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| Biochem Biophys Res Commun, 1994 May 30, 201(1), 346 - 55 Fur (ferric uptake regulation) protein interaction with target DNA: comparison of gel retardation, footprinting and electron microscopy analyses; Frechon D et al.; Fur-DNA interactions were analyzed within the regulatory regions of aerobactin and hemolysin operons by a combination of biochemical and ultrastructural methods . Cartography of the Fur binding sites, carried out from electron micrographs, agreed with the data obtained by DNase I footprinting . Visualization of the complexes confirmed the specificity and metal-dependence of Fur binding and demonstrated that the protein polymerizes on its binding sites . Such a polymerization could be involved in the repression process of the bacterial regulator. Biochem Biophys Res Commun, 1994 May 30, 201(1), 242 - 7 Use of single-cysteine mutants to probe the location of the disulfide bond in LamB protein from Escherichia coli; Ling R et al.; The two cysteine residues of the LamB protein of Escherichia coli outer membrane have been shown to form an intrasubunit disulfide whose location differs greatly in the two current topology models of the LamB protein . This study probes the location of the disulfide by examining conditions for intersubunit disulfide formation in single-cysteine mutants of LamB protein . Formation of an intersubunit bond in the purified mutant proteins, which resulted in a disulfide-linked dimer, only occurred after heat treatment, suggesting the disulfide is not exposed on the surface in the native protein. FEBS Lett, 1994 May 30, 345(2-3), 229 - 32 Chaperonin GroE and ADP facilitate the folding of various proteins and protect against heat inactivation; Kawata Y et al.; In the presence of ADP, the molecular chaperones GroEL and GroES from Escherichia coli not only facilitated the refolding of various proteins, but also prevented their irreversible heat inactivation in vitro . Without nucleotides the refolding reactions were arrested by GroEL . Addition of GroES and ADP to the reaction mixture initiated the refolding reactions and the enzyme activities were regained efficiently . The presence of GroE (GroEL and GroES) and ADP also protected against heat inactivation of native enzymes at various temperatures . These findings suggest that in the presence of GroES, nucleotide binding is an important event in the mechanism of GroEL-facilitated protein folding. FEBS Lett, 1994 May 30, 345(2-3), 181 - 6 The formation of symmetrical GroEL-GroES complexes in the presence of ATP; Llorca O et al.; The incubation of chaperonins cpn60 (GroEL) and cpn10 (GroES) from E . coli in the presence of Mg-ATP and KCl generates the formation, as revealed by electron microscopy, of GroEL-GroES complexes with a symmetrical shape in which one toroidal GroES oligomer is bound to each end of the tetradecameric GroEL aggregate (1:2 GroEL:GroES oligomer molar ratio) . The symmetrical complexes are not observed in the presence of ADP or the non-hydrolyzable ATP analog, ATP gamma S, where only asymmetrical complexes (1:1 GroEL:GroES oligomer molar ratio) are formed . These results suggest that ATP hydrolysis is required for the formation of symmetrical complexes. Biochem Biophys Res Commun, 1994 May 30, 201(1), 8 - 15 Inhibition of metal-catalyzed oxidation systems by a yeast protector protein in the presence of thioredoxin; Kwon SJ et al.; A protector protein from Saccharomyces cerevisiae specifically prevents the inactivation of enzymes caused by a thiol/Fe3+/O2 metal-catalyzed oxidation system but not by an ascorbate/Fe3+/O2 system . Ascorbate/Fe3+/O2-mediated damage of enzymes could be prevented by the protector protein only in the presence of reduced thiol . We demonstrate that two proteins from yeast, thioredoxin plus another protein having properties similar to that expected to thioredoxin reductase, when presented with NADPH and the yeast protector protein prevented inactivation of E . coli glutamine synthetase by the ascorbate/Fe3+/O2 system . This system also removes hydrogen peroxide effectively . We also demonstrate evidence suggesting that the NADPH-dependent thioredoxin system reactivates protector protein by reversible disulfide-dithiols exchange. Biochem Biophys Res Commun, 1994 May 30, 201(1), 436 - 42 Activation of soluble guanylyl cyclase by a factor other than nitric oxide or carbon monoxide contributes to the vascular hyporeactivity to vasoconstrictor agents in the aorta of rats treated with endotoxin; Wu CC et al.; We have examined the role of soluble guanylyl cyclase and possible mediators of its activation in the vascular hyporeactivity caused by bacterial endotoxin (lipopolysaccharide, LPS) ex vivo . Treatment of rats with E . coli LPS (10 mg/kg, i.v . for 3h) resulted in a significant reduction in the contractions elicited by norepinephrine (NE; 10(-9)-10(-6) M) in endothelium-denuded aortic rings ex vivo . Methylene blue or LY-83583, inhibitors of soluble guanylyl cyclase, completely restored contractions to NE, whereas the nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME), caused only a partial restoration . Zinc protoporphyrin-IX, an inhibitor of heme oxygenase, did not enhance NE-induced contraction in rings from LPS-treated rats, indicating that the production of carbon monoxide (CO) does not contribute to this vascular hyporeactivity . Indomethacin, an inhibitor of cyclooxygenase, further suppressed the contractions in rings from LPS-treated rats . These results suggest that hyporesponsiveness to NE caused by LPS is due to the activation of soluble guanylyl cyclase, which is partially mediated by N(O), but not by CO . Moreover, LPS may induce the production of another mediator(s) that activate soluble guanylyl cyclase in the vascular smooth muscle. Gene, 1994 May 27, 143(1), 95 - 100 Structural proteins of mycobacteriophage I3: cloning, expression and sequence analysis of a gene encoding a 70-kDa structural protein; Ramesh GR et al.; The structural proteins of mycobacteriophage I3 have been analysed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), radioiodination and immunoblotting . Based on their abundance the 34- and 70-kDa bands appeared to represent the major structural proteins . Successful cloning and expression of the 70-kDa protein-encoding gene of phage I3 in Escherichia coli and its complete nucleotide sequence determination have been accomplished . A second (partial) open reading frame following the stop codon for the 70-kDa protein was also identified within the cloned fragment . The deduced amino-acid sequence of the 70-kDa protein and the codon usage patterns indicated the preponderance of codons, as predicted from the high G+C content of the genomic DNA of phage I3. Gene, 1994 May 27, 143(1), 1 - 12 The dam and dcm strains of Escherichia coli--a review; Palmer BR et al.; The construction of a variety of strains deficient in the methylation of adenine and cytosine residues in DNA by the methyltransferases (MTases) Dam and Dcm has allowed the study of the role of these enzymes in the biology of Escherichia coli . Dam methylation has been shown to play a role in coordinating DNA replication initiation, DNA mismatch repair and the regulation of expression of some genes . The regulation of expression of dam has been found to be complex and influenced by five promoters . A role for Dcm methylation in the cell remains elusive and dcm- cells have no obvious phenotype . dam- and dcm- strains have a range of uses in molecular biology and bacterial genetics, including preparation of DNA for restriction by some restriction endonucleases, for transformation into other bacterial species, nucleotide sequencing and site-directed mutagenesis . A variety of assays are available for rapid detection of both the Dam and Dcm phenotypes . A number of restriction systems in E . coli have been described which recognise foreign DNA methylation, but ignore Dam and Dcm methylation . Here, we describe the most commonly used mutant alleles of dam and dcm and the characteristics of a variety of the strains that carry these genes . A description of several plasmids that carry dam gene constructs is also included. J Mol Biol, 1994 May 27, 239(1), 15 - 24 Analysis of the N-terminal leucine heptad and hexad repeats of sigma 54; Hsieh M et al.; In order to assess the role of leucine repeat motifs within bacterial protein sigma 54, a series of point mutants were introduced into the many leucine residues near the N terminus . Functional assays in vivo showed that the leucine residues that comprise the previously identified heptad repeat motif are selectively important for function . These heptad leucine residues are critical for mRNA production and also for recognition of the -12 promoter element . An internal proline substitution destroys the function of the heptad repeat region, suggesting a possible alpha-helical structure . Mutants with changes in the distal part of this N-terminal region show the interesting property of allowing nearly full levels of open complex formation, while nonetheless reducing the level of mRNA transcripts produced . All of the above-mentioned properties differ from those exhibited by mutating the interdigitated glutamine residues, which were previously found to be closely involved in the DNA melting reaction . The collection of data suggests that the N-terminal region contains overlapping functional motifs, hydrophobic heptad and glutamine-rich, which together appear to constitute the activation domain of sigma 54. J Biol Chem, 1994 May 27, 269(21), 15362 - 70 Efficient anchoring of RNA polymerase in Escherichia coli during coupled transcription-translation of genes encoding integral inner membrane polypeptides; Ma D et al.; While it has been known that supercoiling of the DNA template can be induced by transcription, the mechanism and the efficiency of this process in vivo is not fully understood . We report here that transcription of genes encoding 16 S rRNA, a stable RNA species, or cytoplasmic polypeptides leads to very little or no detectable DNA supercoiling even under the optimum conditions in Escherichia coli . This indicates that hydrodynamic drag on the transcription complex (including RNA polymerase, nascent RNA, ribosomes, and nascent polypeptides) is not sufficient to anchor RNA polymerase during coupled transcription-translation . On the other hand, transcription of membrane-associated genes encoding integral inner membrane or exported periplasmic polypeptides leads to apparent DNA supercoiling . Transcription of genes encoding integral inner membrane polypeptides leads to significantly greater anchoring of RNA polymerase than does transcription of genes encoding periplasmic polypeptides . This may reflect differences in the coupling of transcription-translation with membrane association during expression of these two classes of polypeptides . Evidence is further presented to suggest that the anchoring of RNA polymerase is probably achieved through the interaction of nascent polypeptides with the cytoplasmic surface of the inner membrane during coupled transcription-translation . Moreover, transcriptions of a membrane-associated gene can, under certain circumstances, induce topological anchoring of an RNA polymerase transcribing a neighboring gene that ordinarily is not membrane-associated . Finally, the potential biological consequences of our findings are discussed. J Biol Chem, 1994 May 27, 269(21), 15217 - 22 Cloning and expression of Ole e I, the major allergen from olive tree pollen . Polymorphism analysis and tissue specificity; Villalba M et al.; Ole e I, the major allergen from the olive tree (Olea europaea), is one of the main causes of allergy in Mediterranean countries and some areas of North America . The cloning and sequencing of several cDNAs coding for the olive allergen have been achieved . cDNA has been synthesized from total pollen RNA and amplified by using the polymerase chain reaction . The nucleotide sequence data demonstrate the existence of microheterogeneities in at least 37 positions out of the 145 amino acids of Ole e I, thus explaining the high degree of polymorphism exhibited by the natural protein . One of the sequenced cDNAs encoding a full-length isoform was inserted into the plasmid vector pGEX-2T and overexpressed . The recombinant Ole e I has been produced in Escherichia coli as a fusion protein with glutathione S-transferase of Schistosoma japonicum . This chimeric protein was purified by affinity chromatography on a glutathione-Sepharose 4B column and digested with thrombin to release the recombinant allergen . Both the fusion protein and the recombinant Ole e I were recognized in Western blot analysis by rabbit polyclonal and mouse monoclonal antisera raised against native Ole e I as well as by the IgE of olive pollen-sensitive human sera . This indicates that the recombinant production of individual isoforms may be useful for the improvement of reagents to be used in diagnosis and therapy of IgE-mediated disorders . In addition, Ole e I mRNA has been observed to be pollen-specific as shown in a Northern blot analysis. J Biol Chem, 1994 May 27, 269(21), 15210 - 6 Mutagenesis of cardiac troponin I . Role of the unique NH2-terminal peptide in myofilament activation; Guo X et al.; Phosphorylation of Ser residues in the NH2-terminal extension unique to cardiac troponin I (cTnI) is known to occur through protein kinase A and to alter myofilament Ca2+ activation (Robertson, S . P., Johnson, J . D., Holroyde, M . J., Kranias, E . G., Potter, J . D., and Solaro, R . J . (1982) J . Biol . Chem . 257, 260-263) . Yet, how the NH2-terminal extension may itself affect thin filament Ca2+ signaling is unknown . To approach this question we have used molecular cloning, mutagenesis, and bacterial synthesis of a full-length cTnI and a truncated mutant (cTnI/NH2) missing the 32 amino acids . Using reconstituted preparations we could show no differences between cTnI and cTnI/NH2 either in inhibition of actomyosin ATPase activity, in Ca(2+)-reversible inhibitory activity, or in the relation between pCa and Ca2+ binding to the regulatory site of cTnC at either pH 7.0 or 6.5 . There were also no significant differences at either pH in the pCa-MgATPase activity relation of myofibrils into which the various species of TnI has been exchanged . Our results indicate: 1) that phosphorylation most likely induces a new state of TnI activity rather than altering an intrinsic effect of the NH2-terminal peptide on Ca2+ activation; and 2) that domains outside the NH2-terminal extension are important with regard to differences in effects of acidic pH on Ca2+ activation on cardiac and skeletal myofilaments. J Biol Chem, 1994 May 27, 269(21), 15132 - 9 Structural requirements of substrate DNA for binding to and cleavage by RuvC, a Holliday junction resolvase; Takahagi M et al.; To elucidate the molecular mechanism of the resolution of Holliday junctions by Escherichia coli RuvC protein, we studied biochemical properties of the protein using various synthetic DNA junctions as model substrates . RuvC cleaves not only a four-way junction but also three-way junctions efficiently . The central core of homology in the junction is essential for the substrates to be cleavable by RuvC . Although the divalent cations are essential for the endonuclease activity, RuvC efficiently forms specific complexes with four-way junctions in the absence of the cations, irrespective of the presence of homologous core sequences . By using T7 endonuclease I as a probe, we studied the topology of the substrate junctions used in our study . The results suggest that RuvC cleaves the three-way junctions with homology core when they become four-way conformers . From the present studies, we propose that RuvC initially binds mostly nonproductively to four-way junctions, which does not require divalent metals, and subsequently cleaves the junctions by a mechanism dependent on a divalent cation and a particular topological conformer that is induced by the sequences at the mobile junctions. J Biol Chem, 1994 May 27, 269(21), 15118 - 23 Human hepatitis virus X gene encodes a regulatory domain that represses transactivation of X protein; Murakami S et al.; The human hepatitis B virus (HBV) X gene seems to be essential for establishment of viral infection, and the X gene product, HBx, transactivates virus and host genes through a wide variety of cis-elements, whereas regulation of HBx has not been fully understood . We found that transactivation-negative HBx mutants truncated at the C-terminal portion specifically repressed the HBx transactivation in trans . The ability to trans-repress the HBx transactivation is confined to the N-terminal third of HBx . Transactivation-positive constructs of HBx were divided into two groups by their sensitivity to trans-repression due to the presence of the N-terminal third . Thus the regulatory domain, the N-terminal third, is separated from the transacting domain and responsible for the negative regulations, the trans-repression and sensitivity to X trans-repression . A possible direct association between the HBx regulatory domains was tested by far-Western blotting using purified fused forms of HBx proteins . The regulatory domain was found to associate preferentially with the full HBx or the regulatory domain, but not with the transacting domain . Taken together, it is possible that HBx has a self-regulatory mechanism that avoids excessive HBx transactivation and is important for regulation of X gene expression. J Biol Chem, 1994 May 27, 269(21), 14974 - 81 The mRNA (guanine-7-)methyltransferase domain of the vaccinia virus mRNA capping enzyme . Expression in Escherichia coli and structural and kinetic comparison to the intact capping enzyme; Higman MA et al.; The mRNA (guanine-7-)methyltransferase active site of the heterodimeric vaccinia virus mRNA capping enzyme was previously localized to the carboxyl-terminal third of the large subunit, D1R, associated with the small subunit, D12L (Cong, P., and Shuman, S . (1992) J . Biol . Chem . 267, 16424-16429; Higman, M . A., Bourgeois, N., and Niles, E . G . (1992) J . Biol . Chem . 267, 16430-16437) . A plasmid was constructed which directs the coexpression of the carboxyl terminus of the D1R subunit from amino acids 498 to 844 and the D12L subunit in Escherichia coli . The mRNA (guanine-7-)methyltransferase catalytic activity in the isolated domain was found to be kinetically equivalent to that present in the intact enzyme . Through mobility shift and ultraviolet photolinkage analyses, both domains were shown to bind RNA in a saturable fashion . RNA binding was localized predominantly to the large subunit, but a low level of linkage of RNA to D12L was also observed . A low, but reproducible, level of mRNA (guanine-7-)methyltransferase activity was detected in the isolated D1R498-844 subunit demonstrating that the active site resides solely within the large subunit of the capping enzyme . This activity is enhanced 30- to 50-fold by the association of the D12L subunit. J Biol Chem, 1994 May 27, 269(21), 14885 - 91 In vitro asymmetric binding of the pleiotropic regulatory protein, FruR, to the ace operator controlling glyoxylate shunt enzyme synthesis; Cortay JC et al.; The fruR gene of Escherichia coli, which encodes the regulatory protein FruR, was cloned in the pT7-5 expression vector so as to overproduce a protein tagged with 6 histidine residues . By using a one-step chromatographic procedure, FruR was purified to near-homogeneity . Analysis of the protein under both denaturing and nondenaturing conditions indicated that it is a tetramer with a molecular mass of about 150 kilodaltons . The positions of interference between FruR and the operator of the acetate operon were examined . The number and nature of the nucleotides essential for FruR binding were determined by several different techniques: base methylation with dimethyl sulfate, base removal by formic acid and hydrazine, uracil interference, and hydroxyl radical footprinting . It was observed that FruR asymmetrically binds to a 16-base pair DNA sequence located 170 base pairs upstream from the transcriptional start point of the ace operon. J Biol Chem, 1994 May 27, 269(21), 15318 - 24 Substrate specificity of Fpg protein . Recognition and cleavage of oxidatively damaged DNA; Tchou J et al.; The 8-oxoguanine-DNA glycosylase of Escherichia coli, also known as formamidopyrimidine-DNA glycosylase (Fpg protein), has N-glycosylase and AP-lyase activities . This enzyme repairs oxidative DNA damage by efficiently removing formamidopyrimidine lesions and 8-oxoguanine residues from DNA . Defined oligodeoxynucleotides containing various 8-oxopurines were used to examine the substrate specificity of Fpg protein and to establish the role of functional groups in DNA on damage recognition and catalysis . Binding affinities of Fpg protein were established for duplex oligodeoxynucleotides containing 8-oxo-2'-deoxyguanine, 8-oxo-2'-deoxyadenine, 8-oxo-2'-deoxynebularine, 8-oxo-2'-deoxyinosine, abasic sites, and a ring-open adduct of C8-aminofluorene guanine . The C8 keto group of 8-oxodG:dC presents in the major groove and is correlated with tight binding (Kd = 8.9 nM) . Binding is much weaker when the C8 keto functional group is in the minor groove, as in 8-oxodG:dA (Kd = 340 nM) . Km and Vmax were determined for the cleavage reaction . Specificity constants (Kcat/Km) are consistently higher for oligodeoxynucleotide duplexes containing 8-oxopurines with C6 and C8 keto groups, as in 8-oxodG:dC and 8-oxodI:dC, where Kcat/Km are 9.3 and 18 min-1 nM x 10(-3), respectively . 8-oxodN:dC lacks the C6 keto group; the specificity constant is 0.024 min-1 nM x 10(-3) . Taken together, our data suggest that the C8 keto group of 8-oxodeoxyguanine and the carbonyl moiety of formamidopyrimidine enable Fpg protein to recognize and bind duplex DNA containing these modified bases . An enzyme-catalyzed reaction involving the C6 keto group of the substrate leads to removal of these lesions . A mechanism involving protonation at O-6 of 8-oxoguanine is proposed to account for the N-glycosylase activity of this enzyme. J Biol Chem, 1994 May 27, 269(21), 15223 - 8 Localization of vitronectin binding domain in plasminogen activator inhibitor-1; Lawrence DA et al.; Plasminogen activator inhibitor type 1 (PAI-1) is the rapid physiologic inhibitor of tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA) . In plasma and the extracellular matrix, PAI-1 is associated with the adhesive glycoprotein vitronectin . In order to characterize the PAI-1 structural domain responsible for binding to vitronectin, the segment of the PAI-1 cDNA encoding amino acids 13-147 (nucleotides 248-650) was randomly mutagenized and subcloned into a bacterial expression vector containing the mature PAI-1 coding sequence . Recombinant PAI-1 mutants were expressed in Escherichia coli and bacterial lysates assayed in duplicate for uPA inhibitory activity and vitronectin binding . Of 190 clones screened, six consistently demonstrated decreased vitronectin binding relative to uPA inhibitory activity . DNA sequence analysis of four of these clones identified 10 unique missense mutations, all located between base pairs 298 and 641, with each clone containing between one and four substitutions . Each substitution was expressed independently by site-directed mutagenesis and again analyzed for uPA inhibitory activity and vitronectin binding . Five point mutations that selectively disrupt vitronectin binding were identified . All 5 residues are located on the exterior of the PAI-1 structure . These findings appear to define a complex binding surface that bridges alpha-helices C and E to beta-strand 1A and includes amino acids 55, 109, 110, 116, and 123 . These results suggest that vitronectin binding may stabilize the active conformation of PAI-1 by restricting the movement of beta-sheet A and thereby preventing insertion of the reactive center loop. Nucleic Acids Res, 1994 May 25, 22(10), 1897 - 902 Mechanism of mutation on DNA templates containing synthetic abasic sites: study with a double strand vector; Takeshita M et al.; Mutagenesis at abasic sites was investigated in E.coli and simian kidney (COS) cells using a duplex shuttle vector containing synthetic analogs of deoxyribose on the phosphodiester backbone . Lesions were positioned on opposite strands of the vector . When the tetrahydrofuranyl analog was used as the abasic site, AT or TA pairs (65-80%) were introduced at the site of the bistrand lesion . Mutagenesis occurred in the absence of SOS induction . Single base deletions (> 80%) dominated the mutational spectra for propanyl and ethanyl analogs of abasic sites lacking a ring structure . For all abasic site analogs, a small proportion of G/C and C/G pairs (6-10%) were observed . dAMP was incorporated predominantly opposite tetrahydrofuranyl sites positioned in the single strand region of a gapped duplex vector . We conclude from these studies that abasic sites positioned in a bistrand configuration are highly mutagenic in E.coli and COS cells . Repair DNA synthesis may be involved in this process. Mol Gen Genet, 1994 May 25, 243(4), 477 - 81 Mapping of transcriptional start sites of the cea and cei genes of the ColE7 operon; Soong BW et al.; Two transcriptional start sites were identified 77 and 78 nucleotides upstream of the translation initiation codon of the colicin E7 gene (ceaE7) . The guanosine nucleotide located at the fifth position of the SOS box is probably a universal transcriptional start site of all E group colicins . Major and minor transcripts of the immunity gene (cei) are initiated at the 3' end of the cea gene . Relative to the -10 sequence, CAAAAT, of the major ceiE7 promoter, the corresponding region of the cei gene of other E group colicins has an increased content of guanosine nucleotides . However the -10 sequence of the minor ceiE7 promoter, TATGAT, was found to be conserved in other colicin promoters . The results indicate that the structure of the major promoter of the ceiE7 gene is unique among the E group colicins. Mol Gen Genet, 1994 May 25, 243(4), 409 - 16 The catalytic domain of Escherichia coli K-12 adenylate cyclase as revealed by deletion analysis of the cya gene; Crasnier M et al.; In Escherichia coli, adenylate cyclase activity is regulated by phosphorylated EnzymeIIA(Glc), a component of the phosphotransferase system for glucose transport . In strains deficient in EnzymeIIA(Glc), cAMP levels are very low . Adenylate cyclase containing the D414N substitution produces a low level of cAMP and it has been proposed that D414 may be involved in the process leading to activation by EnzymeIIA(Glc) . In this work, spontaneous secondary mutants producing large amounts of cAMP in strains deficient in EnzymeIIA(Glc) were obtained . The secondary mutations were all deletions located in the cya gene around the D414N mutation, generating adenylate cyclases truncated at the carboxyl end . Among them, a 48 kDa protein (half the size of wild-type adenylate cyclase) was shown to produce ten times more cAMP than wild-type adenylate cyclase in strains deficient in EnzymeIIA(Glc) . In addition, this protein was not regulated in strains grown on glucose and diauxic growth was abolished . This allowed the definition of a catalytic domain that is not regulated by the phosphotransferase system and produces levels of cAMP similar to that of regulated wild-type adenylate cyclase in wild-type strains grown in the absence of glucose . Further analysis allowed the characterization of the COOH-terminal regulatory domain, which is proposed to be inhibitory to the activity of the catalytic domain. Mol Gen Genet, 1994 May 25, 243(4), 400 - 8 ADP-glucose pyrophosphorylase in shrunken-2 and brittle-2 mutants of maize; Giroux MJ et al.; The Shrunken-2 (Sh2) and Brittle-2 (Bt2) genes of maize encode subunits of the tetrameric maize endosperm ADPglucose pyrophosphorylase . However, in all sh2 and bt2 mutants so far examined, measurable ADPglucose pyrophosphorylase activity remains . We have investigated the origin of the residual activity found in various sh2 and bt2 mutants as well as tissue specific expression and post-translational modification of the Sh2 and Bt2 proteins . Sh2 and Bt2 cDNAs were expressed in Escherichia coli and antibodies were prepared against the resulting proteins SH2 and BT2 specific antibodies were used to demonstrate that SH2 and BT2 are endosperm specific, are altered or missing in various sh2 or bt2 mutants, and have a mol . wt . of 54 and 51 kDa respectively in the wild type . The Sh2 and Bt2 transcripts are also endosperm specific . Ten sh2 and eight bt2 mutants show varying severity of phenotypes expressed at transcript, protein subunit and kernel level . Synthesis of multiple transcripts and proteins commonly occurs as a result of sh2 or bt2 mutation . While all mutants produce detectable enzymic activity, not all produce detectable transcripts and proteins . To examine the origin of the apparent non-SH2/BT2 endosperm enzymic activity, homologs of Sh2 and Bt2, designated Agp1 and Agp2 respectively, were isolated from an embryo cDNA library and found to hybridize to endosperm transcripts distinct from those of Sh2 and Bt2 . Thus Agp1 and Agp2 or closely related genes may be responsible for the residual activity in some sh2 and bt2 mutants . Surprisingly, no evidence of post-translational modification of the SH2 and BT2 protein subunits was detected. Mol Gen Genet, 1994 May 25, 243(4), 379 - 89 Requirement of the Pro-Cys-His-Arg sequence for O6-methylguanine-DNA methyltransferase activity revealed by saturation mutagenesis with negative and positive screening; Ihara K et al.; O6-Methylguanine-DNA methyltransferase catalyzes transfer of a methyl group from O6-methylguanine and O4-methylthymine of DNA to a cysteine residue of the enzyme protein, thereby repairing the mutagenic and carcinogenic lesions in a single-step reaction . There are highly conserved amino acid sequences around the methyl-accepting cysteine site in eleven molecular species of methyltransferases . To elucidate the significance of the conserved sequence, amino acid substitutions were introduced by site-directed mutagenesis of the cloned DNA for Escherichia coli Ogt methyltransferase, and the activity and stability of mutant forms of the enzyme were examined . When cysteine-139, to which methyl transfer occurs, was replaced by other amino acids, all of the mutants showed the methyltransferase-negative phenotype . Methyltransferase-positive revertants, isolated from one of the negative mutants, had restored codons for cysteine . Thus the cysteine residue is essential for acceptance of the methyl group and is not replaceable by other amino acids . Using this negative and positive selection procedure, the analysis was extended to other residues near the acceptor site . At the histidine-140 and arginine-141 sites, all the positive revertants isolated carried codons for amino acids identical to those of the wild-type protein . At proline-138, five substitutions (serine, glutamine, threonine, histidine, and alanine) exhibited the positive phenotype but levels of methyltransferase activity in extracts of cells harboring these mutant forms were very low . This suggests that the proline residue at this site is important for maintaining the proper conformation of the protein . With valine-142 substitutions there were seven types of positive revertants, among which mutants carrying isoleucine, cysteine, leucine, and alanine showed relatively high levels of methyltransferase activity . These results indicate that the sequence Pro-Cys-His-Arg is a sine qua non for methyltransferase to exert its function. Mol Gen Genet, 1994 May 25, 243(4), 374 - 8 Regulation of replication of plasmid pBR322 in amino acid-starved Escherichia coli strains; Herman A et al.; The stringent response causes inhibition of replication of plasmid pBR322 in amino acid-starved Escherichia coli cells whereas in relaxed mutants the replication of this plasmid proceeds for several hours . On the basis of density shift experiments and pulse-labelling experiments we showed that most of the pBR322 molecules begin replication during the relaxed response and the rate of plasmid DNA synthesis in unstarved and isoleucine-starved relA- bacteria is similar . We found that the Rom function plays a key role in the stringent control of plasmid pBR322 replication, as insertional inactivation of the rom gene causes amplification of pBR322rom- in both relA- and relA+ strains during amino acid starvation . Moreover, pUC19, which is a pBR322-derived plasmid lacking the rom gene, behaves like pBR322rom-, whereas introduction of the rom gene into the pUC19 replicon drives it into the pBR322 mode of replication in amino acid-starved bacteria . A model for the regulation of pBR322 plasmid DNA replication by Rom protein in amino acid-starved Escherichia coli strains is proposed. Nucleic Acids Res, 1994 May 25, 22(10), 1866 - 73 Removal of 3'-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1; Winters TA et al.; A recombinant human AP endonuclease, HAP1, was constructed and characterized with respect to its ability to recognize and act upon a model double-stranded 39-mer oligodeoxyribonucleotide substrate containing a strand break site with 3'-phosphoglycolate and 5'-phosphate end-group chemistries . This oligodeoxyribonucleotide substrate exactly duplicates the chemistry and configuration of a major DNA lesion produced by ionizing radiation . HAP1 was found to recognize the strand break, and catalyze the release of the 3'-phosphoglycolate as free phosphoglycolic acid . The enzyme had a Vmax of 0.1 fmole/min/pg of HAP1 protein, and a Km of 0.05 microM for the 3'-phosphoglycolate strand break lesion . The mechanism of catalysis was hydrolysis of the phosphate ester bond between the 3'-phosphoglycolate moiety and the 3'-carbon of the adjacent dGMP moiety within the oligonucleotide . The resulting DNA contained a 3'-hydroxyl which supported nucleotide incorporation by E . coli DNA polymerase I large fragment . AP endonucleolytic activity of HAP1 was examined using an analogous double-stranded 39-mer oligodeoxyribonucleotide substrate, in which the strand break site was replaced by an apyrimidinic site . The Vmax and Km for the AP endonuclease reaction were 68 fmole/min/pg of HAP1 protein and 0.23 microM, respectively. Mol Gen Genet, 1994 May 25, 243(4), 434 - 41 A ribosomal frameshifting error during translation of the argI mRNA of Escherichia coli; Fu C et al.; Using fusions between the Escherichia coli genes argI and lacZ, it has been demonstrated that ribosomal frameshifting occurs at a frequency of between 3% and 16% within the argI mRNA, soon after the initiation codon . The frameshift involves a phenylalanyl-tRNA shifting into the +1 frame at the sequence UUU-U/C . The shift does not occur if the in-frame phenylalanine codon UUU is replaced by UUC . The level of frameshifting is higher in dense cultures and is not dependent on phenylalanine starvation . In the wild-type argI gene this frameshifting event would be an error, leading to a truncated, non-functional protein . Therefore, it is unlike the numerous examples of required frameshifting events that have been described in other genes. Proc Natl Acad Sci U S A, 1994 May 24, 91(11), 5027 - 9 An algorithm to generate low-resolution protein tertiary structures from knowledge of secondary structure; Monge A et al.; An algorithm is described to assemble the three-dimensional fold of a protein starting from its secondary structure . A reduced representation of the polypeptide chain is used together with a crude potential based on pair hydrophobicities . The method is shown to be successful in locating the native topology for two 4-alpha-helix bundles, myohemerythrin and cytochrome b-562. Proc Natl Acad Sci U S A, 1994 May 24, 91(11), 5017 - 21 Formation of a ternary complex by human XPA, ERCC1, and ERCC4(XPF) excision repair proteins; Park CH et al.; The xeroderma pigmentosum complementation group A (XP-A) protein, XPA, has recently been expressed in Escherichia coli in a soluble and fully functional form . An affinity column was prepared by linking the XPA protein to a solid support . When HeLa cell-free extract capable of excision repair was applied to the column, > 99.9% of the proteins were in the flow-through . However, the flow-through fraction lacked excision activity . The activity was restored by adding the high salt (1 M KCl) eluate of the column to the flow-through fraction . The XPA protein-bound fraction was tested for specific proteins by an in vitro complementation assay with a panel of cell-free extracts from DNA repair-deficient human and rodent cell lines . The XPA-bound fraction complemented cell-free extracts of excision repair cross-complementing 1 (ERCC-1), ERCC-4 (XP-F), and XP-A mutants . We conclude that the XPA damage recognition protein makes a ternary complex with the ERCC1/ERCC4(XPF) heterodimer with a potential nuclease function. Proc Natl Acad Sci U S A, 1994 May 24, 91(11), 4703 - 7 Subunit dynamics in Escherichia coli preprotein translocase; Joly JC et al.; SecY, SecE, and band 1 copurify as the SecY/E integral membrane domain of Escherichia coli preprotein translocase . To measure the in vivo association of these polypeptides and assay possible exchange, plasmid-borne secY and secE genes were placed under control of the ara regulon and fused to DNA encoding the influenza hemagglutinin epitope . Cells were incubated with {35S}methionine, grown for a "chase" period, and then induced with arabinose to express epitope-tagged, nonradioactive SecY and SecE . Both the wild-type and epitope-tagged polypeptides assembled into functional, heterotrimeric SecY/E complex . However, immunoprecipitation with antibody to the epitope tag did not cross-precipitate radiolabeled SecY or SecE . Thus, these subunits normally associate stably in vivo. Biochemistry, 1994 May 24, 33(20), 6356 - 62 Thermodynamic properties of the transition state for the rate-limiting step in the folding of the alpha subunit of tryptophan synthase; Chen X et al.; To gain insight into the physical properties of the transition state for the rate-limiting step in the folding of the alpha subunit of tryptophan synthase from Escherichia coli, the urea dependence of the unfolding reaction was examined as a function of temperature . Consistent with a previous, more limited study {Hurle, M.R., Michelotti, G.A., Crisanti, M.M., & Matthews, C.R . (1987) Proteins 2, 54}, the activation entropy for unfolding was found to be negative above 4 M urea . The present study extends this finding to show that both the activation entropy and enthalpy decrease with increasing urea concentrations between 4 and 7.5 M . The change in the heat capacity from the native to the transition state is positive and appears to increase with the denaturant concentration . The urea and temperature dependences of the unfolding rates were analyzed in terms of the denaturant-binding model of Tanford {Tanford, C . (1970) Adv . Protein Chem . 24, 1} . The values for the activation enthalpy and activation entropy of binding are in good agreement with those obtained from a calorimetric study of urea binding to unfolded proteins {Makhatadze, G.I., & Privalov, P.L . (1992) J . Mol . Biol . 226, 491} . These results show that (1) the binding of urea to the transition state of the alpha subunit has thermodynamic properties which are similar to those for urea binding to unfolded proteins, (2) the transition state is distinct from the unfolded conformation and exposes only a fraction of its urea-binding sites to solvent, and (3) the negative value for the activation entropy for unfolding reflects, in part, the ordering of urea on newly exposed surfaces.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 May 24, 33(20), 6350 - 5 Detection of an intermediate in the folding of the (beta alpha)8-barrel N-(5'-phosphoribosyl)anthranilate isomerase from Escherichia coli; Jasanoff A et al.; We have used thermodynamic and kinetic techniques to monitor the guanidinium chloride induced (GdmCl-induced) denaturation of N-(5'-phosphoribosyl)anthranilate isomerase from Escherichia coli (ePRAI) . Although CD-monitored equilibrium denaturation curves are consistent with cooperative unfolding of the protein centered at 1.45 M GdmCl, fluorescence readings drop by over 25% in the region preceding the CD-monitored transition, suggesting non-two-state behavior . Kinetics experiments measure a slow relaxation rate with negative fluorescence amplitude when protein is diluted from 0 to 0.5 M GdmCl, corroborating results from equilibrium conditions . Detection of several unfolding and refolding rates in final GdmCl concentrations from 0 to 5.0 M indicates the presence of at least one intermediate along unfolding and refolding pathways . GdmCl dependence of the relaxation rates can be explained most easily by a nonsequential mechanism for ePRAI unfolding, though a sequential mechanism cannot be ruled out . The data corroborate the fragment complementation studies of Eder and Kirschner {Eder, J., & Kischner, K . (1992) Biochemistry 31, 3617-3625}, which are consistent with unfolding of the C-terminal portion of a yeast-derived PRAI in its folding intermediate . In ePRAI, such partial unfolding would expose W391 to quenching by solvent molecules; W356, ePRAI's other tryptophan, is buried in the hydrophobic core and is unlikely to be affected by local changes in structure . A C-terminally unfolded folding intermediate has been demonstrated in the folding of tryptophan synthase (alpha-subunit), a related beta alpha-barrel enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 May 24, 33(20), 6213 - 20 Modulation of the oxidation-reduction potential of the flavin in lipoamide dehydrogenase from Escherichia coli by alteration of a nearby charged residue, K53R; Maeda-Yorita K et al.; The epsilon-amino group of a lysine residue occupies a position within bonding distance of the flavin N5 and the bound NADPH pyridinium C4' in glutathione reductase, and it has been suggested that this positive charge influences the redox potential of the FAD {Pai & Schulz (1983) J . Biol . Chem . 258, 1752} . A conserved lysine residue occupies a similar position in lipoamide dehydrogenase . This residue has been replaced by an arginine in lipoamide dehydrogenase from Escherichia coli to give K53R . The spectral and redox properties of the FAD in K53R as well as the interaction of the flavin with bound NAD+ are profoundly affected by the change . K53R does not catalyze either the dihydrolipoamide-NAD+ or the NADH-lipoamide reactions except at very low concentrations of the reducing substrate . The absorbance spectrum of K53R in the visible and near-ultraviolet is little changed from that of wild-type enzyme, but in contrast, the spectrum of K53R is sensitive to pH with an apparent pKa = 7.0 . Unlike the wild-type enzyme, the binding of beta-NAD+ to K53R alters the spectrum and indicates an apparent Kd = 7.0 microM at pH 7.6 . The flavin fluorescence is partially quenched, and the visible and near-ultraviolet circular dichroism spectrum is changed by beta-NAD+ . K53R is extensively reduced (mostly EH4) by 2 equiv of dihydrolipoamide/FAD while the wild-type enzyme is only partially reduced (mostly EH2) . The rate of this reduction is lowered by approximately 3-fold relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 May 24, 33(20), 6177 - 85 Mapping critical residues in eukaryotic DNA-binding proteins: a plasmid-based genetic selection strategy with application to the Oct-2 POU motif; Botfield MC et al.; Discrimination between allowed and disallowed amino acid substitutions provides a powerful method for analysis of protein structure and function . Site-directed mutagenesis allows specific hypotheses to be tested, but its systematic application to entire structural motifs is inefficient . This limitation may be overcome by genetic selection, which allows rapid scoring of thousands of randomly (or pseudorandomly) generated mutants . To facilitate structural dissection of DNA-binding proteins, we have designed two generally applicable bacterial selection systems: pPLUS selects for the ability of a protein to bind to a user-defined DNA sequence, whereas pMINUS selects against such binding . Complementary positive and negative selections allow rapid mapping of critical residues . To test and calibrate the systems, we have investigated the bipartite POU domain of the human B-cell-specific transcription factor Oct-2 . (i) An invariant residue (Asn347) in the DNA-recognition helix of the POU-specific homeodomain (POUHD) was substituted by each of the 19 other possible amino acids . The mutant proteins each exhibited decreased specific DNA binding as defined in vivo by genetic selection and in vitro by gel retardation; relative affinities correlate with phenotypes in the positive and negative selection systems . An essential role for Asn347 in wild-type POUHD-DNA recognition is consistent with homologous Asn-adenine interactions in cocrystal structures of canonical homeodomains . (ii) Extension of pPLUS/pMINUS selection to the POU-specific subdomain (POUs) is demonstrated by analysis of mutations in its putative helix-turn-helix (HTH) element.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 May 24, 33(20), 6167 - 76 Effects of base composition on the negative cooperativity and binding mode transitions of Escherichia coli SSB-single-stranded DNA complexes; Lohman TM et al.; We have examined the ability of the Escherichia coli single-stranded DNA binding protein (SSB) tetramer to form its different binding modes on poly(dC), poly(U), and poly(A) over a range of NaCl and NaF concentrations for comparison with previous studies with poly(dT) . In reverse titrations with poly(U) and poly(A) at 25 degrees C, pH 8.1, SSB forms all four binding modes previously observed with poly(dT), namely, (SSB)35, (SSB)40, (SSB)56, and (SSB)65, where the subscript denotes the site size (i.e., the average number of nucleotides occluded per SSB tetramer) . As with poly(dT), the low site size modes are favored at low monovalent salt concentration (< 10 mM), whereas increasing salt concentration facilitates the transitions to the higher site size modes . Surprisingly, SSB does not form a stable (SSB)35 complex on poly(dC), even at 1 mM NaCl; rather, the (SSB)56 mode is formed under these conditions . Upon raising the {NaCl}, the (SSB)56 complex undergoes a transition to the (SSB)65 complex (transition midpoint, 40 mM NaCl) . On the basis of studies with dC(pC)34, dT(pT)34, and dA(pA)34, the inability of the SSB tetramer to form the (SSB)35 complex with poly(dC) is due mainly to a much lower degree of negative cooperativity for binding oligodeoxycytidylates to the SSB tetramer . At low salt concentration, the negative cooperativity parameter, sigma 35, is lowest for dA(pA)34, intermediate for dT(pT)34, and highest for dC(pC)34, indicating that it is most difficult to saturate the SSB tetramer with two molecules of dA(pA)34 . We have also measured the equilibrium constants for binding the oligodeoxynucleotides dC(pC)34, dC(pC)69, dA(pA)34, and dA(pA)69 as a function of {NaCl} and {NaBr} and find that the salt dependencies of these oligonucleotides are dependent upon base composition . These studies also indicate that ion binding accompanies formation of these SSB-ss-DNA complexes, although there is a net release of ions upon formation of the complex . This influence of both salt concentration and base composition indicates that both electrostatic and nonelectrostatic factors contribute to the negative cooperativity associated with ss-DNA binding to the SSB tetramer. Biochemistry, 1994 May 24, 33(20), 6100 - 9 Attractant- and disulfide-induced conformational changes in the ligand binding domain of the chemotaxis aspartate receptor: a 19F NMR study; Danielson MA et al.; The isolated ligand binding domain of the chemotaxis aspartate receptor is the focus of the present study, which both (a) identifies structural regions involved in the attractant-induced conformational change and (b) investigates the kinetic parameters of attractant binding . To analyze the attractant-induced conformational change within the homodimeric domain, 19F NMR is used to monitor six para-fluorophenylalanine (4-F-Phe) positions within each identical subunit of the homodimer . The binding one molecule of aspartate to the homodimer perturbs three of the 4-F-Phe resonances significantly: 4-F-Phe150 in the attractant binding site, 4-F-Phe107 located 26 A from the site, and 4-F-Phe180 at a distance of 40 A from the site . Comparison of the frequency shifts triggered by aspartate and glutamate reveals that these attractants generate different conformations in the vicinity of the attractant site but trigger indistinguishable long-range conformational effects at distant positions . This long-range conformational change is specific for attractant binding, since formation of the Cys36-Cys36' disulfide bond or the nonphysiological binding of 1,10-phenanthroline to an aromatic pocket distal to the attractant site each yield conformational changes which are significantly more localized . The attractant-triggered perturbations detected at 4-F-Phe107 and 4-F-Phe180 indicate that the structural change includes an intrasubunit component communicated through the domain to its C-terminal region, which, in the full-length receptor, continues through the membrane as the second membrane-spanning helix . It would thus appear that the transmembrane signal is transmitted through this helix.(ABSTRACT TRUNCATED AT 250 WORDS) Proc Natl Acad Sci U S A, 1994 May 24, 91(11), 5133 - 7 Three-dimensional working model of M1 RNA, the catalytic RNA subunit of ribonuclease P from Escherichia coli; Westhof E et al.; A three-dimensional model of M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, was constructed with the aid of a computer . The modeling process took into account data from chemical and enzymatic protection experiments, phylogenetic analysis, studies of the activities of mutants, and the kinetics of reactions catalyzed by the binding of substrate to M1 RNA . The model provides a plausible picture of the binding to M1 RNA of the tRNA domain of a precursor tRNA substrate . The scissile bond and adjacent segments of the aminoacyl acceptor stem of a precursor tRNA substrate can fit into a cleft that leads to the phylogenetically conserved, central part of the structure. Proc Natl Acad Sci U S A, 1994 May 24, 91(11), 5114 - 8 Solution NMR structure of the major cold shock protein (CspA) from Escherichia coli: identification of a binding epitope for DNA; Newkirk K et al.; Sequence-specific 1H and 15N resonance assignments have been determined for the major cold shock protein (CspA) from Escherichia coli with recently developed three-dimensional triple-resonance NMR experiments . By use of these assignments, five antiparallel beta-strands were identified from analysis of NMR data . Strands 1-4 have a classical 3-2-1-4 Greek key beta-sheet topology and there are two beta-bulges, at positions Lys10-Trp11 and Gly65-Asn66 . Three-dimensional structures of CspA were generated from NMR data by using simulated annealing with molecular dynamics . The overall chain fold of CspA is a beta-barrel structure, with a tightly packed hydrophobic core . Two-dimensional isotope-edited pulsed-field gradient 15N-1H heteronuclear single-quantum coherence spectroscopy was used to characterize the 15N-1H fingerprint spectrum with and without a 24-base oligodeoxyribonucleotide, 5'-AACGGTTTGACGTACAGACCATTA-3' . Protein-DNA complex formation perturbs a subset of the amide resonances that are located mostly on one face of the CspA molecule . This portion of the CspA molecular surface includes two putative RNA-binding sequence motifs which contribute to an unusual cluster of eight surface aromatic side chains: Trp11, Phe12, Phe18, Phe20, Phe31, His33, Phe34, and Tyr42 . These surface aromatic groups, and also residues Lys16, Ser44, and Lys60 located on this same face of CspA, are highly conserved in the family of CspA homologues . These isotope-edited pulsed-field gradient NMR data provide a low-resolution mapping of a DNA-binding epitope on CspA. Proc Natl Acad Sci U S A, 1994 May 24, 91(11), 4713 - 7 TAR RNA-binding protein is an inhibitor of the interferon-induced protein kinase PKR; Park H et al.; A cDNA encoding a double-stranded-RNA (dsRNA)-binding protein was isolated by screening a HeLa cell cDNA expression library for proteins that bind the HIV-1 Rev-responsive-element RNA . The cDNA encoded a protein that was identical to TRBP, the previously reported cellular protein that binds the transactivation response element (TAR) RNA of human immunodeficiency virus type 1 . TRBP inhibited phosphorylation of the interferon-induced ribosome-associated protein kinase PKR and of the eukaryotic translation initiation factor eIF-2 alpha in a transient-expression system in which the translation of a reporter gene was inhibited by the localized activation of PKR . TRBP expression in HeLa cells complemented the growth and protein-synthesis defect of a vaccinia virus mutant lacking the expression of the dsRNA-binding protein E3L . These results implicate TRBP as a cellular regulatory protein that binds RNAs containing specific secondary structure(s) to mediate the inhibition of PKR activation and stimulate translation in a localized manner. FEBS Lett, 1994 May 23, 345(1), 23 - 6 Prediction of the structural similarity between spermidine/putrescine-binding protein and maltose-binding protein; Matsuo Y et al.; A structural similarity between spermidine/putrescine-binding protein and maltose-binding protein has been predicted by a sequence-structure compatibility method . The sequence alignment obtained by this method revealed a consensus sequence motif located on the surface loop between the first alpha-helix and the second beta-strand, and the further analysis identified a similar motif in iron-binding protein . The conservation of this motif among certain bacterial periplasmic binding proteins suggests a common functional role for this region as well as an evolutionary relationship between them. J Mol Biol, 1994 May 20, 238(5), 852 - 3 Crystallization and preliminary crystallographic data for an FMN-dependent nitroreductase from Escherichia coli B; Skelly JV et al.; An FMN-dependent nitroreductase enzyme isolated from Escherichia coli B has been crystallized in a form suitable for high-resolution structural analysis . The crystals belong to the tetragonal space group P4(1)2(1)2 or its enantiomorph P4(3)2(1)2 with cell parameters a = b = 57.74 A, c = 275.51 A and two molecules per asymmetric unit . Diffraction extends to beyond 1.9 A. J Mol Biol, 1994 May 20, 238(5), 669 - 81 A thermodynamic study of the trp repressor-operator interaction; Ladbury JE et al.; We have measured the heats of formation of the trp repressor/operator complex by direct titration calorimetry over the temperature range 10 degrees C to 40 degrees C . A primary strong mode of binding displays the characteristic large negative heat capacity change observed by other methods in the formation of specific protein/DNA complexes . Unlike most such reactions, however, the formation of the trp repressor/operator complex is enthalpically driven throughout the physiological temperature range . After saturation of this principal mode, we also detected a secondary weaker binding mode, which we ascribe to a now well documented interaction called "half-site" binding . Although weak, this mode also exhibits an unusually large negative heat capacity change . Since the interface of the proposed secondary half-site binding mode has the same complementary stereochemistry as the primary one (due to internal symmetry), we correlate the negative heat capacity change with the formation of a stereospecific interface and not with high affinity . As in similar cases, the empirical correlation between buried non-polar surfaces and reduction of heat capacity does not account for the large negative delta Cp, nor do crystal structures reveal any further reduction in solvent excluded surfaces within the reactants upon complex formation . We attribute the "unaccounted for" decrement in the heat capacity of the complex to the stereospecific restriction of the hydrated polar elements that form the specific interface . We suggest that the "tightening of soft internal modes" at and near the polar interface of the complex is more important than previously recognized because previous considerations did not take into account the highly hydrated nature of these polar elements and the concomitant reduction in the degrees of freedom of the water structure. J Mol Biol, 1994 May 20, 238(5), 643 - 8 Which nucleotides in the "-10" region are crucial to obtain a fully active MalT-dependent promoter? Danot O, Raibaud O. We have replaced the hexanucleotide corresponding to the "-10" region of malPp, a positively regulated promoter from Escherichia coli, by random nucleotide sequences and isolated 48 different variants that were as active as the wild-type promoter . Analysis of the nucleotide sequence of their "-10" region strongly suggests that the nature of the nucleotide present at three positions plays a crucial role: 46 of the 48 malPp variants contained C or T at position -12, A at position -11 and T at position -7 . The nucleotide composition at the three other positions seems to be much more flexible . The features of these "-10" regions are similar to those of the constitutive promoters, but some significant differences are noticeable and will be discussed . In contrast to other positively regulated promoters, none of the various "-10" regions, including the consensus sequence TATAAT, led to a constitutive promoter. J Biol Chem, 1994 May 20, 269(20), 14620 - 4 Glycoproteins with Gal alpha 4Gal are absent from human erythrocyte membranes, indicating that glycolipids are the sole carriers of blood group P activities; Yang Z et al.; The antigenic determinants of the blood group P family (P1, P, Pk, and LKE antigens) are chemically based on Gal alpha 4Gal . For human erythrocytes it has been claimed that the majority of P1 determinants are expressed in glycoproteins, mainly band 4.5 (Haselberger, C . G., and Schenkel-Brunner, H . (1982) FEBS Lett . 149, 126-128) . In the present work, the existence of Gal alpha 4Gal in glycoproteins of erythrocyte membranes (ghosts) of P1 positive and negative human individuals was carefully analyzed on replicas of sodium dodecyl sulfate-polyacrylamide electrophoresis gels using specific reagents (Escherichia coli HB101/pDC1 expressing the Pap gene and monoclonal antibodies with specificities for P1 and Pk antigens) . No binding to glycoproteins was detected with any of these ligands when the ghosts had been pretreated with butanol to remove glycolipids . Therefore, all antigenic determinants of the P blood group family on human red cells are exclusively expressed in glycolipids and are absent from glycoproteins. Carbohydr Res, 1994 May 20, 258, 233 - 41 Structure of the capsular antigen of Escherichia coli O8:K50:H-; Grue MR et al.; The primary structure of the acidic capsular antigen of Escherichia coli O8:K50:H- was shown by glycose analysis, methylation analysis, and one- and two-dimensional 1H and 13C NMR spectroscopy to be composed of repeating linear tetrasaccharide units having the structure: (formula: see text). J Biol Chem, 1994 May 20, 269(20), 14672 - 80 Comparison of deoxyoligonucleotide and tRNA(Lys-3) as primers in an endogenous human immunodeficiency virus-1 in vitro reverse transcription/template-switching reaction; Arts EJ et al.; We developed an endogenous in vitro reverse transcription assay to study the properties of priming and template switching during human immunodeficiency virus (HIV) replication . Reactions were primed with HIV reverse transcriptase (RT) and either a deoxyoligonucleotide primer (dPR) or tRNA(Lys-3), the natural primer for reverse transcription . The RNA templates utilized were the actual HIV sequences involved in the first template switch, namely a primer binding sequence (PBS)/U5/R RNA donor template and a R/U3 RNA acceptor template . Reverse transcription reactions using the latter templates and dPR or tRNA(Lys-3) as primers yielded four major products: (-)-strong-stop DNA, a partial template-switched DNA, full template-switched DNA, and a pseudo-PBS-primed product . Use of dPR resulted in three times less template switching than was obtained with tRNA(Lys-3) . When reactions were primed with either dPR or tRNA(Lys-3), increases in acceptor:donor template ratios resulted in augmented template switching . Increasing the concentration of RT resulted in increased priming from the PBS but had no effect on the efficiency of template switching . Decreasing the extent of R region overlap resulted in a drop in efficiency of template switching . Decreases in the R region on the donor template also caused a drop in initiation of transcription that was primed by tRNA(Lys-3) from the PBS . In contrast, a corresponding reduction of the R region on the acceptor template had no effect on priming . We conclude that a transcriptional complex of tRNA(Lys-3) and RT may be associated not only with the PBS but also with other cis RNA sequences and secondary structures in a manner essential for efficient priming and template switching. J Biol Chem, 1994 May 20, 269(20), 14566 - 74 Glycogen synthase kinase-3 beta is a dual specificity kinase differentially regulated by tyrosine and serine/threonine phosphorylation; Wang QM et al.; The enzyme glycogen synthase kinase-3 (GSK-3) has been implicated in the control of several metabolic enzymes and transcription factors in response to extracellular signals . In the past, the enzyme has been considered to be a protein Ser/Thr kinase although it was recently reported to contain Tyr(P) (Hughes, K., Nikolakaki, E., Plyte, S . E., Totty, N . F., and Woodgett, J . R . (1993) EMBO J . 12, 803-808) . A cDNA encoding rabbit skeletal muscle GSK-3 beta was cloned and expressed in Escherichia coli as an active protein kinase, with apparent M(r) 46,000, capable of phosphorylating several known GSK-3 substrates . Recombinant GSK-3 beta autophosphorylated on Ser, Thr, and Tyr residues although the enzyme already contained Tyr(P) as judged by its recognition by anti-Tyr(P) antibodies . The net result of the autophosphorylation was a 3-5-fold reduction in enzyme activity . GSK-3 alpha, purified from rabbit muscle, also underwent autophosphorylation but only on Ser and Thr residues . In this case, the autophosphorylation stabilized the enzyme activity compared with the control lacking ATP/Mg2+ . Of several phosphatases tested, the lambda-phage phosphatase was the most effective in dephosphorylating at Ser and Thr residues but did not dephosphorylate at Tyr residues . The action of the lambda-phosphatase caused a reactivation of GSK-3 beta to approximately 80% of the starting activity . The protein tyrosine phosphatase PTP1B was able to dephosphorylate at Tyr residues leading to a reduction in enzyme activity . A truncated form of GSK-3 beta, apparent M(r) 40,000, had a significantly higher specific activity, was defective in autophosphorylation, and was not inactivated in the autophosphorylation reaction . We conclude that GSK-3 beta is a dual specificity protein kinase in the same sense as the mitogen-activated protein kinase/ERK family of enzymes . Phosphorylation at different residues differentially controls enzyme activity, Ser/Thr phosphorylation causing inactivation and Tyr phosphorylation resulting in increased activity. Biochim Biophys Acta, 1994 May 18, 1206(1), 90 - 6 Spectroscopic, electrochemical, and ligand binding properties of the horse heart metmyoglobin His64-Tyr variant; Tang HL et al.; The distal histidine (E7) of horse heart myoglobin (Mb) has been replaced by tyrosine using site-specific mutagenesis . The resulting green Mb variant (His64-Tyr) was expressed in Escherichia coli JM101, isolated and purified to homogeneity . Spectrophotometric pH titrations of the variant exhibit a change in spectrum that occurs with a pKa of 4.7 (25 degrees C) . The midpoint reduction potential of the variant is 20 mV (vs . SHE at pH 7, 25 degrees C) . Cyanide and azide binding measurements indicate that the oxidized variant binds these anionic ligands with much greater affinity at pH 4.0 than at neutral pH . Extended X-ray absorption fine structure (EXAFS) spectroscopy establishes that the variant is six coordinate at pH 7.0 and pH 4.2 . Higher shell contributions to the iron EXAFS observed at pH 7.0 are attributed to tyrosine . These contributions are absent at pH 4.2 . Thus, the sixth heme iron ligand of the oxidized variant Mb at pH 7.0 is attributed to oxygen from the hydroxyl group of tyrosine and the sixth ligand present at pH 4.2 is attributed to the oxygen atom of a coordinated water . The EXAFS spectra, electronic absorption spectra, and ligand binding properties of the His64-Tyr Mb variant are consistent with the binding of Tyr-64 as the sixth heme iron ligand between pH 5 and 12 and with the replacement of Tyr-64 by a water molecule at low pH with a pKa of 4.7. Biochim Biophys Acta, 1994 May 18, 1206(1), 143 - 54 Strong-field and integral spin-ligand complexes of the cytochrome bo quinol oxidase in Escherichia coli membrane preparations; Calhoun MW et al.; The cytochrome bo-type terminal oxidase of Escherichia coli is an analogue of mammalian aa3-type cytochrome c oxidase . The catalytic core of both enzymes is a binuclear site containing a penta-coordinate heme (heme o or a3) and copper (CuB) . Herein we report on UV-visible and magnetic properties of ligand complexes of the binuclear site of cytochrome bo . Cyanide, sulfide, and azide react with the Fe(3+)-Cu+ center to give EPR-detectable low-spin complexes, analogous to those formed by cytochrome aa3 . Analyses of the ligand fields of these complexes indicate that heme o has a single axial histidine ligand . Cyanide and azide react with the Fe(3+)-Cu2+ center to yield forms observable via UV-visible spectroscopy but not EPR . With formate and fluoride, cytochrome bo forms integral spin complexes similar to those of cytochrome aa3 . These complexes have UV-visible characteristics of high-spin species, but EPR spectra show features which appear to correspond to transitions within an integral spin multiplet . Cytochrome bo forms another integral spin complex with azide and NO which is nearly identical to the azide-NO species in cytochrome aa3 . This suggests that the binuclear centers of the two enzymes are quite similar. Biochim Biophys Acta, 1994 May 18, 1206(1), 113 - 9 Catalytic core of rat tyrosine hydroxylase: terminal deletion analysis of bacterially expressed enzyme; Walker SJ et al.; Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in catecholamine biosynthesis . This enzyme is hypothesized to consist of an amino-terminal regulatory domain and a carboxy-terminal catalytic domain . In the present studies, we have utilized recombinant DNA techniques to map the boundaries of the regulatory and catalytic domains of TH . We have isolated a full-length cDNA clone for rat pheochromocytoma TH and have expressed the enzyme in bacteria . Utilizing this bacterial expression system and polymerase chain reaction technology, we have constructed and subcloned genes for five amino-terminal deletion mutants (N delta 40, N delta 155, N delta 165, N delta 175 and N delta 200; N delta denotes amino-terminal deletion and the numerical value denotes the number of amino acids deleted) and two carboxy-terminal deletion mutants (C delta 19 and C delta 50) . The catalytic core of rat tyrosine hydroxylase has been identified to include the region from amino acid #165 to amino acid #479 . The amino-terminal deletion mutants, N delta 40, N delta 155 and N delta 165 are from 1.85 to 2.5-fold more active than unmodified recombinant TH, while the removal of 19 amino acids from the C-terminus (C delta 19) results in a 70% reduction in enzyme activity . Removal of additional sequences (ten more residues from the N-terminus {N delta 175}; or an additional 31 amino acids from the C-terminus {C delta 50}) results in protein that is totally without enzyme activity . As expected, removal of 40 (or more) N-terminal amino acids abolishes the ability of the catalytic subunit of the cAMP-dependent protein kinase to phosphorylate the recombinant enzyme; serine-40 is the phosphorylation site on TH for PKA . We conclude that the N-terminal boundary for the TH catalytic domain resides between residues 165 and 175 and that removal of this N-terminal domain (totally or partially) increases the activity of the enzyme . These findings confirm previous reports that proteolytic cleavage at amino acid #158 produces an active (and activated) catalytic fragment. Biochim Biophys Acta, 1994 May 17, 1218(1), 21 - 6 On the binding of isolated yeast tRNA(Phe) anticodon arm to Escherichia coli 30S and 70S ribosomes . Guanosine-42 is important for the binding; Nekhai SA et al.; Conditions for the reversible binding of isolated anticodon arm of yeast tRNA(Phe) (15-nucleotide, corresponding to nucleotides N28-N42) to the P site of Escherichia coli 30S and 70S ribosomes have been determined . The affinity constant for the anticodon arm binding to 70S ribosomes is shown to be only 30-fold weaker than that for the binding of total tRNA(Phe) . The affinities of yeast tRNA(Phe) and the anticodon arm for 30S subunits and of the anticodon arm for the total 70S ribosomes are shown to be equal . These data imply that the anticodon arm moiety of tRNA(Phe) mainly contributes to the tRNA-70S ribosome interaction, i.e., it contributes for the most part to the total free energy of the deacylated tRNA(Phe) interaction with the P site of the 70S ribosome . By taking into account additional contacts in the 3'-CCA end, other contacts in the region besides the anticodon arm and 3'-CCA end are presumably absent . Within the anticodon arm removal of the 3'-end nucleotide corresponding to guanosine-42 in tRNA(Phe) decreases the association constant of the anticodon arm-ribosome interaction 15-fold . Replacement of this guanosine with other nucleosides as well as modification of the ribose moiety (oxidation and reduction) does not affect the affinity . The replacement data are very likely to indicate that G42 affects the anticodon arm affinity by forming a direct contact with ribosome. Biochim Biophys Acta, 1994 May 17, 1218(1), 119 - 22 Expression and myristoylation of NAP-22 using a baculovirus transfer vector system; Maekawa S et al.; NAP-22 is a brain enriched acidic protein localized in the membrane fraction . The peptide sequence of NAP-22 elucidated from the cDNA sequencing, however, showed that NAP-22 is a very hydrophilic protein having no transmembrane regions . The peptide sequence analysis also showed that NAP-22 has a consensus sequence of N-myristoylation and the presence of polybasic domain in its N-terminal region . These sites could be the anchoring sites to localize to the membrane . In this study, we showed the myristoylation of NAP-22 using a Baculovirus expression system and also showed a liposome binding ability of the expressed protein in Escherichia coli. Biochemistry, 1994 May 17, 33(19), 5974 - 83 Reactivity and ionization of the active site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo; Nelson JW et al.; DsbA is a periplasmic protein of Escherichia coli that appears to be the immediate donor of disulfide bonds to proteins that are secreted . Its active site contains one accessible and one buried cysteine residue, Cys30 and Cys33, respectively, which can form a very unstable disulfide bond between them that is 10(3)-fold more reactive toward thiol groups than normal . The two cysteine residues have normal properties when in a short peptide . In DsbA, the Cys30 thiol group is shown to be reactive toward alkylating reagents down to pH 4 and to be fully ionized, on the basis of the UV absorbance of the thiolate anion at 240 nm . Its reactivity is altered by another, unknown group on the reduced protein titrating with a pKa of about 6.7 . The other cysteine residue is buried and unreactive and has a high pKa value . The ionization properties of the DsbA thiol groups can explain, at least partly, the high reactivity of its disulfide bonds and thiol groups at both neutral and acidic pH values. Biochemistry, 1994 May 17, 33(19), 5942 - 6 Mutagenesis at a highly conserved phenylalanine in cytochrome P450 2E1 affects heme incorporation and catalytic activity; Porter TD; The phenylalanine corresponding to Phe-429 of rabbit cytochrome P450 2E1 is 1 of approximately 10 highly conserved residues in this superfamily of over 200 sequenced enzymes . This nearly invariant residue has been postulated to be involved in electron transfer between the heme of cytochrome P450cam and its redox partners {Stayton, P.S., Poulos, T . L., & Sligar, S.G . (1989) Biochemistry 28, 8201-8205} . To test this hypothesis, oligonucleotide-directed mutagenesis was used to replace this amino acid in rabbit P450 2E1 with aspartate, arginine, leucine, tryptophan, or tyrosine, and the mutant proteins were expressed in Escherichia coli . Although immunoblot analysis of whole cell lysates demonstrated that all P450 proteins (mutants and wild-type) were equally well expressed on a per cell basis, in solubilized membranes only the tryptophan and tyrosine mutants yielded ferrous-CO difference spectra characteristic of P450 . The specific content (nanomoles per milligram of membrane protein) and yield per liter of the Trp mutant holoenzyme were approximately one-third those of the native enzyme, suggesting that heme incorporation was hindered by tryptophan at this position, whereas the specific content and yield per liter of the Tyr mutant were significantly greater than those of the native preparation . The stability of the Trp and Tyr mutants, as judged by thermal denaturation studies, was not different from that of the native enzyme . The Trp mutant had 38% of the aniline hydroxylase activity, 25% of the p-nitrophenol hydroxylase activity, and 39% of the N-nitrosodimethylamine demethylase activity of the native enzyme, demonstrating that this substitution also decreased catalytic activity.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 May 17, 33(19), 5912 - 9 Assignment of disulfide bond location in prothoracicotropic hormone of the silkworm, Bombyx mori: a homodimeric peptide; Ishibashi J et al.; The disulfide bond location of a homodimeric peptide, prothoracicotropic hormone (PTTH) of the silkworm, Bombyx mori, was determined by a combination of partial reduction and sequence analysis of peptide fragments generated through a partial reduction of PTTH followed by alkylation and enzyme digestion . The partial reduction and S-alkylation broke the interchain disulfide bond but did not affect the intrachain disulfide bonds, generating monomeric PTTH whose intrachain disulfide bonds were kept intact . This monomeric PTTH has about one-half the biological activity of intact PTTH . Sequence analysis of the fragments generated by lysyl endopeptidase digestion of this monomeric PTTH after complete reduction and S-alkylation by another S-alkylating reagent showed that only the Cys15 residue was reduced and S-alkylated by the foregoing partial reduction, indicating that this residue formed the interchain disulfide bond . The other disulfide bonds which formed intrachain bridgings were determined by sequence and mass analyses of the fragments generated by two successive enzyme digestions of the monomeric PTTH . In conclusion, the disulfide bond location of PTTH was assigned to Cys15-Cys15' as an interchain disulfide linkage and Cys17-Cys54, Cys40-Cys96, and Cys48-Cys98 as intrachain disulfide linkages. Biochemistry, 1994 May 17, 33(19), 5858 - 66 Kinetic characterization of the chemotactic protein from Escherichia coli, CheY . Kinetic analysis of the inverse hydrophobic effect; Munoz V et al.; CheY, the 129 amino acid chemotactic protein from Escherichia coli, is a good model for studying the folding process of the parallel alpha/beta family of proteins . A study of the folding kinetics of CheY using fluorescence and far-UV circular dichroism (CD) stopped-flow measurements is reported . CheY has three prolines, two of them in the trans conformation and one, Pro110, with a cis Lys-Pro peptide bond . This protein presents a unimolecular, but complex, kinetic mechanism that is dominated by a slow phase compatible with a trans-cis isomerization . Mutation of Pro110 to Gly results in the disappearance of this slow phase, indicating that this cis prolyl bond is responsible for it . The slow phase is catalyzed in a very inefficient way by prolyl isomerase, indicating that the cis bond is poorly accessible to the enzyme during refolding . In agreement with this is the fact that the isomerization of the Lys109-Pro110 bond occurs in an intermediate which contains 96% of the native far-UV CD signal and 80% of the native fluorescence signal . Analysis of the unfolded protein with all its prolines in the native conformation shows the existence of a very stable intermediate in the folding reaction . Mutation of a hyperexposed hydrophobic residue, Phe14, to Asn results in an increase in the free energy of unfolding of the protein of approximately 3 kcal mol-1 . Kinetic analysis of the unfolding and refolding reactions of this mutant indicates that the major stabilization effect comes from the relative destabilization of the unfolded state and the kinetic intermediate with respect to the transition state, providing kinetic evidence for the inverse hydrophobic effect . This could also indicate the existence of nonnative interactions in folding intermediates. Biochemistry, 1994 May 17, 33(19), 5846 - 57 Pentalenene synthase . Purification, molecular cloning, sequencing, and high-level expression in Escherichia coli of a terpenoid cyclase from Streptomyces UC5319; Cane DE et al.; Pentalenene synthase, which catalyzes the cyclization of farnesyl diphosphate (1) to the tricyclic sesquiterpene hydrocarbon pentalenene (2), was purified from Streptomyces UC5319 . A 450-bp hybridization probe, generated by PCR amplification of genomic DNA using primers based on N-terminal and internal tryptic peptide sequence data for pentalenene synthase, was used to screen both plasmid and phage DNA libraries of Streptomyces genomic DNA, resulting in the isolation and sequencing of the complete pentalenene synthase gene . PCR was used to insert the pentalenene synthase gene into the T7 expression vector pLM1 . Cloning of the resulting construct in the expression host Escherichia coli BL21 (DE3) gave transformants that expressed pentalenene synthase as greater than 10% of soluble protein . The recombinant enzyme has been purified, and initial physical and kinetic characterization has been performed . The recombinant enzyme appears to be identical in every respect with the native Streptomyces synthase and exhibits the following steady-state kinetic parameters: Km = 0.31 +/- 0.05 microM, kcat = 0.32 +/- s-1, KI(PPi) = 3.2 +/- 0.6 microM . Both enzymes have an absolute requirement of Mg2+ for catalysis and an optimum pH of 8.2-8.4 . Both proteins have M(r) values of 41-42 kDa, as determined by SDS-PAGE. Biochemistry, 1994 May 17, 33(19), 5813 - 8 Recombinant human CNTF receptor alpha: production, binding stoichiometry, and characterization of its activity as a diffusible factor; Panayotatos N et al.; The primary ligand-binding protein (CNTFR alpha) of the multicomponent receptor for ciliary neurotrophic factor was produced in Escherichia coli . Using novel applications of size-exclusion chromatography and a protein gel-shift assay, we obtained quantitative separation of correctly refolded protein, as well as analytical monitoring of the refolding process and ligand binding . By these and other methods, we determined a 1:1 stoichiometry for the receptor-ligand complex . To investigate the proposed activity and mechanism of soluble CNTFR alpha as a diffusible factor, we studied the response of TF-1 cells which lack CNTFR alpha to various CNTF ligands and the stimulation of this response by sCNTFR alpha . The results show that sCNTFR alpha combines with CNTF and mediates cell survival with the same relative ligand specificity and relative affinity as the cell-surface form . Thus, soluble receptor can reconstitute on a cell surface active complexes that are analogous to the native complexes . Moreover, both the relative ligand potency in the absence of CNTFR alpha and the kinetics of the response to sCNTFR alpha indicate that the other components of the receptor complex contribute little, but measurably, to the specific potency of CNTF. Biochemistry, 1994 May 17, 33(19), 5721 - 7 Structure of the NADPH-binding motif of glutathione reductase: efficiency determined by evolution; Rescigno M et al.; The role of the second glycine residue (Gly-176) of the conserved GXGXXA "fingerprint" motif in the NADPH-binding domain of Escherichia coli glutathione reductase has been studied by means of site-directed mutagenesis . This glycine residue occurs at the N-terminus of the alpha-helix in the beta alpha beta fold that characterizes the dinucleotide-binding domain, in close proximity to the pyrophosphate bridge of the bound coenzyme . Introducing an alanine residue (G176A), the minimum possible change, at this position virtually inactivated the enzyme, as did the introduction of valine, leucine, isoleucine, glutamic acid, histidine, or arginine residues . Only the replacement by serine--a natural substitute for this glycine residue in some forms of mercuric reductase, a related flavoprotein disulfide oxidoreductase--produced a mutant enzyme (G176S) that retained significant catalytic activity . It is conceivable that this is due to a favorable hydrogen bond being formed between the serine hydroxyl and a pyrophosphate oxygen atom . In most of the mutant enzymes, the Km for NADPH was substantially greater than that found for wild-type glutathione reductase, as expected, but this was accompanied by an unexpected decrease in the Km for GSSG . The latter can be explained by the observation that the reduction of the enzyme by NADPH, the first half-reaction of the ping-pong mechanism, had become a rate-limiting step of the overall reaction catalyzed, albeit poorly, by the mutant enzymes.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 May 17, 33(19), 5711 - 20 The mutant Escherichia coli F112W cyclophilin binds cyclosporin A in nearly identical conformation as human cyclophilin; Fejzo J et al.; The periplasmic Escherichia coli cyclophilin is distantly related to human cyclophilin (34% sequence identity) . Peptidyl-prolyl isomerase activity, cyclosporin A binding, and inhibition of the calcium-dependent phosphatase calcineurin are compared for human and E . coli wild-type and mutant proteins . Like human cyclophilin, the E . coli protein is a cis-trans peptidyl-prolyl isomerase . However, while the human protein binds cyclosporin A tightly (Kd = 17 nM), the E . coli protein does not (Kd = 3.4 microM) . The mutant F112W E . coli cyclophilin has enhanced cyclosporin binding (Kd = 170 nM) . As for the human protein, the complex of the E . coli mutant with cyclosporin A inhibits calcineurin . Here we describe the structure at pH 6.2 of cyclosporin A bound to the mutant E . coli cyclophilin as solved with solution NMR methods . Despite the low overall sequence identity, the structure of the bound cyclosporin A is virtually identical in both proteins . To assess differences of the cyclosporin binding site, the solution structure of wild-type E . coli cyclophilin was compared with structures of uncomplexed human cyclophilin A and with cyclosporin bound . Despite the structural similarity of bound cyclosporin A, the architecture of the binding site in the E . coli protein is substantially different at the site most distant to tryptophan 121 (human sequence) . This site is constructed by a five-residue insertion in a loop of the E . coli protein, replacing another loop in the human protein. Biochemistry, 1994 May 17, 33(19), 5674 - 81 Kilobase-range communication between polypurine.polypyrimidine tracts in linear plasmids mediated by triplex formation: a braided knot between two linear duplexes; Hampel KJ et al.; Linear plasmids were constructed containing two pyrimidine tracts that were 0.34 and 0.94 kilobases (kb) from either end and were separated by 2.8 kb . The tracts {d(TCCTTC)n and d(CTTCCT)n where n = 6 or 12} were designed so as to be able to form triplexes with each other but not with themselves . Upon lowering of the pH to 4 in the presence of spermine, these plasmids form intermolecular dimers and intramolecular loops of 2.8 kb, as judged from mobility changes on agarose gels . A tethered loop could also be formed in a linear plasmid containing two identical tracts by adding an homologous single-stranded oligopyrimidine, but not an oligopurine . In plasmids containing different tracts, the formation of both dimers and loops could be blocked by adding either homologous single-stranded oligopyrimidine but not an oligopurine . Together with the requirement of low pH, these results demonstrate that triplex formation is of the pyr.pur.pyr type . The extent of dimer and loop formation was dependent on the length of the pyrimidine tract: dimers could be detected in plasmids containing the 72 base pair (bp) inserts after incubation at pH 6, but in plasmids containing the 36 bp inserts, a pH of 5 was required . Hysteresis was also evident to a remarkable extent . Once formed at pH 4, loops and dimers remained stable indefinitely at pH 8, suggesting that the structures become topologically trapped . However, the structures were resolved into the component linear plasmids by incubation with nuclease P1 . This is the first demonstration of a braided or hydrogen-bonded knot between two linear duplexes and may have implications for chromosomal loop formation. Biochemistry, 1994 May 17, 33(19), 5984 - 6003 Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation; Farrow NA et al.; The backbone dynamics of the C-terminal SH2 domain of phospholipase C gamma 1 have been investigated . Two forms of the domain were studied, one in complex with a high-affinity binding peptide derived from the platelet-derived growth factor receptor and the other in the absence of this peptide . 2-D 1H-15N NMR methods, employing pulsed field gradients, were used to determine steady-state 1H-15N NOE values and T1 and T2 15N relaxation times . Backbone dynamics were characterized by the overall correlation time (tau m), order parameters (S2), effective correlation times for internal motions (tau e), and, if required, terms to account for motions on a microsecond-to-millisecond-time scale . An extended two-time-scale formalism was used for residues having relaxation data and that could not be fit adequately using a single-time-scale formalism . The overall correlation times of the uncomplexed and complexed forms of SH2 were found to be 9.2 and 6.5 ns, respectively, suggesting that the uncomplexed form is in a monomer-dimer equilibrium . This was subsequently confirmed by hydrodynamic measurements . Analysis of order parameters reveals that residues in the so-called phosphotyrosine-binding loop exhibited higher than average disorder in both forms of SH2 . Although localized differences in order parameters were observed between the uncomplexed and complexed forms of SH2, overall, higher order parameters were not found in the peptide-bound form, indicating that on average, picosecond-time-scale disorder is not reduced upon binding peptide . The relaxation data of the SH2-phosphopeptide complex were fit with fewer exchange terms than the uncomplexed form . This may reflect the monomer-dimer equilibrium that exists in the uncomplexed form or may indicate that the complexed form has lower conformational flexibility on a microsecond-to-millisecond-time scale. J Immunol Methods, 1994 May 16, 171(2), 157 - 67 Determination of haptoglobin expression in IL-6 treated HepG2 cells by ELISA and by RNA hybridization--evaluation of a quantitative method to measure IL-6; Boe A et al.; Interleukin-6 (IL-6) is known to be an important modulator of acute phase (AP) protein expression in hepatocytes both in vivo and in vitro . In the present study the inducing activity of IL-6 on the expression of the AP protein haptoglobin (HP) by the human hepatoma cell line HepG2, has been evaluated . HP mRNA inducibility was analysed by Northern and slot-blot hybridization, while HP protein was detected by means of an ELISA procedure . A dose-response relationship from 0.3 to 4.8 ng/ml of a human recombinant IL-6 preparation derived from a Chinese hamster ovary (CHO) cell line was observed after 48 h of treatment . Comparable results were obtained by analysing both HP mRNA expression and HP protein secretion . Detectable induction of HP protein secretion was observed with as little as 25 pg/ml of IL-6 . The effect of IL-6 was potentiated by dexamethasone, while an inhibition on HP mRNA inducibility could be prevented by lowering the foetal calf serum (FCS) concentration to 1% . Preliminary data indicate that neither IL-1 beta nor TNF-alpha were able to induce significantly HP mRNA expression and protein secretion . The activity ratio between two IL-6 preparations (from CHO and E . coli cells) obtained with a conventional IL-6 bioassay (i.e., T1165 cell growth assay) was comparable to that obtained in the induction of HP expression . The nominal specific activity of the CHO-derived IL-6 was two to three times higher with both responses. FEBS Lett, 1994 May 16, 344(2-3), 187 - 90 Intergenic suppression in a beta subunit mutant with defective assembly in Escherichia coli F1ATPase . Second-site mutation in the alpha subunit; Miki J et al.; Substitution of Leu-40 by Pro in the beta subunit (beta L40P) of Escherichia coli F1-ATPase caused a decrease in the amount of the alpha and beta subunits on the membranes . A revertant strain, Re50, carrying no suppression mutations in the uncD gene encoding the beta subunit, was isolated from the beta L40P mutant . The uncA gene from this revertant was amplified by PCR, and cloned into an expression plasmid . The expression plasmid carrying the uncA gene from the revertant was used for genetic suppression assays . The suppression mutation in Re50 was in the alpha subunit, and it recovered the assembly of the alpha and beta subunits into the F1F0 complex and the ATPase activity to 50% that of the wild type . In Re50, Leu-111 was substituted by Gln in the alpha subunit . These results suggest that the regions including Leu-40 in the beta subunit and Leu-111 in the alpha subunit are located close together and interact with each other, either directly or indirectly. FEBS Lett, 1994 May 16, 344(2-3), 181 - 6 Characterization of cytosolic malic enzyme in human tumor cells; Loeber G et al.; Cytosolic NADP(+)-dependent malic enzyme (ME) from human tumor cells was characterized in detail and compared to ME from normal human tissues produced in recombinant E . coli . Kinetic properties, size as seen in SDS gels, and HPLC elution profiles of tryptic digests of human 'normal cell' ME and NADP(+)-ME from tumor cells were identical . Thus, NADP(+)-ME found in tumor cells does not constitute a tumor-specific isoform as suggested by other studies but is identical to the 'housekeeping protein' predominantly expressed in human liver and white adipose tissue. FEBS Lett, 1994 May 16, 344(2-3), 171 - 4 Site-specific incorporation of photofunctional nonnatural amino acids into a polypeptide through in vitro protein biosynthesis; Hohsaka T et al.; Nonnatural amino acids with photofunctional groups were incorporated site-specifically into a polypeptide by using in vitro protein synthesizing system . The nonnatural amino acids were attached to tRNA(CCU) through chemical misacylation method, and added to the in vitro system with a mRNA containing a single AGG codon . L-p-Phenylazophenylalanine, L-2-anthrylalanine, L-1-naphthylalanine, L-2-naphthylalanine and L-p-biphenylalanine were successfully incorporated into a polypeptide, but l-1-pyrenylalanine was not . The polypeptides containing the nonnatural amino acids showed photofunctionalities. FEBS Lett, 1994 May 16, 344(2-3), 166 - 70 Nucleic acid-binding regions of the second-largest subunit of Drosophila RNA polymerase II identified by southwestern blotting; Kontermann RE et al.; Analysing overlapping bacterially expressed fragments of the second-largest subunit of Drosophila melanogaster RNA polymerase II in Southwestern DNA binding assays we have identified regions that have the potential to bind nucleic acids non-specifically . A region exhibiting strong DNA binding is located in the N-terminal part of the molecule (amino acids 357-504) and some weak DNA binding is observed for the C-terminal part (amino acids 860-1160) . The non-specific DNA binding behavior of these regions is similar to that of the native enzyme . Most of the known mutations responsible for rifampicin resistance map to a region of the Escherichia coli beta subunit corresponding to the N-terminal nucleic acid-binding region, indirectly supporting the notion that this region participates in interaction with the RNA transcript in ternary complexes. Biochem Biophys Res Commun, 1994 May 16, 200(3), 1477 - 83 Carboxy-terminal region of Escherichia coli SecA ATPase is important to promote its protein translocation activity in vivo; Rajapandi T et al.; The role of the carboxy-terminal region of E . coli SecA ATPase was investigated by using genetic methods to construct a truncated SecA protein missing the last 66 amino acid residues and by systematically substituting serine for each of four cysteine residues present in this protein . Truncation of SecA or alteration of any of the carboxy-terminal cysteine residues resulted in poor growth and a protein secretion defect, indicating that this region of SecA is important in its protein translocation activity . Biochemical analysis of the altered proteins revealed a modest increase in translocation ATPase activity, suggesting that the carboxy-terminal region of SecA may facilitate the coupling of its ATPase activity to cycles of protein translocation. FEBS Lett, 1994 May 16, 344(2-3), 129 - 35 The stability and hydrophobicity of cytosolic and mitochondrial malate dehydrogenases and their relation to chaperonin-assisted folding; Staniforth RA et al.; mMDH and cMDH are structurally homologous enzymes which show very different responses to chaperonins during folding . The hydrophilic and stable cMDH is bound by cpn60 but released by Mg-ATP alone, while the hydrophobic and unstable mMDH requires both Mg-ATP and cpn10 . Citrate equalises the stability of the native state of the two proteins but has no effect on the co-chaperonin requirement, implying that hydrophobicity, and not stability, is the determining factor . The yield and rate of folding of cMDH is unaffected while that of mMDH is markedly increased by the presence of cpn60, cpn10 and Mg-ATP . In 200 mM orthophosphate, chaperonins do not enhance the rate of folding of mMDH, but in low phosphate concentrations chaperonin-assisted folding is 3-4-times faster. J Immunol Methods, 1994 May 16, 171(2), 211 - 26 Recombinant single-chain antibody peptide conjugates expressed in Escherichia coli for the rapid diagnosis of HIV; Lilley GG et al.; Recombinant single chain Fv (scFv) antibody fragments can form the basis of a rapid, whole-blood diagnostic assay . The scFv described in this study is derived from a monoclonal antibody which has a high affinity for glycophorin A, an abundant glycoprotein on the human red blood cell membrane surface . The prototype reagent built around the scFv was designed to detect, in whole blood samples, the presence of antibodies that have arisen through infection with a foreign organism such as human immunodeficiency virus . The scFv was composed of the antibody heavy-chain variable domain (Vh) joined by a 15 residue linker -(GGGGS)3- to the light-chain variable domain (V1) terminated by either a C-terminal octapeptide tail (FLAG) or a 35 amino acid segment from the gp41 surface glycoprotein of HIV-1 . Constructs were cloned into a Escherichia coli expression vector, pHFA, and expressed in a soluble form into culture supernatant . The product retained anti-glycophorin activity which could be detected directly in culture supernatants by ELISA . Furthermore, the scFv-epitope fusion functioned efficiently in the whole blood agglutination assay and was able to distinguish between HIV-1 positive and negative sera. FEBS Lett, 1994 May 16, 344(2-3), 242 - 6 Receptor-binding domain of human alpha 2-macroglobulin . Expression, folding and biochemical characterization of a high-affinity recombinant derivative; Holtet TL et al.; A recombinant version of the receptor binding domain (RBDv) of human alpha 2-macroglobulin (alpha 2M) has been expressed in E . coli and refolded using a novel iterative procedure . RBDv (Val1299-Ala1451) is extended by 15 residues at the N-terminal side of the Lys1313-Glu papain cleavage site in human alpha 2M . RBDv contains the intra-chain bridge Cys1329-Cys1444 and is soluble and monomeric . Competition experiments with 125I-labelled methylamine-treated alpha 2M reveal that RBDv binds to the placental receptor for transformed alpha 2M with a Kd of 8 nM, i.e . the binding affinity of RBDv is of the same order of magnitude as the intrinsic affinity for binding of one domain in transformed alpha 2M to one receptor molecule. Biochem Biophys Res Commun, 1994 May 16, 200(3), 1221 - 9 Overexpression and characterization of a recombinant form of rat calcineurin A; Haddy A et al.; Overexpression of rat recombinant calcineurin A catalytic subunit in E . coli was achieved using a system under control of the T7 promoter . The specific activity of the purified catalytic subunit was suppressed relative to native bovine calcineurin, with the extent of suppression depending upon the choice of substrate . Addition of calcineurin B subunit stimulated phosphatase activity to one third that of native calcineurin . The metal activators Mn2+ and Ni2+ as well as several anion inhibitors affected both native calcineurin and recombinant calcineurin A activity to the same extent . In addition, calcineurin B was required for inhibition by the immunosuppressive complex FK506-FK506-binding protein. Eur J Biochem, 1994 May 15, 222(1), 75 - 81 No intermediate channelling in stepwise hydrolysis of fluorescein di-beta-D-galactoside by beta-galactosidase; Fieldler F et al.; For the hydrolysis of the two glycosidic bonds of fluorescein di-beta-D-galactoside (FDG) by beta-galactosidase from Escherichia coli, small {Hofmann, J . & Sernetz, M . (1983) Anal . Biochem . 131, 180-186} to dramatic {Huang, Z . (1991) Biochemistry 30, 8535-8540} deviations from simple stepwise substrate-intermediate-product kinetics have been reported . Intermediate channelling, a preferred hydrolysis of the intermediate fluorescein mono-beta-D-galactoside (FMG) formed from FDG at the active site and thus in a favourable position for further reaction, has been postulated . As there were reasons to doubt the previous findings and conclusions, the hydrolysis experiments have been repeated at initial FDG concentrations of 7-200 microM, following the concentrations of FDG, FMG and fluorescein with a reliable method, quantitative HPLC, to completion of the reaction . The transient appearance of substantial amounts of the intermediate FMG also in experiments with 200 microM FDG already rules out the existence of the most efficient intermediate channelling deduced by Huang (1991) from measurements of the initially developing fluorescence, incorrectly ascribed to fluorescein . Redetermination of the Michaelis constants for FDG and FMG led to much higher values than those reported previously . Fitting the progress curves by means of nonlinear regression combined with numerical integration of the rate equations resulted in good fits of the normal stepwise substrate-intermediate-product mechanism, without any necessity of assuming a more complex course of the reaction . So one of the rare examples of the hydrolysis of two bonds at a single enzyme-substrate encounter has been invalidated. Eur J Biochem, 1994 May 15, 222(1), 65 - 73 Mapping of Hsp70-binding sites on protein antigens; Roman E et al.; Hsp70-binding sites were mapped on three antigens, the 16-, 19- and 38-kDa proteins of Mycobacterium tuberculosis, using overlapping synthetic peptides in a competitive-binding assay . In each protein, two or three prominent hsp70-binding sites were identified when peptides 20-amino-acid long were used, predominantly in regions containing clusters of aliphatic amino acids . Although there was an overall concordance in the pattern of peptide binding to hsp70 from bacterial (M . tuberculosis) and mammalian sources (immunoglobulin heavy-chain-binding protein), some differences in the specificity of polypeptide binding and the effect of peptides on ATPase activity were observed. Eur J Biochem, 1994 May 15, 222(1), 49 - 54 Assembly of functional skeletal muscle troponin complex in Escherichia coli; Malnic B et al.; The production of multi-subunit proteins of eukaryotic origin in Escherichia coli usually relies on the different subunits being expressed individually and the protein being reassembled in vitro . Here we describe the construction and characterization of plasmids capable of coexpressing the three subunits of chicken skeletal muscle troponin complex in E . coli . We demonstrate that the troponin subunits assembled in the cytoplasm of E . coli cell are fully functional . The troponin complex was purified to homogeneity in high yields . When reconstituted into actin filaments, the complex assembled in vivo was capable of regulating the myosin ATPase with a calcium dependence that was identical to the complex reconstituted in vitro . These results demonstrate that the coexpression of the subunits of a protein complex can prevent the accumulation of denatured proteins in inclusion granules. Eur J Biochem, 1994 May 15, 222(1), 27 - 32 The NAM1 protein (NAM1p), which is selectively required for cox1, cytb and atp6 transcript processing/stabilisation, is located in the yeast mitochondrial matrix; Wallis MG et al.; The NAM1 nuclear gene was shown to control the stability and/or processing of mitochondrial transcripts of the cytochrome b, cytochrome oxidase subunit I and ATP synthase subunit VI genes {Groudinsky O., Bousquet I., Wallis M . G., Slonimski, P . P . & Dujardin G . (1993) Mol . Gen . Genet . 240, 419-427} . In order to better understand the mode of action of the NAM1 gene product, we have examined its intracellular fate . A fusion plasmid enabling bacterial over-expression of the corresponding protein-A-NAM1 cognate was constructed and subsequently employed as an antigen to raise polyclonal antibodies . These antibodies specifically recognise a 50-kDa protein which purifies along with the mitochondria and corresponds to NAM1p . Submitochondrial localisation experiments show that NAM1p is a soluble protein, located interior to the mitoplasts . Matricial location is a strong argument in favour of a direct interaction of NAM1p with particular mitochondrial transcripts and leads us to propose a model in which NAM1p could be an RNA-convoying protein stabilising and directing mitochondrial transcripts towards the inner face of the inner membrane where translation and assembly seem to occur. Eur J Biochem, 1994 May 15, 222(1), 173 - 81 Solid-phase synthesis, conformational analysis and in vitro cleavage of synthetic human synaptobrevin II 1-93 by tetanus toxin L chain; Cornille F et al.; A 93-residue peptide corresponding to the cytosolic domain of a human vesicle associated membrane protein (VAMP or synaptobrevin) has been prepared by solid-phase peptide synthesis in order to investigate the proteolytic activity of the tetanus toxin light chain (TeTx L chain) . This protein has been recently reported to inactivate the neuronal rat synaptobrevin II by proteolysis . We show in this study that the synthetic human synaptobrevin II 1-93 (Syb II 1-93) as well as an N-terminus-shortened 69-residue peptide (Syb II 25-93) were cleaved selectively at the Gln76-Phe77 peptide bond by TeTx L chain while shorter peptides were not . A Michaelis constant Km = 192 +/- 2 microM and a catalytic constant kcat = 0.5 min-1 were found for the 93-residue peptide . A neutral optimum pH for the cleavage rate, an inhibition by preincubation of the toxin with well known nonspecific inhibitors of metallopeptidases as well as a zinc-dependent enzyme activity suggest that TeTx belongs to the zinc endopeptidase family . Moreover an activation by reducing agents and an inhibition by cysteine-modifying chemical reagents indicate a critical thiol dependency . Among several specific inhibitors of zinc endopeptidases tested, none could inhibit TeTx L chain even at high concentration . Structural studies by 600-MHz 1H-NMR showed that in water or dimethylsulfoxide the peptide Syb II 1-93 and shorter fragments did not present well defined conformations . Nevertheless protein-protein interactions have been shown for the peptides Syb II 1-93 and 25-93 but not for Syb II 51-93, a fragment not cleaved by TeTx L chain. Eur J Biochem, 1994 May 15, 222(1), 137 - 46 Characterization of a polyhistidine-tagged form of human myristoyl-CoA: protein N-myristoyltransferase produced in Escherichia coli; McIlhinney RA et al.; The enzyme myristoyl-CoA:protein N-myristoyltransferase is responsible for the attachment of a myristoyl group to the N-terminal glycine of a number of cell, viral and fungal proteins . In order to overcome the difficulties of purification of this enzyme from tissue sources, we have produced an N-terminally polyhistidine-tagged version of the enzyme and expressed this in Escherichia coli . The resulting enzyme has a molecular mass of 53 kDa and is fully active showing the expected specificity for myristic acid and causing the N-terminal myristoylation of both synthetic peptide and protein substrates in vitro . The enzyme exhibits a broad pH optimum peaking at a pH of 8.0 and has a Km for myristoyl-CoA of 7.6 microM . The two synthetic peptide substrates based on the N-terminal sequence of the catalytic subunit of protein kinase A (GNAAAARR) and of p60src (GSSKSKPKDPSQRRRY) have different kinetic parameters with Km values of 115.2 microM and 44.2 microM and Vmax values of 95 and 120 nmol.min-1.mg-1, respectively . The expressed enzyme is partially inhibited (50%) by iodoacetamide at 5 mM and fully inhibited by diethylpyrocarbonate at 10 mM . This latter inhibition can be prevented by including histidine in the incubation of the enzyme and inhibitor . Antisera raised to synthetic peptides based on sequences derived from the N- and C- terminus of the human enzyme reacted with the expressed protein on Western blots, but only the N-terminal sequence reacted with the native protein suggesting that the C-terminus may be not be accessible . The enzyme can catalyse the removal of a myristoyl group from myristoylated peptides but does so only in the presence of added coenzyme A. Biochem J, 1994 May 15, 300 ( Pt 1), 85 - 90 Cloning and expression in Escherichia coli of a dog thyroid cDNA encoding a novel inositol 1,4,5-trisphosphate 5-phosphatase; Verjans B et al.; In brain and many other tissues, type I inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase is the major isoenzyme hydrolysing the calcium-mobilizing second messenger InsP3 . This protein has been purified to apparent homogeneity from a crude soluble fraction of bovine brain, yielding a single major protein band with a molecular mass of 43 kDa after SDS/PAGE . This material was used to determine internal microsequences . A partial DNA sequence has been amplified by PCR by using degenerate primers deduced from two protein sequences (FKAKKYKKV and DENYKSQE) . A cDNA clone (BVCT) was isolated by screening a dog thyroid cDNA library . The encoded protein of 412 amino acids has a calculated molecular mass of 47,681 Da . Peptide sequences generated from the bovine brain enzyme were found to be 96% conserved compared with the dog thyroid protein . When clone BVCT was expressed in Escherichia coli, the recombinant protein was shown to hydrolyse both InsP3 and inositol 1,3,4,5-tetrakisphosphate, with apparent Km values of 28 and 3 microM respectively . Enzyme activity was inhibited by EDTA and 2,3-bisphosphoglycerate, both inhibitors of native InsP3 5-phosphatase, but not by EGTA and LiCl, as previously shown for the bovine brain enzyme . Our data show the cloning of type I InsP3 5-phosphatase which, interestingly, does not share any significant sequence identity with the previously cloned type III isoenzyme. Biochem J, 1994 May 15, 300 ( Pt 1), 111 - 5 Site-directed mutagenesis of rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: role of Asp-130 in the 2-kinase domain; Rider MH et al.; Asp-130 of the recombinant skeletal-muscle 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase was mutated into Ala in order to study its role in catalysis and/or substrate binding . The D130A mutant displayed a 30- to 140-fold decreased 2-kinase Vmax, depending on the pH, and a 30- and 60-fold increase in Km for MgATP and Fru-6-P respectively at pH 8.5 compared with the wild-type . Mutagenesis of Asp-130 to Ala had no effect on the 2-phosphatase activity, and fluorescence measurements indicated that the changes in kinetic properties of PFK-2 in the D130A mutant were not due to instability . The role of Asp-130 in the 2-kinase reaction is discussed and compared with that of Asp-103 of 6-phosphofructo-1-kinase from Escherichia coli, which binds Mg2+. EMBO J, 1994 May 15, 13(10), 2464 - 71 Many of the conserved nucleotides of tRNA(Phe) are not essential for ternary complex formation and peptide elongation; Nazarenko IA et al.; An RNase protection assay was used to show that the dissociation rate constants and equilibrium constants of unmodified yeast and Escherichia coli phenylalanyl-tRNA(Phes) to elongation factor Tu from E.coli were very similar to each other and to their fully modified counterparts . The affinity of aminoacylated tRNA to elongation factor Tu was substantially lower when GTP analogues were used in place of GTP, emphasizing the importance of the beta-gamma phosphate linkage in the function of G-proteins . Fourteen different mutations in conserved and semi-conserved nucleotides of yeast phenylalanyl-tRNA(Phe) were tested for binding to elongation factor Tu.GTP and assayed for activity in the ribosomal A- and P-sites . Most of the mutations did not severely impair the function of these tRNAs in any of the assays . This suggests that the translational machinery does not form sequence-specific interactions with the conserved nucleotides of tRNA. EMBO J, 1994 May 15, 13(10), 2421 - 31 Functional association of essential splicing factor(s) with PRP19 in a protein complex; Tarn WY et al.; We have previously shown that the yeast PRP19 protein is a spliceosomal component, but is not tightly associated with small nuclear RNAs . It appears to associate with the spliceosome concomitant with or just after dissociation of the U4 small nuclear RNA during spliceosome assembly . We have found that PRP19 is associated with a protein complex in the splicing extract and that at least one of the associated components is essential for splicing . Taking advantage of the epitope tagging technique, we have isolated the PRP19-associated complex by affinity chromatography . The isolated complex is functional for complementation for the heat-inactivated prp19 mutant extract, and consists of at least seven polypeptides in addition to PRP19 . At least three of these can interact directly with the PRP19 protein . We also show that the PRP19 protein itself is in an oligomeric form, which might be a prerequisite for its interaction with these proteins. EMBO J, 1994 May 15, 13(10), 2267 - 72 Membrane protein topology: effects of delta mu H+ on the translocation of charged residues explain the 'positive inside' rule; Andersson H et al.; The membrane electrochemical potential is critical for the export of most periplasmic proteins in Escherichia coli . Its exact role during insertion of integral inner membrane proteins, however, remains obscure . Using derivatives of the inner membrane protein leader peptidase (Lep), we now show that the membrane potential appears to stimulate the membrane translocation of chain segments containing negatively charged residues, that positively charged regions appear to be more easily translocated in the absence of a potential, and that certain Lep constructs insert with different topologies in the presence and absence of a membrane potential, suggesting that the electrochemical potential introduces an asymmetry between the topological effects of positively and negatively charged amino acids during the process of membrane protein insertion in E . coli. Arch Biochem Biophys, 1994 May 15, 311(1), 72 - 8 Isolation and refolding of H-ras from inclusion bodies of Escherichia coli: refold procedure and comparison of refolded and soluble H-ras; DeLoskey RJ et al.; A refolding and purification method for economically producing large quantities of H-ras isolated from Escherichia coli inclusion bodies is described . Experiments were performed to optimize the yield of refolded H-ras for structural analysis by NMR spectroscopy . Protein concentration, temperature, and the presence of 10% glycerol during refolding were varied . The yield of H-ras was highest when the protein was refolded at concentrations less than or equal to 0.1 mg/ml and was independent of the presence of 10% glycerol . The yield was slightly higher at 4 degrees C than at 25 degrees C . The refolded H-ras was purified by anion exchange chromatography to yield protein with a purity of > 90%, as determined by C4 reverse-phase HPLC, and a GDP-binding stoichiometry greater than 0.9 . NMR structural analysis of refolded and soluble H-ras was conducted using {15N}glycine- and {15N}serine-enriched protein . The NMR data indicate that the refolded ras protein is structurally similar to ras isolated from the soluble fraction. Arch Biochem Biophys, 1994 May 15, 311(1), 28 - 34 Substrate specificity of aminopeptidase P from Escherichia coli: comparison with membrane-bound forms from rat and bovine lung; Yoshimoto T et al.; The substrate specificity of recombinant E . coli aminopeptidase P (aminoacylprolylpeptide hydrolase) (EC 3.4.11.9) was studied using about 150 synthetic peptides . E . coli aminopeptidase P released the N-terminal amino acid from most peptides containing a penultimate proline, although the relative rates of hydrolysis varied over two orders of magnitude . Dipeptides (X-Pro) were hydrolyzed relatively slowly . Detailed kinetic analysis using peptides of different lengths suggested that the enzyme has at least four subsites for interaction with substrates, namely S1, S'1, S'2, and S'3 . S1 and S'1 have high stereo-specificity Various Pro-X dipeptides where X is a hydrophobic amino acid were competitive inhibitors of the enzyme . The substrate specificity of E . coli aminopeptidase P was compared to that of purified bovine lung and rat lung membrane-bound aminopeptidase P . The mammalian enzymes had much more restricted substrate specificities . The differences appeared to be due primarily to differences in the S'2 subsite . The E . coli enzyme could accommodate bulky amino acid side chains in the S'2 subsite, whereas the mammalian membrane-bound enzymes could not. Arch Biochem Biophys, 1994 May 15, 311(1), 153 - 9 Lactate-dependent killing of Escherichia coli by nitrite plus hydrogen peroxide: a possible role of nitrogen dioxide; Kono Y et al.; The killing of Escherichia coli by nitrite plus hydrogen peroxide was observed in lactate, but not in phosphate or acetate . Although nitrite or hydrogen peroxide alone caused a slight decrease in bacterial survival, nitrite plus hydrogen peroxide killed bacteria synergistically in time-, dose-, and pH-dependent manners . The killing was increased with decreasing pH . The plot of viable cells versus {nitrous acid} was linear . Among the hydroxyl radical scavengers used, only benzoate and formate at concentrations higher than that of lactate inhibited the killing by nitrite plus hydrogen peroxide, whereas dimethyl sulfoxide enhanced it . The generation of peroxynitrous acid during the reaction of nitrite and hydrogen peroxide was confirmed by the formation of malondialdehyde using deoxyribose as a hydroxyl radical-like oxidant detector . The nitration of glycyl-tryosine was observed only in lactate buffer, but not in phosphate and acetate buffers . Benzoate and formate inhibited the nitration, whereas dimethyl sulfoxide and ethanol enhanced it . No evidence for the formation of nitric oxide and superoxide during the reaction of nitrite and hydrogen peroxide was found . These data suggest that nitrogen dioxide from the decomposition of peroxynitrous acid or secondary oxidants formed from the reaction of peroxynitrous acid with lactate is responsible for the lactate-dependent killing of E . coli induced by the reaction of protonated nitrite and hydrogen peroxide. Virology, 1994 May 15, 201(1), 178 - 81 The diverged copy of the citrus tristeza virus coat protein is expressed in vivo; Febres VJ et al.; Nucleotide sequence analysis of a portion of the citrus tristeza closterovirus (CTV) genome revealed an open reading frame immediately upstream of the coat protein gene that can encode a protein with a calculated M(r) of 27,360 (p27) . The deduced amino acid sequence indicated that this putative nonstructural gene product is highly homologous to the coat protein . To investigate whether p27 was expressed in CTV-infected plants, a fusion protein of p27 produced in Escherichia coli was used to raise polyclonal antibodies . Western blot analysis using the p27 antibodies indicated that p27 is expressed in CTV-infected citrus, but not in uninfected plants . Tissue fractionation studies revealed that p27 accumulates in cell wall enriched fractions. Structure, 1994 May 15, 2(5), 407 - 14 Crystal structure of an ATP-dependent carboxylase, dethiobiotin synthetase, at 1.65 A resolution; Huang W et al.; BACKGROUND: In Escherichia coli, the enzymes of the biotin biosynthesis pathway are encoded by the bio operon . One of these enzymes, ATP-dependent dethiobiotin synthetase, catalyzes the carboxylation of 7,8-diaminopelargonic acid leading to the formation of the ureido ring of biotin . The enzyme belongs to the class of ATP-dependent carboxylases and we present here the first crystal structure determined for this class of enzyme . RESULTS: We have determined the crystal structure of homodimeric dethiobiotin synthetase to 1.65 A resolution . The subunit consists of a seven-stranded parallel beta-sheet, surrounded by alpha-helices . The sheet contains the classical mononucleotide-binding motif with a fingerprint peptide Gly-X-X-X-X-X-Gly-Lys-Thr . The mononucleotide binding part of the structure is very similar to the GTP-binding protein H-ras-p21 and thus all GTP-binding proteins . A comparison reveals that some of the residues, which in H-ras-p21 interact with the nucleotide and the metal ion, are conserved in the synthetase . CONCLUSIONS: The three-dimensional structure of dethiobiotin synthetase has revealed that ATP-dependent carboxylases contain the classical mononucleotide-binding fold . Considerable similarities to the structure of the GTP-binding protein H-ras-p21 were found, indicating that both proteins might have evolved from a common ancestral mononucleotide-binding fold. Structure, 1994 May 15, 2(5), 361 - 9 The three-dimensional structure of N-acetylneuraminate lyase from Escherichia coli; Izard T et al.; BACKGROUND: N-acetylneuraminate lyase catalyzes the cleavage of N-acetylneuraminic acid (sialic acid) to form pyruvate and N-acetyl-D-mannosamine . The enzyme plays an important role in the regulation of sialic acid metabolism in bacteria . The reverse reaction can be exploited for the synthesis of sialic acid and some of its derivatives . RESULTS: The structure of the enzyme from Escherichia coli has been determined to 2.2 A resolution by X-ray crystallography . The enzyme is shown to be a tetramer, in which each subunit consists of an alpha/beta-barrel domain followed by a carboxy-terminal extension of three alpha-helices . CONCLUSIONS: The active site of the enzyme is tentatively identified as a pocket at the carboxy-terminal end of the eight-stranded beta-barrel . Lys165 lies within this pocket and is probably the reactive residue which forms a Schiff base intermediate with the substrate . The sequence of N-acetylneuraminate lyase has similarities to those of dihydrodipicolinate synthase and MosA (an enzyme implicated in rhizopine synthesis) suggesting that these last two enzymes share a similar structure to N-acetylneuraminate lyase. Anal Biochem, 1994 May 15, 219(1), 43 - 8 A microtiter plate assay for factor XIII A-chain-fibrin interactions; Achyuthan KE et al.; Factor XIII A-chain-fibrin interactions regulate factor XIIIa formation and fibrin cross-linking . A microtiter plate assay was developed for studying these interactions . Microtiter plate wells were coated with fibrinogen and converted to fibrin by thrombin . After blocking the wells with bovine serum albumin, factor XIII A-chain was added and binding was monitored by incubating first with anti-factor XIII followed by anti-rabbit IgG-alkaline phosphatase . Enzymatic hydrolysis of p-nitrophenyl phosphate was quantitated by the absorbance at 405 nm . BInding was specific, sensitive, rapid, saturable, and reversible, requiring only nanograms of either factor XIII or fibrin . Binding was time- and concentration-dependent and independent of divalent cations . The bound material was identified as factor XIII A-chain by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting . Factor XIII binding was inhibited > 75% by 250 mM sodium chloride or 250 nM anti-factor XIII IgG . The method was also suitable for demonstrating binding using 0.8% plasma or with r-factor XIII expressed in Saccharomyces cerevisiae or Escherichia coli . This method is suitable for identifying the binding sites that are important for plasma factor XIII activation and factor XIIIa activity. Anal Biochem, 1994 May 15, 219(1), 131 - 8 A differential cloning procedure of complex genomic DNA fragments; Yokota H et al.; We have developed a differential cloning procedure designed for cloning of anonymous restriction DNA fragments whose molecular sizes differ between two genomic DNA preparations from higher organisms . The procedure, which was extensively revised from the original one, consists of several steps as summarized below . (i) Digestion of two DNA preparations (target and reference DNA) with the same restriction enzyme (4 base cutter) . (ii) Biotinylation of target DNA and conversion of reference DNA to nonamplifiable form by terminal dephosphorylation . (iii) Electrophoresis of the two DNA preparations through a synthetic gel with a large excess of reference DNA as a competitor . (iv) In-gel alkaline dissociation of DNA, followed by reassociation (in-gel competitive reassociation) . (v) Elution of DNA from the gel and PCR after adapter ligation and adsorption of DNA onto streptavidin-coated matrix . By repeating these steps, we attained substantial enrichment (approximately 10,000-fold) of DNA fragments which were originally present at one copy or less per complex mammalian genome . The details of the procedure and its unique characteristics in cloning of altered genomic DNA fragments, particularly from mammalian genome, are discussed. FEMS Microbiol Lett, 1994 May 15, 118(3), 227 - 31 Identification of the rpmF-plsX-fabH genes of Rhodobacter capsulatus; Carty SM et al.; The rpmF-plsX-fabH gene cluster of Rhodobacter capsulatus homologous to that of Escherichia coli was identified . rpmF encodes ribosomal protein L32, plsX plays an undefined role in membrane lipid synthesis, and fabH encodes beta-ketoacyl-acyl carrier protein synthase III . The R . capsulatus plsX gene complemented a defect in an E . coli strain with the plsX50 mutation . Overproduction of the fabH gene product of R . capsulatus in E . coli resulted in dramatically increased beta-ketoacyl-acyl carrier protein synthase III activity . These results indicate that plsX and fabH apparently function the same in R . capsulatus as in E . coli. Arch Biochem Biophys, 1994 May 15, 311(1), 55 - 61 Properties of variant forms of human stem cell factor recombinantly expressed in Escherichia coli; Langley KE et al.; The gene for human stem cell factor (SCF) encodes a leader sequence followed by 248 amino acids (Martin et al., 1990, Cell 63, 203) . Of these 248 amino acids, the first 189 correspond to an extracellular domain and the remainder correspond to a hydrophobic transmembrane domain plus a cytoplasmic domain . A naturally occurring soluble form, released by proteolytic cleavage after amino acid 165, has been described . An alternatively spliced mRNA, lacking the codons for exon 6, has also been described . Since the amino acids encoded by exon 6 include the proteolytic cleavage site, the form expressed from the alternatively spliced mRNA tends to remain membrane-bound . In the present study, we have begun to explore structure/function relationships within the extracellular domain of SCF . Forms beginning at amino acid 1 (after the leader sequence) and ranging from 127 to 189 at the C-terminus have been recombinantly expressed in Escherichia coli and purified . In addition, forms missing the amino acids encoded by exon 6, forms missing up to 10 amino acids from the N-terminus, and forms with disulfide bond alterations have been expressed and purified . The forms have been characterized structurally, as well as functionally, in quantitative cell proliferation and receptor-binding assays . The results indicate that amino acids 1-141 comprise a structural and functional core and allow conclusions about the necessity of each of the two disulfide bonds for structure and function. J Biol Chem, 1994 May 13, 269(19), 14323 - 8 A role for the H4 subunit of vaccinia RNA polymerase in transcription initiation at a viral early promoter; Deng L et al.; The vaccinia virus H4 gene encodes an essential subunit of the DNA-dependent RNA polymerase holoenzyme encapsidated within virus particles (Ahn, B., and Moss, B . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 3536-3540; Kane, E . M., and Shuman, S . (1992) J . Virol . 66, 5752-5762) . The role of this protein in transcription of viral early genes was revealed by the effects of affinity-purified anti-H4 antibody on discrete phases of the early transcription reaction in vitro . Anti-H4 specifically prevented the synthesis of a 21-nucleotide nascent RNA chain but had no impact on elongation of the 21-mer RNA by preassembled ternary complexes . Inhibition of initiation but not elongation was also observed with affinity-purified anti-D6 antibody directed against the 70-kDa subunit of the vaccinia early transcription initiation factor (ETF) . Native gel mobility-shift assays showed that anti-H4 prevented the NTP-dependent recruitment of RNA polymerase to the preinitiation complex of ETF bound at the early promoter . Two species of ternary complexes could be resolved by native gel electrophoresis . Addition of anti-H4 to preformed complexes elicited a supershift of both ternary species but not of the preinitiation complex . Supeshift by anti-D6 revealed that the more rapidly migrating species of ternary complex did not contain immunoreactive ETF . Loss of ETF from the ternary complex was time-dependent . Thus, whereas the H4 protein was a stable constituent of the elongation complex, ETF was dissociable . We suggest that H4 functions as a molecular bridge to ETF and thereby allows specific recognition of early promoters by the core RNA polymerase . H4 is unlike bacterial sigma factor in that it remains bound to polymerase after the elongation complex is established. J Biol Chem, 1994 May 13, 269(19), 14314 - 22 Cloning and sequencing of flavin-containing monooxygenases FMO3 and FMO4 from rabbit and characterization of FMO3; Burnett VL et al.; The flavin-containing monooxygenases (FMO) are a family of enzymes that contain putative FAD- and NADPH-binding domains within the first 200 residues of their N termini . The cDNAs encoding these enzymes contain an area of relatively high identity over the 5' half of the coding region . Rabbit genomic DNA was probed under low stringency conditions, with a mixture of 5' cDNA fragments encoding rabbit FMO1, FMO2, or FMO5 . Bands associated specifically with FMO1, FMO2, or FMO5 were resolved by analysis at high stringency with individual probes . Several bands were detected that could not be assigned to FMO1, FMO2, or FMO5 . The behavior of the 5' probes at low versus high stringency was used to facilitate the isolation of cDNAs corresponding to the unknown DNA bands . A cDNA library was constructed from rabbit liver mRNA and screened under low stringency hybridization conditions (30 degrees C, 50% formamide, 1 x SSC, 0.1% SDS) with the mixture of 5' FMO1, FMO2, and FMO5 cDNA probes . A total of 157 clones was detected . Of these, 117 clones remained under high stringency hybridization conditions (65 degrees C, 50% formamide, 0.1 x SSC, 0.1% SDS) and were identified as FMO1 (95 clones) or FMO5 (22 clones) . Of the 40 remaining clones, 36 were characterized by sequence analysis as encoding FMO3, previously identified at the protein level by Ozols (Ozols, J . (1991) Arch . Biochem . Biophys . 290, 103-115) as a second rabbit liver FMO . Four clones were shown to encode an FMO not previously described for the rabbit, FMO4 . No clones encoding FMO2 were isolated from the liver library . Sequence analysis revealed that FMO3 and FMO4 are 56% identical, and analysis of genomic DNA indicated that each is encoded by a single gene . Message distribution was tissue-, species-, and form-specific . The properties of FMO3 cDNA expressed in Escherichia coli were found to be more similar to those of FMO1 than FMO2, but to differ significantly from both . Rabbit genomic DNA was probed under conditions of low stringency with a mixture of 5' cDNA fragments encoding all five FMO forms and produced results consistent with the possibility of one additional FMO. J Biol Chem, 1994 May 13, 269(19), 14303 - 6 The erbB3 gene product is a receptor for heregulin; Carraway KL 3rd et al.; ErbB3 is a member of the epidermal growth factor (EGF) receptor subfamily of receptor tyrosine kinases and is believed to be a receptor for an unknown ligand . We have tested the possibility that heregulin, a growth factor possessing an EGF-like domain, is a ligand for ErbB3 . We have found that the iodinated recombinant EGF-like domain of heregulin-beta 1 (125I-rHRG beta 1(177-244) bound specifically to insect cell-expressed bovine ErbB3 with a dissociation constant of 0.85 nM . Moreover, 125I-rHRG beta 1(177-244) bound to NIH3T3 fibroblasts stably transfected with bovine erbB3 with a dissociation constant of 60 pM, but did not bind to parental cells . 125I-rHRG beta 1(177-244) could be chemically cross-linked to a 170-180 kDa protein in erbB3-transfected fibroblasts, and the cross-linked product could be immunoprecipitated with antibodies specific for ErbB3 . Finally, rHRG beta 1 stimulated the tyrosine phosphorylation of both ErbB3 and endogenous p185erbB2/neu in transfectants but not in parental cells . We conclude that ErbB3 is a receptor for HRG and is capable of mediating HRG-stimulated tyrosine phosphorylation of itself and p185erbB2/neu in cells that express both receptors. J Biol Chem, 1994 May 13, 269(19), 14260 - 7 Purification and identification of a vaccinia virus-encoded intermediate stage promoter-specific transcription factor that has homology to eukaryotic transcription factor SII (TFIIS) and an additional role as a viral RNA polymerase subunit; Rosales R et al.; Enzymes and factors, required for in vitro transcription of templates regulated by vaccinia virus intermediate stage promoters, are present in HeLa cells infected with vaccinia virus in the presence of an inhibitor of DNA replication . Previous studies indicated that in vitro transcription could be reconstituted by adding a partially purified transcription factor to the viral RNA polymerase and capping enzyme . By using an independent purification procedure, we isolated two vaccinia virus intermediate were necessary for transcription of several different intermediate stage promoter templates but not for early or late stage promoter templates . VITF-1 was purified to homogeneity, and the sequences of two tryptic peptides were mapped to the fourth open reading frame within the HindIII E fragment (E4L) of the vaccinia virus genome, which had previously been shown to encode an RNA polymerase subunit of 30 kDa (RPO30) with homology to eukaryotic transcription elongation factor SII . Co-chromatography of VITF-1 with the E4L-derived protein was demonstrated using specific antiserum . In addition, transcriptionally active recombinant VITF-1 was made by expressing the E4L open reading frame in Escherichia coli . Thus, E4L encodes a multifunctional protein, serving as a RNA polymerase subunit and a stage-specific transcription factor . The stepwise binding of capping enzyme, VITF-1, and VITF-2 to a DNA/viral RNA polymerase complex was demonstrated. J Biol Chem, 1994 May 13, 269(19), 14191 - 8 Molecular cloning and characterization of two rab GDI species from rat brain: brain-specific and ubiquitous types; Nishimura N et al.; Rab GDP dissociation inhibitor (GDI) is a regulatory protein for Rab proteins that controls not only the GDP/GTP exchange reaction of Rab proteins but also their translocation between the cytosol and cell membranes . Recently, rab GDI cDNAs have been isolated from human, bovine, rat, and Drosophila, and these Rab GDI proteins indicated an important role in the regulation of intracellular vesicle traffic . In this study, we have isolated two different rab GDI cDNAs, designated rat rab GDI alpha and beta, from a rat brain cDNA library using bovine rab GDI cDNA as a probe . Rat rab GDI alpha and beta show 99 and 86% amino acid identity with bovine rab GDI, respectively, indicating that rat rab GDI alpha is a rat counterpart of bovine rab GDI . Both rat Rab GDI alpha and beta proteins expressed in Escherichia coli showed a similar degree of activity of regulating the GDP/GTP exchange reaction to that of bovine Rab GDI using Rab3A and Rab11 as substrates . Northern blot analysis revealed that rab GDI alpha mRNA was expressed abundantly in brain but much less in other tissues, whereas rat rab GDI beta mRNA was ubiquitously expressed . Reverse transcription-polymerase chain reaction analysis revealed that astrocytes expressed rat rab GDI beta gene but not rat rab GDI alpha gene . These results indicate that Rab GDI alpha is involved in sorting of highly specialized vesicles in brain such as neurosecretory vesicles, whereas Rab GDI beta plays a general role in vesicular trafficking in diverse types of cells. J Biol Chem, 1994 May 13, 269(19), 14140 - 8 Molecular cloning and sequencing of calnexin-t . An abundant male germ cell-specific calcium-binding protein of the endoplasmic reticulum; Ohsako S et al.; A mouse testis cDNA expression library was screened using a monoclonal antibody (1C9) that recognized an abundant testis-specific 101-kDa endoplasmic reticulum-associated protein . The screening resulted in the isolation of a 2.3-kilobase cDNA clone (A2/6) . The sequence encoded 611 amino acids with a calculated mass of 69,454 Da, that was 60% similar to mouse calnexin . A high affinity calcium binding domain, present in both calnexin and calreticulin, and one transmembrane domain similar to that of calnexin were found in the A2/6 protein domain . Northern blot analysis of total RNA from seven different tissues showed hybridization only to testis RNA . Southern blot analysis indicated that A2/6 was a single copy gene . The calculated molecular mass for A2/6 was unexpectedly lower than the 101-kDa protein recognized by 1C9 on Western blot analysis of total testis protein . However, Escherichia coli and in vitro translation products of A2/6 cDNA yielded a similar 100-kDa protein . Finally, using the recombinant protein, calcium binding activity was detected by a 45Ca2+ overlay assay . These results suggest that spermatogenic cell endoplasmic reticulum has a unique calcium binding protein, calnexin-t, which appears to be a calnexin variant. J Biol Chem, 1994 May 13, 269(19), 14032 - 7 Nucleotide binding to the hydrophilic C-terminal domain of the transporter associated with antigen processing (TAP); Muller KM et al.; The gene products of tap1 and tap2 encoded in the major histocompatibility complex (MHC) class II region belong to the ATP binding cassette superfamily of transporters . They are thought to form a heterodimer for the delivery of peptides into the lumen of the endoplasmic reticulum; peptides are required for correct assembly and presentation of the MHC class I molecule peptide complex at the cell surface . To elucidate the ATP binding properties of these proteins in vitro, we expressed the hydrophilic C-terminal part of human transporter associated with antigen processing (TAP1) (nucleotide binding domain (NBD)-TAP1, amino acids 452-748) and TAP2 (NBD-TAP2, amino acids 399-686) fused to a His6 tag in Escherichia coli . The recombinant proteins accumulated exclusively in inclusion bodies and were solubilized under denaturing conditions . After purification by immobilized metal ion affinity chromatography, we were able to refold the domains for functional studies . NBD-TAP1 bound to C-8-ATP-agarose and was specifically eluted with ATP or EDTA . Photoaffinity labeling of NBD-TAP1 with the ATP analogues 8-azido-{gamma-32P}ATP and 3'-O-{(4-azido-3,5-{125I}diiodo-2-hydroxybenzoyl)-beta-alanyl}-ATP was specific . The addition of 50 microM ATP inhibited photoaffinity labeling by 8-azido-ATP down to 8% of controls . Efficiency of inhibition decreased as follows: ATP > GTP > ADP > CTP > AMP . Photolabeling of NBD-TAP2 was not observed . ATP hydrolysis by NBD-TAP1 was not detected . Until now strong but only indirect data of the TAP function existed . The described experiments demonstrate ATP binding to an isolated domain of the antigenic peptide transporter, TAP, and therefore support the theory of ATP-dependent peptide translocation. J Biol Chem, 1994 May 13, 269(19), 13825 - 35 A molecular model of the inducer binding domain of the galactose repressor of Escherichia coli; Hsieh M et al.; The C-terminal inducer binding domain of the Escherichia coli galactose repressor (GalR) is homologous to several periplasmic chemoreceptor proteins whose three-dimensional structures have been determined at high resolution (Vyas, N . K., Vyas, M . N., and Quiocho, F . A . (1991) J . Biol . Chem . 266, 5226-5237; Mowbray, S . L . and Cole, L . B . (1992) J . Mol . Biol . 225, 155-175) . The protein backbone was constructed from the coordinates of glucose/galactose-binding protein using the Homology program (Biosym Technologies, San Diego) . Loops were built by searching for substructures in the structure data base, and the side chains were built using a rotamer-base program . A small amount of energy minimization relieved steric strain within the model . The GalR model that has been constructed is consistent with the principles of protein structure; values obtained for the compactness and buried surface area of the model compare favorably with those determined for the chemoreceptor protein structures . The model is consistent with, and provides structural interpretations for, experimental results obtained from physical and biochemical studies . Predictions are made concerning the residues conferring the specificity of galactose induction of GalR and for self-association to dimers . The model provides a first step toward correlating the structure and regulation of GalR and in determining the chemistry of its homologous and heterologous interactions that are critical to its role as a regulator of transcription initiation. J Mol Biol, 1994 May 13, 238(4), 635 - 7 Crystallization and preliminary X-ray analysis of copper amine oxidase from Escherichia coli K-12; Roh JH et al.; Copper-containing monoamine oxidase (MAO) from Escherichia coli was overproduced in the periplasmic space by expression of the cloned gene . The purified MAO has been crystallized by means of the hanging drop technique using sodium citrate as a precipitant . The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit cell dimensions of a = 136.1 A, b = 168.4 A and c = 81.6 A . The asymmetric unit contains one molecule of MAO, with a crystal volume per protein mass (Vm) of 2.88 A3/Da and a solvent content of 58% by volume . The crystals diffract X-rays to a resolution limit of at least 2.7 A and are resistant to X-ray radiation damage . They appear to be suitable for X-ray structure analysis. J Mol Biol, 1994 May 13, 238(4), 592 - 614 The solution structures of the trp repressor-operator DNA complex; Zhang H et al.; The solution structures of the complex between Escherichia coli trp holorepressor and a 20 base-pair consensus operator DNA were determined . The majority of proton chemical shifts of the trp holorepressor and operator DNA were assigned from homonuclear 2D NOESY spectra of selectively deuterated analog-operator DNA complexes and the 3D NOESY-HMQC spectrum of a uniformly 15N-labeled repressor-operator DNA complex . The structures were calculated using restrained molecular dynamics and sequential simulated annealing with 4086 NOE and other experimental constraints . The root-mean-squared deviation (RMSD) among the calculated structures and their mean is 0.9(+/- 0.3)A for the repressor backbone, 1.1(+/- 0.5)A for the DNA backbone, and 1.3(+/- 0.3)A for all heavy atoms . The DNA is deformed to a significant extent from the standard B DNA structure to fit the helix-turn-helix (HTH) segment of the repressor (helices D and E) into its major grooves . Little change is found in the ABCF core of the repressor on complexation in comparison to the free repressor, but changes in the cofactor L-tryptophan binding pocket and the HTH segment are observed . The N-terminal residues (2 to 17) are found to be disordered and do not form stable interactions with DNA . Direct H-bonding to the bases of the operator DNA is consistent with all of our observed NOE constraints . Hydrogen bonds from NH eta 1 and NH eta 2 of Arg69 to O-6 and N-7 of G2 are compatible with the solution structure, as they are with the crystal structure . Other direct H-bonds from Lys72, Ala80, Ile79, Thr83 and Arg84 to base-pair functional groups can also be formed in our solution structures. J Mol Biol, 1994 May 13, 238(4), 555 - 62 Export and trimerization pathways of maltoporin overlap in the inner membrane of Escherichia coli; Stader J et al.; The production of functional porins involves multiple steps including: export of precursor polypeptides from the cytoplasm, assembly of monomers into trimers, and stabilization of trimers by association with lipopolysaccharide . In this report, a late export/assembly intermediate of the maltoporin (LamB) is found in the inner membrane of Escherichia coli using cell fractionation studies . This processed intermediate is transiently associated with a unique Triton X-100-insoluble subfraction of the inner membrane . The kinetics of appearance and solubility characteristics of this intermediate correspond to those of the metastable trimer form of LamB, suggesting that the export and assembly pathways overlap in the inner membrane prior to final localization in the bulk outer membrane. J Mol Biol, 1994 May 13, 238(4), 514 - 27 DNA-protein cooperativity in the assembly and stabilization of mu strand transfer complex . Relevance of DNA phasing and att site cleavage; Namgoong SY et al.; The requirements for negatively supercoiled DNA substrates, the cis-acting transposition enhancer and the Escherichia coli HU protein during the phage Mu transposition reaction are relaxed under DMSO-assay conditions . We have used these modified assay conditions to extend studies on the transposition pathway . We show here that linear DNA fragments containing the right end of Mu (attR) and Mu A protein mutually promote the assembly of "high-order" complexes held together by non-covalent protein-DNA and protein-protein interactions . A large subset of these complexes is competent in mediating strand transfer . DNA fragments containing the left end of Mu (attL) as well as non-Mu DNA can be used as targets during strand transfer . The R1 and R2 subsites within attR are required, but R3 is dispensable, in the protein-DNA oligomerization steps as well as in the strand transfer reaction . Proper phasing and spacing between R1 and R2 are central to the reaction . A single base-pair change in the terminal nucleotide that renders attR non-cleavable prevents the assembly of stable high-order complexes, showing that strand cleavage and stabilization of high-order complexes are tightly coupled events . Conversely, pre-cleavage at the attL site allows it to function in the assembly process, albeit at a much lower efficiency than attR . In the presence of HU, the reactivity of pre-cleaved attL is enhanced significantly. J Biol Chem, 1994 May 13, 269(19), 13854 - 60 Effects of changing glutamate 487 to lysine in rat and human liver mitochondrial aldehyde dehydrogenase . A model to study human (Oriental type) class 2 aldehyde dehydrogenase; Farres J et al.; Many Oriental people possess a liver mitochondrial aldehyde dehydrogenase where glutamate at position 487 has been replaced by a lysine, and they have very low levels of mitochondrial aldehyde dehydrogenase activity . To investigate the cause of the lack of activity of this aldehyde dehydrogenase, we mutated residue 487 of rat and human liver mitochondrial aldehyde dehydrogenase to a lysine and expressed the mutant and native enzyme forms in Escherichia coli . Both rat and human recombinant aldehyde dehydrogenases showed the same molecular and kinetic properties as the enzyme isolated from liver mitochondria . The E487K mutants were found to be active but possessed altered kinetic properties when compared to the glutamate enzyme . The Km for NAD+ at pH 7.4 increased more than 150-fold, whereas kcat decreased 2-10-fold with respect to the recombinant native enzymes . Detailed steady-state kinetic analysis showed that the binding of NAD+ to the mutant enzyme was impaired, and it could be calculated that this resulted in a decreased nucleophilicity of the active site cysteine residue . The rate-limiting step for the rat E487K mutant was also different from that of the recombinant rat liver aldehyde dehydrogenase in that no pre-steady-state burst of NADH formation was found with the mutant enzyme . Both the rat native enzyme and the E487K mutant oxidized chloroacetaldehyde twice as fast as acetaldehyde, indicating that the rate-limiting step was not hydride transfer or coenzyme dissociation but depended upon nucleophilic attack . Each enzyme form showed a 2-fold activation upon the addition of Mg2+ ions . Substituting a glutamine for the glutamate did not grossly affect the properties of the enzyme . Glutamate 487 may interact directly with the positive nicotinamide ring of NAD+ for the Ki of NADH was the same in the lysine enzyme as it was in the glutamate form . Because of the altered NAD+ binding properties and kcat of the E487K variant, it is assumed that people possessing this form will not have a functional mitochondrial aldehyde dehydrogenase. Am J Physiol, 1994 May, 266(5 Pt 1), G899 - 906 Upregulation of Escherichia coli heat-stable enterotoxin receptor in regenerating rat liver; Laney DW Jr et al.; Guanylate cyclase C (GC-C) is a transmembrane protein that serves as a receptor for the recently characterized endogenous ligand guanylin and for Escherichia coli heat-stable toxin (STa) . Binding of either guanylin or STa to intestinal GC-C results in net chloride secretion . Although GC-C is expressed in the rat intestine throughout life, its expression in the rat liver has previously been shown to occur only during the perinatal period . As a step toward elucidating the role of this receptor in the liver, we tested the hypothesis that GC-C mRNA expression could be induced in the adult rat liver following 1) partial hepatectomy, a stimulus for hepatocyte proliferation; 2) intraperitoneal carbon tetrachloride injection, a model of hepatocyte regeneration in the presence of inflammatory changes; and 3) subcutaneous turpentine injection, which generates an acute phase response without hepatocyte proliferation . We demonstrated expression of GC-C mRNA in the regenerating rat liver following either partial hepatectomy or CCl4-induced hepatic necrosis . We have also shown that GC-C mRNA expression occurred in association with an acute phase reaction . Coordinate with the expression of GC-C mRNA, there was upregulation of radiolabeled STa binding to liver plasma membranes prepared from turpentine-treated rats . Maximal expression of GC-C occurred in preparations enriched for the canalicular domain . Although the function of GC-C in the liver is unknown, localization to the canalicular domain would be consistent with a role for GC-C in hepatic chloride secretion, especially in the perinatal liver and during hepatocyte regeneration. Nucleic Acids Res, 1994 May 11, 22(9), 1712 - 8 A tRNA gene mapping within the chloroplast rDNA cluster is differentially expressed during the development of Daucus carota; Manna F et al.; In vivo analysis of expression of the chloroplast rDNA cluster during somatic embryogenesis of Daucus carota (D.carota) was performed by Northern-blot analysis with different DNA probes, spanning both the 16S rRNA gene, the 16S-23S rRNA spacer, which contains the two mosaic tRNA genes tRNA(Ile) and tRNA(Ala), and the region upstream of the 16S rRNA gene, where a tRNA(Val) maps . We show that expression both of the spacer tRNAs tRNA(Ile) and tRNA(Ala) is not significantly regulated during development whereas the amount of the transcript corresponding to tRNA(Val) is not detectable during early embryonic stages and progressively accumulates during late phases . Multiple transcription start sites have been identified upstream of the tRNA(Val) gene by S1 mapping analysis, which are activated late during the embryogenesis . These data indicate that developmental control mechanisms act on plastid gene expression during embryogenesis in carrot. Nucleic Acids Res, 1994 May 11, 22(9), 1670 - 4 Increased removal of 3-alkyladenine reduces the frequencies of hprt mutations induced by methyl- and ethylmethanesulfonate in Chinese hamster fibroblast cells; Klungland A et al.; We have previously reported the isolation of mammalian cell lines expressing the 3-methyladenine DNA glycosylase I (tag) gene from E . coli . These cells are 2-5 fold more resistant to the toxic effects of methylating agents than normal cells (15) . Kinetic measurements of 3-methyladenine removal from the genome in situ show a moderate (3-fold) increase in Tag expressing cells relative to normal as compared to a high (50-fold) increase in exogenous alkylated DNA in vitro by cell extracts . Excision of 7-methylguanine is as expected, unaffected by the tag+ gene expression . The frequency of mutations formed in the hypoxanthine phosphoribosyl transferase (hprt) locus was investigated after methylmethanesulfonate (MMS), ethylmethanesulfonate (EMS), N-nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) exposure . Tag expression reduced the frequency of MMS and EMS induced mutations to about half the normal rate, whereas the mutation frequency in cells exposed to NMU or NEU is not affected by the tag+ gene expression . These results indicate that after exposure to compounds which produce predominantly N-alkylations in DNA, a substantial proportion of the mutations induced is formed at 3-alkyladenine residues in DNA. Nucleic Acids Res, 1994 May 11, 22(9), 1637 - 9 Systematic sequencing of the Escherichia coli genome: analysis of the 2.4-4.1 min (110,917-193,643 bp) region; Fujita N et al.; The complete sequence analysis of the E . coli genome was initiated as a collaborative study in Japan . Following the initial analysis of the 0-2.4 min region (Yura, T . et al . (1992) Nucleic Acids Res . 20, 3305-3308), a contiguous sequence of 82,727 bp corresponding to the 2.4-4.1 min region (110,917-193,643 bp as counted from 0 min) was determined . The resulting sequence was found to contain at least 33 known genes and 24 putative genes predicted from protein sequence homology. Nucleic Acids Res, 1994 May 11, 22(9), 1626 - 31 An activity gel assay for the detection of DNA helicases and nucleases from cell-free extracts; Shukla SK et al.; An activity gel assay was developed for the detection of DNA helicases in crude extracts . The assay was based on the ability of DNA helicases to unwind radioactive fragments from single-stranded M13 circles that were immobilized in an SDS polyacrylamide gel . The displaced radioactive strands were detected by blotting them to a filter and visualizing the resulting bands by autoradiography . Experiments with purified proteins demonstrated that DNA helicases, endonucleases and exonucleases could produce activity bands . A one-dimensional gel assay was sufficiently sensitive to allow detection of DNA helicase I, DNA helicase II, DNA helicase IV, the RecQ helicase as well as 3 unidentified putative DNA helicases in crude extracts of Escherichia coli . Exonuclease and endonuclease activities from crude extracts could be distinguished from DNA helicase activities by their ATP-independence and from each other by their band morphologies. Nucleic Acids Res, 1994 May 11, 22(9), 1687 - 95 The binding site for ribosomal protein S8 in 16S rRNA and spc mRNA from Escherichia coli: minimum structural requirements and the effects of single bulged bases on S8-RNA interaction; Wu H et al.; Through specific interactions with rRNA and mRNA, ribosomal protein S8 of Escherichia coli plays a central role in both assembly of the 30S ribosomal subunit and translational regulation of spc operon expression . To better understand S8-RNA association, we have measured the affinity of S8 for a number of variants of its rRNA and mRNA binding sites prepared by in vitro transcription or chemical synthesis . With the aid of site-directed deletions, we demonstrate that an imperfect, 33-nucleotide helical stem encompassing nucleotides 588-603 and 635-651 possesses all of the structural information necessary for specific binding of S8 to the 16S rRNA . This segment consists of two short duplexes that enclose a conserved, asymmetric internal loop which contains features crucial for protein recognition . The S8 binding site in spc operon mRNA is very similar in both primary and secondary structure to that in 16S rRNA except for the presence of two single bulged bases in one of the duplex segments . In addition, the apparent association constant for the S8-mRNA interaction is approximately fivefold less than that for the S8-rRNA interaction . We show that the difference in affinity can be attributed to the effects of the bulged bases . Deletion of the bulged bases from the mRNA site increases its affinity for S8 to a level similar to that of the rRNA, whereas insertion of single-base bulges at equivalent positions within the rRNA site reduces its affinity for S8 to a value typical of the mRNA . Single-base bulges in the proximity of essential recognition features are therefore capable of modulating the strength of protein-RNA interactions. Mol Gen Genet, 1994 May 10, 243(3), 270 - 6 Expression of the 3-phosphoglycerate kinase gene (pgkA) of Penicillium chrysogenum; Hoskins IC et al.; The sequence of the Penicillium chrysogenum pgkA gene promoter was determined up to 952 nucleotides (nt) 5' to the major transcriptional start point (position +1), and contains a 38 bp pyrimidine-rich region within which transcription initiates at this and two minor sites (-11, -23) . A 21 bp segment (-99 to -79) closely matches a region which is essential for the expression of the Aspergillus nidulans pgkA gene . A further region was found with similarity to sequences in other A . nidulans promoters possibly effecting response to carbon source . The terminator region of the P . chrysogenum pgkA gene was sequenced as far as 192 nt 3' to the stop codon and three polyadenylation sites were found at 94, 103 and 107 nt from this point, the first preceded by a possible polyadenylation signal . No transcription termination signal was found but several regions potentially forming stem-loop-structures were noted . A single 1.3 kb pgkA mRNA was readily detected by Northern blot analysis of total cellular RNA . Steady-state levels of pgkA mRNA were 1.5 to 2.0 times greater in mycelium harvested at similar stages of growth from medium containing the carbon sources acetate or quinate compared to glucose . A transformed strain of P . chrysogenum containing a fusion of the pgkA promoter to the Escherichia coli lacZ reporter gene integrated at the oliC locus was constructed, and beta-galactosidase activity monitored during growth of batch cultures in defined media . The pgkA promoter activity increased during exponential growth and was 2-3 times greater and increased most rapidly in mycelium grown on quinate or acetate compared to glucose.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Gen Genet, 1994 May 10, 243(3), 261 - 9 Genetic analysis of SecY: additional export-defective mutations and factors affecting their phenotypes; Taura T et al.; A number of secY mutants of Escherichia coli showing protein export defects were isolated by a combination of localized mutagenesis and secA-lacZ screening . Most of them were cold sensitive and contained single base substitutions in secY leading to amino acid replacements in various parts of the SecY protein, mainly in the cytoplasmic and the transmembrane domains . A temperature-sensitive mutant with an export defect had the same base substitution as secY24, which was characterized previously . Many cold-sensitive secY mutants exhibited rapid responses to temperature lowering but their apparent defects varied at the permissive temperature . Others exhibited delayed responses to the temperature shift . Some secY mutations, including secY39, interfered with protein export when expressed from a multicopy plasmid, even in the presence of wild-type secY on the chromosome . Such "dominant negative" mutations, including secY-d1, which was studied previously, were all located in either cytoplasmic domain 5 or 6, which is consistent with our previous proposal that the C-terminal region of SecY is important for its function as a protein translocator . We also studied the phenotypes of strains in which one of the secY mutations was combined with the components of the secD operon . Overexpression of secD partially suppressed the secY39 mutation, while overexpression of secF exacerbated the export defects of secY122 and secY125 mutations . Overexpression of "yajC", located within the secD operon, suppressed secY-d1 . Although yajC itself proved to be dispensable, its disruption impaired the growth of the secY39 mutant at 42 degrees C . These observations suggest that SecY interacts with SecD, SecF, and the product of yajC. Proc Natl Acad Sci U S A, 1994 May 10, 91(10), 4539 - 43 A cytoplasmic domain is important for the formation of a SecY-SecE translocator complex; Baba T et al.; An approach to identifying the interaction site of multicomponent protein assembly has been applied to the membrane-bound SecY-SecE complex, which mediates protein export across the Escherichia coli cytoplasmic membrane . A dominant negative secY allele, secY-d1, inactivates SecY but preserves its ability to interact with SecE . Thus, the mutant protein sequesters SecE in an inactive complex . Second site mutations that disrupt the SecE binding site will suppress the export interference . We introduced insertion/deletion mutations that intragenically suppressed secY-d1 . After eliminating knock-out mutations by virtue of the expression of a LacZ alpha sequence that had been attached to the C terminus, we obtained a striking clustering of mutations in cytoplasmic domain 4 . On the basis of this result, the secY24 (Ts) substitution mutation in this domain was examined for its effects on interaction with SecE . It indeed suppressed secY-d1 . Although the instability associated with excess SecY can be alleviated by overproduction of SecE, the secY24 mutant protein was not stabilized by SecE . The basal-level SecY24 protein was also destabilized at 42 degrees C . SecE was coimmunoprecipitated with SecY+ but not with the SecY24 protein . These results indicate that the secY24 mutation weakens SecY's interaction with SecE . Taken together, we propose that cytoplasmic domain 4 is important for the association between SecY and SecE. Proc Natl Acad Sci U S A, 1994 May 10, 91(10), 4328 - 32 Mutational analysis of yeast mRNA capping enzyme; Schwer B et al.; RNA guanylyltransferase (capping enzyme) catalyzes the transfer of GMP from GTP to the 5'-diphosphate end of mRNA . The capping reaction proceeds via an enzyme-guanylate intermediate in which GMP is linked covalently to a lysine residue of the enzyme . In the capping enzyme of Saccharomyces cerevisiae, GMP is attached to a 52-kDa polypeptide, identified as the product of the essential CEG1 gene . The amino acid sequence of the CEG1 protein includes a motif, Lys70-Thr-Asp-Gly, that is conserved at the active site of vaccinia virus RNA guanylyltransferase and which is similar to the KXDG sequence found at the active sites of RNA and DNA ligases . To evaluate the role of this motif in the function of the yeast enzyme, we have expressed the CEG1 protein in active form in Escherichia coli . Replacement of Lys70 or Gly73 with alanine abrogated enzyme-guanylate formation in vitro; in contrast, alanine substitutions at Thr71 or Asp72 merely reduced activity relative to wild-type enzyme . The K70A and G73A mutations were lethal to yeast, whereas yeast carrying the T71A and D72A alleles of CEG1 were viable . These results implicate Lys70 as the active site of yeast guanylyltransferase and provide evidence that cap formation per se is an essential function in eukaryotic cells. Proc Natl Acad Sci U S A, 1994 May 10, 91(10), 4150 - 4 Effects on protein synthesis produced by pairing depolarization with serotonin, an analogue of associative learning in Aplysia; Noel F et al.; A form of associative plasticity in Aplysia, activity-dependent neuromodulation, involves the convergence of neuronal activity and the effects of a modulatory transmitter . To investigate the role of protein synthesis in associative plasticity, we examined the effects of a biochemical analogue of activity-dependent neuromodulation on the level of incorporation of labeled amino acid into proteins . To mimic associative training, abdominal ganglia were exposed to paired treatments of a depolarizing agent, elevated potassium, and a modulatory transmitter, serotonin . The effects of elevated potassium and serotonin applied alone were also examined . At least two proteins (nos . 9 and 17) were affected in a nonadditive way by the paired procedure . Incorporation of label into protein 9 was increased by the paired procedure but was not affected by either elevated potassium or serotonin . Incorporation of label into protein 17 was significantly affected by elevated potassium or serotonin, but the effect of the paired procedure was significantly less than the summed effects of elevated potassium and serotonin applied alone . These results indicate that changes in protein synthesis may be important in the induction of associative plasticities . Amino acid sequences of two peptides derived from protein 9 were obtained . Then, a partial cDNA clone for protein 9 was obtained by performing PCR with degenerate primers corresponding to portions of the sequences of the two peptides . The sequence of protein 9 is related to sequences previously reported for a family of genes comprising the stringent starvation protein of Escherichia coli, auxin-induced proteins of plants, and glutathione S-transferases of a number of organisms. Biochemistry, 1994 May 10, 33(18), 5630 - 5 Substrate activation of brewers' yeast pyruvate decarboxylase is abolished by mutation of cysteine 221 to serine; Baburina I et al.; Brewers' yeast pyruvate decarboxylase (EC 4.1.1.1), a thiamin diphosphate and Mg(II)-dependent enzyme, isolated from Saccharomyces cerevisiae possesses four cysteines/subunit at positions 69, 152, 221, and 222 . Earlier studies conducted on a variant of the enzyme with a single Cys at position 221 (derived from a gene that was the product of spontaneous fusion) showed that this enzyme is still subject to substrate activation {Zeng, X., Farrenkopf, B., Hohmann, S., Jordan, F., Dyda, F., & Furey, W . (1993) Biochemistry 32, 2704-2709}, indicating that if Cys was responsible for this activation, it had to be C221 . To further test the hypothesis, the C221S and C222S single and the C221S-C222S double mutants were constructed . It is clearly shown that the mutation at C221, but not at C222, leads to abolished substrate activation according to a number of kinetic criteria, both steady state and pre steady state . On the basis of the three-dimensional structure of the enzyme {Dyda, F., Furey, W., Swaminathan, S., Sax, M., Farrenkopf, B., Jordan, F . (1993) Biochemistry 32, 6165-6170}, it is obvious that while C221 is located on the beta domain, whereas thiamin diphosphate is wedged at the interface of the alpha and gamma domains, addition of pyruvate or pyruvamide as a hemiketal adduct to the sulfur of C221 can easily bridge the gap between the beta and alpha domains . In fact, residues in one or both domains must be dislocated by this adduct formation . It is very likely that regulation as expressed in substrate activation is transmitted via this direct contact made between the two domains in the presence of the activator.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 May 10, 33(18), 5600 - 6 The Schiff base counterion of bacteriorhodopsin is protonated in sensory rhodopsin I: spectroscopic and functional characterization of the mutated proteins D76N and D76A; Rath P et al.; Both sensory rhodopsin I (SR-I), a phototaxis receptor, and bacteriorhodopsin (BR), a light-driven proton pump, share residues which have been identified as critical for BR functioning . This includes Asp76, which in the case of bacteriorhodopsin (Asp85) functions both as the Schiff base counterion and proton acceptor . We found that substituting an Asn for Asp76 (D76N) in SR-I has no effect on its visible absorption unlike the analogous mutation (D85N) in BR which shifts the absorption to longer wavelengths . The mutated proteins D76N and D76A are also fully functional as phototaxis receptors in contrast to BR, where the analogous substitutions block proton transport . D76N was also found to exhibit a spectrally normal SR587-->S373 transition . However, FTIR difference spectroscopy reveals that two bands in the SR587-->S373 difference spectrum at 1766/1749 cm-1 (negative/positive), assigned to the C=O stretch mode of a carboxylic acid, disappear in D76N, although no changes are observed in the carboxylate region . In addition, the kinetics and yield of this photoreaction are altered . On this basis, it is concluded that, unlike Asp85 in bacteriorhodopsin, Asp76 is protonated in SR-I and undergoes an increase in its hydrogen bonding during the SR587-->S373 transition . This model accounts for the difference in color of SR-I and BR and the finding that Asn can substitute for Asp76 without greatly altering the SR-I phenotype . Interestingly, parallels exist between this residue and Asp83 in the visual receptor rhodopsin which has recently been found to exist in a protonated form and to undergo an almost identical change in hydrogen bonding during rhodopsin activation. Biochemistry, 1994 May 10, 33(18), 5502 - 9 19F NMR spectroscopy of {6-19F}tryptophan-labeled Escherichia coli dihydrofolate reductase: equilibrium folding and ligand binding studies; Hoeltzli SD et al.; Escherichia coli dihydrofolate reductase contains five tryptophan residues distributed throughout its structure . In order to examine the regions of the protein surrounding these tryptophan residues, we have incorporated 6-fluorotryptophan into the protein . To assign the five resonances observed in the 19F NMR spectrum, five site-directed mutants of the enzyme were made, each with one tryptophan replaced by a phenylalanine . The 19F NMR spectra of the apoprotein, two binary complexes (with NADPH or methotrexate), and one ternary complex (with NADPH and methotrexate) were obtained . The chemical shifts of two of the tryptophan resonances (at positions 22 and 74) are particularly sensitive to ligand binding, while the remaining three (at positions 30, 47, and 133) change, but by less . Since several of the tryptophans are distant from the binding site, these results suggest that 19F NMR can detect ligand-induced changes that are propagated throughout the structure . In the apoprotein, the resonances of the tryptophans at positions 22 and 30 are broadened . In the binary complex with NADPH, the resonances of tryptophans 30 and 74 are broadened while that of tryptophan 22 almost disappears . The line broadening of the tryptophan 22 resonance may reflect motion in that part of the protein, since it is near a region that is disordered in the crystal structure of the apoprotein and its NADP+ complex . In contrast, in the ternary complex this region has a defined structure, and all resonances are of equal intensity and line width . The 19F NMR spectra of the apoprotein and the three ligand complexes were also examined as a function of urea concentration.(ABSTRACT TRUNCATED AT 250 WORDS) Proc Natl Acad Sci U S A, 1994 May 10, 91(10), 4125 - 9 Stem-loop IV of 5S rRNA lies close to the peptidyltransferase center; Dontsova O et al.; A DNA fragment containing the Escherichia coli 5S rDNA sequence linked to a T7 promoter was prepared by PCR from an M13 clone carrying the 5S-complementary sequence . The DNA was transcribed with T7 polymerase using a mixture of {alpha-32P}UTP and 4-thio-UTP, yielding a transcript in which approximately 18% of the uridine residues were randomly replaced by thiouridine . This modified 5S RNA could be reconstituted efficiently into 50S ribosomal subunits or 70S functional complexes . The reconstituted particles were irradiated at wavelengths above 300 nm, and the crosslinked ribosomal components were identified . A crosslink in high yield was reproducibly observed between the modified 5S RNA and 23S RNA, involving residue U-89 of the 5S RNA (at the loop end of helix IV) linked to nucleotide 2477 of the 23S RNA in the loop end of helix 89, immediately adjacent to the peptidyltransferase "ring." On the basis of this result, and in combination with earlier immunoelectron microscopic data, we propose a model for the orientation of the 5S RNA in the 50S subunit. FEBS Lett, 1994 May 9, 344(1), 31 - 4 Recognition of UUN codons by two leucine tRNA species from Escherichia coli; Takai K et al.; Codon recognition by Escherichia coli tRNA(Leu)4 and tRNA(Leu)5 was investigated by analysis of the competition between two aminoacyl-tRNA species in an in vitro protein synthesis . Both tRNA species strictly obey the wobble rule when they are in competition with other tRNA species . This is probably due to the post-transcriptional modifications at the first position of the anticodon of these tRNA(Leu) species, supporting the proposal that the conformational rigidity of post-transcriptionally modified pyrimidine nucleotides guarantees the correct codon recognition. Neuroreport, 1994 May 9, 5(9), 1069 - 72 In vivo transfer of a marker gene to study motoneuronal development; Lisovoski F et al.; Adenovirus vectors containing a marker gene (lacZ from Escherichia coli) are potent for transferring the gene to neurones after intraparenchymal injections . Expression of the marker gene may lead to the synthesis of an enormous amount of beta-galactosidase which diffuses throughout the entire neurone, providing a 'Golgi-like' staining . This suggested that the technique may be used to study the morphology of specific neuronal populations . We have validated this hypothesis by analysing the postnatal development of motoneurones in the rat cervical cord . Injections of the viral suspension into one ventral horn were performed at different ages after birth . Histochemical staining using X-Gal revealed morphological changes occurring within the first 3 weeks with enlargement of the perikaryon and increased dendritic complexity . Immunoreactivity for CGRP was visualized in double-staining experiments . In vivo transfer of a marker gene therefore provides a new way to analyse neuronal morphology which allows selection of the cells to be studied and double-labelling with immunohistochemical markers. J Mol Biol, 1994 May 6, 238(3), 319 - 32 High resolution mapping of UV-induced photoproducts in the Escherichia coli lacI gene . Inefficient repair of the non-transcribed strand correlates with high mutation frequency; Chandrasekhar D et al.; UV-induced DNA photoproduct formation and repair has been examined at the gene and nucleotide level in Escherichia coli using two newly developed quantitative assays . A multiplex quantitative PCR assay was used to measure photoproduct formation and repair at the gene level in both the constitutive lacI gene and the inducible lacZ gene, simultaneously . Both genes displayed similar photoproduct formation frequencies (0.4 lesions/kb per 100 J/m2) . Following a 15 minute recovery period, 36% and 39% of the damage resulting from 100 J/m2 was removed from the lacI and lacZ genes, respectively . Under the growth conditions applied, the lacZ gene was expressed at a very low rate resulting in 0.3% of beta-galactosidase activity as compared to induced cells . A newly developed reiterative primer extension assay has been employed to examine photoproduct formation and repair at the nucleotide level . Analysis of UV-induced DNA photoproducts in the first 184 base-pairs of the lacI gene of genomic E . coli DNA has revealed that photoproducts are induced linearly with dose and the slope is sequence context-dependent . A post-irradiation recovery period revealed differences in the repair efficiency at individual nucleotides . Repair of photoproducts on the transcribed strand was generally twice as efficient as repair of photoproducts on the non-transcribed strand, indicating that strand-specific DNA repair occurs in the constitutively transcribed lacI gene of E . coli . Comparison of the UV-induced DNA photoproduct distribution with an established UV-induced mutation spectrum from wild-type cells revealed that photoproducts form at all mutagenic hotspots . Some sites of low frequency mutations were not observed to be sites of photoproduct formation . However, not all photoproducts appeared to be mutagenic . This was especially true for those on the efficiently repaired transcribed strand . It is hypothesized that the preferential repair of photoproducts on this strand may prevent many of these photoproduct sites from becoming mutagenic hotspots . These data strongly support the hypothesis that mutations arise at inefficiently repaired photoproducts on the nontranscribed strand. J Mol Biol, 1994 May 6, 238(3), 309 - 18 Ligand-induced self-association of the Escherichia coli regulatory protein TyrR; Wilson TJ et al.; Analyses of the sedimentation properties of the Escherichia coli regulatory protein TyrR indicated that it undergoes a ligand-induced hexamerization . This phenomenon was observed at protein concentrations approximating to those found in vivo . In the absence of added ligands, TyrR sedimented as a single molecular species with a sedimentation coefficient of 5.3 S and a relative molecular mass of 113,000 . Given a subunit relative molecular mass of 57,640 for TyrR, it was concluded that this species is a dimer . Similar sedimentation properties were observed when TyrR was sedimented in the presence of either tyrosine, phenylalanine, ATP or ATP gamma S, a non-hydrolysable analogue of ATP . However, in the presence of saturating ATP gamma S and 500 microM tyrosine or 25 mM phenylalanine the sedimentation behaviour of TyrR yielded relative molecular masses of 340,000 and 310,000, respectively, indicative of hexamer formation . The sedimentation data obtained across a range of TyrR concentrations fitted equally well to dimer-hexamer and dimer-tetramer-hexamer models . For the latter model, the predicted overall association constant was 3.2 x 10(13) M-2 at saturating tyrosine, while the relative values of the association constants for the two individual steps indicated a concerted mechanism with the tetramer a minor component . There was no indication of dimer dissociation when dilute TyrR solutions (100 nM) were sedimented . A model to explain the role of hexamerization in tyrosine-mediated repression of transcription by TyrR is proposed . It is suggested that the hexameric form of TyrR is the active repressing species, interacting with two or three specific sequences (TyrR boxes) in the targeted regulatory DNA . The hexamerization reaction that takes place when the tyrosine concentration rises is envisaged as occurring in situ on the DNA, with a TyrR dimer that permanently occupies one of the TyrR boxes acting as a nucleation site for the development of the hexamer-DNA complex. J Mol Biol, 1994 May 6, 238(3), 302 - 8 The concentration of polypeptide chain release factors 1 and 2 at different growth rates of Escherichia coli; Adamski FM et al.; The number of molecules of release factor-1 (RF-1) and release factor-2 (RF-2) per Escherichia coli cell grown at various rates was determined using quantitative Western blotting of total solubilized cell protein . The number of RF-1 molecules per cell increased from 1200 to 4900, and of RF-2 from 5900 to 24,900 as growth rates increased from 0.3 to 2.4 doublings per hour . The cellular concentration of the release factors, and therefore efficient termination of protein synthesis is maintained by the increased expression of both RFs as growth rate increases . The expression of both release factors RF-1 and RF-2 is co-ordinated with that of the rest of the translational apparatus, although the increases are less for RF than that for the ribosomes under the same conditions . A significant proportion of the RF pool was found associated with the ribosome fraction . The percentage of ribosomes containing an RF molecule increased from 21 to 33% as the translational rate increased over the growth rate range . Since the cellular concentration of the release factors and their specific activity does not vary significantly with growth rate, this can not provide for an increase in the rate at any of the steps of termination . The postulated strong stop signals, UAAU and UAAG, in genes that are highly expressed at fast growth rates, may result in an increase in the termination rate as a consequence of increased efficiency of decoding by RFs. J Biol Chem, 1994 May 6, 269(18), 13609 - 13 Beta-turn formation in the processing region is important for efficient maturation of Escherichia coli maltose-binding protein by signal peptidase I in vivo; Barkocy-Gallagher GA et al.; Signal peptidase I (also called leader peptidase) is the endopeptidase that removes the signal peptides of most secreted proteins during or after translocation in Escherichia coli . Precursor recognition is contingent in part on the presence of small, uncharged residues in the -3 and -1 positions relative to the cleavage site, and may also depend on the structure of the processing region . Most precursor processing regions include residues likely to form a beta-turn . Mutations were introduced into the processing region of maltose-binding protein (MBP) that altered the prediction of beta-turn formation in this region . MBP species with a decreased probability of beta-turn formation were processed slowly or not at all, whereas MBP species with an increased probability of beta-turn formation were processed efficiently . Mutations altering the prediction of beta-turn formation in the MBP processing region were also made in cis to a proline in the +1 position . Cleavage at the normal processing site is blocked by proline in the +1 position; this MBP species, MBP27-P, inhibits processing of other proteins by signal peptidase I . Decreasing the probability of beta-turn formation in the processing region of MBP27-P eliminated the inhibition of signal peptidase I, and these MBP27-P derivatives remained unprocessed, suggesting that the formation of a beta-turn in the MBP processing region was necessary for recognition by signal peptidase I . Increasing the probability of beta-turn formation in cis to proline at +1 in MBP did not alter recognition of the protein by the processing enzyme . The results presented here are consistent with the hypothesis that the efficiency of recognition and processing by signal peptidase I is increased by the formation of a beta-turn in the processing region of the MBP signal peptide. J Biol Chem, 1994 May 6, 269(18), 13502 - 10 Overexpression and mutagenesis of the cDNA for rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase . Role of cysteines and tyrosines in catalysis; Pawlowski JE et al.; The overexpression and purification of recombinant rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase (3 alpha-HSD; EC 1.1.1.50) in Escherichia coli are described . The properties of the homogeneous recombinant 3 alpha-HSD (r3 alpha-HSD) confirm that a single polypeptide can function as a HSD, as a dihydrodiol dehydrogenase, and as an aromatic aldehyde, ketone, and quinone reductase . Cys-170, Cys-242, and Cys-217, implicated by bromoacetoxysteroid affinity-labeling agents as points of contact for the C-3, C-11, and C-17 positions of steroid ligands, were mutated to alanines . Unexpectedly, the homogeneous C170A and C242A mutants were kinetically similar to wild-type r3 alpha-HSD . By contrast, the C217A mutant gave Km values that were 4-fold higher for androstanedione and 2-fold higher for NADH . Inspection of the recently solved crystal structure of rat liver 3 alpha-HSD (Hoog, S . S., Pawlowski, J . E., Alzari, P . M., Penning, T . M., and Lewis, M . (1994) Proc . Natl . Acad . Sci . U . S . A . 91, 2517-2521) places Cys-170 and Cys-242 on the periphery of an alpha/beta-barrel so that they cannot be involved in catalysis of steroid recognition . This demonstrates that bromoacetoxysteroid affinity-labeling agents may provide misleading information regarding the topography of steroid hormone binding sites . When NADPH was modeled into the crystal structure of 3 alpha-HSD, Tyr-55 was implicated as the general acid, since it is in close proximity to the C-4 position of the nicotinamide ring and could polarize the substrate carbonyl . In support of this model, the purified Y55F mutant was found to be catalytically inactive, but still formed an E-NADPH complex (measured by fluorescence titration) and an E-NADH-testosterone complex (measured by equilibrium dialysis) . The ability of the Y55F mutant to form binary and ternary complexes, but not aid in hydride transfer, is consistent with Tyr-55 acting as the general acid . 3 alpha-HSD is a member of the aldo-keto reductase superfamily, and Tyr-55 is invariant in members of this family where it may perform a similar function . Tyr-205 is present in a pentapeptide sequence that is conserved in HSDs that belong to the short-chain alcohol dehydrogenase family and has been implicated as the general acid within these enzymes . The Y205F mutant was found to be kinetically similar to wild-type r3 alpha-HSD.(ABSTRACT TRUNCATED AT 400 WORDS) J Biol Chem, 1994 May 6, 269(18), 13465 - 71 ATP hydrolysis-driven structural changes in the gamma-subunit of Escherichia coli ATPase monitored by fluorescence from probes bound at introduced cysteine residues; Turina P et al.; Four mutants of the Escherichia coli F1ATPase, gamma S8-C, gamma T106-C, gamma S179-C, and gamma V286-C, which have a cysteine introduced at different sites in the gamma-subunit by site-directed mutagenesis, were reacted with the fluorescent reagent N-(4-7-(diethylamino)4-methylcoumarin-3-yl)-maleimide (CM) under conditions that selectively label the introduced Cys residue . With each mutant the effect of nucleotide binding on the fluorescence of the probe has been monitored . The results obtained with the mutants gamma S8-C and gamma T106-C are similar . In both cases, there was a spectral shift and change in fluorescence intensity on adding AMP.PNP or ATP to enzyme emptied of nucleotide from catalytic sites, while no change in the fluorescence spectrum was observed upon adding ADP . The fluorescence spectral changes obtained with ATP were transient and involved an initial rapid fluorescence enhancement followed by a subsequent fluorescence quenching . The kinetics of these ATP-induced fluorescence changes and the kinetics of ATP hydrolysis as monitored by the rates of ATP binding and of Pi formation were the same under conditions of unisite catalysis, indicating that the conformational changes in the gamma-subunit being measured by the fluorescent probe are driven by ATP hydrolysis in catalytic sites . No nucleotide-dependent fluorescence changes were observed with CM bound at a Cys at position 179 . Nucleotide-dependent changes in fluorescence were seen with CM bound at position 286, but these appear to reflect structural changes due to binding of ADP or ATP in noncatalytic sites . The fluorescence changes observed in mutants gamma S8-C and gamma T106-C were not seen in subunit epsilon-free E . coli F1ATPase, although such enzyme preparations are highly active ATPases . We conclude that the structural changes monitored by the fluorescent probe are a part of the conformational coupling, whereby catalytic site events are linked to proton channeling. J Biol Chem, 1994 May 6, 269(18), 13375 - 81 Construction and expression of a flavocytochrome b5 chimera; Quinn GB et al.; A gene has been constructed coding for a chimeric flavocytochrome b5 protein that comprises the soluble domain of rat hepatic cytochrome b5 as the NH2-terminal portion of the chimera and the flavin-containing domain of spinach assimilatory NADH:nitrate reductase as the C terminus . The chimeric protein has been expressed in Escherichia coli and purified to homogeneity using a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP-agarose, anion-exchange chromatography, and fast protein liquid chromatography gel filtration with an estimated molecular mass of 43 kDa from polyacrylamide gel electrophoresis . Visible and fluorescence spectroscopy indicated the purified protein contained both a b-type cytochrome and FAD prosthetic groups . The chimeric hemoflavoprotein immunologically cross-reacted with both anti-rat cytochrome b5 and anti-spinach nitrate reductase polyclonal antibodies, indicating the conservation of antigenic determinants from both native domains . NH2-terminal and internal amino acid sequencing of the native and CNBr-digested protein confirmed the presence of peptides derived from both the heme- and flavin-binding portions of the sequence which were identical to the deduced amino acid sequence . The chimera exhibited both NADH: ferricyanide reductase and NADH:cytochrome c reductase activities with Vmax values of 88 and 37 mumol of NADH consumed per min/nmol of heme (mu = 0.05 and pH 7.0) and Km values of 2.1, 32, and 1.4 microM for NADH, ferricyanide, and cytochrome c, respectively . This work represents the first successful bacterial expression of a mammalian-plant chimeric metalloflavoprotein . The chimera exhibited properties extremely similar to those of the native cytochrome b5 heme and spinach nitrate reductase FAD components. J Biol Chem, 1994 May 6, 269(18), 13259 - 65 Role of lysine 758 of Escherichia coli DNA polymerase I as assessed by site-directed mutagenesis; Pandey VN et al.; Lys-758 of Escherichia coli DNA polymerase I has been implicated in the process of substrate dNTP binding (Basu, A., and Modak, M . J . (1987) Biochemistry 26, 1704-1709) . To confirm and define the role of Lys-758 in the catalytic mechanism, we carried out site-directed mutagenesis of this residue . Catalytic activity of the purified mutant enzymes, K758A and K758R, showed severe reduction in the polymerase activity but little difference in the 3'-->5' exonuclease activity . Most interestingly, the catalytic ability of both mutant enzymes was maximally affected (300-1,000-fold decrease in kcat) with poly(dA).(dT)15 as template-primer (TP), whereas the ability to use poly(dC) templates decreased by only 20-fold in K758A and remained nearly unchanged with K758R . Kinetic characterization showed that Km(dNTP) increased moderately only with K758A, whereas Kd(TP) remained unchanged for both the mutants . However, binary complex formation between K758A and dNTP, but not between K758A and TP, was severely reduced . Analysis of the processive mode of DNA synthesis by K758A indicated that the mutant enzyme pauses at dA bases but does not dissociate from TP, suggesting a defect in its translocation ability . Thus, Lys-758 in polymerase I appears to participate in two distinct functions: (a) it facilitates the dNTP binding, and (b) it is required for the translocation along the template polynucleotide. J Biol Chem, 1994 May 6, 269(18), 13179 - 84 A mismatch bubble in double-stranded DNA suffices to direct precise transcription initiation by Escherichia coli RNA polymerase; Aiyar SE et al.; Formation of a transcription-competent "open" complex between Escherichia coli RNA polymerase and a promoter, where base pairing is disrupted over a region of 12 base pairs including the start site of transcription, is a complex process involving at least three steps: recognition of specific DNA sequences, a conformational change in RNA polymerase, and DNA melting . By using synthetic constructs devoid of promoter-specific sequences, we show here that a mismatch bubble of 12 base pairs suffices to direct transcription initiation in divergent directions from its edges, reflecting the absence of polarity determinants for RNA polymerase binding . Bubble transcription is obtained with both core polymerase and holoenzyme, but efficient formation of heparin-resistant initiation complexes requires the sigma (specificity) factor . Based on these results it is likely that the sigma factor blocks access of the heparin to a site on the holoenzyme. J Biol Chem, 1994 May 6, 269(18), 13061 - 4 Bridging the gap . Joining of nonhomologous ends by DNA polymerases; King JS et al.; DNA double strand breaks with noncomplementary ends can be joined by mechanisms of nonhomologous recombination . In some systems a DNA end with a 3'-protruding single strand (PSS), which does not have a recessed 3'-hydroxyl that can allow for fill-in DNA synthesis, is joined to a blunt end with preservation of the 3'-PSS . It has been proposed that this process occurs via single strand ligation or is facilitated by an alignment protein . We were interested in testing the hypothesis that a DNA polymerase could function as this putative alignment protein . To characterize polymerase activities in this type of reaction, we incubated short double-stranded oligonucleotides that had an excess of one of the strands with an exonuclease-free Klenow fragment of Escherichia coli polymerase I, Taq DNA polymerase from Thermus aquaticus, or an exonuclease-free Stoffel fragment of Taq DNA polymerase . Products were analyzed by using biotinylated oligonucleotides separated by denaturing polyacrylamide gel electrophoresis . To further assess the effect of DNA polymerases on the joining of 3'-PSS ends to blunt ends, we incubated linear plasmid DNA with the polymerases and subjected the DNA to Southern blot and sequence analysis . We determined that these DNA polymerases can use a 3'-PSS end as a template after priming off the 3'-hydroxyl of a blunt end . This implies that the joining of noncomplementary ends in eukaryotic cells could proceed by a similar mechanism. J Chromatogr A, 1994 May 6, 668(1), 129 - 37 Separation and purification of recombinant proteins from Escherichia coli with aqueous two-phase systems; Asenjo JA et al.; The partition of the protein thaumatin in the presence of Escherichia coli contaminant proteins has been studied . Extraction of thaumatin was followed by back-extraction of the product into a new phosphate phase and also back extraction combined with recycle of the top polyethylene glycol (PEG) phase . When partitioned in the absence or presence of insoluble cell debris (whole cell homogenate) little effect on the partitioning of thaumatin or the soluble E . coli protein was observed . A back extraction step that allowed for dilution of the NaCl was successful in extracting thaumatin back into a heavy phosphate phase . The PEG top phase was recycled to the first extraction stage . The stability ratio (tie-line length) had an effect on the average partition coefficient (Kapp) of E . coli soluble proteins in PEG-phosphate systems but in PEG sulphate systems Kapp was independent of this ratio . An increase in NaCl resulted in an increase in Kapp but this was always below 1 . A mathematical model that describes the continuous steady-state operation of extraction and back extraction has been developed; it is based on steady state mass balances of the main components and phase equilibrium data and was successfully used to simulate the extraction and back extraction processes. J Chromatogr A, 1994 May 6, 668(1), 117 - 20 Liquid-liquid extraction of a recombinant protein, cytochrome b5, with aqueous two-phase systems of polyethylene glycol and potassium phosphate salts; Sarmento MJ et al.; The partitioning of cytochrome b5 in aqueous two-phase systems of polyethylene glycol (PEG) and potassium phosphate salts was investigated . Cytochrome b5 partitioning is enhanced with decreasing polymer molecular mass and with increasing tieline length and pH . The effect of cytochrome b5 mutation, by substitution of the glutamic acid at positions 56 and 92 of the polypeptide chain by a lysine, on protein partitioning was also studied . Partitioning of cytochrome b5 mutants shows the same dependence on tieline length and pH, following the order cytochrome b5 > mutant 56 > mutant 92. J Biol Chem, 1994 May 6, 269(18), 13670 - 9 Antibodies against F1-ATPase alpha-subunit recognize mitochondrial chaperones . Evidence for an evolutionary relationship between chaperonin and ATPase protein families; Alconada A et al.; Antibodies raised against two synthetic peptides from rat liver F1-ATPase alpha-subunit sequence recognized two main heat-shock proteins from Drosophila (p71 and p56) and rat liver (p74 and p54) cells . One of the antisera showed a 20-fold higher reactivity toward Escherichia coli GroEL chaperonin than toward the alpha-subunit purified from Drosophila . Indirect immunofluorescence microscopy and subcellular fractionation experiments located both mammalian heat-shock proteins in the mitochondria . The recent findings of functional homology between chaperonins and alpha-subunits, together with the unsuspected immunological reactivity of two mitochondrial molecular chaperones toward antisera derived from two different sequence motifs of the alpha-subunit, strongly argue in favor of the existence of an evolutionary relationship between chaperonins and alpha-subunits . The complete sequence alignment of F-type ATPase alpha-subunits and chaperonins revealed the existence of eleven most conserved regions (approximately 30% of each protein sequence) with an overall amino acid identity of 20 +/- 2% and similarity of 39 +/- 4% . A search of protein data bases with three different consensus sequences derived from this alignment identified a significant proportion of proteins belonging only to these two protein families . Since the alpha-subunit protein family is evolutionary related to the other catalytic (A and beta) and regulatory (B) subunits of V- and F-type ATPases, the homology reported herein allowed us to analyze, in the chaperonin sequences, the conservation of critical residues involved in nucleotide binding . These data support the hypothesis that chaperonins and the major subunits of V- and F-type ATPases are evolutionary related. J Biol Chem, 1994 May 6, 269(18), 13629 - 36 The effect of groES on the groEL-dependent assembly of dodecameric glutamine synthetase in the presence of ATP and ADP; Fisher MT; The yields of active dodecameric glutamine synthetase (GS) are significantly increased when in vitro folding is initiated in the presence of the Escherichia coli groE chaperonins and ATP (37 degrees C) . To observe the effects of chaperonins and ATP on GS assembly, the GS assembly intermediates were separated by nondenaturing gel electrophoresis, visualized by Western analysis, and studied as a function of time . The form of GS that was initially released from groEL is monomeric . After the monomers formed dimers, active GS oligomers were assembled by the association of assembly competent dimers with higher order even-numbered oligomers until the dodecamer was formed . When ATP was added to the groEL.GS complex (no groES), a groEL.GS complex remained visible for up to 30 min after the renaturation was initiated . This slow disappearance of the groEL.GS complex is consistent with observed lags in both the GS activity regain profile and the assembly-dependent increase in GS tryptophan fluorescence . When groES was present, the addition of ATP resulted in the disappearance of observable complex at early sample times (< 2 min) . Concomitantly, the rates of the regain of GS activity and the GS-dependent increase in tryptophan fluorescence intensity showed substantial accelerations . These results indicate that groES facilitates GS assembly from groEL by inducing the rapid release of GS from groEL, which in turn increases the concentration of assembly competent GS monomers . In addition, groES can initiate renaturation of GS from the groEL.GS arrested complex in the presence of ADP . When chaperonin-dependent GS renaturation was initiated with ATP or ADP (> or = 2 mM), the rates were identical . Since ATP hydrolysis is not absolutely required, the combined binding energies of groES and ATP (or ADP) appear to be sufficient to weaken the binding affinity of groEL for GS subunits and facilitate the release and refolding of assembly competent GS monomers from groEL. Gene, 1994 May 3, 142(1), 85 - 9 Characterization of the gene encoding superoxide dismutase of Bordetella pertussis and construction of a SOD-deficient mutant; DeShazer D et al.; The Bordetella pertussis gene sodB, encoding superoxide dismutase (SOD), was cloned by complementation of an Escherichia coli sodAsodB double mutant . The nucleotide sequence of sodB predicted a 21-kDa protein with homology to manganese- and iron-containing SODs from other organisms . Examination of SOD activity on gels suggests that B . pertussis extracts have a single SOD containing Fe3+ as a prosthetic group . A SOD-deficient mutant was obtained by insertional inactivation of sodB in B . pertussis, confirming that there is only one SOD in this organism. Gene, 1994 May 3, 142(1), 61 - 6 A new T7 RNA polymerase-driven expression system induced via thermoamplification of a recombinant plasmid carrying a T7 promoter-Escherichia coli lac operator; Lebedeva MI et al.; A new temperature-regulated T7 RNA polymerase-driven transcription system has been developed . This system is based on a hybrid regulatory region: the phage T7 late promoter (PT7) linked to the Escherichia coli lac operator (Olac) {Giordano et al., Gene 84 (1989) 209-219}, which was located in an earlier obtained {Mashko et al., Gene 97 (1991) 259-266} temperature-controlled amplifiable plasmid, carrying cat under the control of PT7-Olac and, in addition, lambda major early promoter-operator regions and gene cIts857 . Plasmids of the pT7-Olac-cat-tsr series were stably maintained at a low-copy-number when grown at low temperature (28 degrees C) . In E . coli BL21(DE3), carrying the Plac-controllable T7 RNA polymerase-encoding gene, efficient repression of cat transcription was observed, that was provided by the LacI repressor and, probably, the thermolabile repressor CIts857 . At low and moderate temperatures (28/37 degrees C), this 'cooperative' repression was so tight that cat expression was not observed in the cells carrying PT7-Olac on the plasmids, even after IPTG-inducible T7 RNA polymerase biosynthesis . As a result of the thermo-amplification of the recombinant plasmids and temperature-inactivation of CIts857, expression of the T7 RNA polymerase-encoding gene was derepressed due to the titration of LacI by the increasing copies of Olac which in turn, led to the highly efficient T7 RNA polymerase-driven accumulation of CAT in the cells. Gene, 1994 May 3, 142(1), 17 - 22 Characterization of the Escherichia coli gcv operon; Stauffer LT et al.; The nucleotide (nt) sequence of the Escherichia coli gcvP gene was determined . The polypeptide deduced from the DNA sequence has an M(r) of 104,375 (957 amino acids) . In a minicell system, gcvP encodes a polypeptide that migrates at 93.3 kDa on sodium dodecyl sulfate-polyacrylamide gels . After the coding region, there is a 39-nt sequence followed by a T-rich sequence within which transcription appears to terminate . This region is preceded by a G/C-rich sequence that could form a stable stem-loop structure once transcribed, and is characteristic of Rho-independent transcription terminators . A Northern analysis identified an approx . 4700-nt RNA molecule, large enough to encode the T-, H-and P-proteins of the glycine cleavage enzyme complex . Analyses of gcvP::lacZ fusions with and without stop codons in gcvT, the first gene in the operon, confirmed gcvT, gcvH and gcvP lie in an operon . RNA slot blot analyses indicated that induction of gcv by glycine, and PurR-mediated repression of gcv occur at the level of transcription. Gene, 1994 May 3, 142(1), 147 - 51 Overproduction and purification of biologically active native fungal alpha-sarcin in Escherichia coli; Lacadena J et al.; An efficient system was developed to produce, in Escherichia coli, large amounts of native alpha-sarcin (alpha Sar), a cytotoxin from the mold Aspergillus giganteus . The protein has been purified to homogeneity with a yield of 1.5 micrograms/ml of original culture . The constructed expression vector (pINPG alpha S) is based on the synthesis of a fusion protein between alpha Sar and a modified version of the OmpA signal peptide . This peptide seems to favour the postranslational processing of the fusion protein . The purified recombinant alpha-sarcin (re-alpha Sar) is structurally identical to the mature fungal protein according to the following criteria: N-terminal amino acid (aa) sequence, aa composition, electrophoretic mobility, chromatographic behaviour, immunoreactivity and spectroscopic features . Indeed, the recombinant protein recovered is completely functional, since it cleaves, in vitro, eukaryotic rRNA and it is able to interact with phospholipid vesicles with the same specificity as the native fungal alpha Sar. Gene, 1994 May 3, 142(1), 1 - 8 CAATTG-specific restriction-modification munI genes from Mycoplasma: sequence similarities between R.MunI and R.EcoRI; Siksnys V et al.; The genes coding for the MunI restriction-modification (R-M) system, which recognize the sequence 5'-CAATTG, have been cloned and expressed in Escherichia coli, and their nucleotide sequences have been determined . The restriction endonuclease (ENase; R.MunI) is encoded by an open reading frame (ORF) of 606 bp, and a 699-bp ORF codes for the methyltransferase (MTase) . The two genes are transcribed divergently from a 355-bp region . The gene encoding the ENase is preceded by a short co-linear ORF of 222 bp . The deduced amino acid (aa) sequence of this short ORF (SORF) closely resembles the sequences of a family of regulatory proteins that are associated with other type-II R-M systems . Comparative analysis of the deduced aa sequence of R.MunI revealed several regions of similarity to the EcoRI and RsrI ENases that recognize the GAATTC sequence . The similar mode of interaction of MunI, EcoRI and RsrI with the tetranucleotide AATT, common to the recognition sequences of these ENases, was suggested. Biochemistry, 1994 May 3, 33(17), 5335 - 46 Kinetic analysis of the coding properties of O6-methylguanine in DNA: the crucial role of the conformation of the phosphodiester bond; Tan HB et al.; Production by N-nitroso compounds of O6-alkylguanine (O6-alkylG) in DNA directs the misincorporation of thymine during DNA replication, leading to G:C to A:T transition mutations, despite the fact that DNA containing O6-alkylG:T base pairs is less stable than that containing O6-alkylG:C pairs . We have examined the kinetics of incorporation by Klenow fragment (KF) of Escherichia coli DNA polymerase I of thymine (T) and of cytosine (C) opposite O6-MeG in the template DNA strand . Both T and C were incorporated opposite O6-MeG much slower than nucleotides forming regular A:T or G:C base pairs . Using various concentrations of dTTP, dCTP, or their phosphorothioate (Sp)-dNTP alpha S analogues, or a mixture of dTTP and dCTP, the progress of incorporation of a single nucleotide in a single catalytic cycle of a preformed KF-DNA complex was measured (pre-steady-state kinetics) . The results were consistent with the kinetic scheme (Kuchta, R . D., Benkovic, P., & Benkovic, S . J . (1988) Biochemistry 27, 6716-6725): (1) binding of dNTP to polymerase-DNA; (2) conformational change in polymerase; (3) formation of phosphodiester between the dNTP and the 3'-OH of the primer; (4) conformational change of polymerase; (5) release of pyrophosphate . The results were analyzed mathematically to identify the steps at which the rate constants differ significantly between the incorporation of T and C . The only significant difference was the 5-fold difference in the rates of formation of the phosphodiester bond (for dTTP, kforward = 3.9 s-1 and kback = 1.9 s-1; for dCTP, kforward = 0.7 s-1 and kback = 0.9 s-1) . These pre-steady-state progress curves were biphasic with a rapid initial burst followed by an apparently steady-state rise . Deconvolution of these curves gave direct evidence for the importance of the conformational change after polymerization by showing that the curves represented the sum of the rapid accumulation of the product of step 3 followed by the slow conversion of that to the product of step 5 (because of the rapidity of the release of pyrophosphate there was no significant accumulation of the product of step 4) . The equilibrium constants for each step suggest that the greatest change in the Gibbs free energy occurs at the conformational change after polymerization and that while the formation of the phosphodiester bond to T is slightly exothermic, that to C is slightly endothermic.(ABSTRACT TRUNCATED AT 400 WORDS) Biochemistry, 1994 May 3, 33(17), 5312 - 8 Region of a conserved sequence motif in a class II tRNA synthetase needed for transfer of an activated amino acid to an RNA substrate; Shi JP et al.; The class II Escherichia coli alanine tRNA synthetase aminoacylates RNA miniduplexes, which reconstruct the acceptor end of alanine tRNA with the critical G3:U70 base pair . A benzophenone photoaffinity label attached adjacent to G3:U70 in a miniduplex substrate was previously cross-linked to a long enzyme peptide that begins at Gly161 between the class-defining motifs 2 and 3 {Musier-Forsyth, K., & Schimmel, P . (1994) Biochemistry 33, 773-779} . To identify side chains in this peptide that potentially contribute hydrogen bonding or catalytic determinants for the RNA-dependent step of the aminoacylation reaction, peptide functional side chains that are conserved among sequenced alanine enzymes (Asp, Asn, Arg, Glu, Gln, and Tyr) were individually replaced . Of the 21 mutant proteins so generated, one was identified that was not viable even though it accumulated in vivo . This Asp235-->Ala mutant enzyme is defective in the rate of transfer of the activated amino acid to the 3'-end of the RNA substrate . The conserved Asp235 is at the beginning of motif 3 . By comparison with the crystal structure of the related class II yeast aspartate tRNA synthetase complexed with tRNA(Asp) (Cavarelli et al., 1993), we suggest that D235 is not in direct contact with acceptor helix base pairs such as G3:U70 . Instead, we propose that D235 contributes to transfer-step interactions at the 3'-end of alanine tRNA . Because D235 in alanine tRNA synthetase is at the beginning of one of the conserved motifs that define class II tRNA synthetases, this region of the structure may in general be important for the transfer step. Biochemistry, 1994 May 3, 33(17), 5285 - 90 The glutamate racemase activity from Escherichia coli is regulated by peptidoglycan precursor UDP-N-acetylmuramoyl-L-alanine; Doublet P et al.; The murI gene product of Escherichia coli was recently identified as the glutamate racemase activity which catalyzes the formation of D-glutamic acid, one of the essential components of bacterial cell-wall peptidoglycan {Doublet et al . (1993) J . Bacteriol . 175, 2970-2979} . We here describe the purification to homogeneity and the kinetic properties of this enzyme . In vitro, the glutamate racemase activity shows an absolute requirement for UDP-N-acetylmuramoyl-L-alanine (UDP-MurNAc-L-Ala), the substrate of the D-glutamic acid-adding enzyme which catalyzes the subsequent step in the pathway for peptidoglycan synthesis . The affinity of the enzyme for this activator is particularly high (KD = 4 microM) and specific, since no other peptidoglycan precursor from UDP-GlcNAc to UDP-MurNAc-pentapeptide is an effector . Minor chemical modifications of the UDP-MurNAc-L-Ala molecule, such as the reduction of the uracyl moiety, suppress its activating effect . This specific in vitro requirement most likely represents the physiological mechanism which regulates the activity of the glutamate racemase in vivo . It adjusts the formation of D-glutamic acid to the requirements of peptidoglycan synthesis and avoids an excessive racemization of the intracellular pool of L-glutamic acid. Biochemistry, 1994 May 3, 33(17), 5155 - 61 Active-site mutations of the diphtheria toxin catalytic domain: role of histidine-21 in nicotinamide adenine dinucleotide binding and ADP-ribosylation of elongation factor 2; Blanke SR et al.; Diphtheria toxin (DT) has been studied as a model for understanding active-site structure and function in the ADP-ribosyltransferases . Earlier evidence suggested that histidine-21 of DT is important for the ADP-ribosylation of eukaryotic elongation factor 2 (EF-2) . We have generated substitutions of this residue by cassette mutagenesis of a synthetic gene encoding the catalytic A fragment (DTA) of DT, and have characterized purified mutant forms of this domain . Changing histidine-21 to alanine, aspartic acid, leucine, glutamine, or arginine diminished ADP-ribosylation activity by 70-fold or greater . In contrast, asparagine proved to be a functionally conservative substitution, which reduced ADP-ribosylation activity by < 3-fold . The asparagine mutant was approximately 50-fold-attenuated in NAD glycohydrolase activity, however . Dissociation constants (Kd) for NAD binding, determined by quenching of the intrinsic protein fluorescence, were 15 microM for wild-type DTA, 160 microM for the asparagine mutant, and greater than 500 microM NAD for the alanine, leucine, glutamine, and arginine mutants . These and previous results support a model of the ADP-ribosylation of EF-2 in which histidine-21 serves primarily a hydrogen-bonding function . We propose that the pi-imidazole nitrogen of His-21 hydrogen-bonds to the nicotinamide carboxamide, orienting the N-glycosidic bond of NAD for attack by the incoming nucleophile in a direct displacement mechanism, and then stabilizing the transition-state intermediate of this reaction. Biochemistry, 1994 May 3, 33(17), 4995 - 9 Flexible loop that is novel catalytic machinery in a ligase . Atomic structure and function of the loopless glutathione synthetase; Kato H et al.; The catalytic mechanism of glutathione synthetase is proposed to proceed via phosphorylation of the dipeptide substrate to yield an acyl phosphate intermediate; this intermediate is subsequently attacked by glycine, followed by loss of inorganic phosphate, leading to glutathione formation . A flexible loop (Ile226-Gly242) in Escherichia coli B glutathione synthetase is proposed to stabilize the acyl phosphate intermediate by preventing its decomposition by hydrolysis with water {Tanaka, T., Kato, H., Nishioka, T., & Oda, J . (1992) Biochemistry 31, 2259-2265; Tanaka, T., Yamaguchi, H., Kato, H., Nishioka, T., Katsube, Y., & Oda, J . (1993) Biochemistry 32, 12398-12404} . To investigate the function of the loop in the E . coli enzyme definitely, a loopless mutant in which the loop (Ile226-Arg241) was replaced with three residues of glycine was constructed . The crystal structure of the loopless mutant enzyme was essentially identical with that of the wild-type enzyme . Kinetic measurements showed that the replacement of the loop led to increases in the Km values, especially for the glycine, and a 930-fold decrease in the k0 value . Hence, the loopless mutant was 3 x 10(4) less active in terms of its specificity constant (k0/Km) for glycine than the wild-type enzyme . Moreover, the loopless mutant showed gamma-L-glutamyl-L-cysteine-dependent ATP hydrolase activity to almost the same extent as its glutathione synthetase activity . These studies support the fact that the loop enhances the recognition of glycine as well as stabilizes the acyl phosphate intermediate so that the intermediate rapidly reacts with glycine. Biochemistry, 1994 May 3, 33(17), 5275 - 84 Individual ionization constants of all the carboxyl groups in ribonuclease HI from Escherichia coli determined by NMR; Oda Y et al.; All of the individual carboxyl groups (the side-chain carboxyl groups of Asp and Glu, and the C-terminal alpha-carboxyl group) in Escherichia coli ribonuclease HI, which is an enzyme that cleaves the RNA strand of a RNA/DNA hybrid, were pH-titrated, and their ionization constants (pKa) were determined from an analysis of the pH-dependent chemical shifts of the carboxyl carbon resonances obtained from 1H-13C heteronuclear two-dimensional NMR . The pKa values in the enzyme varied widely among individual residues, for example, in the unusual pKa values for two important catalytic residues, Asp10 (pKa 6.1) and Asp70 (pKa 2.6) . Moreover, remarkable two-step titrations were observed for these carboxylates . The binding of Mg2+ ion to the enzyme, which is the cofactor necessary for catalytic activity, caused no significant change in the pKa values of the carboxyl groups, except for that of Asp10 . The variations of the pKas that were dependent on the microenvironment in the protein were theoretically reproduced to compare with the experimental results by a numerical calculation, using a continuum electrostatic model . Most of the significant pKa decreases were brought about through strong electrostatic interactions with the neighboring basic amino acids, Arg or Lys . The pKa shifts and the two-step titrations of Asp10 and -70, which are close to each other, were interpreted to be due to the neighboring effect of two functional groups, as observed in the interacting titratable groups of a dicarboxyl compound or in the active site carboxylates of lysozyme and aspartic protease . The role of Asp10 in the catalytic action is either to be the proton donor to the RNA moiety or the binding partner of the Mg2+ ion cofactor . Asp70, on the other hand, is considered to be the proton acceptor from a water molecule. Biochemistry, 1994 May 3, 33(17), 5202 - 11 Effects of DsbA on the disulfide folding of bovine pancreatic trypsin inhibitor and alpha-lactalbumin; Zapun A et al.; DsbA is a protein found in the periplasm of Escherichia coli that is required for the formation of disulfide bonds in secreted proteins . It contains only two cysteine residues, which can form reversibly a very unstable disulfide bond that has been proposed to be the oxidant that introduces disulfide bonds into secreted proteins . The present study investigates the effect of DsbA on the well-characterized disulfide-coupled refolding processes of BPTI and of alpha-lactalbumin . Disulfide-bonded DsbA in stoichiometric amounts proved to be a very potent donor of disulfide bonds to reduced BPTI but showed little catalytic activity at neutral pH in the presence of a glutathione redox buffer . In contrast to the related eukaryotic enzyme protein disulfide isomerase, DsbA did not substantially catalyze the usual intramolecular disulfide bond rearrangements of quasi-native folding intermediates of BPTI . Neither did DsbA catalyze the intramolecular rearrangements observed in the three disulfide-bonded "molten globule" form of alpha-lactalbumin at neutral pH . Thiol-disulfide exchange is normally very slow at acidic pH but occurs rapidly with DsbA; consequently, DsbA catalyzed the disulfide folding of BPTI under acidic conditions . It was then possible to detect some increase in the rates of disulfide rearrangements, but only with stoichiometric amounts of DsbA and on the hour time scale . These results suggest that the primary role of DsbA in the bacterial periplasm is to introduce disulfide bonds into newly secreted proteins. Biochemistry, 1994 May 3, 33(17), 5070 - 6 Thermodynamic studies of tyrosyl-phosphopeptide binding to the SH2 domain of p56lck; Lemmon MA et al.; The interaction between SH2 domains and tyrosine-phosphorylated proteins is essential in several cytosolic signal transduction pathways . Here we report thermodynamic studies of the interaction of the p56lck (lck) SH2 domain with several phosphopeptides, using the technique of isothermal titration calorimetry (ITC) . This is the first report of the use of ITC to study SH2 domain binding reactions . The free energy of binding of the SH2 domain of lck to a phosphopeptide corresponding to the autoregulatory C-terminus of the protein (pY505) was found to be similar to that measured for a phosphopeptide modeled on the C-terminus of the epidermal growth-factor receptor (delta G degrees approximately -7.0 kcal mol-1 at pH 6.8), although significant differences in the enthalpy and entropy were observed . Binding of a phosphopeptide modeled on the C-terminus of p185neu was weaker (delta G degrees approximately -5.4 kcal mol-1 at pH 6.8) . Lowering the pH to 5.5 reduced the binding affinity of pY505 by approximately 1 order of magnitude . We ascribe this to the protonation of a histidine side chain in the SH2 domain (H180), which is involved in a hydrogen-bonding network that optimizes the binding site geometry . No difference in affinity was observed between portions of lck corresponding to the SH3-SH2 (residues 63-228) and SH2 (residues 123-228) domains for the pY505 peptide . We also studied the effect upon pY505 peptide binding of mutations at two highly conserved arginine residues in the lck SH2 domain (R134 and R154).(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1994 May 2, 343(3), 247 - 50 Involvement of lysine-88 of spinach ferredoxin-NADP+ reductase in the interaction with ferredoxin; Aliverti A et al.; A mutant of spinach ferredoxin-NADP+ reductase, in which Lys-88 has been changed to glutamine, has been obtained by site-directed mutagenesis . The mutant enzyme was fully active as a diaphorase, but partially impaired in ferredoxin-dependent cytochrome c reductase activity . By steady-state kinetics, the Km for ferredoxin of the K88Q enzyme was found to have increased 10-fold, whereas the kcat was unaffected by the amino acid replacement . The interaction between oxidized ferredoxin and the enzyme forms was also studied by spectrofluorimetric titration: Kd values of 110 and 10 nM were determined for the mutant and wild-type proteins, respectively . These data point out the importance of a positive charge at position 88 of the reductase for the interaction with ferredoxin, confirming previous cross-linking studies. Ann N Y Acad Sci, 1994 May 2, 721, 268 - 76 An optimization study of a pH-inducible promoter system for high-level recombinant protein production in Escherichia coli; San KY et al.; The unique properties exhibited by the pH-inducible promoter system are clearly demonstrated by the plasmid construct, pSM552-545C- . Step changes of pH substantially increase the expression of beta-galactosidase . Very high expression, a level of around 40% of total cellular protein, can be achieved with superbroth . The high level of induction in rich media, typical of those commonly used to achieve high cell density, suggests the system is versatile enough to be adapted to many specific situations . The variable degree of induction by pH within the range of 8.0 and 5.5 makes possible a degree of expression control not easily accomplished with the existing systems . By precise monitoring of induction pH, a "fine tuning" of foreign gene expression and growth rate to optimum levels is possible . The effect of several operating parameters on recombinant protein production are evaluated . Our results show that operating environments play an extremely important role in achieving high recombinant protein expression levels in a dense culture . Under suboptimal conditions, as are shown in this study, only moderately high levels can be obtained . Even for suboptimal cases, an expression level of about 10 to 15% of total cellular protein while achieving an optical density higher than 25 is routinely obtained . Our results also show that a proper balance between cell growth and recombinant protein synthesis processes are critical in maintaining high expression levels in a dense culture . Any imbalance will most likely lead to more cell growth and poorer protein productivity . We have also demonstrated that reactor operating temperature can be a useful parameter to fine-tune this balance, resulting in significantly improved results. Ann N Y Acad Sci, 1994 May 2, 721, 257 - 67 Strategies in high-level expression of recombinant protein in Escherichia coli; San KY et al.; The accumulation of acetate is one of the most commonly encountered problems in attaining high levels of recombinant protein production using E . coli . Two different approaches are examined to reduce the rate of acetate formation . The effects of reduced acetate accumulation on recombinant protein production were also investigated . In the first approach, E . coli mutant strains deficient in enzymes involved in the acetate synthesis pathways were isolated and characterized . The level of specific production of beta-galactosidase by the mutant strain is three times higher than its parent strain . In another approach, metabolic engineering techniques were employed to fine-tune the central metabolic pathways to reduce the amount of acetate formation . The resulting strain, which carries the acetolactase synthase gene from B . subtilis, is successful in maintaining a very low level of acetate accumulation . The ALS-containing strain is also capable of producing higher levels of recombinant protein than its parent strain. Ann N Y Acad Sci, 1994 May 2, 721, 168 - 77 Starch-binding domain of Aspergillus glucoamylase-I . Interaction with beta-cyclodextrin and maltoheptaose; Kusnadi AR et al.; The characterization is reported of two peptide fragments (SBD106 and SBD122) containing the starch-binding domain (SBD) of Aspergillus sp . glucoamylase I . The starch-binding peptides were produced in Escherichia coli as fusion proteins of the maltose-binding protein (MBP) . SBD106 (11.9 kDa) and SBD122 (13.8 kDa) were purified from the factor Xa digest of MBP fusion proteins . The amino acid compositions were similar to those deduced from their amino acid sequences . The interactions of beta-cyclodextrin and maltoheptaose with purified SBD peptides were investigated by UV difference spectroscopy . SBD106 and SBD122 bound specifically beta-cyclodextrin with a dissociation constant (Kd) of 34 microM and 23.5 microM, respectively . Maltoheptaose binding to SBD106 and SBD122 was weaker than that of beta-cyclodextrin; dissociation constants were 0.57 and 0.50 mM, respectively . The results indicate that the intramolecular disulfide bonding is not required for the domain functioning and that O-glycosylation is not critical for the functioning of the starch-binding domain, but may affect its conformation and dynamics. Food Chem Toxicol, 1994 May, 32(5), 477 - 9 Evaluation of potential genotoxicity of stannous chloride: inactivation, filamentation and lysogenic induction of Escherichia coli; Bernardo-Filho M et al.; Because of the importance of stannous chloride in various fields of human endeavour, the potential genotoxicity of this reducing agent was evaluated by measurement of either the inactivation or the induction of SOS responses in bacteria . Escherichia coli strains used in this work (wild type, uvrA, recA, lexA and uvrA recA) were treated with stannous chloride; the wild type was found to be the most resistant and the double mutant, the most sensitive strain . As these strains present mutations on specific genes for the repair of DNA, stannous chloride would appear to be capable of inducing and/or producing lesions in DNA and, thus, can be considered to be a potential genotoxic agent . This capability was confirmed by the lysogenic induction of E . coli K12 (lambda) (Inductest) and by microscopic observation of E . coli B filamentation. Am J Physiol, 1994 May, 266(5 Pt 2), H1746 - 54 Protective effects of N-acetyl-L-cysteine in endotoxemia; Zhang H et al.; Because oxygen free radicals have been implicated in the endothelial cell damage and in the myocardial depression occurring during severe sepsis, we investigated whether N-acetyl-L-cysteine (NAC) could influence the oxygen extraction capabilities during an acute reduction in blood flow induced by cardiac tamponade after endotoxin challenge . Sixteen anesthetized, saline-infused, and ventilated dogs received Escherichia coli endotoxin (2 mg/kg) 30 min before tamponade was induced by repeated bolus injections of warm saline into the pericardial space . Thirty minutes before endotoxin administration, nine dogs received NAC (150 mg/kg, followed by a 20 mg.kg-1.h-1 infusion); the other seven dogs served as a control group . The NAC group maintained higher cardiac index, oxygen delivery (DO2), and left ventricular stroke work index, but lower systemic and pulmonary vascular resistance, than the control group . The oxygen uptake (VO2) levels at critical DO2 (DO2crit) were identical in the two groups . However, DO2crit was significantly lower in the NAC than in the control group (8.1 +/- 1.7 vs . 10.8 +/- 1.8 ml.kg-1.min-1, P < 0.01) . Critical oxygen extraction ratio and the slope of the VO2-to-DO2-dependent line were higher in the NAC than in the control group (72 +/- 14 vs . 53 +/- 15% and 0.80 vs . 0.56, respectively; both P < 0.05) . The peak lactate and the maximal tumor necrosis factor (TNF) levels were lower in the NAC than in the control group (5.2 +/- 0.4 vs . 7.6 +/- 0.4 mM, and 0.14 +/- 0.03 vs . 1.21 +/- 0.58 ng/ml, respectively; both P < 0.01) . NAC significantly increased glutathione peroxidase activity.(ABSTRACT TRUNCATED AT 250 WORDS) Am J Physiol, 1994 May, 266(5 Pt 2), F713 - 22 Interleukin-1 alpha stimulates KC synthesis in rat mesangial cells: glucocorticoids inhibit KC induction by IL-1; Feng L et al.; To assess the possible role of the production of chemokines by intrinsic glomerular cells in the generation of inflammation in glomerulonephritis, the chemokine, KC, was cloned from a rat macrophage cDNA library . Transfection of rat KC into COS-7 cells resulted in increased neutrophil chemotactic activity . The KC cDNA was expressed as a fusion protein in Escherichia coli for generation of an antibody . By using a riboprobe derived from the cDNA and the antibody, interleukin-1 (IL-1) was found to induce the expression of KC in rat mesangial cells . The induction of KC by IL-1 could be inhibited by dexamethasone (DEX) . The protein synthesis inhibitor cycloheximide reversed the DEX-mediated inhibition, which suggested that new protein synthesis was necessary for the inhibitory effect . A nuclear runoff analysis indicated that DEX inhibited the transcription of KC induced by IL-1 . The stability of KC mRNA was not decreased in the presence of DEX . Furthermore, immunoblots showed that DEX also inhibited KC expression at the level of translation . Together the inhibition of transcription and translation of the KC gene by DEX contribute to decreased KC expression in mesangial cells . The finding that mesangial cells express KC in response to proinflammatory cytokines, such as IL-1, points to a central role for the mesangial cell as a chemotactic source in glomerular inflammation. Am J Physiol, 1994 May, 266(5 Pt 1), E760 - 7 Regulation of metallothionein concentrations in rat brain: effect of glucocorticoids, zinc, copper, and endotoxin; Gasull T et al.; The effects of known inducers of liver metallothionein (MT) synthesis on MT concentrations in the rat brain have been determined using antibodies that are specific for MT I and II and do not cross-react with MT III . There were substantial differences in the MT concentrations in different areas of the brain . Dexamethasone increased MT levels after 24 h in the frontal cortex, cortex, medulla oblongata plus pons, midbrain, striatum, hippocampus, and cerebellum but not in the hypothalamus . Corticosterone produced similar results except in the hippocampus . Long-lasting adrenocorticotropic hormone increased MT concentrations after 12 h in midbrain and striatum but not in the liver . Adrenalectomy decreased MT concentrations after 6 days in the medulla oblongata plus pons, striatum, hippocampus, and hypothalamus but increased concentrations in the liver and kidneys; these effects were reversed by corticosterone . The role of glucocorticoids in the regulation of MT levels therefore differs between tissues and within specific areas of the brain . Injection of zinc or copper intracerebroventricularly and the use of a zinc-deficient diet increased and decreased MT levels, respectively, in some but not all brain areas . Endotoxin increased liver MT but not brain MT I levels after 8 h. Am J Physiol, 1994 May, 266(5 Pt 1), C1400 - 5 ESR spectral transition by arteriovenous cycle in nitric oxide hemoglobin of cytokine-treated rats; Kosaka H et al.; Nitric oxide (NO) generation was induced in rats by Escherichia coli lipopolysaccharide (LPS) as detected by electron spin resonance (ESR) signals of NO hemoglobin (HbNO) . However, there were inconsistencies in ESR spectral shape among them . We have therefore carried out a systematic study to clarify the in vivo spectral changes . First, the spectra of the alpha-NO heme species had the distinct three-line hyperfine structure in venous blood but not in arterial blood in all rats treated with tumor necrosis factor, interleukin-1, and/or LPS, and methemoglobin was not detected at the g = 6 (high-spin methemoglobin) region . Second, when the treated rats died, the three-line hyperfine structure was very distinct even in arterial blood . Third, even if HbNO was formed by injection of nitrite to rats, the three-line hyperfine structure of HbNO in venous blood was more marked than that in arterial blood, independent of the appearance of the methemoglobin signal . Fourth, an ex vivo study using whole blood demonstrated that the three-line hyperfine structure intensified lineally when O2 saturation of hemoglobin decreased but disappeared on reoxygenation of hemoglobin . These results directly demonstrate in vivo quaternary structural transition of the hemoglobin tetramer from the high-affinity state in the arterial cycle to the low-affinity state in the venous cycle . The transition makes the diverse ESR spectra of HbNO in vivo. Carcinogenesis, 1994 May, 15(5), 889 - 99 Mutations induced by aromatic amine DNA adducts in pBR322; Melchior WB Jr et al.; A 276 bp region from the tetracycline resistance gene of the plasmid pBR322 was modified with 2-acetylaminofluorene (AAF), 2-aminofluorene (AF), 4-aminobiphenyl (ABP), N'-acetylbenzidine or 1-aminopyrene (AP) in order to determine the effect of adduct structure upon mutation induction . Each modification reaction gave one major adduct and these adducts had chromatographic properties, as determined by 32P-postlabeling, identical to those in which substitution had occurred at C8 of deoxyguanosine through the amine or amide nitrogen . The types and distribution of mutations were then characterized following introduction of the modified plasmids into SOS-induced Escherichia coli using Hanahan et al.'s procedure (Methods Enzymol., 204, 63-113, 1991) . With AAF-modified plasmid, 60% of the mutations were deletions or additions, and these were detected primarily at NarI sites or in repetitive G sequences . Modification with AF gave -G deletions, primarily in runs of Gs, and base substitution mutations, which were mainly G to T transversions . Substitution with ABP or N'-acetylbenzidine resulted in G to T and G to C transversions, the latter being a mutation not detected with AF; in addition, -G deletions were detected at only very low frequency . AP modification gave both -G frameshift and base substitution mutations, of which G to T transversions predominated . A comparison of the mutation frequencies per adduct indicated that the mutagenic efficiencies of the adducts decreased in the order AP > AF > AAF approximately ABP approximately N'-acetylbenzidine . AAF- and ABP-modified pBR322 were also introduced with a CaCl2 method . The mutation frequency per adduct increased with this transformation procedure, and this appeared to be a reflection of a greater percentage of frameshift mutations . These data indicate that a series of structurally related aromatic amines will induce both base substitution and frameshift mutations when incorporated into pBR322, but that frameshift mutations occur almost exclusively with the planar derivatives . Furthermore, the ability to induce frameshift mutations increases the mutagenic efficiency of an adduct. Carcinogenesis, 1994 May, 15(5), 1013 - 6 The stimulatory effect of nickel chloride on DNA replication in human HeLa cells and Escherichia coli; Chin YE et al.; An investigation was undertaken to study DNA replication in cultured human HeLa cells and Escherichia coli in response to nickel chloride (NiCl2) . Treatment with NiCl2 increased both the rate of DNA replication and total cell number in HeLa cells and E . coli in a time- and concentration-dependent manner . The maximum stimulation of thymidine uptake into DNA was observed with 0.125-0.25 mM NiCl2 for both cell types . In studies of DNA replication using a crude HeLa cellular extract, NiCl2 at concentrations below 0.125 mM also induced a stimulation over the background of MgCl2-dependent {3H}dTMP incorporation into activated calf thymus DNA . However, a similar stimulatory effect from NiCl2 was not observed with either purified HeLa DNA polymerase alpha or E.coli DNA polymerase I Klenow fragment . In the absence of Mg2+, the low response of either DNA polymerase alpha or Klenow fragment to stimulation by Ni2+ was thought to be enhanced by the presence of Ni(2+)-binding proteins presented in the crude HeLa cell extract. Biochem J, 1994 May 1, 299 ( Pt 3), 637 - 44 Threonine synthesis from homoserine as a selectable marker in mammalian cells; Rees WD et al.; The plasmid pSVthrBC expresses the Escherichia coli thrB (homoserine kinase) and thrC (threonine synthase) genes in mouse cells and enables them to synthesize threonine from homoserine . After transfection with pSVthrBC and culture in medium containing homoserine, only cells that have incorporated pSVthrBC survive . Homoserine at concentrations greater than 1 mM is toxic to mammalian cells . Mouse cells selected from medium containing 5 mM homoserine had incorporated 20-100 copies of the plasmid per cell and had homoserine kinase activities of 0.001-0.012 nmol/min per mg of protein per copy . Cells selected from medium containing 10 mM homoserine had incorporated one or two copies of the plasmid per cell and had homoserine kinase activities of 0.06-0.39 nmol/min per mg of protein per copy . By using high concentrations of homoserine, it is possible to use pSVthrBC to select and isolate cell lines that have one or two copies of the plasmid incorporated into an active region of chromatin . CHO and HeLa cells have also been successfully transfected with pSVthrBC . COS-7 cells are naturally resistant to homoserine as they are able to metabolize homoserine. J Cell Biol, 1994 May, 125(4), 843 - 52 Direct interaction between yeast spindle pole body components: Kar1p is required for Cdc31p localization to the spindle pole body; Biggins S et al.; The Saccharomyces cerevisiae genes KAR1 and CDC31 are required for the initial stages of spindle pole body (SPB) duplication in yeast . The Cdc31 protein is most related to caltractin/centrin, a calcium-binding protein present in microtubule organizing centers in many organisms . Because of a variety of genetic interactions between CDC31 and KAR1 (Vallen, E . A., W . Ho . M . Winey, and M . D . Rose . 1994 . Genetics . In press), we wanted to determine whether Cdc31p and Kar1p physically interact . Cdc31p was expressed and purified from Escherichia coli and active for binding calcium . Using a protein blotting technique, Cdc31p bound to Kar1p in vitro via an essential domain in Kar1p required for SPB duplication (Vallen, E . A., M . A . Hiller, T . Y . Scherson, and M . D . Rose . 1992a . J . Cell Biol . 117:1277-1287) . By immunofluorescence microscopy, we determined that the interaction also occurs in vivo . Cdc31p was localized to the SPB in wild-type cells but was mislocalized in a kar1 mutant strain . In a kar1 mutant containing a dominant CDC31 suppressor, Cdc31p was again localized to the SPB . Furthermore, the localization of Cdc31p to the SPB was affected by the overexpression of Kar1p-beta-galactosidase hybrids . Based on these data, we propose that the essential function of Kar1p is to localize Cdc31p to the SPB, and that this interaction is normally required for SPB duplication. J Bacteriol, 1994 May, 176(10), 3069 - 71 The presence of linoleic acid in Escherichia coli cannot be confirmed; Cronan JE Jr et al.; Escherichia coli was recently reported to accumulate significant quantities of linoleic acid in stationary phase (H . Rabinowitch, D . D . Sklan, D . H . Chace, R . D . Stevens, and I . Fridovich, J . Bacteriol . 175:5324-5328, 1993) . Since this finding would have considerable impact on the biochemical mechanisms of type II fatty acid synthases, we have attempted to confirm this observation . We found no evidence for the accumulation of linoleic acid in late-stationary-phase cultures of E . coli and conclude that the results of Rabinowitch et al . are artifactual. J Bacteriol, 1994 May, 176(10), 3033 - 9 Enhancement of in vitro transcription by addition of cloned, overexpressed major sigma factor of Chlamydia psittaci 6BC; Douglas AL et al.; Obligate parasitic bacteria of the genus Chlamydia possess a developmental cycle that takes place entirely within eucaryotic host cells . Because standard methods of genetic analysis are not available for chlamydiae, an in vitro transcription system has been developed to elucidate the mechanisms by which chlamydiae regulate gene expression . The in vitro system is specific for chlamydial promoters but is inefficient, presumably because the RNA polymerase is not saturated with sigma factor . Therefore, we prepared recombinant Chlamydia psittaci 6BC major sigma factor to enhance transcription in the in vitro system . The gene encoding the major sigma factor (sigA) was identified by using an rpoD box oligonucleotide and was subsequently cloned and sequenced . It was found to encode a potential 571-amino-acid protein (sigma 66) that is greater than 90% identical to the previously identified major sigma factors from the L2 and MoPn strains of Chlamydia trachomatis . sigA was recloned into a T7 RNA polymerase expression system to produce large quantities of sigma 66 in Escherichia coli . Overexpressed sigma 66 was identified by immunoblot by using monoclonal antibodies 2G10 (reactive) and 2F8 (nonreactive) generated against E . coli sigma 70 . After purification by polyacrylamide gel electrophoresis, the recombinant protein was found to stimulate, by 10-fold or more, promoter-specific in vitro transcription by C . psittaci 6BC and C . trachomatis L2 RNA polymerases . Transcription was dependent on added chlamydial sigma 66, rather than on potentially contaminating E . coli sigma 70 or other fortuitous activators, since the monoclonal antibody 2G10, and not 2F8, inhibited transcription initiation . Recombinant omega(66) had no effect on transcription by E . coli core polymerase . The addition of recombinant omega(66) to the in vitro system should be useful for distinguishing omega(66)-dependent transcription of developmentally regulated chlamydial genes from omega(66)-independent transcription. J Bacteriol, 1994 May, 176(10), 2970 - 5 Cloning, nucleotide sequence, and expression of the Escherichia coli gene encoding carnitine dehydratase; Eichler K et al.; Carnitine dehydratase from Escherichia coli O44 K74 is an inducible enzyme detectable in cells grown anaerobically in the presence of L-(-)-carnitine or crotonobetaine . The purified enzyme catalyzes the dehydration of L-(-)-carnitine to crotonobetaine (H . Jung, K . Jung, and H.-P . Kleber, Biochim . Biophys . Acta 1003:270-276, 1989) . The caiB gene, encoding carnitine dehydratase, was isolated by oligonucleotide screening from a genomic library of E . coli O44 K74 . The caiB gene is 1,215 bp long, and it encodes a protein of 405 amino acids with a predicted M(r) of 45,074 . The identity of the gene product was first assessed by its comigration in sodium dodecyl sulfate-polyacrylamide gels with the purified enzyme after overexpression in the pT7 system and by its enzymatic activity . Moreover, the N-terminal amino acid sequence of the purified protein was found to be identical to that predicted from the gene sequence . Northern (RNA) analysis showed that caiB is likely to be cotranscribed with at least one other gene . This other gene could be the gene encoding a 47-kDa protein, which was overexpressed upstream of caiB. J Bacteriol, 1994 May, 176(10), 2906 - 13 Maturation pathway of Escherichia coli heat-stable enterotoxin I: requirement of DsbA for disulfide bond formation; Yamanaka H et al.; The Escherichia coli heat-stable enterotoxin STp is synthesized as a precursor consisting of pre, pro and mature regions . Mature STp is released into the culture supernatant and is composed of 18-amino-acid resides which contain three intramolecular disulfide bonds . The involvement of DsbA in the formation of the disulfide bonds of STp was examined in this study . A dsbA mutant was transformed with a plasmid harboring the STp gene, and the ST activity was significantly lower than that of the parent strain harboring the same plasmid . Furthermore, purified DsbA induced the conversion of synthetic STp peptide (inactive form) to the active form and increased the ST activity of the culture supernatant derived from the dsbA transformants . These results showed that DsbA directly catalyzes the formation of the disulfide bonds of STp . DsbA is located in periplasmic space, where STp is released as an intermediate form consisting of pro and mature regions . To examine the effect of the pro region on the action of DsbA, we replaced the cysteine residue at position 39 and tested the effect in vivo . The substitution caused a significant decrease of ST activity in the culture supernatant, the accumulation of inactive ST in periplasmic space, and an alteration in the cleavage site of the intermediate of STp . We conclude that Cys-39 is important for recognition by the processing enzymes required for the maturation of STp. J Bacteriol, 1994 May, 176(10), 2885 - 91 In vitro transcriptional activation of the phage Mu mom promoter by C protein; Gindlesperger TL et al.; The phage Mu gene C encodes a 16.5-kDa site-specific DNA-binding protein that functions as a trans-activator of the four phage "late" operons, including mom . We have overexpressed and purified C and used it for DNase I footprinting and transcription analyses in vitro . The footprinting results are summarized as follows . (i) As shown previously (V . Balke, V . Nagaraja, T . Gindlesperger, and S . Hattman, Nucleic Acids Res . 12:2777-2784, 1992) in vivo, Escherichia coli RNA polymerase (RNAP) bound the wild-type (wt) mom promoter at a site slightly upstream from the functionally active site bound on the C-independent tin7 mutant promoter . (ii) In the presence of C, however, RNAP bound the wt promoter at the same site as tin7 . (iii) C and RNAP were both bound by the mom promoter at overlapping sites, indicating that they were probably on different faces of the DNA helix . The minicircle system of Choy and Adhya (H . E . Choy and S . Adhya, Proc . Natl . Acad . Sci . USA 90:472-476, 1993) was used to compare transcription in vitro from the wt and tin7 promoters . This analysis showed the following . (i) Few full-length transcripts were observed from the wt promoter in the absence of C, but addition of increasing amounts of C greatly stimulated transcription . (ii) RNA was transcribed from the tin7 promoter in the absence of C, but addition of C had a small stimulatory effect . (iii) Transcription from linearized minicircles or restriction fragment templates was greatly reduced (although still stimulated by C) with both the wt and tin7 promoters . These results show that C alone is capable of activating rightward transcription in vitro by promoting RNAP binding at a functionally active site . Additionally, DNA topology plays an important role in transcriptional activation in vitro. J Bacteriol, 1994 May, 176(10), 2869 - 76 Sequencing, chromosomal inactivation, and functional expression in Escherichia coli of ppsR, a gene which represses carotenoid and bacteriochlorophyll synthesis in Rhodobacter sphaeroides; Penfold RJ et al.; Sequencing of a DNA fragment that causes trans suppression of bacteriochlorophyll and carotenoid levels in Rhodobacter sphaeroides revealed two genes: orf-192 and ppsR . The ppsR gene alone is sufficient for photopigment suppression . Inactivation of the R . sphaeroides chromosomal copy of ppsR results in overproduction of both bacteriochlorophyll and carotenoid pigments . The deduced 464-amino-acid protein product of ppsR is homologous to the CrtJ protein of Rhodobacter capsulatus and contains a helix-turn-helix domain that is found in various DNA-binding proteins . Removal of the helix-turn-helix domain renders PpsR nonfunctional . The promoter of ppsR is located within the coding region of the upstream orf-192 gene . When this promoter is replaced by a lacZ promoter, ppsR is expressed in Escherichia coli . An R . sphaeroides DNA fragment carrying crtD', -E, and -F and bchC, -X, -Y, and -Z' exhibited putative promoter activity in E . coli . This putative promoter activity could be suppressed by PpsR in both E . coli and R . sphaeroides . These results suggest that PpsR is a transcriptional repressor . It could potentially act by binding to a putative regulatory palindrome found in the 5' flanking regions of a number of R . sphaeroides and R . capsulatus photosynthesis genes. J Bacteriol, 1994 May, 176(10), 2862 - 8 DNA sequence and characterization of GcvA, a LysR family regulatory protein for the Escherichia coli glycine cleavage enzyme system; Wilson RL et al.; The gene encoding GcvA, the trans-acting regulatory protein for the Escherichia coli glycine cleavage enzyme system, has been sequenced . The gcvA locus contains an open reading frame of 930 nucleotides that could encode a protein with a molecular mass of 34.4 kDa, consistent with the results of minicell analysis indicating that GcvA is a polypeptide of approximately 33 kDa . The deduced amino acid sequence of GcvA revealed that this protein shares similarity with the LysR family of activator proteins . The transcription start site was found to be 72 bp upstream of the presumed translation start site . A chromosomal deletion of gcvA resulted in the inability of cells to activate the expression of a gcvT-lacZ gene fusion when grown in the presence of glycine and an inability to repress gcvT-lacZ expression when grown in the presence of inosine . The regulation of gcvA was examined by constructing a gcvA-lacZ gene fusion in which beta-galactosidase synthesis is under the control of the gcvA regulatory region . Although gcvA expression appears to be autogenously regulated over a two- to threefold range, it is neither induced by glycine nor repressed by inosine. J Bacteriol, 1994 May, 176(10), 2781 - 7 Fluctuation analysis of mutations to nalidixic acid resistance in Escherichia coli; Boe L et al.; Mutations of Escherichia coli from sensitivity to nalidixic acid resistance were studied by fluctuation analysis . The mutant distributions in replicate cultures were not significantly affected either by the age of the carbon-starved preculture used for inocula or by the inoculum size . The data from 23 fluctuation tests (48 cultures each) were pooled . The mean number of mutations per culture was estimated to be 0.71 from the fraction of cultures without mutants or 0.74 and 0.77 by maximum-likelihood estimation based on the two models under consideration . When the pooled data were compared with the theoretical expectations, the fits were unsatisfactory (P < 0.005) . The lack of fit was caused mainly by too high a frequency of cultures with between 17 and 32 mutants and too high a frequency of cultures with more than 128 mutants . Possible reasons for the lack of fit and its implications with respect to estimation of mutation rates from fluctuation tests are discussed. EMBO J, 1994 May 1, 13(9), 2097 - 102 The initiation mass for DNA replication in Escherichia coli K-12 is dependent on growth rate; Wold S et al.; It is widely accepted that the initiation mass of Escherichia coli is constant and independent of growth rate, and therefore is an important parameter in the regulation of initiation of DNA replication . We have used flow cytometry to measure the initiation mass of E . coli K-12 cells as a function of growth rate . The average initiation mass was determined by two methods: (i) from a mathematical relationship between average cell mass, cell age at initiation and number of origins present in the cells, and (ii) directly from the cell mass distribution . The light scattering signal from individual cells and the protein content per cell were employed as measures of cell mass . The initiation mass was found to increase monotonically with decreasing growth rate, being 1.6 times higher (light scattering) or 2.1 times higher (protein content) at 0.3 than at 2.5 doublings per hour . We conclude that the initiation mass is dependent on growth rate . This finding indicates that the control for timing of initiation is not governed by a direct connection between mass accumulation and the molecule(s) determining initiation of replication. Neuron, 1994 May, 12(5), 1097 - 109 Regulation of Shaker K+ channel inactivation gating by the cAMP-dependent protein kinase; Drain P et al.; In response to depolarization of the membrane potential, Shaker K+ channels undergo a series of voltage-dependent conformational changes, from resting to open conformations followed by a rapid transition into a long-lived closed conformation, the N-type inactivated state . Application of phosphatases to the cytoplasmic side of Shaker channels in excised inside-out patches slows N-type inactivation gating . Subsequent application of the purified catalytic subunit of the cAMP-dependent protein kinase (PKA) and ATP reverses the effect, accelerating N-type inactivation back to its initial rapid rate . Macroscopic and single-channel experiments indicate that N-type inactivation is selectively modulated . There was little or no effect on the voltage dependence and kinetics of activation . Comparison of site-directed mutant channels shows that a C-terminal consensus site for PKA phosphorylation is responsible for the modulation . Since a cell's integrative characteristics can be determined by the rate of inactivation of its voltage-dependent channels, modulation of these rates by phosphorylation is likely to have functional consequences. J Neurosci, 1994 May, 14(5 Pt 1), 2455 - 63 Cell-specific expression from the human dopamine beta-hydroxylase promoter in transgenic mice is controlled via a combination of positive and negative regulatory elements; Hoyle GW et al.; The promoter region of the human dopamine beta-hydroxylase (DBH) gene was analyzed in transgenic mice to identify DNA sequences responsible for the tissue- and cell-specific expression of the gene . Transgenic mice were generated that carried the Escherichia coli lacZ gene under control of DBH promoter fragments between 0.6 and 5.8 kilobases (kb) in length . Sequences required for expression in adult and fetal noradrenergic neurons were located between 0.6 and 1.1 kb 5' to the DBH transcriptional start site . Sequences in this region and farther upstream also directed expression to dopaminergic and noncatecholaminergic brain neurons that was repressed by negative elements elsewhere in the gene . The results indicate that the neuron-specific expression of the DBH gene is mediated by positive regulatory elements but that negative elements are required to restrict expression to the proper subset of neurons. Eur J Biochem, 1994 May 1, 221(3), 1003 - 12 Insertion in barnase of a loop sequence from ribonuclease T1 . Investigating sequence and structure alignments by protein engineering; Vuilleumier S et al.; Barnase was mutated by inserting into its active site loop sequences found in the related enzyme ribonuclease T1 (RNase T1), according to either structural or sequential similarity alignments . The barnase/RNase T1 hybrid corresponding to the structural alignment of the two proteins, endo-{RNaseT1-(93-99)}102abarnase, contains RNase T1 residues at positions 93-99 inserted between residues at positions 102 and 103 of barnase . The other constructed mutant, endo-{RNaseT1-(95-98)}104abarnase, has RNase T1 residues at positions 95-98 inserted between residues at positions 104 and 105 in barnase, corresponding to published sequence alignments of the two proteins in this region . The mutants were characterized by absorbance, fluorescence and CD spectroscopy; the stability, folding and unfolding kinetics, and catalytic activity were measured and compared with the wild-type enzyme . Endo-{RNaseT1-(93-99)}102abarnase, the mutant protein corresponding to the structural alignment of barnase with ribonuclease T1, shows a slightly higher stability (approximately 5 kJ/mol) towards urea and heat denaturation than the mutant endo-{RNaseT1-(95-98)}104abarnase, designed according to a sequence alignment between the two enzymes . Both mutants have very low catalytic activity, although the effect of mutation is almost entirely limited to kcat in the case of the mutant corresponding to the structural alignment between barnase and ribonuclease T1, while both kcat and Km are affected in the mutant corresponding to the sequence alignment between the two enzymes . Thus, the superiority of structural over sequential alignments cannot be supported conclusively by direct experiment in the present case. Arch Biochem Biophys, 1994 May 1, 310(2), 475 - 80 Characterization of the putative GTP-binding site residues of Escherichia coli adenylosuccinate synthetase by site-directed mutagenesis; Kang C et al.; Adenylosuccinate synthetase contains amino acid sequences in its GTP-binding domain that are homologous to other G-proteins . This homology includes a glycine-rich phosphate-binding loop, GXXXXGK, and a guanine-specific binding region, (N/T/Q)KXD; however, virtually no other sequence homology exists between other G-proteins and adenylosuccinate synthetase . On the basis of X-ray diffraction studies, the folding topologies of the synthetase and the p21 ras proteins are different . Yet, residues that interact with GTP in the p21 ras proteins are present in the synthetase in nearly identical positions . We chose therefore to study the G15V mutant, a phosphate-binding loop mutant, and K331L and K331R, two mutants of Lys331 that are involved in guanine ring binding . The Km values for GTP of adenylosuccinate synthetase mutants, K331L and K331R, when compared to those of the wild-type enzyme, were 27- and 20-fold increased, respectively, without any significant change in the Km values for IMP . Because both mutations affected the Km values for GTP similarly, whereas the kcat and secondary structure were essentially unchanged, it is suggested that Lys331 is located in the GTP-binding site of adenylosuccinate synthetase and the terminal N zeta of the Lys is not necessarily important in GTP-binding on the enzyme . Therefore, Lys331 may interact with GTP through hydrophobic interactions between its linear side chain and the aromatic ring of the guanine base of GTP . Also, structural characterization of the G15V mutant was carried out using circular dichroism (CD) spectrometry, NMR spectroscopy, and spectrofluorometry . The CD spectral data indicated that the secondary structure of the G15V mutant was significantly altered by GTP and IMP, whereas that of the wild-type enzyme is not changed; however, the two enzymes exhibited similar secondary structures in the absence of substrates . The NMR spectra of both enzymes were also similar in the absence of substrates . The dissociation constant (Kd) for IMP of the G15V mutant was 4.8-fold larger than its Km value which was 1.5-fold increased compared to the wild-type enzyme . From these findings, it was concluded that the phosphate-binding region of adenylosuccinate synthetase is involved in a conformational change induced by GTP and IMP binding, and that GTP and IMP binding depend on the presence of the other substrate at the active site of the enzyme . These results suggest that the Lys331 of adenylosuccinate synthetase may play similar roles in the function and structure to that of GTP-binding proteins.(ABSTRACT TRUNCATED AT 400 WORDS) Am J Pathol, 1994 May, 144(5), 896 - 905 Cell lineage study in the liver using retroviral mediated gene transfer . Evidence against the streaming of hepatocytes in normal liver; Bralet MP et al.; The fate of normal hepatocytes in adult rat liver was studied after genetic labeling using the Escherichia coli beta-galactosidase gene coupled to a nuclear localization signal . The marker gene was introduced by direct in vivo retroviral-mediated gene transfer into hepatocytes 24 hours after partial hepatectomy . Analysis of beta-galactosidase expression in the liver at various time after gene transfer revealed that labeled hepatocytes were distributed throughout the entire lobule with a predominance in the periportal and mediolobular regions . Long-term experiments demonstrated that division of hepatocytes did occur as was revealed by the increasing number of beta-galactosidase-positive cells in isolated clusters . There was no evidence for the participation of stem cells in this process . Moreover, we found that after more than 1 year, the pattern of distribution of positive cells within the lobule was not modified . This suggests that hepatocytes do not migrate from the portal space to the perivenous region, as has been previously hypothesized. Am J Obstet Gynecol, 1994 May, 170(5 Pt 1), 1467 - 75 Systemic and local cytokine profiles in endotoxin-induced preterm parturition in mice; Fidel PL Jr et al.; OBJECTIVE: Our purpose was to determine whether endotoxin-induced preterm parturition is preceded by a change in the maternal serum and amniotic fluid concentrations of tumor necrosis factor-alpha, interleukin-6, and interleukin-1 alpha . STUDY DESIGN: C3H/HeN pregnant mice at 15 days of gestation (70% gestation) were randomized to receive an intraperitoneal injection of phosphate-buffered saline solution or lipopolysaccharide (50 micrograms/mouse) . Blood (n = 93) and amniotic fluid (n = 58) were collected at 1, 4, and 10 hours after lipopolysaccharide injection . Tumor necrosis factor-alpha, interleukin-6, and interleukin-1 alpha were determined with sensitive and specific enzyme-linked immunoassays . RESULTS: The injection-to-delivery interval was shorter in mice injected intraperitoneally with 50 micrograms lipopolysaccharide than in phosphate-buffered saline solution-treated mice (median 15.5 hours, range: 10 to 105 hours vs median 88.5 hours, range: 53 to 105 hours; p < 0.001) . In comparison with phosphate-buffered saline solution-treated mice, a distinct serum cytokine pattern was observed in lipopolysaccharide-treated mice . Concentrations of tumor necrosis factor-alpha were detectable 1 and 4 hours after lipopolysaccharide injection (median 874 pg/ml, range: < 100 to 8000 pg/ml, p < 0.001; and median 263 pg/ml, range: < 100 to 927 pg/ml, p < 0.001, respectively) . Concentrations of interleukin-6 were elevated at 1, 4, and 10 hours (median 11.8 ng/ml, range: 6 to 500 ng/ml, p < 0.001; median 27.1 ng/ml, range: 4.5 to 192 ng/ml, p < 0.001; median 1.95 ng/ml, range: < 0.05 to 35 ng/ml, p < 0.015, respectively) . Concentrations of interleukin-1 alpha were significantly increased 4 hours after lipopolysaccharide injection (median 102 pg/ml, range: < 15 to 306 pg/ml, p < 0.001) . A cytokine pattern distinct from serum was observed in amniotic fluid of lipopolysaccharide-treated mice . In comparison with controls, concentrations of interleukin-6 were significantly elevated 4 and 10 hours after treatment with lipopolysaccharide (median 0.88 ng/ml, range: 0.40 to 2.7 ng/ml, p < 0.025; and median 4 ng/ml, range: 1.9 to 33.6 ng/ml, p < 0.001, respectively) . Interleukin-1 alpha was elevated 10 hours after lipopolysaccharide treatment (median 185.3 pg/ml, range: 38 to 511 pg/ml, p < 0.015) . Tumor necrosis factor-alpha was not significantly increased in amniotic fluid . CONCLUSION: Preterm delivery after lipopolysaccharide administration is preceded by the appearance of dramatic increases in maternal serum concentrations of tumor necrosis factor-alpha, interleukin-6, and interleukin-1 alpha and in amniotic fluid concentrations of interleukin-6 and interleukin-1 alpha. Gastroenterology, 1994 May, 106(5), 1150 - 61 Signal transduction in human epithelial cells infected with attaching and effacing Escherichia coli in vitro; Dytoc M et al.; BACKGROUND/AIMS: Human enteropathogenic Escherichia coli infection of epithelial cells is characterized by attaching and effacing adhesion . To determine if signal transduction responses are involved in this adhesion phenotype, levels of inositol 1,4,5-triphosphate and cytosolic free calcium were measured in tissue culture cells infected with enteropathogenic E . coli strain E2348 (serotype O127:H6) . METHODS: Inositol triphosphate levels were measured by using a commercial binding assay, and intracellular calcium levels were determined by spectrofluorometry . RESULTS: Elevated levels of both inositol triphosphate (182% +/- 52%; P < 0.05) and intracellular calcium (125% +/- 40%, mean +/- SE; P < 0.05) were seen after infection of HEp-2 cells with strain E2348 . In contrast, inositol triphosphate and intracellular calcium levels were not elevated in HEp-2 cells infected with six E . coli strains that did not cause attaching and effacing lesions . Subcellular calcium localization using oxalate precipitation and electron microscopy showed calcium accumulation within the terminal web subjacent to regions of attaching and effacing adhesion . Depleting external calcium did not eliminate formation of attaching and effacing lesions, whereas treatment of HEp-2 cells with an intracellular calcium chelator prevented attaching and effacing lesions . CONCLUSIONS: Enteropathogenic E . coli infection elevates both inositol triphosphate and intracellular calcium levels in cultured epithelial cells. Dev Biol, 1994 May, 163(1), 49 - 65 Identification of a new spore coat protein gene in the cellular slime mold Dictyostelium discoideum; Yoder BK et al.; Genomic DNA encoding the prespore cell-specific PL3 cDNA was cloned and sequenced, revealing that the gene consists of three exons separated by short 100-bp introns . The single long open reading frame predicts a primary translation product of 70 kDa after removal of a cleavable signal peptide, two-thirds of which consists of four kinds of amino acid repeat elements, including two found in other spore coat proteins . The 85-kDa PL3 protein synthesized in vivo accumulates specifically in regulated secretory vesicles of prespore cells (prespore vesicles), as determined microscopically using antibody against a PL3 gene fusion product expressed in Escherichia coli . The protein later accumulates extracellularly in the spore coat, which is formed during sporulation, as determined ultrastructurally and by Western blot analysis of SDS-PAGE gels . In addition to its high proportion of repeat elements, the PL3 protein has the following properties which distinguish it from other spore coat proteins: (1) it is located at the outer extent of the middle layer, beneath the outer layer, (2) its dissociation from the coat requires the presence of protein denaturants and reducing agents at elevated temperature, and (3) a large proportion of the protein is not dissociated from the coat even under these conditions, as determined by ultrastructural analysis of the extracted coat . The PL3 protein may contribute to the structure of the coat at the interface between the middle, cellulosic layer and the outer, electron-dense, proteinaceous layer. Dev Biol, 1994 May, 163(1), 38 - 48 The promoter of a gene encoding a novel Dictyostelium spore coat protein; Yoder BK et al.; The cAMP-inducible prespore gene, PL3, encodes a protein which is a novel component of the spore coat . Unlike the well-characterized spore coat proteins (SP60, SP70, and SP96) which are found in the outer layer of the coat, the PL3 gene product is localized to a subregion of the coat beneath the outer proteinaceous layer . Moreover, a substantial portion of the PL3 protein is tightly associated with the spore coat and not released under the conditions that led to the identification of the other coat proteins . The promoter for this novel spore coat gene is described . Unlike the other coat-protein gene promoters, it lacks the extensive CA-type elements . It contains two short CA boxes and five prominent G-rich regions . Sequential deletions from the 5' end of the promoter which remove both CA boxes as well as two of the G-rich regions reduce the level of expression but do not alter the spatial regulation of expression . Despite the sequence differences, the PL3 promoter still confers correct spatial, temporal, and cell type-specific regulation on a reporter gene . Escherichia coli beta-galactosidase enzyme activity expressed under the control of this PL3 promoter first appears in randomly isolated cells at the loose mound stage . Because of the sensitivity of the assay, beta-galactosidase activity is detectable prior to the appearance of the PL3 protein on Western blots and by immunofluorescence . Later the number of cells staining for beta-galactosidase activity and the intensity of staining increases . During tipped mound, slug, and culminant stages, cells expressing beta-galactosidase under the control of the PL3 promoter are localized to prespore regions and are spatially coincident with cells expressing the PL3 protein. Dev Biol, 1994 May, 163(1), 125 - 32 The E box is essential for activity of the cardiac actin promoter in skeletal but not in cardiac muscle; Skerjanc IS et al.; The sequence CANNTG (E box) is frequently found in the promoters of muscle-specific genes and binds members of the basic helix-loop-helix (bHLH) family of transcription factors . We compared the need for the E box in the expression of a muscle-specific promoter normally expressed in both cardiac and skeletal muscle . The E box was mutated in a construct of the cardiac alpha-actin promoter driving the Escherichia coli lacZ gene . The wild-type and mutant constructs were transfected and stably integrated into the genomes of P19 embryonal carcinoma cells . The wild-type promoter was expressed in both cardiac and skeletal myocytes . The promoter lacking an E box was expressed in cardiac but not in skeletal muscle . Neither promoter was active in nonmuscle cells . Thus the E box is not necessary for the cardiac actin promoter activity in P19-derived cardiac muscle but is essential for its activity in skeletal muscle . This result is consistent with our inability to detect cardiac muscle-specific members of the MyoD family of bHLH transcription factors. Am J Clin Nutr, 1994 May, 59(5), 1045 - 9 Thiamin deficiency impairs endotoxin-induced increases in hepatic glucose output; Molina PE et al.; We addressed the role of thiamin, a cofactor for several enzymes involved in glucose metabolism, in the glucose metabolic response to endotoxin . Characterized by hyperglycemia, increased hepatic glucose production exceeding elevated rates of whole-body glucose utilization, this response is mediated by hormones and cytokines and is dependent on the immune and nutritional status of the host . We hypothesized that a thiamin-deficient state would impair the metabolic response to endotoxin . Rats were fed a thiamin-deficient or control diet for 6 wk before in vivo assessment of glucose kinetics . In control rats, Escherichia coli endotoxin increased the rate of glucose appearance (+76%), disappearance (+70%), and metabolic clearance (+50%) . Thiamin deficiency resulted in increased plasma glucose (18%) and lactate (3- to 4-fold) as well as in a 30% decrease in insulin and an increase in glucagon (2.6-fold) and corticosterone (3.6-fold) . Thiamin deficiency inhibited the endotoxin-induced hyperglycemia and the rise in hepatic glucose production, glucose utilization, and metabolic clearance rate. J Bacteriol, 1994 May, 176(9), 2754 - 8 Guanine nucleotide-dependent assembly of FtsZ into filaments; Mukherjee A et al.; FtsZ is an essential cell division protein that is localized to the leading edge of the bacterial septum in a cytokinetic ring . It contains the tubulin signature motif and is a GTP binding protein with a GTPase activity . Further comparison of FtsZ with eukaryotic tubulins revealed some additional sequence similarities, perhaps indicating a similar GTP binding site . Examination of FtsZ incubated in vitro by electron microscopy revealed a guanine nucleotide-dependent assembly into protein filaments, supporting the hypothesis that the FtsZ ring is formed through self-assembly . FtsZ3, which is unable to bind GTP, does not polymerize, whereas FtsZ2, which binds GTP but is deficient in GTP hydrolysis, is capable of polymerization. J Bacteriol, 1994 May, 176(9), 2743 - 6 5-Aminolevulinic acid synthesis in Escherichia coli requires expression of hemA; Chen W et al.; hemA and hemM, which are 213 bp apart and divergently transcribed, were separately cloned . We found that hemA is required for 5-aminolevulinic acid (ALA) synthesis in two ALA- auxotrophs . Overexpression of hemM alone did not produce ALA . More ALA was produced by strains harboring a plasmid with both hemA and hemM than by those with hemA alone . We conclude that hemA alone is required for ALA synthesis but hemA and hemM are required for maximal ALA synthesis. J Bacteriol, 1994 May, 176(9), 2740 - 2 Location of a potassium tellurite resistance operon (tehA tehB) within the terminus of Escherichia coli K-12; Taylor DE et al.; A tellurite resistance determinant, believed to have been cloned from the IncHII plasmid pHH1508a (E . G . Walter, J . H . Weiner, and D . E . Taylor, Gene 101:1-7, 1991), was shown instead to have originated from the chromosome of Escherichia coli K-12 . The two genes, tehA and tehB, constitute an operon located in the terminus at approximately 32.3 min. J Bacteriol, 1994 May, 176(9), 2627 - 34 Cloning, sequencing, and analysis of the griseusin polyketide synthase gene cluster from Streptomyces griseus; Yu TW et al.; A fragment of DNA was cloned from the Streptomyces griseus K-63 genome by using genes (act) for the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor as a probe . Sequencing of a 5.4-kb segment of the cloned DNA revealed a set of five gris open reading frames (ORFs), corresponding to the act PKS genes, in the following order: ORF1 for a ketosynthase, ORF2 for a chain length-determining factor, ORF3 for an acyl carrier protein, ORF5 for a ketoreductase, and ORF4 for a cyclase-dehydrase . Replacement of the gris genes with a marker gene in the S . griseus genome by using a single-stranded suicide vector propagated in Escherichia coli resulted in loss of the ability to produce griseusins A and B, showing that the five gris genes do indeed encode the type II griseusin PKS . These genes, encoding a PKS that is programmed differently from those for other aromatic PKSs so far available, will provide further valuable material for analysis of the programming mechanism by the construction and analysis of strains carrying hybrid PKS. J Bacteriol, 1994 May, 176(9), 2611 - 8 UTP: alpha-D-glucose-1-phosphate uridylyltransferase of Escherichia coli: isolation and DNA sequence of the galU gene and purification of the enzyme; Weissborn AC et al.; The galU gene of Escherichia coli, thought to encode the enzyme UTP:alpha-D-glucose-1-phosphate uridylyltransferase, had previously been mapped to the 27-min region of the chromosome (J . A . Shapiro, J . Bacteriol . 92:518-520, 1966) . By complementation of the membrane-derived oligosaccharide biosynthetic defect of strains with a galU mutation, we have now identified a plasmid containing the galU gene and have determined the nucleotide sequence of this gene . The galU gene is located immediately downstream of the hns gene, and its open reading frame would be transcribed in the direction opposite that of the hns gene (i.e., clockwise on the E . coli chromosome) . The nucleotide sequences of five galU mutations were also determined . The enzyme UTP:alpha-D-glucose-1-phosphate uridylyltransferase was purified from a strain containing the galU gene on a multicopy plasmid . The amino-terminal amino acid sequence (10 residues) of the purified enzyme was identical to the predicted amino acid sequence (after the initiating methionine) of the galU-encoded open reading frame . The functional enzyme appears to be a tetramer of the galU gene product. Infect Immun, 1994 May, 62(5), 2119 - 21 Differential expression of the cytotoxic and hemolytic activities of the ApxIIA toxin from Actinobacillus pleuropneumoniae; Tu AH et al.; The ApxIIA protein secreted from Actinobacillus pleuropneumoniae is both hemolytic and cytotoxic . However, when the cloned apxII operon is expressed in Escherichia coli, two forms of the ApxIIA protein can be recovered . Toxin which remains intracellular has hemolytic and cytotoxic activities, while toxin that is secreted is cytotoxic with little or no hemolytic activity . This indicates that the cytotoxicity of ApxIIA is independent of its hemolytic activity. Infect Immun, 1994 May, 62(5), 2071 - 8 Assignment of functional domains involved in ADP-ribosylation and B-oligomer binding within the carboxyl terminus of the S1 subunit of pertussis toxin; Krueger KM et al.; The roles of the carboxyl terminus of the S1 subunit (composed of 235 amino acids) of pertussis toxin in the ADP-ribosylation of transducin (Gt) and in B-oligomer binding were defined by analysis of two carboxyl-terminal deletion mutants of the recombinant S1 (rS1) subunit: C204, which is composed of amino acids 1 through 204 of S1, and C219, which is composed of amino acids 1 through 219 of S1 . C204 was expressed in Escherichia coli as a stable, soluble peptide that had an apparent molecular mass of 23.4 kDa . In a linear velocity assay, the specific activity of C180 was 2% and that of C204 was 80% of the activity displayed by rS1 in catalyzing the ADP-ribosylation of Gt . In addition, C204 possessed catalytic efficiencies (kcat/Km) that were 110% at variable Gt concentrations and 40% at variable NAD concentrations of those reported for rS1 . These data showed that the catalytic activity of C204 approached the activity of S1 . C204 and C219 were unable to associate with the B oligomer under conditions which promoted association of rS1 with the B oligomer . Consistent with these results, mixtures of C204 or C219 with the B oligomer did not elicit a clustering phenotype in CHO cells, whereas rS1 which had associated with the B oligomer was as cytotoxic as native pertussis toxin . These data indicate that residues between 219 and 235 are important in the association of the S1 subunit with the B oligomer . These data allow the assignment of functional regions to the carboxyl terminus of S1 . Residues 195 to 204 are required for optimal ADP-ribosyltransferase activity, residues 205 to 219 link the catalytic region of S1 and a B-oligomer-binding region of S1, and residues 220 to 235 are required for association of S1 with the B oligomer. Infect Immun, 1994 May, 62(5), 1920 - 6 Protection of C3H/HeN mice from challenge with Borrelia burgdorferi through active immunization with OspA, OspB, or OspC, but not with OspD or the 83-kilodalton antigen; Probert WS et al.; Recent advances in the development of animal models for Lyme borreliosis have provided means of identifying potential targets for the design of a subunit vaccine to prevent this disease . The C3H/HeN mouse model was used to study several Borrelia burgdorferi antigens from a single isolate for their ability to elicit borreliacidal and protective antibodies . The ospA, ospB, ospC, ospD, and 83-kDa genes from a California isolate, SON 188, were cloned and expressed in Escherichia coli as proteins fused to the C-terminal end of maltose-binding protein . Active immunization of mice with these fusion proteins elicited high titers of antibodies that recognized the homologous SON 188 antigens upon immunoblotting . Antibodies generated to the OspA and OspB fusion proteins, but not to the OspC, OspD, and the 83-kDa fusion proteins, demonstrated in vitro borreliacidal activity . Challenge of all actively immunized mice with 10(7) SON 188 spirochetes resulted in infection in all mice receiving the OspD or 83-kDa immunogens but not in any mice receiving the OspA, OspB, or OspC fusion proteins . These results demonstrate the potential of OspA, OspB, and OspC as components of a subunit vaccine for the prevention of Lyme borreliosis. Infect Immun, 1994 May, 62(5), 1889 - 95 Diversity of cultivable and uncultivable oral spirochetes from a patient with severe destructive periodontitis; Choi BK et al.; To determine the genetic diversity of cultivable and uncultivable spirochetes in the gingival crevice of a patient with severe periodontitis, partial 16S rRNA genes were cloned from PCR-amplified products of DNA and RNA extracted from a subgingival plaque sample . Approximately 500 bp were amplified in PCRs by using universally conserved primers with polylinker tails . Purified PCR products were cloned into Escherichia coli by using the plasmid vector pUC19 . The resultant clone library was screened by colony hybridization with a radiolabeled, treponeme-specific oligonucleotide probe . The 16S rRNA inserts of 81 spirochetal clones were then sequenced by standard procedures . Sequences were compared with 16S rRNA sequences of 35 spirochetes, including the four known cultivable oral treponeme species . The analysis revealed an unexpected diversity of oral treponemes from a single patient . When 98% or greater sequence similarity was used as the definition of a species-level cluster, the clone sequences were found to represent 23 species . When 92% similarity was used as the definition, the clones fell into eight major groups, only two of which contained named species, Treponema vincentii and Treponema denticola, while Treponema pectinovorum and Treponema socranskii were not represented in any cluster . Seven of the 81 spirochetal clones were found to contain chimeric 16S rRNA sequences . In situ fluorescence hybridization with a fluorescein isothiocyanate-labeled oligonucleotide probe specific for one of the new species representing cluster 19 was used to identify cells of the target species directly in clinical samples. Infect Immun, 1994 May, 62(5), 1768 - 75 Evidence that the A2 fragment of Shiga-like toxin type I is required for holotoxin integrity; Austin PR et al.; Escherichia coli Shiga-like toxin type I (SLT-I) is a potent cytotoxin consisting of an enzymatically active A subunit and a pentameric B subunit that mediates toxin binding to susceptible eukaryotic cells . Evidence that the carboxy-terminal 38 amino acids of the A subunit are involved in holotoxin 1A:5B association is presented . We compared the ability of purified recombinant SLT-I B subunit (Slt-IB) to combine in vitro with purified recombinant SLT-I A subunit (Slt-IA; full-length subunit A includes amino acids 1 to 293) and its ability to combine with purified recombinant SLT-I A1 subunit (Slt-IA1; truncated subunit A includes amino acids 1 to 255) . Each mixture was analyzed for biological and physical evidence of toxin assembly . Although Slt-IA successfully combined with Slt-IB to form a molecular species similar to holotoxin that was detectable by nondenaturing polyacrylamide gel electrophoresis and immunoblotting and yielded a molecule which was cytotoxic to cultured Vero cells, Slt-IA1 did not have this ability . Slt-IA1 was 36-fold more active than Slt-IA in an in vitro protein synthesis inhibition assay . These findings suggest that the Slt-IA2 fragment is crucial for formation of SLT holotoxin and stabilizes the interaction between the A and B subunits. Infect Immun, 1994 May, 62(5), 1584 - 92 Adherence characteristics of attaching and effacing strains of Escherichia coli from rabbits; Robins-Browne RM et al.; Twelve strains of Escherichia coli previously reported to cause diarrhea in rabbits were examined for properties associated with virulence . Ten strains met the criteria for classification as enteropathogenic E . coli in that they were diarrheagenic strains that evoked attaching-effacing lesions in the small intestine and did not produce detectable enterotoxins or cytotoxins . These bacteria exhibited a variety of patterns when investigated for adherence to HEp-2 epithelial cells . Although several strains displayed localized and/or diffuse adherence to epithelial cells, they did not hybridize with DNA probes that recognize the genes responsible for these phenotypes in diarrheagenic E . coli from humans . The bacteria also varied in their ability to bind to erythrocytes and intestinal brush borders from various animal species . Six strains adhered to rabbit brush borders; two of these also adhered to brush borders from other animals . Two strains that did not adhere to rabbit brush borders adhered to those from guinea pigs or sheep . Only one of the strains investigated carried AF/R1 fimbriae, which are believed to govern the host specificity of this category of diarrheagenic E . coli . This strain was E . coli RDEC-1, which remains the only E . coli strain to date that is known to carry fimbriae of this type . The results indicate that although diarrheagenic E . coli strains from rabbits may have common properties associated with the ability to produce attaching-effacing lesions, they differ from each other and from enteropathogenic E . coli of humans in terms of some of the adhesins that mediate binding to eukaryotic cells. Mol Cell Biol, 1994 May, 14(5), 3376 - 91 The DNA-bending protein HMG-1 enhances progesterone receptor binding to its target DNA sequences; Onate SA et al.; Steroid hormone receptors are ligand-dependent transcriptional activators that exert their effects by binding as dimers to cis-acting DNA sequences termed hormone response elements . When human progesterone receptor (PR), expressed as a full-length protein in a baculovirus system, was purified to homogeneity, it retained its ability to bind hormonal ligand and to dimerize but exhibited a dramatic loss in DNA binding activity for specific progesterone response elements (PREs) . Addition of nuclear extracts from several cellular sources restored DNA binding activity, suggesting that PR requires a ubiquitous accessory protein for efficient interaction with specific DNA sequences . Here we have demonstrated that the high-mobility-group chromatin protein HMG-1, as a highly purified protein, dramatically enhanced binding of purified PR to PREs in gel mobility shift assays . This effect appeared to be highly selective for HMG-1, since a number of other nonspecific proteins failed to enhance PRE binding . Moreover, HMG-1 was effective when added in stoichiometric amounts with receptor, and it was capable of enhancing the DNA binding of both the A and B amino-terminal variants of PR . The presence of HMG-1 measurably increased the binding affinity of purified PR by 10-fold when a synthetic palindromic PRE was the target DNA . The increase in binding affinity for a partial palindromic PRE present in natural target genes was greater than 10-fold . Coimmunoprecipitation assays using anti-PR or anti-HMG-1 antibodies demonstrated that both PR and HMG-1 are present in the enhanced complex with PRE . HMG-1 protein has two conserved DNA binding domains (A and B), which recognize DNA structure rather than specific sequences . The A- or B-box domain expressed and purified from Escherichia coli independently stimulated the binding of PR to PRE, and the B box was able to functionally substitute for HMG-1 in enhancing PR binding . DNA ligase-mediated ring closure assays demonstrated that both the A and B binding domains mediate DNA flexure . It was also demonstrated in competition binding studies that the intact HMG-1 protein binds to tightly curved covalently closed or relaxed DNA sequences in preference to the same sequence in linear form . The finding that enhanced PRE binding was intrinsic to the HMG-1 box, combined with the demonstration that HMG-1 or its DNA binding boxes can flex DNA, suggests that HMG-1 facilitates the binding of PR by inducing a structural change in the target DNA. Mol Cell Biol, 1994 May, 14(5), 3329 - 38 Ligand modulates the conversion of DNA-bound vitamin D3 receptor (VDR) homodimers into VDR-retinoid X receptor heterodimers; Cheskis B et al.; Protein dimerization facilitates cooperative, high-affinity interactions with DNA . Nuclear hormone receptors, for example, bind either as homodimers or as heterodimers with retinoid X receptors (RXR) to half-site repeats that are stabilized by protein-protein interactions mediated by residues within both the DNA- and ligand-binding domains . In vivo, ligand binding among the subfamily of steroid receptors unmasks the nuclear localization and DNA-binding domains from a complex with auxiliary factors such as the heat shock proteins . However, the role of ligand is less clear among nuclear receptors, since they are constitutively localized to the nucleus and are presumably associated with DNA in the absence of ligand . In this study, we have begun to explore the role of the ligand in vitamin D3 receptor (VDR) function by examining its effect on receptor homodimer and heterodimer formation . Our results demonstrate that VDR is a monomer in solution; VDR binding to a specific DNA element leads to the formation of a homodimeric complex through a monomeric intermediate . We find that 1,25-dihydroxyvitamin D3, the ligand for VDR, decreases the amount of the DNA-bound VDR homodimer complex . It does so by significantly decreasing the rate of conversion of DNA-bound monomer to homodimer and at the same time enhancing the dissociation of the dimeric complex . This effectively stabilizes the bound monomeric species, which in turn serves to favor the formation of a VDR-RXR heterodimer . The ligand for RXR, 9-cis retinoic acid, has the opposite effect of destabilizing the heterodimeric-DNA complex . These results may explain how a nuclear receptor can bind DNA constitutively but still act to regulate transcription in a fully hormone-dependent manner. Mol Cell Biol, 1994 May, 14(5), 2946 - 57 Poly(A) polymerase contains multiple functional domains; Raabe T et al.; Poly(A) polymerase (PAP) contains regions of similarity with several known protein domains . Through site-directed mutagenesis, we provide evidence that PAP contains a functional ribonucleoprotein-type RNA binding domain (RBD) that is responsible for primer binding, making it the only known polymerase to contain such a domain . The RBD is adjacent to, and probably overlaps with, an apparent catalytic region responsible for polymerization . Despite the presence of sequence similarities, this catalytic domain appears to be distinct from the conserved polymerase module found in a large number of RNA-dependent polymerases . PAP contains two nuclear localization signals (NLSs) in its C terminus, each by itself similar to the consensus bipartite NLS found in many nuclear proteins . Mutagenesis experiments indicate that both signals, which are separated by nearly 140 residues, play important roles in directing PAP exclusively to the nucleus . Surprisingly, basic amino acids in the N-terminal-most NLS are also essential for AAUAAA-dependent polyadenylation but not for nonspecific poly(A) synthesis, suggesting that this region of PAP is involved in interactions both with nuclear targeting proteins and with nuclear polyadenylation factors . The serine/threonine-rich C terminus is multiply phosphorylated, including at sites affected by mutations in either NLS. Cancer Res, 1994 May 1, 54(9), 2296 - 8 Induction of resistance to fluorodeoxyuridine cytotoxicity and DNA damage in human tumor cells by expression of Escherichia coli deoxyuridinetriphosphatase; Canman CE et al.; Recent studies from our laboratory suggested that, in some human colorectal tumor cell lines, sensitivity to fluorodeoxyuridine may depend upon the extent of dUTP accumulation that occurs following drug treatment and that elevation of dUTPase activity might be the basis for some instances of resistance to fluoropyrimidines . To test this model, we expressed Escherichia coli dUTPase in an established human tumor cell line (HT29) and measured the effect of this manipulation on response to fluorodeoxyuridine . As predicted, HT29 derivatives containing dUTPase activity 4-5-fold higher than controls were protected from fluorodeoxyuridine-induced loss of clonogenicity and from formation of DNA double strand breaks . These data provide the first direct evidence that alteration in a component of the uracil misincorporation/misrepair pathway can confer resistance to fluoropyrimidines in human tumor cells. J Virol, 1994 May, 68(5), 3386 - 90 Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly; Rodgers RE et al.; A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established . The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli . The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography . Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E . coli-expressed VP1 protein . Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres . Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents. J Virol, 1994 May, 68(5), 2937 - 46 Human cytomegalovirus maturational proteinase: expression in Escherichia coli, purification, and enzymatic characterization by using peptide substrate mimics of natural cleavage sites; Burck PJ et al.; The proteolytic processing of the human cytomegalovirus (HCMV) assembly protein, resulting in truncation of its C terminus, is an essential step in virion maturation . The proteinase responsible for this cleavage is the amino-terminal half of the protein encoded by the UL80a open reading fame . We have obtained high expression levels of this 256-amino-acid HCMV proteinase, assemblin, in Escherichia coli . In addition to the 28-kDa proteinase, a 15-kDa protein comprising the first 143 amino acids and a 13-kDa protein comprising the last 113 amino acids of the 28-kDa HCMV proteinase were present . Both the 28-kDa proteinase and the 15-kDa protein were purified by a two-step chromatographic procedure utilizing anion exchange in urea and dithiothreitol and size exclusion in NaSCN and dithiothreitol . Activation of the purified 28-kDa proteinase required denaturation in urea as well as complete reduction of all five cysteine residues in the molecule . Removal of the urea by dialysis with retention of the reducing agent yielded an active proteinase . Addition of glycerol to 50% enhanced the activity . The HCMV proteinase cleaved the peptides RGVVNASSRLAK and SYVKASVSPE, which are mimics of the maturational (M)- and release (R)-site sequences, respectively, in the UL80a-encoded protein . The cleavage site in the peptides was at the same Ala-Ser scissile bond as observed in the UL80a protein . The Km value for the cleavage of RGVVNASSRLAK (M-site mimic) by the proteinase was similar to that for SYVKASVSPE (R-site mimic), but the turnover (kcat) of the M-site peptide mimic substrate by the proteinase was six to eight times faster . The peptide homologs of the herpes simplex virus type 1 M- and R-site sequences in the UL26-encoded protein were also cleaved by the HCMV proteinase, although at rates slower than those for the HCMV substrates . The HCMV proteinase was inhibited by Zn2+ and by alkylating agents, but only at very high inhibitor concentrations . The purified 15-kDa protein, subjected to the same activation conditions as the 28-kDa proteinase, had no enzymatic activity against the HCMV M- and R-site peptide substrates. Vopr Virusol, 1994 May-Jun, 39(3), 110 - 3 {The production of recombinant polypeptides from the herpes simplex viruses types 1 and 2}; Zvonarev AIu et al.; The genes IE175, IE63, IE68, IE12, UL29 and UL19 of herpes simplex virus types 1 and 2 were cloned . Fragments of these genes were cloned into open reading frame expression vector pEX1-3 . Recombinant proteins containing amino acid sequences of ICP4, ICP27, ICP22, ICP47, major DNA-binding protein and major capsid protein were expressed in E . coli cells by fusion with cro-beta-galactosidase proteins. Thromb Haemost, 1994 May, 71(5), 646 - 50 Platelet shape change in whole blood: differential effects of endotoxin; Nystrom ML et al.; The effect of endotoxic lipopolysaccharide (LPS) on platelet shape change (PSC; a preaggregation event) was investigated . PSC is accompanied by an increase in median platelet volume (MPV), which was measured using a channelyzer . In whole blood, but not in platelet rich plasma (PRP), LPS (final concentration 80 mg/l) caused an increase in MPV that could be detected for 2 h . When PRP (prepared from LPS- and saline-pretreated whole blood) was incubated for 40 min, the LPS-mediated increase in MPV could no longer be detected . Taken together, these data imply that PSC is both initiated and maintained by a labile factor(s) present in whole blood, but not in PRP . PRP was prepared from LPS-pretreated whole blood and incubated for 40 min to allow reversal of the LPS-induced PSC; further stimulation with LPS caused PSC . Platelets from LPS-pretreated whole blood also showed enhanced PSC with serotonin (5-HT), diminished PSC with platelet activating factor (PAF), and no change of response to ADP and collagen . Hence, LPS pretreatment of whole blood differentially alters responses of platelets to further stimulation with LPS and other agonists . A specific PAF antagonist completely abolished the effect of LPS on MPV . These data may contribute to an understanding of the cascading thrombotic events and thrombocytopenia associated with septicaemia. Curr Genet, 1994 May, 25(5), 432 - 7 Homologous transformation of the edible basidiomycete Agrocybe aegerita with the URA1 gene: characterization of integrative events and of rearranged free plasmids in transformants; Noel T et al.; The URA1 gene, encoding dihydroorotate dehydrogenase of the pyrimidine pathway, cloned into pUC18 (pUra1-1) was used to develop an homologous transformation system for the cultivated mushroom Agrocybe aegerita . Protoplasts of a ura1 auxotrophic strain were transformed by electroporation with efficiencies ranging from 1 to 26 transformants per micrograms of DNA . The phenotype of the stable Ura+ transformants suggested a strong nuclear heterogeneity further confirmed by Southern-blot analysis . All transformants acquired extrachromosomal forms derived from pUra1-1 . Integration of pUra1-1 into chromosomal DNA occurred for some transformants . Plasmids containing the integrant of pUC18 recombined to different parts of the URA1 gene were rescued from A . aegerita transformants through transformation of E . coli . Their molecular analysis indicated that they represent products of the continuous excision of primary-integrated vector sequences rather than ARS-dependent autoreplicative forms. Hum Mol Genet, 1994 May, 3(5), 809 - 11 Identification of five novel mutations in the porphobilinogen deaminase gene; Mgone CS et al.; We have studied the porphobilinogen deaminase gene transcripts from seven unrelated patients from the West of Scotland, all suffering from acute intermittent porphyria . This was achieved by reverse transcription and PCR amplification of mRNA followed by asymmetric amplification and direct sequencing . Five novel and two previously described mutations were identified and found to be single base substitutions . Of the five novel mutations, three were missense (R116Q, T2691, G274R) and two were nonsense (Q204 Stop, W283 Stop) . Using Escherichia coli PBGD as a model, it is possible to predict and explain the deleterious effects that these mutations might have on the function and structure of the enzyme. Hum Mol Genet, 1994 May, 3(5), 729 - 34 Molecular genetics of human polymorphic N-acetyltransferase: enzymatic analysis of 15 recombinant wild-type, mutant, and chimeric NAT2 allozymes; Hein DW et al.; Human polymorphic N-acetyltransferase (NAT2) catalyzes the N-acetylation of arylamine drugs and carcinogens . Human acetylator phenotype is regulated at the NAT2 locus and has been associated with differential risk to certain drug toxicities or cancer . We examined arylamine substrate and acetyl coenzyme A cofactor affinities, and the N-acetyltransferase catalytic activities of the wild-type and 14 different mutant or chimeric human NAT2 alleles expressed in an Escherichia coli JM105 expression system . NAT2 alleles contained nucleic acid substitutions at positions 191(G-->A; Arg64-->Gln), 282(C-->T; silent), 341(T-->C; Ile114-->Thr), 481(C-->T; silent), 590(G-->A; Arg197-->Gln), 803(A-->G; Lys268-->Arg), 857(G-->A; Gly286-->Glu) and various combinations (282/590; 282/803; 282/857; 341/481; 341/803; 341/481/803; 481/803) of the 870 base pair NAT2 coding region . Expression of all 15 NAT2 alleles produced immunoreactive NAT2 protein with N-acetylation activity . NAT2 proteins encoded by alleles with nucleic acid substitutions at positions 191, 341, 590, 282/590, 341/481, 341/803, and 341/481/803 exhibited arylamine N-acetyltransferase maximum velocities significantly (P < 0.001) lower than the wildtype NAT2 . Thus, nucleic acid substitutions at positions 191, 341, and 590 either alone or in combination with other silent or conservative amino acid substitutions were sufficient to result in NAT2 proteins with significant reductions in N-acetylation activities . The recombinant NAT2 proteins also showed relative differences in intrinsic stability following incubation at 37 degrees C and 50 degrees C . NAT2 encoded by alleles with nucleotide substitutions at positions 191 and 857 were particularly unstable relative to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS) Biochem Mol Biol Int, 1994 May, 33(1), 195 - 204 Binding of Zn(II) to Escherichia coli DNA topoisomerase I; Zhu CX et al.; Titration of Escherichia coli DNA topoisomerase I with PMPS and 65Zn(II) binding showed independent release and binding of the three Zn(II) in each enzyme molecule . Removal of Zn(II) from topoisomerase I or top85 (truncated topoisomerase I with the Zn(II) binding domain at the carboxyl terminal) affected their sensitivity to Glu-C and Asp-N endoproteases but there was no significant effect on their rate of proteolysis by trypsin or Lys-C endoprotease . This suggested that Zn(II) removal did not result in complete unfolding of topoisomerase enzyme structure but only affected folding of small local regions . Digestion with carboxypeptidase Y further demonstrated that the folding of the zinc binding region itself was altered upon Zn(II) removal. Actas Urol Esp, 1994 May, 18(5), 604 - 6 {Acute peritonitis as a clinical manifestation of xanthogranulomatous pyelonephritis}; Vendrell JR et al.; Xanthogranulomatous pyelonephritis can become clinically apparent in a variety of manners . The paper contributes a very infrequent presentation; a picture of acute peritonitis following non-focal generalized toxic syndrome and haematuria episodes. Stem Cells, 1994 May, 12(3), 339 - 47 Overexpression and characterization of recombinant human fusion protein IL-6/IL-2 (CH925); Zhao C et al.; An expression vector encoding the human recombinant fusion protein interleukin 6/interleukin 2 (IL-6/IL-2) was constructed . When a flexible linker had been synthesized and ligated with the IL-2 gene fragment by polymerase chain reaction (PCR) amplification, the IL-6 gene fragment was unidirectionally inserted into the upstream of the linker-IL-2 sequence . The molecule of the IL-6-linker-IL-2 fusion gene named E . coli DH5 alpha/pfIL-6/2 was cloned and identified by DNA sequencing . The expressed protein named as CH925 showed a strong band on SDS-PAGE and amounted to 32% of total cell protein, and its estimated molecular weight was about 37 kDa . The fusion protein purified by gel filtration and reversed-phase HPLC showed as almost homogeneous . CH925 possesses both IL-2 and IL-6 activities when assayed by CTLL2- and 7TD1-dependent cell lines, respectively . The specific activity of IL-2 was 2.1 x 10(6) U/mg while that of IL-6 was 2.3 x 10(8) U/mg . Our studies exhibited that CH925 exerted a significant augmentative effect on the growth of erythroid colony forming units (CFU-E), and synergized with erythropoietin (EPO) and/or IL-3 in a dose-dependent way . Our experimental results also showed CH925 at a low dose causing active lymphokine-activated killer (LAK) cell proliferation more vigorous than IL-2 and/or IL-6 (p < 0.001) . CH925 is a novel fusion protein, being neither IL-6 nor IL-2, more potent than IL-2 and/or IL-6 and causing non-IL-2 and non-IL-6 functions of strong EPO-like and mild IL-3-like effects on erythroid progenitor cell growth . There is a potential for efficacious clinical application of CH925. Chem Res Toxicol, 1994 May-Jun, 7(3), 434 - 42 Specificity of in vitro covalent binding of tienilic acid metabolites to human liver microsomes in relationship to the type of hepatotoxicity: comparison with two directly hepatotoxic drugs; Lecoeur S et al.; In order to better understand the first steps leading to drug-induced immunoallergic hepatitis, we studied the target of anti-LKM2 autoantibodies appearing in tienilic acid-induced hepatitis, and the target of tienilic acid-reactive metabolites . It was identified as cytochrome P450 2C9, (P450 2C9): indeed, anti-LKM2 specifically recognized P450 2C9, but none of the other P450s tested (including other 2C subfamily members, 2C8 and 2C18) . Tienilic acid-reactive metabolite(s) specifically bound to P450 2C9, and experiments with yeast expressing active isolated P450s showed that P450 2C9 was responsible for tienilic acid-reactive metabolite(s) production . Results of qualitative and quantitative covalent binding of tienilic acid metabolite(s) to human liver microsomes were then compared to those obtained with two drugs leading to direct toxic hepatitis, namely, acetaminophen and chloroform . Kinetic constants (Km and Vmax) were measured, and the covalent binding profile of the metabolites to human liver microsomal proteins was studied . Tienilic acid had both the lowest Km and the highest covalent binding rate at pharmacological doses . For acetaminophen and chloroform, several microsomal proteins were covalently bound, while covalent binding was highly specific for tienilic acid and dihydralazine, another drug leading to immunoallergic hepatitis . Although low numbers of drugs were tested, these results led us to think that there may exist a relationship between the specificity of covalent binding and the type of hepatotoxicity. Res Vet Sci, 1994 May, 56(3), 379 - 85 Effects of weaning and enterotoxigenic Escherichia coli on net absorption in the small intestine of pigs; Nabuurs MJ et al.; The purpose of this study was to measure the net absorption of fluid, sodium, potassium and chloride in the small intestine of weaned pigs and of their unweaned littermates and to correlate these values with villus height and crypt depth . Five pairs of segments of the small intestine were prepared in each of 80 pigs; the cranial segment of each pair was injected with an enterotoxigenic strain of Escherichia coli and the caudal segment with a control solution . Net absorption was measured on the day of weaning and four, seven, 11 and 14 days after weaning . In unweaned pigs the net absorption of fluid, potassium and chloride did not vary with time . In weaned pigs the net absorption of fluid in the control segments was significantly less on days 4, 7 and 14 after weaning and of sodium and chloride on days 4 and 7 than in unweaned littermates . In infected segments of weaned pigs the net absorption of fluid was significantly less than in unweaned littermates on day 11 and 14, of sodium and potassium on day 11 and of chloride on days 4 and 11 after weaning . Net absorption was negatively correlated with villus height but only in the infected segments of weaned pigs; no other correlations were found . It was concluded that after weaning the net absorption of fluid and electrolytes in the small intestine of pigs is temporarily decreased, a condition that may initiate diarrhoea. Protein Eng, 1994 May, 7(5), 673 - 9 The use of alanine scanning mutagenesis to determine the role of the N-terminus of the regulatory chain in the heterotropic mechanism of Escherichia coli aspartate transcarbamoylase; Dembowski NJ et al.; The location of the first seven residues of the regulatory chain of Escherichia coli aspartate transcarbamoylase has been identified by X-ray crystallography to be near the binding site of the regulatory nucleotides . In order to determine the function of the N-terminus of the regulatory chain of aspartate transcarbamoylase in heterotropic regulation, alanine scanning mutagenesis was used . Specifically, Thr2r, His3r, Asp4r, Asn5r, Lys6r and Leu7r were each replaced with alanine . Analyses of these mutant enzymes indicate that none of these substitutions significantly alter the catalytic properties of the enzyme . However, three of the mutant enzymes, Asp4r-->Ala, Lys6r-->Ala and Leu7r-->Ala, exhibited notable changes in their response to the regulatory nucleotides, while mutations at Thr2r, His3r and Asn5r exhibited only minor changes in their heterotropic responses . For the Asp4r-->Ala enzyme, the responses to ATP and CTP were reduced approximately 30 and 40% respectively, compared with the wild-type enzyme . For the Lys6r-->Ala enzyme, the response to ATP was reduced approximately 70%, while the CTP response was reduced approximately 50% . In the case of the Leu7r-->Ala enzyme, a 30 and 20% reduction in response to ATP and CTP respectively, was observed . The synergistic inhibition by UTP in the presence of CTP for the Lys6r-->Ala enzyme was reduced approximately 40% compared with that of the wild type enzyme . For the Leu7r-->Ala enzyme, the synergistic inhibition was abolished . In addition, UTP decreased the CTP binding affinity of the Leu7r-->Ala enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) Protein Eng, 1994 May, 7(5), 663 - 71 A mutational analysis of receptor binding sites of interleukin-1 beta: differences in binding of human interleukin-1 beta muteins to human and mouse receptors; Grutter MG et al.; The 3-D crystal structure of interleukin-1 beta (IL-1 beta) has been used to define its receptor binding surface by mutational analysis . The surface of IL-1 beta was probed by site-directed mutagenesis . A total of 27 different IL-1 beta muteins were constructed, purified and analyzed . Receptor binding measurements on mouse and human cell lines were performed to identify receptor affinities . IL-1 beta muteins with modified receptor affinity were evaluated for structural integrity by CD spectroscopy or X-ray crystallography . Changes in six surface loops, as well as in the C- and N-termini, yielded muteins with lower binding affinities . Two muteins with intact binding affinities showed 10- to 100-fold reduced biological activity . The surface region involved in receptor binding constitutes a discontinuous area of approximately 1000 A2 formed by discontinuous polypeptide chain stretches . Based on these results, a subdivision into two distinct local areas is proposed . Differences in receptor binding affinities for human and mouse receptors have been observed for some muteins, but not for wild-type IL-1 beta . This is the first time a difference in binding affinity of IL-1 beta muteins to human and mouse receptors has been demonstrated. Neuroscience, 1994 May, 60(2), 299 - 309 Grafting of nerve growth factor-producing fibroblasts reduces behavioral deficits in rats with lesions of the nucleus basalis magnocellularis; Dekker AJ et al.; Rats received bilateral lesions of the nucleus basalis magnocellularis by infusion of biotenic acid . Two weeks after the lesion, a suspension of genetically modified primary rat fibroblasts was grafted dorsal to the nucleus basalis magnocellularis (2 x 10(5) cells per side) . The fibroblasts were either infected with the gene for human beta-nerve growth factor or Escherichia coli beta-galactosidase . The nerve growth factor-producing fibroblasts released 67 ng nerve growth factor/10(5) cells per day in vitro . Two weeks after implantation of the fibroblasts, spatial learning was tested in the Morris water-maze . Nerve growth factor-producing fibroblasts, but not beta-galactosidase-producing fibroblasts ameliorated the deficit in acquisition of the water-maze task . In addition, spatial acuity was improved to near-normal levels by the nerve growth factor-producing grafts . Choline acetyltransferase activity in cortical areas and hippocampus was not affected by the nerve growth factor-producing grafts . Both grafted groups showed a similar reduction in the level of dopamine, but not homovanillic acid or 3-methoxytyramine, in the frontal cortex . Levels of norepinephrine, epinephrine and serotonin and their metabolites in the neocortex and hippocampus were not affected by the lesion or the grafts . Nerve growth factor-producing grafts increased the size of remaining nerve growth factor-receptor (p75) immunoreactive neurons in the nucleus basalis magnocellularis by 25% . Nucleus basalis magnocellularis lesions reduced the integrated optic density of choline acetyltransferase-positive fiber staining in the ventral neocortex by 46%, but nerve growth factor-producing grafts restored this area to 86% of control . These data suggest that nerve growth factor-producing grafts can cause a marked behavioral improvement, probably through the partial restoration of the lesioned projection from nucleus basalis magnocellularis to neocortex. J Neurobiol, 1994 May, 25(5), 585 - 94 The survival of rat cerebral cortical neurons in the presence of trophic APP peptides; Yamamoto K et al.; One function of Alzheimer amyloid protein precursor (APP) is the regulation of growth and differentiation in several types of cells, including fibroblasts, PC12 cells, and neurons . This activity is represented by a small stretch of amino acids in the center of the molecule around RERMS . The APP 17-mer peptide containing the RERMS domain supported survival and neurite extension of rat cortical neurons in a dose-dependent and sequence-specific manner . The APP fragment synthesized in Escherichia coli supported the survival and neurite extension of rat cortical neurons, whereas the mutant APP fragment lacking the 30 amino acids around the RERMS domain had drastically reduced activity to support the survival and neurite extension . The current study established APP as a neuron survival factor and determined that the sequence around RERMS is important for this function. J Virol Methods, 1994 May, 47(3), 279 - 95 Investigation of the coxsackievirus B3 nonstructural proteins 2B, 2C, and 3AB: generation of specific polyclonal antisera and detection of replicating virus in infected tissue; Hohenadl C et al.; The coxsackievirus B3 (CVB3) nonstructural proteins 2B and 3AB were synthesized as beta-galactosidase fusion proteins in E . coli in order to generate specific polyclonal antisera . 2B and 3AB fusion proteins were purified by preparative SDS-polyacrylamide gel electrophoresis and inoculated into rabbits . Protein 2C-specific antiserum was produced using synthetic oligopeptides which were defined by computer based amino acid sequence analysis . Specificity of the generated antisera was analysed by immunoblotting, immunofluorescence, immunohistochemistry and immunoelectron microscopy . All antisera allowed specific detection of the viral proteins 2B, 2C, and 3AB in CVB3-infected cells as well as in myocardial and pancreatic tissue of CVB3-infected mice . In addition, the CVB3 2C-specific antiserum was shown to be highly cross-reactive with the analogous protein of other picornaviruses, including cardiotropic enterovirus serotypes such as coxsackievirus A9, coxsackievirus B (types 1-5), and echovirus 11 . Moreover, the immunological detection of nonstructural proteins enables diagnosis of replicating virus in infected tissue . These results demonstrate that the generated antisera are valuable tools for diagnostic approaches . Furthermore, they may help to elucidate the role of the nonstructural proteins 2B, 2C, and 3AB in enteroviral replication and pathogenesis. Int J Pept Protein Res, 1994 May, 43(5), 441 - 7 Human parathyroid hormone: efficient synthesis in Escherichia coli using a synthetic gene, purification and characterization; Oshika Y et al.; Human parathyroid hormone is a peptide hormone consisting of 84 amino acid residues . Production of small proteins by direct expression in Escherichia coli is often unsuccessful owing to susceptibility of the mRNA and/or the product to endogenous enzymes . In this study, direct expression of the hormone at an excellent level (over 100 mg/L) has been achieved by using a suitably designed synthetic gene under the control of the T7 promoter . The protein produced in bacteria was extracted and easily purified in a good yield of 27 mg/L . The purified product was physico-chemically identified as intact human parathyroid hormone from the results of amino acid analysis, N-terminal sequencing, and peptide mapping using fast atom bombardment mass spectrometry . In biological assays the purified product stimulated adenylate cyclase in vitro, promoted bone growth and increased the serum osteocalcin in rats to the same extent as the authentic hormone. Drug Metab Dispos, 1994 May-Jun, 22(3), 486 - 97 N-hydroxylation of the antiprotozoal drug pentamidine catalyzed by rabbit liver cytochrome P-450 2C3 or human liver microsomes, microsomal retroreduction, and further oxidative transformation of the formed amidoximes . Possible relationship to the biological oxidation of arginine to NG-hydroxyarginine, citrulline, and nitric oxide; Clement B et al.; Previous investigations have shown that the antiprotozoal drug pentamidine is N-hydroxylated by rabbit and rat liver microsomal fractions . Indirect evidence for the participation of the cytochrome P-450 enzyme system was obtained . In this study, rabbit liver cytochrome P-450 2C3 is shown by reconstitution experiments with highly purified variants of P-450 2C3 isolated from rabbit liver and purified variants of P-450 2C3 expressed by recombinant Escherichia coli to be a microsomal pentamidine N-hydroxylase . The two variants, P-450 2C3 (6 beta H) and P-450 2C3 (6 beta L), are equally efficient for the formation of the monoamidoxime derivative of pentamidine . N-hydroxypentamidine is further oxidized to the respective amide by reconstituted rabbit liver P-450 enzyme systems involving the oxidase and peroxidase activities of this enzyme . Formation of nitric oxide {(NO); endothelium-derived relaxing factor} during this oxidation is shown by the detection of the cytochrome P-420-Fe(II)-NO complex by visible difference spectroscopy . The possibility for the N-hydroxylation of pentamidine to the corresponding amidoximes and subsequent oxidative conversion to the respective amide derivatives is comparable with the physiological transformation of arginine to citrulline via N-hydroxyarginine with liberation of NO (endothelium-derived relaxing factor) . The N-hydroxylated derivatives of pentamidine are easily retroreduced by microsomal fractions from rabbit liver . NADH is preferred to NADPH as cofactor for this reduction, and the reaction is strongly suppressed by the addition of N-methylylhydroxylamine . The N-hydroxylation of pentamidine and the retroreduction are also catalyzed by human liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS) Chromosome Res, 1994 May, 2(3), 171 - 83 Gene ecology: a cis-acting gene-to-gene interaction due to the spatial arrangement of genes in chromosomes affects neighbouring transfected c-H-ras expression; Naora H et al.; A cis-acting interference between gene activities, which occurs when two genes lie on the same DNA strand and have an intergenic distance less than a defined length, was previously deduced when chromosomal organizations of various higher eukaryote nuclear genes in clusters were compared . In order to investigate such an interference due to arrangement of genes along chromosomes, we have isolated a few cell lines which possessed (i) human mutated c-H-ras fused with the mouse mammary tumour virus long terminal repeat and (ii) the E . coli xanthine-guanine phosphoribosyltransferase (gpt) gene with the SV40 promoter, on the same or on different DNA strands, separated by a short intergenic distance or unlinked . Since the cancerous phenotype of a cell can be readily identified due to c-H-ras expression, we examined in these cell lines whether continuous c-H-ras expression, induced by dexamethasone, is disturbed through a cis-acting gene-to-gene interaction when the expression of the neighbouring gpt gene is enforced and as a result, the cancerous state of a cell is converted to the 'normal' state . The enforced expression of the neighbouring gpt gene was shown to alter c-H-ras expression, and thus reversible conversion of a cell between cancerous and normal states occurred only when the cell possessed an optimum number of the gene pair, in which both c-H-ras and the gpt gene were on the same DNA strand . This implies that the spatial arrangement of genes in chromosomes plays an important role in the regulation of gene expression in a cluster. In Vitro Cell Dev Biol Anim, 1994 May, 30A(5), 300 - 5 Activity assays of nine heterogeneous promoters in neural and other cultured cells; Fukuchi K et al.; To express high levels of proteins encoded by transfected DNA constructs in a variety of cultured cells, including neuronal cells, the activities of nine different promoters were evaluated using Escherichia coli beta-galactosidase (beta-gal) (LacZ) as a reporter gene . These nine promoters were categorized into three distinct groups (high, intermediate, and low expresser), in terms of the levels of beta-gal expression . An expression vector containing the cytomegalovirus enhancer and the chick beta-actin promoter (high expresser) showed the highest levels of expression, followed by vectors containing the cytomegalovirus promoter/enhancer and the SV40 promoter/enhancer (intermediate expresser) . The rest of the promoters (thymidine kinase, adenovirus, murine proliferative sarcoma virus, nerve growth factor receptor, Rous sarcoma and mouse mammary tumor virus, and beta-amyloid precursor protein) expressed low levels of beta-gal . These results were consistent for eight different cell types . A particularly attractive model is the stem cell, P19; cultures differentiating into progeny consisting predominantly of cholinergic neurons could be readily transfected with expression vectors using liposomes and expressed beta-gal without significant morphologic changes of the differentiated neurons . The systems should be useful for the study of promoters and various expressed proteins, including those involved in axonal transport. Biotechniques, 1994 May, 16(5), 916 - 23 A versatile plasmid vector system for the regulated expression of genes in Escherichia coli; Diederich L et al.; A series of plasmid expression vectors, which support the regulated and efficient expression of genes in Escherichia coli, have been constructed . The vectors consist of a DNA replication origin cassette, a promoter cassette, an efficient ribosome binding site together with a polylinker region and a lacZ gene . Several types of replication origins and promoter sequences are each available on cassettes . Fusion of the 5' TG dinucleotide of the gene under consideration to the A nucleotide, present on the vector, results in an ATG start codon and allows, in combination with the plasmid-borne ribosome binding site, the efficient expression of the cloned gene . Additionally, a second fusion of the gene at its 3' end with the lacZ gene, which is available in all three reading frames relative to the polylinker region, allows rapid selection of the correctly fused genes . As an example of the cloning of a regulatory gene, this vector system was used for the expression of the dnaA gene, of Escherichia coli, the initiator protein for DNA replication. Biotechniques, 1994 May, 16(5), 888 - 93 New toxicity determination method that uses fluorescent assay of Escherichia coli; Mariscal A et al.; We describe a new method that uses a fluorogenic bioassay of the beta-glucuronidase conversion of 4-methylumbelliferyl beta-D-glucuronide (MUG) to 4-methylumbelliferone to evaluate the individual toxic effects on Escherichia coli of Al3+, Cr6+, Hg2+ and Li+ . This work was designed to examine the effectiveness of this method to measure the effects of five ionic concentrations of either Al3+, Cr6+, Hg2+ or Li+, on the growth of E . coli in a minimal medium that had MUG as the only source of carbon . This method was simple and fast, and its toxicity detection sensitivity was equal to, or greater than, existing bacterial bioassays . The use of the MUG substrate minimized the danger of interference by bacteria other than E . coli . Evaluations of toxicity in samples of public drinking water proved equally sensitive. Proteins, 1994 May, 19(1), 77 - 9 Expression, purification, and crystallization of restriction endonuclease PvuII with DNA containing its recognition site; Balendiran K et al.; We have overexpressed the type II restriction endonuclease PvuII (R-PvuII) in E . coli, prepared large amounts of the homogeneous enzyme, and crystallized it with an oligonucleotide carrying a PvuII recognition site . The cocrystals are orthorhombic space group P2(1)2(1)2(1) with cell constants a = 95.8 A, b = 86.3 A, c = 48.5 A, and diffract X-rays to at least 2.7 A . There is a complex of two protein subunits and one oligonucleotide duplex in the asymmetric unit. Proteins, 1994 May, 19(1), 48 - 54 Crystallization of a fragment of human fibronectin: introduction of methionine by site-directed mutagenesis to allow phasing via selenomethionine; Leahy DJ et al.; Crystals of a fragment of human fibronectin encompassing the 7th through the RGD-containing 10th type III repeats (FN7-10) have been produced with protein expressed in E . coli . The crystals are monoclinic with one molecule in the asymmetric unit and diffract to beyond 2.0 A Bragg spacings . A mutant FN7-10 was produced in which three methionines, in addition to the single native methionine already present, have been introduced by site-directed mutagenesis . Diffraction-quality crystals of this mutant protein have been grown in which methionine was replaced with selenomethionine . The introduction of methionine by site-directed mutagenesis to allow phasing from selenomethionyl-substituted crystals is shown to be feasible by this example and is proposed as a general approach to solving the crystallographic phase problem . Strategies for selecting propitious sites for methionine mutations are discussed. Naunyn Schmiedebergs Arch Pharmacol, 1994 May, 349(5), 523 - 7 Involvement of neuronal processes and nitric oxide in the inhibition by endotoxin of pentagastrin-stimulated gastric acid secretion; Martinez-Cuesta MA et al.; Administration of E . coli endotoxin (1 mg kg-1, i.v.) abolished the acid response induced by the i.v . infusion of pentagastrin (8 micrograms kg-1 h-1) in the continuously perfused stomach of the anaesthetized rat . Local serosal application of tetrodotoxin (36 ng per rat) completely restored acid responses to pentagastrin in endotoxin-treated rats . However, pretreatment with atropine (0.5 mg kg-1, s.c.), capsaicin (20, 30, and 50 mg kg-1, s.c . 2 weeks before the study) or guanethidine (16 mg kg-1, s.c . 3 and 16h before) did not influence the inhibitory effects of endotoxin . Continuous i.v . infusion with NG-nitro arginine methyl ester (L-NAME, 10 mg kg-1 h-1) restored the secretory responses to pentagastrin in endotoxin treated rats . The effects of L-NAME were reversed by L-arginine (100 mg kg-1 h-1, i.v.), but not by its enantiomer D-arginine (100 mg kg-1 h-1, i.v.) . The secretory responses elicited by pentagastrin (10(-10)-10(-6) M) in the isolated lumen perfused stomach of the rat were not influenced by incubation (100 min) with endotoxin (10 micrograms ml-1) . These observations with tetrodotoxin indicate that inhibition of acid secretion by endotoxin in vivo involves neuronal activity, while inhibition of NO synthesis had a comparable inhibitory action . Activation of a systemic non-adrenergic non-cholinergic neuronal pathway involving NO could thus mediate the acute acid inhibitory effects of endotoxin. Mol Gen Mikrobiol Virusol, 1994 May-Jun, (3), 20 - 2 {Cloning the human cytomegalovirus immediate-early gene and expression of it in Escherichia coli}; Klichko VI et al.; The major immediate early gene of human cytomegalovirus has been cloned . The fragment of the immediate early protein containing 351 amino acids has been fused with cro-beta-galactosidase and expressed in Escherichia coli cells . The obtained recombinant protein reacts in immunoblotting with the antibodies in human sera from patients suffering from acute cytomegalovirus infection. Mol Microbiol, 1994 May, 12(3), 433 - 44 Regulation of transcription at the ndh promoter of Escherichia coli by FNR and novel factors; Green J et al.; FNR is a transcriptional regulator that controls gene expression in response to oxygen limitation in Escherichia coli . The NADH dehydrogenase II gene (ndh) is repressed by FNR under anaerobic conditions . Repression is not simply due to occlusion of the promoter (-35 and -10) region by FNR because adjacent pairs of FNR monomers were found to bind at two sites centred at -50.5 and -94.5 in the ndh promoter region without preventing RNA polymerase binding . However, contact between RNA polymerase and the -132 to -62 region of the non-coding strand of ndh DNA, and RNA polymerase-mediated open complex formation, were prevented by bound FNR . The upstream FNR-binding site (-94.5) was needed for efficient FNR-dependent repression of ndh transcription in vitro, and also for repression of an ndh-lacZ fusion in vivo . Anaerobic ndh repression may thus involve the binding of two pairs of FNR monomers upstream of the -35 region, which prevents essential RNA polymerase-DNA contacts in the upstream region as well as inhibiting RNA polymerase function by direct FNR interaction . Expression of the ndh-lacZ fusion in an fnr deletion strain was enhanced by anaerobic growth in rich medium or minimal medium supplemented with amino acids . Furthermore, two proteins (M(r) 12,000 and 35,000) which interact with and may activate transcription from the ndh promoter under these conditions were detected by gel retardation analysis . These putative amino acid-responsive activators may thus offset FNR-mediated repression and maintain a low level of anaerobic ndh expression for regulating the NAD+/NADH ratio during growth in rich media. J Reprod Fertil, 1994 May, 101(1), 227 - 33 Effect of active immunization against recombinant-derived chicken prolactin fusion protein on the onset of broodiness and photoinduced egg laying in bantam hens; March JB et al.; The hypothesis that the onset of incubation behaviour (broodiness) in the domestic hen is induced by an increase in prolactin secretion was investigated by actively immunizing bantam hens against recombinant-derived chicken prolactin . A second objective was to establish whether active immunization against prolactin affects photoinduced onset of egg laying and the rate of egg production . The immunogen was a fusion protein (beta gals-prolactin, 23 kDa) produced in Escherichia coli, comprising chicken prolactin (without the nine amino-terminal amino acids) fused to 18 amino acids of E . coli beta-galactosidase . A control immunogen was produced in the same strain of E . coli harbouring the same plasmid vector used to produce beta gals-prolactin minus the prolactin gene sequence . Hens were immunized i.m . with 1 mg of protein containing 0.8-0.9 mg of fusion protein in Freund's incomplete adjuvant at 4-8 week intervals beginning before or after egg laying, which was induced by increasing the daily photoperiod . The beta gals-prolactin immunogen, but not the control immunogen, stimulated the production of antibodies to chicken prolactin . In Expts 1, 2 and 3, hens were placed in floor pens with nest boxes after photostimulation to induce broodiness . In these experiments, immunization with beta gals-prolactin reduced the incidence or delayed the development of broodiness . This effect was more pronounced if immunization was initiated before, rather than after, the onset of egg laying . In Expts 1 and 2 hens were immunized with beta gals-prolactin before photostimulation . The presence of antibodies to prolactin in their blood did not affect photoinduced onset of egg laying.(ABSTRACT TRUNCATED AT 250 WORDS) J Dairy Res, 1994 May, 61(2), 191 - 9 Triacylglycerol fatty acid composition of milk from periparturient cows during acute Escherichia coli mastitis; Massart-Leen AM et al.; Changes in fat concentration and triacylglycerol fatty acid (TGFA) composition were studied in milk from six periparturient cows 1 d before and 20 d after an experimentally induced Escherichia coli mastitis in the fore and rear homolateral quarters . Opposite fore and rear heterolateral quarters remained uninfected and were used as controls . Milk was collected from all individual quarters during the experiment . The fat concentration in milk from infected quarters did not change, but total fat production decreased owing to reduced milk production after the Esch . coli challenge . In milk from the heterolateral uninfected quarters fat concentration rose significantly 48 and 72 h after induction of mastitis, the rise being concomitant with a decrease in milk production . Throughout the experiment similar changes in TGFA composition were observed for both infected and uninfected quarters . There was an increase in all the even, odd-numbered, iso and anteiso short-chain TGFA from day +6 on after induction of mastitis . There was little change in the composition of 16:0 and 18:0 fatty acids, while the long-chain unsaturated fatty acids decreased . Using multivariate analysis, the results are presented visually . The observed changes in the TGFA can be ascribed to changes normally observed in cows' milk soon after parturition. Protein Sci, 1994 May, 3(5), 853 - 6 A novel zinc-binding motif found in two ubiquitous deaminase families; Reizer J et al.; Two families of deaminases, one specific for cytidine, the other for deoxycytidylate, are shown to possess a novel zinc-binding motif, here designated ZBS . We have (1) identified the protein members of these 2 families, (2) carried out sequence analyses that allow specification of this zinc-binding motif, and (3) determined signature sequences that will allow identification of additional members of these families as their sequences become available. Protein Sci, 1994 May, 3(5), 799 - 809 Structure of glutathione reductase from Escherichia coli at 1.86 A resolution: comparison with the enzyme from human erythrocytes; Mittl PR et al.; The crystal structure of the dimeric flavoenzyme glutathione reductase from Escherichia coli was determined and refined to an R-factor of 16.8% at 1.86 A resolution . The molecular 2-fold axis of the dimer is local but very close to a possible crystallographic 2-fold axis; the slight asymmetry could be rationalized from the packing contacts . The 2 crystallographically independent subunits of the dimer are virtually identical, yielding no structural clue on possible cooperativity . The structure was compared with the well-known structure of the homologous enzyme from human erythrocytes with 52% sequence identity . Significant differences were found at the dimer interface, where the human enzyme has a disulfide bridge, whereas the E . coli enzyme has an antiparallel beta-sheet connecting the subunits . The differences at the glutathione binding site and in particular a deformation caused by a Leu-Ile exchange indicate why the E . coli enzyme accepts trypanothione much better than the human enzyme . The reported structure provides a frame for explaining numerous published engineering results in detail and for guiding further ones. Protein Sci, 1994 May, 3(5), 730 - 6 Determination of the binding frame within a physiological ligand for the chaperone SecB; Topping TB et al.; The hallmark of the class of proteins called chaperones is the amazing ability to bind tightly to a wide array of polypeptide ligands that have no consensus in sequence; chaperones recognize non-native structure . As a step in the elucidation of the molecular mechanism of such remarkable binding, we have characterized complexes between the bacterial chaperone SecB and a series of ligands related to maltose-binding protein . SecB interacts at multiple sites on its polypeptide ligand . The entire binding region covers approximately half of the primary sequence of maltose-binding protein and comprises contiguous sites positioned around the center of the sequence. Biophys J, 1994 May, 66(5), 1604 - 11 Structures of wild-type and mutant signal sequences of Escherichia coli ribose binding protein; Yi GS et al.; The structure of a chemically synthesized 25-residue-long functional signal peptide of Escherichia coli ribose binding protein was compared with that of a nonfunctional mutant-signal peptide using circular dichroism and two-dimensional 1H NMR in solvents mimicking the amphiphilic environments . The functional peptide forms an 18-residue-long alpha-helix starting from the NH2-terminal region and reaching to the hydrophobic stretch in a solvent consisting of 10% dimethylsulfoxide, 40% water, and 50% trifluoroethanol (v/v) . The nonfunctional mutant peptide, which contains a Pro at position 9 instead of a Leu in the wild-type peptide, does not have any secondary structure in that solvent but forms a 12-residue-long alpha-helix within the hydrophobic stretch in water/trifluoroethanol (50:50, v/v) solvent . It seems that the Pro-9 residue in the nonfunctional peptide disturbs the helix propagation from the hydrophobic stretch to the NH2-terminal region . Because both of these peptides have stable helices within the hydrophobic stretch, it may be concluded that the additional 2 turns of the alpha-helix in the NH2-terminal region of the wild-type signal peptide is important for its function. Z Naturforsch {C}, 1994 May-Jun, 49(5-6), 352 - 8 Resonance effect of low-intensity millimeter waves on the chromatin conformational state of rat thymocytes; Belyaev SYa et al.; The method of anomalous viscosity time dependencies (AVTD) was modified for the study of the changes in the chromatin conformational state (CCS) of rat thymocytes of the Wistar line . The response of the thymocytes of male rats to low-intensity millimeter waves (MMW) was examined . It was shown that MMW at power densities (PD) of 1 microW/cm2 produced a resonance effect on the CCS in the frequency range of 41.56-41.67 GHz . The resonance frequency of the cell response did not vary significantly among five examined rats and was determined to be 41.61 +/- 0.01 GHz . A halfwidth of resonances was averaged to 40 MHz . The power dependence of the resonance effect was measured in the range of 10(-11)-10(-4) W/cm2 . Statistically significant changes in CCS were registered, starting with 10(-9) W/cm2 . Right- and left-handed circularly polarized MMW were shown to differ in efficiency at the resonance frequency . The established regularities in the thymocyte response to low-intensity MMW was very similar to those which have been previously found for E . coli cells. Plasmid, 1994 May, 31(3), 297 - 9 Versatile Escherichia coli expression vectors for production of truncated proteins; Omori K et al.; Several expression vector plasmids containing the tac promoter, the rrnBT1T2 terminator, and the pUC ori sequence were constructed . Some of them, the pES series, have a start codon in all three reading frames and multiple cloning sites downstream of the tac promoter and have stop codons also in all three frames and additional stop codons accompanying restriction sites . They are designed for versatile expression of truncated proteins which are produced by deleting portions of the inserted DNA. Brain Res Mol Brain Res, 1994 May, 23(3), 221 - 34 Identification of two promoter regions in the rat B-50/GAP-43 gene; Eggen BJ et al.; To determine cis-acting elements controlling the rat B-50/GAP-43 gene expression, the genomic DNA encoding exon 1 and the 5' flanking sequence was isolated . Sequence analysis of 1 kb 5' untranslated region (UTR) revealed the presence of a (GA)-repeat and a (GT)-repeat . The size of the (GA)-repeat varied due to both an instability of phage lambda lambda DNA in E . coli and genomic variation between rats . Transcription initiation sites were mapped in 8-day-old rat brain poly(A)+ mRNA . Primer extension indicated multiple transcription start sites at -159 and -339/-342 nt upstream of the translation start site; reverse transcriptase coupled PCR showed that the most 5' transcription start site is located between -465 and -440 . Northern blotting demonstrated that approximately 90% of the B-50 mRNAs initiates at approximately -50 . Promoter analysis by transient transfection assays in undifferentiated and retinoic acid-differentiated P19-EC cells revealed that the rat B-50 gene contains two promoters . P1 (located between -750 and -407) contains commonly observed promoter elements such as a TATA box and CCAAT boxes . P2 (located between -233 and -1) neither contains TATA boxes, CCAAT boxes nor consensus sequences of house-keeping gene promoters like GC-boxes . The activity of P1 is inhibited at neuroectodermal differentiation of P19-EC cells whereas the activity of P2 is stimulated . In 8 day old rat brain the majority of the B-50 mRNA transcripts are derived from P2 . It is concluded that at this developmental stage P2 is the most important promoter. Bioorg Khim, 1994 May, 20(5), 524 - 35 {Expression of the gene coding for the D1-protein of barley photosystem II in Escherichia coli}; Efimov VA et al.; Previously characterized by us barley chloroplast psbA gene, which encodes one of the main Photosystem II components--D1 protein, has been inserted in a set of special plasmid vectors and its expression in vitro and in vivo has been investigated . Experiments on the in vitro expression in the rabbit reticulocyte lysate system revealed a major product with a molecular weight ca . 33.5 kD, which corresponds to the unprocessed D1 barley protein . A lower molecular weight protein (about 29 kD) was also observed . These results are in agreement with the existence of two potential translation start sites in the psbA gene in the same reading frame, the second one starting from Met37 residue . The results fully correlate with the earlier data on the in vitro expression of psbA genes of maize, pea, and tobacco . Experiments on the in vivo expression of psbA gene in E . coli cells with the above constructions also revealed proteins with m . w . about 33.5 and 29 kD . The yield of the target recombinant protein in some cases was about 25-30% of the total E . coli cellular protein . The correspondence of the bands to the desired products was proved by the immunoenzyme analysis with the use of polyclonal antibodies . The data obtained show for the first time the construction of E . coli strains producing recombinant D1 protein of cereals in a high level. Mol Biol (Mosk), 1994 May-Jun, 28(3), 658 - 64 {Binding of a fragment of yeast phenylalanyl tRNA containing an anticodon loop with 30S and 70S ribosomes from Escherichia coli . The role of guanosine-42 in this interaction}; Nekhai SA et al.; Poly(U)-dependent binding of isolated yeast tRNA(Phe) anticodon hairpin (15-nucleotide-long, corresponding to nucleotides 28-42 within the tRNA) and several its derivatives to the P site of Escherichia coli 30S and 70S ribosomes was studied quantitatively . The affinity for the hairpin binding to 70S ribosomes was shown to be only 30-fold weaker than that for the binding of total tRNA(Phe) . Within the anticodon hairpin, removal of the 3'-terminal nucleotide corresponding to guanosine-42 in tRNA(Phe) decreases the association constant for the anticodon arm-ribosome interaction 15-fold . Replacement of this guanosine with other nucleosides does not affect the affinity, regardless of involvement in the hairpin secondary structure . These data indicate that G-42 affects the anticodon arm affinity most likely by forming a direct contact with the ribosome . One can assume that this nucleotide within intact tRNA also forms a contact with the P site . Since the 3'-terminal ribose modifications (oxidation, oxidation and reduction) as well as the presence or absence of the 3'-terminal phosphate does not affect the affinity of the anticodon arm fragment, the latter is obviously involved in the interaction through 3'-terminal nucleotide base groups which does not take part in base pairing. Mol Biol (Mosk), 1994 May-Jun, 28(3), 633 - 40 {Interaction of double-stranded DNA in the Escherichia coli RecA protein system with modified oligonucleotides containing an alkylated terminal group}; Kosaganov IuN et al.; We studied modification of double-stranded DNA with chemically active homologous oligonucleotide derivatives carrying an alkylating 4-{N-2-chloroethyl-N-methylamino)benzyl}aminogroup in DNA recombination reaction promoted by E . coli RecA protein . It was shown that this chemical group does not prevent the formation of a complex between RecA protein and oligonucleotide as well as binding of this complex to double-stranded DNA . Formation of cross-links between DNA and oligonucleotide derivatives was observed at oligonucleotide lengths of no less than 30 . The supercoiled form of the plasmid was alkylated with a higher efficacy than relaxed or linear form . In spite of homology between oligonucleotide and a certain region of double-stranded DNA (pBR322), cross-links were observed throughout the DNA length including nonhomologous regions . The causes of such behavior are discussed. Mol Biol (Mosk), 1994 May-Jun, 28(3), 610 - 8 {Selectivity of A-site on Escherichia coli ribosomes in relation to deacylation of transfer RNA}; Ivanov IuV et al.; To evaluate the equilibrium affinity of several deacylated tRNA from E . coli and yeast tRNA(Phe) for the A site of poly(U)- and poly(A)-programmed E . coli ribosomes, we studied their inhibitory effect on the subsequent binding of the template-cognate ternary complex EFTu*GTP* {14C}aminoacyl-tRNA . At 0 degree C and 10 mM Mg2+, affinities of template-cognate tRNAs were characterized by association constants Ka approximately 1-7.10(7) M-1 . At least for the poly(U)-programmed system, this value practically coincided with the one determined earlier from direct nonenzymatic binding . Splitting off two 3'-terminal nucleotides did not appreciably change the tRNA affinity, that is, these nucleotides do not form direct contacts with the A site . Noncognate tRNAs were 120-1800-fold weaker as inhibitors; there was no considerable correlation between tRNA affinity for the A site and the extent of noncomplementarity of its anticodon to the template . The only except was tRNA(Tyr) in the system with poly(U), which displayed an affinity only 9-fold lower than that of template-cognate tRNA(Phe) . The results obtained here show that the mRNA-programmed ribosomal A site reveals selectivity not only for energy-dependent enzymatic binding of aminoacyl-tRNA, but also for nonenzymatically bound nonacylated tRNA . Even in this simplified model, the selectivity is much higher than that observed when codons interact with anticodons in solution without ribosomes. Mol Biol (Mosk), 1994 May-Jun, 28(3), 595 - 601 {Expression of hybrid genes containing sequences coding for human adrenocorticotropic hormone in Escherichia coli strains}; Karapetian VE et al.; E . coli strains producing a hybrid protein containing human adrenocorticotropic hormone (ACTH) and protein A of S . aureus were obtained . The sequence coding for ACTG was obtained from the bovine one using oligonucleotide-directed mutagenesis . The ACTG gene was linked with the protein A gene and its derivatives by synthetic adaptors . It was shown that each of the constructed plasmids directed the synthesis of hybrid protein in E . coli . This protein was purified on IgG-Sepharose . Then ACTH was obtained by HPLC after specific hydrolysis . The amino acid composition of purified sample was determined. Mol Biol (Mosk), 1994 May-Jun, 28(3), 563 - 73 {The recombination mechanism for precise excision of the IS50 mobile element in Escherichia coli K12 cells}; Kil' IuV et al.; Precise excision of composite transposons of Tn10 type, with long inverted repeats, proceeds according to the slippage mechanism (Brunier et al . parallel Cell . 1988 . V . 52 . P . 883) . An alternative recombinational mechanism proved to be possible for transposons with directly oriented IS elements (Goryshin et al . parallel Mol . Biol . 1991 . V . 25 . P . 614) . Making use of an experimental system with plasmid localization of the Tn5 transposon or its IS50 module, we have shown that the recombination mechanism of precise excision can be realized for these mobile elements . The recombination occurs in-trans between direct short repeats flanking a transposon . Formation of a specialized dimer of the original plasmid takes place in this case . The recombination does not depend on the integrity of RecA protein or transposase . Integration of an ori of replication into the mobile element results in the domination of recombination mechanism of precise excision even if the transposon has long inverted repeats at its ends . This seems to occur as a result of inhibition of the slippage mechanism. Mol Biol (Mosk), 1994 May-Jun, 28(3), 496 - 505 {Heterologous secretion in the Escherichia coli system}; Debabov DB; The mechanisms of protein secretion in Escherichia coli are considered . Vector systems intended for secretion of eukaryotic proteins both into the periplasmic space and the culture medium are discussed. J Clin Microbiol, 1994 May, 32(5), 1295 - 301 Genotypic and phenotypic identification of coli surface antigen 6-positive enterotoxigenic Escherichia coli; Grewal HM et al.; A polynucleotide probe comprising the gene encoding a major structural subunit protein of coli surface antigen 6 (CS6) of enterotoxigenic Escherichia coli (ETEC) was developed . Eighty-nine ETEC isolates were examined in parallel with the probe in a colony hybridization assay and in a recently developed polyclonal-antibody-based inhibition enzyme-linked immunosorbent assay (ELISA) . The two assays showed a high level of concordance in the detection of CS6-positive ETEC (kappa = 0.84, P < 0.00001) . Thus, 36 of the 89 ETEC isolates were identified as CS6-positive by both assays . Six strains that were negative for other colonization factor antigens were positive with the CS6 probe but negative in the ELISA, suggesting lack of surface CS6 expression in these strains . One strain was probe negative but positive in the ELISA, while the remaining 46 strains were negative in both assays . The phenotypic and genotypic assays will prove useful in vaccine-oriented studies of ETEC disease. J Clin Microbiol, 1994 May, 32(5), 1197 - 202 Clonal relationships among Escherichia coli serogroup O78 isolates from human and animal infections; Cherifi A et al.; We investigated the clonal relationships among 63 Escherichia coli strains of antigen serogroup O78 isolated from infections in humans, cattle, sheep, pigs, and chickens . Both septicemic and enterotoxigenic isolates were included in the study . A main group of 55 E . coli strains consisting of 52 septicemic isolates and 3 human enterotoxigenic E . coli isolates were clustered in related clones . The remaining eight strains, four human and four animal isolates, were clonally heterogeneous . The main group of 55 clonally related strains included isolates from human and animal infections . This result indicates that animals are a possible source of serogroup O78 septicemic E . coli infections in humans. J Eukaryot Microbiol, 1994 May-Jun, 41(3), 204 - 9 Ribosomal RNA sequences of Enterocytozoon bieneusi, Septata intestinalis and Ameson michaelis: phylogenetic construction and structural correspondence; Zhu X et al.; The microsporidian species Enterocytozoon bieneusi, Septata intestinalis and Ameson michaelis were compared by using sequence data of their rRNA gene segments, which were amplified by polymerized chain reaction and directly sequenced . The forward primer 530f (5'-GTGCCATCCAGCCGCGG-3') was in the small subunit rRNA (SSU-rRNA) and the reverse primer 580r (5'-GGTCCGTGTTTCAAGACGG-3') was in the large subunit rRNA (LSU-rRNA) . We have utilized these sequence data, the published data on Encephalitozoon cuniculi and Encephalitozoon hellem and our cloned SSU-rRNA genes from E . bieneusi and S . intestinalis to develop a phylogenetic tree for the microsporidia involved in human infection . The higher sequence similarities demonstrated between S . intestinalis and E . cuniculi support the placement of S . intestinalis in the family Encephalitozoonidae . This method of polymerized chain reaction rRNA phylogeny allows the establishment of phylogenetic relationships on limiting material where culture and electron microscopy are difficult or impossible and can be applied to archival material to expand the molecular phylogenetic analysis of the phylum Microspora . In addition, the highly variable region (E . coli numbering 590-650) and intergenic spacer regions in the microsporidia were noted to have structural correspondence, suggesting the possibility that they are coevolving. J Photochem Photobiol B, 1994 May, 23(2-3), 155 - 9 Detection of a reduced-flavin triplet state in free flavins and DNA photolyase; Heelis PF et al.; A transient species was observed upon laser excitation (lambda exc = 355 nm) of reduced flavins (cFIH2) under anaerobic conditions . The transient decayed with a lifetime of about 1 microseconds, was quenched by iodide ions and was assigned as the reduced flavin triplet state 3cFIH2 . The triplet-state absorption spectrum assumed three forms, depending on the pH of the solution, corresponding to 3cFIH2 and its monoprotonated and monodeprotonated forms . The triplet-state lifetime was not sensitive to the presence of pyrimidine cyclobutane dimers, apparently ruling out its involvement in the dimer splitting reaction observed with model systems of reduced flavins . An analogous species is apparently formed in the reduced form of Escherichia coli DNA photolyase. J Antibiot (Tokyo), 1994 May, 47(5), 557 - 65 Novel retrovirus protease inhibitors, RPI-856 A, B, C and D, produced by Streptomyces sp . AL-322; Asano T et al.; Four kinds of retrovirus protease inhibitors (RPI-856 A, B, C and D) were isolated as white powder from the culture filtrate of a soil isolate, Streptomyces sp . AL-322 by column chromatography using Diaion HP-20, Sephadex LH-20, ODS reversed phase HPLC and SP-2SW ion exchange HPLC . The structures of these inhibitors were elucidated by physico-chemical properties, chemical reactions and spectral analyses, as valyl-ADPAA-leucyl-AOPBA-valyl-valyl-aspartic acid (RPI-856 A and B) and valyl-ADPAA-leucyl-AOPBA-valyl-valine (RPI-856 C and D) {ADPAA = 2-amino-2-(3,5-dihydroxyphenyl)acetic acid, AOPBA = 3-amino-2-oxo-4-phenylbutyric acid} . RPI-856 A and B, and RPI-856 C and D were both determined to be diasteromers each other on the asymmetric carbon in AOPBA . These four inhibitors strongly inhibited in vitro HIV-1 protease and HTLV-I protease both derived from recombinant Escherichia coli with IC50 of 10(-7) approximately 10(-8) M. Plant Cell, 1994 May, 6(5), 723 - 35 Gene expression in tobacco low-nicotine mutants; Hibi N et al.; Two nuclear genes, Nic1 and Nic2, regulate nicotine levels in tobacco . nic1 and nic2 are semidominant mutations in Burley 21 that reduce leaf nicotine levels and the activities of multiple enzymes in the nicotine pathway and simultaneously increase polyamine levels in cultured roots . Cultured roots homozygous for both mutations were used to isolate two cDNAs by subtraction hybridization; the transcript levels of these two cDNAs were much lower in the mutant roots than in the wild-type roots . The A411 gene encodes a 41-kD protein with considerable homology to mammalian spermidine synthase, whereas the A622 gene encodes a 35-kD protein with high homology to isoflavone reductase . When these genes were expressed in Escherichia coli, A411 had no spermidine synthase activity but did show putrescine N-methyltransferase activity, which is the first enzyme committed to the nicotine biosynthetic pathway, and A622 did not show isoflavone reductase activity . Both the methyltransferase and A622 genes are predominantly expressed in the root, and their expression levels in cultured roots are coordinately decreased by the nic mutations in the order of wild type > nic2 > nic1 > nic1 nic2 . Removal of tobacco flower heads and young leaves rapidly and coordinately induced both genes in the root . Further, exogenous supply of auxin down-regulated both genes in cultured tobacco roots . These results suggest that Nic1 and Nic2 are regulatory genes for nicotine biosynthesis. Plant Cell, 1994 May, 6(5), 601 - 12 A truncated version of an ADP-glucose pyrophosphorylase promoter from potato specifies guard cell-selective expression in transgenic plants; Muller-Rober B et al.; ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme in starch biosynthesis in higher plants . A 3.2-kb promoter of the large subunit gene of the AGPase from potato has been isolated and its activity analyzed in transgenic potato and tobacco plants using a promoter-beta-glucuronidase fusion system . The promoter was active in various starch-containing cells, including guard cells, tuber parenchyma cells, and the starch sheath layer of stems and petioles . No expression was observed in mesophyll cells . Analysis of various promoter derivatives showed that with respect to expression in petioles and stems, essential elements must be located in the 5' distal region of the promoter, whereas elements important for expression in tuber parenchyma cells are located in an internal fragment comprising nucleotides from positions -500 to -1200 . Finally, a 0.3-kb 5' proximal promoter fragment was identified that was sufficient to obtain exclusive expression in guard cells of transgenic potato and tobacco plants . The implications of our observations are discussed with respect to starch synthesis in various tissues and the use of the newly identified promoter as a tool for stomatal biology. Isr J Med Sci, 1994 May-Jun, 30(5-6), 335 - 8 A review of the effect of human milk fractions on the adherence of diarrheogenic Escherichia coli to the gut in an animal model; Ashkenazi S; We conducted studies showing that nonimmunoglobulin fractions of human milk or colostrum inhibited the adherence of certain diarrheogenic Escherichia coli to the gut . This activity resisted boiling and digestion with trypsin, but was nearly abolished by periodate treatment . The inhibitory activity is therefore related to carbohydrate residues in human milk that probably act as receptor analogues. Plant Physiol, 1994 May, 105(1), 357 - 67 Identification of a light-responsive region of the nuclear gene encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana; Kwon HB et al.; We report here the identification of a cis-acting region involved in light regulation of the nuclear gene (GapB) encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana . Our results show that a 664-bp GapB promoter fragment is sufficient to confer light induction and organ-specific expression of the Escherichia coli beta-glucuronidase reporter gene (Gus) in transgenic tobacco (Nicotiana tabacum) plants . Deletion analysis indicates that the -261 to -173 upstream region of the GapB gene is essential for light induction . This region contains four direct repeats with the consensus sequence 5'-ATGAA(A/G)A-3' (Gap boxes) . Deletion of all four repeats abolishes light induction completely . In addition, we have linked a 109-bp (-263 to -152) GapB upstream fragment containing the four direct repeats in two orientations to the -92 to +6 upstream sequence of the cauliflower mosaic virus 35S basal promoter . The resulting chimeric promoters are able to confer light induction and to enhance leaf-specific expression of the Gus reporter gene in transgenic tobacco plants . Based on these results we conclude that Gap boxes are essential for light regulation and organ-specific expression of the GapB gene in A . thaliana . Using gel mobility shift assays we have also identified a nuclear factor from tobacco that interacts with GapA and GapB DNA fragments containing these Gap boxes . Competition assays indicate that Gap boxes are the binding sites for this factor . Although this binding activity is present in nuclear extracts from leaves and roots of light-grown or dark-treated tobacco plants, the activity is less abundant in nuclear extracts prepared from leaves of dark-treated plants or from roots of greenhouse-grown plants . In addition, our data show that this binding factor is distinct from the GT-1 factor, which binds to Box II and Box III within the light-responsive element of the RbcS-3A gene of pea.
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