Escherichia coli

Eur Surg Res, 1992, 24(5), 298 - 301
Effect of activated protein C on impaired fibrinolysis in rats with obstructive jaundice; Uchino R et al.; We studied the effect of activated protein C (APC) on impaired fibrinolysis using a rat model in which disseminated intravascular coagulation (DIC) is induced by the intravenous injection of endotoxin in rats with obstructive jaundice . An intravenous injection of endotoxin in rats with obstructive jaundice resulted in pulmonary hemorrhages and a marked increase in the plasma levels of tissue-type plasminogen activator (t-PA) antigen and plasminogen activator inhibitor activity . Prophylaxis with APC before the injection of endotoxin resulted in a decrease of the number of lung hemorrhages and an accelerated release of t-PA antigen . Thus, DIC in obstructive jaundice may be due to impairment of fibrinolysis and an increased susceptibility of endothelial cells to endotoxin . APC may be effective as a treatment for patients with obstructive jaundice associated with DIC.

Environ Mol Mutagen, 1992, 20(4), 297 - 306
Assessment of oxidative DNA damage in the oxyR-deficient SOS chromotest strain Escherichia coli PQ300; Muller J et al.; The SOS chromotest is a simple short-term genotoxicity assay measuring the induction of gene sfiA in Escherichia coli K-12 . The recent availability of SOS tester strains with additional mutations in DNA repair or protection systems allows testing of DNA damaging compounds for genotoxic specificity . E . coli PQ300 differs from the standard SOS tester strain PQ37 in that it contains an additional mutation in gene oxyR that renders it more sensitive to oxidative genotoxins . The generation of reactive oxygen intermediates (ROI) by hydroperoxides (H2O2, t-butyl hydroperoxide, cumene hydroperoxide), gamma-radiation, glucose oxidase, and xanthine oxidase resulted in a more vigorous SOS response in strain PQ300 compared to strain PQ37 . PQ300 was also more sensitive than PQ37 for the detection of reducing agents such as ascorbic acid, cysteine, and glutathione, which also alter the redox status of the bacterial cells . However, intercalating agents (adriamycin, bleomycin, and mitomycin C) and the UV- and radiomimetic compound 4-nitroquinoline-1-oxide whose DNA damaging potential are known also to involve ROI did not show significant differences between strains PQ37 and PQ300 . It is concluded that the oxyR-deficient strain PQ300 is useful for detecting certain classes of genotoxins that change the oxidative/antioxidative balance of tester bacteria in the SOS chromotest.

Curr Eye Res, 1992, 11 Suppl, 113 - 7
Pathogenicity and immunogenicity of recombinant human retinal S-antigen fusion protein; Kasp E et al.; A full-length cDNA clone to human S-antigen (HS-ag) was isolated from lambda gt 10 human retinal library and expressed as a fusion protein with glutathione S-transferase (GST) in E . Coli . Uveitogenicity and immunogenicity of recombinant GST-HS-ag fusion protein and native HS-ag were compared in EAU-susceptible Lewis rats . Recombinant HS-ag was found less uveitogenic than native HS-ag . Animals inoculated with recombinant HS-ag developed EAU on day 17, three days later than those inoculated with native HS-ag, the incidence of the disease was reduced from 80% to 58% and the score of clinical severity reduced from 2.2 to 1.3 points respectively . In contrast, rGST-HS-ag was more immunogenic than native HS-ag as it elicited four times higher levels of antibodies which reacted specifically with both antigens.

J Biomol NMR, 1992 Jan, 2(1), 71 - 82
A 1H-15N NMR study of human c-Ha-ras protein: biosynthetic incorporation of 15N-labeled amino acids; Yamasaki K et al.; A 1H-15N NMR study was performed on the GDP-bound form of a truncated human c-Ha-ras oncogene product (171 amino acid residues) . Resonance cross peaks of the backbone amide 1H-15N nuclei of a uniformly 15N-labeled protein were observed with heteronuclear single-quantum coherence spectroscopy (HSQC) . In order to resolve overlapping cross peaks, selective 15N-labeling of one or two types of amino acid residues (Ala, Arg, Asx, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Tyr and/or Val) was carried out using appropriate E . coli mutant strains . By this procedure, all the backbone 1H-15N cross peaks were classified into amino acid types.

Anim Genet, 1992, 23(5), 437 - 41
Characterization of a porcine variable number tandem repeat sequence specific for the glucosephosphate isomerase locus; Davies W et al.; A variable number of tandem repeat from a porcine glucosephosphate isomerase intron has been isolated and sequenced . The repeat has a unit size of 39 bp, is highly conserved and is present in at least 14 copies . Flanking sequences show a sequence periodicity of 53-54 bp and some sequence homology to the 39 bp repeat . A considerable part of the genomic DNA has been lost during subcloning and is considered to be deletion prone or refractory to propagation in E . coli . The tandem repeat is locus specific and detects at least six alleles in BamHI digested porcine DNA . No homology to other tandem repeat sequences has been found.

Am J Nephrol, 1992, 12(1-2), 80 - 5
Inhibition of cytokine synthesis by peritoneal dialysate persists throughout the CAPD cycle; Jorres A et al.; The current study focused on the effect of continuous ambulatory peritoneal dialysis (CAPD) dialysate obtained following different intraperitoneal dwell periods on the release of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF alpha) from mononuclear leukocytes (PBMC) . Aliquots of 5 x 10(6)/ml healthy peripheral PBMC were exposed to fresh or spent CAPD dialysate (10-240 min of intra-peritoneal dwell) and stimulated with Escherichia coli endotoxin (10 micrograms/ml, 2h) . IL-6 and TNF alpha in cell supernatants were determined by specific enzyme immunoassays . Control PBMC in physiological buffer released 361 +/- 70 pg/ml IL-6 and 717 +/- 147 pg/ml TNF alpha (mean +/- SEM, n = 8), whereas exposure to fresh dialysis fluids severely suppressed cytokine release from PBMC (less than 30 pg/ml IL-6 and less than 15 pg/ml TNF alpha) . A significant inhibition of IL-6 and TNF alpha release was also observed in PBMC exposed to spent dialysate . The inhibitory capacity of the spent fluids was pronounced with increasing intra-peritoneal dwell time (10 min: 183 +/- 45 pg/ml IL-6 and 538 +/- 109 pg/ml TNF alpha; 240 min: 26 +/- 5 pg/ml IL-6 and 105 +/- 30 pg/ml TNF alpha; mean +/- SEM, n = 16) . These data indicate that the impairment of cell responsiveness following exposure of PBMC to peritoneal dialysate is not restricted to the unused fluids, but is also observed following intra-peritoneal equilibration . Moreover, our findings suggest the presence of cytokine inhibitory factors in the peritoneal dialysate of CAPD patients which appear to accumulate in the peritoneal effluent during the CAPD cycle.

Zh Mikrobiol Epidemiol Immunobiol, 1992 Jan, (1), 8 - 11
{Sterol transformation by Escherichia}; Panchishina MV; The use of the Liebermann-Burhard reaction and the thin-layer chromatography of nonsaponifiable lipids of culture medium (donor blood serum) permitted the isolation of three biological variants of Escherichia in the process of their 48-hour cultivation in this medium . The cholesterol-destroying variant of Escherichia is characterized by a decrease in the content of total, free, esterified cholesterol and a decrease in the occurrence of fractions corresponding to cholesterol, delta 4-cholestenone-3, delta 5-cholestenone-3), as well as nonsaponifiable lipid, where Rf was equal to 0.36; two fractions of labeled nonsaponifiable lipids, not corresponding to cholesterol, appeared on plasma with sodium acetate-1-14C . Cholesterol-transforming biovars produced insignificant changes in the content of chemically determined cholesterol in the medium, but in plasma nonsaponifiable lipid with Rf = 0.26 and other less polar lipids were found . Escherichia strains increasing the amount of chemically determined cholesterol in the process of their growth more frequently transformed or used nonsaponifiable lipids with Rf = 0.26 and 0.42 . As a rule, the occurrence of cholesterol and less polar lipids increased . The sodium acetate-1-14C was incorporated into 3-4 fractions of nonsaponifiable lipids, one of them being identified as cholesterol.

Ophthalmic Res, 1992, 24(3), 175 - 80
Aqueous humor polyamines and alkaline phosphatase activity in endotoxin-induced uveitis: correlations to diverse leukocyte subsets; Wickstrom K et al.; The polyamines putrescine, spermidine and spermine have been proposed to be a part of the acute phase inflammatory response . They have been shown to be useful markers for cellular kinetics and change with various pathological conditions . The hypothesis that aqueous humor polyamines could be used to follow the time course of an endotoxin-induced inflammation in the eye was investigated . Additional parameters studied were the amount of aqueous leukocytes, alkaline phosphatase activity, distribution of leukocyte subsets and breakdown of the blood-aqueous barrier . Aqueous leukocytes, protein, alkaline phosphatase activity, putrescine and acetylated spermidine increased significantly as a response to inflammation during the first days after uveitis induction . Spermidine decreased 24 h after injection and seemed to rise thereafter . The different polyamines, except spermidine, correlated to the diverse infiltrating leukocyte subsets . These observations indicate that aqueous polyamines may be applied as valid markers for inflammation in the eye.

J Clin Lab Anal, 1992, 6(4), 232 - 8
A new sensitive microplate assay of plasma endotoxin; Tamura H et al.; We have developed a microplate method for determining endotoxin in platelet-rich plasma-using Endospecy, an endotoxin-specific chromogenic Limulus test reagent . Nonspecific activators and inhibitors of the test were eliminated by exposing samples (5 microliters) to the alkali reagent consisting of KOH, CaCl2, Triton X-100, ethyleniminepolymer and N,N-bis(2-hydroxyethyl)glycine . The recoveries of various endotoxins were almost complete and not enhanced by dilution . The dose-response curve was linear over endotoxin concentrations of 2-400 pg/ml with good precision (C.V . less than 5.0%) . Normal human plasmas (n = 30) contained less than 5.0 pg/ml of endotoxin in reference to that of Escherichia coli 0111: B4 . All plasma samples with high concentration of endotoxin by a conventional method showed high values by the microplate assay as well . Since it does not require centrifugation, the new treatment allows the whole reactions to proceed on the same microplate . This permits us to apply the Limulus test to an automated assay system, making plasma endotoxin determination simpler and more rapid than a conventional test tube method.

Hereditas, 1992, 117(1), 1 - 9
RecA-like strand-transfer activity at the meiotic prophase in Bombyx mori; Wischmann B; An ATP-independent strand-transfer activity has been identified in nuclear extracts prepared from Drosophila tissue culture cells and isolated nuclei from Bombyx testes . Extraction of the activity from testes at larval stages where the majority of the cells were in meiotic prophase was only possible when the chromosome scaffold/synaptonemal complex was dissolved by addition of high concentrations of DTT (80 mM) . No cross reaction was detected when partly purified extracts were assayed with antibodies against E . coli RecA protein.

Cytogenet Cell Genet, 1992, 61(2), 128 - 31
A chromosome 1-specific DNA library from the domestic pig (Sus scrofa domestica); Miller JR et al.; Chromosomes were prepared from lymphocytes of a male domestic pig and flow-sorted on a dual-laser FACS . Twenty spots were observed, corresponding to the known pig karyotype of 18 pairs of autosomes plus the X and Y . DNA was isolated from 10,000 copies of the presumed chromosome 1 spot, restricted with Sau3A, ligated into the vector pGEM4z, and PCR amplified using universal primers; the products were then re-ligated into pUC18 . After transformation into Escherichia coli, 210,000 independent colonies were obtained, 5% of which contained only vector DNA . The average insert size of the library was 405 bp . Southern blotting revealed that 36% of the clones contained single-copy DNA and that the remainder contained moderately or highly repetitive DNA . Screening with a (CA)n probe revealed that roughly 1% of the clones contained microsatellite sequences . A bulk insert of the library was biotinylated by PCR and used as a probe for chromosomal in situ suppression hybridization to pig chromosomes, which confirmed that the library is specific for chromosome 1 . However, sequences from the centromeric and telomeric regions seem to be underrepresented in the library.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 1992, 10(2), 82 - 5
{Chemical synthesis and cloning of Plasmodium falciparum 45 peptide antigen gene}; Qiu MY et al.; We have synthesized a 162 bp gene of human Plasmodium falciparum hybrid peptide antigen by the solid-phase phosphoramidite method with ABI 381A DNA synthesizer . The gene encodes three fragments of the relative molecules 83 kDa, 55 kDa and 35 kDa merozoite-specific proteins and two CS repeats or four peptides . The gene with the designed two cohesive ends was divided into 8 fragments to be synthesized . All synthetic fragments were annealed and ligated with T4 DNA ligase to form double DNA chain . This synthetic gene was recombined with P-Blue script as vector and transformed into E . coli JM109 . The positive recombinants were screened out by dot hybridization and enzyme analysis . The DNA sequence analysis showed that the synthesized human Plasmodium falciparum hybrid peptide antigen gene was identical with the designed one . (Figs . 1-4).

Cancer Immunol Immunother, 1992, 35(5), 307 - 14
Significant antitumor effect of a synthetic lipid A analogue, DT-5461, on murine syngeneic tumor models; Kumazawa E et al.; The antitumor effect of a synthetic lipid A analogue, DT-5461, was investigated using syngeneic tumor models in mice . Intravenous injection of DT-5461 into mice transplanted with solid tumors of MethA fibrosarcoma, MH134 hepatoma, MM46 mammary carcinoma, Lewis lung carcinoma (3LL), and colon adenocarcinomas 26 and 38 resulted in significant reductions in the weight of all tumors except Colon 26, with marked hemorrhagic necrosis of tumor tissues . Efficacy was almost equal to that of an Escherichia coli-type synthetic lipid A (compound 506), and also to those of some chemotherapeutics including Adriamycin, mitomycin C, fluorouracil and cisplatin . Furthermore, DT-5461 was more effective than other immunotherapeutics, including picibanil (OK-432) and lentinan . However, its antitumor effects were inferior to those of Adriamycin or OK-432 against the malignant ascites caused by intraperitoneal inoculation with MethA or with MH134 cells; life span was not prolonged by either intraperitoneal or intravenous administration . In addition, although DT-5461 showed direct inhibitory effects on the in vitro growth of MethA or MH134, these were much weaker than those of Adriamycin . These findings clearly indicated that DT-5461 with systemic administration is a highly effective antitumor agent on solid tumors, and suggest that the antitumor effect of DT-5461 with potent necrotizing activity might derive from indirect mechanisms related to the activation of host immune systems and not to the weak direct cytotoxicity.

Biometals, 1992 Spring, 5(1), 37 - 46
Identification of the ferrioxamine B receptor, FoxB, in Escherichia coli K12; Nelson M et al.; The photoreactive p-azidobenzoyl analog of ferrioxamine B was used to show that ferrioxamine-B-mediated iron transport is separate and distinct from coprogen-mediated iron transport in Escherichia coli . Photolysis of this analog inhibited uptake of {59Fe}ferrioxamine B but not {59Fe}ferrichrome . Conversely, photolysis of the p-azidobenzoyl analog of coprogen B inhibited uptake of {59Fe}coprogen but not {59Fe}ferrioxamine B or {59Fe}ferrichrome . Photolabeling of outer membranes with p-azidobenzoyl-{59Fe}ferrioxamine B resulted in the labeling of two iron-regulated peptides with molecular masses of about 66 and 26 kDa . Expression of these peptides was increased when ferrioxamine B was the sole iron source . Both peptides were present in outer membrane preparations of the fhuF mutant H1717, but the 66 kDa peptide was not inducible . These results are evidence for an outer membrane receptor in E . coli unique for linear ferrioxamines.

C R Acad Sci III, 1992, 315(1), 1 - 6
{Total chemical synthesis of natural transfer RNA}; Gasparutto D et al.; New improvements in the chemical synthesis of oligoribonucleotides are reported and they are applied to the first total chemical synthesis of a natural RNA . This E . coli K12 alanine tRNA contains in its sequence dihydrouridine, ribothymidine and pseudo-uridine . The synthetic tRNA was fully sequenced and showed a 42% aminoacyl acceptance activity . When tRNA was used as a template, reverse transcriptase directed the incorporation of adenine opposite dihydrouridine, ribothymidine and pseudouridine.

Cancer Immunol Immunother, 1992, 35(6), 395 - 400
Effects of lipopolysaccharide on interleukin-2-induced cytotoxic activity of murine splenocyte cultures: role of prostaglandin E2 and interferons; Vaillier D et al.; Splenocytes cultured for 24 h in the presence of interleukin-2 (IL-2), lipopolysaccharide (LPS) or both together expressed a cytotoxic activity against the YAC-1 lymphoma cell line and to a lesser extent against P815 mastocytoma cells . The association of IL-2 and LPS had an additive effect on induction of cytotoxicity . The IL-2-induced cytotoxic activity lasted for the whole of the culture; however, the addition of LPS at the initiation of the culture increased the cytotoxic activity during its the early phase, the increment being followed by a fall of lytic activity after 72 h of culture . Assessment of interferon (IFN) in the culture supernatants showed (a) a production of IFN gamma by IL-2-supplemented cultures, (b) a more potent IFN production by cultures treated with IL-2 plus LPS (including 20% IFN alpha/beta, (c) and that indomethacin (1 microM) potentiated the effect of either IL-2 or LPS used alone but did not significantly increase the cytotoxic activity of cultures treated with IL-2 plus LPS (the one that produced a high level of IFN) . When cultures were treated by an anti-IFN gamma antibody we observed no change in the cytotoxic activity; however, in the presence of anti-IFN alpha/beta serum the cytotoxic activity of cultures treated with IL-2 plus LPS was inhibited after 24 h but stimulated after 72 h . When cultures treated with IL-2 plus LPS were supplemented with both indomethacin and anti-IFN alpha/beta the cytotoxic activity assessed after 72 h of culture was maintained at the same level as that of IL-2-treated cultures, hence the fall after 72 h of the cytotoxicity of cultures initiated in the presence of LPS alone was affected by both the immune serum and the cyclooxygenase inhibitor . Altogether these data show that when splenocytes are cultured for more than 72 h in the presence of IL-2 and LPS their cytotoxic activity decreases, and it is likely that this diminution is linked to the endogenous production of prostaglandin E2 and INF alpha/beta.

Biomater Artif Cells Immobilization Biotechnol, 1992, 20(2-4), 1051 - 7
Inhibition of endotoxin-mediated activation of endothelial cells by a perfluorocarbon emulsion; Lane T et al.; Endothelial cell (EC) activation plays a key role in the inflammatory response by promoting the margination of leukocytes in inflamed loci . Augmented leukocyte margination to activated EC is mediated by the increased display of leukocyte adhesion molecules on EC surface membranes . The biocompatibility of synthetic oxygen-transport fluids is intimately linked to EC function, since one of the first tissues encountered by such fluids is the vascular endothelium . We investigated the effect of one such agent, a phospholipid-based perfluorocarbon emulsion containing 90% w/v perflurooctyl bromide (perflubron, PFOB) on EC activation . Human umbilical vein EC (HUVEC) were activated by 5 U/ml interleukin-1 (IL-1), 20 U/ml tumor necrosis factor (TNF), or 50 ng/ml E coli endotoxin (LPS) in the presence or absence of up to 20%, w/v perflubron . HUVEC activation was monitored by the extent of up-regulation of expression of intercellular adhesion molecule-1 (ICAM) and endothelial-leukocyte adhesion molecule-1 (ELAM) . Exposure of HUVEC to perflubron did not alter the up-regulation of ICAM or ELAM in response to IL-1 or TNF (n = 20) . However, at 10% perflubron ICAM up-regulation in response to LPS was inhibited by 95 +/- 6% (n = 9; p less than .05) . ELAM expression was similarly affected . The concentration of perflubron required to diminish LPS-induced up-regulation by 50% was 6.0 +/- 0.6% (n = 3) . The inhibitory effect of 10% perflubron was overcome by greater than 1 ug/ml LPS (n = 3) and the inhibitory effect was attenuated by adding perflubron to the cultures after LPS.(ABSTRACT TRUNCATED AT 250 WORDS)

Annu Rev Biophys Biomol Struct, 1992, 21, 379 - 415
The single-nucleotide addition cycle in transcription: a biophysical and biochemical perspective; Erie DA et al.; This review has summarized the known features of the single-nucleotide addition reaction cycle in transcription . The reader will have noted that the information available is very incomplete, and that, in some cases, related experiments seem to lead to contradictory conclusions . We have tried to point out these discrepancies as they occur and to indicate areas where more experimentation is needed . We look forward to the day when all the microscopic steps of the single-nucleotide addition cycle can be identified and defined in thermodynamic, kinetic, and structural terms . At that point, we can begin to understand the principles that relate these parameters to template position and to the pathway of formation of a specific complex . It should be possible to provide specific molecular interpretations for observed effects on activation barrier heights to elongation and termination (154, 155) and to begin to understand the molecular bases of the regulation in these phases of transcription . Much work remains before this happy situation can be totally realized, but we feel that now the problem can at least be approached at this level . We hope that this review helps to illuminate the difficulties that remain.

Bioorg Khim, 1992 Jan, 18(1), 71 - 7
{Chemical-enzymatic synthesis and cloning in Escherichia coli of a gene coding for human granulocyte-colony stimulating factor}; Kash'ian SK et al.; Two artificial genes, encoding two forms of human granulocyte colony stimulating factor as products of a normal and an alternative splicing, have been by a chemical-enzymatic way synthesized and cloned in Escherichia coli . The genes are supplied with recognition sites of restriction endonucleases to facilitate the further cassette mutagenesis.

Arch Virol, 1992, 126(1-4), 321 - 8
Immunogenicity of recombinant core particles of hepatitis B virus containing epitopes of human immunodeficiency virus 1 core antigen; Ulrich R et al.; A Gag protein segment of human immunodeficiency virus 1 (HIV-1) has been fused to a C terminally truncated core antigen of hepatitis B virus (HBcAg) using an E . coli expression system . Fusion of 90 amino acids of HIV-1 Gag protein to HBcAg still allowed the formation of capsids presenting on their surface epitopes of HIV-1 core protein, whereas fusion of 317, 189, or 100 amino acids of Gag prevented self-assembly of chimeric particles . Mice immunized with recombinant particles emulsified with Freund's complete adjuvant (CFA) or aluminium hydroxide developed high anti-HBcAg titers . However, anti-HIVp24 antibodies were detected only in mice inoculated with immunogen emulsified with CFA.

Mutat Res, 1992, 280(3), 205 - 14
Genotoxicity assessment of pirmenol, a new antiarrhythmic drug; Ciaravino V et al.; The genotoxicity of pirmenol was tested in the E . coli and S . typhimurium mutagenesis assay, an in vitro mammalian cell chromosome-aberration assay and an in vivo mouse micronucleus assay . The E . coli tester strain WP2s was exposed to concentrations of pirmenol as high as 10,000 micrograms/plate both in the absence (S9-) and presence (S9+) of metabolic activation . Five strains of S . typhimurium (TA98, TA100, TA1535, TA1537, TA1538) were exposed to concentrations of pirmenol as high as 5000 micrograms/plate in the absence and presence of S9 . Pirmenol was not mutagenic toward either E . coli or S . typhimurium . Chinese hamster lung V79 cell cultures were exposed to pirmenol at concentrations of 500-2500 micrograms/ml (S9-) and 500-3000 micrograms/ml (S9+) . Pirmenol increased the frequency of structural chromosome aberrations (SCAs) . The minimum clastogenic concentration was 1500 micrograms/ml (both S9- and S9+) with a peak clastogenic response of 6% (S9-) and 34% (S9+) cells with aberrations . Although there were statistically significant results in the S9- experiment, the percent cells with aberration values for treated groups were within the historical control range (0-6%) of this laboratory . The observed effects in both the absence and presence of S9 appear at high concentrations compared to human circulating plasma levels of 1-3 micrograms/ml and the clastogenicity was confined to chromosome gaps and breaks . Consequently, this in vitro effect would not be expected to be reflected by either in vivo clastogenic or carcinogenic activity . This was supported by findings in the mouse micronucleus study of pirmenol in which single oral doses administered to male CD-1 mice at 5, 55, or 115 mg/kg (80% LD50) produced no statistically significant increases in the frequency of micronucleated polychromatic erythrocytes in bone marrow at 24, 48 or 72 h postdosing . Additionally, no evidence of carcinogenicity was seen in a mouse or rat bioassay.

Mutat Res, 1992, 280(3), 195 - 203
The genotoxicity of organotin compounds in SOS chromotest and rec-assay; Hamasaki T et al.; In these days pollution by organotin compounds in the environment extends widely and effects on human health are feared . We studied the genotoxicity of various organotin compounds (butyltins, phenyltins, methyltins) and inorganic tin (SnCl4), which are present in the environment, with the SOS chromotest and the rec-assay . Mono-n-butyltin oxide, n-butyltin trichloride and di-n-butyltin dichloride showed high SOS-inducing potency in the SOS chromotest with Escherichia coli PQ37 . Di-n-butyltin dichloride, tri-n-butyltin chloride, bis(tri-n-butyltin)oxide, dimethyltin dichloride and trimethyltin chloride were recognized as genotoxic chemicals by the rec-assay.

Mol Biol (Mosk), 1992 Jan-Feb, 26(1), 70 - 82
{Cloning and expression in Escherichia coli of reverse transcriptase coded by the mobile genetic element jockey}; Ivanova VA et al.; The mobile element jockey is similar in structural organization and coding potential to the LINEs of various organisms . Current models of the mechanism of transposition involve reverse transcription of an RNA intermediate and utilization of element-encoded proteins . As it is demonstrated here, a 2.23 kb DNA fragment from the region of the jockey encoding the putative reverse transcriptase, was stably introduced into the expression system under inducible control of the Escherichia coli lac regulatory elements . We describe the expression of the 92 kDa protein and identify this polypeptide alone as authentic jockey reverse transcriptase based on some of its physical and enzymic properties . The jockey polymerase demonstrates RNA-directed and DNA-directed DNA polymerase activities, but lacks detectable RNase H, has a temperature optimum at 26 degrees C, requires Mg2+ or Mn2+ as a cofactor and is inactivated by sulfhydryl reagent . The enzyme prefers poly(rC) and poly(rA) as template and "activated" DNA is not effective . The results of this work suggest that the RNA-directed DNA polymerase coded by jockey elements may be involved in the transcription of the elements.

Environ Mol Mutagen, 1992, 20(2), 127 - 33
Effects of Captan on DNA and DNA metabolic processes in human diploid fibroblasts; Snyder RD; The fungicide Captan has been examined for its effects on DNA and DNA processing in order to better understand the genotoxicity associated with this agent . Captan treatment resulted in production of DNA single strand breaks and DNA-protein cross-links and elicited an excision repair response in human diploid fibroblasts . Captan was also shown to inhibit cellular DNA synthesis and to form stable adducts in herring sperm and human cellular DNA . Misincorporation of nucleotides into Captan-treated synthetic DNA templates was significantly elevated in an in vitro assay using E . coli DNA polymerase I, suggesting that DNA adduct formation by Captan could have mutagenic consequences . In sum, these studies demonstrate that Captan is capable of interacting with DNA at a number of levels and that these interactions could provide the basis for Captan's genotoxicity . The extreme cytotoxicity of this fungicide, however, could be due to other cellular effects since at the IC50 for cell killing, approximately 0.8 microM, none of the above genotoxic events could be detected by the methods employed.

Mutagenesis, 1992 Jan, 7(1), 41 - 6
Mutagenesis of Escherichia coli: a method for determining mutagenic specificity by analysis of tRNA suppressors; Sledziewska-Gojska E et al.; A method for estimating mutagenic specificity in Escherichia coli (argE3, hisG4, thr-1, supE44), based upon the isolation of Arg+ or His+ revertants and identification of tRNA suppressors, is described . The method gives an insight not only into mutagenic pathways but also into the functioning of tRNA . With N-methyl-N'-nitro-N-nitrosoguanidine, 98% of mutations are GC----AT transitions . With N4-hydroxycytidine, 100% are AT----GC transitions . With hydroxylamine, apart from GC----AT transitions, approximately 30% of Arg+ revertants are formed by GC (or AT)----TA transversions . When the chemistry of the mutagenic attack is known, the method allows us to discriminate whether mutations occur on the transcribed or non-transcribed strands of DNA . It has been found that reversion of argE3 to Arg+ is a better monitor of mutagenic pathways than reversion of hisG4 to His+.

Genetica, 1992, 85(2), 107 - 17
A nitrate reductase gene of the cyanobacterium Synechococcus PCC6301 inferred by heterologous hybridization, cloning and targeted mutagenesis; Lightfoot DA et al.; DNA probes from the narG gene of Escherichia coli, which encodes the large polypeptide of respiratory nitrate reductase, show cross-hybridization at low stringency to a single region of the genome of the cyanobacterium Synechococcus PCC6301 . This segment of cyanobacterial DNA was cloned as the insert of plasmid pDN1 and characterized . RNA complementary to pDN1 was shown to be substantially more abundant in nitrate grown cells of Synechococcus PCC6301 than in ammonium grown cells, thus parallelling the nitrate induction and ammonium repression of nitrate reductase activity in cultures of this cyanobacterium . A mutant of Synechococcus PCC6301 deficient in nitrate reductase activity was obtained after a potentially mutagenic transformation treatment using pDN1 as a donor . This mutant was restored to the wild type phenotype following stable integrative transformation with pDN1 DNA . Taken together these data suggest that pDN1 might encode a polypeptide of nitrate reductase . pDN1 is distinct from three clones of genes involved in nitrate assimilation that were isolated previously from the related cyanobacterium Synechococcus PCC7942 (Kuhlemeier et al., 1984a, J . Bact . 159, 36-41, and 1984b, Gene 31, 109-116).

J Acquir Immune Defic Syndr, 1992, 5(7), 647 - 57
Epitope mapping of HIV-1 reverse transcriptase with monoclonal antibodies that inhibit polymerase and RNase H activities; Szilvay AM et al.; Lysates from E . coli expressing HIV-1 reverse transcriptase (RT) as a TrpE fusion protein were used for immunization of BALB/c mice . Twenty hybridomas producing monoclonal antibodies (MAbs) recognizing the RT part of the TrpE-RT fusion protein by Western blot analysis were isolated . Of these, 18 were reactive in immunofluorescence assays when tested on HIV-infected cells . Twelve MAbs were reactive with both the p66 and p51 fragments of RT, while 6 of the MAbs were reactive only with the p66 band, indicating specificity for the C-terminal (RNase H) region of RT . Mapping of the monoclonal antibody binding sites was performed using deletion and insertion mutants of recombinant RT . The antibodies bound to five distinct regions within amino acid sequences 190-560 of RT . In order to map functionally important regions of the RT molecule, the MAbs were tested for their ability to interfere with the polymerase and RNase H activities of the polypeptide . MAbs binding to two different epitopes in the polymerase domain were found to inhibit the polymerase activity . Of these, three MAbs also inhibited the RNase H activity . Two MAbs binding to the same epitope in the RNase H region inhibited RNase H activity and further mediated an effect on the polymerase activity.

Autoimmunity, 1992, 11(3), 141 - 9
Mapping epitope specificities of monoclonal antibodies to thyroid peroxidase using recombinant antigen preparations; Ewins DL et al.; Five separate monoclonal antibodies (MoAbs) to human thyroid peroxidase (hTPO) were raised by immunising Balb/c mice with hTPO purified from detergent solubilised thyroid microsomes by high performance liquid chromatography (HPLC) . The epitope specificities of these MoAbs were determined by assessing their ability to bind to purified recombinant fusion protein fragments of human TPO (TPO(r)) generated in E . coli . A total of seven small overlapping fragments (averaging 104 amino acid residues) of hTPO, encompassing over 90% of the extracellular region of the molecule, were generated as glutathione S-transferase (GST) fusion proteins . The sequential epitopes on TPO(r) recognised by these MoAbs were analysed by both immunoblotting and enzyme linked immunosorbent assay (ELISA) . Two different MoAbs (A4 and A5) recognised sequential epitopes within the TPO(r) preparation termed R1a + b (residues 1-160) and more specifically, in the case of MoAb A4, within the subfragment R1b (residues 70-160) . The inability of the other MoAbs (A1-A3) to recognise recombinant fragments, suggests they either recognise conformational determinants on the TPO molecule or epitopes that are present on the small regions of the TPO molecule which have not been expressed as recombinant proteins.

Mol Microbiol, 1992 Jan, 6(2), 247 - 55
Characterization of the antigenic and adhesive properties of FaeG, the major subunit of K88 fimbriae; Bakker D et al.; The two K88 serotypes, K88ab and K88ac, differ in terms of antigenic and adhesive properties . The structural determinants of the serotype-specific epitopes and the identify of the amino acid residues involved in fimbriae-receptor interaction were studied by the construction and analysis of K88 hybrid proteins in which various parts of the K88ab and K88ac fimbrial subunit FaeG were exchanged, and by in vitro mutagenesis of non-conserved amino acid residues . Using a set of monoclonal antibodies, several regions or amino acid residues involved in the formation of serotype-specific antigenic determinants were located . The haemagglutinating activity of the hybrid and mutant proteins revealed several amino acid residues involved in the formation of the receptor binding site . A clear correlation was found between the receptor binding site and the serotype-specific antigenic determinants.

Am J Vet Res, 1992 Jan, 53(1), 36 - 43
Development of monoclonal antibody ELISA for simultaneous detection of bovine coronavirus, rotavirus serogroup A, and Escherichia coli K99 antigen in feces of calves; Thorns CJ et al.; A rapid ELISA was developed for simultaneous detection of bovine coronavirus (BCV), rotavirus (RV) serogroup A, and Escherichia coli K99 antigen in feces of calves . A mixture of 3 monoclonal antibodies specific for BCV, RV, or K99 was used successfully to capture the antigens; the same antibodies labeled with peroxidase were used to detect BCV, RV, or K99 . The triple ELISA was compared with standard reference diagnostic methods by examining feces from experimentally and naturally infected and healthy calves . All the components of the test were highly specific (greater than 90%) and sensitive (BCV, 77%; K99, 93%; RV, 100%) when used in a format requiring short incubation steps at 20 C and visual recording of results.

Mutat Res, 1992 Jan, 281(1), 63 - 6
Inducible stable DNA replication in Escherichia coli uvr+ and uvr- cells, treated with genotoxic chemicals; Masek F et al.; Inducible stable DNA replication (iSDR) provoked by a damaging treatment with MMS, MNU, MNNG, NFAA, NFN, 4NQO, NAL or MMC, was followed in both repair-competent E . coli PQ35 and its uvrA derivative E . coli PQ37 . In contrast to SOS-inducible mutagenesis, which is more pronounced in excision-deficient cells, iSDR was more obvious in repair-competent cells . This may be due to special features of iSDR and need not indicate involvement of the uvrA gene product in it.

Biotechniques, 1992 Jan, 12(1), 104 - 13
Sensitive chemiluminescent detection of digoxigenin-labeled nucleic acids: a fast and simple protocol and its applications; Holtke HJ et al.; A fast and simple protocol for the chemiluminescent detection of digoxigenin-labeled nucleic acids with anti-digoxigenin antibody Fab fragments coupled to alkaline phosphatase and 3-(4-methoxyspiro{1,2-dioxetane-3,2'-tricyclo-{3.3.1.1 (3,7)}decan}-4- yl)phenyl phosphate as substrate is described . The washing and blocking procedure was optimized to yield low background even on positively charged nylon membranes . The sensitivity of the system is equal or better than radioactive methods . Exposure to x-ray or Polaroid film for up to 30 minutes is sufficient for the detection of 70 femtograms of homologous DNA . Human single-copy genes are detected in Southern blots of as low as 0.3 microgram total placental DNA . Blots can be reprobed multiple times very easily . The advantages of the digoxigenin system are high sensitivity, absence of background and ease of reprobing and are illustrated by applications for single-copy gene detection in genomic blots of human DNA, Northern hybridizations to rare mRNA, detection of E . coli genes on blots of genomic digests after pulse field gel electrophoresis, as well as for nonradioactive DNA sequencing blots with digoxigenin-labeled primers.

J Exp Med, 1992 Jan 1, 175(1), 245 - 55
The rat c-kit ligand, stem cell factor, induces c-kit receptor-dependent mouse mast cell activation in vivo . Evidence that signaling through the c-kit receptor can induce expression of cellular function; Wershil BK et al.; Interactions between products of the mouse W locus, which encodes the c-kit tyrosine kinase receptor, and the Sl locus, which encodes a ligand for c-kit receptor, which we have designated stem cell factor (SCF), have a critical role in the development of mast cells . Mice homozygous for mutations at either locus exhibit several phenotypic abnormalities including a virtual absence of mast cells . Moreover, the c-kit ligand SCF can induce the proliferation and maturation of normal mast cells in vitro or in vivo, and also can result in repair of the mast cell deficiency of Sl/Sld mice in vivo . We now report that administration of SCF intradermally in vivo results in dermal mast cell activation and a mast cell-dependent acute inflammatory response . This effect is c-kit receptor dependent, in that it is not observed when SCF is administered to mice containing dermal mast cells expressing functionally inactive c-kit receptors, is observed with both glycosylated and nonglycosylated forms of SCF, and occurs at doses of SCF at least 10-fold lower on a molar basis than the minimally effective dose of the classical dermal mast cell-activating agent substance P . These findings represent the first demonstration in vivo that a c-kit ligand can result in the functional activation of any cellular lineage expressing the c-kit receptor, and suggest that interactions between the c-kit receptor and its ligand may influence mast cell biology through complex effects on proliferation, maturation, and function.

J Clin Invest, 1992 Jan, 89(1), 327 - 31
The susceptibility sequence to rheumatoid arthritis is a cross-reactive B cell epitope shared by the Escherichia coli heat shock protein dnaJ and the histocompatibility leukocyte antigen DRB10401 molecule; Albani S et al.; Immunological responses to bacterial heat shock proteins have been implicated in the pathogenesis of arthritis in animals and humans . The predicted amino acid sequence of dnaJ, a heat shock protein from Escherichia coli, contains an 11-amino acid segment that is homologous to the third hypervariable region of the human histocompatibility antigen (HLA) DRB10401 (formerly known as HLA Dw4), the part of the molecule that carries susceptibility to rheumatoid arthritis . To test the biological significance of this finding, we expressed and purified recombinant dnaJ (rdnaJ), and determined its immunologic cross-reactivity with HLA DRB10401 . A rabbit antipeptide antiserum raised against the sequence of the third hypervariable region of HLA DRB10401 specifically bound to 'dnaJ, thus confirming that a similar sequence is expressed on the bacterial protein . Of greater consequence, an antiserum to the 'dnaJ protein recognized not only a peptide from the third hypervariable region of HLA DRB10401, but also the intact HLA DRB10401 polypeptide . Furthermore, the antibody to 'dnaJ reacted with HLA DRB10401 homozygous B lymphoblasts, but not with HLA DRB11501, DRB10101, DRB10301, and DRB10701 (formerly known as HLA Dw2, DR 1, DR 3, and DR 7, in the same order) homozygous cells . These results demonstrate that exposure to a bacterial heat shock protein can elicit antibodies against the rheumatoid arthritis susceptibility sequence in the third hypervariable region of HLA DRB10401.

J Bacteriol, 1992 Jan, 174(2), 627 - 9
The gene for a 4.5S RNA homolog from Mycoplasma pneumoniae: genetic selection, sequence, and transcription analysis; Simoneau P et al.; In an effort to make an inventory of the tRNA genes of Mycoplasma pneumoniae, a DNA fragment was found to contain a sequence that can be folded into a hairpin structure very similar to that of the 4.5S RNA of Escherichia coli . Recombinant plasmids carrying this region were able to complement E . coli strains that were deficient in 4.5S RNA . S1 mapping showed that the mature transcript is only 79 nucleotides long.

J Bacteriol, 1992 Jan, 174(1), 263 - 8
Location of the basal disk and a ringlike cytoplasmic structure, two additional structures of the flagellar apparatus of Wolinella succinogenes; Schuster SC et al.; The basal body of Wolinella succinogenes consists of a central rod, a set of two rings (L and P rings), a basal disk from 70 to 200 nm in diameter, and a terminal knob . In negatively stained preparations of flagellar hook-basal body complexes, some disks remain fixed perpendicularly to the grid and show that such a disk is located on the distal side of the P ring . The basal disks have been isolated with and without the P ring; in both cases there is a hole in the center of the disk . The diameter of the disk is smaller in the presence of the P ring . The L-P ring complex is therefore assumed to be a bushing for the rod . Thin sections of whole bacteria and spheroplasts reveal that the disk is attached to the inner surface of the outer membrane . At the insertions of the flagellar hook-basal body-basal disk complexes, depressions are visible in negatively stained preparations of whole bacteria and spheroplasts . A new ringlike structure is connected to an elongation of the basal body into the cytoplasm in both preparations . Its diameter (60 nm) is larger than that of the M ring . A heavily stained compartment can be seen in between the new ringlike structure and the basal disk, which may be formed by the energy transducing units.

J Immunol, 1992 Jan 1, 148(1), 218 - 24
Epitopes on the outer surface protein A of Borrelia burgdorferi recognized by antibodies and T cells of patients with Lyme disease; Shanafelt MC et al.; We have characterized immunogenic epitopes of the 31-kDa outer surface protein A (OspA) protein of Borrelia burgdorferi, which is a major surface Ag of the spirochete causing Lyme disease . Full length and truncated forms of rOspA proteins were expressed in Escherichia coli, and their reactivities with antibodies and human T cell clones isolated from patients with Lyme disease were determined . The epitopes recognized by three of four OspA-reactive T cell clones are contained within the 60 COOH-terminal amino acids . Each of the four OspA-reactive T cell clones has a different HLA class II molecule involved in Ag recognition and recognizes a distinct epitope . One T cell clone promiscuously recognized an epitope in the context of different HLA-DQ molecules . In addition, the binding of a murine monoclonal anti-OspA antibody, as well as antibodies in sera of three of five patients with Lyme disease, was dependent upon the amino acids in the carboxy-terminal protion of this protein . Taken together, our results indicate that the 60 COOH-terminal amino acids of OspA contain epitopes recognized by human antibodies and T cells.

J Virol, 1992 Jan, 66(1), 608 - 11
A CD4+ cytotoxic T-lymphocyte clone to a conserved epitope on human immunodeficiency virus type 1 p24: cytotoxic activity and secretion of interleukin-2 and interleukin-6; Littaua RA et al.; A CD4+ cytotoxic T-lymphocyte (CTL) clone, established from the peripheral blood of a human immunodeficiency virus (HIV)-seropositive donor, lysed autologous target cells that were infected with a recombinant vaccinia virus containing the gag gene of HIV type 1 and target cells pulsed with p24gag construct expressed in Escherichia coli . The recognition of the HLA-DQ-restricted epitope by this clone was further defined by using overlapping synthetic peptides . The epitope recognized by this CD4+ CTL clone (amino acids 140 to 148) overlaps with a CD8+ epitope and is highly conserved among all isolates of HIV type 1 that have been sequenced . Production and secretion of lymphokines such as interleukin-2 and interleukin-6 after specific antigenic stimulation were demonstrated by this gag-specific CD4+ CTL clone.

J Virol, 1992 Jan, 66(1), 317 - 24
Colocalization of adeno-associated virus Rep and capsid proteins in the nuclei of infected cells; Hunter LA et al.; The mechanism of adeno-associated virus (AAV) DNA replication was characterized both genetically and biochemically . In this study, we used monoclonal and polyclonal antibodies to examine the AAV p5 (Rep78 and Rep68) and p19 (Rep52 and Rep40) proteins in infected cells . By overexpressing a truncated Rep78 protein in Escherichia coli, we obtained monoclonal antibody anti-78/68, which is specific for the p5 Rep proteins, and monoclonal antibody anti-52/40, which recognized both the p5 and p19 Rep proteins . In single-fluorochrome indirect immunofluorescence labeling experiments, the viral Rep proteins were localized in distinct intranuclear foci . Analysis of AAV proteins by double-fluorochrome indirect immunofluorescence experiments demonstrated that (i) all four AAV Rep proteins occupied the same intranuclear compartments and (ii) the Rep and capsid proteins colocalized in the nuclei of infected cells . These results suggest that replication centers similar to those established by other viruses exist for AAV . These reagents should provide a useful tool for further delineation of the mechanism of AAV replication in vitro.

J Virol, 1992 Jan, 66(1), 106 - 14
The position of heterologous epitopes inserted in hepatitis B virus core particles determines their immunogenicity; Schodel F et al.; The nucleocapsid (HBcAg) of the hepatitis B virus (HBV) has been suggested as a carrier moiety for vaccine purposes . We investigated the influence of the position of the inserted epitope within hybrid HBcAg particles on antigenicity and immunogenicity . For this purpose, genes coding for neutralizing epitopes of the pre-S region of the HBV envelope proteins were inserted at the amino terminus, the amino terminus through a precore linker sequence, the truncated carboxy terminus, or an internal site of HBcAg by genetic engineering and were expressed in Escherichia coli . All purified hybrid HBc/pre-S polyproteins were particulate . Amino- and carboxy-terminal-modified hybrid HBc particles retained HBcAg antigenicity and immunogenicity . In contrast, insertion of a pre-S(1) sequence between HBcAg residues 75 and 83 abrogated recognition of HBcAg by 5 of 6 anti-HBc monoclonal antibodies and diminished recognition by human polyclonal anti-HBc . Predictably, HBcAg-specific immunogenicity was also reduced . With respect to the inserted epitopes, a pre-S(1) epitope linked to the amino terminus of HBcAg was not surface accessible and not immunogenic . A pre-S(1) epitope fused to the amino terminus through a precore linker sequence was surface accessible and highly immunogenic . A carboxy-terminal-fused pre-S(2) sequence was also surface accessible but weakly immunogenic . Insertion of a pre-S(1) epitope at the internal site resulted in the most efficient anti-pre-S(1) antibody response . Furthermore, immunization with hybrid HBc/pre-S particles exclusively primed T-helper cells specific for HBcAg and not the inserted epitope . These results indicate that the position of the inserted B-cell epitope within HBcAg is critical to its immunogenicity.

Bioseparation, 1992-93, 3(6), 343 - 58
Mathematical modelling and simulation of aqueous two-phase continuous protein extraction; Mistry SL et al.; A mathematical model has been developed to describe the continuous, steady-state operation of an aqueous two-phase system for protein extraction . The model is based on steady-state mass balances of the main components and phase equilibrium data . Experimental data on the separation of thaumatin from contaminant proteins of an homogenate of E . coli in a PEG4000/Phosphate system was used . The data shows the effect of the presence and absence of NaCl which was used to carry out the extraction of thaumatin into the PEG phase and back into the PO4(-3) phase . Simulation results showing the sensitivity to key process parameters, and the effect of process variables on performance are presented and discussed . The model can be used to predict performance and thus 'robustness' of process conditions as well as predict protein recovery yield and purity . This model can also be used to implement a suitable control strategy to maintain process stability.

Bioseparation, 1992-93, 3(5), 285 - 9
Shear thickening of DNA in SDS lysates; Stephenson D et al.; Cells of Escherichia coli were subjected to lysis by an alkali detergent treatment (sodium dodecyl sulphate) . The rate of detergent lysis was not first order . The rheological properties of the lysate were measured using a controlled stress rheometer . Detergent-lysed cells produced a lysate which showed marked non-Newtonian properties . The material showed a shear thickening at specific low shear stress conditions . The acceleration of the cone during the ascent stage of a flow curve, influenced the shape of the flow curves . These findings are discussed in relation to the form of bacterial DNA in solution.

Cytotechnology, 1992, 8(2), 103 - 8
High level expression of a frog alpha-amidating enzyme, AE-II, in cultured cells and silkworm larvae using a Bombyx mori nuclear polyhedrosis virus expression vector; Kobayashi J et al.; A Xenopus laevis peptidyl C-terminal alpha-amidating enzyme (AE-II) gene, modified by deletion of a region encoding the putative membrane-spanning domain and the putative C-terminal cytosolic tail, was expressed in BoMo-15 AIIc insect cells and silkworm larvae using a Bombyx mori baculovirus expression vector system . The expressed enzyme was identified predominantly in the culture medium and the hemolymph of silkworm larvae, indicating successful secretion of the expressed AE-II . The level of recombinant enzyme in the larval hemolymph at 4 days post-infection (40 micrograms/ml) was more than 100-fold the peak levels found in the culture medium (250 ng/ml) . The enzyme activity in the larval hemolymph at 4 days post-infection was 3700 units/ml.

Appl Microbiol Biotechnol, 1992 Jan, 36(4), 493 - 8
Promoter constructions for efficient secretion expression in Streptomyces lividans; Schmitt-John T et al.; Promoters from different Streptomyces genes were cloned in front of the Tendamistat gene from S . tendae, in order to study secretion-expression in S . lividans using a pIJ702 plasmid vector system . Besides the promoters we cloned a transcriptional terminator downstream of the Tendamistat gene to improve transcription efficiency . The promoters we selected were: (1) a synthetic Escherichia coli-like consensus promoter; (2) the aphI promoter of the neomycin resistance gene from S . fradiae; (3) an ermE-up promoter mutant from Saccharopolyspora erythraea; (4) the melC promoter of the tyrosinase operon from Streptomyces antibioticus . In addition, we tested the thiostrepton-inducible tipA promoter from S . lividans in our Tendamistat secretion system . The promoters were cloned upstream of the Tendamistat ribosome binding site in order to conserve the original translation initiation . The Tendamistat secretion mediated by the different promoter constructions above varied dramatically in up to 10 mg/l in the case of the synthetic promoter and the aph promoter, and up to 500 mg/l mediated by the ermE-up promoter . The melC promoter allowed about 200 mg/l Tendamistat secretion and the tipA promoter proved to be inducible from less than 0.5 mg/l up to 40 mg/l of Tendamistat secretion . Based on the amount of secreted Tendamistat and on the analysis of mRNA levels, we conclude that transcriptional activity regulates the efficiency of our secretion-expression system.

Scanning Microsc Suppl, 1992, 6, 11 - 20; discussion 20-2
Alignment, classification, and three-dimensional reconstruction of single particles embedded in ice; Frank J et al.; Cryo-electron microscopy of single biological particles poses new challenges to digital image processing due to the low signal-to-noise ratio of the data . New tools have been devised to deal with important aspects of 3-D reconstruction following the random-conical data collection scheme: (a) a new shift-invariant function has been derived, which promises to facilitate alignment and classification of single particle projections; (b) a new method of orientation search is proposed, which makes it possible to relate random-conical data sets to one another prior to reconstruction; and (c) the foundation is laid for a 3-D variance estimation which utilizes the oversampling of 3-D angular space by projections in the random-conical reconstruction scheme.

Biol Res, 1992, 25(2), 73 - 8
The separation and identification of picomole amounts of intermediates of glucose metabolism by high performance liquid chromatography on pellicular resins; Preller A et al.; A column (CarboPac PA1, Dionex) containing an anion-exchange pellicular resin was used for the separation of phosphoryl-hexoses derived from labeled glucose microinjected into individual frog oocytes or from cultures of Escherichia coli . Intermediates were identified by: a) comparison of retention times with those of authentic commercial compounds; b) the use of internal labeled standards; c) incubation of samples with specific enzymes and noting the disappearance of one radioactive peak and appearance of another at a new retention time.

Protein Sci, 1992 Jan, 1(1), 145 - 50
Steric and hydrophobic determinants of the solubilities of recombinant sickle cell hemoglobins; Bihoreau MT et al.; Models for the structure of the fibers of deoxy sickle cell hemoglobin (Hb Hb S, beta 6 Glu-->Val) have been obtained from X-ray and electron microscopic studies . Recent molecular dynamics calculations of polymer formation give new insights on the various specific interactions between monomers . Site-directed mutagenesis with expression of the Hb S beta subunits in Escherichia coli provides the experimental tools to test these models . For Hb S, the beta 6 Val residue is intimately involved in a specific lateral contact, at the donor site, that interacts with the acceptor site of an adjacent molecule composed predominantly of the hydrophobic residues Phe 85 and Leu 88 . Comparing natural and artificial mutants indicates that the solubility of deoxyHb decreases in relation to the surface hydrophobicity of the residue at the beta 6 position with Ile > Val > Ala . We also tested the role of the stereospecific adjustment between the donor and acceptor sites by substituting Trp for Glu at the beta 6 location . Among these hydrophobic substitutions and under our experimental conditions, only Val and Ile were observed to induce polymer formation . The interactions for the Ala mutant are too weak whereas a Trp residue inhibits aggregation through steric hindrance at the acceptor site of the lateral contact . Increasing the hydrophobicity at the axial contact between tetramers of the same strand also contributes to the stability of the double strand . This is demonstrated by associating the beta 23 Val-->Ile mutation at the axial contact with either the beta 6 Glu-->Val or beta 6 Glu-->Ile substitution in the same beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosoma, 1992, 102(1 Suppl), S133 - 41
DNA polymerase delta and epsilon holoenzymes from calf thymus; Podust V et al.; Replication of singly-DNA primed M13 DNA by DNA polymerase (pol) delta completely relies on the simultaneous addition of proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and replication protein A (RP-A) (or E . coli single-strand DNA binding protein, SSB) . Pol epsilon core alone is able to synthesize the products on singly-primed ssDNA . However, DNA synthesis by pol epsilon was stimulated up to 10-fold upon addition of the three auxiliary proteins PCNA, RF-C and SSB . This stimulation of pol epsilon by PCNA/RF-C/SSB appears to be the superposition of two events: pol epsilon holoenzyme (pol epsilon, PCNA, RF-C) synthesized longer products than its pol epsilon core counterpart, but elongated less primers . Furthermore, we analyzed the cooperative action of pol alpha/primase with pol delta or pol epsilon holoenzymes on unprimed M13 DNA . While pol delta displayed higher dNMP incorporation than pol epsilon, when a single primer was preannealed to DNA, pol epsilon was more efficient in the utilization of the primers synthesized by pol alpha/primase . Under these conditions both longer products and a higher amount of dNMP incorporation was found for pol epsilon holoenzyme, than for pol delta . Our data support the hypothesis of pol delta as the leading and pol epsilon as the second lagging strand replication enzyme.

DNA Seq, 1992, 2(4), 257 - 63
The complete nucleotide sequence of region 1 of the CFA/I fimbrial operon of human enterotoxigenic Escherichia coli; Jordi BJ et al.; The production of the plasmid-encoded fimbrial antigen CFA/I of enterotoxigenic Escherichia coli requires two DNA regions: CFA/I region 1 and CFA/I region 2 . These two regions are separated by about 40 kb on the wildtype plasmid . CFA/I region 1 contains the structural genes, whereas CFA/I region 2 contains a positive regulator . The first two genes (cfaA and cfaB) and the cfaD' sequence of region 1 have already been described . Here the total nucleotide sequence of region 1 is presented . Two new genes in region 1 are described, named cfaC and cfaE . The GC content of the genes in region 1 is 33.6% which is substantially lower than normally found in E . coli genes (50%) . The codon usage also differs from the standard codons used in E . coli.

Z Naturforsch {C}, 1992 Jan-Feb, 47(1-2), 77 - 84
Characterization of plastid 5-aminolevulinate dehydratase (ALAD; EC 4.2.1.24) from spinach (Spinacia oleracea L.) by sequencing and comparison with non-plant ALAD enzymes; Schaumburg A et al.; We have sequenced 5-aminolevulinate dehydratase (ALAD; EC 2.4.1.24) of a plant . A full-length cDNA clone (1727 bp) encoding this enzyme has been identified by immunoscreening a lambda gt 11 cDNA library of spinach . ALAD is not a plant-specific enzyme; however, the plant enzyme differs from the well known ALAD enzymes of bacteria, yeast and animals in structural and biochemical properties and in that it is located in the plastid . Differences and homologies can be traced back to the molecular level . The mature ALAD subunit, whose N-terminus was determined by automatic Edman degradation, is a protein of 367 amino acid residues and has a Mr of 40,132 . This figure is in the range of molecular weights of non-plant ALADs . The active centre is highly conserved and the same is true for the ion-binding domain, except that 4 cysteines of the non-plant enzymes (binding Zn2+) have disappeared and a total of 6 aspartic acids meets the demands of Mg(2+)-binding . However, there are more distinct differences . Apart from a transit sequence of 56 amino acids targeting the plastid, the N-terminal part of the mature plant enzyme differs considerably from non-plant ALAD enzymes . It is rich in prolines and hydroxylated amino acids . The apparent Mr on SDS-PAGE is 45,000 or higher, but up to now posttranslational modifications have not been found.

Microbiol Immunol, 1992, 36(3), 243 - 56
Establishment of a mouse model of cystitis and roles of type 1 fimbriated Escherichia coli in its pathogenesis; Liao J et al.; The role of type 1 fimbriae in promoting bladder colonization and the course of Escherichia coli cystitis were examined with type 1 fimbriated strains of clinically isolated E . coli . In the experiments of mice in vivo, intact bladder epithelium showed natural resistance to the adherence of type 1 fimbriated and non-fimbriated E . coli . However, the exfoliation of bladder superficial cells by trypsinization before the bacterial inoculation promoted the adhesion and colonization of type 1 fimbriated E . coli onto bladder epithelium . After colonization of E . coli, maximum numbers of E . coli and leukocytes were observed 3 days after inoculation . Nine days after inoculation, both of E . coli and leukocytes disappeared and the regeneration of superficial cells was observed . On the other hand, superficial cells in mice injected with phosphate-buffered saline or non-fimbriated E . coli regenerated 5 days after trypsinization . The present study demonstrated that the removal of superficial cells is essential for the adhesion and colonization of type 1 fimbriated E . coli onto bladder epithelium in vivo and a new model of E . coli cystitis in mice was established . The model which we established is valuable for histopathological, immunological, and therapeutic studies.

Yi Chuan Xue Bao, 1992, 19(1), 55 - 60
{Location of ctDNA fragment related to CMS in Brassica napus L . var . xiangai}; Sun W et al.; The ctDNA of sterile line and its maintainer from Brassica napus L . var . xiangai were digested by restriction endonucleases EcoRI, BamHI, PstI and SmaI . Only one special fragment E3.2kb was observed in EcoRI restriction pattern of maintainer . After being eluted, this fragment was incubated with plasmid pUC9 and then transformed E . coli JM83 . Through colour screening, clony hybridization and electrophoretic analyses, the special clone carrying E3.2 was obtained . The rRNA gene probe was used to hybridize with the EcoRI restriction pattern . The result showed that E3.2kb fragment is homologous to rRNA gene . And then, we use E3.2 fragment as probe to hybridize with rRNA gene digested by BamHI, EcoRI, SalII, BglII, HindIII and PstI . According to rRNA gene map, E3.2 fragment was located at the leader sequence of 16S rRNA gene, from +2.0kb to +5.4kb . Because this 3.4kb region was in the inverted repeat region of ctDNA and it is homologous to E3.2kb that related to CMS . So probably CMS is related to this 3.4kb region in the inverted repeat region of ctDNA.

Acta Virol, 1992 Jan, 36(1), 90 - 102
Cloning and expression in Escherichia coli of the 37-, 14-, and/or 16-kilodalton antigens genes from Rickettsia prowazekii strain E; Aniskovich LP et al.; A gene bank of Rickettsia prowazekii strain E constructed in the phage vector lambda EMBL4 was screened for antigen production with anti-R . prowazekii serum . One of the immunoreactive clones, grown at 37 degrees C exhibited the expression of at least two antigens of molecular weight (M(r)) 37 kD and 14 kD . Subcloning and further analysis revealed that the antigens (polypeptides) of Mr 37, 14, and/or 16 kD apparently represent structural units of the 138 kD complex antigen . Assembly of the above mentioned polypeptides was found to be thermosensitive as it took place at 30 degrees C but not at 37 degrees C and resulted in an oligomeric structure of M(r) 138 kD . The nucleotide sequence of the gene coding for a precursor of the mature polypeptides of Mr 14 and/or 16 kD was determined.

Ultramicroscopy, 1992 Jan, 40(1), 13 - 32
Detection, classification and 3D reconstruction of biological macromolecules on hypercube computers; Carazo JM et al.; In this work we present results of the mapping on hypercube computers of some of the key steps involved in the procedure for 3D structural determination from transmission electron microscopy images . The goal is the introduction of parallel processing tools in the field of electron microscopy image processing . We show how the rich topology of the hypercube, combined with an efficient programming strategy, allows for order-of-magnitude increase in computational capacity for such time-consuming tasks as calculation of multidimensional FFT's, cross-correlation coefficients, fuzzy partitioning functionals and the filtered back-projection 3D reconstruction method.

Vaccine, 1992, 10(5), 309 - 18
Induction of protective class I MHC-restricted CTL in mice by a recombinant influenza vaccine in aluminium hydroxide adjuvant; Dillon SB et al.; Induction of class I MHC-restricted cytotoxic T lymphocyte (CTL) responses by soluble proteins or peptides requires complex adjuvants or carrier systems which are not licensed for use with human vaccines . The data presented in this report show that vaccination with a highly purified recombinant influenza protein antigen in aluminium hydroxide adjuvant, the only adjuvant currently licensed for clinical use, elicited class I restricted CTL and protection from lethal challenge with H1N1 and H2N2 viruses . The antigen (D protein, SK&F 106160) is produced by expression of H1N1 influenza virus-derived cDNA (strain A/PR/8/34) in Escherichia coli, and is composed of the first 81 N-terminal amino acids (aa) of the non-structural protein 1 (NS1) fused via a nine nucleotide non-viral linker sequence to the 157 C-terminal aa of the haemagglutinin 2 subunit (HA2) . Previous work by Kuwano et al demonstrated that in vitro stimulation of spleen cells from influenza virus-primed mice, with a partially purified preparation of the D protein, selected for CD8+ CTL clones which facilitated lung clearance of H1N1 and H2N2 viruses . In the current study, these results were extended by studying the responses of mice actively immunized with highly purified D protein in the presence or absence of adjuvants . Vaccination of CB6F1 (H-2dxb) mice with D protein in aluminum hydroxide or Freund's complete adjuvant generated H1N1 cross-reactive, H-2d-restricted, CD8+ CTL directed against an immunodominant HA2 epitope (aa 189-199) . D protein without adjuvant did not elicit CTL, regardless of the route of injection . However, long-lived (greater than 6 months) splenic memory CTL were elicited by boosting mice intraperitoneally (i.p.) with the D protein in the absence of adjuvant . In mice injected subcutaneously with D protein in aluminium hydroxide at weeks 0 and 3, survival was increased relative to controls up to 16 weeks beyond the second vaccination, after which time additional boosting was required for protection . Studies in H-2b and H-2k mice vaccinated with the D protein showed that induction of CD4+ T-cell or antibody responses, in the absence of CD8+ CTL, did not correlate with protection . Passive transfer of immune sera from CB6F1 mice was also not protective . This prototype H1N1 recombinant subunit vaccine in aluminium adjuvant should directly address the feasibility of achieving a protective cell-mediated immune response in human influenza.

Anim Genet, 1992, 23(1), 19 - 29
Evidence for linkage between the swine L blood group and the loci specifying the receptors mediating adhesion of K88 Escherichia coli pilus antigens; Vogeli P et al.; Brush borders or enterocytes obtained from the small intestine of 248 pedigreed pigs were tested by adhesion assay in vitro with enterotoxigenic Escherichia (E.) coli strains, each expressing one of the three K88 pilus variants K88ab, K88ac and K88ad . All pigs were classified as belonging to one of the four adhesion phenotypes: I--K88ab(-), ac(-), ad(-); II--K88ab(-), ac(-), ad(+); III--K88ab(+), ac(+), ad(-); and IV--K88ab(+), ac(+), ad(+) . Serum or red cells were typed for 15 blood group systems: A-O, B, C, D, E, F, G, H, I, J, K, L, M, N and O; for 11 biochemical polymorphisms: PI1, PI2, PO1A, A1BG, GPI, PGD, TF, HPX, ADA, PGM and AMY; the polymorphism at the IGHG1 locus . Linkage analysis was performed between the alleles at the locus (loci) specifying K88 receptors able to bind one or more different serological types of K88 E . coli and alleles for markers at other loci . Linkage was demonstrated between the locus for the L blood group system and the locus (loci) for K88 E . coli receptors (Z = 3.24), adding one locus (loci) to the previously identified linkage group IV (LGIV) {L-SLB} . The maximum likelihood estimate of the recombination fraction (theta) was 0.23 . No evidence was found for linkage between any of the other biochemical and immunogenetic markers and the receptor locus (loci) of K88 E . coli.

Zentralbl Bakteriol, 1992 Jan, 276(2), 264 - 72
Restriction fragment length polymorphism and virulence pattern of the veterinary pathogen Escherichia coli O139:K82:H1; Tschape H et al.; Escherichia coli O139:K82:H1 strains originating from outbreaks and single cases of oedema disease in pigs were characterized by their genomic restriction fragment length polymorphism (RFLP), their virulence pattern, and by the occurrence as well as the genomic distribution of the determinants for hemolysin (hly) and verotoxins (shiga-like toxins; sltI, sltII) . Whereas the RFLPs revealed considerable variation among the E . coli O139:K82:H1 isolates depending the origin and epidemic source of the strains, the virulence gene slt II was found to be present in nearly all strains in a particular chromosomal region . Similar to RFLPs, the plasmid profiles are useful for epidemiological analysis.

Zentralbl Bakteriol, 1992 Jan, 276(2), 152 - 64
Restriction fragment length polymorphisms associated with alpha-hemolysin determinants are correlating with the expression of alpha-hemolysin in strains of Escherichia coli; Prada J et al.; Three different phenotypes of hemolysis on blood-agar were detected when 58 isolates of Escherichia coli were investigated for alpha-hemolysin synthesis . In strains with chromosomally encoded alpha-hly genes, phenotype I (large and clear hemolysis zones) corresponded with high hemolytic activity and with a 17.2 kb size BamHI restriction fragment hybridizing with an alpha-hly-specific gene probe . Phenotype II (small and turbid hemolysis zones) was associated with low hemolytic activity and generally with a 12.8 kb size hybridizing BamHI restriction fragment . An intermediate phenotype (type I/II) was found in a few strains with moderate hemolytic activity . This correlation between hemolytic phenotype and activity was not found in E . coli carrying alpha-hly plasmids . Type I strains differed from all others in the promotor region of their 17.2 kb size BamHI fragment associated chromosomal alpha-hly determinant . The relation between hemolytic phenotype and DNA hybridization pattern was found in unrelated E . coli isolates of human and animal origin . An association of alpha-hemolysin with other virulence factors was found in most strains of all three phenotypes.

Vaccine, 1992, 10(1), 10 - 3
Preparation of candidate vaccinia-vectored vaccines for haemorrhagic fever with renal syndrome; Schmaljohn CS et al.; Two vaccinia-vectored candidate vaccines for haemorrhagic fever with renal syndrome were prepared by inserting cDNA, representing the medium (M) genome segment, or the M and the small (S) genome segments of Hantaan virus into the thymidine kinase gene of the Connaught vaccine strain of vaccinia virus . In the single recombinant, the M segment was placed under control of the vaccinia virus 7.5 kDa promoter . In the double recombinant, the M and S segments were placed under control of the vaccinia virus 7.5 kDa and 11 kDa promoters, respectively . An immunoplaque assay technique was developed to select recombinants without the need for expression of irrelevant genes or use of potential mutagens . Proteins indistinguishable from authentic viral envelope glycoproteins and nucleocapsid protein were observed by immunoprecipitation with antibodies to Hantaan virus . The recombinant expressing both the M and the S segments was selected for further development and testing as a human vaccine.

Plant Mol Biol, 1992 Jan, 18(2), 327 - 36
Heat shock protein synthesis of the cyanobacterium Synechocystis PCC 6803: purification of the GroEL-related chaperonin; Lehel C et al.; Synechocystis PCC 6803 cells could be induced to synthesize four major HSPs with apparent molecular sizes of 70, 64, 15 and 14 kDa . Heat stress at 42.5 degrees C appeared to be the optimum temperature for HSP formation in cells grown at 30 degrees C . The relative rate of synthesis of HSP70 and HSP15 reached a maximum at 30 min after the temperature shift-up whereas the capability of cells to accumulate HSP64 and HSP14 continued through 2 h . The two most abundant HSPs, HSP70 and HSP64, were recognized on western blots by antibodies raised against authentic DnaK and GroEL from Escherichia coli . To furnish sufficient evidence for the assumption that HSP64 is a GroEL-related chaperonin, this protein was purified to homogeneity . There was a 76% sequence identity between the amino acid sequence of HSP64 and the corresponding protein in Synechococcus PCC 7942 . Moreover, the purified HSP64 cross-reacted to anti-E . coli GroEL antibody . To our knowledge, this is the first report about the purification and partial protein sequencing of a cyanobacterial chaperonin.

J Bacteriol, 1992 Jan, 174(2), 633 - 7
Mutations at Glu-32 and His-39 in the epsilon subunit of the Escherichia coli F1F0 ATP synthase affect its inhibitory properties; LaRoe DJ et al.; A collection of amino acid substitutions at residues Glu-32 and His-39 in the epsilon subunit of the Escherichia coli F1F0 ATP synthase has been constructed by cassette mutagenesis . Substitutions for residue Glu-32 appeared to cause abnormal inhibition of membrane-bound F1 ATPase activity, and replacement of His-39 by Arg, Val, and Pro affected F1F0 interactions.

Infect Immun, 1992 Jan, 60(1), 206 - 12
Mucosal immune response to RDEC-1 infection: study of lamina propria antibody-producing cells and biliary antibody; McQueen CE et al.; Infection of rabbits with Escherichia coli RDEC-1 is a useful model for diarrheal disease caused by mucosally attaching E . coli . Understanding of the protective immunity induced by RDEC-1 infection in rabbits should provide information useful in the design of vaccines for protection against this infection and other mucosally attaching organisms as well . Thus, to define the time course and location of specific immunoglobulin A secretion in relation to bacterial colonization during primary RDEC-1 infection, we infected rabbits with RDEC-1, which express AF/R1 adherence pili, and compared sites of anti-AF/R1 antibody-containing cells in the intestinal mucosa with the sites of luminal colonization and mucosal attachment of RDEC-1 . Also, anti-AF/R1 antibodies in intestinal fluids and bile were measured by enzyme-linked immunosorbent assay, and attachment sites of RDEC-1 to the intestinal epithelium were determined by immunohistochemical examination . Anti-AF/R1 pilus antibody-containing cells were most numerous in the proximal intestine (duodenum and jejunum) . In contrast, both luminal colonization and attachment of RDEC-1 to epithelial cells were densest in the distal intestine (cecum and colon) . Anti-AF/R1 antibodies were present in approximately equal amounts in fluids collected from all levels of the gut after week 1 postinfection . Anti-AF/R1 antibody levels in undiluted bile exceeded those in gut flushes by at least 2 orders of magnitude . Loss of RDEC-1 attachment to epithelial cells preceded resolution of diarrheal illness despite the presence of large numbers of organisms in the intestinal lumen . Our studies indicate that during RDEC-1 infection (i) sites of greatest mucosal anti-AF/R1 antibody secretion are proximal to sites of maximal RDEC-1 luminal colonization and attachment, (ii) bile is a major source of specific antibodies in the intestinal lumen, and (iii) interference with RDEC-1 attachment to epithelial cells may permit resolution of disease.

Second Messengers Phosphoproteins, 1992-93, 14(3), 163 - 71
Lipopolysaccharide stimulates phosphorylation of eukaryotic initiation factor-4F in macrophages and tumor necrosis factor participates in this event; Haas DW et al.; Bacterial lipopolysaccharide (LPS) produces rapid changes in macrophage protein synthesis and function . Phosphorylation of the 25 kDa mRNA cap-binding protein (eIF-4E) in model systems regulates the efficiency of protein synthesis . We report that both LPS and tumor necrosis factor-alpha (TNF-alpha) stimulate phosphorylation of eIF-4E and the p220 component of eIF-4F in bone marrow-derived macrophages . Moreover, anti-TNF-alpha antibodies inhibit LPS-stimulated phosphorylation of eIF-4E and p220 by 43% (+/- 6%) and 50% (+/- 5%), respectively . Our results indicate that LPS stimulates eIF-4F phosphorylation by a TNF-alpha-dependent mechanism, and suggest that phosphorylation of eIF-4F might play a role in the post-transcriptional regulation of gene expression in macrophages exposed to LPS.

Second Messengers Phosphoproteins, 1992-93, 14(3), 127 - 37
Selection of cDNAs for phosphodiesterases that hydrolyze guanosine 3';5'-monophosphate in Escherichia coli; Van Lookeren Campagne MM et al.; A genetic selection procedure has been developed which makes the growth of E . coli dependent on expression of a cGMP phosphodiesterase cDNA . E . coli does not contain a cGMP phosphodiesterase, and guanine auxotrophs cannot extract the guanine from cGMP . If a functional cGMP phosphodiesterase is introduced, then guaA auxotrophs will grow on cGMP as a guanine source . The method also selects GMP synthetase cDNAs, which complement the guanine auxotrophy directly . Expression of a Dictyostelium discoideum or human heart cyclic nucleotide phosphodiesterase cDNA permits growth of the E . coli guaA auxotroph in the presence of cGMP . Several commercial cDNA libraries were corrupt and contained phosphodiesterase and/or GMP synthetase sequences that were from a contaminating DNA source.

Rev Cubana Med Trop, 1992, 44(1), 50 - 4
{The study and evaluation of a method for identifying Escherichia coli by using fluorescent disks}; Niebla Perez A et al.; A study was carried out with 101 strains, 79 of Escherichia coli and 22 of other genera isolated from clinical samples at several hospitals in Havana form October 1989 to January 1990 . In all the strains, beta-glycuronidase enzyme was detected in conditions established by our laboratory and was compared with the results reached by the ROCHE enterotube method . Of the 79 Escherichia coli strains, 74 were positive to the beta-glycuronidase detection test . Sensitivity was 94% and specificity, was 100%.

Insect Mol Biol, 1992, 1(1), 37 - 43
A Drosophila metallothionein promoter is inducible in mosquito cells; Kovach MJ et al.; Expression from a Drosophila metallothionein promoter (Mtn) was investigated in mosquito cells . Recombinant plasmids carrying a transcription unit comprised of the Escherichia coli beta-galactosidase gene (lacZ) fused to the metallothionein promoter were stably introduced into Aedes albopictus C6/36 cells . A low copy transformant containing approximately 60 copies of plasmid per cell, and a high copy transformant (1-2 x 10(4) copies/cell) were characterized . The expression of beta-galactosidase from the metallothionein promoter could be induced and controlled in this heterologous system, even when the copy number of introduced plasmid was several thousand.

Mem Inst Oswaldo Cruz, 1992, 87 Suppl 3, 413 - 22
Protection of Aotus monkeys after immunization with recombinant antigens of Plasmodium falciparum; Enders B et al.; The genus Aotus spp . (owl monkey) is one of the WHO recommended experimental models for Plasmodium falciparum blood stage infection, especially relevant for vaccination studies with asexual blood stage antigens of this parasite . For several immunization trials with purified recombinant merozoite/schizont antigens, the susceptible Aotus karyotypes II, III, IV and VI were immunized with Escherichia coli derived fusion proteins containing partial sequences of the proteins MSAI (merozoite surface antigen I), SERP (serine-stretch protein) and HRPII (histidine alanine rich protein II) as well as with a group of recombinant antigens obtained by an antiserum raised against a protective 41 kD protein band . The subcutaneous application (3x) of the antigen preparations was carried out in intact animals followed by splenectomy prior to challenge, in order to increase the susceptibility of the experimental hosts to the parasite . A partial sequence of HRPII, the combination of three different fusion proteins of the 41 kD group and a mixture of two sequences of SERP in the presence of a modified Al(OH)3 adjuvant conferred significant protection against a challenge infection with P . falciparum blood stages (2-5 x 10(6)) i . RBC) . Monkeys immunized with the MS2-fusion protein carrying the N-terminal part of the 195 kD precursor of the major merozoite surface antigens induced only marginal protection showing some correlation between antibody titer and degree of parasitaemia . Based on the protective capacity of these recombinant antigens we have expressed two hybrid proteins (MS2/SERP/HRPII and SERP/MSAI/HRPII) in E . coli containing selected partial sequences of SERP, HRPII and MSAI . Antibodies raised against both hybrid proteins in rabbits and Aotus monkeys recognize the corresponding schizont polypeptides . In two independent immunization trials using 13 animals (age 7 months to 3 years) we could show that immunization of Aotus monkeys with either of the two hybrid proteins administered in an oil-based well tolerated formulation protected the animals from a severe experimental P . falciparum (strain Palo Alto) infection.

Mem Inst Oswaldo Cruz, 1992, 87 Suppl 3, 179 - 84
A recombinant hybrid protein as antigen for an anti-blood stage malaria vaccine: a study on the conservation of a protective component; Knapp B et al.; Recently we have shown that two hybrid proteins expressed in Escherichia coli confer protective immunity to Aotus monkeys against an experimental Plasmodium falciparum infection (Knapp et al., 1992) . Both hybrid proteins carry a sequence containing amino acids 631 to 764 of the serine stretch protein SERP (Knapp et al., 1989b) . We have studied the diversity of this SERP region in field isolates of P . falciparum . Genomic DNA was extracted from the blood of six donors from different endemic areas of Brazil and West Africa . The SERP region encoding amino acids 630 to 781 was amplified by polymerase chain reaction (PCR) and sequenced . Only conserved amino acid substitutions in maximally two positions of the analyzed SERP fragment could be detected which supports the suitability of this SERP region as a component of an anti-blood stage malaria vaccine.

Acta Physiol Hung, 1992, 79(4), 399 - 408
Influence of endotoxin on experimental post-alcoholic liver injury; Boron-Kaczmarska A et al.; The morphological and biochemical changes of the liver after endotoxin intake were analyzed in rats receiving 20% ethanol during 60 days . Besides morphological changes, concentration of serotonin and histamine in liver homogenates, the activity of asparagine and alanine aminotransferases (AspAT, ALAT), gamma-glutamyltranspeptidase (GGTP) and alcohol dehydrogenase (ADH) in blood serum were determined, too . The most extensive morphologic changes of the liver were seen in group of animals intoxicated with 20% ethanol during 60 days and single dose of endotoxin E . coli 0127:B8 intraperitoneally . These changes included necrosis most hepatocytes, focal steatosis of liver parenchyma, considerable hyperemia and parenchymatous degeneration of the liver cells . The cells lining liver sinuses showed considerable swelling as well as necrotic changes . Figures of cell division and haemorrhagic focuses were seen, too . The clusters of mononuclear cells, surrounding necrotically changed hepatocytes were seen in the central part of the liver lobule . Among the inflammatory mediators estimated in liver homogenate only serotonin reached a high level in the group of experimental animals receiving only endotoxin . Increased activity of aminotransferases AspAt and ALAT were associated with these morphologic and biochemical changes in liver tissue observed in animals receiving ethanol and endotoxin.

Braz J Med Biol Res, 1992, 25(8), 809 - 12
Inhibition of enteropathogenic Escherichia coli adherence to HeLa cells by immune rabbit sera; Barros HC et al.; We have studied the effect of immune rabbit sera on the localized (LA) and diffuse (DA) adherence to HeLa cells of 10 enteropathogenic Escherichia coli (EPEC) strains belonging to serogroups O55, O86, O111, O119, and O142 . Anti-La1 serum, obtained by rabbit immunization with an E . coli strain harboring a cloned DNA fragment from an EPEC LA plasmid, strongly inhibited the adherence of all serogroups but one (O142) . Similar results were obtained with anti-LA2 serum, which is anti-O111 serum absorbed with a non-adherent O111:H- EPEC strain . In contrast, non-absorbed anti-O55 and anti-O111 sera showed an inhibitory effect mainly on the adherence of homologous strains . Except for one experiment diffuse adherence was not inhibited by any antiserum used . The inhibitory effect of immune sera on localized adherence does not seem to be correlated with plasmid curing since adherence plasmid pMS49 proved to be stable after treatment with anti-O55 and anti-O111 sera . The cross-inhibition of adherence by anti-LA sera suggests that localized adherence-related adhesins of the O55, O86, O111, and O119 strains share similar antigens.

Braz J Med Biol Res, 1992, 25(8), 761 - 76
In vitro activities of a recombinant foot-and-mouth disease virus replicase expressed in Escherichia coli; Pacheco AB et al.; 1 . The replicase gene of foot-and-mouth disease virus (FMDV) was expressed in Escherichia coli under the control of a tac promoter . The recombinant enzyme was purified by inclusion body precipitation, elution, and poly(U) Sepharose chromatography . 2 . The enzyme exhibits poly(A)-dependent oligo(U)-primed poly(U) polymerase activity . The specific activity of the purified replicase is 1.3 x 10(5) . The recombinant replicase synthesizes RNA using FMDV RNA as template, as well as heterologous RNAs, such as globin RNA and synthetic RNAs, polyadenylated or not . In all polymerization reactions, RNA products twice the size of the template are formed, both in the presence and absence of an oligo(U) primer . The enzyme is also capable of incorporating {alpha 32P}UTP in all RNAs tested except the viral template . This activity does not seem to be related to the primer independent polymerization activity . 3 . The products from polymerization reactions were characterized by hybridization . In the absence of primer they consist of the template and a complementary strand covalently attached, while in the presence of primer they consist of two complementary strands synthesized de novo . 4 . We propose mechanisms of RNA synthesis by the recombinant FMDV replicase in the absence and presence of primer . These mechanisms are discussed in terms of models for in vitro RNA synthesis of other picornaviruses.

Braz J Med Biol Res, 1992, 25(7), 667 - 72
Use of plasmid profiles to differentiate strains within specific serotypes of classical enteropathogenic Escherichia coli; Fernandes RM et al.; 1 . The usefulness of plasmid profile analysis to differentiate strains of enteropathogenic Escherichia coli (EPEC) was evaluated by studying 123 strains of the most prevalent serotypes causing infant diarrhea in the city of Sao Paulo, Brazil, i.e., O111ab:H-, O111ab:H2 and O119:H6 . 2 . No common profiles were found among strains of distinct serotypes . However, within each serotype, most of the strains were grouped within a few major profiles . More than 68% of the strains of serotypes O111ab:H- and O111ab:H2 were included in 6 and 9 major profiles, respectively . In serotype O119:H6, about 48% of the strains were included in 3 major profiles . 3 . This analysis suggests that only a few EPEC clones are causing infant diarrhea in Sao Paulo and revealed that the distribution of serotypes O111ab:H- and O111ab:H2 during the one-year study was at least partly determined by small outbreaks of the most common profiles . 4 . We conclude that plasmid profile analysis is very useful to differentiate strains within specific EPEC serotypes.

Braz J Med Biol Res, 1992, 25(7), 659 - 66
The N-terminal amino acid sequence is essential for foot-and-mouth disease virus replicase activity; Pacheco AB et al.; 1 . Foot-and-mouth disease virus replicase was expressed by fusing its cDNA to the OmpA signal peptide coding sequence present in the pIN-III ompA series vectors . 2 . Two constructions were developed to express either a full-length or truncated enzyme lacking the 20 amino acids at the N-terminal end . Bacterial extracts expressing the recombinant proteins were submitted to SDS-PAGE and the presence of the replicase was revealed by immunoblotting . The truncated form exhibited a higher mobility and the relative positions of the proteins show that the signal peptide was removed . 3 . The biological activity of these two molecules was tested using a poly(A)-dependent oligo(U)-primed poly(U)-polymerase assay . The full-length replicase is active . The aminoterminal truncated enzyme had 0.02% activity of the intact one . 4 . This result indicates the importance of the twenty N-terminal amino acids for the activity of FMDV RNA-dependent RNA polymerase.

Braz J Med Biol Res, 1992, 25(5), 467 - 75
Chemical and biological properties of endotoxin from Leptospira interrogans serovars canicola and icterohaemorrhagiae; De-Souza L et al.; 1 . Endotoxin-like activity was extracted with phenol-chloroform-petroleum either (PCP) from Leptospira interrogans serovars icterohaemorrhagiae and canicola . Chemical analysis of leptospiral cells obtained from the PCP extract indicated the following distribution of lipopolysaccharide (LPS), protein and polysaccharide in mg/ml: 3.0, 4.5 and 1.0 for icterohaemorrhagiae and 3.3, 5.6 and 1.5 for canicola . 2 . The preparations presented several biological activities: positive Limulus test (1.0 pg/ml) for icterohaemorrhagiae and canicola PCP extract and 0.5 pg/ml for E . coli O111:B4 LPS, lethality for chicken embryos (LD50 45, 25 and 1.0) for icterohaemorrhagiae, canicola and E . coli O111:B4 LPS, pyrogenicity in rabbits with an average increase in rectal temperature of 0.6 degrees C, 0.9 degrees C and 2.2 degrees C for canicola, icterohaemorrhagiae and E . coli O111:B4 LPS, reacted with complement inhibiting the lysis of sheep red blood cells, 62%, 75% and 90% for 2.0 micrograms/ml of icterohaemorrhagiae, canicola PCP extract and E . coli O111:B4 LPS . The PCP extract showed no cytotoxicity on chicken embryo fibroblasts and epithelial cells . 3 . These results demonstrate that Leptospira endotoxin activity is similar to E . coli O111:B4 lipopolysaccharide.

Brain Pathol, 1992 Jan, 2(1), 31 - 7
Integration site-dependent transgene expression used to mark subpopulations of cells in vivo: an example from the neuromuscular junction; Weis J et al.; To produce transgenic mice, an exogenous gene is inserted into the germ line, usually by injection of the DNA construct into a pronucleus of a fertilized egg . In most cases the transgene is integrated into the genome at a single random site . Frequently, the transgenes are combinations of regulatory elements from one gene and protein coding sequences from another gene, the reporter . As expected, the promoter in the construct usually controls the expression pattern of the reporter . In some cases, however, transgenes have been constructed with regulatory elements that are not able to direct transcription on their own . In animals containing such transgenes, the expression of the reporter is dependent on endogenous regulatory elements near the chromosomal site of transgene integration . In the present study, an Escherichia coli beta-galactosidase (lacZ) reporter gene linked to a weak promoter was selectively expressed in discrete subpopulations of cells in each of eight independently derived lines of mice . In one line (line 42), which we analyzed in detail, a subset of cells in skeletal muscle were lacZ-positive . Specifically, fibroblasts close to neuromuscular junctions expressed the lacZ-protein, whereas skeletal muscle fibroblasts far from synaptic sites and fibroblasts in other organs were lacZ-negative . Moreover, Schwann cells at nerve terminals were lacZ-positive, whereas Schwann cells in extramuscular nerves were lacZ-negative . These results indicate the existence of differences between perisynaptic and extrasynaptic fibroblasts in normal skeletal muscle . They also demonstrate how such transgenic mice can be used to identify and mark discrete cell populations.

Zhen Ci Yan Jiu, 1992, 17(3), 212 - 6
{The effect of acupuncture on rabbits with fever caused by endotoxin}; Kuang X et al.; Forty-eight rabbits were used to investigate the effect of puncturing GV14, LI11 on the change of temperature and the level of plasma endotoxin . The animals were divided into 5 groups: reinforcing manipulation, reducing manipulation, electroacupuncture, endotoxin-control, N.S.-control group . The result shows that there is no influence on the fever caused by endotoxin and the level of plasma endotoxin in the treatments by the different acupuncture techniques.

Protein Sci, 1992 Jan, 1(1), 10 - 21
Differences in hydrogen exchange behavior between the oxidized and reduced forms of Escherichia coli thioredoxin; Kaminsky SM et al.; Amide proton exchange of thioredoxin is used to monitor the structural effects of reduction of its single disulfide . An effective 3-5-proton difference between the oxidized and reduced protein form is observed early in proton out-exchange of the whole protein, which is independent of temperature in the range of 5-45 degrees C, indicating that redox-sensitive changes are probably not due to low-energy structural fluctuations . Medium resolution hydrogen exchange experiments have localized the redox-sensitive amide protons to two parts of the sequence that are distant from each other in the three-dimensional structure: the active-site turn and the first beta-strand . The sum of the proton differences observed in the peptides from these regions is equal to that of the whole protein, indicating that all redox-sensitive hydrogen exchange effects are observed in the peptide experiments . A model combining structural changes within the protein matrix with changes in the surface hydration properties is proposed as a mechanism for the communication between distant sites within the protein . Sound velocity and density measurements of reduced and oxidized thioredoxin are presented in the accompanying paper (Kaminsky, S.M . & Richards, F.M., 1992, Protein Sci . 1, 22-30).

J Nutr Sci Vitaminol (Tokyo), 1992, Spec No, 114 - 7
Serum B12 binding proteins versus seminal plasma binder; Fukuda M et al.; It was well confirmed that B12 binding protein in human serum and body fluids largely consists of two kinds of binding proteins, physiologically B12 transporting transcobalamin and haptocorrins which does not involve in B12 transport . In seminal plasma, very potent B12 binding protein, in quantity as much as 23 times of normal serum, was identified . Separation characteristics, gel filtration, CM cellulose column chromatography and column IEF all agreed as one of the haptocorrins . One peculiar feature of seminal plasma binder is marked heterogeneity of much lower pI regions suggesting the presence of increased amount of sialic residues on the molecule . The preliminary data from this laboratory support the view, however, physiological role such as the relationship to the spermatogenesis is not known . The most recent findings in clinical observation on B12 is the treatment of male infertility using methycobalamin . The detailed analysis of seminal binders may open up new arena of B12 bioeffect.

DNA Seq, 1992, 3(4), 233 - 5
A rapid PCR dependent microtitre plate screening method for DNA sequences altered by site-directed mutagenesis; Trower MK; A simple and rapid method for identifying DNA sequences altered by site-directed mutagenesis is described . The procedure requires that a restriction endonuclease recognition sequence is either created or removed from the target DNA sequence by the site-directed mutagenesis reaction . In the screening method presented, transformant colonies or M13 plaques are directly subjected to PCR using universal primers that flank the region containing the site-directed changes . The double stranded DNA products generated are then digested, with no purification, by the restriction enzyme whose site is modified, allowing mutant clones to be differentiated from those carrying wild type sequences . The protocol also provides for recovery of the bacterial cultures harbouring the mutated plasmid or M13 for further characterisation . All the procedures are carried out in small volumes in microtitre plates, thereby lowering costs and enabling the investigation of large numbers of clones . The technique allows a considerable amount of effort to be saved compared to conventional screening practices by circumventing time consuming DNA template preparations.

Eur Surg Res, 1992, 24(6), 363 - 71
Superoxide production by liver macrophages in a septic rat model--relation to arterial ketone body ratio; Iimuro Y et al.; The relationship between superoxide production by liver macrophages and arterial ketone body ratio (AKBR), which reflects the oxidation-reduction state in the mitochondrial compartment of hepatocytes, was studied in rats with lethal and sublethal septicemia induced by intravenous injection of live Escherichia coli 014 . In the sublethal model, AKBR decreased transiently (p < 0.01) and superoxide production by isolated liver macrophages increased significantly after opsonized zymosan (OZ) stimulation (p < 0.05) . On the other hand, in the lethal model, AKBR decreased markedly (p < 0.01) to below 0.4 without recovery, and superoxide production was not activated by OZ stimulation . Thus, when AKBR decreases to an irreversible level, below about 0.4, superoxide production by liver macrophages is impaired, while as long as AKBR remains reversible, more than about 0.4, it is enhanced . It is suggested that superoxide production by the Kupffer cells is related to the intrahepatic oxidation-reduction state in the septic model.

Folia Microbiol (Praha), 1992, 37(5), 347 - 52
Electron microscopic analysis of two nonconjugative derivatives of plasmid R1drd-19Km- from Escherichia coli; Benada O et al.; The plasmids pON5300 and pON5304, nonconjugative variants of the plasmid R1drd-19Km, were analyzed by electron microscopy . It was found by heteroduplex mapping that a 1.4 kb DNA segment was inserted into EcoRI E fragment of both plasmids, where some tra-genes and oriT are localized . Although this DNA segment was mapped to the same region its orientation was different in each of the two plasmids . The inserted DNA segment was identified as an IS10R sequence on the basis of analysis of self-annealed molecules of pON5304 and their cleavage with EcoRV restriction enzyme . These methods enable us not only to map IS10R sequences on 87 kb pON5300 and 65 kb pON5304 molecules, respectively, but also to define their orientation.

J Basic Microbiol, 1992, 32(5), 309 - 15
The metabolism of gluconate in Escherichia coli: a study in continuous culture; Coello NB et al.; The gluconate metabolism in Escherichia coli involves duplicate activities of transport and phosphorylation for gluconate . In both cases, these activities can be differentiated in vitro by their different affinities for the substrate . In addition, the two gluconokinases can be differentiated by their heat sensitivities . The technique of continuous culture was used to investigate the influence of the growth rate on this metabolism in an E . coli HfrG6 strain during gluconate-limited growth under conditions of high and low oxygen concentrations . The transport and phosphorylation for gluconate, induced when the cells are cultivated in media with gluconate were differently influenced by the culture dilution rate . These activities were induced under the two conditions investigated; however, the low affinity transport system for gluconate and the thermosensitive gluconokinase were not detected under conditions of high and low oxygen concentrations, respectively . The induction of the dehydratase was favoured under conditions of low oxygen concentration . The experimental data suggest that induction and repression work together to regulate the levels of these activities during gluconate-limited growth conditions . Furthermore, that an effector molecule distinct from gluconate might be involved in the induction of the dehydratase.

J Ocul Pharmacol, 1992 Winter, 8(4), 295 - 307
Effects of steroids and immunosuppressive drugs on endotoxin-uveitis in rabbits; Ohia EO et al.; Anti-inflammatory actions of dexamethasone (DEXA), Cyclosporin A (CSA) and Rapamycin (RAPA) were assessed on uveitis induced by intravitreal E-coli Endotoxin (100ng) in rabbits at 24 hrs . In this model, endotoxin caused a breakdown of the blood-aqueous barrier (BAB) and polymorphonuclear neutrophils (PMN) infiltration into the aqueous humor (AH) and iris-ciliary body (ICB) . Intramuscular (I.M.) DEXA (2mg/kg) but not topical DEXA (0.1% 6 x daily) inhibited AH leukocytes and protein level . However, both routes caused an inhibition of AH Prostaglandin E2 (PGE2) and Leukotriene B4 (LTB4) . In the ICB, I.M . DEXA significantly inhibited PGE2 synthesis and myeloperoxidase (MPO) activity . I.M . CSA (25mg/kg) and I.M . RAPA (10mg/kg) inhibited the AH leukocytes and protein content and MPO activity in the ICB . RAPA also inhibited AH protein and eicosanoid (except AH LTB4) levels in both the AH and ICB . Interestingly, castor oil, a vehicle of CSA, also inhibited AH leukocytes and the release of PGE2 into AH and from ICB . In summary, systemic administration of DEXA and other immunosuppressive drugs CSA and RAPA significantly inhibited endotoxin-induced uveitis in rabbits.

Vaccine, 1992, 10 Suppl 1, S48 - 52
Possible approaches to develop vaccines against hepatitis A; D'Hondt E; More than a decade ago, successful replication of hepatitis A virus (HAV) in cell culture opened the way to the development of live attenuated and inactivated vaccine candidates . Serial passages of HAV in cell culture led to attenuation as demonstrated by experiments in non-human primates . Several live vaccine candidates obtained through serial passages have been evaluated in volunteers . Significant improvements in the yield of viral antigen from infected cell cultures stimulated the development of killed vaccine candidates . These formalin-inactivated vaccines contain the viral capsid antigens assembled into viral particles . The immunogenic potential of the vaccine candidates depends strongly on the preservation of the configuration of the capsid proteins . Synthetic peptides covering immunogenic sequences of VP1 as well as soluble capsid proteins expressed as fusion proteins in Escherichia coli were therefore only weakly immunogenic when injected at high concentrations in rabbits . On the other hand, tamarin monkeys immunized with a live recombinant vaccinia expressing P1 were protected against virulent challenge . There are, however, considerable drawbacks related to the use of live vaccinia as a carrier virus . Chimeric polio-HAV VP1 viruses have been constructed . These hybrid viruses were not able to induce an immune response, probably because of configurational constraints of poliovirus on the inserted HAV epitopes . More recently, encouraging data on empty virus particles expressed in baculovirus and vaccinia virus systems have been reported.

Agents Actions Suppl, 1992, 38 ( Pt 1), 340 - 8
Characterization of two members (CST4 and CST5) of the cystatin gene family and molecular evolution of cystatin genes; Saitoh E et al.; Two members (CST4 and CST5) of the cystatin gene family have been characterized partially by DNA analysis . The CST4 clone contained the gene coding for the precursor form(141 amino acids) of cystatin S, and its exon-intron organization is the same as that of other members (the cystatin SN gene at the CST1 locus, the cystatin SA gene at the CST2 locus, the cystatin C gene at the CST3 locus and a cystatin pseudogene at the CSTP1 locus) . The second cystatin pseudogene was elucidated in the clone, CST5, and it was assigned to the CSTP2 locus . Alignment of DNA sequences of cystatin genes with other genes suggested that the genes for cystatins, kininogens, and Bowman-Birk type inhibitors have evolved from an ancient ribonuclease-like gene.

Acta Physiol Scand Suppl, 1992, 607, 23 - 9
Mitochondrial F-type ATPases: the glycine-rich loop of the beta-subunit is a pyrophosphate binding domain; Thomas PJ et al.; The beta-subunit of the mitochondrial ATP synthase complex comprises the bulk, if not all, of the catalytic nucleotide binding site on the enzyme . A region of homologous sequence rich in glycines (G) and containing a basic lysine (K) and a threonine (T) is found in the beta-subunit as well as many other purine nucleotide binding proteins . The consensus sequence of this region is Gx4GKT, where x represents any amino acid, and is called the A region or glycine-rich loop . The related function of these proteins implies that the glycine-rich loop is directly involved in nucleotide binding . Here we directly test the involvement of the beta-subunit's glycine-rich region in adenine nucleotide binding using two independent approaches . A synthetic fifty amino acid peptide, PP-50, containing the glycine-rich region and the surrounding sequence was assessed for secondary structure and interaction with potential ligands . Circular dichroism spectropolarimetry indicates that PP-50 assumes a predominantly beta-sheet conformation in solution . Significantly, the peptide precipitates from solution when ATP, ADP, GTP, ITP, and pyrophosphate are added, but not when AMP or phosphate are included . Magnesium is not required for the interaction with the purine nucleotides . Complimentary to these studies, the sequence around the Gx4GKT motif was deleted from a recombinant rat liver beta-subunit overexpressed in E . coli . While the wild type beta-subunit showed specificity for the tri- and diphosphonucleotides, the deletion mutant bound tri-, di-, and monophosphate nucleotides with equal affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Antisense Res Dev, 1992 Summer, 2(2), 111 - 8
Translation inhibition by phosphorothioate oligodeoxynucleotides in cell-free systems; Ghosh MK et al.; A detailed comparison was made of the concentration dependence of translation inhibition by phosphorothioate and phosphodiester oligodeoxynucleotides of the same anti-beta-globin sequence in cell-free systems using beta-globin mRNA and unrelated mRNAs as controls . The results confirm that at low concentrations the phosphorothioate oligomer is more potent as an antisense compound, while at higher concentration (greater than 4 microM) it exhibits more nonspecific inhibition than the phosphodiester oligomer for RNase H-mediated translation inhibition.

Mol Carcinog, 1992, 6(2), 140 - 7
Kinds of mutations induced by aflatoxin B1 in a shuttle vector replicating in human cells transiently expressing cytochrome P4501A2 cDNA; Trottier Y et al.; Transient expression of rat liver cytochrome P450lA2 cDNA was combined with the use of a shuttle vector as a mutational target to determine the frequency and types of mutation caused by the conversion of aflatoxin B1 into genotoxic metabolites within human cells . Ad293 cells were first transfected with p91-lA2, a rat liver P450lA2 cDNA expression vector, or with p91-lA2(i) (a control vector that has the P450 cDNA in the inverted orientation) and incubated for 24 h to permit P450lA2 accumulation . Cells were then transfected with the pS189 shuttle-vector plasmid, which carries the Escherichia coli supF gene as a mutational target, and incubated for a further 24 h in the presence of aflatoxin B1 to permit promutagen activation and pS189 replication . In shuttle vectors replicated in p91-lA2-transfected cells, the supF point-mutation frequency increased with increasing concentration of aflatoxin B1 . This frequency was nine to 23 times greater than the background point-mutation frequency obtained with aflatoxin B1-treated control (p91-lA2(i)-transfected) cells . The large majority of the aflatoxin B1-induced supF point mutations were base substitutions, mostly G:C----T:A transversions . This mutagenesis system permits the molecular analysis of mutations induced by specific P450/promutagen pairs in a shuttle vector replicating in human cells and will permit the investigation of host cell mechanisms involved in the generation of these mutations.

Protein Eng, 1992 Jan, 5(1), 93 - 100
Biochemical properties of the kringle 2 and protease domains are maintained in the refolded t-PA deletion variant BM 06.022; Kohnert U et al.; BM 06.022 is a t-PA deletion variant which comprises the kringle 2 and the protease domain . Production of BM 06.022 in Escherichia coli leads to the formation of inactive inclusion bodies, which have to be refolded by an in vitro refolding process to achieve activity and proper structure of the domains . We analysed the biochemical properties of BM 06.022 to obtain some information about the structure of kringle 2 and the protease as compared with the structure of these domains in the intact t-PA molecule . The kinetic analysis of the amidolytic activity of BM 06.022 and CHO-t-PA yielded similar values for kcat (13.9 s-1 and 11.4 s-1 for the single chain forms and 33.9 s-1 and 27.1 s-1 for the two chain forms of BM 06.022 and CHO-t-PA, respectively) and for Km (2.5 mM and 2.1 mM for the single chains forms and 0.5 mM and 0.3 mM for the two chain forms of BM 06.022 and CHO-t-PA, respectively) . BM 06.022 and CHO-t-PA have the same plasminogenolytic activity in the absence of CNBr fragments of fibrinogen . However, BM 06.022 has a lower plasminogenolytic activity in the presence of CNBr fragments of fibrinogen and a lower affinity to fibrin as compared with CHO-t-PA . The affinity of BM 06.022 for fibrin is completely suppressed by 0.3 mM epsilon-aminocaproic acid, while the intact t-PA has a residual affinity of approximately 30% . The dissociation constants for the interaction with the lysine analogue epsilon-aminocaproic acid are 0.10 mM and 0.09 mM for BM 06.022 and the intact t-PA, respectively . Furthermore, BM 06.022 and CHO-t-PA are inhibited by PAI-1 in a similar manner.

Kurume Med J, 1992, 39(1), 9 - 12
Construction of an expression vector for human papillomavirus type 16 E7-glutathione S transferase fusion protein; Chinami M et al.; An expression vector for human papillomavirus type 16 (HPV 16) E7-glutathione S transferase fusion protein was constructed . The E7 gene was confirmed by southern blot analysis with a digoxygenin 11-dUTP labeled and simultaneously amplified E7 DNA probe by polymerase chain reaction (PCR) . The fusion protein was expressed in E . coli, recovered as inclusion bodies, and was confirmed by western blot analysis using a monoclonal antibody against HPV 16-E7 protein . A polyclonal antibody against the HPV 16-E7 protein was raised in the serum of mice hyper-immuned with the fusion protein . This antibody will be available for screening of patients with cervical cancer.

Arch Virol, 1992, 124(3-4), 355 - 61
Detection of anti-gag antibodies of feline immunodeficiency virus in cat sera by enzyme-linked immunosorbent assay; Furuya T et al.; Using gag protein of feline immunodeficiency virus (FIV) expressed in Escherichia coli, an enzyme-linked immunosorbent assay (ELISA) system was developed for detection of antibodies to FIV gag protein in cat sera . With serum samples from cats experimentally infected with several strains and an infectious molecular clone of FIV, increases of the antibody titers to FIV gag protein were observed in all cases by the ELISA at early stage of infection . When we examined a total of 415 field cat sera which were previously tested by an indirect immunofluorescence assay (IFA), 9 (12.9%) out of 70 IFA positive sera were judged as negative by the ELISA . However, all 3 serum samples tested among the 9 IFA positive sera had antibodies to gp130 but not to p26 by a radioimmunoprecipitation assay . The results indicated that some IFA positive sera did not have antibodies to the p26 though they have antibodies to other proteins specific for FIV.

Photochem Photobiol, 1992 Jan, 55(1), 39 - 45
Singlet oxygen induced DNA damage and mutagenicity in a single-stranded SV40-based shuttle vector; Ribeiro DT et al.; The effects of singlet oxygen (1O2), generated by the thermal decomposition of water soluble NDPO2 (endoperoxide of the disodium 3,3'-(1,4-naphthylidene) dipropionate), on a single-stranded shuttle vector were analysed . 1O2 induces a much higher level of breaks in the phosphodiester backbone of single-stranded than double-stranded DNA . This may be due to a higher accessibility of guanine residue, primarily damaged by 1O2 . The damaged vector was transfected into monkey COS7 cells where single-stranded DNA was converted to the double-stranded replicative form DNA . After 3 days, extrachromosomal DNA was extracted and the plasmids rescued in E . coli to study mutagenesis . There is a significant increase in mutation frequency of damaged single-stranded DNA in comparison to untreated DNA . It is concluded that 1O2 induces breaks in the backbone of single-stranded DNA and that the 1O2-damaged molecules are mutated after passage through mammalian cells.

Free Radic Biol Med, 1992, 12(5), 353 - 64
Radiation-induced generation of chlorine derivatives in N2O-saturated phosphate buffered saline: toxic effects on Escherichia coli cells; Czapski G et al.; The radiolysis of aqueous chloride solutions has been investigated using pulse and steady-state methods . We have found a correlation between the yields of Cl2- and HOCl formed in pulse-irradiated N2O-saturated solutions . The yields increased with the increasing concentrations of Cl- and phosphate . Phosphate enhanced the yield of Cl2- in neutral solutions because of a proton transfer from H2PO4- to HOCl- with a rate constant of (2.6 +/- 0.5) x 10(8) M-1s-1 . HOCl could not be detected in pulse-irradiated He or air-saturated, phosphate-buffered saline (PBS) solutions or in gamma-irradiated N2O, He, or air-saturated PBS solutions . The results are discussed in light of previously suggested mechanisms for the formation and decay of Cl2- . Pulse-irradiated N2O-saturated PBS solutions have a lethal effect on Escherichia coli cells, which is proportional to the amount of HOCl in the solutions . Gamma-irradiation of cells in N2O-saturated PBS solution also raises the radiosensitivity of the cells, although HOCl does not accumulate in this system . The effects of the radiation-induced toxic products on E . coli cells are similar to the effects of NaOCl . The cell membrane is probably the site of physiological injury induced by the radiation products.

Nephrol Dial Transplant, 1992, 7(1), 16 - 28
Permeability of cellulosic and non-cellulosic membranes to endotoxin subunits and cytokine production during in-vitro haemodialysis; Urena P et al.; The possibility of endotoxin transfer across haemodialysis membranes remains a controversial issue . Additional concern has arisen because of the recent introduction in clinical practice of highly permeable, synthetic dialysis membranes and of bacteria-contaminated bicarbonate concentrate with potential short-term and long-term hazards for haemodialysis (HD) patients . Therefore, we performed experiments in an in-vitro dialysis recirculation system using three different types of HD membranes, namely standard regenerated cellulose (Cuprophan, CU), polyacrylonitrile AN-69 (PAN), and polysulphone F-60 (PS) . When radiolabelled lipopolysaccharide (125I M-LPS) from E . coli, together with 10 micrograms/ml unlabelled LPS, was added to the recirculating solution in the dialysis compartment, radioactivity could be detected in the blood compartment after 15 min and increased progressively with time up to respectively 6.7% (CU), 10.3% (PAN), and 10.3% (PS) of initial activity on the dialysate side . The addition of albumin to the solution on the blood side led to a decreased permeability of radioactivity (7.3% vs 10.3%), compared to the absence of albumin (tested only for PS membrane) . Furthermore, 73% of 125I M-LPS transferred across the PS membrane in the presence of albumin was TCA-precipitable . In contrast, free iodine (Na 125I) incubated in an albumin-containing solution did not precipitate with albumin after the addition of TCA (precipitation of only 0.6%) . Moreover, kinetics of transmembranous transfer of Na-125I were strikingly different from that of 125I M-LPS . Analysis by the method of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the blood side solution, after LPS addition in the dialysis solution and 30 min of back-filtration, revealed the presence of several silver-stainable and autoradiographic bands of low-molecular-weight range, probably LPS fragments . Finally, the presence of LPS in the dialysate compartment led to a moderate increase in interleukin 1 (IL-1) and tumour necrosis factor alpha (TNF) concentrations in plasma as well as in monocyte culture supernatants after isolation from recirculating normal human whole blood exposed to CU, PAN, or PS membrane . In conclusion, our study provides evidence for the permeation of low-molecular-weight LPS subunits across cellulosic and non-cellulosic HD membranes . The clinical significance, if any, of such a transfer has, however, still to be demonstrated.

Arch Virol, 1992, 124(1-2), 1 - 20
Expression of porcine pseudorabies virus genes by a bovine herpesvirus-1 (infectious bovine rhinotracheitis virus) vector; Kit S et al.; Recombinant DNA techniques were used to insert foreign genes into bovine herpesvirus-1 {infectious bovine rhinotracheitis virus (IBRV)} vectors which were attenuated by deletion and/or insertion mutations in the IBRV thymidine kinase (tk) gene . In one recombinant, the regulatory and coding sequences of the late pseudorabies virus (PRV) glycoprotein gIII gene, were inserted into the early IBRV tk gene . This recombinant efficiently expressed the PRV gIII gene indicating that immediate early IBRV proteins were competent to transactivate the late PRV gIII gene . IBRV vector viruses were also prepared in which the coding sequences of the early PRV tk gene, the late PRV gIII gene, and the E . coli beta-galactosidase gene were ligated to the late IBRV gIII promoter . Genotypes and phenotypes of the recombinant viruses were verified by restriction endonuclease and molecular hybridization experiments, thymidine plaque autoradiography, beta-gal plaque assays, and by immunoprecipitation experiments on extracts from 3H-mannose-labelled cells . The recombinant IBRV expressing beta-gal from the IBRV gIII promoter has been useful as an intermediate in the construction of IBRV vectors harboring foreign DNA sequences . The infectivity of the IBRV recombinant that expressed PRV gIII from the IBRV gIII promoter, was neutralized by polyclonal PRV antisera and by monoclonal antibodies to PRV gIII . The PRV gIII glycoprotein synthesized by the preceding recombinant has been used to coat microtiter test plate wells in a PRV gIII differential diagnostic test kit.

Proteins, 1992 Jan, 12(1), 42 - 8
Metal ion stabilization of the conformation of a recombinant 19-kDa catalytic fragment of human fibroblast collagenase; Lowry CL et al.; A recombinant 19-kDa human fibroblast collagenase catalytic fragment modeled on a naturally occurring proteolytic product was purified from E . coli inclusion bodies . Following renaturation in the presence of zinc and calcium, the fragment demonstrated catalytic activity with the same primary sequence specificity against small synthetic substrates as the full-length collagenase . Unlike the parent enzyme, it rapidly cleaved casein and gelatin but not native type I collagen . Intrinsic fluorescence of the three tryptophan residues was used to monitor the conformational state of the enzyme, which underwent a 24-nm red shift in emission upon denaturation accompanied by quenching of the fluorescence and loss of catalytic activity . Low concentrations of denaturant unfolded the fragment while the full-length enzyme displayed a shallow extended denaturation curve . Calcium remarkably stabilized the 19-kDa fragment, zinc less so, while together they were synergistically stabilizing . Among divalent cations, calcium was the most effective stabilizer, EC50 approximately 60 microM, and similar amounts were required for substrate hydrolysis . Catalytic activity was more sensitive to denaturation than was tryptophan fluorescence . Least sensitive was the polypeptide backbone secondary structure assessed by CD . These observations suggest that the folding of the 19-kDa collagenase fragment is a multistep process stabilized by calcium.

J Virol Methods, 1992 Jan, 36(1), 85 - 90
Tn9 CAT gene contains a promoter for vaccinia virus transcription: implications for reverse-genetic techniques; Ryan KW; Vaccinia virus-dependent CAT expression was observed in virus-infected cells cotransfected with a promoterless CAT gene . Restriction endonuclease resection of the CAT plasmid indicated that expression was due to recognition by vaccinia virus RNA polymerase of sequences within the CAT gene itself, probably located within the 5' untranslated region of the gene . This observation is relevant to the design of reverse-genetic systems which use CAT as a reporter gene to detect replication of negative-strand RNA virus pseudogenomes.

Brain Res Mol Brain Res, 1992 Jan, 12(1-3), 95 - 102
Effects of gene transfer into cultured CNS neurons with a replication-defective herpes simplex virus type 1 vector; Johnson PA et al.; Vectors derived from herpes simplex virus type 1 (HSV-1) may provide useful tools for gene transfer to cells of the mammalian nervous system . We have studied the infection of cultured CNS neurons using a vector derived from an HSV-1 mutant deleted for the major HSV-1 transcriptional regulatory protein-encoding gene, IE 3 . This vector, denoted Cgal delta 3, contains the E . coli lacZ gene driven by the strong promotor of the human cytomegalovirus major immediate-early gene inserted into a non-coding portion of the mutant viral genome . We studied the efficiency of Cgal delta 3 infection of rat CNS neurons at various times after cell preparation from embryonic rats, the effect of vector infection on the glia subpopulation of the neuronal cultures, and the stability of lacZ expression in infected neurons cultured under conditions optimized for neuronal differentiation and survival using an astrocyte feeder layer . Under these conditions, an HSV-derived vector is a highly efficient vehicle in vitro for short-term gene transfer to cells of the CNS . Despite the fact that this vector cannot undergo a lytic cycle, it was toxic to cultured CNS neurons and glia . Even with the use of an astrocyte feeder layer to support infected neurons, we have detected only transient expression of the lacZ gene, due either to loss of the infected cells and/or to shut off of transgene expression . Further improvements will be needed in the design of HSV vectors to allow long-term gene transfer to cultured neurons.

Mol Biochem Parasitol, 1992 Jan, 50(1), 57 - 67
Molecular cloning and expression of the gene encoding the kinetoplast-associated type II DNA topoisomerase of Crithidia fasciculata; Pasion SG et al.; A type II DNA topoisomerase, topoIImt, was shown previously to be associated with the kinetoplast DNA of the trypanosomatid Crithidia fasciculata . The gene encoding this kinetoplast-associated topoisomerase has been cloned by immunological screening of a Crithidia genomic expression library with monoclonal antibodies raised against the purified enzyme . The gene CfaTOP2 is a single copy gene and is expressed as a 4.8-kb polyadenylated transcript . The nucleotide sequence of CfaTOP2 has been determined and encodes a predicted polypeptide of 1239 amino acids with a molecular mass of 138,445 . The identification of the cloned gene is supported by immunoblot analysis of the beta-galactosidase-CfaTOP2 fusion protein expressed in Escherichia coli and by analysis of tryptic peptide sequences derived from purified topoIImt . CfaTOP2 shares significant homology with nuclear type II DNA topoisomerases of other eukaryotes suggesting that in Crithidia both nuclear and mitochondrial forms of topoisomerase II are encoded by the same gene.

EMBO J, 1992 Jan, 11(1), 373 - 8
Inter-protein distances within the large subunit from Escherichia coli ribosomes; May RP et al.; Selected pairs of protonated ribosomal proteins were reconstituted into deuterated 50S subunits from Escherichia coli ribosomes . The rRNA of the deuterated ribosomal matrix was derived from cells grown in 76% D2O, the deuterated protein moiety from cells grown in 84% D2O . This procedure warrants that the coherent neutron scattering of deuterated proteins and rRNA is nearly the same and equals that of a D2O solution of approximately 90% . The neutron scattering is recorded in a reconstitution buffer containing approximately 90% D2O . The result is a significant improvement of the coherent signal:noise ratio over traditional methods; due to this dilute solutions can be used, thus preventing unfavorable inter-particle effects . From the diffraction pattern the distance between the mass centers of gravity of the two protonated proteins can be deduced . In this way, 50 distances between proteins within the large subunit have been determined which provide a basis for future models of the large ribosomal subunit describing the spatial distribution of the ribosomal proteins . A model containing seven ribosomal proteins is presented.

EMBO J, 1992 Jan, 11(1), 361 - 6
A ubiquitin conjugating enzyme encoded by African swine fever virus; Hingamp PM et al.; The post-translational modification of proteins by covalent attachment of ubiquitin occurs in all eukaryotes by a multi-step process . A family of E2 or ubiquitin conjugating (UBC) enzymes catalyse one step of this process and these have been implicated in several diverse regulatory functions . We report here the sequence of a gene encoded by African swine fever virus (ASFV) which has high homology with UBC enzymes . This ASFV encoded enzyme has UBC activity when expressed in Escherichia coli since it forms thiolester bonds with {125I}ubiquitin in the presence of purified ubiquitin activating enzyme (E1) and ATP, and subsequently transfers {125I}ubiquitin to specific protein substrates . These substrates include histones, ubiquitin and the UBC enzyme itself . The ASFV encoded UBC enzyme is similar in structure and enzyme activity to the yeast ubiquitin conjugating enzymes UBC2 and UBC3 . This is the first report of a virus encoding a functionally active UBC enzyme and provides an example of the exploitation of host regulatory mechanisms by viruses.

Mol Microbiol, 1992 Jan, 6(1), 5 - 14
Escherichia coli DNA helicases: mechanisms of DNA unwinding; Lohman TM; DNA helicases are ubiquitous enzymes that catalyse the unwinding of duplex DNA during replication, recombination and repair . These enzymes have been studied extensively; however, the specific details of how any helicase unwinds duplex DNA are unknown . Although it is clear that not all helicases unwind duplex DNA in an identical way, many helicases possess similar properties, which are thus likely to be of general importance to their mechanism of action . For example, since helicases appear generally to be oligomeric enzymes, the hypothesis is presented in this review that the functionally active forms of DNA helicases are oligomeric . The oligomeric nature of helicases provides them with multiple DNA-binding sites, allowing the transient formation of ternary structures, such that at an unwinding fork, the helicase can bind either single-stranded and duplex DNA simultaneously or two strands of single-stranded DNA . Modulation of the relative affinities of these binding sites for single-stranded versus duplex DNA through ATP binding and hydrolysis would then provide the basis for a cycling mechanism for processive unwinding of DNA by helicases . The properties of the Escherichia coli DNA helicases are reviewed and possible mechanisms by which helicases might unwind duplex DNA are discussed in view of their oligomeric structures, with emphasis on the E . coli Rep, RecBCD and phage T7 gene 4 helicases.

Mol Gen Genet, 1992 Jan, 231(2), 265 - 75
Alleviation of EcoK DNA restriction in Escherichia coli and involvement of umuDC activity; Hiom KJ et al.; The activity of the EcoK DNA restriction system of Escherichia coli reduces both the plating efficiency of unmodified phage lambda and the transforming ability of unmodified pBR322 plasmid DNA . However, restriction can be alleviated in wild-type cells, by UV irradiation and expression of the SOS response, so that 10(3)- to 10(4)-fold increases in phage growth and fourfold increases in plasmid transformation occurred with unmodified DNA . Restriction alleviation was found to be a transient effect because induced cells, which initially failed to restrict unmodified plasmid DNA, later restricted unmodified phage lambda . Although the SOS response was needed for restriction alleviation, constitutive SOS induction, elicited genetically with a recA730 mutation, did not alleviate restriction and UV irradiation was still needed . A hitherto unsuspected involvement of the umuDC operon in this alleviation of restriction is characterized and, by differential complementation, was separated from the better known role of umuDC in mutagenic DNA repair . The need for cleavage of UmuD for restriction alleviation was shown with plasmids encoding cleavable, cleaved, and non-cleavable forms of UmuD . However, UV irradiation was still needed even when cleaved UmuD was provided . The possibility that restriction alleviation occurs by a general inhibition of the EcoK restriction/modification complex was tested and discounted because modification of lambda was not reduced by UV irradiation . An alternative idea, that restriction activity was competitively reduced by an increase in EcoK modification, was also discounted by the lack of any increase in the modification of lambda Ral-, a naturally undermodified phage . Other possible mechanisms for restriction alleviation are discussed.

Mol Gen Genet, 1992 Jan, 231(2), 217 - 25
Cytochrome c1 from potato: a protein with a presequence for targeting to the mitochondrial intermembrane space; Braun HP et al.; Here we report the primary structure of potato cytochrome c1, a nuclear-encoded subunit of complex III . Using heterologous antibodies directed against cytochrome c1 from yeast two types of clones were isolated from an expression library, suggesting that at least two different genes are present and expressed in the genome . Northern blot analysis reveals that slightly varying levels of cytochrome c1 transcripts are present in all potato tissues analysed . A 1304 bp insert of one of the cDNA clones (pC13II) encodes the entire 320 amino acids of the precursor protein corresponding to a molecular weight of 35.2 kDa . As revealed by direct amino acid sequence determination of the cytochrome c1 protein another cDNA clone (pC18I) encodes the major form of cytochrome c1 present in potato tuber mitochondria . Western blots of subfractionated potato mitochondria show that the mature protein present in the membrane fraction is smaller than the pC13II encoded protein synthesized in Escherichia coli . The transient presequence of the protein is 77 amino acids long and has a bipartite polarity profile characteristic of presequences involved in targeting to the intermembrane space of fungal mitochondria . It consists of a positively charged NH2-terminal part which resembles "matrix targeting domains" and an adjacent hydrophobic region showing sequence similarities to "intramitochondrial sorting domains" . The amino-terminal region of potato cytochrome c1 is the first presequence of a plant protein of the mitochondrial intermembrane space to be determined and may be useful in the study of intramitochondrial sorting in plants.

Mol Gen Genet, 1992 Jan, 231(2), 201 - 11
Lethal overproduction of the Escherichia coli nucleoid protein H-NS: ultramicroscopic and molecular autopsy; Spurio R et al.; The Escherichia coli hns gene, which encodes the nucleoid protein H-NS, was deprived of its natural promoter and placed under the control of the inducible lambda PL promoter . An hns mutant yielding a protein (H-NS delta 12) with a deletion of four amino acids (Gly112-Arg-Thr-Pro115) was also obtained . Overproduction of wild-type (wt) H-NS, but not of H-NS delta 12, resulted in a drastic loss of cell viability . The molecular events and the morphological alterations eventually leading to cell death were investigated . A strong and nearly immediate inhibition of both RNA and protein synthesis were among the main effects of overproduction of wt H-NS, while synthesis of DNA and cell wall material was inhibited to a lesser extent and at a later time . Upon cryofixation of the cells, part of the overproduced protein was found in inclusion bodies, while the rest was localized by immunoelectron microscopy to the nucleoids . The nucleoids appeared condensed in cells expressing both forms of H-NS, but the morphological alterations were particularly dramatic in those overproducing wt H-NS; their nucleoids appeared very dense, compact and almost perfectly spherical . These results provide direct evidence for involvement of H-NS in control of the organization and compaction of the bacterial nucleoid in vivo and suggest that it may function, either directly or indirectly, as transcriptional repressor and translational inhibitor.

Genetics, 1992 Jan, 130(1), 7 - 16
Phage lambda has an analog of Escherichia coli recO, recR and recF genes; Sawitzke JA et al.; The RecF pathway catalyzes generalized recombination in Escherichia coli that is mutant for recBC, sbcB and sbcC . This pathway operating on conjugational recombination requires the recA, recF, recJ, recN, recO, recQ, recR, ruvA, ruvB and ruvC genes . In contrast, lambda mutant for its own recombination genes, int, red alpha and red beta, requires only the recA and recJ genes to recombine efficiently in recBC sbcB sbcC cells . Deletion of an open reading frame in the ninR region of lambda results in an additional requirement for recO, recR and recF in order to recombine in recBC sbcB sbcC mutant cells . This function, designated orf for recO-, recR- and recF-like function, is largely RecF pathway specific.

Arch Virol, 1992, 122(3-4), 383 - 90
Expression of feline immunodeficiency virus gag gene in Escherichia coli; Furuya T et al.; The gag gene of a Japanese feline immunodeficiency virus (FIV) isolate, designated as FIV TM 2, was expressed in Escherichia coli as a fusion protein with TrpE . Using this expressed protein, an enzyme-linked immunosorbent assay was developed for detection of antibodies to FIV gag protein in feline sera . With serum samples from a cat experimentally infected with FIV, it was demonstrated that the period of seroconversion detected by this method corresponded to that by Western blotting.

J Gen Virol, 1992 Jan, 73 ( Pt 1), 103 - 12
The unique N terminus of the herpes simplex virus type 1 large subunit is not required for ribonucleotide reductase activity; Conner J et al.; Using purified bacterially expressed herpes simplex virus type 1 ribonucleotide reductase large subunit (R1) and the proteolytic enzymes chymotrypsin and trypsin, we have generated stable N-terminal truncations . Chymotrypsin removes 246 amino acids from the amino terminus to produce a fragment (dN246R1) which retains full enzymic activity and affinity for the small subunit (R2) . Treatment of R1 with trypsin produces a 120K protein and a cleavage at amino acid residue 305 to produce a fragment (dN305R1) which remains associated with a 33K N-terminal polypeptide . Although this 33K-dN305R1 complex retains full binding affinity for R2 its reductase activity is reduced by approximately 50% . Increasing the concentration of trypsin removes the 33K N-terminal polypeptide resulting in dN305R1 which, when bound to R2, has full ribonucleotide reductase activity . Like R1, dN246R1 and dN305R1 each exist as dimers showing that the first 305 amino acids of R1 are not necessary for dimer formation . These results indicate that, in structural studies of subunit interaction, dN246R1 or dN305R1 can be considered as suitable replacements for intact R1.

Genes Dev, 1992 Jan, 6(1), 117 - 28
Ty3 integrates within the region of RNA polymerase III transcription initiation; Chalker DL et al.; Over 190 independent insertions into target plasmids of the retrovirus-like element Ty3 were recovered and mapped . Ty3 was shown to insert upstream of tRNA, 5S, and U6 genes, all of which are transcribed by RNA polymerase III . Integration sites were within 1-4 nucleotides of the position of transcription initiation, even for one mutant gene where the polymerase III initiation site was shifted to a completely new context . Mutagenesis of a SUP2 tRNA gene target showed that integration required functional promoter elements but that it did not correlate in a simple way with target transcription . This is the first report directly linking a discrete genomic function with preferential insertion of a retrotransposon.

Proc Natl Acad Sci U S A, 1992 Jan 1, 89(1), 392 - 6
Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system; Walker GT et al.; An isothermal in vitro DNA amplification method was developed based upon the following sequence of reaction events . Restriction enzyme cleavage and subsequent heat denaturation of a DNA sample generates two single-stranded target DNA fragments (T1 and T2) . Present in excess are two DNA amplification primers (P1 and P2) . The 3' end of P1 binds to the 3' end of T1, forming a duplex with 5' overhangs . Likewise, P2 binds to T2 . The 5' overhangs of P1 and P2 contain a recognition sequence (5'-GTTGAC-3') for the restriction enzyme HincII . An exonuclease-deficient form of the large fragment of Escherichia coli DNA polymerase I (exo- Klenow polymerase) {Derbyshire, V., Freemont, P . S., Sanderson, M . R., Beese, L., Friedman, J . M., Joyce, C . M . & Steitz, T . A . (1988) Science 240, 199-201} extends the 3' ends of the duplexes using dGTP, dCTP, TTP, and deoxyadenosine 5'-{alpha-thio}triphosphate, which produces hemiphosphorothioate recognition sites on P1.T1 and P2.T2 . HincII nicks the unprotected primer strands of the hemiphosphorothioate recognition sites, leaving intact the modified complementary strands . The exo- Klenow polymerase extends the 3' end at the nick on P1.T1 and displaces the downstream strand that is functionally equivalent to T2 . Likewise, extension at the nick on P2.T2 results in displacement of a downstream strand functionally equivalent to T1 . Nicking and polymerization/displacement steps cycle continuously on P1.T1 and P2.T2 because extension at a nick regenerates a nickable HincII recognition site . Target amplification is exponential because strands displaced from P1.T1 serve as targets for P2 and strands displaced from P2.T2 serve as targets for P1 . A 10(6)-fold amplification of a genomic sequence from Mycobacterium tuberculosis or Mycobacterium bovis was achieved in 4 h at 37 degrees C.

J Bacteriol, 1992 Jan, 174(2), 499 - 507
The oriT region of the conjugative transfer system of plasmid pCU1 and specificity between it and the mob region of other N tra plasmids; Paterson ES et al.; The oriT region of the conjugative IncN plasmid pCU1 has been localized to a 669-bp sequence extending from pCU1 coordinates 8.48 to 9.15 kb . The nucleotide sequence of this region was determined . The region is AT-rich (69% AT residues), with one 19-bp and one 81-bp sequence containing 79% or more AT residues . Prominent sequence features include one set of thirteen 11-bp direct repeats, a second set of two 14-bp direct repeats, six different inverted repeat sequences ranging from 6 to 10 bp in size, and two sequences showing 12 of 13 nucleotides identical to the consensus integration host factor binding sequence . Specificity between this oriT and mobilization (mob) functions encoded by the N tra system was demonstrated . This specificity is encoded by the region lying clockwise of the BglII site at coordinate 3.3 on the pCU1 map . Two N tra plasmids isolated in the preantibiotic era were unable to mobilize recombinant plasmids carrying the oriT region of pCU1 or to complement transposon Tn5 mutations in the mob region of the closely related plasmid pKM101.

J Bacteriol, 1992 Jan, 174(2), 398 - 407
Involvement of Fis protein in replication of the Escherichia coli chromosome; Filutowicz M et al.; We report evidence indicating that Fis protein plays a role in initiation of replication at oriC in vivo . At high temperatures, fis null mutants form filamentous cells, show aberrant nucleoid segregation, and are unable to form single colonies . DNA synthesis is inhibited in these fis mutant strains following upshift to 44 degrees C . The pattern of DNA synthesis inhibition upon temperature upshift and the requirement for RNA synthesis, but not protein synthesis, for resumed DNA synthesis upon downshift to 32 degrees C indicate that synthesis is affected in the initiation phase . fis mutations act synergistically with gyrB alleles known to affect initiation . oriC-dependent plasmids are poorly established and maintained in fis mutant strains . Finally, purified Fis protein interacts in vitro with sites in oriC . These interactions could be involved in mediating the effect of Fis on DNA synthesis in vivo.

J Bacteriol, 1992 Jan, 174(1), 56 - 62
Broad-specificity endoribonucleases and mRNA degradation in Escherichia coli; Srivastava SK et al.; Crude extracts from Escherichia coli were screened for any broad-specificity endoribonuclease after the cell proteins were fractionated by size . In a mutant lacking the gene for RNase I (molecular mass, 27,156 Da), the only such activities were also in the size range of 23 to 28 kDa . Fractionation by chromatography on a strong cation-exchange resin revealed only two activities . One of them eluted at a salt concentration expected for RNase M and had the specificity of RNase M . It preferred pyrimidine-adenosine bonds, could not degrade purine homopolymers, and had a molecular mass of approximately 27 kDa (V . J . Cannistraro and D . Kennell, Eur . J . Biochem . 181:363-370, 1989) . A second fraction, eluting at a higher salt concentration, was active against any phosphodiester bond but was about 100 times less active than are RNase I and RNase I* (a form of RNase I) in the wild-type cell . On the basis of sizing-gel chromatography, this enzyme had a molecular mass of approximately 24 kDa . We call it RNase R (for residual) . RNase R is not an abnormal product of the mutant rna gene; a cell carrying many copies of that gene on a plasmid did not synthesize more RNase R . Our search for broad-specificity endoribonucleases was prompted by the expectation that the primary activities for mRNA degradation are expressed by a relatively small number of broad-specificity RNases . If correct, the results suggest that the endoribonucleases for this major metabolic activity reside in the 24- to 28-kDa size range . Endoribonucleases with much greater specificity must have as primary functions the processing of specific RNA molecules at a very limited number of sites as steps in their biosynthesis . In exceptional cases, these endoribonucleases inactivate a specific message that has such a site, and they can also effect total mRNA metabolism indirectly by a global disturbance of the cell physiology . It is suggested that a distinction be made between these processing and degradative activities.

J Bacteriol, 1992 Jan, 174(1), 205 - 13
The pgpA and pgpB genes of Escherichia coli are not essential: evidence for a third phosphatidylglycerophosphate phosphatase; Funk CR et al.; To further define the genes and gene products responsible for the in vivo conversion of phosphatidylglycerophosphate to phosphatidylglycerol in Escherichia coli, we disrupted two genes (pgpA and pgpB) which had previously been shown to encode gene products which carried out this reaction in vitro (T . Icho and C . R . H . Raetz, J . Bacteriol . 153:722-730, 1983) . Strains with either gene or both genes disrupted had the same properties as the original mutants isolated with mutations in these genes, i.e., reduced in vitro phospholipid phosphatase activities, normal growth properties, and an increase in the level of phosphatidylglycerophosphate (1.6% versus less than 0.1% in wild-type strains) . These results demonstrate that these genes are not required for either normal cell growth or the biosynthesis of phosphatidylglycerol in vivo . In addition, the total phosphatidylglycerophosphate phosphatase activity in the doubly disrupted mutant was reduced by only 50%, which indicates that there is at least one other gene that encodes such an activity and thus accounts for the lack of a dramatic effect on the biosynthesis of anionic phospholipids in these mutant strains . The phosphatidic acid and lysophosphatidic acid phosphatase activities of the pgpB gene product were also significantly reduced in gene-interrupted mutants, but the detection of residual phosphatase activities in these mutants indicated that additional genes encoding such phosphatases exist . The lack of a significant phenotype resulting from disruption of the pgpA and pgpB genes indicates that these genes may be required only for nonessential cell function and leaves the biosynthesis of phosphatidylglycerophosphate as the only step in E . coli phospholipid biosynthesis for which a gene locus has not been identified.

J Bacteriol, 1992 Jan, 174(1), 173 - 8
High-molecular-weight linear multimer formation by single-stranded DNA plasmids in Escherichia coli; Dabert P et al.; We inserted foreign DNA segments into plasmids which replicate by a rolling-circle mechanism in Escherichia coli and observed the appearance of high-molecular-weight plasmid multimers (HMW) . This phenomenon, which occurs more frequently with GC-rich segments, depends on the mode of replication of the plasmid and on host homologous recombination functions . We found that (i) HMW are formed upon insertion of a foreign DNA segment into a single-stranded DNA plasmid, whereas the same DNA insert has no such effect on a theta replicon, and (ii) HMW are not present in a recA mutant strain but are found in a lexA (Ind-) mutant . Enzymatic studies allowed us to define the HMW structure as linear double-stranded tandem head-to-tail plasmid repeats . Use of heteroplasmid strains showed that HMW production by one plasmid does not affect another resident plasmid, indicating that no host functions are phenotypically inactivated . This distinguishes our system from the HMW observed with various replicons in the absence of RecBCD enzyme activity . We propose that the role of the foreign insert is to protect the DNA from RecBCD exonuclease attack.

J Virol, 1992 Jan, 66(1), 458 - 68
The UL5 gene of herpes simplex virus type 1: isolation of a lacZ insertion mutant and association of the UL5 gene product with other members of the helicase-primase complex; Zhu LA et al.; The UL5 gene product is required continuously during viral DNA synthesis (L . Zhu and S . K . Weller, Virology 166:366-378, 1988) and has been shown to be a component of a three protein helicase-primase complex encoded by herpes simplex virus type 1 (J . J . Crute, T . Tsurumi, L . Zhu, S . K . Weller, P.D . Olivo, M . D . Challberg, E . S . Mocarski, and I . R . Lehman, Proc . Natl . Acad . Sci . USA 86:2186-2189, 1989) . The other members of the complex are viral proteins encoded by genes UL8 and UL52 . In this study, we isolated a permissive cell line (L2-5) which contains the wild-type UL5 gene under the control of the strong and inducible promoter for the large subunit of herpes simplex virus type 1 ribonucleotide reductase (ICP6) . An insertion mutant containing a mutation in the UL5 gene (hr99) was isolated by using the insertional mutagen ICP6::lacZ, in which the Escherichia coli lacZ gene is expressed under control of the viral ICP6 promoter . When grown on Vero cells, hr99 does not form plaques or synthesize viral DNA, although both defects are complemented efficiently on the L2-5 cells . These results confirm that the UL5 gene product is essential for viral growth and DNA replication . Furthermore, since no detectable UL5 protein is synthesized in hr99-infected cells, these cells provide a valuable control not only for the detection of the UL5 protein itself but also for the detection of protein-protein interactions with UL8 and UL52 by coimmunoprecipitation . In addition, the lacZ insertion in hr99 provides a convenient screening system for the introduction of site-specific mutations into the viral genome (L . Zhu and S . K . Weller, J . Virol . 66:469-479, 1992) . Thus, hr99 is a valuable tool in the structure-function analysis of the UL5 gene.

J Virol, 1992 Jan, 66(1), 341 - 8
Construction and properties of a mutant of herpes simplex virus type 1 with glycoprotein H coding sequences deleted; Forrester A et al.; A mutant of herpes simplex virus type 1 (HSV-1) in which glycoprotein H (gH) coding sequences were deleted and replaced by the Escherichia coli lacZ gene under the control of the human cytomegalovirus IE-1 gene promoter was constructed . The mutant was propagated in Vero cells which contained multiple copies of the HSV-1 gH gene under the control of the HSV-1 gD promoter and which therefore provide gH in trans following HSV-1 infection . Phenotypically gH-negative virions were obtained by a single growth cycle in Vero cells . These virions were noninfectious, as judged by plaque assay and by expression of beta-galactosidase following high-multiplicity infection, but partial recovery of infectivity was achieved by using the fusogenic agent polyethylene glycol . Adsorption of gH-negative virions to cells blocked the adsorption of superinfecting wild-type virus, a result in contrast to that obtained with gD-negative virions (D . C . Johnson and M . W . Ligas, J . Virol . 62:4605-4612, 1988) . The simplest conclusion is that gH is required for membrane fusion but not for receptor binding, a conclusion consistent with the conservation of gH in all herpesviruses.

Blood Purif, 1992, 10(3-4), 132 - 5
Diaphragmatic lymph vessel drainage of the peritoneal cavity; Drake RE et al.; We have studied the drainage of peritoneal fluid through the diaphragmatic lymph vessels in sheep . To measure the lymphatic flow rate, we cannulated the lymphatic vessels and timed the flow from the cannula . After we infused Escherichia coli endotoxin into awake sheep, the diaphragmatic lymph flow rate increased substantially . However, we found no increase in lymph flow in anesthetized acutely operated sheep . This indicates that studies in anesthetized animals may yield underestimates of diaphragmatic lymph flow . In sheep, many of the diaphragmatic lymph vessels drain to the caudal mediastinal lymph node . We cannulated an efferent vessel from that node in 5 sheep . Several days later we infused 100 ml/kg of Ringer's solution into the abdominal space of each awake sheep . In response, the lymph flow rate increased from 0.15 +/- 0.16 ml/min (mean +/- SD) to 0.50 +/- 0.17 ml/min . Our results are important because they demonstrate that diaphragmatic lymph flow increases substantially after fluid infusions into the abdominal space.

Acta Microbiol Hung, 1992, 39(2), 169 - 73
Modified spot hybridization test using biotinylated DNA probe; Venkateswaran N et al.; A modification that simplifies the spot hybridization technique is described for using biotinylated DNA probes . Plasmid EWD299 having LT gene insert, labelled with biotin either by nick translation or using photobiotin was used as DNA probe for the specific detection of enterotoxigenic Escherichia coli . A simple protocol has been described for easy lysis of test samples by boiling in distilled water followed by detergent treatment and was found to be as efficient as the lysis using lysozyme and protease . Three different solid supports namely DEAE-cellulose paper, nitrocellulose paper and nylon membrane were also compared for their suitability in this spot hybridization test . Nitrocellulose paper was found to give better colour signal with the photobiotinylated DNA probe.

Acta Vet Hung, 1992, 40(4), 267 - 77
The involvement of polymorphonuclear leukocytes in the pathogenesis of bronchopneumonia in calves . VI . Superoxide dismutase and lipoprotein lipase activities; Ledwozyw A et al.; The kinetics of superoxide anion production in polymorphonuclear leukocytes of bronchopneumonic calves, as well as superoxide dismutase and lipoprotein lipase activities in the blood plasma of these animals were studied . Granulocytes of bronchopneumonic calves were found to produce 10 times as much O2- radicals as those of healthy animals . Superoxide dismutase and lipoprotein lipase activities were lower in the plasma of bronchopneumonic calves . Escherichia coli endotoxin was found to stimulate superoxide anion production in polymorphonuclear leukocytes in a dose-dependent manner . These facts suggest that an endogenous mediator, probably cachectin or some other monokine(s), plays a role in granulocyte activation during calf bronchopneumonia.

Protein Sci, 1992 Jan, 1(1), 31 - 45
The importance of surface loops for stabilizing an eightfold beta alpha barrel protein; Urfer R et al.; An important step in understanding how a protein folds is to determine those regions of the sequence that are critical to both its stability and its folding pathway . We chose phosphoribosyl anthranilate isomerase from Escherichia coli, which is a monomeric representative of the (beta alpha)8 barrel family of proteins, to construct a variant that carries an internal tandem duplication of the fifth beta alpha module . This (beta alpha)9 variant was enzymically active and therefore must have a wild-type (beta alpha)8 core . It had a choice a priori to fold to three different folding frames, which are distinguished by carrying the duplicated segment as an insert into one out of three different loops . Steady-state kinetic constants, the fluorescence properties of a crucial tryptophan residue, and limited proteolysis showed that the stable (beta alpha)9 variant carries the insertion between beta-strand 5 and alpha-helix 5 . This preference can be explained by the important role of loops between alpha helices and beta strands in stabilizing the structure of the enzyme.

Protein Sci, 1992 Jan, 1(1), 22 - 30
Reduction of thioredoxin significantly decreases its partial specific volume and adiabatic compressibility; Kaminsky SM et al.; The partial specific volume and adiabatic compressibility were determined at several temperatures for oxidized and reduced Escherichia coli thioredoxin . Oxidized thioredoxin had a partial specific volume of 0.785-0.809 mL/g at the observed upper limit for all proteins whereas the partial specific volume of reduced thioredoxin was 0.745-0.755 mL/g, a value in the range found for a majority of proteins . The adiabatic compressibility of oxidized thioredoxin was also much larger (9.8-18 x 10(-12) cm2 dyne-1) than that of the reduced protein (3.8-7.3 x 10(-12)) . Apart from the region immediately around the small disulfide loop, the structures of the oxidized (X-ray, crystal) and reduced protein (nuclear magnetic resonance, solution) are reported to be very similar . It would appear that alterations in the solvent layer in contact with the protein surface must play a major role in producing these large changes in the apparent specific volumes and compressibilities in this system . Some activities of thioredoxin require the reduced structure but are not electron transfer reactions . The large changes in physical parameters reported here suggest the possibility of a reversible metabolic control function for the SS bond.

Neurochem Int, 1992 Jan, 20(1), 129 - 34
Expression of recombinant human nerve growth factor in Escherichia coli; Dicou E; Nerve growth factor (NGF) is a neurotrophic factor for basal forebrain cholinergic neurons and may be of benefit in neurodegenerative diseases of humans . A method is described to obtain significant amounts of biologically active recombinant human NGF (rhNGF) in one step . RhNGF was expressed in E . coli and the majority of the protein accumulated in inclusion bodies . It was immunoprecipitated by a serum against mouse NGF . Solubilization of the inclusion bodies was done in 3M guanidine HCl and renaturation was effected by dilution and air oxidation in the presence of 6 microM CuSO4 . Recoveries were 10-12 micrograms of rhNGF per ml of bacterial suspension . Its biological activity was tested in a bioassay system employing sympathetic chick embryo ganglia and was inhibited by the monoclonal antibody 27/21 against mouse NGF.

Plant J, 1992 Jan, 2(1), 25 - 34
Identification of a cDNA for the plastid-located geranylgeranyl pyrophosphate synthase from Capsicum annuum: correlative increase in enzyme activity and transcript level during fruit ripening; Kuntz M et al.; Geranylgeranyl pyrophosphate synthase is a key enzyme in plant terpenoid biosynthesis . Using specific antibodies, a cDNA encoding geranylgeranyl pyrophosphate synthase has been isolated from bell pepper (Capsicum annuum) ripening fruit . The cloned cDNA codes for a high molecular weight precursor of 369 amino acids which contains a transit peptide of approximately 60 amino acids . In-situ immunolocalization experiments have demonstrated that geranylgeranyl pyrophosphate synthase is located exclusively in the plastids . Expression of the cloned cDNA in E . coli has unambiguously demonstrated that the encoded polypeptide catalyzes the synthesis of geranylgeranyl pyrophosphate by the addition of isopentenyl pyrophosphate to an allylic pyrophosphate . Peptide sequence comparisons revealed significant similarity between the sequences of the C . annuum geranylgeranyl pyrophosphate synthase and those deduced from carotenoid biosynthesis (crtE) genes from photosynthetic and non-photosynthetic bacteria . In addition, four highly conserved regions, which are found in various prenyltransferases, were identified . Furthermore, evidence is provided suggesting that conserved and exposed carboxylates are directly involved in the catalytic mechanism . Finally, the expression of the geranylgeranyl pyrophosphate synthase gene is demonstrated to be strongly induced during the chloroplast to chromoplast transition which occurs in ripening fruits, and is correlated with an increase in enzyme activity.

Tsitologiia, 1992, 34(11-12), 76 - 83
{Survival and the repair of single-stranded DNA breaks in gamma-irradiated Escherichia coli cells adapted to methylmethane sulfonate}; Zhestianikov VD et al.; The survival and repair of single-strand breaks of DNA in gamma-ray-irradiated E . coli adapted to MMS (20 mkg/ml during 3 hours) have been investigated . It is shown that the survival of adapted bacteria of radioresistant strains B/r, H/r30, AB1157 and W3110 pol+ increases with DMF (dose modification factor) ranging within 1.4-1.8 and in radiosensitive strains Bs-1, AB1157 recA13 and AB1157 lexA3 with DMF ranging within 1.3-1.4, and does not change in strains with mutation in polA gene P3478 polA1 and 016 res-3 . There is no increase in radioresistance during the adaptation to MMS under the action of the protein synthesis inhibitor chloramphenicol . The increase in radioresistance during the adaptation to MMS correlates with the acceleration of repair of gamma-ray-induced single-strand breaks in the radioresistant strains B/r and W3110 pol+ and with the appearance of the ability to repair some part of DNA single-strand breaks in the mutant Bs-1, which beyond the adaptation to MMS does not repair these damages . The incomplete reparability of DNA single-strand breaks in P3478 polA1 strain cells, both adapted and non-adapted to MMS, is equal.

J Nutr Sci Vitaminol (Tokyo), 1992, Spec No, 383 - 6
Thiamin triphosphate synthesis in animals; Kawasaki T; All the evidence obtained indicates that the synthesis of TTP in vitro and in vivo is catalyzed by cytosolic adenylate kinase . Further studies should explore whether adenylate kinase is the only enzyme involved in TTP synthesis, together with physiological roles of TTP in living organisms.

Chin J Biotechnol, 1992, 8(2), 93 - 8
High-level expression of recombinant protein A in Escherichia coli; Cai S et al.; A high-level expression plasmid pPA-3 was constructed, which yielded up to 20% of soluble cell proteins as recombinant Protein A in E . coli DH5 alpha strain by heat shock induction . The recombinant Protein A contained only the five domains of native Protein A that bind with the Fc part of IgG . The molecular weights of the recombinant Protein A expressed in E . coli were determined to be 33kDa, 32.2kDa, 29.5kDa and 28.6kDa by SDS-PAGE and Western-blotting . The diversity of the molecular weights may be due to proteolysis, and a possible cleavage site is proposed . Some of the protein was purified with coupled human IgG by one-step affinity chromatography . The reactivity of the protein was compared with that of native Protein A and found that this protein could bind with more IgG than native Protein A in equal quantities of proteins.

Chin J Biotechnol, 1992, 8(2), 107 - 12
Cloning of par region and the effect of par region on the stability of pUC9; Wang Y et al.; The par region of pSC101 was cloned into pUC9, and pEC302 was obtained . The stability of pEC302 and pUC9 in E . coli HB101 was tested on M9 medium to determine the effect of par region on the stability of plasmid . After 40 generations, pUC9 was almost completely lost, while pEC302 was 100% maintained in host cells.

Folia Microbiol (Praha), 1992, 37(6), 395 - 400
Construction of shuttle vectors for cloning in Escherichia coli and Acetobacter pasteurianus; Grones J et al.; New cloning vectors were prepared with the aid of a large plasmid isolated from Acetobacter pasteurianus and from plasmids pBR322 and pUC4-KAPA . Of the prepared cloning vectors, pACK5 contains a gene coding for kanamycin resistance, pACT7 and pACT71 contain a gene coding for tetracycline resistance and vector pACG3 with a gene coding for both kanamycin and tetracycline resistance . The vectors prepared only contained the beginning of replication from the pAC1 plasmid and possessed the ability to replicate within E . coli and A . pasteurianus . The vectors are highly stable in both strains and during the 5-d cultivation under nonselective conditions are not eliminated.

Vet Res Commun, 1992, 16(6), 453 - 64
Endotoxin-induced microvascular injury in isolated and perfused pig lungs; Urbain B et al.; The lungs of 13 healthy Landrace piglets were isolated, perfused and maintained in an isogravimetric state under zone III conditions . By applying vascular occlusion methods, the total blood flow resistance (Rt) was partitioned into four components: arterial (Ra), pre- (Ra') and post-capillary (Rv'), and venous (Rv) . The capillary filtration coefficient (Kfc) was evaluated using a gravimetric technique . A bolus of 55 micrograms of Escherichia coli endotoxins (LPS) per 100 g of lung was injected into the arterial reservoir of eight lungs, followed by an infusion of LPS at a rate of 55 micrograms per 100 g of lung per hour for 180 min . A bolus of theophylline (85 mg per 100 g of lung weight) was injected into the arterial reservoir after the last determination of the Kfc value . All the parameters were evaluated again when the lungs reached a new steady state . Endotoxin induced a significant increase in Rt from 54.7 +/- 7.0 at zero time to 184.7 +/- 44.2 cmH2O min L-1 (100 g)-1 180 minutes later, which can be ascribed to the increase in Ra' and Rv' . These haemodynamic modifications were related to the increases in the arterial pressure and in the pressure at the distal end of the arterial segment and to the decreases in the pressure at the proximal end of the venous segment and in the blood flow . The capillary pressure and the lung weight remained unchanged . Endotoxin infusion induced an increase in the Kfc value from 0.208 +/- 0.011 (at t = 0) to 0.391 +/- 0.034 ml min-1 (cmH2O)-1 (100 g)-1 (at t = 180) . Administration of theophylline significantly reduced Rt,Ra,Ra' and Rv' towards or under the baseline values and also induced a significant increase in the lung weight and in the Kfc value . It was concluded that the endotoxin-induced increase in the total blood flow resistance can be ascribed to a vasospasm occurring at the level of the pre- and post-capillary small vessels and that changes in the permeability of the endothelium greatly contribute to the development of the pulmonary oedema observed in endotoxaemic pigs.

Chromosoma, 1992, 102(1 Suppl), S157 - 60
Replisome pausing in mutagenesis; Moses RE et al.; E . coli cells containing a temperature-sensitive dnaE mutation, in the alpha-subunit of holoenzyme DNA polymerase III, do not survive at the restrictive temperature . Such cells may survive in the presence of the pcbA1 mutation, an allele of the gyrB gene . Such survival is dependent on an active DNA polymerase I . Evidence indicates that DNA polymerase I interacts directly in the replisome (REP.A) . Despite normal survival for cells using the pcbA replication pathway after some type of DNA damage, we have noted a failure of damage-induced mutagenesis . Here we present evidence supporting a model of replisome pausing in cells dependent upon the pcbA replication pathway . The model argues that the (REP.A) complex pauses longer at the site of the lesion, allowing excision repair to occur completely . In the normal replication pathway (REP.E) bypass of the lesion occurs, fixing the mutation.

Chromosoma, 1992, 102(1 Suppl), S121 - 7
Structural and functional relationships of human DNA polymerases; Hao H et al.; A continuing theme of our laboratory has been the understanding of human DNA polymerases at the structural level . We have purified DNA polymerases delta, epsilon and alpha from human placenta . Monoclonal antibodies to these polymerases were isolated and used as tools to study their immunochemical relationships . These studies have shown that while DNA polymerases delta, epsilon and alpha are discrete proteins, they must share common structural features by virtue of the ability of several of our monoclonal antibodies to exhibit cross-reactivity . A second approach we have taken is the molecular cloning of human DNA polymerase delta and epsilon . We have cloned the DNA polymerase delta cDNA, and this has allowed us to compare its primary structure to those of human polymerase alpha and other members of this polymerase family . Multiple sequence alignments have revealed that human DNA polymerase delta is also closely related to the herpes virus family of DNA polymerases . In situ hybridization has shown that the human DNA polymerase delta gene is localized to chromosome 19 q13.3-q13.4 . In order to further determine the functional regions of the DNA polymerase delta structure we are currently expressing human pol delta in E . coli and baculovirus systems . Other work in our laboratory is directed toward examining the expression of DNA polymerase delta during the cell cycle.

Chromosoma, 1992, 102(1 Suppl), S1 - 6
The complex for replication initiation of Escherichia coli; Messer W et al.; We probed the complex between oriC and DnaA protein using two types of mutants in oriC . Base changes in the DnaA binding sites, DnaA boxes, had little effect on origin function . Mutations which change the distance between DnaA boxes R3 and R4, on the other hand, inactivated oriC unless the mutation deleted or inserted one complete helical turn . Origins with other 10 base pair insertions in the interval between DnaA boxes R2 and R3 were functional, but not insertions in the R1-R2 interval . FIS protein binds to a bipartite site in oriC between DnaA boxes R2 and R3 . A model for the oriC/DnaA complex based on these results suggests an array of DnaA monomers with a 34 A spacing upon which oriC is arranged.

Faraday Discuss, 1992, (93), 269 - 80
Modelling the structure and function of enzymes by machine learning; Sternberg MJ et al.; A machine learning program, GOLEM, has been applied to two problems: (1) the prediction of protein secondary structure from sequence and (2) modelling a quantitative structure-activity relationship in drug design . GOLEM takes as input observations and combines them with background knowledge of chemistry to yield rules expressed as stereochemical principles for prediction . The secondary structure prediction was explored on the alpha/alpha class of proteins; on an unrelated test set it yielded 81% accuracy . The rules from GOLEM defined patterns of residues forming alpha-helices . The system studied for drug design was the activities of trimethoprim analogues binding to E . coli dihydrofolate reductase . The GOLEM rules were a better model than standard regression approaches . More importantly, these rules described the chemical properties of the enzyme-binding site that were in broad agreement with the crystallographic structure.

Urol Radiol, 1992, 14(3), 155 - 8
Lymphangioma presenting as a small renal mass during childhood; Levine E; Renal lymphangioma is a very rare lesion . A case of lymphangioma that presented as a small, hyperechoic renal mass on sonography in a child is reported . On CT, the lesion appeared as a low-density, enhancing renal mass . Despite its rarity, lymphangioma should be considered in the differential diagnosis of such a lesion . A suspected lymphangioma may be evaluated by percutaneous biopsy.

Nucleic Acids Symp Ser, 1992, (27), 197 - 8
Sequence specific block of in vitro DNA synthesis with isopropyl phosphotriesters in template oligodeoxyribonucleotides; Yashiki T et al.; We have synthesized four oligodeoxyribonucleotides each bearing an isopropyl phosphotriester at a defined position . These oligomers were used as templates for in vitro DNA synthesis catalyzed by Escherichia coli DNA polymerase I large fragment . Results showed that the phosphotriester inhibits the DNA chain elongation and the level of the inhibition is dependent on the base 5' to the phosphotriester.

Nucleic Acids Symp Ser, 1992, (27), 189 - 90
Action of 5-trifluoromethyl-2'-deoxyuridine on DNA synthesis; Satake H et al.; The action of 5-trifluoromethyl-2'-deoxyuridine (CF3dUrd) on DNA synthesis was investigated in vitro assay systems with purified DNA polymerases . CF3dUrd was incorporated into the DNA of mammalian cells in culture . We studied the incorporation of CF3dUrd 5'-triphosphate (CF3dUTP) into DNA and effect of CF3dUrd residue on DNA synthesis . Therefore, we synthesized oligonucleotides that allow site specific introduction of a CF3dUrd residue into a synthetic DNA oligonucleotide . After CF3dUTP incorporation, the primer was extended for human DNA polymerase alpha (pol . alpha) . When CF3dUrd residue was located at an internucleotide site in the template, however, pol . alpha was exhibited a strong arrest band one nucleotide after the CF3dUrd residue site, and Escherichia coli polymerase I (Klenow fragment) also exhibited a weaker arrest band one nucleotide before the CF3dUrd residue . These results suggested that a mechanism of antitumor activity of CF3dUrd is inhibition of DNA replication.

Nucleic Acids Symp Ser, 1992, (27), 157 - 8
Topological alteration of plasmid DNA during cell growth of Escherichia coli; Furuno A et al.; Superhelical alteration of plasmid DNA in Escherichia coli cells was observed during cell growth . Plasmid DNAs were being supercoiled while the cells were actively dividing, and they were partially relaxed after the cultures reached stationary . Unknown structural materials of plasmid DNA which migrated faster than usual superhelices in agarose gel electrophoresis were found among the strains regardless of gyrase genotype.

Nucleic Acids Symp Ser, 1992, (27), 149 - 50
Cloning and sequence analysis of the evgAS genes involved in signal transduction of Escherichia coli K-12; Utsumi R et al.; We have cloned and sequenced new Escherichia coli genes which belong to member of the family of environmentally responsive two-component system and named evgA and evgS because their amino acid sequences were found the most homologus to the Bordetella pertussis bvgA and bvgS . They were mapped at 51 min . and extending from 6B9 to 7G9 in the Kohara miniset library of the E . coli chromosome . In fact, both EvgA and EvgS proteins predicted from their DNA sequences were identified in the in vitro coupled transcription translation system . When the evgA and evgS were expressed on multiple copy plasmid in an envZ deletion strain, ompC expression was also regulated by temperature, MgSO4 and nicotinic acid, by which virulence of Bordetella pertussis is controlled via BvgA and BvgS . These results indicate that ompC expression was controlled by in vivo cross-talk via EvgA and EvgS which can work in E . coli the same way as BvgA and BvgS.

Nucleic Acids Symp Ser, 1992, (27), 101 - 2
Molecular mechanisms for controlling spontaneous and induced mutagenesis; Sekiguchi M et al.; Errors in the replication of DNA are a major source of spontaneous mutations, and a number of cellular functions are involved in correction of these errors to keep the frequency of spontaneous mutations very low . We report here a novel mechanism which prevents replicational errors by degrading a potent mutagenic substrate for DNA synthesis . We also deal with suppression of alkylation-induced mutations by O6-methylguanine-DNA methyltransferase.

Implant Dent, 1992 Fall, 1(3), 195 - 202
Fibroblastic growth and attachment on hydroxyapatite-coated titanium surfaces following the use of various detoxification modalities . Part II: Contaminated hydroxyapatite; Zablotsky MH et al.; This study evaluated the ability of various chemotherapeutic and mechanical modalities to detoxify endotoxin-contaminated hydroxyapatite-coated dental implant surfaces as determined by the early attachment and growth of human gingival fibroblasts . Hydroxyapatite-coated test strips were contaminated with purified outer membranes of Escherichia coli and treated with citric acid, hydrogen peroxide, stannous fluoride, chlorhexidine gluconate, tetracycline HCl, polymyxin B, a plastic sonic scaler tip, or left untreated (contaminated and sterile controls) . Human gingival fibroblasts were then seeded onto the test strips and incubated for 48 hours . The citric acid-treated strips showed greater cell growth than the other treatments . The plastic sonic scaler tip and the polymyxin B-treated samples exhibited greater cell coverage than the sterile control specimens . The use of citric acid and/or a modified plastic sonic scaler tip may be a valuable adjunct when surgical repair of an ailing hydroxyapatite-coated dental implant is contemplated.

Prog Lipid Res, 1992, 31(3), 245 - 99
Metabolic regulations and biological functions of phospholipids in Escherichia coli; Shibuya I; Extensive genetic and biochemical studies in the last two decades have elucidated almost completely the framework of synthesis and turnover of quantitatively major phospholipids in E . coli . The knowledge thus accumulated has allowed to formulate a novel working model that assumes sophisticated regulatory mechanisms in E . coli to achieve the optimal phospholipid composition and content in the membranes . E . coli also appears to possess the ability to adapt phospholipid synthesis to various cellular conditions . Understanding of the functional aspects of E . coli phospholipids is now advancing significantly and it will soon be able to explain many of the hitherto unclear cell's activities on the molecular basis . Phosphatidylglycerol is believed to play the central role both in metabolism and functions of phospholipids in E . coli . The results obtained with E . coli should undoubtedly be helpful in the study of more complicated phospholipid metabolism and functions in higher organisms.

Beitr Infusionsther, 1992, 30, 78 - 81
Recombinant antigens in viral diagnosis; Vornhagen R et al.; DNA fragments coding for a variety of different viral antigens have been cloned and expressed in Escherichia coli . Selected purified recombinant antigens were used for detection of specific antibodies by means of the ELISA technique . This approach has been used for the development of four different ELISAs for the detection of HIV- and EBV-specific antibodies.

J Nutr Sci Vitaminol (Tokyo), 1992, Spec No, 337 - 40
Structure of G-CSF: significance of the sugar chain; Ono M et al.; 1 . The carbohydrate chain protects rhG-CSF from polymerization and/or conformational alterations associated with physicochemical changes, elevation of pH or temperature fluctuations . 2 . The carbohydrate chain of rhG-CSF prevents loss of its biological activity in normal human serum by inhibiting proteinase activity . 3 . These facts indicate that the carbohydrate chain of rhG-CSF has a markedly important role in maintaining the stability of the protein itself as well as in effecting the exertion of its biological activity.

Chin J Biotechnol, 1992, 8(2), 82 - 91
Overexpression of rnc gene and purification of RNaseIII; Chen S et al.; The reason for low content of RNaseIII in E . coli is that RNaseIII has a negative feedback action on its own synthesis by cutting off the transcripts of its own gene, rnc, at 5'-terminal . On this basis, the scheme for overproduction of RNaseIII was designed . The 5'-flanking sequence of rnc gene transcribing to form a secondary structure which could be degraded by RNaseIII was removed, and the whole coding sequence, including the translational initiation signal, was reserved and put under the control of lambda PL promoter . The constructed plasmid pCR21, which contain the recombined rnc gene, overproduced RNaseIII, which covered over 65% of the total cell protein and formed inclusive bodies in E . coli after induction at 42 degrees C . By using the characteristics of solubility of the protein, electrophoretic pure RNaseIII was obtained with a simple procedure, including lysis of the bacterial cells, washing precipitates of the lysate repeatedly at low temperature and low salt concentration, and dissolving and passing through Q-Sepharose FF column in high salt concentration at room temperature . The yield of purified RNaseIII was 10-12 mg per 100 ml culture, and lambda sib transcripts were cut at special sites . RNaseIII possessing the activity of binding ATP is reported.

Folia Biol (Praha), 1992, 38(6), 350 - 7
A novel panel of monoclonal antibodies against beta-galactosidase of Escherichia coli and its versatility for detection of recombinant expression products; Slavickova A et al.; A panel of seven mouse monoclonal antibodies (BG-O1 - BG-07) raised against beta-galactosidase (beta-gal) from E . coli was characterized in respect of their binding to beta-gal and to fusion proteins . The antibodies were beta-gal specific, recognized six different antigenic determinants on beta-gal molecule and some of them inhibited catalytical activity of the enzyme . The antibodies reacted with C-terminal beta-gal fusion proteins in the native state as well as after Western blotting . BG-02 antibody was successfully used for immunofluorescence detection of cells transfected with vectors containing the lacZ gene.

Nucleic Acids Symp Ser, 1992, (27), 131 - 2
15N and 13C labeling of Escherichia coli tRNAs toward the NMR analysis; Kawai G et al.; Escherichia coli tRNAs were labeled with stable isotope 15N in vivo . Three species of tRNA, tRNA(Glu), tRNA(Lys) and tRNA(Ile), were purified by an HPLC system and their NMR spectra were observed . In heteronuclear 1H-15N multiple or single quantum coherence (HMQC or HSQC) spectra, the crosspeaks corresponding to NH3 of U and NH1 of G can be distinguished clearly since their 15N chemical shifts are significantly different from each other . Thus, this combination of 15N-labeling and the proton detected heteronuclear experiments are useful for the signal assignment and the conformational analysis of tRNAs . Furthermore, C1'- selective 13C-labeling of nucleotides was examined in vivo in order to resolve the H1' signals of tRNAs . By using a newly constructed E . coli mutant strain, the isotopic enrichments of more than 90% at C1' and of less than 10% for other ribose carbons were achieved.

Vestn Ross Akad Med Nauk, 1992, (9-10), 55 - 9
{Study of antigenic structure of HIV-1 protein p24 using monoclonal antibodies}; Konovalov EE et al.; During the experiments 4 murine and 3 rat hybridomas producing monoclonal antibodies (MAb) against the protein p24 of human immunodeficiency virus type 1 (HIV-1) have been obtained . Using the immunoblotting technique, it was established that all the species of MAb reacted with the same viral proteins which are derivatives of gag gene--p24 and p55 . The properties of MAb have been studied in competitive binding . Their ability of binding to different fragments of the gag protein produced by the recombinant plasmids in E . coli cells have been investigated in ELISA . The analysis of the findings suggests that the HIV-1 protein p24 contains at least 3 antigenic epitopes . All species of MAb reacted with 3 different HIV-1 strains and 2 HIV-1 isolates, but failed with 2 different HIV-2 strains . The only MAb NS5E4 can be used as an immunosorbent in the antigenic capture reaction.

Agents Actions, 1992, Spec No, C3 - 9
Actions of nitric oxide on the acute gastrointestinal damage induced by PAF in the rat; Boughton-Smith NK et al.; The actions of nitric oxide (NO) on the acute gastrointestinal damage induced by platelet-activating factor (PAF) have been investigated in the rat . S-nitroso-N-acetyl penicillamine, which spontaneously generates NO, dose-dependently inhibited PAF-induced gastrointestinal plasma leakage, a measure of the initiation of vascular damage . The inhibitor of NO synthase, NG-monomethyl-L-arginine substantially potentiated gastrointestinal damage and plasma leakage induced by E . coli endotoxin, but had no effect on that induced by intravenous infusion of PAF . Endogenous NO may thus have a protective role in the gastrointestinal vascular that can be mimicked by generators of NO . The protection afforded by endogenous NO may, however, be dependent on the nature of the inflammatory stimulus used to induce gastrointestinal damage.

Biochem Biophys Res Commun, 1991 Dec 31, 181(3), 947 - 54
Amino-terminal presequence of the precursor of peroxisomal 3-ketoacyl-CoA thiolase is a cleavable signal peptide for peroxisomal targeting; Osumi T et al.; To examine the function of the amino-terminal presequence of rat peroxisomal 3-ketoacyl-CoA thiolase precursor, fusion proteins of various amino-terminal regions of the precursor with non-peroxisomal enzymes were expressed in cultured mammalian cells . On immunofluorescence microscopy, all constructs carrying the presequence part exhibited punctate patterns of distribution, identical with that of catalase, a peroxisomal marker . Proteins lacking all or a part of the prepiece were found in the cytosol . These results indicate that the presequence of the thiolase has sufficient information for peroxisomal targeting.

Biochem Biophys Res Commun, 1991 Dec 31, 181(3), 1281 - 7
Expression of human T-cell leukemia virus type I protease in Escherichia coli; Hayakawa T et al.; Human T-cell leukemia virus type I (HTLV-I) genome is believed to encode its own protease, although the protease has not yet been detected . To identify the HTLV-I protease, an in-frame gag (3' portion)-prt region was expressed in Escherichia coli . The 14-kDa product was detected using antisera against a synthetic peptide mimicking the fragment of HTLV-I protease, although the molecular weight of the primary translational product was 27,000 . A cell extract had a proteolytic activity to cleave a synthetic peptide substrate containing the cleavage site of gag p19/p24 at the correct site in vitro . Replacement of the putative active site Asp-64 with Gly abolished both in vivo processing activity and in vitro proteolytic activity . These results suggest that the 14-kDa product is the mature enzymatically active HTLV-I protease generated through posttranslational autoprocessing in E . coli.

Biochem Biophys Res Commun, 1991 Dec 31, 181(3), 1056 - 62
Gene expression from multicopy T7 promoter vectors proceeds at single copy rates in the absence of T7 RNA polymerase; Somerville RL et al.; Three different genes (trpR+, tyrR+ and phi (trpR-lacZ)) were inserted into pET3a, a multicopy transcription-translation vector designed by Rosenberg et al . (1) for the T7 RNA polymerase-driven overexpression of proteins in Escherichia coli . Gene orientation was in the anticlockwise ("silent") direction . Gene expression in the absence of T7 RNA polymerase was evaluated either directly using lacZ reporter systems or indirectly by observing the susceptibility of plasmid-bearing tester strains to inhibition by an aromatic amino acid analog . The production of repressor proteins and of a Trp repressor-LacZ chimera was readily detected, at levels comparable to those of haploid trpR+ or tyrR+ E . coli strains . Such T7 vector constructs thus have two especially useful properties: first, they provide a means for the high-level production of various proteins in E . coli; second, they offer a technically advantageous point of departure for structure-function studies of genes whose overexpression from multicopy plasmids would normally be cytotoxic.

Biochem Biophys Res Commun, 1991 Dec 31, 181(3), 1015 - 21
The regulatory role of EF-hand motifs of pig 80K diacylglycerol kinase as assessed using truncation and deletion mutants; Sakane F et al.; To elucidate the regulatory function of EF-hand motifs of pig 80K diacylglycerol (DG) kinase, we constructed and expressed several truncation and deletion mutants of the enzyme in E . coli or COS-7 cells . The bacterially expressed EF-hand region could bind Ca2+ and was suggested to undergo conformational change like calmodulin . A mutant enzyme lacking EF-hands lost Ca(2+)-binding activity, but could be fully activated by phosphatidylserine (PS) or deoxycholate in the absence of Ca2+ . The full activation of the wild-type enzyme by PS, on the other hand, was totally dependent on Ca2+ . Further, the wild-type enzyme expressed in COS-7 cells was exclusively soluble, whereas the EF-hand-deleted mutant was considerably associated with the membranes . The results suggest that under Ca(2+)-free condition, the EF-hand masks the PS-binding site of the DG kinase, and that the Ca(2+)-binding results in the exposure of the PS-binding site through the conformational change of the EF-hand region.

Gene, 1991 Dec 20, 109(1), 71 - 9
Growth-phase-dependent induction of 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase in the cyanobacterium Synechococcus sp . PCC7942; Broedel SE Jr et al.; In most cyanobacteria, the only known pathway for oxidation of stored carbohydrate in the dark or under energy-limiting conditions is the hexose monophosphate shunt . To determine whether the increased use of the shunt under these conditions derives from an increase in the activity level of the respective enzymes, we measured the effect of growth phase during the growth of batch cultures of Synechococcus sp . strain PCC7942 on the specific activity of 6-phosphogluconate dehydrogenase (6PGD) and glucose 6-phosphate dehydrogenase . The specific activities were constant during the exponential growth phase of the culture, but they increased about fivefold during the transition into stationary phase . As an approach to determining the level of expression at which the growth-phase-dependent regulation of 6PGD level is exerted, we constructed operon and gene fusions between the gnd gene, which encodes 6PGD, and the Escherichia coli lacZ gene, which encodes beta-galactosidase (beta Gal) . Strains harboring the fusions integrated into the cyanobacterial chromosome were prepared, and the growth-phase dependence of beta Gal level was determined . The specific activity of beta Gal in cultures of both types of fusion strains increased during the transition into stationary phase, indicating that the growth-phase-dependent regulation is on the gnd mRNA level . Characterization of the growth-phase-dependent induction of 6PGD in strains carrying differing amounts of DNA upstream from the gnd structural gene led to the localization of the promoter and the regulatory site on the restriction map of the gene, whose sequence has previously been determined.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1991 Dec 20, 222(4), 843 - 9
Escherichia coli transcription factor that both activates fatty acid synthesis and represses fatty acid degradation; Henry MF et al.; The fadR gene of Escherichia coli encodes a protein that acts as a negative regulator (repressor) of the inducible beta-oxidation pathway . We report that the FadR protein also functions as a positive transcriptional activator of the fabA gene, which encodes the enzyme introducing the double bond of the unsaturated fatty acids of E . coli.

Carbohydr Res, 1991 Dec 30, 222, 69 - 82
Synthesis of a 3-ether analogue of lipid A; Shiozaki M et al.; Lipid A 3-ether analogues were synthesized from allyl 2-deoxy-4,6-O-isopropylidene-2-trifluoroacetamido-alpha-D-glucopyr anoside and 3,4,6-tri-O-acetyl-2-trifluoroacetamido-alpha-D-glucopyranosyl bromide . The compound lost completely the endotoxic activity.

Gene, 1991 Dec 30, 109(2), 259 - 63
Expression in mammalian cells of a cloned gene encoding murine DNA methyltransferase; Czank A et al.; Mammalian DNA cytosine-5-methyltransferase (MTase, EC 2.1.1.37) is an essential component for establishing and maintaining cell-type specific methylation patterns in the genome . The cDNA for the murine enzyme was previously cloned in segments . We have reconstructed the entire gene, encoding a protein of 1517 amino acids, from a set of overlapping cDNA clones . We report the assembly of two expression constructs in bacterial/mammalian shuttle vectors . Transcription in the first construct (pEMT) is driven by the cytomegalovirus enhancer/promoter and encodes a fusion protein with 15 additional aa at the N terminus, while the second construct (pJMT) is driven by the simian virus 40 early promoter/enhancer upstream from the natural ATG codon . Immunofluorescence microscopy and immunoblot analysis have shown that both constructs direct the synthesis of MTase in COS-1 cells . Enzyme activity in whole-cell lysates of transfected COS-1 cells transfected with pEMT and pJMT are on average tenfold and fivefold higher than in controls, respectively . The specific activities of the recombinant and endogenous mouse-cell enzyme are similar . These expression constructs will be of use in studies of DNA methylation in mammals.

Ann N Y Acad Sci, 1991 Dec 27, 646, 41 - 52
Cloning of a beta-glucosidase gene from Ruminococcus albus and its expression in Escherichia coli; Ohmiya K et al.; A HindIII fragment of R . albus DNA encoding beta-glucosidase was cloned into E . coli . The DNA sequence (3158 bp) was determined, and the longest potential encoding sequence consisted of 2,841 bp (947 amino acids with the calculated molecular weight of 104,276 . The deduced NH2-terminal amino acid sequence from the first (methionine) to the twentieth (glycine) was identical to that of the purified enzyme, suggesting that the gene for beta-glucosidase does not encode a signal peptide . The enzyme purified from the culture supernatant of the transformant had a molecular weight of 120,000 and its maximum activity was revealed at pH 6.5 and 30 degrees C . Reducing reagents activated the enzyme, whereas the sulfhydryl group-blocking reagents and reaction products (glucose) inhibited the activity . Hydrolyzates of celloorigomers contained glucose as a major product, indicating that the enzyme acts as beta-glucosidase . The enzyme from the transformant revealed similar properties to that from R . albus, and both enzyme proteins were immunologically the same to each other, indicating that the cloned gene encodes beta-glucosidase from R . albus.

Ann N Y Acad Sci, 1991 Dec 27, 646, 115 - 24
Comparison of the Fv fragments of different phosphorylcholine binding antibodies expressed in Escherichia coli; Pluckthun A et al.; The development of general methods to express functional antibody fragments in E . coli greatly facilitates the engineering of antibodies . Some of the essential features of the technology are summarized . As a model system, phosphorylcholine binding antibodies are used . The immune response against this antigen results in three classes of antibodies, exemplified by the myeloma proteins McPC603, TEPC15, and MOPC167 . Fv fragments of these antibodies can now be conveniently prepared in E . coli to aid in understanding the structural logic of this well-characterized immune response.

Ann N Y Acad Sci, 1991 Dec 27, 646, 106 - 14
Cloning and expression of an HIV-1 specific single-chain Fv region fused to Escherichia coli alkaline phosphatase; Kohl J et al.; We have constructed a single-chain Fv fragment representing the variable domain of the human monoclonal antibody 3D6, binding specifically to HIV-1 gp41 . This gene was fused to the coding region of E . coli alkaline phosphatase (EcPhoA) and expressed in E . coli . The EcPhoA signal peptide was used to direct the recombinant fusion protein to the periplasmic space of the bacteria, from where it was purified by hydrophobic interaction chromatography and gel filtration followed by antigen-affinity chromatography using a synthetic HIV-1 peptide as ligand . The purified fusion protein was bifunctional, showing both phosphatase activity as well as antigen-binding specificity identical to that of the original antibody.

Biochem Biophys Res Commun, 1991 Dec 16, 181(2), 650 - 6
Inter-molecular degradation of signal peptidase I in vitro; Talarico TL et al.; Highly purified preparations of signal peptidase I (36 kDa) were found to undergo an apparent inter-autocatalytic degradation at 4 degrees C and 37 degrees C . The disappearance of the 36 kDa protein coincided with the stable appearance of a 31 kDa and a 5 kDa species . Amino-terminal sequencing of the 31 kDa product indicated a site specific cleavage following Ala38-Gln-Ala of signal peptidase I . The 31 kDa fragment was purified and shown to have 100-fold less activity than the native enzyme, with pre-maltose binding protein as a substrate.

J Biol Chem, 1991 Dec 25, 266(36), 24596 - 600
SecA protein needs both acidic phospholipids and SecY/E protein for functional high-affinity binding to the Escherichia coli plasma membrane; Hendrick JP et al.; Translocation of preproteins across the Escherichia coli inner membrane requires acidic phospholipids . We have studied the translocation of the precursor protein proOmpA across inverted inner membrane vesicles prepared from cells depleted of phosphatidylglycerol and cardiolipin . These membranes support neither translocation nor the translocation ATPase activity of the SecA subunit of preprotein translocase . We now report that inner membrane vesicles which are depleted of acidic phospholipids are unable to bind SecA protein with high affinity . These membranes can be restored to translocation competence by fusion with liposomes containing phosphatidylglycerol, suggesting that the defect in SecA binding is a direct effect of phospholipid depletion rather than a general derangement of inner membrane structure . Reconstitution of SecY/E, the membrane-embedded domain of translocase, into proteoliposomes containing predominantly a single synthetic acidic lipid, dioleoylphosphatidylglycerol, allows efficient catalysis of preprotein translocation.

J Biol Chem, 1991 Dec 25, 266(36), 24420 - 7
Characterization of membrane-associated and soluble states of SecA protein from wild-type and SecA51(TS) mutant strains of Escherichia coli; Cabelli RJ et al.; The subcellular localization of SecA, a protein essential for the catalysis of general protein export, was studied to better understand its state(s) and function(s) within Escherichia coli cells . In a wild-type strain approximately half of the cellular SecA content was found to be associated with the inner membrane, while the remainder was soluble . Association of SecA protein with the inner membrane required the presence of anionic phospholipids and was modulated by ATP . A fraction of the membrane-bound SecA was found to be integrally associated with the membrane . In the secA51(Ts) mutant 75-95% of SecA protein was found to be membrane associated, independent of the protein export status of the cell, implying that the partitioning of this protein between the cell membrane and cytoplasm may play an important role in its function . secA-lacZ fusions were used to map a membrane association determinant to the amino-terminal quarter of SecA protein sequence . When this portion of SecA protein was expressed within cells, it was found solely in membrane fractions and complemented the growth and protein secretion defect of the secA51(Ts) mutant . This indicates that the membrane is the site of the limiting defect in this mutant and suggests that either SecA functions can be divided into at least two separable activities or that productive interaction between SecA and the amino-terminal fragment can occur in vivo.

Nucleic Acids Res, 1991 Dec 25, 19(24), 6905 - 11
A novel translation initiation region from Mycoplasma genitalium that functions in Escherichia coli; Loechel S et al.; The tuf gene of Mycoplasma genitalium uses a signal other than a Shine-Dalgarno sequence to promote translation initiation . We have inserted the translation initiation region of this gene in front of the Escherichia coli lacZ gene and shown that it is recognized by the translational machinery of E . coli; the signal operates in vivo at roughly the same efficiency as a synthetic Shine-Dalgarno sequence . The M . genitalium sequence was also used to replace the native translation initiation region of the cat gene . When assayed in E . coli, the M . genitalium sequence is equivalent to a Shine-Dalgarno sequence in stimulating translation of this mRNA also . Site-directed mutagenesis enabled us to identify some of the bases that comprise the functional sequence . We propose that the sequence UUAACAACAU functions as a ribosome binding site by annealing to nucleotides 1082-1093 of the E . coli 16S rRNA . The activity of this sequence is enhanced when it is present in the loop of a stem-and-loop structure . Additional sequences both upstream and downstream of the initiation codon are also involved, but their role has not been elucidated.

Nucleic Acids Res, 1991 Dec 25, 19(24), 6811 - 7
Probing of DNA structure with osmium tetroxide,2,2'-bipyridine . Adduct-specific antibodies; Kuderova-Krejcova A et al.; Antibodies against DNA modified with a single-strand selective probe, OsO4 in complex with 2,2'-bipyridine (Os,bipy), were raised in rabbits . These antibodies were fractionated using affinity column chromatography and fractions S89-II and S89-III characterized as highly specific for DNA-Os,bipy adduct with no cross reactivity to at least 1000-fold excess of unmodified DNA, RNA and Os,bipy-modified and unmodified proteins . Cross-reactivity to Os,bipy-modified RNA was very small . S89-II showed no cross-reactivity to DNA modified with OsO4 complexed with tetramethylethylenediamine or with bathophenanthroline disulphonic acid and to DNA oxidized with KMnO4 . It cross-reacted, however, with DNA modified with OsO4,1,10-phenanthroline complex . The limit of detection of immunodot-blot analysis of extensively Os,bipy-modified DNA was below 0.5 pg . Small extent of Os,bipy-modification of supercoiled and linearized plasmids can be detected by DNA gel retardation and immunoblotting techniques . E . coli cells contain DNA regions in which bases are accessible to the single-strand selective probe.

Nucleic Acids Res, 1991 Dec 25, 19(24), 6751 - 5
Site-specific cleavage of natural mRNA sequences by newly designed hairpin catalytic RNAs; Kikuchi Y et al.; The negative strand of tobacco ringspot virus satellite RNA is a self-cleaving RNA . Its catalytic domain and substrate domain have been identified, and the catalytic domain has been named hairpin catalytic RNA . Here we report the construction of a plasmid containing a modified hairpin catalytic RNA sequence that can be transcribed in vitro . Because this plasmid has two specific restriction enzyme recognition sites at both ends of the substrate binding site in the catalytic RNA sequence, it is possible to construct new plasmids by substituting different sequences in the substrate binding site . Using this plasmid, synthetic DNA, and in vitro transcription, we obtained three ribozymes designed to cleave Escherichia coli prolipoprotein signal peptidase (lsp) mRNA at specific sites . All three ribozymes cleaved the lsp mRNA sequence in vitro at the specific sites, and two of them cleaved it efficiently . Kinetic analyses showed that one had a higher kcat/Km value than that of the well-known hammerhead ribozyme . Problems associated with attaining the goal of expressing these ribozymes in vivo also are discussed.

Nucleic Acids Res, 1991 Dec 25, 19(24), 6743 - 50
In the Escherichia coli lacZ gene the spacing between the translating ribosomes is insensitive to the efficiency of translation initiation; Guillerez J et al.; We have constructed a series of 44 Escherichia coli strains in which the chromosomal region corresponding to the Ribosome Binding Site (RBS) of the lacZ gene, has been replaced by small DNA fragments harboring either RBSs from other genes, or artificial RBSs . The beta-galactosidase expression from these strains ranges from 1 to 130 per cent of that of the parental strain . Using this collection, we demonstrate here that strain-to-strain variations in expression are paralleled by nearly equivalent variations in lacZ mRNA content . We propose that, in this system, polarity and mRNA stability are tightly coupled to translation initiation, so that changes in RBS efficiency are detected mainly as changes in mRNA concentration rather than in the spacing between translating ribosomes . In addition, we show that the mRNA sequence immediately downstream from the initiator codon influences per se the lifetime of the lacZ mRNA . We discuss the mechanism of the interdependence between translation, transcription and degradation in this gene, and speculate about the general role of this interdependence in determining the expression of bacterial genes.

Nucleic Acids Res, 1991 Dec 25, 19(24), 6705 - 12
The role of two surface exposed loops in transcription activation by the Escherichia coli CRP and FNR proteins; Williams R et al.; We have investigated a number of mutations that alter the ability of the E . coli transcription factors CRP and FNR to activate transcription . In CRP, some mutations at position 159 (H159L, H159I and delta 159) prevent transcription activation at a number of naturally-occurring and semi-synthetic CRP-dependent promoters . We suggest that some feature of the surface-exposed turn around residue 159 is recognised by RNA polymerase during transcription activation at these promoters . Mutations at position 52 increase CRP activity and reverse the effects of H159L and delta 159, most likely by creating a new contact with RNA polymerase . However this new contact only gives increased expression when the CRP binding site is located 41 1/2 base pairs upstream of the transcription start site and fails to reverse the effects of H159L and delta 159 at promoters where the CRP site is located further upstream . To explain our results we propose that the two surface-exposed turns around residues 52 and 159 contain elements that are potential RNA polymerase docking sites: in the CRP dimer these two active patches are located on adjacent faces of different subunits . FNR, a related transcription activator, contains amino acid sequences homologous to the CRP sequence around position 52 . Mutations in this zone (from residues 81-88 in FNR) reduce expression from an FNR-dependent promoter without stopping FNR binding to its target . This defines a patch on FNR, which is homologous to the CRP surface-exposed loop around position 52, which is involved in transcription activation, most likely by contacting RNA polymerase.

J Biol Chem, 1991 Dec 25, 266(36), 24748 - 56
Interaction of the UvrABC endonuclease with DNA containing a psoralen monoadduct or cross-link . Differential effects of superhelical density and comparison of preincision complexes; Munn MM et al.; The effect of negative supercoiling on UvrABC incision of covalently closed duplex DNA circles containing either a furan-side monoadduct or a cross-link of 4'-hydroxymethyl-4,5',8-trimethylpsoralen at a unique site was examined . The rate of UvrABC incision of these DNA substrates was measured as a function of superhelical density, sigma, for values of sigma between 0 and -0.050 . The monoadducted DNA substrate was incised at close to the maximum rate at all superhelical densities, with only a slight stimulation of activity between sigma = 0 and -0.035 . In contrast, efficient UvrABC incision of the cross-linked DNA substrate required the DNA to be underwound, and activity showed a linear dependence on superhelical density up to sigma = -0.035 . DNase I protection studies show that in the presence of both UvrA and UvrB a protein complex binds to the site of a psoralen monoadduct or cross-link in linear DNA . This UvrA-UvrB-dependent complex binds with similar affinity to both the monoadducted and the cross-linked DNA helices . However, differences in the DNase I footprint on these two DNA substrates indicate that the interaction of this protein complex is different at these two lesions . The addition of UvrC to linear DNA molecules that are saturated at the site of the lesion with the UvrA-UvrB-dependent complex resulted in efficient nicking of the monoadducted DNA, but not the cross-linked DNA . Thus, the properties of a DNA lesion site that lead to UvrAB recognition and binding are not necessarily sufficient to allow incision when all three Uvr subunits are present . We propose that after recognition and binding of a lesion site by the UvrAB complex and prior to incision, the damaged DNA helix undergoes a conformational change such as unwinding or melting that is induced by the lesion-bound Uvr complex.

J Biol Chem, 1991 Dec 25, 266(36), 24712 - 8
Direct analysis of aminoacylation levels of tRNAs in vivo . Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyl-tRNA synthetase; Varshney U et al.; We describe the use of a gel electrophoretic method for measuring the levels of aminoacylation in vivo of mutant Escherichia coli initiator tRNAs, which are substrates for E . coli glutaminyl-tRNA synthetase (GlnRS) due to an anticodon sequence change . Using this method, we have compared the effects of introducing further mutations in the acceptor stem, at base pairs 1:72, 2:71, and 3:70 and discriminator base 73, on the recognition of these tRNAs by E . coli GlnRS in vitro and in vivo . The effects of the acceptor stem mutations on the kinetic parameters for aminoacylation of the mutant tRNAs in vitro are consistent with interactions seen between this region of tRNA and GlnRS in the crystal structure of tRNA(Gln) . GlnRS complex . Except for one mutant, the observed levels of aminoacylation of the mutant tRNAs in vivo agree with those expected on the basis of the kinetic parameters obtained in vitro . We have also measured the relative amounts of aminoacyl-tRNAs for the various mutants and their activities in suppression of an amber codon in vivo . We find that there is, in general, a good correlation between the relative amounts of aminoacyl-tRNAs and their activities in suppression.

J Biol Chem, 1991 Dec 25, 266(36), 24509 - 13
How does trp repressor bind to its operator?
Carey J, Lewis DE, Lavoie TA, Yang J.
Three explanations have been advanced to account for the unexpected absence of direct hydrogen bonds and presence of a buried water layer in the co-crystal-line complex of Escherichia coli trp repressor with DNA . We present results of physical and biochemical measurements that address the testable predictions of each model . We find that the DNA oligomer used for co-crystallization binds to the repressor with high affinity and specificity, and 1:1 stoichiometry, consistent with other evidence that this sequence represents the true operator target for a single repressor dimer . A proposed alternative DNA sequence binds weaker and with higher stoichiometry, consistent with a cooperative binding mode . The operator DNA in solution has a B-form helical structure in the presence and absence of repressor . Affinity of repressor for operator is altered under the conditions used for cocrystal growth.

J Biol Chem, 1991 Dec 25, 266(36), 24404 - 12
Alteration of the binding specificity of cellular retinol-binding protein II by site-directed mutagenesis; Cheng L et al.; Rat cellular retinol-binding protein II (CRBP II) is an abundant 134-residue intestinal protein that binds all-trans-retinol and all-trans-retinal . It belongs to a family of homologous, 15-kDa cytoplasmic proteins that bind hydrophobic ligands in a noncovalent fashion . These binding proteins include a number of proteins that bind long chain fatty acids . X-ray analyses of the structure of two family members, rat intestinal fatty acid-binding protein and bovine myelin P2 protein, indicate that they have a high degree of conformational similarity and that the carboxylate group of their bound fatty acid interacts with a delta-guanidium group of at least 1 of 2 "buried" arginine residues . These 2 Arg residues are conserved in other family members that bind long chain fatty acids and in cellular retinoic acid-binding protein, but are replaced by Gln109 and Gln129 in CRBP II . We have genetically engineered two amino acid substitutions in CRBP II: 1) Gln109 to Arg and 2) Gln129 to Arg . The purified Escherichia coli-derived CRBP II mutant proteins were analyzed by fluorescence and nuclear magnetic resonance spectroscopy . Both mutants exhibit markedly decreased binding of all-trans-retinol and all-trans-retinaldehyde, but no increased binding of all-trans-retinoic acid . Arg substitution for Gln109 but not for Gln129 produces a dramatic increase in palmitate binding activity . Analysis of the endogenous fatty acids associated with the purified E . coli-derived proteins revealed that E . coli-derived intestinal fatty acid binding protein and the Arg109 CRBP II mutant are complexed with endogenous fatty acids in a qualitatively and quantitatively similar manner . These results provide evidence that this internal Arg may play an important role in the binding of long chain fatty acids by members of this protein family.

J Biol Chem, 1991 Dec 25, 266(36), 24359 - 66
Kinetic studies of human immunodeficiency virus type 1 protease and its active-site hydrogen bond mutant A28S; Ido E et al.; Human immunodeficiency virus type 1 (HIV-1) protease optimally catalyzes in the pH range of 4-6 in contrast to nearly all of the other eukaryotic aspartic proteases, which catalyze best in the pH range of 2-4 . A possible structural reason for the higher optimal pH of HIV-1 protease is the absence of a hydrogen bond to the carboxyl group of active-site Asp25, which is nearly universally present in others . To investigate this hypothesis, we have mutated residue 28 in HIV-1 protease from alanine to serine . Both the wild-type and the mutant A28S enzymes have been overexpressed in Escherichia coli using a chemically synthesized gene and purified for a comparative study in enzyme kinetics . The kcat and Km values were determined by a radiometric assay for the wild-type enzyme from pH 3.2 to 7.0, and for the mutant enzyme from pH 3.2 to 6.0 . The low pK values of the active site of the free enzyme, pKe1, are 3.3 and 3.4 for the wild-type and mutant enzymes, respectively . The low pK values of the active site of the enzyme bound to substrate, pKes1, are 5.1 and 4.3 for the wild-type and mutant enzymes, respectively . The high pK values of the free enzyme, pKe2, are 6.8 and 5.6, and the corresponding ones for the substrate-bound enzyme, pKes2, are 6.9 and 6.0 for the wild-type and mutant enzymes, respectively . The lowering of pK values in mutant HIV-1 protease indicates that the hydroxyl group of Ser28 forms a new hydrogen bond to active-site Asp25 to increase its acidity.

J Biol Chem, 1991 Dec 25, 266(36), 24257 - 9
Cell attachment activity of the carboxyl-terminal domain of human thrombospondin expressed in Escherichia coli; Kosfeld MD et al.; Thrombospondin (TS) is a multidomain, adhesive glycoprotein that associates with cells through multiple cell attachment sites . One of these has been located in or near the globular COOH-terminal region of TS by the monoclonal antibody (mAb) C6.7, which inhibits the attachment of human melanoma cells (G361) to TS . The epitope for C6.7 lies within the last 122 residues of the COOH-terminal domain of TS . This domain is distant from two known cell attachment sites in TS, namely the NH2-terminal heparin-binding domain and the CSVTCG sequences in the type I repeats, but is close to the RGDA sequence, an integrin-dependent cell attachment site . In order to separate the adhesive activity of the TS COOH-terminal domain from that of the RGD sequence, we have expressed the COOH-terminal 212 amino acids (residues 941-1152) of TS in Escherichia coli using the expression vector pRIT2T . The resultant fusion protein is effective in supporting G361 cell attachment even though it lacks the RGD sequence . In addition, the expressed protein inhibits adhesion of G361 cells to intact TS . mAb C6.7 blocks adhesion to the expressed TS COOH-terminal domain whereas GRGDSP and VTCG peptides are not inhibitory . These results show that the TS COOH-terminal domain contains a separate cell adhesion site, defined by mAb C6.7, that is distinct from the other adhesion sites of TS.

J Biol Chem, 1991 Dec 25, 266(36), 24657 - 63
Murine polypyrimidine tract binding protein . Purification, cloning, and mapping of the RNA binding domain; Bothwell AL et al.; A complex of nucleic acid binding proteins (100, 35, and 25 kDa) was purified to apparent homogeneity from nuclear extracts of the murine plasmacytoma J558L . Amino-terminal sequence analysis of the 25-kDa subunit enabled the isolation of a cDNA that encodes a 528-amino acid protein that is highly homologous to the human 62-kDa human polypyrimidine tract binding protein (PTB) (Garcia-Blanco, M . A., Jamison, S . F., and Sharp, P . A . (1989) Genes & Dev . 3, 1874-1886; Gil, A., Sharp, P . A., Jamison, S . F., and Garcia-Blanco, M . A . (1991) Genes & Dev . 5, 1224-1236; Patton, J . G., Mayer, S . A., Tempst, P., and Nadal-Ginard, B . (1991) Genes & Dev . 5, 1237-1251) . Sequence comparison programs suggested the presence of domains related to the RNA recognition motif found in other RNA-binding proteins, and deletion analysis revealed that the carboxyl-terminal 195 amino acids of the recombinant PTB was sufficient for specific binding to pre-mRNAs . Cross-linking experiments identified a 25-kDa protein in crude nuclear extracts of J558L cells that possessed the RNA binding properties of PTB, while a approximately 60-kDa protein is detected in other murine cell lines tested . Thus, the 25-kDa protein found in J558L is likely a proteolytic product of the murine polypyrimidine tract binding protein . A probe derived from the PTB cDNA detected a ubiquitous 3.3-kb mRNA in murine cell lines and a 3.6-kb mRNA in human lines . Southern blot analysis revealed three strongly hybridizing DNA fragments and several more weakly hybridizing bands in mouse, human, and yeast DNA . The role of PTB in pre-mRNA splicing is discussed.

Nucleic Acids Res, 1991 Dec 25, 19(24), 6943 - 8
Potassium permanganate as an in situ probe for B-Z and Z-Z junctions; Jiang H et al.; The availability of DNA structural probes that can be applied to living cells is essential for the analysis of biological functions of unusual DNA structures adopted in vivo . We have developed a chemical probe assay to detect and quantitate left-handed Z-DNA structures in recombinant plasmids in growing E . coli cells . Potassium permanganate selectively reacts with B-Z or Z-Z junction regions in supercoiled plasmids harbored in the cells . Restriction enzyme recognition sites located at these junctions are not cleaved by the corresponding endonuclease after modification with KMnO4 . This inhibition of cleavage allows the determination of the relative amounts of B- and Z-forms of the cloned inserts inside the cell . We have successfully applied this method to monitor the extent of Z-DNA formation in E . coli as a function of the growth phase and mutated topoisomerase or gyrase activities . The assay can in principle be used for any unusual DNA structure that contains a restriction recognition site inside or near the structural alteration . It can be a useful tool to analyze in vivo correlations between DNA structure and gene regulatory events.

Nucleic Acids Res, 1991 Dec 25, 19(24), 6895 - 903
Probing the function of conserved RNA structures in the 30S subunit of Escherichia coli ribosomes; Almehdi M et al.; Ribosomes play an active role in protein biosynthesis . Ribosomal RNA conformation in ribosomal subunits, intramolecular interactions between different rRNA sequences within the confinement of the particles, and intermolecular interactions are presumed necessary to support efficient and accurate protein synthesis . Here we report an analysis of the disposition of 16S rRNA conserved zones centered about positions 525, 1400, and 1500 in 30S subunits . Complementary oligodeoxyribonucleotides in conjunction with nuclease S1 digestion were used to do this . All of the sequences examined in 30S subunits are accessible to DNA probes of 9 to 12 nucleotide residues in length . However, the kinetic characteristics of the respective DNA interactions with 30S particles vary significantly . In addition to the investigation of normal 30S particles, a four base deletion within the 1400 region of 16S rRNA was analyzed . The deletion was made by using synthetic DNAs to target the deletion site for RNase H digestion . The direct in vitro procedure for manipulating rRNA conserves nucleotide modifications . The alteration causes a significant change in the disposition of 16S rRNA in 30S subunits, suggesting a reduction in the freedom of movement of the altered zone in the particle . In a factor-dependent in vitro protein synthesis system primed with MS2 mRNA and altered 30S subunits, there was a 50% decrease in phage coat protein synthesis . The reduction could be due to a decrease in the rate of translation or premature termination of translation . We present evidence here, based on isotopic studies, which supports the latter possibility.

J Biol Chem, 1991 Dec 25, 266(36), 24621 - 6
Characterization of the herpes simplex virus origin binding protein interaction with OriS; Hazuda DJ et al.; The origin binding protein (OBP) of herpes simplex virus (HSV), which is essential for viral DNA replication, binds specifically to sequences within the viral replication origin(s) (for a review, see Challberg, M.D., and Kelly, T . J . (1989) Annu . Rev . Biochem . 58, 671-717) . Using either a COOH-terminal OBP protein A fusion or the full-length protein, each expressed in Escherichia coli, we investigated the interaction of OBP with one HSV origin, OriS . Binding of OBP to a set of binding site variant sequences demonstrates that the 10-base pair sequence, 5' CGTTCGCACT 3', comprises the OBP-binding site . This sequence must be presented in the context of at least 15 total base pairs for high affinity binding, Ka = approximately 0.3 nM . Single base pair mutations in the central CGC sequence lower the affinity by several orders of magnitude, whereas a substitution at any of the other seven positions reduces the affinity by 10-fold or less . OBP binds with high affinity to duplex DNA containing mismatched base pairs . This property is exploited to analyze OBP binding to DNA heteroduplexes containing singly substituted mutant and wild-type DNA strands . For positions 2, 3, 5, 6, 7, 8, and 9, substitutions are tolerated on one or the other DNA strand, indicating that base-mediated interactions are limited to one base of each pair . For both Boxes I and II, these interactions are localized to one face of the DNA helix, forming a recognition surface in the major groove . In OriS, the 31 base pairs which separate Boxes I and II orient the two interaction surfaces to the same side of the DNA.

J Biol Chem, 1991 Dec 25, 266(36), 24314 - 9
Properties of photoreceptor-specific phospholipase C encoded by the norpA gene of Drosophila melanogaster; Schneuwly S et al.; Mutations in the norpA gene drastically affect the phototransduction process in Drosophila . To study the biochemical characteristics of the norpA protein and its cellular and subcellular distributions, we have generated antisera against the major gene product of norpA . The antisera recognize an eye-specific protein of 130-kDa relative molecular mass that is present in wild-type head extracts but not in those of strong norpA mutants . The protein is associated with membranes and can be extracted with high salt . Immunohistochemical analysis at the light and electron microscopic levels indicates that the protein is expressed in all adult photoreceptor cells and specifically localized within the rhabdomeres, preferentially adjacent to, but not within, the rhabdomeric membranes . The results of the present study strongly support the previous suggestion that the norpA gene encodes the major phosphoinositol-specific phospholipase C in the photoreceptors . Moreover, insofar as the rhabdomeres are specialized structures for photoreception and phototransduction, specific localization of the norpA protein within these structures, in close association with the membranes, is consistent with the proposal that it has an important role in phototransduction.

Biochemistry, 1991 Dec 24, 30(51), 11842 - 50
Reexamination of the secondary and tertiary structure of histidine-containing protein from Escherichia coli by homonuclear and heteronuclear NMR spectroscopy; Hammen PK et al.; Analysis of the histidine-containing protein (HPr) from Escherichia coli by two-dimensional homonuclear and heteronuclear nuclear magnetic resonance techniques has been performed, extending the work originally reported {Klevit, R . E., Drobny, G . D., & Waygood, E . B . (1986) Biochemistry 25, 7760-7769; Klevit, R . E., & Drobny, G . P . (1986) Biochemistry 25, 7770-7773; Klevit, R . E., & Waygood, E . B . (1986) Biochemistry 25, 7774-7781} . Two-dimensional homonuclear total coherence spectroscopy (TOCSY) allowed for more complete assignments of the side-chain spin systems than had been possible in the original studies . As well, two-dimensional 15N-1H heteronuclear spectroscopy was used to resolve a number of ambiguities present in the homonuclear spectra due to resonance redundancies . These analyses led to the correction of a number of resonance assignments that were made with the spectra that could be collected with the technology that existed 6 years ago . In addition, amide exchange rates and 3JNH coupling constants have been measured, extending the original analysis and yielding new structural information . All these data have been used to reexamine the folding topology of E . coli HPr . Structure calculations showed that the topology derived from the earlier NMR data, i.e., a four-stranded beta-sheet with three alpha-helices running along one side of the sheet, was essentially unchanged, although at the present level of analysis, a well-defined "helix B" could not be established with high confidence . In addition, the data reported here revealed the existence of two slowly-exchanging side-chain hydroxyl protons belonging to Ser31 and Thr59 . Their behavior strongly suggests that these side chains are involved in hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Dec 24, 30(51), 11801 - 11
Phospholipase A2 engineering . X-ray structural and functional evidence for the interaction of lysine-56 with substrates; Noel JP et al.; Site-directed mutagenesis studies of bovine pancreatic phospholipase A2 (PLA2, overproduced in Escherichia coli) showed that replacement of surface residue Lys-56 by a neutral or hydrophobic amino acid residue resulted in an unexpected and significant change in the function of the enzyme . The kcat for phosphatidylcholine micelles increases 3-4-fold for K56M, K56I, and K56F and ca . 2-fold for K56N and K56T but does not change for K56R . These results suggest that the side chain of residue 56 has significant influence on the activity of PLA2 . In order to probe the structural basis for the enhanced activity, the crystal structures of wild-type and K56M PLA2 were determined by X-ray crystallography to a resolution of 1.8 A . The results suggest that the mutation has not only perturbed the conformation of the side chain of Met-56 locally but also caused conformational changes in the neighboring loop (residues 60-70), resulting in the formation of a hydrophobic pocket by residues Met-56, Tyr-52, and Tyr-69 . Docking of a phosphatidylcholine inhibitor analogue into the active site of K56M, according to the structure of the complex of cobra venom PLA2-phosphatidylethanolamine inhibitor analogue {White, S.P., Scott, D . L., Otwinowski, Z., Gleb, M . H., & Sigler, P . (1990) Science 250, 1560-1563}, showed that the choline moiety {N(CH3)3}+ is readily accommodated into the newly formed hydrophobic pocket with a high degree of surface complementarity . This suggests a possible interaction between residue 56 and the head group of the phospholipid, explaining the enhanced activities observed when the positively charged Lys-56 is substituted by apolar residues, viz., K56M, K56I, and K56F . Further support for this interpretation comes from the 5-fold enhancement in kcat for the mutant K56E with a negatively charged side chain, where there would be an attractive electrostatic interaction between the side chain of Glu-56 and the positively charged choline moiety . Our results also refute a recent report {Tomasselli, A . G., Hui, J., Fisher, J., Zurcher-Neely, H., Reardon, I.M., Oriaku, E., Kezdy, F.J., & Heinrikson, R.L . (1989) J . Biol . Chem . 264, 10041-10047} that substrate-level acylation of Lys-56 is an obligatory step in the catalysis by PLA2.

Biochemistry, 1991 Dec 24, 30(51), 11775 - 81
Use of site-directed mutagenesis to define the limits of sequence variation tolerated for processing of the M13 procoat protein by the Escherichia coli leader peptidase; Shen LM et al.; Leader peptidase cleaves the leader sequence from the amino terminus of newly made membrane and secreted proteins after they have translocated across the membrane . Analysis of a large number of leader sequences has shown that there is a characteristic pattern of small apolar residues at -1 and -3 (with respect to the cleavage site) and a helix-breaking residue adjacent to the central apolar core in the region -4 to -6 . The conserved sequence pattern of small amino acids at -1 and -3 around the cleavage site most likely represents the substrate specificity of leader peptidase . We have tested this by generating 60 different mutations in the +1 to -6 domain of the M13 procoat protein . These mutants were analyzed for in vivo and in vitro processing, as well as for protein insertion into the cytoplasmic membrane . We find that in vivo leader peptidase was able to process procoat with an alanine, a serine, a glycine, or a proline residue at -1 and with a serine, a glycine, a threonine, a valine, or a leucine residue at -3 . All other alterations at these sites were not processed, in accordance with predictions based on the conserved features of leader peptides . Except for proline and threonine at +1, all other residues at this position were processed by leader peptidase . None of the mutations at -2, -4, or -5 of procoat (apart from proline at -4) completely abolished leader peptidase cleavage in vivo although there were large effects on the kinetics of processing.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Dec 24, 30(51), 11767 - 74
Arginine-395 is required for efficient in vivo and in vitro aminoacylation of tRNAs by Escherichia coli methionyl-tRNA synthetase; Ghosh G et al.; We have previously shown that the anticodon of methionine tRNAs contains the major recognition site required for aminoacylation of tRNAs by Escherichia coli methionyl-tRNA synthetase (MetRS) and have located part of the anticodon binding domain on the enzyme at a site close to Trp461 {Schulman, L . H., & Pelka, H . (1988) Science 242, 765-768; Ghosh, G., Pelka, H., & Schulman, L.H . (1990) Biochemistry 29, 2220-2225} . In order to gain information about other possible sites of contact between MetRS and its tRNA substrates, we have examined the effects of mutations at a series of positively charged residues on the surface of the C-terminal domain of the enzyme . Conversion of Arg356, Arg366, Arg380, or Arg453 to Gln had little or no effect on enzyme activity . Similarly, conversion of Lys402 or Lys439 to Asn failed to significantly alter aminoacylation activity . Conversion of Arg380 to Ala or Arg442 to Gln produced a 5-fold reduction in kcat/Km for aminoacylation of tRNAfMet, with no effect on methionine activation, indicating a possible minor role for these residues in interaction of the enzyme with the tRNA substrate . In contrast, mutation of a phylogenetically conserved residue, Arg395, to Gln increased the Km for aminoacylation of tRNAfMet about 30-fold and reduced kcat/Km by 25,000-fold . The mutant enzyme was also shown to be highly defective by its inability to complement a strain of E . coli having an altered chromosomal MetRS gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Dec 24, 30(51), 11751 - 9
Fidelity of mammalian DNA replication and replicative DNA polymerases; Thomas DC et al.; Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells . In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene . Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha . DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions . The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate . When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase . This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity . Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon . In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate . Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta . The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase . Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Dec 24, 30(51), 11760 - 7
Artificial mutants generated by the insertion of random oligonucleotides into the putative nucleoside binding site of the HSV-1 thymidine kinase gene; Dube DK et al.; We have obtained 42 active artificial mutants of HSV-1 thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) by replacing codons 166 and 167 with random nucleotide sequences . Codons 166 and 167 are within the putative nucleoside binding site in the HSV-1 tk gene . The spectrum of active mutations indicates that neither Ile166 nor Ala167 is absolutely required for thymidine kinase activity . Each of these amino acids can be replaced by some but not all of the 19 other amino acids . The active mutants can be classified as high activity or low activity on two bases: (1) growth of Escherichia coli KY895 (a strain lacking thymidine kinase activity) in the presence of thymidine and (2) uptake of thymidine by this strain, when harboring plasmids with the random insertions . E . coli KY895 harboring high-activity plasmids or wild-type plasmids can grow in the presence of low amounts of thymidine (less than 1 microgram/mL), but are unable to grow in the presence of high amounts of thymidine . On the other hand, E . coli KY895 harboring low-activity plasmids can grow at a high concentration of thymidine (greater than 50 microgram/mL) in the media . The high-activity plasmids also have an enhanced {3H}dT uptake . The amounts of thymidine kinase activity in vitro in unfractionated extracts do not correlate with either growth at low thymidine concentration or the rate of thymidine uptake . Heat inactivation studies indicate that the mutant enzymes are without exception more temperature-sensitive than the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1991 Dec 20, 222(4), 937 - 61
1-aminocyclopropane-1-carboxylate synthase in tomato is encoded by a multigene family whose transcription is induced during fruit and floral senescence; Rottmann WH et al.; The key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.1.1.14) . It catalyzes the conversion of S-adenosylmethionine to ACC, the precursor of ethylene . We isolated complementary DNA sequences, ptACC2 and ptACC4, for two distinct and differentially regulated ACC synthase mRNAs expressed in ripe tomato fruit . The authenticity of the clones has been confirmed by expression experiments in E . coli . The predicted size of the encoded polypeptides (54,690 and 53,519 Da) is similar to that of the primary in vitro translation products and to the proteins found in vivo . The sequence of the gene encoding one mRNA, LE-ACC2, has been determined and its transcription initiation site defined . Four additional genes, LE-ACC1A, LE-ACC1B, LE-ACC3 and LE-ACC4, have also been identified and the sequence of their coding regions determined . The LE-ACC1A and LE-ACC1B genes are adjacent to each other and are convergently transcribed . Their encoded polypeptides are 96% identical; the identity of the other polypeptides to each other varies between 50 and 70% . The proteins predicted to be encoded by the ACC synthase genes so far cloned from tomato and zucchini contain 11 of the 12 conserved amino acid residues found in various aminotransferases involved in the binding of the substrate and the cofactor pyridoxal-5'-phosphate . The data indicate that ACC synthase is encoded by a divergent multigene family in tomato that encodes proteins related to aminotransferases.

J Mol Biol, 1991 Dec 20, 222(4), 925 - 36
Mutational specificity of the dnaE173 mutator associated with a defect in the catalytic subunit of DNA polymerase III of Escherichia coli; Mo JY et al.; We developed a system to examine forward mutations that occurred in the rpsL gene of Escherichia coli placed on a multicopy plasmid . Using this system we determined the mutational specificity for a dnaE173 mutator strain in which the editing function of DNA polymerase III is impeded . The frequency of rpsL- mutations increased 32,000-fold, due to the dnaE173 mutator, and 87 independent rpsL- mutations in the mutator strain were analyzed by DNA sequencing, together with 100 mutants recovered from dnaE+ strain, as the control . While half the number of mutations that occurred in the wild-type strain were caused by insertion elements, no such mutations were recovered from the mutator strain . A novel class of mutation, named "sequence substitution" was present in mutants raised in the dnaE173 strain; seven sequence substitutions induced in the mutator strain occurred at six sites, and all were located in quasipalindromic sequences, carrying the GTG or CAC sequence at one or both endpoints . While other types of mutation were found in both strains, single-base frameshifts were the most frequent events in the mutator strain . Thus, the mutator effect on this class of mutation was 175,000-fold . A total of 95% of the single-base frameshifts in the mutator strain were additions, most of which occurred at runs of A or C bases so as to increase the number of identical residues . Base substitutions, the frequency of which was enhanced 25,000-fold by the mutator effect, occurred primarily at several hotspots in the mutator strain, whereas those induced in the wild-type strain were more randomly distributed throughout the rpsL sequence . The dnaE173 mutator also increased the frequency of duplications 28,000-fold . Of the three duplications recovered from the mutator strain, one was a simple duplication, the region of which was flanked by direct repeats . The other duplications were complex, one half part of which was in the inverted orientation of a region containing two sets of inverted repeats . The same duplications were also recovered from the wild-type strain . The present data suggest that dnaE173 is a novel class of mutator that sharply induces sequence-directed mutagenesis, yielding high frequencies of single base frameshifts, duplications with inversions, sequence substitutions and base substitutions at hotspots.

J Mol Biol, 1991 Dec 20, 222(4), 851 - 6
Evidence for horizontal gene transfer in Escherichia coli speciation; Medigue C et al.; After extracting more than 780 identified Escherichia coli genes from available data libraries, we investigated the codon usage of the corresponding coding sequences and extended the study of gene classes, thus obtained, to the nature and intensity of short nucleotide sequence selection, related to constraints operating at the nucleotide level . Using Factorial Correspondence Analysis we found that three classes ought to be included in order to match all data now available . The first two classes, as known, encompass genes expressed either continuously at a high level, or at a low level and/or rarely; the third class consists of genes corresponding to surface elements of the cell, genes coming from mobile elements as well as genes resulting in a high fidelity of DNA replication . This suggests that bacterial strains cultivated in the laboratory have been fixed by specific use of antimutator genes that are horizontally exchanged.

J Mol Biol, 1991 Dec 20, 222(4), 1161 - 71
ms2i6A deficiency enhances proofreading in translation; Diaz I et al.; The hypermodified base 2-methylthio-N6-isopentenyladenosine (ms2i6A) at position 37 occurs frequently in tRNAs that read codons starting with uridine . Here we have studied how ms2i6A affects the accuracy of poly(U) translation in vitro . Deficiency leads to a higher rejection rate of tRNA4(Leu) by more aggressive proofreading on the wild-type ribosome, but with the initial selection step unchanged . Our data indicate that ms2i6A has no effect on codon-anticodon interactions on wild-type ribosomes as long as aminoacyl-tRNA is in ternary complex with EF-Tu and GTP . ms2i6A deficiency in the cognate poly(U) reader tRNA(Phe) leads to increased misreading when the near-cognate competitor tRNA4(Leu) is wild-type . ms2i6A deficiency in tRNA4(Leu) gives a decreased error level in competition with wild-type tRNA(Phe).

Gene, 1991 Dec 20, 109(1), 149 - 54
Cloning and analysis of five mitochondrial tRNA-encoding genes from the fungus Beauveria bassiana; Hegedus DD et al.; Five mitochondrial (mt) tRNA genes from the filamentous fungus, Beauveria bassiana, were cloned and sequenced . The genes encoding the Val-, Ile-, Ser-, Trp- and Pro-accepting tRNAs were found clustered in the region 5' to the lrRNA-encoding gene . The genes were 64-77% homologous with the equivalent genes from other filamentous fungi, 49-58% to yeasts with the exception of the Val-accepting tRNA-encoding gene which was 76%, and only slightly homologous with Escherichia coli . The B . bassiana mt genetic code was found to be similar to that of other fungal mitochondria in that the UGA codon is used as a signal for Trp rather than as a stop codon . Transcript analysis has revealed that the genes present in tRNA cluster are transcribed and processed into tRNA-size products . Secondary structure models proposed for the gene products show that conservation of tRNA secondary structure also exists . The presence of a GGC sequence rather than a GGU sequence in the D-loop of the tRNA(Trp)-encoding gene is a feature unique to the B . bassiana mt tRNA . An unconventional G-A base pair present in the D-stem of the tRNA(Ser)-encoding gene is a feature conserved in the mt tRNA of other filamentous fungi . Comparison of the B . bassiana tRNA-encoding genes with those of two other filamentous fungi and two yeasts revealed that the differences between closely related species favoured transition-type mutations.

Gene, 1991 Dec 20, 109(1), 125 - 9
The oligopeptide (Gly-Pro)2-Ala-(Gly-Pro)2 increases the internal proline level and improves NaCl tolerance when produced in Escherichia coli; Bulow L et al.; We have produced various proline-containing peptides as translational fusions with beta-glucuronidase (beta Glu) in Escherichia coli . When these linkers were introduced in the 5'-end of the beta Glu-encoding gene, the production of the enzyme was increased substantially in vivo . The peptides carrying repetitive Gly-Pro sequences could also stimulate the growth of the transformants in media with inhibitory concentrations of NaCl . Furthermore, the freezing tolerance could be improved.

Gene, 1991 Dec 20, 109(1), 115 - 9
Optimised cDNA size selection and cloning procedure for the construction of representative plasmid cDNA libraries; Kieffer BL; Plasmid libraries are more versatile than phage libraries since they allow expression cloning in eukaryotic systems . However, high numbers of primary clones are sometimes difficult to obtain and more efficient transformation procedures are often required . In this paper, a detailed protocol is presented, for the construction of large plasmid libraries from ng quantities of cDNA, based on a highly efficient transformation step . Drop dialysis and electroporation are optimised: complete ligase removal and yeast tRNA addition before dialysis appear critical, while exclusive use of double-distilled water as well as rapid preparation of fresh cells provide excellent electroporation yields . A novel, simple and flexible cDNA size selection procedure is also presented, based on sucrose density gradient . Two libraries were constructed using an expression vector for mammalian cells (pCDM8) and its Escherichia coli host strain (MC1061{p3}) . Numbers of 2 to 10 x 10(6) primary transformants were obtained from 1 microgram of poly(A)+ RNA . Up to 85% of the clones had inserts and half of the inserts were larger than 1.5 kb.

J Mol Biol, 1991 Dec 20, 222(4), 881 - 4
Crystallization and preliminary X-ray analysis of phosphoporin from the outer membrane of Escherichia coli; Tucker AD et al.; Phosphoporin is a pore-forming transmembrane protein that spans the outer membrane of Escherichia coli and facilitates the diffusion of phosphates and phosphorylated compounds . Phosphoporin has been crystallized in several different crystal forms, although only one appears to be suitable for X-ray analysis . These crystals, which are hexagonal plates, diffract X-rays to 3 A resolution and belong to the space-group P6(3)22, with unit cell dimensions a = b = 121 A and c = 111 A.

J Chromatogr, 1991 Dec 20, 587(2), 161 - 9
Immobilization of Fv antibody fragments on porous silica and their utility in affinity chromatography; Berry MJ et al.; Recent advances in molecular biology have allowed antibody binding domains to be cloned and expressed in Escherichia coli . The use of Fv antibody fragments as ligands in immunoaffinity chromatography is reported . Fv fragments specific for hen-egg lysozyme were immobilized on porous silica and used to recover antigen from spiked serum in a single step . Comparison with a conventional immunoadsorbent (whole antibodies immobilized on silica) showed the Fv-silica to have a fivefold superior capacity . Analysis of sectioned Fv-silica particles by immunoelectron microscopy indicated that captured antigen was evenly distributed throughout the internal porous structure of the particle.

J Mol Biol, 1991 Dec 20, 222(4), 1131 - 47
Chromatin assembly on plasmid DNA in vitro . Apparent spreading of nucleosome alignment from one region of pBR327 by histone H5; Jeong SW et al.; We have found that histone H5 (or H1) induces physiological nucleosome spacings and extensive ordering on some plasmid constructions, but not on others, in a fully defined in vitro system . Plasmid pBR327 containing DNA insertions with lengths close to 300 base-pairs permitted histone H5 to induce a remarkable degree of nucleosome alignment . Seventeen multiples of a unit 210(+/- 4) base-pair repeat, covering the entire plasmid, were detected . Plasmid pBR327, not containing a DNA insert, permitted continuous alignment of only a few nucleosomes . These observations suggest that a necessary requirement in this system for histone H5 (or H1)-induced nucleosome alignment on small (less than 4 kb; 1 kb = 10(3) bases or base-pairs) circular plasmids may be that the total DNA length must be close to an integer multiple of the nucleosome repeat length generated, a type of boundary effect . Consistent with this hypothesis, five deletion constructs of pBR327 (not containing inserts), that spanned 64% of the plasmid, and possessed DNA lengths close to integer multiples of 210 base-pairs, permitted nucleosome alignment by histone H5 . We have also found that plasmid length adjustment is not a sufficient condition for nucleosome alignment . For example, plasmids pBR322 and pUC18 did not permit nucleosome alignment when adjusted to near-integer multiples of 210 base-pairs . Also, for pBR327 that contained a length-adjusted deletion in one particular region, appreciable nucleosome alignment no longer occurred . These data suggest that a contiguous approximately 800 base-pair region of pBR327, interrupted in pBR322 and not present in pUC18, can nucleate histone H5-induced nucleosome alignment, which can then spread to adjacent chromatin . Supporting this idea, a positioned five-nucleosome array appears to originate in the required region . Additionally, on a larger (6.9 kb) plasmid construction, the "chromatin organizing region" of pBR327 and adjacent DNA on one side of it exhibited preferred H5-induced nucleosome alignment.

Nature, 1991 Dec 19-26, 354(6354), 531 - 4
Ets-related protein Elk-1 is homologous to the c-fos regulatory factor p62TCF; Hipskind RA et al.; A key event in the response of cells to proliferative signals is the rapid, transient induction of the c-fos proto-oncogene, which is mediated through the serum response element (SRE) in the fos promoter . Genomic footprinting and transfection experiments suggest that this activation occurs through a ternary complex that includes the serum response factor (SRF) and the ternary complex factor p62 . Interaction of p62TCF with the SRF-SRE binary complex requires a CAGGA tract immediately upstream of the SRE . Proteins of the ets proto-oncogene family bind to similar sequences and we have found that a member of this family, Elk-1, forms SRF-dependent ternary complexes with the SRE . Elk-1 and p62TCF have the same DNA sequence requirements and antibodies against Elk-1 block the binding of both proteins . Furthermore, we show that like p62TCF, Elk-1 forms complexes with the yeast SRF-homologue MCM1 but not with yeast ARG80 . But ARG80 mutants that convey interaction with p62TCF can also form complexes with Elk-1 . The similarity, or even identity, between Elk-1 and p62TCF suggests a novel regulatory role for Ets proteins that is effected through interaction with other proteins, such as SRF . Furthermore, the possible involvement of an Ets protein in the control of c-fos has interesting implications for proto-oncogene cooperation in cellular growth control.

Eur J Biochem, 1991 Dec 18, 202(3), 1313 - 9
Purification and characterization of the F1 portion of the ATP synthase (F1Fo) of Streptomyces lividans; Hensel M et al.; The F1 complex of the ATP synthase of Streptomyces lividans was isolated and purified . The procedure involved the solubilization of F1 from membranes with buffer of low ionic strength in the presence of EDTA, ion-exchange chromatography and gel filtration . The purified F1 complex from S . lividans (SLF1) consists of five subunits alpha, beta, gamma, delta and epsilon with molecular masses of 58,000, 50,000, 36,000, 28,000 and 13,000, respectively and exhibits immunological cross-reactivity with the F1 portion purified from Escherichia coli (ECF1) . The enzymatic properties of SLF1 were determined by the use of microtiter-plate-based assay and compared with data obtained for ECF1 . ATPase activity of SLF1 (specific activity: 20-30 U/mg) was only observed in the presence of high concentrations of Ca2+ (10mM) . Stimulation of the ATPase activity by Mg2+ was not detectable; quite to the contrary, Mg2+ inhibited the Ca(2+)-stimulated activity of SLF1 . SLF1 was re-bound to F1-stripped membranes of S . lividans, but not to F1-stripped membrane vesicles of E . coli . In contrast, ECF1 could be cross-reconstituted with F1-stripped membranes of S . lividans; however, a structural but not a functional reconstitution of the hybrid F1Fo complex was observed.

Eur J Biochem, 1991 Dec 18, 202(3), 1307 - 12
Substitution of the cysteinyl residue (Cys21) of subunit b of the ATP synthase from Escherichia coli; Kauffer S et al.; The Fo complex of the ATP synthase (F1Fo) of Escherichia coli contains only two cysteinyl residues, Cys21, of the two copies of subunit b . Modification of Cys21 with the hydrophobic maleimide N-(7-dimethylamino-4-methyl-coumarinyl)maleimide resulted in impairment of Fo functions {Schneider, E . & Altendorf, K . (1985) Eur . J . Biochim . 153, 105-109} . We replaced this residue (via cassette mutagenesis) by Ser, Gly, Ala, Thr, Asp and Pro . None of the replacements resulted in detectable alterations of the function of the ATP synthase, making a functional role for these sulfhydryl residues unlikely . Due to its high tolerance towards amino acid substitutions, the region around Cys21 seems not to be a protein-protein contact area.

Eur J Biochem, 1991 Dec 18, 202(3), 981 - 4
Base 2661 in Escherichia coli 23S rRNA influences the binding of elongation factor Tu during protein synthesis in vivo; Tapio S et al.; The binding of the EF-Tu.GTP.aminoacyl-tRNA ternary complex (EF, elongation factor) to the ribosome is known to be strengthened by a 2661G-to-C mutation in 23S ribosomal RNA, whereas the binding to normal ribosomes is weakened if the factor is in an appropriate mutant form (Aa) . In this report we describe the mutual effects by the 2661C alteration in 23S rRNA and EF-Tu(Aa) on bacterial viability and translation efficiency in strains with normal or mutationally altered ribosomes . The rrnB(2661C) allele on a multicopy plasmid was introduced by transformation into Escherichia coli K-12 strains, harbouring either the wild-type or the mutant gene (tufA) for EF-Tu as well as normal or mutant ribosomal protein S12 (rpsL) . Together with wild-type EF-Tu, the 2661C mutant ribosomes decreased the translation elongation rate in a rpsL+ strain or a non-restrictive rpsL224 strain . This reduction was not seen in strains which harbored EF-Tu(Aa) instead of EF-Tu(As) (As, wild-type form) . Nonsense codon suppression by tyrT(Su3) suppressor tRNA was reduced by 2661C in a rpsL224 strain in the presence of EF-Tu(As) but not in the presence of EF-Tu(Aa) . The lethal effect obtained by the combination of 2661C and a restrictive ribosomal protein S12 mutation (rpsL282) disappeared if EF-Tu(As) was replaced by EF-Tu(Aa) in the strain . In such a viable strain, 2661C had no effect on either the translation elongation rate or nonsense codon suppression . Our data suggest that the G base at position 2661 in 23S rRNA is important for binding of EF-Tu during protein synthesis in vivo . The interaction between this base and EF-Tu is strongly influenced by the structure of ribosomal protein S12.

Eur J Biochem, 1991 Dec 18, 202(3), 841 - 8
Site-directed mutagenesis of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii . Binding of the peripheral components E1p and E3; Schulze E et al.; Site-directed mutagenesis was performed in the protease-sensitive region, between the lipoyl and catalytic domains and in the catalytic domain, of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii . The interaction of the mutated enzymes with the peripheral components pyruvate dehydrogenase (E1p) and lipoamide dehydrogenase (E3) was studied by gel filtration experiments, analytical ultracentrifugation and reconstitution of the pyruvate dehydrogenase complex . Upon binding of peripheral components, the 24-subunit core of A . vinelandii wild-type E2p dissociates into tetramers . Four E1p or E3 dimers can bind to a tetramer . Binding is mutually exclusive, resulting in an active complex containing one E3 and three E1p dimers . Large deletions of the protease-sensitive region of E2p resulted in a total loss of the E1p and E3 binding . A small deletion (delta P361-R362) or the point mutation K367Q in the protease-sensitive region did not influence E3 binding, but affected E1p binding strongly, although with excess E1p almost complete reconstitution was reached . For E2p with the point mutation R416D in the N-terminal region of the catalytic domain only 16% overall activity could be measured in reconstituted complexes . This is due to a very weak E1p/E2p interaction, whereas the E3 binding was not affected . The point mutation R416D did not influence the catalytic activity of E2p, although a function for this residue in the formation of the active site was predicted from amino acid similarities with chloramphenicol acetyltransferase type III from Escherichia coli . Deletion of the complete Ala + Pro-rich sequence between the protease-sensitive region and the catalytic domain did not affect the enzymological properties of E2p, nor the affinity for E1p or E3 . A further deletion of 20 N-terminal residues from the catalytic domain destroyed the E2p activity . From gel filtration experiments it was concluded that the quaternary structure was unaffected, as was E3 binding . E1p binding was lost and, in contrast to the wild-type enzyme, no dissociation of the core upon addition of E3 was observed . This mutant enzyme possesses, like E . coli E2p, six E3 binding sites and clearly shows that interaction of E3 or E1p with the E1p sites and dissociation are linked processes . It is concluded that the binding site for E3 is located on the N-terminal part of the protease-sensitive region . In contrast, the binding site for E1p consists of two regions, one located on the protease-sensitive region and one of the catalytic domain . These regions are separated by a flexible sequence of about 20 amino acids.

Eur J Biochem, 1991 Dec 18, 202(3), 797 - 803
Site-directed mutagenesis of the conserved histidine residue of phosphoenolpyruvate carboxylase . His138 is essential for the second partial reaction; Terada K et al.; Histidine residues have previously been suggested to be essential for the activity of phosphoenolpyruvate carboxylase as demonstrated by chemical modification of these residues . Although the location of these residues on the primary structure is not known, a comparison of nine phosphoenolpyruvate (P-pyruvate) carboxylases sequenced recently revealed that there are only two conserved histidine residues (His138 and His579, coordinates from the E . coli enzyme) . Site-directed mutagenesis of these residues were undertaken with the E . coli P-pyruvate carboxylase and the properties of purified mutant enzymes were investigated . Mutation of His138 to asparagine (H138N) produced a protein which did not show carboxylase activity . However, this mutant enzyme catalyzed the bicarbonate-dependent dephosphorylation (Vmax = 1.4 mumol.min-1.mg-1) of the P-pyruvate . Since this reaction is due to one of the two partial reactions proposed for this enzyme, the results indicate that His138 is obligatory for the second-step reaction, i.e . the carboxylation of the enolate form of pyruvate by carboxyphosphate . Mutation of His579 to asparagine (H579N) produced an enzyme which had 69% of the wild-type carboxylase activity, but its affinity for P-pyruvate was decreased by 24-fold.

Eur J Biochem, 1991 Dec 18, 202(3), 749 - 57
Characterization of glutamate-1-semialdehyde aminotransferase of Synechococcus . Steady-state kinetic analysis; Smith MA et al.; Synechococcus glutamate-1-semialdehyde aminotransferase was expressed in large amounts in transformed cells of Escherichia coli . The resulting purified enzyme has an absorption spectrum characteristic of B6-containing enzymes and could be converted to the pyridoxal-phosphate form with excess dioxovalerate (O2Val), and back to the pyridoxamine-phosphate form with diaminovalerate (A2Val) . Both enzyme forms are similarly active in the conversion of glutamate 1-semialdehyde (GSA) to 5-aminolevulinate (ALev), suggesting that A2Val and O2Val are intermediates . Initial rates of ALev synthesis at various fixed concentrations of GSA followed typical Michaelis-Menten kinetics (Km of GSA for the pyridoxamine-phosphate form of GSA aminotransferase = 12 microM, kcat = 0.23 s-1) . In submicromolar amounts A2Val stimulates ALev synthesis, and in a series of concentrations with various fixed concentrations of GSA, gives a family of parallel lines in Lineweaver-Burk plots (Km for A2Val = 1.0 microM) . On the other hand, O2Val gives competitive inhibition of the pyridoxamine-phosphate form of GSA-aminotransferase and mixed-type inhibition of the pyridoxal-phosphate form (Ki for O2Val = 1.4 mM) . In general the kinetics were typical of ping-pong bi-bi mechanisms in which A2Val is the second substrate (intermediate) and O2Val is an alternative first substrate . There is no compelling evidence that O2Val accepts an amino group at its C5 position resulting in the direct formation of ALev, or the reverse involving the apparent formation of O2Val from ALev . These results are consistent with the hypothesis that the mechanism of GSA aminotransferase mimics that of other aminotransferases and that A2Val is the intermediate.

Eur J Biochem, 1991 Dec 18, 202(3), 1283 - 90
Mutation of the pseudo-EF-hand of calbindin D9k into a normal EF-hand . Biophysical studies; Johansson C et al.; The two Ca(2+)-binding sites in calbindin D9k, a protein belonging to the calmodulin superfamily of intracellular proteins, have slightly different structure . The C-terminal site (amino acids 54-65) is a normal EF-hand as in the other proteins of the calmodulin superfamily, while the N-terminal site (amino acids 14-27) contains two additional amino acids, one of which is a proline . We have constructed and studied five mutants of calbindin D9k modified in the N-terminal site . In normal EF-hand structures the first amino acid to coordinate calcium is invariantly an Asp . For this reason Ala15, is exchanged by an Asp in all mutants and the mutants also contain various other changes in this site . The mutants have been characterized by 43Ca, 113Cd and 1H NMR and by the determination of the calcium binding constants using absorption chelators . In two of the mutants (one where Ala14 is deleted, Ala15 is replaced by Asp and Pro20 is replaced by Gly, the other where, in addition, Asn21 is deleted), we find that the structure has changed considerably compared to the wild-type calbindin . The NMR results indicate that the calcium coordination has changed to mainly side-chain carboxyls, from being octahedrally coordinated by mainly back-bone carbonyls, and/or that the coordination number has decreased . The N-terminal site has thus been turned into a normal EF-hand, in which the calcium ion is coordinated by side-chain carboxyls . Furthermore, the calcium binding constants of these two mutant proteins are almost as high as in the wild-type calbindin D9k . That is, the extensive alterations in the N-terminal site have not disrupted the calcium binding ability of the proteins.

Eur J Biochem, 1991 Dec 18, 202(3), 1169 - 76
Over-production, purification and properties of the uridine diphosphate N-acetylmuramoyl-L-alanine:D-glutamate ligase from Escherichia coli; Pratviel-Sosa F et al.; The UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase of Escherichia coli was over-produced in strains that harbour recombinant plasmids bearing the murD gene under the control of the lac or PR promoter . Purification to homogeneity was achieved by a two-step procedure from a 181-fold over-producing strain . The N-terminal sequence of the purified protein was determined and correlated with the nucleotide sequence of the murD gene . The purified activity was highly dependent on the concentration of potassium phosphate and Mg2+ . The enzyme also catalysed the reverse reaction . The Km values for UDP-N-acetylmuramoyl-L-alanine; D-glutamate and ATP/Mg2+ were estimated at 7.5, 55 and 138 microM, respectively . Under the most optimal in vitro conditions determined, a turnover number of 931 min-1 was estimated . When considering the plasmid-free parental strain, the copy number of the murD gene product was not more than 1000.cell-1.

Eur J Biochem, 1991 Dec 18, 202(3), 1147 - 55
Inhibitory activity and conformational transition of alpha 1-proteinase inhibitor variants; Schulze AJ et al.; Several variants of alpha 1-proteinase inhibitor (alpha 1-PI) were investigated by spectroscopic methods and characterized according to their inhibitory activity . Replacement of Thr345 (P14) with Arg in alpha 1-PI containing an Arg residue in position 358 (yielding {Thr345----Arg, Met358----Arg}alpha 1-PI) results in complete loss of its inhibitory activity against human alpha-thrombin; whereas an exchange of residue Met351 (P8) by Glu {( Met351----Glu, Met358----Arg}alpha 1-PI) does not alter activity . {Thr345----Arg, Met358----Arg}alpha 1-PI is rapidly cleaved by thrombin, while {Met358----Arg}alpha 1-PI and {Met351----Glu, Met358----Arg}alpha 1-PI form stable proteinase-inhibitor complexes . The stability of {Thr345----Arg, Met358----Arg}alpha 1-PI against guanidinium chloride denaturation is significantly enhanced compared to wild-type alpha 1-PI, and does not change after cleavage, resembling ovalbumin, a serpin with no inhibitory activity, from which the Thr345----Arg amino acid exchange had been derived . {Met351----Glu, Met358----Arg}alpha 1-PI and {Met358----Arg}alpha 1-PI resemble the wild-type protein in this respect . The CD spectra of intact and cleaved alpha 1-PI variants do not compare well with the wild-type protein, probably reflecting local structural differences . Insertion of a synthetic peptide, which corresponds to residues Thr345----Met358 of human alpha 1-PI, leads to the formation of binary complexes with all variants having the characteristic features of the binary complex between peptide and wild-type protein.

Eur J Biochem, 1991 Dec 18, 202(3), 1091 - 100
Redox and flavin-binding properties of recombinant flavodoxin from Desulfovibrio vulgaris (Hildenborough); Curley GP et al.; Flavodoxin from Desulfovibrio vulgaris (Hildenborough) has been expressed at a high level (3-4% soluble protein) in Escherichia coli by subcloning a minimal insert carrying the gene behind the tac promoter of plasmid pDK6 . The recombinant protein was readily isolated and its properties were shown to be identical to those of the wild-type protein obtained directly from D . vulgaris, with the exception that the recombinant protein lacks the N-terminal methionine residue . Detailed measurements of the redox potentials of this flavodoxin are reported for the first time . The redox potential, E2, for the couple oxidized flavodoxin/flavodoxin semiquinone at pH 7.0 is -143 mV (25 degrees C), while the value for the flavodoxin semiquinone/flavodoxin hydroquinone couple (E1) at the same pH is -440 mV . The effects of pH on the observed potentials were examined; E2 varies linearly with pH (slope = -59 mV), while E1 is independent of pH at high pH values, but below pH 7.5 the potential becomes less negative with decreasing pH, indicating a redox-linked protonation of the flavodoxin hydroquinone . D . vulgaris apoflavodoxin binds FMN very tightly, with a value of 0.24 nM for the dissociation constant (Kd) at pH 7.0 and 25 degrees C, similar to that observed with other flavodoxins . In addition, the apoflavodoxin readily binds riboflavin (Kd = 0.72 microM; 50 mM sodium phosphate, pH 7.0, 5 mM EDTA at 25 degrees C) and the complex is spectroscopically very similar to that formed with FMN . The redox potentials for the riboflavin complex were determined at pH 6.5 (E1 = -262 mV, E2 = -193 mV; 25 degrees C) and are discussed in the light of earlier proposals that charge/charge interactions between different parts of the flavin hydroquinone play a crucial role in determining E1 in flavodoxin.

Eur J Biochem, 1991 Dec 18, 202(3), 1049 - 55
The conformational stability of the redox states of lipoamide dehydrogenase from Azotobacter vinelandii; van Berkel WJ et al.; The conformational stability of holo-lipoamide and apo-lipoamide dehydrogenase from Azotobacter vinelandii was studied by thermoinactivation, unfolding and limited proteolysis . The oxidized holoenzyme is thermostable, showing a melting temperature, tm = 80 degrees C . The thermal stability of the holoenzyme drastically decreases upon reduction . Unlike the oxidized and lipoamide two-electron reduced enzyme species, the NADH four-electron reduced enzyme is highly sensitive to unfolding by urea . Loss of energy transfer from Trp199 to flavin reflects the unfolding of the oxidized holoenzyme by guanidine hydrochloride . Unfolding of the monomeric apoenzyme is a rapid fully reversible process, following a simple two-state mechanism . The oxidized and two-electron reduced holoenzyme are resistant to limited proteolysis by trypsin and endoproteinase Glu-C . Upon cleavage of the apoenzyme or four-electron reduced holoenzyme by both proteases, large peptide fragments (molecular mass greater than 40 kDa) are transiently produced . Sequence studies show that limited trypsinolysis of the NADH-reduced enzyme starts mainly at the C-terminus of Arg391 . In the apoenzyme, limited proteolysis by endoproteinase Glu-C starts from the C-terminus at the carboxyl ends of Glu459 and/or Glu435 . From crystallographic data it is deduced that the susceptible amino acid peptide bonds are situated near the subunit interface . Thus, these bonds are inaccessible to the proteases in the dimeric enzyme and become accessible after monomerization . It is concluded that reduction of lipoamide dehydrogenase to the four-electron reduced state(s) is accompanied by conformational changes promoting subunit dissociation.

Eur J Biochem, 1991 Dec 18, 202(3), 1003 - 12
Cloning, sequencing and expression studies of the genes encoding amicyanin and the beta-subunit of methylamine dehydrogenase from Thiobacillus versutus; Ubbink M et al.; The genes encoding amicyanin and the beta-subunit of methylamine dehydrogenase (MADH) from Thiobacillus versutus have been cloned and sequenced . The organization of these genes makes it likely that they are coordinately expressed and it supports earlier findings that the blue copper protein amicyanin is involved in electron transport from methylamine to oxygen . The amino acid sequence deduced from the nucleotide sequence of the amicyanin-encoding gene is in agreement with the published protein sequence . The gene codes for a pre-protein with a 25-amino-acid-long signal peptide . The amicyanin gene could be expressed efficiently in Escherichia coli . The protein was extracted with the periplasmic fraction, indicating that pre-amicyanin is translocated across the inner membrane of E . coli . Sequence studies on the purified beta-subunit of MADH confirm the amino acid sequence deduced from the nucleotide sequence of the corresponding gene . The latter codes for a pre-protein with an unusually long (56 amino acids) leader peptide . The sequencing results strongly suggest that pyrroloquinoline quinone (PQQ) or pro-PQQ is not the co-factor of MADH.

Eur J Biochem, 1991 Dec 18, 202(3), 863 - 72
Lipoamide dehydrogenase from Azotobacter vinelandii: site-directed mutagenesis of the His450-Glu455 diad . Spectral properties of wild type and mutated enzymes; Benen J et al.; Three amino acid residues in the active site of lipoamide dehydrogenase from Azotobacter vinelandii were replaced by other residues . His450, the active-site base, was changed into Ser, Tyr and Phe . Pro451, in cis conformation, was changed into Ala . Glu455 was replaced with Asp and Gln . Absorption, fluorescence and CD spectroscopy of the mutated enzymes in their oxidized state (Eox) showed only minor changes with respect to the wild-type enzyme, whereas considerable changes were observed in the spectra of the two-electron-reduced (EH2) species of the enzymes upon reduction by the substrate dihydrolipoamide . Differences in extent of reduction of the flavin by NADH indicate that the redox potential of the flavin is altered by the mutations . Enzyme Pro451----Ala {corrected} showed the greatest deviation from wild type . The enzyme is very easily over-reduced to the four-electron reduced state (EH4) by dihydrolipoamide . This is probably due to a change in the backbone conformation caused by the cis-trans conversion . From studies on the pH dependence of the thiolate charge-transfer absorption and the relative fluorescence of EH2 of the enzymes, it is concluded that mutation of His450 results in a relatively simple and easily interpreted distribution of electronic species at the EH2 level . For all three His450-mutated enzymes an apparent pKa1 near 5.5 is calculated that is assigned to the interchange thiol . A second apparent pKa2 is calculated of 6.9, 7.5 and 7.1 for the His450----Phe, -Ser and -Tyr enzymes, respectively, and signifies the deprotonation of the tautomeric equilibrium between the interchange and charge-transfer thiols . The difference in apparent pKa2 values between the His450-mutated enzymes is explained by changes in micropolarity . At the EH2 level the wild-type enzyme consists of multiple electronic forms as reported for the Escherichia coli enzyme {Wilkinson, K . D . and Williams C . H . Jr (1979) J . Biol . Chem . 254, 852-862} . Based on the results obtained with the His450-mutated enzymes, it is concluded that the lowest pKa is associated with the interchange thiol . A model for the equilibrium species of the wild-type enzyme at the EH2 level is presented which takes three pKa values into account . The results of the pH dependence of the electronic species at the EH2 level of Glu455-mutated enzymes essentially follow the model proposed for the wild-type enzyme . However mutation of Glu455 shifts the tautomeric equilibrium of EH2 in favor of the charge-transfer species.(ABSTRACT TRUNCATED AT 400 WORDS)

Eur J Biochem, 1991 Dec 18, 202(3), 1321 - 5
Aldehyde dehydrogenase induction by glutamate in Escherichia coli . Role of 2-oxoglutarate; Quintilla FX et al.; In Escherichia coli, an aldehyde dehydrogenase that catalyzes the oxidation of L-lactaldehyde to L-lactate is induced not only by L-fucose, L-rhamnose or D-arabinose, but also by growth in the presence of glutamate or amino acids yielding glutamate, with the exception of proline . Induction by these amino acids requires glutamate accumulation . 4-Aminobutyric acid also induces this aldehyde dehydrogenase through its transamination to glutamate . Growth on 2-oxoglutarate, the tricarboxylic acid cycle intermediate with which glutamate is in equilibrium, also induces this aldehyde dehydrogenase . Conditions in which the conversion of 2-oxoglutarate into glutamate is highly restricted displayed unchanged rates of induction by 2-oxoglutarate, indicating that glutamate induces the aldehyde dehydrogenase through 2-oxoglutarate formation . Evidence is presented showing that L-fucose- and 2-oxoglutarate-inducing systems share the same regulatory protein . Induction by growth on either of these two compounds is repressed both by glucose and by glycerol . Addition of cAMP to these cultures partially recovers the glucose-repressed aldehyde dehydrogenase activity, while this nucleotide has no effect on the glycerol-mediated repression . These results indicate that ald is under carbon regulation mediated by at least two different mechanisms.

Eur J Biochem, 1991 Dec 18, 202(3), 1133 - 40
Carboxy-terminal truncated rhuIFN-gamma with a substitution of Gln133 or Ser132 to leucine leads to higher biological activity than in the wild type; Slodowski O et al.; The biological function of the 20 C-terminal amino acids of human interferon-gamma (IFN-gamma) was examined by recombinant DNA methodology . Six truncated IFN-gamma analogues were produced by modification of the 3' end of the coding sequence of the cloned gene, insertion into a vector with the trc promoter and expression of the recombinant IFN-gamma analogue genes in Escherichia coli strain JM 105 . The IFN-gamma analogue proteins were shortened by 10 (C-10L), 11 (C-11, C-11L), 14 (C-14L), 19 (C-19L) and 20 (C-20) amino acid . Four of these constructs were modified to have a C-terminal leucine . The expression rates of precipitating IFN-gamma variants in E . coli cells (wild type, C-10L, C-11, C-11L) amount to 35-40% of the total protein, in contrast to 14-21% for the mainly soluble ones (C-14L, C-20) . The variant C-19L has an exceptional position in its solubility behaviour with a nearly 1:1 distribution between its soluble and insoluble form by an expression rate of 8% . The purification protocol of the insoluble variants contains a denaturing and a renaturation step . The characteristic step for purification soluble IFN-gamma is HPLC cation-exchange chromatography . The antiviral activities of the variants lacking 14 or more amino acids are less than 2% of the wild-type activity . The variants C-10L, C-11 and C-11L show higher biological activities than wild-type IFN-gamma . The most active variant, C-10L, with leucine as the last C-terminal amino acid, has a fourfold higher specific antiviral activity (A549 cells, encephalomyocarditis virus) . Removal, but not replacement of the leucine, represented by the variant C-11, reduces the biological activity compared with variant C-10L . The activity of C-11 is, nevertheless, higher than in the wild type . Comparing the secondary structures, as judged by CD analyses, no significant differences for C-10L, C-14L and C-20, compared with wild type, are observed . Also, all molecules, including the wild-type protein, exist as dimers under physiological conditions . There is a correlation between the grade of truncation and the pI values, which range from pI = 10.4 (wild type) to pI = 8.0 (C-20) . The variant C-10L demonstrates a higher temperature stability (tm = 55 degrees C) compared with wild type (tm = 53 degrees C) . Perhaps this higher stability will result in a longer half-life in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1991 Dec 17, 30(50), 11700 - 6
A sectored colony assay for monitoring mutagenesis by specific carcinogen-DNA adducts in Escherichia coli; Pauly GT et al.; To study the mutagenicity of various carcinogen-DNA adducts in Escherichia coli, a cassette plasmid was developed that permits positioning of specific carcinogen-modified bases within the ATG initiation codon of the lacZ' alpha-complementation gene . Adduct-induced mutations inactivate the gene and lead to formation of blue and white sectored colonies when transformants from an alpha-complementing version of E . coli strain AB1157 are grown on media containing 5-bromo-4-chloro-3-indolyl beta-D-galactoside . In the absence of mutation, blue colonies are produced . This system has been used to measure the mutagenicity of O6-methyl-, O6-ethyl-, and O6-benzyl-2'-deoxyguanosine residues incorporated in place of the normal 2'-deoxyguanosine of the ATG initiation codon . Although a low percentage of sectored colonies was produced in this repair-proficient strain, pretreatment of the bacteria with N-methyl-N'-nitro-N-nitrosoguanidine to disable DNA repair led to a dose-dependent increase in the percentage of sectored colonies . This percentage increased as a function of modified guanine in the order O6-benzyl- less than O6-methyl- less than O6-ethyl-2'-deoxy-guanosine . The only mutations detected at the site of incorporation of these O6-substituted guanines were G-to-A transitions . This sectored colony assay system permits convenient screening of large numbers of colonies and simplifies quantification of modified-base-induced mutations whether they be single-base changes, frameshifts, insertions, or deletions.

Biochemistry, 1991 Dec 17, 30(50), 11694 - 9
Reconstitution of catalytically competent human zeta-thrombin by combination of zeta-thrombin residues A1-36 and B1-148 and an Escherichia coli expressed polypeptide corresponding to zeta-thrombin residues B149-259; Gan ZR et al.; Human zeta-thrombin, a catalytically competent serine proteinase, arises from a single chymotryptic cleavage at Trp-148 in alpha-thrombin to generate two nonconvalently associated polypeptide segments designated zeta 1-thrombin (the 36-residue A-chain disulfide linked to B-chain residues B1-148) and zeta 2-thrombin (B149-259) . We report here the expression of recombinant zeta 2-thrombin in Escherichia coli and the reconstitution of catalytically competent zeta-thrombin by combination of zeta 1-thrombin with recombinant zeta 2-thrombin . A DNA fragment encoding zeta 2-thrombin was cloned into a pATH2 expression vector as a trpE-zeta 2 fusion gene, in which a factor Xa cleavage site was inserted between the trpE and the zeta 2-thrombin gene . High-level expression of this fusion protein was achieved under the control of the E . coli trp promoter . The expressed zeta 2-thrombin was liberated from the fusion protein by factor Xa cleavage, reduced with DTT, and purified to homogeneity by reverse-phase HPLC . Oxidation of the reduced zeta 2-thrombin in the presence of 80 microM CuSO4 and 6 M urea at pH 8.15 yielded material that was indistinguishable on HPLC from zeta 2-thrombin isolated by resolution of human zeta-thrombin . Catalytically active zeta-thrombin was generated by combination of recombinant zeta 2-thrombin with zeta 1-thrombin that was isolated by resolution of human zeta-thrombin . Recombinant zeta-thrombin displayed catalytic activities, toward a small chromogenic substrate and fibrinogen, that were similar to those of alpha-thrombin prepared from human blood plasma and zeta-thrombin obtained by treatment of alpha-thrombin with chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Dec 17, 30(50), 11609 - 14
Reconstitution of a heat shock effect in vitro: influence of GroE on the thermal aggregation of alpha-glucosidase from yeast; Holl-Neugebauer B et al.; alpha-Glucosidase from yeast is inactivated rapidly at temperatures above 42 degrees C . The thermal inactivation is accompanied by aggregation . The molecular chaperone GroEL suppresses the formation of aggregates by binding the thermally inactivated alpha-glucosidase . Spectroscopic studies suggest that GroEL binds alpha-glucosidase in an intermediately folded state . The complex between alpha-glucosidase and GroEL can be dissolved by MgATP . GroES accelerates the MgATP-dependent dissociation of the alpha-glucosidase-GroEL complex . At elevated temperatures this release leads to the formation of aggregates, while at lower temperatures native, enzymatically active molecules are formed.

Biochemistry, 1991 Dec 17, 30(50), 11707 - 19
Protein-protein interactions of HIV-1 reverse transcriptase: implication of central and C-terminal regions in subunit binding; Becerra SP et al.; Human immunodeficiency virus 1 reverse transcriptase (RT) purified from virions is composed of a approximately 51,000 Mr polypeptide and a approximately 66,000 Mr polypeptide that are thought to be in heterodimer structure (Chandra et al., 1986; Hansen et al., 1988; Starnes & Cheng, 1989) and are identical except for a 15,000 Mr C-terminal truncation in the smaller species (Di Marzo-Veronese et al., 1986) . We prepared individual bacterial-recombinant RTs as the approximately 66,000 Mr polypeptide (p66) or as the approximately 51,000 Mr polypeptide (p51) and then conducted various in vitro protein-protein binding experiments . Analytical ultracentrifugation studies in 0.25 M NaCl at pH 6.5 revealed that p66 was in monomer-dimer equilibrium with KA of 5.1 x 10(4) M-1 . p51 failed to dimerize and behaved as a monomer under these conditions . Mixing of the p66 and p51 polypeptides resulted in a 1:1 heterodimer with KA of 4.9 x 10(5) M-1 . These results on formation of the p66/p66 homodimer and p66/p51 heterodimer were confirmed by gel filtration analysis using FPLC Superose-12 columns . Binding between p66 and individual p66 segment polypeptides also was observed using an immunoprecipitation assay . Binding between p51 and p66 in this assay was resistant to the presence of approximately 1 M NaCl, suggesting that the binding free energy has a large hydrophobic component . C-Terminal truncation of p66 to yield a 29-kDa polypeptide eliminated binding to p66, and N-terminal truncation of p66 to yield a 15-kDa peptide also eliminated binding to p66 . The results indicate that purified individual RT peptides p51 and p66 are capable of binding to form a 1:1 heterodimer and suggest that the central region of p66 is required for this subunit binding; the C-terminal region (15,000 Mr) of p66 appears to be required also, as p51 alone did not dimerize.

Biochem Biophys Res Commun, 1991 Dec 16, 181(2), 884 - 8
Library-independent cloning of genomic fragments adjacent to vector integration sites: isolation of the EB4-PSV gene from a Dictyostelium gene disruption transformant; Hildebrandt M et al.; We have designed an efficient procedure to clone genomic DNA adjacent to the integration site of transformation vectors . Using this method on a Dictyostelium gene disruption transformant, we have cloned a 5kb fragment which has previously escaped isolation by conventional library screening . Our protocol eliminates recloning of the original vectors which are often integrated in multiple tandem copies (1) but specifically recovers vectors containing genomic fragments adjacent to the integration site . The protocol is useful to isolate flanking fragments in gene disruption transformants to characterize flanking regions which may influence the expression of transformed genes by position effects and to identify sites of insertional mutagenesis.

Clin Chim Acta, 1991 Dec 16, 203(2-3), 249 - 57
Phospholipase A2 is associated with albumin in patients with acute pancreatitis; Langton SR et al.; Evidence for the association of serum phospholipase A2 (PLA2) (EC 3.1.1.4) and serum proteins was examined . The effect of this on the PLA2 results from patients diagnosed as having acute pancreatitis was investigated . Two distinct zones of PLA2 activity were found on agarose electrophoresis of purified human PLA2 in the presence of albumin, and in the sera from the acute pancreatitis patients . One of the zones was coincident with albumin . To investigate this finding, a comparison of the PLA2 activity in sera and protein-free ultrafiltrates prepared from the sera of the same patients, showed that PLA2 was not completely ultrafiltered as would be expected from its molecular weight . The PLA2 method used employed a radiolabelled E . coli membrane-phospholipid substrate . It has been previously shown that there is an association between PLA2 and albumin and there is good evidence that albumin interferes with certain methods used for the measurement of PLA2 . The recovery of PLA2 activity from the ultrafiltrates of patients' serum was highly variable and it may be that serum proteins, in particular albumin, provide a protective 'buffer' against small increases in PLA2 activity . Inhibitor proteins such as lipocortin which were originally postulated as binding to the enzyme, have been subsequently shown to be substrate-binding agents . However, direct protein effects may be a factor in the inconsistent PLA2 results reported in studies of patients with acute pancreatic disease.

FEBS Lett, 1991 Dec 16, 295(1-3), 63 - 6
A C-terminal proline is required for bioluminescence of the Ca(2+)-binding photoprotein, aequorin; Nomura M et al.; The requirement for a proline residue at the C-terminus of the Ca(2+)-binding photoprotein, aequorin, was investigated by measuring luminescence activities of a series of C-terminal deletion mutants, substitution mutants and an addition mutant . CD spectral measurements of apoaequorin with the C-terminal proline deleted showed a small change in secondary structure . In all cases studied, the C-terminal proline was required for bioluminescence activity.

FEBS Lett, 1991 Dec 16, 295(1-3), 159 - 62
Fluorine-19 NMR studies of the thermal unfolding of 5-fluorouracil-substituted Escherichia coli valine transfer RNA; Chu WC et al.; 19F NMR spectroscopy was used to monitor the thermal unfolding of E . coli tRNAVal labeled by incorporation of 5-fluorouracil (FUra) . With rising temperatures, resonances in the 19F NMR spectrum of (FUra)tRNAVal gradually shift towards the central region of the spectrum and merge into a single broad peak above 85 degrees C . FU55 and FU12 are the first to shift, beginning at temperatures below 40 degrees C, which suggests that the initial steps of thermal denaturation of tRNAVal involve disruption of the tertiary interactions between the D- and T-arms . The acceptor stem and the FU64-G50 wobble base pair in the T-stem are particularly stable to thermal denaturation . A temperature-dependent splitting of the 19F resonance assigned to FU64, at temperatures above 40 degrees C, suggests that the T-arm of (FUra)tRNAVal exists in two conformations in slow exchange on the NMR time scale.

FEBS Lett, 1991 Dec 16, 295(1-3), 13 - 6
Evidence for two protein-lipoylation activities in Escherichia coli; Brookfield DE et al.; The lipoate acyltransferase subunits of the 2-oxo acid dehydrogenase complexes are post-translationally modified with one or more covalently-bound lipoyl cofactors . Two distinct lipoate-protein ligase activities, LPL-A and LPL-B, have been detected in E . coli by their ability to modify purified lipoyl apo-domains of the bacterial pyruvate dehydrogenase complex . Both enzymes require ATP and Mg2+, use L-lipoate, 8-methyllipoate, lipoyl adenylate and octanoyl adenylate as substrates, and both activate lipoyl-deficient pyruvate dehydrogenase complexes . In contrast, only LPL-B uses D-lipoate and octanoate and there are differences in the metal-ion and phosphate requirements . It is suggested that LPL-B may be responsible for the octanoylation of lipoyl domains observed previously under lipoate-deficient conditions.

FEBS Lett, 1991 Dec 16, 295(1-3), 10 - 2
A novel strategy for production of a highly expressed recombinant protein in an active form; Blackwell JR et al.; Under standard growth conditions, E . coli transformed with the high-level expression vector pMON5525 produces recombinant DMAPP/AMP transferase in inactive, insoluble complexes . We have produced large amounts of active, soluble protein by growing and inducing the cells under osmotic stress in the presence of sorbitol and glycyl betaine . This caused an increase of up to 427-fold in the active yield, and the disappearance of the protein from the pelletable fraction of cell extracts . This treatment may have wide applicability.

Biochem Biophys Res Commun, 1991 Dec 16, 181(2), 818 - 26
Inhibition and enhancement of eukaryotic gene expression by potential non-B DNA sequences; Delic J et al.; In a transient or constitutive expression assay we have examined the effect of non-B DNA sequences d(CA)40 and d(CAAAAATGCC)n on gene expression in eukaryotic cells . These sequences were cloned adjacent to the weak eukaryotic promoter (CGTATTTATTTG) and located upstream from the coding sequence of galactokinase enzyme . Recombinants were micro-injected in cultured cells (Chinese hamster fibroblasts R1610, mutant gal-K-) and expression levels have been determined . The alternating purine-pyrimidine tract found in d(CA)40 able to assume the Z-DNA conformation shows an inhibitory effect on gene expression . In addition, our results suggest a new potential role of Z-DNA motifs in vivo to stimulate recombination . The sequences d(CAAAAATGCC)n able to adopt another non-B structure, corresponding to curved (or bended) helix conformation, strongly enhance gene expression and this enhancement depends on sequence redundancy.

Biochem Biophys Res Commun, 1991 Dec 16, 181(2), 748 - 55
Non-essentiality of cysteine and histidine residues for the activity of human class PI glutathione S-transferase; Kong KH et al.; In order to examine the roles of cysteine and histidine residues in the activity of human class Pi glutathione S-transferase (GST pi), site-directed mutagenesis was used to replace each of the four cysteine residues (at positions 14, 47, 101 and 169) with serine and each of the two histidine residues (at positions 71 and 162) with asparagine using a cDNA for the enzyme (Kano, T . et al . (1987) Cancer Res., 47, 5626-5630) and an E . coli expression system . The replacements of Cys101, Cys169, His71 and His162 did not affect the GSH-conjugating activity toward 1-chloro-2,4-dinitrobenzene and ethacrynic acid . On the other hand, the activities were partly decreased by the replacements of Cys47 and Cys14 . These results indicated that the cysteine and histidine residues in GST pi are not essential for the catalytic activity, although Cys47 and Cys14 may contribute to some extent to the catalytic efficiency.

Biochem Biophys Res Commun, 1991 Dec 16, 181(2), 743 - 7
Enzymatic properties of the protein encoded by newly cloned human alcohol dehydrogenase ADH6 gene; Chen CS et al.; Five non-allelic genes which encode five types of alcohol dehydrogenase subunits have been identified in humans . An additional gene (ADH6) and cDNA, whose coding sequences were not highly analogous to any of the known alcohol dehydrogenase subunits, were recently cloned (Yasunami et al., Proc . Natl . Acad . Sci . USA 88, 7610-7614, 1991) . The full-length ADH6 cDNA was expressed in the E.coli expression system and in the in vitro translation system of rabbit reticulocytes . The protein produced had its isoelectric point at pH 8.6, optimum pH at pH 10, and a lower Km for benzylalcohol than for ethanol and propanol . These characteristics are compatible to the properties of mu- or sigma-alcohol dehydrogenase isozyme existing in human stomach, indicating that ADH6 gene encodes the mu- or sigma-alcohol dehydrogenase subunit.

FEBS Lett, 1991 Dec 16, 295(1-3), 93 - 6
Molecular cloning and expression of the cDNA coding for a new member of the S100 protein family from porcine cardiac muscle; Ohta H et al.; We isolated a new calcium-binding protein from porcine cardiac muscle by calcium-dependent hydrophobic and dye-affinity chromatography . It showed an apparent molecular weight of 11,000 on SDS-PAGE . Amino acid sequence determination revealed that the protein contained two calcium-binding domains of the EF-hand motif . The cDNA gene coding for this protein was cloned from the porcine lung cDNA library . Sequence analysis of the cloned cDNA showed that the protein was composed of 99 amino acid residues and its molecular weight was estimated to be 11,179 . Immunological and functional characterization showed that the recombinant S100C protein expressed in Escherichia coli was identical to the natural protein . Homologies to calpactin light chain, S100 alpha and beta protein were 41.1%, 40.9% and 37.5%, respectively . The protein was expressed at high levels in lung and kidney, and low levels in liver and brain . The tissue distribution was apparently different from those of the other S100 protein family . These results indicate that this protein represents a new member of the S100 protein family, and thus we refer to it as S100C protein.

Biochem Biophys Res Commun, 1991 Dec 16, 181(2), 907 - 14
Mutants of human insulin-like growth factor II: expression and characterization of analogs with a substitution of TYR27 and/or a deletion of residues 62-67; Roth BV et al.; Five structural analogs of human insulin-like growth factor II (IGF II), {Leu27}IGF II, {Glu27}IGF II, des(62-67)IGF II, des(62-67){Leu27}IGF II and des(62-67){Glu27}IGF II were constructed by site-directed mutagenesis and expressed as protein A fusion proteins in E . coli BL21 pLysS cells, cleaved with CNBr and purified by affinity chromatography and HPLC . These mutants were tested for their binding affinities to type 1 and type 2 IGF receptors, to IGF binding protein-3 (IGFBP-3) and for their stimulation of thymidine incorporation into DNA . {Leu27}IGF II exhibits an affinity to the type 2 IGF receptor close to that of wild-type IGF II, but has lost completely the affinity to the type 1 IGF receptor . The results further suggest that the D domain, which is close to Tyr27, forms part of the binding region for the type 1 IGF receptor.

Biochem Biophys Res Commun, 1991 Dec 16, 181(2), 833 - 9
F-actin capping by cap32/34 requires heterodimeric conformation and can be inhibited with PIP2; Haus U et al.; The heterodimeric F-actin capping protein cap32/34 from Dictyostelium discoideum is a typical member of a widely distributed family of cytoskeletal proteins . To analyze its regulation and structure/function relationships we cloned and expressed the subunits separately in Escherichia coli using the ATG-expression vector pT7-7 . Studies on the viscosity of F-actin solutions and the kinetics of actin polymerization in the presence of single subunits or the reconstituted protein showed that capping of F-actin absolutely requires the heterodimeric conformation . This activity can be inhibited by phosphatidyl bisphosphate (PIP2), an important component in signal transduction . The regulation of cap32/34 by PIP2 suggests an involvement of this protein in the re-organization of the actin cytoskeleton upon stimulation of D . discoideum cells with chemoattractant.

Proc Natl Acad Sci U S A, 1991 Dec 15, 88(24), 11182 - 6
Identification of the discontinuous binding site in human interleukin 1 beta for the type I interleukin 1 receptor; Labriola-Tompkins E et al.; Human interleukin 1 beta (IL-1 beta) exerts its diverse biological effects by binding to specific receptors on target cells . Two types of IL-1 receptor (IL-1R) have been identified: the type I IL-1R (p80) and the type II IL-1R (p68) . Using site-specific mutagenesis, we have identified the binding site on IL-1 beta for the murine type I IL-1R . Analogs of the IL-1 beta protein containing defined amino acid substitutions were produced and tested for competitive binding to the two IL-1Rs . Substitutions of the amino acids at seven positions resulted in analogs that had greater than or equal to 100-fold reductions in competitive binding to the type I IL-1R, while maintaining substantial binding to the type II IL-1R . These seven amino acids (Arg-4, Leu-6, Phe-46, Ile-56, Lys-93, Lys-103, and Glu-105) are clustered in the IL-1 beta molecule, forming a discontinuous binding site . The side chains of all seven residues are exposed on the surface of IL-1 beta . The cumulative binding energies contributed by each of the residues predict a binding affinity that is consistent with the observed Kd of the wild-type protein for the type I IL-1R.

Proc Natl Acad Sci U S A, 1991 Dec 15, 88(24), 11143 - 7
A protein kinase is present in a complex with adenovirus E1A proteins; Kleinberger T et al.; A kinase activity can be immunoprecipitated in a complex that includes adenovirus E1A proteins . In vitro, this activity phosphorylated other E1A-associated proteins, as well as added E1A and histone H1 proteins . The E1A-associated kinase activity was cleared from extracts with an antibody to cyclin A, but not with antibody to cyclin B . The formation of a complex that included the kinase activity required amino acids 30-60 and 122-129 on the E1A proteins, sequences needed for association of E1A proteins with cyclin A and the retinoblastoma protein and implicated in control of cell growth . The complex of E1A-associated proteins included a 33-kDa ATP-binding protein, similar in size to a cyclin A-associated cdc2 kinase family member . Sucrose gradient analysis revealed two distinct E1A-containing complexes with the kinase activity . We suggest that E1A proteins may affect cellular proliferation by interacting with a member of the cdc2 kinase family and thereby influencing its activity.

J Biol Chem, 1991 Dec 15, 266(35), 24077 - 83
A compact nucleoprotein structure is produced by binding of Escherichia coli integration host factor (IHF) to the replication origin of plasmid R6K; Filutowicz M et al.; Understanding the role of Escherichia coli histone-like protein integration host factor (IHF) in replication of R6K plasmid (Dellis, S., and Filutowicz, M . (1991) J . Bacteriol . 173, 1279-1286) requires detailed analyses of the interaction of IHF protein with the plasmid's replication origin (gamma ori) . We describe an electron microscopic analysis which shows that a compact structure can be formed in the presence of IHF, in which, on average, a 102-base pair (bp) ori segment is involved . IHF.gamma ori complexes also undergo a two-step conformational change in an IHF concentration-dependent manner when analysed by band shift assay . We believe that the DNA is bent at low IHF concentrations, but folded at high IHF concentrations . This idea is supported by the fact that electrophoretic mobility of the IHF.gamma ori complexes is faster at higher concentrations of IHF . Furthermore, it is shown that the formation of a compact nucleoprotein structure depends on the two regions flanking the AT-rich segment; the iterons to the right and the 106-bp ori domain to the left . Finally we show that IHF protects the entire AT-rich segment of the ori against nuclease cleavage . In addition to the protection, an altered cleavage pattern by DNase I, in the presence of high levels of IHF, was observed within the iterons but not within the 106-bp domain of the ori . Implications of the IHF-mediated gamma ori folding as a possible mechanism protecting the ori from replication inhibition by R6K initiator protein tau are discussed.

FEMS Microbiol Lett, 1991 Dec 15, 69(1), 89 - 93
The ability of rifampin-resistant Escherichia coli to colonize the mouse intestine is enhanced by the presence of a plasmid-encoded aerobactin-iron(III) uptake system; Stojiljkovic I et al.; Rifampin-resistant Escherichia coli are known to be poor colonizers of the animal intestine . In this report, we show that the colonizing ability of rifampin-resistant E . coli cells is increased dramatically in the presence of the aerobactin-mediated iron(III) uptake system . In contrast, the colonization by nalidixic acid-resistant E . coli does neither depend on the aerobactin-iron(III) nor on the dicitrate-iron(III) uptake system . Likewise, it does not depend on the production of the siderophore enterochelin.

FEMS Microbiol Lett, 1991 Dec 15, 69(1), 57 - 62
RNA polymerase heterogeneity in Streptomyces aureofaciens: characterization by antibody-linked polymerase assay; Farkasovsky M et al.; Using antibody-linked polymerase assay we studied the polypeptide composition of DNA-dependent RNA polymerase from Streptomyces aureofaciens and immunological cross-reaction with Escherichia coli RNA polymerase . We identified about 25 'ALPA-reactive' polypeptides which are probably involved in the transcriptional apparatus . We demonstrated that beta' and beta subunits from S . aureofaciens and E . coli are immunologically related and sigma70 (E . coli) shows immunochemical similarity with sigma35 (S . aureofaciens) . According to the reconstitution of RNA polymerase holoenzyme and antibody-linked polymerase assay we identified sigma factors responsible for recognition of two promoters.

FEMS Microbiol Lett, 1991 Dec 15, 69(1), 49 - 52
Colicinogeny of O55 EPEC diarrhoeagenic Escherichia coli; Bradley DE; Approximately 24% of a sample of pathogenic Escherichia coli strains from different serogroups were found to synthesize colicins . Serogroup O55 had an unusually large proportion of such strains (33%) . In a sample of 27 O55 isolates, one synthesized a class A colicin (identified as ColE9), five produced class B colicins (three ColIa, two, unidentifiable), and three a class A and a class B together.

Biochem J, 1991 Dec 15, 280 ( Pt 3), 575 - 9
H.p.l.c . of oligo(sialic acids) . Application to the determination of the minimal chain length serving as exogenous acceptor in the enzymic synthesis of colominic acid; Ferrero MA et al.; A rapid, sensitive and easy h.p.l.c . method was developed for the quantitative analysis of oligosialic acids . This procedure which permits the complete separation (in 23 min) of several sialyloligomers with a degree of polymerization of between 1 and 16, has been employed to establish the minimal chain length of oligomer accepted, as an exogenous acceptor, by Escherichia coli K-235 sialytransferase complex (ST) leading to the synthesis in vitro of colominic acid . We showed that this membrane-bound enzyme catalyses the direct transfer of Neu5Ac residues (one by one) from CMP-Neu5Ac to an exogenous acceptor molecule which contains at least three Neu5Ac residues . Free Neu5Ac or (Neu5Ac)2 were not recognized as substrates, whereas the maximal rate of polymer elongation was achieved when (Neu5Ac)5 was used as substrate.

Proc Natl Acad Sci U S A, 1991 Dec 15, 88(24), 11574 - 8
Escherichia coli mfd mutant deficient in "mutation frequency decline" lacks strand-specific repair: in vitro complementation with purified coupling factor; Selby CP et al.; Mutation frequency decline (MFD) is the rapid decrease in the frequency of certain induced nonsense suppressor mutations occurring when protein synthesis is transiently inhibited immediately after irradiation . MFD is abolished by mutations in the uvrA, -B, or -C genes, which prevent excision repair, or by a mfd mutation, which reduces the rate of excision but does not affect survival . Using an in vitro repair synthesis assay we found that although wild-type cells repair the transcribed (template) strand preferentially, mfd- cells are incapable of strand-specific repair . The deficiency in strand-selective repair of mfd- cell extract was corrected by adding highly purified "transcription-repair coupling factor" to the reaction mixture . We conclude that mfd is, most likely, the gene encoding the transcription-repair coupling factor.

Proc Natl Acad Sci U S A, 1991 Dec 15, 88(24), 11445 - 9
Interaction of the human T-cell lymphotrophic virus type I (HTLV-I) transcriptional activator Tax with cellular factors that bind specifically to the 21-base-pair repeats in the HTLV-I enhancer; Zhao LJ et al.; The human T-cell lymphotrophic virus type I (HTLV-I) Tax protein activates transcription from three 21-base-pair (bp) repeat sequences in the viral enhancer . Using gel electrophoretic mobility-shift assays, we now show that Tax interacts directly with the nuclear proteins, Tax activation factors (TAFs), that bind the 21-bp repeats . This interaction is demonstrated by decreased electrophoretic mobilities of the TAFs-21-bp-repeats complexes upon supply of Tax exogenously . Formation of the TAFs-21-bp-repeats and Tax-TAFs-21-bp-repeats complexes correlates with in vivo transactivation by Tax . Furthermore, interaction of Tax with TAFs enhances their binding to the 21-bp repeats . These data indicate that trans-activation by Tax is most likely mediated by interaction of Tax with TAFs.

Proc Natl Acad Sci U S A, 1991 Dec 15, 88(24), 11373 - 7
RAM2, an essential gene of yeast, and RAM1 encode the two polypeptide components of the farnesyltransferase that prenylates a-factor and Ras proteins; He B et al.; In the yeast Saccharomyces cerevisiae, mutations in either of two unlinked genes, RAM1 or RAM2, abolish the farnesyltransferase activity responsible for prenylation of Ras proteins and the a-factor mating pheromone . Here we report that the function of RAM1 and RAM2 genes is required for the membrane localization of Ras proteins and a-factor . The RAM2 gene was sequenced and can encode a 38-kDa protein . We examined the functional interaction of RAM2 and RAM1 by expressing the genes in Escherichia coli . Extracts derived from an E . coli strain that coexpressed RAM1 and RAM2 efficiently farnesylated a-factor peptide and Ras protein substrates . In contrast, extracts derived from E . coli strains that expressed either RAM gene alone were devoid of activity; however, when the latter extracts were mixed, protein farnesyltransferase activity was reconstituted . These results indicate that the yeast farnesyl-protein transferase is comprised of Ram1 and Ram2 polypeptides . Although Ram1 is a component of the enzyme, disruption of the RAM1 gene in yeast was not lethal, indicating that the Ram1-Ram2 farnesyltransferase is not essential for viability . In contrast, disruption of RAM2 was lethal, suggesting that Ram2 has an essential function in addition to its role with Ram1 in protein farnesylation.

Proc Natl Acad Sci U S A, 1991 Dec 15, 88(24), 11325 - 9
Positioning of a peptide in the cleft of HLA-A2 by complementing amino acid changes; Latron F et al.; Several mutant HLA-A2 molecules have been constructed and expressed in the mutant human B-cell line C1R, which lacks HLA-A and HLA-B antigens, and examined for presentation of a previously defined peptide epitope derived from the influenza matrix protein to appropriate human cytotoxic T-lymphocyte lines . When leucine residue 66 in this matrix peptide containing residues 57-68 (matrix peptide 57-68) was replaced by arginine, the resulting matrix peptide 57-68 R66 was not presented to HLA-A2, but the mutation Y116D (tyrosine to aspartic acid at residue 116) in the floor of the peptide binding cleft near its right end dramatically restored peptide presentation . A similar result was obtained by substitution of ornithine for leucine at residue 66 . These data provide strong support for a model in which the peptide is orientated with its amino terminus at the left end of the cleft of HLA-A2 and its carboxyl terminus at the right.

J Biol Chem, 1991 Dec 15, 266(35), 24070 - 6
Purified estrogen receptor DNA binding domain expressed in Escherichia coli activates transcription of an estrogen-responsive promoter in cultured cells; Nardulli AM et al.; The region of the Xenopus laevis estrogen receptor responsible for interaction with DNA, the DNA binding domain (DBD), has been cloned and overexpressed in Escherichia coli using a T7 RNA polymerase expression system . Extracts from cells transformed with the DBD expression vector contain a single protein which reacts with polyclonal antibodies to estrogen receptor and exhibits sequence-specific binding to a DNA fragment containing a consensus estrogen response element . The DBD protein has been purified to near homogeneity . Determination of the rotational relaxation time of the dansylated DBD by fluorescence polarization and size fractionation by Superdex column chromatography indicate that the DBD is a monomer in solution . The DBD forms a single protein-estrogen response element complex in gel mobility shift assays at DBD concentrations of 18-3,600 nM, suggesting that the DBD is bound to both halves of the palindromic estrogen response element . To investigate the ability of the DBD expressed in bacteria to activate gene expression, we have developed a simple liposome-based system for delivery of protein into cultured cells . Transfected DBD protein elicited large, concentration-dependent increases in transcription of an estrogen receptor regulated reporter gene . These data demonstrate that the bacterially expressed DNA binding domain, which represents a small portion of the Xenopus laevis estrogen receptor, retains significant ability to activate transcription of an estrogen-responsive promoter in vertebrate cells.

J Biol Chem, 1991 Dec 15, 266(35), 24031 - 7
Expression of human aldose and aldehyde reductases . Site-directed mutagenesis of a critical lysine 262; Bohren KM et al.; Human aldose reductase (EC 1.1.1.21) and aldehyde reductase (EC 1.1.1.2) are implicated in the development of diabetic complications by a variety of mechanisms, and a number of drugs to inhibit these enzymes have been proposed for the therapy and prevention of these complications . To probe the structure and function of these two enzymes, we used site-directed mutagenesis in the cDNAs of both enzymes to replace lysine 262 with methionine . Wild-type and mutant enzymes were overexpressed in Escherichia coli and purified by anion exchange and affinity chromatography . N-terminal sequence analysis, Western blots, and kinetic studies confirmed the identity of the recombinant wild-type enzymes with the native human placental and liver enzymes . Recombinant aldose reductase (hAR) and aldehyde reductase (hGR) have apparent kinetic constants virtually identical to their respective native enzymes . The mutant aldose reductase (hARK262 greater than M) shows a 66-fold increase in Km for NADPH with respect to the wild type (1.9 +/- 0.4 microM versus 125 +/- 14 microM), whereas the Km for DL-glyceraldehyde increased 35-fold (20 +/- 2 versus 693 +/- 41 microM) . The same constants for the mutant aldehyde reductase (hGRK262 greater than M) increased 97- and 86-fold, respectively (from 2.0 +/- 0.4 to 194 +/- 16 microM and from 1.6 +/- 0.4 to 137 +/- 3 mM) . These results indicate that lysine 262 in aldose reductase and aldehyde reductase is crucial to their catalytic activity by affecting co-factor binding.

J Biol Chem, 1991 Dec 15, 266(35), 23953 - 8
Purification, characterization, cloning, and amino acid sequence of the bifunctional enzyme 5,10-methylenetetrahydrofolate dehydrogenase/5,10-methenyltetrahydrofolate cyclohydrolase from Escherichia coli; D'Ari L et al.; We have purified the enzyme 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) from Escherichia coli to homogeneity by a newly devised procedure . The enzyme has been purified at least 2,000-fold in a 31% yield . The specific activity of the enzyme obtained is 7.4 times greater than any previous preparation from this source . The purified enzyme is specific for NADP . The protein also contains 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9) activity . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behavior on a molecular sieving column suggest that the enzyme is a dimer of identical subunits . We have cloned the E . coli gene coding for the enzyme through the use of polymerase chain reaction based on primers designed from the NH2 terminal analysis of the isolated enzyme . We sequenced the gene . The derived amino acid sequence of the enzyme contains 287 amino acids of Mr 31,000 . The sequence shows 50% identity to two bifunctional mitochondrial enzymes specific for NAD, and 40-45% identity to the presumed dehydrogenase/cyclohydrolase domains of the trifunctional C1-tetrahydrofolate synthase of yeast mitochondria and cytoplasm and human and rat cytoplasm . An identical sequence of 14 amino acids with no gaps is present in all 7 sequences.

J Biol Chem, 1991 Dec 15, 266(35), 23927 - 31
Mapping of the priming substrate contacts in the active center of Escherichia coli RNA polymerase; Mustaev A et al.; The active center of DNA-dependent RNA polymerase performs the principal biochemical reaction of gene expression . Using cross-linkable substrate analogs and site-directed mutations, two evolutionarily invariant amino acids in the beta subunit of the Escherichia coli enzyme (Lys1065 and His1237) were mapped close to the binding site of the priming substrate of the reaction . Surprisingly, the mutational substitution of these residues (Lys1065----Arg and His1237----Ala) did not inactivate the catalytic function, but inhibited transition from the initiation to the elongation stage of transcription.

J Biol Chem, 1991 Dec 15, 266(35), 23904 - 8
Epimerization of 3-hydroxy-4-trans-decenoyl coenzyme A by a dehydration/hydration mechanism catalyzed by the multienzyme complex of fatty acid oxidation from Escherichia coli; Smeland TE et al.; The mechanism of 3-hydroxyacyl-CoA epimerase (EC 5.1.2.3), which is associated with the multienzyme complex of fatty acid oxidation from Escherichia coli, was studied with D-3-hydroxy-4-trans-decenoyl-CoA as a substrate . The E . coli complex catalyzes the rapid and direct dehydration of D-3-hydroxy-4-trans-decenoyl-CoA to 2-trans,4-trans-decadienoyl-CoA, which is slowly hydrated to L-3-hydroxy-4-trans-decenoyl-CoA . A kinetic analysis of the epimerase and its partial reactions established that epimerization of 3-hydroxyacyl-CoAs occurs solely by a dehydration/hydration mechanism . The results of a substrate competition study with L-3-hydroxy-4-trans-decenoyl-CoA and its D-isomer, together with the conclusion from a sequence analysis of the large subunit of the E . coli complex (Yang, X.-Y., Schulz, H., Elzinga, M., and Yang, S.-Y . (1991) Biochemistry 30, 6788-6795), prompt the suggestion that a single active site is responsible for the dehydration of the D- and L-isomers of 3-hydroxyacyl-CoAs.

J Biol Chem, 1991 Dec 15, 266(35), 23654 - 9
Structural and functional consequences of amino acid substitutions in the second conserved loop of Escherichia coli adenylate kinase; Rose T et al.; All known nucleoside monophosphate kinases contain an invariant sequence Asp-Gly-Phe(Tyr)-Pro-Arg . In order to understand better the structural and functional role of individual amino acid residues belonging to the above sequence, three mutants of Escherichia coli adenylate kinase (D84H, G85V, and F86L) were produced by site-directed mutagenesis . Circular dichroism spectra revealed that the secondary structure dichroism spectra revealed that the secondary structure of all three mutant proteins is very similar to that of the wild-type enzyme . However, each of the substitutions resulted in a decreased thermodynamic stability of the protein, as indicated by differential scanning calorimetry measurements and equilibrium unfolding experiments in guanidine HCl . The destabilizing effect was most pronounced for the G85V mutant, in which case the denaturation temperature was decreased by as much as 11 degrees C . The catalytic activity of the three mutants represented less than 1% of that of the wild-type enzyme . Furthermore, for the D84H-modified form of adenylate kinase, the impaired binding of nucleotide substrates was accompanied by a markedly decreased affinity for magnesium ion . These observations support the notion that Asp84 is directly involved in binding of nucleotide substrates and that this binding is mediated by interaction of the aspartic acid residue with divalent cation . The two remaining residues probed in this study, Gly85 and Phe86, belong to a beta-turn which appears to play a major role in stabilizing the three-dimensional structure of adenylate kinase.

J Biol Chem, 1991 Dec 15, 266(35), 23558 - 60
Substantial increase of the inhibitory activity of Mirabilis antiviral protein by an elimination of the disulfide bond with genetic engineering; Habuka N et al.; Mirabilis antiviral protein (MAP) is a rigid, heat-stable protein composed of 250 amino acids with an intramolecular disulfide bond . MAP inhibits the in vitro protein synthesis of rabbit reticulocyte with approximately one-thirtieth the activity of the ricin A chain, a homologous protein with no such bond (Habuka, N., Murakami, Y., Noma, M., Kudo, T., and Horikoshi, K . (1989) J . Biol . Chem . 264, 6629-6637; Habuka, N., Akiyama, K., Tsuge, H., Miyano, M., Matsumoto, T., and Noma, M . (1990) J . Biol . Chem . 265, 10988-10992) . The bond is presumed to induce some structural perturbation that alters the mode of interaction with the substrate ribosome and thus lowers the activity . To confirm this hypothesis, a mutant MAP gene in which the codons of both cysteines were replaced by those of serines was constructed and expressed in Escherichia coli, and its product (C36/22OS) was purified . In a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, C36/220S showed the same mobility as that of MAP reduced by 2-mercaptoethanol, whereas nonreduced MAP showed faster migration . The inhibitory activity of C36/220S was approximately 22 times higher than that of native MAP, that is the mutant had an IC50 of 0.16 nM for the protein synthesis of the rabbit reticulocyte system, whereas the native MAP had an IC50 of 3.5 nM . The results indicate that the activity of MAP is increased by the elimination of the disulfide bond, and this supports the hypothesis.

J Immunol Methods, 1991 Dec 15, 145(1-2), 127 - 36
Human group II phospholipase A2 . Characterization of monoclonal antibodies and immunochemical quantitation of the protein in synovial fluid; Stoner CR et al.; Murine monoclonal and rabbit polyclonal antibodies were generated against human group II phospholipase A2 (PLA2) in order to study the role of this enzyme in inflammatory disease, the source of its synthesis, and the interaction of PLA2 with its substrate . Monoclonal antibody PLA187 exhibits potent inhibitory activity toward human PLA2 using autoclaved E . coli membranes as the substrate . Three other monoclonal antibodies (PLA184, PLA185, and PLA186) also inhibit enzyme activity, but with about 50-fold less potency . Based on the results of double-antibody competition experiments and enzyme inhibition profiles, PLA184 and PLA185 appear to recognize the same epitope . Monoclonal antibody PLA186 recognizes an epitope which is spatially distinct from that recognized by PLA184/185 . The results also suggest that the epitope recognized by PLA187 may overlap with both epitopes recognized by PLA186 and PLA184/185 . A double-antibody sandwich ELISA was developed using a combination of PLA185 and rabbit polyclonal antibody against PLA2 . The ELISA provides a sensitive and quantitative method for monitoring specifically group II PLA2 in various biological sources, independent of factors which may affect enzyme activity . We have utilized this assay to quantitate PLA2 levels in synovial fluid from the joints of individuals with rheumatoid arthritis as well as from non-arthritic joints . Our results indicate that elevated levels of group II PLA2 in synovial fluid are not necessarily associated with arthritis.

J Biol Chem, 1991 Dec 15, 266(35), 24140 - 8
Rho-dependent transcription termination . Characterization of the requirement for cytidine in the nascent transcript; Hart CM et al.; By substituting template segments encoding AU-rich, GU-rich, and CA-rich transcripts for natural sequences upstream of the phage lambda rho-dependent tR1 termination site, we demonstrate that cytidines are required in the upstream RNA for rho-dependent termination to occur . These results are extended through in vitro mutagenesis of a template encoding an inactive AU-rich upstream sequence: certain mutant templates encoding new cytidines are able to activate rho-dependent termination . Cytidines must be dispersed over a region of the transcript in order for rho to be activated, although no specific pattern of cytidines appears to be required . The results show that no local clustering or regular spacing of cytidines is necessary and that cytidines are not used as a "ruler" to determine the location of termination sites . Rho is somewhat sensitive to the relative positions of cytidines since slightly different nascent transcripts activate rho termination activity to various degrees . A model is presented in which hexameric rho binds 78 nucleotides of contiguous RNA in a primary site, such that each monomer interacts with at least one cytidine somewhere in the 13 nucleotides allotted to the monomeric primary site.

J Biol Chem, 1991 Dec 15, 266(35), 23932 - 5
Mapping of a contact for the RNA 3' terminus in the largest subunit of RNA polymerase; Borukhov S et al.; Stalled elongation complexes of Escherichia coli RNA polymerase were prepared carrying the photo-cross-linkable 8-azido derivative of adenine at the 3'-terminus of the nascent RNA chain . Ultraviolet irradiation of such complexes resulted in the cross-linking of radiolabeled RNA exclusively to the beta' subunit of RNA polymerase . The adduct was mapped between Met932 and Trp1020 in the linear sequence of the beta' polypeptide using specific chemical degradation of the cross-linked species.

J Biol Chem, 1991 Dec 15, 266(35), 23834 - 9
Transcriptional regulation of p90 with sequence homology to Escherichia coli glycerol-3-phosphate acyltransferase; Shin DH et al.; We have previously isolated cDNA clones for several mRNAs that are dramatically increased in livers of fasted mice refed a high carbohydrate diet . We report here the sequence and regulation of one such mRNA; the 6.8-kilobase mRNA has an open reading frame of 2481 nucleotides, and the coded protein contains 827 amino acid residues (Mr of 90,000) with a 30% identity and an additional 42% similarity in an approximately 300-amino acid stretch to Escherichia coli glycerol-3-phosphate acyltransferase . The p90 mRNA is highly expressed in liver and in adipose tissue . When previously fasted mice were refed a high carbohydrate, fat-free diet, the liver mRNA level for p90 was increased about 20-fold at 8 h . Administration of dibutyryl cAMP at the time of refeeding prevented the increase in the p90 mRNA by 70% . In addition, there was no increase in the p90 mRNA level when previously starved streptozotocin-diabetic mice were refed . In diabetic animals, the p90 mRNA level increased by 2-fold 1 h after insulin injection and reached a maximum of 19-fold after 6 h . The increase in transcription rate of the p90 gene preceded that of steady state mRNA level caused by fasting/refeeding, and cAMP abolished the increase in transcription . Transcription of the p90 gene was not detectable in either fasted or refed streptozotocin-diabetic mice, but increased 4-fold 30 min after insulin administration and further increased up to 8-fold at 2 h . On-going protein synthesis was necessary for this increase.

J Biol Chem, 1991 Dec 15, 266(35), 23648 - 53
Effect of amino acid substitution by sited-directed mutagenesis on the carbohydrate recognition and stability of human 14-kDa beta-galactoside-binding lectin; Hirabayashi J et al.; The roles of selected amino acid residues of human 14-kDa beta-galactoside-binding lectin were studied by site-directed mutagenesis . Ten mutant lectin proteins were produced, in each of which one of the residues regarded as possibly related to the stability of the lectin (6 cysteine residues) or one of those highly conserved in the vertebrate beta-galactoside-binding lectin family (Asn46, Trp68, Glu71, and Arg73), was substituted . All the mutant lectins in which one of the cysteine residues had been substituted with serine (C2S, C16S, C42S, C60S, C88S, and C130S) proved to have sugar binding ability comparable with that of the wild-type lectin . In addition, one of the mutants in which Cys2 was substituted (C2S) was found to have become considerably more stable under non-reducing conditions . It retained asialofetuin binding activity for over a week in the absence of beta-mercaptoethanol, while the wild-type lectin lost it within a day . This suggests that oxidation of Cys2 could be a key process in the inactivation of human 14-kDa lectin . Substitution of highly conservative Trp68 to tyrosine (W68Y) slightly reduced lactose binding ability, but the mutant was still adsorbed strongly on asialofetuin-agarose . Other mutant lectins in which conservative hydrophilic amino acids were substituted (N46D, E71Q, and R73H) failed to bind to the asialofetuin agarose, with no sign of retardation . Thus, conservative hydrophilic residues proved to be more important in carbohydrate recognition than the cysteine and tryptophan residues, contrary to the widely accepted concept that these latter residues are essential.

What Is Genetic Engineering?, What Is Activated Sludge?, What Is Water Purification?, What Is Staphylococcus Aureus?, What Is Functional Genomics?, n, Bacteriology, r, Bacterium, c, Microbe, c, Microbes, a, Microorganisms, r, Microbiological, o, Vibriosis, r, Bactericidal, i, Bacteriological, n, Staphylococcus aureus, a, Candida albicans, o, Antimicrobials, e, S. cerevisiae, o, Haemophilus, n, Microorganisms, s, Fermentations, s, Escherichia coli, o, Rhodotorula, o, Escherichia coli, o, Escherichia coli, a, Escherichia coli, c, Gram negative, a, Denitrificans, n, Streptococci, n, Antimicrobial, n, Petri dish

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005