Biochemistry, 1992 Jan 14, 31(1), 32 - 9 Contribution to catalysis and stability of the five cysteines in Escherichia coli aspartate aminotransferase . Preparation and properties of a cysteine-free enzyme; Gloss LM et al.; The five cysteines, at positions 82, 191, 192, 270, and 401, of Escherichia coli aspartate aminotransferase (AATase) were, individually and in some combinations, converted to alanine by site-directed mutagenesis (C82A, C191A, C192A, C270A, C401A) . Cys-191, which is conserved in all AATase isozymes, was mutated to serine as well (C191S) . A quintuple mutant, with all cysteines converted to alanines (Quint), was also constructed . The effects of these single and multiple mutations were examined by steady-state kinetics and urea denaturation . The thermal stabilities of Quint and of the wild-type enzyme (WT) were determined by differential scanning calorimetry . The mutants had kcat values up to 50% greater than that of WT and KMAsp and KM alpha-KG values up to 1.5- and 3.3-fold higher than that of WT . The mutants C82A and C191A exhibit nearly the same CM in urea denaturation experiments as WT, while the other single mutants and Quint are less stable, with CM differences of up to 0.7 M urea . Quint is also less thermostable than WT, with a delta TM of 3.3-4.4 degrees C . Thus the five cysteine replacements yield small, but significant, changes in catalytic and denaturation parameters, but none of the cysteines was found to be essential . The changes manifested in the mutation of the conserved Cys-191 to alanine are no greater than those observed with the four nonconserved cysteines . We consider the evolutionary implications of these findings. Biochemistry, 1992 Jan 14, 31(1), 155 - 62 Serine hydroxymethyltransferase: origin of substrate specificity; Angelaccio S et al.; All forms of serine hydroxymethyltransferase, for which a primary structure is known, have five threonine residues near the active-site lysyl residue (K229) that forms the internal aldimine with pyridoxal phosphate . For Escherichia coli serine hydroxymethyltransferase each of these threonine residues has been changed to an alanine residue . The resulting five mutant enzymes were purified and characterized with respect to kinetic and spectral properties . The mutant enzymes T224A and T227A showed no significant changes in kinetic and spectral properties compared to the wild-type enzyme . The T225A and T230A enzymes exhibited differences in Km and kcat values but exhibited the same spectral properties as the wild-type enzyme . The four threonine residues at positions 224, 225, 227, and 230 do not play a critical role in the mechanism of the enzyme . The T226A enzyme had nearly normal affinity for substrates and coenzymes but had only 3% of the catalytic activity of the wild-type enzyme . The spectrum of the T226A enzyme in the presence of amino acid substrates showed a large absorption maximum at 343 nm with only a small absorption band at 425 nm, unlike the wild-type enzyme whose enzyme-substrate complexes absorb at 425 nm . Rapid reaction studies showed that when amino acid substrates and substrate analogues were added to the T226A enzyme, the internal aldimine absorbing at 422 nm was rapidly converted to a complex absorbing at 343 nm in a second-order process . This was followed by a very slow first-order formation of a complex absorbing at 425 nm.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1992 Jan 14, 31(1), 106 - 10 Substrate specificity of the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase): excision of purine lesions in DNA produced by ionizing radiation or photosensitization; Boiteux S et al.; We have investigated the excision of a variety of modified bases from DNA by the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase) {Boiteux, S., O'Connor, T . R., Lederer, F., Gouyette, A., & Laval, J . (1990) J . Biol . Chem . 265, 3916-3922} . DNA used as a substrate was modified either by exposure to ionizing radiation or by photosensitization using visible light in the presence of methylene blue (MB) . The technique of gas chromatography/mass spectrometry, which can unambiguously identify and quantitate pyrimidine- and purine-derived lesions in DNA, was used for analysis of hydrolyzed and derivatized DNA samples . Thirteen products resulting from pyrimidines and purines were detected in gamma-irradiated DNA, whereas only the formation of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 8-hydroxyguanine (8-OH-Gua) was observed in visible light/MB-treated DNA . Analysis of gamma-irradiated DNA after incubation with the Fpg protein followed by precipitation revealed that the Fpg protein significantly excised 4,6-diamino-5-formamidopyrimidine (FapyAde), FapyGua, and 8-OH-Gua . The excision of a small but detectable amount of 8-hydroxyadenine was also observed . The detection of these products in the supernatant fractions of the same samples confirmed their excision by the enzyme . Nine pyrimidine-derived lesions were not excised . The Fpg protein also excised FapyGua and 8-OH-Gua from visible light/MB-treated DNA . The presence of these products in the supernatant fractions confirmed their excision.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1992 Jan 14, 31(1), 133 - 8 Catalytic domains of the LAR and CD45 protein tyrosine phosphatases from Escherichia coli expression systems: purification and characterization for specificity and mechanism; Cho H et al.; The cytoplasmic domains of two human transmembrane protein tyrosine phosphatases (PTPases), LAR and CD45, have been expressed in Escherichia coli, purified to near-homogeneity, and compared for catalytic efficiency toward several phosphotyrosine-containing peptide substrates . A 615-residue LAR fragment (LAR-D1D2) containing both tandemly repeated PTPase domains shows almost identical specific activity and high catalytic efficiency as the 40-kDa single-domain LAR-D1 fragment, consistent with a single functional active site in the 70-kDa LAR-D1D2 enzyme . A 90-kDa fragment of the human leukocyte CD45 PTPase, containing two similar tandemly repeated PTPase domains, shows parallel specificity to LAR-D1 and LAR-D1D2 with a high kcat/Km value for a phosphotyrosyl undecapeptide . Sufficient purified LAR-D1 and LAR-D1D2 PTPases were available to demonstrate enzymatic exchange of 18O from 18O4 inorganic phosphate into H2(16)O at rates of approximately 1 x 10(-2) s-1 . The oxygen-18 exchange probably proceeds via a phosphoenzyme intermediate . Brief incubation of all three PTPase fragments with a {32P}phosphotyrosyl peptide substrate prior to quench with SDS sample buffer and gel electrophoresis led to autoradiographic detection of 32P-labeled enzymes . Pulse/chase studies on the LAR 32P-enzyme showed turnover of the labeled phosphoryl group. Biochemistry, 1992 Jan 14, 31(1), 121 - 32 Physical properties of the Escherichia coli transcription termination factor rho . 2 . Quaternary structure of the rho hexamer; Geiselmann J et al.; Under approximately physiological conditions, the transcription termination factor rho from Escherichia coli is a hexamer of planar hexagonal geometry {Geiselmann, J., Yager, T . D., Gill, S . C., Calmettes, P., & von Hippel, P . H . (1992) Biochemistry (preceding paper in this issue)} . Here we describe studies that further define the quaternary structure of this hexamer . We use a combination of chemical cross-linking and treatment with mild denaturants to show that the fundamental unit within the rho hexamer is a dimer stabilized by an isologous (or pseudoisologous) bonding interface . Three identical dimers of rho interact via a second type of isologous bonding interface to yield a hexamer with C3 or D3 symmetry . Cross-linking and denaturation experiments definitely rule out C6 and C2 symmetry for the rho hexamer . Data from fluorescence quenching, lifetime, and energy transfer experiments also argue against C2 symmetry . The simplest symmetry assignment that is not contradicted by any experimental data is D3; thus we conclude that the rho hexamer has D3 symmetry . We also consider the positioning of the binding sites for RNA and ATP relative to the coordinate reference frame of the D3 hexamer . Fluorescence energy transfer data are presented and integrated with data from the literature to arrive at a self-consistent model for the quaternary structure of the rho hexamer. Biochemistry, 1992 Jan 14, 31(1), 111 - 21 Physical properties of the Escherichia coli transcription termination factor rho . 1 . Association states and geometry of the rho hexamer; Geiselmann J et al.; To function as a DNA-RNA helicase in rho-dependent transcript termination, six genetically identical subunits of the Escherichia coli transcription termination protein rho must first assemble into a hexameric complex . To help determine the quaternary structure of this complex, we have studied the association equilibria of the rho protomers . Sedimentation equilibrium, sedimentation velocity, diffusion, X-ray scattering, and neutron-scattering data have been combined to create a "phase diagram" of the association states of this protein as a function of protein concentration and ionic environment . The results show that rho exists predominantly as a hexamer under approximately physiological conditions and that this hexamer is in equilibrium with both lower and higher states of association that may also have physiological relevance . Small-angle X-ray scattering measurements and theoretical calculations indicate that the rho hexamer has a radius of gyration of 50 +/- 3 A . The radius of gyration measured by small-angle neutron scattering in 2H2O is 47 +/- 3 A . These scattering studies also support earlier models of rho as a planar hexagon which have been developed on the basis of electron microscopy . In the following paper in this issue {Geiselmann, J., Seifried, S . E., Yager, T . D., Liang, C., & von Hippel, P . H . (1992)}, these results are combined with information on symmetry, subunit interactions, and packing geometry to obtain a model of the quaternary structure of the functional rho hexamer. Biochemistry, 1992 Jan 14, 31(1), 22 - 6 Circular DNA molecules imaged in air by scanning force microscopy; Bustamante C et al.; Routine and reproducible imaging of DNA molecules in air with the scanning force microscope (SFM) has been accomplished . Circular molecules of plasmid DNA were deposited onto red mica and imaged under various relative humidities . In related experiments, the first images of the Escherichia coli RNA polymerase-DNA complex have also been obtained . This has been possible by (1) the use of specially modified SFM tips with a consistent radius of curvature of 10 nm or less, to minimize the amount of image distortion introduced by the finite dimensions of commercially available tips, (2) the optimization of a method to deposit and bind DNA molecules to the mica surface in a stable fashion, and (3) careful control of the sample humidity, to prevent solvation of the molecules and detachment from the surface by the scanning tip or stylus . Contact forces in the range of a few nanonewtons are routinely possible in air and in the presence of residual humidity . The spatial resolution of the images appears determined by the radius of curvature of the modified styli, which can be estimated directly from the apparent widths of the DNA molecules in the images. Biochemistry, 1992 Jan 14, 31(1), 12 - 6 Affinity purification of functional receptors for Escherichia coli heat-stable enterotoxin from rat intestine; Hugues M et al.; Active receptors for Escherichia coli heat-stable enterotoxin (ST) were partially purified by ligand-affinity chromatography . The affinity column was prepared by coupling ST to biotin derivatized with an extended N-hydroxysuccinylated spacer arm prior to binding to monomeric avidin immobilized on agarose . Detergent extracts of rat intestinal mucosa membranes were quantitatively depleted of ST binding activity when chromatographed on this affinity matrix . Biotinylated ST-receptor complexes were eluted from affinity columns with 2 mM biotin and these complexes quantitatively dissociated with bile salts . Using this technique, functional ST receptors were purified maximally about 2000-fold, with about 3% of the total activity in crude extracts recovered in these purified preparations . Analysis of affinity-purified preparations by polyacrylamide gel electrophoresis and silver staining demonstrated a major protein subunit of 74 kDa . Affinity cross-linking of these preparations to 125I-ST demonstrated specific labeling predominantly of the 74-kDa subunit . In addition, lower amounts of labeled ST were incorporated into subunits of 164 and 45 kDa, confirming the heterogeneous nature of ST receptors . Purified receptors bound ST in a concentration-dependent fashion, with an IC50 of 10(-9) M . These studies demonstrate that ligand-affinity chromatography can be employed to purify ST receptors . The availability of purified receptors will facilitate further studies of mechanisms underlying ST-induced intestinal secretion. Biotechniques . 1992 Jan;12(1):40, 42, 44. A rapid PCR method of screening for small mutations; Major JG Jr; We report a modified method of screening for point mutations using a PCR approach based upon the sensitivity of PCR to the 3' terminus of the primer . This method provides a sensitive screen when using either plasmid DNA or bacterial cell lysates . We have optimized the technique for general use to allow rapid screening of mutants with good discrimination . Unlike previous similar methods, this technique has no inherent limitation in primer design on the 3'-terminal base chosen. J Bacteriol, 1992 Jan, 174(2), 623 - 6 Molecular characterization of mutations affecting expression level and growth rate-dependent regulation of the Escherichia coli zwf gene; Rowley DL et al.; We characterized three cis dominant mutations which elevate glucose 6-phosphate dehydrogenase level . Growth rate-dependent regulation and oxidative stress control of enzyme level were altered by the mutations . DNA sequencing and transcript mapping showed that the "up" mutations created new promoters whose hyperactive expression overrides the normal regulation of the native promoter. J Bacteriol, 1992 Jan, 174(1), 92 - 101 Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations; Puziss JW et al.; It is believed that one or more basic residues at the extreme amino terminus of precursor proteins and the lack of a net positive charge immediately following the signal peptide act as topological determinants that promote the insertion of the signal peptide hydrophobic core into the cytoplasmic membrane of Escherichia coli cells with the correct orientation required to initiate the protein export process . The export efficiency of precursor maltose-binding protein (pre-MBP) was found to decrease progressively as the net charge in the early mature region was increased systematically from 0 to +4 . This inhibitory effect could be further exacerbated by reducing the net charge in the signal peptide to below 0 . One such MBP species, designated MBP-3/+3 and having a net charge of -3 in the signal peptide and +3 in the early mature region, was totally export defective . Revertants in which MBP-3/+3 export was restored were found to harbor mutations in the prlA (secY) gene, encoding a key component of the E . coli protein export machinery . One such mutation, prlA666, was extensively characterized and shown to be a particularly strong suppressor of a variety of MBP export defects . Export of MBP-3/+3 and other MBP species with charge alterations in the early mature region also was substantially improved in E . coli cells harboring certain other prlA mutations originally selected as extragenic suppressors of signal sequence mutations altering the hydrophobic core of the LamB or MBP signal peptide . In addition, the enzymatic activity of alkaline phosphatase (PhoA) fused to a predicted cytoplasmic domain of an integral membrane protein (UhpT) increased significantly in cells harboring prlA666 . These results suggest a role for PrlA/SecY in determining the orientation of signal peptides and possibly other membrane-spanning protein domains in the cytoplasmic membrane. J Gen Microbiol, 1992 Jan, 138 ( Pt 1), 23 - 30 Identification of expression signals of the mycobacteriophages Bxb1, L1 and TM4 using the Escherichia-Mycobacterium shuttle plasmids pYUB75 and pYUB76 designed to create translational fusions to the lacZ gene; Barletta RG et al.; Mycobacterial expression signals were cloned using specially constructed gene fusion shuttle plasmid probes carrying a truncated Escherichia coli lacZ (beta-galactosidase) gene which lacked a promoter, a ribosome binding site, and an ATG start codon . Libraries of mycobacteriophage Bxb1, L1 and TM4 DNAs were constructed, and introduced by electroporation into Mycobacterium smegmatis and the 'bacille Calmette-Guerin' (BCG) . Clones carrying mycobacterial expression sequences were detected by their blue colour or characteristic fluorescence when plated on media containing chromogenic or fluorogenic substrates . Varying degrees of beta-galactosidase expression were observed, and one Bxb1 expression signal was identified where beta-galactosidase expression is repressed in phage lysogens. Prostate, 1992, 20(2), 113 - 6 Prostate specific antigen and prostatitis . II . PSA production and release kinetics in vitro; Moon TD et al.; Elevations in serum prostate specific antigen (PSA) levels in patients with prostatitis are well known, but the pathophysiologic mechanisms involved with this phenomenon are poorly understood . We have recently evaluated the effect of prostatitis on PSA levels in primates . This data demonstrated a rapid rise in PSA, which subsequently fell along the biological decay curve . This suggested a release of sequestered PSA . We evaluated the effect of infection upon PSA production for the prostatic adenocarcinoma cell line LNCaP . The cell line was determined to produce 9.6 +/- 2.7 fg/cell/hr PSA with essentially linear kinetics . No effect was seen with dead bacteria, bacterial supernatant or complement upon the PSA production . Live E . coli had no effect for 4-8 hours at which time the cells sloughed and PSA production ceased . No release of stored PSA was seen . These data do not elucidate the reasons for increased serum PSA levels found with acute bacterial prostatitis, but indicate that it is not a storage phenomenon. J Ind Microbiol, 1992 Jan, 9(1), 1 - 9 Effect of the levels of dissolved oxygen on the expression of recombinant proteins in four recombinant Escherichia coli strains; Li X et al.; Four recombinant strains of Escherichia coli were examined for the effects of the dissolved oxygen level on the level of biomass, the plasmid content, and the level of recombinant protein at the stationary phase of batch growth . Strains JM101/pYEJ001, and TB-1/pYEJ001 (encoding chloramphenicol acetyltransferase), and strain TB-1/p1034, and TB-1/pUC19 (encoding beta-galactosidase) were grown at the constant dissolved oxygen levels of 0, 50, and 100% air saturation, as well as in the absence of dissolved oxygen control . The biomass of all strains under constant aerobic conditions was 12-36 times higher than that under anaerobic conditions, but was the same as or slightly higher than that without dissolved oxygen control . The plasmid content in all strains under aerobic conditions was 2.9-11.7 times higher than that under aerobic conditions . The optimal dissolved oxygen concentration for the specific activity of recombinant proteins was dependent upon the strain . In no strain were constant aerobic conditions optimal . However, because of the effect on biomass, controlled aerobic conditions were optimal for the volumetric activity of recombinant protein in all but one strain. J Bacteriol, 1992 Jan, 174(2), 514 - 24 Translational control of pyrC expression mediated by nucleotide-sensitive selection of transcriptional start sites in Escherichia coli; Wilson HR et al.; Expression of the pyrC gene, which encodes the pyrimidine biosynthetic enzyme dihydroorotase, is negatively regulated by pyrimidine availability in Escherichia coli . To define the mechanism of this regulation, an essential regulatory region between the pyrC promoter and the initial codons of the pyrC structural gene was identified . Mutational analysis of this regulatory region showed that the formation of a hairpin at the 5' end of the pyrC transcript, which overlaps the pyrC ribosome binding site, is required for repression of pyrC expression . Formation of the hairpin appears to be controlled by nucleotide-sensitive selection of the site of pyrC transcriptional initiation . When the CTP level is high, the major pyrC transcript is initiated with this nucleotide at a position seven bases downstream of the pyrC -10 region . This transcript is capable of forming a stable hairpin at its 5' end . When the CTP level is low and the GTP level is high, conditions found in cells limited for pyrimidines, the major pyrC transcript is initiated with GTP at a position two bases further downstream . This shorter transcript appears to be unable to form a stable hairpin at its 5' end . These results suggest a model for regulation in which the longer pyrC transcripts are synthesized predominantly under conditions of pyrimidine excess and form the regulatory hairpin, which blocks pyrC translational initiation . In contrast, the shorter pyrC transcripts are synthesized primarily under conditions of pyrimidine limitation, and they are readily translated, resulting in a high level of dihydroorotase synthesis . The data also indicate that a low level of pyrimidine-mediated regulation may occur at the level of transcriptional initiation. Acta Chir Hung, 1992-93, 33(1-2), 197 - 208 Protective effect of radio-detoxified endotoxin (Tolerin) on the ultrastructure of pancreas in experimental endotoxin shock of rats; Bende S Jr et al.; The ultrastructural changes of pancreas exocrine cells were studied after the intravenous administration of endotoxin (LPS) or radio-detoxified endotoxin (150 kGy 60Co-gamma irradiated: RD-LPS or Tolerin) . The LPS (1 mg/rat) induces an autolytic destruction in the membranes of the mitochondria of the pancreas exocrine cells . The RD-LPS given in similar dose does not produce any autolytic change . However, a small dose (100/micrograms/rat) of RD-LPS (Tolerin) as a pretreatment can protect the autolytic destruction of the mitochondria induced by LPS . This may be attributed to the membrane stabilizing effect of RD-LPS. Res Microbiol, 1992 Jan, 143(1), 5 - 14 Effect of Bdellovibrio bacteriovorus infection on the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli: evidence for activation of cytoplasmic proteolysis; Romo AJ et al.; Intact cells of Bdellovibrio bacteriovorus strain 109J were found to be incapable of taking up 14C-methyl alpha-glucoside, mannitol or fructose, and extracts derived from these cells exhibited negligible activities of the protein components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) . Escherichia coli strain ML35 cells exhibited high in vivo sugar uptake activities that were progressively lost over a period of 2 h at 30 degrees C following the entry of B . bacteriovorus into the periplasm of E . coli . In vitro complementation assays revealed that the E . coli PTS enzymes, enzyme I, HPr, and the glucose- and mannitol-specific enzymes II, were all lost almost in parallel with the disappearance of uptake activity . Thus, loss of activity in vivo was not due to membrane leakiness, energy depletion, or preferential inhibition or inactivation of any one protein component of the PTS . Instead, loss of PTS activity was attributed to digestion of the protein constituents of the system by proteases present in the cytoplasm of the host cell after bdellovibrio entry . Both ethylenediaminetetraacetate and phenylmethylsulphonyl fluoride partially protected against inactivation in vitro, and the two inhibitors together gave full protection, suggesting that both metallo- and seryl-proteases were responsible for the inactivation . Protease activity increased progressively with time following bdellovibrio entry and appeared to degrade the E . coli PTS enzymes in vivo . Preliminary evidence suggested that the proteases responsible for PTS enzyme degradation may be encoded by the B . bacteriovorus chromosome. J Membr Biol, 1992 Jan, 125(1), 81 - 91 Evidence that plasma membrane electrical potential is required for vesicular stomatitis virus infection of MDCK cells: a study using fluorescence measurements through polycarbonate supports; Akeson M et al.; We used fluorescence microscopy of Madin-Darby Canine Kidney (MDCK) cells grown on polycarbonate filters to study a possible link between plasma membrane electrical potential (delta psi pm) and infectivity of vesicular stomatitis virus (VSV) . Complete substitution of K+ for extracellular Na+ blocks VSV infection of MDCK cells as well as baby hamster kidney (BHK) cells . When we independently perfused the apical and basal-lateral surfaces of high resistance monolayers, high K+ inhibited VSV infection of MDCK cells only when applied to the basal-lateral side; high K+ applied apically had no effect on VSV infection . This morphological specificity correlates with a large decrease in delta psi pm of MDCK cells when high K+ buffer is perfused across the basal-lateral surface . Depolarization of the plasma membrane by 130 mM basal K+ causes a sustained increase of cytosol pH in MDCK cells from 7.3 to 7.5 as reported by the fluorescent dye BCECF . Depolarization also causes a transient increase of cytosol Ca2+ from 70 to 300 nM as reported by the dye Fura-2 . Neither increase could explain the block of VSV infectivity by plasma membrane depolarization . One alternative hypothesis is that delta psi pm facilitates membrane translocation of viral macromolecules as previously described for colicins, mitochondrial import proteins, and proteins secreted by Escherichia coli. Nucleic Acids Res, 1992 Jan 11, 20(1), 41 - 8 In vitro cleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins; Scherzinger E et al.; We have used purified RSF1010 mobilization proteins to reproduce in vitro a strand-specific nicking at the plasmid origin of transfer, oriT . In the presence of Mg2+, the proteins MobA (78-kDa form of RSF1010 DNA primase), MobB, and MobC and supercoiled or linear duplex oriT DNA form large amounts of a cleavage complex, which is characterized by its sensitivity to protein-denaturant treatment . Upon addition of SDS to such a complex, a single strand break is generated in the DNA, and MobA is found linked to the 5' nick terminus, presumably covalently . The double-strand nicking activity of MobA requires, in addition to Mg2+, the presence of MobC and is stimulated by the presence of MobB . The nick site has been shown by DNA sequencing to lie at the position cleaved in vivo during transfer, between nucleotides 3138/3139 in the r strand of RSF1010 . We have found that MobA will also cleave DNA at sites other than oriT if the DNA is present in single-stranded form . Breakage in this case occurs in the absence of denaturing conditions, and after prolonged incubation, reclosure can be demonstrated. Nucleic Acids Res, 1992 Jan 11, 20(1), 105 - 9 Inhibition of DNA synthesis at the hemimethylated pBR322 origin of replication by a cell membrane fraction; Malki A et al.; The replication of both ColE1-type plasmids and plasmids bearing the origin of replication of the Escherichia coli chromosome (oriC) has been shown to be inhibited by hemimethylation of adenine residues within GATC sequences . In the case of oriC plasmids, this inhibition was previously shown to be mediated by the specific affinity of the hemimethylated origin DNA for an outer cell membrane fraction . Here, we suggest that a similar mechanism is operating in the case of the ColE1-like plasmid pBR322 as (i) a hemimethylated DNA fragment carrying the promoter for the RNA which primes DNA synthesis (RNAII) is specifically bound by the same membrane fraction and, (ii) the addition of the membrane fraction to a soluble assay of pBR322 replication results in preferential inhibition of initiation on the hemimethylated template . We suggest that membrane sequestration of hemimethylated origin DNA and/or associated replication genes following replication may be a common element restricting DNA replication to precise moments in the cell cycle. Cell, 1992 Jan 10, 68(1), 133 - 42 A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomains of an alpha 2 dimer; Smith DL et al.; DNA recognition sequences for dimeric proteins typically contain two types of information . The first is the DNA sequence of each half-site, and the second is the arrangement of these half-sites . We show that dimers of the yeast homeodomain protein alpha 2, although able to read the first type of information, lack the ability to assess the second type . Rather, alpha 2 dimers bind with equal affinity to artificial operators in which the two half-sites are arrayed as inverted repeats, as direct repeats, or as everted (inside-out) repeats . We show that a second protein-MCM1-sets the exact spacing and orientation of the homeodomains in the alpha 2 dimer so that they accommodate only the geometry of the naturally occurring operators . These experiments show directly how the target specificity of a homeodomain protein is raised by an auxiliary protein, allowing it to distinguish the biologically correct operators from closely related sequences in the cell. Science, 1992 Jan 10, 255(5041), 203 - 6 Dimerization of a specific DNA-binding protein on the DNA; Kim B et al.; Many specific DNA-binding proteins bind to sites with dyad symmetry, and the bound form of the protein is a dimer . For some proteins, dimers form in solution and bind to DNA . LexA repressor of Escherichia coli has been used to test an alternative binding model in which two monomers bind sequentially . This model predicts that a repressor monomer should bind with high specificity to an isolated operator half-site . Monomer binding to a half-site was observed . A second monomer bound to an intact operator far more tightly than the first monomer; this cooperativity arose from protein-protein contacts. Science, 1992 Jan 10, 255(5041), 197 - 200 Site-specific incorporation of novel backbone structures into proteins; Ellman JA et al.; A number of unnatural amino acids and amino acid analogs with modified backbone structures were substituted for alanine-82 in T4 lysozyme . Replacements included alpha,alpha-disubstituted amino acids, N-alkyl amino acids, and lactic acid, an isoelectronic analog of alanine . The effects of these electronic and structural perturbations on the stability of T4 lysozyme were determined . The relatively broad substrate specificity of the Escherichia coli protein biosynthetic machinery suggests that a wide range of backbone and side-chain substitutions can be introduced, allowing a more precise definition of the factors affecting protein stability. Biochim Biophys Acta, 1992 Jan 9, 1118(2), 107 - 15 Renaturation of cobra venom phospholipase A2 expressed from a synthetic gene in Escherichia coli; Kelley MJ et al.; Cobra venom (Naja naja naja) phospholipase A2 (PLA2) contains 14 cysteines in the form of 7 disulfide bonds amongst its 119 amino acids . A gene encoding the PLA2 was synthesized and inserted into a bacterial expression vector containing the phage lambda pL promoter . In order to obtain protein without the initiating methionine at the N-terminus, a Factor Xa site was engineered upstream from the PLA2 gene . Upon heat-induction of the cells transformed with the expression plasmid, the protein is produced as insoluble inclusion bodies . The enzyme was partially purified by washing the inclusion bodies with Triton X-100 and urea . The expressed protein was first denatured with 8 M guanidine-HCl and 10 mM DTT . After digestion with Factor Xa, formation of disulfide bonds and refolding into the fully active form was carried out in the presence of cysteine and Ca2+ . The renatured recombinant protein was purified by Affi-gel blue column chromatography . The purified recombinant enzyme had the same specific activity as the native enzyme when assayed on a variety of substrates and cross-reacted with antisera prepared against the native enzyme . This is the first report of the expression of a recombinant PLA2 from any venom. J Theor Biol, 1992 Jan 7, 154(1), 91 - 107 Phospholipid domains determine the spatial organization of the Escherichia coli cell cycle: the membrane tectonics model; Norris V; Escherichia coli normally divides at its equator between segregated nucleoids . Such division is inhibited during perturbations of chromosome replication (even in the absence of inducible division inhibitors); eventually, division resumes at sites which are not at this equator . Escherichia coli will also divide at its poles to generate minicells following overproduction of the FtsZ or MinE proteins . The mechanisms underlying the division inhibition and the positioning of the division sites are unknown . In the membrane tectonics model, I propose that the formation of phospholipid domains within the cytoplasmic membrane positions division sites . The particular phospholipid composition of a domain attracts particular proteins and determines their activity; conversely, particular proteins change the composition of domains . Principally via such proteins, the interaction of the chromosome with the membrane creates a chromosomal domain . The development of chromosomal domains during replication and nucleoid formation contributes to the formation and positioning of a septal domain between them . During septation (cell division), this septal domain matures into a polar domain . Each domain attracts and activates different enzymes . The septal domain attracts and activates enzymes necessary for septation . Preventing the formation of the septal domain by preventing chromosome replication prevents normal division . Altering the composition of the polar domain may allow septation enzymes to function there and generate minicells . A corollary of the model explains how the formation of an origin domain by the attachment of hemi-methylated origin DNA to the membrane may underlie the creation and migration of structures within the envelope, the periseptal annuli. Biochim Biophys Acta, 1992 Jan 6, 1129(2), 177 - 82 Selective inhibition of the polypeptide chain elongation in eukaryotic cells; Tujebajeva RM et al.; The effect of Cephalotaxus alkaloids--homoharringtonine and cephalotaxine--on translation in a cell-free system from rabbit reticulocytes and on phenylalanine polymerisation by human ribosomes was studied . The effect of the alkaloids on the nonenzymatic and the eEF-1-dependent Phe-tRNA(Phe) binding to poly(U)-programmed 80S ribosomes, diphenylalanine synthesis accompanying nonenzymatic Phe-tRNA(Phe) binding and acetylphenylalanyl-puromycin formation was examined . Homoharringtonine was shown to inhibit the formation of diphenylalanine and acetylphenylalanyl-puromycin catalysed by human and rat liver ribosomes, but was inactive as an inhibitor on the E . coli elongation system . Neither nonenzymatic nor enzymatic Phe-tRNA(Phe) binding was noticeably affected by the alkaloid . It has been proposed that the site of homoharringtonine binding to 80S ribosomes should overlap or coincide with the acceptor site of the ribosomal peptidyl transferase centre . The association constant of homoharringtonine for 80S human ribosomes was estimated to be (2.57 +/- 0.33).10(7) M-1 in the presence of puromycin . Cephalotaxine did not exert a significant influence on the polypeptide chain elongation. Biochim Biophys Acta, 1992 Jan 6, 1129(2), 161 - 5 Enhancement of DNA transfection efficiency by heat treatment of cultured mammalian cells; Takai T et al.; The expression of genes introduced into various mammalian cell lines was enhanced by raising the temperature of the cells to 42 degrees C for a few hours after DNA transfection . This heat treatment resulted in an up to 10-fold increase in the frequency of the cells that transiently expressed a foreign gene such as that of beta-galactosidase, whereas it had only a limited enhancing effect on the development of stable transformants . By immunotitration analysis, it was confirmed that the enhanced expression of beta-galactosidase activity correlated well with the increase of the enzyme protein . This procedure may have an applicability for augmenting the frequency of transient gene expression in many cell types. Biochim Biophys Acta, 1992 Jan 6, 1129(2), 149 - 54 Expression of enzymatically active rat liver and human placental catechol-O-methyltransferase in Escherichia coli; purification and partial characterization of the enzyme; Lundstrom K et al.; To produce sufficient amounts of recombinant catechol-O-methyltransferase (COMT) for structural and functional studies the coding regions of the rat liver and human placental COMT genes have been introduced into a bacterial expression vector pKEX14 . Recombinant COMT was produced in Escherichia coli up to 10% of total bacterial protein after the induction of the T7 RNA polymerase gene with isopropyl-beta-D-thiogalactopyranoside . Both the rat and human enzymes were enzymatically active, soluble and reacted with anti-COMT antiserum in Western blotting . Both enzymes were purified from E . coli cells and partially characterized by determining their specific activity, apparent molecular weight and pI. Biochim Biophys Acta, 1992 Jan 6, 1129(2), 145 - 8 An inhibitor of elongation factor G (EF-G) GTPase present in the ribosome wash of Escherichia coli: a complex of initiation factors IF1 and IF3? Nagel K, Voigt J. An inhibitor of elongation factor G (EF-G) GTPase isolated from the ribosome wash of Escherichia coli was shown to stimulate the poly(A,U,G)- and initiation factor 2 (IF2)-dependent binding of N-formyl-{35S}Met-tRNAfMet to ribosomes . In the presence of saturating amounts of the EF-G GTPase inhibitor, neither addition of initiation factor 1 (IF1) nor addition of initiation factor 3 (IF3) caused a further stimulation of the formation of N-formyl-{35S}Met-tRNAfMET/poly(A,U,G)/ribosome complexes . Both IF1 and IF3 were shown to inhibit ribosome-dependent EF-G GTPase, especially when both initiation factors were added either in absence or in the presence of initiation factor 2 (IF2), poly(A,U,G) and N-formyl-Met-tRNAfMet . Therefore, we conclude that the EF-G GTPase inhibitor consisting of two polypeptide subunits with apparent molecular masses of 23,000 and 10,000 Da is a complex of initiation factors IF1 and IF3 . The inhibition of EF-G GTPAse by IF3, but not the effects of IF1 in the presence or absence of IF3 could be reversed by increasing the Mg(2+)-concentration as already shown for the EF-G GTPase inhibitor . Therefore, IF1 as well as the EF-G GTPase inhibitor do not influence the ribosome-dependent EF-G GTPase by affecting the association of ribosomal subunits. FASEB J, 1992 Jan 6, 6(2), 759 - 64 High-level expression of functional human cytochrome P450 1A2 in Escherichia coli; Fisher CW et al.; Enzymatically active human cytochrome P450 1A2 was expressed in Escherichia coli utilizing the pCWori+ vector containing a modified cDNA . The coding sequence for the NH2-terminal region of the protein was modified by the alignment and substitution of a 27 bp segment from a modified bovine P450 17A1 cDNA onto the 5' end of the open reading frame of P450 1A2 at amino acid 21 . The expressed chimeric P450 was produced at a high level in a functionally intact form, as assayed by the formation in vivo of the 449 nm absorbance band of the CO complex of the reduced hemoprotein . E . coli membrane preparations were shown to contain P450 1A2, which was active in the 2-hydroxylation of estradiol, and the O-deethylation of 7-ethoxycoumarin and 7-ethoxyresorufin, when reconstituted with recombinant rat liver NADPH-cytochrome P450 reductase. Biochim Biophys Acta, 1992 Jan 6, 1129(2), 219 - 22 The 16S rRNA gene of Streptomyces lividans TK64 contains internal promoters; Chang SC et al.; A 632-bp Sau3AI fragment of Streptomyces lividans TK64 genome was found to confer promoter activity in Streptomyces and Escherichia coli . This fragment showed almost identical sequence (97.8%) to the S . coelicolor 16S rRNA segment encompassing from nucleotide 706 to 1337 region . The transcription start points of this fragment were identified by the primer extension method . Analysis of the nucleotide sequence upstream the transcription start points revealed two putative E . coli-like promoters resided within this fragment . The occurrence of internal promoters active in Streptomyces and E . coli was also confirmed in the 16S rRNA gene of rrnE operon from TK64. Biochim Biophys Acta, 1992 Jan 6, 1129(2), 172 - 6 Induction by psychotropic drugs and local anesthetics of DnaK and GroEL proteins in Escherichia coli; Tanji K et al.; We examined effects of psychotropic drugs and local anesthetics on the synthesis of heat shock proteins in Escherichia coli . Chlorpromazine, a phenothiazine derivative, was shown to induce DnaK and GroEL proteins, major heat shock proteins in E . coli . The inductions of these proteins were not observed in an rpoH (= htpR) amber mutant strain, indicating that the heat shock sigma factor sigma 32 was required for their inductions . Northern blot hybridization analysis revealed that chlorpromazine induced increases of messenger RNAs for the DnaK and GroEL proteins . Thus, the induction occurred at the level of transcription . Chlorpromazine also induced non-heat shock proteins with molecular masses of 21 kDa, 20 kDa, and 17 kDa, even in the rpoH mutant strain . Other psychotropic drugs and local anesthetics, namely, dibucaine, lidocaine, imipramine, tetracaine and procaine, also induced DnaK and GroEL proteins and the small molecular weight proteins. Biochim Biophys Acta, 1992 Jan 6, 1129(2), 215 - 8 Partite expression of the bovine papillomavirus E1 open reading frame in Escherichia coli; Wilson VG et al.; Six recombinants were constructed which expressed portions of the bovine papillomavirus E1 open reading frame as OmpF/E1/beta-galactosidase tribrid fusion proteins in Escherichia coli . Rabbit sera containing E1-specific antibodies were generated against five of these six fusion proteins (which together constitute 74% of the full-length E1 open reading frame) . The individual fusion proteins and their cognate antisera will be useful reagents for defining the structure and function of the BPV E1 protein(s). J Mol Biol, 1992 Jan 5, 223(1), 31 - 40 Leftward ribosome frameshifting at a hungry codon; Gallant JA et al.; Previous experiments have shown that limitation for certain aminoacyl-tRNA species results in phenotypic suppression of a subset of frameshift mutant alleles, including members in both the (+) and (-) incorrect reading frames . Here, we demonstrate that such phenotypic suppression can occur through a ribosome reading frame shift at a hungry AAG codon calling for lysyl-tRNA in short supply . Direct amino acid sequence analysis of the product and DNA sequence manipulation of the gene demonstrate that the ribosome frameshift occurs through a movement of one base to the left, so as to decode the triplet overlapping the hungry codon from the left or 5' side, followed by continued normal translation in the new, shifted reading frame. J Mol Biol, 1992 Jan 5, 223(1), 159 - 70 Expectation maximization algorithm for identifying protein-binding sites with variable lengths from unaligned DNA fragments; Cardon LR et al.; An Expectation Maximization algorithm for identification of DNA binding sites is presented . The approach predicts the location of binding regions while allowing variable length spacers within the sites . In addition to predicting the most likely spacer length for a set of DNA fragments, the method identifies individual sites that differ in spacer size . No alignment of DNA sequences is necessary . The method is illustrated by application to 231 Escherichia coli DNA fragments known to contain promoters with variable spacings between their consensus regions . Maximum-likelihood tests of the differences between the spacing classes indicate that the consensus regions of the spacing classes are not distinct . Further tests suggest that several positions within the spacing region may contribute to promoter specificity. J Mol Biol, 1992 Jan 5, 223(1), 131 - 44 Direct evidence for the effect of transcription on local DNA supercoiling in vivo; Rahmouni AR et al.; The B-to-Z structural transition of varying lengths (74 to 14 base-pairs) of (CG) tracts has been used as a superhelicity probe to examine the local topological changes induced by transcription at defined genetic loci in vivo . The local-topology reporter sequences indicate that under steady-state transcription the region upstream from the promoter experiences an increase in negative supercoiling whereas the region downstream from the terminator displays a decrease in negative superhelicity . This result provides direct in vivo evidence for the notion that the translocation of an RNA polymerase elongation complex along the double-helical DNA generates positive supercoils in front of it and negative supercoils behind it . Also, this twin-supercoiled domain model was tested inside a transcribed region where a high degree of negative supercoiling generated by the passage of each individual RNA polymerase was detected . Hence, these data indicate that the induced supercoils are confined to the vicinity of each RNA polymerase complex in a multipolymerase system. J Mol Biol, 1992 Jan 5, 223(1), 115 - 29 C-terminal truncated Escherichia coli RecA protein RecA5327 has enhanced binding affinities to single- and double-stranded DNAs; Tateishi S et al.; RecA5327 is a truncated RecA protein that is lacking 25 amino acid residues from the C-terminal end . The expression of RecA5327 protein in the cell resulted in the constitutive induction of SOS functions without damage to the DNA . Purified RecA5327 protein effectively promoted the LexA repressor cleavage reaction and ATP hydrolysis at a lower concentration of single-stranded DNA than that required for wild-type RecA protein . A DNA binding study showed that RecA5327 has about ten times higher affinity for single-stranded DNA than does the wild-type RecA protein . Moreover RecA5327 protein binds stably to double-stranded (ds) DNA in conditions where the wild-type RecA protein could not bind . The binding of RecA5327 protein to dsDNA was associated with the unwinding of dsDNA, suggesting that RecA5327 binds to dsDNA in the same manner as does the wild-type protein . The fact that RecA5327 does not bind stoichiometrically but forms short filaments on dsDNA suggests that it nucleates to dsDNA much more frequently than does the wild-type protein . The role of the 25 C-terminal residues, in the regulation of RecA binding to DNA, is discussed. J Mol Biol, 1992 Jan 5, 223(1), 105 - 14 Inhibitory effects of N- and C-terminal truncated Escherichia coli recA gene products on functions of the wild-type recA gene; Horii T et al.; The effects of the expression of Escherichia coli truncated RecA protein on the host recA functions were examined . The recA gene on a multicopy plasmid was manipulated to express the truncated RecA protein from its carboxyl (C) and amino (N) terminal ends where a maximum of four extra amino acid residues was added . The regulatory part of the recA gene was substituted by the lacUV5 promoter in the plasmid to facilitate the artificial control of recA expression . Enzyme-linked immunosorbent assay and Western blot analyses revealed great differences in accumulation of the truncated RecA proteins in the cell, depending on the location of the site of truncation . The expression of truncated proteins lacking 62, 77, 93 or 149 amino acid residues from the C-terminal end caused the host recA+ wild-type cell to become sensitive to ultraviolet irradiation and interfered with chromosomal recombination but did not interfere with the induction of lambda prophage . The expression of truncated RecA protein with 25 amino acid residues deleted from the C-terminal end caused the host cell to induce SOS functions constitutively . Truncated RecA proteins with 15 or 28 amino acid residues missing from the N-terminal end severely interfered with all of the host recA functions examined here . The effect of the loss of 41 amino acid residues from the N-terminal end of RecA was significant but less than the effect of proteins lacking 15 or 28 amino acid residues from the N-terminal end . A protein lacking 59 amino acid residues from the N-terminal end showed little interference with any measured recA functions, suggesting that the deletion of the region from around residues 41 to 59, which is rich in hydrophobic side-chains, influenced the ability of the truncated protein to interfere with the functions of wild-type RecA protein . We also constructed a mutant gene with an internal deletion whose product was missing a region from residues 184 to 204 . That mutant RecA protein was stably accumulated in the cell . This protein had little effect on the function of host wild-type recA gene product . The possible function of the regions at the N and C termini are discussed. J Biol Chem, 1992 Jan 5, 267(1), 91 - 5 Vanadate-dependent photomodification of serine 319 and 321 in the active site of isocitrate lyase from Escherichia coli; Ko YH et al.; Vanadate was used as a substrate analogue to modify and subsequently localize active site serine residues of isocitrate lyase from Escherichia coli . Irradiation of the enzyme on ice with UV light in the presence of vanadate resulted in inactivation . Inactivation was prevented by the substrates glyoxylate or Ds-isocitrate and to a much lesser extent by succinate . Reduction of photoinactivated isocitrate lyase by NaBH4 partially restored enzyme activity . The photomodified enzyme was labeled by reduction with NaB{3H}4 in the presence and absence of the substrates succinate plus glyoxylate . Highly differential labeling of serine residues 319 and 321 in the absence of substrates suggests their importance in the action of isocitrate lyase . These residues are highly conserved in all five known sequences of this enzyme. J Biol Chem, 1992 Jan 5, 267(1), 542 - 5 GTPase-mediated activation of ATP sulfurylase; Leyh TS et al.; GTP stimulates the synthesis of APS (adenosine 5'-phosphosulfate) by the enzyme ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) via a GTPase mechanism . The activation of the enzyme, purified from Escherichia coli, is titratable with GTP . The initial rate of APS formation is increased 116-fold at a saturating concentration of GTP . The enzyme exhibits a GTPase activity that is stimulated by ATP and further enhanced by SO4; however, SO4 alone does not significantly stimulate GTP hydrolysis . The larger subunit of ATP sulfurylase, encoded by cysN, contains a GTP-binding consensus sequence common to other known GTP-binding proteins . This is the first evidence that the sulfate activation pathway is a metabolic target for regulation by a GTPase. J Biol Chem, 1992 Jan 5, 267(1), 419 - 25 (A-C-B) human proinsulin, a novel insulin agonist and intermediate in the synthesis of biosynthetic human insulin; Heath WF et al.; The hormone insulin is synthesized in the beta cell of the pancreas as the precursor, proinsulin, where the carboxyl terminus of the B-chain is connected to the amino terminus of the A-chain by a connecting or C-peptide . Proinsulin is a weak insulin agonist that possesses a longer in vivo half-life than does insulin . A form of proinsulin clipped at the Arg65-Gly66 bond has been shown to be more potent than the parent molecule with protracted in vivo activity, presumably as a result of freeing the amino terminal residue of the A-chain . To generate a more active proinsulin-like molecule, we have constructed an "inverted" proinsulin molecule where the carboxyl terminus of the A-chain is connected to the amino terminus of the B-chain by the C-peptide, leaving the critical Gly1 residue free . Transformation of Escherichia coli with a plasmid coding for A-C-B human proinsulin led to the stable production of the protein . By a process of cell disruption, sulfitolysis, anion-exchange chromatography, refolding, and reversed-phase high-performance liquid chromatography, two forms of the inverted proinsulin differing at their amino termini as Gly1 and Met0-Gly1 were identified and purified to homogeneity . Both proteins were shown by a number of analytical techniques to be of the inverted sequence, with insulin-like disulfide bonding . Biological analyses by in vitro techniques revealed A-C-B human proinsulin to be intermediate in potency when compared to human insulin and proinsulin . The time to maximal lowering of blood glucose in the fasted normal rat appeared comparable to that of proinsulin . Additionally, we were able to generate fully active, native insulin from A-C-B human proinsulin by proteolytic transformation . The results of this study lend themselves to the generation of novel insulin-like peptides while providing a simplified route to the biosynthetic production of insulin. J Biol Chem, 1992 Jan 5, 267(1), 35 - 8 Differential phosphorylation and localization of the transcription factor UBF in vivo in response to serum deprivation . In vitro dephosphorylation of UBF reduces its transactivation properties; O'Mahony DJ et al.; We have analyzed the expression, phosphorylation, and localization of the ribosomal DNA transcription factors UBF1 and UBF2 in Chinese hamster ovary cells in response to serum deprivation . In vivo labeling experiments demonstrate that UBF1 and UBF2 are phosphoproteins . Phosphoamino acid analysis of the in vivo labeled proteins demonstrate that UBF is phosphorylated on serine residues . Following serum deprivation there is no alteration in the cellular levels of UBF1 and UBF2 as determined by Western blotting, but there is an 80% reduction in the level of phosphorylation of UBF compared with logarithmically growing cells . Following serum deprivation there is a redistribution of UBF between the nucleolus, the nucleus, and the cytoplasm . Phosphatase-treated UBF demonstrated a reduced ability to rescue transcription by RNA polymerase I from the rDNA spacer promoter in vitro . These findings suggest that phosphorylation of UBF is a prerequisite for transactivation of RNA polymerase I. J Biol Chem, 1992 Jan 5, 267(1), 150 - 8 Mechanism of the irreversible inactivation of mouse ornithine decarboxylase by alpha-difluoromethylornithine . Characterization of sequences at the inhibitor and coenzyme binding sites; Poulin R et al.; Mouse ornithine decarboxylase (ODC) was expressed in Escherichia coli and the purified recombinant enzyme used for determination of the binding site for pyridoxal 5'-phosphate and of the residues modified in the inactivation of the enzyme by the enzyme-activated irreversible inhibitor, alpha-difluoromethylornithine (DFMO) . The pyridoxal 5'-phosphate binding lysine in mouse ODC was identified as lysine 69 of the mouse sequence by reduction of the purified holoenzyme form with NaB{3H}4 followed by digestion of the carboxymethylated protein with endoproteinase Lys-C, radioactive peptide mapping using reversed-phase high pressure liquid chromatography and gas-phase peptide sequencing . This lysine is contained in the sequence PFYAVKC, which is found in all known ODCs from eukaryotes . The preceding amino acids do not conform to the consensus sequence of SXHK, which contains the pyridoxal 5'-phosphate binding lysine in a number of other decarboxylases including ODCs from E . coli . Using a similar procedure to analyze ODC labeled by reaction with {5-14C}DFMO, it was found that lysine 69 and cysteine 360 formed covalent adducts with the inhibitor . Cysteine 360, which was the major adduct accounting for about 90% of the total labeling, is contained within the sequence -WGPTCDGL(I)D-, which is present in all known eukaryote ODCs . These results provide strong evidence that these two peptides form essential parts of the catalytic site of ODC . Analysis by fast atom bombardment-mass spectrometry of tryptic peptides containing the DFMO-cysteine adduct indicated that the adduct formed in the enzyme was probably the cyclic imine S-(2-(1-pyrroline)methyl)cysteine . This is readily oxidized to S-((2-pyrrole)methyl)cysteine or converted to S-((2-pyrrolidine)methyl)cysteine by NaBH4 reduction . This adduct is consistent with spectral evidence showing that inactivation of the enzyme with DFMO does not entail the formation of a stable adduct between the pyridoxal 5'-phosphate, the enzyme, and the inhibitor. J Mol Biol, 1992 Jan 5, 223(1), 79 - 93 RecA protein-promoted homologous pairing and strand exchange between intact and partially single-stranded duplex DNA; Chow SA et al.; In the pairing reaction between circular gapped and fully duplex DNA, RecA protein first polymerizes on the gapped DNA to form a nucleoprotein filament . Conditions that removed the formation of secondary structure in the gapped DNA, such as addition of Escherichia coli single-stranded DNA binding protein or preincubation in 1 mM-MgCl2, optimized the binding of RecA protein and increased the formation of joint molecules . The gapped duplex formed stable joints with fully duplex DNA that had a 5' or 3' terminus complementary to the single-stranded region of the gapped molecule . However, the joints formed had distinct properties and structures depending on whether the complementary terminus was at the 5' or 3' end . Pairing between gapped DNA and fully duplex linear DNA with a 3' complementary terminus resulted in strand displacement, symmetric strand exchange and formation of complete strand exchange products . By contrast, pairing between gapped and fully duplex DNA with a 5' complementary terminus produced a joint that was restricted to the gapped region; there was no strand displacement or symmetric strand exchange . The joint formed in the latter reaction was likely a three-stranded intermediate rather than a heteroduplex with the classical Watson-Crick structure . We conclude that, as in the three-strand reaction, the process of strand exchange in the four-strand reaction is polar and progresses in a 5' to 3' direction with respect to the initiating strand . The present study provides further evidence that in both three-strand and four-strand systems the pairing and strand exchange reactions share a common mechanism. J Mol Biol, 1992 Jan 5, 223(1), 9 - 15 Bar to normal UGA translation by the selenocysteine tRNA; Li WQ et al.; The selC gene product, tRNA(Sec), inserts selenocysteine at UGA (opal) codons in a specialized mRNA context . We have investigated the action of the tRNA at ordinary UGA codons, normally not translated, by changing the unusual structural features of tRNA(Sec) . Sequences in the D arm, CCA arm and variable arm of the tRNA all contribute to the prohibition against translation of ordinary UGA codons . One multiple mutant is a moderately efficient serine-inserting UGA suppressor tRNA. J Mol Biol, 1992 Jan 5, 223(1), 41 - 54 Mechanism of post-segregational killing by the hok/sok system of plasmid R1 . Sok antisense RNA regulates hok gene expression indirectly through the overlapping mok gene; Thisted T et al.; The hok/sok locus of plasmid R1, which mediates plasmid stabilization by killing of plasmid-free segregants, codes for two RNAs, Hok mRNA and Sok antisense RNA . Hok mRNA encodes the Hok killer protein of 52 amino acid residues . Expression of hok is regulated post-transcriptionally by Sok antisense RNA . Killing of plasmid-free daughter-cells by the hok/sok system is accomplished through differential decay of the Hok and Sok-RNAs: Hok mRNA is very stable while Sok-RNA decays rapidly, thus leading to derepression of Hok mRNA translation in plasmid-free segregants, ensuring a rapid and selective killing of these cells . Sok antisense RNA is complementary to the leader region of the Hok mRNA . However, the region of complementarity does not overlap with the hok Shine-Dalgarno sequence . Thus, Sok-RNA regulates hok translation indirectly by an as yet unknown mechanism . We show here that Sok antisense RNA regulates the translation of another reading frame located in the hok/sok locus . This new reading frame, which overlaps with almost the entire hok gene, was denoted mok (mediation of killing) . Point-mutations that prevent mok translation through the hok translational initiation region abolish efficient expression of hok . Furthermore, these mutations abolish the Sok-RNA-mediated control of hok gene expression . Hence, the antisense-RNA-mediated regulation of the hok gene seems to occur via translational coupling between the hok and mok reading-frames. J Biol Chem, 1992 Jan 5, 267(1), 144 - 9 A relationship between asparagine synthetase A and aspartyl tRNA synthetase; Hinchman SK et al.; A highly conserved protein motif characteristic of Class II aminoacyl tRNA synthetases was found to align with a region of Escherichia coli asparagine synthetase A . The alignment was most striking for aspartyl tRNA synthetase, an enzyme with catalytic similarities to asparagine synthetase . To test whether this sequence reflects a conserved function, site-directed mutagenesis was used to replace the codon for Arg298 of asparagine synthetase A, which aligns with an invariant arginine in the Class II aminoacyl tRNA synthetases . The resulting genes were expressed in E . coli, and the gene products were assayed for asparagine synthetase activity in vitro . Every substitution of Arg298, even to a lysine, resulted in a loss of asparagine synthetase activity . Directed random mutagenesis was then used to create a variety of codon changes which resulted in amino acid substitutions within the conserved motif surrounding Arg298 . Of the 15 mutant enzymes with amino acid substitutions yielding soluble enzyme, 13 with changes within the conserved region were found to have lost activity . These results are consistent with the possibility that asparagine synthetase A, one of the two unrelated asparagine synthetases in E . coli, evolved from an ancestral aminoacyl tRNA synthetase. J Biol Chem, 1992 Jan 5, 267(1), 526 - 41 Genetic and biochemical characterization of the trpB8 mutation of Escherichia coli tryptophan synthase . An amino acid switch at the sharp turn of the trypsin-sensitive "hinge" region diminishes substrate binding and alters solubility; Zhao GP et al.; The trpB8 mutation of Escherichia coli tryptophan synthase is unique in that the cells bearing this lesion are not only capable of utilizing indole for growth, but they also accumulate indole, under conditions of tryptophan limitation . The lesion was shown by DNA sequencing to be a G to C transversion at nucleotide 5528 of the trp operon, resulting in a Gly to Arg switch at codon 281 . Gly-281, within the trypsin-sensitive "hinge" region, is invariant among all known beta polypeptides . The catalytic activity of the mutant beta 2(B8) protein is dramatically stimulated by alpha subunit, both in vivo and in vitro . In the absence of alpha subunit, ammonium ion effectively stimulated the activity in an apparently cooperative manner . The pH optimum for the mutant subunit was 9.8, which is 2 units higher than that of wild type . In contrast to the wild-type subunit, beta(B8) partially aggregated within cells upon overexpression . At the optimal concentration of ammonium ions (2.25 M), the beta 2(B8) mutant enzyme displayed lower affinity than wild-type enzyme toward indole and L-serine, but the Vmax was almost unchanged . The physicochemical behavior of beta 2(B8) is supported by computer graphic modeling studies . An open versus closed model of conformational change within the beta 2 protein is proposed . A plausible role for the hinge region is discussed. J Biol Chem, 1992 Jan 5, 267(1), 413 - 8 In vitro translocation of secretory proteins possessing no charges at the mature domain takes place efficiently in a protonmotive force-dependent manner; Kato M et al.; The effect of charges existing on the mature domain of secretory proteins on the efficiency and protonmotive force dependence of translocation into everted membrane vesicles of Escherichia coli was studied . Model secretory proteins devoid of charges on the mature domain were constructed at the DNA level using proOmpF-Lpp as the starting protein . The chargeless presecretory proteins thus constructed were translocated and processed for the signal peptide much faster than proOmpF-Lpp and the rate of translocation was appreciably enhanced by imposition of the protonmotive force . Not only the membrane potential but also delta pH were effective in stimulating the rate of translocation of the chargeless proteins . The results indicate that the mature domain does not have to be charged for the secretory translocation and that the major requirement of the protonmotive force for the secretory translocation is not for the movement, including an electrophoretic one, of charged regions of the mature domain . All of the proOmpF-Lpp derivatives thus constructed were translocated efficiently into everted membrane vesicles in a SecA-dependent manner, irrespective of their size . The mature domain of the smallest one was 45 amino acid residues in length . Contrary to the views previously presented by other workers, these results suggest that there is no sharp boundary at the reported regions for the translocation of presecretory proteins across the cytoplasmic membrane or for the requirement of SecA. Biochemistry, 1991 Dec 24, 30(51), 11788 - 95 Properties of lipoamide dehydrogenase altered by site-directed mutagenesis at a key residue (I184Y) in the pyridine nucleotide binding domain; Maeda-Yorita K et al.; The binding of pyridine nucleotide to human erythrocyte glutathione reductase, an enzyme of known three-dimensional structure, requires some movement of the side chain of Tyr197 . Moreover, this side chain lies very close to the isoalloxazine ring of the FAD cofactor . The analogous residue, Ile184, in the homologous enzyme Escherichia coli lipoamide dehydrogenase has been altered by site-directed mutagenesis to a tyrosine residue (I184Y) {Russell, G . C., Allison, N., Williams, C . H., Jr., & Guest, J.R . (1989) Ann . N.Y . Acad . Sci . 573, 429-431} . Characterization of the altered enzyme shows that the rate of the pyridine nucleotide half-reaction has been markedly reduced and that the spectral properties have been changed to mimic those of glutathione reductase . Therefore, Ile184 is shown to be an important residue in modulating the properties of the flavin in lipoamide dehydrogenase . Turnover in the dihydrolipoamide/NAD+ reaction is decreased by 10-fold and in the NADH/lipoamide reaction by 2-fold in I184Y lipoamide dehydrogenase . The oxidized form of I184Y shows remarkable changes in the fine structure of the visible absorption and circular dichroism spectra and also shows nearly complete quenching of FAD fluorescence . The spectral properties of the altered enzyme are thus similar to those of glutathione reductase and very different from those of wild-type lipoamide dehydrogenase . On the other hand, spectral evidence does not reveal any change in the amount of charge-transfer stabilization at the EH2 level . Stopped-flow data indicate that, in the reduction of I184Y by NADH, the first step, reduction of the flavin, is only slightly slowed but the subsequent two-electron transfer to the disulfide is markedly inhibited.(ABSTRACT TRUNCATED AT 250 WORDS) Science, 1992 Jan 3, 255(5040), 85 - 7 Interaction of p107 with cyclin A independent of complex formation with viral oncoproteins; Ewen ME et al.; The p107 protein and the retinoblastoma protein (RB) both bind specifically to two viral oncoproteins, the SV40 T antigen (T) and adenoviral protein E1A (E1A) . Like RB, p107 contains a segment (the pocket) that, alone, can bind specifically to T, E1A, and multiple cellular proteins . Cyclin A bound to the p107 pocket, but not the RB pocket . Although both pockets contain two, related collinear subsegments (A and B), the unique sequence in the p107 pocket that occupies the space between A and B is required for the interaction with cyclin A. Nature, 1992 Jan 2, 355(6355), 87 - 9 Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR; Ansari AZ et al.; Positive control of transcription often involves stimulatory protein-protein interactions between regulatory factors and RNA polymerase . Critical steps in the activation process itself are seldom ascribed to protein-DNA distortions . Activator-induced DNA bending is typically assigned a role in binding-site recognition, alterations in DNA loop structures or optimal positioning of the activator for interaction with polymerase . Here we present a transcriptional activation mechanism that does not require a signal-induced DNA bend but rather a receptor-induced untwisting of duplex DNA . The allosterically modulated transcription factor MerR is a repressor and an Hg(II)-responsive activator of bacterial mercury-resistance genes . Escherichia coli RNA polymerase binds to the MerR-promoter complex but cannot proceed to a transcriptionally active open complex until Hg(II) binds to MerR (ref . 6) . Chemical nuclease studies show that the activator form, but not the repressor, induces a unique alteration of the helical structure localized at the centre of the DNA-binding site . Data presented here indicate that this Hg-MerR-induced DNA distortion corresponds to a local underwinding of the spacer region of the promoter by about 33 degrees relative to the MerR-operator complex . The magnitude and the direction of the Hg-MerR-induced change in twist angle are consistent with a positive control mechanism involving reorientation of conserved, but suboptimally phased, promoter elements and are consistent with a role for torsional stress in formation of an open complex. Gene, 1992 Jan 2, 110(1), 95 - 9 The nucleotide sequence of recG, the distal spo operon gene in Escherichia coli K-12; Kalman M et al.; A gene is identified in the Escherichia coli K-12 spo operon as recG . Previously identified genes in the spo operon were spoS, alias rpoZ, encoding the omega (omega) subunit of RNA polymerase, as well as the spoT gene encoding the major cellular source of guanosine 3',5'-bispyrophosphate hydrolase activity . The gene order within the spo operon is: spoS (rpoZ), spoT, spoU, recG . A convergent gltS gene is present beyond the spo operon . Mutants bearing recG deletion-insertion alleles display mild sensitivities to both ultraviolet irradiation and to mitomycin C, which is expected to be due to a known recG insertion allele . Deletion-insertion mutations in upstream operon genes (spoT and spoU) show polar effects on these assays of recG function . The deduced 693-amino acid (aa) RecG sequence shows a weak, but significant, relatedness to aa sequence motifs previously reported for putative helicases involved in replication, recombination, and DNA repair. Gene, 1992 Jan 2, 110(1), 119 - 22 Multifunctional yeast high-copy-number shuttle vectors; Christianson TW et al.; A set of four yeast shuttle vectors that incorporate sequences from the Saccharomyces cerevisiae 2 mu endogenous plasmid has been constructed . These yeast episomal plasmid (YEp)-type vectors (pRS420 series) differ only in their yeast selectable markers, HIS3, TRP1, LEU2 or URA3 . The pRS420 plasmids are based on the backbone of a multifunctional phagemid, pBluescript II SK+, and share its useful properties for growth in Escherichia coli and manipulation in vitro . The pRS420 plasmids have a copy number of about 20 per cell, equivalent to that of YEp24 . During non-selective yeast growth, pRS420 plasmids are lost through mitotic segregation at rates similar to other YEp vectors and yeast centromeric plasmid (YCp) vectors, in the range of 1.5-5% of progeny per doubling . The pRS420 series provides high-copy-number counterparts to the current pRS vectors {Sikorski and Hieter, Genetics 122 (1989) 19-27}. Gene, 1992 Jan 2, 110(1), 101 - 3 Two cat expression vectors for cloning and generation of 3'- and 5'-deletion mutants; Kumar G; The construction of two versatile cat vectors, pGK0CAT and pGKA10CAT, is reported . These vectors possess multiple cloning sites derived from the Bluescript pKS(+) plasmid that allow the cloning of diverse DNA fragments . From a single cloned insert, i.e., a putative promoter element, one can use the exonuclease III (ExoIII) and S1 or mung-bean nuclease method to generate sequential deletion mutants of the 3' and 5' region . Linker-scanning and internal deletion mutants can thus be created by using appropriate 3'- and 5'-deletion mutants . These plasmids could thus be used for the identification of cis-acting promoter or enhancer elements by either in vivo or in vitro transcriptional analyses . The ability of these vectors to generate deletion mutants from the 3' end make them suitable to identify cis-acting elements in the 5'-noncoding region of the mRNA involved in the translational regulation of protein synthesis . Single-stranded circular mutant plasmids could also be generated from these vectors to study various protein-DNA interactions. Gene, 1992 Jan 2, 110(1), 115 - 8 An Escherichia coli-Mycobacterium shuttle cosmid vector, pMSC1; Hinshelwood S et al.; A shuttle cosmid vector, pMSC1, has been constructed which replicates in Escherichia coli and Mycobacterium smegmatis . The vector was mainly derived from the lambda ori cosmid, Lawrist4, and the Mycobacterium fortuitum cryptic plasmid, pAL5000, which replicates in M . smegmatis and Mycobacterium bovis BCG . The vector contains two cos sites which facilitates library construction, unique BamHI and HindIII sites for cloning, and a kanamycin-resistance-encoding gene for selection in mycobacteria . After packaging, the vector sequences comprise 10.3 kb, so that the theoretical size limits for inserts are 30-42 kb . A genomic library from M . smegmatis was constructed in E . coli; clones from this library were transferred into M . smegmatis by electroporation, and back again to E . coli, without any apparent rearrangements . This vector will be useful in cloning genes encoding complex pathways in mycobacteria. Gene, 1992 Jan 2, 110(1), 1 - 7 A novel method for converting common restriction enzymes into rare cutters: integration host factor-mediated Achilles' cleavage (IHF-AC); Kur J et al.; Integration host factor (IHF)-mediated protection against enzymatic methylation at ihf-overlapping sites provides the basis for this novel application of the Achilles' cleavage (AC) technique {Koob et al., Science 241 (1988) 1084-1086} for generating rare natural cleavage sites . When applying IHF-AC to plasmid, phage lambda, Escherichia coli and yeast genomes, only a few of the EcoRI, HinfI, and MboI sites (which overlapped the ihf sites) remained cleavable after prior methylation with the cognate M.EcoRI, M.HinfI, or Dam methyltransferases in the presence of IHF . Thus, IHF-AC essentially converted these enzymes into very rare cutters . The extent of cleavage could be controlled by varying the IHF:DNA ratio and temperature . Moreover, the method permits the genomic location and strength of the ihf sites to be determined. Oncogene, 1992 Jan, 7(1), 9 - 17 c-ets-1 DNA binding to the PEA3 motif is differentially inhibited by all the mutations found in v-ets; Leprince D et al.; The proto-oncogene c-ets-1, one of the two cellular sequences transduced by the avian retrovirus E26, encodes for two transcription factors that activate through a purine-rich motif . The v-ets oncogene differs from its cellular progenitor p68c-ets-1 (i) by its fusion to gag- and myb-derived sequences in the E26 P135gag-myb-ets fusion protein, (ii) by two point mutations, and (iii) by the replacement of the 13 C-terminal amino acids present in c-ets-1 by 16 unrelated residues in v-ets . A 35 kDa protein which binds to the purine-rich PEA3 motif in a sequence-specific manner has been obtained by expression in Escherichia coli of the 311 carboxy-terminal amino acids of c-ets-1 . Using various v-/c-ets-1 chimeric 35 kDa proteins expressed in bacteria, we have shown that all the mutations found in v-ets, when introduced into this c-ets-1 protein, diminish or even abolish its sequence-specific DNA binding . These results demonstrate that, in addition to the previously defined 85 amino acids located near the carboxy terminus of the c-ets-1 protein (the ETS domain), other sequences are required for sequence-specific DNA binding . In addition, the c-ets-1 35 kDa polypeptide carrying the two point mutations and the viral-specific carboxy terminus, and thus similar to the v-ets-encoded domain of the E26 P135gag-myb-ets, does not bind to the PEA3 motif. Life Sci, 1992, 50(11), 807 - 11 Serum phospholipase A2 enzyme activity and immunoreactivity in a prospective analysis of patients with septic shock; Vadas P et al.; Massive elevations of serum phospholipase A2 activity have been documented in patients with septic shock . Serum PLA2 activity correlated to the degree and duration of circulatory collapse, while purified native PLA2 reproduced hypotension in experimental animals . In a prospective study of patients with septic shock, we have determined the relationship of PLA2 enzyme activity to PLA2 immunoreactivity using radiolabelled E . coli phospholipid substrate and an ELISA specific for group II human nonpancreatic PLA2 . In all patients, there was a clear concordance of the two assays . Maximal PLA2 concentration was increased a mean of 554-fold over normal levels . We found no evidence to support the presence of activating or inhibitory proteins . These data confirm that the observed increase in serum PLA2 activity in septic shock is due to intravascular release of group II nonpancreatic PLA2. EMBO J, 1992 Jan, 11(1), 71 - 7 Involvement of the chaperonin dnaK in the rapid degradation of a mutant protein in Escherichia coli; Sherman MYu et al.; The ability of Escherichia coli rapidly to degrade abnormal proteins is inhibited by mutations affecting any of several heat shock proteins (hsps) . We therefore tested whether a short-lived mutant protein might become associated with hsps as part of its degradation . At 30 degrees C, the non-secreted mutant form of alkaline phosphatase, phoA61, is relatively stable, and very little phoA61 is found associated with the hsp dnaK . However, raising the temperature to 37 degrees C or 41 degrees C stimulated the degradation of this protein, and up to 30% of cellular phoA61 became associated with dnaK, as shown by immunoprecipitation and Western blot analysis . Also found in complexes with phoA61 were the hsps, protease La and grpE (but no groEL, or groES) . The rapid degradation of phoA61 at 37 degrees C and 41 degrees C is in part by protease La, since it decreased by 50% in lon mutants . This process also requires dnaK, since deletion of this gene prevented phoA61 degradation almost completely (unless a wild-type dnaK gene was introduced) . In contrast, the missense mutation, dnaK756, enhanced phoA61 degradation . The dnaK756 protein also was associated with phoA61, but this complex, unlike that containing wild-type dnaK could not be dissociated by ATP addition . Furthermore, in a grpE mutant, the degradation of phoA61 and the amount associated with dnaK increased, while in a dnaJ mutant, phoA61 degradation and its association with dnaK decreased . Thus, complex formation with dnaK appears essential for phoA61 degradation by protease La and some other cell proteases, and a failure of the dnaK to dissociate normally may accelerate proteolytic attack.(ABSTRACT TRUNCATED AT 250 WORDS) EMBO J, 1992 Jan, 11(1), 57 - 62 Identification and characterization of an Escherichia coli gene required for the formation of correctly folded alkaline phosphatase, a periplasmic enzyme; Kamitani S et al.; Tn5 insertion mutations of Escherichia coli were isolated that impaired the formation of correctly folded alkaline phosphatase (PhoA) in the periplasm . The PhoA polypeptide synthesized in the mutants was translocated across the cytoplasmic membrane but not released into the periplasmic space . It was susceptible to degradation by proteases in vivo and in vitro . The wild-type counterpart of this gene (named ppfA) has been sequenced and shown to encode a periplasmic protein with a pair of potentially redox-active cysteine residues . PhoA synthesized in the mutants indeed lacked disulfide bridges . These results indicate that the folding of PhoA in vivo is not spontaneous but catalyzed at least at the disulfide bond formation step. EMBO J, 1992 Jan, 11(1), 215 - 23 Biochemical and genetic analysis of operator contacts made by residues within the beta-sheet DNA binding motif of Mnt repressor; Knight KL et al.; Residues 2, 6, 8 and 10 of Mnt repressor are the major determinants of operator DNA binding and recognition . Here, we investigate the interaction of wild-type Mnt and mutants bearing the Arg2----Lys, His6----Ala, Asn8----Ala and Arg10----Lys mutations with operator DNA modified by methylation or by symmetric base substitutions . The wild-type pattern of methylation interference is altered in specific ways for each of the mutant proteins . In addition, some of the mutant proteins show a 'loss of contact' phenotype with specific mutant operators . Taken together, these and previous results predict the following contacts between side chains in the Mnt tetramer and operator DNA: Arg2 recognizes the guanines at operator positions 10 and 12; His6 contacts the guanines at operator positions 5 and 17; Asn8 contacts operator positions 4, 7, 15 and 18; Arg10 contacts the guanines at operator positions 8 and 14 . The proposed contacts can be accommodated in a structural model in which the anti-parallel beta-sheet motifs of Mnt dimers lie in the major grooves of each operator half-site, centered over pseudo-symmetry axes that are 5.5 bp from the central dyad axis of the operator. Mol Microbiol, 1992 Jan, 6(1), 115 - 21 Haemolysin-derived synthetic peptides with pore-forming and haemolytic activity; Oropeza-Wekerle RL et al.; Escherichia coli haemolysin (Hlya) is a pore-forming protein which belongs to the family of 'Repeat-toxins' (RTX) (Lo et al., 1987; Lally et al., 1989; Kraig et al., 1990) . A model for the pore-forming structure of HlyA has been proposed (Ludwig et al., 1991) which consists of eight transmembrane segments all present in this hydrophobic region of HlyA . We report here that two synthetic peptides of 10 and 8 amino acids in length (Pep1 and Pep2, respectively), which are derived from transmembrane segment V, are able to form pores in an artificial lipid bilayer . In addition, Pep1 exhibits strong haemolytic activity when tested on human red blood cells (HRBCs) . The haemolytic activity of Pep1 and of E . coli haemolysin is completely inhibited by antibodies raised against Pep1. JPEN J Parenter Enteral Nutr, 1992 Jan-Feb, 16(1), 25 - 31 Protein malnutrition alone and in combination with endotoxin impairs systemic and gut-associated immunity; Deitch EA et al.; Because protein-malnourished or endotoxemic patients are at an increased risk of developing nosocomial infections, this study was performed to investigate the effects of protein malnutrition and endotoxemia, alone and in combination, on systemic and intestinal immunity . Protein malnutrition was created by feeding the animals a solid diet containing 0.03% protein . Subgroups of these protein-malnourished mice were killed after being challenged with saline or endotoxin on days 0, 7, 14, or 21 . At death, the animals were weighed, tissues were harvested for histologic analysis (ileum, mesenteric lymph node {MLN}, liver, and spleen), mitogen responsiveness (MLN, Peyer's patches, and spleen), and xanthine oxidase measurements (ileum and cecum) . Separate groups were evaluated for survival . Both the saline and endotoxin-challenged mice had lost about 30% of their body weight after 21 days on the low-protein diet . The protein-malnourished mice were more susceptible to endotoxin-induced mortality (70% at 21 days) than the normally nourished mice (0%) (p less than .001) . The mitogen responsiveness of the protein-malnourished mice to the T-cell mitogens (PHA and Con-A) progressively decreased the longer the mice were protein malnourished, and this decreased in blastogenic responsiveness was associated with histologic evidence of lymphoid atrophy . In contrast, the blastogenic response to the primarily B-cell mitogen, PWM, was largely preserved . The endotoxin challenge further depressed the immune state of mice tested after 0, 7, or 14 (but not 21) days of protein malnutrition . Thus, both protein malnutrition and endotoxin impaired systemic and gut-associated immune responsiveness to mitogens . However, in the protein-malnourished mice, the degree of immune suppression did not correlate with endotoxin-induced mortality. J Antimicrob Chemother, 1992 Jan, 29(1), 19 - 25 Uptake of minocycline by Escherichia coli; Chopra I et al.; Uptake of tetracyclines into Escherichia coli was assessed with a strain carrying a tetA-lacZ translational fusion, in which expression of the enzyme is controlled by the pSC101 tetR repressor gene, by examining beta-galactosidase induction . The ability of tetracycline analogues to induce beta-galactosidase synthesis was correlated with their hydrophobicity, such that hydrophobic analogues were poor enzyme inducers . Treatment of E . coli with polymyxin B nonapeptide (PMBN) rendered cells more permeable to minocycline, but not to tetracycline. Mol Gen Genet, 1992 Jan, 231(2), 256 - 64 Regulation of the gua operon of Escherichia coli by the DnaA protein; Tesfa-Selase F et al.; The guaBA operon determines production of the two enzymes required to convert hypoxanthine to guanine at the nucleotide level during guanine nucleotide biosynthesis . Two DnaA boxes, binding sites for the DNA replication-initiating DnaA protein, are present in the gua operon, one at the gua promoter (guaP) and the other within the guaB coding sequence . Regulation of the guaBA operon by DnaA protein was studied using strains carrying chromosomal gua-lacZ fusions . In these strains beta-galactosidase acts as a reporter enzyme for transcription initiated at guaP . When the intracellular levels of DnaA were increased (by induction of a multicopy plasmid carrying the dnaA gene fused to the tac promoter) transcription from the gua promoter was repressed . Reducing the intracellular level of DnaA, either by sequestration with an oriC plasmid or by placing a temperature-sensitive dnaA mutant at the restrictive temperature, resulted in increased transcription from guaP . Thus the transcriptional activity of the gua operon is coupled, through the DnaA protein, to the DNA replication cycle . Repression of guaP by DnaA was dependent on the presence of both boxes in the gua-lacZ fusion; constructs containing only the box at guaP were unaffected by DnaA. Mol Gen Genet, 1992 Jan, 231(2), 248 - 55 In vitro interactions of integration host factor with the ompF promoter-regulatory region of Escherichia coli; Ramani N et al.; Previous work has shown that integration host factor (IHF) mutants have increased expression and altered osmoregulation of OmpF, a major Escherichia coli outer membrane protein . By in vitro analysis the possibility was investigated that IHF interacts directly with the ompF promoter region . Gel retardation assays and DNase I protection experiments showed that IHF binds to two sites in the ompF promoter region centered at positions -180 and -60 relative to the start of transcription . Gel electrophoresis studies with circularly permuted ompF promoter fragments indicated that IHF binding strongly increased a small intrinsic bend in the ompF promoter region . The addition of IHF to a purified in vitro transcription system strongly and specifically inhibited ompF transcription . This inhibition was reversed by increasing the concentration of OmpR, a positive activator required for ompF expression, suggesting that IHF may inhibit ompF transcription by altering how OmpR interacts with the ompF promoter. Mol Gen Genet, 1992 Jan, 231(2), 169 - 78 Transcription in vivo within the replication origin of the Escherichia coli chromosome: a mechanism for activating initiation of replication; Asai T et al.; Within the replication origin, oriC, of the Escherichia coli chromosome, novel in vivo transcripts were detected which proceeded rightward and whose production was activated by DnaA protein . In contrast, DnaA protein repressed the previously described ori-L leftward transcription . The former should introduce negative supercoiling, and the latter positive supercoiling, into the 13-mers . The effects of transcription on the initiation of replication were also investigated by making constructs with promoters placed near oriC . Transcription was found to enhance the origin activity only when it was oriented in such a way as to introduce negative supercoiling into the 13-mers . From these results, we propose that transcription within oriC regulates replication initiation by altering the topology of the 13-mer region. Biotechniques . 1992 Jan;12(1):28, 30. An efficient method for blunt-end ligation of PCR products; Liu ZG et al.; This report presents data demonstrating a simple method that can potentially be extended to a wide range of cloning strategies to increase the yield of insert-containing recombinants . The method requires that the ligation of an insert to a vector does not regenerate the original restriction enzyme recognition sequence . In the example presented, PCR products were blunt-end ligated to a SmaI-cut vector, in the presence of SmaI endonuclease . The addition of the restriction enzyme to the ligation reaction dramatically favored the ligation of insert to vector rather than vector self-ligation. Scand J Immunol, 1992 Jan, 35(1), 53 - 62 Regulation of the immune response to hepatitis B virus and human serum albumin . III . Induction of anti-albumin antibody secretion in vitro by C-gene-derived proteins in peripheral B cells from chronic carriers of HBsAg; Hellstrom UB et al.; The circulatory pool of B cells from the majority (11/13) of chronic hepatitis B surface antigen (HBsAg) carriers contained sensitized B cells with the capacity to secrete IgG antibodies with specificity for human serum albumin (HSA), when stimulated with E . coli-derived core protein at low concentrations in vitro . The IgG anti-HSA secretion was dependent upon and regulated by T cells, and optimal secretion was obtained at T/B-cell ratios of 1.0-4.0, varying for different individuals . The level of anti-HSA secretion was higher for patients with on-going viral replication as assessed by hepatitis B virus (HBV)-DNA in serum . Culture supernatants containing anti-HSA antibodies also contained anti-HBc antibodies, as detected by enzyme-linked immunosorbent assay (ELISA) where the solid phase was charged with E . coli-derived core protein, or the synthetic peptides corresponding to the 75-84 and 132-147 sequences in the C region of HBV . In contrast, IgG anti-HBc (E . coli-derived), but no anti-HSA or anti-HBc 75-84, 132-147 antibodies, were detected at similar T/B-cell ratios in cell cultures from 5/6 individuals with naturally acquired immunity to hepatitis B . These data indicate that peripheral B cells from the majority of HB-immune donors are sensitized to unique (e.g . non-albumin associated) structures in the nucleocapsid of HBV, while B cells in the majority of chronic HBsAg carriers are sensitized to linear C-gene-derived structures in association with the host 'self'-component HSA. Hum Genet, 1992 Jan, 88(3), 320 - 4 Heterogeneity of mutations in the uroporphyrinogen III synthase gene in congenital erythropoietic porphyria; Boulechfar S et al.; Congenital erythropoietic porphyria (CEP) or Gunther's disease is an inborn error of heme biosynthesis transmitted as an autosomal recessive trait and characterized by a profound deficiency of uroporphyrinogen III synthase (UROIIIS) activity . We have previously described two missense mutations in the UROIIIS gene, confirming that the primary defect responsible for CEP is a structural alteration of this gene . We have extended our work to 5 additional unrelated families . Two new point mutations, a deletion and an insertion have been found in the messenger RNA . Our study shows that a molecular heterogeneity of the mutations exists in Gunther's disease . One mutation (C73R), however, appears to be more frequent than the others . Finally, the different normal and mutated proteins have been expressed in Escherichia coli to determine the consequence of the mutations on the enzyme activity. Clin Chem, 1992 Jan, 38(1), 44 - 7 New enzymatic method with tryptophanase for determining potassium in serum; Kimura S et al.; We established a simple and rapid enzymatic method for measuring potassium ion in serum by using tryptophanase (EC 4.1.99.1) purified from Escherichia coli K12 strain (E . coli K12 IFO 3301) . The presence of pyridoxal 5-phosphate promotes this enzymatic reaction, and potassium and (or) ammonium ions further accelerate it, with ammonium and potassium ions providing equivalent acceleration . We eliminated endogenous ammonium ion by using glutamate dehydrogenase (GLDH; EC 1.4.1.4), then produced ammonium ion in the presence of tryptophanase, tryptophan, and pyridoxal 5-phosphate . The concentration of formed ammonium ion, which was proportional to that of potassium ion in sample, was determined by adding GLDH to produce NADP+ in the presence of 2-oxoglutarate and NADPH; we then read the change of absorbance at 340 nm . The standard curve was linear for potassium ion concentrations up to 7.00 mmol/L . The within-assay variation (CV) was 0.89% at 5.51 mmol/L and 1.32% at 3.37 mmol/L . The day-to-day CVs were 0.99% at 6.85 mmol/L and 1.71% at 3.52 mmol/L . Analytical recoveries ranged from 98.7% to 108.9% . The correlation coefficient between values obtained with this enzymatic assay (y) and by flame photometry (x) was 0.995: y = 0.984x + 0.091 mmol/L (Sy.x = 0.105, n = 100) . The presence of hemoglobin, bilirubin, or other cations little affects this system. Am J Surg, 1992 Jan, 163(1), 100 - 3; discussion 103-4 Influence of levamisole on pancreatic infection in acute pancreatitis; Widdison AL et al.; We investigated the effect of levamisole on pancreatic infection in a model of acute pancreatitis (AP) in cats . Animals with and without AP received Escherichia coli intravenously . Blood was then taken at intervals for culture . AP reduced phagocytic function by 28% as measured by the rate of bacterial disappearance from the blood (p less than 0.03) . In other cats, AP was induced, and E . coli were placed into the pancreatic duct . Levamisole was given orally in some cats; the remainder were untreated . Control cats (neither AP nor levamisole) also received E . coli . Seven days later, pancreases from all control cats were sterile . In AP cats, the pancreatic infection rate was 73% . Levamisole reduced the rate of infection to 22% (p less than 0.03) . We concluded that phagocytic function was impaired in cats with AP . Levamisole reduced the rate of pancreatic infection. Am J Obstet Gynecol, 1992 Jan, 166(1 Pt 1), 14 - 5 Abruptio placentae associated with perforated appendicitis and generalized peritonitis; Yaron Y et al.; A primigravid woman at 35 weeks' gestation was admitted with abdominal pain, fever, and vomiting . Forceful contractions and signs of fetal distress suggested abruptio placentae . During caesarean section, seropurulent exudate and a perforated appendix were found; an appendectomy was performed . A mechanism linking appendicitis with abruptio placentae is suggested. Genetics, 1992 Jan, 130(1), 37 - 49 Restriction-stimulated homologous recombination of plasmids by the RecE pathway of Escherichia coli; Nussbaum A et al.; To test the double-strand break (DSB) repair model in recombination by the RecE pathway of Escherichia coli, we constructed chimeric phages that allow restriction-mediated release of linear plasmid substrates of the bioluminescence recombination assay in infected EcoRI+ cells . Kinetics of DSB repair and expression of recombination products were followed by Southern hybridization and by the bioluminescence recombination assay, respectively . Plasmid recombinants were analyzed with restriction endonucleases . Our results indicate that a DSB can induce more than one type of RecE-mediated recombination . A DSB within the homology induced intermolecular recombination that followed the rules of the DSB repair model: (1) Recombination was enhanced by in vivo restriction . (2) Repair of the break depended on homologous sequences on the resident plasmid . (3) Break-repair was frequently associated with conversion of alleles that were cis to the break . (4) Conversion frequency decreased as the distance from the break increased . (5) Some clones contained a mixture of plasmid recombinants as expected by replication of a heteroduplex in the primary recombinant . The rules of the DSB repair model were not followed when recombination was induced by a DSB outside the homology . Both the cut and the uncut substrates were recipients in conversion events . Recombination events were associated with deletions that spanned the break site, but these deletions did not reach the homology . We propose that a break outside the homology may stimulate a RecE-mediated recombination pathway that does not involve direct participation of DNA ends in the homologous pairing reaction. Plant Mol Biol, 1992 Jan, 18(1), 65 - 78 Activation of a truncated PR-1 promoter by endogenous enhancers in transgenic plants; Beilmann A et al.; PR-1 genes are induced by various environmental stimuli such as pathogen attack or exposure of the plants to certain chemicals . To examine the regulation of these genes, the 5' flanking regions of the PR-la gene and of two PR-1 pseudogenes were joined by a transcriptional fusion to the Escherichia coli beta-glucuronidase (GUS) gene . These constructs were stably integrated into the tobacco genome and independent primary transformants were monitored for the expression of the reporter gene . Unexpectedly, out of 55 transformants analysed, four plants exhibited considerable GUS activities without any inductive treatment of the plants . Expression of the endogenous PR-1 genes, however, could not be detected in these plants . Primer extension analyses revealed correct initiation of the PR1/GUS hybrid transcripts from the PR-1a TATA box . When the plants were analysed at the cellular level, clear differences regarding the tissue specificity of expression of the reporter gene were observed . These results strongly suggest that the PR1/GUS hybrid promoter expression cassettes may be activated when integrated in the vicinity of heterologous enhancer elements dispersed in the tobacco genome . In order to support this hypothesis, domain B of the enhancer of the 35S RNA promoter from cauliflower mosaic virus (CaMV) was fused to various PR1/GUS hybrid genes upstream as well as downstream from the RNA start site . These constructs were stably introduced into the tobacco genome . In any primary transformant analysed, strong GUS activities were observed with the PR1/GUS hybrid RNAs originating from the normal transcription start site of the PR-1a gene . The tissue specificity of gene expression was identical to that described previously for the CaMV 35S domain B enhancer element . Thus, modulations of the transcriptional activity of the PR-1 promoter can be achieved by heterologous enhancers in transgenic plants and may be encountered upon random integration of PR-1 promoter constructs into the tobacco genome. Biochem J, 1992 Jan 1, 281 ( Pt 1), 57 - 65 Purification and analysis of proteinase-resistant mutants of recombinant platelet-derived growth factor-BB exhibiting improved biological activity; Cook AL et al.; Recombinant platelet-derived growth factor (PDGF)-BB was expressed and secreted from yeast in order to study the structure-function relationships of this mitogen . A simple purification scheme has been developed which yields greater than 95% pure PDGF-BB . Analysis of this recombinant PDGF-BB shows partial proteolysis after arginine-32 . Substitution of this arginine residue, or arginine-28 {a potential KEX2 (lysine-arginine endopeptidase) cleavage site}, prevents or reduces cleavage of PDGF-BB respectively . These mutations result in a 5-fold increase in expression levels of PDGF-BB, and the resulting mutant proteins show higher activity in a number of biological assays than the cleaved wildtype PDGF-BB . These data are in accord with previous work by Giese, LaRochelle, May-Siroff, Robbins & Aaronson {(1990) Mol . Cell Biol . 10, 5496-5501} suggesting that the region isoleucine-25-phenylalanine-37 is involved in PDGF-receptor binding. Biochem J, 1992 Jan 1, 281 ( Pt 1), 255 - 9 Stimulation of the dithiol-dependent reductases in the vitamin K cycle by the thioredoxin system . Strong synergistic effects with protein disulphide-isomerase; Soute BA et al.; It has been shown previously that the thioredoxin system (thioredoxin + thioredoxin reductase + NADPH) may replace dithiothreitol (DTT) as a cofactor for vitamin KO and K reductase in salt-washed detergent-solubilized bovine liver microsomes . Here we demonstrate that the system can be improved further by adding protein disulphide-isomerase (PDI) to the components mentioned above . Moreover, NADPH may be replaced by reduced RNAase as a hydrogen donor . In our in vitro system the various protein cofactors were required at concentrations 2-5 orders of magnitude lower than that of DDT, whereas the maximal reaction rate was about 3-fold higher . PDI stimulated the thioredoxin-driven reaction about 10-fold, with an apparent Km value of 8 microM . These data suggest that in the vitro system the formation of disulphide bonds is somehow linked to the vitamin K-dependent carboxylation of glutamate residues . In vivo, both disulphide formation and vitamin K-dependent carboxylation are post-translational modifications taking place at the luminal side of the endoplasmic reticulum of mammalian secretory cells . The possibility that the reactions are also coupled in vivo is discussed. Arch Virol, 1992, 122(3-4), 223 - 35 Identification and sequence determination of the capsid protein gene of feline calicivirus; Carter MJ et al.; We have determined 4380 bases of the sequence from a cDNA clone containing the 3' end of feline calicivirus strain F9 . We find four candidate open reading frames of which three are complete and comprise 245, 317 and 2012 nucleotides . The fourth continues toward the 5' end . We have expressed the largest complete open reading frame in E . coli . Sera raised to this antigen react specifically with the capsid protein and its intracellular precursor molecule . N-terminal sequence analysis of purified, mature capsid protein confirms this assignment and has identified the position at which precursor is cleaved. J Med Microbiol, 1992 Jan, 36(1), 37 - 40 A comparative study of specific gene probes and standard bioassays to identify diarrhoeagenic Escherichia coli in paediatric patients with diarrhoea in Bangladesh; Faruque SM et al.; We compared the usefulness of gene probes with standard bioassays to identify diarrhoeagenic Escherichia coli amongst isolates from Bangladeshi children under 1 year of age with diarrhoea . E . coli isolates were analysed with specific gene probes for localised adhesiveness (LA), diffuse adhesiveness (DA), heat-labile toxin (LT), heat-stable toxin (ST), Shiga-like toxins (SLT I and SLT II), and enteroinvasiveness, and in bioassays for production of enterotoxins and cytotoxins, and for cell adherence . With 1136 isolates from 387 patients, there was general agreement between the two assay methods . When there was disparity, gene-probe-positive isolates gave negative results in the corresponding bioassay . In the HeLa cell adherence assay, 94% of the LA probe-positive isolates and 91.6% of the DA probe-positive isolates gave positive bioassay results for LA and DA respectively . Thirty-six of 39 LT probe-positive isolates and 73 of 86 ST probe-positive isolates gave positive results in the bioassays . Of 28 isolates that gave negative results in the suckling mouse assay but were initially positive with the probe for ST, 15 were later found to hybridize with the cloning vector for the ST probe . Addition of denatured vector DNA at a concentration of 10 micrograms/ml in the hybridisation solution eliminated these false positive results . None of the other probe-positive isolates hybridised with any of the cloning vectors used . The DNA hybridisation assay appeared to be a convenient alternative to bioassays for screening large numbers of isolates in epidemiological investigation. J Exp Med, 1992 Jan 1, 175(1), 275 - 84 A major T cell antigen of Mycobacterium leprae is a 10-kD heat-shock cognate protein; Mehra V et al.; Several mycobacterial antigens, identified by monoclonal antibodies and patient sera, have been found to be homologous to stress or heat-shock proteins (hsp) defined in Escherichia coli and yeast . A major antigen recognized by most Mycobacterium leprae-reactive human T cell lines and cell wall-reactive T cell clones is a 10-kD protein that has now been cloned and sequenced . The predicted amino acid sequence of this protein is 44% homologous to the hsp 10 (GroES) of E . coli . The purified native and recombinant 10-kD protein was found to be a stronger stimulator of peripheral blood T cell proliferation than other native and recombinant M . leprae proteins tested . The degree of reactivity paralleled the response to intact M . leprae throughout the spectrum of leprosy . Limiting-dilution analysis of peripheral blood lymphocytes from a patient contact and a tuberculoid patient indicated that approximately one third of M . leprae-reactive T cell precursors responded to the 10-kD antigen . T cell lines derived from lepromin skin tests were strongly responsive to the 10-kD protein . T cell clones reactive to both the purified native and recombinant 10-kD antigens recognized M . leprae-specific epitopes as well as epitopes crossreactive with the cognate antigen of M . tuberculosis . Further, the purified hsp 10 elicited strong delayed-type hypersensitivity reactions in guinea pigs sensitized to M . leprae . The strong T cell responses against the M . leprae 10-kD protein suggest a role for this heat-shock cognate protein in the protective/resistant responses to infection. J Cell Biol, 1992 Jan, 116(1), 1 - 14 Localization of the nucleolar protein NO38 in amphibian oocytes; Peculis BA et al.; To examine the role of primary amino acid sequence in the localization of proteins within the nucleus, we studied the nucleolar protein NO38 of amphibian oocytes . We synthesized NO38 transcripts in vitro, injected them into the oocyte cytoplasm, and followed the distribution of the translation products . The injected RNA contained a short sequence encoding an epitope derived from the human c-myc protein . We used an mAb against this epitope to detect translation products from injected RNAs by Western blots and by immunofluoresent staining of cytological preparations . When full-length transcripts of NO38 were injected into oocytes, the translation products accumulated efficiently in the germinal vesicle, and a major fraction was localized in the multiple nucleoli . To identify protein domains involved in this nucleolus-specific accumulation, we prepared a series of carboxy-terminal deletions of the cDNA . Oocytes injected with RNA encoding truncated forms of NO38 were examined for altered patterns of protein accumulation . We defined a domain of about 24 amino acids near the carboxy terminus that was essential for nucleolar localization of NO38 . This domain is separated by more than 70 amino acids from two putative nuclear localization signals near the middle of the molecule . Hybrid constructs were made which encoded part of Escherichia coli beta-galactosidase or pyruvate kinase fused to a long segment of NO38 containing the essential domain . Injection of RNA from these constructs showed that the essential domain was not sufficient to target the hybrid proteins to the nucleolus . We suggest that nucleolar accumulation of NO38 requires more than a single linear domain. Genes Dev, 1992 Jan, 6(1), 129 - 34 In vitro selection of active hairpin ribozymes by sequential RNA-catalyzed cleavage and ligation reactions; Berzal-Herranz A et al.; In vitro selection methods provide rapid and extremely powerful tools for elucidating interactions within and between macromolecules . Here, we describe the development of an in vitro selection procedure that permits the rapid isolation and evaluation of functional hairpin ribozymes from a complex pool of sequence variants containing an extremely low frequency of catalytically proficient molecules . We have used this method to analyze the sequence requirements of two regions of the ribozyme-substrate complex: a 7-nucleotide internal loop within the ribozyme that is essential for catalytic function and substrate sequences surrounding the cleavage-ligation site . Results indicate that only 3 of the 16,384 internal loop variants examined have high cleavage and ligation activity and that the ribozyme has a strong requirement for guanosine immediately 3' to the cleavage-ligation site. Proc Natl Acad Sci U S A, 1992 Jan 1, 89(1), 56 - 9 parB: an auxin-regulated gene encoding glutathione S-transferase; Takahashi Y et al.; We have isolated an auxin-regulated cDNA, parB, from the early stage of cultured tobacco mesophyll protoplasts . The expression of parB was observed during transition from G0 to the S phase of tobacco mesophyll protoplasts cultured in vitro . The predicted amino acid sequence of parB cDNA has 213 amino acid residues with a relative molecular weight of 23,965 . Nucleotide sequence analysis revealed that parB cDNA has homology to glutathione S-transferase (GST; RX:glutathione R-transferase, EC 2.5.1.18) from several sources including plant and animal cells . When we introduced expression vector pKK233-2, which retains parB cDNA, into Escherichia coli, we could detect GST activity in the parB gene product . Accordingly a significant increase of GST activity was detected in the tobacco mesophyll protoplasts cultured in the presence of 2,4-dichlorophenoxyacetic acid . This is an example in which the function of auxin-regulated gene product is shown to be ascribed to a specific enzymatic activity . As GST, and its substrate glutathione, are shown to be related to cell proliferation as well as detoxification of xenobiotics in plant and animal cells, the role of parB is discussed in relation to the induction of proliferative activity in differentiated and nondividing mesophyll protoplasts of tobacco. Proc Natl Acad Sci U S A, 1992 Jan 1, 89(1), 397 - 401 Class II (B) general transcription factor (TFIIB) that binds to the template-committed preinitiation complex is different from general transcription factor BTF3; Moncollin V et al.; A class II (B) general transcription factor of 34 kDa has been purified from HeLa cells to apparent homogeneity . This factor appears to be transcription factor IIB (TFIIB), since it binds in vitro to template-committed preinitiation complexes formed between a template containing the TATA box/cap-site elements of the adenovirus type 2 major late promoter (Ad2MLP) and recombinant human or yeast TFIID (previously called BTF1) expressed in Escherichia coli . DNase I footprint studies show an extended pattern of protection of Ad2MLP TATA box/cap-site sequences when TFIIB is bound to template-committed complexes, even though TFIIB does not bind on its own to the template in the absence of TFIID . We also show that TFIIB is different from BTF3 by a number of criteria. Proc Natl Acad Sci U S A, 1992 Jan 1, 89(1), 295 - 9 Tsp: a tail-specific protease that selectively degrades proteins with nonpolar C termini; Silber KR et al.; An Escherichia c