J Biol Chem, 1996 Mar 29, 271(13), 7559 - 67 Purification and characterization of intact and truncated forms of the Escherichia coli biotin carboxyl carrier subunit of acetyl-CoA carboxylase; Nenortas E et al.; Biotin biosynthesis and retention in Escherichia coli is regulated by the multifunctional protein, BirA . The protein acts as both the transcriptional repressor of the biotin biosynthetic operon and as a ligase for covalent attachment of biotin to a unique lysine residue of the acetyl-CoA carboxylase . Biotinyl-5'-AMP is the activated intermediate for the ligase reaction and the allosteric effector for DNA binding . We have purified and characterized apoBCCP and a truncated form containing the COOH-terminal 87 residues (apoBCCP87) . Molecular masses of the proteins measured using matrix-assisted laser desorption ionization time-of-flight mass spectrometry conformed to the expected values . The assembly states of apoBCCP and apoBCCP87 were determined using sedimentation equilibrium ultracentrifugation . Nearly quantitative enzymatic transfer of biotin from BirA-biotinyl-5'-AMP to the apoBCCP forms was assessed using two methods, mass spectrometric analysis of acceptor proteins after incubation with BirA-bio-5'-AMP and a steady state fluorescence assay . The BirA catalyzed rates of transfer of biotin from bio-5'-AMP to apoBCCP and apoBCCP87 were measured by stopped-flow fluorescence . Kinetic parameters estimated from these measurements indicate that the intact and truncated forms of the acceptor protein are functionally identical. J Biol Chem, 1996 Mar 29, 271(13), 7535 - 43 Cloning and structure of delta-latroinsectotoxin, a novel insect-specific member of the latrotoxin family: functional expression requires C-terminal truncation; Dulubova IE et al.; The venom of the black widow spider (BWSV) (Latrodectus mactans tredecimguttatus) contains several potent, high molecular mass (>110 kDa) neurotoxins that cause neurotransmitter release in a phylum-specific manner . The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown, and they have not been functionally expressed . This study reports on the primary structure of delta-latroinsectotoxin (delta-LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids . delta-LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain . The domain organization of delta-LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins . The predicted molecular mass and apparent mobility of the protein (approximately 130 kDa) encoded in the delta-LIT gene differs from that of native delta-LIT purified from BWSV (approximately 100 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor . MALDI-MS of purified native delta-LIT revealed a molecular ion with m/z+ of 110916 +/- 100, indicating that the native delta-LIT is 991 amino acids in length . When the full-length delta-LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C-terminally truncated protein containing 991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar concentrations . Channels formed in locust muscle membrane and artificial lipid bilayers by the native delta-LIT have a high Ca2+ permeability, whereas those formed by truncated, recombinant protein do not. J Biol Chem, 1996 Mar 29, 271(13), 7494 - 500 Identification of the high affinity receptor binding region in human immunoglobulin E; Helm BA et al.; We have investigated the capacity of N- and C-terminally truncated and chimeric human (h) IgE-derived peptides to inhibit the binding of 125I-labeled hIgE, and to engage cell lines expressing high and low affinity receptors (Fc-epsilon-RI/II) . The peptide sequence Pro343-Ser353 of the hC-epsilon-3 domain is common to all h-epsilon-chain peptides that recognize hFc-epsilon-RI . This region in IgE is homologous to the A loop in C-gamma-2 that engages the rat neonatal IgG receptor . Optimum Fc-epsilon-RI occupancy by hIgE occurs at pH 6.4, with a second peak at 7.4 . N- or C-terminal truncation has little effect on the association rate of the ligands with this receptor . Dissociation markedly increases following C-terminal deletion, and hFc-epsilon-RI occupancy at pH 6.4 is diminished . His residue(s) in the C-terminal region of the epsilon-chain may thus contribute to the high affinity of interaction . Grafting the homologus rat epsilon-chain sequence into hIgE maintains hFc-epsilon-RI interaction without conferring binding to rat Fc-epsilon-RI . hFc-epsilon-RII interaction is lost, suggesting that these residues also contribute to hFc-epsilon RII binding . h-epsilon-chain peptides comprising only this sequence do not block hIgE/hFc-epsilon-RI interaction or engage the receptor . Therefore, sequences N- or C-terminal to this core peptide provide structures necessary for receptor recognition. J Biol Chem, 1996 Mar 29, 271(13), 7423 - 8 Selective screening of a large phage display library of plasminogen activator inhibitor 1 mutants to localize interaction sites with either thrombin or the variable region 1 of tissue-type plasminogen activator; van Meijer M et al.; Phage display technology has been exploited to study in detail the interaction between plasminogen activator inhibitor 1 (PAI-1) and either thrombin or an essential positively charged "loop" of tissue-type plasminogen activator (t-PA), denoted variable region 1 (VR1) . For this purpose, a PAI-1 mutant phage library was used that served as a reservoir of PAI-1 proteins potentially deficient in the interaction with either VR1 or thrombin . A stringent two-step selection procedure was developed . (i) A negative selection was performed by incubating the pComb3/PAI-1 mutant library with an excess of a thrombin mutant with its VR1 domain substituted with that of t-PA (thrombin-VR1) . (ii) The remaining phages were complexed with t-PA (positive selection) and selected by panning with an immobilized anti-t-PA monoclonal antibody . Four consecutive panning rounds yielded an enrichment of pComb3/PAI-1 mutant phages of approximately 50-fold . Sequence analysis of 16 different cDNAs, encoding PAI-1 mutants that are hampered in the binding to thrombin-VR1, revealed the following mutations . Four independent variants share a mutation of the P4' residue (Glu350 --> Lys) . Nine independent PAI-1 variants share a substitution of P1' (Met347 --> Lys), whereas three others share a P2 substitution (Ala345 --> Asp) . Kinetic analysis of representative PAI-1 mutants provides evidence that the P4' residue is essential for the interaction with the VR1 domain, consistent with the data of Madison et al . (Madison, E.L., Goldsmith, E.J., Gething, M.J., Sambrook, J.F., and Gerard, R.D . (1990) J . Biol . Chem . 265, 21423-21426), whereas the P1' and P2 residues confer thrombin specificity . Concordant with the design of the selection procedure, mutants were obtained that inhibit thrombin-VR1 at least 100-fold slower than wild-type PAI-1, identifying residues that are central to the interaction with either thrombin or VR1 . This study demonstrates that phage technology can be used to analyze large numbers of mutants defective in their interaction with other (domains of) proteins, provided an adequate selection scheme is devised. J Biol Chem, 1996 Mar 29, 271(13), 7404 - 11 A novel cytoplasmic hemimethylated oriC binding activity; Garwood J et al.; Using hemimethylated, fully methylated, and unmethylated oligonucleotide probes corresponding to part of the origin of Escherichia coli DNA replication, oriC (+81-136), we have characterized a novel hemimethylated DNA-specific protein binding activity . This activity appears to be located in the cytoplasm rather than in membrane fractions . It has been partially purified and, in DNase footprinting analysis, found to preferentially protect only a subset of the hemimethylated GATC sites present in the minimal oriC . These sites are found adjacent to the DnaA binding box, R1, and overlap the integration host factor binding site . The activity does not correspond to known hemimethylated binding proteins, although in the seqA deletion mutant, there is a 3-fold reduction of the activity . The stage of the cell cycle in synchronized PC2 cultures does not seem to significantly affect thte relative levels of this binding activity . A possible role in sequestration of the newly replicated hemimethylated origin is discussed. J Biol Chem, 1996 Mar 29, 271(13), 7387 - 91 Site-directed mutagenesis of recombinant sulfite oxidase: identification of cysteine 207 as a ligand of molybdenum; Garrett RM et al.; Each of the four cysteines in rat sulfite oxidase was altered by site-directed mutagenesis to serine, and the mutant proteins were expressed in Escherichia coli . Three of the replacements proved to be silent mutations, while a single cysteine, Cys-207, was found to be essential for enzyme activity . The C207S mutation was also generated in cloned human sulfite oxidase . The mutant human enzyme also displayed severely attenuated activity but was expressed at higher levels allowing purification and spectroscopic analysis . The absorption spectrum of the isolated molybdenum domain of the human C207S mutant displayed marked attenuation of the peak at 350 nm and a lesser decrease in absorbance from 450-600 nm as compared with the native human molybdenum domain . The molybdenum and molybdopterin contents of the two samples were comparable . These data suggest that the major features in the absorption spectrum of the native molybdenum domain arise from the binding of Cys-207 to the molybdenum and indicate that this residue functions as a ligand of the metal. J Biol Chem, 1996 Mar 29, 271(13), 7343 - 50 Adenovirus-mediated transfer of CCAAT/enhancer-binding protein-alpha identifies a dominant antiproliferative role for this isoform in hepatocytes; Diehl AM et al.; CCAAT/enhancer-binding protein (C/EBP) isoforms are thought to be important regulators of the hepatocyte phenotype . However, the specific physiological roles of different isoforms are poorly understood because hepatocytes express multiple C/EBPs, and various isoforms have overlapping functions . To identify the functions of C/EBPalpha in mature hepatocytes, replication-defective adenovirus vectors were used to efficiently and homogeneously overexpress the mouse C/EBPalpha gene in a SV40 virus-conditionally transformed rat hepatocyte line that can be induced to express C/EBPbeta and C/EBPdelta but that has little endogenous C/EBPalpha expression . Hepatocytes were infected with a recombinant adenovirus vector carrying the cDNA for C/EBPalpha driven by Rous sarcoma virus promoter elements (AdCEBPalpha) or a similar vector carrying the Escherichia coli lacZ gene (Adbetagal) . Staining for beta-galactosidase demonstrated an infection efficiency of 100% at a multiplicity of infection of 25 plaque-forming units/cell and persistence of foreign gene expression for at least 9 days . Cultures infected with AdCEBPalpha had 50-fold higher levels of C/EBPalpha mRNA and protein than those infected with Ad-beta-gal, but similar expression of C/EBP-beta . Infection with AdCEBPalpha inhibited proliferation in cells expressing little C/EBPbeta, even when proliferation was driven by the SV40 transforming antigen, and also blunted mitogenic induction of the c-myc proto-oncogene in nontransformed cells with high levels of C/EBPbeta . Although overexpression of C/EBPalpha consistently increased C/EBPalpha DNA binding activity, it was not sufficient for albumin expression . Infection with AdCEBPalpha only increased albumin mRNA levels in nontransformed cells that also expressed relatively high levels of C/EBPbeta . Thus, in hepatocytes, C/EBPalpha has a dominant antiproliferative function, but must interact with other factors to regulate hepatocyte-specific gene expression. J Biol Chem, 1996 Mar 29, 271(13), 7269 - 72 Activation of SoxR-dependent transcription in vitro by noncatalytic or NifS-mediated assembly of {2Fe-2S} clusters into apo-SoxR; Hidalgo E et al.; SoxR is a transcriptional activator that senses superoxide and nitric oxide stress in Escherichia coli . The active protein isolated from E . coli contains a pair of {2Fe-2S} clusters per SoxR dimer . We previously demonstrated that the iron-free protein (apo-SoxR), isolated during purification in thiol-containing buffers, binds soxS promoter DNA with an affinity equal to that of the metalloprotein (Fe-SoxR), but lacks significant ability to activate transcription in vitro . Here we demonstrate the reversibility of this process: the full transcriptional activity of SoxR can be restored by in vitro assembly of iron-sulfur clusters into the apoprotein . Two methods were used to synthesize the metallocenters of SoxR: (i) nonenzymatic, in which apo-SoxR, incubated in the presence of iron, inorganic sulfide, and a reducing agent, regained full transcriptional activity in 5-6 h; (ii) enzymatic, in which NifS protein of Azotobacter vinelandii regenerated active Fe-SoxR in as little as 2 min . Analysis by electron paramagnetic resonance spectroscopy indicated that binuclear {2Fe-2S} clusters were restored by both the enzymatic and nonenzymatic reconstitutions . A mutant SoxR protein missing one of its four cysteine residues failed to undergo either transcriptional activation or the formation of {2Fe-2S} centers, even in the presence of NifS . Thus, only the presence of an iron-sulfur center is required to restore transcriptional activity to apo-SoxR . Moreover, the catalytic generation of {2Fe-2S} centers extends the known specificity of this enzyme beyond that already shown for {4Fe-4S} centers . Catalytic generation of {2Fe-2S}-containing SoxR could allow for rapid activation of this transcription factor in vivo. J Mol Biol, 1996 Mar 29, 257(2), 233 - 45 Selection of Streptomyces griseus protease B mutants with desired alterations in primary specificity using a library screening strategy; Sidhu SS et al.; Streptomyces griseus protease B (SGPB) has primary specificity for large hydrophobic residues . The protease is secreted in a promature form, and autocatalytic removal of the propeptide is essential for activity . We genetically substituted the P1 Leu at the promature junction of SGPB with Phe, Met, or Val and monitored expression levels in Escherichia coli . Substitution with Phe had no effect on active SGPB production; substitution with Met or Val abolished proteolytic activity . An E . coli expression library containing 29,952 possible SGPB mutants was constructed with variations at seven sites involved in conferring primary specificity . A rapid, visual screening strategy was used to detect active protease secretion . The expression library was screened, in conjunction with the different promature junction sequences, for those variants producing increased proteolytic activity . The sequences of the isolated mutant genes were determined; the substrate specificities and thermostabilities of the corresponding protease were investigated . Mutants isolated from the screen with the wild-type promature junction exhibited substrate specificities and thermostabilities similar to wild-type . The screen with Phe at the promature junction P1 site resulted in the isolation of mutant proteases with increased thermostabilities (up to an order of magnitude increase in half-life at 55 degrees C), while a protease with broad substrate specificity was isolated from Val screen . Proteases isolated from the screen with Met at the promature junction P1 site exhibited dramatic increases in activity towards a synthetic substrate with Met at P1 site . The results suggests that the substrate specificity of recombinant SGPB is constrained by the sequence of the promature junction; active protease production is dependent on the efficiency of the self-processive promature junction cleavage . With an efficient screening strategy, this relationship can be used to isolate catalytically active proteases with desired specificities engineered at the promature junction. Eur J Pharmacol, 1996 Mar 28, 299(1-3), 179 - 86 A glucocorticoid receptor-independent mechanism for neurosteroid inhibition of tumor necrosis factor production; Di Santo E et al.; We investigated the effect of two neurosteroids, pregnenolone and dehydroepiandrosterone sulfate on lipopolysaccharide-induced tumor necrosis factor (TNF) production in vivo and in vitro . Dehydroepiandrosterone sulfate (0.3-30 mg/kg, i.p.) inhibited serum TNF induced by lipopolysaccharide (2.5 micrograms/mouse, i.p.), without affecting the induction of serum corticosterone . Intracerebroventricular (i.c.v.) administration of dehydroepiandrosterone sulfate (0.2-5 micrograms/mouse) also inhibited brain TNF induced by i.c.v . lipopolysaccharide (2.5 micrograms/mouse) . Dehydroepiandrosterone sulfate and pregnenolone (10(-6)-10(-4) M) inhibited TNF production in vitro by lipopolysaccharide-stimulated human peripheral blood mononuclear cells or by the human THP-1 cell line, suggesting that this action might also be relevant in humans . We obtained two lines of evidence that neurosteroids do not inhibit TNF via the glucocorticoid receptor . (1) Dehydroepiandrosterone sulfate and pregnenolone did not activate the alpha 1-acid glycoprotein promoter, a typical effect of glucocorticoids mediated by the glucocorticoid receptor, while strong activation of this promoter was observed with dexamethasone . (2) The inhibitory effect of dehydroepiandrosterone sulfate and pregnenolone on TNF production was not reversed by the glucocorticoid receptor antagonist, mifepristone (RU38486) . On the contrary the inhibitory effect of dexamethasone, a classical glucocorticoid and inhibitor of TNF synthesis, was completely reversed by RU38486. Eur J Pharmacol, 1996 Mar 28, 299(1-3), 153 - 9 Inhibition of lipopolysaccharide-induced bowel erythrocyte extravasation in rats, and of mesenteric hypoperfusion in dogs, by phosphodiesterase inhibitors; Cardelus I et al.; Sepsis is intricately associated with mesenteric ischemia . The remote complications of mesenteric ischemia are essentially those of sepsis, whether as a cause or as a consequence . Experimental endotoxic shock induces bowel hypoperfusion, erythrocyte extravasation and intestinal necrosis . The effects of pentoxifylline, rolipram and denbufylline, three phosphodiesterase inhibitors, were studied on endotoxin-induced bowel erythrocyte extravasation and intestinal and renal hypoperfusion, in conscious rats and anaesthetized dogs, respectively . Two hours after lipopolysaccharide i.v . injection in rats, erythrocyte extravasation was evident throughout the intestinal musculature and mucosa, apparently without affecting lungs, heart, kidneys, liver or pancreas . Pretreatment with the non-selective phosphodiesterase inhibitor, pentoxifylline, or selective phosphodiesterase IV inhibitors such as denbufylline or rolipram reduced intestinal haemoconcentration . In the anaesthetized dog, pentoxifylline and denbufylline both inhibited the E . coli lipopolysaccharide-induced mesenteric blood flow fall, without affecting renal blood flow or cardiac index . In conclusion, phosphodiesterase inhibitors protected from intestinal damage and bowel hypoperfusion after lipopolysaccharide challenge . This action may thus play a role in the protective effects against endotoxin-induced lethal toxicity previously described for phosphodiesterase inhibitors. Eur J Pharmacol, 1996 Mar 28, 299(1-3), 119 - 26 A new and potent calmodulin antagonist, HF-2035, which inhibits vascular relaxation induced by nitric oxide synthase; Win NH et al.; HF-2035, 2-{N-(2-aminoethyl)-N-(2,4,5-trichlorobenzenesulfonyl)} amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, was synthesized and its effects on calmodulin-dependent enzymes were investigated . HF-2035 inhibited calmodulin kinase I, calmodulin kinase II and myosin light-chain kinase with IC50 values of 1.3 microM, 1.6 microM and 68 microM, respectively . HF-2035 also inhibited the activity of recombinant rat neuronal nitric oxide synthase, one of the calmodulin-dependent enzymes, with a Ki of 0.78 microM . Partially purified nitric oxide synthase of rat brain was also inhibited by HF-2035 with an IC50 of 3.2 microM . Kinetic analysis indicated that this inhibitory effect of HF-2035 was competitive with respect to calmodulin . We examined the effects of HF-2035 on constitutive nitric oxide synthase in a bioassay using vascular strips of rabbit carotid artery with and without endothelium . HF-2035 inhibited acetylcholine- and calcium ionophore, A23187 (6S-{6 alpha (2S*,3S*),8 beta (R*),9 beta, 11 alpha}-5- (methylamino)-2-{{3,9,11-trimethyl-8-{1-methyl-2-oxo-2-(1H-pyrrol-2-yl)- ethyl}-1,7-dioxaspiro{5.5}undec-2-yl}methyl}-4-benzoxazol ecarboxylic acid)-induced relaxation of endothelium-intact strips with an ED50 of 1.5 +/- 0.5 microM and 2.8 +/- 1 microM, respectively . This compound, however, did not inhibit N-nitroso-N-morpholinoaminoacetonitrile (SIN-1A), an exogenous nitric oxide donor, -induced relaxation of endothelium-denuded strips . W-7 (N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide) inhibited acetylcholine-induced relaxation with an ED50 of 46 +/- 7 microM, which was 30-fold less potent than HF-2035 . HF-2035 was unable to inhibit the activity of the inducible form of nitric oxide synthase in isolated thoracic aorta of rat treated with Escherichia coli lipopolysaccharide . These findings suggest that HF-2035 is a new and potent calmodulin antagonist, and may be used as a mother compound to develop more selective inhibitors of constitutive nitric oxide synthase. Biochim Biophys Acta, 1996 Mar 28, 1273(3), 207 - 16 K+-dependent Na+ transport driven by respiration in Escherichia coli cells and membrane vesicles; Verkhovskaya ML et al.; Respiration-driven Na+ transport from Escherichia coli cells and right-side-out membrane vesicles is strictly dependent on K+ . Cells from an E . colic mutant deficient in three major K+ transport systems were incapable of accumulating K+ or expelling Na+ unless valinomycin was added . Membrane vesicles from an E . coli mutant from which the genes encoding the two known electrogenic Na+/nH+ antiporters nhaA and nhaB were deleted transported Na+ as well as did vesicles from wild-type cells . Quantitative analysis of Delta psi and Delta pH showed a high driving force for electrogenic Na+/nH+ antiport whether K+ was present or not, although Na+ transport occurred only in its presence . These results suggest that an Na+/nH+ antiporter is not responsible for the Na+ transport . Respiration-driven efflux of Na+ from vesicles was found to be accompanied by primary uphill efflux of K+ . Also, no respiration-dependent efflux of K+ was observed in the absence of Na+ . Such coupling between Na+ and K+ fluxes may be explained by the operation of an Na+, K+/H+ antiporter previously described in E . coli membrane vesicles (Verkhovskay, M.L., Verkhovsky, M.I . and Wikstrom, M . (1995) FEBS Lett . 363, 46-48) . Active Na+ transport is abolished when delta mu H+ is eliminated by a protonophore, but at low concentrations the protonophore actually accelerated Na+ transport . Such an effect may be expected if the Na+, K+/H+ antiporter normally operates in tight conjunction with respiratory chain complexes, thus exhibiting some phenomenological properties of a primary redox-linked sodium pump. Biochim Biophys Acta, 1996 Mar 28, 1273(3), 191 - 4 Identification of an aspartic acid residue in the beta subunit which is essential for catalysis and proton pumping by transhydrogenase from Escherichia coli; Meuller J et al.; Based on the alignment of 7 unknown amino acid sequences, including the recently determined sequences for the mouse and human enzymes, a highly conserved acidic domain was identified which in the Escherichia coli enzyme is located close to the C-terminal end of the predicted NADP(H)-binding site of the beta subunit . The effect of replacing the four conserved acidic residues, betaE361, betaE374, betaD383 and betaD392, in this domain on catalytic and proton-pumping activity was tested by site-directed mutagenesis . In addition, betaE371, which is not conserved but located in the same domain, was also mutated . Of these residues, betaAsp 392 proved to be the only residue which is essential for both activities . However, two betaAsp 392 mutants were still partly active in catalyzing the cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADPH, suggesting that the mutations did not cause a global change but rather a subtle local change influencing the dissociation of NADP(H) . It is proposed that betaAsp 392 together with th previously identified betaHis91 form part of a proton wire in transhydrogenase. J Immunol Methods, 1996 Mar 28, 190(1), 143 - 5 Comparison of protocols for depleting anti-E . coli antibody in immunoblotting of recombinant antigens; Covini G et al.; A common problem in immunoblotting for the detection of specific antibodies to recombinant proteins synthesized in E . coli is the presence of contaminating antibodies to E . coli proteins . Four protocols for the efficient depletion of anti-E . coli antibodies from human sera are provided and their uses are discussed. Nature, 1996 Mar 28, 380(6572), 360 - 4 Structural basis of calcium-induced E-cadherin rigidification and dimerization; Nagar B et al.; The cadherins mediate cell adhesion and play a fundamental role in normal development . They participate in the maintenance of proper cell-cell contacts: for example, reduced levels of epithelial cadherin (E-cadherin) correlate with increased invasiveness in many human tumour cell types . The cadherins typically consist of five tandemly repeated extracellular domains, a single membrane-spanning segment and a cytoplasmic region . The N-terminal extracellular domains mediate cell-cell contact while the cytoplasmic region interacts with the cytoskeleton through the catenins . Cadherins depend on calcium for their function: removal of calcium abolishes adhesive activity, renders cadherins vulnerable to proteases (reviewed in ref . 4) and, in E-cadherin, induces a dramatic reversible conformational change in the entire extracellular region . We report here the X-ray crystal structure at 2.0 A resolution of the two N-terminal extracellular domains of E-cadherin in the presence of calcium . The structure reveals a two-fold symmetric dimer, each molecule of which binds a contiguous array of three bridged calcium ions . Not only do the bound calcium ions linearize and rigidify the molecule, they promote dimerization . Although the N-terminal domain of each molecule in the dimer is aligned in a parallel orientation, the interactions between them differ significantly from those found in the neural cadherin (N-cadherin) N-terminal domain (NCD1) structure . The E-cadherin dual-domain structure reported here defines the role played by calcium in the cadherin-mediated formation and maintenance of solid tissues. Nature, 1996 Mar 28, 380(6572), 316 - 22 Structural similarity between TAFs and the heterotetrameric core of the histone octamer; Xie X et al.; A complex of two TFIID TATA box-binding protein-associated factors (TA FIIs) is described at 2.0A resolution . The amino-terminal portions of dTAFII42 and dTAFII62 from Drosophila adopt the canonical histone fold, consisting of two short alpha-helices flanking a long central alpha-helix . Like histones H3 and H4, dTAFII42 and dTAFII62 form an intimate heterodimer by extensive hydrophobic contacts between the paired molecules . In solution and in the crystalline state, the dTAFII42/dTAFII62 complex exists as a heterotetramer, resembling the (H3/H4)2 heterotetrameric core of the histone octamer, suggesting that TFIID contains a histone octamer-like substructure. Biochem Biophys Res Commun, 1996 Mar 27, 220(3), 816 - 23 The dimerization domain upstream binding factor contains multiple helical structures; Lai YS et al.; The upstream binding factor, UBF, is an RNA polymerase I transcription factor which contains multiple DNA binding domains and a novel protein dimerization domain . Active UBF forms homodimers in vivo through the intramolecular interactions of its dimerization domain, which spans a hundred amino-terminal residues . In the presence of both UBF dimerization domain and its immediately adjacent lysine-rich basic DNA binding domain, the E . coli expressed recombinant polypeptide, dbUBF (dimerization plus basic motifs of UBF), forms homodimers in vitro and binds to double-stranded DNA nonselectively . In gel retardation assay, dbUBF dimers make multiple shift-ladders corresponding to numerous protein dimer-DNA complexes . The UBF dimerization domain contains multiple helical structures, as predicted by EMBO-PHD program . Most of hydrophobic residues in the dimerization domain are confined in the hydrophobic phase of these hypothetic helices . Mutating these hydrophobic residues to glutamate prohibits dbUBF association and gives a different shift pattern in gel retardation assay . The results we present here argue that UBF association is largely exerted by the hydrophobic interactions between the multiple helices to bring two molecules together. Biochem Biophys Res Commun, 1996 Mar 27, 220(3), 633 - 6 Depth-targeted efficient gene delivery and expression in the skin by pulsed electric fields: an approach to gene therapy of skin aging and other diseases; Zhang L et al.; The ability to target genes to the various layers, cell types, and appendages of the skin could be used to correct disorders, including those of aging such as wrinkling, as well as utilize specific cell types for production molecules useful elsewhere in the body . However, the stratum corneum acts as a significant physical barrier to gene transfer into the skin . In this report we describe the ability to target and express the lacZ reporter gene to various depths of the dermis region in hairless mice . Skin-depth targeting is achieved by varying pulsed electrical fields and subsequent pressure from caliper-type electrodes on topically applied naked lacZ gene constructs . With electric pulses and extended pressure, the maximum depth of lacZ expression in the dermis and transfected cells was achieved at 370 micron and 457 cells/mm2, respectively . Gene expression was observed only the hair follicles in the case of the control. Biochem Biophys Res Commun, 1996 Mar 27, 220(3), 496 - 501 Mutagenic specificity of ultraviolet light in the tonB gene on the chromosome of Escherichia coli uvrA cells; Kitamura K et al.; We have analyzed the DNA sequence changes in a total of 60 ultraviolet-induced mutations in the endogenous tonB gene of Escherichia coli uvrA strain . Of the mutations 82% were base substitutions among which G:C-->A:T transition predominated . Three GG-->AA tandem double-base substitutions, which are thought to originate from UV damage, were also observed . The sites where base substitutions occurred were correlated with sequences of adjacent pyrimidines, indicating mutation-targeted UV photoproducts . G:C-->A:T transition in the tonB gene mutation can be exclusively observed at either the 3' side of the TC site which is on the template for the lagging strand of DNA replication or the 5'/3' sides of the CC site on the template for the leading strand . We hypothesize that this extreme strand specificity is due to a difference in fidelity of DNA replication of the leading and the lagging strand. Biochem Biophys Res Commun, 1996 Mar 27, 220(3), 1002 - 7 High reductase activity of recombinant NOS2 flavoprotein domain lacking the calmodulin binding regulatory sequence; Rafferty S et al.; Nitric oxide synthases (NOSs) represent a special case of the cytochrome P450: cytochrome P450 reductase system in which the two redox partners occur as distinct domains on the same polypeptide chain and are linked by a calmodulin binding regulatory sequence . We expressed the carboxy-terminal, flavoprotein domain (residues 527-1144) of murine NOS2 in E . coli . The UV-visible spectrum of this domain resembles those of other flavoproteins, and the protein catalyses the reduction of ferricytochrome c by NADPH {Vmax = 3.1 +/- .1 mol cytochrome reduced/minute/mmol flavoprotein domain, Km (cytochrome c) = 23 +/- 2 microM, Km (NADPH) 0.30 +/- .06 microM} . The high reductase activity of this NOS2 flavoprotein domain, which lacks a calmodulin binding site, precludes a role for this site in mediating electron transfer within the flavoprotein domain of the intact enzyme . This is in contrast to the case of NOS1 and suggests that electron transfer is regulated differently in the two isoforms. Biochemistry, 1996 Mar 26, 35(12), 3837 - 44 Zinc stimulates Mg2+-dependent 3'-processing activity of human immunodeficiency virus type 1 integrase in vitro; Lee SP et al.; Human immunodeficiency virus type 1 integrase (HIV-1 IN) catalyzes both 3'-donor processing and strand transfer reactions . Previous studies have determined that the N-terminal region, a putative zinc finger, is capable of binding Zn2+ . The function of zinc coordination to this domain, however, is still unknown . In this report, we present evidence that Mg2+-dependent 3'-donor processing by HIV-1 IN is enhanced by the addition of Zn2+ in vitro . This activity is inhibited in the presence of the chelator 1,10-phenanthroline (OP) . In addition, the Mg2+-dependent 3'-donor processing activity is more sensitive to the concentration of IN than is the Mn2+-dependent activity . A combination of dimethyl sulfoxide (DMSO) and poly(ethylene glycol) (PEG) was found to further activate the Mg2+-dependent 3'-donor processing activity while diminishing the Mn2+-dependent activity . These results suggest factors such as substrate-length, concentration of IN, Zn2+ coordination, and protein-protein interactions are important for efficient and specific donor processing activity with Mg2+ in vitro. Biochemistry, 1996 Mar 26, 35(12), 3735 - 45 Interactions between RNA polymerase and the positive and negative regulators of transcription at the Escherichia coli gal operon; Dalma-Weiszhausz DD et al.; The simultaneous binding of Gal repressor (GalR), catabolite activator protein (CAP or CRP), and RNA polymerase (RNAP) to the promoter region of the Escherichia coli gal operon has been analyzed thermodynamically, by quantitative DNase I "footprint" titration analysis, and structurally, by the use of hydroxyl radical (.OH) and 5-phenylphenanthroline (5OPP) "footprinting." In the absence of regulatory proteins, the preference of RNAP for one (P1) of the two gal operon overlapping promoters (P1 and P2) is -0.4 +/- 0.2 kcal/mol, indicating only a small energetic preference for P1 . The simultaneous binding of CAP and RNAP occurs with 10-fold cooperativity, with greater than 99% of the CAP-RNAP complex present at the P1 promoter . This cooperativity is inhibited by the binding of GalR to the upstream operator, OE, but does not result in the repartitioning of RNAP between the P1 and P2 promoters . These results suggest that the CAP-RNAP cooperativity and promoter partitioning are not linked and are consistent with a mechanism by which GalR binding to OE represses transcription by inhibiting the CAP-RNAP cooperativity . It is suggested that the CAP-RNAP cooperativity is dependent upon contacts made by the complex with the upstream DNA and that GalR binding to OE prevents these contacts from occurring . Changes in nuclease reactivity at the internal operator OI (centered at +53.5) take place upon RNAP binding . These changes are dependent on the DNA sequence present at OI and on the presence or absence of CAP . They are independent of the helical phasing between the promoters and OI and of the distance between them . These results suggest that RNAP can directly communicate with events occurring at both the external and the internal operator sequences without direct contact between repressor molecules bound at their cognate sites. Biochemistry, 1996 Mar 26, 35(12), 3704 - 11 Phosphorylation of beta III-tubulin; Khan IA et al.; There is considerable evidence that mammalian beta-tubulin is phosphorylated . Specifically, of the seven beta isotypes, the phosphorylated one is beta III, the isotype found almost entirely in neurons . The phosphate is added at a serine and perhaps a tyrosine near the C-terminus . All the evidence to date has been gathered by growth of cells and tissues in the presence of radioactive inorganic phosphate followed by tubulin isolation and determination of the labeled tubulin; thus, the actual extent of phosphorylation of beta III is unknown . Nor is it known if alpha-tubulin and the other beta isotypes are phosphorylated by a mechanism which would not be revealed by previous experiments . In addition, the role of tubulin phosphorylation is unknown . We have purified the alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers from bovine brain and have determined their phosphate content chemically . We have found that alpha-tubulin is not phosphorylated and neither are the beta II or beta IV isotypes . However, beta III is phosphorylated with a stoichiometry of about 1.52 mol/mol . We have found that the phosphate on beta III is resistant to a wide variety of phosphatases except for human erythrocyte phosphatase 2A and that removal of the phosphate inhibits microtubule assembly in vitro stimulated by microtubule-associated protein 2 (MAP 2) . However such an inhibition was not evident when microtubule assembly was induced in the absence of microtubule-associated proteins . Our results suggest the possibility that beta III phosphorylation may play a role in regulating microtubule assembly in vivo. Mutat Res, 1996 Mar 26, 351(1), 33 - 43 Mutagenicity of acridines in a reversion assay based on tetracycline resistance in plasmid pBR322 in Escherichia coli; Hoffman GR et al.; The mutagenicity of a series of acridine compounds was studied in an assay based on the reversion of mutations in the tetracycline-resistance gene (tet) of plasmid pBR322 in Escherichia coli . Mutations that restore the tetracycline-resistant phenotype were detected in tetracycline-sensitive strains carrying mutant plasmids . Mutations that revert by +2, +1, -1 and -2 frameshift mutations and by base-pair substitutions were used to analyze the mutagenicity of two simple acridines, two acridine mustards, and a nitroacridine . The simple acridines (9-aminoacridine and quinacrine) effectively induced -1 frameshifts and weakly induced +1 frameshifts . The acridine mustards (quinacrine mustard and ICR-191) were more potent inducers of -1 and +1 frameshifts than the simple acridines . Reactive acridines, including both the mustards and the nitroacridine Entozon, were effective inducers of -2 frameshifts but the simple acridines were not . The two classes of reactive acridines differed from one another, in that the mustards were better inducers of +1 frameshifts than Entozon, whereas Entozon was a particularly potent inducer of -2 frameshifts . None of the compounds induced +2 frameshifts, and the induction of base-pair substitutions was negligible . These results confirm and extend studies showing that adduct-forming acridines are stronger frameshift mutagens than simple intercalating acridines and that the acridines differ from one another not only in overall mutagenic potency but also in the prevalence of different classes of frameshift mutations. FEBS Lett, 1996 Mar 25, 383(1-2), 51 - 4 Potato yellow mosaic geminivirus AC2 protein is a sequence non-specific DNA binding protein; Sung YK et al.; The AC2 protein of potato yellow mosaic geminivirus (PYMV) is by analogy with related geminiviruses thought to be a transcriptional activator protein . We have over-expressed the AC2 open reading frame in E . coli and purified the protein from bacterial extracts to near homogeneity . We have studied the interaction of the AC2 protein with DNA and from gel retardation assays shown that it binds both double-stranded (ds) and single-stranded (ss) DNA non-specifically . The binding to PYMV intergenic region ds DNA appeared to be independent of the presence of zinc ions and did not require the protein to be phosphorylated. FEBS Lett, 1996 Mar 25, 383(1-2), 124 - 8 Characterization of the dystrophin-syntrophin interaction using the two-hybrid system in yeast; Castello A et al.; The carboxy-terminal region of dystrophin has previously been shown to interact directly with alpha1 syntrophin, a cytoplasmic component of the dystrophin-glycoprotein complex, by in vitro biochemical studies such as overlay assay or immunoprecipitation . Using the two-hybrid system, we have isolated from a human heart cDNA library the entire coding sequence of human alpha1 syntrophin, therefore confirming for the first time this interaction via an in vivo approach . In addition, we have reduced the interaction domain to the distal half of alpha1 syntrophin. FEBS Lett, 1996 Mar 25, 383(1-2), 119 - 23 Production and crystallization of MHC class I B allele single peptide complexes; Reid SW et al.; Major histocompatibility complex class I B alleles, HLA B8, B53 and B3501 have been cloned, expressed, refolded and crystallized in specific complexes with a number of different 8-mer and 9-mer peptides . For some of these crystallization was initiated by cross-seeding between different B allele complexes . All crystallize in the space group P212121, with similar unit cell dimensions of approximately 52 A X 81 A X 112 A, contain one complex per asymmetric unit and diffract to approximately 2.0 A resolution. FEBS Lett, 1996 Mar 25, 383(1-2), 105 - 8 Conformational changes in subdomain I of actin induced by proteolytic cleavage within the DNase I-binding loop: energy transfer from tryptophan to AEDANS; Kuznetsova I et al.; Alteration of the actin polypeptide chain within the DNase I-binding loop by cleavage with E . coli A2 protease or subtilisin was shown to increase the efficiency of energy transfer from tryptophan residues to AEDANS attached to Cys-374 . Analysis of structural and fluorescence data suggested that only two of four actin tryptophan residues, namely, Trp-340 and/or Trp-356, can be energy transfer donors . It was also found that labelling with AEDANS induces perturbations in the environment of the tryptophan residues, these perturbations being smaller in the cleaved actin . These changes are consistent with a shift of the C-terminal segment of actin monomer upon cleavage and confirm the existence of high conformational coupling between subdomains 1 and 2 of actin monomer . We also suggest that tryptophan residues 340 and/or 356 are located in the focus of this coupling. Mol Cell Biochem, 1996 Mar 23, 156(2), 117 - 24 Reduction of methylglyoxal in Escherichia coli K12 by an aldehyde reductase and alcohol dehydrogenase; Misra K et al.; Two enzymes, one NADPH-dependent and another NADH-dependent which catalyze the reduction of methylglyoxal to acetol have been isolated and substantially purified from crude extracts of Escherichia coli K12 cells . Substrate specificity and formation of acetol as the reaction product by both the enzymes, reversibility of NADH-dependent enzyme with alcohols as substrates and inhibitor study with NADPH-dependent enzyme indicate that NADPH-dependent and NADH-dependent enzymes are identical with an aldehyde reductase (EC 1.1.1.2) and alcohol dehydrogenase (EC 1.1.1.1) respectively . The K(m) for methylglyoxal have been determined to be 0.77 mM for NADPH-dependent and 3.8 mM for NADH-dependent enzyme . Stoichiometrically equimolar amount of acetol is formed from methylglyoxal by both NADPH- and NADH-dependent enzymes . In phosphate buffer, both the enzymes are active in the pH range of 5.8-6.6 with no sharp pH optimum . Molecular weight of both the enzymes were found to be 100,000 +/- 3,000 by gel filtration on a Sephacryl S-200 column . Both NADPH- and NADH-dependent enzymes are sensitive to sulfhydryl group reagents. Carbohydr Res, 1996 Mar 22, 283, 27 - 51 Syntheses of 1-O-carboxyalkyl GLA-60 analogues; Shiozaki M et al.; As part of our ongoing study to survey potent LPS antagonists, the following six compounds were synthesized in an efficient manner: 3-carboxypropyl and carboxymethyl 2-deoxy-2-(2,2-difluorotetradecanamido)-4-O-phosphono-3-O-{(R)-3- (tetradecanoyloxy)tetradecanoyl}-alpha- and beta-D-glucopyranosides (11 and 23; 32 and 36), as well as the non-fluorinated equivalents, carboxymethyl 2-deoxy-4-O-phosphono-2-tetradecanamido-3-O-{(R)-3-(tetradecano yloxy)- tetradecanoyl}-alpha-D-glucopyranoside (44) and carboxymethyl 2-deoxy-2-{(R)-3-(hydroxy)tetradecanamido}-4-O-phosphono-3-O-{(R)- 3- (tetradecanoyloxy)tetradecanoyl}-alpha-D-glucopyranoside (48) . Of these compounds, 32 was most pronounced in terms of LPS-antagonistic activity. J Biol Chem, 1996 Mar 22, 271(12), 7212 - 7 GroEL binds to and unfolds rhodanese posttranslationally; Reid BG et al.; The Escherichia coli chaperone GroEL is a member of a class of molecular chaperones that possesses a stacked double ring structure containing seven subunits per ring, with approximately 60-kDa subunits . It has been suggested that newly synthesized proteins may interact with a eukaryotic homolog of GroEL co-translationally, thereby sequestering the unfolded protein from other proteins in the cell . To test whether it is essential for GroEL to form a stable interaction with a nascent polypeptide co-translationally, we translated the well studied GroEL substrate rhodanese in bacterial and wheat germ translation extracts . We found that rhodanese formed stable complexes with GroEL solely posttranslationally . Upon binding to GroEL, the protease resistant N-terminal domain of rhodanese unfolds . This interaction with GroEL leads to productive folding of the full-length rhodanese . We conclude that GroEL is able to assist in the folding of newly synthesized proteins following release from the ribosome and that GroEL can unfold a trapped protein folding intermediate of rhodanese. J Biol Chem, 1996 Mar 22, 271(12), 7177 - 86 Analysis of incision sites produced by human cell extracts and purified proteins during nucleotide excision repair of a 1,3-intrastrand d(GpTpG)-cisplatin adduct; Moggs JG et al.; Nucleotide excision repair by mammalian enzymes removes DNA damage as part of approximately 30-mer oligonucleotides by incising phosphodiester bonds on either side of a lesion . We analyzed this dual incision reaction at a single 1,3-intrastrand d(GpTpG)-cisplatin cross-link in a closed circular duplex DNA substrate . Incisions were formed in the DNA with human cell extracts in which DNA repair synthesis was inhibited . The nicks were mapped by restriction fragment end labeling and primer extension analysis . Principal sites of cleavage were identified at the 9th phosphodiester bond 3' to the lesion and at the 16th phosphodiester bond 5' to the lesion . The predominant product was found to be a 26-mer platinated oligonucleotide by hybridization to a 32P-labeled complementary DNA probe . Oligonucleotides were formed at the same rate as the 3' cleavage, suggesting that both incisions are made in a near-synchronous manner . There was, however, a low frequency of 5' incisions in the absence of 3' cleavage . The dual incision reaction was reconstituted using the purified mammalian proteins XPA, RPA, XPC, TFIIH, XPG, and a fraction containing ERCC1-XPF and IF7 . All of these components were required in order to observe any cleavage. J Biol Chem, 1996 Mar 22, 271(12), 7072 - 8 The plasmid RK2 initiation protein binds to the origin of replication as a monomer; Toukdarian AE et al.; The TrfA protein encoded by the broad host range bacterial plasmid RK2 specifically binds to eight direct repeats (iterons) present at the plasmid replication origin to initiate DNA replication . Purified TrfA protein is largely in the form of a dimer, and using a dimerization test system that involves the fusion of the amino-terminal domain of the lambda cI repressor protein to TrfA, we show that the TrfA protein forms dimers in vivo . Because of the high stability of the dimer form of TrfA, the formation of heterodimers between the wild-type and different sized TrfA proteins requires in vivo de novo folding of the primary protein sequence or in vitro denaturation and renaturation . The results of gel mobility shift assays using in vitro or in vivo formed heterodimers indicated that the TrfA protein binds to the iteron DNA as a monomer . Furthermore, when the monomeric and dimeric forms of TrfA are separated by gel filtration chromatography, only the protein in the chromatographic position of the monomeric form demonstrated significant DNA binding activity . These results indicate that only the monomer form of the TrfA protein is active for binding to the iterons at the RK2 replication origin. J Biol Chem, 1996 Mar 22, 271(12), 7052 - 60 A novel plant calmodulin-binding protein with a kinesin heavy chain motor domain; Reddy AS et al.; Calmodulin, a ubiquitous calcium-binding protein, regulates many diverse cellular functions by modulating the activity of the proteins that interact with it . Here, we report isolation of a cDNA encoding a novel kinesin-like calmodulin-binding protein (KCBP) from Arabidopsis using biotinylated calmodulin as a probe . Calcium-dependent binding of the cDNA-encoded protein to calmodulin is confirmed by 35S-labeled calmodulin . Sequence analysis of a full-length cDNA indicates that it codes for a protein of 1261 amino acids . The predicted amino acid sequence of the KCBP has a domain of about 340 amino acids in the COOH terminus that shows significant sequence similarity with the motor domain of kinesin heavy chains and kinesin-like proteins and contains ATP and microtubule binding sites typical of these proteins . Outside the motor domain, the KCBP has no sequence similarity with any of the known kinesins, but contains a globular domain in the NH2 terminus and a putative coiled-coil region in the middle . By analyzing the calmodulin binding activity of truncated proteins expressed in Escherichia coli, the calmodulin binding region is mapped to a stretch of about 50 amino acid residues in the COOH terminus region of the protein . Using a synthetic peptide, the calmodulin binding domain is further narrowed down to a 23-amino acid stretch . The synthetic peptide binds to calmodulin with high affinity in a calcium-dependent manner as judged by electrophoretic mobility shift assay of calmodulin-peptide complex . The KCBP is coded by a single gene and is highly expressed in developing flowers and suspension cultured cells . Although many kinesin heavy chains and kinesin-like proteins have been extensively characterized at the biochemical and molecular level in evolutionarily distant organisms, none of them is known to bind calmodulin . The plant kinesin-like protein with a calmodulin binding domain and a unique amino-terminal region is a new member of the kinesin superfamily . The presence of a calmodulin-binding motif in a kinesin heavy chain-like protein suggests a role for calcium and calmodulin in kinesin-driven motor function(s) in plants. J Biol Chem, 1996 Mar 22, 271(12), 7038 - 42 A mutation in which alanine 128 Is replaced by aspartic acid abolishes dimerization of the b-subunit of the F0F1-ATPase from Escherichia coli; Howitt SM et al.; Site-directed mutagenesis was used to investigate the roles of a short series of hydrophobic amino acids in the b-subunit of the Escherichia coli F0F1-ATPase . A mutation affecting one of these, G131D, had been previously characterized and was found to interrupt assembly of the F0F1-ATPase (Jans, D . A., Hatch, L., Fimmel, A . L., Gibson, D., and Cox, G . B . (1985) J . Bacteriol . 162, 420-426) . To extend this work, aspartic acid was substituted for each one of the residues from positions 124 to 132 . The properties of mutants in this series are consistent with the region from Val124 to Gly131 forming an alpha-helix . Two of the mutations, V124D and A128D, resulted in a similar phenotype to the G131D mutation . This suggested that Val124, Ala128, and Gly131 form a helical face which may have a role in inter- or intrasubunit interactions . This was tested by overexpressing and purifying the cytoplasmic domains of the wild type and A128D mutant b-subunits . Sedimentation equilibrium centrifugation indicated that the wild type domain formed a dimer whereas the mutant was present as a monomer. J Biol Chem, 1996 Mar 22, 271(12), 6947 - 51 Activation of Gsalpha by the epidermal growth factor receptor involves phosphorylation; Poppleton H et al.; Previous studies from our laboratory have shown that epidermal growth factor (EGF) stimulates cAMP accumulation in the heart via a process involving Gsalpha and the EGF receptor (EGFR) protein tyrosine kinase activity (Nair, B . G., Parikh, B., Milligan, G., and Patel, T . B . (1990) J . Biol . Chem . 265, 21317-21322; Nair, B . G., and Patel, T . B . (1993) Biochem . Pharmacol . 46, 1239-1245) . Therefore, studies were performed to investigate the hypothesis that the EGFR protein tyrosine kinase phosphorylates Gsalpha and activates this protein . Employing purified EGFR and Gsalpha, we have demonstrated that the EGFR kinase phosphorylates Gsalpha in a time-dependent manner with a stoichiometry of 2 mol of phosphate incorporated/mol of Gsalpha . As determined by phosphoamino acid analysis, the phosphorylation of Gsalpha by the EGFR kinase was exclusively on tyrosine residues . Interestingly, GDP and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) inhibited the phosphorylation of Gsalpha without altering EGFR autophosphorylation . However, G protein betagamma subunits protected against GDP- and GTPgammaS-mediated inhibition of phosphorylation of Gsalpha . In functional studies, phospho-Gsalpha demonstrated a greater GTPase activity and also a greater capacity to bind GTPgammaS as compared to the nonphosphorylated Gsalpha . Moreover, the phospho-Gsalpha augmented adenylyl cyclase activity in S49 cyc- cell membranes to a greater extent than its nonphosphorylated counterpart . Therefore, we conclude that phosphorylation of Gsalpha on tyrosine residues by the EGFR kinase activates this G protein and increases its ability to stimulate adenylyl cyclase. J Biol Chem, 1996 Mar 22, 271(12), 6895 - 902 Human stem cell factor dimer forms a complex with two molecules of the extracellular domain of its receptor, Kit; Philo JS et al.; Stem cell factor (SCF) is a cytokine that is active toward hematopoietic progenitor cells and other cell types, including germ cells, melanocytes, and mast cells, which express its receptor, the tyrosine kinase, Kit . SCF exists as noncovalently associated dimer at concentrations where it has been possible to study its quaternary structure; it stimulates dimerization and autophosphorylation of Kit at the cell surface . We have used recombinant versions of human SCF and human Kit extracellular domain (sKit) to study SCF-Kit interactions . By size exclusion chromatography, plus various physical chemical methods including light scattering, sedimentation equilibrium, and titration calorimetry, we demonstrate the formation of complexes containing a dimer of SCF (unglycosylated SCF1-165) plus two molecules of sKit . The concentrations of SCF and sKit in these studies were in the range of 0.35-16.2 microM . The data are analyzed and discussed in the context of several possible models for complex formation . In particular, the sedimentation data are not consistent with a model involving cooperative binding . The Kd estimate for SCF-sKit interaction, obtained by sedimentation equilibrium, is about 17 nm at 25 degrees C . With glycosylated SCF1-165, the Kd is considerably higher. J Biol Chem, 1996 Mar 22, 271(12), 6827 - 31 The free radical of the anaerobic ribonucleotide reductase from Escherichia coli is at glycine 681; Sun X et al.; The anaerobic ribonucleoside triphosphate reductase of Escherichia coli is an iron-sulfur protein carrying an oxygen-sensitive organic radical, which is essential for catalysis . The radical was tentatively proposed to be on glycine 681, based on a comparison with the glycyl radical-containing enzyme pyruvate formate-lyase . By EPR spectroscopy of selectively 2H- and 13C-labeled anaerobic ribonucleotide reductase, the radical was now unambiguously assigned to carbon-2 of a glycine residue . The large 1H hyperfine splitting (1.4 millitesla) was assigned to the alpha-proton . Site-directed mutagenesis was used to change glycine 681 into an alanine residue . In separate experiments, the two adjacent residues, cysteine 680 and tyrosine 682, were changed into serine and phenylalanine, respectively . All mutated proteins were retained on dATP-Sepharose, indicating that the mutant proteins had intact allosteric sites . They also contained amounts of iron comparable with the wild type reductase and showed the same iron-sulfur-related spectrum, suggesting that the mutant proteins were properly folded . Of the three mutant proteins only the G681A protein completely lacked the detectable glycyl radical as well as enzyme activity . Our results identify glycine 681 as the stable free radical site in E . coli anaerobic ribonucleotide reductase. J Biol Chem, 1996 Mar 22, 271(12), 6801 - 9 Wild-type Escherichia coli cells regulate the membrane lipid composition in a "window" between gel and non-lamellar structures; Morein S et al.; Escherichia coli strain K12 was grown at 17, 27, and 37 degrees C . The acyl chain composition of the membrane lipids varied with the growth temperature; the fraction of cis-vaccenoyl chains decreased, and the fraction of palmitoyl chains increased, when the growth temperature was increased . However, the polar head group composition did not change significantly . The equilibria between lamellar and reversed non-lamellar phases of lipids extracted from the inner membrane (IM), and from both the membranes (IOM), were studied with NMR and x-ray diffraction . At temperatures above the growth temperature the lipid extracts formed a reversed hexagonal phase, or a bicontinuous cubic phase, depending on the degree of hydration of the lipids . It was observed that: 1) at equal elevations above the growth temperature, IM lipid extracts, as well as IOM lipid extracts, have a nearly equal ability to form non-lamellar phases; 2) IM extracts have a stronger tendency than IOM extracts to form non-lamellar phases; 3) non-lamellar phases are formed under conditions that are relatively close to the physiological ones; the membrane lipid monolayers are thus "frustrated"; and 4) as a consequence of the change of the acyl chain structures, the temperature for the lamellar gel to liquid crystalline phase transition is changed simultaneously, and in the same direction, as the temperature for the lamellar to non-lamellar phase transition . With a too large fraction of saturated acyl chains the membrane lipids enter a gel state, and with a too large fraction of unsaturated acyl chains the lipids transform to non-lamellar phases . It is thus concluded that the regulation of the acyl chain composition in wild-type cells of E . coli is necessary for the organism to be able to grow in a "window" between a lamellar gel phase and reversed non-lamellar phases. J Biol Chem, 1996 Mar 22, 271(12), 6789 - 93 Cloning, sequencing, and expression in escherichia coli of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes; Abe K et al.; OxlT is the oxalate/formate exchange protein that represents the vectorial component of a proton-motive metabolic cycle in Oxalobacter formigenes . Here we report the cloning and sequencing of OxlT and describe its expression in Escherichia coli . The OxlT amino acid sequence specifies a polytopic hydrophobic protein of 418 residues with a mass of 44,128 daltons . Analysis of hydropathy and consideration of the distribution of charged residues suggests an OxlT secondary structure having 12 transmembrane segments, oriented so that the N and C termini face the cytoplasm . Expression of OxlT in E . coli coincides with appearance of a capacity to carry out the self-exchange of oxalate and the heterologous, electrogenic exchange of oxalate with formate . The unusually high velocity of OxlT-mediated transport is also preserved in E . coli . We conclude that the essential features of OxlT are retained on its expression in E . coli. J Biol Chem, 1996 Mar 22, 271(12), 6736 - 45 Glutaredoxin-3 from Escherichia coli . Amino acid sequence, 1H AND 15N NMR assignments, and structural analysis; Aslund F et al.; The primary and secondary structure of glutaredoxin-3 (Grx3), a glutathione-disulfide oxidoreductase from Escherichia coli, has been determined . The amino acid sequence of Grx3 consists of 82 residues and contains a redox-active motif, Cys-Pro-Tyr-Cys, typical of the glutaredoxin family . Sequence comparison reveals a homology (33% identity) to that of glutaredoxin-1 (Grx1) from E . coli as well as to other members of the thioredoxin superfamily . In addition to the active site cysteine residues, Grx3 contains one additional cysteine (Cys65) corresponding to one of the two non-active site (or structural) cysteine residues present in mammalian glutaredoxins . The sequence-specific 1H and 15N nuclear magnetic resonance assignments of reduced Grx3 have been obtained . From a combined analysis of chemical shifts, 3JHNalpha coupling constants, sequential and medium range NOEs, and amide proton exchange rates, the secondary structure of reduced Grx3 was determined and found to be very similar to that inferred from amino acid sequence comparison to homologous proteins . The consequences of the proposed structural similarity to Grx1 are that Grx3, while possessing a largely intact GSH binding cleft, would have a very different spatial distribution of charged residues, most notably surrounding the active site cysteine residues and occurring in the proposed hydrophobic protein-protein interaction area . These differences may contribute to the observed very low Kcat of Grx3 as a reductant of insulin disulfides or as a hydrogen donor for ribonucleotide reductase . Thus, despite an identical active site disulfide motif and a similar secondary structure and tertiary fold, Grx3 and Grx1 display large functional differences in in vitro protein disulfide oxido-reduction reactions. J Biol Chem, 1996 Mar 22, 271(12), 6713 - 9 Equilibrium and kinetic measurements reveal rapidly reversible binding of Ras to Raf; Gorman C et al.; Raf is a serine/threonine kinase that binds through its amino-terminal regulatory domain to the GTP form of Ras and thereby activates the mitogen-activated protein kinase pathway . In this study, we have characterized the interaction of the Ras-binding domain of Raf with Ras using equilibrium binding methods (scintillation proximity assay and fluorescence anisotropy), rather than with more widely used nonequilibrium procedures (such as enzyme-linked immunosorbent assay and affinity precipitation) . Initial studies using glutathione S-transferase fusion proteins with either residues 1-257 or 1-190 of Raf showed that although it was possible to detect Ras binding using an enzyme-linked immunosorbent assay or affinity precipitation, it was substoichiometric; under equilibrium conditions with only a small excess of Raf almost no binding was detected . This difference was probably due to the presence of a high percentage of inactive Raf protein . Further studies used protein containing residues 51-131 of Raf, which expressed in Escherichia coli as a stable glutathione S-transferase fusion . With this protein, binding with Ras could readily be measured under equilibrium conditions . The catalytic domain of neurofibromin inhibited binding of Ras to Raf, and Raf inhibited the binding of Ras to neurofibromin showing that Raf and neurofibromin cannot be bound simultaneously to Ras . The affinities of interaction of neurofibromin and Raf with Harvey-RasLeu-61 were similar . The rate constant for dissociation of Raf from Ras was estimated to be >1 min-1, suggesting that Ras, Raf, and neurofibromin may be in rapid equilibrium in the cell . In contrast to previous reports, under equilibrium conditions there was no evidence for a difference in affinity between the minimal Ras binding domain of Raf (residues 51-131) and a region containing an additional 16 carboxyl-terminal amino acids, suggesting that residues 132-147 do not form a critical binding determinant. J Biol Chem, 1996 Mar 22, 271(12), 6686 - 93 Selective sugar binding to the carbohydrate recognition domains of the rat hepatic and macrophage asialoglycoprotein receptors; Iobst ST et al.; Asialoglycoprotein receptors on the surfaces of both hepatocytes and peritoneal macrophages bind terminal galactose residues of desialylated glycoproteins and mediate endocytosis and eventual degradation of these ligands . The hepatic receptor binds oligosaccharides with terminal N-acetylgalactosamine residues more tightly than ligands with terminal galactose residues, but the macrophage receptor shows no such differential binding affinity . Carbohydrate recognition domains from the macrophage receptor and the major subunit of the hepatic receptor have been expressed in a bacterial system and have been shown to retain the distinct binding selectivities of the receptors from which they derive . Binding of a series of N-acyl derivatives of galactosamine suggests that the 2-substituent of these sugars interacts with the surface of the hepatic receptor with highest affinity binding observed for the N-propionyl derivative . Chimeric sugar-binding domains have been used to identify three regions of the hepatic receptor that are essential for establishing selectivity for N-acetylgalactosamine over galactose . Based on these results and the orientation of N-acetylgalactosamine when bound to an homologous galactose-binding mutant of rat serum mannose-binding protein, a fourth region likely to interact with N-acetylgalactosamine has been identified and probed by site-directed mutagenesis . The results of these studies define a binding pocket for the 2-substituent of N-acetylgalactosamine in the hepatic asialoglycoprotein receptor. J Biol Chem, 1996 Mar 22, 271(12), 6611 - 7 The leucine-responsive regulatory protein (Lrp) from Escherichia coli . Stoichiometry and minimal requirements for binding to DNA; Cui Y et al.; Lrp (Leucine-responsive regulatory protein) regulates the expression of a number of operons in Escherichia coli . A recent study of DNA sequences recognized by Lrp established the consensus as a 15-bp sequence, YAGHAWATTWTDCTR (Y = C/T, H = "not G," W = A/T, D ="not C," R = A/G) (Cui, Y., Wang, Q., Stormo, G . D., and Calvo, J . M . (1995) J . Bacteriol . 177, 4872-4880) . Here we report the stoichiometry of Lrp binding (an Lrp dimer binds to a single binding site) and studies that define the minimal length of DNA required for binding . A double-stranded 15 mer having a sequence that closely matches the consensus does not show measurable binding to Lrp . One or two base pairs of DNA flanking each end are not sufficient for binding, but constructs having 3-5 additional base pairs (21 mer) show relatively strong binding . Single-stranded flanking DNA also contributes to strong binding . The extent of the contribution to binding is dependent upon whether the single strand is on the left or right of the double-stranded region and whether the polarity of the single-stranded DNA is 5' to 3' or 3' to 5'. J Mol Biol, 1996 Mar 22, 257(1), 9 - 20 Identification of functional regions of the Nun transcription termination protein of phage HK022 and the N antitermination protein of phage lambda using hybrid nun-N genes; Henthorn KS et al.; Phages lambda and HK022 express proteins N and Nun, respectively, each of which acts with a number of Escherichia coli host Nus factors at lambda NUT RNA sites, to influence transcription elongation . The lambda nut sites, nearly identical sequences located downstream of the early promoters, pL and pR, were first identified as cis-acting signals required for the action of N in forming termination-resistant transcription complexes . Surprisingly, the Nun protein, resembling N and expressed by another lambdoid phage, HK022, also acts with Nus proteins to terminate specifically transcription initiating at pL and pR near the lambda nut sites . Based on structural considerations of the amino acid sequences, we have constructed nine hybrid N-nun genes and used these hybrids to identify functional regions of the N and Nun proteins . Three classes of hybrid gene products were identified: (1) those that, like N, support antitermination, (2) those that, like Nun, terminate transcription, and (3) those that block N action but do not terminate transcription . We find that, similar to N, the amino-terminal portion of Nun is involved in RNA recognition . The more carboxy portions influence transcription elongation, antitermination (N) and termination (Nun) . Depending on the derivations of the more carboxy regions, hybrids with either the N or Nun amino portions support either termination or antitermination . The activity of a hybrid protein may be influenced by the host strain depending on the nature of the rpoC locus, a locus encoding the beta' subunit of RNA polymerase . One of the hybrid proteins blocks antitermination when the rpoC locus is wild-type . The same hybrid in the presence of the rpoC100 mutation, which alters the beta' subunit, has antitermination activity . This result supports the argument that the beta' subunit plays an essential role in determining the progress of transcription elongation. J Mol Biol, 1996 Mar 22, 257(1), 21 - 9 Repression of lac promoter as a function of distance, phase and quality of an auxiliary lac operator; Muller J et al.; The tetrameric Lac repressor can bind simultaneously to two lac operators on the same DNA molecule, thereby including the formation of a DNA loop . We investigated the phasing dependence of DNA loop formation between lac operator O1 and an auxiliary ideal lac operator (O(id)) on the bacterial chromosome, with inter-operator distances varying from 57.5 to 1493.5 bp . Repression of a CAP-independent lac UV5 promoter by O1 at its natural position increased up to 50-fold in the presence of an optimally positioned auxiliary O(id)) . Repression values alternated between local maxima and minima with a periodicity of 11.0 to 11.3 bp, suggesting that the chromosomal helical repeat is in this range in vivo . Repression increased significantly with decreasing inter-operator DNA length, indicating that the local Lac repressor concentration at O1 is crucial for tight repression . Maximal repression, attributed to stable DNA loop formation, was obtained at an operator spacing of 70.5 bp . Other repression maxima occurred at operator distances of 92.5 and 115.5 bp, corresponding to natural operator spacings in the lac and in the gal operon, respectively . Substitution of the auxiliary O(id) with the weaker binding lac operator O3 lowered repression efficiency, presumably due to the reduced local concentration of Lac repressor. J Mol Biol, 1996 Mar 22, 257(1), 116 - 28 The three-dimensional structure of two mutants of the signal transduction protein CheY suggest its molecular activation mechanism; Bellsolell L et al.; The three-dimensional crystal structures of the single mutant M17G and the triple mutant F14G-S15G-M17G of the response regulator protein CheY have been determined to 2.3 and 1.9 angstrom, respectively . Both mutants bind the essential Mg2+ cation as determined by the changes in stability, but binding does not cause the intrinsic fluorescence quenching of W58 observed in the wild-type protein . The loop beta4-alpha4 appears to be very flexible in both mutants and helix alpha4, which starts at N94 in the native Mg2+-CheY and at K91 in the native apo-CheY, starts in both mutants at residue K92 . The side-chain of K109 appears to be more mobile because of the space freed by the M17G mutation . In the triple mutant the main chain of K109 and adjacent residues (loop beta5-alpha5) is displaced almost by 2 angstrom affecting the main chain at residues T87 to E89 (C terminus of beta4) . The triple mutant structure has a Mg2+ bound at the active site, but although the Mg2+ coordination is similar to that of the native Mg2+-CheY, the structural consequences of the metal binding are quite different . It seems that the mutations have disrupted the mechanism of movement transmission observed in the native protein . We suggest that the side-chain of K109, packed between V86, A88 and M17 in the native protein, slides forwards and backwards upon activation and deactivation dragging the main chain at the loop beta5-alpha5 and triggering larger movements at the functional surface of the protein. Nature, 1996 Mar 21, 380(6571), 268 - 70 Kinetic trapping of oxygen in cell respiration; Verkhovsky MI et al.; Cell respiration in eukaryotes is catalysed by mitochondrial enzyme cytochrome c oxidase . In bacteria there are many variants of this enzyme, all of which have a binuclear haem iron-copper centre at which O2 reduction occurs, and a low-spin haem, which serves as the immediate electron donor to this centre . It is essential that the components of the cell respiratory system have a high affinity for oxygen because of the low concentration of dissolved O2 in the tissues; however, the binding of O2 to the respiratory haem-copper oxidases is very weak . This paradox has been attributed to kinetic trapping during fast reaction of O2 bound within the enzyme's binuclear haem iron-copper centre . Our earlier work indicated that electron transfer from the low-spin haem to the oxygen-bound nuclear centre may be necessary for such kinetic oxygen trapping . Here we show that specific decrease of the haem-haem electron transfer rate in the respiratory haem-copper oxidase from Escherichia coli leads to a corresponding decrease in the enzyme's operational steady-state affinity for O2 . This demonstrates directly that fast electron transfer between the haem groups is a key process in achieving the high affinity for oxygen in cell respiration. Hum Gene Ther, 1996 Mar 20, 7(5), 589 - 93 In vivo gene transfer and expression in rat stomach by submucosal injection of plasmid DNA; Takehara T et al.; Gastrointestinal nonepithelial tissue is a useful target for in vivo gene transfer . The aim of this study was to investigate whether gene transfer into this organ could be achieved by submucosal injection of plasmid DNA . Plasmid DNA carrying either the firefly luciferase or Escherichia coli LacZ reporter gene was injected directly into the gastric submucosa of adult rats . Gene expression was characterized by quantitative luciferase assay and qualitative in situ beta-galactosidase (beta-Gal) staining . Luciferase activity was detected as early as 1 day after injection, increased markedly at 2 days, and then decreased . Some of the rats showed detectable levels of luciferase expression at 14 and 21 days postinjection . Histochemical staining for beta-Gal demonstrated that expression of the recombinant genes was localized in smooth muscle cells of the muscularis mucosae and the muscular layer and mesenchymal cells in the lamina propria . Our results indicate that gene transfer into the gastrointestinal tract can be achieved by simple needle insertion of naked plasmid DNA into the submucosa. Mol Gen Genet, 1996 Mar 20, 250(5), 626 - 34 RNA facilitates RecA-mediated DNA pairing and strand transfer between molecules bearing limited regions of homology; Kotani H et al.; The RecA protein of Escherichia coli catalyzes homologous pairing and strand exchange between a wide range of molecules showing nucleotide sequence complementarity, including a linear duplex and a single-stranded DNA molecule . We demonstrate that RecA can promote formation of joint molecules when the duplex contains an RNA/DNA hairpin and a single-stranded circle serves as the pairing partner . A chimeric RNA/DNA hairpin can be used to form stable joint molecules with as little as 15 bases of shared homology as long as the RNA stretch contains complementarity to the circle . The joint molecule bears some resemblance to a triple helical structure composed of RNA residues surrounded by two DNA strands which are in a parallel orientation . Evidence is presented that supports the notion that short stretches of RNA can be used in homologous pairing reactions at lengths below that required for DNA-DNA heteroduplex formation. Mol Gen Genet, 1996 Mar 20, 250(5), 593 - 600 Co-induction of DNA relaxation and synthesis of DnaK and GroEL proteins in Escherichia coli by expression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor; Kaneko T et al.; We examined the influence of overexpression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor of Escherichia coli, on DNA supercoiling and induction of heat shock proteins . Cells were transformed with a plasmid carrying the structural gene for LetD protein under control of the tac promoter, and LetD protein was induced by adding isopropyl beta-D-thiogalactopyranoside (IPTG) to the culture medium . Analysis by agarose gel electrophoresis in the presence of chloroquine revealed relaxation of plasmid DNA in cells depending on the concentration of IPTG employed for induction . Protein pulse-labeling experiments with {35S}methionine and cysteine revealed that synthesis of DnaK and GroEL proteins was also induced by IPTG, and concentrations necessary for DNA relaxation and induction of the heat shock proteins were much the same . Expression of mutant LetD protein lacking two amino acid residues at the C-terminus induced neither DNA relaxation nor the synthesis of DnaK and GroEL proteins . Induction of wild-type LetD protein but not mutant LetD protein markedly enhanced synthesis of sigma32 . We interpret these results to mean that DNA relaxation in cells caused by the expression of LetD protein induces heat shock proteins via increased synthesis of sigma32. Mol Gen Genet, 1996 Mar 20, 250(5), 547 - 57 Self-incompatibility (S) alleles of the Rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily; Sassa H et al.; Stylar ribonucleases (RNases) are associated with gametophytic self-incompatibility in two plant families, the Solanaceae and the Rosaceae . The self-incompatibility-associated RNases (S-RNases) of both the Solanaceae and the Rosaceae were recently reported to belong to the T2 RNase gene family, based on the presence of two well-conserved sequence motifs . Here, the cloning and characterization of S-RNase genes from two species of Rosaceae, apple (Malus x domestica) and Japanese pear (Pyrus serotina) is described and these sequences are compared with those of other T2-type RNases . The S-RNases of apple specifically accumulated in styles following maturation of the flower bud . Two cDNA clones for S-RNases from apple, and PCR clones encoding a further two apple S-RNases as well as two Japanese pear S-RNases were isolated and sequenced . The deduced amino acid sequences of the rosaceous S-RNases contained two conserved regions characteristic of the T2/S-type RNases . The sequences showed a high degree of diversity, with similarities ranging from 60.4% to 69.2% . Interestingly, some interspecific sequence similarities were higher than those within a species, possibly indicating that diversification of S-RNase alleles predated speciation in the Rosaceae . A phylogenetic tree of members of the T2/S-RNase superfamily in plants was obtained . The rosaceous S-RNases formed a new lineage in the tree that was distinct from those of the solanaceous S-RNases and the S-like RNases . The findings suggested that self-incompatibility mechanisms in Rosaceae and Solanaceae are similar but arose independently in the course of evolution. Mol Gen Genet, 1996 Mar 20, 250(5), 523 - 32 Requirement for two conserved cysteine residues in the Ada protein of Escherichia coli for transactivation of the ada promoter; Taketomi A et al.; Cysteine residue 69 of the Escherichia coli Ada transcription factor, which accepts a methyl group from methylphosphotriester in methylated DNA, was substituted by each of 19 other amino acids . Only the mutant Ada (C69H), carrying a histidine substitution of Cys69, exhibited a limited degree of transactivating potential for the ada promoter in E . coli cells although the mutant protein was completely devoid of methylphosphotriester-DNA methyltransferase activity . Using a multicopy plasmid system for the expression of Ada protein, we have shown that Ada C69H has a transactivating capacity equivalent to that of wild-type Ada protein in the absence of an alkylating agent . This indicates that the zinc-binding capacity of histidine at residue 69 is likely to be sufficient for Ada to recognize and bind to the ada promoter . Furthermore, transactivation of the ada promoter by Ada C69H was enhanced up to 6-fold by treatment with methylating agents . An additional substitution was made with alanine in Ada C69H, replacing Cys321, the site for acceptance of a methyl group from O6-methylguanine and O4-methylthymine residues in DNA, with alanine . This renders the protein completely inactive as a methyltransferase but this derivative is constitutively active as a transactivator for the ada promoter . Therefore, acquisition of a methyl group at Cys321 apparently enhances the transactivating capacity of Ada protein on the ada promoter . We propose that the transcription-regulating function of Ada protein is under dual control by methylation of cysteine residues at positions 69 and 321; the former enhances DNA binding, while the latter enhances the transactivating capacity of the protein. Biochemistry, 1996 Mar 19, 35(11), 3563 - 71 Differential proximity probing of two DNA binding sites in the Escherichia coli recA protein using photo-cross-linking methods; Wang Y et al.; The DNA strand-exchange reaction catalyzed by the Escherichia coli RecA protein occurs between the two DNA binding sites that are functionally distinct . Site I is the site to which a DNA molecule (normally single-stranded DNA) binds first; this first binding makes site II available for additional DNA-binding (normally double- stranded DNA) . Photo-cross linking was employed to identify the amino acid residues located close to the bound DNA molecule(s) . A ssDNA oligo containing multiple 5-iodouracil residues (IdU) was cross-linked to RecA by irradiation with a XeC1 pulse laser (308 nm), and the cross-linked peptides were purified and sequenced . To differentiate the two DNA binding sites, we used two protocols for making RecA-ssDNA complexes: (1) IdU-containing oligo was mixed with a stoichiometric excess of RecA, a condition which favors the binding of the oligo to site I, and (2) RecA was first allowed to bind to a nonphotoreactive oligo and then chased with the IdU-containing oligo, a condition which favors the binding of the IdU-oligo to site II . We observed that when RecA was in excess (site I probing), cross-linking occurred to Met-164 which is located in the disordered loop 1 of the RecA crystal structure {Story, R.M., Weber, I.T., & Steitz, T.A . (1992) Nature 355, 318-325} . When site II was probed, the majority of cross-linking occurred to Met-202 or Phe-203, located in loop 2 . These results support the idea that, as predicted by Story and co-workers (1992), the disordered loops are involved in DNA binding . The results also suggest that the two sites are not only functionally but also physically distinct. Biochemistry, 1996 Mar 19, 35(11), 3525 - 33 Sequence-specific actinomycin D binding to single-stranded DNA inhibits HIV reverse transcriptase and other polymerases; Rill RL et al.; Primer extension assays using recombinant templates constructed to contain all 256 possible base quartets in a minimum length sequence were used to examine binding of the anticancer drug actinomycin D to single-stranded DNA . Single-stranded templates were generated by digestion of linearized plasmid with the double-strand-specific T7 gene 6 exonuclease . Actinomycin D formed high-affinity, kinetically stable complexes that paused primer elongation at specific sites by HIV-1 reverse transcriptase, Sequenase (modified T4 DNA polymerase), the Klenow fragment of Escherichia coli DNA polymerase, and Vent (exo-) DNA polymerase . Pauses occurred most commonly near G+C-rich nucleotide clusters, including GpC steps, the preferred sites of double-stranded DNA binding . Complexes were stable for several minutes at temperatures over 50 degrees C as determined by their abilities to pause Vent polymerase at elevated temperatures . Significant variations were noted in pause patterns of different polymerases, demonstrating differential responses of polymerases to a bound actinomycin . Covalent adducts formed on template DNA by a photoaffinity analog of actinomycin D completely stopped primer extension . These results support the possibility that actinomycin D inhibits transcription elongation by complexing single-stranded DNA in the open transcription complex . Single-stranded DNA binding by actinomycin D or analogs may also provide routes for combating HIV or other viruses which replicate through single-stranded intermediates. Proc Natl Acad Sci U S A, 1996 Mar 19, 93(6), 2517 - 22 The translational function of nucleotide C1054 in the small subunit rRNA is conserved throughout evolution: genetic evidence in yeast; Chernoff YO et al.; Mutations at position C1054 of 16S rRNA have previously been shown to cause translational suppression in Escherichia coli . To examine the effects of similar mutations in a eukaryote, all three possible base substitutions and a base deletion were generated at the position of Saccharomyces cerevisiae 18S rRNA corresponding to E . coli C1054 . In yeast, as in E . coli, both C1054A (rdn-1A) and C1054G (rdn-1G) caused dominant nonsense suppression . Yeast C1054U (rdn-1T) was a recessive antisuppressor, while yeast C1054-delta (rdn-1delta) led to recessive lethality . Both C1054U and two previously described yeast 18S rRNA antisuppressor mutations, G517A (rdn-2) and U912C (rdn-4), inhibited codon-nonspecific suppression caused by mutations in eukaryotic release factors, sup45 and sup35 . However, among these only C1054U inhibited UAA-specific suppressions caused by a UAA-decoding mutant tRNA-Gln (SLT3) . Our data implicate eukaryotic C1054 in translational termination, thus suggesting that its function is conserved throughout evolution despite the divergence of nearby nucleotide sequences. Proc Natl Acad Sci U S A, 1996 Mar 19, 93(6), 2488 - 92 The response regulator SprE controls the stability of RpoS; Pratt LA et al.; In Escherichia coli, the sigma factor, RpoS, is a central regulator in stationary-phase cells . We have identified a gene, sprE (stationary-phase regulator), as essential for the negative regulation of rpoS expression . SprE negatively regulates the rpoS gene product at the level of protein stability, perhaps in response to nutrient availability . The ability of SprE to destabilize RpoS is dependent on the ClpX/ClpP protease . Based on homology, SprE is a member of the response regulator family of proteins . SprE is the first response regulator identified that is implicated in the control of protein stability . Moreover, SprE is the first reported protein that appears to regulate rpoS in response to a specific environmental parameter. Proc Natl Acad Sci U S A, 1996 Mar 19, 93(6), 2459 - 63 Non-iron porphyrins cause tumbling to blue light by an Escherichia coli mutant defective in hemG; Yang H et al.; Previously we showed that an Escherichia coli hemH mutant, defective in the ultimate step of heme synthesis, ferrochelatase, is somewhat better than 100-fold more sensitive than its wild-type parent in tumbling to blue light . Here we explore the effect of a hemG mutant, defective in the penultimate step, protoporphyrinogen oxidase . We found that a hemG mutant also is somewhat better than 100-fold more sensitive in tumbling to blue light compared to its wild-type parent . The amount of non-iron porphyrins accumulated in hemG or hemH mutants was more than 100-fold greater than in wild type . The nature of these accumulated porphyrins is described . When heme was present, as in the wild type, the non-iron (non-heme) porphyrins were maintained at a relatively low concentration and tumbling to blue light at an intensity effective for hemG or hemH did not occur . The function of tumbling to light is most likely to allow escape from the lethality of intense light. Proc Natl Acad Sci U S A, 1996 Mar 19, 93(6), 2348 - 52 Baculovirus-mediated gene transfer into mammalian cells; Boyce FM et al.; This paper describes the use of the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) as a vector for gene delivery into mammalian cells . A modified AcMNPV virus was prepared that carried the Escherichia coli lacZ reporter gene under control of the Rous sarcoma virus promoter and mammalian RNA processing signals . This modified baculovirus was then used to infect a variety of mammalian cell lines . After infection of the human liver cell lines HepG2, >25% of the cells showed high-level expression of the transduced gene . Over 70% of the cells in primary cultures of rat hepatocytes showed expression of beta-galactosidase after exposure to the virus . Cell lines from other tissues showed less or no expression of lacZ after exposure to the virus . The block to expression in less susceptible cells does not appear to result from the ability to be internalized by the target cell but rather by events subsequent to viral entry . The onset of lacZ expression occurred within 6 hr of infection in HepG2 cells and peaked 12-24 hr postinfection . Because AcMNPV is able to replicate only in insect hosts, is able to carry large (>15 kb) inserts, and is a highly effective gene delivery vehicle for primary cultures of hepatocytes, AcMNPV may be a useful vector for genetic manipulation of liver cells. Proc Natl Acad Sci U S A, 1996 Mar 19, 93(6), 2328 - 32 Genes encoding the same three subunits of respiratory complex II are present in the mitochondrial DNA of two phylogenetically distant eukaryotes; Burger G et al.; Although mitochondrial DNA is known to encode a limited number (<20) of the polypeptide components of respiratory complexes I, III, IV, and V, genes for components of complex II {succinate dehydrogenase (ubiquinone); succinate:ubiquinone oxidoreductase, EC 1.3.5.1} are conspicuously lacking in mitochondrial genomes so far characterized . Here we show that the same three subunits of complex II are encoded in the mitochondrial DNA of two phylogenetically distant eukaryotes, Porphyra purpurea (a photosynthetic red alga) and Reclinomonas americana (a heterotrophic zooflagellate) . These complex II genes, sdh2, sdh3, and sdh4, are homologs, respectively, of Escherichia coli sdhB, sdhC, and sdhD . In E . coli, sdhB encodes the iron-sulfur subunit of succinate dehydrogenase (SDH), whereas sdhC and sdhD specify, respectively, apocytochrome b558 and a hydrophobic 13-kDa polypeptide, which together anchor SDH to the inner mitochondrial membrane . Amino acid sequence similarities indicate that sdh2, sdh3, and sdh4 were originally encoded in the protomitochondrial genome and have subsequently been transferred to the nuclear genome in most eukaryotes . The data presented here are consistent with the view that mitochondria constitute a monophyletic lineage. Biochem Biophys Res Commun, 1996 Mar 18, 220(2), 437 - 42 Cloning, sequencing, and expression of equinatoxin II; Anderluh G et al.; Equinatoxin II (EqtII), a basic protein of 179 amino acids lacking cysteine residues, is the most abundant cytolysin isolated from the sea anemone Actinia equina . Its mode of action is still poorly understood . In order to initiate further structure-function studies by protein engineering, cDNA library was prepared from the whole animal and hybridized with a PCR-derived probe, deduced from the EqtII primary structure . The longest positive clone of 899 bp was shown to encode a 214 residue precursor of EqtII . The mature protein region was amplified by PCR, cloned into a T7 RNA polymerase-based expression vector and expressed in Escherichia coli . Recombinant toxin was isolated by a simple, two-step isolation procedure including separation on CM-cellulose and gel filtration using an FPLC system . Its biochemical properties and hemolytic activity were practically indistinguishable from those of native toxin. Biochem Biophys Res Commun, 1996 Mar 18, 220(2), 330 - 3 Site-directed mutagenesis of the conserved serine 138 of human placental NAD+-dependent 15-hydroxyprostaglandin dehydrogenase to an alanine results in an inactive enzyme; Ensor CM et al.; Human placental NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a member of the short-chain dehydrogenase family of enzymes . It has been proposed that a highly conserved serine residue (corresponding to serine 138 of 15-PGDH) may be involved in the catalytic mechanism of many of these enzymes . Site-directed mutagenesis was used to change serine 138 of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase to an alanine . The mutant protein was then expressed in E . coli . Western blot analysis indicated that the S138A mutant protein was expressed at levels similar to the wild type enzyme; however, the mutant protein was found to be inactive . These results support the proposed role of this highly conserved serine in enzyme activity. Biochem Biophys Res Commun, 1996 Mar 18, 220(2), 280 - 4 A new plasmid-encoded proteic killer gene system: cloning, sequencing, and analyzing hig locus of plasmid Rts1; Tian QB et al.; A new proteic killer gene system, hig, was identified on the plasmid Rts1 . The hig locus consisting of a higA and higB is directly related to the temperature sensitive host cell growth conferred by Rts1 . We proved that higB encoding presumably a 92-amino-acid polypeptide inhibited segregation of plasmid free cells, and higA encoding a 104-amino-acid polypeptide suppressed the higB function both in cis and in trans. Biochem Biophys Res Commun, 1996 Mar 18, 220(2), 255 - 63 Expression and functional characterization of a single chain Fv antibody directed against secretions involved in plant nematode infection process; Rosso MN et al.; Expression in plants of antibodies directed against proteins essential for pathogenesis could provide an alternative approach to engineer new resistance traits into crops . Salivary secretions of the root-knot nematode Meloidogyne incognita are known to play a key role during this nematode infection process . From a hybridoma cell line producing an IgM monoclonal antibody specific to these secretions, we have constructed a synthetic gene that encodes an antigen-binding single-chain Fv protein (scFv) . The scFv gene was created by polymerase chain reaction amplification of variable domain encoding regions from the IgM antibody . The cloned scFv was initially expressed in Escherichia coli as a 33-kDa protein which could be purified to near homogeneity by immobilized metal affinity chromatography . The produced scFv is fully functional since it shows the same specificity towards a crude extract of M . incognita infective larvae as the corresponding parental IgM . Transient expression assays with tobacco leaf protoplasts using different targeting signals resulted in a high intracellular accumulation of scFv, especially when fused to the tetrapeptide KDEL retention signal . Activity analysis and stability characterization of this scFv in tobacco protoplast represent the first step before plant transformation in order to test a new form of resistance to root-knot nematode in plants. FEBS Lett, 1996 Mar 18, 382(3), 285 - 8 Cloning of rat 92-kDa type IV collagenase and expression of an active recombinant catalytic domain; Xia Y et al.; A full-length cDNA for rat 92-kDa type IV collagenase was isolated and sequenced . RNase protection assay revealed tissue specific differential expression of the 92-kDa type IV collagenase in the rat during development . Natural and modified forms of the 92-kDa type IV collagenase were expressed . One active protein, 92-CD, contained only the putative catalytic domain . Large quantities of the 92-CD were expressed in Escherichia coli, extracted from inclusion bodies, purified, and refolded to an active form . This recombinant protein was able to cleave denatured and native collagen and was inactivated by known MMP inhibitors. FEMS Microbiol Lett, 1996 Mar 15, 137(1), 85 - 9 The structural gene for rusticyanin from Thiobacillus ferrooxidans: cloning and sequencing of the rusticyanin gene; Hall JF et al.; The rusticyanin gene from the acidophilic chemolithotroph Thiobacillus ferrooxidans has been cloned and sequenced . A central portion of the gene was identified by PCR reactions utilising primers optimised for codon bias followed by nested PCR with degenerate primers . The 5' and 3' ends of the rusticyanin gene were then cloned using degenerate primers to each end and anchor sequences to the known internal sequence . The entire gene was amplified using Tli DNA polymerase and specific primers to the 5' and 3' ends and the sequence confirmed after cloning into Bluescript and transformation of XL-1 Blue Escherichia coli. FEMS Microbiol Lett, 1996 Mar 15, 137(1), 51 - 6 A putative signal recognition particle receptor alpha subunit (SR alpha) homologue is expressed in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius; Moll R et al.; A 1.64 kb genomic DNA sequence from the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius is composed of two adjacent genes . The first functionally unassigned open reading frame (orf-1) comprises 450 base pairs . The second 1.1 kb large open reading frame encodes the putative signal recognition particle receptor alpha subunit (SR alpha) . Both genes are expressed under the heterotrophic growth conditions of the organism . The main transcript of orf-1 appears as a monocistronic RNA in Northern hybridization . With regard to SR alpha the transcription pattern was investigated by reverse transcription polymerase chain reaction and primer extension analysis . A polyclonal antiserum directed against E . coli lacZ'/Sulfolobus SR alpha fusion protein detects a 40.5 kDa protein (p41) in agreement with the 41.4 kDa as deduced from the nucleotide sequence. Anal Biochem, 1996 Mar 15, 235(2), 195 - 201 Tetracycline-controlled gene expression system achieves high-level and quantitative control of gene expression; Yin DX et al.; The tetracycline-controlled gene expression system utilizes the control elements of the tetracycline resistance operon encoded in TnlO of Escherichia coli to control gene expression in eukaryotic cells . Here we demonstrate the quantitative control of the expression of the luciferase gene, dihydrofolate reductase gene, and bcl-2 gene in HeLa S3 or Chinese hamster ovary AA8 cells using the tetracycline-controlled gene expression system . Regardless of the host cell lines or the genes being expressed, there is a common range of tetracycline concentration within which the expression of genes is most sensitively regulated . In addition, the maximal gene expression level of the tetracycline-controlled gene expression system is higher than that of the wild-type CMV promoter/enhancer-driven system . Nonetheless, careful selection of stably transfected clones is necessary to achieve the optimally regulated gene expression using this system. Structure, 1996 Mar 15, 4(3), 219 - 22 Lac repressor at last; Sauer RT; Crystal and cocrystal structures of the LacI and PurR repressors reveal a novel use of hinge alpha helices which bind in the minor groove of the operator and mediate transmission of the allosteric signals that modulate DNA-binding activity. Eur J Biochem, 1996 Mar 15, 236(3), 991 - 5 Identification, nuclear localization, and binding activities of OZF, a human protein solely composed of zinc-finger motifs; Ferbus D et al.; The OZF cDNA was identified in a human mammary cell line and encodes a polypeptide solely composed of ten zinc-finger motifs which belongs to the Kruppel family of zinc-finger proteins . The OZF protein produced in Escherichia coli binds zinc ions, DNA and heparin . These binding activities are characteristic of zinc-finger proteins . Immunochemical analysis using antibodies produced against the recombinant protein detected its expression in human mammary epithelial cells but not in stroma cells, which is consistent with the pattern of expression observed at the RNA level in cell cultures . Western blot analysis demonstrated the expression of a 33-kDa nuclear protein similar in size to the predicted protein and therefore excluded the presence of an additional trans-acting domain . These data establish the unique structure of the OZF protein which is distinct from previously identified zinc-finger proteins . In addition, OZF protein overexpression was found in a tumor cell line, which suggests a possible involvement in carcinogenesis. Eur J Biochem, 1996 Mar 15, 236(3), 937 - 46 Properties of the recombinant beta subunit of glutamate synthase; Vanoni MA et al.; Glutamate synthase is a complex iron-sulfur flavoprotein containing one molecule each of FAD and FMN and three distinct iron-sulfur centers/alpha beta protomer . Production of the beta subunit was observed in total extracts of Escherichia coli BL21 (DE) cells harbouring a pT7-7 derivative carrying gltD, the gene encoding the Azospirillum brasilense glutamate synthase beta subunit . The protein was soluble, and the identity of the purified protein with the Azospirillum glutamate synthase beta subunit was confirmed by N-terminal sequence analysis . The kinetic and spectroscopic characterization of the glutamate synthase beta subunit confirmed that it contains the NADPH binding site, but, in contrast with earlier proposals that assigned both FAD and FMN binding sites to the alpha subunit of glutamate synthase, the beta subunit was shown to contain stoichiometric amounts of FAD . No iron-sulfur centers were detected by EPR spectroscopy measurements of the recombinant beta subunit . Under steady-state conditions, the glutamate synthase beta subunit can catalyze the NADPH-dependent reduction of several synthetic electron acceptors but no glutamate synthase or glutamate dehydrogenase reactions in either direction . The results are in agreement with previous data from our laboratory and, together with the absence of amino acid sequence similarity between glutamate synthase beta subunit and glutamate dehydrogenases, are against the hypothesis that glutamate synthase is evolutionarily derived from the association of an ancestral glutamate dehydrogenase (the beta subunit) and an amidotransferase (the alpha subunit) . The protein-bound FAD is reduced by NADPH at a rate much faster than turnover with synthetic electron acceptors, leading to formation of a stable reduced flavin-NADP+ charge-transfer complex . The rate of reduction of the bound FAD by NADPH is also similar to the rate at which one of the flavins is reduced in the native glutamate synthase, as measured in a stopped-flow spectrophotometer under pre-steady-state conditions . The ability of FAD bound to the beta subunit of glutamate synthase to react with NADPH and the lack of reactivity with sulfite lead us to conclude that FAD is Flavin 1 of glutamate synthase {Vanoni, M.A., Edmondson, D.E., Zanetti, G . & Curti, B . (1992) Biochemistry 31, 4613-4623}. Eur J Biochem, 1996 Mar 15, 236(3), 911 - 21 Solution structure of the DNA-binding domain of the tomato heat-stress transcription factor HSF24; Schultheiss J et al.; Two-dimensional-NMR and three-dimensional-NMR experiments were performed to determine the solution structure of the DNA-binding domain of the tomato heat-stress transcription factor HSF24 . Samples of uniformly 15N-labeled and 15N, 13C-labeled recombinant proteins were used in the investigation . A near-complete assignment of the backbone 1H, 15N, and 13C resonances was obtained by three-dimensional triple-resonance experiments, whereas three-dimensional 15N-TOCSY-heteronuclear-single-quantum-correlation-spectroscopy, HCCH-COSY and HCCH-TOCSY spectra were recorded for side-chain assignments, 885 non-redundant distance constraints from two-dimensional-homonuclear and three-dimensional-15N-edited and 13C-edited NOESY spectra and 40 hydrogen-bond constraints from exchange experiments were used for structure calculations . The resulting three-dimensional structure contains a three-helix bundle and a small four-stranded antiparallel beta-sheet that forms a hydrophobic core . The two C-terminal helices are parts of a highly conserved helix-turn-helix motif that is probably involved in DNA recognition and binding . In contrast to heat-stress factors from yeast and animals, the plant heat-stress factors lack a loop of 11 amino acid residues inserted between beta3 and beta4 . This leads to a tight turn between these beta-strands. Cancer Res, 1996 Mar 15, 56(6), 1361 - 6 Treatment of microscopic pulmonary metastases with recombinant autologous tumor vaccine expressing interleukin 6 and Escherichia coli cytosine deaminase suicide genes; Mullen CA et al.; Poorly immunogenic tumor cells genetically transduced to simultaneously express the cytokine interleukin 6 (IL-6) and the bacterial metabolic suicide gene cytosine deaminase (205-IL6-CD) become highly immunogenic . They are rejected by normal mice without 5-fluorocytosine prodrug treatment . Mice with preexisting wild-type pulmonary micrometastases exhibit prolonged survival and an increased rate of cure when treated with live 205-IL6-CD cells as a therapeutic vaccine . Treatment with these autologous tumor cells producing both the cytokine and the bacterial protein was more effective than treatment with exogenous IL-6 and/or irradiated wild-type tumor cells . Irradiation of the 205-IL6-CD cells significantly reduced their therapeutic efficacy . Therapeutic vaccination with 205-IL6-CD was more effective in animals with wild-type 205 tumor than in animals bearing an unrelated syngeneic tumor . Vaccine efficacy was significantly reduced in animals pretreated with high-dose cyclophosphamide . The results indicate that genetically engineered autologous tumor vaccines may be capable of inducing significant antitumor immunity in hosts of preexisting micrometastatic disease. Cancer Res, 1996 Mar 15, 56(6), 1315 - 23 Comparison of the effects of three different toxin genes and their levels of expression on cell growth and bystander effect in lung adenocarcinoma; Hoganson DK et al.; Transduction of malignant cells with toxin genes provides a novel means to promote tumor cell destruction . The efficacy of a toxin gene is dependent on the cell type targeted, the quantity of exogenous protein synthesized, and the mechanisms of growth inhibition and bystander killing . To develop gene therapy for targeting metastatic lung adenocarcinoma, the toxic activity of herpes simplex virus type 1-thymidine kinase, Escherichia coli cytosine deaminase, and human deoxycytidine kinase were investigated in metastatic human lung adenocarcinoma cell lines H1437 and H2122 . Cells were transduced stably with retroviral vectors containing the toxin gene cDNA under the control of either a strong {cytomegalovirus (CMV) immediate early promotor and enhancer} or an intermediate strength (Moloney murine leukemia virus long terminal repeat) promotor . A comparison of toxin gene efficacy was based on the level of specific enzyme activity, the concentration of prodrug required to inhibit cell growth by 50%, and the magnitude of the bystander effect . In lung adenocarcinoma cell lines, cytosine deaminase, driven by the CMV promoter, was superior to thymidine kinase and deoxycytidine kinase in its ability to achieve high levels of specific enzyme activity, to induce growth inhibition, and to affect neighboring cell growth . Therefore, cytosine deaminase expressed from the CMV promotor seems to be the most promising toxin gene for human lung adenocarcinoma gene therapy. EMBO J, 1996 Mar 15, 15(6), 1340 - 9 Escherichia coli protein analogs StpA and H-NS: regulatory loops, similar and disparate effects on nucleic acid dynamics; Zhang A et al.; Expression of the Escherichia coli StpA protein was investigated and a functional comparison undertaken with the structurally analogous nucleoid protein H-NS . Analysis of stpA and hns expression indicated that although stpA transcript levels are much lower than those of hns, the two gene products are capable of both negative autogenous control and cross-regulation . Examination of cellular proteins in stpA, hns, or stpA-hns backgrounds revealed that StpA can repress and activate a subset of H-NS-regulated genes . Mechanistic parallels in regulation of gene expression are indicated by the ability of both proteins to inhibit transcription from promoters containing curved DNA sequences, and to form nucleoprotein structures that constrain DNA supercoils . Despite their functional similarities, each molecule is capable of independent activities . Thus, H-NS regulates a class of genes that are unaffected by StpA in vivo, whereas StpA has much stronger RNA chaperone activity in vitro . We therefore propose that in addition to its role as a molecular back-up of H-NS, StpA's superior effect on RNA may be exploited under some specific cellular conditions to promote differential gene expression. EMBO J, 1996 Mar 15, 15(6), 1333 - 9 The response regulator RssB controls stability of the sigma(S) subunit of RNA polymerase in Escherichia coli; Muffler A et al.; The rpoS-encoded sigma(S) subunit of RNA polymerase is a central regulator in a regulatory network that governs the expression of many stationary phase-induced and osmotically regulated genes in Escherichia coli . sigma(S) is itself induced under these conditions due to an increase in rpoS transcription (only in rich media) and rpoS translation as well as a stabilization of sigma(S) protein which in growing cells is subject to rapid turnover . We demonstrate here that a response regulator, RssB, plays a crucial role in the control of the cellular sigma(S) content . rssB null mutants exhibit nearly constitutively high levels of sigma(S) and are impaired in the post-transcriptional growth phase-related and osmotic regulation of sigma(S) . Whereas rpoS translational control is not affected, sigma(S) is stable in rssB mutants, indicating that RssB is essential for sigma(S) turnover . RssB contains a unique C-terminal output domain and is the first known response regulator involved in the control of protein turnover. EMBO J, 1996 Mar 15, 15(6), 1238 - 46 Linking microfilaments to intracellular membranes: the actin-binding and vesicle-associated protein comitin exhibits a mannose-specific lectin activity; Jung E et al.; Comitin is a 24 kDa actin-binding protein from Dictyostelium discoideum that is located primarily on Golgi and vesicle membranes . We have probed the molecular basis of comitin's interaction with both actin and membranes using a series of truncation mutants obtained by expressing the appropriate cDNA in Escherichia coli . Comitin dimerizes in solution; its principle actin-binding activity is located between residues 90 and 135 . The N-terminal 135 'core' residues of comitin contain a 3-fold sequence repeat that is homologous to several monocotyledon lectins and which retains key residues that determine these lectins' three-dimensional structure and mannose binding . These repeats of comitin appear to mediate its interaction with mannose residues in glycoproteins or glycolipids on the cytoplasmic surface of membrane vesicles from D.discoideum, and comitin can be released from membranes with mannose . Our data indicate that comitin binds to vesicle membranes via mannose residues and, by way of its interaction with actin, links these membranes to the cytoskeleton. EMBO J, 1996 Mar 15, 15(6), 1203 - 10 Exploring the functional robustness of an enzyme by in vitro evolution; Martinez MA et al.; The evolution of natural proteins is thought to have occurred by successive fixation of individual mutations . In vitro protein evolution seeks to accelerate this process . RNA hypermutagenesis, cDNA synthesis in the presence of biased dNTP concentrations, delivers elevated mutant and mutation frequencies . Here lineages of active enzymes descended from the homotetrameric 78 residue dihydrofolate reductase (DHFR) encoded by the Escherichia coli R67 plasmid were generated by iterative RNA hypermutagenesis, resulting in >20% amino acid replacement . The 22 residue N-terminus could be deleted yielding a minimum functional entity refractory to further changes, designating it as a determinant of R67 robustness . Complete substitution of the segment still allowed fixation of mutations . By the facile introduction of multiple mutations, RNA hypermutagenesis allows the generation of active proteins derived from extant genes through a mode unexplored by natural selection. J Med Chem, 1996 Mar 15, 39(6), 1293 - 302 Angular furoquinolinones, psoralen analogs: novel antiproliferative agents for skin diseases . Synthesis, biological activity, mechanism of action, and computer-aided studies; Rodighiero P et al.; With the aim of obtaining new potential photochemotherapeutic agents, having increased antiproliferative activity and decreased u