J Mol Biol, 1989 Oct 20, 209(4), 623 - 34 Plasmid-mediated lethality and plasmid multimer formation in an Escherichia coli recBC sbcBC mutant . Involvement of RecF recombination pathway genes; Kusano K et al.; Apparent plasmid instability, i.e . progressive plasmid loss in a bacterial culture growing in the absence of selection for the plasmid, in an Escherichia coli recBC sbcBC mutant was investigated with two different ColE1 derivatives (pMB9 and pBR322) and a mini-F plasmid . The instability was most striking for pMB9 and much less, but still significant, for pBR322 and the mini-F . It was also dependent upon a subset of the genes involved in the RecF recombination pathway: in addition to the previously reported recA, recF and recJ mutations, a recO and a recQ mutation showed a total and a partial suppression, respectively, of the instability . Other recF-family mutations, recN and ruv, were without such an effect . Population analyses of the recBC sbcBC strain carrying pMB9 or the mini-F, as carried out by plating and Coulter counting, revealed marked loss of viability in plasmid-carrying cells, strongly implicating plasmid-mediated cell death in the apparent defect in plasmid maintenance . Analysis of intracellular plasmid DNA by pulsed-field gel electrophoresis combined with the in-agarose cell lysis technique showed that the instability was associated with the formation of plasmid multimers, with a good correlation between the degree of the instability and the amount of the multimers . The multimer formation was also dependent on the same subset of the RecF pathway genes as in the instability phenomenon . These results strongly suggest that the lethality is somehow caused by the multimer formation . Various DNase treatments of cell lysates showed that such multimers of pMB9 DNA comprised molecules of exonuclease-sensitive and exonuclease-resistant types . It was inferred that the former class, which showed electrophoretic mobilities corresponding to plain linear duplexes of approximately 200 x 10(3) to 2200 x 10(3) base-pairs, represented linear multimers possibly carrying circular structures at one end . The latter class, which remained in the origin, was thought to consist of circular multimers and/or linear multimers protected by circular structures at both ends against exonucleolytic attack. Science, 1989 Oct 20, 246(4928), 358 - 63 Stabilization of Z DNA in vivo by localized supercoiling; Rahmouni AR et al.; Biological processes such as transcription may generate domains of supercoiling on a circular DNA . The existence of these domains in Escherichia coli was investigated by the ability of different lengths of (CG) tracts, cloned upstream or downstream from the tetracycline resistance gene (tet) of pBR322, to adopt the Z structure in vivo . Segments as short as 12 base pairs adopt the Z form when cloned upstream from the tet gene (Eco RI site), whereas no Z DNA was detected when this sequence was cloned downstream (Sty I site), even with a 74-base pair (CG) tract that requires less supercoiling than shorter tracts for the B-Z transition . Hence the localized supercoil density in pBR322 can be as high as -0.038 and as low as -0.021 at different loci . These data demonstrate the existence of the Z structure for commonly found natural sequences and support the notion of domains of negative supercoiling in vivo. J Mol Biol, 1989 Oct 20, 209(4), 599 - 606 Cloning, over-expression and the catalytic properties of the EcoP15 modification methylase from Escherichia coli; Rao DN et al.; The EcoP15 modification methylase gene from the p15B plasmid of Escherichia coli 15T-has been cloned and expressed at high levels in a plasmid vector system . We have purified the enzyme to near homogeneity in large amounts and have studied some of its enzymatic properties . Initial rates of methyl transfer are first order in methylase concentration and, with pUC19 DNA as substrate, the reaction proceeds by a random mechanism in which either DNA or S-adenosylmethionine can bind to the free enzyme . After methyltransfer to DNA, the methylated DNA and S-adenosylhomocysteine appear to dissociate in random order . As expected in such a mechanism, S-adenosylhomocysteine is a non-competitive inhibitor by S-adenosylmethionine at concentrations not much above its KM suggests that release of methylated DNA may be the rate-limiting step . This suggestion is strengthened by the fact that a mutant of the closely related EcoP1 does not show such substrate inhibition. Eur J Biochem, 1989 Oct 20, 185(1), 163 - 71 The role of subunit 4, a nuclear-encoded protein of the F0 sector of yeast mitochondrial ATP synthase, in the assembly of the whole complex; Paul MF et al.; The yeast nuclear gene ATP4, encoding the ATP synthase subunit 4, was disrupted by insertion into the middle of it the selective marker URA3 . Transformation of the Saccharomyces cerevisiae strain D273-10B/A/U produced a mutant unable to grow on glycerol medium . The ATP4 gene is unique since subunit 4 was not present in mutant mitochondria; the hypothetical truncated subunit 4 was never detected . ATPase was rendered oligomycin-insensitive and the F1 sector of this mutant appeared loosely bound to the membrane . Analysis of mitochondrially translated hydrophobic subunits of F0 revealed that subunits 8 and 9 were present, unlike subunit 6 . This indicated a structural relationship between subunits 4 and 6 during biogenesis of F0 . It therefore appears that subunit 4 (also called subunit b in beef heart and Escherichia coli ATP synthases) plays at least a structural role in the assembly of the whole complex . Disruption of the ATP4 gene also had a dramatic effect on the assembly of other mitochondrial complexes . Thus, the cytochrome oxidase activity of the mutant strain was about five times lower than that of the wild type . In addition, a high percentage of spontaneous rho- mutants was detected. Cell, 1989 Oct 20, 59(2), 385 - 94 A specific class of IS10 transposase mutants are blocked for target site interactions and promote formation of an excised transposon fragment; Haniford DB et al.; We report the identification and characterization of a class of IS10 transposase mutants that carry out only some of the steps required for transposition . These mutants were identified among transposition-defective mutants as a specific subclass that retains the wild-type ability to induce SOS functions in the presence of transposon ends . Mutants of this class successfully promote excision of the element from its donor site, but do not promote transfer of the transposon sequences to a target site . SOS induction presumably results from the degradation of the donor site . Uniquely among transposition-defective mutants, SOS+ Tnsp- mutants promote the formation of a new product, the excised transposon fragment (ETF), which consists of the transposon excised from the original donor molecule by double-strand breaks at the transposon ends . SOS+ Tnsp- mutants identified thus far define two patches of amino acids that might correspond to regions of different function . A single additional mutation maps within a region that is highly conserved among IS element transposases . The existence of SOS+ Tnsp- mutants and the structure of the ETF provide strong support for the previously proposed nonreplicative model of Tn10/IS10 transposition. Cell, 1989 Oct 20, 59(2), 373 - 83 Intramolecular transposition by Tn10; Benjamin HW et al.; Transposon Tn10 promotes the formation of a circular product containing only transposon sequences . We show that these circles result from an intramolecular transposition reaction in which all of the strand cleavage and ligation events have occurred but newly created transposon/target junctions have not undergone repair . The unligated strand termini at these junctions are those expected according to a simple model in which the target DNA is cleaved by a pair of staggered nicks 9 bp apart, transposon sequences are separated from flanking donor DNA by cleavage at the terminal nucleotides on both strands (at both ends) of the element, and 3' transposon strand ends are ligated to 5' target strand ends . The stability of the unligated junctions suggests that they are protected from cellular processing by transposase and/or host proteins . We propose that the nonreplicative nature of Tn10 transposition is determined by the efficiency with which the nontransferred transposon strand is separated from flanking donor DNA and by the nature of the protein-DNA complexes present at the strand transfer junctions. J Mol Biol, 1989 Oct 20, 209(4), 607 - 22 Determining residue-base interactions between AraC protein and araI DNA; Brunelle A et al.; Depurination/depyrimidation binding-interference experiments (missing contact probing) identified specific candidate residue-base interactions lost by mutants of Escherichia coli L-arabinose operon regulatory protein, AraC, to one of its binding sites, araI . These candidates were then checked more rigorously by comparing the affinities of wild-type and alanine-substituted AraC protein to variants of araI with alterations in the candidate contacted positions . Residues 208 and 212 apparently contact DNA and support, but do not prove the existence of a helix-turn-helix structure in this region of AraC protein whereas contacts by mutants with alterations at positions 256, 257 and 261 which are within another potential helix-turn-helix region do not support the existence of such a structure there . The missing contacts displayed by three AraC mutants are found within two major groove regions of the DNA and are spaced 21 base-pairs apart in a pattern indicating a direct repeat orientation for the subunits of AraC. Eur J Biochem, 1989 Oct 20, 185(1), 63 - 71 Vascular anticoagulant beta: a novel human Ca2+/phospholipid binding protein that inhibits coagulation and phospholipase A2 activity . Its molecular cloning, expression and comparison with VAC-alpha; Hauptmann R et al.; A cDNA was cloned coding for a new member of the human Ca2+-modulated phospholipid-binding protein family termed annexins . Due to its 56% identity to the human vascular anticoagulant (VAC) the new protein is named VAC-beta, renaming the previous VAC as VAC-alpha . Northern analysis detects one hybridizing mRNA species of 2.2 kb in human placenta . Genomic Southern blot analysis shows a VAC-beta gene of comparable complexity to the VAC-alpha gene . The cDNA was expressed in Escherichia coli and the recombinant protein purified to homogeneity . Antiserum raised against VAC-beta weakly cross-reacts with VAC-alpha . The properties of VAC-beta as an anticoagulant and as an inhibitor of phospholipase A2 activity were analyzed and compared to those of VAC-alpha. Biochemistry, 1989 Oct 17, 28(21), 8479 - 84 Insertion of new sequences into the catalytic domain of an enzyme; Starzyk RM et al.; Activities of enzymes can be modified by the replacement of active-site amino acids with residues that strengthen specific interactions with substrates or that alter the specificity . The scope for engineered enzymes would be broadened if additional, new sequences could be inserted into a catalytic domain . Properly designed, these sequences could encode new ligand binding sites, be intermediates in the construction of chimeric enzymes, or alter the internal flexibility and "breathing" modes of the active-site region . As a first step toward this objective, we inserted oligopeptides of up to 14 amino acids into various locations within an 82 amino acid region of the adenylate synthesis domain of Escherichia coli methionyl-tRNA synthetase . These sites include ones that are flanked by sequences that are conserved between the proteins from E . coli and the yeast Saccharomyces cerevisiae and those that are essential for activity and stability . We found that all of the insertional mutants are stable and some have catalytic parameters for adenylate synthesis that are comparable to those of the wild-type enzyme . Thus, such an approach may provide for a variety of novel applications. Biochemistry, 1989 Oct 17, 28(21), 8287 - 92 Repair of pyrimidine dimer ultraviolet light photoproducts by human cell extracts; Wood RD; A newly developed method allows human cell extracts to carry out repair synthesis on ultraviolet light irradiated closed circular plasmid DNA {Wood, R . D., Robins, P., & Lindahl, T . (1988) Cell 53, 97-106} . The identity of the photodamage that leads to this repair replication was investigated . Removal of stable pyrimidine hydrates from irradiated plasmid pAT153 did not significantly affect the amount of repair replication in the fluence range of 0-450 J/m2, because of the low yield of these products and their short DNA repair patch size . Photoreactivation of irradiated DNA using purified Escherichia coli DNA photolyase to remove more than 95% of the cyclobutane dimers from the DNA reduced the observed repair synthesis by 20-40% . The greater part of the repair synthesis is highly likely to be caused by (6-4) pyrimidine dimer photoproducts . This class of lesions is rapidly repaired by mammalian cells, and their removal is known to be important for cell survival after ultraviolet irradiation. Biochemistry, 1989 Oct 17, 28(21), 8454 - 9 Investigation of the mechanism of CTP synthetase using rapid quench and isotope partitioning methods; Lewis DA et al.; The UTP-dependent ATPase reaction and the glutamine-dependent overall reaction of Escherichia coli CTP synthetase have been studied by rapid quench and isotope partitioning kinetics . The effect of GTP, an allosteric effector, on the pre-steady-state kinetics of both reactions has also been examined . The time courses of the UTP-dependent ATPase reaction in the presence and absence of GTP are both characterized by a burst of acid-labile phosphate equivalent to 0.93 and 0.43 subunits, respectively . The time course of the glutamine-dependent reaction in the absence of GTP is also characterized by a burst of acid-labile phosphate corresponding to 0.8 subunit; however, in the presence of GTP, no burst was observed . These results along with positional isotope exchange experiments {von der Saal, W., Anderson, P . M., & Villafranca, J . J . (1985) J . Biol . Chem . 260, 14997} provide evidence that the mechanism of CTP formation involves phosphorylation of UTP followed by attack of NH3, and finally release of phosphate, producing CTP, ADP, and Pi . A kinetic model for the first stages of the enzymatic reaction was developed from the rapid quench data, and the internal equilibrium constant for the formation of the phosphorylated UTP intermediate was determined . The internal equilibrium constants for the UTP-dependent reaction in the presence and absence of GTP were found to be 1.1 and 18, respectively . By contrast, the internal equilibrium constant for the reaction in the presence of glutamine was 50 . Thus, the presence of glutamine shifts the internal equilibrium constant to favor formation of the phosphorylated UTP intermediate.(ABSTRACT TRUNCATED AT 250 WORDS) Biochem Biophys Res Commun, 1989 Oct 16, 164(1), 503 - 11 Primary structure and functional expression of h-caldesmon complementary DNA; Hayashi K et al.; Recently, the two Mr forms of caldesmon (Mr's in the range of 120-150kDa and 70-80kDa as judged by SDS-PAGE) have been identified . h-Caldesman (high Mr 120-150kDa caldesmon) is predominantly expressed in smooth muscles, and l-caldesmon (low Mr 70-80kDa caldesmon) in non-muscle cells . In this paper, we report the nucleotide sequence of chick embryo gizzard h-caldesmon cDNA and its translation into amino acid sequence . This sequence predicts a protein of 771 amino acids with a Mr of 88,743 . The central portion of this sequence is composed of a 10-fold repeat of conserved amino acid sequence containing 13-15 amino acids . Further, a recombinant protein produced in Escherichia coli containing the full-length h-caldesmon cDNA has been characterized . Although the Mr of h-caldesmon predicted from amino acid sequence is 88,743, native and recombinant proteins show the same mol . wt . with 150kDa as measured by SDS-PAGE . This discrepancy may be due to the acidic amino acid-rich sequences at the N-terminal and central portions . A recombinant protein produced in E . coli possesses calmodulin-, F-actin- and tropomyosin-binding abilities in common with the native h-caldesmon. Biochem Biophys Res Commun, 1989 Oct 16, 164(1), 233 - 7 Ecori DNA methylase activity is eliminated upon histidine residue modification; Everett EA et al.; The E . coli EcoRI DNA methylase activity is completely eliminated in five minutes upon incubation with the histidine residue specific reagent diethyl pyrocarbonate . In that two moles of N-ethoxyformylimidazole per mole of methylase are detected spectroscopically upon inactivation and activity is not restored by hydroxylamine, it is likely that activity loss is due to double modification of a single histidine residue . This information is critical in determining the enzymatic mechanism, causes of the pH-activity curve, designing protein mutants and interpreting previous structure-function data. Biochem Biophys Res Commun, 1989 Oct 16, 164(1), 88 - 93 Observation of arginyl-deoxyoligonucleotide interactions in Taq I endonuclease by detection of specific 1H NMR signals from 140kD {N eta 1, N eta 2, 15N Arg}Taq I/oligomer complexes; Glushka J et al.; Proton and nitrogen signals of the guanidinium amines in {N eta 1, N eta 2 15N Arg}Taq I endonuclease were observed using isotope filtered experiments and proton detected 1H{15N} heterocorrelated two dimensional NMR spectroscopy . These rapidly exchanging protons could be detected in the free enzyme only at pH 4.5; at pH 8.5, no signals were measured after extensive signal averaging . Addition of deoxyribonucleotide oligomers resulted in the appearance of two groups of signals at about 6.8 and 7.5 ppm . Since these signals are independent of the presence of cognate sequence or Mg2+, it is assumed they represent nonspecific arginyl-DNA interactions . This labeling/NMR approach provides a new method for investigating the role of arginine in protein-DNA interactions. Biochem Biophys Res Commun, 1989 Oct 16, 164(1), 30 - 8 Substitution mutations of the highly conserved arginine 87 of HIV-1 protease result in loss of proteolytic activity; Louis JM et al.; The 297bp gene coding for the HIV-1 protease was chemically synthesized and expressed in E . coli . Single amino acid substitutions (Arg 87 - greater than Lys; Arg 87 - greater than Glu) were introduced in the C-terminally located conserved region GlyArgAsn of the protease gene in the wild-type clone . The products of the mutant and the wild-type clones were expressed at approximately similar levels at 30 minutes of induction but the mutant protease proteins accumulated as a function of time of induction unlike the wild-type protease which declined after 60 minutes . The mutants were completely devoid of proteolytic activity as determined in assays employing as substrates a synthetic nonapeptide and a gag related recombinant polyprotein. Biochem Biophys Res Commun, 1989 Oct 16, 164(1), 580 - 6 In vitro phosphorylation of the tumor suppressor gene RB protein by mitosis-specific histone H1 kinase; Taya Y et al.; The major components of the mitosis-specific histone H1 kinase are CDC2 kinase and cyclin and the consensus amino acid sequence for phosphorylation by this enzyme has been proposed . We have noted the presence of such sequences in six sites of the tumor suppressor gene RB protein and determined whether or not RB protein is in fact phosphorylated by this kinase . Highly purified enzyme was used for this purpose . HeLa cell extracts immunoprecipitated with anti-RB antiserum as well as RB proteins expressed in E . coli cells were shown to be phosphorylated by this kinase in vitro . Synthetic peptides for the six expected sites were also phosphorylated . These results suggest the possibility that the function of RB protein is regulated by CDC2 kinase. Biochem Biophys Res Commun, 1989 Oct 16, 164(1), 22 - 9 The biological activity of interferon alpha is influenced by two distinct regions in the protein; Bosveld J et al.; With the aim to assign differences in activity between murine interferon-alpha 1 and -alpha 4 to specific amino acids, we have constructed hybrid genes and analysed the antiviral properties of the corresponding hybrid proteins . The hybrid genes were constructed by means of homologous recombination between the alpha 1 and alpha 4 genes in Escherichia coli . Hybrids in which the N-terminal part is derived from alpha 1 show that two regions have a major effect on the activity: amino acid 10-20 and 55-67 . When comparing hybrids with N-terminal alpha 4 sequences, transitions in activity are found in the same regions . Interestingly, the curves for the two sets of hybrids are exactly each others mirror image. J Biol Chem, 1989 Oct 15, 264(29), 17395 - 400 RecA protein promoted homologous pairing in vitro . Pairing between linear duplex DNA bound to HU Protein (nucleosome cores) and nucleoprotein filaments of recA protein-single-stranded DNA; Ramdas J et al.; RecA protein promotes two distinct types of synaptic structures between circular single strands and duplex DNA; paranemic joints, where true intertwining of paired strands is prohibited and the classically intertwined plectonemic form of heteroduplex DNA . Paranemic joints are less stable than plectonemic joints and are believed to be the precursors for the formation of plectonemic joints . We present evidence that under strand exchange conditions the binding of HU protein, from Escherichia coli, to duplex DNA differentially affects homologous pairing in vitro . This conclusion is based on the observation that the formation of paranemic joint molecules was not affected, whereas the formation of plectonemic joint molecules was inhibited from the start of the reaction . Furthermore, introduction of HU protein into an ongoing reaction stalls further increase in the rate of the reaction . By contrast, binding of HU protein to circular single strands has neither stimulatory nor inhibitory effect . Since the formation of paranemic joint molecules is believed to generate positive supercoiling in the duplex DNA, we have examined the ability of positive superhelical DNA to serve as a template in the formation of paranemic joint molecules . The inert positively supercoiled DNA could be converted into an active substrate, in situ, by the action of wheat germ topoisomerase I . Taken collectively, these results indicate that the structural features of the bacterial chromosome which include DNA supercoiling and organization of DNA into nucleosome-like structures by HU protein modulate homologous pairing promoted by the nucleoprotein filaments of recA protein single-stranded DNA. Biochem J, 1989 Oct 15, 263(2), 485 - 90 Prostaglandin-dependent muscle wasting during infection in the broiler chick (Gallus domesticus) and the laboratory rat (Rattus norvegicus); Tian S et al.; Systemic infection with Escherichia coli significantly decreased feed intake, slowed growth of the whole body and skeletal muscles, and severely inhibited muscle protein accumulation in both chicks and rats . Treatment with naproxen (6-methoxy-alpha-methyl-2-naphthaleneacetic acid), an inhibitor of prostaglandin production, decreased weight losses of body and muscle, and significantly inhibited muscle protein wasting in infected chicks and rats . E . coli infection increased net protein degradation by 44.8% (P less than 0.05) and prostaglandin E2 production by 148% (P less than 0.05) in isolated extensor digitorum communis muscle from chicks on day 2 after infection . Naproxen treatment significantly decreased net protein degradation and prostaglandin E2 production in infected chicks to values seen in muscles of healthy controls . Quantitatively and qualitatively similar results were seen in isolated rat epitrochlearis muscle. J Immunol, 1989 Oct 15, 143(8), 2740 - 4 Thymosin-beta 4 gene . Preliminary characterization and expression in tissues, thymic cells, and lymphocytes; Gomez-Marquez J et al.; A cDNA for rat thymosin-beta 4 was used to investigate the expression of this gene in different tissues, thymic cells, and lymphocytes . Hybridization analysis of total RNA from 13 rat tissues demonstrated the presence of an 800 nucleotides-long mRNA in all the tissues surveyed, with the highest levels in spleen, thymus, and lung . Examination of thymic cells showed that the thymosin-beta 4 gene is predominantly expressed in thymocytes . The thymosin-beta 4 mRNA was also studied in Ig+ and Ig- lymphocytes, being fourfold more abundant in Ig- than Ig+ splenic lymphocytes, whereas similar levels were found in both types of blood cells . The analysis of RNA from T cells at different maturation stages evidenced slight differences in their thymosin-beta 4 mRNA content, indicating that thymosin-beta 4 gene expression is not clearly related to the differentiation process of T cells . All these results do not support the roles for thymosin-beta 4 in cellular immunity and differentiation of lymphoid cells, suggesting a more general function for this peptide . Preliminary characterization of the human beta 4 gene by restriction analysis disclosed a complicated pattern consistent with multiple genes and/or introns . The analysis of genomic DNA from different species ranging from humans to Escherichia coli showed that this gene is only highly conserved in mammals. J Biol Chem, 1989 Oct 15, 264(29), 17309 - 15 Purification of receptor protein Trg by exploiting a property common to chemotactic transducers of Escherichia coli; Burrows GG et al.; The methyl-accepting chemotactic transducers of Escherichia coli were found to bind strongly to Cibacron blue-Sepharose . Among potential elutants tested, only S-adenosylmethionine at moderate concentrations and NaCl at concentrations greater than 1.5 M caused dissociation of these detergent-solubilized transmembrane proteins from the dye . Release by S-adenosylmethionine may be a generalized effect rather than the result of a specific binding site for that compound on transducers . A truncated trg gene was created that coded for the carboxyl-terminal three-fifths of the transducer, which constitutes the cytoplasmic domain common to all four transducers in E . coli . This domain bound to Cibacron blue-Sepharose and was eluted in a pattern similar to that exhibited by intact Trg, indicating that interaction with the dye occurred in this conserved domain . Adherence to Cibacron blue and elution by high salt formed the core of an efficient purification scheme, developed for Trg but applicable to all transducers in E . coli and perhaps to methyl-accepting chemotaxis proteins in other species . Determination of the amino acid sequence at the beginning of purified Trg confirmed that it contained a longer hydrophilic segment at its amino terminus than other transducers of E . coli . The initial methionine of Trg is neither cleaved nor modified, in contrast to the Tar transducer in which the amino terminus was found previously to be blocked . Circular dichroic measurements of purified Trg indicated that the secondary structural organization of the protein is predominantly alpha-helix. J Biol Chem, 1989 Oct 15, 264(29), 17293 - 7 Functional and nonfunctional LamB signal sequences can be distinguished by their biophysical properties; McKnight CJ et al.; The role of the signal sequence in the secretion of proteins remain unclear despite extensive research . We have examined properties of synthetic peptides corresponding to a family of signal sequences derived from the lamB gene of Escherichia coli, including five examples of known phenotype that contain mutations in the signal sequence . By circular dichroism spectroscopy, the wild type and export-component mutant signal peptides show a high alpha-helix content in membrane mimetic environments (sodium dodecyl sulfate micelles and phospholipid vesicles) . Tendency to adopt helical conformations is clearly not sufficient to define a functional signal sequence, however, as some nonfunctional mutant signal peptides also contain a relatively high proportion of alpha-helix . The affinity of these peptides for phospholipid monolayers as assessed by surface tensiometry reveals further distinguishing properties . Export-competent peptides show an increased affinity for and greater perturbation of phospholipid monolayers and bilayers than to export-defective peptides . These results suggest a lipid binding role for the signal sequence during protein export in addition to its recognition by proteins of the export pathway. J Biol Chem, 1989 Oct 15, 264(29), 17266 - 74 Regulatory behavior of Escherichia coli aspartate transcarbamylase altered by site-specific mutation of Tyr240----Phe in the catalytic chain; Wedler FC et al.; Isotopic exchange kinetics at chemical equilibrium have been used to identify changes in the regulatory properties of aspartate transcarbamylase (ATCase) caused by site-specific mutation of Tyr240----Phe (Y240F) in the catalytic chain . With both wild-type and the mutant enzymes, ATP activates both {14C}Asp in equilibrium N-carbamyl-L-aspartate (C-Asp) and the {32P}carbamyl phosphate (C-P) in equilibrium Pi exchanges . In contrast, with wild-type enzyme, CTP inhibits both exchanges, but with Y240F mutant enzyme CTP inhibits Asp in equilibrium C-Asp exchange and activates C-P in equilibrium Pi exchange . The bisubstrate analog N-(phosphonacetyl-L-aspartate), PALA, activates Asp in equilibrium C-Asp at a lower concentration with the Y240F enzyme, but the extent of activation is decreased, relative to wild-type enzyme . PALA activation of C-P in equilibrium Pi observed with wild-type enzyme disappears completely with the Y240F mutant enzyme . Analysis of perturbations of exchange rates by ATP and CTP were carried out by systematic methods plus computer-based simulations with the ISOBI program . These analyses indicate that (a) ATP increases the rates of association and dissociation for both C-P and Asp, but (b) CTP differentially increases the rate of C-P association to a greater degree than dissociation, but also decreases the rates for Asp association and dissociation in equal proportion . In addition, Arrhenius plots for Y240F ATCase suggest that ATP and CTP act by different mechanisms: ATP increases Vmax (decreases delta G not equal to) uniformly at all temperatures, whereas CTP does not alter either Vmax (delta G not equal to) or the Arrhenius slope (delta H not equal to). J Biol Chem, 1989 Oct 15, 264(29), 17259 - 65 Site-specific mutation of Tyr240----Phe in the catalytic chain of Escherichia coli aspartate transcarbamylase . Consequences for kinetic mechanism; Hsuanyu Y et al.; In the catalytic chain of Escherichia coli aspartate transcarbamylase, Tyr240 helps stabilize the T-state conformation by an intrachain hydrogen bond to Asp271 . Changes in kinetic characteristics of ATCase that result from disruption of this bond by site-specific mutation of Tyr240----Phe have been investigated by isotopic exchanges at chemical equilibrium . The Tyr240----Phe (Y240F) mutation caused the rate of the {32P} carbamyl phosphate (C-P) in equilibrium Pi exchange to decrease by 2-8-fold, without altering the {14C}Asp in equilibrium N-carbamyl-L-aspartate (C-Asp) rate . The mutation also caused the S0.5 and Hill nH values to decrease in virtually every substrate saturation experiment . Upon increasing the concentrations of the C-P,Pi or C-P,C-Asp reactant-product pairs, inhibition effects observed with the C-P in equilibrium Pi exchange for wild-type enzyme were not apparent with the Y240F mutant enzyme . In contrast, upon increasing the concentrations of the Asp,C-Asp and Asp,Pi pairs, inhibition effects on C-P in equilibrium Pi observed with wild-type enzyme became stronger with the Y240F mutant enzyme . These data indicate that the Tyr240----Phe mutation alters the kinetic mechanism in two different ways: on the reactant side, C-P binding prior to Asp shifts from preferred to compulsory order, and, on the product side, C-Asp and Pi release changes from preferred to nearly random order . These conclusions were also confirmed on a quantitative basis by computer simulations and fitting of the data, which also produced an optimal set of rate constants for the Y240F enzyme . The Arrhenius plot for wild-type holoenzyme was biphasic, but those for catalytic subunits and Y240F enzyme were linear (monophasic) . Taken together, the data indicate that the Tyr240----Phe mutation destabilizes the T-state and shifts the equilibrium for the T-R allosteric transition toward the R-state by increasing the rate of T----R conversion. Cell Immunol, 1989 Oct 15, 123(2), 294 - 306 Intravenous endotoxin recruits a distinct subset of human neutrophils, defined by monoclonal antibody 31D8, from bone marrow to the peripheral circulation; Brown CC et al.; Human neutrophils label with fluorochrome-labeled monoclonal antibody 31D8 as bright or dull . We determined the source and fate of 31D8 dull neutrophils by studying volunteers injected with endotoxin, epinephrine, or hydrocortisone, by examining bone marrow, and by examining skin blister exudate . We find that 31D8 dull neutrophils are normally not present in significant numbers in the circulation, are present in large numbers in normal marrow, and are recruited from the marrow by endotoxin, to a lesser extent by steroid, but not at all by epinephrine . 31D8 dull pattern correlates with morphologic immaturity in postendotoxin peripheral blood and bone marrow; however, blister exudate neutrophils contain only morphologically mature neutrophils, of which a significant number are 31D8 dull . We conclude that 31D8 dull neutrophils reside primarily in bone marrow and are released by agents which enhance bone marrow release of neutrophils . Their accumulation in skin blister exudate is unexplained, but suggests a special role in the inflammatory process. FEMS Microbiol Lett, 1989 Oct 15, 52(3), 291 - 5 The outer membrane protein of enteropathogenic Escherichia coli, described as the 'localised adherence factor', is OmpF and probably not involved in adhesion to HEp-2 cells; Chart H et al.; Strains of enteropathogenic Escherichia coli (EPEC) were examined for a factor, described as an outer membrane protein (OMP) of 32 kilodaltons (kDa) and reported to be involved in the adhesion of EPEC to HeLa cells . A comparable OMP of 35 kDa was detected in strains of EPEC, although expression of this protein was not related to the ability of strains to adhere to HEp-2 cells . The 35 kDa OMP was found to be heat-modifiable and peptidoglycan associated, and considered to be the porin protein OmpF. J Biol Chem, 1989 Oct 15, 264(29), 17322 - 8 Role of tryptophan 26 in the NAD glycohydrolase reaction of the S-1 subunit of pertussis toxin; Cortina G et al.; The gene encoding a catalytically active deletion peptide, the C180 peptide, of the S-1 subunit of pertussis toxin was engineered to facilitate mutagenesis at the Trp-26 (wild-type) coding sequence . A synthetic double-stranded oligonucleotide was inserted into the C180 gene such that all possible codons would be introduced into position 26 . Seven individual mutants of the C180 peptide which possessed amino acid substitutions at residue 26 (collectively termed C180W26n peptides) were purified from periplasmic extracts of Escherichia coli . Each C180W26n peptide was present as a single major peptide that had an apparent molecular mass of between 20.9 and 24.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and each showed similar immunoreactivity relative to the C180 peptide . The C180W26n peptides demonstrated marked reduction of both ADP-ribosyltransferase and NAD glycohydrolase activities at 25 nM and 10 microM NAD, respectively . Kinetic analysis of the two most active mutants, C180W26F and C180W26Y, revealed that the major perturbation of NAD glycohydrolase activity was due to an increase (approximately 20-fold) in the Km for NAD between these mutants and the C180 peptide. J Biol Chem, 1989 Oct 15, 264(29), 17078 - 83 Synthesis of peptides as cloned ubiquitin extensions; Yoo Y et al.; Oligonucleotides encoding four peptides ranging in length from 10 to 21 amino acids were cloned between the Afl2 and KpnI sites in the ubiquitin expression vector, pNMHUb . Escherichia coli AR13, a strain that contains a temperature-sensitive lambda repressor, was transformed by the plasmids, and upon shift to 42 degrees C, the cells produced large amounts of ubiquitin extended at its carboxyl terminus by each of the four peptides . Following a simple three-step purification of the ubiquitin fusion proteins, the peptides were cleaved from ubiquitin using a ubiquitin-alpha-protein hydrolase isolated from rabbit reticulocytes . The released ubiquitin molecules were shown to be competent in conjugation assays, and amino acid analyses of the purified peptides revealed that full length products were obtained in all cases . Since peptide yields varied from 2 to 4 mg/liter of culture medium, ubiquitin extensions provide an attractive alternative to solid phase synthesis for the production of peptides. J Biol Chem, 1989 Oct 15, 264(29), 17502 - 12 Escherichia coli helicase II (uvrD) protein can completely unwind fully duplex linear and nicked circular DNA; Runyon GT et al.; We have examined the duplex DNA unwinding (helicase) properties of the Escherichia coli helicase II protein (uvrD gene product) over a wide range of protein concentrations and solution conditions using a variety of duplex DNA substrates including fully duplex blunt ended and nicked circular molecules . We find that helicase II protein is able to initiate on and completely unwind fully duplex DNA molecules without the requirement for a covalently attached 3' single-stranded DNA tail . This DNA unwinding activity is dependent upon Mg2+ and ATP and requires that the amount of protein be in excess of that needed to saturate the resulting single-stranded DNA . Unwinding experiments on fully duplex blunt ended DNA with lengths of 341, 849, 1625, and 2671 base pairs indicate that unwinding occurs at the same high ratios of helicase II protein/nucleotide, independent of DNA length (50% unwinding requires approximately 0.6 helicase II monomers/nucleotide in 2.5 mM MgCl2, 10% glycerol, pH 7.5, 37 degrees C) . Helicase II protein is also able to unwind completely a nicked circular DNA molecule containing 2671 base pairs . At lower but still high molar ratios of helicase II protein to DNA, duplex DNA molecules containing a single-stranded (ss) region attached to a 3' end of the duplex are preferentially unwound in agreement with the results obtained by S . W . Matson {1986) J . Biol . Chem . 261, 10169-10175) . This preferential unwinding of duplex DNA with an attached 3' ssDNA most likely reflects the availability of a high affinity site (ssDNA) with the proper orientation for initiation; however, this may not reflect the type of DNA molecule upon which helicase II protein initiates DNA unwinding in vivo . The effects of changes in NaCl, NaCH3COO, and MgCl2 concentration on the ability of helicase II protein to unwind fully duplex DNA and duplex DNA with a 3' ssDNA tail have also been examined . Although the unwinding of fully duplex and nicked circular DNA molecules reported here occurs at higher helicase II protein to DNA ratios than have been previously used in most studies of this protein in vitro, this activity is likely to be relevant to the function of this protein in vivo since very high levels of helicase II protein accumulate in E . coli during the SOS response to DNA damage (approximately 2-5 x 10(4) copies/cell).(ABSTRACT TRUNCATED AT 400 WORDS) J Biol Chem, 1989 Oct 15, 264(29), 17583 - 8 Purification and characterization of neutral trehalase from the yeast ABYS1 mutant; App H et al.; Neutral trehalase was purified from stationary yeast ABYS1 mutant cells deficient in the vacuolar proteinases A and B and the carboxypeptidases Y and S . The purified electrophoretically homogeneous preparation of phosphorylated neutral trehalase exhibited a molecular mass of 160,000 Da on nondenaturing gel electrophoresis and of 80,000 Da on sodium dodecyl sulfate-gel electrophoresis . Maximal activity (114 mumol of trehalose min-1 x mg-1 at 37 degrees C) was observed at pH 6.8-7.0 . The apparent Km for trehalose was 34.5 mM . Among seven oligosaccharides studied, the enzyme formed glucose only from trehalose . Neutral trehalase is located in the cytosol . A polyclonal rabbit antiserum raised against neutral trehalase precipitates the enzyme in the presence of protein A . The antiserum does not react with acid trehalase . Dephosphorylation by alkaline phosphatase from Escherichia coli of the active phosphorylated enzyme is accompanied by greater than or equal to 90% inactivation . Rephosphorylation by incubation with the catalytic subunit of beef heart protein kinase is accompanied by reactivation and incorporation of 0.85 mol of phosphate/mol subunit (80,000 Da) . The phosphorylated amino acid residue was identified as phosphoserine. J Biol Chem, 1989 Oct 15, 264(29), 17349 - 54 Characterization of the catalytic subunit of an anion pump; Hsu CM et al.; The ArsA protein, the 63-kDa catalytic subunit of an oxyanion-translocating ATPase, was purified by successive chromatography using Q-Sepharose, red agarose, and phenyl-Sepharose to a specific activity in excess of 1 mumol of ATP hydrolyzed per min per mg of protein . ATPase activity was dependent on the presence of the oxyanionic substrates . Inhibitors of other classes of ion-translocating ATPases had no effect on ArsA ATPase activity, including N,N'-dicyclohexyl-carbodiimide, azide, vanadate, and nitrate . The apparent Km for ATP was determined to be 0.13 mM . The optimal pH range for ATP hydrolysis was 7.5 to 7.8 . ATPase activity required Mg2+ at a molar ratio of 2 ATP:1 Mg2+ . Limited proteolysis by trypsin was used to study conformational changes produced upon binding of substrates to the ArsA protein . In the absence of substrates, the ArsA protein was rapidly cleaved by trypsin to a major product of 30 kDa . ATP was partially protected from trypsin digestion, while the anionic substrate antimonite alone had no effect on proteolysis . Combination of the two substrates nearly completely protected the ArsA protein from proteolysis . Proteolytic cleavage correlated with loss of anion-stimulated ATPase activity and substrate protection from cleavage correlated with retention of activity . These results demonstrate that ATP and antimonite together produce a conformational change which is different from that of the ArsA protein in the presence of either substrate alone and suggest interaction between the oxyanion and ATP binding sites. Nature, 1989 Oct 12, 341(6242), 544 - 6 Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli; Ward ES et al.; In antibodies, a heavy and a light chain variable domain, VH and VL, respectively, pack together and the hypervariable loops on each domain contribute to binding antigen . We find, however, that isolated VH domains with good antigen-binding affinities can also be prepared . Using the polymerase chain reaction, diverse libraries of VH genes were cloned from the spleen genomic DNA of mice immunized with either lysozyme or keyhole-limpet haemocyanin . From these libraries, VH domains were expressed and secreted from Escherichia coli . Binding activities were detected against both antigens, and two VH domains were characterized with affinities for lysozyme in the 20 nM range . Isolated variable domains may offer an alternative to monoclonal antibodies and serve as the key to building high-affinity human antibodies . We suggest the name 'single domain antibodies (dAbs)' for these antigen binding demands. Nature, 1989 Oct 12, 341(6242), 503 - 7 Phosphorylation of large tumour antigen by cdc2 stimulates SV40 DNA replication; McVey D et al.; Simian virus 40 large tumour antigen (T) is a replication origin binding protein required for viral DNA synthesis . Unphosphorylated T antigen is deficient in promoting DNA replication in vitro but can be activated by phosphorylation at residue threonine 124 by the cdc2 protein kinase . This observation demonstrates that T is regulated by phosphorylation and provides a model for cdc2 function in the control of DNA replication. Nucleic Acids Res, 1989 Oct 11, 17(19), 7855 - 63 Role of the extra G-C pair at the end of the acceptor stem of tRNA(His) in aminoacylation; Himeno H et al.; All sequenced histidine tRNAs have one additional nucleotide at the 5' end compared with other tRNA species . To investigate the role of this unique structure in aminoacylation, we constructed in vitro transcripts corresponding to the E . coli histidine tRNA sequence and its variants at the G-1-C73 base pair, by using T7 RNA polymerase transcription system . A transcript having a wild-type sequence with no modified bases was a good substrate for histidyl-tRNA synthetase (HisRS), and aminoacylation activity was affected by introduction of a triphosphate at the 5' terminus . Base replacements at position 73 caused a marked decrease of Vmax, and deletion and substitution of the G-1 had a remarkable effect on the aminoacylation . A mutant having an A-1-U73 pair was also not a good substrate for HisRS . Comparison among G-1-deficient mutants showed that A was preferable rather than C as the base at position 73 . These data demonstrate that the set of the G-1-C73 pair at the end of the acceptor stem of histidine tRNA is crucial for the catalytic process of aminoacylation. Nucleic Acids Res, 1989 Oct 11, 17(19), 7771 - 8 A cis-acting transcription element of the c-myc gene can assume an H-DNA conformation; Kinniburgh AJ; I have used chemical probes and an oligonucleotide-association assay to determine the structure of a nuclease-sensitive, c-myc DNA region . I find that this DNA region can form a triplex-single stranded conformer in vitro--the H-DNA conformer . This DNA region has been shown previously to be a positive, cis-acting transcription element of the c-myc gene and to bind nuclear factors, including a base-paired ribonucleoprotein . Therefore, H-DNA may be a functionally important in vivo topoisomer where the H-DNA and B-DNA conformers have different transcriptional activities. Nucleic Acids Res, 1989 Oct 11, 17(19), 7671 - 80 The c1 genes of P1 and P7; Osborne FA et al.; The c1 genes of the heteroimmune phages P1 and P7 were sequenced and their products were compared . P7c1 expression was correlated with the translation in vitro of a protein whose predicted molecular weight (33,000 daltons) is indistinguishable from that of the P1c1 repressor . The c1 regions from both P1 and P7 were found to contain open reading frames capable of coding for a 283-amino acid protein whose predicted secondary structure lacks the helix-turn-helix motif commonly associated with repressor proteins . Two P1c1 amber mutations were localized to the 283-amino acid open reading frame . The P1c1 and P7c1 sequences were found to differ at only 18 positions, all but two of which alter the third position of the affected codon and do not alter the amino acid sequence of the protein . Plasmids expressing the c1 gene from either phage cause the repression of transcription from a cloned promoter situated upstream of P1c1. Nucleic Acids Res, 1989 Oct 11, 17(19), 7833 - 42 Deoxynucleotide-containing oligoribonucleotide duplexes: stability and susceptibility to RNase V1 and RNase H; Wyatt JR et al.; Oligoribonucleotide duplexes containing one to four 2'-deoxynucleotide residues were used as substrates for ribonuclease V1 and RNase H . Either deoxyadenosine and/or deoxythymidine were incorporated into the duplex, 5'GGCCGGAUCCGCGC3'-5'GCGCGGAUCCGGCC3' by substitution of the appropriate deoxynucleoside triphosphate into a transcription reaction with T7 RNA polymerase . The melting temperature, Tm, of the duplex (1.8 microM in strands in 50 mM NaCl) containing only ribonucleotides was 79.9 degrees C . Substitution of deoxyadenosine in both strands of the duplex lowered the Tm by 2.4 degrees C . Substitution of deoxythymidine had no measurable effect on the Tm . Comparison of RNase V1 digestion patterns of fully ribonucleotide and deoxy-substituted duplexes suggest that any distortion is localized to the site of the substitution . An oligoribonucleotide containing two deoxy residues directs specific cleavage of RNA by E . coli RNase H . Structural requirements for cleavage are proposed for RNase V1 and RNase H. Nucleic Acids Res, 1989 Oct 11, 17(19), 7735 - 48 Hydroxyl radical footprints reveal novel structural features around the NF I binding site in adenovirus DNA; Zorbas H et al.; We have identified a number of as yet unknown structural abnormalities of the NF I-DNA binding site within the inverted terminal repetition of adenovirus DNA by probing it with a hydroxyl radical footprinting technique . NF I binding alters the accessibility of the deoxyribose moieties to hydroxyl radicals both at the 3' and at the 5' side of the recognition sequence 5'-TGG(N)6GCCAA-3' . A smooth bend at the 5' side of the binding sequence is already present in naked linear DNA and it is further enhanced by protein binding . This could be demonstrated not only by hydroxyl radical footprinting but also by studying the temperature dependent mobility during gel electrophoresis of DNA fragments carrying the NF I binding site at circularly permutated positions . We propose that the bent conformation at this site is responsible for facilitating protein/DNA interactions. Nucleic Acids Res, 1989 Oct 11, 17(19), 7591 - 608 Euglena gracilis chloroplast ribosomal protein operon: a new chloroplast gene for ribosomal protein L5 and description of a novel organelle intron category designated group III; Christopher DA et al.; We describe the structure (3840 bp) of a novel Euglena gracilis chloroplast ribosomal protein operon that encodes the five genes rpl16-rpl14-rpl5-rps8-rpl36 . The gene organization resembles the spc and the 3'-end of the S10 ribosomal protein operons of E . coli . The rpl5 is a new chloroplast gene not previously reported for any chloroplast genome to date and also not described as a nuclear-encoded, chloroplast protein gene . The operon contains at least 7 introns . We present evidence from primer extension analysis of chloroplast RNA for the correct in vivo splicing of five of the introns . Two of the introns within the rps8 gene flank an 8 bp exon, the smallest exon yet characterized in a chloroplast gene . Three introns resemble the classical group II introns of organelle genomes . The remaining 4 introns appear to be unique to the Euglena chloroplast DNA . They are uniform in size (95-109 nt), share common features with each other and are distinct from both group I and group II introns . We designate this new intron category as 'group III'. Tidsskr Nor Laegeforen, 1989 Oct 10, 109(28), 2887 - 9 {Gene technology in clinical medicine--DNA sequencing}; Kristensen T; There are two established procedures for sequencing cloned DNA fragments . In Maxam-Gilbert sequencing, or sequencing by chemical degradation, end-labeled DNA is treated with various chemicals which induce base specific chain breaks with a low frequency . In dideoxy sequencing, or sequencing by the Sanger procedure, a single-stranded DNA is used as a template for synthesis of a labeled complementary strand by a DNA polymerase . Addition of dideoxynucleoside triphosphates will induce base-specific chain termination . In both procedures the nucleotide sequence of the cloned DNA can be deduced after fractionation of the labeled products by polyacrylamide gel electrophoresis . As yet, radioactive labeling of the reaction products is most common, but fluorescence labeling and computer-assisted automated sequence interpretation has become as a powerful alternative during the last years . Further automation of the various processes involved in DNA sequencing will be necessary for the planned sequencing of large genomes, such as the Escherichia coli genome, the yeast genome, and the human genome. FEBS Lett, 1989 Oct 9, 256(1-2), 79 - 84 Biochemical properties of the YPT-related rab1B protein . Comparison with rab1A; Touchot N et al.; We recently identified a novel rat cDNA: rab1B, closely related to the rab1A cDNA and to the yeast YPT1 gene . The rab1B cDNA encodes a 202 amino acid protein (22.1 kDa) that was produced in Escherichia coli under the control of the phi 10 promoter for the T7 RNA polymerase . The rab1B protein was purified in large amounts to near homogeneity in a simplified procedure . We studied the biochemical properties of rab1B and rab1A proteins . They both bind specifically GTP and GDP and possess intrinsic GTPase activities . The rab1B Lys21----Met mutant protein does not bind GTP, whereas the Ala65----Thr mutant has a reduced GTPase activity and is competent for autophosphorylation in the presence of GTP. FEBS Lett, 1989 Oct 9, 256(1-2), 135 - 8 Orientation of the carboxyl terminus of the Na+/proline symport carrier in Escherichia coli; Komeiji Y et al.; The orientation of the carboxyl terminal region of the Escherichia coli proline carrier in the cytoplasmic membrane was studied . The beta-galactosidase moiety of the PutP-LacZ fusion protein {(1987) J . Biol . Chem . 262, 14100-14104} was exposed outside the inside-out vesicles and inside the right-side-out vesicles . A site-directed antibody raised against a synthetic peptide corresponding to the putative carboxyl terminal region of the carrier reacted preferentially with the inside-out vesicles prepared from a wild-type proline carrier overproducing strain and less with the right-side-out vesicles . These results indicate that the carboxyl terminus of the proline carrier is exposed to the cytoplasmic side of the membrane. J Biol Chem, 1989 Oct 5, 264(28), 16591 - 7 Circular dichroism studies on synthetic signal peptides indicate beta-conformation as a common structural feature in highly hydrophobic environment; Reddy GL et al.; The conformations of synthetic peptides corresponding to signal sequences of chicken lysozyme and Escherichia coli proteins alkaline phosphatase and lipoprotein (wild-type) and their "variants" with a charged amino acid in the hydrophobic region, have been studied by circular dichroism spectroscopy in trifluoroethanol and micelles of sodium dodecyl sulfate, Brij 35, and sodium deoxycholate . In trifluoroethanol and aqueous mixtures of trifluoroethanol, the "wild-type" and variant signal sequences show similar conformational behavior . The wild-type signal peptides show increasing amounts of beta-structure going from sodium dodecyl sulfate to deoxycholate micelles (i.e . increasing order of hydrophobicity) . The variant signal sequences, however, are largely unordered in micelles . The absence of beta-structure in variant signal sequences which do not initiate protein translocation across membranes, strongly suggests that the ability of signal sequences to adopt beta-structure in a highly hydrophobic environment is important for function. J Mol Biol, 1989 Oct 5, 209(3), 503 - 4 Crystallization and preliminary X-ray studies of glutathione synthetase from Escherichia coli B; Kato H et al.; The glutathione synthetase from Escherichia coli B has been crystallized from 27% saturated ammonium sulfate solution (pH 5.5) . The crystals are hexagonal, space group P6(2)22 or P6(4)22 . The cell dimensions are a = b = 88.0 A, c = 164.2 A, and gamma = 120 degrees . The enzyme is a tetramer (Mr = 143,000) with 222 symmetry, and the asymmetric unit contains one subunit molecule (Mr = 35,600) . The crystals diffract to at least 2.5 A resolution. J Mol Biol, 1989 Oct 5, 209(3), 499 - 501 Crystallization and preliminary X-ray studies of an aspartate aminotransferase mutant from Escherichia coli; Jager J et al.; Mutant aspartate aminotransferase V39L (Val39 replaced by Leu) from Escherichia coli has been crystallized into a monoclinic cell from a polyethylene glycol solution (pH 7.5) by vapor diffusion . The space group and the unit cell dimensions have been determined using a precession camera, a CAD4 diffractometer and a Nicolet Xentronics area detector to be P2(1) with a = 86.8 A, b = 79.9 A, c = 89.4 A, beta = 118.74 degrees . The crystals diffract to better than 2.3 A and are suitable for X-ray structure analysis. J Biol Chem, 1989 Oct 5, 264(28), 16613 - 9 A cysteine-histidine-aspartate catalytic triad is involved in glutamine amide transfer function in purF-type glutamine amidotransferases; Mei B et al.; A family of four glutamine amidotransferases has a homologous glutamine amide transfer domain, designated purF-type, that is named after purF-encoded glutamine phosphoribosylpyrophosphate amidotransferase . The glutamine amide transfer domain of approximately 194 amino acid residues is at the NH2 terminus of the protein chain . Site-directed mutagenesis was used to replace several of the 9 invariant amino acids in the glutamine amide transfer domain of glutamine phosphoribosylpyrophosphate amidotransferase . The results indicate that a Cys1-His101-Asp29 catalytic triad is involved in the glutamine amide transfer function of this enzyme . The evidence suggests that His101 functions to increase the nucleophilicity of Cys1, which is used to form a glutamine-enzyme covalent intermediate . Asp29 has a role subsequent to formation of the covalent intermediate . The Cys-His-Asp catalytic triad is implicated in the glutamine amide transfer function of purF-type amidotransferases. J Biol Chem, 1989 Oct 5, 264(28), 16502 - 6 Thioredoxin reductase-dependent insulin disulfide reduction by phage T7 DNA polymerase reflects dissociation of the enzyme into subunits; Slaby I et al.; Phage T7 DNA polymerase contains Escherichia coli thioredoxin as a subunit and is a 1:1 complex with T7 gene 5 protein . The enzyme showed high thioredoxin activity in assays at 37 degrees C using reduction of insulin disulfides with NADPH and thioredoxin reductase, leading Randahl (Randahl, H . (1982) FEBS Lett . 150, 109-113) to propose that the thioredoxin dithiol active site is exposed in T7 DNA polymerase . However, T7 DNA polymerase and free thioredoxin differ in reactivity with iodoacetic acid after preincubation with dithiothreitol or incubation with insulin . Insulin reduction assays work at low temperatures even at 0 degrees C . The time and temperature dependence of the thioredoxin activity of T7 DNA polymerase demonstrated that dissociation into subunits at 25 or 37 degrees C accounts for the previously observed activity . Thus, T7 DNA polymerase contains the reduced form of thioredoxin with its active site SH groups masked by the subunit contact with the gene 5 protein in agreement with the results of Adler and Modrich (Adler, S., and Modrich, P . (1983) J . Biol . Chem . 258, 6956-6962) . The subunit interaction of thioredoxin and gene 5 protein is salt-insensitive, but markedly temperature-dependent consistent with involvement of a hydrophobic surface area in reduced thioredoxin. J Biol Chem, 1989 Oct 5, 264(28), 16465 - 9 Translocation of pro-OmpA across inner membrane vesicles of Escherichia coli occurs in two consecutive energetically distinct steps; Geller BL et al.; The rate of energy-dependent transfer of pro-OmpA across Escherichia coli inner membrane vesicles in vitro was found to be a function of the ATP concentration . At concentrations above 0.1 mM ATP, the addition of a transmembrane electrochemical potential (proton motive force or pmf) increased the rate of pro-OmpA translocation . Additional experiments demonstrated that the overall reaction proceeded by at least two distinct energy-requiring steps . The first step required only ATP, was nearly unaffected by the pmf, and resulted in the insertion of the amino-terminal domain of pro-OmpA across the membrane . The insertion exposed the signal sequence cleavage site to the periplasmic side of the membrane, as measured by the appearance of a mature length translocation intermediate . However, this intermediate was partially exposed to the cytoplasmic side of the membrane . In a second energy-dependent step, either ATP or the pmf was sufficient to complete the translocation of mature length OmpA across the membrane. J Biol Chem, 1989 Oct 5, 264(28), 16403 - 10 K+-transport protein TrkA of Escherichia coli is a peripheral membrane protein that requires other trk gene products for attachment to the cytoplasmic membrane; Bossemeyer D et al.; The TrkA protein, which is essential for the activity of the constitutive Trk K+-uptake system of Escherichia coli, is a peripheral membrane protein . The protein was detected in immunoblots by polyclonal antibodies to sodium dodecyl sulfate-denatured TrkA protein . In extracts from wild-type cells equal amounts of TrkA were found in the membrane and soluble fractions, suggesting that membrane binding is relatively weak . When the protein was moderately overproduced it appeared mainly in the soluble fraction; stronger overproduction led to the formation of aggregates that could not be solubilized by nonionic detergents . Mutations in the three other genes implicated in Trk activity, trkE, trkG, and trkH, reduced or abolished the binding of TrkA to the membrane . These results support the model, previously based solely on genetic data, that Trk is a multisubunit complex and implicates the products of the other trk genes in the normal binding of TrkA to the complex in the cytoplasmic membrane. J Biol Chem, 1989 Oct 5, 264(28), 16552 - 6 A mutation altering the kinetic responses of the yeast mitochondrial F1-ATPase; Mueller DM; Nucleotide-binding proteins, including the mitochondrial F1-ATPase, the ras proteins, and the G-proteins, contain a homologous glycine-rich sequence that is thought to constitute part of the active site . This study reports the effects of a single amino acid replacement of Thr197 to Ser197, which is located at the hinge region of this putative loop, in the yeast Saccharomyces cerevisiae F1-ATPase . This replacement resulted in a 3-fold increase in the specific activity of the enzyme, eliminated the stimulatory effects of oxyanions, and modulated the effects of the inhibitor NaN3 while having little effect on the uni-site ATPase . These results indicate a role of the glycine-rich loop in many of the kinetic responses of the F1-ATPase. J Mol Biol, 1989 Oct 5, 209(3), 359 - 78 Codon contexts from weakly expressed genes reduce expression in vivo; Folley LS et al.; Nucleotides that neighbor codons in Escherichia coli genes are highly non-random . Furthermore, these context biases are stronger and extend farther from the codon in weakly expressed than in highly expressed genes . We therefore suggested that codon contexts are selected to reduce gene expression levels . We now compare the expression levels of lacZ genes containing two specific coding sequences (context inserts) . One context insert represents contexts seen in weakly expressed genes (low variant); the other represents contexts seen in highly expressed genes (high variant) . The two variants have identical nucleotide and codon compositions, and encode the same protein . A permutation of four nucleotides, which changes eight codon:codon interfaces of 1043, comprises the only difference between the high and low context variant genes . In three different lacZ mRNAs, the low variant was expressed at a level significantly below that of the high variant . This context effect depends entirely on translation of the contexts in the correct frame; its magnitude depends in part on the placement of other features (e.g . transcriptional pauses and terminators, or perhaps other slow codons or contexts) in the mRNAs . Changing the ribosome density on the message by changing the ribosome binding site distinguishes between dropoff, interference and polarity, three fundamentally different types of models for the context effect . The expression difference between context variants is eliminated by both increases and decreases in the ribosome initiation frequency, as uniquely predicted by the polarity model . In fact, data from all constructions are accommodated by a model in which slow translation of the low context insert increases rho-dependent transcriptional termination within the test gene . The data suggest that the rates of translational initiation and elongation are poised with respect to the rate of transcriptional elongation so that all are influential in setting the expression level of wild-type lacZ . We conclude that context-induced polarity will exist in genes wherever low and reproducible gene product levels have been selected. Nature, 1989 Oct 5, 341(6241), 462 - 4 The alpha-lytic protease pro-region does not require a physical linkage to activate the protease domain in vivo; Silen JL et al.; alpha-Lytic protease, an extracellular serine protease of Lysobacter enzymogenes 495, is synthesized as a pre-pro-protein . Previously it has been shown that when expressed in Escherichia coli, the protein is autocatalytically processed in the periplasmic space, and that the functional protease domain accumulates extracellularly . Engineered proteins lacking the 166 amino-acid pro-region were enzymatically inactive and remained cell-associated . By independently expressing the pro- and protease domains in vivo, evidence is provided here that direct covalent linkage is not required for production of active protease . We postulate that the pro-region acts as a template to promote the folding of the protease domain into an active configuration . Our results, combined with recent experiments on the evolutionarily unrelated subtilisin E (ref . 3), suggest that the ability of the pro-region of these bacterial proteases to facilitate folding of their protease domains is not a curiosity of a single system, but may reflect a general property of extracellular bacterial serine proteases. J Biol Chem, 1989 Oct 5, 264(28), 16700 - 12 Universality and structure of the N-end rule; Gonda DK et al.; Our previous work has shown that, in the yeast Saccharomyces cerevisiae, any of the eight stabilizing amino-terminal residues confers a long (greater than 20 h) half-life on a test protein beta-galactosidase (beta gal), whereas 12 destabilizing amino-terminal residues confer on beta gal half-lives from less than 3 min to 30 min . We now show that an analogous single-residue code (the N-end rule) operates in an in vitro system derived from mammalian reticulocytes . We also show that the N-end rule has a hierarchical structure . Specifically, amino-terminal Glu and Asp (and also Cys in reticulocytes) are secondary destabilizing residues in that they are destabilizing through their ability to be conjugated to primary destabilizing residues such as Arg . Amino-terminal Gln and Asn are tertiary destabilizing residues in that they are destabilizing through their ability to be converted, via selective deamidation, into secondary destabilizing residues Glu and Asp . Furthermore, in reticulocytes, distinct types of the N-end-recognizing activity are shown to be specific for three classes of primary destabilizing residues: basic (Arg, Lys, His), bulky hydrophobic (Phe, Leu, Trp, Tyr), and small uncharged (Ala, Ser, Thr) . Features of the N-end rule in reticulocytes suggest that the exact form of the N-end rule may depend on the cell's physiological state, thereby providing a mechanism for selective destruction of preexisting proteins upon cell differentiation. J Biol Chem, 1989 Oct 5, 264(28), 16489 - 95 Evidence for the essential function of 2,4-dienoyl-coenzyme A reductase in the beta-oxidation of unsaturated fatty acids in vivo . Isolation and characterization of an Escherichia coli mutant with a defective 2,4-dienoyl-coenzyme A reductase; You SY et al.; For the purpose of assessing in vivo the importance of 2,4-dienoyl-CoA reductase (EC 1.3.1.34) in the beta-oxidation of unsaturated fatty acids, reductase mutants of Escherichia coli were isolated by selecting cells that were able to grow on oleate but not on petroselinic acid (6-cis-octadecenoic acid) . One mutant (fadH) exhibited 12% of the 2,4-dienoyl-CoA reductase activity present in the parental strain with other beta-oxidation enzymes being essentially unaffected . Antireductase antibodies were used to show that the mutant contains a fadH gene product at a level similar to that observed in the parental strain . Thus, the mutation seems to have resulted in the synthesis of a fadH gene product with lower specific activity . The mutation was mapped in the 71-75-min region of the E . coli chromosome where no other gene for beta-oxidation enzymes has so far been located . Complementation of the mutation by F'141, which carries the 67-75.5-min region of the E . coli genome, resulted in an increase in the 2,4-dienoyl-CoA reductase activity to 80% of the level found in the parental strain . Measurements of respiration with petroselinic acid as the substrate showed rates to be linearly dependent on the 2,4-dienoyl-CoA reductase activity up to levels found in wild-type E . coli . 2,4-Dienoyl-CoA reductase, like other enzymes of beta-oxidation, was induced when E . coli was grown on a long chain fatty acid as the sole carbon source . It is concluded that 2,4-dienoyl-CoA reductase is required in vivo for the beta-oxidation of unsaturated fatty acids with double bonds extending from even-numbered carbon atoms. Nature, 1989 Oct 5, 341(6241), 410 - 4 Structure and associated DNA-helicase activity of a general transcription initiation factor that binds to RNA polymerase II; Sopta M et al.; RAP30/74 is a heteromeric general transcription initiation factor which binds to RNA polymerase II . Here we report that preparations of RAP30/74 contain an ATP-dependent DNA helicase whose probable function is to melt the DNA at transcriptional start sites . The sequence of the RAP30 subunit of RAP30/74 indicates that RAP30 may be distantly related to bacterial sigma factors. J Biol Chem, 1989 Oct 5, 264(28), 16393 - 8 One single lysine residue is responsible for the special interaction between polyphosphate and the outer membrane porin PhoE of Escherichia coli; Bauer K et al.; Site-directed mutagenesis was performed with the phosphate starvation-inducible outer membrane porin PhoE of Escherichia coli K-12 to study the molecular basis of its anion selectivity . Lysines 18, 29, 64, and 125 were replaced by glutamic acids, and the properties of the mutant porins were investigated in in vivo and in vitro experiments . Lipid bilayer experiments showed that all these mutations had no influence on the pore structure because PhoE and the mutants had the same single channel conductance in KCl solution . Selectivity measurements revealed that the mutations changed the ionic selectivity of PhoE, but the change was dependent on the location of the lysine . Replacement of Lys18 and Lys29 by glutamic acid had a relatively small influence . The effect of the Lys64 substitution was somewhat larger, and the effect of the replacement of Lys125 resulted in the most drastic change in selectivity and in the loss of the interaction of PhoE with polyphosphate, whereas the replacement of the other lysines had no effect on the polyphosphate interaction behavior . The results are consistent with the assumption that the charge spot in PhoE consists of only 1 lysine per monomer, located in position 125 of the primary sequence and probably close to the pore interior. Biochemistry, 1989 Oct 3, 28(20), 8167 - 74 Mutations at the interdomain hinge region of the DadB alanine racemase: effects of length and conformational constraint of the linker sequence on catalytic efficiency; Galakatos NG et al.; The pentapeptide "hinge" region of the DadB alanine racemase links two structural domains of the protein {Galakatos, N . G., & Walsh, C . T . (1987) Biochemistry 26, 8475} . The presence of substrate markedly reduces the rate of hinge-specific proteolysis of this racemase and induces a conformational change observed by circular dichroism . To evaluate the possible contribution of the proteolytically sensitive hinge residues (-Y253GGGY257-) on catalytic efficiency, site-directed mutations were generated to probe the effects of size and conformational rigidity of that region . A bacterial overproducing system for the dadB gene was constructed that expresses the enzyme as 4.5% of total soluble protein . On this construct, a four-part ligation allowed the engineering of two unique and proximal restriction sites required for cassette mutagenesis at the hinge region . For two of the eight mutants generated, expressed protein could not be detected (deletion of -GGGY-; termination codon at position 255) . Deletion of one or two of the three Gly residues had no effect on catalytic efficiency . Insertion of a fourth Gly resulted in a 5-fold drop in Vmax/Km . For G254P, G255P, and G256P, Vmax/Km was 60%, 126%, and 26% of the native enzyme, respectively . In all cases, the Km's remained essentially constant, suggesting that the hinge region is not involved in substrate binding . The rate of hinge-specific proteolysis of the mutants was faster than that of wild-type DadB except for the G255P protein for which it was equivalent. Biochemistry, 1989 Oct 3, 28(20), 8149 - 53 Reaction of Escherichia coli and yeast photolyases with homogeneous short-chain oligonucleotide substrates; Jordan SP et al.; Similar rates have been observed for dimer repair with Escherichia coli photolyase and the heterogeneous mixtures generated by UV irradiation of oligothymidylates {UV-oligo(dT)n, n greater than or equal to 4} or DNA . Comparable stability was observed for ES complexes formed with UV-oligo(dT)n, (n greater than or equal to 9) or dimer-containing DNA . In this paper, binding studies with E . coli photolyase and a series of homogeneous oligonucleotide substrates (TpT, TpTp, pTpT, TpTpT, TpTpT, TpTpTpT, TpTpTpT, TpTpTpT, TpTpTpT) show that about 80% of the binding energy observed with DNA as substrate (delta G approximately 10 kcal/mol) can be attributed to the interaction of the enzyme with a dimer-containing region that spans only four nucleotides in length . This major binding determinant (TpTpTpT) coincides with the major conformational impact region of the dimer and reflects contributions from the dimer itself (TpT, delta G = 4.6 kcal/mol), adjacent phosphates (5'p, 0.8 kcal/mol; 3'p, 1.1 kcal/mol), and adjacent thymine residues (5'T, 0.8 kcal/mol; 3'T, 1.3 kcal/mol) . Similar turnover rates (average kcat = 6.7 min-1) are observed with short-chain oligonucleotide substrates and UV-oligo(dT)18, despite a 25,000-fold variation in binding constants (Kd) . In contrast, the ratio Km/Kd decreases as binding affinity decreases and appears to plateau at a value near 1 . Turnover with oligonucleotide substrates occurs at a rate similar to that estimated for the photochemical step (5.1 min-1), suggesting that this step is rate determining . Under these conditions, Km will approach Kd when the rate of ES complex dissociation exceeds kcat.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1989 Oct 3, 28(20), 8072 - 7 Purification of recombinant human tissue factor; Paborsky LR et al.; Tissue factor (TF) is a 263 amino acid membrane-bound procoagulant protein that serves as a cofactor for the serine protease factor VII (fVII) . Recombinant human TF (rTF) produced in both human kidney 293 cells and Escherichia coli has been immunoaffinity purified by using a TF-specific monoclonal antibody . Recombinant TF produced in 293 cells is glycosylated and migrates on reducing SDS-PAGE with an apparent molecular weight (Mr) of 45K . Some interchain disulfide-bonded rTF dimers are observed under nonreducing conditions . The E . coli produced rTF has a molecular weight of 33K and 35K, with the 33K band missing nine amino acids at the carboxy terminus . Although the E . coli produced rTF does not contain any carbohydrate, it is fully functional in both a chromogenic assay and a one-stage prothrombin time assay . A variant has been constructed wherein the cytoplasmic cysteine (residue 245) has been mutagenized to a serine residue . The amount of disulfide-linked aggregates is dramatically reduced following immunoaffinity purification of this four-cysteine variant (C2455), which is active in the chromogenic and prothrombin time assays. Biochemistry, 1989 Oct 3, 28(20), 7979 - 84 A new mechanism for repairing oxidative damage to DNA: (A)BC excinuclease removes AP sites and thymine glycols from DNA; Lin JJ et al.; Escherichia coli (A)BC excinuclease is the major enzyme responsible for removing bulky adducts, such as pyrimidine dimers and 6-4 photoproducts, from DNA . Mutants deficient in this enzyme are extremely sensitive to UV and UV-mimetic agents, but not to oxidizing agents, or ionizing radiation which damages DNA in part by generating active oxygen species . DNA glycosylases and AP1 endonucleases play major roles in repairing oxidative DNA damage, and thus it has been assumed that nucleotide excision repair has no role in cellular defense against damage by ionizing radiation and oxidative damage . In this study we show that the E . coli nucleotide excision repair enzyme (A)BC excinuclease removes from DNA the two major products of oxidative damage, thymine glycol and the baseless sugar (AP site) . We conclude that nucleotide excision repair is an important cellular defense mechanism against oxidizing agents. Biochemistry, 1989 Oct 3, 28(20), 7985 - 91 Direct observation of the enzyme-intermediate complex of 5-enolpyruvylshikimate-3-phosphate synthase by 13C NMR spectroscopy; Barlow PN et al.; The interaction of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, 4,5-dideoxyshikimate 3-phosphate (ddS3P), and {2-13C}-and {3-13C}phosphoenolpyruvate (PEP) has been examined by 13C NMR spectroscopy . Although no resonances due to a dead-end intermediate complex could be detected, an enzyme active site specific formation of pyruvate was observed . The interaction of EPSP synthase with shikimate 3-phosphate (S3P) and {2-13C}- or {3-13C}PEP has been examined by 13C NMR spectroscopy . With {2-13C}PEP, in addition to the resonances due to {2-13C}PEP and {8-13C}EPSP, new resonances appeared at 164.8, 110.9, and 107.2 ppm . The resonance at 164.8 ppm has been assigned to enzyme-bound EPSP . The resonance at 110.9 ppm has been assigned to C-8 of an enzyme-free tetrahedral intermediate of the sort originally proposed by Levin and Sprinson {Levin, J . G., & Sprinson, D . B . (1964) J . Biol . Chem . 239, 1142-1150} and recently independently observed by Anderson et al . {Anderson, K . S., Sikorski, J . A., Benesi, A . J., & Johnson, K . A . (1988) J . Am . Chem . Soc . 110, 6577-6579} . The resonance at 107.2 ppm has been assigned to an enzyme-bound intermediate whose structure is closely related to that of the tetrahedral intermediate . With {3-13C}PEP, new resonances appeared at 88.9, 26.2, 25.5, and 24.5 ppm . The resonance at 88.9 ppm has been assigned to enzyme-bound EPSP . The resonance at 26.2 ppm, which was found to correlate with 1.48 ppm by isotope-edited multiple quantum coherence 1H NMR spectroscopy, has been assigned to the methyl group 4-hydroxy-4-methylketoglutarate.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1989 Oct 3, 28(20), 8142 - 8 Expression of cDNAs encoding wild-type and mutant neuromodulins in Escherichia coli: comparison with the native protein from bovine brain; Au DC et al.; Murine cDNA that encodes neuromodulin, a neurospecific calmodulin binding protein, was inserted into the plasmid pKK223-3 for expression in Escherichia coli . After being transformed into E . coli strain SG20252 (lon-), the expression vector directed the synthesis of a protein that was recognized by polyclonal antibodies raised against bovine neuromodulin . The recombinant protein expressed in E . coli was found to be tightly associated with insoluble cell material and was extractable only with guanidine hydrochloride or sodium dodecyl sulfate . Following solubilization with guanidine hydrochloride, the protein was purified to apparent homogeneity by a single CaM-Sepharose affinity column step with a yield of 0.2 mg of protein/L of E . coli culture . The availability of the purified recombinant neuromodulin made it possible to answer several specific questions concerning the structure and function of the protein . Despite the fact that murine neuromodulin is 12 amino acid residues shorter than the bovine protein and the recombinant protein expressed in E . coli may lack any posttranslational modifications, the two proteins displayed similar biochemical properties in almost all respects examined . They both had higher affinity for CaM-Sepharose in the absence of Ca2+ than in its presence; they were both phosphorylated in vitro by protein kinase C in a Ca2+- and phospholipid-dependent manner; neither form of the proteins was autophosphorylated, and the phosphorylated form of the proteins did not bind calmodulin . The recombinant neuromodulin and neuromodulin purified from bovine brain had similar, but not identical, affinities of calmodulin, indicating that the palmitylation of the protein that occurs in animal cells is not crucial for calmodulin interactions.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1989 Oct 3, 28(20), 8161 - 7 2.8-A-resolution crystal structure of an active-site mutant of aspartate aminotransferase from Escherichia coli; Smith DL et al.; The three-dimensional structure of a mutant of the aspartate aminotransferase from Escherichia coli, in which the active-site lysine has been substituted by alanine (K258A), has been determined at 2.8-A resolution by X-ray diffraction . The mutant enzyme contains pyridoxamine phosphate as cofactor . The structure is compared to that of the mitochondrial aspartate aminotransferase . The most striking differences, aside from the absence of the lysine side chain, occur in the positions of the pyridoxamine group and of tryptophan 140. Biochemistry, 1989 Oct 3, 28(20), 8067 - 72 Mapping the lacZ ribosome binding site by RNA footprinting; Murakawa GJ et al.; The ribosome binding site of the Escherichia coli lacZ mRNA has been characterized by using an RNA footprinting technique . Purified E . coli 70S ribosomes and fMet-tRNA were incubated with mRNA, and the complex was treated with RNA-reactive reagents or RNases as probes . The protected sites on the mRNA were then mapped by extending a radioactive primer with reverse transcriptase . Dimethyl sulfate, diethyl pyrocarbonate, and 1,10-phenanthroline-copper ion oxidative complex were used as reagent probes; they detected interaction sites within the ribosome binding site . A region of approximately 35 nucleotides was protected by the ribosome, specifically across the Shine-Dalgarno region, around the fMet initiation codon, and at a region 7-12 nucleotides distal to the fMet codon . In addition, an enhanced reaction occurred between the fMet codon and the distal site . These results imply an internally selective interaction between the ribosome and the mRNA sequence . The enhanced reactivity of a site distal to the initiation site--flanked by the AUG codon and a site previously identified as conserved in a study of initiation sequences--may indicate a region where the mRNA is specifically exposed. Biochim Biophys Acta, 1989 Oct 2, 985(1), 9 - 18 Electrical properties and molecular architecture of the channel formed by Escherichia coli hemolysin in planar lipid membranes; Ropele M et al.; A 107 kDa hemolysin from Escherichia coli is able to open pores in lipid membranes . By studying its interaction with planar phospholipid bilayers we have derived some structural information on the organization of the pore . We measured the current-voltage characteristic and the ion selectivity of the channel both in neutral membranes, made of egg phosphatidylcholine (PC) and in negatively charged membranes, made of a 1:1 mixture of PC with phosphatidylserine (PS) . Experiments were performed varying both the pH and the salt concentration of the bathing KCl solution . In neutral membranes the pore is ohmic and its conductance increases almost linearly with the salt concentration . The channel is cation-selective at high pH but nearly unselective at low pH . We interpret these results in terms of a minimal model based on classical electro-diffusional theories assuming that the pore is wide and bears a negative charge at its entrances . In membranes containing the acidic lipid the current-voltage curve is non-linear in such a way to suggest that the trans (but not the cis) entrance of the pore is affected by the surface potential of the membrane . Applying our model we find that the trans and cis entrances are located, respectively, about 0.5 nm and more than 5 nm apart from the plane of the membrane . We confirmed the asymmetric disposition of the channel by enzymatic digestion of preformed pores . This was effective only when the enzyme was applied on the cis side. Proc Natl Acad Sci U S A, 1989 Oct, 86(20), 7682 - 6 Developmentally regulated gene from Leishmania encodes a putative membrane transport protein; Cairns BR et al.; We have cloned a developmentally regulated gene from the parasitic protozoan Leishmania enrietti . The mRNA from this gene accumulates to a much higher level in the promastigote stage of the parasite life cycle that lives in the gut of the insect vector than in the amastigote stage of the parasite that lives inside the macrophages of the mammalian host . The predicted protein encoded by this gene is homologous to the human erythrocyte glucose transporter and to several sugar-transport proteins from Escherichia coli . These structural similarities strongly suggest that the cloned gene encodes a membrane transport protein that is developmentally induced when the parasite enters its insect vector . Regulated membrane transporters may be required for the parasite to adapt to the environment of the insect gut. Eur J Biochem, 1989 Oct 1, 184(3), 497 - 501 Isolation of a cDNA encoding the rat liver S-adenosylmethionine synthetase; Horikawa S et al.; We have isolated cDNA clones encoding the rat liver S-adenosylmethionine synthetase by means of immunological screening from a phage lambda gt 11 expression library containing cDNA synthesized from adult rat liver poly(A)-RNA . The amino acid sequence deduced from the cDNA indicates that the rat liver enzyme for this protein contains 397 amino acid residues and has a molecular mass of 43697 Da . The deduced amino acid sequence of rat liver S-adenosylmethionine synthetase was 68% similar to those of yeast S-adenosylmethionine synthetases encoded by two unlinked genes SAM1 and SAM2 . The rat liver S-adenosylmethionine synthetase also shows 52% similarity with the deduced amino acid sequence of the MetK gene encoding the S-adenosylmethionine synthetase in Escherichia coli. Proc Natl Acad Sci U S A, 1989 Oct, 86(19), 7537 - 41 Molecular characterization of a 23-kilodalton major antigen secreted by Toxoplasma gondii; Cesbron-Delauw MF et al.; The strategy chosen for cloning potential vaccine antigens of Toxoplasma gondii was based on the hypothesis that the definitive protection observed in natural infection is due to the presence of encysted bradyzoite forms in host tissues throughout life . The antigens released by the bradyzoites would maintain an immune response against the invading tachyzoites . This led us to identify in tachyzoite in vitro translation products a polypeptide of 24 kDa that is an excreted-secreted antigen (ESA) and is cross-reactive with bradyzoites . In addition, the detection of anti-P24 IgG antibodies is correlated with the chronic infection in man . The gene encoding P24 has been isolated, sequenced, and expressed in Escherichia coli and eukaryotic cells . The recombinant proteins were immunogenic in mice, producing anti-native P23 antibodies . Immunocytochemical analysis located the native antigen in the dense granules of both tachyzoite and bradyzoite forms and showed that it is secreted within host-cell-modified phagosome . Moreover 45Ca2+ labeling as well as regional homologies indicate that this protein has Ca2+-binding properties, suggesting its physiological importance in host-cell invasion . P23 is of diagnostic interest as a marker of chronic toxoplasmosis and is proposed as a vaccine component. Crit Care Med, 1989 Oct, 17(10), 1010 - 4 Amino acid losses during continuous high-flux hemofiltration in the critically ill patient; Davenport A et al.; Total amino acid losses were measured daily in the ultrafiltrate collected from eight patients with acute renal failure treated by continuous high-flux venovenous hemofiltration . All patients had type-1 respiratory failure and required mechanical ventilation . Four patients also suffered cardiogenic shock and were dependent on two or more inotropic drugs; the other four had Escherichia coli septicemia . All patients received identical daily parenteral nutrition as a continuous infusion . The overall mean serum amino acid values were greater in the group with cardiogenic shock (180 +/- 36 {SEM} mumol/L) compared to those with septicemia (131 +/- 26 mumol/L; p less than .05) . The daily ultrafiltrate losses of amino acids were also greater, 4.2 +/- 0.7 mmol/24 h (1.1 g N) compared to 2.1 +/- 0.3 mmol/24 h (0.6 g N, p less than .05) . For all patients there was a positive correlation between the serum amino acid value and ultrafiltrate loss (r = .84, p less than .001) . However, the slopes of the regression curves for the two groups differed (p less than .001), suggesting that for the same plasma value the ultrafiltrate loss was greater for those patients with cardiogenic shock. J Immunol, 1989 Oct 1, 143(7), 2153 - 9 IL-6/IFN-beta 2 in synovial effusions of patients with rheumatoid arthritis and other arthritides . Identification of several isoforms and studies of cellular sources; Bhardwaj N et al.; We have looked for IL-6, a cytokine that has immunomodulating and inflammation-associated activities, in joint exudates (fluid and mononuclear cells) from patients with rheumatoid arthritis and other arthritides using both biologic and biochemical assays . IL-6 was assessed by its ability to stimulate alpha 1-antichymotrypsin secretion from the human hepatoma cell line Hep3B clone 2, an activity which is blocked by an antiserum to Escherichia coli derived IL-6, and by the growth of the IL-6-dependent murine hybridoma 7TD1 cell line . IL-6 isoforms in synovial fluid were characterized by immunoaffinity chromatography followed by Western blotting . The presence of IL-1 in synovial fluids and its production by synovial fluid mononuclear cells was monitored by Western blotting and indirect immunofluorescence with polyclonal anti-IL-1 beta antisera . In an analysis of 30 effusions from 27 rheumatoid patients with acutely inflamed joints, abundant quantities of IL-6 (greater than 2 ng/ml) were detected in 23 by the alpha 1-antichymotrypsin bioassay . Several rheumatoid synovial fluids also had elevated IL-6 levels in the 7TD1 bioassay . Seven of nine nonrheumatoid effusions also contained high levels of IL-6 (greater than 2 ng/ml) . No IL-1 (less than 0.25 ng/ml) could be detected by Western blotting in 10 rheumatoid effusions even though eight of these contained high levels of IL-6 . The IL-6 activity could be neutralized with a rabbit antiserum to rIL-6 . Multiple IL-6 isoforms (25, 30, 45 kDa) were present in two rheumatoid and one traumatic effusion studied . Fresh mononuclear cells isolated from various synovial effusions did not appear to make IL-6 constitutively, as no IL-6 could be detected in the media of cells cultured for 12 to 18 h after isolation . Similarly, there was no constitutive production of IL-1 by these cells . However, synovial fluid mononuclear cells could be induced to secrete both IL-6 and IL-1 after stimulation with LPS . The LPS-responsive cells were monocytes and not lymphocytes or dendritic cells . These findings suggest that IL-6 is involved in inflammatory joint disease . However, the primary cells synthesizing it may be located in the synovial lining instead of the joint exudate. Gastroenterology, 1989 Oct, 97(4), 990 - 8 Detection of hepatitis B virus X gene protein and antibody in type B chronic liver disease; Katayama K et al.; The genome of the hepatitis B virus contains a sequence (X gene) whose role is unclear . The almost complete region of the hepatitis B virus X gene was expressed in Escherichia coli, with the resulting protein being approximately 17 kilodaltons in molecular weight . Sera from 139 subjects were analyzed by Western blot analysis . Of the hepatitis B surface antigen-positive patients, anti-X was not found in 4 patients with acute hepatitis and in 12 healthy carriers, but was present in 41% (21/51) of the patients with chronic hepatitis, 63% (15/24) of those with liver cirrhosis, and 46% (12/26) of those with hepatocellular carcinoma . The expression of the X product in the liver tissues (43 hepatitis B surface antigen-positive patients) was investigated using an indirect immunohistochemical method . The X protein was observed in 64% (21/33) of the patients with chronic hepatitis and 50% (5/10) of those with liver cirrhosis, and was found when the serum was negative for anti-X . Hepatitis B core antigen was frequently expressed together with the X protein in the liver . The conclusions reached were that the frequency of anti-X increases with the length of chronic hepatitis B virus infection, that anti-X may suppress the expression of the X protein in the liver, and that the X protein may be related to hepatitis B virus replication. J Gen Microbiol, 1989 Oct, 135 ( Pt 10), 2589 - 99 Mutants of Escherichia coli O9:K30 with altered synthesis and expression of the capsular K30 antigen; Whitfield C et al.; Escherichia coli K30 produces a thermostable group I capsular polysaccharide . Two classes of mutants were isolated with defects in the synthesis or expression of capsule . The most common mutant phenotype was acapsular (K-), with no K-antigen synthesized . A second class of mutants, termed Ki or intermediate forms, produced colonies which were indistinguishable from those of acapsular forms yet K-antigenicity was expressed . Previous studies had demonstrated that E . coli strains that produce K30 antigen synthesize a lipopolysaccharide (LPS) fraction that is recognised by monoclonal antibodies against the K30 antigen . Synthesis of this LPS fraction was not affected in Ki forms . The results of morphological examination, LPS analysis and phage sensitivity studies are consistent with the interpretation that the defect in Ki strains results from an inability to polymerize the K30 antigen . Using plasmid pULB113 (RP4::mini-Mu), mutations resulting in both K- and Ki phenotypes were localized near the his region of the chromosome. J Gen Microbiol, 1989 Oct, 135 ( Pt 10), 2577 - 87 Uncoupler resistance in Escherichia coli: the role of cellular respiration; Quirk PG et al.; Bioenergetic properties of a mutant strain of Escherichia coli K12 designated TUV, which is resistant to the protonophoric uncoupling agent 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidiazole (TTFB) have been compared with those of its non-resistant parent, E . coli K12 Doc-S . Strain TUV grew and respired some 20-30% faster than strain Doc-S, and was cross-resistant to carbonylcyanide p-(trifluoromethoxy)phenylhydrazone and triphenyltin, but not to 2,4-dinitrophenol . Phosphorus nuclear magnetic resonance demonstrated the TTFB-mediated collapse of the transmembrane pH gradient at identical rates in starved cells of both strains, indicating that uncoupler access and function were unimpaired in the mutant under these conditions . Strain TUV displayed enhanced uncoupler resistance and maintained intracellular pH and ATP levels only when respiring . On the other hand, strain TUV also showed increased resistance to novobiocin, implying that its outer wall permeability had been lowered . We suggest that the active resistance of strain TUV results from the exclusion of uncoupler by the interaction of inner and outer membrane components in a manner modulated by the degree of cellular energization. Biotechniques, 1989 Oct, 7(9), 1026 - 8 A new oxygen-regulated promoter for the expression of proteins in Escherichia coli; Hughes DE et al.; Several properties of a new oxygen-regulated promoter, OXYPRO, were tested in small-scale Escherichia coli cultures . Using OXYPRO, maximal activity of a reporter gene encoding chloramphenicol acetyltransferase (CAT) occurred in cultures that were tightly capped immediately after inoculation . This is probably a result of the reduced oxygen concentration attained in capped cultures, a condition known to be required for OXYPRO induction . CAT levels were significantly higher when the cells were grown in a glycerol-based medium . Similar levels of CAT expression were obtained when OXYPRO was compared to the trp-lac (tac) promoter . In addition, regulated expression of CAT occurred in a wild type strain of E . coli, suggesting that OXYPRO will be useful in most E . coli strains . Thus, OXYPRO provides a simple, inexpensive, and unobtrusive method to achieve high levels of cloned protein expression in most strains of E . coli . OXYPRO is available in a high copy plasmid with a convenient multiple cloning site for the insertion of genes for direct expression in E . coli. Biotechniques, 1989 Oct, 7(9), 1000 - 6, 1008-10 Site-directed deletion mutagenesis using phagemid vectors and genetic selection; Wang LM et al.; Oligonucleotide-directed mutagenesis was used along with the dut and ung genetic selection method of Kunkel to introduce large site-specific deletions into cDNAs cloned into phagemid vectors . We find that large deletions can be achieved with an efficiency equal to that of single point mutations, with a very low frequency of aberrent clones . To facilitate screening of clones, E . coli strain DH5 alpha was used as the recipient host cell to genetically select for deletion mutants . Comparisons were made to deletion mutagenesis without genetic selection, and to reactions utilizing two oligonucleotide primers simultaneously . The low frequency of deletion mutants observed without genetic selection renders random screening for deletion mutant clones cumbersome . The results provide representative expectations and a useful guide for those contemplating the construction of deletion mutants. Bioorg Khim, 1989 Oct, 15(10), 1394 - 410 {Synthesis of 2-acrylamidoethylglycosides of 3-O-(beta-D- glucopyranosyluronic acid)-alpha-L-rhamnose and 3-O-(alpha-L- rhamnopyranosyl)-beta-D-glucopyranuronic acid and copolymeric artificial antigens based on them}; Cherniak AIa et al.; The synthesis of disaccharide repeating units, D-GlcA-(beta 1----3)-L-Rha (fragment A) and L-Rha-(alpha 1----3)-D-GlcA (fragment B), of the K54-antigenic polysaccharide from uropathogenic Escherichia coli 06:K54:H10 is described . Essential stages of the synthesis of fragment A involved the glycosylation of methyl 2,4-di-O-benzoyl-alpha-L-rhamnopyranoside followed by acetolysis of the methyl bioside obtained and further transformation into 2-(benzyloxycarbonylamino)ethyl glycoside; deprotection and, finally, conversion into 2-(acrylamido)ethyl glycoside . Selective opening of lactone ring in 2-azidoethyl 2,4-di-O-acetyl-beta-D-glucopyranoside-6,3-lactone was used for deprotection of 3-OH group in the synthesis of fragment B . Rhamnosylation of the glucuronic acid derivative thus obtained followed by transformation into 2-(acrylamido)ethyl glycoside and deprotection gave fragment B . Both fragments A and B were converted into artificial antigens of copolymer type. Curr Genet, 1989 Oct, 16(4), 225 - 39 A yeast telomere binding activity binds to two related telomere sequence motifs and is indistinguishable from RAP1; Longtine MS et al.; Telomere Binding Activity (TBA), an abundant protein from Saccharomyces cerevisiae, was identified by its ability to bind to telomeric poly(C1-3A) sequence motifs . The substrate specificity of TBA has been analyzed in order to determine whether the activity binds to a unique structure assumed by the irregularly repeating telomeric sequences or whether the activity recognizes and binds to subset of specific sequences found within the telomere repeat tracts . Deletion analysis and DNase I protection assays demonstrate that TBA binds specifically to two poly-(C1-3A) sequences that differ by one nucleotide . The methylation of four guanine residues, located at identical relative positions within these two binding sequences, interferes with TBA binding to the substrates . A synthetic olignucleotide containing a single TBA binding site can function as a TBA binding substrate . The TBA binding site shares homology with the binding sites reported for the Repressor/Activator Protein 1 (RAP1), Translation Upshift Factor (TUF) and General Regulatory Factor (GRFI) transcription factors, and TBA binds directly to RAP1/TUF/GRFI substrate sequences . Yeast TBA preparations and the RAP1 gene product expressed in E . coli cells are both similarly sensitive to in vitro protease digestion . Affinity-purified TBA extracts include a protein indistinguishable from RAP1 in binding specificity, size, and antigenicity . The binding affinity of TBA for the two telomeric poly(C1-3A) binding sites is higher than its affinity for any of the other binding substrates used for its identification . In extracts of yeast spheroplasts prepared by incubation of yeast cells with Zymolyase, an altered, proteolyzed form, of TBA (TBA-S) is present . TBA-S has a faster mobility in gel retardation assays and SDS-PAGE gels, yet it retains the DNA binding properties of standard TBA preparations: it binds to RAP1/TUF/GRFI substrates with the same relative binding affinity and protects poly(C1-3A) tracts from DNase I digestion with a "footprint" identical to that of standard TBA preparations. Res Microbiol, 1989 Oct, 140(8), 517 - 30 Activation of amino acid transport during steady-state growth of Escherichia coli; Kubitschek HE et al.; Accumulation of amino acids in exponentially increasing cultures of Escherichia coli was linear, supporting the interpretation that the biphasic response observed when cultures grew without these acids reflects a transient perturbation in accumulation . Rates of accumulation of glutamine, histidine and glycine were compared in steady-state and non-steady-state cultures . Their uptake rates were markedly enhanced in steady-state cultures at low exogenous concentrations, 10 microM or less . The results support the activation of amino acid transport systems by low concentrations of the particular amino acid present during growth . This activation was decreased when exogenous concentrations of the amino acid were markedly increased or when cells were washed free of the amino acid . Upon readdition of the amino acid after washing, recovery of enhanced transport required several generations, supporting a process of recovery other than enzymatic induction . The observation of amino acid enhancement of transport for eight other amino acids examined in steady-state culture suggests that this enhancement is a common process. Mol Gen Genet, 1989 Oct, 219(1-2), 69 - 74 Methylation strongly enhances DNA bending in the replication origin region of the Escherichia coli chromosome; Kimura T et al.; Two-dimensional gel electrophoresis, at high and low temperatures, and gel mobilities of circularly permuted DNA segments showed a large bending locus about 50 bp downstream from the right border of the 245 bp oriC box, a minimal essential region of autonomous replication on the Escherichia coli chromosome . Bending was strongly enhanced by Dam methylation . In DNA from a Dam- strain, the mobility anomaly arising from altered conformation was much reduced, but was raised to the original level by methylation in vivo or in vitro . Enhancement of the mobility anomaly was also observed by hybrid formation of the Dam- strand with the Dam+ strand . Near the bending center, GATC, the target of Dam methylase, occurs seven times arranged essentially on the same face of the helix with 10.5 bp per turn . We concluded that small bends at each Dam site added up to the large bending detectable by gel electrophoresis. Mol Gen Genet, 1989 Oct, 219(1-2), 328 - 31 Involvement in DNA repair of the ruvA gene of Escherichia coli; Iwasaki H et al.; The ruv operon of Escherichia coli consists of two genes, orf1 and ruv, which encode 22 and 37 kilodalton proteins, respectively, and are regulated by the SOS system . Although the distal gene, ruv, is known to be involved in DNA repair, the function of orf1 has not been studied . To examine whether orf1 is also involved in DNA repair, we constructed a strain with a deletion of the entire ruv operon . The strain was sensitive to UV even after introduction of low copy number plasmids carrying either orf1 or ruv, but UV resistance was restored by introduction of a plasmid carrying both orf1 and ruv . These results suggest that orf1 as well as ruv is involved in DNA repair . Therefore, orf1 and ruv should be renamed ruvA and ruvB, respectively. Mol Gen Genet, 1989 Oct, 219(1-2), 249 - 55 Replication of ColE2 and ColE3 plasmids: in vitro replication dependent on plasmid-coded proteins; Itoh T et al.; We developed an in vitro replication system for ColE2 and ColE3 plasmids using cell extracts prepared from bacteria with or without these plasmids . DNA synthesis depended on host DNA polymerase I and was sensitive to rifampicin and chloramphenicol . Preincubation of the extracts with plasmid DNA, however, allowed replication of template DNA added subsequently in a plasmid-specific manner in the presence of rifampicin and chloramphenicol . The plasmid-specified trans-acting factor(s) was detected in cell extracts from bacteria carrying a recombinant plasmid with the region of ColE2 or ColE3 encoding the Rep protein . The plasmid-specified factor(s) consisted at least in part of protein, probably the Rep protein . In vitro replication started within a region of ColE2 or ColE3 containing the smallest cis-acting segment essential for in vivo replication and proceeded in a fixed direction. Mol Gen Genet, 1989 Oct, 219(1-2), 204 - 8 Presence in the stroma of chloroplasts of a large pool of a ribosomal protein not structurally related to any Escherichia coli ribosomal protein; Zhou DX et al.; A search was made for the presence of a pool of free ribosomal proteins in the stroma of the spinach chloroplast . The results showed that a relatively large amount of one protein, CS-S5, is present in the stroma . Immunoprecipitation experiments showed that this protein is encoded by the nuclear genome . Clones were isolated from a cDNA library constructed in the expression vector lambda gt11, using specific antibodies raised against the CS-S5 protein . A full-length cDNA was sequenced which contains an open reading frame (ORF) for the precursor of the CS-S5 protein, as shown by immunoprecipitation . This precursor contains a putative transit peptide of 66 amino acids and the mature product has no significant homology with any of the Escherichia coli ribosomal proteins, in contrast to the other ribosomal protein gene products so far identified in spinach chloroplasts. Mol Gen Genet, 1989 Oct, 219(1-2), 177 - 86 Nucleotide sequence of the leading region adjacent to the origin of transfer on plasmid F and its conservation among conjugative plasmids; Loh S et al.; The leading region of the Escherichia coli K12 F plasmid is the first segment of DNA to be transferred into the recipient cell during conjugal transfer . We report the nucleotide sequence of the 64.20-66.77F portion of the leading region immediately adjacent to the origin of transfer, oriT . The 2582 bp region encodes three open reading frames, ORF95, ORF169 and ORF273; the product of ORF273, is equivalent in size and map location to the 35 kDa protein, 6d, previously described (Cram et al . 1984) . S1 nuclease analyses of mRNA transcripts have identified a potential promoter for ORF95 and ORF273 and indicated that these ORFs are transcribed as a single transcript; in contrast, ORF169 appears to be transcribed from two overlapping promoters on the complementary DNA strand . The products of ORF95 and ORF273 are mainly hydrophilic and are probably located in the cytoplasm . ORF273 shares some homology with DNA-binding proteins . There is a signal peptide sequence at the NH2-terminus of ORF169 and the mature form of ORF169 probably resides in the periplasm due to its hydrophilic nature . Both ORF273 and ORF169 are well conserved among conjugative F-like and a few non-F-like plasmids . On the other hand, ORF95 sequences are only present on some of these plasmids . Several primosome and integration host factor recognition sites are present implicating this region in DNA metabolism and/or replication functions. Mol Microbiol, 1989 Oct, 3(10), 1397 - 404 Transcription antitermination in an Escherichia coli haemolysin operon is directed progressively by cis-acting DNA sequences upstream of the promoter region; Koronakis V et al.; Export of haemolysin protein (HlyA) directed by the Escherichia coli pHly152 hly determinant is dependent upon transcriptional activation, primarily strong intraoperon transcript antitermination imposed between the haemolysin structural genes hlyC and hlyA and the contiguous downstream export genes hlyB and hlyD . Transcript elongation was dictated by a DNA sequence several kb upstream of the rho-independent terminator but could not be assigned to a discrete locus; on the contrary, it was progressive, increasing with the addition of up to 3.5 kbp of operon-proximal sequence containing the insertion elements IS2 and IS91 . Antitermination was prominent throughout logarithmic growth but absent in stationary phase, and was effective only in cis but not in trans . Primer extension indicated that transcription activation utilized the native transcriptional start sites of the unactivated hly operon. Mol Microbiol, 1989 Oct, 3(10), 1329 - 36 Compartmentalization of the periplasm at cell division sites in Escherichia coli as shown by fluorescence photobleaching experiments; Foley M et al.; Morphological evidence has previously indicated that the periplasmic space of Escherichia coli is compartmentalized at sites corresponding to future sites of cell division . The borders of these morphological compartments are formed by localized zones of adhesion (periseptal annuli) . In the present study, the technique of fluorescence recovery after photobleaching was used to determine whether these structures act as barriers to the free movement of proteins within the periplasm . The recovery of fluorescence in the ftsA filaments was found to be uniformly low over at potential sites of cell division and at the cell poles, indicating that these regions are biochemically sequestered from the remainder of the periplasmic space . Our results provide direct evidence for local compartments within the periplasm, primarily located at the sites of past or future cell divisions . The implications of this finding for cell division and other periplasmic processes are discussed. Zhonghua Liu Xing Bing Xue Za Zhi, 1989 Oct, 10(5), 299 - 301 {A study on phage analysis of two episodes of food poisoning caused by the same serotype of enteroinvasive Escherichia coli}; Yang ZS; The strains of EIEC serotype O28ac:K73 (B):H-were isolated from two episodes of food poisoning in Beijing during July to September in 1984 . All strains were examined by E . coli K12 and found that they possessed the ability to produce the same colicin and same phage sensitivity pattern to eight phages isolated from contaminated water in Beijing area . These characteristics were different from reserved strains O28ac and other strains of E . coli . It indicated that two episodes were caused by the same phage type of O28ac:K73 (B):H-and proved the local outbreak was caused by same strain in western area of Beijing . The authors discussed in detail . The differentiation methods for plaque formation produced by colicin and phage. J Protein Chem, 1989 Oct, 8(5), 619 - 28 A novel concatenated dimer of recombinant bovine somatotropin; Violand BN et al.; A novel protein concatenated dimer structure was generated during the folding/oxidation of inclusion bodies of recombinant bovine somatotropin synthesized in Escherichia coli . The structure of this dimeric molecule was determined by peptide mapping with trypsin, and limited proteolysis by thrombin . Peptide mapping demonstrated that the two disulfide pairs in bovine somatotropin dimer were identical to those in monomer . Limited p