J Bacteriol, 1992 Aug, 174(16), 5424 - 9 Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding; Rinken R et al.; Infection of Escherichia coli with phage T4 gene 2am was used to transport 3H-labeled linear duplex DNA into cells to follow its degradation in relation to the cellular genotype . In wild-type cells, 49% of the DNA was made acid soluble within 60 min; in recB or recC cells, only about 5% of the DNA was made acid soluble . Remarkably, in recD cells about 25% of the DNA was rendered acid soluble . The DNA degradation in recD cells depended on intact recB and recC genes . The degradation in recD cells was largely decreased by mutations in recJ (which eliminates the 5' single-strand-specific exonuclease coded by this gene) or xonA (which abolishes the 3' single-strand-specific exonuclease I) . In a recD recJ xonA triple mutant, the degradation of linear duplex DNA was roughly at the level of a recB mutant . Results similar to those with the set of recD strains were also obtained with a recC++ mutant (in which the RecD protein is intact but does not function) and its recJ, xonA, and recJ xonA derivatives . The observations provide evidence for a recBC-dependent DNA-unwinding activity that renders unwound DNA susceptible to exonucleolytic degradation . It is proposed that the DNA-unwinding activity causes the efficient recombination, DNA repair, and SOS induction (after application of nalidixic acid) in recD mutants . The RecBC helicase indirectly detected here may have a central function in Chi-dependent recombination and in the recombinational repair of double-strand breaks by the RecBCD pathway. Virology, 1992 Aug, 189(2), 803 - 7 Selective inhibition of the 3'-to-5' exonuclease activity associated with Epstein-Barr virus DNA polymerase by ribonucleoside 5'-monophosphates; Tsurumi T; Epstein-Barr virus (EBV) DNA polymerase possesses a proofreading 3'-to-5' exonuclease activity (Tsurumi, T . (1991) Virology 182, 376-381) . The 3'-to-5' exonuclease activity can be selectively inhibited by ribonucleoside 5'-monophosphates, while no inhibition of the DNA polymerase activity can be observed even when the template/primer concentrations are rate-limiting . Deoxynucleoside monophosphates except 5'dGMP have almost no effect on the exonuclease activity . Of the four ribonucleoside monophosphates, 5'GMP is the most potent (62% inhibition at 5 mM) . The kinetic study shows that 5'-GMP inhibits the exonuclease activity competitively with respect to DNA template/primer . During DNA polymerization process the EBV DNA polymerase catalyzes the DNA-dependent conversion of complementary deoxynucleoside triphosphate to monophosphate form . With poly(dT).oligo(rA) as a template primer, selective inhibition of the exonuclease activity by 5'-GMP results in a decrease in the amount of free dAMP generated which is complementary to the template DNA, suggesting the functional relationship between the editing exonuclease activity and the chain elongation activity of the EBV DNA polymerase molecule. Virology, 1992 Aug, 189(2), 629 - 39 An in vivo study of a glycoprotein gIII-negative bovine herpesvirus 1 (BHV-1) mutant expressing beta-galactosidase: evaluation of the role of gIII in virus infectivity and its use as a vector for mucosal immunization; Liang X et al.; We constructed a recombinant BHV-1 in which the glycoprotein gIII gene was replaced by the Escherichia coli lacZ gene . The resultant virus mimics the simple gIII deletion mutant in its growth characteristics in cell culture; however, it expresses beta-galactosidase in virus-infected cells . Further characterization of its virulence and the immune responses elicited by it was conducted in cattle . The mutant virus retained the ability to establish an infection when administered intranasally . Infected animals were also capable of transmitting virus to sentinel penmates . However, the mutant virus showed a reduced replication efficiency in the respiratory tract of cattle, as manifested by significantly lower virus shedding and a shorter duration of shedding when compared to wild-type (wt) BHV-1 infections . The mutant virus induced an efficient anti-BHV-1 antibody response and convalescent cattle were fully protected from subsequent wt virus challenge . In addition, cattle infected with the lacZ-expressing virus developed antibodies to beta-galactosidase . Our results demonstrate that the presence of gIII is not a prerequisite for BHV-1 infection; however, gIII does play an important role in maintaining virus replication efficacy in its natural host . With respect to developing BHV-1 as a vaccine vector, our results indicate that deletion of the gIII gene, which partially attenuates the virus and serves as a vaccine virus marker, does not compromise immunogenicity to BHV-1 . Most importantly, this vector is effective in delivering foreign antigens to mucosal surfaces of the respiratory tract. Virology, 1992 Aug, 189(2), 568 - 82 Temperature-sensitive polioviruses containing mutations in RNA polymerase; Burns CC et al.; Site-directed mutagenesis was performed to change the wild-type residue (asparagine) to aspartate, histidine, or tyrosine at amino acid 424 of the poliovirus RNA polymerase, 3Dpol . The mutations were introduced into plasmids containing full-length viral cDNA and plasmids which direct the expression of 3Dpol in Escherichia coli . Mutant viruses, recovered after transfection of HeLa cells with RNA transcripts of the full-length clones, produced small plaques at 32 degrees . In addition, the plaquing efficiency was decreased for all three mutants at 37 degrees, compared to 32 degrees . The polyprotein processing of all mutant viruses was normal at the temperatures tested, suggesting that the mutant plaque phenotypes were not due to incorrect processing of viral proteins . Analyses of viral RNA synthesis in infected cells and of the polymerase activities of mutant enzymes produced in E . coli suggested the following: (1) The his424 mutant enzyme appeared to be defective in the initiation of plus-strand RNA synthesis in HeLa cells . (2) The asp424 mutant enzyme appeared unable to assume proper conformation for active polymerase function when synthesized at 37 degrees . (3) The tyr424 mutant enzyme was totally inactive when synthesized in E . coli at 37 degrees. Exp Cell Res, 1992 Aug, 201(2), 462 - 9 Ultraviolet mutagenesis in human lymphocytes: the effect of cellular transformation; Parris CN et al.; We have assessed the role of cellular transformation in ultraviolet (uv)-induced mutagenic events in human cells . To maintain uniformity of genetic background and to eliminate the effect of DNA repair, primary nontransformed lymphocytes (T-cells) and Epstein-Barr virus-transformed lymphocytes (B-cells) from one patient (XP12Be) with the DNA repair-deficient disorder xeroderma pigmentosum (group A) were transfected with the mutagenesis shuttle vector pZ189 . Parallel control experiments were performed with primary, nontransformed lymphocytes from a normal individual and with a repair-proficient Epstein-Barr virus-transformed lymphocyte line (KR6058) . pZ189 was treated with uv and introduced into the four cell lines by electroporation . Plasmid survival and mutations inactivating the marker supF suppressor tRNA gene in the recovered pZ189 were scored by transforming an indicator strain of Escherichia coli . Plasmid survival was reduced and mutation frequency elevated equally with both XP-A cell lines compared to both normal cell lines . Base sequence analysis of more than 250 independent plasmids showed that while the G:C----A:T base substitution mutation was found in at least 60% of plasmids with single or tandem mutations with all four cell lines, the frequency with the transformed XP-A (93%) cells was significantly higher (P less than 0.01) than that with the nontransformed XP-A cells (77%) . In addition, with the transformed XP-A cells, there were significantly fewer plasmids with transversions and with mutations at a transversion hotspot (base pair 134) than with plasmids recovered from nontransformed XP-A cells . Interleukin-2 and phytohemagglutinin (used to maintain growth of the nontransformed lymphocytes) treatment of transformed XP12Be cells did not change overall plasmid survival or mutation frequency, but increased the transversion frequency and induced a mutational hotspot (at base pair 159), while another mutational hotspot (at base pair 123) disappeared . Thus we have demonstrated that in repair-deficient human cells, cellular transformation, while not affecting overall postuv plasmid survival and mutation frequency, does increase the susceptibility to G:C----A:T transition mutations, a type of mutation associated with uv-induced neoplasia. J Virol, 1992 Aug, 66(8), 4884 - 92 The UL8 subunit of the herpes simplex virus helicase-primase complex is required for efficient primer utilization; Sherman G et al.; The herpes simplex virus (HSV) type 1 helicase-primase is a three-protein complex, consisting of a 1:1:1 association of UL5, UL8, and UL52 gene products (J.J . Crute, T . Tsurumi, L . Zhu, S . K . Weller, P . D . Olivo, M . D . Challberg, E . S . Mocarski, and I . R . Lehman, Proc . Natl . Acad . Sci . USA 86:2186-2189, 1989) . We have purified this complex, as well as a subcomplex consisting of UL5 and UL52 proteins, from insect cells infected with baculovirus recombinants expressing the appropriate gene products . In confirmation of previous reports, we find that whereas UL5 alone has greatly reduced DNA-dependent ATPase activity, the UL5/UL52 subcomplex retains the activities characteristic of the heterotrimer: DNA-dependent ATPase activity, DNA helicase activity, and the ability to prime DNA synthesis on a poly(dT) template . We also found that the primers made by the subcomplex are equal in length to those synthesized by the UL5/UL8/UL52 complex . In an effort to uncover a role for UL8 in HSV DNA replication, we have developed a model system for lagging-strand synthesis in which the primase activity of the helicase-primase complex is coupled to the activity of the HSV DNA polymerase on ICP8-coated single-stranded M13 DNA . Using this assay, we found that the UL8 subunit of the helicase-primase is critical for the efficient utilization of primers; in the absence of UL8, we detected essentially no elongation of primers despite the fact that the rate of primer synthesis on the same template is undiminished . Reconstitution of lagging-strand synthesis in the presence of UL5/UL52 was achieved by the addition of partially purified UL8 . Essentially identical results were obtained when Escherichia coli DNA polymerase I was substituted for the HSV polymerase/UL42 complex . On the basis of these findings, we propose that UL8 acts to increase the efficiency of primer utilization by stabilizing the association between nascent oligoribonucleotide primers and template DNA. J Virol, 1992 Aug, 66(8), 4803 - 13 Transcriptional synergy by the Epstein-Barr virus transactivator ZEBRA; Carey M et al.; ZEBRA is an Epstein-Barr virus (EBV) transcriptional activator that mediates a genetic switch between the latent and lytic states of the virus by binding to the promoters of genes involved in lytic DNA replication and activating their transcription . A computer survey revealed that 9 of 23 potential or known ZEBRA-responsive EBV genes contained two or more upstream binding sites; this suggested that ZEBRA can stimulate transcription synergistically . By using a series of synthetic promoters bearing one, two, three, five, and seven upstream recognition sites, we showed that ZEBRA activates transcription synergistically when templates bearing multiple sites were compared with a template bearing a single site . This phenomenon was observed in both uninfected and EBV-infected B-lymphoid cells and in vitro in a HeLa cell nuclear extract . DNase I footprinting was used to show that the synergy was not due to cooperative DNA binding mediated by direct contact between ZEBRA dimers . The in vitro experiments revealed two manifestations of synergy . One was seen when the levels of transcription observed with the same amounts of ZEBRA added to templates bearing different numbers of sites were compared . The other was observed when the two lowest concentrations of ZEBRA that stimulated measurable transcription from any given template were compared . On the basis of both the number of sites and the calculated Kd of ZEBRA for a single site, we estimated that the critical concentration of ZEBRA needed to elicit transcriptional synergy corresponds to a site occupancy of two or three bound ZEBRA dimers . Our results have biologic implications for both the EBV lytic cycle and other processes in which the concentration of an activator changes either temporally or spatially. J Bacteriol, 1992 Aug, 174(15), 5168 - 70 Cloning and sequencing of Escherichia coli mutR shows its identity to topB, encoding topoisomerase III; Schofield MA et al.; We have cloned and sequenced the mutR gene from Escherichia coli, which results in an increased frequency of spontaneous deletions, by using a strain carrying a Tn10 derivative inserted into mutR . The analysis of 1,286 bp of mutR sequence shows that this gene is identical to the topB gene, which encodes topoisomerase III . The increased deletion formation is the first reported phenotype for cells lacking topoisomerase III, and this suggests that topoisomerase III is involved in reactions that normally reduce the levels of spontaneous deletions. J Bacteriol, 1992 Aug, 174(15), 5079 - 85 IncN plasmid pKM101 and IncI1 plasmid ColIb-P9 encode homologous antirestriction proteins in their leading regions; Belogurov AA et al.; The IncN plasmid pKM101 (a derivative of R46), like the IncI1 plasmid ColIb-P9, carries a gene (ardA, for alleviation of restriction of DNA) encoding an antirestriction function . ardA was located about 4 kb from the origin of transfer, in the region transferred early during bacterial conjugation . The nucleotide sequence of ardA was determined, and an appropriate polypeptide with the predicted molecular weight of about 19,500 was identified in maxicells of Escherichia coli . Comparison of the deduced amino acid sequences of the antirestriction proteins of the unrelated plasmids pKM101 and ColIb (ArdA and Ard, respectively) revealed that these proteins have about 60% identity . Like ColIb Ard, pKM101 ArdA specifically inhibits both the restriction and modification activities of five type I systems of E . coli tested and does not influence type III (EcoP1) restriction or the 5-methylcytosine-specific restriction systems McrA and McrB . However, in contrast to ColIb Ard, pKM101 ArdA is effective against the type II enzyme EcoRI . The Ard proteins are believed to overcome the host restriction barrier during bacterial conjugation . We have also identified two other genes of pKM101, ardR and ardK, which seem to control ardA activity and ardA-mediated lethality, respectively . Our findings suggest that ardR may serve as a genetic switch that determines whether the ardA-encoded antirestriction function is induced during mating. J Bacteriol, 1992 Aug, 174(15), 4907 - 12 Mutations in a central highly conserved non-DNA-binding region of OmpR, an Escherichia coli transcriptional activator, influence its DNA-binding ability; Brissette RE et al.; OmpR is a transcriptional activator for the expression of outer membrane porin genes ompF and ompC in Escherichia coli . Its C-terminal half has been identified as the DNA-binding domain (K . Tsung, R . Brissette, and M . Inouye, J . Biol . Chem . 264:10104-10109, 1989) . Recent studies have indicated that the N-terminal non-DNA-binding domain of OmpR is involved in modulating OmpR function through interaction with the EnvZ protein, a kinase and phosphatase for OmpR . We isolated and characterized two mutations, G94D and E111K, in the N-terminal domain of OmpR and one mutation, R182C, in the DNA-binding domain of OmpR . All three mutations abolished the ability of OmpR to bind to the ompF and ompC promoters in vivo, thus giving an OmpF- OmpC- phenotype . The decreased DNA-binding ability of the mutant OmpRs was not due to diminished phosphorylation of their N termini, since all the mutant OmpRs were found to be normally phosphorylated by EnvZ in vitro . The mutant OmpRs produced from multicopy plasmids were also found to inhibit completely the production of OmpF and OmpC in wild-type cells, and the complete inhibition depended on the function of EnvZ which was produced in cis or in trans from plasmids . The relationship of the possible alterations in OmpR by the mutations with the observed diminished binding ability is discussed. Protein Sci, 1992 Aug, 1(8), 1007 - 13 Mapping of the catalytic site of CHO-t-PA and the t-PA variant BM 06.022 by synthetic inhibitors and substrates; Sturzebecher J et al.; BM 06.022 is a t-PA deletion variant that is produced as inactive inclusion bodies in Escherichia coli and transformed into the native form by an in vitro refolding process . Until now, no X-ray and NMR structures of BM 06.022 were available . Therefore a detailed kinetic analysis of the hydrolysis of peptide substrates and of the inhibition by several benzamidine-derived inhibitors was carried out in order to assess that the active site region of the protease domain of BM 06.022 is correctly structured in comparison with t-PA . Our data reveal that the single-chain as well as the two-chain form of BM 06.022 and native t-PA are similar in catalytic and in inhibitor binding properties . This indicates that the active site and the highly complex rearrangement of t-PA upon cleavage of the Arg275-Ile276 bond are maintained in BM 06.022. Nat Genet, 1992 Aug, 1(5), 372 - 8 Adenovirus-mediated in vivo gene transfer and expression in normal rat liver; Jaffe HA et al.; Replication deficient, recombinant adenovirus (Ad) vectors do not require target cell replication for transfer and expression of exogenous genes and thus may be useful for in vivo gene therapy in hepatocytes . In vitro, primary cultures of rat hepatocytes infected with a recombinant Ad containing a human alpha 1-antitrypsin cDNA (Ad-alpha 1AT) synthesized and secreted human alpha 1AT for 4 weeks . In rats, in vivo intraportal administration of a recombinant Ad containing the E . coli lacZ gene, was followed by expression of beta-galactosidase in hepatocytes 3 days after infection . Intraportal infusion of Ad-alpha 1AT produced detectable serum levels of human alpha 1AT for 4 weeks . Thus, targeted gene expression has been achieved in the liver, albeit at low levels, suggesting that adenovirus vectors may be a useful means for in vivo gene therapy in liver disorders. Cell Biophys, 1992 Aug-Dec, 21(1-3), 1 - 12 Expression of a Chlamydia anticarbohydrate single-chain antibody as a maltose binding fusion protein; Malinowski DP et al.; A single-chain antibody fragment has been constructed for an antibody that binds to the Chlamydia specific carbohydrate structure of the lipopolysaccharide . Single-chain protein was expressed and secreted into the periplasmic space of E . coli as a fusion protein with the maltose binding protein . The fusion protein was purified in one step by virtue of its ability to bind to maltose . In a sandwich ELISA, the eluted protein bound Chlamydia lipopolysaccharide, which demonstrates that the single-chain protein domain will function as part of a fusion protein . The expression of maltose binding fusion proteins into the periplasmic space could be used for production of other single-chain antibodies or protein fragments requiring appropriate folding and disulfide bond formation. Acta Virol, 1992 Aug, 36(4), 337 - 46 Bacterially expressed core and envelope proteins of human immunodeficiency virus type-1 (HIV-1): comparative evaluation in detection of type-specific antibodies; Kovac J et al.; Recombinant proteins derived from immunodominant conserved domains of HIV-1 env and gag genes were synthesized in E . coli . An immunoblot system using total cell lysates was employed for the analysis of recombinant bacterial clones . Together 427 serum samples obtained from asymptomatic anti-HIV seropositive individuals, AIDS patients, healthy donors and persons suffering from various conditions were comparatively evaluated for the presence of HIV-1 antibodies using recombinant peptides and commercially available western blot (WB) and ELISA assays . The recombinant antigen product of plasmid pEX41 was found to be superior, with respect to sensitivity and specificity, to the viral gp41 which represents a diagnostically important constituent of the WB. Biochim Biophys Acta, 1992 Jul 31, 1122(2), 212 - 8 Identification of the histidine residue in Escherichia coli isocitrate lyase that reacts with diethylpyrocarbonate; Rua J et al.; Escherichia coli isocitrate lyase was inactivated by diethylpyrocarbonate in a pseudo-first-order process . The enzyme was completely inactivated by modification of a single histidine residue, but slower modification of further residues also occurred . The substrate, isocitrate, and products, glyoxylate and succinate, protected against inactivation by diethylpyrocarbonate but this was not simply due to binding at the active site . Treatment of the inactivated enzyme with hydroxylamine led to only partial recovery of activity . Diethylpyrocarbonate also reacted with sulphydryl groups in isocitrate lyase, as judged by titrations with Nbs2, but this reaction was not responsible for the failure of hydroxylamine to reactivate the enzyme fully . The reactivity of isocitrate lyase to diethylpyrocarbonate declined with pH, following a titration curve for a group of pKa 6.1 . Isolation and sequencing of ethoxyformylated peptides showed that the major site of modification by diethylpyrocarbonate was histidine residue 306. Biochem Biophys Res Commun, 1992 Jul 31, 186(2), 811 - 8 Signal peptide for peroxisomal targeting: replacement of an essential histidine residue by certain amino acids converts the amino-terminal presequence of peroxisomal 3-ketoacyl-CoA thiolase to a mitochondrial signal peptide; Osumi T et al.; The role of the histidine residue at position -17 of the amino-terminal signal peptide of rat peroxisomal 3-ketoacyl-CoA thiolase was studied in vivo, employing site-directed mutagenesis . Among the nine amino acids tested, only glutamine could partially substitute for the histidine . Mutants carrying basic amino acids, arginine and lysine, and hydrophobic residues, leucine and valine, in place of histidine were all translocated to mitochondria, but not to peroxisomes . These results indicate that the signal peptide of the thiolase is recognized by a mechanism totally different from that for the SKL motif, a known peroxisomal targeting signal . Relationship of the thiolase signal peptide to those of mitochondrial proteins is discussed. Biochem Biophys Res Commun, 1992 Jul 31, 186(2), 686 - 9 Identification of the iron-binding histidine residues in soybean lipoxygenase L-1; Steczko J et al.; Lipoxygenases constitute a class of non-heme, non-sulfur iron dioxygenases acting upon lipids possessing a 1,4-cis-cis-pentadiene moiety . The iron is known to be essential for activity . A motif of six histidine residues has been found in all of the thirteen lipoxygenases, from both plant and animal sources, whose sequences have been reported . We had previously obtained mutant proteins in which each of the 6 conserved histidines of soybean lipoxygenase L-1 had been replaced and found that the mutants H499Q, H504Q (or H504S) and H690Q had no detectable enzymatic activity . We have now found that these inactive proteins contain no Fe, although they have the same electrophoretic mobility as wild-type L-1 under both denaturing and non-denaturing conditions and react with anti-L-1 antibodies. Biochem Biophys Res Commun, 1992 Jul 31, 186(2), 1081 - 6 Phosphorylation of the recombinant spliced variants of the alpha-sub-unit of the stimulatory guanine-nucleotide binding regulatory protein (Gs) by the catalytic sub-unit of protein kinase A; Pyne NJ et al.; Both GS alpha-1 and GS alpha-4 were phosphorylated by the purified catalytic sub-unit of protein kinase A . Phosphate incorporation into 220 pmol and 190 pmol of GS alpha-4 and GS alpha-1 after a 1 hour incubation with kinase was 14 pmol and 10 pmol, respectively . These low levels of phosphorylation are due to the thermal lability of purified recombinant GS alpha . However, the phosphorylation was inhibited by guanine nucleotides (GDP-beta-S, GppNHp and GTP) and is, therefore, a specific event . We suggest that, as for GS alpha phosphorylation by protein kinase C (Pyne et al., 1992), the guanine nucleotide-free form of GS alpha is the most likely substrate . Guanine-nucleotides reduce the lifetime and, therefore availability for phosphorylation, of guanine-nucleotide free GS alpha . GS alpha phosphorylation by protein kinase A in vitro provides preliminary evidence that a similar phosphorylation of GS alpha may be an important regulatory event in cells. Biochemistry, 1992 Jul 28, 31(29), 6683 - 91 Unfolding of trp repressor studied using fluorescence spectroscopic techniques; Fernando T et al.; The unfolding properties of the trp repressor of Escherichia coli have been studied using a number of different time-resolved and steady-state fluorescence approaches . Denaturation by urea was monitored by the average fluorescence emission energy of the intrinsic tryptophan residues of the repressor . These data were consistent with a two-state transition from dimer to unfolded monomer with a free energy of unfolding of 19.2 kcal/mol . The frequency response profiles of the fluorescence emission brought to light subtle urea-induced modifications of the intrinsic tryptophan decay parameters both preceding and following the main unfolding transition . The increase of lifetime induced by urea required higher concentrations of urea than the increase in the total intensity described by Gittelman and Matthews {(1990) Biochemistry 29, 7011} . This indicates that the intensity increase has both dynamic and static origins . To assess the effect of tryptophan binding upon repressor stability, and to determine whether repressor oligomerization would be detectable in an unfolding experiment, we examined denaturation profiles of repressor labeled with the long-lived fluorescence probe 5-(dimethylamino)naphthalene-1-sulfonyl (DNS), by monitoring the average rotational correlation time of the probe . These experiments revealed a protein concentration dependent transition at low urea concentrations . This transition was promoted by tryptophan binding . We ascribe this transition to urea-induced dissociation of repressor tetramers . The main unfolding transition of the dimer to unfolded monomer was also observable using this technique, and the free energies associated with this transition were 18.3 kcal/mol in the absence of tryptophan and 24.1 kcal/mol in its presence, demonstrating that co-repressor binding stabilizes the repressor dimer against denaturation.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1992 Jul 28, 307(2), 185 - 9 Cloning, sequence analysis and expression in Escherichia coli of the cDNA encoding a precursor of peanut agglutinin; Rodriguez-Arango E et al.; The cDNA coding for pre-peanut agglutinin (PNA) was isolated from a bacterial expression library . It codes for a polypeptide of 273 amino acids composed of a hydrophobic signal peptide of 23 amino acids and a mature protein of 250 amino acids . The sequence of the latter is identical to that of native PNA, determined very recently by conventional methods, except that it contains 14 additional amino acids at the C-terminus . Bacterial cells harboring a plasmid with the prePNA-cDNA, produced two PNA cross-reacting proteins: one migrated on SDS-PAGE identically with the native lectin (apparent mol . wt . 31 kDa); the other, at 35 kDa, was a beta-galactosidase pre-PNA fusion protein . The former protein possessed an N-terminal sequence identical to that of the mature, native PNA, suggesting that it was processed from the 35 kDa prePNA precursor . Only the 31 kDa protein was exported into the bacterial periplasmic space, and had the ability to bind to galactose-Sepharose . The isolated processed protein had the same hemagglutinating activity as the native lectin, when assayed with sialidase-treated human erythrocytes . Like the native lectin, it did not agglutinate the untreated cells, was not inhibited by N-acetylgalactosamine, and was inhibited by Gal beta 1----3GalNAc 30-times more strongly than by galactose. FEBS Lett, 1992 Jul 28, 307(2), 229 - 32 Serine residues responsible for tetracycline transport are on a vertical stripe including Asp-84 on one side of transmembrane helix 3 in transposon Tn10-encoded tetracycline/H+ antiporter of Escherichia coli; Yamaguchi A et al.; Putative transmembrane helix 3 of the tetracycline/H+ antiporter encoded by a transposon, Tn10, contains four serine residues, Ser-77, Ser-82, Ser-91 and Ser-92 . Each of these serine residues was replaced by site-directed mutagenesis . Of these four serine residues, Ser-77 was important for the transport function, and a bulky side chain at position 91 hindered substrate translocation, whereas Ser-82 and Ser-92 did not play any role . Ser-77 and Ser-91 are on the same vertical stripe, that includes the essential Asp-84, on the hydrophilic side of putative helix 3 . These observations suggest that helix 3 is part of the tetracycline translocation channel across the membrane. J Biol Chem, 1992 Jul 15, 267(20), 14443 - 50 A novel site-directed mutant of myoglobin with an unusually high O2 affinity and low autooxidation rate; Carver TE et al.; Mutants of sperm whale myoglobin were constructed at position 29 (B10 in helix notation) to examine the effects of distal pocket size on the rates of ligand binding and autooxidation . Leu29 was replaced with Ala, Val, and Phe using the synthetic gene and Escherichia coli expression system of Springer and Sligar (Springer, B . A., and Sligar, S . G . (1987) Proc . Natl . Acad . Sci . U . S . A . 84, 8961-8965) . Structures of the ferric forms of Val29 and Phe29, and the oxy form of Phe29 myoglobin were determined to 1.7 A by x-ray crystallography . The ferric mutant proteins are remarkably isomorphous with the wild type protein except in the immediate vicinity of residue 29 . Thus, the protein structure in the distal pocket of myoglobin can accommodate either a large "hole" (i.e . Ala or Val) or a large side chain (i.e . Phe) at position 29 without perturbation of tertiary structure . Phe29 oxymyoglobin is also identical to the native oxy protein in terms of overall structure and interactions between the bound O2 and His64, Val68, Phe43, and Ile107 . The distance between the nearest side chain atom of residue 29 and the second atom of the bound oxygen molecule is 3.2 A in the Phe29 protein and 4.9 A in native myoglobin . The equilibrium constants for O2 binding to Ala29, Val29, and Leu29 (native) myoglobin are the same, approximately 1.0 x 10(6) M-1 at 20 degrees C, whereas that for the Phe29 protein is markedly greater, 15 x 10(6) M-1 . This increase in affinity is due primarily to a 10-fold decrease in the O2 dissociation rate constant for the Phe29 mutant and appears to be the result of stabilizing interactions between the negative portion of the bound O2 dipole and the partially positive edge of the phenyl ring . Increasing the size of residue 29 causes large decreases in the rate of autooxidation of myoglobin: k(ox) = 0.24, 0.23, 0.055, and 0.005 h-1 for Ala29, Val29, Leu29 (native), and Phe29 myoglobin, respectively, in air at 37 degrees C . Thus, the Leu29----Phe mutation produces a reduced protein that is remarkably stable and is expressed in E . coli as 100% MbO2 . The selective pressure to conserve Leu29 at the B10 position probably represents a compromise between reducing the rate of autooxidation and maintaining a large enough O2 dissociation rate constant to allow rapid oxygen release during respiration. J Biol Chem, 1992 Jul 15, 267(20), 14227 - 32 Purification and characterization of heterodimeric human immunodeficiency virus type 1 (HIV-1) reverse transcriptase produced by in vitro processing of p66 with recombinant HIV-1 protease; Chattopadhyay D et al.; Active recombinant reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) with an amino-terminal extension containing a hexa-histidine sequence has been prepared in milligram quantities in a pure heterodimeric (p66/p51) form by coordinated applications of immobilized metal affinity chromatography (IMAC) and HIV-1 protease treatment . The precursor protein, isolated from extracts of recombinant Escherichia coli by IMAC in a predominantly unprocessed form (p66), migrated on sodium dodecyl sulfate-polyacrylamide gels as a 66-kDa band with minor heterogeneity at lower relative molecular mass . Incubation of this protein with recombinant HIV-1 protease produced a stable heterodimeric RT that was purified in a single step by IMAC . The purified protein retained both RT and RNase H activity, and kinetic parameters (Km and Vmax) were measured with both RNA-dependent DNA polymerization and RNase H activity assays . Carboxyl-terminal sequencing of purified heterodimeric RT indicated that one subunit is intact p66, whereas the other, p51, is a truncated form of p66 that terminates at residue Phe440 . Analysis of the HIV-1 protease digest revealed two cleavage sites, at Tyr483-Leu484 and Tyr532-Leu533, in addition to the site at Phe440-Tyr441 that is cleaved to produce p51. Nucleic Acids Res, 1992 Jul 25, 20(14), 3693 - 700 Expression and characterisation of the korB gene product from the Streptomyces lividans plasmid pIJ101 in Escherichia coli and determination of its binding site on the korB and kilB promoters; Zaman S et al.; A 0.5kb Spel-BclI fragment containing the pIJ101 korB ORF was cloned into pUC8 under the control of the lacZ promoter, creating pQR206 . In vitro coupled transcription-translation of pQR206 identified a protein product of approximately 10kDa, which corresponds to the predicted molecular weight deduced from the korB sequence . pQR206 was used to express the 10kDa KorB protein in vivo in E . coli . Crude E . coli protein extracts containing KorB were shown to bind to a 0.8kb kilB fragment and a 0.5kb korB fragment in gel retardation assays . DNasel footprinting indicated that the DNA recognition sequence of the KorB protein lies within a 60bp protected region encompassing the kilB promoter and a 36bp region encompassing the korB promoter. Nucleic Acids Res, 1992 Jul 25, 20(14), 3651 - 7 The application of Markov chain analysis to oligonucleotide frequency prediction and physical mapping of Drosophila melanogaster; Cuticchia AJ et al.; Here we compare several methods for predicting oligonucleotide frequencies in 691 kb of Drosophila melanogaster DNA . As in previous work on Escherichia coli and Saccharomyces cerevisiae, a relatively simple equation based on tetranucleotide frequencies can be used in predicting frequencies of higher order oligonucleotides . For example, the mean of observed/expected abundances of 4,096 hexamers was 1.07 with a sample standard deviation of .55 . This simple predictor arises by considering each base on the sense strand of D . melanogaster to depend only on the three bases 5' to it (a 3rd order Markov chain) and is more accurate than the random predictor . This equation is useful in predicting restriction enzyme fragment sizes, selecting restriction enzymes that cut preferentially in coding vs noncoding regions, and in selecting probes to fingerprint clones in contig mapping . Once again, this equation well predicts the occurrence of higher order oligonucleotides, supporting our hypothesis that this predictor holds in evolutionarily diverse organisms . When ranked from highest to lowest abundance, the observed frequencies of oligomers of a given length are closely tracked by the predicted abundances of a 3rd order Markov chain . Through use of the dependence of oligomer frequencies on base composition, we report a list of oligomers that will be useful for the completion of a cosmid physical map of D . melanogaster . Presently, the library is such that it will be possible to construct large contigs using only 30 oligonucleotide probes to fingerprint cosmids. Nucleic Acids Res, 1992 Jul 25, 20(14), 3631 - 7 A comparison of several similarity indices used in the classification of protein sequences: a multivariate analysis; Landes C et al.; The present work describes an attempt to identify reliable criteria which could be used as distance indices between protein sequences . Seven different criteria have been tested: i and ii) the scores of the alignments as given by the BESTFIT and the FASTA programs; iii) the ratio parameter, i.e . the BESTFIT score divided by the length of the aligned peptides; iv and v) the statistical significance (Z-scores) of the scores calculated by BESTFIT and FASTA, as obtained by comparison with shuffled sequences; vi) the Z-scores provided by the program RELATE which performs a segment-by-segment comparison of 2 sequences, and vii) an original distance index calculated by the program DOCMA from all the pairwise dotplots between the sequences . These 7 criteria have been tested against the aminoacid sequences of 39 globins and those of the 20 aminoacyl-tRNA synthetases from E . coli . The distances between the sequences were analyzed by the multivariate analysis techniques . The results show that the distances calculated from the scores of the pairwise alignments are not adequately sensitive . The Z-score from RELATE is not selective enough and too demanding in computer time . Three criteria gave a classification consistent with the known similarities between the sequences in the sets, namely the Z-scores from BESTFIT and FASTA and the multiple dotplot comparison distance index from DOCMA. J Biol Chem, 1992 Jul 25, 267(21), 14799 - 803 Multiple forms of ubiquitin-activating enzyme E1 from wheat . Identification of an essential cysteine by in vitro mutagenesis; Hatfield PM et al.; Ubiquitin-activating enzyme, E1, directs the ATP-dependent formation of a thiol ester linkage between itself and ubiquitin . The energy in this bond is ultimately used to attach ubiquitin to various intracellular proteins . We previously reported the isolation of multiple E1s from wheat and the characterization of a cDNA encoding this protein (UBA1) . We now report the derived amino acid sequence of two additional members of this gene family (UBA2 and UBA3) . Whereas the amino acid sequence of UBA2 is nearly identical to UBA1, the sequence of UBA3 is significantly different . Nevertheless, the protein encoded by UBA3 catalyzes the ATP-dependent activation of ubiquitin in vitro . Comparison of derived amino acid sequences of genes encoding E1 from plant, yeast, and animal tissues revealed 5 conserved cysteine residues, with one potentially involved in thiol ester bond formation . To identify this essential residue, codons corresponding to each of the 5 cysteines in UBA1 were individually altered using site-directed mutagenesis . The mutagenized enzymes were expressed in Escherichia coli and assayed for their ability to activate ubiquitin . Only substitution of the cysteine at position 626 abolishes E1 activity, suggesting that this residue forms the thiol ester linkage with ubiquitin. J Biol Chem, 1992 Jul 25, 267(21), 14611 - 5 Functional interactions of stimulatory and inhibitory GDP/GTP exchange proteins and their common substrate small GTP-binding protein; Kikuchi A et al.; smg GDS and rho GDI are stimulatory and inhibitory GDP/GTP exchange proteins, respectively, for a group of ras p21-related small GTP-binding proteins (G proteins) . rho p21 is a common substrate small G protein for both GDP/GTP exchange proteins . We examined here the functional interactions of these GDP/GTP exchange proteins with rho p21 as a substrate . smg GDS and rho GDI interacted with the GDP-bound form of rho p21 and thereby stimulated and inhibited, respectively, the dissociation of GDP . The inhibitory effect of rho GDI was much stronger than the stimulatory effect of smg GDS . The GDP-bound form of rho p21 formed a complex with rho GDI but not with smg GDS in their simultaneous presence . Since the content of smg GDS was generally less than that of rho GDI in cells, these results suggest that there is some mechanism to release the inhibitory action of rho GDI and to make rho p21 sensitive to the smg GDS action during the conversion of rhoA p21 from the GDP-bound inactive form to the GTP-bound active form in intact cells . On the other hand, rho p21 was previously shown to be ADP-ribosylated by bacterial ADP-ribosyltransferases, named C3 and EDIN, at Asn41 in the putative effector region of rho p21 . This ADP-ribosylation was inhibited by rho GDI much more efficiently than by smg GDS . These results suggest that rho GDI may mask the putative effector region of rho p21 and thereby inhibit its interaction with the target protein even in the presence of smg GDS . Thus, both smg GDS and rho GDI are important to regulate the rho p21 activity and action in cooperation with each other. Nucleic Acids Res, 1992 Jul 25, 20(14), 3645 - 50 RNA binding specificity of a Drosophila snRNP protein that shares sequence homology with mammalian U1-A and U2-B" proteins; Harper DS et al.; We have characterized a recombinant Drosophila melanogaster RNA binding protein, D25, by virtue of its antigenic relationship to mammalian U1 and U2 small nuclear ribonucleoprotein (U snRNP) proteins . Sequence analysis revealed that D25 bears strong similarity to both the human U1 snRNP-A (U1-A) and U2 snRNP-B" (U2-B") proteins . However, at residues known to be critical for the RNA binding specificities of U1-A and U2-B" D25 sequence is more similar to U2-B" . Using direct RNA binding assays D25 selected U1 RNA from either HeLa or Drosophila Kc cell total RNA . Furthermore, D25 bound U1 RNA when transfected into mammalian cells . Thus, D25 appears to be a Drosophila homolog of the mammalian U1-A protein, despite its sequence similarity to U2-B". J Biol Chem, 1992 Jul 25, 267(21), 15184 - 92 Deep penetration of a portion of Escherichia coli SecA protein into model membranes is promoted by anionic phospholipids and by partial unfolding; Ulbrandt ND et al.; SecA protein, a principal component of the protein export machinery of Escherichia coli, is found both in the cytoplasm and inner membrane of cells . Previous in vitro and in vivo studies demonstrated that the interaction of SecA with the inner membrane requires the presence of physiological levels of anionic (acidic) phospholipids . In this report the degree of SecA insertion into model membranes and the conformational changes associated with this event have been examined . The extent of association of SecA with model membranes was determined by photolabeling with a hydrophobic reagent, and the depth of insertion of the protein into the phospholipid bilayer was determined by the amount of quenching of SecA fluorescence by both brominated and spin-labeled phospholipids . These methods demonstrated that SecA penetrates deep within the acyl chain region of the phospholipid bilayer . It was also found that SecA penetration into vesicles was associated with a major conformational change in the protein . This change can be induced by higher temperatures and involves a partial unfolding event as judged by differential scanning calorimetry, SecA fluorescence and increased sensitivity to proteolysis . These properties suggest the induction of a molten-globule-like conformation in a portion of the SecA polypeptide . This change was also induced at lower temperatures by the presence of membranes containing a physiological amount of the anionic phospholipid, phosphatidylglycerol . The partial unfolding and concomitant deep insertion of SecA into membranes may aid in the insertion of precursor proteins into the inner membrane and may influence possible interactions between SecA and the integral membrane export machinery components. J Biol Chem, 1992 Jul 25, 267(21), 14713 - 20 DNA sequence determinants for binding of the Escherichia coli catabolite gene activator protein; Gunasekera A et al.; The consensus DNA site for binding of the Escherichia coli catabolite gene activator protein (CAP) is 22 base pairs in length and is 2-fold symmetric: 5'-AAATGTGATCTAGATCACATTT-3' . Positions 4 to 8 of each half of the consensus DNA half-site are the most strongly conserved . In this report, we analyze the effects of substitution of DNA base pairs at positions 4 to 8, the effects of substitution of thymine by uracil and by 5-methylcytosine at positions 4, 6, and 8, and the effect of dam methylation of the 5'-GATC-3' sequence at positions 7 to 10 . All DNA sites having substitutions of DNA base pairs at positions 4 to 8 exhibit lower affinities for CAP than does the consensus DNA site, consistent with the proposal that the consensus DNA site is the ideal DNA site for CAP . Specificity for T:A at position 4 appears to be determined solely by the thymine 5-methyl group . Specificity for T:A at position 6 and specificity for A:T at position 8 appear to be determined in part, but not solely, by the thymine 5-methyl group . dam methylation has little effect on CAP.DNA complex formation . The thermodynamically defined consensus DNA site spans 28 base pairs . All, or nearly all, DNA determinants required for maximal affinity for CAP and for maximal thermodynamically defined CAP.DNA ion pair formation are contained within a 28-base pair DNA fragment that has the 22-base pair consensus DNA site at its center . The quantitative data in this report provide base-line thermodynamic data required for detailed investigations of amino acid-base pair and amino acid-phosphate contacts in this protein-DNA complex. J Biol Chem, 1992 Jul 25, 267(21), 14559 - 62 pH dependence of proton translocation by Escherichia coli; Verkhovskaya M et al.; Proton translocation in spheroplasts from Escherichia coli has been studied in two mutants, one of which expresses cytochrome o and the other cytochrome d as the terminal oxidase . Using the O2 pulse method, the H+/e- ratio of proton translocation associated with cytochrome o was confirmed to be near 2 at neutral pH, but was found to decrease considerably when the medium pH was raised above 8 . At high pH there was an increase in H+/OH- permeability of the cell membrane, but this was not sufficient to explain the decline in proton ejection . The pH effect was confined to cytochrome o-linked activity . It was not present when cytochrome d generated the electrochemical proton gradient . This makes it improbable that the Na+/H+ antiporter is responsible . The most likely explanation for our finding is that there is a "slip" in the proton-pumping mechanism of cytochrome o at high pH. Biochem Pharmacol, 1992 Jul 22, 44(2), 199 - 204 Substrate specificity of Escherichia coli thymidine phosphorylase for pyrimidine nucleosides with anti-human immunodeficiency virus activity; Schinazi RF et al.; Various nucleoside antiviral agents and their metabolites were examined for their ability to be cleaved across the glycosidic bond by Escherichia coli thymidine phosphorylase . The increasing order of susceptibility to cleavage was U greater than T much greater than C derivatives . Nucleosides that were unsaturated in the sugar moiety were more susceptible than saturated ones . 3'-Deoxy-2',3'-didehydrothymidine was a substrate, whereas 3'-azido-, 3'-fluoro-, 3'-oxo- and 3'-thiapyrimidine nucleosides were resistant to this enzyme. Biochemistry, 1992 Jul 21, 31(28), 6598 - 602 13C isotope effect studies of Escherichia coli aspartate transcarbamylase in the presence of the bisubstrate analog N-(phosphonoacetyl)-L-aspartate; Parmentier LE et al.; 13C isotope effects have been measured for the aspartate transcarbamylase holoenzyme (ATCase) and catalytic subunit catalyzed reactions in the presence of the bisubstrate analog N-(phosphonoacetyl)-L-aspartate (PALA) . For holoenzyme-catalyzed reactions in the physiological direction with very low levels of L-aspartate as substrate, or with L-cysteine sulfinate as substrate, or in the reverse direction with carbamyl-L-aspartate and phosphate as substrates, the isotope effect data show a slight dependence on PALA concentration . Under these conditions, PALA first stimulates the rate and then inhibits it at higher concentrations . The observed isotope effect at maximum stimulation by PALA is slightly smaller than in the absence of the analog, but as the PALA concentration is increased to reduce the rate to its original value, the observed isotope effect also increases and approaches the value of the isotope effect determined in the absence of PALA . These data suggest that the kinetic properties of the active enzyme are affected by the number of active sites occupied by PALA, indicating communication between subunits, and a mathematical model is proposed which explains our experimental observations . In contrast to these results with the holoenzyme, isotope effects measured for the reaction catalyzed by the isolated catalytic subunits are not altered in the presence of PALA . Taken together, these data are consistent with the two-state model for the homotropic regulation of ATCase. Biochemistry, 1992 Jul 21, 31(28), 6577 - 84 13C and 15N isotope effects as a probe of the chemical mechanism of Escherichia coli aspartate transcarbamylase; Parmentier LE et al.; 13C and 15N isotope effects have been measured for the aspartate transcarbamylase (ATCase) reaction in an effort to elucidate the chemical mechanism of this highly regulated enzyme . The observed 15(V/K(asp))H2O value for the ATCase holoenzyme at saturing carbamyl phosphate and 12 mM L-aspartate is 1.0045 at pH 7.5, and this value remains unchanged in the presence of 5 mM ATP (activator) or 5 mM CTP (inhibitor) . The fact that the isotope effect is not changed by the allosteric modifiers supports the conclusion that the kinetic properties of the active form of ATCase are not influenced by ATP or CTP . The observed 15(V/K(asp)) values for the catalytic subunit of ATCase are also the same as those determined for the holoenzyme, suggesting that the chemical mechanisms of both enzyme species are the same . Quantitative analysis of 13C and 15N isotope effects in both H2O and D2O has led to the proposal of a chemical model for the ATCase reaction which involves a precatalytic conformational change to form an activated complex that facilitates deprotonation of L-aspartate by an enzyme functional group . Nucleophilic attack on the carbonyl carbon of carbamyl phosphate by the alpha-amino group of L-aspartate results in the formation of a tetrahedral intermediate . An intramolecular proton transfer leads to formation of products N-carbamyl-L-aspartate and inorganic phosphate. Biochemistry, 1992 Jul 21, 31(28), 6570 - 6 13C isotope effects as a probe of the kinetic mechanism and allosteric properties of Escherichia coli aspartate transcarbamylase; Parmentier LE et al.; 13C kinetic isotope effects have been measured in carbamyl phosphate for the reaction catalyzed by aspartate transcarbamylase . For the holoenzyme, the value was 1.0217 at zero aspartate, but unity at infinite aspartate, with 4.8 mM aspartate eliminating half of the isotope effect . This pattern proves an ordered kinetic mechanism, with carbamyl phosphate adding before aspartate . The same parameters were observed in the presence of ATP or CTP, showing that there is only one form of active enzyme present, regardless of the presence or absence of allosteric modifiers . These data support the Monod model of allosteric behavior in which the equilibrium between fully active and inactive enzyme is perturbed by selective binding interactions of substrates and modifiers, and there are no enzyme forms having partial activity . Isolated catalytic subunits of the enzyme showed similar 13C isotope effects (1.0240 at zero aspartate, 1.0039 at infinite aspartate, 3.8 mM aspartate causing half of the change from one value to the other), but the finite isotope effect at infinite aspartate shows that the kinetic mechanism is now partly random . With the very slow and poorly bound aspartate analog cysteine sulfinate, the 13C isotope effects were 1.039 for both holoenzyme and catalytic subunits and were not decreased significantly by high levels of cysteine sulfinate . The value of 1.039 is probably close to the intrinsic isotope effect on the chemical reaction, while the kinetic mechanism with this substrate is now fully random because the chemistry is so much slower than release of either reactant from the enzyme. Biochemistry, 1992 Jul 21, 31(28), 6562 - 9 Ionization of amino acid residues involved in the catalytic mechanism of aspartate transcarbamoylase; Turnbull JL et al.; The chemical and kinetic mechanisms of the reaction catalyzed by the catalytic trimer of aspartate transcarbamoylase have been examined . The variation of the kinetic parameters with pH indicated that at least four ionizing amino acid residues are involved in substrate binding and catalysis . The pH dependence of K(ia) for carbamoyl phosphate and the K(i) for N-(phosphonoacetyl)-L- aspartate revealed that a protonated residue with a pK value of 9.0 is required for the binding of carbamoyl phosphate . However, the variation with pH of K(i) for succinate, a competitive inhibitor of aspartate, and for cysteine sulfinate, a slow substrate, showed that a single residue with a pK value of 7.3 must be protonated for binding these analogues and, by inference, aspartate . The profile of log V against pH displayed a decrease in reaction rate at low and high pH, suggesting that two groups associated with the Michaelis complex, a deprotonated residue with a pK value of 7.2 and a protonated group with a pK value of 9.5, are involved in catalysis . By contrast, the catalytically productive form of the enzyme-carbamoyl phosphate complex, as illustrated in the bell-shaped pH dependence of log (V/K)(asp), is one in which a residue with a pK value of 7.0 must be protonated while a group with a pK value of 9.1 is deprotonated . This interpretation is supported by the results from the temperature dependence of the V and V/K profiles and from the pH dependence of pK(i) for the aspartate analogues.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1992 Jul 21, 31(28), 6402 - 13 Phospholipase A2 engineering . Structural and functional roles of highly conserved active site residues tyrosine-52 and tyrosine-73; Dupureur CM et al.; Site-directed mutagenesis was used to probe the structural and functional roles of two highly conserved residues, Tyr-52 and Tyr-73, in interfacial catalysis by bovine pancreatic phospholipase A2 (PLA2, overproduced in Escherichia coli) . According to crystal structures, the side chains of these two active site residues form H-bonds with the carboxylate of the catalytic residue Asp-99 . Replacement of either or both Tyr residues by Phe resulted in only very small changes in catalytic rates, which suggests that the hydrogen bonds are not essential for catalysis by PLA2 . Substitution of either Tyr residue by nonaromatic amino acids resulted in substantial decreases in the apparent kcat toward 1,2-dioctanoyl-sn-glycero-3-phosphocholine (DC8PC) micelles and the v(o) (turnover number at maximal substrate concentration, i.e., mole fraction = 1) toward 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DC14PM) vesicles in scooting mode kinetics {Berg, O . G., Yu, B.-Z., Rogers, J., & Jain, M . K . (1991) Biochemistry 30, 7283-7297} . The Y52V mutant was further analyzed in detail by scooting mode kinetics: the E to E* equilibrium was examined by fluorescence; the dissociation constants of E*S, E*P, and E*I (KS*, KP*, and KI*, respectively) in the presence of Ca2+ were measured by protection of histidine-48 modification and by difference UV spectroscopy; the Michaelis constant KM* was calculated from initial rates of hydrolysis in the absence and presence of competitive inhibitors; and the turnover number under saturating conditions (kcat, which is a theoretical value since the enzyme may not be saturated at the interface) was calculated from the vo and KM* values . The results indicated little perturbation in the interfacial binding step (E to E*) but ca . 10-fold increases in KS*, KP*, KI*, and KM* and a less than 10-fold decrease in kcat . Such changes in the function of Y52V are not due to global conformational changes since the proton NMR properties of Y52V closely resemble those of wild-type PLA2; instead, it is likely to be caused by perturbed enzyme-substrate interactions at the active site . Tyr-73 appears to play an important structural role . The conformational stability of all Tyr-73 mutants decreased by 4-5 kcal/mol relative to that of the wild-type PLA2 . The proton NMR properties of Y73A suggested significant conformational changes and substantially increased conformational flexibility . These detailed structural and functional analyses represent a major advancement in the structure-function study of an enzyme involved in interfacial catalysis. Biochemistry, 1992 Jul 21, 31(28), 6447 - 53 Proximity between nucleotide/dinucleotide and metal ion binding sites in DNA-dependent RNA polymerase from Escherichia coli; Tyagi SC et al.; In order to understand translocation in transcription, it is important to develop a continuous functional assay for RNA polymerase (RNAP) activity in vitro . Fluorescent derivatives of ATP, UTP, UpA, and CpA with aminonaphthalene-5-sulfonic acid (AmNS) attached to the nucleotide triphosphates via a gamma-phosphoramidate bond or to the dinucleotide monophosphates via a 5'-secondary amine linkage were synthesized {Tyagi, S.C., & Wu, F.Y.-H . (1987) J . Biol . Chem . 262, 10684-10688} . The fluorescent emission spectra of (5'-AmNS)UpA, (5'-AmNS)CpA, (gamma-AmNS)ATP, and (gamma-AmNS)UTP overlap the absorption spectrum of co-substituted RNA polymerase (Co-RNAP) and ensure fluorescence resonance energy transfer (FRET) between the fluorescent analog and Co(II) in Co-RNAP . The binding constants at a single site for (gamma-AmNS)ATP, (gamma-AmNS)UTP, (5'-AmNS)UpA, and (5'-AmNS)CpA were observed to be 7.11, 5.26, 0.52, and 0.61 microM, respectively, in Co-RNAP and 5.70, 3.42, 0.12, and 0.21 microM, respectively, in Zn-RNAP . (8-AmTEMPO)ATP, with the spin probe AmTEMPO attached to the C-8 position at ATP {Tyagi, S.C . (1991) J . Biol . Chem . 266, 17936-17940}, and Mn(3'-OCH3)UTP were synthesized . Mn-(II)-substituted RNA polymerase (Mn-RNAP) is prepared . The single site binding constants for (8-Am-TEMPO)ATP and Mn(3'-OCH3)UTP were 3.58 and 2.35 microM in Zn-RNAP and 5.77 and 3.43 microM in Mn-RNAP, respectively . These results indicate that dinucleotides bind much more tightly than mononucleotides to RNAP and that the binding constants are roughly the same for both Co- and Zn-substituted RNAP.(ABSTRACT TRUNCATED AT 250 WORDS) J Mol Biol, 1992 Jul 20, 226(2), 387 - 97 Binding of the arginine repressor of Escherichia coli K12 to its operator sites; Tian G et al.; In the arginine regulon of Escherichia coli K12 each of the eight operator sites consists of two 18-base-pair-long palindromic sequences called ARG boxes . In the operator sites for the structural genes of the regulon the two ARG boxes are separated by three base-pairs, in the regulatory gene argR they are separated by two base-pairs . The hexameric arginine repressor, the product of argR, binds to the two ARG boxes in an operator in the presence of L-arginine . From the results of various kinds of in vitro footprinting experiments with the ARG boxes of argF and argR (DNase I protection, hydroxyl radical, ethylation and methylation interference, methylation protection) it can be concluded that: (1) the repressor binds simultaneously to two adjacent ARG boxes; (2) that it binds on one face of the double helix; and (3) that it forms contacts with the major and minor grooves of each ARG box, but not with the central three base-pairs . The repressor can bind also to a single ARG box, but its affinity is about 100-fold lower than for two ARG boxes . From gel retardation experiments with 3H-labeled repressor and 32P-labeled argF operator DNA, it is concluded that the retarded DNA-protein complex contains no more than one repressor molecule per operator site and that most likely one hexamer binds to two ARG boxes . The bound repressor was shown to induce bending of argF operator DNA . The bending angle calculated from the results of gel retardation experiments is about 70 degrees and the bending center was located within the region encompassing the ARG boxes . The main features that distinguish the arginine repressor from other repressors studied in E . coli are its hexameric nature and the simultaneous binding of one hexameric molecule to two palindromic ARG boxes that are close to each other. J Mol Biol, 1992 Jul 20, 226(2), 367 - 86 Arginine regulon of Escherichia coli K-12 . A study of repressor-operator interactions and of in vitro binding affinities versus in vivo repression; Charlier D et al.; The 12 genes which in E . coli K-12 constitute the arginine regulon are organized in nine transcriptional units all of which contain in their 5' non-coding region two 18 bp partially conserved imperfect palindromes (ARG boxes) which are the target sites for binding of the repressor, a hexameric protein . In vitro binding experiments with purified repressor (a gift from W . K . Maas) were performed on the operator sites of four genes, argA, argD, argF, argG, and of two operons, carAb and the bipolar argECBH cluster . A compilation of results obtained by DNase I and hydroxyl radical footprinting clearly indicates that in each case the repressor binds symmetrically to four helical turns covering adjacent pairs of boxes separated by 3 bp, but to one face of the DNA only . Methylation protection experiments bring to light major base contacts with four highly conserved G residues symmetrically distributed in four consecutive major grooves . Symmetrical contacts in the minor groove with A residues have also been identified . Stoichiometry experiments suggest that a single hexameric repressor molecule binds to a pair of adjacent ARG boxes . Although the wild-type operator consists of a pair of adjacent ARG boxes separated by 3 bp (except argR where there are only 2 bp), repressor can bind to a single box but with a greatly reduced affinity . Therefore, adjacent boxes behave co-operatively with respect to the Arg repressor binding, in the sense that the presence of one box largely stimulates the binding of the properly located second box . The optimal distance separating two boxes is 3 bp, but one bp more or less does not abolish this stimulation effect . However, it is completely abolished by the introduction of two or more additional bp unless a full helical turn is introduced . Large variations in the in vivo repression response between individual arginine genes or a wild-type gene and cognate Oc type mutants are not reflected by similar differences in the in vitro binding results where only small differences are observed . The significance of this lack of correlation is discussed. FEBS Lett, 1992 Jul 20, 306(2-3), 251 - 6 Fast and slow kinetics of porin channels from Escherichia coli reconstituted into giant liposomes and studied by patch-clamp; Berrier C et al.; E . coli porins (OmpF and OmpC) were purified and reconstituted into liposomes which were enlarged to giant proteoliposomes by dehydration-rehydration and studied by patch-clamp . The porins could be closed by voltage pulses under -100 mV . The kinetics of closure was slow, with closure events of about 200 pS in 0.1 M KCl . Rapid fluctuations (in the millisecond range) of about one third (60-70 pS) of the large closure steps were also observed . The data are interpreted as follows: an increase in membrane potential favours the cooperation transition of multimers towards an inactivated state, while monomers which have not been inactivated can flicker rapidly between an open and a short-lived closed state. FEBS Lett, 1992 Jul 20, 306(2-3), 103 - 7 A functional tomato ACC synthase expressed in Escherichia coli demonstrates suicidal inactivation by its substrate S-adenosylmethionine; Li N et al.; 1-Aminocyclopropane-1-carboxylate (ACC) synthase is a key enzyme in the biosynthesis of the plant hormone, ethylene . We have isolated, sequenced and expressed a functional tomato (cv Pik-Red) ACC synthase gene in Escherichia coli . ACC synthase expressed in E . coli was inactivated by incubation with S-adenosylmethionine (SAM), the half-time of which was concentration dependent . Mixing the tomato fruit protein extract with the cell-free extract from transformed E . coli did not affect SAM-dependent inactivation of ACC synthase activity . Thus, single isoforms of the ACC synthase enzyme, which demonstrate the biochemical features expected of the tomato fruit enzyme, can be expressed in E . coli and their structure-function relationships investigated. Carbohydr Res, 1992 Jul 20, 232(1), 77 - 87 Studies of the interaction of the maltose-binding protein of Escherichia coli, a closed-groove binder, with 4,6-O-ethylidenemalto-oligosaccharides (dp 2-5) and its regioselective labelling with 3-azibutyl 1-thio-alpha-(6-3H)maltoside; Lehmann J et al.; Four malto-oligosaccharides (dp 2-5), each with a 4,6-O-ethylidene group on the glucosyl unit at the non-reducing terminus, were synthesised and used to prove that the maltose-binding protein (MBP) of E . coli is a closed-groove binder . alpha-D-Glucosylation of 3-azibutyl 1-thio-alpha-D-(6-3H)glucopyranoside yielded a 3H-labelled, photolabile 1-thiomaltoside derivative that was used to chemically modify the binding site of MBP . The 3H-labelled peptide containing 83% of the total radioactivity, which was isolated after tryptic cleavage of the modified MBP and sequenced, is part of the closed end of the MBP groove. Carbohydr Res, 1992 Jul 20, 232(1), 33 - 45 The synthesis and characterisation of 2-O-(6-O-L-glycero-alpha,beta-D-manno-heptopyranosyl-alpha-D-glucopyran osyl)-alpha,beta-D-glucopyranose; Nepogodev SA et al.; The title compounds were synthesised, and appropriate derivatives were characterised by GLC, GLC-MS, and NMR spectroscopy . The GLC and GLC-MS data proved 2-O-(6-O-L-glycero-alpha-D-manno-heptopyranosyl-alpha-D-glucopyranosyl)- D- glucopyranose to be a constituent of the outer-core region of the lipopolysaccharide from Escherichia coli K-12, indicating the heptosyl residue to be linked to the terminal glucopyranose residue. J Mol Biol, 1992 Jul 20, 226(2), 425 - 32 Electron microscopic study of (A)BC excinuclease . DNA is sharply bent in the UvrB-DNA complex; Shi Q et al.; Nucleotide excision repair in Escherichia coli is initiated by the UvrA, UvrB and UvrC proteins . UvrA is the damage recognition subunit, makes an A2B1 complex with the targeting subunit UvrB, and the complex binds to the lesion site; UvrA dissociates leaving behind a very stable UvrB-DNA complex that is recognized by the trigger subunit, UvrC, and the ensuing UvrB-UvrC heterodimer makes two incisions, one on either side of the lesion . Using electron microscopy, we investigated the structures of these early A, A-B intermediates on DNA containing ultraviolet light photoproducts . UvrA, which is known to bind to DNA as a dimer and produce a DNase I footprint of 33 base-pairs does not change the trajectory of DNA appreciably . The A2B1 complex clearly shows a bipartite structure and its effect on the trajectory of the DNA was not consistently straight or kinked . In contrast, the DNA in the preincision UvrB-DNA complex appears to be severely kinked; 43% of the molecules are bent by 80 degrees or more, with an average bending angle of 127 degrees . It appears that protein-induced bending is an important step on the pathway leading to excision of the damaged nucleotide by (A)BC excinuclease. J Mol Biol, 1992 Jul 20, 226(2), 399 - 409 Important 2'-hydroxyl groups in model substrates for M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli; Perreault JP et al.; The role of 2'-hydroxyl groups in a model substrate for RNase P from Escherichia coli was studied using mixed DNA/RNA derivatives of such a substrate . The presence of the 2'-hydroxyl groups of nucleotides at positions -1 and -2 in the leader sequence and at position 1, as well as at the first C in the 3'-terminal CCA sequence, are important but not absolutely essential for efficient cleavage of the substrate by RNase P or its catalytic RNA subunit, M1 RNA . The 2'-hydroxyl groups in the substrate that are important for efficient cleavage also participate in the binding of Mg2+ . An all-DNA external guide sequence (EGS) can efficiently render a potential substrate, derived from the model substrate, susceptible to cleavage by the enzyme or its catalytic RNA subunit . Furthermore, both DNA and RNA EGSs turn over during the reaction with RNase P in vitro . The identity of the nucleotide at position 1 in the substrate, the adjacent Mg(2+)-binding site in the leader sequence, and the junction of the single and double-stranded regions are the important elements in the recognition of model substrates, as well as in the identification of the sites of cleavage in those model substrates. FEBS Lett, 1992 Jul 20, 306(2-3), 185 - 8 3'-Mercapto-2',3'-dideoxynucleotides are high effective terminators of DNA synthesis catalyzed by HIV reverse transcriptase; Yuzhakov AA et al.; Four 3'-mercapto-2',3'-dideoxynucleoside 5'-triphosphates (A, G, C and T) were tested as DNA chain terminator substrates for calf thymus alpha-DNA polymerase, E . coli DNA polymerase I Klenow fragment, terminal deoxynucleotidyl transferase and reverse transcriptases of AMV, HIV and MLV viruses . It was shown that the analogues selectively and irreversibly terminated DNA chain elongation by AMV and HIV reverse transcriptases and the terminal transferase . Other DNA polymerases tested did not use the nucleotide analogues as chain terminator substrate. Biochem J, 1992 Jul 15, 285 ( Pt 2), 537 - 40 Participation of the phenolic hydroxyl group of Tyr-8 in the catalytic mechanism of human glutathione transferase P1-1; Kolm RH et al.; The coding region of cDNA corresponding to human class Pi glutathione transferase P1-1 was amplified by the PCR, subcloned into an expression vector, pKHP1, expressed in Escherichia coli, and characterized . The physicochemical and catalytic properties of recombinant glutathione transferase P1-1 were indistinguishable from those of the enzyme previously isolated from human placenta . The active-site residue Tyr-8 of the wild-type enzyme was converted into Phe by means of oligonucleotide-directed mutagenesis . The mutant enzyme Y8F displayed a 300-fold decrease in specific activity, ascribable mainly to a lowered k(cat.) (or V) value . Kinetic parameters reflecting binding affinity, S0.5 (substrate concn . giving 1/2V) and I50 (concn . of inhibitor giving 50% remaining activity), were only moderately elevated in the mutant enzyme . These results indicate that Tyr-8 contributes primarily to catalysis as such, rather than to binding of the substrates . The dependence of k(cat.)/Km on pH shows an optimum at pH 7.0, defined by acidic and basic ionic dissociation constants with pKa1 = 6.7 and pKa2 = 7.3 respectively . The mutant enzyme Y8F does not display the basic limb of the k(cat.)/Km versus pH profile, but shows a monotonic increase of k(cat.)/Km with an apparent pKa1 of 7.1 . The results indicate that the phenolic hydroxyl group of Tyr-8 in un-ionized form, but not the phenolate of Tyr-8, contributes to catalysis by glutathione transferase P1-1. Biochem J, 1992 Jul 15, 285 ( Pt 2), 377 - 81 Contribution of five amino acid residues in the glutathione-binding site to the function of human glutathione transferase P1-1; Widersten M et al.; Five amino acids in proximity to GSH bound in the active-site cavity of human Class Pi glutathione transferase (GST) P1-1 were mutated by oligonucleotide-directed site-specific mutagenesis . The following mutations gave catalytically active mutant proteins with the proper dimeric structure: Arg14----Ala, Lys45----Ala, Gln52----Ala, Gln65----His and Asp99----Asn . The mutation Gln65----Ala was also made, but the protein was not characterized because of its poor catalytic activity . Residues Arg14, Lys45, Gln52 and Gln65 all contribute to binding of glutathione, and the substitutions caused an approx . 10-fold decrease in affinity, corresponding to 5 kJ/mol, except for Arg14, for which the effect was larger . In addition, Arg14 appears to have an important structure role, since the Arg14----Ala mutant demonstrated a significantly lower stability as compared with the wild-type and the other mutant enzymes . Asp99 primarily contributes to catalysis rather than to binding . The kcat./Km-versus-pH profile for the Asp99----Asn mutant is shifted by 0.5 pH unit in the alkaline direction, and it is proposed that Asp99 may participate in proton transfer in the catalytic mechanism . The possibility of redesigning the substrate specificity for GSTs was shown by the fact that the mutant Lys45----Ala displayed a higher catalytic efficiency with GSH monoethyl ester than with its natural substrate, GSH. Gene, 1992 Jul 15, 116(2), 139 - 50 Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody; Trirawatanapong T et al.; Envelope glycoprotein E of flaviviruses is exposed at the surface of the virion, and is responsible for eliciting a neutralizing antibody (Ab) response, as well as protective immunity in the host . In this report, we describe a method for the fine mapping of a linear sequence of the E protein of dengue virus type-2 (DEN-2), recognized by a type-specific and neutralizing monoclonal Ab (mAb), 3H5 . First, an Escherichia coli expression vector containing a heat-inducible lambda pL promoter was used to synthesize several truncated, and near-full length E polypeptides . Reactivities of these polypeptides with polyclonal mouse hyperimmune sera, as well as the 3H5 mAb revealed the location of the 3H5-binding site to be within a region of 166 amino acids (aa) between aa 255 and 422 . For fine mapping, a series of targeted deletions were made inframe within this region using the polymerase chain reaction (PCR) . The hydrophilicity pattern of this region was used as a guide to systematically delete the regions encoding the various groups of surface aa residues within the context of a near-full-length E polypeptide by using PCR . The 3H5-binding site was thus precisely mapped to a region encoding 12 aa (between aa 386 and 397) . A synthetic peptide containing this sequence was able to bind to the 3H5 mAb specifically, as shown by enzyme-linked immunosorbent assay . In addition, we show that rabbit Abs raised against the synthetic peptide of 12 aa were able to bind to the authentic E protein, and to neutralize DEN-2 virus in a plaque reduction assay. Eur J Biochem, 1992 Jul 15, 207(2), 733 - 9 A mutation at Gly314 of the beta subunit of the Escherichia coli pyridine nucleotide transhydrogenase abolishes activity and affects the NADP(H)-induced conformational change; Ahmad S et al.; Escherichia coli RH1 contains a mutation causing complete loss of pyridine nucleotide transhydrogenase activity . A single base change in the chromosomal DNA resulted in the replacement of Gly314 of the beta subunit by a Glu residue . The mutant enzyme was partially purified and its trypsin cleavage products examined . The distinct pattern of polypeptides given by proteolysis of the normal transhydrogenase in the presence of NADP(H) was absent when the mutant enzyme was treated with trypsin . However, the beta subunit of the mutant enzyme retained its ability to bind to NAD-agarose . Further substitutions were made at Gly314 converting it to Ala, Val or Cys by the use of site-directed mutagenesis . All substitutions for Gly314 abolished the activity completely . The enzyme containing the Gly314----Ala mutation was studied in detail and behaved exactly as the enzyme containing the Gly314----Glu mutation . It is concluded that the mutation in the beta subunit abolished the NADP(H)-induced conformational change in the mutant enzyme . This conformational change, caused by NADP(H) binding, is required to cleave the normal beta subunit at Arg265 by trypsin . The genes encoding the pyridine nucleotide transhydrogenase were completely resequenced and several corrections have been made to the previously published sequence {Clarke et al . (1986) Eur . J . Biochem . 158, 647-653}. Eur J Biochem, 1992 Jul 15, 207(2), 541 - 7 S100P, a novel Ca(2+)-binding protein from human placenta . cDNA cloning, recombinant protein expression and Ca2+ binding properties; Becker T et al.; A novel member of the S100 protein family, present in human placenta, has been characterized by protein sequencing, cDNA cloning, and analysis of Ca(2+)-binding properties . Since the placenta protein of 95 amino acid residues shares about 50% sequence identity with the brain S100 proteins alpha and beta, we proposed the name S100P . The cDNA was expressed in Escherichia coli and recombinant S100P was purified in high yield . S100P is a homodimer and has two functional EF hands/polypeptide chain . The low-affinity site (Kd = 800 microM), which, in analogy to S100 beta, seems to involve the N-terminal EF hand, can be followed by the Ca(2+)-dependent decrease in tyrosine fluorescence . The high-affinity site, provided by the C-terminal EF hand, influences the reactivity of the sole cysteine which is located in the C-terminal extension (Cys85) . Binding to the high-affinity site (Kd = 1.6 microM) can be monitored by fluorescence spectroscopy of S100P labelled at Cys85 with 6-proprionyl-2-dimethylaminonaphthalene (Prodan) . The Prodan fluorescence shows a Ca(2+)-dependent red shift of the maximum emission wavelength from 485 nm to 502 nm, which is accompanied by an approximately twofold loss in integrated fluorescence intensity . This indicates that Cys85 becomes more exposed to the solvent in Ca(2+)-bound S100P, making this region of the molecule, the so-called C-terminal extension, an ideal candidate for a putative Ca(2+)-dependent interaction with a cellular target . In p11, a different member of the S100 family, the C-terminal extension which contains a corresponding cysteine (Cys82 in p11), is involved in a Ca(2+)-independent complex formation with the protein ligand annexin II . The combined results support the hypothesis that S100 proteins interact in general with their targets after a Ca(2+)-dependent conformational change which involves hydrophobic residues of the C-terminal extension. Eur J Biochem, 1992 Jul 15, 207(2), 521 - 31 Structural studies by proton-NMR spectroscopy of plant horseradish peroxidase C, the wild-type recombinant protein from Escherichia coli and two protein variants, Phe41----Val and Arg38----Lys; Veitch NC et al.; Wild-type recombinant horseradish peroxidase isoenzyme C and two protein variants, Phe41----Val and Arg38----Lys, have been characterised using both one- and two-dimensional NMR spectroscopy . Proton NMR spectra recorded in both resting and cyanide-ligated states of the proteins were compared with those of the corresponding plant peroxidase . The latter contains 18% carbohydrate in eight N-linked oligosaccharide side chains whereas the recombinant proteins are expressed in nonglycosylated form . The spectra of the plant enzyme and refolded recombinant protein are essentially identical with the exception of carbohydrate-linked resonances in the former, indicating that their solution structures are highly similar . This comparison also identifies classes of carbohydrate resonances in the plant enzyme which provides new information on the local environment and mobility of the oligosaccharide side chains . Comparison of the spectra of the cyanide-ligated states of the two variants and those of plant horseradish peroxidase C indicated that there were significant differences with respect to haem and haem-linked resonances . These could not be rationalised simply on the basis of the local perturbation expected from a single-site substitution . The two substitutions made to residues on the distal side of the haem apparently influenced the degree of imidazolate character of the proximal His170 imidazole ring thus perturbing the magnetic environment of the haem group . Inspection of the spectra of the Phe41----Val variant also showed that the resonances of a phenylalanine residue in the haem pocket had been incorrectly assigned to Phe41 in a previous study . A new assignment, based on additional information from two-dimensional nuclear Overhauser enhancement spectroscopy, was made to Phe152 . The assignments made for the Phe41----Val variant were also used as a basis to investigate the structure of the complex formed with the aromatic donor molecule, benzhydroxamic acid. Eur J Biochem, 1992 Jul 15, 207(2), 441 - 7 Inhibition of triosephosphate isomerase from Trypanosoma brucei with cyclic hexapeptides; Kuntz DA et al.; Two series of oligopeptides have been synthesized . Their effects on the activity of purified triosephosphate isomerase from Trypanosoma brucei and various other organisms have been studied . Using detailed three-dimensional structure information, the first series consisted of both cyclic and linear hydrophilic peptides that were designed to mimic the beta turns of the subunit interface loops of the trypanosome triosephosphate isomerase dimer . None of these exerted any inhibitory effect . The second series consisted of more hydrophobic cyclic peptides, originally designed to inhibit a hepatic transport system . Several of these were very effective in inhibiting the trypanosome triosephosphate isomerase, but not the homologous enzymes from rabbit, dog, yeast or Escherichia coli . The most active peptide, cyclo{-Trp-Phe-D-Pro-Phe-Phe-Lys(Z)-}, exerted 50% inhibitory activity at a concentration of 3 microM . The nature of the inhibitory action of one of these compounds cyclo{-Trp-Tyr(OSO3Na)-D-Pro-Phe-Thr(OSO3Na)-Lys(Z)-} was studied in more detail . Its inhibition was noncompetitive and reversible and more than one peptide was able to bind/active site. Proc Natl Acad Sci U S A, 1992 Jul 15, 89(14), 6535 - 9 Introducing mutations into the single-copy chromosomal 23S rRNA gene of the archaeon Halobacterium halobium by using an rRNA operon-based transformation system; Mankin AS et al.; A vector-transformation system is described that permits replacement of a portion of the single rRNA operon of the archaeon Halobacterium halobium with a homologous fragment from a vector-borne gene . The vector construct contains three functional sections: (i) an entire H . halobium rRNA operon with two selective mutations in the 23S rRNA gene, the substitutions of A----G at position 1159 conferring resistance to thiostrepton and C----U at position 2471 conferring resistance to anisomycin; (ii) the complete pHSB1 plasmid from Halobacterium sp . SB3, which interferes with vector maintenance in the transformed halobacterial cells; and (iii) a segment of the pBR322 plasmid that permits vector replication in Escherichia coli . Transformation of H . halobium with the vector plasmid generates cells resistant to both anisomycin and thiostrepton that can be selected for, and discriminated from spontaneous mutants, by a two-step selection procedure . After transformation, the plasmid recombines homologously with the chromosome so that the plasmid-borne rDNA segment with resistance markers substitutes for the corresponding region of the chromosomal rRNA operon, and the transforming plasmid is lost . Eventually, this leads to a homogeneous population of the mutant ribosomes in the cell . Other mutations that are engineered in the vector-borne rRNA sequences can be transferred to the chromosomal rRNA operon concomitantly with the selective markers . The system has considerable potential for ribosomal engineering. Proc Natl Acad Sci U S A, 1992 Jul 15, 89(14), 6492 - 6 The synapsis event in the homologous pairing of DNAs: RecA recognizes and pairs less than one helical repeat of DNA; Hsieh P et al.; A key step in homologous recombination is the alignment and pairing of homologous DNAs . The Escherichia coli RecA protein initiates pairing by binding to single-strand DNA, forming a helical nucleoprotein filament . We demonstrate that in the presence of the nonhydrolyzable ATP analogue adenosine 5'-{gamma-thio}triphosphate and ADP, RecA can pair a homologous oligonucleotide 15 bases long with a duplex DNA to yield synaptic complexes consisting of the oligonucleotide and duplex DNA stabilized by RecA . RecA can pair as few as eight bases of homology to form such synaptic complexes . The homologous DNAs remain paired to each other upon removal of RecA provided that the length of shared homology is at least 26 base pairs . Based on our findings and the work of others, we propose that in vitro, one helical turn of a RecA nucleoprotein filament containing approximately six RecA monomers and 15 bases of single-strand DNA is the functional unit sufficient to carry out the homology search. J Biol Chem, 1992 Jul 15, 267(20), 14328 - 34 Precursor for the light-harvesting chlorophyll a/b-binding protein synthesized in Escherichia coli blocks import of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase; Oblong JE et al.; When synthesized in Escherichia coli, the light-harvesting chlorophyll a/b-binding protein (LHCP) precursor accumulates in inclusion-like bodies (Abad, M . S., Oblong, J . E., and Lamppa, G . K . (1991) Plant Physiol . 96, 1220-1227) . In this study we show that after solubilization in 6 M urea and dialysis into 20 mM Tris-HCl (pH 8.0) the recombinant LHCP precursor (preLHCP) was not found as a monomer (31 kDa), but instead produced a heterogeneous population of oligomeric complexes, ranging from 60-300 kDa as determined by gel filtration chromatography . Circular dichroism analysis indicated that the oligomers had folded structure, and that it was composed of both alpha-helix and beta-sheet . Approximately half of recombinant preLHCP found in these complexes was cleavable at the transit peptide-mature protein junction by a soluble chloroplast-processing enzyme in an organelle-free reaction . At 1.5 microM the recombinant precursor inhibited the import of radiolabeled preLHCP and the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase generated by reticulocyte lysate translations . When chloroplasts were preincubated with the precursor, followed by their reisolation, import was still blocked, providing evidence that competition between recombinant preLHCP and these substrates occurred at the chloroplast per se . Recombinant preLHCP was visualized on the envelope by immunofluorescence microscopy, and its presence there was mediated by a thermolysin-sensitive factor. J Biol Chem, 1992 Jul 15, 267(20), 14167 - 74 Biochemical and biophysical characterization of human recombinant IgE-binding protein, an S-type animal lectin; Hsu DK et al.; IgE-binding protein (epsilon BP) was originally identified by virtue of its affinity for IgE . It is now known to be a beta-galactoside-binding lectin with the characteristic of an S-type carbohydrate recognition domain . The protein is composed of two domains: the amino-terminal domain consisting of tandem repeats and the carboxyl-terminal domain containing sequences shared by other S-type carbohydrate recognition domains . The amino-terminal domain also contains a number of potential recognition sites for collagenase cleavage . In this study, human epsilon BP was first expressed in Escherichia coli, and the carboxyl-terminal domain (epsilon BP-C) was then generated by collagenase digestion of epsilon BP . By equilibrium dialysis, the association constants of epsilon BP and epsilon BP-C for lactose were found to be similar (6.0 +/- 0.70) x 10(4) M-1 and (4.7 +/- 0.27) x 10(4) M-1, respectively . Both polypeptides contain only one lactose-binding site/molecule . By an assay involving binding of 125I-labeled epsilon BP or epsilon BP-C to solid phase IgE, and inhibition of this binding by saccharides, it was determined that epsilon BP-C retains the saccharide specificity of epsilon BP . Importantly, although unlabeled epsilon BP-C inhibited the binding of the radiolabeled epsilon BP to IgE, unlabeled epsilon BP caused increased binding to IgE, suggesting self-association among epsilon BP molecules . Oligomeric structures resulting from self-association of epsilon BP were confirmed by chemical cross-linking studies . Furthermore, epsilon BP possesses hemagglutination activity on rabbit erythrocytes, whereas epsilon BP-C lacks such activity . Based on these results, we propose a structural model for multivalency of epsilon BP: dimerization or oligomerization of epsilon BP occurs through intermolecular interaction involving the amino-terminal domain. J Biol Chem, 1992 Jul 15, 267(20), 14122 - 8 Upstream sequence elements required for NarL-mediated activation of transcription from the narGHJI promoter of Escherichia coli; Dong XR et al.; Transcription of the narGHJI operon (encoding nitrate reductase) in Escherichia coli is primarily dependent on the activation of the pleiotropic transacting factor Fnr, which interacts with the promoter through a cis element (Fnr box) located near the transcription start site . Further stimulation of transcription occurs in the presence of nitrate and is dependent on activation of the transacting factor NarL and a cis-acting sequence (NarL box) located approximately 200 base pairs upstream from the transcription start site . To define the structure of the NarL box, alterations in the NarL box region, generated by saturation mutagenesis of the sequence from positions -184 to -202 in the narGHJI promoter of a narG::lacZ fusion-bearing plasmid, were analyzed for their effects on NarL-mediated stimulation of transcription . Single base substitutions that significantly reduced the NarL-mediated stimulation were restricted to a 6-base sequence, TACTCC, located at positions -193 to -198 in the narGHJI promoter . When 2 bases were modified, NarL-mediated stimulation was severely reduced when one or both alterations were located within the 6-base sequence . Attempts to restore NarL-mediated stimulation with an inverted NarL box were not successful . Although previous studies suggested that NarL-mediated stimulation of transcription may occur by a DNA looping mechanism, the results presented here demonstrate that it does not involve the passive formation of a simple DNA loop . Replacement of 94 or 108 bases of the approximately 150 base sequence between the Fnr box and the NarL box with an unrelated sequence resulted in elimination of NarL-mediated stimulation of transcription . Furthermore, shifting of most of the intervening sequence or defined segments of the sequence by 4 bases while maintaining the position of the NarL box relative to sequences required for Fnr-dependent, anaerobic transcription also eliminated the NarL-mediated stimulation . We conclude that in addition to the 6-base NarL box located on a specific face of the promoter DNA, the stimulation of transcription by NarL requires some specific sequences and/or higher order structure specified by the DNA that separates the NarL box from the Fnr box. J Biol Chem, 1992 Jul 15, 267(20), 14077 - 83 Evidence that a salt bridge in the light chain contributes to the physical stability difference between heavy and light human ferritins; Santambrogio P et al.; Human ferritin, a multimeric iron storage protein, is composed by various proportions of two subunit types: the H- and L-chains . The biological functions of these two genic products have not been clarified, although differences in reactivity with iron have been shown . Starting from the hypothesis that the high stability typical of ferritin is an important property which may be relevant for its iron storage function, we studied ferritin homopolymers of H- and L-chains in different denaturing conditions . In addition we analyzed 13 H-chain variants with alterations in regions conserved within mammalian H-chains . In all the denaturation experiments H-chain ferritin showed lower stability than L-chain ferritin . The difference was greater in guanidine HCl denaturation experiments, where the end products are fully unfolded peptides, than in acidic denaturation experiments, where the end products are peptides with properties analogous to "molten globule." The study on H-chain variants showed: (i) ferritin stability was not affected by alterations of regions exposed to the inner or outer surface of the shell and not involved in intra- or inter-chain interactions; (ii) stability was reduced by alterations of sequences involved in inter-subunit interactions such as the deletion of the N-terminal extension or substitutions along the hydrophobic and hydrophilic channels; (iii) stability was increased by the substitution of 2 amino acids inside the four-helix bundle with those of the homologous L-chain . One of the residues is involved in a salt bridge in the L-chain, and we concluded that the stability difference between H- and L-ferritins is to a large extent due to the stabilizing effect of this salt bridge on the L-subunit fold. J Biol Chem, 1992 Jul 15, 267(20), 13986 - 92 cDNA cloning and molecular characterization of MSE55, a novel human serum constituent protein that displays bone marrow stromal/endothelial cell-specific expression; Bahou WF et al.; Hemonectin is a lineage-specific cytoadhesive protein that may be involved in the developmentally regulated adhesion of granulocytic cells to bone marrow stroma . Immunoblot analysis using an anti-hemonectin antibody recognizes two distinct immunoreactive species in endothelial cell lysates (approximately M(r) 65,000) and human serum (approximately M(r) 55,000) . Initial characterization of the 55-kDa protein has now been completed by isolating the cDNA from a human endothelial cell expression library . Sequence analysis of overlapping clones identifies a composite sequence spanning 2030 nucleotides with an open reading frame of 1173 base pairs . No significant sequence similarity was observed on analysis of current GenBank databases . The open reading frame was expressed as a recombinant protein in Escherichia coli and used as an immunogen for the production of a specific polyclonal antibody . Immunoblotting with this antibody identifies a single immunoreactive species of apparent M(r) 55,000 in HUVEC lysates and human serum, confirming that a secreted form normally circulates as a serum constituent protein . This antibody fails to recognize purified hemonectin, suggesting that the M(r) 55,000 protein is not hemonectin . Cross-species Southern blot analysis reveals persistent hybridizing fragments in all species tested, suggestive of a developmentally conserved function . Northern blot analysis demonstrates expression limited to endothelial and bone marrow stromal cells, but not poly(A) RNA from monkey liver, spleen, brain, lung, and kidney . On this basis, we have designated this novel protein MSE55, for marrow stromal/endothelial cell protein with a molecular mass of 55,000 daltons . Its tissue-specific expression may suggest a functional role in hematopoiesis. J Biol Chem, 1992 Jul 15, 267(20), 13843 - 50 Deletion of lactose repressor carboxyl-terminal domain affects tetramer formation; Chen J et al.; The carboxyl-terminal sequence of the lac repressor protein contains heptad repeats of leucines at positions 342, 349, and 356 that are required for tetramer assembly, as substitution of these leucine residues yields solely dimeric species (Chakerian, A . E., Tesmer, V . M., Manly, S . P., Brackett, J . K., Lynch, M . J., Hoh, J . T., and Matthews, K . S . (1991) J . Biol . Chem . 266, 1371-1374; Alberti, S., Oehler, S., von Wilcken-Bergmann, B., Kramer, H., and Muller-Hill, B . (1991) New Biol . 3, 57-62) . To further investigate this region, which may form a leucine zipper motif, a family of lac repressor carboxyl-terminal deletion mutants eliminating the last 4, 5, 11, 18, and 32 amino acids (aa) has been constructed . The -4 aa mutant, in which all of the leucines in the presumed leucine zipper are intact, is tetrameric and displays operator and inducer binding properties similar to wild-type repressor . The -5 aa, -11 aa, -18 aa, and -32 aa deletion mutants, depleted of 1, 2, or all 3 of the leucines in the heptad repeats, are all dimeric, as demonstrated by gel filtration chromatography . Circular dichroism spectra and protease digestion studies indicate similar secondary/tertiary structures for the mutant and wild-type proteins . Differences in reaction with a monoclonal antibody specific for a subunit interface are observed for the dimeric versus tetrameric proteins, indicative of exposure of the target epitope as a consequence of deletion . Inducer binding properties of the deletion mutants are similar to wild-type tetrameric repressor at neutral pH . Only small differences in affinity and cooperativity from wild-type are evident at elevated pH; thus, the cooperative unit within the tetramer appears to be the dimer . "Apparent" operator binding affinity for the dimeric proteins is diminished, although minimal change in operator dissociation rate constants was observed . The diminution in apparent operator affinity may therefore derive from either 1) dissociation of the dimeric mutants to monomer generating a linked equilibrium or 2) alterations in intrinsic operator affinity of the dimers; the former explanation is favored . This detailed characterization of the purified mutant proteins confirms that the carboxyl-terminal region is involved in the dimer-dimer interface and demonstrates that cooperativity for inducer binding is contained within the dimer unit of the tetramer structure. Biochim Biophys Acta, 1992 Jul 15, 1131(3), 307 - 10 A rapid procedure for production of human basic fibroblast growth factor in Escherichia coli cells; Ke Y et al.; Human basic fibroblast growth factor has been expressed in Escherichia coli cells at a level of 2-3 mg/l culture, using a rapid procedure which requires only simple DNA manipulative work . The recombinant material has the same potency as natural basic fibroblast growth factor from bovine pituitary glands. J Immunol, 1992 Jul 15, 149(2), 454 - 60 Cloning and expression of recombinant Aspergillus fumigatus allergen I/a (rAsp f I/a) with IgE binding and type I skin test activity; Moser M et al.; Aspergillus fumigatus secretes an 18-kDa nonglycosylated IgE-binding protein . This protein was previously shown to be a ribotoxin, like alpha-sarcin and mitogillin . A 686-bp long A . fumigatus cDNA encoding an 18-kDa ribotoxin was cloned and expressed in Escherichia coli as a fusion protein with six adjacent histidines (rAsp f I/a) . rAsp f I/a was purified to homogeneity by Ni(2+)-chelate affinity chromatography and refolded . The recombinant protein was enzymatically active resulting in the cleavage of 28S rRNA within a universally conserved region . rAsp f I/a was cytotoxic for EBV immortalized or PHA stimulated human PBMC . Furthermore, rAsp f I/a was recognized by murine mAb made against an 18-kDa ribotoxin . IgE of individuals allergic to A . fumigatus bound to rAsp f I/a as shown by ELISA, dot blots, and Western blots . rAsp f I/a elicited positive immediate type I skin reactions in individuals allergic to A . fumigatus but not in healthy control individuals . The results show that rAsp f I/a has similar functional characteristics when compared to the native 18-kDa ribotoxin . rAsp f I/a expressed in E . coli can therefore be used as a standardized Ag/allergen for serologic and clinical diagnosis of A . fumigatus-associated diseases. Eur J Biochem, 1992 Jul 15, 207(2), 463 - 70 Cloning, sequencing and in vivo expression of genes encoding the F0 part of the sodium-ion-dependent ATP synthase of Propionigenium modestum in Escherichia coli; Kaim G et al.; A DNA fragment containing the genes encoding subunits of the F0 part of the sodium-translocating ATPase of Propionigenium modestum was cloned in Escherichia coli and sequenced . The predicted amino acid sequences of subunits a, b and c of the P . modestum ATPase were compared with those of the corresponding subunits of proton-translocating ATPases from other bacteria and chloroplasts . Deletion mutants of E . coli, lacking different genes for ATPase subunits, were transformed with a recombinant plasmid, containing the genes for the subunits a, c, b, delta and part of alpha of the ATPase of P . modestum . Functionally reconstituted ATPase activity could be demonstrated for the transformants . The identity of the vector containing P . modestum genes was verified by restriction analysis of plasmid DNA. J Biol Chem, 1992 Jul 15, 267(20), 14175 - 82 Protein-protein interaction studied by site-directed mutagenesis . Characterization of the annexin II-binding site on p11, a member of the S100 protein family; Kube E et al.; p11, a member of the S100 protein family, forms a stable heterotetrameric complex with annexin II . The p11-binding site of annexin II resides in the N-terminal 14 residues, which form an amphiphatic alpha-helix with the hydrophobic face representing the contact site for p11 (Johnsson, N., Marriott, G., and Weber, K . (1988) EMBO J . 7, 2435-2442) . We show that a corresponding peptide can be used to purify recombinant p11 by affinity chromatography . To map the annexin II-binding site on p11, we have produced progressively truncated p11 derivatives by site-directed mutagenesis . Our analysis reveals that a highly hydrophobic region between residues 85 and 91 is indispensable for annexin II-binding . It is located in the C-terminal extension, following the second distorted EF-hand . Using a series of single amino acid replacements, we have identified individual hydrophobic residues, which seem to represent contact points for annexin II . Most notably, substitution of tyrosine 85 or phenylalanine 86 by alanine drastically reduces the affinity of p11 for annexin II, whereas replacement of these residues by tryptophan has no or only a marginal effect . Thus, hydrophobic side chains on both annexin II and p11 are involved in complex formation. Biochem Biophys Res Commun, 1992 Jul 15, 186(1), 483 - 90 A gradient in expression of the Escherichia coli heat-stable enterotoxin receptor exists along the villus-to-crypt axis of rat small intestine; Cohen MB et al.; Binding of Escherichia coli heat-stable enterotoxin to its receptor is critical to the initiation of toxin-induced secretion and diarrheal disease; it is also likely, however, that this receptor binds an endogenous ligand . In order to characterize the expression of the heat-stable enterotoxin receptor in the small intestine, we isolated epithelial cells from villus tip to crypt in rat jejunum and ileum . Binding of radiolabeled toxin was maximal in the villus preparations and gradually decreased along the villus-to-crypt axis, paralleling the decline of sucrase activity . Northern blots of total RNA identified a single heat stable enterotoxin receptor transcript (3.8 kb), predominantly in the villus cell fractions . In situ hybridization demonstrated clear signal in the villus cells with no apparent signal in the crypt cells, lamina propria or muscularis . Expression of this receptor was greatest after enterocytes leave the proliferative cycle and enter villi . This pattern of gene and protein expression may reflect a role of this receptor in binding endogenous ligands which in turn may regulate intestinal ion flux along the villus-to-crypt axis. J Biol Chem, 1992 Jul 15, 267(20), 14429 - 35 Excision of 5'-terminal deoxyribose phosphate from damaged DNA is catalyzed by the Fpg protein of Escherichia coli; Graves RJ et al.; Homogeneous Fpg protein of Escherichia coli has DNA glycosylase activity which excises some purine bases with damaged imidazole rings, and an activity excising deoxyribose (dR) from DNA at abasic (AP) sites leaving a gap bordered by 5'- and 3'-phosphoryl groups . In addition to these two reported activities, we show that the Fpg protein also catalyzes the excision of 5'-terminal deoxyribose phosphate (dRp) from DNA, which is the principal product formed by the incision of AP endonucleases at abasic sites . Moreover, the rate of the Fpg protein catalysis for the 2,6-diamino-4-hydroxy-5-formamidopyrimidine-DNA glycosylase activity is slower than the activities excising dR from abasic sites and dRp from abasic sites preincised by endonucleases . The product released by the Fpg protein in the excision of 5'-terminal dRp from an abasic site preincised by an AP endonuclease is a single base-free unsaturated dRp, suggesting that the excision results from beta-elimination . The release of 5'-terminal dRp by crude extracts of E . coli from wild type and fpg-mutant strains shows that the Fpg protein is one of the major EDTA-resistant activities catalyzing this reaction. J Biol Chem, 1992 Jul 15, 267(20), 13998 - 4004 In vitro expression of thyroxine-binding globulin (TBG) variants . Impaired secretion of TBGPRO-227 but not TBGPRO-113; Janssen OE et al.; Thyroxine-binding globulin (TBG) is a glycoprotein that transports thyroid hormones in blood . Of two naturally occurring variants in man that harbor single proline substitutions (TBG-CD5 and TBG-Montreal), only TBG-CD5 manifests as complete TBG deficiency . In order to determine the pathophysiology of these TBG disorders, we expressed TBG-CD5 and TBG-Montreal (TBG-M), as well as the common type TBG (TBG-C) in reticulocyte lysate and Xenopus oocytes . Vectors encoding the three TBG types were constructed, transcribed in vitro, and their products of cell-free translation and processing by canine microsomal membranes were analyzed . TBG-C and TBG-M had identical mobility on denaturing polyacrylamide gel electrophoresis but could be distinguished by differences in thyroxine (T4) binding . TBG-CD5 had altered electrophoretic mobility and did not bind T4 . TBG-C and TBG-M expressed in microinjected Xenopus oocytes showed properties similar to their respective serum forms, whereas TBG-CD5 was found in small amounts only intracellularly . Our results confirm that the previously described alanine 113 to proline substitution is responsible for the altered properties of TBG-M . The substitution of leucine 227 by proline in TBG-CD5 appears to impair its cotranslational processing and secretion. J Biol Chem, 1992 Jul 15, 267(20), 13879 - 83 The effects of cysteine mutations on the catalytic activities of the reverse transcriptase of human immunodeficiency virus type-1; Loya S et al.; The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) has only 2 cysteine residues at positions 38 and 280 . In order to investigate the role of these cysteines in the structure and function of the enzyme, we have previously modified each of the cysteines to serines employing site-directed mutagenesis . Two of the mutant forms of HIV-1 RT, the single mutant of cysteine 280 and a double mutant with both cysteines modified, were purified . In the present study we have compared the catalytic properties of the DNA-polymerizing and the ribonuclease H (RNase H) functions of the two mutant RTs to those of the native enzyme . The results indicate that the single mutant RT closely resembles the wild type enzyme in almost all the catalytic functions tested . The double cysteine mutant RT, on the other hand, exhibits several unique features . First, the specific activities of the RNA- and DNA-directed DNA synthesis are significantly lower than the corresponding activities of the other two enzymes . This probably results from the lower Vmax values exhibited by the double mutant RT, since the Km values calculated for all enzymes were similar . Second, the most outstanding differences are associated with the RNase H activity of the double mutant RT . The specific activity of RNase H is about 4-fold higher than the wild type and the single mutant RTs . Furthermore, the heat stability of the RNase H function of the double mutated RT is at least 15-fold higher than that of the other two RTs . The substantial resistance to heat denaturation is apparent only for the RNase H activity, since the DNA polymerizing function of the double mutant RT is as sensitive to heat denaturation as the other two proteins. J Biol Chem, 1992 Jul 15, 267(20), 13823 - 9 Cell-free synthesis of the branched RNA-linked msDNA from retron-Ec67 of Escherichia coli; Hsu MY et al.; msDNA-Ec67 is produced in a clinical strain of Escherichia coli and composed of a 67-base single-stranded DNA, which is linked to the 2'-OH group of the 15th rG residue of a 58-base RNA molecule by a 2',5'-phosphodiester linkage (Lampson, B . C., Sun, J., Hsu, M.-Y., Vallejo-Ramirez, J., Inouye, S., and Inouye, M . (1989) Science 243, 1033-1038) . The production of msDNA-Ec67 is dependent upon retron-Ec67, which consists of the msr-msd region and the gene for reverse transcriptase (RT) . These two elements were separately cloned into plasmids; p67-BHO.6 contained the msr-msd region and pRT-67 contained the RT gene under the lpp-lac promoter-operator . msDNA-Ec67 was produced only when cells were transformed with both plasmids . In addition, msDNA-Ec67 was synthesized in a cell-free system using total RNA prepared from cells harboring plasmid p67-BHO.6 and purified Ec67-RT . Using this cell-free system, the priming reaction, during initiation of DNA synthesis, was demonstrated to be a specific template-directed event; only dTTP was incorporated into a 132-base precursor RNA yielding a 133-base compound . This specific dT addition could be altered to dA or dC by simply substituting the 118th A residue of the putative msr-msd transcript with a T or G residue . The priming reaction was blocked when A was substituted for G at the 15th residue of the precursor RNA transcript, which corresponds to the branched rG residue in msDNA . DNA chain elongation could be terminated by adding ddNTP in the cell-free system, forming a sequence ladder . The DNA sequence determined from this ladder completely agreed with the msDNA sequence . The RT extension reaction was completely blocked when the RNA preparation was treated with RNase A but not when the preparation was treated with DNase . This clearly demonstrates that RNA but not DNA is responsible for the msDNA production . A part of the fully extended cell-free product contained a 13-base RNA strand resistant to RNase A, which is consistent with the previously proposed model . In this model, the 5'-end sequence of the msr-msd transcript (a2; bases 1-13) forms a duplex with the 3'-end sequence (a1) of the same transcript, thus serving as a primer, as well as a template for msDNA synthesis by RT . Our results are inconsistent with a model recently proposed by Lease and Yee (Lease, R . A., and Yee, T . (1991) J . Biol . Chem . 266, 14497-14503). Biochem J, 1992 Jul 15, 285 ( Pt 2), 503 - 6 Inhibition of Escherichia coli DNA topoisomerase I activity by phospholipids; Mizushima T et al.; The DNA relaxation activity of Escherichia coli DNA topoisomerase I in vitro was greatly inhibited by cardiolipin . Inhibition also occurred to some extent with phosphatidylglycerol from egg yolk . Analysis with synthetic phospholipid revealed that phosphatidylglycerol containing unsaturated fatty acids exhibited a strong inhibitory effect, whereas inhibition by phosphatidylglycerol containing saturated fatty acids was weak . Phosphatidylethanolamine showed no inhibitory effect . Chlorpromazine, which interacts with phospholipids, suppressed the inhibitory effect of cardiolipin . Cardiolipin and phosphatidylglycerol with unsaturated fatty acid precipitated topoisomerase I even at low concentrations, whereas phosphatidylglycerol from egg yolk and a synthetic phosphatidylglycerol containing saturated fatty acids precipitated this enzyme only at high concentrations . One-third of the total topoisomerase I in E . coli was found in the membrane fraction . Treatment of E . coli cells with chlorpromazine resulted in relaxation of plasmid DNA . This DNA relaxation was not observed in a topA mutant, suggesting that this relaxation by chlorpromazine in vivo is catalysed by topoisomerase I. Eur J Biochem, 1992 Jul 15, 207(2), 479 - 85 Escherichia coli Rep protein and helicase IV . Distributive single-stranded DNA-dependent ATPases that catalyze a limited unwinding reaction in vitro; Yancey-Wrona JE et al.; Rep protein and helicase IV, two DNA-dependent adenosine 5'-triphosphatases with helicase activity, have been purified from Escherichia coli and characterized . Both enzymes exhibit a distributive interaction with single-stranded DNA as DNA-dependent ATPases in a reaction that is relatively resistant to increasing NaCl concentration and sensitive to the addition of E . coli single-stranded DNA binding protein (SSB) . The helicase reaction catalyzed by each protein has been characterized using a direct unwinding assay and partial duplex DNA substrates . Both Rep protein and helicase IV catalyzed the unwinding of a duplex region 71 bp in length . However, unwinding of a 119-bp or 343-bp duplex region was substantially reduced compared to unwinding of th