Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7341 - 5 Genetic construction and properties of a diphtheria toxin-related substance P fusion protein: in vitro destruction of cells bearing substance P receptors; Fisher CE et al.; We have genetically replaced the native receptor binding domain of diphtheria toxin with an extended form of substance P (SP): SP-glycine (SP-Gly) . The resulting fusion protein, DAB389SP-Gly, is composed of the catalytic and transmembrane domains of diphtheria toxin genetically coupled to SP-Gly . Because native SP requires a C-terminal amide moiety to bind with high affinity to the SP receptor, the precursor form of the fusion toxin, DAB389SP-Gly, was converted to DAB389SP by treatment with peptidylglycine-alpha-amidating monooxygenase . We demonstrate that following conversion, DAB389SP is selectively cytotoxic for cell lines that express either the rat or the human SP receptor . We also demonstrate that the cytotoxic action of DAB389SP is mediated via the SP receptor and dependent upon passage through an acidic compartment . To our knowledge, this is the first reported use of a neuropeptide as the targeting ligand for a fusion toxin; and the first instance in which an inactive precursor form of a fusion toxin is converted to the active form by a posttranslational modification. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7305 - 9 Formation of stable cationic lipid/DNA complexes for gene transfer; Hofland HE et al.; Stable cationic lipid/DNA complexes were formed by solubilizing cationic liposomes with 1% octylglucoside and complexing a DNA plasmid with the lipid in the presence of detergent . Removal of the detergent by dialysis yielded a lipid/DNA suspension that was able to transfect tissue culture cells up to 90 days after formation with no loss in activity . Similar levels of gene transfer were obtained by mixing the cationic lipid in a liposome form with DNA just prior to cell addition . However, expression was completely lost 24 hr after mixing . The transfection efficiency of the stable complex in 15% fetal calf serum was 30% of that obtained in the absence of serum, whereas the transient complex was completely inactivated with 2% fetal calf serum . A 90-day stability study comparing various storage conditions showed that the stable complex could be stored frozen or as a suspension at 4 degrees C with no loss in transfection efficiency . Centrifugation of the stable complex produced a pellet that contained approximately 90% of the DNA and 10% of the lipid . Transfection of cells with the resuspended pellet and the supernatant showed that the majority of the transfection activity was in the pellet and all the toxicity was in the supernatant . Formation of a stable cationic lipid/DNA complex has produced a transfection vehicle that can be stored indefinitely, can be concentrated with no loss in transfection efficiency, and the toxicity levels can be greatly reduced when the active complex is isolated from the uncomplexed lipid. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7300 - 4 Molecular cloning of a rat chromosome putative recombinogenic sequence homologous to the hepatitis B virus encapsidation signal; Aoki H et al.; Previously, we reported that a 61-bp subgenomic HBV DNA sequence (designated as 15AB, nt 1855-1915) is a hot spot for genomic recombination and that a cellular protein binding to 15AB may be the putative recombinogenic protein . In the present study, we established the existence of a 15AB-like sequence in human and rat chromosomal DNA by Southern blot analysis . The 15AB-like sequence isolated from the rat chromosome demonstrated a 80.9% identity with 5'-CCAAGCTGTGCCTTGGGTGGC-3', at 1872-1892 of the hepatitis B virus genome, thought to be the essential region for recombination . Interestingly, this 15AB-like sequence also contained the pentanucleotide motifs GCTGG and CCAGC as an inverted repeat, part of the chi known hot spot for recombination in Escherichia coli . Importantly, a portion of the 15AB-like sequence is homologous (82.1%, 23/28 bp) to break point clusters of the human promyelocytic leukemia (PML) gene, characterized by a translocation {t(15;17)}, and to rearranged mouse DNA for the immunoglobulin kappa light chain . Moreover, 15AB and 15AB-like sequences have striking homologies (12/15 = 80.0% and 13/15 = 86.7%, respectively) to the consensus sequence for topoisomerase II . Our present results suggest that this 15AB-like sequence in the rat genome might be a recombinogenic candidate triggering genomic instability in carcinogenesis. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7258 - 62 Cloning and expression of human deoxyguanosine kinase cDNA; Johansson M et al.; A human cDNA sequence homologous to human deoxycytidine kinase (dCK; EC 2.7.1.74) was identified in the GenBank sequence data base . The longest open reading frame encoded a protein that was 48% identical to dCK at the amino acid level . The cDNA was expressed in Escherichia coli and shown to encode a protein with the same substrate specificity as described for the mitochondrial deoxyguanosine kinase (dGK; EC 2.7.1.113) . The N terminus of the deduced amino acid sequence had properties characteristic for a mitochondrial translocation signal, and cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein size of 28 kDa . Northern blot analysis determined the length of dGK mRNA to 1.3 kbp with no cross-hybridization to the 2.8-kbp dCK mRNA . dGK mRNA was detected in all tissues investigated with the highest expression levels in muscle, brain, liver, and lymphoid tissues . Alignment of the dGK and herpes simplex virus type 1 thymidine kinase amino acid sequences showed that five regions, including the substrate-binding pocket and the ATP-binding glycine loop, were also conserved in dGK . To our knowledge, this is the first report of a cloned mitochondrial nucleoside kinase and the first demonstration of a general sequence homology between two mammalian deoxyribonucleoside kinases . Our findings suggest that dCK and dGK are evolutionarily related, as well as related to the family of herpes virus thymidine kinases. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7178 - 83 Direct binding of a soluble natural killer cell inhibitory receptor to a soluble human leukocyte antigen-Cw4 class I major histocompatibility complex molecule; Fan QR et al.; Natural killer (NK) cells expressing specific p58 NK receptors are inhibited from lysing target cells that express human leukocyte antigen (HLA)-C class I major histocompatibility complex molecules . To investigate the interaction between p58 NK receptors and HLA-Cw4, the extracellular domain of the p58 NK receptor specific for HLA-Cw4 was overexpressed in Escherichia coli and refolded from purified inclusion bodies . The refolded NK receptor is a monomer in solution . It interacts specifically with HLA-Cw4, blocking the binding of a p58-Ig fusion protein to HLA-Cw4-expressing cells, but does not block the binding of a p58-Ig fusion protein specific for HLA-Cw3 to HLA-Cw3-expressing cells . The bacterially expressed extracellular domain of HLA-Cw4 heavy chain and beta2-microglobulin were refolded in the presence of a HLA-Cw4-specific peptide . Direct binding between the soluble p58 NK receptor and the soluble HLA-Cw4-peptide complex was observed by native gel electrophoresis . Titration binding assays show that soluble monomeric receptor forms a 1:1 complex with HLA-Cw4, independent of the presence of Zn2+ . The formation of complexes between soluble, recombinant molecules indicates that HLA-Cw4 is sufficient for specific ligation by the NK receptor and that neither glycoprotein requires carbohydrate for the interaction. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7120 - 4 Stabilization of diverged tandem repeats by mismatch repair: evidence for deletion formation via a misaligned replication intermediate; Lovett ST et al.; A functional methyl-directed mismatch repair pathway in Escherichia coli prevents the formation of deletions between 101-bp tandem repeats with 4% sequence divergence . Deletions between perfectly homologous repeats are unaffected . Deletion in both cases occurs independently of the homologous recombination gene, recA . Because the methyl-directed mismatch repair pathway detects and excises one strand of a mispaired duplex, an intermediate for RecA-independent deletion of tandem repeats must therefore be a heteroduplex formed between strands of each repeat . We find that MutH endonuclease, which in vivo incises specifically the newly replicated strand of DNA, and the Dam methylase, the source of this strand-discrimination, are required absolutely for the exclusion of "homeologous" (imperfectly homologous) tandem deletion . This supports the idea that the heteroduplex intermediate for deletion occurs during or shortly after DNA replication in the context of hemi-methylation . Our findings confirm a "replication slippage" model for deletion formation whereby the displacement and misalignment of the nascent strand relative to the repeated sequence in the template strand accomplishes the deletion. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 6953 - 8 Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinity of the enzyme; Ibba M et al.; Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs both ensure accurate RNA recognition and prevent the binding of noncognate substrates . Here we show for Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) that the accuracy of tRNA recognition also determines the efficiency of cognate amino acid recognition . Steady-state kinetics revealed that interactions between tRNA identity nucleotides and their recognition sites in the enzyme modulate the amino acid affinity of GlnRS . Perturbation of any of the protein-RNA interactions through mutation of either component led to considerable changes in glutamine affinity with the most marked effects seen at the discriminator base, the 10:25 base pair, and the anticodon . Reexamination of the identity set of tRNA(Gln) in the light of these results indicates that its constituents can be differentiated based upon biochemical function and their contribution to the apparent Gibbs' free energy of tRNA binding . Interactions with the acceptor stem act as strong determinants of tRNA specificity, with the discriminator base positioning the 3' end . The 10:25 base pair and U35 are apparently the major binding sites to GlnRS, with G36 contributing both to binding and recognition . Furthermore, we show that E . coli tryptophanyl-tRNA synthetase also displays tRNA-dependent changes in tryptophan affinity when charging a noncognate tRNA . The ability of tRNA to optimize amino acid recognition reveals a novel mechanism for maintaining translational fidelity and also provides a strong basis for the coevolution of tRNAs and their cognate synthetases. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 6935 - 40 Increased accommodation of nascent RNA in a product site on RNA polymerase II during arrest; Gu W et al.; RNA polymerases encounter specific DNA sites at which RNA chain elongation takes place in the absence of enzyme translocation in a process called discontinuous elongation . For RNA polymerase II, at least some of these sequences also provoke transcriptional arrest where renewed RNA polymerization requires elongation factor SII . Recent elongation models suggest the occupancy of a site within RNA polymerase that accommodates nascent RNA during discontinuous elongation . Here we have probed the extent of nascent RNA extruded from RNA polymerase II as it approaches, encounters, and departs an arrest site . Just upstream of an arrest site, 17-19 nucleotides of the RNA 3'-end are protected from exhaustive digestion by exogenous ribonuclease probes . As RNA is elongated to the arrest site, the enzyme does not translocate and the protected RNA becomes correspondingly larger, up to 27 nucleotides in length . After the enzyme passes the arrest site, the protected RNA is again the 18-nucleotide species typical of an elongation-competent complex . These findings identify an extended RNA product groove in arrested RNA polymerase II that is probably identical to that emptied during SII-activated RNA cleavage, a process required for the resumption of elongation . Unlike Escherichia coli RNA polymerase at a terminator, arrested RNA polymerase II does not release its RNA but can reestablish the normal elongation mode downstream of an arrest site . Discontinuous elongation probably represents a structural change that precedes, but may not be sufficient for, arrest by RNA polymerase II. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 6892 - 7 Mutational analysis of NM23-H2/NDP kinase identifies the structural domains critical to recognition of a c-myc regulatory element; Postel EH et al.; NM23-H2, a presumed regulator of tumor metastasis in humans, is a hexameric protein with both enzymatic (NDP kinase) and regulatory (transcriptional activation) activity . While the structure and catalytic mechanisms have been well characterized, the mode of DNA binding is not known . We examined this latter function in a site-directed mutational study and identified residues and domains essential for the recognition of a c-myc regulatory sequence . Three amino acids, Arg-34, Asn-69, and Lys-135, were found among 30 possibilities to be critical for DNA binding . Two of these, Asn-69 and Lys-135, are not conserved between NM23 variants differing in DNA-binding potential, suggesting that DNA recognition resides partly in nonconserved amino acids . All three DNA-binding defective mutant proteins are active enzymatically and appear to be stable hexamers, suggesting that they perform at the level of DNA recognition and that separate functional domains exist for enzyme catalysis and DNA binding . In the context of the known crystal structure of NM23-H2, the DNA-binding residues are located within distinct structural motifs in the monomer, which are exposed to the surface near the 2-fold axis of adjacent subunits in the hexamer . These findings are explained by a model in which NM23-H2 binds DNA with a combinatorial surface consisting of the "outer" face of the dimer . Chemical crosslinking data support a dimeric DNA-binding mode by NM23-H2. Biochemistry, 1996 Jul 9, 35(27), 8942 - 7 Essential cysteines in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase from Escherichia coli: analysis by chemical modification and site-directed mutagenesis; Salleh HM et al.; The enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase (EC 4.1 . 2.16) (KDO 8-P synthase) that catalyzes the condensation of D-arabinose 5-phosphate (A 5-P) with phosphoenolpyruvate (PEP) to give 3-deoxy-D-manno-octulosonic acid 8-phosphate (KDO 8-P) and inorganic phosphate (Pi) was inactivated by the thiol-modifying reagents 5,5-dithiobis (2-nitrobenzoate) (DTNB) and methyl methanethiosulfonate (MMTS) . Reaction of cloned native KDO 8-P synthase with DTNB correlated with modification of two of the four cysteine sulfhydryls per monomer of enzyme and total loss of enzymatic activity which could be partially restored by treatment with dithiothreitol (DTT) . Cyanolysis of the DTNB-inactivated enzyme with KCN led to the elimination of 2 equiv of 5-thio-2-nitrobenzoate and partial recovery of activity . The presence of either substrate(s) or product(s) provided no protection against inactivation nor affected the number of cysteines modified, indicating that the cysteines modified are most likely not at the active site of KDO 8-P synthase . Titration of denatured enzyme with DTNB resulted in the modification of all four cysteines . After treatment of native enzyme with MMTS, no cysteines could be titrated with DTNB and no enzymatic activity could be detected . Treatment of the MMTS-inactivated KDO 8-P synthase with DTT resulted in restoration of enzymatic activity and the presence of two DTNB-titratable cysteine residues . Based on these observations and a report that KDO 8-P synthase is inactivated in a time-dependent manner with 3-bromopyruvate and that the substrate PEP protects against this inactivation, all four cysteines (38, 166, 206, and 249) were individually mutated to alanines via a modified PCR methodology . The C206A and C249A mutants were both enzymatically active with K(m) and Vmax values approximately identical to those of wild-type KDO 8-P synthase, and both native mutants reacted with DTNB to modify only one of the three remaining cysteine sulfhydryls per monomer of enzyme . Titration of denatured C206A and C249A mutants resulted in the modification of three cysteines . The C38A and C166A mutants were both for the most part enzymatically inactive . Titration of native C38A and C166A with DTNB resulted in modification of two cysteines while titration of the denatured mutant protein resulted in modification of the three remaining cysteines . Circular dichroism measurements of wild-type KDO 8-P synthase and the four C --> A mutants indicate modest but significant changes in the structure of the mutants . These results indicate that C206 and C249 in native KDO 8-P synthase are readily accessible to the modification reagent DTNB and therefore inactivation may result from structural changes in the DTNB-modified KDO 8-P synthase or blockage of access of substrates to the active site . The C38 and C166 in native KDO 8-P synthase are inaccessible to the modification reagent DTNB, indicating that they are located in the interior of KDO 8-P synthase, and loss of activity in the C38A and C166A mutants suggests their essentiality in the KDO 8-P synthase reaction. Biochemistry, 1996 Jul 9, 35(27), 8934 - 41 Active site of 5-aminolevulinate synthase resides at the subunit interface . Evidence from in vivo heterodimer formation; Tan D et al.; 5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthetic pathway of animals, fungi and some bacteria . It functions as a homodimer and requires pyridoxal 5'-phosphate as an essential cofactor . In mouse erythroid 5-aminolevulinate synthase, lysine 313 has been identified as the residue involved in the Schiff base linkage with pyridoxal 5'-phosphate {Ferreira, G . C., et al . (1993) Protein Sci . 2, 1959-1965}, while arginine 149, a conserved residue among all known 5-aminolevulinate synthase sequences, is essential for function {Gong & Ferreira (1995) Biochemistry 34, 1678-1685} . To determine whether each subunit contains an independent active site (i.e., intrasubunit arrangement) or whether the active site resides at the subunit interface (i.e., intersubunit arrangement), in vivo complementation studies were used to generate heterodimers from site-directed, catalytically inactive mouse 5-aminolevulinate synthase mutants . When R149A and K313A mutants were co-expressed in a hem A- Escherichia coli strain, which can only grow in the presence of 5-aminolevulinate or when it is transformed with an active 5-aminolevulinate synthase expression plasmid, the hem A- E . coli strain acquired heme prototrophy . The purified K313A/R149A heterodimer mixture exhibited K(m) values for the substrates similar to those of the wild-type enzyme and approximately 26% of the wild-type enzyme activity which is in agreement with the expected 25% value for the K313A/R149A coexpression system . In addition, DNA sequencing of four Saccharomyces cerevisiae 5-aminolevulinate synthase mutants, which lack ALAS activity but exhibit enzymatic complementation, revealed that mutant G101 with mutations N157Y and N162S can complement mutant G220 with mutation T452R, and mutant G205 with mutation C145R can complement mutant Ole3 with mutation G344C . Taken together, these results provide conclusive evidence that the 5-aminolevulinate synthase active site is located at the subunit interface and contains catalytically essential residues from the two subunits. Biochemistry, 1996 Jul 9, 35(27), 8855 - 62 A rapid screen of active site mutants in glycinamide ribonucleotide transformylase; Warren MS et al.; Specific and saturation site-directed mutageneses have been used to alter each polar residue within 6 A of the catalytic center of glycinamide ribonucleotide transformylase (EC 2.1.2.2) . These mutants were rapidly screened for catalytic activity using functional complementation of auxotrophic cells . This screen allows a rapid qualitative estimate of enzyme activity for each of these mutants . These results have shown that none of the polar residues close to the catalytic center of the enzyme are irreplaceable, although several are important for full catalytic activity, namely, Asn106, His108, Ser135, and Asp144 . A mechanism is proposed in which a fixed water molecule mediates the required proton transfers between substrate and cofactor, while the formyl group is transferred from 10-formyltetrahydrofolate by direct nucleophilic attack by the amine of glycinamide ribonucleotide . The active site polar residues may act to alter the pKa values of the attacking and leaving amino groups within a putative tetrahedral intermediate in order to facilitate the transfer of the formyl group. Biochemistry, 1996 Jul 9, 35(27), 8805 - 14 Structural influence of cation binding to recombinant human brain S100b: evidence for calcium-induced exposure of a hydrophobic surface; Smith SP et al.; The dimeric calcium-binding protein S100b is proposed to undergo a calcium-induced structural change allowing it to interact, via a hydrophobic surface, with other proteins . Previously it has been suggested that calcium binding to S100b leads to the exposure of at least one phenylalanine residue (Mani et al., 1982, 1983) . This effect appears to be "reversed" at higher ionic strength, leading to a possible reburying of phenylalanine residues (Mani et al., 1982, 1983) . To study these effects, we monitored calcium binding to recombinant human S100b by NMR spectroscopy under different salt (KCI) conditions . 15N-Labeled glycine residues in S100b showed calcium-induced chemical shift changes similar to those reported for the related monomeric protein calbindin D9k, suggesting similar conformational changes are occurring in the calcium-binding loops of these two proteins . Calcium binding to S100b also resulted in a shifting and broadening of several 1H resonances from the Ca-S100b form only including those from the side chains of residues F14, F70, and F73 but not those of residue Y17 . This broadening was enhanced with increased ionic strength (KCI) . However, small additions ( < 15% v/v) of the hydrophobic solvent trifluoroethanol relieved this phenomenon, leading to narrower line widths . These observations are consistent with the calcium-induced exposure of at least one of these hydrophobic residues, resulting in self-association of the S100b dimer . Trifluoroethanol serves to dissociate these complexes back to the dimeric calcium species . We propose that this cluster of hydrophobic residues which include F14, F73, and F88 may be important for interactions with a target protein. FEBS Lett, 1996 Jul 8, 389(3), 297 - 303 Characterization of human presenilin 1 using N-terminal specific monoclonal antibodies: Evidence that Alzheimer mutations affect proteolytic processing; Mercken M et al.; The majority of cases of early-onset familial Alzheimer disease are caused by mutations in the recently identified presenilin 1 (PS1) gene, located on chromosome 14 . PS1, a 467 amino acid protein, is predicted to be an integral membrane protein containing seven putative transmembrane domains and a large hydrophilic loop between the sixth and seventh membrane-spanning domain . We produced 7 monoclonal antibodies that react with 3 non-overlapping epitopes on the N-terminal hydrophilic tail of PS1 . The monoclonal antibodies can detect the full-size PS1 at Mr 47000 and a more abundant Mr 28000 product in membrane extracts from human brain and human cell lines . PC12 cells transiently transfected with PS1 constructs containing two different Alzheimer mutations fail to generate the 28 kDa degradation product in contrast to PC12 cells transfected with wild-type PS1 . Our results indicate that missense mutations in this form of familial Alzheimer disease may act via a mechanism of impaired proteolytic processing of PS1. Biochem Biophys Res Commun, 1996 Jul 5, 224(1), 164 - 8 Iron metabolism-related genes and mitochondrial genes are induced during involution of mouse mammary gland; Lee M et al.; To understand molecular mechanisms of mammary gland involution, several clones were isolated after the primary differential screening of a total 40,000 pfu of involution-specific cDNA library, and further characterized . The partial sequences and Northern analysis revealed that iron metabolism-related genes and mitochondrial genes were induced during mammary gland involution . The expression of the lactoferrin gene was induced at involution days 1, 2, and 3 . The expression of ferritin heavy chain gene was induced at involution days 1, 2, 3 and 4 . Cytochrome oxidase subunit 1 and cytochrome oxidase subunit 2 genes were induced at involution days 4 and 7 . The expression of cytochrome b gene was induced at involution day 7 . These results imply that iron metabolism and mitochondrial function may be altered during mammary gland involution. Biochem Biophys Res Commun, 1996 Jul 5, 224(1), 103 - 7 Association of a 14-3-3 protein with CMP-NeuAc:GM1 alpha 2,3-sialyltransferase; Gao L et al.; CMP-NeuAc:GM1 alpha 2,3-sialyltransferase (ST-IV) was purified to homogeneity from rat brain . Microsequencing of the tryptic peptides derived from the purified enzyme revealed two amino acid sequences homologous to the 14-3-3 proteins . A polyclonal antibody was raised against purified ST-IV . A 33 kDa protein was co-immunoprecipitated from rat brain extracts with the anti-(ST-IV) antibody as detected by Western blot analysis . This protein was identified as a subtype of 14-3-3 family by an anti-(14-3-3) antibody . Screening of a rat brain lambda gt11 library using the anti-(ST-IV) antibody resulted in the identification of a cDNA clone coding for the subtype of 14-3-3 protein . These results indicate an association of the 14-3-3 protein with the sialyltransferase . Since the 14-3-3 protein has PKC inhibitor activities and the activity of sialyltransferases is, at least in part, regulated by PKC, the association of the 14-3-3 protein with ST-IV may indicate a role for this protein in the post-translational regulation of the sialyltransferase activity through the processes of phosphorylation and dephosphorylation. Mutat Res, 1996 Jul 5, 354(1), 95 - 101 Specificity of spontaneous and t-butyl hydroperoxide-induced mutations in delta oxyR strains of Escherichia coli differing with respect to the SOS mutagenesis proficiency and to the MutY and MutM functions; Urios A et al.; Mutations induced by oxidative DNA damage appear to occur by two pathways, differing in their dependence on SOS mutagenesis . We have analysed the specificity of mutations produced by each pathway . Base substitutions generating extragenic suppressors were characterized in Trp+ revertants of Escherichia coli strains carrying the trpE65 ochre mutation, which were hypersensitive to oxidative mutagenesis due to a deletion of the oxyR gene . In strain IC3821, containing MucA/B proteins and therefore proficient for SOS mutagenesis, the more frequently scored base substitutions, either spontaneous or induced by t-butyl hydroperoxide (BuOOH), were T:A-A:T transversions, followed by G:C-A:T transitions, while the frequency of G:C-T:A transversions was lower . This SOS-dependent mutability could be promoted by abasic sites . In strains IC3894 (mutY) and IC3981 (mutY mutM), lacking mutagenesis proteins, SOS-independent revertants arose almost exclusively via G:C-T:A transversions probably derived from oxidatively damaged 8-oxoguanine/adenine mispairs . Formation of these mispairs in IC3894 and IC3981 would be enhanced by BuOOH treatment since it caused a significant increase in the revertant number . Strains IC3894 and IC3981 could have a complementary role to that of IC3821 to analyse the mutagenicity and the mutational specificity of oxidants. J Biol Chem, 1996 Jul 5, 271(27), 16000 - 7 Specific interaction of DNA polymerase beta and DNA ligase I in a multiprotein base excision repair complex from bovine testis; Prasad R et al.; Base excision repair (BER) is a cellular defense mechanism repairing modified bases in DNA . Recently, a G:U repair reaction has been reconstituted with several purified enzymes from Escherichia coli (Dianov, G., and Lindahl, T.(1994) Curr . Biol . 4, 1069-1076) . Using bovine testis crude nuclear extract, we have shown that G:U is repaired efficiently in vitro, and DNA polymerase beta (beta-pol) is responsible for the single nucleotide gap-filling synthesis (Singhal, R . K., Prasad, R., and Wilson, S . H.(1995) J . Biol . Chem . 270, 949-957) . To investigate potential interaction of beta-pol with other BER protein(s), we developed affinity chromatography matrices by cross-linking purified rat beta-pol or antibody against beta-pol to solid supports . Crude nuclear extract from bovine testis was applied to these affinity columns, which were then extensively washed . Proteins that bound specifically to the affinity columns were co-eluted in a complex with beta-pol . This complex had a molecular mass of approximately 180 kDa and was able to conduct the complete uracil-initiated BER reaction . The BER complex contained both beta-pol and DNA ligase I . An antibody to beta-pol was able to shift the complex in sucrose gradients to a much larger molecular mass (>300 kDa) that again contained both beta-pol and DNA ligase I . Furthermore, DNA ligase I and beta-pol were co-immunoprecipitated from the testis nuclear extract with anti beta-pol IgG . Thus, we conclude that beta-pol and DNA ligase I are components of a multiprotein complex that performs BER. J Biol Chem, 1996 Jul 5, 271(27), 16180 - 6 Fluorescence detection of symmetric GroEL14(GroES7)2 heterooligomers involved in protein release during the chaperonin cycle; Torok Z et al.; The GroEL14 chaperonin from Escherichia coli was labeled with 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (I-AEDANS), a hydrophobic probe whose fluorescent emission is sensitive to structural changes within the protein . Increasing concentrations of ATP or adenylyl imidodiphosphate but not ADP caused two successive GroES7-dependent changes in the fluorescence intensity of AEDANS-GroEL14, corresponding to the sequential binding of two GroES7 heptamers and the formation of two types of chaperonin heterooligomers, GroEL14GroES7 and GroEL14(GroES7)2 . The binding of thermally denatured malate dehydrogenase (MDH) caused a specific increase in fluorescence intensity of AEDANS-GroEL14 that allowed the direct measurement in solution at equilibrium of ATP- and GroES7-dependent protein release from the chaperonin . Structure/function analysis during the generation of ATP from ADP indicated the following sequence of events: 1) ADP-stabilized MDH-GroEL14GroES7 particles bind newly formed ATP . 2) MDH-GroEL14GroES7 particles bind a second GroES7 . 3) MDH-GroEL14(GroES7)2 particles productively release MDH . 4) Released MDH completes folding . Therefore, the symmetrical GroEL14(GroES7)2 heterooligomer is an intermediate after the formation of which the protein substrate is productively released during the chaperonin-mediated protein folding cycle. J Biol Chem, 1996 Jul 5, 271(27), 16409 - 15 Structural differences in the minimal catalytic domains of the GTPase-activating proteins p120GAP and neurofibromin; Ahmadian MR et al.; The kinetic properties for the enzymatic stimulation of the GTPase reaction of p21(ras) by the two GTPase-activating proteins (GAPs) p120(GAP) and neurofibromin are different . In order to understand these differences and since crystallization attempts have only been successful with truncated fragments, structure/function requirements of the catalytic core of these proteins were investigated . Differences in size of the minimal catalytic domains of these two proteins were found as determined by limited proteolysis . The minimal catalytic domain has a molecular mass of 30 kDa in the case of p120(GAP) and of 26 kDa in the case of neurofibromin . Both catalytic domains contain the homology boxes as well as the residues perfectly conserved among all Ras GAPs . The C termini of these fragments are identical, whereas the N-terminal part of the minimal p120(GAP) domain is 47 amino acids longer . These newly identified minimal catalytic fragments were as active in stimulating GTPase activity toward p21(ras) as the corresponding larger fragments GAP-334 and NF1-333 from which they had been generated via proteolytic digestion . Recently it was postulated that a fragment of 91 amino acids from neurofibromin located outside the conserved domain contains catalytic activity . In our hands this protein is unstable and has no catalytic activity . Thus, we believe that we have defined the true minimal domains of p120(GAP) (GAP-273, residues Met714-His986) and neurofibromin (NF1-230, residues Asp1248-Phe1477), which can be expressed via LMM fusion vectors in Escherichia coli and isolated in high purity. J Biol Chem, 1996 Jul 5, 271(27), 16288 - 93 Cloning of a cDNA encoding an aldehyde dehydrogenase and its expression in Escherichia coli . Recognition of retinal as substrate; Wang X et al.; The biosynthesis of the hormone retinoic acid from retinol (vitamin A) involves two sequential steps, catalyzed by retinol dehydrogenases and retinal dehydrogenases, respectively . This report describes the cloning of a cDNA encoding a heretofore unknown aldehyde dehydrogenase from a rat testis library and its expression in Escherichia coli . This enzyme has been designated retinal dehydrogenase, type II, RalDH(II) . The deduced amino acid sequence of RalDH(II) had the highest identity with mammalian aldehyde dehydrogenases that feature low Km values (microM) for retinal: human ALDH1 (72.2%), rat retinal dehydrogenase, type I (71.5%), bovine retina (72.7%), and mouse AHD-2 (71.5%) . RalDH(II) expressed in E . coli recognizes as substrates free retinal, with a Km of approximately 0.7 microM, and cellular retinol-binding protein-bound retinal, with a Km of approximately 0.2 microM . RalDH(II) also can utilize as substrate retinal generated in situ by microsomal retinol dehydrogenases, from the physiologically most abundant substrate: retinol bound to cellular retinol-binding protein . Rat testis expresses RalDH(II) mRNA most abundantly, followed by (relative to testis): lung (6.7%), brain (6.3%), heart (5.2%), liver (4.4%), and kidney (2.7%) . RalDH(II) does not recognize citral, benzaldehyde, acetaldehyde, and propanal efficiently as substrates, but does metabolize octanal and decanal efficiently . These data support a function for RalDH(II) in the pathway of retinoic acid biogenesis. J Biol Chem, 1996 Jul 5, 271(27), 15905 - 10 An isoleucine to valine substitution in Escherichia coli acyl carrier protein results in a functional protein of decreased molecular radius at elevated pH; Keating DH et al.; Escherichia coli acyl carrier protein (ACP) has been reported to exist in at least two distinct conformers in solution . A novel form of ACP having an increased electrophoretic mobility on polyacrylamide gel electrophoresis was noted previously during work on beta-ketoacyl-acyl carrier protein synthase II (fabF) mutants of E . coli (Jackowski, S., and Rock, C . O.(1987) J . Bacteriol . 169, 1469-1473) . These workers reported that the increased electrophoretic mobility of the ACP from fabF strains occurred irrespective of prosthetic group attachment or the state of acylation of the prosthetic group . Since these workers were unable to detect a difference between the amino acid sequence of the ACP from the fabF mutants and that of wild type ACP, they suggested that the increased electrophoretic mobility was due to an unknown post-translational modification of the polypeptide chain . We have reinvestigated these mutants and report that the increased electrophoretic mobility is due to a mutation within the gene (acpP) that encodes ACP . This mutation results in substitution of isoleucine for valine 43 of ACP . Site-directed mutagenesis of a synthetic ACP gene demonstrated that the amino acid substitution at residue 43 is the cause of the increased electrophoretic mobility . Gel filtration experiments indicated that the increased electrophoretic mobility results from the more compact structure of V43I ACP at high pH . The altered residue lies within the ACP region of greatest conformational lability, and thus the V43I substitution may shift the equilibrium toward the more compact conformation(s) . The disulfide-linked dimer of V43I ACP was readily formed and had an electrophoretic migration greater than the dimer of wild type ACP, suggesting that formation of ACP.ACP dimers does not require structural deformation of the protein. J Biol Chem, 1996 Jul 5, 271(27), 16218 - 26 Identification of the structural and functional domains of MutY, an Escherichia coli DNA mismatch repair enzyme; Manuel RC et al.; The linear amino acid sequences of the Escherichia coli DNA repair proteins, MutY and endonuclease III, show significant homology, even though these enzymes recognize entirely different substrates . In this study, proteolysis and molecular modeling of MutY were used to elucidate its domain organization . Proteolysis by trypsin cleaved the enzyme into 26- and 13-kDa fragments . NH2-terminal sequencing showed that the p13 domain begins at Gln226, indicating that the COOH-terminal portion of MutY, absent in endonuclease III, is organized as a separate domain . The large p26 domain is almost equivalent to the size of endonuclease III . Binding activity of the p26 domain to a DNA substrate containing an A.G mismatch was comparable with that of the intact enzyme . In vitro studies show that the p26 domain retains adenine glycosylase and AP lyase activity on DNA containing undamaged adenine opposite guanine or 8-oxo-7,8-dihydro-2'-deoxyguanine . Although the activity was somewhat reduced, the above results show that the critical amino acid residues involved in substrate binding and catalysis are present in this domain . The structure predicted by molecular modeling indicates that the region of MutY (Met1-Trp216), which is homologous to endonuclease III exhibits a two domain structure, even though this portion is resistant to proteolysis by trypsin. J Biol Chem, 1996 Jul 5, 271(27), 15942 - 9 Biochemical characterization of p16INK4- and p18-containing complexes in human cell lines; Ragione FD et al.; The regulation of the D-type cyclin-dependent kinase (CDK4 and CDK6) activity appears to be the key step in the progression of eukaryotic cells through the G1 cell cycle phase . One of the mechanisms involved in this process is the binding of some small proteic inhibitors, with a molecular mass ranging between 14 and 20 kDa, to these CDKs . We have evaluated the amount of two such inhibitors, namely p16(INK4) and p18, in normal and transformed cells, as well as the biochemical features of the macromolecular complexes containing these proteins . The results obtained indicated that (i) p18 gene expression, unlike p16(INK4) gene, is not regulated by pRb status, (ii) no evident relationship exists between the expression of p16(INK4) and p18 genes, (iii) significant amounts of the two proteins are not bound to CDKs but occur as free molecules, (iv) each inhibitor forms a complex with the CDK protein with a 1:1 stoichiometry, and (v) a competition exists between cyclin D and the inhibitor protein toward the CDK protein resulting in the absence of detectable cellular free kinase . Moreover, employing the human native partially purified p16(INK4)or the pure recombinant protein, we have been able to demonstrate in vitro the dissociation of CDK4-cyclin D1 complex and the formation of CDK4-p16(INK4) bimolecular complex . Our findings suggest that during the cell division cycle the members of the p16(INK4) protein family and cyclin Ds compete for binding to CDK4/CDK6 and that their quantitative ratio is essential for G1 --> S transition. J Biol Chem, 1996 Jul 5, 271(27), 16294 - 9 Molecular cloning and identification of N-acyl-D-glucosamine 2-epimerase from porcine kidney as a renin-binding protein; Maru I et al.; N-Acetylneuraminic acid (NeuAc) is an important molecule in biological recognition systems . NeuAc is known to be biosynthesized either from UDP-N-acetyl-D-glucosamine by an action of UDP-N-acetyl-D-glucosamine 2-epimerase or from N-acetyl-D-glucosamine by N-acyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) . However, the physiological function of the GlcNAc 2-epimerase in NeuAc biosynthesis has not been fully evaluated . To clarify the role of GlcNAc 2-epimerase in NeuAc biosynthesis, the enzyme and its gene were isolated from porcine kidney cortex . Escherichia coli cells transformed with the gene expressed the GlcNAc 2-epimerase having the same properties as those of the GlcNAc 2-epimerase from porcine kidney . Sequence analysis indicated that the gene was capable of synthesizing a 46.5-kDa protein (402 amino acids) with a conserved leucine zipper motif . Homology search for the cloned gene revealed that the GlcNAc 2-epimerase was identical with renin-binding protein (RnBP) in porcine kidney (Inoue, H., Fukui, K., Takahashi, S., and Miyake, Y.(1990) J . Biol . Chem . 265, 6556-6561) (identity: 99.6% in nucleotide sequence, 99.0% in amino acid sequence) . That GlcNAc 2-epimerase is a RnBP was confirmed by its ability to bind porcine kidney renin and mask its protease activity . These findings provide unequivocal evidence that the enzyme GlcNAc 2-epimerase is a RnBP. J Biol Chem, 1996 Jul 5, 271(27), 16068 - 74 Escherichia coli contains a protein that is homologous in function and N-terminal sequence to the protein encoded by the nifS gene of Azotobacter vinelandii and that can participate in the synthesis of the Fe-S cluster of dihydroxy-acid dehydratase; Flint DH; In this paper, I report the purification of a protein from Escherichia coli that is very similar in sequence, molecular weight, and the reactions it can catalyze to the protein encoded by the Azotobacter vinelandii nifS gene . This E . coli protein contains pyridoxal phosphate as a cofactor and catalyzes the removal of sulfur from cysteine to form alanine and S0 . When dithiothreitol is present along with cysteine, the S0 formed is reduced to S2- . This protein has a reactive sulfhydryl group that is essential for activity . As isolated, this sulfhydryl group appears to be in a disulfide linkage with the sulfhydryl group from the phosphopantetheine moiety of the acyl carrier protein . The purified E . coli protein can mobilize the sulfur from cysteine and contribute it to the formation of a {4Fe-4S} cluster on the apoprotein of E . coli dihydroxy-acid dehydratase . A mechanism is proposed for the early stages of the synthesis of Fe-S clusters using this protein and sulfur in the S0 oxidation state. J Biol Chem, 1996 Jul 5, 271(27), 16053 - 67 Studies on the synthesis of the Fe-S cluster of dihydroxy-acid dehydratase in escherichia coli crude extract . Isolation of O-acetylserine sulfhydrylases A and B and beta-cystathionase based on their ability to mobilize sulfur from cysteine and to participate in Fe-S cluster synthesis; Flint DH et al.; The apoprotein of Escherichia coli dihydroxy-acid dehydratase, which contains a catalytically essential {4Fe-4S} cluster in its active form, has been used as a substrate to investigate Fe-S cluster synthesis . The inactive apoprotein could be reactivated in vitro by factors present in the crude extract of E . coli and to a much smaller extent in the presence of Fe3+, S2-, and dithiothreitol . This reactivation occurs as a result of Fe-S cluster synthesis . It is anticipated that the Fe-S cluster synthesis observed in crude extracts in vitro may involve some of the components that participate in Fe-S cluster synthesis in vivo . The origin of the sulfur used to form Fe-S clusters was investigated . Four enzymatic activities in the crude extract of E . coli were found that can provide sulfur for Fe-S cluster synthesis in vitro by mobilizing the sulfur from cysteine . The purification of the proteins responsible for three of these activities is reported in this paper . The three proteins have been identified as O-acetylserine sulfhydrylase A, O-acetylserine sulfhydrylase B, and beta-cystathionase . The rate and extent of sulfide mobilization from cysteine in the reaction catalyzed by O-acetylserine sulfhydrylases A and B depend on the presence of nucleophiles that can add to the aminoacrylate formed on the enzyme following the removal of sulfide from cysteine . A new amino acid is formed when the nucleophiles add to the aminoacrylate . Sulfur mobilization by beta-cystathionase does not require a nucleophile, and the reaction is a minor variation on the cleavage of beta-cystathionine, with pyruvate, ammonia, and sulfide being the products . Once sulfur is mobilized by these enzymes, its efficient use in Fe-S cluster synthesis seems to be affected by the presence of yet unidentified factors present in crude extract . In crude extract and partially purified preparations from E . coli where these factors are present, the rapidity with which Fe-S clusters are formed and the efficiency with which sulfur is used imply an orderly controlled formation of Fe-S clusters that is generally typified by enzymatic reactions. Science, 1996 Jul 5, 273(5271), 107 - 9 Mapping of catalytic residues in the RNA polymerase active center; Zaychikov E et al.; When the Mg2+ ion in the catalytic center of Escherichia coli RNA polymerase (RNAP) is replaced with Fe2+, hydroxyl radicals are generated . In the promoter complex, such radicals cleave template DNA near the transcription start site, whereas the beta' subunit is cleaved at a conserved motif NADFDGD (Asn-Ala-Asp-Phe-Asp-Gly-Asp) . Substitution of the three aspartate residues with alanine creates a dominant lethal mutation . The mutant RNAP is catalytically inactive but can bind promoters and form an open complex . The mutant fails to support Fe2+-induced cleavage of DNA or protein . Thus, the NAD-FDGD motif is involved in chelation of the active center Mg2+. Biochemistry, 1996 Jul 2, 35(26), 8786 - 93 Thermodynamic stabilization of nucleotide binding to thymidylate synthase by a potent benzoquinazoline folate analogue inhibitor; Chen CH et al.; The stabilization of dUMP, FdUMP, and dGMP binding to Escherichia coli thymidylate synthase (TS) in the presence and absence of a folate analogue inhibitor of TS, 1843U, was determined by differential scanning calorimetry . When the enzyme is thermally unfolded in the presence of dUMP, two separate temperature transitions are evident, although only one binding site/dimer was detected in equilibrium dialysis experiments . In the absence of dUMP, TS shows a major peak of unfolding at 45 degrees C with a shoulder at 47 degrees C . In the presence of increasing amounts of dUMP progressive changes in the size of each peak occur, each associated with a higher temperature of unfolding . At a ratio of dUMP/TS of 100, a major peak predominates with an unfolding temperature (Td) of 60 degrees C . FdUMP shows a similar profile, while dGMP does not alter the Td of the enzyme since dGMP alone does not bind to TS . Despite the fact that 1843U binds tightly to TS in the absence of nucleotide ligands {Dev, I . K., Dallas, W.S., Ferone, R., Hanlon, H., McKee, D.D., & Yates, B . B . (1994) J.Biol . Chem . 269, 1873-1882}, it exhibits only a small effect on the Td profile of TS . However, when 1843U is present, in addition to the nucleotides (dUMP, FdUMP, or dGMP), a Td of 72 degrees C is achieved and the enthalpy of unfolding is increased by one-third . The stabilizing effect of substrate binding to TS by 1843U examined by thermodynamic parameters can be attributed to the considerable extra amount of free energy released on formation of the ternary complex of TS-1843U-nucleotide . The tightness of this complex is due to the stacking energy that results from Van der Waals contacts between the nucleotide purine or pyrimidine ring and the benzoquinazoline ring of 1843U {Weichsel, A., Montfort, W . R., Ciesla, J., & Maley, F . (1995) Proc . Natl . Acad . Sci . U.S.A . 92, 3493-3497}, which induces a local conformational change in the protein . This conformational change is associated with a significant positive entropy change, which suggests that water is expelled from the active site region. Biochemistry, 1996 Jul 2, 35(26), 8610 - 8 Escherichia coli diacylglycerol kinase is an alpha-helical polytopic membrane protein and can spontaneously insert into preformed lipid vesicles; Sanders CR 2nd et al.; Escherichia coli diacylglycerol kinase (DAGK) is a 13.2 kDa enzyme which spans the cytoplasmic membrane three times . Functional DAGK was purified to homogeneity using a polyhistidine tag and Ni(II)-chelate chromatography . Transmission Fourier transform infrared spectroscopy (FT-IR) of DAGK in phosphatidylcholine multilayers led to the conclusion that > or = 90 of DAGK's native 121 residues are alpha-helical, consistent with a model in which DAGK consists of two amphipathic alpha-helices and three transmembrane helices . Polarized attenuated total reflection FT-IR studies of DAGK in oriented multilamellae yielded data consistent with a topological arrangement in which the three transmembrane helices are well-aligned with the bilayer normal while the two amphipathic helices are approximately parallel with the membrane plane . The ability of DAGK to spontaneously insert into preformed lipid vesicles was examined using a novel assay system involving DAGK-catalyzed phosphorylation of a fluorescently tagged diacylglycerol . When micellar DAGK is diluted into L alpha-phase vesicles spontaneous insertion of the enzyme is fairly efficient (ca . 30%) . DAGK refolding and insertion from delipidated urea-solubilized DAGK into lipid vesicles is also modestly efficient (3.8 +/- 2.1%) above the gel to liquid crystalline phase transition temperature . The insertion studies indicate that the difference in energy barriers (delta delta G++) between pathways leading to catalytically productive folding and insertion of DAGK relative to unproductive pathways is < 4 kcal/mol . However, additional studies carried out with mutant forms of DAGK indicated that the differences between refolding/insertion pathways for DAGK in vivo and in vitro can be significant. Biochemistry, 1996 Jul 2, 35(26), 8595 - 602 Inactivation of Escherichia coli ribonucleotide reductase by 2'-deoxy-2'-mercaptouridine 5'-diphosphate . Electron paramagnetic resonance evidence for a transient protein perthiyl radical; Coves J et al.; Ribonucleotide reductase catalyzes a key step in DNA biosynthesis and repair, supplying the cell with the four common deoxyribonucleotides . It is thus the target of antiproliferative agents . The enzyme consists of two subunits named protein R1 and protein R2 . R1 provides the sites for the nucleotide substrates and redox-active cysteines required for catalysis . R2 harbors a tyrosyl radical essential for activity . We show here that 2'-deoxy-2'-mercaptouridine 5'-diphosphate, a substrate analog, is a very efficient inactivator of ribonucleotide reductase (Ki = 35 microM, Kinact = 0.18 s-1) . Inactivation is due to specific scavenging of the protein R2 tyrosyl radical . This unique feature sets this compound apart from other mechanism-based inhibitors such as 2'-azido-or 2'-chloro-2'-deoxyribonucleotide which induce partial or total protein R1 inactivation . During reaction, a transient organic radical was detected by EPR spectroscopy . Its g anisotropy (gz = 2.0620, gy = 2.0265, and gx = 2.0019) and its hyperfine structure are consistent with a perthiyl RSS . radical . The loss of the hyperfine structure by deuterium labeling of the beta protons of R1 cysteines unambiguously shows that the perthiyl radical is located on protein R1 . We thus conclude that inactivation of ribonucleotide reductase by 2'-deoxy-2'-mercaptouridine 5'-diphosphate is due to an irreversible transfer of the radical located on protein R2 to a cysteine residue of protein R1. Biochemistry, 1996 Jul 2, 35(26), 8587 - 94 Functional properties of the histidine-aspartate ion pair of flavocytochrome b2 (L-lactate dehydrogenase): substitution of Asp282 with asparagine; Gondry M et al.; The FMN prosthetic group of flavocytochrome b2 or L-lactate dehydrogenase oxidizes lactate to pyruvate . The reducing equivalents are then transferred one by one, intramolecularly, to heme b2 and then to external acceptors . Substrate oxidation is thought to begin with abstraction of the substrate alpha-hydrogen as a proton by an enzyme base . It has been proposed that this role is played by His373, which lies close to the flavin in the crystal structure and interacts with Asp282 . It has also been shown before, using hydrogen exchange measurements, that the pKa of His373 is substantially increased in the wild-type reduced enzyme compared to that in the oxidized state . We report here the enzymatic properties of the D282N mutant flavocytochrome b2 . Steady-state rate measurements with {2-1H}lactate and {2-2H}-lactate indicate that, as predicted, the Michaelis complex stability is hardly affected, whereas the transition state for proton abstraction increases in energy by 2.8 kcal/mol . Steady-state inhibition studies were conducted with a number of active-site ligands: sulfite, D-lactate, pyruvate, and oxalate . Binding was found to be most affected for oxalate, but kinetic patterns indicated oxalate and pyruvate were still capable of binding to the enzyme both at the oxidized and semiquinone stages, whereas inhibition by excess substrate, due to lactate binding at the semiquinone stage, was lost . Finally, analysis of the intermolecular hydrogen transfer catalyzed by the enzyme between {2-3H}lactate and fluoropyruvate indicated that the substitution with asparagine facilitates exchange of the histidine-bound proton and hence induces a decrease in the pKa value of H373 in the reduced enzyme of about 1.4 pH units . Nevertheless, the rate constant value for exchange with the solvent of the enzyme-bound substrate alpha-proton indicates that H373 is still protonated in the reduced mutant enzyme at neutral pH . Thus, the D282N mutation destabilizes the transition state for proton abstraction and decreases the pKa of H373 in the reduced enzyme but is insufficient to bring it back to a normal value. Vopr Virusol, 1996 Jul-Aug, 41(4), 150 - 3 {Immunochemical properties and specificity of monoclonal antibodies against N- and C-terminal sites of the recombinant protein of the nucleocapsid of hepatitis C virus (HCcAg)}; Masalova OV et al.; Highly affine murine monoclonal antibodies (MAB) to recombinant nucleocapsid (core) protein of hepatitis C virus (rHCcAg) expressed in E . coli were obtained . The MABs were analyzed by solid-phase enzyme immunoassay (EIA), immunodot, immunoblotting, and competitive immunochemical analysis . For estimating the epitope specificity of MAB, several immunoreactive fragments of different length were cloned from the HCcAg region overlapping 160 N-terminal amino acid (a . a.) residues . Use of these fragments and the competitive EIA demonstrated that MAB recognize 4 non-overlapping epitopes, 2 of which are localized in the 1-80 a . a . and 2 other in the 80-150 a . a . regions . A protocol of EIA for detecting HCcAg using MABs to two nonoverlapping HCcAg epitopes has been designed . The sensitivity of double-site sandwich is 1 ng/ml for the recombinant protein. Mol Gen Mikrobiol Virusol, 1996 Jul-Sep, (3), 36 - 40 {Synthesis, cloning, and expression in Escherichia coli cells of human alpha1-antitrypsin cDNA}; Tikunova NV et al.; cDNA coding for the full-length human alpha 1-antitrypsin (AAT) and its leader sequence has been cloned and sequenced . DNA sequences encoding the deletion variants and a full-length copy of AAT were cloned and expressed under trp-promoter control . It has been shown that guanine replacing right upstream and downstream the ATG of deletion variant increases the expression to ten-fold . The synthesis of deletion AAT in E . coli was evaluated immunologically and the level of the synthesis was shown to be 4-5% of total cellular protein. Mol Gen Mikrobiol Virusol, 1996 Jul-Sep, (3), 23 - 6 {Biosynthesis of alpha2-interferon and tumor necrosis factor at different stages of culturing}; Andreeva IS et al.; Study of the biosynthesis of human alpha 2-interferon and tumor necrosis factor in derivatives of E . coli SG200-50 recipient strain containing pIF16, pTNF311, and pLT21 plasmids demonstrated elimination of the plasmids from the cells without degradation thereof in the course of culturing . Methods increasing the content of the end products in the biomass are proposed. Bioorg Khim, 1996 Jul, 22(7), 489 - 502 {Biosynthesis and conformational state of 17-kDa and 27-kDa N-terminal fragments of elongation factor EF-2 in solution}; Plotnikov AN et al.; N-Terminal fragments of the rat liver elongation factor EF-2 containing 162 (17 kDa) and 244 (27 kDa) amino acid residues of 857 (95 kDa) residues of the native protein were synthesized in E . coli cells and in a wheat germ cell-free translation system, and their conformations were studied . Both fragments were synthesized as inclusion bodies (nonspecific molecular aggregates) . The conformations of the fragments in a solution were studied at neutral pH values by CD, fluorescence spectroscopy, scanning microcalorimetry, viscosimetry, gel-filtration, limited proteolysis, and interaction with monospecific anti-EF-2 antibodies and GroEL/ES molecular chaperone . Under nondenaturing conditions, both fragments existed in a solution as associates within a broad range of molecular masses, contained a considerable amount of elements of the intramolecular secondary structure, and represented globules without rigid tertiary structure (molten globules) . A rigid tertiary structure was not formed even after the interaction of the fragments with the GroEL/ES molecular chaperone, thus indicating that the C-terminal fragment is essential for the formation of the rigid tertiary structure . Both fragments contained conformational antigenic determinants similar to those in the whole protein; i.e., despite the absence of the rigid tertiary structure, the fragments contained elements whose structure was similar to that of the corresponding regions in the whole protein. Mikrobiologiia, 1996 Jul-Aug, 65(4), 481 - 7 {Catabolism of methylphosphonic acid and its physiological regulation in Escherichia coli}; Matys SV et al.; It was found that methyl phosphonic acid (Pn) was degraded by different Escherichia coli strains, which utilized it as the sole phosphorus source with resulting methane formation . This ability was influenced by mutations in the regulatory genes of the pho regulon . Thus, Pn was not degraded by an E . coli mutant defective in the regulatory phoB gene, responsible for the induction of pho-regulon proteins during phosphorus starvation . The intensity of Pn degradation depended on the age and concentration of the inoculum . Preincubation of bacteria in the presence of Pn accelerated subsequent degradation of both methyl phosphonic acid and its esters . Cultures developing from a small amount of inoculum degraded Pn more efficiently than heavily inoculated cultures that underwent only one cell division . However, cultures heavily inoculated with adapted cells degraded Pn as efficiently as cultures developing from a small amount of inoculum . Aeration was an important factor regulating Pn degradation: Pn was degraded more efficiently under anaerobic conditions regardless of the amount of inoculum. J Ind Microbiol, 1996 Jul, 17(1), 47 - 52 A direct comparison of approaches for increasing carbon flow to aromatic biosynthesis in Escherichia coli; Gosset G et al.; Different approaches to increasing carbon commitment to aromatic amino acid biosynthesis were compared in isogenic strains of Escherichia coli . In a strain having a wild-type PEP:glucose phosphotransferase (PTS) system, inactivation of the genes encoding pyruvate kinase (pykA and pykF) resulted in a 3.4 fold increase in carbon flow to aromatic biosynthesis . In a strain already having increased carbon flow to aromatics by virtue of overexpression of the tktA gene (encoding transketolase), the pykA and/or pykf mutations had no effect . A PTS- glucose+ mutant showed a 1.6-fold increase in carbon flow to aromatics compared to the PTS+ control strain . In the PTS- glucose+ host background, overexpression of tktA caused a further 3.7-fold increase in carbon flow, while inactivation of pykA and pykF caused a 5.8-fold increase . When all of the variables tested (PTS-glucose+, pykA, pykF, and overexpressed tktA) were combined in a single strain, a 19.9-fold increase in carbon commitment to aromatic biosynthesis was achieved. Biotechnol Prog, 1996 Jul-Aug, 12(4), 572 - 4 A quantitative immunoassay utilizing Escherichia coli cells possessing surface-expressed single chain Fv molecules; Chen G et al.; A facile, quantitative immunoassay is described that utilizes Escherichia coli (E . coli) bacteria expressing single chain Fv (scFv) antibody fragments attached to the cell surface . A Scatchard analysis demonstrated that the antibodies on the surface of the cells retained full binding activity (Kd = 2.2 x 10(-9) M) and that there are 60,000 scFv molecules per cell . The cells are used as the antibody reagent in the assay, and, following incubation with analyte, simple centrifugation is used to separate the antibody-bound from unbound analyte . The immunoassay is rapid and accurate down to the nanomolar level . In addition, a variety of detection strategies can be used, and the immunoassay is not adversely affected by the presence of animal serum . A key advantage of the new immunoassay is that the antibody reagent can be inexpensively produced in a "ready to use" form by simply growing cultures of the bacteria. Appl Microbiol Biotechnol, 1996 Jul, 45(6), 755 - 63 The effect of pre- and pro-sequences and multicopy integration on heterologous expression of the Fusarium solani pisi cutinase gene in Aspergillus awamori; van Gemeren IA et al.; A synthetic derivative of the cutinase cDNA of Fusarium solani pisi was expressed in Aspergillus awamori using the A . awamori endoxylanase II (exlA) promoter and terminator . The influence of the origin of the pre-sequence and the presence of a pro-sequence on the efficiency of extracellular cutinase production was analysed in single-copy transformants containing an expression cassette integrated at the pyrG locus . Transformants containing a construct encoding a direct, inframe fusion of the xylanase pre-peptide to the mature cutinase showed a 2-fold higher cutinase production level compared to strains containing constructs with an additional cutinase pro-peptide . The effect of multicopy integration of the expression cassette on cutinase production was analysed in strains with different numbers of a cutinase construct containing its own pre-pro-sequence . The multicopy strains showed a 6-to 12-fold increased production of extracellular cutinase relative to the single-copy strains . No linear dose response relation to the number of expression cassettes present in the strains was observed . The amount of active enzyme produced by the strains correlated with the amount of cutinase-specific mRNA, suggesting that cutinase overproduction is not limited at the level of translation or secretion. Exp Eye Res, 1996 Jul, 63(1), 35 - 50 Morphological and physiological consequences of the selective elimination of rod photoreceptors in transgenic mice; McCall MA et al.; We have produced transgenic mice (rdta mice) that express the gene for an attenuated diphtheria toxin under the control of a portion of the rhodopsin promotor . Morphologically, expression of this transgene results in the elimination of the majority of cell bodies in the outer nuclear layer (ONL) of the retina . This cell loss is evident as early as postnatal day 7 (P7), which corresponds closely to the onset of expression of rhodopsin in the mouse retina that occurs about P5 . Reverse transcription-PCR (RT-PCR) analysis of mRNA from the retinae of rdta mice shows that the level of rhodopsin mRNA is reduced by 50% as early as P14 and by P28, has declined to approximately 15% of that in the retinae of control mice . Electroretinographic recordings from the dark-adapted rdta mice at P17 reveal that their retinae do not generate any rod-mediated signals . The majority of the cell bodies that persist in the ONL of the rdta retinae have the morphological features of cone photoreceptors, although these cells never develop normal inner and outer segments . To confirm that the surviving cells are cones we crossed the rdta mice to a different line of transgenic mice that express the E . coli beta-galactosidase (lacZ positive) reporter gene in all cone photoreceptors . In retinae from mice that inherit both transgenes, nearly every cell that remains in the ONL expresses lacZ and, thus, is a cone . This finding also is consistent with RT-PCR analyses, which show that cone opsin mRNAs persist in the retinae of our rdta mice at ages when rhodopsin mRNA is significantly reduced . Electroretinograms can be obtained from the rdta mice under conditions that saturate the rod response and, thus, providing evidence that the cones that remain are functional, even though they lack inner and outer segments . Finally, we have examined the inner nuclear layer for changes that result from rod photorecptors ablation . We show that, while the elimination of the rod photoreceptors has little or no effect on the morphology of the post-synaptic neurons, this deletion does alter their laminar position. Genetika, 1996 Jul, 32(7), 1013 - 6 {Attenuation of type I restriction in Escherichia coli: effect of the ard gene in UV-irradiated cells}; Zavil'gel'skii GB et al.; The effect of conjugative plasmids ColIb-P9 (incI1) and pKM 101 (incN), containing the active ard gene, on the efficiency of EcoK restriction of nonmodified phage lambda .0 in UV-irradiated Escherichia coli cells was studied . ard-Dependent antirestriction enzyme activity was shown to decrease in UV-irradiated cells . The efficiency of action of the ard plasmid gene on lambda .0 was also shown not to depend on cell helicases RecBCD and UvrD, in contrast to the UV-induced alleviation of EcoK restriction (SOS alleviation). Gac Med Mex, 1996 Jul-Aug, 132(4), 367 - 75 {Importance of questioning and physical examination in pediatric clinical diagnosis}; Rendon-Macias ME et al.; In order to determine the certainty of the nosologic as well as the etiologic diagnosis of a complication that a group of physicians can achieve with the use of the interrogatory and physical examination in solving pediatric cases, a comparative survey was carried out with 36 pediatricians . Three clinical cases were chosen (A, B and C) in three different versions (1 = interrogatory, 2 = interrogatory plus physical examination, and 3 = the same plus clinical laboratory tests) . Randomly, the physicians reached a diagnosis in all three cases in any one of its versions . Generally, through interrogatory, 66.6% of the physicians (24/36) reached a nosologic diagnosis, 8.3% (3/26) diagnosed the complication and 41% (15/36) the etiological . Together with the physical examination, the percentages increased to 67.5% (25/ 37), 18.9% (7/37) and 43.2% (16/37), respectively (p < 0.05) . In Case A, the main nosologic diagnosis was reached by 71, 90 and 91% of the physicians, V = 1, 2 and 3 (p = ns) . In Case B, the main nosologic diagnosis was reached by all of the physicians, and in C, 10, 21 and 90% (p < 0.05) of the physicians, respectively . No differences were found in determining the etiologic diagnosis in versions one and two of the three cases . Differences were found in V3 (p < 0.001) . More physicians reached the diagnosis of the complications in Cases A and C . Having previous experience in similar cases allowed for a greater percentage of physicians to reach the main nosologic diagnosis (75%, 18/24 vs . 50%, 6/ 112, p = 0.03) . The interrogatory and the physical examinations in pediatrics continue to be a useful tool, allowing for a certain diagnosis in 70% to 100% of the cases of common ambulatory pathology . Previous experience in similar cases is a determining factor in reaching a correct diagnosis. Plasmid, 1996 Jul, 36(1), 26 - 35 Molecular analysis of the replication elements of the broad-host-range RepA/C replicon; Llanes C et al.; RepA/C is a replicon specific to the IncA/C incompatibility group of plasmids and was isolated recently from plasmid RA1 . The sequence of this autoreplicative region was established; it contains 13 repeats, suggesting that the replicon uses iterons to control its copy number . The sequence contains two ORFs, one potentially coding for a 33-kDa protein (ORF1) and a second potentially coding for a 14-kDa protein (ORF2) (Llanes et al., 1994b) . In this work, using an in vitro transcription/translation system, we detected a polypeptide whose size corresponded well to that of the deduced product of ORF1 . Deletion and insertion mutation analysis showed that ORF1 is essential for replication; it encodes an initiator protein (called RepA) . ORF2 was not essential for replication in Escherichia coli and its function remains to be determined . Using complementation experiments, the replication origin (ori) of RepA/C was defined . The ori was located in a 600-bp fragment downstream from repA, containing 10 direct repeats . To study the control of repA expression, a transcriptional fusion PrepA::lacZ was constructed . Its analysis showed that repA is transcriptionally autoregulated as are most repA genes of replicons controlled by iterons. J Endod, 1996 Jul, 22(7), 358 - 61 Regulation of pulp cell matrix metalloproteinase production by cytokines and lipopolysaccharides; Panagakos FS et al.; Little information is currently known regarding the effects of cytokines and lipopolysaccharides (LPS's) on matrix metalloproteinase (MMP) production by pulp cells in vitro . In this study, human pulp cells (HPC's) and clonal rat pulp cells RPC-C2A were treated with interleukin (IL)-1 alpha, IL-1 beta, tumor necrosis factor (TNF)-alpha, and LPS for 24 h . Conditioned medium and cell lysates were collected and analyzed by gelatin zymography . RPC-C2A cells treated with IL-1 beta and TNF-alpha displayed elevated levels of MMP's in conditioned medium fractions . LPS's at increasing concentrations had a similar effect . HPC's treated with either cytokines or LPS's had no change in the pattern of MMP's produced or secreted in either cellular or conditioned medium fractions . These studies indicate that the effects of cytokines and LPS's on pulp cells are not identical for cells from different species and requires further investigation to clarify these variations. Chirurg, 1996 Jul, 67(7), 754 - 6 {Paranephritic abscess . Local late complication after laparoscopic cholecystectomy}; Sichardt G et al.; Spilled gallstones abandoned intraperitoneally may cause serious complications . The following case history describes a patient who had undergone a laparoscopic cholecystectomy elsewhere 2 years previously, and who had to have a laparotomy because of a pararenal abscess caused by spilled gallstones . fatal sepsis ensued and the patient died on the 6th postoperative day. Biol Chem, 1996 Jul-Aug, 377(7-8), 535 - 8 Characterization of the preprotein translocase of the outer mitochondrial membrane by blue native electrophoresis; Dekker PJ et al.; The mitochondrial outer membrane contains import receptors for nuclear-encoded preproteins and a general import pore responsible for membrane translocation of preproteins . Receptors and the general import pore have been suggested to assemble into a loose complex . However, biochemical characterization of the complex has been limited so far . We report that blue native electrophoresis separates two complexes . One complex of approximately 400 kDa contains the receptor Tom22 and the general import pore component Tom40, the other complex of approximately 120 kDa contains the receptor Tom70 . A preprotein accumulated at the general import pore apparently co-migrates with the larger complex, suggesting the functionality of the complex . We conclude that the translocase of the outer membrane consists of at least two subcomplexes and that blue native electrophoresis will be a powerful tool for biochemical analysis of the complexes. Biol Chem, 1996 Jul-Aug, 377(7-8), 505 - 12 Three extremely thermostable proteins from Sulfolobus and a reappraisal of the 'traffic rules'; Schafer T et al.; Three cytosolic enzymes from the extremely thermoacidophilic archaeon Sulfolobus acidocaldarius (DSM 639) have been investigated: adenylate kinase, pyrophosphatase and superoxide dismutase . The latter was isolated from S . acidocaldarius cells, the others were heterologically overproduced in Escherichia coli . Long-term thermostability, flexibility, catalytic activity, and thermal denaturation were investigated by biochemical and physical methods . Superoxide dismutase is hyperthermostable over several days . The other enzymes have Tm values between 87 degrees C - 98 degrees C depending on conditions and reveal long-term stability in the range of hours . On the basis of sequence alignments, core structures were defined and compared to mesophilic homologues selected by growth temperature of organisms from 25 degrees C to 88 degrees C . The data set confirms none of the simple sequence based 'traffic rules' previously proposed by others . Some aspects of thermostability based on molecular modeling studies are discussed which remain to be proved by the 3D structures . All three enzymes could be crystallized. Ultramicroscopy, 1996 Jul, 63(3-4), 205 - 18 A common-lines based method for determining orientations for N > 3 particle projections simultaneously; Penczek PA et al.; A method is proposed for determining the directions of projections . An arbitrary number of projections of unknown three-dimensional structure are simultaneously used as input . The method is based on common lines and uses a new discrepancy measure accounting for the uneven distribution of common lines in angular space . An application to the 70S Escherichia coli ribosome data obtained from an energy-filtering electron microscope is described. J Protein Chem, 1996 Jul, 15(5), 491 - 9 The carboxy-terminal region of human interleukin-5 is essential for maintenance of tertiary structure but not for dimerization; Proudfoot AE et al.; The C-terminal region of interleukin-5 has previously been suggested to be important for biological activity {Mackenzie et al., (1991), Mol . Immunol . 28, 155-158; Kodama et al . (1991), Biochem . Biophys . Res . Commun . 178, 514-519} . We have investigated this region by making a series of truncation mutants . The proteins were expressed in Escherichia coli, purified from inclusion bodies, and were able to refold with the disulfide homodimeric topology typical of interleukin-5 . Analysis of the truncated carboxy-terminal proteins in an interleukin-5-dependent proliferation assay on TF-1 cells showed a rapid loss of activity as the C-terminal was shortened by more than two amino acids . This loss of biological activity correlated with a drop in binding affinity to both the alpha chain of the receptor and the high-affinity complex consisting of the alpha and beta subunits . Analysis of the proteins by 1H-NMR showed that the truncated mutants have higher exchange rates with solvent, indicating a less rigid structure . The carboxy-terminal region is therefore necessary to maintain the stability of the four-helix bundle and to orient correctly the important residues of the fourth helix . Inspection of the structure determined by X-ray crystallography shows that Trp-110 acts as the major residue in anchoring the fourth helix. J Protein Chem, 1996 Jul, 15(5), 481 - 9 Splase: a new class IIS zinc-finger restriction endonuclease with specificity for Sp1 binding sites; Huang B et al.; A new restriction endonuclease, named Splase, was constructed by genetically fusing the DNA-cleavage domain of the restriction endonuclease Fok1 with the zinc-finger DNA-binding domain of the transcription factor Sp1 . The resulting protein was expressed in Escherichia coli., partially purified, and shown to selectively digest plasmid DNA harboring consensus Sp1 sites . Splase was also shown to selectively digest the long terminal repeat of the HIV-1 DNA at Sp1 sites . Splase recognizes a 10-bp DNA sequence and hydrolyzes phosphodiester bonds upstream of the binding sequence . The binding specificity of Splase makes this a "rare cutter" restriction enzyme which could be valuable in creating large DNA fragments for genome sequencing projects . The result also presents the opportunity to create other restriction enzymes by altering the binding specificity of the zinc-finger recognition helix. J Invest Surg, 1996 Jul-Aug, 9(4), 313 - 9 Secretory phospholipase A2 levels in rat small bowel; Sonnino RE et al.; Preliminary studies on ischemia/reperfusion injury in transplanted small bowel grafts showed that secretory phospholipase A2 (sPLA2) may play a substantial role by breaking down membrane phospholipids . This study sought to determine the normal values of sPLA2 in the rat small bowel as a function of site and length as a baseline for future studies . The entire small bowel of male Lewis rats (200 g) was flushed with normal saline to eliminate solid contents . In group 1, the entire small bowel was divided into 5-cm segments (numbered 1-9), which were snap frozen and processed the same day for sPLA2 . In group 2, a 25-cm segment of bowel (corresponding to segments 2-6 in group 1) was harvested from each animal, snap frozen, and immediately processed for sPLA2 . To assess the effect of bowel storage on enzyme content, group 3 and group 4 grafts were stored for 7 and 14 days, respectively, at -85 degrees C prior to processing . All samples were homogenized in buffer, extracted with H2SO4 and assayed for sPLA2 activity using {1-14C}oleate-labeled autoclaved Escherichia coli as substrate . Results were analyzed statistically by ANOVA . sPLA2 activity rose from 85.46 +/- 14.46% hydrolysis/min fraction-1 in segment 1, to 476.38 +/- 176.75% hydrolysis/min fraction-1 in segment 9 . The increase was linear and statistically significant (p < .0001) . There was no significant difference in enzymatic activity between groups 2, 3, and 4 . Group 2 activity was 263.02 +/- 43.74% hydrolysis/min fraction-1 . This value was not statistically different from the mathematically calculated mean of segments 2-6 in group 1 (237.75) . The results show that (1) sPLA2 activity increases predictably with distance from the ligament of Treitz (2) storage at -85 degrees C does not affect sPLA2, activity, and (3) 25-cm grafts may be evaluated in toto with reproducible baseline enzyme activity . Given the variability of enzyme activity along the course of the rat small bowel, it is imperative that exact location be identified in any studies evaluating sPLA2 activity. Hepatogastroenterology, 1996 Jul-Aug, 43(10), 914 - 8 Influence of endotoxemia on hepatic energy metabolism in rats with obstructive jaundice; Takada T et al.; BACKGROUND/AIMS: A secondary insult in patients with obstructive jaundice can lead to multiple system organ failure . We evaluated the influence of endotoxin on hepatic energy metabolism and hepatic tissue blood flow in obstructive jaundiced rats . MATERIAL AND METHODS: Male Sprague-Dawley rats were divided into a control group, an endotoxin administration group, an obstructive jaundice group, and an obstructive jaundice with endotoxin administration group . To evaluate hepatic energy metabolism, we have measured arterial blood ketone body ratio, and arterial blood total ketone body concentration . Hepatic tissue blood flow was determined by laser Doppler velocimetry . RESULTS: In the endotoxin administration group, no change was observed in hepatic energy metabolism . However, the obstructive jaundice group was associated with decreased hepatic tissue blood flow shortly after the outset of jaundice, while no change was observed in hepatic energy metabolism until 3 weeks later . In the obstructive jaundice with endotoxin administration, a significant decrease in hepatic tissue blood flow and an increase in hepatic energy metabolism were measured . CONCLUSION: Endotoxin administration alone had no influence on hepatic energy metabolism, while endotoxin administration in the presence of obstructive jaundice results in a rapid decrease in hepatic energy metabolism . This occurred as a result of the secondary insult of endotoxin in the setting of decreased hepatic tissue blood flow caused by obstructive jaundice. Avian Dis, 1996 Jul-Sep, 40(3), 690 - 8 Description of cellulitis lesions and associations between cellulitis and other categories of condemnation; Elfadil AA et al.; A total of 110 broiler flocks processed in a single processing plant in southern Ontario were studied for purposes of describing the cellulitis lesions and investigating possible associations between cellulitis and other categories of condemnation at the processing plant . Two hundred and ninety-five carcasses condemned for cellulitis were examined . They came from 65 of the 110 flocks . The lesions tended to be unilateral with most carcasses (87%) having one lesion . The majority of the lesions (92%) were located on the abdomen . Almost 65% of the lesions were large (> or = 8.1 cm2), and 27% were medium (2.1-8.0 cm2) . On the basis of gross appearance, 69% of the lesions were classified as severe, 26% moderate, and 5% mild . Of 149 lesions examined histologically, 74% were classified as chronic, 21% ongoing, and 5% mild-acute . Condemnation data from the 110 broiler flocks were analyzed using Poisson regression . Simple relationships were examined between a count outcome (number of cellulitis-condemned carcasses per flock) and other categories of condemnation and average bird weight . Cellulitis was significantly associated with average bird weight (P = 0.0018), Escherichia coli-related conditions (SEROSITIS; P < or = 0.0001), ascites (P = 0.0004), cyanosis (P < or = 0.0001), valgus varus deformity (P < or = 0.0001), REJECT (combined carcass condemnations for bruising, mutilation, and contamination; P = 0.0003), and the interaction terms "average bird weight and ascites" (AVWT*ASCIT; P < or = 0.0001) and "average bird weight and cyanosis" (AVWT*CYAN; P < or = 0.0001) . Average bird weight, SEROSITIS, ascites, cyanosis, valgus varus deformity, and AVWT*ASCIT were the only significant factors after adjusting for clustering . No association was observed between cellulitis and emaciation and dead on arrival . Variables significantly associated with cellulitis in the multivariate analysis could be considered as potential predictors . These predictors may share common risk factors predisposing broiler chickens to cellulitis. Avian Dis, 1996 Jul-Sep, 40(3), 677 - 89 A prospective study of cellulitis in broiler chickens in southern Ontario; Elfadil AA et al.; This study investigated the associations among cellulitis and hatchery, farm, and abattoir factors . Forty-four broiler flocks from 24 farms located in southern Ontario were followed from hatching to processing . Poisson regression was used to analyze the data . Cellulitis as a count outcome (CELLCOUNT) was significantly associated (P < or = 0.05) with the hatchery of origin, strain of birds, farm size, type of litter, lighting system, total down time, prevalence of abdominal scratches, Escherichia coli-related conditions (SEROSITIS), ascites, and valgus varus deformity . However, only farm size, abdominal scratches, SEROSITIS, ascites, and valgus varus deformity were significant (P < or = 0.05) after adjusting for clustering . No significant associations were found between cellulitis and source of eggs, sex, average bird weight, feed company, growth promoter, or stocking density . Factors significantly associated with cellulitis in this study could be considered as potential risk factors for cellulitis in broiler chickens in southern Ontario. Avian Dis, 1996 Jul-Sep, 40(3), 634 - 44 Divergent antibody responses to vaccines and divergent body weights of chicken lines selected for high and low humoral responsiveness to sheep red blood cells; Parmentier HK et al.; Primary and secondary antibody responses to intramuscularly administered proteins of Eschericia coli (F11), Newcastle disease virus (NCD), infectious bronchitis virus (IB), and infectious bursal disease virus (IBD), respectively, were measured at weekly intervals in two chicken lines . The latter had been divergently selected for high and low antibody responses to sheep red blood cells (SRBC), and in a random-bred control line . An oil-based adjuvant was required to induce primary and secondary antibody responses to NCD, IB, and IBD . With respect to F11, elevated antibody responses were found in birds sensitized and boosted to F11 with and without adjuvant . The humoral response to F11 and to all viral antigens was significantly higher in the high (H) line than in the low (L) line, whereas the control (C) line showed intermediate titers . At 5 and 17 weeks of age, L line birds were significantly heavier than birds of the H and the C lines . A negative phenotypic correlation within lines between body weight at 17 weeks of age and antibody titers at 1 week after sensitization was found, but no further correlations between humoral responses and body weight or growth could be established . The present results suggest that selection for enhanced humoral responsiveness to SRBC resulted in enhanced responsiveness to components of several vaccines . Mechanisms underlying the relationship between divergent selection for immune responsiveness and body weight are discussed. Avian Dis, 1996 Jul-Sep, 40(3), 540 - 5 Lethality, hemagglutination and adhesion of Escherichia coli strains (serotype 01) isolated in Morocco from chickens with colibacillosis; Amara A et al.; Six strains of Escherichia coli, isolated from chickens with colibacillosis and belonging to serotype 01, were studied . All of them were lethal for 1-day-old chicks and were strongly adherent in vitro . Hemagglutination tests showed a strong reaction to horse and chicken erythrocytes . This reaction was inhibited by the addition of 5% D-mannose and also by overnight incubation at 18 C . When the strains were heated at 65 C or at 85 C for 1 hr, agglutination of horse and chicken erythrocytes was conserved only at 65 C . Purification of the subunit of fimbriae by several cycles of solubilization-crystallization using MgCl2 gave good results . The sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the molecular weight for one strain was about 18.4 kD. Genet Anal, 1996 Jul, 13(2), 45 - 7 Isolation of genes encoding tRNA binding proteins by probing an expression library with unmodified tRNA; Gustafsson C; A rapid method for isolating Escherichia coli genes encoding proteins which bind unmodified, but not modified, tRNA is described . The method is generally applicable, and can be used to clone many RNA binding proteins where a structured RNA ligand is known. Eur J Surg, 1996 Jul, 162(7), 561 - 5 Quantification of intestinal blood flow by ultrasonic transit time flowmetry in fed and endotoxaemic rats; Haque SM et al.; OBJECTIVE: To compare the dye dilution method with ultrasonic transit time flowmetry (UTTF) for quantifying intestinal blood flow in the same experimental animals . DESIGN: Open experimental study . SETTING: University hospital, Osaka, Japan . MATERIAL: 11 (experiment 1) and 25 (experiment 2) adult male Sprague-Dawley rats . INTERVENTIONS: In experiment 1, the rats were fasted overnight . In experiment 2, endotoxin, Escherichia coli lipopolysaccharide (endotoxin) or saline (sham) was injected intraperitoneally . MAIN OUTCOME MEASURES: Superior mesenteric venous and abdominal aortic blood flow . RESULTS: Experiment 1: intestinal blood flow measured by UTTF was significantly decreased by 17% (p < 0.05) and 56% (p < 0.05) after 30 minutes infusion through the tertiary branch of the mesenteric vein and during simultaneous drawing of blood samples from both the carotid artery and the superior mesenteric vein over a 1.5 minute period, respectively . The intestinal blood flow measurements obtained by UTTF at different intervals before simultaneous drawing of blood from the carotid artery and the superior mesenteric vein differed significantly (p < 0.05) from those obtained by the dye dilution method . Experiment 2: intestinal blood flow was also decreased by 21%-34% (p < 0.05) during similar simultaneous drawing of blood . Aortic blood flow in the endotoxin group was reduced by 66% (p < 0.05) compared with fed animals and 63% (p < 0.05) compared with sham animals . Simultaneously, intestinal blood flow in the endotoxin group was also reduced by 57% (p < 0.05) compared with fed or 48% (p < 0.05) compared with sham treated animals . CONCLUSION: Real intestinal blood flow might not be measured by the procedure of simultaneously drawing blood from the carotid artery and the superior mesenteric vein in rats as is usually done in the dye dilution method . Intraperitoneal endotoxin reduced aortic as well as intestinal blood flow . We propose that UTTF is an alternative method for quantifying intestinal blood flow in fed and endotoxaemic animals. Vaccine, 1996 Jul, 14(10), 949 - 58 Construction, purification and immunogenicity of antigen-antibody-LTB complexes; Green EA et al.; An oligonucleotide, encoding a short epitope peptide tag, termed Pk, was inserted at the 3'-end of the gene coding B-subunit of Escherichia coli heat-labile enterotoxin (LTB) . The presence of the Pk epitope on LTB-Pk was used to construct novel macromolecular assemblies comprising LTB-Pk, an anti-Pk mAb, (mAb SV5-P-k) and Pk-linked recombinant SIV proteins . The 1:1:1 stoichiometry of such complexes was ensured by binding LTB-Pk to one arm of mAb SV5-P-k and an SIV-Pk antigen to the other arm of the antibody . Such SIV-mAb-LTB macromolecular complexes bound to GM1-ganglioside in vitro, and when immunized systemically into mice were highly immunogenic, inducing both humoral and cell-mediated responses to the recombinant SIV antigens. Exp Lung Res, 1996 Jul-Aug, 22(4), 449 - 65 Neutrophil activation and lung injury associated with chronic endotoxemia in rabbits; Klut ME et al.; Chronic endotoxemia produces emphysematous lung destruction in several animal models . The present study was designed to examine changes in the polymorphonuclear leukocytes (PMN) and the lung parenchyma of rabbits that received either saline (control, n = 6) or Escherichia coli endotoxin (LPS, n = 6) 2-3 times weekly for 15 to 28 weeks . Peripheral blood was collected just before and after each intravenous injection and lung tissue was processed at the end of the experiment . PMN myeloperoxidase was stained with diaminobenzidine tetrahydrochloride (DAB)-H2O2, and CD11/CD18 was detected with immunogold . The changes in the PMN and the lung parenchyma were quantitatively analyzed . The results show that each dose of LPS produced an initial fall, followed by a rise in the circulating mature and immature PMN cell counts . Repeated doses of LPS induced PMN activation, degranulation, and an increase in the mean thickness of the alveolar wall (control, 4.1 +/- 0.2; LPS, 5.1 +/- 0.5; p < .05) at 28 weeks without evidence of alveolar septa destruction . Morphometric analysis of intravascular PMN showed an increase in the volume (V) of myeloperoxidase-containing azurophil granules (control, 6.1 +/- 1.3 microns3; LPS, 13.1 +/- 2.8 microns3; p < .05); a trend for a decrease in the V of specific granules (control, 15.8 +/- 3.4 microns3; LPS, 10.2 +/- 1.5 microns3; p = .09); an increase in the V of the cytoplasm (control, 37.3 +/- 6.4 microns3; LPS, 54.5 +/- 7.1 microns3; p < .05); and an increase in CD11/CD18 expressed as the number of gold particles per micrometer of cell surface membrane (G/micron) (control, 7.1 +/- 1.4 G/micron; LPS, 18.1 +/- 7.8 G/micron; p < .05) . The results indicate that chronic endoxemia in rabbits, accelerates the release of PMN from the bone marrow, enhances the retention of both mature and immature activated PMN in the pulmonary microvessels, and causes alveolar wall thickening rather than emphysematous lung destruction. J Perinatol, 1996 Jul-Aug, 16(4), 281 - 4 Pentoxifylline does not delay bacterially induced preterm delivery in rabbits; Walton DL et al.; OBJECTIVE: Our purpose was to determine whether pentoxifylline, a potent cytokine inhibitor, would delay bacterially induced preterm delivery in rabbits . STUDY DESIGN: The study was a randomized, blinded, prospective trial . Twenty-seven rabbits underwent laparotomy . Of these, five (shams) received an intrauterine injection of endotoxin-free water, and the remaining 22 received an injection of 10(5) Escherichia coli into the lower uterine segment . Postoperatively the animals that received the Escherichia coli were divided into two groups . The placebo group (n = 11) received subcutaneous injections of saline solution three times a day and the treatment group (n = 11) received pentoxifylline 20 mg/kg/day in three divided doses . The following parameters were evaluated: (1) time until delivery, (2) time until death, and (3) intrauterine pathologic features . RESULTS: All five of the sham rabbits were delivered at term without any evidence of infection . There were no differences in the preterm delivery rates between the placebo group (eight of 11) and the pentoxifylline-treated group (seven to 11) . However, there was a trend toward prolonging time until death in the treatment group . There were no differences in intrauterine pathologic feature between the placebo and treatment groups . CONCLUSION: Pentoxifylline does not delay Escherichia coli induced preterm delivery in rabbits. J Biochem (Tokyo), 1996 Jul, 120(1), 153 - 9 Human homolog of Drosophila heterochromatin-associated protein 1 (HP1) is a DNA-binding protein which possesses a DNA-binding motif with weak similarity to that of human centromere protein C (CENP-C); Sugimoto K et al.; Heterochromatin-associated protein 1 (HP1) is a nonhistone chromosomal component tightly associated with the pericentromeric heterochromatic region of fruit fly, mouse, and human throughout the cell cycle . Drosophila HP1 has been shown to be involved in position effect variegation and to be required for the correct chromosome segregation in vivo, while the biological activity of human homolog (HP1Hsa) has not yet been characterized . We previously reported that human CENP-B and CENP-C, two major centromere heterochromatin autoantigens often recognized by autosera in scleroderma patients, possess DNA-binding activity in vitro . Here, we show that human HP1, which is also an autoantigen targeted by some types of anticentromere autosera, is a DNA-binding protein . Human HP1 was expressed as a GST-fusion in Escherichia coli and purified with glutathione-Sepharose . The DNA-binding activity of the recombinant HP1 was demonstrated by gel mobility shift assay and South-Western-type blotting . The minimum DNA-binding region was further limited to the internal 64-amino acid stretch that is less-conserved between human and fruit fly but retains a helix-enriched motif with weak similarity to CENP-C . This suggests that HP1 is involved in the pericentromeric heterochromatin formation by directly associating with genomic DNA. J Biochem (Tokyo), 1996 Jul, 120(1), 14 - 7 Three-dimensional structure of porcine kidney D-amino acid oxidase at 3.0 A resolution; Mizutani H et al.; The X-ray crystallographic structure of porcine kidney D-amino acid oxidase, which had been expressed in Escherichia coli transformed with a vector containing DAO cDNA, was determined by the isomorphous replacement method for the complex form with benzoate . The known amino acid sequence, FAD and benzoate were fitted to an electron density map of 3.0 A resolution with an R-factor of 21.0% . The overall dimeric structure exhibits an elongated ellipsoidal framework . The prosthetic group, FAD, was found to be in an extended conformation, the isoalloxazine ring being buried in the protein core . The ADP moiety of FAD was located in the typical beta alpha beta dinucleotide binding motif, with the alpha-helix dipole stabilizing the pyrophosphate negative charge . The substrate analog, benzoate, is located on the re-face of the isoalloxazine ring, while the si-face is blocked by hydrophobic residues . The carboxylate group of benzoate is ion-paired with the Arg283 side chain and is within interacting distance with the hydroxy moiety of Tyr228 . The phenol ring of Tyr224 is located just above the benzene ring of benzoate, implying the importance of this residue for catalysis . There is no positive charge or alpha-helix dipole near N(1) of flavin . Hydrogen bonds were observed at C(2) = O, N(3)-H, C(4) = O, and N(5) of the flavin ring. Exp Nephrol, 1996 Jul-Aug, 4(4), 222 - 30 Lipopolysaccharide-induced glomerulonephritis develops in the absence of interferon-gamma signaling; Haas C et al.; IFN gamma is a costimulator of macrophage activation and it plays an important role as a proinflammatory cytokine by upregulation of adhesion molecules and MHC antigens . In this study we tested the role of IFN gamma in a model of endotoxin-induced glomerulonephritis . A systemic lupus-like disease was induced by injection of 50 micrograms bacterial LPS twice a week for 4 weeks in wild-type and in IFN gamma receptor-deficient (IFN gamma R-/-) mice . The renal cortex was examined by immunofluorescence and by light microscopy . LPS treatment induced an increase in serum levels of IgG and anti-dsDNA antibodies . A mild glomerulonephritis was characterized morphologically, but proteinuria was not observed . The main histological features of glomerulonephritis were an increase in ICAM-1 expression, deposition of immune complexes and of complement in the glomeruli, increased mesangial matrix and mesangial hypercellularity . The number of intraglomerular leukocytes, detected by MHC class-II and LFA-1 expression increased roughly 4-fold . All those alterations took place in a similar manner in wild-type and in IFN gamma R-/-mice . Therefore it is concluded that IFN gamma does not play an important role in the development of endotoxic glomerulonephritis. Cell Calcium, 1996 Jul, 20(1), 63 - 72 The calcium-binding protein calretinin-22k, an alternative splicing product of the calretinin gene is expressed in several colon adeno carcinoma cell lines; Gander JC et al.; An alternatively spliced mRNA for the calcium-binding protein calretinin (CR) is present in the colon adenocarcinoma cell line WiDr . As a consequence of a frame shift, the resulting protein, calretinin-22k (CR-22k), consists of the first 178 amino acids of calretinin followed by a carboxy-terminal peptide of 14 amino acids that is not present in full-length calretinin . Antibodies specific for this C-terminal region have been generated by 2 different methods . A peptide corresponding to the specific C-terminal region of CR-22k was either chemically synthesized and coupled to a carrier protein or was expressed in Escherichia coli as a carboxyterminal fusion to a carrier protein applying recombinant techniques . Both antisera produced in rabbits were tested in Western blots and immuno-histochemical experiments . The antisera recognized human recombinant CR-22k overexpressed in E . coli, but not fulllength calretinin and stained fixed WiDr cells . The presence of CR-22k was also confirmed in the colon cell lines CO115/3 in which mRNA coding for CR-22k mRNA coding for CR-22k mRNA is present as well as in the lines COLO205 and LS-180, all of which also express full-length calretinin . Although the intracellular distribution of CR-22k and CR are similar as evidenced by immunohistochemical stainings, CR-22k is preferentially localized in the nucleus in the cell lines LS-180 and Co115/3 suggesting potentially different roles for the two proteins. Mol Microbiol, 1996 Jul, 21(2), 409 - 19 Fucosylation and arabinosylation of Nod factors in Azorhizobium caulinodans: involvement of nolK, nodZ as well as noeC and/or downstream genes; Mergaert P et al.; The DNA region downstream of the nodABCSUIJ operon of Azorhizobium caulinodans was further characterized and two new genes, nodZ and noeC were identified in the same operon . The A . caulinodans wild-type strain produces a population of Nod factors that, at the reducing end, are either unmodified or carry a D-arabinosyl and/or an L-fucosyl branch . Nod factors produced by Tn5-insertion mutants in nodZ, noeC, and the separate nolK locus, were analysed by thin-layer chromatography and mass spectrometry . Fucosylation of Nod factors depended on both nodZ and nolK . Arabinosylation depended on noeC and/or downstream genes . Protein extracts of A . caulinodans contained an enzymatic activity for fucose transfer from GDP-fucose to chitooligosaccharides and to Nod factors . By mutant analysis and expression of nodZ in Escherichia coli, the fucosyltransferase activity was ascribed to the protein encoded by nodZ . In addition, a Nod factor fucosyltransferase activity, independent of nodZ or other known nod genes, was detected in A . caulinodans . Finally, on the basis of sequence similarity of the nolK gene product, and mass spectrometric analysis of Nod factors produced by a nolK mutant, we propose that this gene is involved in the synthesis of GDP-fucose. Mol Microbiol, 1996 Jul, 21(2), 361 - 72 The coupling between ftsZ transcription and initiation of DNA replication is not mediated by the DnaA-boxes upstream of ftsZ or by DnaA; Smith RW et al.; The DnaA protein of Escherichia coli is a multi-functional protein which, In addition to promoting initiation of replication, can regulate the initiation or termination of transcription of a variety of genes . It acts by binding to DNA at a defined sequence, termed a DnaA-box . Three candidate DnaA-boxes which occur within the essential cell-division genes, ftsQ and ftsA, have been hypothesized to mediate the response of the downstream ftsZ gene to intracellular levels of DnaA, and thus to couple the processes of initiation and cell division . We show here that, although transcription from promoters upstream of ftsZ is increased when initiation of chromosome replication is blocked by DnaA inactivation, this response is not mediated by the DnaA-boxes near these promoters, nor is it specific to DnaA . We show, furthermore, that mutational inactivation of the putative DnaA-binding sites in the fts region of the chromosome does not lead to impaired growth or reduced survival of cells. Mol Microbiol, 1996 Jul, 21(2), 347 - 60 Escherichia coli translation initiation factor 3 discriminates the initiation codon in vivo; Sussman JK et al.; In a genetic selection designed to isolate Escherichia coli mutations that increase expression of the IS 10 transposase gene (tnp), we unexpectedly obtained viable mutants defective in translation initiation factor 3 (IF3) . Several lines of evidence led us to conclude that transposase expression, per se, was not increased . Rather, these mutations appear to increase expression of the tnp'-'lacZ gene fusions used in this screen, by increasing translation initiation at downstream, atypical initiation codons . To test this hypothesis we undertook a systematic analysis of start codon requirements and measured the effects of IF3 mutations on initiation from various start codons . Beginning with an efficient translation initiation site, we varied the AUG start codon to all possible codons that differed from AUG by one nucleotide . These potential start codons fall into distinct classes with regard to translation efficiency in vivo: Class I codons (AUG, GUG, and UUG) support efficient translation; Class IIA codons (CUG, AUU, AUC, AUA, and ACG) support translation at levels only 1-3% that of AUG; and Class IIB codons (AGG and AAG) permit levels of translation too low for reliable quantification, importantly, the IF3 mutations had no effect on translation from Class I codons, but they increased translation from Class II codons 3-5-fold, and this same effect was seen in other gene contexts . Therefore, IF3 is generally able to discriminate between efficient and inefficient codons in vivo, consistent with earlier in vitro observations . We discuss these observations as they relate to IF3 autoregulation and the mechanism of IF3 function. Mol Microbiol, 1996 Jul, 21(2), 331 - 46 The role of the AUU initiation codon in the negative feedback regulation of the gene for translation initiation factor IF3 in Escherichia coli; Sacerdot C et al.; The expression of the infC gene encoding translation initiation factor IF3 is negatively autoregulated at the level of translation, i.e . the expression of the gene is derepressed in a mutant infC background where the IF3 activity is lower than that of the wild type . The special initiation codon of infC, AUU, has previously been shown to be essential for derepression in vivo . In the present work, we provide evidence that the AUU initiation codon causes derepression by itself, because if the initiation codon of the thrS gene, encoding threonyl-tRNA synthetase, is changed from AUG to AUU, its expression is also derepressed in an infC mutant background . The same result was obtained with the rpsO gene encoding ribosomal protein S15 . We also show that derepression of infC, thrS, and rpsO is obtained with other 'abnormal' initiation codons such as AUA, AUC, and CUG which initiate with the same low efficiency as AUU, and also with ACG which initiates with an even lower efficiency . Under conditions of IF3 excess, the expression of infC is repressed in the presence of the AUU or other 'abnormal' initiation codons . Under the same conditions and with the same set of 'abnormal' initiation codons, the repression of thrS and rpsO expression is weaker . This result suggests that the infC message has specific features that render its expression particularly sensitive to excess of IF3 . We also studied another peculiarity of the infC message, namely the role of a GC-rich sequence located immediately downstream of the initiation codon and conserved through evolution . This sequence was proposed to interact with a conserved region in 16S RNA and enhance translation initiation . Unexpectedly, mutating this GC-rich sequence increases infC expression, indicating that this sequence has no enhancing role . Chemical and enzymatic probing of infC RNA synthesized in vitro indicates that this GC-rich sequence might pair with another region of the mRNA . On the basis of our in vivo results we propose, as suspected from earlier in vitro results, that IF3 regulates the expression of its own gene by using its ability to differentiate between 'normal' and 'abnormal' initiation codons. Mol Microbiol, 1996 Jul, 21(2), 301 - 11 A comprehensive set of DnaA-box mutations in the replication origin, oriC, of Escherichia coli; Langer U et al.; We probed the complex between the replication origin, oriC, and the initiator protein DnaA using different types of mutations in the five binding sites for DnaA, DnaA boxes R1-R4 and M: (i) point mutations in individual DnaA boxes and combinations of them; (ii) replacement of the DnaA boxes by a scrambled 9 bp non-box motif; (iii) positional exchange; and (iv) inversion of the DnaA boxes . For each of the five DnaA boxes we found at least one type of mutation that resulted in a phenotype . This demonstrates that all DnaA boxes in oriC have a function in the initiation process . Most mutants with point mutations retained some origin activity, and the in vitro DnaA-binding capacity of these origins correlated well with their replication proficiency . Inversion or scrambling of DnaA boxes R1 or M inactivated oriC-dependent replication of joint replicons or minichromosomes under all conditions, demonstrating the importance of these sites . In contrast, mutants with inverted or scrambled DnaA boxes R2 or R4 could not replicate in wild-type hosts but gave transformants in host strains with deleted or compromised chromosomal oriC at elevated DnaA concentrations . We conclude that these origins require more DnaA per origin for initiation than does wild-type oriC . Mutants in DnaA box R3 behaved essentially like wild-type oriC, except for those in which the low-affinity box R3 was replaced by the high-affinity box R1 . Apparently, initiation is possible without DnaA binding to box R3, but high-affinity DnaA binding to DnaA box R3 upsets the regulation . Taken together, these results demonstrate that there are finely tuned DnaA binding requirements for each of the individual DnaA boxes for optimal build-up of the initiation complex and replication initiation in vivo. Mol Microbiol, 1996 Jul, 21(2), 281 - 92 The clp (CS31A) operon is negatively controlled by Lrp, ClpB, and L-alanine at the transcriptional level; Martin C; Biosynthesis of the Escherichia coli CS31A surface antigen was subject to phase-variation control and repressed by L-alanine . Nucleotide sequence analysis of the clp operon, encoding the biosynthesis of CS31A, revealed the presence of a regulatory gene, clpB . The amino acid sequence of the regulatory protein ClpB showed similarity to the primary structure of PapB, FaeB and AfaA, involved in the regulation of expression of Pap, K88, and Afa-3 fimbriae, respectively . The clp regulatory region contained two deoxyadenosine methylase sites (GATC-I and GATC-II) . The leucine-responsive regulatory protein (Lrp) was required for specific methylation inhibition of the GATC-II site . The activity of the clp promoter was monitored in a clp-lacZYA single-copy fusion . The cloned DNA used in this study did not contain a related papl gene . In these conditions, we showed, as expected, that phase variation did not occur . However, transcription of the clp operon was negatively controlled by ClpB and Lrp, and was maximal in the absence of Dam methylase . In the presence of AfaF, a Papl equivalent, the phase-variation control was restored . We concluded that two regulatory mechanisms were superimposed to control the clp expression . Phase variation, mediated by Lrp and a Papl equivalent, controlled the number of cells producing CS31A in a single colony . The second mechanism, described in this report, was mediated by ClpB and Lrp and controls the level of CS31A produced by a single cell . Furthermore, we showed that L-alanine reduced, by about twofold, the clp promoter activity independently of a Papl equivalent, ClpB, Lrp or Dam methylase . In addition, the presence of L-alanine prevented the phase-variation control mediated by AfaF. Mol Microbiol, 1996 Jul, 21(2), 257 - 66 Definition of a consensus DNA-binding site for the Escherichia coli pleiotropic regulatory protein, FruR; Negre D et al.; The FruR regulator of Escherichia coli controls the initiation of transcription of several operons encoding a variety of proteins involved in carbon and energy metabolism . The sequence determinants of the FruR-binding site were analysed by using 6x His-tagged FruR and a series of double-stranded randomized oligonucleotides . FruR consensus binding sites were selected and characterized by several consecutive rounds of the polymerase chain reaction-assisted binding-site selection method (BSS) using nitrocellulose-immobilized DNA-binding protein . FruR was demonstrated to require, for binding, an 8 bp left half-site motif and a 3 bp conserved right half-site with the following sequence: 5'-GNNGAATC/GNT-3' . In this sequence, the left half-site AATC/ consensus tetranucleotide is a typical motif of the DNA-binding site of the regulators of the GalR-Lacl family . On the other hand, the high degree of degeneracy found in the right half-site of this palindrome-like structure indicated that FruR, which is a tetramer in solution, interacts asymmetrically with the two half-sites of its operator . However, potentially FruR-target sites showing a high degree of symmetry were detected in 13 genes/operons . Among these, we have focused our interest on the pfkA gene, encoding phosphofructo-kinase-1, which is negatively regulated by FruR. Mol Microbiol, 1996 Jul, 21(2), 213 - 9 Three, four or more: the translational stop signal at length; Tate WP et al.; Translational stop signals are defined in the genetic code as UAA, UAG and UGA, although the mechanism of their decoding via protein factors is clearly different from that of the other codons . There are strong biases in the upstream and downstream nucleotides surrounding stop codons . Experimental tests have shown that termination-signal strength is strongly influenced by the identity of the nucleotide immediately downstream of the codon (+4), with a correlation between the strength of this four-base signal and its occurrence at termination sites . The +4 nucleotide and other biases downstream of the stop codon may reflect sites of