Gene, 1990 Sep 28, 94(1), 9 - 14 Use of the lac repressor in constructing sequential deletions and a new sequencing vector; Johnson DF et al.; Large sequencing projects require an efficient strategy to generate a series of overlapping clones . This can be accomplished by protecting one end of a linear DNA molecule while sequential deletions are introduced into the other end by exonuclease digestion . We demonstrate that the lac repressor can protect the ends of linear nucleotide sequences from digestion by exonuclease if these ends contain the lac operator sequence . To exploit this, we have inserted the lac operator sequence between the primer-binding site and multiple cloning site of an M13 sequencing vector . Linearizing the replicative form and binding lac repressor protein protects the end next to the vector sequences . Sequential deletions are then introduced into the insert by digesting with exonuclease III or BAL 31 . Because the rate and time of digestion are readily controlled, the region brought next to the sequencing primer site, after religation, can be selected in a timed series of reactions . This minimizes the screening needed to isolate an overlapping series of clones and facilitates sequencing of long regions. Gene, 1990 Sep 28, 94(1), 109 - 13 Construction of a shuttle vector consisting of the Escherichia coli plasmid pACYC177 inserted into the Streptomyces cattleya phage TG1; Foor F et al.; The Escherichia coli plasmid, pACYC177, was inserted into the single PstI site of a deletion derivative of the Streptomyces cattleya phage, TG1 . The hybrid molecule can be propagated as a phage in S . cattleya and as a plasmid in E . coli and is readily transferred between the two species by transfection and transformation . The kanamycin-resistance-encoding gene derived from pACYC177 is not expressed in lysogens of the hybrid phage . Analysis of deletion mutants of the hybrid phage indicated that at least 7.5 kb of phage DNA is dispensable . Some of the deletion mutants fail to lysogenize S . cattleya (Lyg- phenotype) . The locations of these deletions are consistent with the location of the phage att site as previously established by Southern hybridization analysis . The thiostrepton-resistance-encoding gene derived from Streptomyces azureus was inserted into Lyg+ and Lyg- deletion derivatives and is expressed in S . cattleya. Gene, 1990 Sep 28, 94(1), 15 - 22 Chromosome rearrangements induced by recombinant coliphage lambda placMu; Barr GC et al.; Operon fusions to lacZ, commonly used to study bacterial gene expression in vivo, are normally constructed using phage derivatives such as lambda placMu53 or Mud-1 . These derivatives contain a part of trp operon, and we have found that, when integrated into the chromosome, recombination can occur at high frequency between this trp DNA and the chromosomal trp operon leading to chromosomal inversions which fuse lacZ to the trp promoter . Large segments of the chromosome can be inverted by such rearrangements and their occurrence can seriously complicate the isolation of regulatory mutations and other studies unless appropriate precautions are taken . This phenomenon provides a simple means of isolating inversions of defined chromosomal segments and determining the direction of transcription of some lacZ operon fusions. Gene, 1990 Sep 28, 94(1), 1 - 7 A phasmid optimised for protein design projects: pMAMPF; Szardenings M et al.; Protein design requires the rapid production of recombinant genes and active recombinant proteins, the latter in sufficient amounts for functional and physical studies . We present here the construction and application of a new phasmid vector system, using the fd phage origin, lambda pL promoter, ompA-leader sequence and pMB1 origin, which allows the preparation of secretable proteins in active form, mutagenesis and gene sequencing, without subcloning steps . The vector can be used in plasmid form in a stably transformed culture to induce product formation, or as a packaged single-stranded phasmid, which, via batch transduction in a growing culture, leads directly to recombinant protein formation . This latter method has the advantage that, during the short period required for phasmid amplification, little counterselection against clones with high rDNA-protein synthesis potential occurs . The total sequence of pMAMPF-1 and pMAMPF-3 can be assembled from known sequences of constituent fragments . Mutated regions were directly sequenced. Gene, 1990 Sep 28, 94(1), 89 - 94 Isolation and sequencing of a new beta-galactosidase-encoding archaebacterial gene; Cubellis MV et al.; The gene lacS coding for a beta-galactosidase (beta Gal; EC 3.2.1.23) has been cloned from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4 . It encodes a polypeptide chain of 489 amino acids (aa) (56,764 Da) in good agreement with the value directly measured for the enzyme (60 +/- 2 kDa per subunit) . The aa composition of the enzyme and, in particular, its peculiarly low cysteine content (one Cys per subunit) has been confirmed; at the same time, it has been observed that the very low G + C content of the S . solfataricus genome strongly influences the codon usage preferences in the lacS sequence . There appears to be no evident similarity between this and the Escherichia coli lacZ sequence, thus suggesting that the two enzymes have analogous function, but are not homologous . By comparison with the published sequences of archaebacterial promoters, terminators and ribosome-binding sites, potential regulatory sites have been identified in the flanking regions of the S . solfataricus lacS gene. Biochem Biophys Res Commun, 1990 Sep 28, 171(3), 1064 - 70 Effects of sequence selective drugs on the gel mobility of a bent DNA fragment; Cons BM et al.; The effects of various drugs on the structure of a bent DNA fragment have been investigated by studying DNA mobility in polyacrylamide gels . This DNA fragment has an anomalously slow rate of migration on account of its phased runs of adenines . Nogalamycin and echinomycin increase the gel mobility of kinetoplast DNA suggesting that the bending has been removed . Mithramycin, actinomycin, distamycin and ethidium have either no effect or cause a further reduction in mobility . These results are compared with other, non-bent DNA species which always show a decrease in gel mobility in the presence of DNA binding drugs. Nature, 1990 Sep 27, 347(6291), 382 - 6 Expression and characterization of the cystic fibrosis transmembrane conductance regulator; Gregory RJ et al.; Cystic fibrosis (CF) is a common lethal genetic disease that manifests itself in airway and other epithelial cells as defective chloride ion absorption and secretion, resulting at least in part from a defect in a cyclic AMP-regulated, outwardly-rectifying Cl- channel in the apical surface . The gene responsible for CF has been identified and predicted to encode a membrane protein termed the CF transmembrane conductance regulator (CFTR) . Identification of a cryptic bacterial promoter within the CFTR coding sequence led us to construct a complementary DNA in a low-copy-number plasmid, thereby avoiding the deleterious effects of CFTR expression on Escherischia coli . We have used this cDNA to express CFTR in vitro and in vivo . Here we demonstrate that CFTR is a membrane-associated glycoprotein that can be phosporylated in vitro by cAMP-dependent protein kinase . Polyclonal and monoclonal antibodies directed against distinct domains of the protein immunoprecipitated recombinant CFTR as well as the endogenous CFTR in nonrecombinant T84 cells . Partial proteolysis fingerprinting showed that the recombinant and non-recombinant proteins are indistinguishable . These data, which establish several characteristics of the protein responsible for CF, will now enable CFTR function to be studied and will provide a basis for diagnosis and therapy. J Biol Chem, 1990 Sep 25, 265(27), 16699 - 703 A ribosomal protein is encoded in the chloroplast DNA in a lower plant but in the nucleus in angiosperms . Isolation of the spinach L21 protein and cDNA clone with transit and an unusual repeat sequence; Smooker PM et al.; The distribution of chloroplast ribosomal protein genes between the organelle DNA and the nuclear DNA is highly conserved in land plants, but a notable exception is rpl21 . This gene has been found in the completely sequenced chloroplast genome of a lower plant but not in that of two higher plants . We describe the purification and characterization of the spinach chloroplast ribosomal protein L21 and the isolation and nucleotide sequence of a cDNA clone that encodes its cytoplasmic precursor . The mature protein, identified by NH2-terminal sequencing, has 201 residues (Mr 22,766) and is thus substantially larger than either its Escherichia coli (103 residues) or the lower plant homologue (116 residues) . The extra length is in peptide extensions at both amino and carboxyl termini . The COOH-terminal extension is unusual in that it comprises seven Ala-Glu repeats, a feature not found in any other ribosomal proteins described so far . The cDNA clone also encodes a 55-residue long transit peptide (with a high proportion of the polar residues, threonine and serine), to target the L21 protein into chloroplasts . The identification of rpl21 as a nuclear gene in a higher plant (spinach) and chloroplast gene in a lower plant (liverwort) suggests an organelle-to-nucleus gene relocation during the evolution of the former. J Biol Chem, 1990 Sep 25, 265(27), 16366 - 72 Expression and kinetic characterization of variants of human beta 1 beta 1 alcohol dehydrogenase containing substitutions at amino acid 47; Hurley TD et al.; Arg-47 of human beta 1 beta 1 alcohol dehydrogenase has been replaced with Lys, His, Gln, and Gly by site-directed mutagenesis . The mutated enzymes were expressed in Escherichia coli and purified to homogeneity . The recombinant enzymes with Arg and His at position 47 exhibit kinetic constants and stability which are similar to beta 1 beta 1 and beta 2 beta 2, respectively . The substitution of Lys, His, or Gln for Arg-47 resulted in active enzymes with lower affinity for coenzyme and higher Vmax values than beta 1 beta 1 . The substitution of Gln at position 47 resulted in an enzyme with the highest Vmax for ethanol oxidation of any mammalian alcohol dehydrogenase . In this series of enzymes, the affinity for coenzyme decreases with decreasing pKa of the substituted amino acid side chains . The substitution of Gly at position 47 resulted in an enzyme with a Vmax that was one-half that of the low activity beta 1 beta 1 and coenzyme affinities that are lower than beta 1 beta 1, but are equal to or greater than the affinities exhibited by the His-47 or Gln-47 enzymes . Product inhibition studies indicated a change in mechanism from ordered Bi Bi for beta 1 beta 1 to rapid equilibrium random Bi Bi for the Gly-47 enzyme . The kinetic properties of the Gly-47 enzyme are substantially different from human liver alpha alpha which also has Gly at position 47. Biochemistry, 1990 Sep 25, 29(38), 9023 - 8 Specific binding of lac repressor to linear versus circular polyoperator molecules; Sasmor HM et al.; Gel shift assays were used to examine the binding of the lactose (lac) repressor to polyoperator DNA molecules . Specific binding was differentiated from nonspecific DNA association by (i) equilibrating repressor-operator complexes below the nonspecific association constant and (ii) demonstrating the effects of the inducer isopropyl beta-D-thiogalactoside (IPTG) on the formation of repressor-operator complexes . With the linear polyoperator molecules, all eight operator sites could be simultaneously bound by distinct repressors . However, with circular molecules, the eight operator sites were saturable by repressor only in the nicked circular state and not in the covalently closed circular form . Under the experimental conditions used, there was no evidence of bifunctional repressor binding or loop formation . The results suggest that the conformational perturbation of DNA that occurs upon specific repressor binding was retained in topologically closed molecules and could modify other operator sites so as to make them unavailable for specific binding. Nucleic Acids Res, 1990 Sep 25, 18(18), 5381 - 6 A single mutation in 16S rRNA that affects mRNA binding and translation-termination; Prescott CD et al.; A single base change in 16S rRNA (C726 to G) has previously been shown to have a dramatic effect on protein synthesis in E . coli (1) . This paper more specifically details the effects of the mutation on mRNA binding and translation-termination . The in vitro technique of toeprinting (2) was used to demonstrate that 30S subunits containing the mutation 726G had an altered binding affinity for mRNA by comparison to the wild type . In addition, expression of the mutant ribosomes in vivo resulted in exclusive suppression of the UGA nonsense codon . This effect was supported by in vitro studies that showed the mutant ribosomes to have an altered binding affinity for Release Factor-2. Nucleic Acids Res, 1990 Sep 25, 18(18), 5347 - 51 IPTG-dependent vaccinia virus: identification of a virus protein enabling virion envelopment by Golgi membrane and egress; Rodriguez JF et al.; A novel method has been developed to study the functional roles of individual vaccinia virus gene products that is neither limited by the possible essentiality of the target gene nor by the availability of conditional lethal mutants . The system utilises the E . coli lac repressor protein, the operator sequence to which it binds and the specific inducer IPTG . It allows the generation of recombinant viruses in which the expression of any chosen gene, and hence virus replication, can be externally controlled . In principle, this system is broadly applicable to the functional analysis of genes in any large DNA virus . This approach has demonstrated that the gene encoding the 14 kDa membrane protein of vaccinia virus is non-essential for the production of infectious intracellular virus particles, but essential for the envelopment of intracellular virions by Golgi membrane and for egress of mature extracellular viral particles . This is the first vaccinia virus protein shown to be specifically required for these processes . In vivo this system may prove useful as a means of attenuating recombinant vaccinia virus vaccines by preventing virus spread without reducing the amount of the foreign antigen expressed in each infected cell . Attenuation of other live virus vaccines may be developed in a similar way. J Biol Chem, 1990 Sep 25, 265(27), 16676 - 82 Ribosomal proteins L15 and L16 are mere late assembly proteins of the large ribosomal subunit . Analysis of an Escherichia coli mutant lacking L15; Franceschi FJ et al.; The (minus L15) character from the Escherichia coli strain AM16.98 was transduced to an RNase-deficient strain in order to enable a reconstitution analysis . The following results were obtained . 1) The strain lacking L15 showed a 2-3-fold prolonged generation time and the 70 S ribosomes a reduced tendency toward dissociation . 2) Active particles could not be reconstituted unless L15 was added . Addition of L15 regained activity, even if L15 was added after the two-step procedure during a third incubation . However, a modification of the standard two-step reconstitution procedure (lowering NH4+ from 400 to 240 mM and the incubation temperature of the second step from 50 to 47 degrees C) yielded 100% active particles in the absence of L15 . Active particles could be formed which even lacked L15, L16, and L30 . Addition of either L15 or L16 accelerated the formation of active particles in the second step by a factor of five, and both proteins together by a factor of more than 20 . 3) The activation energy of the rate-limiting step of the second incubation was surprisingly reduced for about 20 kcal/mol in the absence of L15, although the corresponding rates were two to five times slower . We conclude 1) that L15 and L16 are late assembly proteins which accelerate the formation of active particles during the late assembly but are neither needed for the early assembly nor essential for ribosomal functions; 2) that some routes of the late assembly (e.g . incorporation of L16) are changing their significance depending on the NH4+ concentration and the absence and presence of L15; and 3) that different reactions are rate limiting during the second step incubation in the presence and absence of L15, respectively, and that the corresponding reaction rates exhibit a different temperature dependence. J Biol Chem, 1990 Sep 25, 265(27), 16592 - 603 A Pro to Gly mutation in the hinge of the arabinose-binding protein enhances binding and alters specificity . Sugar-binding and crystallographic studies; Vermersch PS et al.; The L-arabinose-binding protein (ABP) of Escherichia coli consists structurally of two distinct globular domains connected by a hinge of three separate peptide segments . Arabinose is bound and completely sequestered within the deep cleft between the two domains . With reduced affinity, ABP also binds D-galactose (approximately 2-fold reduction) and D-fucose (approximately 40-fold reduction) . Experiments have been conducted to explore the role in sugar binding of the hinge connecting the two domains of ABP . To increase the flexibility of the hinge region, a glycine was substituted for a proline at position 254 by site-directed mutagenesis . Unexpectedly, this mutation resulted in the dramatic enhancement of galactose binding over that of arabinose . The affinity of the mutant ABP for galactose increased by over 20-fold, while that for arabinose and fucose remained relatively unchanged . We have measured association and dissociation rates of the Gly-254 ABP with L-arabinose, D-galactose, and D-fucose and have determined the crystallographic structure of the protein complexed with each of the three sugars . Both the ligand-binding kinetic measurements and structure analysis indicate that the altered specificity is due to an effective increase in the rigidity of the hinge in the closed conformation which is induced upon galactose binding . Stabilizing contacts are formed between the strands of the hinge in the Gly-254 ABP when galactose is bound which are not found in complexes with the other sugars or the liganded wild-type protein. Biochemistry, 1990 Sep 25, 29(38), 9006 - 14 Reduction of the potent DNA polymerase III holoenzyme 3'----5' exonuclease activity by template-primer analogues; Griep MA et al.; The DNA polymerase III holoenzyme of Escherichia coli contains a potent 3'----5' exonuclease that removes the terminal nucleotide from a synthetic deoxyoligonucleotide primer with a half-life of approximately 2 s . Degradation of primers could not be effectively prevented by permitting the holoenzyme to "idle" at the primer terminus in the presence of limited deoxynucleoside triphosphates . To further characterize this exonuclease and to develop stable primers to facilitate experimental manipulations, we synthesized a series of twelve 25-mer oligonucleotides that differed only in the two 3'-terminal residues . The penultimate position contained either a CMP or a dCMP residue, while at the terminal position either AMP, dAMP, 2',3'-dideoxyAMP, cordycepin (3'-dAMP), dAMP alpha S, or 2',3'-dideoxyAMP alpha S was incorporated . No single change at either the 3'-penultimate or 3'-terminal positions resulted in a decrease in the exonuclease rate greater than 10-fold; however, combined changes at these two sites resulted in a strong synergistic effect . Placing a ribonucleotide at the penultimate position coupled by a phosphorothioate linkage to a terminal 2',3'-dideoxynucleotide reduced the rate of exonucleolytic activity almost 30,000-fold (half-life approximately 16 h) . If only the ribonucleotide and phosphorothioate substitutions were made, a primer capable of being efficiently elongated was generated that exhibited a 500-fold increase in stability (half-life = 40 min) . The elemental effect observed by substituting a nonbridging oxygen in the terminal phosphodiester bond for sulfur increased from 1.5 to 200 as other substitutions were made that decreased the exonuclease rate . This was consistent with a change in the rate-limiting step of the exonuclease reaction from a conformational change to the chemical step where the covalent bond is cleaved . At least part of this effect appears to be due to perturbations within the enzyme's active site and not solely due to changes in electrophilicity. J Biol Chem, 1990 Sep 25, 265(27), 16478 - 83 Expression of the phospholipid-dependent Escherichia coli sn-1,2-diacylglycerol kinase in COS cells perturbs cellular lipid composition; Ramer JK et al.; The Escherichia coli sn-1,2-diacylglycerol (DAG) kinase has been successfully expressed in COS cells . The E . coli dgkA locus which contains the coding sequences for DAG kinase was subcloned into an eukaryotic expression vector, pMT2 . COS cells transfected with the vector pMT2dgk expressed the DAG kinase as shown by Western analysis . Immunofluorescence studies revealed that the E . coli DAG kinase was prominently but not exclusively located in the endoplasmic reticulum . In addition, mixed micellar assays in beta-octyl glucoside revealed that membranes prepared from pMT2dgk-transfected COS cells contained over a 1500-fold increase in DAG kinase activity: 107 nmol/min/mg compared with only 0.067 nmol/min/mg for controls . DAG kinase activity from the E . coli enzyme was distinguished from endogenous COS cell activity based on differences in thermolability and the ability of the E . coli enzyme to use ceramide as a substrate . No ceramide kinase activity was detected in control COS cells, so the activity detected in pMT2dgk transfectants must have resulted from the expressed E . coli DAG kinase . The Km values for DAG kinase derived from E . coli and COS cells were nearly identical . Finally, transfected COS cells were labeled with {32P}Pi to investigate possible perturbations in lipid composition induced by the action of the E . coli DAG kinase . Ceramide (generated by the action of sphingomyelinase) was also used to clearly implicate the E . coli enzyme . Levels of ceramide phosphate increased more than 150-fold in pMT2dgk-transfected cells relative to controls . The results of these studies show that the E . coli enzyme expressed in COS cells is active and perturbs lipid composition in the intact cell system; the absolute lipid cofactor requirement of E . coli DAG kinase can be satisfied in COS cells. J Biol Chem, 1990 Sep 25, 265(27), 16389 - 93 Yeast cytochrome c oxidase subunit VII is essential for assembly of an active enzyme . Cloning, sequencing, and characterization of the nuclear-encoded gene; Aggeler R et al.; The gene COX VII coding for yeast cytochrome c oxidase subunit VII has been cloned by a two-step procedure . Two degenerate oligonucleotides corresponding to amino- and carboxyl-terminal protein segments were used in a polymerase chain reaction for the amplification of a major portion of subunit VII (residues 1-52), which was then used for the cloning of complete COX VII . From the nucleotide sequence, an additional amino-terminal and two additional carboxyl-terminal amino acids are predicted as compared with the described primary sequence (Power, S . D., Lochrie, M . A., and Poyton, R . O . (1986) J . Biol . Chem . 261, 9206-9209) . Beside subunit VIIa the subunit described here is the only nuclear encoded subunit of cytochrome c oxidase in yeast without a leader sequence . COX VII exists as a single copy per haploid genome as shown by Southern blot and gene disruption . Null mutants produced by gene disruption at the COX VII locus were respiratory-deficient . No cytochrome c oxidase activity was detectable nor was there an assembly of the oxidase complex. J Biol Chem, 1990 Sep 25, 265(27), 16527 - 33 Synapsin II . Mapping of a domain in the NH2-terminal region which binds to small synaptic vesicles; Thiel G et al.; The synapsins are a family of neuron-specific phosphoproteins that selectively bind to small synaptic vesicles in the presynaptic nerve terminal . Using the cDNA encoding rat synapsin IIb, we employed an Escherichia coli expression system to synthesize a variety of fusion proteins containing a truncated protein A linked to different portions of the NH2-terminal region of synapsin II . The recombinant proteins were purified by IgG-Sepharose chromatography and tested in vitro for their ability to bind to purified synaptic vesicles . These experiments identified a region between amino acids 43 and 121 of the amino-terminal portion of synapsin II which binds to synaptic vesicles . Mild trypsinization of synaptic vesicles reduces binding of recombinant proteins to synaptic vesicles, suggesting that the interaction between synapsin II and the vesicles is in part mediated by a synaptic vesicle protein . The 42 NH2-terminal amino acids of synapsin II are not necessary for binding to synaptic vesicles, although this domain contains the phosphorylation site for cAMP-dependent protein kinase. Biochemistry, 1990 Sep 25, 29(38), 9064 - 72 Histidine-40 of ribonuclease T1 acts as base catalyst when the true catalytic base, glutamic acid-58, is replaced by alanine; Steyaert J et al.; Mechanisms for the ribonuclease T1 (RNase T1; EC 3.1.27.3) catalyzed transesterification reaction generally include the proposal that Glu58 and His92 provide general base and general acid assistance, respectively {Heinemann, U., & Saenger, W . (1982) Nature (London) 299, 27-31} . This view was recently challenged by the observation that mutants substituted at position 58 retain high residual activity; a revised mechanism was proposed in which His40, and not Glu58, is engaged in catalysis as general base {Nishikawa, S., Morioka, H., Kim, H., Fuchimura, K., Tanaka, T., Uesugi, S., Hakoshima, T., Tomita, K., Ohtsuka, E., & Ikehara, M . (1987) Biochemistry 26, 8620-8624} . To clarify the functional roles of His40, Glu58, and His92, we analyzed the consequences of several amino acid substitutions (His40Ala, His40Lys, His40Asp, Glu58Ala, Glu58Gln, and His92Gln) on the kinetics of GpC transesterification . The dominant effect of all mutations is on Kcat, implicating His40, Glu58, and His92 in catalysis rather than in substrate binding . Plots of log (Kcat/Km) vs pH for wild-type, His40Lys, and Glu58Ala RNase T1, together with the NMR-determined pKa values of the histidines of these enzymes, strongly support the view that Glu58-His92 acts as the base-acid couple . The curves also show that His40 is required in its protonated form for optimal activity of wild-type enzyme . We propose that the charged His40 participates in electrostatic stabilization of the transition state; the magnitude of the catalytic defect (a factor of 2000) from the His40 to Ala replacement suggests that electrostatic catalysis contributes considerably to the overall rate acceleration . For Glu58Ala RNase T1, the pH dependence of the catalytic parameters suggests an altered mechanism in which His40 and His92 act as base and acid catalyst, respectively . The ability of His40 to adopt the function of general base must account for the significant activity remaining in Glu58-mutated enzymes. J Biol Chem, 1990 Sep 25, 265(27), 16604 - 13 Proteasomes are essential for yeast proliferation . cDNA cloning and gene disruption of two major subunits; Fujiwara T et al.; The cDNAs encoding two major subunits, named YC1 and YC7-alpha, of yeast proteasomes (multicatalytic proteinase complexes) were isolated and sequenced . As deduced from their nucleotide sequences, YC1 and YC7-alpha consist of 288 and 252 amino acid residues with calculated molecular weights of 31,534 and 27,999, respectively . They showed marked sequence homology to other eukaryotic proteasome components, suggesting that proteasomes are composed of a family of subunits with the same evolutional origin . To obtain information on the physiological role of proteasomes, we disrupted the chromosomal genes of YC1 and YC7-alpha of yeast cells independently, using isolated cDNA clones . Disruption of the coding region of one copy of the YC1 gene in diploid yeast created a recessive lethal mutation, but disruption of the 3'-noncoding region of the gene had no effect on cell proliferation . Disruption of the YC7-alpha gene also had a lethal effect on haploid yeast cells . These findings demonstrated that YC1 and YC7-alpha are both encoded by a single copy gene and that these genes are essential for proliferation of yeast cells. Eur J Biochem, 1990 Sep 24, 192(3), 695 - 701 Electron microscopic analysis of DNA forks generated by Escherichia coli DNA helicase II; Wessel R et al.; T7 phage DNA eroded with lambda exonuclease (to create 3'-protruding strands) or exonuclease III (to create 5'-protruding strands) was treated under unwinding assay conditions with DNA helicase II . Single-stranded DNA-binding protein (of Escherichia coli or phage T4) was added to disentangle the denatured DNA and the complexes were examined in the electron microscope . DNA helicase II complexes filtered through a gel column before assay retain the ability to generate forks suggesting that DNA helicase II unwinds in a preformed complex by translocating along the bound DNA strand . The enzyme initiates preferentially at the ends of the lambda-exonuclease-treated duplexes and is found at a fork on the initially protruding strand . It also initiates at the ends of the exonuclease-III-treated duplexes where, as with approximately 5% of the forks traceable back to a single-stranded gap, it is found on the initially recessed strand . The results are consistent with the view that DNA helicase II unwinds in the 3'-5' direction relative to the bound strand . They also confirm that the enzyme can initiate at the end of a fully base-paired strand . At a fork, DNA helicase II is bound as a tract of molecules of approximately 110 nm in length . Tracts of enzyme assemble from non-cooperatively bound molecules in the presence of ATP . During unwinding, DNA helicase II apparently can translocate to the displaced strand which conceivably can deplete the leading strand of the enzyme . Continued adsorption of enzyme to DNA might replenish forks arrested by strand switch of the unwinding enzyme. Eur J Biochem, 1990 Sep 24, 192(3), 689 - 93 Direction of the DNA-unwinding reaction catalysed by Escherichia coli DNA helicase II; Georgi-Geisberger P et al.; The direction of the DNA-unwinding reaction catalysed by Escherichia coli DNA helicase II was studied using gapped linear DNA molecules with short duplex ends as substrate . The results suggest that DNA helicase II unwinds with 3'-5' polarity relative to the single strand of the DNA partial duplex . At high enzyme DNA ratio the enzyme also unwinds the duplex connected to the 3' end of the single strand and, as further studies show, fully duplex linear DNA . The fraction of DNA unwound decreases as the length of the duplex substrate increases . The preference of DNA helicase II for a short duplex can obscure the fact that the typical substrate is duplex connected to the 5' end of a single strand. Eur J Biochem, 1990 Sep 24, 192(3), 583 - 9 Reconstitution of translocation activity for secretory proteins from solubilized components of Escherichia coli; Tokuda H et al.; The protein translocation system of Escherichia coli was solubilized and reconstituted, using the octylglucoside dilution method, into liposomes prepared from E . coli phospholipids . SecA, ATP, phospholipids and membrane proteins were found to be essential for the translocation of a model secretory protein, uncleavable OmpF-Lpp . Phospholipids were found to play roles not only in liposome formation but also in the stabilization of membrane proteins during the octylglucoside extraction . The effects of IgGs specific to five distinct regions of the SecY molecule on protein translocation into proteoliposomes were examined . IgGs specific to the amino- and carboxyl-terminal regions of the SecY molecule strongly inhibited the translocation activity, indicating the participation of SecY in the translocation . Generation of a proton motive force due to the simultaneous reconstitution of F0F1-ATPase was also observed in the presence of ATP . An ATP-generating system consisting of creatine phosphate and creatine kinase significantly enhanced the formation of the proton motive force and the protein translocation activity of the proteoliposomes . Collapse of the proton motive force thus generated partially inhibited the translocation. Proc R Soc Lond B Biol Sci, 1990 Sep 22, 241(1302), 179 - 86 Directed mutagenesis of the redox-active disulphide bridge in glutathione reductase from Escherichia coli; Deonarain MP et al.; Directed mutagenesis of the gor gene from Escherichia coli encoding the flavoprotein glutathione reductase was used to convert the two cysteine residues that comprise its redox-active disulphide bridge to alanine (C42A) and serine (C47S) residues . A double mutant (C42AH439A) was also created in which His-439, the proton donor/acceptor in the glutathione-binding site, was additionally converted into an alanine residue . The C42A and C47S mutants were both unable to catalyse the reduction of glutathione by NADPH . The C42A mutant retained the transhydrogenase activity of the wild-type enzyme, whereas the C47S mutant was also inhibited in this reaction . These results support the view that in the catalytic mechanism of E . coli glutathione reductase, the thiolate form of Cys-42 acts as a nucleophile to initiate disulphide exchange with enzyme-bound glutathione and that the thiolate form of Cys-47 generates an essential charge-transfer complex with enzyme-bound FAD . Titration of the C42A and C42AH439A mutants indicated that the imidazole side-chain of His-439 lowered the pKa of the charge-transfer thiol (Cys-47) from 7.7 to 5.7, enhancing its ability to act as an anion at neutral pH . Several important differences between these mutants of E . coli glutathione reductase and similar mutants (or chemically modified forms) of other members of the flavoprotein disulphide oxidoreductase family were noted, but these could be explained in terms of the different redox chemistries of the enzymes concerned. Science, 1990 Sep 21, 249(4975), 1398 - 405 Structure of ribonuclease H phased at 2 A resolution by MAD analysis of the selenomethionyl protein; Yang W et al.; Ribonuclease H digests the RNA strand of duplex RNA.DNA hybrids into oligonucleotides . This activity is indispensable for retroviral infection and is involved in bacterial replication . The ribonuclease H from Escherichia coli is homologous with the retroviral proteins . The crystal structure of the E . coli enzyme reveals a distinctive alpha-beta tertiary fold . Analysis of the molecular model implicates a carboxyl triad in the catalytic mechanism and suggests a likely mode for the binding of RNA.DNA substrates . The structure was determined by the method of multiwavelength anomalous diffraction (MAD) with the use of synchrotron data from a crystal of the recombinant selenomethionyl protein. Cell, 1990 Sep 21, 62(6), 1135 - 41 Fine-tuning the topology of a polytopic membrane protein: role of positively and negatively charged amino acids; Nilsson I et al.; The effects of positively and negatively charged residues on the membrane topology of a model E . coli protein with two transmembrane segments have been studied . We show that addition or removal of as little as a single positively charged lysine residue in one of two critical regions can be sufficient to reverse the transmembrane topology of the molecule from Nout-Cout to Nin-Cin . Negatively charged residues are much less potent and significantly affect the topology only if present in high numbers . Finally, we provide data to suggest that sec-independent and sec-dependent translocation mechanisms differ in their sensitivity to positively charged amino acids. J Mol Biol, 1990 Sep 20, 215(2), 267 - 76 Changes in conserved region 2 of Escherichia coli sigma 70 affecting promoter recognition; Waldburger C et al.; We describe a mutation in rpoD, the gene encoding the sigma 70 subunit of RNA polymerase, which alters the promoter specificity of the holoenzyme in vivo . The mutant sigma causes a substantial and specific increase in the activity of mutant ant and lac promoters with a T.A to C.G substitution at position -12, the first position of the -10 hexamer . The rpoD mutation is a single base-pair substitution causing a Gln----His change at position 437, which is in a domain of conserved region 2.4 that is predicted to form an alpha-helix . Gln437 would lie one turn of the alpha-helix away from Thr440, which was previously implicated in recognition of position -12 . The rpoD-QH437 mutation described here lends further support to the model that region 2.4 of sigma is involved in recognition of the 5' end of the -10 hexamer . In addition, two rpoD mutations with non-specific effects on promoter recognition are described. J Mol Biol, 1990 Sep 20, 215(2), 257 - 65 Escherichia coli minichromosomes: random segregation and absence of copy number control; Jensen MR et al.; Minichromosomes, i.e . plasmids that can replicate from an integrated oriC, have been puzzling because of their high copy numbers compared to that of the chromosomal oriC, their lack of incompatibility with the chromosome and their high loss frequencies . Using single cell resistance to tetracycline or ampicillin as an indicator of copy number we followed the development of minichromosome distributions in Escherichia coli cells transformed with minichromosomes and then allowed to grow towards the steady state . The final copy number distribution was not reached within 15 to 20 generations . If the minichromosome carried the sop (partitioning) genes from plasmid F, the development of the copy number distribution was further drastically delayed . We conclude that E . coli cells have no function that directly controls minichromosomal copy numbers, hence the absence of incompatibility in the sense of shared copy number control . We suggest that minichromosomes are subject to the same replication control as the chromosome but segregate randomly in the absence of integrated partitioning genes . This, combined with evidence that the lowest copy number classes are normally present despite high average copy numbers, can account for the high loss frequencies. J Mol Biol, 1990 Sep 20, 215(2), 215 - 6 Crystallization and preliminary X-ray study of a recombinant cutinase from Fusarium solani pisi; Abergel C et al.; Recombinant cutinase from Fusarium solani pisi is expressed and excreted with very high yields in Escherichia coli cultures . Cutinase was crystallized at 20 degrees C using the vapour diffusion technique, with polyethylene glycol 6000 as precipitant . Best crystals were obtained at pH 7.0 with polyethylene glycol 6000 as precipitant . Best crystals were obtained at pH 7.0 with polyethylene glycol at 15 to 20% . They are monoclinic, with space group P2(1) and cell dimensions a = 35.1 A, b = 67.4 A, c = 37.05 A and beta = 94.0 degrees; they diffract beyond 1.5 A resolution . The asymmetric unit contains one molecule of 22,000 Da (Vm = 1.98 A3/Da; 38% water). Nature, 1990 Sep 20, 347(6290), 249 - 55 A second class of synthetase structure revealed by X-ray analysis of Escherichia coli seryl-tRNA synthetase at 2.5 A; Cusack S et al.; The three-dimensional crystal structure of seryl-transfer RNA synthetase from Escherichia coli, refined at 2.5 A resolution, is described . It has an N-terminal domain that forms an antiparallel alpha helical coiled-coil, stretching 60 A out into the solvent and stabilized by interhelical hydrophobic interactions and an active-site alpha-beta domain based around a seven-stranded antiparallel beta sheet . Unlike the three other known synthetase structures, the enzyme contains no classical nucleotide-binding fold, and is the first representative of a second class of aminoacyl-tRNA synthetase structures. Carbohydr Res, 1990 Sep 19, 205, 93 - 103 Photolabile, spacer-modified oligosaccharides for probing malto-oligosaccharide binding sites in proteins; Lehmann J et al.; O-Deacylation and S-deacylation of the diastereomers of 2-azido-4-S-benzoyl-4-mercaptobutyl 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranoside (9) with methanolic sodium methoxide and coupling of the resulting thiol to methyl 3,4-anhydro-6-deoxy-beta-L-arabino-hex-5-enopyranoside (2) gave the corresponding diastereomers of the spacer-modified disaccharide methyl 4-S-(3-azido-4-alpha-D-glucopyranosyloxybutyl)-6-deoxy-4-thio-alph a-D-xylo-hex-5-enopyranoside (10) . Glucosylation of the diastereomers of 10 with alpha-cyclo-dextrin-CGTase and treatment of the products with beta-amylase gave the diastereomers of the spacer-modified oligosaccharides methyl 4-S-(3-azido-4-alpha-maltosyloxybutyl)-6-deoxy-4-thio-alpha-D-xylo -hex-5-enopyranosides (11) and 4-S-(3-azido-4-alpha-maltotriosyloxybutyl)-6-deoxy-4-thio-alpha-D- hex-5-enopyranosides (12) . The diastereomers of 10 each had a good affinity for pancreatic alpha-amylase and the maltose-binding protein from E . coli . The affinities of the diastereomers of 11 and 12 were higher by at least one order of magnitude. Carbohydr Res, 1990 Sep 19, 205, 361 - 70 The structure of the capsular antigen from Escherichia coli O8:K87:H19; Parolis H et al.; The structure of the capsular polysaccharide from Escherichia coli O8:K87:H19 was investigated by methylation analysis and by one- and two-dimensional 1H- and 13C-n.m.r . spectroscopy . The repeating unit was shown to be a branched pentasaccharide with the structure (formula; see text) Carbohydr Res, 1990 Sep 19, 205, 347 - 59 Structure of the amino acid-containing capsular polysaccharide from Escherichia coli O8:K49:H21; Beynon LM et al.; The structure of the capsular antigen of E . coli K49 and the oligosaccharides derived from it by partial acid hydrolysis were studied by 1D- and 2D-n.m.r . spectroscopy, g.l.c.-c.i.-mass spectrometry, and methylation analysis . The K49 polysaccharide consists of the repeating unit----4)-beta-D-GlcpA-(1----6)-beta-D-Galp-(1----6)-beta-D-Glcp- (1----3)-beta - D-GalpNAc-(1---- . The glucuronic acid residues are substituted, in the apparent molar ratio of 4:1, with L-threonine and L-serine linked amidically to the carboxyl group. Biochemistry, 1990 Sep 18, 29(37), 8627 - 31 Role of the zinc(II) ions in the structure of the three-finger DNA binding domain of the Sp1 transcription factor; Kuwahara J et al.; The transcription factor Sp1 from Hela cells contains near the C-terminus of this protein of 778 amino acids three contiguous repeats of an amino acid sequence, -Cys-X4-Cys-X12-His-X3-His-, typical of the Cys2His2-type zinc-finger DNA binding domain first found in transcription factor TFIIIA . A DNA sequence corresponding to the condons from residue 614 to residue 778 of Spl (encompassing the three zinc-finger motifs) has been cloned and overproduced in Escherichia coli . The fragment of Sp1 containing the C-terminal 165 residues plus 2 from the cloning vector, designated Sp1(167*), can be extracted with 5 M urea and then refolded in the presence of Zn(II) to a protein of specific conformation containing 3.0 +/- 0.2 mol of tightly bound Zn(II)/mol of protein . Gel retardation assays using a labeled 14-bp DNA sequence containing a consensus Sp1 binding site show that the refolded Zn(II) protein specifically recognizes the "GC box" sequence in the presence of a large excess of calf thymus DNA . Treatment of Zn(II)Sp1(167*) with 10 mM EDTA results in removal of Zn(II) and the formation of an apoprotein which does not specifically recognize DNA . Cd(II) can be exchanged for Zn(II) in the refolded protein with full retention of specific DNA recognition . This is the first Cys2His2-type "finger" protein where this substitution has been accomplished . Titration of the Zn(II) protein with 6 mol of p-mercuribenzenesulfonate/mol of protein results in the complete release of the three Zn(II) ions . Release of Zn(II) is highly cooperative.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1990 Sep 18, 29(37), 8608 - 14 Mechanism of binding of substrate analogues to tryptophan indole-lyase: studies using rapid-scanning and single-wavelength stopped-flow spectrophotometry; Phillips RS et al.; We have examined the binding of oxindolyl-L-alanine, (3R)-2,3-dihydro-L-tryptophan, L-homophenylalanine, and N1-methyl-L-tryptophan to tryptophan indole-lyase (tryptophanase) from Escherichia coli by using rapid-scanning and single-wavelength stopped-flow kinetic techniques . Rate constants for the reactions were determined by fitting the concentration dependencies of relaxations to either linear (pseudo-first-order) or hyperbolic (rapid second-order followed by slow first-order) equations . The reaction with oxindolyl-L-alanine forms a quinonoid intermediate that exhibits a strong peak at 506 nm . This species is formed more rapidly than with the other analogues (84.5 s-1) and is reprotonated very slowly (0.2 s-1) . Reaction with L-homophenylalanine also forms a quinonoid intermediate with a strong peak at 508 nm, but the rate constant for its formation is slower (6.9 s-1) . The reaction with L-homophenylalanine exhibits a transient intermediate absorbing at about 340 nm that decays at the same rate as the quinonoid peak forms and that may be a gem-diamine . Tryptophan indole-lyase reacts with (3R)-2,3-dihydro-L-tryptophan much more slowly to form a moderately intense quinonoid peak at 510 nm, and a transient intermediate absorbing at about 350 nm is also formed . The species formed in the reaction of N1-methyl-L-tryptophan exhibits a peak at 425 nm and a very weak quinonoid absorption peak at 506 nm, which is formed at less than 4 s-1.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1990 Sep 18, 29(37), 8569 - 76 Dihydrofolate reductase from Escherichia coli: probing the role of aspartate-27 and phenylalanine-137 in enzyme conformation and the binding of NADPH; Dunn SM et al.; In the absence of ligands, dihydrofolate reductase from Escherichia coli exists in at least two interconvertible conformations, only one of which binds NADPH with high affinity . This equilibrium is pH dependent, involving an ionizable group of the enzyme (pK approximately 5.5), and the proportion of the NADPH-binding conformer increases from 42% at pH 5 to 65% at pH 8 . The role of specific amino acids in enzyme conformation has been investigated by studying the kinetics of NADPH binding to three dihydrofolate reductase mutants: (i) a mutant in which Asp-27, a residue that is directly involved in the binding of folates and antifolates but not NADPH, has been replaced by a serine, (ii) a mutant in which Phe-137 on the exterior of the molecule and distant from the binding sites has been replaced by a serine, and (iii) a mutant in which both Asp-27 and Phe-137 have been replaced by serines . Mutation of the Asp-27 residue reduces the affinity for NADPH by approximately 7-fold . Kinetic measurements have suggested that this is due mainly to an increase in the rate of dissociation of the initial complex and a slight shift in the enzyme equilibrium to favor the nonbinding conformation . The pH dependence of the conformer equilibrium is also shifted by approximately one pH unit to higher pH (pK approximately 6.5) . In addition, the pH profile suggests the involvement of a second ionizable group having a pK of about 8 since, above pH 7, the proportion of the NADPH-binding form decreases.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1990 Sep 18, 29(37), 8561 - 9 A second-site mutation at phenylalanine-137 that increases catalytic efficiency in the mutant aspartate-27----serine Escherichia coli dihydrofolate reductase; Howell EE et al.; The adaptability of Escherichia coli dihydrofolate reductase (DHFR) is being explored by identifying second-site mutations that can partially suppress the deleterious effect associated with removal of the active-site proton donor aspartic acid-27 . The Asp27----serine mutant DHFR (D27S) was previously characterized and the catalytic activity found to be greatly decreased at pH 7.0 {Howell et al . (1986) Science 231, 1123-1128} . Using resistance to trimethoprim (a DHFR inhibitor) in a genetic selection procedure, we have isolated a double-mutant DHFR gene containing Asp27----Ser and Phe137----Ser mutations (D27S+F137S) . The presence of the F137S mutation increases kcat approximately 3-fold and decreases Km(DHF) approximately 2-fold over D27S DHFR values . The overall effect on kcat/Km(DHF) is a 7-fold increase . The D27S+F137S double-mutant DHFR is still 500-fold less active than wild-type DHFR at pH 7 . Surprisingly, Phe137 is approximately 15 A from residue 27 in the active site and is part of a beta-bulge . We propose the F137S mutation likely causes its catalytic effect by slightly altering the conformation of D27S DHFR . This supposition is supported by the observation that the F137S mutation does not have the same kinetic effect when introduced into the wild-type and D27S DHFRs, by the altered distribution of two conformers of free enzyme {see Dunn et al . (1990)} and by a preliminary difference Fourier map comparing the D27S and D27S+F137S DHFR crystal structures. Biochim Biophys Acta, 1990 Sep 18, 1046(2), 111 - 9 Acyl carrier protein interacts with melittin; Ernst-Fonberg ML et al.; Acyl carrier protein (ACP) from Escherichia coli has been shown to form complexes with melittin, a cationic peptide from bee venom . ACP is a small (Mr 8847), acidic, Ca2(+)-binding protein, which possesses some characteristics resembling those of regulatory Ca2(+)-binding proteins including interaction with melittin . Complexing between melittin and ACP which occurred both in the presence and absence of Ca2+ was evident by chemical cross-linking the two peptides, fluorescence changes (including anisotropy measurements), and inhibition by melittin of the activity of a nonaggregated fatty acid synthetase from Euglena . Also, anti-Apis mellifera antibodies which contained antibodies against melittin specifically inhibited the same enzyme system activity relative to non-immune IgG. Biochemistry, 1990 Sep 18, 29(37), 8797 - 804 Comparative proton NMR analysis of wild-type cytochrome c peroxidase from yeast, the recombinant enzyme from Escherichia coli, and an Asp-235----Asn-235 mutant; Satterlee JD et al.; Proton NMR spectra of cytochrome c peroxidase (CcP) isolated from yeast (wild type) and two Escherichia coli expressed proteins, the parent expressed protein {CcP(MI)} and the site-directed mutant CcP(MI,D235N) (Asp-235----Asn-235), have been examined . At neutral pH and in the presence of only potassium phosphate buffer and potassium nitrate, wild-type Ccp and CcP(MI) demonstrate nearly identical spectra corresponding to normal (i.e., "unaged") high-spin ferric peroxidase . In contrast, the mutant protein displays a spectrum characteristic of a low-spin form, probably a result of hydroxide ligation . Asp-235 is hydrogen-bonded to the proximal heme ligand, His-175 . Changing Asp-235 to Asn results in alteration of the pK for formation of the basic form of CcP . Thus, changes in proximal side structure mediate the chemistry of the distal ligand binding site . All three proteins bind F-, N3-, and CN- ions, although the affinity of the mutant protein (D235N) for fluoride ion appears to be much higher than that of the other two proteins . Analysis of proton NMR spectra of the cyanide ligated forms leads to the conclusion that the mutant protein (D235N) possesses a more neutral proximal histidine imidazole ring than does either wild-type CcP or CcP(MI) . It confirms that an important feature of the cytochrome c peroxidase structure is at least partial, and probably full, imidazolate character for the proximal histidine (His-175). Biochemistry, 1990 Sep 18, 29(37), 8793 - 7 Secondary structure in formylmethionine tRNA influences the site-directed cleavage of ribonuclease H using chimeric 2'-O-methyl oligodeoxyribonucleotides; Hayase Y et al.; In order to cleave RNA at specific positions in Escherichia coli formylmethionine tRNA, RNase H and complementary chimeric oligonucleotides consisting of DNA and 2'-O-methyl-RNA (Inoue et al . (1987) FEBS Lett . 215, 327} were used . Specific cleavages in the D loop, anticodon loop, T psi C loop, anticodon stem, and acceptor stem were investigated . Virtually unique hydrolyses with RNase H were observed at the T psi C loop, anticodon stem, and acceptor stem when relatively longer chimeric oligonucleotides (20-mer) were used . An efficient cleavage at the anticodon was obtained with a chimeric 13-mer when the higher structure of the tRNA was broken by hybridization with a 20-mer at the acceptor as well as the T psi C stem region . It was found that stabilities of hybrids with chimeric oligonucleotides and the presence of minor nucleosides affect the cleavage of tRNA by this approach. Biochemistry, 1990 Sep 18, 29(37), 8620 - 7 Nucleotide regulation of Escherichia coli glycerol kinase: initial-velocity and substrate binding studies; Pettigrew DW et al.; Substrate binding to Escherichia coli glycerol kinase (EC 2.7.1.30; ATP-glycerol 3-phosphotransferase) was investigated by using both kinetics and binding methods . Initial-velocity studies in both reaction directions show a sequential kinetic mechanism with apparent substrate activation by ATP and substrate inhibition by ADP . In addition, the Michaelis constants differ greatly from the substrate dissociation constants . Results of product inhibition studies and dead-end inhibition studies using 5'-adenylyl imidodiphosphate show the enzyme has a random kinetic mechanism, which is consistent with the observed formation of binary complexes with all the substrates and the glycerol-independent MgATPase activity of the enzyme . Dissociation constants for substrate binding determined by using ligand protection from inactivation by N-ethylmaleimide agree with those estimated from the initial-velocity studies . Determinations of substrate binding stoichiometry by equilibrium dialysis show half-of-the-sites binding for ATP, ADP, and glycerol . Thus, the regulation by nucleotides does not appear to reflect binding at a separate regulatory site . The random kinetic mechanism obviates the need to postulate such a site to explain the formation of binary complexes with the nucleotides . The observed stoichiometry is consistent with a model for the nucleotide regulatory behavior in which the dimer is the enzyme form present in the assay and its subunits display different substrate binding affinities . Several properties of the enzyme are consistent with negative cooperativity as the basis for the difference in affinities . The possible physiological importance of the regulatory behavior with respect to ATP is considered. Biochemistry, 1990 Sep 18, 29(37), 8643 - 51 Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine; Wilson BA et al.; Glutamic acid-148, an active-site residue of diphtheria toxin identified by photoaffinity labeling with NAD, was replaced with aspartic acid, glutamine, or serine by directed mutagenesis of the F2 fragment of the toxin gene . Wild-type and mutant F2 proteins were synthesized in Escherichia coli, and the corresponding enzymic fragment A moieties (DTA) were derived, purified, and characterized . The Glu----Asp (E148D), Glu----Gln (E148Q), and Glu----Ser (E148S) mutations caused reductions in NAD:EF-2 ADP-ribosyltransferase activity of ca . 100-, 250-, and 300-fold, respectively, while causing only minimal changes in substrate affinity . The effects of the mutations on NAD-glycohydrolase activity were considerably different; only a 10-fold reduction in activity was observed for E148S, and the E148D and E148Q mutants actually exhibited a small but reproducible increase in NAD-glycohydrolytic activity . Photolabeling by nicotinamide-radiolabeled NAD was diminished ca . 8-fold in the E148D mutant and was undetectable in the other mutants . The results confirm that Glu-148 plays a crucial role in the ADP-ribosylation of EF-2 and imply an important function for the side-chain carboxyl group in catalysis . The carboxyl group is also important for photochemical labeling by NAD but not for NAD-glycohydrolase activity . The pH dependence of the catalytic parameters for the ADP-ribosyltransferase reaction revealed a group in DTA-wt that titrates with an apparent pKa of 6.2-6.3 and is in the protonated state in the rate-determining step.(ABSTRACT TRUNCATED AT 250 WORDS) Neurosci Lett, 1990 Sep 18, 117(3), 259 - 63 Plasmid DNAs directly injected into mouse brain with lipofectin can be incorporated and expressed by brain cells; Ono T et al.; In this study, we demonstrated that lipofectin-treated DNAs which were injected into mouse brain could be incorporated and expressed by brain cells . When L7RH-beta gal plasmid DNA harboring E . coli beta-galactosidase gene fused with the nuclear location signal of SV40 T-antigen gene was injected into brains of 1-week-old mice, cells whose nuclei appeared to be densely stained with the chromogenic substrate X-gal were detected in several portions of the brain till 9 days after injection . Injection of pMLV-CAT plasmid DNA which contains the E . coli chloramphenicol acetyltransferase (CAT) gene also resulted in cells immunoreactive to the anti-CAT antibody. FEBS Lett, 1990 Sep 17, 270(1-2), 53 - 6 The functional properties of full length and mutant chicken gizzard smooth muscle caldesmon expressed in Escherichia coli; Redwood CS et al.; Wild type chicken gizzard caldesmon (756 amino acids) was expressed in a T7 RNA polymerase-based bacterial expression system at a yield of 1 mg pure caldesmon per litre bacterial culture . A mutant composed of amino acids 1-578 was also constructed and expressed . The wild type and mutant caldesmon were purified and compared with native chicken gizzard caldesmon . Native and wild type expressed caldesmon were indistinguishable in assays for inhibition of actin-tropomyosin activation of myosin ATPase, reversal of inhibition by Ca2(+)-calmodulin and binding to actin, actin-tropomyosin, Ca2(+)-calmodulin, tropomyosin and myosin . The mutant missing the C-terminal 178 amino acids had no inhibitory effect and did not bind to actin or Ca2(+)-calmodulin . It bound to tropomyosin with a 5-fold reduced affinity and to myosin with a greater than 10-fold reduced affinity. FEBS Lett, 1990 Sep 17, 270(1-2), 76 - 80 Purification and characterization of the RNase H domain of HIV-1 reverse transcriptase expressed in recombinant Escherichia coli; Becerra SP et al.; The ribonuclease H (RNase H) domain of human immuno-deficiency virus (HIV-1) reverse transcriptase has been produced with the aim of providing sufficient amounts of protein for biophysical studies . A plasmid vector is described which directs high level expression of the RNase H domain under the control of the lambda PL promoter . The domain corresponds to residues 427-560 of the 66 kDa reverse transcriptase . The protein was expressed in Escherichia coli and was purified using ion-exchange and size exclusion chromatography . The purified protein appears to be in a native-like homogeneous conformational state as determined by 1H-NMR spectroscopy and circular dichroism measurements . HIV-protease treatment of the RNase H domain resulted in cleavage between Phe-440 and Tyr-441. Biochem J, 1990 Sep 15, 270(3), 697 - 703 Expression of human liver arginase in Escherichia coli . Purification and properties of the product; Ikemoto M et al.; Arginase is an enzyme that catalyses the hydrolysis of arginine to urea and ornithine . It is abundantly present in the liver of ureotelic animals (i.e . those whose excretion is characterized by the excretion of uric acid as the chief end-product of nitrogen metabolism), but its purification has hitherto not been simple, and the yield not high . Starting with a partially truncated cDNA for human liver arginase recently made available, we constructed an expression plasmid that had tandemly linked tac promotors placed upstream of a full-length cDNA . By selecting Escherichia coli strain KY1436 as the host micro-organism, we established an efficient system for the production of human liver arginase protein . Chromatographies on CM-Sephadex G-150, DEAE-cellulose and Sephadex G-150, followed by preparative agar-gel electrophoresis, yielded 10 mg of apparently homogeneous enzyme protein from 1 g (wet wt.) of E . coli cells . E . coli-expressed human liver arginase had chemical, immunological and most catalytic properties indistinguishable from those of purified human erythrocyte arginase . However, E . coli-expressed arginase was a monomer of Mr 35,000, whereas the purified erythrocyte arginase was trimer of Mr 105,000 . They differed also in pH- and temperature-stabilities . Gel-filtration experiments with these two purified arginases under various conditions, as well as with unfractionated human liver and erythrocyte cytosol preparations, indicated that the native form of human arginase should be of Mr 35,000, and that the trimeric appearance of human erythrocyte arginase after purification was an artifact of the purification procedures . It was thus concluded that, in Nature, the liver and erythrocyte arginases are identical proteins. Experientia, 1990 Sep 15, 46(9), 929 - 40 Migratory patterns of clonally related cells in the developing central nervous system; Gray GE et al.; Neurons and glioblasts that arise in the ventricular zone migrate to form discrete nuclei and laminae as the central nervous system develops . By stably labeling precursor cells in the ventricular zone, pathways taken by different cells within an individual clone can be described . We have used recombinant retroviruses to label precursor cells with a heritable marker, the E . coli lacZ gene; clones of lacZ-positive cells are later mapped histochemically . Here we review results from three regions of the chicken central nervous system--the optic tectum, spinal cord, and forebrain--and compare them with previous results from mammalian cortex and other regions of the vertebrate CNS . In particular, we consider the relationship between migratory patterns and functional organization, the existence of multiple cellular sources of migratory guidance, and the issue of whether a cell's choice of migratory pathway influences its ultimate phenotype. J Biol Chem, 1990 Sep 15, 265(26), 15813 - 7 Cloning of ubiquitin activating enzyme from wheat and expression of a functional protein in Escherichia coli; Hatfield PM et al.; The initial step in the conjugation of ubiquitin to substrate proteins involves the activation of ubiquitin by ubiquitin activating enzyme, E1 . Previously, we purified and characterized multiple species of E1 from wheat germ . We now describe the isolation and characterization of a cDNA clone encoding E1 from wheat . This clone (UBA1) was isolated from a cDNA expression library with anti-wheat E1 antibodies . It contained an open reading frame coding for 1051 amino acids and directed the synthesis of a protein that comigrated with a wheat germ E1 of 117 kDa . UBA1 was confirmed as encoding E1 by (i) comparison of the peptide map of the protein product of UBA1 synthesized in Escherichia coli with that of purified E1 from wheat, and (ii) amino acid sequence identity of peptides generated from purified E1 with regions of the derived amino acid sequence of UBA1 . The isolation of two additional cDNAs closely related to UBA1 indicated that E1 was encoded by a small gene family in wheat . Nonetheless, a single poly(A+) mRNA size class of 4 kilobases hybridized with UBA1 . When expressed in E . coli, the product of UBA1 catalyzed the formation of a thiol ester linkage between ubiquitin and an ubiquitin carrier protein . The ability of E . coli containing UBA1 to synthesize an active protein will allow us to identify domains important for E1 function using in vitro mutagenesis. J Biol Chem, 1990 Sep 15, 265(26), 15410 - 7 Biosynthesis of lipid A in Escherichia coli . Acyl carrier protein-dependent incorporation of laurate and myristate; Brozek KA et al.; In previous studies we described enzyme(s) from Escherichia coli that transfer two 3-deoxy-D-manno-octulosonate (KDO) residues from two CMP-KDO molecules to a tetraacyldisaccharide-1,4'-bis-phosphate precursor of lipid A, termed lipid IVA (Brozek, K . A., Hosaka, K., Robertson, A . D., and Raetz, C . R . H . (1989) J . Biol . Chem . 264, 6956-6966) . The product, designated (KDO)2-IVA, can be prepared in milligram quantities and/or radiolabeled with 32P at position 4' of the IVA moiety . We now demonstrate the presence of enzymes in E . coli extracts that transfer laurate and/or myristate residues from lauroyl or myristoyl-acyl carrier protein (ACP) to (KDO)2-IVA . Thioesters of coenzyme A are not substrates . The cytosolic fraction catalyzes rapid acylation with lauroyl-ACP, but not with myristoyl, R-3-hydroxymyristoyl, palmitoyl, or palmitoleoyl-ACP . The membrane fraction transfers both laurate and myristate to (KDO)2-IVA . Evidence for the enzymatic acylation of (KDO)2-IVA is provided by (a) conversion of {4'-32P}(KDO)2-IVA to more rapidly migrating products in the presence of the appropriate acyl-ACP, (b) incorporation of {1-14C}laurate or {1-14C}myristate into these metabolites in the presence of (KDO)2-IVA, (c) fast atom bombardment-mass spectrometry, and (d) 1H NMR spectroscopy . At protein concentrations less than 0.5 mg/ml, the acylation of (KDO)2-IVA by the cytoplasmic fraction is absolutely dependent upon the addition of exogenous acyl-ACP . These acyltransferases cannot utilize lipid IVA as a substrate, demonstrating that they possess novel KDO recognition domains . The unusual substrate specificity of these enzymes provides compelling evidence for their involvement in lipid A biosynthesis . Depending on the conditions it is possible to acylate (KDO)2-IVA with 1 or 2 lauroyl residues, with 1 or 2 myristoyl residues, or with 1 of each. J Biol Chem, 1990 Sep 15, 265(26), 15920 - 31 Species-specific substrate interaction of picornavirus 3C proteinase suballelic exchange mutants; Lawson MA et al.; The substrate recognition properties of the polio-virus type 1 and coxsackievirus B3 3C proteinases have been examined in vitro by allelic and suballelic exchange of 3C between the cloned virus genomes . The activity of the altered 3C proteinases was examined by translation of synthetic RNA in a rabbit reticulocyte lysate/HeLa cell extract translation system . Analysis of the subsequent processing of virus polyproteins by the altered 3C proteinases showed that all of the mutant proteinases maintained some catalytic activity . The disruption of polyprotein cleavages mediated by 3C followed a distinct pattern, suggesting a specific order of events in processing the polyprotein . Differences in cleavage activity of mutant proteinases when tested on coxsackievirus or poliovirus protein substrates suggest that, although structural elements throughout the proteinase play a role in efficient substrate utilization, the carboxyl-terminal region of the 3C proteinase contains elements most important in species-specific substrate recognition. J Biol Chem, 1990 Sep 15, 265(26), 15854 - 9 Expression of human parathyroid hormone-(1-84) in Escherichia coli as a factor X-cleavable fusion protein; Gardella TJ et al.; Recombinant human parathyroid hormone (hPTH)-(1-84) was obtained from Escherichia coli using a cleavable fusion protein strategy . The fusion protein contains residues 1-138 of human growth hormone as the amino-terminal region and residues 1-84 of hPTH as the carboxyl-terminal region . A 7-residue linker containing the recognition/cleavage sequence of the site-specific blood coagulation protease activated factor X (factor Xa) joins the two regions . Intact hPTH-(1-84) is released from this fusion protein by cleavage in vitro with factor Xa . The fusion protein was produced at a high level and formed inclusion bodies which allowed it to be easily purified by low speed centrifugation, with a yield of approximately 50 mg/liter of culture . After factor Xa cleavage and high performance liquid chromatography purification, highly purified hPTH was obtained, with a final yield of 1.5-3 mg/liter . Physical and biological characterization of the purified hormone demonstrated that it was intact and active hPTH-(1-84). J Biol Chem, 1990 Sep 15, 265(26), 15796 - 803 Formation and enzymatic properties of the UvrB.DNA complex; Orren DK et al.; The UvrA, UvrB, and UvrC proteins collectively catalyze the dual incision of a damaged DNA strand in an ATP-dependent reaction . We previously reported (Orren, D . K., and Sancar, A . (1989) Proc . Natl . Acad . Sci . U.S.A . 86, 5237-5241) that UvrA delivers UvrB to damaged sites in DNA; upon addition of UvrC to these UvrB.DNA complexes, the DNA is incised . In the present study, we have further characterized both the delivery of UvrB to DNA and the subsequent incision process, with emphasis on the role of ATP in these reactions . The UvrA-dependent delivery of UvrB onto damaged DNA is relatively slow (kon approximately 6 x 10(4) M-1 s-1) and requires ATP hydrolysis (Km = 120 microM) . Although ATP enhances the stability of UvrB.DNA complexes (koff = 8.5 x 10(-5) s-1), the isolated UvrB.DNA complexes do not contain any covalently attached or stably bound nucleotide . However, ATP binding is required for the UvrC-dependent dual incision of DNA bound by UvrB . Interestingly, adenosine 5'-(3-O-thio)triphosphate can substitute for ATP at this step . The Km for ATP during incision is 2 microM, but ATP is not hydrolyzed at a detectable level during the incision reaction . The incisions made by UvrB-UvrC are on both sides of the adduct and result in the excision of the damaged nucleotide. J Biol Chem, 1990 Sep 15, 265(26), 15638 - 43 Mutations in the aspartate receptor of Escherichia coli which affect aspartate binding; Mowbray SL et al.; The effects of five mutations at arginines 64, 69, and 73 of the Tar protein were analyzed using swarm assays, aspartate binding in purified membranes, and methylation both in vitro and in vivo . The defects in the responses of these mutant receptors to aspartate were shown to be directly attributable to reduced binding of aspartate to the receptor rather than to defects in their signaling characteristics . Mutations at residues 64, 69, and 73 reduced aspartate binding by factors of greater than 10(-4), 10(-3), and 10(-2), respectively . Once aspartate was bound, the mutants exhibited normal signaling properties . No cooperativity was observed in the coupling of aspartate binding to methylation, indicating that the monomers of the receptor dimer act independently . The in vitro methylation system was thus shown to be an effective way of measuring aspartate binding constants and examining the functional integrity of the proteins . The maltose responses of the receptor proteins were affected slightly, or not at all, in an in vivo methylation assay . Two models for the roles of these arginine residues in receptor function are discussed. J Biol Chem, 1990 Sep 15, 265(26), 15617 - 22 Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase . Use of site-directed mutagenesis to evaluate the roles of His-258 and His-392 in catalysis; Tauler A et al.; The current model for hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase divides the protein into two functional domains: an N-terminal kinase domain and a carboxyl-terminal bisphosphatase domain . Site-directed mutagenesis was used to evaluate the role of two putative bisphosphatase active site histidyl residues in catalysis . His-258 has been implicated as a phosphoacceptor (Pilkis, S . J., Lively, M . O., and El-Maghrabi, M . R . (1987) J . Biol . Chem . 262, 12672-12675), and the importance of this residue was confirmed when it was mutated to alanine and neither bisphosphatase activity nor a phosphoenzyme intermediate could be detected . Mutation of His-392 to alanine produced an enzyme which had five percent of wild-type fructose 2,6-bisphosphatase activity, and the rate of phosphoenzyme formations was decreased from 4800 nmol/min/mg to 2.9 nmol/min/mg . Mutation of His-392 to phenylalanine, lysine, or aspartic acid also produced proteins that did not hydrolyze fructose 2,6-bisphosphate or form a phosphoenzyme intermediate . These results are consistent with an important role for His-392 in the bisphosphatase reaction, probably as a proton donor, and with its designation as an active site residue based on homology modeling (Bazan (1989) Proc . Natl . Acad . Sci . U.S.A . 86, 9642-9646) . H258A had the same Vmax for 6-phosphofructo-2-kinase as the wild-type enzyme, and the mutant's kinase was inhibited by cAMP-dependent phosphorylation . In addition, H392F and H392K did not catalyze the kinase reaction, although H392D had normal kinase activity which was also modulated by cAMP-dependent phosphorylation in the same manner as the wild-type enzyme . Thus, an active bisphosphatase domain is not a necessary condition for phosphorylation-induced changes in 6-phosphofructo-2-kinase activity . The results also suggest that structural and/or active site interactions exist between the two domains of the enzyme. J Biol Chem, 1990 Sep 15, 265(26), 15525 - 30 Metal-tetracycline/H+ antiporter of Escherichia coli encoded by a transposon, Tn10 . The role of the conserved dipeptide, Ser65-Asp66, in tetracycline transport; Yamaguchi A et al.; The transposon Tn10-encoded tetracycline resistance protein functions as a metal-tetracycline/H+ antiporter (Yamaguchi, A., Udagawa, T., and Sawai, T . (1990) J . Biol . Chem . 265, 4809-4813) . The Ser65-Asp66 dipeptide is conserved in all known tetracycline antiporter proteins and is an important target for site-directed mutagenesis . When Asp66 was replaced by Asn, the transport activity was completely lost, whereas when it was replaced by Glu, the activity was reduced to 10% of the wild-type level, indicating that a negative charge at position 66 is essential for tetracycline transport . Replacement of Ser65 by Cys or Ala, in contrast, caused only a minor change in tetracycline transport activity . However, the Cys65 mutant antiporter was sensitive to sulfhydryl reagents . Complete inactivation of the Cys65 antiporter by N-ethylmaleimide was not prevented by the substrate . A less bulky reagent, methyl methanethiosulfonate, caused partial inactivation of the Cys65 antiporter without changing its affinity to the substrate . These results indicate that a region including the dipeptide plays an important role in metal-tetracycline transport except for substrate binding . It may act as a gate which opens on the charge-charge interaction between Asp66 and the metal-tetracycline. Int J Cancer, 1990 Sep 15, 46(3), 500 - 7 Analysis of cell-surface sugar receptor expression by neoglycoenzyme binding and adhesion to plastic-immobilized neoglycoproteins for related weakly and strongly metastatic cell lines of murine tumor model systems; Gabius S et al.; Recognition of the carbohydrate part of cellular glycoconjugates by cell-surface sugar receptors may contribute to interactions, essential to the establishment of metastases . Comparison of the properties of strongly metastatic variants to their related, less metastatic counterparts offers a generally accepted approach to the discovery of metastasis-associated characteristics . The chemically induced murine lymphoma line Eb and its spontaneously arising variant ESb with increased potential for lung and liver colonization, the virally induced lymphosarcoma cell line RAW117-P and its in vivo selected variant H10 with increased potential for liver colonization, and the B16-F1 melanoma line and its in vivo selected variant F10 with increased potential for lung colonization, were chosen . A panel of 12 types of chemically glycosylated E . coli beta-galactosidase, exposing the pivotal carbohydrate residues for specific carbohydrate-dependent cell binding, was employed to study the expression of respective cell-surface sugar receptors on these cell lines . Specific binding occurred in a non-uniform manner for the individual probes . Systematic measurements at a non-saturating ligand concentration revealed quantitative differences between the 2 cell lines of each system . However, there were no consistent changes associated with the metastatic phenotype . A similar result was obtained employing Scatchard analyses for quantitative evaluation of binding characteristics in several cases . Surface receptor expression was responsive to chemical induction of differentiation in the lymphosarcoma model . Analyses of sugar-inhibitable cell adhesion to neoglycoprotein-coated plastic wells for the lymphoma and lymphosarcoma cells revealed that the presence of cell-surface sugar receptors, even at similar densities to those defined by neoglycoenzyme binding, will not necessarily translate into an identical adhesive response . Several carbohydrates, especially N-acetyl-D-galactosamine, can differentially affect this interaction at a non-toxic concentration in both model systems. Biochem Pharmacol, 1990 Sep 15, 40(6), 1405 - 10 DNA damaging effects and voltammetric studies on the hypoxic cell toxin 3-amino-1,2,4-benzotriazine-1,4-dioxide, SR4233, as a function of pH; Tocher JH et al.; The compound 3-amino-1,2,4-benzotriazine-1,4-dioxide, SR4233, has recently attracted considerable attention as a possible hypoxic cell radiation sensitizer and cytotoxic agent . The present study examines the influence of pH on the DNA damaging ability of SR4233 upon electrolytic reductive activation, and the corresponding changes in electrochemistry . A phi X174 double transfection assay has been employed to assess the DNA damaging ability of SR4233 between pH 4 to 7 . Upon electrolytic reduction the drug was found to be more effective in damaging DNA at acidic pH than at neutral conditions . This indicated that the damaging species was probably protonated . The DNA damaging ability of SR4233, as measured by a viral transfection assay, was linearly related to pH between the values of 4 and 7, and this feature has implications for its potential efficacy in the treatment of hypoxic tumors . The electrochemistry of SR4233 has been examined as a function of pH between the ranges 2 and 10.5 . Three investigation techniques have been employed, cyclic voltammetry and differential pulse and dc polarographies . A general shift towards less negative potentials with increasing acidity was found between pH 2 and 8.5 giving a linear relationship . The behaviour was found to be relatively invariant at alkaline pH. Blood, 1990 Sep 15, 76(6), 1201 - 8 Retroviral mediated transfer and expression of exogenous genes in primary lymphoid cells: assaying for a viral transactivator activity in normal and malignant cells; Strair RK et al.; In this report we describe the use of recombinant retroviruses to characterize the activity of an exogenous promoter in primary cells obtained from patients with lymphoproliferative disorders . The infection of a variety of cultured and primary lymphoid cells with a recombinant retrovirus containing a histone promoter-driven beta-galactosidase gene is shown to result in the expression of beta-galactosidase in 50% to 100% of the cells . A similar infection with a recombinant retrovirus containing the beta-galactosidase gene with an adenovirus E2 promoter, results in beta-galactosidase activity in a limited number of cultured and primary cells . Since the adenovirus E2 promoter has been well characterized and is known to be regulated by transactivators encoded by many viruses, the activity of this promoter in specific cell types is discussed in reference to both the biology of the cell and the possible presence of as yet undetected viral gene products. J Biol Chem, 1990 Sep 15, 265(26), 15560 - 3 Endotoxin induction of plasminogen activator and plasminogen activator inhibitor type 1 mRNA in rat tissues in vivo; Quax PH et al.; The tissue-specific distribution of tissue-type and urokinase-type plasminogen activator (t-PA and u-PA) and their inhibitor type 1 (PAI-1) was analyzed at mRNA level in five major rat organ tissues . t-PA mRNA was detected in lung, kidney, heart, and liver . u-PA mRNA was detected in kidney and lung . Presence of PA mRNA correlated with the detection of PA activity in extracts of these tissues . PAI-1 mRNA was detected predominantly in heart and lung . Although PAI activity could not be measured directly in tissue extracts, the presence of PAI-1 mRNA correlated with the occurrence of PA.PAI complex in fibrin autography of tissue extracts . Endotoxin injection caused a very large increase in plasma PAI activity . This increase correlated with a marked increase in PAI-1 mRNA in nearly all tissues studied . The increase in PAI-1 mRNA is most pronounced in lung and liver . Endotoxin injection also caused an increased level of t-PA mRNA in heart and kidney, and an increased u-PA mRNA level in kidney . mRNA analysis of freshly isolated and separated subfractionated liver cells showed that the marked increase in PAI-1 mRNA in the liver after endotoxin injection may be due mainly to a strong increase of PAI-1 mRNA in the liver endothelial cells. J Immunol, 1990 Sep 15, 145(6), 1968 - 73 The allorecognition of H-2Kb-specific CD4-CD8- T cell hybridomas is influenced by the substitution at residue 256 of MHC class I molecules; Tanabe M et al.; Our previous studies demonstrated that allorecognition of HTB176.10 and HTB177.2, H-2Kb-reactive CD4-CD8- T cell hybridomas is markedly influenced by the exchange of the alpha 3 domain between H-2Kb and H-2Dp . The recombinant genes of the exon 4 between H-2Kb and H-2Dp were constructed to determine the residues of the alpha 3 domain that influence the allorecognition of these T cell hybridomas . Seven recombinant genes of the exon 4 were generated by in vivo recombination in Escherichia coli . Chimeric genes containing these recombinants were transfected into L cells and the transfectants expressing equivalent amounts of chimeric molecules were selected by flow cytometry . Studies on responses of these T cell hybridomas to the chimeric molecules confirmed our previous observation that the primary structure of the alpha 3 domain influences the allorecognition by the hybridomas . Moreover, it was indicated that residue 256 on the alpha 3 domain markedly affects the allorecognition by the T cell hybridomas, although substitutions at residues 184, 193, 195, 197, 262, and 264 exerted some effects on the T cell recognition . Further studies with the use of a single amino acid mutant of H-2Kb at residue 256 confirmed the effect of substitution at residue 256 on allorecognition of the T cell hybridomas . Taken together, results of this study demonstrated that polymorphism of the alpha 3 domain is indeed involved in the formation of allodeterminants recognized by TCR. Gene, 1990 Sep 14, 93(2), 229 - 34 A novel expression vector for high-level synthesis and secretion of foreign proteins in Escherichia coli: overproduction of bovine pancreatic phospholipase A2; Deng TL et al.; A novel expression plasmid (pTO-N) has been constructed that allows for the production of large quantities of foreign proteins (or fragments thereof) in an unfused state . The vector has a strong and tightly regulated T7 gene 10 promoter and the ompA Shine-Dalgarno (SD) sequence, followed by the ompA sequence and a cloning linker region . The mRNAs produced by the vector are protected by secondary structures at both ends of the mRNAs . The OmpA signal peptide directed the synthesized proteins into the periplasmic space of Escherichia coli . Phospholipase A2 and prophospholipase A2 from bovine pancreas have been produced to a high level by using this expression vector . One additional feature, which is essential for the stable maintenance of the plasmid in the E . coli expression host, BL21 (DE3){pLysS}, is the shortened distance between the 5' secondary structure sequence (immediately following the gene 10 promoter) and the SD sequence . This vector could be particularly useful for synthesis of toxins in E . coli. Gene, 1990 Sep 14, 93(2), 183 - 8 Biologically active A-chain of the plant toxin ricin expressed from a synthetic gene in Escherichia coli; Shire D et al.; To assess the biological activity and pharmacokinetic properties of nonglycosylated ricin A-chain (RA), we have obtained the polypeptide following expression of a synthetic 842-bp RA gene in Escherichia coli . Expression of the gene was carried out using the phage T5 PN25 promoter fused to the E . coli lac operator . The RA polypeptide was synthesized in a completely soluble form and was purified in one step by immunoabsorption . It was shown to be as cytotoxic for a human cell line as both native RA and chemically deglycosylated native RA . Reconstituted whole ricin and an immunotoxin containing the recombinant RA were also biologically active . Immunotoxins made with recombinant and deglycosylated RA had similar clearance rates in vivo showing, after a short period of rapid elimination, stabilities far higher than that of an immunotoxin made with native RA . Our results show that the complete elimination of sugar side chains from the RA is not sufficient to entirely eradicate the rapid initial in vivo clearance of RA-based biologicals. Biochem Biophys Res Commun, 1990 Sep 14, 171(2), 684 - 9 Chemo-enzymatic synthesis of optically pure l-leucovorin, an augmentor of 5-fluorouracil cytotoxicity against cancer; Uwajima T et al.; Optically pure l-leucovorin was synthesized on a large scale by the combination of chemical and enzymatic processes . After reduction of folate with zinc, dihydrofolate was reduced asymmetrically to (6)-tetra-hydrofolate by use of dihydrofolate reductase from E . coli C600/pTP600, with simultaneous NADPH cofactor recycling using glucose dehydrogenase from Gluconobacter scleroideus KY3613 . Calcium l-leucovorin.4H2O (113 g) was obtained from (6S)-tetrahydrofolate via 5,10-methyenyltetrahydrofolate by formylation, reflux, addition of calcium ions and floricil column chromatography, with an overall yield of 50% based on folate . The l-leucovorin showed optical purity of 99.9% de as (6S)-form. Gene, 1990 Sep 14, 93(2), 205 - 12 Synthesis of Drosophila melanogaster alcohol dehydrogenase in yeast; Atrian S et al.; Expression systems for the heterologous expression of Drosophila melanogaster alcohol dehydrogenase (ADH) in Saccharomyces cerevisiae have been designed, analyzed and compared . Four different yeast/Escherichia coli shuttle vectors were constructed and used to transform four different yeast strains . Expression was detectable in ADH- yeast strains, from either a constitutive promoter, yeast ADH1 promoter (ADCp), or a regulated promoter, yeast GALp . The highest amount of D . melanogaster ADH was obtained from a multicopy plasmid with the D . melanogaster Adh gene expressed constitutively under the control of yeast ADCp promoter . The D . melanogaster enzyme was produced in cell extracts, as assessed by Coomassie blue staining and Western blotting after polyacrylamide-gel electrophoresis and it was fully active and able to complement the yeast ADH deficiency . Results show that D . melanogaster ADH subunits synthesized in yeast are able to assemble into functional dimeric forms . The synthesized D . melanogaster ADH represents up to 3.5% of the total extracted yeast protein. Gene, 1990 Sep 14, 93(2), 189 - 98 A modular set of lacZ fusion vectors for studying gene expression in Caenorhabditis elegans; Fire A et al.; We describe a series of plasmid vectors which contain modular features particularly useful for studying gene expression in eukaryotic systems . The vectors contain the Escherichia coli beta-galactosidase (beta Gal)-encoding region (the lacZ gene) flanked by unique polylinker segments on the 5' and 3' ends, and several combinations of a variety of modules: a selectable marker (an amber suppressor tRNA), a translational initiation region, a synthetic intron segment, the early polyadenylation signal from SV40, and 3' regions from two nematode genes . A segment encoding the nuclear localization peptide from the SV40 T antigen is incorporated into many of the constructs, leading to beta Gal accumulation in nuclei, which can facilitate identification of producing cells in complex tissues . To make functional beta Gal fusions to secreted proteins, we constructed plasmids with an alternate module encoding a synthetic transmembrane domain upstream from lacZ . This domain is designed to stop transfer of secreted proteins across the membrane during secretion, allowing the beta Gal domain of the fusion polypeptide to remain in the cytoplasm and thus function in enzymatic assays . We have used the vectors to analyze expression of several genes in the nematode Caenorhabditis elegans, and have demonstrated in these studies that lacZ can be expressed in a wide variety of different tissues and cell types . These vectors should be useful in studying gene expression both in C . elegans and in other experimental systems. Nature, 1990 Sep 13, 347(6289), 203 - 6 Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs; Eriani G et al.; The aminoacyl-transfer RNA synthetases (aaRS) catalyse the attachment of an amino acid to its cognate transfer RNA molecule in a highly specific two-step reaction . These proteins differ widely in size and oligomeric state, and have limited sequence homology . Out of the 18 known aaRS, only 9 referred to as class I synthetases (GlnRS, TyrRS, MetRS, GluRS, ArgRS, ValRS, IleRS, LeuRS, TrpRS), display two short common consensus sequences ('HIGH' and 'KMSKS') which indicate, as observed in three crystal structures, the presence of a structural domain (the Rossman fold) that binds ATP . We report here the sequence of Escherichia coli ProRS, a dimer of relative molecular mass 127,402, which is homologous to both ThrRS and SerRS . These three latter aaRS share three new sequence motifs with AspRS, AsnRS, LysRS, HisRS and the beta subunit of PheRS . These three motifs (motifs 1, 2 and 3), in a search through the entire data bank, proved to be specific for this set of aaRS (referred to as class II) . Class II may also contain AlaRS and GlyRS, because these sequences have a typical motif 3 . Surprisingly, this partition of aaRS in two classes is found to be strongly correlated on the functional level with the acylation occurring either on the 2' OH (class I) or 3' OH (class II) of the ribose of the last nucleotide of tRNA. Biochemistry, 1990 Sep 11, 29(36), 8491 - 8 The catalytic site of Escherichia coli aspartate transcarbamylase: interaction between histidine 134 and the carbonyl group of the substrate carbamyl phosphate; Xi XG et al.; Previous pKa determinations indicated that histidine 134, present in the catalytic site of aspartate transcarbamylase, might be the group involved in the binding of the substrate carbamyl phosphate and, possibly, in the catalytic efficiency of this enzyme . In the present work, this residue was replaced by an asparagine through site-directed mutagenesis . The results obtained show that histidine 134 is indeed the group of the enzyme whose deprotonation increases the affinity of the catalytic site for carbamyl phosphate . In the wild-type enzyme this group can be titrated only by those carbamyl phosphate analogues that bear the carbonyl group . In the modified enzyme the group whose deprotonation increases the catalytic efficiency is still present, indicating that this group is not the imidazole ring of histidine 134 (pKa = 6.3) . In addition, the pKa of the still unknown group involved in aspartate binding is shifted by one unit in the mutant as compared to the wild type. Eur J Biochem, 1990 Sep 11, 192(2), 487 - 98 The multifunctional 6-methylsalicylic acid synthase gene of Penicillium patulum . Its gene structure relative to that of other polyketide synthases; Beck J et al.; 6-Methylsalicylic acid synthase (MSAS) from Penicillium patulum is a homomultimer of a single, multifunctional protein subunit . The enzyme is induced, at the transcriptional level, during the end of the logarithmic growth phase . After approximately 150-fold purification, a homogeneous enzyme preparation was obtained exhibiting, upon SDS gel electrophoresis, a subunit molecular mass of 188 kDa . By immunological screening of a genomic P . patulum DNA expression library, the MSAS gene together with its flanking sequences was isolated; 7131 base pairs of the cloned genomic DNA were sequenced . Within this sequence the MSAS gene was identified as a 5322-bp-long open reading frame coding for a protein of 1774 amino acids and 190,731 Da molecular mass . Transcriptional initiation and termination sites were determined both by primer extension studies and from cDNA sequences specially prepared for the 5' and 3' portions of the gene . The same cDNA sequences revealed the presence of a 69-bp intron within the N-terminal part of the MSAS gene . The intron contains the canonical GT and AG dinucleotides at its 5'- and 3'-splice junctions . An internal TACTGAC sequence, resembling the TACTAAC consensus element of Saccharomyces cerevisiae introns is suggested to represent the branch point of the lariat splicing intermediate . When compared to other known polyketide synthases, distinct amino acid sequence similarities of limited lengths were observed with some, though not all, of them . A comparatively low degree of similarity was detected to the yeast and Penicillium FAS or to the plant chalcone and resveratrol synthases . In contrast, a significantly higher sequence similarity was found between MSAS and the rat fatty acid synthase, especially at their transacylase, 2-oxoacyl reductase, 2-oxoacyl synthase and acyl carrier protein domains . Besides several dissimilar, interspersed regions probably coding for MSAS- and FAS-specific functions, the sequential order of the similar domains was colinear in both enzymes . The low similarity between the two P . patulum polyketide synthases, MSAS and FAS, possibly supports a convergent rather than a divergent evolution of both multienzyme proteins. Eur J Biochem, 1990 Sep 11, 192(2), 441 - 9 Sequence of proton abstraction and stereochemistry of the reaction catalyzed by naphthoate synthase, an enzyme involved in menaquinone (vitamin K2) biosynthesis; Igbavboa U et al.; The enzymic conversion of the coenzyme A ester of 4-(2'-carboxyphenyl)-4-oxobutyric acid (i.e . o-succinylbenzoic acid) to 1,4-dihydroxy-2-naphthoic acid is a cyclization reaction which is part of menaquinone (vitamin K2) biosynthesis . This conversion, which is probably a two-step process, was investigated using chirally labelled samples of the coenzyme A ester of 4-(2'-carboxyphenyl)-4-oxobutyric acid . To synthesize these, the following enzymes were employed: isocitrate: NADP+ oxidoreductase (EC 1.1.1.42), isocitrate glyoxylate-lyase (EC 4.1.3.1), 2-oxoglutarate dehydrogenase complex (which includes EC 1.2.4.2), 4-(2'-carboxyphenyl)-4-oxobutyrate synthase system and 4-(2'-carboxyphenyl)-4-oxobutyrate: CoA ligase . Isocitrate: NADP+ oxidoreductase was employed to generate the two enantiomeric samples of 2-oxoglutarate enantiotopically labelled at C-3 . These samples were converted enzymically to succinate with retention of configuration at C-2 and C-3, and to 4-(2'-carboxyphenyl)-4-oxobutyric acid with retention of configuration at C-3 . Isocitrate glyoxylate-lyase and isocitrate NADP+ oxidoreductase were employed to generate samples of 2-oxoglutarate enantiotopically tritiated at C-4 or at C-3 and C-4 . The four variously labelled samples of 2-oxoglutarate were enzymically converted to the coenzyme A ester of 4-(2'-carboxyphenyl)-4-oxobutyric acid . The resulting variously labelled coenzyme A esters were incubated with naphthoate synthase to investigate the ring closure reaction . In the first step the 2HRe atom of the oxobutyric moiety of the coenzyme A ester is equilibrated with solvent protons in a fast and reversible reaction . Subsequently the 2HSi and 3HSi atoms are removed whereas the 3HRe atom becomes the proton at C-3 of 1,4-dihydroxy-2-naphthoic acid . The second step in this ring closure reaction is the rate-limiting step. Eur J Biochem, 1990 Sep 11, 192(2), 401 - 9 On the roles of magnesium and spermidine in the isoleucyl-tRNA synthetase reaction . Analysis of the reaction mechanism by total rate equations; Airas RK; The reaction of isoleucyl-tRNA synthetase from Escherichia coli B was analysed by deriving total steady-state rate equations for the ATP/PPi exchange reaction and for the aminoacylation of tRNA, and by fitting these rate equations to series of experimental results . The analysis suggests that (a) a Mg2+ inhibits the aminoacylation of tRNA but not the activation of the amino acid . In the chosen mechanism, this enzyme-bound Mg2+ is required at the activation step . (b) Another Mg2+ is required at ATP, but the MgATP apparently can be replaced by the spermidine.ATP complex . Spermidine.ATP is a weaker substrate . The role of spermidine.ATP is especially suggested by the relative rates of the aminoacylation of tRNA when the spermidine and magnesium concentrations are varied . The aminoacylation measurements still suggest that (c) two (or more) Mg2+ are bound to the tRNA molecule and are required for enzyme activity at the transfer step, and that these Mg2+ can be replaced by spermidines. Eur J Biochem, 1990 Sep 11, 192(2), 305 - 9 Fluoroaluminates do not affect the guanine-nucleotide binding centre of the peptide chain elongation factor EF-Tu; Kraal B et al.; EF-Tu is often referred to as a model for guanine-nucleotide-binding regulatory proteins (G-proteins), since X-ray diffraction analysis of its GTP-binding domain shows a detailed location of the 'consensus' amino acid sequences involved in nucleotide binding . Fluoroaluminates are thought to mimick the gamma-phosphate in the GTPase centre on account of their activating effect on a variety of GTP binding proteins . In the case of EF-Tu, we could find no such effects on the basis of at least three independent functional assays . We did notice, however, complicating interactions between free nucleotides, fluoroaluminates and other ligands . By consequence, if indeed AlF4- behaves as a gamma-phosphate analogue in G-proteins, then EF-Tu must have a different GDP/GTP binding site, despite of the conserved consensus sequences. Nucleic Acids Res, 1990 Sep 11, 18(17), 5107 - 12 Solid phase in vitro mutagenesis using plasmid DNA template; Hultman T et al.; Site-specific mutagenesis was accomplished using a solid support to generate single stranded vector and insert fragments which can be used to form gap-duplex plasmids through flanking, complementary double stranded regions . More than 80% mutants were obtained in both a single and a double primer approach . No special vectors or strains are needed and mismatch repair is avoided as the mutagenesis region is in a single stranded form when transformed into the Escherichia coli host cell . The fragments to be immobilized can be produced either by a polymerase chain reaction using general primers or by a site-specific restriction followed by a fill-in reaction . This novel method is rapid, simple and flexible and well suited for both manual and semi-automated in vitro mutagenesis protocols. Nucleic Acids Res, 1990 Sep 11, 18(17), 4993 - 5000 Searching for and predicting the activity of sites for DNA binding proteins: compilation and analysis of the binding sites for Escherichia coli integration host factor (IHF); Goodrich JA et al.; An analysis of the sequence information contained in a compilation of published binding sites for E . coli integration host factor (IHF) was performed . The sequences of twenty-seven IHF sites were aligned; the base occurrences at each position, the information content, and an extended consensus sequence were obtained for the IHF site . The base occurrences at each position of the IHF site were used with a program written for the Apple Macintosh computers in order to determine the similarity scores for published IHF sites . A linear correlation was found to exist between the logarithm of IHF binding and functional data (relative free energies) and similarity scores for two groups of IHF sites . The MacTargsearch program and its potential usefulness in searching for other sites and predicting their relative activities is discussed. Biochemistry, 1990 Sep 11, 29(36), 8410 - 6 Electron paramagnetic resonance spectroscopic characterization of dimethyl sulfoxide reductase of Escherichia coli; Cammack R et al.; The electron transfer centers in dimethyl sulfoxide reductase were examined by EPR spectroscopy in membranes of the overproducing Escherichia coli strain HB101/pDMS159, and in purified enzyme . Iron-sulfur clusters of the {4Fe-4S} type and a molybdenum center were detected in the protein, which comprises three different subunits: DmsA, -B, and -C . The intensity of the reduced iron-sulfur clusters corresponded to 3.82 +/- 0.5 spins per molecule . The dithionite-reduced clusters were reoxidized by DMSO or TMAO . The enzyme, as prepared, showed a spectrum of Mo(V), which resembles the high-pH form of E . coli nitrate reductase . The Mo(V) detected by EPR was absent from a mutant which does not assemble the molybdenum cofactor . In these cases, the levels of EPR-detectable iron-sulfur clusters in the cells were increased . Extracts from HB101/pDMS159 enriched in DmsA showed more Mo(V) signals and considerably less iron-sulfur . These results are in agreement with predictions from amino acid sequence comparisons, that the molybdenum center is located in DmsA, while four iron-sulfur clusters are in DmsB . The midpoint potentials of the molybdenum and iron-sulfur clusters in the various preparations were determined by mediator titrations . The iron-sulfur signals could be best fitted by four clusters, with midpoint potentials spread between -50 and -330 mV . The midpoint potentials of the iron-sulfur clusters and Mo(V) species were pH dependent . In addition, all potentials became less negative in the presence of the detergent Triton X-100 . Observation of relaxation enhancement of the Mo(V) species by the reduced {4Fe-4S} clusters indicated that the centers are in proximity within the protein. Nucleic Acids Res, 1990 Sep 11, 18(17), 5157 - 62 Site-specific insertion and deletion mutants in the mer promoter-operator region of Tn501; the nineteen base-pair spacer is essential for normal induction of the promoter by MerR; Parkhill J et al.; We have made site-specific mutations in the promoter-operator region of the mercury-resistance (mer) operon of transposon Tn501 . Mutations were selected to alter the spacing between the -10 and -35 promoter elements without altering the sequence of a 7 bp dyad symmetrical sequence, which is the site of binding of the regulatory protein, MerR . (MerR acts as a repressor in the absence of mercuric salts and as an inducer in their presence, and binds to the same site in each case) . Transcription from the mutant promoters was measured in vivo in the presence and absence of MerR and of mercuric salts; and the relative affinities of the mutant promoters for partially purified MerR were determined in vitro by gel-shift assay in the presence and absence of mercuric salts . The 19 bp spacer was found to be essential for correct induction and repression of the operon; a spacer size of 20 or 21 bp prevents induction, and a spacer size of 18 or 17 bp causes the promoter to be highly active under all conditions . Double mutations, which alter the position of the 7 bp dyad relative to the -10 and -35 sequences without altering their spacing prevent induction by the MerR-Hg(II) complex, demonstrating the tight constraints on the positions of MerR and RNA polymerase in the transcriptional complex . The data are compatible with a model for induction of the mer promoter involving a local conformational change in the DNA structure caused by the MerR-Hg(II) complex. Nucleic Acids Res, 1990 Sep 11, 18(17), 5045 - 50 DNA sequence analysis of the imp UV protection and mutation operon of the plasmid TP110: identification of a third gene; Lodwick D et al.; The sequence of the imp operon of the plasmid TP110 (which belongs to the Incl1 incompatibility group) has been determined, and is shown to contain three open reading frames . This operon, involved in UV protection and mutation, is functionally analogous to the umuDC operon of E . coli and the mucAB operon of the plasmid pKM101, which belongs to the quite unrelated IncN incompatibility group . The umu and muc operons however contain only two open reading frames, coding for proteins of approximately 16kD and 46kD . The high degree of homology between the two 16kD proteins (UmuD and MucA) and between the two 46kD proteins (UmuC and MucB) clearly shows their relatedness . This is shown also to extend to the imp gene products, with ImpA sharing homology with UmuD and MucA, and ImpB sharing homology with UmuC and MucB . However, the two imp genes are preceded in the operon by a third gene, impC, which encodes a small protein of 9.5kD and which has no equivalent in the umu and muc operons. Nucleic Acids Res, 1990 Sep 11, 18(17), 5069 - 75 Analysis of class II (hydrolytic) and class I (beta-lyase) apurinic/apyrimidinic endonucleases with a synthetic DNA substrate; Levin JD et al.; We have developed simple and sensitive assays that distinguish the main classes of apurinic/apyrimidinic (AP) endonucleases: Class I enzymes that cleave on the 3' side of AP sites by beta-elimination, and Class II enzymes that cleave by hydrolysis on the 5' side . The distinction of the two types depends on the use of a synthetic DNA polymer that contains AP sites with 5'-{32P}phosphate residues . Using this approach, we now show directly that Escherichia coli endonuclease IV and human AP endonuclease are Class II enzymes, as inferred previously on the basis of indirect assays . The assay method does not exhibit significant interference by nonspecific nucleases or primary amines, which allows the ready determination of different AP endonuclease activities in crude cell extracts . In this way, we show that virtually all of the Class II AP endonuclease activity in E . coli can be accounted for by two enzymes: exonuclease III and endonuclease IV . In the yeast Saccharomyces cerevisiae, the Class II AP endonuclease activity is totally dependent on a single enzyme, the Apn1 protein, but there are probably multiple Class I enzymes . The versatility and ease of our approach should be useful for characterizing this important class of DNA repair enzymes in diverse systems. Biochim Biophys Acta, 1990 Sep 10, 1087(1), 55 - 60 Lac repressor-operator interaction: DNA length dependence; Khoury AM et al.; The interaction of the E . coli lac operon repressor with its operator DNA has been directly examined as a function of the length of operator-containing DNA . The apparent bimolecular association rate constants were calculated as ka = (kd/KD), where the dissociation equilibrium constant, KD and the dissociation rate constant, kd, were measured by nitrocellulose filter adsorption assays . The values obtained for the overall association rate constants are compared with theoretical association rate curves for specific mechanisms . Association of the repressor with short operator containing DNA fragments (less than 70 base pairs) occurs at rates expected of three-dimensional diffusion . Our data also imply that at longer DNA lengths a combination of three-dimensional diffusion with one-dimensional sliding along with hopping and/or intersegment transfer must be involved to facilitate the repressor operator association. Biochim Biophys Acta, 1990 Sep 10, 1087(1), 39 - 48 Search of the 5' untranslated region of the human cardiac actin gene for segments controlling translation; Colledge WH et al.; We have examined the possibility that the 5'-UT region of the human cardiac actin (CH-actin) mRNA is responsible for regulating translation of this transcript during skeletal myogenesis . Genes were constructed which consisted of the murine leukaemia virus promoter driving the Escherichia coli LacZ coding region with and without the CH-actin 5'-UT region . These constructs were transfected into L6 myoblasts that were subsequently differentiated into myotubes . The presence of the CH-actin 5'-UT region appeared to have no effect on expression of the LacZ reporter gene . EGTA blocks myogenesis and inhibits translation of muscle-specific transcripts (Endo, T . and Nadal-Ginard, B . (1987) Cell 49, 515-526) but EGTA had no effect on expression of the chimaeric LacZ transcripts . Thus, if the CH-actin transcript is subject to translational regulation, it must be mediated by sequences other than those of the 5'-UT region. Biochim Biophys Acta, 1990 Sep 7, 1027(3), 238 - 44 Mechanism of penetration and of action of local anesthetics in Escherichia coli cells; Collura V et al.; Escherichia coli cells were used to study the mechanism of penetration of local anesthetics and the relationship between permeation and functional properties . We show that both the neutral and the protonated form of dibucaine can be accumulated in the cells . Accumulation of the protonated form occurs in response to a transmembrane electrical potential (negative inside) and results in high trapped concentrations (70 mM) . Accumulation can lead to an alkalinization of the internal pH . Low concentrations of dibucaine stimulate the respiration, increase the transmembrane electrical potential and raise the accumulation of solutes . Inhibition of these functions occurs at higher concentrations of the drug . Furthermore, the drug concentration required to inhibit these functions is smaller at alkaline external pH than at acidic external pH, suggesting that the inhibition is mainly due to the neutral form of the anesthetics . Other hydrophobic amines also stimulate and inhibit different membrane functions, their efficiency being correlated to their lipophilicity. J Mol Biol, 1990 Sep 5, 215(1), 67 - 71 DNA replication in Escherichia coli is initiated by membrane detachment of oriC . A model; Norris V; An adequate model for the initiation of chromosome replication in Escherichia coli should explain why the introduction of multiple copies of the chromosomal origin of replication, oriC, does not perturb cells seriously and why such multiple origins are replicated synchronously; it should explain why the key initiator protein, DnaA, is activated in vitro by binding specifically to acidic phospholipids and why the Dam methyltransferase is essential for the correct timing of initiation; it should explain why phospholipid synthesis and fluidity are necessary for initiation . In the detachment model, presented here, cyclical changes in the phospholipid composition of the cytoplasmic membrane activate initiator proteins such as DnaA protein and cause origins to detach; this detachment allows torsional stresses to open 13mer sequences in oriC; DnaA assists in the serial opening of these sequences and guides the entry of the helicase to form a pre-priming complex and trigger initiation; the greater affinity of hemi-methylated origin for membrane is re-interpreted as a mechanism for preventing re-initiation. J Biol Chem, 1990 Sep 5, 265(25), 15308 - 15 DNA structural polymorphism modulates the kinetics of superhelical DNA cleavage by BamHI restriction endonuclease; Nardone G et al.; A compartmental model developed by Hensley (Hensley, P., Nardone, G., Chirikjian, J.G., and Wastney, M . E., (1990) J . Biol . Chem . 265, 15300-15307) for analysis of the time courses of the cleavage of superhelical DNA substrates by the restriction endonuclease, BamHI, has been used to quantify the effects of changes in temperature, ionic strength, superhelical density, and the DNA substrate on the binding and strand cleavage processes . Studies reported here indicate that changes in topology may be introduced into the DNA substrate solely as a result of the plasmid preparation process and in the absence of covalent bond cleavage and ligation . These changes in topology have qualitatively different effects on the kinetics than those promoted by changes in the superhelical density . The former are removed by briefly warming the DNA prior to assay, suggesting that they are only kinetically stable, while the latter changes are not affected by heating . Increasing the {NaCl} from 0.01 M to 0.1 M increases the overall rate of plasmid cleavage by increasing both the rates of cleavage and enzyme DNA association . To describe the decrease in the overall cleavage rate observed in 0.15 M NaCl, an ionic strength-dependent rate-determining structural transition in the DNA substrate was incorporated into the model . The largest changes in the rate of the cleavage process resulted from changes in the DNA substrate .