Escherichia coli

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 enzymic membrane . In such a situation the apparent enzyme activity and the product distribution in the system have been studied versus all the possibilities of combination of ATP and glycerol supply . Our approach has lead us to differentiate two different roles played by a diffusion layer adjacent to a permeable enzymic membrane . Depending on the spatial origin of the enzymic substrates (i.e . from which compartment they derive), the diffusion layer can play either the role of a passive additional resistance to that of the membrane or the role of a third compartment in which the reaction product can partially accumulate before splitting on both parts of the membrane . Our results mainly demonstrate that a membrane-bound enzyme activity and the resulting product distribution occurring in a compartmentalized system may be regulated by the cumulative effect due to the asymmetry in volumes of the compartments, the presence of a diffusion layer and the different possibilities of substrate supply . With the topography studied, which is close to that reported for many 'in vivo' situations, the product may be diffused lead to vectorial metabolism processes.

Proc Natl Acad Sci U S A, 1991 Mar 15, 88(6), 2578 - 82
Primary structures of the precursor and mature forms of stearoyl-acyl carrier protein desaturase from safflower embryos and requirement of ferredoxin for enzyme activity; Thompson GA et al.; Stearoyl-acyl carrier protein (ACP) desaturase (EC 1.14.99.6) catalyzes the principal conversion of saturated fatty acids to unsaturated fatty acids in the synthesis of vegetable oils . Stearoyl-ACP desaturase was purified from developing embryos of safflower seed, and extensive amino acid sequence was determined . The amino acid sequence was used in conjunction with polymerase chain reactions to clone a full-length cDNA . The primary structure of the protein, as deduced from the nucleotide sequence of the cDNA, includes a 33-amino-acid transit peptide not found in the purified enzyme . Expression in Escherichia coli of a gene encoding the mature form of stearoyl-ACP desaturase did not result in an altered fatty acid composition . However, active enzyme was detected when assayed in vitro with added spinach ferredoxin . The lack of significant activity in vitro without added ferredoxin and the lack of observed change in fatty acid composition indicate that ferredoxin is a required cofactor for the enzyme and that E . coli ferredoxin functions poorly, if at all, as an electron donor for the plant enzyme.

Proc Natl Acad Sci U S A, 1991 Mar 15, 88(6), 2510 - 4
Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally unrelated to the animal and fungal homologs; Shanklin J et al.; Stearoyl-acyl-carrier-protein (ACP) desaturase (EC 1.14.99.6) was purified to homogeneity from avocado mesocarp, and monospecific polyclonal antibodies directed against the protein were used to isolate full-length cDNA clones from Ricinus communis (castor) seed and Cucumis sativus (cucumber) . The nucleotide sequence of the castor clone pRCD1 revealed an open reading frame of 1.2 kilobases encoding a 396-amino acid protein of 45 kDa . The cucumber clone pCSD1 encoded a homologous 396-amino acid protein with 88% amino acid identity to the castor clone . Expression of pRCD1 in Saccharomyces cerevisiae resulted in the accumulation of a functional stearoyl-ACP desaturase, demonstrating that the introduction of this single gene product was sufficient to confer soluble desaturase activity to yeast . There was no detectable identity between the deduced amino acid sequences of the castor delta 9-stearoyl-ACP desaturase and either the delta 9-stearoyl-CoA desaturase from rat or yeast or the delta 12 desaturase from Synechocystis, suggesting that these enzymes may have evolved independently . However, there was a 48-residue region of 29% amino acid sequence identity between residues 53 and 101 of the castor desaturase and the proximal border of the dehydratase region of the fatty acid synthase from yeast . Stearoyl-ACP mRNA was present at substantially higher levels in developing seeds than in leaf and root tissue, suggesting that expression of the delta 9 desaturase is developmentally regulated.

Proc Natl Acad Sci U S A, 1991 Mar 15, 88(6), 2500 - 4
High level expression in Escherichia coli of soluble, enzymatically active schistosomal hypoxanthine/guanine phosphoribosyltransferase and trypanosomal ornithine decarboxylase; Craig SP 3rd et al.; The bacterial alkaline phosphatase (phoA) promoter and signal peptide have been used previously to control recombinant expression and secretion of eukaryotic proteins in Escherichia coli . Other reports have shown that this expression system can generate relatively modest levels of active hypoxanthine/guanine phosphoribosyltransferase (HPRT; hypoxanthine phosphoribosyltransferase; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), which carries part of the signal peptide but remains in the cytosol of the bacteria . Herein, the phoA promoter without its associated signal peptide is used to regulate expression of the HPRT of Schistosoma mansoni and the ornithine decarboxylase (ODC; L-ornithine carboxy-lyase, EC 4.1.1.17) of Trypanosoma brucei, two enzymes that have been identified as potential targets for antiparasitic chemotherapy . The levels of recombinant expression range from 20% to 60% of the total bacterial protein, and the majority of both recombinant enzymes was soluble . The specific activity for the recombinant trypanosomal ODC was one-third to two-thirds that of the authentic native enzyme and yields were predicted to be 15-30 mg of active enzyme per liter of bacterial culture . The specific activity for the recombinant schistosomal HPRT was equivalent to that for the native enzyme purified from schistosomes and up to 10 mg of enzymatically active HPRT has been purified from a 0.5-liter culture of treated bacteria . These results represent a break-through in recombinant expression of HPRT and ODC.

J Immunol, 1991 Mar 15, 146(6), 1900 - 8
The presence of sialidase-sensitive sialosylgangliotetraosyl ceramide (GM1b) in stimulated murine macrophages . Deficiency of GM1b in Escherichia coli-activated macrophages from the C3H/HeJ mouse; Yohe HC et al.; The stimulated murine macrophage was found to contain 11 major gangliosides of which 8 were determined to be monosialylated . The thin-layer chromatographic patterns were complicated by the presence of both sialic acid and ceramide fatty acid heterogeneity . N-glycolyl and N-acetylneuraminic acid-containing species were present for each ganglioside characterized . Although C18 sphingosine was the only long chain base detected, ceramide fatty acid ranged from C16 to C24 carbon moieties . Based on gas-liquid chromatographic and antibody analyses, all major tetraosyl structure gangliosides were ganglio series types . Comprising 43 to 60% of thioglycollate-stimulated cells and 60 to 70% of Escherichia coli-activated cells, monosialosyl-gangliotetraosyl ceromides (Gm1 gangliosides) were the major monosialo species of which four were present: sialidase-resistant NeuGc-GM1a and NeuAc-GM1a and sialidase sensitive NeuGc-GM1b and NeuAc-GM1b . Analyses of thioglycollate-elicited murine peritoneal macrophage ganglioside patterns from four strains of mice, including the C3H/HeJ strain, indicated that, in the absence of any expression of a genetic defect, the pattern is conserved . However, when E . coli was used as the activating agent, the normal C3H/HeN macrophage contained little Gm1a with the sialidase-sensitive Gm1b predominant; the converse was true for the congenic endotoxin hyporesponsive C3H/HeJ strain . Therefore, C3H/HeJ mice are not defective in ganglioside metabolism per se but in the processing of an endotoxin stimulus such that one manifestation is an altered macrophage ganglioside pattern deficient in Gm1b.

J Biol Chem, 1991 Mar 15, 266(8), 5323 - 32
Sequence and inactivation of the pss gene of Escherichia coli . Phosphatidylethanolamine may not be essential for cell viability; DeChavigny A et al.; Phosphatidylethanolamine is the only zwitterionic phospholipid in Escherichia coli and accounts for 70-80% of the total glycerophospholipids of this organism . To investigate the function of phosphatidylethanolamine in E . coli, we constructed an inactivated allele (pss93::kan) of the gene encoding the phosphatidylserine synthase which catalyzes the committed step to the synthesis of phosphatidylethanolamine . Growth of this mutant was dependent on a plasmid-borne copy of the wild type gene . After curing the mutant of the wild type gene, growth stopped when the content of phosphatidylethanolamine reached 30% of the total phospholipid . Divalent metal ions at millimolar concentrations suppressed the growth phenotype of the mutant in the following order of efficiency: Ca2+ greater than Mg2+ greater than Sr2+ . Although phosphatidylserine synthase activity was not detectable, phosphatidylethanolamine was still present at 0.007% of the total phospholipid after growth for many generations in rich medium containing 20 mM Mg2+ . The remainder of the phospholipid was primarily phosphatidylglycerol and cardiolipin with no other unique phosphate-containing chloroform-soluble material present . The phospholipid to protein ratio and the fatty acid composition were very similar to the parental strain . The broad divalent metal ion auxotrophy brought about by the lack of phosphatidylethanolamine suggests a primarily structural role for this phospholipid in E . coli.

J Biol Chem, 1991 Mar 15, 266(8), 5018 - 24
Anomalous stimulation of Escherichia coli alkaline phosphatase activity in guanidinium chloride . Modulation of the rate-limiting step and negative cooperativity; Rao NM et al.; Guanidinium chloride stimulates the activity of alkaline phosphatase from Escherichia coli, by 3-4-fold . Structural parameters of the enzyme, monitored by fluorescence and circular dichroism, indicate progressive denaturation . This unusual stimulation is shown to be independent of the nature of the substrate and source of the enzyme . Profiles of pH dependence and transphosphorylation reaction indicate that the dephosphorylation step of the catalysis is enhanced in the presence of guanidinium chloride . We demonstrate, by fast-flow kinetics and inhibitor titrations, that guanidinium chloride enhances activity by abolishing negative cooperativity and by accelerating the dissociation of rate-limiting enzyme and substrate (E.P) complex.

J Biol Chem, 1991 Mar 15, 266(8), 4878 - 82
Proofreading activity of DNA polymerase III responds like elongation activity to auxiliary subunits; Reems JA et al.; A comparison of the 3'----5' proofreading properties between Escherichia coli DNA polymerase III holoenzyme and DNA polymerase III' was conducted . This study indicated that the influence of the holoenzyme auxiliary subunits on the proofreading exonuclease parallels their effect on the elongation reaction . At physiological ionic strengths the auxiliary subunits markedly stimulated the exonuclease rate in an ATP-dependent reaction, while the exonuclease rate of DNA polymerase III' was not affected by ATP . E . coli single-stranded DNA binding protein stimulated the 3'----5' exonuclease activity of holoenzyme and inhibited DNA polymerase III' . Similarly, the auxiliary subunits and ATP converted the proofreading activity to a highly processive exonuclease . Our observation, that the exonuclease activity of the DNA polymerase III holoenzyme responded to ATP, salt, and E . coli single-stranded DNA-binding protein like the elongation activity, is consistent with the polymerase and exonuclease subunits acting within the same complex in a coordinated reaction.

J Biol Chem, 1991 Mar 15, 266(8), 4686 - 91
Expression of cloned bovine adrenal rhodanese; Miller DM et al.; A cDNA for the enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) has been cloned from a bovine adrenal library . An initiator methionine codon precedes the amino-terminal amino acid found in the isolated protein . Rhodanese is synthesized in the cytoplasm and transferred to the mitochondrial matrix . Thus, any amino-terminal sequence required for organelle import is retained in the mature protein . Furthermore, the DNA sequence shows that there are three additional amino acids, Gly-Lys-Ala, at the carboxyl terminus that are not found by protein sequencing . Additionally, comparison of the published amino acid sequence with that encoded by the open reading frame revealed three differences in the amino acid sequence . Comparison of the bovine and chicken liver sequences shows an overall level of 70% sequence homology, but there is complete identity of all residues that have been implicated in the function of the enzyme . When two mammalian cells, cos-7 and 293 cells, were transiently transfected with a plasmid containing the rhodanese coding region, rhodanese activity in lysates increased approximately 20-fold . Fluorograms of denaturing polyacrylamide gels detected a large increase in a polypeptide that co-migrated with the native protein and reacted with anti-rhodanese antibodies . Nondenaturing gels showed two active species that co-migrated with the two major electrophoretic forms purified by current procedures . Escherichia coli, transformed with a plasmid containing the rhodanese coding region, showed a 15-fold increase in rhodanese activity over baseline values . When the E . coli recombinant protein was analyzed on a nondenaturing gel, only one species was observed that co-electrophoresed with the more electropositive variant seen in purified bovine liver rhodanese . This single variant could be converted by carboxypeptidase B digestion to a form of the enzyme that co-migrated with the more electronegative species isolated from bovine liver . Thus, two major, enzymatically active electrophoretic variants, commonly observed in mammalian cells, can be accounted for by carboxyl-terminal processing without recourse to other post-translational modifications.

Biochem J, 1991 Mar 15, 274 ( Pt 3), 707 - 13
Albino mutants of Streptomyces glaucescens tyrosinase; Jackman MP et al.; Site-directed mutagenesis was used to determine the functional role of several residues of Streptomyces glaucescens tyrosinase . Replacement of His-37, -53, -193 or -215 by glutamine yields albino phenotypes, as determined by expression on melanin-indicator plates . The purified mutant proteins display no detectable oxy-enzyme and increased Cu lability at the binuclear active site . The carbonyl derivatives of H189Q and H193Q luminesce, with lambda max . displaced more than 25 nm to a longer wavelength compared with native tyrosinase . The remaining histidine mutants display no detectable luminescence . The results are consistent with these histidine residues (together with His-62 and His-189 reported earlier) acting as Cu ligands in the Streptomyces glaucescens enzyme . Conservative substitution of the invariant Asn-190 by glutamine also gives an albino phenotype, no detectable oxy-enzyme and labilization of active-site Cu . The luminescence spectrum of carbonyl-N190Q, however, closely resembles that of the native enzyme under conditions promoting double Cu occupancy of the catalytic site . A critical role for Asn-190 in active-site hydrogen-bonding interactions is proposed.

J Biol Chem, 1991 Mar 15, 266(8), 4979 - 86
A highly reactive beta-galactosidase (Escherichia coli) resulting from a substitution of an aspartic acid for Gly-794; Martinez-Bilbao M et al.; The beta-galactosidases of several mutagenized strains of Escherichia coli K12 which grew on lactobionate were found to be heat labile . Sequence analysis of the lacZ gene (ligated into Bluescript) of one of these strains (E . coli REH4) showed that the only change in the amino acid sequence was a substitution of an Asp for Gly-794 . This change caused a dramatic increase of the activity when lactose was the substrate . The kcat of the purified enzyme from E . coli REH4 (G794D-beta-galactosidase) with lactose as the substrate was five to six times as large as the kcat of the normal enzyme with lactose . Purified G794D-beta-galactosidase was, however, less stable to heat and also to chymotrypsin (which cleaves next to Trp-585) than was normal beta-galactosidase . G794D-beta-Galactosidase bound substrates and substrate analog inhibitors less well than did normal beta-galactosidase while planar transition state analog inhibitors were more strongly bound . The ability to bind 2-amino-D-galactose (a positively charged transition state analog inhibitor) was either unaltered or was decreased somewhat . The data showed that the alteration in structure caused an increase in the value of k2 (the rate constant for the step in which the glycosidic bond is cleaved) with each substrate tested (the increase was at least 25-fold when lactose was the substrate) while k3 was decreased about 4-fold (k3 is the rate constant for the common hydrolysis step with each substrate) . Since k2 is rate determining when lactose is the substrate of the normal enzyme, the increase in k2 resulted in a large increase in rate despite the fact that the value of k3 decreased . Large rate increases were not found with the other two substrates because the k2 values were not increased by large factors and because the decrease in the value of k3 negated the effects of the increased k2 values . The destabilization of the substrate binding coupled with a stabilization of the binding of a planar transition state is a possible cause of the significant increase in the value of k2 and of the enhanced activity with lactose.

Biochem J, 1991 Mar 15, 274 ( Pt 3), 807 - 12
A comparative study of the kinetics and stereochemistry of the serine hydroxymethyltransferase- and tryptophan synthase-catalysed exchange of the pro-2R and pro-2S protons of glycine; Malthouse JP et al.; The stereospecificity of the serine hydroxymethyltransferase (EC 2.1.2.1)- and tryptophan synthase (EC 4.2.1.20)- catalysed exchange of the pro-2R and pro-2S alpha-protons of glycine was investigated by using 13C n.m.r . The exchange process is described in terms of a minimal four-step mechanism, and a method for analysing the exchange process by complete progress curves is presented . It is shown that serine hydroxymethyltransferase does not have absolute stereospecificity for the pro-2S-proton of glycine, but it catalyses the exchange of this proton 7400 times faster than the pro-2R proton of glycine . Tryptophan synthase is shown preferentially to catalyse the exchange of the pro-2R proton of glycine at a rate 380 times faster than the pro-2S proton of glycine . The exchange rates for the rapidly exchanged alpha-protons of glycine are similar for both enzymes . However, the exchange rates of the slowly exchanged alpha-protons differ by an order of magnitude . The structural features that may be responsible for the differences in the stereospecificity of the two enzymes are discussed.

Biochem J, 1991 Mar 15, 274 ( Pt 3), 723 - 30
Redox analysis of the cytochrome o-type quinol oxidase complex of Escherichia coli reveals three redox components; Bolgiano B et al.; Potentiometric analyses of the cytochrome o-type oxidase of Escherichia coli, using membranes from a strain containing amplified levels of the cytochrome bo complex, were conducted to resolve the redox centres of the oxidase . The cytochrome o-type oxidase of E . coli, a quinol oxidase, contains 2 mol of b-type haem per mol of complex and copper . Detailed analysis of potentiometric titrations, based on the absorbance of the Soret band, suggests that there are three contributions with midpoint potentials (Em,7) around +55 mV, +211 mV and +408 mV, all with maxima at 426-430 nm in the reduced state . In the alpha region of the spectra, a component with Em,6.85 = +58 mV has a maximal peak at 557 nm, and twin peaks at 556 and 564 nm nitrate with Em,6.85 = +227 mV . A feature corresponding to the highest potential Soret contribution was not observed . These data can be explained either by a model incorporating haem-haem interaction or by attributing the shorter-wavelength band (557 nm) to haem b and a split alpha-band (556, 564 nm) to the haem o (oxygen-binding haem b) . Absolute spectra of oxidized membranes show continuous absorbance from 460 to 530 nm and suggest the presence of a high-spin haem component in the membranes . Monitoring absorbance at 635 minus 672 nm, contributions with midpoints (Em,7) around +52 mV, +234 mV and +371 mV are observed . This latter contribution is possibly the highest-potential component which titrates with Em greater than +400 mV in the Soret region and may represent copper-haem coupling in the cytochrome o complex.

J Biol Chem, 1991 Mar 15, 266(8), 5226 - 37
Comparison of the periplasmic receptors for L-arabinose, D-glucose/D-galactose, and D-ribose . Structural and Functional Similarity; Vyas NK et al.; The primary sequence of the receptor for L-arabinose or Ara-binding protein (ABP) composed of 306 residues is very different from the D-glucose/D-galactose-binding protein (GGBP) which consists of 309 residues . Nevertheless, superimpositioning of the well-refined high resolution structures of ABP in complex with D-galactose and the GGBP in complex with D-glucose shows very similar structures; 220 of the residues (or about 70%) have a root mean square deviation of 2.0 A . From the superpositioning, nine pairs of continuous segments (consisting of 8-51 residues), mainly alpha-helices and beta-strands that form the core of the two lobes of the bilobate proteins were found to exhibit strong sequence homology . The equivalenced structures and aligned sequences show that many of the polar, as well as aromatic residues, in the sugar-binding sites located in the cleft between the two lobes are highly conserved . Surprisingly, however, the exact mode of binding of the D-galactose in ABP is totally different from that of the D-glucose in GGBP . Using the structurally aligned sequences of the ABP and GGBP as a template, we have matched the sequence of the ribose-binding protein (RBP) which consists of 271 residues with the ABP/GGBP pair . Although the nine aligned segments of all three proteins show little sequence identity, they have significant homology . Four additional segments of RBP were matched only with GGBP, leading to the alignment of about 90% of the RBP sequence with the GGBP sequence . Many of the conserved residues in the binding sites of ABP and GGBP matched with similar residues in RBP . Additional observations indicate that the GGBP/RBP pair is more closely related than the ABP/RBP or ABP/GGBP pair . All three binding proteins, which may have diverged from a common ancestor, serve as primary receptors for bacterial high affinity active transport systems . Moreover, GGBP and RBP, but not ABP, also act as receptors for chemotaxis . An exposed site located in one domain, which includes Gly74, for interacting with the trg transmembrane signal transducer that is involved in triggering chemotaxis has been located in the structure of GGBP (Vyas, N.K., Vyas, M.N., and Quiocho, F.A . (1988) Science 242, 1290-1295) . Whereas the site is absent in the structure of ABP, it is strongly predicted to be present in RBP which shares the same trg transducer with GGBP . The knowledge-based alignment of RBP further revealed two possible additional peripheral chemotactic sites that show high structural and sequence similarity between GGBP and RBP only . At least one of these sites, together with the one proven to exist in the other domain, could be used by the signal transducer with which both binding proteins interact in a way which the substrate-loaded "closed cleft" structure could be discriminated from the unliganded "open cleft" form by the transducer.

J Biol Chem, 1991 Mar 15, 266(8), 5104 - 12
Ubiquitin conjugation by the yeast RAD6 and CDC34 gene products . Comparison to their putative rabbit homologs, E2(20K) AND E2(32K); Haas AL et al.; The recombinant yeast RAD6 and CDC34 gene products were expressed in Escherichia coli extracts and purified to apparent homogeneity . The physical and catalytic properties of RAD6 and CDC34 were similar but distinct from their putative rabbit reticulocyte homologs, E2(20k) and E2(32k), respectively . Like their reticulocyte counterparts, RAD6 and CDC34 are bifunctional enzymes competent in both ubiquitin:protein ligase (E3)-independent and E3-dependent conjugation reactions . RAD6 and E2(20k) exhibit marked specificity for the conjugation of core histones and catalyze the processive ligation of up to three ubiquitin moieties directly to such model substrates . RAD6 differed from its putative E2(20k) homolog in exhibiting simple saturation behavior in the kinetics of histone conjugation and in being unable to distinguish kinetically between core histones H2A and H2B, yielding identical values of kcat (1.9 min-1) and Km (20 microM) . A slow rate of multiubiquitination involving formation of extended ubiquitin homopolymers on the histones was also observed with RAD6 and E2(20k) . Comparison of conjugate patterns among native, reductively methylated, and K48R ubiquitin variants demonstrated that the linkage between ubiquitin moieties formed by E2(20k) and RAD6 was not through Lys-48 of ubiquitin, the site previously demonstrated as a strong signal for degradation of the target protein . In contrast, CDC34 differs from its putative homolog, E2(32k), in showing a specificity for conjugation to bovine serum albumin rather than to core histones . Both CDC34 and E2(32k) exhibit a marked kinetic selectivity for processive multiubiquitination via Lys-48 of ubiquitin . Calculations based on a model ubiquitin conjugation reaction indicated that E2(32k) and CDC34 preferentially catalyzed multiubiquitination over ligation of the polypeptide directly to target proteins . Formation of such multiubiquitin homopolymers by E2(32k) and CDC34 suggests these enzymes may commit their respective target proteins to degradation via an E3-independent pathway.

J Biol Chem, 1991 Mar 15, 266(8), 4951 - 8
A range of catalytic efficiencies with avian retroviral protease subunits genetically linked to form single polypeptide chains; Bizub D et al.; Molecular modeling based on the crystal structure of the Rous sarcoma virus (RSV) protease dimer has been used to link the two identical subunits of this enzyme into a functional, single polypeptide chain resembling the nonviral aspartic proteases . Six different linkages were selected to test the importance of different interactions between the amino acids at the amino and carboxyl termini of the two subunits . These linkages were introduced into molecular clones of fused protease genes and the linked protease dimers were expressed in Escherichia coli and purified . Catalytically active proteins were obtained from the inclusion body fraction after renaturation . The linked protease dimers exhibited a 10-20-fold range in catalytic efficiencies (Vmax/Km) on peptide substrates . Both flexibility and ionic interactions in the linkage region affect catalytic efficiency . Some of the linked protease dimers were 2-3-fold more active than the nonlinked enzyme purified from bacteria, although substrate specificities were unchanged . Similar relative efficiencies were observed using a polyprotein precursor as substrate . Mutation of one catalytic Asp in the most active linked protease dimer inactivated the enzyme, demonstrating that these proteins function as single polypeptide chains rather than as multimers.

Proc Natl Acad Sci U S A, 1991 Mar 15, 88(6), 2495 - 9
RNA-binding domain of the A protein component of the U1 small nuclear ribonucleoprotein analyzed by NMR spectroscopy is structurally similar to ribosomal proteins; Hoffman DW et al.; An RNA recognition motif (RRM) of approximately 80 amino acids constitutes the core of RNA-binding domains found in a large family of proteins involved in RNA processing . The U1 RNA-binding domain of the A protein component of the human U1 small nuclear ribonucleoprotein (RNP), which encompasses the RRM sequence, was analyzed by using NMR spectroscopy . The domain of the A protein is a highly stable monomer in solution consisting of four antiparallel beta-strands and two alpha-helices . The highly conserved RNP1 and RNP2 consensus sequences, containing residues previously suggested to be involved in nucleic acid binding, are juxtaposed in adjacent beta-strands . Conserved aromatic side chains that are critical for RNA binding are clustered on the surface of the molecule adjacent to a variable loop that influences recognition of specific RNA sequences . The secondary structure and topology of the RRM are similar to those of ribosomal proteins L12 and L30, suggesting a distant evolutionary relationship between these two types of RNA-associated proteins.

Proc Natl Acad Sci U S A, 1991 Mar 15, 88(6), 2471 - 5
Proton transfer is rate-limiting for translocation of precursor proteins by the Escherichia coli translocase; Driessen AJ et al.; The protonmotive force stimulates translocation in vivo, in crude in vitro reactions, and in a purified, reconstituted reaction . Translocation activity is a function of the pH at the inner face of the membrane . Both the transmembrane pH gradient and the transmembrane electrical potential stimulate translocation . A late-stage translocation intermediate of the proOmpA preprotein completes its translocation in the absence of ATP when a protonmotive force is imposed . This completion of translocation is retarded by a factor of greater than 3 in deuterium oxide relative to water, demonstrating that translocation involves proton-transfer reactions in rate-limiting steps.

Proc Natl Acad Sci U S A, 1991 Mar 15, 88(6), 2432 - 6
Generation of diverse high-affinity human monoclonal antibodies by repertoire cloning; Persson MA et al.; Combinatorial libraries of antibody heavy and light chains derived from the peripheral blood lymphocytes of an individual immunized with tetanus toxoid have been expressed in Escherichia coli by using phage lambda vectors . Screening of the libraries allowed identification of a large number of human monoclonal Fab fragments specific for tetanus toxoid . Initial studies suggested considerable sequence diversity in these antibodies . The method should allow the generation of many human monoclonal antibodies of interest and the dissection of human humoral immune responses.

Gene, 1991 Mar 15, 99(2), 151 - 6
Synthesis and characterization of a recombinant myohemerythrin protein encoded by a synthetic gene; Alexander H et al.; The antigenic epitopes of the myohemerythrin (MHr) molecule have been studied extensively . The critical amino acid residues responsible for its immune recognition have been identified by using synthetic peptides and the technique of epitope scanning . To assess the true relevance of these techniques for determining the molecular mechanism of antigenic recognition and immunogenicity, the results obtained with isolated peptides should be tested in the context of the folded protein . To this end, we have designed and constructed a synthetic MHr gene, in modular form, which will allow subsequent alterations of nucleotide sequence encoding epitopes of interest . We have produced the recombinant protein at high level, and have shown by several criteria that it possesses the chemical, physical and immunological properties of the native worm protein . Thus, we have developed a valuable system for detailed immunological studies of the structure and chemistry required for antibody binding to protein.

Proc Natl Acad Sci U S A, 1991 Mar 15, 88(6), 2307 - 11
Transcript elongation and termination are competitive kinetic processes; von Hippel PH et al.; In this paper, we develop a kinetic approach to predict the efficiency of termination at intrinsic (factor independent) terminators of Escherichia coli and related organisms . In general, our predictions agree well with experimental results . Our analysis also suggests that termination efficiency can readily be modulated by protein factors and environmental variables that shift the kinetic competition toward either elongation or termination . A quantitative framework for the consideration of such regulatory effects is developed and the strengths and limitations of the approach are discussed.

Proc Natl Acad Sci U S A, 1991 Mar 15, 88(6), 2122 - 6
Isolation and characterization of a cDNA clone encoding an alternative oxidase protein of Sauromatum guttatum (Schott); Rhoads DM et al.; Polyclonal and monoclonal antibodies that recognize the 35-, 36-, and 37-kDa alternative oxidase proteins of Sauromatum guttatum (Schott) were used to isolate a cDNA clone, pAOSG81, from an S . guttatum cDNA expression library . A fusion protein with an apparent molecular mass of 48 kDa was expressed from a pUC119 derivative of pAOSG81 (pAOSG81-119) in Escherichia coli cells and was recognized by the monoclonal antibodies . When the in vitro translated and immunoprecipitated products made from mRNA hybrid-selected by pAOSG81 were analyzed, a single band corresponding to a protein with an apparent molecular mass of 42 kDa was observed . DNA sequence characterization showed that pAOSG81 contains the entire coding region of a protein with a calculated molecular mass of 38.9 kDa, a putative 63-amino acid transit peptide, and a 9-amino acid match to the authentic N-terminal sequence of the 36-kDa alternative oxidase protein . Analyses of the deduced amino acid sequence indicate: (i) that the transit peptide is predicted to form amphiphilic helices, and (ii) that three regions of the processed protein are likely to form transmembrane alpha-helices . We conclude from these data that pAOSG81 represents a nuclear gene, aox1, encoding a precursor protein of one or more of the alternative oxidase proteins of S . guttatum.

Proc Natl Acad Sci U S A, 1991 Mar 15, 88(6), 2046 - 50
Design, synthesis, and functional expression of a gene for charybdotoxin, a peptide blocker of K+ channels; Park CS et al.; A gene encoding charybdotoxin (CTX), a K+ channel blocker from scorpion venom, was designed, synthesized, and expressed as a cleavable fusion protein in Escherichia coli . A sequence-specific protease, factor Xa, was used to cleave the fusion protein and thus release the toxin peptide . The recombinant toxin was purified, oxidized to form disulfide bonds, and treated to form N-terminal pyroglutamate . Recombinant CTX is identical to the native venom CTX with respect to high-performance liquid chromatography mobility, amino acid composition, and N-terminal modification . With single Ca2(+)-activated K+ channels as an assay system, recombinant CTX shows blocking and dissociation kinetics identical to the native venom toxin . The synthetic gene and high-level expression of functionally active CTX make it possible to study the fundamental mechanism of the toxin-ion channel interaction.

Gene, 1991 Mar 15, 99(2), 211 - 6
Synthesis of the nonconserved dihydroorotase domain of the multifunctional hamster CAD protein in Escherichia coli; Musmanno LA et al.; CAD is the multifunctional protein of higher eukaryotes which catalyzes the first three steps of pyrimidine biosynthesis . Its enzymatic activities exist as independent domains in the order: N terminus-carbamylphosphate synthetase II(CPSase)-dihydroorotase(DHOase)-aspartate transcarbamylase(ATCase)-C terminus . To functionally define the minimum hamster cDNA region required to encode an active DHOase, expression constructs were generated . Many such constructs complement Escherichia coli mutants defective not only in DHOase but also in ATCase . Constructs deleted for most of the sequence encoding the ATCase domain continue to complement E . coli mutants defective in DHOase . All of these smaller constructs also lack the region encoding CPSase . Therefore, a 'genetic cassette', containing information for neither the CPSase nor the ATCase domain, can direct the synthesis of a polypeptide with DHOase activity . Interestingly, inclusion of a portion of the DHOase-ATCase interdomain bridge appears to be required for optimum activity.

Brain Res, 1991 Mar 15, 543(2), 194 - 200
Increase in omega 3 (peripheral type benzodiazepine) binding sites in the rat cortex and striatum after local injection of interleukin-1, tumour necrosis factor-alpha and lipopolysaccharide; Bourdiol F et al.; The possible involvement of lymphokines in the glial reaction/proliferation that follows brain injury has been investigated by measuring the density of omega 3 (peripheral type benzodiazepine) binding sites associated to glial cells and macrophages after local injection of lymphokines in the rat cerebral cortex and striatum . omega 3 Site densities were measured either by quantitative autoradiography in brain sections or by conventional binding in membrane using {3H}PK 14105 or {3H}PK 11195 as ligands . Intracortical or intrastriatal infusion of interleukin-1 (10 and 20 units) caused a marked increase in the density of omega 3 sites (+83% and +80%, respectively, when compared to saline-infused animals) around the injection site at 7 days postinjection . There was a good spatial correspondence between the autoradiographic distribution of omega 3 sites and the distribution of reactive astrocytes (as assessed by GFAP immunostaining) or acid phosphatase rich cells (phagocytes) . Significant increases in omega 3 site densities were also observed in striatal homogenates 1 week after local administration of tumor necrosis factor-alpha (TNF-alpha) . The maximal increase (+80%) was observed after the administration of 3 units, higher and lower doses resulting in smaller increases . Intrastriatal injection of E . coli lipopolysaccharide (LPS), a bacterial endotoxin known to stimulate interleukin-1 and TNF-alpha production by microglial cells in culture, also resulted in significant increases in omega 3 site densities in striatal homogenates (maximal increase, +170% 1 week after the injection of 200 ng).(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1991 Mar 14, 196(2), 469 - 74
Isolation and characterization of catalytic and calmodulin-binding domains of Bordetella pertussis adenylate cyclase; Munier H et al.; A truncated Bordetella pertussis cya gene product was expressed in Escherichia coli and purified by affinity chromatography on calmodulin-agarose . Trypsin cleavage of the 432-residue recombinant protein (Mr = 46,659) generated two fragments of 28 kDa and 19 kDa . These fragments, each containing a single Trp residue, were purified and analyzed for their catalytic and calmodulin-binding properties . The 28-kDa peptide, corresponding to the N-terminal domain of the recombinant adenylate cyclase, exhibited very low catalytic activity, and was still able to bind calmodulin weakly, as evidenced by using a fluorescent derivative of the activator protein . The 19-kDa peptide, corresponding to the C-terminal domain of the recombinant adenylate cyclase, interacted only with calmodulin as indicated by a shift in its intrinsic fluorescence emission spectrum or by the enhancement of fluorescence of dansyl-calmodulin . T28 and T19 fragments exhibited an increased sensitivity to denaturation by urea as compared to uncleaved adenylate cyclase, suggesting that interactive contacts between ordered portions of T28 and T19 in the intact protein participate both in their own stabilization and in stabilization of the whole tertiary structure . The two fragments reassociated into a highly active calmodulin-dependent species . Reassociation was enhanced by calmodulin itself, which 'trapped' the two complementary peptides into a stable, native-like, ternary complex, which shows similar catalytic properties to intact adenylate cyclase.

Eur J Biochem, 1991 Mar 14, 196(2), 415 - 21
Functional analysis of the pathogenesis-related 1a protein gene minimal promoter region . Comparison of reporter gene expression in transient and in stable transfections; Beilmann A et al.; Pathogenesis-related (PR) proteins are a heterogeneous group of host encoded, low-molecular-mass proteins that are induced in plants by various external stimuli, such as pathogen attack or exposure of the plants to certain chemicals . To examine the regulation of these genes, the 5'-flanking region of the tobacco PR-1a gene {Pfitzner U.M., Pfitzner, A.J.P . & Goodman, H.M . (1988) Mol . Gen . Genet . 211, 290-295} was joined by a transcriptional fusion to the Escherichia coli beta-glucuronidase (GUS) gene . Expression of the reporter gene was monitored in transient expression assays as well as in stable transformants . The PR-1a 5'-flanking sequences from -335, -149 or -71 to +28 are functional promoter elements in tobacco and carrot protoplasts, as determined by transient expression . These constructs direct correct initiation at the normal transcription-start site of the PR-1a gene . The level of gene expression was about twofold less than that obtained with the cauliflower mosaic virus 35S RNA promoter . Regulation of gene expression by acetylsalicylic acid, however, could not be detected in the transient assays . When the same constructs were stably integrated into the tobacco genome, neither constitutive nor induced beta-glucuronidase activity was observed . A comparison of the results from the transient and the stable transfection experiments suggests that expression of the reporter gene may be due to a constitutive transcriptional activity of the PR-1a 5'-flanking regions under the conditions of the transient assays and that the PR-1a promoter may contain at least two functional domains.

Nature, 1991 Mar 14, 350(6314), 165 - 7
Function of DnaJ and DnaK as chaperones in origin-specific DNA binding by RepA; Wickner S et al.; Heat-shock proteins are normal constituents of cells whose synthesis is increased on exposure to various forms of stress . They are interesting because of their ubiquity and high conservation during evolution . Two families of heat-shock proteins, hsp60s and hsp70s, have been implicated in accelerating protein folding and oligomerization and also in maintaining proteins in an unfolded state, thus facilitating membrane transport . The Escherichia coli hsp70 analogue, DnaK, and two other heat-shock proteins, DnaJ and GrpE, are required for cell viability at high temperatures and are involved in DNA replication of phage lambda and plasmids P1 and F . These three proteins are involved in replication in vitro of P1 DNA along with many host replication proteins and the P1 RepA initiator protein . RepA exists in a stable protein complex with DnaJ containing a dimer each of RepA and DnaJ . We report here that DnaK and DnaJ mediate an alteration in the P1 initiator protein, rendering it much more active for oriP1 DNA binding.

Eur J Biochem, 1991 Mar 14, 196(2), 357 - 61
Expression of three human beta-adrenergic-receptor subtypes in transfected Chinese hamster ovary cells; Tate KM et al.; The genes coding for three pharmacologically distinct subtypes of human beta-adrenergic receptors (beta 1 AR, beta 2 AR and beta 3 AR) were transfected for expression in Chinese hamster ovary (CHO) cells . Stable cell lines expressing each receptor were analyzed by ligand binding, adenylate cyclase activation and photoaffinity labeling, and compared to beta AR subtypes observed in previously described tissues, primary cultures and transfected cell lines . Each of the three receptor subtypes displayed saturable {125I}iodocyanopindolol-binding activity . They showed the characteristic rank order of potencies for five agonists, determined by measuring the accumulation of intracellular cAMP . These recombinant cell lines express a homogeneous population of receptors and display the known pharmacological properties of beta 1 AR and beta 2 AR, in human tissues . It is therefore likely that the pattern of ligand binding and adenylate cyclase activation, mediated by the new beta 3 AR in CHO cells, also reflects the yet-undetermined pharmacological profile in humans.

Eur J Biochem, 1991 Mar 14, 196(2), 287 - 94
Mutation of a negatively charged amino acid in thioredoxin modifies its reactivity with chloroplastic enzymes; de Lamotte-Guery F et al.; A new over-expression system has been set up for Escherichia coli thioredoxin, yielding 55 mg purified protein/10 g fresh cells . This system has been used to produce thioredoxin modified by site-directed mutagenesis . Taking advantage of the structural and enzymatic similarity between E . coli and spinach m-type thioredoxin, Asp61 of E . coli thioredoxin has been changed into Asn in order to investigate the impact of the suppression of a charged residue on the interaction of thioredoxin with target enzymes . The modification did not significantly alter the structure of the protein . Neither the rate of reduction of insulin and 5,5'-dithio-bis(2-nitrobenzoic acid) by the reduced thioredoxin, nor the reduction by NADPH-dependent thioredoxin reductase, have been modified . The major effect of the mutation was observed for chloroplast enzyme activation with thioredoxin reduced by dithiothreitol and with thioredoxin reduced by ferredoxin-dependent thioredoxin reductase in a light-activation reconstituted chloroplast system . The substitution of the negatively charged Asp61 by the neutral Asn led to an increase in the efficiency of spinach fructose-1,6-bisphosphatase activation by the dithiothreitol-reduced thioredoxin, and to an increase in both spinach fructose-1,6-bisphosphatase and corn NADP-dependent malate dehydrogenase activities in the light-activation system . This suggests that the suppression of the negative charge improves the reactivity of thioredoxin with chloroplast enzymes such as fructose-1,6-bisphosphatase and ferredoxin-dependent thioredoxin reductase.

Eur J Biochem, 1991 Mar 14, 196(2), 423 - 30
Induced plant responses to pathogen attack . Analysis and heterologous expression of the key enzyme in the biosynthesis of phytoalexins in soybean (Glycine max L . Merr . cv . Harosoy 63); Welle R et al.; In soybean (Glycine max L.), pathogen attack induces the formation of glyceollin-type phytoalexins . The biosynthetic key enzyme is a reductase which synthesizes 4,2', 4'-trihydroxychalcone in co-action with chalcone synthase . Screening of a soybean cDNA library from elicitor-induced RNA in lambda gt11 yielded two classes of reductase-specific clones . The deduced proteins match to 100% and 95%, respectively, with 229 amino acids sequenced in the purified plant protein . Four clones of class A were expressed in Escherichia coli, and the proteins were tested for enzyme activity in extracts supplemented with chalcone synthase . All were active in 4,2',4'-trihydroxychalcone formation, and the quantification showed that shorter lengths of the cDNAs at the 5' end correlated with progressively decreasing enzyme activities . Genomic blots with DNA from plants capable of 4,2',4'-trihydroxychalcone synthesis revealed related sequences in bean (Phaseolus vulgaris L.) and peanut (Arachis hypogaea L.), but not in pea (Pisum sativum L.) . No hybridization was observed with parsley (Petroselinum crispum) and carrot (Daucus carota) which synthesize other phytoalexins . The reductase protein contains a leucine-zipper motif and reveals a marked similarity with other oxidoreductases most of which are involved in carbohydrate metabolism.

Eur J Biochem, 1991 Mar 14, 196(2), 255 - 60
Modification of histidine residues on proteins from the 50S subunit of the Escherichia coli ribosome . Effects on subunit assembly and peptidyl transferase centre activity; Sumpter VG et al.; L2, L3, L4, L16 and L20 are proteins of the 50S ribosomal subunit of Escherichia coli which are essential for the assembly and activity of the peptidyl transferase centre . These proteins have been modified with the histidine-specific reagent, diethylpyrocarbonate, while L17 and L18 were treated as controls . Each modified protein tested was able to participate in the reconstitution of a 50S particle when replacing its normal counterpart, although the particles assembled with modified L2 were heterogeneous . However, although they could support assembly, modified L16 and L20 were not themselves reconstituted stably, and modified L2 and L3 were found in less than stoichiometric amounts . Particles assembled in the presence of modified L16 retained significant peptidyl transferase activity (60-70% at 10 mM diethylpyrocarbonate) whereas those reconstituted with modified L2, L3, L4 or L20 had low activity (10-30% at 10 mM diethylpyrocarbonate) . The particles assembled with the modified control protein, L17, retained 80% of their peptidyl transferase activity under the same conditions . The histidine residues within the essential proteins therefore contribute to ribosome structure and function in three significant ways; in the correct assembly of the ribosomal subunit (L2), for the stable assembly of the proteins within the ribosomal particle (L20 and L16 in particular), and directly or indirectly for the subsequent activity of the peptidyl transferase centre (L2, L3, L4 and L20) . The essential nature of the unmodified histidines for assembly events precludes the use of the chemical-modification strategy to test the proposal that a histidine on one of the proteins might participate in the catalytic activity of the centre.

Int J Cancer, 1991 Mar 12, 47(5), 665 - 9
Presence of circulating anti-c-myb oncogene product antibodies in human sera; Sorokine I et al.; Bacterially expressed mouse c-myb protein was purified from E . coli extracts and used as antigen to screen human sera for circulating anti-c-myb oncogene product antibodies . Using Western blotting, we have found that human sera contain IgG antibodies to the c-myb gene product . The percentage of positive sera in cancer patients appears to be dependent upon cancer type and is significantly higher in breast-cancer patients than in normal donors: 43% of sera from breast-cancer patients are positive, whereas in neuroblastoma cancer patients the production of IgG anti-c-myb appears to be rare . No significant correlation was observed between the presence of circulating anti-c-myb antibodies and the expression of the c-myb gene in breast tumors.

Biochemistry, 1991 Mar 12, 30(10), 2685 - 98
High-resolution three-dimensional structure of reduced recombinant human thioredoxin in solution; Forman-Kay JD et al.; The solution structure of recombinant human thioredoxin (105 residues) has been determined by nuclear magnetic resonance (NMR) spectroscopy combined with hybrid distance geometry-dynamical simulated annealing calculations . Approximate interproton distance restraints were derived from nuclear Overhauser effect (NOE) measurements . In addition, a large number of stereospecific assignments for beta-methylene protons and torsion angle restraints for phi, psi, and chi 1 were obtained by using a conformational grid search on the basis of the intraresidue and sequential NOE data in conjunction with 3JHN alpha and 3J alpha beta coupling constants . The structure calculations were based on 1983 approximate interproton distance restraints, 52 hydrogen-bonding restraints for 26 hydrogen bonds, and 98 phi, 71 psi, and 72 chi 1 torsion angle restraints . The 33 final simulated annealing structures obtained had an average atomic rms distribution of the individual structures about the mean coordinate positions of 0.40 +/- 0.06 A for the backbone atoms and 0.78 +/- 0.05 A for all atoms . The solution structure of human thioredoxin consists of a five-stranded beta-sheet surrounded by four alpha-helices, with an active site protrusion containing the two redox-active cysteines . The overall structure is similar to the crystal and NMR structures of oxidized {Katti, S . K., LeMaster, D . M., & Eklund, H . (1990) J . Mol . Biol . 212, 167-184} and reduced {Dyson, J . H., Gippert, G . P., Case, D . A., Holmgren, A., & Wright, P . (1990) Biochemistry 29, 4129-4136} Escherichia coli thioredoxin, respectively, despite the moderate 25% amino acid sequence homology . Several differences, however, can be noted . The human alpha 1 helix is a full turn longer than the corresponding helix in E . coli thioredoxin and is characterized by a more regular helical geometry . The helix labeled alpha 3 in human thioredoxin has its counterpart in the 3(10) helix of the E . coli protein and is also longer in the human protein . In contrast to these structural differences, the conformation of the active site loop in both proteins is very similar, reflecting the perfect sequence identity for a stretch of eight amino acid residues around the redox-active cysteines.

Biochemistry, 1991 Mar 12, 30(10), 2664 - 73
Differential sequence dynamics of homopolymeric and alternating AT tracts in a small plasmid DNA; Sullivan JK et al.; The location of OsO4 bispyridine hyper- and hyporeactivity in a small deletion derivative of plasmid ColE1 (PTC12, 1727 bp) has been determined for approximately 70% of the molecule . Thymine bases in homopolymeric (dA)n.(dT)n tracts (n greater than or equal to 4) were always found to be resistant toward OsO4 modification . DNA supercoiling did not destabilize these tracts . The extent of OsO4 bispyridine reactivity of homopolymeric (dA)n.(dT)n tracts, where n = 3, was found to be dependent on the rate of base unpairing of the sequence immediately 5' and 3' to the tract . Repressed OsO4 reactivity of thymine bases in (dA)3.(dT)3 tracts was observed if immediately both 5' and 3' to the tract were stable DNA sequences composed of GC base pairs and/or a homopolymeric (dA)n.(dT)n tract (n greater than or equal to 4) . Homopolymeric tracts of n = 3 not having adjacent sequences with repressed unpairing rates did not show reduced levels of OsO4 bispyridine reactivity . Alternating d(TA)n tracts (n greater than or equal to 2) were found to exhibit hyperreactivity with OsO4 . The extent of this hyperreactivity was dependent on the length of the tract and superhelical torsional stress . The distribution and frequency of homopolymeric (dA)n.(dT)n (n greater than or equal to 4) tracts in Escherichia coli promoter sequences were examined, and the possible implications of these tracts on promoter function are discussed.

Biochemistry, 1991 Mar 12, 30(10), 2655 - 64
Characterization of DNA metabolizing enzymes in situ following polyacrylamide gel electrophoresis; Longley MJ et al.; We have detected the in situ activities of DNA glycosylase, endonuclease, exonuclease, DNA polymerase, and DNA ligase using a novel polyacrylamide activity gel electrophoresis procedure . DNA metabolizing enzymes were resolved through either native or SDS-polyacrylamide gels containing defined 32P-labeled oligonucleotides annealed to M13 DNA . After electrophoresis, these enzymes catalyzed in situ reactions and their {32P}DNA products were resolved from the gel by a second dimension of electrophoresis through a denaturing DNA sequencing gel . Detection of modified (degraded or elongated) oligonucleotide chains was used to locate various enzyme activities . The catalytic and physical properties of Novikoff hepatoma DNA polymerase beta were found to be similar under both in vitro and in situ conditions . With 3'-terminally matched and mismatched {32P}DNA substrates in the same activity gel, DNA polymerase and/or 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I (large fragment), DNA polymerase III (holoenzyme), and exonuclease III were detected and characterized . In addition, use of matched and mismatched DNA primers permitted the uncoupling of mismatch excision and chain extension steps . Activities first detected in nondenaturing activity gels as either multifunctional or multimeric enzymes were also identified in denaturing activity gels, and assignment of activities to specific polypeptides suggested subunit composition . Furthermore, DNA substrates cast within polyacrylamide gels were successfully modified by the exogenous enzymes polynucleotide kinase and alkaline phosphatase before and after in situ detection of E . coli DNA ligase activity, respectively . Several restriction endonucleases and the tripeptide (Lys-Trp-Lys), which acts as an apurinic/apyrimidinic endonuclease, were able to diffuse into gels and modify DNA . This ability to create intermediate substrates within activity gels could prove extremely useful in delineating the steps of DNA replication and repair pathways.

Biochemistry, 1991 Mar 12, 30(10), 2635 - 41
Mutant aminoacyl-tRNA synthetase that compensates for a mutation in the major identity determinant of its tRNA; Miller WT et al.; A single G3.U70 base pair in the acceptor helix is the major determinant for the identity of alanine transfer RNAs (Hou & Schimmel, 1988) . Introduction of this base pair into foreign tRNA sequences confers alanine acceptance on them . Moreover, small RNA helices with as few as seven base pairs can be aminoacylated with alanine, provided that they encode the critical base pair (Francklyn & Schimmel, 1989) . Alteration of G3.U70 to G3.C70 abolishes aminoacylation with alanine in vivo and in vitro . We describe here the mutagenesis and selection of a single point mutation in Escherichia coli Ala-tRNA synthetase that compensates for a G3.C70 mutation in tRNAAla . The mutation maps to a region previously implicated as proximal to the acceptor end of the bound tRNA . In contrast to the wild-type enzyme, the mutant charges small RNA helices that encode a G3.C70 base pair . However, the mutant enzyme retains specificity for alanine tRNA and can serve as the sole source of Ala-tRNA synthetase in vivo . The results demonstrate the capacity of an aminoacyl-tRNA synthetase to compensate through a single amino acid substitution for mutations in the major determinant of its cognate tRNA.

Biochemistry, 1991 Mar 12, 30(10), 2600 - 12
Communication between the active sites in dimeric mercuric ion reductase: an alternating sites hypothesis for catalysis; Miller SM et al.; Mercuric reductase, a flavoprotein disulfide oxidoreductase, catalyzes the two-electron reduction of Hg(II) to Hg(0) by NADPH . As with all the members of this class of proteins, the enzyme is a dimer of identical subunits with two active sites per dimer, each composed of one FAD and catalytically essential residues from both subunits . In the enzyme from Tn501, these residues include, at a minimum, FAD and cysteines 135 and 140 from one subunit and cysteines 558' and 559' from the other . With this sort of active site arrangement, the enzyme seems perfectly set up for some type of subunit communication . In this report, we present results from several titrations, as well as kinetics studies, that, taken together, are consistent with the occurrence of subunit communication . In particular, the results indicate that pyridine nucleotide complexed dimers of the enzyme are asymmetric . Since the EH2-NADPH complex of the enzyme is the relevant reductant of Hg(II), these observations suggest that the enzyme may function asymmetrically during catalysis . An alternating sites model is proposed for the catalytic reduction of Hg(II), where both subunits of the dimer function in catalysis, but the steps are staggered and the subunits reverse roles after part of the reaction . An attractive feature of this proposal is that it provides a reasonable solution to the thermodynamic dilemma the enzyme faces in needing to both bind Hg(II) very tightly and reduce it.

Biochemistry, 1991 Mar 12, 30(10), 2642 - 50
Conversion of a mitochondrial gene for mammalian cytochrome c oxidase subunit II into its universal codon equivalent and expression in vivo and in vitro; Cao JL et al.; To begin to assess the independent structural and functional characteristics of the mitochondrially encoded subunits of mammalian cytochrome c oxidase, we have converted the cloned mitochondrial gene for rat subunit II (coxII) into its universal codon equivalent (ucoxII) by oligonucleotide-directed, site-specific mutagenesis . This involved synthesizing 12 oligodeoxynucleotides to achieve the 13 ATA to ATG and the 5 TGA to TGG changes needed . To express ucoxII in Escherichia coli, we used a number of different expression vectors in which the promoters and ribosome-binding sequences of the messenger RNA were varied . While ucoxII alone was expressed at a low level, a striking increase in the level of expression resulted when the ucoxII gene was fused to other E . coli genes . The COXII peptide was identified by proteolytic digestion, partial sequencing, and reaction with specific antisera . A cro-beta-galactosidase-COXII fusion protein has been purified, characterized, and used to produce polyclonal antibodies to the COXII peptide . The ucoxII gene was also expressed in a cell-free translation system and in Xenopus oocytes, yielding a nondenatured, membrane-associated peptide with the same apparent molecular weight as authentic subunit II . In oocytes and in a reticulocyte lysate in vitro system supplemented with microsomal membranes, the protein is glycosylated and coisolates with the washed membrane fraction . In both cases, the COXII peptide is soluble under mild conditions in a nonionic detergent and is precipitable by antibodies to subunit II . The production of subunit II in the in vitro translation system is stimulated as strongly by addition of soybean phospholipid vesicles as by microsomal membranes, providing further evidence of membrane insertion and stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Mar 12, 30(10), 2625 - 8
Ligand interactions in the ArsA protein, the catalytic component of an anion-translocating adenosinetriphosphatase; Karkaria CE et al.; The ars operon of the conjugative R-factor R773 produces resistance to arsenicals in cells of Escherichia coli . The operon encodes an oxyanion pump which is composed of a membrane subunit, the 45.5-kDa ArsB protein, and a catalytic subunit, the 63-kDa ArsA protein . Purified ArsA protein is an arsenite(antimonite)-stimulated ATPase . From its amino acid sequence, as deduced from the nucleotide sequence, the ArsA protein has four tryptophanyl residues which could serve as intrinsic fluorescent probes for the study of substrate-induced conformational changes . Both static and dynamic measurements of tryptophan fluorescence were performed with the ArsA protein . Results from static anisotropy measurements indicated differences in molecular motion with addition of ATP, SbO2-, or Mg2+ . These results were supported by time decay measurements of fluorescence anisotropy . The results of time decay measurements indicated a shorter correlation time, reflecting localized motion in the vicinity of the probe, and a longer correlation time, which could have arisen from rotation of the major portion of the molecule . The longer correlation time changed with addition of the various effectors, especially MgCl2, suggesting that binding of Mg2+ decreases probe mobility.

Biochem Pharmacol, 1991 Mar 15-Apr 1, 41(6-7), 985 - 93
Hemolytic and microbicidal actions of diethyldithiocarbamic acid; Agar NS et al.; Micromolar concentrations of diethyldithiocarbamic acid (DDC) kill fungi, bacteria and malaria . DDC forms chelates with copper and the microbicidal effectiveness of this drug is enhanced greatly by small amounts of copper . DDC, in the presence of at least 1 molar equivalent of copper, also causes lysis of human erythrocytes . To explore the cytocidal actions of DDC and copper, we have used human erythrocytes and Escherichia coli as models . We found that: (1) the combination of DDC and copper also lysed E . coli spheroplasts, suggesting a possible common mechanism of hemolytic and microbicidal action; (2) higher ratios of drug: metal (greater than 4:1) diminished hemolytic and, as observed earlier, microbicidal effects; (3) cobalt, known to suppress the microbicidal effects of DDC:Cu, also prevented red cell lysis; (4) despite the necessary involvement of copper in DDC-mediated hemolysis, there was no evidence of oxidative damage to erythrocytes, and both lysis of erythrocytes and killing of E . coli were undiminished in the absence of oxygen; (5) the DDC:Cu chelate preferentially located in organic solvents and in membranes of erythrocytes . The chelate was quite soluble in chloroform but much less so in a C-16 hydrocarbon (hexadecane) which resembled erythrocyte membrane lipid . In hexadecane and at greater than 10(-4) M DDC and 5 x 10(-5) copper, an amphipathic drug:metal complex accumulated at the organic:aqueous interface; and (6) this amphipathic complex may permeabilize the lipid bilayer, causing leakage of ions and cell water and eventuating in colloid osmotic lysis . Red cells and E . coli exposed to the chelate showed early loss of intracellular rubidium (86Rb+) . Furthermore, lysis of erythrocytes and E . coli spheroplasts was suppressed by the inclusion of either dextran or sucrose . Thus, it appears that DDC:Cu chelates are cytocidal by virtue of concentrating in the lipid bilayer and, perhaps, forming amphipathic complexes which disrupt membrane integrity . Drugs with similar behavior hold promise for therapy of malaria because metals capable of forming such complexes may accumulate within parasitized red cells.

FEBS Lett, 1991 Mar 11, 280(1), 125 - 8
Identification of the C2-1H histidine NMR resonances in chloramphenicol acetyltransferase by a 13C-1H heteronuclear multiple quantum coherence method; Derrick JP et al.; Chloramphenicol acetyltransferase (CAT) was used to assess the feasibility of study of specific proton resonances in an enzyme of overall molecular mass 75,000, {ring 2-13C}Histidine was selectively incorporated into the type III chloramphenicol acetyltransferase (CATIII) using a histidine auxotroph of E . coli . Heteronuclear multiple and single quantum experiments were used to select the C2 protons in the histidyl imidazole ring . One- and two-dimensional spectra revealed six signals out of a total of seven histidine residues in CATIII . pH titration, chemical modification and ligand binding were used to demonstrate that the signal from H195, the histidine at the active site, is not among those observed . Nevertheless, this work demonstrates that selective isotopic enrichment and multiple quantum coherence techniques can be used to distinguish proton resonances in a protein of high molecular mass.

Nucleic Acids Res, 1991 Mar 11, 19(5), 1049 - 56
Stepwise cloning and molecular characterization of the HgiDI restriction-modification system from Herpetosiphon giganteus Hpa2; Dusterhoft A et al.; The restriction-modification system HgiDI from Herpetosiphon giganteus strain Hpa2 has been cloned in E . coli in a two-step procedure . Selection of the methyltransferase (M.HgiDI) gene in vitro was performed using the heterologous restriction endonuclease AhaII, an isoschizomer of Acyl and HgiDI (GRCGYC) . Cloning of the complete HgiDI endonuclease (R.HgiDI) gene could only be achieved in recipient cells harbouring a recombinant plasmid, which was expressing the corresponding methyltransferase and could thereby prevent the host from self-destruction of its genetic material . The HgiDI restriction-modification system was sequenced and functionally correlated with two open reading frames of 309 (M) and 359 (R) codons . In homology studies M.HgiDI showed significant similarities to 20 other m5C-methyltransferases and turned out to be the most compact enzyme of this group described so far . Initial attempts for overexpression of M.HgiDI and partial purification of R.HgiDI have been successful.

Nucleic Acids Res, 1991 Mar 11, 19(5), 1021 - 8
Computer simulations and experimental studies of gel mobility patterns for weak and strong non-cooperative protein binding to two targets on the same DNA: application to binding of tet repressor variants to multiple and single tet operator sites; Kleinschmidt C et al.; A series of computer simulations of gel patterns assuming non-cooperative binding of a protein to two targets on the same DNA fragment was performed and applied to interprete gel mobility shift experiments of Tet repressor-tet operator binding . While a high binding affinity leads to the expected distribution of free DNA, DNA bound by one repressor dimer and DNA bound by two repressor dimers, a lower affinity or an increased electrophoresis time results in the loss of the band corresponding to the singly occupied complex . The doubly occupied complex remains stable under these conditions . This phenomenon is typical for protein binding to DNA fragments with two identical sites . It results from statistical disproportionation of the singly occupied complex in the gel . The lack of the singly occupied complex is commonly taken to indicate cooperative binding, however, our analysis shows clearly, that cooperativity is not needed to interprete these results . Tet repressor proteins and small DNA fragments with two tet operator sites have been prepared from four classes of tetracycline resistance determinants . The results of gel mobility shift analyses of various complexes of these compounds confirm the predictions . Furthermore, calculated gel patterns assuming different gel mobilities of the two singly occupied complexes show discrete bands only if the electrophoresis time is shorter than the inverse of the microscopic dissociation rate constant . Simulations assuming increasing dissociation rates predict that the two bands first merge into one, which then disappears . This behavior was verified by gel mobility analyses of Tet repressor-tet operator titrations at increased salt concentrations as well as by direct footprinting of the complexes in the gel . It is concluded that comparison of the intensities of the single and the double occupation bands allow a rough estimation of the dissociation rate constant . On this basis the sixteen possible Tet repressor-tet operator combinations can be ordered with decreasing binding affinities by a simple gel shift experiment . The implications of these results for gel mobility analyses of other protein-DNA complexes are discussed.

Nucleic Acids Res, 1991 Mar 11, 19(5), 1007 - 13
Cloning and characterization of the MboII restriction-modification system; Bocklage H et al.; The two genes encoding the class IIS restriction-modification system MboII from Moraxella bovis were cloned separately in two compatible plasmids and expressed in E . coli RR1 delta M15 . The nucleotide sequences of the MboII endonuclease (R.MboII) and methylase (M.MboII) genes were determined and the putative start codon of R.MboII was confirmed by amino acid sequence analysis . The mboIIR gene specifies a protein of 416 amino acids (MW: 48,617) while the mboIIM gene codes for a putative 260-residue polypeptide (MW: 30,077) . Both genes are aligned in the same orientation . The coding region of the methylase gene ends 11 bp upstream of the start codon of the restrictase gene . Comparing the amino acid sequence of M.MboII with sequences of other N6-adenine methyltransferases reveals a significant homology to M.RsrI, M.HinfI and M.DpnA . Furthermore, M.MboII shows homology to the N4-cytosine methyltransferase BamHI.

FEBS Lett, 1991 Mar 11, 280(1), 163 - 6
Tyr-426 of the Escherichia coli asparaginyl-tRNA synthetase, an amino acid in a C-terminal conserved motif, is involved in ATP binding; Anselme J et al.; Sequence comparisons of the E . coli asparaginyl-tRNA synthetase (NRSEC) with aminocyl-tRNA synthetase sequences of class II enzymes show significant homologies with aspartyl- and lysyl-tRNA synthetases . Three conserved regions were found, one of which is located in the C-terminal part of the NRSEC sequence . Site-directed mutagenesis was performed in this conserved region . A single point mutation Tyr-426----Ser results in a 15-fold increase in the Km for ATP, while all the other kinetic parameters remain unchanged . The replacement of this Tyr-426 by a Phe does not affect the kinetic behaviour of the enzyme . These data indicate that Tyr-426 is part of the ATP binding site.

Nucleic Acids Res, 1991 Mar 11, 19(5), 1129 - 37
A segment of a plasmid gene required for conjugal transfer encodes a site-specific, single-strand DNA endonuclease and ligase; Bhattacharjee MK et al.; The polypeptide encoded by a segment of a gene required for the conjugal mobilization of the broad host-range plasmid R1162 has been purified as a beta-galactosidase fusion protein . The hybrid protein binds specifically to a small, double-stranded DNA fragment containing the origin of transfer (oriT), and specifically cleaves oriT single-stranded DNA at the position cleaved during transfer . Only one of the two DNA strands is a substrate . A fraction of the digested DNA is resistant to lambda exonuclease digestion, indicating that some molecules have protein covalently attached at the 5' end . After prolonged incubation with fusion protein, some of the cleaved molecules are religated . In vivo, M13 phage DNA containing two, directly-repeated copies of oriT recombine in cells containing the fusion protein . The single-stranded viral DNA forms are the probable substrates for the protein, the cleaved DNA being subsequently religated to form recombinant molecules . Cleavage of the DNA might be the reverse reaction of the ligation that normally takes place after conjugative transfer of a single, linear plasmid DNA strand.

FEBS Lett, 1991 Mar 11, 280(1), 91 - 3
The inhibition of restriction endonucleases due to Z-DNA in negatively supercoiled plasmid; Lesnik EA et al.; Plasmid pGC20 containing the (dGC)9 insert in SmaI recognition site has been used to study the inhibition of cleavage by different restriction endonuclease due to Z-DNA formation in (dCG)10 sequence of the negatively supercoiled plasmid . Data obtained indicate the different sensitivity of restriction endonucleases to DNA conformational perturbations resulted from the Z-DNA formation . Therefore, the inhibition of DNA cleavage by a particular restriction endonuclease cannot serve as a criterion for the estimation of the length of B-Z junctions in circular supercoiled DNAs.

FEBS Lett, 1991 Mar 11, 280(1), 65 - 9
Molecular cloning and expression of a protein-tyrosine phosphatase showing homology with transcription factors Fos and Jun; Swarup G et al.; A cDNA clone coding for a protein-tyrosine phosphatase (PTPase) was isolated from a rat spleen cDNA library . Nucleotide sequence of the clone showed an open reading frame coding for a polypeptide of 363 amino acids . Expression of this clone in E . coli in an expression vector showed PTPase activity . The non-catalytic region of this PTPase located at the carboxy terminus shows homology with the basic domains of transcription factors Fos and Jun . Northern blot analysis showed that a 1.7 kb transcript was present in many tissues and cells, the highest level being in macrophages . This PTPase is a rat homolog of human T-cell PTPase although it shows 3 large deletions in the carboxy terminal non-catalytic region.

FEBS Lett, 1991 Mar 11, 280(1), 167 - 70
A Drosophila nuclear localisation signal included in an 18 amino acid fragment from the serendipity delta zinc finger protein; Noselli S et al.; Sequence analysis of the nuclear Drosophila serendipity delta Cys-2/His-2 finger protein indicated the presence of a short motif of positively charged amino acids, with homology to the SV40 large T and c-myc nuclear localisation signals . Using P-element mediated transformation we constructed transgenic Drosophila lines expressing beta-galactosidase fusion proteins, containing (or not) an 18 residue segment of sry delta including this basic, PTKKRVK, motif . Histochemical detection of fusion proteins on dissected tissues showed that this segment of sry delta can act autonomously to drive the beta-galactosidase in nuclei.

Nucleic Acids Res, 1991 Mar 11, 19(5), 1073 - 9
Cloning, expression and characterization of the human transcription elongation factor, TFIIS; Yoo OJ et al.; The cDNA for the human elongation factor, TFIIS, has been cloned and expressed in E . coli with the T7 expression system . This 280-amino acid TFIIS protein is shorter by 21 residues than that of the mouse . The missing 21 residues are located in the amino-terminal region, which is not thought to be required for transcriptional stimulation . Apart from this gap, human and mouse proteins reveal 96% overall identity and 98.5% sequence similarity if conservative substitutions are taken into account . The bacterially expressed human protein and the purified calf thymus proteins are indistinguishable in their ability to stimulate transcript elongation by purified RNA polymerase II . Estimation of the native molecular size of the human protein in solution indicates that it exists as a dimer.

Nucleic Acids Res, 1991 Mar 11, 19(5), 1067 - 72
Specific inhibition of human DNA ligase adenylation by a distamycin derivative possessing antitumor activity; Montecucco A et al.; The antiviral distamycin A and its phenyl mustard derivative FCE24517 possessing antitumor activity were tested for their ability to inhibit macromolecular synthesis in three human and one murine cell line . While distamycin A was poorly active in these systems, FCE24517 inhibited DNA synthesis efficiently, RNA synthesis to a lower extent and had little effect on protein synthesis . These findings suggest that the in vivo activity of FCE24517 derives from the specific inhibition of DNA synthesis . When the two drugs were tested on several enzymes involved in human DNA metabolism a strikingly similar pattern of inhibition appeared, with distamycin A being the more potent . Both drugs showed: A), no inhibitory activity against thymidine kinase and DNA primase; B), low activity against DNA topoisomerases I and II and the 3'-5' exonuclease associated with the DNA polymerase epsilon; C), high activity against DNA polymerases alpha and epsilon, uracil-DNA glycosylase and the joining activity of the replicative DNA ligase; D), the highest inhibitory activity against the AMP-dependent DNA relaxing activity of DNA ligase . The strong in vitro inhibition of several DNA enzymatic activities, including DNA ligase, do not match with the in vivo activities of the two drugs . However a unique difference was observed: only FCE24517 inhibited the DNA-independent reaction of adenylation of human DNA ligase while the adenylation reaction of T4 and E . coli DNA ligase was unaffected by either drug . It is still unclear whether these properties are relevant for modulating the killing activity of FCE24517 against proliferating cells both in culture and in vivo . Nevertheless FCE24517 is the first known molecule capable of interacting directly and specifically with human DNA ligase.

Biochim Biophys Acta, 1991 Mar 8, 1077(1), 35 - 46
Purification and characterization of recombinant mouse thymidylate synthase; Zhang HC et al.; Recombinant mouse thymidylate synthase (TS) expressed at high levels in Escherichia coli was purified to homogeneity in greater than 70% yield by a rapid three-step procedure . Both 0.1% Triton X-100 and 10% glycerol were required to stabilize the enzyme whose activity remained unchanged after 1 month when stored at -20 degrees C . Thermal inactivation of the enzyme was a first-order process at 37 degrees C, with t1/2 values of 6.9, 15.6 and 3.0 min at pH 5.5, 7.0 and 8.5, respectively . The presence of saturating levels of dUMP at pH 8.5 increased the t1/2 of inactivation of 38 min . The pH profile for enzyme activity showed a narrow optimum region centered at pH 7.0, which was mirrored by the shape of the Km, dUMP/Vmax plot . The pH dependence of Kd for the covalent inhibitory ternary complex of enzyme, 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate exhibited a broad minimum between pH 5.5 and 8.5, and ranged between 3.1, 0.8 and 1.1 nM at pH 5.5, 7.0 and 8.5, respectively . The UV/VIS spectrum of the native enzyme exhibited a maximum at 280 nm (epsilon = 98,200 M-1 cm-1), while that of the inhibitory ternary complex showed an additional maximum at 320 nm . The 19F-NMR spectrum of the mouse enzyme:FdUMP binary complex revealed two new resonances at -2.8 and -34.8 ppm . The most deshielded resonance represented the noncovalent binary complex while the other resonance was assigned to the nucleotide covalently bound to the enzyme . The alteration of nucleotide binding equilibria produced by addition of H4 folate was exemplified by both an increase in intensity and a 5 ppm deshielding of the resonance attributed to the covalent FdUMP-enzyme complex . Addition of formaldehyde to the latter mixture produced the covalent ternary complex which resulted in the collapse of the resonances at -2.8 and -39.5 ppm and the appearance of a new resonance at -12.4 ppm.

Science, 1991 Mar 8, 251(4998), 1211 - 7
Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc; Blackwood EM et al.; The myc protooncogene family has been implicated in cell proliferation, differentiation, and neoplasia, but its mechanism of function at the molecular level is unknown . The carboxyl terminus of Myc family proteins contains a basic region helix-loop-helix leucine zipper motif (bHLH-Zip), which has DNA-binding activity and has been predicted to mediate protein-protein interactions . The bHLH-Zip region of c-Myc was used to screen a complementary DNA (cDNA) expression library, and a bHLH-Zip protein, termed Max, was identified . Max specifically associated with c-Myc, N-Myc, and L-Myc proteins, but not with a number of other bHLH, bZip, or bHLH-Zip proteins . The interaction between Max and c-Myc was dependent on the integrity of the c-Myc HLH-Zip domain, but not on the basic region or other sequences outside the domain . Furthermore, the Myc-Max complex bound to DNA in a sequence-specific manner under conditions where neither Max nor Myc exhibited appreciable binding . The DNA-binding activity of the complex was dependent on both the dimerization domain and the basic region of c-Myc . These results suggest that Myc family proteins undergo a restricted set of interactions in the cell and may belong to the more general class of eukaryotic DNA-binding transcription factors.

Cell, 1991 Mar 8, 64(5), 971 - 81
Mechanism of action of an acidic transcriptional activator in vitro; Lin YS et al.; Transcription of a eukaryotic structural gene by RNA polymerase II requires the ordered assembly of general transcription factors on the promoter to form a pre-initiation complex . Here we analyze affinity-purified complexes at various stages of assembly to determine the mechanism of action of an acidic transcriptional activator . We show that the activator can function in the absence of ATP and stimulates transcription by increasing the number of functional preinitiation complexes . The activator effects this increase by recruiting the general transcription factor TFIIB to the promoter . Using protein affinity chromatography we demonstrate a specific interaction between an acidic activating region and TFIIB . Based on these combined results, we propose that TFIIB is a direct target of an acidic activator.

Cell, 1991 Mar 8, 64(5), 927 - 39
Delta mu H+ and ATP function at different steps of the catalytic cycle of preprotein translocase; Schiebel E et al.; Preprotein translocation in E . coli requires ATP, the membrane electrochemical potential delta mu H+, and translocase, an enzyme with an ATPase domain (SecA) and the membrane-embedded SecY/E . Studies of translocase and proOmpA binds to the SecA domain . Second, SecA binds ATP . Third, ATP-binding energy permits translocation of approximately 20 residues of proOmpA . Fourth, ATP hydrolysis releases proOmpA . ProOmpA may then rebind to SecA and reenter this cycle, allowing progress through a series of transmembrane intermediates . In the absence of delta mu H+ or association with SecA, proOmpA passes backward through the membrane, but moves forward when either ATP and SecA or a membrane electrochemical potential is supplied . However, in the presence of delta mu H+ (fifth step), proOmpA rapidly completes translocation . delta mu H(+)-driven translocation is blocked by SecA plus nonhydrolyzable ATP analogs, indicating that delta mu H+ drives translocation when ATP and proOmpA are not bound to SecA.

Biochim Biophys Acta, 1991 Mar 8, 1077(1), 91 - 8
Distance changes at the regulatory and catalytic sites on Escherichia coli glutamine synthetase: a spin label study on the effect of substrate(s) binding; Ubom GA et al.; A spin-labeled ATP analogue, 2,2,6,6-tetramethylpiperidine-1-oxyl adenosine triphosphatase (Tempo-ATP) is used to adenylate Escherichia coli glutamine synthetase (L-glutamine: ammonia ligase (ADP-forming), EC 6.3.1.2) . The Tempo adenylylated glutamine synthetase (Tempo-GS) exhibits similar catalytic properties, pH profile and inhibitor susceptibility as those of glutamine synthetase adenylylated with normal ATP . Using the spin label on the enzyme as a probe and employing the spin-spin interactions between the label probe and paramagnetic Mn2+, the distances from the nitroxyl moiety of the covalently bound Tempo-AMP to the two Mn2+ binding sites, n1 and n2 were determined . The n1 site is the structural site and n2 is located at the catalytic site . The distances from Mn2+ at n1 and n2 sites to the nitroxyl radical are 19 and 16 A, respectively . Binding of the substrate, L-Glu, causes a protein conformational change which is reflected by the reduction of approximately 2 A for the n1 to Tempo-AMP distance and lengthening of approximately 2 A for the n2 to the Tempo-AMP distance . Addition of ATP to the Tempo-GS/L-Glu complex increases the distance between n1 and Tempo-AMP, and n2 and Tempo-AMP by 4 and 3 A, respectively.

J Biol Chem, 1991 Feb 25, 266(6), 3709 - 14
Full-length chicken parathyroid hormone . Biosynthesis in Escherichia coli and analysis of biologic activity; Lim SK et al.; Chicken parathyroid hormone (cPTH) has been reported to stimulate adrenal steroidogenesis and to have unusual potency on traditional PTH target tissues . To evaluate these properties, chicken PTH-(1-88) has been expressed in Escherichia coli using a plasmid encoding a fusion protein which links together growth hormone, a factor Xa recognition site, and chicken PTH-(1-88) . The growth hormone-cPTH fusion protein required the presence of 0.02% sodium dodecyl sulfate to remain in solution and be cleaved by factor Xa . The high performance liquid chromatography-purified recombinant cPTH-(1-88) and chemically synthesized cPTH-(1-34) had similar potency in rat osteosarcoma (ROS 17/2.8) cells, opossum kidney (OK) cells, and dispersed primary chicken kidney cells . The biologic potencies of cPTH-(1-34) and cPTH-(1-88) in radioreceptor binding and cAMP generation in both bone- and kidney-derived cell lines were less than those of human (h)PTH-(1-34) . In dispersed chicken kidney cells, cAMP production by cPTH-(1-34) and cPTH-(1-88) was similar to that stimulated by human PTH-(1-34) . No stimulation of steroidogenesis could be detected when recombinant chicken PTH-(1-88) was added to dispersed chicken adrenal cells . The biologic activity of recombinant chicken PTH-(1-88) purified from E . coli was comparable with that of chicken PTH-(1-88) expressed by mammalian COS cells . Thus, the full-length chicken PTH did not exhibit enhanced potency, when compared with human PTH in ROS 17/2.8, OK cell lines, and dispersed chicken kidney cells and did not demonstrate the novel steroidogenic action previously reported in adrenal cells . The successful production of chicken PTH-(1-88) will enhance our understanding of the structure-activity relationships for PTH, particularly the sequence-dependent metabolism of the hormone.

J Mol Biol, 1991 Mar 5, 218(1), 99 - 105
Detection of Escherichia coli ribosome binding at translation initiation sites in the absence of tRNA; Hartz D et al.; Binary complexes between messenger RNA and E . coli ribosomes were examined . A ribosome-mRNA binary complex on T4 gene 32 mRNA withstood inhibition by antibodies against ribosomal protein S1 . Anti-S1 blocks ternary complex formation, as measured by "extension inhibition" or "toeprinting" analysis, only when preincubated with ribosomes prior to mRNA addition and not when anti-S1 was added after preincubation of ribosomes and mRNA . The ribosome was directly localized in a binary complex on two translation initiation sites by toeprinting analysis . In the absence of tRNA the ribosome halted cDNA synthesis by reverse transcriptase close to the Shine and Dalgarno sequence . Binary complex formation was inhibited by an oligodeoxynucleotide competitor of the Shine and Dalgarno sequence.

J Mol Biol, 1991 Mar 5, 218(1), 119 - 28
Regulation of the F plasmid traY promoter in Escherichia coli by host and plasmid factors; Silverman PM et al.; F plasmid DNA transfer (tra) gene expression in Escherichia coli is regulated by chromosome- and F-encoded gene products . To study the relationship among these regulatory factors, we constructed low-copy plasmids containing a phi(traY'-'lacZ)hyb gene that couples beta-galactosidase and Lac permease synthesis to the F plasmid traY promoter . Wild-type transformants maintained high levels of beta-galactosidase over a broad range of culture densities . Primer extension analysis of tra mRNA from F'lac and phi(traY'-'lacZ)hyb strains indicated very similar, though not identical, transcription initiation sites . Moreover, phi(traY'-'lacZ)hyb gene expression required both TraJ and SfrA, as does tra gene expression in F+ strains . beta-Galactosidase activity was reduced approximately 30-fold in the absence of TraJ, which could be supplied in cis or in trans . In a two-plasmid system in which TraJ was supplied in trans by a lac-traJ operon fusion, phi(traY'-'lacZ)hyb expression was a linear, saturable function of traJ expression . Enzyme activity was reduced approximately tenfold in sfrA mutants . That reduction could not be attributed to an effect on the TraJ level . Several other cellular or environmental variables had only a modest effect on phi(traY'-'lacZ)hyb expression . Hyperexpression was observed at high cell density (twofold) and in anaerobic cultures (1.2- to 1.5-fold) . In contrast, expression was reduced twofold in integration host factor mutants.

J Mol Biol, 1991 Mar 5, 218(1), 107 - 18
Tyr60 variants of Flp recombinase generate conformationally altered protein-DNA complexes . Differential activity in full-site and half-site recombinations; Chen JW et al.; The tyrosine at position 60 of the Flp recombinase of the Saccharomyces cerevisiae plasmid, 2 mu circle, is invariant among site-specific recombinases of the "yeast plasmid family" . Alterations of this residue give rise to Flp variants that show no recombination activity when assayed in vivo in Escherichia coli . Upon purification, they bind substrate, execute DNA cleavage and catalyze recombination . The efficiency of strand cleavage follows the order: Flp(Y60F) greater than Flp greater than Flp(Y60S) greater than Flp(Y60D); efficiency of recombination between Flp sites on a linear substrate and a circular one follows the order: Flp greater than Flp(Y60F) greater than Flp(Y60S) greater than Flp(Y60D) . Methylation footprints of the DNA-protein complexes formed by two of the Flp variants, Flp(Y60S) and Flp(Y60D), do not show hypermethylation of the G residues within the substrate core that is characteristic of complexes formed by wild-type Flp . The third variant, Flp(Y60F), causes significant distortion (although less than wild-type Flp) of the substrate core, as indicated by enhanced G-methylation . Binding profiles with circularly permuted substrates indicate that Flp(Y60S) and Flp(Y60D), but not Flp(Y60F), are defective in bending substrate DNA . In recombination between two Flp half-sites, the variant proteins are significantly more active than in normal full-site recombination.

J Mol Biol, 1991 Mar 5, 218(1), 1 - 6
Identification of a subunit assembly domain in the alpha subunit of Escherichia coli RNA polymerase; Igarashi K et al.; The alpha subunit of Escherichia coli DNA-dependent RNA polymerase is encoded by the rpoA gene and plays a major role in enzyme assembly . A set of C-terminal deletion mutations of the rpoA gene was constructed . The results of mixed reconstitution experiments in vitro, using the truncated alpha polypeptides encoded by the rpoA deletion mutants, suggest that the amino-terminal two-thirds of alpha subunit is sufficient for the formation of pseudo-core complexes containing both beta and beta' subunits.

Biochemistry, 1991 Mar 5, 30(9), 2501 - 8
Characterization of a cDNA for chicken osteopontin: expression during bone development, osteoblast differentiation, and tissue distribution; Moore MA et al.; The chicken bone phosphoprotein (approximately 66-kDa BPP) is a major noncollagenous component of bone and is the major phosphoprotein synthesized by cultured chicken embryo osteoblasts {Gotoh, Y., Gerstenfeld, L . C., & Glimcher, M . J . (1990) Eur . J . Biochem . 87, 49-58} . A cDNA clone for this protein was isolated from an expression library made from embryonic chicken bone mRNA . The complete primary protein sequence of 264 amino acids was deduced from the cDNA sequence inclusive of a 16 amino acid signal peptide sequence and terminated by 4 in-frame stop sequences . A sequence alignment indicated an approximate 35% overall similarity in protein sequence between the avian approximately 66-kDa BPP and the mammalian protein osteopontin, while at the nucleotide level 60% similarity was observed . Features of this sequence which showed the greatest similarity to mammalian osteopontin included a region in which seven of nine consecutive residues are aspartic acid, a recognition sequence for integrin-mediated cell binding (-Arg-Gly-Asp), and four possible recognition sequences for phosphorylation by casein kinase II . Hybridization analysis indicated a message of 1.5 kb found predominantly in bone and kidney . The mRNA was inducible in phorbol ester treated primary cultures of chondrocytes which show no expression under normal growth conditions . A temporal induction was seen during osteoblastic differentiation both in vivo and in vitro, thus suggesting that regulation of the approximately 66-kDa BPP is under transcriptional control during osteoblast development . In summary, both the protein's primary structure and its biological features suggest that it is the avian homologue to mammalian protein osteopontin.

Biochemistry, 1991 Mar 5, 30(9), 2306 - 10
Human and Escherichia coli cyclophilins: sensitivity to inhibition by the immunosuppressant cyclosporin A correlates with a specific tryptophan residue; Liu J et al.; The human T-cell protein cyclophilin shows high affinity for and is the proposed target of the major immunosuppressant drug cyclosporin A (CsA) . Cyclophilin also has peptidyl prolyl cis-trans isomerase activity that is inhibited by CsA with an IC50 of 6 nM, while by contrast a homologous PPIase from Escherichia coli has been found to be much less sensitive to CsA, shown here to be 500-fold less potent at an IC50 of 3000 nM . This E . coli rotamase lacks the single highly conserved tryptophan residue of eukaryotic cyclophilins, and we show here that mutation of the natural F112 to W112 enhances E . coli rotamase susceptibility to CsA inhibition by 23-fold . Correspondingly, the human W121 mutations to F121 or A121 yield cyclophilins with 75- and 200-fold decreased sensitivity to CsA, while kcat/Km values of rotamase activity in a tetrapeptide assay drop only 2- and 13-fold, respectively . This complementary gain and loss of CsA sensitivity to mutation to or from tryptophan validate the indole side chain as a major determinant in immunosuppressant drug recognition and the separation of PPIase catalytic efficiency from CsA affinity.

J Biol Chem, 1991 Mar 5, 266(7), 4622 - 30
Site-directed mutagenesis of rat cellular retinol-binding protein . Alteration in binding specificity resulting from mutation of glutamine 108 to arginine; Stump DG et al.; Cellular retinol-binding protein (CRBP) is a retinol-specific binding protein . A rat cDNA clone of CRBP was expressed in Escherichia coli . In order to determine amino acid residues in CRBP which may be important for the binding of all-trans-retinol, comparative model-building studies were performed in which strong sequence similarities were identified between CRBP and several other binding proteins . Based on this analysis, specific amino acids were predicted to be important in retinol binding, and these predictions were tested using the technique of site-directed mutagenesis to subtly alter the protein's structure and function . Specifically, site-directed mutagenesis was performed to alter the Gln-108 to Arg-108 (Q108R) . Making use of fluorescence, Q108R was found to have a 3-fold lower affinity for all-trans-retinol, and the fine structure of the excitation spectrum of the Q108R.all-trans-retinol complex was also different than for the wild type.all-trans-retinol complex . The mutant bound 13-cis-retinol with an excitation spectrum identical to wild type bound to 13-cis-retinol, but with only one-half of the fluorescence intensity . In competition binding experiments, the Q108R mutant was found to have similar binding affinities for all-trans-retinol, all-trans-retinoic acid, 13-cis-retinoic acid, and retinal, while wild type CRBP was only able to bind to all-trans-retinol . Thus, altering a single amino acid in CRBP (Gln-108 to Arg-108) caused a significant change in the ligand binding specificity of the protein.

J Biol Chem, 1991 Mar 5, 266(7), 4425 - 9
Isolation of BamHI variants with reduced cleavage activities; Xu SY et al.; Derivation of the bamhIR sequence (Brooks, J . E., Nathan, P.D., Landry, D., Sznyter, L.A., Waite-Rees, P., Ives, C . C., Mazzola, L . M., Slatko, B . E., and Benner, J . S . (1991) Nucleic Acids Res., in press), the gene coding for BamHI endonuclease, has facilitated construction of an Escherichia coli strain that overproduces BamHI endonuclease (W . E . Jack, L . Greenough, L . F . Dorner, S . Y . Xu, T . Strezelecka, A . K . Aggarwal, and I . Schildkraut, submitted for publication) . As expected, low-level constitutive expression of the bamhIR gene in E . coli from the Ptac promotor construct is lethal to the host unless the bamHIM gene, which encodes the BamHI methylase, is also expressed within the cell . We identified four classes of BamHI endonuclease variants deficient in catalysis by selecting for survival of a host deficient for bamHIM gene, transformed with mutagenized copies of the bamhIR gene, and then screening the surviving cell extracts for DNA cleavage and binding activities . Class I variants (G56S, G91S/T153I, T114I, G130R, E135K, T153I, T157I, G194D) displayed 0.1-1% of the wild-type cleavage activity; class II variant (D94N) lacked cleavage activity but retained wild-type DNA binding specificity; class III variants (E77K, E113K) lacked cleavage activity but bound DNA more tightly; class IV variants (G56D, G90D, G91S, R122H, R155H) lacked both binding and cleavage activities . Variants with residual cleavage activities induced the E . coli SOS response and thus are presumed to cleave chromosomal DNA in vivo . We conclude that Glu77, Asp94, and Glu113 residues are essential for BamHI catalytic function.

J Biol Chem, 1991 Mar 5, 266(7), 4139 - 44
An analysis of lactose permease "sugar specificity" mutations which also affect the coupling between proton and lactose transport . II . Second site revertants of the thiodigalactoside-dependent proton leak by the Val177/Asn319 permease; Eelkema JA et al.; The double mutant of the lactose permease containing Val177/Asn319 exhibits proton leakiness by two pathways (see Brooker, R . J . (1991) J . Biol Chem . 266, 4131-4138) . One type of H+ leakiness involves the uncoupled influx of H+ (leak A pathway) while a second type involves the coupled influx of H+ and galactosides in conjunction with uncoupled galactoside efflux (leak B pathway) . In the current study, 14 independent lactose permease mutants were isolated from the Val177/Asn319 parent which were resistant to thiodigalactoside growth inhibition but retained the ability to transport maltose . All of these mutants contained a third mutation (besides Val177/Asn319) at one of two sites . Eight of the mutants had Ile303 changed to Phe, while six of the mutants had Tyr236 changed to Asn or His . Each type of triple mutant was characterized with regard to sugar transport, H+ leakiness, and sugar specificity . Like the parental strain, all three types of triple mutant showed moderate rates of downhill lactose transport and were defective in the uphill accumulation of sugars . However, with regard to proton leakiness, the triple mutants fell into two distinct categories . The mutant containing Phe303 was generally less H+ leaky than the parent either via the leak A or leak B pathway . In contrast, the triple mutants containing position 236 substitutions (Asn or His) were actually more H+ leaky via the leak A pathway and exhibited similar H+ leakiness via the leak B pathway at high thiodigalactoside concentrations . The ability of the position 236 mutants to grow better than the parent in the presence of low concentrations of thiodigalactoside appears to be due to a decrease in affinity for this particular sugar rather than a generalized defect in H+ leakiness . Finally, the triple mutants showed a sugar specificity profile which was different from either the Val177/Asn319 parent, the single Val177 mutant, or the wild-type strain . These results are discussed with regard to the effects of mutations on both the sugar and H+ transport pathways.

J Biol Chem, 1991 Mar 5, 266(7), 4131 - 8
An analysis of lactose permease "sugar specificity" mutations which also affect the coupling between proton and lactose transport . I . Val177 and Val177/Asn319 permeases facilitate proton uniport and sugar uniport; Brooker RJ; The sugar specificity mutants of the lactose permease containing Val177 or Val177/Asn319 were analyzed with regard to their ability to couple H+ and sugar co-transport . Both mutants were able to transport lactose downhill to a significant degree . The Val177 mutant was partially defective in the active accumulation of galactosides, whereas the Val177/Asn319 mutant was completely defective in the uphill accumulation of sugars . With regard to coupling, the Val177 mutant was shown to catalyze the uncoupled transport of H+ to a substantial degree . This led to a decrease in the H+ electrochemical gradient under aerobic conditions and also resulted in faster H+ uptake when a transient H+ electrochemical gradient was generated under anaerobic conditions . Interestingly, galactosides were shown to diminish the rate of uncoupled H+ transport in the Val177 strain . The Val177/Asn319 strain also catalyzed uncoupled H+ transport, but to a lesser degree than the single Val177 mutant . In addition, the Val177/Asn319 mutant was shown to transport galactosides with or without H+ . The observed H+/lactose stoichiometry was 0.30 in the double mutant compared to 0.98 in the wild-type strain . When an H+ electrochemical gradient was generated across the membrane, the Val177/Asn319 mutant permease was shown to facilitate an extremely rapid net H+ leak if nonmetabolizable galactosides had been equilibrated across the membrane . The mechanism of this leak is consistent with a circular pathway involving H+/galactoside influx and uncoupled galactoside efflux . The magnitude of the H+ leak in the presence of nonmetabolizable galactosides was so great in the double mutant that low concentrations of certain galactosides (i.e . 0.5 mM thiodigalactoside) resulted in a complete inhibition of growth . These results are discussed with regard to the possibility that cation and sugar binding to the lactose permease may involve a direct physical coupling at a common recognition site.

J Biol Chem, 1991 Mar 5, 266(7), 4099 - 105
Characterization of a limited trypsin digestion form of eukaryotic elongation factor 1 alpha; Kinzy TG et al.; The involvement of the first 69 amino acids of eukaryotic elongation factor 1 alpha (EF-1 alpha) from rabbit reticulocyte in GTP and aminoacyl-tRNA binding has been analyzed by a variety of techniques . EF-1 alpha was subjected to limited trypsin digestion, which cleaved predominantly at residues 36 and 69 . A digested form of Escherichia coli EF-Tu, similar to the one used for this study, has been characterized by x-ray crystallography and is used as a structural model for EF-1 alpha . This form of EF-1 alpha bound E . coli Phe-tRNAPhe similar to the wild type protein, but lacked activity in phenylalanine polymerization with poly(U)-programmed ribosomes . These results were obtained regardless of whether or not loosely associated N-terminal peptides were removed by gel filtration chromatography . The digested EF-1 alpha also shows reduced GTPase activity, but the activity is stimulated by both ribosomes and aminoacyl-tRNA . Binding of EF-1 alpha to the 80 S ribosome, as determined by association of reductively methylated protein through Sepharose 6B chromatography, is reduced approximately 7-fold for the limited digested form of the protein . Limited digested EF-1 alpha can, however, be photo-cross-linked with GTP and 3'-p-azido-GTP similar to intact EF-1 alpha . Chemical cross-linking with oxidized GTP, fluorosulfonylbenzoyl-GTP, or with trans-diaminedichloroplatinum(II) and GPT, shows a similar modification of both intact and limited digested EF-1 alpha . In order to further localize the modification site with the GTP reagents and assure that modification was not occurring in the first 69 amino acids, intact EF-1 alpha was modified with these same reagents . Limited trypsin digestion of modified protein indicates that none of these reagents cross-links GTP to the first 69 amino acids of EF-1 alpha, which includes the first GTP binding consensus element, GXXXXGK.

Biochemistry, 1991 Mar 5, 30(9), 2535 - 42
Function of serine-52 and serine-80 in the catalytic mechanism of Escherichia coli aspartate transcarbamoylase; Xu W et al.; Carbamoyl phosphate is held in the active site of Escherichia coli aspartate transcarbamoylase by a variety of interactions with specific side chains of the enzyme . In particular, oxygens of the phosphate of carbamoyl phosphate interact with Ser-52, Thr-53 (backbone), Arg-54, Thr-55, and Arg-105 from one catalytic chain, as well as Ser-80 and Lys-84 from an adjacent chain in the same catalytic subunit . In order to define the role of Ser-52 and Ser-80 in the catalytic mechanism, two mutant versions of the enzyme were created with Ser-52 or Ser-80 replaced by alanine . The Ser-52----Ala holoenzyme exhibits a 670-fold reduction in maximal observed specific activity, and a loss of both aspartate and carbamoyl phosphate cooperativity . This mutation also causes 23-fold and 5.6-fold increases in the carbamoyl phosphate and aspartate concentrations required for half the maximal observed specific activity, respectively . Circular dichroism spectroscopy indicates that saturating carbamoyl phosphate does not induce the same conformational change in the Ser-52----Ala holoenzyme as it does for the wild-type holoenzyme . The kinetic properties of the Ser-52----Ala catalytic subunit are altered to a lesser extent than the mutant holoenzyme . The maximal observed specific activity is reduced by 89-fold, and the carbamoyl phosphate concentration at half the maximal observed velocity increases by 53-fold while the aspartate concentration at half the maximal observed velocity increases 6-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Mar 5, 30(9), 2448 - 53
Interaction of the tRNA(Phe) acceptor end with the synthetase involves a sequence common to yeast and Escherichia coli phenylalanyl-tRNA synthetases; Sanni A et al.; Modified lysines resulting from the cross-linking of the 3' end of tRNA(Phe) to yeast phenylalanyl-tRNA synthetase (an enzyme with an alpha 2 beta 2 structure) have been characterized by sequencing the labeled chymotryptic peptides that were isolated by means of gel filtration and reversed-phase chromatography . The analysis showed that Lys131 and Lys436 in the alpha subunit are the target sites of periodate-oxidized tRNA(Phe) . Mutant protein with a Lys----Asn substitution established that each lysine contributes to the binding of the tRNA but is not essential for catalysis . The major labeled lysine (K131) belongs to the sequence IALQDKL (residues 126-132), which shares three identities with the peptide sequence ADKL found around the tRNAox-labeled Lys61 in the large subunit of Escherichia coli phenylalanyl-tRNA synthetase {Hountondji, C., Schmitter, J . M., Beauvallet, C., & Blanquet, S . (1987) Biochemistry 26, 5433-5439}.

J Biol Chem, 1991 Mar 5, 266(7), 4654 - 9
Lipid modifications of G protein subunits . Myristoylation of Go alpha increases its affinity for beta gamma; Linder ME et al.; Myristoylated recombinant proteins can be synthesized in Escherichia coli by concurrent expression of the enzyme myristoyl-CoA:protein N-myristoyl-transferase with its protein substrates (Duronio, R.J., Jackson-Machelski, E., Heuckeroth, R.O., Olins, P . O., Devine, C.S., Yonemoto, W., Slice, L . W., Taylor, S . S., and Gordon, J . I . (1990) Proc . Natl . Acad . Sci . U . S.A . 87, 1506-1510) . Expression of the G protein subunit Go alpha in this system results in the synthesis of two forms of the protein; these were separated on a column of heptylamine-Sepharose . Purification of the more abundant form of Go alpha yielded a product that has a blocked amino terminus . Chemical analysis of the fatty acids released by acid hydrolysis of the protein revealed myristic acid . The second form of the protein was not myristoylated . Myristoylated and nonmyristoylated recombinant Go alpha were compared with brain Go alpha (which is myristoylated) for their ability to interact with G protein beta gamma subunits . The nonmyristoylated recombinant protein clearly had a reduced affinity for beta gamma, while the myristoylated recombinant protein was indistinguishable from native Go alpha in its subunit interactions . Thus, myristoylation increases the affinity of alpha subunits for beta gamma . We propose that the function of myristoylation of G protein alpha subunits is, at least in part, to facilitate formation of the heterotrimer and the localization of alpha to the plasma membrane.

J Biol Chem, 1991 Mar 5, 266(7), 4375 - 80
Antisera specific for rap 1 proteins distinguish between processed and nonprocessed rap 1b; Winegar DA et al.; Polyclonal antisera were generated against synthetic peptides corresponding to distinct regions of the rap 1 protein sequences . A "rap 1-common" antiserum, prepared against an 18-amino acid segment of the rap 1a protein near the proposed GTP-binding region, reacted with both rap 1a and rap 1b recombinant proteins expressed in Escherichia coli and with two low molecular weight GTP-binding proteins of 22 and 24 kDa in unstimulated human platelets . An antiserum raised against a carboxyl-terminal peptide of rap 1b containing the putative site of post-translational processing reacted strongly with bacterial-expressed recombinant rap 1b and with a 24-kDa GTP-binding protein in platelets, but not with recombinant rap 1a or a 22-kDa GTP-binding protein . The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this rap 1b immunoreactive protein coincided with that of bacterial-expressed rap 1b and not with the faster migrating form of rap 1b that incorporates radioactivity from {3H}mevalonic acid in the insect/baculovirus system . This suggests that our rap 1b-specific antiserum recognizes only one form of rap 1b, that which has not undergone carboxyl-terminal post-translational processing.

J Biol Chem, 1991 Mar 5, 266(7), 4315 - 21
The product of the rap2 gene, member of the ras superfamily . Biochemical characterization and site-directed mutagenesis; Lerosey I et al.; The human rap2 gene encodes a 183 amino acid protein that shares 46% identity with the K-ras p21 . Its cDNA was engineered and inserted into the bacterial expression vector ptac; this allowed the production of high levels of soluble recombinant protein in Escherichia coli that was purified to near homogeneity . The rap2 protein binds GTP and exhibits a low intrinsic GTPase activity (rate constant of 0.5 x 10(-2) min-1) . It exchanges its bound GDP with a half-life of 18 min at 37 degrees C in the presence of 10 mM Mg2+ . Under the same conditions, the dissociation of bound GTP was at least 25-fold slower showing that the rap2 protein has a much higher affinity for GTP than GDP . The contribution of individual domains of the protein to its biochemical activities was investigated by site-directed mutagenesis . Substitution of Val for Gly at position 12 results in a 2-fold decrease in the GDP dissociation rate constant and GTPase activity . Replacement of the Ser at position 17 by Asn severely impairs the GTP binding ability of the protein and points to an important role of this residue in the coordination of Mg2+ . Mutation of Thr-35 to Ala results in a decreased affinity for GTP and a reduction (3-fold) of the GTPase activity . Finally, substitution of Thr-145 by Ile leads to an imperfect binding of guanyl nucleotides as exemplified by an increase in their dissociation rate constants and reduction of the GTPase activity of the protein . These properties of the normal and mutant rap2 proteins are compared with those of ras p21 carrying similar substitutions and are discussed in relation to the structural models proposed for ras p21.

J Mol Biol, 1991 Mar 5, 218(1), 45 - 54
AraC-DNA looping: orientation and distance-dependent loop breaking by the cyclic AMP receptor protein; Lobell RB et al.; The arabinose operon promoter, pBAD, is negatively regulated in the absence of arabinose by AraC protein, which forms a DNA loop by binding to two sites separated by 210 base-pairs, araO2 and araI1 . pBAD is also positively regulated by AraC-arabinose and the cyclic AMP receptor protein, CRP . We provide evidence that CRP breaks the araO2-araI1 repression loop in vitro . The ability of CRP to break the loop in vitro and to activate pBAD in vivo is dependent upon the orientation and distance of the CRP binding site relative to araI1 . An insertion of one DNA helical turn, 11 base-pairs, between CRP and araI only partially inhibits CRP loop breaking and activation of pBAD, while an insertion of less than one DNA helical turn, 4 base-pairs, not only abolishes CRP activation and loop breaking, but actually causes CRP to stabilize the loop and increases the araO2-mediated repression of pBAD . Both integral and non-integral insertions of greater than one helical turn completely abolish CRP activation and loop breaking in vitro.

J Mol Biol, 1991 Mar 5, 218(1), 129 - 40
Molecular and functional characterization of a carbon starvation gene of Escherichia coli; Schultz JE et al.; Escherichia coli induces the synthesis of at least 30 proteins at the onset of carbon starvation, two-thirds of which are positively regulated by the cyclic AMP (cAMP) and cAMP receptor protein (CRP) complex . Two of the cAMP-CRP-dependent genes mapped to 14 and 93 minutes of the chromosome and are designated cstA and cstB, respectively . The cstA promoter region was cloned and localized to a 600 base-pair fragment downstream from the iron-regulated entCEBA-P15 operon . Carbon starvation-inducible transcription initiated at three sites spaced one turn of the DNA helix apart . All had--10 sequences similar to consensus E sigma 70 promoters and poor--35 sequences . Deletion of a putative CRP binding site abolished carbon starvation-mediated induction . Sequence analysis of the cstA coding region revealed the presence of three sequential open reading frames potentially encoding two hydrophobic proteins of 60,223 Da and 15,201 Da and a hydrophilic protein of 7467 Da . Overexpression of the cstA region produced starvation-inducible proteins of the expected sizes . Suggestive evidence was obtained that cstA is involved in peptide utilization.

J Mol Biol, 1991 Mar 5, 218(1), 23 - 31
In vivo analysis of overlapping transcription units in the rplKAJLrpoBC ribosomal protein-RNA polymerase gene cluster of Escherichia coli; Steward KL et al.; Transcription of the rplKAJLrpoBC ribosomal protein (rpl) RNA polymerase (rpo) gene cluster is governed by a complex set of signals . To dissect the transcription units active in vivo and to quantify the relative contribution of each, an extensive array of rplKAJLrpoB/lacZ gene fusions were constructed on lambda phage derivatives and introduced in single copy into the chromosomes of lac- cells . Measurements of beta-galactosidase production from fusions containing wild-type and/or mutagenized rplrpo DNA fragments permitted the establishment of high-resolution transcription profiles of the gene cluster . The results show that transcription initiated at the upstream rplKp promoter (located just before rplK) does not terminate before the rplJp promoter (located upstream from rplJ), but instead reads through into the distal genes . In addition, rplJp continues to function efficiently in the presence of readthrough transcription from rplKp . As a result the rplJL genes are transcribed at almost twice the frequency of the upstream rplKA genes . However, the transcription of rpoB, which is situated downstream from the previously identified attenuator (rpoBa), is only marginally increased (20%) when both promoters are present . This suggests that although both transcription units overlap, transcriptional termination at rpoBa is modulated in response to the frequency of initiation from both promoters.

J Mol Biol, 1991 Mar 5, 218(1), 83 - 97
Influence of mRNA determinants on translation initiation in Escherichia coli; Hartz D et al.; We have studied the classic initiation elements of mRNA sequence and structure to better understand their influence on translation initiation rates in Escherichia coli . Changes introduced in the initiation codon, the Shine and Dalgarno sequence, the spacing between those two elements, and in the secondary structures within initiation domains each change the rate of 30 S ternary complex formation . We measured these differences using extension inhibition analysis, a technique we have called "toeprinting" . The rate of 30 S initiation complex formation in the absence of initiation factors agrees well with in vivo translation rates in some instances, although in others a regulatory role of initiation factors in 30 S complex formation is likely . Nucleotides 5' to the Shine and Dalgarno domain facilitate ternary complex formation.

J Biol Chem, 1991 Mar 5, 266(7), 4562 - 7
Higher order structure of the ribosomal 5 S RNA; Nazar RN; The structure of the ribosomal 5 S RNA was examined using Fe(II)-EDTA, a solvent-based reagent that cleaves the phosphodiester backbone of both double- and single-stranded RNA but is restricted by the three-dimensional structure . In the yeast 5 S RNA, cleavages were significantly restricted in six specific regions of the molecule; restrictions in only two of these regions were clearly dependent on a high salt/magnesium ion environment . A comparison of four RNAs of diverse origin revealed strong similarities in the cleavage profiles supporting a highly conserved higher order structure . Taken together with previous studies these data provide a more detailed modeling of the three-dimensional structure.

Anal Biochem, 1991 Mar 2, 193(2), 280 - 6
Assay of HIV-1 proteinase: a colorimetric method using small peptide substrates; Broadhurst AV et al.; A colorimetric assay for HIV proteinase using small protected peptide substrates is described . Substrates are cleaved to release N-terminal prolyl peptides which react with isatin to form a blue product which is measured spectrophotometrically . The assay is suitable for use with pure enzyme or crude extracts derived from genetically engineered Escherichia coli.

Anal Biochem, 1991 Mar 2, 193(2), 200 - 3
A colorimetric assay for a pyridoxal phosphate-dependent beta-replacement reaction with L-cysteine: application to studies of wild-type and mutant tryptophan synthase alpha 2 beta 2 complexes; Kayastha AM et al.; We present an improved and simple direct assay for formation of inorganic sulfide from L-cysteine in a beta-replacement reaction catalyzed by tryptophan synthase . This method provides a useful enzymatic assay for pyridoxal phosphate-dependent beta-replacement reactions in which the amino acid substrate is L-cysteine and the cosubstrate is 2-mercaptoethanol . The assay should be applicable to similar reactions with L-cysteine and other cosubstrates . The method has several advantages over other methods which have been used to assay similar beta-replacement reactions . The assay is highly reproducible and sensitive and is conveniently carried out in disposable 1.5-ml centrifuge tubes . The color remains stable for several hours . The thiol compounds L-cysteine and 2-mercaptoethanol do not interfere at the concentrations used . The method has useful applications to studies of the rates and reaction specificities of several other pyridoxal phosphate enzymes which catalyze beta-replacement reactions . We demonstrate the use of the method to study the effects of site-directed mutagenesis on the reaction specificity and mechanism of the tryptophan synthase alpha 2 beta 2 complex.

Anal Biochem, 1991 Mar 2, 193(2), 220 - 4
Use of DNA-protein interaction to isolate specific genomic DNA sequences; Hobson K et al.; We describe a simple and rapid method for the isolation of specific genomic DNA sequences recognized by DNA-binding proteins . This procedure consists of four steps: (1) restriction enzyme digestion and size fractionation of genomic DNA; (2) DNA--protein binding using the gel mobility-shift assay; (3) ligation of isolated DNA fragments followed by transformation of Escherichia coli; and (4) screening of recombinant clones for inserts containing specific DNA--protein binding sequences . We have used this protocol to isolate human DNA sequences, 100-200 bp in size, that are recognized by both partially purified and affinity purified proteins . Unlike other procedures designed to identify genomic target sequences, the method described does not require polymerase chain reaction or successive immunoprecipitations.

J Dairy Sci, 1991 Mar, 74(3), 819 - 25
Intramammary challenge with Escherichia coli following immunization with a curli-producing Escherichia coli; Todhunter DA et al.; Holstein and Jersey cattle were immunized with a curli-producing strain of Escherichia coli (pCRL65/A012) or a noncurli-producing strain (pUC18/HB101) to determine differences in resistance to establishment of experimental intramammary infection . Cows (n = 6 per group) were immunized at 14 d prior to drying off, 7 d of involution, and at calving with 3 x 10(10) E . coli in Freund's Incomplete Adjuvant . At 30 d of lactation, one mammary quarter of each cow was infused with a wild strain of E . coli (727) . Escherichia coli 727 was isolated from a naturally occurring intramammary infection and produced curli . All challenged quarters became infected, and all cows developed acute clinical mastitis . Geometric mean duration of intramammary infections was 6 d for both immunization groups . All infections were spontaneously eliminated within 10 d . No differences occurred between immunization groups in blood selenium and glutathione peroxidase activity, plasma selenium, number of E . coli 727 isolated from secretion after challenge, rectal temperature and SCC response, clinical status of mammary quarters, or DMI . Reduction in milk production after challenge was greater for cows immunized with E . coli pCRL65/A012 . Immunization of dairy cattle with a curli-producing strain of E . coli did not protect against experimental intramammary challenge during lactation.

Physiol Behav, 1991 Mar, 49(3), 647 - 9
Endotoxin treatment of pregnant rats affects sexual behavior of the male offspring; Wijkstra S et al.; The offspring of endotoxin-infused pregnant rats (0.2 micrograms endotoxin, 53.3 min, day 18 of pregnancy) did not exhibit different behavior in the Hebb-Williams-type maze test, but the males showed aberrations in the sexual behavior test . Because endotoxin did not cross the placental barrier, it was concluded that the effect reflects abnormal brain development, caused by endotoxin-induced placental malfunction, notably impaired oxygen transport.

Pathol Biol (Paris), 1991 Mar, 39(3), 185 - 90
{Factors intervening in the variations of in vitro adhesion power of enterotoxinogenic colibacillus (ETEC) to human enterocytes}; Darfeuille-Michaud A et al.; Human enterotoxigenic Escherichia coli adhere to the brush border of human enterocytes . The mean number of bacteria adhering to one enterocyte (adhesion index) varied from 0.5 to 3.1 when the strains produce adhesins . Different factors related to enterocytes and to bacteria are involved in this variability . The number of bacteria which adhered to enterocytes issued from the same donor varied from from 0 to 12 . Moreover the proportion of enterocytes on which several bacteria sticked did not exceed 20% . This variability might be due to the disparity in the maturation of the enterocytes . On the other hand, whatever the adhesion factors considered, the adhesion index varied according to the donors . ETEC strains did not express adhesion when bacteria were grown in a liquid medium but this capacity could be restored after transfer on solid medium . This phenomenon seemed like a phase-variation and appeared to be linked to a 4 to 6 kilobases (kb) plasmid . On the other hand, when the bacteria were grown on agar medium (CFA-agar or Mueller-Hinton agar) two phenotypes of colonies could be observed: large colonies (LC) which were composed of non-adhesive bacteria and small colonies (SC) which were composed of a majority of adhesive bacteria; when the number of subcultures was not too great, a majority of colonies presented the small colonies phenotype . The plasmid content analysis showed the segregation of a high molecular weight plasmid DNA (approximately 100 kb) for the bacteria issued from large colonies phenotype.

Rinsho Byori, 1991 Mar, 39(3), 321 - 7
{Detection of anti double-stranded DNA antibodies by RECOMBIGEN anti DNA kit}; Miyawaki S et al.; Anti double-stranded DNA antibodies were detected in patients with various connective tissue diseases by using RECOMBIGEN Anti DNA kit (Japan DPC Co), newly developed utilizing highly purified double-stranded DNA obtained from E . coli plasmid by recombinant technique . High levels of anti double-stranded DNA antibodies were mostly found and distributed in patients with systemic lupus erythematosus and the allied disorders . Because of antibody excess, the final antibody titers could not be determined in most sera which had antibodies titers of more than 100 units per ml . In such sera the expression of antibody titer by DNA binding activities was found to be clinically more useful . RECOMBIGEN Anti DNA kit, thus, is a highly sensitive and reproducible method to detect anti double-stranded DNA antibodies.

Poult Sci, 1991 Mar, 70(3), 539 - 47
Protective immunization against the intestinal parasite Eimeria acervulina with recombinant coccidial antigen; Jenkins MC et al.; The gene encoding an immunodominant Eimeria acervulina merozoite surface antigen (EAMZ250) was expressed in bacteria as a fusion peptide with the galactose-binding protein (GBP) of Escherichia coli . Recombinant and control antigens were administered to 1-wk-old chickens by peroral inoculation with live nonpathogenic bacteria that were expressing GBP-EAMZ250 or GBP protein . The immunization elicited antigen-specific humoral and cellular immune responses as measured by ELISA and T-cell blastogenesis assay . In addition, chickens immunized with recombinant GBP-EAMZ250 exhibited significant protection against weight loss and intestinal lesions after E . acervulina challenge . Bacterial transformants were recoverable from the upper and middle intestine of inoculated chickens for various times after immunization . These data indicate that oral administration of live E . coli expressing a recombinant E . acervulina antigen is an effective means of inducing resistance to coccidiosis.

Mol Microbiol, 1991 Mar, 5(3), 607 - 16
The cloning and DNA sequence of the gene for the glutathione-regulated potassium-efflux system KefC of Escherichia coli; Munro AW et al.; The kefC gene of Escherichia coli encodes a potassium-efflux system that is regulated by glutathione metabolites . The close proximity of the E . coli kefC gene to the folA gene, encoding dihydrofolate reductase, has been utilized to clone the structural gene for the system from a Clarke-Carbon plasmid . The cloned gene has been refined to a region of DNA approximately 2.1 kb in length using exonuclease III-generated deletions and random MudII1734 (lacZ) insertions . The direction of transcription has been deduced from the orientation of the Mu insertions in the cloned DNA . A hybrid protein consisting of approximately two thirds of the KefC protein fused to beta-galactosidase has been shown to be membrane-located . The DNA sequence of the gene has been determined and an open reading frame of 1.86 kb has been located which could encode a protein of 620 amino acids (79010 Da) . Using the T7 expression system a membrane protein, of apparent molecular mass 55-60 kDa, has been shown to be encoded by the kefC gene . The predicted protein sequence shows a highly hydrophobic amino-terminus and a strongly hydrophilic carboxy-terminus . Comparison of the amino acid sequence of the kefC gene product with those of two glutathione-utilizing enzymes, glyoxalase and dehalogenase, has revealed some similarities.

Circ Shock, 1991 Mar, 33(3), 178 - 82
Survival factors in a canine septic shock model; Chung TW et al.; Physiological indicators of tissue perfusion in a canine septic shock model have been examined . An early death (ED) group and a combined late death and survivor group (LDS) were defined and the corresponding data compared . It was found that the LDS group had less reductions in mean systemic arterial pressure (P less than 0.05), transcutaneous oxygen pressure (P less than 0.05), and red blood cell deformability (P less than 0.001); a smaller increase in hematocrit (P less than 0.05); and a lower concentration of white blood cells (P less than 0.05), relative to the ED group, at 6 hr after an infusion of Escherichia coli organisms . These results suggest that dogs in the LDS group have better tissue perfusion than those in the early death ED group . Post-treatment of dogs with pentoxifylline did not improve survival time or enhance flow factors.

Pediatr Infect Dis J, 1991 Mar, 10(3), 248 - 50
An outbreak of Cryptosporidium diarrhea in a pediatric hospital; Navarrete S et al.; PIP: Physicians investigated a nosocomial diarrhea outbreak among 11 2 year old undernourished children in the nutrition service of the pediatric teaching hospital, Hospital Infantile, in Mexico City, Mexico in April 1988 . Health practitioners took at least 2 stool samples from each ill child to be analyzed for Cryptosporidium oocysts . The attack rate stood st 82% . The hospital admitted a malnourished child with chronic diarrhea and pneumonia on March 22 . Laboratory tests revealed that he had many Cryptosporidium oocysts and was positive for HIV . Hospital staff did not isolate him . He died on May 9 of Escherichia coli and Candida septicemia . The outbreak ended 1 week later . Laboratory tests detected Cryptosporidium oocysts in 9 cases all of whom were 3-13 months old . Further the symptoms (mean duration 14 days, fever {mean peak 38.6 degrees Celsius, and vomiting} matched those of other reported Cryptosporidium diarrhea outbreaks . The epidemic curve suggested a common source of the outbreak . Since the infants received intravenous feedings or sterilized formula, food and water could not have been the source . The physicians believed the AIDS case was that source . Direct person to person transmission was probably not responsible since each infant had his/her own separate crib . Even though the physicians could not conclusively identify the vehicle of transmission, it was most likely the hands of hospitals staff either directly by touching the infants or by contaminating the nasogastric tubes . After the outbreak, the physicians observed that only 30% of medical personnel indeed washed their hands before caring for an infant . 4 previous studies on nosocomial Cryptosporidium diarrhea outbreaks also reported the source case as immunodeficient, but these studies only included adults .

FEMS Microbiol Lett, 1991 Mar 1, 62(2-3), 347 - 53
Construction and transduction of a shuttle vector bearing the cos site of Streptomyces phage phi C31 and determination of its cohesive ends; Kobler L et al.; A plasmid has been constructed which is a bifunctional vector for Escherichia coli and for streptomycetes, containing the cos site of Streptomyces phage phi C31 . It can be efficiently transduced into S . lividans 66 and into several other Streptomyces species . The nucleotide sequence of a 311-bp DNA fragment containing the cohesive ends has been determined . The cohesive ends consist of 10 bases protruding at the 3'-end of the phage genome.

FEMS Microbiol Lett, 1991 Mar 1, 62(2-3), 297 - 300
A regulatory mutant of the trimethylamine N-oxide reductase of Escherichia coli K12; Pascal MC et al.; A mutant of Escherichia coli was isolated in which the trimethylamine N-oxide (TMAO) reductase activity is lost and the inducible TMAO reductase protein is absent . The lack of the gene product specifically prevents the expression of torA::lacZ fusion indicating a transcriptional control of torA, the TMAO reductase structural gene . The gene designated torR was mapped at 22 min on the chromosome.

FEMS Microbiol Lett, 1991 Mar 1, 62(2-3), 189 - 93
Isolation and characterization of a plasmid from Treponema denticola; Ivic A et al.; Agarose gel electrophoresis of whole genomic DNA of the oral spirochaete Treponema denticola has revealed a plasmid-like fraction . Purification and restriction enzyme analysis has confirmed the presence of a 2.6-kb circular plasmid, which has been mapped for restriction sites and cloned into the Escherichia coli plasmid pUC18 . Southern blot analysis of genomic T . denticola DNA, using the plasmid as a probe, has shown that the plasmid is present only as an extra-chromosomal element . No plasmid-coded recombinant gene product from a PstI insert in pUC18 has been detected in host cells of E . coli by SDS-PAGE or immunoblotting with polyclonal immune rabbit serum to T . denticola . The discovery of this plasmid may provide a useful tool in the application of new molecular approaches in spirochaetal biology.

FEMS Microbiol Lett, 1991 Mar 1, 62(2-3), 177 - 81
Molecular cloning of the genes for anthranilate synthetase from Streptomyces venezuelae ISP 5230; Paradkar AS et al.; Fragments of genomic DNA from Streptomyces venezuelae ISP5230 were cloned in the Escherichia coli expression vector pTZ18R and the plasmids were used to transform E . coli JA194 (trpE) . The transformants included a prototrophic strain containing a recombinant plasmid, pDQ181, with an approximately 6.8-kb insert . Subcloning located the trpE-complementing DNA in a 2.4-kb segment . Transformation of E . coli ED23 (lacking both trpE and trpG functions) with plasmids containing the 2.4-kb DNA segment gave prototrophic strains exhibiting both the ASI and ASII activities of anthranilate synthetase . The results indicated that trpE and trpG are clustered in S . venezuelae . Regions hybridizing to the pDQ181 insert were present in the genomic DNA of other streptomycetes.

FEMS Microbiol Lett, 1991 Mar 1, 62(2-3), 149 - 52
Mutations permitting the anaerobic growth of Escherichia coli on trehalose; Mat-Jan F et al.; Wild-type strains of Escherichia coli K-12 do not grow anaerobically on trehalose or galactose . We isolated two operon fusion mutants of E . coli which gained the ability to grow on trehalose anaerobically (tan) . The tanA-lac mutation was located at 41 min on the E . coli genetic map and also abolished growth on glucuronic acid both aerobically and anaerobically . The tanB-lac mutation was mapped to 68 min and permitted anaerobic growth on galactose as well as trehalose . The tanB-lac fusion was induced anaerobically whereas tanA-lac showed more or less constitutive beta-galactosidase expression.

Appl Environ Microbiol, 1991 Mar, 57(3), 665 - 71
Construction of a hybrid plasmid capable of replication in Amycolatopsis mediterranei; Lal R et al.; A new plasmid, pA387, has been isolated from "Amycolatopsis sp." (DSM 43387) . This plasmid could be isolated from liquid culture as well as mycelium from agar plates by a modified procedure . Plasmid pA387 is about 29.6 kb and can be cured at low frequency by protoplasting and ethidium bromide and heat treatment . Hybridization experiments showed that this plasmid is present in free form and does not integrate into the chromosome . A hybrid plasmid was constructed by cloning a 5.1-kb fragment of pA387 into the Escherichia coli vector pDM10 . This hybrid plasmid, termed pRL1, could be transformed into Amycolatopsis mediterranei and A . orientalis by electroporation . A transformation frequency of 2.2 x 10(3) transformants per micrograms of DNA at 12.5 kV/cm and a pulse duration of 10.8 ms was obtained in A . mediterranei, whereas 1.1 x 10(5) transformants per microgram of DNA were obtained at a field strength of 7.5 kV/cm and a pulse duration of 7.6 ms in A . orientalis . Plasmid pRL1 is the first hybrid plasmid which could be used successfully for the transformation of A . mediterranei . The plasmid has a rather high copy number, is genetically stable, and can be easily reisolated from A . mediterranei . Plasmid pRL1 will be useful for further construction of a shuttle vector for E . coli and A . mediterranei and becomes the basis for the development of gene cloning techniques in Amycolatopsis spp.

Antimicrob Agents Chemother, 1991 Mar, 35(3), 519 - 23
Mechanisms of quinolone resistance in a clinical isolate of Escherichia coli highly resistant to fluoroquinolones but susceptible to nalidixic acid; Moniot-Ville N et al.; Two associated resistance mechanisms were found in a nalidixic acid-susceptible (4 micrograms/ml) but fluoroquinolone-resistant (8 to 16 micrograms/ml) strain of Escherichia coli Q2 selected under norfloxacin therapy . As compared with the susceptible E . coli Q1 isolated before treatment, changes in outer membrane proteins and lipopolysaccharides in Q2 were associated with a 1.5- to 3-fold decrease in the uptake of fluoroquinolones but not nalidixic acid . A 50% inhibition of DNA synthesis in toluene-permeabilized cells of the resistant strain E . coli Q2 required up to 500-fold increased quantities of fluoroquinolones, whereas such inhibition was obtained in both E . coli Q1 and Q2 with similar amounts of nalidixic acid . Selection from E . coli Q1 on norfloxacin of one-step resistant mutants resembling E . coli Q2 was unsuccessful . From these results we infer that a decrease in outer membrane permeability, associated with a peculiar alteration of the DNA gyrase, was responsible for the unusual quinolone resistance phenotype of E . coli Q2.

J Clin Microbiol, 1991 Mar, 29(3), 656 - 9
Characterization of Escherichia coli strains producing heat-stable enterotoxin b (STb) isolated from humans with diarrhea; Lortie LA et al.; Two of 49 cytolethal distending toxin-producing strains of Escherichia coli isolated from human stools contained the gene coding for heat-stable enterotoxin b (STb), as detected by a colony hybridization assay . The STb gene was found to be on a 70-kb plasmid also coding for heat-labile enterotoxin (pLT-I) . Restriction endonuclease analysis showed the STb gene from human isolates to be similar to the STb gene found in porcine strains . Moreover, by enzymatic amplification based on oligonucleotide primers designed from a porcine STb sequence, the expected portion of the STb gene was amplified for the human E . coli strains . The STb enterotoxin from these strains was bioactive in rat jejunal loops and was detected with an enzyme-linked immunosorbent assay by using polyclonal antiserum raised against purified porcine STb toxin.

Am J Trop Med Hyg, 1991 Mar, 44(3), 306 - 13
Recombinant Plasmodium falciparum glutamate rich protein; purification and use in enzyme-linked immunosorbent assay; Dziegiel M et al.; A method for purification of a recombinant Plasmodium falciparum protein produced in E . coli and its use in an enzyme-linked immunosorbent assay (ELISA) is described . The cloned gene fragment encodes GLURP,489-1271 the carboxy-terminal 783 amino acid residue portion of a 1271 amino acid residue P . falciparum glutamate rich protein (GLURP), with a molecular weight of 220 kilodalton . The protein is associated with all parasite stages in the human host . Examination of sera from 105 adult Liberians living in a malaria endemic area revealed anti-GLURP IgG antibodies in 98% of the sera . The recombinant GLURP489-1271 was expressed as a chimeric protein, fused with E . coli beta-galactosidase . However, antibodies in sera were directed only against the malaria part of the fusion protein and not against beta-galactosidase . Antigen from in vitro P . falciparum cultures of isolates from Tanzania (F32), Papua New Guinea (MAD20) and Honduras (HB3) completely absorbed specific antibodies, indicating the presence of conserved epitopes produced by all isolates of P . falciparum . Recombinant GLURP489-1271 ELISA is sensitive and rapid, and therefore well-suited for sero-epidemiological studies, and for control of the immunogenicity of a possible future P . falciparum vaccine utilizing epitopes from GLURP.

Res Vet Sci, 1991 Mar, 50(2), 131 - 3
Detection of foreign DNA in transgenic chicken embryos using the polymerase chain reaction; Savva D et al.; Chicken primordial germ cells were infected with a defective retrovirus containing the Escherichia coli lacZ gene and injected into the heart of stage 15 embryos . DNA samples were isolated from various tissues of the injected embryos at different stages of development and were examined for the presence of the lacZ gene using the polymerase chain reaction . Integration of the retrovirus DNA was demonstrated with a 32P-labelled oligonucleotide in five-, 10- and 18-day embryos . This quick procedure provides an opportunity for the early detection of foreign DNA in small numbers of transfected cells and is a valuable tool in the detection of transgenic animals.

Radiobiologiia, 1991 Mar-Apr, 31(2), 275 - 8
{Functional changes in the small intestine under the influence of sorbents in rats with a radiation-thermal lesion}; Nesterenko VS et al.; Major small intestine functions of rats were inhibited after whole-body exposure to radiation and heat . The injection of sorbents varying in the chemical structure favored the recovery of the small intestine function.

J Gen Microbiol, 1991 Mar, 137 ( Pt 3), 685 - 91
Control of methionine biosynthesis in Escherichia coli K12: a closer study with analogue-resistant mutants; Chattopadhyay MK et al.; Control of methionine biosynthesis in Escherichia coli K12 was reinvestigated by using methionine-analogue-resistant mutants . Norleucine (NL) and alpha-methylmethionine (MM) were found to inhibit methionine biosynthesis directly whereas ethionine (Et) competitively inhibited methionine utilization . Adenosylation of Et to generate S-adenosylethionine (AdoEt) by cell-free enzyme from E . coli K12 was demonstrated . Tolerance of increasing concentrations of NL by E . coli K12 mutants is expressed serially as phenotypes NLR, NLREtR, NLRMMR and finally NLREtRMMR . All spontaneous NLR mutants had a metK mutation, whereas NTG-induced mutants had mutations in both the metK and metJ genes . The kinetics of methionine adenosylation by the E . coli K12 cell-free enzyme were found to be similar to those reported for the yeast enzyme, showing the typical lag phase at low methionine concentration and disappearance of this phase when AdoMet was included in the incubation mixture . NL extended the lag phase, and lowered the rate of subsequent methionine adenosylation, but did not affect the shortening of the lag phase of adenosylation by AdoMet.

Zhonghua Zheng Xing Shao Shang Wai Ke Za Zhi, 1991 Mar, 7(1), 51 - 3, 77
{Pulmonary microcirculatory changes in acute lung injury in rats induced by burns and endotoxin administration}; Gong XQ; An animal model of acute pulmonary injury similar to adult respiratory distress syndrome (ARDS) was produced by intravenous injection of E . coli endotoxin to burned rats . The changes in pulmonary microcirculation were observed in vivo with "pleura window" method and the cast of the lung capillaries was studied using scanning electron microscope . The histological changes in the lung in these rats were observed with transmission electron microscope . It was shown that lung vessels constricted, microvascular permeability increased, pulmonary edema occurred and leukocytes and aggregated platelets accumulated in lung capillaries . This study suggested that pulmonary microcirculatory changes are the pathological basis of acute lung injury induced by burns accompanied with endotoxemia.

Mol Gen Genet, 1991 Mar, 225(3), 510 - 3
Cloning and sequencing of an Escherichia coli K12 gene which encodes a polypeptide having similarity to the human ferritin H subunit; Izuhara M et al.; Using lambda phage clones containing segments of the Escherichia coli K12 chromosome as hybridization probes, we found one gene at 42 min on the E . coli chromosome map, the expression of which was affected by RNase III . The sequence of the DNA fragment containing this gene (gen-165) revealed the presence of an open reading frame encoding a polypeptide of 165 amino acid residues . The amino acid sequence deduced from the nucleotide sequence exhibited a remarkable similarity to that of the human ferritin H chain.

Mol Gen Genet, 1991 Mar, 225(3), 448 - 52
Repair of uracil residues closely spaced on the opposite strands of plasmid DNA results in double-strand break and deletion formation; Dianov GL et al.; The role of closely spaced lesions on both DNA strands in the induction of double-strand breaks and formation of deletions was studied . For this purpose a polylinker sequence flanked by 165 bp direct repeats was inserted within the tet gene of pBR327 . This plasmid was used to construct DNA containing one or two uracil residues which replaced cytosine residues in the KpnI restriction site of the polylinker . Incubation of the plasmid DNA construct with Escherichia coli cell-free extracts showed that double-strand breaks occurred as a result of excision repair of the opposing uracil residues by uracil-DNA glycosylase (in extracts from ung+ but not in extracts from ung- E . coli strains) . Recombination of direct repeats, induced by double-strand breakage of plasmid DNA, can lead to the deletion of the polylinker and of one of the direct repeats, thus restoring the tet+ gene function which can be detected by the appearance of tetracycline-resistant colonies of transformants . Transformation of E . coli cells with single or double uracil-containing DNAs demonstrated that DNA containing two closely spaced uracil residues was tenfold more effective in the induction of deletions than DNA containing only a single uracil residue . The frequency of deletions is increased tenfold in an ung+ E . coli strain in comparison with an ung- strain, suggesting that deletions are induced by double-strand breakage of plasmid DNA which occurs in vivo as a result of the excision of opposing uracil residues.

Mol Gen Genet, 1991 Mar, 225(3), 420 - 6
In vitro roles of Escherichia coli DnaJ and DnaK heat shock proteins in the replication of oriC plasmids; Malki A et al.; Heat shock proteins have been shown to be involved in many cellular processes in procaryotic and eucaryotic cells . Using an in vitro DNA replication assay, we show that DNA synthesis initiated at the chromosomal origin of replication of Escherichia coli (oriC) is considerably reduced in enzyme extracts isolated from cells bearing mutations in the dnaK and dnaJ genes, which code for heat shock proteins . Furthermore, unlike DNA synthesis in wild-type extracts, residual DNA synthesis in dnaK and dnaJ extracts is thermosensitive . Although thermosensitivity can be complemented by the addition of DnaK and DnaJ proteins, restoration of near wild-type replication levels requires supplementary quantities of purified DnaA protein . This key DNA synthesis initiator protein is shown to be adsorbed to DnaK affinity columns . These results suggest that at least one of the heat shock proteins . DnaK, exerts an effect on the initiation of DNA synthesis at the level of DnaA protein activity . However, our observation of normal oriC plasmid transformation ratios and concentrations in heat shock mutants at permissive temperatures would suggest that heat shock proteins play a role in DNA replication mainly at high temperatures or under other stressful growth conditions.

Mol Gen Genet, 1991 Mar, 225(3), 379 - 86
Characterization of the Escherichia coli K12 gltS glutamate permease gene; Kalman M et al.; The gltS gene is known to encode a sodium-dependent, glutamate-specific permease . We have localized the Escherichia coli K12 gltS gene with respect to the spoT gene, sequenced it, and recombined a null insertion-deletion allele into the chromosome without loss of viability . The gltS null allele gives a Glt- phenotype, i.e . it abolishes the ability of a gltCc host to grow on glutamate as sole carbon and nitrogen source and also confers alpha-methylglutamate resistance . A multicopy plasmid expressing the gltS gene can reverse the Glt- phenotype of gltS- or wild-type strains while other plasmids show host-dependent complementation patterns . Induction of gltS gene overexpression under control of isopropyl-beta-D-thiogalactoside (IPTG)-inducible promoters severely inhibits growth . The GltS protein is deduced to be a 42425 dalton hydrophobic protein with 2 sets of 5 possible integral protein domains, each flanking a central hydrophilic, flexible region.

Biochem J, 1991 Mar 1, 274 ( Pt 2), 549 - 55
Effects of directed mutagenesis on conserved arginine residues in a human Class Alpha glutathione transferase; Stenberg G et al.; Glutathione transferase (GST) epsilon (also known as GST2 or GST B1B1), the major Class Alpha GST in human liver has been subjected to oligonucleotide-directed site-specific mutagenesis . Four arginine residues, R13, R20, R69 and R187, of which all but R69 are strictly conserved through GST Classes Alpha, Mu and Pi have been replaced by Ala . The mutant enzymes have been expressed in Escherichia coli, purified by affinity chromatography and characterised . Compared with the wild-type enzyme, all mutant GSTs had altered catalytic properties . All mutants had decreased specific activity with 1-chloro-2,4-dinitrobenzene (CDNB) . Mutants R13A, R69A and R187A also showed decreased activities with other substrates such as cumene hydroperoxide (CuOOH) and androstenedione . In contrast, mutant R20A had an increased peroxidase activity and an isomerase activity essentially the same as that of the wild-type GST . With the substrates used, kcat./Km values were decreased for all mutant GSTs . Increases in the {S0.5} values were most significant for glutathione (GSH), while values for CDNB and CuOOH were less markedly affected . Thus, various kinetic data indicate that the GSH affinity has been reduced by the mutations and that this loss of affinity is linked to the decreased specific activities . Inhibition studies showed an increased sensitivity towards S-hexyl-GSH; this was particularly marked for mutant R69A . Mutant R20A had a lowered {I50} value but, in contrast, also the highest {I80} value as compared with the wild-type enzyme . Towards bromosulphophthalein, mutants R20A and R69A had a markedly increased sensitivity, about 35-fold in comparison with the wild-type . The inhibition properties of mutant R187A were similar to those of the wild-type enzyme and the properties of mutant R13A were in between . The increased sensitivity to S-hexyl-GSH, in contrast with the decreased affinity for GSH, was suggested to be due to an altered distribution between conformational states of the enzyme induced by the mutations . The arginine residues in positions 13, 20 and 69 all seem to be important for the catalytic properties of GST . Further, the inhibition studies indicate a role of arginine residues in the stabilisation of conformational states of the enzyme.

Heart Lung, 1991 Mar, 20(2), 202 - 5
Neuroleptic malignant syndrome and Escherichia coli urosepsis; Teglia MQ et al.; Neuroleptic malignant syndrome is a rare but potentially life-threatening reaction to neuroleptic drugs . The syndrome develops rapidly, and may occur at the initiation of neuroleptic therapy or after long-term use; its pathogenesis is unclear . The signs and symptoms associated with the syndrome are hyperpyrexia, extreme muscle rigidity, an altered level of consciousness, and autonomic dysfunction . We describe a case of neuroleptic malignant syndrome in a patient who had Escherichia coli urosepsis caused by thioridazine.

Mutat Res, 1991 Mar, 262(3), 183 - 8
Identification and cloning of a umu locus in Streptomyces coelicolor A3(2); Misuraca F et al.; The umuDC operon of Escherichia coli is required for efficient mutagenesis by UV and many other DNA-damaging agents . E . coli umu mutants are defective in mutagenesis and slightly more sensitive to DNA-damaging agents . The existence of a umuDC analogue in Streptomyces coelicolor was suggested by data of our previous works . We cloned from Streptomyces coelicolor a fragment of DNA homologous to the E . coli umuDC region that is able to complement the E coli umuC122::Tn5 mutation . Therefore our data suggest that S . coelicolor contains a functional umu-like operon.

Mutat Res, 1991 Mar, 254(2), 175 - 84
Establishment of a monoclonal antibody recognizing cyclobutane-type thymine dimers in DNA: a comparative study with 64M-1 antibody specific for (6-4)photoproducts; Mizuno T et al.; We obtained a monoclonal antibody (TDM-1) binding to 313-nm UV-irradiated DNA in the presence of acetophenone . The binding of TDM-1 to 254-nm UV-irradiated DNA was not reduced with the subsequent irradiation of 313-nm UV . Furthermore, the treatment of UV-irradiated DNA with photolyase from E . coli and visible light exposure reduced both the antibody binding and the amount of thymine dimers in the DNA . A competitive inhibition assay revealed that the binding of TDM-1 to UV-irradiated DNA was inhibited with photolyase, but not with 64M-1 antibody specific for (6-4)photoproducts . These results suggest that TDM-1 antibody recognizes cyclobutane-type thymine dimers in DNA . Using TDM-1 and 64M-1 antibodies, we differentially measured each type of damage in DNA extracted from UV-irradiated mammalian cells . Repair experiments confirm that thymine dimers are excised from UV-irradiated cellular DNA more slowly than (6-4)photoproducts, and that the excision rates of thymine dimers and (6-4)photoproducts are lower in mouse NIH3T3 cells than in human cells.

Mutat Res, 1991 Mar, 247(1), 5 - 18
In vitro mutagenesis in the lacI gene of Escherichia coli: fate of 3'-terminal mispairs versus internal base mispairs in a transfection assay; Maldonado-Rodriguez R et al.; The fate of G.T mismatches and frameshifts, present at the 3'-terminus of primer-template or internally, has been studied with a combined transfection and electrophoretic assay following in vitro polymerization by DNA polymerase I (Klenow enzyme) of Escherichia coli . Several synthetic oligodeoxynucleotide primers were synthesized and annealed to uracil-containing single-stranded DNA of M13 phage bearing the lacI gene, to produce 1-3 consecutive G.T mismatches in the middle of the duplex region or at the 3'-OH end of the primer . Additional mismatched primer-templates were prepared, in which the primer had a deleted nucleotide, an extra nucleotide or both G.T mismatch and an extra nucleotide . The extension or degradation of these primers during in vitro DNA synthesis in the presence of all 4 dNTPs ('complete' reaction) or in the absence of dATP ('-A' reaction) was monitored by gel electrophoresis . Duplex DNA products were used in a transfection assay and the nucleotide changes in i-mutant progeny were determined by sequence analysis . The results suggest that whereas a single 3'-terminal G.T mismatch is relatively stable in chain elongation by Klenow enzyme, multiple terminal G.T mismatches are degraded by the 3'-exonuclease activity of this polymerase prior to primer extension . This editing activity is increased with the number of 3'-terminal mispairs . Single, double and triple T----C base substitutions were efficiently recovered when the mismatches occurred internally . Also, single-base eliminations or additions were readily recovered when the mutagenic primers contained an internal base deletion or addition, respectively . When products of the '-A' misincorporation reaction (catalyzed by Klenow enzyme) were assayed by transfection, base substitutions (exclusively T----C), but no frameshifts, were recovered . The results indicate that the absence of multiple tandem base substitutions among i- mutants recovered following primer elongation under mutagenic 'minus' conditions was due to the efficient action of the 3'-exonuclease activity of the Klenow enzyme on multiple terminal mismatches during in vitro polymerization, rather than to in vivo events (lack of expression or occurrence of mismatch repair) in the M13-lacI transfection assay.

J Nutr, 1991 Mar, 121(3), 395 - 400
Decreased resistance and immune response to Escherichia coli infection in chicks with low or high intakes of vitamin A; Friedman A et al.; The effects of vitamin A excess of insufficiency on resistance to Escherichia coli infection and subsequent anti-E . coli immune response were examined in chicks . Chicks receiving depleted (0 microgram/kg), sufficient (0.85 mg/kg) or excess (1000 mg/kg) levels of vitamin A in their feed were inoculated by a subcutaneous injection of pathogenic E . coli (1 x 10(9) and 2 x 10(9) cfu per chick) . Susceptibility to E . coli was determined by mortality, morbidity and immune responses (antibody production and T lymphocyte proliferation) . Excess of insufficient vitamin A led to increased susceptibility of chicks of E . coli infection; this was accompanied by depressed immune responses . Chicks receiving excess vitamin A were more sensitive to E . coli than vitamin A-depleted chicks . This was reflected in higher mortality and morbidity rates and in severely depressed immune responses . In contrast to chicks receiving excess vitamin A, T lymphocyte responses (though not antibody responses) of vitamin A-depleted chicks achieved levels similar to those of vitamin A-sufficient birds with a lag period of 6 to 10 d . Therefore, reduction in resistance to E . coli infection, resulting from vitamin A excess or deficiency, probably was compounded by a delayed immune response.

J Bacteriol, 1991 Mar, 173(6), 2116 - 9
Tandem translation starts in the cheA locus of Escherichia coli; Kofoid EC et al.; The cheA locus of Escherichia coli encodes two protein products, CheAL and CheAS . The nucleotide sequences of the wild-type cheA locus and of two nonsense alleles confirmed that both proteins are translated in the same reading frame from different start points . These start sites were located on the coding sequence by direct determination of the amino-terminal sequences of the two CheA proteins . Both starts are flanked by inverted repeats that may play a role in regulating the relative expression rates of the CheA proteins through alternative mRNA secondary structures.

J Bacteriol, 1991 Mar, 173(6), 2068 - 76
Increased spontaneous mutation and alkylation sensitivity of Escherichia coli strains lacking the ogt O6-methylguanine DNA repair methyltransferase; Rebeck GW et al.; Escherichia coli expresses two DNA repair methyltransferases (MTases) that repair the mutagenic O6-methylguanine (O6MeG) and O4-methylthymine (O4MeT) DNA lesions; one is the product of the inducible ada gene, and here we confirm that the other is the product of the constitutive ogt gene . We have generated various ogt disruption mutants . Double mutants (ada ogt) do not express any O6MeG/O4MeT DNA MTases, indicating that Ada and Ogt are probably the only two O6MeG/O4MeT DNA MTases in E . coli . ogt mutants were more sensitive to alkylation-induced mutation, and mutants arose linearly with dose, unlike ogt+ cells, which had a threshold dose below which no mutants accumulated; this ogt(+)-dependent threshold was seen in both ada+ and ada strains . ogt mutants were also more sensitive to alkylation-induced killing (in an ada background), and overexpression of the Ogt MTase from a plasmid provided ada, but not ada+, cells with increased resistance to killing by alkylating agents . The induction of the adaptive response was normal in ogt mutants . We infer from these results that the Ogt MTase prevents mutagenesis by low levels of alkylating agents and that, in ada cells, the Ogt MTase also protects cells from killing by alkylating agents . We also found that ada ogt E . coli had a higher rate of spontaneous mutation than wild-type, ada, and ogt cells and that this increased mutation occurred in nondividing cells . We infer that there is an endogenous source of O6MeG or O4MeT DNA damage in E . coli that is prevalent in nondividing cells.

J Bacteriol, 1991 Mar, 173(6), 1992 - 6
Role of RpoH, a heat shock regulator protein, in Escherichia coli carbon starvation protein synthesis and survival; Jenkins DE et al.; Escherichia coli starvation proteins include several heat shock proteins whose induction by heat is controlled by the minor sigma factor, sigma 32 . The level of sigma 32 increased in wild-type E . coli upon starvation, and three sigma 32-controlled heat shock proteins (DnaK, GroEL, and HtpG) were not induced during starvation in an isogenic delta rpoH strain, which is unable to synthesize sigma 32 . Thus, sigma 32 plays a role in the induction of these proteins during both heat shock and starvation . The delta rpoH strain was more sensitive to starvation but could develop starvation-mediated cross protection against heat and oxidation.

J Bacteriol, 1991 Mar, 173(6), 1965 - 70
Partial characterization of a lysU mutant of Escherichia coli K-12; Hassani M et al.; The Escherichia coli K-12 strain GNB10181 shows no inducible lysyl-tRNA synthetase (LysRS) activity . Two-dimensional gel electrophoretic analysis of the polypeptides synthesized by this strain indicates that the normal lysU gene product, LysU, is absent . When both GNB10181 and its parent, MC4100, were grown at elevated temperatures (42 to 45 degrees C) no significant difference between their growth rates was observed . The lysU mutation was transferred to other E . coli K-12 backgrounds by using P1 transduction . The lysU transductants behaved comparably to their lysU+ parents at different growth temperatures . Therefore, the LysU proteins does not appear to be essential for growth at high temperatures, at least under the conditions examined here . In addition, lysU transductants were found to be defective for inducible lysine decarboxylase, (LDC), inducible arginine decarboxylase (ADI), and melibiose utilization (Mel), which are all missing in GNB10181 . Complementation of the above missing functions was achieved by using the Clarke-Carbon plasmids pLC4-5 (LysU LDC) and pLC17-38 (LysU Mel ADI) . From these experiments, it appears that GNB10181 has suffered a chromosomal deletion between 93.4 and 93.7 min, which includes the lysU gene . By using plasmid pLC17-38, the position of ADI on two-dimensional gels was identified . Finally, lysS delta lysU double mutants were constructed which can potentially be used as positive selection agents for the isolation of LysRS genes from other sources.

J Bacteriol, 1991 Mar, 173(6), 1902 - 10
Nucleotide sequence of the Escherichia coli micA gene required for A/G-specific mismatch repair: identity of micA and mutY; Tsai-Wu JJ et al.; The Escherichia coli methylation-independent repair pathway specific for A/G mismatches has been shown to require the gene product of micA . Extracts prepared from micA mutants do not form an A/G mismatch-specific DNA-protein complex and do not contain an A/G mismatch-specific nicking activity . Moreover, a partially purified protein fraction containing both A/G mismatch-specific nicking and binding activities restores repair activity in micA mutant extracts . The DNA sequence of a 2.3-kb fragment containing the micA gene has been determined . There are two open reading frames (ORF) in this DNA fragment: one ORF encodes a 25.7-kDa protein whose function is still unknown, the other ORF codes for a protein with an Mr of 39,147, but this ORF can be transcribed and the mRNA can be translated to yield a protein with an apparent Mr of 36 kDa on a sodium dodecyl sulfate-polyacrylamide gel . Deletion analysis showed that this 39.1-kDa ORF is the micA gene as judged by the capacity of the encoded protein to restore the A/G mismatch-specific nicking activity of micA mutant extracts . Furthermore, our results suggest that micA is the same gene as the closely mapped mutY, which encodes the A/G mismatch-specific glycosylase.

J Bacteriol, 1991 Mar, 173(6), 1873 - 8
Stoichiometry of maltodextrin-binding sites in LamB, an outer membrane protein from Escherichia coli; Gehring K et al.; We have directly measured the stoichiometry of maltodextrin-binding sites in LamB . Scatchard plots and computer fitting of flow dialysis (rate-of-dialysis) experiments clearly establish three independent binding sites per LamB trimer, with a dissociation constant of approximately 60 microM for maltoheptaose . The current model for LamB's function as a specific pore is discussed with respect to the symmetry in LamB's kinetic properties and the implications of our results.

EMBO J, 1991 Mar, 10(3), 687 - 96
HU and IHF, two homologous histone-like proteins of Escherichia coli, form different protein-DNA complexes with short DNA fragments; Bonnefoy E et al.; Using the gel retardation technique we have studied the protein-DNA complexes formed between HU--the major histone-like protein of Escherichia coli--and short DNA fragments . We show that several HU heterodimers bind DNA in a regularly spaced fashion with each heterodimer occupying about 9 base pairs . The alpha 2 and beta 2 HU homodimers form the same structure as the alpha beta heterodimer on double stranded DNA . However when compared to the heterodimer, they bind single stranded DNA with higher affinity . We also show that HU and the Integration Host Factor of E . coli (IHF) form different structures with the same DNA fragments . Moreover, HU seems to enhance the DNA-binding capacity of IHF to a DNA fragment which does not contain its consensus sequence.

EMBO J, 1991 Mar, 10(3), 675 - 9
Regulation of the acetate operon in Escherichia coli: purification and functional characterization of the IclR repressor; Cortay JC et al.; Growth of Escherichia coli on acetate requires operation of the anaplerotic sequence known as the glyoxylate bypass . In this pathway three different enzymes are activated: malate synthase, isocitrate lyase and isocitrate dehydrogenase kinase/phosphatase which are encoded by genes aceB, aceA and aceK, respectively . These three genes are clustered, in that order, in the same acetate (ace) operon whose expression is under the transcriptional control of the iclR gene located downstream from aceK . We have cloned the iclR gene in the pKK233-2 vector which allows optimization of both transcription and translation initiation . The IclR repressor has been overproduced, then purified to homogeneity in a one-step procedure by cation exchange chromatography after ammonium sulfate fractionation . Its specific interaction with the operator/promoter region of the ace operon has been analyzed by gel retardation and DNase I footprinting experiments . The IclR repressor has been shown to recognize a 35 bp palindromic sequence which largely overlaps the -35 recognition site of RNA polymerase . Moreover, the formation of the complex between IclR and the operator/promoter region has been found to be impaired by phosphoenol pyruvate but insensitive to acetate, acetyl-CoA, pyruvate, and oxaloacetate . These results are discussed in terms of primary regulation of the expression of the ace operon.

EMBO J, 1991 Mar, 10(3), 617 - 24
The maize regulatory locus Opaque-2 encodes a DNA-binding protein which activates the transcription of the b-32 gene; Lohmer S et al.; The maize locus, Opaque-2, controls the expression in developing endosperm of structural genes encoding a family of storage proteins, the 22 kd zeins, and an abundant albumin, termed b-32 . It is shown that the promoter of the b-32 gene is activated in vivo in the presence of the O2 gene product and that the information necessary for this activation resides in a 440 bp DNA fragment containing five O2 binding sites (GATGAPyPuTGPu) . Two of these sites are embedded in copies of the 'endosperm box', a motif thought to be involved in endosperm-specific expression, which is also represented in 22 kd zein promoters . The O2 protein is also shown to be capable of binding in vitro and activating in vivo, its own promoter.

EMBO J, 1991 Mar, 10(3), 547 - 53
Identification of the structural gene for glucose-6-phosphate dehydrogenase in yeast . Inactivation leads to a nutritional requirement for organic sulfur; Thomas D et al.; Cloning of the MET19 gene revealed that it encodes the glucose-6-phosphate dehydrogenase from yeast . Sequence analysis showed a high degree of similarity between the yeast and the human enzymes . The cloned gene has allowed the construction of a glucose-6-phosphate dehydrogenase null mutant . The only phenotype of such a strain is an absolute requirement for an organic sulfur source, i.e . methionine, S-adenosylmethionine (AdoMet), cysteine, glutathione or homocysteine . The phenotype of this null mutant raises some new questions about the exact functions of the pentose phosphate pathway which was usually considered as the main cellular source of NADPH . Moreover, results reported here show that an increase of the AdoMet pool represses the transcription of the glucose-6-phosphate dehydrogenase gene . This regulation acts on the glucose-6-phosphate dehydrogenase biosynthesis but does not affect the synthesis of 6-phosphogluconate dehydrogenase . That AdoMet controls a part of the pentose phosphate pathway sheds new light on mechanisms regulating the relative fluxes of carbon utilization through the pentose phosphate pathway and glycolysis.

Proc Natl Acad Sci U S A, 1991 Mar 1, 88(5), 1903 - 7
Human cyclophilin B: a second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence; Price ER et al.; We report the cloning and characterization of a cDNA encoding a second human cyclosporin A-binding protein (hCyPB) . Homology analyses reveal that hCyPB is a member of the cyclophilin B (CyPB) family, which includes yeast CyPB, Drosophila nina A, and rat cyclophilin-like protein . This family is distinguished from the cyclophilin A (CyPA) family by the presence of endoplasmic reticulum (ER)-directed signal sequences . hCyPB has a hydrophobic leader sequence not found in hCyPA, and its first 25 amino acids are removed upon expression in Escherichia coli . Moreover, we show that hCyPB is a peptidyl-prolyl cis-trans isomerase which can be inhibited by cyclosporin A . These observations suggest that other members of the CyPB family will have similar enzymatic properties . Sequence comparisons of the CyPB proteins show a central, 165-amino acid peptidyl-prolyl isomerase and cyclosporin A-binding domain, flanked by variable N-terminal and C-terminal domains . These two variable regions may impart compartmental specificity and regulation to this family of cyclophilin proteins containing the conserved core domain . Northern blot analyses show that hCyPB mRNA is expressed in the Jurkat T-cell line, consistent with its possible target role in cyclosporin A-mediated immunosuppression.

Proc Natl Acad Sci U S A, 1991 Mar 1, 88(5), 1631 - 5
Role of integration host factor in the regulation of the glnHp2 promoter of Escherichia coli; Claverie-Martin F et al.; The glnHPQ operon of Escherichia coli encodes components of the high-affinity glutamine transport system . One of the two promoters of this operon, glnHp2, is responsible for expression of the operon under nitrogen-limiting conditions . The general nitrogen regulatory protein (NRI) binds to two overlapping sites centered at -109 and -122 from the transcription start site and, when phosphorylated, activates transcription of glnHp2 by catalyzing isomerization of the closed sigma 54-RNA polymerase promoter complex to an open complex . The DNA-bending protein integration host factor (IHF) binds to a site immediately upstream of glnHp2 and enhances the activation of open complex formation by NRI phosphate . The NRI-binding sites can be moved several hundred base pairs further upstream without altering the ability of NRI phosphate to activate open complex formation . We propose that the IHF-induced bend can facilitate or obstruct the interaction between NRI phosphate and the closed complex depending on the relative positions of NRI phosphate and sigma 54-RNA polymerase on the DNA.

J Surg Res, 1991 Mar, 50(3), 234 - 9
Contribution of portal systemic shunt to Kupffer cell dysfunction in obstructive jaundice; Dunn CW et al.; Previous animal models of biliary tract obstruction have shown that hepatic phagocytic activity is impaired secondary to Kupffer cell dysfunction . Biliary tract obstruction leads to portal hypertension and an associated portal systemic shunt . Forty-eight Sprague-Dawley rats were studied to determine the contribution of portal systemic shunt to Kupffer cell dysfunction after 21 days of obstructive jaundice or sham operation . Liver uptake of radiolabeled Escherichia coli decreased from 76.1 +/- 1.4% (sham) to 63.1 +/- 6.1% in the common duct ligation (CDL) rats (P less than 0.05); lung uptake increased from 4.0 +/- 0.6% (sham) to 20.2 +/- 4.5 (CDL) (P less than 0.05) . Portal systemic shunt, determined using radioactive microspheres, increased from 2.0 +/- 1.0% (sham) to 46.6 +/- 13.1% (CDL), P less than 0.05 . Although a significant portal systemic shunt does exist in this 21-day model of obstructive jaundice, it does not appear to be the only mechanism underlying Kupffer cell dysfunction.

J Bacteriol, 1991 Mar, 173(5), 1711 - 21
Structure of Escherichia coli K-12 miaA and characterization of the mutator phenotype caused by miaA insertion mutations; Connolly DM et al.; Previously, we reported several unusual relationships between the 2-methylthio-N6-(delta 2-isopentenyl)adenosine-37 (ms2i6A-37) tRNA modification and spontaneous mutagenesis in Escherichia coli K-12 (D . M . Connolly and M . E . Winkler, J . Bacteriol . 171:3233-3246, 1989) . To confirm and extend these observations, we determined the structure of miaA, which mediates the first step of ms2i6A-37 synthesis, and characterized the miaA mutator phenotype . The most likely translation start of miaA overlaps the last two codons of mutL, which encodes a protein required for methyl-directed mismatch repair . This structural arrangement confirms that miaA and mutL are in the same complex operon . The miaA gene product, delta 2-isopentenylpyrophosphate transferase, shows extensive homology with the yeast MOD5 gene product, and both enzymes contain a substrate binding site found in farnysyl pyrophosphate synthetase and a conserved putative ATP/GTP binding site . Insertions in miaA cause exclusively GC----TA transversions, which contrasts with the GC----AT and AT----GC transitions observed in mutL mutants . To correlate the absence of the ms2i6A-37 tRNA modification directly with the mutator phenotype, we isolated a unique suppressor of a leaky miaA(ochre) mutation . The miaD suppressor mapped to 99.75 min, restored the ms2i6A-37 tRNA modification to miaA(ochre) mutants, and abolished the miaA mutator phenotype . We speculate that miaD causes a decrease in ms2i6A-37 tRNA demodification or an increase in miaA gene expression but not at the level of operon transcription . Together, these observations support the idea that the ms2i6A-37 tRNA modification acts as a physiological switch that modulates spontaneous mutation frequency and other metabolic functions.

J Bacteriol, 1991 Mar, 173(5), 1663 - 9
Isolation and molecular characterization of the ribosomal protein L6 homolog from Chlamydia trachomatis; Gray GJ et al.; The cloning of a Chlamydia trachomatis eukaryotic cell-binding protein reported earlier from our laboratory (R . Kaul, K . L . Roy, and W . M . Wenman, J . Bacteriol . 169:5152-5156, 1987) represents an artifact generated by nonspecific recombination of chromosomal DNA fragments . However, the amino terminus of this plasmid-encoded fusion product demonstrated significant homology to Escherichia coli ribosomal protein L6 . By using a 458-bp PstI-HindIII fragment of recombinant pCT161/18 (representing the 5' end of the cloned gene), we isolated and characterized a C . trachomatis homolog of the ribosomal protein L6 gene of E . coli . Sequence analysis of an 1,194-bp EcoRI-SacI fragment that encodes chlamydial L6 (designated CtaL6e) revealed a 552-bp open reading frame comprising 183 amino acids and encodes a protein with a molecular weight of 19,839 . Interestingly, complete gene homology between C . trachomatis serovars L2 and J, each of which exists as a single copy per genome, was observed . Expression of a plasmid-encoded gene product is dependent on the lac promoter, since no product was obtained if the open reading frame was oriented in opposition to the lac promoter . Immunoblotting of purified ribosomes revealed functional, as well as antigenic, homology between the E . coli and C . trachomatis ribosomal L6 proteins.

Infect Immun, 1991 Mar, 59(3), 829 - 35
Mapping the minimal contiguous gene segment that encodes functionally active Shiga-like toxin II; Perera LP et al.; Shiga-like toxin type II (SLT-II) is one of two antigenically distinct cytotoxins produced by enterohemorrhagic Escherichia coli that are believed to play a central role in the pathogenesis of enterohemorrhagic E . coli-induced disease . SLT-II is a bipartite toxin with an enzymatically active A subunit that inhibits protein synthesis and an oligomeric B subunit that binds to the glycolipid globotriaosylceramide on eukaryotic cells . In this study, functional boundaries of the slt-II operon were mapped . Mutant proteins lacking the last four amino acids from the carboxy terminus of the 70-amino-acid mature SLT-II B polypeptide had no cytotoxic activity . However, when only two amino acids were removed from the carboxy terminus of the B subunit, the cytotoxic activity of the holotoxin was not altered drastically . Furthermore, a 21-amino-acid extension to the carboxy terminus of the SLT-II B polypeptide was tolerated with a minimum reduction in cytotoxic activity of the holotoxin . Deletion of the region coding for amino acids 3 through 18 of the 296-amino-acid mature SLT-II A polypeptide resulted in complete ablation of the cytotoxic activity of the holotoxin as well as abolition of the enzymatic activity of the A subunit . Thus, it appears that both 5'- and 3'-terminal coding sequences are essential for function of the slt-II operon.

Infect Immun, 1991 Mar, 59(3), 814 - 21
Experimental infection of newborn pigs with an attaching and effacing Escherichia coli O45:K"E65" strain; Helie P et al.; The ability of a nonenterotoxigenic, K88-negative porcine Escherichia coli strain of serogroup O45:K"E65" to induce attaching-effacing lesions was investigated in newborn pigs . Typical attaching-effacing lesions, characterized by intimate adherence of bacteria to mature enterocyte brush borders with effacement of the microvilli, were observed on light and electron microscopy . Bacteria were also seen in intracytoplasmic vacuoles of mature enterocytes and, in areas of heavier colonization, in the lamina propria of the intestinal mucosa . A moderate inflammatory response with mild focal ulceration of the intestinal mucosa was observed . In a sequential study, we observed that the attaching-effacing lesions were well established in the duodenum, jejunum, and ileum at 12 h postinoculation but did not develop in the cecum and colon until 24 to 48 h postinoculation, although bacteria had colonized the latter areas as early as 12 h postinoculation . Initially, bacteria were very intimately attached, with an irregular arrangement on the enterocyte apical cell membrane, and subsequently reoriented to form a typical palisade arrangement with a narrow regular gap between the bacterial cell wall and the enterocyte apical cell membrane . This phenomenon of early intimate attachment of irregularly disposed bacteria has not been reported for human enteropathogenic attaching and effacing E . coli and could represent a new and different mechanism of attachment and effacement to intestinal epithelial cells.

Infect Immun, 1991 Mar, 59(3), 1074 - 8
Expression and deletion analysis of the Trypanosoma brucei rhodesiense cysteine protease in Escherichia coli; Pamer EG et al.; Trypanosoma brucei, the cause of African sleeping sickness, differentiates in the mammalian bloodstream from a long, slender trypanosome into a short, stumpy trypanosome . This event is necessary for infection of the tsetse fly and maintenance of the life cycle . We have previously shown that the stumpy form contains 10- to 15-fold-greater cysteine protease activity than either the slender form or the insect midgut procyclic, and we have isolated a cDNA encoding the protease . In order to determine whether the cDNA encodes the developmentally regulated cysteine protease, we have purified the protease from trypanosomes and have made a polyclonal antiserum against it . The trypanosomal protease gene was then expressed in Escherichia coli with three different methionines within the pre- and propeptides acting as initiation sites . In each case, a protein was synthesized that was recognized by an antiserum specific for the developmentally regulated trypanosomal cysteine protease . The protein synthesized from the more upstream initiation site within the propeptide was proteolytically active . The recombinant protease and the trypanosomal enzyme were identical with respect to peptide substrates and protease inhibitors . The protein remained active when synthesized in a truncated form lacking the nine consecutive prolines and carboxy-terminus extension, indicating that the terminal 108 amino acids are not necessary for proteolytic activity.

Cancer Res, 1991 Mar 1, 51(5), 1359 - 65
Further studies of the action of a partially purified bacteriocin against a murine fibrosarcoma; Hill RP et al.; We have reported previously that a partially purified bacteriocin (PPB) from Escherichia coli HSC10 is toxic to KHT cells growing in vivo as micrometastases but apparently has no activity against a tumor growing i.m . We report here experiments to investigate possible reasons for this difference . The PPB was shown to become less effective against micrometastases, initiated by i.v . injection of KHT cells, as the time between cell injection and PPB treatment increased . The kinetics of the loss of efficacy did not, however, correlate exactly with the growth kinetics of the nodules as assessed by survival following radiation treatment at different times after cell injection . This suggests the possibility of a diffusion limitation although it was found that s.c . injections of PPB were nearly as effective against micrometastases as i.p . injections . We also demonstrated that the lifetime of the majority of the toxic activity of PPB in vivo was relatively short (less than 1 day) and that the majority of its effect was not caused by stimulating macrophages to act against the tumor cells . The PPB was found to be cytotoxic to KHT cells in vitro but the effect was reduced at high cell density (approximately 10(6) cells/ml) . The PPB did not induce an immune reaction against itself in C3H mice nor was it toxic to either bone marrow stem cells or jejunal crypt cells at doses which were effective against KHT micrometastases . We conclude that PPB may have potential as a cytotoxic agent to act against circulating tumor cells or very small deposits of tumor cells but is limited in its efficacy against larger tumor masses probably because of diffusional and/or cell density effects.

J Virol, 1991 Mar, 65(3), 1325 - 31
A specific inhibitor of cysteine proteases impairs a Vif-dependent modification of human immunodeficiency virus type 1 Env protein; Guy B et al.; The Vif protein of human immunodeficiency virus type 1 (HIV-1) regulates viral infectivity . Virions produced in cell culture after transfection by a Vif-negative molecular clone show a dramatic decrease in infectivity for susceptible CD4+ cell lines, although the Vif protein does not appear to be a constituent of the viral particle . The exact mechanism by which Vif affects HIV-1 infectivity is so far unknown . We report the existence of structural homologies between Vif and a family of cysteine proteases and present evidence which suggests that one of the targets of Vif is the Env protein and more precisely the cytoplasmic domain of gp41 . Vif was found to modify both the processing and conformation of the Env protein . Ethyl(25, 35)- 3{(5)-3-methyl-1-(3-methylbutylcarbamoyl)}oxirane-2-carboxylate, a specific inhibitor of cysteine proteases, inhibits the effect of Vif, as does the mutation of Cys-114 to Leu in Vif . Furthermore, Cys-114 of Vif produced in Escherichia coli, interacts directly with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane . These observations suggest that a cysteine protease activity is associated with Vif and that this activity plays a role in Env maturation.

J Infect Dis, 1991 Mar, 163(3), 507 - 13
Diffuse-adhering Escherichia coli (DAEC) as a putative cause of diarrhea in Mayan children in Mexico; Giron JA et al.; Diarrhea is a major cause of infantile morbidity and mortality in developing countries . A community-based, case control study was conducted in a southern Mexican Mayan village for 3 weeks during the peak diarrhea period to prospectively identify the infectious agents associated with childhood diarrheal disease . Several enteropathogens were isolated from stools of 34 of 58 cases, although none was significantly associated with diarrhea . For the 24 cases from which no enteropathogens were isolated, diffuse-adhering Escherichia coli (DAEC) strains were significantly associated with diarrheal disease (P less than .02; odds ratio = 6; 95% confidence limit, 1.08-99.0) . DAEC were highly heterogeneous with respect to plasmid content and serotype . Three DNA probes designed to differentiate E . coli exhibiting localized, diffuse, or aggregative adherence were compared with results from a standard HeLa cell binding assay to assess the utility of these probes in the field . This study provides evidence for the potential pathogenic capacity of DAEC and underscores the variety of diarrheal agents operating within a community.

Virology, 1991 Mar, 181(1), 378 - 81
Vaccinia virus encodes a protein with similarity to glutaredoxins; Johnson GP et al.; Recently, we have reported the complete nucleotide sequence of vaccinia virus (Goebel, S . J., Johnson, G . P., Perkus, M . E., Davis, S . W., Winslow, J . P., and Paoletti, E . 1990, Virology 179, 247-266) . Approximately 2.2 kbp leftward of the large subunit of ribonucleotide reductase resides a 108-amino acid open reading frame, O2L (nt 62,851-62,528) with significant similarity to known glutaredoxins . The deduced amino acid sequence of open reading frame O2L is 28.7% identical to the yeast and Escherichia coli proteins and greater than 40% identical to various mammalian glutaredoxins . Similar patterns of hydrophobicity as well as alpha-helix and beta-sheet potentials suggest that O2L and the glutaredoxins share a similar secondary structure . Furthermore, a common function is inferred by the presence of a highly conserved redox-active site.

Virology, 1991 Mar, 181(1), 258 - 72
Genetic and molecular biological characterization of a vaccinia virus temperature-sensitive complementation group affecting a virion component; Fathi Z et al.; The gene affected by five previously isolated temperature-sensitive (ts) mutants (ts 10, ts 18, ts 38, ts 39, ts 44) of vaccinia virus strain WR constituting a single "normal" complementation group has been characterized . Marker rescue and DNA sequence analysis show that the five members of the complementation group map in an open reading frame, ORF 18R, which spans the HindIII I-G junction and has the capacity to encode a 77.6-kDa protein . The nucleotide sequence change responsible for temperature sensitivity in each of the five mutants was determined . Two of the mutants, ts 38 and ts 44, have the identical nucleotide change and may therefore be sisters . Northern blot analysis demonstrates that ORF 18R is transcribed at both early and late times during infection . Two distinct early transcripts have been observed which are 5' coterminal and which contain a 518 nucleotide 5' untranslated region . The long early transcript spans the entire 18R gene while the 3' end of the shorter early transcript maps to an early transcription termination signal contained within the 18R coding sequence . The 5' ends of the late transcripts have been mapped to a family of AUG proximal sites using both S1 nuclease and primer extension analysis . Primer extension analysis also identifies additional late 5' ends which map between nucleotides -500 and -1000 relative to the ORF 18R AUG.

Virology, 1991 Mar, 181(1), 251 - 7
Protein p22 of African swine fever virus: an early structural protein that is incorporated into the membrane of infected cells; Camacho A et al.; The open reading frame K'177, located at the left end of the African swine fever virus genome, codes for an early induced structural protein of 22,000 Da (p22), which is released from the viral particle by the nonionic detergent n-octyl-beta-D-glucopyranoside under conditions that solubilize external viral structural proteins . The predicted amino acid sequence of the protein contains a hydrophobic region at its N-terminus with characteristics of a signal peptide and, at early times after virus infection of Vero cells, the protein can be detected in the cell membrane by immunolabeling.

J Immunol, 1991 Mar 1, 146(5), 1541 - 6
Lipopolysaccharide, tumor necrosis factor-alpha, and IL-1 beta prevent programmed cell death (apoptosis) in human peripheral blood monocytes; Mangan DF et al.; Human peripheral blood monocytes progressively lose viability when cultured in the absence of serum, cytokines, or other stimuli . In this study, we investigated whether monocyte death results from membrane damage (i.e., necrosis) or internally regulated processes {i.e., programmed cell death (PCD) or apoptosis} . Our results clearly indicated that monocytes die by PCD when cultured without stimulation . Death was associated with fragmentation of DNA into integer multiples of approximately 200 bp, a decrease in cell size, condensation of the nucleus and cytoplasmic organelles, and membrane blebbing, all of which are cardinal features of PCD . Monocytes exposed to nonphysiologic conditions such as acidic media (pH 4.2), 56 degrees C for 30 min, or freezing and thawing were killed without concomitant DNA fragmentation, indicating that DNA fragmentation was not a result of cell death per se . Addition of Escherichia coli LPS, a potent monocyte activating agent, in concentrations as low as 0.1 ng/ml caused a marked increase in monocyte survival and prevented DNA fragmentation . Moreover, exogenous human rTNF-alpha or IL-1 beta also prevented PCD, suggesting that PCD is regulated by certain cytokines released from LPS-stimulated monocytes . The results indicate that in the absence of appropriate stimulation, monocytes are programmed to undergo a sequence of molecular events leading to cell death . Regulation of PCD may be an important homeostatic mechanism for controlling the number of monocytes available to respond to infection, wound healing, and tumor growth.

J Immunol, 1991 Mar 1, 146(5), 1534 - 40
Generation of monoclonal antibodies to murine IL-1 beta and demonstration of IL-1 in vivo; Hogquist KA et al.; The role of murine IL-1 beta in vitro and in vivo has not been defined . We describe here the production of neutralizing and immunoprecipitating mAb and polyclonal antibodies specific for murine IL-1 beta and their application to a characterization of the murine IL-1 beta protein . Immunization of either hamsters or rabbits with the recombinant mature form of murine IL-1 beta emulsified in CFA elicited antisera and hamster mAb that only recognized denatured IL-1 beta . In contrast, immunization with rIL-1 beta adsorbed to alum resulted in the generation of neutralizing and immunoprecipitating rabbit and hamster antisera and hamster mAb . All of the mAb recognize both the pro-form of IL-1 beta and the mature bioactive form produced by cultures of murine peritoneal macrophages . Using these antibodies, we demonstrate that approximately half of the IL-1 activity present in supernatants of LPS-treated cultured mouse macrophages is composed of IL-1 beta . Additionally, IL-1 beta as well as IL-1 alpha can be detected in the plasma of LPS-treated mice . These studies, therefore, demonstrate the production of IL-1 beta both in vitro and in vivo.

Cytokine, 1991 Mar, 3(2), 134 - 40
Murine interferon-gamma/interleukin-1 fusion proteins used as antigens for the generation of hybridomas producing monoclonal anti-interleukin-1 antibodies; Dijkmans R et al.; In several biological systems interferon-gamma (IFN-gamma) and interleukin-1 (IL-1) act synergistically . We therefore examined whether it would be possible to construct IFN-gamma/IL-1 hybrid proteins that would be more active than the individual components . Hybrid proteins were examined that consisted of the amino-terminal 118 residues of mouse IFN-gamma and the 156 or 152 carboxyl-terminal residues of mouse IL-1 alpha or IL-1 beta, respectively . They were obtained by ligation of the respective coding sequences and expression of the fused genes under control of the PL promotor in Escherichia coli . Both the IFN-gamma/IL-1 alpha and the IFN-gamma/IL-1 beta fusion proteins were purified by affinity chromatography on an anti-IFN-gamma monoclonal antibody column . Analysis of biological activities showed that these fusion proteins were less active than the individual cytokines . Specific antiviral activity of the IFN-gamma/IL-1 beta hybrids was less than 0.1% that of IFN-gamma and D10.G4.1 T-cell proliferative (IL-1) activity amounted to 0.1% that of mouse IL-1 . Affinity-purified preparations of the IFN-gamma/IL-1 alpha hybrid were found to contain variable proportions of a Mr 14,000 degradation product possessing IFN-gamma activity, whereas the undegraded Mr 30,000 fusion protein, while being devoid of detectable IFN-gamma activity, did possess IL-1 activity (1%) . Serum from rats immunized with the IFN-gamma/IL-1 alpha hybrid contained high levels of IL-1 alpha-binding and -neutralizing antibodies and IFN-gamma-binding antibodies, but no detectable levels of IFN-gamma-neutralizing antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Biol (Mosk), 1991 Mar-Apr, 25(2), 396 - 404
{Molecular biological study of the vaccinia virus genome . IV . The late nonstructural 36K protein of vaccinia virus is vitally important}; Shchelkunov SN et al.; Two genetics markers: the herpes simplex virus thymidine kinase and Escherichia coli beta-galactosidase genes were inserted into the 36K protein gene of vaccinia virus located in a HindIII-P DNA fragment . An unstability of recombinant viruses with Lac(+)-phenotype were discovered . A mechanism of viruses unstable variants formation was proposed, it was confirmed by the results of hybridisation analyses of virus recombinant genomes . The importance of a late nonstructural 36K protein gene for virus reproduction was demonstrated.

Metab Brain Dis, 1991 Mar, 6(1), 19 - 32
Aflatoxin B1 alters central and systemic tryptophan and tyrosine metabolism: influence of immunomodulatory drugs; Weekley LB; Semi-chronic exposure of ICR male Mice to Aflatoxin B1 (AFB1) in non-toxic doses results in elevated lung tryptophan (TRP) levels without change in serotonin (5-HT) or 5-hydroxyindole-3-acetic acid (5-HIAA) levels . This change is organ specific in that TRP levels are not altered in spleen, duodenum, heart or central nervous system (CNS) . Acute (48 hour) flunixin treatment decreases lung TRP levels and reverses the AFB1 mediated increase in lung TRP levels . On the other hand, flunixin treatment decreases CNS TRP levels in control mice but not in AFB1 treated mice . Aflatoxin B1 treated mice have an increase in splenic serotonin (5-HT) content . Acute (48 hour) treatment of mice with E . coli lipopolysaccharide (LPS) also increases splenic 5-HT, and AFB1 treatment followed by LPS have a slightly additive effect on spleen 5-HT content . Treatment of mice with LPS increases heart 5-HT, an effect which is not altered in AFB1 pretreated mice . Both LPS and AFB1 per se increases lung TYR levels although the combination of treatments is not significantly different from the control value . Flunixin treatment increases lung tyrosine (TYR) levels, an effect which is not altered by AFB1 pretreatment . Acute treatment with either LPS or flunixin decreases the CNS TRP/TYR ratio; pretreatment with AFB1 prevents those changes in the CNS TRP/TYR ratio . Central nervous system catecholamines are reduced in AFB1 pretreated mice . However, CNS catecholamine changes in AFB1 treated mice are normalized by vitamin E supplementation during the treatment period.

Biol Chem Hoppe Seyler, 1991 Mar, 372(3), 225 - 34
An approach to the functional analysis of lecithin-cholesterol acyltransferase . Activation by recombinant normal and mutagenized apolipoprotein AI; Bruhn H et al.; Apolipoprotein AI (apo AI) of human serum high-density lipoprotein functions as an activator of lecithin-cholesterol acyltransferase (LCAT) and therefore plays an important role in reversed cholesterol transport . The mechanism of the acyltransfer, the activating polypeptide domains of apo AI and the active site of LCAT in this transesterification are not yet known . Synthetic peptides of the apo AI sequence have been designed to determine the activating structure, but did not yet lead to conclusive results . This also applies to spontaneous apo AI mutants . We therefore used the method of site-directed mutagenesis of apo AI cDNAs using the overlap extension approach by the polymerase chain reaction . These constructs were cloned into the procaryotic vector pET8c and expressed under the inducible T7 promoter . The engineered apo AI polypeptides were isolated and purified by affinity chromatography and assayed for their activator activity . The essentials of this approach to the structure and function of activators in general have successfully been exemplified for the LCAT activation by engineering apo AI mutant polypeptides a) by the deletion of two adjacent amphipathic helices (amino acid residues 146-186) and b) by introducing a point mutation (Glu111----Gln).

J Photochem Photobiol B, 1991 Mar, 8(4), 371 - 83
Synergism between electricity and ionizing radiation; Capella MA et al.; Weak direct electric currents which produce little (or no) lethal damage to Escherichia coli bacteria are shown to act synergistically with ionizing radiation, both electromagnetic radiation (X-ray) and charged particles (beta radiation) . This synergism greatly enhances the lethal effect of ionizing radiation on bacteria . This is possibly due to increased single-strand breaks in DNA, as detected by the alkaline sucrose gradient method . It is also shown that in cells with thymidine-3H incorporated into their DNA and treated with electricity, the radioactivity is released from the acid-insoluble fraction to the acid-soluble fraction, so that the ratio of radioactivity in the soluble fraction to that in the insoluble fraction increases from 0.47 in the non-treated control cells to 3.46 in the cells treated with an electric current of 1.0 mA (3.0 V) for 30 min, which indicates extensive degradation of cellular DNA . No synergism is detected between electricity and 254 nm UV radiation nor between electricity and X-rays, when these two agents are used sequentially in any order . Electricity alone produces lesions in cell membranes, as shown by electron microscopy.

Circ Shock, 1991 Mar, 33(3), 142 - 55
Sequential renal alterations in septic shock in the primate; Voss BL et al.; This is a descriptive sequential study of the response of the baboon to LD100 Escherichia coli . The response was found to consist of three stages based on electron microscopic, physiologic, and clinical laboratory data . This study associates the inflammatory, coagulant, and cell injury (stage 1-3) responses with markers of activation of inflammatory cells (tumor necrosis factor) and of the vascular endothelium (tissue plasminogen activator) . This work also shows that in contrast to the underlying parenchymal cells of the organ, the vascular endothelium remains intact throughout the response to LD100 E . coli . The possible role of the vascular endothelium in mediation of events at both its luminal (blood) and antiluminal (parenchymal) surfaces is discussed.

Gene, 1991 Mar 1, 99(1), 121 - 6
Adsorption to starch of a beta-galactosidase fusion protein containing the starch-binding region of Aspergillus glucoamylase; Chen LJ et al.; We have constructed and purified by affinity chromatography three beta-galactosidase (beta Gal) fusion proteins (BSB133, BSBCD8, and BGA134) containing amino acid (aa) sequences from Aspergillus glucoamylase (GA) . BSB133, containing the C-terminal 133 aa of GA (aa 484-616), adhered to native starch granules with a much higher affinity (Kad = 18 ml/g starch) than a beta Gal control (Kad = 0.9 ml/g starch) . Two other fusion proteins, BSBCD8 and BGA134, similar in size to BSB133, adhered to starch with a relatively low affinity (Kad = 7 ml/g starch, and Kad = 4 ml/g starch, respectively) . BSBCD8 differs from BSB133 by a truncation of 8 aa at the C terminus . BGA134 contains 134 aa from an overlapping region of GA (aa 380-513) . These results confirm the presence of a strong starch-binding region (SBR) included in the C-terminal 133 aa of GA and indicate that the SBR can confer starch-binding activity on a fusion protein produced in Escherichia coli . In the presence of crude soluble cell extracts, the fusion proteins adsorbed by native starch granules with an affinity similar to that of the purified enzymes . BSB133 that had been adsorbed by starch from crude extracts could be eluted at a high level of purity, similar to that achieved by affinity chromatography . These results suggest that it may be feasible to use native starch as an adsorbent for the recovery and purification of recombinant fusion proteins containing the SBR . Starch has many favorable qualities for this application: it is inexpensive, stable, nontoxic, and easy to recover by centrifugation.

Cell Immunol, 1991 Mar, 133(1), 95 - 108
T cell activation by mycobacterial antigens in inflammatory synovitis; Pope RM et al.; To define which mycobacterial antigens were responsible for the activation of synovial fluid T lymphocytes, acetone-precipitated Mycobacterium tuberculosis (AP-MT) antigens were separated into five fractions following polyacrylamide gel electrophoresis and added to the mononuclear cell cultures of patients with inflammatory synovitis . Fractions 2 (50 to 70 kDa) and 5 (less than 28 kDa) resulted in significantly more proliferation than that of fractions 1, 3, and 4 . The response to a purified mycobacterial 65-kDa heat shock protein (hsp), which migrated in fraction 2, was highly correlated (r = 0.89, P less than 0.001) with the response to the crude AP-MT . The proliferative response to a different hsp . the Escherichia coli DnaK, by synovial fluid lymphocytes was marginal . Analysis of the synovial fluid T cell response to mycobacterial culture filtrates by T cell Western blotting revealed dominant responses to antigen(s) in the range of 31 to 21 kDa in each responding patient, although no other consistent pattern of T cell activation was noted . Three lines of evidence suggested that the response to the low molecular weight fractions was directed against degradation fragments of the 65-kDa protein . These observations suggest that the activation of T lymphocytes obtained from inflammatory synovial fluids by crude mycobacterial antigens was due in large part to recognition of the 65-kDa mycobacterial hsp.

Biochem Int, 1991 Mar, 23(5), 915 - 22
Substrate analogues and divalent cations as inhibitors of glutamate decarboxylase from Escherichia coli; Youngs TL et al.; To examine the idea that glutamate decarboxylase from E . coli can be a convenient source for the study of the effects of compounds on GABA synthesis in the nervous system, a series of substrate analogues and divalent cations were tested as potential inhibitors of the bacterial enzyme . Those analogues exhibiting inhibitor activity did so in a competitive manner . The most effective inhibitors were 3-mercaptopropionic acid, 4-bromoisophthalic acid and isophthalic acid which exhibited Ki values of 0.13 mM, 0.22 mM and 0.31 mM, respectively . Eight other analogues produced lesser degrees of inhibition . In addition, seven divalent metal cations were tested as inhibitors of the enzyme . However, only Hg2+, Cd2+, Cu2+ and Zn2+ were effective at a concentration of 0.1mM . When these results were compared to the patterns of inhibition of glutamate decarboxylase from mouse brain, certain differences in the manner in which the enzymes responded to the inhibitors, emerged . Consequently, the bacterial decarboxylase may not be a good model for the study of drug action on brain GABA synthesis.

Mol Biol (Mosk), 1991 Mar-Apr, 25(2), 358 - 67
{Affinity modification of DNA polymerase I from Escherichia coli and its Klenow fragment with nucleotide imidazolides}; Doronin SV et al.; Affinity modification of E . coli DNA polymerase I and its Klenow fragment by imidazolides of dNMP (Im-dNMP) and dNTP was studied . DNA polymerase activity of DNA polymerase I was reduced by both Im-dNMP and Im-dNTP . However Im-dNTP does not inactivate of the Klenow fragment . The level of covalent labelling of both enzymes by radioactive Im-dNTP did not exceed 0.01 mol of reagent per mol of enzyme . But the deep inactivation of DNA polymerase I by Im-dNTP was observed . It is likely that this inactivation is due to the formation of intramolecular ether followed by phosphorylation of the carboxyl group . This assumption is strongly supported by the increase of the isoelectrical point of DNA polymerase I after its incubation with Im-dNTP in conditions of enzyme inactivation . All data permit us to suggest that the affinity modification of both enzymes by Im-dNMP and covalent labeling by Im-dNTP takes place without complementary binding of dNTP moiety with the template . However inactivation of DNA polymerase I by Im-dNTP occurs only if the dNTP-moiety is complementary to the template in the template.primer complex . It was shown that His residue was phosphorylated by Im-dNMP and Tyr or Ser residues between Met-802 and Met-848 were phosphorylated by Im-dNTP . We suppose that there are two states of DNA polymerase active site for the binding of dNTPs . One of them is independent on the template, in the other state the dNTP hydrogen bond with the template is formed.

J Med Virol, 1991 Mar, 33(3), 181 - 7
Expression and characterization of the preS1 peptide of hepatitis B surface antigen in Escherichia coli; Lin Y et al.; The infectious particles of hepatitis B virus (HBV) contain 3 related surface antigens, i.e., small, medium, and large, all of which are encoded by one large open reading frame with multiple initiation codons . The large surface antigen (L-Ag) contains preS1, preS2, and S regions while both the middle and small surface antigens lack preS1 . Several lines of evidence suggested that the preS1 region is involved in the binding of HBV to human hepatocytes as shown by its binding to HepG2 cells and isolated human hepatocyte membranes . To obtain large quantity of preS1 peptide, an expression vector was constructed containing a lac promoter, the 5' half of the beta-galactosidase gene, the Factor Xa tetrapeptide recognition sequence, and the coding region of preS1 plus preS2 . This recombinant plasmid constitutively produced high concentration of a fusion protein in inclusion bodies in Escherichia coli . When the fusion protein was treated with Factor Xa, a peptide consisting of the N-terminal 91 amino acids of the preS1 region was released . This preS1 fragment purified by anion exchange chromatography was able to bind specifically to the isolated plasma membranes from human liver . Hence, this recombinant preS1 peptide can be used to identify and isolate hepatocyte receptors for HBV.

J Biochem (Tokyo), 1991 Mar, 109(3), 404 - 9
Renaturation, purification, and characterization of human truncated macrophage colony-stimulating factor expressed in Escherichia coli; Yamanishi K et al.; A human truncated macrophage colony-stimulating factor (M-CSF) encoding the amino acid residues from 3 to 153 of the native M-CSF was expressed by using a two-cistron expression system in Escherichia coli . The truncated M-CSF found in inclusion bodies was renatured and had CSF activity . Purification, which included a QAE-ZeTa preparative cartridge concentration step followed sequentially by HPLC on TSK-gel Phenyl-5PW and TSK-gel DEAE-5PW columns, gave an overall yield of 63.8% . The purified truncated M-CSF had a specific activity of 4 x 10(7) units/mg of protein . Peptide mapping of a lysylendopeptidase digest by reversed-phase HPLC confirmed the amino acid sequence predicted from the cDNA sequence . SDS-PAGE of the purified truncated M-CSF gave a single band at 17 kDa under reducing conditions and at 32 kDa under non-reducing conditions . Activated Thiol-Sepharose 6B column chromatography and other experiments failed to detect any free cysteine residue in spite of the existence of 7 cysteine residues in the truncated M-CSF subunit . These results indicate that it is a dimeric structure linked by one or more intermolecular disulfide bonds.

Development, 1991 Mar, 111(3), 647 - 55
Colonization of the post-umbilical bowel by cells derived from the sacral neural crest: direct tracing of cell migration using an intercalating probe and a replication-deficient retrovirus; Pomeranz HD et al.; Experiments were done to test the hypothesis that the avian gut is colonized by cells derived from both vagal and sacral regions of the neural crest . A fluorescent dye, diI (1,1-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), and a replication-deficient retrovirus (LZ10; Galileo et al . 1990) were employed as tracers . Since LZ10 was constructed with lacZ of E . coli as a reporter gene, infected cells were identified by demonstrating beta-galactosidase immunoreactivity . DiI and LZ10 were injected between the neural tube and surface ectoderm (before the migration of crest cells away from the injection sites) at vagal, truncal (diI only), or sacral axial levels . The bowel was examined 4 days later in order to allow crest-derived cells sufficient time to migrate to the gut . Following injections of either tracer into the vagal crest, labelled cells were found in the gizzard and duodenum . When diI or LZ10 was injected into the sacral crest, labelled cells were seen in the post-umbilical bowel and ganglion of Remak . In the hindgut, marked cells were concentrated in the mesenchyme, just internal to the serosa, and were never observed rostral to the umbilicus . No fluorescent cells were ever found in the bowel following truncal injections of diI, although such cells were observed in sympathetic ganglia . Labelled cells were always found in dorsal root ganglia, no matter which tracer or level of the crest was injected . In embryos injected with LZ10, infected cells in the gut and dorsal root ganglia displayed a neural crest marker (NC-1 immunoreactivity) . These observations confirm that the gut is colonized by cells from the sacral as well as the vagal region of the neural crest and that the emigres from the sacral crest are confined to the post-umbilical bowel.

Biopolymers, 1991 Mar, 31(4), 385 - 95
An estimate of the nearest neighbor base-pair content of 5S RNA using CD and absorption spectroscopy; Johnson KH et al.; We analyzed the CD and uv absorption spectra of 5S RNA from Escherichia coli using the method developed in the preceding paper . The analysis of spectra of 5S RNA at 20 degrees C in 0.1M NaClO4, 2.5 mM Na+ (phosphate), pH 7.0, and 0.5 mM MgSO4 gave 7 +/- 3.6 A.U base pairs, 25 +/- 3.6 G.C base pairs, and 7.5 +/- 3.6 G.U base pairs . Estimates of nearest neighbor base pairs were more consistent with the Pieler-Erdmann and the Gewirth-Moore secondary structure models than with the Fox-Woese or the Burns-Luoma-Marshall models . We also examined the structure of 5S RNA as a function of temperature . The melting profile exhibited two transitions--one at about 35 degrees C and one above 50 degrees C . Our spectral data showed that helices I and II were stable during the first transition, and agreed with other data that helix III was the most likely helix to have melted . The results from this in-depth study of 5S RNA indicate that our method of analysis should be useful for studying the secondary structures of other small, unmodified RNAs.

Mol Gen Mikrobiol Virusol, 1991 Mar, (3), 24 - 30
{Marburg virus: the first determined nucleotide sequence of two genes}; Bukreev AA et al.; The preparations of purified Marburg virus were isolated from blood plasma of infected guinea pigs and characterized . Viral RNA was extracted from the virions . The cDNA was synthesized on the isolated RNA matrix by the reverse transcriptase with the use of dissipated priming . The obtained cDNA was inserted into the plasmid pBR322 by the connector technique and the resulting recombinant plasmids were cloned in Escherichia coli cells . The specific clones selected by molecular hybridization method were analyzed by the restriction mapping and cross-hybridization . Four overlapping cDNA clones were found and the virus specific 5012 bp fragment of the viral genome was sequenced . Three open reading frames were found and the preliminary analysis of the coded amino acid sequence and corresponding genes was fulfilled.

Cardiovasc Intervent Radiol, 1991 Mar-Apr, 14(2), 106 - 8
Percutaneous drainage of postappendectomy abscesses complicated by enteric communication; Peer A et al.; Four patients with postappendectomy abscesses complicated by enteric fistulae were treated by percutaneous drainage . Sinograms, obtained at the time of the initial drainage, demonstrated communication to the cecum in 3 patients and to the small bowel in 1 patient . Complete cure was attained in 3 patients by percutaneous drainage . In the fourth patient, surgery was performed after 7 days of catheter drainage . Percutaneous drainage of abscesses with enteric communication requires a modified technique, which includes longer-term drainage than for simple noncommunicating abscesses.

Gene, 1991 Mar 1, 99(1), 25 - 9
The association of thymidine kinase activity and thymidine transport in Escherichia coli; Dube DK et al.; We have constructed a series of mutants within the putative nucleoside-binding site of the herpes simplex type-1 virus (HSV-1) thymidine kinase (TK)-encoding gene (tk), contained within an expression vector . While most mutations within this sequence produce an inactive protein, we find no absolute requirement for the wild-type Ile166 and Ala167 . The uptake of thymidine (dT) into Escherichia coli tdk-, lacking functional endogenous TK activity, is proportional to the amount of TK activity expressed from the heterologous HSV-1 tk gene . In contrast, there is no enhancement in deoxycytidine uptake into E . coli producing (HSV-1) TK . These results imply a specific role for TK in the active transport of dT into E . coli.

Mol Gen Genet, 1991 Mar, 225(3), 387 - 94
P element excision in Drosophila melanogaster and related drosophilids; O'Brochta DA et al.; The frequency of P element excision and the structure of the resulting excision products were determined in three drosophilid species . Drosophila melanogaster, D . virilis, and Chymomyza procnemis . A transient P element mobility assay was conducted in the cells of developing insect embryos, but unlike previous assays, this mobility assay permitted the recovery of excision products from plasmids regardless of whether the excision event was precise or imprecise . Both quantitative and qualitative differences between the products of excision in the various species studied were observed . The frequency with which P element excision products were recovered from D . melanogaster was 10-fold greater than from D . virilis and C . procnemis; however, the proportion of all excision events resulting in the reversion of a P-induced mutant phenotype was the same . Virtually all excision products recovered, including those resulting in a reversion of the mutant phenotype, did not result in the exact restoration of the original target sequence . Sequence analysis suggested that duplex cleavage at the 3' and 5' termini of the P element, or their subsequent modification, occurred asymmetrically and interdependently . P element-encoded transposase was not absolutely required for P element excision.

Somat Cell Mol Genet, 1991 Mar, 17(2), 185 - 90
Gene transfer into rat airway epithelial cells using retroviral vectors; Stanley C et al.; Primary cultures of epithelial cells from adult rat tracheas were maintained in vitro on collagen matrices and were exposed to a murine retrovirus vector expressing the E . coli beta-galactosidase gene . Infection was carried out on cells grown as monolayers under medium and on cells grown on raised platforms . Cells maintained at an air-medium interface were highly susceptible to infection with the vector, showing an efficiency of infection of 20-25%, compared with an efficiency of less than 1% for cells grown under medium . Infected beta-galactosidase-expressing cells were seeded into denuded tracheas and were capable of partially repopulating the denuded tracheas grafted subcutaneously into host rats . The susceptibility of these cells to retroviral infection suggests an approach to the treatment of some pulmonary genetic disorders such as cystic fibrosis.

J Nucl Med, 1991 Mar, 32(3), 453 - 60
Endotoxin reduces specific pulmonary uptake of radiolabeled monoclonal antibody to angiotensin-converting enzyme; Muzykantov VR et al.; The biodistribution of radiolabeled monoclonal antibody (Mab) to angiotensin-converting enzyme (ACE) was examined in normal and endotoxin-treated rats . Endotoxin administration at a dose of 4 mg/kg induced mild or middle pulmonary edema . The ACE activity in lung homogenate remained virtually unchanged, while the activity of serum ACE increased 15 hr after endotoxin infusion . In normal rats, anti-ACE Mab accumulates specifically in the lung after i.v . injection . Endotoxin injection induces reduction of specific pulmonary uptake of this antibody . Even in non-edematous endotoxemia, the accumulation of anti-ACE Mab antibody (Mab 9B9) decreased from 19.02 to 11.91% of ID/g of tissue without any change in accumulation of control nonspecific IgG . The antibody distribution in other organs and its blood level were almost the same as in the control . In a case of endotoxemia accompanied by increased microvascular permeability, the lung accumulation of Mab 9B9 was reduced to 9.17% of ID/g of tissue, while the accumulation of nonspecific IgG increased to 1.44% versus 0.89% in the control.

J Gen Virol, 1991 Mar, 72 ( Pt 3), 731 - 4
Human papillomavirus type 6 and 11 E4 gene products in condyloma acuminata; Tomita Y et al.; The human papillomavirus type 6 (HPV-6) E4 gene was expressed in Escherichia coli as a fusion protein with E . coli beta-galactosidase (E4-beta-Gal), and rabbit antibody against the E4-beta-Gal was prepared . By Western blotting with this antibody, we detected E4 gene products in six out of 18 condyloma acuminata specimens . In four specimens (C-1, C-13, C-14 and C-19), the E4 protein was found as a 10K/11K doublet, but in other specimens (C-8 and C-23), only the 11K protein was detected . By Southern blot analysis, it was found that C-13 harboured HPV-6 DNA but that C-1 and C-8 harboured HPV-11 DNA, indicating that the E4 proteins of HPV-6 and -11 have cross-reactive antigenicity . After incubation at 37 degrees C of the C-23 tissue specimen, the 10K protein was clearly detected . These results suggest that the 10K protein may be derived from the 11K protein by a modification such as proteolytic cleavage before and/or after specimens were taken.

J Gen Virol, 1991 Mar, 72 ( Pt 3), 677 - 85
Sequence of the major nucleocapsid protein gene of pneumonia virus of mice: sequence comparisons suggest structural homology between nucleocapsid proteins of pneumoviruses, paramyxoviruses, rhabdoviruses and filoviruses; Barr J et al.; The complete nucleotide sequence of gene 3 of pneumonia virus of mice has been determined, and the 5' end of the mRNA mapped using a modification of the polymerase chain reaction technique . The gene contains a single open reading frame, beginning with a 5'-proximal AUG initiation codon, encoding a polypeptide with a predicted Mr of 43141 . Expression of the gene 3 protein in Escherichia coli and in vitro showed that it reacted with virus-specific antiserum and comigrated with the major nucleocapsid (N) polypeptide . The predicted amino acid sequence has extensive identity with that of the N protein of human respiratory syncytial virus . Comparisons with the amino acid sequences of N proteins of other paramyxoviruses, vesicular stomatitis virus and Ebola virus suggest that these proteins may have retained much of the same structure . These regions of conserved structure would most likely have the common functions of RNA binding and protein/protein interactions in the virus nucleocapsid.

EMBO J, 1991 Mar, 10(3), 633 - 9
The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen; Rihs HP et al.; We have previously demonstrated {Rihs, H.-P . and Peters, R . (1989) EMBO J., 8, 1479-1484} that the nuclear transport of recombinant proteins in which short fragments of the SV40 T-antigen are fused to the amino terminus of Escherichia coli beta-galactosidase is dependent on both the nuclear localization sequence (NLS, T-antigen residues 126-132) and a phosphorylation-site-containing sequence (T-antigen residues 111-125) . While the NLS determines the specificity, the rate of transport is controlled by the phosphorylation-site-containing sequence . The present study furthers this observation and examines the role of the various phosphorylation sites . Purified, fluorescently labeled recombinant proteins were injected into the cytoplasm of Vero or hepatoma (HTC) cells and the kinetics of nuclear transport measured by laser microfluorimetry . By replacing serine and threonine residues known to be phosphorylated in vivo, we identified the casein kinase II (CK-II) site S111/S112 to be the determining factor in the enhancement of the transport . Either of the residues 111 or 112 was sufficient to elicit the maximum transport enhancement . The other phosphorylation sites (S120, S123, T124) had no influence on the transport rate . Examination of the literature suggested that many proteins harboring a nuclear localization sequence also contain putative CK-II sites at a distance of approximately 10-30 amino acid residues from the NLS . CK-II has been previously implicated in the transmission of growth signals to the nucleus . Our results suggest that CK-II may exert this role by controlling the rate of nuclear protein transport.

J Bacteriol, 1991 Mar, 173(5), 1801 - 6
Isocitrate dehydrogenase kinase/phosphatase: aceK alleles that express kinase but not phosphatase activity; Ikeda T et al.; For Escherichia coli, growth on acetate requires the induction of the enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase . The branch point between the glyoxylate bypass and the Krebs cycle is controlled by phosphorylation of isocitrate dehydrogenase (IDH), inhibiting that enzyme's activity and thus forcing isocitrate through the bypass . This phosphorylation cycle is catalyzed by a bifunctional enzyme, IDH kinase/phosphatase, which is encoded by aceK . We have employed random mutagenesis to isolate novel alleles of aceK . These alleles were detected by the loss of ability to complement an aceK null mutation . The products of one class of these alleles retain IDH kinase activity but have suffered reductions in IDH phosphatase activity by factors of 200 to 400 . Selective loss of the phosphatase activity also appears to have occurred in vivo, since cells expressing these alleles exhibit phenotypes which are reminiscent of strains lacking IDH; these strains are auxotrophic for glutamate . Assays of cell-free extracts confirmed that this phenotype resulted from nearly quantitative phosphorylation of IDH . The availability of these novel alleles of aceK allowed us to assess the significance of the precise control which is a characteristic of the IDH phosphorylation cycle in vivo . The fractional phosphorylation of IDH was varied by controlled expression of one of the mutant alleles, aceK3, in a wild-type strain . Reduction of IDH activity to 50% of the wild-type level did not adversely affect growth on acetate . However, further reductions inhibited growth, and growth arrest occurred when the IDH activity fell to 15% of the wild-type level . Thus, although wild-type cells maintain a precise effective IDH activity during growth on acetate, this precision is not critical.

J Virol, 1991 Mar, 65(3), 1584 - 8
Isolation of a Marek's disease virus (MDV) recombinant containing the lacZ gene of Escherichia coli stably inserted within the MDV US2 gene; Cantello JL et al.; We have isolated a stable, recombinant Marek's disease virus (MDV) containing the lacZ gene of Escherichia coli inserted into the unique short region of the genome . The nucleotide sequence of the insertion site indicates that it lies within a sequence homologous to the US2 gene of herpes simplex virus . Stable insertion of the lacZ gene into the MDV US2 gene indicates that the site is nonessential for MDV growth in cell culture.

J Virol, 1991 Mar, 65(3), 1420 - 6
Isolation of cDNAs for DNA-binding proteins which specifically bind to a tax-responsive enhancer element in the long terminal repeat of human T-cell leukemia virus type I; Tsujimoto A et al.; One of the gene products of human T-cell leukemia virus type I (HTLV-I), p40tax, activates its own viral transcription in trans through tax-responsive enhancers in viral long terminal repeats . Five species of cDNA clones for proteins that bind to the tax-responsive enhancer element in HTLV-I were isolated from the Jurkat cell library . The beta-galactosidase fusion protein prepared from the lysogen of a clone specifically recognized the cyclic AMP-responsive element in HTLV-I enhancer . The nucleotide sequence of a full-length cDNA clone (TAXREB67) had a coding capacity of 351 amino acids, which contained a basic motif followed by a leucine zipper structure near the carboxy terminus . Its mRNA was detected in human cell lines, including HTLV-I-infected or noninfected hematopoietic cell lines . The mRNA level in Jurkat cells was decreased temporarily by increasing cyclic AMP concentration but increased by increasing Ca2+ concentration . Polyclonal antibodies against the fusion protein specifically recognized a 52-kDa protein in Jurkat cells . Analyses of the function of this protein and its interactions with other cellular factors will be useful to help understand the regulatory mechanism through tax-responsive enhancers in HTLV-I.

J Virol, 1991 Mar, 65(3), 1168 - 76
Characterization of the DNA-binding properties of the polyomavirus capsid protein VP1; Moreland RB et al.; The major capsid protein of polyomavirus, VP1, has been expression cloned in Escherichia coli, and the recombinant VP1 protein has been purified to near homogeneity (A . D . Leavitt, T . M . Roberts, and R . L . Garcea, J . Biol . Chem . 260:12803-12809, 1985) . With this recombinant protein, a nitrocellulose filter transfer assay was developed for detecting DNA binding to VP1 (Southwestern assay) . In optimizing conditions for this assay, dithiothreitol was found to inhibit DNA binding significantly . With recombinant VP1 proteins deleted at the carboxy and amino termini, a region of the protein affecting DNA binding was identified within the first 7 amino acids (MAPKRKS) of the VP1 amino terminus . Southwestern analysis of virion proteins separated by two-dimensional gel electrophoresis demonstrated equivalent DNA binding among the different VP1 isoelectric focusing subspecies, suggesting that VP1 phosphorylation does not modulate this function . By means of partial proteolysis of purified recombinant VP1 capsomeres for assessing structural features of the protein domain affecting DNA binding, a trypsin-sensitive site at lysine 28 was found to eliminate VP1 binding to DNA . The binding constant of recombinant VP1 to polyomavirus DNA was determined by an immunoprecipitation assay (R . D . G . McKay, J . Mol . Biol . 145:471-488, 1981) to be 1 x 10(-11) to 2 x 10(-11) M, which was not significantly different from its affinity for plasmid DNA . McKay analysis of deleted VP1 proteins and VP1-beta-galactosidase fusion proteins indicated that the amino terminus was both necessary and sufficient for DNA binding . As shown by electron microscopy, DNA inhibited in vitro capsomere self-assembly into capsidlike structures (D . M . Salunke, D . L . D . Caspar, and R . L . Garcea, Cell 46:895-904, 1986) . Thus, VP1 is a high-affinity, non-sequence-specific DNA-binding protein with the binding function localized near its trypsin-accessible amino terminus . The inhibitory effects of disulfide reagents on DNA binding and of DNA on capsid assembly suggest possible intermediate steps in virion assembly.

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