Mol Microbiol, 1998 Apr, 28(2), 395 - 401 High-affinity binding sites for the initiator protein DnaA on the chromosome of Escherichia coli; Roth A et al.; The initiator protein DnaA of Escherichia coli binds with unusually high affinity to five regions on the chromosome, in addition to the replication origin, oriC . Using a solid-phase DNA binding assay, in which the DNA binding C-terminal domain of DnaA is bound via a biotin tag to magnetic beads, we could fish only fragments with these six regions from different chromosomal digests . Except for oriC, these fragments contain only one or two consensus DnaA binding sites, DnaA boxes . The distribution of these high-affinity DnaA boxes on the chromosome is random. Mol Microbiol, 1998 Apr, 28(2), 383 - 93 Metronidazole resistance in Helicobacter pylori is due to null mutations in a gene (rdxA) that encodes an oxygen-insensitive NADPH nitroreductase; Goodwin A et al.; Metronidazole (Mtz) is a critical component of combination therapies that are used against Helicobacter pylori, the major cause of peptic ulcer disease . Many H . pylori strains are Mtz resistant (MtzR), however, and here we show that MtzR results from loss of oxygen-insensitive NADPH nitroreductase activity . The underlying gene (called 'rdxA') was identified in several steps: transformation of Mtz-susceptible (MtzS) H . pylori with cosmids from a MtzR strain, subcloning, polymerase chain reaction (PCR) and DNA sequencing . We also found that (i) E . coli (normally MtzR) was rendered MtzS by a functional H . pylori rdxA gene; (ii) introduction of rdxA on a shuttle vector plasmid into formerly MtzR H . pylori rendered it MtzS; and (iii) replacement of rdxA in MtzS H . pylori with an rdxA::camR null insertion allele resulted in a MtzR phenotype . The 630 bp rdxA genes of five pairs of H . pylori isolates from infections that were mixed (MtzR/MtzS), but uniform in overall genotype, were sequenced . In each case, the paired rdxA genes differed from one another by one to three base substitutions . Typical rdxA genes from unrelated isolates differ by 5% in DNA sequence . Therefore, the near identity of rdxA genes from paired MtzR and MtzS isolates implicates de novo mutation, rather than horizontal gene transfer in the development of MtzR . Horizontal gene transfer could readily be demonstrated under laboratory conditions with mutant rdxA alleles . RdxA is a homologue of the classical nitroreductases (CNRs) of the enteric bacteria, but differs in cysteine content (6 vs . 1 or 2 in CNRs) and isoelectric point (pI=7.99 vs . 5.4-5.6), which might account for its reduction of low redox drugs such as Mtz . We suggest that many rdxA (MtzR) mutations may have been selected by prior use of Mtz against other infections . H . pylori itself is an early risk factor for gastric cancer; the possibility that its carcinogenic effects are exacerbated by Mtz use, which is frequent in many societies, or the reduction of nitroaromatic compounds to toxic, mutagenic and carcinogenic products, may be of significant concern in public health. Mol Microbiol, 1998 Apr, 28(2), 371 - 81 Regulation of type 1 fimbrial expression in uropathogenic Escherichia coli: heterogeneity of expression through sequence changes in the fim switch region; Leathart JB et al.; Over 80% of uropathogenic Escherichia coli express type 1 fimbriae . Expression is phase variable, and regulation of phase switching can differ between isolates . Previously, this was explained by differences in the expression of the fim recombinases, FimB and FimE . Our study of 50 uropathogenic E . coli isolates confirms variation in the regulation of type 1 fimbriae but, in many cases, the variation could be accounted for by sequence changes within and adjacent to the fim switch, rather than by differences in recombinase expression . This was demonstrated by moving the switch from the isolates into an isogenic background and comparing the switching behaviour with that of the original isolate . Isolates could be arranged into groups based on fim switch regulation and sequence similarity . In certain cases, the altered regulation was located to specific basepair changes within the fim switch . Sequence changes were found that had a marked effect on the activity of either FimB or FimE switching, while others affected FimB switching in only one direction . These results emphasize the value of using naturally selected sequence variation to further the understanding of gene regulation. Mol Microbiol, 1998 Apr, 28(2), 333 - 42 Structure of the chromosomal insertion site for pSAM2: functional analysis in Escherichia coli; Raynal A et al.; The element pSAM2 from Streptomyces ambofaciens integrates into the chromosome through site-specific recombination between the element (attP) and the chromosomal (attB) sites . These regions share an identity segment of 58bp extending from the anti-codon loop through the 3' end of a tRNA(Pro) gene . To facilitate the study of the attB site, the int and xis genes, expressed from an inducible promoter, and attP from pSAM2 were cloned on plasmids in Escherichia coil . Compatible plasmids carrying the different attB regions to be tested were introduced in these E . coli strains . Under these conditions, Int alone could promote site-specific integration; Int and Xis were both required for site-specific excision . This experimental system was used to study the sequences required in attB for efficient site-specific recombination . A 26 bp sequence, centred on the anti-codon loop region and not completely included in the identity segment, retained all the functionality of attB; shorter sequences allowed integration with lower efficiencies . By comparing the 26-bp-long attB with attP, according to the Lambda model, we propose that B and B', C and C' core-type Int binding sites consist of 9 bp imperfect inverted repeats separated by a 5 bp overlap region. Mol Microbiol, 1998 Apr, 28(2), 305 - 13 Three genes encode distinct AP33 proteins involved in Trichomonas vaginalis cytoadherence; Engbring JA et al.; Adherence to host cells is essential for the initiation and maintenance of infection by mucosal pathogens . The protozoan Trichomonas vaginalis colonizes the human urogenital tract via four surface proteins (AP65, AP51, AP33 and AP23) . To characterize AP33 further, six cDNA clones were examined . Restriction mapping indicated that the six clones represented three similar genes . Southern analysis confirmed the existence of three single-copy AP33 genes and suggested a semi-conservative genomic arrangement between T . vaginalis isolates . Analysis of full-length sequences determined that each contained a 930bp open reading frame encoding a protein of approximately 33,000 Da . Sequence comparisons revealed a high degree of identity at both the DNA and the protein levels . N-terminal protein sequencing established the presence of leader peptides . Each of the three full-length recombinant proteins had a predicted pI of approximately 10, which was verified experimentally for the T . vaginalis AP33 adhesin . A database search revealed that AP33 had significant identity to the succinyl-CoA synthetase alpha-subunit of several different organisms and virtually 100% identity to the reported T . vaginalis subunit . Unlike commercially purchased enzyme, the recombinant proteins retained adhesive properties equal to the natural T . vaginalis AP33 . The characteristics of the AP33 protein are similar to those of the other adhesins and emphasize a complex host-parasite relationship. Mol Microbiol, 1998 Apr, 28(2), 227 - 33 Immunity proteins and their specificity for endonuclease colicins: telling right from wrong in protein-protein recognition; Kleanthous C et al.; Immunity proteins inhibit colicins, protein toxins released by bacteria during times of environmental stress, by binding and inactivating their cytotoxic domains . This protects the producing organism as it attempts to kill off competing bacteria . The cytotoxic domains of related colicins share a high degree of sequence identity, as do their corresponding immunity proteins, yet specificity and affinity are also high, with little non-cognate biological cross-protection evident under physiological conditions . We review recent work on DNase-specific immunity proteins, which shows that, although both cognate and non-cognate proteins can bind a single toxin, their affinities can differ by as much as 12 orders of magnitude . We have termed this mode of binding dual recognition, because the DNase-binding surface of an immunity protein is made up of two components, one conserved and the other variable . The strength of the binding interaction is dominated by the conserved residues, while neighbouring variable residues control specificity . Similar dual recognition systems may exist in other biological contexts, particularly where a protein must discriminate the right binding partner from numerous, structurally homologous alternatives. RNA, 1998 Jun, 4(6), 658 - 68 New features of 23S ribosomal RNA folding: the long helix 41-42 makes a "U-turn" inside the ribosome; Baranov PV et al.; 23S rRNA from Escherichia coli was cleaved at single internucleotide bonds using ribonuclease H in the presence of appropriate chimeric oligonucleotides; the individual cleavage sites were between residues 384 and 385, 867 and 868, 1045 and 1046, and 2510 and 2511, with an additional fortuitous cleavage at positions 1117 and 1118 . In each case, the 3' terminus of the 5' fragment was ligated to radioactively labeled 4-thiouridine 5'-,3'-biphosphate ("psUp"), and the cleaved 23S rRNA carrying this label was reconstituted into 50S subunits . The 50S subunits were able to associate normally with 30S subunits to form 70S ribosomes . Intra-RNA crosslinks from the 4-thiouridine residues were induced by irradiation at 350 nm, and the crosslink sites within the 23S rRNA were analyzed . The rRNA molecules carrying psUp at positions 867 and 1117 showed crosslinks to nearby positions on the opposite strand of the same double helix where the cleavage was located, and no crosslinking was detected from position 2510 . In contrast, the rRNA carrying psUp at position 384 showed crosslinking to nt 420 (and sometimes also to 416 and 425) in the neighboring helix in 23S rRNA, and the rRNA with psUp at position 1045 gave a crosslink to residue 993 . The latter crosslink demonstrates that the long helix 41-42 of the 23S rRNA (which carries the region associated with GTPase activity) must double back on itself, forming a "U-turn" in the ribosome . This result is discussed in terms of the topography of the GTPase region in the 50S subunit, and its relation to the locations of the 5S rRNA and the peptidyl transferase center. RNA, 1998 Jun, 4(6), 639 - 46 Recognition of the universally conserved 3'-CCA end of tRNA by elongation factor EF-Tu; Liu JC et al.; Escherichia coli tRNA(Val) with pyrimidine substitutions for the universally conserved 3'-terminal adenine can be readily aminoacylated . It cannot, however, transfer valine into polypeptides . Conversely, despite being a poor substrate for valyl-tRNA synthetase, tRNA(Val) with a 3'-terminal guanine is active in in vitro polypeptide synthesis . To better understand the function of the 3'-CCA sequence of tRNA in protein synthesis, the effects of systematically varying all three bases on formation of the Val-tRNA(Val):EF-Tu:GTP ternary complex were investigated . Substitutions at C74 and C75 have no significant effect, but replacing A76 with pyrimidines decreases the affinity of valyl-tRNA(Val) for EF-Tu:GTP, thus explaining the inability of these tRNA(Val) variants to function in polypeptide synthesis . Valyl-tRNA(Val) terminating in 3'-guanine is readily recognized by EF-TU:GTP . Dissociation constants of the EF-Tu:GTP ternary complexes with valine tRNAs having nucleotide substitutions at the 3' end increase in the order adenine < guanine < uracil; EF-Tu has very little affinity for tRNA terminating in 3' cytosine . Similar observations were made in studies of the interaction of 3' end mutants of E . coli tRNA(Ala) and tRNA(Phe) with EF-Tu:GTP . These results indicate that EF-Tu:GTP preferentially recognizes purines and discriminates against pyrimidines, especially cytosine, at the 3' end of aminoacyl-tRNAs. Biomed Mater Eng, 1997, 7(6), 391 - 400 Immunogold electron microscopy in situ end-labeling (EM-ISEL): assay for biomaterial DNA damage detection; Assad M et al.; We have evaluated a genotoxicity assay that combines in situ end-labeling, colloidal gold tagging and electron microscopy in order to adapt it to the measurement of in vitro biomaterial-induced genotoxicity . Human lymphocytes were cultured in semi-physiological medium which had been previously exposed to biomaterial extracts of commercially pure titanium following ISO standards . In order to visualize the location of induced DNA strand breaks, cells were then exposed to exonuclease III which partially digests and amplifies lesions by releasing nucleotides at free 3' hydroxyl ends from nicked double-stranded DNA . The resulting single-stranded DNA was allowed to hybridize with short oligonucleotides of random sequences including biotinylated dUTP . After random priming using Escherichia coli DNA polymerase I, incorporation of biotin-dUTP was detected by immunogold binding to the chromatin . Cells exposed to a mutagenic concentration of methyl methanesulfonate, as a positive control, showed a significantly higher and stronger gold staining than both titanium-exposed and unexposed specimens . This assay allows a precise localization and quantification of both in vitro DNA breakage and DNA repair . It could provide a powerful tool for rapid assessment of the genotoxic potential of new biomaterials. Cancer Gene Ther, 1998 May-Jun, 5(3), 144 - 9 Purine salvage rescue by xanthine-guanine phosphoribosyltransferase (XGPRT) potentiates methotrexate resistance conferred by transfer of a mutated dihydrofolate reductase gene; Mineishi S et al.; We have previously shown that successful gene transfer of a mutated dihydrofolate reductase (DHFR) cDNA confers resistance to methotrexate (MTX) upon infected cells . We constructed a retrovirus vector, DC/SV6S31GPT, which carries both the Escherichia coli xanthine-guanine phosphoribosyltransferase gene and the mutated Serine 31 DHFR gene . Mouse fibroblast NIH3T3 cells infected with DC/SV6S31 GPT are more resistant to MTX than cells infected with DC/SV6S31, which carries the Serine 31 DHFR and the neomycin resistance gene cDNA . The mechanism of this augmented resistance is the increased salvaging of purines due to expression of xanthine-guanine phosphoribosyltransferase, as the augmentation does not occur when dialyzed serum, containing little xanthine or guanine, is used for cytotoxicity assays . These results indicate that coexpression of a metabolically related gene can potentiate the resistance carried by a drug resistance gene . This vector may be useful in clinical gene therapy to protect bone marrow from the toxic effects of MTX. J Am Vet Med Assoc, 1998 Jun 1, 212(11), 1746 - 50 Randomized controlled trial of effects of Escherichia coli antiserum on serum immunoglobulin G concentrations and morbidity and mortality rates in foals; Chaffin MK et al.; OBJECTIVE: To determine whether administration of commercially available Escherichia coli antiserum to neonatal foals would affect serum IgG concentration or morbidity and mortality rates during the first 60 days of life . DESIGN: Randomized controlled trial . ANIMALS: 271 neonatal foals on 4 well-managed farms . PROCEDURE: Foals were randomly assigned to a treatment or control group . All foals were allowed to suckle colostrum normally . In addition, treatment-group foals were given E coli antiserum (10 micromilligrams) orally between 0 and 8 hours after birth . Serum samples were obtained between 18 and 36 hours after birth, and serum IgG concentration was determined . Foals were monitored for the first 60 days after birth, and causes of disease or death were recorded . RESULTS: Groups did not differ significantly in regard to breed, sex, month of birth, season of birth, age of dams, parity of dams, duration of gestation, or specific gravity of colostrum before suckling . In addition, groups did not differ significantly in regard to mean serum IgG concentration, prevalence of complete or partial failure of passive transfer of immunity, frequency or causes of disease, or frequency of death from infectious causes . CLINICAL IMPLICATIONS: In this group of foals on well-managed farms, administration of E coli antiserum did not alter serum IgG concentrations or morbidity and mortality rates during the first 60 days of life. J Hum Genet, 1998, 43(2), 135 - 7 A one-base deletion (183delC) and a missense mutation (D276H) in the T-protein gene from a Japanese family with nonketotic hyperglycinemia; Kure S et al.; Two novel mutations in the gene encoding T-protein, a component of the glycine cleavage system, were identified in a Japanese family with nonketotic hyperglycinemia . The proband had two affected sibs, and enzymatic analysis of the liver sample from the proband revealed the T-protein deficiency . The first mutation, 183delC, was found in exon 1 . One of six cytidine residues (base position 183-188) was deleted . The deletion was located in a coding region of the mitochondrial leader peptide and was deduced to create a truncated peptide with 94 amino acids . The second mutation was a base substitution from G to C at position 955 in exon 7 . The G955C substitution caused an amino acid change from aspartate to histidine at position 276 (D276H) . Aspartic acid at position 276 is evolutionarily conserved among human, bovine, chicken, and pea genes, and replaced by glutamic acid in Escherichia coli, suggesting that the presence of an acidic amino acid at 276 may be crucial for the enzymatic function . No base change other than the 183delC and the G955C was observed in the sequencing analysis . Familial analysis revealed that the 183delC and the D276H mutations were inherited from the father and the mother, respectively . This is the first report of T-protein gene mutation in Oriental patients with nonketotic hyperglycinemia. J Hematother, 1998 Jun, 7(3), 225 - 39 Retroviral-mediated marker gene transfer in hematopoiesis-supportive marrow stromal cells; Bulabois CE et al.; A Moloney-derived retrovirus containing both LacZ and NeoR genes (G1BgSVNa from Genetic Therapy, Inc.), was used to transduce human and murine bone marrow stromal cells . Different kinds of stromal cells that were able to support hematopoiesis were transduced by incubation for 24 h in the presence of virus-containing supernatant . Semiconfluent layers of MRC-5 (human, myofibroblastic, fetal, pulmonary) and MS-5 (murine, myofibroblastic, medullary) cells were successfully transduced after one 24-h incubation, as demonstrated by G418 resistance and Escherichia coli beta-galactosidase staining . In contrast, human stromal cells, purified from primary confluent layers grown for 3-4 weeks, could not be transduced . However, stromal cells generated after 10-12 days in culture from Stro-1+ and 1B10+ stromal precursors were successfully transduced in the presence of basic fibroblast growth factor . Transduced stromal cells maintained a myofibroblastic phenotype, although with a decreased number of alpha-SM actin-positive microfilaments in MS-5 cells . The ability to support the generation of stroma-adherent colony-forming cells from cocultured cord blood CD34+ cells after 4 weeks in culture was similar before and after transduction and G418 selection . In conclusion, human primary stromal precursors can be efficiently transduced, and the stromal cell phenotype and function are not significantly altered after retroviral-mediated transfer of marker genes. Emerg Infect Dis, 1998 Apr-Jun, 4(2), 251 - 61 Enteroaggregative Escherichia coli; Nataro JP et al.; Enteroaggregative Escherichia coli (EAEC), an increasingly recognized cause of diarrhea in children in developing countries, has been particularly associated with persistent diarrhea (more than 14 days), a major cause of illness and death . Recent outbreaks implicate EAEC as a cause of foodborne illness in industrialized countries . The pathogenesis of EAEC infection is not well understood, but a model can be proposed in which EAEC adhere to the intestinal mucosa and elaborate enterotoxins and cytotoxins, which result in secretory diarrhea and mucosal damage . EAEC's ability to stimulate the release of inflammatory mediators may also play a role in intestinal illness. Glycobiology, 1998 Jul, 8(7), 719 - 24 Ion-spray mass spectrometry for identification of the nonreducing terminal sugar of glycosaminoglycan; Takagaki K et al.; Various oligosaccharides from hyaluronic acid, which have glucuronic acid or N- acetylglucosamine at the nonreducing terminal, were prepared by digestion with a combination of testicular hyaluronidase and beta-glucuronidase . These oligo saccharides were analyzed by negative-mode ion-spray mass spectrometry (MS) with an atmospheric pressure ion source . Introduction of collisionally activated dissociation tandem mass spectrometry (CAD-MS/MS) produced ions derived from cleavage of the glycosidic bonds, allowing the structure to be analyzed . The CAD-MS/MS spectrum showed an intense and characteristic fragment ion at m/z 193 for oligosaccharides having glucuronic acid at the nonreducing terminal . On the other hand, this ion was not observed in the spectra of oligosaccharides having N- acetylglucosamine at the nonreducing terminal . Therefore, the fragmentation pattern revealed by CAD-MS/MS provides useful information for distinguishing glucuronic acid and N- acetylglucosamine at the nonreducing terminal of oligosaccharides derived from hyaluronic acid and other glycosaminoglycans . This ion-spray CAD-MS/MS technique was also applied successfully to the characterization of glycosaminoglycans reconstructed by glycotechnology. J Bacteriol, 1998 Jun, 180(12), 3245 - 9 Appropriate expression of filamentous phage f1 DNA replication genes II and X requires RNase E-dependent processing and separate mRNAs; Kokoska RJ et al.; The products of in-frame overlapping genes II and X carried by the filamentous phage f1 genome are proteins with required but opposing functions in phage DNA replication . Their normal relative levels are important for continuous production of phage DNA without killing infected Escherichia coli hosts . Here we identify several factors responsible for determining the relative levels of pII and pX and that, if perturbed, alter the normal distribution of the phage DNA species in infected hosts . Translation of the two proteins is essentially relegated to separate mRNAs . The mRNAs encoding genes II and X are also differentially sensitive to cleavage dependent on rne, the gene encoding the only E . coli endo-RNase known to have a global role in mRNA stability . Whereas pII levels are limited at the level of mRNA stability, normal pX levels require transcription in sufficient amounts from the promoter for the smaller mRNA encoding only pX. J Bacteriol, 1998 Jun, 180(12), 3237 - 40 An autonomously replicating transforming vector for Sulfolobus solfataricus; Cannio R et al.; A plasmid able to transform and to be stably maintained both in Sulfolobus solfataricus and in Escherichia coli was constructed by insertion into an E . coli plasmid of the autonomously replicating sequence of the virus particle SSV1 and a suitable mutant of the hph (hygromycin phosphotransferase) gene as the transformation marker . The vector suffered no rearrangement and/or chromosome integration, and its copy number in Sulfolobus was increased by exposure of the cells to mitomycin C. J Bacteriol, 1998 Jun, 180(12), 3218 - 21 The MTCY428.08 gene of Mycobacterium tuberculosis codes for NAD+ synthetase; Cantoni R et al.; The product of the MTCY428.08 gene of Mycobacterium tuberculosis shows sequence homology with several NAD+ synthetases . The MTCY428.08 gene was cloned into the expression vectors pGEX-4T-1 and pET-15b . Expression in Escherichia coli led to overproduction of glutathione S-transferase fused and His6-tagged gene products, which were enzymatically assayed for NAD synthetase activity . Our results demonstrate that the MTCY428.08 gene of M . tuberculosis is the structural gene for NAD+ synthetase. J Bacteriol, 1998 Jun, 180(12), 3205 - 8 Effects of carbon source on expression of F0 genes and on the stoichiometry of the c subunit in the F1F0 ATPase of Escherichia coli; Schemidt RA et al.; Expression of the genes for the membrane-bound F0 sector of the Escherichia coli F1F0 proton-translocating ATPase can respond to changes in metabolic conditions, and these changes are reflected in alterations in the subunit stoichiometry of the oligomeric F0 proton channel . Transcriptional and translational lacZ fusions to the promoter and to two F0 genes show that, during growth on the nonfermentable carbon source succinate, transcription of the operon and translation of uncB, encoding the a subunit of F0, are higher than during growth on glucose . In contrast, translation of the uncE gene, encoding the c subunit of F0, is higher during growth on glucose than during growth on succinate . Translation rates of both uncB and uncE change as culture density increases, but transcription rates do not . Quantitation of the c stoichiometry shows that more c subunits are assembled into the F1F0 ATPase in cells grown on glucose than in cells grown on succinate . E . coli therefore appears to have a mechanism for regulating the composition and, presumably, the function of the ATPase in response to metabolic circumstances. J Bacteriol, 1998 Jun, 180(12), 3120 - 30 Folding-based suppression of extracytoplasmic toxicity conferred by processing-defective LamB; Cosma CL et al.; We have utilized processing-defective derivatives of the outer membrane maltoporin, LamB, to study protein trafficking functions in the cell envelope of Escherichia coli . Our model proteins contain amino acid substitutions in the consensus site for cleavage by signal peptidase . As a result, the signal sequence is cleaved with reduced efficiency, effectively tethering the precursor protein to the inner membrane . These mutant porins are toxic when secreted to the cell envelope . Furthermore, strains producing these proteins exhibit altered outer membrane permeability, suggesting that the toxicity stems from some perturbation of the cell envelope (J . H . Carlson and T . J . Silhavy, J . Bacteriol . 175:3327-3334, 1993) . We have characterized a multicopy suppressor of the processing-defective porins that appears to act by a novel mechanism . Using fractionation experiments and conformation-specific antibodies, we found that the presence of this multicopy suppressor allowed the processing-defective LamB precursors to be folded and localized to the outer membrane . Analysis of the suppressor plasmid revealed that these effects are mediated by the presence of a truncated derivative of the polytopic inner membrane protein, TetA . The suppression mediated by TetA' is independent of the CpxA/CpxR regulon and the sigma E regulon, both of which are involved in regulating protein trafficking functions in the cell envelope. J Bacteriol, 1998 Jun, 180(12), 3114 - 9 Changes in ribosomal activity of Escherichia coli cells during prolonged culture in sea salts medium; Kalpaxis DL et al.; The activity of ribosomes from a clinical isolate of Escherichia coli, exposed to starvation for 7 days in sea salts medium, was investigated by measuring the kinetic parameters of ribosomal peptidyltransferase, by using the puromycin reaction as a model reaction . No alterations in the extent of peptide bond formation were observed during starvation . In contrast, a 50% reduction in the kmax/Ks ratio could be seen after 24 h of starvation; an additional 6 days of starvation resulted in a progressive but less abrupt decline in the kmax/Ks value . (kmax is the apparent catalytic rate constant of peptidyl transferase, and Ks is the dissociation constant of the encounter complex between acetyl (Ac){3H}Phe-tRNA-poly(U)-ribosome and puromycin.) Although the distribution of ribosomal particles remained constant, a substantial decrease in the number of ribosomes per starved cell and a clear decline in the ability of ribosomes to bind AcPhe-tRNA were observed, particularly during the first day of starvation . Further analysis indicated that rRNA in general, but especially 23S rRNA, was rapidly degraded during the starvation period . In addition, the L12/L7 molar ratio decreased from 1.5 to 1 during the initial phase of starvation (up to 24 h) but remained constant during the subsequent starvation period . Ribosomes isolated from 24-h-starved cells, when artificially depleted of L7/L12 protein and reconstituted with L7/L12 protein from mid-logarithmic-phase cells, regenerated an L12/L7 molar ratio of 1.5 and restored the peptidyltransferase activity to a substantial level . An analogous effect of reconstitution on the efficiency of ribosomes in binding AcPhe-tRNA was evident not only during the initial phase but throughout the starvation period. J Bacteriol, 1998 Jun, 180(12), 3070 - 9 Promoter region of the Escherichia coli O7-specific lipopolysaccharide gene cluster: structural and functional characterization of an upstream untranslated mRNA sequence; Marolda CL et al.; We report the identification of the promoter region of the Escherichia coli O7-specific lipopolysaccharide (LPS) gene cluster (wbEcO7) . Typical -10 and -35 sequences were found to be located in the intervening region between galF and rlmB, the first gene of the wbEcO7 cluster . Data from RNase protection experiments revealed the existence of an untranslated leader mRNA segment of 173 bp, including the JUMPStart and two ops sequences . We characterized the structure of this leader mRNA by using the program Mfold and a combination of nested and internal deletions transcriptionally fused to a promoterless lac operon . Our results indicated that the leader mRNA may fold into a series of complex stem-loop structures, one of which includes the JUMPStart element . We have also found that one of the ops sequences resides on the predicted stem and the other resides on the loop region, and we confirmed that these sequences are essential for the RfaH-mediated regulation of the O polysaccharide cluster . A very similar stem-loop structure could be predicted in the promoter region of the LPS core operon encoding the waaQGPSBIJYZK genes . We observed another predicted stem-loop, located immediately downstream from the wbEcO7 transcription initiation site, which appeared to be involved in premature termination of transcription . This putative stem-loop is common to many other O polysaccharide gene clusters but is not present in core oligosaccharide genes . wbEcO7-lac transcriptional fusions in single copy numbers were also used to determine the effects of various environmental cues in the transcriptional regulation of O polysaccharide synthesis . No effects were detected with temperature, osmolarity, Mg2+ concentration, and drugs inducing changes in DNA supercoiling . We therefore conclude that the wbEcO7 promoter activity may be constitutive and that regulation takes place at the level of elongation of the mRNA in a RfaH-mediated manner. J Bacteriol, 1998 Jun, 180(12), 3039 - 48 Target choice and orientation preference of the insertion sequence IS903; Hu WY et al.; We have examined the targeting preference of the bacterial insertion element IS903 by determining the sites of insertion of a large number of transposition events into the 55-kb conjugative plasmid pOX38 . Despite the large target size, all the insertions were clustered in four small distinct regions associated with conjugal DNA transfer . Within these regions, many different sites were used for insertion; however, there were a few sites that IS903 inserted into more than once . Alignment of the insertion sites showed that there was no consensus sequence within the 9-bp target duplication but that there were preferred sequences located symmetrically on either side of the target . This is consistent with target recognition by a dimer or multimer of transposase, with either sequence-specific or structure-specific interactions on both sides of the target . We show further that when one of these preferred regions was cloned into a second conjugative plasmid, pUB307, it was still a preferred target, implying that all the sequences necessary for target selection are contained within this DNA segment . Also, we observed a very strong preference for insertion in a single orientation in pUB307 . We examined the possibility that either DNA replication from the origin of vegetative replication, oriV, or the origin of transfer, oriT, might determine this orientation effect . We find that reversing the direction of vegetative replication had no effect on the orientation of transposon insertions; however, reversing the direction of DNA transfer abolished the orientation effect . This supports the idea that conjugal DNA transfer imparts a polarity on the target that is sensed by the transposon. J Bacteriol, 1998 Jun, 180(12), 3026 - 30 In vivo analysis of sequence requirements for processing and degradation of the colicin A lysis protein signal peptide; Howard SP et al.; The lipid modification and processing of a number of colicin lysis proteins take place exceedingly slowly and result in the release of a stable signal peptide . It is possible that this peptide or the presence of lipid-modified precursors which result from the slow processing plays a role in the release of colicins and in the quasilysis that occurs in induced colicinogenic cultures . We used in vitro mutagenesis and pulse-chase radiolabeling and immunoprecipitation to examine the reasons for the slow processing and signal peptide degradation reactions for the colicin A lysis protein (Cal) . In one mutant, isoleucine 13 was replaced with serine, and in another, alanine 18, the last residue of the signal peptide, was replaced with glycine . In each case, the mutation caused a striking increase in the rate of maturation of the precursor, and in the case of the serine 13 derivative, the mutation also destabilized the signal peptide . A precursor containing both of these mutations was completely matured and its signal sequence degraded within seconds of its synthesis . The release of colicin A and the quasilysis of producing cultures were unchanged for each of these mutants, indicating that neither the stable signal peptide nor lipid-modified processing intermediates of Cal are required for either of these events in wild-type cells. Biochem J, 1998 Jun 15, 332 ( Pt 3), 651 - 9 Structure and activity of mouse S-adenosylmethionine decarboxylase gene promoters and properties of the encoded proteins; Nishimura K et al.; The promoter regions of two S-adenosylmethionine decarboxylase genes (AMD genes) were isolated from a mouse genomic library . One promoter was that of the bona fide mouse AMD gene (AMD1) whereas the other was that of the intronless AMD gene (AMD2) . There was no sequence identity between the two promoters . The sequence of the AMD1 promoter was highly homologous to the human AMD1 and rat Amd1B promoters . After transient transfection in various cell lines, the AMD1 promoter was one to two orders of magnitude stronger than the AMD2 promoter . Similar results were obtained by using stably transfected mouse FM3A cells . In S-adenosylmethionine decarboxylase (AdoMetDC)-overproducing SAM-1 cells, the AMD1 gene was amplified over 5-fold . AdoMetDC encoded by the intronless AMD2 gene had two amino acid replacements (Met to Ile at codon 70 and Ala to Val at codon 139), compared with the protein encoded by the AMD1 gene, and exhibited decreased catalytic activity (<50%) and decreased processing activity when expressed in AdoMetDC-deficient Escherichia coli cells . When Ile-70 of the protein encoded by AMD2 was converted into Met, both the catalytic and processing activities recovered markedly, indicating that Met-70 adjacent to the proenzyme-processing site is important for both activities . The third AMD locus (AMD3) in FM3A cells contains a pseudogene, in which deletion of two bases generates a premature termination codon at position 57 . Since the AMD2 promoter had only 1-10% of the strength of the bona fide AMD1 gene and AMD2 protein possessed lower specific activity, the relative contribution of the AMD2-encoded enzyme to total AdoMetDC activity is small . Thus AdoMetDC activity in murine cells is thought to be due mainly to the product of the AMD1 gene. J Biol Chem, 1998 Jun 5, 273(23), 14582 - 7 Molecular cloning and expression of GDP-D-mannose-4,6-dehydratase, a key enzyme for fucose metabolism defective in Lec13 cells; Ohyama C et al.; Subsets of mammalian cell surface oligosaccharides contain specific fucosylated moieties expressed in lineage- and/or temporal-specific patterns . The functional significance of these fucosylated structures is incompletely defined, although there is evidence that subsets of them, represented by the sialyl Lex determinant, are important participants in leukocyte adhesion and trafficking processes . Genetic deletion of these fucosylated structures in the mouse has been a powerful tool to address functional questions about fucosylated glycans . However, successful use of such approaches can be problematic, given the substantial redundancy in the mammalian alpha-1,3-fucosyltransferase and alpha-1,2-fucosyltransferase gene families . To circumvent this problem, we have chosen to clone the genetic locus encoding a mammalian GDP-D-mannose-4,6-dehydratase (GMD) . This enzyme generates GDP-mannose-4-keto-6-D-deoxymannose from GDP-mannose, which is then converted by the FX protein (GDP-4-keto-6-D-deoxymannose epimerase/GDP-4-keto-6-L-galactose reductase) to GDP-L-fucose . GMD is thus imperative for the synthesis of all fucosylated oligosaccharides . An expression cloning approach and the GMD-deficient CHO host cell line Lec13 were used to generate a population of cDNA molecules enriched in GMD cDNAs . This enriched plasmid population was then screened using a human expressed sequence tag (EST AA065072) with sequence similarity to an Arabidopsis thaliana GMD cDNA . This approach, together with 5'-rapid amplification of cDNA ends, yielded a human cDNA that complements the fucosylation defect in the Lec13 cell line . Northern blot analyses indicate that the GMD transcript is absent in Lec13 cells, confirming the genetic deficiency of this locus in these cells . By contrast, the transcript encoding the FX protein, which forms GDP-L-fucose from the ketosugar intermediate produced by GMD, is present in increased amounts in the Lec13 cells . These results suggest that metabolites generated in this pathway may participate in the transcriptional regulation of the FX protein and possibly the GMD protein . The results also suggest that the genomic structure encoding GMD in Lec13 cells likely has a defect different from a point mutation in the coding region. J Biol Chem, 1998 Jun 5, 273(23), 14269 - 76 The phosphate carrier from yeast mitochondria . Dimerization is a prerequisite for function; Schroers A et al.; Wild type phosphate carrier (PIC) from Saccharomyces cerevisiae and recombinant PIC proteins with different C-terminal extensions were expressed in Escherichia coli as inclusion bodies . From these, PIC was isolated with the detergent sodium lauroyl sarcosinate in a form, partially monomeric and unfolded . This PIC associates to stable dimers after exchanging the detergent to the polyoxyethylene detergent C12E8 and dialysis . Combining two differently tagged monomers of PIC and following this with affinity chromatography yields defined homo- and heterodimeric forms of PIC, which are all fully active after reconstitution . As a member of the mitochondrial carrier family PIC is supposed to function as a homodimer . We investigated its dimeric nature in the functionally active state after reconstitution . When reconstituting PIC monomers a sigmoidal dependence of transport activity on the amount of inserted protein is observed, whereas insertion of PIC dimers leads to a linear dependence . Heterodimeric PIC constructs consisting of both an active and an inactivated subunit do not catalyze phosphate transport . In contrast, reconstitution of a mixture of active and inactive monomeric subunits led to partially active carrier . These experiments prove (i) that PIC does not function in monomeric form, (ii) that PIC dimers are stable both in the solubilized state and after membrane insertion, and (iii) that transport catalyzed by PIC dimers involves functional cross-talk between the two monomers. J Biol Chem, 1998 Jun 5, 273(23), 14172 - 8 Pressure-induced dissociation of carbamoyl-phosphate synthetase domains . The catalytically active form is dimeric; Guy HI et al.; Carbamoyl-phosphate synthetase consists of an amidotransferase domain or subunit (GLN) that hydrolyzes glutamine and transfers the ammonia to the synthetase component (CPS) where the biosynthetic reaction occurs . The CPS domain is composed of two homologous subdomains, CPS.A and CPS.B, that catalyze different ATP-dependent reactions involved in carbamoyl phosphate synthesis . When the individual CPS.A and CPS.B subdomains were individually cloned and expressed in Escherichia coli (Guy, H . I., and Evans, D . R . (1996) J . Biol . Chem . 271, 13762-13769), they were found to be functionally equivalent and could each independently catalyze carbamoyl phosphate synthesis . The proposal was advanced that, although the monomers could catalyze the individual partial reactions, overall synthesis of carbamoyl phosphate required a homodimer of CPS.A or CPS.B . To test this hypothesis, the GLN-CPS.B dimer was reversibly dissociated at 1500 bar in a high pressure cell . Dissociation was accompanied by a loss of both glutamine- and ammonia-dependent CPSase activity . Activity was recovered once the protein was returned to atmospheric pressure . If the sample was cross-linked before exposure to high pressure, there was no dissociation and no loss of biosynthetic activity . In contrast, the bicarbonate-dependent ATPase and the carbamoyl phosphate-dependent ATP synthetase activities were largely unaffected by pressure-induced dissociation . These experiments confirmed the hypothesis that the synthesis of carbamoyl phosphate requires the concerted action of the two active sites within the homodimer. Biochem J, 1998 Apr 15, 331 ( Pt 2), 459 - 64 The reaction of halides with pulsed cytochrome bo from Escherichia coli; Moody AJ et al.; Cytochrome bo forms complexes with chloride, bromide and iodide in which haem o remains high-spin and in which the '630 nm' charge-transfer band is red-shifted by 7-8 nm . The chloride and bromide complexes each have a characteristic set of integer-spin EPR signals arising from spin coupling between haem o and CuB . The rate and extent of chloride binding decreases as the pH increases from 5.5 to 8.5 . At pH 5.5 the dissociation constant for chloride is 2 mM and the first-order rate constant for dissociation is 2 x 10(-4) s-1 . The order of rate of binding, and of affinity, at pH 5.5 is chloride (1) > bromide (0.3) >iodide (0.1) . It is suggested that the halides bind in the binuclear site but, unlike fluoride, they are not direct ligands of the iron of haem o . In addition, both the stability of the halide complexes and the rate of halide binding seem to be increased by the co-binding of a proton. Biochem J, 1998 Apr 15, 331 ( Pt 2), 437 - 45 The dhnA gene of Escherichia coli encodes a class I fructose bisphosphate aldolase; Thomson GJ et al.; The gene encoding the Escherichia coli Class I fructose-1, 6-bisphosphate aldolase (FBP aldolase) has been cloned and the protein overproduced in high amounts . This gene sequence has previously been identified as encoding an E . coli dehydrin in the GenBanktrade mark database {gene dhnA; entry code U73760; Close and Choi (1996) Submission to GenBanktrade mark} . However, the purified protein overproduced from the dhnA gene shares all its properties with those known for the E . coli Class I FBP aldolase . The protein is an 8-10-mer with a native molecular mass of approx . 340 kDa, each subunit consisting of 349 amino acids . The Class I enzyme shows low sequence identity with other known FBP aldolases, both Class I and Class II (in the order of 20%), which may be reflected by some novel properties of this FBP aldolase . The active-site peptide has been isolated and the Schiff-base-forming lysine residue (Lys236) has been identified by a combination of site-directed mutagenesis, kinetics and electrospray-ionization MS . A second lysine residue (Lys238) has been implicated in substrate binding . The cloning of this gene and the high levels of overexpression obtained will facilitate future structure-function studies. Biochem J, 1998 Apr 15, 331 ( Pt 2), 423 - 30 Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies; Uhlein M et al.; The translational apparatus is a highly complex structure containing three to four RNA molecules and more than 50 different proteins . In recent years considerable evidence has accumulated to indicate that the RNA participates intensively in the catalysis of peptide-bond formation, whereas a direct involvement of the ribosomal proteins has yet to be demonstrated . Here we report the functional and structural conservation of a peptidyltransferase centre protein in all three phylogenetic domains . In vivo replacement studies show that the Escherichia coli L2 protein can be replaced by its homologous proteins from human and archaebacterial ribosomes . These hybrid ribosomes are active in protein biosynthesis, as proven by polysome analysis and poly(U)-dependent polyphenylalanine synthesis . Furthermore, we demonstrate that a specific, highly conserved, histidine residue in the C-terminal region of L2 is essential for the function of the translational apparatus . Replacement of this histidine residue in the human and archaebacterial proteins by glycine, arginine or alanine had no effect on ribosome assembly, but strongly reduced the translational activity of ribosomes containing these mutants. Biochem J, 1998 Apr 15, 331 ( Pt 2), 409 - 15 Functional effects of single amino acid substitutions in the region of Phe113 to Asp138 in the plasminogen activator inhibitor 1 molecule; Sui GC et al.; Thirteen amino acid substitutions have been introduced within the stretch Phe113 to Asp138 in the plasminogen activator inhibitor 1 (PAI-1) molecule by site-directed mutagenesis . The different proteins and wild-type (wt) PAI-1 have been overexpressed in Escherichia coli and purified by chromatography on heparin-Sepharose and on anhydrotrypsin-agarose . The PAI-1 variants have been characterized by their reactivity with tissue plasminogen activator (tPA), interactions with vitronectin or heparin, and stability . Most PAI-1 variants, except for Asp125-->Lys, Phe126-->Ser and Arg133-->Asp, displayed a high spontaneous inhibitory activity towards tPA, which did not change greatly on reactivation with 4 M guanidinium chloride, followed by dialysis at pH 5.5 . The variants Asp125-->Lys and Arg133-->Asp became much more active after reactivation and they were also more rapidly transformed to inactive forms (t12 22-31 min) at physiological pH and temperature than the other variants . However, in the presence of vitronectin they were both almost equally stable (t12 2.3 h) as wtPAI-1 (t12 3.0 h) . The mutant Glu130-->Lys showed an increased stability, both in the absence and in the presence of vitronectin compared with wtPAI-1 . Nevertheless a similar affinity between all the active PAI-1 variants and vitronectin was observed . Further, all mutants, including the three mutants with low activity, were to a large extent adsorbed on anhydrotrypsin-agarose and were eluted in a similar fashion . In accordance with these data, the three variants with a low activity were all to a large extent cleaved as a result of their reaction with tPA, suggesting that they occurred predominantly in the substrate conformation . Our results do not support the presence of a binding site for vitronectin in this part of the molecule, but rather that it might be involved in controlling the active PAI-1 to substrate transition . Partly, this region of the PAI-1 molecule (Arg115 to Arg118) seems also to be involved in the binding of heparin to PAI-1. Nature, 1998 May 28, 393(6683), 392 - 6 Atomic structure of progesterone complexed with its receptor; Williams SP et al.; The physiological effects of progestins are mediated by the progesterone receptor, a member of the steroid/nuclear receptor superfamily . As progesterone is required for maintenance of pregnancy, its receptor has been a target for pharmaceuticals . Here we report the 1.8 A crystal structure of a progesterone-bound ligand-binding domain of the human progesterone receptor . The nature of this structure explains the receptor's selective affinity for progestins and establishes a common mode of recognition of 3-oxy steroids by the cognate receptors . Although the overall fold of the progesterone receptor is similar to that found in related receptors, the progesterone receptor has a quite different mode of dimerization . A hormone-induced stabilization of the carboxy-terminal secondary structure of the ligand-binding domain of the progesterone receptor accounts for the stereochemistry of this distinctive dimer, explains the receptor's characteristic pattern of ligand-dependent protease resistance and its loss of repression, and indicates how the anti-progestin RU486 might work in birth control . The structure also indicates that the analogous 3-keto-steroid receptors may have a similar mechanism of action. J Clin Microbiol, 1998 Jun, 36(6), 1795 - 7 Multiplex PCR for enterotoxigenic, attaching and effacing, and Shiga toxin-producing Escherichia coli strains from calves; Franck SM et al.; A multiplex PCR was developed to identify enterotoxigenic, attaching and effacing, and Shiga toxin-producing Escherichia coli strains by amplifying genes encoding K99 and F41 fimbriae, heat-stable enterotoxin a, intimin, and Shiga toxins 1 and 2 . This multiplex PCR was specific and sensitive . It will be useful for identification of E . coli strains which cause diarrhea in calves. J Clin Microbiol, 1998 Jun, 36(6), 1604 - 7 Virulence properties of Shiga toxin-producing Escherichia coli (STEC) strains of serogroup O118, a major group of STEC pathogens in calves; Wieler LH et al.; Shiga toxin-producing Escherichia coli (STEC) strains of serogroup 0118 are the most prevalent group among STEC strains in diarrheic calves in Germany (L . H . Wieler, Ph.D . thesis, University of Giessen, 1997) . To define their virulence properties, 42 0118 (0118:H16 {n = 38} and 0118:H- {n = 4}) strains were characterized . The strains displayed three different Stx combinations (Stx1 {36 of 42}, Stx1 and Stx2 {2 of 42}, and Stx2 {4 of 42}) . A total of 41 strains (97.6%) harbored a large virulence-associated plasmid containing hlyEHEC (hly from enterohemorrhagic E . coli) . The strains' adhesive properties varied in relation to the eukaryotic cells tested . Only 28 of 42 strains (66.7%) showed localized adhesion (LA) in the human HEp-2 cell line . In contrast, in bovine fetal calf lung (FCL) cells, the number of LA-positive strains was much higher (37 of 42 {88.1%}) . The locus of enterocyte effacement (LEE) was detected in 41 strains (97.6%) . However, not all LEE-positive strains reacted positively in the fluorescence actin-staining (FAS) test, which indicated the attaching and effacing (AE) lesion . In HEp-2 cells, only 22 strains (52.4%) were FAS positive, while in FCL cells, the number of FAS-positive strains was significantly higher (38 of 42 {90.5%; P < 0.001}) . In conclusion, the vast majority of the 0118 STEC strains from calves (41 of 42 {97.6%}) have a high virulence potential (stx, hlyEHEC, and LEE) . This virulence potential and the high prevalence of STEC 0118 strains in calves suggest that these strains could be a major health threat for humans in the future . In addition, the poor association between results of the geno- and phenotypical tests to screen for the AE ability of STEC strains calls the diagnostic value of the FAS test into question. J Interferon Cytokine Res, 1998 May, 18(5), 287 - 95 The glycosylation heterogeneity of recombinant human IFN-gamma; Hooker A et al.; The cloning of the cDNA for human interferon-gamma (IFN-gamma) has resulted in its expression in Escherichia coli, baculovirus-infected insect cells, Chinese hamster ovary (CHO) cells, and the mammary gland of transgenic mice . Large quantities of highly purified recombinant IFN-gamma have been generated, aided by the use of highly specific neutralizing monoclonal antibodies, with a view to its production as a human therapeutic protein . The primary source of structural heterogeneity for IFN-gamma during its production in mammalian expression systems is glycosylation, which can profoundly affect the three-dimensional structure of a glycoprotein and its biological function . A number of analytical approaches have been developed recently to allow a detailed analysis of the carbohydrate structures associated with IFN-gamma, the principal advances being in the areas of capillary electrophoresis and mass spectrometry . The implementation of these high-resolution analytical tools to determine the glycosylation profile of IFN-gamma makes it one of the best characterized recombinant glycoproteins . Recombinant human IFN-gamma acts as a model secretory glycoprotein, typifying the intrinsic glycosylation processing events associated with production of a potential therapeutic glycoprotein. Plant Mol Biol, 1998 May, 37(1), 179 - 85 Isolation, characterisation and expression of a cDNA for pea cholinephosphate cytidylyltransferase; Jones PL et al.; In plants, phosphatidylcholine is the major phospholipid in extra-plastid membranes and is synthesised mainly by the CDP-choline pathway . Evidence from studies in animals, as well as in plants, suggests that the intermediate step catalysed by cholinephosphate cytidylyltransferase (CPCT) has a major control in carbon flux to this lipid . We have isolated a full-length CPCT cDNA (designated PCT2) from Pisum sativum cv . Feltham First using an Arabidopsis probe and the polymerase chain reaction (PCR) . The deduced amino acid of PCT2 is 48%, 43% and 76% identical to the rat, yeast and Brassica napus amino acid sequences, respectively . Expression of the CPCT protein in Escherichia coli confirmed the activity of the enzyme . Expression of the PCT2 mRNA in pea roots and stems was increased by treatment with 0.1 microM indole-3-acetic acid. Plant Mol Biol, 1998 May, 37(1), 131 - 40 Molecular cloning and characterization of an inorganic pyrophosphatase from barley; Visser K et al.; A cDNA clone with sequence homology to soluble inorganic pyrophosphatase (IPPase) was isolated from a library of developing barley grains . The protein encoded by this clone was produced in transgenic Escherichia coli, and showed IPPase activity . In nondormant barley grains, the gene appeared to be expressed in metabolically active tissue such as root, shoot, embryo and aleurone . During inhibition, a continuous increase of the steady state mRNA level of IPPase was observed in embryos of non-dormant grains . In the embryos of dormant grains its production declined, after an initial increase . With isolated dormant and nondormant embryos, addition of recombinant IPPase, produced by E . coli, enhanced the germination rate . On the other hand, addition of pyrophosphate (PPi), substrate for this enzyme, appeared to reduce the germination rate . A role for this IPPase in germination is discussed. Plant Mol Biol, 1998 May, 37(1), 99 - 108 Characterization of a single soybean cDNA encoding cytosolic and glyoxysomal isozymes of aspartate aminotransferase; Gebhardt JS et al.; A soybean cDNA clone, pSAT1, which encodes both the cytosolic and glyoxysomal isozymes of aspartate aminotransferase (AAT; EC 2.6.1.1) was isolated . Genomic Southern blots and analysis of genomic clones indicated pSAT1 was encoded by a single copy gene . pSAT1 contained an open reading frame with ca . 90% amino acid identity to alfalfa and lupin cytosolic AAT and two in-frame start codons, designated ATG1 and ATG2 . Alignment of this protein with other plant cytosolic AAT isozymes revealed a 37 amino acid N-terminal extension with characteristics of a peroxisomal targeting signal, designated PTS2, including the modified consensus sequence RL-X5-HF . The second start codon ATG2 aligned with previously reported start codons for plant cytosolic AAT cDNAs . Plasmids constructed to express the open reading frame initiated by each of the putative start codons produced proteins with AAT activity in Escherichia coli . Immune serum raised against the pSAT1-encoded protein reacted with three soybean AAT isozymes, AAT1 (glyoxysomal), AAT2 (cytosolic), and AAT3 (subcellular location unknown) . We propose the glyoxysomal isozyme AAT1 is produced by translational initiation from ATG1 and the cytosolic isozyme AAT2 is produced by translational initiation from ATG2 . N-terminal sequencing of purified AAT1 revealed complete identity with the pSAT1-encoded protein and was consistent with the processing of the PTS2 . Analysis of cytosolic AAT genomic sequences from several other plant species revealed conservation of the two in-frame start codons and the PTS2 sequence, suggesting that these other species may utilize a single gene to generate both cytosolic and glyoxysomal or peroxisomal forms of AAT. Plant Mol Biol, 1998 May, 37(1), 25 - 37 Intraspecific variability of the tandem repeats in Nicotiana putrescine N-methyltransferases; Hashimoto T et al.; The putrescine N-methyltransferase (PMT) cDNA clone previously isolated from tobacco encodes a spermidine synthase-like protein with an 11 amino acid element repeated four times in tandem at the amino terminus . Genomic Southern blot analyses indicated that this N-terminal repeat array is found in tobacco PMTs but absent in Hyoscyamus and Atropa PMTs . A truncated tobacco PMT in which this repeat array was entirely removed still retained full enzymatic activity when expressed in Escherichia coli . Three PMT genes (NsPMT1, NsPMT2, NsPMT3) isolated from Nicotiana sylvestris encode two, five, and nine tandem repeats, respectively, in the first exon, but otherwise encode highly conserved proteins . Analysis of PCR fragments amplified from the genomes of N . tabacum and its two probable progenitors shows that one of the nine repeat elements in NsPMT3 was precisely deleted in the corresponding N . tabacum gene . These results indicate that direct tandem repeats of a 33 bp sequence that encodes 11 amino acids of no obvious function were added to the ancestral Nicotiana PMT gene, and that the tandem repetition was genetically very unstable, contracting or expanding during evolution of the Nicotiana species. J Cancer Res Clin Oncol, 1997, 123(11-12), 609 - 13 Expression and purification of single-chain anti-HBx antibody in Escherichia coli; Zhou G et al.; Monoclonal antibodies have been widely used in tumor targeting studies with promising results . However, their clinical application has been limited by heterogeneity and macro-molecular movement of murine antibody . In this study, the variable-region (heavy- and light-chain) fragments of anti-HBx monoclonal antibody were enriched by the polymerase chain reaction . The expression vector, which included a 6x histidine sequence in the 3' terminus of the HBx single-chain antibody (sFv) was recombined with a linker sequence (KLGGGGFSGA) between the variable regions . The expression product from Escherichia coli fused with 6xHis was purified by nickel (Ni2+) nitrilotriacetate chelating resin . The results of enzyme-linked immunosorbent assay and Western blotting showed that sFv had binding affinity with HBxAg, suggesting that it could become a novel targeting carrier in clinical trials. Virus Res, 1998 Feb, 53(2), 141 - 9 Expression of recombinant hepatitis C virus non-structural protein 5B in Escherichia coli; Al RH et al.; The hepatitis C virus (HCV) represents a major public health problem that can produce liver failure and hepatocellular carcinoma in chronically infected patients . Our goal was to express the HCV non-structural protein 5B (NS5B) protein of HCV genotype 1a in Escherichia coli and initiate studies of its role in HCV genomic replication . In this report we demonstrate that a recombinant NS5B protein with an amino terminal sequence of ASMSYSWTG has RNA-dependent RNA polymerase (RDRP) activity . This recombinant enzyme was active in poly(U) polymerase assays and produced template-sized RNA products when globin mRNA was used as a template . The polymerase activity of recombinant NS5B was primer-dependent and was active for at least 60 min of incubation at 30 degrees C . Deletion of the carboxyl terminal region of HCV NS5B resulted in a loss of RDRP activity indicating that the enzymatic activity observed was due to the full-length recombinant enzyme . Recombinant NS5B (RDRP) should assist in understanding the mechanism of HCV replication and the identification of specific enzyme inhibitors. Genes Cells, 1998 Mar, 3(3), 145 - 56 Rad52 forms ring structures and co-operates with RPA in single-strand DNA annealing; Shinohara A et al.; BACKGROUND: The RAD52 epistasis group in Saccharomyces cerevisiae is involved in various types of homologous recombination including recombinational double-strand break (DSB) repair and meiotic recombination . A RecA homologue, Rad51, plays a pivotal role in homology search and strand exchange . Genetic analysis has shown that among members of its epistasis group, RAD52 alone is required for recombination between direct repeats yielding deletions . Very little has been discovered about the biochemical roles and structure of the Rad52 protein . RESULTS: Purified Rad52 protein binds to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) . Electron microscope observations revealed that Rad52 molecules form multimeric rings . An increase in the intensity of fluorescence when Rad52 is bound to epsilonDNA showed an alteration of the structure of ssDNA . RPA was binding to Rad52 and enhanced the annealing of complementary ssDNA molecules . This enhancement was not observed in Escherichia coli SSB protein or T4 phage gp32 protein . CONCLUSION: Rad52 forms a ring-like structure and binds to ssDNA . Its structure and DNA binding properties are different from those of Rad51 . The interaction of Rad52 with RPA plays an important role in the enhancement of annealing of complementary ssDNAs . We therefore propose that Rad52 mediates the RAD51-independent recombination through an ssDNA annealing, assisted by RPA. FASEB J, 1998 Jun, 12(9), 653 - 63 Prediction-based threading of the hMSH2 DNA mismatch repair protein; de las Alas MM et al.; Mutations in the genes whose products participate in DNA mismatch repair underlie the increased risk of cancer in families with hereditary nonpolyposis colon carcinoma . Mutations in hMSH2 account for approximately 50% of the mutations found in these families . We sought to predict the 3-dimensional structure of hMSH2 by identifying structural homologues using prediction-based threading and by computer modeling using information from these putative structurally related proteins . Prediction-based threading identified three candidate structural homologues: glycogen phosphorylase (gpb), a 70 kDa soluble lytic transglycosylase, and ribonucleotide reductase protein R1 . An independent approach utilizing a potential-based threading program also identified gpb as a structural homologue . The models based on the structures of these proteins suggest that the ATP binding domain and helix-turn-helix domain are exposed on the outside of the protein . All known bacterial MutS and hMSH2 mutations appear to be clustered in similar vicinities in the theoretical models of hMSH2; the major site is within the ATP binding domain and near the carboxyl-terminal end, whereas a smaller number map to the region coding for exon 5 and the amino-terminal domain . All point mutations also appear to affect amino acids that are exposed on the outside surface of the protein. Cytokine, 1998 May, 10(5), 390 - 4 Different roles of brain interleukin 1 in the adrenocorticotropin response to central versus peripheral administration of lipopolysaccharide in the rat; Habu S et al.; Although it is well established that peripheral administration of endotoxin activates the hypothalamic-pituitary-adrenal (HPA) axis, information is very limited regarding whether central administration of endotoxin can similarly stimulate the endocrine axis . Moreover, it is also unknown whether a difference exists in the mode of involvement of brain-derived cytokines in determining the HPA response to peripheral vs central administration of endotoxin . In the present study, the authors attempted to gain more knowledge on these issues focusing on interleukin (IL) 1 in the brain, one of key pro-inflammatory cytokines mediating the immuno-endocrine network . In male rats, both intravenous (i.v., 100 micrograms/kg body weight) and intracerebroventricular {i.c.v . (the 3rd ventricle), 10 micrograms} injections of Escherichia coli lipopolysaccharide (LPS) caused a significant elevation of adrenocorticotropin (ACTH) levels in plasma, even though peaked ACTH responses occurred earlier after the i.v . (60 min post-injection) than the i.c.v . (120 min post-injection) LPS . Although the ACTH response to i.c.v . LPS was significantly suppressed by a prior (5 min) i.c.v . administration of IL-1 receptor antagonist (IL-1Ra, 1 microgram), the hormonal response to i.v . LPS was not . That this dose of IL-1Ra was not biologically a small dose was indicated by another experiment that the same dose of i.c.v . IL-1Ra was able to significantly suppress the ACTH response to an i.c.v . injection of recombinant human IL-1 beta (50 ng) . These results suggest that i.c.v . LPS, as i.v . LPS, can stimulate ACTH secretion in the rat, and this hormonal response may, at least in part, be mediated by brain-derived IL-1 . Although there is one previous study reporting an important role of central IL-1 in mediating the HPA response to systemic LPS treatment, our present data suggest that such a mechanism may not operate before and during an early, peak phase of ACTH secretion after i.v . LPS. Cytokine, 1998 May, 10(5), 319 - 30 Expression of active thrombopoietin and identification of its key residues responsible for receptor binding; Hou J et al.; In this report expression of the biologically active N-terminal half (amino acids 1-153) of thrombopoietin (TPO153) in Escherichia coli is described and the structure-function relationships in TPO are explored . TPO153 was chosen for expression because of its full biological activity . Since natural TPO153 cDNA expressed poorly, synthetic cDNA was constructed with a unique polymerase chain reaction to enhance the expression . In addition, the 5'-end codons of the synthetic cDNA were altered to maximize the expression . The expressed TPO153 was refolded and then purified to homogeneity . The protein is biologically active, and interestingly, the EC50 of this protein is 8-10-fold smaller in a TPO-dependent cell proliferation assay than that of full-length wild-type TPO . In order to identify the amino acid residues that are involved in the interaction between TPO and its receptor, all charged residues and some of the uncharged residues on the four putative helices of TPO were mutated and biological activities of the mutant proteins were examined . The mutagenesis studies suggest that there are at least two clusters of residues that are vital for TPO to be able to interact with its receptor . These residues are centred respectively around arginine 10 on helix 1 and around lysine 138 on helix IV . The successful expression of the protein in E . coli will greatly facilitate biochemical and crystallographic studies of TPO, and the structure-function relationship studies suggest that TPO has two binding sites which may interact with two individual receptors, resulting in dimerization of the receptors. Parasite Immunol, 1998 Apr, 20(4), 183 - 95 Mapping of B epitopes in GRA4, a dense granule antigen of Toxoplasma gondii and protection studies using recombinant proteins administered by the oral route; Mevelec MN et al.; GRA4, a dense granule protein of Toxoplasma gondii elicits both mucosal and systemic immune responses following oral infection of mice with cysts . We studied the antigenicity and immunogenicity of truncated and soluble forms of GRA4 expressed as glutathione S-transferase fusion proteins in Escherichia coli . Protein C (amino-acids 297-345) was particularly well recognized by serum IgG antibodies, milk IgA antibodies and intestinal IgA antibodies from T . gondii infected mice and by serum IgG antibodies from T . gondii infected humans and T . gondii infected sheep . One major B epitope was localized within the last 11 C-terminal residues of GRA4 . A second epitope, recognized with lower frequency, was mapped within the region 318-334 . In contrast, the N domain of GRA4 (amino acids 25-276) was poorly recognized . Oral immunization of C57BL/6 mice with N, C or NC (amino acids 25-276 fused to 297-345) in association with cholera toxin induced a significant production of serum anti-GRA4 IgG antibodies but a weak and inconsistent intestinal anti-GRA4 IgG antibody response and afforded partial resistance to oral infection with T . gondii . These results provide new molecular and immunological understanding of GRA4 and indicate that it is a potential candidate for oral vaccination against T . gondii. Parasite Immunol, 1998 Apr, 20(4), 169 - 74 Suppressive effect of Ixodes ricinus salivary gland extract on mechanisms of natural immunity in vitro; Kopecky J et al.; Tick saliva has been shown to contain a variety of pharmacologically active molecules, including those with immunosuppressive activities . There is increasing evidence that the nonspecific suppression of host immunity by tick saliva is exploited by tick-borne pathogens, e.g . the saliva-activated transmission (SAT) of some tick-borne viruses . We have demonstrated the suppressive effect of the salivary gland extract (SGE) derived from partially fed (five days) Ixodes ricinus females on important mechanisms of innate immunity: natural killer (NK) cells, interferon and nitric oxide production . SGE reduced the interferon induction by polyinosinic-polycytidylic acid (poly I:C) or Escherichia coli lipopolysaccharide (LPS) in a culture of Balb/c mouse splenocytes by 94% and 62%, respectively . SGE suppressed the cytotoxicity of nonstimulated and in vivo poly I:C-stimulated mouse NK cells by up to 31% and 26%, respectively . The induction of NK activity in vitro by LPS but not by Concanavalin-A (Con-A) was also downregulated in the presence of SGE . The addition of SGE to cultures of mouse macrophages partially inhibited the production of nitric oxide, induced by LPS . These data suggest that the facilitating effect of SGE on the transmission of some tick-borne pathogens might be associated with the suppression of the host innate resistance mechanisms, represented by interferon, nitric oxide and NK cells. Curr Genet, 1998 May, 33(5), 368 - 77 Paxilline-negative mutants of Penicillium paxilli generated by heterologous and homologous plasmid integration; Young C et al.; Using a monoclonal antibody based ELISA, 600 pAN7-1 plasmid-tagged mutants of Penicillium paxilli were screened for paxilline accumulation and one paxilline-negative mutant, YI-20, was identified . A molecular analysis of this mutant showed that pAN7-1 was inserted at a single site but was present as 4-6 copies arranged in a head-to-tail tandem array . Rescue of flanking sequences and analysis of the corresponding genomic region revealed that YI-20 has an extensive deletion at the site of pAN7-1 integration . Probing of a CHEF gel with the same sequences showed that associated with the deletion is a rearrangement of chromosome Va . Targeted gene disruption of wild-type sequences adjacent to the site where pAN7-1 inserted, resulted in the generation of two additional paxilline-negative mutants; both were single crossovers with deletions extending outside the region mapped . Neither of these new mutants had a rearrangement of chromosome Va, suggesting that deletion of genes on this chromosome is responsible for the paxilline-negative phenotype . Telomeric fingerprinting of genomic digests of P . paxilli, combined with pulsed-field gel electrophoresis of chromosomal DNA, established that there are a minimum of eight chromosomes in this fungus. Microbiol Mol Biol Rev, 1998 Jun, 62(2), 309 - 33 Acylation of Escherichia coli hemolysin: a unique protein lipidation mechanism underlying toxin function; Stanley P et al.; The pore-forming hemolysin (HlyA) of Escherichia coli represents a unique class of bacterial toxins that require a posttranslational modification for activity . The inactive protoxin pro-HlyA is activated intracellularly by amide linkage of fatty acids to two internal lysine residues 126 amino acids apart, directed by the cosynthesized HlyC protein with acyl carrier protein as the fatty acid donor . This action distinguishes HlyC from all bacterial acyltransferases such as the lipid A, lux-specific, and nodulation acyltransferases, and from eukaryotic transferases such as N-myristoyl transferases, prenyltransferases, and thioester palmitoyltransferases . Most lipids directly attached to proteins may be classed as N-terminal amide-linked and internal ester-linked acyl groups and C-terminal ether-linked isoprenoid groups . The acylation of HlyA and related toxins does not equate to these but does appear related to a small number of eukaryotic proteins that include inflammatory cytokines and mitogenic and cholinergic receptors . While the location and structure of lipid moieties on proteins vary, there are common effects on membrane affinity and/or protein-protein interactions . Despite being acylated at two residues, HlyA does not possess a "double-anchor" motif and does not have an electrostatic switch, although its dependence on calcium binding for activity suggests that the calcium-myristoyl switch may have relevance . The acyl chains on HlyA may provide anchorage points onto the surface of the host cell lipid bilayer . These could then enhance protein-protein interactions either between HlyA and components of a host signal transduction pathway to influence cytokine production or between HlyA monomers to bring about oligomerization during pore formation. Biochem Biophys Res Commun, 1998 May 29, 246(3), 797 - 804 Expression systems for study of mycobacterial gene regulation and development of recombinant BCG vaccines; DasGupta SK et al.; Successful genetic engineering of mycobacteria is crucial for developing new approaches to combat tuberculosis as well as for dissecting out the molecular basis of pathogenesis of Mycobacterium tuberculosis . We have constructed a Mycobacterium-Escherichia coli shuttle expression vector pSD5 . It carries a modular expression cassette which provides sites for cloning of promoters, a ribosome binding site (RBS) with an appropriately placed initiation codon and multiple cloning sites for cloning the genes of interest . We also constructed pDK20, an integration proficient derivative of pSD5, by incorporating mycobacteriophage L5 integration signals in lieu of the origin of DNA replication for mycobacteria . This vector permits stable expression of genes in M.bovis BCG, M.smegmatis, and M.tuberculosis under the transcriptional control of a mycobacterial promoter . These vectors enable the expression of a gene to be regulated by several hundred fold depending upon the strength of mycobacterial promoter . We propose that expression of protective antigens using an appropriate promoter derivative of pDK20 should help in development of recombinant BCG vaccines that can induce an optimal immune response from the host . We have further employed the integration proficient expression system for comparing the efficiency and specificity of transcriptional recognition in M.bovis BCG, M.tuberculosis, and M.smegmatis . We show that fast growing M.smegmatis and slow growing M.tuberculosis and M.bovis BCG recognize mycobacterial promoters with comparable efficiency inspite of differences in their growth rates. Biochem Biophys Res Commun, 1998 May 29, 246(3), 740 - 5 New paramagnetic species formed at the expense of the transient tyrosyl radical in mutant protein R2 F208Y of Escherichia coli ribonucleotide reductase; Liu A et al.; The highly conserved residue F208 in protein R2 of E . coli ribonucleotide reductase is close to the binuclear iron center, and found to be involved in stabilizing the tyrosyl radical Y122 . in wild type R2 . Upon the reconstitution reaction of the mutant R2 F208Y with ferrous iron and molecular oxygen, we observed a new EPR singlet signal (g = 2.003) formed concomitantly with decay of the transient tyrosyl radical Y122 . (g = 2.005) . This new paramagnetic species (denoted Z) was stable for weeks at 4 degrees C and visible by EPR only below 50 K . The EPR singlet could not be saturated by available microwave power, suggesting that Z may be a mainly metal centered species . The maximum amount of the compound Z in the protein purified from cells grown in rich medium was about 0.18 unpaired spin/R2 . An identical EPR signal of Z was found also in the double mutant R2 F208Y/Y122F . In the presence of high concentration of sodium ascorbate, the amounts of both the transient Y122 . and the new species Z increased considerably in the reconstitution reaction . The results suggest that Z is most likely an oxo-ferryl species possibly in equilibrium with a Y208 ligand radical. Biochem Biophys Res Commun, 1998 May 29, 246(3), 602 - 5 The flavoprotein component of the Escherichia coli sulfite reductase can act as a cytochrome P450c17 reductase; Zeghouf M et al.; The flavoprotein component (SiR-FP) of the E . coli sulfite reductase was found to support 17 alpha-hydroxylation of pregnenolone in the presence of cytochrome P450c17 . Half maximum activity is obtained for a 1:1 ratio of SiR-FP, expressed as monomer concentration, to P450c17 . When compared to bovine NADPH-cytochrome P450 reductase, SiR-FP is about 12-15 times less efficient . P450c17 was demonstrated to interact specifically with the FMN-binding domain of the protein and the N-terminal part of SiR-FP is suspected to play a role in electron transfer . A cluster of negatively charged residues was found in SiR-FP by amino acid sequence comparison with rat cytochrome P450 reductase . These results argue in favour of the flavodoxin origin of the FMN-binding domain of SiR-FP. Anal Biochem, 1998 Jun 1, 259(2), 258 - 64 A sandwich hybridization assay employing enzyme amplification for termination of specific ribosomal RNA from unpurified cell lysates; Wicks B et al.; We have employed the power of the cyclic NAD-based enzyme amplification system to the determination of 16S rRNA . This generally applicable system employs two oligonucleotide probes, one of which is captured on a microtiter well surface and the other labeled with alkaline phosphatase . The detection of very low levels of hybridization of the capture probe is then achieved by the means of the ultrasensitive enzyme-amplified assay system, resulting in a highly sensitive, convenient, and rapid technology which can be directly employed on unpurified samples . We have been able to demonstrate the detection of 20 amol (10(7) molecules) of pure rRNA, and specific signals from as few as 2000 bacterial cells have also been demonstrated . The total procedural time can be short-5 to 18 h-depending on the dynamic range and sensitivity required . RNA target in the range of 10(12)-10(8) molecules can be assayed within 5 h . Extending the substrate incubation time enables between 10(11) and 10(7) molecules to be determined within 18 h . The system has great potential use with respect to studying the distribution and physiological states of cellular organisms . EMBO J, 1998 May 15, 17(10), 2877 - 85 Regulation of crp transcription by oscillation between distinct nucleoprotein complexes; Gonzalez-Gil G et al.; FIS belongs to the group of small abundant DNA-binding proteins of Escherichia coli . We recently demonstrated that, in vivo, FIS regulates the expression of several genes needed for catabolism of sugars and nucleic acids, a majority of which are also transcriptionally regulated by cAMP-cAMP-receptor protein (CRP) complex . Here we provide evidence that FIS represses transcription of the crp gene both in vivo and in vitro . Employing crp promoter-lacZ fusions, we demonstrate that both FIS and cAMP-CRP are required to keep the crp promoter in a repressed state . We have identified in the crp promoter other transcription initiation sites which are located 73, 79 and 80 bp downstream from the previously mapped start site . Two CRP- and several FIS-binding sites with different affinities are located in the crp promoter region, one of them overlapping the downstream transcription initiation sites . We show that initiation of transcription at the crp promoter is affected by the composition of nucleoprotein complexes resulting from the outcome of competition between proteins for overlapping binding sites . Our results suggest that the control of crp transcription is achieved by oscillation in the composition of these regulatory nucleoprotein complexes in response to the physiological state of the cell. Nucleic Acids Res, 1998 May 15, 26(10), 2508 - 10 New positive/negative selectable markers for mammalian cells on the basis of Blasticidin deaminase-thymidine kinase fusions; Karreman C; Two positive and negative selectable markers were created for use in mammalian cells . They are based on two genes for the resistance to Blasticidin S (BlaS) and on the thymidine kinase (Tk) gene of herpes simplex virus (HSV) . The markers can be selected positively by their ability to induce BlaS resistance and negatively on the induced sensitivity towards gancyclovir (GANC) . Both constructs are also expressed in Escherichia coli and transfer BlaS resistance to this organism as well, making these markers very suitable for the construction of shuttle vectors. Nucleic Acids Res, 1998 May 15, 26(10), 2505 - 7 Direct visualization of site-specific and strand-specific DNA methylation patterns in automated DNA sequencing data; Rao BS et al.; We report here a simple method of directly visualizing in automated DNA sequencing chromatograms DNA methylations of different types including cytosine methylations in Hpa II and dcm sites as well as adenine methylations in dam sites . This is made possible by the observation that the extent of incorporation of fluorescently labeled dideoxynucleotides is influenced by the methylated bases in template DNA . This simple approach involves routine automated DNA sequencing without any prior treatment of DNA specific for detecting DNA methylation. Nucleic Acids Res, 1998 May 15, 26(10), 2500 - 1 Preparation of active tRNA gene transcripts devoid of 3'-extended products and dimers; Kholod N et al.; Significant amounts (10-30%) of 3'-extended products with one or two extra nucleotides are synthesized in the course of run-off tRNA gene transcription with T7 RNA polymerase . Denaturing polyacrylamide gel electrophoresis appeared to be insufficient to provide preparative amounts of pure correct-size transcripts . Formation of dimers by tRNA gene transcripts as side products in the course of their activation is also another obstacle in preparation of biologically active transcripts . Here, we have shown that EF-Tu affinity chromatography and/or non-denaturing electrophoresis are simple and efficient tools for isolation of highly active correct-size transcripts . Conditions for transcript activation in vitro should be carefully controlled to prevent dimer formation and obtain reliable data on tRNA transcript structure and function. Nucleic Acids Res, 1998 May 15, 26(10), 2353 - 8 Cloned human FMR1 trinucleotide repeats exhibit a length- and orientation-dependent instability suggestive of in vivo lagging strand secondary structure; Hirst MC et al.; The normal human FMR1 gene contains a genetically stable (CGG) n trinucleotide repeat which usually carries interspersed AGG triplets . An increase in repeat number and the loss of interspersions results in array instability, predominantly expansion, leading to FMR1 gene silencing . Instability is directly related to the length of the uninterrupted (CGG) n repeat and is widely assumed to be related to an increased propensity to form G-rich secondary structures which lead to expansion through replication slippage . In order to investigate this we have cloned human FMR1 arrays with internal structures representing the normal, intermediate and unstable states . In one replicative orientation, arrays show a length-dependent instability, deletions occurring in a polar manner . With longer arrays these extend into the FMR1 5'-flanking DNA, terminating at either of two short CGG triplet arrays . The orientation-dependent instability suggests that secondary structure forms in the G-rich lagging strand template, resolution of which results in intra-array deletion . These data provide direct in vivo evidence for a G-rich lagging strand secondary structure which is believed to be involved in the process of triplet expansion in humans. Nucleic Acids Res, 1998 May 15, 26(10), 2273 - 8 Selective inhibition of cell-free translation by oligonucleotides targeted to a mRNA hairpin structure; Le Tinevez R et al.; Using an in vitro selection approach we have previously isolated oligodeoxy aptamers that can bind to a DNA hairpin structure without disrupting the double-stranded stem . We report here that these oligomers can bind to the RNA version of this hairpin, mostly through pairing with a designed 6 nt anchor . The part of the aptamer selected against the DNA hairpin did not increase stability of the RNA-aptamer complex . However, it contributed to the binding site for Escherichia coli RNase H, leading to very efficient cleavage of the target RNA . In addition, a 2'- O -methyloligoribonucleotide analogue of one selected sequence selectively blocked in vitro translation of luciferase in wheat germ extract by binding to the hairpin region inserted upstream of the initiation codon of the reporter gene . Therefore, non-complementary oligomers can exhibit antisense properties following hybridization with the target RNA . Our study also suggests that in vitro selection might provide a means to extend the repertoire of sequences that can be targetted by antisense oligonucleotides to structured RNA motifs of biological importance. Nucleic Acids Res, 1998 May 15, 26(10), 2265 - 72 Characterization of protein-binding to the spinach chloroplast psbA mRNA 5' untranslated region; Alexander C et al.; RNA-binding proteins play a major role in regulating mRNA metabolism in chloroplasts . In this work we characterized two proteins, of 43 and 47 kDa, which bind to the spinach psbA mRNA 5' untranslated region (psbA encoding the D1 protein of photosystem II) . The 43 kDa protein, which is present in the stroma and in membranes, co-sediments with a complex of 68S . It was purified, and the N-terminal sequence was determined . Upon homology search it was identified as the chloroplast homologue of the Escherichia coli ribosomal protein S1 . The 47 kDa protein, which, in contrast with the 43 kDa protein, sediments with a small sedimentation coefficient, is only detected in the stromal fraction . It is soluble in an uncomplexed form . By deletion analysis, an element within the psbA mRNA 5' untranslated region was identified that is necessary but not sufficient for binding of stromal proteins . The 'central protein binding element' ranges from nucleotide -49 to -9 of the psbA mRNA 5' untranslated region . It comprises the Shine-Dalgarno-like GGAG motif and, 7 nucleotides upstream, an endonucleolytic cleavage site involved in psbA mRNA degradation in vitro . The mechanistic impacts of this region in relation to RNA-binding proteins are discussed. J Mol Biol, 1998 May 1, 278(2), 307 - 16 Combinatorial effects of NusA and NusG on transcription elongation and Rho-dependent termination in Escherichia coli; Burns CM et al.; The transcription factors NusA and NusG from Escherichia coli are modulators of the RNA polymerase elongation reaction and Rho-dependent transcription termination . NusA decreases the elongation rate and termination efficiency while NusG increases both activities . Both Nus factors are able to physically interact with Rho and with RNA polymerase . Experiments with purified components designed to determine whether these factors act independently or competitively showed that the change in elongation rate was a composite of their individual effects, that the combined effect on termination was dependent on the reaction conditions and that the two factors do not compete for their sites of action for either effect . The two factors were also found not to enhance significantly the slight (20%) inhibition of elongation caused by 200 microM guanosine 3',5'-bisdiphosphate (ppGpp) during transcription in vitro . The results also show that the effects of NusA and NusG on RNA polymerase elongation and Rho function are contrary to the inverse relationship between elongation and termination that is expected for a kinetic coupling of Rho action to RNA polymerase elongation . This property suggests that in addition to their known actions on RNA polymerase that influence the length of pausing, these factors act on some other rate-limiting step of the Rho-dependent termination process . Shock, 1998 May, 9(5), 364 - 8 Etiology of metabolic acidosis during saline resuscitation in endotoxemia; Kellum JA et al.; We sought to understand the mechanism of metabolic acidosis that results in acute resuscitated endotoxic shock . In six pentobarbital-anesthetized dogs, shock was induced by Escherichia coli endotoxin infusion (1 mg/kg) and was treated with saline infusion to maintain mean arterial pressure > 80 mmHg . Blood gases and strong ions were measured during control conditions and at 15, 45, 90, and 180 min after endotoxin infusion . The mean saline requirement was 1833+/-523 mL over a 3 h period . The total acid load from each source was calculated using the standard base deficit . The mean arterial pH decreased from 7.32 to 7.11 (p < .01); pCO2 and lactate were unchanged . Saline accounted for 42% of the total acid load . However, 52% of the total acid load was unexplained . Although serum Na+ did not change, serum Cl-increased (127.7+/-5.1 mmol/L vs . 137.0+/-6.1 mmol/L; p=.016) . We conclude that saline resuscitation alone accounts for more than one-third of the acidosis seen in this canine model of acute endotoxemia, whereas lactate accounts for less than 10% . A large amount of the acid load can be attributed to differential Na+ and Cl- shifts from extravascular to vascular spaces. Shock, 1998 May, 9(5), 329 - 35 Selective inhibition of the activity of inducible nitric oxide synthase prevents the circulatory failure, but not the organ injury/dysfunction, caused by endotoxin; Wray GM et al.; Inhibitors of nitric oxide synthase (NOS) attenuate the circulatory failure caused by endotoxin, but the role of NO in the development of multiple organ dysfunction and the relative contribution of NO produced by endothelial NOS and inducible NOS (iNOS) to organ injury remains unclear . Here we report for the first time that 1400W, a novel and highly selective inhibitor of iNOS activity, attenuates the delayed hypotension as well as the rise in the plasma levels of nitrite/nitrate caused by endotoxin in the rat . Inhibition of iNOS activity with 1400W administered either before or 2 h after endotoxin injection did not, however, attenuate the hepatocellular injury, renal dysfunction, or pancreatic injury in this model . Similarly, administration of another selective inhibitor of iNOS activity, L-NIL, 2 h after endotoxin injection abolished the rise in nitrite/nitrate and attenuated the delayed hypotension caused by endotoxin, but failed to ameliorate organ injury . Thus, selective inhibition of iNOS activity with 1400W attenuates the circulatory failure induced by endotoxin in the rat, but fails to influence the degree of organ injury/dysfunction. Plant Mol Biol, 1998 Jun, 37(3), 495 - 504 Biochemical and immunological characterization of rice homologues of the human immunodeficiency virus-1 Tat binding protein and subunit 4 of human 26S proteasome subunits; Suzuka I et al.; Previously, we isolated two cDNA clones, TBPOs-1 and TBPOs-2, encoding putative ATPases that are the rice homologues of human immunodeficiency virus-1 (HIV-1) Tat binding protein-1 and subunit 4 of human 26S proteasome . In order to determine the RNA-dependent ATPase activity of these putative proteins, the subclones from these cDNA clones were expressed in Escherichia coli as fusion proteins with maltose-binding protein . The recombinant proteins stimulated ATP hydrolysis in the presence of poly(U) and rice total RNA . In contrast, single- and double-stranded forms of HindIII-digested lambda phage DNA are less effective at stimulating ATP hydrolysis . Western blot analysis using antisera against the TBPOs proteins showed a widespread appearance of these proteins in rice tissues and cultured cells . The TBPOs proteins were also found around the region where rice proteasomes would sediment . In addition, the TBPOs-1 protein bound to tobacco TATA-binding protein in vitro . Thus, we suggest that the TBPOs proteins are novel RNA-dependent ATPases characteristic of DEAD-box proteins and propose that the TPBOs proteins can exist in rice proteasomes . Further, the TBPOs-1 protein is thought to play a role in transcriptional events. Plant Mol Biol, 1998 May, 37(2), 337 - 47 Engineering in vivo instability of firefly luciferase and Escherichia coli beta-glucuronidase in higher plants using recognition elements from the ubiquitin pathway; Worley CK et al.; The ubiquitin pathway targets proteins for degradation through the post-translational covalent attachment of the 76 amino acid protein ubiquitin to epsilon-amino lysyl groups on substrate proteins . Two instability determinants recognized by the ubiquitin pathway in Saccharomyces cerevisiae have been identified . One is described by the N-end rule and requires specific destabilizing residues at the substrate protein N-termini along with a proximal lysyl residue for ubiquitin conjugation . The second is a linear uncleavable N-terminal ubiquitin moiety . The ability of these two determinants to function in higher plants was investigated in tobacco protoplast transient transfection assays using DNA encoding variants of well characterized reporter enzymes as substrates: firefly luciferase that is localized to peroxisomes (pxLUC), a cytosolic version of LUC (cLUC), and Escherichia coli beta-glucuronidase (GUS) . cLUC with phenylalanine encoded at its mature N-terminus was 10-fold less abundant than cLUC with methionine at its mature N-terminus . GUS with phenylalanine encoded at its mature N-terminus was 3-fold less abundant than GUS with methionine at its mature N-terminus . The presence of a uncleavable N-terminal ubiquitin fusion resulted in 50-fold lower protein accumulation of cLUC, but had no effect on GUS . Both instability determinants had a much larger effect on cLUC than on pxLUC, suggesting that these degradation signals are either unrecognized or poorly recognized in the peroxisomes. Plant Mol Biol, 1998 May, 37(2), 205 - 15 Cloning, characterization and expression of carbonic anhydrase from the cyanobacterium Synechocystis PCC6803; So AK et al.; A 3.3 kb HindIII restriction-digest DNA fragment was isolated from a Synechocystis sp . strain PCC6803 subgenomic plasmid library which strongly hybridized to a 349 bp fragment of the icfA (ccaA) gene from Synechococcus sp . strain PCC7942 . DNA sequence analysis of the fragment revealed three open reading frames (ORFs), two of which potentially coded for pantothenate synthetase (ORF275) and cytidylate kinase (ORF230) . The third, ORF274, was 825 bp in length, encoding a deduced polypeptide of 274 aa (Mr, 30747) that bears 55% sequence identity to the Synechococcus icfA (ccaA) translation product, a beta-type carbonic anhydrase (CA) . A 932 bp EcoRI fragment containing ORF274 was subcloned into an expression vector and the construct was transformed into Escherichia coli for overexpression . Electrometric assays for CA activity revealed that whole cell extracts containing the recombinant protein significantly enhanced the rate of conversion of CO2 to HCO3- and that 98% of this catalytic activity was inhibited by ethoxyzolamide, a well-characterized CA inhibitor . Antisera derived against the overexpressed protein recognized a 30.7 kDa protein that was predominantly associated with the isolated carboxysome fraction from Synechocystis . These results provide molecular and physiological evidence for the identification of a ccaA homologue in Synechocystis PCC6803 that encodes a carboxysomal beta-type CA. Plant Mol Biol, 1998 May, 37(2), 197 - 204 Identification of a 37 kDa plant protein that interacts with the turnip mosaic potyvirus capsid protein using anti-idiotypic-antibodies; McClintock K et al.; Experimental data are provided for the presence of a plant protein that interacts with the capsid protein (CP) of turnip mosaic potyvirus (TuMV) . The receptor-like protein was identified by exploiting the molecular mimicry potential of anti-idiotypic antibodies . A single-chain Fv molecule derived from the monoclonal antibody 7A (Mab-7A), which recognizes the CP of TuMV, was produced in Escherichia coli and the recombinant protein was used to raise rabbit antibodies . The immune serum reacted with Mab-7A but not with a monoclonal antibody of the same isotype, indicating that anti-idiotypic antibodies were produced . These anti-idiotypic antibodies recognized a 37 kDa protein from Lactuca sativa . Complex formation between the anti-idiotypic antibodies and the plant protein was inhibited by the CP of TuMV which indicates that the plant protein interacts with the viral protein . The 37 kDa protein was localized in chloroplasts and was detected in other plant species. Curr Eye Res, 1998 May, 17(5), 501 - 5 The role of platelet-activating factor in cell infiltration in endotoxin-induced uveitis in guinea pigs; Tsuji F et al.; PURPOSE: We investigated the role of platelet-activating factor (PAF) in cell infiltration in endotoxin-induced uveitis (EIU) in guinea pigs . METHODS: To elicit EIU, lipopolysaccharide (LPS) was injected into the anterior chamber of the eye . Cell numbers in the aqueous humor after LPS injection were determined by flow cytometry . PAF and prostaglandin E2 (PGE2) production after LPS injection were also examined . RESULTS: Intracameral injection of LPS induced cell infiltration into the anterior chamber, and platelet-activating factor (PAF) was detected in the aqueous humor . In addition, topical apafant (PAF antagonist) partially inhibited cell infiltration . Intracameral injection of PAF scarcely induced cell infiltration but the reaction with EIU was accelerated by intracameral injection of a small amount of PAF . CONCLUSIONS: These results suggest that PAF has weak direct activity on cell infiltration in intraocular inflammation but enhances intraocular inflammation. Respir Med, 1998 Feb, 92(2), 162 - 6 Budesonide but not terbutaline decreases phagocytosis in alveolar macrophages; Zetterlund A et al.; Alveolar macrophages are the most common cells in bronchoalveolar lavage fluid . The macrophages participate in the inflammatory response and defence of the airways by secretion of mediators and by phagocytizing foreign particles such as bacteria and viruses . beta-Agonists and glucocorticosteroids are the most frequently used drugs in asthma . Alveolar macrophages have beta 2-adrenoceptors on their surface but the functional role of these receptors is unknown . Glucocorticosteroids interact with mediator release from macrophages . However, nothing is known about the effects of those drugs on the phagocytic capacity of alveolar macrophages . Therefore, the present study has investigated phagocytosis of alveolar macrophages from nine healthy volunteers after incubation with a beta 2-agonist, terbutaline (10(-8), 10(-6) and 10(-4) M) and a glucocorticosteroid, budesonide (10(-9), 10(-7) and 10(-5) M) . Alveolar macrophages were incubated with FITC-labelled Escherichia coli, and the drugs and phagocytosis was assessed by flow cytometry . Phagocytosis was measured as the proportion of phagocytizing cells and mean fluorescence intensity (MFI) . MFI was highly correlated with phagocytized E . coli per cell assessed by fluorescence microscopy (r = 0.996) . The proportion of phagocytizing macrophages (control) was {median (25th-75th) percentiles} 46% (40-63) and 29% (18-60), and MFI were 174 (154-205) and 122 (90-271) in the terbutaline and budesonide experiments, respectively . Terbutaline did not affect the phagocytosis significantly, while budesonide decreased the phagocytic capacity (percent phagocytizing cells and MFI) in a dose-dependent manner (P < 0.01) . At the highest budesonide concentration (10(-5) M), phagocytosis was approximately half of the control situation . In conclusion, this in vitro study indicate that a glucocorticosteroid decreases phagocytosis in alveolar macrophages in a concentration that may be relevant in the airway lining fluid . Further investigations regarding the effect on other micro-organisms and in vivo effects are necessary to further elucidate these findings. Gut, 1998 Apr, 42(4), 460 - 1 Viral vectors expressing immunoregulatory cytokines to treat inflammatory bowel disease; Macdonald TT; Inflammatory bowel disease (IBD) is characterised by altered immunoregulation and augmented synthesis of nitric oxide . The purpose of this study was to determine the effects of exogenous IL-4, introduced by a recombinant human type 5 adenovirus (Ad5) vector, on the tissue injury associated with an experimental model of colonic immune activation and inflammation . Colitis was induced in rats by the intrarectal administration of trinitrobenzene sulfonic acid (TNB) dissolved in 50% ethanol, and control rats received saline via the same route . 1 h later, all rats were randomized into two groups . The first group was injected intraperitoneally (i.p.) with 3.0 x 10(6) plaque forming units (PFUs) of Ad5 transfected with murine interleukin-4 (Ad5IL-4) and the second group was injected i.p . with the same amount of Ad5 expressing the Escherichia coli Lac Z gene (Ad5LacZ) . One-half of the colitic and controls rats were injected again with 3.0 x 10(6) PFUs of Ad5IL-4 or Ad5LacZ on day 3 of the 6-d study . When introduced once or twice via the peritoneal route into control rats Ad5LacZ was localised to the serosal lining of the peritoneal cavity, the diaphragm and the liver on day 6 . One or two injections of Ad5IL-4 into rats also produced measurable levels of circulating IL-4 . TNB-colitis in both Ad5LacZ-treated groups was associated with pronounced elevations in serum IFN-gamma, and mucosal ulceration of the distal colon . Myeloperoxidase and inducible nitric oxide synthase II (NOS II) synthetic activity were also increased by 30- and fivefold, respectively, above control levels in the distal colon . However, two injections of AD5IL-4 into colitic rats caused the overexpression of IL-4, and significantly inhibited tissue damage, serum and colon IFN-gamma levels and myeloperoxidase activity in the distal colon . In addition, NOS II gene expression and NOS II nitric oxide synthesis was significantly inhibited . No therapeutic effect was observed in rats injected once with AD5IL-4 . Thus, IL-4, introduced by Ad5, is therapeutic during acute inflammation in the rat colon . The therapeutic effect of IL-4 was associated with an inhibition of inducible nitric oxide expression and a reduction in nitric oxide synthesis. Vet Parasitol, 1998 Apr 15, 76(3), 189 - 202 Studies with recombinant proteins of Ehrlichia risticii: identification of strain-specific antigen as a protective antigen; Vemulapalli R et al.; Ehrlichia risticii is the causative agent of Potomac horse fever, an acute infectious disease of equines . To study the role of major antigens of E . risticii in protective immune response, we have expressed the genes of the 55 kDa, 51 kDa and 85/50 kDa-strain-specific antigens of the 90-12 (85 kDa antigen) and 25-D (50 kDa antigen) strains in Escherichia coli using pRSET A, B, C system (Invitrogen, San Diego, CA) . Mice immunized with these purified recombinant proteins of E . risticii developed strong and specific humoral immune response . The recombinant 85 kDa antigen of the 90-12 strain protected mice against challenge infection with both E . risticii strains, whereas its homologue from the 25-D strain, the recombinant 50 kDa antigen, protected mice against only the homologous strain challenge, but not against the heterologous 90-12 strain . Sera from mice immunized with the 85- or 50-kDa antigens did not inhibit the replication of cell-free Ehrlichiae in in vitro neutralization assays . Sera from normal mice and mice immunized with other antigens caused non-specific neutralization of E . risticii . Immunoglobulin G from mice immunized with the 51 kDa protein of the 90-12 strain caused partial in vitro neutralization of both strains of E . risticii . These studies demonstrate that the 85/50-kDa-strain-specific antigen of E . risticii is involved in immunoprotection against PHF. Anticancer Res, 1998 Mar-Apr, 18(2B), 1255 - 60 GM-CSF in chemotherapy-induced febrile neutropenia--a double-blind randomized study; Arnberg H et al.; Modern chemotherapy programmes render patients susceptible to bacterial and fungal infections, and the risk of developing febrile neutropenia after a chemotherapy course is in proportion to the severity and duration of the neutropenia thus caused . This double-blind randomized study presents details of 29 patients who developed febrile neutropenia an average of 10 days after their course of chemotherapy for different types and stages of malignancy . Fourteen received granulocyte/macrophage colony stimulating factor (GM-CSF) and 15 placebo during 7 consecutive days as subcutaneous injections . The GM-CSF group demonstrated significant increases in total white blood cell count (TWBC) and absolute neutrophil count (ANC) from the morning of the third day of the study . The study concludes that GM-CSF has an important therapeutic role in the treatment of febrile neutropenia that arises during intensive chemotherapy programmes but further studies of dosage and therapy duration are required, as is the development of methods of assessing bone marrow vitality. Genomics, 1998 May 1, 49(3), 411 - 8 An evolutionarily conserved gene on human chromosome 5q33-q34, UBH1, encodes a novel deubiquitinating enzyme; Hansen-Hagge TE et al.; While cloning breakpoint sequences of a leukemia patient exhibiting a t(5; 14) translocation, we identified a pseudogenic variant of a novel multigene family in proximity to the breakpoint . Chromosomal in situ hybridization suggested that the gene family is clustered on human chromosome 5q33-q34 . The gene family is evolutionarily conserved . Northern blot analysis of mouse tissues revealed low-level expression of a functional member of this gene family in almost all samples . Marked levels of transcripts were detected by in situ hybridization in the retina, the olfactory epithelium, the peripheral neuronal ganglia, and distinct areas of the gut . The predicted protein displays striking similarity to a hypothetical protein of Caenorhabditis elegans (R10E11.3.) and to two yeast deubiquitinating enzymes, Ubp9 and Ubp13, albeit to a lesser extent . We expressed the putative coding region of the human gene in Escherichia coli and demonstrated that it indeed bears deubiquitinating activity based on its ability to cleave ubiquitin from a ubiquitin-beta-galactosidase fusion protein . This new deubiquitinating enzyme has been named UBH1, for ubiquitin hydrolyzing enzyme 1. J Mol Biol, 1998 May 15, 278(4), 815 - 25 Triple helix-directed psoralen crosslinks are recognized by Uvr(A)BC excinuclease; Duval-Valentin G et al.; Pyrimidine oligonucleotides bind to the major groove of an oligopyrimidine-oligopurine DNA sequence by triple helix formation . A 14-mer oligopyrimidine 3'-psoralen-conjugate (P) and a doubly modified 5'-acridine/3'-psoralen-oligonucleotide (PA) were photo-crosslinked to their target site . The crosslinked complexes were tested regarding their sensitivity to Uvr(A)BC excinuclease/DNA complex formation and excision, and compared to free psoralen crosslinked to the same site (M) . An electrophoretic mobility-shift assay showed that the crosslinked triple-helix did not hamper formation of the (A)2B complex under conditions where the third strand was bound to its target . In vitro excision experiments performed on damaged DNA fragments containing crosslinked 5-methoxypsoralen (M-target) confirmed that the psoralen photoadduct was recognized by Uvr(A)BC and that excision occurred at the crosslinked site . The major cleavage reaction took place on the 5'-side of oligopurine strand . The excision was less efficient on the 5'-side of the pyrimidine strand . The 3'-side incision either on the purine or pyrimidine strand was even weaker . With optimal Uvr(A) concentrations, it was observed that the incision reaction on (P)- and (PA)-modified targets was clearly inhibited compared to the (M)-modified target, reflecting an effect of the oligonucleotide on the recognition/excision process . These results demonstrate that a triple helix is efficient in promoting inhibition of Uvr(A)BC excision nuclease activity . These results could account for divergent findings concerning the effects of triple helix-forming oligonucleotides on repair systems and open new perspectives to study DNA repair processes through the use of bi-substituted triple helix-forming oligonucleotides. J Mol Biol, 1998 May 15, 278(4), 801 - 13 tRNA anticodon recognition and specification within subclass IIb aminoacyl-tRNA synthetases; Commans S et al.; Subclass IIb aminoacyl-tRNA synthetases (Asn-, Asp- and LysRS) recognize the anticodon triplet of their cognate tRNA (GUU, GUC and UUU, respectively) through an OB-folded N-terminal extension . In the present study, the specificity of constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli was analyzed by cross-mutagenesis of the tRNA(Lys) anticodon, on the one hand, and of the amino acid residues composing the anticodon binding site on the other . From this analysis, a tentative model is deduced for both the recognition of the cognate anticodon and the rejection of non-cognate anticodons . In this model, the enzyme offers a rigid scaffold of amino acid residues along the beta-strands of the OB-fold for tRNA binding . Phe85 and Gln96 play a critical role in this spatial organization . This scaffold can recognize directly U35 at the center of the anticodon . Specification of the correct enzyme:tRNA complex is further achieved through the accommodation of U34 and U36 . The binding of these bases triggers the conformationnal change of a flexible seven-residue loop between strands 4 and 5 of the OB-fold (L45) . Additional free energy of binding is recovered from the resulting network of cooperative interactions . Such a mechanism would not depend on the modifications of the anticodon loop of tRNA(Lys) (mnm5s2U34 and t6A37) . In the model, exclusion by the synthetase of non-cognate anticodons can be accounted for by a hindrance to the positioning of the L45 loop . In addition, Glu135 would repulse a cytosine base at position 35 . Sequence comparisons show that the composition and length of the L45 loop are markedly conserved in each of the families composing subclass IIb aminoacyl-tRNA synthetases . The possible role of the loop is discussed for each case, including that of archaebacterial aspartyl-tRNA synthetases. J Mol Biol, 1998 May 15, 278(4), 787 - 800 A map of the biotin repressor-biotin operator interface: binding of a winged helix-turn-helix protein dimer to a forty base-pair site; Streaker ED et al.; The Escherichia coli biotin repressor is a member of the "winged helix-turn-helix" class of site-specific DNA binding proteins . The protein binds as a dimer to the 40 bp biotin operator sequence . Although the structure of the aporepressor has been solved by X-ray crystallographic techniques, no structure of the holorepressor-DNA complex is yet available . In order to characterize the structural features of the biotin repressor-biotin operator interface we have applied a number of solution techniques including DNase I, hydroxyl radical and dimethyl su