Biochemistry, 1993 Jul 20, 32(28), 7065 - 8 DNA photolyase repairs the trans-syn cyclobutane thymine dimer; Kim ST et al.; DNA photolyases catalyze the splitting of the cyclobutane ring joining the two dihydropyrimidines of a pyrimidine dimer by a photoinduced electron-transfer reaction . Previous studies concluded that photolyase repairs only the cis-syn form of the eight stereoisomers of the cyclobutane pyrimidine dimer (Pyr{ }Pyr) . In this study we found that Escherichia coli photolyase binds to the trans-syn-I isomer of T{ }T with about 10(4)-fold lower affinity than the cis-syn isomer but it repairs it relatively efficiently. J Mol Biol, 1993 Jul 20, 232(2), 680 - 92 Thermodynamic and structural analysis of the folding/unfolding transitions of the Escherichia coli molecular chaperone DnaK; Montgomery D et al.; The thermal unfolding of the Escherichia coli 70 kDa heat shock protein, DnaK, exhibits three well defined transitions . At pH 7.6, these transitions are centered at 45.2, 58.0 and 73.3 degrees C . High sensitivity calorimetric scans as a function of pH indicate that the folding/unfolding behavior is well described by a four-state model which includes a delta H, tm and delta Cp for each state . Calorimetric scans of a 44 kDa N-terminal proteolytic fragment show a major transition centered at 47.5 degrees C (N1) and a minor transition at 79.4 degrees C (N2) . A calorimetric scan of a 23 kDa C-terminal proteolytic fragment exhibits a low temperature peak at 58.5 degrees C (C1) and a high temperature peak at 70.6 degrees C (C2) . Deconvolution analysis of the low temperature peak reveals that it is actually composed of two transitions of roughly equal delta H centered at 50.4 degrees C (C1a) and 58.2 degrees C(C1b) . These experiments have allowed us to assign the transitions of the intact protein as follows . The low temperature transition of DnaK can be assigned to the N-terminal region on the basis of the similarity between the delta H and tm values for the low temperature transition and those obtained for the N1 transition of the isolated N-terminal fragment . This assignment is also supported by measurements of the intrinsic fluorescence emission as a function of temperature . DnaK contains a single tryptophan localized at residue 102 in the N-terminal domain of the protein . Additionally, calorimetric scans show that the tm of the low temperature transition increases by 9.2 degrees C in the presence of excess ADP, which is known to bind to the N-terminal domain . The middle transition can be assigned to the C1a and C1b transitions of the C-terminal fragment on the basis of the similarity of delta H and tm . In the intact protein C1a and C1b form a single cooperative unit; however, the cooperative interactions between these folding/unfolding domains are disrupted in the isolated fragment . The high temperature transition of the intact protein is composed of contributions from both the N-terminal and C-terminal regions of the protein . These studies have allowed us to develop a quantitative model of the folding/unfolding behavior of DnaK. Biochemistry, 1993 Jul 20, 32(28), 7310 - 6 Activity of recombinant alpha and beta subunits of casein kinase II from Xenopus laevis; Hinrichs MV et al.; Casein kinase II (CKII) is a ubiquitous protein kinase, found predominantly in cell nuclei, which has two subunits in a tetrameric alpha 2 beta 2 or alpha alpha' beta 2 conformation . The catalytic center is present in the alpha subunit which is active by itself while beta is a regulatory subunit that can greatly enhance the activity of alpha . The cDNA genes of Xenopus laevis coding for the alpha and beta subunits of CKII have been expressed in Escherichia coli and extensively purified . The recombinant subunits reconstitute a fully active holoenzyme when incubated in stoichiometric amounts . Mutations that change serines in positions 2 and 3 of the beta subunit for glycines completely eliminate the autophosphorylation site present in this subunit but do not significantly affect the capacity of beta to activate alpha . A fusion protein composed of glutathione transferase linked to the X . laevis CKII beta subunit can also activate alpha . This fusion protein binds to glutathione-agarose beads and can mediate the binding of the alpha subunit to this matrix . Conversely, the alpha subunit was found to bind to glass fiber filters in an active form that can still be activated by beta to an extent similar to that seen in solution . Using peptides containing tyrosine and glutamic acid as inhibitors of the activity of the isolated alpha subunit and of the holoenzyme, the effect of beta on the specificity of inhibition was studied.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1993 Jul 20, 32(28), 7172 - 80 Functional effects of base changes which further define the decoding center of Escherichia coli 16S ribosomal RNA: mutation of C1404, G1405, C1496, G1497, and U1498; Cunningham PR et al.; The existence and functional importance of the tertiary base pair G1401:C1501, which brings together two universally present and highly sequence-conserved single-stranded segments of small subunit ribosomal RNA, was proven recently by mutational analysis {Cunningham, P . R., Nurse, K., Bakin, A., Weitzmann, C . J., Pflumm, M., & Ofengand, J . (1992) Biochemistry 31, 12012-12022} . Here we show that the additional nearby tertiary base pairs C1404:G1497 and G1405:C1496 also exist and are functionally important for tRNA binding to the ribosomal A and P sites . Breakage of the base pairs in turn led to a loss of activity at both A and P sites, whereas restoration in the reverse orientation led to recovery of activity . Recovery was incomplete, indicating that base pairing alone is insufficient for full restoration of function . Mutation of U1498 to G created the potential for the tertiary base pair C1403:G1498, which could stack on the aforementioned double base pair, creating a more stable helix longer by one residue . This mutation did not affect subunit association, A- and P-site binding of tRNA to 70S, fMet-tRNA binding to 30S, or poly(Phe) synthesis but did block formation of the first peptide bond, fMet-Val . Mutation of U1498 to A or C did not show this effect . Since the G1498 mutant could make both the 70S initiation complex and the peptide bond, as shown by its ability to form fMet-puromycin, the block in fMet-Val synthesis appears to involve some aspect of A-site function.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1993 Jul 20, 32(28), 7152 - 61 A physical assay for and kinetic analysis of the interactions between M1 RNA and tRNA precursor substrates; Guerrier-Takada C et al.; A gel-shift assay was devised to detect stable enzyme-substrate (E-S) complexes between M1 RNA, the catalytic subunit of RNase P from Escherichia coli, and its tRNA precursor substrates . The use of deletion derivatives of M1 RNA in the gel-shift assay has allowed us to identify regions of the enzyme that are involved in the binding of the substrate or that are necessary for catalytic activity . Fragments of substrates that contain the 3' CCA sequence bind preferentially to regions in the 5' half of M1 RNA, while 5' leader sequences interact primarily with regions in the 3' half of M1 RNA . The 5' leader sequence present in the precursor to tRNA(Tyr)su3 from E . coli plays an important role in the formation of stable E-S complexes with M1 RNA . The CCA sequence at the 3' end of precursor tRNA substrates is involved in the product-release step of the reaction that is catalyzed by M1 RNA . Direct measurements of the concentrations of all the components in the reaction catalyzed by M1 RNA facilitated a new approach to the kinetic analysis of the action of the enzyme. FEBS Lett, 1993 Jul 19, 327(1), 17 - 20 Hydrolysis of the IciA protein, an inhibitor of DNA replication initiation, by protease Do in Escherichia coli; Yoo SJ et al.; The 33 kDa IciA protein, an inhibitor of replication initiation of the Escherichia coli chromosome, was found to be specifically cleaved to 27 kDa fragment by protease Do, the htrA gene product . The 27 kDa polypeptide could no longer interact with the oriC region, and therefore the cleavage-site is likely to reside within the N-terminal DNA-binding domain of the IciA protein . In addition, protease Do was found to localize primarily to the cytoplasm although it also could bind to membranes through an ionic interaction . These results suggest that intracellular breakdown of the IciA protein by protease Do may provide a potential mechanism involving the regulation of initiation of DNA replication in Escherichia coli. FEBS Lett, 1993 Jul 19, 327(1), 45 - 8 Modified nucleoside 5'-triphosphates containing 2',3'-fused three-membered rings as substrates for different DNA polymerases; Semizarov DG et al.; 5'-Triphosphates of 1-(2',3'-epithio-2',3'-dideoxy-beta-D- lyxofuranosyl)thymine, 1-(2',3'-epithio-2',3'-dideoxy-beta-D-ribofuranosyl)thymine and 2',3'-lyxoanhydrothymidine have been shown to be termination substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) reverse transcriptases as well as DNA polymerase I from E . coli and DNA polymerase beta from rat liver . At the same time they do not terminate DNA synthesis catalysed by DNA polymerase epsilon from human placenta . Km values of ltTTP, rtTTP and laTTP incorporation into the DNA chain during catalysis by AMV reverse transcriptase agree closely with each other being 1.5-2.5 times higher than Km value for dTTP . Furthermore, Vmax values for modified substrates are only 2-3 times lower than Vmax for dTTP . The evidence favours the hypothesis of high affinity of modified nucleotides with a flattened furanosyl ring for DNA polymerase active sites. Biochim Biophys Acta, 1993 Jul 18, 1174(1), 114 - 6 Human cDNA encoding DnaJ protein homologue; Oh S et al.; We have cloned a cDNA encoding a DnaJ-like protein from the human fibrosarcoma HT-1080 cDNA library . This cDNA encodes a protein of 397 amino acid residues whose sequence shows 38.2% identity with the Escherichia coli DnaJ protein and 47.2% with the yeast DnaJ homologue along the entire length . Since the sequence contains the N-terminal domain region conserved in DnaJ family members and the four repeats of the Cys-X-X-Cys-X-Gly-X-Gly motif which are characteristic of DnaJ proteins, we conclude that this cDNA clone encodes the human homologue of DnaJ. Biochim Biophys Acta, 1993 Jul 18, 1174(1), 111 - 3 Cloning of a unique human homologue of the Escherichia coli DNAJ heat shock protein; Chellaiah A et al.; A human homologue of the bacterial DNAJ heatshock protein, HDJ-2, was isolated from a human umbilical vein endothelial cDNA library using a monoclonal antibody which reacts specifically to human endothelial cells and monocytes . This cDNA clone consists of 1469 nucleotides with an open reading frame of 1191 nucleotides . HDJ-2 shares significant homology with Escherichia coli heat shock protein DNAJ, as well as the yeast homologues Sec63, YDJ1, SCJ1 and SIS1 . This homology suggests HDJ-2 may be involved in protein folding and/or transport. Biochim Biophys Acta, 1993 Jul 18, 1174(1), 27 - 30 Effects of magnesium ions on ribosomes: a fluorescence study; Bonincontro A et al.; Fluorescence intensity measurements of ethidium bromide (EB) bound to ribosomal RNA (rRNA) in suspensions of 30S and 50S subunits, of 70S ribosomal particles and of protein-free extracted rRNA are presented . Changes in the intercalation of EB reflect changes in conformation and degree of exposure of rRNA . The effect of removal of magnesium ions on the binding of EB is compared in protein-free rRNA and in ribosomal particles by a Scatchard plot analysis . In free ribosomal RNA the number of bound EBs do not depend on magnesium content, only the association constant is affected . In intact 70S particles and both in the separated 50S and 30S subunits the presence of magnesium greatly reduces binding of EB and no saturation of the fluorescence intensity with rRNA concentration is observed, preventing a Scatchard plot analysis . Removal of magnesium restores a strong EB intercalation . Then magnesium ions induce a conformational change in the 70S particles as well as in the separated subunits . The different behavior of the free-rRNA and of the ribosomal particles indicates that ribosomal proteins are relevant to the structural changes induced by magnesium ions . The comparison of the number of excluded sites and of the association constant in the 30S, 50S subunits and in the 70S particles indicates that even without Mg2+ ions the two subunits still interact, at variance with the commonly shared opinion that subunits dissociation takes place at low magnesium concentration. J Biol Chem, 1993 Jul 15, 268(20), 15118 - 26 Phosphorylation of the human vitamin D receptor by protein kinase C . Biochemical and functional evaluation of the serine 51 recognition site; Hsieh JC et al.; We have reported previously that the human vitamin D receptor (hVDR) is selectively phosphorylated by protein kinase C-beta (PKC-beta), in vitro, on a serine residue in the sequence RRS51MKRK, which is located between the two zinc fingers of hVDR and is potentially important to its transacting function (Hsieh, J.-C., Jurutka, P.W., Galligan, M.A., Terpening, C.M., Haussler, C.A., Samuels, D.S., Shimizu, Y., Shimizu, N., and Haussler, M.R . (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 9315-9319) . In the present experiments we evaluated this phosphorylation event using a series of hVDR mutants in which serine 51 or its flanking residues were modified . Alteration of serine 51 to a non-phosphorylatable residue resulted in an approximately 60% reduction in basal hVDR phosphorylation in intact cells but did not diminish 1,25-dihydroxyvitamin D3-stimulated phosphorylation . Such mutations also abolished subsequent phosphorylation of immunoprecipitated hVDR by purified PKC-beta, in vitro, as did replacement of basic residues on either side of serine 51 . Mutation of serine 51 to glycine (S51G) or to aspartic acid (S51D), as well as altering the basic residues flanking serine 51, abolished the interaction of hVDR with the vitamin D-responsive element (VDRE) as monitored by gel mobility shift analysis . Thus, we conclude that unmodified serine 51 and its surrounding basic residues are crucial not only for PKC-beta substrate recognition but also for the optimal VDRE binding of native hVDR . In transactivation assays, S51G and S51D possessed only 35 and 10% of wild-type hVDR activity, respectively . Mutation of serine 51 to threonine (S51T) restored phosphorylation by PKC-beta, in vitro, to about 40% of wild-type and transactivation to 45% of that of wild-type hVDR . Alteration of serine 51 to alanine, which is the residue in the corresponding position of the glucocorticoid, progesterone, mineral-ocorticoid, and androgen receptors, eliminated PKC-beta phosphorylation but completely preserved the specific DNA binding activity and transactivation capacity of hVDR . Thus, phosphorylation of hVDR at serine 51 is not required for either VDRE binding or transactivation . Finally, incubation of Escherichia coli-expressed hVDR with PKC-beta elicits marked phosphorylation of the receptor and significantly inhibits its ability to complex with the VDRE . We therefore speculate that posttranslational modification of hVDR at serine 51 may constitute a negative regulatory loop which could be operative when target cells are subject to PKC activation events. J Biol Chem, 1993 Jul 15, 268(20), 14576 - 8 ATP hydrolysis-linked structural changes in the N-terminal part of the gamma subunit of Escherichia coli F1-ATPase examined by cross-linking studies; Aggeler R et al.; A mutant of Escherichia coli F1-ATPase (ECF1) in which the serine residue in position 8 of the gamma subunit has been replaced by a cysteine residue (gamma S8C) has been used to study nucleotide-dependent cross-linking of the gamma subunit to a beta subunit . When examined in the presence of ADP+Mg2+, either supplied directly or as produced during catalytic turnover of ATP+Mg2+, the main cross-linked product generated using the heterobifunctional, photoactivatable, cross-linker tetrafluorophenylazide maleimide-6 had a M(r)(app) of 108,000 . When ATP hydrolysis was inhibited, either by cold or by reaction with sodium azide, or when ATP hydrolysis was prevented by the use of adenyl-5'-yl beta,gamma-imidodiphosphate, the main cross-linked products were species with M(r)(app) of 102,000 and 84,000 . The nucleotide-dependent switching from one cross-linking pattern to another could only be observed when the epsilon subunit was bound to ECF1; it was not seen in ECF1*, an enzyme preparation missing delta and epsilon subunits, but was observed in preparations selectively depleted of the delta subunit . We conclude that the changes detected in these cross-linking experiments are occurring during the hydrolysis of ATP when the beta-gamma phosphate bond is cleaved and that they are related to the coupling of ATP hydrolysis to proton translocation. Cancer Res, 1993 Jul 15, 53(14), 3270 - 5 Fidelity of DNA replication by extracts of normal and malignantly transformed human cells; Boyer JC et al.; To test the hypothesis that a mutator phenotype may be associated with carcinogenesis (L . A . Loeb, Cancer Res., 51: 3074-3079, 1991), we have compared the fidelity of double-stranded DNA replication and the efficiency of mismatch repair in extracts from normal diploid and malignantly transformed human cells . Included was a diploid fibroblast strain and its transformed derivative, as well as a second diploid fibroblast strain and HeLa cells . The fidelity of DNA replication by cytoplasmic extracts in the presence of simian virus 40 large tumor antigen (SV40 T-antigen) was measured using a forward mutagenesis assay . The replicated DNA consisted of double-stranded M13 mp2 DNA containing the SV40 origin of replication and the lacZ alpha complementation gene as a target sequence for scoring mutations . T-antigen-dependent replication was detected in all cell extracts, with those from transformed cells having the greatest activity . No differences in replication fidelity were detected between normal and transformed cell extracts . Using a heteroduplex containing a G.G mispair, we also detected mismatch repair activity in the cell extracts, including efficient repair in extracts from malignantly transformed cells . While these data do not eliminate the possibility that a mutator phenotype may be associated with carcinogenesis, they do suggest that genetic instability associated with transformation does not involve reduced fidelity of replication of undamaged DNA or reduced mismatch repair efficiency. Cancer Res, 1993 Jul 15, 53(14), 3250 - 2 Potent intracellular oxidative stress exerted by the carcinogen 4-nitroquinoline-N-oxide; Nunoshiba T et al.; Oxidative stress exerted by superoxide-generating (redox-cycling) agents such as paraquat triggers the soxRS regulon of Escherichia coli . In this system, SoxR protein is the redox-sensitive activator of the soxS gene, the product of which then activates the approximately 10 promoters of this regulon . We found that 4-nitroquinoline-N-oxide (4NQO) is a powerful inducer of soxS, > 10-fold more potent than paraquat . The transcriptional induction of the soxS gene by 4NQO was tightly dependent on a functional soxR gene and on the presence of molecular oxygen, as found previously for several well characterized redox-cycling agents . Two 4NQO-related compounds were also shown to induce soxS:4-nitropyridine-N-oxide, with an efficiency only slightly less than 4NQO, and 4-hydroxyaminoquinoline-N-oxide, at approximately 50-fold lower potency than 4NQO . E . coli strains that are hypersensitive to oxidative stress (owing to deficiency in either superoxide dismutases or oxidative DNA repair enzymes) were hypersensitive to killing by 4NQO . Thus, considerable oxidative stress is induced in cells by 4NQO, which might contribute to the carcinogenic potency of this compound. FEMS Microbiol Lett, 1993 Jul 15, 111(1), 129 - 34 Identification of the mcrC gene product in Methanococcus vannielii; Stroup D et al.; The polypeptide encoded by the mcrC gene has been identified in Methanococcus vannielii by immunoblotting using rabbit antibodies raised against the product of a lacZ-mcrC gene fusion synthesized and purified from Escherichia coli . The mcrC gene product (gpmcrC) was located in both the supernatant and pellet fractions after centrifugation of Mc . vannielii cell extracts for 2 h at 100,000 x g . When anaerobic reducing conditions were maintained during purification, gpmcrC co-sedimented through sucrose gradients to the same position as molecules of the methyl coenzyme M reductase holoenzyme (approx . 300 kDa) . This co-sedimentation was lost under aerobic, nonreducing conditions. Eur J Biochem, 1993 Jul 15, 215(2), 481 - 6 Refolding and single-step purification of porcine interferon-gamma from Escherichia coli inclusion bodies . Conditions for reconstitution of dimeric IFN-gamma; Vandenbroeck K et al.; Recombinant porcine interferon-gamma, overexpressed in Escherichia coli, was found to accumulate in cytoplasmic inclusion bodies . The influence of various physicochemical parameters on refolding was investigated using 6 M guanidine/HCl-solubilised inclusion bodies which had been purified by ultracentrifugation on a sucrose step gradient . It appeared that the yield of reconstitution of denatured protein reached 60-70% under optimum conditions, i.e . at an intermediary guanidine/HCl concentration of 0.5 M and at a protein concentration of 10-20 microM (0 degrees C) . Since intermediary guanidine/HCl concentrations at 0.5-1.65 M increasingly promoted off-pathway formation of soluble aggregates and at 0.5-0.2 M progressively promoted precipitation, maximal recovery of biologically active protein required a twofold transition in the surrounding guanidine/HCl concentration (6 M-->0.5 M-->0 M) . A single additional size-exclusion chromatographic step yielded a final product that was > 99.5% pure, had specific antiviral activity > 10(7) U/mg protein and contained < or = 25 pg/ml endotoxin . Cross-linking by means of disulfosuccinimidyl tartarate revealed that the refolded protein possessed a dimeric structure . Furthermore, we have characterized three different molecular species of recombinant porcine interferon-gamma that are formed under non-optimal refolding conditions (1 M guanidine/HCl) and that differ from each other in specific activity, size and stability . One of these converts irreversibly into dimeric interferon-gamma in a temperature-dependent manner and is therefore considered as a productive folding intermediate. Proc Natl Acad Sci U S A, 1993 Jul 15, 90(14), 6776 - 80 An unusual RNA tertiary interaction has a role for the specific aminoacylation of a transfer RNA; Hou YM et al.; The nucleotides in a tRNA that specifically interact with the cognate aminoacyl-tRNA synthetase have been found largely located in the helical stems, the anticodon, or the discriminator base, where they vary from one tRNA to another . The conserved and semiconserved nucleotides that are responsible for the tRNA tertiary structure have been shown to have little role in synthetase recognition . Here we report that aminoacylation of Escherichia coli tRNA(Cys) depends on the anticodon, the discriminator base, and a tertiary interaction between the semiconserved nucleotides at positions 15 and 48 . While all other tRNAs contain a purine at position 15 and a complementary pyrimidine at position 48 that establish the tertiary interaction known as the Levitt pair, E . coli tRNA(Cys) has guanosine -15 and -48 . Replacement of guanosine -15 or -48 with cytidine virtually eliminates aminoacylation . Structural analyses with chemical probes suggest that guanosine -15 and -48 interact through hydrogen bonds between the exocyclic N-2 and ring N-3 to stabilize the joining of the two long helical stems of the tRNA . This tertiary interaction is different from the traditional base pairing scheme in the Levitt pair, where hydrogen bonds would form between N-1 and O-6 . Our results provide evidence for a role of RNA tertiary structure in synthetase recognition. Proc Natl Acad Sci U S A, 1993 Jul 15, 90(14), 6646 - 50 Homologous recognition promoted by RecA protein via non-Watson-Crick bonds between identical DNA strands; Rao BJ et al.; The RecA protein of Escherichia coli forms a nucleoprotein filament that promotes homologous recognition and subsequent strand exchange between a single strand and duplex DNA via a three-stranded intermediate . Recognition of homology within three-stranded nucleoprotein complexes, which is probably central to genetic recombination, is not well understood as compared with the mutual recognition of complementary single strands by Watson-Crick base pairing . Using oligonucleotides, we examined the determinants of homologous recognition within RecA nucleoprotein filaments . Filaments that contained a single strand of DNA recognized homology not only in a complementary oligonucleotide but also in an identical oligonucleotide, whether their respective sugar-phosphate backbones were antiparallel or parallel, and a filament that contained duplex DNA showed the same polymorphic versatility in the recognition of homology . Recognition of self by a filament that contains a single strand reveals that RecA filaments can recognize homology via non-Watson-Crick hydrogen bonds . Recognition of multiple forms of the same sequence by duplex DNA in the filament shows that it primarily senses base-sequence homology, and suggests that recognition can be accomplished prior to the establishment of new Watson-Crick base pairs in heteroduplex products . However, unlike the initial recognition of homology, strand exchange is stereospecific, requiring the proper antiparallel orientation of complementary strands. Proc Natl Acad Sci U S A, 1993 Jul 15, 90(14), 6606 - 10 Transcriptional arrest of yeast RNA polymerase II by Escherichia coli rho protein in vitro; Wu SY et al.; A promoter-independent assay utilizing poly(dC)-tailed DNA templates has revealed that Saccharomyces cerevisiae whole-cell extracts can be proficient for transcription by the endogenous yeast RNA polymerase II as well as for correct 3'-end RNA processing . Our attempts to examine the fate of polymerase II itself were inconclusive, because only trace transcription products corresponded to the expected size of terminated RNA species . Transcription in our processing-proficient extract was thus insufficient to cause termination . To test our system with a known, albeit heterologous, signal, we examined a dC-tailed template carrying the E . coli rho-dependent termination signal trp t' in the yeast extract . Transcripts from this template were not susceptible to processing, but addition of rho protein resulted in two distinct truncated transcripts that could not be chased by excess unlabeled nucleotides . These RNA species thus represented stably paused or terminated polymerase II products, and their absence when a mutated unresponsive trp t' template was used affirmed that they were due to the effects of rho . E . coli RNA polymerase added to a yeast extract pretreated with alpha-amanitin was also halted by rho at these same two sites . A mutated rho protein, while only partly defective with E . coli polymerase, failed to provoke arrest when transcription was carried out by RNA polymerase II . Thus, functional rho and its cognate site, trp t', appear necessary and sufficient to elicit the production of truncated transcripts by RNA polymerase II in a yeast whole-cell extract . The ability of rho to halt the eukaryotic enzyme strengthens the likelihood that a rho-like helicase may be involved in RNA polymerase II transcription termination. Proc Natl Acad Sci U S A, 1993 Jul 15, 90(14), 6542 - 6 Transmutation of a heme protein; Barker PD et al.; Residue Asn57 of bovine liver cytochrome b5 has been replaced with a cysteine residue, and the resulting variant has been isolated from recombinant Escherichia coli as a mixture of four major species: A, BI, BII, and C . A combination of electronic spectroscopy, 1H NMR spectroscopy, resonance Raman spectroscopy, electrospray mass spectrometry, and direct electrochemistry has been used to characterize these four major cytochrome derivatives . The red form A (E(m) = -19 mV) is found to possess a heme group bound covalently through a thioether linkage involving Cys57 and the alpha carbon of the heme 4-vinyl group . Form BI has a covalently bound heme group coupled through a thioether linkage involving the beta carbon of the heme 4-vinyl group . Form BII is similar to BI except that the sulfur involved in the thioether linkage is oxidized to a sulfoxide . The green form C (E(m) = 175 mV) possesses a noncovalently bound prosthetic group with spectroscopic properties characteristic of a chlorin . A mechanism is proposed for the generation of these derivatives, and the implications of these observations for the biosynthesis of cytochrome c and naturally occurring chlorin prosthetic groups are discussed. Proc Natl Acad Sci U S A, 1993 Jul 15, 90(14), 6498 - 502 A general two-metal-ion mechanism for catalytic RNA; Steitz TA et al.; A mechanism is proposed for the RNA-catalyzed reactions involved in RNA splicing and RNase P hydrolysis of precursor tRNA . The mechanism postulates that chemical catalysis is facilitated by two divalent metal ions 3.9 A apart, as in phosphoryl transfer reactions catalyzed by protein enzymes, such as the 3',5'-exonuclease of Escherichia coli DNA polymerase I . One metal ion activates the attacking water or sugar hydroxyl, while the other coordinates and stabilizes the oxyanion leaving group . Both ions act as Lewis acids and stabilize the expected pentacovalent transition state . The symmetry of a two-metal-ion catalytic site fits well with the known reaction pathway of group I self-splicing introns and can also be reconciled with emerging data on group II self-splicing introns, the spliceosome, and RNase P . The role of the RNA is to position the two catalytic metal ions and properly orient the substrates via three specific binding sites. Proc Natl Acad Sci U S A, 1993 Jul 15, 90(14), 6493 - 7 Kinetic control of Ca(II) signaling: tuning the ion dissociation rates of EF-hand Ca(II) binding sites; Renner M et al.; EF-hand Ca(II) binding sites share a conserved architecture and are prevalent in Ca(II) signaling pathways . The ion binding kinetics of these sites are carefully tuned to provide the physiologically appropriate activation and inactivation time scales . Here we examine kinetic tuning by the side chain at the ninth position of the EF-loop . A model is proposed in which both the size and charge of the side chain contribute to kinetic tuning . To test this model, the ninth loop position of the EF-hand-like site in the Escherichia coli D-galactose binding protein has been engineered and the Tb(III) dissociation kinetics of the resulting sites have been analyzed . Substitutions at this position are observed to generate up to 10(4)-fold changes in Tb(III) dissociation rates, with little effect on Tb(III) affinity . Furthermore, the observed pattern of rate changes confirm the model's predictions; long side chains at the ninth loop position yield slow dissociation kinetics as predicted for a steric block, whereas acidic side chains yield slow dissociation kinetics as expected for an electrostatic barrier. Proc Natl Acad Sci U S A, 1993 Jul 15, 90(14), 6468 - 72 O6-methylguanine-DNA methyltransferase protects against nitrosamine-induced hepatocarcinogenesis; Nakatsuru Y et al.; We previously generated transgenic C3H/HeN mice by introducing the Escherichia coli O6-methylguanine-DNA methyltransferase (MGMT, DNA-O6-methylguanine:protein-L-cysteine S-methyltransferase, EC2.1.1.63) gene, ada, attached to the Chinese hamster metallothionein I gene promoter . One transgenic mouse line expressing both ada-specific mRNA and Ada protein could be propagated over many generations in a homozygous state with respect to the integrated DNA . Liver extracts from transgenic homozygous mice have consistently demonstrated about 3 times the control activity of normal mice . Furthermore, in the transgenic homozygotes treated with ZnSO4, activity is increased to 6-8 times the normal level in mice and is equivalent to that for man . To examine whether these increased levels of MGMT activity can actually decrease the susceptibility of animals to N-nitroso compounds, we studied liver carcinogenesis in our transgenic mice expressing high amounts of MGMT . Groups of transgenic and nontransgenic mice, each comprising about 200 suckling animals (14 +/- 1 days old), were divided each into eight subgroups, providing paired groups of transgenic and nontransgenic mice . They received an i.p . injection of ZnSO4 to induce MGMT, and 10 hr thereafter were given an i.p . injection of either dimethylnitrosamine or diethylnitrosamine . Liver tumor development was quantitatively assessed at 7-11 months . Here, we report statistically significant reduction of tumor formation in transgenic mice of four of the six paired groups that received treatment . The remaining two demonstrated results in line with dose dependence . Therefore, our data indicate that MGMT can indeed protect animals from low-dose exposure to environmental alkylating carcinogens. Proc Natl Acad Sci U S A, 1993 Jul 15, 90(14), 6444 - 8 "Diabodies": small bivalent and bispecific antibody fragments; Holliger P et al.; Bivalent and bispecific antibodies and their fragments have immense potential for practical application . Here we describe the design of small antibody fragments with two antigen-binding sites . The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) on the same polypeptide chain (VH-VL) . By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites . As indicated by a computer graphic model of the dimers, the two pairs of domains can pack together with the antigen-binding sites pointing in opposite directions . The dimeric antibody fragments, or "diabodies," can be designed for bivalent or bispecific interactions . Starting from the monoclonal antibodies NQ11.7.22 (NQ11) and D1.3 directed against the hapten phenyloxazolone and hen egg lysozyme, respectively, we built bivalent fragments (VHNQ11-VLNQ11)2 and (VHD1.3-VLD1.3)2 and bispecific fragments VHNQ11-VLD1.3 and VHD1.3-VLNQ11 . The fragments were expressed by secretion from bacteria and shown to bind specifically to the hapten and/or antigen . Those with 5- and 15-residue linkers had similar binding affinities to the parent antibodies, but a fragment with the VH domain joined directly to the VL domain was found to have slower dissociation kinetics and an improved affinity for hapten . Diabodies offer a ready means of constructing small bivalent and bispecific antibody fragments in bacteria. Gene, 1993 Jul 15, 129(1), 27 - 32 Evidence for a fourteen-gene, phnC to phnP locus for phosphonate metabolism in Escherichia coli; Metcalf WW et al.; The Escherichia coli phn (psiD) locus consists of a large gene cluster encoding proteins necessary for the use of phosphonates (Pn) as a sole phosphorus source . On the basis of nucleotide (nt) sequence analysis, the phn locus contains a 12.6-kb operon of seventeen genes named, in alphabetical order, phnA to phnQ {Chen et al., J . Biol . Chem . 265 (1990) 4461-4471} . New Pn+ plasmids were made which are suitable for mutational analysis of this gene cluster . These plasmids contain the R6K origin for DNA replication, can be conjugatively transferred, contain the tetAR genes, and therefore provide a way for allele replacement . The construction of these plasmids showed that phnA and phnB have no role in Pn metabolism . Also, these plasmids were employed to introduce nonpolar phnD::lacZ and phnD::uidA fusions into the chromosome, which allowed us to show that phnD probably has a role in transport . In addition, it was shown that phnP is the most distal gene required for Pn use . This was done by testing the effect of phn::uidA insertions in or near the 3' end of phnP on Pn use . Altogether, these results show that all genes required for Pn use are in the 10.9-kb, fourteen-gene, phnCDEFGHIJKLMNOP locus. Gene, 1993 Jul 15, 129(1), 153 - 4 The cleavage sites and localization of genes encoding the restriction endonucleases Eco1831I and EcoHI; Kravetz AN et al.; The restriction endonucleases Eco1831I and EcoHI cleave before the first 5'-cytosine in the recognition sequence 5'-decreases CCSGG--3'/3'--GGSCC increases-5' (where S = G or C), generate 5-base 5' cohesive ends, and are encoded by homologous plasmids that are restricted in McrA+ hosts . Thus, they differ in their cleavage specificity from that of the BcnI isoschizomer, which cleaves after the second 5' cytosine. Gene, 1993 Jul 15, 129(1), 135 - 9 Measurement of recombination frequencies between two homologous DNA segments embedded in a YAC vector; Yasui H et al.; We measured the frequencies of recombination in a yeast host between two homologous segments of DNA that had been inserted with the same polarity in a yeast artificial chromosome (YAC) vector . Three kinds of YAC clones were constructed in which the gene encoding neomycin(Nm) resistance was sandwiched between two homologous segments of DNA, such as the IS3 elements of Escherichia coli or human Alu sequences . Frequencies of homologous recombination in yeast were measured in terms of loss of resistance to Nm . In the case of IS3 fragments, homologous recombination between them did occur at a relatively high frequency (5 x 10(-4) . In contrast, recombination between two Alu sequences did not occur at a detectable level during a 30-day incubation . Thus, the frequency was less than 10(-5) . These results indicate that the Alu sequences do not sufficiently promote the frequency of recombination between two homologous fragments in yeast as to induce rearrangements of DNA in a substantial fraction of YAC clones in libraries. Biochem Biophys Res Commun, 1993 Jul 15, 194(1), 483 - 9 The effect of depletion of nucleotide and of delta and epsilon subunits on ATP synthesis in dimethyl sulfoxide by F1-ATPase of Escherichia coli; Beharry S et al.; The F1-ATPase of Escherichia coli contains 3 mol bound adenine nucleotide/mol F1 . It is of the F1{2,1} type based on the ability of GTP to displace 1 mol adenine nucleotide/mol F1 (Kironde, F.A.S . and Cross, R.L . (1986) J . Biol . Chem . 261, 12544-12549) . A portion of the adenine nucleotide (2 mol/mol F1) is not displaceable by GTP . F1{2,1} was converted to F1{1,0} . This form of the enzyme synthesized ATP from endogenous ADP and inorganic phosphate in a medium containing 30% (v/v) dimethyl sulfoxide (Me2SO) . A delta,epsilon subunit-depleted form of the F1-ATPase was shown to be predominantly in the F1{0,1} form . ATP was not synthesized from endogenous ADP by the subunit-depleted enzyme in Me2SO unless additional molecules of ADP were bound . It is concluded that ATP synthesis from endogenous ADP in Me2SO occurs at GTP-nondisplaceable adenine nucleotide binding sites. J Biol Chem, 1993 Jul 15, 268(20), 15285 - 90 Structure-function analysis of interleukin-6 utilizing human/murine chimeric molecules . Involvement of two separate domains in receptor binding; van Dam M et al.; As an approach to understanding the interaction of interleukin-6 (IL-6) and its 80-kDa receptor (gp80), we have constructed chimeric human/murine IL-6-molecules, which were expressed in Escherichia coli and analyzed for biological activity and receptor binding . This experimental strategy was based on the observation that human IL-6 acts on human and murine cells, whereas murine IL-6 stimulates only murine cells . The regions to be exchanged were chosen according to the four antiparallel helix model of the hematopoietic cytokine family . All 14 chimeras constructed showed biological activity on murine cells . From the differential biological activities on human cells we deduced that three out of four domains of IL-6 are involved in species specificity, whereas only two domains are necessary for specific recognition by the gp80 IL-6-receptor protein. J Biol Chem, 1993 Jul 15, 268(20), 15200 - 4 Structure/function studies of human beta-cell glucokinase . Enzymatic properties of a sequence polymorphism, mutations associated with diabetes, and other site-directed mutants; Takeda J et al.; Glucokinase plays a key role in the regulation of glucose metabolism in insulin-secreting pancreatic beta-cells and in the liver . Recent studies have shown that mutations in this enzyme can lead to the development of a form of non-insulin-dependent diabetes mellitus that is characterized by an autosomal dominant mode of inheritance and onset during childhood . Here, we report the catalytic properties of five additional missense mutations associated with diabetes (Glu70-->Lys, Ser131-->Pro, Ala188-->Thr, Trp257-->Arg and Lys414-->Glu), one polymorphism present in both normal and diabetic subjects (Asp4-->Asn), and three site-directed mutations (Glu177-->Lys, Glu256-->Ala, and Lys414-->Ala) . The Trp257-->Arg mutation generated an enzyme that had an activity that was less than 0.5% of that for native human beta-cell glucokinase . By contrast, the Glu70-->Lys, Ser131-->Pro, Ala188-->Thr, and Lys414-->Glu mutations had a Vmax that was 20-100% of normal but a Km for glucose that was 8-14-fold greater than the native enzyme . There was no effect of the Asp4-->Asn polymorphism or the Glu177-->Lys substitution on glucokinase activity . The Lys414-->Ala substitution had no effect on Vmax but increased the Km for glucose 2-fold and the Glu256-->Ala substitution caused a approximately 200-fold decrease in Vmax . These studies have led to the identification of additional residues involved in glucokinase catalysis and substrate binding. J Biol Chem, 1993 Jul 15, 268(20), 15033 - 8 Structure of mouse Mx1 protein . Molecular assembly and GTP-dependent conformational change; Nakayama M et al.; Mouse Mx1 protein is an interferon-inducible nuclear protein and confers resistance to influenza virus infection . The Mx1 protein purified from interferon-induced A2G mouse liver exhibited GTPase activity as did the Mx1 protein purified from the Mx1 cDNA-expressing Escherichia coli (Nakayama, M., Nagata, K., Kato, A., and Ishihama, A . (1991) J . Biol . Chem . 266, 21404-21408; Nakayama, M., Nagata, K., and Ishihama, A . (1992) Virus Res . 22, 227-234) . The Mx1 protein purified from both mouse liver and Mx1-cDNA expressing E . coli was found to exist as assembled polymeric states judged from gel filtration pattern . By making a set of deletion derivatives of the Mx1 cDNA, the main motif for self-assembly of the Mx1 protein was mapped between amino acid residues 51-99 . This motif is highly conserved not only in the Mx family of proteins but also in Mx-related proteins . The polymeric form of Mx1 from E . coli was observed as "horseshoe"-like structure by negative staining microscopy . When the Mx1 protein was incubated with GTP, this horseshoe structure was transformed to larger and tightly stacked helical forms . Electron microscopic analysis of immunostained liver of the interferon-induced mice indicated that the Mx1 protein exists in nuclei, forming giant complexes of about half the size of nucleoli. J Biol Chem, 1993 Jul 15, 268(20), 15004 - 16 A reverse DNA strand exchange mediated by recA protein and exonuclease I . The generation of apparent DNA strand breaks by recA protein is explained; Bedale WA et al.; The combined action of exonuclease I and recA protein leads to a kind of reverse DNA strand exchange in which joint molecules formed on the "wrong" or distal end of a linear duplex in the presence of ATP are stabilized by exonuclease I degradation of the displaced (+) strand . Continued pairing and degradation of the displaced strand leads to strand exchange that appears to progress with a polarity opposite that of the normal recA protein promoted reaction (i.e . 3'-5' with respect to the (+) strand) . However, in contrast to the normal 5'-3' strand exchange, the displaced strand is completely degraded in the process . When the linear duplex DNA substrate has a heterologous region at the 5' (proximal) end, the major product (described in a previous study (Bedale, W . A., Inman, R . B., and Cox, M . M . (1991) J . Biol . Chem . 266, 6499-6510)) is a circular duplex DNA molecule with a double-stranded tail whose length corresponds closely to the heterologous segment of the substrate . The origin of this product is here shown to be the result of the exonuclease activity of exonuclease I (either added exogenously or present as a trace contaminant of recA protein or SSB protein preparations), as opposed to endonucleolytic or mechanical breakage . The levels of exonuclease I required to generate these products are sufficiently low that they are undetected by assays for exonuclease contamination in recA protein preparations . These results demonstrate that the interplay of recA protein with other enzymes can have a profound effect on both the mechanism and outcome of recA protein-promoted DNA strand exchange . They also demonstrate that the (+) strand of the duplex DNA substrate is at least transiently displaced in recA protein-mediated pairing even when joint molecules are limited to the distal end. J Biol Chem, 1993 Jul 15, 268(20), 14921 - 31 A single amino acid switch within the "hinge" region of the tryptophan synthase beta subunit of Escherichia coli that leads to diminished association with alpha subunit and arrested conversion of ESII to product; Zhao GP et al.; The trpB8 mutation of Escherichia coli causes a major conformational change within the beta subunit of tryptophan synthase . The basis of this effect is a replacement of glycine 281 by arginine within a structurally important "hinge" region . The mutant subunit, beta(B8), is catalytically active only under certain conditions, both in vivo and in vitro . Physiologically, the availability of wild type alpha subunit is the most important determinant of catalytic proficiency (Zhao, G.-P., and Somerville, R . L . (1992) J . Biol . Chem . 267, 526-541; Zhao, G.-P., and Somerville, R . L . (1993) J . Biol . Chem . 268, 14912-14920) . Through enzyme activity titration experiments it was shown that the alpha subunit of tryptophan synthase dramatically stimulates catalysis by the beta 2(B8) mutant enzyme . However, by size exclusion high performance liquid chromatography, the stability of the alpha.beta 2(B8) complex was markedly reduced in comparison with wild type . The alpha-mediated stimulation of catalysis by the beta 2(B8) mutant enzyme was enhanced by polyethylene glycol, a volume excluder . By absorption spectroscopy, it was shown that catalysis by the beta(B8) mutant protein is blocked in at least one step after the formation of a particular Schiff base intermediate (ESII) . Either the alpha subunit or ammonium ion was able to overcome this block . The microenvironment of the ESII catalytic intermediate was examined by fluorescence spectroscopy . The data are consistent with a less hydrophobic environment for ESII in the beta 2(B8) mutant protein than in the wild type protein . These lines of evidence not only support a conformational switch model of open versus closed states within the beta subunit during the catalytic cycle but also suggest a functional role for the hinge region in the process of conformational switching. J Biol Chem, 1993 Jul 15, 268(20), 14912 - 20 An amino acid switch (Gly281-->Arg) within the "hinge" region of the tryptophan synthase beta subunit creates a novel cleavage site for the OmpT protease and selectively diminishes affinity toward a specific monoclonal antibody; Zhao GP et al.; The in vitro susceptibility to endogenous proteases of the beta subunit of Escherichia coli tryptophan synthase was studied immunochemically . Whereas the wild-type beta subunit was apparently very stable, the missense mutant beta(B8), carrying an amino acid switch from Gly to Arg at residue 281, underwent specific proteolytic cleavage . Polyclonal chicken antibodies and monoclonal antibodies specific for the N terminus (monoclonal antibody (mAb) 15-1), the C terminus (mAb 93-6), and the "hinge" region (mAb 164-2) were used to study the hydrolysis of the beta(B8) polypeptide . Cleavage products of 30 kDa, from the N terminus, and 13 kDa, from the C terminus, were observed . These two polypeptides correspond to the well characterized F1 (N-terminal) and F2 (C-terminal) fragments that are generated during the limited tryptic proteolysis of the wild-type beta subunit . The outer membrane-associated protease OmpT was shown to be responsible for the cleavage of the beta(B8) mutant protein . Proteolytic cleavage, observed only under neutral non-denaturing conditions, was specific for the peptide bond between Arg281 and Met282 . The Arg-Met peptide bond has not previously been reported to be susceptible to cleavage by the OmpT protease . The beta(B8) polypeptide had dramatically reduced affinity for mAb 164-2 . This antibody interacted more strongly with the OmpT-generated F1-like fragment than with the intact beta(B8) protein . These results strongly suggest that the G281R mutation alters the conformation of the hinge region of the mutant beta subunit, particularly the beta-turn around Gly281 . The implications with respect to the epitope recognized by mAb 164-2 are discussed. J Biol Chem, 1993 Jul 15, 268(20), 14820 - 5 Rifampicin region revisited . New rifampicin-resistant and streptolydigin-resistant mutants in the beta subunit of Escherichia coli RNA polymerase; Severinov K et al.; Mutations to rifampicin resistance (RifR) in Escherichia coli alter the beta subunit of RNA polymerase (RNAP) . A series of new point RifR mutations was isolated with the aid of random polymerase chain reaction-mediated mutagenesis followed by selection on rifampicin (Rif) . All of the new RifR mutants, including changes in two novel positions, fell into the two principal clusters previously identified in the middle section of beta . Disruption of the spacer between the two clusters with deletions and insertions led to RNAP that was functional and sensitive to Rif in vitro, indicating that the spacer is not directly involved in Rif binding . However, most of the spacer mutants were strongly resistant to streptolydigin, suggesting that this area is involved in the binding of streptolydigin . An insertion of six consecutive histidines into the spacer was constructed that could be used to anchor RNAP on a Nichelate matrix without the loss of enzymatic activity, indicating that this region is looped out of the RNAP molecule. J Biol Chem, 1993 Jul 15, 268(20), 14794 - 8 Analysis of heterodimer formation by the Escherichia coli trp repressor; Hurlburt BK et al.; The trp repressor of Escherichia coli is a dimeric DNA-binding protein that regulates transcription of several operons concerned with tryptophan metabolism . Although heterodimer formation between mutant and wild type subunits occurs readily in vivo, comparable heterodimers could be formed in vitro only under extreme conditions . To explain this difference we analyzed trp repressor dimer formation and dissociation using an in vitro transcription/translation system . Nascent wild type or mutant repressor polypeptides, synthesized in the presence of an excess of a second repressor, were invariably incorporated into heterodimers . In contrast, previously synthesized and assembled wild type dimers appeared to be refractory to dissociation, since they did not form heterodimers . However, previously synthesized mutant dimeric repressors that were defective in tryptophan binding readily dissociated and formed heterodimers . We noted that the ability of a dimeric repressor to dissociate under our conditions correlated inversely with its affinity for tryptophan . Consistent with this conclusion, we found that dissociation of the wild type aporepressor (no added tryptophan) was appreciably more rapid than dissociation of the tryptophan-saturated wild type repressor. J Biol Chem, 1993 Jul 15, 268(20), 14788 - 93 Functional characterization of recombinant human red cell alpha-spectrin polypeptides containing the tetramer binding site; Kotula L et al.; Spectrin, a heterodimer composed of alpha and beta subunits, interacts with itself head-to-head to form tetramers in the erythrocyte membrane cytoskeleton . The NH2-terminal region of alpha-spectrin, encompassing the alpha I 80-kDa domain, was expressed in Escherichia coli . In addition to the correctly initiated polypeptide, four smaller polypeptides were produced by initiation at internal codons . Only the full-length polypeptide was able to bind to spectrin dimers, beta monomers, and to a recombinant polypeptide containing the COOH terminus of beta-spectrin . The head-to-head interaction with beta-spectrin was also retained by a recombinant polypeptide containing the NH2-terminal 158 amino acids of the alpha subunit . Deletion of the first 27 or 49 NH2-terminal amino acids abolished binding of this polypeptide to the beta monomer . The phasing used to design these recombinant polypeptides was based on a conformational model recently refined by Speicher et al . (Speicher, D . W., DeSilva, T . M., Speicher, K . D., Ursitti, J . A., Hembach, P., and Weglarz, L . (1993) J . Biol . Chem . 268, 4227-4235), where the structural unit begins and terminates around residue 30 of the repeat unit . The binding properties, mobility on gel filtration, and circular dichroism data of the recombinant polypeptides indicated that most polypeptides were able to assume their native conformation. J Biol Chem, 1993 Jul 15, 268(20), 14732 - 42 The role and properties of the iron-sulfur cluster in Escherichia coli dihydroxy-acid dehydratase; Flint DH et al.; Dihydroxy-acid dehydratase has been purified from Escherichia coli and characterized as a homodimer with a subunit molecular weight of 66,000 . The combination of UV visible absorption, EPR, magnetic circular dichroism, and resonance Raman spectroscopies indicates that the native enzyme contains a {4Fe-4S}2+,+ cluster, in contrast to spinach dihydroxy-acid dehydratase which contains a {2Fe-2S}2+,+ cluster (Flint, D . H., and Emptage, M . H . (1988) J . Biol . Chem . 263, 3558-3564) . In frozen solution, the reduced {4Fe-4S}+ cluster has a S = 3/2 ground state with minor contributions from forms with S = 1/2 and possibly S = 5/2 ground states . Resonance Raman studies of the {4Fe-4S}2+ cluster in E . coli dihydroxy-acid dehydratase indicate non-cysteinyl coordination of a specific iron, which suggests that it is likely to be directly involved in catalysis as is the case with aconitase (Emptage, M . H., Kent, T . A., Kennedy, M . C., Beinert, H., and Munck, E . (1983) Proc . Natl . Acad . Sci . U.S.A . 80, 4674-4678) . Dihydroxy-acid dehydratase from E . coli is inactivated by O2 in vitro and in vivo as a result of oxidative degradation of the {4Fe-4S}cluster . Compared to aconitase, the oxidized cluster of E . coli dihydroxy-acid dehydratase appears to be less stable as either a cubic or linear {3Fe-4S} cluster or a {2Fe-2S} cluster . Oxidative degradation appears to lead to a complete breakdown of the Fe-S cluster, and the resulting protein cannot be reactivated with Fe2+ and thiol reducing agents. J Biol Chem, 1993 Jul 15, 268(20), 14687 - 93 Characterization of rabbit skeletal muscle glycogenin . Tyrosine 194 is essential for function; Cao Y et al.; The biogenesis of glycogen involves a specific initiation event mediated by the initiator protein, glycogenin, which undergoes self-glucosylation to generate an oligosaccharide primer from which the glycogen molecule grows . Rabbit muscle glycogenin was expressed at high levels in Escherichia coli and purified close to homogeneity in a procedure that involved binding to a UDP-agarose affinity column . The resulting protein had subunit molecular weight of 38,000 as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate . Analysis of peptide fragments by mass spectroscopy indicated that the recombinant glycogenin was already glucosylated at Tyr-194 and contained from 1 to 8 glucose residues attached . The enzyme was active as a glucosyl transferase and could incorporate a further approximately 5 mol of glucose/mol . The apparent Km for the glucosyl donor UDP-glucose was 4.5 microM, and the pH optimum was pH 8 . Of a number of nucleotides and related compounds surveyed, UDP and UTP were the most effective inhibitors . There was also a correlation between inhibition and the presence of a pyrophosphate group . Of several oligosaccharides of glucose, only maltose caused significant inhibition . The glucosylation reaction was first order with respect to glycogenin suggesting that it was intramolecular . The efficacy of the purified glycogenin as a substrate for the elongation reaction catalyzed by glycogen synthase was significantly enhanced if glycogenin was first allowed to undergo self-glucosylation . The length of the priming oligosaccharide is thus important for glycogen synthase action . A mutant of glycogenin, in which Tyr-194 was changed to Phe, behaved identically to the wild-type through purification and in particular bound to the UDP-agarose affinity matrix . Despite these indications of the protein's overall structural integrity, it was unable to self-glucosylate . This result indicates that Tyr-194 is necessary for glycogenin function and is consistent with Tyr-194 being the sole site of glucosylation. Nature, 1993 Jul 15, 364(6434), 249 - 52 Protein kinase C alpha activates RAF-1 by direct phosphorylation; Kolch W et al.; The kinase Raf-1 can be activated by treatment of cells with mitogens and by the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (reviewed in refs 1,2) . Activated Raf-1 triggers a protein kinase cascade by direct phosphorylation of MAP kinase kinase, resulting in phosphorylation of ternary complex factor and Jun by MAP kinase . Here we investigate the molecular mechanism and biological consequences of PKC alpha-mediated Raf-1 activation in NIH3T3 fibroblasts . PKC alpha directly phosphorylates and activates Raf-1 both in vitro and in vivo . PKC alpha induces Raf-1 phosphorylation at several sites, including a serine residue at position 499 . Mutation of serine at this position or at residue 259 does not abrogate Raf-1 stimulation by a combination of Ras plus the src tyrosine kinase Lck, but severely impedes Raf-1 activation by PKC alpha . Consistent with such a direct interaction is the observation that Raf-1 and PKC alpha cooperate in the transformation of NIH3T3 cells . The Ser499 phosphorylation site is necessary for this synergism. Int J Cardiol, 1993 Jul 15, 40(3), 251 - 6 Intracardiac thrombosis diagnosed by echocardiography in childhood: predisposing and etiological factors; Ozkutlu S et al.; Eleven cases of intracardiac thrombi caused by different factors including protein-C deficiency are presented for discussion of the etiology and predisposing factors of intracardiac thrombi during infancy and childhood, and to stress the importance of protein-C deficiency as an etiological factor . Thrombi were localised in the left heart in five patients and right heart in five patients . One patient had both-sided thrombi . Four of our patients had dilated cardiomyopathy, one had mitral valve hypoplasia, and one had pulmonary valvar stenosis as the predisposing factors for thrombus formation . In three patients whose cardiac anatomies were completely normal, we determined protein-C deficiency as an etiological factor of thrombus formation . One of these had congenital protein-C deficiency and the other two had acquired temporary protein-C deficiency due to sepsis . In conclusion we recommend that protein-C deficiency should be investigated as an etiological factor in all cases of intracardiac thrombi irrespective of whether or not another predisposing factor is identified. Eur J Pharmacol, 1993 Jul 15, 246(2), 129 - 33 Functional expression of rat alpha 2B-adrenoceptor in Escherichia coli; Xia Y et al.; Rat alpha 2B-adrenoceptor was expressed in Escherichia coli using 'ATG vector' containing cDNA encoding the 'non-glycosylated rat alpha 2-adrenoceptor' (RNG alpha 2) . The highest receptor binding activity (using the alpha 2-adrenoceptor ligand {3H}MK 912) was found when transfected bacteria cultures were grown at 30 degrees C for about 4 h after induction with isopropyl beta-D-thiogalactopyranoside (IPTG) . Saturation experiments showed that the radioligand bound to a single saturable site with a Kd of 1.42 +/- 0.09 nM and capacity of 281 +/- 6 fmol/mg protein . Binding constants of 14 compounds for the rat alpha 2B-adrenoceptor expressed in E . coli were determined and compared to the values previously obtained for the rat alpha 2B-adrenoceptor when expressed in COS cells as well as for native neonatal rat lung alpha 2B-adrenoceptors . The results indicate that when the rat alpha 2B-adrenoceptor is expressed in E . coli it retains identical ligand binding properties to those found when the receptor is present in the eukaryotic system . Expressing alpha 2B-adrenoceptors in E . coli would, therefore, seem to constitute a valid alternative in, e.g., drug screening and structure analysis of the alpha 2B-adrenoceptors. Nature, 1993 Jul 15, 364(6434), 255 - 8 Characterization of a functionally important mobile domain of GroES; Landry SJ et al.; Although genetic and biochemical evidence has established that GroES is required for the full function of the molecular chaperone, GroEL, little is known about the molecular details of their interaction . GroES enhances the cooperativity of ATP binding and hydrolysis by GroEL (refs 4, 5) and is necessary for release and folding of several GroEL substrates . Here we report that native GroES has a highly mobile and accessible polypeptide loop whose mobility and accessibility are lost upon formation of the GroES/GroEL complex . In addition, lesions present in eight independently isolated mutant groES alleles map in the mobile loop . Studies with synthetic peptides suggest that the loop binds in a hairpin conformation at a site on GroEL that is distinct from the substrate-binding site . Flexibility may be required in the mobile loops on the GroES seven-mer to allow them to bind simultaneously to sites on seven GroEL subunits, which may themselves be able to adopt different arrangements, and thus to modulate allosterically GroEL/substrate affinity. Proc Natl Acad Sci U S A, 1993 Jul 15, 90(14), 6756 - 60 The Escherichia coli pcnB gene promotes adenylylation of antisense RNAI of ColE1-type plasmids in vivo and degradation of RNAI decay intermediates; Xu F et al.; Previous work has shown that RNase E-mediated cleavage of RNAI, an antisense repressor of the replication of ColE1-type plasmids, relieves repression in vivo by endonucleolytically converting RNAI to a rapidly decaying product . We report that mutations in the Escherichia coli pcnB gene result in a 10-fold prolongation of the half-life of RNAI decay intermediates and also of truncated RNAI primary transcripts lacking sites attacked by RNase E . Using Northern blotting, primer extension analysis, {32P}GTP capping of 5'-triphosphate termini, and PCR amplification methods, we show that pcnB-mediated acceleration of RNAI degradation is associated with posttranscriptional 3' addition of adenosine residues in vivo to native and processed forms of RNAI . Accumulation of antisense RNAI decay products in pcnB mutants potentially explains the reduced copy number of ColE1-type plasmids seen in the mutated bacteria. Biochemistry, 1993 Jul 13, 32(27), 7054 - 63 Folding and stability of a tryptophan-containing mutant of ubiquitin; Khorasanizadeh S et al.; To provide a fluorescence probe for equilibrium and kinetic folding studies on ubiquitin, cassette mutagenesis in an Escherichia coli expression plasmid was used to replace the largely buried Phe 45 by a tryptophan . Under native conditions, the tryptophan fluorescence spectrum of this F45W mutant exhibits a blue-shifted emission maximum at 336 nm indicative of a largely solvent-shielded tryptophan environment . In contrast, the unfolded protein in 6 M guanidine hydrochloride (GuHCl) shows a 4-fold more intense emission band at 353 nm matching that of free tryptophan . The two-dimensional 1H NMR spectrum of F45W ubiquitin was assigned by comparison with published assignments of the wild type . The mutation results in only limited chemical shift changes for residues in the immediate vicinity of residue 45 . The structural similarity of F45W with wild-type ubiquitin was confirmed by a preliminary analysis of the nuclear Overhauser spectrum . NMR and circular dichroism measurements of the reversible GuHCl-induced unfolding transition show that the F45W mutation lowers the stability of the folded ubiquitin structure by less than 0.4 kcal/mol . The biological activity of the mutant was found to be indistinguishable from that of wild-type in terms of its reaction with the ubiquitin activating enzyme E1 and an in vitro assay of ATP-dependent protein degradation . The kinetics of folding and unfolding of F45W ubiquitin was studied at two temperatures (8 and 25 degrees C) in a series of fluorescence-detected stopped-flow measurements over a wide range of GuHCl concentrations (0.5-6 M) . The measurements at 25 degrees C are consistent with a two-state model with strongly denaturant-dependent folding and unfolding rates above about 2 M GuHCl . However, at lower denaturant concentrations, the rate of the major folding phase becomes GuHCl-independent, and up to 60% of the total fluorescence change occurs during the 2-ms dead time of the stopped-flow measurement . These observations provide clear evidence for the formation of an early folding intermediate during the first few milliseconds of refolding with a partially developed hydrophobic core involving Trp 45 . The sigmoid denaturant dependence of the initial amplitude with an apparent midpoint of 1.3 M GuHCl suggests the presence of a discrete state that is destabilized at higher denaturant concentrations . In contrast, there is no evidence for an early intermediate in the folding kinetics at 8 degrees C . The destabilization of the intermediate at low temperature is consistent with a collapsed state stabilized primarily by hydrophobic interactions. Biochemistry, 1993 Jul 13, 32(27), 6815 - 20 Escherichia coli rep helicase unwinds DNA by an active mechanism; Amaratunga M et al.; DNA helicases unwind duplex DNA to form the single-stranded (ss) DNA intermediates required for replication, recombination, and repair in reactions that require nucleoside 5'-triphosphate hydrolysis . Helicases generally require a ss-DNA flanking the duplex in order to initiate unwinding in vitro; however, the precise function of the ss-DNA is not understood . If a helicase unwinds DNA by a "passive" mechanism, it would bind to and translocate unidirectionally along the ss-DNA and facilitate duplex unwinding by translocating onto the ss-DNA that is formed transiently by thermal fluctuations in the duplex . We have examined the kinetics of DNA unwinding by Escherichia coli Rep protein (a 3' to 5' helicase) by rapid quench-flow methods using a series of novel, nonnatural DNA substrates possessing 3' flanking ss-DNA within which is embedded either a segment of ss-DNA possessing reversed backbone polarity or a non-DNA {poly(ethylene glycol)} spacer, either of which should block unwinding by a passive helicase . The E . coli Rep helicase effectively unwinds these DNA substrates, ruling out a passive mechanism of unwinding . Instead, the results are consistent with an "active" rolling mechanism during which Rep binds to ss-DNA and duplex DNA simultaneously. Biochemistry, 1993 Jul 13, 32(27), 6951 - 6 Fluorescence study of a temperature-induced conversion from the "loose" to the "tight" binding form of membrane-bound cytochrome b5; Ladokhin AS et al.; Cytochrome b5 is a liver integral membrane protein that has now been expressed in, and isolated from, Escherichia coli . The structure-function relationships of the 43 amino acid membrane-binding domain (nonpolar peptide) have been examined in both native and mutant forms of the protein; in the latter, tryptophan residues at positions 108 and 112 were replaced by leucine . The temperature dependence of the fluorescence quantum yield of the Trp residues in the isolated membrane-binding domain was examined while the domain was bound to lipid vesicles . Both the lipid-bound mutant domain and lipid-bound native domain showed an irreversible increase in fluorescence above 50 degrees C . When the whole cytochrome b5 molecule, bound to lipid vesicles, was heated to this temperature, there was a conversion of the metastable, intermembrane-exchangeable ("loosely" bound), conformation to a final, virtually unexchangeable ("tightly" bound), conformation . It has been suggested previously that the protein exists in a "looped back" conformation and a "bilayer penetrating" conformation . Although the present studies are not designed to determine the absolute conformations of the loose and tight forms, the changes observed in steady-state and frequency-modulated fluorescence and the lack of change in depth of Trp 109 in the bilayer are consistent with a movement of the C-terminal segment from a looped back to a bilayer penetrating conformation as the tight form is generated. FEBS Lett, 1993 Jul 12, 326(1-3), 275 - 80 Cloning, expression and purification of a recombinant poly-histidine-linked HIV-1 protease; Leuthardt A et al.; The gene coding for the HIV-1 protease was cloned in an Escherichia coli expression vector adding three-histidine codons to the amino and carboxy terminus of the protease sequence . Expression of the protease from this construct led to the accumulation of high amounts of insoluble histidine-linked protease entrapped in inclusion bodies . The histidine-linked protease could be efficiently released from purified inclusion bodies with 6 M guanidine hydrochloride and further purified by metal chelate affinity chromatography . The refolded protease cleaved synthetic peptide substrates and the viral polyprotein p55 with the same specificity as the wild type protease . It displays a specific activity of 4.4 mumol/min/mg. FEBS Lett, 1993 Jul 12, 326(1-3), 192 - 8 The nuclear-encoded polypeptide Cfo-II from spinach is a real, ninth subunit of chloroplast ATP synthase; Herrmann RG et al.; Proton-translocating F-ATP synthases from chloroplasts contain a nuclear-coded subunit, CFo-II, that lacks an equivalent in the corresponding E . coli complex . Three recombinant phages that code for the entire precursor of this subunit have been isolated from lambda gt11 cDNA expression libraries made from polyadenylated spinach RNA using a two-step strategy . The reading frame of 222 amino acid residues includes 147 residues for the mature protein (M(r) 16.5 kDa) and a transit sequence of 75 residues (M(r) 8.0 kDa) . Secondary structure predictions indicate a bitopic protein, anchored by a single N-terminal transmembrane segment and a C-terminal hydrophilic region that probably reaches into CF1 . CFo-II precursor made in vitro can be imported into isolated, intact chloroplasts and assembled into ATP synthase . This protein is a real subunit of the plastid enzyme and a distinctive characteristic of ATP synthases involved in photosynthetic processes . Unique features are (i) that the gene for CFo-II (atpG) appears to be a duplication of atpF encoding CFo-I, the homologues of the genes for subunits b' and b in photosynthetic bacteria, (ii) that it represents the first instance that one copy of the various duplicated loci found in plastid chromosomes has been phylogenetically translocated to the nucleus, and (iii) that it operates with a bipartite (import/thylakoid-targeting) transit peptide but without an intermediate cleavage site for the stroma protease, suggestive of a way of membrane integration different from that of its plastome-encoded counterpart CFo-I . With these data, the first complete sequence for a chloroplast ATP synthase of a higher plant (spinach) is available. Nucleic Acids Res, 1993 Jul 11, 21(14), 3281 - 6 Isolation, sequencing and overexpression of the gene encoding the theta subunit of DNA polymerase III holoenzyme; Carter JR et al.; The gene encoding the theta subunit of DNA polymerase III holoenzyme, designated holE, was isolated using a strategy in which peptide sequence was used to derive a DNA hybridization probe . Sequencing of the gene, which maps to 41.43 centisomes of the chromosome, revealed a 76-codon open reading frame predicted to produce a protein of 8,846 Da . When placed in a tac promoter expression vector, the open reading frame directed expression of a protein, that comigrated with authentic theta subunit from purified holoenzyme, to 6% of total soluble protein. Nucleic Acids Res, 1993 Jul 11, 21(14), 3239 - 43 RNase MRP and RNase P share a common substrate; Potuschak T et al.; RNase MRP is a site-specific ribonucleoprotein endoribonuclease that processes RNA from the mammalian mitochondrial displacement loop containing region . RNase P is a site-specific ribonucleoprotein endoribonuclease that processes pre-tRNAs to generate their mature 5'-ends . A similar structure for the RNase P and RNase MRP RNAs and a common cleavage mechanism for RNase MRP and RNase P enzymes have been proposed . Experiments with protein synthesis antibiotics have shown that both RNase MRP and RNase P are inhibited by puromycin . We also show that E . coli RNase P cleaves the RNase MRP substrate, mouse mitochondrial primer RNA, exactly at a site that is cleaved by RNase MRP. Biochim Biophys Acta, 1993 Jul 10, 1164(2), 124 - 32 Hybrid enzymes for structure-function analysis of cytochrome P-450 2B11; Kedzie KM et al.; Previous work has shown that P-450 2B11 is responsible for the unique ability of dogs to metabolize and eliminate certain highly-chlorinated biphenyls such as 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB), whereas the related P-450 2B forms in rat and rabbit are unable to metabolize the compound to any significant degree . To determine the structural basis for this functional diversity, hybrid enzymes were generated . Success with this approach required a careful choice of second enzyme and common substrate with which to assess the functional integrity of the hybrid proteins . The choices of P-450 2B5 from rabbit as the second enzyme and androstenedione as the substrate were based in part on the finding that P-450 2B11 and P-450 2B5 hydroxylate androstenedione with similar overall activities but distinct profiles . Enzymatic studies with eight hybrid enzymes provided evidence for two regions of P-450 2B11 and 2B5, between residues 95-239 and 240-370, that appear to be involved in defining substrate specificity for androstenedione, and three regions of P-450 2B11, between residues 95-239, 240-370, and 371-494, that contain amino acids necessary for metabolism of 245-HCB . This deliberate approach to the creation of hybrid cytochromes P-450 has generated a series of enzymes that will be central to further structure-function studies of the cytochromes P-450 2B. Biochemistry, 1993 Jul 6, 32(26), 6643 - 8 Characterization of recombinant and endogenous ADP-ribosylation factors synthesized in Sf9 insect cells; Kunz BC et al.; ADP-ribosylation factors (ARFs) are a family of highly conserved, 20-kDa guanine nucleotide-binding proteins that participate in protein trafficking and enhance cholera toxin-catalyzed ADP-ribosylation . ARF 2 from bovine retinal cDNA was expressed in Sf9 insect cells using recombinant baculovirus and compared to the major insect cell ARF (Sf9 ARF) and to recombinant ARF 2 expressed in Escherichia coli (E . coli rARF 2) . The 150000g supernatant and particulate fractions of freeze-thawed, recombinant ARF 2 baculovirus-infected cells contained immunoreactive proteins of 20 and 21 kDa at significantly higher levels than were found in uninfected cells . Infected Sf9 cells incorporated {3H}myristate only into the 20-kDa protein . Sf9 cell recombinant ARF 2 (Sf9 rARF 2) and Sf9 ARF were separated by isoelectric focusing or ion-exchange chromatography and identified by microsequencing of HPLC-purified tryptic peptides . Sf9 ARF displayed considerable sequence identity to mammalian class I ARFs . Both Sf9 ARF and Sf9 rARF 2 stimulated in a GTP-dependent manner cholera toxin-catalyzed ADP-ribosylation . The Ka for GTP of Sf9 ARF was, however, significantly lower than that of Sf9 rARF 2 or E . coli rARF 2 . Myristoylation did not significantly affect the ability of ARF 2 to enhance cholera toxin-catalyzed ADP-ribosylation or the Ka for GTP . Despite the sequence identities and the fact that both were synthesized in insect cells, the endogenous Sf9 ARF was functionally different from Sf9 rARF 2. Biochemistry, 1993 Jul 6, 32(26), 6680 - 7 Secondary structure and topology of Acanthamoeba profilin I as determined by heteronuclear nuclear magnetic resonance spectroscopy; Archer SJ et al.; The protein profilin binds to both actin and the head groups of poly)phosphoinositide)s and may regulate both actin assembly and the phosphoinositide signaling pathway . As a first step in understanding the activity of profilin at the molecular level, we have determined the secondary structure of Acanthamoeba profilin I in solution using multidimensional, heteronuclear NMR spectroscopy . Using a combination of triple-resonance (1H, 13C, 15N) experiments, we obtained virtually complete backbone and side-chain resonance assignments based solely on scalar couplings . 3D and 4D NOESY experiments were then used to determine the secondary structure and global fold of Acanthamoeba profilin I . The central feature of the protein structure is a five-stranded antiparallel beta-sheet flanked by three helices and a short two-stranded antiparallel beta-sheet. Biochemistry, 1993 Jul 6, 32(26), 6649 - 55 Determination of the reduction-oxidation potential of the thioredoxin-like domains of protein disulfide-isomerase from the equilibrium with glutathione and thioredoxin; Lundstrom J et al.; Protein disulfide-isomerase (PDI) contains two thioredoxin-like domains with the active-site sequence: Cys-Gly-His-Cys . Reduction of the two active-site disulfides in PDI by NADPH and bovine thioredoxin reductase was not reversible by addition of excess NADP+, consistent with a redox potential (E0') above -200 mV . Redox states of PDI and a mutated Escherichia coli thioredoxin, P34H Trx, were determined by quantitative analysis of cysteine residues by alkylation in equilibrium mixtures of oxidized and reduced forms of the two proteins . From the known E0' of P34H Trx (-235 mV), an E0' value of -190 +/- 10 mV was calculated for PDI . Similarly, with defined redox buffers of glutathione, the redox-active dithiols in PDI were shown to have an equilibrium constant of 3 mM (E0' = -175 +/- 15 mV) . The results showed that PDI has a high redox potential and therefore is a good oxidant of nascent protein thiols . Direct transfer of reducing equivalents from PDI to NADP+ via thioredoxin reductase during protein disulfide formation seems unlikely due to the unfavorable equilibrium . The thioredoxin domains in PDI have a widely different redox potential compared with that of thioredoxin . A Pro to His exchange in the active site contributes to half of the change; the other half remains to be identified in the structure of PDI. Biochemistry, 1993 Jul 6, 32(26), 6523 - 30 Cytosine deamination in mismatched base pairs; Frederico LA et al.; The rate of deamination of cytosine in mismatched base pairs has been determined . Incubation of M13mp2 nicked heteroduplex DNA molecules containing T.C or C.C mispairs in the lacZ alpha-complementation gene results in deamination of cytosine to uracil, producing T.U or C.U mispairs . Strands which have undergone deamination at the target site to produce uracil will yield dark blue plaque revertants, while all other strands yield faint blue or colorless plaque phenotypes upon transfection of an ung- alpha-complementation Escherichia coli host strain . Rate constants were calculated from the reversion frequencies for several different heteroduplexes incubated at either 60 or 37 degrees C . For the 60 degrees C incubations, the hydrolytic deamination rate constants for mispairs in three different local sequence environments ranged from 8 x 10(-10) to 40 x 10(-10) s-1 . For incubations at 37 degrees C, the rate constants were between 0.4 x 10(-10) and 1.3 x 10(-10) sec-1 . At both temperatures and for all mispairs, these rate constants are significantly greater than deamination rate constants in properly matched Watson-Crick G.C base pairs and are similar to those constants determined for cytosine deamination in single-stranded DNA . Since deamination most likely occurs via a single-stranded intermediate, the data suggest that, at 37 degrees C, the T.C and C.C mispairs exhibit from 20% to 100% single-stranded character . We conclude that cytosine residues involved in a mispair in DNA are 1-2 orders of magnitude more prone to deaminate to uracil than are cytosines in double-stranded DNA.(ABSTRACT TRUNCATED AT 250 WORDS) J Immunol Methods, 1993 Jul 6, 163(1), 77 - 83 Production and characterization of monoclonal antibodies raised against recombinant human granzymes A and B and showing cross reactions with the natural proteins; Kummer JA et al.; The human serine proteases granzymes A and B are expressed in cytoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells . Recombinant granzyme A and granzyme B proteins were produced in bacteria, purified and then used to raise specific mouse monoclonal antibodies . Seven monoclonal antibodies (mAb) were raised against granzyme A, which all recognized the same or overlapping epitopes . They reacted specifically in an immunoblot of interleukin-2 (IL-2) stimulated PBMNC with a disulfide-linked homodimer of 43 kDa consisting of 28 kDa subunits . Seven mAb against granzyme B were obtained, which could be divided into two groups, each recognizing a different epitope . On an immunoblot, all mAb reacted with a monomer of 33 kDa protein . By immunohistochemistry, these mAb could be used to detect granzymes A and B expression in activated CTL and NK cells . The availability of these mAb may facilitate studies on the role of human cytotoxic cells in various immune reactions and may contribute to a better understanding of the role of granzymes A and B in the cytotoxic response in vivo. Biochemistry, 1993 Jul 6, 32(26), 6555 - 62 AP sites are not significantly involved in mutagenesis by the (+)-anti diol epoxide of benzo{a}pyrene: the complexity of its mutagenic specificity is likely to arise from adduct conformational polymorphism; Drouin EE et al.; In previous work, mutations induced by the (+)-anti diol epoxide of benzo{a}pyrene {(+)-anti-B{a}PDE} were scored in the supF gene of the Escherichia coli plasmid pUB3 {Rodriguez & Loechler (1993) Biochemistry 32, 1759} . pUB3 was reacted with (+)-anti-B{a}PDE and then either (1) transformed immediately into E . coli or (2) heated at 80 degrees C for 10 min prior to transformation . Heating only released a small fraction of adducts (approximately 5%) and did not significantly affect the mutagenic pattern at most sites in supF . However, at the major base substitution hotspot, G115, principally G-->T mutations (87%) were obtained prior to heating, while after heating, G-->T mutations decreased (45%) and G-->A (21%) and G-->C (33%) mutations became more prevalent . One model for this result is that prior to heating a heat-labile adduct at G115 causes one pattern of mutagenesis, but after heating the labile adduct is hydrolyzed to an apurinic site (AP site), which causes a second mutational pattern . To test this, a role for AP sites generated from labile adducts by heating at 80 degrees C for 10 min is investigated . It is shown that when plasmid pUB3 contains 22 (+)-anti-B{a}PDE adducts, 0.6% (or fewer) are converted to AP sites as determined in an assay based upon the action of an AP-endonuclease . In a separate line of investigation not involving (+)-anti-B{a}PDE adducts, mutation frequency (MF) per AP site is estimated . (In these experiments, AP sites were introduced into pUB3 by the classic procedure of heating at 70 degrees C/pH 5.0 to hydrolyze purines.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1993 Jul 5, 268(19), 14316 - 21 Guanylate kinase of Escherichia coli K-12; Gentry D et al.; We have identified the gene gmk, in the same operon as rpoZ, spoT, and recG at about 82 minutes on the Escherichia coli chromosome . The gmk (GMP kinase) gene encodes a peptide of 23,592 Da, possessing extensive similarity to the amino acid sequence of guanylate kinase from yeast . To confirm that gmk truly encodes guanylate kinase and to explore some of its enzymatic features, we have overproduced the product of gmk and purified it to homogeneity . Unlike guanylate kinases purified from eukaryotic sources, E . coli guanylate kinase is multimeric, and ionic conditions dictate its protomeric state; under low ionic conditions it appears to be a tetramer while under high ionic conditions it is a dimer . Kinetic analysis reveals that guanylate kinase, again, unlike eukaryotic guanylate kinases, binds GMP cooperatively and that the observed cooperatively changes with ionic strength . These results indicate that, despite extensive sequence similarity to its eukaryotic counterparts, E . coli guanylate kinase is structurally and enzymatically different. J Biol Chem, 1993 Jul 5, 268(19), 14056 - 64 Regulation of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase . Role of the NH2-terminal region; Kurland IJ et al.; The role of the NH2-terminal region of the liver and skeletal muscle 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatases was investigated, as well that of a mutant of the liver isoform lacking the first 22 amino acids, by the overexpression of these enzymes in Escherichia coli and the comparison of their kinetic properties . The muscle isoform and the deletion mutant had Km values for fructose 6-phosphate which were 50- and 20-fold higher, respectively, than that of the liver isoform, and the bisphosphatase maximal velocity of the liver deletion mutant was 4-fold higher than that of the native liver isoform . Phosphorylation of the liver isoform increased bisphosphatase activity by 2-3-fold and the Km for fructose 6-phosphate of the 6-phosphofructo-2-kinase by 10-15-fold, but these kinetic effects were greatly diminished for the deletion mutant despite equivalent phosphorylation by cAMP-dependent protein kinase . Arg-173 of the skeletal muscle isoform was found to be functionally equivalent to the residue corresponding to the essential fructose 6-phosphate binding residue of the liver kinase domain, Arg-195 . The results suggest that 1) the NH2-terminal regions of the liver and skeletal muscle isoforms are important determinants of fructose 6-phosphate affinity, and 2) the initial 22 amino acids of the liver isoform exert an inhibitory influence on the bisphosphatase and mediate, at least in part, the response of both activities of the enzyme to cAMP-dependent phosphorylation. J Mol Biol, 1993 Jul 5, 232(1), 23 - 34 Activation of DNA binding by the monomeric form of the P1 replication initiator RepA by heat shock proteins DnaJ and DnaK; DasGupta S et al.; RepA protein of plasmid P1 binds to arrays of 19 bp repeat sequences (iterons) and mediates initiation of replication and its control . Escherichia coli heat shock proteins DnaJ and DnaK can stimulate iteron binding activity of RepA in an ATP-dependent fashion . It has been proposed that RepA binds to DNA as monomers and that the stimulation in binding involves monomerization of RepA dimers which are inactive in the binding reaction . RepA-iteron and RepA-RepA interactions have been measured in this study to determine the equilibrium constants of the two reactions . The apparent KD value for RepA-iteron binding decreased from 10 nM to no more than 0.2 nM at increasing concentrations of the heat shock proteins . The stimulation of binding appears to be due to an increase in active RepA fraction and not to a change in the maximum binding capacity of the active species . This view was deduced from measurements of active RepA fraction, which increased in the presence of heat shock proteins, and from measurements of dissociation rate constants, which were independent of the heat shock protein concentrations . Accounting for the active fractions, the true KD value was estimated to be 0.10(+/- 0.09) nM in 20 mM Tris.HCl (pH 8), 100 mM NaCl, 40 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, 0.1 mM EDTA, ATP (50 microM), bovine serum albumin (50 micrograms/ml), calf thymus DNA (50 micrograms/ml) and glycerol (5%) . The dissociation rate constant was 1.5 x 10(-2) s-1 and the calculated association rate constant was 1.5 x 10(8) M-1 s-1 . Ultracentrifugation analyses of RepA at 15,000 r.p.m . in the above buffer but without ATP, bovine serum albumin, calf thymus DNA and glycerol, revealed that the protein was in monomer-dimer equilibrium with a KD of 2.6(+/- 0.2) microM at 5 degrees C . Therefore, at protein concentrations used in the binding reactions, RepA is monomeric (> 99.5%), in confirmation of the earlier result that RepA binds as a monomer . It follows that the species that is stimulated to bind by the heat shock proteins is also a monomeric form of RepA. J Mol Biol, 1993 Jul 5, 232(1), 213 - 22 Crystal structure of a ternary complex of Escherichia coli malate dehydrogenase citrate and NAD at 1.9 A resolution; Hall MD et al.; The structure of malate dehydrogenase from Escherichia coli complexed with the substrate analog, citrate and the cofactor NAD, has been determined by X-ray crystallography . A monoclinic crystal of the malate dehydrogenase, grown in citrate buffer, was soaked in 10 mM NAD solution and found to be isomorphous with the apo-form . The X-ray data extended to 1.9 A, nearly the same resolution limit as the apo-enzyme crystals . The ternary complex of malate dehydrogenase has very few conformational differences from that of the pseudo binary complex of enzyme with bound citrate . In addition, the NAD molecule has a very similar conformation to the NAD as found in the crystal structure of the cytosolic eukaryotic malate dehydrogenase . Similar hydrogen bond interactions are made by both enzymes from polar groups belonging to the NAD . Such interactions include hydrogen bonds from the ribose oxygens and the phosphate oxygens, to backbone amide and carbonyl atoms of the protein and to side-chains of a select few conserved hydrophilic residues . The only notable difference occurs in the active site region where the nicotinamide moiety is obstructed from further entering the active site by the C-6 carbonyl atoms of citrate . In this position there are no direct polar interactions between the protein and the nicotinamide moiety . Energy minimization of the structure with malate substituted for citrate in the active site shows that the nicotinamide moiety assumes the same position in the active site as the NAD in cytosolic malate dehydrogenase . The carboxamide atoms of the energy minimized model make significant hydrogen bond interactions with the catalytic residue, H177, and with the main-chain atoms of I117 and V146 in the vicinity of the active site, while the position of the rest of the cofactor remains unchanged. J Mol Biol, 1993 Jul 5, 232(1), 123 - 64 Structure and function of the Escherichia coli ribonucleotide reductase protein R2; Nordlund P et al.; The crystal structure of the ribonucleotide reductase free radical protein R2 from Escherichia coli has been determined by multiple isomorphous replacement and twofold molecular averaging . The structure has been refined at 2.2 A resolution to R = 0.175 . The subunit structure of the R2 protein has a novel fold where the basic motif is a bundle of eight long helices . The R2 dimer has two equivalent dinuclear iron centers . Each iron center is well buried in the subunit . The iron atoms have both histidine and carboxyl acid ligands and are bridged by an oxide ion and the carboxylate group of Glu115 . One iron atom is octahedrally coordinated with small deviations from ideal values, while the coordination of the other iron ion is more distorted, mainly due to the fact that Asp84 is a bidental ligand to this iron atom . The oxidation of the enzymatically essential tyrosine residue (Tyr122) and the dinuclear iron center by molecular oxygen is suggested to take part in a suitable conserved oxygen-binding pocket between the iron center and the tyrosine zeta-oxygen 5.3 A away from the closest iron ion . The tyrosine proton can be abstracted by the dioxygen and the deprotonated tyrosine residue is then more easily oxidized to a radical species . Tyr122 is buried inside the protein about 10 A from the surface . This has the consequence that the tyrosyl radical cannot participate directly in hydrogen abstraction from the substrate ribose at the active site of the holoenzyme located on the R1 subunit . The radical must then be indirectly involved in the mechanism of the enzyme and an electron transfer reaction between the active site and the tyrosine must take place . Based on the analysis of the available ribonucleotide reductase sequences, the binding surface for the large ribonucleotide reductase protein R1, and a possible route for an electron transport between the buried radical and this surface is described. J Biol Chem, 1993 Jul 5, 268(19), 14011 - 7 Expression of rat microsomal epoxide hydrolase in Escherichia coli . Identification of a histidyl residue essential for catalysis; Bell PA et al.; The cDNA containing the complete coding region for rat microsomal epoxide hydrolase (EC 3.3.2.3) was cloned into the expression/secretion vector pIN-III-OmpA3 and expressed in Escherichia coli strain TG1 . Recombinant epoxide hydrolase was found to represent 4-9% of total bacterial protein and catalyzed the hydrolysis of styrene oxide and benzo{a}pyrene 4,5-oxide with specific activities of 421 and 734 nmol min-1 mg of epoxide hydrolase-1, respectively . Previous work implicated a histidyl residue at or near the active site of the enzyme (DuBois, G . C., Appella, E., Levin, W., Lu, A . Y . H., and Jerina, D . M . (1978) J . Biol . Chem . 253, 2932-2939) . Comparison of the amino acid sequences of rat, human, and rabbit epoxide hydrolases revealed the presence of 14 conserved histidyl residues . To investigate the role of these residues in epoxide hydrolysis, site-specific mutants were generated and expressed in E . coli . Mutants H64L, H82L, H115N, H126N, H129L, H148N, H170L, H176L, H242L, H247L, H301L, H385L, K386M-H387L, delta 385-391, and H407L catalyzed the hydrolysis of benzo{a}pyrene 4,5-oxide with specific activities between 115 and 830 nmol min-1 mg-1 . Mutants H431L, H431N, and H431R were all found to have activities of < 5 nmol min-1 mg-1, which is at least 150-fold less than the activity of the wild type enzyme . A Vm versus pH profile for the recombinant wild type epoxide hydrolase revealed a broad pH optimum of 6.5 to 8.5 and the presence of three ionizable groups with pKa values of 5.8 +/- 0.2, 9.2 +/- 0.1, and 9.7 +/- 0.4 . The group with a pKa of 5.8 is preferentially unprotonated, while the other two groups are preferentially protonated for catalysis . We propose that histidine 431 corresponds to the group with a pKa of 5.8, while the others, with pKa values of 9.2 and 9.7 likely represent lysyl, cysteinyl, or tyrosyl residues . Thus, the data are consistent with a model where His-431 acts as a general base, abstracting a proton from water, while another residue(s), perhaps lysine, act as a general acid protonating the alkoxide anion that forms upon cleavage of the carbon-oxygen bond. J Biol Chem, 1993 Jul 5, 268(19), 13947 - 55 Escherichia coli transcription termination factor rho . II . Binding of oligonucleotide cofactors; Wang Y et al.; The relative binding affinities for rho of the oligonucleotide rho ATPase cofactors studied in the accompanying paper (Wang, Y., and von Hippel, P . H . (1993) J . Biol . Chem . 268, 13940-13946) have been determined by gel mobility shift and ultrafiltration binding analyses . We find that each rho hexamer carries three strong and three weak RNA-binding sites that differ approximately 10-fold in their affinities for oligonucleotide cofactors . Furthermore, in contrast to the sequence dependence of ATPase activation, we find that the binding affinities of these oligonucleotide cofactors for rho depend only on their cytosine content . In addition, we show that changes in the positions of rU residues in the oligo(rU,rC) cofactors (which significantly modulate the ATPase activity of rho) have no effect on binding affinities and that the addition of ATP, ADP, or the nonhydrolyzable ATP analog adenosine 5'-(beta,gamma-methylene)triphosphate also does not change the binding affinities of the oligonucleotide cofactors for rho . Considered in the context of the coupling of the rho ATPase and RNA binding and release cycles, these results suggest that rC residues are required for the formation of stable rho-RNA complexes, whereas rU residues at the 5' termini of cofactors bound to rho initiate or facilitate the release of the RNA from the individual cofactor site as a consequence of ATP hydrolysis . Thus, both the tightness of the binding of RNA segments to the individual RNA-binding sites of rho and the rate of release of these segments from these sites are critical in controlling the ATPase rate of rho and probably also in modulating the function of this protein in transcript termination. J Biol Chem, 1993 Jul 5, 268(19), 13940 - 6 Escherichia coli transcription termination factor rho . I . ATPase activation by oligonucleotide cofactors; Wang Y et al.; Rho protein is required to bring about RNA release from Escherichia coli transcription complexes paused at specific (rho-dependent) termination sites . Rho functions in termination as a hexamer of identical subunits arranged in D3 symmetry, with each rho subunit carrying an RNA- and an ATP-binding site . The detailed mechanism of rho-catalyzed transcript release remains to be determined, but it is clear that the RNA-dependent ATPase activity that is stimulated by interaction with the nascent transcript is essential to the termination function of rho . In this study, we have used short (8-10 nucleotide residues) synthetic ribo-oligonucleotides to model the interaction of segments of the RNA cofactor with rho . A poly(dC) enhancement procedure was used to permit the measurement of steady state ATPase parameters . We show that (i) ATPase activation is cofactor composition- and sequence-dependent; (ii) at least 60% of the residues of these short RNA cofactors must be cytosine to produce maximal rho ATPase activation; (iii) oligo(rU,rC) cofactors with the rU residues located at the 5' termini of the oligomer are much better ATPase cofactors than oligomers containing rC residues only; (iv) this enhanced stimulation is not observed if the rU residues are replaced by rA residues; (v) this cofactor activity relative to oligo(rC) is reversed if the rU residues are placed at the 3' terminus of RNA oligomer; and (vi) these nucleotide sequence and composition effects do not appear to be functions of K+ or Mg2+ concentration . These ATPase activation results are correlated with the binding to rho of oligonucleotide cofactors in the accompanying paper (Wang, Y., and von Hippel, P . H . (1993) J . Biol . Chem . 268, 13947-13955). J Biol Chem, 1993 Jul 5, 268(19), 13799 - 804 Functional interactions between K+ pore residues located in different subunits; Kirsch GE et al.; The aqueous pore (P-region) of homotetrameric voltage-gated K+ channels has been modeled as a radially symmetrical eight-stranded antiparallel beta-barrel to which each of the four subunits contributes equally . This model has hydrogen bonding between residues located on adjacent subunits and predicts that subunit interactions might have functional consequences . Previously we have used point mutations and an electrophysiological assay to detect functional interactions between a pair of residues at positions 369 and 374 in the P-region, but we could not distinguish between intra- and intersubunit interactions . In the present paper, we present evidence for interaction across subunit boundaries after co-injecting two cRNAs encoding subunits differing from each other at either position 369 or 374 . Comparison of the phenotypes of homo- and heterotetrameric channels suggests that pore residues residing in adjacent subunits form a closely packed structure which determines both ion conductance and stability of the open state of the channel . Our results are consistent with a structure in which pore residues 369 and 374 are located in close proximity on adjacent antiparallel strands to allow both intra- and intersubunit interactions. J Biol Chem, 1993 Jul 5, 268(19), 14125 - 30 Identification of N,N'-dicyclohexylcarbodiimide-reactive glutamic and aspartic acid residues in Escherichia coli transhydrogenase and the exchange of these by site-specific mutagenesis; Glavas N et al.; Pyridine nucleotide transhydrogenase (EC 1.6.1.1) from Escherichia coli was investigated with respect to the role of glutamic and aspartic acid residues reactive to N,N'-dicyclohexylcarbodiimide (DCCD) and potentially involved in the proton-pumping mechanism of the enzyme . The E . coli transhydrogenase consists of an alpha (510 residues) and a beta (462 residues) subunit . DCCD reacts with the enzyme to inhibit catalytic activity and proton pumping . This reagent modifies Asp alpha 232, Glu alpha 238, and Glu alpha 240 as well as amino acid residue(s) in the beta subunit . Using the cloned and overexpressed E . coli transhydrogenase genes (Clarke, D . M., and Bragg, P . D . (1985) J . Bacteriol . 162, 367-373), Asp alpha 232 and Glu alpha 238 were replaced independently by site-specific mutagenesis . In addition, Asp alpha 232, Glu alpha 238, and Glu alpha 240 were replaced to generate triple mutants . The specific catalytic activities of the mutant transhydrogenases alpha D232N, alpha D232E, alpha D232K, alpha D232H, alpha E238K, and alpha E238Q as well as of the triple mutants alpha D232N, alpha E238Q, alpha E240Q and alpha D232H, alpha E238Q, alpha E240Q were in the range of 40-90% of the wild-type activity . Proton-pumping activity was present in all mutants . Examination of the extent of subunit modification by {14C}DCCD revealed that the label was still incorporated into both alpha and beta subunits in the Asp alpha 232 mutants, but that the alpha subunit was not labeled in the triple mutants . Catalytic and proton-pumping activities were nearly insensitive to DCCD in the triple mutants . This suggests that loss of catalytic and proton-pumping activities is associated with modification of the aspartic and glutamic acid residues of the alpha subunit . In the presence of the substrate NADPH, the rate of modification of the beta subunit by {14C}DCCD was increased, and there was a greater extent of enzyme inactivation . By contrast, NADH and 3-acetylpyridine-NAD+ protected the catalytic activity of the transhydrogenase from inhibition by DCCD . The protection was particularly marked in the E238Q and E238K mutants . It is concluded that the Asp alpha 232, Glu alpha 238, and Glu alpha 240 residues are not essential for catalytic activity or proton pumping . The inactivation by DCCD is likely due to the introduction of a sterically hindering group that reacts with the identified acidic residues close to the NAD(H)-binding site. J Biol Chem, 1993 Jul 5, 268(19), 13777 - 9 On the assembly of dodecameric glutamine synthetase from stable chaperonin complexes; Fisher MT; For many in vitro protein-folding reactions, the fraction of correctly folded product declines as the initial protein concentration increases due primarily to misfolding and aggregation reactions . Under optimal conditions and in the presence of ATP, chaperonins (groEL and groES) enhanced the renaturation of dodecameric glutamine synthetase (GS) with yields of active enzyme between 75 and 85% of the original activity (Fisher, M.T . (1992) Biochemistry 31, 3955-3963) . In spite of this enhancement, a concentration-dependent decline in recoverable activity was observed when increasing concentrations of unfolded GS were rapidly mixed with renaturation buffer containing a 2-fold molar excess (GS subunits:groEL oligomer) of chaperonins . When a stable groEL-GS complex, formed under optimal conditions, was concentrated 4-fold by centrifugal ultrafiltration prior to ATP addition, the amount of total active GS (percent of the original activity) recovered remained at optimal levels and no longer showed a concentration-dependent decline . The GS subunits that are initially bound and then released from groEL by ATP are assembly-competent . It is proposed that the subunits are no longer able to kinetically equilibrate with folding intermediates that misfold or aggregate . If a stable groEL-protein substrate complex can be amassed without loss of activity, this will facilitate studies on molecular aspects of chaperonin release mechanisms and oligomeric protein assembly. J Mol Biol, 1993 Jul 5, 232(1), 165 - 91 Quadratic minimization of predictors for protein secondary structure . Application to transmembrane alpha-helices; Edelman J; Sliding-window averaging of amino acid properties is often used for predicting protein secondary structure . Such a scheme (linear convolutional recognizer, LCR) assigns a number (weight) to each type of monomer, and then convolutes a window function with the sequence of weights to yield a decision function . Features, regions having the property of interest, are predicted to occur where the decision function exceeds some threshold . A general method for approximating the best possible window and weights is presented . The needed data are the sequences of some chains and the locations of their features . The method is applied to transmembrane helices (TMH) of membrane proteins . Optimal weights and windows are calculated, using bacteriorhodopsin and photosynthetic reaction centers as the reference chains . The predictor is then tested on other proteins . No TMH are predicted in porin, whose transmembrane segments are beta-sheets . This shows that the predictor is specific for helical segments . Few segments are predicted for non-membrane globular proteins . The predictor thus correctly rejects their hydrophobic helices . Finally, the predictor is tested with some membrane proteins whose transmembrane topology is partially known . Among their TMH, the LCR is unable to resolve 6% which are closely spaced . Taking 17 as the minimum allowed length of a predicted TMH, 4% of the known ones are missed and 6% of the predicted ones are false . For a minimum length of 10, 0.5% are missed and 14% are false . The mean magnitude of the endpoint error is about four residues . Alternative prediction methods make more errors. J Infect Dis, 1993 Jul, 168(1), 135 - 42 Lipopolysaccharide and peptidoglycan share binding sites on human peripheral blood monocytes; Rabin RL et al.; p73, a binding site for lipopolysaccharide (LPS) on human peripheral blood monocytes was identified using the radiolabeled photoaffinity cross-linker sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD) . The 125I-labeled conjugate of SASD and LPS (125I-labeled ASD-LPS) was bound to monocytes and UV cross-linked, and the cellular extracts were analyzed with two-dimensional SDS-PAGE and autoradiography . In addition to the major binding site on human monocytes at 73 kDa, isoelectric point 5.95, there were multiple minor binding sites that recognized both smooth and rough LPS . Binding of 125I-labeled ASD-LPS to monocytes is concentration dependent, decreased in the absence of calcium and magnesium, and inhibited by either excess LPS or the low-molecular-weight soluble isolate of bacterial cell wall peptidoglycan (sPGN) . However, sPGN only minimally stimulates tumor necrosis factor (TNF) secretion by human peripheral blood mononuclear cells . In contrast, the relatively insoluble high-molecular-weight peptidoglycan significantly stimulates TNF secretion. Infect Immun, 1993 Jul, 61(7), 2827 - 33 Primary structure of and immunoglobulin E response to the repeat subunit of gp15/400 from human lymphatic filarial parasites; Paxton WA et al.; We have isolated and sequenced clones encoding the repeated subunit of the surface-associated glycoprotein gp15/400 from the two nematode species predominantly responsible for lymphatic filariasis in humans: Brugia malayi and Wuchereria bancrofti . The amino acid sequence of the 15-kDa subunit, derived from the nucleotide sequence of the gene fragment from B . malayi, is identical to that previously reported for B . pahangi, whereas the derived W . bancrofti protein sequence differs in only 7 of 132 residues . The identity of the protein in the two Brugia species allowed us to use a recombinant from B . pahangi to examine the serological response of adult Indonesian subjects infected with B . malayi . The polymerase chain reaction-amplified subunit was expressed in Escherichia coli via the pDS56/RBS11 plasmid and purified by nickel-chelating chromatography . A significant proportion of individuals produced antigen-specific immunoglobulin E (IgE) . This was most pronounced in the individuals with elephantiasis, with 14 of 15 showing elevated titers and a mean of 3.2 ng of specific IgE ml-1 . Only 2 of 15 microfilaremic individuals possessed elevated titers of specific IgE, with a mean of 0.045 ng ml-1 for the group as a whole . Asymptomatic amicrofilaremic residents showed approximately equal numbers of responders (defined as having a value in the radioimmunoassay greater than two standard deviations above controls) and nonresponders, with a group mean of 1.2 ng of antigen-specific IgE ml-1. Infect Immun, 1993 Jul, 61(7), 2780 - 5 Identification of a carbohydrate recognition domain in filamentous hemagglutinin from Bordetella pertussis; Prasad SM et al.; The adherence of Bordetella pertussis to ciliated cells and macrophages is critical to colonization and infection of the respiratory tract . Adherence to both types of cells involves the recognition of eukaryotic carbohydrates by the bacterial adhesin filamentous hemagglutinin (Fha) . The carbohydrate recognition domain (CRD) of Fha is considered an important antigen for subcomponent vaccines to maximize the generation of antiadherence antibodies capable of protecting against colonization . For identification of the CRD of Fha, a bank of eight monoclonal antibodies (MAbs) that mapped to four contiguous regions were tested for their ability to block Fha binding to lactosylceramide or to block bacterial binding to ciliated cells . Only MAb 12.5A9, which maps to amino acid residues 1141 to 1279, blocked both Fha binding to lactosylceramide and bacterial binding to ciliated cells . An 18-kDa polypeptide corresponding to this region was expressed in Escherichia coli . Cell lysates containing this protein bound to lactosylceramide in a manner identical to that of native Fha . Mutant strains of B . pertussis that contained an in-frame deletion of the coding sequence for this region produced a truncated Fha that showed negligible cross-reactivity with MAb 12.5A9 . In an adherence assay, these mutant strains failed to bind efficiently to either ciliated cells or macrophages . The numbers of adherent bacteria for these strains were reduced to the number obtained with a nonadherent strain . We conclude that the region defined by residues 1141 to 1279 of Fha constitutes a CRD critical for bacterial adherence and represents a potential candidate for a subcomponent vaccine. Infect Immun, 1993 Jul, 61(7), 2755 - 62 Enteropathogenic Escherichia coli decreases the transepithelial electrical resistance of polarized epithelial monolayers; Canil C et al.; The mechanisms whereby enteropathogenic Escherichia coli (EPEC) causes diarrhea remain undefined . We found that EPEC caused a decrease in transepithelial electrical resistance across polarized monolayers of Caco-2 and MDCK epithelial cells . This occurred approximately 6 to 10 h after bacterial addition and was reversible if the monolayers were treated with tetracycline or gentamicin . Although significant alterations in host actin occurred beneath adherent EPEC, actin filaments supporting tight junctions were not noticeably