J Biol Chem, 1991 Dec 15, 266(35), 23529 - 36 Psychotrine and its O-methyl ether are selective inhibitors of human immunodeficiency virus-1 reverse transcriptase; Tan GT et al.; Psychotrine dihydrogen oxalate and O-methylpsychotrine sulfate heptahydrate (MP), the salts of isoquinoline alkaloids from ipecac, were found to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) . We currently report the results of additional studies designed to characterize the mechanism of inhibition facilitated by MP . The inhibition was noncompetitive with respect to TTP and uncompetitive with respect to poly(rA) and oligo(dT)12-18 (4:1) at low template-primer concentrations but competitive at high concentrations (greater than 200 microM) . Identical non-Michaelis-type kinetics were observed when activated DNA was used as the template . The biphasic nature of the double-reciprocal plots and Hill coefficients of less than 1 indicate that MP functions as an allosteric inhibitor of the enzyme which appears to possess multiple active sites that interact in a cooperative (negative) fashion in the presence of the inhibitor . MP was selective for the recombinant HIV-1 RT (p66) utilizing poly(rA) and oligo(dT)12-18 (4:1) as template-primer . Greater inhibition was observed with this template primer as compared with other natural and synthetic template-primers tested . MP had significantly less effect on avian myeloblastosis virus RT as well as mammalian or bacterial DNA and RNA polymerases . Other members of the ipecac class of alkaloids, e.g . emetine hydrochloride, were inactive against all of these enzymes, including HIV-1 RT . Conversely, MP did not inhibit in vitro protein synthesis, a property manifested by all the other ipecac alkaloids tested . Studies conducted with structural analogs revealed that the imine functionality at positions 1' and 2' of MP is the key structural requirement for HIV-1 RT inhibitory activity . Therefore, MP appears to possess unique structural properties that enable interaction with HIV-1 RT in a manner that can be differentiated from other polymerases . Use of these alkaloids for the definition of this viral enzyme-specific topology may lead to the development of therapeutically useful chemotherapeutic agents. Gene, 1991 Dec 15, 108(2), 265 - 7 Cloning and sequencing of a jack bean urease-encoding cDNA; Riddles PW et al.; A cDNA which encodes the entire amino acid (aa) sequence of the mature jack bean urease has been cloned in Escherichia coli from a library prepared from the mRNA of developing jack beans . It was necessary to use reverse transcriptase in the cDNA was obtained in the form of two contiguous DNA fragments, each of which was completely sequenced . The conceptual translation of the nt sequence gave an 840-aa sequence which was identical to the directly determined sequence except for one conservative aa substitution (Takashima et al., Eur . J . Biochem . 175 (1988) 151-165) . These data constitute the first report on the cloning and sequence of the cDNA encoding a urease from any higher plant. Biochem J, 1991 Dec 15, 280 ( Pt 3), 703 - 8 Primer terminus recognition and highly processive replication by Epstein-Barr virus DNA polymerase; Tsurumi T; The Epstein-Barr virus (EBV) DNA polymerase is essential for viral DNA replication in the lytic phase of the EBV life cycle . It efficiently extends RNA primers on the template DNA, suggesting the possible involvement of the EBV DNA polymerase in synthesizing Okazaki fragments from RNA primers on the lagging strand template . Competition experiments revealed that the EBV DNA polymerase had significantly higher affinity for primer termini hybridized to the template DNA than for the single-stranded DNA template or the single-stranded primer itself . ATP was not required either for primer terminus recognition or for sustainment of polymerization . The stimulation of the enzyme by (NH4)2SO4 was dependent on the template/primers utilized . These observations suggest that the primary and secondary structure of the template/primers are important factors for primer terminus recognition by the EBV DNA polymerase . The enzyme elongated synthetic RNA primer annealed to circular single-stranded M13 DNA coated with Escherichia coli single-stranded DNA-binding protein without dissociation . The processivity of the EBV DNA polymerase was strikingly high (greater than 7200 nucleotides) and the rate of polymerization was 12 nucleotides/s per polymerase molecule . The high processing capacity is a desirable feature in the synthesis of multiple copies of the EBV genome in rolling-circle DNA replication. Proc Natl Acad Sci U S A, 1991 Dec 15, 88(24), 11535 - 9 How does RNase H recognize a DNA.RNA hybrid? Nakamura H, Oda Y, Iwai S, Inoue H, Ohtsuka E, Kanaya S, Kimura S, Katsuda C, Katayanagi K, Morikawa K, et al. The mechanism of RNase H substrate recognition is proposed from a model of a chemically modified DNA.RNA hybrid Escherichia coli RNase H complex . Site-directed mutagenesis of the enzyme and substrate titration observed by heteronuclear two-dimensional NMR spectra have been carried out . A model complex has been built, based on free structures of the enzyme and the substrate independently determined by x-ray crystallography and NMR distance geometry, respectively . In addition to steric and electrostatic complementarities between the molecular surfaces of the enzyme and the minor groove of the hybrid in the model, putative hydrogen bonds between the polar groups in the enzyme and 2'-oxygens of the RNA strand of the hybrid fix the hybrid close to the active site of the enzyme . The enzymatic activities of the mutant proteins and the changes in NMR spectra during the course of substrate titration are consistent with the present model . Moreover, the specific cleavage of the RNA strand in DNA.RNA hybrids can be explained as well as cleavage modes in modified heteroduplexes . A mechanism of enzymatic action is proposed. Proc Natl Acad Sci U S A, 1991 Dec 15, 88(24), 11057 - 61 Intermolecular complementation between two defective mutant signal-transducing receptors of Escherichia coli; Yang Y et al.; Taz1 is a hybrid signal-transducing membrane receptor between Tar, an aspartate chemoreceptor, and EnvZ, an osmosensor of Escherichia coli that is able to induce ompC expression by phosphorylating OmpR (a transcriptional activator) in response to aspartate . When the Taz1 His-277, the proposed autophosphorylation site in the cytoplasmic EnvZ domain, was replaced with a valine residue, the mutant Taz1 was unable to induce ompC expression . Similarly, when approximately two-thirds of the EnvZ domain was deleted, Taz1 was nonfunctional . However, when these two defective Taz1 proteins were coexpressed in a cell, ompC was constitutively expressed . Coinciding with this result, two mutant Taz1 molecules were able to intermolecularly complement each other to restore the OmpR kinase activity but not phosphatase activity in vitro . The identical result was also obtained with EnvZ . The present results suggest that the autophosphorylation of Taz1 and EnvZ is an intermolecular phosphorylation reaction, requiring formation of a dimer (or oligomer), and that ligand-dependent ompC expression requires not only kinase but also phosphatase activity. J Biol Chem, 1991 Dec 15, 266(35), 24212 - 9 Purification, characterization, and comparison of poliovirus RNA polymerase from native and recombinant sources; Neufeld KL et al.; Poliovirus RNA encodes an RNA-dependent RNA polymerase, designated 3Dpol, that catalyzes the synthesis of both plus and minus strand viral RNA . This enzyme was purified to near homogeneity from poliovirus-infected HeLa cells, recombinant baculovirus-infected insect cells, and from Escherichia coli transformed with an expression plasmid containing poliovirus 3D sequences . The two recombinant expression systems produced significantly higher yields of active enzyme than could be attained from virus-infected HeLa cells . All preparations contained a 52-kDa protein, recognized by antisera raised against 3D expressed as a fusion protein in E . coli . Immunoreactive protein resolved into 3-4 species on isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional gels . Efforts to demonstrate that the multiple spots resulted from phosphorylation were negative . Furthermore, no evidence for autophosphorylation of purified 3Dpol was obtained . Purified 3Dpol from recombinant sources manifested the same specific activities as enzyme from poliovirus-infected HeLa cells in both a poly(A)-dependent poly(U) polymerase assay and a poliovirus RNA-dependent RNA polymerase assay . The products of the latter reaction reached the length of the template (7.5 kilobases) in 20-30 min, indicating an elongation rate of approximately 300 nucleotides/min at 30 degrees C . No products exceeded the length of the template . Intermediate length products were detected, which presumably resulted from pauses in transcription due to template structure . All transcription was dependent on primer . The kinetic parameters of all three purified enzyme preparations were the same; the Km for UTP was 2.4 +/- 0.1 microM in an RNA polymerase activity assay . Product formation was linear for up to 45 min, except for a 3-5-min lag before synthesis began . The lag was independent of enzyme concentration, and independent of the template used . The lag was eliminated by preincubating enzyme, template, primer, and three of the four nucleotide triphosphates, but not by preincubating any subset of these components . This suggested that a preinitiation complex must form as a prerequisite to RNA synthesis . Partially purified preparations of 3Dpol from the three sources showed significant differences in activities and products, including the appearance of primer-independent polymerase activity and production of dimer-length RNA products . These variable properties are likely due to different contaminating activities provided by the different cellular hosts, since upon further purification, all three enzymes exhibited identical properties. J Biol Chem, 1991 Dec 15, 266(35), 23878 - 85 Cloning of a 16-kDa ubiquitin carrier protein from wheat and Arabidopsis thaliana . Identification of functional domains by in vitro mutagenesis; Sullivan ML et al.; Ubiquitin carrier proteins (E2s) are involved in the covalent attachment of ubiquitin to a variety of cellular target proteins in eukaryotes . Here, we report the cloning of genes from wheat and Arabidopsis thaliana that encode 16-kDa E2s and a domain analysis of E2s by in vitro mutagenesis . The genes for E216kDa, which we have designated wheat and At UBC1, encode proteins that are only 33% identical (58% similar) with a 23-kDa E2 from wheat (encoded by the gene now designated wheat UBC4), but are 63% identical (82% similar) with the E2 encoded by the Saccharomyces cerevisiae DNA repair gene, RAD6 . Unlike the proteins encoded by RAD6 and wheat UBC4, the UBC1 gene products lack acidic C-terminal domains extending beyond the conserved core of the proteins and are incapable of efficient in vitro ligation of ubiquitin to histones . From enzymatic analysis of the UBC1 and UBC4 gene products mutagenized in vitro, we have identified several domains important for E2 function, including the active site cysteine and N-terminal and C-terminal domains . Cysteine residues 88 and 85 in the UBC1 and UBC4 gene products, respectively, are necessary for formation of the ubiquitin-E2 thiol ester intermediate . Whereas the UBC1 gene product does not require its additional cysteine residue at position 116 for thiol ester formation, alteration of cysteine 143 in the UBC4 gene product greatly diminishes this ability . The N terminus of UBC1 contains two domains that affect activity: a proximal region containing hydroxylated and uncharged residues whose removal increases the rate of thiol ester formation and a distal tract rich in basic residues . Deletion or substitution of these basic residues with neutral residues diminishes the rate of thiol ester formation . We have demonstrated also that C-terminal extensions can function to confer substrate specificity to E2s . When the acidic extension was deleted from UBC4, the protein was unable to efficiently conjugate ubiquitin to histones in vitro . Furthermore, fusion of the UBC4 acidic extension to the C terminus of UBC1 resulted in a chimeric protein capable of efficient histone conjugation, as did fusion of short tracts of alternating aspartate and glutamate residues . This result suggests that the target protein specificity of E2s can be altered by the addition of appropriate C-terminal extensions, thus providing a way to modify the selectivity of the ubiquitin system. J Biol Chem, 1991 Dec 15, 266(35), 23641 - 7 EPR characterization of the stereochemistry of the distal heme pocket of the engineered human myoglobin mutants; Ikeda-Saito M et al.; Recombinant human myoglobin mutants with the distal histidine residue replaced by Leu, Val, or Gln residues have been prepared by site-directed mutagenesis and expression in Escherichia coli . The recombinant apomyoglobin proteins have been successfully reconstituted with cobaltous protoporphyrin IX to obtain cobalt myoglobin mutant proteins, and the role of the distal histidine residue on the interaction between the bound ligand and the myoglobin molecule has been studied by EPR spectroscopy . We found that the distal histidine residue is significant in the orientation of the bound oxygen molecule . Low temperature photolysis experiments on both oxy cobalt proteins and ferric nitric oxide complexes indicated that the nature of the photolyzed form depends on the steric crowding of the distal heme pocket . To our surprise, the distal Leu mutant has a less restricted, less sterically crowded distal heme pocket than that of the distal Val mutant myoglobin, despite the fact that Leu has a larger side chain volume than Val . Our results demonstrate that the distal heme pocket steric crowding is not necessarily related to the side chain volume of the E7 residue. Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10895 - 9 Crystals of a ternary complex of human immunodeficiency virus type 1 reverse transcriptase with a monoclonal antibody Fab fragment and double-stranded DNA diffract x-rays to 3.5-A resolution; Jacobo-Molina A et al.; Two crystal forms of complexes have been grown that contain human immunodeficiency virus type 1 reverse transcriptase and a monoclonal antibody Fab fragment . One of the crystal forms (form II, space group P3112, a = 168.7 A, c = 220.3 A) diffracts x-rays to 3.5-A resolution and appears suitable for moderate-resolution structure determination . The form II crystals have the unusual property that their maximum resolution of diffraction and resistance to radiation damage are enhanced by either crystallization in the presence of or soaking with double-stranded DNA primer-template mimics . These crystals may permit structural studies of catalytically relevant complexes and eventually enable us to experimentally observe successive steps in the reverse transcription process. Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10402 - 6 Disulfide cross-linking studies of the transmembrane regions of the aspartate sensory receptor of Escherichia coli; Lynch BA et al.; The Escherichia coli aspartate receptor, a dimer of identical subunits, has two transmembrane regions (TM1, residues 7-30; TM2, residues 189-212) of 24 residues each . To study the relative placement and orientation of the regions, cysteine residues were introduced individually into the center of each: at positions 17, 18, and 19 in TM1; and at positions 198, 199, 200, and 201 in TM2 . Based on the patterns of disulfide cross-linking observed between subunits in the mutant receptors, there appears to be close contact between the TM1 and TM1' regions at the dimer interface but no such direct interaction between the TM2 and TM2' regions . The cross-linking results are consistent with an alpha-helical structure extending across the transmembrane region up through at least residue 36, which lies on the periplasmic side of TM1 . The ability of an 18-18' cross-linked dimer to transmit an aspartate-induced transmembrane signal is also supportive of such an extended helix . The changes in relative rates of disulfide cross-linking provide experimental evidence of a conformational change transmitted through the transmembrane domain during signaling . Once formed, disulfides between the transmembrane regions are unusually resistant to reduction by low molecular weight thiols in the presence of denaturants like SDS . These targeted disulfide cross-links can be used to reveal structural and dynamic aspects of protein function. Biochim Biophys Acta, 1991 Dec 11, 1118(1), 1 - 5 Synthesis and kinetic study of transition state analogs for ribonuclease T1; Georgalis Y et al.; Based on the proposal that ribonucleases cleave the RNA phosphodiester bond with a mechanism involving pentacovalent phosphorous as transition state, complexes of guanosine and inosine with vanadate-(IV, V), molybdate-(VI), tungstate-(VI), chromate-(VI) and hexacyanochromate-(III) were synthesized and probed as inhibitors of recombinant ribonuclease T1, obtained from an E . coli . overproducing strain . The apparent dissociation constants of these inhibitors and RNase T1, as determined by Michaelis-Menten kinetics, vary between 0.5-0.9 microM and indicate very strong binding, 100- to 1000-fold stronger than the binding of guanosine (Kd = 545 microM) and inosine (Kd = 780 microM), and 50-100-fold stronger than the binding of the product 3' GMP (Kd = 55 microM) . Therefore the synthesized inhibitors may be considered as genuine transition state analogs for the enzyme. Nucleic Acids Res, 1991 Dec 11, 19(23), 6505 - 9 Genetic organization of the KpnI restriction--modification system; Chatterjee DK et al.; The KpnI restriction-modification (KpnI RM) system was previously cloned and expressed in E . coli . The nucleotide sequences of the KpnI endonuclease (R.KpnI) and methylase (M . KpnI) genes have now been determined . The sequence of the amino acid residues predicted from the endonuclease gene DNA sequence and the sequence of the first 12 NH2-terminal amino acids determined from the purified endonuclease protein were identical . The kpnIR gene specifies a protein of 218 amino acids (MW: 25,115), while the kpnIM gene codes for a protein of 417 amino acids (MW: 47,582) . The two genes transcribe divergently with a intergeneic region of 167 nucleotides containing the putative promoter regions for both genes . No protein sequence similarity was detected between R.KpnI and M.KpnI . Comparison of the amino acid sequence of M.KpnI with sequences of various methylases revealed a significant homology to N6-adenine methylases, a partial homology to N4-cytosine methylases, and no homology to C5-methylases. Nucleic Acids Res, 1991 Dec 11, 19(23), 6465 - 8 Stereochemistry of methyl transfer catalyzed by tRNA (m5U54)-methyltransferase--evidence for a single displacement mechanism; Kealey JT et al.; tRNA (m5U54)-methyltransferase (RUMT) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the 5-carbon of uridine 54 of tRNA . We have determined the steric course of methyl transfer, using (methyl-R)- and (methyl-S)-{methyl-2H1,3H}-AdoMet as the chiral methyl donors, and tRNA lacking the 5-methyl group at position 54 as the acceptor . Following methyl transfer, ribothymidine was isolated and degraded to chiral acetic acid for configurational analysis . Transfer of the chiral methyl group to U54 proceeded with inversion of configuration of the chiral methyl group, suggesting that RUMT catalyzed methyl transfer occurs by a single SN2 displacement mechanism. Nucleic Acids Res, 1991 Dec 11, 19(23), 6573 - 8 Use of single-stranded DNA oligonucleotides in programming ribosomes for translation; Ricker RD et al.; Single-stranded DNA (ssDNA) oligomers were compared to synthetic RNA oligomers in their ability to program E . coli ribosomes in vitro . AUG and dATG-containing oligomers promoted the non-enzymatic binding of fmet-tRNA to ribosomes, with similar dependence on time and magnesium concentration; only at 10 mM Mg++ or at low oligomer concentration was RNA slightly preferred in complex formation . These initiation complexes were biologically active in that fmet-tRNA, bound in response to ssDNA or RNA, was fully reactive with puromycin . While dAUG could not function as an initiation codon, p-dAUG functioned as well as AUG or dATG . However, dUAA and p-dUAA could not replace UAA in directing release-factor (RF) activity, and dTAA functioned only to a slight extent . Release factors had specificity for termination complexes containing dATGTAA, dATGTAG, or dATGTGA . At Mg++ concentrations of 15 mM or higher, these hexamers directed peptidyl transferase-dependent fmet-tRNA hydrolysis in the absence of RF . We suggest this RF-independent activation of peptidyl transferase as a unique system for studying the mechanism of termination . Overall, these results indicate that ssDNA can be used in place of RNA for certain studies of protein synthesis. Nucleic Acids Res, 1991 Dec 11, 19(23), 6541 - 5 Isolation and mapping of EVX1, a human homeobox gene homologous to even-skipped, localized at the 5' end of HOX1 locus on chromosome 7; Faiella A et al.; We isolated and mapped the human homeobox gene EVX1 . This gene encodes a protein of 407 amino acid residues containing a homeodomain closely related to the Drosophila even-skipped (eve) segmentation gene of the pair-rule class . EVX1 belongs to a small family of vertebrate eve-related homeobox genes including human EVX1 and EVX2 genes, their murine homologs, Evx 1 and Evx 2, and the frog Xhox-3 gene . We previously reported that EVX2 is localized at the 5' end of the HOX4 locus on chromosome 2 . We show here that EVX1 is localized at the 5' end of the HOX1 locus on chromosome 7, 48 kb upstream from the most 5' of the eleven HOX1 genes, namely HOX1J . Both EVX genes are transcribed in an opposite orientation as compared to that of adjacent HOX genes . Human HOX1 and HOX4 complex loci appear to be both closely linked to a homeobox gene of the EVX family. Biochemistry, 1991 Dec 10, 30(49), 11595 - 9 Kinetics of extension of O6-methylguanine paired with cytosine or thymine in defined oligonucleotide sequences; Dosanjh MK et al.; The frequency of extending m6G.C or m6G.T pairs, when the 3' and 5' flanking neighbors of m6G are either cytosines or thymines, was investigated using primed 25-base-long oligonucleotides and the Klenow fragment of Escherichia coli DNA polymerase I (Kf) . The efficiency, Vmax/Km, of extension to the following normal base pair was up to 40-fold greater than for the formation of the m6G.T or m6G.C pair . The frequencies of inserting either dCMP or dTMP opposite these m6G bases did not appear to be different in the two sequences, C-m6G-C and T-m6G-T, but extension was favored in the C-m6G-C sequence . The m6G.T pair extended to a C.G pair most efficiently, indicating that it was not a strong block to continued replication past the template lesion . Thus, m6G.T flanked by cytosines replicates more readily than when flanked by thymines, increasing G----A transitions . These data lend further support to the importance of sequence context in mutagenesis. Biochemistry, 1991 Dec 10, 30(49), 11567 - 79 Analysis of hydride transfer and cofactor fluorescence decay in mutants of dihydrofolate reductase: possible evidence for participation of enzyme molecular motions in catalysis; Farnum MF et al.; A remarkable correlation has been discovered between fluorescence lifetimes of bound NADPH and rates of hydride transfer among mutants of dihydrofolate reductase (DHFR) from Escherichia coli . Rates of hydride transfer from NADPH to dihydrofolate change by a factor of 1,000 for the series of mutant enzymes . Since binding constants for the initial complex between coenzyme and DHFR change by only a factor of 10, the major portion of the change in hydride transfer must be attributed to losses in transition-state stabilization . The time course of fluorescence decay for NADPH bound to DHFR is biphasic . Lifetimes ranging from 0.3 to 0.5 ns are attributed to a solvent-exposed dihydronicotinamide conformation of bound coenzyme which is presumably not active in catalysis, while decay times (tau 2) in the range of 1.3 to 2.3 ns are assigned to a more tightly bound species of NADPH in which dihydronicotinamide is sequestered from solvent . It is this slower component that is of interest . Ternary complexes with three different inhibitors, methotrexate, 5-deazafolate, and trimethoprim, were investigated, along with the holoenzyme complex; 3-acetylNADPH was also investigated . Fluorescence polarization decay, excitation polarization spectra, the temperature variation of fluorescence lifetimes, fluorescence amplitudes, and wavelength of absorbance maxima were measured . We suggest that dynamic quenching or internal conversion promotes decay of the excited state in NADPH-DHFR . When rates of hydride transfer are plotted against the fluorescence lifetime (tau 2) of tightly bound NADPH, an unusual correlation is observed . The fluorescence lifetime becomes longer as the rate of catalysis decreases for most mutants studied . However, the fluorescence lifetime is unchanged for those mutations that principally alter the binding of dihydrofolate while leaving most dihydronicotinamide interactions relatively undisturbed . The data are interpreted in terms of possible dynamic motions of a flexible loop region in DHFR which closes over both substrate and coenzyme binding sites . These motions could lead to faster rates of fluorescence decay in holoenzyme complexes and, when correlated over time, may be involved in other motions which give rise to enhanced rates of catalysis in DHFR. Biochemistry, 1991 Dec 10, 30(49), 11485 - 9 Identification of a ferryl intermediate of Escherichia coli cytochrome d terminal oxidase by resonance Raman spectroscopy; Kahlow MA et al.; The 680-nm-absorbing "peroxide state" of the Escherichia coli cytochrome d terminal oxidase complex, obtained by addition of excess hydrogen peroxide to the enzyme, is shown to be a ferryl intermediate in the catalytic cycle of the enzyme . This ferryl intermediate is also created by aerobic oxidation of the fully reduced enzyme . Resonance Raman spectra with 647.1-nm excitation show an FeIV = O stretching band at 815 cm-1, a higher frequency than noted in any other ferryl-containing enzyme to date . The band shows an 16O/18O frequency shift of -46 cm-1, larger than that observed for any porphyrin ferryl species . The FeIV = O formulation was unambiguously established by oxidations of the reduced enzyme with 16O2, 18O2, and 16O18O . Only the use of a mixed-isotope gas permitted discrimination between a ferryl and a peroxo structure . A catalytic cycle for the cytochrome d terminal oxidase complex is proposed, and possible reasons for the high v(Fe = O) frequency are discussed. Biochem Biophys Res Commun, 1991 Nov 27, 181(1), 337 - 41 Intrapeptide sequence homology in rubrerythrin from Desulfovibrio vulgaris: identification of potential ligands to the diiron site; Kurtz DM Jr et al.; Two regions in the amino acid sequence of the 21.5 kDa subunit of rubrerythrin from Desulfovibrio vulgaris (Hildenborough) are shown to be homologous . Rubrerythrin contains a non-heme, non-sulfur diiron site, and the internally homologous regions share homology with at least one proposed iron binding region of the component A alpha subunit of methane monooxygenase, which also contains a non-heme, non-sulfur diiron site . Comparison of the rubrerythrin sequences with those of the B2 subunit of E . coli ribonucleotide reductase, whose diiron site ligands have been identified, suggests that two glutamate and two histidine residues at positions 53, 56, 129, and 131 within the rubrerythrin sequence furnish ligands to the diiron site . A pair of EXXH sequences appears to represent a diiron binding motif in all three aforementioned proteins . No propene monooxygenase activity was detected with rubrerythrin using the assay designed to test activity of methane monooxygenase component A in the absence of other protein components. Lancet, 1991 Dec 7, 338(8780), 1423 - 4 Production of interleukin-1-receptor antagonist during experimental endotoxaemia; Granowitz EV et al.; Interleukin-1 (IL-1) has been implicated in the pathogenesis of sepsis . IL-1-receptor antagonist (IL-1ra) is a naturally occurring inhibitor of IL-1 activity that competes with IL-1 for occupancy of cell-surface receptors but possesses no agonist activity . We induced endotoxaemia in 9 healthy human volunteers by injection of Escherichia coli endotoxin, and measured plasma concentrations of IL-1 and IL-1ra by radioimmunoassay during the next 24 h . Peak plasma concentrations of IL-1ra were about a hundred-fold greater than those of IL-1 beta . No IL-1 or IL-1ra were detectable in the plasma of 4 volunteers injected with saline . Our results suggest that the predominant natural response to endotoxin in man is the production of antagonist rather than agonist. Science, 1991 Dec 6, 254(5037), 1509 - 12 Systemic delivery of human growth hormone by injection of genetically engineered myoblasts; Dhawan J et al.; A recombinant gene encoding human growth hormone (hGH) was stably introduced into cultured myoblasts with a retroviral vector . After injection of genetically engineered myoblasts into mouse muscle, hGH could be detected in serum for 3 months . The fate of injected myoblasts was assessed by coinfecting the cells with two retroviral vectors, one encoding hGH and the other encoding beta-galactosidase from Escherichia coli . These results provide evidence that myoblasts, which can fuse into preexisting multinucleated myofibers that are vascularized and innervated, may be advantageous as vehicles for systemic delivery of recombinant proteins. Science, 1991 Dec 6, 254(5037), 1494 - 7 Selective cleavage of human DNA: RecA-assisted restriction endonuclease (RARE) cleavage; Ferrin LJ et al.; Current methods for sequence-specific cleavage of large segments of DNA are severely limited because of the paucity of possible cleavage sites . A method is described whereby any Eco RI site can be targeted for specific cleavage . The technique is based on the ability of RecA protein from Escherichia coli to pair an oligonucleotide to its homologous sequence in duplex DNA and to form a three-stranded complex . This complex is protected from Eco RI methylase; after methylation and RecA protein removal, Eco RI restriction enzyme cleavage was limited to the site previously protected from methylation . When pairs of oligonucleotides are used, a specific fragment can be cleaved out of genomes . The method was tested on lambda phage, Escherichia coli, and human DNA . Fragments exceeding 500 kilobases in length and yields exceeding 80 percent could be obtained. J Biol Chem, 1991 Nov 25, 266(33), 22096 - 101 Fine balance in the regulation of DnaB helicase by DnaC protein in replication in Escherichia coli; Allen GC Jr et al.; The DnaC protein of Escherichia coli is essential for replication in vivo and in vitro . In the initiation of replication of a minichromosome at its origin, DnaC delivers the DnaB helicase from a DnaB.DnaC complex to the future replication fork and then departs . However, if an excess of DnaC was present in subsequent steps, it severely inhibited replication by slowing the DnaB helicase at the replication fork . When DnaB was present at a level equimolar with the excess DnaC, the inhibition was relieved, implying that the ratio of DnaC to DnaB is critical for achieving optimal replication activity and avoiding inhibition by DnaC . In vivo, overproduction of DnaC slowed cell growth . This slowing was alleviated by overproducing DnaB at the same time . E . coli strains with a dnaCts gene defective in chromosomal initiation were complemented by the wild-type gene in trans . On the other hand, strains with an elongation-defective dnaCts gene were not complemented by the wild-type dnaC gene . The dominance of the mutant protein suggests that it remains tightly complexed with DnaB at the replication fork, inhibiting elongation even in the presence of the wild-type DnaC. Nature, 1991 Dec 5, 354(6352), 369 - 73 Structure and functional properties of human general transcription factor IIE; Peterson MG et al.; The general transcription factor IIE (TFIIE) is an essential component of the eukaryotic RNA polymerase II initiation complex . We have isolated human complementary DNA clones for both the subunits of TFIIE . Using purified recombinant proteins we find that both subunits are essential to form a stable preinitiation complex and to reconstitute basal-level and Sp1-activated transcription in vitro . Analysis of their predicted amino-acid sequences reveals several intriguing structural motifs that could provide insight into the role of TFIIE in transcription initiation. Eur J Biochem, 1991 Dec 5, 202(2), 479 - 84 Mechanism of action of deoxyribonuclease II from human lymphoblasts; Harosh I et al.; Deoxyribonuclease II has been purified through five fractionation steps from the human lymphoblast cell line K562 . Isolation included DEAE-cellulose and heparin-agarose chromatography followed by fractionation on Mono-S, Mono-Q and Superose-12 FPLC columns . In an extension of previous studies, deoxyribonuclease II was found to introduce a much higher proportion of single-strand nicks relative to double-strand breaks into supercoiled DNA than has been reported for linear DNA . Application of DNA sequencing techniques has further revealed a unique resistance of 3' termini to hydrolysis by this enzyme . Deoxyribonuclease II cleaves at every available site along the duplexed portion of a paired oligonucleotide substrate with the exception of the last four nucleotides . Consistent with previous results, this deoxyribonuclease II is active at low pH in the absence of Mg2+ and is not inhibited by EDTA, but complete inhibition is observed with 100 microM Fe3+ . Likewise we confirmed the presence of 3'-phosphoryl termini on the DNA cleavage products since they failed to function as primers for DNA synthesis catalyzed by Escherichia coli DNA polymerase I. Eur J Biochem, 1991 Dec 5, 202(2), 309 - 13 Cloning and expression of the gene encoding the soluble cytochrome b562 of Escherichia coli; Nikkila H et al.; The gene for the soluble cytochrome b562 from Escherichia coli B has been cloned on a SalI fragment . The analysis of the gene reveals the presence of a leader sequence in front of the sequence encoding the mature protein . Expression of cytochrome b562 using the lac-promoter produced the protein to a level of 3-5% of total protein . This over-production enables employment of a simple, high-yield purification protocol to obtain homogeneous cytochrome b562 . Spectroscopic and N-terminal sequence analyses of the purified protein demonstrate that it is identical to the chromosomally expressed cytochrome b562 purified and characterized from E . coli B {Itagaki, E . & Hager, L.P . (1966) J . Biol . Chem . 241, 3687-3695} . It is demonstrated that the genomic sequence codes for a classic N-terminal signal sequence and that mature cytochrome b562 is translocated to the periplasmic space. J Mol Biol, 1991 Dec 5, 222(3), 763 - 85 Construction of new ligand binding sites in proteins of known structure . I . Computer-aided modeling of sites with pre-defined geometry; Hellinga HW et al.; We have devised a molecular model building computer program (DEZYMER) which builds new ligand binding sites into a protein of known three-dimensional structure . It alters only the sequence and the side-chain structure of the protein, leaving the protein backbone fold intact by definition . The program searches for a constellation of backbone positions arranged such that if appropriate side-chains were placed there, they would bind the ligand according to a pre-defined geometry of interaction specified by the experimentalist . These binding sites are introduced by the program by taking into account simple rules such as steric hindrance, atomic close-packing and hydrogen bond patterns, which are known to maintain the integrity of a protein structure to a first approximation . A test case is presented in this paper where the copper binding site found in blue-copper proteins such as plastocyanin, azurin and cupredoxin is introduced into Escherichia coli thioredoxin . The model building of one of the solutions found by the program is presented in some detail . The experimental construction and properties of this new protein are described in an accompanying paper . It is hoped that this program provides a general method for the design of ligand binding sites and enzyme active sites, which can then be tested experimentally. J Mol Biol, 1991 Dec 5, 222(3), 599 - 620 Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli; Zimmerman SB et al.; The very high concentration of macromolecules within cells can potentially have an overwhelming effect on the thermodynamic activity of cellular components because of excluded volume effects . To estimate the magnitudes of such effects, we have made an experimental study of the cytoplasm of Escherichia coli . Parameters from cells and cell extracts are used to calculate approximate activity coefficients for cytoplasmic conditions . These calculations require a representation of the sizes, concentrations and effective specific volumes of the macromolecules in the extracts . Macromolecule size representations are obtained either by applying a two-phase distribution assay to define a related homogeneous solution or by using the molecular mass distribution of macromolecules from gel filtration . Macromolecule concentrations in cytoplasm are obtained from analyses of extracts by applying a correction for the dilution that occurs during extraction . That factor is determined from experiments based upon the known impermeability of the cytoplasmic volume to sucrose in intact E . coli . Macromolecule concentrations in the cytoplasm of E . coli in either exponential or stationary growth phase are estimated to be approximately 0.3 to 0.4 g/ml . Macromolecule specific volumes are inferred from the composition of close-packed precipitates induced by polyethylene glycol . Several well-characterized proteins which bind to DNA (lac repressor, RNA polymerase) are extremely sensitive to changes in salt concentration in studies in vitro, but are insensitive in studies in vivo . Application of the activity coefficients from the present work indicates that at least part of this discrepancy arises from the difference in excluded volumes in these studies . Applications of the activity coefficients to solubility or to association reactions are also discussed, as are changes associated with cell growth phase and osmotic or other effects . The use of solutions of purified macromolecules that emulate the crowding conditions inferred for cytoplasm is discussed. J Mol Biol, 1991 Dec 5, 222(3), 495 - 508 Control regions of an archaeal gene . A TATA box and an initiator element promote cell-free transcription of the tRNA(Val) gene of Methanococcus vannielii; Hausner W et al.; To identify the DNA sequences required for initiation of transcription in archaea, the 5'-flanking region of the tRNA(Val) gene of Methanococcus vannielii was modified by deletions, restructuring and site-directed mutagenesis, and the tRNA encoding sequence was replaced by a fortuitous Escherichia coli sequence . The effects of these mutations on promoter function were tested in an homologous cell-free transcription system . The DNA region from position -35 to +9 relative to the transcription start site was sufficient for maximal initiation of cell-free transcription . Removal of the DNA region between -35 and -30 reduced initiation by a factor of 2 . Deletions extending to position -24 almost completely abolished specific transcription . Analysis of 16 site-specific mutations in the region from -33 to +2 provided evidence that a conserved A + T-rich sequence (TATA box), centered at -25, is essential for initiation of transcription . Single point mutations in six positions of the TATA box reduced initiation of transcription from 0.2 to 0.01 of wild-type levels . A second conserved motif at the transcription start site (consensus ATGC) could be replaced by some sequences containing a pyrimidine-purine dinucleotide but appeared necessary for a maximal rate of gene transcription . Mutations altering the spacing between the two conserved elements demonstrated that initiation occurs at a strictly defined distance of 22 to 27 base-pairs downstream from the TATA box . Our results support the conclusion that the TATA box is the major DNA region mediating promoter recognition, influencing the efficiency of transcription and specifying the site of transcription initiation . This Methanococcus promoter element closely resembles in structure and function the TATA box of promoters of eukaryotic protein-encoding genes transcribed by RNA polymerase II. J Biol Chem, 1991 Dec 5, 266(34), 23499 - 504 Study of the endoproteolytic cleavage of platelet glycoprotein IIb using oligonucleotide-mediated mutagenesis; Kolodziej MA et al.; The precursor of platelet membrane glycoprotein IIb (GPIIb) undergoes endoproteolytic cleavage into heavy and light chains post-translation . Endoproteolysis occurs within a 17-amino acid stretch of the precursor that contains 4 arginine residues, 3 in dibasic sequences {Lys-Arg (855-856) and Arg-Arg (858-859)} and a single arginine at 871 . To determine the site of GPIIb cleavage and its role in the function of the glycoprotein IIb/IIIa heterodimer, we mutated arginine 856, the di-arginine sequence 858-859, and arginine 871 and coexpressed the mutants with glycoprotein IIIa (GPIIIa) in COS-1 cells . Each GPIIb mutant formed recombinant GPIIb-IIIa heterodimers, but mutants lacking arginine at 856 or 858-859 failed to undergo cleavage . Nevertheless, heterodimers containing the uncleaved GPIIb were expressed on the cell surface . Because endoproteolysis most often occurs after arginines in dibasic sequences, we next expressed GPIIb mutants containing lysine at 856 or aspartic acid at 855 with GPIIIa . Both mutants were cleaved and surface-expressed, indicating that the dibasic sequence at 858-859, but not at 855-856, is required for GPIIb cleavage . Lastly, we tested the function of GPIIb-IIIa containing uncleaved GPIIb by measuring adhesion of transfected cells to immobilized fibrinogen . We found no difference in the adhesion of cells expressing either wild-type or mutant GPIIb, indicating GPIIb-IIIa heterodimers containing uncleaved GPIIb maintain their ability to interact with fibrinogen. J Biol Chem, 1991 Dec 5, 266(34), 23204 - 14 The cyclophilin multigene family of peptidyl-prolyl isomerases . Characterization of three separate human isoforms; Bergsma DJ et al.; Cyclophilin (CyP), a major cytosolic protein possessing peptidyl-prolyl cis-trans isomerase activity, has been implicated as the specific receptor of the immunosuppressive drug cyclosporin A (CsA) . To identify other potential CsA receptors related to CyP, two human cDNA libraries were screened under low stringency conditions using human CyP cDNA (encoding hCyP1) as a probe . Two cDNAs were identified which encode distinct proteins related to human hCyP1 . These two novel proteins, designated hCyP2 and hCyP3, share 65 and 76% amino acid sequence homology with hCyP1, respectively . Both hCyP2 and hCyP3 contain NH2-terminal hydrophobic extensions of 32 and 42 amino acids, respectively . Protein-specific antibodies revealed the predominant association of hCyP2 and hCyP3 with membranes and subcellular organelles, which suggests that the amino-terminal leader sequences of the two CyP isoforms may act as signal peptides . In contrast to the results with hCyP1, Southern blot analysis indicated that both hCyP2 and hCyP3 gene sequences are represented infrequently in the human genome . Northern and Western blot analysis showed that the distribution of mRNA and proteins of the three hCyPs in differing tissues and cell types was similar . Each hCyP protein was expressed in Escherichia coli, purified, and shown to be an active peptidyl-prolyl isomerase . Substrate specificity was examined with 11 synthetic peptides (Suc-Xaa-Yaa-Pro-Phe-4-nitroanilide), and inhibition of the peptidyl-prolyl isomerase activities associated with hCyP1, hCyP2, and hCyP3 was studied with CsA, MeAla6-CsA and MeBm2t1-CsA . From both equilibrium considerations and the results of kinetic characterizations it is proposed that of these three CyP proteins, hCyP1 is the most likely intracellular target for CsA. J Biol Chem, 1991 Dec 5, 266(34), 23097 - 102 Role of disulfide bonds in biologic activity of human interleukin-6; Snouwaert JN et al.; We have examined the functional importance of the two disulfide bonds formed by the four conserved cysteines of human interleukin (IL-6) . Using a bacterial expression system, we have synthesized a series of recombinant IL-6 mutants in which the constituent cysteines of the first (Cys45-Cys51), second (Cys74-Cys84), or both disulfide bonds of recombinant human interleukin-6 were replaced by other amino acids . Each mutant was partially purified and tested in four representative bioassays . While mutants lacking Cys45 and Cys51 retained activity similar to nonmutated recombinant IL-6, the activity of mutants lacking Cys74 and Cys84 was significantly reduced, especially in assays involving human cell lines . These results indicate that the first disulfide bond of human interleukin-6 is not required for maintenance of normal biologic activity . However, the fact that mutants lacking Cys45 and Cys51 were more active than corresponding cysteine-free mutants indicates that the disulfide bond formed by these residues contributes to biologic activity in the absence of the second disulfide bond . Competition binding studies with representative mutants indicate that their affinity for the human IL-6 receptor parallels their biologic activities on human cells. J Biol Chem, 1991 Dec 5, 266(34), 23022 - 6 The effect of carbohydrate on the structure and stability of erythropoietin; Narhi LO et al.; Erythropoietin is a glycoprotein hormone that stimulates the maturation of late erythroid progenitor cells . It has three N-linked and one O-linked carbohydrates which play an important role in the biosynthesis and biological activities of the protein . To determine the role the carbohydrate might have in maintaining the conformational stability of the protein, the protein expressed in mammalian cells (fully glycosylated), the asialo mammalian-expressed protein, and the protein expressed in Escherichia coli (no carbohydrate) were compared for their stability to guanidine HCl, pH, and temperature . Circular dichroism was used to follow protein unfolding . Both the intact and asialo mammalian-expressed proteins unfolded with a cooperative transition in guanidine HCl, with a midpoint at 1.75 M guanidine HCl . The E . coli-expressed material unfolded with a midpoint of 1.2 M guanidine HCl, and a delta G of unfolding which was 1.4 kcal/mol less than that of the two glycosylated molecules . The E . coli-derived protein was also significantly less stable to pH-induced conformational changes, showing a cooperative transition in 35% glycerol with a midpoint at pH 4.4, while both the intact and asialo mammalian-expressed molecules had a transition midpoint of pH 3.75 in the absence of glycerol, and approximately pH 3 in the presence of 35% glycerol . The E . coli-expressed molecule unfolded and precipitated upon heating to 44 degrees C, while the asialo and intact mammalian-expressed proteins remained soluble, with a Tm of 56 degrees C . From these experiments, the carbohydrate appears to play a critical role in stabilizing the erythropoietin molecule to denaturing conditions, and this increased stability does not depend on the presence of sialic acid. Eur J Biochem, 1991 Dec 5, 202(2), 643 - 8 Complete primary structure of porcine tenascin . Detection of tenascin transcripts in adult submaxillary glands; Nishi T et al.; Tenascin is an extracellular matrix protein that is postulated to modulate tissue differentiation and cell migration during development . cDNA clones for tenascin were isolated from a cDNA library of adult porcine submaxillary glands . Three forms of tenascin clones were observed which varied with the number (8-10) of fibronectin type III (FN-III) domains . A major form consists of the N-terminal domain involved in the hexamer formation of tenascin subunits, 14 epidermal-growth-factor-like domains, nine FN-III domains, and the fibrinogen-like domain . A minor form with ten FN-III domains has never been described . Another striking feature is the lack of an RGD sequence that has been implicated to be crucial for cell adhesion, whereas RGD is present in both chicken and human tenascin sequences . In the adult, tenascin is expressed in very restricted tissues such as brain and chicken gizzard . A survey of tenascin transcripts in various adult rat normal tissues, including brain, revealed that the transcripts were detected only in submaxillary glands where tenascin expression has never been reported. Eur J Biochem, 1991 Dec 5, 202(2), 605 - 16 Site-directed mutagenesis and epitope-mapped monoclonal antibodies define a catalytically important conformational difference between human placental and germ cell alkaline phosphatase; Hoylaerts MF et al.; Placental (PLAP) and germ cell (GCAP) alkaline phosphatases were probed immunologically with a library of 18 murine monoclonal antibodies reacting with different conformational epitopes on PLAP . Three main antigenic domains (I, II and III) were mapped by antibody competition experiments and the relative binding of the antibodies to site-directed PLAP mutants . Relative affinities of each of the antibodies for the wild type (wt) GCAP were 2-3-fold lower than the values found for wt PLAP . Relative affinity was determined for a series of PLAP mutants, in which one, two or three amino acids were substituted for the corresponding wt GCAP residues by site-directed mutagenesis . Substitutions at residues 15, 38, 67, 241 or 254 induced a major decrease in affinity (6-10-fold) primarily for those antibodies reacting within domain I, whereas changes at positions 84 and 297 led to a 2-3-fold enhancement of affinities as measured with antibodies reacting within the three domains . Arg209 was found to constitute the only difference between the S and F allelic phenotypes of PLAP and to structure the epitope for the F/S allotype-discriminating antibodies . Arg241 was found to constitute the epitope for the antibody 17E3 that discriminates between PLAP and GCAP . Mutagenesis at position 68 or 133 had little effect on the overall reactivity with the antibody panel . Substitution in wt PLAP of Glu429 for Gly429 or even for His429 (found at this position in tissue-nonspecific alkaline phosphatase) and Ser429 (found in the intestinal alkaline phosphatase) induced a general decrease in affinities as detected by 16 of the 18 antibodies . The conformational change accompanying mutagenesis of Glu429 in PLAP, is important in view of the recent identification of Gly429 as the major determinant of the unique GCAP inhibition by the uncompetitive inhibitor L-Leu . Relative affinity values determined for the rare L-Leu sensitive heterodimeric FD and SD PLAP phenotypes, suggested that the reactivity pattern of the D homodimer with the antibody panel, would resemble more closely that of wt GCAP than wt PLAP . Our data suggest that the uncompetitive inhibition of GCAP by L-Leu is due to an enzymatically critical conformational change in a loop region proximal to the active site of the enzyme, induced by substitution of a single amino acid residue. J Biol Chem, 1991 Dec 5, 266(34), 23329 - 33 Characterization of Escherichia coli SecA protein binding to a site on its mRNA involved in autoregulation; Dolan KM et al.; In order to understand further the autogenous regulation of Escherichia coli secA translation, we have set up a purified system to study the binding of SecA protein to portions of its mRNA . Specific SecA protein-RNA binding was demonstrated by UV cross-linking, filter binding, and gel shift assays . Use of the filter binding assay allowed optimization of binding, which was influenced by Mg2+ and ATP concentrations, and a measurement of the affinity of this interaction . A nested series of RNAs lacking either 5' or 3' portions of geneX-secA sequences were used to localize the SecA protein binding site to sequences around the geneX-secA intergenic region . These studies imply that SecA protein directly regulates its own translation by a specific RNA binding activity that presumably blocks translational initiation. J Biol Chem, 1991 Dec 5, 266(34), 23003 - 9 Reconstitution and properties of homologous and chimeric HIV-1.HIV-2 p66.p51 reverse transcriptase; Howard KJ et al.; Metal chelate affinity chromatography has been used to follow reconstitution of the 66- and 51-kDa human immunodeficiency (HIV)-1 and HIV-2 reverse transcriptase (RT) subunits into heterodimer, as well as chimeric enzymes comprised of heterologous subunits . By adding a small N-terminal polyhistidine extension to the 51-kDa subunit of either enzyme, reconstituted RT could be recovered from a cell lysate by chromatography on Ni(2+)-nitrilotriacetic acid-Sepharose . Homologous RT subunits rapidly associated to form the respective heterodimers (1-p66.1-p51 and 2-p66.2-p51) when bacterial lysates containing the individual components were mixed . Under the same conditions, association of p66 HIV-2 and p51 HIV-1 RT was inefficient and could be improved slightly by prolonged incubation of the respective p66 and p51 subunits . In contrast, HIV-1 p66 RT rapidly associated with the 51-kDa subunit of the HIV-2 enzyme . RNA-dependent DNA polymerase activity was associated with all reconstituted enzymes, and the response of each chimeric RT to an inhibitor selective for the HIV-1 enzyme indicated that sensitivity to inhibition was determined by the source of its 66-kDa subunit. J Mol Biol, 1991 Dec 5, 222(3), 787 - 803 Construction of new ligand binding sites in proteins of known structure . II . Grafting of a buried transition metal binding site into Escherichia coli thioredoxin; Hellinga HW et al.; In an accompanying paper a computational procedure is described, which introduces new ligand-binding sites into proteins of known structure . Here we describe the experimental implementation of one of the designs, which is intended to introduce a copper-binding site into Escherichia coli thioredoxin . The new binding site can be introduced with a minimum of four amino acid changes . The binding site is buried so that structural rules for making mutations in the hydrophobic core of a protein, as well as for the introduction of new functions, are being tested in this experiment . The mutant protein is folded even in the absence of metals, and variants that retain the original activity of thioredoxin can be isolated . The protein has gained a metal-binding site specific for transition metals . The metal co-ordination chemistry at the binding site varies depending on the metal that is introduced into it . Mercury(II) is co-ordinated in the expected manner . Copper(II) binds in a way that was not anticipated in the original design . It appears to use two of the four residues intended to form the co-ordination sphere, and two other residues that were not part of the original set of mutations . It is therefore necessary not only to introduce new functional groups to form a new site, but also to consider and remove alternative modes of binding. J Mol Biol, 1991 Dec 5, 222(3), 567 - 80 EnvZ controls the concentration of phosphorylated OmpR to mediate osmoregulation of the porin genes; Russo FD et al.; Osmoregulation of the bacterial porin genes ompF and ompC is controlled by a two-component regulatory system . EnvZ, the sensor component of this system, is capable both of phosphorylating and dephosphorylating OmpR, the effector component . Mutations were isolated in envZ that abolish the expression of both porin genes . These mutants appear to have lost the kinase activity of EnvZ while retaining their phosphatase activity, so that in their presence OmpR is completely unphosphorylated . The behavior of these mutants in haploid, and in diploid with other envZ alleles, is consistent with a model in which EnvZ mediates osmoregulation by controlling the concentration of a single species . OmpR-P. J Biol Chem, 1991 Dec 5, 266(34), 23169 - 74 Transcriptional regulation of cytochrome d in nitrogen-fixing Azotobacter vinelandii . Evidence that up-regulation during N2 fixation is independent of nifA but dependent on ntrA; Moshiri F et al.; Cytochrome d has been postulated to be the "respiratory protection" oxidase of Azotobacter vinelandii, allowing this organism to fix nitrogen under aerobic growth conditions . We have previously cloned and characterized the structural genes for the A . vinelandii cytochrome d (cydA and cydB) . The cyd genes are co-transcribed, yielding an mRNA of approximately 3.6 kilobase pairs . The level of the cyd message was 2-3-fold higher in cells that were fixing nitrogen, as compared with non-nitrogen-fixing cells . RNase protection analysis was used to determine the transcriptional start site at 275 bases upstream of the initiator ATG of cydA, and this start site was the same for nitrogen-fixing and non-nitrogen-fixing cells . The cyd promoter has sequence similarities to the canonical Escherichia coli promoters, which are transcribed by the major sigma 70 form of RNA polymerase . Plasmid-borne lacZ transcriptional fusions were constructed, using approximately 650 base pairs of 5'-upstream sequences of the cyd structural genes . This region had a strong promoter activity which was further up-regulated 1.5-2.5-fold upon the induction of nitrogen fixation . The cyd-lacZ fusions were characterized in a nifA- as well as an ntrA- background . Mutations in neither of these nif regulatory genes affected the constitutive expression of cyd under non-nitrogen-fixing conditions . However, the up-regulation of this promoter during the induction of nitrogen fixation was abolished only in the ntrA- background . Based on these results, the cytochrome d promoter of A . vinelandii belongs to a new class of nitrogen-regulated promoters which, unlike the authentic nif genes, does not require the ntrA gene product for its expression . The up-regulation of this promoter during nitrogen fixation, however, requires the ntrA gene product. Eur J Pharmacol, 1991 Dec 3, 205(3), 271 - 6 Protective effect of SR 27417, a novel PAF antagonist, on lethal anaphylactic and endotoxin-induced shock in mice; Herbert JM et al.; In anaphylactic shock, SR 27417, the first member of a newly developed series of PAF (platelet-activating factor) antagonists, inhibited in a dose-dependent manner the lethal effect of antigen (ovalbumin) rechallenge in actively sensitized mice . It protected mice when given i.v . 5 min before ovalbumin challenge (ED50 = 50 micrograms/kg) or when given p.o . 1 h before ovalbumin administration (ED50 = 1.25 mg/kg) . After i.v . or oral administration, SR 27417 (2.5 and 10 mg/kg, respectively) greatly improved the survival rate of mice after antigen challenge and had an extremely long duration of action (48 and 30 h, respectively) . Similarly, i.v . or oral doses of SR 27417 afforded in mice complete protection against endotoxin-induced lethality (ED50 values were 100 and 150 micrograms/kg, respectively) . SR 27417 (1 mg/kg) inhibited endotoxin-induced death in mice with impressive oral or i.v . durations of action of 66 and 110 h, respectively . These results confirm that PAF plays a major role in anaphylactic and endotoxin-induced shock and that SR 27417 may be an effective preventative drug. Biochemistry, 1991 Dec 3, 30(48), 11412 - 20 Interaction of ribosomal protein S1 and initiation factor IF3 with the 3' major domain and the decoding site of the 30S subunit of Escherichia coli; Laughrea M et al.; We have studied the effect of the binding of ribosomal protein S1 and initiation factor IF3 on the accessibility of nucleotide residues 584-1506 in the small subunit of the Escherichia coli ribosome . Protein S1 strongly decreases RNase V1 attack at G1164, in hairpin 40 of the 3' major domain, and weakly decreases DMS attack at C1302, in the central loop of the 3' major domain, and at A1503, in the 3' minor domain . It also weakly increases the DMS reactivity of A1004, in the 3' major domain, and of A901, in the central domain . Factor IF3 strongly decreases RNase V1 attack (but not dimethyl sulfate attack) at A1408, in the decoding site, and weakly protects A1500, in the 3' minor domain and near the colicin E3 cleavage site . Neomycin does not interfere with this effect of IF3, but IF3 interferes with the protective effect of neomycin against dimethyl sulfate attack at A1408. Biochemistry, 1991 Dec 3, 30(48), 11403 - 12 Stimulation of DNA polymerase alpha activity by microtubule-associated proteins; Shioda M et al.; Microtubule-associated protein 2 (MAP2) isolated from porcine brains stimulated the activity of DNA polymerase alpha immunopurified from calf thymus or human lymphoma cells, in a dose-dependent manner . This stimulation was pronounced when activated DNA or poly(dA).(dT)10 was used as the template-primer . DNA polymerase alpha bound to a MAP2-immobilized column, whereas preincubation of the enzyme with unbound MAP2 prevented binding to the column . These events suggested that a physical binding occurred between the polymerase and MAP2 . Kinetic analyses revealed that MAP2 decreased the Km value of the polymerase for deoxyribonucleotides, irrespective of the species of template-primer . A concomitant increase in Vmax was observed; however, the extent of the increase depended on the species of template-primer . MAP2 also decreased the Km value of the polymerase for template-primers when activated DNA of poly(dA).(dT)10 was used as the template-primer . Product analyses showed that MAP2 did not significantly alter the processivity of the polymerase and the increment of Vmax is considered to be due to an increase in the frequency of initiation of DNA synthesis . The stimulation by MAP2 occurred specifically in the activity of DNA polymerase alpha, but not DNA polymerases beta, gamma, and I from Escherichia coli . Other MAPs, tau and 190-kDa MAP, could substitute for MAP2 . Thus, the specific stimulation of DNA polymerase alpha by MAPs supports the notion of a possible involvement of MAPs or MAP-like proteins in DNA replication, in vivo. Eur J Pharmacol, 1991 Dec 3, 205(3), 277 - 82 Elevation of plasma endothelin concentrations during endotoxin shock in dogs; Nakamura T et al.; The effect of endotoxin on the release of endothelin, a novel potent vasoconstrictor peptide, was examined in anesthetized dogs and in cultured endothelial cells . Administration of 2.63 mg lipopolysaccharide, E . coli 0111:B4/kg body weight caused shock in the animals and produced a long-lasting increase in the plasma immunoreactive endothelin-1 level that remained higher than the basal level (1.83 pg/ml as mean level) from 30 to 120 min after the injection, with a peak at 90 min (8.15 pg/ml as mean level) . In vitro immunoreactive endothelin-1 in a culture medium, in which calf pulmonary artery endothelial cells were incubated in the presence of 10% fetal bovine serum, increased dose dependently with the concentration of added lipopolysaccharide between 0.01 and 10 micrograms/ml . These data indicate that plasma endothelin increases during endotoxin shock and that stimulation by endotoxin, per se, in the presence of serum participates at least partially in the mechanism for its release. Biochim Biophys Acta, 1991 Dec 2, 1129(1), 64 - 72 Thermal stability of turnip yellow mosaic virus RNA: effect of pH and multivalent cations on RNA deaggregation and degradation; Sam T et al.; Light scattering studies of RNA isolated from turnip yellow mosaic virus (TYMV) revealed a molar mass of 1.9.10(6) g mol-1, which is close to the value of 2.0.10(6) g mol-1 published for intact genomic TYMV RNA (2M RNA) . However, gel electrophoresis under denaturing conditions demonstrated that only 30-40% of this native RNA was 2M RNA . Sucrose gradient centrifugation revealed the occurrence of a series of smaller RNA size classes, the mass ratios of which were greatly influenced by the pH of the solution and the presence of EDTA . These results suggest that native TYMV RNA preparations originally contain a mixture of intact RNA particles and of aggregates of RNA fragments with the same molar mass of about 2.10(6) g mol-1, and that the size classes are intermediates in the deaggregation process of the degraded genomic TYMV RNA . The native RNA displayed pH-dependent deaggregation and degradation . The degradation process of 2M RNA followed (pseudo) first-order kinetics . Lower degradation rates were observed for RNA depleted of divalent cations and polyamines . For depleted 2M RNA an enthalpy of activation of about 100 kJ mol-1 and an almost zero entropy of activation was calculated . Similar values were also found for depleted E . coli ribosomal RNAs and depleted MS2 RNA, demonstrating that all RNAs are equally vulnerable to degradation . In the presence of multivalent cations the activation enthalpy for 2M TYMV RNA degradation increased to 150 kJ mol-1 and the entropy of activation to 150 J K-1 mol-1, indicative for a different degradation mechanism. Biochim Biophys Acta, 1991 Dec 2, 1129(1), 135 - 8 Cloning and sequence analysis of membrane-bound alkaline phosphatase cDNA of the silkworm, Bombyx mori; Itoh M et al.; The nucleotide sequence (1974 bp) of cDNA coding for membrane-bound alkaline phosphatases (m-ALP) of Bombyx mori was isolated . The cDNA clone contained an open reading frame encoding a polypeptide (547 amino acids), which contains a hydrophobic signal peptide of 36 amino acids and the mature protein of 511 amino acids (Mr = 56,163) . We found a highly hydrophobic domain presumed to be a membrane anchoring region at the C-terminus . Comparing analysis between Bombyx m-ALP and mammalian and Escherichia coli ALPs suggested an evolutionary relationship of sharing a common ancestral gene. Biochim Biophys Acta, 1991 Dec 2, 1129(1), 109 - 11 The nucleotide sequence of the Escherichia coli crp divergent RNA and an overlapping ORF; Bhasin R et al.; The nucleotide sequence specifying the crp divergent RNA of Escherichia coli was determined . An open reading frame (ORF) is located at +135 to +536 relative to the initiation site of the divergent RNA . Potential factor independent transcription terminators were found at +257 to +294 and +544 to +576 . These putative termination sites may account for the two RNAs of approximately 300 and 550 nucleotides previously identified as originating from the crp divergent promoter. FEBS Lett, 1991 Dec 2, 294(1-2), 56 - 8 The 3' promoter region involved in RNA synthesis directed by the turnip yellow mosaic virus genome in vitro; Gargouri-Bouzid R et al.; We have previously shown that the last 100 nucleotides from the 3' end of turnip yellow mosaic virus (TYMV) RNA compete in vitro with genomic RNA for the TYMV-specific RNA-dependent RNA polymerase (RdRp) . To further characterize the promoter on genomic RNA that produces complementary RNA strands, shorter fragments corresponding to the 3' region of the viral RNA were generated and used in in vitro assays . Fragments as short as 38 nucleotides corresponding to the 3' end of TYMV RNA compete with the viral RNA for the RdRp suggesting that the 3' promoter on plus strand RNA is probably less than or equal to 38 nucleotides long . These transcripts are themselves used as templates in vitro. FEBS Lett, 1991 Dec 2, 294(1-2), 59 - 63 Insertional inactivation of the psbO gene encoding the manganese stabilizing protein of photosystem II in the cyanobacterium Synechococcus PCC7942 . Effect on photosynthetic water oxidation and L-amino acid oxidase activity; Bockholt R et al.; A Synechococcus PCC7942 mutant in which the psbO gene was inactivated by insertion of a chloramphenicol interposon and which did not contain any detectable manganese stabilizing protein in immunoblot experiments, was constructed . Such a Synechococcus mutant was able to grow under photoautotrophic conditions . Isolated thylakoid membranes from the mutant required addition of CaCl2 and MnCl2 for photosynthetic O2 evolution, and the detectable L-amino acid oxidase activity in the isolated thylakoid membranes from the mutant was approximately four times higher than in wild-type thylakoids . The results are discussed with respect to our model suggesting that the water-oxidizing enzyme may have evolved from a flavoprotein with L-amino acid dehydrogenase/oxidase activity. FEBS Lett, 1991 Dec 2, 294(1-2), 16 - 8 Diverse proteins homologous to inositol monophosphatase; Neuwald AF et al.; Bovine inositol monophosphatase (IMP) and several homologous proteins were found to share two sequence motifs with bovine inositol polyphosphate 1-phosphatase (IPP) . These motifs may correspond to binding sites within IMP and IPP for inositol phosphates or for lithium, since both substances are bound by these proteins . This suggests that the proteins homologous to IMP, which have diverse biological roles but whose function is not clear, may act by enhancing the synthesis or degradation of phosphorylated compounds. Virology, 1991 Dec, 185(2), 605 - 14 Structural similarities between the RNAs of two satellites of tobacco necrosis virus; Danthinne X et al.; The complete nucleotide sequence of satellite tobacco necrosis virus 2 (STNV-2) RNA has been determined . It has the same organization as the previously studied STNV-1 RNA . The 5' untranslated regions (about 30 nt) are nearly identical, while the coat protein coding regions (about 600 nt) have 55% nucleotide sequence similarity . The 620-nt-long trailer sequences, with 64% nucleotide sequence conservation, can fold into a phylogenetically conserved secondary structure consisting of three pseudoknots followed by a long-range interaction-born hairpin structure . The significance of these elements is discussed in view of the particular properties (stability, translational competitiveness, and replication) that characterize these RNAs. Virology, 1991 Dec, 185(2), 572 - 9 Two newly detected nonstructural viral proteins in potyvirus-infected cells; Rodriguez-Cerezo E et al.; The existence of two viral RNA-encoded proteins in cells infected with tobacco vein mottling potyvirus (TVMV) has been demonstrated . One of the proteins (named 34K) maps at the N-terminus of the TVMV polyprotein and the other (42K) between the helper component and cylindrical inclusion proteins; both had previously been predicted in the consensus potyviral genetic map . The 34K and 42K coding regions of TVMV were cloned separately in a bacterial expression vector and the proteins were isolated from transformed Escherichia coli . These were used to raise polyclonal antibodies which reacted specifically with proteins of the expected size in immunoblots of extracts of TVMV-infected tobacco leaves and protoplasts . In addition to 42K, the anti-42K serum detected similar amounts of a second protein of apparent size 37 kDa that was absent in 42K-expressing bacteria . Both 34K and 42K were present predominantly in membrane-enriched fractions of extracts of TVMV-infected tobacco leaves . Computer analysis of the deduced amino acid sequence of 42K suggests that this viral protein may be an integral transmembrane protein. Radiat Res, 1991 Dec, 128(3), 251 - 7 Ionizing radiation at low doses induces inflammatory reactions in human blood; Vicker MG et al.; Irradiation of whole blood with 137Cs gamma rays intensifies the oxidative burst . Oxidant production was used as an indicator of inflammatory cell reactions and was measured by luminol-amplified chemiluminescence after treatment with inflammatory activators including bacteria, the neutrophil taxin formyl-Met-Leu-Phe, the Ca2+ ionophore A23187, the detergent saponin, and the tumor promoter phorbol ester . The irradiation response is dose-dependent up to about 100 microGy, is detectable within minutes, persists at least 1 h, and is transmitted intercellularly by a soluble mediator . The response is completely inhibited by Ca2+ sequestration in the presence of A23187 or by adenosine, indicating its Ca2+ dependency, and by the phospholipase A2 blocker p-bromphenacyl bromide . However, inhibition by the cyclooxygenase blocker aspirin is sporadic or absent . Blood taken after diagnostic examination of lungs with X rays also exhibited intensified chemiluminescence . These reactions implicate a role for specific amplifying mediator pathways, especially metabolites of the arachidonic acid cascade, in the response: "damage and repair" to cells or DNA plays little or no role . Our results provide evidence for a new mechanism of radiation action with possible consequences for the homeostasis of reactions involving inflammation and second messengers in human health and early development. Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10907 - 11 Identification of a cold shock transcriptional enhancer of the Escherichia coli gene encoding nucleoid protein H-NS; La Teana A et al.; The hns (27 min) gene encoding the 15.4-kDa nucleoid protein H-NS was shown to belong to the cold shock regulon of Escherichia coli, its expression being enhanced 3- to 4-fold during the growth lag that follows a shift from 37 degrees C to 10 degrees C . A 110-base-pair (bp) DNA fragment containing the promoter of hns fused to a promoterless cat gene (hns-cat fusion) conferred a similar cold shock response to the expression of chloramphenicol acetyltransferase (CAT) activity in vivo and in coupled transcription-translation systems prepared with extracts of cold-shocked cells . Extracts of the same cells produce a specific gel shift of the 110-bp DNA fragment and this fragment, immobilized on a solid support, specifically retains a single 7-kDa protein present only in cold-shocked cells that was found to be identical to F10.6 (CS7.4), the product of cspA . This purified protein, which is homologous to human DNA-binding protein YB-1, recognizes some feature of the 110-bp promoter region of hns and acts as a cold shock transcriptional activator of this gene since it stimulates the expression of CAT activity and of cat transcription in in vitro systems programmed with plasmid DNA carrying the hns-cat fusion. Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10865 - 9 URF13, a maize mitochondrial pore-forming protein, is oligomeric and has a mixed orientation in Escherichia coli plasma membranes; Korth KL et al.; URF13, an inner mitochondrial membrane protein of the maize Texas male-sterile cytoplasm (cms-T), has one orientation in the inner membrane of maize mitochondria but two topological orientations in the plasma membrane when expressed in Escherichia coli . Antibodies specific for the carboxyl terminus of URF13 and for an amino-terminal tag fused to URF13 in E . coli were used to determine the location of each end of the protein following protease treatments of right-side-out and inside-out vesicles derived from cms-T mitochondria and the E . coli plasma membrane . Cross-linking studies indicate that a portion of the URF13 population in mitochondria and E . coli exists in membranes in an oligomeric state and, in combination with proteolysis studies, show that individual subunits within a given multimer have the same orientation . A three-membrane-spanning helical model for URF13 topology is presented. Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10691 - 5 Transcription factor requirement for multiple rounds of initiation by human RNA polymerase II; Szentirmay MN et al.; We have investigated conditions that allow multiple rounds of transcription initiation from the adenovirus major late promoter in an in vitro system derived from HeLa cell nuclear extracts . Templates containing guanine-free cassettes provided a direct assay for discriminating between reinitiated transcripts and transcripts generated by a first-round of transcription initiations . When reactions were reconstituted with the previously characterized class II transcription factors (TFIIA, TFIIB, TFIID, TFIIE/F), transcription by human RNA polymerase II from the adenovirus major late promoter was essentially restricted to a single round of initiations . Reinitiations at previously transcribed major late templates required an additional activity, designated reinitiation transcription factor (RTF) . The RTF activity could be separated from the required transcription initiation factors . Semipurified human RTF also promoted transcription reinitiations at minimal promoters derived from the human c-myc, histone H4, and heat shock 70-kDa protein genes, indicating that the same reinitiation factor may be utilized by many, if not all, genes . The possible role of RTF in regulating the transcription rate of various class II genes is discussed. Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10573 - 7 Evaluating the effects of a single amino acid substitution on both the native and denatured states of a protein; Lin TY et al.; For proteins that contain a disulfide bond, stability is linked thermodynamically to thiol-disulfide exchange . We use this relationship to obtain unfolding free energies for both the reduced and oxidized forms of Escherichia coli thioredoxin from measurements of the effective concentrations of protein thiols . We then evaluate the effect of an amino acid substitution on disulfide bond formation in both the native and denatured states of the protein . Although the Pro-34----Ser substitution in thioredoxin results in a decrease of the effective concentration of protein thiols in the native state, the effective concentration increases in the denatured state . The net effect of the amino acid substitution is to increase the stability of reduced thioredoxin by approximately 2.4 kcal/mol, whereas the stability of the oxidized protein remains the same . By assuming a two-state unfolding equilibrium and a mutation free energy of -7.7 kcal/mol for the Pro-34----Ser substitution in the reduced, urea-unfolded state (based on estimates of solvation and entropic changes), we obtained relative free energies for the native and denatured states of the mutant and wild-type proteins, in both the reduced and oxidized forms. Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10568 - 72 A 70-amino acid zinc-binding polypeptide from the regulatory chain of aspartate transcarbamoylase forms a stable complex with the catalytic subunit leading to markedly altered enzyme activity; Markby DW et al.; In an effort to clarify effects of specific protein-protein interactions on the properties of the dodecameric enzyme aspartate transcarbamoylase (carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), we initiated studies of a simpler complex containing an intact catalytic trimer and three copies of a fragment from the regulatory chain . The partial regulatory chain was expressed as a soluble 9-kDa zinc-binding polypeptide comprising 11 amino acids encoded by the polylinker of pUC18 fused to the amino terminus of residues 84-153 of the regulatory chain; this polypeptide includes the zinc domain detected in crystallographic studies of the holoenzyme . In contrast to intact regulatory chains, the zinc-binding polypeptide is monomeric in solution because it lacks the second domain responsible for dimer formation and assembly of the dodecameric holoenzyme . The isolated 9-kDa protein forms a tight, zinc-dependent complex with catalytic trimer, as shown by the large shift in electrophoretic mobility of the trimer in nondenaturing polyacrylamide gels . Enzyme assays of the complex showed a hyperbolic dependence of initial velocity on aspartate concentration with Vmax and Km for aspartate approximately 50% lower than the values for free catalytic subunit . A mutant catalytic subunit containing the Lys-164----Glu substitution exhibited a striking increase in enzyme activity at low aspartate concentrations upon interaction with the zinc domain because of a large reduction in Km upon complex formation . These changes in functional properties indicate that the complex of the zinc domain and catalytic trimer is an analog of the high-affinity R ("relaxed") state of aspartate transcarbamoylase, as proposed previously for a transiently formed assembly intermediate composed of one catalytic and three regulatory subunits . Conformational changes at the active sites, resulting from binding the zinc-containing polypeptide chains, were detected by difference spectroscopy with trinitrophenylated catalytic trimers . Isolation of the zinc domain of aspartate transcarbamoylase provides a model protein for study of oligomer assembly, communication between dissimilar polypeptides, and metal-binding motifs in proteins. Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10563 - 7 Phosphorylation controls binding of acidic proteins to the ribosome; Naranda T et al.; The replacement of each one of the eight serine residues present in the amino acid sequence of the Saccharomyces cerevisiae acidic ribosomal phosphoprotein YP2 beta (L45) by different amino acids has been performed by heteroduplex site-directed mutagenesis in the cloned gene . The mutated DNA was used to transform a yeast strain previously deprived of the original protein YP2 beta (L45) by gene disruption . The replacement of serine in position 19 by either alanine, aspartic acid, or threonine prevents in vivo phosphorylation of the protein and its interaction with the ribosome . The serine-19 mutated gene is unable to rescue the negative effect on the growth rate caused by elimination of the original protein in YP2 beta (L45) gene disrupted strains . The mutation of any one of the other seven serine residues has no effect on either the phosphorylation or the ribosome binding capacity of the protein, although replacement of serine-72 seems to increase the sensitivity of the polypeptide to degradation . These results provide strong evidence indicating that ribosomal protein phosphorylation plays an important part in the activity of the particle and that it supports the existence of a control mechanism of protein synthesis, which would regulate the level of phosphorylation of acidic proteins. Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10553 - 7 Functional analysis of an oxygen-regulated transcriptional enhancer lying 3' to the mouse erythropoietin gene; Pugh CW et al.; Erythropoietin, the major hormone controlling red-cell production, is regulated in part through oxygen-dependent changes in the rate of transcription of its gene . Using transient transfection in HepG2 cells, we have defined a DNA sequence, located 120 base pairs 3' to the poly(A)-addition site of the mouse erythropoietin gene, that confers oxygen-regulated expression on a variety of heterologous promoters . The sequence has the typical features of a eukaryotic enhancer . Approximately 70 base pairs are necessary for full activity, but reiteration restores activity to shorter inactive sequences . This enhancer operates in HepG2 and Hep3B cells, but not in Chinese hamster ovary cells or mouse erythroleukemia cells, and responds to cobalt but not to cyanide or 2-deoxyglucose, thus reflecting the physiological control of erythropoietin production accurately. Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10515 - 9 Interplay of two cis-acting mRNA regions in translational control of sigma 32 synthesis during the heat shock response of Escherichia coli; Nagai H et al.; When Escherichia coli cells are transferred from 30 degrees C to 42 degrees C, transcription from specific promoters recognized by RNA polymerase containing sigma 32 (the rpoH gene product) is transiently activated, resulting in induction of heat shock proteins . Transcription from heat shock promoters is activated by an increased cellular concentration of sigma 32 due to enhanced synthesis and stabilization . We have constructed and examined the expression of mutant derivatives (deletions and base substitutions) of rpoH-lacZ gene fusion . Synthesis of a sigma 32-beta-galactosidase fusion protein was found to be regulated at the translational level involving two distinct 5'-proximal rpoH coding regions . A small region immediately downstream of the initiation codon is required for potentially high-level expression, whereas a much larger internal region is required for thermal regulation--namely, repression at low temperature or nonstress conditions . The two mRNA regions act as positive and negative cis elements, respectively, in controlling rpoH translation . We propose that an interplay between these RNA regions involving secondary structure formation is important in regulating translation initiation and that transient disruption of secondary structure represents a primary step of the heat shock response. Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10500 - 4 p55CDC25 is a nuclear protein required for the initiation of mitosis in human cells; Millar JB et al.; The cdc25+ gene of fission yeast encodes a phosphotyrosine phosphatase that dephosphorylates tyrosine-15 of p34cdc2 and thereby activates p34cdc2/cyclin to bring about entry into M phase . We have recently cloned a human homolog, CDC25, which rescues the M-phase initiation defect of yeast cdc25 temperature-sensitive mutants . Antibodies raised against the CDC25 gene product specifically recognize human proteins of approximately 55 and approximately 52 kDa . Microinjection of affinity-purified anti-CDC25 antibodies into HeLa cells inhibits entry into mitosis . These observations suggest that the CDC25 gene products are essential for the initiation of mitosis in human cells, similar to their homologs in fission yeast and Drosophila . CDC25 gene products, like p34CDC2, are localized primarily in the nucleus during interphase, suggesting that activation of p34CDC2/cyclin by p52/p55CDC25 occurs within the nucleus. Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10392 - 6 In vitro assembly of apophytochrome and apophytochrome deletion mutants expressed in yeast with phycocyanobilin; Deforce L et al.; Recombinant pea type I phytochrome apoprotein expressed in yeast is shown to assemble in vitro with phycocyanobilin to produce a photoreversible phytochrome-like adduct . As an initial investigation of the amino acid sequence requirements for chromophore incorporation, three phyA gene product deletion mutants were produced in yeast . Truncation of the N-terminal tail to residue 46 demonstrates that this region is not critical to bilin attachment, but a deletion mutant lacking 222 amino acids from the N terminus failed to yield holophytochrome in vitro, under the same conditions . A mutant comprising a deletion of the C terminus to residue 548 showed bilin incorporation and red/far-red photoreversibility, indicating that bilin-apophytochrome assembly still occurred even when the entire C-terminal domain was truncated. Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10382 - 6 Three-dimensional structure of the Escherichia coli phosphocarrier protein IIIglc; Worthylake D et al.; The crystal structure of a proteolytically modified form of the Escherichia coli phosphocarrier and signal transducing protein IIIglc has been determined by multiple isomorphous and molecular replacement . The model has been refined to an R-factor of 0.166 for data between 6- and 2.1-A resolution with an rms deviation of 0.020 A from ideal bond lengths and 3.2 degrees from ideal bond angles . The molecule is a beta-sheet sandwich, with six antiparallel strands on either side . Several short distorted helices line the periphery of the active site, which is a shallow extremely hydrophobic depression approximately 18 A in diameter near the center of one face . The side chains of the active site histidine residues 75 and 90 face each other at the center of the depression, with the N3 positions exposed to solvent, separated by 3.3 A in an excellent position to form adducts with phosphate . Chloroplatinate forms a divalent adduct with both histidyl side chains, suggesting that the phosphodonor reaction might proceed through a similar transition state . The hydrophobic patch forms the primary crystal contact, suggesting a mode of association of IIIglc with other components of the phosphoenolpyruvate-dependent phosphotransferase system. Metabolism, 1991 Dec, 40(12), 1337 - 45 Intestinal absorption of aspartame decomposition products in adult rats; Lipton WE et al.; The dipeptide sweetener aspartame (N-L-alpha-aspartyl-L-phenylalanine, 1-methyl ester; alpha-APM) is relatively stable in dry powder form . However, when exposed to elevated temperature, extremes of pH and/or moisture, alpha-APM is converted into a variety of products . In aqueous solution alpha-APM decomposes to yield methanol, two isomeric forms of L-aspartyl-L-phenylalanine (Asp-Phe) {alpha-Asp-Phe and beta-Asp-Phe}, and APM's diketopiperazine cyclo-Asp-Phe . Depending on beverage storage conditions, individuals drinking alpha-APM-sweetened beverages may consume small quantities of these three compounds . Relatively little has been published about the metabolism of beta-Asp-Phe and cyclo-Asp-Phe . We compared the absorption and metabolism of alpha-Asp-Phe, beta-Asp-Phe, and cyclo-Asp-Phe with that of L-phenylalanine (Phe) in adult rats . Steady-state perfusion studies of rat jejunum indicated rapid carrier-assisted uptake of Phe and alpha-Asp-Phe, but only slow passive diffusion of beta-Asp-Phe and cyclo-Asp-Phe from the lumen . Homogenates of rat intestinal mucosa, liver, and cecal contents, as well as homogenates of pure cultures of Escherichia coli B, catalyzed the hydrolysis of alpha-Asp-Phe, but not cyclo-Asp-Phe . Homogenates of E coli and rat cecal contents, but not homogenates of rat liver or intestinal mucosa catalyzed the hydrolysis of beta-Asp-Phe. Crit Care Med, 1991 Dec, 19(12), 1552 - 60 Systemic and regional oxygen uptake and delivery and lactate flux in endotoxic dogs infused with dopexamine; Cain SM et al.; OBJECTIVE: To test whether dopexamine, a dopaminergic and beta 2-adrenergic receptor agonist, would: a) direct a greater share of cardiac output to gut than to muscle when used to increase systemic oxygen delivery (DO2) in endotoxic dogs; and b) enhance the ability of peripheral tissues to extract oxygen . DESIGN: Two groups of eight dogs infused for 1 hr with 2 mg/kg Escherichia coli endotoxin . One group was continually infused with dopexamine (12 micrograms/min.kg) and the other group was not (control group) . After 2 hrs, oxygen extracting ability was challenged by changing inspired gas to 12% oxygen for 30 mins . SUBJECTS: Anesthetized, paralyzed, pump-ventilated mongrel dogs . INTERVENTIONS: Donor RBCs and dextran used during endotoxin infusion to maintain cardiac output while preserving hematocrit near 40% . MEASUREMENTS AND MAIN RESULTS: In the dopexamine-treated group, cardiac output, systemic DO2, and oxygen consumption (VO2) were higher than in the control group during the first 90 mins, but were not thereafter . Gut and muscle blood flow did not differ between groups, but the fraction of cardiac output going to each region tended to be less in the dopexamine-treated dogs . Arterial lactate values increased to about 6 mmol/L in all dogs . In both groups, limb muscle first produced lactate but then took up lactate after the first hour . The gut in controls converted from lactate uptake in the first hour to producing about 20 mumol/min.100 g, whereas the gut never produced lactate in the dopexamine-treated group . During hypoxia, systemic DO2 and VO2 decreased only in the dopexamine-treated group, even though oxygen extraction was only slightly above 40% . Oxygen extraction was not demonstrably improved by dopexamine treatment . CONCLUSIONS: Dopexamine temporarily increased systemic DO2 and VO2 in volume-expanded endotoxic dogs during normoxia and may have caused gut mucosa to be preferentially perfused and thus to be kept better oxygenated. J Infect Dis, 1991 Dec, 164(6), 1224 - 7 Protection of mice from Lyme borreliosis by oral vaccination with Escherichia coli expressing OspA; Fikrig E et al.; Mice immunized with recombinant outer surface protein A (OspA) in Freund's adjuvant or with intraperitoneal injections of live Escherichia coli expressing OspA have been shown to be protected from infection with Borrelia burgdorferi . To investigate the efficacy of oral vaccination, C3H/He mice were inoculated with 10(8) live E . coli expressing recombinant OspA by gavage and boosted in a similar manner on days 10, 20, 30, and 40 . The animals developed serum IgG antibodies to OspA by immunoblot and were protected from infection when challenged with 10(4) B . burgdorferi intradermally 14 days after the last boost . Control mice did not develop antibodies to OspA and were not protected against challenge infection . These results suggest that an oral preparation of recombinant OspA could potentially be used for vaccination. Endocrinology, 1991 Dec, 129(6), 3027 - 33 Effects of zinc and other divalent metals on deoxyribonucleic acid binding and hormone-binding activity of human alpha 1 thyroid hormone receptor expressed in Escherichia coli; Miyamoto T et al.; Full-length human thyroid hormone receptor alpha 1 (hTR alpha 1) was expressed in Escherichia coli using a T7 expression system . While present in large amounts, the receptor was highly enriched in the insoluble fraction after cell lysis . We describe here the successful solubilization and refolding of the expressed receptor in a functional form in the presence of Zn2+ . Using a DNA-cellulose binding assay and gel shift assay, we found that treatment of expressed receptor with 1 mM EDTA in the denaturing agent (5 M guanidine-HCl) results in the formation of aporeceptor that does not specifically recognize target DNA, while it does retain T3-binding activity . This aporeceptor recovered DNA-binding activity by adding Zn2+ during refolding . Zinc-induced restoration of DNA-binding activity occurred in a dose- and time-dependent manner . Moreover, once recovered, this DNA-binding activity persisted without Zn2+, even in the presence of 1 mM EDTA . These data indicate that the hTR alpha 1 molecule has a high affinity for Zn2+, and this metal coordination is essential for proper folding of TR protein into its native active structure. Endocrinology, 1991 Dec, 129(6), 2862 - 6 Interleukin-1 beta induces interleukin-1 alpha messenger ribonucleic acid expression in primary cultures of Leydig cells; Wang DL et al.; Previously, we have reported that interleukin-1 (IL-1) can modulate Leydig cell steroidogenesis . Recently, IL-1-like material has been shown to be present in the testis; however, the cellular source of this material remains unclear . In the present study we found that human recombinant IL-1 beta (1-100 ng/ml) caused dose-dependent increases in IL-1 alpha mRNA expression in Leydig cells . Similar to that reported in other tissues, IL-1 alpha mRNA from Leydig cells is mainly 2.2 kilobases . IL-1 alpha mRNA expression in Leydig cells was detectable as early as 2 h after the addition of IL-1 beta (10 ng/ml) and persisted for up to 24 h . Lipopolysaccharide also stimulated IL-1 alpha mRNA expression in these cells, but phorbol ester had no effect . Our results indicate that Leydig cells are a potential source of IL-1, which has both autocrine and paracrine effects. Arch Biochem Biophys, 1991 Dec, 291(2), 395 - 400 Primary structure of the two variants of Xenopus laevis mtSSB, a mitochondrial DNA binding protein; Ghrir R et al.; The primary structure of the single-stranded DNA binding protein from Xenopus laevis oocyte mitochondria (mtSSB) has been determined by Edman degradation of the intact molecule and peptides derived from partial alpha-chymotrypsin proteolysis and enzymatic cleavage with trypsin and endoproteinase Glu-C . The native mtSSB is composed of two related polypeptide chains, mtSSBs and mtSSBr . The sequence of mtSSBs consists of 129 amino acids with a calculated molecular mass of 14,627 Da . Comparison of the first 80 residues of the two chains reveals 91% identity . A high degree of similarity is found between mtSSB and Escherichia coli SSB or F sex factor SSB. Arch Biochem Biophys, 1991 Dec, 291(2), 307 - 10 1H NMR study of the interaction of ATP with Escherichia coli RNA polymerase containing in vivo-incorporated Co(II); Panth H et al.; The DNA-dependent RNA polymerase containing two intrinsic cobalt ions (Co2-RPase) instead of the naturally occurring zinc was purified from Escherichia coli cells grown in zinc-depleted, cobalt-enriched media . Longitudinal NMR relaxation rates of the H2 and H8 protons of ATP were measured in the absence and presence of up to 92 microM Co2-RPase . No enhancement of the proton relaxation rates was observed in the presence of cobalt-containing enzyme, suggesting that the ATP substrate does not undergo rapid exchange at a site close to either of the intrinsic cobalt ions . This result is in contrast to that previously observed when Co2+ was incorporated into RPase by an in vitro procedure involving partial urea denaturation of the protein. J Immunol, 1991 Dec 1, 147(11), 3988 - 93 Gene expression of the A- and B-chain of mouse C1q in different tissues and the characterization of the recombinant A-chain; Petry F et al.; Immunoscreening of a mouse macrophage cDNA library with an anti-mouse C1q-antibody resulted in the isolation of cDNA clones . The deduced amino acid sequence was homologous to the A-chain of human C1q . Homology on the DNA level was found to be 76% and on the protein level 72% thus it appeared the clones coded for the mouse C1q A-chain . An immunoblot of murine serum C1q separated by SDS-PAGE was detected with an A-chain specific antibody that had been affinity purified on recombinant mouse C1q A-chain expressed in Escherichia coli . The antibody preparation reacted exclusively with the mouse C-chain (as defined by SDS-PAGE) . Northern blot analysis with strand-specific cDNA probes coding for the A- and B-chain of murine C1q showed that mouse peritoneal macrophages produced the highest concentration of C1q gene transcripts . RNA from mouse spleen, thymus, heart, and brain gave substantial hybridization signals, whereas RNA preparations from liver, kidney, lung, and small intestine appeared to contain only trace amounts of C1q mRNA . In a Northern blot analysis of different guinea pig cells and tissues, only RNA preparations from peritoneal macrophages hybridized with the mouse C1q probes . These results indicate that macrophages are a major site of C1q biosynthesis. J Immunol, 1991 Dec 1, 147(11), 3926 - 34 Biochemical and functional analysis of extracellular stress proteins of Mesocestoides corti; Estes DM et al.; Previous studies of the serum antibody response in mice to Mesocestoides corti infection indicated that molecules released by the parasite influenced the production of IgM and IgG1 to the exclusion of other isotypes . Two proteins isolated from M . corti culture supernatants were found to be homologous to the 70-kDa heat shock proteins (hsp70) and Escherichia coli GroEL families of stress proteins . The proliferative responses of splenic lymphocytes from infected mice were assessed to unfractionated M . corti supernatants as well as the 70- and 60-kDa stress protein homologs isolated from supernatants . Lymphocytes from infected mice respond to complete supernatant and both of the isolated p70 and p60 stress protein homologs . In addition, supernatant from M . corti cultures stimulates an in vitro antibody response restricted to IgM and IgG1; the same isotypes induced during infection . These results suggest that stress proteins play an integral part in the immune response to M . corti and the associated isotype restriction. J Bacteriol, 1991 Dec, 173(23), 7719 - 22 Escherichia coli alkaline phosphatase fails to acquire disulfide bonds when retained in the cytoplasm; Derman AI et al.; The cysteines of the Escherichia coli periplasmic enzyme alkaline phosphatase, which are involved in disulfide bonds in the native enzyme, were found to be fully reduced when the protein was retained in the cytoplasm . Under these circumstances the cysteines remained reduced for at least several minutes after the synthesis of the protein was completed . This contrasted with the normally exported protein, wherein disulfide bonds formed rapidly . Disulfide bond formation accompanied export and processing . The implications of these findings for the inactivity of the enzyme in the cytoplasm are discussed. J Bacteriol, 1991 Dec, 173(23), 7701 - 4 Molecular analysis of the TyrR protein-mediated activation of mtr gene expression in Escherichia coli K-12; Sarsero JP et al.; Expression of the mtr gene, which encodes a tryptophan-specific transport system in Escherichia coli K-12, is activated by the TyrR protein . Two TyrR protein binding sites (TYR R boxes) are positioned upstream of the -35 promoter region . Mutational and DNase protection studies indicate that TyrR protein binds preferentially to the TYR R box closest to the promoter, and this is essential for activation of gene expression . In the presence of tyrosine and ATP, a second TyrR molecule is able to cooperatively bind to the second box and cause a further increase in the level of activation. J Bacteriol, 1991 Dec, 173(23), 7673 - 83 Sequence analysis of the clpG gene, which codes for surface antigen CS31A subunit: evidence of an evolutionary relationship between CS31A, K88, and F41 subunit genes; Girardeau JP et al.; The clpG gene coding for the CS31A subunit was localized on a 0.9-kb SphI fragment from the recombinant plasmid pAG315 . This was established by testing the ability of subclones to hybridize with a 17-meric oligonucleotide probe obtained from N-terminal analysis of the CS31A subunit . The nucleotide sequence of the region coding for CS31A was determined . From primer extension analysis, two initiation translation start sites were detected . Two possible promoterlike sequences were identified; the ribosome binding site and the translation terminator are proposed . Inverted repeat sequences leading to the formation of possible hairpin structures of the transcripts were found on the 5' untranslated region of clpG . The deduced amino acid composition was in close agreement with the chemical amino acid composition and sequence match with the first 25 N-terminal amino acids from the published N-terminal sequence of the purified CS31A subunit . The clpG gene codes for a mature protein of 257 amino acids with a molecular size of 26,777 Da . An obvious homology was observed when the amino acid sequence of CS31A was compared with those of K88 and F41 . This homology includes five different conserved sequences of up to 19 identical amino acids, which is associated with conserved proline . An extensive change in the CS31A region homologous to that identified to contain the K88 receptor binding site might be responsible for the functional divergence between CS31A and K88. J Bacteriol, 1991 Dec, 173(23), 7565 - 72 Nucleotide sequence and mutational analysis of the vnfENX region of Azotobacter vinelandii; Wolfinger ED et al.; The nucleotide sequence (3,600 bp) of a second copy of nifENX-like genes in Azotobacter vinelandii has been determined . These genes are located immediately downstream from vnfA and have been designated vnfENX . The vnfENX genes appear to be organized as a single transcriptional unit that is preceded by a potential RpoN-dependent promoter . While the nifEN genes are thought to be evolutionarily related to nifDK, the vnfEN genes appear to be more closely related to nifEN than to either nifDK, vnfDK, or anfDK . Mutant strains (CA47 and CA48) carrying insertions in vnfE and vnfN, respectively, are able to grow diazotrophically in molybdenum (Mo)-deficient medium containing vanadium (V) (Vnf+) and in medium lacking both Mo and V (Anf+) . However, a double mutant (strain DJ42.48) which contains a nifEN deletion and an insertion in vnfE is unable to grow diazotrophically in Mo-sufficient medium or in Mo-deficient medium with or without V . This suggests that NifE and NifN substitute for VnfE and VnfN when the vnfEN genes are mutationally inactivated . AnfA is not required for the expression of a vnfN-lacZ transcriptional fusion, even though this fusion is expressed under Mo- and V-deficient diazotrophic growth conditions. J Bacteriol, 1991 Dec, 173(23), 7458 - 63 Transformation system for an asporogenous methylotrophic yeast, Candida boidinii: cloning of the orotidine-5'-phosphate decarboxylase gene (URA3), isolation of uracil auxotrophic mutants, and use of the mutants for integrative transformation; Sakai Y et al.; An integrative transformation system was established for an asporogenous methylotrophic yeast, Candida boidinii . This system uses a uracil auxotrophic mutant of C . boidinii as the host strain in combination with its URA3 gene as the selectable marker . First, the C . boidinii URA3 gene coding for orotidine-5'-phosphate decarboxylase (ODCase) was cloned by using complementation of the pyrF mutation of Escherichia coli . Next, the host ODCase-negative mutant strains (ura3 strains) were isolated by mutagenesis and selection for 5-fluro-orotic acid (5-FOA) resistance . Five ura3 host strains that exhibited both a low reversion rate and good methylotrophic growth were obtained . All of these strains could be transformed to Ura+ phenotype with a C . boidinii URA3-harboring plasmid linearized within the Candida DNA . The transformants had a stable Ura+ phenotype after nonselective growth for 10 generations . These results and extensive Southern analysis indicated that the linearized plasmid was integrated into the host chromosomal DNA by homologous recombination at the URA3 locus in C . boidinii. Infect Immun, 1991 Dec, 59(12), 4621 - 7 Transformation of Actinobacillus actinomycetemcomitans by electroporation, utilizing constructed shuttle plasmids; Sreenivasan PK et al.; Actinobacillus actinomycetemcomitans, a periodontal pathogen, has been strongly implicated in human periodontal disease . Advances in the molecular analysis of A . actinomycetemcomitans virulence factors have been limited due to the unavailability of systems for genetic transfer, transposon mutagenesis, and gene complementation . Slow progress can be traced almost exclusively to the lack of gene vector systems and methods for the introduction of DNA into A . actinomycetemcomitans . An electrotransformation system that allowed at least five strains of A . actinomycetemcomitans to be transformed with stable shuttle plasmids which efficiently replicated in both Escherichia coli and A . actinomycetemcomitans was developed . One plasmid, a potential shuttle vector designated pDL282, is 5.7 kb in size, has several unique restriction enzyme sites, and codes for resistance to spectinomycin and ampicillin . E . coli and A . actinomycetemcomitans were transformed with equal efficiencies of approximately 10(5) transformants per micrograms of DNA . Similar transformation efficiencies were obtained whether the plasmid DNA was isolated from A . actinomycetemcomitans or E . coli . In addition, frozen competent cells of A . actinomycetemcomitans yielded comparable efficiencies of transformation . Restriction enzyme analysis of pDL282 isolated after transformation confirmed the presence of intact donor plasmids . A plasmid isolated from A . pleuropneumoniae was also capable of transforming some isolates of A . actinomycetemcomitans, although generally at a lower frequency . The availability of these shuttle plasmids and an efficient transformation procedure should significantly facilitate the molecular analysis of virulence factors of A . actinomycetemcomitans. Infect Immun, 1991 Dec, 59(12), 4343 - 8 Immunization of guinea pigs with recombinant TmpB antigen induces protection against challenge infection with Treponema pallidum Nichols; Wicher K et al.; Treponema pallidum-susceptible guinea pigs of strain C4D were immunized with recombinant T . pallidum antigens TmpA, TmpB, TmpC, and TmpA plus TmpB plus TmpC; with Escherichia coli membranes; or with adjuvant alone . Animals in groups of five received six immunizing injections, each of 100 micrograms of antigen incorporated in RIBI adjuvant . After the sixth immunization, all experimental and nonimmunized controls were intradermally challenged with 3 x 10(6) T . pallidum Nichols freshly extracted from infected rabbit testes . Although high titers of antitreponemal antibodies in the fluorescent-treponemal-antibody test or an enzyme-linked immunosorbent assay were evoked in all animals immunized with recombinant antigens, only guinea pigs receiving TmpB antigen demonstrated protection expressed by the development of significantly (P less than 0.01) smaller, atypical lesions of significantly (P less than 0.01) shorter duration and devoid of or containing fewer T . pallidum organisms than lesions in the remaining immunized and control animals. EMBO J, 1991 Dec, 10(12), 3897 - 904 AppppA binds to several proteins in Escherichia coli, including the heat shock and oxidative stress proteins DnaK, GroEL, E89, C45 and C40; Johnstone DB et al.; The dinucleotide AppppA (5',5'''-P1, P4-diadenosine tetraphosphate) is rapidly synthesized in cells exposed to heat stress or oxidative stress . Stress-induced AppppA accumulation has been observed in all cell types studied to date . In order to study the function(s) of AppppA, we created a mutation in the Escherichia coli gene that encodes the sole AppppN hydrolase (apaH) . High levels of AppppA have subsequently been shown to affect many cellular processes, including expression of catabolite repressible genes and the ability to survive starvation, oxidative stress and near-UV irradiation . Nevertheless, the precise role of AppppA remains undefined . In order to better understand the mechanism by which AppppA exerts its effects, we attempted to determine which proteins bind to AppppA by synthesizing (alpha'-32P) 8-N3AppppA for use in photocrosslinking experiments with extract derived from cells with different genetic backgrounds and exposed to various stress conditions . We report here that several E . coli proteins bind AppppA, including the heat shock and oxidative stress proteins DnaK, GroEL, E89, C45 and C40 . In addition, we show that apaH mutants, which have high basal levels of AppppA, are hypersensitive to killing by heat. Plant Mol Biol, 1991 Dec, 17(6), 1203 - 15 cDNA cloning, sequence analysis and seasonal expression of lignin-bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase of aspen; Bugos RC et al.; A cDNA clone (Ptomt 1) encoding a lignin-bispecific O-methyltransferase (OMT) was isolated by immunological screening of a lambda gt11 expression library prepared from mRNA of developing secondary xylem of aspen (Populus tremuloides) . Nucleotide sequence analysis of Ptomt1 revealed an open reading frame of 1095 bp which encodes a polypeptide with a predicted molecular weight of 39,802, corresponding well with the size of the OMT polypeptide estimated by SDS-PAGE . Authenticity of Ptomt1 was demonstrated in part by detection of OMT activity and protein in extracts of Escherichia coli cultures transformed with a plasmid construct containing Ptomt1 . In addition, peptides produced from a proteolytic digest of purified OMT and sequenced by automated Edman degradation matched to portions of the deduced amino acid sequence of Ptomt1 . Comparison of this sequence to amino acid sequences of OMTs of diverse species identified regions of similarity which probably contribute to the binding site of S-adenosyl-L-methionine . Tissue-specific expression was demonstrated by northern analysis which showed that Ptomt1 hybridized to a 1.7 kb transcript from aspen developing secondary xylem and by tissue printing of aspen stems in which only the outer layer of xylem bound the antibody . A biphasic pattern of gene expression and enzyme activity for OMT was observed from xylem samples of aspen during the growing season which suggests linkage between gene expression for a monolignol biosynthetic enzyme and seasonal regulation of xylem differentiation in woody plants. Mol Microbiol, 1991 Dec, 5(12), 2935 - 45 Response of Escherichia coli cell membranes to induction of lambda cl857 prophage by heat shock; Kucharczyk K et al.; Heat shock induces protein aggregation in Escherichia coli and E . coli (lambda cl857) . The aggregates (S fraction) appear 15 min post-induction and are separable from membranes by sucrose density-gradient centrifugation . The S fraction quickly disappears in wild type strains but persists in rpoH mutant with concomitant quick inner membrane destruction . We propose that: (1) the disappearance of