Eur J Biochem, 1991 Jun 15, 198(3), 723 - 32 Preproabrin: genomic cloning, characterisation and the expression of the A-chain in Escherichia coli; Wood KA et al.; Synthetic oligonucleotides representing all possible sequences of an N-terminal and an internal region of the A-chain of abrin C were used to generate a probe specific for abrin-related sequences using the polymerase chain reaction on Abrus precatorius genomic DNA . A lambda phage library constructed from genomic DNA isolated from leaf tissue of A . precatorius was screened and positive hybridising clones were characterised by restriction enzyme analysis . The coding regions of unique clones were characterised by DNA sequencing . One clone encodes a preproprotein closely related to abrin C with 83% similarity between the A-chain sequences . Based on similarity with the ricin toxins and Ricinus communis agglutinin, the preproabrin consists of an A-chain of 251 amino acids preceded by 34 amino acids containing an N-terminal signal peptide, followed by a 14-amino-acid linker and a B-chain of 263 amino acids . The mature A-chain of the preproabrin has been expressed cytoplasmically in Escherichia coli and the soluble recombinant protein was produced at levels exceeding 6% of total cell protein . The recombinant A-chain has been purified to homogeneity and its ability to depurinate 28S rRNA in rat liver ribosomes has been demonstrated in vitro. Eur J Biochem, 1991 Jun 15, 198(3), 571 - 9 Structural organization of the multifunctional animal fatty-acid synthase; Witkowski A et al.; The amino acid sequence of the multifunctional fatty-acid synthase has been examined to investigate the exact location of the seven functional domains . Good agreement in predicting the location of interdomain boundaries was obtained using three independent methods . First, the sites of limited proteolytic attack that give rise to relatively stable, large polypeptide fragments were identified; cryptic sites for protease attack at the subunit interface were unmasked by first dissociating the dimer into its component subunits . Second, polypeptide regions exhibiting higher-than-average rates of non-conservative mutation were identified . Third, the sizes of putative functional domains were compared with those of related monofunctional proteins that exhibit similar primary or secondary structure . Residues 1-406 were assigned to the oxoacyl synthase, residues 430-802 to the malonyl/acetyl transferase, residues 1630-1850 to the enoyl reductase, residues 1870-2100 to the oxyreductase, residues 2114-2190 to the acyl-carrier protein and residues 2200-2505 to the thioesterase . The 47-kDa transferase and 8-kDa acyl-carrier-protein domains, which are situated at opposite ends of the multifunctional subunit, were nevertheless isolated from tryptic digests as a non-covalently associated complex . Furthermore, a centrally located domain encompassing residues 1160-1545 was isolated as a nicked dimer . These findings, indicating that interactions between the head-to-tail juxtaposed subunits occur in both the polar and equatorial regions, are consistent with previously derived electron-micrograph images that show subunit contacts in these areas . The data permit refinement of the model for the fatty-acid synthase dimer and suggest that the malonyl/acetyl transferase and oxoacyl synthase of one subunit cooperate with the reductases, acyl carrier protein and thioesterase of the companion subunit in the formation of a center for fatty-acid synthesis. J Biol Chem, 1991 Jun 15, 266(17), 11328 - 34 Mechanism of the sliding beta-clamp of DNA polymerase III holoenzyme; Stukenberg PT et al.; DNA polymerase III holoenzyme (holoenzyme), the multiprotein replicase of Escherichia coli, is essentially unlimited in processive DNA synthesis . Processive activity can be reconstituted from two components . One component, the beta preinitiation complex, is a beta dimer clamped onto primed DNA . The beta preinitiation complex is formed by the five-protein gamma complex, which hydrolyzes ATP to chaperone beta onto primed DNA . The other component is the alpha epsilon polymerase . The alpha epsilon polymerase itself is not processive, but is endowed with extremely high processive activity upon assembly with the beta preinitiation complex . Here we examine the mechanism by which the beta preinitiation complex confers processivity onto the alpha epsilon polymerase . We find the beta preinitiation complex to be mobile on DNA . Diffusion of beta on DNA is specific to duplex DNA, is bidirectional, does not require ATP, and appears to diffuse linearly along the duplex . Furthermore, beta directly binds the alpha epsilon polymerase through contact with alpha, the DNA polymerase subunit . Hence, the high processivity of the holoenzyme is rooted in a "sliding clamp" of beta on DNA that tethers the polymerase to the primed template . Implications for transcription and translation are discussed. J Biol Chem, 1991 Jun 15, 266(17), 11295 - 300 Trimerization of an in vitro synthesized OmpF porin of Escherichia coli outer membrane; Sen K et al.; The assembly of outer membrane proteins of Escherichia coli was examined using the OmpF porin as a model . Since this protein is made as a precursor, which is processed to a protein of Mr 37,000 before being assembled into trimers in the outer membrane, we synthesized a modified OmpF, which lacked 16 out of 22 amino acid residues from its signal sequence, in a coupled transcription-translation system . This modified protein resembled the unfolded, monomeric OmpF in its electrophoretic behavior, but much of the protein apparently existed in a more tightly folded conformation as it was recognized by a monoclonal antibody specific to a surface epitope of the native, trimeric OmpF porin . At least some conformers of this protein could be further incorporated into outer membrane or lipopolysaccharide bilayers, and assembled into trimers . The trimers formed were trypsin-resistant and heat-stable in sodium dodecyl sulfate up to 70 degrees C, thus showing the characteristics of the native trimeric protein . These results extend our earlier observation that OmpF monomer secreted by spheroplasts of E . coli can be trimerized in vitro (Sen, K., and Nikaido, H (1990) Proc . Natl . Acad . Sci . U.S . A 87, 743-747) and show that the trimerization can occur, albeit at a low efficiency, with porin monomers synthesized in vitro, presumably not contaminated by membrane fragments or other components of the cell envelope . However, comparison of trimerization efficiency of the nascent in vitro product with that of the same product already exposed to aqueous medium, as well as with that of the spheroplast-secreted product, leads us to the working hypothesis that the trimerization process in intact cells is accelerated either by accessory components or by the conformational changes accompanying the secretion through the cytoplasmic membrane and that the reactions observed in this study represent only part of the physiological process. J Biol Chem, 1991 Jun 15, 266(17), 11213 - 20 The C terminus of mouse ornithine decarboxylase confers rapid degradation on dihydrofolate reductase . Support for the pest hypothesis; Loetscher P et al.; Several years ago, we proposed that polypeptide regions rich in proline (P), glutamic acid (E), serine (S), and threonine (T) (PEST) target intracellular proteins for destruction (Rogers, S., Wells, R., and Rechsteiner, M . (1986) Science 234, 364-368) . To test the PEST hypothesis, we have produced chimeric proteins in which the N or C terminus of mouse dihydrofolate reductase is extended by the PEST-containing C terminus of mouse ornithine decarboxylase . Oligonucleotides encoding the 37 C-terminal residues of mouse ornithine decarboxylase (mODC) or equivalent lengths of dissimilar amino acids were inserted at appropriate sites in a dihydrofolate reductase (DHFR) expression vector . The various fusion proteins were expressed in Escherichia coli and purified to homogeneity by enzyme affinity chromatography . All purified fusion proteins exhibited similar abilities to convert dihydrofolate to tetrahydrofolate, thereby demonstrating that the attachment of peptide extensions to either terminus did not prevent the proper folding of DHFR . Metabolic stabilities of the radioiodinated fusion proteins were assayed in rabbit reticulocyte lysate or Xenopus egg extract . Proteolysis was found to be energy-dependent with mODC-DHFR fusion proteins being degraded from 2 to almost 40-fold faster than the parental DHFR molecule or DHFR fusion proteins bearing non-PEST extensions . Deletion of most of the PEST region from the mODC extension resulted in a significantly more stable fusion protein . Rapid proteolysis of DHFR proteins containing intact mODC extensions provides support for the PEST hypothesis. J Biol Chem, 1991 Jun 15, 266(17), 11116 - 21 Probing the functional role and localization of Escherichia coli ribosomal protein L16 with a monoclonal antibody; Nag B et al.; A monoclonal antibody specific for Escherichia coli ribosomal protein L16 was prepared to test its effects on ribosome function and to locate L16 by immunoelectron microscopy . The antibody recognized L16 in 50 S subunits, but not in 70 S ribosomes . It inhibited association of ribosomal subunits at 10 mM Mg2+, but not at 15 mM Mg2+ . Poly(U)-directed polyphenylalanine synthesis and peptidyltransferase activities were completely inhibited when the L16 antibody was bound to 50 S subunits at a molar ratio of 1 . There was no inhibitory effect on the binding of elongation factors or on the associated GTPase activities . Fab fragments of the antibody gave the same result as the intact antibody . Chemical modification of the single histidine (His13) by diethyl pyrocarbonate destroyed antibody binding . Electron microscopy of negatively stained antibody subunit complexes showed antibody binding beside the central protuberance of the 50 S particle on the side away from the L7/L12 stalk and on or near the interface between the two subunits . This site of antibody binding is fully consistent with its biochemical effects that indicate that protein L16 is essential for the peptidyltransferase activity activity of protein biosynthesis and is at or near the subunit interface. J Biol Chem, 1991 Jun 15, 266(17), 10982 - 8 Mutational analysis and protein engineering of receptor-binding determinants in human placental lactogen; Lowman HB et al.; Human placental lactogen (hPL) shares 85% sequence identity to human growth hormone (hGH) yet has some very different receptor-binding properties . For example, hPL binds 2300-fold weaker than hGH to the hGH receptor, yet these two hormones have similar affinities for prolactin receptors . We have expressed hPL in Escherichia coli, and we show that, like hGH, hPL requires zinc for tight binding to the extracellular domain of the human prolactin receptor (hPRLbp) . In fact, hPL contains virtually the same receptor-binding determinants and zinc ligands (His-18, His-21, and Glu-174) that hGH uses for coordinating zinc in the hGH.hPRLbp complex . As with hGH, mutation of Glu-174 to Ala in hPL reduces the affinity for the hPRLbp by 1400-fold . We can increase the affinity of hPL by over 200-fold for the hGHbp by installing four hGH receptor determinants that are not conserved in hPL . By simultaneously introducing E174A, we produced a pentamutant whose binding affinity for the hGHbp is only 1.6-fold weaker than hGH, but whose binding affinity for the hPRLbp is weaker by greater than 1000-fold relative to wild-type hPL . Thus, we have identified an hPRLbp epitope in hPL, "recruited" an hGHbp epitope into hPL, and produced receptor selective analogs of hPL that are designed to bind tightly to either, neither, or both receptors . Such variants should be important molecular probes to link specific receptor-binding, activation, and biological events. J Biol Chem, 1991 Jun 15, 266(17), 10959 - 66 Role of the tetrameric structure of Escherichia coli pyruvate oxidase in enzyme activation and lipid binding; Wang AY et al.; Pyruvate oxidase of Escherichia coli, an enzyme greatly activated by phospholipids, is a tetramer of a Mr 62,000 subunit . We have utilized the differing electrophoretic mobilities of several mutant oxidases on native polyacrylamide gels to study the role of the quaternary structure of the enzyme in the activation process . We found that when two poxB gene alleles coexisted in cells, heterotetrameric species were formed in addition to homotetramers . The concentration of each tetrameric species varied according to the concentration of the different subunits present, and the distribution seemed virtually identical to those expected from random mixing . We showed that the intrinsic activity of pyruvate oxidase was not affected by interactions among the four subunits . However, binding of the enzyme to lipids, a property required for function in vivo, required that a tetramer contain at least two subunits capable of lipid binding . Our data fit the model proposed previously (Grabau, C., Chang, Y.-Y., and Cronan, J . E., Jr . (1989) J . Biol . Chem . 264, 12510-12519) in which the carboxyl termini of two subunits interact to form a functional lipid-binding domain . We also have detected oxidase activity in a form of oxidase of unusually high electrophoretic mobility . This form seems to be either a monomeric or a dimeric form (more probably the former) of the oxidase subunit. J Biol Chem, 1991 Jun 15, 266(17), 10906 - 11 Expression in Escherichia coli and characterization of the heat-stable inhibitor of the cAMP-dependent protein kinase; Thomas J et al.; Pure heat-stable inhibitor of the cAMP-dependent protein kinase (PKI) has been isolated in high yield by using a bacterial expression vector constructed to synthesize the complete sequence of the rabbit muscle protein kinase inhibitor, plus an amino-terminal initiator methionine and glycine . Bacterially expressed PKI has an inhibitory activity identical to that of the protein isolated from rabbit skeletal muscle and, by gel filtration and gel electrophoresis, has the same physicochemical characteristics as the native physiological form of PKI . Fourier transformed infrared spectroscopy and CD establish that PKI has unusually large amounts of random coil and turn structures, with significantly smaller amounts of alpha-helix and beta structures. J Biol Chem, 1991 Jun 15, 266(17), 10893 - 8 Stabilization of the shikimate pathway enzyme dehydroquinase by covalently bound ligand; Kleanthous C et al.; Reversible binding of a ligand to an enzyme active site can elicit a variety of changes in the protein, such as conformational changes (close to the site of binding or communicated over long distances), changes in the ionization state of surrounding amino acid side chains, changes in the interaction of the target protein with other subunits (or other proteins), or even changes in the thermodynamic stability of the protein . Relatively little attention has been given to studying these effects in proteins to which the ligand has been irreversibly bound, yet this can be a convenient way of studying the effects of ligand binding in the absence of association/dissociation equilibria . We report the dramatic changes which occur to the shikimate pathway enzyme dehydroquinase when ligand is attached to its active site after borohydride reduction of the mechanistically important Schiff's base intermediates . The effects of this modification have been characterized by limited proteolysis, circular dichroism, guanidine hydrochloride denaturation, and differential scanning calorimetry . The conclusions from these studies are that although anchoring the ligand at the active site does not cause a gross change in conformation, it does increase markedly the conformational stability of the protein . This is conclusively established by three separate experiments: 1) the modified protein is completely resistant to proteases, whereas the unmodified protein is very susceptible to proteolysis; 2) the concentration of guanidine hydrochloride required to unfold the ligand-linked dehydroquinase is 3-4-fold greater than that of the unmodified protein; 3) the melting temperature (Tm) of the modified protein is 40 degrees C higher than that of the unmodified protein . These results are a very clear example of the thermodynamic link between ligand binding, conformational stability, and proteolytic susceptibility in vitro and will be a useful system for dissecting the contributions of individual protein-ligand interactions to these parameters. J Biol Chem, 1991 Jun 15, 266(17), 10830 - 8 In vivo interactions of the NahR transcriptional activator with its target sequences . Inducer-mediated changes resulting in transcription activation; Huang JZ et al.; The nahR gene from the NAH7 naphthalene degradation plasmid encodes a LysR-type transcriptional activator of the nah and sal promoters (Pnah and Psal, respectively) that responds to the inducer salicylate . In vivo methylation protection experiments with dimethyl sulfate showed that in the absence of inducer, NahR interacts in a similar manner with its target sites at Psal and Pnah . Both target sites also have very similar sequences comprised of a 4-base pair interrupted dyad containing two symmetrical guanines (-73 and -64 of Pnah; -71 and -62 of Psal), each located in adjacent major grooves on the same helical face, and both strongly protected by NahR . When inducer was present, several additional guanines of Pnah (-35, -45, and -58) and Psal (-42 and -40) became protected from methylation, while a guanine at -52 of Pnah became markedly enhanced for methylation, indicating that inducer and NahR-dependent interactions with these downstream sites of each promoter are quite different . Deletion of Psal sequences downstream of -30 did not affect its methylation patterns suggesting that NahR alone is responsible for the altered reactivities of these nucleotides . Similar in vivo methylation analyses with inducer-insensitive or inducer-independent NahR mutants also suggested that all alterations in methylation sensitivity are directly caused by NahR . It is more probable that the salicylate-induced reactivity changes result from direct NahR-guanine contacts which are required for, but not sufficient for transcription activation; however, they could also result from NahR-induced DNA contortions caused by upstream protein-DNA contacts. J Biol Chem, 1991 Jun 15, 266(17), 10820 - 4 Inactivation of DNA polymerase beta by in vitro phosphorylation with protein kinase C; Tokui T et al.; The Mr = 38,300 polypeptide of the purified recombinant rat DNA polymerase beta served as an excellent substrate for protein kinase C (PKC) in vitro but not for the catalytic subunit of cAMP-dependent protein kinase . The phosphorylation by PKC resulted in inactivation of DNA polymerase beta activity, and recovery was achieved by dephosphorylation with alkaline phosphatase . Since the phosphorylated DNA polymerase beta was retained with use of a single-stranded DNA-cellulose column, inactivation might occur at a site different from that for the DNA binding . Amino acid sequence analysis of the phosphopeptides revealed that the phosphorylated sites were 2 serine residues at positions 44 and 55 from the NH2 terminus, either or both of which might be involved in the catalytic activity of DNA polymerase beta . Thus, the inactivation of the DNA repair enzyme, DNA polymerase beta, by PKC may be an important process in the modification of DNA metabolism in the nucleus through signal transduction processes. J Biol Chem, 1991 Jun 15, 266(17), 10781 - 6 Structural and catalytic properties of a deletion derivative (delta 133-157) of Escherichia coli adenylate kinase; Rose T et al.; Escherichia coli adenylate kinase (AKe) as well as the enzyme from yeast and mitochondria differs from the muscle cytosolic variant (AK1) by an insertion of 25 amino acid residues that are missing in AK1 . The extra sequence, highly homologous in "large" size variants, is situated between residues 133 and 157 in AKe . Removal of 25 codons in the corresponding adk gene resulted in expression of a modified form of adenylate kinase (delta 133-157 AKe) which still conserved 7% of the maximal activity of the wild-type protein . The apparent Km for nucleotide substrates was increased by a factor of 4.6 (ADP), 23 (ATP) or 43 (AMP) in delta 133-157 AKe when compared with the wild-type enzyme . The secondary structure of delta 133-157 AKe, as well as its thermal stability were very similar to the parent protein . However, the deleted protein was much more sensitive than the wild-type enzyme to inactivation by trypsin . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of trypsin digested delta 133-157 AKe revealed accumulation of several well defined fragments which were not observed in the case of wild-type enzyme . We conclude that the additional sequence, although necessary for expression of full activity in AKe, is not critical for catalysis . It is perhaps responsible for interaction of enzyme with other cellular components although a different mechanism of water shielding for large and small size variants of AK can be also envisaged. J Biol Chem, 1991 Jun 15, 266(17), 10775 - 80 Signal transduction and osmoregulation in Escherichia coli . A novel type of mutation in the phosphorylation domain of the activator protein, OmpR, results in a defect in its phosphorylation-dependent DNA binding; Nakashima K et al.; The transcriptional factors, OmpR and EnvZ, are crucially involved in the osmotic regulation of ompF and ompC expression in Escherichia coli . The DNA binding ability of the positive regulator, OmpR, is modulated through its phosphorylation and dephosphorylation mediated by EnvZ in response to the medium osmolarity . In this study, two examples of a novel type of mutant ompR allele, ompR96A and ompR115S, whose phenotype is OmpF- OmpC- irrespective of the medium osmolarity, were characterized . These mutations result in amino acid conversions, Glu96 to Ala and Arg115 to Ser, respectively, within the phosphorylation domain of OmpR . Nevertheless, these mutant proteins were capable of undergoing phosphorylation and dephosphorylation normally, just like wild-type OmpR . However, the phosphorylation-dependent enhancement of their in vitro DNA binding ability was found to be severely affected . It was thus revealed that these mutant OmpR represent a novel type in terms of the mechanism of phosphorylation-dependent activation of the function of OmpR, i.e . those are normally phosphorylated but not activated to bind to the cognate promoter DNAs . In this respect, it was further suggested that OmpR oligomerization may be involved in the mechanism underlying the phosphorylation-dependent enhancement of the DNA binding ability of OmpR . The mutant proteins characterized in this study seem to be defective in this particular oligomerization process observed in vitro. J Biol Chem, 1991 Jun 15, 266(17), 10768 - 74 Characterization of Lrp, and Escherichia coli regulatory protein that mediates a global response to leucine; Willins DA et al.; Exogenous leucine affects the expression of a number of different operons in Escherichia coli . For at least some of these operons, the leucine-related effect is mediated by a protein called Lrp (Leucine-responsive regulatory protein) . The purification of Lrp to near homogeneity is described . Lrp is a moderately abundant, basic protein composed of two subunits of molecular mass 18.8 kDa each . In addition, the corresponding protein was purified from a strain having a mutation within the gene that encodes Lrp (lrp) . This mutation (lrp-1) causes high constitutive expression of ilvIH, one of the operons controlled by Lrp (Platko, J . V., Willins, D.A., and Calvo, J.M . (1990) J . Bacteriol . 172, 4563-4570) . The Lrp-1 and Lrp proteins have similar physical properties, but they show some differences in the characteristics with which they bind DNA upstream of the ilvIH promoter . The nucleotide sequences of the lrp and lrp-1 genes differ by only a single nucleotide, a C to G change that would substitute a Glu for an Asp at amino acid 114 . Lrp has some amino acid sequence similarity to AsnC, a protein that regulates asnA expression (Kolling, R., and Lother, H . (1985) J . Bacteriol . 164, 310-315). J Biol Chem, 1991 Jun 15, 266(17), 10735 - 8 A novel secreted cyclophilin-like protein (SCYLP); Spik G et al.; A novel cyclosporin A binding glycoprotein of 21 kDa was isolated from human milk by several steps of cation exchange chromatography . The corresponding gene was cloned from human T cells, expressed in Escherichia coli and the recombinant protein purified . The protein shares 58% amino acid identity with the cytosolic cyclophilin and is initially synthesized with a hydrophobic leader sequence . The cyclophilin-like protein has also peptidyl-prolyl cis/trans-isomerase activity, although less efficient, that is inhibited by cyclosporin A . The existence of a secreted form of cyclophilin-like protein in addition to the previously known cytosolic cyclophilin implies that these proteins act on different in vivo targets. J Biol Chem, 1991 Jun 15, 266(17), 10715 - 8 The tryptophan synthase alpha 2 beta 2 complex . Cleavage of a flexible loop in the alpha subunit alters allosteric properties; Miles EW; This study explores the catalytic and allosteric roles of a flexible loop in tryptophan synthase . Trypsin is known to cleave the tryptophan synthase alpha 2 beta 2 complex in an alpha subunit loop at Arg-188 . Cleavage yields an active "nicked" alpha 2 beta 2 derivative . The new results provide evidence that the alpha subunit loop serves two important roles: substrate binding and communicating the effects of substrate binding to the beta subunit . A role for the loop in substrate binding is supported by our finding that addition of a substrate analogue of the alpha subunit, alpha-glycerol 3-phosphate, decreases the rate of cleavage by trypsin . An allosteric role for the loop is supported by the finding although the native alpha 2 beta 2 complex is strongly inhibited by alpha-glycerol 3-phosphate, the nicked alpha 2 beta 2 complex is desensitized to this inhibition . The time course of proteolysis in the presence and absence of alpha-glycerol 3-phosphate is followed by sodium dodecyl sulfate-gel electrophoresis and by assays of activity in the presence and absence of alpha-glycerol 3-phosphate . We use spectroscopic measurements of the pyridoxal phosphate-L-tryptophan intermediates at the active site of the beta subunit to determine the affinity of the native and nicked enzymes for L-tryptophan and alpha-glycerol 3-phosphate . Although cleavage alters the equilibrium distribution of intermediates and reduces the affinity for alpha-glycerol 3-phosphate, it has little effect on the affinity for amino acids bound to the beta subunit . We conclude that the loop in the alpha subunit is important for ligand binding and for communicating the effects of ligand binding from the alpha subunit to the beta subunit in the alpha 2 beta 2 complex. FEMS Microbiol Lett, 1991 Jun 15, 65(2), 209 - 13 Escherichia coli accumulates the eukaryotic osmolyte taurine at high osmolarity; McLaggan D et al.; Escherichia coli accumulated taurine at high osmolarity via the ProU and ProP transport systems . Taurine accumulation was shown to be osmotically active as it displaced cytoplasmic K+ . In contrast to betaine and proline, taurine only modestly enhanced the growth rate of E . coli at high osmolarity and only if the cell was unable to synthesise trehalose . These studies show that taurine cannot be used as a tracer for the extra-cytoplasmic space of bacteria grown at high osmolarity. Gene, 1991 Jun 15, 102(1), 33 - 7 Characterization of phosphinothricin acetyltransferase and C-terminal enzymatically active fusion proteins; Botterman J et al.; Enhanced expression of the bialaphos resistance (bar) from Streptomyces hygroscopicus, which confers resistance to the herbicides bialaphos and phosphinothricin (PPT), has been obtained in Escherichia coli using a vector system based on translational coupling . The gene product, PPT acetyltransferase, was purified to homogeneity and its enzymatic properties were analyzed . Hybrid gene constructs with gene fragments fused to the 3'-terminus of bar yield fusion proteins having acetyltransferase activity, with a Michaelis constant for the PPT substrate comparable to the unmodified enzyme . The bar gene represents a selectable and assayable reporter gene especially suitable for 3'-terminal gene fusions. Gene, 1991 Jun 15, 102(1), 123 - 7 Sequence and expression in Escherichia coli of a Mycoplasma hominis gene encoding elongation factor Tu; Luneberg E et al.; We describe the molecular cloning and the complete nucleotide (nt) sequence of a Mycoplasma hominis gene common to a broad range of Mycoplasma species, as defined by hybridization analysis with the cloned gene . Production of M . hominis protein in Escherichia coli was assayed by use of a monoclonal antibody . The nt sequence analysis revealed a 1194-bp open reading frame that could encode a 43 516-Da protein . Computer-aided sequence comparison indicated that the gene codes for elongation factor Tu. Gene, 1991 Jun 15, 102(1), 1 - 6 Escherichia coli RecBCD enzyme: inducible overproduction and reconstitution of the ATP-dependent deoxyribonuclease from purified subunits; Boehmer PE et al.; The intracellular levels of the Escherichia coli RecBCD proteins have been amplified by fusing the recBCD genes to the strong tac promoter/operator in the expression vector, pKK223-3 . The overproduced proteins occur at levels amounting to approx . 10% of total cellular protein . Strains harbouring these overexpression plasmids have been used to purify the RecB . RecC and RecD protein subunits, as well as the RecBCD holoenzyme . The individually purified protein subunits can be used to reconstitute the ATP-dependent DNase activity of the RecBCD enzyme. Gene, 1991 Jun 15, 102(1), 19 - 26 Cloning of an aminoglycoside-resistance-encoding gene, kamC, from Saccharopolyspora hirsuta: comparison with kamB from Streptomyces tenebrarius; Holmes DJ et al.; An aminoglycoside-resistance-encoding gene (kamC) has been isolated from the sporaricin producer, Saccharopolyspora (Sac.) hirsuta, and expressed both in Streptomyces lividans and Escherichia coli . The pattern of resistance conferred by this gene was identical to that given by another gene (kamB) previously isolated from Streptomyces tenebrarius . In accordance with the known action of the kamB product, the Sac, hirsuta determinant also encodes a methyltransferase that modifies 16S rRNA, thereby rendering ribosomes refractory to certain aminoglycosides . The nucleotide sequences of both genes have been determined and comparison of the deduced amino acid sequences reveals a high degree of similarity. Proc Natl Acad Sci U S A, 1991 Jun 15, 88(12), 5124 - 8 Direct interaction between adenovirus E1A protein and the TATA box binding transcription factor IID; Horikoshi N et al.; Adenovirus E1A has long been known to activate/repress cellular and viral transcription . The transcriptional activity of nuclear extracts was depleted after chromatography on immobilized E1A protein columns that specifically retained the transcription factor (TF) IID . Stronger direct interactions between E1A and human TFIID than between E1A and yeast TFIID suggest that the unique sequences of the human protein may be involved . We have demonstrated that this interaction occurs directly between bacterially produced E1A and bacterially produced human TFIID in a protein blot assay . We propose that E1A protein may transduce regulatory signals from upstream activators to basal elements of the transcriptional machinery by contacting TFIID. J Biol Chem, 1991 Jun 15, 266(17), 11395 - 403 Both ATPase sites of Escherichia coli UvrA have functional roles in nucleotide excision repair; Thiagalingam S et al.; The roles of the two tandemly arranged putative ATP binding sites of Escherichia coli UvrA in UvrABC endonuclease-mediated excision repair were analyzed by site-directed mutagenesis and biochemical characterization of the representative mutant proteins . Evidence is presented that UvrA has two functional ATPase sites which coincide with the putative ATP binding motifs predicted from its amino acid sequence . The individual ATPase sites can independently hydrolyze ATP . The C-terminal ATPase site has a higher affinity for ATP than the N-terminal site . The invariable lysine residues at the ends of the glycine-rich loops of the consensus Walker type "A" motifs are indispensable for ATP hydrolysis . However, the mutations at these lysine residues do not significantly affect ATP binding . UvrA, with bound ATP, forms the most favored conformation for DNA binding . The initial binding of UvrA to DNA is chiefly at the undamaged sites . In contrast to the wild type UvrA, the ATPase site mutants bind equally to damaged and undamaged sites . Dissociation of tightly bound nucleoprotein complexes from the undamaged sites requires hydrolysis of ATP by the C-terminal ATPase site of UvrA . Thus, both ATP binding and hydrolysis are required for the damage recognition step enabling UvrA to discriminate between damaged and undamaged sites on DNA. J Biol Chem, 1991 Jun 15, 266(17), 11388 - 94 Deletion mutagenesis of the Escherichia coli UvrA protein localizes domains for DNA binding, damage recognition, and protein-protein interactions; Claassen LA et al.; The UvrA protein is the DNA binding and damage recognition subunit of the damage-specific UvrABC endonuclease . In addition, it is an ATPase/GTPase, and the binding energy of ATP is linked to dimerization of the UvrA protein . Furthermore, the UvrA protein interacts with the UvrB protein to modulate its activities, both in solution and in association with DNA, where the UvrAB complex possesses a helicase activity . The domains of the UvrA protein that sponsor each of these activities were localized within the protein by studying the in vitro properties of a set of purified deletion mutants of the UvrA protein . A region located within the first 230 amino acids was found to contain the minimal region necessary for interactions with UvrB, the UvrA dimerization interface was localized to within the first 680 amino acids, and the DNA binding domain lies within the first 900 amino acids of the 940-amino acid UvrA protein . Two damage recognition domains were detected . The first domain, which coincides with the DNA binding region, is required to detect the damage . The second domain, located on or near the C-terminal 40 amino acids, stabilizes the protein-DNA complex when damage is encountered. J Biol Chem, 1991 Jun 15, 266(17), 11163 - 9 Specific 33-residue repeat(s) of erythrocyte ankyrin associate with the anion exchanger; Davis LH et al.; Erythrocyte ankyrin contains an 89-kDa domain (residues 2-827) comprised almost entirely of 22 tandem repeats of 33 amino acids which are responsible for the high affinity interaction of ankyrin with the anion exchanger (Davis, L., and Bennett, V . (1990) J . Biol . Chem . 265, 10589-10596) . The question of whether the repeats are equivalent with respect to binding to the anion exchanger was addressed using defined regions of erythrocyte and brain ankyrins expressed in bacteria . The conclusion is that the repeats are not interchangeable and that the 44 residues from 722 to 765 are essential for high affinity binding between erythrocyte ankyrin and the anion exchanger . Residues 348-765 were active whereas a polypeptide of the same size (residues 305-721) but missing the 44 residues was not active . The difference between the active and inactive polypeptides was not caused by the degree of folding based on circular dichroism spectra . The 44 residues from 722 to 765 were not sufficient for binding since deletions of residues from 348 to 568 resulted in a 10-fold loss of activity . However, the role of residues 348-568 may be at the level of folding rather than a direct contact since the deleted sequences were not active in the absence of 722-765 and since circular dichroism spectra revealed significant loss of structure in the smaller polypeptides . Further evidence that the 33-residue repeats are not equivalent in ability to bind to the anion exchanger is that a region of human brain ankyrin containing 18 33-residue repeats with 67% overall sequence identity to erythrocyte ankyrin was 8-fold less active than a region of erythrocyte ankyrin containing only 12 repeats . The fact that the anion exchanger binds to certain repeats suggests that the other 33-amino acid repeats could interact with proteins distinct from the anion exchanger and provide ankyrin with the potential for considerable diversity in association with membrane proteins as well as cytoplasmic proteins . Tubulin was identified as one example of a protein that can interact with ankyrin repeats that are not recognized by the anion exchanger. Proc Natl Acad Sci U S A, 1991 Jun 15, 88(12), 5462 - 6 Identification of an immunodominant epitope within the capsid protein of hepatitis C virus; Nasoff MS et al.; We have isolated cDNA clones from the 5' end of the Hutchinson strain of hepatitis C virus . Sequences encoding various segments of the HCV structural region were fused to the gene for glutathione S-transferase and analyzed for the expression of hepatitis C virus-capsid fusion proteins . With a set of these fusion proteins, both human and chimpanzee immune responses to capsid were studied . An immunodominant epitope was located within the amino-terminal portion of capsid that is preferentially recognized by antibodies in both human and chimpanzee hepatitis C virus-positive sera . In addition, analyses of sequential serum samples taken from humans and chimpanzees with either chronic or apparently self-limited infections revealed that a strong anti-capsid response develops rapidly after onset of infection. Proc Natl Acad Sci U S A, 1991 Jun 15, 88(12), 5262 - 66 The p15 carboxyl-terminal proteolysis product of the human immunodeficiency virus type 1 reverse transcriptase p66 has DNA polymerase activity; Hafkemeyer P et al.; The reverse transcriptase of human immunodeficiency virus type 1 is a heterodimeric protein consisting of two polypeptides with masses of 66 and 51 kDa and has, as a second enzymatic activity, RNase H activity . The 66-kDa polypeptide can be cleaved by the virus-encoded protease to yield polypeptides of 51 and 15 kDa . The latter has been characterized as possessing RNase H activity {Hansen, J., Schultze, T., Mellert, W . & Moelling, K . (1988) EMBO J . 7, 239-243} . We have purified simultaneously the heterodimeric reverse transcriptase/RNase H containing the 66/51-kDa polypeptides and the 15-kDa RNase H from Escherichia coli containing the expression vector pJS 3.7 by a procedure including chromatography on DEAE-cellulose, phosphocellulose, and heparin-Sepharose . Two RNase H and reverse transcriptase peaks were separated on phosphocellulose, one coinciding with the heterodimeric protein and the other with the 15-kDa protein . On the basis of the following findings it appears that the 15-kDa polypeptide has both RNase H and reverse transcriptase activities: (i) it copurified with both activities; (ii) it functioned as a reverse transcriptase in an in situ assay after SDS/polyacrylamide gel electrophoresis; (iii) polyclonal antibodies raised against the 66-kDa polypeptide reacted in immunoblots exclusively with a 15-kDa polypeptide, reacted in immunoblots exclusively with a 15-kDa polypeptide, while no immunoreactive bands in the range of 51-66 kDa were seen in the 15-kDa polypeptide preparation; (iv) the p15 and the p66/51 reverse transcriptase could be quantitatively pelleted in an enzymatically active form only when antibodies specific for the p66 carboxyl terminus were used; and (v) the p15 protein had bona fide properties of a reverse transcriptase and could enzymatically synthesize a high molecular weight, alkali-resistant product . The two reverse transcriptases appear to have different behaviors on various template/primer systems tested . Conceivably different forms of human immunodeficiency virus type 1 reverse transcriptases might be used in individual steps of (+)- and (-)-strand replication. Eur J Biochem, 1991 Jun 15, 198(3), 793 - 9 Rat liver dimethylglycine dehydrogenase . Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone; Lang H et al.; Dimethylglycine dehydrogenase (Me2GlyDH), an enzyme of choline catabolism specifically expressed in the mammalian liver, was analyzed in rat hepatocytes in culture . This mitochondrial enzyme carries the FAD cofactor covalently attached to the polypeptide chain by its riboflavin 8 alpha position to N pi of histidine {Cook, R., Misono, K.S . & Wagner, C . (1980) J . Biol . Chem . 259, 12475-12480} . Subcellular fractionation of {14C}riboflavin-labelled hepatocytes and immunoprecipitation with Me2GlyDH-specific antiserum identified a {14C}riboflavin-labelled polypeptide of the size of mature Me2GlyDH only in the mitochondrial fraction . Immunoprecipitation of extracts from {35S}Met-labelled hepatocytes revealed a putative precursor protein to the mature Me2GlyDH in the cytoplasmic fraction . These Me2GlyDH polypeptides were not expressed in cells of the rat hepatoma cell line FAO . A Me2GlyDH cDNA clone of apparent full length was isolated from a rat liver cDNA bank constructed in the plasmid vector pcD-X {Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota, T . & Arai, K . (1987) Methods Enzymol . 154, 3-28} . The nucleotide sequence of the cDNA contains an open reading frame encoding a protein of 96059 Da . This molecular mass agrees well with the migration on SDS/PAGE of the assumed Me2GlyDH precursor immunoprecipitated from the cytoplasm of {35S}Met-labelled cells . Proteolytic cleavage at the putative mitochondrial processing protease-recognition site Arg(-2)-Ala(-1)-Glu(+1) would lead to the formation of a protein of 91391 Da, which is in good agreement with the estimated 90 kDa of mature Me2GlyDH {Wittwer, A.J . & Wagner, C . (1981) J . Biol . Chem . 256, 4102-4108}, and a 43-amino-acid leader peptide . The N-terminus of Me2GlyDH contains a conserved amino acid sequence which forms the dinucleotide-binding site in many enzymes with noncovalently bound FAD . Close to the modified histidine there is an amino acid sequence resembling a sequence conserved in thymidylate synthases and shown in these enzymes to be involved in the binding of the pteroyl polyglutamate cofactor. FEMS Microbiol Lett, 1991 Jun 15, 65(2), 233 - 7 Production of a mannose-resistant fibrillar haemagglutinin by strains of Escherichia coli of EPEC serotype O111:H12; Yakubu DE et al.; Two strains of Escherichia coli of serotype O111:H12 produced a mannose-resistant haemagglutinin (MREHA) of Duguid's pattern 7 that reacted strongly with the red cells of ox and sheep . These strains also adhered to HEp2-epithelial cells and formed fibrillae demonstrable by negative staining and immunogold labelling. Gene, 1991 Jun 15, 102(1), 87 - 91 Two promoters control the aroH gene of Escherichia coli; Hudson GS et al.; The aroH gene from Escherichia coli encodes 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (Trp), one of three isoenzymes which catalyse the first committed step in the biosynthesis of aromatic amino acids and vitamins . S1 mapping and primer extension analysis of in vivo transcripts revealed the presence of two nonoverlapping promoters for aroH . The more distal of these has been described previously and is negatively regulated by the trp repressor . The second promoter is active under conditions of growth in rich medium, and may be involved in ensuring sufficient levels of precursors for the biosynthesis of aromatic vitamins under these growth conditions. Gene, 1991 Jun 15, 102(1), 141 - 2 Polymorphism in the dgt-dapD-tsf region of Escherichia coli K-12 strains; Degryse E; Restriction analysis of the dapD region cloned from several strains of Escherichia coli, revealed a restriction-fragment length polymorphism (RFLP) . This RFLP, which includes the BamHI, EcoRI and SalI sites, may be useful in classification of various E . coli strains. Proc Natl Acad Sci U S A, 1991 Jun 15, 88(12), 5457 - 61 A Tn3 derivative that can be used to make short in-frame insertions within genes; Hoekstra MF et al.; A Tn3 derivative was constructed to make small in-frame insertions within genes . The transposon contains the URA3 gene, the tetA gene, a truncated lacZ, and phage P1 loxP recombination sites at either end . Insertions that have fused lacZ to an open reading frame are lac+ because they express the truncated lacZ . In the presence of the phage P1 cyclization recombinase cre, the transposon can delete the URA3, tetA, and lacZ genes between the two loxP sites . The remaining short imperfect palindrome contains the ends of Tn3 and a loxP site and does not contain a translational termination codon in the correct reading frame . We have analyzed several insertions within the yeast HO gene . Several insertions inactivate HO and prohibit initiation of mating-type switching . In contrast, an epitope inserted in the central portion encodes a functional HO endonuclease. Eur J Biochem, 1991 Jun 15, 198(3), 789 - 92 Orientation of the haems of the ubiquinol oxidase:O2 reductase, cytochrome bo of Escherichia coli; Salerno JC et al.; The orientation of the two haems of the Escherichia coli ubiquinol oxidase:O2 reductase, cytochrome bo, has been determined by electron paramagnetic resonance studies on oriented multilayer preparations of cytoplasmic membrane fragments . The enzyme contains a low-spin b-like haem and a high-spin b-like haem, designated cytochromes b and o respectively . Both haems are oriented with their planes perpendicular to the membrane plane, further extending the catalogue of structural and functional similarities between this enzyme and the mammalian cytochrome c oxidase, cytochrome aa3. J Biol Chem, 1991 Jun 15, 266(17), 11380 - 7 Construction of deletion mutants of the Escherichia coli UvrA protein and their purification from inclusion bodies; Claassen LA et al.; The functions of each of the three subunits of the damage-specific UvrABC endonuclease is currently being studied by systematically mutagenizing the corresponding genes to generate mutant proteins for characterization in vitro . In this communication, we describe the construction of C-terminal deletion mutants of the UvrA protein and a procedure to purify the mutant and wild-type UvrA proteins from inclusion bodies in cells overexpressing the recombinant proteins . The method yields highly purified proteins with between 10 and 50% of the specific activity of wild-type UvrA purified by conventional techniques from the soluble fraction . The wild-type UvrA protein purified by this method had the properties of significant and selective loss of activity in assays of incision of damaged DNA, while still retaining high levels of the other unique molecular phenotypic properties associated with intact UvrA . Furthermore, the demonstration of the absolute requirement for zinc during refolding for recovery of activity is the first evidence that the zinc previously shown to be associated with the UvrA protein is in fact a necessary component for its function. J Biol Chem, 1991 Jun 15, 266(17), 11372 - 9 Site-specific interaction of vaccinia virus topoisomerase I with duplex DNA . Minimal DNA substrate for strand cleavage in vitro; Shuman S; Purified vaccinia virus DNA topoisomerase I forms a cleavable complex with duplex DNA at a conserved sequence element 5'(C/T)CCTTdecreases in the incised DNA strand . DNase I footprint studies show that vaccinia topoisomerase protects the region around the site of covalent adduct formation from nuclease digestion . On the cleaved DNA strand, the protected region extends from +13 to -13 (+1 being the site of cleavage) . On the noncleaved strand, the protected region extends from +13 to -9 . Similar nuclease protection is observed for a mutant topoisomerase (containing a Tyr ---- Phe substitution at the active site amino acid 274) that is catalytically inert and does not form the covalent intermediate . Thus, vaccinia topoisomerase is a specific DNA binding protein independent of its competence in transesterification . By studying the cleavage of a series of 12-mer DNA duplexes in which the position of the CCCTTdecreases motif within the substrate is systematically phased, the "minimal" substrate for cleavage has been defined; cleavage requires six nucleotides upstream of the cleavage site and two nucleotides downstream of the site . An analysis of the cleavage of oligomer substrates mutated singly in the CCCTT sequence reveals a hierarchy of mutational effects based on position within the pentamer motif and the nature of the sequence alteration. J Biol Chem, 1991 Jun 15, 266(17), 11289 - 94 Overproduction and purification of a functional Na+/H+ antiporter coded by nhaA (ant) from Escherichia coli; Taglicht D et al.; A Na+/H+ antiporter coded by the nhaA (ant) gene of Escherichia coli has been overproduced and purified . The amino-terminal sequence of the protein has been determined and shown to correlate with initiation at a GUG codon, 75 bases upstream from the previously suggested AUG initiation codon . The purified protein, when reconstituted into proteoliposomes, has Na+/H+ antiport activity . It can mediate sodium uptake when a transmembrane pH gradient is applied . Downhill sodium efflux is shown to be highly dependent on pH and is accelerated by a transmembrane pH gradient . An imposed membrane potential negative inside accelerates Na+ efflux at all pH values tested . These findings suggest that the antiporter is electrogenic both at acid and alkaline pH . The activation at alkaline pH values (2000-fold increase) is consistent with the proposed role of the antiporter in regulation of internal pH at the alkaline pH range. J Biol Chem, 1991 Jun 15, 266(17), 11058 - 62 Characterization of an extended form of recombinant human insulin-like growth factor II; Hammarberg B et al.; To investigate the biological role of variants of human insulin-like growth factor II (IGF-II), an extended form designated IGF-IIE21, with a molecular mass of 9.8 kDa, was produced in Escherichia coli as a stable and soluble secreted fusion protein . After site-specific cleavage of the affinity purified fusion protein, followed by purification using ion exchange and reversed phase chromatography, it could be demonstrated that IGF-IIE21 and IGF-II have similar or identical activities according to radioimmunoassay and radioreceptor assay . However, IGF-IIE21 showed only 1% growth promotion activity as compared with IGF-II in a clonal expansion assay using human K562 cells which lacks IGF-I receptors . These results suggest that this extended variant of IGF-II can bind to the receptor but has limited growth promoting activity. J Biol Chem, 1991 Jun 15, 266(17), 10967 - 73 The use of gene fusions to examine the membrane topology of the L-subunit of the photosynthetic reaction center and of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides; Yun CH et al.; The topology of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides has been examined by generating gene fusions with alkaline phosphatase . Gene fusions were generated at random locations within the fbcB gene encoding the cytochrome b subunit . These fusion products were expressed in Escherichia coli and were screened for alkaline phosphatase activity on chromogenic plates . 33 in-frame fusions which showed activity were further characterized . The fusion junctions of all those fusions which had a high specific activity were clustered in three regions of the cytochrome b polypeptide, and thus these regions were tentatively assigned as being near the periplasmic surface . The data are consistent with a model containing eight transmembrane helices . In order to explore the validity of the gene fusion approach for a protein not normally expressed in E . coli, the topology of the L-subunit of the photosynthetic reaction center from R . sphaeroides was also explored using phoA gene fusions . A similar protocol was used as with the cytochrome b subunit . The gene fusions with high specific activity were shown to be in regions of the L-subunit polypeptide known to be at or near the periplasmic surface, as defined by the high resolution structure determined by X-ray crystallography . These data demonstrate the utility of this approach for determining membrane protein topology and extend potential applications to include at least some proteins not normally expressed in E . coli. Biochem Pharmacol, 1991 Jun 15, 41(12), 1839 - 48 Molecular features necessary for the uptake of diamines and related compounds by the polyamine receptor of rat lung slices; O'Sullivan MC et al.; The influence of 17 putrescine analogues on the uptake of putrescine and/or paraquat by rat lung slices has been determined . Most of these compounds are competitive inhibitors of putrescine and/or paraquat uptake, but three show no inhibiting activity . Apparent Ki values of the putrescine derivatives increase, and thus the inhibitory effects decrease, with increasing N-methylation . Comparison of N-methyl-1,4-diaminobutane (Ki = 8 microM) with N,N'-bis-methyl-1,4-diaminobutane (Ki = 25.5 microM) shows that a single primary amino group is desirable for high inhibiting activity . Dimethylation at one amino function does not greatly decrease inhibitory potential (thus N,N-dimethyl-1,4-diaminobutane has Ki = 11.5 microM) . Increasing the size of N-alkyl substituents in putrescine derivatives, decreased their inhibitory action on the uptake of putrescine . Investigation of the effect of conformationally-restricted analogues of putrescine shows that both (E) and (Z) isomers of 1,4-diaminobut-2-ene are poor inhibitors of putrescine uptake . Analogues of putrescine with bulky substituents on the butyl chain, i.e . the meso- and rac-isomers of 1,1-dichloro-2,3-diaminomethylcyclopropane, do not inhibit putrescine uptake . Inhibiting putrescine derivatives which contain aziridine groups are competitive inhibitors of putrescine and paraquat uptake . Surprisingly, N-(4-aminobutyl)aziridine is the most effective inhibitor of putrescine uptake studied, and is a better inhibitor of paraquat uptake than the endogenous polyamine, putrescine . N-(4-Aminobutyl)aziridine binds reversibly to the polyamine transporter and its inhibitory effects do not appear to be due to any cytotoxic activity of the aziridine . The parameter A (mM)-1 defined as 1000/Ki (where Ki units are microM) was taken as a measure of the affinity of a compound for the polyamine receptor in this paper. Biochem Biophys Res Commun, 1991 Jun 14, 177(2), 745 - 50 Correlation between spermine stimulation of rat liver Ile-tRNA formation and structural change of the acceptor stem by spermine; Kusama-Eguchi K et al.; We have recently reported that the interaction of spermine with the acceptor and anticodon stems may be important for spermine stimulation of rat liver Ile-tRNA formation {Peng, Z . et al . (1990) Arch . Biochem . Biophys . 279, 138-145} . To pinpoint which interaction of spermine is more important for spermine stimulation of Ile-tRNA formation, Ile-tRNA formation and ribonuclease V1 sensitivity of tRNA(Ile) were studied using purified tRNAs(Ile) from rat liver, wheat germ, brewer's yeast, torula yeast and Escherichia coli . The results indicate that spermine stimulation of rat liver Ile-tRNA formation correlated with the structural change of the acceptor stem by spermine . The nucleotide sequence of wheat germ tRNA(Ile) was also determined. Biochem Biophys Res Commun, 1991 Jun 14, 177(2), 597 - 602 cDNA cloning of the rat O6-methylguanine-DNA-methyltransferase; Rahden-Staron I et al.; A cDNA expression library was constructed from a rat hepatoma cell line ( H4 cells ) and introduced into an Escherichia coli strain ( BK2110 ) deficient in the repair of O6-methylguanine residues . Following three exposures to N-methyl-N'-nitro-N-nitrosoguanidine, a resistant colony harboring a plasmid named RMGMT was isolated . Extracts of BK2210 cells hosting the RMGMT plasmid expressed a O6-methylguanine-DNA-methyltransferase (transferase) activity and this protein had the same molecular weight as the transferase from H4 cells . The cDNA sequence of 763 bp contains an open reading frame of 630 bp encoding a protein of 209 amino acids with a calculated molecular weight of 22.2 kd . The rat protein shows 68% homology with the human transferase. Genes Dev, 1991 Jun, 5(6), 1009 - 21 The Neurospora mitochondrial tyrosyl-tRNA synthetase is sufficient for group I intron splicing in vitro and uses the carboxy-terminal tRNA-binding domain along with other regions; Kittle JD Jr et al.; Neurospora mitochondrial tyrosyl-tRNA synthetase (mt tyrRS), which is encoded by nuclear gene cyt-18, functions in splicing of group I introns in mitochondria . Here, we overproduced functional cyt-18 protein in Escherichia coli and purified it to near homogeneity . The purified protein has splicing and tyrRS activities similar to those of cyt-18 protein isolated from mitochondria and is by itself sufficient to splice the mitochondrial large rRNA intron in vitro . Structure-function relationships in the cyt-18 protein were analyzed by in vitro mutagenesis . We confirmed that a small amino-terminal domain not found in bacterial tyrRSs is required for splicing activity, but not tyrRS activity . Two linker insertion mutations, which disrupt the predicted ATP-binding site, completely inhibit tyrRS activity but leave substantial splicing activity . Finally, deletions or linker insertion mutations in the putative carboxy-terminal tRNA-binding domain inhibit both tyrRS and splicing activities, although some have differential effects on the two activities . Our results show that the normal catalytic activity of the cyt-18 protein is not required for splicing and are consistent with the hypothesis that the protein functions by binding to the precursor RNA and facilitating formation of the correct RNA structure . Regions required for splicing are distributed throughout the cyt-18 protein and overlap, but are not identical to, regions required for tyrRS activity . The finding that the putative carboxy-terminal tRNA-binding domain is required for both tyrRS and splicing activities suggests that the mechanism for binding the intron has similarities to the mechanism for binding tRNA(Tyr). Genetics, 1991 Jun, 128(2), 225 - 32 Selection for Tn10 tet repressor binding to tet operator in Escherichia coli: isolation of temperature-sensitive mutants and combinatorial mutagenesis in the DNA binding motif; Wissmann A et al.; We have constructed a genetic assay which selects positively for a functional interaction between Tet repressor and its cognate operator in Escherichia coli . In this strain Tet repressor blocks expression of lacI and lacZ . This leads to derepression of a lacPO controlled galK gene . The strain can be selected by growth on galactose as the sole carbon source and screened for the beta-galactosidase phenotype . These features allow the identification of one candidate among 10(8) false clones on a single plate . The assay was applied to select mutants with a ts DNA binding phenotype and to screen oligonucleotide generated Tet repressor mutants . Analysis of these mutations revealed that they affect DNA and inducer binding and possibly the dimerization domains . These mutations are located at residues 21, 48, 49, 89 and at the C terminus of the protein (193), respectively. Nucleic Acids Res, 1991 Jun 11, 19(11), 3139 - 41 Eliminating primers from completed polymerase chain reactions with exonuclease VII; Li HH et al.; Single-stranded oligonucleotide primers can be efficiently removed after PCR using E.coli exonuclease VII . Even only a few molecules of double stranded PCR product are unaffected by a treatment which eliminates 20 picomoles of primer in the presence of 500 ng of denatured genomic DNA . Exonuclease VII treatment is rapid and could simplify complicated multistep PCR protocols. Nucleic Acids Res, 1991 Jun 11, 19(11), 3115 - 21 Recombinant human chromosomal proteins HMG-14 and HMG-17; Bustin M et al.; Vectors for expressing human chromosomal proteins HMG-14 and HMG-17 in bacterial cultures under the control of the temperature-inducible lambda PL promoter have been constructed . The open reading frames of the cDNAs have been amplified by the polymerase chain reaction (PCR), utilizing amplimers containing desired restriction sites, thereby facilitating precise location of the initiation codon downstream from a ribosomal binding site . Expression of the recombinant proteins does not significantly affect bacterial growth . The rate of synthesis of the recombinant proteins is maximal during the initial stages of induction and slows down appreciably with time . After an initial burst of protein synthesis, the level of the recombinant protein in the bacterial extracts remains constant at different times following induction . Methods for rapid extraction and purification of the recombinant proteins are described . The recombinant proteins are compared to the proteins isolated from eucaryotic cells by electrophoretic mobility, Western analysis and nucleosome core mobility-shift assays . The ability of the proteins to shift the mobility of the nucleosome cores, but not that of DNA, can be used as a functional assay for these HMG proteins . A source for large quantities of human chromosomal proteins HMG-14 and HMG-17 will facilitate studies on their structure, cellular function and mechanism of interaction with nucleosomes. Nucleic Acids Res, 1991 Jun 11, 19(11), 2947 - 53 Effect of DNA bending in various regions of a consensus-like Escherichia coli promoter on its strength in vivo and structure of the open complex in vitro; Lozinski T et al.; A series of E . coli promoters made of the consensus -35 and -10 hexamers separated by 17 bp spacer with variously located bending dTn.dAn, n = 5 or 6, sequences was constructed and cloned into the plasmid pDS3 . Electrophoretic gel mobilities of restriction fragments containing these promoters correlated with the number of the T tracts encoded in the promoter sequences . The open complexes formed by E . coli RNA polymerase on promoters containing the T5(-34...-38) tract exhibited gel retardation indicative of their different gross geometry . The strength of these promoters measured in vivo in relation to an internal transcriptional standard was shown to be significantly lower than that of the group without the T5(-34...-38) tract . Within both these groups the promoters with two T6 tracts in the spacer, aligned in phase with the B-DNA helix repeat, had lower transcriptional activity, while the T6 tract encoded in the -7...-2 promoter region apparently had no influence on the strength of the respective promoters. Biochemistry, 1991 Jun 11, 30(23), 5777 - 84 Site-directed mutagenesis to probe protein folding: evidence that the formation and aggregation of a bovine growth hormone folding intermediate are dissociable processes; Lehrman SR et al.; Bovine growth hormone (bGH) forms a stable folding intermediate that aggregates at elevated concentrations (greater than 10 microM) . Thermodynamic and kinetic studies have shown that the formation of this bGH folding intermediate and its aggregation are separate processes, implying that selective modifications of bGH can lead to their independent modulation . In addition, a bGH region that includes amino acid residues 109-133 appears to be directly involved in this aggregation process . Human growth hormone (hGH), which is unable to aggregate via this mechanism, differs from the bovine primary sequence at eight positions within this protein region . We have characterized the folding of a bGH analogue that contains the hGH sequence between amino acid residues 109-133 (8H-bGH) at low and high concentrations . The equilibrium folding characteristics of bGH and 8H-bGH are similar when monitored at low protein concentrations (less than or equal to 2 microM) . The wild-type and analogue proteins have equivalent denaturation midpoints when equilibrium unfolding is monitored by the use of far-UV circular dichroism, second-derivative UV, or fluorescence . In addition, the enhanced fluorescence that is associated with the formation of the bGH monomeric folding intermediate (Havel, H . A., et al . (1988) Biochim . Biophys . Acta 955, 154-163) is observed for 8H-bGH under similar conditions . In contrast, partial denaturation of 8H-bGH at higher concentrations (greater than 2 microM) leads to significantly less aggregation than is observed for bGH . This result is obtained from near-UV CD spectroscopy, kinetic folding, size-exclusion chromatography, and dynamic light-scattering data.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1991 Jun 11, 30(23), 5750 - 4 pH dependence of the kinetic properties of allosteric phosphofructokinase from Escherichia coli; Deville-Bonne D et al.; The pH dependence of the activity of the allosteric phosphofructokinase from Escherichia coli has been studied in the pH range from 6 to 9, in the absence or presence of allosteric effectors . The sigmoidal cooperative saturation of phosphofructokinase by fructose 6-phosphate has been analyzed according to the Hill equation, and the following results have been obtained: (i) the apparent affinity for Fru-6P, as measured by the half-saturating concentration, {Fru-6P}0.5, does not change with pH; (ii) the cooperativity, as measured empirically by the Hill coefficient, nH, increases markedly with pH and reaches a value of 5.5-6 at pH 9; (iii) the catalytic rate constant, kcat, is controlled by the ionization of a critical group which has a pK of 7 in the absence of effector and must be deprotonated for phosphofructokinase to be active . The observation that pH affects both the cooperativity and the maximum velocity suggests that the catalytic efficiency of a given active site could be modified by the binding of fructose 6-phosphate to other remote sites . Finding values of the cooperativity coefficient larger than the number of substrate binding sites indicates that slow conformational changes may occur in phosphofructokinase . The cooperative saturation of phosphofructokinase by fructose 6-phosphate appears more complex than described by the classical concerted model at steady state and could involve two slowly interconverting states which differ in both their turnover rate constants and their affinities for fructose 6-phosphate . The presence of GDP shifts the pK of the critical group which controls kcat from 7 to 6.6.(ABSTRACT TRUNCATED AT 250 WORDS) Lancet, 1991 Jun 8, 337(8754), 1368 - 72 Recognition of human 60 kD heat shock protein by mononuclear cells from patients with juvenile chronic arthritis; De Graeff-Meeder ER et al.; A postulated mechanism for autoimmune disorders is that the immunoreactivity develops against bacterial antigens which show a high degree of sequence homology with mammalian proteins . The mycobacterial 65 kD heat shock protein (hsp) has been implicated in several forms of arthritis . Substantial amounts of the human 60 kD homologue (hsp60) were produced by insertion of the gene into Escherichia coli . To investigate the hypothesis that T-cell reactivity is directed against the endogenous hsp, T-cell proliferation of synovial-fluid and peripheral-blood mononuclear cells in response to hsp60 was studied in samples from six patients with juvenile chronic arthritis (JCA) and nine adult patients with rheumatoid arthritis (RA) . There was no T-lymphocyte proliferative response to purified fractions of hsp60 in mononuclear cells from RA patients or healthy children and young adults . However, both synovial-fluid and peripheral-blood mononuclear cells from JCA patients showed substantial proliferative responses . There was a significant correlation between the stimulation indices for human hsp60 and for mycobacterial hsp65 (r = 0.948, p less than 0.02) . A similar correlation for hsp60 and mycobacterial hsp70 did not achieve significance . Immunohistochemistry showed that hsp65 and hsp70 homologues were expressed in the synovial membrane in these patients but not in controls . These findings suggest a sequence of events in which hsps become expressed during synovial inflammation and function as autoantigens . In JCA this may be manifested by specific T-cell reactivity which apparently is lost in the more bone-eroding and non-remitting adult disease. J Mol Biol, 1991 Jun 5, 219(3), 393 - 8 Isolation and characterization of visible light-sensitive mutants of Escherichia coli K12; Miyamoto K et al.; Six mutants of Escherichia coli K12 that are sensitive to visible light have been isolated . Five of them, including an amber mutant, are defective in a gene that maps near 11 minutes on the linkage map of the chromosome, and this gene has been designated visA . The sixth mutant, which was isolated from bacteria that carried the visA+/visA+ diploid allele, is defective in a gene that maps near 63 minutes on the linkage map, which has been designated visB . These mutant strains of bacteria are killed by illumination with visible light . The effective wavelength of the light is around 460 nm . The nucleotide sequence of the visA gene was determined . As a result of a search for homologous products, we found that visA may be identical to hemH, the structural gene for ferrochelatase which catalyzes a final step in the biosynthesis of heme . A possible mechanism for the killing of the visA mutant bacteria is discussed. J Biol Chem, 1991 Jun 5, 266(16), 10686 - 93 Selective metal binding to Cys-78 within endonuclease V causes an inhibition of catalytic activities without altering nontarget and target DNA binding; Prince MA et al.; T4 endonuclease V is a pyrimidine dimer-specific DNA repair enzyme which has been previously shown not to require metal ions for either of its two catalytic activities or its DNA binding function by virtue of its ability to function in the presence of metal-chelating agents . However, we have investigated whether the single cysteine within the enzyme was able to bind metal salts and influence the various activities of this repair enzyme . A series of metals (Hg2+, Ag+, Cu+) were shown to inactivate both endonuclease Vs pyrimidine dimer-specific DNA glycosylase activity and the subsequent apurinic nicking activity . The binding of metal to endonuclease V did not interfere with nontarget DNA scanning or pyrimidine dimer-specific binding . The Cys-78 codon within the endonuclease V gene was changed by oligonucleotide site-directed mutagenesis to Thr-78 and Ser-78 in order to determine whether the native cysteine was directly involved in the enzyme's DNA catalytic activities and whether the cysteine was primarily responsible for the metal binding . The mutant enzymes were able to confer enhanced ultraviolet light (UV) resistance to DNA repair-deficient Escherichia coli at levels equal to that conferred by the wild type enzyme . The C78T mutant enzyme was purified to homogeneity and shown to be catalytically active on pyrimidine dimer-containing DNA . The catalytic activities of the C78T mutant enzyme were demonstrated to be unaffected by the addition of Hg2+ or Ag+ at concentrations 1000-fold greater than that required to inhibit the wild type enzyme . These data suggest that the cysteine is not required for enzyme activity but that the binding of certain metals to that amino acid block DNA incision by either preventing a conformational change in the enzyme after it has bound to a pyrimidine dimer or sterically interfering with the active site residue's accessibility to the pyrimidine dimer. J Biol Chem, 1991 Jun 5, 266(16), 10624 - 31 Structure-activity relationship study of human interleukin-3 . Identification of residues required for biological activity by site-directed mutagenesis; Lokker NA et al.; Recombinant human interleukin-3 (rhuIL-3) variants were generated by site-directed mutagenesis and expression in Escherichia coli . Amino acid deletions and substitutions were made in the previously identified epitopes of two huIL-3-specific neutralizing monoclonal antibodies (mAbs) . The rhuIL-3 variants were analyzed for their ability to bind to the IL-3 receptor and to induce the proliferation of the human IL-3-dependent cell line M-O7 . Several deletion mutants spanning the epitopes of these neutralizing mAbs indicated the importance of residues Pro33 and Leu34 for biological activity . Further, substitution of Pro33 with Asn (Asn33) showed an enhanced proliferative activity (4-fold) and a moderate increase in receptor binding (2-fold) compared to wild-type (wt) rhuIL-3 . The most remarkable change, however, was seen with variant Gly33, which showed a 14-fold increase in promoting the growth of M-O7 cells without a significant modification in its receptor binding capacity . In contrast, substitution of Leu34 with Gly (Gly34) yielded an IL-3 variant that had a 25-fold decreased receptor binding capacity and proliferative activity, while Glu34 had properties similar to wild-type rhuIL-3 . Analysis of the binding of these variants to different rhuIL-3-specific monoclonal antibodies suggested that no major modification had occurred in their conformations . These results indicate that both residues, Pro33 and Leu34, play a critical role in modulating the activity of rhuIL-3. J Biol Chem, 1991 Jun 5, 266(16), 10485 - 9 Inhibition of actin polymerization by a synthetic dodecapeptide patterned on the sequence around the actin-binding site of cofilin; Yonezawa N et al.; Cofilin is an F-actin side-binding and -depolymerizing protein with an apparent molecular mass of 21 kDa . By means of the end label fingerprinting method, the amino acid residue on cofilin sequence cross-linked to actin by zero length cross-linker, 1-ethyl-3-(3-dimethylamino propyl)carbodiimide, was identified as Lys112 and/or Lys114 . A synthetic dodecapeptide patterned on the sequence around the actin-cross-linking site of cofilin (Trp104-Met115) inhibited the binding of cofilin to actin . Moreover, the dodecapeptide was found to be a potent inhibitor of actin polymerization . Thus, we conclude that the dodecapeptide sequence constitutes the region essential for the actin-binding and -depolymerizing activity of cofilin . A sequence similar to the dodecapeptide is found in other actin-depolymerizing proteins, destrin, actin-depolymerizing factor, and depactin . Therefore, the dodecapeptide sequence may be a consensus sequence essential for actin-binding and -depolymerizing activity in actin-depolymerizing proteins. J Biol Chem, 1991 Jun 5, 266(16), 10400 - 5 Induction of glycinebetaine uptake into Xenopus oocytes by injection of poly(A)+ RNA from renal cells exposed to high extracellular NaCl; Robey RB et al.; Madin-Darby canine kidney (MDCK) cells accumulate glycinebetaine via Na(+)-dependent transport in response to hypertonic stress . When extracellular tonicity is increased by the addition of NaCl, Vmax for glycinebetaine transport increases without an associated change in Km, consistent with an increase in the number of functioning transporters . To test whether increased transport activity results from increased gene expression, we injected poly(A)+ RNA (mRNA) from MDCK cells into Xenopus oocytes and assayed for glycinebetaine uptake in ovo . RNA-induced Na(+)-dependent uptake is observed in oocytes injected with mRNA from cells exposed to high extracellular NaCl, but not in oocytes injected with either water or mRNA from cells maintained in isotonic medium . Unfractionated mRNA induces glycinebetaine uptake in ovo at a rate which is approximately 3-fold higher than in water-injected controls . Size-fractionated mRNA (median size 2.8 kilobases) induces uptake at a rate which is approximately 7-fold higher than controls . Such RNA-induced transport activity in ovo is consistent with heterologous expression of Na(+)/glucinebetaine cotransporters encoded by renal mRNA . Increased transporter mRNA in cells exposed to hypertonicity probably underlies the pattern of expression observed in ovo . This can account for the observed rise in MDCK cell glycinebetaine transport during hypertonic stress. J Biol Chem, 1991 Jun 5, 266(16), 10351 - 7 Evidence for one or more Raf-1 kinase kinase(s) activated by insulin and polypeptide growth factors; Lee RM et al.; The protein product of the Raf-1 proto-oncogene is a protein serine/threonine kinase that is activated after stimulation of cells with insulin and other mitogens . To investigate the mechanism of this activation, we used purified Raf-1 expressed in E . coli as a substrate for a putative Raf-1 protein kinase kinase . In three different insulin-sensitive cell types, insulin activated Raf-1 kinase kinase activity in crude cytosolic cellular fractions . The insulin stimulation of this activity was evident as early as 2 min after exposure to insulin, maximal at 5-8 min, and inapparent at 15 min . Phosphoamino acid analysis of phosphorylated Raf-1 revealed that serine was the primary phosphate acceptor for the insulin-activated kinase or kinases; small amounts of phosphothreonine were also detected . The insulin effect occurred in cells depleted of protein kinase C, and in extracts depleted of endogenous Raf-1 kinase by immunodepletion; these data argue against protein kinase C or Raf-1 kinase itself being the insulin-stimulated activity . The insulin-activated kinase or kinases phosphorylated the Raf-1 protein on multiple sites in vitro, as evidenced by tryptic mapping; at least some of these appeared to overlap with sites phosphorylated in response to serum in intact cells . Several other mitogens and growth factors stimulated Raf-1 kinase kinase activity, including epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, serum, and phorbol 12-myristate 13-acetate . This insulin- and mitogen-stimulated Raf-1 kinase kinase activity may play a role in mediating the phosphorylation and possibly the activation of the Raf-1 kinase by insulin and other growth factors. J Biol Chem, 1991 Jun 5, 266(16), 10168 - 73 Activation of Escherichia coli pyruvate oxidase enhances the oxidation of hydroxyethylthiamin pyrophosphate; Bertagnolli BL et al.; The catalytic efficiency (kcat/Km) of Escherichia coli flavin pyruvate oxidase can be stimulated 450-fold either by the addition of lipid activators or by limited proteolytic hydrolysis . Previous studies have shown that a functional lipid binding site is a mandatory prerequisite for the in vivo functioning of this enzyme (Grabau, C., and Cronan, J . E., Jr . (1986) Biochemistry 25, 3748-3751) . The effect of activation on the transient state kinetics of partial reactions in the overall oxidative conversion of pyruvate to acetate and CO2 has now been examined . The rate of decarboxylation of pyruvate to form CO2 and hydroxyethylthiamin pyrophosphate for both activated and unactivated forms of the enzyme is identical within experimental error . The decarboxylation step was measured using substrate concentrations of the enzyme in the absence of an electron acceptor . The pseudo-first order rate constant for the decarboxylation step is 60-80 s-1 . The rate of oxidation of hydroxyethylthiamin pyrophosphate and concomitant enzyme-bound flavin reduction was analyzed by stopped-flow methods utilizing synthetic hydroxyethylthiamin pyrophosphate . The pseudo-first order rate for this step with unactivated enzyme was 2.85 s-1 and increased 145-fold for lipid-activated enzyme to 413 s-1 and 61-fold for the proteolytically activated enzyme to 173 s-1 . The analysis of a third reaction step, the reoxidation of enzyme-bound FADH, was also investigated by stopped-flow techniques utilizing ferricyanide as the electron acceptor . The rate of oxidation of enzyme.FADH is very fast for both unactivated (1041 s-1) and activated enzyme (645 s-1) . The data indicate that the FAD reduction step is the rate-limiting step in the overall reaction for unactivated enzyme . Alternatively, the rate-limiting step in the overall reaction with the activated enzyme shifts to one of the partial steps in the decarboxylation reaction. J Biol Chem, 1991 Jun 5, 266(16), 10112 - 21 DNA substrate requirements for stable joint molecule formation by the RecA and single-stranded DNA-binding proteins of Escherichia coli; Konforti BB et al.; In reactions between linear single-stranded DNAs (ssDNAs) and circular double-stranded DNAs (dsDNAs), stable joint molecule formation promoted by the recA protein (RecA) requires negative superhelicity, a homologous end, and an RecA-ssDNA complex . Linear ssDNAs with 3'-end homology react more efficiently than linear ssDNAs with 5'-end homology . This 3'-end preference is explained by the finding that 3'-ends are more effectively coated by RecA than 5'-ends, as judged by exonuclease VII protection, and are thus more reactive . The ability of linear ssDNAs with 5'-end homology to react is improved by the presence of low concentrations of exonuclease VII . In reactions between ssDNAs and linear dsDNAs with end homology, stable joint molecule formation occurs more efficiently when the homology is at the 3'-end rather than at the 5'-end of the complementary strand . In addition, linear dsDNAs with homology at the 3'-end of the complementary strand react more efficiently with linear ssDNAs with 3'-end homology than with linear ssDNAs with 5'-end homology . The ability of linear ssDNAs with 5'-end homology to react, in the absence of single-stranded DNA-binding protein, is improved by adding 33-46 nucleotides of heterologous sequence to the 5'-end of the linear ssDNA . The poor reactivity of linear ssDNAs with 5'-end homology is explained by a lack of RecA at the 5'-ends of linear ssDNAs, which is a consequence of the polar association and dissociation of RecA. J Biol Chem, 1991 Jun 5, 266(16), 10201 - 9 Enhancement of transcription termination factor rho activity with potassium glutamate; Zou LL et al.; The efficiencies of rho action as a termination factor during transcription in vitro of several DNA templates were determined as a function of the concentration and type of electrolyte ions . The termination efficiencies with lambda-tR1 and the promoter proximal lacZ intragenic terminators were significantly higher with 0.1-0.2 M potassium glutamate as the major electrolyte than with the optimal concentrations of KCl (approximately 0.05 M) or potassium acetate (approximately 0.15 M) . Similar high efficiencies were obtained with salts of other acidic amino acids but not with a salt of N-acetylglutamic acid or with a mixture of 0.15 M potassium acetate and 0.15 M glycine, and termination was inhibited completely when 0.12 M KCl was present along with 0.12 M potassium glutamate . The salts that give high termination efficiencies have two properties in common; they consist of anions that are also zwitterions, and they are weak chelators of Mg2+ ions . The increase in termination efficiency with potassium glutamate can be ascribed mainly to a facilitation of the reactions of rho with RNA that are coupled to ATP hydrolysis, as the rate of ATP hydrolysis with isolated transcripts as cofactors was about five times higher with 0.15 M potassium glutamate than with 0.05 M KCl, whereas the rates of chain elongation, the general stability of the transcription complexes, and the binding affinity of rho with the transcripts were all very similar under the two conditions . Further analysis revealed that the activation of ATP hydrolysis is an outcome of a shift in the optimum magnesium salt concentration from 0.5 mM with 0.05 M KCl to 4 mM with 0.15 M potassium glutamate . Since glutamate is a relatively weak counterion for cationic groups in proteins, potassium glutamate can be used at 0.15 M without inhibiting the binding of rho to RNA . At that concentration, it serves to buffer the level of free Mg2+ available to stabilize RNA secondary structures that are known to impede rho action on RNA . The two special properties of glutamate together create conditions that allow rho to terminate transcription in vitro at an efficiency that matches the in vivo efficiency with use of a physiological level of K+ ions. J Biol Chem, 1991 Jun 5, 266(16), 10534 - 43 Isolation and characterization of temperature-sensitive and thermostable mutants of the human receptor-like protein tyrosine phosphatase LAR; Tsai AY et al.; Human LAR is a transmembrane receptor-like protein whose cytoplasmic region contains two tandemly duplicated domains homologous to protein tyrosine phosphatases (PTPases) . Whereas the membrane-proximal domain I has enzymatic activity, the membrane-distal domain II has no apparent catalytic activity but seems to have a regulatory function . In order to study structure-function relationships of the LAR PTPase, LAR domain I was expressed in Escherichia coli, and mutants that have reduced catalytic activity or reduced thermostability were isolated and characterized . We isolated 18 unique hydroxylamine-induced missense mutations in the LAR domain I segment, of which three were temperature-sensitive . Five additional temperature-sensitive mutations were isolated using N-methyl-N'-nitro-N-nitrosoguanidine . All eight temperature-sensitive mutations are confined within a short segment of the LAR domain I sequence between amino acid positions 1329 and 1407 . To examine whether this region is particularly prone to temperature-sensitive mutations, tyrosine at amino acid position 1379 was changed to a phenylalanine by oligonucleotide-directed mutagenesis . This mutant, Y1379-F, was indeed temperature-sensitive . We also isolated a revertant of a temperature-sensitive mutant . The revertant contained a second-site mutation (C1446-Y) that suppresses several temperature-sensitive mutations and also enhances the folding of LAR protein produced in E . coli. J Biol Chem, 1991 Jun 5, 266(16), 10086 - 92 Site- and strand-specific nicking in vitro at oriT by the traY-traI endonuclease of plasmid R100; Inamoto S et al.; We developed an in vitro system to reproduce a site- and strand-specific nicking at the oriT region of plasmid R100 . The nicking reaction was dependent on the purified TraY protein and on the lysate, which was prepared from cells overproducing the TraI protein . This supports the idea that the protein products of two genes, traY and traI, constitute an endonuclease that introduces a specific nick in vivo in the oriT region of the conjugative plasmids related to R100 . The products were the "complex" DNA molecules with a protein covalently linked with the 5'-end of the nick . The nick was introduced in the strand, which is supposed to be transferred to recipient cells during conjugation, and was located at the site 59 base pairs upstream of the TraY protein binding site, sbyA. J Biol Chem, 1991 Jun 5, 266(16), 10070 - 2 Disulfide pairing of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli; Vlahos CJ et al.; The kringle-2 domain of tissue plasminogen activator, cloned and expressed in Escherichia coli (Wilhelm, O.G., Jaskunas, S.R., Vlahos, C.J., and Bang, N.U . (1990) J . Biol . Chem . 265, 14606-14611), was internally radiolabeled using {35S}methionine-cysteine . Following refolding and isolation, the labeled polypeptide was further purified by reverse-phase high performance liquid chromatography . The purified kringle-2 domain was digested with thermolysin, and the resulting peptides were purified by high performance liquid chromatography . Five major peptides containing 35S were obtained . Amino acid sequence analysis showed that these peptides represented various cleavage products containing one or more of the following disulfides: Cys180-Cys261, Cys201-Cys243, Cys232-Cys256 (sequence numbering based on Pennica et al . (Pennica, D., Holmes, W.E., Kohr, W.J., Hakins, R.N., Vehar, G . A., Ward, C.A., Bennett, W.F., Yelverton E., Seeburg, P.H., Heynecker, H.L., Goeddel, E.V., and Collen, D . (1983) Nature 301, 214-221)) . These results confirm that the refolding methodology used produced kringle-2 with the predicted disulfide linkage and, thus, yielded material suitable for structural and functional studies. Biochemistry, 1991 Jun 4, 30(22), 5608 - 15 Mutations in the heparin-binding domains of human basic fibroblast growth factor alter its biological activity; Heath WF et al.; Eleven structural analogues of human basic fibroblast growth factor (bFGF) have been prepared by site-directed mutagenesis of a synthetic bFGF gene to examine the effect of amino acid substitutions in the three putative heparin-binding domains on FGF's biological activity . After expression in Escherichia coli, the mutant proteins were purified to homogeneity by use of heparin-Sepharose chromatography and analyzed for their ability to stimulate DNA synthesis in human foreskin fibroblasts . Recombinant human bFGF 1-146 and {Ala69,Ser87}bFGF, an analogue where two of the four cysteines had been replaced by alanine and serine, were equipotent to standard bovine basic fibroblast growth factor . Substitution of aspartic acid-19 by arginine in the first heparin-binding domain yielded a molecule that stimulated a higher total mitogenic response in fibroblasts as compared to bFGF . In addition, replacement of either arginine-107 in the second domain or glutamine-123 in the third domain with glutamic acid resulted in compounds that were 2 and 4 times more potent than bFGF . In contrast, substitution of arginine-107 with isoleucine reduced the activity of the molecule by 100-fold . Combination of domain substitutions to generate the {Glu107,123}bFGF and {Arg19,Lys123,126}bFGF mutants did not show any additivity of the mutations on biological activity . Alterations in the biological activity of the analogues was dependent on both the site of and the type of modification . Increased positive charge in the first domain and increased negative charge in the second and third domains enhanced biological potency . The altered activities of the derivatives appear to be due in part to changes in the affinity of the analogues for heparin . We conclude that changes in all three of the putative heparin-binding domains result in altered mitogenic activity and heparin interaction of basic fibroblast growth factor. Biochemistry, 1991 Jun 4, 30(22), 5539 - 46 Mechanism of adenylate kinase . Demonstration of a functional relationship between aspartate 93 and Mg2+ by site-directed mutagenesis and proton, phosphorus-31, and magnesium-25 NMR; Yan HG et al.; Earlier magnetic resonance studies suggested no direct interaction between Mg2+ ions and adenylate kinase (AK) in the AK.MgATP (adenosine 5'-triphosphate) complex . However, recent NMR studies concluded that the carboxylate of aspartate 119 accepts a hydrogen bond from a water ligand of the bound Mg2+ ion in the muscle AK.MgATP complex {Fry, D.C., Kuby, S.A., & Mildvan, A.S . (1985) Biochemistry 24, 4680-4694} . On the other hand, in the 2.6-A crystal structure of the yeast AK.MgAP5A {P1,P5-bis(5'-adenosyl)pentaphosphate} complex, the Mg2+ ion is in proximity to aspartate 93 {Egner, U., Tomasselli, A.G., & Schulz, G.E . (1987) J . Mol . Biol . 195, 649-658} . Substitution of Asp-93 with alanine resulted in no change in dissociation constants, 4-fold increases in Km, and a 650-fold decrease in kcat . Notable changes have been observed in the chemical shifts of the aromatic protons of histidine 36 and a few other aromatic residues . However, the results of detailed analyses of the free enzymes and the AK.MgAP5A complexes by one- and two-dimensional NMR suggested that the changes are due to localized perturbations . Thus it is concluded that Asp-93 stabilizes the transition state by ca . 3.9 kcal/mol . The next question is how . Since proton NMR results indicated that binding of Mg2+ to the AK.AP5A complex induces some changes in the proton NMR signals of WT but not those of D93A, the functional role of Asp-93 should be in binding to Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1991 Jun 4, 30(22), 5524 - 31 Tritium NMR spectroscopy of ligand binding to maltose-binding protein; Gehring K et al.; Tritium-labeled alpha- and beta-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein . Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied . Tritium NMR spectroscopy of the labeled sugars showed large upfield chemical shift changes upon binding and strong anomeric specificity . At 10 degrees C, MBP bound alpha-maltose with 2.7 +/- 0.5-fold higher affinity than beta-maltose, and, for longer maltodextrins, the ratio of affinities (KD beta/KD alpha) was even larger (between 10 and 30) . The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound alpha-maltodextrin makes close contact with an aromatic residue in the MBP-binding site . Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound beta-maltotriose resonances in rapid exchange . We interpret these two resonances as arising from two distinct sugar-protein complexes . In one complex, the beta-maltodextrin is bound by its reducing end, and, in the other complex, the beta-maltodextrin is bound by the middle glucose residue(s) . This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins. Biochemistry, 1991 Jun 4, 30(22), 5475 - 84 Escherichia coli glutaredoxin: cloning and overexpression, thermodynamic stability of the oxidized and reduced forms, and report of an N-terminal extended species; Sandberg VA et al.; Escherichia coli glutaredoxin (MW 9700) catalyzes intracellular redox reactions utilizing a disulfide/dithiol enzymatic mechanism involving the active-site residues -Cys-Pro-Tyr-Cys- . It is functionally related to the thioredoxin family and is expected to share similar three-dimensional structure {Eklund, H., Cambillau, C., Sjoberg, B.-M., Holmgren, A., Jornvall, H., Hoog, J.-O., & Branden, C.-I . (1984) EMBO J . 3, 1443-1449} . We constructed an overexpression system in which production of glutaredoxin is controlled by temperature-sensitive expression of the phage T7 promoter . In addition to glutaredoxin, a second gene product is observed; this species, which we call glutaredoxin N, is glutaredoxin extended by the sequence Met-Arg-Arg-Glu-Ile- at the N terminus . We have begun characterization of the structure and stability of the oxidized and reduced forms of glutaredoxin (grx-S2 and grx-(SH)2, respectively) . Secondary structure calculated from CD data agrees with that predicted from the three-dimensional model of Eklund et al . The cooperative denaturation reactions of oxidized and reduced glutaredoxin were measured in temperature-induced and guanidine hydrochloride induced unfolding experiments . Surprisingly, oxidized and reduced glutaredoxins are very similar in stability . In heat-induced denaturation, monitored by CD, Tm is 55 and 57 degrees C for oxidized and reduced, respectively . In GuHCl denaturation, monitored by fluorescence, the midpoint denaturant concentrations are 2 M for both oxidized and reduced . It follows that the redox potentials of the disulfide bond are similar in unfolded and folded glutaredoxin . This is unexpected because in E . coli thioredoxin the oxidized form is far more stable than the reduced {Kelley, R.F., Shalongo, W., Jagannadham, M.V., & Stellwagen, E . (1987) Biochemistry 26, 1406-1411} and the redox potential of folded thioredoxin is significantly more negative than that of unfolded thioredoxin {Lin, T.-Y., & Kim, P . (1989) Biochemistry 28, 5282-5287}. FEBS Lett, 1991 Jun 3, 283(2), 267 - 9 Methotrexate binds in a non-productive orientation to human dihydrofolate reductase in solution, based on NMR spectroscopy; Stockman BJ et al.; Dihydrofolate reductase (DHFR) is an intracellular target enzyme for folate antagonist drugs, including methotrexate . In order to compare the binding of methotrexate to human DHFR in solution with that observed in the crystalline state, NMR spectroscopy has been used to determine the conformation of the drug bound to human DHFR in solution . In agreement with what has been observed in the crystalline state, NOE's identified protein and methotrexate protons indicate that methotrexate binds in a non-productive orientation . In contrast to what has been reported for E . coli DHFR in solution, only one bound conformation of methotrexate is observed. FEBS Lett, 1991 Jun 3, 283(2), 180 - 4 Mutating P2 and P1 residues at cleavage junctions in the HIV-1 pol polyprotein . Effects on hydrolysis by HIV-1 proteinase; Jupp RA et al.; Mutations were introduced into the P2 and P1 positions of the junctions, (a) linking reverse transcriptase (RT) and integrase (IN) (-Leu*Phe-) and (b) between the p51 and RNase H domain (-Phe*Tyr-) within p66 of RT in the HIV-1 pol polyprotein . Processing by HIV proteinase (PR) in cis was monitored upon expression of these constructs in E . coli . Whereas the presence of Leu or Phe in P1 permitted rapid cleavage at either junction, substitution of a beta-branched (Ile) hydrophobic residue essentially abolished hydrolysis . By contrast, placement of a beta-branched (Val) residue in the P2 position flanking such -Hydrophobic*Hydrophobic- junctions resulted in effective cleavage of the scissile peptide bond . Gly in P2, however, abrogated cleavage . The significance of these findings in terms of PR specificity, polyprotein processing and the generation of homodimeric (p51/p51) RT for crystallisation purposes is discussed. FEBS Lett, 1991 Jun 3, 283(2), 291 - 4 Recombinant poliovirus 3C protease . The enzyme application to protein specific fragmentation; Sablina EP et al.; Active 3C protease of poliovirus 1(M) was obtained when cloning and expressing fragment HindII-HindIII (bases from 5240 to 6056) of cDNA in vector pTTQ8 in E.coli cells . As shown, fragment 3D of polymerase covalently bound to 3C does not deprive the enzyme of its specific proteolytic activity . The absence of 26 N-terminal amino acids in 3C entails its inactivation . The recombinant 3C protease cleaved peptide bond Gln-Gly not only in virus polyprotein, but also in molecules of beta-galactosidase and bovine catalase. FEBS Lett, 1991 Jun 3, 283(2), 251 - 4 RNA-RNA and RNA-protein interactions in 30 S ribosomal subunits . Association of 16 S rRNA fragments in the presence of ribosomal proteins; Afonina EI et al.; The E . coli 16 S rRNA with single-site breaks centered at position 777 or 785 was obtained by RNase H site-specific cleavage of rRNA . Spontaneous dissociation of the cleaved 16 S rRNA into fragments occurred under 'native' conditions . The reassociation of the 16 S rRNA fragments was possible only in the presence of ribosomal proteins . The combination of S4 and S16(S17) ribosomal proteins interacting mainly with the 5'-end domain of 16 S rRNA was sufficient for reassociation of the fragments . The 30 S subunits with fragmented RNA at ca . 777 region retained some poly(U)-directed protein synthetic activity. FEBS Lett, 1991 Jun 3, 283(2), 247 - 50 After microinjection hemimethylated DNA is converted into symmetrically methylated DNA before DNA replication; Sandberg G et al.; In this investigation we analysed the maintenance methylation activity of the mammalian cell DNA methyltransferase by microinjection of hemimethylated HSV-tk DNA into thymidine kinase-negative rat 2 cells . We found that the hemimethylated DNA was efficiently converted into symmetrical methylated molecules before DNA replication . Furthermore, integration of the trans-DNA into the host genome is an early event after gene transfer. Mol Gen Genet, 1991 Jun, 227(2), 260 - 6 Molecular cloning of a glutathione S-transferase overproduced in an insecticide-resistant strain of the housefly (Musca domestica); Wang JY et al.; We report the cloning and sequencing of a glutathione S-transferase (GST) gene from the housefly Musca domestica . A cDNA lambda gt11 library was prepared from the organophosphate insecticide-resistant housefly strain Cornell-R--a variant that has elevated GST activity . The lambda phage GST clone was identified on the basis of its ability to cross-hybridize to a GST DNA probe from Drosophila melanogaster . Based on amino acid homology to other GSTs and expression of GST activity in Escherichia coli, the Musca GST gene (MdGST-1) belongs to the GST gene family . Although organophosphate resistance in Cornell-R is largely due to one of the GSTs, MdGST-1 is probably not the enzyme responsible for resistance . The mutation that controls resistance to organophosphate insecticides in Cornell-R is highly unstable and we isolated spontaneous variants to both insecticide sensitivity and to even higher levels of resistance . This provided us with an isogenic set of three strains . We found that MdGST-1 transcript levels as measured by Northern assays are higher in all three Cornell-R strains relative to the sensitive wild type, but that the sensitive Cornell-R strain has more MdGST-1 transcript than does the highly resistant Cornell-R strain . These data as well as Southern analysis of genomic DNA allow us to conclude: (1) there are multiple GST genes in M . domestica; (2) the natural variant Cornell-R overproduces excess transcript from two and probably more of these genes; and (3) the unstable mutation in Cornell-R influences the levels of multiple GSTs. Mol Gen Genet, 1991 Jun, 227(2), 252 - 9 Mutagenic specificity of N-methyl-N'-nitro-N-nitrosoguanidine in the gpt gene on a chromosome of Chinese hamster ovary cells and of Escherichia coli cells; Sockett H et al.; DNA base sequence changes induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis have been determined for the Escherichia coli gpt gene stably incorporated in a chromosome of Chinese hamster ovary cells and in the chromosome of both growing and starving E . coli cells, instead of on a plasmid as in most previous studies . In the three cases, nearly all mutations were G:C to A:T transitions, with a 2- to 4-fold higher mutation rate, compared to other sites, at guanines flanked on the 5' side by another guanine . Mutagenic hot spots in these experiments were less prominent than in published results for MNNG mutagenesis of gpt and of other genes . A suggested explanation involves repair of O6meG . At low levels of mutagenic products, most are repaired and even small differences in the repair rates leads to large differences in the relative amounts of residual O6meG at various sites; in contrast, at high levels of mutagenic products there is little effect of repair on the distribution. J Med Chem, 1991 Jun, 34(6), 1879 - 84 2'-Deoxy-2'-methylenecytidine and 2'-deoxy-2',2'-difluorocytidine 5'-diphosphates: potent mechanism-based inhibitors of ribonucleotide reductase; Baker CH et al.; It has been found that 2'-deoxy-2'-methyleneuridine (MdUrd), 2'-deoxy-2'-methylenecytidine (MdCyd), and 2'-deoxy-2',2'-difluorocytidine (dFdCyd) 5'-diphosphates (MdUDP (1) MdCDP (2) and dFdCDP (3), respectively) function as irreversible inactivators of the Escherichia coli ribonucleoside diphosphate reductase (RDPR) . 2 is a much more potent inhibitor than its uridine analogue 1 . It is proposed that 2 undergoes abstraction of H3' to give an allylic radical that captures a hydrogen atom and decomposes to an active alkylating furanone species . RDPR also accepts 3 as an alternative substrate analogue and presumably executes an initial abstraction of H3' to initiate formation of a suicide species . Both 2 and 3 give inactivation results that differ from those of previously studied inhibitors . The potent anticancer activities of MdCyd and dFdCyd indicate a significant chemotherapeutic potential . The analogous RDPR of mammalian cells should be regarded as a likely target and/or activating enzyme for these novel mechanism-based inactivators. Am J Physiol, 1991 Jun, 260(6 Pt 2), R1058 - 65 Ethanol oxidation is not required to attenuate endotoxin-enhanced glucose metabolism; Molina PE et al.; Previous studies from our laboratory demonstrated that acute ethanol (EtOH) intoxication, through an unknown mechanism, blunts the endotoxin-enhanced carbohydrate metabolism . The purpose of the present study was to determine whether oxidation of the ethanol moiety is required for the inhibition of the endotoxin-induced changes in carbohydrate metabolism . In vivo glucose kinetics were assessed by the intravenous administration of D-{3-3H}glucose in catheterized conscious unrestrained rats . Escherichia coli endotoxin (200 micrograms/100 g body wt) increased glucose rate of appearance (Ra) and metabolic clearance rate (MCR) by 75 and 50%, respectively . A primed-constant infusion of EtOH (275 mg/100 g + 25 mg.100 g-1.h-1) initiated 2 h before endotoxin challenge attenuated the endotoxin-enhanced glucose kinetics . EtOH intoxication did not prevent endotoxin-induced hyperglycemia but delayed the hyperlactacidemic response . The importance of EtOH metabolism in suppressing the glucose metabolic response to endotoxin was studied by administering 4-methyl-pyrazole (4-MP; 8 mg/100 g), an inhibitor of alcohol dehydrogenase activity . After administration of 4-MP and a bolus injection of EtOH (275 mg/100 g), the plasma EtOH concentration remained constant and matched the level of EtOH in rats receiving a primed-constant infusion of EtOH . Inhibition of EtOH metabolism with 4-MP did not abrogate the ability of EtOH to suppress endotoxin-induced increases in glucose Ra or MCR . Furthermore, the injection of the nonmetabolized alcohol tert-butanol abolished the endotoxin-induced increase in glucose Ra and MCR without preventing the endotoxin-induced hyperglycemia and hyperlactacidemia.(ABSTRACT TRUNCATED AT 250 WORDS) Proc Natl Acad Sci U S A, 1991 Jun 1, 88(11), 4887 - 91 Isolation of TFC1, a gene encoding one of two DNA-binding subunits of yeast transcription factor tau (TFIIIC); Swanson RN et al.; Transcription factor TFIIIC mediates tRNA and 5S RNA gene activation by binding to intragenic promoter elements . The factor from Saccharomyces cerevisiae, also called tau, is a large, multisubunit protein (550-650 kDa) containing two polypeptides that interact directly with DNA encoding tRNA (tDNA) . We have obtained peptide sequences from the 95-kDa DNA-binding subunit (tau 95) and cloned the corresponding gene, called TFC1 . The gene encodes a polypeptide of calculated Mr 73,500 . However, when TFC1 was transcribed and translated in vitro, the gene product comigrated with tau 95 in SDS/polyacrylamide gels . A fusion protein expressed in bacteria was able to prevent the binding of anti-tau 95 antibodies to tau-tDNA complexes . The TFC1 gene is present in single copy on yeast chromosome II and is essential for growth . Spores containing a disrupted gene germinate but only proceed through a few cell divisions before ceasing to grow . The TFC1-encoded protein contains a potential helix-turn-helix structure and an acidic carboxyl-terminal domain, a feature characteristic of some DNA-binding proteins and transcriptional regulators. Proc Natl Acad Sci U S A, 1991 Jun 1, 88(11), 4690 - 4 8-oxoguanine (8-hydroxyguanine) DNA glycosylase and its substrate specificity; Tchou J et al.; Substrate specificities of FPG protein (also known as formamidopyrimidine DNA glycosylase) and 8-hydroxyguanine endonuclease were compared by using defined duplex oligodeoxynucleotides containing single residues of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA), and 2,6-diamino-4-hydroxy-5-(N-methyl)formamidopyrimidine (Me-Fapy) . Duplexes containing 8-oxodG positioned opposite dC, dG, or dT were cleaved, whereas single-stranded DNA and duplexes containing 8-oxodG.dA or 8-oxodA positioned opposite any of the four DNA bases were relatively resistant . Both enzymes cut duplexes containing 8-oxoG.dC 3' and 5' to the modified base but failed to cleave duplex DNA containing synthetic abasic sites, mismatches containing dG, or unmodified DNA . 8-Oxoguanine, identified by HPLC-electrochemical detection techniques, was released during the enzymatic reaction . Apparent Km values for FPG protein acting on duplex substrates containing a single Me-Fapy or 8-oxodG residue positioned opposite dC were 41 and 8 nM, respectively, and those for 8-hydroxyguanine endonuclease were 30 and 13 nM, respectively . Comparison of the properties of the two enzyme activities suggest that they are identical . In view of the widespread distribution of 8-oxodG in cellular DNA, the demonstrated miscoding and mutagenic properties of this lesion, and the existence of a bacterial gene coding for FPG protein, we propose that 8-oxodG DNA is the primary physiological substrate for a constituent glycosylase found in bacteria and mammalian cells. Proc Natl Acad Sci U S A, 1991 Jun 1, 88(11), 4636 - 40 Localization in human interleukin 2 of the binding site to the alpha chain (p55) of the interleukin 2 receptor; Sauve K et al.; Human interleukin 2 (IL-2) analogs with defined amino acid substitutions were used to identify specific residues that interact with the 55-kDa subunit (p55) or alpha chain of the human IL-2 receptor . Analog proteins containing specific substitutions for Lys-35, Arg-38, Phe-42, or Lys-43 were inactive in competitive binding assays for p55 . All of these analogs retained substantial competitive binding to the intermediate-affinity p70 subunit (beta chain) of the receptor complex . The analogs varied in ability to interact with the high-affinity p55/p70 receptor . Despite the lack of binding to p55, all analogs exhibited significant biological activity, as assayed on the murine CTLL cell line . The dissociation constants of Arg-38 and Phe-42 analogs for p70 were consistent with intermediate-affinity binding; the Kd values were not significantly affected by the presence of p55 in binding to the high-affinity IL-2 receptor complex . These results confirm the importance of the B alpha-helix in IL-2 as the locus for p55-receptor binding and support a revised model of IL-2-IL-2 receptor interaction. J Bacteriol, 1991 Jun, 173(12), 3918 - 20 Overproduction and purification of McrC protein from Escherichia coli K-12; Zheng L et al.; The McrC protein, encoded by one of the two genes involved in the McrB restriction system, was produced in Escherichia coli cells by using a T7 expression system . Following sequential DEAE-Sepharose and hydroxylapatite column chromatography, the protein was purified to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The N-terminal amino acid sequence of the purified McrC protein agreed exactly with the one deduced from the DNA sequence by Ross et al . (J . Bacteriol . 171:1974-1981, 1989). J Bacteriol, 1991 Jun, 173(12), 3904 - 6 Translational regulation of sigma 32 synthesis: requirement for an internal control element; Kamath-Loeb AS et al.; We have investigated the sequence requirements for the translational regulation of sigma 32 by examining the behavior of a new rpoH-lacZ protein fusion containing a short N-terminal fragment of sigma 32 fused to beta-galactosidase . Although the fusion retains rpoH translational initiation signals, it lacks translational regulation, implicating coding sequences within rpoH in this regulatory process. J Bacteriol, 1991 Jun, 173(12), 3888 - 93 Growth rate regulation of translation initiation factor IF3 biosynthesis in Escherichia coli; Liveris D et al.; infC, the gene encoding translation initiation factor IF3 in Escherichia coli, can be transcribed from three promoters . Two of these promoters, PI1 and PI2, are located in the upstream thrS sequence which codes for threonyl-tRNA synthetase . Previous studies had shown that PI2 was the major promoter for infC . In the present study, the extent of transcription from PI1 and/or PI2 at a variety of steady-state growth rates was analyzed by promoter fusion studies . PI2 was the more active promoter (two- to threefold stronger than PI1) at all growth rates tested . A fusion plasmid containing both PI1 and PI2 exhibited a transcription level approximately equal to the sum of those observed with the fusion plasmids containing the individual promoters . The transcriptional activities of PI1 and PI2 did not change as the growth rate was varied from 0.3 to 1.7 doublings per h . In contrast, a fusion plasmid carrying the rrnB P1 promoter displayed the expected growth rate response . The steady-state concentrations of infC mRNA in cells grown at different rates were measured and found not to vary . These results indicate that the previously reported growth rate regulation of IF3 biosynthesis neither is accomplished by transcriptional control nor is a result of differential mRNA stability . In view of these results, the steady-state levels of IF3 in cells grown at a number of different growth rates were determined by quantitative immunoblotting . IF3 levels were found to vary with growth rate in a manner essentially identi