Escherichia coli

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 . For the SV40 substrate compared to pBR322, the rate constants describing the two association processes and the first bond cleavage event were increased 6- to 7-fold . The rate of the second bond cleavage process was not affected . These changes may be due to differences in the flanking sequences.

J Biol Chem, 1990 Sep 5, 265(25), 15300 - 7
The time-resolved kinetics of superhelical DNA cleavage by BamHI restriction endonuclease; Hensley P et al.; The kinetics of the cleavage of superhelical plasmid DNA (pBR322) by the restriction endonuclease, BamHI, have been analyzed in terms a compartmental model consistent with the chemistry first proposed by Rubin and Modrich (Rubin, R . A., and Modrich, P . (1978) Nucleic Acids Res . 5, 2991-2997) for analysis of the kinetics of the restriction endonuclease, EcoRI . The model was defined in terms of two compartments representing DNA substrate (bound and free), two compartments representing nicked intermediate (bound and free), one compartment representing linear product, and one compartment for free enzyme . A simultaneous analysis of concentration changes over time of the three DNA forms (superhelical, nicked, and linear) at six different enzyme concentrations was undertaken employing this compartmental model using SAAM (Simulation Analysis And Modeling) software . Results showed that rate constants characterizing the association of enzyme with superhelical DNA (6.0 x 10(5) M-1 s-1) and nicked DNA (2.8 x 10(5) M-1 s-1) were similar in magnitude and rate constants characterizing cleavage of the first (1.2 x 10(-2) s-1) and second phosphodiester bonds (3.1 x 10(-2) s-1) were also similar . The analysis yields a kinetically determined equilibrium constant of 12.9 nM for the dissociation of nicked intermediate from the enzyme . The rate constant describing the release of the nicked intermediate from the enzyme has a value of 3.7 x 10(-3) s-1 . By comparing the value of this release rate constant to the value of the constant describing the second cleavage event, it can be determined that only 10% of the nicked intermediate bound to the enzyme is released as free nicked DNA and that 90% of the nicked intermediate is processed to the linear form without being released . Hence, most of the DNA is cleaved as the result of a single enzyme-DNA recognition event . No steady state assumptions were made in the analysis . The approach was to directly solve the differential equations which described the kinetic processes using an interactive method . This study demonstrates the usefulness of this approach for the analysis of kinetics of protein-DNA interactions for the restriction endonucleases.

J Biol Chem, 1990 Sep 5, 265(25), 15267 - 74
Endotoxin induction of murine metallothionein gene expression; De SK et al.; Bacterial endotoxin-lipopolysaccharide (LPS) rapidly induced hepatic metallothionein (MT) mRNA levels in the LPS-sensitive CD-1 strain of mice . This LPS effect was severely attenuated in the LPS-resistant C3H/HeJ strain of mice, but could be mimicked by injection of human recombinant interleukin-1 alpha (IL-1 alpha) or human recombinant tumor necrosis factor (TNF-alpha) . In the CD-1 strain, LPS induction of MT gene expression occurred in each of 10 organs examined (liver, kidney, pancreas, intestine, lung, heart, brain, ovary, uterus, and spleen) . Solution hybridization with probes specific for MT-I or MT-II mRNA established that these genes were co-induced in each of the organs and that the liver and kidney contained the highest absolute levels of these mRNAs, whereas in the intestine and spleen they were 10-20-fold lower . LPS and cytokine induction of hepatic MT gene expression occurred in hypophysectomized mice, which suggests a lack of significant involvement of glucocorticoids . Several recombinant cytokines (TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, interferon-gamma (IFN-gamma), as well as poly(rI.rC) were effective inducers of hepatic MT-I and MT-II genes . As an attempt to determine which of these cytokines may mediate LPS effects on MT gene expression in vivo, CD-1 mice were injected with LPS or various cytokines, and RNA from liver, ovary, and uterus was extracted at various times postinjection and analyzed by Northern blotting using probes specific for IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, and MT mRNA . In each organ examined, LPS, IL-1 alpha, or IL-1 beta injection caused a rapid, coordinate, transient increase in the levels of each of the cytokine mRNAs which peaked by 1 h and declined to low levels by 4 h . In contrast, levels of MT mRNA did not reach a peak until 4-6 h postinjection . TNF-alpha had minimal effects on expression of cytokine and MT genes in organs other than liver . IL-6 had no effect on hepatic cytokine mRNA levels, and induced MT mRNA only in the liver which suggests a direct effect of IL-6 on hepatic MT gene expression . These data suggest that the acute effects of LPS on MT gene expression may include complex paracrine interactions between a variety of cytokines and the cells expressing MT genes in each organ, and tissue-specific cytokine effects on the MT genes.

J Biol Chem, 1990 Sep 5, 265(25), 15239 - 44
Minimal deletion of amino acids from the carboxyl terminus of the B subunit of heat-labile enterotoxin causes defects in its assembly and release from the cytoplasmic membrane of Escherichia coli; Sandkvist M et al.; Minimal alterations at the carboxyl terminus of the B subunit (EtxB) of heat-labile enterotoxin from Escherichia coli were found to have a marked effect on the assembly and release of this polypeptide into the periplasm . Nine mutant EtxB polypeptides were obtained by genetic manipulation of the 3'-end of the etxB gene using Bal31 nuclease digestion and codon substitution . A correlation was observed between the magnitude of the changes introduced at the carboxyl terminus and the extent to which the mutant polypeptides were defective in assembly and release . Some of the mutant B subunits, exemplified by those in which the last 2 amino acids had been deleted or in which the last 4 residues had been replaced by three different ones, were found to be only partially defective, with a proportion being associated with the periplasmic face of the cytoplasmic membrane and the remainder being exported to the periplasm . The portion associated with membranes was detected as monomers on sodium dodecyl sulfate-polyacrylamide gels, whereas the portion exported to the periplasm were detected as assembled oligomers . We conclude that the last few amino acids at the carboxyl terminus of EtxB exert a profound influence on the assembly and release of the B subunit from the cytoplasmic membrane during export in E . coli.

J Biol Chem, 1990 Sep 5, 265(25), 15023 - 7
Ligand-induced isomerizations of Escherichia coli ornithine transcarbamoylase . An ultraviolet difference analysis; Miller AW et al.; Ligand-induced ultraviolet difference spectra have been determined for Escherichia coli ornithine transcarbamoylase . The most prominent feature of the spectra is an absorbance difference which resembles a single period of a sine wave spanning the 245-320 nm region with a maximum at approximately 270 nm and a minimum at around 295-300 nm . This broad absorbance difference is typical of a blue-shift 1La band of tryptophan . Superimposed on the broad band in the 275-310 nm region is a series of smaller, narrow peaks resulted from red-shifted 1Lb bands of tryptophan and tyrosine residues . At pH 8.5, only carbamoyl phosphate and its analog phosphonacetamide yield a large ultraviolet difference absorbance (approximately 1800 M-1 cm-1) when bound to the enzyme . The spectra obtained are essentially the same in lineshape to and 80% in intensity of that produced by the bisubstrate analogy, N-(phosphonacetyl)-L-ornithine . In contrast, inorganic phosphate, a product of the reaction, induces small protein absorbance changes (approximately 300 M-1 cm-1) mainly in the 275-310 nm range . When complexed to the free enzyme, L-ornithine yields a marginally discernible ultraviolet difference spectrum in the 275-310 nm region, and its analogs L-norvaline and L-citrulline provide no absorbance change . However, inorganic phosphate in combination with any of the L-amino acids produces a difference spectrum similar to that given by carbamoyl phosphate alone . Collectively, these spectra suggest that carbamoyl phosphate elicits an isomerization required for the formation of the ternary complex and are consistent with the compulsory ordered mechanism of the enzyme at pH 8.5 with carbamoyl phosphate being the first substrate bound . Below pH 8, there is a kinetically discernible amount of random binding, but ordered addition is still the preferred pathway (Wargnies B., Legrain, C., and Stalon, V . (1978) Eur J . Biochem . 89, 203-212) . Reflecting this change, the difference absorbance of the enzyme bound with carbamoyl phosphate is also pH dependent . The 1La band in the carbamoyl phosphate difference spectrum diminishes by approximately 20% at low pH . The PALO-induced changes, however, are pH invariant suggesting that full extent of the induced-fit isomerization is always reached in the ternary complex.

J Biol Chem, 1990 Sep 5, 265(25), 14763 - 9
Ligand-specific transactivation of gene expression by a derivative of the human glucocorticoid receptor expressed in yeast; Wright AP et al.; In this study we have reconstituted transactivation of gene expression by the human glucocorticoid receptor in the yeast, Saccharomyces cerevisiae . We have expressed the C-terminal half of the human glucocorticoid receptor (residues 415-777), the smallest derivative that can be expected to function as a ligand-dependent activator of transcription, in yeast cells . The function of the expressed protein has been assayed using a reporter gene consisting of the beta-galactosidase gene from Escherichia coli fused to the yeast iso-1-cytochrome c promoter with a glucocorticoid-responsive element from the rat tyrosine aminotransferase gene upstream . Transactivation of expression from the reporter gene by the expressed receptor is seen only in the presence of steroid hormones with glucocorticoid activity and occurs via specific interaction of receptor with the glucocorticoid-responsive element upstream of the reporter gene . This result is different from those obtained for the estrogen receptor in which a similar derivative was not functional in yeast . This suggests that the well documented conservation of structure and function between steroid receptors may not extend to the transactivation domains . Our results also suggest that the mechanism by which receptors are sequestered in an inactive, non-DNA binding state in the absence of ligand may be functionally conserved in yeast . In support of this we show evidence that the expressed receptor is associated with the yeast molecular weight 90,000 heat shock protein as seen in mammalian cells.

J Biol Chem, 1990 Sep 5, 265(25), 14701 - 4
Polyisoprenylation of Ras in vitro by a farnesyl-protein transferase; Schaber MD et al.; Farnesylation of Ras occurs in vivo on a Cys residue in the C-terminal sequence -Cys-Val-Leu-Ser (termed a CAAX box) . This modification is required for Ras membrane localization and cell transforming activity . Using {3H}farnesyl-PPi as precursor and Escherichia coli-expressed Ras, forms of Ras having the CAAX sequence were radiolabeled upon incubation with the cytosolic fraction of bovine brain . Forms of Ras having a deletion of the CAAX sequence or a Cys to Ser substitution in this sequence were not substrates . Radioactivity incorporated into Ras by bovine brain cytosol was released by treatment with iodomethane but not with methanolic KOH indicating a thioether linkage . High pressure liquid chromatography analysis of the cleavage products on a C-18 column showed a major peak of radioactivity that co-eluted with a farnesol standard . The enzyme responsible for Ras farnesylation in bovine brain was approximately 190 kDa as estimated by gel filtration and required a divalent cation for activity . Nonradioactive farnesyl-PPi, geranylgeranyl-PPi, and Ras peptides having the C-terminal sequence -Cys-Val-Leu-Ser competed in the assay with IC50 values of 0.7, 1.4, and 1-3 microM, respectively . Farnesol and Ras peptides having the sequence -Ser-Val-Leu-Ser were not inhibitory . These results identify a farnesyl-protein transferase activity that may be responsible for the polyisoprenylation of Ras in intact cells.

J Biol Chem, 1990 Sep 5, 265(25), 15134 - 44
The ABC-primosome . A novel priming system employing dnaA, dnaB, dnaC, and primase on a hairpin containing a dnaA box sequence; Masai H et al.; A priming mechanism requiring dnaA, dnaB, and dnaC proteins operates on a single-stranded DNA coated with single-stranded DNA-binding protein . This novel priming, referred to as "ABC-priming," requires a specific hairpin structure whose stem carries a dnaA protein recognition sequence (dnaA box) . In conjunction with primase and DNA polymerase III holoenzyme, ABC-priming can efficiently convert single-stranded DNA into the duplex replicative form . dnaA protein specifically recognizes and binds the single-stranded hairpin and permits the loading of dnaB protein to form a prepriming protein complex containing dnaA and dnaB proteins which can be physically isolated . ABC-priming can replace phi X174 type priming on the lagging strand template of pBR322 in vitro, suggesting a possible function of ABC-priming for the lagging strand synthesis and duplex unwinding . Similar to the phi X174 type priming, a mobile nature of ABC-priming was indicated by helicase activity in the presence of ATP of a prepriming protein complex formed at the hairpin . The implications of this novel priming in initiation of replication at the chromosomal origin, oriC, and in its contribution to the replication fork are discussed.

J Biol Chem, 1990 Sep 5, 265(25), 15124 - 33
Roles of phi X174 type primosome- and G4 type primase-dependent primings in initiation of lagging and leading strand syntheses of DNA replication; Masai H et al.; Eleven single strand initiation sequences (ssi) were isolated from various plasmid genomes using a plaque-morphology assay . Out of seven ssi that require dnaB and dnaC functions for replication in a crude in vitro system, six use a phi X174 type priming mechanism, and a phi X174 type primosome is assembled at these sequences from the purified proteins, n'(priA), n(priB), n"(priC), dnaT, dnaB, dnaC, and primase . The same ssi potentiate dATPase activity of n' protein, and thus represent new n' protein recognition sequences (n'-pas) . Based on sequence homology, two structural groups are evident . Two sequences show a strong homology with the phi X174 site, whereas three share extensive homology with the previously characterized n'-pas of ColE1, ssiA(ColE1) . All the n'-pas have a potential to form stem and loop structures, although sequence homology between the two classes is absent . In addition to the phi X174 type priming, three ssi do not require either dnaB or dnaC function for replication, and use a G4 type priming, requiring only SSB and primase . The 5' ends of primer RNA synthesized by primase are localized within the vicinity of one of the three blocks of highly conserved nucleotide sequences . Deletions of parts of these conserved sequences result in loss of priming activity, suggesting that they are important for priming on the G4 type ssi, which are termed G site . The general significance of these two types of priming in initiation of lagging or leading strand synthesis as well as various modes of initiation at origins of replication are proposed.

J Biol Chem, 1990 Sep 5, 265(25), 15154 - 9
The purification and preliminary X-ray diffraction studies of recombinant Synechococcus ribulose-1,5-bisphosphate carboxylase/oxygenase from Escherichia coli; Newman J et al.; X-ray crystallographic diffraction data has been collected for recombinant hexadecameric ribulose-P2 carboxylase from the cyanobacterium Synechococcus PCC6301 expressed in Escherichia coli . The enzyme has been purified and then crystallized in a number of crystal forms from polyethylene glycol solutions . The best crystals were obtained with enzyme that was first activated with the cofactors CO2 and Mg2+ in the presence of the tight-binding intermediate analogue, 2'-carboxyarabinitol 1,5-bisphosphate . One crystal form with plate-like morphology diffracts beyond 2.5 A but has one axis greater than 350 A . A second crystal form that diffracts to similar resolution grows with space group P212121 and unit cell dimensions of a = 223.9 A, b = 111.9 A, and c = 199.7 A . The crystal forms used to collect the diffraction data have been redissolved to determine that the recombinant ribulose-P2 carboxylase L8S8 molecule is indeed composed of equal numbers of large and small subunits and also that a quaternary complex between activated ribulose-P2 carboxylase E.CO2.Mg2+, and the analogue was present in the crystals . Denaturation of the redissolved enzyme in the absence of thiol-reducing agents established that the L-subunits of the L8 core are substantially dimeric, cross-linked by a disulfide bridge . Crystals of spinach ribulose-P2 carboxylase were likewise analyzed to show that dimers of the L-subunit were also predominant . This report identifies a single cysteine residue in the L-subunit that forms a bridge between those L-monomers that compose the four putative functional dimers of the L8 core.

J Biol Chem, 1990 Sep 5, 265(25), 15145 - 53
Transcription pausing by Escherichia coli RNA polymerase is modulated by downstream DNA sequences; Lee DN et al.; Escherichia coli RNA polymerase pauses immediately after transcription of certain sequences that can form stable secondary structures in the nascent RNA transcript; pausing appears to be essential for several types of bacterial transcription attenuation mechanisms . Because base changes that weaken the RNA secondary structures reduce the half-life of pausing by RNA polymerase, nascent transcript RNA hairpins are thought to cause pausing at these sites . We show here that, for the well characterized trpL pause site, the determinants of transcription pausing are not limited to the RNA hairpin, but include the not-yet-transcribed sequence of DNA immediately downstream from the pause site . We show that this effect extends to bases up to fourteen nucleotides downstream from the pause site, that placement of a oligo(dT) tract in the nontranscribed strand in this region does not convert the pause site to a termination site, and that shifting the position of pausing by one nucleotide downstream almost eliminates pausing . From an analysis of many variants of this downstream sequence, we argue that the effect of downstream sequence is not related simply to its GC content . We suggest that these effects are mediated by altered interactions between RNA polymerase and the DNA template downstream from the enzyme's active site.

Biochemistry, 1990 Sep 4, 29(35), 8184 - 9
Study of the binding of single-stranded DNA-binding protein to DNA and poly(rA) using electric field induced birefringence and circular dichroism spectroscopy; Kuil ME et al.; Binding of the single-stranded DNA-binding protein (SSB) of Escherichia coli to single-stranded (ss) polynucleotides produces characteristic changes in the absorbance (OD) and circular dichroism (CD) spectra of the polynucleotides . By use of these techniques, complexes of SSB protein and poly(rA) were shown to display two of the binding modes reported by Lohman and Overman {Lohman, T.M., & Overman, L . (1985) J . Biol . Chem . 260, 3594-3603} . The circular dichroism spectra of the "low salt" (10 mM NaCl) and "high salt" (greater than 50 mM NaCl) binding mode are similar in shape, but not in intensity . SSB binding to poly(rA) yields a complexed CD spectrum that shares several characteristics with the spectra obtained for the binding of AdDBP, GP32, and gene V protein to poly(rA) . We therefore propose that the local structure of the SSB-poly(rA) complex is comparable to the structures proposed for the complexes of these three-stranded DNA-binding proteins with DNA (and RNA) and independent of the SSB-binding mode . Electric field induced birefringence experiments were used to show that the projected base-base distance of the complex is about 0.23 nm, in agreement with electron microscopy results . Nevertheless, the local distance between the successive bases in the complex will be quite large, due to the coiling of the DNA around the SSB tetramer, thus partly explaining the observed CD changes induced upon complexation with single-stranded DNA and RNA.

Biochemistry, 1990 Sep 4, 29(35), 8164 - 71
Effect of phosphorylation on hydrogen-bonding interactions of the active site histidine of the phosphocarrier protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system determined by 15N NMR spectroscopy; van Dijk AA et al.; The phosphocarrier protein HPr of the phosphoenolpyruvate-dependent sugar transport system of Escherichia coli can exist in a phosphorylated and a nonphosphorylated form . During phosphorylation, the phosphoryl group is carried on a histidine residue, His15 . The hydrogen-bonding state of this histidine was examined with 15N NMR . For this purpose we selectively enriched the histidine imidazole nitrogens with 15N by supplying an E . coli histidine auxotroph with the amino acid labeled either at the N delta 1 and N epsilon 2 positions or at only the N delta 1 position . 15N NMR spectra of two synthesized model compounds, phosphoimidazole and phosphomethylimidazole, were also recorded . We show that, prior to phosphorylation, the protonated His15 N epsilon 2 is strongly hydrogen bonded, most probably to a carboxylate moiety . The H-bond should strengthen the nucleophilic character of the deprotonated N delta 1, resulting in a good acceptor for the phosphoryl group . The hydrogen bond to the His15 N delta 1 breaks upon phosphorylation of the residue . Implications of the H-bond structure for the mechanism of phosphorylation of HPr are discussed.

Biochemistry, 1990 Sep 4, 29(35), 8126 - 30
Intrinsic fluorescence of a truncated Bordetella pertussis adenylate cyclase expressed in Escherichia coli; Gilles AM et al.; A truncated, 432 residue long, Bordetella pertussis adenylate cyclase expressed in Escherichia coli was analyzed for intrinsic fluorescence properties . The two tryptophans (Trp69 and Trp242) of adenylate cyclase, each situated in close proximity to residues important for catalysis or binding of calmodulin (CaM), produced overlapping fluorescence emission bands upon excitation at 295 nm . CaM, alone or in association with low concentrations of urea, induced important modifications in the spectra of adenylate cyclase such as shifts of the maxima and change in the shape of the bands . From these changes and from the fluorescence spectrum of a modified form of adenylate cyclase, in which a valine residue was substituted for Trp242, it was deduced that, upon binding of CaM to the wild-type adenylate cyclase, only the environment of Trp242 was affected . The fluorescence maximum of this residue, which is more exposed to the solvent than Trp69 in the absence of CaM, is shifted by 13 nm to shorter wavelength upon interaction of protein with its activator . Trypsin cleaved adenylate cyclase into two fragments, one carrying the catalytic domain, and the second carrying the CaM-binding domain (Ladant et al., 1989) . The isolated peptides conserved most of the environment around their single tryptophan residues, as in the intact adenylate cyclase, which suggests that the two domains of truncated B . pertussis adenylate cyclase also conserved most of their three-dimensional structure in the isolated forms.

Biochemistry, 1990 Sep 4, 29(35), 8131 - 7
Tryptophan fluorescence study on the interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with model membranes; Killian JA et al.; The interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with different phospholipid vesicles was investigated by fluorescence techniques, using a synthetic mutant signal peptide in which valine at position -8 in the hydrophobic sequence was replaced by tryptophan . First it was established that this mutation in the signal sequence of prePhoE does not affect in vivo and in vitro translocation efficiency and that the biophysical properties of the synthetic mutant signal peptide are similar to those of the wild-type signal peptide . Next, fluorescence experiments were performed which showed an increase in quantum yield and a blue shift of the emission wavelength maximum upon interaction of the signal peptide with lipid vesicles, indicating that the tryptophan moiety enters a more hydrophobic environment . These changes in intrinsic fluorescence were found to be more pronounced in the presence of phosphatidylglycerol (PG) or cardiolipin (CL) than with phosphatidylcholine (PC) . In addition, quenching experiments demonstrated a shielding of the tryptophan fluorescence from quenching by the aqueous quenchers iodide and acrylamide upon interaction of the signal peptide with lipid vesicles, a shielding in the case of acrylamide that was more pronounced in the presence of negatively charged lipids . Finally it was found that acyl chain brominated lipids incorporated into phospholipid bilayers were able to quench the tryptophan fluorescence of the signal peptide, with the quenching efficiency in CL vesicles being much higher than in PC vesicles . The results clearly demonstrate that the PhoE signal peptide interacts strongly with different lipid vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1990 Sep 4, 29(35), 8190 - 8
Mapping of the active site of Escherichia coli methionyl-tRNA synthetase: identification of amino acid residues labeled by periodate-oxidized tRNA(fMet) molecules having modified lengths at the 3'-acceptor end; Hountondji C et al.; Initiator tRNA molecules modified at the 3'-end and lacking either the A76 (tRNA-C75), the C75-A76 (tRNA-C74), the C74-C75-A76 (tRNA-A73), or the A73-C74-C75-A76 (tRNA-A72) nucleotides were prepared stepwise by repeated periodate, lysine, and alkaline phosphatase treatments . When incubated with trypsin-modified methionyl-tRNA synthetase (MTST), excess amounts of the dialdehyde derivative of each of these shortened tRNAs (tRNA-C75ox, tRNA-C74ox, tRNA-A73ox, and tRNA-A72ox) abolished both the isotopic {32P}PPi-ATP exchange and the tRNA aminoacylation activities of the enzyme . In the presence of limiting concentrations of the various tRNAox species, the relative extents of inactivation of the enzyme were consistent with the formation of 1:1 complexes of the reacting tRNAs with the monomeric modified synthetase . Specificity of the labeling was further established by demonstrating that tRNA-C75ox binds the enzyme with an equilibrium constant and stoichiometry values in good agreement with those for the binding of nonoxidized tRNA-C75 . The peptides of MTST labeled with either tRNA-C75ox or tRNA-C74ox were identified . The chymotryptic digestion of the covalent MTST.{14C}tRNA-C75ox complex yielded four peptides (A-D) . In the case of tRNA-C74ox, only two of the above peptides (C and D) were identified . Peptides A, B, C, and D corresponded to fragments Ser334-Phe340, Lys61-Leu65, Val141-Tyr165, and Glu433-Phe437, respectively, in the MTST primary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1990 Sep 4, 29(35), 8144 - 51
Binding of initiation factor 2 and initiator tRNA to the Escherichia coli 30S ribosomal subunit induces allosteric transitions in 16S rRNA; Wakao H et al.; The specific effect of the binding of IF2 and initiator fMET-tRNA(fMet) on Escherichia coli 16S rRNA has been probed by phosphate alkylation with ethylnitrosourea . The results show that IF2 does not significantly shield portions of 16S rRNA but induces both reductions and enhancements of reactivity scattered in the entire molecule . Most of them are topographically constrained in a region corresponding to the cleft, the lateral protrusion, and the part of the head facing the protrusion (positions 694, 771, 791, 1225, 1268, 1398, 1401, 1504, and 1527) . Weak effects are also observed in distant parts of the subunit (positions 301, 302, 492, and 1428) . All the reactivity changes induced by the binding of IF2 are still observed in the presence of the initiator tRNA and AUG as messenger . The additional changes induced by the tRNA are mostly centered around the cleft-head-lateral protrusion region, near positions affected by IF2 binding . Most of the changes correspond to reduced reactivities (positions 791, 1222, 1263, 1393, 1395, 1430, 1431, 1504, 1528, and 1529), while enhanced reactivities are observed at positions 708, 709, and 1398 . Functional implications are discussed, which stress the dynamic properties of the ribosome.

FEBS Lett, 1990 Sep 3, 269(2), 398 - 401
Determination of tRNA nucleotide residues directly interacting with proteins in the post- and pretranslocated ribosomal complexes; Abdurashidova GG et al.; Nucleotide residues of E . coli tRNA interacting directly with proteins in pre- and posttranslocated ribosomal complexes have been identified by analysis of photo-induced tRNA-protein cross-links . A9, G18, A26 and U59 residues of NAcPhePhe-tRNA, located in the Ab-site (pretranslocated complex) have been cross-linked with proteins S10, L27, S7 and L2 respectively . In deacylated tRNA, located in the Pb-site, residues C17, G44, C56 and U60 have been cross-linked with proteins L2, L5, L27 and S9 respectively . The G44-L5 cross-link disappeared after translocation (NAc-PhePhe-tRNA located in the Pt-site).

Biochim Biophys Acta, 1990 Sep 3, 1040(2), 153 - 8
The fidelity of protein synthesis: can mischarging by aspartyl-tRNA(Asp) synthetase lead to the formation of isoaspartyl residues in proteins?
Momand JA, Clarke S.
We have tested the hypothesis that isoaspartic acid residues in proteins can arise via errors that occur during protein synthesis . One such error involves a mischarging step in which the aspartic acid side-chain beta-carboxyl group is linked to the tRNA(Asp) instead of the main chain alpha-carboxyl group . If this altered Asp-tRNA(Asp) is a substrate for the ribosomal elongation reactions, a polypeptide will be made with an isoaspartic acid, or beta-linkage, in which the peptide chain is branched at the side chain of the aspartic acid residue . Using an ammonium sulfate fraction of aspartyl-tRNA(Asp) synthetase from Escherichia coli and {3H}aspartic acid, we have prepared {3H}aspartyl-tRNA(Asp) complexes and directly analyzed the linkage of the {3H}aspartate to the tRNA by identifying the products of ammonolysis . Normal attachment of the alpha-carboxyl group of aspartate to the tRNA produces {3H}isoasparagine, while the mischarging reaction leads to {3H}asparagine formation after ammonolysis . We have separated {3H}isoasparagine from {3H}asparagine and found an upper limit of 1 asparagine per 10,000 isoasparagines . These results show that the bacterial aminoacyl-tRNA synthetase can very accurately distinguish between the alpha- and beta-carboxyl groups of aspartic acid and suggest that only a very small fraction of the isoaspartic acid residues found to occur in cellular proteins may be the result of mischarging steps.

Biochim Biophys Acta, 1990 Sep 3, 1040(2), 137 - 44
Antigenic conservation of primary structural regions of S-adenosylmethionine synthetase; Kotb M et al.; Although the physical and kinetic properties of S-adenosylmethionine (AdoMet) synthetases from different sources are quite different, it appears that these enzymes have structurally or antigenically conserved regions as demonstrated by studies with AdoMet synthetase specific antibodies . Polyclonal anti-human lymphocyte AdoMet synthetase crossreacted with enzyme from rat liver (beta isozyme), Escherichia coli and yeast . In addition, polyclonal anti-E . coli enzyme and antibodies to synthetic peptides copying several regions of the yeast enzyme reacted with the human gamma and rat beta isozymes . Antibodies to yeast SAM1 encoded protein residues 6-21, 87-113 and 87-124 inhibited the activity of human lymphocyte AdoMet synthetase, while antibodies to residues 272-287 had no effect on the enzyme activity . Our results suggest that these conserved regions may be important in enzyme activity.

Am J Obstet Gynecol, 1990 Sep, 163(3), 728 - 32
Transplacental transfer of zidovudine in the near-term pregnant baboon; Hankins GD et al.; Approximately one third of infants born to human immunodeficiency virus type 1 seropositive mothers have evidence of infection or of acquired immunodeficiency syndrome by the age of 18 months . One fifth of infected infants also have died by age 18 months . This prevalence, combined with the demonstration that zidovudine (formerly azidothymidine) can decrease mortality and the frequency of opportunistic infections in patients with acquired immunodeficiency syndrome or acquired immunodeficiency syndrome--related complex, may lead to increasing use of azidothymidine in pregnancy despite a paucity of information regarding its pharmacokinetics . To further investigate the distribution of azidothymidine and its inactive metabolite 5'-glucuronide azidothymidine in the mother, fetus, and amniotic fluid, 12 near-term pregnant baboons were given oral azidothymidine (21 mg/kg/day in four divided doses every 6 hours, equivalent to the usual nonpregnant human dose of 1500 mg/day) . Specimens of maternal blood, fetal arterial blood obtained by percutaneous umbilical cord blood sampling, and amniotic fluid were obtained after from one to 17 doses of azidothymidine . Azidothymidine levels were measured by radioimmunoassay with the INCSTAR commercial radioimmunoassay kit and using Escherichia coli beta-glucuronidase for determination of 5'-glucuronide azidothymidine levels . Paired analyses revealed significant concentration gradients between amniotic fluid, fetal serum, and maternal serum for both azidothymidine (p less than 0.019) and 5'-glucuronide azidothymidine (p less than 0.002) . The amniotic fluid 5'-glucuronide azidothymidine level increased with increasing doses of azidothymidine despite the fact that the maternal azidothymidine and 5'-glucuronide azidothymidine concentrations were unchanged . This accumulation of amniotic fluid 5'-glucuronide azidothymidine may provide a functional drug reservoir and contribute to the higher fetal concentrations of the medication and its metabolite . Alternatively, the higher fetal levels may represent slower clearance in the fetus than in the mother . Further studies appear warranted with respect to possible adverse fetal effects, especially bone marrow suppression with prolonged and chronic exposure to azidothymidine.

Mol Cell Biol, 1990 Sep, 10(9), 4795 - 806
Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica; Xuan JW et al.; Mutants affected at the LYS5 locus of Yarrowia lipolytica lack detectable dehydrogenase (SDH) activity . The LYS5 gene has previously been cloned, and we present here the sequence of the 2.5-kilobase-pair (kb) DNA fragment complementing the lys5 mutation . Two large antiparallel open reading frames (ORF1 and ORF2) were observed, flanked by potential transcription signals . Both ORFs appear to be transcribed, but several lines of evidence suggest that only ORF2 is translated and encodes SDH . (i) The global amino acid compositions of Saccharomyces cerevisiae SDH and of the putative ORF2 product are similar and that of ORF1 is dissimilar . (ii) An in-frame translational fusion of ORF2 with the Escherichia coli lacZ gene was introduced into yeast cells and resulted in a beta-galactosidase activity regulated similarly to SDH; no beta-galactosidase activity was obtained with an in-frame fusion of ORF1 with lacZ . (iii) The introduction of a stop codon at the beginning of ORF2 prevented SDH expression in yeast cells, whereas no phenotypic effect was observed when ORF1 translation was blocked.

Exp Cell Res, 1990 Sep, 190(1), 141 - 4
DNA single-strand breaks in adult and embryonic avian erythrocytes; Guy SP et al.; We have investigated the possibility that the reactivation rate of adult avian erythrocytes, which is slower than that of embryonic erythrocytes, after fusion with metabolically active cells, is due to a greater number of single-strand breaks (ssb) in the DNA of the former . We have assayed ssb by measuring the template activity of the erythrocyte nuclei for added Escherichia coli DNA polymerase . We have found that differences in the numbers of ssb within polymerase-accessible regions between adult and embryonic cells are within experimental error . We conclude that, unless very localized clusters of damage exist within the DNA (which would not be detectable by this or other techniques), the difference in reactivation rate is not attributable to differences in ssb numbers.

Mol Biol (Mosk), 1990 Sep-Oct, 24(5), 1230 - 40
{Directed reconstruction of the influenza virus hemagglutinin gene}; Petrenko VA et al.; A new approach to create chimeric genes by directed exchange of oligonucleotide fragments was developed . By oligonucleotide-directed mutagenesis a few deletion mutants of the influenza virus hemagglutinin (HA) gene were obtained . These variants of HA gene contain unique restriction sites in DNA regions coding for the A and B epitopes of the HA molecule . The obtained special vectors may be used for cloning DNA fragments coding for new amino acid sequences in internal sites of the HA gene.

Mol Microbiol, 1990 Sep, 4(9), 1523 - 33
Import-defective colicin B derivatives mutated in the TonB box; Mende J et al.; The pore-forming colicin B is taken up into Escherichia coli by a receptor and TonB-dependent process . The receptor and colicin B both contain a similar amino acid sequence, close to the N-terminal end, termed the TonB box . Point mutations were introduced into the TonB-box region of the colicin B structural gene cba resulting in colicin B derivatives which were partially or totally inactive against E . coli cells . All derivatives still bound to the receptor . An inactive derivative killed cells when translocated across the outer membrane by osmotic shock treatment, and formed pores in planar lipid bilayer membranes identical to the wild-type colicin . Some of the mutations were partially suppressed by mutations in the tonB structural gene . It was concluded that the TonB-box mutations define a region that is involved in the uptake of colicin B across the outer membrane.

Mol Microbiol, 1990 Sep, 4(9), 1465 - 75
How Escherichia coli RNA polymerase can negatively regulate transcription from a constitutive promoter; Duval-Valentin G et al.; We previously described the structures and functions of specific complexes between the bla promoter from Tn3 (present in pBR322) and RNA polymerase (RNAP), showing that, at excess RNAP, complexes can form in which one or two RNAPs bind to the same promoter (1:1 and 2:1 complexes) (Duval-Valentin and Ehrlich, 1988) . We report here that the 2:1 complex cannot be detected below 25 degrees C; above that temperature, a 1:1 complex forms at a rate one order of magnitude faster than that of the 2:1 complex, and above 30 degrees C, the amounts of both species become equal for RNAP/promoter ratio r30 less than or equal to r less than or equal to 70 . The 2:1 complex decays back to a 1:1 complex losing the last RNAP at a rate about three times that of the 1:1 complex decay . Functional assays of the complexes formed at excess RNAP show that both 1:1 and 2:1 complexes are immediately and permanently inhibited, even when the promoters are pre-incubated with ribonucleotide selections potentially enabling entrance into abortive cycling or formation of a stressed complex . We conclude that the inhibition step probably takes place in the complex formation pathway between RPi and RPo, at a novel stable intermediate isomer, RPj, formed above 25 degrees C . A possible mechanism of formation of the 2:1 complex is outlined . In vivo studies, in which r was modified by varying the bacterial growth rate, show a reduction of bla expression as r values are upshifted, specific to the bla promoter from Tn3.

Mol Microbiol, 1990 Sep, 4(9), 1443 - 54
Escherichia coli molecular genetic map (1000 kbp): update I; Medigue C et al.; The sequenced genes from Escherichia coli that are available in the EMBL library (release 21) have been localized on an updated and corrected version of the restriction map of the chromosome generated by Kohara et al . (1987) . One thousand kbp of sequenced DNA are incorporated in this update; this is equivalent to 23% of the total genome . The accuracy of the map is assessed, and it is corrected and updated where appropriate . A significant number of sites were missing from the original map, mainly involving two of the eight enzymes used by Kohara et al . (1987), ie . PvuII and EcoRV . The nucleotide environment of such missing sites was examined and, using an Artificial Intelligence approach, it appears that the site for these enzymes is sensitive to context effects . Several genes of known position on the E . coli chromosome could not be placed on the restriction map; this suggests that additional gaps are likely to exist on the restriction map, in addition to the original seven identified by Kohara et al . We have also obtained information about the probable direction of transcription of chromosomal genes with respect to the map . Most genes are transcribed in the same direction as the replication forks, particularly around oriC at 84 min.

Mol Microbiol, 1990 Sep, 4(9), 1433 - 8
Towards an understanding of the structural basis of 'forbidden' transport pathways in the Escherichia coli lactose carrier: mutations probing the energy barriers to uncoupled transport; King SC et al.; Recent progress in the analysis of mutants of the Escherichia coli lactose carrier function is reviewed, with special emphasis on the structural basis for energy barriers which prevent 'forbidden' conformational changes . Mutations which break down the barriers to forbidden isomerizations involving the binary carrier:sugar (CS) and carrier:proton (CH) complexes have been obtained in several laboratories . These mutants allow uncoupled transport of H+ or galactoside in the lactose carrier which normally couples cation and sugar movement in a 1:1 stoichiometry . These uncoupled mutants appear to be associated with changes in both sugar and cation recognition, suggesting that the physical interactions forming the basis for co-substrate recognition and uncoupling are not independently variable . By postulating that translocation involves transformation of the stable intermediate of the co-transport cycle to unstable transition state conformations of the carrier, it is possible to consider the consequences of mutagenesis in terms of transition state theory . Consistent with several experimental observations, the analysis predicts in each mutant the occurrence of more than one abnormality in the transport cycle (such as changes in sugar recognition, cation recognition or the coupling reaction) . We have called the general phenomenon a 'mutational double-effect' because any mutation which alters the Gibbs free energy change of one reaction in the transport cycle must affect the free energy change of at least one other reaction in this cycle.

Photochem Photobiol, 1990 Sep, 52(3), 533 - 40
Photobiological activity of 3,4'-dimethyl-8-methoxypsoralen, a linear furocoumarin with unusual DNA-binding properties; Palumbo M et al.; The furocoumarin derivative 3,4'-dimethyl-8-methoxypsoralen (DMe-8-MOP) exhibits remarkable antiproliferative activity, but is devoid of skin phototoxicity . To gain insight into this peculiar behaviour we investigated non-covalent and covalent binding of DMe-8-MOP to calf thymus DNA, along with DNA-synthesis inhibition and mutagenic activity . The non-covalent interaction of DMe-8-MOP with the nucleic acid is quite poor as shown by equilibrium dialysis, spectroscopic, chiroptical and hydrodynamic techniques . However, it exhibits relevant photobinding ability to DNA using both isolated nucleic acid samples and cellular systems . Unlike the large majority of congeners, DMe-8-MOP undergoes predominantly photochemical monoaddition to the double helical polynucleotide . Upon examination of the products obtained by enzymatic hydrolysis of DMe-8-MOP photomodified DNA, the formation of an unusual furan side adduct is proposed, which could account for the peculiar photochemical and photobiological properties of the 3,4'-dimethyl furocoumarin derivative.

Pharmazie, 1990 Sep, 45(9), 678 - 9
Some studies on the preservation of indometacin suspensions intended for ophthalmic use; Vulovic N et al.; Suspensions of indometacin (1% w/v), buffered to pH 5.6, may be satisfactorily preserved by 0.002% w/v phenylmercuric nitrate in the presence of hydroxypropylmethylcellulose (0.5% w/v), despite 90% of the preservative being adsorbed to the indometacin powder . Polyvinyl alcohol (1.4%) could be used as an alternative suspending agent.

J Inorg Biochem, 1990 Sep, 40(1), 23 - 35
The new antitumor compound, cis-{Pt(NH3)2(4-methylpyridine)Cl}Cl, does not form N7,N7-d(GpG) chelates with DNA . An unexpected preference for platinum binding at the 5'G in d(GpG); Lempers EL et al.; The reaction of the antitumor active agent cis-{Pt(NH3)2(4-mepy)Cl}Cl (4-mepy stands for 4-methylpyridine) with d(GpG) has been investigated by 1H magnetic resonance spectroscopy . Initially, two mononuclear complexes cis-Pt(NH3)2(4-mepy){d(GpG)-N7(1)} 1 and cis-Pt(NH3)2(4-mepy){d(GpG)-N7(2)} 2 are formed in an unexpected ratio 65:35, as determined by 1H NMR and enzymatic digestion techniques . Both products react further with a second equivalent of cis-{Pt(NH3)2(4-mepy)Cl}Cl forming the dinuclear platinum complex {cis-Pt(NH3)2(4-mepy)}2{mu-d(GpG)- N7(1),N7(2)} 3 . With {Pt(dien)Cl}Cl and {Pt(NH3)3Cl}Cl similar complexes are formed . No evidence was found for the formation of chelates cis-Pt(NH3)(4-mepy) {d(GpG)-N7(1),N7(2)}, which would be formed upon ammonia release from the mononuclear complexes 1 and 2 . Even addition of strong nucleophiles, like sodium diethyldithiocarbamate, thiourea, cysteine, or methionine, before or after reaction, do not induce the formation of a chelate . Under all conditions the N-donor ligands remain coordinated to Pt in 1,2 and 3 . In addition, the results of bacterial survival and mutagenesis experiments with E . coli strains show that the in vivo formation of bifunctional adducts in DNA, comparable to those induced by cis-Pt(NH3)2Cl2, by treatment of cells with cis-{Pt(NH3)2(4-mepy)Cl}Cl is unlikely . Also, a mechanism of binding and intercalation is not supported by experimental data . All experiments suggest that the mechanism of action of this new class of antitumor agents must be different from that of cis-Pt(NH3)2Cl2.

New Biol, 1990 Sep, 2(9), 818 - 27
The requirement of IHF protein for extrachromosomal replication of the Escherichia coli oriC in a mutant deficient in DNA polymerase I activity; Filutowicz M et al.; It is shown here that plasmids containing the replication origin of Escherichia coli (oriC) cannot replicate in an extrachromosomal state in E . coli cells with the polA1hip3 double mutation . This E . coli mutant is deficient in the polymerizing function of DNA polymerase I (Pol I) and is unable to produce functional IHF protein . The inability of the oriC minichromosomes to replicate in the absence of IHF is dependent on the absence of Pol I; cells with the polA+himA- or polA+hip- mutation, which are deficient in the alpha and beta subunits of the IHF heterodimer, respectively, can support replication of the oriC replicons . We propose that IHF-deficient cells utilize an alternative pathway of the DNA replication in which Pol I is required . In vitro DNA binding assays revealed that the IHF binding site resides between the oriC coordinates 110 and 122 and is adjacent to the DnaA "box" 1 . Within the area protected by IHF we found at least 1 out of 11 GATC methylation sites present in oriC . The consequences of lack of IHF protein binding to the oriC and the indirect effects of the IHF deficiency on the oriC replication are discussed.

New Biol, 1990 Sep, 2(9), 812 - 7
Participation of the Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE in autorepression of the P1 plasmid repA promoter; Tilly K et al.; The replicon of the low copy number plasmid P1 uses the three Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE for the efficient initiation of its DNA replication . The only P1-encoded protein required for plasmid replication is the initiator, RepA . Binding of RepA to the origin also represses the promoter for the repA gene, which is located within the origin . We found that repression is incomplete in E . coli strains with mutations in the dnaJ, dnaK, or grpE genes . Since there is no decrease in RepA concentration in the mutant strains, the mutations are likely to affect the protein-DNA or protein-protein reactions required for repression, thereby decreasing RepA binding at its promoter . We also showed that the deficit in repression can be overcome by providing excess RepA, implying that the mechanism of repression is not altered in the mutant strains . Since repression requires RepA binding to the origin, a binding deficit might account for the replication defect in the heat shock mutants.

J Biochem (Tokyo), 1990 Sep, 108(3), 488 - 93
Transmembrane signal transduction and osmoregulation in Escherichia coli: II . The osmotic sensor, EnvZ, located in the isolated cytoplasmic membrane displays its phosphorylation and dephosphorylation abilities as to the activator protein, OmpR; Tokishita S et al.; The EnvZ protein is presumably a membrane-located osmotic sensor, which specifically phosphorylates the activator protein, OmpR, involved in expression of the ompF and ompC genes in Escherichia coli . In this study, we developed an in vitro system for analyzing the intact form of the EnvZ protein located in the isolated cytoplasmic membrane . This particular form of the EnvZ protein exhibited its in vitro ability not only as to OmpR-phosphorylation but also OmpR-dephosphorylation . It was found that when a high concentration of a mono-cation (K+, Na-, or Li+) was present during the in vitro reactions, OmpR-dephosphorylation was preferentially inhibited and consequently the phosphorylated from of the OmpR protein was accumulated under the in vitro conditions used, although the K+ ion appears to be essential for the OmpR-phosphorylation reaction . Procaine, a local anesthetic, is known to affect the osmotic regulation of the ompF and ompC genes in vivo . In this study, procaine was also found to preferentially inhibit OmpR-dephosphorylation mediated by the EnvZ protein in vitro.

J Biochem (Tokyo), 1990 Sep, 108(3), 483 - 7
Transmembrane signal transduction and osmoregulation in Escherichia coli: I . Analysis by site-directed mutagenesis of the amino acid residues involved in phosphotransfer between the two regulatory components, EnvZ and OmpR; Kanamaru K et al.; Previously, the transfer of a phosphoryl group between the EnvZ and OmpR proteins, which are involved in expression of the ompF and ompC genes in response to the medium osmolarity, was demonstrated in vitro . In this study, the histidine (His) residue at position 243 of the EnvZ protein, and the aspartate (Asp) residues at positions 12 and 55 of the OmpR protein were changed, respectively, by means of site-directed mutagenesis . We characterized the mutant proteins in terms of not only their in vitro phosphotransfer reactions but also their in vivo osmoregulatory phenotypes . The mutant EnvZ protein was defective in its in vitro ability not only as to EnvZ-autophosphorylation but also OmpR-phosphorylation and OmpR-dephosphorylation . This particular mutant EnvZ protein seemed to exhibit null functions as to the in vivo osmoregulatory phenotype . The mutant OmpR protein with the amino acid change at position 12 was clearly phosphorylated in vitro, but at a very low rate as compared with the wild-type OmpR protein . In vitro phosphorylation of the mutant OmpR protein with the amino acid change at position 55 was more severely affected . This mutant OmpR protein appeared to exhibit null functions as to the in vivo osmoregulatory phenotype . These results suggest that the histidine residue at position 243 of the EnvZ protein and the aspartate residues at positions 12 and 55 of the OmpR protein are deeply involved in the phosphotransfer between the EnvZ and OmpR proteins.

J Biochem (Tokyo), 1990 Sep, 108(3), 388 - 92
Cloning and sequencing of the endo-cellulase cDNA from Robillarda sp . Y-20; Yoshigi N et al.; An endo-cellulase cDNA has been screened from a Robillarda sp . Y-20 cDNA expression library using polyclonal antibodies (immunoscreening) . Western blot analysis showed that recombinant CMCase I fused to beta-galactosidase with the molecular weight of approximately 150,000 was expressed in Escherichia coli Y1090 . Sequence analysis of the cloned cDNA revealed that it consisted of 1,136 nucleotides . By comparison of the amino acid sequence deduced from the cDNA and the N-terminal amino acid sequence of the purified protein (residues 1 to 18, determined by protein sequencing), the cDNA was found to lack 44 nucleotides at its 5' end corresponding to residues 1 to 15 . Therefore, the mature cellulase (CMCase I) was deduced to be composed of 375 amino acid residues and the molecular weight of its protein was calculated to be 41,004 . Yaguchi et al . reported that the N-terminal amino acid sequence of an endo-beta-1,4-glucanase (endo-cellulase) from Schizophyllum commune was homologous with the active site of various hen egg-white type lysozymes, and the homology offered evidence for a lysozyme-type mechanism in enzymatic hydrolysis of cellulase {Biochem . Biophys . Res . Commun . (1983) 116, 408-411} . Pentilla et al . also pointed out that some amino acid homology was found between endo-glucanase I from Trichoderma reesei and the lysozyme of the phage T4 {Gene (1986) 45, 253-263} . From the result of sequence alignment of the endo-cellulase from Robillarda sp . Y-20 and four kinds of lysozymes, there was a possibility that the endo-cellulase from Robillarda sp . Y-20 also hydrolyzes carboxymethyl cellulose by a lysozyme-type mechanism.

FEMS Microbiol Lett, 1990 Sep 1, 59(1-2), 31 - 4
The cloning and expression of a gene encoding haemolytic activity from the fish pathogen Renibacterium salmoninarum; Evenden AJ et al.; A gene encoding haemolytic activity from Renibacterium salmoninarum (strain PPD) was cloned into Escherichia coli using the cosmid vector pHC79, and subsequently subcloned on a 1.6 kbp SAlI fragment into pBR328 . Southern blot hybridisation revealed that a homologous sequence is found in other strains of R . salmoninarum.

FEMS Microbiol Lett, 1990 Sep 1, 59(1-2), 215 - 9
Cloning, nucleotide sequence and amplified expression of the gene encoding the extracellular metallo (Zn) DD-peptidase of Streptomyces albus G; Duez C et al.; The gene encoding the extracellular metallo (Zn) DD-peptidase of Streptomyces albus G has been cloned in Escherichia coli DH5 alpha MCR via pBR322 or 325, and then transferred into Streptomyces lividans TK24 via pIJ486, with substantial amplification of the expressed DD-peptidase . The gene has the information for the synthesis of a 255 amino acid precursor, the amino terminal region of which has the characteristic features of a signal peptide . The primary structure as deduced from nucleotide sequencing confirms that previously determined by chemical methods except for the occurrence of an Asp instead of Asn at position 1 and an additional Ala immediately downstream of Pro67.

FEMS Microbiol Lett, 1990 Sep 1, 59(1-2), 193 - 8
Cytolethal distending toxin (CLDT) production by enteropathogenic Escherichia coli (EPEC); Bouzari S et al.; Culture supernates of 16 strains of EPEC belonging to 6 different serogroups, when assayed on Chinese Hamster Ovary (CHO) cells up to 96-120 h, induced distinct morphological changes . The supernate activities were heat-labile, unrelated to heat-labile enterotoxin (LT), verotoxin (VT), or hemolysin, did not show necrosis in rabbit skin and caused no fluid secretion in the rabbit ileal loop assay (RILA) . Simultaneous production of CLDT and heat-stable enterotoxin (ST) were detected in four EPEC strains.

FEMS Microbiol Lett, 1990 Sep 1, 59(1-2), 173 - 7
Characterization of two plasmids arising spontaneously in phosphate-limited continuous cultures of Escherichia coli HB101{pAT153}; Brownlie L et al.; Two derivatives of plasmid pAT153 which arose spontaneously in phosphate-limited continuous cultures of Escherichia coli HB101{pAT153} have been characterised . The smaller plasmid, pLAB-446, resulted from a deletion of 446 base pairs of DNA covering the translation start and codon sequence of the tetracycline resistance gene . The other plasmid, pLAB135, differed from pAT153 only by an A:T to G:C transition in the transcribed but untranslated leader region of the tet gene which decreased tet transcription by 84%, possibly due to the formation of a more stable stem-loop structure in the mRNA.

Zhonghua Zheng Xing Shao Shang Wai Ke Za Zhi, 1990 Sep, 6(3), 164 - 6, 235
{Experimental study on pathogenesis of endotoxin-induced gut origin infection}; Ma L; It has been documented that endotoxin could induce gut origin infection . Consequently, experiments were performed to correlate endotoxin-induced gut origin infection with changes in intestinal mucosal structure and xanthine dehydrogenase and oxidase activity . Bacteria infection from the intestines to extraintestinal organs in 70% of the mice receiving endotoxin . Endotoxin injured primarily the ileal and cecal mucosa and increased ileal and hepatic xanthine dehydrogenase and cecal oxidase activities (P less than 0.05) . These results suggest that xanthine oxidase-induced mucosal damage plays a role in endotoxin-induced gut origin infection.

Appl Environ Microbiol, 1990 Sep, 56(9), 2915 - 8
Survival in seawater of Escherichia coli cells grown in marine sediments containing glycine betaine; Gauthier MJ et al.; Considering both the protective effect of glycine betaine (GB) on enteric bacteria grown at high osmolarity and the possible presence of GB in marine sediments, we have analyzed the survival, in nutrient-free seawater, of Escherichia coli cells incubated in sediments supplemented with GB or not supplemented and measured the efficiency of GB uptake systems and the expression of proP and proU genes in both seawater and sediments . We did this by using strains harboring proP-lacZ and proU-lacZ operon or gene fusions . We found that the uptake of GB and the expression of both proP and proU were very weak in seawater . The survival ability of cells in seawater supplemented with GB was a linear function of GB concentration, although the overall protection by the osmolyte was low . In sediments, proP expression was weak and GB uptake and proU expression were variable, possibly depending on the availability of organic nutrients . In a sediment with a high total organic carbon content, GB uptake was very high and proU expression was enhanced; cells previously incubated in this sediment showed a higher resistance to decay in seawater . GB might therefore play a significant role in the long-term maintenance of enteric bacterial cells in some marine sediments.

Izv Akad Nauk SSSR Biol, 1990 Sep-Oct, (5), 782 - 5
{The role of oxygen in the inhibition of Escherichia coli respiration by copper ions}; Lebedev VS et al.; Effect of Cu2+ on E . coli respiration and the role of oxygen in toxic action of copper has been studied . Stimulation of respiration is observed at initial time after introduction of Cu2+ . It is based on a nonspecific cell response to membrane damage . After finishing of transitory processes, Cu2+ influenced respiration by noncompetitive inhibition, i.e . copper-sensitive enzyme can be oxygenated or nonoxygenated, and only the latter form of the enzyme is inhibited.

Mol Gen Genet, 1990 Sep, 223(3), 508 - 12
The glutamate dehydrogenase structural gene of Escherichia coli; Helling RB; The glutamate dehydrogenase structural gene, gdhA, was mapped at 38.6 min on the genetic map and at 1860 kb on the physical map . A detailed map of this region is presented.

Mol Gen Genet, 1990 Sep, 223(3), 481 - 6
DNA sequence analysis of spontaneous histidine mutations in a polA1 strain of Escherichia coli K12 suggests a specific role of the GTGG sequence; Jankovic M et al.; Spontaneously arising histidine mutations in an Escherichia coli K12 strain deficient for DNA polymerase I were analysed at the DNA sequence level . We screened approximately 150,000 colonies and isolated 106 histidine auxotrophs . Of these, 98 were unstable hisC mutations; 12 representative mutants analysed were shown to have arisen by the excision of a single quadruplet repeat in the sequence 5'-GCTGGCTGGCTGGCTG-3' . Of the eight mutations at other sites, three hisA deletions and one hisD deletion occurred as a consequence of misalignment of tandemly repeated pentamers (hisD) or decamers (hisA) . A single hisA point mutation was found to be a missense mutation . Two extended deletions, covering the his operon were not analysed . We could not identify the hisC deletion by sequencing . We conclude that polA1 is a strong mutator that induces mutations mostly of the minus frameshift and deletion type by a Streisinger-type of mispairing in repetitive DNA sequences . Finally, the possible role of a 5'-GTGG-3' sequence and its inverted or direct complements, which are found in the vicinity of all the deletions and frameshifts, is discussed.

Mol Gen Genet, 1990 Sep, 223(3), 433 - 7
Mutant of the glutamine transfer RNA gene as UGA suppressor in Escherichia coli; Inokuchi H et al.; A UGA suppressor derived from a glutamine tRNA gene of Escherichia coli K12 was isolated and characterized . Phages carrying the suppressor su+2UGA could be obtained only from a hybrid transducing phage, h80cI857psu+2oc, but not from the original transducing phage lambda cI857psu+2oc . By DNA sequence analysis, it was found that the su+2 UGA suppressor obtained has two mutations; one is in the anticodon (TTA----TCA), as expected, and the other (C----T) is at the 7th position from the 3' end of tRNA(2Gln) . The significance of these mutations and the lethal effect on phage lambda of the increased amounts of UGA suppressor tRNAs are discussed.

Mol Gen Genet, 1990 Sep, 223(3), 379 - 84
Iron(III) hydroxamate transport of Escherichia coli: restoration of iron supply by coexpression of the N- and C-terminal halves of the cytoplasmic membrane protein FhuB cloned on separate plasmids; Koster W et al.; Transport of iron(III) hydroxamates across the inner membrane into the cytoplasm of Escherichia coli cells is mediated by the FhuC, FhuD and FhuB proteins . We studied the extremely hydrophobic FhuB protein (70 kDa) which is located in the cytoplasmic membrane . The N- and C-terminal halves of the protein {FhuB(N) and FhuB(C)} show homology to each other and to the equivalent polypeptides involved in uptake of ferric dicitrate and of vitamin B12 . Various plasmids carrying only one-half of the fhuB gene were expressed in fhuB- mutants . Only combinations of FhuB(N) and FhuB(C) polypeptides restored sensitivity to albomycin and growth on iron hydroxamates as sole iron source; no activity was obtained with either half of FhuB alone . These results indicate that both halves of FhuB are essential for substrate translocation and that they combine to form an active permease when expressed separately . In addition, a FhuB derivative with a large internal duplication of 271 amino acids was found to be partially active in transport, indicating that the extra portion did not perurb proper insertion of the active FhuB segments into the cytoplasmic membrane.

Mol Gen Genet, 1990 Sep, 223(3), 361 - 8
Possible involvement of the ugpA gene product in the stable maintenance of mini-F plasmid in Escherichia coli; Ezaki B et al.; The seg-3 mutant Escherichia coli does not support the maintenance of mini-F plasmid at 42 degrees C . We cloned the chromosomal DNA segment of the wild-type strain W3110 that complements the Seg- phenotype of this mutant . Cleavage mapping of this segment showed that it was derived from the 76-min region of the E . coli chromosome map . Complementation tests using plasmids carrying subcloned DNA segments suggested that the seg-3 mutant carried two mutations that additively affected the maintenance of mini-F plasmid; one was in the ugpA gene and the other was presumably in the rpoH gene . We generated a disrupted ugpA null mutant and found that the mini-F plasmid was unstable in this ugpA null mutant even at 30 degrees C . This suggests that the ugpA gene product is required for the stable maintenance of mini-F plasmid.

Indian J Med Res, 1990 Sep, 91, 386 - 92
Cloning & expression of chikungunya virus genes coding structural proteins in Escherichia coli; Ranadive SN et al.; DNA complementary to the single stranded RNA genome of Chikungunya (CHIK) virus with poly A tract was cloned into the plasmid pGEM-3Zf(-) and 5Zf(+) by blunt end ligation strategy . Clones containing the cDNA inserts were selected by X-gal, IPTG system . They were tested for the expression of structural protein(s) of CHIK virus by in situ enzyme immunoassay and Western blot . The former assay system showed the presence of expressed viral proteins . Analysis of Western blot shows that three structural proteins, E1, E2 and capsid (C) are expressed in Esch . coli . The molecular weights of envelope proteins E1 and E2 were 44-46 Kd and 42-44 Kd respectively, which are lesser than the actual molecular weights of virional proteins (50-52 Kd) . This may be due to the absence of glycosylation of these proteins in Esch . coli . In clone no . 382, a high molecular weight protein (56-58 Kd) was observed, which was probably the unglycosylated form of P62 polyprotein coded by the virus during its multiplication . A small protein of MW 6-8 Kd was also expressed in clone nos . 382 and 504, and this appeared to be the unglycosylated form of E3 protein of CHIK virus.

Indian J Med Res, 1990 Sep, 91, 368 - 71
Human enterocyte adhesion of enteroadherent Escherichia coli; Raj P et al.; Esch . coli strains manifesting localised (17), diffuse (8) or aggregative (17) phenotypes of adherence to HEp-2 cells were tested for their ability to adhere to human enterocytes isolated from duodenal biopsies of adult volunteers to obtain further evidence of their enteropathogenecity . Esch . coli strains H10407+; CFAI+ and LT+ STp+ STh+, F 294 B; a localised adherent strain positive with entero-adherent factor probe reported previously to attach to small intestinal enterocytes and F 582 C; LT- STp+ STh+ were the positive controls: H10407P (CFAI- mutant of H10407+) and K12 served as negative control strains . Adherence of variable degree was seen with 35.3 per cent of enteroaggregative Esch . coli (EAggEc) and with 58.8 per cent of enteroadherent Esch . coli localised (EAEC-L); EAEC-diffuse (EAEC-D) did not adhere to the human enterocytes . The possibility that EAgg EC and diffuse phenotypes may adhere better to lower small intestine or the large intestine, needs to be investigated.

AIDS Res Hum Retroviruses, 1990 Sep, 6(9), 1099 - 105
Antibodies to HIV-1 nef(p27): prevalence, significance, and relationship to seroconversion; Cheingsong-Popov R et al.; A sensitive and specific enzyme-linked immunoassay for antibodies to the human immunodeficiency virus type 1 (HIV-1) nef gene product, p27, has been developed using recombinant Escherichia coli-derived protein from the LAV-1-Bru sequence . Of 92 HIV-1 infected hemophiliacs, 72 (78%) produced anti-nef antibodies in this assay; the early appearance of anti-nef prior to full seroconversion was a rare event in this population, occurring in only one subject (approximately 1%) . Anti-nef antibodies were not detected in any of 500 sera from 98 repeatedly HIV seronegative subjects who had been exposed to sexually transmitted modes of HIV infection (45 subjects) or through blood products (53 subjects) . There was no significant association of titer or anti-nef antibody with protection from disease in HIV infection (p = 0.1) . Although the nef protein is relatively immunogenic in natural infection, this study cannot confirm the previously reported high prevalence of anti-nef antibodies prior to seroconversion, nor the finding of anti-nef antibodies in HIV seronegative but exposed subjects.

Clin Rheumatol, 1990 Sep, 9(3), 356 - 61
Follow-up with OM-8980 after a double-blind study of OM-8980 and auranofin in rheumatoid arthritis; Vischer TL; A 6-month double-blind study of OM-8980 and auranofin in 145 patients with rheumatoid arthritis was followed by an open observation period of 6 months for which 100 OM-8980-treated patients could be assessed . At the end of this second phase, the Ritchie index, number of swollen joints, pain scale, morning stiffness, grip strength and ESR had all improved further with respect to the significant improvements already recorded under OM-8980 and auranofin in the double-blind phase . The statistical analysis of the Ritchie index, pain scale and ESR showed significant changes in these 3 parameters during both the 6-month follow-up phase and the entire 12-month period . As regards the tolerance, 2 patients reported gastrointestinal disorders during the follow-up . The investigators' final assessment of efficacy indicated an improvement in 76% of the patients during the follow-up phase and in 95% during the entire 12-month period.

Vet Microbiol, 1990 Sep, 24(3-4), 391 - 408
Development of a recombinant Anaplasma marginale DNA probe; Aboytes-Torres R et al.; An Anaplasma marginale DNA probe has been developed by using an improved method for the isolation of genomic DNA . Purified genomic A . marginale DNA from the St . Croix isolate was partially digested with Sau 3A1 into fragments (greater than or equal to 5.0 kb) . The restriction fragments were cloned using standard techniques in the pBR322 vector and used to transform E . coli (DH5) host cells . The recombinant A . marginale DNA library was screened by the colony lifting procedure . Colonies containing plasmids with A . marginale DNA inserts were identified by hybridization with a genomic A . marginale DNA radiolabeled probe (32P) . Seven recombinant A . marginale DNA probes were evaluated by dot-blot in vitro hybridization assays to identify candidates as diagnostic tools in bovine anaplasmosis studies . Specificity and sensitivity experiments were carried out by using heterologous and homologous DNAs . The heterologous panel contained bovine DNA (WBC) and blood parasites DNA from Babesia bovis (Bb), Babesia bigemina (Bbi), Eperythrozoon suis (Es) and Eperythrozoon wenyoni (Ew) . The homologous DNA panel included A . marginale DNAs of 12 different isolates which were isolated in the Caribbean, Mexico, and the U.S.A . The selected diagnostic probe was identified as pSt . Croix A1, and labeled with 32P by using in vitro nick translation and random primer techniques . The pSt . Croix A1 probe demonstrated 100% specificity and high sensitivity by hybridization in dot blotting and Southern blotting . The probe can detect 500-1000 infected erythrocytes per microliters which corresponds to a parasitemia of less than 0.01% . The A . marginale DNA insert was approximately 6.4 kb in size and a partial restriction map has been constructed.

Mol Gen Genet, 1990 Sep, 223(2), 349 - 52
Integration host factor bends the DNA in the Escherichia coli ilvBN promoter region; Tsui P et al.; Integration host factor (IHF) of Escherichia coli is a site-specific DNA binding protein involved in a wide variety of physiological activities in E . coli and its phages and plasmids . We have previously found that IHF binds specifically to a site just upstream from the ilvBN promoter and strongly decreases transcriptional pausing and termination in the ilvBN leader . In this work we show by gel retardation analysis that IHF binds to bent ilvBN DNA and greatly enhances the bend located within or near the IHF binding site . These data are consistent with the hypothesis that IHF-induced alterations in the conformation of ilvBN promoter-leader DNA is a key to its antitermination activity in this system.

Mol Gen Genet, 1990 Sep, 223(2), 297 - 304
Successive binding of raf repressor to adjacent raf operator sites in vitro; Aslanidis C et al.; The raf repressor negatively regulates the transcription of the raf operon which encodes functions required for the uptake and hydrolysis of raffinose in Escherichia coli . Overexpression of the repressor gene under lac promoter control led to the formation of inclusion bodies . These were partially purified by centrifugation, solubilized in 0.1% SDS and reactivated by dilution . DNase I protection and gel retardation experiments demonstrated the specific binding of raf repressor to DNA fragments that contained the previously identified raf operator, an element comprising two 18 bp palindromic nucleotide sequences that flank the -35 raf promoter box . By using DNA fragments with one, two, or four copies of the 18 bp palindrome, these experiments revealed concentration dependent, successive occupation of all available binding sites by raf repressor . Melibiose released the repressor from the operator complexes, whereas raffinose and other alpha-galactosides did not, indicating that melibiose is the actual inducer in vivo . We suggest that successive occupation by repressor of two strategically located operator sites is a specific type of stepwise down-regulation of gene expression in response to repressor concentration.

Lymphology, 1990 Sep, 23(3), 145 - 8
The effect of anesthesia and surgery on diaphragmatic lymph vessel flow after endotoxin in sheep; Elk JR et al.; Increases in diaphragmatic lymph vessel flow (Qdi) may be important in preventing ascites because diaphragmatic lymph vessels drain the peritoneal space . However, lymphatic vessel function may be depressed in anesthetized, open chested animals . To test this hypothesis, we cannulated diaphragmatic lymph vessels in five sheep which were anesthetized with 1-2% halothane . We performed a thoracotomy and cannulated a diaphragmatic lymph vessel in each sheep . Then we infused 0.75-1.0 micrograms/kg of E . coli endotoxin intravenously and we measured Qdi and the lymph protein concentration for 2-4 hrs . The data were compared to previously reported data for five unanesthetized sheep (J . Appl . Physiol . 62:706-710, 1987) . At baseline Qdi = 0.8 +/- 0.7 (SD) in the anesthetized sheep and 1.0 +/- 0.8 ml/hr in the unanesthetized sheep . After endotoxin, Qdi increased to 4.5 +/- 3.1 ml/hr in the unanesthetized sheep (p less than 0.05) but Qdi did not change in the anesthetized sheep . However, the lymph protein concentration increased similarly in each group, indicating that endotoxin caused the same degree of injury in each group . Our results indicate that diaphragmatic lymph vessel function is depressed in anesthetized, open chested sheep.

Zentralbl Bakteriol, 1990 Sep, 273(4), 492 - 500
In vitro cytotoxic effect of alpha-hemolytic Escherichia coli on human blood granulocytes . Inhibition by alpha-hemolysin antibody; Gadeberg OV et al.; The influence of alpha-hemolysin antibody on the in vitro cytotoxic effect of alpha-hemolytic Escherichia coli bacteria and culture filtrates was investigated . Damage to human blood granulocytes was quantified by measuring the release of chromium 51 from labelled cells in the presence of whole or fractionated plasma containing alpha-hemolysin antibody . Anti-alpha-hemolysin activity was found exclusively in the IgG fraction of plasma . Human plasma contained "natural" alpha-hemolysin antibody to various titers . Vaccination of rabbits resulted in only modest rises in alpha-hemolysin antibody titers . The cytotoxic effect of alpha-hemolytic E . coli bacteria as well as that of bacterial culture filtrates was reduced in a titer-dependent way in the presence of plasma containing alpha-hemolysin antibody . These results indicate that the cytotoxic effect of alpha-hemolytic E . coli is inhibited by alpha-hemolysin antibody.

Somat Cell Mol Genet, 1990 Sep, 16(5), 477 - 86
Replication-dependent mutagenesis by 5-bromodeoxyuridine: identification of base change and sequence effects on mutability; Xu FM et al.; The molecular mechanism of reversion induced by 5-bromodeoxyuridine (BrdU) replication-dependent mutagenesis in mammalian cells was studied . Murine cells with single mutant copies of the E . coli gpt gene integrated chromosomally as part of a shuttle vector were mutagenized with BrdU, and GPT+ revertants were selected . Thirteen mutant cell lines (each of which had a gpt gene that varied from the wild-type gene by a different GC----AT base transition in the coding region) were mutagenized, and only four were found to be effectively reverted . All revertant gpt genes that were analyzed had reverted via AT----GC base transition at the original site of mutation, thus demonstrating that replication-dependent mutagenesis by BrdU causes AT----GC transitions . The nine cell lines that were nonrevertible by BrdU replication-dependent mutagenesis could be mutated by this protocol to ouabain resistance as effectively as the four revertible lines, indicating that the nonrevertible lines were susceptible to such mutagenesis . Thus, differences among the cell lines in frequencies of HATr revertants generated by BrdU replication-dependent mutagenesis could not be attributed to differences in general susceptibility of the lines to the mutagenic protocol . The revertible and nonrevertible lines could not be separated according to the position of the original GC----AT transition in the gpt coding region . However, there was evidence that the DNA base sequence flanking the site of mutation affected the susceptibility of that site to BrdU replication-dependent mutagenesis . For example, six of the cell lines tested had gpt genes in which the mutant T residue was immediately adjacent on its 3' side to an A residue, and all six were found to be nonrevertible by BrdU replication-dependent mutagenesis . Furthermore, a target AT base pair flanked by GC base pairs in opposite orientation and either immediately adjacent to or one base removed from the target site on both the 5' and 3' sides appeared to have an increased susceptibility to BrdU replication-dependent mutagenesis.

J Infect, 1990 Sep, 21(2), 191 - 3
Infected cephalhaematoma and neonatal osteomyelitis; Lee PY; Neonatal osteomyelitis of the skull secondary to cephalhaematoma has been reported infrequently . The case reported here is that of a 7-week-old infant with a previously resolving cephalhaematoma who presented with cranial osteomyelitis . Cephalhaematomas should be considered potential sites of infection in any infant, even without a history of scalp trauma.

Gene, 1990 Sep 1, 93(1), 129 - 34
The pAX plasmids: new gene-fusion vectors for sequencing, mutagenesis and expression of proteins in Escherichia coli; Markmeyer P et al.; A family of plasmid cloning vectors have been constructed, allowing both the sequencing and mutagenesis of foreign genes and the easy isolation of their expression products via fusion proteins in Escherichia coli . Fusion proteins can be inducibly expressed and isolated by affinity chromatography on APTG-Sepharose . The fusion protein consists of beta-galactosidase at the N-terminus, linked by a collagen 'hinge' region containing blood coagulation factor Xa cleavage site to the foreign protein at the C terminus . The factor Xa cleavage site at the N-terminal side of the foreign protein allows the release of the desired amino acid sequence under mild conditions . A multiple cloning site in all three reading frames and stop codons followed by the strong lambda t0 terminator facilitate simple gene insertions and manipulations . The intergenic region of the phage f1 inserted in both orientations allows the isolation of single-stranded DNA from either plasmid-strand for sequencing and mutagenesis . This vector family has been successfully used for the expression and purification of the isoleucyl-tRNA synthetase from Saccharomyces cerevisiae and the histidyl-tRNA synthetase from E . coli.

Genetics, 1990 Sep, 126(1), 5 - 16
Spontaneous point mutations that occur more often when advantageous than when neutral; Hall BG; Recent reports have called into question the widespread belief "that mutations arise continuously and without any consideration for their utility" (in the words of J . Cairns) and have suggested that some mutations (which Cairns called "directed" mutations) may occur as specific responses to environmental challenges, i.e., they may occur more often when advantageous than when neutral . In this paper it is shown that point mutations in the trp operon reverted to trp+ more frequently under conditions of prolonged tryptophan deprivation when the reversions were advantageous, than in the presence of tryptophan when the reversions were neutral . The overall mutation rate, as determined from the rates of mutation to valine resistance and to constitutive expression of the lac operon, did not increase during tryptophan starvation . The trp reversion rate did not increase when the cells were starved for cysteine for a similar period, indicating that the increased reversion rate was specific to conditions where the reversions were advantageous . Two artifactual explanations for the observations, delayed growth of some preexisting revertants and cryptic growth by some cells at the expense of dying cells within aged colonies, were tested and rejected as unlikely . The trp+ reversions that occurred while trp- colonies aged in the absence of tryptophan were shown to be time-dependent rather than replication-dependent, and it is suggested that they occur by mechanisms different from those that have been studied in growing cells . A heuristic model for the molecular basis of such mutations is proposed and evidence consistent with that model is discussed . It is suggested that the results in this and previous studies can be explained on the basis of underlying random mechanisms that act during prolonged periods of physiological stress, and that "directed" mutations are not necessarily the basis of those observations.

Genetics, 1990 Sep, 126(1), 17 - 24
Context effects in the formation of deletions in Escherichia coli; Kazic T et al.; We have examined the frequency with which identical deletions are formed in different chromosomal contexts . A panel of six mutant bla genes containing palindrome/direct repeat structures were moved from pBR322 to three locations: at lambda att, at chromosomal lac, and at F'lac . Deletion of the palindromes and one of the direct repeats results in reversion to Ampr . The frequency of deletion for all alleles declines beyond the reduction in copy number when they are moved from the multicopy plasmid environment to a single-copy chromosome . The magnitude of the declines varies in an allele-specific and location-specific manner . Our data support the hypothesis that context can influence the frequency of mutation independent of the immediate DNA sequence.

J Gen Virol, 1990 Sep, 71 ( Pt 9), 2157 - 62
Terminal redundancy and circular permutation of mycoplasma virus L3 DNA; Just W et al.; This communication reports the physical map of mycoplasma virus L3 (MV-L3) DNA derived from restriction patterns obtained by digestion with seven different restriction endonucleases . The length of the restriction map is 36,200 bp in contrast to the contour length of native MV-L3 DNA molecules which is 39,400 bp as determined by electron microscopy . The difference in length of 3,200 bp (corresponding to 8.1% of the native viral DNA contour length) is explained by terminal redundancy . It was possible to clone all fragments from particular restriction patterns into Escherichia coli vector pAT153, an indication of circular permutation within a population of MV-L3 DNA . However clear evidence has been obtained from the molar ratios of fragments and from hybridization experiments . We suppose that viral DNA is packaged from a concatemeric precursor molecule starting at a specific site called pac.

Circ Shock, 1990 Sep, 32(1), 77 - 81
Effects of endothelin on the lethality induced by platelet-activating factor or endotoxin in the mouse; Etienne A et al.; Platelet-activating factor (PAF) is a mediator that decreases cardiac output and total peripheral resistances leading to profound hypotension . It seems to be involved in shock states and in the deleterious effects of endotoxin . As the natural peptide endothelin (ET) shows potent vasoconstrictor properties, we evaluate its activity towards PAF and endotoxin-induced lethality in mice . ET, at doses which per se did not exert any effects on mice vitality, produced a dose-dependent decrease in the PAF-induced lethality with a total protection at 5 micrograms/kg . But conversely to these results, ET potentiated the mortality induced by endotoxin . These results suggest that ET or endothelin analogs could be worthy for therapeutic use in some types of shock, at least when endotoxin is not involved . Complementary studies are necessary to strengthen these preliminary results.

Biochem J, 1990 Sep 1, 270(2), 357 - 61
Preparation and characterization of human interleukin-5 expressed in recombinant Escherichia coli; Proudfoot AE et al.; The gene coding for human interleukin-5 was synthesized and expressed in Escherichia coli under control of a heat-inducible promoter . High-level expression, 10-15% of total cellular protein, was achieved in E . coli . The protein was produced in an insoluble state . A simple extraction, renaturation and purification scheme is described . The recombinant protein was found to be a homodimer, similar to the natural murine-derived protein . Despite the lack of glycosylation, high specific activities were obtained in three 'in vitro' biological assays . Physical characterization of the protein showed it to be mostly alpha-helical, supporting the hypothesis that a conformational similarity exists among certain cytokines.

Biochem J, 1990 Sep 1, 270(2), 319 - 23
Modification of uridine phosphorylase from Escherichia coli by diethyl pyrocarbonate . Evidence for a histidine residue in the active site of the enzyme; Drabikowska AK et al.; Uridine phosphorylase from Escherichia coli is inactivated by diethyl pyrocarbonate at pH 7.1 and 10 degrees C with a second-order rate constant of 840 M-1.min-1 . The rate of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.6 . Hydroxylamine added to the inactivated enzyme restores the activity . Three histidine residues per enzyme subunit are modified by diethyl pyrocarbonate . Kinetic and statistical analyses of the residual enzymic activity, as well as the number of modified histidine residues, indicate that, among the three modifiable residues, only one is essential for enzyme activity . The reactivity of this histidine residue exceeded 10-fold the reactivity of the other two residues . Uridine, though at high concentration, protects the enzyme against inactivation and the very reactive histidine residue against modification . Thus it may be concluded that uridine phosphorylase contains only one histidine residue in each of its six subunits that is essential for enzyme activity.

Mutat Res, 1990 Sep-Nov, 236(2-3), 203 - 11
Structure and function of the (A)BC excinuclease of Escherichia coli; Selby CP et al.; (A)BC excinuclease is the enzymatic activity resulting from the mixture of E . coli UvrA, UvrB and UvrC proteins with damaged DNA . This is a functional definition as new evidence suggests that the three proteins never associate in a ternary complex . The UvrA subunit associates with the UvrB subunit in the form of an A2B1 complex which, guided by UvrA's affinity for damaged DNA binds to a lesion in DNA and delivers the UvrB subunit to the damaged site . The UvrB-damaged DNA complex is extremely stable (t1/2 congruent to 100 min) . The UvrC subunit, which has no specific affinity for damaged DNA, recognizes the UvrB-DNA complex with high specificity and the protein complex consisting of UvrB and UvrC proteins makes two incisions, the 8th phosphodiester bond 5' and the 5th phosphodiester bond 3' to the damaged nucleotide . (A)BC excinuclease recognizes DNA damage ranging from AP sites and thymine glycols to pyrimidine dimers, and the adducts of psoralen, cisplatinum, mitomycin C, 4-nitroquinoline oxide and interstrand crosslinks.

Proc Natl Acad Sci U S A, 1990 Sep, 87(17), 6798 - 802
Cloning and cDNA sequence of a bovine submaxillary gland mucin-like protein containing two distinct domains; Bhargava AK et al.; A lambda gt11 cDNA library prepared from bovine submaxillary gland mRNA was screened with polyclonal anti-apo-bovine submaxillary mucin antibodies with the aim of obtaining the deduced amino acid sequence of the mucin core protein . One of the positive clones had a 1.8 kilobase (kb) cDNA insert and coded for an incomplete protein . A 2.0-kb cDNA clone was isolated by rescreening the library with the 1.8-kb cDNA . Nucleotide sequencing of the full-length 2.0-kb cDNA revealed an open reading frame that coded for a 563-amino acid protein . A striking feature of the cloned protein is the skewed distribution of the amino acids, most notably that of the hydroxy amino acids and cysteine . The amino-terminal domain of 339 residues is very rich in threonine, serine, and glycine and poor in cysteine, aspartic acid, tyrosine, phenylalanine, and tryptophan . In contrast, the carboxyl-terminal domain of 224 residues is rich in cysteine, aspartic acid, tyrosine, lysine, and asparagine and relatively poor in threonine, serine, and glycine . A search of the protein data bank for homologies to the deduced amino acid sequence revealed statistically significant matches to several proteins, including the porcine submaxillary apomucin fragment . The cysteine-rich domain by itself was not statistically homologous with any of the registered polypeptide sequences . RNA blot analysis using DNA probes corresponding to the mucin-like and cysteine-rich regions detected a nearly identical pattern of transcripts, demonstrating that the characterized clones are not artifacts of cDNA library construction . The blots also showed the presence of polydisperse transcripts in bovine submaxillary gland but no detectable hybridization signals in liver or brain RNA.

Proc Natl Acad Sci U S A, 1990 Sep, 87(17), 6659 - 63
Substrate specificity of trypsin investigated by using a genetic selection; Evnin LB et al.; The structural determinants of the primary substrate specificity of rat anionic trypsin were examined by using oligonucleotide-directed mutagenesis coupled to a genetic selection . A library was created that encoded trypsins substituted at amino acid positions 189 and 190 at the base of the substrate binding pocket . A genetic selection, with a dynamic range of 5 orders of proteolytic activity, was used to search 90,000 transformants of the library . Rapid screening for arginyl amidolysis and esterolysis confirmed the activity of the purified isolates . Trypsin and 15 mutant trypsins with partially preserved function were identified and characterized kinetically on arginyl and lysyl peptide substrates . Alternative arrangements of amino acids in the substrate binding pocket sustained efficient catalysis . A negative charge at amino acid position 189 or 190 was shown to be essential for high-level catalysis . With the favored aspartic acid residue at position 189, several amino acids could replace serine at position 190 . Modulation of the specificity for arginine and lysine substrates was shown to depend on the amino acid at position 190 . The regulatory effect of the amino acid side chain at position 190 on the substrate specificity is also reflected in substrate binding pockets of naturally occurring trypsin homologs.

Proc Natl Acad Sci U S A, 1990 Sep, 87(17), 6644 - 8
Structure-based design of nonpeptide inhibitors specific for the human immunodeficiency virus 1 protease; DesJarlais RL et al.; By using a structure-based computer-assisted search, we have found a butyrophenone derivative that is a selective inhibitor of the human immunodeficiency virus 1 (HIV-1) protease . The computer program creates a negative image of the active site cavity using the crystal structure of the HIV-1 protease . This image was compared for steric complementarity with 10,000 molecules of the Cambridge Crystallographic Database . One of the most interesting candidates identified was bromperidol . Haloperidol, a closely related compound and known antipsychotic agent, was chosen for testing . Haloperidol inhibits the HIV-1 and HIV-2 proteases in a concentration-dependent fashion with a Ki of approximately 100 microM . It is highly selective, having little inhibitory effect on pepsin activity and no effect on renin at concentrations as high as 5 mM . The hydroxy derivative of haloperidol has a similar effect on HIV-1 protease but a lower potency against the HIV-2 enzyme . Both haloperidol and its hydroxy derivative showed activity against maturation of viral polypeptides in a cell assay system . Although this discovery holds promise for the generation of nonpeptide protease inhibitors, we caution that the serum concentrations of haloperidol in normal use as an antipsychotic agent are less than 10 ng/ml (0.03 microM) . Thus, concentrations required to inhibit the HIV-1 protease are greater than 1000 times higher than the concentrations normally used . Haloperidol is highly toxic at elevated doses and can be life-threatening . Haloperidol is not useful as a treatment for AIDS but may be a useful lead compound for the development of an antiviral pharmaceutical.

Laryngoscope, 1990 Sep, 100(9), 995 - 1000
Structural changes in the round window membrane following exposure to Escherichia coli lipopolysaccharide and hydrocortisone; Spandow O et al.; The present study focused on structural changes of the round window membrane (RWM) from agents that evoke transient or permanent impairment of the auditory brainstem response when applied into the RW niche . Escherichia coli (E . coli) lipopolysaccharide (LPS) in sterile water (SW) and a 2% suspension of hydrocortisone (CORT), micronized in SW, were instilled into the round window (RW) niche of Sprague-Dawley rats . The morphology of the RWM was analyzed 3 to 21 days after instillation of either substance . Both substances caused minor structural alterations at the light microscopic level . The RWM showed a slight thickening and an invasion of inflammatory cells . At the ultrastructural level, the CORT-treated specimens showed an increased epithelial height and numerous microvilli, whereas the epithelium of the LPS-treated specimens was extended and contained few microvilli resembling those in the normal RWM . We postulate that the RWM may undergo dynamic structural changes when exposed to various agents . The structural alterations per se can influence the passage of substances from the middle ear to the inner ear.

J Surg Res, 1990 Sep, 49(3), 256 - 61
Tumor necrosis factor production by Kupffer cells requires protein kinase C activation; Bankey P et al.; Tumor necrosis factor (TNF) has been proposed as a primary inflammatory mediator of septic shock . In vitro and in vivo studies indicate that endotoxin- or lipopolysaccharide (LPS)-activated macrophages are a principle source of TNF; however, membrane signal transduction and intracellular pathways by which LPS triggers TNF production in macrophages are unclear . Recent evidence indicates that specific protein phosphorylation via activation of protein kinase C (PKC) is an early, critical step in the signaling of macrophage TNF production by phorbol esters . We hypothesize that PKC activation is also required in LPS-signaled Kupffer cell (KC) TNF production . Murine KCs were obtained by liver perfusion and digestion and then stimulated with LPS (Escherichia coli O111:B4) or LPS in the presence of H-7, a selective PKC inhibitor . Conditioned media was collected at 3 hr for assay of TNF utilizing the L929 cytolysis bioassay standardized to murine-rTNF-alpha . We found that H-7 inhibited significantly LPS signaled TNF release at a concentration of 10 microM, while H-8 (a cyclic nucleotide specific inhibitor) had no effect . The effect of H-7 was dose dependent and present at varying concentrations of LPS . Down regulation of PKC activity by preincubation of KCs with phorbol myristate acetate (PMA, a direct activator of PKC) also resulted in significantly reduced TNF release after LPS stimulation . The inhibitor H-7 (10 microM) also significantly inhibited LPS signaled prostaglandin E2 release in Kupffer cells . Total and specific intracellular protein phosphorylation was determined by trichloroacetic acid precipitation and SDS-polyacrylamide gel electrophoresis after labeling stimulated Kupffer cells with 32Pi . Total protein phosphorylation was not significantly altered by LPS stimulation; however, autoradiograms from PMA- and LPS-stimulated KCs demonstrate enhanced phosphorylation of a 40-kDa protein (2.7 +/- 0.9-fold) and a 33-kDa protein (3.1 +/- 1.0-fold) which were inhibited by H-7 . We conclude that activation of PKC and protein phosphorylation are required steps in the signal transduction pathway of LPS-stimulated TNF production in Kupffer cells.

J Clin Endocrinol Metab, 1990 Sep, 71(3), 740 - 7
Modulation of HLA-DR expression in epithelial cells by interleukin 1 and estradiol-17 beta; Tabibzadeh SS et al.; Both ultrapure human interleukin-1 (IL-1) and Escherichia coli derived recombinant IL-1 alpha and beta consistently induced the expression of major histocompatibility class II (HLA-DR) molecules in a human endometrial and a breast carcinoma cell line . {35S}Methionine incorporation into IL-1 induced, immunoprecipitable HLA-DR molecules demonstrated de novo synthesis of both light and heavy chains of the HLA-DR molecules . Lipopolysaccharide, recombinant interleukin-2 and recombinant interleukin-6 failed to induce HLA-DR expression in these epithelial cells . In contrast to the dramatic effect on HLA-DR expression, IL-1 had no effect on the epithelial cell proliferation . Pretreatment of T47D cells with estradiol-17 beta significantly decreased the IL-1 induced HLA-DR expression, and pretreatment of IL-1 with an IL-1 specific antibody, neutralized IL-1 action . These studies demonstrate that a cytokine (IL-1) and a sex steroid hormone estradiol-17 beta can interact to regulate the expression of HLA-DR molecules in epithelial cells.

J Bacteriol, 1990 Sep, 172(9), 5514 - 5
Oxygen regulation of L-1,2-propanediol oxidoreductase activity in Escherichia coli; Cabiscol E et al.; Regardless of the respiratory conditions of the culture, Escherichia coli synthesizes an active propanediol oxidoreductase . Under anaerobic conditions, the enzyme remained fully active and accomplished its physiological role, while under aerobic conditions, it was inactivated in a process that did not depend on protein synthesis or on the presence of a carbon source.

J Bacteriol, 1990 Sep, 172(9), 5490 - 3
The heat-stable toxin I gene from Escherichia coli 18D; Dallas WS; The heat-stable toxin I gene in the human Escherichia coli isolate 18D is the estA1 allele . The gene is not part of a composite transposon, but inspection of the flanking DNA sequences suggests that it was at one time part of a transposon . The hypothetical transposon originated from an event other than the occurrence that formed Tn1681.

J Bacteriol, 1990 Sep, 172(9), 5374 - 81
Reconstitution of an active lactose carrier in vivo by simultaneous synthesis of two complementary protein fragments; Wrubel W et al.; Escherichia coli lactose permease mediates the proton-driven translocation of galactosides across the cytoplasmic membrane . To define regions important for membrane insertion as well as for biological function, we constructed plasmids encoding different portions of the lactose carrier . Among several lacY deletions, two were obtained that encoded mutant proteins with complementary amino acid sequences . The truncated polypeptide Y71/1 (amino acid residues 1 to 71) comprises the first two alpha-helices predicted for the intact protein, and polypeptide delta Y4-69 carries an internal deletion of this region . Regulated coexpression of these lacY-DNA segments governed by separate but identical lacOP control regions resulted in functional complementation with the following characteristics . (i) Simultaneous synthesis of both incomplete proteins restored transport activity in transport-negative cells, measured as accumulation of {14C}lactose . (ii) Under complementing conditions, but not in the absence of the smaller N-terminal protein, specific radiolabeling of the larger polypeptide by N-ethylmaleimide was prevented by substrate . (iii) The presence of the complementing N-terminal polypeptide was also required for the detection of the larger C-terminal protein by antibodies directed against the C terminus of lactose permease, indicating a stabilizing effect contributed by the smaller N-terminal fragment . Thus, coexpression of lacY mutant genes encoding two nonoverlapping portions of the lactose carrier resulted in reconstitution of a two-subunit protein in the cytoplasmic membrane exhibiting biological properties of intact lactose permease.

J Bacteriol, 1990 Sep, 172(9), 5278 - 85
Uracil-DNA glycosylase causes 5-bromodeoxyuridine photosensitization in Escherichia coli K-12; Yamamoto Y et al.; An Escherichia coli uracil-DNA glycosylase-defective mutant (ung-1 thyA) was more resistant than its wild-type counterpart (ung+ thyA) to the killing effect of UV light when cultured in medium containing 5-bromouracil or 5-bromo-2'-deoxyuridine (BrdUrd) . The phenotype of resistance to BrdUrd photosensitization and the uracil-DNA glycosylase deficiency appeared to be 100% cotransduced by P1 phage . During growth with BrdUrd, both strains exhibited similar growth rates and 5-bromouracil incorporation into DNA . The resistant phenotype of the ung-1 mutant was observed primarily during the stationary phase . In cells carrying 5-bromouracil-substituted DNA, mutations causing resistance to rifampin and valine were induced by UV irradiation at a higher frequency in the wild type than in the ung-1 mutant . This Ung-dependent UV mutagenesis required UmuC function . These results suggest that the action of the uracil-DNA glycosylase on UV-irradiated 5-bromouracil-substituted DNA produces lethal and mutagenic lesions . The BrdUrd photosensitization-resistant phenotype allowed us to develop a new, efficient method for enriching and screening ung mutants.

J Bacteriol, 1990 Sep, 172(9), 5260 - 5
Synthesis of Escherichia coli heat-stable enterotoxin STp as a pre-pro form and role of the pro sequence in secretion; Okamoto K et al.; Escherichia coli heat-stable enterotoxin STp is presumed from its DNA sequence to be synthesized in vivo as a 72-amino-acid residue precursor that is cleaved to generate mature STp consisting of the 18 carboxy-terminal amino acid residues . There are two methionine residues in the inferred STp sequence in addition to the methionine residue at position 1 . In order to confirm production of the STp 72-amino-acid residue precursor, we substituted the additional methionine residues by oligonucleotide-directed site-specific mutagenesis . Since these substitutions did not cause a significant change in STp production, it can be concluded that STp is normally synthesized as the 72-amino-acid residue precursor . The length of the STp precursor indicated the existence of a pro sequence between the signal peptide and the mature protein . In order to identify the pro sequence and determine its role in protein secretion, deletion and fusion proteins were made . A deletion mutant in which the gene fragment encoding amino acid residues 22 to 53 of STp was removed was made . STp activity was found in the culture supernatant of cells . Amino acid sequence analysis of the purified STp deletion mutant revealed that the pro sequence encompasses amino acid residues 20 to 54 . A hybrid protein consisting of STp amino acids 1 to 53 fused in frame from residue 53 to nuclease A was not secreted into the culture supernatant . These results indicate that the pro sequence does not function to guide periplasmic protein into the extracellular milieu.

J Bacteriol, 1990 Sep, 172(9), 5218 - 24
Repellents for Escherichia coli operate neither by changing membrane fluidity nor by being sensed by periplasmic receptors during chemotaxis; Eisenbach M et al.; A long-standing question in bacterial chemotaxis is whether repellents are sensed by receptors or whether they change a general membrane property such as the membrane fluidity and this change, in turn, is sensed by the chemotaxis system . This study addressed this question . The effects of common repellents on the membrane fluidity of Escherichia coli were measured by the fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene in liposomes made of lipids extracted from the bacteria and in membrane vesicles . Glycerol, indole, and L-leucine had no significant effect on the membrane fluidity . NiSO4 decreased the membrane fluidity but only at concentrations much higher than those which elicit a repellent response in intact bacteria . This indicated that these repellents are not sensed by modulating the membrane fluidity . Aliphatic alcohols, on the other hand, fluidized the membrane, but the concentrations that elicited a repellent response were not equally effective in fluidizing the membrane . The response of intact bacteria to alcohols was monitored in various chemotaxis mutants and found to be missing in mutants lacking all the four methyl-accepting chemotaxis proteins (MCPs) or the cytoplasmic che gene products . The presence of any single MCP was sufficient for the expression of a repellent response . It is concluded (i) that the repellent response to aliphatic alcohols can be mediated by any MCP and (ii) that although an increase in membrane fluidity may take part in a repellent response, it is not the only mechanism by which aliphatic alcohols, or at least some of them, are effective as repellents . To determine whether any of the E . coli repellents are sensed by periplasmic receptors, the effects of repellents from various classes on periplasm-void cells were examined . The responses to all the repellents tested (sodium benzoate, indole, L-leucine, and NiSO4) were retained in these cells . In a control experiment, the response of the attractant maltose, whose receptor is periplasmic, was lost . This indicates that these repellents are not sensed by periplasmic receptors . In view of this finding and the involvement of the MCPs in repellent sensing, it is proposed that the MCPs themselves are low-affinity receptors for the repellents.

J Bacteriol, 1990 Sep, 172(9), 5097 - 102
The native form of FtsA, a septal protein of Escherichia coli, is located in the cytoplasmic membrane; Pla J et al.; Antisera able to recognize FtsA, one of the septal proteins of Escherichia coli, have been obtained and used to show that native FtsA, when expressed at levels ranging from physiological to induced from lambda pR, is located in the inner membrane . Experiments of trypsin accessibility to FtsA in membranes, spheroplasts, and vesicles indicated that FtsA is located such that it faces the cytoplasm . This location is consistent with current knowledge about the participation of FtsA in a molecular complex active in cell division called septator.

J Bacteriol, 1990 Sep, 172(9), 5001 - 10
Rhodobacter capsulatus genes involved in early steps of the bacteriochlorophyll biosynthetic pathway; Yang ZM et al.; Three open reading frames in the Rhodobacter capsulatus photosynthesis gene cluster, designated F0, F108, and F1025, were disrupted by site-directed mutagenesis . Mutants bearing insertions in these reading frames were defective in converting protoporphyrin IX to magnesium-protoporphyrin monomethyl ester, protochlorophyllide to chlorophyllide a, and magnesium-protoporphyrin monomethyl ester to protochlorophyllide, respectively . These results demonstrate that the genes examined most likely encode enzyme subunits that catalyze steps common to plant and bacterial tetrapyrrole photopigment biosynthetic pathways . The open reading frames were found to be part of a large 11-kilobase operon that encodes numerous genes involved in early steps of the bacteriochlorophyll a biosynthetic pathway.

J Bacteriol, 1990 Sep, 172(9), 4870 - 6
Electrochemical potential releases a membrane-bound secretion intermediate of maltose-binding protein in Escherichia coli; Geller BL; A secretionary intermediate of the Escherichia coli maltose-binding protein accumulated in the inner membrane when the membrane electrochemical potential was reduced and the cytosolic ATP concentration was normal . The intermediate was mature in size, but maintained a conformation similar to the cytosolic precursor form, and not the mature periplasmic protein, as measured by differences in susceptibility to proteinase K in vitro . The intermediate was located on the periplasmic side of the inner membrane . Restoration of the membrane electrochemical potential resulted in the movement of the intermediate from the inner membrane to the periplasm . In other experiments in which the ATP concentration was reduced by 96% and the electrochemical potential remained normal, no intermediate accumulated . Thus, the final step in the export of maltose-binding protein requires the electrochemical potential of the inner membrane and does not require ATP.

Am J Hum Genet, 1990 Sep, 47(3), 562 - 7
Hereditary fructose intolerance caused by a nonsense mutation of the aldolase B gene; Kajihara S et al.; The nucleotide sequence of a patient's aldolase B gene was determined and showed a substitution of a single nucleotide (C----A) at position 720 in the coding region, which resulted in the 240th amino acid, a cysteine, being changed to a stop codon (TGC----TGA) . By an allele-specific oligonucleotide probe and polymerase chain reaction, the patient was shown to be homozygous for the mutation . To examine whether this mutation causes functional defect of the enzyme, the activity of the aldolase B from the patient, expressed in Escherichia coli by using expression plasmid, was measured . No activity was observed, and the predicted product was recovered from E . coli expression plasmid, indicating that this nonsense mutation was the cause of aldolase B deficiency.

EMBO J, 1990 Sep, 9(9), 2717 - 22
Mapping of catalytically important domains in Escherichia coli leader peptidase; Bilgin N et al.; Leader peptidase (Lep) is a central component of the secretory machinery of Escherichia coli, where it serves to remove signal peptides from secretory proteins . It spans the inner membrane twice with a large C-terminal domain protruding into the periplasmic space . To investigate the importance of the different structural domains for the catalytic activity, we have studied the effects of a large panel of Lep mutants on the processing of signal peptides, both in vivo and in vitro . Our data suggest that the first transmembrane and cytoplasmic regions are not directly involved in catalysis, but that the second transmembrane region and the region immediately following it may be in contact with the signal peptide and/or located spatially close to the active site of Lep.

Virology, 1990 Sep, 178(1), 52 - 61
Immunodetection in vivo of beet necrotic yellow vein virus-encoded proteins; Niesbach-Klosgen U et al.; Open reading frames identified on the four genomic RNAs of beet necrotic yellow vein virus were cloned into bacterial expression vectors and resulting cl-fusion proteins expressed in Escherichia coli were used to raise polyclonal antibodies . This set of antisera was used to show the presence of 7 of 9 predicted viral proteins in mechanically inoculated Chenopodium quinoa leaves by the Western blot technique . Viral coat protein (p22) and its readthrough protein p85 encoded by RNA-2 could be detected in all subcellular fractions . Two other RNA-2-encoded proteins, p42 and p13, are predominantly associated with membranous structures . Another RNA-2-encoded protein, p14, as well as the two polypeptides p25 and p31, encoded by RNA-3 and -4, respectively, are soluble proteins . The viral proteins could first be detected about the time lesions became visible and increased thereafter except for p85, in which case the amount of the soluble form decreased with time . No protein could be detected corresponding to the RNA-1-encoded p237 protein or to the p15 species encoded by open reading frame V of RNA-2.

Mol Cell Biol, 1990 Sep, 10(9), 4872 - 85
Multiple factors bind the upstream activation sites of the yeast enolase genes ENO1 and ENO2: ABFI protein, like repressor activator protein RAP1, binds cis-acting sequences which modulate repression or activation of transcription; Brindle PK et al.; Binding sites for three distinct proteins were mapped within the upstream activation sites (UAS) of the yeast enolase genes ENO1 and ENO2 . Sequences that overlapped the UAS1 elements of both enolase genes bound a protein which was identified as the product of the RAP1 regulatory gene . Sequences within the UAS2 element of the ENO2 gene bound a second protein which corresponded to the ABFI (autonomously replicating sequence-binding factor) protein . A protein designated EBF1 (enolase-binding factor) bound to sequences which overlapped the UAS2 element in ENO1 . There was a good correlation among all of the factor-binding sites and the location of sequences required for UAS activity identified by deletion mapping analysis . This observation suggested that the three factors play a role in transcriptional activation of the enolase genes . UAS elements which bound the RAP1 protein or the ABFI protein modulated glucose-dependent induction of ENO1 and ENO2 expression . The ABFI-binding site in ENO2 overlapped sequences required for UAS2 activity in wild-type strains and for repression of ENO2 expression in strains carrying a null mutation in the positive regulatory gene GCR1 . These latter results showed that the ABFI protein, like the RAP1 protein, bound sequences required for positive as well as negative regulation of gene expression . These observations strongly suggest that the biological functions of the RAP1 and ABFI proteins are similar.

Mol Cell Biol, 1990 Sep, 10(9), 4638 - 49
Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis; Banta LM et al.; vps33 mutants missort and secrete multiple vacuolar hydrolases and exhibit extreme defects in vacuolar morphology . Toward a molecular understanding of the role of the VPS33 gene in vacuole biogenesis, we have cloned this gene from a yeast genomic library by complementation of a temperature-sensitive vps33 mutation . Gene disruption demonstrated that VPS33 was not essential but was required for growth at high temperatures . At the permissive temperature, vps33 null mutants exhibited defects in vacuolar protein localization and vacuole morphology similar to those seen in most of the original mutant alleles . Sequence analysis revealed a putative open reading frame sufficient to encode a protein of 691 amino acids . Hydropathy analysis indicated that the deduced product of the VPS33 gene is generally hydrophilic, contains no obvious signal sequence or transmembrane domains, and is therefore unlikely to enter the secretory pathway . Polyclonal antisera raised against TrpE-Vps33 fusion proteins recognized a protein in yeast cells of the expected molecular weight, approximately 75,000 . In cell fractionation studies, Vps33p behaved as a cytosolic protein . The predicted VPS33 gene product possessed sequence similarity with a number of ATPases and ATP-binding proteins specifically in their ATP-binding domains . One vps33 temperature-sensitive mutant contained a missense mutation near this region of sequence similarity; the mutation resulted in a Leu-646----Pro substitution in Vps33p . This temperature-sensitive mutant strain contained normal vacuoles at the permissive temperature but lacked vacuoles specifically in the bud at the nonpermissive temperature . Our data suggest that Vps33p acts in the cytoplasm to facilitate Golgi-to-vacuole protein delivery . We propose that as a consequence of the vps33 protein-sorting defects, abnormalities in vacuolar morphology and vacuole assembly result.

Infect Immun, 1990 Sep, 58(9), 2977 - 82
A system for production and rapid purification of large amounts of the Shiga toxin/Shiga-like toxin I B subunit; Calderwood SB et al.; We have constructed a plasmid expression vector (pSBC32) that encodes the B subunit of Shiga toxin/Shiga-like toxin I under control of the inducible trc promoter . The encoded B subunit is transported to the periplasmic space, allowing single-step purification of milligram amounts of this protein from periplasmic extracts by using receptor analog affinity chromatography . The purified B subunit interacts normally with both polyclonal antiserum to Shiga toxin and a monoclonal antibody specific for B subunit . B subunit purified in this system is pentameric (as in native holotoxin) and biologically active in blocking binding of Shiga holotoxin to HeLa cells . This expression system may allow rapid purification of sufficient amounts of Shiga toxin B subunit to attempt crystallization or to study its efficacy as a vaccine, either by itself or coupled to an appropriate polysaccharide antigen.

Infect Immun, 1990 Sep, 58(9), 2935 - 9
Cloning, expression, and occurrence of the Brucella Cu-Zn superoxide dismutase; Bricker BJ et al.; Recently, the complete amino acid sequence of a protein expressed in Escherichia coli from cloned Brucella abortus DNA was reported . On the basis of amino acid homology, this protein was identified as a copper-zinc superoxide dismutase (Cu-Zn SOD) (B . L . Beck, L . B . Tabatabai, and J . E . Mayfield, Biochemistry 29:372-376, 1990) . We demonstrate in this paper that the sequenced protein is the same as the previously studied salt-extractable protein BCSP20 . The plasmid-encoded protein expressed from recombinant E . coli is identical to the Brucella-derived BCSP20 in molecular mass, N-terminal amino acid sequence, and cross-reactivity with homologous and heterologous rabbit sera against either the recombinant gene product or the Brucella-derived protein . A survey of the expression of the Cu-Zn SOD protein in Brucella biovars representing all species was done by Western blotting (immunoblotting) using antisera raised against the recombinant E . coli-derived protein . With the exception of B . neotomae and B . suis biovar 2, the Cu-Zn SOD protein was detectable in all Brucella species and biovars tested, including eight biovars of B . abortus.

Cancer Res, 1990 Sep 1, 50(17), 5328 - 32
Alpha-interferon structure and natural killer cell stimulatory activity; Li BL et al.; Expression vectors for human alpha-interferon (Hu-IFN-alpha) J1, a site-specific mutant {Ser116}Hu-IFN-alpha J1, and Hu-IFN-alpha J/C or Hu-IFN-alpha C/J hybrids were constructed and expressed in Escherichia coli . These interferons and others were purified by immunoaffinity chromatography with a monoclonal antibody against human alpha-interferon . Their antiviral activity and ability to stimulate natural killer cell activity were determined in comparison to several other human interferons . These results provide some insight into structure-activity relationships for stimulating natural killer cells and confirm our previous conclusions that antiviral activity cannot be used to predict other activities for an individual IFN-alpha species . The observations suggest that the tertiary structure rather than any specific linear sequence of amino acids regulates the ability of the interferons to stimulate natural killer cell activity.

J Virol, 1990 Sep, 64(9), 4428 - 37
Function of the human immunodeficiency virus types 1 and 2 Rev proteins is dependent on their ability to interact with a structured region present in env gene mRNA; Dillon PJ et al.; The interaction of the human immunodeficiency virus type 1 (HIV-1) Rev protein with a structured region in env mRNA (the Rev-responsive element {RRE}) mediates the export of structural mRNAs from the nucleus to the cytoplasm . We demonstrated that unlike HIV-1 Rev, which functions with both the HIV-1 and HIV-2 RREs, HIV-2 Rev functions only with the HIV-2 RRE . Rev-RRE binding studies suggested that the lack of nonreciprocal complementation stems from the inability of HIV-2 Rev to interact with HIV-1 RRE RNA . Maintenance of RNA secondary structure, rather than the primary nucleotide sequence, appeared to be the major determinant for interaction of both HIV-1 and HIV-2 Rev with the HIV-2 RRE . Moreover, the binding domain of the HIV-2 RRE recognized by HIV-1 Rev was dissimilar to the binding domain of the HIV-1 RRE, in terms of both secondary structure and primary nucleotide sequence . Our results support the hypothesis that function of HIV Rev proteins and possibly the functionally similar Rex proteins encoded by the human T-cell leukemia viruses (HTLVs) HTLV-I and HTLV-II is controlled by the presence of RNA secondary structure generated within the RRE RNA.

J Virol, 1990 Sep, 64(9), 4051 - 8
Characterization of hepatitis delta antigen: specific binding to hepatitis delta virus RNA; Lin JH et al.; It has previously been shown that human hepatitis virus delta antigen has an RNA-binding activity (Chang et al., J . Virol . 62:2403-2410, 1988) . In the present study, the specificity of such an RNA-protein interaction was demonstrated by expressing various domains of the delta antigen in Escherichia coli as TrpE fusion proteins and testing their RNA-binding activities in a Northwestern protein-RNA immunoblot assay and RNA gel mobility shift assay . Hepatitis delta virus (HDV) RNA bound specifically to the delta antigen in the presence of an excess amount of unrelated RNAs and a relatively high salt concentration . Both genome- and antigenome-sense HDV RNAs and at least two different regions of HDV genomic RNA bound to the delta antigen . Surprisingly, these two different regions of HDV genomic RNA could compete with each other for delta antigen binding, although they do not have common nucleotide sequences . In contrast, this binding could not be competed with by other viral or cellular RNA . Since both the genomic and antigenomic HDV RNAs had strong intramolecular complementary sequences, these results suggest that the binding of delta antigen is probably specific for a secondary structure unique to the HDV RNA . By expressing different subdomains of the delta antigen, we found that the middle one-third of delta antigen was responsible for binding HDV RNA . Neither the N-terminal nor the C-terminal domain bound HDV RNA . Binding between the delta antigen and HDV RNA was also demonstrated within the HDV particles isolated from the plasma of a human delta hepatitis patient . This in vivo binding resisted treatment with 0.1% sodium dodecyl sulfate and 0.5% Nonidet P-40 . In addition, we showed that the antiserum from a human patient with delta hepatitis reacted with all three subdomains of the delta antigen, indicating that all of the domains are immunogenic in vivo . These studies demonstrated the specific interaction between delta antigen and HDV RNA.

J Gen Microbiol, 1990 Sep, 136 ( Pt 9), 1825 - 31
Characterization of Escherichia coli adenylate cyclase mutants with modified regulation; Crasnier M et al.; In Escherichia coli there is a large increase of cAMP synthesis in crp strains, which are deficient in the catabolite gene activator protein . In this work it was shown that this increase in cAMP synthesis does not occur in crp crr strains, deficient in both the catabolite gene activator protein and enzymeIII-glucose, a component of the phosphotransferase system . It was also shown that the other components of the phosphotransferase system are required to obtain the increase of cAMP synthesis in a crp background . Adenylate cyclase mutants were obtained, by random mutagenesis, which had partial adenylate cyclase activity but which did not exhibit increased levels of cAMP in a crp background . For three mutants the mutation was identified as a single point mutation . This allowed the identification of residues arginine 188, aspartic acid 414 and glycine 463 which could be involved in the catabolite gene activator protein dependent activation process.

Biull Eksp Biol Med, 1990 Sep, 110(9), 298 - 301
{Genetic organization of transfer region of F-like plasmid pAP18-1}; Buianova NI et al.; The results of complementation analysis of nitrosoguanidine-induced mutants of F-like drd-plasmid pAP18-1 (Tc, ColV) testified to the existence of at least 3 tra regions (tra1, tra2, tra3) and regulating locus fin V in the genome of this plasmid . By means of molecular cloning of tra2 region and locus fin V of plasmid pAP18-1drd were located in Sall-fragment f5 (3.9 MD).

Nihon Kyobu Shikkan Gakkai Zasshi, 1990 Sep, 28(9), 1214 - 9
{Influence of lipopolysaccharide on angiotensin converting enzyme activity expressed by human umbilical vein endothelial cells in culture}; Watanabe K et al.; The activity of angiotensin converting enzyme (ACE) released into the culture medium of human umbilical cord vein endothelial cells in culture (HUVEC) for 24-hour incubation with or without various serotypes of lipopolysaccharide (LPS) was unchanged . ACE activity, however, in cell lysate or monolayer HUVEC after 24-hour incubation of various serotypes of LPS except LPS from E . coli, 026:B6, showed significant decrease, compared with no LPS treatment . Cell lysate contained much higher ACE activity than monolayer HUVEC after 24-hour incubation with or without LPS . These findings give rise to speculation that ACE or ACE-like substance might be located not only on the luminal surface, but also in cytoplasm of HUVEC, and also that decrease of ACE activity in the serum of septic adult respiratory distress syndrome (ARDS) patients might be due to reduced synthesis by vascular endothelial cells.

Vet Microbiol, 1990 Sep, 24(3-4), 307 - 26
Development of specific nucleic acid probes for the differentiation of porcine rotavirus serotypes; Johnson ME et al.; A dot blot hybridization assay is described for the detection and differentiation of porcine rotavirus serotypes . Recombinant complementary DNA (cDNA) representing gene 9 (the gene encoding the neutralization antigens in VP7 glycoprotein) from OSU (porcine rotavirus serotype 1) and Gottfried (porcine rotavirus serotype 2) strains were used to determine the optimal hybridization conditions which allow specific detection of group A porcine rotaviruses . Probes were prepared by excision of the inserts from the recombinant plasmids and radiolabeling of cDNA with 32P by the random primer extension method . Probes were hybridized at various stringencies with viral RNA from different rotavirus serotypes bound to nylon membranes . Hybridization at low stringency (26% base pair mismatch for stable hybrid formation) had high sensitivity but low specificity . Hybridization at high stringency (16% base pair mismatch for stable hybrid formation) produced high specificity but decreased the sensitivity observed at low stringency . Probes were specific for rotavirus at both stringencies and did not hybridize with nucleic acids from other porcine viruses . Subgenomic gene 9 fragments were then tested to provide more specific probes . A 322 bp fragment from OSU gene 9 between nucleotides 382 and 704 and a 266 bp fragment from Gottfried gene 9 between nucleotides 230 and 496 were found to be specific as hybridization probes . These studies demonstrated the feasibility of the dot blot hybridization assay using subgenomic fragments of gene 9 to detect and differentiate serotypes of porcine rotavirus . Additional studies are warranted to further evaluate the sensitivity and the capability of these probes to detect porcine field isolates of the same serotype.

Vet Immunol Immunopathol, 1990 Sep, 26(1), 13 - 30
B-congenic chickens differ in macrophage inflammatory responses; Puzzi JV et al.; The influence of the chicken major histocompatibility (B) complex (MHC) on monocyte and macrophage recruitment and activation was examined using fully developed 15I5-B congenic White Leghorn lines (ten backcross generations) . The phagocytic activity of Sephadex-elicited peritoneal macrophages for sheep red blood cells (SRBCs) was highest in lines 15.7-B2 and 15.P-B13 and lowest in 15.15I-B5 and 15.N-B21 . The same pattern of phagocytic activity was obtained when LPS (E . coli) was used as the in vivo elicitor-activator of peritoneal macrophages . Lines with B2 and B13 haplotypes had elevated percentages of phagocytic macrophages and a higher internalization activity per cell than did B5 and B21 congenic chickens . Differential peritoneal macrophage function between congenic lines was further supported by quantitation of superoxide anion release . B2 and B13 haplotypes were associated with high activity in contrast with B5, which was low, and 15I5 (B15) and B21 which were intermediate for superoxide anion release by macrophages . In vitro activation of blood monocytes with LPS resulted in similar line differences for SRBC phagocytic activity as were observed with in vivo Sephadex and LPS activation . In contrast, chemotaxis of blood mononuclear leukocytes to f-met-leu-phe produced a reciprocal response pattern among the haplotypes . Cells from lines with haplotypes B5 and B21 were superior to those of B2, B13, and B15 congenic lines in their directed migration towards this chemoattractant . All functional differences occurred despite similarities among lines in the cellular profiles of both elicited peritoneal exudate cells and isolated blood mononuclear cells.

Gene, 1990 Sep 1, 93(1), 67 - 72
Identification of an insertion-sequence-like genetic element in the newly recognized human pathogen Mycoplasma incognitus; Hu WS et al.; Cloned 2.2-kb DNA (plasmid psb-2.2) of Mycoplasma incognitus, a pathogen in AIDS and non-AIDS patients {Lo et al., Am . J . Trop . Med . Hyg . 41 (1989) 364-376; 601-616}, contains a 1405-bp genetic element closely resembling bacterial insertion sequence (IS) elements . This IS-like element has 29-bp terminal inverted repeats with seven mismatches, is immediately flanked by 3-bp direct repeats, and has typical stem-and-loop structures at or near both the termini . Two potential open reading frames (ORF-1 and ORF-2) encode 143 amino acids (aa) and 103 aa, respectively, in this IS-like element . Part (57 aa) of the deduced aa sequence of ORF-2 has a significant homology (43%) with the putative transposase of Escherichia coli IS3 . In this study, a series of synthetic oligodeoxyribonucleotides each containing a specific sequence of a selected segment in psb-2.2, have been used as probes which reveal that the IS-like element occurs more than ten times in the genome of M . incognitus . This potentially transposable element has many characteristic features in common with bacterial IS elements.

Gene, 1990 Sep 1, 93(1), 139 - 42
Cloning of the DNA gyrase genes under tac promoter control: overproduction of the gyrase A and B proteins; Hallett P et al.; The construction of plasmids which over-produce the Escherichia coli DNA gyrase A and B proteins (GyrA and GyrB) is described . Both plasmids are based on the pTTQ vectors of Stark {Gene 51 (1987) 255-267} and contain either the gyrA or gyrB gene under the tight control of the hybrid tac promoter . Expression of the gyrase genes is shown to be repressed in the absence of the inducer IPTG, but in its presence, strains containing these plasmids synthesise the A and B proteins to about 40% of soluble cell protein.

Genetics, 1990 Sep, 126(1), 25 - 40
Genetic dissection of the biochemical activities of RecBCD enzyme; Amundsen SK et al.; RecBCD enzyme of Escherichia coli is required for the major pathway of homologous recombination following conjugation . The enzyme has an ATP-dependent DNA unwinding activity, ATP-dependent single-stranded (ss) and double-stranded (ds) DNA exonuclease activities, and an activity that makes a ss DNA endonucleolytic cut near Chi sites . We have isolated and characterized ten mutations that reduced recombination proficiency and inactivated some, but not all, activities of RecBCD enzyme . One class of mutants had weak ds DNA exonuclease activity and lacked Chi-dependent DNA cleavage activity, a second class lacked only Chi-dependent DNA cleavage activity, and a third class retained all activities tested . The properties of these mutants indicate that the DNA unwinding and ss DNA exonuclease activities of the RecBCD enzyme are not sufficient for recombination . Furthermore, they suggest that the Chi-dependent DNA cleavage activity or another, as yet unidentified activity or both are required for recombination . The roles of the RecBCD enzymatic activities in recombination and exclusion of foreign DNA are discussed in light of the properties of these and other recBCD mutations.

J Gen Virol, 1990 Sep, 71 ( Pt 9), 2185 - 90
Increased antibody responses to human papillomavirus type 16 L1 protein expressed by recombinant vaccinia virus lacking serine protease inhibitor genes; Zhou J et al.; The L1 gene of human papillomavirus type 16 (HPV-16) driven by the vaccinia virus major late 4b gene promoter has been inserted into three different sites of the vaccinia virus genome . Insertion into the thymidine kinase (TK) gene was achieved by selection of TK- mutants in BUdR on TK- cells . Insertion into two vaccinia virus serine protease inhibitor (serpin) genes was achieved by co-insertion of the Escherichia coli xanthine guanine phosphoribosyltransferase gene linked to the vaccinia virus 7.5K promoter and selection of mycophenolic acid-resistant recombinant viruses . Each recombinant virus expressed a 57K L1 protein at similar levels and with similar kinetics . However, immunization of mice with these recombinant viruses induced different levels of antibody to the L1 protein . Viruses lacking serpin genes B13R and B24R induced significantly higher antibody levels than did viruses lacking the TK gene . The presence of functional B13R and B24R gene products is therefore somehow immunosuppressive at least for antibody responses to the L1 protein of HPV-16.

J Gen Virol, 1990 Sep, 71 ( Pt 9), 2005 - 11
Cloning, sequencing and expression in Escherichia coli of cDNA for a non-A, non-B hepatitis-associated microtubular aggregates protein; Takahashi K et al.; A 1.7 kb cDNA encoding a novel antigen (p44; apparent Mr 44K) associated with non-A, non-B (NANB) hepatitis, was isolated from the hepatic cDNA library of a chimpanzee infected with NANB hepatitis . The library was screened with a monoclonal antibody against this antigen . The cDNA cloned contained an open reading frame encoding a 444 amino acid protein with an Mr calculated to be 50,468 . The cDNA hybridized to a 1.9 kb mRNA obtained from chimpanzee hepatocytes infected with either the NANB or hepatitis delta viruses . It hybridized weakly to mRNA from hepatitis B virus-infected hepatocytes, and not at all to mRNA from normal chimpanzee hepatocytes . Southern blot analysis revealed that p44 is a host protein in chimpanzees, and that an identical gene exists in the human genome.

Neuron, 1990 Sep, 5(3), 353 - 60
A latent, nonpathogenic HSV-1-derived vector stably expresses beta-galactosidase in mouse neurons; Dobson AT et al.; A genetically engineered herpes simplex virus variant was constructed for use as a stable gene vector for neurons . To inhibit replication, the agent possessed a deletion in the immediate early gene ICP4, and to minimize reactivation from the latent state, the gene encoding the latency-associated transcript was deleted . The E . coli beta-galactosidase gene under the control of the Maloney murine leukemia virus long terminal repeat promoter was inserted into the ICP4 region . When introduced into the peripheral nervous system, this virus established latent infections and stably expressed beta-galactosidase in primary sensory neurons . Expression of beta-galactosidase over a more limited time period was observed when the latent infection was established in motor neurons of the hypoglossal nucleus . Agents of this general design have considerable potential for use as gene vectors for studies of neuronal function and correction of genetic defects affecting neurons.

Am J Physiol, 1990 Sep, 259(3 Pt 1), G410 - 9
Phosphoinositide metabolism in intestinal brush borders: stimulation of IP3 formation by guanine nucleotides and Ca2+; Vaandrager AB et al.; The potential role of polyphosphoinositide (PPI) metabolism as a signal-transduction mechanism in apical membranes of polarized epithelial cells was evaluated by examining the formation and breakdown of PPI in rat intestinal brush-border membranes (BBM) prelabeled by intraluminal injection of {3H}inositol in vivo or by {gamma-32P}ATP in vitro . Freshly isolated BBM prelabeled with {3H}inositol contained higher amounts of {3H}phosphatidylinositol 4,5-diphosphate compared with a basolateral membrane (BLM) preparation (approximately 14 and 6.8% of total {3H}PPIs, respectively) and were enriched in inositol lipid kinases, diacylglycerol (DAG) kinase, and phosphomonoesterases degrading PPI, inositol bisphosphate, and inositol triphosphate (IP3) . In the presence of nonhydrolysable GTP analogues and low Ca2+ (pCa 6-8) or at high Ca2+ alone (pCa 4) endogenous pools of PPI were rapidly depleted by an intrinsic PPI-specific phospholipase C apparently coupled to a GTP-binding protein (G protein) . Surprisingly, despite the assignment of most G protein-coupled hormone receptors to the BLM, the capacity of isolated BBM to release {3H}IP3 in response to Ca2+ or GTP gamma S appeared comparable to that in a BLM preparation . Intestinal secretagogues acting through apical membrane receptors (adenosine, heat-stable Escherichia coli toxin), however, were unable to promote {3H}IP3 release in isolated BBM in the presence of GTP . PPI metabolism in BBM may be coupled to receptors for as yet unidentified secretagogues or may serve as an amplification mechanism for hormone-stimulated PPI breakdown in BLM . The local release of DAG and IP3 at the interior of the intestinal microvilli likely plays a role in the regulation of ion transport systems in microvillar membranes.

Am J Vet Res, 1990 Sep, 51(9), 1370 - 4
Blood biochemical response to sodium bicarbonate infusion during sublethal endotoxemia in ponies; Gossett KA et al.; Hypertonic NaHCO3 infusion caused blood volume expansion, increased blood bicarbonate concentration, and delayed the onset of hypophosphatemia in ponies with endotoxemia . However, NaHCO3 infusion did not normalize blood pH, and it increased blood L-lactate concentration, and caused hypokalemia, hypernatremia, and hyperosmolality . The deleterious effects of NaHCO3 infusion in endotoxemia ponies outweighed the beneficial effects . The role of hypertonic NaHCO3 given IV for treatment of endotoxemia in equids must be reevaluated.

Proc Natl Acad Sci U S A, 1990 Sep, 87(17), 6555 - 9
In vitro assembly of relaxosomes at the transfer origin of plasmid RP4; Pansegrau W et al.; During initiation of conjugative transfer of DNA containing the transfer origin (oriT) of the promiscuous plasmid RP4, the proteins TraI, TraJ, and TraH interact and assemble a specialized nucleoprotein complex (the relaxosome) at oriT . The structure can be visualized on electron micrographs . Site- and strand-specific nicking at the transfer origin in vitro is dependent on the proteins TraI and TraJ and on Mg2+ ions . Substrate specificity is directed exclusively towards the cognate transfer origin: the RP4-specified TraJ protein cannot recognize the closely related oriT of plasmid R751 . After nicking, TraI protein remains attached to the 5'-terminal 2'-deoxycytidyl residue at the nic site {Pansegrau, W., Ziegelin, G . & Lanka, E . (1990) J . Biol . Chem . 265, 10637-10644} . Nicking and relaxosome formation require supercoiled DNA . Thus, a complicated structure involving multiple plasmid-specified proteins and a defined region of DNA must be formed at the transfer origin to prepare the plasmid for generating the single strand to be transferred.

J Bacteriol, 1990 Sep, 172(9), 5516 - 9
Membrane association of the Tnp and Inh proteins of IS50R; DeLong A et al.; Using a radioimmunoassay for the IS50R proteins Tnp and Inh, we found that both proteins were present primarily in the cytoplasm, but 3 to 11% of Tnp and 3 to 5% of Inh were found in association with the inner membrane . The fractions of total Tnp and Inh that became membrane bound were unaffected by the amount of Tnp and Inh synthesized in whole cells, provided that the ratio of total Tnp to total Inh was not changed . In addition, Inh was not found in the membrane fraction in Tnp- IS50R mutants, indicating that Tnp is required for Inh localization.

J Bacteriol, 1990 Sep, 172(9), 5511 - 3
Efficient incorporation of galactose into lipopolysaccharide by Escherichia coli K-12 strains with polar galE mutations; Schnaitman CA et al.; Efficient incorporation of exogenous galactose into lipopolysaccharide was observed in a strain with a galE::Tn10 insertion and a strain with a deletion of galOPE . No incorporation was observed in a strain with a longer (galETK) deletion, indicating that incorporation into the galE mutants was due to a low level of expression of galTK from internal or fortuitous promoters . These galE mutants provided a convenient and specific way to radiolabel lipopolysaccharide.

J Bacteriol, 1990 Sep, 172(9), 5501 - 2
Osmotic regulation of PhoE porin synthesis in Escherichia coli; Meyer SE et al.; In Escherichia coli, adaptation to hyperosmotic conditions alters the expression of the outer membrane porins OmpF and OmpC . The amount of PhoE porin, which is normally induced by phosphate deprivation, was greatly reduced in cells adapted to high-osmolarity conditions . Osmoregulation of PhoE operated independently of the activity of the PhoR phosphate sensor and did not involve cross-talk from the homologous osmosensor EnvZ . PhoE synthesis was partially restored by additional copies of the positive regulator phoB+ and by the osmoprotectant glycine betaine.

J Bacteriol, 1990 Sep, 172(9), 5497 - 500
The complete nucleotide sequence of the Escherichia coli gene appA reveals significant homology between pH 2.5 acid phosphatase and glucose-1-phosphatase; Dassa J et al.; The whole nucleotide sequence of Escherichia coli gene appA, which encodes periplasmic phosphoanhydride phosphohydrolase (optimum pH, 2.5), and its flanking regions was determined . The AppA protein is significantly homologous to the product of the nearby gene agp, acid glucose-1-phosphatase . Because identical amino acids are distributed over the whole lengths of the proteins, it is likely that appA and agp originate from the same ancestor gene.

J Bacteriol, 1990 Sep, 172(9), 5445 - 9
Isolation and properties of acyl carrier protein phosphodiesterase of Escherichia coli; Fischl AS et al.; The acyl carrier protein (ACP) phosphodiesterase of Escherichia coli catalyzes the hydrolytic cleavage of the 4'-phosphopantetheine residue from ACP, with the generation of apo-ACP (P . R . Vagelos and A . R . Larrabee, J . Biol . Chem . 242:1776-1781, 1967) . Although it has been postulated to play a role in the regulation of fatty acid synthesis, presently available evidence makes this unlikely, and its physiological function requires further investigation . We have now purified the enzyme from E . coli more than 3,000-fold and have identified it as a protein of Mr 25,000, as judged from its migration during electrophoresis in gels containing sodium dodecyl sulfate . The enzyme has remarkable thermostability, being protected against irreversible inactivation at 90 degrees C by the presence of sodium dodecyl sulfate . A partial sequence of the amino terminus of the enzyme is as follows: H2N-Ser-Lys-Val-Leu-Val-Leu-Lys-Ser-?-Ile-Leu-Ala-Gly-Tyr-Ser- . Other properties of the enzyme are also described.

J Bacteriol, 1990 Sep, 172(9), 4959 - 63
Uncoupling of transpositional immunity from gamma delta transposition by a mutation at the end of gamma delta; Wiater LA et al.; The transposon gamma delta, in common with other members of the Tn3 family, confers transpositional immunity, a phenomenon by which plasmids containing a single transposon end show reduced activity as targets for further insertion by the same element . We found that a copy of a mutant delta end, in which the two terminal base pairs (5' GG) were substituted with cytosines, conferred the same degree of immunity as the unaltered delta end . However, a transposon analog with the mutant delta end as its termini could not transpose . These results suggest that the binding of transposase to a site on a target replicon is sufficient to confer immunity and that immunity does not involve subsequent DNA transactions at the bound target site, analogous to the catalytic processes that occur at the transposon ends during transposition.

J Bacteriol, 1990 Sep, 172(9), 4951 - 8
Integration host factor increases the transpositional immunity conferred by gamma delta ends; Wiater LA et al.; The ends of the bacterial transposon gamma delta contain adjacent binding sites for gamma delta transposase and integration host factor (IHF) . IHF+ and IHF- strains were used in conjunction with gamma delta transposon ends containing or lacking the site for IHF binding to determine the role that IHF plays in various gamma delta-mediated transposition events . IHF was not essential for the transposition of gamma delta and seemed to decrease its frequency of transposition about threefold . IHF played no role in determining the distribution of gamma delta inserts into a target replicon, nor did it significantly alter the frequency of simple transpositions . The only clear role discerned for IHF and the terminal IHF-binding sites was in transposition immunity . IHF stimulated the immunity of those plasmids that contain an end of gamma delta, provided the end included the terminal IHF-binding site . For both ends, the degree of stimulation of immunity was similar to the stimulation of binding of transposase by IHF.

J Bacteriol, 1990 Sep, 172(9), 4919 - 26
Interdependence of calcium and cobalamin binding by wild-type and mutant BtuB protein in the outer membrane of Escherichia coli; Bradbeer C et al.; The binding of calcium and cobalamin to outer membranes from cells of Escherichia coli that contained amplified levels of wild-type or mutant btuB was studied . The mutant (BBam50) had an aspartyl-prolyl dipeptide inserted after the original 50th amino acid residue of the mature BtuB protein, which is within a region that shows extensive homology with the ferric siderophore receptors . This insertion resulted in cleavage of the BtuB in two places . The larger form retained the insertion but had lost 11 amino acid residues from the amino terminus . The smaller form was cut at the insertion site . Both the wild-type protein and the larger form of mutant BtuB showed calcium-dependent cobalamin binding with the same affinity for cobalamin, although the mutant had a much lower affinity for calcium . The smaller form of the mutant BtuB protein had a greatly reduced affinity for cobalamin, which was probably the result of inactivation of the cobalamin-dependent calcium-binding site . Cobalamin-dependent calcium binding was measured in wild-type BtuB preparations and was found to have the same corrinoid specificity and response to various corrinoid concentrations as shown previously for cobalamin binding . The results are consistent with a role for calcium in the cobalamin pump of the outer membrane of E . coli and show that a conserved part of the BtuB protein is required for the cobalamin-dependent binding of calcium.

EMBO J, 1990 Sep, 9(9), 2679 - 84
Mutational analysis of ligand binding activity of beta 2 adrenergic receptor expressed in Escherichia coli; Breyer RM et al.; A novel in situ screening procedure was used to identify neutral mutations in the human beta 2 adrenergic receptor (beta 2AR) . The coding region of the human beta 2AR gene was subcloned under transcriptional control of an inducible T7 promoter and used to transform Escherichia coli . Colonies expressing the beta 2AR bound the radiolabeled antagonist {125I}iodocyanopindolol and could be identified by autoradiography after transfer to nitrocellulose filters . The region of the beta 2AR between residues 76 and 83, in the second transmembrane helix, was mutagenized by a saturation mutagenesis technique, so that virtually all of the beta 2AR genes contained at least one mutation . Colonies retaining ligand binding activity were isolated using the in situ screen . Sequence analysis of the active mutant receptor genes allowed the identification of individual amino acid side chains which are essential for either ligand binding or structural integrity of the beta 2AR receptor.

Virology, 1990 Sep, 178(1), 238 - 46
Coexpression of the human papillomavirus type 16 E4 and L1 open reading frames in early cervical neoplasia; Crum CP et al.; Although the E4 open reading frame (ORF) of human papillomaviruses (HPV) encodes an abundant protein in cutaneous warts, the location and extent of HPV E4 expression in genital precancers, specifically those associated with HPV-16, has not been described . Expression plasmids (pATH) containing segments of the HPV-16 E4 (3401-3620) and L1 (6151-6792) open reading frames (ORFs) were induced and expressed in bacteria and the resulting fusion proteins were used to elicit antisera in rabbits . Antisera reacting to the E4 and L1 components of the fusion proteins were used to screen biopsies from 150 cervical precancers (cervical intraepithelial neoplasia) and condylomata . Six biopsies exhibiting specific immunostaining with the anti-E4 sera . Staining was cytoplasmic, and occurred virtually always in foci containing immunostaining for L1 proteins . Moreover, analysis of these 6 cases and 22 others for HPV-16 RNA by RNA-RNA in situ hybridization demonstrated a similar correlation between E4 immunostaining and the presence of abundant transcripts specific to HPV-16 . These data are consistent with the hypothesis that expression of the HPV-16 E4 ORF is dependent upon viral replication and epithelial differentiation, similar to L1 expression, and that the E4 epitopes identified by the rabbit antisera may be unique to HPV-16 relative to other common cervical papillomaviruses.

Mol Cell Biol, 1990 Sep, 10(9), 4974 - 7
A cellular factor binds to the herpes simplex virus type 1 transactivator Vmw65 and is required for Vmw65-dependent protein-DNA complex assembly with Oct-1; Xiao P et al.; The herpes simplex virus transactivator Vmw65 assembles into a multicomponent protein-DNA complex along with the octamer binding protein Oct-1 . Using affinity chromatography on columns conjugated with purified Vmw65 fusion protein expressed in Escherichia coli, we demonstrate that a cellular factor, distinct from Oct-1, binds to Vmw65 in the absence of target DNA and is necessary for Vmw65-mediated complex assembly with Oct-1.

Mol Cell Biol, 1990 Sep, 10(9), 4778 - 87
A single amino acid change in CUP2 alters its mode of DNA binding; Buchman C et al.; CUP2 is a copper-dependent transcriptional activator of the yeast CUP1 metallothionein gene . In the presence of Cu+ and Ag+) ions its DNA-binding domain is thought to fold as a cysteine-coordinated Cu cluster which recognizes the palindromic CUP1 upstream activation sequence (UASc) . Using mobility shift, methylation interference, and DNase I and hydroxyl radical footprinting assays, we examined the interaction of wild-type and variant CUP2 proteins produced in Escherichia coli with the UASc . Our results suggest that CUP2 has a complex Cu-coordinated DNA-binding domain containing different parts that function as DNA-binding elements recognizing distinct sequence motifs embedded within the UASc . A single-amino-acid substitution of cysteine 11 with a tyrosine results in decreased Cu binding, apparent inactivation of one of the DNA-binding elements and a dramatic change in the recognition properties of CUP2 . This variant protein interacts with only one part of the wild-type site and prefers to bind to a different half-site from the wild-type protein . Although the variant has about 10% of wild-type DNA-binding activity, it appears to be completely incapable of activating transcription.

Mol Cell Biol, 1990 Sep, 10(9), 4582 - 9
Selection and analysis of galactose metabolic pathway variants of a mouse liver cell line; Zaret KS et al.; To study the genetic expression and regulation of galactose-metabolizing enzymes, we mutagenized the mouse liver H2.35 cell line and selected for cell clones resistant to the toxic galactose analog, 2-deoxy-D-galactose (2-DOG) . One cloned line, designated H12.10, was stably resistant to high levels of 2-DOG and was completely deficient in galactokinase activity . Galactokinase activity and growth sensitivity to 2-DOG could be restored by transfecting H12.10 cells with a plasmid containing the Escherichia coli galactokinase (galK) gene fused to a eucaryotic promoter; thus, the 2-DOG selection could be directed against transfected recombinant constructs in a liver cell line . We also found that H2.35 cells could not utilize galactose as a primary carbon source because of a deficiency in galactose-1-phosphate uridyltransferase; a variant line of H2.35 cells selected in galactose medium expressed higher levels of uridyltransferase activity . Finally, we found that in all mammalian cell lines tested, galactokinase expression was the same whether the medium contained glucose, galactose, or both sugars . These studies demonstrate differences between mammalian cells and yeast cells in the regulation of gal enzymes, and they define different schemes for obtaining altered expression of genes in the galactose metabolic pathway . The isogenic liver cell lines described here can also serve as model systems for studying galactosemias, which are inherited disorders of galactose metabolism in humans.

Cancer Res, 1990 Sep 1, 50(17 Suppl), 5649S - 5652S
Induction of topoisomerase II gene expression in human lymphocytes upon phytohemagglutinin stimulation; Hwong CL et al.; The carboxyl-terminal one-third of human topoisomerase II polypeptide expressed in Escherichia coli was used as antigen to generate polyclonal antibodies in rabbits . With the use of antiserum, DNA topoisomerase II levels of phytohemagglutinin-stimulated human lymphocytes were measured by immunoblotting . Our results showed that the increase in intracellular topoisomerase II level paralleled the entry of cells into proliferation . We also found that the increase in the topoisomerase II level resulted from an increase in the amount of topoisomerase II mRNA . The time course study indicated that the appearance of topoisomerase II mRNA was first observed at 36 h after phytohemagglutinin stimulation . The maximal level of topoisomerase II mRNA was seen at 45 h after stimulation . The same RNA blot was rehybridized with a thymidine kinase probe . The maximal level of thymidine kinase mRNA was observed at 39 h after phytohemagglutinin stimulation . In a comparison of the time course of topoisomerase II gene expression with that of {3H}thymidine incorporation and thymidine kinase gene expression, it was found that the expression of the topoisomerase II gene was later than the onset of DNA replication . Thus, this study suggests that topoisomerase I, which is constantly expressed throughout the cell cycle, might participate in the initiation of DNA replication, while topoisomerase II is involved in solving the DNA topological problems accompanying DNA strand separation during DNA replication.

J Virol, 1990 Sep, 64(9), 4099 - 107
Lack of myristoylation of poliovirus capsid polypeptide VP0 prevents the formation of virions or results in the assembly of noninfectious virus particles; Marc D et al.; We previously described the generation of a set of mutations into a cDNA of poliovirus type 1 in the myristoylation signal of the capsid polypeptide VP4 (D . Marc, G . Drugeon, A.-L . Haenni, M . Girard, and S . van der Werf, EMBO J . 8:2661, 1989) . Genomic transcripts synthesized in vitro from the mutated cDNAs were found to be noninfectious upon transfection of permissive cells, and this property correlated with the lack of VP0 myristoylation in vivo . In the study presented here, we analyzed the assembly intermediates that could be recovered from cells transfected with the mutated transcripts . We found that 14S pentamers could still assemble to a certain extent with an unmyristoylated VP0 . Furthermore, viral particles sedimenting at 150S and containing capsid polypeptides VP1 to VP4 and virus-specific RNA were detected in the transfected cells . However, these mature virions were less abundant than those recovered after transfection with an infectious transcript, and they were devoid of infectivity . The results suggest that VP0 myristoylation plays a role in the late steps of poliovirus assembly and that the myristate moiety of VP4 may be required in the early steps of poliovirus infection.

Plant Cell, 1990 Sep, 2(9), 857 - 66
A metal-dependent DNA-binding protein interacts with a constitutive element of a light-responsive promoter; Lam E et al.; We have used DNase I footprinting to characterize nuclear factors that bind to the light-responsive promoter of pea rbcS-3A, one member of the gene family encoding the small subunit of ribulose-1,5-bisphosphate carboxylase . A sequence-specific binding activity, designated 3AF1, binds to an AT-rich sequence present at the -45 region of the rbcS-3A promoter . A tetramer of the 3AF1 binding site, designated as Box VI, can form multiple complexes with tobacco leaf and root nuclear extracts . Mutations of 3 base pairs in Box VI severely reduce DNA-protein complex formation in vitro . The wild-type Box VI tetramer, but not the mutant tetramer, is active in transgenic tobacco plants when placed upstream of the cauliflower mosaic virus 35S promoter truncated at -90 . These results correlate binding of 3AF1 to the in vivo function of Box VI . The Box VI tetramer/35S chimeric construct confers expression in diverse cell types and organs and its activity is not dependent on light . By using the Box VI tetramer as a probe to screen a cDNA expression library, we have obtained a putative cDNA clone for the 3AF1 DNA-binding activity . Lysogen extracts of Escherichia coli expressing the cDNA clone give sequence-specific complexes with Box VI . The deduced amino acid sequence of the protein encoded by the cDNA contains two stretches of about 100 residues that are 80% homologous . Moreover, in each of the two repeats, there is an arrangement of histidines and cysteines, which may be related to the two known types of zinc-finger motifs found in many DNA-binding proteins . Consistent with the expectation that metal coordination plays an important role in DNA binding by this protein, we found that 1,10-phenanthroline can abolish the formation of DNA-protein complexes . Interestingly, we found that the same treatment did not abolish the DNA binding activity of 3AF1 in crude nuclear extracts of tobacco . These data indicate that the nuclear 3AF1 activity is likely due to multiple DNA-binding proteins all interacting with Box VI in vitro . RNA gel blot analysis shows that multiple transcripts homologous to this cDNA clone are expressed in different tobacco organs.

Mutat Res, 1990 Sep-Nov, 236(2-3), 213 - 21
The UvrABC endonuclease system of Escherichia coli--a view from Baltimore; Grossman L et al.; Nucleotide excision is initiated by the UvrABC endonuclease system in which the initial DNA interaction is with UvrA which was dimerized in the presence of ATP . Nucleoprotein formation most likely takes place on undamaged regions of DNA by (UvrA)2 which has been dimerized in the presence of ATP . Topological unwinding of DNA, driven by ATP binding, is increased by the presence of UvrB to approximately a single helical turn . The Uvr(A)2B complex translocates to a damaged site by the combined Uvr(A)2B helicase in which the driving force is provided by the UvrB-associated ATPase . The dual incision reaction is initiated by the binding of the UvrC protein to the Uvr(A)2B-nucleoprotein complex . The proteins in this post-incision nucleoprotein complex do not turn over and require the presence of the UvrD protein and DNA polymerase I under polymerizing conditions . The final integrity of the DNA strands is restored with polynucleotide ligase.

Glycobiology, 1990 Sep, 1(1), 93 - 100
Biosynthesis of the polysialic acid capsule in Escherichia coli K1 . Cold inactivation of sialic acid synthase regulates capsule expression below 20 degrees C; Merker RI et al.; When neuroinvasive Escherichia coli K1 cells are grown at temperatures below 20 degrees C, they fail to synthesize the alpha-2,8-linked polysialic acid (polySia) capsule . The objective of this study was to use a genetic and biochemical approach to analyse why capsule expression was defective at cold temperatures . The strategy was to construct E.coli K1-derived mutants with defects in activation and degradation of Sia . The inability to degrade Sia because of a defect in the Sia-specific aldolase permitted accurate quantitation of Sia and CMP-Sia . Strains EV5 and EV90 possessed a defective CMP-Sia synthetase and were unable to activate Sia . These mutants were then used to study how synthesis of Sia, CMP-Sia, and the polySia capsule was affected by growth at 15 degrees C . In contrast to wild type strains, the mutants accumulated Sia in considerable quantities (up to 100 nmol mg protein-1) at 37 degrees C . However, no Sia was detected after growth at 15 degrees C . A temperature upshift experiment showed that the intracellular concentration of Sia increased ca . 3-fold within 5-10 min after shift from 15 to 37 degrees C, even in the presence of inhibitors of protein synthesis or transcription initiation . An in vitro assay for Sia synthase showed that Sia was synthesized at 37 degrees C in cell-free extracts from both 37 and 15 degrees C grown cells, but that no synthesis occurred when the same extracts were assayed at 15 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Biol (Mosk), 1990 Sep-Oct, 24(5), 1339 - 50
{Construction and properties of a hybrid operon coding the HBcAG and beta-galactosidase of the hepatitis B virus in Escherichia coli}; Makeeva IV et al.; A set of plasmids was constructed, that carries the hybrid operons with an artificial region for translation initiation of the second cistron . The SD-sequence situated close to the termination signal of the previous cistron facilitates the reinitiation of translation . Both HBcAg and beta-galactosidase coding cistrons are functionally active . The analysis of expression efficacy shows: 1) The second cistron possesses its own initiation region; ii) the opportunity of translation reinitiation increases of the protein synthesis level . The correlation between the translation initiation efficacy and the structure of the initiator codon was investigated . AUG and UUG provide comparable protein synthesis levels, AUG being 1.5-3 times more effective . Probably, there exists a different efficacy of recognition of initiator codons by ribosomes for the systems with independent and connected initiation of translation . The influence of mRNA secondary structure in the translation initiation region on expression is discussed.

Biochimie, 1990 Sep, 72(9), 639 - 51
Antibody engineering and perspectives in therapy; Lefranc G et al.; Techniques of genetic engineering, homologous recombination, and gene transfection make it feasible to produce antigen-binding molecules with widely varying structures . Novel proteins which possess the binding specificity of antibody associated with sequences such as an enzyme or toxin have potential use in immunoassays, in imaging, in immunotherapy . Antibody fusion proteins can also be used as a means to purify proteins or to study the function of surface protooncogenes . This paper reviews the recent data on the obtention and utilisation of the genetically engineered antibody molecules, as well as the approach which consists on the expression in vitro, in Escherichia coli, of a practically unlimited repertoire of Fab fragments and antibody sites.

J Biochem (Tokyo), 1990 Sep, 108(3), 420 - 5
An Escherichia coli protein that preferentially binds to sharply curved DNA; Yamada H et al.; We attempted to find Escherichia coli proteins which preferentially bind to a curved DNA sequence even in the presence of an excess amount of a non-curved DNA sequence as a competitor, mainly by means of a DNA-binding gel retardation assay . Since the two sequences used had nearly the same nucleotide compositions, including consecutive dA5 stretches, we reasoned that this strategy would allow us to identify proteins which preferentially recognize an overall DNA curvature . We purified such a protein from E . coli . Its preferential binding to the curved DNA was found to be inhibited by distamycin, which removes the curvature from appropriate DNA sequences . The purified protein was identified as the E . coli nucleoid protein, H-NS.

Gene, 1990 Sep 1, 93(1), 101 - 9
Functional elements in the promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase; Punt PJ et al.; Analysis of the promoter region of the highly expressed Aspergillus nidulans gpdA gene is described . The nucleotide (nt) sequence of a 1.3-kb region upstream from the ATG was determined . Comparison with promoter regions of other Aspergillus and Neurospora genes revealed several regions of similar sequence . Both random and site-specific mutations were introduced into the promoter region of the gpdA gene, and the resulting mutant promoters were fused to the Escherichia coli lacZ gene . The constructed fusions were introduced into A . nidulans and transformants that contained one copy of these fusions at the argB locus were analysed . beta-Galactosidase assays and primer extension experiments were used to identify sequence elements involved in transcription activation and transcription initiation . Two elements, located around 650 and 250 nt upstream from the major transcription start point (tsp), were identified as transcription activation elements . These elements coincide with regions of putative secondary structure (direct or inverted repeats) . A third element, a C + T-rich region directly upstream from the major tsp, was shown to be involved in correct initiation of transcription.

Oncogene, 1990 Sep, 5(9), 1321 - 8
Molecular cloning and characterization of a novel type of regulatory protein (GDI) for the rho proteins, ras p21-like small GTP-binding proteins; Fukumoto Y et al.; We have recently purified to near homogeneity a novel type of regulatory protein for the rho proteins, ras p21-like small GTP-binding proteins, from bovine brain cytosol . This regulatory protein, named GDP dissociation inhibitor for the rho proteins (rho GDI), regulates the GDP/GTP exchange reaction of the rho proteins by inhibiting the dissociation of GDP from them, and the subsequent binding of GTP to them . In the present studies, we have isolated the cDNA of rho GDI from a bovine brain cDNA library using oligonucleotide probes designed from the partial amino acid sequences of the purified rho GDI and determined its complete nucleotide and deduced amino acid sequences . The cDNA contains an open reading frame encoding a protein of 204 amino acids with a calculated Mr value of 23,421 . This Mr value is similar to those of the purified rho GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultra-centrifugation, both of which are about 27,000 . The rho GDI cDNA is expressed in Escherichia coli and COS7 cells and the encoded protein exhibits rho GDI activity . The 1.9-kilobase rho GDI mRNA corresponding to the isolated cDNA is detected in various rat tissues by Northern blot analysis . Hydropathy analysis indicates that rho GDI is overall hydrophilic except for one hydrophobic region . Computer homology search has revealed that rho GDI is a novel protein that does not share a high amino acid sequence homology with any known protein.

Biochem J, 1990 Sep 1, 270(2), 463 - 8
Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-beta-galactosidase fusion proteins; Brown JC et al.; Antisera raised against fusion proteins consisting of murine laminin B1 and B2 subunit sequences fused to the C-terminus of Escherichia coli beta-galactosidase were tested for their subunit specificity on Western blots of deglycosylated murine Engelbreth-Holm-Swarm (EHS) laminin . The antisera raised against B2 subunit sequences (anti-XLB2.1 and anti-XLB2.2) bound only to the EHS laminin B2 subunit . One of the antisera raised against B1 subunit sequences (anti-XLB1.2) was specific for the B1 subunit, whereas two others (anti-XLB1.1 and anti-XLB1.3) cross-reacted with the EHS laminin B2 subunit . Gold-labelled heparin-albumin was shown to bind specifically to the A subunit of deglycosylated EHS laminin on Western blots . These reagents were used to identify the homologous subunits in rat parietal-yolk-sac laminin and human placental laminin . The anti-(fusion protein) antisera identified the B1 and B2 subunits of the rat laminin, and these were similar in size to the murine EHS B subunits . Human placental laminin gave bands of 400, 340, 230, 190 and 180 kDa on reducing SDS/PAGE . The anti-(fusion protein) antisera identified the 230 and 190 kDa bands as the B1 and B2 subunits respectively . Gold-labelled heparin-albumin bound to the 400, 340 and 190 kDa bands of human placental laminin and so did not unambiguously identify a single A subunit . The human placental laminin may contain a mixture of isoforms, with alternative subunits substituting for the A subunit.

Proc Natl Acad Sci U S A, 1990 Sep, 87(17), 6569 - 73
General colorimetric method for DNA diagnostics allowing direct solid-phase genomic sequencing of the positive samples; Wahlberg J et al.; A system for rapid colorimetric detection of specific genome DNA fragments amplified by the polymerase chain reaction (PCR) is described that has been designed to allow direct solid-phase sequencing of positive samples . The amplified material is immobilized on magnetic beads by using the biotin streptavidin system . An Escherichia coli lac operator DNA sequence is incorporated in the amplified material during the second step of a nested primer procedure . This 21-base-pair sequence is used for a general colorimetric detection with a fusion protein consisting of the E . coli Lac repressor and beta-galactosidase . Positive samples can be treated subsequently with alkali to obtain a single-stranded DNA template suitable for direct genomic sequencing . This method to detect immobilized amplified nucleic acids (DIANA) is well adapted for automated or semiautomated clinical assays . Here, we show that it can be used to detect and sequence Chlamydia trachomatis genomic DNA in clinical samples.

Proc Natl Acad Sci U S A, 1990 Sep, 87(17), 6527 - 31
RAP2B: a RAS-related GTP-binding protein from platelets; Ohmstede CA et al.; A platelet cDNA expression library was screened with the monoclonal antibody M90, which recognizes a specific epitope on RAS-encoded p21 proteins (amino acids 107-130) . DNA sequence analysis of one clone revealed that it encoded a partial amino acid sequence of a protein closely related to RAP2, which we have named RAP2B . A repeated screening of the platelet cDNA library with an internal Ava I fragment of the RAP2B cDNA allowed the isolation of a full-length cDNA for the RAP2B sequence . RAP2B is 90% identical to RAP2 at the amino acid level with the most variability at the carboxyl terminus of the protein . Oligonucleotides were synthesized to complete the amino acid sequence of the RAP2B protein and the entire sequence was expressed in Escherichia coli . Analysis of crude soluble extracts indicated that RAP2B was a Mr 22,000 protein that specifically bound GTP on blots . Moreover, incubation of similar extracts with the catalytic subunit of cAMP-dependent protein kinase did not cause phosphorylation of RAP2B, as had been observed for the closely homologous proteins, RAP1A and RAP1B . These results suggest that RAP2B, like the other RAP proteins, is a low molecular weight GTP-binding protein in human platelets.

Am J Clin Nutr, 1990 Sep, 52(3), 548 - 52
Short-term TPN containing n-3 fatty acids ameliorate lactic acidosis induced by endotoxin in guinea pigs; Pomposelli JJ et al.; We evaluated the effect of total parenteral nutrition (TPN) enriched with n-3 fatty acids on the physiologic response to endotoxin in guinea pigs . Animals were randomly assigned to receive TPN differing only in lipid source for 3.5 d . Group 1 received soybean fat emulsion (Intralipid) whereas group 2 received fish (menhaden) oil . During the last 7 h of TPN, animals were further randomized to have either saline or E coli endotoxin added to the infusate . Acid-base status and serum lactate concentrations were determined . Animals infused with soybean fat emulsions and endotoxin developed a significant metabolic acidosis, lactic acidemia, and decrease in mixed venous O2 compared with controls and fish-oil-treated animals (p less than 0.05) . The significantly reduced serum lactate and higher mixed venous O2 in fish-oil-infused animals suggests that the underlying mechanism involves improvement in endotoxin-induced tissue hypoperfusion, presumably through alterations in prostaglandin metabolism.

Mol Cell Biol, 1990 Sep, 10(9), 4912 - 9
Upstream elements repress premature expression of an Aspergillus developmental regulatory gene; Adams TH et al.; The Aspergillus nidulans abaA gene regulates intermediate steps in asexual reproductive development and is itself developmentally regulated . An 822-base-pair DNA fragment from the abaA 5'-flanking region is sufficient to drive developmentally appropriate expression of the Escherichia coli lacZ gene . Deletion analysis showed that this fragment contains elements that repress transcription in vegetative cells and immature conidiophores and that activate transcription later during development . A 45-base-pair region encompassing the major and minor abaA transcription initiation sites contains directly repeated sequences related to the mammalian initiator (Inr) element (S . T . Smale and D . Baltimore, Cell 57:103-113, 1989) . This element or sequences in the untranslated leader were sufficient for correct transcription initiation and for measurable developmental induction . Similar elements were present at or near the initiation sites of other developmentally regulated genes . We propose that the temporal and spatial specificity of expression of these genes results from modulation of the activity of Inr elements.

Infect Immun, 1990 Sep, 58(9), 3154 - 7
Nucleotide sequence of htpB, the Legionella pneumophila gene encoding the 58-kilodalton (kDa) common antigen, formerly designated the 60-kDa common antigen; Sampson JS et al.; Gene htpB, which encodes the 58-kilodalton protein of Legionella pneumophila, was cloned in Escherichia coli and its complete nucleotide sequence was determined . Analysis of this sequence revealed an open reading frame of 1,644 nucleotides encoding a protein with a predicted molecular mass of 57,952 daltons . Data obtained by amino-terminal sequencing of the purified 58-kilodalton protein agreed, except for one amino acid residue, with the predicted amino acid sequence, identifying this open reading frame as htpB . A comparison of the primary structure of this protein to other proteins of similar molecular weights from E . coli, Mycobacterium leprae, M . tuberculosis, and Coxiella burnetii revealed significant regions of sequence similarity, which are discussed.

Infect Immun, 1990 Sep, 58(9), 2760 - 9
A protective protein antigen of Rickettsia rickettsii has tandemly repeated, near-identical sequences; Anderson BE et al.; The nucleotide sequence of a Rickettsia rickettsii gene that encodes a high-molecular-mass surface antigen (190 kilodaltons), which elicits protective immunity, was determined . The 6,747-nucleotide gene coded for a 2,249-amino-acid protein with a calculated molecular weight of 224,321 . A 3.8-kilobase PstI fragment proximal to the 5' end of the gene was found to consist of 13 highly related tandem repeats which constituted over 40% of the coding region . The repeated sequences could be divided into either a 225-nucleotide, 75-amino-acid unit (type I) or a 216-nucleotide, 72-amino-acid unit (type II), with extensive homology between the two types of repeating units . The deduced amino acid sequence for these repeat units, overall, was slightly hydrophobic with short hydrophilic domains . The carboxy-terminal (nonrepetitive) portion of the deduced protein sequence was hydrophilic, with potential surface-exposed epitopes . The full-length reading frame was reconstructed in Escherichia coli, and transient expression of the 190-kilodalton antigen was demonstrated; however, the protein appeared to be severely degraded by proteases and was apparently toxic to E . coli . The conservation of this unique repetitive gene structure, coupled with results from previous reports showing the protective properties of the 190-kilodalton antigen, suggests that this protein plays an important role in the pathogenesis of and immunity to Rocky Mountain spotted fever.

Res Microbiol, 1990 Sep-Oct, 141(7-8), 931 - 9
Recombinant BCG as a candidate oral vaccine vector; Barletta RG et al.; Bacille Calmette-Guerin (BCG), currently the most widely used vaccine in the world, was originally administered for many years as an oral vaccine . The low frequency of serious complications, inexpensive production, and adjuvanticity make BCG an ideal candidate for a recombinant vaccine vehicle . Although mycobacteria are slow growing and not yet well characterized genetically, we have recently developed technology for the genetic manipulation of BCG and other mycobacteria . Phage and plasmid systems based on a shuttle strategy to manipulate DNA in Escherichia coli and transfer it to mycobacteria have been developed . We have established that the aminoglycoside phosphotransferase gene can be used as an effective selectable marker in the mycobacteria and that a foreign antigen from Mycobacterium leprae can be expressed in BCG . Furthermore, a thorough analysis of mycobacterial expression sequences has been undertaken to optimize the expression of foreign antigens in BCG . We constructed an expression probe shuttle plasmid with beta-galactosidase as reporter gene, and have used it successfully to identify multiple mycobacteriophage DNA sequences with varying levels of constitutive or regulable promoter activity . Further genetic advances required for development of recombinant BCG into an effective recombinant vaccine vehicle, including possibilities for oral administration, are adumbrated.

Res Microbiol, 1990 Sep-Oct, 141(7-8), 785 - 6
Diffuse adherence of enteropathogenic Escherichia coli strains; Benz I et al.; For the identification and characterization of the factor(s) responsible for the diffuse adherence (DA) pattern of enteropathogenic Escherichia coli strains, E . coli strain 2787 isolated from a case of infantile diarrhoea was employed . A plasmid-derived 11-kb fragment was cloned into pBR322 . The recombinant plasmid pIB6 was shown to confer the diffuse adherence phenotype on different E . coli K12 strains as well as pIB4, a plasmid with a 9.2-kb insert . The DNA fragment necessary for the expression of the DA phenotype could be reduced to 6.0 kb . Antiserum obtained against pIB4-encoded proteins recognized a surface-associated protein of about 100 kDa in Western blotting . The isolated 100-kDa protein was found to bind to HeLa cells . The antiserum against C600(pIB4) inhibits adherence of E . coli 2787 and C600(pIB6) to HeLa cells . For this reason, the protein is called adhesin involved in diffuse adherence (AIDA-I).

Plasmid, 1990 Sep, 24(2), 119 - 31
The sequences of genes bordering oriT in the enterotoxin plasmid P307: comparison with the sequences of plasmids F and R1; Graus-Goldner A et al.; The nucleotide sequences of the enterotoxin plasmid P307 transfer genes traM, finP, traJ, traY, and gene 19 were determined . Gene 19 is highly conserved; its product is very similar to that coded by the F and R1 plasmids . The TraM protein is similar in P307 and in F; the R1 sequence shows differences in the 40 N-terminal amino acids . The traJ product is very different in P307, F, and R1 . The traY gene from P307, which in F is almost twice as long, is similar in size to that from R1 . The finP RNA shows a high degree of homology with that from R1 and F, except for the two loop regions where base changes were observed . The genes coding for proteins, except traY, could be expressed in minicell- and T7 promoter-driven expression systems, whereas traJ and gene 19 could be expressed only in the latter system.

Hua Xi Yi Ke Da Xue Xue Bao, 1990 Sep, 21(4), 362 - 5
{The study on genome size of leptospires}; Xiao J et al.; Determination of genome size is very important in genetic studies and the molecular cloning of leptospires . With pulsed field gel electrophoresis (PFGE), which can be used in the analysis of large DNA molecules, we studied the genome size of leptospires and analysed it with rare cutter restrict endonuclease Not I . The PFGE system used was CHEF-DR II which produced parallel bands in agarose gel . The results showed that the genome size of leptospires was 2000kb and there was no dramatic difference between the 5 strains of 2 genus leptospira . Rare cutter Not I digestion of genome DNA of leptospira resulted in 11 bands and its bands pattern was quite different from that of E . coli, S . choleraesuis, S . typhimurium, and S . dysenteriae genome DNA . It is thought that more information can be obtained about leptospiral DNA with PFGE technique.

J Pharmacobiodyn, 1990 Sep, 13(9), 549 - 57
The pharmacokinetic pattern of glycosylated human recombinant lymphotoxin (LT) in rats after intravenous administration; Kawatsu M et al.; We have examined the pharmacokinetics of glycosylated recombinant human lymphotoxin (LT) after intravenous bolus injection in rats and compared them with those of tumor necrosis factor (TNF) or LT species . The results are as follows . 1) The mean half-life of glycosylated LT in serum increases for each increase in dose, and the distribution volume (V) and total body clearance {(Cl (total)} tend to decrease for increase in dose . On the other hand, the half-life of TNF also increases for increase in dose, but the V tends to increase for increase in dose and Cl (total) does not change . 2) The glycosylated LT distributes to all organs so far tested except brain, and tends to accumulate to kidney more than other tissues at 6 h after the injection . 3) Nonglycosylated LT produced by E . coli and the glycosylated LT species carrying both N-type and mucin-type sugar moieties (25 kDa LT) have shorter half-lives and higher Cl (total)s than 23 kDa LT carrying N-type sugar moieties alone . The 21 kDa LT, the same species as 23 kDa LT except that it lacks 15 amino acid residues at the N-terminus, disappears much faster than 23 kDa LT and shows higher V and Cl (total) . Thus, glycosylated LT shows nonlinear pharmacokinetics like TNF, but the deposition is quite different from that of TNF . The high serum concentration of glycosylated LT depends upon the presence of N-type sugar moieties, but not mucin-type sugar moieties . The N-terminal protein chain of LT also correlates with the serum concentration.

Development, 1990 Sep, 110(1), 141 - 9
The closely related Drosophila sry beta and sry delta zinc finger proteins show differential embryonic expression and distinct patterns of binding sites on polytene chromosomes; Payre F et al.; Serendipity (sry) beta (beta) and delta (delta) are two finger protein genes resulting from a duplication event . Comparison of their respective protein products shows interspersed blocks of conserved and divergent amino-acid sequences . The most extensively conserved region corresponds to the predicted DNA-binding domain which includes 6 contiguous fingers; no significant sequence conservation is found upstream and downstream of the protein-coding region . We have analysed the evolutionary divergence of the sry beta and delta proteins on two separate levels, their embryonic pattern of expression and their DNA-binding properties in vitro and in vivo . By using specific antibodies and transformant lines containing beta-galactosidase fusion genes, we show that the sry beta and sry delta proteins are maternally inherited and present in embryonic nuclei at the onset of zygotic transcription, suggesting that they are transcription factors involved in this process . Zygotic synthesis of the sry beta protein starts during nuclear division cycles 12-13, prior to cellularisation of the blastoderm, while the zygotic sry delta protein is not detectable before germ band extension (stage 10 embryos) . Contrary to sry delta, the zygotic sry beta protein constitutes only a minor fraction of the total embryonic protein . The sry beta and delta proteins made in E . coli bind to DNA, with partly overlapping specificities . Their in vivo patterns of binding to DNA, visualised by immunostaining polytene chromosomes, differ both in the number and position of their binding sites . Thus changes in expression pattern and DNA-binding specificity have contributed to the evolution of the sry beta and delta genes.

Glycobiology, 1990 Sep, 1(1), 101 - 9
Intracellular accumulation of the cellular prion protein after mutagenesis of its Asn-linked glycosylation sites; Rogers M et al.; The cellular isoform of the prion protein (PrPC) is a sialoglycoprotein bound almost exclusively on the external surface of the plasma membrane by a glycosyl phosphatidylinositol anchor . The deduced amino acid sequence of Syrian hamster PrPC identifies two potential sites for the addition of Asn-linked carbohydrates at amino acids 181-183 (Asn-Ile-Thr) and 197-199 (Asn-Phe-Thr) . We have altered these sites by replacing the threonine residues with alanine and expressed the mutant proteins transiently in CV1 cells utilizing a mutagenesis vector with the T7 promoter located upstream from the PrP gene . The T7 RNA polymerase was supplied by infection with a recombinant vaccinia virus . The 3 mutant proteins (PrPAla183, PrPAla199 and PrPAla183/199) have a reduced relative molecular weight compared to wild-type (wt) PrP . Deglycosylation as well as synthesis in the presence of tunicamycin reduced the relative molecular weight of all the PrP species to that of the double mutant PrPAla183/199 . Our results indicate that both single-site mutant prion proteins are glycosylated at non-mutated sites and they suggest that both potential sites for Asn-linked glycosylation are utilized in wt PrPC . Immunofluorescence studies demonstrate that while wt PrPC localizes to the cell surface, all the mutant PrP molecules accumulate intracellularly . The site of accumulation of PrPAla183 is probably prior to the mid-Golgi stack since this protein does not acquire resistance to endoglycosidase H . Whether the intracellular locations of the mutant PrPC species are the same as those identified for the scrapie isoform of the prion protein (PrPSc) remains to be established.

Appl Environ Microbiol, 1990 Sep, 56(9), 2784 - 90
Genetic relatedness of Legionella longbeachae isolates from human and environmental sources in Australia; Lanser JA et al.; The genetic relatedness of Legionella longbeachae isolated in Australia since 1987 was investigated by restriction fragment length polymorphism (RFLP) analysis and allozyme electrophoresis . Three radiolabeled probes were used in Southern hybridizations for the RFLP studies . They were Escherichia coli 16S and 23S rRNA and cloned fragments of L . longbeachae selected empirically from genomal banks in lambda and a cosmid . The legionellae included in the study comprised 11 Legionella longbeachae serogroup 1 organisms isolated from humans, 28 L . longbeachae serogroup 1 isolates from environmental sources, and 3 L . longbeachae serogroup 2 environmental isolates . These were compared with the American Type Culture Collection reference strains of both serogroups and some other related Legionella species . Results of allozyme and RFLP analysis showed that all the isolates from humans and all but three of the environmental L . longbeachae serogroup 1 isolates were closely related . They were also closely related to L . longbeachae serogroup 1 ATCC 33462 . There was wider variation among the three L . longbeachae serogroup 2 environmental isolates . One of these was closely related to L . longbeachae serogroup 2 ATCC 33484 . RFLP studies with the rRNA probe provided the most discrimination among isolates but did not distinguish between the two serogroups.

J Clin Microbiol, 1990 Sep, 28(9), 2006 - 11
Adhesive fimbriae associated with porcine enterotoxigenic Escherichia coli of the O141 serotype; Kennan RM et al.; Enterotoxigenic Escherichia coli of the O141 serotype, isolated from piglets with postweaning coliform enteritis but producing none of the characterized adhesive fimbriae, was examined for fimbrial production by transmission electron microscopy . Two strains that produced numerous fimbriae were chosen for further characterization . The fimbriae were isolated and purified and had a subunit molecular weight of 17,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Using antiserum raised against this protein, we have shown it to be specific for the 17,000-molecular-weight band by immunoblotting and to be directed against the fimbriae by immunoelectron microscopy . These fimbriae were not produced when the bacteria were grown at 18 degrees C and did not show any mannose-resistant hemagglutination of the erythrocytes tested . We propose that these are a new type of adhesive fimbriae associated with porcine enterotoxigenic E . coli of the O141 serotype.

Int J Radiat Biol, 1990 Sep, 58(3), 449 - 61
Superoxide dismutase and media dependence of far-UV radiation resistance in thiol-treated cells; Claycamp HG et al.; Pretreatment of wild-type Escherichia coli K12 cells with dithiothreitol (DTT) induces far-UV radiation resistance after the thiol is removed (Claycamp 1988) . The present study shows that a 1 h treatment of cells with DTT in minimal medium followed by a 0.5 h incubation in buffer (37 degrees C) results in a dose reduction factor (DRF) calculated at F37 of 1.81 . When the thiol pretreatment was in rich medium, sensitization occurs with DRF = 0.729 . This sensitization could be reversed to protection by inhibiting extracellular thiol oxidation in rich medium with the chelator, DETAPAC, such that the thiol oxidation rate was equivalent to that of DTT in minimal medium . Both thiol-induced resistance and sensitization produced changes predominantly in the shoulders of the survival curves . Furthermore, for either protection or sensitization, at least one form of endogenous superoxide dismutase (SOD) was required: in SOD-deficient cells (sodAsodB) the DRFs were 1.08 and 0.882 for minimal and rich media, respectively . These results suggest that different targets are involved in thiol-induced UV protection and sensitization: DNA and extracellular targets (e.g . the membrane), respectively . The results augment observations of alternate and multiple repair pathways inducible by oxygen radicals and may help understanding non-physicochemical thiol protection mechanisms.

J Bacteriol, 1990 Sep, 172(9), 5335 - 42
Molecular cloning, sequencing, and expression of the glutamine synthetase II (glnII) gene from the actinomycete root nodule symbiont Frankia sp . strain CpI1; Rochefort DA et al.; In common with other plant symbionts, Frankia spp., the actinomycete N2-fixing symbionts of certain nonleguminous woody plants, synthesize two glutamine synthetases, GSI and GSII . DNA encoding the Bradyrhizobium japonicum gene for GSII (glnII) hybridized to DNA from three Frankia strains . B . japonicum glnII was used as a probe to clone the glnII gene from a size-selected KpnI library of Frankia strain CpI1 DNA . The region corresponding to the Frankia sp . strain CpI1 glnII gene was sequenced, and the amino acid sequence was compared with that of the GS gene from the pea and glnII from B . japonicum . The Frankia glnII gene product has a high degree of similarity with both GSII from B . japonicum and GS from pea, although the sequence was about equally similar to both the bacterial and eucaryotic proteins . The Frankia glnII gene was also capable of complementing an Escherichia coli delta glnA mutant when transcribed from the vector lac promoter, but not when transcribed from the Frankia promoter . GSII produced in E . coli was heat labile, like the enzyme produced in Frankia sp . strain CpI1 but unlike the wild-type E . coli enzyme.

J Bacteriol, 1990 Sep, 172(9), 5079 - 88
Regulation and sequence of the Synechococcus sp . strain PCC 7942 groESL operon, encoding a cyanobacterial chaperonin; Webb R et al.; The molecular chaperonins such as GroEL are now widely regarded as essential components for the stabilization of integral membrane or secretory proteins before membrane insertion or translocation, as well as for the assembly of macromolecular complexes such as ribulose bisphosphate carboxylase-oxygenase . The groESL operon of Synechococcus sp . strain PCC 7942 was cloned as two independent lacZ-groEL translational fusions by immunoscreening a lambda ZAP genomic expression library and then sequenced . The derived amino acid sequences of the GroES and GroEL proteins demonstrated very high levels of amino acid identity with cognate chaperonins from bacteria and chloroplasts . The bicistronic 2.4-kilobase transcript from this operon, barely detectable in RNA preparations from cells grown at 30 degrees C, accumulated approximately 120-fold in preparations from cells grown for 20 min at 45 degrees C . Under these conditions, GroEL protein accumulated to 10-fold-higher levels . Primer extension analysis was used to identify a cyanobacterial heat shock promoter located at -81 base pairs from the groES initiation codon . The transcriptional -10 and -35 sequences differ slightly from Escherichia coli consensus heat shock promoter sequences.

Genetika, 1990 Sep, 26(9), 1690 - 3
{Transposon Tn5 and its derivatives effectively transpose over a broad temperature range}; Borovok IA; It was shown that the 1st class composite transposon Tn5 (5.8 Kb) and its synthetic derivatives--TnV (Tn5-ReppSC101; 6.1 Kb) and Tn5-MobRP4 (about 7.7 Kb) transpose in Escherichia coli K-12 cells (RecA strain HB101) with similar efficiency both at 28 and at 42 degrees C as well as at 37 degrees C . This property of Tn5-like elements distinguishes them from the class II transposons (such as Tn3, Tn21 etc.), whose transposition, as is well known, is strongly suppressed even at 37 degrees C . It was also demonstrated that transposition frequency of Tn5-derived elements depends on their copy number.

Mol Biol (Mosk), 1990 Sep-Oct, 24(5), 1419 - 28
{Transposition of the composite synthetic transposon TnV (Tn5-Rep(pSC101)) is accompanied by the formation of the mini-plasmid pTnV, containing defective Is50-elements}; Borovok IA; Using thermoelimination (at 42 degrees C) of the thermoinducible coliphage P1tsCmr omega::TnV (TnV is a Tn5 derivative which contains the replication origin (Rep) of plasmid pSC101), more than 110 KmrCms Escherichia coli K12 clones were selected . It was supposed that the KmrCms phenotype could result only from insertion of TnV (Kmr) into E . coli chromosome and the loss of phage (Cms) . It was found that the majority of KmrCms clones (35-90%) contained miniplasmids . Their molecular sizes did not exceed the TnV size (6.1 kb) . The formation of miniplasmids called pTnV was observed both in RecA+ cells (C600) and in RecA- (HB101), more often in the latters . Interestingly, that miniplasmids of only several molecular sizes were detected: from 6.1 kb (pTnV60) to 4.35 kb (pTnV43) . A restriction analysis showed that DNA of the majority of pTnV plasmids had varying deletions (0.3-1.3 kb) of mainly IS50L element which together with IS50R flank TnV . Very low transposition frequencies (approx . 10(-8) Kmr transconjugants per transferred R388) of all pTnV types (including pTnV60 plasmids containing probably microdeletions of the joining "outside" IS50's ends) suggest that pTnV plasmids are not intermediates in TnV transposition . Possibly the circularized TnV derivatives (pTnV's) are side products of the transposition resulting from the abortive attempts of an excised and autonomous transposon molecule to insert into itself . In the present paper the possible mechanisms of the origin of limited pTnV type numbers are also discussed.

Mol Biol (Mosk), 1990 Sep-Oct, 24(5), 1219 - 29
{Dependence of 3'-5-exonuclease activity of a fragment of Klenow DNA polymerase I from Escherichia coli on the length and structure of the cleaved oligonucleotide}; Khalabuda OV et al.; The Km and vmax values for oligothymidylates d(pT)2-16 in reaction of 3'-5'-exonuclease hydrolysis catalyzed by Klenow fragment were measured in the absence and presence of poly(dA) template without the poly(dA), the Km values for oligonucleotides are slightly dependent on their length . The rate of oligothymidylates hydrolysis increases with their length and for d(pT)16 it is about 190-times higher than for d(pT)2 . The addition on poly(dA) does not lead to an essential change of the Km values for d(pT)2-16, but raises the rate of d(pT)2-7 hydrolysis 2-17-fold and at the same time lowers the efficiency of d(pT)8-16 hydrolysis . The Km values for d(pC)10, d(pA)19 and d(pT)10 are nearly the same . However the velocity of d(pC)10 hydrolysis is approximately 1,2 and 7,8-times higher than for d(pA)10 and d(pC)10, respectively d(pC)10, d(pA)10 and d(pT)10 under conditions of interaction with the template-binding site raise the rate of hydrolysis of d(pT)2 combined with the exonuclease center, with various efficiency . Under similar conditions, d(pT)8, d(pT)10 and d(pT)16 as templates activated hydrolysis of d(pT)2 . The dependence of the Klenow fragment exonuclease activity both on the length and structure of the template and on the length of the hydrolyzed oligonucleotide was suggested.

Mol Microbiol, 1990 Sep, 4(9), 1477 - 86
Functional organization of the ends of IS1: specific binding site for an IS 1-encoded protein; Zerbib D et al.; The IS 1-encoded protein InsA binds specifically to both ends of IS1, and acts as a repressor of IS1 gene expression and may be a direct inhibitor of the transposition process . We show here, using DNasel 'foot-printing' and gel retardation, that the InsA binding sites are located within the 24/25 bp minimal active ends of IS1 and that InsA induces DNA bending upon binding . Conformational modification of the ends of IS1 as a result of binding of the host protein integration host factor (IHF) to its site within the minimal ends has been previously observed . Using a collection of synthetic mutant ends we have mapped some of the nucleotide sequence requirements for InsA binding and for transposition activity . We show that sequences necessary for InsA binding are also essential for transposition activity . We demonstrate that InsA and IHF binding sites overlap since some sequence determinants are shared by both InsA and IHF . The data suggest that these ends contain two functional domains: one for binding of InsA and IHF, and the other for transposition activity . A third region, when present, may enhance transposition activity with an intact right end . This 'architecture' of the ends of IS1 is remarkably similar to that of IS elements IS10, IS50 and IS903.

Res Microbiol, 1990 Sep-Oct, 141(7-8), 995 - 1001
Insertion of myoglobin T-cell epitopes into the Escherichia coli alkaline phosphatase; Freimuth P et al.; We are interested in antigen processing mechanisms of antigen-presenting cells, and to what extent the susceptibility of protein antigens to degradation contributes to immunogenicity . Understanding the biochemistry of antigen processing may be essential for reliable prediction of T-cell epitopes and for the design of vaccines that are optimized for T-cell priming . To examine possible effects of protein structural context on antigen presentation, we used genetic engineering techniques to insert helper T-cell epitopes derived from sperm whale myoglobin into surface loops of the highly stable Escherichia coli alkaline phosphatase, with the expectation that presentation of the myoglobin guest epitopes might vary with their position in the carrier protein . Levels of recombinant protein expression in E . coli cells and residual enzyme activity depended on the location of the guest peptides in the alkaline phosphatase carrier . Mutants with insertions between residues 189-190 of the carrier were recovered with yields and activities similar to the wild type protein; however, insertion of the same peptides at a second site, between residues 165-166, led to low recoveries and diminished phosphatase activities . Subcutaneous injection of mice with one of the purified recombinant proteins in complete Freund's adjuvant induced T cells that responded to in vitro challenge with myoglobin . The potential use of this system to dissect processing mechanisms is discussed.

J Korean Med Sci, 1990 Sep, 5(3), 137 - 43
Studies on rat liver nuclear DNA damaged by chemical carcinogen (3'-Me DAB) and AP DNA endonuclease . II . Kinetic properties of AP DNA endonucleases in rat liver chromatin; Kim YS et al.; An experiment was designed to investigate the reaction mechanism of AP (apurinic or apyrimidinic) DNA endonucleases (APcI, APcII, APcIII) purified from rat liver chromatin . Sulfhydryl compounds (2-mercaptoethanol, dithiothreitol) brought about optimal activities of AP DNA endonucleases and N-ethylmaleimide or HgCl2 inhibited the enzyme activities, indicating the presence of sulfhydryl group at or near the active sites of the enzymes . Mg2+ was essential and 4mM of Mg2+ was sufficient for the optimal activities of AP DNA endonucleases . Km values of APcI, APcII and APcIII for the substrate (E . coli chromosomal AP DNA) were 0.53, 0.27 and 0.36 microM AP sites, respectively . AMP was the most potent inhibitor among adenine nucleotides tested and the inhibition was uncompetitive with respective to the substrate . The Ki values of APcI, APcII and APcIII were 0.35, 0.54 and 0.41mM, respectively . The degree of nick translation of AP DNAs nicked by APcI, APcII and APcIII with Klenow fragment in the presence and absence of T4 polynucleotide kinase or alkaline phosphatase were the same, suggesting that all 3 AP DNA endonucleases excise the phosphodiester bond of AP DNA strand to release 3-hydroxyl nucleotides and 5-phosphomonoester nucleotides.

Genomics, 1990 Sep, 8(1), 149 - 54
Human aminoacylase-1: cloning, regional assignment to distal chromosome 3p21.1, and identification of a cross-hybridizing sequence on chromosome 18; Miller YE et al.; Aminoacylase-1 (ACY1, EC 3.5.1.14) is a cytosolic enzyme with a wide range of tissue expression and has been postulated to function in the catabolism and salvage of acylated amino acids . ACY1 has been assigned to chromosome 3p21, a region reduced to homozygosity in small-cell lung cancer and renal cell carcinoma, and has been reported to exhibit reduced or absent expression in small-cell lung cancer cell lines and tumors . Using monoclonal antibodies to human ACY1, we have isolated cDNA clones from a liver lambda gt11 cDNA library . As proof of identity, the fusion protein encoded by a putative ACY1 cDNA displayed ACY1 enzymatic activity . Additionally, it was determined that the putative ACY1 cDNA clones hybridize to an EcoR1 restriction fragment that has been mapped to chromosome 3p . Both ACY1 activity and this restriction fragment have been further demonstrated to be syntenic to distal 3p21.1 through the use of a panel of human-rodent somatic cell hybrids containing fragments of chromosome 3 . An additional EcoR1 restriction fragment to which the probe hybridizes has been assigned to chromosome 18 . The major mRNA species to which the ACY1 cDNA hybridizes is 0.9 kb; faint hybridization to a 4.2-kb mRNA species is also detected . These studies further refine a region of interest in the investigation of gene inactivation in small-cell lung cancer and provide a new marker on chromosome 18.

J Nat Prod, 1990 Sep-Oct, 53(5), 1234 - 40
Differential inhibition of reverse transcriptase and various DNA polymerases by digallic acid and its derivatives; Nakane H et al.; Digallic acid (gallic acid 5,6-dihydroxy-3-carboxyphenyl ester) {4} was found to be a potent inhibitor of the activities of the reverse transcriptases from murine leukemia virus (MLV) and human immunodeficiency virus (HIV) . Under the reaction conditions specified for each of MLV and HIV reverse transcriptases, both enzymes were inhibited by approximately 90% in the presence of 0.5 micrograms/ml digallic acid . Under the same conditions, however, gallic acid had no effect on the reverse transcriptase activity . The mode of the inhibition by digallic acid was partially competitive with respect to the template.primer, (rA)n.(dT)12-18', and noncompetitive to the triphosphate substrate, dTTP . The Ki value of digallic acid for HIV-reverse transcriptase was determined to be 0.58 microM . Examination of several derivatives of digallic acid have shown that all three hydroxyl groups at the 3, 4, and 5 positions seem to be required for the inhibitory activity of these compounds . Besides reverse transcriptase, DNA polymerases alpha and beta were moderately inhibited by digallic acid, whereas DNA polymerase gamma, terminal deoxynucleotidyltransferase, and E . coli DNA polymerase I were virtually insensitive to inhibition by this compound.

Anal Biochem, 1990 Sep, 189(2), 254 - 61
A quantitative assessment for transcriptional pausing of DNA-dependent RNA polymerases in vitro; Theissen G et al.; A simple definition for pause strength (tau i) has been given, quantitatively describing transcriptional pausing of RNA polymerases . It permits derivation of practical assessments, based on single-round transcription reactions, which measure the average time a polymerase stops in vitro at certain sites during transcription elongation . We demonstrate that pause strengths can be determined with high accuracy when transcription elongation is started simultaneously from radioactively labeled and purified ternary complexes and transcripts are separated on sequencing gels . Our concept is exemplified by measuring pause strengths on supercoiled templates in the leader region of the Escherichia coli rrnB operon in the presence and the absence of the transcription factor NusA.

Trop Med Parasitol, 1990 Sep, 41(3), 294 - 6
Transfer RNA genes in mycobacteria: organization and molecular cloning; Tyagi JS et al.; DNAs from nine mycobacterial species were cleaved with different restriction endonucleases and hybridized with cDNA probes synthesized to total transfer RNAs (tRNAs) from Mycobacterium smegmatis and M . tuberculosis . The hybridization data indicate that tRNA genes are conserved but their gross genomic organization has diverged in six of the nine species examined . Species of the MTB complex appeared to have identical tRNA gene organization . Hybridization with cDNAs synthesized to 23S plus 16S rRNAs from Escherichia coli indicate that the tRNA genes map near the rRNA genes . Recombinant plasmids, pSB1, pSB2, pSB4 and pSB8 encoding tRNA(s) and rRNA(s) were isolated from a gene bank of M . tuberculosis H37Rv . Using pSB2 as probe, a SalI RFLP was observed that distinguishes the virulent and avirulent strains of M . tuberculosis H37Rv and H37Ra, respectively.

Mol Gen Mikrobiol Virusol, 1990 Sep, (9), 18 - 23
{Cloning of Brucella genes in Escherichia coli K12 cells and analysis of products of the cloned genes}; Gorelov VN et al.; The libraries of Brucella melitensis 565 and Brucella abortus 99 in Escherichia coli cells have been constructed . Some clones of Escherichia coli producing the specific brucella antigens have been found in immunological tests with brucella antiserum . Two strains producing antigens have been characterized, one being from Brucella melitensis 565 and another from Brucella abortus 99 clone libraries . Both strains synthesize two antigens that were studied by immunoelectrophoresis, immunoblotting after treatment of antigen preparations with different physical and chemical agents substrate specific enzymes . Both strains are found to synthesize the specific brucella antigens of protein nature . One of them has the mol mass about 15 kD, another--31-32 kD . The 31-32 kD antigen can be, evidently, referred to as the main protein of an outer membrane of brucella.

Mol Gen Genet, 1990 Sep, 223(2), 249 - 57
Antibodies against a fused gene product identify the protein encoded by a group II yeast mitochondrial intron; Bergantino E et al.; In the mitochondrial genome of Saccharomyces cerevisiae, introns aI1 and aI2 of the gene encoding the COX1 subunit are the only group II introns with open reading frames (ORFs); these can be translated into two homologous proteins, the maturase aI1 and aI2 . These proteins are structurally related to viral reverse transcriptases and have been shown genetically to be involved in pre-mRNA splicing and in the deletion of introns from mitochondrial DNA . To identify these mitochondrial proteins and study their properties more directly, we raised antibodies against a part of the intron aI2 ORF translation product . For this purpose, we constructed series of fusion genes, by joining parts of the genes for protein A or lacZ to different portions of the intron aI2 . These were expressed in Escherichia coli as hybrid polypeptides, which were used for the production and identification of specific antibodies against the yeast mitochondrial protein . The antibodies recognized the 57 kDa protein (maturase aI2) that accumulates in two yeast mutants deficient in the splicing of aI2 . This protein corresponds to the translation product of the 3' part of intron aI2 and accumulates unaltered in the two cis-acting mutants.

J Clin Microbiol, 1990 Sep, 28(9), 1906 - 12
Monoclonal antibodies against the different subcomponents of colonization factor antigen II of enterotoxigenic Escherichia coli; Lopez-Vidal Y et al.; Monoclonal antibodies (MAbs) against the different coli surface antigens CS1, CS2, and CS3 of colonization factor antigen II (CFA/II) of enterotoxigenic Escherichia coli (ETEC) were generated by fusing F/O myeloma cells with spleen cells from BALB/c mice immunized with different preparations of purified CFA/II . Five hybrids that produced antibodies specific for CS1, CS2, or CS3 in high titer were cloned and propagated . All the anti-CS MAbs were of the immunoglobulin G1 isotype, and all gave single precipitation lines in immunodiffusion tests when reacting with CFA/II-positive E . coli extracts containing the corresponding CS factor . The binding of all the MAbs to solid-phase-bound CFA/II could be completely inhibited by purified CFA/II containing the corresponding CS factor . However, whereas one MAb against CS3 was inhibited by all of 18 different CFA/II-positive strains tested, another anti-CS3 MAb was inhibited by bacteria expressing CS1 and CS3 (CS1 + CS3 strains) or CS3 alone but not by CS2 + CS3 strains, suggesting antigenic differences in CS3 when expressed by different strains . Use of the anti-CS MAbs in slide agglutination, immunodiffusion, or a CFA inhibition enzyme-linked immunosorbent assay revealed differences in the relative distribution of the various CS factors of CFA/II in clinical ETEC isolates from different geographic areas . By using the anti-CS MAbs in an enzyme-linked immunosorbent assay-nitrocellulose replica method, CFA/II-positive colonies could be detected in stool cultures from infected animals without prior isolation of the ETEC organisms.

Hokkaido Igaku Zasshi, 1990 Sep, 65(5), 510 - 6
{Cloning and expression of rat alpha-fetoprotein cDNA in Escherichia coli}; Soda M; AFP is a major serum protein during ontogeny and is synthesized mainly by the mammalian fetal liver and yolk sac . Its synthesis ceases early in postnatal life and its reappearance in the serum in adult is a sign of hepatoma or yolk sac tumor, since these tumors produce AFP . This paper describes the cloning of rat AFP cDNA spanning complete coding region, its expression in E . coli and characterization of this recombinant AFP . The determination of the nucleotide sequence and cell-free translation of purified AFPmRNA suggested that rat AFP was synthesized as a precursor with a signal peptide of 24 amino acids followed by mature AFP of 587 amino acids . An expression vector was constructed with the cDNA and the introduction of the plasmid into E . coli resulted in the production of immunologically reactive AFP with a molecular weight of 65,000 . The recombinant AFP was highly purified by immunoaffinity chromatography followed by SDS-PAGE . Analysis of the amino acids sequence indicated that the product was AFP lacking N-terminal 53 amino acid residues of preAFP.

J Gen Virol, 1990 Sep, 71 ( Pt 9), 2107 - 14
The antigenic structure of dengue type 1 virus envelope and NS1 proteins expressed in Escherichia coli; Mason PW et al.; The antigenic structures of the envelope protein, E, and the non-structural protein, NS1, of dengue type 1 virus (DEN1) have been studied in the form of recombinant fusion proteins expressed in Escherichia coli . Deletion analysis was used to identify two distinct antigenic domains in E that reacted with subsets of antiviral monoclonal antibodies (MAbs) . Domain I of E extends from amino acid residues (aa) 76 to 93 of E; domain II extends from aa 293 to 402 and contains an essential disulphide bridge . MAbs also reacted with several determinants clustered near the N terminus of the NS1 protein (aa 57 to 126) . Recombinant fusion proteins containing E . coli trpE sequences and most of the sequences for either E or NS1 were immunogenic in mice . The antibodies elicited by the E fusion protein reacted with a portion of the protein containing domain II, whereas antibodies elicited by the NS1 fusion protein did not react with the antigenic determinants defined by our MAbs.

Immunology, 1990 Sep, 71(1), 127 - 32
Effects of major histocompatibility genes and antigen delivery on induction of protective mucosal immunity to E . acervulina following immunization with a recombinant merozoite antigen; Lillehoj HS et al.; Intramuscular immunization with the recombinant p250 surface antigen of Eimeria acervulina merozoite (rEAMZp250) or oral inoculation with live recombinant Escherichia coli expressing the rEAMZp250 protein resulted in antigen-specific T-cell and humoral responses and conferred a significant reduction in mucosal parasitism compared to immunization with the negative control antigen preparation . Among the major histocompatibility complex (MHC) (B)-congenic chickens receiving intramuscular immunization, strain .6-2 (B2-B2) showed significant (P less than 0.05) protection to live E . acervulina challenge compared to the other strains examined . In contrast, strains .C-12(B12B12) and .P-13 (B13B13) showed significant protection among the groups given live recombinant E . coli . In general, strains showing enhanced T-cell responses to the rEAMZp250 protein were better protected compared to those showing minimal or no T-cell responses . Thus the results suggest that the B haplotypes of the host and the mode of antigen presentation influence the outcome of protection following an immunization of chickens with recombinant coccidial antigen.

Scand J Immunol, 1990 Sep, 32(3), 297 - 311
Monocyte function in IDDM patients and healthy individuals; Molvig J et al.; Interleukin 1 beta (IL-1 beta) and tumour necrosis factor alpha (TNF-alpha) may be pathogenetically important in insulin-dependent diabetes mellitus (IDDM), which is associated with genes of the HLA region . Since a regulatory role of HLA region genes on monokine production may exist, we looked for an association between the monokine and prostaglandin E2 (PGE2) responses of monocytes (Mo) from 20 healthy males (18-50 years) with HLA-DR types relevant for IDDM susceptibility and resistance (DR1,2, DR1,3, DR1,4, DR3,4) . Monokine assays were established and evaluated and the secretions of IL-1 beta, TNF-alpha, and PGE2 measured in Mo cultures (2h, 6h, 20h) prepared by endotoxin-free techniques and stimulated by low-dose E . coli lipopolysaccharides (LPS) . There were no significant associations between Mo responses and HLA-DR phenotype . Likewise, Mo from DR2 (n = 5) and DR4 (n = 5) homozygous healthy males demonstrated no significant differences in monokine and PGE2 responses of Mo . In the HLA class III region a diallelic TNF-beta gene NcoI polymorphism consisting of alleles of 5.5 kb and 10.5 kb was recently described and associated with susceptibility to autoimmune diseases including IDDM . We report that IL-1 beta and TNF-alpha responses of Mo from TNF-beta 10.5 kb homozygous healthy individuals were significantly higher than for TNF-beta 5.5/10.5 kb heterozygotes . IL-1 beta and TNF-alpha responses of Mo from males (18-35 years) with newly diagnosed (n = 10) and long-standing IDDM (n = 10) and from age- and HLA-DR-matched healthy males (n = 10) were studied . LPS, gamma interferon (IFN), and TNF-alpha-stimulated Mo cultures were investigated . No significant differences were found between Mo responses of IDDM patients and controls . IFN (1000 U/ml) in the presence of LPS significantly potentiated LPS-stimulated Mo TNF-alpha secretion and reduced the levels of IL-1 beta immunoreactivity in Mo lysates . IFN and TNF-alpha did not have any effects on LPS-stimulated Mo secretion of IL-1 beta immunoreactivity . We conclude that Mo IL-1 beta and TNF-alpha production is normal in patients with recent-onset and long-standing IDDM . The interindividual differences in monokine responses may be accounted for by the diallelic human TNF-beta gene polymorphism rather than by HLA class II genes . This observation may be important for understanding the association of certain HLA haplotypes with autoimmune phenomena and disease.

Mutat Res, 1990 Sep-Nov, 236(2-3), 173 - 201
The enzymology of apurinic/apyrimidinic endonucleases; Doetsch PW et al.; Studies on the enzymology of apurinic/apyrimidinic (AP) endonucleases from procaryotic and eucaryotic organisms are reviewed . Emphasis will be placed on the enzymes from Escherichia coli from which a considerable portion of our knowledge has been derived . Recent studies on similar enzymes from eucaryotes will be discussed as well . In addition, we will discuss the chemical and physical properties of AP sites and review studies on peptides and acridine derivatives which incise DNA at AP sites.

Mol Cell Biol, 1990 Sep, 10(9), 4506 - 17
Characterization of a cDNA encoding a cysteine-rich cell surface protein located in the flagellar pocket of the protozoan Trypanosoma brucei; Lee MG et al.; We have characterized a cDNA encoding a cysteine-rich, acidic integral membrane protein (CRAM) of the parasitic protozoa Trypanosoma brucei and Trypanosoma equiperdum . Unlike other membrane proteins of T . brucei, which are distributed throughout the cell surface, CRAM is concentrated in the flagellar pocket, an invagination of the cell surface of the trypanosome where endocytosis has been documented . Accordingly, CRAM also locates to vesicles located underneath the pocket, providing evidence of its internalization . CRAM has a predicted molecular mass of 130 kilodaltons and has a signal peptide, a transmembrane domain, and a 41-amino-acid cytoplasmic extension . A characteristic feature of CRAM is a large extracellular domain with a roughly 66-fold acidic, cysteine-rich 12-amino-acid repeat . CRAM is conserved among different protozoan species, including Trypanosoma cruzi, and CRAM has structural similarities with eucaryotic cell surface receptors . The most striking homology of CRAM is to the human low-density-lipoprotein receptor . We propose that CRAM functions as a cell surface receptor of different trypanosome species.

Infect Immun, 1990 Sep, 58(9), 3116 - 21
Cloning and expression in Escherichia coli of a protective antigen of Erysipelothrix rhusiopathiae; Galan JE et al.; Erysipelothrix rhusiopathiae is a primary pathogen of swine and turkeys and sporadic cause of disease in a variety of other hosts, including humans . A genomic library of the highly virulent strain of E . rhusiopathiae E1-6P was constructed in the expression-cloning vector lambda gt11 and screened with serum from a pig convalescent from an E . rhusiopathiae experimental infection . Immunoreactive clones were screened for their ability to protectively immunized mice . Two clones, lambda gt11/ersA and lambda gt11/ersB, were obtained that protected mice against challenge with E . rhusiopathiae E1-6P . Antisera against the recombinant clones reacted with polypeptides of molecular weights 66,000, 64,000, and 43,000 in detergent-solubilized surface antigen preparations and whole-cell lysates of E . rhusiopathiae . These polypeptides were also the major antigens recognized by convalescent pig serum when reacted with the same preparations . Western immunoblot and Southern blot analysis revealed that the cloned genes and gene products were present in all of the E . rhusiopathiae strains tested.

Infect Immun, 1990 Sep, 58(9), 3029 - 35
Epitopes of Escherichia coli alpha-hemolysin: identification of monoclonal antibodies that prevent hemolysis; Ji GE et al.; The antigenic regions of Escherichia coli alpha-hemolysin were determined by antibody binding to cyanogen bromide (CnBr) fragments of this protein under denatured conditions . Alpha-hemolysin was isolated from filtered culture supernatants of a recombinant strain by a combination of trichloroacetic acid precipitation and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Alpha-hemolysin was used to (i) produce polyclonal rabbit antisera and murine monoclonal immunoglobulin G (IgG) antibodies and (ii) generate CnBr fragments . Rabbit IgG and 13 murine IgG monoclonal antibodies (MAbs) were elicited to alpha-hemolysin as determined by enzyme-linked immunosorbent and immunoprecipitation assays . Antibodies bound to three specific CnBr fragments of alpha-hemolysin in Western blots (immuno-blots) from sodium dodecyl sulfate-polyacrylamide gels: CnBrII (encompassing residues {R} 2 to 160), CnBrV (R 425 to 892), and CnBrVI (R 893 to 1023) . Five MAbs bound to CnBrII, seven MAbs bound to CnBrV, and one MAb bound to CnBrVI . These specific CnBr fragments are predicted to be hydrophilic and charged . There was no antibody binding to the highly hydrophobic CnBrIII (R 161 to 416) . Similar binding patterns were observed when rabbit polyclonal anti-alpha-hemolysin IgG was used . Polyclonal antibodies to alpha-hemolysin readily inhibited hemolysis and its neutralization capacity was 4- to 64-fold more potent than neutralizing MAbs . The five MAbs that bind to CnBrII possessed hemolytic neutralizing activity to various degrees . In contrast, only three of seven MAbs that bind to CnBrV fragment exhibited neutralization capacity to various degrees; the MAb to CnBrVI did not exhibit this capacity . Based on these data, we predict that denatured alpha-hemolysin and its CnBrII and CnBrV fragments might be worthwhile immunoprophylactic candidates for the prevention of hemolysin-mediated E . coli tissue injury.

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