J Gen Microbiol, 1992 Apr, 138 ( Pt 4), 685 - 91 Identification, characterization and sequence analysis of the gene encoding phosphoenolpyruvate carboxylase in Anabaena sp . PCC 7120; Luinenburg I et al.; The gene (ppc) encoding phosphoenolpyruvate carboxylase (PEPCase) in the cyanobacterium Anabaena sp . PCC 7120 has been isolated, characterized and its nucleotide sequence determined . Heterologous hybridization using the Synechococcus sp . PCC 7942 ppc gene as a probe of an Anabaena genomic DNA library identified an 8.2 kb HindIII DNA fragment that contained a 3.08 kb open reading frame encoding the cyanobacterial PEPCase . Deletion analysis of the 8.2 kb DNA fragment was used to determine sequences required for expression of enzyme activity in Escherichia coli cells . Primer extension data have been used to identify the cyanobacterial transcription initiation site and the position of the ppc start codon . Comparisons of the Anabaena deduced amino acid sequence with the Synechococcus sp . PCC 6301, E . coli and higher-plant ppc sequences have also been performed and the data are discussed with respect to conservation of specific regions of the protein. Mol Cell Endocrinol, 1992 Apr, 84(3), 209 - 17 Expression and function of a human thyroid hormone receptor-derived DNA-binding domain protein; Hu RM et al.; DNA binding domain proteins (DBDP) were prepared using a pET construct containing an insert coding for amino acids 49-122 of human thyroid hormone receptor (hTR) alpha and 103-179 of hTR beta . These proteins were expressed in Escherichia coli strain BL21 (DE3)-plysS after induction by isopropyl-D-thiogalactopyranoside (IPTG) . The hTR alpha and hTR beta DBDP contain respectively 79 and 82 amino acids, including an amino terminal 4 amino acid extension derived from pET-3a or the synthesized initiation codon . Using a gel shift assay, both DBDPs were found to bind to a DNA oligonucleotide containing a thyroid hormone response element (TRE) . The DBDPs competed with full length hTR alpha 1 for binding to the oligonucleotide . Apo-DBDPs (Zn2+ released by low pH) failed to bind to the palindromic TRE . DNA binding is restored however if apo-DBDP is preincubated in 500 microM Zn2+ . When the DBDPs were expressed in COS-7 cells using a pCB6+ expression vector, they did not induce expression of a TRE-CAT fusion gene . hTR DBDPs thus can bind to DNA, presumably as monomers, since they do not contain the leucine zipper-like motif for dimerization . In COS-7 cells, they fail to cause transactivation of a TRE-CAT fusion gene . It is inferred that this may be because the DBDPs are not translocated to the nucleus or lack a transactivation domain. Mol Cell Endocrinol, 1992 Apr, 84(3), 167 - 74 The Atlantic salmon prepro-gonadotropin releasing hormone gene and mRNA; Klungland H et al.; Screening for the gene encoding salmon gonadotropin releasing hormone (sGnRH) in an Atlantic salmon (Salmo salar) genomic library resulted in isolation of a positive clone designated lambda sGnRH-1 . An anchor polymerase chain reaction (PCR) technique was used to amplify GnRH cDNA derived from salmon hypothalamic mRNA . The cDNA sequence was aligned to the 7607 base pair genomic sequence which was shown to encode the entire prepro-GnRH gene . The cDNA proved that the cloned gene is expressed in the hypothalamus of mature salmon . The coding domain of sGnRH differs from the mammalian GnRH by six nucleotide changes which allow the two amino acid differences between the two GnRH variants . Salmon GnRH associated peptide (GAP) differs extensively in sequence and size from the mammalian counterpart . Compared to the GnRH cDNA of a cichlid species the similarity is 69.3% in the protein coding sequence. Thorax, 1992 Apr, 47(4), 288 - 91 Relation between the bronchial obstructive response to inhaled lipopolysaccharide and bronchial responsiveness to histamine; Michel O et al.; BACKGROUND: Bronchoconstriction has developed after inhalation of lipopolysaccharide in a dose of 20 micrograms in asthmatic patients and of 200 micrograms in normal subjects . This study set out to determine whether the bronchial response to lipopolysaccharide was related to non-specific bronchial responsiveness and atopy . METHODS: Sixteen subjects with a fall in specific airway conductance of 40% (PD40sGaw) after inhaling up to 900 micrograms histamine inhaled 20 micrograms lipopolysaccharide (from Escherichia coli type 026:B6) a week after bronchial challenge with a control solution of saline . The bronchial response over five hours was measured as change in FEV1 and area under the FEV1-time curve . RESULTS: FEV1 fell significantly more after lipopolysaccharide than after diluent inhalation, the difference in mean (SE) FEV1 being 4.6% (5.4%); response was maximal 60 minutes after lipopolysaccharide inhalation and lasted more than five hours . Histamine PD20FEV1 and PD40sGaw correlated with the fall in FEV1 after lipopolysaccharide inhalation . There was no difference in the proportions of responders and non-responders to lipopolysaccharide who were atopic . CONCLUSION: Lipopolysaccharide induced bronchial obstruction is associated with non-specific responsiveness but not with atopy. Mol Endocrinol, 1992 Apr, 6(4), 502 - 14 Capacity for cooperative binding of thyroid hormone (T3) receptor dimers defines wild type T3 response elements; Brent GA et al.; Thyroid hormone response elements (T3REs) have been identified in a variety of promoters including those directing expression of rat GH (rGH), alpha-myosin heavy chain (rMHC), and malic enzyme (rME) . A detailed biochemical and genetic analysis of the rGH element has shown that it consists of three hexamers related to the consensus {(A/G)GGT(C/A)A} . We have extended this analysis to the rMHC and rME elements . Binding of highly purified thyroid hormone receptor (T3R) to T3REs was determined using the gel shift assay, and thyroid hormone (T3) induction was measured in transient tranfections . We show that the wild type version of each of the three elements binds T3R dimers cooperatively . Mutational analysis of the rMHC and rME elements identified domains important for binding T3R dimers and allowed a direct determination of the relationship between T3R binding and function . In each element two hexamers are required for dimer binding, and mutations that interfere with dimer formation significantly reduce T3 induction . Similar to the rGH element, the rMHC T3RE contains three hexameric domains arranged as a direct repeat followed by an inverted copy, although the third domain is weaker than in rGH . All three are required for full function and T3R binding . The rME T3RE is a two-hexamer direct repeat T3RE, which also binds T3R monomer and dimer . Across a series of mutant elements, there was a strong correlation between dimer binding in vitro and function in vivo for rMHC (r = 0.99, P less than 0.01) and rME (r = 0.67, P less than 0.05) T3REs . Our results demonstrate a similar pattern of T3R dimer binding to a diverse array of hexameric sequences and arrangements in three wild type T3REs . Addition of nuclear protein enhanced T3R binding but did not alter the specificity of binding to wild type or mutant elements . Binding of purified T3R to T3REs was highly correlated with function, both with and without the addition of nuclear protein . T3R dimer formation is the common feature which defines the capacity of these elements to confer T3 induction. Mol Microbiol, 1992 Apr, 6(8), 963 - 8 Effect of lac repressor oligomerization on regulatory outcome; Chakerian AE et al.; Regulatory outcome in a bacterial operon depends on the interactions of all the components which influence mRNA production . Levels of mRNA can be altered profoundly by both negative and positive regulatory elements which modulate initiation of transcription . The occupancy of regulatory sites on the DNA by repressors and activators is determined not only by the affinity of these proteins for their cognate site(s) but also by the oligomeric state of the regulatory protein . The lac operon in Escherichia coli provides an excellent prototypic example of the influence of protein assembly on the transcriptional status of the associated structural genes . DNA loop formation is essential for maximal repression of the lac operon and is contingent upon the presence of multiple operator sites in the DNA and the ability of the repressor to self-associate to form a bidentate tetramer . The stability of this looped complex is enhanced significantly by DNA supercoiling . Tetramer assembly from dimers apparently occurs via interactions of a 'leucine zipper' motif in the C-terminal domain of the protein, and the tetramer is essential to formation of looped complexes . Furthermore, analysis of the DNA-binding characteristics of dimeric mutants has established that the monomer-dimer association and dimer-DNA binding (monomer does not bind to DNA) are coupled equilibria . Thus, dimer assembly is essential for generating a DNA-binding unit, and tetramer assembly is required for formation of the stable looped DNA structure that maximally represses mRNA synthesis . Protein-protein interactions therefore play a pivotal role in the regulatory activities of the lac repressor and must be considered when analysing the activities of any oligomeric DNA-binding protein. Mol Microbiol, 1992 Apr, 6(8), 1061 - 71 Mobilization protein-DNA binding and divergent transcription at the transfer origin of the Thiobacillus ferrooxidans pTF1 plasmid; Drolet M et al.; The possible interaction of the trans-acting mobilization proteins, MobL and MobS, at the cognate origin of transfer (oriT) region of the Thiobacillus ferrooxidans plasmid pTF1 has been investigated . In gel retardation assays with crude protein extracts from overproducing strains, a truncated MobL (c . 28 kDa) as well as its native protein (42 kDa), but not the 11 kDa MobS protein, were found to bind specifically to a 42-mer oligonucleotide which represents the transferred DNA strand of the minimal oriT fragment of pTF1 . In vivo, the binding of MobL was studied by monitoring catechol 2,3-dioxygenase (xylE) activities driven by promoters of the divergently transcribed mobL and mobS genes . The mob promoter sequences were found to resemble the Escherichia coli sigma 70-dependent consensus promoter elements . The '-10' recognition sequences of mobL and one of the two mobS promoters overlap except for one base and they are positioned within the putative 'hairpin' structure in the minimal oriT sequence . In accordance with the twin supercoil-domain model of Liu and Wang (1987) which suggests that transcription can generate local variations in DNA superhelicity, we propose a possible physiological role of DNA supercoiling in the transfer origin with reference to divergent transcription of mobL and mobS genes. Proteins, 1992 Apr, 12(4), 372 - 81 The molecular structure of UDP-galactose 4-epimerase from Escherichia coli determined at 2.5 A resolution; Bauer AJ et al.; UDP-galactose 4-epimerase catalyzes the conversion of UDP-galactose to UDP-glucose during normal galactose metabolism . The molecular structure of UDP-galactose 4-epimerase from Escherichia coli has now been solved to a nominal resolution of 2.5 A . As isolated from E . coli, the molecule is a dimer of chemically identical subunits with a total molecular weight of 79,000 . Crystals of the enzyme used for this investigation were grown as a complex with the substrate analogue, UDP-benzene, and belonged to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 76.3 A, b = 83.1 A, c = 132.1 A, and one dimer per asymmetric unit . An interpretable electron density map calculated to 2.5 A resolution was obtained by a combination of multiple isomorphous replacement with six heavy atom derivatives, molecular averaging, and solvent flattening . Each subunit of epimerase is divided into two domains . The larger N-terminal domain, composed of amino acid residues 1-180, shows a classic NAD+ binding motif with seven strands of parallel beta-pleated sheet flanked on either side of alpha-helices . The seventh strand of the beta-pleated sheet is contributed by amino acid residues from the smaller domain . In addition, this smaller C-terminal domain, consisting of amino acid residues 181-338, contains three strands of beta-pleated sheet, two major alpha-helices and one helical turn . The substrate analogue, UDP-benzene, binds in the cleft located between the two domains with its phenyl ring in close proximity to the nicotinamide ring of NAD+ . Contrary to the extensive biochemical literature suggesting that epimerase binds only one NAD+ per functional dimer, the map clearly shows electron density for two nicotinamide cofactors binding in symmetry-related positions in the dimer . Likewise, each subunit in the dimer also binds one substrate analogue. J Interferon Res, 1992 Apr, 12(2), 67 - 74 A whole blood immunoassay for the interferon-inducible human Mx protein; Towbin H et al.; Mx protein, an intracellular protein induced by type I interferons (IFNs), is useful as a marker for the IFN-induced state . It is detectable, for example, in leukocytes of patients undergoing IFN-alpha treatment as well as in patients suffering from viral or autoimmune diseases . For immunizations and standardizations, recombinant human MxA protein was expressed in Escherichia coli and purified from inclusion bodies by several steps of chromatography . Two monoclonal antibodies against nonoverlapping epitopes and specific for human Mx protein were selected to establish a simple two-site immunometric enzyme assay . In addition, a monoclonal antibody also reacting with Mx proteins of other species was identified . Prior to assay, whole blood samples were lysed with a nonionic detergent . The sample was incubated on wells coated with a first monoclonal antibody (1304.5.32) together with a second biotinylated monoclonal (1302.34.16), which, after washing, was revealed by an avidin-alkaline phosphatase system . Limit of detection was 5 ng/ml . In two-thirds of normal blood samples (n = 87), Mx protein levels were below 5 ng/ml; 25 samples (29%) had Mx levels between 5 and 50 ng/ml; and 4 samples (5%) were above 50 ng/ml . No Mx was found in plasma, and the mononuclear cell fraction accounted for the bulk of Mx in blood . In vitro, as determined by flow cytometry, monocytes and lymphocytes accumulated Mx protein for 24 h with similar kinetics and remained at plateau levels for more than 70 h . Monocytes contained around eight times more Mx than lymphocytes . The immunoassay was also suitable for detecting Mx after IFN induction in heparinized blood. J Interferon Res, 1992 Apr, 12(2), 139 - 43 Engineered disulfide bond greatly increases specific activity of recombinant murine interferon-beta; Day C et al.; Unlike other species of interferon-beta (IFN-beta) mouse (Mu) IFN-beta has no naturally occurring intramolecular disulfide bond . When expressed in Escherichia coli, MuIFN-beta appears to exhibit instability and low activity . To increase its activity, we engineered a pair of cysteines into recombinant MuIFN-beta to test whether this change would improve its antiviral activity . In the absence of detailed structural data, the optimal placement of cysteines was determined by sequence comparison with other species of IFN-beta . While MuIFN-beta has only a single cysteine (at position 17), other species of IFN-beta have three cysteines, at positions 17, 31, and 141 (numbering based on consensus sequence), a disulfide bond being formed between the latter two residues . Thus, we introduced cysteines into MuIFN-beta at positions 29 and 136, which correspond to positions 31 and 141 of the consensus sequence . When the variant form of MuIFN-beta was expressed in bacteria and purified, we found that the additional cysteines greatly increased the antiviral activity . Further improvement was obtained by replacement of the Cys-17 with a serine . In this manner a specific activity approximately 15-fold that of the wild-type recombinant molecule was achieved. Gastroenterol Jpn, 1992 Apr, 27(2), 212 - 21 Pathogenesis of hepatic atrophy in canine model of hepatolithiasis; Ueda N et al.; The pathogenesis of the hepatic atrophy that accompanies hepatolithiasis was investigated pathomorphologically using a canine model . Two groups were evaluated: infected and noninfected . In the infected group, inflammation in Glisson's capsule caused by cholangitis involved the portal vein at the region of the large bile duct . At this region, the periportal fibrosis ratio was significantly greater in the infected group than in the noninfected group both at 1 and 3 months . At the regions of the septal and interlobular bile ducts, the caliber ratio of the portal vein in the two experimental groups was less than in the normal liver both at 1 and 3 months . At both regions, the caliber ratio of the portal vein in the infected group was less than in the noninfected group at 3 months . The rate of atrophy was significantly greater in the infected group than in the noninfected group at 3 months . These results suggest that disturbance of the portal venous blood flow attributed to cholangitis of the large bile ducts is one of the most important factors leading to hepatic atrophy in hepatolithiasis. J Clin Microbiol, 1992 Apr, 30(4), 981 - 6 A cloned DNA probe for Cowdria ruminantium hybridizes with eight heartwater strains and detects infected sheep; Mahan SM et al.; The DNA probe pCS20, which was cloned from the DNA of the Crystal Springs heartwater strain from Zimbabwe, cross-reacted with DNAs of heartwater strains from all endemic areas, including four heartwater strains from Zimbabwe, two strains from South Africa, one strain from Nigeria, and the Gardel strain from the Caribbean island of Guadeloupe . By nucleic acid hybridization, the pCS20 DNA probe detected Cowdria ruminantium DNA in all DNA preparations made from plasma samples from infected sheep before and during the febrile reaction . Synthetic oligonucleotides were prepared for amplification of specific C . ruminantium DNA sequences by the polymerase chain reaction (PCR) . Amplification of two DNA products (181 and 279 bp) from pCS20 DNA and C . ruminantium genomic DNA of heartwater strains was demonstrated . In contrast, amplification of these products or any other products was not possible from genomic DNAs of Anaplasma marginale, Babesia bigemina, Trypanosoma brucei brucei, Escherichia coli, and bovine endothelial cells . The cross-reactivities of the 32P-labeled PCR products with genomic DNAs from several heartwater strains were similar to those with the pCS20 DNA probe . A nucleic acid-based test that uses hybridization assays and PCR provides a sensitive method for the detection of heartwater in both animals and ticks and has applications in epidemiological studies for the disease, which may allow for improved disease control. J Steroid Biochem Mol Biol, 1992 Apr, 42(2), 131 - 9 Zinc coordination scheme for the C-terminal zinc binding site of nuclear hormone receptors; Zilliacus J et al.; The DNA-binding domain of the glucocorticoid receptor contains two zinc ions which are important for the structure and function of the protein . The zinc ions are tetrahedrally coordinated by cysteine residues within the DNA-binding domain . The DNA-binding domain of the glucocorticoid receptor, as well as of the other nuclear hormone receptors, contains nine highly conserved cysteine residues . It has not been clearly established which of these nine cysteine residues are involved in the coordination of zinc . Two models have been proposed for the zinc coordination scheme . We present evidence in favour of the model which excludes the most C-terminal cysteine residue (Cys-481 of the human glucocorticoid receptor) from the zinc coordination scheme . Mutation of this residue in the context of the glucocorticoid receptor DNA-binding domain expressed in E . coli does not significantly reduce the structural integrity of the protein or its DNA-binding properties . These in vitro results are also confirmed by in vivo transactivation assays in yeast. Biochem J, 1992 Apr 1, 283 ( Pt 1), 91 - 8 Structure-function analysis of human transforming growth factor-alpha by site-directed mutagenesis; Feild JA et al.; Site-directed mutants of transforming growth factor-alpha (TGF-alpha) were expressed in an Escherichia coli outer membrane protein A (ompA) expression/secretion vector under the transcriptional control of the lambda PL promoter . TGF-alpha mutant proteins were isolated from cell pellets using alkaline extraction with 0.1 M-Tris (pH 10.5) . The levels of protein expression of 23 TGF-alpha mutants were comparable with those of wild-type TGF-alpha, as determined by immunoblotting and radioimmunoassay . An analysis of biological activity using as assays radioreceptor binding competition and colony formation in soft agar showed that the following mutations destroy the activity of TGF-alpha: Gly-19 to Val, Val-33 to Pro and Gly-40 to Val . Mutations of Arg-42 to Lys, Leu-48 to Ala, Tyr-38 to Trp or Phe-17 to Tyr significantly decrease, but do not destroy, biological activity when compared with the wild-type . Mutations in 14 other residues did not significantly alter receptor binding or colony-forming activity . These studies suggest that two domains localized at the surface of TGF-alpha are important in receptor binding and colony-forming activity . Domain I involves amino acid residues which include Tyr-38 and Leu-48; domain II includes residues Phe-15, Phe-17 and Arg-42. Gene, 1992 Apr 1, 113(1), 95 - 9 High-level temperature-induced synthesis of an antibody VH-domain in Escherichia coli using the PelB secretion signal; Power BE et al.; We have constructed a temperature-inducible Escherichia coli expression vector (pPOW) for enhanced secretion of antibody (Ab) domains and other foreign proteins . The vector contains the lambda pRpL promoters in tandem, and the cI857 gene encoding the temperature-sensitive repressor which provide tight control over protein production . The PelB secretion signal directs the synthesized foreign protein through the cytoplasmic membrane . A mouse Ab fragment (the variable heavy (VH) domain of NC41) was synthesized efficiently by this vector and accumulated with the cell membranes (not as inclusion bodies) at levels of 30 mg/l . This represents the highest yields reported to date for Ab fragments with a native N terminus . An octapeptide (FLAG) tail was fused to the C terminus of the VH domain to aid in purification, and remained intact throughout the protein purification process . The optimum conditions for protein production were controlled by the type of culture medium used, the age of the bacterial population at the time of induction, and the period of synthesis of the protein product . The purified Ab VH fragment showed binding affinity (Ka less than 10(4)/M) to its target antigen (neuraminidase). Gene, 1992 Apr 1, 113(1), 135 - 6 Purification of restriction endonuclease EcoO128I produced by an enteropathogenic Escherichia coli O128Ly3; Miyahara M et al.; Restriction endonuclease EcoO128I, an isoschizomer of BstEII, was purified from a rough mutant of Escherichia coli O128Ly3 . EcoO128I should be more convenient for recombinant DNA applications than BstEII, because of its improved cleavage activity at 37 degrees C. Gene, 1992 Apr 1, 113(1), 101 - 6 Replication and copy number control of the broad-host-range plasmid RSF1010; Frey J et al.; Initiation of replication of the broad-host-range plasmid RSF1010, is accurately controlled by the plasmid-encoded proteins, RepB (MobA), RepB', RepA and RepC {Haring et al., Proc . Natl . Acad . Sci . USA 82 (1985) 6090-6094; Scherzinger et al., Nucleic Acids Res . 19 (1991) 1203-1211} . The genes encoding these proteins which are essential for replication and conjugative mobilization are transcribed from a cluster of promoters, P1/P3 and P2, which partly overlap with the origin of conjugal transfer, oriT . Three regions were found where deletion mutations affect the mobilization of RSF1010 and increase its copy number in Escherichia coli . A deletion in the mobC gene increased the copy number of RSF1010 four-fold . Another deletion, that removed oriT and part of the promoter believed to be responsible for the expression of mobC, results in a three-fold increase in copy number . The third type of deletions affect the N-terminal part of RepB (MobA) . A deletion that created a frame-shift results in a three-fold increase in copy number . A smaller, in-frame deletion of this region only affected the mobilization of RSF1010, but not its copy number . The extent by which RSF1010 or its deletion derivatives could repress the P1/P3 and P2 promoters has indicated that these promoters are negatively regulated by MobC and RepB (MobA), presumably by their attachment to the oriT region of RSF1010 . Both MobC and RepB are required for the maximal repression of the rep operon . Optimal function of RSF1010 thus involves not only overlapping genes, but also proteins that exert multiple functions, mobilization, replication and regulation. FASEB J, 1992 Apr, 6(7), 2422 - 7 Antibody engineering: the use of Escherichia coli as an expression host; Ward ES; The hypervariable loops of an antibody molecule are supported on the relatively conserved beta-sheeted frameworks of the heavy- and light-chain variable domains (designated VH and VL domains, respectively) . Residues within and flanking these loops interact with antigen and confer the specificity and affinity of antigen binding on the immunoglobulin molecule . Thus, the isolation and expression of VH and VL domain genes are of particular interest both for analysis of the determinants of antibody specificity and for generation of fragments with binding affinities for use in therapy and diagnosis . The PCR can now be used to isolate diverse repertoires of antibody VH and VL domain genes from antibody-producing cells from different species, including humans and mice . The genes can be expressed as either secreted or surface-bound Fv or Fab fragments, using Escherichia coli expression systems, and the desired antigen-binding specificity screened for or, preferably, selected . The use of E . coli as an expression host allows the required antigen-binding specificity to be isolated in clonal form in a matter of days . The VH and VL domain genes can also be hypermutated and higher-affinity variants isolated by screening or selection . Thus, the use of this technology should allow the isolation of novel binding specificities or specificities that are difficult to generate by hybridoma technology . It will also facilitate the isolation of human-derived Fv/Fab fragments that may be less immunogenic in therapy . This approach therefore has almost unlimited potential in the generation of therapeutics with binding specificities to order . The fragments can be used either alone or linked to effector functions in the form of antibody-constant domains or toxins . The new technology could prove to be a method of choice for the rapid and convenient production of designer antibodies. EMBO J, 1992 Apr, 11(4), 1303 - 7 Non-glycosylated recombinant pro-concanavalin A is active without polypeptide cleavage; Min W et al.; The complex post-translational processing of concanavalin A (Con A) in maturing jackbeans is unique because the non-glycosylated mature active protein is circularly permuted in primary sequence relative to its own inactive precursor (glycosylated pro-Con A) and to other legume lectins . We show here that non-glycosylated pro-Con A expressed in bacteria from recombinant cDNA (rec-pro-Con A) folds in vivo and in vitro to a stable form which is active without further processing . N-glycosylation alone must therefore be sufficient to inactivate pro-Con A--a novel role for glycosylation in regulating activity during protein maturation. Invest Ophthalmol Vis Sci, 1992 Apr, 33(5), 1642 - 9 Morphologic correlations with fluorophotometric data from monkey eyes with anterior uveitis; Freddo TF et al.; Acute anterior uveitis was induced in monkeys by unilateral intravitreal injection of 1.0 ng of Escherichia coli endotoxin . Twenty-four hours later, each animal received an intravenous injection of 250 mg/kg body weight of fluoresceinated horseradish peroxidase (F-HRP), and fluorophotometric measurements were taken for 90 min . The animals were killed, and both eyes were processed for HRP demonstration . In the anterior chamber aqueous humor of normal control eyes, F-HRP concentrations were less than 0.002 mg/ml at 90 min . The F-HRP concentration was elevated consistently in the endotoxin-injected eyes; however, the magnitude of the effect varied . By fluorophotometry, inflamed eyes fell into two distinct groups . At 90 min, most had an anterior chamber F-HRP concentration of 0.014-0.06 mg/ml, although others had 0.39 mg/ml . In the latter group, an appreciably shorter latency was observed between the time of tracer injection and its detection in the anterior chamber . Aqueous humor protein concentrations, although highest in the most F-HRP-permeable eyes, followed more of a continuum in their distribution and identified less clearly the subpopulations seen by fluorophotometry . Normal eyes had no tracer leakage across either the ciliary epithelial or iris vascular endothelial barriers . All inflamed eyes had HRP leakage across the ciliary epithelium, but the subpopulation of eyes with shorter latencies and higher F-HRP concentrations by fluorophotometry also had iris vascular leakage. Proc Natl Acad Sci U S A, 1992 Apr 1, 89(7), 2980 - 4 Cloning and expression of a cytosolic megakaryocyte protein-tyrosine-phosphatase with sequence homology to retinaldehyde-binding protein and yeast SEC14p; Gu M et al.; Protein tyrosine phosphorylation is important in the regulation of cell growth, the cell cycle, and malignant transformation . We have cloned a cDNA that encodes a cytosolic protein-tyrosine-phosphatase (PTPase), MEG2, from MEG-01 cell and human umbilical vein endothelial cell cDNA libraries . The 4-kilobase cDNA sequence of PTPase MEG2 corresponds in length to the mRNA transcript detected by Northern blotting . The predicted open reading frame encodes a 68-kDa protein composed of 593 amino acids and has no apparent signal or transmembrane sequences, suggesting that it is a cytosolic protein . The C-terminal region has a PTPase catalytic domain that has 30-40% amino acid identity to other known PTPases . The N-terminal region has 254 amino acids that are 28% identical to cellular retinaldehyde-binding protein and 24% identical to yeast SEC14p, a protein that has phosphatidylinositol transfer activity and is required for protein secretion through the Golgi complex in yeast . Recombinant PTPase MEG2 expressed in Escherichia coli possesses PTPase activity . PTPase MEG2 mRNA was detected in 12 cell lines tested, which suggests that this phosphatase is widely expressed . The structure of PTPase MEG2 implies that a tyrosine phosphatase could participate in the transfer of hydrophobic ligands or in functions of the Golgi apparatus. Proc Natl Acad Sci U S A, 1992 Apr 1, 89(7), 2839 - 43 Isolation and characterization of a cDNA encoding Drosophila transcription factor TFIIB; Yamashita S et al.; A Drosophila cDNA encoding a human transcription factor TFIIB homologue was isolated by PCR methods . The deduced amino acid sequence indicates 85% sequence similarity with human TFIIB, and the corresponding cDNA product expressed in Escherichia coli is interchangeable with human TFIIB for both basal and GAL4-VP16-induced transcription . Structural motifs including the direct repeats, basic repeats, and sigma sequence similarities are well conserved among Drosophila, human, and Xenopus TFIIB . However, the N-terminal region of each direct repeat is less conserved among the three species, suggesting the presence of two structural subdomains in the direct repeat . Moreover, the amino acid changes in the N-terminal subdomain produce altered positions of the conserved amino acids between the direct repeats . An overall similarity in general structural features between TFIIB and TFIID tau (the TATA-binding subunit of TFIID) was previously noted . However, in contrast to the sequence divergence reported for the N-terminal domains of TFIID tau from different species, the N-terminal sequence of TFIIB was highly conserved among the species . This suggests that TFIIB has a more rigid structure, consistent with its function as a "bridging" protein between TFIID and RNA polymerase II . Further implications of the TFIIB structure are discussed. Proc Natl Acad Sci U S A, 1992 Apr 1, 89(7), 2829 - 33 Thymidine-induced mutations in mammalian cells: sequence specificity and implications for mutagenesis in vivo; Kresnak MT et al.; Imbalances in the intracellular nucleotide precursor pools in mammalian cells can result in the induction of mutations during the DNA replication process . By using a shuttle vector system developed in our laboratory, we have analyzed the sequence specificity of mutations induced in mouse A9 cells by exposure of the cells to a high concentration of thymidine . The target for mutagenesis in these studies was the bacterial gpt gene stably integrated into the chromosomal DNA of the mouse cells . Previous studies in this laboratory had generated a large panel of xanthine guanine phosphoribosyl-transferase (EC 2.4.2.22)-negative mutant lines that possess single-base mutations within the gpt coding sequence . This study utilized four xanthine guanine phosphoribosyltransferase-negative mutant lines to assess the frequency of mutation induced by thymidine at guanine residues in four sequence contexts: the 5' and 3' guanine residues of a GG doublet, the middle guanine residue of a GGG triplet, and the 3' guanine residue of a GGGG quartet . The results of this study demonstrate that treatment of cultured cells with a high concentration of thymidine can result in G.C----A.T transition mutations that occur preferentially at the 3' guanine residue of a run of two or more adjacent guanines . Guanine residues flanked on their 3' side by other guanine residues are severalfold less mutable by thymidine than are guanine residues flanked on their 3' side by a different base . This study demonstrates a sequence-specific mode for thymidine-induced mutations and suggests implications for mutagenesis in vivo. J Bacteriol, 1992 Apr, 174(8), 2688 - 93 Characterization of an autonomously replicating region from the Streptomyces lividans chromosome; Zakrzewska-Czerwinska J et al.; The chromosomal replication origin of the plasmidless derivative (TK21) from Streptomyces lividans 66 has been cloned as an autonomously replicating minichromosome (pSOR1) by using the thiostrepton resistance gene as a selectable marker . pSOR1 could be recovered as a closed circular plasmid which shows high segregational instability . pSOR1 was shown to replicate in Streptomyces coelicolor A3(2) and in S . lividans 66 and hybridized with DNA from several different Streptomyces strains . Physical mapping revealed that oriC is located on a 330-kb AseI fragment of the S . coelicolor A3(2) chromosome . DNA sequence analyses showed that the cloned chromosomal oriC region contains numerous DnaA boxes which are arranged in two clusters . The preferred sequence identified in the oriC region of Escherichia coli and several other bacteria is TTATCCACA . In contrast, in S . lividans, which has a high GC content, the preferred sequence for DnaA boxes appears to be TTGTCCACA. J Bacteriol, 1992 Apr, 174(8), 2670 - 8 Regulation of the Escherichia coli cad operon: location of a site required for acid induction; Meng SY et al.; The cad operon encodes lysine decarboxylase and a protein homologous to amino acid antiporters . These two genes are induced under conditions of low pH, anaerobiosis, and excess lysine . The upstream regulatory region of the cad operon has been cloned into lacZ expression vectors for analysis of the sequences involved in these responses . Deletion analysis of the upstream region and cloning of various fragments to make cadA::lacZ or cadB::lacZ protein fusions or operon fusions showed that cadA was translated more efficiently than cadB and localized the pH-responsive site to a region near an upstream EcoRV site . Construction of defined end points by polymerase chain reaction further localized the left end of the regulatory site . The presence of short fragments bearing the regulatory region on high-copy-number plasmids greatly reduced expression from the chromosomal cad operon, suggesting that titration of an essential activator protein was occurring . With nonoptimal polymerase chain reaction conditions, a set of single point mutants were made in the upstream regulatory region . Certain of these altered regulatory regions were unable to compete for the regulatory factor in vivo . The locations of these essential bases indicate that a sequence near the EcoRV site is very important for the activator-DNA interaction . In vivo methylation experiments were conducted with cells grown at pH 5.5 or at pH 8, and a difference in protection was observed at specific G residues in and around the region defined as important in pH regulation by the mutation studies . This work defines essential sequences for acid induction of this system involved in neutralization of extracellular acid. J Bacteriol, 1992 Apr, 174(8), 2517 - 24 Overproduction of the beta subunit of DNA polymerase III holoenzyme reduces UV mutagenesis in Escherichia coli; Tadmor Y et al.; Overproduction of the beta subunit of DNA polymerase III holoenzyme caused a 5- to 10-fold reduction of UV mutagenesis along with a slight increase in sensitivity to UV light in Escherichia coli . The same effects were observed in excision-deficient cells, excluding the possibility that they were mediated via changes in excision repair . In contrast, overproduction of the alpha subunit of the polymerase did not influence either UV mutagenesis or UV sensitivity . The presence of the mutagenesis proteins MucA and MucB expressed from a plasmid alleviated the effect of overproduced beta on UV mutagenesis . We have previously suggested that DNA polymerase III holoenzyme can exist in two forms: beta-rich form unable to bypass UV lesions and a beta-poor form capable of bypassing UV lesions (O . Shavitt and Z . Livneh, J . Biol . Chem . 264:11275-11281, 1989) . The beta-poor form may be related to an SOS form of DNA polymerase III designed to perform translesion polymerization under SOS conditions and thereby generate mutations . On the basis of this model, we propose that the overproduced beta subunit affects the relative abundance of the regular replicative beta-rich polymerase and the SOS bypass-proficient polymerase by sequestering the polymerase molecules to the beta-rich form and blocking the SOS form. J Bacteriol, 1992 Apr, 174(8), 2511 - 6 Export of the outer membrane lipoprotein is defective in secD, secE, and secF mutants of Escherichia coli; Sugai M et al.; The export of major outer membrane lipoprotein has been found to be affected in secD, secE, and secF mutants of Escherichia coli, which are defective in protein export in general . After a shift to the nonpermissive temperature, the kinetics of accumulation of prolipoprotein and pre-OmpA protein was indistinguishable from that of pre-OmpA protein accumulation in the secD and secF mutants but different in the secE mutant . The prolipoprotein accumulated in the secD, secE, and secF mutants at the nonpermissive temperature was not modified with glyceride . We conclude from these results and those of previous studies that the export of lipoprotein requires all common sec gene products except the SecB protein, i.e., the SecA, SecD, SecE, SecF, and SecY proteins. J Bacteriol, 1992 Apr, 174(8), 2431 - 9 Heterologous expression and secretion of a Streptomyces scabies esterase in Streptomyces lividans and Escherichia coli; Hale V et al.; The esterase gene from Streptomyces scabies FL1 was cloned and expressed in Streptomyces lividans on plasmids pIJ486 and pIJ702 . In S . lividans, the esterase gene was expressed during later stages of growth and was regulated by zinc, as is seen with S . scabies . The 36-kDa secreted form of the esterase was purified from S . lividans . N-terminal amino acid sequencing indicated that the processing site utilized in S . lividans for the removal of the signal sequence was the same as that recognized for processing in S . scabies . Western blots (immunoblots) revealed the presence of a 40-kDa precursor form of the esterase in cytoplasmic extracts . A 23-amino-acid deletion was introduced into the putative signal sequence for the esterase . When this deleted form of the esterase was expressed in S . lividans, a cytoplasmic 38-kDa precursor protein was produced but no secreted esterase was detected, suggesting the importance of the deleted sequence for efficient processing and secretion . The esterase gene was also cloned into the pUC119 plasmid in Escherichia coli . By using the lac promoter sequence, the esterase gene was expressed, and the majority of the esterase was localized to the periplasmic space. Eur J Biochem, 1992 Apr 1, 205(1), 71 - 6 Pigment-binding properties of mutant light-harvesting chlorophyll-a/b-binding protein; Paulsen H et al.; Light-harvesting chlorophyll-a/b-binding protein (LHCP), overexpressed in Escherichia coli, can be reconstituted with pigments to yield complexes that are structurally very similar to light-harvesting complex II (LHCII) isolated from thylakoids {Paulsen, H., Rumler, U . & Rudiger, W . (1990) Planta 181, 204-211} . In order to analyze which domains of the protein are involved in pigment binding, we reconstituted deletion mutants of LHCP with pigments and characterized the resulting complexes regarding their pigment composition and spectroscopic properties . Series of progressive deletions from either end of the protein revealed that most of the N-terminal and part of the C-terminal hydrophilic regions of LHCP are dispensible for pigment binding . In either deletion series, the deletions that completely abolished reconstitution could be narrowed down to segments of five amino acids that do not contain histidine, asparagine, or glutamine . All mutants either formed complexes with both pigment composition and spectroscopic properties very similar to those of light-harvesting complex II isolated from thylakoids, or they did not form any stable complexes at all . There is no indication of a segment of LHCP binding a subset of LHCII pigments . We conclude that the stabilization of LHCP-pigment complexes is highly synergetic rather than based on individual pigment-binding sites provided by the protein. Eur J Biochem, 1992 Apr 1, 205(1), 425 - 31 Leucine aminopeptidase from Arabidopsis thaliana . Molecular evidence for a phylogenetically conserved enzyme of protein turnover in higher plants; Bartling D et al.; Leucine aminopeptidases are exopeptidases which are presumably involved in the processing and regular turnover of intracellular proteins; however, their precise function in cellular metabolism remains to be established . Towards this goal, a full-length complementary DNA encoding a plant leucine aminopeptidase was isolated from a cDNA library of Arabidopsis thaliana and sequenced . The nucleotide sequence showed 49.5% identity to the Escherichia coli xerB-encoded leucine aminopeptidase . Sequence analysis revealed that the cDNA encodes a polypeptide of 520 amino acids with a calculated molecular mass of 54,506 Da . The C-terminal part (amino acids 200-520) of the deduced amino acid sequence showed 43.8% sequence identity to the xerB-encoded leucine aminopeptidase and 42.6% sequence identity to the amino acid sequence of bovine lens leucine aminopeptidase (EC 3.4.11.1) . No sequence similarity (not even over short sequence elements) was observed with any other known peptidase or proteinase sequence . The cDNA was expressed as a fusion protein from the lacZ promoter in E . coli . Enzymatic analysis proved that the cloned cDNA encoded an active leucine aminopeptidase . The properties of this enzyme, including metal requirements, inhibitor sensitivity, pH optimum and the remarkable temperature stability, are very similar to those reported for leucine aminopeptidases from other tissues . Amino acids involved in metal and substrate binding in bovine lens aminopeptidase are completely conserved in the plant enzyme as well as in the XerB protein . Our results show that leucine aminopeptidases form a superfamily of highly conserved enzymes, spanning the evolutionary period from the bacteria to animals and higher plants . This is the first aminopeptidase cloned from a plant. Eur J Biochem, 1992 Apr 1, 205(1), 375 - 81 Purification and properties of the mercuric-ion-binding protein MerP; Sahlman L et al.; The gene merP, coding for a mercuric-ion-binding periplasmic protein (P protein), was cloned into the expression vector pCA . In an Escherichia coli strain bearing the resulting plasmid, the P protein constitutes about 20% of total soluble protein . P protein was purified using ammonium sulfate precipitation and two chromatography steps . Typical yields were 20-30 mg from 7.5 l bacterial culture . The protein is a monomer with a molecular mass of 7500 Da . The periplasmic signal peptide was processed identically in both the recombinant and the wild-type proteins . CD spectra of both proteins were identical and indicated that the structure is highly ordered, containing approximately 80% alpha-helix . Purification in the presence of excess cysteine resulted in a form of the protein containing two reduced thiols, in agreement with the published sequence which has two cysteine residues . When cysteine was omitted from the purification buffers, no reduced thiol groups could be detected suggesting that the cysteine residues are oxidized . Both of these forms of the protein were found to bind approximately five Hg2+ ions/protein molecule in an apparently non-specific manner . However, in the presence of external thiol compounds, the protein with reduced thiols bound only one Hg2+ ion/protein molecule with an apparent Kd of 3.7 +/- 1.3 microM . Under these conditions, the protein with oxidized thiols did not bind Hg2+ . The possible physiological role of this protein in Hg2+ detoxification is discussed. Eur J Biochem, 1992 Apr 1, 205(1), 347 - 53 Incorporation and degradation by kidney lysosomes of cystatin alpha injected intravenously; Ohshita T et al.; Cystatin alpha was purified from Escherichia coli transfected with a recombinant cystatin alpha gene and injected into the tail vein of rats . Its fate was then followed using a sensitive enzyme immunoassay for cystatin alpha . Results showed that it was rapidly removed from the blood and taken up by the kidney . Percoll density-gradient analysis showed that it was rapidly incorporated into lysosomes in the kidney . Its level in the kidney was maximal 30 min after its injection then rapidly decreased . The activity of cathepsin H in the kidney lysosomal fraction was markedly decreased 30 min after injection of cystatin alpha but recovered rapidly . Anti-(cystatin alpha) antibody precipitated cathepsin H and anti-(cathepsin H) antibody precipitated cystatin alpha in an extract of the lysosome-rich fraction of the kidney 30 min after injection of cystatin alpha . These results indicate that cystatin alpha was taken up by kidney lysosomes, formed a complex with cathepsin H and initially decreased the activity of cathepsin H, but that later the level of cystatin alpha in the kidney rapidly decreased. Eur J Biochem, 1992 Apr 1, 205(1), 111 - 5 The third subunit of desulfoviridin-type dissimilatory sulfite reductases; Pierik AJ et al.; In addition to the 50-kDa (alpha) and 40-kDa (beta) subunits, an 11-kDa polypeptide has been discovered in highly purified Desulfovibrio vulgaris (Hildenborough) dissimilatory sulfite reductase . This is in contrast with the hitherto generally accepted alpha 2 beta 2 tetrameric subunit composition . Purification, high-ionic-strength gel-filtration, native electrophoresis and isoelectric focussing do not result in dissociation of the 11-kDa polypeptide from the complex . Densitometric scanning of SDS gels and denaturing gel-filtration indicate a stoichiometric occurrence . A similar 11-kDa polypeptide is present in the desulfoviridin of D . vulgaris oxamicus (Monticello), D . gigas and D . desulfuricans ATCC 27774 . We attribute an alpha 2 beta 2 gamma 2 subunit structure to desulfoviridin-type sulfite reductases . N-terminal sequences of the alpha, beta and gamma subunits are reported. J Exp Med, 1992 Apr 1, 175(4), 1139 - 42 Differentiation factor/leukemia inhibitory factor protection against lethal endotoxemia in mice: synergistic effect with interleukin 1 and tumor necrosis factor; Alexander HR et al.; Differentiation factor (D factor), also called leukemia inhibitory factor (LIF), is a glycoprotein that has been increasingly recognized to possess a wide range of physiological activities . We examined the possibility that the administration of D factor may confer beneficial effects and enhance host resistance against lethal endotoxemia . A single intravenous dose of recombinant human D factor completely protected C57/Bl6 mice from the lethal effect of Escherichia coli endotoxin (lipopolysaccharide {LPS}) . The protective effects were dose dependent and observed when administered 2-24 h before LPS . Previous work has shown that interleukin 1 (IL-1) and tumor necrosis factor (TNF) also protect against a subsequent LPS challenge in a dose-dependent manner . When human D factor was combined with sub-protective doses of IL-1 beta or TNF-alpha, there was dramatic synergistic protection against a subsequent lethal LPS challenge. J Infect Dis, 1992 Apr, 165(4), 695 - 701 Treatment of severe infectious purpura in children with human plasma from donors immunized with Escherichia coli J5: a prospective double-blind study . J5 study Group; Revised nucleotide sequence of the gltP gene et al.; Department of Microbiology, University of Groningen, Haren, The NetherlandsThe gene encoding the proton-glutamate carrier (GltP) of Escherichia coli K-12 was sequenced, and the primary structure of the protein was analyzed . The nucleotide sequence was found to differ in several aspects from the previously published sequence (B . Wallace, Y . Yang, J . Hong, and D . Lum, J . Bacteriol . 172:3214-3220, 1990) . The corrected open reading frame encodes a protein of 437 (instead of 395) amino acids . Hydropathy analysis predicts 12 membrane-spanning alpha-helical regions . The complementary strand does contain an open reading frame possibly encoding a highly hydrophilic polypeptide of 272 amino acids. J Bacteriol, 1992 Apr, 174(7), 2305 - 11 Iron(III) hydroxamate transport in Escherichia coli K-12: FhuB-mediated membrane association of the FhuC protein and negative complementation of fhuC mutants; Schultz-Hauser G et al.; Iron(III) hydroxamate transport across the cytoplasmic membrane is catalyzed by the very hydrophobic FhuB protein and the membrane-associated FhuC protein, which contains typical ATP-binding domains . Interaction between the two proteins was demonstrated by immunoelectron microscopy with anti-FhuC antibodies, which showed FhuB-mediated association of FhuC with the cytoplasmic membrane . In addition, inactive FhuC derivatives carrying single amino acid replacements in the ATP-binding domains suppressed wild-type FhuC transport activity, which arose either from displacement of active FhuC from FhuB by the mutated FhuC derivatives or from the formation of mixed inactive FhuC multimers between wild-type and mutated FhuC proteins . Inactive FhuC derivatives containing internal deletions and insertions showed no phenotypic suppression, indicating conformational alterations that rendered the FhuC derivatives unable to displace wild-type FhuC . It is concluded that the physical interaction between FhuC and FhuB implies a coordinate activity of both proteins in the transport of iron(III) hydroxamates through the cytoplasmic membrane. J Bacteriol, 1992 Apr, 174(7), 2124 - 30 Involvement of phosphotransacetylase, acetate kinase, and acetyl phosphate synthesis in control of the phosphate regulon in Escherichia coli; Wanner BL et al.; Two controls of the phosphate (PHO) regulon require sensor proteins that are protein kinases that phosphorylate the regulator, PhoB, which in turn activates transcription only when phosphorylated . Pi control requires the Pi sensor PhoR; the other control is Pi independent and requires the sensor CreC (formerly called PhoM) . Here we describe an additional control of the PHO regulon which is Pi independent and requires neither PhoR nor CreC . This control is regulated by a two-step pathway in carbon metabolism in which acetyl coenzyme A, Pi, and ADP are converted into acetate, coenzyme A, and ATP via the enzymes phosphotransacetylase (Pta) and acetate kinase (AckA) . It responds to the synthesis of acetyl phosphate, an intermediate in the Pta-AckA pathway . Since the synthesis of acetyl phosphate via this pathway leads to the incorporation of Pi into ATP, the primary phosphoryl donor in metabolism, we propose that a regulatory coupling(s) may exist between the PHO regulon, which encodes genes for Pi uptake, and genes for enzymes in central metabolism for incorporation of Pi into ATP . Regulatory interactions of this sort may be important in global control . Further, it provides a functional basis for the concept of cross-regulation in the PHO regulon . This is also the first evidence that acetyl phosphate may have a role as an effector of gene regulation. J Bacteriol, 1992 Apr, 174(7), 2121 - 3 Nonrandom F-plasmid replication in Escherichia coli K-12; Koppes LJ; Both the autonomous and chromosomally integrated F plasmids were found to replicate in a nonrandom fashion after a density transfer from heavy medium ({13C}glucose, 15NH4) to light medium ({12C}glucose, 14NH4) . The data are consistent with the hypothesis that both the chromosome and the F plasmid are replicated in a cell cycle-specific manner . Thus, these data support the proposal (J . D . Keasling, B . O . Palsson, and S . Cooper, J . Bacteriol . 173:2673-2680, 1991) that plasmids replicate in a cell cycle-specific manner. J Bacteriol, 1992 Apr, 174(7), 2102 - 10 Proteins encoded by the Escherichia coli replication terminus region; Moir PD et al.; The replication terminus region (31 to 35 min) of the Escherichia coli chromosome contains very few mapped genes (two per min) compared with the remainder of the chromosome, and much of the DNA appears dispensable . In order to determine whether, despite this, the terminus region consists of protein-coding sequences, we cloned 44 kb (1 min) of terminus region DNA (that surrounding trg at 31.4 min) and examined its ability to catalyze protein synthesis in vitro or in minicells . We were able to account for more than half the coding capacity of the cloned DNA with proteins synthesized in these systems, indicating that the sparsity of mapped genes in the terminus region does not result from a lack of identifiable coding sequences . We can therefore conclude that the terminus region is composed mainly of expressable, albeit inessential, protein-encoding genes. J Bacteriol, 1992 Apr, 174(7), 2072 - 7 Oxygen sensitivity of an Escherichia coli mutant; Adler H et al.; Genetic evidence indicates that Oxys-6, an oxygen-sensitive mutant of Escherichia coli AB1157, is defective in the region of the hemB locus . Oxys-6 is capable of growth under aerobic conditions only if cultures are initiated at low-inoculum levels . Aerobic liquid cultures are limited to a cell density of 10(7) cells per ml by the accumulation of a metabolically produced, low-molecular-weight, heat-stable material in complex organic media . Both Oxys-6 and AB1157 cells produce the material, but only aerobic cultures of the mutant are inhibited by it . The material is produced by both intact cells and cell extracts in complex media . This reaction also occurs when the amino acid L-lysine is substituted for complex media. Arch Biochem Biophys, 1992 Apr, 294(1), 282 - 90 Expression, purification, and characterization of the recombinant kringle 1 domain from tissue-type plasminogen activator; DeSerrano VS et al.; A novel fusion protein expression plasmid that allows ready purification and subsequent facile release of the target molecule has been constructed and employed to express in Escherichia coli and purify the tissue plasminogen activator kringle 1 domain ({K1tPA} residues C92-C173) . The resulting plasmid encodes the tight lysine-binding kringle (K)1 domain of human plasminogen ({K1HPg}) followed by a peptide (PfXa) containing a factor Xa-sensitive bond, downstream of which {K1tPA} was inserted . The recombinant (r) {K1HPg}PfXa{K1tPA} fusion polypeptide was purified from various cell fractions in one step by Sepharose-lysine affinity chromatography . After cleavage with fXa, the mixture was repassaged over Sepharose-lysine, whereupon the r-{K1tPA}-containing polypeptide passed unretarded through the column . A homogeneous preparation of this material was then obtained after a simple step employing fast protein liquid chromatography . The purified r-{K1tPA}, which contained the amino acid sequence SNAS{K1tPA}S, provided an amino-terminal amino acid sequence, through at least 20 amino acid residues, that was identical to that predicted from the cDNA sequence . The molecular mass of r-SNAS{K1tPA}S, determined by electrospray mass spectrometry, was 9621.9 +/- 4.0 (expected molecular mass, 9623.65) . 1H-NMR spectroscopy and thermal stability studies of r-SNAS{K1tPA}S revealed that the purified material was properly folded and similar to other isolated kringle domains . Additionally, employment of this methodology revealed that only a very weak interaction between epsilon-aminocaproic acid and the isolated r-{K1tPA} domain occurred. Arch Biochem Biophys, 1992 Apr, 294(1), 279 - 81 Crystallization and preliminary analysis of oxidized, recombinant, heterocyst {2Fe-2S} ferredoxin from Anabaena 7120; Jacobson BL et al.; The {2Fe-2S} ferredoxin produced in the heterocyst cells of Anabaena 7120 plays a key role in nitrogen fixation, where it serves as an electron acceptor from various sources and an electron donor to nitrogenase . Crystals of recombinant heterocyst ferredoxin, coded for by the fdx H gene from Anabaena 7120 and overproduced in Escherichia coli, have been grown from ammonium sulfate solutions and are suitable for high resolution X-ray crystallographic analysis . They belong to the hexagonal space group P6(1) or P6(5) with unit cell dimensions of a = b = 44.2 A and c = 80.6 A . The crystals contain one molecule per asymmetric unit and diffract to a nominal resolution of 1.6 A . The molecular structure of this heterocyst ferredoxin is of special interest in that 4 of the 22 amino acid positions thought to be absolutely conserved in nonhalophilic ferredoxins are different and, based on amino acid sequence alignments, three of these positions are located in the metal-cluster binding loop . Consequently, a high-resolution X-ray analysis of this {2Fe-2S} ferredoxin, and subsequent three-dimensional comparisons with other known ferredoxin models, will provide new insight into structure/function relationships for this class of redox proteins. Mol Cell Biol, 1992 Apr, 12(4), 1605 - 12 Generation of single-nucleotide repair patches following excision of uracil residues from DNA; Dianov G et al.; The extent and location of DNA repair synthesis in a double-stranded oligonucleotide containing a single dUMP residue have been determined . Gently prepared Escherichia coli and mammalian cell extracts were employed for excision repair in vitro . The size of the resynthesized patch was estimated by restriction enzyme analysis of the repaired oligonucleotide . Following enzymatic digestion and denaturing gel electrophoresis, the extent of incorporation of radioactively labeled nucleotides in the vicinity of the lesion was determined by autoradiography . Cell extracts of E . coli and of human cell lines were shown to carry out repair mainly by replacing a single nucleotide . No significant repair replication on the 5' side of the lesion was observed . The data indicate that, after cleavage of the dUMP residue by uracil-DNA glycosylase and incision of the resultant apurinic-apyrimidinic site by an apurinic-apyrimidinic endonuclease activity, the excision step is catalyzed usually by a DNA deoxyribophosphodiesterase rather than by an exonuclease . Gap-filling and ligation complete the repair reaction . Experiments with enzyme inhibitors in mammalian cell extracts suggest that the repair replication step is catalyzed by DNA polymerase beta. J Virol, 1992 Apr, 66(4), 2435 - 42 Reversion of Q beta RNA phage mutants by homologous RNA recombination; Palasingam K et al.; Q beta phage RNAs with inactivating insertion (8-base) or deletion (17-base) mutations within their replicase genes were prepared from modified Q beta cDNAs and transfected into Escherichia coli spheroplasts containing Q beta replicase provided in trans by a resident plasmid . Replicase-defective (Rep-) Q beta phage produced by these spheroplasts were detected as normal-sized plaques on lawns of cells containing plasmid-derived Q beta replicase, but were unable to form plaques on cells lacking this plasmid . When individual Rep- phage were isolated and grown to high titer in cells containing plasmid-derived Q beta replicase, revertant (Rep+) Q beta phage were obtained at a frequency of ca . 10(-8) . To investigate the mechanism of this reversion, a point mutation was placed into the plasmid-derived Q beta replicase gene by site-directed mutagenesis . Q beta mutants amplified on cells containing the resultant plasmid also yielded Rep+ revertants . Genomic RNA was isolated from several of the latter phage revertants and sequenced . Results showed that the original mutation (insertion or deletion) was no longer present in the phage revertants but that the marker mutation placed into the plasmid was now present in the genomic RNAs, indicating that recombination was one mechanism involved in the reversion of the Q beta mutants . Further experiments demonstrated that the 3' noncoding region of the plasmid-derived replicase gene was necessary for the reversion-recombination of the deletion mutant, whereas this region was not required for reversion or recombination of the insertion mutant . Results are discussed in terms of a template-switching model of RNA recombination involving Q beta replicase, the mutant phage genome, and plasmid-derived replicase mRNA. Infect Immun, 1992 Apr, 60(4), 1653 - 61 Characterization of hybrid toxins produced in Escherichia coli by assembly of A and B polypeptides from type I and type II heat-labile enterotoxins; Connell TD et al.; The genes encoding the individual A and B polypeptides of the type I enterotoxin LTp-I and type II enterotoxins LT-IIa and LT-IIb were cloned and tested for complementation in Escherichia coli . Each gene encoding an A polypeptide was cloned into pACYC184, and each gene encoding a B polypeptide was cloned into the compatible plasmid Bluescript KS+ . In addition, operon fusions representing all combinations of A and B genes were constructed in Bluescript KS+ . Extracts from strains of E . coli expressing each combination of A and B genes, either from compatible plasmids or from operon fusions, were tested for immunoreactive holotoxin by radioimmunoassays and for toxicity by Y1 adrenal cell assays . Biologically active holotoxin was detected in each case, but the toxicity of extracts containing the hybrid toxins was usually less than that of extracts containing the wild-type holotoxins . The ganglioside-binding activity of each holotoxin was tested, and in each case, the B polypeptide determined the ganglioside-binding specificity . The A and B polypeptides of the type II heat-labile enterotoxins were also shown to form holotoxin in vitro without exposure to denaturing conditions, in contrast to the polypeptides of the type I enterotoxins that failed to form holotoxin in vitro under comparable conditions . These findings suggest that type I and type II enterotoxins have conserved structural features that permit their A and B polypeptides to form hybrid holotoxins, although the B polypeptides of the type I and type II enterotoxins have very little amino acid sequence homology. Endocrinology, 1992 Apr, 130(4), 1844 - 51 Demonstration of relaxin precursors in pregnant rat ovaries with antisera against bacterially expressed rat prorelaxin; Soloff MS et al.; The existence of rat 18-kilodalton (kDa) prorelaxin, which has been postulated from the coding sequence of cloned cDNA and the results of cell-free translation studies, has been directly demonstrated in rat ovaries with antibodies against bacterially expressed rat prorelaxin . The peptide expressed in E . coli from a rat prorelaxin cDNA construct was comprised of the B- and A-chains of relaxin and a 105-amino acid connecting region . Immunoreactive bands of 18 and 16.5 kDa were shown in ovaries from day 20 pregnant rats . Partial amino acid sequence analysis of both peptides revealed that they had identical N-terminal sequences, corresponding to rat prorelaxin . Both 18- and 16.5-kDa bands were present only from midpregnancy until near term, when they declined sharply . These changes in the concentration of 18-kDa prorelaxin match changes in preprorelaxin mRNA levels, suggesting that relaxin synthesis is regulated at the transcriptional level and not by protein processing . Prorelaxin was transiently secreted by COS-1 cells transfected with preprorelaxin cDNA . Treatment of culture medium with trypsin resulted in the appearance of material corresponding in size to mature relaxin . Thus, correctly folded prorelaxin appears to be a suitable precursor for relaxin . The combined concentrations of 18- and 16.5-kDa peptides in ovaries on day 20 of pregnancy were considerably more than 30 times greater than that of relaxin, however, suggesting that prorelaxin might also be more than a precursor per se. Virology, 1992 Apr, 187(2), 643 - 53 Immature viral envelope formation is interrupted at the same stage by lac operator-mediated repression of the vaccinia virus D13L gene and by the drug rifampicin; Zhang Y et al.; Specific missense mutations of the vaccinia virus D13L gene confer resistance to the effects of rifampicin on virion morphogenesis . We constructed a recombinant vaccinia virus in which elements of the Escherichia coli lac operator system were used to regulate the D13L gene . Replication of the recombinant vaccinia virus was dependent on addition of the inducer isopropyl beta-D-thiogalactoside (IPTG) and the virus yield was decreased by more than 99% when IPTG was omitted . Under the nonpermissive condition, transcription of the D13L gene was reduced and synthesis of the 65,000-Da protein product was inhibited by more than 95% . Consequently, virion morphogenesis was blocked at an early stage and uncoated membrane precursors of the immature viral envelope and uncleaved precursors of the major core proteins accumulated . The phenotype of the conditional lethal mutant virus, in the absence of IPTG, closely resembled that of wild-type virus in cells treated with rifampicin. Mol Gen Genet, 1992 Apr, 232(3), 489 - 97 A partially deficient mutant, recA1730, that fails to form normal nucleoprotein filaments; Dutreix M et al.; The phenotype of the recA1730 mutant is highly dependent on the level of expression of the RecA1730 protein . If the recA1730 gene was expressed from its own promoter, the cells were deficient in recombination and SOS induction . In contrast, when the recA1730 gene was expressed under the control of recAo98, a constitutive operator that increased the RecA1730 concentration 20-fold, cells became proficient in recombination and SOS induction . Likewise, in crude extracts, fivefold more RecA1730 than RecAwt was required to produce full cleavage of LexA protein . The requirement for a high RecA1730 concentration for recombination and LexA cleavage suggests that the recA1730 defect alters a common reaction step . In fact, in vitro data show that the impaired assembly of RecA1730 protein on single-stranded DNA (ssDNA) can account for the mutant phenotype . Purified RecA1730 protein was assayed in vitro for ssDNA binding and ATPase activities . RecA1730, like RecAwt, retained ssDNA equally well on nitrocellulose filters; this activity was specifically inhibited by a monoclonal anti-RecA antibody . However, RecA1730 protein did not form complete filaments on ssDNA, as shown by two observations: (i) most of the protein did not elute with ssDNA during gel filtration; and (ii) binding of RecA1730 to ssDNA did not protect it from being digested by DNaseI . RecA1730 hydrolysed ATP in high salt but was defective in ssDNA-dependent ATP hydrolysis . These results strongly suggest that RecA1730 binds to ATP and ssDNA but does not form normal nucleoprotein filaments. Mol Gen Genet, 1992 Apr, 232(3), 394 - 8 A novel mutation FruS, altering synthesis of components of the phosphoenolpyruvate: fructose phosphotransferase system in Escherichia coli K12; Bolshakova TN et al.; A novel mutation, FruS localised in the fru operon was obtained . It uncouples expression of the genes determining synthesis of the fructose-specific transport proteins and fructose-1-phosphate kinase . In FruS bacteria the fruA and fruF genes (coding for Enzyme IIfru and FPr, respectively) are constitutive by expressed while fruK (encoding fructose-1-phosphate kinase) remains inducible . In contrast to other mutations, which render expression of the whole fru operon constitutive, the FruS mutation: (1) does not lead to D-xylitol sensitivity; (2) does not inhibit growth on D-lactate, pyruvate and L-alanine; (3) does not decrease phosphoenolpyruvate (PEP) synthase activity. Genetics, 1992 Apr, 130(4), 737 - 48 Analysis of junction sequences resulting from integration at nonhomologous loci in Neurospora crassa; Asch DK et al.; We have analyzed the junctions involved in two examples of ectopic integration of plasmids containing the am+ (glutamate dehydrogenase) gene into a strain of Neurospora crassa bearing a complete deletion of the am locus . In one transformed strain a single copy of plasmid DNA had been integrated into linkage group (LG) III DNA without the loss of chromosomal DNA . In contrast, 450 bp had been lost from plasmid sequences at the site of integration . The transforming DNA used was circular, so we postulate that the plasmid was linearized and truncated prior to its integration by end joining into a break in LG III DNA . There was no significant homology between the incoming DNA and DNA at the site of integration . The second transformed strain resulted from transformation with a linearized plasmid . It contained multiple integrated copies of plasmid DNA, one of which was recloned, together with adjacent chromosomal DNA, by plasmid rescue in Escherichia coli . Prior to integration into chromosomal DNA, the linear plasmid had been truncated by 64 bp on one end and 3.2 kbp on the other end . One end of the integrated DNA was adjacent to DNA from the right arm of LG I, while the other end was integrated into a copy of a repetitive sequence . Restriction fragment length polymerism mapping showed that integration was in a copy of the repetitive sequence that is linked to the previously unassigned telomere M11 and is distantly linked to the LG VI marker con-11 . Genetic analysis revealed that a long segment of LG I containing all markers from un-1 to the right tip has been translocated to the right end of LG VI . Tetrad analysis showed that the integrated DNA was closely linked to the translocation . We conclude that the transforming DNA was truncated and joined to DNA from two different chromosomes by end joining during the formation of a quasiterminal translocation, T(IR----VIR) UK-T12 . We also conclude that the previously unassigned telomere, M11, is the right end of LG VI. Clin Infect Dis, 1992 Apr, 14(4), 927 - 32 Prevention of preterm delivery and low birth weight associated with asymptomatic bacteriuria; Mittendorf R et al.; Since the first report of an association between asymptomatic bacteriuria and low birth weight (less than 2,500 g) in 1962, greater than 30 other studies on the same subject have been published . Some of these confirmed this association while others disputed it . Now, however, by using meta-analysis (a technique considered valid by many but not all statisticians) one may conclude with increased certainty that true associations between asymptomatic bacteriuria and preterm delivery (less than 37 weeks of gestation) and asymptomatic bacteriuria and low birth weight do exist . Because asymptomatic bacteriuria in pregnancy remains prevalent and preventable, a review of this important subject is relevant at this time. Arch Ophthalmol, 1992 Apr, 110(4), 547 - 9 Activity of an interleukin 1 receptor antagonist in rabbit models of uveitis; Rosenbaum JT et al.; Interleukin 1 has been implicated in intraocular inflammation . The availability of a cloned, recombinant interleukin 1 receptor antagonist has enabled us to test the role of interleukin 1 in specific models of uveitis in New Zealand white rabbits . Seventy-five micrograms of interleukin 1 receptor antagonist injected intravitreally resulted in a 97% reduction in aqueous humor cells present 6 hours after intravitreal injection of 10 ng of human interleukin 1 alpha . Disruption of the blood aqueous barrier was prevented by the receptor antagonist (mean +/- SD aqueous humor protein of 0.6 +/- 0.1 g/L in rabbits treated with interleukin 1 receptor antagonist vs 32.2 +/- 9.9 g/L in controls) . Lower doses of interleukin 1 produced more modest but significant inhibition . Despite the activity of interleukin 1 receptor antagonist in inhibiting interleukin 1-induced inflammation, interleukin 1 receptor antagonist did not produce significant reduction in inflammation subsequent to an active Arthus reaction or subsequent to the intravitreal injection of 125 ng of endotoxin . A potential explanation of these observations is that cytokines in addition to interleukin 1 may be present in sufficient quantities to produce intraocular inflammation or that the effects of interleukin 1 may be primarily intracellular (intracrine) and therefore resistant to the activity of exogenously administered receptor antagonist. J Bacteriol, 1992 Apr, 174(7), 2152 - 9 KdpD and KdpE, proteins that control expression of the kdpABC operon, are members of the two-component sensor-effector class of regulators; Walderhaug MO et al.; The Kdp system of Escherichia coli, a transport ATPase with high affinity for potassium, is expressed when turgor pressure is low . Expression requires KdpD, a 99-kDa membrane protein, and KdpE, a 25-kDa soluble cytoplasmic protein . The sequences of KdpD and KdpE show they are members of the sensor-effector class of regulatory proteins: the C-terminal half of KdpD is homologous to sensors such as EnvZ and PhoR, and KdpE is homologous to effectors such as OmpR and PhoB . The predicted structure of KdpD suggests that it is anchored to the membrane by four membrane-spanning segments near its middle, with both C- and N-terminal portions in the cytoplasm . Subcellular fractionation confirms the expected location of the protein in the inner membrane . The N-terminal region has no homology to known proteins and is the site of mutations that make Kdp expression partially constitutive; this portion may serve to sense turgor pressure . Since several other sensor-effectors have been shown to mediate control through phosphorylation, this mechanism is proposed to control expression of Kdp. J Bacteriol, 1992 Apr, 174(7), 2145 - 51 The products of the kdpDE operon are required for expression of the Kdp ATPase of Escherichia coli; Polarek JW et al.; The expression of the Kdp system for K+ uptake in Escherichia coli requires the products of two genes, kdpD and kdpE . These genes constitute an operon adjacent to the kdpABC operon that encodes the three membrane protein subunits of Kdp . Both operons are transcribed in the same direction and overlap; the kdpDE promoter is in kdpC, the last gene of the kdpABC operon . Transcription of the kdpDE operon is at a low level when Kdp is not expressed; transcription increases about 10-fold when kdpABC is turned on, indicating significant read-through of the kdpDE operon by transcripts beginning at the promoter of kdpABC operon . The proximal region of the kdpD gene is the site of most mutations that lead to constitutive expression of the kdpABC operon. Eur J Clin Chem Clin Biochem, 1992 Apr, 30(4), 187 - 91 Effect of ascorbic acid on neutrophil functions and hypoxanthine/xanthine oxidase-generated, oxygen-derived radicals; Dwenger A et al.; The chemiluminescence of isolated neutrophils, stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine, latex, lipopolysaccharide from Escherichia coli, zymosan A, or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate was inhibited up to 99% by the dose-dependent oxygen radical scavenging activity of 6 mmol/l ascorbic acid . The chemiluminescence of neutrophils in blood, stimulated with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, or with zymosan A was inhibited 35% or 48%, respectively, by 6 mmol/l ascorbic acid . Ascorbic acid, up to 6 mmol/l, did not inhibit the release of beta-N-acetylglucosaminidase and elastase from isolated neutrophils activated by the above stimulatory agents . During neutrophil/nylon fibre interaction ascorbic acid reduced the oxygen radical production dose-dependently (77% inhibition of the chemiluminescence response at 6 mmol/l ascorbic acid), whereas the adherence was unaffected . Hypoxanthine/xanthine oxidase-generated oxygen radicals were scavenged by ascorbic acid in a dose-dependent manner (99% inhibition of the chemiluminescence response at 100 mumol/l ascorbic acid) . From these results, ascorbic acid can highly be recommended for animal experiments and clinical studies in patients with trauma, shock and sepsis and for studies to prevent or reduce reperfusion injuries. Kobe J Med Sci, 1992 Apr, 38(2), 79 - 92 Function of the post-translationally modified C-terminal region of rho p21; Hori Y; rhoA p21, a ras p21-like small GTP-binding protein, purified from bovine aortic smooth muscle is similarly modified at its C-terminal region as described for ras p21s . In this study, I examined the role of the post-translational modifications of the C-terminal region of rhoA p21 by comparing bovine rhoA p21 with bacterially-produced rhoA p21 because the bacterial protein was not modified . Bovine rhoA p21 bound to plasma membranes, but bacterial rhoA p21 did not . Both the stimulatory and inhibitory GDP/GTP exchange proteins for bovine rhoA p21 were inactive for bacterial rhoA p21 . On the other hand, the GTPase activating protein for bovine rhoA p21 was also active for bacterial rhoA p21 . These results indicate that the post-translational modifications of the C-terminal region of bovine rhoA p21, which are absent in bacterial rhoA p21, are essential for its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins but not with the GTPase activating protein. Acta Crystallogr B, 1992 Apr 1, 48 ( Pt 2), 241 - 4 Crystallization studies of the catalytic subunit of cAMP-dependent protein kinase: crystals of murine recombinant catalytic subunit and a mutant, Cys 343----Ser, diffract to 2.7 A resolution; Zheng JH et al.; The recombinant mouse catalytic subunit of cAMP-dependent protein kinase, expressed and purified from E . coli, has been successfully cocrystallized as a binary complex with an inhibitor peptide and as a ternary complex with an inhibitor peptide and MgATP . In contrast to the catalytic subunit obtained from porcine heart, the recombinant catalytic subunit lacks a myristoyl group at the amino terminus and differs in sequence at nine positions out of 350 amino acids . The catalytic activities of the two enzymes, however, are nearly identical . Both enzymes cocrystallized with a 20-amino-acid inhibitor and MgATP; however, the porcine-heart enzyme crystallized in a hexagonal space group (P6(1)22) while the recombinant murine catalytic subunit crystallized in an orthorhombic space group (P2(1)2(1)2(1), a = 73.70, b = 76.26, c = 80.74 A) . The orthorhombic crystals of the recombinant catalytic subunit exhibit the best diffraction characteristics of all catalytic subunit crystals obtained so far: 2.7 A resolution . Unlike the mammalian porcine-heart enzyme, no crystals of the recombinant apo-enzyme were obtained under the same crystallization conditions . These results are consistent with earlier conclusions that the catalytic subunit exists in at least two distinct conformational states and furthermore suggests that the inhibitor peptide alone is sufficient to induce the major conformational changes that distinguish the two states . A mutant form of the catalytic subunit where Cys343 was replaced with Ser was also cocrystallized with the 20-amino-acid peptide inhibitor and MgATP, and resulted in an orthorhombic crystal isomorphous to crystals of the unmutated enzyme with a similar diffraction of 2.7 A. J Mol Endocrinol, 1992 Apr, 8(2), 165 - 72 Recombinant mouse prolactin: expression in Escherichia coli, purification and biological activity; Yamamoto M et al.; Transformation of Escherichia coli cells with a recombinant plasmid containing modified mouse prolactin (mPRL) cDNA and a pKK223-3 vector resulted in efficient expression of mPRL protein . Cloned mPRL cDNA was modified by removing the 5' non-translating sequence as well as the sequence which encoded the signal peptide of preprolactin for recombination . In addition, approximately 100 nucleotides of the 5'-terminal region of the cDNA, which include the ATG initiation codon and the following 31 codons of mature mPRL, were replaced by a chemically synthesized oligonucleotide duplex . The sequence of this duplex was chosen to be rich in AT without changing the amino acid sequence of the protein . The modified cDNA was finally inserted into the multicopy plasmid, pUC19, before high-level expression of mPRL in E . coli cells was obtained . Western blotting analysis of total protein from transformed E . coli cells showed that both 23 and 16 kDa peptides were recognized by specific mPRL antisera . The purified and refolded 23 kDa protein exhibited a growth-stimulating effect on rat Nb 2 Node lymphoma cells, and was very similar to that of natural pituitary PRL. J Mol Endocrinol, 1992 Apr, 8(2), 137 - 44 Expression of a human thyrotrophin receptor fragment in Escherichia coli and its interaction with the hormone and autoantibodies from patients with Graves' disease; Huang GC et al.; Graves' disease is an autoimmune thyroid disease characterized by the presence of pathogenic autoantibodies to the TSH receptor (TSH-R) . By using polymerase chain reaction, the extracellular region of the human TSH-R cDNA has been amplified and used to prepare recombinant TSH-R (extracellular) protein fused with glutathione-S-transferase (GST) . Purification of the recombinant TSH-R (extracellular)-GST fusion protein was achieved by preparative gel electrophoresis in SDS or by preparative isoelectric focusing in urea . Following removal of SDS by detergent exchange or urea by dialysis, the purified recombinant receptor preparations were assessed for binding to the hormone or to autoantibodies from Graves' disease patients . The purified recombinant receptor preparations fail to show any binding to the hormone or autoantibodies either by inhibition of binding assays or by immunoblotting . The results imply that the correct folding and/or post-translational modifications of the polypeptide chain which are not achieved in recombinant proteins produced in Escherichia coli may be important for the binding of the hormone or Graves' disease autoantibodies to the TSH-R . The recombinant receptor prepared in this manner will be useful for immunological and cellular investigations in patients with Graves' disease. Jpn J Cancer Res, 1992 Apr, 83(4), 351 - 7 Ubiquitous presence in mammalian cells of enzymatic activity specifically cleaving 8-hydroxyguanine-containing DNA; Yamamoto F et al.; Here we report the finding of enzymatic activity that specifically cleaves DNA containing 8-hydroxyguanine (oh8Gua) residues in various mammalian cells . To detect this activity, we used a synthetic double-stranded DNA containing a single oh8Gua at a defined position as the substrate, and analyzed the products of enzymatic digestion by polyacrylamide gel electrophoresis . Two cleavage sites near the oh8Gua residue were detected with partially purified fractions from cow brain and rat liver, and also with preparations from all mammalian tissues examined . These results suggest that enzymatic activity for the removal of oh8Gua from DNA is widely distributed in mammalian cells. Biotechniques, 1992 Apr, 12(4), 558 - 63 Production of recombinant rat interleukin-6 in Escherichia coli using a novel highly efficient expression vector pGEX-3T; Frorath B et al.; Interleukin-6 (IL-6) is one of the most important mediators of the acute phase reaction in liver . For the production of recombinant rat IL-6 in Escherichia coli, a previously isolated cDNA coding for the rat IL-6 was cloned into the modified novel expression vector pGEX-3T . The IL-6 cDNA was highly expressed as a fusion protein with the glutathione S-transferase (GST) at its C-terminus and rat IL-6 at its N-terminus . The GST-IL-6 fusion protein was controlled by a tac-promoter and could be induced very efficiently by isopropyl-beta-D-thiogalactopyranoside . The synthesized GST-IL-6 fusion protein was insoluble and precipitated intracellularly in E . coli . Using an advanced technique, the insoluble protein was solubilized and purified to homogeneity by affinity chromatography using immobilized glutathione in a one-step procedure. Biotechniques, 1992 Apr, 12(4), 544 - 9 Recombinant peptides as immunogens: a comparison of protocols for antisera production using the pGEX system; Oettinger HF et al.; Using an inducible vector system that directs high-level production and rapid purification of recombinant protein, we have immunized mice with peptides prepared by several methods: 1) samples fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) subsequently transferred to PVDF membrane and subcutaneously implanted in mice; 2) samples cut directly from SDS-PAGE gels and injected intraperitoneally; 3) injection of recombinant protein bound to agarose beads; and 4) injection of log-phase E . coli transformed with recombinant vector . All four strategies yielded specific antisera reacting with both the parental fusion protein and the recombinant fragment as determined by enzyme-linked immunosorbent assay and immunoblot analysis . Specific recognition of the recombinant fragment was demonstrated by a competitive inhibition assay in which the parental fusion protein abrogated reactivity of serum with the isolated recombinant fragment. Biotechniques, 1992 Apr, 12(4), 528 - 30, 532, 534-5 Recombinant circle PCR and recombination PCR for site-specific mutagenesis without PCR product purification; Jones DH et al.; Two simple methods for site-specific mutagenesis are described and compared . In each method, the PCR is used in two separate amplifications to mutate the site of interest and to add ends to one PCR product that are homologous to the ends of the other PCR product . In the first method, the two products are combined, denatured and reannealed prior to transformation of E . coli in order to form recombinant circles in vitro, while in the second method, the two linear products are co-transfected directly into E . coli without prior manipulation, resulting in transformation of E . coli with the recombinant of interest by recombination in vivo . Each PCR amplification uses a plasmid template that has been linearized by restriction enzyme digestion outside the region to be amplified . This permits use of unpurified PCR products in these two protocols and generation of the mutant of interest with no other enzymatic manipulation in vitro apart from PCR amplification . In each protocol greater than or equal to 50% of the resulting clones contained the mutation of interest without detected errors. Plant Cell, 1992 Apr, 4(4), 451 - 61 Cytokinins and auxins control the expression of a gene in Nicotiana plumbaginifolia cells by feedback regulation; Dominov JA et al.; Both cytokinin (N6-benzyladenine {BA}) and auxin (2,4-dichlorophenoxyacetic acid {2,4-D}) stimulate the accumulation of an mRNA, represented by the cDNA pLS216, in Nicotiana plumbaginifolia suspension culture cells . The kinetics of RNA accumulation were different for the two hormones; however, the response to both was transient, and the magnitude of the response was dose dependent . Runoff transcription experiments demonstrated that the transient appearance of the RNA could be accounted for by feedback regulation of transcription and not by the induction of an RNA degradation system . The feedback mechanism appeared to desensitize the cells to further exposure of the hormone . In particular, cells became refractory to the subsequent addition of 2,4-D after the initial RNA accumulation response subsided . A very different response was observed when the second hormone was added to cells that had been desensitized to the first hormone . Under such conditions, BA produced a heightened response in cells desensitized to 2,4-D and vice versa . These findings support a model in which cytokinin further enhances the auxin response or prevents its feedback inhibition . The hormone-induced RNA accumulation was blocked by the protein kinase inhibitor staurosporin . On the other hand, the protein phosphatase inhibitor okadaic acid stimulated expression, and, in particular, okadaic acid was able to stimulate RNA accumulation in cells desensitized to auxin . This suggests that hormone activation involves phosphorylation of critical proteins on the hormone signaling pathway, whereas feedback inhibition may involve dephosphorylation of these proteins . The sequence of pLS216 is similar to genes in other plants that are stimulated by multiple agonists such as auxins, elicitors, and heavy metals, and to the gene encoding the stringent starvation protein in Escherichia coli . It is proposed that this gene family in various plants be called multiple stimulus response (msr) genes. Zhongguo Zhong Xi Yi Jie He Za Zhi, 1992 Apr, 12(4), 226 - 7, 197 {Antagonistic effect of re du qing on damage of hepatocytic microsomes in rabbits with endotoxin-induced disseminated intravascular coagulation}; Tu SH et al.; In this study, the generalized Shwartzman reaction of rabbit induced by endotoxin of Escherichia Coli was built as DIC models . The experiment showed that the levels of lipid peroxide (LPO) in the hepatocytic microsomes of model group were increased significantly, whereas the cytochrome P-450 (Cyto . P-450) contents and aniline hydroxylase activities were obviously decreased . In the Re Du Qing (RDQ) group and dexamethasone group the decrease of LPO in hepatocytic microsomes as well as the reduction of Cyto . P-450 contents and aniline hydroxylase activities were alleviated . Furthermore, the correlation analysis indicated a significant negative correlation between levels of LPO in microsomes and the Cyto . P-450 contents as well as aniline hydroxylase activities . This study indicates the LPO may play an important role in the damage of hepatocytic microsomes and RDQ could prevent hepatocytic microsomes from injury of rabbits with endotoxin-induced DIC. Zh Mikrobiol Epidemiol Immunobiol, 1992 Apr, (4), 44 - 7 {The correction of the functional activity of alveolar macrophages in inducing a primary immune response in influenza}; Pozdeev OK et al.; The functional activity of alveolar macrophages obtained from mice, both healthy and infected with influenza virus A/Aichi 2/68 (H3N2), as manifested by their capacity to initiate the development of primary immune response to sheep red blood cells and Escherichia coli lipopolysaccharide after the transfer of these macrophages to intact syngeneic recipients was studied . The capacity of alveolar macrophages to perform antigen-presenting functions in the induction of humoral immune response was shown, and at the same time the development of experimental influenza infection was found to essentially decrease these properties . The injection of the immunomodulating agent diuciphon into experimental mice somewhat enhanced the immune response after the syngeneic transfer of alveolar macrophages from infected mice to intact recipients. Indian J Pathol Microbiol, 1992 Apr, 35(2), 125 - 8 Vaginitis in non pregnant women in Haryana; Saini S et al.; Study was carried out in 100 patients of non-specific vaginitis (NSV) to find out the incidence of vaginitis due to G . vaginalis . Out of a total of 100 subjects 20 were positive for G . vaginalis as compared to only 6 in equal number of normal matched controls . One positive specimen showed concomitant presence of C . albicans and E . coli was found in another positive specimen . Presence of amines and clue cells in the discharge did not correlate with the isolation rate of G . vaginalis, thus emphasizing the necessity of culture to diagnose NSV due to G . vaginalis. Zhonghua Wai Ke Za Zhi, 1992 Apr, 30(4), 244 - 7, 256 {Protective effect of antiserum to Re-LPS on experimental multiple organ failure}; Yao YM; We determined the possible beneficial effect of administering antiserum against Re-LPS(F515) on experimental multiple system organ failure (MSOF) in rabbits . The results showed that there were a more significant decrease of the plasma LPS level and a shorter period to recovery than in the control group after receiving antiserum to Re-LPS . Pretreatment with antiserum can remarkably improve the function of liver, lung, kidney, blood and gastrointestinal tract . The incidence of MSOF in rabbits receiving immune sera was only 11.2% and the survival rate was increased by 40.0% . The results suggest that early passive immunotherapy may neutralize gut-derived endotoxin, inhibit endotoxin-induced mediator release and prevent development of severe complication due to sepsis . Prophylactic application of antiserum to LPS core region may provide protective effect on experimental MSOF. Protein Expr Purif, 1992 Apr, 3(2), 93 - 100 Purification and biochemical characterization of recombinant rat liver phenylalanine hydroxylase produced in Escherichia coli; Citron BA et al.; Phenylalanine hydroxylase, important in phenylalanine metabolism in mammals, is regulated through short-term (activation) and long-term (induction) mechanisms . To help elucidate the structure-function relationships involved in the activation of this enzyme, we have isolated and characterized full-length cDNA clones to rat phenylalanine hydroxylase . Recombinant rat phenylalanine hydroxylase was placed into an expression vector in Escherichia coli . The enzyme has been purified to homogeneity and its physical and catalytic properties have been characterized . The molecular weight and the fluorescence emission spectrum of the recombinant enzyme were identical to those of the native enzyme . The recombinant enzyme could be activated by incubation with phenylalanine or lysolecithin or by phosphorylation, as is the rat liver enzyme . The extent of activation is the same as that for the native enzyme in each case except for phenylalanine, which activates the recombinant enzyme only 5- to 10-fold rather than the 15- to 30-fold activation observed with the native enzyme . The kinetic constants determined for the recombinant enzyme are also essentially the same as those reported for the native enzyme . We conclude that this enzyme is essentially identical to the native enzyme and should be very useful in the future study of this important hydroxylase. Am J Physiol Imaging, 1992 Apr-Jun, 7(2), 66 - 72 Multiple organ platelet sequestration, hemodynamics, and gas exchange in endotoxin shock and the effects of aspirin-terbutaline treatment; Sigurdsson GH et al.; We studied the effects of two different drugs on multiple organ platelet sequestration, hemodynamics, and respiratory function during endotoxin shock . Twenty-eight sheep (four groups) were anesthetized and ventilated . Group AT-E received aspirin and terbutaline 30 min before and group E-AT 30 min after Escherichia coli endotoxin . Group E also received endotoxin but no drug treatment (shock controls), and group C received neither endotoxin nor drug treatment . There was a marked platelet trapping in the lungs and in the liver immediately after administration of endotoxin in groups E and E-AT, but after 4 hr it was less pronounced in group E-AT than in the endotoxin controls (P < 0.05) . In the pretreated animals (group AT-E) there was no increase in platelet sequestration until almost 2 hr after endotoxin both in the lungs and the liver, but at the end of the study (240 min) there was no difference between the pre- and posttreated groups . No significant changes occurred in the kidneys and spleen in any of the groups . In groups E and E-AT the endotoxin infusion resulted in 200% rise in pulmonary artery pressure (PAP) and a sharp decrease in mean arterial pressure (MAP; 25-30%), respiratory compliance (CT; 50%), arterial oxygen tension (PaO2; 70%) as well as in oxygen delivery index (DO2; 30-40%) within 30 min . After 4 hr the PAP had decreased significantly in group E-AT, but remained high in group E (> 100% higher than in group C) . Also MAP, PaO2, DO2 and CT improved slightly in group E-AT, while they remained low in group E.(ABSTRACT TRUNCATED AT 250 WORDS) Antibiot Khimioter, 1992 Apr, 37(4), 11 - 4 {Consumption of amino acids by Escherichia coli--a producer of recombinant proteins}; Ustiuzhanina SV et al.; The dynamics of amino acid consumption from the medium by Escherichia coli 1864, a strain producing recombinant protein was studied . It was shown that the strain actively used glutamate and aspartate from the medium, which was determined by the leading role of the amino acids in nitrogen metabolism . The strain also consumed threonine, glycine and alanine capable of effectively providing the culture with metabolic energy. Protein Eng, 1992 Apr, 5(3), 253 - 7 A point mutation of human interferon gamma abolishes receptor recognition; Lunn CA et al.; We identified a single amino acid mutation that abolished the bioactivity of human IFN gamma . The mutation was identified by screening a mutagenized IFN gamma expression library for molecules with altered biological activity . The mutant protein was expressed at high levels in Escherichia coli, and remained soluble upon purification . However, the protein was completely inactive in all IFN gamma assays investigated, exhibiting less than 0.0006% of the specific activity of native IFN gamma antiviral activity . Sequencing the plasmid DNA encoding this mutant protein showed that the histidine at position 111 of native human IFN gamma is changed to aspartic acid (IFN gamma/H111D) . Other mutations at this site showed that only hydrophobic amino acids at position 111 maintain significant, though low, biological activity . Structural characterization of the IFN gamma/H111D protein by NMR as well as CD spectroscopy demonstrated that the protein has limited conformational differences from native IFN gamma . Models of the X-ray crystal structure of human IFN gamma {Ealick, P.E., W.J . Cook, S . Vijay-Kumar, M . Carson, T.L . Nagabhushan, P.P . Trotta and C.E . Bugg (1991) Science, 252, 698-702} suggest that this histidine residue is located at a severe 55 degrees bend in the C-terminal F helix . We conclude that H111 lies within or affects the receptor binding domain of human IFN gamma. Protein Eng, 1992 Apr, 5(3), 249 - 52 A point mutation that decreases the thermal stability of human interferon gamma; Lunn CA et al.; We have identified a mutation of human gamma-interferon (IFN gamma) causing a temperature-sensitive phenotype . We used a randomized oligonucleotide to mutagenize a synthetic human IFN gamma gene, then screened the resulting mutants produced in Escherichia coli for proteins with altered biological activity . One mutant protein selected for detailed characterization exhibited less than 0.3% of the specific biological activity of native IFN gamma in an antiviral activity assay performed at 37 degrees C . However, the protein bound the human IFN gamma receptor with native efficiency at 4 degrees C . Sequencing the plasmid DNA encoding this protein showed that the mutation changed the lysine residue at amino acid 43 to glutamic acid (IFN gamma/K43E) . Site-specific mutagenesis at amino acid 43 showed that this protein's phenotype resulted from positioning a negative charge at position 43 . Structural characterization of IFN gamma/K43E using CD demonstrated that the protein had native conformation at 25 degrees C, but assumed an altered conformation at 37 degrees C . IFN gamma/K43E in this altered conformation bound poorly to the IFN gamma receptor at 37 degrees C, providing a rationale for the mutant's decreased antiviral activity. Protein Eng, 1992 Apr, 5(3), 241 - 8 Phe496 and Leu497 are essential for receptor binding and cytotoxic action of the murine interleukin-4 receptor targeted fusion toxin DAB389-mIL-4; Lakkis F et al.; DAB389-mIL-4 is a murine interleukin-4 (mIL-4) diphtheria toxin-related fusion protein which has been shown to be selectively toxic to cells expressing the mIL-4 receptor . In this report, we have used site-directed and in-frame deletion mutagenesis to study the role of the putative C-terminal alpha-helix (helix E) of the mIL-4 component of DAB389-mIL-4 in the intoxication process . We demonstrate that deletion of the C-terminal 15 amino acids of the fusion toxin leads to loss of cytotoxicity . The substitution of Phe496 with either Pro, Ala or Tyr, results in a greater than 20-fold decrease in cytotoxic activity of the respective mutant fusion toxins . In addition, substitution of Leu497 with either Ala or Glu results in a similar loss of cytotoxic activity . All of these mutant forms of the mIL-4 fusion toxin demonstrate a significant decrease in binding affinity (Ki) to the mIL-4 receptor in a competitive radioligand binding assay . In marked contrast, however, the substitution of Asp495 with Asn results in a 4-fold increase in cytotoxic potency and binding affinity to mIL-4 receptor bearing cells in vitro. Protein Eng, 1992 Apr, 5(3), 235 - 40 Fluorescent properties of the Escherichia coli D-xylose isomerase active site; Jamieson AC et al.; The consequences of active site mutations of the Escherichia coli D-xylose isomerase (E.C . 5.3.1.5) on substrate binding were examined by fluorescence spectroscopy . Site-directed mutagenesis of conserved tryptophan residues in the E . coli enzyme (Trp49 and Trp188) reveals that fluorescence quenching of these residues occurs during the binding of xylose by the wild-type enzyme . The fluorescent properties of additional active site substitutions at His101 were also examined . Substitutions of His101 which inactivate the enzyme were shown to have altered spectral characteristics, which preclude detection of substrate binding . In the case of H101S, a mutant protein with measurable isomerizing activity, substrate binding with novel fluorescent properties was observed, possibly the bound pyranose form of xylose under steady-state conditions. Indian J Biochem Biophys, 1992 Apr, 29(2), 183 - 8 Partial amino acid sequence of sheep liver serine hydroxymethyltransferase and comparison of peptide maps of the enzyme from human, ox livers and Escherichia coli; Usha R et al.; A comparison of the tryptic peptide maps of serine hydroxymethyltransferase from sheep, human, ox livers and Escherichia coli revealed that the mammalian enzymes were similar, while the bacterial enzyme exhibited differences in the primary structure . N-terminus of the reduced carboxymethylated sheep liver enzyme was acetylated . Serine hydroxymethyltransferase was hydrolyzed with trypsin and fragments of peptides were separated by high performance liquid chromatography using a combination of gel permeation, reverse phase and ion-pair chromatography . The peptides were sequenced manually using the 4-N,N'-dimethyl aminoazobenzene-4'-isothiocyanate/phenyl isothiocyanate double coupling method . The tryptic peptides with 80% homology or above were aligned on the rabbit liver enzyme sequence. Indian J Biochem Biophys, 1992 Apr, 29(2), 148 - 53 Mechanism of ribosomal subunit association: oligodeoxynucleotides as probes; Agrawal RK et al.; From the kethoxal treatment data {Herr, W.; Chapman, N.M.; Noller, H.F . (1979) J . Mol . Biol . 130, 433-439} some regions of ribosomal RNAs are thought to be responsible for the association of 30S and 50S ribosomes of E . coli to form 70S ribosomes . In order to test this possibility about a dozen oligodeoxynucleotides complementary to the suspected regions of rRNAs were synthesised . Their association with ribosomes and naked rRNAs was tested by the gel filtration technique . In order to check the effects on the ribosomal subunit association or rRNA association either intact 30S and 50S ribosomes or naked 16S and 23S rRNAs were preincubated with the individual oligodeoxynucleotide and its effect was checked by density gradient centrifugation followed by UV absorbance monitoring . Some oligodeoxynucleotides interfered with either subunit association or 16S RNA and 23S RNA association, some with both . These data clearly indicate that RNA-RNA interaction plays the major role in ribosomal subunit association. Indian J Biochem Biophys, 1992 Apr, 29(2), 128 - 34 Molecular anatomy of the transcription complex of Escherichia coli during initiation; Chatterji D et al.; Transcription is the foremost event in gene expression in which the enzyme RNA polymerase copies the genetic information from DNA to RNA . Much of our understanding of this process have come from studies carried out in Escherichia coli . A faithful and efficient transcription machinery of E . coli can be reconstituted in vitro with purified RNA polymerase and promoter-containing DNA . It is generally believed that in E . coli and most other organisms, the control of gene expression lies with the initiation of transcription . In this review, an attempt has been made to understand the mechanistic details of the initiation of transcription from the structural point of view of the promoter and the RNA polymerase . Allosteric nature of the enzyme has also been discussed at the end. Endocrinol Jpn, 1992 Apr, 39(2), 203 - 7 Recognition of a 56 kDa protein in partially purified rat hepatic nuclear thyroid hormone receptor by anti-human c-erb A beta antibody; Ichikawa K et al.; Human beta thyroid hormone receptor (c-erb A beta protein) produced by an Escherichia coli expression system was purified by sequential column chromatography followed by electroelution from an electrophoresis gel and an antibody was prepared . The antibody recognized a 56 kDa protein band in a partially purified rat hepatic nuclear thyroid hormone receptor fraction on Western blotting . Although multiple bands appeared on Western blotting of crude rat hepatic receptor preparations, a 56 kDa band was the most prominent and preadsorption of the antibody by purified c-erb A protein resulted in almost complete disappearance of the 56 kDa band, indicating that the 56 kDa band was formed by a specific antigen-antibody interaction . Furthermore, the 56 kDa protein appeared to co-elute with 3, 5, 3'-triiodo-L-thyronine binding activity in hydroxylapatite, Sephacryl S-200, and DNA-cellulose column chromatography of rat hepatic nuclear receptor, and sequential column purification resulted in selective enrichment of the 56 kDa band . These results suggest that the 56 kDa protein may be the major component of the rat hepatic thyroid hormone receptor. Zhonghua Jie He He Hu Xi Za Zhi, 1992 Apr, 15(2), 86 - 8, 126-7 {Changes and significance of chemiluminescence of polymorphonuclear leukocytes in endotoxin-induced lung injury in conscious sheep}; Chen JK; The chemiluminescence (CL) of polymorphonuclear leukocytes and its relation with pulmonary microvascular permeability after endotoxin-induced lung injury in conscious sheep with lung lymph fistula were observed . Four hours after the injury the CL of PMNs increased from 0.27 cpm/PMN of baseline to 0.69 cpm/PMN (P < 0.05) . The increment of the CL had positive correlation with the increment of lung lymph flow or permeability index (r = 0.632 0.638 P < 0.05), suggesting that the increase of pulmonary microvascular permeability after the endotoxin injury had relation with the increase of the respiratory tract of PMNs. Jpn J Genet, 1992 Apr, 67(2), 85 - 95 Isolation and characterization of a new acetate-requiring mutant strain, ace-9, of Neurospora crassa; Santosa S et al.; A new acetate-requiring mutant strain of Neurospora crassa, ace-9, has been isolated . The mutant gene was mapped between nuc-2 and arg-12 on the right arm of the second linkage group . The ace-9 mutant strain shows very weak activity of pyruvate dehydrogenase complex (PDHC) . Three strains that show no activity of PDHC had already been found, i.e., ace-2, ace-3, and ace-4 . Thus the ace-9 is the fourth gene that causes the deficiency in PDHC activity by a mutation . Deficiency of PDHC activity in ace-9 strain seems to be due to defective E1 component, because (1) the activity of E1 component enzyme is very weak in ace-9 mutant strain, and (2) normal PDHC activity was resumed when a preparation of ace-9 was mixed with E1-E2 fraction of wild type or with E1 component of wild type E . coli . Difference in thermostability of both E1 component enzyme and PDHC between ace-9 and the wild type strains supports this conclusion. Genetika, 1992 Apr, 28(4), 39 - 45 {Cloning and complementation analysis of the Escherichia coli gpr locus, influencing DNA replication of certain lamdoid phages}; Samsonov VV et al.; Escherichia coli DNA fragments which suppressed gpr27 and gpr2 mutations in the earlier proposed gprA and gprB genes, respectively, were cloned within phage vectors . Mutations gpr2 and gpr27 restrict DNA replication of some lambdoid phages and are located in the region of dnaK, J genes . DNA-DNA hybridization showed that the cloned fragments correspond to the region where gpr mutations were genetically mapped and, in some cases, do not include dnaK, J genes . These results provide the evidence that gprA and gprB genes may be physically separated from dnaK, J genes. Biochimie, 1992 Apr, 74(4), 319 - 26 Structure-function correlations (and discrepancies) in the 16S ribosomal RNA from Escherichia coli; Brimacombe R; The published model for the three-dimensional arrangement of E coli 16S RNA is re-examined in the light of new experimental information, in particular cross-linking data with functional ligands and cross-links between the 16S and 23S RNA molecules . A growing body of evidence suggests that helix 18 (residues 500-545), helix 34 (residues 1046-1067/1189-1211) and helix 44 (residues 1409-1491) are incorrectly located in the model . It now appears that the functional sites in helices 18 and 34 may be close to the decoding site of the 30S subunit, rather than being on the opposite side of the 'head' of the subunit, as hitherto supposed . Helix 44 is now clearly located at the interface between the 30S and 50S subunits . Furthermore, almost all of the modified bases in both 16S and 23S RNA appear to form a tight cluster near to the upper end of this helix, surrounding the decoding site. Biochimie, 1992 Apr, 74(4), 307 - 17 Mapping the three-dimensional locations of ribosomal RNA and proteins; Scheinman A et al.; Seven regions of 16S rRNA have been located on the surface of the 30S ribosomal subunit by DNA hybridization electron microscopy in our laboratory . In addition, we have recently mapped the three-dimensional locations of an additional seven small ribosomal proteins by immunoelectron microscopy . The information from the direct mapping of the sites on rRNA has been incorporated into a model for the tertiary structure of 16S rRNA, accounting for approximately 40% of the total 16S rRNA . A novel structure, the platform ring, is proposed for a region of rRNA within the central domain . This structure rings the edges of the platform and includes regions 655-751 and 769-810 . Another region, the recognition complex, consists of nucleotides 500-545, and occupies a region on the exterior surface of the subunit, near the EF-Tu binding site . In addition, 19 of the 21 small subunit ribosomal proteins have been mapped by immunoelectron microscopy in our laboratory . In order to evaluate the reliability of