J Biol Chem, 1993 Feb 25, 268(6), 4259 - 66 Isolation and characterization of a novel member of the gene family encoding the cAMP response element-binding protein CRE-BP1; Nomura N et al.; Among multiple CRE (cyclic AMP response element)-binding proteins, CRE-BP1 (also designated ATF-2) has two unique characteristics: it mediates the adenovirus E1A-induced trans-activation and forms a heterodimer with c-Jun . Two structures, a putative metal finger and a leucine zipper, in CRE-BP1 are responsible for these capacities . As a new member of a CRE-BP1 family that has similar metal finger and leucine zipper structures, we have isolated cDNA clones of CRE-BPa by cross-hybridization with CRE-BP1 cDNA . CRE-BPa protein consists of 508 amino acids and has a molecular weight of 56,840 . CRE-BPa protein is highly homologous with CRE-BP1 in four regions: two of them are the regions containing the putative metal finger or the DNA-binding domain consisting of the basic amino acid cluster and the leucine zipper . Like CRE-BP1, CRE-BPa binds to CRE with higher affinity than to the 12-O-tetradecanoylphorbol-13-acetate response element as a homodimer or a CRE-BPa/c-Jun or CRE-BPa/CRE-BP1 heterodimer . However, using the c-Myb-CRE-BPa fusion protein, it was show that CRE-BPa could not mediate the E1A-induced trans-activation . Expression of CRE-BPa mRNA was found in a limited number of cell lines, and multiple sizes of CRE-BPa mRNA species were detected in some cell lines and tissues . CRE-BPa will be useful to clarify the mechanism of CRE-mediated transcriptional activation by E1A or c-Jun. J Biol Chem, 1993 Feb 25, 268(6), 3925 - 37 Protein folding in a cell-free translation system . The fate of the precursor to mitochondrial aspartate aminotransferase; Mattingly JR Jr et al.; The precursor to rat mitochondrial aspartate aminotransferase (pmAspAT) can be expressed in and purified from Escherichia coli as a fully active enzyme with remarkable trypsin resistance . Only two sites within the presequence are readily hydrolyzed (Martinez-Carrion, M., Altieri, F., Iriarte, A., Mattingly, J . R., Youssef, J., and Wu, T . (1990) Ann . N.Y . Acad . Sci . 585, 346-356) . In contrast, pmAspAT freshly synthesized in rabbit reticulocyte lysate is significantly less resistant to proteolysis and is completely digested by trypsin . Extended incubation of the pmAspAT translation product slowly converts it to a species with qualitatively the same trypsin resistance as the purified pmAspAT . In addition, this species binds pyridoxal 5'-phosphate, exhibits catalytic activity, and loses its ability to be imported into mitochondria . This process appears to reflect protein folding . The rate of folding is unaffected by the addition of cofactor or the depletion of endogenous cofactor and is not significantly affected by the concentration of translation product in the reaction . Agents that decrease the availability of ATP partially inhibit the folding, whereas the sulfhydryl alkylating reagent N-ethylmaleimide and the detergent Triton X-100 completely prevent the conversion . Although the folding of pmAspAT in reticulocyte lysate is slow, folding is rapid once the translation product is sequestered within the mitochondria as the mature form of the enzyme . These results are presented as a model for the in vivo folding of pyridoxal-dependent, oligomeric mitochondrial precursors in the presence of cytoplasmic components and for the fate of true mitochondrial precursor proteins when not imported. J Biol Chem, 1993 Feb 25, 268(6), 3911 - 9 Molecular cloning, DNA sequencing, and biochemical analyses of Escherichia coli glyoxylate carboligase . An enzyme of the acetohydroxy acid synthase-pyruvate oxidase family; Chang YY et al.; Glyoxylate carboligase (Gcl) (EC 4.1.1.47) of Escherichia coli catalyzes the condensation of two molecules of glyoxylate to give tartronic semialdehyde, a key intermediate in glyoxylate catabolism . We report the cloning, genomic location, and DNA sequence of the gene (called gcl) encoding E . coli Gcl and isolation of mutants lacking the enzyme . Gcl is a protein of 593 amino acid residues (64,738 Da) that has a high level (30%) of sequence similarity to the acetohydroxy acid synthases (AHAS) of branched chain amino acid synthetic pathway . Significant sequence identity (26%) was also observed with E . coli pyruvate oxidase, a redox flavoprotein, previously shown to be related to the AHAS enzymes (Chang, Y.-Y., and Cronan, J . E., Jr . (1988) J . Bacteriol . 170, 3937-3945) . Consistent with a grouping of Gcl with the AHAS and pyruvate oxidase enzymes . Gcl contains a quinone binding site as well as binding site for thiamine pyrophosphate and FAD . We also found that a gene (orf258) immediately downstream of the gcl gene encoded a protein (Orf258) of 258 residues . Although the gene organization of gcl and orf258 is analogous to that of the ilv gene operons which encode the E . coli AHAS isozyme large and small subunits, Orf258 does not function as a Gcl subunit . Moreover, disruption of the chromosomal copy of orf258 did not affect growth on glyoxylate or glycolate. J Biol Chem, 1993 Feb 25, 268(6), 3897 - 902 Characterization of the DNA-melting function of the Rickettsia prowazekii RNA polymerase; Ding HF et al.; In vitro specific transcription by the Rickettsia prowazekii RNA polymerase was investigated . The purified rickettsial RNA polymerase, in striking contrast to that of Escherichia coli, could specifically transcribe two R . prowazekii genes (ATP/ADP translocase and citrate synthase genes) and one E . coli gene (RNA-I) on negative supercoiled plasmids but not the same genes on linear plasmids . Following the specific binding of the rickettsial RNA polymerase to the translocase gene promoter on a linear plasmid, there was no detectable open complex formation . Both the E . coli and the R . prowazekii RNA polymerases worked well when poly(dA-dT).poly(dA-dT) or poly(dI-dC).poly-(dI-dC) was used as template for generalized transcription . However, the rickettsial RNA polymerase, in contrast to the E . coli enzyme, had little activity on poly(dG-dC).poly(dG-dC), a template with a larger number of hydrogen bonds . These data indicate that the rickettsial RNA polymerase is weak, at least relative to E . coli, in the function required for the opening of DNA duplex . It appears that this operation in R . prowazekii is aided by the negative supercoiling and the high 72% AT composition of the rickettsial genome. J Biol Chem, 1993 Feb 25, 268(6), 3845 - 9 Reduction of mutant phage T4 glutaredoxins by Escherichia coli thioredoxin reductase; Nikkola M et al.; Fifteen mutant T4 glutaredoxins (previously T4 thioredoxin) have been assayed for activity with Escherichia coli thioredoxin reductase . The mutations include substitutions in the region of the active site, in the 2 cysteines, and in the 2 residues between the cysteines forming the active-site disulfide bridge . Mutant thioredoxins where substitutions have been made in charged residues around the active site show the biggest differences in activity . The positive residues Lys-13 and Lys-21 were found to be important for efficient binding to thioredoxin reductase . Substitution of the aspartic acid at position 80 with a serine produced a glutaredoxin with superior activity . This mutant glutaredoxin has earlier been shown to be more efficient than the wild type in thiol transferase activity (Nikkola, M., Gleason, F . K., Saarinen, M., Joelson, T., Bjornberg, O., and Eklund, H . (1991) J . Biol . Chem . 266, 16105-16112) . Even the glutaredoxin P66A, where the active-site cis-proline has been substituted, could be efficiently reduced by thioredoxin reductase . Glutaredoxins lacking one or both cysteines were not active. J Biol Chem, 1993 Feb 25, 268(6), 4441 - 6 Structural requirement of CRK SH2 region for binding to phosphotyrosine-containing proteins . Evidence from reactivity to monoclonal antibodies; Matsuda M et al.; The SH2 region of signal-transducing proteins mediates binding to phosphotyrosine-containing proteins . We analyzed the structure-function relationship of the SH2 region of the human CRK protein by using a series of monoclonal antibodies (mAbs) . Seventeen mAbs against the CRK SH2 region were classified into 5 groups according to the reactivity with mutant CRK proteins expressed in COS7 cells and in Escherichia coli and by epitope scanning with synthetic nonapeptides . Two groups of mAbs (groups A and B) were reactive only with intact SH2 . Mutation(s) in either the amino-terminal B box or the carboxyl-terminal C box, which are the two subdomains of SH2, abolished the reactivity of the CRK mutants to the mAbs of groups A and B . Group A mAbs competed the binding of the CRK SH2 region to the phosphotyrosine-containing proteins . Moreover, the spectrum of the CRK mutants which were recognized by group A mAbs coincided with that of the CRK mutants which could bind phosphotyrosine-containing proteins, suggesting that group A mAbs were directed against the site of binding to phosphotyrosine-containing proteins . Group C mAbs, directed against the region between the B and C boxes, were reactive with both wild-type and mutant CRK proteins and did not affect the capacity of SH2 to bind phosphotyrosine-containing proteins . Contrary to group A and B mAbs, mAbs belonging to groups D and E, which were mapped onto the C box, did not bind well to the native CRK proteins, but bound to CRK mutants with mutation(s) in either the B or the C box . These results suggest that the B and C boxes, which are separated by a hinge region, coordinately form the functional SH2 domain that binds to the phosphotyrosine-containing proteins and to the group A mAbs. J Chromatogr, 1993 Feb 24, 633(1-2), 273 - 80 Evaluation of several affinity chromatographic supports for the purification of maltose-binding protein from Escherichia coli; Kroviarski Y et al.; To obtain affinity adsorbents with good mechanical resistance, suitable for the purification of maltose-binding protein (MBP) from Escherichia coli and genetically engineered proteins fused to MBP, a series of supports were prepared by grafting amylose on to agarose by different chemistries . Their capacities for MBP and their abilities to be used at relatively high flow-rates were examined . Efficient supports were most conveniently prepared by coupling amylose to epoxy-activated agarose in an aqueous-organic mixture. Biochim Biophys Acta, 1993 Feb 24, 1166(2-3), 301 - 4 Lecithin-cholesterol acyltransferase: effects of mutagenesis at N-linked oligosaccharide attachment sites on acyl acceptor specificity; Francone OL et al.; Site-directed mutagenesis was used to generate lecithin-cholesterol acyltransferase (LCAT) species in which individual attachment sites for N-linked oligosaccharide residues were replaced with residues that prevent the attachment of carbohydrate . Mutants at three of four sites retained significant acyltransferase activity, and phospholipase activity in the absence of cholesterol . Mutation at one site (asn272) converted LCAT to a phospholipase generating fatty acids not cholesteryl esters. Biochemistry, 1993 Feb 23, 32(7), 1810 - 5 A hydrogen-bonding network modulating enzyme function: asparagine-194 and tyrosine-225 of Escherichia coli aspartate aminotransferase; Yano T et al.; The electron distribution within the coenzyme or coenzyme-substrate conjugate needs to be properly regulated during the catalytic process of aspartate aminotransferase (AspAT) . Asn194 and Tyr225 may function in regulating the electron distribution through hydrogen-bonding to O(3') of the coenzyme, pyridoxal 5'-phosphate (PLP) or pyridoxamine 5'-phosphate (PMP) . The roles of Tyr225 have already been explored by site-directed mutagenesis (Inoue et al., 1991; Goldberg et al., 1991) . In the present studies, the mutant enzymes Asn194-->Ala and Asn194-->Ala + Tyr225-->Phe were analyzed kinetically and spectroscopically and were compared with the wild-type and Tyr225-->Phe enzymes . The kinetic studies showed that Asn194 is not essential for AspAT catalysis, although the Kd values for the substrates were increased by 10- to 50-fold upon the replacement of Asn194 . The measurements of the absorption and fluorescence excitation spectra revealed that the ratio of an enolimine to a ketoenamine form was considerably increased as a tautomeric form of the protonated PLP in the active site of the double mutant enzyme . The pH-pKd relationship for the binding of maleate to AspAT could be explained by a simple thermodynamic cycle where only one ionizing group (the imine nitrogen of the internal aldimine bond) affects the binding of maleate . The analyses of the pH-pKd curves for the wild-type and mutant enzymes showed that (i) the hydrogen bond between O(3') of PLP and Asn194 is weakened by the binding of maleate to AspAT, while the hydrogen bond between O(3') and Tyr225 is not changed, and that (ii) the replacement of Asn194 causes some effect hampering the binding of maleate.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1993 Feb 23, 32(7), 1759 - 69 Mutagenesis by the (+)-anti-diol epoxide of benzo{a}pyrene: what controls mutagenic specificity? Rodriguez H, Loechler EL. Mutagenesis by (+)-anti-benzo{a}pyrene-7,8-dihydrodiol-9,10-epoxide {(+)-anti-B{a}PDE}, an important mutagenic/carcinogenic metabolite of benzo{a}pyrene (B{a}P), is being studied in order to understand the factors that influence mutagenesis both quantitatively and qualitatively . A new mutational system, which permits the selection of supF- mutations in an Escherichia coli plasmid, pUB3, was used . The work described herein is an extension of previous work, which involved plasmid adduction and then immediate transformation (Rodriguez & Loechler, 1993), and began with the observation that mutation frequency (MF) decreased approximately 2-fold when the (+)-anti-B{a}PDE-adducted plasmid pUB3 is either (1) frozen and then thawed prior to transformation or (2) heated at 80 degrees C for 10 min prior to transformation . Several results suggest that this decrease is not due to the loss of labile adducts . To begin to understand this phenomenon, the mutagenic spectra are compared for (+)-anti-B{a}PDE in supF for the unheated (187 mutants), the freeze/thawed (134 mutants), and the heated (254 mutants) samples . In general, freeze/thawing and heating cause a decrease in all classes of mutations . Considering substitution mutations at G.C base pairs, which predominate, the mutagenic specificity for the combined data sets is GC-->TA (57%), GC-->AT (23%), and GC-->CG (20%) . This raises the question, how does (+)-anti-B{a}PDE generate this complex mutagenic specificity, which contrasts with the situation for, e.g., simple methylating agents? One factor is that mutagenic specificity at a particular guanine residue can be influenced by the base on its immediate 5'-side, most notably where mutations are virtually exclusively restricted to GC-->TA in 5'-TG-3' sequence contexts . One unexpected finding may provide additional insight . G115 in supF, which is the major hot spot for base-pairing mutagenesis, is the only site where the qualitative pattern of mutagenesis is significantly affected by heating the (+)-anti-B{a}PDE-adducted plasmid prior to transformation . Without heating, G115-->T mutations predominate, but following heating there is a statistically significant increase in the fraction of G115-->A and G115-->C mutations . The most likely model to explain this and other results is (1) a particular DNA adduct can adopt multiple conformations, (2) the conformation adopted by an adduct can be influenced by various factors, including DNA sequence context, as well as heating and freeze/thawing, and (3) each of these conformations can cause a different pattern of mutation.(ABSTRACT TRUNCATED AT 400 WORDS) Biochemistry, 1993 Feb 23, 32(7), 1689 - 94 A pre-transition-state mimic of an enzyme: X-ray structure of adenosine deaminase with bound 1-deazaadenosine and zinc-activated water; Wilson DK et al.; The refined 2.4-A structure of adenosine deaminase, recently discovered to be a zinc metalloenzyme {Wilson, D . K., Rudolph, F . B., & Quiocho, F . A . (1991) Science 252, 1278-1284}, complexed with the ground-state analog 1-deazaadenosine shows the mode of binding of the analog and, unexpectedly, a zinc-activated water (hydroxide) . This structure of a pre-transition-state mimic, combined with that previously determined for the complex with 6(R)-hydroxy-1,6-dihydropurine ribonucleoside, a nearly ideal transition-state analog, sheds new understanding of the precise stereospecificity and hydrolytic catalysis of an important and well-characterized member of a large group of zinc metalloenzymes . As both of these excellent mimics were generated in the active site, they demonstrate a powerful means of dissecting the course of an enzymatic reaction by direct crystallographic analysis. Biochemistry, 1993 Feb 23, 32(7), 1770 - 3 Photo-cross-linking of CRP to nonspecific DNA in the absence of cAMP . DNA interacts with both the N- and C-terminal parts of the protein; Katouzian-Safadi M et al.; Adenosine cyclic 3',5'-phosphate receptor protein (CRP or CAP) is a regulatory protein involved in the transcription of several operons in Escherichia coli . cAMP-independent, nonspecific complexes of CRP and DNA were investigated by photochemical cross-linking of the protein to nonspecific DNA, whose thymines are substituted by 5-bromouracil (BrUra) . The cross-linked protein was completely digested by trypsin, and the covalently bound peptides were sequenced . We identified two regions of the protein in close contact with DNA: one in the C-terminal part, overlapping the canonical helix-turn-helix motif, and the other one in the N-terminal part, which is usually not considered to belong to the DNA-interacting domain of CRP . This result lead us to propose models for nonspecific interaction, where the DNA is in contact with both the N- and C-terminal parts of the protein. FEBS Lett, 1993 Feb 22, 318(1), 11 - 6 Identification of zinc-binding ligands in the class II fructose-1,6-bisphosphate aldolase of Escherichia coli; Berry A et al.; An expression and mutagenesis system for the E . coli Class II fructose-1,6-bisphosphate aldolase has been created by modification of the vector pKfda (Biochem . J . 257 (1989) 529-534) . Large amounts of Class II aldolase (about 1 g/l in crude extracts), with properties consistent with those previously reported for the naturally occurring enzyme (Biochem . J . 169 (1978) 633-641) are obtained . The enzyme contains 2 zinc ions per enzyme dimer . We have investigated the nature of the zinc-binding site of the enzyme by site-directed mutagenesis . His-108, His-111, Cys-112 and His-142 were identified as possible zinc-binding ligands by sequence alignments and comparisons with other known zinc-containing enzymes . Mutation of these residues identified His-108 and His-111 as two of the ligands directly responsible for the tight binding of zinc . Mutation of the other two residues results in only a small effect on the amount of zinc bound per monomer and a corresponding change in specific activity . These residues are, therefore, unlikely to be directly involved in zinc binding, but may be indirectly involved in some manner in the zinc-binding environment. J Mol Biol, 1993 Feb 20, 229(4), 833 - 48 Significant dispersed recurrent DNA sequences in the Escherichia coli genome . Several new groups; Blaisdell BE et al.; New computer and statistical methods were used to determine significant direct and inverted repeats in the Escherichia coli contig sequence collection of aggregate 1.6 x 10(6) base-pairs . Eight groups of mostly new structural repeat identities were uncovered . Apart from the high statistical significance of these repeat sequences, there are suggestive relationships of the group matches in terms of neighboring genes, of genomic distributions, of their texts, and of their potentials for secondary structure . Four of these groups are relatively numerous, 11 to 26 members, one is in coding sequences and three are in non-coding . The coding group consists of the ATP-activated transmembrane component of a typical high-affinity protein-binding transport system . One of the non-coding groups consists of a special rho-independent transcription termination signal closely following an operon . The gene neighbors of this group often appear to be involved in some way in processing RNA or DNA . A second non-coding group has, for one or both neighboring genes, a component of a system responding to stress or starvation for some nutrient. J Mol Biol, 1993 Feb 20, 229(4), 1159 - 62 Formation in vivo, purification and crystallization of a complex of the gamma and epsilon subunits of the F0F1-ATPase of Escherichia coli; Cox GB et al.; A complex comprising the epsilon subunit of Escherichia coli F1-ATPase (ECF1-ATPase) and a glutathione-S-transferase gamma subunit (of ECF1-ATPase) fusion protein was formed in vivo and purified from cell extracts by binding to glutathione-agarose beads . The glutathione-S-transferase was released from the complex by digestion with thrombin and the gamma/epsilon complex purified by cation-exchange chromatography . Crystals of the complex were grown by vapour diffusion using PEG8000 as precipitant . The crystals are orthorhombic, space-group P2(1)2(1)2 with a = 161.9 A, b = 44.1 A and c = 63.4 A . The volume of the asymmetric unit is consistent with the presence of a complex of one gamma subunit and one epsilon subunit. J Mol Biol, 1993 Feb 20, 229(4), 1157 - 8 Crystallization and preliminary X-ray crystallographic analysis of the protease inhibitor ecotin; Shin DH et al.; Ecotin, a novel serine protease inhibitor isolated from Escherichia coli, has been crystallized using polyethylene glycol 1500 as the precipitating agent . The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters of a = 39.22 A, b = 84.86 A, and c = 98.74 A . The asymmetric unit contains one dimeric molecule of ecotin, with a crystal volume per protein mass (Vm) of 2.55 A3/Da and a solvent content of 51.8% by volume . The crystals diffract to at least 2.2 A using a conventional X-ray source, and X-ray data have been collected to 2.7 A Bragg spacing from a native crystal. J Mol Biol, 1993 Feb 20, 229(4), 1150 - 2 Crystallization and preliminary X-ray diffraction studies of recombinant human interleukin-5; Hassell AM et al.; Recombinant human interleukin-5 (rhIL-5) has been crystallized by the hanging drop vapor diffusion method using 0.1 M-Tris.HCl buffer (pH 8.5) containing 0.2 to 0.25 M-sodium acetate and 26 to 30% PEG 4000 at 22 degrees C . The parallel-piped crystals belong to the space group C2 with unit cell dimensions of a = 122.1 A, b = 36.11 A, c = 56.42 A, beta = 98.59 degrees . They diffract to at least 2.0 A resolution on a rotating anode X-ray source . The molecular mass weight of the protein and the volume of the unit cell suggest that the asymmetric unit contains one intermolecular disulfide-bonded homodimer. J Mol Biol, 1993 Feb 20, 229(4), 1083 - 100 Three-dimensional structure of the glutathione synthetase from Escherichia coli B at 2.0 A resolution; Yamaguchi H et al.; Glutathione synthetase (gamma-L-glutamyl-L-cysteine: glycine ligase (ADP-forming) EC 6.3.2.3: GSHase) catalyzes the synthesis of glutathione from gamma-L-glutamyl-L-cysteine and Gly in the presence of ATP . The enzyme from Escherichia coli is a tetramer with four identical subunits of 316 amino acid residues . The crystal structure of the enzyme has been determined by isomorphous replacement and refined to a 2.0 A resolution . Two regions, Gly164 to Gly167 and Ile226 to Arg241, are invisible on the electron density map . The refined model of the subunit includes 296 amino acid residues and 107 solvent molecules . The crystallographic R-factor is 18.6% for 17.914 reflections with F > 3 sigma between 6.0 A and 2.0 A . The structure consists of three domains: the N-terminal, central, and C-terminal domains . In the tetrameric molecule, two subunits that are in close contact form a tight dimer, two tight dimers forming a tetramer with two solvent regions . The ATP molecule is located in the cleft between the central and C-terminal domains . The ATP binding site is surrounded by two sets of the structural motif that belong to those respective domains . Each motif consists of an anti-parallel beta-sheet and a glycine-rich loop. Biochim Biophys Acta, 1993 Feb 20, 1172(1-2), 12 - 6 Identification and characterization of a yeast gene encoding an adenylate kinase homolog; Konrad M; Screening for genes homologous to adenylate kinase in the yeast Saccharomyces cerevisiae resulted in the isolation of a homolog of the previously characterized ADK1 . The derived protein sequence is most closely related to mammalian GTP:AMP phosphotransferase (adenylate kinase isozyme 3; AK3); this novel gene is therefore named ADK3 . Its deletion from the yeast genome does not lead to an observable change in cellular phenotype . A strain defective for both ADK1 and ADK3 is viable . When introduced on a multicopy plasmid into an ADK1-deficient yeast strain, which shows a reduced proliferation rate, ADK3 did not rescue this growth defect . The protein was also highly overexpressed in E . coli cells . However, no change in enzymatic activity was detected in cellular extracts of yeast or bacteria. J Mol Biol, 1993 Feb 20, 229(4), 1007 - 21 X-ray analysis and spectroscopic characterization of M121Q azurin . A copper site model for stellacyanin; Romero A et al.; The dependence of the properties of the azurin blue copper site on the nature of the axial ligand at position 121 was tested by site-directed mutagenesis . This residue was substituted for a glutamine, the purported fourth copper ligand in the related protein stellacyanin . M121Q azurin was isolated and purified from Escherichia coli and characterized by spectroscopic methods . The mutant copper site has the ultra-violet-vis and electron paramagnetic resonance (EPR) characteristics of a type I site, but the spectroscopic details differ significantly from wild-type (wt) azurin . The X and S-band EPR spectra of M121Q azurin can be well stimulated with the parameters for stellacyanin, indicating that the copper sites of both proteins in the oxidized state are similar . The midpoint potential of M121Q is 263 mV, 25 mV lower than for wt azurin . The reactivity of the mutant was probed by measuring the electron self exchange rate by nuclear magnetic resonance spectroscopy . The rate was 8 x 10(3) mol-1 s-1, almost two orders of magnitude lower than the value for wt azurin (5 x 10(5) mol-1 s-1) . Detailed structural information on the M121Q Cu(II)-site was obtained by X-ray analysis of M121Q azurin crystals at 1.9 A resolution . The histidine and cysteine copper ligand distances and angles in the equatorial plane around the copper are very similar to the wt protein . Gln121 is co-ordinated in a monodentate fashion via its side-chain oxygen atom at a distance of 2.26 A . The distance between copper and the carbonyl group of Gly45 is increased from 3.13 A (wt) to 3.37 A resulting in a distorted tetrahedral N2SO copper co-ordination . The possible significance of these results for the structure of the copper site of stellacyanin, the only small blue copper protein lacking a methionine ligand, is discussed . Conformational changes with respect to the wt azurin are seen in some of the connecting loops between secondary structure elements, in the mutation site and in the beta-strand 2a . The side-chains involved in the hydrophobic patch surrounding His117 are subject to large changes in their conformations . In contrast to wt azurin, the copper site in M121Q azurin undergoes significant structural changes on reduction.(ABSTRACT TRUNCATED AT 400 WORDS) J Mol Biol, 1993 Feb 20, 229(4), 860 - 72 Quantitative model of ColE1 plasmid copy number control; Brendel V et al.; Initiation of replication of the Escherichia coli plasmid ColE1 is inhibited by formation of a complex between a small plasmid RNA (RNA I) and the pre-primer for DNA synthesis (RNA II) . Complex formation (and inhibition of replication) is enhanced by the plasmid-encoded Rom protein . The in vitro kinetics of complex formation were previously studied both experimentally and theoretically . The in vivo concentrations and half-lives of RNA I, RNA II and Rom protein have been measured recently . We present a dynamic model for the in vivo replication control mechanism that accounts for the measured concentration values . From the model we deduce a simple formula for the steady-state plasmid concentration . Our results agree with a previous simple steady-state analysis done by Brenner and Tomizawa, in that plasmid copy number is most strongly dependent on the per plasmid rate of RNA I synthesis . However, our model predicts other parameter dependencies that are not evident from or at variance with the previous analysis . Accordingly, we predict that plasmid copy number is greatly influenced by changes in the rate constant describing the formation of an initial unstable RNA I-RNA II complex, but is only slightly influenced by changes in the dissociation rate of this complex . Plasmid copy number per average cell volume is predicted to increase linearly with increases in the RNA II synthesis rate and with increases in the generation time of the host culture . Rom protein, which promotes conversion of the unstable RNA I-RNA II complex to a stable complex, serves to decrease copy number; however, its presence or absence does not seem to qualitatively alter the copy number control mechanism . Our model predicts the quantitative increase of plasmid copy number in rom- mutants . Several experiments are suggested to investigate the predictions of the model. Biochim Biophys Acta, 1993 Feb 20, 1172(1-2), 161 - 6 Characterization of a cDNA encoding a human kidney, cytochrome P-450 4A fatty acid omega-hydroxylase and the cognate enzyme expressed in Escherichia coli; Palmer CN et al.; A cDNA encoding a cytochrome P-450 4A (CYP4AII) was cloned from a human kidney cDNA library . Northern blot analysis and RNase protection assays indicate that related mRNAs occur in kidney and liver with the highest abundance found in kidney . The enzyme was expressed from its cDNA in Escherichia coli . A solubilized preparation of the enzyme reconstituted with cytochrome P-450 reductase catalyzed the omega-hydroxylation of lauric acid, palmitic acid, and arachidonic acid with turnover numbers of 9.8, 2.2 and 0.55 min-1, respectively . Little or no activity was detected toward prostaglandins A1 and E1. Science, 1993 Feb 19, 259(5098), 1154 - 7 The nonhelical structure of antifreeze protein type III; Sonnichsen FD et al.; Antifreeze proteins (AFPs) are present in the blood of some marine fishes and inhibit the growth of ice crystals at subzero temperatures by adsorption to the ice lattice . The solution structure of a Type III AFP was determined by two-dimensional nuclear magnetic resonance spectroscopy . These measurements indicate that this 66-residue protein has an unusual fold in which eight beta strands form two sheets of three antiparallel strands and one sheet of two antiparallel strands, and the triple-stranded sheets are packed orthogonally into a beta sandwich . This structure is completely different from the amphipathic, helical structure observed for Type I AFPs. Biochemistry, 1993 Feb 16, 32(6), 1601 - 9 Magnesium in the active site of Escherichia coli alkaline phosphatase is important for both structural stabilization and catalysis; Janeway CM et al.; Site-specific mutagenesis was used to explore the roles of the side chains of residues Lys-328 and Asp-153 in Escherichia coli alkaline phosphatase . The D153H enzyme exhibits a 3.5-fold decrease in activity at pH 8.0 compared to that of the wild-type enzyme, while a double mutant D153H/K328H exhibits a 16-fold decrease in activity under these conditions . However, the Km values for both enzymes, employing the substrate p-nitrophenyl phosphate, are lower than the value for the wild-type enzyme . The Ki for phosphate, which is pH- and Mg(2+)-dependent, is decreased for the D153H enzyme and increased for the D153H/K328H enzyme . Relative to the wild-type enzyme, both mutant enzymes bind Mg2+ more weakly and undergo a time-dependent activation induced by Mg2+ . The half-time of the activation process is independent of the Mg2+ concentration, indicating that the activation most probably involves a conformational change . The pH versus activity profiles of both enzymes are altered relative to that of the wild-type enzyme and exhibit greatly enhanced activity, relative to that of the wild-type enzyme, at high pH values . The pre-steady-state kinetics for the D153H and D153H/K328H enzymes exhibit a transient burst of product formation at pH 8.0, under conditions at which the wild-type enzyme exhibits no transient burst, indicating that at pH 8.0 the hydrolysis of the covalent enzyme-phosphate complex is rate-determining and not the release of phosphate from the noncovalent enzyme-phosphate complex as is observed for the wild-type enzyme . Therefore, these mutations are directly influencing catalysis.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1993 Feb 16, 32(6), 1548 - 54 Use of adenosine (5')polyphospho(5')pyridoxals to study the substrate-binding region of glutathione synthetase from Escherichia coli B; Hibi T et al.; Adenosine(5')polyphospho(5')pyridoxals (APn-PLs, n = 2, 3, 4) were examined for affinity labeling of glutathione synthetase (EC 6.3.2.3) from Escherichia coli B . When the enzyme was incubated with an APn-PL or pyridoxal phosphate in the presence of Mg2+ and then reduced with sodium borohydride, it was most rapidly inactivated by AP4-PL . AP4-PL had a high affinity to the enzyme . The dissociation constant of AP4-PL in the inactivation process was 23 microM . The enzyme was almost completely protected from inactivation by addition of either ATP or gamma-glutamylcysteine . Complete inactivation corresponded to the incorporation of 1 mol of AP4-PL/mol of subunit of the tetrameric enzyme . Proteolytic digestion and sequence analysis of the AP4-PL-labeled enzyme revealed that only Lys-18 was modified . In contrast, the less efficient AP3-PL was found attached to Lys-17, Lys-18, Lys-144, and Lys-148 . In the three-dimensional structure of the enzyme, Lys-18 is located close to the putative gamma-glutamylcysteine-binding site, but Lys-17, Lys-144, and Lys-148 are in the mouth of the inner-solvent region, at the bottom of which is the active-site cleft . Furthermore, difference Fourier analysis with the AP4-PL-soaked crystal of the enzyme showed that the adenosine moiety of the bound AP4-PL was in the crevice, which is the ATP-binding site of the enzyme . These results demonstrate the bivalent binding of AP4-PL lying across the gamma-glutamylcysteine- and ATP-binding sites. Biochemistry, 1993 Feb 16, 32(6), 1541 - 7 Human nucleotide excision nuclease incises synthetic double-stranded DNA containing a pyrimidine dimer at the fourth phosphodiester linkage 3' to the pyrimidine dimer; Tateishi S et al.; Linear 75mer double-stranded DNA containing a single pyrimidine dimer at a unique site was used to investigate pyrimidine dimer-dependent endonuclease activities from human cells . HeLaS3 cell extract incised the target DNA at the fourth phosphodiester linkage 3' to the pyrimidine dimer . However, incision of the DNA at 5' side of the pyrimidine dimer was not detected . The incision was also detected in cell extracts prepared from other excision repair-proficient cell lines . Incision was detected only on the DNA strand containing a pyrimidine dimer in the presence of poly(dI-dC)-poly(dI- dC) double strand . The reaction required Mg2+ but not ATP . The extract prepared from excision repair-deficient xeroderma pigmentosum (XP) cells belonging to the complementation group A was unable to incise the DNA . Extracts from the complementation groups C, D, and G incised the DNA very weakly at the third phosphodiester linkage 3' to the pyrimidine dimer, a site different from that incised by normal human cell extract . These results suggest that the observed incision reaction is associated with excision repair in human cells. Biochemistry, 1993 Feb 16, 32(6), 1510 - 8 Engineering the zinc binding site of human carbonic anhydrase II: structure of the His-94-->Cys apoenzyme in a new crystalline form; Alexander RS et al.; The structure of the His-94-->Cys variant of human carbonic anhydrase II (CAII) has been determined by X-ray crystallographic methods to a resolution of 2.3 A with a final crystallographic R factor of 0.155 . This variant of CAII crystallizes in orthorhombic space group P2(1)2(1)2(1) which is the first example of a new crystal form for this important zinc hydrase (the wild-type enzyme crystallizes in monoclinic space group P21 under similar crystallization conditions) . Although the overall structure of the enzyme in the orthorhombic crystal form is similar to that of the wild-type protein in the monoclinic crystal form, the rms deviation of C alpha atoms between the two structures is 0.5 A . Larger structural deviations occur in regions of the protein molecule involved in crystal lattice contacts, and significant structural changes are found in the polypeptide strand containing Cys-94 . Surprisingly, no electron density corresponding to a zinc ion is found in the active site of crystalline His-94-->Cys CAII, even though the stoichiometry of zinc binding to this variant in solution is confirmed by atomic absorption spectroscopy . However, the KD for zinc dissociation from the variant is increased 10(4)-fold compared with wild-type enzyme; furthermore, under the crystallization conditions of high ionic strength (1.75-2.5 M ammonium sulfate), the observed KD is increased further, which leads to zinc dissociation . Spectroscopic analysis of Co(2+)-substituted His-94-->Cys CAII indicates that the metal binds in a tetrahedral geometry with a new thiolate bond.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1993 Feb 16, 32(6), 1454 - 65 Further characterization of the psbH locus of Synechocystis sp . PCC 6803: inactivation of psbH impairs QA to QB electron transport in photosystem 2; Mayers SR et al.; The psbH gene encodes a small protein which copurifies with photosystem 2 . In the cyanobacterium Synechocystis sp . PCC 6803, psbH is located upstream of the cytochrome b6-f complex genes petC and petA . In striking contrast, in the genomes of plant chloroplasts, psbH is cotranscribed with petB and petD, encoding the other two major subunits of the cytochrome b6-f complex . We report that in Synechocystis sp . PCC 6803 monocistronic psbH and dicistronic petCA transcripts are probably initiated separately, each from DNA regions bearing some similarity to Escherichia coli sigma 70 promoters . Synechocystis sp . PCC 6803 psbH null mutants were generated by cartridge mutagenesis . Studies using a rapid screening procedure involving in situ complementation showed that the PsbH protein is not absolutely required for the assembly of a functionally active photosystem 2 complex since psbH insertion and deletion strains were able to grow photoautotrophically . The rate of photoautotrophic growth was, however, slower than the wild type, and studies of oxygen evolution, chlorophyll fluorescence, and thermoluminescence indicated that this reduction in growth rate is probably due mainly to an impairment in electron flow from QA to QB . We conclude, therefore, that the PsbH protein is not an absolute requirement for photosystem 2 activity but that it functions to optimize electron flow between the two secondary plastoquinone acceptors by interacting with the QB site on the D1 protein. Biochemistry, 1993 Feb 16, 32(6), 1396 - 400 Mannitol-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli: physical size of enzyme IImtl and its domains IIBA and IIC in the active state; Lolkema JS et al.; The size of enzyme IImtl solubilized in the active state has been determined by size-exclusion chromatography under conditions that favor the association of the enzyme . The contribution of the detergent bound to the enzyme was determined by solubilizing the enzyme and running the TSK250 column in a number of detergents with decreasing micellar sizes . The size, expressed as the equivalent molecular mass of a globular protein, decreased from 315 kDa in decylPEG, to 275 kDa in octylPEG and octyl glucoside, and then to 245 kDa in cholate . Enzyme IImtl is not active in the latter three detergents when at concentrations above their cmc values but still binds mannitol with high affinity without significant loss of sites . This, together with the full reversibility of the inactivation, is taken as evidence that the enzyme does not unfold or dissociate in these detergents . The sizes of the separated domains IIBA and IIC of enzyme IImtl were 38 and 175 kDa, respectively . The cytoplasmic domain, IIBA, was monomeric at high concentration, whereas the membrane-bound domain, IIC, was associated at much lower concentration . Apparently, the sites that interact to keep enzyme IImtl in the associated state are exclusively located in the membrane-bound domain. Biochemistry, 1993 Feb 16, 32(6), 1519 - 27 Cloning, overexpression, and characterization of the functional dihydroorotase domain of the mammalian multifunctional protein CAD; Zimmermann BH et al.; Mammalian dihydroorotase (DHOase) is part of a large multidomain protein called CAD, which initiates the first three steps in the de novo pyrimidine biosynthetic pathway . DHOase activity is carried out by a 44-kDa structural domain which could be isolated in active form from elastase digests . A core domain the same size as monofunctional dihydroorotases was defined, although the domain borders were uncertain . Two recombinants were overexpressed in Escherichia coli . The first encoded the core domain with 55 and 13 residues added to the amino and carboxyl ends, respectively, and was expressed in insoluble form . The recombinant protein was refolded from urea into a soluble form which was resistant to protease digestion but was catalytically inactive . In contrast, the proteolytic fragment from CAD could be unfolded and refolded with recovery of 40-100% catalytic activity . The second construct, which approximated the proteolytic fragment, had 21 residues on the amino end and 65 residues on the carboxyl end of the core domain . A 46-kDa soluble protein was expressed at 4% of the total soluble protein . The recombinant protein was catalytically active and had the expected amino-terminal sequence . The protein was purified to homogeneity . Dihydroorotase saturation curves gave a Km = 41.9 +/- 3.5 microM and a kcat = 2.79 +/- 0.06 s-1, parameters that were similar to those obtained for the proteolytic fragment . The Km was 6-fold higher and the kcat 2-fold lower than the values obtained for the parent protein, which suggests that interdomain interactions stabilize the active conformation.(ABSTRACT TRUNCATED AT 250 WORDS) FEMS Microbiol Lett, 1993 Feb 15, 107(1), 71 - 6 Temperature-sensitive amber suppression of ompF'-'lacZ fused gene expression in a supE mutant of Escherichia coli K12; Kuriki Y; In an ompF'-'lacZ fusion system carried by the open reading frame vector pORF1 in a supE mutant of Escherichia coli K12, read-through of an amber codon was decreased at temperatures higher than 40 degrees C . This effect of temperature was dependent on the nucleotide sequence surrounding the amber codon, which was inserted into a site between the ompF and lacZ cistrons . Upon a temperature shift-up from 30 to 42 degrees C, beta-galactosidase synthesis directed by this fusion showed a transient arrest. FEMS Microbiol Lett, 1993 Feb 15, 107(1), 25 - 30 A yeast gene for trehalose-6-phosphate synthase and its complementation of an Escherichia coli otsA mutant; McDougall J et al.; A Saccharomyces cerevisiae gene for trehalose-6-phosphate synthase (TPS1) was sequenced . The gene appeared to code for a protein of 495 amino acid residues, giving the protein a molecular mass of 56 kDa . The TPS1 gene was able to restore both osmotolerance and trehalose accumulation during salt stress in an Escherichia coli strain mutated in the otsA gene encoding trehalose-6-phosphate synthase . Complementation studies with E . coli galU mutants showed that the TPS1-encoded trehalose-6-phosphate synthase is UDP-glucose-dependent . Sequence analysis and data base searches showed that TPS1 is allelic to GGS1, byp1, cif1 and fdp1 . A possible gene for trehalose-6-phosphate synthase in Methanobacterium thermoautotrophicum was identified. FEMS Microbiol Lett, 1993 Feb 15, 107(1), 111 - 4 Purification of a fibroblast-inhibitory factor from Actinobacillus actinomycetemcomitans Y4; Kataoka M et al.; A factor showing inhibitory activity against human gingival fibroblasts was extracted from the cytosol fraction of Actinobacillus actinomycetemocimitans Y4 . The activity markedly inhibited the proliferation of human gingival fibroblasts, but had no effect on cell viability or gross morphology . No such activity was found in cytosol fractions from either Porphyromonas gingivalis 381 or Escherichia coli HB101 . The extract from A . actinomycetemocomitans Y4 was then purified by anion-exchange chromatography, hydroxyapatite chromatography and gel-filtration chromatography to give a single band on SDS-PAGE with an apparent molecular mass of 65 kDa . The purification ratio was 183-fold with a recovery rate of 5% compared with the crude extract (starting material) when the activity was assessed by direct cell counts. FEMS Microbiol Lett, 1993 Feb 15, 107(1), 101 - 5 Non-specific hole formation in the Escherichia coli inner membrane by lambda S proteins in independent of cellular secY and secA functions and of the proportion of membrane acidic phospholipids; Rietsch A et al.; Formation of the lesion in the Escherichia coli inner membrane caused by lambda lysis protein S was examined by electron microscopy . We also show that macromolecules exceeding the size of the lambda R transglycosylase can pass through the S-dependent hole and that assembly of the S-dependent hole is independent of the proportion of acidic phospholipids in the inner membrane and of components of the cellular transport machinery. Anal Biochem, 1993 Feb 15, 209(1), 1 - 5 Enzymatic synthesis of 6R-{U-14C}tetrahydrobiopterin from {U-14C}GTP; Hatakeyama K et al.; An enzymatic method for preparing 6R-{U-14C}-tetrahydrobiopterin from {U-14C}GTP is presented . This method utilizes purified preparations of three biosynthetic enzymes for 6R-tetrahydrobiopterin, i.e., Escherichia coli GTP cyclohydrolase I, rat 6-pyruvoyl-tetrahydropterin synthase, and rat sepiapterin reductase . Based on the catalytic properties of these enzymes, the reaction conditions were optimized to complete the entire conversion reaction from GTP to 6R-tetrahydrobiopterin in a single reaction solution without the need to isolate unstable intermediates after each enzymatic reaction . The reaction conditions thereby established yielded {U-14C}biopterin in an amount equivalent to 75%, on a molar basis, of the initial amount of {U-14C}GTP . The product was subsequently isolated by high-performance liquid chromatography . The method produced labeled 6R-tetrahydrobiopterin with a specific activity of 450 Ci/mol and an overall yield of more than 60%. Eur J Biochem, 1993 Feb 15, 212(1), 27 - 34 Isolation of a ripening and wound-induced cDNA from Cucumis melo L . encoding a protein with homology to the ethylene-forming enzyme; Balague C et al.; A cDNA clone (pMEL1) was isolated from a climacteric melon fruit cDNA library using the tomato ethylene-forming-enzyme (EFE) cDNA, pTOM13, as a probe . Northern analysis of RNA isolated from wounded leaf and fruit tissue and from preclimacteric and climacteric pericarp tissue was used to determine the pattern of pMEL1 RNA expression . pMEL1 hybridized to a 1.3-kb transcript in climacteric fruit and wounded leaf tissue but was undetectable in preclimacteric fruit and unwounded leaves . Maximal expression of pMEL1 RNA occurred in wounded ripe fruit . pMEL1 contained a 1230-bp insert containing a predicted open reading frame of 318 amino acids and molecular mass of 35.3 kDa . The predicted amino acid sequence of pMEL1 was 73-81% identical to the deduced amino acid sequences of tomato (pTOM13) EFE and EFE-related genes isolated from tomato and avocado fruit and senescent carnation petals . Genomic Southern analysis indicated that pMEL1 hybridized to a number of genomic fragments consistent with the presence of more than one pMEL1-related gene in melon . On Western analysis of total protein extracts from ripe tomato and melon fruit, using an antibody raised against tomato EFE (pTOM13) expressed in Escherichia coli, a single 35.5-kDa protein was detected . A 35-kDa product translated from in-vitro transcribed pMEL1 and immunoadsorbed by anti-EFE serum was very similar in size to the predicted 35.3-kDa pMEL1 cDNA protein product . These results indicate that pMEL1 may encode an EFE gene involved in ethylene biosynthesis during fruit ripening and may be identical to or share extensive sequence similarity with an EFE expressed in response to tissue wounding. Eur J Biochem, 1993 Feb 15, 212(1), 201 - 10 Thymidine phosphorylase activity of platelet-derived endothelial cell growth factor is responsible for endothelial cell mitogenicity; Finnis C et al.; Recombinant human platelet-derived endothelial cell growth factor, expressed in the yeast Saccharomyces cerevisiae was purified to greater than 98% purity by anion-exchange and hydroxyapatite chromatography . It was shown to possess thymidine phosphorolytic activity in vitro (pH optimum, pH 5.3; Km, 0.11 mM; Vmax, 12.5 mmol min-1 mg-1; turnover number, 9.4 s-1) . Covalent modification simultaneously inhibited the enzymatic and mitogenic properties of the protein, while interaction with a cell-surface receptor was not required to stimulate mitogenesis . Purified Escherichia coli thymidine phosphorylase was also mitogenic toward endothelial cells . It is proposed that platelet-derived endothelial cell growth factor is human thymidine phosphorylase which promotes endothelial cell proliferation by reducing thymidine levels that would otherwise be inhibitory to endothelial cell growth. Arch Biochem Biophys, 1993 Feb 15, 301(1), 64 - 70 Regulation of the mitochondrial ATP synthase/ATPase complex: cDNA cloning, sequence, overexpression, and secondary structural characterization of a functional protein inhibitor; Lebowitz MS et al.; The ATPase inhibitor protein of the rat liver mitochondrial ATP synthase/ATPase complex has been cloned from a rat liver cDNA library, and its nucleotide sequence determined . The sequence is highly homologous to both the bovine heart (approximately 70%) and the yeast inhibitor proteins (approximately 40%) . The deduced protein sequence is 107 amino acids in length, and based on homology to the bovine heart protein, the first 25 N-terminal amino acids encode a putative mitochondrial targeting sequence . The "mature" protein (without the targeting sequence) fused to the maltose binding protein has been overexpressed in Escherichia coli . The maltose binding protein was used as a handle for the development of a rapid one-step purification of the fusion protein by affinity chromatography on an amylose resin . The purified fusion protein was cleaved with Factor Xa protease at the fusion junction, and the resulting ATPase inhibitor protein was purified to > 90% purity . The purified, overexpressed inhibitor protein displays normal inhibitor activity . The protein inhibits ATP hydrolysis catalyzed by the ATP synthase/ATPase complex in submitochondrial particles in a manner kinetically indistinguishable from the same protein purified from rat liver mitochondria, and exhibits a specific activity of approximately 10,000 units/mg . The secondary structure of the inhibitor protein was determined by circular dichroism spectropolarimetry . The experimentally determined structure shows a high content of alpha-helix and is in good agreement with sequence-based structural predictions . As the function of the inhibitor protein is known to exhibit a high dependence on pH, a study of the pH dependence of inhibitor secondary structure was performed . It is shown that as pH is lowered, conditions which activate inhibitory capacity, the protein loses significant alpha-helical structure . This is the first report of the overexpression in E . coli of a functional ATPase inhibitor protein . Secondary structural analysis of this protein indicates that conversion from its active to its inactive form involves a significant conformational change. Arch Biochem Biophys, 1993 Feb 15, 301(1), 58 - 63 Production and characterization of polyclonal antibodies in rabbits to 4S-limonene synthase from spearmint (Mentha spicata); Alonso WR et al.; Limonene synthase, a monoterpene cyclase from the oil glands of spearmint (Mentha spicata) leaves that catalyzes the conversion of geranyl pyrophosphate to (-)-4S-limonene, was purified, and polyclonal antibodies were generated in rabbits against the sodium dodecyl sulfate-denatured protein . Immunoblotting analysis revealed that the antibodies were very specific for denatured limonene synthase from all Mentha species tested . However, no immunological cross-reactivity was observed with denatured limonene synthases from Valencia oranges (Citrus sinensis, Rutaceae) or wormseed (Chenopodium ambrosioides, Chenopodiaceae) . Furthermore, the antibody preparation did not detectably cross-react with other monoterpene cyclases from related angiosperm species of the Lamiaceae, Asteraceae, and Umbellifereae, or from conifer species, and no cross-reactivity was demonstrated toward several sesquiterpene cyclases of higher plant and fungal origin . Although the antibody preparation was highly selective for denatured limonene cyclase from Mentha, the antibodies did not recognize the native protein in several different types of experiments . Nevertheless, specificity for the target enzyme was unambiguously demonstrated when the antibody preparation was shown to cross-react with the cyclase protein expressed in Escherichia coli that harbored the corresponding limonene synthase cDNA gene from M . spicata. Biochem Biophys Res Commun, 1993 Feb 15, 190(3), 794 - 800 Membrane association of leucyl-tRNA synthetase during leucine starvation in Escherichia coli; Williamson RM; The influence of leucine starvation on the subcellular location of leucyl-tRNA synthetase in Escherichia coli was examined in a leucine auxotrophic strain by sucrose density sedimentation analysis . Analysis of the subcellular distribution of leucyl-tRNA synthetase activity revealed that during unrestricted growth, the leucine synthetase enzyme activity was found to be localized in the soluble protein fraction of the gradient . However, during restricted growth on low levels of leucine, leucyl-tRNA synthetase activity was localized in the cytoplasmic membrane fraction of the gradient . The transition from soluble to membrane-association of the enzyme also occurred following inhibition of protein synthesis by treatment of cells with chloramphenicol . These results collectively suggest that leucyl-tRNA synthetase may be recruited to the cytoplasmic membrane in response to shortages of leucine or perturbation of protein synthesis. Biochem Biophys Res Commun, 1993 Feb 15, 190(3), 1066 - 72 pH-dependent decarboxylation of 2-amino-3-ketobutyrate, the unstable intermediate in the threonine dehydrogenase-initiated pathway for threonine utilization; Marcus JP et al.; 2-Amino-3-ketobutyrate can be readily formed enzymatically by the action of L-threonine dehydrogenase . A convenient assay for determining the half-life of this beta-keto acid is afforded by its rapid and quantitative conversion to glycine (+ acetyl CoA), as catalyzed by 2-amino-3-ketobutyrate CoA lyase . Using this system, we have found the half-life of 2-amino-3-ketobutyrate varies with pH from 8.6 minutes at pH 5.9 to 140 minutes at pH 11.1 yielding a theoretical titration curve that predicts a pKa value of 8.15 for the alpha-amino group of this intermediate . These data are considered relevant to discussions pertaining to a threonine dehydrogenase/2-amino-3-ketobutyrate CoA lyase enzyme complex in the threonine utilization pathway and to mechanistic aspects of the 5-aminolevulinate synthase-catalyzed reaction where 2-amino-3-ketoadipate is involved. Biochem J, 1993 Feb 15, 290 ( Pt 1), 279 - 87 Purification and characterization of 5-aminolaevulinic acid dehydratase from Escherichia coli and a study of the reactive thiols at the metal-binding domain; Spencer P et al.; 5-Aminolaevulinic acid dehydratase (ALAD) from a recombinant strain of Escherichia coli was purified to homogeneity . The enzyme is a homo-octamer of subunit M(r) 36554 +/- 17 . Enzyme activity was dependent on the presence of Zn2+ ions and an exogenous thiol . Two molar equivalents of Zn2+ are bound/mol of subunit under reducing conditions . On exposure to the metal chelator EDTA, the two Zn2+ ions are removed, giving an inactive metal-depleted apo-ALAD . On oxidation of holo-ALAD, two disulphide bonds are formed with the loss of 1 mol of Zn2+/mol of subunit . The formation of the first disulphide led to the loss of catalytic activity . Replacement of the two bound Zn2+ ions with Co2+ resulted in the formation of a green protein with a spectrum indicative of the presence of charge-transfer bands from one or more cysteine-Co2+ ligands . While Mg2+ could not activate apo-ALAD alone, it was able to substitute for the second molar equivalent of bound Zn2+, leading to a further 4-fold stimulation in activity . The four cysteine residues involved in the formation of the two disulphide bonds were identified by protein-chemistry studies and were all located in a region of the protein extending from amino acid residues 120-134 . Protein sequence data obtained in the present study has permitted the resolution of several differences between the published gene-derived protein sequences for ALAD from E . coli. Biochem J, 1993 Feb 15, 290 ( Pt 1), 15 - 9 The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase; Lewendon A et al.; A catalytically essential histidine residue (His-195) of chloramphenicol acetyltransferase (CAT) acts as a general base in catalysis, abstracting a proton from the primary hydroxy group of chloramphenicol . The pKa of His-195 has been determined from the pH-dependence of chemical modification . Both methyl 4-nitrobenzenesulphonate and iodoacetamide inactivate CAT by irreversible modification of His-195 . The kinetics of inactivation by methyl 4-nitrobenzenesulphonate are pseudo-first-order, and the pH-dependence of inactivation yields a pKa value of 6.60 . Iodoacetamide inactivation proceeds with second-order kinetics and a pKa value of 6.80 . An alternative site of modification at the active site of CAT is the thiol group of Cys-31, a residue which has no catalytic role . On replacement of Cys-31 with alanine (Ala-31 CAT), the pH-dependence of iodoacetamide inactivation gives a pKa value of 6.66 . The pKa values derived from chemical-modification experiments directed at His-195 are in agreement with the pKa values of 6.62 and 6.61 determined for wild-type and Ala-31 CAT respectively from the pH-dependence of kcat/Km. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1576 - 9 In vitro selection of optimal DNA substrates for T4 RNA ligase; Harada K et al.; We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 RNA ligase . We find that the ensemble of selected sequences ligated about 10 times as efficiently as the random mixture of sequences used as the input for selection . Surprisingly, the majority of the selected sequences approximated a well-defined consensus sequence. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1556 - 60 5' sequences are important positive and negative determinants of the longevity of Chlamydomonas chloroplast gene transcripts; Salvador ML et al.; We have found that sequences in the 5' leader of the Chlamydomonas chloroplast rbcL gene, when fused 5' to foreign genes, destabilize transcripts of these chimeric genes in the chloroplast of transgenic Chlamydomonas but that 5' sequences of the rbcL structural gene prevent this destabilization . Transcripts of the chloroplast rbcL gene are about equally abundant at all times in Chlamydomonas reinhardtii growing on an alternating 12-h light/12-h dark cycle . However, Chlamydomonas chloroplast transformants, harboring chimeric genes containing the same rbcL promoter with 63 or 92 bp of the rbcL 5' leader sequence fused upstream of the Escherichia coli uidA (beta-glucuronidase, GUS) gene, accumulated GUS transcripts only in the dark . Transcripts disappeared rapidly upon illumination of the cells . The same phenomenon was exhibited by transcripts of chimeric genes in which the GUS gene coding sequence was replaced by other unrelated genes . The precipitous light-induced drop in GUS transcript abundance was found to be due to an approximately 16-fold increase in the rate of degradation of GUS transcripts in light rather than to a decrease in the rate of transcription of the GUS gene . Transcripts of a chimeric rbcL-GUS construct in which the leader sequence of the rbcL gene was replaced by 103 bp of the leader sequence of the atpB gene were stable in illuminated cells . The destabilizing effect of the rbcL 5' leader sequence was reversed by adding 257 bp of the 5' coding region of the rbcL gene . The results show that chloroplast transcript levels in illuminated Chlamydomonas cells--and perhaps in other cases--can be determined, at least to some extent, by sequences and interactions of sequences transcribed from the 5' ends of genes. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1536 - 40 A Drosophila photoreceptor cell-specific protein, calphotin, binds calcium and contains a leucine zipper; Ballinger DG et al.; The calphotin protein, encoded by the calphotin (cap) gene, is expressed in the soma and axons of all Drosophila photoreceptor cells . It is expressed early in photo-receptor cell development, at the time when cell-type decisions are being made . Expression of calphotin is not altered by the glass mutation, which blocks photoreceptor cell development . The calphotin protein binds calcium and contains a long C-terminal leucine zipper . Potential implications of these properties are discussed. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1526 - 30 Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family; Steele FR et al.; Cultured pigment epithelial cells of the fetal human retina secrete a protein, pigment epithelium-derived factor (PEDF), that induces a neuronal phenotype in cultured human retinoblastoma cells . Morphological changes include the induction of an extensive neurite meshwork and the establishment of corona-like cellular aggregates surrounding a central lumen . The differentiated cells also show increases in the expression of neuron-specific enolase and the 200-kDa neurofilament subunit . Amino acid and DNA sequence data demonstrate that PEDF belongs to the serine protease inhibitor (serpin) family . The PEDF gene contains a typical signal-peptide sequence, initiator methionine codon, and polyadenylylation signal and matches the size of other members of the serpin superfamily (e.g., alpha 1-antitrypsin) . It lacks homology, however, at the putative serpin reactive center . Thus, PEDF could exert a paracrine effect in the embryonic retina, influencing neuronal differentiation by a mechanism that does not involve classic inhibition of serine protease activity. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1518 - 22 The short form of the CheA protein restores kinase activity and chemotactic ability to kinase-deficient mutants; Wolfe AJ et al.; Escherichia coli expresses two forms of the chemotaxis-associated CheA protein, CheAL and CheAS, as the result of translational initiation at two distinct, in-frame initiation sites in the gene cheA . The long form, CheAL, plays a crucial role in the chemotactic signal transduction mechanism by phosphorylating two other chemotaxis proteins: CheY and CheB . CheAL must first autophosphorylate at amino acid His-48 before transferring its phosphono group to these other signal transduction proteins . The short form, CheAS, lacks the N-terminal 97 amino acids of CheAL and, therefore, does not possess the site of autophosphorylation . Here we demonstrate that although it lacks the ability to autophosphorylate, CheAS can mediate phosphorylation of kinase-deficient variants of CheAL each of which retains a functional autophosphorylation site . This transphosphorylation enables these kinase-deficient CheAL variants to phosphorylate CheY . Because it mediates this activity, CheAS can restore to kinase-deficient E . coli cells the ability to tumble and, thus, to perform chemotaxis in swarm plate assays. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1474 - 8 Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy; Swain AL et al.; The crystal structure of Escherichia coli asparaginase II (EC 3.5.1.1), a drug (Elspar) used for the treatment of acute lymphoblastic leukemia, has been determined at 2.3 A resolution by using data from a single heavy atom derivative in combination with molecular replacement . The atomic model was refined to an R factor of 0.143 . This enzyme, active as a homotetramer with 222 symmetry, belongs to the class of alpha/beta proteins . Each subunit has two domains with unique topological features . On the basis of present structural evidence consistent with previous biochemical studies, we propose locations for the active sites between the N- and C-terminal domains belonging to different subunits and postulate a catalytic role for Thr-89. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1470 - 3 Identification of C-terminal amino acid residues of cauliflower mosaic virus open reading frame III protein responsible for its DNA binding activity; Mougeot JL et al.; We cloned in Escherichia coli truncated versions of the protein p15 encoded by open reading frame III of cauliflower mosaic virus . We then compared the ability of the wild-type p15 (129 amino acids) and the deleted p15 to bind viral double-stranded DNA genome . Deletions of > 11 amino acids in the C-terminal proline-rich region resulted in loss of DNA binding activity of wild-type p15 . Moreover, a point mutation of the proline at position 118 sharply reduced the interaction between the viral protein and DNA . These results suggest that cauliflower mosaic virus p15 belongs to the family of DNA binding proteins having a proline-rich motif involved in interaction with double-stranded DNA. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1397 - 401 Sequence conversion during postreplicative adenovirus overlap recombination; Bennett KL et al.; Sequence conversion efficiently transfers genetic information in high yield during postreplicative adenovirus overlap recombination . This process is intrinsically nonreciprocal, depends on adenovirus-specific strand-displacement replication by both partner molecules, and requires that complementary sequences on displaced strands must exceed a minimal length to form a heteroduplex intermediate. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1364 - 8 Evidence for functional interaction between elongation factor Tu and 16S ribosomal RNA; Powers T et al.; Translation of the genetic code requires the accurate selection of elongation factor (EF)-Tu.GTP.tRNA ternary complexes at the ribosomal acceptor site, or A site . Several independent lines of evidence have implicated the universally conserved 530 loop of 16S rRNA in this process; yet its precise role has not been identified . Using an allele-specific chemical probing strategy, we have examined the functional defect caused by a dominant lethal G-->A substitution at position 530 . We find that mutant ribosomes are impaired in EF-Tu-dependent binding of aminoacyl-tRNA in vitro; in contrast, nonenzymatic binding of tRNA to the A and P sites is unaffected, indicating that the defect involves an EF-Tu-related function rather than tRNA-ribosome interactions per se . In vivo, the mutant ribosomes are found in polysomes at low levels and contain reduced amounts of A-site-bound tRNA, but normal levels of P-site tRNA, in agreement with the in vitro results; thus the dominant lethal phenotype of mutations at G530 can be explained by impaired interaction of mutant ribosomes with ternary complex . These results provide evidence for a newly defined function of 16S rRNA--namely, modulation of EF-Tu activity during translation. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1315 - 9 RuvA and RuvB proteins of Escherichia coli exhibit DNA helicase activity in vitro; Tsaneva IR et al.; The SOS-inducible ruvA and ruvB gene products of Escherichia coli are required for normal levels of genetic recombination and DNA repair . In vitro, RuvA protein interacts specifically with Holliday junctions and, together with RuvB (an ATPase), promotes their movement along DNA . This process, known as branch migration, is important for the formation of heteroduplex DNA . In this paper, we show that the RuvA and RuvB proteins promote the unwinding of partially duplex DNA . Using single-stranded circular DNA substrates with annealed fragments (52-558 nucleotides in length), we show that RuvA and RuvB promote strand displacement with a 5'-->3' polarity . The reaction is ATP-dependent and its efficiency is inversely related to the length of the duplex DNA . These results show that the ruvA and ruvB genes encode a DNA helicase that specifically recognizes Holliday junctions and promotes branch migration. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1166 - 71 Two light-transducing membrane proteins: bacteriorhodopsin and the mammalian rhodopsin; Khorana HG; Site-directed mutagenesis has provided insight into the mechanisms of action of bacteriorhodopsin and rhodopsin . These studies are summarized here. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1146 - 53 Molecular recognition analyzed by docking simulations: the aspartate receptor and isocitrate dehydrogenase from Escherichia coli; Stoddard BL et al.; Protein docking protocols are used for the prediction of both small molecule binding to DNA and protein macromolecules and of complexes between macromolecules . These protocols are becoming increasingly automated and powerful tools for computer-aided drug design . We review the basic methodologies and strategies used for analyzing molecular recognition by computer docking algorithms and discuss recent experiments in which (i) substrate and substrate analogues are docked to the active site of isocitrate dehydrogenase and (ii) maltose binding protein is docked to the extracellular domain of the receptor, which signals maltose chemotaxis. J Biol Chem, 1993 Feb 15, 268(5), 3715 - 9 The regulatory domain of protein kinase C beta 1 contains phosphatidylserine- and phorbol ester-dependent calcium binding activity; Luo JH et al.; Certain isoforms of protein kinase C (PKC) require both Ca2+ and phospholipid for optimum activity . However, little is known about the nature of the interaction between PKC and Ca2+ . The present study demonstrates that the isolated regulatory domain of PKC beta 1, when synthesized as a fusion protein in Escherichia coli, binds 45Ca2+ with high affinity, but only in the presence of phosphatidylserine or 12-O-tetradecanoyl-phorbol-13-acetate . This binding is highly selective for Ca2+ since it is preferentially inhibited by excess non-radioactive Ca2+ when compared with the cations Mg2+, Mn2+, Na+, or K+ . It appears, therefore, that the binding of Ca2+ to PKC requires a complex tertiary structure in the regulatory domain. J Biol Chem, 1993 Feb 15, 268(5), 3616 - 24 The P1 plasmid partition complex at parS . II . Analysis of ParB protein binding activity and specificity; Funnell BE et al.; The P1 plasmid prophage is partitioned by a very high affinity protein complex at its partition site, parS, that contains the P1 ParB protein and Escherichia coli integration host factor (IHF) . ParB binds to regions of parS that flank the IHF binding site . In this report, we have examined the sequences to which ParB binds, the spatial relationship between them, and the effect of IHF on ParB binding patterns . Methylation protection and interference experiments were performed on supercoiled plasmids . Mutations that interfered with the action of both proteins in vivo were identified following random mutagenesis of parS . These studies revealed that ParB binds to a complicated, nonsymmetrical region in the right side of parS . ParB recognizes a partial copy of this sequence, TCGCCA, in the left side of parS with much lower affinity . The presence of IHF greatly facilitates the interaction of ParB with parS such that both sides bind with an equal affinity that is much greater than to either side alone . The stimulation by IHF is strongly influenced by helical phasing . These observations support the proposal that ParB is directed, by the bend created by IHF, to bind simultaneously to properly placed sequences flanking the IHF site. J Biol Chem, 1993 Feb 15, 268(5), 3586 - 93 Translocation of conjugated presecretory proteins possessing an internal non-peptide domain into everted membrane vesicles in Escherichia coli; Kato M et al.; Polypeptides comprising 20 amino acid residues (Y2) were covalently bound to the carboxyl terminus of a truncated proOmpA (proOmpA-D72C) through N,N'-bis(3-maleimidopropionyl)-2-hydroxy-1,3-propanediamine (X) . The length of the inverted linker domain was 2.8 nm . proOmpA-D72C-X-Y2 thus synthesized was subjected to in vitro translocation into everted membrane vesicles of Escherichia coli . The conjugated protein was translocation-competent in terms of both proteinase K resistance and signal peptide cleavage, when a proton motive force (delta microH+) was imposed . The translocation was ATP-dependent . The proteinase K-treatment resulted in the digestion of SecA, SecE, and SecY in the membrane, suggesting that the proteinase K resistance of the Y2 domain was not due to its interaction with these Sec proteins in the secretory machinery . In the absence of delta microH+, the translocation ceased at the linker domain . Upon the imposition of delta microH+, the linker-Y2 domain underwent translocation, which did not require ATP hydrolysis as in the case of the translocation of the latter portion of usual secretory proteins . The translocation was prevented by anti-Y2 IgG even when delta microH+ was imposed . Another conjugated protein, which possesses a polypeptide comprising 61 amino acid residues after the linker (proOmpA-D72C-X-Lpp'), was synthesized . This compound was also translocated into everted membrane vesicles with cleavage of the signal peptide . These results suggest that substances to be translocated through the secretory machinery need not necessarily be solely held together by polypeptide bonds. J Biol Chem, 1993 Feb 15, 268(5), 3507 - 13 Characterization of a DNA mismatch-binding activity in yeast extracts; Miret JJ et al.; An activity present in nuclear extracts of the yeast Saccharomyces cerevisiae binds specifically to oligonucleotides containing DNA mismatches, as judged by a band shift assay . The specificity of this activity for mismatched DNA was confirmed by competition experiments; binding to radiolabeled heteroduplexes was abolished in the presence of excess unlabeled heteroduplex but not when excess unlabeled homoduplex was added . Both T/G and T/- (single base deletion) mispairs were recognized in each of two sequence contexts . Binding was also observed with G/G, G/A, A/C, and T/C mismatches, but recognition of a C/C mispair was very weak . Competition studies with the various mismatches were consistent with the idea that a single activity recognizes all mispairs tested . Extracts from strains mutant in either or both of two putative mismatch recognition functions, MSH2 and MSH3, were also tested . Mismatch-binding activity was present in extracts of msh3- strains but completely absent in msh2- strains . The molecular weight of the major binding protein was estimated by UV cross-linking experiments to be approximately 110 kDa, in good agreement with the size predicted for Msh2 protein (Reenan, R . A . and Kolodner, R . D . (1992) Genetics 132, 963-973). J Biol Chem, 1993 Feb 15, 268(5), 3414 - 9 Functional organization of microtubule-associated protein tau . Identification of regions which affect microtubule growth, nucleation, and bundle formation in vitro; Brandt R et al.; Tau protein is a microtubule-associated protein that is almost exclusively expressed in the brain and is enriched in the axon . Determination of tau's sequence has revealed three to four tandem repeats that have been shown to constitute the microtubule binding site . In order to study the functional organization of tau, we prepared a series of truncated tau fragments using an Escherichia coli expression system . We assayed each fragment's activity in promoting growth of microtubules and in nucleating free microtubules . We found that tau's ability to nucleate microtubules requires the presence of additional sequence amino-terminal to that required for growth . We demonstrate that tau's carboxyl and amino termini differentially affect microtubule growth and nucleation . Finally, we show that in vitro microtubule bundle formation occurs when tubulin is assembled in the presence of an amino- and carboxyl-terminally truncated tau protein, whereas almost no bundling is observed in the presence of full-length tau or tau fragments that contain the amino terminus in addition to the repeat domain . We conclude that although the presence of the repeat domain promotes the growth of microtubules, the structural requirements for nucleation activity are more stringent . The differentiation between the growth promoting and nucleation activities on the structural level makes it possible for the two activities to be differentially regulated in vivo. J Biol Chem, 1993 Feb 15, 268(5), 3268 - 71 Expression and characterization of the heme-binding domain of Chlorella nitrate reductase; Cannons AC et al.; A recombinant protein corresponding to the putative heme-binding domain of assimilatory NADH:nitrate reductase from Chlorella vulgaris has been expressed and purified from transformed Escherichia coli BL21 cells . The recombinant protein, exhibited a subunit molecular mass of approximately 10 kDa with a N-terminal sequence beginning with the residues PAGA in agreement with that predicted by cDNA analysis . The UV-visible spectrum of the protein confirmed the incorporation of heme with maxima at 413 nm and 423, 528, and 557 nm for the oxidized and reduced forms, respectively . Circular dichroism spectra indicated the environment of the heme chromophore was very similar to that of the native enzyme . Potentiometric titrations of the recombinant heme domain yielded a midpoint potential of +16 mV (n = 1, pH 7), substantially higher than the values of -160 mV obtained for the native enzyme and -28 mV obtained for a previously expressed recombinant heme domain that contained part of the Mo-pterin domain . These results indicate that portions of the amino acid sequence that are involved in the formation of the Mo-pterin domain of Chlorella nitrate reductase influence the redox potential of the heme prosthetic group. J Biol Chem, 1993 Feb 15, 268(5), 3222 - 5 Only one of the charged amino acids located in the transmembrane alpha-helices of the gamma-aminobutyric acid transporter (subtype A) is essential for its activity; Pantanowitz S et al.; The gamma-aminobutyric acid (GABA) transporter (subtype A) is located in nerve terminals and catalyses coupled electrogenic uptake of the neurotransmitter with two or three sodium and one chloride ions . It contains 599 amino acids and 12 putative membrane spanning alpha-helices and is the first described member of a neurotransmitter transporter superfamily . The membrane domain contains 5 charged amino acids which are basically conserved . Using site-directed mutagenesis, we show that only one of them, arginine 69, is absolutely essential for activity . It is located in a highly conserved region encompassing parts of helices 1 and 2 . The three other positively charged amino acids and the only negative charged one, glutamate 467, are not critical . These results suggest that the translocation pathway of the sodium ions through the membrane does not involve charged amino acid residues and underline the importance of the highly conserved stretch between amino acids 66 and 86. J Biol Chem, 1993 Feb 15, 268(5), 3216 - 21 Mutagenesis of acidic residues in putative membrane-spanning segments of the melibiose permease of Escherichia coli . II . Effect on cationic selectivity and coupling properties; Zani ML et al.; Individual substitution of Cys or Asn for Asp-31, Asp-51, Asp-55, or Asp-120, distributed in different membrane spanning segments of the NH2-terminal domain of melibiose (mel) permease partially or completely inactivates Na(+)-linked sugar transport and stimulation of sugar binding on mel permease by Na+ ions (Pourcher, T., Zani, M.-L., and Leblanc, G . (1993) J . Biol . Chem . 268, 3209-3215) . To investigate further the effect of these substitutions on the cationic selectivity and coupling properties of mel permease, H(+)-melibiose coupled transport, coupling between H+ and melibiose movements, sugar counterflow, and zero-trans sugar efflux by the mutant permeases were analyzed . The results provide additional evidence indicating that manipulation of some of these Asp in the membrane-spanning segments of mel permease alters its cationic selectivity properties . The results also indicate that the individual mutations diversely affect mel permease-coupling properties . For example, only permease with Asn in place of Asp-31 or Cys in place of Asp-51 retains the capacity to actively transport melibiose . On the other hand, replacing Asp-55 by Cys produces uncoupling of cosubstrate flows by the carrier but does not hamper sugar translocation . These and other features of the mutant permeases are used to discuss the relative participation of Asp-31, Asp-51, Asp-55, or Asp-120 to the mel symport mechanism and to its ionic selectivity and also the existence of a possible gating mechanism that may contribute the obligatory coupling of cosubstrate flows by the symporter. J Biol Chem, 1993 Feb 15, 268(5), 3209 - 15 Mutagenesis of acidic residues in putative membrane-spanning segments of the melibiose permease of Escherichia coli . I . Effect on Na(+)-dependent transport and binding properties; Pourcher T et al.; Four aspartic acids, distributed in different putative membrane-spanning segments of the NH2-terminal domain of melibiose (mel) permease (D31 in helix I, D51 and D55 in helix II, and D120 in helix IV) were individually replaced by either Asn or Cys using site-directed mutagenesis . mel permease with either neutral residues at position 51, 55, or 120 or permease with a Cys in place of D31 does not catalyze significant Na(+)-linked methyl-1-thio-beta-D-galactopyranoside (TMG) accumulation . Binding studies of a high affinity ligand (p-nitrophenyl-alpha-D-galactopyranoside (NPG)) on de-energized membrane vesicles indicate that these modified transporters (i) retain the ability to bind the alpha-galactosides NPG or melibiose and the beta-galactoside TMG and (ii) exhibit a Na(+)-independent sugar-binding phenotype . In contrast, mel permease with an Asn residue at position 31 mediates Na(+)-coupled TMG transport and displays a Na(+)-dependent sugar binding phenotype, but requires a higher concentration of sodium than wild-type permease to produce maximal stimulation of sugar binding . The observation that individual mutation of the Asp residue at position 31, 51, 55, or 120 systematically and selectively modifies the contribution of the coupling ion to the early step of the transport reaction, i.e . cosubstrate binding, raises the possibility that (i) these 4 aspartic residues are at or near the cationic binding site of mel permease, (ii) the NH2-terminal domain of mel permease in which they are distributed accommodates or is part of the cationic binding site, and (iii) the oxygen atoms of these Asp side chains contribute to coordination of the coupling ion. J Biol Chem, 1993 Feb 15, 268(5), 3156 - 60 Domains near ATP gamma phosphate in the catalytic site of H+-ATPase . Model proposed from mutagenesis and inhibitor studies; Iwamoto A et al.; The beta Gly-149 residue is in a glycine-rich sequence (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr; residues 149-156) of the Escherichia coli H(+)-ATPase (ATP synthase) beta subunit . Substitution of beta Gly-149 by Ser suppressed the effect of the beta Ser-174-->Phe mutation (Iwamoto, A., Omote, H., Hanada, H., Tomioka, N., Itai, A., Maeda, M., and Futai, M . (1991) J . Biol . Chem . 266, 16350-16355), suggesting that beta Gly-149 is located near beta Ser-174 . In this study, we introduced different residues at position 149 and found that a single mutant beta Cys-149 was defective . The effect of beta Cys-149 mutation was suppressed by beta Gly-172-->Glu, beta Ser-174-->Phe, beta Glu-192-->Val, or beta Val-198-->Ala replacement . These results suggest that beta Gly-149, beta Gly-172, beta Ser-174, beta Glu-192, and beta Val-198 residues are located close together in the catalytic site . From these findings we propose a model of the catalytic site of the enzyme near the gamma phosphate moiety of ATP . F1 enzymes with the double mutations beta Cys-149/beta Glu-172, beta Cys-149/beta Phe-174, beta Cys-149/beta Val-192, and beta Cys-149/beta Ala-198 were less sensitive than wild-type F1 to dicyclohexylcarbodiimide and adenosine triphosphopyridoxal (an affinity analogue of ATP forming a Schiff base with the epsilon-amino group of beta Lys-155 or beta Lys-201), and became sensitive to N-ethylmaleimide in an ATP-protected manner . These results of inhibitor studies are consistent with the proposed model. J Biol Chem, 1993 Feb 15, 268(5), 3107 - 13 Inactivation of the recA protein by mutation of histidine 97 or lysine 248 at the subunit interface; Nguyen TT et al.; We have used site-directed mutagenesis to prepare two new mutant recA proteins, one in which histidine 97 has been replaced by alanine, and another in which lysine 248 has been replaced by alanine . Although these mutant proteins were originally designed from different considerations, they turned out to have remarkably similar properties . Both the {H97A}recA protein and the {K248A}recA protein bind poorly to single-stranded DNA, have no single-stranded DNA-dependent ATP hydrolysis activity, and do not promote renaturation of complementary single-stranded DNA molecules or the ATP-dependent three-strand exchange reaction . Furthermore, both mutant proteins are defective in Mg(2+)-induced helical filament formation . To account for these results, we propose that the mutation of either histidine 97 or lysine 248 alters subunit interactions between recA monomers and that this leads to the loss of cooperative single-stranded DNA binding and DNA pairing activities . This proposal is consistent with the recently determined x-ray structure of the recA protein, which shows that although histidine 97 and lysine 248 are distant from one another in the monomer structure, these two residues are on the opposing complementary faces of the recA subunit and pack against each other at the interface between adjacent recA monomers in the helical filament (Story, R . M., Weber, I . T., and Steitz, T . A . (1992) Nature 355, 318-325). J Biol Chem, 1993 Feb 15, 268(5), 3099 - 106 Glutamate synthase genes of the diazotroph Azospirillum brasilense . Cloning, sequencing, and analysis of functional domains; Pelanda R et al.; A 10-kilobase EcoRI fragment of Azospirillum brasilense genomic DNA was cloned in Escherichia coli . Two open reading frames of 4548 and 1446 base pairs (bp) were identified within the fragment as the structural genes for the alpha and beta subunits (gltB and gltD, respectively) of A . brasilense GltS . The organization of the gltBD region of A . brasilense differs from that of the corresponding region in E . coli: in A . brasilense, gltD is upstream relative to gltB, and its stop codon is separated by 141 bp from the first ATG of gltB . The deduced amino acid sequences reveal a high similarity with GltS from E . coli and with the ferredoxin-dependent GltS from maize . Binding domains for flavin cofactors and NADPH, a domain for glutamine binding and activation, and cysteine clusters for iron-sulfur centers formation were tentatively identified on the basis of sequence comparison with flavoproteins, pyridine nucleotide-dependent enzymes, amidotransferases, and iron-sulfur proteins. FEBS Lett, 1993 Feb 15, 317(3), 267 - 70 ATP-driven Na+ transport and Na(+)-dependent ATP synthesis in Escherichia coli grown at low delta mu H+; Avetisyan AV et al.; In inverted subcellular vesicles of Escherichia coli grown at high delta mu H+ (neutral pH, no protonophorous uncoupler), ATP-driven Na+ transport and oxidative phosphorylation are completely inhibited by the protonophore CCCP . If E . coli was grown at low delta mu H+, i.e . at high pH or in the presence of uncoupler, some oxidative phosphorylation was observed in the vesicles even in CCCP-containing medium, and Na+ transport was actually stimulated by CCCP . The CCCP-resistant transport and phosphorylation were absent from the unc mutant lacking F0F1 ATPase . Both processes proved to be sensitive to (i) the Na+/H+ antiporter monensin, (ii) the Na+ uniporter ETH 157, (iii) the F0 inhibitors DCCD and venturicidin, and (iv) the F1 inhibitor aurovertin . The CCCP-resistant oxidative phosphorylation was stimulated by Na+ and arrested by oppositely directed delta pNa . These data are consistent with the assumption that, under appropriate growth conditions, the F0F1-type ATPase of E . coli becomes competent in transporting Na+ ions. FEBS Lett, 1993 Feb 15, 317(3), 237 - 40 Synthesis of homocysteine thiolactone by methionyl-tRNA synthetase in cultured mammalian cells; Jakubowski H et al.; Homocysteine thiolactone is a product of an error-editing reaction, catalyzed by Escherichia coli and Saccharomyces cerevisiae methionyl-tRNA synthetases, which prevents incorporation of homocysteine into tRNA and protein both in vitro and in vivo . Here, homocysteine thiolactone is also shown to be synthesized by cultured mammalian cells such as human cervical carcinoma (HeLa), mouse renal adenocarcinoma (RAG), and Chinese hamster ovary (CHO) cells labeled with {35S}methionine, but not by normal human and mouse (Balb/c 3T3) fibroblasts . A temperature-sensitive methionyl-tRNA synthetase mutant of CHO cells, Met-1, does not make the thiolactone at the non-permissive temperature . The data indicate that methionyl-tRNA synthase is involved in synthesis of homocysteine thiolactone in CHO cells, thereby extending this important proofreading mechanism to mammalian cells. Arch Biochem Biophys, 1993 Feb 15, 301(1), 98 - 102 Effect of glutathione on aconitase in Escherichia coli; Gardner PR et al.; The effect of glutathione (GSH) on the superoxide-sensitive {4Fe-4S}-containing aconitase of Escherichia coli was explored . A mutant deficient in GSH biosynthesis, designated gshA, grew slower in a defined medium than did the parental strain and this effect was more pronounced when succinate was supplied as the carbon source in place of glucose . This suggested that the citric acid cycle was compromised in the gshA strain . Aconitase activity was approximately 25% lower in GSH-deficient cells growing on either glucose or succinate, and was lower still in strains producing less superoxide dismutase . Addition of GSH to the medium stimulated growth of the gshA strain on succinate . It also elevated the aconitase activity in the presence of chloramphenicol, which was added to block protein synthesis . Dithiothreitol and 2-mercaptoethanol were much less effective in this regard than was GSH . Exposure of cultures to 4.2 atm O2 caused a rapid decline in aconitase activity and this was the case in both GSH-proficient and GSH-deficient E . coli; however, the reactivation which was seen when the hyperoxic exposure was terminated was significantly impaired in the gshA strain . There is a dynamic balance between inactivation of aconitase by superoxide and reactivation by Fe(II) and this balance is altered in GSH-deficient E . coli . GSH may facilitate reactivation of aconitase, and of other {4Fe-4S}-containing dehydratases, by increasing the rate of transfer of Fe(II) to the {3Fe-4S} site. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1212 - 6 Histidine-226 is part of the pH sensor of NhaA, a Na+/H+ antiporter in Escherichia coli; Gerchman Y et al.; The nhaA gene of Escherichia coli, which encodes a pH-activated Na+/H+ antiporter, has been modified; six of its eight histidine codons were mutated to arginine codons by site-directed mutagenesis, yielding the mutations H254R-H257R (a double mutant), H226R, H39R, H244R, and H319R . In addition a deletion (delta nhaA1-14) lacking the remaining two histidines, His-3 and His-5, has been constructed . By comparing the phenotypes conferred by plasmids bearing the various mutations to the phenotype of the wild type upon transformation of strains NM81 (delta nhaA) or EP432 (delta nhaA, delta nhaB) we found that none of the NhaA histidines are essential for the Na+/H+ antiporter activity of the NhaA protein . However, the replacement of His-226 by Arg markedly changes the pH dependence of the antiporter . All mutants except H226R confer to NM81 and EP432 Na+ resistance up to pH 8.5 as well as Li+ resistance . Cells bearing H226R are resistant to Li+ and to Na+ at neutral pH, but they become sensitive to Na+ above pH 7.5 . Analysis of the Na+/H+ antiporter activity of membrane vesicles derived from H226R cells shows that the mutated protein is activated by pH to the same extent as the wild type . However, whereas the activation of the wild-type NhaA occurs between pH 7 and pH 8, that of H226R antiporter occurs between pH 6.5 and pH 7.5 . Furthermore, while the wild-type antiporter remains almost fully active at least up to pH 8.5, H226R is reversibly inactivated above pH 7.5, reaching 10-20% of the maximal activity at pH 8.5 . We suggest that His-226 is part of a pH-sensitive site that regulates the activity of NhaA. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1455 - 9 mRNA processing independent of RNase III and RNase E in the expression of the F1845 fimbrial adhesin of Escherichia coli; Bilge SS et al.; F1845, the fimbrial adhesin of a diarrhea-associated Escherichia coli, confers upon the bacteria the ability to adhere to cultured epithelial cells in a diffuse pattern . The fimbrial subunit gene, daaE, is encoded on a polycistronic mRNA which is processed endoribonucleolytically to produce a stable message encoding only daaE . The processing event occurs in bacterial strains with mutations in RNase III or RNase E, the only endoribonucleases which have been implicated in the processing of E . coli mRNA . Sequences encoding a stem-loop structure downstream of daaE play an essential role in determining the stability of the daaE mRNA . Rapid degradation of the sequences upstream of the cleavage site occurs upon processing, suggesting that processing of the F1845 polycistronic mRNA results in differential expression of genes involved in the biogenesis of fimbriae. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1160 - 5 A genetic approach to the generation of antibodies with enhanced catalytic activities; Lesley SA et al.; A hydrolytic catalytic antibody, generated against a nitrophenyl phosphonate transition state analogue, has been cloned and expressed in Escherichia coli for use as a model system to demonstrate the feasibility of using genetic selections to enhance catalytic activity . Conditions were found that permit the secretion of active recombinant antibody into the periplasm of a strain of E . coli deficient in the biotin biosynthetic genes (delta bio-gal) . A number of substrates were synthesized that, upon hydrolysis by the antibody, yield free biotin, which is required for cell growth . The substrates and selections can be used to identify mutants of the antibody with altered activities . This approach should be generalizable to a wide number of hydrolytic reactions including the selective cleavage of peptide, polysaccharide, phosphodiester, and ester bonds. Arch Biochem Biophys, 1993 Feb 15, 301(1), 77 - 84 Topological switching of the COOH-terminal domain of peptidylglycine alpha-amidating monooxygenase by alternative RNA splicing; Yun HY et al.; Proteins encompassing the two catalytic domains (monooxygenase and lyase) and the COOH-terminal domain of rat peptidylglycine alpha-amidating monooxygenase (rPAM)3 were purified from recombinant Escherichia coli overexpressing each domain and used to raise domain-specific polyclonal antibodies . Four alternatively spliced forms of PAM RNA (PAM-1, -2, -3, and -4) were transcribed in vitro and used to synthesize PAM proteins in a cell-free translation system . The orientation of the proteins in microsomal membrane vesicles was analyzed using trypsin protection assays and immunoprecipitation with the domain-specific antibodies . Only one of the two potential N-glycosylation sites (Asn765-Phe-Ser) in PAM-1 was efficiently utilized by microsomal membranes . PAM-1 and PAM-2 were shown to be type Ia membrane proteins with their two catalytic domains residing within microsomal vesicles and their COOH-terminal domains exposed to the cytosol . In contrast, PAM-3 and PAM-4 were shown to be soluble proteins contained entirely within vesicles . Thus, the COOH-terminal domain underwent topological switching between the cytosolic (PAM-1 and -2) and luminal (PAM-3) compartments as a function of alternative splicing of exons Ba/Bb . Computer analyses of the PAM protein sequence correlated the exons encoding PAM-1 with a model for the structural and functional domains of the PAM protein . The dual topologies of the PAM proteins confer an important means of functional regulation to this secretory granule associated neuropeptide processing enzyme. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1345 - 9 Vitamin D and adaptation to dietary calcium and phosphate deficiencies increase intestinal plasma membrane calcium pump gene expression; Cai Q et al.; The effect of vitamin D and other variables on the synthesis of the chicken intestinal plasma membrane calcium pump (PMCA) mRNA was assessed . The DNA probe for Northern analysis was obtained by reverse transcription and PCR with intestinal poly(A)+ RNA, using two 20-mer oligonucleotide primers homologous to the 3' coding region of the human teratoma PMCA . An EcoRI restriction fragment of the PCR product was cloned into the pBluescript II KS(-) phagemid vector, and the chimeric plasmid was used to transform Escherichia coli . The amino acid sequence deduced from the nucleotide DNA sequence of the PCR product and the cloned DNA were 96% homologous with the teratoma sequence . Northern blots of intestinal poly(A)+ RNA with 32P-labeled DNA showed the presence of three major species of chicken PMCA mRNAs at about 6.6, 5.4, and 4.5 kb . Northern analysis with the chicken PMCA DNA indicated that repletion of vitamin D-deficient chickens with vitamin D increased PMCA mRNAs in the duodenum, jejunum, ileum, and colon . After injection of 1,25-dihydroxyvitamin D3 intravenously into vitamin D-deficient chickens, duodenal PMCA mRNA tended to increase by 2 hr, reached a maximum at about 16 hr, and returned to baseline levels at 48 hr . Adaptation of chickens to either a calcium- or phosphorus-deficient diet resulted in a 2- to 3-fold increase in duodenal PMCA mRNA . These results indicate that vitamin D and specific variables that affect calcium absorption through the vitamin D-endocrine system increase intestinal PMCA gene expression. J Inorg Biochem, 1993 Feb 15, 49(3), 171 - 5 Metallobiochemistry of RNA . Co(NH3)6(3+) as a probe for Mg2+(aq) binding sites; Cowan JA; Co(NH3)6(3+) may serve as a probe of outer sphere complexation by Mg(H2O)6(2+) with RNA . The number of metal binding sites may be quantitatively assessed by optical spectroscopy (55-130, depending on background {K+}) . Novel coordination-induced features have been observed in circular dichroism spectra. J Biol Chem, 1993 Feb 15, 268(5), 3670 - 6 Molecular cloning of chicken myosin-binding protein (MyBP) H (86-kDa protein) reveals extensive homology with MyBP-C (C-protein) with conserved immunoglobulin C2 and fibronectin type III motifs; Vaughan KT et al.; The complete nucleotide sequence of a cDNA clone encoding the chicken skeletal muscle myosin-binding protein H (MyBP-H), formerly termed 86-kDa protein, has been established and the predicted amino acid sequence compared with other proteins entered into the GenBank data base . The full-length cDNA of 2066 base pairs contains a single open reading frame of 1611 base pairs encoding a muscle-specific protein of 58,487 Da . The predicted molecular weight differs significantly from the relative mobility of 86-kDa protein in reducing sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) . The full-length protein expressed in Escherichia coli also exhibits an anomalously slow mobility in SDS-PAGE; this gel retardation is a property of the N-terminal 24 kDa of the protein which contain two extended motifs of alternating alanine and proline residues, resembling the N terminus of skeletal muscle myosin light chain 1 (Nabeshima, Y . I., Fujii-Kuriyama, Y., Muramatsu, M., and Ogata, K . (1984) Nature 308, 333-338) . The C-terminal 40 kDa share 49.6% sequence identity and 17% conservative substitutions with chicken skeletal muscle MyBP-C (C-protein) (Einheber, S., and Fischman, D . A . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 2157-2161) . The protein contains four internal repeats of approximately 100 amino acids each, two of which bear significant resemblance to the C2 set of the immunoglobulin superfamily, and the other two are related to the type III fibronectin repeat . The arrangement of these repeats, -III-C2-III-C2-, is identical to that seen in the C-terminal 40-kDa section of MyBP-C . This repeat structure is implicated in myosin binding for the MyBP family . Finally, genomic Southern blots indicate that a single gene encodes fast skeletal muscle MyBP-H. J Biol Chem, 1993 Feb 15, 268(5), 3625 - 31 Mechanism of platelet-derived growth factor (PDGF) AA, AB, and BB binding to alpha and beta PDGF receptor; Fretto LJ et al.; The biological effects of platelet-derived growth factor (PDGF) are mediated by cell surface alpha and beta PDGF receptors, which, as a result of ligand binding, undergo dimerization in a manner consistent with PDGF being bivalent . In order to directly demonstrate PDGF bivalency and to define the binding of PDGF AB to isolated beta receptor, we developed solid-phase binding assays using purified recombinant extracellular domain of human PDGF receptors . PDGF AA, AB, and BB were prepared from the monomeric chains expressed in Escherichia coli, and each was purified to homogeneity; PDGF AB contained < 0.5% of either homodimer . The interactions of these isoforms with immobilized PDGF receptors were examined by several approaches . Scatchard analysis revealed high affinity binding (Kd = 0.5-1.0 nM) of radiolabeled PDGF AA and AB to alpha receptor and of PDGF BB to both receptor subtypes . Contrary to previous reports, PDGF AB also bound beta receptor with high affinity (Kd = 0.9 nM) . When a B-chain-specific monoclonal antibody that recognizes the putative binding domain of PDGF BB was used for ligand detection, we found that PDGF AB binding to beta receptor occurred exclusively through the B-chain subunit, whereas binding to alpha receptor occurred through either subunit . In addition, site-directed mutagenesis was used to specifically inactivate the B chain of PDGF AB, which eliminated binding to the beta receptor without affecting alpha receptor binding . These results establish that PDGF is bivalent and that monovalent ligand retains high affinity receptor binding. J Biol Chem, 1993 Feb 15, 268(5), 3282 - 8 Properties and sequence of a female-specific, juvenile hormone-induced protein from locust hemolymph; Zhang J et al.; In the fat body of Locusta migratoria, an RNA transcript of about 800 nucleotides has been detected that is specific to the adult female and dependent on induction by juvenile hormone (JH) or an analog . The corresponding cDNA has been cloned (lambda 21) and a 718-base pair sequence determined . It encodes a 196-amino acid polypeptide, including a signal peptide . An NH2-terminal sequence has 24 out of 28 amino acids identical with those of a previously described 19K locust hemolymph protein, but the remainder of the sequence shows no similarity . From adult female hemolymph, a 21-kDa protein, designated 21K protein, has been purified, with an NH2-terminal sequence exactly matching that deduced from clone lambda 21 . This 21K protein is found only in the adult female, is dependent on induction by JH, and is assumed to represent the product of the lambda 21 gene . It shows no immunochemical cross-reaction with locust 19K protein, apolipophorin III, nor with vitellogenin (Vg) . Its isoelectric point is pH 5.4; it contains some carbohydrate . 21K protein is synthesized in adult female fat body, accumulates in hemolymph, and is taken up into the developing oocytes in parallel with Vg . In locusts deprived of JH with precocene, production of 21K protein and of lambda 21-hybridizing transcripts is induced by the JH analog, methoprene, in parallel with Vg and its mRNA . Because of its sex-, stage-, and JH-dependent regulation, coordinate with Vg, the 21K protein will be valuable for analysis of gene expression. Blood, 1993 Feb 15, 81(4), 973 - 9 Endotoxin-induced tissue factor messenger RNA in human monocytes is negatively regulated by a cyclic AMP-dependent mechanism; Ollivier V et al.; Tissue factor (TF) is a transmembrane receptor that serves as the major cofactor for factor VIIa-catalyzed proteolytic activation of factors IX and X . In response to bacterial lipopolysaccharide (LPS), monocytes transcribe, synthesize, and express TF on their surface, thereby conveying to activated monocytes the ability to initiate the blood coagulation protease cascades . Agents that elevate cellular cyclic AMP (cAMP) inhibit the functional expression of TF by LPS-stimulated monocytes . In this study, we investigated the mechanism of this suppression . Northern blot analysis of total RNA from LPS-stimulated monocytes showed a concentration-dependent decrease in TF messenger RNA (mRNA) levels in response to dibutyryl-cAMP (dBt-cAMP) . TF mRNA and procoagulant activity were inhibited as early as 1 hour after the addition of dBt-cAMP and the inhibition persisted through 4 hours . Suppression of specific mRNA abundance was also observed with agents, including forskolin and iso-butyl-methyl-xanthine (IBMX), that increase cAMP levels by independent mechanisms . Flow immunocytometric analysis confirmed that cell-surface TF protein levels declined in parallel with TF functional activity . The rate of decay of TF mRNA after the arrest of transcription by actinomycin D was not altered by the addition of dBt-cAMP, IBMX, or forskolin, thus excluding effects on TF mRNA stability . We conclude that elevated cAMP levels suppress TF mRNA by reducing the rate of TF gene transcription. Gene, 1993 Feb 14, 124(1), 87 - 92 Physical and functional characterization of the ColS8 plasmid; Garcia ME et al.; The restriction map of the 5.1-kb colicinogenic plasmid ColS8 is reported . Transposon-insertion mutagenesis has been carried out to investigate the location of the various functional regions of this plasmid . Twenty-six independent ColS8::Tn1 insertions and six different deletion mutant plasmids were isolated, and the locations of the insertions and deletions were determined . The mapping of the transposon-insertion sites, together with characterization of the phenotypes of these mutants, permitted the localization of the regions of DNA involved in colicin production, colicin S8 immunity and mobilization by other plasmids . There is a polar effect in some mutants in which single insertions result in the non-expression of all three functions . In minicell preparations, the ColS8 plasmid directed the synthesis of colicin S8 (60 kDa) and a 14-kDa immunity protein . One deletion mutant with a colicin-less phenotype can synthesize colicin S8 in minicells, which placed the DNA region involved in colicin release between the colicin production and immunity regions . Both the 60-kDa and 14-kDa proteins are expressed from pColS8 in maxicell preparations. Gene, 1993 Feb 14, 124(1), 83 - 5 The pRSET family of T7 promoter expression vectors for Escherichia coli; Schoepfer R; A family of eight T7 promoter-based expression plasmids is presented . These are high-copy-number vectors featuring translational start and stop elements and a multiple cloning site (polylinker) with eleven unique restriction sites in all six reading frames . Depending on the cloning strategy used, recombinant proteins may contain either short vector-encoded fusion fragments or no fusion fragments at all . Following promoter induction, proteins are usually produced at a high level. Gene, 1993 Feb 14, 124(1), 57 - 65 Cloning, sequencing, and heterologous expression of a cellulase-encoding cDNA (cbh1) from Penicillium janthinellum; Koch A et al.; From a Penicillium janthinellum cDNA library, two clones with 1.8- and 1.9-kb inserts were isolated by hybridization to a Trichoderma reesei cellulase-encoding gene probe (egl1) . Both cDNAs have identical 5' ends and coding sequences, but different polyadenylation start points in their 3' untranslated regions . In the nucleotide (nt) sequence, one open reading frame of 537 amino acids was detected which shows 56% homology with endoglucanase I of T . reesei and 70% homology with cellobiohydrolase I of T . reesei, Phanerochaete chrysosporium, and Humicola grisea . Expression of the 1.9-kb cDNA in the Escherichia coli T7 system led to the detection of a 57-kDa protein, in agreement with the theoretical value . Fusion to the promoter of the yeast phosphoglycerokinase-encoding gene led to efficient expression and partial secretion of the cDNA-encoded cellulase with cellobiohydrolase I activity in Saccharomyces cerevisiae. Gene, 1993 Feb 14, 124(1), 29 - 35 Escherichia coli host strains SURE and SRB fail to preserve a palindrome cloned in lambda phage: improved alternate host strains; Doherty JP et al.; We have attempted to produce Escherichia coli strains with the optimal combination of host mutations required for the construction of genomic libraries in lambda and cosmid vectors . For lambda vectors, we defined this as a strain that combined high efficiency of phage plating with optimal tolerance to DNA methylation and the ability to propagate recombinants containing regions of potential secondary structure . To optimize this latter property, we have tested a series of strains for the ability to propagate a lambda phage containing a palindromic sequence . These included an mcr- derivative of a strain shown by Ishiura et al . {J . Bacteriol . 171 (1989) 1068-1074} to allow optimal stability of inserts in cosmid clones . All the sbcC strains allowed plaque formation of the palindrome-containing lambda phage . However, while the palindrome-containing phage plated with reasonable efficiency on SURE (recB sbcC recJ umuC uvrC) and SRB (sbcC recJ umuC uvrC), the majority of phage recovered from these strains no longer required an sbcC host for subsequent plating . These two strains also gave poorer titres with a low-yielding phage clone from the human Prader-Willi chromosome region . Optimal phage hosts appear to be those that are mcrA delta(mcrBC-hsd-mrr) combined with mutations in sbcC plus recBC or recD and without mutations in additional recombination functions such as recJ or recJ umuC uvrC (all of our E . coli strains are available on request). Gene, 1993 Feb 14, 124(1), 115 - 20 Isolation of a yeast essential gene, COF1, that encodes a homologue of mammalian cofilin, a low-M(r) actin-binding and depolymerizing protein; Iida K et a