FEMS Microbiol Lett, 1992 Sep 15, 75(2-3), 155 - 9 Serogroups of Escherichia coli strains producing cytotoxic necrotizing factors CNF1 and CNF2; Blanco J et al.; The serogroups of 396 necrotizing Escherichia coli of human and bovine origin isolated in Spain between 1979 and 1991 have been determined . The 270 cytotoxic necrotizing factor strains belonged to 22 different O serogroups; however, 84% (226 of 270) were of one of seven serogroups (O2, O4, O6, O14, O22, O75 and O83) . Although necrotizing E . coli producing cytotoxic necrotizing factor 2 belonged to 28 different serogroups, only six of them (O1, O3, O15, O55, O88 and O123) accounted for 60% (76 of 126) of cytotoxic necrotizing factor 2 strains . Furthermore, only 3% (4 of 126) of cytotoxic necrotizing factor 2 strains belonged to serogroups most common among strains producing cytotoxic necrotizing factor 1 . The majority of necrotizing E . coli producing cytotoxic necrotizing factor 1 were obtained from human extraintestinal infections, whereas cytotoxic necrotizing factor 2 strains were isolated from stools of healthy and diarrhoeic calves. Eur J Biochem, 1992 Sep 15, 208(3), 699 - 704 Precursor of mitochondrial aspartate aminotransferase synthesized in Escherichia coli is complexed with heat-shock protein DnaK; Schmid D et al.; On expression of the cDNA encoding the precursor of chicken mitochondrial aspartate aminotransferase (pmAspAT) in Escherichia coli, the bulk of pmAspAT was found to be associated with the 70-kDa heat-shock protein DnaK which is closely related to mitochondrial 70-kDa heat-shock protein (HSP70) . Purification protocols for the DnaK/pmAspAT complex and its individual components were elaborated . The complex dissociated on treatment with MgATP or at pH 5.5 . Like the mature enzyme, pmAspAT is a dimer (2 x 47 kDa) and exhibits about a third of its enzyme activity . In the DnaK/pmAspAT complex, one DnaK molecule is bound to each subunit of pmAspAT; this tetramer may further aggregate to an octamer . The complex is catalytically almost as active as free pmAspAT . It could be reconstituted from isolated DnaK and pmAspAT . No complex was formed with mAspAT . Apparently, DnaK binds to the solvent-exposed presequence of folded pmAspAT without significantly changing the structure and functional properties of its mature moiety. Eur J Biochem, 1992 Sep 15, 208(3), 635 - 42 Binding of the competitive inhibitor dCDP to ribonucleoside-diphosphate reductase from Escherichia coli studied by 1H NMR . Different properties of the large protein subunit and the holoenzyme; Allard P et al.; Ribonucleoside-diphosphate reductase (EC 1.17.4.1) from Escherichia coli consists of two nonidentical subunits, proteins R1 and R2 . The binding of the product dCDP to protein R1 and to the holoenzyme R1R2 has been studied by means of 1H-NMR spectroscopy . In presence of the effector dTTP at 25 degrees C, dCDP was found to be in rapid exchange between the binding sites and the solvent which results in a broadening of the dCDP resonances . When both proteins R1 and R2 are present, so that the complex R1R2 is formed, a smaller broadening is observed than with protein R1 alone . No further linewidth decrease was observed when the {R2}/{R1} ratio exceeded 1 . The binding constant of dCDP to R1 or R1R2 is the same, Kd = 0.9 mM . The smaller broadening of the dCDP resonances observed with the complex R1R2 as compared with R1 may be explained by the combination of two effects: (a) the overall tumbling time of the protein will increase when going from R1 to R1R2, which will cause the broadening to increase correspondingly, and (b) a twofold decrease of the number of binding sites in rapid exchange, which will decrease the broadening by a factor of 0.5 . The effect of R2 without iron (apoR2) is reduced compared with native R2, probably because of some denatured proteins, while a C-terminal peptide from R2 did not cause any narrowing at all. Eur J Biochem, 1992 Sep 15, 208(3), 631 - 4 Studies on ribonucleoside-diphosphate reductase from Escherichia coli . The product dCDP is a competitive inhibitor and functions as a spectroscopic probe for the substrate binding site; demonstration by enzyme kinetics and 1H NMR; Shen B et al.; Ribonucleoside-diphosphate reductase (EC 1.17.4.1) from Escherichia coli consists of two protein subunits, R1 of 171.5 kDa and R2 of 86.8 kDa, and catalyzes the reduction of all four common ribonucleoside diphosphates . In a search for ligands that bind weakly to the enzyme active site and may be in fast exchange suitable for NMR studies, we have found that the product dCDP is a competitive inhibitor . Kinetics with CDP as substrate shows Km = 4.8 x 10(-5) M and dCDP inhibits with Ki = 1.6 x 10(-4) M . With an assumed diffusion limited binding rate approximately less than 10(9) M-1s-1, the dissociation rate of dCDP would be approximately less than 10(5) s-1 . In 1H-NMR experiments studying linewidths, i.e . spin-spin relaxation, dCDP is indeed demonstrated to be in fast exchange . Enzyme subunit R1 causes a line broadening of dCDP resonances . Unexpectedly less broadening was observed when subunit R2 combined with R1 . No paramagnetic interaction from the tyrosyl radical of R2 could be detected . It is concluded that dCDP is a promising NMR probe for studies of active-site properties of the enzyme. Biochemistry, 1992 Sep 15, 31(36), 8648 - 53 Purification, crystallization, and preliminary X-ray diffraction analysis of an M.HhaI-AdoMet complex; Kumar S et al.; The type-II DNA-(cytosine-5)-methyltransferase M.HhaI was overexpressed in Escherichia coli and purified to apparent homogeneity . The purification scheme exploits a unique high salt back-extraction step to solubilize M.HhaI selectively, followed by FPLC chromatography . The yield of purified protein was 0.75-1.0 mg per gram of bacterial paste . M.HhaI could be isolated in two forms: bound with its cofactor S-adenosylmethionine (AdoMet) or devoid of the cofactor . The AdoMet-bound form was capable of methylating DNA in vitro in the absence of exogenous AdoMet . From kinetic studies of the purified enzyme, values for KmAdoMet (60 nM), KiAdoHye (0.4 nM), and Kcat (0.22 s-1) were determined . The purified enzyme bound with its cofactor was crystallized by the hanging drop vapor diffusion technique . Crystals were of monoclinic space group P2(1) and had unit-cell dimensions of a = 55.3 A, b = 72.7 A, c = 91.0 A, and beta = 102.5 degrees, with two molecules of M.HhaI in each of the two asymmetric units . The crystals diffract beyond 2.5 A and are suitable for structure determination. Biochemistry, 1992 Sep 15, 31(36), 8516 - 22 Energetic limits of phosphotransfer in the catalytic subunit of cAMP-dependent protein kinase as measured by viscosity experiments; Adams JA et al.; Viscosogenic agents were used to test the diffusion limits of the reaction catalyzed by the catalytic subunit of the cAMP-dependent protein kinase . The effects of glycerol and sucrose on the maximum rate (kcat) and the apparent second-order rate constants (kcat/Kpeptide) for the phosphorylation of four peptidic substrates were measured at their pH optima . The agents were found to have moderate to no effect on kcat/Kpeptide for good and poor substrates, respectively . Conversely, kcat was highly sensitive to solvent viscosity for three of the four peptides at high concentrations of ATP . Taken together, these data indicate that enzymatic phosphorylation by the catalytic subunit proceeds with rapid or near rapid equilibrium binding of substrates and that all steps following the central substrate complex (i.e., chemical and conformational events) are fast relative to the rate-determining dissociation of product, ADP, when ATP levels are high . Under saturating concentrations of peptide I, LRRASLG, an unproductive form of the enzyme is populated . The observed phosphorylation rate from this complex is involved in rate limitation owing to a slow step separating unproductive and productive enzyme forms . The data are used to establish a kinetic mechanism for the catalytic subunit that predicts initial reaction velocities under varying concentrations of ATP and substrate. Biochemistry, 1992 Sep 15, 31(36), 8508 - 15 Effects of replacement of active site residue glutamine 231 on activity and allosteric properties of aspartate transcarbamoylase; Peterson CB et al.; Since crystallographic studies on Escherichia coli aspartate transcarbamoylase (ATCase) indicate that Gln 231 is in the active site of the enzyme and participates in the binding of the substrate, aspartate, it seemed of interest to examine mutant enzymes in which Gln 231 was replaced by Asn or Ile . The two mutant forms containing amino acid substitutions were characterized by a combination of steady-state kinetics, hydrodynamic measurements, and equilibrium ligand binding techniques . Both mutant forms exhibited a dramatic reduction in the affinity of the protein for substrates and substrate analogues as well as a very large decrease in catalytic activity . Moreover, the amino acid substitutions introduced within the active site of the enzyme led to unusual allosteric properties in the mutant enzymes . Although the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate promotes the characteristic global conformational change in the mutant forms that is observed with the wild-type enzyme, the combination of substrate and substrate analogue does not . Cooperativity with respect to substrate binding is largely reduced compared to wild-type ATCase . Also, the effector molecules ATP and CTP which bind to the regulatory chains have dramatic effects on the activity of the mutant enzymes containing replacements for Gln 231 in the catalytic chains . In stark contrast to the wild-type enzyme, in which effects of nucleotides are manifested primarily by changes in the K0.5 of the enzyme, ATP and CTP have large effects on the Vmax of the mutant enzymes.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1992 Sep 15, 31(36), 8482 - 7 Segmental movement: definition of the structural requirements for loop closure in catalysis by triosephosphate isomerase; Sampson NS et al.; To determine what drives the closure of the active-site loop in the reaction catalyzed by triosephosphate isomerase, several residues involved in hydrogen bonding between the loop and the bulk of the protein have been altered . It was known from earlier work that the loop serves two functions: to stabilize the reaction intermediate (and the two transition states that flank it) and to prevent the loss of this unstable species into free solution . To discover what elements of the protein are necessary for proper closure of the loop, selective destabilization of the "open" and the "closed" forms of the enzyme with respect to one another has been attempted . The mutant Y164F isomerase has been prepared to evaluate the importance of the structure of the "open" form, and the mutant E129Q, Y208F, and S211A enzymes have allowed investigation of the "closed" form . The integrity of the loop itself has been destabilized by making the T172A isomerase . We have found that only those mutations that destabilize the "closed" form of the enzyme significantly perturb the catalytic properties of the isomerase . The second-order rate constants (kcat/Km) of the S211A and E129Q enzymes are reduced 30-fold, and that of the mutant Y208F enzyme is reduced 2000-fold, from the level of the wild-type enzyme . The dramatic drop in activity of the Y208F enzyme is accompanied by a 200-fold increase in the dissociation constant of the intermediate analogue phosphoglycolohydroxamate . The most important property of the mobile loop of triosephosphate isomerase lies, therefore, in the stability of the system when the active site contains ligand and the loop is closed. Biochemistry, 1992 Sep 15, 31(36), 8437 - 41 Effect of 5-deazaflavin on energy transduction during catalysis by Escherichia coli DNA photolyase; Ramsey AJ et al.; DNA photolyase from Escherichia coli contains 1,5-dihydroFAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate . The action spectrum observed for apoenzyme reconstituted with 5-deazaFADH2 (EdFADH2) matched its absorption spectrum after correction for the presence of a small amount of inactive 5-deazaFADox . The quantum yield for dimer repair with EdFADH2 (phi EdFADH2 = 0.110) was 6-fold lower than that observed with apoenzyme reconstituted with FADH2 . Excited-state redox potential calculations indicate that 5-deazaFADH2 singlet is a better one-electron donor (E = -3.5 V) than FADH2 singlet (E = -2.7 V) . Other studies indicate that the quantum yield for electron transfer from reduced flavin singlet to pyrimidine dimer (0.88) is unaffected when FADH2 is replaced by 5-deazaFADH2 . Enhanced back electron transfer from pyrimidine dimer radical to flavin radical may account for the decreased quantum yield observed with EdFADH2 since, in the ground state, 5-deazaFADH . is a better oxidant than FADH. . The action spectrum observed for apoenzyme reconstituted with 5-deazaFADH2 plus 5,10-CH(+)-H4folate (EPtedFADH2) matched the absorption spectrum determined for enzyme-bound 5-deazaFADH2, indicating that the pterin chromophore was inactive as a sensitizer . This differs from results obtained with native enzyme, where pterin acts as a sensitizer via efficient singlet-singlet energy transfer to FADH2 . The quantum yield for dimer repair by 5-deazaFADH2 bound to EPtedFADH2 (phi EPtedFADH2 = 0.0318) was 28.9% of that observed for EdFADH2 . Spectroscopic studies indicate that singlet-singlet energy transfer in EPtedFADH2 is very efficient but only occurs in the "wrong" direction, i.e., from excited 5-deazaFADH2 to pterin.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1992 Sep 15, 31(36), 8429 - 36 Differences and similarities in the repair of two benzo{a}pyrene diol epoxide isomers induced DNA adducts by uvrA, uvrB, and uvrC gene products; Tang MS et al.; We have determined the role of the uvrA, uvrB, and uvrC genes in Escherichia coli cells in repairing DNA damage induced by three benzo{a}pyrene diol epoxide isomers . Using the phi X174 RF DNA-E . coli transfection system, we have found that BPDE-I or BPDE-II modified phi X174 RF DNA has much lower transfectivity in uvrA, uvrB, and uvrC mutant cells compared to wild type cells . In contrast, BPDE-III modification of phi X174 RF DNA causes much less difference in transfectivity between wild type and uvr- mutant cells . Moreover, BPDE-I and -II-DNA adducts are much more genotoxic than are BPDE-III-DNA adducts . Using purified UVRA, UVRB, and UVRC proteins, we have found that these three gene products, working together, incise both BPDE-I- and BPDE-III-DNA adducts quantitatively and, more importantly, at the same rate . In general, UVRABC nuclease incises on both the 5' (six to seven nucleotides) and 3' (four nucleotides) sides of BPDE-DNA adducts with similar efficiency with few exceptions . Quantitation of the UVRABC incision bands indicates that both of these BPDE isomers have different sequence selectivities in DNA binding . These results suggest that although UVR proteins can efficiently repair both BPDE-I- and BPDE-III-DNA adducts, in vivo the uvr system is the major excision mechanism for repairing BPDE-I-DNA adducts but may play a lesser role in repairing BPDE-III-DNA adducts . It is possible the low lethality of BPDE-III-DNA adducts is due to less complete blockage of DNA replication.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1992 Sep 15, 267(26), 18953 - 60 Determination of the 1-ethyl-3-{(3-dimethylamino)propyl}-carbodiimide- induced cross-link between the beta and epsilon subunits of Escherichia coli F1-ATPase; Dallmann HG et al.; The zero-length cross-link between the inhibitory epsilon subunit and one of three catalytic beta subunits of Escherichia coli F1-ATPase (alpha 3 beta 3 gamma delta epsilon), induced by a water-soluble carbodiimide, 1-ethyl-3-{(3-dimethylamino) propyl}-carbodiimide (EDC), has been determined at the amino acid level . Lability of cross-linked beta-epsilon to base suggested an ester cross-link rather than the expected amide . A 10-kDa cross-linked CNBr fragment derived from beta-epsilon was identified by electrophoresis on high percentage polyacrylamide gels . Sequence analysis of this peptide revealed the constituent peptides to be Asp-380 to Met-431 of beta and Glu-96 to Met-138 of epsilon . Glu-381 of beta was absent from cycle 2 indicating that it was one of the cross-linked residues, but no potential cross-linked residue in epsilon was identified in this analysis . A form of epsilon containing a methionine residue in place of Val-112 (epsilon V112M) was produced by site-directed mutagenesis . epsilon V112M was incorporated into F1-ATPase which was then cross-linked with EDC . An 8-kDa cross-linked CNBr fragment of beta-epsilon V112M was shown to contain the peptide of epsilon between residues Glu-96 and Met-112 and the peptide of beta between residues Asp-380 and Met-431 . Again residue Glu-381 of beta was notably reduced and no missing residue from the epsilon peptide could be identified, but the peptide sequence limited the possible choices to Ser-106, Ser-107, or Ser-108 . Furthermore, an epsilon mutant in which Ser-108 was replaced by cysteine could no longer be cross-linked to a beta subunit in F1-ATPase by EDC . Both mutant forms of epsilon supported growth of an uncC-deficient E . coli strain and inhibited F1-ATPase . These results indicate that the EDC-induced cross-link between the beta and epsilon subunits of F1-ATPase is an ester linkage between beta-Glu-381 and, likely, epsilon-Ser-108 . As these residues must be located immediately adjacent to one another in F1-ATPase, our results define a site of subunit-subunit contact between beta and epsilon. Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8779 - 83 Escherichia coli MutY protein has both N-glycosylase and apurinic/apyrimidinic endonuclease activities on A.C and A.G mispairs; Tsai-Wu JJ et al.; In Escherichia coli the mutY (or micA)-dependent DNA mismatch repair pathway can convert A degrees G and A degrees C mismatches to C.G and G.C base pairs, respectively, through a short repair-tract mechanism . The MutY protein has been purified to near homogeneity from an E . coli overproducer strain . Purified MutY has been shown to contain both N-glycosylase and 3' apurinic/apyrimidinic (AP) endonuclease activities . The N-glycosylase removes the mispaired adenines of A degrees G and A degrees C mismatches, and the AP endonuclease acts on the first phosphodiester bond 3' to the AP sites . The N-glycosylase and the nicking (combined N-glycosylase and AP endonuclease) activities copurified through multiple chromatographic steps without a change in relative specific activities . Furthermore, both N-glycosylase and AP endonuclease activities can be recovered by renaturation of a single polypeptide band from an SDS/polyacrylamide gel . Renaturation required the presence of iron and sulfide . These findings suggest that the MutY protein, like endonuclease III, is an iron-sulfur protein . DNA fragments with A degrees C mismatches were 20-fold less active than DNA with A degrees G mispairs as a substrate for purified MutY. J Biol Chem, 1992 Sep 15, 267(26), 18255 - 8 Functional analysis of novel selective mutants of the reverse transcriptase of human immunodeficiency virus type 1; Hizi A et al.; We have generated by site-directed mutagenesis plasmids that induce the synthesis of specific mutants of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) . These recombinant mutants of HIV-1 RT, designed on the basis of our previous studies of HIV-1 and HIV-2 RTs, were analyzed for structure-function relationship by assessing their RNA-dependent and DNA-dependent DNA polymerase as well as the ribonuclease H activities . Three groups of mutants were studied . 1) We have investigated the importance of the only two sets of highly conserved double prolines found in the sequence of HIV-1 RT . The results indicate that the conversion of either one or both prolines (at positions 225 and 226) to threonines have no significant effect on all catalytic activities of the enzyme . The mutants in which prolines 419 and 420 were individually modified to threonines exhibit full activities, whereas the double proline 419/420 mutant lost most of its RNase H activity (although the DNA polymerase function was fully retained) . 2) We have deleted phenylalanine 346 from HIV-1 RT, which is absent in wild type HIV-2 RT . This mutant of HIV-1 RT lost practically all catalytic activities . 3) A mutant of HIV-1 RT in which a cysteine residue substituted for alanine 446, was found to be slightly hyperactive for both DNA polymerase and RNase H activities. Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8696 - 700 Identification, characterization, and DNA sequence of a functional "double" groES-like chaperonin from chloroplasts of higher plants; Bertsch U et al.; Chloroplasts of higher plants contain a nuclear-encoded protein that is a functional homolog of the Escherichia coli chaperonin 10 (cpn10; also known as groES) . In pea (Pisum sativum), chloroplast cpn10 was identified by its ability to (i) assist bacterial chaperonin 60 (cpn60; also known as groEL) in the ATP-dependent refolding of chemically denatured ribulose-1,5-bisphosphate carboxylase and (ii) form a stable complex with bacterial cpn60 in the presence of Mg.ATP . The subunit size of the pea protein is approximately 24 kDa--about twice the size of bacterial cpn10 . A cDNA encoding a spinach (Spinacea oleracea) chloroplast cpn10 was isolated, sequenced, and expressed in vitro . The spinach protein is synthesized as a higher molecular mass precursor and has a typical chloroplast transit peptide . Surprisingly, however, attached to the transit peptide is a single protein, comprised of two distinct cpn10 molecules in tandem . Moreover, both halves of this "double" cpn10 are highly conserved at a number of residues that are present in all cpn10s that have been examined . Upon import into chloroplasts the spinach cpn10 precursor is processed to its mature form of approximately 24 kDa . N-terminal amino acid sequence analysis reveals that the mature pea and spinach cpn10 are identical at 13 of 21 residues. Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8803 - 7 Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA; Kuspa A et al.; Introduction of restriction enzyme along with linearized plasmid results in integration of plasmid DNA at genomic restriction sites in a high proportion of the resulting transformants . We have found that electroporating BamHI or EcoRI together with pyr5-6 plasmids cut with the same enzyme stimulates the efficiency of transformation in Dictyostelium discoideum more than 20-fold over the rate seen when plasmid DNA alone is introduced . Restriction enzyme-mediated integration generates insertions into genomic restriction sites in an apparently random manner, some of which cause mutations . About 1 in 400 of the Dictyostelium transformants displayed arrested or aberrant development . The integrated plasmid, along with flanking genomic DNA, was excised from some of these mutants, cloned in Escherichia coli, and used to transform other Dictyostelium cells . Homologous recombination within the flanking sequences resulted in the same phenotypes displayed by the original mutants, directly demonstrating that the affected genes were responsible for the specific morphological phenotypes . This method of insertional mutagenesis should be useful for tagging, and subsequent cloning, of many developmentally important genes that can be identified by their mutant phenotypes. J Biol Chem, 1992 Sep 15, 267(26), 18683 - 8 A polymerase chain reaction strategy to identify and clone cyclic nucleotide phosphodiesterase cDNAs . Molecular cloning of the cDNA encoding the 63-kDa calmodulin-dependent phosphodiesterase; Repaske DR et al.; Multiple isozymes of cyclic nucleotide phosphodiesterases (PDEs) are expressed simultaneously in mammalian tissues . To identify and clone these PDEs, a polymerase chain reaction (PCR) strategy was developed using degenerate oligonucleotide primers designed to hybridize with highly conserved PDE DNA domains . Both known and novel PDEs were cloned from rat liver, the mouse K30a-3.3 lymphoma cell line, and a human hypothalamus cDNA library, demonstrating that these PCR primers can be used to amplify the cDNA of multiple PDE isozymes . One unique mouse PDE clone was found to encode a polypeptide identical with the corresponding portion of the bovine brain 63-kDa calmodulin-dependent PDE as reported in the companion article (Bentley, J . K., Kadlecek, A., Sherbert, C . H., Seger, D., Sonnenburg, W . K., Charbonneau, H., Novack, J . P., and Beavo, J . A . (1992) J . Biol . Chem . 267, 18676-18682) . This mouse clone was used as a probe to screen a rat brain cDNA library for a full-length clone . The conceptual translation of the nucleotide sequence of the resulting rat clone has an open reading frame of 535 amino acids and maintains a high degree of homology with the bovine 63-kDa calmodulin-dependent PDE, indicating that this protein is likely to be the rat homolog of the 63-kDa calmodulin-dependent PDE . Expression of the full-length clone in Escherichia coli yielded a cGMP hydrolyzing activity that was stimulated severalfold by calmodulin . Northern blot analysis demonstrated that the mRNA encoding this PDE is highly expressed in rat brain and also in the S49.1 T-lymphocyte cell line . These data demonstrate that the PCR method described is a viable strategy to isolate cDNA clones of known and novel members of different families of PDE isozymes . Molecular cloning of these PDEs will provide valuable tools for investigating the roles of these isozymes in regulation of intracellular concentrations of the cyclic nucleotides. FEBS Lett, 1992 Sep 14, 309(3), 363 - 70 Studies on antisense inhibition of translation in vitro . Anomalies and re-evaluation; Ricker RD et al.; Experiments were carried out to better characterize antisense control of translation . Results in an E . coli system confirmed specific inhibition of poly(U) translation . At low concentrations, certain homopolymers (including poly(rA)) stimulated translation . Oligo(dA(n)) was inhibitory at n less than or equal to 8 . Translation of globin mRNA in reticulocyte lysates indicated that ssDNA 15-mers targeted at beta-globin mRNA inhibited both alpha- and beta-globin production . Sequences targeted immediately downstream of the AUG were the least effective in inhibition . These and other anomalies are discussed here in relation to those of others, emphasizing caution in performing antisense experiments. Biochemistry, 1992 Sep 1, 31(34), 7815 - 25 Roles of Mg2+ in the mechanism of formation and dissociation of open complexes between Escherichia coli RNA polymerase and the lambda PR promoter: kinetic evidence for a second open complex requiring Mg2+; Suh WC et al.; Comparative studies of the effects of Mg2+ vs Na+ and of acetate (OAc-) vs Cl- on the kinetics of formation and dissociation of E . coli RNA polymerase (E sigma 70)-lambda PR promoter open complexes have been used to probe the mechanism of this interaction . Composite second-order association rate constants ka and first-order dissociation rate constants kd, and their power dependences on salt concentration SKa (SKa identical to d log ka/d log {salt}) and Skd (Skd identical to d log kd/d log {salt}), were determined in MgCl2 and NaOAc to compare with the results of Roe and Record (1985) in NaCl . Replacement of NaCl by MgCl2 reduces the magnitude of Ska 2-fold (Ska = -11.9 +/- 1.1 in NaCl; Ska = -5.2 +/- 0.3 in MgCl2) and (by extrapolation) drastically reduces the magnitude of ka at any specified salt concentration (e.g., approximately 10(6)-fold at 0.2 M) . Replacement of NaCl by NaOAc does not significantly affect Ska (Ska = -12.0 +/- 0.7 in NaOAc) and (by extrapolation) increased ka by approximately 80-fold at any fixed {Na+} . In the absence of Mg2+, replacement of NaCl by NaOAc is found to increase the half-life of the open complex by approximately 560-fold at fixed {Na+} without affecting Skd {Skd = 7.6 +/- 0.1 in NaOAc; in NaCl, Skd = 7.7 +/- 0.2 (Roe & Record, 1985)} . Replacement of NaCl by MgCl2 drastically reduces both Skd and the half-life of the open complex at any salt concentration below approximately 0.2 M . Strikingly, Skd = 0.4 +/- 0.1 in MgCl2, indicating that the net uptake of Mg2+ ions in the kinetically significant steps in dissociation of the open complex is much smaller than that expected by analogy with the uptake of approximately 8 Na+ ions in the corresponding steps in NaCl . In NaCl/MgCl2 mixtures, at a constant {NaCl} in the range 0.1-0.2 M, initial addition of MgCl2 (0.5 mM less than or equal to {MgCl2} less than or equal to 1 mM) increases the half-life of the open complex; further addition of MgCl2 causes the half-life to decrease, though the effect of {MgCl2} on kd is always less than that predicted by a simple competitive model . The observed effects of MgCl2 on Skd and kd differ profoundly from those expected from the behavior of kd and Skd in NaCl and NaOAc and indicate that the role of Mg2+ in dissociation is not merely that of a nonspecific divalent competitor with RNAP for interactions with DNA phosphates and of a DNA helix-stabilizer, both of which should cause kd to increase monotonically with increasing {Mg2+}.(ABSTRACT TRUNCATED AT 400 WORDS) Mol Biol Evol, 1992 Sep, 9(5), 787 - 805 Metabolic compartmentation of vertebrate glutamine synthetase: putative mitochondrial targeting signal in avian liver glutamine synthetase; Campbell JW et al.; The evolution of uricoteley as a mechanism for hepatic ammonia detoxication in vertebrates required targeting of glutamine synthetase (GS) to liver mitochondria in the sauropsid line of descent leading to the squamate reptiles and archosaurs . Previous studies have shown that in birds and crocodilians, sole survivors of the archosaurian line, hepatic GS is translated without a transient, N-terminal targeting signal common to other mitochondrial matrix proteins . To identify a putative internal targeting sequence in the avian enzyme, the amino acid sequence of chicken liver GS was derived by a combination of sequencing of cloned cDNA, direct sequencing of mRNA, and sequencing of polymerase chain reaction (PCR) products amplified from reverse-transcribed mRNA . Analysis of the first 20 or so N-terminal amino acids of the derived sequence for the chicken enzyme shows that they are devoid of acidic amino acids, contain several hydroxy amino acids, and can be predicted to form a positively charged, amphipathic helix, all of which are characteristic properties of mitochondrial targeting signals . A comparison of the N-terminus of chicken GS with the N-termini of cytosolic mammalian GSs indicates that at least three amino acid replacements may have been responsible for converting the N-terminus of the cytosolic mammalian enzyme into a mitochondrial targeting signal . Two of these, His15 and Lys19, result in additional positive charges, as well as in changes in hydrophilicity . Both could have resulted from third-base-codon substitutions . A third replacement, Ala12, may contribute to the helicity of the N-terminus of the chicken enzyme . The N-terminus of the cytosolic chicken brain GS (positions 1-36) was found to be identical to that of the liver enzyme . The complete sequence of chicken retinal GS is also identical to that of the liver enzyme . GS is coded by a single gene in birds, so these sequence data suggest that, unlike the situation in other tissue-specific compartmental isozymes, differential targeting of avian GS to the mitochondrial or cytosolic compartments is not dependent on the sequence of the primary translation product of its mRNA but may involve some other tissue-specific factor(s). Nucleic Acids Res, 1992 Sep 11, 20(17), 4599 - 606 Integration of image analysis and robotics into a fully automated colony picking and plate handling system; Jones P et al.; We describe here the integration of image analysis and robotics to produce a fully automated colony picking/plate handling system . Biological tests were performed to verify its performance in terms of sterilisation and accuracy of picking . The machine was then used by a single operative to pick a 36,000 clone cDNA library in approximately 42 hrs over 5 days. Nucleic Acids Res, 1992 Sep 11, 20(17), 4591 - 8 Construction and use of lambda PL promoter vectors for direct cloning and high level expression of PCR amplified DNA coding sequences; Cheng X et al.; A set of plasmid vectors which allow single-step cloning and expression of PCR-amplified DNA coding sequences has been constructed . The vectors contain the phage lambda PL promoter, a synthetic translation initiation region (TIR), and convenient cloning sites . The cloning sites provide all or part of an AUG translation initiation codon and facilitate the precise fusion of target DNA sequences to vector transcriptional and translational signals . The vectors were constructed with synthetic TIRs because there is evidence which suggests that the efficiency of the phage lambda cII gene TIR present in the parental vector depends strongly on information contained within the cII N-terminal coding sequence . Bovine brain 14-3-3 eta chain cDNA was PCR-amplified and used to demonstrate the expression capacity of the newly constructed vectors . A significant increase in expression of 14-3-3 protein was observed when synthetic TIRs were used in the place of the cII TIR . Expression levels vary from 15% to 48% of total cell protein . The effects of a reported translational enhancer from phage T7 on expression of the 14-3-3 protein are also discussed . The vectors should be generally useful for high level heterologous protein expression in Escherichia coli. Nucleic Acids Res, 1992 Sep 11, 20(17), 4547 - 51 Chemical and enzymatic incorporation of N2-(p-n-butylphenyl)-2'-deoxyguanosine into an oligodeoxyribonucleotide; Misra HK et al.; An 18mer oligodeoxyribonucleotide containing a N2-(p-n-butylphenyl)-2'-deoxyguanosine (BuPdG) residue at the 3' end has been synthesized by both chemical and enzymatic methods . Chemical synthesis involved attachment of 5'-DMT-BuPdG as the 3'-H-phosphonate to uridine-controlled pore glass (CPG), followed by extension via H-phosphonate chemistry . After oxidation of the backbone, deprotection of bases, and removal from CPG, the uridine residue was removed by periodate cleavage and beta-elimination . The resulting oligomer 3'-phosphate was digested with alkaline phosphatase to give the free BuPdG-18mer . E.coli DNA polymerase I (Klenow) incorporated BuPdGTP at the 3' end of the corresponding 17mer primer annealed to a complementary 29mer template, and the properties of this product were identical to those of chemically synthesized BuPdG-18mer . E.coli DNA polymerase I (Klenow) was unable to extend the BuPdG-18mer, and the 3' to 5' exonuclease activity of the enzyme was unable to remove the modified nucleotide. Nucleic Acids Res, 1992 Sep 11, 20(17), 4507 - 13 Preparation of 13C and 15N labelled RNAs for heteronuclear multi-dimensional NMR studies; Nikonowicz EP et al.; A procedure is described for the efficient preparation of isotopically enriched RNAs of defined sequence . Uniformly labelled nucleotide 5'triphosphates (NTPs) were prepared from E.coli grown on 13C and/or 15N isotopically enriched media . These procedures routinely yield 180 mumoles of labelled NTPs per gram of 13C enriched glucose . The labelled NTPs were then used to synthesize RNA oligomers by in vitro transcription . Several 13C and/or 15N labelled RNAs have been synthesized for the sequence r(GGCGCUUGCGUC) . Under conditions of high salt or low salt, this RNA forms either a symmetrical duplex with two U.U base pairs or a hairpin containing a CUUG loop respectively . These procedures were used to synthesize uniformly labelled RNAs and a RNA labelled only on the G and C residues . The ability to generate milligram quantities of isotopically labelled RNAs allows application of multi-dimensional heteronuclear magnetic resonance experiments that enormously simplify the resonance assignment and solution structure determination of RNAs . Examples of several such heteronuclear NMR experiments are shown. Nucleic Acids Res, 1992 Sep 11, 20(17), 4417 - 21 Structure of the human DNA repair gene HAP1 and its localisation to chromosome 14q 11.2-12; Robson CN et al.; Apurinic/apyrimidinic (AP) sites are pre-mutagenic DNA lesions which occur spontaneously and following exposure of cells to ionising radiation or chemical mutagens . HAP1 (Human AP endonuclease 1), the major enzyme in human cells initiating repair of AP sites, shows strong sequence homology to DNA repair enzymes from bacteria, Drosophila and other mammalian species . We have cloned the HAP1 gene and determined its complete nucleotide sequence . The site of transcription initiation has been mapped to 452 bp upstream of the ATG initiation codon in the genomic DNA . The HAP1 gene consists of five exons and is unusually small (less than 2.6 kb from transcription initiation site to polyadenylation sequence) with 54% of the protein coding region and the entire 3' untranslated region contained within a single exon . The first exon is non-coding . Regions of three exons show sequence homology to the E.coli xth (exonuclease III) gene . Using in situ hybridisation, the HAP1 gene has been localised to human chromosome 14q 11.2-12. Nucleic Acids Res, 1992 Sep 11, 20(17), 4397 - 400 Conformity of RNAs that interact with tetranucleotide loop binding proteins; Zwieb C; A group of RNA binding proteins, termed tetraloop binding proteins, includes ribosomal protein S15 and protein SRP19 of signal recognition particle . They are primary RNA binding proteins, recognize RNA tetranucleotide loops with a GNAR consensus motif, and require a helical region located adjacent to the tetraloop . Closely related RNA structures that fit these criteria appear in helix 6 of SRP RNA, in helices 22 and 23A of 16 S ribosomal RNA, and, as a pseudoknot, in the regulatory region of the rpsO gene. Gene, 1992 Sep 10, 118(2), 239 - 45 Production of mature bovine pancreatic ribonuclease in Escherichia coli; Tarragona-Fiol A et al.; The coding sequence for the bovine pancreatic ribonuclease (RNase) precursor has been cloned and produced in Escherichia coli using the polymerase chain reaction (PCR) technique . A PCR amplification has been carried out utilizing as template the recombinant plasmid, pQR138, which contains the coding sequence for the RNase precursor, and primers that allow for the addition of new sequences at the 5' and 3' ends of the coding sequence . The resultant fragment contains two coding sequences, one for a hexapeptide and the other for pre-RNase . This fragment has been cloned into the expression vector, pKK223.3, under the control of the tac promoter, to form a two-cistron vector . Upon induction with IPTG, E . coli cells harboring this construct generate a bicistronic mRNA which upon translation produces a hexapeptide and pre-RNase . The RNase precursor is efficiently translocated into the periplasmic space of E . coli . Upon translocation, the signal sequence is removed generating mature RNase . Formation of the disulfide bridges in RNase is facilitated by the oxidative environment of the periplasm and a fully active protein is obtained . RNase produced in E . coli has been purified to homogeneity by cation-exchange chromatography, and the removal of the signal sequence has been verified by N-terminal sequencing . The total process from inoculation of media to obtaining pure and fully active recombinant RNase is achieved in 48 h. Gene, 1992 Sep 10, 118(2), 171 - 9 Identification of a genomic DNA fragment containing the Drosophila melanogaster ovarian tumor gene (otu) and localization of regions governing its expression; Comer AR et al.; We have identified a genomic DNA fragment which restores fertility to mutants of the ovarian tumor locus (otu) of Drosophila melanogaster . Germ-line transformants bearing this fragment express otu mRNA with the same tissue specificity as, and at levels comparable to, the wild-type otu gene . Transcription from the otu promoter, P(otu), which lacks a TATA element, appears to be initiated at multiple transcription start points (tsp) within an 80-bp region . Deletion of sequences upstream of the tsp indicates that a region between nucleotides -190 and -310 is required for proper expression from the otu gene . A DNA fragment containing 452 bp upstream and 126 bp downstream from the tsp is able to direct expression of the Escherichia coli lacZ gene in the germ cells of the ovary and testis, indicating that cis-acting regulatory elements governing these expression patterns are located in a 578-bp region surrounding the multiple tsp. Gene, 1992 Sep 10, 118(2), 153 - 62 The yptV1 gene encodes a small G-protein in the green alga Volvox carteri: gene structure and properties of the gene product; Fabry S et al.; Small G-proteins encoded by ras-like genes are ubiquitous in eukaryotic cells . These G-proteins are believed to play a role in central processes, such as signal transduction, cell differentiation and membrane vesicle transport . By screening genomic and cDNA libraries of the colonial alga, Volvox carteri f . nagariensis, with ypt DNA probes from Zea mays, we have identified the first member of a ypt gene family, yptV1, within a green alga . The 1538-bp yptV1 gene of V . carteri consists of nine exons and eight introns and has three potential polyadenylation sites 210, 420 and 500 bp downstream from the UGA stop codon . The derived 203-amino-acid polypeptide, YptV1, exhibits 81% similarity with Ypt1 from mouse, with the corresponding genes sharing four identical intron positions . Recombinant YptV1 (reYptV1) produced in Escherichia coli retains the ability to bind GTP after SDS-PAGE and immobilization on nitrocellulose . Immunological studies using polyclonal antibodies against reYptV1 indicate that the protein is present in the membrane fraction of a V . carteri extract and is expressed throughout the whole life-cycle of the alga . Similar to other Ras-like proteins, YptV1 contains two conserved C-terminal cysteine residues suggesting post-translational modification(s), such as isoprenylation or palmitoylation, required for membrane anchoring . The presumptive role of YptV1 in cytoplasmic vesicle transport is briefly discussed. Biochemistry, 1992 Sep 8, 31(35), 8196 - 200 Enhanced catalysis by active-site mutagenesis at aspartic acid 153 in Escherichia coli alkaline phosphatase; Matlin AR et al.; Bacterial alkaline phosphatase catalyzes the hydrolysis and transphosphorylation of phosphate monoesters . Site-directed mutagenesis was used to change the active-site residue Asp-153 to Ala and Asn . In the wild-type enzyme Asp-153 forms a second-sphere complex with Mg2+ . The activity of mutant enzymes D153N and D153A is dependent on the inclusion of Mg2+ in the assay buffer . The steady-state kinetic parameters of the D153N mutant display small enhancements, relative to wild type, in buffers containing 10 mM Mg2+ . In contrast, the D153A mutation gives rise to a 6.3-fold increase in kcat, a 13.7-fold increase in kcat/Km (50 mM Tris, pH 8), and a 159-fold increase in Ki for Pi (1 M Tris, pH 8) . In addition, the activity of D153A increases 25-fold as the pH is increased from 7 to 9 . D153A hydrolyzes substrates with widely differing pKa's of their phenolic leaving groups (PNPP and DNPP), at similar rates . As with wild type, the rate-determining step takes place after the initial nucleophilic displacement (k2) . The increase in kcat for the D153A mutant indicates that the rate of release of phosphate from the enzyme product complex (k4) has been enhanced. Biochemistry, 1992 Sep 8, 31(35), 8221 - 8 Conformational changes of HIV reverse transcriptase subunits on formation of the heterodimer: correlation with kcat and Km; Anderson SF et al.; The reverse transcriptase (RT) from the human immunodeficiency virus (HIV) is initially expressed as a 66-kDa protein and is subsequently proteolytically processed in vivo to form a 66-kDa/51-kDa heterodimer . Comparison of circular dichroism spectra of the 66-kDa, 51-kDa, and heterodimeric forms of RT indicates that the conversion is accompanied by dramatic changes in subunit conformation . The mean residue ellipticity per subunit at 220 nm decreases from -10.7 x 10(3) deg cm2 dmol-1 for the 66-kDa protein to -6 x 10(3) deg cm2 dmol-1 for the heterodimer . The same loss of ellipticity is observed whether the heterodimer is produced by proteolysis or by mixing a separately-expressed cloned 51-kDa subunit with the 66-kDa protein . Comparison with the spectrum of the cloned 51-kDa protein suggests that much of the conformational change arises from formation of the 51-kDa subunit but substantial changes occur in the remaining 66-kDa subunit as well . A kinetic analysis was performed to correlate these conformational changes with changes in enzyme function . Application of an integrated Michaelis-Menten equation to the catalysis of poly(dT) formation using a d(pT)20-poly(rA) primer-template shows that the kcat for the heterodimer is approximately half that of the 66 kDa enzyme, decreasing from 2.9 to 1.2 nucleotides/s upon formation of the heterodimer . However, km values for the primer-template decrease from 0.54 to 0.12 microM upon heterodimer formation . Thus, kcat/Km is 2-fold larger for the heterodimer, giving it a distinct catalytic advantage at undersaturating concentrations of enzyme and primer-template.(ABSTRACT TRUNCATED AT 250 WORDS) Thromb Haemost, 1992 Sep 7, 68(3), 306 - 9 Effect of DDAVP on endotoxin-induced intravascular coagulation in rabbits; Paloma MJ et al.; We have evaluated the effect of 1-Deamino-8D-arginine vasopressin (DDAVP) on an experimental model of intravascular coagulation (DIC) induced in rabbits by injection of 20 micrograms kg-1 h-1 during 6 h of E . coli lipopolysaccharide . DDAVP significantly ameliorated the platelet drop and fibrinogen decrease (p less than 0.01) induced by endotoxin in control animals . A significant reduction in factor XII consumption (p less than 0.01) and a decrease in the generation of endotoxin induced PAI-1 activity in rabbits circulation was also observed (p less than 0.005) . Moreover, fibrin deposition in kidneys of rabbits receiving DDAVP was significantly reduced as compared to control animals . Finally, the mortality rate in the control group was significantly higher than in DDAVP-treated rabbits (p less than 0.01) . The hemostatic changes induced by DDAVP correlated with lower fibrin deposition and reduction in mortality rates. FEBS Lett, 1992 Sep 7, 309(2), 157 - 60 Identification of heme macrocycle type by near-infrared magnetic circular dichroism spectroscopy at cryogenic temperatures; Peng Q et al.; The electron paramagnetic resonance (EPR) and near-infrared magnetic circular dichroism (MCD) spectra of the azide and cyanide adducts of nitrimyoglobin and hydroperoxidase II from Escherichia coli have been measured at cryogenic temperatures . For the first time, ligand-to-metal charge-transfer transitions in the near-infrared have been observed for an Fe(III)-chlorine system . It is shown that near-ultraviolet-to-visible region electronic spectra of 'green' hemes such as these are an unreliable indicator of macrocycle type . However, the combined application of EPR and near-infrared MCD spectroscopies clearly distinguishes between the porphyrin-containing nitrimyoglobin and the chlorine-containing hydroperoxidase II. FEBS Lett, 1992 Sep 7, 309(2), 127 - 9 The formate complex of the cytochrome bo quinol oxidase of Escherichia coli exhibits a 'g = 12' EPR feature analogous to that of 'slow' cytochrome oxidase; Calhoun MW et al.; The cytochrome bo quinol oxidase of Escherichia coli is homologous in sequence and in structure to cytochrome aa3 type cytochrome oxidase in subunit I, which contains the catalytic core . The cytochrome bo enzyme forms a formate complex which exhibits 'g = 12' and 'g = 2.9' EPR signals at X band; similar signals have previously been observed only in association with the 'slow' and formate-ligand states of cytochrome oxidase . These signals arise from transitions within integral spin multiples identified with the homologous heme-copper binuclear catalytic centers in both enzymes. J Mol Biol, 1992 Sep 5, 227(1), 72 - 80 Effect of terminal non-homology on intramolecular recombination of linear plasmid substrates in Escherichia coli; Luisi-DeLuca C et al.; Circular dimer plasmids linearized with a restriction endonuclease undergo intramolecular recombination to yield recombinant circular monomers at high efficiency by a recA-independent mechanism in Escherichia coli recB recC sbcA mutants . The rate of this reaction is at least 1000-fold higher than the recombination rate observed for circular plasmid recombination substrates in the same mutants . Three potential models have been previously proposed to explain the recombination events observed . The validity of these models was tested in recA recB recC sbcA mutants using additional recombination substrates . These substrates, when linearized by incubation with an appropriate restriction enzyme, contain non-homologous adenovirus 2 DNA on one or both ends . The data indicate that terminal non-homology does not significantly affect the efficiency of recovering recombinants . In contrast to many recombination models proposed that involve the invasion of homologous duplex DNA by single-stranded DNA ends, the intramolecular recombination reaction studied here does not appear to involve direct pairing from the end(s) of the substrate DNA . Furthermore, the results are consistent with a model proposing that pairing and strand exchange occur between two homologous duplex regions within the linear dimer molecule. J Mol Biol, 1992 Sep 5, 227(1), 347 - 51 Crystallization and crystallographic characterization of the iron-sulfur-containing DNA-repair enzyme endonuclease III from Escherichia coli; Kuo CF et al.; Endonuclease III from Escherichia coli is an iron-sulfur enzyme possessing both DNA N-glycosylase and apurinic/apyrimidinic lyase activities . It could serve to repair damaged thymine residues in DNA via base excision-repair . We have crystallized endonuclease III by a combination of dialysis and seeding techniques after exploration of a wide variety of precipitants which failed to yield macroscopic crystals . Important features of the optimized crystallization include: the use of 5 to 10% glycerol, a temperature of 15 degrees C, controlled dialysis to decrease ionic strength and macroseeding using a 200 mM-NaCl transfer buffer to dissolve microcrystalline contamination . The crystals belong to space group P2(1)2(1)2(1) with unit cell dimensions of a = 48.5 A, b = 65.8 A, c = 86.8 A, alpha = beta = gamma = 90 degrees, have one 23 kDa monomer per asymmetric unit, and diffract to 1.84 A . A native anomalous Patterson map located the iron-sulfur cluster and reaffirmed its existence . The reported crystallization procedures ensure an ample supply of crystals for the extensive heavy-atom derivative search necessary for this labile iron-sulfur enzyme . The elucidation of endonuclease III structure will facilitate not only the understanding of glycosylase and lyase mechanisms but also the structure and function of this new class of iron-sulfur proteins. J Mol Biol, 1992 Sep 5, 227(1), 334 - 46 Structural data suggest that the active and inactive forms of the RecA filament are not simply interconvertible; Yu X et al.; We have used electron microscopy to examine the two major conformational states of the helical filament formed by the RecA protein of Escherichia coli . The compressed filament, formed in the absence of a nucleotide cofactor either as a self-polymer or on a single-stranded DNA molecule, is characterized in solution by about 6.1 subunits per turn of a 76 A pitch helix, and appears to be inactive with respect to all RecA activity . The active state of the filament, formed with ATP or an ATP analog on either a single or double-stranded DNA substrate, has about 6.2 subunits per turn of a 94 A pitch helix . Measurements of the contour length of RecA-covered single-stranded DNA circles in ice, formed in the absence of nucleotide cofactor, indicate that each RecA subunit binds five bases, in contrast to the three bases or base-pairs per subunit in the active state . The different stoichiometries of DNA binding suggests that the two polymeric forms are not interconvertible, as has been suggested on biochemical grounds . A three-dimensional reconstruction of the inactive state shows the same general features as the 83 A pitch filament present in the RecA crystal . This structural similarity and the fact that the crystal does not contain ATP or DNA suggests that the crystal structure is more similar to the compressed filament than the active, extended filament. J Mol Biol, 1992 Sep 5, 227(1), 283 - 92 Crystal structure of glycinamide ribonucleotide transformylase from Escherichia coli at 3.0 A resolution . A target enzyme for chemotherapy; Chen P et al.; The atomic structure of glycinamide ribonucleotide transformylase, an essential enzyme in purine biosynthesis, has been determined at 3.0 A resolution . The last three C-terminal residues and a sequence stretch of 18 residues (residues 113 to 130) are not visible in the electron density map . The enzyme forms a dimer in the crystal structure . Each monomer is divided into two domains, which are connected by a central mainly parallel seven-stranded beta-sheet . The N-terminal domain contains a Rossmann type mononucleotide fold with a phosphate ion bound to the C-terminal end of the first beta-strand . A long narrow cleft stretches from the phosphate to a conserved aspartic acid, Asp144, which has been suggested as an active-site residue . The cleft is lined by a cluster of residues, which are conserved between bacterial, yeast, avian and human enzymes, and likely represents the binding pocket and active site of the enzyme . GAR Tfase binds a reduced folate cofactor and glycinamide ribonucleotide for the catalysis of one of the initial steps in purine biosynthesis . Folate analogs and multi-substrate inhibitors of the enzyme have antineoplastic effects and the structure determination of the unliganded enzyme and enzyme-inhibitor complexes will aid the development of anti-cancer drugs. J Mol Biol, 1992 Sep 5, 227(1), 197 - 213 Domain closure in mitochondrial aspartate aminotransferase; McPhalen CA et al.; The subunits of the dimeric enzyme aspartate aminotransferase have two domains: one large and one small . The active site lies in a cavity that is close to both the subunit interface and the interface between the two domains . On binding the substrate the domains close together . This closure completely buries the substrate in the active site and moves two arginine side-chains so they form salt bridges with carboxylate groups of the substrate . The salt bridges hold the substrate close to the pyridoxal 5'-phosphate cofactor and in the right position and orientation for the catalysis of the transamination reaction . We describe here the structural changes that produce the domain movements and the closure of the active site . Structural changes occur at the interface between the domains and within the small domain itself . On closure, the core of the small domain rotates by 13 degrees relative to the large domain . Two other regions of the small domain, which form part of the active site, move somewhat differently . A loop, residues 39 to 49, above the active site moves about 1 A less than the core of the small domain . A helix within the small domain forms the "door" of the active site . It moves with the core of the small domain and, in addition, shifts by 1.2 A, rotates by 10 degrees, and switches its first turn from the alpha to the 3(10) conformation . This results in the helix closing the active site . The domain movements are produced by a co-ordinated series of small changes . Within one subunit the polypeptide chain passes twice between the large and small domains . One link involves a peptide in an extended conformation . The second link is in the middle of a long helix that spans both domains . At the interface this helix is kinked and, on closure, the angle of the kink changes to accommodate the movement of the small domain . The interface between the domains is formed by 15 residues in the large domain packing against 12 residues in the small domain and the manner in which these residues pack is essentially the same in the open and closed structures . Domain movements involve changes in the main-chain and side-chain torsion angles in the residues on both sides of the interface . Most of these changes are small; only a few side-chains switch to new conformations.(ABSTRACT TRUNCATED AT 400 WORDS) J Biol Chem, 1992 Sep 5, 267(25), 17809 - 19 Membrane topology of the pBR322 tetracycline resistance protein . TetA-PhoA gene fusions and implications for the mechanism of TetA membrane insertion; Allard JD et al.; The tetracycline resistance gene of pBR322 encodes a 41-kDa inner membrane protein (TetA) that acts as a tetracycline/H+ antiporter . Based on hydrophobicity profiles, we identified 12 potential transmembrane segments in TetA . We used oligonucleotide deletion mutagenesis to fuse alkaline phosphatase (PhoA) to the C-terminal edge of each of the predicted periplasmic and cytoplasmic segments of TetA . In general, the PhoA activities of the TetA-PhoA fusions support a TetA topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm . However, several TetA-PhoA fusions have unexpected properties . One PhoA fusion to a predicted cytoplasmic segment (C6) has high activity . However, previous protease accessibility studies on the related Tn10 TetA protein indicated that C6 is cytoplasmically localized as predicted (Eckert, B., and Beck, C . F . (1989) J . Biol . Chem . 264, 11663-11670) . PhoA fusions to three predicted periplasmic segments (P1, P2, and P5) have low to intermediate activity . In each case, the preceding transmembrane segment (TM1, TM3, and TM9) contains an aspartate (Asp17, Asp86, and Asp287) . We show that these aspartates act like signal sequence mutations for PhoA export: (i) Asp----Ala mutations increase the PhoA activity of fusions to P1, P2, and P5 . (ii) The signal sequence mutation suppressor prlA402 increases the PhoA activity of these same fusions . We also show that the aspartates in TM1, TM3, and TM9 are critical for wild-type TetA function; they are conserved in related TetA proteins and Asp----Ala mutations reduce or eliminate tetracycline resistance . The properties of the anomalous TetA-PhoA fusions suggest that TetA sequences C-terminal to some cytoplasmic and periplasmic segments are required for the proper localization of those segments, i.e . long range interactions may be more important in determining the membrane topology of TetA than suggested in some general models. J Biol Chem, 1992 Sep 5, 267(25), 17766 - 72 Effects of phospholipid and GTP on recombinant ADP-ribosylation factors (ARFs) . Molecular basis for differences in requirements for activity of mammalian ARFs; Price SR et al.; ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that were first identified based on their ability to stimulate the cholera toxin-catalyzed ADP-ribosylation of Gs alpha and thus activate adenylyl cyclase . Proteins with ARF activity have been characterized from different mammalian tissues and exhibited different requirements for activity, stability, and phospholipid . Based on molecular cloning and mRNA distribution, at least six mammalian ARFs, which fall into three classes, have been identified . To test whether individual ARFs might have different requirements for optimal activity, as judged by their ability to enhance cholera toxin ADP-ribosyltransferase activity, four ARFs from classes I, II, and III were produced as recombinant proteins in Escherichia coli and characterized . Recombinant bovine ARF 2 (rARF 2) and human ARF 3 (rARF 3) (class I), human ARF 5 (rARF 5, class II), and human ARF 6 (rARF 6, class III) differed in the effects of phospholipid and detergent on their ability to enhance cholera toxin activity; rARFs 2, 3, and 5 required dimyristoylphosphatidylcholine (DMPC) and cholate, whereas rARF 6 did not require phospholipid/detergent for activity . Further characterization of two of the more divergent ARFs (ARFs 2 and 6) showed that both exhibited guanosine 5'-O-(3-thio)triphosphate binding which was enhanced by DMPC/cholate . In the transferase assay, rARF 2 required approximately 4 microM GTP for half-maximal stimulation of toxin activity, whereas rARF 6 required 0.05 microM GTP . rARF 6 exhibited a delay in activation of toxin not detected with rARF 2 that may be related to a requirement for guanine nucleotide exchange and/or GTP binding . These findings are consistent with the conclusion that the highly conserved members of the ARF family have different requirements for optimal activity. J Biol Chem, 1992 Sep 5, 267(25), 17679 - 87 Escherichia coli serine hydroxymethyltransferase . The role of histidine 228 in determining reaction specificity; Stover P et al.; Serine hydroxymethyltransferase has a conserved histidine residue (His-228) next to the lysine residue (Lys-229) which forms the internal aldimine with pyridoxal 5'-phosphate . This histidine residue is also conserved at the equivalent position in all amino acid decarboxylases and tryptophan synthase . Two mutant forms of Escherichia coli serine hydroxymethyltransferase, H228N and H228D, were constructed, expressed, and purified . The properties of the wild type and mutant enzymes were studied with substrates and substrate analogs by differential scanning calorimetry, circular dichroism, steady state kinetics, and rapid reaction kinetics . The conclusions of these studies were that His-228 plays an important role in the binding and reactivity of the hydroxymethyl group of serine in the one-carbon-binding site . The mutant enzymes utilize substrates and substrate analogs more effectively for a variety of alternate non-physiological reactions compared to the wild type enzyme . As one example, the mutant enzymes cleave L-serine to glycine and formaldehyde when tetrahydropyteroylglutamate is replaced by 5-formyltetrahydropteroylglutamate . The released formaldehyde inactivates these mutant enzymes . The loss of integrity of the one-carbon-binding site with L-serine in the two mutant forms of the enzyme may be the result of these enzymes not undergoing a conformational change to a closed form of the active site when serine forms the external aldimine complex. J Biol Chem, 1992 Sep 5, 267(25), 17920 - 4 An active covalently linked dimer of human interferon-gamma . Subunit orientation in the native protein; Lunn CA et al.; We have constructed and expressed a covalently linked head to tail dimer of human interferon-gamma (IFN-gamma) in which two monomers are joined head to tail via a rigid peptide hinge using genetic engineering techniques . The hinge was derived from the human immunoglobin IgA1 sequence (Hallewell, R.A., Laria, I., Tabrizi, A., Carlin, G., Getzoff, E.D., Tainer, J.A., Cousens, L.S., and Mullenbach, G.T . (1989) J . Biol . Chem . 264, 5260-5268) . Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the polypeptide produced by this construction migrates as a 30,000 polypeptide species . The protein elutes as a single species by molecular sieve chromatography under native conditions . The covalently linked dimer exhibits one-half the antiviral activity of native dimeric IFN-gamma; receptor binding assays show the covalently linked dimer binds to the IFN-gamma receptor with one-half the avidity of native IFN-gamma . This difference is not due to conformational differences between the two molecules, as the aromatic region of the NMR spectrum of the purified covalently linked dimer is identical with that of the wild type protein . From these data, we suggest that human IFN-gamma associates in a head to tail dimer in its active configuration . Regions of IFN-gamma are contiguous with the amino and carboxyl termini and are obscured by the hinge peptide in the covalently linked dimer . Our studies demonstrate that these regions may be important for receptor-ligand interaction. J Biol Chem, 1992 Sep 5, 267(25), 17693 - 700 Active site of (A)BC excinuclease . II . Binding, bending, and catalysis mutants of UvrB reveal a direct role in 3' and an indirect role in 5' incision; Lin JJ et al.; UvrB plays a central role in (A)BC excinuclease . To study its role in the incision reactions, conserved His and Asp residues in this subunit were mutagenized . All His and the majority of Asp mutants behaved like wild-type protein in vivo and in vitro . However, three mutants, D337A, D478A, and D510A, either completely or partially abolished UvrB activity . All three mutant proteins associate with UvrA normally but D337A and D510A were unable to bind to DNA specifically . The UvrB-D478A mutant bound to DNA specifically but failed to denature and kink the DNA . However, UvrB-D478A was efficiently loaded onto DNA preincised at the 3' site and promoted near-normal incision by UvrC at the 5' site . We propose that D478 is involved in bending DNA and catalysis of the 3' incision and that the 3' incision precedes the 5' incision . UvrB which is missing the carboxyl-terminal 43 amino acids binds to, and kinks DNA but is unable to make the 3' incision suggesting that it is missing a residue involved in catalysis . This residue was identified to be E639 by site-specific mutagenesis. J Biol Chem, 1992 Sep 5, 267(25), 17688 - 92 Active site of (A)BC excinuclease . I . Evidence for 5' incision by UvrC through a catalytic site involving Asp399, Asp438, Asp466, and His538 residues; Lin JJ et al.; (A)BC excinuclease of Escherichia coli removes damaged nucleotides from DNA by hydrolyzing the 8th phosphodiester bond 5' and the 15th phosphodiester bond 3' to the modified base . The activity results from the ordered action of UvrA, UvrB, and UvrC proteins . The role of UvrA is to help assemble the UvrB.DNA complex, and it is not involved in the actual incision reactions which are carried out by UvrB and UvrC . To investigate the role of UvrC in the nuclease activity a subset of His, Asp, and Glu residues in the C-terminal half of the protein were mutagenized in vitro . The effect of these mutations on UV resistance in vivo and incision activity in vitro were investigated . Mutations, H538F, D399A, D438A, and D466A conferred extreme UV sensitivity . Enzyme reconstituted with these mutant proteins carried out normal 3' incision but was completely defective in 5' incision activity . Our data suggest that UvrC makes the 5' incision by employing a mechanism whereby the three carboxylates acting in concert with H538 and a Mg2+ ion facilitate nucleophilic attack by an active site water molecule. J Biol Chem, 1992 Sep 5, 267(25), 18175 - 81 Characterization and epitope mapping of monoclonal antibodies directed against the beta' subunit of the Escherichia coli RNA polymerase; Luo J et al.; Monoclonal antibodies (mAbs) raised against the beta' subunit of the Escherichia coli RNA polymerase were used to probe the structure and function of this subunit . Of the five anti-beta' monoclonal antibodies studied, only mAb 311G2 is a strong inhibitor of RNA polymerase activity . This antibody binds to an epitope which is exposed in both the assembled holoenzyme and isolated beta' subunit . In contrast, the null antibodies bind to the free beta' subunit but very weakly to native RNA polymerase . It would appear that the beta' domain in which their epitopes reside is either conformationally altered or blocked due to interaction with other subunits in native RNA polymerase . In order to locate the positions of the epitopes for these five monoclonal antibodies, a series of overlapping deletion mutants have been constructed by partial restriction and religation of the beta' gene present in pT7 beta' (Zalenskaya, K., Lee, J., Gujuluva, C . N., Shin, Y . K., Slutsky, M., nd Goldfarb, A . (1990) Gene 89, 7-12) . The presence of the epitopes for each of the anti-beta' monoclonal antibodies was assessed by Western blotting . The results indicate that the epitopes for mAb 340F11, mAb 370F3, mAb 371D6, and mAb 372B2 are located between amino acids 817-876 . This region may be important in enzyme assembly or subunit-subunit interaction . The epitope for the inhibitory antibody, mAb 311G2, is located between amino acids 1047-1093 . This region may be involved in the catalytic function of RNA polymerase. J Biol Chem, 1992 Sep 5, 267(25), 17773 - 9 Effects of the chaperonin GroE on the refolding of tryptophanase from Escherichia coli . Refolding is enhanced in the presence of ADP; Mizobata T et al.; The refolding of the tetrameric enzyme tryptophanase was facilitated by the chaperonin GroE . Maximum refolding yield of tryptophanase molecules (about 80%) was attained in the presence of a 15-fold excess of GroE 21-mer over tryptophanase monomer . The GroEL subunit was required for this improvement in refolding yield, whereas the GroES subunit was not . Light scattering experiments of the refolding reaction revealed that GroE bound to tryptophanase folding intermediates and suppressed their aggregation . The presence of ATP was required for the efficient dissociation of tryptophanase from GroEL . However, our experiments indicated that tryptophanase dissociated readily from GroEL in the presence of not only ATP, but also in the presence of non-hydrolyzable ATP analogues such as ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)) and AMP-PNP (adenyl-5'-yl imidodiphosphate) as well . Surprisingly, the release of tryptophanase from GroEL was facilitated in the presence of ADP as well . We concluded that the binding of nucleotides such as ATP and ADP changed the conformation of GroEL and facilitated the dissociation of tryptophanase molecules . The conformation formed in the presence of ADP was distinct from the conformation formed in the presence of ATP, as shown by the selective dissociation of various folding proteins from the two conformations. J Biol Chem, 1992 Sep 5, 267(25), 17631 - 4 Chaperonin cpn60 from Escherichia coli protects the mitochondrial enzyme rhodanese against heat inactivation and supports folding at elevated temperatures; Mendoza JA et al.; The chaperonin protein cpn60 from Escherichia coli protects the monomeric, mitochondrial enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) against heat inactivation . The thermal inactivation of rhodanese was studied for four different states of the enzyme: native, refolded, bound to cpn60 in the form of a binary complex formed from unfolded rhodanese, and a thermally perturbed state . Thermal stabilization is observed in a range of temperatures from 25 to 48 degrees C . Rhodanese that had been inactivated by incubation at 48 degrees C, in the presence of cpn60 can be reactivated at 25 degrees C, upon addition of cpn10, K+, and MgATP . A recovery of about 80% was achieved after 1 h of the addition of those components . Thus, the enzyme is protected against heat inactivation and kept in a reactivable form if inactivation is attempted using the binary complex formed between rhodanese folding intermediate(s) and cpn60 . The chaperonin-assisted refolding of urea-denatured rhodanese is dependent on the temperature of the refolding reaction . However, optimal chaperonin assisted refolding of rhodanese observed at 25 degrees C, which is achieved upon addition of cpn10 and ATP to the cpn60-rhodanese complex, is independent of the temperature of preincubation of the complex, that was formed previously at low temperature . The results are in agreement with a model in which the chaperonin cpn60 interacts with partly folded intermediates by forming a binary complex which is stable to elevated temperatures . In addition, it appears that native rhodanese can be thermally perturbed to produce a state different from that achieved by denaturation that can interact with cpn60. J Mol Biol, 1992 Sep 5, 227(1), 38 - 53 ATP hydrolysis and the displaced strand are two factors that determine the polarity of RecA-promoted DNA strand exchange; Konforti BB et al.; When the recA protein (RecA) of Escherichia coli promotes strand exchange between single-stranded DNA (ssDNA) circles and linear double-stranded DNAs (dsDNA) with complementary 5' or 3' ends a polarity is observed . This property of RecA depends on ATP hydrolysis and the ssDNA that is displaced in the reaction since no polarity is observed in the presence of the non-hydrolyzable ATP analog, ATP gamma S, or in the presence of single-strand specific exonucleases . Based on these results a model is presented in which both the 5' and 3' complementary ends of the linear dsDNA initiate pairing with the ssDNA circle but only one end remains stably paired . According to this model, the association/dissociation of RecA in the 5' to 3' direction on the displaced strand determines the polarity of strand exchange by favoring or blocking its reinvasion into the newly formed dsDNA . Reinvasion is favored when the displaced strand is coated with RecA whereas it is blocked when it lacks RecA, remains covered by single-stranded DNA binding protein or is removed by a single-strand specific exonuclease . The requirement for ATP hydrolysis is explained if the binding of RecA to the displaced strand occurs via the dissociation and/or transfer of RecA, two functions that depend on ATP hydrolysis . The energy for strand exchange derives from the higher binding constant of RecA for the newly formed dsDNA as compared with that for ssDNA and not from ATP hydrolysis. Biochemistry, 1992 Aug 25, 31(33), 7736 - 40 Pertussis toxin-catalyzed ADP-ribosylation of G(o) alpha with mutations at the carboxyl terminus; Avigan J et al.; The guanine nucleotide-binding protein G(o alpha) has been implicated in the regulation of Ca2+ channels in neural tissues . Covalent modification of G(o alpha) by pertussis toxin-catalyzed ADP-ribosylation of a cysteine (position 351) four amino acids from the carboxyl terminus decouples G(o alpha) from receptor . To define the structural requirements for ADP-ribosylation, preparations of recombinant G(o alpha) with mutations within the five amino acids at the carboxyl terminus were evaluated for their ability to serve as pertussis toxin substrates . As expected, the mutant in which cysteine 351 was replaced by glycine (C351G) was not a toxin substrate . Other inactive mutants were G352D and L353 delta/Y354 delta . Mutations that had no significant effect on toxin-catalyzed ADP-ribosylation included G350D, G350R, Y354 delta, and L353V/Y354 delta . Less active mutants were L353G/Y354 delta, L353A/Y354 delta, and L353G . ADP-ribosylation of the active mutants, like that of wild-type G(o alpha), was enhanced by the beta gamma subunits of bovine transducin . It appears that three of the four terminal amino acids critically influence pertussis toxin-catalyzed ADP-ribosylation of G(o alpha). J Biol Chem, 1992 Aug 25, 267(24), 16841 - 7 The genes encoding the two carboxyltransferase subunits of Escherichia coli acetyl-CoA carboxylase; Li SJ et al.; We report characterization of the component proteins and molecular cloning of the genes encoding the two subunits of the carboxyltransferase component of the Escherichia coli acetyl-CoA carboxylase . Peptide mapping of the purified enzyme component indicates that the carboxyltransferase component is a complex of two nonidentical subunits, a 35-kDa alpha subunit and a 33-kDa beta subunit . The alpha subunit gene encodes a protein of 319 residues and is located immediately downstream of the polC gene (min 4.3 of the E . coli genetic map) . The deduced amino acid composition, molecular mass, and amino acid sequence match those determined for the purified alpha subunit . Six sequenced internal peptides also match the deduced sequence . The amino-terminal sequence of the beta subunit was found within a previously identified open reading frame of unknown function called dedB and usg (min 50 of the E . coli genetic map) which encodes a protein of 304 residues . Comparative peptide mapping also indicates that the dedB/usg gene encodes the beta subunit . Moreover, the deduced molecular mass and amino acid composition of the dedB/usg-encoded protein closely match those determined for the beta subunit . The deduced amino acid sequences of alpha and beta subunits show marked sequence similarities to the COOH-terminal half and the NH2-terminal halves, respectively, of the rat propionyl-CoA carboxylase, a biotin-dependent carboxylase that catalyzes a similar carboxyltransferase reaction reaction . Several conserved regions which may function as CoA-binding sites are noted. Science, 1992 Sep 4, 257(5075), 1392 - 5 Cloning of the 62-kilodalton component of basic transcription factor BTF2; Fischer L et al.; Cloning of the mammalian basic transcription factors serves as a major step in understanding the mechanism of transcription initiation . The 62-kilodalton component (p62) of one of these transcription factors, BTF2 was cloned and overexpressed . A monoclonal antibody to this polypeptide inhibited transcription in vitro . Immunoaffinity experiments demonstrated that the 62-kilodalton component is closely associated with the other polypeptides present in the BTF2 factor . Sequence similarity suggests that BTF2 may be the human counterpart of RNA polymerase II initiation factor b from yeast. Science, 1992 Sep 4, 257(5075), 1395 - 8 Modulation of the dimerization of a transcriptional antiterminator protein by phosphorylation; Amster-Choder O et al.; The transcriptional antiterminator protein BglG inhibits transcription termination of the bgl operon in Escherichia coli when it is in the nonphosphorylated state . The BglG protein is now shown to exist in two configurations, an active, dimeric nonphosphorylated form and an inactive, monomeric phosphorylated form . The migration of BglG on native polyacrylamide gels was consistent with it existing as a dimer when nonphosphorylated and as a monomer when phosphorylated . Only the nonphosphorylated dimer was found to bind to the target RNA . When the dimerization domain of the lambda repressor was replaced with BglG, the resulting chimera behaved like an intact lambda repressor in its ability to repress lambda gene expression, which suggests that BglG dimerizes in vivo . Repression by the lambda-BglG hybrid was significantly reduced by BglF, the BglG kinase, an effect that was relieved by conditions that stimulate dephosphorylation of BglG by BglF . These results suggest that the phosphorylation and the dephosphorylation of BglG regulate its activity by controlling its dimeric state. Eur J Pharmacol, 1992 Sep 4, 219(3), 445 - 50 5-HT receptor antagonists and heat-stable Escherichia coli enterotoxin-induced effects in the rat; Beubler E et al.; The effect of heat-stable E . coli enterotoxin on intestinal fluid secretion is commonly considered to be mediated by stimulation of mucosal cyclic guanosine monophosphate (cGMP) . It was demonstrated recently that 5-hydroxytryptamine (5-HT) acts as an important mediator in cholera toxin-induced fluid secretion . To elucidate the possible involvement of 5-HT in the secretory response to heat-stable E . coli enterotoxin, in vivo experiments were performed in the rat jejunum . The inhibitory effects of the 5-HT2 receptor antagonist ketanserin, the 5-HT3 receptor antagonist tropisetron and indomethacin were studied in heat-stable E . coli enterotoxin-induced fluid secretion . Tropisetron and ketanserin (100 micrograms/kg each) alone only partially reduced the secretory effect of the toxin . However, in combination, the two blockers (100 plus 100 micrograms/kg) significantly reduced and at 200 plus 200 micrograms/kg totally abolished heat-stable E . coli enterotoxin-induced secretion without influencing the enterotoxin-induced increase in cGMP . Pretreatment with indomethacin (10 mg/kg) reduced the secretory response to the enterotoxin by about 50% . These results support the concept that 5-HT is an important mediator in intestinal fluid secretion induced by heat-stable E . coli enterotoxin . The enterotoxin may use 5-HT to stimulate prostaglandin formation via 5-HT2 receptors and to activate neuronal structures via 5-HT3 receptors. Bull Math Biol, 1992 Sep, 54(5), 785 - 812 Poisson, compound Poisson and process approximations for testing statistical significance in sequence comparisons; Goldstein L et al.; DNA and protein sequence comparisons are performed by a number of computational algorithms . Most of these algorithms search for the alignment of two sequences that optimizes some alignment score . It is an important problem to assess the statistical significance of a given score . In this paper we use newly developed methods for Poisson approximation to derive estimates of the statistical significance of k-word matches on a diagonal of a sequence comparison . We require at least q of the k letters of the words to match where 0 less than q less than or equal to k . The distribution of the number of matches on a diagonal is approximated as well as the distribution of the order statistics of the sizes of clumps of matches on the diagonal . These methods provide an easily computed approximation of the distribution of the longest exact matching word between sequences . The methods are validated using comparisons of vertebrate and E . coli protein sequences . In addition, we compare two HLA class II transplantation antigens by this method and contrast the results with a dynamic programming approach . Several open problems are outlined in the last section. Virology, 1992 Sep, 190(1), 522 - 6 Introduction of foreign DNA into the vaccinia virus genome by in vitro ligation: recombination-independent selectable cloning vectors; Merchlinsky M et al.; Homologous recombination has been the exclusive means of introducing foreign DNA into the genomes of large DNA viruses . Here we demonstrate that direct in vitro ligation can be used to efficiently insert DNA fragments of up to 26,000 bp into the genome of vaccinia virus modified to contain a single NotI site either in the Escherichia coli lacZ gene or in the vaccinia virus thymidine kinase gene . Viruses containing chimeric genomes can be identified by chromogenic screening or thymidine-kinase-negative selection. Virology, 1992 Sep, 190(1), 510 - 4 Release of a 22-kDa protein derived from the amino-terminal domain of the 49-kDa NIa of turnip mosaic potyvirus in Escherichia coli; Laliberte JF et al.; The coding region for the precursor 6K-small nuclear inclusion a (NIa) protein and for the NIa protein of turnip mosaic potyvirus (TuMV) were introduced into the plasmid pET-11d for high-level expression in Escherichia coli . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses of E . coli proteins showed that the NIa protein underwent endoproteolysis and released a 22-kDa polypeptide . NH2-terminal amino acid sequencing of the recombinant 22-kDa protein was performed and was identical to the predicted amino end of the NIa protein . Site-directed mutagenesis confirmed that the hydrolysis was associated with the NIa proteolytic activity and that the proteinase recognized a Glu residue within an amino acid sequence found in the NIa protein which fitted the TuMV consensus cleavage site sequence . Fusion of the 6K protein with the NIa protein partially inhibited the hydrolytic reaction . The recombinant 22-kDa protein is likely the VPg of TuMV. FEMS Microbiol Lett, 1992 Sep 1, 75(1), 37 - 42 Chemotaxis of Leptospirillum ferrooxidans and other acidophilic chemolithotrophs: comparison with the Escherichia coli chemosensory system; Acuna J et al.; Ni2+, Fe2+ and Cu2+ were attractants and aspartate was an apparent repellent for Leptospirillum ferrooxidans, a behaviour opposite to that for Escherichia coli . Membranes from L . ferrooxidans contained proteins with a molecular mass in the range of 80 kDa which were methylated in vitro . Methylation was stimulated in the presence of a membrane-free extract from E . coli, showing the response pattern expected for L . ferrooxidans, increased methylation by Ni2+, and demethylation by aspartate . This suggests the existence of sensory transducers having a common methylation domain with the E . coli methyl-accepting chemotaxis proteins . Total chromosomal DNA digests from L . ferrooxidans, Thiobacillus ferrooxidans and T . thiooxidans hybridized with probes containing different domains of the tar gene from E . coli, implying the presence of tar type genes in the acidophilic bacteria studied. Clin Chem, 1992 Sep, 38(9), 1824 - 9 Immunochemical detection of group I and group II phospholipases A2 in human serum; Nevalainen TJ et al.; Time-resolved fluoroimmunoassay was developed for the detection of synovial-type phospholipase A2 (s-PLA2) in human serum . This solid-phase, sandwich assay uses a polyclonal rabbit antibody raised against synovial-type group II PLA2 produced in Escherichia coli . No cross-reactions were detected between s-PLA2 and PLA2 from human or porcine pancreas, human ascitic fluid, or bee or cobra venom . In healthy individuals, the average concentration of s-PLA2 is 3.7 micrograms/L, with a 95% reference interval from 1.3 to 10.8 micrograms/L . We investigated pancreatic PLA2, which is a group I PLA2, and synovial-type group II PLA2 in sera of patients with hematological malignancies and septic fever . The concentration of s-PLA2 was increased in patient sera and correlated significantly with the catalytic activity of PLA2 and the concentration of C-reactive protein . No correlation with the concentration of pancreatic PLA2 was found . The results suggest that the increased catalytic activity of PLA2 in sera of patients with septic fever results from synovial-type group II PLA2. J Steroid Biochem Mol Biol, 1992 Sep, 42(8), 803 - 12 Association of the 90-kDa heat shock protein does not affect the ligand-binding ability of androgen receptor; Nemoto T et al.; An N-terminal truncated androgen receptor with putative DNA- and ligand-binding domains (AR438) and that with a ligand-binding domain (AR612) were expressed under control of the T7 promoter in E . coli or translated in vitro with rabbit reticulocyte lysate, and their ligand-binding properties and the interaction with HSP90 were investigated . Bacterially expressed AR438 and AR612 bound a synthetic androgen, {3H}R1881, with apparent dissociation constants of 2.6 +/- 0.2 and 3.1 +/- 0.7 nM, respectively, values which are comparable to those of androgen receptor in target tissues . The recombinant androgen receptors sedimented at the 4-5 S region irrespective of the presence of 10 mM tungstate, indicating that the receptor exists free from HtpG, which is the bacterial homolog of eukaryotic HSP90 . The apparent dissociation constant of truncated androgen receptors translated in vitro was 0.1 nM for AR438 and 0.2 nM for AR612 . Sedimentation coefficients of in vitro translated molecules were converted from 7-8 S in the presence of tungstate to 3 S in the absence of tungstate . Both AR438 and AR612 translated in vitro were retained by anti-rat HSP90 antibody-protein A Sepharose . Exposure to 0.3 M NaCl in the presence of ligand caused dissociation of AR438 and AR612 from HSP90, and concomitantly, the DNA-cellulose binding ability of AR438 was enhanced . Thus, we conclude that the androgen receptor associates with HSP90 through the ligand-binding domain and that this association prevents the interaction of the androgen receptor with DNA . However, HSP90 seems to have little effect on the ligand-binding characteristics of the androgen receptor. J Protozool, 1992 Sep-Oct, 39(5), 609 - 12 Amphotericin B-induced carbohydrate changes on the Trypanosoma cruzi surface membrane; Haido RM et al.; Changes in the cell surface carbohydrates of Trypanosoma cruzi epimastigotes induced by Amphotericin B (AmB) were assessed by chemical methods and by agglutination assay employing a panel of highly purified lectins of various sugar specificities . Escherichia coli K12 with mannose-sensitive fimbriae was also used as an agglutination probe . Amphotericin B caused a decrease in the total carbohydrate content of all glycoconjugate fractions isolated . Exposure to AmB strongly affected the mannose/galactose ratio (1:5) in the CHCl3/methanol/H2O soluble fraction . These sugars in 1.4:1 ratio were the major hexose components of control cells . The decrease in the mannose content (48 to 15%) after AmB treatment agrees with the marked decrease in the T . cruzi cell surface receptors for fimbriated E . coli K12 . Also, an increase in the galactose content (74%) as compared with control cells (34%) is in agreement with the peanut agglutinin and Euonymus europaeus lectins agglutination results . Differences in the cell surface carbohydrates induced by AmB could be associated with alterations in the membrane structure and organization. J Bacteriol, 1992 Sep, 174(18), 5961 - 70 Cloning, sequencing, and enhanced expression of the dihydropteroate synthase gene of Escherichia coli MC4100; Dallas WS et al.; The Escherichia coli gene coding for dihydropteroate synthase (DHPS) has been cloned and sequenced . The protein has 282 amino acids and a compositional molecular mass of 30,314 daltons . Increased expression of the enzyme was realized by using a T7 expression system . The enzyme was purified and crystallized . A temperature-sensitive mutant was isolated and found to express a DHPS with a lower specific activity and lower affinities for para-aminobenzoic acid and sulfathiazole . The allele had a point mutation that changed a phenylalanine codon to a leucine codon, and the mutation was in a codon that is conserved among published DHPS sequences. J Bacteriol, 1992 Sep, 174(18), 5953 - 60 Determination of the mechanism of retrotransfer by mechanistic mathematical modeling; Top E et al.; Two mathematical models to elucidate the mechanism of retromobilization (or retrotransfer), that is, the ability of conjugative plasmids to mobilize genes into the cell containing the conjugative plasmid, were developed . This study deals with retromobilization of nonconjugative plasmids (Tra-Mob+) . Plasmid transfer was modeled by two mass action models . The first is based on the hypothesis that retromobilization of the Tra-Mob+ vector occurs in one step, by means of the pilus formed by the Tra+ plasmid in the original host . In the second model, retromobilization is considered to be a two-step process involving two transfer events . The first step involves the transfer of the Tra+ plasmid from the recipient cell to the donor of the nonconjugative vector, and during the second encounter the nonconjugative vector is mobilized toward the recipient . Since the relationships between the number of transconjugants and the number of recipients for the two models are different, filter matings were performed for short time periods with different initial densities of the recipient population . Comparison of the numbers of transconjugants with the results of the mathematical equations confirmed the hypothesis that retromobilization is a one-step conjugation process. Eur J Biochem, 1992 Sep 1, 208(2), 475 - 80 Site-specific mutagenesis of Escherichia coli asparaginase II . None of the three histidine residues is required for catalysis; Wehner A et al.; Site-specific mutagenesis was used to replace the three histidine residues of Escherichia coli asparaginase II (EcA2) with other amino acids . The following enzyme variants were studied: {H87A}EcA2, {H87L}EcA2, {H87K}EcA2, {H183L}EcA2 and {H197L}EcA2 . None of the mutations substantially affected the Km for L-aspartic acid beta-hydroxamate or impaired aspartate binding . The relative activities towards L-Asn, L-Gln, and l-aspartic acid beta-hydroxamate were reduced to the same extent, with residual activities exceeding 10% of the wild-type values . These data do not support a number of previous reports suggesting that histidine residues are essential for catalysis . Spectroscopic characterization of the modified enzymes allowed the unequivocal assignment of the histidine resonances in 1H-NMR spectra of asparaginase II . A histidine signal previously shown to disappear upon aspartate binding is due to His183, not to the highly conserved His87 . The fact that {H183L}EcA2 has normal activity but greatly reduced stability in the presence of urea suggests that His183 is important for the stabilization of the native asparaginase tetramer . 1H-NMR and fluorescence spectroscopy indicate that His87 is located in the interior of the protein, possibly adjacent to the active site. Eur J Biochem, 1992 Sep 1, 208(2), 443 - 9 Mechanistic studies of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase from Escherichia coli; Kohen A et al.; The anomeric specificity and the steady-state kinetic mechanism of homogeneous 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase were investigated . The open-chain 4-deoxy analogue of arabinose-5-phosphate (Ara5P), which is structurally prohibited from undergoing ring closure, was synthesized and tested as a substrate for the synthase . It was found that the analogue functions as a substrate with a similar kcat value to that of the original substrate . The kcat/Km value for the natural substrate is seven-times greater than that of the 4-deoxy analogue . However, taking into account the 9.5% and approximately 1% concentrations of the aldehyde forms of the 4-deoxy analogue and Ara5P in solution, then the 'true' Km values must be in the range 31.5 microM and 0.26 microM, respectively, requiring about a 3 kcal/mol contribution to the binding energy by the 4-hydroxyl group of Ara5P . The data provides evidence that the enzyme acts upon the acyclic form of the natural substrate . The steady-state kinetic study of KDO8P synthase was analyzed via inhibition using the products KDO8P and inorganic phosphate, and D-ribose-5-phosphate as a dead-end inhibitor . First, intersecting lines in double-reciprocal plots of initial-velocity data at substrate concentrations in the micromolar range suggest a sequential mechanism for the enzyme-catalyzed reaction . The inhibition by D-ribose-5-phosphate is competitive for Ara5P and uncompetitive for phosphoenolpyruvate (P-pyruvate) . These inhibition patterns are consistent with the model wherein P-pyruvate binding precedes that of Ara5P binding . Furthermore, this order of substrate binding was supported by the observations that KDO8P is a competitive inhibitor for P-pyruvate binding, supporting the concept that KDO8P and P-pyruvate bind to the same enzyme form, and noncompetitively with respect to Ara5P . In addition, the inhibition by inorganic phosphate is noncompetitive with respect to both P-pyruvate and Ara5P, suggesting an apparent ordered release of products such that Pi first, followed by KDO8P . In conclusion, these data suggest a steady-state kinetic mechanism for KDO8P synthase where P-pyruvate binding precedes that of Ara5P, followed by the ordered release of inorganic phosphate and KDO8P. Eur J Biochem, 1992 Sep 1, 208(2), 351 - 7 Protein structure of pig liver 4-aminobutyrate aminotransferase and comparison with a cDNA-deduced sequence; De Biase D et al.; The amino acid sequence of pig liver 4-aminobutyrate aminotransferase has been determined by gas-phase sequencing of proteolytically derived peptide fragments . The sequence differs substantially from that predicted for the same enzyme on the basis of the sequence of cDNA derived from pig brain in recently published work {Kwon, O., Park, J . & Churchich, J . E . (1992) J . Biol . Chem . 267, 7215-7216} . Apart from a few minor differences, the two sequences are completely different in the segment of protein comprising the 36 residues at positions 107-142 . Insertion of a cytosine between bases 402 and 403 in the cDNA sequence, together with deletion of the guanine at position 510, results in a DNA sequence which predicts exactly the amino acid sequence determined by peptide analysis in the present work . The mammalian enzyme has approximately 44% sequence identity with the same enzyme from two unicellular eukaryotes (Saccharomyces cerevisiae, Aspergillus nidulans) and 22% identity with that from Escherichia coli. Eur J Biochem, 1992 Sep 1, 208(2), 251 - 7 Site-directed mutagenesis of elongation factor Tu . The functional and structural role of residue Cys81; Anborgh PH et al.; A Cys residue located in the second consensus sequence element (DCPG) of the GTP-binding region is highly conserved in bacterial elongation factors (EF) Tu . Chemical modification of this Cys81 in EF-Tu from Escherichia coli by N-tosyl-L-phenylalanine chloromethane {Jonak, J., Petersen, T . E., Clark, B . F . C . & Rychlik, I . (1982) FEBS Lett . 150, 485-488}, and of homologous Cys residues in other bacterial EF-Tu, selectively blocks the binding of Xaa-tRNA . We have substituted Cys81 with Gly using site-directed mutagenesis of the EF-Tu-encoding tuf A gene . This substitution induces a partial inhibition (20-70%) of: (a) poly(U)-directed poly(Phe) synthesis; (b) EF-Tu/Xaa-tRNA interaction, determined as protection by EF-Tu of the non-enzymic deacylation of Xaa-tRNA; (c) EF-Tu-dependent binding of Xaa-tRNA to the mRNA/ribosome complex and (d) the intrinsic GTPase reaction, that is also less sensitive to stimulation by Xaa-tRNA . Our results thus provide evidence that Cys81, though important, is not essential for the binding of Xaa-tRNA to EF-Tu . The accuracy in poly(Phe) synthesis, measured as misincorporation of Leu, was increased . Both the binding affinity of {C81G}EF-Tu for the nucleotide and the resistance against thermal denaturation are more strongly decreased in the case of the GDP-bound state than in the case of the GTP-bound state, suggesting that Cys81 plays a more specific role in the former conformation . The sensitivity to N-tosyl-L-phenylalanine chloromethane is decreased by 80% but not totally lost . The inhibition by N-tosyl-L-phenylalanine chloromethane treatment of the function of EF-Tu appears to be a consequence of steric hindrance and/or of an altered conformation of EF-Tu.GTP . The lower activities of {C81G}EF-Tu are probably due to long-range effects, mediated by an overall destabilization of the molecule that is particularly pronounced for the GDP-bound state. Am Rev Respir Dis, 1992 Sep, 146(3), 730 - 4 Effects of N-acetylcysteine on diaphragmatic function and malondialdehyde content in Escherichia coli endotoxemic rats; Van Surell C et al.; We evaluated the effects of sublethal Escherichia coli endotoxemia with or without concomitant administration of N-acetylcysteine, an antioxidant agent, on diaphragmatic strength, endurance, and malondialdehyde (MDA) content in rats . One hundred ninety rats were inoculated subcutaneously on 2 successive days with 0.6 and 1.2 mg/100 g body weight of E . coli lipopolysaccharide respectively (E animals, n = 100) or saline (C group, n = 90) . E and C animals were divided into two groups based on administration of endotoxin or saline alone (E group, n = 55; C group, n = 47, respectively) or endotoxin or saline plus N-acetylcysteine (1 g/kg body weight/day intraperitoneally) (E-NAC group, n = 45; C-NAC group, n = 43, respectively) . Diaphragmatic strength was assessed in vivo 48 h after the first endotoxin or saline administration by measuring the transdiaphragmatic pressure (Pdl) generated during electrical stimulation of the phrenic nerves at 0.5, 10, 20, 30, 50, and 100 Hz . Endurance index was calculated as the percent ratio of Pdl generated after 30 s of phrenic stimulation at 10 Hz divided by the initial force . Diaphragmatic MDA (fluorometric technique) was measured 0, 6, 18, 30, 42, and 48 h after the first dose of endotoxin or saline . Pdl for 50 and 100 Hz was significantly reduced in Group E as compared with group C . This phenomenon was associated with a reduced endurance performance as assessed by a lower diaphragmatic endurance index in E as compared with C animals (90.9 +/- 4.2 versus 114.3 +/- 4.1 respectively; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) Scand J Immunol, 1992 Sep, 36(3), 371 - 84 Antibodies against secreted and non-secreted antigens in mice after infection with live Mycobacterium tuberculosis; Verbon A et al.; Mice from four different inbred strains were infected with live Mycobacterium tuberculosis and the immune response to M . tuberculosis was followed for 24 weeks, using Western blotting . Nearly all mice, irrespective of H-2 type, reacted with the 38-kDa protein band . Antibodies against this secreted 38-kDa protein were the first to appear, 4 weeks after infection . Thereafter the secreted 19-kDa protein and non-secreted antigens, such as the 65-kDa and 33-kDa proteins, were recognized . The immune response against the non-secreted antigens was influenced by the mouse strain . However, the 33-kDa protein band was recognized by all mouse strains after a second injection with live M . tuberculosis . The specificity of the antibodies was analysed in Western blot using sonicates of M . tuberculosis, M . kansasii, M . avium, M . terrae, M . gordonae and Escherichia coli . Antibodies against the 38-kDa and 33-kDa protein bands seemed to be specific for M . tuberculosis, while antibodies against the 19-kDa protein band showed limited cross-reactivity . Antibodies against the 65-kDa protein were strongly cross-reactive . These results suggest that the 38-kDa protein is secreted in vivo and, therefore, may be available to the humoral immune system at an early stage of infection . The non-secreted 33-kDa protein is only recognized by all mouse strains after prolonged contact with M . tuberculosis. Proc Natl Acad Sci U S A, 1992 Sep 1, 89(17), 8073 - 7 A plant cDNA that partially complements Escherichia coli recA mutations predicts a polypeptide not strongly homologous to RecA proteins; Pang Q et al.; A plant (Arabidopsis thaliana) cDNA previously selected for its ability to partially complement the UV sensitivity of Escherichia coli RecA-UvrC-Phr- mutants and designated DRT100 (DNA-damage repair/toleration) was subcloned into a high-copy-number plasmid and expressed via a bacterial promotor . It increased resistance of RecA-UvrB-Phr- bacteria to mitomycin C and methyl methanesulfonate as well as to UV light . This lack of specificity, and its ability to increase resistance in both UvrB- and UvrC- mutants, suggested that Drt100 activity might be complementing RecA- phenotypes . DRT100 partially complemented three RecA- phenotypes thought to reflect deficiencies in homologous recombination--namely, inability to plate lambda red-gam- phages and P1 phages and to recombinationally integrate donor DNA during conjugal crosses--but did not complement inability to induce E . coli SOS functions . The 395-amino acid DRT100 open reading frame encodes an apparent N-terminal chloroplast transit peptide and a putative 322-residue mature protein with a conserved nucleotide binding motif, but otherwise little global homology with bacterial RecA proteins . There are several tandemly repeated leucine-rich motifs . DNA from two closely related plants, but not from maize, hybridized strongly to a DRT100 cDNA probe. Proc Natl Acad Sci U S A, 1992 Sep 1, 89(17), 8068 - 72 A homolog of Escherichia coli RecA protein in plastids of higher plants; Cerutti H et al.; Studies of chloroplast DNA variations, and several direct experimental observations, indicate the existence of recombination ability in algal and higher plant plastids . However, no studies have been done of the biochemical pathways involved . Using a part of a cyanobacterial recA gene as a probe in Southern blots, we have found homologous sequences in total DNA from Pisum sativum and Arabidopsis thaliana and in a cDNA library from Arabidopsis . A cDNA was cloned and sequenced, and its predicted amino acid sequence is 60.7% identical to that of the cyanobacterial RecA protein . This finding is consistent with our other results showing both DNA strand transfer activity and the existence of a protein of the predicted molecular mass crossreactive with antibodies to Escherichia coli RecA in the stroma of pea chloroplasts. Proc Natl Acad Sci U S A, 1992 Sep 1, 89(17), 7890 - 4 Ligand occupancy mimicked by single residue substitutions in a receptor: transmembrane signaling induced by mutation; Yaghmai R et al.; We used mixed, mutagenic oligonucleotides to create single amino acid substitutions in the bacterial chemoreceptor Trg . Mutagenesis was directed at a 20-residue segment of the periplasmic domain implicated in ligand recognition . Transmembrane signaling by the mutant receptors was assayed in vivo by monitoring adaptational covalent modification . Among 20 functionally altered but stable receptors there were two distinct signaling phenotypes . Insensitive receptors did not signal upon stimulation and thus appeared defective in productive ligand interaction . Mimicked-occupancy receptors exhibited transmembrane signaling without ligand . Many mimicked-occupancy receptors produced additional signaling upon ligand binding and in appropriate conditions mediated effective chemotaxis; most insensitive receptors did not . Like normal receptors with one binding site occupied, mimicked-occupancy proteins adapted to persistent transmembrane signaling by increased methylation and thus could respond to other stimuli . Signaling phenotypes were strikingly segregated by residue position . Substitutions mimicking ligand occupancy occurred in half the segment, and those creating insensitive phenotypes occurred in the other half . These observations could be related to the three-dimensional structure of the periplasmic domain of the Tar(s) chemoreceptor . Insensitive substitutions occurred near the distal end of helix 1, where bulky protein ligands could interact; occupancy-mimicking substitutions were on the same helix at positions buried in the subunit interface between helices 1 and 1' . Thus perturbation of the interface induced transmembrane signaling, implicating changes at that interface in signal transduction, a conclusion consistent with differences in crystal structures of unoccupied and ligand-occupied Tar(s). Genes Dev, 1992 Sep, 6(9), 1679 - 94 Stable synapsis of homologous DNA molecules mediated by the Escherichia coli RecA protein involves local exchange of DNA strands; Adzuma K; Escherichia coli RecA protein promotes stable synapsis between a single-stranded DNA and a homologous duplex DNA, resulting in the formation of a complex of RecA with three DNA strands . To gain insight into the molecular interactions responsible for DNA synapsis, the base-pairing status within the synaptic complex was analyzed by using dimethylsulfate and potassium permanganate as probes . The results indicate that the original base pairs in the parental duplex are disrupted; one strand is displaced and the other strand appears to be involved in Watson-Crick base-pairing with the incoming single-stranded DNA . The state of base-pairing thus resembles that of the end products of strand exchange and not a canonical DNA triple helix involving non-Watson-Crick base-pairing . The results also indicate that this local strand exchange can occur without homology at the ends of the DNA substrates (i.e., when axial rotation of the product heteroduplex with respect to the axis of the parental duplex is obstructed) . Taken together, these results suggest that exchange of DNA strands mediated by RecA occur at or before the stage of stable DNA synapsis by a process that does not require DNA rotation. J Cell Biol, 1992 Sep, 118(5), 1085 - 95 Role of the COOH-terminal nonhelical tailpiece in the assembly of a vertebrate nonmuscle myosin rod; Hodge TP et al.; A short nonhelical sequence at the COOH-terminus of vertebrate nonmuscle myosin has been shown to enhance myosin filament assembly . We have analyzed the role of this sequence in chicken intestinal epithelial brush border myosin, using protein engineering/site-directed mutagenesis . Clones encoding the rod region of this myosin were isolated and sequenced . They were truncated at various restriction sites and expressed in Escherichia coli, yielding a series of mutant myosin rods with or without the COOH-terminal tailpiece and with serial deletions from their NH2-termini . Deletion of the 35 residue COOH-terminal nonhelical tailpiece was sufficient to increase the critical concentration for myosin rod assembly by 50-fold (at 150 mM NaCl, pH 7.5), whereas NH2-terminal deletions had only minor effects . The only exception was the longest NH2-terminal deletion, which reduced the rod to 119 amino acids and rendered it assembly incompetent . The COOH-terminal tailpiece could be reduced by 15 amino acids and it still efficiently promoted assembly . We also found that the tailpiece promoted assembly of both filaments and segments; assemblies which have different molecular overlaps . Rod fragments carrying the COOH-terminal tailpiece did not promote the assembly of COOH-terminally deleted material when the two were mixed together . The tailpiece sequence thus has profound effects on assembly, yet it is apparently unstructured and can be bisected without affecting its function . Taken together these observations suggest that the nonhelical tailpiece may act sterically to block an otherwise dominant but unproductive molecular interaction in the self assembly process and does not, as has been previously thought, bind to a specific target site(s) on a neighboring molecule. J Bacteriol, 1992 Sep, 174(17), 5661 - 8 Enhanced export of beta-galactosidase fusion proteins in prlF mutants is Lon dependent; Snyder WB et al.; We have used fusions of the outer membrane protein LamB to beta-galactosidase (encoded by lacZ) to study the protein export process . This LamB-LacZ hybrid protein blocks export when synthesized at high levels, as evidenced by inducer (maltose) sensitivity, a phenomenon termed LacZ hybrid jamming . The prlF1 mutation relieves LacZ hybrid jamming and allows localization of the fusion protein to a noncytoplasmic compartment . prlF1 and similar alleles are gain-of-function mutations . Null mutations in this gene confer no obvious phenotypes . Extragenic suppressors of a gain-of-function prlF allele have been isolated in order to understand how this gene product affects the export process . The suppressors are all lon null mutations, and they are epistatic to all prlF phenotypes tested . Lon protease activity has been measured in prlF1 cells and shown to be increased . However, the synthesis of Lon is not increased in a prlF1 background, suggesting a previously unidentified mechanism of Lon activation . Further analysis reveals that prlF1 activates degradation of cytoplasmically localized precursors in a Lon protease-dependent manner . It is proposed that accumulation of precursors during conditions of hybrid protein jamming titrates an essential export component(s), possibly a chaperone . Increased Lon-dependent precursor degradation would free this component, thus allowing increased protein export under jamming conditions. J Bacteriol, 1992 Sep, 174(17), 5617 - 23 Purification and phosphorylation of the Arc regulatory components of Escherichia coli; Iuchi S et al.; In Escherichia coli, a two-component signal transduction system, consisting of the transmembrane sensor protein ArcB and its cognate cytoplasmic regulatory protein ArcA, controls the expression of genes encoding enzymes involved in aerobic respiration . ArcB belongs to a subclass of sensors that have not only a conserved histidine-containing transmitter domain but also a conserved aspartate-containing receiver domain of the regulator family . 'ArcB (a genetically truncated ArcB missing the two transmembrane segments on the N-terminal end) and ArcA were purified from overproducing cells . Autophosphorylation of 'ArcB was revealed when the protein was incubated with {gamma-32P}ATP but not with {alpha-32P}ATP or {gamma-32P}GTP . When ArcA was incubated in the presence of 'ArcB and {gamma-32P}ATP, ArcA acquired radioactivity at the expense of the phosphorylated protein 'ArcB-32P . When a limited amount of 'ArcB was incubated with excess ArcA and {gamma-32P}ATP, ArcA-32P increased linearly with time . Under such conditions, for a given time period the amount of ArcA phosphorylated was proportional to the concentration of 'ArcB . Thus, 'ArcB acted as a kinase for ArcA . Chemical stabilities of the phosphorylated proteins suggested that 'ArcB-32P contained both a histidyl phosphate and an aspartyl phosphate(s) and that ArcA-32P contained only an aspartyl phosphate(s). J Bacteriol, 1992 Sep, 174(17), 5597 - 603 Mini-F plasmid mutants able to replicate in Escherichia