECDs) . Little is currently known about the energetics governing the important regulatory step in receptor signaling (step 2) because of previously existing experimental barriers in characterizing the binding of the second receptor (ECD2) . A further complication is that ECD2 binds through contacts from two spatially distinct sites: through its N-terminal domain to hGH, and to ECD1 through its C-terminal domain, which forms a pseudo-2-fold symmetrical interaction between the stems of the two receptors . We report here a detailed evaluation of the energetics of step 2 binding using a modified surface plasmon resonance method that is able to measure accurately the kinetics of the trimolecular binding process and separate the effects of the two binding sites . The binding kinetics of 23 single and 126 ECD1-ECD2 pair-wise alanine mutations was measured . Although both of the ECD2 binding interfaces were found to be important, the ECD1-ECD2 stem-stem contact is the stronger of the two . It was determined that most residues in the binding interfaces act in additive fashion, and that the six residues common in both ECDs contribute very differently to homodimerization depending on which ECD they reside in . This interface is characterized by a binding "hot-spot" consisting of a core of three residues in ECD1 and two in ECD2 . There is no similar hot-spot in the N-terminal domain of ECD2 binding to Site2 of hGH . This study suggests ways to engineer ECD molecules that will bind specifically to either Site1 or Site2 of hGH, providing novel reagents for biophysical and biological studies. Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 856 - 61 Epub 2003 Jan 24. Structural evidence for substrate strain in antibody catalysis; Yin J et al.; The crystal structure of the Michaelis complex between the Fab fragment of ferrochelatase antibody 7G12 and its substrate mesoporphyrin has been solved to 2.6-A resolution . The antibody-bound mesoporphyrin clearly adopts a nonplanar conformation and reveals that the antibody catalyzes the porphyrin metallation reaction by straining/distorting the bound substrate toward the transition-state configuration . The crystal structures of the Fab fragment of the germ-line precursor antibody to 7G12 and its complex with the hapten N-methylmesoporphyrin have also been solved . A comparison of these structures with the corresponding structures of the affinity-matured antibody 7G12 reveals the molecular mechanism by which the immune system evolves binding energy to catalyze this reaction. J Biol Chem, 2003 Mar 28, 278(13), 11237 - 45 Epub 2003 Jan 27. C termini of the Escherichia coli mechanosensitive ion channel (MscS) move apart upon the channel opening; Koprowski P et al.; Heptameric YggB is a mechanosensitive ion channel (MscS) from the inner membrane of Escherichia coli . We demonstrate, using the patch clamp technique, that cross-linking of the YggB C termini led to irreversible inhibition of the channel activities . Application of Ni(2+) to the YggB-His(6) channels with the hexahistidine tags added to the ends of their C termini also resulted in a marked but reversible decrease of activities . Western blot revealed that YggB-His(6) oligomers are more stable in the presence of Ni(2+), providing evidence that Ni(2+) is coordinated between C termini from different subunits of the channel . Intersubunit coordination of Ni(2+) affecting channel activities occurred in the channel closed conformation and not in the open state . This may suggest that the C termini move apart upon channel opening and are involved in the channel activation . We propose that the as yet undefined C-terminal region may form a cytoplasmic gate of the channel . The results are discussed and interpreted based on the recently released quaternary structure of the channel. J Biol Chem, 2003 Apr 11, 278(15), 13235 - 43 Epub 2003 Jan 27. Functional roles of loops 3 and 4 in the cyclic nucleotide binding domain of cyclic AMP receptor protein from Escherichia coli; Chen R et al.; Cyclic AMP is a ubiquitous secondary message that regulates a large variety of functions . The protein structural motif that binds cAMP is highly conserved with the exception of loops 3 and 4, whose structure and length are variable . The cAMP receptor protein of Escherichia coli, CRP, was employed as a model system to elucidate the functional roles of these loops . Based on the sequence differences between CRP and cyclic nucleotide gated channel, three mutants of CRP were constructed: deletion (residues 54-56 in loop 3 were deleted), insertion (loop 4 was lengthened by 5 residues between Glu-78 and Gly-79) and double mutants . The effects of these mutations on the structure and function of CRP were monitored . Results show that the deletion and insertion mutations do not significantly change the secondary structure of CRP, although the tertiary and quaternary structures are perturbed . The functional data indicate that loop 3 modulates the binding affinities of cAMP and DNA . Although the lengthened loop 4 may have some fine-tuning functions, the specific function of the original loop 4 of CRP remains uncertain . The function consequences of mutation in loop 3 of CRP are similar to that of site A and site B in the regulatory subunits of cyclic AMP-dependent protein kinases . Thus, the roles played by loop 3 in CRP may represent a more common mechanism employed by cyclic nucleotide binding domain in modulating ligand binding affinity and intramolecular communication. Life Sci, 2003 Feb 21, 72(14), 1627 - 33 Marathon running alters the DNA base excision repair in human skeletal muscle; Radak Z et al.; Reactive oxygen and nitrogen species generated either as products of aerobic metabolism or as a consequence of environmental mutagens, oxidatively modify DNA . Formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (endo III) or their functional mammalian homologues repair 7,8-dihydro-8-oxoguanine (8-oxoG) and damaged pyrimidines, respectively, to curb the deleterious effects of oxidative DNA alterations . A single bout of physical exercise can induce oxidative DNA damage . However, its effect on the activity of repair enzymes is not known . Here we report that the activity of a functional homolog of Fpg, human 8-oxoG DNA glycosylase (hOGG1), is increased significantly, as measured by the excision of 32P labeled damaged oligonucleotide, in human skeletal muscle after a marathon race . The AP site repair enzyme did not change significantly . Despite the large individual differences among the six subjects measured, data suggest that a single-bout of aerobic exercise increases the activity of hOGG1 which is responsible for the excision of 8-oxoG . The up-regulation of DNA repair enzymes might be an important part of the regular exercise induced adaptation process. Biochem J, 2003 May 1, 371(Pt 3), 965 - 72 Chaperone properties of Escherichia coli thioredoxin and thioredoxin reductase; Kern R et al.; Thioredoxin, thioredoxin reductase and NADPH form the thioredoxin system and are the major cellular protein disulphide reductase . We report here that Escherichia coli thioredoxin and thioredoxin reductase interact with unfolded and denatured proteins, in a manner similar to that of molecular chaperones that are involved in protein folding and protein renaturation after stress . Thioredoxin and/or thioredoxin reductase promote the functional folding of citrate synthase and alpha-glucosidase after urea denaturation . They also promote the functional folding of the bacterial galactose receptor, a protein without any cysteines . Furthermore, redox cycling of thioredoxin/thioredoxin reductase in the presence of NADPH and cystine stimulates the renaturation of the galactose receptor, suggesting that the thioredoxin system functions like a redox-powered chaperone machine . Thioredoxin reductase prevents the aggregation of citrate synthase under heat-shock conditions . It forms complexes that are more stable than those formed by thioredoxin with several unfolded proteins such as reduced carboxymethyl alpha-lactalbumin and unfolded bovine pancreatic trypsin inhibitor . These results suggest that the thioredoxin system, in addition to its protein disulphide isomerase activity possesses chaperone-like properties, and that its thioredoxin reductase component plays a major role in this function. Biochemistry, 2003 Feb 4, 42(4), 1160 - 9 The initiating steps of a type II fatty acid synthase in Plasmodium falciparum are catalyzed by pfACP, pfMCAT, and pfKASIII; Prigge ST et al.; Malaria, a disease caused by protozoan parasites of the genus Plasmodium, is one of the most dangerous infectious diseases, claiming millions of lives and infecting hundreds of millions of people annually . The pressing need for new antimalarials has been answered by the discovery of new drug targets from the malaria genome project . One of the early findings was the discovery of two genes encoding Type II fatty acid biosynthesis proteins: ACP (acyl carrier protein) and KASIII (beta-ketoacyl-ACP synthase III) . The initiating steps of a Type II system require a third protein: malonyl-coenzyme A:ACP transacylase (MCAT) . Here we report the identification of a single gene from P . falciparum encoding pfMCAT and the functional characterization of this enzyme . Pure recombinant pfMCAT catalyzes malonyl transfer from malonyl-coenzyme A (malonyl-CoA) to pfACP . In contrast, pfACP(trans), a construct of pfACP containing an amino-terminal apicoplast transit peptide, was not a substrate for pfMCAT . The product of the pfMCAT reaction, malonyl-pfACP, is a substrate for pfKASIII, which catalyzes the decarboxylative condensation of malonyl-pfACP and various acyl-CoAs . Consistent with a role in de novo fatty acid biosynthesis, pfKASIII exhibited typical KAS (beta-ketoacyl ACP synthase) activity using acetyl-CoA as substrate (k(cat) 230 min(-1), K(M) 17.9 +/- 3.4 microM) . The pfKASIII can also catalyze the condensation of malonyl-pfACP and butyryl-CoA (k(cat) 200 min(-1), K(M) 35.7 +/- 4.4 microM) with similar efficiency, whereas isobutyryl-CoA is a poor substrate and displayed 13-fold less activity than that observed for acetyl-CoA . The pfKASIII has little preference for malonyl-pfACP (k(cat)/K(M) 64.9 min(-1)microM(-1)) over E . coli malonyl-ACP (k(cat)/K(M) 44.8 min(-1)microM(-1)) . The pfKASIII also catalyzes the acyl-CoA:ACP transacylase (ACAT) reaction typically exhibited by KASIII enzymes, but does so almost 700-fold slower than the KAS reaction . Thiolactomycin did not inhbit pfKASIII (IC(50) > 330 microM), but three structurally similar substituted 1,2-dithiole-3-one compounds did inhibit pfKASIII with IC(50) values between 0.53 microM and 10.4 microM . These compounds also inhibited the growth of P . falciparum in culture. Biochemistry, 2003 Feb 4, 42(4), 1109 - 17 MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli; Kambampati R et al.; Thionucleosides are uniquely present in tRNA . In many organisms, tRNA specific for Lys, Glu, and Gln contain hypermodified 2-thiouridine (s(2)U) derivatives at wobble position 34 . The s(2) group of s(2)U34 stabilizes anticodon structure, confers ribosome binding ability to tRNA and improves reading frame maintenance . Earlier studies have mapped and later identified the mnmA gene (formerly asuE or trmU) as required for the s(2)U modification in Escherichia coli . We have prepared a nonpolar deletion of the mnmA gene and show that it is not required for viability in E . coli . We also cloned mnmA from E . coli, and overproduced and purified the protein . Using a gel mobility shift assay, we show that MnmA binds to unmodified E . coli tRNA(Lys) with affinity in the low micromolar range . MnmA does not bind observably to the nonsubstrate E . coli tRNA(Phe) . Corroborating this, tRNA(Glu) protected MnmA from tryptic digestion . ATP also protected MnmA from trypsinolysis, suggesting the presence of an ATP binding site that is consistent with analysis of the amino acid sequence . We have reconstituted the in vitro biosynthesis of s(2)U using unmodified E . coli tRNA(Glu) as a substrate . The activity requires MnmA, Mg-ATP, l-cysteine, and the cysteine desulfurase IscS . HPLC analysis of thiolated tRNA digests using {(35)S}cysteine confirms that the product of the in vitro reaction is s(2)U . As in the case of 4-thiouridine synthesis, purified IscS-persulfide is able to provide sulfur for in vitro s(2)U synthesis in the absence of cysteine . Small RNAs that represent the anticodon stem loops for tRNA(Glu) and tRNA(Lys) are substrates of comparable activity to the full length tRNAs, indicating that the major determinants for substrate recognition are contained within this region. Biochemistry, 2003 Feb 4, 42(4), 1095 - 100 Role of glutamate-126 and arginine-144 in the lactose permease of Escherichia coli; Johnson JL et al.; Several previous studies have suggested that glutamate-126 and arginine-144 in the lactose permease of Escherichia coli form an ion pair that is essential for sugar binding . To further investigate the role of these residues, E126Q, R144Q, and R144S mutants were made . The R144Q and R144S strains, which had negligible levels of transport, were used as parental strains to isolate suppressor mutations that partially restored sugar transport . The R144Q parent only yielded first-site revertants, but the R144S strain produced three types of second-site replacements: E126Q, V229A, and L330R . In downhill transport assays, the E126Q strain was able to transport lactose at low levels, with an apparent K(m) 3-fold higher than the wild-type strain but a severely depressed apparent V(max) . A triple mutant, E126Q/R144S/V229A, showed a relatively robust V(max) value for downhill transport and could actively accumulate lactose against a concentration gradient . Taken together, these results indicate that Glu-126 and Arg-144 are not essential for sugar binding . An alternative explanation for their role in maintaining secondary structure is discussed. Biochemistry, 2003 Feb 4, 42(4), 1078 - 85 Structural/functional characterization of the alpha 2-plasmin inhibitor C-terminal peptide; Frank PS et al.; The alpha(2)-plasmin inhibitor (A2PI) is a main physiological regulator of the trypsin-like serine proteinase plasmin . It is composed of an N-terminal 15 amino acid fibrin cross-linking polypeptide, a 382-residue serpin domain, and a flexible C-terminal segment . The latter, peptide Asn(398)-Lys(452), and its Lys452Ala mutant were expressed as recombinant proteins in Escherichia coli (r-A2PIC and r-A2PICmut, respectively) . CD and NMR analyses indicate that r-A2PIC is flexible, loosely folded, and with low content of regular secondary structure . Functional characterization via intrinsic fluorescence ligand titrations shows that r-A2PIC interacts with the isolated plasminogen kringle 1 (r-K1) (K(a) approximately 69.9 mM(-)(1)), K4 (K(a) approximately 45.7 mM(-)(1)), K5 (K(a) approximately 4.3 mM(-)(1)), and r-K2 (K(a) approximately 3.2 mM(-)(1)), all of which are known to exhibit lysine-binding capability . The affinities of these kringles for r-A2PIC are consistently larger than those reported for the ligand N(alpha)-acetyllysine, a mimic of a C-terminal Lys residue . The r-A2PICmut, with a C-terminal Ala residue, also interacts with r-K1 and K4, although with approximately 5-fold lesser affinities relative to r-A2PIC, demonstrating that while Lys(452) plays a major role in the binding, internal residues in r-A2PIC tether the kringles . (1)H NMR spectroscopy shows that key aromatic residues within the K4 lysine-binding site (LBS), namely, Trp(25), Trp(62), Phe(64), Trp(72), and Tyr(74), selectively respond to the addition of r-A2PIC and r-A2PICmut, indicating that these interactions proceed via the kringles' canonical LBS . We conclude that r-A2PIC docks to kringles primarily through lysine side chains and that Lys(452) most definitely enhances the binding . This suggests that multiple Lys residues within A2PI could contribute, perhaps in a zipper-like fashion, to its binding to the in-tandem, multikringle array that configures the plasmin heavy chain. Biochemistry, 2003 Feb 4, 42(4), 1024 - 30 Binding of Zn-chlorin to a synthetic four-helix bundle peptide through histidine ligation; Razeghifard MR et al.; We have used a two histidine-containing synthetic peptide (Sharp et al . (1998) Proc . Natl . Acad . Sci . U.S.A . 95, 10465-10470) as a scaffold to bind Zn(II) chlorin e6 (ZnCe6) through histidine ligation . Protocols for the preparation and purification of the peptide using an Escherichia coli expression system are presented . Size-exclusion chromatography and circular dichroism measurements indicate that the peptide self-assembles into a four-helix bundle protein . Two variants of the peptide lacking either one or both of the histidine residues were used to demonstrate the stoichiometry of ZnCe6 binding . Comparison of the titration profiles determined by UV-vis spectroscopy for the purified one- and two-histidine peptides suggests that the two-histidine peptide can bind two ZnCe6 . The binding stoichiometry of ZnCe6 was verified by gel chromatography and native gel electrophoresis using the peptide variant lacking histidine residues as the control . Like many other chlorophyll analogue molecules, ZnCe6 can be photooxidized . The light-induced electron transfer between the ZnCe6-peptide complex and the added phenyl-p-benzoquinone was measured using time-resolved EPR spectroscopy and shown to be faster and have a higher yield than the electron transfer between unbound ZnCe6 and quinone . The implications of constructing a ZnCe6-peptide complex in terms of artificial photosynthesis are discussed. Biochemistry, 2003 Feb 4, 42(4), 864 - 9 Tautomeric rearrangement of a dihydroflavin bound to monomeric sarcosine oxidase or N-methyltryptophan oxidase; Khanna P et al.; Monomeric sarcosine oxidase (MSOX) and N-methyltryptophan oxidase (MTOX) are homologous bacterial flavoenzymes that contain covalently bound flavin {8alpha-(S-cysteinyl)FAD} . Reaction of MSOX or MTOX with a small excess of sodium borohydride results in immediate flavin reduction to a species that exhibits spectral properties (lambda(max) = 405 nm with a second broad peak at 332 nm) similar to those of 3,4-dihydroflavin . The borohydride-reduced enzymes retain full catalytic activity . Substrate reduction converts the 405 nm species to an air-sensitive tetrahydroflavin that reacts with oxygen to yield unmodified oxidized enzyme . Unexpectedly, the putative 3,4-dihydroflavin bound to MSOX or MTOX is unstable in the absence of substrate . An isosbestic conversion of the 405 nm species to yield unmodified, oxidized flavin is observed when the reaction is conducted under aerobic conditions (k(obs) = 4.9 x 10(-2) min(-1)) . Under anaerobic conditions, an oxygen-sensitive species resembling 1,5-dihydroflavin is formed in an isosbestic reaction that occurs at a rate similar to that of the aerobic reaction (k(obs) = 5.3 x 10(-2) min(-1)) . Possible reaction of the 3,4-dihydroflavin with a second molecule of borohydride to yield an air-sensitive tetrahydroflavin is unlikely since prior scavenging of residual borohydride with excess formaldehyde had no effect on the aerobic conversion to unmodified oxidized flavin . The observed instability is attributed to a tautomeric rearrangement of the 3,4-dihydroflavin to generate 1,5-dihydroflavin, a species that is also air-sensitive . Evidence in favor of an active site facilitated tautomerization reaction is provided by the fact that the stability of the 405 nm species formed with MSOX is enhanced 200-fold upon denaturation with urea or heat . The observed tautomeric rearrangement of 3,4-dihydroflavin may provide insight regarding a related flavin tautomerization reaction that has been proposed as a key step in the biosynthesis of covalent flavin linkages. Wei Sheng Wu Xue Bao, 1998 Feb, 38(1), 26 - 31 {Research on the regulation of glucoamylase gene(glaA) expression in A . niger . II . Analysis of the function of 5'-regulatory region of A . niger T21 and 3.795 glaA gene}; Qiao D et al.; Two plasmid vectors pXH2 and pGH1 were constructed through the fusion of E . coli hph gene, the report gene and the 5' upstream regions of A . niger T21 and 3.795 respectively, as well as the terminator of A . nidulans trpC gene . The plasmid vectors were than used to transform A . niger T21 to functionally identify those different basic groups between the two 5' upstream regions responsible for high-level expression of the glaA gene . Southern analysis of two transformants XH2C and GH1C revealed that pXH2 and pGH1 were integrated respectively into the chromosome at same site with two copies in tandem array . The level resistant to HmB(3000 micrograms/ml) of XH2C was twice as high as that (1500 micrograms/ml) of GH1C, indicating that the changes of basic groups through mutation result in twice increase of functional level of region responsible for transcription and regulation of A . niger T21 glaA gene compared with that of 3.795. Wei Sheng Wu Xue Bao, 1998 Apr, 38(2), 103 - 7 {Studies on the selection of strains producing colominic acid and culture conditions}; Guo L et al.; One strain E . coli C-8, the highest yield of colominic acid, was selected from 40 E . coli strains in the medium in which glucose and ammonium sulfate were the only carbon and nitrogen resources . An optimum medium for the growth and colominic acid production of E . coli C-8 was studied . The optimum carbon resources for colominic acid production was sorbitol selected from 16 kinds of carbons, and its optimum concentration was 2.5% . The optimum inorganic and organic nitrogen resources for colominic acid production were ammonium sulfate (0.5%) and tryptone (1.5%), respectively . The optimum concentration of K2 HPO4 was 90 mmol/L . The optimum culture temperature was 37 degrees C, but no colominic acid was produced below 20 degrees C . The optimum pH range was 7.5-8.2 . The strain growth in the optimum medium kept logarithmic phase in 40 h (maxim A = 22) . The colominic acid secreted into medium was much lower before 20 h, but high biosynthetic rate of colominic acid was detected after 40 h, the colominic acid reached the highest level (1,200 micrograms/ml) at 65 h. Wei Sheng Wu Xue Bao, 1998 Jun, 38(3), 193 - 6 {Expression, purification and characterization of recombinant human cyclophilin A}; Li F et al.; Plasmid-derived expression of the human CyPA in E . coli would make it possible to obtain ample protein quantities and to avoid difficult task of obtaining human tissues for protein purification . The cDNA encoding human CyPA from MT4 lymph cell line has been cloned and an expression vector (pET11/CyPA) has been constructed under control of the T7 promoter for efficient expression in E . coli . The recombinant CyPA is produced at 41% of total soluble cell protein, and showed the peptidyl-prodyl cis-trans isomerase activity. Wei Sheng Wu Xue Bao, 2001 Feb, 41(1), 65 - 9 {Construction of plasmid gene bank of V . cholerae O139 and detection of O-antigen genes}; Qu D et al.; Because O-antigen biosynthesis genes are a tandem gene cluster . Gnomic fragments of 4-20 kilobases (kb) were obtained by digesting genomic DNA of V . cholerae O139 with restriction endonuclease EcoRI, then plasmid gene bank was constructed . Recombinant colony, E . coli DH5 alpha (pMG320), expressing O-antigen of V . cholerae O139 was detected from the bank by immunological agglutinative reaction . The futher analysis showed O-antigen expressed by recombinant colony had both immunogenicity and reactogenicity, and the size of O-antigen biosynthesis genes was about 4.6 kb. Wei Sheng Wu Xue Bao, 2001 Feb, 41(1), 9 - 15 {Subcloning and sequencing of DNA fragment related to salt tolerance in Sinorhizobium meliloti 042B}; Ge S et al.; A 4 kb ClaI DNA fragment related to salt tolerance from S . meliloti 042B was digested by HindIII down 2.4 kb fragment, and a 1.6 kb ClaII-HindIII fragment was retained on plasmid pML122 . Then, the 2.4 kb DNA fragment was ligated with plasmid pBBR1-MCS2, and the recombinant plasmid was transformed to E . coli DH5 alpha, and transformant GS2 was obtained . Three-parental mating experiments were carried out with transformant GS2 as donor, salt sensitive strains GZ17 as recipient and pRK2013 as helper plasmid, then the transconjugant GG2 was selected on FY plates containing kanamycin and 0.4 mol/L NaCl . The remaining DNA fragment was self ligated with pML122 and then transformed into E . coli S17-1 and transformat GS0 was obtained . Two-parental mating experiment was carried out with transformant GS0 as donor and salt sensitive strain GZ17 as recipient, but no transconjugant was obtained on the FY plates . Then, the 2.4 kb HindIII DNA fragment was ligated into sequencing vector pGEM-7Zf(+) for sequencing . The result of sequencing and analysis showed that the 2.4 kb DNA fragment contained three ORFs . According to the result of sequencing, further subcloning was conducted and 1.9 kb HindIII-Sac II DNA fragment related to salt tolerance was obtained. Wei Sheng Wu Xue Bao, 2000 Dec, 40(6), 605 - 9 {The construction and application of Streptomyces-E . coli shuttle plasmid pSGLgpp}; Zhang H et al.; The high-copy-number plasmid pSGL1(7.4 kb) was isolated from Streptomyces geobisporous . Its minimal replicon had been determined and sequenced . With the total DNA of S . geobisporus as template, the DNA fragment involving C1027 apoprotein signal peptide encoding sequence (gpp) was amplified by PCR . After this fragment was inserted into the plasmid pSGLN, a derivative plasmid of pSGL1, one new Streptomyces-E . coli shuttle plasmid was obtained namely as pSGLgpp . Using the new vector, we carried out the expression of hsIL-1RI in streptomyces. Shi Yan Sheng Wu Xue Bao, 2000 Sep, 33(3), 229 - 35 {Cloning, expression of a segment of hTERT gene and detection of telomerase and hTERT by anti-hTERT polyclonal antibody}; Saleh SA et al.; Telomerase is an important biomarker in cancer cells . It is active in germline cells, most of cancer tissues and cell lines, but not in most somatic tissues . Telomerase is composed of two components, and while hTER is present in normal and tumor cells, expression of hTERT appears to be highly regulated and correlates with telomerase activity . In order to detect the telomerase enzyme and hTERT protein, anti-hTERT polyclonal antibodies were produced in this study . A segment of hTERT cDNA was amplified by RT-PCR and cloned into the multi-cloning site of the GST gene fusion vector pGEX-5X-3 . After the recombinant plasmid was expressed in E . coli BL21, the fusion protein was purified for immunization . Extracts from several cultured cells were analyzed by Western blot, and the results indicated that telomerase enzyme and hTERT protein could be specifically detected by this anti-hTERT antibod' . Thus, a simple and effective method was primarily established for the immunodetection of telomerase enzyme and hTERT protein. Wei Sheng Wu Xue Bao, 2001 Apr, 41(2), 248 - 51 {Cloning and expression of D-arabitol dehydrogenase gene from Acetobacter suboxydans in Escherichia coli}; He X et al.; The partial genomic library of Acetobacter suboxydans was constructed using Yeast-E . coli shuttle plasmid YEp352 as vector . Two positive transformants, designated as DH5 alpha(pAD91) and DH5 alpha(pAD98), were obtained by screening the growth of transformants on the agar plate in which D-arabitol was used as the sole carbon source . The results of Southern blot and restriction endonuclease analysis showed that the two recombinants are identical . The insert is about 2.3 kb . Arabitol dehydrogenase activity assay indicated that the transformants could produce D-xylulose-forming D-arabitol dehydrogenase . Hence, the gene encoding D-arabitol dehydrogenase exists in the cloned DNA fragment. Wei Sheng Wu Xue Bao, 2001 Apr, 41(2), 186 - 90 {Purification and gene cloning of a novel fibrinolytic protease from Streptomyces sp . C3662}; Gong Y et al.; A novel fibrinolytic protease from Streptomyces sp . C3662 was purified by (NH4)2SO4 fractionation, DEAE-Sepharose and CM-Sepharose chromatography . The molecular weight of the protease was indicated to be 30 kD by SDS-PAGE . Using a E . coli/Streptomyces shuttle plasmid pIJ699 as the vector, shot-gun cloning was performed to clone the gene of the protease . One clone with fibrinolytic activity harboring a plasmid that contains a DNA fragment of 6 kb was obtained from 3000 clones . Sequence analysis reveals that the open reading frame of the gene of the protease is 903 bp in size, encoding a putative protein of 300 amino acids with 30 kD . The overall GC% and the third codon GC% of the ORF were 68.33% and 95.6%, respectively . Comparison of homologue showed that the purative protein is highly homologous with other proteases of Streptomyces sp. Wei Sheng Wu Xue Bao, 2001 Apr, 41(2), 181 - 5 {Cloning of promelittin cDNA and its expression in Escherichia coli}; Wang G et al.; The cDNA encoding promelittin was obtained from the total RNA of bee poison gland by RT-PCR and was cloned to pT7Blu-T vector . The expression vector of promelittin fused with partial sequence of beta-galactosidase was constructed by ligating the fragment inserted to pUC118 . Moreover, it was expressed in the strain DH5 alpha of Escherichia coli . The result of DNA sequence analysis demonstrated that the obtained cDNA sequence was same with the published one and the reading frame of fusion gene was correct. Wei Sheng Wu Xue Bao, 2001 Apr, 41(2), 173 - 80 {Analysis and expression of Hyphantria cunea nuclear polyhedrosis virus sod gene}; Cao G et al.; The sequencing results indicated that Hyphantria cunea nuclear polyhedrosis virus (HcNPV) sod gene open reading frame of 456 nt encoding protein of 151 amino acid, was identified to that of Bombyx mori nuclear polyhedrosis virus (BmNPV), and exhibited 97.2% homology at nucletde level to that of Autographa californica nuclear polyhedrosis virus (AcNPV), three amino acid residues difference in amino acid level with AcNPV sod . The essential amino acid residues for the construction and active could be detected in HcNPV sod . Activity of the SOD is 147.09 U per milliliter E . coli. Wei Sheng Wu Xue Bao, 2000 Jun, 40(3), 284 - 9 {Study on production of poly-beta-hydroxybutyrate by recombinant strain VG1(pTU14)}; Yu H et al.; Cloning and expression of Vitreoscilla hemoglobin gene (vgb) and lambda phage lytic genes (S-RRz) in production of Poly-beta-hydroxybutyrate(PHB) was studied in different Escherichia coli hosts such as E . coliJM105, E . coliJM109 and VG1 . In the recombinant strains, VG1(pTU14) was a superior one which simultaneously contained three exogenes including vgb, S-RRz and PHB biosynthesis genes(phbCAB) . The experimental results showed that after 82 hours fed-batch cult ure in LBG medium in shaking flask, cell concentration of VG1(pTU14) could reach 25.9 g/L, which was the highest PHB production ever reported, and till 52 h PHB content could be higher than 95% . Additionally, inducible cell lysis was also attained successfully in recombinant VG1(pTU14) accompanying with its high cell density culture . Therefore, VG1(pTU14) is a novel potential strain with promising prospect in industrial production of PHB. Wei Sheng Wu Xue Bao, 2000 Jun, 40(3), 270 - 6 {Selection and recombinant expression of an immunodominant B cell epitope of HIV gp41}; Du Y et al.; A novel method was designed for disease-specific B cell epitope mapping and epitope expression in E . coli . A phage library displaying random dodecamers was biopanned first with human total IgG antibodies against HIV-1, and then non-specific phages were subtracted by HIV(-) polyclonal antibodies . After three rounds of screening, the positive phages were tested in an ELISA for their reactivity with HIV(+)-IgG and HIV(-)-IgG antibodies . Phages that showed positive reactivity with HIV(+)-IgG, but negative to HIV(-)-IgG, were selected and their displayed peptides were determined by DNA sequencing . All the 13 positive clones sequenced displayed five kinds of peptides (SPKCLGKLLCAF, THQCLGKLQCGV, SCSAKFTCTTQI, KSDCSARFMCSV, DCLKQWACEWSR) that have homology to the HIV-1 gp41(602GCSGKLICTTNV613), demonstrating there is a dominant epitope in the region. Wei Sheng Wu Xue Bao, 2000 Aug, 40(4), 384 - 8 {Cloning and expression of metallothionein gene of Bombyx mori}; He X et al.; Yeast MTI gene from vector pCMI-1 was used as a probe . It appeared strong hybridization signals when the total DNA of Bombyx mori Huishu eggs hybridizes with the probe . The 1-6 kb DNA fragments were isolated from the EcoR I digested total DNA of Bombyx mori Huishu eggs and ligated with M13- vector digested by restriction EcoR I . The ligation mixtures were used to transform E . coli DH5 alpha . blue/White colonies selection was used to identify colonies with insert . Approximately 4000 white colonies were selected, so the part genomic library of Bombyx mori was constructed . Three positive colonies were gained from the genomic library by southern blotting analysis, designated T1 (pZHC-1), T5 (pZHC-5), T7 (pZHC-7) . Digesting the recombinant plasmid pZHC-5 with 12 restriction enzymes, the results suggested that the inserted fragment was about 1.2 kb and there was only a Hind III site . The experiment of resistant to CuSO4 proved that the DH5 alpha cells contain recombinant plasmids were more resistance than the recepient DH5 alpha cells . According to these results, the inserted fragment possibly contains the gene encoding Metallothionein of Bombyx mori . The sequence analysis of the inserted fragment and its high-expressions in E . coli are in progress. Wei Sheng Wu Xue Bao, 2000 Aug, 40(4), 359 - 64 {Overproduction and partial characterization of the Ssh7a and Ssh7b proteins from Sulfolobus shibatae}; Chen X et al.; The Sulfolobus shibatae Ssh7a and Ssh7b proteins have been separately overproduced in Escherichia coli and purified using a simple procedure which includes a heat treatment step . The recombinant and native Ssh7 proteins are similar in the ability to bind both negatively supercoiled and relaxed DNAs . In addition, the recombinant Ssh7a resembles the native Ssh7 protein in constraining negative DNA supercoils . Our data suggest that the two isoforms of Ssh7 interact with DNA in a similar fashion, and the methylation state of Ssh7 interact with DNA in a similar fashion, and the methylation state of Ssh7 does not affect DNA binding and supercoil constraining by the protein. Wei Sheng Wu Xue Bao, 2000 Apr, 40(2), 155 - 60 {Construction of a novel Bm NPV Bac to Bac system}; Deng X et al.; A Bi-Shuttle vector Bm-Bacmid was constructed by co-infecting Bm N cells with wild type genomic DNA from BmNPV and Ac-Bacmid DNA . It could not only replicate in E . coli cells as a large plasmid and but also remain infectious when induced into Bm N or Sf9 cells . Recombinant virus rBmHBe was obtained after transposition of a donor plasmid carrying Hepatitis Be antigen gene (HBeAg) into att Tn7, and was demonstrated by Southern blotting . SDS-PAGE analysis showed that HBeAg gene were highly expressed in Bm N cells . By ELISA testing, the highest antigenecity titer of HBeAg protein in cell cultural medium was up to a dilution of 1:32,000 . Although HBeAg protein also presented in the Bm N cells the titer was only 1:2000 . The HBcAg protein was much fewer than HBeAg (< 1:160) whatever in culture medium and in cells . The results showed that Bm N cells was able to recognize the signal peptide sequence and cut it correctly for HBeAg protein's excreting production. Wei Sheng Wu Xue Bao, 2000 Apr, 40(2), 121 - 5 {Expression, purification and characterization of fruA, a transcription factor in Myxococcus xanthus}; Mao X et al.; FruA is a transcription factor essential for the development of Myxocoxccus xanthus . Gene encoding fruA with a poly-histidine tag was expressed in E . coli and simply purified by chromatography on nickel column . Data from gel retardation assay suggest that FruA regulates transcription of target genes in collaboration with other factors. Wei Sheng Wu Xue Bao, 2000 Feb, 40(1), 50 - 6 {Expression of Vitreoscilla hemoglobin gene in Streptomyces avermitilis}; Wen Y et al.; Two expression vectors, pWY101 and pWY102, were constructed by cloning Vitreoscilla hemoglobin gene(vhb) with its native oxygen-regulated promoter into E . coli-Streptomyces shuttle vector pIJ653 . They were introduced into Streptomyces avermitilis, but Western blotting experiment failed to detect vhb gene expression . pHZ1252 is another shuttle vector for expressing VHb, in which vhb structural gene is controlled by a strong, thiostrepton-inducible Streptomyces promoter PtipA . pHZ1252 was transformed into S . avermitilis and expressed VHb which had biological activity after thiostrepton induction . pHZ1252 was structurally unstable in S . avermitilis, occurring deletion recombination . However, the rest part of pHZ1252 was stable in S . avermitilis and still contained vhb gene and PtipA . Plasmid pHZ1252 isolated from S . avermitilis was unable to transform E . coli, showing the loss of part of E . coli plasmid from pHZ1252. Proteomics, 2003 Jan, 3(1), 36 - 44 A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard; Alban A et al.; The comparison of two-dimensional (2-D) gel images from different samples is an established method used to study differences in protein expression . Conventional methods rely on comparing images from at least 2 different gels . Due to the high variation between gels, detection and quantification of protein differences can be problematic . Two-dimensional difference gel electrophoresis (Ettan trade mark DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples . In the application of DIGE different samples are labelled with mass and charge matched spectrally resolvable fluorescent dyes and are then separated on the same 2-D gel . Using an Escherichia coli lysate "spiked" with varying amounts of four different known proteins, we have tested a novel experimental design that exploits the sample multiplexing capabilities of DIGE, by including a standard sample in each gel . The standard sample comprises equal amounts of each sample to be compared and was found to improve the accuracy of protein quantification between samples from different gels allowing accurate detection of small differences in protein levels between samples. Biopolymers, 2003 Feb, 68(2), 139 - 49 Stepwise induced fit in the pico- to nanosecond time scale governs the complexation of the even-skipped transcriptional repressor homeodomain to DNA; Flader W et al.; Induced fit effects in the complex of a DNA decamer with two even-skipped transcriptional repressor homeodomain molecules were investigated by means of molecular dynamics simulations . Dynamics of these effects are found to be in the time scale from pico- to nanoseconds . First steps are made by the fast-moving DNA backbone phosphates, which upon binding change their B(I)/B(II) substate distribution . Further rearrangements in the DNA double helix induced upon complexation, like bending of the helix axis, changes of the minor groove width, and of different helical parameters, are slower and occur within a few nanoseconds . The flexibility of the DNA, especially of its backbone, seems thereby to play an important role for specific DNA ligand recognition . J Pediatr Gastroenterol Nutr, 2003 Feb, 36(2), 253 - 60 Enteropathogenic Escherichia coli: stimulating neutrophil migration across a cultured intestinal epithelium without altering transepithelial conductance; Michail SK et al.; INTRODUCTION: Migration of neutrophils across the intestinal epithelium is the hallmark of inflammatory conditions of the bowel . In cultured intestinal epithelial monolayer models, neutrophils can be induced to migrate along a chemotactic gradient such as n-formyl-methionyl-leucyl-phenylalanine (fMLP) . Physical passage of the neutrophils across the epithelium could disrupt the tight-junctions, possibly leading to a large increase in the transepithelial conductance (G(t)) . The goal of this study is to determine whether transepithelial migration of neutrophils induced by enteropathogenic (EPEC) causes changes in G(t) comparable with those seen with fMLP . METHODS: The apical side of T84 monolayers were rapidly infected with EPEC E2348/69 or exposed to 1 microM fMLP . A third group of monolayers exposed to neither EPEC nor fMLP served as control . Indium-labeled neutrophils were added to the serosal side of monolayers grown on a cell culture insert membrane (12 microm pores) . G(t) was measured at fixed intervals up to 4 hours . After a 150-minute incubation, radioactivity of the neutrophils that migrated to the apical side was assayed and the number of migrating neutrophils was calculated . RESULTS: At 150 minutes, EPEC induced similar neutrophil chemotactic capability compared to fMLP (231 +/- 34.10(3) and 193 +/- 48.10(3), respectively, n = 13, P > 0.05) . However, EPEC-induced neutrophil migration was not associated with significant increase in G(t), 1.13 +/- 0.16 fold of baseline G(t), in distinction with fMLP groups, 13.3 +/- 0.48 fold, n = 7 (P< 0.05) . G(t) changes with EPEC were seen after 4 hours of infection, but were not different in the presence or absence of neutrophil migration (1.37 +/- 0.12 fold and 1.42 +/- 0.17 fold of baseline G(t), respectively) . CONCLUSIONS: The results indicate that EPEC-induced neutrophil migration can occur without significant disruption of barrier function . In addition, the chemo-attractant recruiting neutrophils during EPEC infection is unlikely to be fMLP; and, the G(t) increase seen with fMLP-driven recruitment may indicate a discretionary compromise of barrier function during neutrophil migration. Biophys J, 2003 Feb, 84(2 Pt 1), 1131 - 45 Fluorescence resonance energy transfer over approximately 130 basepairs in hyperstable lac repressor-DNA loops; Edelman LM et al.; Lac repressor (LacI) binds two operator DNA sites, looping the intervening DNA . DNA molecules containing two lac operators bracketing a sequence-directed bend were previously shown to form hyperstable LacI-looped complexes . Biochemical studies suggested that orienting the operators outward relative to the bend direction (in construct 9C14) stabilizes a positively supercoiled closed form, with a V-shaped LacI, but that the most stable loop construct (11C12) is a more open form . Here, fluorescence resonance energy transfer (FRET) is measured on DNA loops, between fluorescein and TAMRA attached near the two operators, approximately 130 basepairs apart . For 9C14, efficient LacI-induced energy transfer ( approximately 74% based on donor quenching) confirms that the designed DNA shape can force the looped complex into a closed form . From enhanced acceptor emission, correcting for observed donor-dependent quenching of acceptor fluorescence, approximately 52% transfer was observed . Time-resolved FRET suggests that this complex exists in both closed- and open form populations . Less efficient transfer, approximately 10%, was detected for DNA-LacI sandwiches and 11C12-LacI, consistent with an open form loop . This demonstration of long-range FRET in large DNA loops confirms that appropriate DNA design can control loop geometry . LacI flexibility may allow it to maintain looping with other proteins bound or under different intracellular conditions. Ann N Y Acad Sci, 2002 Dec, 981, 111 - 34 Genome organization and reorganization in evolution: formatting for computation and function; Shapiro JA; This volume deals with the role of epigenetics in life and evolution . The most dynamic forms of functional genome formatting involve DNA interacting with cellular complexes that do not alter sequence information . Such important epigenetic phenomena are the main subjects of other articles in this volume . This article focuses on the long-lived form of genome formatting that lies within the DNA sequence itself . I argue for a computational view of genome function as the long-term information storage organelle of each cell . Structural formatting consists of organizing various signals and coding sequences into computationally ready systems facilitating genome expression and genome transmission . The basic features of genome organization can be understood by examining the E . coli lac operon as a paradigmatic genomic system . Multiple systems are connected through distributed signals and repetitive DNA to form higher-order genome system architectures . Molecular discoveries about mechanisms of DNA restructuring show that cells possess the natural genetic engineering functions necessary for evolutionary change by rearranging genomic components and reorganizing system architectures . The concepts of cellular computation and decision-making, genome system architecture, and natural genetic engineering combine to provide a new way of framing evolutionary theories and understanding genome sequence information. Fish Shellfish Immunol, 2003 Jan, 14(1), 1 - 23 The rainbow trout classical MHC class I molecule Onmy-UBA*501 is expressed in similar cell types as mammalian classical MHC class I molecules; Dijkstra JM et al.; Onmy-UBA is a polymorphic classical major histocompatibility (MHC) class I locus in rainbow trout (Oncorhynchus mykiss) . A common allomorph is Onmy-UBA*501, which has been detected in several wildtype strains, in the clonal homozygous rainbow trout C25 and, in the current study, in the rainbow trout gonad cell line RTG-2 . The extracellular domain of this allomorph was expressed in E . coli and a murine monoclonal antibody designated H9 was generated against the recombinant protein . In Western blot analysis Mab H9 specifically recognised an n-glycosylated protein of 45 kDa in leucocytes and erythrocytes of C25 fish and in RTG-2 cells . The level of Onmy-UBA*501 expression in erythrocytes was very low . Immunocytochemistry of isolated cells indicated expression in lymphocytes, macrophages, neutrophils, erythrocytes, RTG-2 cells and Onmy-UBA *501 transfected CHO cells, but not in untransfected CHO cells . Immunohistochemistry using frozen sections of C25 fish indicated that Onmy-UBA*501 expression is strong in the lymphoid organs (thymus, head kidney and spleen) and in the epithelia and endothelia of several organs . No significant expression was observed in muscle fibres, hepatocytes or neurons . These observations demonstrate that in jawed fish, the lowest phylogenetic group possessing an MHC system, the classical MHC class I molecules are expressed in similar cell types as in higher vertebrates. Vaccine, 2003 Feb 14, 21(9-10), 836 - 42 The adjuvant OM-174 induces both the migration and maturation of murine dendritic cells in vivo; Pajak B et al.; The aim of this study was to test the capacity of the novel adjuvant OM-174, a lipid A analog, to induce the migration and the maturation of murine dendritic cells (DC) in vivo, a step which is considered as the initiation of the adaptive immune response . BALB/c mice were injected intravenously or subcutaneously with OM-174 . The spleen and popliteal lymph nodes were harvested, and analyzed for DC localization and phenotype . The data presented here clearly show that, OM-174 induces the migration of DC from the periphery to the T cell areas of lymphoid organs, and their maturation into cells expressing high levels of MHC class II and co-stimulatory molecules, with a potency close to that of Escherichia coli lipopolysaccharide (LPS). Mutat Res, 2003 Feb 5, 535(1), 55 - 72 Mutagenicity test system based on a reporter gene assay for short-term detection of mutagens (MutaGen assay); Schmid C et al.; The construction of a bacterial mutation assay system detecting reversions of base substitutions and frameshifts in tetracycline (tet) and ampicillin resistance genes located on low copy plasmids is described . Frameshift mutations were introduced into repetitive GC-sequences and G-repeats known to be mutagenic hot-spots . Base pair substitutions were inserted in or around the active site of the ampicillinase gene thus generating reversibility of the ampicilline sensitivity . The plasmids carry genes to enable sensitive, fast and specific detection of mutagens in bacteria . MucAB was cloned into the test plasmid to enhance error-prone DNA-repair . The conventional reversion principle has been combined with the luminometric measurement of an inducible reporter gene . The revertants are detected after induction of the beta-galactosidase-producing lacZ-gene either controlled by its natural lac-promotor or by the more stringently repressed (anhydrotetracyclin inducible) tetA promotor . The tester strains containing the tetA/lacZ reporter gene construct can grow in full medium over the complete assay . This test procedure enables screening for mutations within one working day . Incubation for 16 h reveals high sensitivity . J Mol Biol, 2003 Feb 7, 326(1), 217 - 23 Solution structure of the R3H domain from human Smubp-2; Liepinsh E et al.; The R3H domain is a conserved sequence motif, identified in over 100 proteins, that is thought to be involved in polynucleotide-binding, including DNA, RNA and single-stranded DNA . In this work the 3D structure of the R3H domain from human Smubp-2 was determined by NMR spectroscopy . It is the first 3D structure determination of an R3H domain . The fold presents a small motif, consisting of a three-stranded antiparallel beta-sheet and two alpha-helices, which is related to the structures of the YhhP protein and the C-terminal domain of the translational initiation factor IF3 . The similarities are non-trivial, as the amino acid identities are below 10% . Three conserved basic residues cluster on the same face of the R3H domain and could play a role in nucleic acid recognition . An extended hydrophobic area at a different site of the molecular surface could act as a protein-binding site . A strong correlation between conservation of hydrophobic amino acids and side-chain solvent protection indicates that the structure of the Smubp-2 R3H domain is representative of R3H domains in general. J Mol Biol, 2003 Feb 7, 326(1), 203 - 16 Aspartate transcarbamylase from the hyperthermophilic archaeon Pyrococcus abyssi: thermostability and 1.8A resolution crystal structure of the catalytic subunit complexed with the bisubstrate analogue N-phosphonacetyl-L-aspartate; Van Boxstael S et al.; The Pyrococcus abyssi aspartate transcarbamylase (ATCase) shows a high degree of structural conservation with respect to the well-studied mesophilic Escherichia coli ATCase, including the association of catalytic and regulatory subunits . The adaptation of its catalytic function to high temperature was investigated, using enzyme purified from recombinant E.coli cells . At 90 degrees C, the activity of th