Biochemistry, 1995 Aug 8, 34(31), 9936 - 43 Divalent cation modulation of the ribonuclease functions of human immunodeficiency virus reverse transcriptase; Cirino NM et al.; The stimulatory effect of Mg2+ and Mn2+ on the ribonuclease H (RNase H) functions of HIV-1 reverse transcriptase (RT) has been evaluated using a model 90-nt RNA template/36-nt DNA primer . Wild type enzyme exhibits similar endonuclease and directional processing activities in response to both cations, while RNase H activity (hydrolysis of double-stranded RNA) is only evident in the presence of Mn2+ . Enzyme altered at the p66 residue Glu478 (Glu478-->Gln478), which participates in metal ion binding, is completely inactive in Mg2+ . However, Mn2+ restores specifically its endoribonuclease activity . In the presence of Mn2+, mutant RT also catalyzes specific removal of the tRNA replication primer, eliminating the possibility of contaminating Escherichia coli RNase H in our recombinant enzyme . However, the efficiency with which mutant RT catalyzes transfer of nascent DNA between RNA templates (an event mandating RNase H activity) is severely reduced . These findings raise the possibility that directional processing activity is required to accelerate transfer of nascent DNA between templates during retroviral replication. Biochemistry, 1995 Aug 8, 34(31), 9834 - 9 Redesigning the topology of a four-helix-bundle protein: monomeric Rop; Predki PF et al.; The topology of alpha-helices and beta-sheets in folded proteins is largely specified by the connectivities of the loops and turns which join them . We have used the protein Rop to test the feasibility of using short glycine-rich linkers to reconnect the alpha-helices within a four-helix-bundle protein . In wild-type Rop the four-helix-bundle structure is formed by the association of two identical helix-turn-helix monomers . Our redesigns encode Rop as a single chain to create a monomeric, rather than a dimeric, form of the protein . Characterization of a series of such variants demonstrates that new connections of this type can be used to generate stable, native-like proteins . The length of the connections is of key importance; if the loops are too short, correct association of the helices is prevented, and misfolded, higher order oligomers occur . Designs with sufficiently long loop connections, however, generate exclusively the desired monomeric form of the protein . Moreover, the successful monomeric designs bind Rop's RNA substrate with affinities that are equal to that of the wild-type protein . This result provides strong confirmation that the positioning of the helices in the monomeric variants is closely similar to that in wild-type Rop. Biochemistry, 1995 Aug 8, 34(31), 10078 - 85 Diffusion-limited interaction between unfolded polypeptides and the Escherichia coli chaperone SecB; Fekkes P et al.; SecB is a chaperone dedicated to protein translocation in Escherichia coli . SecB binds to a subset of precursor proteins, and targets them in a translocation-competent state to the SecA subunit of the translocase . The nature and kinetics of the interaction of SecB with polypeptides were studied by spectroscopic techniques using the reduced form of bovine pancreatic trypsin inhibitor (BPTI) as a model substrate . Binding of SecB to BPTI resulted in an increase in the fluorescence of the surface-exposed tryptophan residue 36 of SecB . SecB reversibly binds BPTI in stoichiometric amounts . Labeling of BPTI with the fluorophore acrylodan allowed the analysis of the binding reaction at nanomolar concentrations . High-affinity binding (KD of 5.4 nM) of labeled BPTI to SecB resulted in a blue shift of the acrylodan emission maximum and an increase in the fluorescence quantum yield, suggesting that BPTI binds in an apolar environment . Stopped-flow acquisition of rate constants of complex formation between SecB and BPTI yielded a second-order binding rate constant of 5 x 10(9) M-1 s-1, and a dissociation rate constant of 48 s-1 . These data demonstrate that in vitro, the association of SecB with polypeptide substrates is limited by the rate of collision . In vivo, SecB binding is selective, and predominantly occurs with nascent polypeptides . Since these chains are not expected to fold into stable structures, SecB association may be governed by "more or less" specific interactions and be limited by the rate of chain elongation rather than the rate of folding. Brain Res, 1995 Aug 7, 688(1-2), 143 - 8 Changes of growth inhibitory factor after stab wounds in rat brain; Hozumi I et al.; The growth inhibitory factor (GIF) is a new metallothionein (MT)-like protein that is downregulated in Alzheimer's disease (AD) brain . The biological function of GIF has not been fully clarified yet . We have raised an antibody to the synthetic polypeptide that is specific for rat GIF . The purified antibody reacted to recombinant GIF and native rat GIF but not to MT or maltose-binding protein . Using the antibody and GIF cDNA probe, we investigated changes of GIF and GIF mRNA by Western and Northern blotting techniques in rat brains after stab wounds . The levels of GIF and GIF mRNA began to increase 4 days postoperation, reached a maximum at 14-21 days and sustained the increased level at least through 28 days . While both glial fibrillary acidic protein (GFAP) and GIF were recognized in astrocytes, the increases of these 2 proteins after stab wounds showed different patterns . The results indicated that GIF could play an important role in the repair after brain damage and also produce new insights into the mechanism of gliosis investigated mainly from the viewpoint of GFAP. FEBS Lett, 1995 Aug 7, 369(2-3), 283 - 6 The role of ATP hydrolysis in the function of the chaperonin GroEL: dynamic complex formation with GroES; Kawata Y et al.; In order to understand the role of ATP hydrolysis of the chaperonin GroEL during protein folding, we have studied GroEL-GroES complex formation in the presence of ATP or ADP by using capillary electrophoresis and surface plasmon resonance . Capillary electrophoresis analysis showed that the GroEL 14-mer and GroES 7-mer formed a 1:1 complex in the presence of ATP . In the presence of ADP, both the association and dissociation rates of the complex were slower by about one order of magnitude than the rates in the presence of ATP at 25 degrees C . The implications of such a stable complex on the overall mechanism of chaperonin function are discussed. FEBS Lett, 1995 Aug 7, 369(2-3), 197 - 201 Purification and structural characterization of the CD11b/CD18 integrin alpha subunit I domain reveals a folded conformation in solution; Fairbanks MB et al.; The alpha subunits of the leukocyte CD11/CD18 integrins contain an approximately 200 amino acid 'inserted' or I domain . The I domain of the cell-surface Mac-1 (CD11b/CD18) integrin has been shown to be the major recognition site for several adhesion ligands, including iC3b, fibrinogen, factor X, and ICAM-1 . The I domain from the Mac-1 alpha subunit has been expressed in Escherichia coli as a soluble GST-fusion protein containing a factor Xa sensitive cleavage site . Analytical characterization of the purified I domain reveals that it is obtained in very high quality at high yields . CD and NMR spectra indicate that I domain adopts a predominantly folded structure in solution, independent of the remainder of the alpha subunit . Addition of Ca2+ and Mg2+ did not significantly perturb the structural conformation. FEBS Lett, 1995 Aug 7, 369(2-3), 149 - 52 Identification and localization of the first glutaredoxin in leaves of a higher plant; Morell S et al.; Glutaredoxin(thioltransferase) has been identified and purified to homogeneity from spinach leaves . Its cytosolic localization was demonstrated by chromatographic and immunological analysis of extracts from isolated spinach chloroplasts and mitochondria, respectively . Spinach glutaredoxin shows a significant crossreactivity with antibodies raised against E . coli glutaredoxin and possesses a specific thioltransferase activity comparable to that of the E . coli protein . Minor thioltransferase activities (less than 10% of total leaf activity) have been observed in spinach chloroplasts which are probably due to the presence of trypsin inhibitor and thioredoxins (TRf and TRm). J Mol Biol, 1995 Aug 4, 251(1), 59 - 75 The capsid size-determining protein Sid forms an external scaffold on phage P4 procapsids; Marvik OJ et al.; Although the phages P2 and P4 build their capsids from the same precursor, the product of the P2 N gene, the two capsids differ in size: P2 builds a 60 nm, T = 7 capsid from 420 subunits, whereas P4 makes a 45 nm, T = 4 capsid from 240 subunits . This difference leads to substantial changes in shell geometry and subunit interactions . Previous results have demonstrated that the P4 sid gene is responsible for the assembly of P4-sized shells . We have used cryo-electron microscopy and image reconstruction to determine the structure of a putative assembly intermediate of P4 capsids, produced in vivo from cloned genes . We demonstrate that Sid forms a P4-specific scaffold with icosahedral symmetry on the outside of the procapsid-like particles . The Sid molecules (60 or 120 copies) form lofty arches that interact with the gpN hexamers on the icosahedral 2-fold axes, and connect as trimers over the 3-fold axes, forming a continuous dodecahedrally shaped outer cage . The gpN shell inside the Sid cage is approximately 40 nm wide, consistent with the previously suggested maturational expansion . The main difference with respect to the mature P4 capsids is found in the hexamers, which appear strongly elongated and more protruding than in the mature shell . These and previous results are discussed in the light of a model for regulation of capsid size. J Mol Biol, 1995 Aug 4, 251(1), 15 - 29 Nitrate and nitrite regulation of the Fnr-dependent aeg-46.5 promoter of Escherichia coli K-12 is mediated by competition between homologous response regulators (NarL and NarP) for a common DNA-binding site; Darwin AJ et al.; The NarL and NarP proteins are homologous response regulators that function to regulate anaerobic respiratory gene expression in response to nitrate and nitrite in Escherichia coli . Expression of the aeg-46.5 operon (anaerobically expressed gene at 46.5 minutes on the genetic map) is induced during anaerobic growth by the global transcriptional regulatory protein Fnr . aeg-46.5 operon expression is further induced by the NarP protein in response to nitrate or nitrite and this induction is antagonized by NarL . We used in vivo and in vitro techniques to investigate how these three transcriptional regulatory proteins control the activity of a single promoter . Deletion and mutational analysis of the aeg-46.5 operon control region identified two distinct cis-acting elements . A sequence with similarity to the Fnr-binding site consensus, centered at position -64.5, was essential for Fnr-dependent anaerobic induction of aeg-46.5 operon expression . In all other naturally occurring Fnr-dependent promoters the primary Fnr-binding site is centered between -40 and -50 . The second cis-acting element, a region of perfect symmetry centered at -44.5, shares sequence similarity with the NarL-binding site consensus . This region was required for nitrate and nitrite induction of aeg-46.5 operon expression . We purified the NarP and NarL proteins as maltose-binding protein (MBP) fusion proteins and investigated their interaction with the aeg-46.5 operon control region . Incubation with the phospho-donor, acetyl phosphate, allowed both MBP-NarP and MBP-NarL to protect the -44.5 region of the aeg-46.5 operon control region from DNase I cleavage . Single and double nucleotide substitutions in the -44.5 region reduced or abolished nitrate and nitrite induction of aeg-46.5 operon expression in vivo and prevented the binding of MBP-NarP and MBP-NarL to the control region in vitro . Presumably, the NarP and NarL proteins compete for the -44.5 binding site to regulate aeg-46.5 operon expression in response to nitrate and nitrite . Apparently, only the NarP protein is competent to activate transcription of the aeg-46.5 operon when bound to the -44.5 region. Biochem Biophys Res Commun, 1995 Aug 4, 213(1), 74 - 80 Mutational specificity of the ferrous ion in a supF gene of Escherichia coli; Akasaka S et al.; A plasmid, pZ189, was treated with Fe2+/EDTA, and mutagenesis was determined by DNA sequencing . In the fgp+ Escherichia coli host, 78% were base substitutions, with G:C- > C:G transversion (58.7%) predominant, followed by G:C- > T:A transversion (28.3%) . In the fpg-1 mutant, 88% were base substitutions among which 46% were G:C- > C:G and 42% G:C- > T:A . Fe2+ resulted in increased formation of 8-hydroxydeoxyguanosine (8-ohdG) in pZ189 DNA . The origin of Fe(2+)-induced G:C- > T:A transversion may be 8-ohdG; on the other hand, the origin of G:C- > C:G is neither 8-ohdG nor 2,6-diamino-4-hydroxy-5-formamidopyrimidine. Biochem Biophys Res Commun, 1995 Aug 4, 213(1), 154 - 60 Molecular cloning of the human gene encoding lanosterol synthase from a liver cDNA library; Baker CH et al.; Lanosterol synthase {(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7} catalyzes the cyclization of (S)-2,3-oxidosqualene to lanosterol in the reaction that forms the sterol nucleus . We report herein the cloning and characterization of the human gene (OSC) encoding lanosterol synthase, a predicted 83 kDa protein of 732 amino acids . The deduced amino acid sequence is 36-40% identical to known yeast and plant homologues and 83% identical to Rattus norvegicus lanosterol synthase . The new gene was shown to encode lanosterol synthase . The yeast lanosterol synthase deficient mutant SMY8 was complemented by the human gene, and a cell-free homogenate of SMY8 expressing the human gene was shown to convert 2,3-oxidosqualene to lanosterol. J Biol Chem, 1995 Aug 4, 270(31), 18558 - 62 Heparin binding by fibronectin module III-13 involves six discontinuous basic residues brought together to form a cationic cradle; Busby TF et al.; The thirteenth type III domain of fibronectin binds heparin almost as well as fibronectin itself and contains a so-called heparin-binding consensus sequence, Arg6-Arg7-Ala8-Arg9 (residues 1697-1700 in plasma fibronectin) . Barkalow and Schwarzbauer (Barkalow, F.J., and Schwarzbauer, J.E . (1991) J . Biol . Chem . 266, 7812-7818) showed that mutation of Arg6-Arg7 in domain III-13 of recombinant truncated fibronectins abolished their ability to bind heparin-Sepharose . However, synthetic peptides containing this sequence have negligible affinity for heparin (Ingham, K.C., Brew, S.A., Migliorini, M . M., and Busby, T.F . (1993) Biochemistry 32, 12548-12553) . We generated a three-dimensional model of fibronectin type III-13 based on the structure of a homologous domain from tenascin . The model places Arg23, Lys25, and Arg54 parallel to and in close proximity to the Arg6-Arg7-Ala8-Arg9 motif, suggesting that these residues may also contribute to the heparin-binding site . Domain III-13 and six single-site mutants containing Ser in place of each of the above-mentioned basic residues were expressed in Escherichia coli . All of the purified mutant domains melted reversibly with a Tm near that of the wild type indicating that they were correctly folded . When fluorescein-labeled heparin was titrated at physiological ionic strength, the wild type domain increased the anisotropy in a hyperbolic fashion with a Kd of 5-7 microM, close to that of the natural domain obtained by proteolysis of fibronectin . The R54S mutant bound 3-fold weaker and the remaining mutants bound at least 10-fold weaker than wild type . The results point out that the Arg6-Arg7-Ala8-Arg9 consensus sequence by itself has little affinity for heparin under physiological conditions, even when presented in the context of a folded domain . Thus, the heparin-binding site in fibronectin is more complex than previously realized . It is formed by a cluster of 6 positively charged residues that are remote in the sequence but brought together on one side of domain III-13 to form a "cationic cradle" into which the anionic heparin molecule could fit. J Biol Chem, 1995 Aug 4, 270(31), 18551 - 7 Role of Tyr residues in the contact region of anti-lysozyme monoclonal antibody HyHEL10 for antigen binding; Tsumoto K et al.; It has been shown that Tyr residues are unusually localized in the regions of antibodies responsible for contact with antigens (Padlan, E . A . (1990) Proteins Struct . Funct . Genet . 7, 112-124) . In order to clarify the role of these Tyr residues in antigen binding, the interaction between hen egg white lysozyme (HEL) and its monoclonal antibody HyHEL10, whose structure has been well studied in complex with its antigen, was investigated . Four Tyr residues in the VH chain (HTyr-33, HTyr-50, HTyr-53, and HTyr-58) were replaced with Ala, Leu, Phe, or Trp, and the interactions between these mutant Fv fragments and HEL were studied by inhibition assay of the enzymatic activity of HEL and isothermal titration calorimetry . Twelve mutant Fv fragments could be expressed, but two mutants (HY50W and HY58W) could not be obtained in the Escherichia coli expression system, and a further two mutants (HY33A and HY50A) could not be purified by affinity chromatography . It was shown by inhibition assay that Tyr residues at each mutated site made positive contributions to the interaction to different degrees . Thermodynamic studies showed that the role of Tyr residues in antigen binding was to obtain enthalpic energy . The roles of Tyr residues in antibody HyHEL10 for the association with antigen, HEL, can be summarized as follows: 1) formation of hydrogen bonds by the hydroxyl group, 2), creating more favorable interactions through the aromatic ring and decreasing the entropic loss upon binding, and 3) allowing hydrophobic interaction through the side chain . The four Tyr residues studied here were found to play significant roles in the association in various ways. J Biol Chem, 1995 Aug 4, 270(31), 18484 - 90 Structural and functional roles of cysteine 90 and cysteine 240 in S-adenosylmethionine synthetase; Reczkowski RS et al.; Site-specific mutagenesis was performed on the structural gene for Escherichia coli S-adenosylmethionine (AdoMet) synthetase to introduce mutations at cysteines 90 and 240, residues previously implicated by chemical modification studies to be catalytically and/or structurally important . The AdoMet synthetase mutants (i.e . MetK/C90A, MetK/C90S, and MetK/C240A) retained up to approximately 10% of wild type activity, demonstrating that neither sulfhydryl is required for catalytic activity . Mutations at Cys-90 produced a mixture of noninterconverting dimeric and tetrameric proteins, suggesting a structural significance for Cys-90 . Dimeric Cys-90 mutants retained approximately 1% of wild type activity, indicating a structural influence on enzyme activity . Both dimeric and tetrameric MetK/C90A had up to a approximately 70-fold increase in Km for ATP, while both dimeric and tetrameric MetK/C90S had Km values for ATP similar to the wild type enzyme, suggesting a linkage between Cys-90 and the ATP binding site . MetK/C240A was isolated solely as a tetramer and differed from wild type enzyme only in its 10-fold reduction in specific activity, suggesting that the mutation affects the rate-limiting step of the reaction, which for the wild type enzyme is the joining of ATP and L-methionine to yield AdoMet and tripolyphosphate . Remarkably all of the mutants are much more thermally stable than the wild type enzyme. J Biol Chem, 1995 Aug 4, 270(31), 18335 - 40 Effects of the antiterminator BoxA on transcription elongation kinetics and ppGpp inhibition of transcription elongation in Escherichia coli; Vogel U et al.; It has been shown previously that two different mRNA chains (lacZ and infB) are elongated at a rate of approximately 40 nucleotides (nt)/s during steady state growth on minimal medium and that the rate of mRNA chain elongation is inhibited by ppGpp in vivo . On the other hand, it was found that a truncated ribosomal RNA chain was elongated at a rate of approximately 80 nt/s, independent of growth condition (Vogel, U., and Jensen, K . F . (1994) J . Biol . Chem . 269, 16236-16241) . We reasoned that the different transcriptional behavior of mRNA genes and rRNA operons might be caused by the antiterminator sequences present in the rRNA operons . To test this possibility, we have (a) inserted the minimal antiterminator boxA sequence between the promoter and the lacZ and infB genes and (b) deleted the antiterminator sequences from the rRNA transcription unit and measured transcription elongation rates in vivo on the resulting hybrid genes . We found that insertion of boxA in front of the coding region of lacZ increased the transcription elongation rate from 42 nt/s to 69 nt/s during steady state growth and that it eliminated the ppGpp-dependent decrease in the transcription elongation rate during the stringent response . On the other hand, deletion of the antiterminator sequences from the rRNA operon resulted in a reduced transcription elongation rate, but the elongation rate was still insensitive to changes in the ppGpp pool . These results are consistent with the hypothesis that the antiterminator boxA is a primary determinant of the rate of transcription elongation rate. J Biol Chem, 1995 Aug 4, 270(31), 18295 - 300 A string of enzymes, purification and characterization of a fusion protein comprising the four subunits of the glucose phosphotransferase system of Escherichia coli; Mao Q et al.; A multidomain protein comprising the four subunits of the glucose phosphotransferase system of Escherichia coli was constructed by fusion of the transmembrane subunit IICBGlc and the three cytoplasmic proteins, IIAGlc, HPr, and enzyme I . The subunits were linked in the above order with Ala-Pro-rich linkers; the fusion protein was overexpressed in E . coli and purified by Ni2+ chelate affinity chromatography . Approximately 3 mg of the fusion protein could be purified from 1 liter of culture . The phosphotransferase activity of the purified fusion protein was 3-4 times higher than that of an equimolar mixture of the isolated subunits . The mannose transporter, which also requires enzyme I and HPr, was not an effective competitor in the overall phosphoryltransfer reaction when the fusion protein was used, whereas it was a competitor when an equimolar mixture of the separate subunits was employed . Transphosphorylation activity of the fusion protein was almost indistinguishable from the wild-type IICBglc . Addition of extra IICBGlc subunit could significantly stimulate the phosphotransferase activity of the fusion protein, addition of extra IIAGlc subunit and enzyme I, in contrast, was slightly inhibitory, and HPr had almost no effect . An optimal detergent-lipid ratio is required for maximum activity of the fusion protein . Our results suggest that Ala-Pro-rich linker sequences may be of general use for the construction of catalytically active fusion proteins with novel properties. J Biol Chem, 1995 Aug 4, 270(31), 18277 - 84 Investigation of monovalent cation activation of S-adenosylmethionine synthetase using mutagenesis and uranyl inhibition; McQueney MS et al.; S-Adenosylmethionine (AdoMet) synthetase catalyzes the formation of AdoMet from ATP and L-methionine with subsequent hydrolysis of the bound tripolyphosphate intermediate . Maximal activity requires the presence of two divalent and one monovalent cation per active site . Recently, the x-ray structure of the Escherichia coli AdoMet synthetase was solved, and the positions of the two Mg2+ binding sites were identified . Based on additional spherical electron density, the K+ binding site was postulated to be a nearby site where the uranyl heavy atom derivative also bound in the crystal . The side chain of glutamate 42 is within ligation distance of the metals . Mutagenesis of glutamate 42 to glutamine (E42QMetK) abolished monovalent cation activation and produced an enzyme that has kinetic properties virtually identical to those of K(+)-free wild type AdoMet synthetase in both the overall AdoMet synthetase reaction and in the hydrolysis of tripolyphosphate . Thus, there is a approximately 100-fold decrease in the Vmax for AdoMet synthesis and large increases in the Km values for both substrates . In contrast there is only a 2-fold decrease in Vmax for tripolyphosphate hydrolysis . The uranyl ion, UO2(2+), is a competitive inhibitor with respect to K+ (Ki = 350 nM) and is the first ion to bind at this site and inhibit the enzyme . The UO2(2+) inhibition is reversible and tight-binding, and results from UO2(2+) and not UO2(2+)-ATP . Analogous to K+ activation, UO2(2+) predominantly inhibits AdoMet formation rather than tripolyphosphate hydrolysis . The kinetic results indicate that UO2(2+) inhibition is likely to result from interference with productive ATP binding . UO2(2+) remains a tight-binding inhibitor of the E42Q mutant, which suggests that K+ and UO2(2+) have different ligation preferences when bound in the monovalent cation binding pocket . The results support the model that glutamate 42 provides ligands to the K+ and has a major role in monovalent cation binding. J Biol Chem, 1995 Aug 4, 270(31), 18329 - 34 Purification and functional reconstitution of the recombinant large mechanosensitive ion channel (MscL) of Escherichia coli; Hase CC et al.; The large mechanosensitive ion channel (MscL) of Escherichia coli was expressed on a plasmid encoding MscL as a fusion protein with glutathione S-transferase in an Escherichia coli strain containing a disruption in the chromosomal mscL gene . After purification of the fusion protein using glutathione-coated beads, thrombin cleavage allowed recovery of the MscL protein . The purified protein was reconstituted into artificial liposomes and found to be fully functional when examined with the patch-clamp technique . The reconstituted recombinant MscL protein formed ion channels that exhibited characteristic conductance and pressure sensitivity and were blocked by the mechanosensitive ion channel inhibitor gadolinium . The recombinant MscL protein was also used to raise specific anti-MscL polyclonal antibodies which abolished channel activity when preincubated with the MscL protein. J Chromatogr B Biomed Appl, 1995 Aug 4, 670(1), 167 - 72 Determination of muramic acid in organic dust by gas chromatography-mass spectrometry; Mielniczuk Z et al.; A method is described for the quantitation of muramic acid, a marker of bacterial peptidoglycan, in organic dust . House dust samples were hydrolysed in hydrochloric acid and then extracted with hexane to remove hydrophobic compounds . The aqueous phase was evaporated, heated in a silylation reagent to form trimethylsilyl derivatives, and analysed by gas chromatography--mass spectrometry . The muramic acid derivative gave two peaks upon injection into the gas chromatograph--mass spectrometer . Injection of 10 pg of the derivative gave a signal-to-noise ratio of 17 for the dominating peak when using selected ion monitoring in the electron impact mode, and a linear calibration curve was achieved upon analysis of samples containing 5-1500 ng of muramic acid . In a house dust sample, 40 ng of muramic acid was found per mg of dust; the coefficient of variation was 8.2% (n = 6, 1.2 mg of dust analysed) . The described method is rapid and simple to apply, and should therefore become widely used for measuring peptidoglycan in many types of environmental samples, including organic dust. Mol Microbiol, 1995 Aug, 17(4), 757 - 67 The deletion of 70 amino acids near the N-terminal end of the sucrose-specific porin ScrY causes its functional similarity to LamB in vivo and in vitro; Schulein K et al.; A deletion mutant ScrY delta 3-73 of the sucrose-specific porin ScrY was constructed in which 70 amino acids of the mature protein were deleted near the N-terminal end . ScrY delta 3-72 was still able to oligomerize and inserted properly into the outer membrane of an Escherichia coli strain . The protein was isolated and purified by standard procedures . The mutant protein showed, in contrast to wild-type ScrY, a tight association with the murein . Reconstitution experiments with artificial lipid bilayer membranes demonstrated that ScrY delta 3-72 produced defined cation-selective channels in planar lipid bilayers . Its single-channel conductance was reduced to about half of the value of wild-type ScrY . The deletion had a relatively small influence on the stability constants for carbohydrate binding . However, in contrast to wild-type ScrY, {14C}-maltopentaose was efficiently taken up into whole E . coli cells containing ScrY delta 3-72 . The sequence of the N-terminus of mature ScrY was identified as starting with glutamine 23 . The possible structure of ScrY and ScrY delta 3-72 in the outer membrane is discussed. Mol Microbiol, 1995 Aug, 17(4), 727 - 35 Localized denaturation of oriT DNA within relaxosomes of the broad-host-range plasmid R1162; Zhang S et al.; The broad-host-range, multicopy plasmid R1162 is efficiently mobilized during conjugation by the self-transmissible plasmid R751 . The relaxosome, a complex of plasmid DNA and R1162-encoded proteins, forms at the origin of transfer (oriT) and is required for mobilization . Transfer is initiated by strand- and site-specific nicking of the DNA within this structure . We show by probing with potassium permanganate that oriT DNA is locally melted within the relaxosome, in the region from the inverted repeat to the site that is nicked . Mutations in this region of oriT, and in genes encoding the protein components of the relaxosome, affect both nicking and melting of the DNA . The nicking protein in the relaxosome is MobA, which also ligates the transferred linear, single strand at the termination of a round of transfer . We propose that there is an underlying similarity in the substrates for these two MobA-dependent, DNA-processing reactions . We also show that MobA has an additional role in transfer, beyond the nicking and resealing of oriT DNA. Mol Microbiol, 1995 Aug, 17(4), 633 - 41 NusG overexpression inhibits Rho-dependent termination in Escherichia coli; Burova E et al.; The Escherichia coli NusG protein binds Rho in vitro and is required for efficient Rho-dependent transcription termination in vivo . In this work we examine transcription termination in cells that over-express NusG . Termination at the Rho-dependent lambda tL1 and lambda tR1 sites was significantly inhibited by excess NusG, whereas the Rho-independent lambda tl site was fully functional . Although Western analysis showed no reduction in the levels of soluble Rho, termination was restored when Rho was also overexpressed . Our data indicated that the ratio of NusG and Rho proteins affects the efficiency of transcription termination. Mol Microbiol, 1995 Aug, 17(4), 611 - 20 Region 2 of the Escherichia coli K5 capsule gene cluster encoding proteins for the biosynthesis of the K5 polysaccharide; Petit C et al.; The nucleotide sequence of region 2 of the Escherichia coli K5 capsule gene cluster has been determined . This region, essential for the biosynthesis of the K5 polysaccharide, contained four genes, termed kfiA-D . The G + C ratio was 33.4%, which was lower than the typical G + C ratio for E . coli and that of the flanking regions 1 and 3 in the K5 capsule gene cluster . Three major RNA transcripts were detected within region 2 by Northern blotting and three promoters located by transcript mapping . Promoter activity was confirmed by promoter-probe analysis . The predicted amino acid sequence of KfiC had homology to a number of glycosyl transferase enzymes and overexpression of the KfiC gene resulted in increased K5 transferase activity . The predicted amino acid sequence of KfiD had homology to a number of NAD-dependent dehydrogenase enzymes and was demonstrated to be a UDP-glucose dehydrogenase that catalyses the information of UDP-glucuronic acid from UDP-glucose. Antibiot Khimioter, 1995 Aug, 40(8), 32 - 5 {Survival of Escherichia coli in human blood serum as a function of R-plasmid content}; Evdokimova OV et al.; Thirty four strains of Escherichia coli of the pathogenic and nonpathogenic serogroups were tested for their survival in normal human blood serum as dependent on the content of the R plasmids . It was shown that the capacity for the survival of the E . coli strains of different origin was widely distributed . The value of the survival associated with the content of the R plasmids was higher in the E . coli strains of the pathogenic serogroup by comparison to that in the ordinary cultures . The capacity for the survival of the E . coli strains in the serum was more frequently accompanied by the tetracycline resistance. Rev Med Chil, 1995 Aug, 123(8), 961 - 6 {Prevention of surgical wound infections after appendectomy: intravenous versus rectal metronidazole}; Anselmi M et al.; AIM: To compare the efficacy of rectal and intravenous metronidazole in the prevention of anaerobic wound infections after appendicectomy . PATIENTS AND METHODS: One hundred patients subjected to appendicectomy were randomly assigned to receive, 2 hours before operation, gentamycin 80 mg i.v . and metronidazole 1 g i.v . or the same amount of gentamycin and 1 g of metronidazole as a suppository . Surgical wounds were observed for infections until the tenth day of the postoperative period . RESULTS: Seven of 45 patients receiving intravenous metronidazole and six of 44 receiving the drug as suppositories had wound infection . The frequency of infections was higher among patients with gangrenous or perforated appendices . They were detected at the fifth postoperative day in 8 patients and the most frequently isolated bacteria were E coli and S aureus . CONCLUSIONS: Rectal metronidazole is equally effective than intravenous metronidazole in the prevention of would infections after appendicectomy. Indian J Biochem Biophys, 1995 Aug, 32(4), 171 - 8 Primase structure and function; Griep MA; Primase is the ssDNA-dependent RNA polymerase that synthesizes RNA primers during DNA replication . In common with all DNA and RNA polymerases, primase has structural and functional features involved in polymer elongation . As RNA polymerase, it has structural and functional features for initiating chain synthesis . As a primase, it has structural and functional features for initiating chain synthesis on ssDNA . Using amino acid sequence analysis the structure of Escherichia coli primase responsible for binding zinc, at least three magnesium, and DnaB helicase has been identified . One of the magnesium binding motifs resembles the inverted question markactive magnesium inverted question mark motif found in all DNA and RNA polymerases . This motif can be considered to be involved in phosphodiester bond formation . The region with the putatuve zinc binding motif is the most highly conserved portion, including more than 25% of identical residues among bacterial primases . The function of the zinc finger may be to bind ssDNA in a sequence-specific manner . Primase has inverted question markRNAP inverted question mark motif, a sequence found in all RNA polymerases which may be involved in chain initiation . Many of the observations concerning primer synthesis initiation in vivo have been reproduced by several of the in vitro assay systems . Important among these is that Okazaki fragments are initiated in vivo from d(CTG) most of the time . This trinucleotide initiation specificity has been shown to be an intrinsic property of pure primase in vitro . Using artificial ssDNA templates, primase has been shown to be the slowest and most error-prone polymerase yet studied . The rate-determining step is the first phosphodiester bond formed . Any protein which can influence either the dinucleotide synthesis rate or primase-ssDNA binding affinity will also play a key role in the regulation of primer synthesis initiation. Protein Eng, 1995 Aug, 8(8), 843 - 8 Recombinant calpain II: improved expression systems and production of a C105A active-site mutant for crystallography; Elce JS et al.; The bacterial production of recombinant rat calpain II has been improved greatly by the use of two compatible plasmids for the two subunits . The calpain small subunit C-terminal fragment (21 kDa) was expressed from a new A15-based vector created by cloning T7 control elements into pACYC177 . This vector is compatible with the ColE1-based pET-24d(+) vector containing the calpain large subunit, and the yield of calpain activity was increased at least 16-fold by co-expression from these two vectors . A high level of activity was also obtained from a bicistronic construct containing both subunit cDNAs under the control of one T7 promoter . The addition of a C-terminal His-tag to the large subunit simplified purification without affecting subunit association or enzyme activity . The active-site cysteine 105 was mutated to alanine, causing complete loss of activity . The yield of purified C105A-calpain II (80 + 21 kDa) dimer following three column chromatography steps was 10 mg/l of cell culture . This provides a purified calpain, stable to autolysis and oxidation, which is likely to facilitate crystallization in both the presence and absence of calcium. J Bioenerg Biomembr, 1995 Aug, 27(4), 437 - 45 URF13, a ligand-gated, pore-forming receptor for T-toxin in the inner membrane of cms-T mitochondria; Rhoads DM et al.; URF13 is the product of a mitochondrial-encoded gene (T-urf13) found only in maize plants containing the Texas male-sterile cytoplasm (cms-T), and it is thought to be responsible for both cytoplasmic male sterility and the susceptibility of cms-T maize to the fungal pathogens Bipolaris maydis race T and Phyllosticata maydis . Mitochondria isolated from cms-T maize are uniquely sensitive to pathotoxins (T-toxin) produced by these fungi and to methomyl (a commercial insecticide) . URF13 acts as a receptor that specifically binds T-toxin to produce hydrophilic pores in the inner mitochondrial membrane . When expressed in Escherichia coli cells, URF13 also forms hydrophilic pores in the plasma membrane if exposed to T-toxin or methomyl . Topological studies established that URF13 contains three membrane-spanning alpha-helices, two of which are amphipathic and can contribute to pore formation . Chemical cross-linking of URF13 was used to demonstrate the existence of URF13 oligomers in cms-T mitochondria and E . coli cells . The ability of the carboxylate-specific reagent, N,N'-dicyclohexycarbodiimide, to cross-link URF13 was used in conjunction with site-directed mutagenesis to establish that the URF13 tetramer has a central core consisting of a four-alpha-helical bundle which undergoes a conformational change after interaction with T-toxin or methomyl . Overall, the experimental evidence indicates that URF13 functions as a ligand-gated, pore-forming T-toxin receptor in cms-T mitochondria. Thromb Haemost, 1995 Aug, 74(2), 722 - 9 Native and non-glycosylated recombinant single-chain urokinase-type plasminogen activator are recognized by different receptor systems on rat parenchymal liver cells; van der Kaaden ME et al.; The recognition systems mediating the clearance of glycosylated high molecular weight single-chain urokinase-type plasminogen activator (HMW-scu-PA, produced in human embryonic kidney cells) and recombinant non-glycosylated scu-PA (rscu-PA, produced in E . coli) were analyzed by studying their binding characteristics to freshly isolated rat parenchymal liver cells . The binding of 125I-HMW-scu-PA at 4 degrees C was calcium-dependent and of high affinity (Kd = 37.6 nM) and could be inhibited by low molecular weight two-chain u-PA (LMW-tcu-PA) and lactose, but not by the low density lipoprotein receptor-related protein (LRP)-associated 39-kDa protein (RAP), rscu-PA or a mutant form lacking amino acids 11-135 (Delta 125-rscu-PA) . Removal of the carbohydrate side chain of HMW-scu-PA by treatment with N-glycosidase F, completely reduced the specific binding to the parenchymal cells and strongly reduced its competition with 125I-HMW-scu-PA in cell binding . Recombinant scu-PA also bound with high affinity (Kd = 38.7 nM) to the parenchymal liver cells . The binding of 125I-rscu-PA could be competed for by unlabeled rscu-PA while Delta 125-rscu-PA, LMW-tcu-PA or lactose were ineffective . In contrast to HMW-scu-PA, binding of 125I-rscu-PA could be effectively inhibited by RAP (Ki = 1.1 nM), while also its association and degradation, as determined at 37 degrees C, were inhibited by RAP . Pretreatment of the parenchymal cells with proteinase K supplied further evidence for the involvement of two different receptor systems.(ABSTRACT TRUNCATED AT 250 WORDS) Immunopharmacol Immunotoxicol, 1995 Aug, 17(3), 493 - 509 Survival to lipopolysaccharide, cytokine release and phagocyte functions in mice treated with different total parenteral nutrition regimens; Tufano MA et al.; Effects on host defenses of Total Parenteral Nutrition (TPN) with long- (LCT) and medium-chain triglycerides (MCT) were studied . Survival to lipopolysaccharide (LPS) challenge, blood clearance of Escherichia coli, in vivo and in vitro production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were investigated . In BALB/c mice, LCTs produced a 25% reduction in mortality, compared with controls . TPN performed with a LCT plus MCT mixture reduced mortality by 50% . Spasms appeared after 18 h and 12 h respectively in mice treated with LCT-MCT mixture or LCTs alone, respect to controls (8 h) . The LCT-MCT mixture produced a 67% blood clearance of E . coli after 1 h, while the treatment with LCTs alone had no significant effects compared to controls (about 40%) . The LCT-MCT mixture induced a 50% increase in chemiluminescence respect to controls . A 33% increase was observed in rats treated with LCTs alone . TNF-alpha serum levels after challenge with LPS were not modified by any of the triglycerides or their combinations . IL-6 increased by 43% with LCT-MCT mixture and by 39% with LCTs alone versus controls . After a 3 h in vitro treatment with LCTs, human monocytes were stimulated to release TNF-alpha at levels higher than those stimulated with the LCT-MCT mixture, and respect to controls . In contrast after 3 h the stimulation with LCT-MCT mixture induced a higher IL-6 release than controls and cells stimulated with LCTs alone, or with LPS. Vet Immunol Immunopathol, 1995 Aug, 47(3-4), 323 - 31 Development and application of an antibody ELISA for the marker protein of ovine pulmonary carcinoma; Kwang J et al.; Ovine pulmonary carcinoma (OPC) is a contagious pulmonary neoplasia with a suspected retroviral etiology . The major core protein (P27) of the putative OPC virus cross-reacts with antibodies to P27 of the Mason-Pfizer monkey virus (MPMV), a type-D retrovirus . This serological reactivity serves as the only accepted biological marker for OPC . In order to make a useful reagent for the detection of the OPC marker for serodiagnosis and epidemiological studies, the MPMV-P27 coding region was cloned and expressed in Escherichia coli . Gel purified recombinant MPMV-P27 protein was used to develop an immunoassay . This recombinant enzyme-linked immunosorbent assay (ELISA) was then used to screen 223 sera from US sheep and 176 sera from Italian sheep . In this study, we found: (1) a high prevalence of infection with the putative OPC retrovirus in sheep with chronic pneumonia; (2) a subclinical infection with OPC virus may be more common in US sheep than indicated by the rare recorded occurrence of pulmonary carcinoma; (3) an apparent association between ovine lentivirus (OLV) and OPC infection. Jpn J Med Sci Biol, 1995 Aug, 48(4), 193 - 8 Study of hemagglutinating property of enteroinvasive Escherichia coli from various geographical locations; Bhuiyan SH et al.; Thirty-four strains of enteroinvasive Escherichia coli (EIEC) were examined for their ability to agglutinate erythrocytes from different animal species . All strains cultured in Casamino acid-yeast extract medium in the presence of 1 mM CaCl2 at 37 C for 16-22 hr induced maximal expression of hemagglutination (HA) of broad spectrum erythrocytes . The strongest HA was observed with guinea-pig erythrocytes followed by human (O type), rat, mouse, rabbit and sheep erythrocytes . All the strains failed to agglutinate chicken erythrocytes . HA was resistant to D-mannose, D-glucose, D-galactose, L-fucose, and D-fructose . Also HA was resistant to ethylene diamine tetraacetic acid (EDTA) and Na metaperiodic acid, an oxidizing agent . However, it was heat labile and completely inhibited by proteolytic enzymes such as proteinase K and trypsin, suggesting that the possible hemagglutinin of EIEC associated with the cell surface is a proteinaceous substance. Eur J Clin Microbiol Infect Dis, 1995 Aug, 14(8), 713 - 6 Use of a new oligonucleotide probe for detection of colonization factor antigen III gene in enterotoxigenic Escherichia coli; Taniguchi T et al.; An alkaline phosphatase-labeled 30-mer oligonucleotide probe was designed to detect the gene for pilus colonization factor antigen III (CFA/III) of the human type of enterotoxigenic Escherichia coli (ETEC) . The CFA/III probe was used to identify CFA/III-producing ETEC among 303 Escherichia coli obtained from subjects with traveler's diarrhea . Six isolates positive for the CFA/III gene were found . This result was confirmed immunologically by using a specific monoclonal antibody developed against CFA/III . These six isolates, isolated from travelers returning from India, Pakistan and China, were all positive for the gene of heat-labile enterotoxin and possessed an identical serotype (025:H-). Mol Microbiol, 1995 Aug, 17(3), 565 - 73 Identification of base pairs important for OmpR-DNA interaction; Pratt LA et al.; OmpR, the transcriptional regulator of the ompF and ompC porin genes, is a member of a novel class of DNA-binding proteins . The mechanism(s) by which this class of proteins interacts with target DNA sites is not understood . To address this issue, we investigated the nature of the DNA sequences recognized by OmpR . A 36 bp DNA fragment was identified that is capable of supporting OmpR-DNA interaction in vivo . The base pairs within this region of DNA that are critical to this interaction were identified by isolating mutations within the fragment that hinder normal OmpR-DNA binding . The results obtained provide insights concerning the nature of the sequences recognized by OmpR and also support a model in which co-operative binding is involved in OmpR-DNA interaction. Mol Microbiol, 1995 Aug, 17(3), 493 - 504 Transcription-induced deletions in Escherichia coli plasmids; Vilette D et al.; Characterization of functions that render DNA susceptible to rearrangement is important for a better understanding of genome instability . In a previous work, we showed that sequences located downstream of a strong promoter are particularly prone to deletion . In this paper, the parameters that influence transcription-induced deletions were studied . pBR322 derivatives carrying the M13 (+) replication origin and a PTac-dependent transcription region were used . Deletion formation was analysed in the presence of the replication protein of M13, which introduces a nick at the phage replication origin, and in a rep- strain to avoid M13-driven replication . Our study showed that: (i) 4 h after induction of transcription, a few per cent of the plasmids have experienced a deletion; (ii) these deletions result in joining of the M13 replication origin to a nucleotide located in or downstream of the transcribed region; (iii) deletion formation strongly depends on the orientation of transcription, on promoter strength and transcript length, but is independent of translation; (iv) formation of transcription-induced supercoiling domains does not induce deletion formation . We propose that deletions in the transcribed region result from collisions between converging replication and transcription machineries. Mol Microbiol, 1995 Aug, 17(3), 483 - 92 Evidence for two aromatic amino acid-binding sites, one ATP-dependent and the other ATP-independent, in the Escherichia coli regulatory protein TyrR; Wilson TJ et al.; In Escherichia coli, genetic regulation of aromatic amino acid biosynthesis and uptake is effected by the protein TyrR, which acts via ligand-mediated repression and activation . Characterization of the interactions of tyrosine, phenylalanine and tryptophan with TyrR revealed the presence of two separate aromatic amino acid-binding sites, one ATP-dependent, the other ATP-independent . Binding to the ATP-dependent site induces the self-association of TyrR . Using sedimentation equilibrium analyses, dissociation constants for this site in the dimeric and hexameric forms of TyrR were determined to be 330 microM and 24 microM, respectively, for tyrosine, and 55 mM and 3.7 mM, respectively, for phenylalanine . Tryptophan bound with a strength similar to that of phenylalanine, and both phenylalanine and tryptophan competed with the binding of tyrosine . The ATP-independent site, which has not been observed previously, was characterized by ultraviolet (u.v.) difference spectroscopy and a sedimentation-velocity meniscus-depletion method . Phenylalanine bound co-operatively to this site, exhibiting half-saturation at 260 microM . Tryptophan competed weakly with phenylalanine, half-saturation occurring at 1.2 mM . No binding of tyrosine to this site could be detected . We propose that the binding of phenylalanine or tryptophan to this ATP-independent site is responsible for phenylalanine- and tryptophan-mediated regulation by TyrR. Mol Microbiol, 1995 Aug, 17(3), 471 - 81 Analysis of an Escherichia coli mutant TyrR protein with impaired capacity for tyrosine-mediated repression, but still able to activate at sigma 70 promoters; Kwok T et al.; In Escherichia coli, TyrR represses and activates transcription of operons required for tyrosine, phenylalanine and tryptophan biosynthesis and uptake . The TyrR central domain is homologous with NtrC and some other bacterial regulatory proteins, although TyrR regulates sigma 70, not sigma 54, promoters . We isolated a central domain TyrR mutant (TyrR E274Q) by substitution of a normally conserved amino acid . The mutant was unable to bring about tyrosine-mediated repression of aroF, aroL, tyrB, and tyrP and had diminished capability for tyrosine- and phenylalanine-mediated repression of aroP . In contrast, it was able to effect wild-type levels of phenylalanine-mediated repression of aroG, tryptophan-mediated repression of aroP and transcriptional activation of mtr and tyrP . The binding of purified TyrR E274Q to ATP (a requirement for tyrosine binding) and to the strong TyrR box of tyrP operator DNA were normal, but tyrosine binding and tyrosine-dependent hexamerization were significantly impaired . These properties are consistent with the proposal that self association is essential for tyrosine-mediated repression by TyrR but not for tyrosine- or phenylalanine-mediated activation . E274 of TyrR must participate in either the binding of tyrosine, or the coupling of ATP binding with a conformational change that alters the affinity of the ATP-dependent aromatic amino acid-binding site. Mol Microbiol, 1995 Aug, 17(3), 411 - 20 RNase P--a 'Scarlet Pimpernel'; Kirsebom LA; RNase P is responsible for the maturation of the 5'-termini of tRNA molecules in all cells studied to date . This ribonucleoprotein has to recognize and identify its cleavage site on a large number of different precursors . This review covers what is currently known about the function of the catalytic subunit of Escherichia coli RNase P, M1 RNA, and the protein subunit, C5, in particular with respect to cleavage-site selection . Recent genetic and biochemical data show that the two C residues in the 3'-terminal CCA sequence of a precursor interact with the enzyme through Watson-Crick base-pairing . This is suggested to result in unfolding of the amino acid acceptor-stem and exposure of the cleavage site . Furthermore, other close contact points between M1 RNA and its substrate have recently been identified . These data, together with the two existing three-dimensional structure models of M1 RNA in complex with its substrate, establish a platform that will enable us to seek an understanding of the underlying mechanism of cleavage by this elusive enzyme. J Biochem (Tokyo), 1995 Aug, 118(2), 435 - 41 Efficient replication of polyomavirus DNA in a cell-free system supplemented with Escherichia coli single-stranded DNA binding protein, which exhibits species-specificity in the requirement for DNA polymerase alpha-primase; Eki T et al.; We established a modified cell-free system for polyomavirus (PyV) DNA replication, which was supplemented with Escherichia coli single-stranded DNA binding protein (SSB) . DNA synthesis in this system was enhanced by 1.4- to over 15-fold depending upon the amount of cell extracts contained in the reaction mixture . By supplementing with E . coli SSB, we were able to reduce the amount of cell extracts in the reaction mixture, and to lower the concentrations of creatine phosphate and Tris, rendering this system more resistant to salts than the conventional PyV DNA replication system . The modified system was characterized using mutant cell extracts which had heat-inactivated DNA polymerase alpha . DNA synthesis in the system was dependent on PyV T antigen, the PyV origin of DNA replication, mutant cell extracts, and DNA polymerase alpha-primase complex purified from wild-type cells . The DNA polymerase alpha-primase complex was not replaced by DNA polymerase alpha, indicating that this system requires a functional DNA polymerase alpha-primase complex . This system exhibited species-specificity in the requirement for DNA polymerase alpha-primase; only mouse DNA polymerase alpha-primase but not human DNA polymerase alpha-primase functioned in this system. J Biochem (Tokyo), 1995 Aug, 118(2), 364 - 70 Comparison of Ca2+/calmodulin-dependent protein kinase IV from rat brain, expressed in insect cells, and expressed in Escherichia coli; Kitani T et al.; Calmodulin-dependent protein kinase IV (CaM-kinase IV) is thought to play crucial roles in the functioning of Ca2+ in the central nervous system and immune system, and the regulation of its activity is therefore very important . Recombinant CaM-kinase IV is invaluable for studies of its regulatory mechanism, because of its large-amount availability and ready site-specific mutagenesis . In the present study, rat CaM-kinase IV was expressed in Sf9 cells and Escherichia coli, and the kinetic properties were examined with syntide-2 and peptide-gamma as substrates . The recombinant enzymes were produced highly efficiently, comprising as much as about 15% of the total protein in Sf9 cells and 9% in E . coli . The brain enzyme shows two Km values for syntide-2 in the presence of Ca2+/calmodulin, but the recombinant enzymes showed normal kinetic behavior . The brain enzyme and Sf9 enzyme showed Km values for peptide-gamma of 53 and 82 microM, respectively, but the Km of the E . coli enzyme was as high as 1.7 mM, in the presence of Ca2+/calmodulin . Thus, the three enzymes differed in their kinetic properties, but all the three were markedly activated upon incubation with CaM-kinase IV kinase under the Ca2+/calmodulin-dependent protein phosphorylation conditions. J Biochem (Tokyo), 1995 Aug, 118(2), 285 - 90 Expression and kinetic properties of a recombinant 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver; Deyashiki Y et al.; Human liver cytosol contains multiple forms of 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase with hydroxysteroid dehydrogenase activity, and multiple cDNAs for the enzymes have been cloned from human liver cDNA libraries . To understand the relationship of the multiple enzyme froms to the genes, a cDNA, which has been reported to code for an isoenzyme of human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase, was expressed in Escherichia coli . The recombinant enzyme showed structural and functional properties almost identical to those of the isoenzyme purified from human liver . In addition, the recombinant isoenzyme efficiently reduced 5 alpha-dihydrotestosterone and 5 beta-dihydrocortisone, the known substrates of human liver 3 alpha-hydroxysteroid dehydrogenase and chlordecone reductase previously purified, which suggests that these human liver enzymes are identical . Furthermore, the steady-state kinetic data for NADP(+)-linked (S)-1-indanol oxidation by the recombinant isoenzyme were consistent with a sequential ordered mechanism in which NADP+ binds first . Phenolphthalein inhibited this isoenzyme much more potently than it did the other human liver dihydrodiol dehydrogenases, and was a competitive inhibitor (Ki = 20 nM) that bound to the enzyme-NADP+ complex. Bioseparation, 1995 Aug, 5(4), 217 - 23 Analysis of some operating parameters of novel adsorbents for recovery of proteins in expanded beds; Hjorth R et al.; We have analysed some operational parameters for two novel adsorbents intended for recovery of proteins from particle-containing feedstocks using expanded bed adsorption . The adsorbents tested were STREAMLINE DEAE and STREAMLINE SP, ion exchangers based on an agarose/crystalline quartz composite . Parameters analysed included bed expansion, adsorption efficiency, washing and elution . Bed expansion was considerably lower for STREAMLINE adsorbents compared to conventional agarose based media, higher flow velocities were thus possible during the expanded bed process . Breakthrough capacity was 63 mg ml-1 for lysozyme on STREAMLINE SP and 36 mg ml-1 for bovine serum albumin on STREAMLINE DEAE at a flow velocity of 300 cm h-1 . To achieve high breakthrough capacity, the sedimented bed height should be at least 10 cm . Furthermore, breakthrough capacity increased to some extent when temperature was increased from room temperature to 36 degrees C, a phenomenon which can be useful in some processes . The number of living E . coli cells in the effluent was reduced by a factor of 10(5) after washing with 15 sedimented bed volumes . The optimal flow velocity for elution was 100 cm h-1 considering time for elution and volume of the eluted fraction . Flow direction during elution in packed bed mode had little impact on the elution volume, however, elution in expanded bed mode increased the volume by approx . 40% . The data presented on the performance of STREAMLINE adsorbents show that they are very useful for recovery of proteins from particle-containing feedstocks using expanded bed adsorption. Bioorg Khim, 1995 Aug, 21(8), 608 - 11 {Expression of a synthetic gene for human angiogenin in a recombinant vaccinia virus}; Netesova NA et al.; The gene for angiogenin was cloned into vaccinia virus genome . The recombinant virus expressing angiogenin was obtained . The level of protein synthesis directed by the recombinant virus was analyzed by immunoblotting using monoclonal antibodies against human angiogenin. Biochem Mol Biol Int, 1995 Aug, 36(6), 1255 - 61 Purification of LamB proteins using continuous elution electrophoresis: a comparison with immunoaffinity chromatography; Flamminio G et al.; LamB is a membrane protein that allows the exposition of a foreign peptide on the surface of a recombinant E . coli cells . An immunopurified hybrid LamB protein has been used to elicit high-titre antibodies to a foreign epitope . Looking for a simpler purification procedure we have compared the traditional approach, which includes affinity chromatography, to continuous elution electrophoresis, in the purification of two different hybrid LamB proteins as foreign epitopes . The results obtained showed that both methods yielded the same purification, although the electrophoretic procedure had a higher yield . Continuous-elution electrophoresis could be a useful tool for the purification of membrane proteins. Biochem Mol Biol Int, 1995 Aug, 36(6), 1187 - 95 Cloning and high-level expression of chicken apocytochrome c gene in Escherichia coli; Tong JC et al.; Chicken apocytochrome c gene with correct reading frame was easily cloned through excision by polymerase chain reaction of the intron in the genomic clone of chicken cytochrome c gene, and was successfully overexpressed in Escherichia coli by cloning into expression vector pET-3d under the control of T7 promoter . Expressed protein can amount to as high as 40% of the total protein and mainly presents as inclusion body . Purification of chicken apocytochrome c from the inclusion body and characterization by SDS-PAGE, isoelectric focusing electrophoresis, and amino acid analysis showed that the purified apocytochrome c is identical to that prepared from chicken heart cytochrome c by chemically depletion of heme. Virus Res, 1995 Aug, 37(3), 305 - 15 Epitope mapping of cross-reactive monoclonal antibodies specific for the influenza A virus PA and PB2 polypeptides; Ochoa M et al.; Characterization of the epitopes recognized by 21 monoclonal antibodies (MAbs) specific for the influenza A virus PA (13 MAbs) and PB2 (8 MAbs) polypeptides (Barcena et al . (1994) J . Virol . 68, 6900-6909) raised against denatured polypeptides produced in E . coli is described . MAbs were characterized by: (1) competitive binding ELISAs; (2) mapping of the protein regions that specify their binding sites; and (3) analyses of their ability to recognize the corresponding viral protein in a number of viral isolates . Five and three non-overlapping antigenic areas were defined by the anti-PA and anti-PB2 MAbs, respectively . Five of the anti-PA MAbs recognized antigenic determinants located within the amino-terminal 157 amino acids of the PA protein, and 6 others reacted strongly with a PA fragment comprising the first 236 amino acids . All 8 anti-PB2 antibodies reacted strongly with a polypeptide fragment containing amino acids 1-113 of the PB2 protein . Analyses of the reactivities of 4 anti-P antibodies with 23 influenza A virus reference strains isolated over a period of 61 years and recovered from humans, pigs, birds and horses, showed that the epitopes were conserved among all viral isolates . The application of these antibodies as research and diagnostic tools is discussed. Protein Expr Purif, 1995 Aug, 6(4), 555 - 8 Rapid purification of recombinant green fluorescent protein using the hydrophobic properties of an HPLC size-exclusion column; Deschamps JR et al.; The green fluorescent protein (GFP) of the jelly fish Aequoria victoria was cloned into an Escherichia coli cell line that is a methionine auxotroph . The recombinant GFP (rGFP) was isolated from the cells and purified using a simple procedure consisting of only two chromatographic steps: size-exclusion chromatography and ion-exchange HPLC . Due to the hydrophobic nature of the protein, the surface characteristics of the HPLC size column, and the high initial salt concentration, the rGFP sticks to the size column and is eluted by reducing the salt concentration . Due to this unique behavior the purification procedure can readily be scaled to handle larger quantities of rGFP. Protein Expr Purif, 1995 Aug, 6(4), 546 - 54 Characterization of nuclear pore protein p62 produced using baculovirus; Bailer SM et al.; Nuclear pore glycoproteins are essential components of the nuclear import apparatus in eukaryotes . In vertebrates, the most abundant of these glycoproteins is a molecule called p62 . Like other O-linked N-acetylglucosamine glycoproteins, p62 is normally modified in the cytoplasm and cannot be overexpressed and conveniently collected in a secreted form . We devised an efficient scheme for expression and purification of recombinant p62 from Sf9 cells that may have general applicability for this class of glycoproteins . The purified rat p62 bound to wheat germ agglutinin, consistent with modification by O-linked N-acetylglucosamine . Carbohydrate analysis, in conjunction with amino acid analysis, revealed that baculovirus-expressed rat p62 contains 5-6 mol of N-acetylglucosamine/mol of p62 . As observed by circular dichroism, purified p62 expressed in the baculovirus system or in Escherichia coli share essentially the same secondary structure . Purified glycosylated rat p62 will be critical in determining the role of N-acetylglucosamine in both nuclear transport and assembly of the nuclear pore complex. Protein Expr Purif, 1995 Aug, 6(4), 472 - 80 An efficient system for active bovine pancreatic ribonuclease expression in Escherichia coli; Okorokov AL et al.; Bovine pancreatic ribonuclease (RNase A) is a member of a homologous group of extensively studied proteins . It is a small, basic protein, containing 124 amino acid residues and four stabilizing disulfide bridges . Ribonuclease A catalyzes the hydrolysis of the phosphodiester bonds in ribonucleic acids . Since this degradation of RNA interferes with normal cell functions, the signal peptide of alkaline phosphatase (phoA, Escherichia coli) was cloned onto the gene coding for RNase A, directing the protein to the periplasm . Several expression systems have been evaluated which use T7, trc, or PR promoters to transcribe the RNase A gene . Also, variation in host strains was tested to optimize the protein yield . It was found that the PR system gave better expression than the two other systems . E . coli strain BL21 was shown to be the strain in which export to the periplasm was most effective and recombinant RNase A could be isolated from the periplasmic fraction of these cells . The system provides a stable yield of active recombinant bovine pancreatic RNase of about 45-50 mg/liter of cell culture. Protein Expr Purif, 1995 Aug, 6(4), 423 - 30 Cloning, expression, purification, and characterization of 2'-deoxyuridylate hydroxymethylase from phage SPO1; Schellenberger U et al.; 2'-Deoxyuridylate hydroxymethylase (dUMP-hmase) from phage SPO1 has been cloned and expressed in Escherichia coli . In crude extracts, the enzyme represents about 25% of the soluble protein and has a higher specific activity than the most purified preparation yet reported . The enzyme was purified to homogeneity by ion-exchange and hydrophobic chromatography . The subunits of dUMP-hmase are 45 kDa by SDS-PAGE and form dimers with a molecular mass of 89.2 kDa by analytical centrifugation . In addition to the normal reaction, dUMP-hmase catalyzes the 5,10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate)-independent tritium exchange of {5-3H}dUMP for protons of water and dehalogenation of 5-bromo-2'-deoxy-uridine-5'-monophosphate; the enzyme also forms a covalent binary adduct with pyridoxal 5'-monophosphate and a covalent ternary complex with 5-fluoro-2'-deoxyuridine-5'-monophosphate and CH2H4folate . Folic acid inhibits the tritium release catalyzed by dUMP-hmase in the presence of cofactor but has no effect on the catalysis of cofactor-independent tritium exchange. Protein Expr Purif, 1995 Aug, 6(4), 411 - 6 Expression, purification, and characterization of human cytosolic serine hydroxymethyltransferase; Kruschwitz H et al.; A cDNA which codes for human cytosolic serine hydroxymethyltransferase (Garrow et al., 1993, J . Biol . Chem . 268, 11910-11916) has been cloned into a pT7-7 vector as a NdeI-EcoRI insert . HMS174 (de3) cells were transformed with this plasmid and, after induction with isopropyl thiogalactoside, expressed a catalytically active serine hydroxymethyltransferase . The enzyme was purified and shown to be the expressed human enzyme by N-terminal amino acid sequencing . About 225 mg of pure enzyme can be obtained from a 20-liter culture . Spectral characteristics of the bound pyridoxal phosphate were essentially identical to the spectral properties of rabbit cytosolic serine hydroxymethyltransferase . Kinetic constants for the natural substrates L-serine and tetrahydrofolate were also similar to the values obtained previously for the rabbit cytosolic enzyme. Comput Appl Biosci, 1995 Aug, 11(4), 379 - 87 Identification of common motifs in unaligned DNA sequences: application to Escherichia coli Lrp regulon; Fraenkel YM et al.; We describe a relatively simple method for the identification of common motifs in DNA sequences that are known to share a common function . The input sequences are unaligned and there is no information regarding the position or orientation of the motif . Often such data exists for protein-binding regions, where genetic or molecular information that defines the binding region is available, but the specific recognition site within it is unknown . The method is based on the principle of 'divide and conquer'; we first search for dominant submotifs and then build full-length motifs around them . This method has several useful features: (i) it screens all submotifs so that the results are independent of the sequence order in the data; (ii) it allows the submotifs to contain spacers; (iii) it identifies an existing motif even if the data contains 'noise'; (iv) its running time depends linearly on the total length of the input . The method is demonstrated on two groups of protein-binding sequences: a well-studied group of known CRP-binding sequences, and a relatively newly identified group of genes known to be regulated by Lrp . The Lrp motif that we identify, based on 23 gene sequences, is similar to a previously identified motif based on a smaller data set, and to a consensus sequence of experimentally defined binding sites . Individual Lrp sites are evaluated and compared in regard to their regulation mode. Mol Biotechnol, 1995 Aug, 4(1), 45 - 54 Epitope mapping of recombinant antigens by transposon mutagenesis; Morris GE et al.; We describe a method for generating a plasmid library expressing random truncations of a recombinant protein and for epitope mapping by screening the library with monoclonal antibodies . The key step is the random introduction of the transposon, Tn1000, which carries stop codons in all three reading frames, into a bacterial expression plasmid by using a simple bacterial mating procedure . Antibody-positive clones are then selected and the point of protein truncation is determined by sequencing the plasmid DNA at the point of transposon insertion . One advantage of the method is that no subcloning or in vitro manipulation of DNA is necessary. Protein Sci, 1995 Aug, 4(8), 1658 - 60 Crystallization and preliminary X-ray diffraction studies of the human adenovirus serotype 2 proteinase with peptide cofactor; Keefe LJ et al.; Recombinant human adenovirus serotype 2 proteinase (both native and selenomethionine-substituted) has been crystallized in the presence of the serotype 12, 11-residue peptide cofactor . The crystals (space group P3(1)21 or P3(2)21, one molecule per asymmetric unit, a = b = 41.3 angstrum, c = 197.0 angstrum) grew in solutions containing 20-40% 2-methyl-2,4-pentanediol (MPD), 0.1-0.2 M sodium citrate, and 0.1 M sodium HEPES, pH 5.0-7.5 . Diffraction data (84% complete to 2.2 angstrum resolution with Rmerge of 0.0335) have been measured from cryopreserved native enzyme crystals with the Argonne blue (1,024 x 1,024 pixel array) charge-coupled device detector at beamline X8C at the National Synchrotron Light Source (operated by Argonne National Laboratory's Structural Biology Center) . Additionally, diffraction data from selenomethionine-substituted proteinase, 65% complete to 2.0 angstrum resolution with Rmerge values ranging 0.05-0.07, have been collected at three X-ray energies at and near the selenium absorption edge . We have determined three of the six selenium sites and are initiating a structure solution by the method of multiwavelength anomalous diffraction phasing. Protein Sci, 1995 Aug, 4(8), 1651 - 3 Crystallization of the chaperone protein SecB; Vrielink A et al.; The secretory protein SecB found in Escherichia coli is a molecular chaperone that binds to precursor forms of a number of proteins targeted for export to the periplasmic space . SecB maintains these proteins in a translocation-competent conformation facilitating the translocation process . The material has been cloned and expressed in E . coli . Crystals have been grown from polyethylene glycol 8000 by vapor diffusion using the hanging drop technique . These crystals are monoclinic, belonging to space group C2 with unit cell dimensions a = 56.0 A, b = 111.1 A, c = 134.7 A, and beta = 104 degrees . The crystals diffract to 8 A resolution on a Rigaku imaging plate detector . Dynamic light scattering experiments suggest that SecB exhibits aggregation behavior with a number of different precipitating agents . These results may explain resistance of SecB to forming ordered crystals. Protein Sci, 1995 Aug, 4(8), 1648 - 50 Purification and preliminary X-ray crystallographic studies of recombinant L-ribulose-5-phosphate 4-epimerase from Escherichia coli; Andersson A et al.; The araD gene from Escherichia coli, coding for L-ribulose-5-phosphate 4-epimerase, was overexpressed and the resulting enzyme was purified to homogeneity . Crystals of L-ribulose-5-phosphate 4-epimerase, obtained with 4.0 M sodium formate as precipitant, belong to space group P4212 with unit cell dimensions a = b = 107.8 A and c = 281.4 A and diffract to at least 2.2 A resolution . Density measurements of these crystals are consistent with eight subunits in the asymmetric unit. Protein Sci, 1995 Aug, 4(8), 1544 - 52 Urea-induced dissociation and unfolding of dodecameric glutamine synthetase from Escherichia coli: calorimetric and spectral studies; Zolkiewski M et al.; Urea-induced dissociation and unfolding of manganese.glutamine synthetase (Mn.GS) have been studied at 37 degrees C (pH 7) by spectroscopic and calorimetric methods . In 0 to approximately 2 M urea, Mn.GS retains its dodecameric structure and full catalytic activity . Mn.GS is dissociated into subunits in 6 M urea, as evidenced by a 12-fold decrease in 90 degrees light scattering and a monomer molecular weight of 51,800 in sedimentation equilibrium studies . The light scattering decrease in 4 M urea parallels the time course of Trp exposure but occurs more rapidly than changes in secondary structure and Tyr exposure . Early and late kinetic steps appear to involve predominantly disruption of intra-ring and inter-ring subunit contacts, respectively, in the layered hexagonal structure of Mn.GS . The enthalpies for transferring Mn.GS into urea solutions have been measured by titration calorimetry . After correcting for the enthalpy of binding urea to the protein, the enthalpy of dissociation and unfolding of Mn.GS is 14 +/- 4 cal/g . A net proton uptake of approximately 50 H+/dodecamer accompanies unfolding reactions . The calorimetric data are consistent with urea binding to multiple, independent sites in Mn.GS and the number of binding sites increasing approximately 9-fold during the protein unfolding. Protein Sci, 1995 Aug, 4(8), 1507 - 15 C-terminal specific protein degradation: activity and substrate specificity of the Tsp protease; Keiler KC et al.; The activity of Tsp, a periplasmic endoprotease of Escherichia coli, has been characterized by assaying the cleavage of protein and peptide substrates, determining the cleavage sites in several substrates, and investigating the kinetics of the cleavage reaction . Tsp efficiently cleaves substrates that have apolar residues and a free alpha-carboxylate at the C-terminus . Tsp cleaves its substrates at a discrete number of sites but with rather broad primary sequence specificity . In addition to preferences for residues at the C-terminus and cleavage sites, Tsp displays a preference for substrates that are not stably folded: unstable variants of Arc repressor are better substrates than a hyperstable mutant, and a peptide with little stable structure is cleaved more efficiently than a protein substrate . These data are consistent with a model in which Tsp cleavage of a protein substrate involves binding to the C-terminal tail of the substrate, transient denaturation of the substrate, and then recognition and hydrolysis of specific peptide bonds. Protein Sci, 1995 Aug, 4(8), 1498 - 506 Escherichia coli alkaline phosphatase: X-ray structural studies of a mutant enzyme (His-412-->Asn) at one of the catalytically important zinc binding sites; Ma L et al.; The X-ray structure of a mutant version of Escherichia coli alkaline phosphatase (H412N) in which His-412 was replaced by Asn has been determined at both low (-Zn) and high (+Zn) concentrations of zinc . In the wild-type structure, His-412 is a direct ligand to one of the two catalytically critical zinc atoms (Zn1) in the active site . Characterization of the H412N enzyme in solution revealed that the mutant enzyme required high concentrations of zinc for maximal activity and for high substrate and phosphate affinity (Ma L, Kantrowitz ER, 1994, J Biol Chem 269:31614-31619) . The H412N enzyme was also inhibited by Tris, in contrast to the wild-type enzyme, which is activated more than twofold by 1 M Tris . To understand these kinetic properties at the molecular level, the structure of the H412N (+Zn) enzyme was refined to an R-factor of 0.174 at 2.2 A resolution, and the structure of the H412N(-Zn) enzyme was refined to an R-factor of 0.166 at a resolution of 2.6 A . Both indicated that the Asn residue substituted for His-412 did not coordinate well to Zn1 . In the H412N(-Zn) structure, the Zn1 site had very low occupancy and the phosphate was shifted by 1.8 A from its position in the wild-type structure . The Mg binding site was also affected by the substitution of Asn for His-412 . Both structures of the H412N enzyme also revealed a surface-accessible cavity near the Zn1 site that may serve as a binding site for Tris.(ABSTRACT TRUNCATED AT 250 WORDS) J Vet Med Sci, 1995 Aug, 57(4), 751 - 3 Effect of platelet activating factor antagonist (TCV-309) on lung injury in dogs with experimentally endotoxin-induced shock; Okano S et al.; The effect of TCV-309, a newly developed platelet activating factor (PAF) antagonist, on the wet/dry weight ratio of the lung (index of pulmonary edema) and the pulmonary surface activity (index of pulmonary compliance) was evaluated in comparison with that of CV-3988 (PAF-antagonist) . Administration of TCV-309 (1 mg/kg) or CV-3988 (10 mg/kg) significantly reduced the wet/dry weight ratio which was increased by endotoxin administration (3 mg/kg) . It also augmented the pulmonary surface activity . Administration of either TCV-309 or CV-3988 alleviated the histologic lesions caused by endotoxic shock . These results suggest that lung injury during endotoxic shock can be controlled by TCV-309 as by CV-3988. J Trauma, 1995 Aug, 39(2), 179 - 85; discussion 185-6 Effects of inhaled nitric oxide on right ventricular function in endotoxin shock; Offner PJ et al.; Sepsis has been shown to cause right ventricular (RV) dysfunction, which may be related to pulmonary hypertension and increased RV afterload . This study evaluates the effects of inhaled nitric oxide (NO), a selective pulmonary vasodilator, on RV function in a porcine model of endotoxemia . After an infusion of Escherichia coli lipopolysaccharide (LPS; 200 micrograms/kg), animals were resuscitated with saline (1 mL/kg/min) and observed for 3 hours while being mechanically ventilated (Fio2 = 0.6, tidal volume = 12 mL/kg, and peak end-expiratory pressure = 5 cm H2O) . The LPS group (n = 5) received no additional treatment . The NO group (n = 5) received inhaled NO (40 ppm) for the last 2 hours . The control group (n = 5) received only saline without LPS . Hemodynamic data and blood gases were collected hourly for 3 hours . LPS resulted in pulmonary hypertension and RV dysfunction as indexed by a decreased RV ejection fraction and increased RV end-diastolic volume . Inhaled NO significantly decreased pulmonary hypertension and significantly increased RV ejection fraction and oxygen delivery without adverse effects . In conclusion, inhaled NO significantly improved pulmonary hypertension and RV dysfunction in a porcine model of endotoxemia and should be a useful therapeutic modality in selected septic patients. Plant J, 1995 Aug, 8(2), 283 - 94 Biochemical and molecular characterization of a novel starch synthase from potato tubers; Edwards A et al.; An isoform of starch synthase from potato tubers which is present both in the stroma of the plastid and tightly bound to starch granules has been identified biochemically and a cDNA has been isolated . The protein encoded by the cDNA is 79.9 kDa and has a putative transit peptide and a distinct N-terminal domain which is predicted to be highly flexible . It is similar in both amino acid sequence and predicted structure to the granule-bound starch synthase II (GBSSII) of pea embryos . When expressed in Escherichia coli, the mature protein has starch synthase activity . The importance of the isoform has been assessed by biochemical measurements and antisense transformation experiments in which the amount of the isoform in the tuber is severely and specifically reduced . Both approaches indicate that the isoform contributes a maximum of 15% of the total starch synthase activity of the tuber . It is suggested that this isoform and the GBSSII of pea embryos represent a widely distributed class of isoforms of starch synthase . The contribution to total starch synthase activity of members of this class probably varies considerably from one type of storage organ to another. Nat Genet, 1995 Aug, 10(4), 430 - 5 Gene therapy of metastatic cancer by in vivo retroviral gene targeting; Hurford RK Jr et al.; We have achieved efficient transduction of tumour metastases in vivo by the vascular delivery of retroviral producer cells . Experimental liver metastases in mice were created by intrasplenic injection of tumour cells into the portal venous circulation . Following the establishment of micrometastases, delivery of retroviral producer cells by the same route with a vector containing the Escherichia coli beta-galactosidase (lacZ) gene demonstrated selective in vivo gene transfer to tumour deposits . By this approach, two retroviral producer cell lines encoding cytokines (IL-4 and IL-2) directed tumoricidal inflammatory responses to established metastases . Cytokine gene targeting inhibited metastasis formation and caused significant overall reduction in tumour burden . These results suggest a novel therapeutic approach for the treatment of disseminated cancer. FEMS Microbiol Rev, 1995 Aug, 17(1-2), 207 - 12 Two-stage model for integration of the lysis protein E of phi X174 into the cell envelope of Escherichia coli; Schon P et al.; As a tool for determining the topology of the small, 91-amino acid phi X174 lysis protein E within the envelope complex of Escherichia coli, a lysis active fusion of protein E with streptavidin (E-FXa-StrpA) was used . The E-FXa-StrpA fusion protein was visualised using immune electron microscopy with gold-conjugated anti-streptavidin antibodies within the envelope complex in different orientations . At the distinct areas of lysis characteristic for protein E, the C-terminal end of the fusion protein was detected at the surface of the outer membrane, whereas at other areas the C-terminal portion of the protein was located at the cytoplasmic side of the inner membrane . These results suggest that a conformational change of protein E is necessary to induce the lysis process, an assumption supported by proteinase K protection studies . The immune electron microscopic data and the proteinase K accessibility studies of the E-FXa-StrA fusion protein were used for the working model of the E-mediated lysis divided into three phases: phase 1 is characterised by integration of protein E into the inner membrane without a cytoplasmic status in a conformation with its C-terminal part facing the cytoplasmic side; phase 2 is characterised by a conformational change of the protein transferring the C-terminus across the inner membrane; phase 3 is characterised by a fusion of the inner and outer membranes and is associated with a transfer of the C-terminal domain of protein E towards the surface of the outer membrane of E . coli. FEMS Microbiol Rev, 1995 Aug, 17(1-2), 109 - 19 Replication of coliphage lambda DNA; Taylor K et al.; A general scheme of lambda phage and plasmid DNA replication in Escherichia coli is presented, and results of in vivo experiments from the authors' laboratory are superimposed . The initiator lambda O functions in the assembly of the replication complex (RC) at ori lambda, making it a stable component of this structure . ClpP/ClpX protease-specific action on lambda O does not affect the regulation of replication; it only degrades the surplus of synthesized lambda O . The initiator lambda O becomes protected from proteolysis at a distinct step of the pathway of RC assembly . The host DnaA initiator-regulated transcriptional activation of ori lambda seems to be coupled with RC assembly at the step of chaperone-mediated rearrangement of the pre-primosome . The once-assembled RC is inherited by one of two lambda plasmid daughter copies at each round of circle-to-circle (theta) replication . The inherited, old RC-driven replication is also dependent on RNA polymerase and DnaA functions . It seems that DnaA licenses lambda plasmid DNA for only one replication round, resembling the putative eukaryotic licensing factor in this respect . The lambda O binding to ori lambda does not seem to play any role in regulation of lambda plasmid replication, and the Cro-autoregulatory loop may be deleted . The emerging picture shows lambda plasmid circles with RCs bound to their ori, awaiting a signal triggering initiation of replication . The host DnaA initiator-regulated transcriptional activation of ori lambda may be involved in signal transmission . Inactivation of DnaA function blocks initiation of lambda phage DNA replication, but the lambdoid prophage Rac compensates this defect and all parental phage DNA molecules, after one round of theta replication switch to the sigma mode and produce progeny in high yield . We suspect that DnaA-regulated transcriptional activation is involved in installation and adequate positioning of two RCs, required for bidirectional replication, but in the Rac-promoted process only one RC may be installed, leading to unidirectional replication continued in the sigma mode . In wild-type cells consumption of DnaA function by the rapidly replicating lambda phage DNA may switch replication from bidirectional theta to unidirectional theta, and later to the sigma mode; the lambda circles produced earlier may play the role of Rac, which is required only when DnaA function has been inactivated prior to phage infection. J Egypt Soc Parasitol, 1995 Aug, 25(2), 303 - 10 A survey of intestinal parasites in students of Adult Educational Center in Sivas, Turkey; Saygi G et al.; The prevalence of intestinal parasites in students of Adult Educational Center (Halk Egitim Merkezi) in Sivas, Turkey was assessed . All of the students were females and their age ranged from 12 to 46 with a mean age of 19 . Diagnosis was made following examination of stool specimens and cellophane tape preparations (CTP) . From each stool specimen only one preparation prepared in saline was searched for both protozoon and helminth parasites . A total of 524 stool specimens and 504 CTP were examined . One or more parasites were detected in 34.5% the former and 23.8% of the latter . When the findings in both specimens were combined the infection rate raised to 48.0% . 176 of the 485 students harboured one, whereas 57 had more than one parasite; 87 had only protozoa, 104 only helminths whereas 42 were infected with parasites of the two groups . The rate of parasites found was as follows: G . intestinalis 8.2%, E . histolytica 1.2%, E . coli 17.9%, A . lumbricoides 2.3%, T . trichiura 0.8%, E . vermicularis 12.6%, T . saginata 16.7%, H . nana 1.0%, and others 0.6 . Findings were evaluated from several points. Anim Genet, 1995 Aug, 26(4), 237 - 42 The porcine intestinal receptor for Escherichia coli K88ab, K88ac: regional localization on chromosome 13 and influence of IgG response to the K88 antigen; Edfors-Lilja I et al.; The loci encoding the porcine intestinal receptors for Escherichia coli K88ab and K88ac (K88abR and K88acR) were firmly assigned to chromosome 13 by linkage analysis using a three-generation pedigree . The linear order of these loci and seven other markers on chromosome 13 was determined by multipoint analyses . The K88abR and K88acR loci were tightly linked (theta = 0.01, zeta = 41.06) with the K88abR locus localized 7.4 cM (sex average) proximal to the transferrin locus . The results, together with previous reports from two other groups, provide an unequivocal assignment of the K88 receptor loci to chromosome 13, and reject a previous assignment to chromosome 4 . Pigs possessing the receptor had a slightly higher specific IgG response to the K88 antigen after an intramuscular immunization with an E . coli vaccine. Am J Physiol, 1995 Aug, 269(2 Pt 1), E199 - 207 Impact of infection on hepatic disposal of a peripheral glucose infusion in the conscious dog; McGuinness OP et al.; The effect of infection on hepatic uptake and disposal of a continuous (180-min) intravenous glucose infusion (8 mg.kg-1.min-1) was examined in conscious, 54-h-fasted, chronically catheterized dogs . Thirty-six hours before a study, either infection was induced by implantation of an Escherichia coli-containing (INF; 2 x 10(9) organisms/kg body wt; n = 6) fibrinogen clot, or a sterile (SH; n = 6) clot was implanted into the peritoneal cavity . Hepatic glucose metabolism was assessed using tracer ({3-3H}glucose and {U-14C}glucose) and arteriovenous difference techniques . Infection increased the basal rate of glucose appearance (45%); glucose levels were not altered . In response to glucose infusion, average blood glucose levels increased to similar levels (140 +/- 9 vs . 147 +/- 11 mg/dl in INF and SH, respectively), whereas arterial insulin levels were higher in the infected group during the last hour of the glucose infusion (77 +/- 10 vs . 41 +/- 5 microU/ml in INF vs . SH) . Infection impaired net hepatic glucose uptake (0.6 +/- 0.5 and 2.7 +/- 0.7 mg.kg-1.min-1 in INF and SH; P < 0.05) . The liver remained a persistent lactate consumer (4.1 +/- 1.8 mumol.kg-1.min-1), whereas the sham group became a net producer of lactate (-3.8 +/- 1.3 mumol.kg-1.min-1) . Infection decreased net hepatic glycogen deposition by 53% . In conclusion, infection impairs net hepatic glucose uptake and glycogen deposition despite an exaggerated increase in insulin levels. Genes Dev, 1995 Aug 1, 9(15), 1846 - 56 Apoptotic phenotype induced by overexpression of wild-type gas3/PMP22: its relation to the demyelinating peripheral neuropathy CMT1A; Fabbretti E et al.; Although the Gas3/PMP22 protein is expressed at highest levels in differentiated Schwann cells, its presence, albeit at lower levels, in non-neuronal tissues and in NIH-3T3 growth-arrested fibroblasts argues for a more general function of this protein that is uncoupled to myelin structure . We show that gas3/PMP22 overexpression in NIH-3T3 growing cells leads to an apoptotic-like phenotype, which is suppressed by antioxidants and characterized by typical membrane blebbing, rounding up, and chromatin condensation, but with no evidence of DNA fragmentation . REF-52 fibroblasts seem to be completely refractive to gas3/PMP22 overexpression . Recently, several point mutations of the human gas3/PMP22 gene have been associated with Charcot-Marie-Tooth type 1A (CMT1A), a common hereditary demyelinating neuropathy . When gas3/PMP22 point mutations (L16P, S79C, T118M, and G150D) are similarly overexpressed in NIH-3T3 cells, the induced apoptotic-like phenotype as compared to the wild-type is significantly reduced . Both of the dominant mutations (L16P, S79C) for CMT1A behave as dominant negatives with respect to the wild type, whereas T118M, the only recessive mutant described, behaves as recessive under the same coexpression experiments . These data suggest a role for altered Schwann cell apoptosis in the pathogenesis of CMT1A. FEMS Microbiol Lett, 1995 Aug 1, 130(2-3), 259 - 65 Involvement of prostaglandin E2 synthesis in the intestinal secretory action of Escherichia coli heat-stable enterotoxin II; Fujii Y et al.; The prostaglandin response of mouse intestinal epithelial cells after exposure to Escherichia coli heat-stable enterotoxin II was examined . The quantity of prostaglandin E2 produced by the intestinal cells was directly related to the dose of heat-stable enterotoxin II . The change in the amount of prostaglandin E2 over time correlated to that of the volume of fluid released into the intestinal lumen . We then demonstrated that administration of heat-stable enterotoxin II into the intestinal loops of mice induced elevation of arachidonic acid and phosphatidic acid levels in intestinal epithelial cells . These results show that heat-stable enterotoxin II stimulates arachidonic acid metabolism in intestinal epithelial cells and that the synthesized prostaglandin E2 functions as a mediator of fluid secretion induced by this enterotoxin. FEMS Microbiol Lett, 1995 Aug 1, 130(2-3), 237 - 44 The role of endonucleases in the expression of ribonuclease II in Escherichia coli; Zilhao R et al.; Ribonuclease II (RNase II), encoded by the rnb gene, is one of the two major Escherichia coli exonucleases involved in mRNA degradation . Some of the ribonucleases implicated in this process have recently been shown to be inter-regulated . In this paper we studied the effects of the endonucleases RNase E and RNase III in rnb expression . We have shown that RNase E cleaves the rnb message internally: when this ribonuclease is inactivated rnb mRNA accumulates with a concomitant increase in RNase II activity . RNase III also affects RNase II expression but in an indirect way . We discuss these implications for the regulation of mRNA degradation. FEMS Microbiol Lett, 1995 Aug 1, 130(2-3), 215 - 20 Isolation and characterization of inhibitory factors of DNA polymerase III holoenzyme from Escherichia coli; Hase M et al.; We isolated fractions by Mono Q chromatography that inhibited the activity of Escherichia coli DNA polymerase III holoenzyme using an assay system with a primed single-stranded DNA template coated with single-stranded DNA binding protein (SSB) . The inhibitory activities were inactivated by heat-treatment at 100 degrees C for 10 min, suggesting that they are proteins . The factors did not inhibit the activity of RNA polymerase of Escherichia coli . The inhibitory effects were less potent for the activities of the large (Klenow) fragment of DNA polymerase I and T4 DNA polymerase than for DNA polymerase III holoenzyme . No degradation of single- or double-stranded DNA was observed in the fractions, indicating that inhibition was not due to degradation of the DNA. FASEB J, 1995 Aug, 9(11), 1013 - 22 Protein Motifs . 7 . The four-helix bundle: what determines a fold? Kamtekar S, Hecht MH. The four-helix bundle motif occurs in many structural contexts and in proteins that are functionally diverse . The motif can be classified into individual folds on the basis of topological and geometric properties . It has been thoroughly investigated structurally by both nuclear magnetic resonance and x-ray crystallography . Many mutants of four-helix bundles have been generated, and the motif has also been the target of de novo design studies . Taken together, these studies provide an opportunity to examine many of the forces governing protein folding . In this article we consider the relative importance of the burial of hydrophobic residues, loss of conformational entropy, packing interactions, interhelical turn composition, and helical dipole interactions all within the context of a single folding motif . We conclude by examining why de novo designed four-helix bundle proteins possess flexible interiors, and possible mechanisms by which natural proteins may lock their cores into rigid structures. Eur J Biochem, 1995 Aug 1, 231(3), 823 - 30 Isolation and structural characterization of trimeric cyanobacterial photosystem I complex with the help of recombinant antibody fragments; Tsiotis G et al.; A monoclonal antibody was derived from mice immunized with the native trimeric, photosystem I (PSI) complex from the cyanobacterium Synechococcus PCC 7002 which reacts with a conformational epitope of the PSI complex . As seen by immunoelectron microscopy, the mAb bound to the stromal side of the thylakoid membranes . The DNA sequence encoding variable regions of the mAb was cloned into recombinant plasmids, sequenced and expressed in Escherichia coli . ELISA, Western blots and immunoelectron microscopy provided evidence that the expressed paired variable domain (Fv) fragments bind to the antigen in the same way as the parent mAb . A one-step purification was applied to purify the trimeric PSI complex using an affinity tag attached to the Fv fragment . Analysis by gel electrophoresis and N-terminal sequencing revealed the presence of the psaA, psaB, psaC, psaD, psaE, psaF and psaL gene products . The antenna size of the isolated PSI/Fv was 139 +/- 9 chlorophyll a/primary electron donor . Flash-induced absorption-change measurements showed that the complex exhibited electron transfer from the primary electron donor, P700, to the Fe-S center, FA/FB . The position of the bound Fv fragment on the trimeric PSI surface was determined by high-resolution electron microscopy and digital image processing. Eur J Biochem, 1995 Aug 1, 231(3), 673 - 81 Comparison of the specificity of bacterially expressed cytoplasmic protein-tyrosine phosphatases SHP and SH-PTP2 towards synthetic phosphopeptide substrates; Dechert U et al.; SHP and SH-PTP2 are related cytoplasmic protein-tyrosine phosphatases having two tandem amino-terminal src homology 2 domains linked to a single catalytic domain . There is growing evidence that these two molecules may exhibit opposing effects within specific signaling pathways . However, the relative contributions of the src homology 2 domains or the catalytic domains to these opposing effects are not well known . To evaluate the potential contribution of the catalytic domains, we compared the substrate specificity of the two phosphatases . As seen previously, the catalytic activities of bacterially expressed SHP and SH-PTP2 were regulated by the presence of the linked src homology 2 domains . In addition, we characterized a cryptic thrombin cleavage site within the carboxy-terminus of SHP that led to a striking increase in the activity of the catalytic domain . Employing a panel of phosphopeptide substrates whose sequences were modeled after intracellular phosphorylation sites, both SHP and SH-PTP2 demonstrated a similar specificity pattern . Similar to SH-PTP2, SHP failed to elicit detectable phosphate release from several phosphopeptide substrates, while displaying catalytic efficiencies that ranged over approximately 40-1.6 x 10(3) M-1 s-1 towards other substrates . In contrast, the PTP-1B phosphatase dephosphorylated all of the phosphopeptide substrates tested with approximately equal ease . The overall similarity demonstrated by the catalytic domains of SHP and SH-PTP2 suggested that differences in the in vivo behavior of these two molecules might not stem from differences in the substrate specificity of the catalytic domains, suggesting instead that the specificity of the src homology 2 domains is more important in this regard. Eur J Biochem, 1995 Aug 1, 231(3), 639 - 43 Overproduction in Escherichia coli, purification and properties of human prothymosin alpha; Evstafieva AG et al.; A bacterial strain overproducing human prothymosin alpha was constructed based on the efficient T7 RNA polymerase transcription of human prothymosin alpha cDNA . The highest yield of the human prothymosin alpha, up to 30% of the total bacterial protein, was achieved with constructions containing 6-10 nucleotides between the Shine-Dalgarno sequence and initiation ATG codon . Unexpectedly, cells grown in the presence of inducer of T7 RNA polymerase synthesis produced substantially lower levels of prothymosin alpha than those grown in the absence of inducer . A simple procedure for prothymosin alpha isolation was elaborated, resulting in large amounts of electrophoretically pure and immunoactive protein. Eur J Biochem, 1995 Aug 1, 231(3), 544 - 50 Recombinant and chemical derivatives of apamin . Implication of post-transcriptional C-terminal amidation of apamin in biological activity; Devaux C et al.; The use of the colicin A lysis protein to direct the extracellular release of a fusion protein from Escherichia coli was investigated as an approach for the preparation of recombinant animal toxins . Apamin, a bee venom neurotoxin, was used as the model toxin . It is reticulated by two disulfide bridges and interacts with small conductance Ca(2+)-activated K+ channels . Substantial amounts of free recombinant apamin were obtained by CNBr cleavage of the fusion protein {col-(1-171)-apa} and HPLC purification . It was recognized by conformation-dependent monoclonal antibodies with a K0.5 value close to that for natural apamin, indicating that folding was correct . In toxicity and binding experiments, the recombinant apamin displayed low activity . The recombinant and natural molecules differed by the amidation of the C-terminal histidine residue . Previous structure/activity relationship studies do not implicate this C-terminal residue in activity but the role of its amidation was not investigated . An apamin analog with a non-amidated C-terminal residue was then chemically synthesized . The biological properties of both recombinant and chemical molecules were determined . Amidation of the C-terminal alpha-carboxyl of apamin appears to be essential for full expression of its biological activity. Eur J Biochem, 1995 Aug 1, 231(3), 528 - 34 Evidence for a catalytic role of tyrosine 383 in the peptidase reaction of leukotriene A4 hydrolase; Blomster M et al.; Leukotriene A4 (LTA4) hydrolase is a bifunctional zinc metalloenzyme which catalyzes the final step in the biosynthesis of the proinflammatory leukotriene B4 and which also possesses a peptidase activity . From sequence comparisons with aminopeptidases, a tyrosine at position 383 in LTA4 hydrolase has been suggested as a possible catalytic amino acid . To explore the potential role of this amino acid in catalysis, we replaced the tyrosine residue with phenylalanine, histidine or glutamine residues by site-directed mutagenesis . The mutated cDNAs were expressed in Escherichia coli and the resulting recombinant proteins, named {Y383F}LTA4 hydrolase, {Y383H}LTA4 hydrolase and {Y383Q}LTA4 hydrolase, were purified to homogeneity to allow assays of both the epoxide hydrolase activity, i.e . the conversion of LTA4 into leukotriene B4, and the peptidase activity . None of the mutated proteins exhibited significant peptidase activities, all of them showing activities less than 0.3% that of the wild-type enzyme . The epoxide hydrolase activity was not affected to the same degree and corresponded to 11, 16 and 17% that of the unmutated enzyme for {Y383F}LTA4 hydrolase, {Y383H}LTA4 hydrolase and {Y383Q}LTA4 hydrolase, respectively . Kinetic analysis was performed with the mutant {Y383Q}LTA4 hydrolase, which revealed an approximately 10-fold increase in Km for leukotriene A4 compared to that for the unmutated enzyme . At high concentrations of substrate, the difference in enzyme velocity was only moderate, with Vmax values of 600 nmol.mg-1.min-1 and 1000 nmol.mg-1.min-1 for {Y383Q}LTA4 hydrolase and the wild-type enzyme, respectively . No such effect of substrate concentration could be observed on the peptidase activity . As a positive control, we exchanged a glycine residue in position 386 for an alanine residue, and the recombinant protein, {G386A}LTA4 hydrolase retained 19% and 77% of the peptidase and epoxide hydrolase activities, respectively . The results from this study are consistent with a role for Tyr383 in the peptidase reaction of LTA4 hydrolase, where it may act as a proton donor in a general base mechanism . However, our data do not allow a similar interpretation for the mechanism involved in the hydrolysis of LTA4 into LTB4. Clin Exp Immunol, 1995 Aug, 101(2), 383 - 6 Both VH and VL chains of polyreactive IgM antibody are required for polyreactivity: expression of Fab in Escherichia coli; Cheung SC et al.; Monoclonal polyreactive antibodies can bind to many structurally dissimilar self and non-self antigens . Neither the precise antigen-binding site on the polyreactive antibody molecule nor the molecular basis of polyreactivity has been elucidated . The present study was initiated to see whether antibody genes encoding the Fab fragment of a human monoclonal polyreactive IgM antibody (MoAb 67) could be efficiently expressed in Escherichia coli, and whether the bacterially expressed Fab fragments possessed biological activity . cDNA encoding the variable domains of the heavy and light chains of MoAb 67 were cloned, amplified by polymerase chain reaction (PCR) and expressed in E . coli . Neither the recombinant heavy nor light chain showed antigen-binding activity . In contrast, the recombinant Fab 67 fragment showed the same antigen-binding reactivity profile as the native IgM antibody . It is concluded that the antigen-binding activity of polyreactive antibodies resides in the Fab fragment, and that both the heavy and light chains are required for activity. Mol Carcinog, 1995 Aug, 13(4), 213 - 9 How are potent bulky carcinogens able to induce such a diverse array of mutations? Loechler EL. Mutations induced by activated benzo{a}pyrene ((+)-anti-B{a}PDE) in Escherichia coli are being investigated, by using both random and adduct-site-specific mutagenesis approaches . A working hypothesis was proposed that the major adduct of (+)-anti-B{a}PDE (formed at N2-Gua) is able to induce different base-substitution mutations (e.g., GC-->TA vs . GC-->AT) depending upon its conformation in DNA, which can be influenced by various factors, notably DNA sequence context . Frameshift mutations are also common with (+)-anti-B{a}PDE, and other work suggested that the frameshift and base-substitution mutagenesis pathways are coupled . The simplest hypothesis to rationalize this interrelationship is that a single (+)-anti-B{a}PDE adduct in a single conformation can be bypassed via either a frameshift or a base-substitution pathway . This counterintuitive notion can be reconciled if there are two different kinds of conformations on the pathway to mutagenesis: a class I conformation, which is the initial conformation of a DNA adduct in double-stranded DNA before its encounter with a DNA polymerase, and a class II conformation, which is the conformation that forms at a single-strand/double-strand DNA junction during replication by a DNA polymerase . Thus, GC-->TA and GC-->AT mutations may be induced by different class I conformations, whereas base substitution and frameshift mutations may be induced by the same class I conformation but by different class II conformations . The pathway of mutagenesis would be dictated by the relevant class I and II conformations, which in turn would be controlled by various factors, notably DNA sequence context. Virology, 1995 Aug 1, 211(1), 73 - 83 Mutational analysis of vaccinia DNA ligase defines residues essential for covalent catalysis; Shuman S et al.; DNA ligation entails AMP transfer from ATP to the 5' end of DNA to form a DNA-adenylate structure, A(5')pp(5')N . A similar reaction involving GMP transfer occurs during 5' capping of eukaryotic mRNA . In both cases, nucleotidyl transfer occurs through a covalent lysyl-NMP intermediate . There is local sequence conservation among ligases and capping enzymes in the vicinity of the active site lysine (KxDG) and at three other collinear motifs . The role of these motifs in DNA ligation was tested by mutating individual conserved residues in the vaccinia virus DNA ligase . Wild-type and mutated versions of vaccinia ligase were expressed in bacteria as His-tagged fusion proteins and purified by Ni-affinity and phosphocellulose chromatography steps . We found that Ala substitution for Lys-231 (the presumptive active site) abrogated enzyme-adenylate formation and DNA ligation activities . Ala mutations at conserved residues Glu-283, Glu-377, and Lys-397 also resulted in loss of ligation activity, which correlated with a defect in ligase-AMP formation . These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a common structural basis for covalent nucleotidyl transfer. Virology, 1995 Aug 1, 211(1), 218 - 26 Involvement of cellular casein kinase II in the phosphorylation of measles virus P protein: identification of phosphorylation sites; Das T et al.; The phosphoprotein P gene of measles virus (Edmonston strain) has been cloned in the Escherichia coli expression vector pET-3a with a histidine tag at the C-terminal end . The expressed protein was soluble, unphosphorylated, and constituted 10 to 20% of total cellular protein . Recombinant P protein purified by Ni-affinity chromatography was found to be efficiently phosphorylated in vitro by recombinant casein kinase II (CKII) or by the CKII activity present in the uninfected cell extract . A comparison of phosphopeptide analyses between the in vivo- and the in vitro-32P-labeled P proteins revealed that both proteins share common phosphorylation sites . In an attempt to identify the exact site of the CKII-mediated phosphorylation, we altered specific serine residues located within the CKII consensus motif to alanine by site-directed mutagenesis . The results indicate that Ser 86, Ser 151, and Ser 180 located within the N-terminal half of the P protein are involved in the CKII-mediated phosphorylation of the P protein. J Bacteriol, 1995 Aug, 177(16), 4676 - 80 Identification of transcriptional start sites and the role of ppGpp in the expression of rpoS, the structural gene for the sigma S subunit of RNA polymerase in Escherichia coli; Lange R et al.; rpoS is the structural gene for the sigma S subunit of RNA polymerase which controls the expression of a large number of genes in Escherichia coli that are induced during entry into stationary phase or in response to increased medium osmolarity . Using a combination of primer extension experiments and a 5' deletion analysis of the region upstream of rpoS, we show that rpoS transcription is mainly driven by a single promoter (rpoSp1) located within the nlpD gene upstream of rpoS (the two relatively weak nlpD promoters contribute to the low level of rpoS expression during early exponential phase) . In addition, we demonstrate that the expression of both transcriptional and translational rpoS::lacZ fusions as well as the level of rpoS mRNA originating at rpoSp1 is strongly reduced in ppGpp-deficient relA spoT mutants . However, experiments with the 5' deletion constructs indicate that a lack of ppGpp does affect transcriptional elongation rather than initiation. J Bacteriol, 1995 Aug, 177(16), 4587 - 92 Aerobic inactivation of fumarate reductase from Escherichia coli by mutation of the {3Fe-4S}-quinone binding domain; Cecchini G et al.; Fumarate reductase from Escherichia coli functions both as an anaerobic fumarate reductase and as an aerobic succinate dehydrogenase . A site-directed mutation of E . coli fumarate reductase in which FrdB Pro-159 was replaced with a glutamine or histidine residue was constructed and overexpressed in a strain of E . coli lacking a functional copy of the fumarate reductase or succinate dehydrogenase complex . The consequences of these mutations on bacterial growth, assembly of the enzyme complex, and enzymatic activity were investigated . Both mutations were found to have no effect on anaerobic bacterial growth or on the ability of the enzyme to reduce fumarate compared with the wild-type enzyme . The FrdB Pro-159-to-histidine substitution was normal in its ability to oxidize succinate . In contrast, however, the FrdB Pro-159-to-Gln substitution was found to inhibit aerobic growth of E . coli under conditions requiring a functional succinate dehydrogenase, and furthermore, the aerobic activity of the enzyme was severely inhibited upon incubation in the presence of its substrate, succinate . This inactivation could be prevented by incubating the mutant enzyme complex in an anaerobic environment, separating the catalytic subunits of the fumarate reductase complex from their membrane anchors, or blocking the transfer of electro