Biochemistry, 1997 May 20, 36(20), 6000 - 8 Solution structure of the actinorhodin polyketide synthase acyl carrier protein from Streptomyces coelicolor A3(2); Crump MP et al.; The solution structure of the actinorhodin acyl carrier protein (act apo-ACP) from the polyketide synthase (PKS) of Streptomyces coelicolor A3(2) has been determined using 1H NMR spectroscopy, representing the first polyketide synthase component for which detailed structural information has been obtained . Twenty-four structures were generated by simulated annealing, employing 699 distance restraints and 94 dihedral angle restraints . The structure is composed, principally, of three major helices (1, 2, and 4), a shorter helix (3) and a large loop region separating helices 1 and 2 . The structure is well-defined, except for a portion of the loop region (residues 18-29), the N-terminus (1-4), and a short stretch (57-61) in the loop connecting helices 2 and 3 . The RMS distribution of the 24 structures about the average structure is 1.47 A for backbone atoms, 1.84 A for all heavy atoms (residues 5-86), and 1.01 A for backbone atoms over the helical regions (5-18, 41-86) . The tertiary fold of act apo-ACP shows a strong structural homology with Escherichia coli fatty acid synthase (FAS) ACP, though some structural differences exist . First, there is no evidence that act apo-ACP is conformationally averaged between two or more states as observed in E . coli FAS ACP . Second, act apo-ACP shows a disordered N-terminus (residues 1-4) and a longer flexible loop (19-41 with 19-29 disordered) as opposed to E . coli FAS ACP where the N-terminal helix starts at residue 3 and the loop region is three amino acids shorter (16-35) . Most importantly, however, although the act apo-ACP structure contains a hydrophobic core, there are in addition a number of buried hydrophilic groups, principally Arg72 and Asn79, both of which are 100% conserved in the PKS ACPs and not the FAS ACPs and may therefore play a role in stabilizing the growing polyketide chain . The structure-function relationship of act ACP is discussed in the light of these structural data and recent genetic advances in the field. FEBS Lett, 1997 May 19, 408(2), 182 - 6 Antibodies to rat soluble IL-6 receptor stimulate B9 hybridoma cell proliferation; Thibault V et al.; Interleukin-6 mediates its pleiotropic effects by interacting with its membrane bound receptor (gp80) or the soluble counterpart gp54, resulting in activation of a complex that includes the transducer protein gp130 . We have generated a polyclonal antibody against the rat soluble IL-6 receptor (anti-rat sIL-6R) in rabbits . By Western blot analysis we show that purified anti-rat sIL-6R IgG antibody reacts specifically with recombinant rat sIL-6R generated from E . coli, baculovirus or adenovirus expression systems . Anti-rat sIL-6R inhibited IL-6-induced acute phase protein synthesis in rat (H35) but not human (HepG2) hepatoma cells, and did not affect stimulation of those cells by Oncostatin-M . Conversely, on the mouse hybridoma B9 cell line, IgG anti-rat sIL-6R showed a dose-dependent stimulation of proliferation . Fab fragments of this antibody did not stimulate, but abrogated IL-6-mediated hepatoma cell stimulation and B9 cell proliferation . Gel shift analysis of STAT nuclear factors showed activation of STAT DNA binding in nuclei of B9 cells treated with IgG anti-rat sIL-6R, whereas in H35, NIH-3T3 and M1 cells, only IL-6 could trigger a similar STAT activation . Our data suggest that mechanisms of IL-6 receptor activation and signalling in mouse B9 hybridoma cells show subtle but important differences from other IL-6-responsive cells. FEBS Lett, 1997 May 19, 408(2), 156 - 60 Purification and characterization of a fusion protein of plant acetohydroxy acid synthase and acetohydroxy acid isomeroreductase; Dumas R et al.; The nucleotide sequence coding for the Arabidopsis thaliana acetohydroxy acid synthase was genetically fused in frame with the nucleotide sequence coding for the Spinacia oleracea acetohydroxy acid isomeroreductase and expressed in Escherichia coli . This construction allowed the production of large amounts of soluble fusion protein . The pure chimeric enzyme exhibits high acetohydroxy acid synthase and acetohydroxy acid isomeroreductase specific activities . Fusion and native enzymes exhibit similar Km values for their substrates and for most cofactors . Furthermore, whereas native plant acetohydroxy acid synthase is highly unstable, the stability of this enzyme in the fusion has been increased . Thus, the chimeric enzyme appears to be a useful tool for the determination of kinetic and structural properties of plant acetohydroxy acid synthase. Biochim Biophys Acta, 1997 May 17, 1346(1), 25 - 9 Nucleotide sequence of human alkyl-dihydroxyacetonephosphate synthase cDNA reveals the presence of a peroxisomal targeting signal 2; de Vet EC et al.; Two overlapping clones were isolated from a human liver cDNA library in lambda gt11 that coded for human alkyl-dihydroxyacetonephosphate synthase using guinea pig and PCR-derived human cDNA probes . The open reading frame encodes a protein of 658 amino acids that shows a homology of 92% with the guinea pig homolog and a similarity of 98% . The peroxisomal targeting signal 2 that was recently identified in the presequence of the guinea pig enzyme appeared to be completely preserved in the human enzyme . Supportive confirmation for parts of the sequence of the mature protein was obtained from the Expressed Sequence Tags database of the National Center for Biotechnology Information . This database contained nine cDNA sequences, derived from seven independent clones, that correspond exactly to parts of the cDNA of human alkyl-dihydroxyacetonephosphate synthase . One of these clones most likely represents a not fully processed RNA with a putative intron containing an Alu sequence . An unexpected homology with D-lactate dehydrogenase (cytochrome C) precursor from Saccharomyces cerevisiae and with glycolate oxidase subunit D from Escherichia coli was also revealed. Carbohydr Res, 1997 May 16, 300(3), 259 - 64 Preparation and biological activity of manno- and galacto-validamines, new 5a-carba-glycosylamines as alpha-glycosidase inhibitors; Kameda Y et al.; Manno- and galacto-validamines, which are epimers of validamine, were semi-synthesized by the configurational inversion of validamine, a pseudo-sugar analogue of alpha-D-gluco-pyranose that has inhibitory activity for alpha-glucosidases . The inhibitory activities of these analogues were determined against several mannosidases and galactosidase . Manno-validamine shows potent inhibition for the alpha-mannosidases (competitive . K(i) = 4.6 x 10(-5) M for jack beans, and competitive, Ki = 2.8 x 10(-5) M for almonds), and galacto-validamine shows weak inhibition for the alpha-galactosidases (coffee bean and E . coli) . The inhibitory effect of the epimers on the N-linked oligosaccharide-processing mannosidases involved in glycoprotein biosynthesis and lysosomal mannosidase from rat liver were also examined . Mannovalidamine shows potent inhibition on the endoplasmic reticulumal alpha-mannosidase (competitive, K(i) = 1.2 x 10(-6) M), Golgi mannosidases IA, II (competitive, K(i) = 2.8 x 10(-5) M), and lysosomal alpha-mannosidase (competitive, Ki = 1.7 x 10(-5) M). Carbohydr Res, 1997 May 16, 300(3), 251 - 8 Lectin-deficient ricin toxin intoxicates cells bearing the D-mannose receptor; Frankel AE et al.; Ricin toxin with genetic or chemical modification of lectin sites has been previously reported to show markedly reduced cytotoxicity to cells following uptake by several receptors including the mannose receptor . Investigators have hypothesized that an intracellular galactoside-binding function was required for optimal intracellular targeting of ricin for these receptors . We have prepared insect-derived mutant ricin toxin B chain (RTB) with modifications of three lectin side domains (1 alpha, 1 beta, and 2 gamma) yielding a 1000-fold reduced galactoside avidity . After reassociation with plant RTA, the recombinant heterodimer and plant ricin were tested for cytotoxicity on mammalian cells expressing (mouse peritoneal macrophages, J774E cells, and MMR61 cells) or not expressing (KB cells) the D-mannose receptor . Receptor expression was confirmed by immunofluorescence microscopy . Lactose was included in the media to block cell-surface galactoside binding, and mannan was added as a control in each experiment to confirm mannose receptor-specific targeting . Plant ricin A chain (RTA) and E . coli-derived RTA were also tested for cytotoxicity on J774E and KB cells . Both wild-type and lectin-deficient ricin displayed mannose-receptor mediated cell cytotoxicity . This is the first report of a genetically modified ricin showing that RTB intracellular galactose binding activity is not required for ricin cytotoxicity . Sensitivity of mannose-receptor bearing cells, but not control cells, to mannosylated RTA, but not unglycosylated RTA, confirmed these observations . These results imply fusion toxins employing ricin can be prepared with maximal reductions in normal tissue binding. Biochim Biophys Acta, 1997 May 16, 1320(1), 83 - 94 The reduction of acetylpyridine adenine dinucleotide by NADH: is it a significant reaction of proton-translocating transhydrogenase, or an artefact? Stilwell SN, Bizouarn T, Jackson JB. Transhydrogenase is a proton pump . It has separate binding sites for NAD+/NADH (on domain I of the protein) and for NADP+/NADPH (on domain III) . Purified, detergent-dispersed transhydrogenase from Escherichia coli catalyses the reduction of the NAD+ analogue, acetylpyridine adenine dinucleotide (AcPdAD+), by NADH at a slow rate in the absence of added NADP+ or NADPH . Although it is slow, this reaction is surprising, since transhydrogenase is generally thought to catalyse hydride transfer between NAD(H)--or its analogues and NADP(H)--or its analogues, by a ternary complex mechanism . It is shown that hydride transfer occurs between the 4A position on the nicotinamide ring of NADH and the 4A position of AcPdAD+ . On the basis of the known stereospecificity of the enzyme, this eliminates the possibilities of transhydrogenation(a) from NADH in domain I to AcPdAD+ wrongly located in domain III; and (b) from NADH wrongly located in domain III to AcPdAD+ in domain I . In the presence of low concentrations of added NADP+ or NADPH, detergent-dispersed E . coli transhydrogenase catalyses the very rapid reduction of AcPdAD+ by NADH . This reaction is cyclic; it takes place via the alternate oxidation of NADPH by AcPdAD+ and the reduction of NADP+ by NADH, while the NADPH and NADP+ remain tightly bound to the enzyme . In the present work, it is shown that the rate of the cyclic reaction and the rate of reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH, have similar dependences on pH and on MgSO4 concentration and that they have a similar kinetic character . It is therefore suggested that the reduction of AcPdAD+ by NADH is actually a cyclic reaction operating, either with tightly bound NADP+/NADPH on a small fraction (< 5%) of the enzyme, or with NAD+/NADH (or AcPdAD+/AcPdADH) unnaturally occluded within the domain III site . Transhydrogenase associated with membrane vesicles (chromatophores) of Rhodospirillum rubrum also catalyses the reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH . When the chromatophores were stripped of transhydrogenase domain I, that reaction was lost in parallel with 'normal reverse' transhydrogenation (e.g., the reduction of AcPdAD+ by NADPH) . The two reactions were fully recovered upon reconstitution with recombinant domain I protein . However, after repeated washing of the domain I-depleted chromatophores, reverse transhydrogenation activity (when assayed in the presence of domain I) was retained, whereas the reduction of AcPdAD+ by NADH declined in activity . Addition of low concentrations of NADP+ or NADPH always supported the same high rate of the NADH-->AcPdAD+ reaction independently of how often the membranes were washed . It is concluded that, as with the purified E . coli enzyme, the reduction of AcPdAD+ by NADH in chromatophores is a cyclic reaction involving nucleotides that are tightly bound in the domain III site of transhydrogenase . However, in the case of R . rubrum membranes it can be shown with some certainty that the bound nucleotides are NADP+ or NADPH . The data are thus adequately explained without recourse to suggestions of multiple nucleotide-binding sites on transhydrogenase. Int J Cancer, 1997 May 16, 71(4), 675 - 9 Transduction of cytosine deaminase gene makes rat glioma cells highly sensitive to 5-fluorocytosine; Ge K et al.; To investigate the potential use of E . coli cytosine deaminase (CD) gene instead of the commonly used HSV-TK gene in the gene therapy of brain tumors, we constructed a retrovirus vector carrying the CD gene . We then transduced a rat glioma cell line C6 with CD gene by the retrovirus vector . Transduction of the CD gene made C6 cells become highly sensitive to the anti-fungi drug 5-fluorocytosine (5FC) . IC50 for 5FC was 6,000 microM in CD-negative cells, while it was 3 microM in CD-positive cells . Mixed cellular assay showed that CD-positive cells had a strong "bystander effect" on CD-negative cells when exposed to 5FC . Significant anti-tumor effects were observed in nude mice bearing s.c . tumors derived from CD-positive cells when these animals were given 250 mg/kg 5FC twice a day for 20 consecutive days . A marked decrease in tumor weight occurred when a mixture containing 50% CD-positive and 50% CD-negative C6 cells was injected s.c., followed by 5FC treatment, suggesting the bystander effect in vivo . Concerning the pharmacokinetics of 5FC, especially its high oral bio-availability and good penetration into cerebrospinal fluid, we suppose that the combination of CD-gene transfer and 5FC oral administration may have potential use in the gene therapy of brain tumors. Int J Cancer, 1997 May 16, 71(4), 638 - 44 Cloning and expression of the recombinant FAb fragment of monoclonal antibody K1 that reacts with mesothelin present on mesotheliomas and ovarian cancers; Brinkmann U et al.; The monoclonal antibody (MAb) K1 recognizes an approximate 40 kDa glycoprotein, mesothelin, that is present on the surface of human mesothelial cells, mesotheliomas and ovarian cancers . We have cloned the cDNAs encoding the variable regions of MAb K1 and constructed plasmids for expression of recombinant K1(FAb) . Recombinant FAb was produced in Escherichia coli in inclusion bodies that were solubilized and refolded to active protein . Binding of K1 MAb and FAb was compared by radioactive binding and competition assays and by surface plasmon resonance (BIAcore) . Recombinant K1(FAb) binds to cells expressing K1-antigen with a similar affinity as papain derived FAb from K1(IgG) and with a 4- to 10-fold reduced affinity compared with bivalent IgG . The cloned FAb can be used to make higher affinity antibodies and immunoconjugates that could be useful for various types of immunotherapies. J Mol Biol, 1997 May 16, 268(4), 712 - 23 GroE modulates kinetic partitioning of folding intermediates between alternative states to maximize the yield of biologically active protein; Fedorov AN et al.; The central issue of chaperone function is the mechanism whereby partitioning of folding polypeptides along the productive pathway may be maximized, while non-productive folding pathways are minimized . We have found that the GroE chaperone is capable of accelerating the rate of the productive pathway of bacterial luciferase alphabeta heterodimer formation . At intermediate temperatures at which the productive pathway and non-productive pathways leading to dimerization-incompetent monomeric forms of the subunits coexist, GroE enhances the yield of native enzyme while minimizing the yield of misfolded protein . These results suggest that GroE releases the subunits in forms capable of achieving the native structure faster than the forms initially bound by the chaperone . At higher temperatures, at which the native enzyme is stable but the dimerization reaction is diminished, GroE is unable to force the productive folding reaction to occur . However, the chaperone decreases the rate of formation of the heterodimerization-incompetent species, thereby enhancing the final yield of active enzyme when the temperature is reduced to the permissive range . Our results suggest a mechanism by which the chaperone functions to maximize the yield of the biologically active form of the protein while maintaining or even accelerating the essential rapid kinetics of folding reactions. J Biol Chem, 1997 May 16, 272(20), 13355 - 64 Interaction of MutS protein with the major and minor grooves of a heteroduplex DNA; Biswas I et al.; Thermus aquaticus MutS protein is a DNA mismatch repair protein that recognizes and binds to heteroduplex DNAs containing mispaired or unpaired bases . Using enzymatic and chemical probe methods, we have examined the binding of Taq MutS protein to a heteroduplex DNA having a single unpaired thymidine residue . DNase I footprinting identifies a symmetrical region of protection 24-28 nucleotides long centered on the unpaired base . Methylation protection and interference studies establish that Taq MutS protein makes contacts with the major groove of the heteroduplex in the immediate vicinity of the unpaired base . Hydroxyl radical and 1, 10-phenanthroline-copper footprinting experiments indicate that MutS also interacts with the minor groove near the unpaired base . Together with the identification of key phosphate groups detected by ethylation interference, these data reveal critical contact points residing in the major and minor grooves of the heteroduplex DNA. J Biol Chem, 1997 May 16, 272(20), 13302 - 8 acrB mutation located at carboxyl-terminal region of gyrase B subunit reduces DNA binding of DNA gyrase; Funatsuki K et al.; Mutations that exhibit susceptibility to acriflavine have been isolated and classified as acr mutations in Escherichia coli . We cloned the acrB gene, which has been identified as a mutation of the gyrB gene, and found a double point mutation altering two consecutive amino acids (S759R/R760C) in the COOH-terminal region of the gyrase B subunit . The mutant B subunit was found to associate with the A subunit to make the quaternary structure, and the reconstituted gyrase showed an 80-fold reduction of specific activity in DNA supercoiling assay; the sensitivity to acriflavine was not different in the same unit of wild-type and mutant gyrases . The mutant enzyme retained intrinsic ATPase activity, but DNA-dependent stimulation was observed infrequently . A gel shift assay showed that acriflavine inhibited the DNA binding of gyrase . The acrB mutation also reduced significantly the DNA binding of gyrase but did not change the sensitivity to acriflavine . These results revealed that the acrB mutation is related to the inhibitory mechanism of acriflavine; and the acriflavine sensitivity of the mutant, at least in vitro, is caused mainly by reduction of the enzyme activity . Further, our findings suggest that the COOH-terminal region of the B subunit is essential for the initial binding of gyrase to the substrate DNA. J Biol Chem, 1997 May 16, 272(20), 13171 - 9 Mutation of residue Phe97 to Leu disrupts the central allosteric pathway in Scapharca dimeric hemoglobin; Pardanani A et al.; Residue Phe97, which is thought to play a central role in the cooperative functioning of Scapharca dimeric hemoglobin, has been mutated to leucine to test its proposed role in mediating cooperative oxygen binding . This results in an 8-fold increase in oxygen affinity and a marked decrease in cooperativity . Kinetic measurements of ligand binding to the Leu97 mutant suggest an altered unliganded (deoxy) state, which has been confirmed by high resolution crystal structures in the unliganded and carbon monoxide-liganded states . Analysis of the structures at allosteric end points reveals them to be remarkably similar to the corresponding wild-type structures, with differences confined to the disposition of residue 97 side chain, F-helix geometry, and the interface water structure . Increased oxygen affinity results from the absence of the Phe97 side chain, whose tight packing in the heme pocket of the deoxy state normally restricts the heme from assuming a high affinity conformation . The absence of the Phe97 side chain is also associated with diminished cooperativity, since Leu97 packs in the heme pocket in both states . Residual cooperativity appears to be coupled with observed structural transitions and suggests that parallel pathways for communication exist in Scapharca dimeric hemoglobin. J Biol Chem, 1997 May 16, 272(20), 13073 - 83 Regulation of substrate recognition by the MiaA tRNA prenyltransferase modification enzyme of Escherichia coli K-12; Leung HC et al.; We purified polyhistidine (His6)-tagged and native Escherichia coli MiaA tRNA prenyltransferase, which uses dimethylallyl diphosphate (DMAPP) to isopentenylate A residues adjacent to the anticodons of most tRNA species that read codons starting with U residues . Kinetic and binding studies of purified MiaA were performed with several substrates, including synthetic wild-type tRNAPhe, the anticodon stem-loop (ACSLPhe) of tRNAPhe, and bulk tRNA isolated from a miaA mutant . Gel filtration shift and steady-state kinetic determinations showed that affinity-purified MiaA had the same properties as native MiaA and was completely active for tRNAPhe binding . MiaA had a Kmapp (tRNA substrates) approximately 3 nM, which is orders of magnitude lower than that of other purified tRNA modification enzymes, a Kmapp (DMAPP) = 632 nM, and a kcatapp = 0.44 s-1 . MiaA activity was minimally affected by other modifications or nonsubstrate tRNA species present in bulk tRNA isolated from a miaA mutant . MiaA modified ACSLPhe with a kcatapp/Kmapp substrate specificity about 17-fold lower than that for intact tRNAPhe, mostly due to a decrease in apparent substrate binding affinity . Quantitative Western immunoblotting showed that MiaA is an abundant protein in exponentially growing bacteria (660 monomers per cell; 1.0 microM concentration) and is present in a catalytic excess . However, MiaA activity was strongly competitively inhibited for DMAPP by ATP and ADP (Kiapp = 0.06 microM), suggesting that MiaA activity is inhibited substantially in vivo and that DMAPP may bind to a conserved P-loop motif in this class of prenyltransferases . Band shift, filter binding, and gel filtration shift experiments support a model in which MiaA tRNA substrates are recognized by binding tightly to MiaA multimers possibly in a positively cooperative way (Kdapp approximately 0.07 microM). J Biol Chem, 1997 May 16, 272(20), 13026 - 32 Characterization of TreR, the major regulator of the Escherichia coli trehalose system; Horlacher R et al.; The pathway of trehalose utilization in Escherichia coli is different at low and high osmolarity . The low osmolarity system takes up trehalose as trehalose 6-phosphate which is hydrolyzed to glucose and glucose 6-phosphate . treB and treC, the genes for the enzymes involved, form an operon that is controlled by TreR (encoded by treR), the repressor of the system, for which trehalose 6-phosphate is the inducer . We have cloned and sequenced treR . The protein contains 315 amino acids with a molecular weight of 34,508 . TreR was purified and shown to bind as a dimer trehalose 6-phosphate and trehalose with a Kd of 10 and 280 microM, respectively . The conformations of the protein differ from each other with either one or the other substrate-bound . Protease treatment removed the DNA-binding domain from the intact protein leaving the dimerization domain (a 29-kDa carboxyl-terminal fragment) intact . Nuclease protection experiments revealed a palindromic sequence located directly upstream of the -35 promoter sequence of treB that functions as the operator of the system. J Biol Chem, 1997 May 16, 272(20), 13013 - 8 Expression and characterization of the small subunit of human DNA polymerase delta; Sun Y et al.; DNA polymerase delta is a heterodimer consisting of a catalytic subunit of 125 kDa and a small subunit of 50 kDa (p50) . We have overexpressed p50 in Escherichia coli and have characterized the recombinant protein . p50 was readily overexpressed using the pET vector as an insoluble protein . A procedure was developed for its purification and renaturation . Examination of the physicochemical properties of renatured p50 showed that it is a monomeric protein with an apparent molecular weight of 60,000, a Stokes radius of 34 A, and a sedimentation coefficient of 4.1 S . Its physical properties were indistinguishable from p50 expressed as a soluble protein using the pTACTAC vector . Examination of the effects of recombinant p50 on the activity of DNA polymerase delta showed that p50 is able to slightly stimulate (about 2-fold) the activity of the recombinant 125-kDa catalytic subunit using poly(dA).oligo(dT) as a template in the absence of proliferating cell nuclear antigen . In the presence of proliferating cell nulear antigen, activity is stimulated about 5-fold . Seven stable hybridoma cell lines were established that produced monoclonal antibodies against p50 . One of these antibodies (13D5) inhibited the activity of calf thymus DNA polymerase delta . This antibody, when coupled to a solid support, also was found to provide a method for the immunoafffinity purification of recombinant p50 and of DNA polymerase delta from calf thymus or HeLa extracts . Immunoprecipitation and enzyme-linked immunosorbent assays also confirmed that p50 interacts with the catalytic subunit of DNA polymerase delta. J Biol Chem, 1997 May 16, 272(20), 12889 - 92 The yeast JEM1p is a DnaJ-like protein of the endoplasmic reticulum membrane required for nuclear fusion; Nishikawa S et al.; DnaJ-like proteins are functional partners for Hsp70 molecular chaperones . Complete nucleotide sequencing of yeast chromosome X has revealed that an open reading frame YJL073w encodes a novel member of the DnaJ-like protein family . The open reading frame represents a protein of 692 amino acids with a J-domain and one putative membrane-spanning segment . An epitope-tagged version of the protein was anchored in the endoplasmic reticulum (ER) membrane and its J-domain faced the ER lumen . We therefore propose to designate this gene JEM1 (DnaJ-like protein of the ER membrane) and to designate its gene product JEM1p . The JEM1 gene is not essential for cell growth, but double disruption of the JEM1 gene and the SCJ1 gene, which encodes another DnaJ-like protein in the ER lumen, causes growth arrest at elevated temperature . The Deltajem1 mutant is defective in nuclear fusion, karyogamy, during mating . A mutant JEM1p carrying a mutation in the highly conserved His-Pro-Asp sequence in the J-domain could not complement either temperature-sensitive growth of the Deltajem1 Deltascj1 double mutant or defects in karyogamy of the Deltajem1 mutant . JEM1p likely assists the functions of BiP, Hsp70 in the ER, including karyogamy. Eur J Biochem, 1997 May 15, 246(1), 243 - 51 Domain-specific N-glycosylation of the membrane glycoprotein dipeptidylpeptidase IV (CD26) influences its subcellular trafficking, biological stability, enzyme activity and protein folding; Fan H et al.; Dipeptidyl peptidase IV (DPPIV, CD26) is an N-glycosylated type II plasma membrane protein . The primary structure of rat wild-type DPPIV contains eight potential N-glycosylation sites . To investigate the role of N-glycosylation in the function of DPPIV, three of its asparagine residues were separately converted to glutamine by site-directed mutagenesis . The resulting N-glycosylation mutants of rat DPPIV were studied in stable transfected Chinese hamster ovary cells . All three N-glycosylation mutants of DPPIV showed a reduced half-life, as well as differing degrees of inhibition of the processing of their N-glycans . Mutation of the first (Asn83-->Gln) or eighth (Asn686-->Gln) N-glycosylation site had only a small effect on its enzymatic activity, cell-surface expression and dimer formation, whereas the mutation of the sixth N-glycosylation site (Asn319-->Gln) abolished the enzymatic activity, eliminated cell-surface expression and prevented the dimerization of the DPPIV protein . The mutant {Gln319}DPPIV is retained in the cytoplasm and its degradation was drastically increased . Our data suggest that the N-glycosylation at Asn319 is involved in protein trafficking and correct protein folding. Eur J Biochem, 1997 May 15, 246(1), 181 - 5 The complete cDNA sequence and expression of the first major allergenic protein of Malassezia furfur, Mal f 1; Schmidt M et al.; For the first time the complete cDNA encoding a major allergen and novel protein of the yeast Malassezia furfur, Mal f 1, has been sequenced and expressed . The amino acid sequences of nine tryptic peptides of the protein were determined . Oligonucleotides were designed from these amino acid sequences . The cDNA sequence was obtained by hybridizing these primers to mRNA and enhancement by reverse-transcriptase PCR techniques . The cDNA is 1176 bp in length . It shows an open reading frame of 1050 bp coding for a protein of 38178 Da and a deduced amino acid sequence containing 350 residues . The hydropathy plot and the tryptic digest indicate that the first 22 amino acids represent a leader sequence determining a mature protein of 35 988 Da . The complete encoding cDNA was expressed as a maltose-binding protein fusion protein in Escherichia coli . The recombinant fusion protein reacted with our specific monoclonal antibody and with IgE from patients with atopic dermatitis. Eur J Biochem, 1997 May 15, 246(1), 166 - 72 The N-terminal region of alpha-dystroglycan is an autonomous globular domain; Brancaccio A et al.; The structure of the N-terminal region of mouse alpha-dystroglycan (DGN) was investigated by expression of two protein fragments (residues 30-180 and 30-438) in Escherichia coli cells . Trypsin susceptibility experiments show the presence of a stable alpha-dystroglycan N-terminal region (approximately from residue 30 to 315) . In addition, guanidinium hydrochloride (Gdn/HCl) denaturation of DGN-(30-438)-peptide, monitored by means of tryptophan fluorescence, produces a cooperative transition typical of folded protein structures . These results strongly suggest that the alpha-dystroglycan N-terminal is an autonomous folding unit preluding a flexible mucin-like region and that its folding is not influenced by the absence of glycosylation . In order to obtain more information on the structural features of the N-terminal domain we have also used circular dichroism, analytical sedimentation and electron microscopy analysis . Circular dichroic spectra show the absence of typical secondary structure (e.g . alpha-helix or beta-sheet) and closely resemble those recorded for loop-containing proteins . This is consistent with a sequence similarity of the alpha-dystroglycan domain with the loop-containing protein elastase . Analytical ultracentrifugation and electron microscopy analysis reveal that the N-terminal domain has a globular structure . DGN-(30-438)-peptide does not bind in the nanomolar range to an iodinated agrin fragment which binds with high affinity to tissue purified alpha-dystroglycan . No binding was detected also to laminin . This result suggests that the alpha-dystroglycan N-terminal domain does not contain the binding site to its extracellular matrix binding partners . It is less likely than the lack of glycosylation reduces its binding affinity, because the N-terminal globular domain only contains two glycosylation sites. Eur J Biochem, 1997 May 15, 246(1), 127 - 32 Activation of partially folded mitochondrial malate dehydrogenase by thioredoxin; Li W et al.; CD spectroscopy reveals that mitochondrial malate dehydrogenase in 3M guanidinium chloride shows little residual secondary structure . Refolding of the denatured protein by dilution with buffer of pH 7.5 does not restore the CD spectrum of the native enzyme . A partially folded intermediate, possessing 25% of the alpha-helix content of the native enzyme, is formed upon dilution . The partially folded intermediate binds the extrinsic probe 1-anilinonaphtalene-8-sulfonate, and the increase in fluorescence (tenfold) is accompanied by a blue shift in the band position of the emission spectrum . Partially folded malate dehydrogenase is devoid of catalytic activity . In vitro refolding of the denatured protein takes place in the presence of dithiotreitol and thioredoxin . In the presence of micromolar concentrations of thioredoxin, a recovery of approximately 70% of the catalytic activity was observed . Emission-anisotropy titrations of oxidized thioredoxin, tagged with a fluorescent probe, revealed that the oxidoreductase recognizes partially folded intermediates of malate dehydrogenase with a dissociation constant of 6 microM . Moreover, a covalently linked complex formed by thioredoxin and monomeric malate dehydrogenase was detected by SDS/PAGE . A general mechanism is postulated for the reactivation of denatured proteins by thioredoxin. Eur J Biochem, 1997 May 15, 246(1), 119 - 26 Characterisation of the molybdenum-responsive ModE regulatory protein and its binding to the promoter region of the modABCD (molybdenum transport) operon of Escherichia coli; Anderson LA et al.; Molybdenum-dependent repression of transcription of the Escherichia coli modABCD operon, which encodes the high-affinity molybdate transporter, is mediated by the ModE protein . This regulatory protein was purified as an N-terminal His6-tagged derivative and characterised both with and without the N-terminal oligohistidine extension . Equilibrium centrifugation showed that ModE is at least a 57-kDa homodimer . Circular dichroism spectroscopy indicated that when molybdate or tungstate bind to ModE there is little change in its alpha-helical content, but a major change in the environment of tryptophan and tyrosine residues occurs . Addition of molybdate or tungstate to the protein results in almost 50% quenching of the fluorescence attributed to tryptophan . Titration of fluorescence quenching showed that two molecules of molybdenum bind to each dimer of ModE with a Kd of 0.8 microM . DNA mobility-shift assays showed that ModE requires molybdenum, or tungstate, to bind with high affinity (approximate Kd of 30 nM ModE) to the modABCD promoter region . In accord with ModE's role as a molybdenum-dependent transcriptional repressor, DNase I footprinting experiments showed that the ModE-molybdenum complex binds to a single 31-bp region around the transcription start of the modABCD promoter . This region contains a 6-base palindromic sequence CGTTAT-N12-ATAACG. Eur J Biochem, 1997 May 15, 246(1), 103 - 11 The nuclear ABC1 gene is essential for the correct conformation and functioning of the cytochrome bc1 complex and the neighbouring complexes II and IV in the mitochondrial respiratory chain; Brasseur G et al.; The nuclear ABC1 gene was isolated as a multicopy suppressor of a cytochrome b mRNA translation defect . Its inactivation leads to a respiratory deficiency suggesting a block in the bc1 segment of the respiratory chain {Bousquet, I., Dujardin, G . & Slonimski, P . P . (1991) EMBO J . 10, 2023-2031} . In the present study, we established that deleting the ABC1 chromosomal gene from Saccharomyces cerevisiae does not prevent the assembly of the bc1 complex (complex III) but markedly impairs the kinetics of its high-potential electron transfer pathway occurring on the positive, outer, side of the membrane, which results in reduced activity of the bc1 complex . In addition, the activity of complex II and its cytochrome b560 decrease drastically and complex IV activity is halved . It is also observed that the binding of the quinol to the bc1 complex ubiquinol oxidation site is affected and that adding exogenous quinones partially compensates for the respiratory deficiency in vitro, although the quinone content of mutant and wild-type mitochondria are similar . Lastly, complexes II, III and IV are found to be thermosensitive and the bc1 complex exhibits greater sensitivity than the wild-type strain to center N and P inhibitors, suggesting that the three multisubunit complexes have undergone structural modifications . The data suggest that the ABC1 gene product acts as a chaperone-like protein essential for the proper conformation and efficient functioning of the bc1 complex and the effects of the Abc1 protein on the complexes II and IV might result from interactions with the modified bc1 complex. Eur J Biochem, 1997 May 15, 246(1), 38 - 44 The N-terminal Arg-rich region of human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus Nef is involved in RNA binding; Echarri A et al.; Comparison of the amino acid sequences of human immunodeficiency virus (HIV) Nef protein and several RNA-binding proteins shows similarities in some regions of these proteins . Thus, poliovirus protein 2C, an RNA-binding protein, shares with Nef the sequence YXQQ...MDD...DXXD . In addition, both proteins contain an Arg-rich motif that, in the case of poliovirus 2C, is involved in RNA-binding activity . Moreover, the RNA-binding, anti-terminator N proteins of lambda, phi21 and P22 phages show sequence similarities with HIV Nef at the Arg-rich motif . To assess the significance of this motif, native and deletion variants of Nef protein were assayed for RNA-binding activity . The N-terminal 35 amino acids of HIV-1 Nef that comprise the Arg-rich motif are sufficient for RNA binding . Point mutations engineered at the Arg-rich motif of HIV-1 Nef revealed that basic amino acid residues are essential for RNA-binding activity . The Nef proteins from HIV-2 and SIV can also interact with RNA, while the same proteins with the N-terminal Arg-rich domain truncated fail to interact with RNA . These findings indicate that all three Nef proteins from HIV-1, HIV-2 and simian immunodeficiency virus belong to the RNA-binding family of proteins . The three proteins contain an Arg-rich region at the N-terminus which is necessary to interact with RNA. EMBO J, 1997 May 15, 16(10), 2968 - 74 Rescuing an essential enzyme-RNA complex with a non-essential appended domain; Whelihan EF et al.; Certain protein-RNA complexes, such as synthetase-tRNA complexes, are essential for cell survival . These complexes are formed with a precise molecular fit along the interface of the reacting partners, and mutational analyses have shown that amino acid or nucleotide substitutions at the interface can be used to disrupt functional or repair non-functional complexes . In contrast, we demonstrate here a feature of a eukaryote system that rescues a disrupted complex without directly re-engineering the interface . The monomeric yeast Saccharomyces cerevisiae glutaminyl-tRNA synthetase, like several other class I eukaryote tRNA synthetases, has an active-site-containing 'body' that is closely homologous to its Escherichia coli relative, but is tagged at its N-terminus with a novel and dispensable appended domain whose role has been obscure . Because of differences between the yeast and E . coli glutamine tRNAs that presumably perturb the enzyme-tRNA interface, E . coli glutaminyl-tRNA synthetase does not charge yeast tRNA . However, linking the novel appended domain of the yeast to the E . coli enzyme enabled the E . coli protein to function as a yeast enzyme, in vitro and in vivo . The appended domain appears to contribute an RNA interaction that compensates for weak or poor complex formation . In eukaryotes, extra appended domains occur frequently in these proteins . These domains may be essential when there are conditions that would otherwise weaken or disrupt formation of a critical RNA-protein complex . They may also be adapted for other, specialized RNA-related functions in specific instances. EMBO J, 1997 May 15, 16(10), 2682 - 92 rqh1+, a fission yeast gene related to the Bloom's and Werner's syndrome genes, is required for reversible S phase arrest; Stewart E et al.; In eukaryotic cells, S phase can be reversibly arrested by drugs that inhibit DNA synthesis or DNA damage . Here we show that recovery from such treatments is under genetic control and is defective in fission yeast rqh1 mutants . rqh1+, previously known as hus2+, encodes a putative DNA helicase related to the Escherichia coli RecQ helicase, with particular homology to the gene products of the human BLM and WRN genes and the Saccharomyces cerevisiae SGS1 gene . BLM and WRN are mutated in patients with Bloom's syndrome and Werner's syndrome respectively . Both syndromes are associated with genomic instability and cancer susceptibility . We show that, like BLM and SGS1, rqh1+ is required to prevent recombination and that in fission yeast suppression of inappropriate recombination is essential for reversible S phase arrest. Anal Biochem, 1997 May 15, 248(1), 94 - 101 Di-fluoresceinthiocarbamyl-insulin: a fluorescent substrate for the assay of protein disulfide oxidoreductase activity; Heuck AP et al.; We have developed a novel method for the continuous assay of protein disulfide oxidoreductase activity using as substrate bovine pancreas insulin in which both N-terminal amino groups are chemically modified with fluorescein isothiocyanate . The reduction of intercatenary disulfide bonds of di-fluoresceinthiocarbamyl-insulin with dithiothreitol initially lowers but subsequently enhances the emission intensity . In this biphasic kinetics, the rate of increase is sensitive enough for the estimation of Escherichia coli thioredoxin concentrations from 5 nM (0.06 microgram/ml) to 500 nM (6 micrograms/ml) . Neither changes of pH over a range of 6.2 to 8.4 nor neutral salts (K+, Mg2+, and Ca2+) at concentrations lower than 100 mM affect this simple reaction system . Moreover, the fluorometric method is functional for measuring the reductive capacity of Brassica napus protein disulfide isomerase . Hence, a highly reproducible and accurate one-state assay for protein disulfide oxidoreductase activity not only greatly improves the sensitivity compared to the commonly used turbidimetric assay but also represents a reliable alternative to assays based on accessory enzymes or radiolabeled substrates. Anal Biochem, 1997 May 15, 248(1), 50 - 62 Accurate topological comparison of two recombinant human growth hormones by optical surface plasmon resonance; Mani JC et al.; A strategy for the comparison of two recombinant derived human growth hormones (r-hGH) has been developed using surface plasmon resonance (SPR) . Statistical analysis was systematically used on the results obtained with several batches derived from two different Escherichia coli strains . Monoclonal antibodies (MAb) directed against four different domains in the tertiary structure of natural human growth hormone were used to compare the epitopic maps of the three (two recombinant and one natural) hGH by SPR analysis . Topological studies show the homogeneity of the epitopic maps of the three hGH . The kinetic parameters, association rate, and dissociation rate constants were also analyzed for the binding of each hGH batch to all MAbs . They were found to be homogeneous between the three hormones . Furthermore, the two r-hGH were compared by more classical approaches examining recognition of lactogenic or somatogenic receptors using, respectively, a bioassay of Nb2 cell proliferation and binding to rat liver microsomes . Specific bioactivities and IC50 values calculated in radioreceptor assays did not significantly differ between different r-hGH . The method was sensitive enough to show slight differences on koff value for one MAb (3C11) between (natural) hormone and two r-hGH . These differences are discussed in relation to previous observation made in the literature and the presence of isoforms in the natural product . The strategy developed here was very useful as a new tool to establish the equivalence of the two r-hGH. J Physiol, 1997 May 15, 501 ( Pt 1), 125 - 48 ATPase kinetics on activation of rabbit and frog permeabilized isometric muscle fibres: a real time phosphate assay; He ZH et al.; 1 . The rate of appearance of inorganic phosphate (Pi) and hence the ATPase activity of rabbit psoas muscle in single permeabilized muscle fibres initially in rigor was measured following laser flash photolysis of the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP) in the presence and absence of Ca2+ . Pi appearance was monitored from the fluorescence signal of a Pi-sensitive probe, MDCC-PBP, a coumarin-labelled A197C mutant of the phosphate-binding protein from Escherichia coli . Fibres were immersed in oil to optimize the fluorescence signal and to obviate diffusion problems . The ATPase activity was also measured under similar conditions from the rate of NADH disappearance using an NADH-linked coupled enzyme assay . 2 . On photolysis of NPE-caged ATP in the presence of Ca2+ at 20 degrees C, the fluorescence increase of MDCC-PBP was non-linear with time . ATPase activity was 41 s-1 in the first turnover based on a myosin subfragment 1 concentration of 150 microM . This was calculated from a linear regression of the fluorescence signal reporting 20-150 microM of Pi release . Tension was at 67% of its isometric level by the time 150 microM Pi was released . ATPase activities were 36 and 31 s-1 for Pi released in the ranges of 150-300 microM and 300-450 microM, respectively . The ATPase activity had a Q10 value of 2.9 based on measurements at 5, 12 and 20 degrees C . 3 . An NADH-linked assay showed the ATPase activity had a lower limit of 12.7 s-1 at 20 degrees C . The response to photolytic release of ADP showed that the rate of NADH disappearance was partially limited by the flux through the coupled reactions . Simulations indicated that the linked assay data were consistent with an initial ATPase activity of 40 s-1 . 4 . On photolysis of NPE-caged ATP in the absence of Ca2+ the ATPase activity was 0.11 s-1 at 20 degrees C with no discernible rapid transient phase of Pi release during the first turnover of the ATPase . 5 . To avoid the rigor state, the ATPase rate in the presence of Ca2+ was also measured on activation from the relaxed state by photolytic release of Ca2+ from a caged Ca2+ compound, nitrophenyl-EGTA . At 5 degrees C the ATPase rate was 5.8 and 4.0 s-1 in the first and second turnovers, respectively . These rates are comparable to those when NPE-caged ATP was used . 6 . The influence of ADP and Pi on the ATPase activities was measured using the MDCC-PBP and NADH-linked assays, respectively . ADP (0.5 mM) decreased the initial ATPase rate by 23% . Pi (10 mM) had no significant effect . Inhibition by ADP, formed during ATP hydrolysis, contributed to the decrease of ATPase activity with time . 7 . The MDCC-PBP assay and NPE-caged ATP were used to measure the ATPase rate in single permeabilized muscle fibres of the semitendinosus muscle of the frog . At 5 degrees C in the presence of Ca2+ the ATPase activity was biphasic being 15.0 s-1 during the first turnover (based on 180 microM myosin subfragment 1) . Tension was 74% of its isometric level by the time 180 microM Pi was released . During the third turnover the ATPase rate decreased to about 20% of that during the first turnover . 8 . ATPase activity in isometric rabbit muscle fibres during the first few turnovers is about an order of magnitude greater than that when a steady state is reached . Possible reasons and the consequences for understanding the mechanism of muscular contraction are discussed. FEMS Microbiol Lett, 1997 May 15, 150(2), 297 - 301 Immotile phenotype of an Escherichia coli mutant lacking the histone-like protein HU; Nishida S et al.; The histone-like protein HU in Escherichia coli are encoded by the hupA and hupB genes . A hupA-hupB double deletion mutant has now been shown to express an immotile phenotype . The motility of hupA or hupB single mutants was similar to that of wild-type cells . SDS-polyacrylamide gel electrophoresis revealed that the amount of flagellin in the hupA-hupB double deletion mutant was markedly reduced compared with the wild-type strain suggesting that the immotile phenotype of the double deletion mutant is caused by a loss of flagella. FEMS Microbiol Lett, 1997 May 15, 150(2), 283 - 8 Nucleotide sequence of a nicking site of the Streptomyces plasmid pSN22 replicating by the rolling circle mechanism; Suzuki I et al.; A putative nicking site in the double strand origin (DSO) of the Streptomyces plasmid pSN22 was identified by comparing the nucleotide sequence of the DSO region with those of two other Streptomyces plasmids, pIJ101 and pJVI . A 7-bp sequence of this putative nicking site, 5'-CTTGGGA-3', was similar to the consensus sequence of the nicking site of the pC194 group of plasmids . When several point mutations were introduced into this 7-bp sequence, the transformation abilities of the mutant plasmid molecules for Streptomyces lividans were either reduced or lost . Southern hybridization analysis indicated that these mutant plasmids could not replicate in S . lividans, but were integrated into the chromosomal DNA. FEMS Microbiol Lett, 1997 May 15, 150(2), 239 - 47 Characterization of dnaA gene expression in Mycoplasma capricolum; Seto S et al.; Expression of the dnaA gene in Mycoplasma capricolum was studied . The transcriptional start site was located 10 bp upstream from the putative translational initiation codon . Immunoblotting analysis revealed that DnaA protein was expressed at the levels significantly larger than those in Escherichia coli, and was localized mainly in the membrane . The transcriptional level of dnaA gene was reduced by inhibition of DNA synthesis with methyl methanesulfonate or mitomycin C, while the level of DnaA protein did not change . Reduction of protein synthesis did not significantly affect the total amount of DnaA protein, suggesting that the rates of synthesis and degradation of the protein are slow . These observations showed that the expression pattern of dnaA gene in M . capricolum is different from those of walled bacteria in some aspects. Arch Biochem Biophys, 1997 May 15, 341(2), 353 - 9 Selenophosphate synthetase: enzyme labeling studies with {gamma-32P}ATP, {beta-32P}ATP, {8-14C}ATP, and {75Se}selenide; Liu SY et al.; Selenophosphate synthetase catalyzes a reaction in which ATP and selenide are converted to H3SeP03, H3P04, and AMP in a 1:1:1 ratio . Selenophosphate is derived from the gamma phosphoryl group and orthophosphate from the beta phosphoryl group of ATP . In the absence of selenide, a slow reaction in which ATP is converted quantitatively to 2 H3P04 and AMP occurs . Labeling experiments carried out to detect a putative enzyme-bound pyrophosphate intermediate in the overall reaction showed that up to 0.6 equivalent of the 32P label from {gamma-32P}ATP was bound to protein under enzyme turnover conditions, but only a negligible amount of 32P from {beta-32P}ATP was present . Thus, no Enz-PP intermediate was present in a detectable amount under the experimental conditions used . Isolated enzyme samples contained 75Se from 75Se-labeled selenide and {14C}AMP from {8-14C}ATP in amounts similar to the bound 32P from {gamma-32P}ATP, suggesting that two of the final products, selenophosphate and AMP, were the radioactive compounds detected in these experiments. Arch Biochem Biophys, 1997 May 15, 341(2), 329 - 36 Mapping the mechanism-based modification sites in L-aspartase from Escherichia coli; Giorgianni F et al.; Inactivation of the enzyme L-aspartase from Escherichia coli by the substrate analog aspartate beta-semialdehyde has previously been shown to occur by the mechanism-based conversion to the corresponding product aldehyde, followed by covalent modification of cysteine-273 (F . Giorgianni et al . (1995) Biochemistry 34, 3529) . Inactivation by the product analog, fumaric acid aldehyde (FAA), has now been examined directly by adding a reduction step to the modification protocol in order to stabilize the resulting enzyme-FAA derivative(s) . HPLC and mass spectrometric analyses of proteolytic digests of inactivated L-aspartase have confirmed the modification at cysteine-273, and have also identified an additional modified peptide . The inactivation at this additional site involves a crosslink between cysteine-140 and an adjacent lysine . Site-directed mutagenesis studies have shown that cysteine-140 is a very reactive and accessible nucleophile that is not, however, directly involved in enzyme activity . The adjacent lysine-139 that is modified does appear to play a role in substrate binding . A double mutant in which both of the reactive cysteines have been replaced is almost completely insensitive to modification by these substrate and product analogs. Arch Biochem Biophys, 1997 May 15, 341(2), 309 - 14 Construction and expression of chimeric rat liver hydroxysteroid sulfotransferase isozymes; Tamura H et al.; The St-20 and ST-40 cDNAs encode rat liver hydroxysteroid sulfotransferases (HS-ST) that are 90% identical in amino acid sequence but exhibit different substrate preferences for dehydroepiandrosterone (DHEA), androsterone (AD), and cortisol (CS) . ST-40 is active for all three substrates, whereas ST-20 is mainly active for cortisol . To determine the domain responsible for the substrate preferences of the HS-STs, 20 chimeric HS-STs were constructed by reciprocal exchanges of DNA fragments derived from the cDNAs and were expressed in Escherichia coli . Some chimeric enzymes were enzymatically active for all three substrates, and some displayed reduced or lost CS-ST activity, with retention of DHEA- and AD-ST activities . Others lost all HS-ST activity . Analysis revealed that a central region (region III spanning amino acids 102-164 with five amino acid differences between ST-20 and ST-40) is essential for HS-ST activity, whereas regions II (amino acids 65-101) and IV (amino acids 165-219) are unimportant with regard to substrate preference . It was also shown that the parental combination of regions I (amino acids 1-64) and V (amino acids 220-284) is essential for CS-ST activity . Photoaffinity labeling with {35S}3'-phosphoadenosine 5'-phosphosulfate (PAPS) revealed that some inactive chimeras lost affinity for PAPS . These results suggested that an ordered structure formed by regions I, III, and V is required for HS-ST activity, especially for substrate preference and PAPS binding. Biochem J, 1997 May 15, 324 ( Pt 1), 273 - 81 Biochemical characteristics of a rice (Oryza sativa L., IR36) G-protein alpha-subunit expressed in Escherichia coli; Seo HS et al.; A cDNA encoding the alpha-subunit of the heterotrimeric G-protein in rice (RGA1) was overexpressed in Escherichia coli and then isolated by Ni2+-nitrilotriacetic acid affinity chromatography . The molecular mass of RGA1 bearing a His tag was approx . 49 kDa . Immunoblot analysis using anti-RGA1 revealed that the RGA1 protein is most abundant in seedling leaves and least abundant in mature roots . It exists at particularly high levels in the immature embryo after pellicle extrusion . In addition, the RGA1 antiserum exhibited a difference in binding affinity for Galpha proteins from monocots (maize and rice) and dicots (Arabidopsis, pea, soya bean and tomato); whereas it cross-reacted with Galpha proteins of monocots, it did not with those of dicot plants . When bound to guanosine 5'-(gamma-thio)triphosphate (GTP{S}), the RGA1 protein was partially protected from tryptic proteolysis . In the presence of GTP{S}, trypsin cleaved the RGA1 protein into four fragments 24, 14, 11 and 5 kDa in size . When RGA1 was bound to GDP, only the 5 kDa polypeptide was seen on SDS/PAGE after trypsin digestion . Photoaffinity labelling with {alpha-32P}GTP and a GTP{S}-binding assay revealed that RGA1 incorporated 32P and showed specific binding to a guanine nucleotide . Guanidine binding of RGA1 was affected by the concentration of MgCl2 (maximum at 2 mM) . The rate of guanine nucleotide binding of RGA1 (kon,GTP{S}=0.0141+/-0.0014 min-1) and, at steady state, the kcat value for GTP hydrolysis (0.0075+/-0.0001 min-1) were very low even at 2 mM MgCl2 . The binding affinity for the nucleotides examined was in the order GTP-S- >/= GTP > GDP > CTP > ATP >/= dTTP. Biochem J, 1997 May 15, 324 ( Pt 1), 97 - 102 Cloning and characterization of two glutathione S-transferases from a DDT-resistant strain of Anopheles gambiae; Ranson H et al.; Two cDNA species, aggst1-5 and aggst1-6, comprising the entire coding region of two distinct glutathione S-transferases (GSTs) have been isolated from a 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) resistant strain (ZANDS) of Anopheles gambiae . The nucleotide sequences of these cDNA species share 80.2% identity and their derived amino acid sequences are 82.3% similar . They have been classified as insect class I GSTs on the basis of their high sequence similarity to class I GSTs from Drosophila melanogaster and Musca domestica and they are localized to a region of an An . gambiae chromosome known to contain further class I GSTs . The genes aggst1-5 and aggst1-6 were expressed at high levels in Escherichia coli and the recombinant GSTs were purified by affinity chromatography and characterized . Both agGST1-5 and agGST1-6 showed high activity with the substrates 1-chloro-2,4-dinitrobenzene and 1, 2-dichloro-4-nitrobenzene but negligible activity with the mammalian theta class substrates, 1,2-epoxy-3-(4-nitrophenoxy)propane and p-nitrophenyl bromide . Despite their high level of sequence identity, agGST1-5 and agGST1-6 displayed different kinetic properties . Both enzymes were able to metabolize DDT and were localized to a subset of GSTs that, from earlier biochemical studies, are known to be involved in insecticide resistance in An . gambiae . This subset of enzymes is one of three in which the DDT metabolism levels are elevated in resistant insects. Biochem J, 1997 May 15, 324 ( Pt 1), 25 - 8 Glutathione transferases catalyse the detoxication of oxidized metabolites (o-quinones) of catecholamines and may serve as an antioxidant system preventing degenerative cellular processes; Baez S et al.; o-Quinones are physiological oxidation products of catecholamines that contribute to redox cycling, toxicity and apoptosis, i.e . the neurodegenerative processes underlying Parkinson's disease and schizophrenia . The present study shows that the cyclized o-quinones aminochrome, dopachrome, adrenochrome and noradrenochrome, derived from dopamine, dopa, adrenaline and noradrenaline respectively, are efficiently conjugated with glutathione in the presence of human glutathione transferase (GST) M2-2 . The oxidation product of adrenaline, adrenochrome, is less active as a substrate for GST M2-2, and more efficiently conjugated by GST M1-1 . Evidence for expression of GST M2-2 in substantia nigra of human brain was obtained by identification of the corresponding PCR product in a cDNA library . Glutathione conjugation of these quinones is a detoxication reaction that prevents redox cycling, thus indicating that GSTs have a cytoprotective role involving elimination of reactive chemical species originating from the oxidative metabolism of catecholamines . In particular, GST M2-2 has the capacity to provide protection relevant to the prevention of neurodegenerative diseases. Cancer Res, 1997 May 15, 57(10), 2035 - 41 The human ALL-1/MLL/HRX antigen is predominantly localized in the nucleus of resting and proliferating peripheral blood mononuclear cells; Ennas MG et al.; The ALL-1 gene is an important regulator of embryonal and hematopoietic development, and structural variants of the human gene generated by chromosomal translocations and other genomic alterations presumably act as oncogenes in the pathogenesis of acute leukemias and other hematological malignancies . Antisera against two different epitopes of the human ALL-1 protein (anti-ALL1-N and anti-ALL1-C) were produced . Both sera revealed indistinguishable patterns of antigen localization in human peripheral blood mononuclear cells (PBMCs) . In resting PBMCs, the antigen was distributed in a speckled pattern across the nuclei, with an increased density at the nuclear envelope and the nuclear indentation . In mitotically stimulated PBMCs, the antigen surrounded the condensing chromosomes but did not colocalize with chromatin or the nuclear scaffold . The antigen is considered a marker for a novel nuclear subcompartment, a perichromosomal area termed the "chromosomal envelope." In Western blot experiments, the anti-ALL1-N serum reacted with a polypeptide corresponding to the expected full-length 430-kDa ALL-1 protein . Recombinant proteins representing the AT-hook and zinc binding subdomains of the ALL-1 protein interacted in vitro with a degenerate mixture of double-stranded oligodeoxynucleotides . Thus, the ALL-1 protein probably is a DNA-binding protein with both a sequence-unspecific (AT-hook) and a sequence-specific (zinc binding subdomains) double-stranded DNA binding mode. Cancer Res, 1997 May 15, 57(10), 2007 - 12 Creation of human alkyltransferases resistant to O6-benzylguanine; Christians FC et al.; O6-benzylguanine (BG), an inhibitor of O6-alkylguanine-DNA alkyltransferase, is being tested clinically for its ability to chemosensitize tumors to alkylating agents . Although this drug may increase the killing of tumors that express high levels of alkyltransferase, it would also be expected to reduce the already low alkyltransferase levels of hematopoietic stem cells and, thus, exacerbate the dose-limiting side effect of myelosuppression . One way to overcome this problem would be to transduce hematopoietic stem cells with a gene encoding a BG-resistant alkyltransferase prior to BG/alkylation treatment . We used the technique of random mutagenesis followed by positive genetic selection to create such a mutant gene . A pool of 6.5 x 10(6) human alkyltransferases that were randomly mutated at six amino acids near the alkyl-accepting cysteine was transformed into alkyltransferase-deficient Escherichia coli . Five mutants were selected based on their ability to provide the bacteria with resistance to both N-methyl-N'-nitro-N-nitrosoguanidine and BG . One mutant, V139F/P140R/L142M, not only had the highest BG resistance (50% inhibitory concentration, >500 microM) but also offered E . coli the best protection from N-methyl-N'-nitro-N-nitrosoguanidine and, thus, is a promising gene therapy candidate. Nucleic Acids Res, 1997 May 15, 25(10), 2025 - 9 Angle and locus of the bend induced by the msp I DNA methyltransferase in a sequence-specific complex with DNA; Dubey AK et al.; Bending of DNA induced by M.Msp I, one of the m5C-DNA methyltransferases, has been investigated using circular permutation analysis . The M.Msp I MTase induced sharp bends in DNA containing its recognition sequence 5'-CCGG-3'which was estimated to be 142+/-4 degrees and 132+/-4 degrees for circularly permuted DNA fragments of 127 and 1459 bp respectively . The bend centre was found to be asymmetric with respect to the CCGG sequence and appeared to exclude the 'target cytosine' . An estimate of approximately 15 kcal/mol was obtained for the free energy associated with M.Msp I-induced DNA bending. Nucleic Acids Res, 1997 May 15, 25(10), 2012 - 9 Transcription factor Sp3 antagonizes activation of the ornithine decarboxylase promoter by Sp1; Kumar AP et al.; Ornithine decarboxylase (ODC) expression is important for proliferation and is elevated in many tumor cells . We previously showed that Sp1 is a major positive regulator of ODC transcription . In this paper we have investigated transcriptional regulation of rat ODC by the closely related factor Sp3 . While over-expression of Sp1 caused a dramatic activation of the ODC promoter, over-expression of Sp3 caused little or no activation in either Drosophila SL2 cells (lacking endogenous Sp1 or Sp3) or in H35 rat hepatoma cells . Furthermore, co-transfection studies demonstrated that Sp3 abolished trans -activation of the ODC promoter by Sp1 . DNase I footprint studies and electrophoretic mobility shift assays demonstrated that both recombinant Sp1 and Sp3 bind specifically to several sites within the ODC promoter also protected by nuclear extracts, including overlapping GC and CT motifs located between -116 and -104 . This CT element is a site of negative ODC regulation . Mutation of either element reduced binding, but mutation of both sites was required to eliminate binding of either Sp1 or Sp3 . These results demonstrate that ODC is positively regulated by Sp1 and negatively regulated by Sp3, suggesting that the ratio of these transcription factors may be an important determinant of ODC expression during development or transformation. Nucleic Acids Res, 1997 May 15, 25(10), 2005 - 11 Analyses of frameshifting at UUU-pyrimidine sites; Schwartz R et al.; Others have recently shown that the UUU phenylalanine codon is highly frameshift-prone in the 3'(rightward) direction at pyrimidine 3'contexts . Here, several approaches are used to analyze frameshifting at such sites . The four permutations of the UUU/C (phenylalanine) and CGG/U (arginine) codon pairs were examined because they vary greatly in their expected frameshifting tendencies . Furthermore, these synonymous sites allow direct tests of the idea that codon usage can control frameshifting . Frameshifting was measured for these dicodons embedded within each of two broader contexts: the Escherichia coli prfB (RF2 gene) programmed frameshift site and a 'normal' message site . The principal difference between these contexts is that the programmed frameshift contains a purine-rich sequence upstream of the slippery site that can base pair with the 3'end of 16 S rRNA (the anti-Shine-Dalgarno) to enhance frameshifting . In both contexts frameshift frequencies are highest if the slippery tRNAPhe is capable of stable base pairing in the shifted reading frame . This requirement is less stringent in the RF2 context, as if the Shine-Dalgarno interaction can help stabilize a quasi-stable rephased tRNA:message complex . It was previously shown that frameshifting in RF2 occurs more frequently if the codon 3'to the slippery site is read by a rare tRNA . Consistent with that earlier work, in the RF2 context frameshifting occurs substantially more frequently if the arginine codon is CGG, which is read by a rare tRNA . In contrast, in the 'normal' context frameshifting is only slightly greater at CGG than at CGU . It is suggested that the Shine-Dalgarno-like interaction elevates frameshifting specifically during the pause prior to translation of the second codon, which makes frameshifting exquisitely sensitive to the rate of translation of that codon . In both contexts frameshifting increases in a mutant strain that fails to modify tRNA base A37, which is 3'of the anticodon . Thus, those base modifications may limit frameshifting at UUU codons . Finally, statistical analyses show that UUU Ynn dicodons are extremely rare in E.coli genes that have highly biased codon usage. Nucleic Acids Res, 1997 May 15, 25(10), 1984 - 90 A novel genetic system to isolate a dominant negative effector on DNA-binding activity of Oct-2; Terunuma A et al.; Recent studies have revealed that interactions between transcription factors play an important role in regulation of gene expression in eukaryotic cells . To isolate cDNA clones that dominantly inhibit the DNA-binding activity of Oct-2, chosen as a representative factor, we have developed a novel screening system . This employs an Escherichia coli tester strain carrying a modified lac operon as a reporter gene, with the lac operator sequence replaced by an octamer sequence . Oct-2 expressed in this tester strain represses the expression of the reporter gene and changes the phenotype of the cell from Lac+to Lac- . Introduction of a cDNA expression library prepared from a human T-cell line into the Oct-2-harboring tester strain allowed selection of three Lac+clones out of 1 x 10(5) transformants . One of them, hT86, encoding a putative zinc finger protein was found to derepress beta-galactosidase activity in the Oct-2-harboring tester strain at the transcriptional level . In gel mobility shift assays, hT86 attenuated the intensity of the retarded band composed of the octamer probe and Oct-2, suggesting a dominant negative effect on the DNA-binding activity of Oct-2 . The strategy described here provides a new approach for studying protein-protein interactions that govern the complex regulation of gene expression. Nucleic Acids Res, 1997 May 15, 25(10), 1920 - 9 A truncation in the 14 kDa protein of the signal recognition particle leads to tertiary structure changes in the RNA and abolishes the elongation arrest activity of the particle; Thomas Y et al.; The signal recognition particle (SRP) provides the molecular link between synthesis of polypeptides and their concomitant translocation into the endoplasmic reticulum . During targeting, SRP arrests or delays elongation of the nascent chain, thereby presumably ensuring a high translocation efficiency . Components of the Alu domain, SRP9/14 and the Alu sequences of SRP RNA, have been suggested to play a role in the elongation arrest function of SRP . We generated a truncated SRP14 protein, SRP14-20C, which forms, together with SRP9, a stable complex with SRP RNA . However, particles reconstituted with SRP9/14-20C, RC(9/14-20C), completely lack elongation arrest activity . RC(9/14-20C) particles have intact signal recognition, targeting and ribosome binding activities . SRP9/14-20C therefore only impairs interactions with the ribosome that are required to effect elongation arrest . This result provides evidence that direct interactions between the Alu domain components and the ribosome are required for this function . Furthermore, SRP9/14-20C binding to SRP RNA results in tertiary structure changes in the RNA . Our results strongly indicate that these changes account for the negative effect of SRP14 truncation on elongation arrest, thus revealing a critical role of the RNA in this function. Nucleic Acids Res, 1997 May 15, 25(10), 1875 - 82 Strong, specific, monodentate G-C base pair recognition by N7-inosine derivatives in the pyrimidine.purine-pyrimidine triple-helical binding motif; Marfurt J et al.; The nucleoside analogs 7-(2'-deoxy-alpha-D-ribofuranosyl)hypoxanthine (alpha7H,1), 7-(2'-deoxy-beta-D-ribofuranosyl)hypoxanthine (beta7H,2) and 7-7-(2'-O-methyl-beta-D- ribofuranosyl)hypoxanthine (beta7HOMe,3) were prepared and incorporated into triplex forming oligodeoxynucleotides, designed to bind to DNA in the parallel (pyrimidine.purine-pyrimidine) motif . By DNase I footprinting techniques and UV-melting curve analysis it was found that, at pH 7 . 0, the 15mer oligonucleotides d(TTTTTMeCTXTMeCTMeCTMeCT) (MeC = 5-methyl-deoxycytidine, X =beta7H,beta7HOMe) bind to a DNA target duplex forming a H.G-C base triple with equal to slightly increased (10-fold) stability compared to a control oligodeoxynucleotide in which the hypoxanthine residue is replaced by MeC . Remarkably, triple-helix formation is specific to G-C base pairs and up to 40 microM third strand concentration, no stable triplex exhibiting H.A-T, H.T-A or H.C-G base arrangements could be found (target duplex concentration approximately 0.1 nM) . Multiply substituted sequences containing beta7H residues either in an isolated {d(TTTTTbeta7HTbeta7HTbeta7HTbeta7HTbeta7HT)} or in a contiguous {d(TTTbeta7Hbeta7Hbeta7Hbeta7HTTTTbeta7HTTT)} manner still form triplexes with their targets of comparable stability as the control (MeC-containing) sequences at pH 7.0 and high salt or spermine containing buffers . General considerations lead to a structural model in which the recognition of the G-C base pair by hypoxanthine takes place via only one H-bond of the N-H of hypoxanthine to N7 of guanine . This model is supported by a molecular dynamics simulation . A general comparison of the triplex forming properties of oligonucleotides containing beta7H with those containing MeC or N7-2'-deoxyguanosine (N7G) reveals that monodentate recognition in the former case can energetically compete with bidentate recognition in the latter two cases. Biochemistry, 1997 May 13, 36(19), 5884 - 92 A method for assessing the stability of a membrane protein; Lau FW et al.; The integral membrane protein diacylglycerol kinase (DGK) from Escherichia coli has been reversibly unfolded in a protein/detergent/mixed micelle system by varying the molar ratio of n-decyl beta-D-maltoside (DM) and sodium dodecyl sulfate (SDS) . Unfolding was monitored by circular dichroism (CD) and ultraviolet (UV) absorbance spectroscopy . When unfolding is monitored by measuring changes in absorbance at 294 nm, two distinct denaturation phases are observed, indicative of a stable intermediate . When CD is used as a conformational probe, the resulting denaturation curve contains only one major transition, which corresponds to the first unfolding phase observed by absorbance changes . The unfolding behavior of several mutant proteins in which the tryptophan residues were selectively replaced made it possible to assign the first unfolding phase to a denaturation event in a cytoplasmic domain and the second phase to denaturation of the membrane-embedded portion of the protein . The denaturation curves fit well to a model which assumes two cooperative transitions and a linear relationship between unfolding free energy and SDS concentration . Extrapolation back to zero denaturant indicates an unfolding free energy of 6 kcal/mol for the cytoplasmic domain and 16 kcal/mol for the transmembrane domain . The high apparent stability of the transmembrane domain could explain the high degree of tolerance to amino acid substitutions seen for DGK and other membrane proteins . The approach described in this paper may be applicable to other membrane protein systems. Biochemistry, 1997 May 13, 36(19), 5846 - 52 Two reactive site locations and structure-function study of the arrowhead proteinase inhibitors, A and B, using mutagenesis; Xie ZW et al.; The arrowhead (Sagittaria sagittifolia, Linn.) proteinase inhibitor A and B are double-headed and multifunctional, consisting of 179 amino acid residues with three disulfide bridges . Both their primary structures and cDNA sequences have been elucidated {Yang, H . L., Luo, R . S., Wang, L . X., Zhu, D . X., & Chi, C . W . (1992) J . Biochem . 111, 537; Xu, W . F., Tao, W . K., Gong, Z . Z., & Chi, C . W . (1993) J . Biochem . 113, 153; Luo, M . J., Lu, W . Y., & Chi, C . W . (1997) J . Biochem . (in press)} . Though they share 91% homology, they are different in inhibitory activities . Sequence analysis of their full-length cDNAs showed that there are seven extra residues in the C-terminal part which might be cleaved off by proteinase post-processing . To locate the reactive sites and study the structure-function relationship of the two forms A and B, the genes coding for the mature inhibitor B and its extended form were respectively cloned into the secretion expression vector, pVT102U/alpha, and expressed in Saccharomyces cerevisiae strain S-78 . Both of the gene products were purified and characterized to have the same inhibitory activities as the natural one . The gene product of the extended form was a mixture with the extended C-terminal part of the inhibitor either completely or partially removed . The two previously predicted reactive site residues, Lys-44 and Arg-76 of inhibitor B, were then respectively substituted with Ala by site-directed mutagenesis and expressed . As compared with the natural inhibitor, each of the mutants could only inhibit one molecule, instead of two molecules of trypsin, and displayed an inhibitory activity against elastase, thus confirming the location of the two reactive sites in the inhibitors . The gene coding for inhibitor A, which for some reason could not be expressed in S . cereviciae, was successfully expressed in the reconstructed plasmid pET-1522bx in Escherichia coli strain BL21 with the expressed product existing in the inclusion body . After denaturation and renaturation, the active inhibitor A was obtained and purified by anhydrotrypsin affinity chromatography . Using site-directed mutagenesis, two residues of inhibitor A, namely, Ser-82 and Leu-87, prominently different from Leu-82 and Arg-87 in inhibitor B, were replaced by these two corresponding residues, respectively . As compared with the natural inhibitor A, its S82L mutant showed a lower inhibitory activity toward trypsin, whereas a higher activity was found in the L87R mutant . Meanwhile, both of their chymotrypsin inhibitory activities became weaker than the natural one . The important accessary role of the residue of position 87 in causing the difference in inhibitory properties between inhibitor A and B was discussed. Biochemistry, 1997 May 13, 36(19), 5769 - 76 Specific recognition of O6-methylguanine in DNA by active site mutants of human O6-methylguanine-DNA methyltransferase; Hazra TK et al.; O6-Methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, acts as a monomer in removing the mutagenic DNA adduct O6-alkylguanine (induced by alkylating carcinogens) via a stoichiometric reaction . The alkyl group is transferred without a cofactor to a specific cysteine acceptor residue of MGMT, Cys-145 in the case of human MGMT, containing 207 amino acid residues and thereby inactivates the protein . As a prelude to the investigation of the reaction mechanism of human MGMT by elucidation of its structure in free and substrate-bound forms via NMR spectroscopy and X-ray crystallography, two types of MGMT mutants were generated and characterized . First, systematic deletion analysis of the protein was carried out to determine the smallest size at which it is active or inactive but forms a stable complex with the substrate and so may be useful for NMR spetroscopic analysis . Deletion of more than 8 or 31 residues from the amino or carboxyl terminus, respectively, led to the loss of both activity and substrate binding . Removal of Arg-9 or Leu-176 and distal residues inactivated the protein, presumably by altering its tertiary structure . On the basis of the criteria of bacterial overexpression and solubility, the mutant MGMT with deletion of 28 residues at the carboxyl terminus should be suitable for NMR studies . In the second approach, we examined mutants at the active site (Cys-145) that retain substrate binding . Inactive C145A and C145S substitution mutants were found to form specific and stable complexes with an O6-methylguanine (m6G)-containing oligonucleotide substrate . Wild type MGMT also formed a similar complex, but only as a transient intermediate . Footprinting studies indicated a strong discriminatory effect of the base adduct on the binding of C145A to substrate DNA; 17-18 nucleotides on the m6G-containing strand and 13-14 nucleotides in the complementary strand spanning the base adduct were protected from DNase I digestion by the mutant protein . These results, as well as the identical protease sensitivity of the wild type and mutant proteins, suggest minimal structural change due to conservative mutations at the active site . Thus, the mutant proteins may be utilized for solving the structure and mechanism of human MGMT. Biochemistry, 1997 May 13, 36(19), 5612 - 23 Urea and thermal equilibrium denaturation studies on the dimerization domain of Escherichia coli Trp repressor; Gloss LM et al.; The urea-induced equilibrium unfolding of the Escherichia coli Trp repressor (TR) is a two-state process, involving the native dimeric and unfolded monomeric species . Kinetic studies, however, reveal the presence of transient intermediates that appear only during the folding of the 107-residue protein {Gittelman, M . G., & Matthews, C . R . (1990) Biochemistry 29, 7011-7020} . In order to gain insight into the complex kinetic folding mechanism, the sequence of TR was reduced to the amino-terminal 66 residues, corresponding to the dimerization domain . Two polypeptides, 2-66 and NHis-7-66, were shown to be dimeric at 25 degrees C by size exclusion chromatography and to retain native-like spectroscopic features as evidenced by near- and far-UV circular dichroism and fluorescence spectroscopy . The equilibrium properties of the urea-induced folding of these core fragments were examined by intrinsic tryptophan fluorescence and circular dichroism and found to be well described by a two-state model . At 25 degrees C, the stabilities of both fragments are 14 kcal mol(-1), as compared to the 24 kcal mol(-1) observed for full-length TR . In contrast, the thermal denaturation of {2-66}2 and full-length TR are three-state processes; the midpoint of the transition monitored by absorbance at 292 nm precedes that monitored by circular dichroism at 222 nm . Global analysis of the thermal data as a function of monomer concentration suggests that both the full-length and {2-66}2 TR variants unfold via a dimeric intermediate . Taken together, these results demonstrate that the {2-66}2 fragment constitutes a well-structured, independently folding subdomain of TR that may be useful in elucidating the properties of the transient intermediates observed in the folding of the full-length protein . The dimeric intermediate observed in the thermal denaturation of {2-66}2 suggests that it may be possible to further reduce the core sequence while maintaining the ability to dimerize. Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 5461 - 6 A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine; Li L et al.; S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin . The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants . In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied . However, little is known about lignin pathway OMTs in gymnosperms . We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT) . The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences . However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis . The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem . Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem. Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 5417 - 21 Novel form of crosstalk between G protein and tyrosine kinase pathways; Diverse-Pierluissi M et al.; Neuronal Ca2+ channels are inhibited by a variety of transmitter receptors coupled to Go-type GTP-binding proteins . Go has been postulated to work via a direct interaction between an activated G protein subunit and the Ca2+ channel complex . Here we show that the inhibition of sensory neuron N-type Ca2+ channels produced by gamma-aminobutyric acid involves a novel, rapidly activating tyrosine kinase signaling pathway that is mediated by Galphao and a src-like kinase . In contrast to other recently described G protein-coupled tyrosine kinase pathways, the Galphao-mediated modulation requires neither protein kinase C nor intracellular Ca2+ . The results suggest that this pathway mediates rapid receptor-G protein signaling in the nervous system and support the existence of a previously unrecognized form of crosstalk between G protein and tyrosine kinase pathways. Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 5405 - 10 Interaction of the synprint site of N-type Ca2+ channels with the C2B domain of synaptotagmin I; Sheng ZH et al.; N-type Ca2+ channels mediate Ca2+ influx, which initiates fast exocytosis of neurotransmitters at synapses, and they interact directly with the SNARE proteins syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) through a synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their alpha1B subunits . Introduction of peptides containing the synprint site into presynaptic neurons reversibly inhibits synaptic transmission, confirming the importance of interactions with this site in synaptic transmission . Here we report a direct interaction of the synprint peptide from N-type Ca2+ channels with synaptotagmin I, an important Ca2+ sensor for exocytosis, as measured by an affinity-chromatography binding assay and a solid-phase immunoassay . This interaction is mediated by the second C2 domain (C2B) of synaptotagmin I, but is not regulated by Ca2+ . Using both immobilized recombinant proteins and native presynaptic membrane proteins, we found that the synprint peptide and synaptotagmin competitively interact with syntaxin . This interaction is Ca2+-dependent because of the Ca2+ dependence of the interactions between syntaxin and these two proteins . These results provide a molecular basis for a physical link between Ca2+ channels and synaptotagmin, and suggest that N-type Ca2+ channels may undergo a complex series of Ca2+-dependent interactions with multiple presynaptic proteins during neurotransmission. Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 5290 - 5 Prevention of autoimmune disease due to lymphocyte modulation by the B-subunit of Escherichia coli heat-labile enterotoxin; Williams NA et al.; We demonstrate that the receptor binding moiety of Escherichia coli heat-labile enterotoxin (EtxB) can completely prevent autoimmune disease in a murine model of arthritis . Injection of male DBA/1 mice at the base of the tail with type II collagen in the presence of complete Freund's adjuvant normally leads to arthritis, as evidenced by inflammatory infiltration and swelling of the joints . A separate injection of EtxB at the same time as collagen challenge prevented leukocyte infiltration, synovial hyperplasia, and degeneration of the articular cartilage and reduced clinical symptoms of disease by 82% . The principle biological property of EtxB is its ability to bind to the ubiquitous cell surface receptor GM1 ganglioside, and to other galactose-containing glycolipids and galactoproteins . The importance of receptor interaction in mediating protection from arthritis was demonstrated by the failure of a non-receptor-binding mutant of EtxB to elicit any protective effect . Analysis of T cell responses to collagen, in cultures of draining lymph node cells, revealed that protection was associated with a marked increase in interleukin 4 production concomitant with a reduction in interferon gamma levels . Furthermore, in protected mice there was a significant reduction in anti-collagen antibody levels as well as an increase in the IgG1/IgG2a ratio . These observations show that protection is associated with a shift in the Th1/Th2 balance as well as a general reduction in the extent of the anti-type II collagen immune response . This suggests that EtxB-receptor-mediated modulation of lymphocyte responses provides a means of preventing autoimmune disease. Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 5278 - 83 High-affinity binding of bioactive glycosylation-inhibiting factor to antigen-primed T cells and natural killer cells; Sugie K et al.; High-affinity binding was demonstrated between suppressor-T-cell-derived bioactive glycosylation-inhibiting factor (GIF) and helper T hybridomas and natural killer cell line cells . Inactive GIF present in cytosol of suppressor T cells and Escherichia coli-derived recombinant human GIF (rhGIF) failed to bind to these cells . However, affinity of rhGIF for the target cells was generated by replacement of Cys-57 in the sequence with Ala or of Asn-106 with Ser or binding of 5-thio-2-nitrobenzoic acid to Cys-60 in the molecule . Such mutations and the chemical modification of rhGIF synergistically increased the affinity of GIF molecules for the target cells . The results indicated that receptors on the target cells recognize conformational structures of bioactive GIF . Equilibrium dissociation constant (Kd) of the specific binding between bioactive rGIF derivatives and high-affinity receptors was 10-100 pM . Receptors for bioactive GIF derivatives were detected on Th1 and Th2 T helper clones and natural killer NK1.1(+) cells in normal spleen but not on naive T or B cells . Neither the inactive rGIF nor bioactive rGIF derivatives bound to macrophage and monocyte lines or induced macrophages for tumor necrosis factor alpha production. Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 4982 - 7 RNA polymerase sigma factor determines start-site selection but is not required for upstream promoter element activation on heteroduplex (bubble) templates; Fredrick K et al.; Sequence-selective transcription by bacterial RNA polymerase (RNAP) requires sigma factor that participates in both promoter recognition and DNA melting . RNAP lacking sigma (core enzyme) will initiate RNA synthesis from duplex ends, nicks, gaps, and single-stranded regions . We have used DNA templates containing short regions of heteroduplex (bubbles) to compare initiation in the presence and absence of various sigma factors . Using bubble templates containing the sigmaD-dependent flagellin promoter, with or without its associated upstream promoter (UP) element, we demonstrate that UP element stimulation occurs efficiently even in the absence of sigma . This supports a model in which the UP element acts primarily through the alpha subunit of core enzyme to increase the initial association of RNAP with the promoter . Core and holoenzyme do differ substantially in the template positions chosen for initiation: sigmaD restricts initiation to sites 8-9 nucleotides downstream of the conserved -10 element . Remarkably, sigmaA also has a dramatic effect on start-site selection even though the sigmaA holoenzyme is inactive on the corresponding homoduplexes . The start sites chosen by the sigmaA holoenzyme are located 8 nucleotides downstream of sequences on the nontemplate strand that resemble the conserved -10 hexamer recognized by sigmaA . Thus, sigmaA appears to recognize the -10 region even in a single-stranded state . We propose that in addition to its described roles in promoter recognition and start-site melting, sigma also localizes the transcription start site. Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 4978 - 81 Trigger factor is induced upon cold shock and enhances viability of Escherichia coli at low temperatures; Kandror O et al.; Trigger factor (TF) in Escherichia coli is a molecular chaperone with remarkable properties: it has prolyl-isomerase activity, associates with nascent polypeptides on ribosomes, binds to GroEL, enhances GroEL's affinity for unfolded proteins, and promotes degradation of certain polypeptides . Because the latter effects appeared larger at 20 degrees C, we studied the influence of temperature on TF expression . Unlike most chaperones (e.g., GroEL), which are heat-shock proteins (hsps), TF levels increased progressively as growth temperature decreased from 42 degrees C to 16 degrees C and even rose in cells stored at 4 degrees C . Upon temperature downshift from 37 degrees C to 10 degrees C or exposure to chloramphenicol, TF synthesis was induced, like that of many cold-shock proteins . We therefore tested if TF expression might be important for viability at low temperatures . When stored at 4 degrees C, E . coli lose viability at exponential rates . Cells with reduced TF content die faster, while cells overexpressing TF showed greater viability . Although TF overproduction protected against cold, it reduced viability at 50 degrees C, while TF deficiency enhanced viability at this temperature . By contrast, overproduction of GroEL/ES, or hsps generally, while protective against high temperatures, reduced viability at 4 degrees C, which may explain why expression of hsps is suppressed in the cold . Thus, TF represents an example of an E . coli protein which protects cells against low temperatures . Moreover, the differential induction of TF at low temperatures and hsps at high temperatures appears to provide selective protection against these opposite thermal extremes. Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 4913 - 8 Structural basis of DNA bending and oriented heterodimer binding by the basic leucine zipper domains of Fos and Jun; Leonard DA et al.; Interactions among transcription factors that bind to separate sequence elements require bending of the intervening DNA and juxtaposition of interacting molecular surfaces in an appropriate orientation . Here, we examine the effects of single amino acid substitutions adjacent to the basic regions of Fos and Jun as well as changes in sequences flanking the AP-1 site on DNA bending . Substitution of charged amino acid residues at positions adjacent to the basic DNA-binding domains of Fos and Jun altered DNA bending . The change in DNA bending was directly proportional to the change in net charge for all heterodimeric combinations between these proteins . Fos and Jun induced distinct DNA bends at different binding sites . Exchange of a single base pair outside of the region contacted in the x-ray crystal structure altered DNA bending . Substitution of base pairs flanking the AP-1 site had converse effects on the opposite directions of DNA bending induced by homodimers and heterodimers . These results suggest that Fos and Jun induce DNA bending in part through electrostatic interactions between amino acid residues adjacent to the basic region and base pairs flanking the AP-1 site . DNA bending by Fos and Jun at inverted binding sites indicated that heterodimers bind to the AP-1 site in a preferred orientation . Mutation of a conserved arginine within the basic regions of Fos and transversion of the central C:G base pair in the AP-1 site to G:C had complementary effects on the orientation of heterodimer binding and DNA bending . The conformational variability of the Fos-Jun-AP-1 complex may contribute to its functional versatility at different promoters. Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 4907 - 12 A sigma32 mutant with a single amino acid change in the highly conserved region 2.2 exhibits reduced core RNA polymerase affinity; Joo DM et al.; sigma32, the product of the rpoH gene in Escherichia coli, provides promoter specificity by interacting with core RNAP . Amino acid sequence alignment of sigma32 with other sigma factors in the sigma70 family has revealed regions of sequence homology . We have investigated the function of the most highly conserved region, 2.2, using purified products of various rpoH alleles . Core RNAP binding analysis by glycerol gradient sedimentation has revealed reduced core RNAP affinity for one of the mutant sigma32 proteins, Q80R . This reduced core interaction is exacerbated in the presence of sigma70, which competes with sigma32 for binding of core RNAP . When a different but more conserved amino acid was introduced at this position by site-directed mutagenesis (Q80N), this mutant sigma factor still displayed a significant reduction in its core RNAP affinity . Based on these results, we conclude that at least one specific amino acid in region 2.2 is involved in core RNAP interaction. Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 4872 - 7 Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position; Cahoon EB et al.; Acyl-acyl carrier protein (ACP) desaturases introduce double bonds at specific positions in fatty acids of defined chain lengths and are one of the major determinants of the monounsaturated fatty acid composition of vegetable oils . Mutagenesis studies were conducted to determine the structural basis for the substrate and double bond positional specificities displayed by acyl-ACP desaturases . By replacement of specific amino acid residues in a Delta6-palmitoyl (16:0)-ACP desaturase with their equivalents from a Delta9-stearoyl (18:0)-ACP desaturase, mutant enzymes were identified that have altered fatty acid chain-length specificities or that can insert double bonds into either the Delta6 or Delta9 positions of 16:0- and 18:0-ACP . Most notably, by replacement of five amino acids (A181T/A200F/S205N/L206T/G207A), the Delta6-16:0-ACP desaturase was converted into an enzyme that functions principally as a Delta9-18:0-ACP desaturase . Many of the determinants of fatty acid chain-length specificity in these mutants are found in residues that line the substrate binding channel as revealed by x-ray crystallography of the Delta9-18:0-ACP desaturase . The crystallographic model of the active site is also consistent with the diverged activities associated with naturally occurring variant acyl-ACP desaturases . In addition, on the basis of the active-site model, a Delta9-18:0-ACP desaturase was converted into an enzyme with substrate preference for 16:0-ACP by replacement of two residues (L118F/P179I) . These results demonstrate the ability to rationally modify acyl-ACP desaturase activities through site-directed mutagenesis and represent a first step toward the design of acyl-ACP desaturases for the production of novel monounsaturated fatty acids in transgenic oilseed crops. Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 4866 - 71 Crystal structure of glutamate-1-semialdehyde aminomutase: an alpha2-dimeric vitamin B6-dependent enzyme with asymmetry in structure and active site reactivity; Hennig M et al.; The three-dimensional structure of glutamate-1-semialdehyde aminomutase (EC 5.4.3.8), an alpha2-dimeric enzyme from Synechococcus, has been determined by x-ray crystallography using heavy atom derivative phasing . The structure, refined at 2.4-A resolution to an R-factor of 18.7% and good stereochemistry, explains many of the enzyme's unusual specificity and functional properties . The overall fold is that of aspartate aminotransferase and related B6 enzymes, but it also has specific features . The structure of the complex with gabaculine, a substrate analogue, shows unexpectedly that the substrate binding site involves residues from the N-terminal domain of the molecule, notably Arg-32 . Glu-406 is suitably positioned to repel alpha-carboxylic acids, thereby suggesting a basis for the enzyme's reaction specificity . The subunits show asymmetry in cofactor binding and in the mobilities of the residues 153-181 . In the unliganded enzyme, one subunit has the cofactor bound as an aldimine of pyridoxal phosphate with Lys-273 and, in this subunit, residues 153-181 are disordered . In the other subunit in which the cofactor is not covalently bound, residues 153-181 are well defined . Consistent with the crystallographically demonstrated asymmetry, a form of the enzyme in which both subunits have pyridoxal phosphate bound to Lys-273 through a Schiff base showed biphasic reduction by borohydride in solution . Analysis of absorption spectra during reduction provided evidence of communication between the subunits . The crystal structure of the reduced form of the enzyme shows that, despite identical cofactor binding in each monomer, the structural asymmetry at residues 153-181 remains. J Immunol Methods, 1997 May 12, 204(1), 51 - 6 A flow cytometric method for the rapid detection of beta-galactosidase transfected cells: an in vitro and in vivo study; Mouawad R et al.; A flow cytometric method has been developed for the rapid analysis of lacZ transduced cells . The method described is based on an indirect immunofluorescence staining procedure using a monoclonal antibody which binds specifically to beta-galactosidase from E . coli and to beta-galactosidase fusion proteins . This technique was used for the quantification in vitro as well as in vivo of beta-galactosidase expression in B16 melanoma cells . The described method is appropriate for a variety of cell types (species, lineage), is simple, quantitative, reliable, rapid and applicable to all constructs containing the lacZ selectable markers . It should prove to be very helpful (1) for the quantification of cells expressing the lacZ reporter gene and (2) for studying gene regulation, including transfection modality, promoter efficacy, enhancer activity, and other regulatory factors. FEBS Lett, 1997 May 12, 408(1), 99 - 104 Regulation of the DNA binding of p53 by its interaction with protein kinase CK2; Prowald A et al.; Some of the numerous functions of the growth suppressor protein p53 are regulated by its interaction with viral and cellular proteins . C-terminal sequences of p53 are implicated in binding to the regulatory beta-subunit of protein kinase CK2 . Using a p53-specific DNA binding element we found that the beta-subunit of CK2 inhibited the DNA binding of p53 whereas the alpha-subunit had no influence . The CK2 holoenzyme consisting of two alpha- and two beta-subunits led to a supershift in DNA binding of p53 similar to the p53-specific monoclonal antibody PAb421 as well as the C-terminus of p53 . Thus, our results showed an individual role of the free beta-subunit of CK2 on the DNA binding activity of p53. FEBS Lett, 1997 May 12, 408(1), 62 - 6 Cloning, expression and characterisation of murine procathepsin E; Tatnell PJ et al.; The cDNA encoding murine procathepsin E was isolated and sequenced and recombinant enzyme was produced in Escherichia coli . The activity of the purified recombinant mouse cathepsin E was characterised quantitatively using two synthetic peptide substrates and naturally occurring inhibitors . The majority of the recombinant enzyme was present as a homodimer (Mr approximately 80) in which the two monomers were linked by an intermolecular disulfide bond . By analogy to previous studies with human cathepsin E, this is most likely a consequence of the presence of a unique cysteine residue near the N-terminus of the mature proteinase . The availability of (i) recombinant murine enzyme in reasonable quantities and (ii) a full-length cDNA now enables structural investigations and attempts to generate 'knock-out' mice deficient in this important aspartic proteinase to be undertaken. FEBS Lett, 1997 May 12, 408(1), 52 - 6 Molecular heterogeneity of the cDNA encoding a 74-kDa regulatory subunit (B" or delta) of human protein phosphatase 2A; Tanabe O et al.; Two cDNAs for possible splicing variants of a 74-kDa regulatory subunit (B" or delta) of human protein phosphatase 2A, were isolated . These variants were identified from human cerebral cortex by library screening and PCR, and designated delta1 and delta3 isoforms, while the previously reported isoform {Tanabe et al . (1996) FEBS Lett . 379, 107-1111 was designated delta2 . Compared with the delta2 isoform, the delta1 isoform contained a 32-residue insertion beginning at residue 84, and consisted of 602 amino acids in all . The delta3 isoform lacked a 74-residue sequence corresponding to residues 1083 of the delta2 isoform, and consisted of 496 amino acids . Using isoform-specific antipeptide antisera, the 74-kDa subunit (B" or delta) originally purified from human erythrocytes was identified as the delta1 isoform. FEBS Lett, 1997 May 12, 408(1), 25 - 9 DNA binding sites recognised in vitro by a knotted class 1 homeodomain protein encoded by the hooded gene, k, in barley (Hordeum vulgare); Krusell L et al.; The homeodomain of the knotted classes of transcription factors from plants differs from the well characterized Antp/En type homeodomains from Drosophila at key amino acid residues contributing to the DNA binding . A cDNA, Hvh21, derived from the hooded gene and encoding a full length homolog of knotted1 from maize was isolated from barley seedlings and expressed as a maltose binding protein fusion in E . coli . The purified HvH21-fusion protein selected DNA fragments with 1-3 copies of the sequence TGAC . Gel shift experiments showed that the TGAC element was required for binding and the results further indicate that the HvH21-fusion protein binds DNA as a monomer. FEBS Lett, 1997 May 12, 408(1), 11 - 5 Subunit interactions in the Escherichia coli protein translocase: SecE and SecG associate independently with SecY; Homma T et al.; We used hexahistidine-tagged SecE and SecY to study how the core subunits (SecY, SecE and SecG) of Escherichia coli protein translocase interact with each other . Detergent extracts were prepared from the plasma membranes and fractionated by Ni2+-NTA agarose affinity binding . Although His6-SecE, expressed in wild-type cells, brought down both SecY and SecG, neither of them was brought down when the same protein was expressed in the secY24 mutant cells . His6-SecY brought down both SecE and SecG, as expected . Interestingly, His6-SecY24 was able to bring down SecG but not SecE . These results confirm our previous conclusion that the secY24 alteration impairs the SecY-SecE interaction, and demonstrate that SecY and SecG can form a complex that does not contain SecE . Likewise, SecY-SecE complex could be isolated from the secG-deleted strain . The trimeric complex, in detergent extracts, dissociated at a critical temperature between 23 and 26 degrees C, whereas the SecY-SecE complex without SecG dissociated at a slightly lower temperature (20-23 degrees C) . We conclude that each of SecE and SecG independently binds to SecY, the central subunit of protein translocase, although the trimeric complex is more stable than the binary complexes. Biochem Pharmacol, 1997 May 9, 53(9), 1323 - 32 Compartmentalisation and characteristics of a Ca2+-dependent phospholipase A2 in human colon mucosa; Lamura E et al.; The biochemical properties of the phospholipase A2 (PLA2) found in the 100,000 x g centrifugate cytosol or particulate fractions of human colonic mucosa have been investigated using both deoxycholate-solubilized and Escherichia coli (E . coli) phospholipids as substrates . PLA2 activity was present in both subcellular fractions and the profiles of biochemical activites were similar . Activity in the particulate fraction was approximately twofold greater than the cytosol fraction when expressed on the basis of protein concentration . The PLA2 is Ca2+ dependent and using EGTA-regulated buffers cytosolic or particulate fraction activity was similar at both 10 microm or 10 mm Ca2+ concentrations . Using deoxycholate-phospholipid micelles as substrate a small but statistically significant twofold preference for glycero-phosphatidylcholine bearing sn-2-arachidonate compared with sn-2-oleate was seen, but this preference was not noted using arachidonate or oleate labelled E . coli membranes . Dithiothreitol (10 mM) reduced colon mucosal cytosol PLA2 activity significantly by 63.5 +/- 1.90% in cytosol and by 30.54 +/- 1.27% in microsomes using micelles as substrate or by 84.3 +/- 2.30% in cytosol and by 69.33 +/- 11.30% in microsomes using oleate-labelled E . coli as substrates . Warming at 57 degrees C reduced activity significantly by 35.0 +/- 5.80% in microsomes and by 40.0 +/- 7.08% in cytosol . Acid treatment increased PLA2 activity to 148 +/- 16.3% in microsomes and 145 +/- 18.6% in cytosol . When mucosal preparations were subjected to heparin-Sepharose chromatography, it bound tightly and eluted in the same position on a salt gradient as authentic human group II PLA2 . Further purification by gel-permeation chromatography gave activity in the 14 kDa region of the elution profile . These features have many of the characteristics expected of a 14 kDa isoform of PLA2 but exhibit activity at concentrations of Ca2+ that are relevant in the intracellular environment and may participate in cellular lipid metabolism. J Biol Chem, 1997 May 9, 272(19), 12847 - 53 Unique structural features of a novel class of small heat shock proteins; Leroux MR et al.; Small heat shock proteins (smHSPs) and alpha-crystallins constitute a family of related molecular chaperones that exhibit striking variability in size, ranging from 16 to 43 kDa . Structural studies on these proteins have been hampered by their tendency to form large, often dynamic and heterogeneous oligomeric complexes . Here we describe the structure and expression of HSP12.6, a member of a novel class of smHSPs from the nematode Caenorhabditis elegans . Like other members of its class, HSP12.6 possesses a conserved alpha-crystallin domain but has the shortest N- and C-terminal regions of any known smHSP . Expression of HSP12.6 is limited to the first larval stage of C . elegans and is not significantly up-regulated by a wide range of stressors . Unlike other smHSPs, HSP12.6 does not form large oligomeric complexes in vivo . HSP12.6 was produced in Escherichia coli as a soluble protein and purified . Cross-linking and sedimentation velocity analyses indicate that the recombinant HSP12.6 is monomeric, making it an ideal candidate for structure determination . Interestingly, HSP12.6 does not function as a molecular chaperone in vitro, since it is unable to prevent the thermally induced aggregation of a test substrate . The structural and functional implications of these findings are discussed. J Biol Chem, 1997 May 9, 272(19), 12816 - 23 DNA topoisomerases regulate R-loop formation during transcription of the rrnB operon in Escherichia coli; Masse E et al.; Recent in vivo and in vitro studies have suggested an important role for DNA topoisomerases in regulating R-loop formation during transcription in Escherichia coli . In the present report we present genetic and biochemical evidence strongly suggesting that R-loop formation can occur during transcription of a portion of the rrnB operon and that it is regulated by DNA topoisomerase activity . We found that a multicopy plasmid (pBR322) carrying an heavily transcribed portion of the rrnB operon cannot be transformed in topA mutants unless RNase H is overproduced . Transcription of the 567-base pair HindIII fragment from the rrnB operon allows the extraction of large amount of R-looped plasmid DNAs from a topA mutant, in a manner that depends on the intracellular level of RNase H activity . When DNA gyrase is sufficiently active, hypernegatively supercoiled plasmid DNA is produced if the same DNA fragment is transcribed in a topA mutant . The formation of such topoisomers most likely reflect the presence of extensive R-loops since it is sensitive to the intracellular level of RNase H activity . Finally, the formation of R-looped plasmid DNAs in an in vitro transcription system using phage RNA polymerases is also detected when the 567-base pair HindIII fragment is transcribed on a negatively supercoiled DNA template. J Biol Chem, 1997 May 9, 272(19), 12771 - 7 The DNA binding pattern of the retinoid X receptor is regulated by ligand-dependent modulation of its oligomeric state; Kersten S et al.; The retinoid X receptor (RXR) regulates target gene transcription via its association with cognate DNA response elements either as a homodimer or as a heterodimer with a number of other nuclear receptors . We previously demonstrated that, in solution, RXR forms tetramers with a high affinity and that ligand binding leads to dissociation of receptor tetramers to smaller species . Here it is shown that RXR tetramers form stable complexes with direct repeats (DR-1 or DR-5) or palindromic (TREpal) response elements . Binding of RXR tetramers to cognate DNA occurs with a significantly higher affinity as compared with dimers . Ligand binding by DNA-bound RXR tetramers results in their dissociation to DNA-bound dimers, a process that is completely reversed upon removal of the ligand . Formation of stable tetramer-DNA complexes requires binding of two oligonucleotides/tetramer . It is proposed that ligand-dependent modulation of the oligomeric state of RXR is a regulatory feature of this nuclear receptor. J Biol Chem, 1997 May 9, 272(19), 12723 - 9 Cloning, expression, and catalytic mechanism of murine lysophospholipase I; Wang A et al.; A lysophospholipase (LysoPLA I) has been purified and characterized from the mouse macrophage-like P388D1 cell line (Zhang, Y . Y, and Dennis, E . A . (1988) J . Biol . Chem . 263, 9965-9972) . This enzyme has now been sequenced, cloned, and expressed in Escherichia coli cells . The enzyme contains 230 amino acid residues with a calculated molecular mass of 24.7 kDa . It has a high helical content in its predicated secondary structure, which is also indicated in its CD spectrum . The cloned LysoPLA I was purified to homogeneity from the transformed E . coli cells by a gel filtration column and an ion exchange column . The specific activity of the purified protein is 1 . 47 micromol/min.mg toward 1-palmitoyl-sn-glycero-3-phosphorylcholine at pH 8.0 and 40 degrees C, corresponding to the reported value of 1.3-1.7 micromol/min.mg for the protein purified from the P388D1 cells . In addition, the cloned protein cross-reacted with an antibody raised against LysoPLA I also purified from the P388D1 cells . The deduced LysoPLA I sequence contains a well conserved GXSXG motif found in the active site of many serine enzymes, and the activity of the LysoPLA I was irreversibly inhibited by the classical serine protease inhibitor diisopropyl fluorophosphate . Furthermore, site