Bioorg Khim, 1991 Apr, 17(4), 461 - 9 {Synthesis, cloning, and expression of artificial genes, coding antigenic determinants of the foot-and-mouth virus substrain A22}; Korobko VG et al.; Chemical-enzymatic synthesis and cloning in Escherichia coli of double-stranded DNAs, coding for simple and complex antigenic determinants of foot-and-mouth disease virus (FMDV) strain A22, have been carried out . The simple antigenic determinants are a part of the viral coat protein VP1 (amino acid sequence 131-152 or 131-160) whereas the complex antigenic determinants comprise additionally the amino acid sequence 200-213 of VP1 linked to N-terminus of simple antigenic determinants through a tetrapeptide spacer Pro-Pro-Ser-Pro . Recombinant DNAs containing genes for antigenic determinants of FMDV fused with C-terminus of gene for human tumor necrosis factor (hrTNF) have been constructed . Expression of the hybrid genes and properties of the proteins coded were studied . All recombinant proteins were shown to interact specifically with polyclonal antibodies both against hrTNF and FMDV strain A22 . The recombinant proteins produced by bacteria are perspective for study as a vaccine against FMDV. Plant Mol Biol, 1991 Apr, 16(4), 615 - 25 Effect of light on the NADPH-protochlorophyllide oxidoreductase of Arabidopsis thaliana; Benli M et al.; A cDNA encoding the NADPH-protochlorophyllide oxidoreductase (Pchlide reductase) of Arabidopsis thaliana has been isolated and sequenced . The cDNA contains the complete reading frame for the precursor of the Pchlide reductase . The deduced amino acid sequence of the Arabidopsis enzyme closely resembles the corresponding sequences of barley and oat . The cDNA has been used as a template for the synthesis of the enzyme protein in Escherichia coli . An antiserum was raised against this enzyme protein and both the antiserum and the cDNA were used as experimental tools to study the effects of light on the Pchlide reductase in A . thaliana . When etiolated seedlings of Arabidopsis were exposed to light the enzyme activity and the concentration of the enzyme protein rapidly declined . Similar light effects have been described previously for other angiosperms . In contrast to most of these species, however, in Arabidopsis only minor changes in Pchlide reductase mRNA content could be observed when etiolated seedlings were exposed to light. Mol Microbiol, 1991 Apr, 5(4), 875 - 86 Structure and function of periplasmic chaperone-like proteins involved in the biosynthesis of K88 and K99 fimbriae in enterotoxigenic Escherichia coli; Bakker D et al.; The nucleotide sequence of faeE and fanE, two genes involved in the biosynthesis of K88 and K99 fimbriae, respectively, was determined and the amino acid sequence of the FaeE and FanE proteins was deduced . Immunoblotting of subcellular fractions with an antiserum raised against purified FaeE confirmed that FaeE is located in the periplasm . Indications were obtained that FaeE functions as a chaperone-like protein . Its interaction with the fimbrial subunit (FaeG) in the periplasm stabilized this polypeptide and prevents its degradation by the cell-envelope protease DegP . Furthermore, FaeE prevents the formation of FaeG multimers which cannot be incorporated into fimbriae . The reactions of the FaeE/FaeG dimers with a set of monoclonal antibodies directed against the various epitopes present on K88 fimbriae revealed that the fimbrial subunits associated with FaeE were present in a conformation resembling their native configuration . Indications about the domains in FaeG involved in the interaction with FaeE are discussed. Mol Microbiol, 1991 Apr, 5(4), 857 - 64 Genetic studies of cleavage-initiated mRNA decay and processing of ribosomal 9S RNA show that the Escherichia coli ams and rne loci are the same; Melefors O et al.; We show in the present paper that the cleavages initiating decay of the ompA mRNA are suppressed both in the Escherichia coli ams(ts) strain (originally defined by a prolonged bulk mRNA half-life) and in the me(ts) strain (originally defined by aberrant 9S RNA processing) . The temperature-sensitive defects of both these strains are complemented by a recombinant lambda phage containing a genomic segment that carries the putative ams locus . A 5.8 kb fragment from this genomic DNA segment was cloned into a low-copy plasmid and used to transform the ams(ts) and rne(ts) strains . This resulted in growth at the non-permissive temperature and a reoccurrence of the cleavages initiating decay of the ompA mRNA . Deletion analyses of this 5.8 kb fragment indicated that the putative ams open reading frame could complement both the Ams(ts) and the Rne(ts) phenotype with regard to the ompA cleavages . In addition we showed that the ams(ts) strain suppresses 9S RNA processing to 5S RNA to the same extent as the rne(ts) strain, and that the rne(ts0 strain has a prolonged bulk mRNA half-life, as was reported for the ams(ts) strain . Therefore we suggest that ams and rne reflect the same gene locus; one which is involved both in mRNA decay and RNA processing . We discuss how this gene locus may related to the previously characterized endoribonucleolytic activities of RNase E and RNase K. Mol Microbiol, 1991 Apr, 5(4), 851 - 5 The gene specifying RNase E (rne) and a gene affecting mRNA stability (ams) are the same gene; Taraseviciene L et al.; A DNA clone complementing the rne-3071 mutation has been expressed and localized in the physical map of Escherichia coli . The DNA fragment from this clone was localized to the region of the E . coli chromosome where the rne-3071 mutation has been mapped . The position of this DNA fragment in the E . coli chromosome, the size of the product directed by this DNA fragment (110,000 Da), the restriction map of this fragment, the fact that the same clone complements the ams mutation, and the observation that the rne-3071 and the ams mutations cause similar patterns of RNA synthesis, show that the rne gene--a gene specifying the processing endonuclease RNase E--and the ams gene--a gene that affects mRNA stability--are identical. J Virol Methods, 1991 Apr, 32(1), 1 - 10 Competition ELISA using a human monoclonal antibody for detection of antibodies against human immunodeficiency virus type 1; Bugge TH et al.; A novel competition ELISA for detection of antibodies against HIV-1 was developed . The assay is based on competition at the single epitope level and utilises a human monoclonal antibody and an E . coli-produced fragment of the transmembrane glycoprotein gp41 . The sensitivity of the assay was 100% in tests on 247 serum samples obtained from 219 individuals previously shown to be HIV-1 antibody positive by both conventional indirect ELISA and the immunoblotting test . The patients represented various clinical and immunological stages of HIV-1 infection . Likewise, the specificity of the assay was 100% in tests on 105 serum samples from normal individuals previously tested negative by indirect ELISA . Further, among 105 serum samples selected due to consistent false positive reactions in the indirect ELISA only 2 samples (1.9%) demonstrated false positive reactions in the competition ELISA, i.e . 98.1% specificity . Finally, only 2 of 57 (3.5%) serum samples from HIV-2 infected individuals showed positive reactions in the assay, while 54 (94.7%) had absorbance values similar to the negative controls . These results demonstrate that human monoclonal antibodies may form the basis for highly sensitive and specific assays for detection of antibodies to HIV-1. J Pharmacol Exp Ther, 1991 Apr, 257(1), 107 - 13 Effect of atrial natriuretic factor and 8-bromo cyclic guanosine 3':5'-monophosphate on {3H}acetylcholine outflow from myenteric-plexus longitudinal muscle of the guinea pig; Matusak O et al.; We report that atrial natriuretic factor (ANF) inhibits electrically induced cholinergic twitches of longitudinal muscle in whole intestinal segments and myenteric-plexus longitudinal muscle (MPLM) strips from the guinea pig ileum . To elucidate the possible presynaptic mechanism of ANF's action, we studied spontaneous and stimulation-evoked radiolabeled acetylcholine (ACh) outflow from MPLM after incubation with {3H}choline . We developed a method of mounting and treating MPLM preparations, which allowed us, at the same time, to record isometric contractions and to determine {3H}ACh outflow upon electrical stimulation by a train of three pulses . ANF (5 x 10(-8) M), norepinephrine (2 x 10(-7) M) and 8-bromoguanosine 3':5'-cyclic monophosphate (10(-3) M) in nearly equieffective concentrations caused a similar inhibition of cholinergic twitches . However, ANF had no effect on {3H}ACh outflow, whereas norepinephrine was found to suppress {3H}ACh outflow and 8-bromoguanosine 3':5'-cGMP to enhanced {3H}ACh outflow . ANF (5 x 10(-8) M) caused a 7.0-fold increase of cGMP over control values, predominantly in muscle layers, whereas Escherichia coli heat-stable toxin (12.5 U/ml) elicited a 35-fold increment of cGMP in the extramuscular layer . Thus, ANF is able to elevate cGMP in intestinal smooth muscle and to inhibit cholinergic contractions of MPLM . This inhibition is mimicked by exogenous cGMP and by endogenously generated cyclic nucleotides . We suggest that the depressive action of ANF on cholinergic contractions of MPLM is mediated via its postsynaptic impact implicating elevation of cGMP in smooth muscle. Virology, 1991 Apr, 181(2), 671 - 86 A prominent antigenic surface polypeptide involved in the biogenesis and function of the vaccinia virus envelope; Gordon J et al.; Polypeptides of the vaccinia virus envelope exposed on the surface were identified by means of sulfo-N-hydroxysuccinimidobiotin as a surface tag . Among surface expressed polypeptides is the 35-kDa antigen, previously designated Ag35 . Both monoclonal (mAb) and monospecific affinity pure antibodies directed against Ag35 neutralized vaccinia infectiousness, indicating that this prominent surface antigen has a function during early virus-host cell interactions . The binding of several monoclonal antibodies to various regions of Ag35 was tested by reacting CNBr fragments, derived from the polypeptide, employing Western blotting . All mAbs tested reacted with the same region of Ag35 . Estimation of the molecular weights (MW), based on migration of the CNBr peptides in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that those partial digestion products which contained a proline-rich 99 amino acid limit digest fragment were present at a position approximately 12.5 kDa larger than that predicted from the DNA sequence . By contrast, partial and limit digest products lacking the proline-rich fragment migrated to the MW position expected from the length of the DNA sequence . This observation demonstrates that departure from a predicted 22.3 kDa to an anomalous MW of Ag35 is conferred by the proline-rich peptide . The surface location of Ag35 was confirmed by immune electron microscopy . In a competition test the binding specificity of mAb and affinity-purified antibodies at the surface of virions could be demonstrated . Evidence for an association of Ag35 with the virus envelope at various stages during biogenesis of vaccinia was obtained by immune electron microscopy of whole mounts and thin sections . Presence of Ag35 as an early component of immature and mature virions, probably residing in the bilayer membrane structure was detected . A distinction can, therefore, be made between Ag35 and several other vaccinia envelope polypeptides which are synthesized as late functions and added during late stages of envelope assembly. EMBO J, 1991 Apr, 10(4), 893 - 902 The dimensions of the T lymphocyte glycoprotein leukosialin and identification of linear protein epitopes that can be modified by glycosylation; Cyster JG et al.; Leukosialin (CD43) is a major glycoprotein of T lymphocytes whose extracellular domain of 224 amino acids contains on average one O-linked carbohydrate unit per three amino acids . This suggests an unfolded structure for the extracellular domain which has now been established to extend to a length of 45 nm by transmission electron microscopy following low angle rotary shadowing . The antigenicity of rat leukosialin has been studied using nine monoclonal antibodies (MAbs) whose binding is differentially affected by the cell type on which leukosialin is expressed and by the removal of sialic acid . From these observations it appears that the epitopes are affected by glycosylation, yet seven of the nine MAbs reacted clearly with the extracellular domain of leukosialian expressed in an unglycosylated form in Escherichia coli . The MAbs showing this positive reaction included three of the four antibodies whose epitopes were affected by neuraminidase treatment of leukosialin . It thus appears that linear protein epitopes are recognized and that some of these can be modified in the native structure by glycosylation . The positions of the antigenic determinants have been mapped by expressing fusion proteins of different lengths and the identity of one epitope was proven by the binding of two MAbs to an octapeptide expressed as a fusion protein . For three MAbs, the location of epitopes in the native protein was confirmed by electron microscopy of shadowed leukosialin--Fab complexes . Overall it is concluded that leukosialin is a major component at the periphery of the T lymphocyte and that despite its high level of glycosylation, protein determinants are exposed that could be ligands in cell interactions. J Bacteriol, 1991 Apr, 173(7), 2378 - 84 Transcription of the stability operon of IncFII plasmid NR1; Min YN et al.; The stability (stb) locus of IncFII plasmid NR1 is composed of an essential cis-acting DNA site located upstream from two tandem genes that encode essential stability proteins . The stb locus was found to be transcribed from a promoter site just upstream from the first gene, stbA . This promoter was active for transcription both in vivo and in vitro and was located within the region that includes the essential cis-acting site . Transcripts initiated from this site were approximately 1,500 to 1,600 nucleotides in length . Northern (RNA) blot analysis indicated that the transcripts traversed both stbA and the downstream gene, stbB . Mutants from which the promoter had been deleted failed to produce detectable transcripts from either stbA or stbB . Transcription of a third open reading frame, stbC, which is contained within the stbB gene in the opposite DNA strand, could not be detected . For a mutant in which a transposon had been inserted in stbA, no transcription of stbB was detected . After deletion of most of the transposon, which left behind a 35-bp frameshift insertion in stbA, transcription of stbB was restored, although the insertion still had a polar effect on stbB function . The rate of in vivo transcription of the stb locus was measured by pulse-labeling of RNA followed by quantitative RNA-DNA hybridization . Mutants deleted of stbB had an approximately 10-fold increase in the rate of transcription, whereas those deleted of the promoter region had at least a 10-fold reduction in transcription rate . The half-life of stb mRNA was approximately 2 min . These data suggest that stbA and stbB are cotranscribed as an operon that may be autoregulated. J Bacteriol, 1991 Apr, 173(7), 2328 - 40 Specificity of attenuation control in the ilvGMEDA operon of Escherichia coli K-12; Chen JW et al.; Three different approaches were used to examine the regulatory effects of the amino acids specified by the peptide-coding region of the leader transcript of the ilvGMEDA operon of Escherichia coli K-12 . Gene expression was examined in strains carrying an ilvGMED'-lac operon fusion . In one approach, auxotrophic derivatives were starved of single amino acids for brief periods, and the burst of beta-galactosidase synthesis upon adding the missing amino acid was determined . Auxotrophic derivatives were also grown for brief periods with a limited supply of one amino acid (derepression experiments) . Finally, prototrophic strains were grown in minimal medium supplemented with single and multiple supplements of the chosen amino acids . Although codons for arginine, serine, and proline are interspersed among the codons for the three branched-chain (regulatory) amino acids, they appeared to have no effect when added in excess to prototrophs or when supplied in restricted amounts to auxotrophs . Deletions removing the terminator stem from the leader removed all ilv-specific control, indicating that the attenuation mechanism is the sole mechanism for ilv-specific control. J Bacteriol, 1991 Apr, 173(7), 2256 - 64 Structure and organization of Escherichia coli genes involved in biosynthesis of the deazaguanine derivative queuine, a nutrient factor for eukaryotes; Reuter K et al.; The plasmid pPR20 contains the gene tgt, which encodes tRNA guanine transglycosylase (Tgt), on a 33-kbp DNA insert from a region around 9 min on the Escherichia coli linkage map . The plasmid was subcloned to determine the sequence and organization of the tgt gene . Tgt is a unique enzyme that exchanges the guanine residue with 7-aminomethyl-7-deazaguanine in tRNAs with GU(N) anticodons . After this exchange, a cyclopentendiol moiety is attached to the 7-aminomethyl group of 7-deazaguanine, resulting in the hypermodified nucleoside queuosine (Q) . Here we give the complete sequence of a 3,545-bp StuI-BamHI DNA fragment where we found the tgt gene and three previously unknown genes encoding proteins with calculated molecular masses of 42.5 (Tgt), 14, 39, and 12 kDa . The gene products were characterized on sodium dodecyl sulfate gels after synthesis in a combined transcription-translation system . The mRNA start sites of the open reading frames (ORFs) were determined by primer extension analysis . Plasmids containing the ORF encoding the 39-kDa protein (ORF 39) complemented a mutation in Q biosynthesis after the Tgt step . This gene was designated queA . The genes are arranged in the following order: ORF 14 (transcribed in the counterclockwise direction), queA, tgt, and ORF 12 (all transcribed in the clockwise direction) . The organization of the promoter sequences and the termination sites suggests that queA, tgt, and ORF 12 are localized on a putative operon together with the genes secD and secF. J Bacteriol, 1991 Apr, 173(7), 2225 - 30 First step toward a virus-derived vector for gene cloning and expression in spiroplasmas, organisms which read UGA as a tryptophan codon: synthesis of chloramphenicol acetyltransferase in Spiroplasma citri; Stamburski C et al.; Spiroplasmas are wall-less procaryotes in which the UGA codon serves not as a stop signal but as a code for the amino acid tryptophan . Spiroplasma genes that contain UGA codons thus cannot be studied in the usual Escherichia coli cloning and expression systems . Although this problem can be circumvented by using UGA-suppressor strains of E . coli, spiroplasmas themselves would provide a more efficient cloning and expression host . We have now successfully employed the replicative form (RF) of a filamentous spiroplasma virus (SpV1) to clone and express the E . coli-derived chloramphenicol acetyltransferase (CAT) gene in Spiroplasma citri . The CAT gene was inserted in one of the four intergenic regions of the SpV1 RF and introduced into cells by electroporation . Both the RF and the virion DNA produced by the transfected cells contained the CAT gene sequences . Northern blot analysis, primer extension, and S1 mapping showed that transcription of the CAT gene started from a promoter located on the SpV1 RF and was terminated downstream of the CAT gene, still within the viral RF . Expression of the CAT gene was demonstrated by acetylation of chloramphenicol by cell-free extracts from the transfected spiroplasmas. J Bacteriol, 1991 Apr, 173(7), 2167 - 72 Multiple determinants of functional mRNA stability: sequence alterations at either end of the lacZ gene affect the rate of mRNA inactivation; Petersen C; The Escherichia coli lacZ gene was used as a model system to identify specific sequence elements affecting mRNA stability . Various insertions and substitutions at the ribosome-binding site increased or decreased the rate of mRNA inactivation by up to fourfold . Deletion of a dyad symmetry, which may give rise to a very stable secondary structure in the mRNA immediately downstream of the gene, decreased the functional stability of the lacZ message . The magnitude of the latter effect was strongly dependent on the sequences at the ribosome-binding site, ranging from practically no effect for the most labile transcripts to a threefold decrease in stability for the most stable one . The results suggest that the wild-type lacZ message is inactivated predominantly by attacks near the ribosome-binding site, presumably in part because the putative secondary structure downstream of the gene protects against 3'-exonucleolytic attack . Taken together, the data for all of the modified variants of lacZ were shown to be quantitatively compatible with a general model of mRNA inactivation involving multiple independent target sites. Cancer Res, 1991 Apr 1, 51(7), 1876 - 82 Preparation of synthetic polypeptide domains of carcinoembryonic antigen and their use in epitope mapping; Hass GM et al.; Genes encoding the four principal polypeptide domains (N, A1-B1, A2-B2, and A3-B3) of carcinoembryonic antigen (CEA) were synthesized and expressed in Escherichia coli as fusion products with bacterial CMP-KDO synthetase (CKS) . The four synthetic fusion proteins were purified in high yield and used as targets in Western blots for 11 anti-CEA MAbs and to compete with immobilized CEA for binding to four of these MAbs . Each of the MAbs showed strong binding to one or more of the fusion proteins . In Western blots, MAbs H19C91 and 4230 bound only to CKS-N . MAbs H8C2 and H11C35 bound only CKS-A1-B1, and MAbs T84.66, H46C136, and H21C83 appeared to be specific for CKS-A3-B3 . None of the MAbs tested bound only to CKS-A2-B2 . However, two MAbs bound both CKS-A1-B1 and CKS-A3-B3 and one MAb (3519) bound to all three of the repeated domains . Since these three domains exhibit over 90% amino acid sequence homology, the latter results were not surprising . The competition studies largely confirmed the results of Western blots but did show some MAb-fusion protein interactions not observed in Western blots . These competition studies also allowed estimation of the relative affinities of the MAbs for the synthetic domains and for native CEA . These studies demonstrated that epitopes in CEA recognized by the MAbs in this study are peptide in nature and that the fusion proteins are of utility in the localization of the epitopes on the polypeptide chain of CEA. Microb Pathog, 1991 Apr, 10(4), 271 - 80 Serological conservation and location of the adhesin of avian Escherichia coli type 1 fimbriae; Chanteloup NK et al.; By inoculation of mice with purified type 1-like fimbriae isolated from an avian Escherichia coli strain, a monoclonal antibody (mAb G5) was obtained . mAb G5 reacted in an enzyme-linked immunosorbent assay (ELISA) with type 1-like and type 1A fimbriae differing in the molecular masses of their major fimbrial subunit and isolated from several avian E . coli strains . The specificity of mAb G5 for type 1 fimbriae was assessed in a whole bacteria ELISA with 16 reference E . coli strains expressing different types of fimbriae . Immunoblotting experiments showed that mAb G5 recognized the 29 kDa minor component of reference type 1A fimbriae which has been identified as the adhesin . mAb G5 also recognized the 29 kDa component of type 1-like and type 1A fimbriae expressed by avian E . coli strains, suggesting that the adhesin is antigenically conserved among these fimbriae . Immunoelectron microscopic studies gave evidence that the adhesin could be located mainly at the tip or both at the tip and along the fimbriae, depending on the strain. J Vet Diagn Invest, 1991 Apr, 3(2), 115 - 8 Comparison of seroagglutination, ELISA, and indirect fluorescent antibody staining for the detection of K99, K88, and 987P pilus antigens of Escherichia coli; Mullaney CD et al.; Seroagglutination (SAT), enzyme-linked immunosorbent assay (ELISA), and indirect fluorescent antibody staining tests (IFAT) were compared for reliability in the detection of pilus antigens K99, K88, and 987P of Escherichia coli . Test sensitivities were compared using mixtures of piliated bacteria of several strains diluted to a constant optical density with a nonpiliated strain . Relative sensitivities and specificities of the 3 tests were also compared using 55 E . coli strains that had previously been serotyped and characterized for pilus genes by DNA probe . Although specificity was not a serious problem with any of the tests, the SAT was relatively nonsensitive . The IFAT showed the greatest sensitivity of the 3 tests in detecting K88, K99, and 987P E . coli. Int J Food Microbiol, 1991 Apr, 12(4), 333 - 8 Use of probes to detect virulence factor DNA sequences in Escherichia coli strains isolated from foods; Franco BD et al.; Escherichia coli strains were isolated from 96 food samples (32 milks, 4 dairy products, 36 raw meats, 7 meat products, 7 sandwiches and 10 ready-to-eat meals) . A total of 306 colonies was submitted to hybridization assays with DNA probes for the following virulence factors: heat-labile toxins (LT-I and LT-II), heat-stable toxins (ST-h and ST-p) . Shiga-like toxins (SLT-I and SLT-II), adherence factor of enteropathogenic E . coli (EAF) and invasive factor (INV) . Six colonies isolated from 4 food samples hybridized with the probes for LT-II (3 colonies isolated from a milk sample), SLT-I and SLT-II (1 colony isolated from raw bovine meat) or EAF (2 colonies isolated from two raw chicken meat samples). Biochem Cell Biol, 1991 Apr, 69(4), 232 - 8 A mutant of Escherichia coli citrate synthase that affects the allosteric equilibrium; Anderson DH et al.; We describe a mutant of Escherichia coli citrate synthase, CS R319L, in which the arginine residue at position 319 of the sequence has been replaced by leucine . In this mutant, saturation by the substrate acetyl-CoA is changed from sigmoid (Hill parameter = 1.75 +/- 0.2) to hyperbolic (Hill parameter = 1.0 +/- 0.1) and dependence on the activator KCl is greatly reduced . Further mutations at this site and at position 343 (which model building predicts is close enough to allow a side-chain interaction with position 319) are also described . In the wild-type enzyme, the model suggests the possibility of a salt-bridge interaction between Arg-319 (located on the P helix in the small domain) and Glu-343 (in the Q helix in the same domain), but mutation of Glu-343 to Ala (CS E343A) produced a much smaller difference in the kinetic properties than the ARg-319 to Leu mutation did . Small changes in kinetic properties were also obtained with an Arg-319----Glu (CS R319E) mutation . In CS R319L, oxaloacetate, the first substrate to bind, induces an ultraviolet difference spectrum which is obtained with wild-type enzyme only in the presence of KCl . To account for these observations we postulate that wild-type E . coli citrate synthase exists in two conformational states, T and R, which are equilibrium; T state binds NADH, the allosteric inhibitor, while R state binds substrates and can be converted to another substrate-binding state, R', by KCl.(ABSTRACT TRUNCATED AT 250 WORDS) Am J Vet Res, 1991 Apr, 52(4), 523 - 7 Influence of an omega-3 fatty acid-enriched ration on in vivo responses of horses to endotoxin; Henry MM et al.; Because certain inflammatory processes are dependent on the fatty acid composition of the cellular membrane, dietary manipulations that replace omega-6 fatty acids with omega-3 fatty acids may modify inflammatory responses . We investigated the effect of supplemental dietary linseed oil, containing the omega-3 fatty acid, alpha-linolenic acid, on in vivo responses of horses to endotoxin . One group of horses (n = 6) was fed a control pelleted ration (0% linseed oil), and another group of horses (n = 6) was fed an 8% linseed oil pelleted ration . After 8 weeks of consuming these rations, all horses were given 0.03 micrograms of Escherichia coli 055:B5 endotoxin/kg of body weight, infused over 30 minutes . Horses were monitored over 24 hours . Compared with baseline values within each ration group, endotoxin infusion caused significant (P less than 0.05) increase in rectal temperature, heart rate, and plasma concentration of thromboxane B2, 6-keto-prostaglandin F1 alpha, and fibrinogen and significant (P less than 0.05) decrease in total WBC count . Compared with baseline values within each ration group, endotoxin infusion failed to cause significant changes in prothrombin, activated partial thromboplastin, thrombin, or whole blood recalcification times, serum concentration of fibrin degradation products, PCV, or plasma total protein concentration . Before and after endotoxin infusion, horses given the linseed oil ration had longer mean whole blood recalcification time and activated partial thromboplastin time than did horses fed the control ration. Int J Radiat Biol, 1991 Apr, 59(4), 941 - 9 Negative supercoiling increases the sensitivity of plasmid DNA to single-strand break induction by X-rays; Miller JH et al.; Negatively supercoiled topoisomers of the plasmid pIBI 30 were irradiated with 250 kV X-rays and assayed for strand scission by agarose gel electrophoresis . The survival of supercoiled molecules (Form I) decreased exponentially with increasing X-ray exposure and the dose required to reduce the fraction of DNA in Form I to 37% of its value in unirradiated controls (D37) decreased with increasing negative superhelicity . This enhanced radiation sensitivity of underwound DNA is tentatively attributed to the transient denaturation of the double helix that increases the susceptibility of individual strands to free radical attack. Biochem J, 1991 Apr 1, 275 ( Pt 1), 29 - 34 Activation of intestinal brush border guanylate cyclase by aromatic disulphide compounds; elDeib MM et al.; Guanylate cyclase in pig intestinal brush border membranes was stimulated by certain aromatic disulphides . The most effective were 6-thioguanine disulphide {(TGS)2}, 6-mercaptopurine disulphide, 6,6'-dithiodinicotinic acid, 5,5'-dithiobis-(2-nitrobenzoic acid) and 5-carboxy-2-thiouracil disulphide . (TGS)2 stimulated activity 15-fold when present at 0.1 mM . The optimum concentration for each disulphide was different, and higher concentrations were inhibitory . There was no activation by alkyl disulphides or by N-ethylmaleimide . Activation by 50 microM-(TGS)2 was partially reversed by later addition of 0.1 mM-dithiothreitol, whereas activation by the Escherichia coli heat-stable enterotoxin STa was relatively unaffected . Pretreatment of the membranes with (TGS)2 produced a concentration-dependent inhibition of STa-stimulated activity, while stimulating basal activity, until the activities were equal at 50 microM . Activity was {Mg2+}-dependent, the optimal {Mg2+} progressively increasing as the enzyme was stimulated by (TGS)2, STa and Lubrol PX respectively . However, (TGS)2 pretreatment prevented the shift to higher {Mg2+}optima induced by STa or Lubrol alone . Substitution of Mn2+ for Mg2+ in the reaction elevated basal activity and eliminated by activation (TGS)2 . (TGS)2 only inhibited Mn2(+)-dependent activity (both basal and stimulated) . The affinity of 125I-STa for its receptor was slightly increased by (TGS)2 . We propose that (TGS)2 undergoes thiol-disulphide exchange with at least three different protein thiols of decreasing reactivity . The first is associated with Mg2(+)-dependent activation, the second is associated with a tonic inhibition of activity and the third is associated with the catalytic activity, although probably not at the active site. Infect Immun, 1991 Apr, 59(4), 1552 - 7 Activation of particulate guanylate cyclase by Escherichia coli heat-stable enterotoxin is regulated by adenine nucleotides; Gazzano H et al.; Guanylate cyclase is regulated by adenine nucleotides in membranes of intestinal mucosal cells . Basal guanylate cyclase was activated about twofold by adenine nucleotides . Activation was specific for adenine, as compared with the pyrimidine nucleotides UTP and CTP . In addition, enzyme activation was obtained in the presence of saturating concentrations of GTP, the substrate for guanylate cyclase . The most potent adenine nucleotide was the nonhydrolyzable analog of ATP, adenosine 5'-O-(3-thiotriphosphate) . Adenine nucleotide activation was specific for the particulate form of guanylate cyclase, as compared with the soluble form . Also, adenine nucleotides potentiated the activation of guanylate cyclase by the heat-stable enterotoxin produced by Escherichia coli . Indeed, enzyme activation by adenine nucleotides and toxin was greater than the sum of individual activations by these agents . Adenine nucleotides regulate guanylate cyclase by increasing the maximum velocity of the enzyme without altering its affinity for substrate or its cooperativity . In addition to stimulating guanylate cyclase, adenine nucleotides decreased the specific binding of the heat-stable enterotoxin to receptors in intestinal membranes . The coordinated regulation of the toxin-receptor interaction and guanylate cyclase activity by a process utilizing nonhydrolyzable analogs of a purine nucleotide is similar to the mechanisms involved in the hormone regulation of adenylate cyclase by guanine nucleotide-binding proteins . These data suggest that an adenine nucleotide-dependent protein may couple the toxin-receptor interaction to the regulation of particulate guanylate cyclase in intestinal membranes. Gastroenterology, 1991 Apr, 100(4), 946 - 53 Role of free radicals and platelet-activating factor in the genesis of intestinal motor disturbances induced by Escherichia coli endotoxins in rats; Pons L et al.; The effects of IV administration of Escherichia coli endotoxin on intestinal myoelectric activity was investigated in conscious fasted rats chronically implanted with nichrome electrodes in the duodenojejunum . These effects were compared with those of platelet-activating factor and were evaluated in animals pretreated with a specific platelet-activating factor antagonist, BN 52021, indomethacin, a selective prostaglandin E2 antagonist, SC 19220, and several free radical scavengers . Intravenous administration of endotoxin (E . coli S.O111:B4) at a dose of 50 micrograms/kg suppressed the migrating myoelectric complexes, which were replaced by continuous rhythmic clusters of rapidly propagated spike bursts for 114.7 +/- 19.9 minutes . Intraperitoneal platelet-activating factor (25 micrograms/kg) also inhibited the migrating myoelectric complex pattern for 146.1 +/- 24.1 minutes . Previous IV administration of BN 52021 (50 mg/kg-1) abolished the motor alterations induced by platelet-activating factor and significantly reduced to 43.1 +/- 12.2 minutes those induced by endotoxin (P less than 0.01) . Indomethacin (10 mg/kg IP), injected before endotoxin or platelet-activating factor, also significantly reduced the duration of migrating myoelectric complex inhibition to 45.6 +/- 7.8 and 47.7 +/- 8.3 minutes, respectively (P less than 0.01) . SC 19220 significantly reduced the effects of platelet-activating factor from 151.8 +/- 26.4 to 67.4 +/- 14.7 min (P less than 0.01) . Superoxide dismutase (15,000 U/kg IV) injected before either endotoxin or platelet-activating factor shortened the migrating myoelectric complex inhibition to 45.7 +/- 9.9 and 72.9 +/- 10.4 minutes, respectively (P less than 0.01) . Allopurinol and dimethylsulfoxide administered orally at 50 mg/kg 1 hour before endotoxin reduced the migrating myoelectric complex inhibition to 42.5 +/- 6.5 and 38.2 +/- 6.4 minutes, respectively (P less than 0.01) . They also reduced platelet-activating factor-induced intestinal myoelectric alterations to 68.5 +/- 10.6 and 31.7 +/- 6.1 minutes, respectively (P less than 0.01) . It is concluded that endogenous release of platelet-activating factor is partly responsible for the intestinal motor alterations induced by endotoxin, these effects being also mediated through the release of prostaglandins and free radicals . However, prostaglandins, as well as free radicals, appear to be partly involved in the platelet-activating factor-induced action of E . coli endotoxin on intestinal motility. Protein Expr Purif, 1991 Apr-Jun, 2(2-3), 175 - 8 Refolding and purification of human papillomavirus type 16 E7-lacZ fusion protein expressed in Escherichia coli; Chinami M et al.; Human papillomavirus (HPV) type 16 E7-lacZ fusion protein was produced in Escherichia coli, extracted as inclusion bodies, refolded with reducing reagents, and subjected to gel filtration . The refolded protein was purified by ion-exchange column chromatography, resulting in a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . 1H nuclear magnetic resonance spectral changes were observed in the high field methyl region in the presence of Zn2+ ion, suggesting that the refolded form of the fusion protein is possibly renaturated into the putative zinc finger motif (C . Edmond and K . H . Vousden, 1989, J . Virol . 63, 2650-2656) and supporting the data of J . A . Rawls, R . Pusztai, and M . Green (1990, J . Virol . 64, 6121-6129) on zinc binding to E7 protein using radioisotopically labeled zinc ion. Endocrinol Jpn, 1991 Apr, 38(2), 205 - 12 Evidence for the presence of protein kinases which stimulate phosphorylation of c-erb A protein in rat kidney nuclei; Hashizume K et al.; Protein kinases were separated from rat kidney nuclear extract by hydroxylapatite column chromatography . Five (I-V) different protein kinases were isolated when histone was used as a substrate . Two (I and III) of them stimulated phosphorylation of c-erb A-beta protein (50 kDa) expressed in Escherichia coli . The c-erb A product has an activity of high affinity T3 binding . One (I) of the kinases was dependent on cyclic adenosine 3',5'-monophosphate (cyclic AMP) . The other kinase (III) was not dependent on cyclic nucleotides . The latter kinase was eluted from hydroxylapatite column with 0.05 M PO4 at pH 7.4 . The sedimentation coefficient(s) estimated by continuous sucrose density gradient centrifugation was approximately 6.0 Km values for ATP were estimated by double reciprocal analyses, which gave 110.0 microM in the protein kinase I (in the presence of 10(-6) M cyclic AMP) and 25 microM in the protein kinase III, respectively . The data showed that 1.0 mol phosphate was incorporated into 80 mol of c-erb A protein (50 kDa) either in the presence of protein kinase I (with 10(-6) M cyclic AMP) or in the presence of protein kinase III . These results suggested that there are protein kinases for c-erb A protein, whose functional properties are similar to those of nuclear T3 receptor, in rat kidney nuclei. Biochimie, 1991 Apr, 73(4), 501 - 3 Interaction of lambda Gam protein with the RecD subunit of RecBCD enzyme increases radioresistance of the wild-type Escherichia coli; Marsic N et al.; By making use of the gam(+)-plasmid, the so-called gam-dependent radioresistance was studied . This resistance is the result of the interaction between Gam protein (encoded by the gam gene of lambda) and RecBCD enzyme of Escherichia coli . gam-dependent radioresistance is observed in recB+ recC+ recD+ but not in recB+ recC+ recD- cells . It is suggested that Gam protein interacts specifically with the RecD subunit of RecBCD enzyme; the RecBC complex probably retains its activity in the presence of this viral protein. Biochimie, 1991 Apr, 73(4), 399 - 405 Different mechanisms for SOS induced alleviation of DNA restriction in Escherichia coli; Hiom K et al.; The alleviation of DNA restriction during the SOS response in Escherichia coli has been further investigated . With the EcoK DNA restriction system UV irradiated wild-type cells show a 10(4)-fold increase in ability to plate non-modified lambda phage and a 3-4 fold increase in transformation by non-modified plasmid DNA . A role for the umuDC genes of E coli in the process of SOS-induced restriction alleviation was identified by showing that a umuC122::Tn5 mutant could alleviate EcoK restriction to only 5% that of wild-type levels . Although umuDC are better characterized for their pivotal role in SOS induced mutagenesis, it is demonstrated here that umu-dependent alleviation of EcoK restriction is a transient process in which umu-dependent mutagenesis plays little part . A second form of SOS induced alleviation of DNA restriction is described in this paper involving the McrA restriction system . The mcrA gene is shown to be encoded within a defective prophage called e14 situated at the 25 min region on the Escherichia coli genetic map . e14 is known to abortively excise from the chromosome after SOS induction and it is demonstrated in this report that mcrA is lost from the genome after SOS induction as part of e14 . This results in co-ordinate decrease in the level of McrA restriction within a population of cells. J Clin Microbiol, 1991 Apr, 29(4), 778 - 81 Seroreactive recombinant herpes simplex virus type 2-specific glycoprotein G; Parkes DL et al.; The herpes simplex virus type 2 (HSV-2) genome codes for an envelope protein, glycoprotein G (gG), which contains predominantly type 2-specific epitopes . A portion of this gG gene has been expressed as a fusion protein in Escherichia coli . Expression was regulated by a lambda phage pL promoter . The 60,000-molecular-weight recombinant protein was purified by ion-exchange chromatography . Amino acid sequence analysis confirmed the N terminus of the purified protein . Mice immunized with recombinant gG developed antibodies reactive with native HSV-2 protein, but not with HSV-1 protein, in an indirect immunofluorescence assay . The serological activity of this purified recombinant gG protein was evaluated by immunoblot assay . This protein was reactive with an HSV-2 gG monoclonal antibody . It was also reactive with HSV-2 rabbit antiserum but not with HSV-1 rabbit antiserum . Of 15 patient serum samples known to have antibody to HSV-2, 14 were reactive with this recombinant type 2-specific gG protein, and none of 15 HSV antibody-negative patient serum samples showed reactivity . In agreement with the expected prevalence of HSV-2 infection, 27.6% of 134 serum samples from random normal individuals had antibodies reactive with recombinant gG . This recombinant gG protein may be of value in detecting HSV-2-specific antibody responses in patients infected with HSV-2. Bioorg Khim, 1991 Apr, 17(4), 456 - 60 {Use of uracil-repair selection for extensive DNA sequence deletions}; Shekhter II et al.; A procedure for extensive deletion mutagenesis of DNA using the uracil repair system is exemplified by reconstruction of the pBR322 replication regulatory region cloned into M13tg131 . By means of an oligonucleotide primer the 116-nucleotide fragment was excised and four nucleotides were introduced to form a BglII restriction site . Use of the uracil repair selection provided a 30-fold increase in the deletion mutagenesis efficiency. Prostaglandins Leukot Essent Fatty Acids, 1991 Apr, 42(4), 209 - 16 Sequential release of eicosanoids during endotoxin-induced shock in anesthetized pigs; Mozes T et al.; The release of eicosanoids during endotoxin shock was investigated in anesthetized pigs receiving 5 micrograms/kg Escherichia coli lipopolysaccharide (LPS) over 60 min into the superior mesenteric artery . TXB2, 6-keto PGF1 alpha and LTB4 concentrations in blood obtained from the superior mesenteric vein (SMV), right ventricle (RV) and aorta, during LPS infusion and an additional period of 2 h, were assessed along with hemodynamic variables, blood gases and pH and laboratory parameters . Half of the animals died within 30 min after termination of LPS infusion (non-survivors, n = 8), while the other half survived the experimental period of 3 h, though in a shock state (survivors, n = 9) . The non-surviving pigs demonstrated progressively reduced cardiac output, hypotension and hypoperfusion in all organs . The surviving pigs demonstrated also a reduced cardiac output, which however was compensated by an elevated systemic vascular resistance resulting in a maintenance of arterial blood pressure . After exhausting this compensation the flow to non-vital organs increased and consequently arterial blood pressure was reduced resulting in hypoperfusion . In survivors a marked, though, transient increase was measured in concentrations of TXB2 and 6-keto PGF1 alpha level . A significant increase was measured in plasma concentration of LTB4 in SMV without any elevation in RV and aorta . LTB4 production started when prostanoid release had decreased . In contrast to survivors, no changes could be observed in eicosanoid release for non-survivors . A correlation was observed between systemic vascular resistance and TXB2 to 6-keto PGF1 alpha ratio.(ABSTRACT TRUNCATED AT 250 WORDS) Anal Biochem, 1991 Apr, 194(1), 9 - 15 Ligase-free subcloning: a versatile method to subclone polymerase chain reaction (PCR) products in a single day; Shuldiner AR et al.; Often, it is convenient to subclone polymerase chain reaction (PCR) products into a plasmid vector for subsequent replication in bacteria, but conventional subcloning methods often fail . We report a rapid and versatile method to subclone PCR products directionally into a specific site of virtually any plasmid vector . The procedure requires only four primers, does not require DNA ligase, and may be accomplished in a single day . Ligase-free subcloning is performed by incorporating into the PCR primers sequences at the 5' ends that result in PCR products whose 3' ends are complementary to the 3' ends of the recipient linearized plasmid . The PCR product and the linearized plasmid are spliced together in a second PCR reaction in which Taq polymerase extends the complementary overlapping 3' ends (ligation by overlap extension) . Denaturation followed by heterologous reannealing and cyclization results in a cyclic recombinant plasmid with two nicks that may be used directly to transform competent Escherichia coli . In our hands, ligase-free subcloning is rapid, and offers many advantages over existing strategies. Mol Microbiol, 1991 Apr, 5(4), 969 - 75 A novel function of the cAMP-CRP complex in Escherichia coli: cAMP-CRP functions as an adaptor for the CytR repressor in the deo operon; Sogaard-Andersen L et al.; Unlike classical bacterial repressors, the CytR repressor of Escherichia coli cannot independently regulate gene expression . Here we show that CytR binding to the deoP2 promoter relies on interaction with the master gene regulatory protein, CRP, and, furthermore, that cAMP-CRP and CytR bind co-operatively to deoP2 . Using mutant promoters we show that tandem, properly spaced DNA-bound cAMP-CRP complexes are required for this co-operative binding . These data suggest that CytR forms a bridge between tandem cAMP-CRP complexes, and that cAMP-CRP functions as an adaptor for CytR . The implications of this new version of negative control in E . coli on bacterial gene expression and on combinatorial gene regulation in higher organisms are discussed. Appl Environ Microbiol, 1991 Apr, 57(4), 955 - 61 Use of inosine-containing oligonucleotide primers for enzymatic amplification of different alleles of the gene coding for heat-stable toxin type I of enterotoxigenic Escherichia coli; Candrian U et al.; A method which employs the polymerase chain reaction (PCR) to identify Escherichia coli strains containing the estA gene was developed . This gene codes for heat-stable enterotoxin type I . The use of an inosine-containing pair of amplification primers allowed the amplification of a specific 175-bp DNA fragment from several different estA alleles . The amplified fragments were identified and distinguished by allele-specific oligonucleotide hybridization and characterized by restriction endonuclease analysis . An extension of the classical two-primer PCR proved to be a very simple and rapid method to identify and characterize the estA alleles . Besides the inosine-containing pair of primers, which recognized all described alleles, additional oligonucleotides were used as primers . The sequence of each of these primers was allele specific, and each was amplification compatible with one of the inosine-containing primers . Thus, in one PCR the 175-bp fragment typical for all estA alleles and an allele-specific fragment of different size were produced . These fragments could be separated by agarose gel electrophoresis and were recognized by ethidium bromide staining . Twenty-seven E . coli strains were tested with this amplification system . The presence or lack of the genetic information for production of heat-stable enterotoxin type I was perfectly consistent with the ability of these strains to produce this enterotoxin, as determined by enzyme-linked immunosorbent assay. Appl Environ Microbiol, 1991 Apr, 57(4), 1057 - 61 Adsorption of plasmid DNA to mineral surfaces and protection against DNase I; Romanowski G et al.; The adsorption of {3H}thymidine-labeled plasmid DNA (pHC314; 2.4 kb) of different conformations to chemically pure sand was studied in a flowthrough microenvironment . The extent of adsorption was affected by the concentration and valency of cations, indicating a charge-dependent process . Bivalent cations (Mg2+, Ca2+) were 100-fold more effective than monovalent cations (Na+, K+, NH4+) . Quantitative adsorption of up to 1 microgram of negatively supercoiled or linearized plasmid DNA to 0.7 g of sand was observed in the presence of 5 mM MgCl2 at pH 7 . Under these conditions, more than 85% of DNA adsorbed within 60 s . Maximum adsorption was 4 micrograms of DNA to 0.7 g of sand . Supercoil molecules adsorbed slightly less than linearized or open circular plasmids . An increase of the pH from 5 to 9 decreased adsorption at 0.5 mM MgCl2 about eightfold . It is concluded that adsorption of plasmid DNA to sand depends on the neutralization of negative charges on the DNA molecules and the mineral surfaces by cations . The results are discussed on the grounds of the polyelectrolyte adsorption model . Sand-adsorbed DNA was 100 times more resistant against DNase I than was DNA free in solution . The data support the idea that plasmid DNA can enter the extracellular bacterial gene pool which is located at mineral surfaces in natural bacterial habitats. Gene, 1991 Apr, 100, 247 - 50 The 3'-terminal region of the hygromycin-B-resistance gene is important for its activity in Escherichia coli and Nicotiana tabacum; Bilang R et al.; We have modified the 3'-coding region of the hygromycin B (Hy) phosphotransferase-encoding gene (hph) . The level of Hy resistance conferred by the modified hph genes was determined in Escherichia coli and Nicotiana tabacum . Polypeptides up to 12 amino acids shorter or longer than the wild-type (wt) protein remained active in both pro- and eukaryotic cells . A gene construct of wt length, but with a modified 3' sequence, was less active as measured by the level of Hy resistance . Therefore, the nature of the C terminus of the hph gene product, rather than the length of the polypeptide, influences the ability to confer resistance to Hy. Zhonghua Yi Xue Za Zhi (Taipei), 1991 Apr, 47(4), 280 - 3 Hepatic microabscesses caused by Escherichia coli--US and CT appearance; Chou YH et al.; Hepatic microabscesses have been described in immunosuppressed patients . However, there has been no previous report concerning hepatic microabscesses caused by Escherichia coli (E . coli) . Recently, we experienced a 75-year-old male patient who had suffered from fever and upper abdominal pain for 4 days . His laboratory tests revealed an increased erythrocyte sedimentation rate (55 mm/hr), the white cell count was 7500/cumm with 82% segmented leukocytes, minimally elevated serum alkaline phosphatase and serum glutamic-pyruvic transaminase . Ultrasonography (US) showed multiple tiny hypo- or nearly anechoic lesions (3-8mm) diffusely scattered in both hepatic lobes . Some lesions were too small to be demonstrated and only distal acoustic enhancement posterior to the lesions could be noted . Contrast-enhanced computed tomography (CT) scan subsequently demonstrated the tiny hypodense and cystic lesions and confirmed the US diagnosis of microabscesses . Cultures of blood and liver aspirates showed E . coli . Although US and CT appearance of hepatic microabscesses caused by E . coli may be characteristic, it is not specific . Differential diagnosis should include multiple biliary hamartomas, and definite diagnosis should be made by needle aspiration. Agric Biol Chem, 1991 Apr, 55(4), 1075 - 80 Production of a site specifically cleavable P-glycoprotein-beta-galactosidase fusion protein; Shimabuku AM et al.; We have fused full length and the carboxyl-half of human MDR1 cDNA with the E . coli lacZ gene via a collagen linker and allowed their expression in yeast Saccharomyces cerevisiae . Using antibodies against beta-galactosidase we partially purified the fusion proteins by immunoprecipitation and show here that the full length fusion protein has ATPase activity . By contrast, the fusion protein containing the carboxyl-half of P-glycoprotein did not show ATPase activity, indicating that both domains of P-glycoprotein are necessary . By treatment of the immunoprecipitated fusion protein with collagenase, P-glycoprotein was released from the beta-galactosidase moiety . The results shown here open the possibility for a large scale purification of P-glycoprotein using this site specifically cleavable fusion protein. Curr Opin Biotechnol, 1991 Apr, 2(2), 238 - 46 Antibody engineering; Pluckthun A; Antibody engineering has received a boost from the development of an Escherichia coli expression system that now allows the screening of libraries with bacteria or phages . These random selection techniques can be applied using knowledge obtained from new X-ray structures of recombinant antibody domains, and anti-peptide antibodies . The first crystal structure of an anti-idiotype complex has also been solved . Additionally, the engineering of binding sites for metals and haptens, and the design of new immunotoxins have been reported. Trends Biotechnol, 1991 Apr, 9(4), 132 - 7 Single chain antibody variable regions; Bird RE et al.; The use of antibodies or antibody fragments for targeting tumors (either for tumor imaging or as carriers for drugs or toxins), has encountered problems of clearance, and non-specific or inefficient binding in clinical trials . A novel approach, linking two antibody variable fragments (Fvs), with a short peptide to generate a continuous polypeptide chain, may be able to overcome some of these problems . Since these single chain antibody variable regions (scFvs), are transcribed from constructed 'genes', large-scale production in, for example, E . coli, should be straightforward. J Biotechnol, 1991 Apr, 18(1-2), 55 - 68 Temperature and induction effects on the degradation rate of an abnormal beta-galactosidase in Escherichia coli; Kosinski MJ et al.; Intracellular protein degradation was investigated using an unstable fragment of Escherichia coli beta-galactosidase, the CSH11 mutant, as a model protein . This abnormal protein was expressed from a single copy gene in the chromosome and is converted to a detectable degradable intermediate . The in vivo degradation rates of both beta-galactosidase fragments were measured using pulse-chase radioactive labeling techniques, and their intracellular concentrations were determined using alpha-complementation assays . In the physiological range of 30 to 37 degrees C, the apparent degradation rate constant for the CSH11 fragment follows Arrhenius behavior; while the intermediate's apparent degradation rate constant is nearly unchanged . However, above 37 degrees C the degradation rates of both fragments increase significantly . Analysis of the labeled intermediate's rate of change above 40 degrees C reveals that the CSH11 fragment is being degraded by a second pathway which does not produce the intermediate . When the induction level of the abnormal beta-galactosidase was varied the degradation rates of both fragments behaved similarly, but they unexpectedly decreased with increasing IPTG concentration . The two parallel degradation pathways for CSH11 apparently operated at only the lower IPTG levels . The measured degradation rates did not correlate directly with the intracellular concentration of abnormal proteins. Appl Microbiol Biotechnol, 1991 Apr, 35(1), 38 - 45 Integrative transformation of the ascomycete Podospora anserina: identification of the mating-type locus on chromosome VII of electrophoretically separated chromosomes; Osiewacz HD et al.; Protoplasts of wild-type strain s and a long-lived extrachromosomal mutant (AL2) of the ascomycete Podospora anserina were transformed using a plasmid (pAN7-1) which contains the hygromycin B phosphotransferase gene (hph) of Escherichia coli under the control of Aspergillus nidulans regulatory sequences . After optimizing the transformation procedure, transformation efficiencies of 15-21 transformants/micrograms plasmid DNA were obtained . Using a second selectable vector (pBT3), which contains the beta-tubuline gene of a benomyl-resistant Neurospora crassa mutant, the co-transformation rate was determined . Southern blot hybridization experiments revealed that the transforming plasmid became integrated into the genome of the recipient either as a single copy or as multiple copies . In addition, the data from molecular as well as from classical genetic analyses indicated that in independent transformants vector integration occurred at different positions . The mitotic and meiotic stability of transformants proved to be dependent on the number of integrated plasmid copies . Genetic analyses revealed a transformant in which the integrated vector is closely linked to the mating-type locus . Fractionation of whole chromosomes by pulsed field gel electrophoresis and subsequent hybridization of the immobilized DNAs against radiolabelled vector sequences indicated the largest of seven chromosomes as the chromosome containing the integrated vector and thus the mating-type locus. Appl Microbiol Biotechnol, 1991 Apr, 35(1), 32 - 7 Secretion of active truncated CD4 into Escherichia coli periplasm; Rockenbach SK et al.; A truncated molecule containing the first 183 amino acid residues of the HIV-1 receptor, CD4, was made by periplasmic secretion in Escherichia coli . The signal sequence from the E . coli proteins OmpA, PhoA, or OmpF was fused to the truncated CD4, under the control of either the trp or the lac promoter . The processed material secreted into the periplasm reacted with monoclonal antibodies and exhibited binding activity to the HIV-1 envelope protein gp120 . Not all of the processed product was recovered in the periplasm by osmotic shock, suggesting that either the material aggregated in the periplasm or, during secretion, the molecule assumed some transient conformation that interfered with its translocation across the inner membrane . A mutation in prlA (a gene involved in secretion) increased the level of processing, suggesting that secretion of a heterologous protein in E . coli can be optimized by manipulating the host secretion apparatus. Biochem Biophys Res Commun, 1991 Mar 29, 175(3), 784 - 94 Expression of an autoprocessing CAT-HIV-1 proteinase fusion protein: purification to homogeneity of the release 99 residue proteinase; Montgomery DS et al.; The 99 residue human immunodeficiency virus type 1 proteinase has been expressed in Escherichia coli as part of an autocleaving fusion protein . Expression of the fusion protein is toxic to the host cells, however yields of the released proteinase have been improved by optimising induction nad harvest times to increase culture biomass, and decrease degradation of the proteinase . Soluble proteinase was extracted from these cells by a simple and highly efficient three step process . N-terminal sequence analysis confirms that the enzyme preparation is highly pure and correctly autoprocessed . The proteinase cleaves peptide substrate IGCTLNFPISPIETV between F and P at pH 6.0 with a Km of 310 microM and a Kcat of 14s-1 . The enzyme is sensitive to its ionic environment, showing stimulation of activity at high salt concentrations, and shows a pH optimising 5.5. Biochem Biophys Res Commun, 1991 Mar 29, 175(3), 1131 - 8 Active recombinant C3a of human anaphylatoxin produced in Escherichia coli; Fukuoka Y et al.; DNA sequence coding for the complete human C3a with 77 amino acids was divided into three portions, synthesized separately and constructed for expression in Escherichia coli . High expression of the recombinant C3a was achieved by an expression system using T7 polymerase . Purified recombinant C3a showed the same activities of ileum contraction and platelet aggregation of guinea pig as C3a purified from human serum. Biochem Biophys Res Commun, 1991 Mar 29, 175(3), 1057 - 63 Transcriptional start and MetR binding sites on the Escherichia coli metH gene; Marconi R et al.; The 5' upstream region of the Escherichia coli metH gene has been sequenced . Primer extension analysis revealed a transcription start site at 324 bases upstream of the initiator codon . An 8 base sequence homologous to the MetR binding region on the E . coli metE gene is present 217 bp downstream of the transcription start site . Gel retardation experiments showed that purified MetR protein could bind to a 30 base oligonucleotide containing the putative MetR binding region . No "met box" was present which explains the relative lack of regulation of the expression of the metH gene by methionine. Science, 1991 Mar 29, 251(5001), 1597 - 600 Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR; Gatignol A et al.; Human immunodeficiency virus type 1 (HIV-1) gene expression is activated by Tat, a virally encoded protein . Tat trans-activation requires viral (trans-activation--responsive; TAR) RNA sequences located in the R region of the long terminal repeat (LTR) . Existing evidence suggests that Tat probably cooperates with cellular factors that bind to TAR RNA in the overall trans-activation process . A HeLa complementary DNA was isolated and characterized that encodes a TAR RNA-binding protein (TRBP) . TRBP activated the HIV-1 LTR and was synergistic with Tat function. Eur J Biochem, 1991 Mar 28, 196(3), 717 - 24 Domain structure and interaction within the pentafunctional arom polypeptide; Hawkins AR et al.; The AROM locus of Aspergillus nidulans specifies a pentafunctional polypeptide catalysing five consecutive steps leading to the production of 5-enolpyruvylshikimate 3-phosphate in the shikimate pathway . Aided by oligonucleotide-mediated site-directed mutagenesis, the whole AROM locus and various overlapping subfragments from within it have been fused to the powerful hybrid trc promoter in the Escherichia coli plasmid pKK233-2 . Expression of these subfragments in appropriate aro mutants of E . coli has (a) allowed the delineation of functional domains within the arom polypeptide, (b) shown that the arom polypeptide falls in two independently folding and functioning regions, the N-terminal half specifying 3-dehydroquinate (DHQ) synthase and EPSP synthase and the C-terminus specifying shikimate kinase, biosynthetic 3-dehydroquinase (DHQase) and shikimate dehydrogenase, and (c) strongly suggested an interaction between the DHQ synthase and EPSP synthase domains to stabilise the EPSP synthase activity . In addition an isoenzyme of biosynthetic DHQase, catabolic DHQase, encoded by the QUTE gene of A . nidulans has been transcribed from the trc promoter and upon isopropyl-thio-beta-D-galactoside induction produces up to 20% of the total soluble cell protein. Biochim Biophys Acta, 1991 Mar 26, 1088(3), 439 - 41 The beta subunit of casein kinase II: cloning of cDNAs from murine and porcine origin and expression of the porcine sequence as a fusion protein; Boldyreff B et al.; cDNAs encoding the beta subunit of pig and mouse CKII were isolated . The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies. Biochemistry, 1991 Mar 26, 30(12), 3041 - 8 Two distinct forms of peptidylprolyl-cis-trans-isomerase are expressed separately in periplasmic and cytoplasmic compartments of Escherichia coli cells; Hayano T et al.; Peptidylprolyl-cis-trans-isomerase (PPIase) is thought to be essential for protein folding in the cell . Two forms, a and b, of PPIase and their corresponding genes were isolated from Escherichia coli cells . Despite their insensitivity to cyclosporin A (CsA), both amino acid sequences were homologous and related to that of pig cyclophilin, a protein that has PPIase activity sensitive to CsA (Takahashi et al., 1989) . PPIase a is found to be identical with the E . coli ORF 190 gene product that was sequenced by Kawamukai et al . (1989) and overexpressed by Liu and Walsh (1990) . It is translocated into E . coli periplasmic space with the signal sequence . PPIase b lacks a hydrophobic amino acid stretch which could serve as a signal sequence or a transmembrane domain, and it is detected mainly in the bacterial cytoplasm . These findings indicate that proteins with the ability to assist folding of various polypeptides are located on both sides of the inner membrane . Thus, we propose that the folding of some exported proteins may be catalyzed by the periplasmic proline isomerase and, in turn, that some proteins which have isomerized may not be translocated efficiently. Biochemistry, 1991 Mar 26, 30(12), 2999 - 3002 The T-arm of tRNA is a substrate for tRNA (m5U54)-methyltransferase; Gu XR et al.; Fragments of Escherichia coli FUra-tRNA(1Val) as small as 15 nucleotides form covalent complexes with tRNA (m5U54)-methyltransferase (RUMT) . The sequence essential for binding includes position 52 of the T-stem and the T-loop and extends toward the 3' acceptor end of FUra-tRNA . The in vitro synthesized 17mer T-arm of E . coli tRNA(1Val), composed of the seven-base T-loop and 5-base-pair stem, is a good substrate for RUMT . The Km is decreased 5-fold and kcat is decreased 2-fold compared to the entire tRNA . The T-arm structure could be further reduced to an 11mer containing the loop and two base pairs and still retain activity; the Km was similar to that of the 17mer T-arm, whereas kcat was decreased an additional 20-fold . The data indicate that the primary specificity determinants for the RUMT-tRNA interaction are contained within the primary and secondary structure of the T-arm of tRNA. Biochemistry, 1991 Mar 26, 30(12), 3088 - 98 Wild-type and mutant bacteriorhodopsins D85N, D96N, and R82Q: purification to homogeneity, pH dependence of pumping, and electron diffraction; Miercke LJ et al.; Bacterioopsin, expressed in Escherichia coli as a fusion protein with 13 heterologous residues at the amino terminus, has been purified in the presence of detergents and retinylated to give bacteriorhodopsin . Further purification yielded pure bacteriorhodopsin, which had an absorbance ratio (A280/A lambda max) of 1.5 in the dark-adapted state in a single-detergent environment . This protein has a folding rate, absorbance spectrum, and light-induced proton pumping activity identical with those of bacteriorhodopsin purified from Halobacterium halobium . Protein expressed from the mutants D85N, D96N, and R82Q and purified similarly yielded pure protein with absorbance ratios of 1.5 . Proton pumping rates of bacteriorhodopsins with the wild-type sequence and variants D85N, D96N, and R82Q were determined in phospholipid vesicles as a function of pH . D85N was inactive at all pH values, whereas D96N was inactive from pH 7.0 to pH 8.0, where wild type is most active, but had some activity at low pH . R82Q showed diminished proton pumping with the same pH dependence as for wild type . Bacteriorhodopsin purified from E . coli crystallized in two types of two-dimensional crystal lattices suitable for low-dose electron diffraction, which permit detailed analysis of structural differences in site-directed variants . One lattice was trigonal, as in purple membrane, and showed a high-resolution electron diffraction pattern from glucose-sustained patches . The other lattice was previously uncharacterized with unit cell dimensions a = 127 A, b = 67 A, and symmetry of the orthorhombic plane group pgg. FEBS Lett, 1991 Mar 25, 280(2), 383 - 6 Cleavage of the precursor of pea chloroplast cytochrome f by leader peptidase from Escherichia coli; Anderson CM et al.; Leader peptidase from Escherichia coli was able to process the precursor of pea cytochrome f synthesised in vitro . N-Terminal sequencing established that cleavage by leader peptidase generated the same mature sequence as in pea chloroplasts . Processing by leader peptidase was much more efficient co-translationally rather than post-translationally, and the extent of post-translational processing declined with time suggesting that the cytochrome f precursor folded to an uncleavable conformation . Detergent extracts of pea thylakoid membranes were unable to process the cytochrome f precursor co- or post-translationally. FEBS Lett, 1991 Mar 25, 280(2), 347 - 50 Purification of HIV-1 wild-type protease and characterization of proteolytically inactive HIV-1 protease mutants by pepstatin A affinity chromatography; Wondrak EM et al.; Recombinant wild-type protease of human immunodeficiency virus, type 1 (HIV-1) expressed in E . coli was purified by pepstatin A affinity chromatography . An 88-fold purification was achieved giving a protease preparation with a specific enzymatic activity of approximately 3700 pmol/min/micrograms . Two proteolytically inactive HIV-1 mutant proteases (Arg-87----Lys; Asn-88----Glu) were found to bind to pepstatin A agarose, and and they were purified as the wild-type protease . A third mutant protease Arg-87----Glu) was apparently unable to bind to pepstatin A under similar conditions . Binding to pepstatin A indicates the binding ability of the substrate binding site and the ability to form dimers . These features may be used to purify and to characterize other mutated HIV-1 proteases. FEBS Lett, 1991 Mar 25, 280(2), 344 - 6 The effect of salt on the Michaelis Menten constant of the HIV-1 protease correlates with the Hofmeister series; Wondrak EM et al.; The effect of different types of salt on the proteolytic activity of HIV-1 protease was studied . At a similar ionic strength, the enzyme activity changed according to the salting out effect of the ions used (Hofmeister series) . Kinetic studies showed that a stronger salting out effect of the ions rather than the higher ionic strength per se increased the affinity to the substrate (Km) but in general did not alter the Kcat value. J Biol Chem, 1991 Mar 25, 266(9), 5991 - 9 Escherichia coli ppGpp synthetase II activity requires spoT; Hernandez VJ et al.; Escherichia coli has two enzymes catalyzing the synthesis of guanosine tetraphosphate (ppGpp), designated ppGpp synthetase I (PSI = RelA) and II (PSII), whose activities are regulated differently . Until now, the gene for PSII had not been identified . Here, an E . coli relA1 strain that expresses lacZ from an rrnB P1 promoter was used to screen mutants with increased beta-galactosidase activity on 5-bromo-4-chloro-3-indoyl beta-D-galactoside indicator plates at 30 degrees C . About 15% of the mutants obtained in this manner had reduced levels of ppGpp at 30 degrees C and no detectable ppGpp at 43 degrees C . These mutants did not form colonies at 42 degrees C on minimal medium plates and had elevated ribosome concentrations and higher growth rates at 30 degrees C . Genetic mapping by phage P1 transduction and complementation analyses showed that the mutations were located in spoT and that they were recessive . Specific inhibition of SpoT-dependent ppGpp degradation activity with picolinic acid showed that two of the mutants tested were deficient in ppGpp synthesis activity . These results indicate that spoT is required for PSII activity, suggesting that spoT encodes both ppGpp degradation and synthesis activities and that these two functions can be affected independently by mutation. J Biol Chem, 1991 Mar 25, 266(9), 5980 - 90 Residual guanosine 3',5'-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations; Xiao H et al.; It was known previously that 1) the relA gene of Escherichia coli encodes an enzyme capable of guanosine 3',5'-bispyrophosphate (ppGpp) synthesis, 2) an uncharacterized source of ppGpp synthesis exists in relA null strains, and 3) cellular degradation of ppGpp is mainly due to a manganese-dependent ppGpp 3'-pyrophosphohydrolase encoded by the spoT gene . Here, the effects of spoT gene insertions and deletions are compared with analogous alterations in neighboring genes in the spo operon and found to be lethal in relA+ strains as well as slower growing in relAl backgrounds than delta relA hosts . Cells with null alleles in both the relA and spoT genes are found no longer to accumulate ppGpp after glucose exhaustion or after chelation of manganese ions by picolinic acid addition; the inability to form ppGpp is reversed by a minimal spoT gene on a multicopy plasmid . Strains apparently lacking ppGpp show a complex phenotype including auxotrophy for several amino acids and morphological alterations . We propose that the SpoT protein can either catalyze or control the alternative pathway of ppGpp synthesis in addition to its known role as a (p)ppGpp 3'-pyrophosphohydrolase . We favor the possibility that the SpoT protein is a bifunctional enzyme capable of catalyzing either ppGpp synthesis or degradation. J Biol Chem, 1991 Mar 25, 266(9), 5801 - 7 Cloning, expression, purification, and characterization of biosynthetic threonine deaminase from Escherichia coli; Eisenstein E; Feedback inhibition of the regulatory enzyme threonine deaminase by isoleucine provides an important level of enzymic control over branched chain amino acid biosynthesis in Escherichia coli . Cloning ilvA, the structural gene for threonine deaminase, under control of the trc promoter results in expression of active enzyme upon induction by isopropyl 1-thio-beta-D-galactoside to levels of approximately 20% of the soluble protein in cell extracts . High level expression of threonine deaminase has facilitated the development of a rapid and efficient protocol for the purification of gram quantities of enzyme with a specific activity 3-fold greater than previous preparations . The catalytic activity of threonine deaminase is absolutely dependent on the presence of pyridoxal phosphate, and the tetrameric molecule is isolated containing 1 mol of cofactor/56,000-Da chain . Wild-type threonine deaminase demonstrates a sigmoidal dependence of initial velocity on threonine concentration in the absence of isoleucine, consistent with a substrate-promoted conversion of the enzyme from a low activity to a high activity conformation . The enzymic dehydration of threonine to alpha-ketobutyrate measured by steady-state kinetics, performed at 20 degrees C in 0.05 M potassium phosphate, pH 7.5, is described by a Hill coefficient, nH, of 2.3 and a K0.5 of 8.0 mM . The negative allosteric effector L-isoleucine strongly inhibits the enzyme, yielding a value for nH of 3.9 and K0.5 of 74 mM whereas enzyme activity is greatly increased by L-valine, which yields nearly hyperbolic kinetics characterized by a value for nH of 1.0 and a K0.5 of 5.7 mM . Thus, these effectors promote dramatic and opposing effects on the transition from the low activity to the high activity conformation of the tetrameric enzyme. J Biol Chem, 1991 Mar 25, 266(9), 5634 - 42 Substitution of basic amino acids within endonuclease V enhances nontarget DNA binding; Nickell C et al.; Several DNA-interactive proteins, including the DNA repair enzyme T4 endonuclease V, have been shown to locate their target recognition sites utilizing an electrostatically mediated facilitated diffusion mechanism . Previous work indicates that a decrease in the affinity of endonuclease V for nontarget DNA results in an increased nontarget dissociation rate . This study was designed to investigate the effect of an increase in the affinity of endonuclease V for nontarget DNA . Using a working structural model of the enzyme as a guide, the electrostatic character of endonuclease V was altered . Substitution of Thr-7 with Lys-7 resulted in an enzyme with wild type in vitro characteristics . Mutations which increased the positive charge along a proposed solvent-exposed alpha-helical face had significant effects . The mutants Ala-30, Val-31----Lys-30, Leu-31 and Asn-37----Lys-37 displayed wild type in vitro apurinic-specific and dimer-specific nicking activities . Although the processive dimer-specific nicking rate of the Lys-37 mutant resembled that of wild type, the rate of the Lys-30, Leu-31 mutant was reduced by 60% . In addition, the salt concentration range over which these mutants processively nick dimer-containing DNA has been greatly expanded . Both mutants are shown to have an increased affinity for nontarget DNA. J Biol Chem, 1991 Mar 25, 266(9), 5359 - 62 Glucose phosphorylation . Site-directed mutations which impair the catalytic function of hexokinase; Arora KK et al.; Recent studies from this and other laboratories have resulted in the cloning and sequencing of hexokinases from a variety of tissues including yeast, human kidney, rat brain, rat liver, and mouse hepatoma . Significantly, studies on the hepatoma enzyme conducted in this laboratory (Arora, K.K., Fanciulli, M., and Pedersen, P.L . (1990) J . Biol . Chem . 265, 6481-6488) resulted also in its overexpression in Escherichia coli in active form . We have now used site-directed mutagenesis for the first time in studies of hexokinase to evaluate the role of amino acid residues predicted to interact with either glucose or ATP . Four amino acid residues (Ser-603, Asp-657, Glu-708, and Glu-742) believed to interact with glucose were mutated to alanine or glycine, whereas a lysine residue (Lys-558) thought to be directly involved in binding ATP was mutated to either methionine or arginine . Of all the mutations in residues believed to interact with glucose, the Asp-657----Ala mutation is the most profound, reducing the hexokinase activity to a level less than 1% of the wild type . The relative Vmax values for Ser-603----Ala, Glu-708----Ala, and Glu-742----Ala enzymes are 6, 10, and 6.5%, respectively, of the wild-type enzyme . Glu-708 and Glu-742 mutations increase the apparent Km for glucose 50- and 14-fold, respectively, while the Ser-603----Ala mutation decreases the apparent Km for glucose 5-fold . At the putative ATP binding site, the relative Vmax for Lys-558----Arg and Lys-558----Met enzymes are 70 and 29%, respectively, of the wild-type enzyme with no changes in the apparent Km for glucose . No changes were observed in the apparent Km for ATP with any mutation . These results support the view that all 4 residues predicted to interact with glucose from earlier x-ray studies may play a role in binding and/or catalysis . The Asp-657 and Ser-603 residues may be involved in both, while Glu-708 and Glu-742 clearly contribute to binding but are not essential for catalysis . In contrast, Lys-558 appears to be essential neither for binding nor catalysis. J Biol Chem, 1991 Mar 25, 266(9), 5664 - 9 Characterization of the catalytic subunit of casein kinase II expressed in Escherichia coli and regulation of activity; Lin WJ et al.; The catalytic (alpha) subunit of casein kinase II from Drosophila, cloned and expressed in Escherichia coli (Saxena, A., Padmanabha, R., and Glover, C . V . C., (1987) Mol . Cell . Biol . 7, 3409-3417), has been purified and characterized, and the properties have been compared to those of the holoenzyme . The catalytic subunit exhibits protein kinase activity with casein as substrate and is autophosphorylated . The specific activity of the purified subunit is 6% of the activity of the holoenzyme from reticulocytes or from Drosophila . The alpha subunit is a monomer, eluting at Mr = 40,000 upon gel filtration in high salt, but as part of an aggregate in low salt . The alpha subunit has been purified to apparent homogeneity by sequential chromatography on DEAE-cellulose, Mono S, and Mono Q . A single band, Mr = 37,000, is detected by silver staining following polyacrylamide gel electrophoresis . The isolated alpha subunit displays apparent Km values for beta casein, ATP, and GTP similar to those of the holoenzyme . The activity of the alpha subunit is inhibited by heparin with an I50 of 0.1-0.3 micrograms/ml, a value similar to that observed for the holoenzyme; autophosphorylation is also inhibited by heparin . Polylysine has no stimulatory effect on the activity of the catalytic subunit, as measured with casein and by autophosphorylation, but stimulates both activities with the holoenzyme . When physiological substrates for casein kinase II are examined, glycogen synthase and eukaryotic initiation factor 3 (eIF-3) (p120) are phosphorylated by the alpha subunit at a rate equivalent to that of the holoenzyme, while phosphorylation of eIF-3 (p67) is reduced 9-fold and eIF-2 beta is not modified . From these data, it can be concluded that the alpha subunit of casein kinase II is sufficient for catalysis, is autophosphorylated, and can be directly inhibited by heparin, whereas the beta subunit mediates the effects of basic stimulatory compounds and is involved in recognition and/or binding to specific physiological substrates. Nucleic Acids Res, 1991 Mar 25, 19(6), 1203 - 11 Plasmid RSF1010 DNA replication in vitro promoted by purified RSF1010 RepA, RepB and RepC proteins; Scherzinger E et al.; We have constructed and analyzed an in vitro system that will efficiently replicate plasmid RSF1010 and its derivatives . The system contains a partially purified extract from E.coli cells and three purified RSF1010-encoded proteins, the products of genes repA, repB (or mobA/repB), and repC . Replication in this system mimics the in vivo mechanism in that it (i) is initiated at oriV, the origin of vegetative DNA replication, (ii) proceeds in a population of plasmid molecules in both directions from this 396-base-pair origin region, and (iii) is absolutely dependent on the presence of each of the three rep gene products . In addition, we find that E.coli DNA gyrase, DnaZ protein (gamma subunit of poIIII holoenzyme) and SSB are required for in vitro plasmid synthesis . The bacterial RNA polymerase, the initiation protein DnaA, and the primosomal proteins DnaB, DnaC, DnaG and DnaT are not required . Furthermore, the replicative intermediates seen in the electron microscope suggest that replication in vitro begins with the simultaneous or non-simultaneous formation of two displacement loops that expand for a short stretch of DNA toward each other, and form a theta-type structure when the two displacing strands pass each other. J Biol Chem, 1991 Mar 25, 266(9), 5412 - 6 Site-directed mutagenesis of the putative catalytic triad of poliovirus 3C proteinase; Hammerle T et al.; Based on predictions of the structure of proteinase 3C of poliovirus, mutations have been made at residues that are supposed to constitute the catalytic triad . Wild-type and mutant 3C were expressed in Escherichia coli, purified to homogeneity, and characterized by the ability to cleave a synthetic peptide substrate or an in vitro translated polypeptide consisting of part of the polyprotein of poliovirus . Additionally, the ability of autocatalytic processing of a precursor harboring wild-type or mutant 3C sequences was tested . Single substitutions of the residues His-40, Glu-71, and Cys-147 by Tyr, Gln, and Ser, respectively, resulted in an inactive enzyme . Replacement of Asp-85 by Asn resulted in an enzyme that was as active as wild-type enzyme in trans cleavage assays but whose autoprocessing ability was impaired . Our results are consistent with the proposal that residues His-40, Glu-71, and Cys-147 constitute the catalytic triad of poliovirus 3C proteinase . Furthermore, residue Asp-85 is not required for proper proteolytic activity despite being highly conserved between different picornaviruses . This indicates that Asp-85 might be involved in a different function of 3C. J Biol Chem, 1991 Mar 25, 266(9), 5703 - 10 Modified calcium-dependent regulatory function of troponin C central helix mutants; Dobrowolski Z et al.; Mutations have been made in the exposed region of the avian troponin C central helix, the D/E linker, which change its length and the orientation of the Ca2(+)-binding domains relative to each other . The region 87Glu-Asp-Ala-Lys-Gly-Lys-Ser-Glu-Glu-Glu97 has been altered in five deletion (d) mutants: dEDA, dKG, dKGK, dSEEE, and dKEDAKGK . The recombinant troponin Cs were expressed in Escherichia coli, purified, and assayed for function . All mutants retained basic troponin C function . They all bound Ca2+ to the low and high affinity sites, and they all were able to confer Ca2+ sensitivity on the regulated actomyosin ATPase . However, the regulatory function of all mutants except dSEEE was defective in one part of the Ca2+ switch or the other . In certain conditions dKGK and dKEDAKGK failed to inhibit fully whereas dEDA and dKG failed to activate the regulated actomyosin ATPase fully . The following general conclusions have been made . (a) The length of the D/E linker per se (assuming the linker is helical) and the orientation of the two Ca2(+)-binding domains relative to each other are not crucial for regulation . (b) The conserved charge cluster 95Glu-Glu-Glu97, in a region of troponin C known to bind to troponin I and postulated to be required for regulation, appears to be unimportant for function . (c) Deletion of 88Glu-Asp-Ala90 resulted in a troponin C that could not activate the actomyosin (or S1) ATPase over the level of actomyosin alone, thus defining a role for troponin C in this aspect of thin filament regulation . The results have been interpreted in terms of the crystallographic structure of troponin C and related to results with analogous calmodulin mutants. J Biol Chem, 1991 Mar 25, 266(9), 5450 - 8 Asymmetry in the recA protein-DNA filament; Lauder SD et al.; The apparent DNA site size obtained from an assay monitoring the ATPase activity of Escherichia coli recA protein (n = 3.5) differs from that determined from a direct DNA binding assay (n = 7) done under identical conditions . Investigation of this discrepancy indicates that at a DNA:protein ratio of 3.5:1, one-half of the recA protein population is less sensitive to ATPase activity inhibition by the nonhydrolyzable ATP analogue adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), suggesting that the recA protein filament is asymmetric with respect to NTP affinity . This asymmetry does not depend on the presence of ATP gamma S since the apparent Km for ATP derived from single-stranded DNA-dependent ATP hydrolysis activity is dependent on the DNA:protein ratio . Three models are proposed to account for the observed site size discrepancy and the NTP binding affinity asymmetry . They differ mainly in the intrinsic site size for each recA protein monomer and in the number of DNA-binding sites/recA molecule . Gel filtration of recA-single-stranded DNA complexes at different DNA:protein ratios complements the enzymological data and provides an additional method of distinguishing among the proposed models . The phenomenon of subunit nonequivalence within the recA protein presynaptic filament may provide a molecular basis for understanding how recA protein can discriminate between different DNA molecules during homologous pairing. J Biol Chem, 1991 Mar 25, 266(9), 5424 - 9 Catalytic site of F1-ATPase of Escherichia coli . Lys-155 and Lys-201 of the beta subunit are located near the gamma-phosphate group of ATP in the presence of Mg2+; Ida K et al.; The catalytic site of Escherichia coli F1 was probed using a reactive ATP analogue, adenosine triphosphopyridoxal (AP3-PL) . For complete loss of enzyme activity, about 1 mol of AP3-PL bound to 1 mol of F1 was estimated to be required in the presence or absence of Mg2+ . About 70% of the label was bound to the alpha subunit and the rest to the beta subunit in the absence of Mg2+, and the alpha Lys-201 and beta Lys-155 residues, respectively, were the major target residues (Tagaya, M., Noumi, T., Nakano, K., Futai, M., and Fukui, T . (1988) FEBS Lett . 233, 347-351) . Addition of Mg2+ decreased the AP3-PL concentration required for half-maximal inhibition, and predominant labeling of the beta subunit (beta Lys-155 and beta Lys-201) with the reagent . ATP and ADP were protective ligands in the presence and absence of Mg2+ . The alpha subunit mutation (alpha Lys-201----Gln or alpha Lys-201 deletion) were active in oxidative phosphorylation . However, purified mutant F1s showed impaired low multi-site activity, although their uni-site catalyses were essentially normal . Thus alpha Lys-201 is not a catalytic residue, but may be important for catalytic cooperativity . Mutant F1s were inhibited less by AP3-PL in the absence of Mg2+, and consistent with this, modifications of their alpha subunits by AP3-PL were reduced . AP3-PL was more inhibitory to the mutant enzymes in the presence of Mg2+, and bound to the beta Lys-155 and beta Lys-201 residues of mutant F1 (alpha Lys-201----Gln) . These results strongly suggest that alpha Lys-201, beta Lys-155, and beta Lys-201 are located close together near the gamma-phosphate group of ATP bound to the catalytic site, and that the two beta residues and the gamma-phosphate group become closer to each other in the presence of Mg2+. J Theor Biol, 1991 Mar 21, 149(2), 257 - 63 The study of stacking energy for natural DNA sequences; Zhang CT et al.; The stacking energies between bases of DNA for both A and B conformation have been calculated by Aida & Nagata (1986, Int . J . Quantum Chem . 29, 1253-1261) . For naturally occurring DNA sequences, by assuming that the average stacking energy of A conformation is completely identical with that of B, the lowest average stacking energy for double strand DNA has been calculated and found to be equal to -7.38 kcal M-1 . This conclusion has been confirmed by the data of stacking energy of 112 promoter sequences of Escherichia coli and some other natural DNA sequences . Our result shows that the natural DNA sequences are bi-stable . One stable-state is the A conformation, the other is B . It is shown that the result is only applicable to the transcription processes that regulate the gene expression . Through the interaction with RNA polymerase in the processes of transcription, the conformation of DNA might undergo a transition of B----A----B. Nature, 1991 Mar 21, 350(6315), 250 - 2 Generating yeast transcriptional activators containing no yeast protein sequences; Ruden DM et al.; We previously reported that roughly 1% of the short peptides encoded by Escherichia coli genomic DNA fragments act as transcriptional activating regions in yeast when fused to GAL4(1-147), a DNA-binding portion of the yeast transcriptional activator GAL4 (ref . 1) . Struhl questioned the conclusion that we had identified new transcriptional activating sequences that function in the absence of yeast transcriptional activating sequences . His criticism was based on two considerations: first, GAL4(1-147) contains an acidic segment (and subsequent experiments have shown that this region contains a weak activating region in vitro); second, attempts to isolate new activating regions failed when the DNA-binding domain of a bacterial repressor, LexA(1-87), was used as the DNA-binding unit . We report here a repeat of our original experiment using the complete LexA molecule LexA(1-202) as the DNA-binding region, instead of GAL4(1-147) or LexA(1-87) . We find that, as in the original experiment, about 1% of the short peptides encoded by E . coli genomic fragments act as transcriptional activating regions when fused to intact LexA . All of the new activating regions whose sequences we determined bore an excess of acidic amino acids (see Table 1). J Mol Biol, 1991 Mar 20, 218(2), 449 - 64 Reaction mechanism of alkaline phosphatase based on crystal structures . Two-metal ion catalysis; Kim EE et al.; Alkaline phosphatase (AP) is a widely distributed non-specific phosphomonoesterase that functions through formation of a covalent phosphoseryl intermediate (E-P) . The enzyme also catalyzes phosphoryl transfer reaction to various alcohols . Escherichia coli AP is a homodimer with 449 residues per monomer . It is a metalloenzyme with two Zn2+ and one Mg2+ at each active site . The crystal structure of native E . coli AP complexed with inorganic phosphate (Pi), which is a strong competitive inhibitor as well as a substrate for the reverse reaction, has been refined at 2.0 A resolution . Some parts of the molecular have been retraced, starting from the previous 2.8 A study . The active site has been modified substantially and is described in this paper . The changes in the active site region suggest the need to reinterpret earlier spectral data, and suggestions are made . Also presented are the structures of the Cd-substituted enzyme complexed with inorganic phosphate at 2.5 A resolution, and the phosphate-free native enzyme at 2.8 A resolution . At pH 7.5, where the X-ray data were collected, the Cd-substituted enzyme is predominantly the covalent phosphoenzyme (E-P) while the native Zn/Mg enzyme exists in predominantly noncovalent (E.P) form . Implication of these results for the catalytic mechanism of the enzyme is discussed . APs from other sources are believed to function in a similar manner. J Mol Biol, 1991 Mar 20, 218(2), 313 - 21 The roles of residues 5 and 9 of the recognition helix of Lac repressor in lac operator binding; Sartorius J et al.; We constructed expression libraries for Lac repressor mutants with amino acid exchanges in positions 1, 2, 5 and 9 of the recognition helix . We then analysed the interactions of residues 5 and 9 with operator variants bearing single or multiple symmetric base-pair exchanges in positions 3, 4 and 5 of the ideal fully symmetric lac operator . We isolated 37 independent Lac repressor mutants with five different amino acids in position 5 of the recognition helix that exhibit a strong preference for particular residues in position 2 and, to a lesser extent, in position 1 of the recognition helix . Our results suggest that residue 5 of the recognition helix (serine 21) contributes to the specific recognition of base-pair 4 of the lac operator . They further suggest that residue 9 of the recognition helix (asparagine 25) interacts non-specifically with a phosphate of the DNA backbone, possibly between base-pairs 2 and 3. J Mol Biol, 1991 Mar 20, 218(2), 335 - 47 Mutations in the inverted repeats of Tn3 affect binding of transposase and transposition immunity; Nissley DV et al.; In order to better understand the interaction between the inverted repeats (IRs) of the transposon Tn3 and Tn3 transposase, we have looked at the effects of mutations within the IRs on binding of transposase and transposition immunity . Binding of transposase to mutated IRs was measured using a site-specific nitrocellulose filter binding assay and by DNase I protection studies . Transposition immunity was measured in vivo using a transposition mating-out assay . The most important determinants for binding of transposase are present within the inside 21 base-pairs of the IR and several single base-pair mutations significantly reduce binding . Base-pair mutations which do not effect binding have strong negative effects on transposition immunity indicating that simple binding of transposase to the IR is not sufficient for the establishment of transposition immunity. J Mol Biol, 1991 Mar 20, 218(2), 293 - 311 Six group I introns and three internal transcribed spacers in the chloroplast large subunit ribosomal RNA gene of the green alga Chlamydomonas eugametos; Turmel M et al.; The chloroplast large subunit rRNA gene of Chlamydomonas eugametos and its 5' flanking region encoding tRNA(Ile) (GAU) and tRNA(Ala) (UGC) have been sequenced . The DNA sequence data along with the results of a detailed RNA analysis disclosed two unusual features of this green algal large subunit rRNA gene: (1) the presence of six group I introns (CeLSU.1-CeLSU.6) whose insertion positions have not been described previously, and (2) the presence of three short internal transcribed spacers that are post-transcriptionally excised to yield four rRNA species of 280, 52, 810 and 1720 nucleotides, positioned in this order (5' to 3') in the primary transcript . Together, these RNA species can assume a secondary structure that is almost identical to that proposed for the 23 S rRNA of Escherichia coli . All three internal transcribed spacers map to variable regions of primary sequence and/or potential secondary structure, whereas all six introns lie within highly conserved regions . The first three introns are inserted within the sequence encoding the 810 nucleotide rRNA species and map within domain II of the large subunit rRNA structure; the remaining introns, found in the sequence encoding the 1720 nucleotide rRNA species, lie within either domain IV or V, as is the case for all other large subunit rDNA introns that have been documented to date . CeLSU.5 and CeLSU.6 each contain a long open reading frame (ORF) of more than 200 codons . While the CeLSU.6 ORF is not related to any known ORFs, the CeLSU.5 ORF belongs to a family of ORFs that have been identified in Podospora and Neurospora mitochondrial group I introns . The finding that a polymorphic marker showing unidirectional gene conversion during crosses between C . eugametos and Chlamydomonas moewusii is located within the CeLSU.5 ORF makes it likely that this intron is a mobile element and that its ORF encodes a site-specific endonuclease promoting the transfer of the intron DNA sequence. J Mol Biol, 1991 Mar 20, 218(2), 269 - 70 Crystallization and preliminary X-ray diffraction studies of the human erythrocyte bisphosphoglycerate mutase; Cherfils J et al.; Bisphosphoglycerate mutase (EC 2.7.5.4) catalyzes the synthesis and breakdown of 2,3-diphosphoglycerate in red cells . The human enzyme, cloned and expressed in Escherichia coli has been crystallized in the rhombohedral space group R32 with a = b = c = 100.4 A and alpha = beta = gamma = 81.2 degrees . The asymmetric unit contains either a dimeric enzyme molecule, or a monomer. Biochim Biophys Acta, 1991 Mar 19, 1092(1), 1 - 6 Intracellular calcium and pH alterations induced by Escherichia coli endotoxin in rat hepatocytes; Portoles MT et al.; In this study, the fluorescent Ca2+ probe fura-2 and the fluorescent pH indicator BCECF have been used to monitor cytosolic free Ca2+ and intracellular pH (pHi), respectively, in isolated and cultured hepatocytes treated with Escherichia coli O111:B4 endotoxin . Uptake of 45Ca2+ was also measured to study the effect of endotoxin on the extracellular calcium influx . Endotoxin treatment produced a progressive increase of cytosolic Ca2+ in a dose-dependent manner caused by both induction of a significant release of Ca2+ from intracellular stores and stimulation of the extracellular calcium influx . The perturbation of Ca2+ homeostasis by endotoxin may cause an abnormal stimulation of physiological processes, developing lethal cell injury . Endotoxin also produced a significant decrease in the pHi of hepatocytes which can justify important metabolic alterations during endotoxicosis. Biochemistry, 1991 Mar 19, 30(11), 2940 - 6 Structural and functional analysis of EcoRI DNA methyltransferase by proteolysis; Reich NO et al.; Native EcoRI DNA methyltransferase (Mtase, Mr 38,050) is proteolyzed by trypsin to generate an intermediate 36-kDa fragment (p36) followed by the formation of two polypeptides of Mr 23,000 and 13,000 (p23 and p13, respectively) . Protein sequence analysis of the tryptic fragments indicates that p36 results from removal of the first 14 or 16 amino acids, p23 spans residues 15-216, and p13 spans residues 217-325 . The relative resistance to further degradation of p23 and p13 suggests stable domain structures . This is further supported by the generation of similar fragments with SV8 endoprotease which has entirely different peptide specificities . Our results suggest the Mtase is a two-domain protein connected by a highly flexible interdomain hinge . The putative hinge region encompasses previously identified peptides implicated in AdoMet binding {Reich, N.O., & Everett, E . (1990) J . Biol . Chem . 265, 8929-8934} and catalysis {Everett et al . (1990) J . Biol . Chem . 265, 17713-17719} . Protection studies with DNA, S-adenosylmethionine (AdoMet), S-adenosylhomocysteine (AdoHcy), and sinefungin (AdoMet analogue) show that the Mtase undergoes significant conformational changes upon ligand binding . Trypsinolysis of the AdoMet-bound form of the Mtase generates different fragments, and the AdoMet-bound form is over 800 times more stable than unbound Mtase . The sequence-specific ternary complex (Mtase-DNA-sinefungin) is 2000 times more resistant to degradation by trypsin; cleavage eventually generates 26- and 12-kDa fragments which span residues 104-325 and 1-103, respectively (p26 and p12) . The first 14 or 16 amino acids of the Mtase are not essential since p36 retains activity . Activity analysis of the p26 and p12 mixture also indicates retention of activity.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1991 Mar 19, 30(11), 2933 - 9 Kinetic mechanism of the EcoRI DNA methyltransferase; Reich NO et al.; We present a kinetic analysis of the EcoRI DNA N6-adenosine methyltransferase (Mtase) . The enzyme catalyzes the S-adenosylmethionine (AdoMet)-dependent methylation of a short, synthetic 14 base pair DNA substrate and plasmid pBR322 DNA substrate with kcat/Km values of 0.51 X 10(8) and 4.1 X 10(8) s-1 M-1, respectively . The Mtase is thus one of the most efficient biocatalysts known . Our data are consistent with an ordered bi-bi steady-state mechanism in which AdoMet binds first, followed by DNA addition . One of the reaction products, S-adenosylhomocysteine (AdoHcy), is an uncompetitive inhibitor with respect to DNA and a competitive inhibitor with respect to AdoMet . Thus, initial DNA binding followed by AdoHcy binding leads to formation of a ternary dead-end complex (Mtase-DNA-AdoHcy) . We suggest that the product inhibition patterns and apparent order of substrate binding can be reconciled by a mechanism in which the Mtase binds AdoMet and noncanonical DNA randomly but that recognition of the canonical site requires AdoMet to be bound . Pre-steady-state and isotope partition analyses starting with the binary Mtase-AdoMet complex confirm its catalytic competence . Moreover, the methyl transfer step is at least 10 times faster than catalytic turnover. Biochemistry, 1991 Mar 19, 30(11), 2916 - 27 Biosynthesis of the Escherichia coli siderophore enterobactin: sequence of the entF gene, expression and purification of EntF, and analysis of covalent phosphopantetheine; Rusnak F et al.; The sequence of the entF gene which codes for the serine activating enzyme in enterobactin biosynthesis is reported . The gene encodes a protein with a calculated molecular weight of 142,006 and shares homologies with the small subunits of gramicidin S synthetase and tyrocidine synthetase . We have subcloned and overexpressed entF in a multicopy plasmid and attempted to demonstrate L-serine-dependent ATP-{32P}PPi exchange activity and its participation in enterobactin biosynthesis, but the overexpressed enzyme appears to be essentially inactive in crude extract . A partial purification of active EntF from wild-type Escherichia coli, however, has confirmed the expected activities of EntF . In a search for possible causes for the low level of activity of the overexpressed enzyme, we have discovered that EntF contains a covalently bound phosphopantetheine cofactor. J Biol Chem, 1991 Mar 5, 266(7), 4568 - 73 Escherichia coli DNA polymerase II is stimulated by DNA polymerase III holoenzyme auxiliary subunits; Hughes AJ Jr et al.; DNA polymerase III of Escherichia coli requires multiple auxiliary factors to enable it to serve as a replicative complex . We demonstrate that auxiliary components of the DNA polymerase III holoenzyme, the gamma delta complex and beta subunit, markedly stimulate DNA polymerase II on long single-stranded templates . DNA polymerase II activity is enhanced by single-stranded DNA binding protein, but the stimulation by gamma delta and beta can be observed either in the absence or presence of single-stranded DNA binding protein . In contrast with DNA polymerase III, the requirement of DNA polymerase II for gamma delta cannot be bypassed by large excesses of the beta subunit at low ionic strength in the absence of the single-stranded DNA binding protein . The product of the DNA polymerase II-gamma delta-beta reaction on a uniquely primed single-stranded circle is of full template length; the reconstituted enzyme apparently is incapable of strand displacement synthesis . The possible biological implications of these observations are discussed. FEMS Microbiol Lett, 1991 Mar 15, 63(1), 51 - 6 Cloning and sequencing of the fus-gene encoding elongation factor 2 in the archaebacterium Thermoplasma acidophilum; Pechmann H et al.; We have cloned a 1.6-kb region of chromosomal DNA from Thermoplasma acidophilum into Escherichia coli using as a probe part of the Methanococcus vannielii fus-gene . The sequence of the clone was highly homologous to part of the corresponding Methanococcus vannielii gene . By chromosome walking, a 4.7-kb EcoRI fragment containing the complete gene was isolated . Nucleotide sequencing revealed an open reading frame of 2196 nucleotides . The deduced amino acid sequence contains the known peptide sequence around the ADP-ribosylation site of T . acidophilum elongation factor 2, which unequivocally confirms that the fus-gene has been cloned . The amino acid sequence was compared to that of hamster and E . coli, as well as to known archaebacterial EF-2 sequences. FEMS Microbiol Lett, 1991 Mar 15, 63(1), 27 - 30 Analysis of the antigenic difference between Vero toxin 2 (VT2) and VT2 variant (VT2vh) of Verotoxin-producing Escherichia coli by a site-directed mutagenesis; Ito H et al.; Ouchterlony double gel diffusion analysis of the A and B subunits of purified Vero toxin 2 (VT2) and a variant of VT2 (VT2vh) demonstrated that the difference in antigenicity between VT2 and VT2vh is due to the difference in the B subunit of the two toxins . Analysis of mutants of VT2vh prepared by site-directed mutagenesis attributed the antigenic dissimilarity to the difference in the amino acid residue at position 24 of the B subunit. Gene, 1991 Mar 15, 99(2), 205 - 9 Production of rat Spot14 protein in Escherichia coli; Planells R et al.; Hormonal, nutritional and developmental factors modulate, in rat lipogenic tissues, the transcription of the mRNA coding for a protein of unknown function, called Spot14 (S14) . The corresponding protein has never been purified from tissues . In this paper, we describe the production of S14 in Escherichia coli . In the absence of available antibodies (Ab) directed against S14 protein, our strategy was to produce this protein by constructing two different recombinant expression vectors . The first recombinant plasmid produced a S14::protein A fusion which was easily purified and then rabbit Ab could be raised against it . The second expression vector directly produced S14 . This expression was demonstrated by specific binding of polyclonal Ab directed against the fusion protein . These Ab also recognized a rat-liver protein sharing characteristics of S14. Gene, 1991 Mar 15, 99(2), 141 - 50 Targeting the Escherichia coli lac repressor to the mammalian cell nucleus; Hu MC et al.; We have previously shown that about 90% of total Escherichia coli lac repressor synthesized in mammalian cells is located in the cytoplasm {Hu and Davidson, Cell 48 (1987) 555-566} . To target a functional lac repressor to the nucleus, we mutated 10 nucleotides at the 3' end of the coding sequence, thus adding the nuclear localization signal of the simian virus 40 large-T antigen to the C terminus of the repressor . The mutant lacI gene and the wild-type (wt) gene, both in standard animal cell expression vectors, driven by the promoter of the Rous sarcoma virus long terminal repeat, were stably transfected into three rodent cell lines . In confirmation of our previous results, only about 10% of the wt repressor, but all of the mutant protein, was localized in the nucleus . DNase I footprint analyses showed that the mutant repressor retained the same operator DNA-binding specificity as wt repressor . Furthermore, both repressor-operator complexes could be dissociated by addition of isopropyl-beta-D-thiogalactopyranoside in vitro . However, the ratio of number of repressor molecules per nucleus that, by in vitro assay, could bind to the operator sequence to the number of monomer repressor polypeptides per nucleus, as determined by Western blotting, was about 1:4 for the wt repressor and about 1:30 for the mutant repressor . This suggests that: (a) the mutant repressor assembles into tetramers inefficiently; and/or (b) it has reduced binding affinity to the operator sequence; and/or (c) it has higher binding affinity to nonspecific DNA. Biochem Biophys Res Commun, 1991 Mar 15, 175(2), 437 - 43 A spectroscopic study of the conformations of active and latent forms of recombinant plasminogen activator inhibitor-1; Dwivedi AM et al.; Recombinant plasminogen activator inhibitor-1 (rPAI-1) purified from Escherichia coli, like its natural counterpart, can exist in either active or latent form . To elucidate the structural basis for these two forms, both active and latent rPAI-1 have been studied using ultra-violet (UV), circular dichroism (CD), and fluorescence spectroscopy . The secondary structures determined by CD show no significant differences and indicate that both the forms are predominantly alpha helical and random . The UV spectra are also very similar with absorption maxima around 278 nm . The structures of the two forms were further characterized by measuring tryptophan fluorescence emissions and their quenching with ionic (iodide) and neutral (acrylamide) quenchers . These data indicate clear differences in the tertiary structures of the two forms with the latent form being more compact and folded in comparison with the active form. Biochem J, 1991 Mar 15, 274 ( Pt 3), 885 - 9 Histidines, histamines and imidazoles as glycosidase inhibitors; Field RA et al.; This present study reports the ability of a range of derivatives of L-histidine, histamine and imidazole to act as inhibitors of sweet-almond beta-glucosidase, yeast alpha-glucosidase and Escherichia coli beta-galactosidase . The addition of a hydrophobic group to the basic imidazole nucleus greatly enhances binding to both the alpha- and beta-glucosidases . L-Histidine (beta-naphthylamide (Ki 17 microM) is a potent competitive inhibitor of sweet-almond beta-glucosidase as is omega-N-acetylhistamine (K1 35 microM), which inhibits the sweet-almond beta-glucosidase at least 700 times more strongly than either yeast alpha-glucosidase or Escherichia coli beta-galactosidase, and suggests potential for the development of selective reversible beta-glucosidase inhibitors . A range of hydrophobic omega-N-acylhistamines were synthesized and shown to be among the most potent inhibitors of sweet-almond beta-glucosidase reported to date. Biochem J, 1991 Mar 15, 274 ( Pt 3), 819 - 24 Compartmentalized system with membrane-bound glycerol kinase . Activity and product distribution versus asymmetrical substrate supply; Girard A et al.; An artificial-membrane-bound glycerokinase chosen as a membrane-bound two-substrate-enzyme model has been used to separate two unequal compartments of a specially designed diffusion cell . An interesting feature is the asymmetry of compartments and the existence of a diffusion layer adjacent to only one face of the