Escherichia coli

Life Sci, 1994, 55(15), PL277 - 85
Octreotide does not alter endotoxin lethality in mice or endotoxin-induced suppression of human leukocyte migration; Ogden BE et al.; Octreotide, a somatostatin analog, was evaluated for its effects on long-term survival in a mouse model of endotoxemia and for its effects on endotoxin-induced suppression of human leukocyte migration . Swiss Webster mice were simultaneously rendered endotoxemic with a single intraperitoneal injection of 800 micrograms E . coli Lipopolysaccharide (LPS) and treated with one of four doses of subcutaneous (s.c.) octreotide (1.0 mg/kg in 0.4 ml saline, 0.1 mg/kg in 0.4 ml saline, 0.1 mg/kg in 0.04 ml saline, or 0.001 mg/kg in 0.04 ml saline) or saline alone (fluid-resuscitated control group: 0.4 ml saline s.c.; or non-fluid-resuscitated control group: 0.04 ml saline s.c.) . Octreotide was continued with or without supplemental s.c . fluid resuscitation (0.4 ml saline) at eight hour intervals for either twenty-four or forty hours . There was no statistical significance to differences in long-term survival between comparable groups of octreotide treated vs saline treated animals during the entire fourteen day period of observation . Fluid resuscitation during the first forty hours following endotoxemia induction delayed death, but did not significantly improve long-term survival . In vitro work was conducted to determine the effect of octreotide on endotoxin-induced suppression of human leukocyte migration . Octreotide at concentrations ranging from 3.05 x 10(-5) Molar to 3.05 x 10(-11) Molar had no significant effect on leukocyte migration . In this study octreotide treatment failed to improve long-term survival in mice with endotoxemia and did not alter endotoxin-induced suppression of leukocyte migration.

Curr Biol, 1994 Jan 1, 4(1), 73 - 5
Transcriptional termination . Charging it cancels the order; Zahler SA et al.; In a newly discovered mechanism for regulating transcription termination, a cognate uncharged tRNA acts directly to promote transcription readthrough.

Arch Med Res, 1994 Summer, 25(2), 229 - 33
Actinobacillus pleuropneumoniae: virulence and gene cloning; Negrete-Abascal E et al.; Actinobacillus pleuropneumoniae is the causal agent of porcine contagious pleuropneumonia (PCP) . The infection produces important economic losses in porciculture due to its high morbidity and mortality . Survivors are asymptomatic carriers infectious to other pigs and have low alimentary conversion . The causative agent possesses several virulence factors: adhesion fimbriae, lipopolysaccharide of the outer membrane, capsule, and cytolysins . In addition, our group has reported secretion proteases of a wide pH range of activity . These proteases degrade different substrates such as porcine gelatin, hemoglobin and IgA, and bovine or human hemoglobin . To control PCP dissemination, farmers require serodiagnostic tests which detect carriers and discriminate between vaccinated and infected animals . Bacterines used as immunogens are serotype specific and do not prevent the infection . Genes have been cloned that codify a cohemolysin, cytolysins, and an iron-binding protein . We have cloned A . pleuropneumoniae genes using the expression plasmids pUC19 and Bluescript, in Escherichia coli Q358 and DH5 alpha; the screening for antigen production was made in four groups of pigs (vaccinated, experimentally infected, naturally infected, and from slaughterhouses); two E . coli clones expressed polypeptides recognized by sera from all the groups.

Biomed Pharmacother, 1994, 48(1), 35 - 9
The enhancement of the extracellular carboxyl-terminal domain of human growth hormone receptor on growth hormone dependent responses of 3T3-F442A cells; Asakura A et al.; We have expressed the carboxyl-terminal domain (C domain) of the cytokine receptor homologous (CRH) region of human growth hormone receptor (hGHR) as a protein fused with maltose binding protein (MBP) in E . coli . Following proteolytic cleavage by restriction protease factor Xa, the C domain was purified to homogeneity as a monomeric form . The purified C domain appears to be folded properly judged by NMR spectrum and the far-UV circular dichroism (CD) spectrum . The C domain did not exhibit ligand binding activity . However, the C domain enhanced the human growth hormone (hGH) dependent differentiation of preadipose 3T3-F442A cells into adipose cells and the phosphorylation of a 34 kDa membrane protein.

Prog Mol Subcell Biol, 1994, 14, 176 - 97
The 2-5A system and HIV infection; Schroder HC et al.; 2',5'-Oligoadenylates (2-5A) have an essential role in the establishment of the antiviral state of a cell exposed to virus infection . The key enzymes of the 2-5A system are the 2-5A forming 2',5'-oligoadenylate synthetase (2-5OAS), the activity of which depends on the presence of viral or cellular double-stranded RNA (dsRNA), and the 2-5A-activated ribonuclease (RNase L) . Basic research in recent years has shown that the 2-5A system is a promising target for anti-HIV chemotherapy, particularly due to its interaction with double-stranded segments within HIV RNA . Two new strategies have been developed which yield a selective antiviral effect of 2-5A against HIV-1 infection: (1) development of 2-5A analogues displaying a dual mode of action (activation of RNase L and inhibition of HIV-1 RT) and (2) intracellular immunization of cells against HIV-1 infection by application of the HIV-1-LTR--2-5OAS hybrid gene . A further strategy is the inhibition of DNA topoisomerase I by longer 2-5A oligomers.

Microb Pathog, 1994 Jan, 16(1), 15 - 25
Analysis of genes coding for the major and minor fimbrial subunits of the Prs-like fimbriae F165(1) of porcine septicemic Escherichia coli strain 4787; Maiti SN et al.; Septicemic Escherichia coli 4787 of porcine origin produces a Prs-like fimbrial antigen, F165(1) which agglutinates sheep erythrocytes in a similar manner to Prs fimbriae, but unlike the latter, also agglutinates pig erythrocytes . In a previous study, we reported the cloning of the f165(1) operon and showed that it encodes a Prs-like adhesin . Here, we report the sequence of the f165(1)A, and f165(1)EFG genes . f165(1)A encodes a protein of 161 amino acids preceded by a signal peptide of 21 amino acids . A size of 19.3 kDa was calculated for the processed F165(1)A protein . The E, F, and G open reading frames potentially give rise to mature proteins of 149, 148 and 313 amino acids respectively . The F165(1)A protein showed significant similarity with the major subunit protein of P-fimbriae of F11 serotype, differing only in four positions . F165(1)E and F165(1)G were found to be closely related to the PrsE and PrsG proteins of Prs-fimbriae variants, whereas F165(1)F was found to be almost completely identical to the F protein of various P and Prs fimbriae . Our results indicated that the f165(1) is a mosaic operon consisting of sequences related to both the pap operon and the prs operon.

Mol Membr Biol, 1994 Jan-Mar, 11(1), 9 - 16
Role of glutamate-269 in the lactose permease of Escherichia coli; Ujwal ML et al.; Glu-269, which is located on the hydrophilic face of putative helix VIII in the lactose permease of Escherichia coli, has been replaced with Asp, Gln or Cys by oligonucleotide-directed, site specific mutagenesis . Cells expressing Asp-269 permease exhibit no lactose accumulation or lactose-induced H+ translocation, but retain some ability to mediate lactose influx down a concentration gradient at high substrate concentrations . Furthermore, right-side-out membrane vesicles containing Asp-269 permease do not catalyse active lactose transport, facilitated lactose efflux or equilibrium exchange . Remarkably, however, Asp-269 permease accumulates beta, D-galactopyranosyl 1-thio-beta,D-galactopyranoside in a partially uncoupled fashion, whereas no transport of methyl-beta,D-thiogalactopyranoside, sucrose or maltose is detectable . Mutant permeases containing neutral replacements (Gln or Cys) or Glu-269 are completely devoid of activity, although the proteins are present in the membrane at concentrations comparable with wild-type or Asp-269 permease . The observations demonstrate that a carboxylate at position 269 is essential for transport activity, and Glu-269 is important for substrate binding and/or recognition.

Vet Microbiol, 1994 Jan, 38(3), 217 - 25
Hybridization of clinical Escherichia coli isolates from calves and piglets in New York State with gene probes for enterotoxins (STaP, STb, LT), Shiga-like toxins (SLT-1, SLT-II) and adhesion factors (K88, K99, F41, 987P); Shin SJ et al.; Six hundred and sixty-six bovine and fifty-seven swine clinical isolates of E . coli from New York state were examined for the presence of enterotoxins (STaP, STb, LT, SLT-I, and SLT-II) and adhesins (K88, K99, F41, and 987P) using colony hybridization techniques . Three hundred and sixty-seven of the bovine isolates (45.2%) hybridized with at least one gene probe . Of these, two hundred and twenty-three (33.2%) hybridized with F41, one hundred twelve (16.7%) with K99, eighty-two (12.2%) with 987P, ninety-six (14.3%) with STaP, seven (1.1%) with STb, and none (0.0%) with LT and K88 . A total of thirty-three (4.7%) of the isolates hybridized with SLT-I, and one (0.1%) with SLT-II . The major pathotypes among the 666 isolates from bovine were K99/F41/StaP (9.8%), K99/F41 (2.5%), p87P/F41 (2.1%) and 987P/K99/F41/StaP (1.4%) . Of the swine clinical isolates, twenty-two hybridized with at least one gene probe . The major pathotypes among the isolates from piglets were K88/K99/F41/StaP (5.3%) and K88/F41 (5.3%).

Vet Microbiol, 1994 Jan, 38(3), 203 - 15
The distribution and stability of Escherichia coli K88 receptor in the gastrointestinal tract of the pig; Chandler DS et al.; The levels of Escherichia coli K88 receptor were measured at various sites within the pig intestinal tract using an enzyme immunoassay . The amount of receptor in samples taken from K88-adhesive phenotype animals was found to vary widely along the length of the intestinal tract, but was usually highest in mucosal scrapings taken from the mid-small intestine . Receptor was evident in material collected near either end of the small intestine and was not apparent in material collected from the caecum or lower bowel . The ability of receptor-containing intestinal material to react with immobilized K88 adhesin was inhibited by exposure of the material to either trypsin or contents from the lower bowel, if the receptor-containing material was reacted with the immobilised K88 adhesin prior to exposure to trypsin or lower bowel contents, the bound material remained evident for 24 to 48 h . The possible implications of variable receptor activity in proteolytic environments in relation to pathogenesis and the determination of K88 phenotype in live pigs is discussed.

Appl Biochem Biotechnol, 1994 Spring, 45-46, 233 - 44
Cell separations using targeted monoclonal antibodies against overproduced surface proteins; Tadikonda KR et al.; The capacity of polystyrene microspheres with immobilized antibodies against type 1 pili of E . coli was measured . Using pure IgG-type monoclonal antibodies obtained by affinity purification, it was found that, at high concentrations of antibody in solution, 2.6 +/- 0.4 mg of antibody immobilized/m2 of surface area . The binding of piliating E . coli cells on incubation with antibody-coated microspheres at various microsphere-to-cell number ratios ranging from 1-3750 was studied . It was found that a maximum of 290 +/- 50 cells were bound/microsphere, equivalent to a binding capacity of 2.9 +/- 0.7 x 10(10) cells/m2 of surface area.

Chirality, 1994, 6(2), 76 - 85
Isomeric-activity ratios of trimetoquinol enantiomers on beta-adrenergic receptor subtypes: functional and biochemical studies; Fraundorfer PF et al.; Trimetoquinol {1-(3',4',5'-trimethoxybenzyl)-6,7-dihydroxy-1,2,3,4- tetrahydroisoquinoline , TMQ} exists as two enantiomers, and the (-)-(S)-isomer is a potent beta-adrenergic receptor (beta-AR) agonist . Experiments were conducted to examine the functional and biochemical potencies of the (S)-and (R)-enantiomers of TMQ for interaction with beta-AR subtypes in tissues, membrane fractions, and cell systems . The isomeric-activity ratios (IARs) of the TMQ isomers {(S)-isomer > > (R)-isomer} for stimulation of beta 1- and beta 2-AR of guinea pig right atria and trachea were 224 and 1585, respectively; these IARs were similar to those observed on atypical beta-AR systems of rat distal colon (575), rat brown adipocytes (398), but differed from that of rat esophageal smooth muscle (2884) in the presence of pindolol . In the absence of pindolol, the potencies for the TMQ enantiomers were slightly increased; however, the IARs remained unchanged in rat distal colon, rat brown adipocytes, and rat esophageal smooth muscle . Similarly, radioligand binding studies demonstrated that the TMQ isomer beta-AR affinities were stereoselective for the (-)-(S)-isomer in membranes of guinea pig left ventricle (beta 1) and lung (beta 2) giving IARs of 115 and 389, respectively; and in E . coli expressing human beta 1- and beta 2-AR giving IARs of 661 and 724, respectively . Corresponding IARs of receptor affinities and stimulation of cAMP accumulation in Chinese hamster ovary cells expressing human beta 2-AR and rat beta 3-AR were 331 and 282, and 118 and 4678, respectively . These results indicate that the (-)-(S)-isomer of TMQ exhibits high affinity, and is a potent and highly stereoselective agonist for each beta-AR subtype, that the isomers generally fail to differentiate between the beta-AR subtypes, and that, based upon differences in IAR within beta 3-AR containing systems, subtypes of atypical beta (or beta 3)-AR may exist in adipose tissue and smooth muscle.

Am J Trop Med Hyg, 1994, 50(4 Suppl), 27 - 32
Immunity to erythrocytic stages of malarial parasites; Long CA et al.; In those individuals who live in endemic areas, immunity to malaria is slow to develop and stage-specific . The nature and antigenic specificity of this response, which may involve components of both cell-mediated and humoral immunity, is not well understood . Rodent models provide useful systems to explore the spectrum of host responses that may contribute to resolution of erythrocytic-stage infection or possibly to pathogenesis . Moreover, these models allow identification of plasmodial molecules that can induce different types of host responses . Two different mouse model systems, Plasmodium yoelii yoelii and P . chabaudi adami are presented . These have been selected because resolution of infection by P . yoelii yoelii has been shown to require B cell-dependent mechanisms, while control of acute P . chabaudi adami infection can be achieved by T cell-dependent mechanisms . A monoclonal antibody that provides passive protection to P . yoelii challenge infection has been shown to recognize the cysteine-rich, carboxyl-terminal region of the merozoite surface protein-1 . This region, obtained in an appropriate configuration from recombinant Escherichia coli, can induce significant protective immune responses in naive mice . In contrast, cell-mediated immune mechanisms make a major contribution to resolution of asexual-stage P . chabaudi adami infection . An empirical approach using continuous flow electrophoresis has identified several low molecular weight plasmodial proteins that can induce partial protective responses in susceptible hosts . These observations are briefly discussed with respect to human malaria.

Microbios, 1994, 77(312), 141 - 52
Asparagine synthetase: an oxidant-sensitive enzyme in Escherichia coli; Draczynska-Lusiak B et al.; Oxidant stress inhibits the growth of Escherichia coli, which is partially relieved by adding asparagine to the culture medium . Asparagine synthetase (AS), assayed using hydroxylamine as an amino donor, was decreased in a concentration-dependent manner by exposure of cultures to oxygen from near-anaerobic to hyperbaric oxygen (HBO) and by aerobic, but not by anaerobic, paraquat . The specific activity of AS was not decreased when cells were exposed to HBO without a carbon and energy source . HBO caused less AS inactivation in cells containing mutations in both superoxide dismutase (SOD) genes and producing no active SOD . Whether or not cells had catalase had no effect on HBO sensitivity of AS . Aerobic paraquat depressed AS less in cells lacking either catalase or superoxide dismutases . Cells which were decompressed following HBO poisoning had AS restored to normal activity whether or not chloramphenicol was present . These results indicate that asparagine synthetase is oxidant-sensitive; paraquat requires aerobic conditions and HBO requires energy metabolism for AS inactivation; and cells can repair oxidatively-damaged enzyme molecules . The failure of superoxide dismutase or catalase to protect AS suggests that its oxidant-inactivation in cells is not a simple effect of superoxide or hydrogen peroxide.

Arch Surg, 1994 Jan, 129(1), 59 - 65
Glucocorticoids regulate intestinal glutamine synthetase gene expression in endotoxemia; Sarantos P et al.; PURPOSE: Although glutamine is required to maintain gut mucosal metabolism and function, intestinal glutamine uptake from the gut lumen and from the bloodstream is decreased during sepsis . We hypothesized that endogenous mucosal glutamine biosynthesis is increased during endotoxemia, and we attempted to define the "stress" mediators that regulate the activity of small intestinal glutamine synthetase (GS), the principal enzyme of de novo glutamine biosynthesis in the gut . METHODS: Adult rats received Escherichia coli lipopolysaccharide (LPS) (7.5 mg/kg intraperitoneally), RU 38486 (a glucocorticoid antagonist; 10 mg/kg by gavage) 2 hours prior to LPS administration, antibody to tumor necrosis factor (TNF) (4 mg/kg intraperitoneally) prior to LPS administration, or ketorolac tromethamine (a prostaglandin synthesis inhibitor; 1 mg/kg intraperitoneally) followed by LPS administration . Mucosal GS activity was assayed 12 hours after LPS administration . In a separate set of studies, cultured intestinal mucosal cells (Caco-2) were exposed to LPS, interleukin 1 (IL-1), IL-6, TNF-alpha, interferon-gamma, prostaglandin E2, or dexamethasone . Twelve hours later, GS activity was assayed and messenger RNA was extracted . The GS transcripts were labeled with a GS complementary DNA probe radiolabeled with phosphorus 32, were quantitated by phosphoimaging, and were normalized to beta-actin . RESULTS: In vivo LPS treatment increased mucosal GS activity by 250% . Pretreatment with antibody to TNF or ketorolac did not inhibit the LPS-induced increase in mucosal GS, whereas pretreatment with RU 38486 attenuated the increase in gut GS activity by 60% . Lipopolysaccharide, IL-1, IL-6, TNF-alpha, gamma-interferon, and prostaglandin E2 did not increase GS activity in Caco-2 cells, whereas dexamethasone increased GS activity and messenger RNA 2.5-fold and threefold, respectively . These data indicate that cytokines and prostaglandins (prostaglandin E2) do not regulate mucosal GS expression during endotoxemia . Glucocorticoids, however, stimulate GS gene expression directly . CONCLUSIONS: This hormonally mediated response may support de novo mucosal GS during septic states when uptake of glutamine from the lumen and blood is decreased.

Infect Immun, 1994 Jan, 62(1), 41 - 7
Epithelial cell invasion by bovine septicemic Escherichia coli; Korth MJ et al.; Little is known regarding the pathogenesis of Escherichia coli-induced septicemic colibacillosis of calves . To understand the mechanism by which these strains penetrate the intestinal epithelium and gain access to the bloodstream, we examined the potential of bovine septicemic E . coli to invade cultured epithelial cells . By using a gentamicin survival assay, we demonstrated bacterial invasion of Madin-Darby canine kidney (MDCK) cells . Transcytosis of polarized MDCK cell monolayers was also observed, but only when bacteria were added to the basolateral surface . Electron microscopy confirmed the presence of intracellular organisms which appeared to be within membrane-bound vacuoles . The bovine septicemic isolate used in this study expressed the fimbrial adhesion CS31A . To examine the role of CS31A-mediated adherence in invasion and transcytosis of MDCK cell monolayers, a CS31A-deficient mutant was constructed by suicide vector-mediated insertional mutagenesis . Although nonadherent, the mutant showed a level of invasion similar to that of the wild-type parent . E . coli DH5 alpha carrying the cloned CS31A determinant was noninvasive . These findings suggest that expression of CS31A is neither required nor sufficient to mediate invasion.

Virology, 1994 Jan, 198(1), 245 - 56
Nef 27, but not the Nef 25 isoform of human immunodeficiency virus-type 1 pNL4.3 down-regulates surface CD4 and IL-2R expression in peripheral blood mononuclear cells and transformed T cells; Greenway AL et al.; Continuing controversy surrounds the cellular effects of the Nef protein of HIV-1, a nonstructural protein expressed by most isolates . Highly purified protein isoforms of MW 27 kDa (Nef 27) and 25 kDa (Nef 25), produced in Escherichia coli by translation from the first and second start codons of HIV-1 nef clone pNL4.3, respectively, were introduced into cells by a sophisticated electroporation technique which uses electric field rather than electric charge to transfer macromolecules across cell membranes . Electroporation of Nef 27 reduced the expression of cell surface CD4 by 30-50%, as measured by flow cytometry, on phytohemagglutinin (PHA)-activated PBMC as well as on a variety of CD4+ T-cell lines (MT-2, CEM, and Jurkat) . Reduction in surface CD4 was observed in all cells of the CD4+ T-cell lines but only in the CD4+ cells of the mixed PBMC population . Electroporation of Nef 27 into MT-2 cells and PHA-activated PBMC also reduced the expression of IL-2R to background levels . Other cell surface antigens analyzed such as CD2, CD7, or transferrin receptor (TfR) were not affected by the introduction of HIV-1 Nef 27 . In contrast to the effects of Nef 27, electroporation of Nef 25 into cells at equivalent concentrations did not affect the surface expression of CD4 and IL-2R . These data show that the HIV-1 clone pNL4.3 Nef 27 but not the Nef 25 isoform specifically decreases expression of two cell surface receptors important for antigen recognition of MHC class II antigens and for cell proliferation . Production of Nef 27 during HIV-1 infection of cells of the immune system may contribute to immunodeficiency even in the absence of direct viral cytopathic effects.

DNA Seq, 1994, 5(1), 17 - 24
DNA sequence of a gene in Escherichia coli encoding a putative tripartite transcription factor with receiver, ATPase and DNA binding domains; Ramseier TM et al.; We have sequenced downstream of the last previously sequenced gene of the glucitol operon (gutABDMRQ) in E . coli and have found that gutQ is the last gene of this operon . Downstream of the gutQ gene is found a palindromic unit (PU or REP sequence), followed by a large open reading frame of 1515 (or possibly 1590) bps transcribed in the direction opposite to that of the gut operon . This open reading frame encodes a protein of 504 (or possibly 529) amino acids with a tripartite structure . The N-terminal "receiver" domain of 187 (or possibly 212) residues is homologous to the FhlA protein of E . coli, a transcriptional activator of formate hydrogen lyase . It may possess a short domain at its extreme N-terminus exhibiting sequence similarity to carbohydrate binding proteins . The central ATPase domain (236 residues) exhibits greatest sequence similarity to the HydG protein of E . coli, a transcriptional activator of labile hydrogenase . The C-terminal DNA binding domain (81 residues) is homologous to NtrX of Azorhizobium caulinodans, a protein involved in transcriptional regulation of nitrogen fixation . Sequence comparisons with well-characterized transcription factors suggest that ORF504 encodes a protein that hydrolyzes ATP to generate the open transcriptional initiation complex of sigma 54-dependent promoters, possibly in response to redox conditions and/or ligand binding . We propose that this tripartite transcription factor arose by fusion of gene fragments encoding its three constituent modules.

Chin J Biotechnol, 1994, 10(3), 169 - 78
Characterization of polyketide ketoreductase gene (MPKR) from midecamycin-producing strain (Streptomyces mycarofaciens 1748); Xia H et al.; This paper presents the results about the expression of the polyketide ketoreductase gene from midecamycin-producing strain (S . mycarofaciens 1748), gene localization and nucleotide sequences analysis . A BamHI-BamHI 4.0 kg fragment isolated from a genomic library of midecamycin-producing strains containing the actIII homologous DNA was inserted into E . coli-Streptomyces shuttle vector pWHM3 . A recombinant plasmid pCB4 was obtained and introduced into a 2-hydroxyaklavinone producer S . galilaeus ATCC 31671 that was a polyketide ketoreductase gene deficient mutant . The transformant produced aklavinone according to TLC and HPLC analyses . The BamHI-BamHI 4.0-kb fragment was inserted into pWHM3 in the reverted orientation with pCB4 and a recombinant plasmid pCBR4 was obtained . Introduction pCBR4 into S . galilaeus ATCC 31671 also resulted in the production of aklavinone . Thus we demonstrated that this gene encoded a polyketide ketoreductase which results in deoxygenation of 2-hydroxyaklavinone in C-2 position and that this gene has its own promoter . Restriction map analysis of pCB4 indicated that there were no sites for EcoRI and HindIII, but there were sites for BssHII, SalI, SphI and XhoI on the cloned gene fragment . The polyketide ketoreductase gene was located on a BssHII-BamHI 1.3-kb fragment from Southern hybridization result, using actIII gene as a probe, and that was confirmed by gene expression in S . galilaeus ATCC 31671 . The nucleotide sequence analysis showed that the BssHII-BamHI 1.3-kb fragment contained an open reading frame (ORF) . The protein coding region was assumed to start with an ATG start codon, end at an TAG stop codon . There was a 5nt (GGAGG) SD sequence at the upstream of start codon . A presumptive promoter consisted of 7 nt AACCGGA at the -10 region and 5 nt TTCGA at the -35 region . The deduced amino acid sequences of MPKR gene consisted of 261 aa residues . Its amino acid were compared with actIII gene coding protein sequences . The similarity was 77.4% and the identity was 66.7%.

Chin J Biotechnol, 1994, 10(3), 153 - 61
Fusion expression vectors for recombinant gene products processed easily and purified rapidly by affinity chromatography; Li B et al.; A DNA fragment encoding IgG-binding domain B,C (PABC) was separated from protein A gene, cloned into phage M13 and modified by oligo-directed mutagenesis at the hydroxylamine-cleaved site from Asn-Gly to Asn-Ala in domain B and C, respectively . The modified PABCm gene fragment was used to construct one set of fusion expression vectors in different reading frames . Processing sequences such as those recognized by enterkinase, collagenase, thrombin, activated factor X and cleaved by the hydroxylamine, N-chlorosuccinimide etc . can be created in the fusion site . Using the above vectors, fusion proteins such as PABCm-IGF-I, -hGRF, -bGRF and their derivatives were highly expressed in E . coli . The yield of fusion proteins is over 100 mg per liter cultured by analysis of SDS-PAGE . The PABC fusion proteins can be rapidly purified by the affinity chromatography with a IgG-sepharose column.

Annu Rev Genet, 1994, 28, 49 - 70
Chi and the RecBC D enzyme of Escherichia coli; Myers RS et al.; The products of genes recB and recC are responsible for conjugal recombination and for the repair of chromosomal double chain breaks in Escherichia coli . The product of the recD gene, which combines with the RecB and RecC proteins to comprise the RecBCD enzyme, is not required for either recombination or repair . On the contrary, RecBCD enzyme is a potent exonuclease which inhibits recombination by destroying linear DNA . The RecD Ejection model supposes that RecBCD enzyme enters DNA at a double-chain end and travels destructively along the DNA until (typically) it encounters the recombination hotspot sequence chi . Chi then alters the RecBCD enzyme by weakening the affinity of the RecD subunit for the RecBC heterodimer . With the loss of the RecD subunit from the enzyme, the resulting protein, RecBC(D-), becomes deficient for exonuclease activity and proficient as a recombinagenic helicase . Thus, the RecD Ejection model proposes that chi participates in recombination by acting as a toggle to convert RecBCD (a powerful exonuclease) to RecBC(D-) (a recombinase) . We review the properties of RecBCD and its cognate site chi, including recent results that support both the RecD Ejection model and the view that chi plays only an indirect role in recombination.

Acta Physiol Pharmacol Bulg, 1994, 20(1), 19 - 24
Changes in rabbit cardiac function during fulminant endotoxin shock; Velkov Z; Septic shock in humans is usually characterized by severe changes in the left ventricular function, while little is known about the dysfunction of the right ventricle . Rabbits were injected intraventricularly with endotoxin from Escherichia coli at a dose of 2 mg/kg following catheterization of both heart ventricles with the purpose of measuring their pressure, and determining a number of contractility indices . In the animals in which endotoxin shock developed very rapidly and death occurred between the 60th and the 90th min (shock development is called fulminant), we established significant changes in the function of both ventricles . At the 20th min, the intraventricular pressure, EDP and P-(dP/dtmax) increased, indicating a tension lead of the right ventricle . At the same time, there was a decrease in plus dP/dtmax, minus dP/dtmax, (dP/dtmax)/P and {(dP/dt)/P}max, considered evidence for impaired contractility and relaxation of the right ventricular myocardium . During the subsequent observation periods, some of the contractility indices returned to normal . At the 20th min, a reduction of intraventricular pressure, P-(dP/dtmax), plus dP/dtmax, minus dP/dtmax and (dP/dt)P was registered in the left ventricle . Pronounced bradycardia was observed from the 30th min to the end of the experiments . Fulminant endotoxin shock in rabbits occurs as a result of the impaired function of the right ventricle attributed to tension leading and endotoxin-induced biventricular myocardial dysfunction.

Microbios, 1994, 80(324), 189 - 202
Acid pH responses of Escherichia coli: inhibition of colicin V synthesis and activity at pH 5.0; Rowbury RJ et al.; On solid medium, streaks of ColV+ Escherichia coli strains produced much larger inhibitory zones on sensitive indicators at pH 7.0 and 9.0 than at pH 5.0 . Such an acid pH effect also occurred in liquid medium, with sensitive strains being killed by ColV+ ones at pH 7.0 or 9.0 but not at pH 5.0 . Colicin V was responsible for the killing effect . Culture filtrates from PAP1401 (ColV+) were used to examine the basis for the pH effect and it was found that acid pH interfered directly or indirectly with colicin V production but that colicin V was not irreversibly inactivated at pH 5.0 . Additionally, colicin V formed at pH 7.0 was less active when tested on sensitive strains at pH 5.0 than at pH 7.0 . Neither reduced colicin synthesis nor its reduced activity can be attributed to increased availability of iron at pH 5.0.

Dev Biol Stand, 1994, 83, 87 - 92
Use of polymerase chain reaction (PCR) in the development of a recombinant organism; Helder JC et al.; Tryptic mapping of a purified recombinant antibody expressed in a mammalian cell line demonstrated low level heterogeneity in the heavy chain . Amino acid analysis and N-terminal sequencing of these fragments revealed that a conversion of tyrosine (Y) to glutamine (Q) had occurred at residue 376 . DNA sequencing of 30 clones of the original expression construct indicated that the chance of the Y376Q variant being present in the original plasmid was low, < 5% . Once the gene had been cloned into a mammalian cell system, sequencing of 30 clones is considered ineffective for screening large numbers of subclones . Consequently, an alternative method was developed to directly probe total mammalian DNA for the presence of cloned variant . The method described here uses a polymerase chain reaction (PCR) based assay optimizing the ability of Taq polymerase to distinguish whether the 3' end of primers have completely annealed with template DNA during PCR cycling . Ten percent of the subclones producing high levels of the variant were identified and experiments indicated that the Y to Q conversion had developed during transfection of the antibody gene into the mammalian cell system . PCR was shown to be useful as a quick screen for verification of a cloned variant . Tryptic mapping is still considered the most sensitive and appropriate analytical tool for assessing genetic heterogeneity of recombinant cell lines.

Breast Cancer Res Treat, 1994, 31(2-3), 349 - 56
Breast cancer gene therapy: transgenic immunotherapy; Su N et al.; A number of studies have demonstrated that potent anti-tumor immunity can be induced using cytokine gene transfer, a strategy termed transgenic immunotherapy . Our aim is to express cytokine genes in the vicinity of tumor cells, either by transducing tumor cells themselves, or by delivering cytokine-expressing endothelial cells to tumor sites . We compared the ability of cytokine-expressing tumor cells or endothelial cells to inhibit the tumorigenesis of MDA-MB-435 breast cancer cells in athymic nude mice . Retroviral vectors containing either human interleukin 2 (hIL-2) or interleukin 1 (hIL-1 alpha) were used to transduce MDA-MB-435 cells or human umbilical vein endothelial cells (HUVEC) . Using a modified MTT bioassay and an ELISA specific for hIL-2, 43 of 70 MDA-MB-435 clones transduced with IL-2 were found to secrete between 100-800 units of IL-2/10(6) cells/24 hr . hIL-2 and hIL-1 alpha-transduced HUVEC secreted 40 ng/IL-2/10(6)/24 hr and 1.8 ng/10(6)/24 hr, respectively . To facilitate in vivo tracking of tumor cells, both nontransduced and IL-2-expressing MDA-MB-435 cells were genetically-marked with the E . coli lacZ gene and selected using flow cytometry . To study in vivo tumorigenicity, cells were injected into the mammary fat pad of athymic nude mice: (1) lacZ/MDA-MB-435 cells injected alone formed tumors in all animals; (2) IL-2-expressing lacZ/MDA-MB-435 cells did not form any tumors; (3) co-inoculation of MDA-MB-435/IL-2, or HUVEC/IL-2, or HUVEC/IL-1 alpha with lacZ/MDA-MB-435 cells prevented or delayed tumor growth.(ABSTRACT TRUNCATED AT 250 WORDS)

J Gastroenterol Hepatol, 1994, 9 Suppl 1, S45 - 9
Involvement of endogenous nitric oxide in the inhibition by endotoxin and interleukin-1 beta of gastric acid secretion; Esplugues JV et al.; Administration of Escherichia coli endotoxin abolished the acid secretory response induced by a bolus injection of pentagastrin in the continuously perfused stomach of the anaesthetized rat . Likewise, acid secretion stimulated by the continuous intravenous perfusion of pentagastrin was inhibited by administration of interleukin-1 beta (IL-1 beta) . In both cases pretreatment with NG-nitro-L-arginine methyl ester (L-NAME) but not dexamethasone or indomethacin substantially restored the secretory responses to pentagastrin . The actions of L-NAME were reversed by the prior administration of L-arginine but not by its enantiomer D-arginine . Even though L-NAME increased blood pressure, this does not seem to be the mechanism by which endotoxin-induced acid inhibition was prevented, since similar systemic pressor responses induced by phenylephrine had no such effect . The secretory response elicited by pentagastrin in the isolated lumen perfused stomach of the rat was not influenced by incubation (100 min) with IL-1 beta . These observations suggest that the acute inhibition of acid responses to pentagastrin by endotoxin and IL-1 beta involves nitric oxide (NO) synthesis from L-arginine.

Biochimie, 1994, 76(6), 554 - 60
Molecular biology of c-type cytochromes from Desulfovibrio vulgaris Hildenborough; Pollock WB et al.; Sulfate reducing bacteria of the genus Desulfovibrio harbor a wide variety of redox proteins . Three different c-type cytochromes, cytochrome c-553, cytochrome c3 and the high molecular mass cytochrome have been isolated from these bacteria . The high molecular mass cytochrome is part of an operon that encodes a transmembrane protein complex that mediates electron transfer across the cytoplasmic membrane . The physiological function of the other two cytochromes is less clear . They are encoded by monocistronic genes and their redox partners can thus not be identified by gene sequencing . Expression of genes for c-type cytochromes in a foreign host are complicated due to the requirement for covalent heme insertion . Cytochrome c-553 is readily expressed in Escherichia coli in functional form, but cytochrome c3 and the high molecular mass cytochrome are for reasons that are presently not clear.

J Tongji Med Univ, 1994, 14(1), 16 - 9, 41
The production of strains highly expressing human interleukin-2 cDNA; Zhang DJ et al.; The plasmid pTLIL-2, which only directed a low-level expression of human interleukin 2(IL-2) cDNA in E . coli, was reconstructed: a series of deletions were made in 3' non-coding region of human IL-2 cDNA, and 7 recombinant plasmids with different deletions were selected, on the other hand, a Tac promoter sequence from pDR540 was inserted to upstream of IL-2 cDNA in pTLIL-2 so that pTLIL-2DT, which contains double Tac promoters, was constructed . And then, E . coli JM103 was transformed with the above 8 recombinant plasmids respectively . The expression efficiency of IL-2 cDNA in E . coli transformed with different plasmids was evaluated by means of SDS-PAGE and measuring 3H-TdR incorporation of IL-2-dependent activated T lymphocytes in the presence of bacterial extracts . Three engineering strains with high efficiency of IL-2 expression were selected, and all of these strains could produce recombinant IL-2(rIL-2) 4 times more than E . coil JM103/pTLIL-2.

Nephron, 1994, 68(4), 433 - 6
Increased incidence of HLA-B40 group antigens in children with hemolytic-uremic syndrome; Sheth KJ et al.; Hemolytic uremic syndrome (HUS) develops in 25-30% of children infected with Escherichia coli strains that produce Shiga-like toxins, also known as verocytotoxins . Mild HUS also occurs in 1 in 4 of the other family members, suggesting a familial predisposition to HUS . To understand the possible genetic predisposition, the frequency of HLA antigens was evaluated in 30 children (12 boys, 18 girls; mean age 3.8 years) with HUS following a prodrome of bloody diarrhea . When compared to a blood donor population from the same geographic area and ethnic background, no significant differences were noted in the frequency of HLA-A, HLA-C, HLA-DR, and HLA-DQ antigens . However, the frequency of HLA-B40 and its splits (B60, 61, 41, 47) was significantly higher in the study population (corrected p < 0.005) . The relative risk of developing HUS was 6.04 when HLA-B40 and HLA-B40 split products were present, and the risk increased to 8.5 when the analysis was extended to include the cross-reactive antigens B44 and B13 . These HLA-B antigens share common amino acid sequences at positions 41-45 and 67-74 on the alpha-1 domain of the HLA class I molecule . Our data suggest that the inheritance of HLA-B40, its splits, and cross-reactive antigens increases the risk of developing HUS.

Microbiol Immunol, 1994, 38(10), 797 - 800
Use of the recombinant 38-kDa antigen of Mycobacterium tuberculosis as an immunogen for specific antisera production; D'Souza CD et al.; The Mycobacterium tuberculosis 38-kDa protein antigen is one of the secreted immunodominant antigens showing high immunogenicity at B-cell and T-cell levels . Although monoclonal antibodies to this antigen have been produced, specific polyclonal antisera is required for standardization of specific immunodiagnostic assays . This protein has been overexpressed and purified from recombinant Escherichia coli using an inducible vector system . During each stage of expression and purification, the recombinant protein was used to immunize mice and rabbits by several methods: 1) as overexpressed protein present as inclusion bodies in recombinant E . coli; 2) embedded in a polyacrylamide gel; 3) fixed to a solid-phase nitrocellulose membrane and 4) emulsified with an adjuvant . All strategies yielded specific antisera as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses . The results obtained, both quantitative (ELISA) and qualitative (immunoblot) demonstrate that the purified recombinant antigen retains its antigenicity and immunogenicity throughout the various steps in the process of expression and purification and serves as a potent antigen for production of specific antisera to be used in immunoassays.

Acta Microbiol Immunol Hung, 1994, 41(4), 473 - 4
Production of antibodies against adenovirus hexons in Escherichia coli by recombinant phages (a note); Ruzsics Z et al.; For the study of complex antigen structures, such as the adenovirus hexon capsomer, monoclonal antibodies proved to be very useful tools . Production and stabilization of monoclonal antibodies in a larger amount is complicated and requires special infrastructural background, if we apply the convential hybridoma techniques . Recombinant DNA and gene amplification technology, however, has made it possible to clone antibody genes in bacteria, and to produce antibodies in bacterial cultures.

Acta Microbiol Immunol Hung, 1994, 41(4), 465 - 71
Effect of radiodetoxified endotoxin and trace elements on reticulo-endothelial system damaged by ethanol in rats; Gal K et al.; For the demonstration of RES activity 99-mTc labelled nano-albumon micro-colloid solution (prepared by OSSKI, Budapest, Hungary) was injected into the prepared jugular vein of narcotized rats and the clearance rate was investigated . The effects of various materials have been observed on RES in vivo . Continuous consumption of 20% ethanol solution for 15 weeks decreased the phagocytic activity . One hundred microgram/100 g body weight (i.v.) of the radio-detoxified endotoxin preparation (RD-LPS, TOLERIN) prepared from Escherichia coli endotoxin by 60Co-gamma irradiation (150 KGy) increased the granulopectic activity of RES of intoxicated animals . The preparation containing trace elements also increased the former activity and the treatment consisting of the use of both trace elements and RD-LPD was as effective as the RD-LPD per sc.

Acta Microbiol Immunol Hung, 1994, 41(4), 457 - 63
The effect of X-radiation on reticulo-endothelial system and its treatment with radiodetoxified-endotoxin and trace elements in rats; Gal K et al.; A new in vivo method has been developed for the precise observation of RES activity . Both Escherichia coli endotoxin 100 micrograms/100 g i.v . (LPS) and radiodetoxified endotoxin 100 micrograms/100 g body weight i.v . (RD-LPS, TOLERIN) both increased the granulopectic activity of RES . The RD-LPS was more effective . The preparation containing trace elements also increased the activity of RES . The treatment consisting of the use of both trace elements and RD-LPS proved to be the most effective . The activity of RES was inversely proportional to various doses of X-ray irradiation (7, 8, 9 Gy) . Trace elements and RD-LPS even improved the immunity system of animals having deteriorated RES.

Cell Mol Biol Res, 1994, 40(4), 351 - 7
Existence of multiple phosphorylated forms of human platelet actin binding protein; Wu MP et al.; Platelet actin binding protein (ABP) as isolated from human platelets exists in at least four phosphorylated forms which we have designated ABP-0, ABP-1, ABP-2, and ABP-3 whose phosphate content ranges from 18 (ABP-0) to 40 (ABP-3) moles Pi/mole ABP . These forms differ in their resistance to calpain cleavage and ability to cross-link F-actin with ABP-3 being the best in each of these properties . Attempts to phosphorylate ABP-1, two or three with protein kinase C (PKC) were unsuccessful except if the proteins were pretreated with Escherichia coli alkaline phosphatase . All of the forms could be phosphorylated with cAMP-dependent kinase (PKA) and subsequent resistance to calpain cleavage conferred . Phosphorylation/dephosphorylation of ABP may be an important regulatory mechanism by which the cytoskeletal architecture is stabilized or transformed.

Oncol Res, 1994, 6(6), 237 - 42
Antisense phosphorothioate oligonucleotides direct both site-specific and nonspecific RNAse H cleavage of in vitro synthesized p120 mRNA; Weidner DA et al.; To determine the sequence specificity with which various antisense phosphorothioate oligonucleotides to the p120 gene could direct RNAse H-dependent cleavage of p120 mRNA, an in vitro RNAse H assay was done on in vitro synthesized p120 mRNA . Three oligonucleotides tested (ISIS 3462, coding region; ISIS 3466, 3' untranslated region; and ISIS 3782, 3' untranslated region) directed cleavage of p120 mRNA at the target sites . In addition, several additional cleavage products from nontarget sites were detected . A computer sequence homology search revealed that these additional bands probably result from cleavage at regions of complementarity of eight or nine contiguous bases of the oligonucleotide and the p120 mRNA . These findings suggest that such nontarget specific cleavages may account for multiple effects of phosphorothioate oligonucleotides.

Rev Elev Med Vet Pays Trop, 1994, 47(2), 169 - 75
{Factors and markers of virulence of Escherichia coli strains isolated from diarrhea in calves aged 4-45 days in Algeria}; Mohamed Ou Said A et al.; The study involved 492 strains of Escherichia coli isolated from the feces of 44 diarrhoeal calves and 4 healthy calves in 7 wilayates in Algeria (Tipaza, Ain Defla, Bejaia, Borj Bou Arreridj, Bouira, Medea and Algiers) . The authors looked for the surface proteins K99, CS31A, Vir, F17 (FY), 20K and certain factors or markers of virulence such as the production of colicins, particularly colicin V, the aerobactin siderophore, alpha hemolysin and enterohemolysin . They also studied the frequency of certain 0 serogroups and evaluated the resistance of the E . coli strains to 10 antibiotics . The results showed that the majority of diarrhoeal calves were colonized by Escherichia coli expressing one or more factors of virulence and that the stocks which produce the antigen CS31A are usually resistant to 4 or 6 antibiotics.

Growth Factors, 1994, 11(2), 93 - 104
Identification of functional domains of interleukin-3 by construction of primate interspecies chimera; Dorssers LC et al.; Interleukin-3 (IL-3) is involved in regulation of proliferation and differentiation of multipotent hemopoietic cells and stimulates the production of most blood cell types . The observed functional specificity across species concurs with an extreme rate of IL-3 amino acid substitutions during mammalian evolution . Tamarin IL-3 exhibited 70.5% sequence identity with human IL-3 and was severely impaired in supporting proliferation of human IL-3-dependent cells . In contrast, chimpanzee IL-3 displayed high amino acid sequence homology (98.5%) and could substitute for human IL-3 . A panel of interspecies chimera between the chimpanzee and tamarin IL-3 genes has been constructed and expressed in Escherichia coli and eukaryotic cells to investigate the role of substitutions in different protein domains on the functional species specificity . Our analyses show that substitutions at residues encoded by the first two exons appear crucial in the functional species specificity, whereas C-terminal alterations show only moderate effects.

Microbiol Immunol, 1994, 38(9), 695 - 701
Prevalence of colonization factor antigens (CFAs) and adherence to HeLa cells in enterotoxigenic Escherichia coli isolated from feces of children in São Paulo; Guth BE et al.; Fifty-eight enterotoxigenic Escherichia coli (ETEC) strains, isolated from children with and without diarrhea in Sao Paulo, were examined for the presence of colonization factor antigens (CFAs) and their ability to adhere to HeLa cells . Antisera to CFA/I, the coli surface (CS) antigens CS1CS3, CS2CS3, and CS2 of CFA/II, CFA/III, and CS5CS6 and CS6 of CFA/IV were used . CFAs were identified in 43% of the ETEC strains: 40% of the CFAs strains with CFAs harbored CFA/I, 24% carried CFA/II (CS1CS3), 24% carried CFA/IV (CS6), and 12% carried CFA/IV (CS5CS6) . CFAs occurred mainly among ETEC strains producing only heat-stable (ST-I) enterotoxin and in strains also producing heat-labile toxin (LT-I) . No ETEC strains tested expressed CFA/III . A marked change in serotypes of ST-I-producing strains was found in Sao Paulo between 1979 and 1990 . Adherence to HeLa cells was detected in 14% of the ETEC strains . All of them had a diffuse adherence pattern and produced only ST-I, and 88% carried CS6 antigen.

Biochimie, 1994, 76(5), 398 - 403
High level expression of rat gamma-D-crystallin in Escherichia coli; Ooki K et al.; Gamma-crystallins have been implicated in various kinds of cataracts . In order to facilitate studies elucidating the molecular mechanism of cataractogenesis, large quantities of rat recombinant gamma-D-crystallin were produced in E coli . A full length cDNA clone coding for gamma-D-crystallin was isolated from a rat lens lambda gt11 cDNA library using a synthetic oligonucleotide as a probe . The coding region of this cDNA was inserted into a cloning vector pKK233-2 under the control of the trc promoter . The resulting construct, pKKCR91, was transfected into E coli to produce rat gamma-D-crystallin in an amount of 10-15% of the total bacterial proteins . The crystallin produced was purified to an apparent homogeneity as judged by SDS gel electrophoresis . The sequence of the N-terminal 11 amino acids of the purified crystallin was determined, showing that it is completely identical to that predicted from the cDNA sequence . Measurements of the far-UV CD spectra also revealed that recombinant rat gamma-D-crystallin thus produced retains a native conformational structure.

Biochimie, 1994, 76(5), 389 - 93
Transfer and isomerization of the ribose moiety of AdoMet during the biosynthesis of queuosine tRNAs, a new unique reaction catalyzed by the QueA protein from Escherichia coli; Slany RK et al.; The enzyme QueA of E coli is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine . The enzyme catalyzes the synthesis of an epoxycyclopentane moiety and transfers this compound to specific tRNAs containing the queuosine precursor 7-(aminomethyl)-7-deazaguanine (preQ1) . S-adenosylmethionine (AdoMet) is the sole cofactor that is required for this reaction (Slany et al, 1993, Biochemistry 32, 7811-7817) . To proof that the ribose moiety of AdoMet is the precursor of the epoxycyclopentane moiety, labeled AdoMet, was generated from different types of 3H ATP and methionine by the AdoMet synthetase enzyme (MetK) from E coli . The resulting 3H labeled AdoMet was directly used as the cofactor for the QueA reaction . Using {2,5', 8-3H}ATP, containing tritium at C5' of the ribose ring, resulted in an incorporation of radioactivity into preQ1 tRNA, whereas this was not the case when {2,8-3H}ATP was applied . A model for the reaction catalyzed by the S-adenosylmethionine:tRNA ribosyltransferase-isomerase QueA is proposed.

Biochimie, 1994, 76(5), 376 - 83
The N-terminal half of initiation factor IF3 is folded as a stable independent domain; Fortier PL et al.; Initiation factor IF3 plays an essential role in the initiation of protein translation by binding to the 30S ribosomal subunit and selecting a proper tRNA(fMet)/initiation codon complex . The domain structure of IF3 from Escherichia coli has been investigated by limited proteolysis followed by mass spectrometry and protein sequencing of the resulting peptides . This analysis revealed a highly segmented structure with two independent domains connected by a charged linker peptide, highly susceptible to proteolytic cleavage . The N-terminal domain is very stable and comparison of its 2-D NMR spectrum with that of intact IF3 revealed that it retains its three-dimensional fold.

Yi Chuan Xue Bao, 1994, 21(5), 350 - 5
{Localization of GH gene to bovine chromosome 5q22-26 by in situ hybridization}; Li X et al.; The bGH entire gene (3.0kb), as a probe which was cloned into recombinant plasmid pbGH was used for in situ hybridization . The E . coli RR1, as a receptor, was transformed by recombinant plasmid pbGH . Recombinant plasmid DNAs were amplified, extracted and purified, the bGH fragments were collected and labelled by nicktranslation . The metaphase and early-metaphase chromosome spreads were prepared from TdR-BrdU-synchronized peripheral blood lymphocytes in Beijing Black-white dairy cattle . After in situ hybridization and autoradiography, chromosomal G-bands were stained with FPG method . The number of silver grains on every chromosome was calculated . The result showed that bGH gene is located at chromosome 5q22-26 . This assignment of the GH gene in cattle differs from those of previous assignments . Finally, the relationships between probe, mapping method and mapping accuracy were discussed.

Ciba Found Symp, 1994, 180, 97 - 104; discussion 105-10
Structural studies on porphobilinogen deaminase; Lambert R et al.; The X-ray crystallographic analysis of porphobilinogen deaminase (hydroxymethylbilane synthase, EC 4.3.1.8) shows the polypeptide chain folded into three domains, (1) N-terminal, (2) central and (3) C-terminal, of approximately equal size . Domains 1 and 2 have a similar overall topology, a modified doubly wound parallel beta-sheet . Domain 3 is an open-faced three-stranded antiparallel beta-sheet, with one face covered by three alpha-helices . The active site is located between domains 1 and 2 . The dipyrromethane cofactor linked to cysteine 242 protrudes from domain 3 into the mouth of the cleft . Flexible segments between domains 1 and 2 are thought to have a role in a hinge mechanism, facilitating conformational changes . The cleft is lined with positively charged, highly conserved, arginine residues which form ion pairs with the acidic side chains of the cofactor . Aspartic acid 84 has been identified as a critical catalytic residue both by its proximity to the cofactor pyrrole ring nitrogen and by structural and kinetic studies of the Asp-84-->Glu mutant protein . The active site arginine residues have been altered by site-directed mutagenesis to histidine residues . The mutant proteins have been studied crystallographically in order to reconcile the functional changes in the polymerization reaction with structural changes in the enzyme.

Ciba Found Symp, 1994, 180, 70 - 89; discussion 89-96
The biosynthesis of uroporphyrinogen III: mechanism of action of porphobilinogen deaminase; Jordan PM; The biosynthesis of the uroporphyrinogen III macrocycle from porphobilinogen requires the sequential participation of two enzymes--porphobilinogen deaminase (1-hydroxymethylbilane synthase, EC 4.3.1.8) and uroporphyrinogen III synthase (cosynthase, EC 4.2.1.75) . The product of the deaminase-catalysed reaction is a highly unstable 1-hydroxymethylbilane called preuroporphyrinogen which acts as the substrate for the uroporphyrinogen III synthase, resulting in the exclusive formation of uroporphyrinogen III . In the absence of the synthase, preuroporphyrinogen cyclizes spontaneously to give uroporphyrinogen I . Porphobilinogen deaminase contains a dipyrromethane cofactor that acts as a primer onto which the tetrapyrrole chain is built . The assembly process occurs in stages through enzyme-intermediate complexes, ES, ES2, ES3 and ES4 . The negatively charged carboxylates of the cofactor, substrate and intermediate complexes interact with positively charged amino acid side chains in the catalytic cleft . Mutagenesis of conserved arginines has dramatic effects on the assembly of the dipyrromethane cofactor and on the tetrapolymerization process . During the polymerization, the enzyme changes conformation to accommodate the elongating pyrrole chain . The structure of the deaminase from Escherichia coli has been determined by X-ray crystallography at 1.9A resolution and gives important insight into the enzymic mechanism . Aspartate 84 plays a key role in catalysis and its substitution by glutamate reduces kcat by two orders of magnitude.

Ciba Found Symp, 1994, 180, 50 - 64; discussion 64-9
5-Aminolaevulinic acid dehydratase: characterization of the alpha and beta metal-binding sites of the Escherichia coli enzyme; Spencer P et al.; The alpha and beta metal-binding sites of 5-aminolaevulinic acid dehydratase (ALAD) (porphobilinogen synthase, EC 4.2.1.24) from Escherichia coli were investigated to determine the function of each metal ion and the role of the reactive cysteines in metal binding . Occupancy of the alpha site by Zn2+ restored virtually all catalytic activity to the inactive metal-depleted ALAD (apoALAD) . Occupancy of the alpha site by Co2+ also yielded an active enzyme and resulted in a charge-transfer band indicative of a single cysteine amongst the metal ligands . Subsequent labelling of this cysteine residue with 14C-labelled N-ethylmaleimide, followed by peptide analysis, indicated the involvement of Cys-130 . The metal ion at the alpha site is thought to be essential for binding of the second molecule of substrate at the A substrate-binding site that forms the acetic acid side of the product, porphobilinogen . Binding of Zn2+ to the beta site restored little activity if the alpha site was unfilled . Metal ion binding to the beta site could be monitored by following the change in protein fluorescence with Zn2+ titration of apoALAD at pH 6 . A conformational change upon beta site occupancy may explain why binding of Mg2+ at the alpha site can occur only if Zn2+ is bound at the beta site . The binding of Co2+ at the beta site produced an inactive enzyme that exhibited a charge-transfer band indicative of at least three cysteine ligands.

Ciba Found Symp, 1994, 180, 26 - 40; discussion 40-9
5-Aminolaevulinic acid synthase and uroporphyrinogen methylase: two key control enzymes of tetrapyrrole biosynthesis and modification; Warren MJ et al.; Two enzymes which play an important role in regulation and flux control through the tetapyrrole biosynthetic pathway are considered . The Rhodobacter sphaeroides 5-aminolaevulinic acid synthase isoenzymes are discussed and the progress being made on their recombinant expression and isolation is reported . The Escherichia coli uroporphyrinogen methylase, which is encoded by the cysG gene, is also examined . In this case evidence is provided which demonstrates that the gene product is responsible for the complete synthesis of sirohaem from uroporphyrinogen III . The enzyme is thus capable of performing two S-adenosylmethionine-dependent methylation reactions, an NADP(+)-dependent dehydrogenation and iron chelation . The uroporphyrinogen methylase is thus a small multifunctional enzyme.

Physiol Chem Phys Med NMR, 1994, 26(3), 215 - 26
DNA damage by various forms of active oxygens and its inhibition by different scavengers using plasmid DNA; Takehara Y et al.; The degree of DNA damage by the treatment with various molecular species of active oxygen and its inhibition by pretreatment with different scavengers were evaluated using pUC19 plasmid DNA . DNA damage caused by O2- . generated by xanthine-xanthine oxidase system (X-XOD), .OH by Fenton reactions, and OCl- by NaOCl involved the generation of open circle DNA demonstrating single strand breaks . Catalase (Cat), diethylenetriaminepentaacetic acid (DETAPAC), desferroxiamine (Desferal), dimethyl sulfoxide (DMSO) and ethanol (EtOH) all inhibited 60-80% of DNA damage by the generated O2-. . Superoxide dismutase (SOD) inhibited all DNA damages by O2-. . Cat, DETAPAC and Desferal effectively inhibited DNA break by .OH; complete inhibition of .OH-induced DNA break was achieved by addition of DMSO and EtOH . Desferal and EtOH completely inhibited DNA damage by OCl- . These findings suggested that metal ions are associated with the mechanism of DNA damage by all forms of active oxygen species.

Microbios, 1994, 79(321), 241 - 4
Near-UV light protection effect against lethality induced by stannous chloride in Escherichia coli; Silva FC et al.; Stannous chloride (SnCl2) is a reducing agent largely employed in industry and in medical procedures . To evaluate its potential genotoxicity, several Escherichia coli strains were treated with SnCl2 and their survival rates determined . Results showed that the double mutant on specific genes for the repair of deoxyribonucleic acid damage was the most sensitive strain . Simultaneous near-UV illumination inhibited the lethal effect of SnCl2 in the wild type strain . Although the nature of the induced lesions are not known these results indicate the potential genotoxicity of SnCl2.

Cell Motil Cytoskeleton, 1994, 29(1), 29 - 45
Differential regulation of skeletal muscle myosin-II and brush border myosin-I enzymology and mechanochemistry by bacterially produced tropomyosin isoforms; Fanning AS et al.; In this report, we have compared the physical properties and actin-binding characteristics of several bacterially produced nonmuscle and striated muscle tropomyosins, and we have examined the effects of these isoforms on the interactions of actin with two structurally distinct classes of myosin: striated muscle myosin-II and brush border (BB) myosin-I . All of the bacterially produced nonmuscle tropomyosins bind to F-actin with the expected stoichiometry and with affinities comparable to that of a tissue produced alpha-tropomyosin, although the striated muscle tropomyosin CTm7 has a lower affinity for F-actin than a tissue-purified striated muscle alpha tropomyosin . The bacterially produced isoforms also protect F-actin from severing by villin as effectively as tissue-purified striated muscle alpha-tropomyosin . The bacterially produced 284 amino acid striated muscle tropomyosin isoform CTm7, the 284 amino acid nonmuscle tropomyosin isoform CTm4, and two chimeric tropomyosins (CTm47 and CTm74) all inhibit the actin-activated MgATPase activity of muscle myosin S1 by approximately 70-85%, comparable to the inhibition seen with tissue-purified striated muscle alpha tropomyosin . The 248 amino acid tropomyosin XTm4 stimulated the actin-activated MgATPase activity of muscle myosin S1 approximately two- to threefold . The in vitro sliding of actin filaments translocated by muscle myosin-II (2.4 microns/sec at 19 degrees C, 5.0 microns/s at 24 degrees C) increased 25-65% in the presence of XTm4 . Tropomyosins CTm4, CTm7, CTm47, and CTm74 had no detectable effect on myosin-II motility . The actin-activated MgATPase activity of BB myosin-I was inhibited 75-90% by all of the tropomyosin isoforms tested, including the 248 amino acid tropomyosin XTm4 . BB myosin-I motility (50 nm/s) was completely inhibited by both the 248 and 284 amino acid tropomyosins . These results demonstrate that bacterially produced tropomyosins can differentially regulate myosin enzymology and mechanochemistry, and suggest a role for tropomyosin in the coordinated regulation of myosin isoforms in vivo.

Biochimie, 1994, 76(3-4), 234 - 40
N-arginine dibasic convertase (NRD convertase): a newcomer to the family of processing endopeptidases . An overview; Chesneau V et al.; N-arginine dibasic convertase (NRD convertase) (accession number L27124) is a metalloendopeptidase from rat brain cortex and testis which cleaves peptide substrates on the N-terminus of arginine residues in basic doublets . Its predicted amino acid sequence contains the putative zinc binding motif HXXEH in a region which exhibits 35% and 48% similarity with E coli protease III (pitrilysin E.C 3.4.99.44) and rat or human insulinase (E.C 3.4.99.45) respectively . This feature clearly classifies this endopeptidase as a member of the pitrilysin family of zinc-metalloproteases . However, the NRD convertase sequence contains a distinctive additional feature consisting of a 71 acidic amino acid stretch . Its substrate selectivity and the characteristic motifs of its amino acid sequence allow us to propose this new metalloendopeptidase as the first member of a new class of processing enzymes.

J Basic Microbiol, 1994, 34(6), 363 - 70
Mutations affecting gluconate catabolism in Escherichia coli . Genetic mapping of loci for the low affinity transport and the thermoresistant gluconokinase; de Rekarte UD et al.; The isolation and properties of strains of Escherichia coli carrying mutations affecting either the low affinity transport for gluconate (gene gntU) or the thermoresistant gluconokinase (gene gntK) are described . A lesion of each type was genetically characterized by transduction experiments . Both mutations mapped in the asd region, and the order was malA-glpD-asd-gntU-gntK, with the last two markers at about min 75.78 and 75.86 on the map, respectively . Mutations altering specifically gntU have not been previously reported.

Genetica, 1994, 93(1-3), 149 - 60
Genome and stresses: reactions against aggressions, behavior of transposable elements; Arnault C et al.; The action of stresses on the genome can be considered as responses of cells or organisms to external aggressions . Stress factors are of environmental origin (climatic or trophic) or of genomic nature (introduction of foreign genetic material, for example) . In both cases, important perturbations can occur and modify hereditary potentialities, creating new combinations compatible with survival; such a situation may increase the variability of the genome, and allow evolutive processes to take place . The behavior of transposable elements under stress conditions is thus of particular interest, since these sequences are sources of mutations and therefore of genetic variability; they may play an important role in population adaptation . The survey of the available experimental results suggest that, although some examples of mutations and transposable elements movements induced by external factors are clearly described, environmental injuries or introduction of foreign material into a genome are not systematically followed by drastic genomic changes.

Exp Brain Res, 1994, 100(2), 287 - 92
Expression of major histocompatibility complex class II antigen on amoeboid microglial cells in early postnatal rat brain following intraperitoneal injections of lipopolysaccharide; Xu J et al.; In rats given two single intraperitoneal injections of lipopolysaccharide (LPS) at 1 and 4 days of age and killed at 7 days of age, 11.5--12% of amoeboid microglial cells (AMC) in the supraventricular corpus callosum were induced to express major histocompatibility complex (MHC) class II antigen, as detected with monoclonal antibody OX-6 . The MHC class II antigen induced was colocalized with MHC class I antigen and type 3 complement receptors on the same cells . The expression of MHC class II antigen on the plasma membrane of AMC was confirmed in immunoelectron microscopy . Although OX-6-positive AMC often assumed a perivascular position, the majority of them, however, were far removed from the blood vessels . The cytoplasmic processes of the perivascular OX-6-positive AMC appeared to rest directly on the vascular lamina, and in some section profiles they were in contact with a large surface area of the outer wall of small blood vessels . It is concluded from this study that although MHC class II antigen is not constitutively present on AMC, it is, however, inducible under stimulation with LPS . It is, therefore, suggested that the OX-6-positive AMC, especially the perivascular AMC, may have the potentiality to function as antigen-presenting cells in the developing brain when challenged by LPS.

Tsitologiia, 1994, 36(2), 194 - 9
{The repair of UV-induced postreplication DNA gaps in Escherichia coli cells adapted to methylmethane sulfonate and ethylmethane sulfonate}; Zhestianikov VD et al.; The survival (only after the adaptation to methylmethane sulfonate, MMS) and repair of DNA postreplication gaps in UV-irradiated Escherichia coli, adapted to MMS (20 mkg/ml for 3 h) and ethylmethane sulfonate (EMS, 100 mkg/ml for 3 h), have been investigated . The survival of MMS-adapted bacteria of wild strains B/r and K12 AB1157 somewhat increased, whereas the survival of AB1886 uvrA mutant, which unlike the wild type bacteria is unable to excise cyclobutane pyrimidine dimers, was seen to decrease . The repair of postreplicative gaps in MMS-adapted bacteria correlates qualitatively with changes in survival: in B/r and AB1157 strains the repair is somewhat more effective (10-15%), while in AB1886 uvrA mutant significantly slower (near 30%) than in non-adapted bacteria . Similar changes of postreplicative repair (PRR) of DNA are observed in AB1157 and AB1886 uvrA strains adapted to EMS . It is suggested that the decreased efficiency of PRR in bacteria AB1886 uvrA, adapted to alkylating agent, may be due to the interference between the two inducible repair processes: adaptive response and SOS response . The latter process is involved in the repair of some part of postreplicative gaps of DNA . Different results of PRR of DNA in bacteria of wild types, adapted to MMS and EMS, may be associated with the intrinsic PRR in uvr+ strains . Due to this process in uvr+ bacteria SOS component of PRR of DNA is not formed . It is suggested that PRR in uvr+ bacteria adapted to alkylating agents is accelerated by enzymes of adaptive response in the absence of antagonism between the SOS response and the adaptive response.

Arch Med Res, 1994 Autumn, 25(3), 303 - 6
Frequency of enterotoxigenic Escherichia coli in infants during the first three months of life; Flores-Abuxapqui JJ et al.; To know at what age infants begin to excrete enterotoxigenic Escherichia coli from feces, we studied 30 infants belonging to low socioeconomic status during the first 3 months of life, taking 11 samples of feces from each infant, beginning at the day of birth . We detected LT and ST enterotoxins of E . coli using ELISA test . From the samples studied, 34 samples from 21 (70%) infants were positive for LT . In four samples (1.2%), the presence of LT-producing E . coli coincided with diarrhea, corresponding to three (10.0%) from the infants studied . The earliest age that we observed LT-producing E . coli was day 0 (day of birth) . We did not find ST + or LT/ST-producing E . coli strains . We concluded that ETEC strains are frequent findings in healthy infants, and its presence is too early in persons of low socioeconomic status.

DNA Seq, 1994, 4(5), 343 - 6
Nucleotide sequence of mouse spermidine synthase cDNA; Myohanen S et al.; The nucleotide sequence of mouse cDNA for spermidine synthase appeared to contain 75 nucleotides of 5' untranslated region, an open reading frame of 909 nucleotides and 297 nucleotides of 3' untranslated region . The open reading frame encoded a polypeptide of 302 amino acids, displaying 95% similarity to human and 33% similarity to E . coli spermidine synthase . The 3' flanking region contained an unusual polyadenylation signal AATACA.

Chin J Biotechnol, 1994, 10(2), 121 - 8
Preparation, characterization and application of monoclonal antibodies against PAI-1; Ding H et al.; Six hybridoma cell lines (AP1-AP6) secreting monoclonal antibodies (McAb) against PAI-1 were obtained by fusing the murine myeloma cell line SP2/0 with the spleen cells from Balb/c mouse immunized with recombinant PAI-1 expressed in E . coli . These antibodies were purified by SPA affinity chromatography . All McAbs recognized rPAI-1 and PAI-1 from the human hepatoma cell line HepG2 . The titers of ascites were more than 10(6) . The antibody-antigen affinity constants (Kaff) for anti-PAI-1 McAb measured by EIA were between 3.45 x 10(7)-1.05 x 10(10) M . AP2 and AP3 McAbs were effective in quenching the activity of PAI-1 . Partial quenching of PAI-1 activity was achieved with AP4, AP5 and AP6 McAbs respectively . AP1 McAb had no effect upon PAI-1 activity . Three of the six McAbs (AP1, AP4 and AP5) bound to the PAI-1/t-PA complex, while the others did not . The PAI-1 was purified 51 folds to homogeneity from serum free medium of HepG2 with the recovery rate of 92% by one-step procedure using Sepharose 4B conjugated with anti-PAI-1 McAb (AP1, AP3 and AP4) . A sandwich ELISA for the measurement of PAI-1 antigen in human plasma was developed, based on anti-PAI-1 McAb against non-overlapping epitopes . The mean value of plasma PAI-1 for the healthy donors was 24.7 +/- 7.75 ng/ml measured by ELISA.

Bioelectromagnetics, 1994, 15(5), 377 - 83
Frequency-dependent alterations in enolase activity in Escherichia coli caused by exposure to electric and magnetic fields; Dutta SK et al.; Some neurochemical effects of low-intensity electric and magnetic fields have been shown to be nonlinear functions of exposure parameters . These effects occurred within narrow ranges of frequency and intensity . Previous studies on membrane-associated endpoints in cell culture preparations demonstrated changes in calcium efflux and in acetylcholinesterase activity following exposure to radiofrequency radiation, amplitude modulated (AM) at 16 and at 60 Hz, at a specific absorption rate of 0.05 W/kg . In this study, these modulation frequencies were tested for their influence on the activity of a cytoplasmic enzyme, enolase, which is being tested clinically for detection of neoplasia . Escherichia coli cultures containing a plasmid with a mammalian gene for enolase were exposed for 30 min, and cell extracts were assayed for enolase activity by measuring absorbance at 240 nm . The enolase activity in exposed cultures was compared to the activity in paired control cultures . Exposure to 147 MHz carrier waves at 0.05 W/kg, AM at 16 Hz showed enolase activity enhanced by 62%, and AM at 60 Hz showed enolase activity reduced by 28% . Similarly, exposure to 16 Hz fields alone, at 21.2 V/mrms (electric) and 97 nTrms (magnetic), showed enhancement in enolase activity by 59%, whereas exposure to 60 Hz fields alone, at 14.1 V/mrms (electric) and 65 nTrms (magnetic), showed reduction in activity by 24% . Sham exposures as well as exposure to continuous-wave 147 MHz radiation at 0.05 W/kg showed no change in enolase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Microbiol Immunol, 1994, 38(8), 669 - 71
Localization of apoptosis (programmed cell death) in mice by administration of lipopolysaccharide; Zhang XM et al.; Localization of apoptotic cells by administration of lipopolysaccharide into mice was studied by using the in situ specific labeling of fragmented DNA . This method clearly stained the nuclei of thymocytes at the cortex of the thymus . The nuclei of cells in the bone marrow and in the spleen were also positively stained . It was suggested that the cortex in the thymus is where the LPS-induced programmed cell death occurs.

Cell Mol Biol Res, 1994, 40(7-8), 693 - 8
Cloning of the chandipura virus phosphoprotein encoding gene and its expression in Escherichia coli; Chattopadhyay D et al.; Chandipura (CHP) virus, a member of the vesiculovirus genus within the Rhabdoviridae family, was first isolated from human patients in India . The full length phosphoprotein P gene of CHP have been cloned and expressed in Escherichia coli using the T7 polymerase-based pET-3 series of expression vectors . Under optimal conditions of induction with IPTG, the recombinant P protein constituted 35% of the total bacterial protein . The bacterially expressed protein was found to be phosphate-free . Deletion analysis suggested that the anomalous mobility of the P protein was due to its high acidity . The expressed protein can be phosphorylated in vitro by the extracts prepared from baby hamster kidney cells or rabbit reticulocytes . The cellular kinase involved in phosphorylation appears to be casein kinase II.

Cell Mol Biol Res, 1994, 40(7-8), 671 - 82
Characterization of the interactions between double-stranded RNA and the double-stranded RNA binding domain of the interferon induced protein kinase; Patel RC et al.; The interferon-inducible protein kinase, PKR, requires double-stranded (ds) RNA for its activation . We have previously mapped its dsRNA-binding domain (DRBD) to the amino terminal 170 residues (Patel and Sen, 1992) . In the present study, we have characterized in detail the interactions between dsRNA and DRBD . For this purpose, DRBD was produced in bacteria as a polyhistidine-tagged protein and purified by affinity chromatography . A polyclonal antibody was raised against purified DRBD . For studying dsRNA-DRBD interactions, a Northwestern assay and an electrophoretic mobility shift assay (EMSA) using a radiolabeled in vitro transcribed 82 bp dsRNA probe was developed . The antiserum reacted with both DRBD and PKR but did not prevent their interactions with dsRNA . DRBD, on the other hand, blocked the activation of PKR by dsRNA . DRBD and the dsRNA probe formed multimeric complexes which were separable by EMSA . The antibody could interact with these complexes and supershift their mobility . Competition with unlabeled dsRNA revealed that the dimeric DRBD-dsRNA complex was much more stable than the monomeric complex . Similar competition assays using 11 different synthetic and natural RNA molecules revealed that only authentic dsRNA molecules could effectively compete with the probe for binding DRBD in a sequence-independent fashion.

Chin J Biotechnol, 1994, 10(4), 249 - 56
Expression and immunogenicity of tripeptide repeat region on P190 of Plasmodium falciparum; Pan W et al.; A DNA fragment, designated as P190TR, encoding amino acid residues of the tripeptide region of the P190 antigen was amplified by polymerase chain reaction from genomic DNA of FCC1/HN Plasmodium falciparum isolated from Hainan Province, China . Upon comparison with the nucleotide sequences of MAD20 allele, it was found that there were five bases substitution in the P190TR which cause amino acid changes . The DNA fragment sequenced were ligated to BamHI and XbaI-digested pGEX-2T vector . Competent E . coli JM109 (DE3) were transformed with either parental or recombinant pGEX-2T for expression . Analysis of soluble cellular proteins revealed the high level expression of GST-P190TR as fusion proteins . Affinity purification of the fusion protein under nondenaturing condition resulted in the removal of almost all other E . coli proteins . The purified P190TR protein was highly immunogenic in rabbits . The antibodies against the recombinant protein recognized the malaria parasite with the titers at 1:320 measured by IFA and antisera from malarial patients reacted with the expressed protein in Western Blot.

Growth Factors, 1994, 11(4), 271 - 6
NMR studies of a murine-human chimera of leukaemia inhibitory factor (LIF) . Comparison with human LIF; Maurer T et al.; Leukaemia inhibitory factor (LIF) is a polyfunctional cytokine active on many cell types . We present here 1H NMR studies on the solution properties and stability of MH35, a chimera of murine and human LIF which can be expressed at high levels in Escherichia coli, thus enabling efficient labelling of the protein with the stable isotopes 13C and 15N . MH35 has 85% sequence identity with human LIF and similar activity in biological assays . The 1H chemical shifts of the 12 conserved aromatic residues and the pKa values of the five conserved histidine residues in MH35 and human LIF are very similar . Temperature dependence studies indicate that both proteins are stable, with significant conformational changes occurring only above 70 degrees C . These results show that these proteins have a similar overall structure and stability and that MH35 is therefore a suitable analogue of human LIF for structural studies in solution.

Cytotechnology, 1994, 15(1-3), 129 - 38
Maximization of recombinant protein yield in the insect cell/baculovirus system by one-time addition of nutrients to high-density batch cultures; Bedard C et al.; Suspension cultures of Sf-9 cells at different stages of growth were infected with a recombinant baculovirus expressing beta-galactosidase, using a range of multiplicities of infection (MOI) of 0.05 to 50 . Following infection, the cells were resuspended either in the medium in which they had been grown or in fresh medium . Specific beta-galactosidase yields were not markedly affected by either MOI or medium change in cultures infected in early exponential phase (< or = 3 x 10(6) cells mL-1) . In cultures infected at later growth stages, beta-galactosidase yields could only be maintained by medium replacement . The possibility that this requirement for medium replacement is due either to the accumulation of an inhibitory byproduct or nutrient limitation was examined . Alanine, a major byproduct of cultured insect cell metabolism, did not significantly reduce recombinant protein yield when added to infected cultures in concentrations of up to 40 mM . Following a factorial design, various nutrient concentrates were added alone or in combination to cultures infected in late exponential phase . Additions that included both yeastolate ultrafiltrate and an amino acid mixture restored specific beta-galactosidase yields to levels observed at earlier growth stages or in late stages with medium replacement; the addition of these concentrates, by permitting production at higher cell density, led to increases in the volumetric yield of recombinant protein . Together or separately, the concentrates when added to uninfected late exponential phase cultures, lead to a doubling of the maximum total cell protein level normally supported by unamended medium.

J Ind Microbiol, 1994 Jan, 13(1), 24 - 9
Intracellular aminopeptidases in Streptomyces lividans 66; Butler MJ et al.; We have investigated the aminopeptidase activities present in Streptomyces lividans strains . The majority of these activities proved to be intracellular with multiple active species . Two aminopeptidase P genes were identified to be responsible for the ability to hydrolyze amino terminal peptide bonds adjacent to proline residues . Two other broad spectrum aminopeptidases were found to display homology at both the DNA and protein levels . One showed significant homology to PepN proteins, particularly around the putative zinc-binding residues which are important for catalysis . The second broad spectrum activity was not analyzed in detail but showed a different spectrum of substrate specificity to that of PepN.

Planta, 1994, 194(2), 230 - 40
Functional analysis of a leucine aminopeptidase from Solanum tuberosum L; Herbers K et al.; A protein encoded by a potato cDNA homologous to a leucine aminopeptidase (LAP) from bovine lens (Hildmann et al . 1992) was expressed in Escherichia coli cells and biochemically characterized by hydrolysis of leucine p-nitroanilide . Activity was highest under alkaline conditions with an optimum at about pH 10 . Maximal activities were measured at 65 degrees C . Apart from leucine p-nitroanilide the enzyme could also efficiently hydrolyze the p-nitroanilides of arginine and methionine . Complete inhibition of the enzyme was achieved by incubating bacterial extracts with bestatin and EDTA, which classifies the enzyme as a metalloprotease belonging to the same group as the homohexameric LAPs from mammals . Protein blots showed low constitutive expression of the LAP in all organs of potato plants: buds, flowers, tubers, roots and leaves . An increase in steady-state protein that was paralleled by an increase in total LAP activity was observed in leaf extracts after supplying jasmonic acid via the petioles . Plants containing the cDNA in antisense orientation behind the constitutive Cauliflower Mosaic Virus 35S promoter showed nearly complete reduction of the corresponding mRNA in leaves . However, in these plants LAP activities were only decreased by about 20% as compared to non-transgenic potato plants, while after feeding with jasmonic acid the activity of transgenic plants was reduced to about 5% of that of non-transgenic plants also induced by jasmonic acid . There was no phenotypic difference between wild-type and LAP antisense plants.

Planta, 1994, 193(4), 494 - 501
Cloning, structure and expression of a pea cDNA clone coding for a photosynthetic fructose-1,6-bisphosphatase with some features different from those of the leaf chloroplast enzyme; Carrasco JL et al.; A positive clone against pea (Pisum sativum L.) chloroplast fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) antibodies was obtained from a copy DNA (cDNA) library in lambda gt11 . The insert was 1261 nucleotides long, and had an open reading frame of 1143 base pairs with coding capability for the whole FBPase subunit and a fragment of a putative processing peptide . An additional 115 base pairs corresponding to a 3'-untranslated region coding for an mRNA poly(A)+ tail were also found in the clone . The deduced sequence for the FBPase subunit was a 357-amino-acid protein of molecular mass 39,253 daltons (Da), showing 82-88% absolute homology with four chloroplastic FBPases sequenced earlier . The 3.1-kilobase (kb) KpnI-SacI fragment of the lambda gt11 derivative was subcloned between the KpnI-SacI restriction sites of pTZ18R to yield plasmid pAMC100 . Lysates of Escherichia coli (pAMC100) showed FBPase activity; this was purified as a 170-kDa protein which, upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, displayed a 44-kDa band . As occurs with native FBPases, this indicates a homotetrameric structure for the expressed FBPase . When assayed under excess Mg2+ (10 mM), the expressed enzyme had a higher affinity for the substrate than the native pea leaf FBPase; this parameter appears to be substantiated by a tenfold higher specific activity than that of the native enzyme . However, when activated with dithiothreitol plus saturating concentrations of pea thioredoxin (Td) f, both FBPase had similar activities, with a 4:1 Td f-FBPase stoichiometry . In contrast to the native pea chloroplast FBPase, the E . coli-expressed enzyme did not react with the monoclonal antibody GR-PB5.(ABSTRACT TRUNCATED AT 250 WORDS)

Planta, 1994, 192(4), 453 - 60
Immunocytolocalization of 1-aminocyclopropane-1-carboxylic acid oxidase in tomato and apple fruit; Rombaldi C et al.; The subcellular localization of 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase), an enzyme involved in the biosynthesis of ethylene, has been studied in ripening fruits of tomato (Lycopersicum esculentum Mill.) . Two types of antibody have been raised against (i) a synthetic peptide derived from the reconstructed pTOM13 clone (pRC13), a tomato cDNA encoding ACC oxidase, and considered as a suitable epitope by secondary-structure predictions; and (ii) a fusion protein overproduced in Escherichia coli expressing the pRC13 cDNA . Immunoblot analysis showed that, when purified by antigen affinity chromatography, both types of antibody recognized a single band corresponding to ACC oxidase . Superimposition of Calcofluor white with immunofluorescence labeling, analysed by optical microscopy, indicated that ACC oxidase is located at the cell wall in the pericarp of breaker tomato and climacteric apple (Malus x domestica Borkh.) fruit . The apoplasmic location of the enzyme was also demonstrated by the observation of immunogold-labeled antibodies in this region by both optical and electron microscopy . Transgenic tomato fruits in which ACC-oxidase gene expression was inhibited by an antisense gene exhibited a considerable reduction of labeling . Immunocytological controls made with pre-immune serum or with antibodies pre-absorbed on their corresponding antigens gave no staining . The discrepancy between these findings and the targeting of the protein predicted from sequences of ACC-oxidase cDNA clones isolated so far is discussed.

Biotechnol Prog, 1994 Jan-Feb, 10(1), 109 - 13
A simplified model for the disruption of Escherichia coli: the effect of cell septation; Middelberg AP et al.; A new model for the disruption of Escherichia coli by high-pressure homogenization has been previously presented . Initial model development assumed a bimodal distribution of effective cell strengths to allow for a possible difference in strength between septated and nonseptated cells . A considerably simpler model is obtained when any difference in strength is neglected and a normal distribution is employed . In this article, the disruption of a culture with an abnormally high septated fraction is examined . Disruption versus pressure curves are predicted using both the bimodal and normal approximations to the strength distribution . An examination of disrupted cultures by optical and electron microscopy suggests that septated cells are indeed weaker, thus implying that a bimodal approximation is strictly correct . However, comparison of the model predictions with the experimental results suggests that the simple normal distribution provides sufficient predictive accuracy even for cultures with a high septated fraction.

J Chem Technol Biotechnol, 1994 Jan, 59(1), 67 - 72
Purification and renaturation of recombinant human lymphotoxin (tumour necrosis factor beta) expressed in Escherichia coli as inclusion bodies; Jin H et al.; High level expression of recombinant human tumour necrosis factor beta (rhTNF-beta) in Escherichia coli results in the formation of two portions of protein, namely soluble active protein and insoluble protein which is inactive and aggregates in the form of inclusion bodies (IBs) . In this study, a procedure for purification and renaturation of rhTNF-beta from inclusion bodies has been designed and verified experimentally with a product purity of more than 90% and a recovery of about 30% . The procedure includes washing of IBs with specific wash buffer (Triton X-100/EDTA/lysozyme/PMSF), their solubilization with 8 mol dm-3 alkaline urea, purification with ion-exchange columns, refolding with renaturation buffer and finally concentration and desalination with an ultrafiltration membrane . The characteristics of the renatured protein were identical with those of purified protein from the soluble fraction as demonstrated by (1) SDS-PAGE, (2) cytotoxic activity on mouse L929 cells, (3) N-terminal amino acid sequence, and (4) gel filtration chromatography.

Planta, 1994, 192(2), 221 - 31
The proteins encoded by two tapetum-specific transcripts, Sa tap35 and Sa tap44, from Sinapis alba L . are localized in the exine cell wall layer of developing microspores; Staiger D et al.; By differential screening of a copy DNA (cDNA) library from flowering Sinapis alba L . apices against cDNAs from vegetative apices, two cDNA clones were isolated representing transcripts that are expressed transiently at an early stage of tapetum development . The Sa tap35 cDNA encodes a polypeptide with a predicted molecular weight of 12.7 kDa and an isoelectric point of 10.4 . The sa tap44 cDNA codes for a putative 12.4-kDa polypeptide with an isoelectric point of 7.5 . The deduced amino-acid sequences display 76% sequence identity and contain an N-terminal stretch of hydrophobic amino acids which has characteristics of secretory signal sequences . In-vitro transcription of the cDNAs and translation of the resulting RNAs in the presence of canine pancreatic microsomes demonstrates that the two proteins are translocated into the microsomes and that the putative preproteins are proteolytically processed to the mature forms . By immunoelectron microscopy the Sa TAP35 and Sa TAP44 proteins were detected at the developing peritapetal membrane between the tapetal cytoplasm and the adjacent middle layer of the anther wall . Furthermore, labelling was observed within the locule in association with globules resembling pro-Ubisch bodies which appeared at the tetrad stage . During the early vacuolate stage of microspore development the young exine was strongly labelled . The exine and the peritapetal membrane both are composed of sporopollenin, and the pro-Ubisch bodies are thought to contain sporopollenin precursors . Thus, Sa TAP35 and Sa TAP44 might be involved in sporopollenin formation and/or deposition.

J Tongji Med Univ, 1994, 14(4), 230 - 4
Effect of increasing biliary tract pressure on house rabbit blood dynamics in acute cholangitis of severe type; Zheng QC et al.; In this study 12 Japanese long ear rabbits were used as models of acute cholangitis of severe type (ACST), and also an increasing pressure apparatus of self-made caecus to form high pressure of the biliary tract . The animals were observed for changes in blood dynamics in an attempt to explore the effect and relation of high pressure of biliary tract and infective element in pathogenesis of ACST . It was found that when the biliary pressure was increased within 120 min in the 20 kPa group, the blood endotoxin level showed no obvious increment (P > 0.05), but the decreased range of average MAP (mean artery pressure) was over 4 kPa, and the cardiac output also decreased evidently (P < 0.05), and that when the biliary pressure was decreased, MAP and cardiac output were restored to normal gradually . Of these animals 3 didn't restore their normal condition when the blood pressure decreased to zero and died finally . Meanwhile the electric discharge frequency of the right greater splanchnic nerves increased (P < 0.05), but when pressure was reduced, the frequency slowed down . From the above findings, the authors came to the conclusion that the rapid increase of the biliary tract pressure is the important factor leading to a decrease in blood pressure of ACST, and even bringing about irreversible shock, which is involved in the activity of splanchnic nerves.

Biochimie, 1994, 76(12), 1183 - 91
tRNA-guanine transglycosylase from Escherichia coli: recognition of dimeric, unmodified tRNA(Tyr); Curnow AW et al.; In order to probe the interaction between tRNA and the tRNA hypermodifying enzyme, tRNA-guanine transglycosylase (TGT) from Escherichia coli, we have undertaken the generation of E coli tRNA(Tyr) and analogues . During efforts to adapt currently available in vitro transcription techniques we encountered difficulties attributable to dimerization of the tRNA products . E coli tRNA(Tyr) has previously been characterized for its ability to form a dimer in solutions of suitable salt concentrations at appropriate temperatures (Yang SK, Soll DG, Crothers DM (1972) Biochemistry 11, 2311-2320; Rordorff BF, Kearns DR (1976) Biochemistry 15, 3320-3330) . We have applied similar techniques to our unmodified analogue of E coli tRNA(Tyr) and produced both monomeric and dimeric forms of E coli tRNA(Tyr) . In this report we find that the dimer does serve as a substrate for modification by TGT . While both the conformers are equal in terms of Vmax (within experimental error) a 2.5-fold increase in KM occurs when going from monomer to dimer . This suggests that TGT preferentially binds the monomer but once either conformer is bound will catalyze the modification reaction equally well . We have also compared the results for the two conformers to our previous data of an RNA minihelix corresponding to the anticodon arm of E coli tRNA(Tyr) . Here we find that our earlier conclusion, that the recognition elements for TGT are localized within the anticodon arm of cognate tRNAs, is supported.

Biochimie, 1994, 76(12), 1168 - 77
Transcriptional patterns of the mutL-miaA superoperon of Escherichia coli K-12 suggest a model for posttranscriptional regulation; Tsui HC et al.; The complex amiB-mutL-miaA-hfq-hflX-hflK-hflC superoperon of E coli contains important genes for several fundamental cellular processes, including cell-wall hydrolysis (amiB), DNA repair (mutL), tRNA modification (miaA) and proteolysis (hflX-hflK-hflC) . We report here the transcriptional pattern and possible posttranscriptional regulation of mutL, miaA and hfq genes of this superoperon . RNase protection analysis of mRNA transcribed from the bacterial chromosome demonstrated that there is co-transcription of mutL and miaA . In addition, two internal promoters, PmiaA and P1hfq were identified and mapped to 201 and 837 nucleotides upstream from the respective translation start sites . PmiaA contains poor matches to the -10 and -35 regions of the sigma-70 RNA polymerase consensus sequences, but it contains multiple potential Fis-binding sites and an upstream AT-rich region with poly(A) sequences . The basic arrangement of Fis-binding sites followed by an AT rich region is shared with promoters for rRNA operons and some of the tRNA and tRNA modification genes . As part of an initial study of mutL and miaA regulation, we measured transcript amounts in isogenic rne, rnc and rne rnc double mutants which are deficient in RNase E, RNase III or both . The amounts of steady state level mutL-miaA cotranscript, PmiaA transcript and P1hfq transcript increased eight-, nine- and three-fold respectively in an rne3071 mutant when compared to the rne+ parent . In contrast, amounts of the three transcripts were the same in an rnc105 mutant and its rnc+ parent . These results indicate that mutL, miaA, and hfq expression could be regulated by multiple mechanisms, including degree of cotranscription from upstream genes, modulation of internal promoter strength, and by RNase E activity . A model is presented for RNase E-mediated posttranscriptional regulation that may coordinate mutL expression with replication and miaA with tRNA amounts under different growth conditions, especially during nutrient upshifts.

Biochimie, 1994, 76(12), 1143 - 51
Aminoacyl-tRNA synthetase and U54 methyltransferase recognize conformations of the yeast tRNA(Phe) anticodon and T stem/loop domain; Guenther RH et al.; The enzyme-catalyzed posttranscriptional modification of tRNA and the contributions of modified nucleosides to tRNA structure and function can be investigated with chemically synthesized domains of the tRNA molecule . Heptadecamer RNAs with and without modified nucleosides and DNAs designed as analogs to the anticodon and T stem/loop domains of yeast tRNA(Phe) were produced by automated chemical synthesis . The unmodified T stem/loop domain of yeast tRNA(Phe) was a substrate for the E coli m5U54-tRNA methyltransferase activity, RUMT . Surprisingly, the DNA analog of the T stem/loop domain composed of d(A,U,G,C) was also a substrate . In addition, the DNA analog inhibited the methylation of unfractionated, undermodified E coli tRNA lacking the U54 methylation . RNA anticodon domains and DNA analogs differentially and specifically affected aminoacylation of the wild type yeast tRNA(Phe) . Three differentially modified tRNA(Phe) anticodon domains with psi 39 alone, m1G37 and m5C40, or psi 39 with m1G37 and m5C40,stimulated phenylalanyl-tRNA synthetase (FRS) activity . However, one anticodon domain, with m5C40 as the only modified nucleoside and a closed loop conformation, inhibited FRS activity . Modified and unmodified DNA analogs of the anticodon, tDNA(PheAC), inhibited FRS activity . Analysis of the enzyme activity in the presence of the DNA analog characterized the DNA/enzyme interaction as either partial or allosteric inhibition . The disparity of action between the DNA and RNA hairpins provides new insight into the potential allosteric relationship of anticodon binding and open loop conformational requirements for active site function of FRS and other aaRSs . The comparison of the stimulatory and inhibitory properties of variously modified RNA domains and DNA analogs demonstrates that conformation, in addition to primary sequence, is important for tRNA-protein interaction . The enzyme recognition of various DNA analogs as substrate and/or inhibitors of activity demonstrates that conformational determinants are not restricted to ribose and the standard A-form RNA structure.

Biochimie, 1994, 76(10-11), 958 - 67
Interaction of Fis protein with DNA: bending and specificity of binding; Betermier M et al.; The Escherichia coli Fis protein is a dimeric DNA-binding protein whose specific binding sites share a weak consensus sequence . Use of the gel retardation technique indicates that binding of Fis on a linear DNA fragment leads to the formation of a ladder of defined retarded complexes, independently of the presence of a specific site . This non-specific binding of Fis is consistent with a model where equivalent low-affinity sites on a given fragment would be bound randomly and independently of each other by consecutive Fis dimers . Evidence is presented that non-specific binding of Fis can, however, induce an apparent site-specific conformational change in the DNA . This observation is discussed in terms of a model in which each Fis:DNA complex detected in gel retardation experiments actually represents a dynamic equilibrium of a fixed number of Fis dimers distributed on the fragment.

Biochimie, 1994, 76(10-11), 941 - 50
Genetic and biochemical analysis of IHF/HU hybrid proteins; Goldenberg D et al.; Integration host factor (IHF) is a small heterodimeric DNA-binding protein of E coli composed of two subunits, alpha and beta, encoded by the himA and hip genes, respectively . IHF binds to DNA at a consensus sequence and bends DNA . HU protein, encoded by the hupA and hupB genes, is similar to IHF except that it does not bind to a specific DNA sequence . To investigate the protein determinants for IHF specificity we exchanged progressively longer segments from the C-terminus of Hip with those of HupA, and followed the activity in vivo and in vitro of four such IHF/HU hybrids . Replacement of 11 residues from the C-terminal alpha helix of Hip by the complementary eight residues of HupA (hybrid 1), had only minor effects on the DNA binding activity of the protein . As progressively longer segments of Hip were replaced by HupA, a precipitous decrease in IHF activity was observed . The hybrid with the longest substitution, hybrid 4, was totally inactive in vivo and could not be purified . None of the hybrid proteins could complement HU activity . Comparing the activities of hybrid 1, hybrid 2 and IHF point mutants, led us to conclude that the structural integrity of the C-terminal alpha helix and its spatial position, but not its amino acid sequence, are important for DNA binding specificity . We favor the hypothesis that alpha helices 3 of both IHF subunits interact with the body of IHF so as to anchor the arms . This interaction stabilizes the arms to permit DNA binding specificity . Thus the C-termini of IHF influence, in an indirect way, the recognition of specific sites on DNA.

Biochimie, 1994, 76(10-11), 924 - 32
P1 plasmid partition: binding of P1 ParB protein and Escherichia coli integration host factor to altered parS sites; Funnell BE et al.; The Escherichia coli integration host factor (IHF) participates in P1 plasmid partition by assisting the interaction of P1 ParB protein with its specific site, parS . Together they form an extremely high-affinity protein-DNA complex, in which parS DNA is wrapped around a core of ParB and IHF protein in a precise three-dimensional conformation . We have investigated the interaction of ParB and IHF with mutant DNA sites, to examine protein specificity and cooperativity . The results indicate that ParB specifically recognizes two separate types of sequence repeats in its minimal binding site in one half of the parS site . The affinity of ParB or IHF for parS is much greater in the presence of the other protein . Mutations that decrease ParB or IHF binding to parS have relatively minor defects in vivo, because each protein still binds well to parS in the presence of the other protein . We observed that ParB acts better when provided in cis than in trans to parS in vivo . Our experiments suggest that in vivo, the local concentration of ParB protein near the plasmid is high, so that ParB can act reasonably well to promote partition in cells without IHF . However, this activity is lower than in wild-type cells, indicating that IHF is essential for long-term plasmid stability.

Biochimie, 1994, 76(10-11), 917 - 23
Functions of histone-like proteins in the initiation of DNA replication at oriC of Escherichia coli; Roth A et al.; Using methidiumpropyl-EDTA (MPE) footprinting we found one specific binding site for FIS protein in the E coli replication origin, oriC . We mutagenized the binding sites for FIS and IHF in oriC and analyzed the effect of the mutations on protein binding and oriC function . The replication efficiency of oriC plasmids paralleled the ability of the mutated DNA fragments to bind IHF or FIS . We conclude that these histone-like proteins function in cis in the initiation of DNA replication at oriC.

Biochimie, 1994, 76(10-11), 901 - 8
Histones, HMG, HU, IHF: Même combat; Oberto J et al.; In this review article we present a compilation of the proteins homologous to Escherichia coli HU: the HU-like family . Two of these, HU and IHF from E coli have been extensively characterized genetically and biochemically . Due to their DNA binding activities, these proteins confer a condensed shape to the chromosome and regulate the transcription of selected sets of its genes . The parallel between the dual function of the HU-like proteins and the roles described for eukaryotic histone and HMG proteins is striking, especially in the view that they are evolutionary unrelated.

Biochimie, 1994, 76(10-11), 1082 - 9
Protein HU from the cyanobacterium Anabaena; Nagaraja R et al.; Protein HU was purified from the cyanobacterium Anabaena 7120 . Its complete amino acid sequence was determined by automated Edman degradation of the whole protein and of CNBr and chymotryptic peptides . The active DNA-binding protein is a homodimer of 94-amino acid subunits . Approximately half of the residues are identical to those of the two subunits of HU protein from E . coli . The protein binds to both supercoiled and relaxed double-stranded DNA, cooperatively . The contour lengths of circular DNAs were reduced up to six-fold by HU binding at low ratios of HU to DNA . At higher ratios, highly condensed aggregates were observed . Heterocysts are cells specialized for nitrogen fixation that differentiate at regular intervals along the filaments of Anabaena when they are transferred to a medium lacking combined nitrogen . Protein HU, labeled with 35S in cells growing on ammonia, disappears from developing heterocysts, although it is stably maintained in the intervening strings of vegetative cells . Following establishment of the heterocyst pattern, in which the differentiated cells are spaced about ten cells apart, HU is synthesized in the vegetative cells but not in the heterocysts . Several other vegetative cell DNA-binding proteins are also degraded during the differentiation . The major DNA-binding protein in heterocysts is a new one of subunit molecular mass around 12,000, whose relationship to other DNA-binding proteins is unknown . The gene encoding protein HU was cloned from Anabaena DNA and sequenced . The gene sequence is consistent with the amino acid sequence determined previously . Low stringency hybridization to Anabaena DNA digests suggest that there is a single gene for HU, consistent also with the unique amino acid sequence . S1 nuclease protection experiments suggest that the HU gene promoter differs from those of other Anabaena genes determined to date.

Biochimie, 1994, 76(10-11), 1071 - 4
Characterisation of mutant alleles of the cell division protein FtsA, a regulator and structural component of the Escherichia coli septator; Sanchez M et al.; Two alleles of ftsA, a gene that encodes an essential cell division protein in Escherichia coli, have-been mapped at the nucleotide level . The mutations are located inside domains that are conserved in an ATP-binding protein family . The ftsA2 mutation lies in the adenine-binding domain, and the ftsA3 in the ribose-binding domain . The defect in ampicillin binding to PBP3 described for allele ftsA3 is allele-specific . This supports the hypothesis of the existence of different domains in FtsA having different functions.

Biochimie, 1994, 76(10-11), 1063 - 70
The role of H-NS in one carbon metabolism; Landgraf JR et al.; The H-NS protein of Escherichia coli regulates the expression of genes involved in many general processes such as osmoregulation and virulence . More recently, H-NS was shown to exert an effect on ilvIH gene expression in conjunction with the leucine responsive regulatory protein (Lrp) . We show that H-NS is involved in the transcriptional regulation of the kbl/tdh operon, which is also Lrp regulated . Insertional inactivation of the hns gene results in two-fold derepression of the kbl/tdh operon . This level of expression is sufficient to suppress the auxotrophic requirements imposed by a glyA mutation . We show that expression of the kbl/tdh operon is temperature controlled and that this control is not mediated through H-NS action as has been shown for some other temperature controlled genes.

Biochimie, 1994, 76(10-11), 1052 - 4
Regions of the CFA/I promoter involved in the activation by the transcriptional activator CfaD and repression by the histone-like protein H-NS; Jordi BJ et al.; Expression of CFA/I fimbriae of Escherichia coli requires the transcriptional activator CfaD . The mechanism by which CfaD activates the CFA/I promoter is to overcome the repression by H-NS, one of the histone-like proteins in E coli . This study addresses the question of which sequences in the promoter region of CFA/I interact with CfaD and H-NS . In order to determine this, deletion mutants of the CFA/I promoter were constructed and cloned upstream of the promoterless lacZ gene . The effect of CfaD and H-NS on the expression of these constructs was determined.

Biochimie, 1994, 76(10-11), 1041 - 51
On the role of the Escherichia coli integration host factor (IHF) in repression at a distance of the pyrimidine specific promoter P1 of the carAB operon; Charlier D et al.; Binding of integration host factor to its target site, centered around nucleotide -305 upstream of the transcription startpoint, exerts antagonistic effects on the expression of P1, the upstream pyrimidine specific promoter of the E coli and S typhimurium carAB operons . IHF stimulates P1 promoter activity in minimal medium, but also increases the repressibility of this promoter by pyrimidines . We present evidence strongly suggesting that IHF exerts these effects by modulating the binding of another pyrimidine specific regulatory molecule, probably the product of gene carP . The carAB control region contains a GATC Dam methylation site, 106 bp upstream of the P1 transcription startpoint, which can be protected in vivo against methylation . This protection requires at least the regulatory carP gene product and a high pyrimidine nucleotide pool and, as shown here, the integration host factor . Whether CarP directly binds to this site or exerts its protective effect indirectly is not yet known . In the absence of IHF (himA) or in mutants affected in the IHF target site this protection is strongly impaired, suggesting that IHF positively influences the formation or the stability of the protective protein-DNA complex some 200 bp downstream . Furthermore, we have demonstrated that the distance separating the IHF and GATC Dam methylase target sites is crucial for the in vivo protection and for pyrimidine mediated regulation of P1 promoter expression . Indeed, shortening this distance by 6 bp, and more surprisingly also by 11 bp, results in a severe reduction of the degree of in vivo protection of the GATC site against methylation and concomitantly of the repressibility by pyrimidines of P1 promoter activity . The absence of both these effects in a double, deletion-duplication, mutant resulting in a net increase of the intervening sequence by 1 bp, clearly demonstrates that these effects are not due to the disruption of an important regulatory site, but must be attributed to variations in the distance separating different protein binding sites.

Antonie Van Leeuwenhoek, 1994, 66(1-3), 57 - 88
The hydrogenases and formate dehydrogenases of Escherichia coli; Sawers G; Escherichia coli has the capacity to synthesise three distinct formate dehydrogenase isoenzymes and three hydrogenase isoenzymes . All six are multisubunit, membrane-associated proteins that are functional in the anaerobic metabolism of the organism . One of the formate dehydrogenase isoenzymes is also synthesised in aerobic cells . Two of the formate dehydrogenase enzymes and two hydrogenases have a respiratory function while the formate dehydrogenase and hydrogenase associated with the formate hydrogenlyase pathway are not involved in energy conservation . The three formate dehydrogenases are molybdo-selenoproteins while the three hydrogenases are nickel enzymes; all six enzymes have an abundance of iron-sulfur clusters . These metal requirements alone invoke the necessity for a profusion of ancillary enzymes which are involved in the preparation and incorporation of these cofactors . The characterisation of a large number of pleiotropic mutants unable to synthesise either functionally active formate dehydrogenases or hydrogenases has led to the identification of a number of these enzymes . However, it is apparent that there are many more accessory proteins involved in the biosynthesis of these isoenzymes than originally anticipated . The biochemical function of the vast majority of these enzymes is not understood . Nevertheless, through the construction and study of defined mutants, together with sequence comparisons with homologous proteins from other organisms, it has been possible at least to categorise them with regard to a general requirement for the biosynthesis of all three isoenzymes or whether they have a specific function in the assembly of a particular enzyme . The identification of the structural genes encoding the formate dehydrogenase and hydrogenase isoenzymes has enabled a detailed dissection of how their expression is coordinated to the metabolic requirement for their products . Slowly, a picture is emerging of the extremely complex and involved path of events leading to the regulated synthesis, processing and assembly of catalytically active formate dehydrogenase and hydrogenase isoenzymes . This article aims to review the current state of knowledge regarding the biochemistry, genetics, molecular biology and physiology of these enzymes.

Antonie Van Leeuwenhoek, 1994, 66(1-3), 47 - 56
Nitrate reductases in Escherichia coli; Bonnefoy V et al.; Escherichia coli expresses two different membrane-bound respiratory nitrate reductases, nitrate reductase A (NRA) and nitrate reductase Z (NRZ) . In this review, we compare the genetic control, biochemical properties and regulation of these two closely related enzyme systems . The two enzymes are encoded by distinct operons located within two different loci on the E . coli chromosome . The narGHJI operon, encoding nitrate reductaseA, is located in the chlC locus at 27 minutes, along with several functionally related genes: narK, encoding a nitrate/nitrite antiporter, and the narXL operon, encoding a nitrate-activated, two component regulatory system . The narZYWV operon, encoding nitrate reductase Z, is located in the chlZ locus located at 32.5 minutes, a region which includes a narK homologue, narU, but no apparent homologue to the narXL operon . The two membrane-bound enzymes have similar structures and biochemical properties and are capable of reducing nitrate using normal physiological substrates . The homology of the amino acid sequences of the peptides encoded by the two operons is extremely high but the intergenic regions share no related sequences . The expression of both the narGHJI operon and the narK gene are positively regulated by two transacting factors Fnr and NarL-Phosphate, activated respectively by anaerobiosis and nitrate, while the narZYWV operon and the narU gene are constitutively expressed . Nitrate reductase A, which accounts for 98% of the nitrate reductase activity when fully induced, is clearly the major respiratory nitrate reductase in E . coli while the physiological role of the constitutively expressed nitrate reductase Z remains to be defined.

Cell Biophys, 1994, 24-25, 27 - 36
Recombinant proteins L and LG . Two new tools for purification of murine antibody fragments; Vola R et al.; Several bacterial cell wall proteins, like proteins A and G, with valuable affinity for immunoglobulins have been discovered and are currently employed in immunological techniques . In 1988, protein L, a bacterial cell wall protein with Ig-binding capacity, was isolated from the anaerobic bacterial species Peptostreptococcus magnus . Binding data with immunoglobulin fragments suggested that protein L could selectively bind the variable region of human kappa light chains . More recently a recombinant LG fusion protein was expressed in E . coli containing four repeated Ig-binding domains of protein L (fragment B1-4) and two IgG Fc-binding protein G domains (fragment CDC) . Recombinant L and LG proteins were tested in the purification of murine monoclonal IgG and their fragments . After affinity-constant evaluation in different buffer systems, high-pressure affinity-chromatography columns were prepared by coupling the proteins to Affi-prep 10 resin and tested with eight different murine monoclonal antibodies and their fragments of various isotypes . Affinity-chromatography experiments confirming radioimmunoassay results showed 75% fragment-binding capacity of protein L and 100% of protein LG . These results evidenced protein LG as the most powerful Ig-binding tool so far described . Therefore, application of these proteins may be suggested in the purification of murine immunoglobulins and their fragments, including the engineered ones.

Cell Biophys, 1994, 24-25, 185 - 92
Comparison of two anthracycline-based prodrugs for activation by a monoclonal antibody-beta-glucuronidase conjugate in the specific treatment of cancer; Haisma HJ et al.; Antibody-directed enzyme prodrug therapy (ADEPT) may improve the therapeutic index of cytostatic agents . We compared two prodrugs, epirubicin-glucuronide (Epi-glu) and doxorubicin-spacer-glucuronide (Dox-sp-glu), for their cytotoxicity on activation by a monoclonal antibody-enzyme conjugate bound to tumor cells . The results showed that the prodrugs were 10 (Dox-sp-glu) and 100 (Epi-glu) times less toxic than the parent drugs against OVCAR-3 cells . This difference was a result of the hydrophilic property of the prodrugs resulting in a reduced cellular uptake . The enzyme-catalyzed hydrolysis of Dox-sp-glu by E . coli-derived beta-glucuronidase (GUS) (Km 500 microM, Vmax 21,000 mumol/min/g) was much more efficient than that of Epi-glu (Km 10 microM, Vmax 40 mumol/min/g) . Incubation of OVCAR-3 cells with an enzyme-immunoconjugate prepared from monoclonal antibody 323/A3 and E . coli-derived GUS before treatment with prodrugs completely restored the cytotoxicity of the prodrugs to the level of the parent drugs.

Cell Mol Biol Res, 1994, 40(5-6), 421 - 9
Modes of regulation of casein kinase II; Jakobi R et al.; Casein kinase II is unique when compared to other protein kinases; it utilizes GTP with almost the same effectiveness as ATP and exists as an active holoenzyme which does not need to be activated by dissociation of regulatory subunits or unfolding of regulatory domains . In vitro, the activity of casein kinase II is inhibited by acidic compounds and stimulated by basic compounds . Casein kinase II activity is inhibited by 2,3-bisphosphoglycerate and stimulated by polyamines at levels which are physiological in red cells . To examine the effects of autophosphorylation of the beta subunit on activity, two mutants of the Drosophila beta subunit have been constructed in which Ser-4 or Ser-(2-4) are changed to alanine residues . Analysis of autophosphorylation with wild-type and mutant recombinant holoenzymes reveals Ser-2 and Ser-3 as the major autophosphorylation sites . Autophosphorylation does not affect the phosphorylation of casein, but reduces the rate of phosphorylation of glycogen synthase by 30%, elongation factor I by 50-70%, and calmodulin by 20-40% . The data indicate that autophosphorylation of the beta subunit can negatively regulate the phosphotransferase activity of casein kinase II with physiological substrates . To examine regulation of casein kinase II activity by the beta subunit, recombinant alpha and beta subunits from human and Drosophila were expressed in Escherichia coli . Upon formation of the holoenzyme, the beta subunit stimulated the catalytic activity 4- to 5-fold . The catalytic alpha subunit contains the eleven conserved subdomains characteristic of all protein kinases.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Mol Biol Res, 1994, 40(5-6), 391 - 9
Protein kinase CK2 structure-function relationship: effects of the beta subunit on reconstitution and activity; Boldyreff B et al.; Protein kinase CK2 subunits alpha and beta were expressed either separately or together in a bacterial expression system (pT7-7/BL21(DE3)) and purified to homogeneity . After mixing the subunits, a CK2 holoenzyme (alpha 2 beta 2) was spontaneously reconstituted, which displays identical features as the native enzyme . The alpha subunit alone, although catalytically active by itself, has different biochemical and biophysical properties than the holoenzyme, e.g., it is extremely salt sensitive, already 50 mM monovalent salt can lead to a 50% inhibition of the catalytic activity . Furthermore, it is readily inactivated through urea, protease, and heat treatment . In contrast, the holoenzyme, either reconstituted or native, is much more stable when similar negative insults prevail . The beta subunit has at least three functions: (a) it is necessary for maximum activity of the enzyme under physiological salt conditions, (b) it protects the alpha subunit against denaturing agents or conditions, and (c) it alters the substrate specificity of the alpha subunit . By site-directed mutagenesis, certain functions of the beta subunit could be assigned to specific amino acids or domains . Twenty one mutants of the beta subunit have been prepared and assayed for their ability to assemble with the catalytic alpha subunit to give a fully competent CK2 holoenzyme . The beta subunit contains an acidic stretch (amino acid 55-64), which is obviously responsible for a negative control of enzyme activity since mutations of certain acidic amino acids within this stretch to alanine lead to a hyperactive CK2 holoenzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Biol Cell, 1994, 82(1), 39 - 44
Molecular and immunological characterization of a Trypanosoma cruzi protein homologous to mammalian elongation factor 1 gamma; Billaut-Mulot O et al.; In previous studies, we reported the characterization of three Trypanosoma cruzi proteins with molecular masses of 45, 30 and 25 kDa eluted from a glutathione agarose column (these proteins were named TcGBP) . Using antibodies against TcGBP native proteins we could isolate from a lambda ZAPII epimastigote cDNA library cDNA clones encoding the 30 and 25 kDa proteins . Comparison of the two sequences with amino acid sequences in several data banks revealed that both protein sequences were highly homologous to human and Artemia salina elongation factor 1 beta . Thus, the proteins were named TcEF-1 beta 25 and TcEF-1 beta 30 . In the present study we used a double immunoscreening strategy that allowed us to isolate a cDNA clone corresponding to the 45 kDa protein . The protein sequence revealed 31% identity and 61% homology with human and Artemia salina EF1 gamma and therefore was named TcEF-1 gamma . Moreover, three putative phosphorylation sites at position 51 (CSPC), at position 90 (RTPL) and at position 265 (PSPF) were found in the TcEF-1 gamma sequence . These sites are compatible with the notion that TcEF-1 gamma could be the target of phosphorylation by protein kinase(s) . Random primed cDNA hybridized with a single 1.4 kb mRNA found in epimastigote, trypomastigote and amastigote forms . In addition, Southern blot analysis of genomic DNA suggested that the protein is encoded by a single gene . The TcEF-1 gamma cDNA was subcloned into the pGEX-4T-3 vector for expression in Escherichia coli.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth Factors, 1994, 11(3), 163 - 74
Involvement of basic fibroblast growth factor NH2 terminus in nuclear accumulation; Patry V et al.; The human basic Fibroblast Growth Factor (bFGF) gene was shown to encode four polypeptides by an alternative use of initiation codons (three CUG and one AUG) . In this report, we present a comparative study of the fate and intracellular localization of individual bFGF isoforms . For this purpose, we have produced the various bFGF isoforms in E . coli and purified them to homogeneity: the 210 amino acid form initiated at CUG1 that contains a nuclear localization sequence (NLS), the 155 amino acid form (AUG-mediated initiation) and the 146 amino acid form (processed form extracted from tissues) . While the different bFGFs were taken up by the cell with equal efficiency, more of the 210 amino acid form accumulated in the nucleus and represented 36% of the internalized bFGF compared with 15% in the others . A chimeric protein containing the minimal SV40 Large T NLS (SV40NLS) fused to the 155 amino acid bFGF form (SVbFGF) behaves like the native 155 amino acid form, indicating that nuclear accumulation of exogenous bFGF is not mediated by the NLS-associated function . These results suggest that the amino-terminal part of the 210 amino acid bFGF contains a sequence responsible for its nuclear retention . Bioactivities of the different forms were tested on adult bovine aortic endothelial (ABAE) cells . The bFGF degradation pathways, mitogenic activity and stimulation of rRNA synthesis appeared to be the same for all bFGFs but the stimulation of plasminogen activator was enhanced by the 210 amino acid form and correlated with nuclear accumulation.

Ann Fr Anesth Reanim, 1994, 13(5), 675 - 84
{Intestinal mucosa injury during experimental endotoxin-induced shock}; Vallet B et al.; To ascertain tissue oxygenation during conversion from hypo to hyperdynamic state with vascular volume expansion, venous outflow from a segment of ileum was isolated in anesthetized and pump-ventilated endotoxic dogs to measure gut oxygen uptake (VO2), lactate metabolism, intramucosal PCO2 and tissue PO2 (PtiO2) . Tissue PO2 was measured by multipoint surface Mehrdraht Dortmund Oberflache electrodes placed on mucosal and serosal surfaces of gut . Six dogs were infused with 2 mg.kg-1 E . coli lipopolysaccharide (LPS) in one hour followed by a two hour 0.5 mL.kg-1.min-1 dextran infusion . Two dogs were used as controls and received dextran infusion in order to assess time and hemodilution-dependent effects . LPS infusion resulted in an hypodynamic sepsis with supply limited VO2, increased arterial lactate and increased lactate output by gut . Resuscitation resulted in an hyperdynamic sepsis with improvement of whole-body VO2 . In the gut, VO2 remained low and intramucosal PCO2 as well as lactate output remained high, despite increased flow . Gut PtiO2 results suggested blood flow maldistribution with tissue hypoxia in the mucosa despite increased total flow to the gut . Gut VO2, lactate flux, intramucosal PCO2, and tissue PO2 were consistent with regulatory responses that shut down mucosal perfusion and oxygenation in spite of increased blood flow to gut.

Acta Biochim Pol, 1994, 41(4), 415 - 9
Effect of disulfide and sulfhydryl reagents on abortive and productive elongation catalyzed by Escherichia coli RNA polymerase; Radlowski M et al.; The effect of disulfide and sulfhydryl reagents on the rate of abortive and productive elongation has been studied using Escherichia coli RNA polymerase holoenzyme and poly{d(A-T)} as template . In the presence of UTP as a single substrate and UpA as a primer, the enzyme catalyzed efficiently the synthesis of the trinucleotide product UpApU . Incubation of RNA polymerase with 1 mM 2-mercaptoethanol resulted in a 5-fold increase of the rate of UpApU synthesis . In contrast, incubation of the enzyme with 1 mM 5,5'-dithio-bis(2-nitrobenzoic) acid resulted in a 6-fold decrease of the rate of abortive elongation . Determination of the steady state kinetic constants associated with UpApU synthesis disclosed that the disulfide and sulfhydryl reagents mainly affected the rate of UpApU release from the ternary transcription complexes and therefore influenced the stability of such complexes.

Folia Microbiol (Praha), 1994, 39(4), 255 - 60
Direct selection shuttle plasmid vector, pPW264, used for cloning the alpha-amylase gene of Schwanniomyces occidentalis; Puta F et al.; We constructed a novel cloning system with positive selection for inserted fragments . The gene for tetracycline resistance (tetR) originally used in plasmid pTR262 was replaced with the gene for chloramphenicol acetyltransferase (cat) and terminator sequences were introduced downstream of the cat gene . The terminator sequences stop transcription originating on strong PR promoter that would otherwise proceed through the region of replication origin and interfere with plasmid replication . Thus the copy number of recombinant plasmid molecules is stabilized . The cloning system has been constructed in a new YEp type shuttle vector, pPW264 . The 8.1 kb-vector carries two unique cloning sites, BglII and HindIII . The maintenance of the vector and selection in yeast is ensured by URA3 Saccharomyces cerevisiae gene . The vector was employed in cloning of the gene for alpha-amylase from Schwanniomyces occidentalis.

Genetica, 1994, 94(1), 17 - 25
A temperature-sensitive mutant of Escherichia coli with an alteration in ribosomal protein L22; Burnette-Vick B et al.; A temperature-sensitive, protein synthesis-defective mutant of Escherichia coli exhibiting an altered ribosomal protein L22 has been investigated . The temperature-sensitive mutation was mapped to the rplV gene for protein L22 . The genes from the wild type and mutant strains were amplified by the polymerase chain reaction and the products were sequenced . A cytosine to thymine transition at position 22 of the coding sequence was found in the mutant DNA, predicting an arginine to cysteine alteration in the protein . A single cysteine residue was found in the isolated mutant protein . This amino acid change accounts for the altered mobility of the mutant protein in two-dimensional gels and during reversed-phase HPLC . The temperature-sensitive phenotype was fully complemented by a plasmid carrying the wild type L22 gene . Ribosomes from the complemented cells showed only wild type protein L22 by two dimensional gel analysis and were as heat-resistant as control ribosomes in a translation assay . The point mutation in the L22 gene is uniquely responsible for the temperature-sensitivity of this strain.

J Mol Neurosci, 1994 Summer, 5(2), 121 - 32
Increased globotriaosylceramide on plasma membranes of synchronized familial dysautonomia cells . Verotoxin binding studies; Pereira J et al.; Familial dysautonomia is an autosomal recessive genetic disease found almost exclusively among Ashkenazi Jews, characterized by deficits in autonomic, sensory, and central functions . Although the gene has been localized to chromosome 9, the biochemical defect remains elusive . We previously reported an increase in globotriaosylceramide in dysautonomic fibroblasts and lymphoblasts, and unusual fibroblast growth patterns suggesting plasma membrane abnormalities . Globotriaosylceramide is a plasma membrane component, and the natural receptor for verotoxin derived from E . coli . In Vero and HeLa cells, which are susceptible to verotoxin, the expression of globotriaosylceramide on the cell surface is maximal at the G1/S boundary of the cell cycle . Measurement of toxin binding at 0 degrees C at this boundary is indicative of the amount of globotriaosylceramide exposed on the cell surface . Above 0 degrees C, verotoxin enters, and is toxic to, the cell . We analyzed verotoxin-globotriaosylceramide interactions in synchronized FD and normal cells at this boundary . 125I-toxin binding was much more marked to lymphoblasts from patients than from controls . When cells were grown in the presence of verotoxin, at 10(-2)-10(-7) micrograms/mL, 70% of dysautonomic lymphoblasts died, compared to 25% of controls . The CD50 was 10 ng/mL for dysautonomic fibroblasts vs 450 for controls . These results may be exploited to create a biological assay to differentiate between FD and normal cells.

Genet Anal Tech Appl, 1994, 11(5-6), 171 - 80
Three new developments in P1 cloning . Increased cloning efficiency, improved clone recovery, and a new P1 mouse library; Sternberg N et al.; In this report, we describe three new P1 cloning developments . Two of these developments represent improvements in cloning efficiency and clone recovery, and the third is the production and partial characterization of a new P1 mouse library . To increase cloning efficiency, we have produced a new lysis-defective (delta lydAB) P1 lysogen (NS3690) for the production of the stage II head-tail-P1 packaging extract that is easier to use than the original stage II lysogen (NS3210), and that produces stage II extracts that are five- to eightfold more efficient than the original extracts . We believe the increased efficiency is due to the more concentrated packaging components in the NS3690 extract . Regarding P1 clone recovery, we demonstrate here that the less than optimal recovery of P1 plasmid DNA from P1 clones is due to the continuous presence of the P1 Cre recombinase in the host strain containing those clones (NS3529) . Consequently, a simple method of P1 plasmid clone transduction is described to transfer clone DNA from NS3529 (Cre+) to its Cre- parent (NS3516) . Yields of P1 plasmid DNA from NS3516 are as much as tenfold higher than from NS3529 . Finally, we document here the production of a new P1 mouse library that was generated using genomic DNA from embryonic stem cell line E14 (a 129/0la mouse) . The library contains 182,000 independent clones whose average insert size is 80 kb and, based on > 100 polymerase chain reaction screens, has an average unique sequence-hit size of 4.6.

Genet Anal Tech Appl, 1994, 11(5-6), 117 - 28
Direct sequencing of terminal regions of genomic P1 clones . A general strategy for the design of sequence-tagged site markers; Kimmerly WJ et al.; A method for the preparation of P1 DNA is presented, which allows the direct sequencing of ends of inserts in genomic P1 clones using the Applied Biosystems 373A DNA Sequencer and the Dye Terminator sequencing methodology . We surveyed several common methods of DNA preparation including alkaline lysis, Triton-lysozyme lysis, CsCl density-gradient purification, and a commercial column matrix DNA purification kit manufactured by Qiagen . We found that a modified alkaline lysis preparation of P1 DNA was most successful for generating P1 DNA that could be sequenced directly . We also noted that the host bacterial strain from which the P1 DNA was purified dramatically affected the quality of sequencing templates . The bacterial strains NS3145 and NS3529, in which the Drosophila melanogaster and human P1 genomic libraries are harbored, routinely yielded poor-quality sequencing templates . However, the bacterial strain DH10B routinely yielded P1 DNA that was sequenced successfully . A bacterial mating scheme is presented that exploits gamma delta transposition events to allow the transfer of P1 clones from the library host strain to DH10B . Using either an SP6 or a T7 primer, an average of 350 base pairs of DNA sequence was obtained with an uncalled base frequency of approximately 2% . About 4% of P1 end sequences generated corresponded to unique Drosophila loci present in the Genbank database . These single-pass DNA sequences were used to design sequence-tagged site markers for physical mapping studies in both humans and Drosophila.

Adv Biophys, 1994, 30, 1 - 35
Structure of RecA-DNA complex and mechanism of DNA strand exchange reaction in homologous recombination; Takahashi M et al.; The importance of filament formation of RecA for the DNA strand exchange reaction, both in vivo and in vitro, is established . RecA forms a very long and relatively stiff filament by binding around DNA with high cooperativity . The monomer units are assembled in the filament in a head-to-tail arrangement in a helical manner, similar to the organization of RecA molecules found in the crystal of pure RecA or including ADP . This filament of RecA, containing a DNA molecule in its interior, can bind another DNA molecule and yet a third one in the presence of cofactor (ATP or its analogs) . Each filament may have three DNA binding sites, each able to bind one DNA strand of either ss or ds DNA . According to linear dichroism and fluorescence spectroscopies, the DNA molecules in the RecA filament are well organized with a well defined but modified structure . This organization and modification of DNA by RecA probably has the purpose of facilitating Watson-Crick base-pair recognition and strand exchange reaction . RecA is thus actively involved in the reaction . The phosphoribose backbone of DNA follows the RecA helix and the DNA is stretched 50% and unwound . The nucleobases are destacked but still firmly oriented almost perpendicular to the axis for the first DNA and are immobile even in the case of ssDNA . Even in the second DNA the motion of DNA bases is very restricted although their orientation appears to be less perpendicular . All DNA strands in the complex show some sequence dependent interaction with each other . It could be a non-conventional base-base pairing, that could provide a mechanism of search for homologous DNA . In order to understand how RecA organizes DNA, a 3-D model building of the RecA-DNA complex is in progress undertaken based on the crystal structure of RecA and results obtained using chemical interference and protein engineering techniques . A characterization of structure of the second DNA and the mode of DNA-DNA interaction may further clarify the reaction mechanism of strand exchange.

Kurume Med J, 1994, 41(4), 165 - 9
Transformation assays of the cell mitotic and proliferation activities of purified oncogene products; Yuge K et al.; Using two direct introduction methods, DNA synthesis or cell proliferation activities of three purified proteins from E . coli, namely, human papillomavirus (HPV) E7 proteins of type 16, a mutant type 16 (24 C-G) (transformation defective) and type 6b, were measured in mouse fibroblast, C127 cells . By a microinjection method, the order of the cell mitotic indexes for the three E7 proteins as determined by 5-bromo-2'-deoxy-uridine (BrdU) staining was type 16, 6b and 16 (24 C-G) . By the osmotic shock method, the 3H-TdR incorporation and coloration by (3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetolazorium (MTS) for the three proteins correlated with the pRb binding and focus forming activities previously reported (Munger et al . 1991) . These results indicate that the simple osmotic shock method for direct protein introduction may be generally useful for transformation assays of oncoproteins.

Biol Cell, 1994, 81(3), 215 - 22
Expression of the beta-subunit isoforms of the Na,K-ATPase in rat embryo tissues, inner ear and choroid plexus; Gonzalez-Martinez LM et al.; We report evidence of the apical localization of the two Na,K-ATPase beta-subunit isoforms in cells of the inner ear and of the choroid plexus of the rat . To this end, we generated isoform-specific antisera against the human Na,K-ATPase beta 1 and beta 2 subunits . These polyclonal rabbit antisera were raised against truncated beta-isoform proteins that were made in E coli with pET expression vectors . Deglycosylation of the native antigen with N-endoglycosidase F shows four bands in the beta 1 isoform and five bands in the beta 2 isoform immunoblots . In E15 rat embryos, the beta 1 isoform was detected in brain, heart and kidney and the beta 2 isoform only in brain . While beta-subunit mRNA expression (Watts AG, Sanchez-Watts G, Emanuel JR, Levenson R (1991) Proc Natl Acad Sci USA 88, 7425-7429), immunoblotting and enzymatic activity have been determined (Zlokovic BV< mackic JB, Wang L, McComb JG, McDonough A (1993) J Biol Chem 268, 8019-8025), very little is known about the specific localization of each beta-isoform in the epithelia of choroid plexus and inner ear . Immunocytochemical preparations of 15-day-old whole rat embryos and adult rat brain showed an enhanced staining for the beta 1 and beta 2 isoforms in the apical membrane of the ampullary crests of the inner ear's semicircular ducts and in the cuboidal cells of the choroid plexus.

Biosens Bioelectron, 1994, 9(9-10), 719 - 23
Reconstitution of functional electron transfer between membrane biological elements in a two-dimensional lipidic structure at the electrode interface; Torchut E et al.; These studies develop a methodology to form supported phospholipid bilayers at an electrode/solution interface that models biological membrane systems . Two kinds of electrode were used, a planar gold electrode and a microporous aluminium oxide electrode on which octadecanethiol or octadecyltrichlorosilane was self-assembled . The supported lipidic structures were produced by transfer of a phospholipid monolayer by the Langmuir-Blodgett technique or by direct fusion of phospholipid vesicles . Ubiquinone was introduced into the lipidic structures during their formation; electrochemical measurements demonstrated the mobility of ubiquinone along the plane of the bilayer . A membrane enzyme, pyruvate oxidase from E . coli, was successfully incorporated into this artificial bilayer and was found to be able to exchange electrons with ubiquinone present in the bilayer.

Nat Struct Biol, 1994 Jan, 1(1), 30 - 5
Solution structure of apocytochrome b562; Feng Y et al.; The apoprotein is an important intermediate on the folding pathways of many haem proteins, yet a detailed structure of such an intermediate has remained elusive . Here we present the structure of apocytochrome b562 obtained by NMR spectroscopy . The apoprotein has a topology similar to the holoprotein . Nevertheless, significant differences in helix-helix packing between the two are evident . Much of the haem binding pocket in the apoprotein is preserved but exposed to solvent creating a large cavern . As apocytochrome b562 displays many of the physical characteristics ascribed to the molten globule state, these results help ellucidate the origin of several properties of the protein molten globule.

J Mol Neurosci, 1994 Fall, 5(3), 133 - 48
Rapid purification, site-directed mutagenesis, and initial characterization of recombinant RC3/neurogranin; Gerendasy DD et al.; RC3/Neurogranin is a postnatal-onset, forebrain-specific, thyroid hormone-regulated, protein kinase C (PKC) substrate that binds calmodulin (CaM) and accumulates in dendritic spines . We bacterially expressed and purified RC3 and, for comparison, GAP-43/neuromodulin to near homogeneity using relatively simple procedures . We then raised antisera against recombinant RC3 that does not crossreact with GAP-43 and is suitable for immunohistochemical analysis of brain slices . We also constructed over 30 RC3 sequence variants by PCR-mediated, site-directed mutagenesis, and purified four of these to near homogeneity . The elution profiles displayed by RC3 and sequence variants during purification on CaM-Sepharose columns suggest that two different affinity forms of the RC3.CaM complex coexist when Ca2+ is absent and that GAP-43.CaM interactions are far more sensitive to salt than those that occur between recombinant RC3 and CaM . Variant proteins in which serine 36 was changed failed to serve as a substrate for PKC, implicating this as the target residue.

Acta Physiol Pharmacol Bulg, 1994, 20(3-4), 95 - 102
Disorders of ventricular contractility and electrogenesis in the early stage of endotoxin shocked rabbits; Lolov R et al.; This is a report on ventricular contractility and electrogenesis disorders in rabbits, following intravenous injection of E . coli endotoxin at a dose of 2 mg.kg-1 . At the 30th min, the right ventricular contractility indices (dP/dtmax)/P and {(dP/dt)/P}max had lower values, whereas end diastolic pressure (EDP), right ventricular systolic pressure (RVSP) and P(dP/dtmax) showed higher values compared to the initial ones . Most of the left ventricular contractility indices tested showed significantly lower values at the 30th and 60th min of the registration . In the scalar orthogonal ECG leads, at the 5th min an increased Qz amplitude, and at the 60th min an increased Rz amplitude, a decreased Ry amplitude, and QRS complex widening and bradicardia, were registered . In the spatial magnitude curve an increased amplitude of the main vectors of ventricular depolarization was documented . The changes in electrogenesis are interpreted first and foremost by the presence of hemodynamic disorders . The inference is reached that both left and right ventricular dysfunction have been already formed during the initial stage of endotoxin shock.

Proc Int Conf Intell Syst Mol Biol, 1994, 2, 53 - 61
A generalized profile syntax for biomolecular sequence motifs and its function in automatic sequence interpretation; Bucher P et al.; A general syntax for expressing biomolecular sequence motifs is described, which will be used in future releases of the PROSITE data bank and in a similar collection of nucleic acid sequence motifs currently under development . The central part of the syntax is a regular structure which can be viewed as a generalization of the profiles introduced by Gribskov and coworkers . Accessory features implement specific motif search strategies and provide information helpful for the interpretation of predicted matches . Two contrasting examples, representing E . coli promoters and SH3 domains respectively, are shown to demonstrate the versatility of the syntax, and its compatibility with diverse motif search methods . It is argued, that a comprehensive machine-readable motif collection based on the new syntax, in conjunction with a standard search program, can serve as a general-purpose sequence interpretation and function prediction tool.

Proc Int Conf Intell Syst Mol Biol, 1994, 2, 188 - 94
Neural networks for determining protein specificity and multiple alignment of binding sites; Heumann JM et al.; We use a quantitative definition of specificity to develop a neural network for the identification of common protein binding sites in a collection of unaligned DNA fragments . We demonstrate the equivalence of the method to maximizing Information Content of the aligned sites when simple models of the binding energy and the genome are employed . The network method subsumes those simple models and is capable of working with more complicated ones . This is demonstrated using a Markov model of the E . coli genome and a sampling method to approximate the partition function . A variation of Gibbs' sampling aids in avoiding local minima.

DNA Res, 1994, 1(3), 123 - 7
The nature of the nucleotide at the 5' side of the tRNA Su9 anticodon affects UGA suppression in Escherichia coli; Goldman-Levi R et al.; In Escherichia coli a UGA codon can be efficiently suppressed by a suppressor tRNA(Trp) called Su9 . Here, we show that the level of UGA suppression is determined by the nature of the nucleotide at the 5' side of the anticodon of the suppressor (position 33) . UGA suppression occurs when a pyrimidine residue is located in position 33 of the tRNA, and suppression is more efficient with a U than with a C in this position . On the other hand, when a purine residue is located at this position UGA suppression is extremely low . These results show that in the case of tRNA Su9, the UGA codon context effect does not require base pairing between the nucleotide at the 3' side of the codon and the 5' side of the anticodon.

DNA Res, 1994, 1(2), 97 - 8
Nucleotide sequence of a putative sensory kinase gene from the cyanobacterium, Anacystis nidulans 6301; Fukuta M et al.; The nucleotide sequence of a 2,146 bp portion of the Anacystis nidulans (Synechococcus PCC6301) genome has been determined . This region contains an open reading frame (ORF) of 392 codons, whose predicted protein sequence shows partial homology to those of E . coli phoM and envZ . Hence ORF392 is suggested to be a sensory kinase gene in cyanobacteria.

Biochimie, 1994, 76(12), 1192 - 204
Effect of nucleoside modifications on the structure and thermal stability of Escherichia coli valine tRNA; Kintanar A et al.; Transfer RNA transcribed in vitro lacks the base modifications found in native tRNA . To understand the effect of base modifications on the structure of tRNA, the downfield region of the 1H NMR spectrum of in vitro transcribed E coli tRNAVal in aqueous phosphate buffer in the presence of excess Mg2+ was investigated . The resonances of all imino protons involved in hydrogen bonds in the helical stem regions and in tertiary interactions were assigned using two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) and one-dimensional difference nuclear Overhauser effect (NOE) methods . In addition, some aromatic C2 and C8 proton resonances as well as one amino proton resonance were assigned . The chemical shifts of the assigned resonances of unmodified E coli tRNAVal were compared with those of the native tRNA molecule under similar solution conditions . The similarity of the NMR data for unmodified and modified tRNA indicates that the in vitro transcribed tRNA has nearly the same solution structure as the native molecule in the presence of excess Mg2+ . The only significant differences were the chemical shifts of resonances corresponding to protons in (or interacting with) bases, indicating the possibility of local structural perturbations . The thermal stability of E coli modified and unmodified tRNAVal in the presence of Mg2+ was also investigated by analyzing the temperature dependence of the imino proton spectra . Several tertiary interactions involving modified nucleosides in native E coli tRNAVal are less stable in the absence of base modifications.

Biochimie, 1994, 76(12), 1123 - 8
Methylation of the conserved A1518-A1519 in Escherichia coli 16S ribosomal RNA by the ksgA methyltransferase is influenced by methylations around the similarly conserved U1512.G1523 base pair in the 3' terminal hairpin; Formenoy LJ et al.; An in vitro system developed for the site-specific mutagenesis of 16S rRNA of Escherichia coli ribosomes was used to make five mutations around the highly conserved U1512.G1523 base pair in the 3' terminal hairpin . Each of the mutant RNAs was reconstituted with a complete mixture of 30S proteins to yield 30S ribosomal particles, which were tested for the ability of the ksgA methylase to form m6(2)A1518 and m6(2)A1519 . Dimethylation of A1518 and A1519 in the hairpin loop was inhibited 20-80% by the mutations . The results indicate that G1523 and C1524 in the stem are important determinants for the dimethylation of A1518 and A1519 in the loop . Either the enzyme recognition region extends that far or the effect of mutations in the stem are propagated in some manner to the loop . The conserved U.G base pair does not of itself appear to play a major role in ksgA methylase recognition.

Mol Biol Rep, 1994, 20(2), 85 - 95
Carbohydrate specificity of IgM autoantibodies to CD45 in systemic lupus erythematosus; Fernsten PD et al.; Patients with SLE develop IgM autoantibodies to different isoforms of CD45, the major surface membrane protein tyrosine phosphatase on lymphocytes and other nucleated hemopoietic cells . Because such autoantibodies could have a potential role in the development of immune dysfunction in this disorder, we performed a series of experiments to characterize their antigenic specificity further . Blots of recombinant E . coli fusion proteins encoded by exons 3-7 of the p220 and p180 isoforms were uniformly non-reactive with SLE IgM, suggesting that anti-CD45 autoantibodies in SLE are directed against conformational and/or carbohydrate epitopes, rather than linear polypeptide epitopes . This issue was examined further using chemically and enzymatically modified CD45 purified from T cells by lectin affinity chromatography as substrates . Treatment of CD45 with 25 mM sodium-m-periodate, sufficient to abrogate binding to various lectins, abolished the reactivity with SLE anti-CD45 autoantibodies . On the other hand, digestion of CD45 with neuraminidase enhanced the binding of anti-CD45 autoantibodies from some of the SLE sera . This result probably reflects decreased steric hindrance or charge repulsion because the binding of mouse monoclonal antibodies directed against linear polypeptide epitopes of CD45 was similarly enhanced . Digestion of CD45 with N-glycosidase F had no effect on autoantibody staining . Taken together, these data suggest that IgM anti-CD45 autoantibodies in SLE recognize non-sialylated carbohydrate determinants in the highly O-glycosylated polymorphic domains of CD45.

Biol Cell, 1994, 81(2), 95 - 119
Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium; Callen AM et al.; Ciliates are very good models for studying post-translationally generated tubulin heterogeneity because they exhibit highly differentiated microtubular networks in combination with reduced genetic diversity . We have approached the analysis of tubulin heterogeneity in Paramecium through extensive isolation and characterization of monoclonal antibodies using various antigens and several immunization protocols . Eight monoclonal antibodies and 10 hybridoma supernatants were characterized by: i) immunoblotting on ciliate and pig brain tubulins as well as on peptide maps of Paramecium axonemal tubulin; ii) immunoblotting on ciliate tubulin fusion peptides generated in E coli, a procedure which allows in principle to discriminate antibodies that are directed against tubulin sequence (reactive on fusion peptides) from those directed against a post-translational epitope (non-reactive); and iii) immunofluorescence on Paramecium, 3T3 and PtK2 cells . Twelve antibodies labeled all microtubules in Paramecium cells and were found to be directed against tubulin primary sequences (nine of them being located in the alpha N-terminal domain, one in the beta C-terminal one, and two in alpha and beta central stretches) . The remaining ones decorated only a specific subset of microtubules within the cell and were presumably directed against post-translational modifications . Among these, three antibodies are directed against an N-terminal acetylated epitope of alpha-tubulin whereas the epitopes of three other ones (TAP 952 degrees, AXO 58 and AXO 49 degrees) apparently correspond to still unidentified post-translational modifications, located in the C-terminal domain of both alpha- and beta-tubulins . The AXO 49 degrees specificity is similar to that of a previously described polyclonal serum raised against Paramecium axonemal tubulin {2} . The results are discussed in terms of identification and accessibility of the epitopes and immunogenicity of ciliate tubulin with reference to mammalian and ciliate tubulin sequences.

Acta Microbiol Pol, 1994, 43(2), 233 - 9
Methods of introduction of foreign DNA into mycobacteria; Dziadek J et al.; Two methods: triparental conjugation and electrotransformation were used for introduction of plasmid DNA into mycobacterial cells . The introduction of shuttle plasmid pMY10 into M . fortuitum mutant caused the activation of its chromosomal cryptic KmR gene . The used of integration vector pUS 903 allowed to obtain a collection of mutants interesting for studies of genetic determination of steroid biotransformation and drug-resistance in mycobacteria.

Acta Microbiol Pol, 1994, 43(2), 229 - 31
Improvement of the strain for the rapid identification of genes encoding restriction and modification enzymes; Piekarowicz A et al.; The E . coli AP1-200-9 strain for rapid identification of genes encoding restriction and modification enzymes carries a temperature sensitive lacZ gene fused to the damage-inducible dinD locus . A derivative of this strain was constructed that has a wild-type form of this locus which allows for a more efficient identification of recombinant plasmids encoding restriction and modifications enzymes.

Vestn Ross Akad Med Nauk, 1994, (7), 49 - 53
{Effects of external biophysical factors on reproducibility of Escherichia coli characteristics}; Kolpakova SD et al.; The paper gives the results of examining the influence of external biophysical factors (inoculate's age, inoculative dose, cultivating temperature) on the reproducibility of changes in the formation of intercellular biophysical processes in the bacterial population . The conditions under which there is a high reproducibility (the error being 5-7%) of development dynamic parameters for E . coli are shown to be the age of an inoculate, 24 hours, the cultivating temperature, 24 degrees C, the inoculative dose is the dose corresponds to the baseline optical density of working liquor in the range of 0.025-0.050.

Ann Trop Paediatr, 1994, 14(2), 105 - 10
Enteropathogenic and enteroadherent-aggregative Escherichia coli in children with persistent diarrhoea and malnutrition; Sullivan PB et al.; Our investigation of 61 children with persistent diarrhoea and malnutrition (PDM) aimed to characterize in them the range of enteropathogenic E . coli . Age- and sex-matched control groups consisted of 42 healthy children and 16 children with marasmus but without diarrhoea . E . coli isolates from stool cultures were serotyped, examined for Vero cells cytotoxicity, tested for enterotoxin production (LT, ST, VTI, and VTII) . Synthetic oligonucleotide probes were used to test for enteroinvasivity, EPEC adherence factor, and for EAggEC . Classical E . coli serotypes commonly associated with diarrhoea (O18, O26, O119, O126) were isolated from six of 60 (10%) children with PDM . Serotype O126 was isolated from two of 42 (4.8%) healthy children and serotype O119 from one of 16 (6%) marasmic controls . Testing for Vero toxin production was negative in all isolates . Classical ETEC were confirmed in four of 60 (ST 2; LT 2) cases of PDM; no ETEC were recovered from 58 control patients . EAggEC were identified in five children with PDM and in five healthy controls without diarrhoea or malnutrition . This controlled study has shown that, in The Gambia, E . coli carrying known virulence factors are prevalent but, with the exception of enterotoxigenic E . coli, the various forms of pathogenic E . coli do not seem to be important pathogens in children with persistent diarrhoea.

Growth Factors, 1994, 10(2), 89 - 98
Mitogenic and in vitro angiogenic activity of human recombinant heparin affin regulatory peptide; Laaroubi K et al.; We have previously described the purification of a heparin binding growth factor from adult bovine brain named heparin affin regulatory peptide (HARP), which was identical to an uterus derived growth factor named pleiotrophin and to a developmentally regulated neurite promoting factor named heparin-binding growth associated molecule . However, for yet unclear reasons, the mitogenic activity of this purified polypeptide following isolation from animal tissue extracts is a subject of controversy, due to conflicting and irreproducible data when produced by recombinant DNA technologies in E . coli or insect cells . The purified protein was inactive in mitogenic assays but the natural molecule was active in assay of neurite outgrowth . In order to clarify these conflicting results and to obtain a recombinant protein free from other contaminating heparin-binding growth factors, we have cloned human cDNA encoding human HARP, engineered its expression in NIH 3T3 cells and characterised the resulting recombinant polypeptide . Purified recombinant HARP displayed mitogenic activity for capillary endothelial cells with half-maximal stimulation at approximately 1 ng/ml (55 pM) and induced angiogenesis in an in vitro model . Interestingly, while the NH2 terminal sequence of tissue purified HARP was NH2-GKKEKPEKK, the NH2 terminal sequence of the biologically active recombinant protein was NH2-AEAGKKEKPEKK, corresponding to a three amino acid extended form.

Methods Enzymol, 1994, 235, 657 - 67
Assays of hemolytic toxins; Rowe GE et al.; The ability to produce a cytolytic toxin contributes to the success of many organisms in a particular niche by such diverse means as lysis of a phagolysosomal membrane of the macrophage by hemolysin from the intracellular parasite Trypanosoma cruzi, disruption of leukocyte activity by the Escherichia coli hemolysin, and destruction of invading bacteria by hemolysin from the annelid Glycera dibranchiata . The relative contribution of erythrocyte lysis to survival of the cytolysin producer is still under investigation . Nevertheless, the hemolytic phenotype is both a powerful tool for identifying novel cytolysins and a convenient marker for studying cytolytic activity in established toxins.

J Clin Lab Anal, 1994, 8(3), 163 - 6
Mapping of antigenic epitopes within the recombinant human preproinsulin related to insulin-dependent diabetes mellitus; Walter M et al.; The human insulin domains, signal peptide, B-chain, C-peptide, and A-chain, were highly expressed in Escherichia coli as recombinant proteins N-terminally fused to glutathione-S-transferase and a histidine-hexapeptide . The recombinant proteins were purified from insoluble cell fraction by affinity chromatography using metal chelating matrix, which was charged with Ni+2 ions . ELISA screening for autoantibodies directed to preproinsulin were performed with sera from patients with recently diagnosed insulin-dependent diabetes mellitus in order to localize the antigenic epitopes within the human preproinsulin . Of the patients, 14% had developed autoantibodies that recognized either the recombinant C-peptide or the signal peptide . No reaction was observed with the A-chain or B-chain.

Biochimie, 1994, 76(2), 165 - 70
Production of anti-peptide antibodies directed against the first and the second extracellular loop of the human serotonin 5-HT1A receptor; Verdot L et al.; The second extracellular loop of the beta-adrenergic and muscarinic acetylcholine receptors was shown to be an autoimmune target for antibodies in several autoimmune diseases . These autoantibodies and the antibodies induced against synthetic peptides corresponding to this loop have pharmacological and physiological properties upon receptor recognition which could explain their pathophysiological role . We here describe the immune properties of the first and second extracellular loops of another G protein-coupled receptor, the serotonin 5-HT1A receptor . The injection in rabbits of the free peptides Y16L and G21G corresponding to the first and second extracellular loops respectively induced anti-peptide antibodies with high titer, demonstrating the presence of a T-cell epitope on each peptide . Interestingly, in contrast to the G21G peptide that induced only anti-G21G antibodies (Ab-2 antibodies), the Y16L peptide induced two populations of antibodies . One recognized only the Y16L peptide (Ab-1 antibodies), the other recognized both peptides (Ab-12 antibodies) . This reflects the presence on the two peptides of at least two B-cell epitopes . The fact that the G21G peptide induces only one antibody population might indicate that it possesses one immunodominant epitope involved in the Ab-2 antibody production and one cryptic epitope involved in the cross-reaction with the anti-Y16L antibodies . But only Ab-2 antibodies were able to recognize specifically the human protein receptor expressed in E coli in immunoblot.

Biochimie, 1994, 76(2), 159 - 64
Epitope analysis of T- and B-cell response against the human beta 1-adrenoceptor; Tate K et al.; Several reports have recently raised the possible significance of the presence of autoantibodies against the beta 1-adrenoceptor in patients with idiopathic dilated cardiomyopathy . An investigation was thus initiated to study the immune response against this receptor at the T-cell and the B-cell level . Using membranes of E coli transfected with the human beta 1-adrenoceptor gene as immunogen, T-helper cells of the immunized mice were stimulated with synthetic peptides derived from the receptor and predicted to be immunogenic to assess the T-cell immunodominant regions of the receptor . Three peptides derived from the second transmembrane region, from the second extracellular loop and from the C-terminal domain were shown to be stimulatory . Synthetic peptides, derived from two domains of the receptor which could be potential targets for autoantibodies, yielded an antibody response after immunization with the free peptides . The peptide derived from the N-terminal region yielded antibodies which recognized the receptor in immunoblot and by immunoprecipitation but they had no functional effect on the receptor . The peptide derived from the second extracellular loop yielded antibodies which recognized the receptor in immunoblot and by immunoprecipitation of the free receptor and which had a pharmacological effect on the receptor . The second extracellular loop thus contains T- and B-cell epitopes which could be involved in the autoimmune process.

Arch Virol Suppl, 1994, 9, 543 - 58
Assembly of tobacco mosaic virus and TMV-like pseudovirus particles in Escherichia coli; Hwang DJ et al.; High-level expression of plant viral proteins, including coat protein (CP), is possible in Escherichia coli . Native tobacco mosaic virus (TMV) CP expressed in E . coli remains soluble but has a non-acetylated N-terminal Ser residue and following extraction, is unable to package TMV RNA in vitro under standard assembly conditions . Changing the Ser to Ala or Pro by PCR-mutagenesis did not confer assembly competence in vitro, despite these being non-acetylated N-termini present in two natural strains of TMV . All TMV CPs made in E . coli formed stacked cylindrical aggregates in vitro at pH 5.0 and failed to be immunogold-labelled using a mouse monoclonal antibody specific for helically assembled TMV CP . TMV self-assembly has been studied extensively in vitro, and an origin of assembly sequence (OAS) mapped internally on the 6.4 kb ssRNA genome . Pseudovirus particles can be assembled mono- or bi-directionally in vitro using virus-derived CP and chimeric ssRNAs containing the cognate TMV OAS, but otherwise of unlimited length and sequence . Studies on plant virus assembly in vivo would be facilitated by a model system amenable to site-directed mutagenesis and rapid recovery of progeny particles . When chimeric transcripts containing the TMV OAS were co-expressed with TMV CP in vivo for 2-18 h, helical TMV-like ribonucleoprotein particles of the predicted length were formed in high yield (up to 7.4 micrograms/mg total bacterial protein) . In addition to providing a rapid, inexpensive and convenient system to produce, protect and recover chimeric gene transcripts of any length or sequence, this E . coli system also offers a rapid approach for studying the molecular requirements for plant virus "self-assembly" in vivo . Transcription of a full-length cDNA clone of TMV RNA also resulted in high levels of CP expression and assembly of sufficient intact genomic RNA to initiate virus infection of susceptible tobacco plants.

Biochimie, 1994, 76(1), 83 - 7
Characterization and expression of a gene encoding serine tRNA5 from Escherichia coli; Cummings HS et al.; The genes for translational components frequently are located together on the Escherichia coli genome . We have reported previously that the gene for a serine tRNA lies directly downstream from infA, the gene encoding initiation factor IF1 . Here we characterize this tRNA gene, named serW . The serW gene expresses a minor form of serine tRNA(GGA) which recognizes the most frequently used serine codons, UCC and UCU . Two promoters were identified by S1 nuclease mapping: P1, which lies about 72 bp upstream from the structural gene; and P2, which lies about 35 bp upstream . Expression from P1 and P2 is comparable under conditions of rapid growth . The P2 promoter is followed by a GC-rich element characteristic of promoters regulated by ppGpp . A putative hairpin structure followed by a stretch of U residues about 25 nucleotides following the mature tRNA sequence resembles a rho-independent termination signal . The upstream gene, infA, is followed by a transcriptional terminator, but S1 mapping shows considerable readthrough . This serW expression appears to rely both on its own promoters and on promoters further upstream . The downstream gene, encoding an unidentified protein of about 100 kDa, is expressed in the opposite orientation and also is followed by a termination signal . Therefore serW is expressed both as a monocistronic gene and in combination with infA.

Biochimie, 1994, 76(1), 51 - 7
Effect of the nucleotide-37 on the interaction of tRNA(Phe) with the P site of Escherichia coli ribosomes; Katunin V et al.; The method of anticodon loop replacement has been used to make derivatives of yeast tRNA(Phe) with the substitution at position 37 (tRNA(Phe)GAAA) and at the anticodon(tRNA(Phe)GCAG) . A quantitative study of the interaction of various types of deacylated yeast tRNA(Phe) (tRNA(Phe)+Y, tRNA(Phe)GAAA, tRNA(Phe)-Y) with the P site of the {70S ribosome*poly(U)}-complex was carried out at different Mg2+ concentrations and temperatures . The presence and nature of the nucleotide situated at the 3'-end of the anticodon are essential for such interaction in E coli ribosomes . Replacement of the Y base with the unmodified adenosine decreases the interaction enthalpy from 39 kcal/mol to 24 kcal/mol, whereas its removal reduces the interaction enthalpy to 16 kcal/mol . Replacement of the second anticodon nucleotide, adenosine, with cytosine further reduces the enthalpy to 6 kcal/mol, which is typical of tRNA-P site interaction in the absence of poly(U) . In the absence of poly(U) the affinity of tRNA(PheY) for the P site of the 70S ribosome is five times lower than the affinity of tRNA(Phe+Y) or tRNA(Phe)GCAG . Thus, in the ribosome the modified nucleotide stabilizes the codon-anticodon interaction through its stacking interaction with the codon-anticodon base stack . In addition, this decreases the free energy of binding as a result of the interaction of the modified nucleotide itself with the hydrophobic center of the P site.

Biol Chem Hoppe Seyler, 1994 Jan, 375(1), 11 - 20
The Escherichia coli ribosomal RNA leader: a structural and functional investigation; Pardon B et al.; The structure of the E . coli ribosomal RNA leader was analyzed by treatment with single and double strand specific ribonucleases and by chemical modification . The experimentally derived data together with secondary structure calculations according to minimum free energy was used to construct a secondary structure model . The binding of purified Nus proteins to the ribosomal leader RNA was further tested . Contrary to the recently reported interactions of NusB and NusE with a nut RNA sequence we obtained evidence that the presence of NusA and NusE resulted in protection against hydroxyl radical reaction of the leader nut elements boxA, boxB and boxC . The possible significance of this interaction is discussed . In the second part of the study we analyzed effects of leader mutations, which are known to affect cell growth, on the activity of ribosomes in vivo . A system was used able to distinguish the proportion of ribosomes assembled from rRNA of chromosomal origin (wild type) and plasmid origin (mutant) . It turned out that the amount of 16S RNA transcribed from genes with point mutations in the leader region decreased if ribosomal pools with different translational activities were compared . High amounts of transcripts from mutant operons were present in the free ribosomal RNA and the free 30S fractions . Significantly less 16S RNA transcripts from the mutated genes were detected in the functionally active and homogeneous 70S tight couple preparations, and even less in the polysome fraction involved in active translation . The results allow a better understanding of the function of rRNA leader sequences in structure formation and correct ribosome biogenesis.

Eur Biophys J, 1994, 23(1), 29 - 38
Statistics of RNA melting kinetics; Tacker M et al.; We present and study the behavior of a simple kinetic model for the melting of RNA secondary structures, given that those structures are known . The model is then used as a map that assigns structure dependent overall rate constants of melting (or refolding) to a sequence . This induces a "landscape" of reaction rates, or activation energies, over the space of sequences with fixed length . We study the distribution and the correlation structure of these activation energies.

Microbiology, 1994 Jan, 140 ( Pt 1), 49 - 57
Escherichia coli K12 regains its O antigen; Liu D et al.; Extant Escherichia coli K12 strains are phenotypically rough, their lipopolysaccharide having a complete core structure, but no O antigen . We used DNA hybridization and DNA sequencing to show that the rough phenotype of this strain is due to the presence of one of two independent mutations in the rfb gene cluster . The rfb-50 mutation, consisting of an IS5 insertion at the downstream end of rfb, is present in strain EMG2, which is representative of most K12 derivatives . The rfb-51 mutation is a deletion at the upstream end of rfb, and was found in strain WG1 . A gene cloned from strain WG1 could complement the rfb-50 mutation in strain EMG2, and the complemented strain produced O antigen which was typed as O16 with cross reaction to O17.

Microbiology, 1994 Jan, 140 ( Pt 1), 123 - 32
Nucleotide sequence and secondary structures of precursor 16S rRNA of slow-growing mycobacteria; Ji YE et al.; Slow-growing mycobacteria have a single ribosomal RNA (rrn) operon, with the genes for 16S, 23S and 5s rRNA being present in that order . The transcription start site of the rrn operon of Mycobacterium tuberculosis was identified in Escherichia coli . PCR methodology was used to amplify parts of the rrn operon, namely the leader region and the spacer-1 region separating the 16S rRNA and 23S rRNA genes of Mycobacterium avium, Mycobacterium paratuberculosis, Mycobacterium intracellulare, 'Mycobacterium lufu', Mycobacterium simiae and Mycobacterium marinum . The amplified DNA was sequenced . The sequence data, together with those obtained previously for Mycobacterium leprae and M . tuberculosis, were used to identify putative antitermination signals and RNase III processing sites within the leader region . Notable features include a highly conserved Box B element and a sequence of 31 nucleotides which is common to all eight slow-growers which were scrutinized . A secondary structure for mycobacterial precursor-16S rRNA was devised, based on sequence homologies and homologous nucleotide substitutions . The 18 nucleotides at the 5'-end of spacer-1 have the capacity of binding sequences close to the 5'- and 3'-ends of mature 16S rRNA, suggesting that secondary structure is important to the maturation process . All the slow-growers, including M . leprae, conform to the same scheme of secondary structure . The scheme proposed for M . tuberculosis is a variant of the main theme . The leader and spacer sequences may prove a useful supplement to 16S rRNA sequences in establishing phylogenetic relationships between very closely related species . 'M . lufu' appears to be a close relative of M . intracellulare.

Mol Biol (Mosk), 1994 Jan-Feb, 28(1), 113 - 26
{Modified nucleoside-5'-triphosphates with an additional conjugated through the 2'-3'-fused ring as DNA polymerase substrates}; Semizarov DG et al.; 5'-Triphosphates of 1-(2',3'-epithio-2',3'-dideoxy-beta-D-lyxofuranosyl) thymine, 1-(2',3'-epithio-2',3'-dideoxy-beta-D-ribofuranosyl) thymine and 2',3'-lyxoanhydrothymidine have been shown to be terminator substrates of human immunodeficiency virus and avian myeloblastosis virus reverse transcriptases as well as DNA polymerase I from E . coli . At the same time they do not terminate DNA synthesis catalysed by DNA polymerase epsilon from human placenta . The KM values of ltTTP, rtTTP and laTTP incorporation into DNA chain agree closely with each other, being 1.5-2.5 times higher than KM for dTTP . Furthermore, Vmax values for modified substrates are only 2-3 times less than Vmax for dTTP . The evidence favours the hypothesis of a great affinity of modified nucleosides with flattened ribose ring of glycone for DNA polymerases active sites.

Alcohol, 1994 Jan-Feb, 11(1), 53 - 60
Escherichia coli-induced inhibition of endothelium-dependent relaxation and gene expression and release of nitric oxide is attenuated by chronic alcohol ingestion; Greenberg SS et al.; We examined the effect of chronic administration of ETOH on Escherichia coli-mediated suppression of relaxation and nitric oxide (NO) production by the rat thoracic aorta (RTA) and gene expression for constitutive NO synthase (cNOS) by the adrenal gland . Chronic alcoholic rats ("alcoholic") were fed a diet containing ETOH as 36% of the caloric intake for 8-10 weeks . Nonalcoholic control rats ("control") were fed an isocaloric equivalent diet containing 36% dextrin . Alcoholic rats were given an injection of approximately approximately 10(10) live E . coli through a dorsal SC catheter 24 and 19 h before experimentation ("alcoholic-septic"), and control rats were treated in an identical manner ("septic") . The next day the rats were anesthetized with ketamine-xylazine (0.1 ml/100 g rat) and rings of RTA were mounted in muscle chambers for isometric recording of force development . Rings of RTA were precontracted with an EC50 concentration of phenylephrine, and relaxation to acetylcholine (ACh), A23187, and nitroglycerin were obtained . A23187- and ACh-induced relaxation was attenuated in RTA obtained from septic rats, whereas the relaxation to nitroglycerin was slightly enhanced . Chronic administration of ETOH attenuated the effects of E . coli on endothelium-dependent relaxation in alcoholic-septic rats . NO was measured with ozone chemiluminescence . Basal and stimulated NO production was attenuated in RTA obtained from septic rats and unaffected in RTA obtained from alcoholic or alcoholic-septic rats . cNOS was unmeasurable in adrenals from septic rats . ETOH increased mRNA for cNOS, an effect amplified in alcoholic-septic rats . Thus, E . coli inhibits endothelium-dependent relaxation and NO production, and ETOH attenuates these effects of E . coli on the endothelium-NO system, possibly by upregulating gene expression for cNOS.

Eur Surg Res, 1994, 26(1), 10 - 8
Is endotoxin-induced hypotension related to nitric oxide formation?
Preiser JC, Zhang H, Wachel D, Boeynaems JM, Buurman W, Vincent JL.
Nitric oxide (NO), an endothelium-derived relaxing factor (EDRF), is released by different types of cells under the influence of endotoxin and various cytokines: a causative role of endothelium-derived NO in the endotoxin-induced hypotension has thus been suggested . To test the hypothesis that NO may be involved in the acute hypotension following endotoxin challenge, we administered a competitive inhibitor of NO synthase, L-N-monomethylarginine (L-NMMA) to anesthetized dogs in the presence and absence of endotoxin . Dogs were randomly allocated to three groups . Group 1 (n = 3) was given Escherichia coli endotoxin (3 mg/kg, i.v.), group 2 (n = 3) was given L-NMMA (5 mg/kg, i.v . bolus) 15 min after endotoxin and group 3 (n = 3) was given L-NMMA only . One additional dog was given L-arginine (100 mg/kg, i.v . bolus) after L-NMMA and endotoxin to reverse the inhibition of NO synthase . In each animal, saline was infused intravenously throughout the experiment to restore and maintain pulmonary artery occluded pressure at baseline level . After L-NMMA, the increases in mean arterial pressure were similar in group 2 (from 55 +/- 18 to 75 +/- 15 mm Hg, p < 0.01) and in group 3 (from 107 +/- 27 to 128 +/- 24 mm Hg, p < 0.01) . Systemic vascular resistance increased from 2,994 +/- 72 to 3,658 +/- 673 dyn.s.cm-5 (p < 0.01) in group 3 . Group 1 had lower plasma lactate levels than group 2 (3.5 +/- 2.3 +/- vs . 2.0 +/- 1.6 mEq/l, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Int Arch Allergy Immunol, 1994, 103(4), 396 - 9
Immunization of guinea pigs with Treponema pallidum recombinant antigens reveals the presence of novel epitopes; Wicher K et al.; The DNA technology employed in the construction and purification of recombinant antigens has the potential of creating epitopes with specificities other than those of native antigens . Such a phenomenon has been observed when guinea pigs were immunized with Treponema pallidum recombinant antigens, TmpA and TmpB, expressed in Escherichia coli K12 . Adsorption of the immune sera with E . coli K12 and T . pallidum revealed the presence of antibodies directed against epitopes not present or exposed in the native antigens of the organisms from which the DNA has been cloned.

FEMS Microbiol Lett, 1994 Jan 1, 115(1), 1 - 6
The alpha subunit of RNA polymerase specifically inhibits expression of the porin genes ompF and ompC in vivo and in vitro in Escherichia coli; Bowrin V et al.; Overproduction of the alpha subunit of RNA polymerase in Escherichia coli resulted in inhibition of transcription of two osmoregulated porin genes, ompF and ompC, but not of constitutively expressed housekeeping genes . Overproduction of the sigma subunit did not have any inhibitory effects . The specific inhibitory effect of the alpha subunit was also found to depend upon the OmpR protein, the transcriptional activator for ompF and ompC . These results are in general agreement with other biochemical and genetic evidence suggesting that the alpha subunit is the subunit of RNA polymerase that directly interacts with certain transcriptional activators to initiate transcription.

Environ Mol Mutagen, 1994, 23(1), 32 - 6
Transcription by T7 RNA polymerase of DNA containing abasic sites; Sanchez G et al.; The effects of abasic (AP) sites on RNA synthesis were studied in vitro, using T7 RNA polymerase and a plasmid template containing a T7 promoter . The presence of increasing numbers of AP sites caused a progressive decline in RNA synthesis . The average RNA chain length, calculated from the ratio of initiation to chain elongation, decreased with increasing numbers of AP sites, revealing that complete blocks must occur during synthesis . The probability that RNA polymerase would be blocked at an AP site in the DNA template strand was estimated to be 0.3 in our experimental conditions . These results demonstrate that RNA synthesis by T7 RNA polymerase is inhibited by AP sites and that readthrough of the lesion occurs more frequently than premature chain termination . Chemical reduction of AP sites in the template did not change the block/bypass pattern.

Hypertension, 1994 Jan, 23(1 Suppl), I45 - 8
Expression of nitric oxide synthase by cytokines in vascular smooth muscle cells; Koide M et al.; In cultured vascular smooth muscle cells, the baseline mRNA and protein levels of an inducible type of nitric oxide synthase were barely detectable . Interferon gamma, tumor necrosis factor-alpha, and interleukin-1 beta each markedly increased mRNA and protein levels of this enzyme in parallel with the production of nitrite, a stable oxidative metabolite of nitric oxide . Actinomycin D abolished the cytokine-induced increases in mRNA levels and nitrite production . Cycloheximide, which abolished the cytokine-induced increase in nitrite production, had no effect on the interferon-gamma-induced increase in mRNA levels but partially inhibited that induced by interleukin-1 beta and markedly inhibited that induced by tumor necrosis factor-alpha . Transforming growth factor-beta 1, which inhibited the interferon gamma-, interleukin-1 beta-, and tumor necrosis factor-alpha-induced nitrite production, did not affect the increases in mRNA levels caused by these cytokines . Transforming growth factor-beta 1, however, significantly inhibited the increase in protein levels caused by these cytokines . These findings suggest that interferon gamma directly induces the expression of the inducible nitric oxide synthase gene, whereas tumor necrosis factor-alpha and interleukin-1 beta induce it, at least in part, via the induction of intermediary protein(s), and that transforming growth factor-beta 1 inhibits cytokine-induced nitric oxide production by blocking the posttranscriptional synthesis of inducible nitric oxide synthase.

Hypertension, 1994 Jan, 23(1 Suppl), I126 - 30
Angiotensinogen: an acute-phase protein?
Soden M, Klett C, Hasmann T, Hackenthal E.
Angiotensinogen has been assumed to be an acute-phase protein, because some forms of acute inflammation, eg, the injection of lipopolysaccharide or cellite or partial hepatectomy, increased the hepatic synthesis of angiotensinogen . In addition, the well-characterized nephrectomy-induced stimulation of angiotensinogen was thought to represent an acute-phase reaction . To evaluate this hypothesis, we examined changes in angiotensinogen secretion by the isolated perfused rat liver after the systemic administration of turpentine or lipopolysaccharide as well as in response to nephrectomy or sham nephrectomy . Comparison was made with the secretion of two typical acute-phase proteins, alpha 1-acid glycoprotein and alpha 2-macroglobulin, and with the secretion of the negative acute-phase protein albumin . All forms of experimental surgery stimulated the secretion of both control acute-phase proteins several-fold . In contrast, the response of angiotensinogen was not uniform; lipopolysaccharide and bilateral nephrectomy stimulated secretion twofold to threefold, sham nephrectomy had no effect, and turpentine decreased the secretion to 30% of the control level . A similar inhomogeneity was found in an additional experiment performed to analyze the direct effects of interleukin-1 or interleukin-6 on the secretion of angiotensinogen by freshly isolated hepatocytes . Interleukin-6 increased but interleukin-1 decreased the mRNA and secretion of angiotensinogen, whereas both cytokines increased the secretion of both acute-phase proteins . Because of this nonuniform behavior of angiotensinogen, it is premature to classify angiotensinogen as an acute-phase protein until a specific function for angiotensinogen during acute inflammation is known.

J Exp Med, 1994 Jan 1, 179(1), 71 - 80
Interleukin 1 activates soluble guanylate cyclase in human vascular smooth muscle cells through a novel nitric oxide-independent pathway; Beasley D et al.; Recent demonstration of cytokine-inducible production of nitric oxide (NO) in vascular smooth muscle cells (VSMC) from rat aorta has implicated VSMC-derived NO as a key mediator of hypotension in septic shock . Our studies to determine whether an inducible NO pathway exists in human VSMC have revealed a novel cytokine-inducible, NO-independent pathway of guanylate cyclase activation in VSMC from human saphenous vein (HSVSMC) . Interleukin 1 (IL-1), tumor necrosis factor (TNF), interferon gamma (IFN-gamma) and Escherichia coli lipopolysaccharide (LPS) increased cGMP at 24 h, whereas IL-2 and IL-6 were ineffective . The effect of IL-1 on cyclic guanosine 3',5'-monophosphate (cGMP) was delayed, occurring after 6 h of exposure, and was maximal after 10 h . Methylene blue and LY83583 reversed the IL-1-induced increase in cGMP, suggesting that it was mediated by activation of soluble guanylate cyclase . However, IL-1-induced cGMP in HSVSMC was not inhibited by extracellular hemoglobin . Also, the effect of IL-1 on cGMP was not reversed by nitro- or methyl-substituted L-arginine analogs, aminoguanidine, or diphenyleneiodonium, all of which inhibit IL-1-induced NO synthase in rat aortic VSMC (RAVSMC) . IL-1-induced cGMP in HSVSMC was also independent of tetrahydrobiopterin and extracellular L-arginine, as it was not affected by 2,4-diamino-6-hydroxyprytimidine, an inhibitor of tetrahydrobiopterin biosynthesis, and was similar in L-arginine-free and L-arginine-containing media . Analysis of NO synthase mRNA with the use of polymerase chain reaction indicates that levels of mRNA for inducible NO synthase are several orders of magnitude lower in IL-1-treated human HSVSMC than in IL-1-treated RAVSMC . IL-1-induced cGMP was also NO independent in human umbilical artery VSMC, and NO dependent in rat vena cava VSMC . Together these results indicate that IL-1 activates a novel NO-independent pathway of soluble guanylate cyclase activation in human VSMC.

Virology, 1994 Jan, 198(1), 92 - 9
Mechanism of interferon action motif I of the interferon-induced, RNA-dependent protein kinase (PKR) is sufficient to mediate RNA-binding activity; McCormack SJ et al.; The interferon-induced P1/eIF-2 alpha protein kinase cDNA, designated PKR, was expressed both in Escherichia coli and in transfected monkey COS cells . TrpE-PKR fusion proteins and PKR nonfusion proteins were examined for their RNA-binding activity by Northwestern blot analysis . PKR is a RNA-binding protein that possesses two copies of a highly conserved motif, RI and RII, within the N-terminal region of the protein . Amino acid residues between 11 and 243 of PKR, which includes both copies of the R motif, displayed RNA-binding activity comparable to that of the full-length 551-amino-acid PKR protein . Analysis of substitution and deletion mutant PKR proteins revealed that motif RI was both necessary and sufficient for RNA-binding activity, whereas motif RII was not . Substitution of the highly conserved lysine at position 64 within the RI motif abolished RNA-binding activity, both of full-length PKR and the N-terminal 243-amino-acid truncated PKR . Finally, PKR substitution and deletion mutant cDNAs deficient for kinase function were expressed to much higher levels in transfected monkey cells than was the full-length wild-type PKR cDNA.

Mutat Res, 1994 Jan, 314(1), 87 - 97
Restoration of the DNA damage resistance of Deinococcus radiodurans DNA polymerase mutants by Escherichia coli DNA polymerase I and Klenow fragment; Gutman PD et al.; Deinococcus radiodurans and other species of this genus share extreme resistance to ionizing radiation and many other agents that damage DNA . D . radiodurans mutant strains defective in a deinococcal DNA polymerase that is homologous with E . coli DNA polymerase I are highly sensitive to DNA damage . In the current work we have inquired whether E . coli DNA Pol I can substitute for D . radiodurans Pol in partially or fully restoring to pol- D . radiodurans mutants the extreme DNA damage-resistance typical of this organism . The E . coli polA gene or a 5'-truncated polA gene that encodes the Klenow fragment were introduced and expressed in two different D . radiodurans pol- mutants: Strain 303, which is a chemically mutagenized derivative, and strain 6R1A, which is isogenic with wild-type D . radiodurans except for an insertional mutation within the pol gene . Expression of E . coli polA in both of these mutants fully restored wild-type resistance to ionizing- and UV254-radiation and mitomycin-C exposure . Expression of the Klenow fragment-encoding gene restored wild-type resistance to D . radiodurans strain 303, but only partial resistance to strain 6R1A . The observation that E . coli DNA Pol I is as effective as D . radiodurans Pol in restoring damage resistance, indicates that D . radiodurans DNA Pol per se does not have special properties that are essential or prerequisite for expression of the extreme resistance of D . radiodurans.

Mutat Res, 1994 Jan, 314(1), 51 - 6
Induction of the alkylation-inducible aidB gene of Escherichia coli by cytoplasmic acidification and N-ethylmaleimide; Smirnova GV et al.; We have studied the effects of changes in intracellular pH and the influence of thiol reagents on the induction of the DNA damage-inducible genes of Escherichia coli, aidB, alkA, and alkB . Under aerobic conditions in the absence of alkylating agents aidB, but not alkA or alkB, was induced by an acidification of cytoplasm or by treatment with the sulfhydryl reagent N-ethylmaleimide . Alkaline shift and thiosalicyclic acid did not affect the induction of aidB and alkB . The induction of alkA increased under the alkaline shift but not in the case of treatment with reducing agents . Compared with the aidB gene, a component of the SOS system, the sulA (sfiA) gene, responded to changes in cytoplasmic pH and in the level of intracellular thiols in an opposite way . SulA induction was observed under alkaline shift and after treatment with thiosalicylic acid.

Mutat Res, 1994 Jan, 314(1), 39 - 49
Effects of the umuDC, mucAB, and samAB operons on the mutational specificity of chemical mutagenesis in Escherichia coli: II . Base substitution mutagenesis; Watanabe M et al.; Mutational spectra induced by different classes of chemical mutagens including two ultraviolet-mimetic mutagens, an alkylating agent, intercalators, a crosslinking agent, and base analogs were characterized by means of a set of mutant lacZ genes in E . coli . These strains can be used to detect each of two types of transition and four types of transversion, simply by measuring the number of Lac+ revertant colonies . 4-Nitroquinoline 1-oxide induced G.C-->A.T, G.C-->C.G, or G.C-->T.A changes almost equally, whereas furylfuramide and mitomycin C induced only G.C-->A.T transitions and G.C-->T.A transversions, respectively . No base substitutional mutations were detected by the treatment with 9-aminoacridine . A weak stimulation of G.C-->A.T transitions by ICR-191 was observed . Both the G.C-->A.T and A.T-->G.C transitions were induced by N-methyl-N'-nitro-N-nitrosoguanidine and N4-aminocytidine . 5-Azacytidine was a specific inducer of G.C-->C.G transversions . In addition, a comparative study of mutational specificity was performed in the strains bearing either the umuDC, mucAB, or the samAB operon on a multicopy plasmid . Regardless of the kind of mutagen, G.C-->T.A transversions were greatly potentiated by the introduction of plasmids in the order of pGW1700 (mucAB) > pSE117 (umuDC) > or = pYG8011 (samAB) . Besides G.C-->T.A transversions, the introduction of pGW1700, but not pSE117 and pYG8011, enhanced the mutations of A.T-->C.G and A.T-->T.A transversions . The mucAB plasmid also enhanced the G.C-->A.T transitions and G.C-->C.G transversions induced by some mutagens.

Mutat Res, 1994 Jan, 314(1), 27 - 37
Effects of the umuDC, mucAB, and samAB operons on the mutational specificity of chemical mutagenesis in Escherichia coli: I . Frameshift mutagenesis; Watanabe M et al.; The specificity of frameshift mutations induced by several classes of chemical mutagens was determined using a collection of mutant E . coli lacZ genes . This collection can detect each of five kinds of specific frameshift events by scoring Lac+ revertant colonies . In addition, the mutational spectra were characterized in backgrounds carrying plasmids that encode the umuDC, mucAB, or samAB operon . 4-Nitroquinoline 1-oxide (4-NQO) and furylfuramide (AF-2) induced efficiently -1G, -2(C-G), and +1A frameshift mutations . 4-NQO and AF-2 differed in the ability for the induction of -1A and +1G frameshifts . +1A and -1A frameshift mutations induced by 4-NQO or AF-2 were enhanced by the introduction of the mucAB plasmid, and, to a lesser extent, the umuDC plasmid . The enhancing effect of the umuDC or mucAB plasmid on -1G and -2(C-G) frameshifts was weak or else not observed . 9-Aminoacridine was a potent inducer of +1G, -1G and -1A frameshifts, whereas ICR-191 induced all types of frameshift mutations . A mutation enhancing effect was observed only on ICR-191-induced +1A frameshift mutations by the introduction of the mucAB plasmid . Mitomycin C caused no appreciable induction of frameshift mutations to the tester strains without plasmid . However, all types of frameshifts, except -1G, were induced in the strains carrying the mucAB plasmid . N-methyl-N'-nitro-N-nitrosoguanidine induced all types of frameshift mutations . The mucAB plasmid enhanced mutagenesis in strains designed to detect the addition or loss of A.T base pair, indicating that the formation of +1A and -1A frameshifts was partly dependent on an error-prone SOS repair . Any frameshift mutagenesis was not affected by the samAB plasmid . In general, frameshifts in adenine runs were enhanced more preferentially by the mucAB and umuDC plasmids than frameshifts at runs of guanine were.

Mutat Res, 1994 Jan, 314(1), 1 - 9
Spectrum of proton-induced mutagenesis of the Escherichia coli crp gene; Takimoto K et al.; Mutation of the adenosine 3',5'-cyclic monophosphate receptor protein gene (crp) of Escherichia coli induced by protons, ionizing radiation of charged particles, was analyzed to determine the specificity of the mutational spectrum . The majority, 44 of 49 mutations detected, were base substitutions, and three frameshifts and two gross structural changes were also found . Base substitutions included 35 transversions and nine transitions . G:C to T:A transversions were the dominant type of base substitution, followed by G:C to C:G and A:T to T:A transversions . Almost all transitions were eight G:C to A:T changes . The spectrum of proton mutagenesis was quite different from that of X-ray mutagenesis of the crp gene, in which G:C to A:T transitions dominated.

Rocz Akad Med Bialymst, 1994, 39, 74 - 80
The effect of endotoxin on alcohol dehydrogenase (ADH) activity in the serum of ethanol intoxicated rats; Chrostek L et al.; Serum alcohol dehydrogenase activity at different pH-values (8.8, 9.6 and 10.4) was investigated in induced endotoxemia (3 mg/kg) after chronic alcohol consumption in rats (60 days, 11.5 mg/kg, per day) . It has been shown that ADH activity is higher after the single than after the triple injection of endotoxin given the healthy rats . Such a difference was not observed in the ethanol-intoxicated rats . The single dose of endotoxin did not maintain the increased ADH activity after alcohol administration, however a triple injection of endotoxin maintained the activity but did not increase . This results show, that the highest ADH activity in all tested rats was observed at pH 9.6 and that chronic ethanol consumption did not cause an increase of the toxic effects of endotoxin on liver cells, measured by serum alcohol dehydrogenase activity.

Bull Soc Pathol Exot, 1994, 87(5), 323 - 9
Trypanosoma brucei products activate components of the reactive response in astrocytes in vitro; Pentreath VW et al.; A study was made of the effects of T . b . brucei and the disrupted parasite material on different components of the reactive response of astrocytes in primary cultures prepared from neonatal rats and C6 glioma cells . The effects were compared with lipopolysaccharide (LPS) and fragments of the C6 cells . The disrupted trypanosome material, LPS and C6 fragments caused dose--and time--dependent alterations in morphology of the primary cultures from flat to stellate shape, increases in levels of glial fibrillary acidic protein (GFAp) and enhanced MHC class I and II expression . Exposure to trypanosome material and C6 fragments caused marked increases in phagolysosomes and lysosomes in the primary cultures . The findings demonstrate that T . b . brucei products and astrocyte cell debris are internalized and initiate astrogliosis in vitro.

Arch Immunol Ther Exp (Warsz), 1994, 42(4), 313 - 7
Effect of macrophage-modulating agents on in vivo growth of transplantable Lewis lung cancer in mice; Nowicki A et al.; C57Bl/6 mice, bearing transplantable Lewis lung cancer (non-metastatic subline) implanted either subcutaneously or intraperitoneally were treated with macrophage colony stimulating factor (M-CSF, 10(6) units per mouse, per day for 19 days), Escherichia coli lipopolysaccharide or both . Lipopolysaccharide (5 micrograms per mouse) administered daily once a day for up to 30 days impaired both subcutaneous and intraperitoneal tumor growth and prolonged survival of tumor bearing mice . Macrophage colony stimulating factor, administered daily, inhibited only subcutaneous tumor growth, both when administered alone and in combination with with lipopolysaccharide, and had no effect on intraperitoneal tumor . Moreover, it did not prolong survival of tumor bearing mice, when administered alone, and nullified the effects of lipopolysaccharide when administered concomitantly . These data suggest that macrophage colony stimulating factor, at least in this tumor model and in this dose schedule, offers little benefit . In contrast, the present data confirm earlier suggestions on therapeutic usefulness of bacterial lipopolysaccharide in neoplastic disease, which makes this compound an interesting candidate for future clinical trials.

Gene, 1993 Dec 31, 137(2), 309 - 14
Cloning and characterization of a cDNA encoding chicken liver alpha-N-acetylgalactosaminidase; Zhu A et al.; Chicken liver alpha-N-acetylgalactosaminidase (alpha AGA) specifically removes terminally alpha-linked alpha-N-acetylgalactosamine from oligosaccharide chains on the surface of group A erythrocytes . Here, we report the molecular cloning of a alpha AGA cDNA by both library screening and PCR amplification . The clone contains a 1.2-kb 3'-untranslated region and 1.2-kb coding region which encodes a 45-kDa protein . The protein was produced in bacteria and in rabbit reticulocyte lysate, and is specifically recognized on Western blot by an antibody raised against the purified chicken liver enzyme . Enzymatic activity was detected when alpha AGA was produced in Saccharomyces cerevisiae . The deduced amino acid sequence shows 80% homology with human alpha AGA and about 60% homology with alpha-galactosidases from a number of sources, indicating that these two families of exoglycosidases are evolutionarily related.

Gene, 1993 Dec 31, 137(2), 275 - 80
The 1629-bp open reading frame of the Autographa californica multinucleocapsid nuclear polyhedrosis virus encodes a virion structural protein; Pham DQ et al.; A 1629-bp open reading frame (ORF) of Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) is shown to encode a 78-kDa virion structural protein . To determine this, polyclonal antibody was made to a fusion protein synthesized in Escherichia coli from a chimeric gene that contained 1415 bp of the 1629-bp gene . In Western blot analyses, this antibody cross-reacted with a protein of about 78 kDa in both extracellular virions (ECV) and virions isolated from polyhedra (PDV), and with a 78-kDa protein in PDV envelope preparations, but not with PDV nucleocapsids . This suggests that the protein encoded by the 1629-bp ORF is a virion envelope protein or a protein that occurs in the virion intermediate layer between the envelope and nucleocapsid.

Gene, 1993 Dec 31, 137(2), 259 - 64
Cloning, sequence and expression of the gene (aprV5) encoding extracellular serine acidic protease V5 from Dichelobacter nodosus; Riffkin MC et al.; The acidic protease V5-encoding gene (aprV5) from Gram- Dichelobacter nodosus virulent strain 198 was isolated from a cosmid bank by activity screening and sequenced . The 2371-bp nucleotide (nt) sequence contained an open reading frame coding for a protein precursor of 595 amino acid (aa) residues composed of a signal peptide, a pro-region, a mature active protease of 347 aa and a C-terminal extension region of 120 aa . The deduced aa sequence of the pre-pro-mature protease regions showed about 65% similarity to that of D . nodosus basic protease while the C-terminal extension region showed only about 26% similarity . The aprV5 gene, without its C-terminal extension region, was cloned and expressed in Escherichia coli . The acidic protease B5-encoding gene (aprB5) from non-virulent strain 305 was also cloned and sequenced . The aprB5 nt sequence showed 99% homology to that of aprV5 with two single-aa changes occurring in the precursor.

Gene, 1993 Dec 31, 137(2), 217 - 22
Construction of Tn4001lac derivatives to be used as promoter probe vectors in mycoplasmas; Knudtson KL et al.; Studies of gene expression in mycoplasmas has been difficult, because the elements involved in gene expression remain relatively undefined . In order to be able to examine these regulatory elements in vivo, derivatives of Tn4001 containing the promoterless Escherichia coli lacZ reporter gene have been constructed . A Tn4001lac derivative, Tn4001.2062.2lac, transforms Mycoplasma gallisepticum and Acholeplasma strain ISM1499 . Approximately 3% of the M . gallisepticum and 8% of the Acholeplasma ISM1499 transformants appeared to generate lacZ fusions based on blue colony formation on XGal-containing media . However, placement of lacZ into an IS256 arm of Tn4001 resulted in a derivative that transformed at a frequency about 3-7-fold lower than that of wild-type Tn4001 . Another Tn4001lac derivative, Tn4001.2065, possesses a plasmid, as well as lacZ, in the IS256 arm . This construct was designed to permit the direct rescue of adjacent chromosomal sequences that are driving the expression of lacZ contained within the transposon . These constructs should be useful in locating and defining the upstream elements involved in mycoplasma gene expression.

Gene, 1993 Dec 31, 137(2), 163 - 9
Novel lacZ-based recombination vectors for mammalian cells; Herzing LB et al.; We have constructed two sets of Escherichia coli lacZ-based vectors for use in studies of general mitotic recombination, both in somatic mammalian cells grown in culture and in transgenic animals . The vectors use two mutant copies of the E . coli lacZ gene as their recombination substrates and contain a neo gene for selection of stable transformants . In one vector, pLrec, an SV40 promoter drives lacZ, while the other vector, pArec, utilizes a human non-muscle beta-actin promoter for lacZ expression . Gene conversions, unequal sister chromatid exchanges and reciprocal exchanges between the two lacZ genes result in expression of beta-galactosidase, which can be detected in situ by histochemical staining . These vectors yield rates and frequencies of mitotic intrachromosomal recombination in human and rodent cell lines which are similar to rates reported using conventional recombination vectors . Molecular analysis of recombinational events involving the lacZ-based vectors can be carried out on genomic DNA isolated from clonally expanded populations and individual LacZ+ cells and cell clusters can be analyzed using PCR amplification . These reporter gene-based vectors may facilitate the study of recombination in cells with limited proliferative capacities, allow for analysis of both products of an unequal sister chromatid exchange, and permit in situ detection of recombination in the tissues of transgenic animals.

J Virol Methods, 1993 Dec 31, 45(3), 291 - 301
Transactivation of the early SV40 promoter by avian infectious laryngotracheitis virus in avian hepatoma cells; Scholz E et al.; An avian hepatoma cell line has been reported to be suitable for the cultivation of avian laryngotracheitis virus (ILTV) (Scholz et al . (1993) J . Virol . Methods, 273-286; Guo et al . (1993) Am . J . Vet . Res., in press) . To provide information for the establishment of avian expression systems and for the construction of avian recombinant viruses, five expression plasmids were constructed to test two avian viral and two mammalian viral promotors for their suitability and strength for gene expression in this cell line . Chicken hepatoma cells were transfected with plasmids carrying the bacterial beta-galactosidase (beta-gal) gene as a reporter gene . The beta-gal gene of three plasmid constructs expressed in both E . coli and avian hepatoma cells, while the beta-gal gene of two other constructs expressed only in avian hepatoma cells . The beta-gal gene expressed independently of any viral infection when under the control of the early Rous sarcoma virus (RSV) promoter or the immediate-early cytomegalovirus (CMV) promoter . However, expression of beta-gal gene under the control of the SV40 early promoter/enhancer and the ILTV TK promoter was greatly potentiated when the transfected cells were co-infected with ILTV . This finding provides a system for the enhancement of gene expression in avian cells, especially when ILTV is used as vector.

Biochem Biophys Res Commun, 1993 Dec 30, 197(3), 1524 - 9
Molecular cloning and identification of two types of hamster cyclin-dependent kinases: cdk2 and cdk2L; Noguchi E et al.; We isolated two types of hamster cyclin-dependent kinase 2 (cdk2) cDNAs from BHK21 cells derived from Golden hamsters . One type of cdk2 (cdk2hm) encodes the 32 kDa protein consisting of 298 predicted amino acids and shows strong homology to the cdk2 cDNAs of humans and Xenopus . The other cdk2 (cdk2Lhm) encodes the 38 kDa protein containing the insertion of 48 amino acids in the cdk2hm protein . Immunoblotting analysis suggested that these two types of cdk2 protein exist in mammalian cells . The cdk2hm has the activity of protein kinase, while the cdk2Lhm does not, however, both bind with cyclin E.

Biochem Biophys Res Commun, 1993 Dec 30, 197(3), 1415 - 23
Production and properties of recombinant C3-type phosphoenolpyruvate carboxylase from Sorghum vulgare: in vitro phosphorylation by leaf and root PyrPC protein serine kinases; Pacquit V et al.; In this work, the C3-type form of Sorghum phosphoenolpyruvate carboxylase (PyrPC) was produced in PyrPC-deficient strains of Escherichia coli transformed by a plasmid bearing the corresponding full-length cDNA (CPR1) . The full-sized protein was purified to homogeneity by immunoaffinity chromatography . Some functional and regulatory properties were described; notably, the immunopurified PyrPC could be phosphorylated in reconstituted assay by 1) both a mammalian PKA and the PyrPC protein serine kinase purified from Sorghum leaves and 2) a novel protein kinase affinity-purified from Sorghum roots . In all cases phosphorylation was accompanied by a marked reduction in its malate sensitivity.

Biochem Biophys Res Commun, 1993 Dec 30, 197(3), 1326 - 33
Endotoxin protects the gastric mucosa against ulcerogenic stimuli; Tsuji K et al.; It is well-documented that large amounts of endotoxin produce hemorrhagic mucosal lesions in the stomach . To determine whether endotoxin, when injected at small doses, similarly exerts ulcerogenic actions, endotoxin (0.4-40 micrograms/kg) was injected into 24 hr-fasted rats . These small doses of endotoxin did not affect the integrity of the gastric mucosa . Unexpectedly, however, pretreatment with these minute amounts of endotoxin protected the gastric mucosa against various ulcerogenic stimuli such as stress, nonsteroidal anti-inflammatory drugs and ethanol . The anti-ulcer actions of endotoxin were not observed in endotoxin-insensitive animals (C3H/HeJ mice), thereby suggesting that endogenous cytokines such as interleukin-1 may mediate these protective actions . These findings stand in contrast to the toxic effect of endotoxin as an ulcerogen and indicate that endotoxin, albeit its term "toxin," may have a beneficial effect for the host.

Biochem Biophys Res Commun, 1993 Dec 30, 197(3), 1154 - 66
In vitro processing by signal peptidase I of precursor maltose-binding protein species with alterations in and around the signal peptide; Talarico TL et al.; Processing of 37 precursor maltose-binding protein (preMBP) species by purified signal peptidase I (SPase I) was assayed . The in vitro reaction was inefficient compared to processing in Escherichia coli cells . The extent of preMBP processing in vitro was higher when SPase I was present during translation as compared to processing after translation was arrested by chloramphenicol . Complete conversion of wild-type (wt) preMBP (greater than 90%) to mature protein required 4300-fold more enzyme than substrate during a 15 min reaction . Most preMBP species with alterations in the signal peptide processing region that were efficiently processed (greater than 85%) in vivo were also processed in vitro, although the efficiency of processing was usually lower than the corresponding in vivo value . Increasing the level of SPase I in the in vitro reaction often increased the extent of preMBP processing . A number of amino acid substitutions in the processing region that drastically reduced or eliminated processing in vivo also eliminated processing in vitro . Processing occurred at an alternate site in some mutant preMBP species in vivo, but this event occurred very inefficiently in vitro . Amino acid substitutions in the hydrophobic core or in the charged regions at the N-terminus of the signal peptide and early mature region of preMBP slightly reduced in vitro processing as compared to processing of wt preMBP, regardless of their effect on secretion in vivo.

Biochem Biophys Res Commun, 1993 Dec 30, 197(3), 1111 - 7
Evidence for identity of beta-pyrazolealanine synthase with cysteine synthase in watermelon: formation of beta-pyrazole-alanine by cloned cysteine synthase in vitro and in vivo; Noji M et al.; The responsibility of cysteine synthase (EC 4.2.99.8) from watermelon (Citrullus vulgaris) for the formation of beta-(pyrazole-1-yl)-L-alanine, a non-protein amino acid specifically accumulated in Curcubitaceae plants, was confirmed in vitro and in vivo by the cloned cDNA on expression vectors, pCCS11 and pCEN1 . The cDNA sequence derived from pCCS11, an expression vector driven by the lacZ promoter, was placed under the transcriptional control of strong T7 promoter of pET3d to yield an over-expression vector, pCEN1, in Escherichia coli . The concentration of the exogenous cysteine synthase protein was increased up to approximately 10% of the total soluble protein of E . coli cells by the expression of cDNA on pCEN1 . beta-(Pyrazole-1-yl)-L-alanine was formed in vitro from O-acetyl-L-serine and pyrazole by the action of cysteine synthase expressed in E . coli carrying pCCS11 or pCEN1 . To confirm the responsibility of cysteine synthase for the formation of beta-(pyrazole-1-yl)-L-alanine in vivo, the feeding experiments of pyrazole and serine or O-acetyl-L-serine were carried out using the transformed E . coli culture . beta-(Pyrazole-1-yl)-L-alanine was produced in vivo by feeding the substrates to the culture of E . coli carrying pCEN1 . These results provide the confirming evidence that the cloned cysteine synthase of watermelon catalyzes the formation of beta-(pyrazole-1-yl)-L-alanine, indicating that beta-pyrazolealanine synthase is identical with cysteine synthase in Cucurbitaceae plants.

Biochem Biophys Res Commun, 1993 Dec 30, 197(3), 1609 - 14
Association of RNA with human papillomavirus E7 protein of type 16 but not type 6b; Chinami M et al.; Nonfused human papillomavirus (HPV) type 16 and 6b E7 proteins were expressed in E . coli and fractionated by ion exchange and gel filtration chromatography . The E7 protein of type 6b was purified, but that of type 16 was found to be associated with RNA molecules which could not be excluded by repeated chromatography . The type 16 E7 protein in CaSki cells was also associated with RNA molecules of the same size.

FEBS Lett, 1993 Dec 28, 336(3), 530 - 4
Calcium transport mediated by NhaA, a Na+/H+ antiporter from Escherichia coli; Dibrov PA; In everted membrane vesicles of E . coli strain EP432/pGM42, which has only one Na+/H+ antiporter (NhaA), external CaCl2 inhibits dissipation of the respiration-dependent delta pH in response to the addition of NaCl at pH 7.5, and decreases equilibrium concentration of the intravesicular Na+ . In the NhaA proteoliposomes, imposition of an artificial delta pH (acid inside) leads to the several-fold accumulation of calcium . The apparent Km for this delta pH-driven Ca2+ uptake at pH 8.5 is 2 mM, and the Vmax is 1.79 mumol/min/mg of protein . Dissipation of delta pH causes release of calcium from the vesicles . CaCl2 was found to inhibit the delta pH-driven Na+ uptake mediated by reconstituted NhaA, and vice versa . Further, heterological Ca2+/Na+ exchange has been demonstrated in proteoliposomes containing NhaA . Transmembrane electric potential difference proved to drive this process . All these data are consistent with the assumption that NhaA can also catalyze Ca2+/H+ exchange.

FEBS Lett, 1993 Dec 28, 336(3), 525 - 9
Mechanism of Na+/H+ exchange by Escherichia coli NhaA in reconstituted proteoliposomes; Dibrov PA et al.; Purified NhaA, a Na+/H+ antiporter from Escherichia coli, reconstituted into proteoliposomes was used to study partial reactions catalyzed by this protein . Homologous Na+/Na+ exchange as well as Na+/Li+ exchange via NhaA were detected by monitoring the effects of external Li+ and Na+ ions on the delta pH-driven sodium uptake into NH4 Cl-loaded vesicles . Furthermore, a sodium counterflow reaction was demonstrated in proteoliposomes preloaded with non-radioactive Na+ and placed into the experimental buffer containing low amounts of 22Na+ under experimental conditions when both components of protonmotive force generated by the antiporter . delta psi and delta pH, were dissipated by corresponding ionophores . The apparent Km for sodium counterflow is 1.1 mM, and Vmax is 80 mumol/min/mg of protein . External Na+ accelerates the downhill efflux of 22Na+ suggesting that the translocation of the Na(+)-loaded form of the carrier is faster than the rest of the catalytic cycle.

Biochemistry, 1993 Dec 28, 32(51), 14125 - 31
Preparation of active recombinant TIMP-1 from Escherichia coli inclusion bodies and complex formation with the recombinant catalytic domain of PMNL-collagenase; Kleine T et al.; TIMP-1 is a member of the family of tissue inhibitors of metalloproteinases involved in regulating the activity of extracellular matrix degrading metalloproteinases . The TIMP-1 cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) amplification of the corresponding mRNA from human fibroblasts . Cloning and expression of the TIMP-1 cDNA were performed in Escherichia coli . In the host vector system chosen, rTIMP-1 is stored intracellularly in its denatured, insoluble form in inclusion bodies . We report a new method for the purification and renaturation of rTIMP-1 from E . coli inclusion bodies to an active inhibitor of matrix metalloproteinases (80% yield), presumably containing the correct assignment of the six disulfide bonds . A resin with the covalently bound recombinant catalytic domain of the PMNL-collagenase as the affinity ligand provided an effective means for the separation of correctly folded, active rTIMP-1 from inactive forms with mismatched disulfides . TIMP-1 and TIMP-2, the two most extensively examined members of the family of tissue inhibitors of metalloproteinases, are known to form a complex with the activated forms of most matrix metalloproteinases and the latent forms of the 92-kDa and 72-kDa gelatinases, respectively . In this study, we report on the complex formation of the recombinant catalytic domain of the PMNL-collagenase with TIMP-1, nonglycosylated recombinant TIMP-1, and recombinant TIMP-2 . The Ki values for the different inhibitors were determined in a kinetic assay using a fluorogenic substrate peptide . In this assay, rTIMP-2 had a more effective inhibitory capability against the recombinant catalytic domain of the PMNL-collagenase than TIMP-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Dec 28, 32(51), 14102 - 10
Molecular properties of pyruvate formate-lyase activating enzyme; Wong KK et al.; Pyruvate formate-lyase is a radical-containing enzyme that catalyzes the nonoxidative cleavage of pyruvate via a postulated homolytic mechanism . The formation of this enzymic radical in vitro requires an activating system composed of PFL-activating enzyme, S-adenosylmethionine, ferrous ion, a reduced flavin, DTT, and pyruvate as an allosteric effector . The need for large quantities of PFL-activating enzyme for biochemical and biophysical studies on the mechanism of protein radical formation has prompted us to clone the act gene and overexpress the gene product in Escherichia coli . Using PCR technology, the act gene was isolated and subcloned into various expression vectors . The overexpression of the protein was as high as 30-50% of the total cellular protein . However, the majority of the protein resided in the form of insoluble inclusion bodies . A procedure was developed to denature and isolate the inclusion bodies followed by refolding under anaerobic conditions . This purification method affords 5 mg of purified protein from 1 g of cells . Biochemical characterization demonstrated that the enzyme can bind one Fe(II) per protein monomer, and the protein did not exhibit any visible chromophore as previously observed . Co(II) and Cu(II) can be reconstituted into the protein with similar stoichiometries . Kinetic studies showed that the rate of radical formation was independent of ionic strength and the Km's for SAM and inactive PFL were determined to be 2.8 and 1.2 microM, respectively . Fluorescent binding data revealed that the Kd for SAM binding to the activating enzyme alone was comparable to the Km for SAM in the PFL activation indicating that the binding site for SAM resides on AE.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Dec 28, 32(51), 14095 - 101
Crystal structures of the Klenow fragment of DNA polymerase I complexed with deoxynucleoside triphosphate and pyrophosphate; Beese LS et al.; Crystal structures of the Klenow fragment (KF) of DNA polymerase I from Escherichia coli complexed with deoxynucleoside triphosphate (dNTP) or with pyrophosphate (PPi) determined to 3.9-A resolution by X-ray crystallography show these molecules binding within the cleft of the polymerase domain and surrounded by residues previously implicated in dNTP binding . The dNTP binds adjacent to the O-helix {Ollis, D . L., Brick, P., Hamlin, R., Xuong, N . G., & Steitz, T . A . (1985a) Nature 313, 762-766} with its triphosphate moiety anchored by three positively charged residues, Arg 754, Arg 682, and Lys 758, plus His 734 and Gln 708 . The dNTP binding site observed in the crystal is consistent with the results of chemical modification including cross-linking and is also near many of the amino acid residues whose mutation affects catalysis {Polesky, A . H., Steitz, T . A., Grindley, N . D . F., & Joyce, C . M . (1990) J . Biol . Chem . 265, 14579-14591; Polesky, A . H., Dahlberg, M . E., Benkovic, S . J., Grindley, N . D . F., & Joyce, C . M . (1992) J . Biol . Chem . 267, 8417-8428} . However, we conclude that the position of at least the dNMP moiety of dNTP in the binary complex is not likely to be the same as in its catalytically relevant complex with primer-template DNA.

Biochemistry, 1993 Dec 28, 32(51), 14089 - 94
Solution structure of the DNA methyl phosphotriester repair domain of Escherichia coli Ada; Myers LC et al.; The Escherichia coli Ada protein repairs methyl phosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteine residues . The methyl-transfer process appears to be autocatalyzed by coordination of the acceptor residue, Cys-69, to a tightly bound zinc ion . Upon methyl transfer, Ada acquires the ability to bind DNA sequence-specifically and thereby to induce genes that confer resistance to methylating agents . The solution structure of an N-terminal 10-kDa fragment of Ada, which retains zinc binding and DNA methyl phosphotriester repair activities, was determined using multidimensional heteronuclear nuclear magnetic resonance techniques . The structure reveals a zinc-binding motif unlike any observed thus far in transcription factors or zinc-containing enzymes and provides insight into the mechanism of metalloactivated DNA repair.

Biochemistry, 1993 Dec 28, 32(51), 14210 - 9
Discrimination among tRNAs intermediate in glutamate and glutamine acceptor identity; Rogers KC et al.; The set of nucleotides in Escherichia coli tRNA(Gln) which facilitate aminoacylation by glutaminyl-tRNA synthetase (GlnRS) has been defined {Hayase et al . (1992), EMBO J . 11, 4159-4165} . To determine whether the glutamine "identity set" is sufficient to confer acceptance on a noncognate tRNA, we constructed tRNA(Glu) mutants with the set of glutamine recognition elements . These mutants were examined for aminoacylation in vitro with GlnRS and also with glutamyl-tRNA synthetase (GluRS) to correlate gains in glutamine acceptance with losses of glutamate acceptance . Incorporating glutamine recognition elements in only the acceptor stem or anticodon loop of tRNA(Glu) improved the specificity constant (kcat/KM) for aminoacylation by GlnRS . However, the introduction of all defined glutamine recognition elements in tRNA(Glu) resulted in a substrate with a specificity constant 100-fold below that for aminoacylation of tRNA(Gln) . Including the tertiary framework of tRNA(Gln) (in addition to the glutamine recognition elements) in the tRNA(Glu) context further improved aminoacylation by GlnRS, but the specificity was still reduced compared with that of tRNA(Gln) . The increase in glutamine acceptance was correlated for all mutants with a decrease in glutamate acceptance, indicating that GluRS also recognizes acceptor stem and anticodon sequences in cognate tRNA . The inability to completely convert tRNA(Glu) to glutamine acceptance with these mutations suggests that tRNA(Glu) contains antideterminants to glutamine identity . The analysis of these mutants with both enzymes revealed that there is a strong element of discrimination between glutamate and glutamine tRNAs associated with the anticodon . To test this dependence, mutants of both tRNAs were made to effect anticodon switches to the possible glutamate and glutamine isoacceptors . The kinetic evaluation of the anticodon switch mutants suggests that overlap in anticodon recognition is avoided through specificity for the third anticodon position coupled with divergent preferences for the wobble base.

Gene, 1993 Dec 27, 137(1), 41 - 7
Selection of new biologically active molecules from random nucleotide sequences; Dube DK et al.; Genetic diversity can be achieved in vitro by inserting random nucleotide (nt) sequences into cloned genes . In the case of enzymes, subsequent genetic complementation can be used to select for new mutants that exhibit different substrate specificities, altered catalytic activities, or altered temperature sensitivities . Using this technique, one can also analyze the contribution of different amino acid residues to the structure and function of enzyme . Selecting biologically active DNA sequences from large random populations provides a new method for identifying nt sequences with unique functions . Analogous random sequence selection techniques have been applied to determine the consensus sequence of the Escherichia coli promoters, DNA and RNA sequences that bind specific protein(s), DNA regulatory sequences, ribozyme(s) and ligand-specific RNA(s) . In this manuscript, we will consider recent data obtained in our laboratory as a result of inserting random sequences into the putative nucleoside-binding site of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) . We have obtained over 2000 new mutant HSV-1 TKs, some of which are stable at higher temperatures or have altered substrate specificity and/or catalytic rates when compared to those of the wild-type enzyme.

Gene, 1993 Dec 27, 137(1), 121 - 6
A genetic selection elucidates structural determinants of arginine versus lysine specificity in trypsin; Perona JJ et al.; A genetic selection has been used to isolate variants of the serine protease, trypsin (Tsn), altered in specificity toward lysine- and arginine-containing substrates . Growth of a lysine auxotroph of Escherichia coli was coupled to activation by Tsn of a non-nutritive source of lysine present in selective media . Nine Tsn variants possessing partial activities were isolated from a random library encompassing amino acids 189 and 190 at the base of the primary specificity pocket . Functional analysis of these isolates indicates that preservation of activity toward lysine-containing substrates is more tolerant to mutation than is activity toward equivalent arginine-containing substrates . Both the position, as well as the accessibility to substrate, of a negatively charged group in the binding pocket appear critical to maintenance of high-level catalytic potency by Tsn.

FEBS Lett, 1993 Dec 27, 336(2), 236 - 8
Crystallization and preliminary X-ray analysis of leukemia inhibitory factor; Betzel C et al.; Leukemia inhibitory factor (LIF) is a polyfunctional molecule with significant and diverse biological activities . LIF is a glycoprotein secreted by a number of different cell types in vitro . It is induced in fibroblasts, lymphocytes, monocytes and astrocytes by various inducers such as serum, TNF, interleukin-IP and EGF . Due to extensive and variable glycosylation the molecular weight can range from 38 to 67 kDA . The biological functions of LIF are mediated through a receptor and a signal transducer, gp130, which is also used by factors like interleukin-6 (IL-6), cilliary neurotropic factor (CNTF), and oncostatin M (OSM) . Here, we report the crystallization of the non-glycosylated human-like LIF expressed in E . coli . The present crystals diffract to 2.0 A using synchrotron radiation . They belong to the monoclinic space group C2, and the cell dimensions are a = 61.5 A, b = 45.3 A, c = 77.7 A and beta = 112.3 degrees.

FEBS Lett, 1993 Dec 27, 336(2), 221 - 4
Active increase in cardiolipin synthesis in the stationary growth phase and its physiological significance in Escherichia coli; Hiraoka S et al.; Activity of the Escherichia coli cardiolipin synthase, encoded by cls, increased about 10-fold in the stationary growth phase, while other committed-step enzymes in phospholipid biosynthesis rather decreased . A null cls mutant lost viability to 10(-4) of the wild-type cells during the prolonged incubation for 5 days . Cardiolipin was most stable among membrane phospholipids during the incubation . Accordingly, cardiolipin should play a role in survival of the cell and E . coli employs a sophisticated way to form cardiolipin according to need even under non-growing conditions.

FEBS Lett, 1993 Dec 27, 336(2), 363 - 7
Truncated GroEL monomer has the ability to promote folding of rhodanese without GroES and ATP; Makino Y et al.; Similar to chaperonins from other sources, intact chaperonin from Escherichia coli (GroEL) exists as a tetradecamer, and the ability to promote folding of other proteins has been considered to be dependent on this oligomeric structure . However, the peptide fragments of GroEL of molecular size 34-50 kDa, which are produced by limited proteolysis of monomeric GroEL and are unable to assemble into an oligomer, retain the ability to promote folding of rhodanese even though the yield of productive folding is lower than the intact GroEL/GroES/ATP system . This promotion by truncated GroEL obeys rapid kinetics and does not require GroES and ATP.

Gene, 1993 Dec 27, 137(1), 133 - 7
Recombinant RNA phage Q beta capsid particles synthesized and self-assembled in Escherichia coli; Kozlovska TM et al.; The Escherichia coli RNA phage Q beta coat protein-encoding gene (C) was amplified from native Q beta RNA using a reverse transcription-PCR technique . Gene C contains sequences coding for both the 133-amino acid (aa) Q beta coat protein (CP) and the 329-aa read-through protein (A1) consisting of CP and an additional 196-aa C-terminal sequence, separated from CP within the C gene by an opal (UGA) stop codon . Primers ensuring the natural environment for gene C, especially within the ribosome-binding site, and supplying C with unique restriction sites at both ends have been prepared . An amplified 1062-bp PCR fragment was positioned under the control of the strong E . coli trp promoter (Ptrp) within a pGEM-derived plasmid . The synthesis of gene C products was confirmed electrophoretically and immunologically . An immunodiffusion test with anti-Q beta phage antibodies and electron microscopy evaluation of the purified recombinant products showed that when expressed, the Q beta C gene was responsible for high-level synthesis and correct self-assembly of Q beta CP monomers into capsids indistinguishable morphologically and immunologically from Q beta phage particles, which we plan to use as surface display vectors.

Nucleic Acids Res, 1993 Dec 25, 21(25), 5817 - 23
E1 protein of human papillomavirus is a DNA helicase/ATPase; Hughes FJ et al.; Replication of human papillomavirus (HPV) DNA requires the viral proteins E1 and E2 . Amino acid similarities to SV40 large-T antigen had suggested that E1 is a DNA helicase/ATPase involved in initiating viral DNA replication, and this has recently been shown for bovine papillomavirus type 1 (BPV-1) E1 protein . However, in vitro analysis of HPV E1 has been hampered by the inability to produce purified protein using heterologous expression systems . We have succeeded in demonstrating ATPase and DNA helicase activities in purified HPV E1, expressed in E . coli as a maltose-binding protein fusion (MBP-E1), for the first time . As further confirmation that the ATPase and DNA helicase activities are due to E1 and not contaminating E . coli enzymes, we have shown that a fusion protein containing an amino acid change (E1 Pro-479 to Ser), predicted to inactivate ATP-binding, has impaired activities . We have carried out a structure prediction analysis which suggests that E1 may form two domains: a relatively open N-terminal domain (residues 1-125), and a highly structured C-terminal domain (170-649), with an intermediate region (125-170) predicted to form an inter-domain linker . This is consistent with the proteolytic susceptibility of MBP-E1 at a site 15-20 kD from the N-terminus of E1, and the accumulation of a 58 kD C-terminal fragment of E1 . We speculate that the N-terminal domain is involved in DNA-binding, while the C-terminal 58 kD may constitute a distinct enzymatic domain . HPV E1 is of interest as a therapeutic target and the availability of pure enzyme will be invaluable in the search for antiviral compounds.

J Biol Chem, 1993 Dec 25, 268(36), 27428 - 39
The ethanol-inducible YAT1 gene from yeast encodes a presumptive mitochondrial outer carnitine acetyltransferase; Schmalix W et al.; The gene YAT1 from Saccharomyces cerevisiae encodes a protein of 688 amino acids which displays significant sequence similarity to vertebrate L-carnitine acyltransferases and yeast inner mitochondrial L-carnitine acetyltransferase . Steady state levels of the respective mRNA are low during growth on glucose or galactose, derepressed on glycerol, and significantly induced when ethanol or acetate are the only carbon sources . The YAT1 promotor region confers the identical carbon source dependence also on the expression of beta-galactosidase when cloned 5' to the Escherichia coli lacZ-coding region . An antiserum directed against a beta-galactosidase/Yat1p fusion protein recognizes a protein of 78-kDa molecular mass in the mitochondrial fraction from yeast . In vitro translated Yat1p, which lacks a cleavable presequence, binds to mitochondria in a protease-sensitive location in a standard in vitro import assay . Deletion of the single copy gene in a haploid yeast strain yields no obvious phenotype with any carbon source . Carnitine acetyltransferase activities of intact mitochondria from a YAT1 deletion mutant are slightly lower than wild type, but are approximately alike in lysed mitochondria from mutant and control cells . Thus, the novel gene is nonessential and likely to code for a minor mitochondrial outer carnitine acetyltransferase.

J Biol Chem, 1993 Dec 25, 268(36), 27349 - 54
A serine and a lysine residue implicated in the catalytic mechanism of the Escherichia coli leader peptidase; Tschantz WR et al.; We report that a thiol leader peptidase, produced by replacing the critical serine at position 90 with a cysteine residue, is enzymatically active . In contrast to the wild-type leader peptidase, the thiol enzyme can be inactivated with N-ethylmaleimide, a cysteine-specific reagent . This strongly suggests that the serine 90 is involved in catalysis and is located at the active site . Of the three conserved basic residues in the signal peptidase family, only lysine 145 appears to be critical for catalysis; when lysine 145 was mutated to an alanine residue, leader peptidase K145A protein was inactive both in vitro and in vivo . A control experiment showed that the K145A mutant competes with the wild-type leader peptidase for substrate binding, confirming that the K145A mutation did not cause a global conformational change . The data provides evidence that catalysis of leader peptidase is carried out by a serine-lysine dyad.

J Biol Chem, 1993 Dec 25, 268(36), 27291 - 8
The human active breakpoint cluster region-related gene encodes a brain protein with homology to guanine nucleotide exchange proteins and GTPase-activating proteins; Tan EC et al.; GTPase-activating proteins (GAPs) modulate the activity of the ras superfamily of proteins by converting active GTP-bound to inactive GDP-bound p21s . Employing a novel GAP overlay assay (Manser, E., Leung, T., Monfries, C., Teo, M., Hall, C., and Lim, L . (1992) J . Biol . Chem . 267, 16025-16028), we demonstrated a diversity of proteins with GAP activities in different tissues . Using a polymerase chain reaction strategy exploiting conserved residues in the GAP domains of n-chimaerin and the product of the breakpoint cluster region gene (BCR), we isolated a human brain 5.3-kilobase cDNA containing a 486-base pair region with complete identity to a previously reported active BCR-related (ABR) gene sequence on human chromosome 17 . The brain cDNA encoded a 98-kDa protein (ABR) resembling BCR (68% identity), containing both the oncogene dbl-related domain at the N terminus and the GAP domain at the C terminus; however, it lacks the N-terminal BCR protein kinase domain . The ABR GAP domain expressed as an Escherichia coli fusion protein was active against Rac1 and Cdc42 of the rho subfamily . The ABR mRNA is highly enriched in the brain . ABR probably corresponds to the brain-enriched 100-kDa GAP for Rac and Cdc42Hs previously detected . The relationship of ABR to Miller-Dieker syndrome, a neurological disorder co-mapping to 17p13.3, is discussed.

J Biol Chem, 1993 Dec 25, 268(36), 27094 - 9
The mannose transporter of Escherichia coli . Structure and function of the IIABMan subunit; Stolz B et al.; The mannose transporter of the bacterial phosphotransferase system consists of two transmembrane subunits (IICMan and IIDMan) and a hydrophilic subunit (IIABMan) . IIABMan has two flexibly linked domains containing one phosphorylation site each and occurs as a dimer . Substrate transport is coupled to phosphorylation . The phosphoryl group is transferred from a phosphoryl carrier protein to His10 on IIA, hence to His175 on IIB and finally to the substrate . IIABMan mutants were analyzed in vitro for complementation, negative dominance, cysteine cross-linking and reactivity . Conclusions: (i) His10, Trp12, Lys48, and Ser72 form a functional unit (phosphorylation site 1); (ii) His86 on the IIA domain and His175 on the IIB domain of the same subunit form a functional unit (phosphorylation site 2); (iii) phosphoryl transfer can occur between His10 and His175 of the same as well as of different subunits and His86 is necessary for this transfer; (iv) the subunits in the dimer are interdependent; (v) The phosphorylation site mutant H175C is highly reactive toward thiol reagents and it forms extensive homo- and heterocross-links with other surface-exposed cysteines . The phosphorylation site mutant H10C is 1000-fold less reactive . The two residues might be in complementary locations, His10 buried in a concave, His175 exposed on a convex surface.

J Biol Chem, 1993 Dec 25, 268(36), 27020 - 5
Biochemical analysis of Escherichia coli selenophosphate synthetase mutants . Lysine 20 is essential for catalytic activity and cysteine 17/19 for 8-azido-ATP derivatization; Kim IY et al.; A labile selenium donor compound, selenophosphate, is formed from selenide and ATP by selenophosphate synthetase . A cysteine residue (Cys-17) that is essential for catalytic activity of the enzyme (Kim, I.Y., Veres, Z., and Stadtman, T . C . (1992) J . Biol . Chem . 267, 19650-19654) is located in a glycine-rich segment near the N terminus of the protein . The possibility that this peptide sequence (HGAGCGCK) defines the ATP-binding site of the enzyme, as does a conserved ATP or GTP binding sequence (GXXXXGKS/T) found in several other proteins, was tested by site-specific mutagenesis . Thus His-13 and Gly-18 were changed to Asn and Val, respectively, and Lys-20 to Arg or Gln . Catalytic activity was markedly decreased by mutation of Lys-20 to Arg and abolished by mutation of Lys-20 to Gln . The mutation of Cys-19 and His-13 did not substantially alter the ATP Km and Vmax values, whereas the Gly-18 mutation resulted in a 4-fold increase in the ATP Km value compared with that of the wild type . ATP binding properties of the mutant enzymes were determined using Mn-{32P}ATP or Mn-{14C}ATP and gel filtration . Photoaffinity labeling of the proteins with {gamma-32P}8-azido-ATP showed that all mutant enzymes could be labeled with the ATP analog except those in which Cys-17 or Cys-19 were replaced with serine.

J Biol Chem, 1993 Dec 25, 268(36), 26950 - 5
Kinetics of factor Xa inhibition by tissue factor pathway inhibitor; Huang ZF et al.; Tissue factor pathway inhibitor is a multivalent, Kunitz-type proteinase inhibitor . It directly inhibits factor Xa and, in a factor Xa-dependent fashion, produces feedback inhibition of the factor VIIa/tissue factor catalytic complex which is responsible for the initiation of coagulation . Human recombinant TFPI (rTFPI) produced in Escherichia coli was used to define the kinetic constants describing the human factor Xa:TFPI interaction . The inactivation of factor Xa by E . coli-rTFPI is indistinguishable from that of rTFPI produced in mammalian SK-hepatoma cells, suggesting that post-translational modifications such as glycosylation and phosphorylation do not play a major role in the inhibitory process . The slow, tight-binding inhibition of factor Xa follows the scheme: {formula: see text} Where the enzyme (E) and inhibitor (I) form an initial, immediate collision complex (EI) that then isomerizes slowly to a tightened final EI* complex . In the absence of other additions, the initial Ki (=k2/k1) and final Ki* for the inhibition of factor Xa by E . coli-rTFPI are 1.24 nM and 26.4 pM, respectively . In the presence of calcium ions (5 mM) the interaction between factor Xa and rTFPI is substantially weaker, with a Ki of 42.7 nM and Ki* of 85.2 pM . The addition of other components of the prothrombinase complex produces enhanced factor Xa inhibition predominantly through an effect on the initial Ki . In the presence of calcium ions and saturating concentrations of phospholipids and factor Va, the Ki and Ki* for factor Xa inactivation are 2.04 nM and 52.3 pM . The enhancing effect of heparin on the inhibitory process is concentration dependent and exhibits an optimum, reminiscent of the "template" model for heparin's acceleration of thrombin and factor IXa inhibition by antithrombin III . At optimal concentrations, the major mechanism of heparin action is also a reduction in the Ki of the initial encounter complex between factor Xa and rTFPI.

J Biol Chem, 1993 Dec 25, 268(36), 26927 - 34
Identification of the functional domains in heme O synthase . Site-directed mutagenesis studies on the cyoE gene of the cytochrome bo operon in Escherichia coli; Saiki K et al.; The cytochrome bo complex is a terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and is encoded by the cyoABCDE operon . Recently, we have demonstrated that heme O at the high-spin heme-binding site is essential for redox-coupled proton pumping by the oxidase and suggested that the cyoE gene encodes a novel enzyme for heme O biosynthesis, protoheme IX farnesyltransferase (heme O synthase) (Saiki, K., Mogi, T., and Anraku, Y . (1992) Biochem . Biophys . Res . Commun . 189, 1491-1497) . This study was focused to define the catalytic domain(s) of the CyoE protein via a site-directed mutagenesis approach . We have individually substituted 40 amino acid residues including 22 invariant residues with alanines and found that 23 mutant oxidases were nonfunctional and exhibited a specific loss of the CO binding activity at the site of the high-spin heme . Characterizations of the purified D65A, Y120A, and W172A mutant oxidases, which represent the mutations of different topological domains, revealed that their defects are attributable to substitution of protoheme IX for heme O present in the high-spin heme-binding site . Based on the above observations, we suggest that the conserved amino acid residues present in the cytoplasmic loops II/III and IV/V are part of the catalytic center of heme O synthase.

J Biol Chem, 1993 Dec 25, 268(36), 26842 - 9
Functional expression and properties of the tRNA(Lys)-specific core anticodon nuclease encoded by Escherichia coli prrC; Morad I et al.; Escherichia coli carrying the optional locus prr harbor a latent, tRNA(Lys)-specific anticodon nuclease, activated by the product of phage T4 stp . Anticodon nuclease latency is ascribed to the masking of prrC, implicated with the enzymatic activity, by flanking, type Ic DNA restriction modification genes (prrA, B&D-hsdM, S&R) . Overexpression of plasmid-borne prrC elicited anticodon nuclease activity in uninfected E . coli . In vitro, the prr-C-coded core activity was indifferent to a synthetic Stp polypeptide, GTP, ATP, and endogenous DNA, effectors that synergistically activate the latent enzyme . Several facts suggested that PrrC is highly labile in the absence of the masking proteins . The core activity decayed with t1/2 below 1 min at 30 degrees C, and the PrrC portion of a fusion protein was unstable . Moreover, expression of prrC from its own promoter at low plasmid copy number did not allow detection of core activity . Yet, it sufficed for establishment of a latent, T4-inducible enzyme when complemented by the masking Hsd proteins, which were provided by another replicon . Interaction between the antagonistic components of latent anticodon nuclease was also demonstrated immunochemically . The coupling of anticodon nuclease with a DNA restriction modification system may serve to ward off its inadvertent toxicity and maintain it as an antiviral contingency.

J Biol Chem, 1993 Dec 25, 268(36), 26836 - 41
The crystallographic structure of a human dihydropteridine reductase NADH binary complex expressed in Escherichia coli by a cDNA constructed from its rat homologue; Su Y et al.; A human dihydropteridine reductase (EC 1.6.99.10) has been created from a rat cDNA clone by a single five-oligonucleotide mutagenesis reaction and expressed in good yield in Escherichia coli . The enzyme has been purified to homogeneity, and kinetic identity to the naturally occurring enzyme has been proven . Crystallization has also been achieved, and the crystal structure was solved using 2.5 A data that was refined to an R value of 16.9% . The structure described in this report represents the first complete structural characterization of this important human enzyme.

J Biol Chem, 1993 Dec 25, 268(36), 26827 - 35
Directed evolution of biosynthetic pathways . Recruitment of cysteine thioethers for constructing the cell wall of Escherichia coli; Richaud C et al.; We report that expansion of thioether biosynthesis in Escherichia coli generates sulfur-containing amino acids that can replace meso-diaminopimelate, the essential amino acid used for cross-linking the cell wall . This was accomplished by jointly overexpressing the metB gene coding for L-cystathionine gamma-synthase and disrupting the metC gene, whose product, L-cystathionine beta-lyase, is responsible for the destruction of L-cystathionine and other L-cysteine thioethers . As a result, meso-lanthionine and L-allo-cystathionine were produced endogenously and incorporated in the peptidoglycan, thereby enabling E . coli strains auxotrophic for diaminopimelate to grow in its absence . Thus, current techniques of metabolic engineering can be applied to evolving the chemical constitution of living cells beyond its present state.

J Biol Chem, 1993 Dec 25, 268(36), 27286 - 90
Characterization of human type I and type II IMP dehydrogenases; Carr SF et al.; Human IMP dehydrogenase, a target for anticancer and immunosuppressive chemotherapy, exists as two isoforms, types I and II . Nonfusion sequences of each isoform were overexpressed in an IMP dehydrogenase-deficient strain of Escherichia coli and purified to homogeneity . Both recombinant isoforms were tetramers, which was in agreement with the subunit structure of the native mammalian enzyme . The results of initial velocity and product inhibition studies were consistent with an Ordered Bi Bi kinetic mechanism for both isoforms . Substrate affinities were similar for types I and II with Km values of 18 and 9.3 microM, respectively, for IMP, and 46 and 32 microM, respectively, for NAD.kcat values were 1.5 and 1.3 s-1 at 37 degrees C for types I and II, respectively . Xanthosine 5'-monophosphate and NADH inhibited the two isoforms with identical inhibition patterns and inhibition constants . Mycophenolic acid, however, inhibited the type II enzyme with a 4.8-fold lower K than the type I . Selective inhibitors of the inducible type II isoform may mitigate toxicity caused by inhibition of the constitutively expressed type I isoform.

J Biol Chem, 1993 Dec 25, 268(36), 26990 - 5
Second-site mutation of Ala-220 to Glu or Asp suppresses the mutation of Asp-285 to Asn in the transposon Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli; Yamaguchi A et al.; A carboxyl group of Asp-285 is essential for tetracycline/H+ antiport mediated by the transposon Tn10-encoded metal-tetracycline/H+ antiporter (TetA) of Escherichia coli (Yamaguchi, A., Akasaka, T., Ono, N., Someya, Y., Nakatani, M., and Sawai, T . (1992) J . Biol . Chem . 267, 7490-7498) . Spontaneous tetracycline resistance revertants were isolated from E . coli cells carrying the Asn-285 mutant tetA gene . All of the revertants were due to the second-site mutation at codon 220 of GCG (Ala) to GAG (Glu) . The Km value of the tetracycline transport mediated by the revertant TetA protein was about 4-fold higher than that of the wild-type, indicating that the revertant is a low affinity mutant . A Glu-220 and Asn-285 double mutant constructed by site-directed mutagenesis showed the same properties as the revertants, confirming that the mutation of Ala-220 is solely responsible for the suppression . The Asp-220 mutation of the Asn-285 mutant resulted in a lower level of restoration of the tetracycline resistance and the transport activity than in the case of the Glu-220 mutation . A single mutation replacing Ala-220 with Glu or Asp caused about a 2-4-fold decrease in the tetracycline resistance, but no crucial change in the transport activity . It is not likely that Glu-220 is required for a charge-neutralizing salt bridge because an unpaired negative charge in a Glu-220 or Asp-220 single mutant did not cause a serious change in the activity . An alternative explanation is reasonable; Asp-285 directly contributes to the binding of a cationic substrate, metal-tetracycline chelation complex, or proton, and an acidic residue at position 220 can take over the role of Asp-285.

Science, 1993 Dec 24, 262(5142), 2030 - 3
A covalent enzyme-substrate intermediate with saccharide distortion in a mutant T4 lysozyme; Kuroki R et al.; The glycosyl-enzyme intermediate in lysozyme action has long been considered to be an oxocarbonium ion, although precedent from other glycosidases and theoretical considerations suggest it should be a covalent enzyme-substrate adduct . The mutation of threonine 26 to glutamic acid in the active site cleft of phage T4 lysozyme (T4L) produced an enzyme that cleaved the cell wall of Escherichia coli but left the product covalently bound to the enzyme . The crystalline complex was nonisomorphous with wild-type T4L, and analysis of its structure showed a covalent linkage between the product and the newly introduced glutamic acid 26 . The covalently linked sugar ring was substantially distorted, suggesting that distortion of the substrate toward the transition state is important for catalysis, as originally proposed by Phillips . It is also postulated that the adduct formed by the mutant is an intermediate, consistent with a double displacement mechanism of action in which the glycosidic linkage is cleaved with retention of configuration as originally proposed by Koshland . The peptide part of the cell wall fragment displays extensive hydrogen-bonding interactions with the carboxyl-terminal domain of the enzyme, consistent with previous studies of mutations in T4L.

J Chromatogr A, 1993 Dec 24, 657(1), 55 - 61
Single-step purification of a bacterially expressed antibody Fv fragment by immobilized metal affinity chromatography in the presence of betaine; Essen LO et al.; A procedure was developed for the rapid isolation of an antibody Fv fragment expressed in Escherichia coli via immobilized metal affinity chromatography . Metal affinity was mediated by fusing hexahistidine tails to both the VL and the VH domain and was thus independent of the antigen-binding specificity . Unexpectedly, it was not possible to isolate the Fv fragment with correct stoichiometric composition of the two variable domains under standard chromatographic conditions . Proper non-covalent association of VL and VH was, however, maintained when using glycine betaine as electrolyte, thus permitting purification of the intact Fv fragment to homogeneity in a single step.

Gene, 1993 Dec 22, 136(1-2), 337 - 40
Overproduction of the toxic protein, bovine pancreatic DNaseI, in Escherichia coli using a tightly controlled T7-promoter-based vector; Doherty AJ et al.; A synthetic gene coding for bovine pancreatic DNaseI has been cloned under the control of a T7 promoter present on the plasmid pET11 . This construct yields a stable Escherichia coli transformant only when transcription from this promoter is tightly controlled . Production of recombinant DNaseI (reDNaseI) is achieved by infection of the cells with a mutant lambda phage, CE6, which carries the gene encoding T7 RNA polymerase . Induced bacterial cultures yield in excess of 2 mg per litre of reDNaseI after purification.

Gene, 1993 Dec 22, 136(1-2), 287 - 90
A PCR method for the sequence analysis of the gyrA, polA and rnhA gene segments from mycobacteria; Mizrahi V et al.; Internal segments of the gyrA and polA genes involved in DNA replication of Mycobacterium tuberculosis and rnhA of M . smegmatis, have been amplified by the polymerase chain reaction (PCR) using degenerate oligodeoxyribonucleotide primers based on conserved sequences . The deduced amino acid sequences were 54-66% homologous to the corresponding segments of their Escherichia coli counterparts . This method provides a useful means of cloning genes encoding DNA replication enzymes of mycobacteria.

Gene, 1993 Dec 22, 136(1-2), 243 - 6
Construction and expression of a synthetic streptavidin-encoding gene in Escherichia coli; Thompson LD et al.; A synthetic gene encoding 'core' streptavidin (SAV) {amino acid (aa) residues 13-140 of Streptomyces avidinii SAV} has been efficiently expressed in Escherichia coli from the IPTG-inducible lac promoter of plasmid pET3a . In this system, expression levels are nearly tenfold greater for the synthetic gene than for the corresponding native gene . The synthetic gene was constructed from overlapping oligodeoxyribonucleotides whose sequences were optimized to incorporate codons preferred by highly expressed E . coli genes . Biochemical characterizations by gel methods, aa analysis, N-terminal sequencing, and size exclusion chromatography show that the synthetic gene product purified by affinity chromatography possesses the properties expected for core SAV.

Gene, 1993 Dec 22, 136(1-2), 237 - 42
Controlled high-level expression of the lon gene of Escherichia coli allows overproduction of Lon protease; Thomas CD et al.; Lon protease from Escherichia coli is an ATP-dependent protease which plays important roles in regulating the levels of specific proteins and in eliminating abnormal proteins . A major problem of working with Lon protease, the inability to substantially overproduce the enzyme, has been overcome by placing the lon gene under the control of an inducible trp promoter within a copy-number-controllable plasmid . Induction resulted in higher levels of production of the protease (approximately 100 micrograms/ml of cell culture) than were previously possible . The enzyme has been purified to apparent homogeneity and shown to possess the characteristic ATP-dependent proteolytic activity . Sequence verification during DNA manipulations revealed differences from two previously published sequences for the lon gene.

Gene, 1993 Dec 22, 136(1-2), 227 - 30
The deduced amino-acid sequence of the cloned cpxR gene suggests the protein is the cognate regulator for the membrane sensor, CpxA, in a two-component signal transduction system of Escherichia coli; Dong J et al.; The cpxA gene of Escherichia coli K-12 encodes a membrane-associated sensor element of a two-component signal transduction system in bacteria . The cognate regulator element, however, has not yet been definitively identified . A 2.1-kb segment upstream from cpxA was amplified by polymerase chain reaction, cloned and sequenced . An open reading frame encoding 232 amino acids was found . It showed high homology to the regulator elements of two-component transduction systems . The newly identified gene, designated as cpxR, may encode the cognate protein receiving signals from CpxA.

Gene, 1993 Dec 22, 136(1-2), 215 - 9
Tropist3: a cosmid vector for simplified mapping of both G+C-rich and A+T-rich genomic DNA; De Smet KA et al.; We have constructed a cosmid vector, Tropist3, based on the lambda origin double-cos-site vector Lawrist4, which is designed for efficient cloning and mapping of genomic DNA . Tropist3 contains two cloning sites in addition to the HindIII and BamHI sites present in Lawrist4; a SalI site allows cloning of Sau3AI partial digests following partial filling-in of the ends, and a PmlI site is suitable for blunt-end cloning . Both these strategies reduce the chance of co-cloning two inserts . Tropist3 also contains NotI, PacI, SacII and KpnI sites flanking the cloning region; these allow most inserts to be excised cleanly and mapped by partial digestion followed by hybridization with short vector sequences which lie adjacent to the cloning sites . This will also be useful for recloning inserts into different vectors, or for cosmid sequencing projects.

Gene, 1993 Dec 22, 136(1-2), 199 - 203
A versatile and highly repressible Escherichia coli expression system based on invertible promoters: expression of a gene encoding a toxic product; Wulfing C et al.; A very flexible and tightly regulatable expression system has been constructed . It uses the principle of invertible promoters {Podhajska et al., Gene 40 (1985) 163-168} . Here, we describe the construction of a plasmid that provides the integrase, which causes promoter inversion in a tightly regulated fashion, as well as modified plasmids carrying the invertible module . The way the integrase is provided on a separate plasmid closely mimicks expression of the integrase from a lambda lysogen . Thus, the flexibility of the original system is considerably extended by making it strain-independent without compromising the tight regulation . We present the expression of a single-chain T-cell receptor fragment as an example of application, in order to illustrate the properties of this expression system.

Gene, 1993 Dec 22, 136(1-2Che), 185 - 92
Cloning, expression and characterisation of the cDNA encoding human hepatic squalene synthase, and its relationship to phytoene synthase; Summers C et al.; The reaction catalysed by squalene synthase (SQS) shows many similarities to that performed by another polyisoprene synthase, phytoene synthase (PhS) . By identifying sequences conserved between yeast SQS (ySQS) and PhS, we have cloned a 2-kb cDNA (hSQS) encoding human SQS, a protein of 417 amino acids with a predicted M(r) of 48,041, which has only limited homology to ySQS . When expressed in E . coli, the hSQS cDNA directed the production of active enzyme . Two hSQS mRNA species of 2.0 and 1.55 kb have been identified which differ in their 3' untranslated sequences . The two mRNAs are present in roughly equal amounts in heart, placenta, lung, liver, kidney and pancreas, but the 2-kb mRNA predominates in brain and skeletal muscle . In HepG2 cells, both mRNAs are induced 2-4-fold by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, lovastatin . In contrast, Northern blot analysis of rat tissues reveals only a 2.0-kb mRNA, which is considerably up-regulated in vivo by lovastatin.

Gene, 1993 Dec 22, 136(1-2), 129 - 36
Stable transformation of mosquito cell lines using a hsp70::neo fusion gene; Lycett GJ et al.; Mosquito cell culture transfection will allow the advancement of genetic studies of these important disease-transmitting insects . Towards this end, we report the generation of stably transformed Aedes aegypti Mos20 cells using a plasmid construct containing the Tn5 neo gene, the Drosophila melanogaster hsp70 promoter, an SV40 intron and poly adenylation sequence, and a pBR 322 backbone . The apparent frequency of transfection, as measured by transient resistance of cell colonies to Geneticin (G418), ranged between 1 x 10(-4) and 1 x 10(-5), whereas the mean frequency of transformation, as assessed by establishment of cloned lines, was 3.3 x 10(-6) . The stable cell lines display typical characteristics common to mammalian cell lines transformed with plasmids, including stable resistance to G418 after removal of selection, and co-transformation with unlinked plasmids . However, in contrast to the report of transformation of Ae . albopictus cells {Monroe et al., Proc . Natl . Acad . Sci . USA 89 (1992) 5725-5729}, the plasmids within transformed Ae . aegypti cells have a wide range of copy number (3 to 5000), are extensively rearranged, and are only found integrated into the chromosome.

Gene, 1993 Dec 22, 136(1-2), 323 - 8
HIV1 integrase expressed in Escherichia coli from a synthetic gene; Holler TP et al.; Human immunodeficiency virus type 1 (HIV1) integrase is cleaved from the gag-pol precursor by the HIV1 protease . The resulting 32-kDa protein is used by the infecting virus to insert a linear, double-stranded DNA copy of its genome, prepared by reverse transcription of viral RNA, into the host cell's chromosomal DNA . In order to achieve high levels of expression, to minimize an internal initiation problem and to facilitate mutagenesis, we have designed and synthesized a gene encoding the integrase from the infectious molecular clone, pNL4-3 . Codon usage was optimized for expression in Escherichia coli and unique restriction sites were incorporated throughout the gene . A 905-bp cassette containing a ribosome-binding site, a start codon and the integrase-coding sequence, sandwiched between EcoRI and HindIII sites, was synthesized by overlap extension of nine long synthetic oligodeoxyribonucleotides {90-120 nucleotides (nt)} and subsequent amplification using two primers (28-30 nt) . The cassette was subcloned into the vector pKK223-3 for expression under control of a tac promoter . The protein produced from this highly expressed gene has the expected N-terminal sequence and molecular mass, and displays the DNA processing, DNA joining and disintegration activities expected from recombinant integrase . These studies have demonstrated the utility of codon optimization, and lay the groundwork for structure-function studies of HIV1 integrase.

Biochemistry, 1993 Dec 21, 32(50), 14053 - 61
Triple aminoacylation specificity of a chimerized transfer RNA; Frugier M et al.; We report here the rational design and construction of a chimerized transfer RNA with tripartite aminoacylation specificity . A yeast aspartic acid specific tRNA was transformed into a highly efficient acceptor of alanine and phenylalanine and a moderate acceptor of valine . The transformation was guided by available knowledge of the requirements for aminoacylation by each of the three amino acids and was achieved by iterative changes in the local sequence context and the structural framework of the variable loop and the two variable regions of the dihydrouridine loop . The changes introduced to confer efficient acceptance of the three amino acids eliminate aminoacylation with aspartate . The interplay of determinants and antideterminants for different specific aminoacylations, and the constraints imposed by the structural framework, suggest that a tRNA with an appreciable capacity for more than three efficient aminoacylations may be inherently difficult to achieve.

Biochemistry, 1993 Dec 21, 32(50), 14015 - 22
Physiological inhibitors of the catalytic subunit of cAMP-dependent protein kinase: effect of MgATP on protein-protein interactions; Herberg FW et al.; The catalytic (C) subunit of cAMP-dependent protein kinase interacts with two classes of inhibitors . The regulatory (R) subunits, types I and II, associate to form an inactive holoenzyme complex that is activated in response to cAMP . The C-subunit is also inhibited by small heat-stable protein kinase inhibitors (PKI's) . Inhibition by both PKI and RI-subunit requires the synergistic high-affinity binding of MgATP . The stabilizing effect of ATP was quantitated by using analytical gel chromatography . Both the type I holoenzyme and the C.PKI complex in the presence of MgATP show apparent Kd's for subunit association that are below 0.1 nM, while in the absence of MgATP the apparent Kd's are 125 nM and 2.3 microM, respectively, for the two complexes . In the absence of MgATP both complexes also can be dissociated readily and, hence, activated by salt-induced dissociation . Under physiological salt concentrations, salt-induced dissociation would be substantial in the absence of the high-affinity binding of MgATP . In both complexes, the ATPase activity of the free C-subunit is abolished . The off rates for MgATP also indicate that the type I holoenzyme is more stable than the C.PKI complex . The off rate (t1/2) for MgATP from the C.PKI complex is 17 min, while the off rate for the type I holoenzyme is 11.7 h . When the C.PKI complex is incubated with RI-subunit in the presence or absence of MgATP, the C-subunit preferentially reassociates with the RI-subunit, forming holoenzyme . In contrast, free PKI cannot compete for the C-subunit when it is part of a holoenzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Dec 21, 32(50), 13991 - 9
Membrane-bound conformation of a signal peptide: a transferred nuclear Overhauser effect analysis; Wang Z et al.; We have determined the conformation of an analogue of the Escherichia coli LamB signal peptide inserted into a model membrane using the transferred nuclear Overhauser effect (trNOE) NMR technique . In order to make NMR analysis feasible, a water-soluble LamB signal peptide analogue was designed by inserting three basic residues (KRR) into the N-terminal region of the wild-type sequence (with a Val-->Trp mutation for fluorescence measurements), viz., MMITLRKRRKLPLAVAVAAGWMSAQAMA-NH2 . For the purpose of the trNOE study, the binding affinity of the peptide for phospholipid vesicles was tuned by adjusting the proportion of acidic lipid in the vesicle . Circular dichroism and fluorescence measurements showed that the KRR-LamB signal peptide spontaneously inserted into the lipid bilayer with a conformational transition from a mostly random coil to a predominantly alpha-helical structure . The trNOE analysis revealed that the alpha-helix extended from approximately the beginning of the hydrophobic core (residue Leu8) to the C-terminus . The continuity of the helix was somewhat disrupted at the end of the hydrophobic core (around residue Gly17) . Furthermore, the topological arrangement of the peptide within the lipid bilayer was explored by NMR line broadening induced by a paramagnetic nitroxide-labeled lipid . The line-broadening results demonstrated that the residues in the helical region are well integrated into the acyl chain region of the bilayer . The N-terminal part of the peptide showed many trNOEs, but without any indication of a helical conformation . The line-broadening analysis indicates that this part of the peptide primarily interacts with the membrane surface.

Biochemistry, 1993 Dec 21, 32(50), 13981 - 90
Urea-induced unfolding of the alpha subunit of tryptophan synthase: one-dimensional proton NMR evidence for residual structure near histidine-92 at high denaturant concentration; Saab-Rincon G et al.; The urea-induced unfolding reaction of the alpha subunit of tryptophan synthase was monitored by examining the chemical shifts and peak areas of the C epsilon protons of the four histidine residues with 1D NMR spectroscopy . In a native base-line region defined by tyrosine absorbance and far-UV circular dichroism spectroscopy, histidine-146 appears to undergo a rapid, local unfolding reaction at increasing denaturant concentrations . As the native form is converted to a previously detected stable intermediate between 2 and 3 M urea {Matthews, C . R., & Crisanti, M . M . (1981) Biochemistry 20, 784}, histidines-92 and -146 in the amino folding unit (residues 1-188) and histidines-195 and -244 in the carboxy folding unit (residues 189-268) all experience a change in their environments which is slow on the NMR time scale . The subsequent conversion of this intermediate to a newly detected, stable, partially folded form populated at 5 M urea appears to have no effect on any of the histidines at 25 degrees C when an intermolecular association process involving His-244 is taken into account . Strikingly, a slow exchange process involving only His-92 is observed to begin at 5 M urea where the unfolding transitions monitored by absorbance or far-UV circular dichroism spectroscopy are essentially complete . This residual tertiary structure unfolds in a cooperative fashion as the urea concentration is increased to 8 M.

Biochemistry, 1993 Dec 21, 32(50), 13925 - 32
Fluorescence lifetimes of the tryptophan residues in ornithine transcarbamoylase; Shen WH; Multifrequency (2-230 MHz) phase-modulation fluorescence measurements and site-directed mutagenesis have been employed to assign fluorescence lifetimes, quantum yields, and emission maxima to the four tryptophans in the enzyme ornithine transcarbamoylase from Escherichia coli (OTCase) (Trp-125, -92, -233, and -243) . OTCase displays two apparent fluorescence lifetimes, 7.2 and 3.2 ns . Results on specific mutants show that Trp-233 has a lifetime of 7.1 ns, while TRP-125, -192, and -243 have lifetimes of 4.0, 3.6, and 4.9 ns, respectively . Thus, the specific conformational changes of the polypeptide segment involving Trp-233 may be monitored conveniently in the wild-type enzyme . On the basis of quantum yield values, Trp-233 is calculated to contribute approximately 43% of the fluorescence intensity of the enzyme, while direct measurements of the enzyme show that up to 65% of the total intensity is really emitted by this tryptophan . The discrepancy may arise from energy transfer from Trp-125 to Trp-233, with an efficiency of 20% . Application of the assigned tryptophan lifetimes to probe ligand-induced protein conformational changes has also been demonstrated.

Biochemistry, 1993 Dec 21, 32(50), 13809 - 17
Tertiary structures of class I ubiquitin-conjugating enzymes are highly conserved: crystal structure of yeast Ubc4; Cook WJ et al.; The three-dimensional structure of a yeast ubiquitin-conjugating enzyme, encoded by the Saccharomyces cerevisiae UBC4 gene, has been determined at 2.7 A . The structure was solved using molecular replacement techniques and refined by simulated annealing to an R-factor of 0.198 . Bond lengths and angles in the molecule have root mean square deviations from ideal values of 0.018 A and 4.0 degrees, respectively . Ubc4 is an alpha/beta protein with four alpha-helices and a four-stranded antiparallel beta-sheet . The ubiquitin-accepting cysteine is located in a cleft between two loops . Comparison with the recently determined structure of a different plant enzyme suggests that class I ubiquitin-conjugating enzymes are highly conserved in their three-dimensional folding . Except for two extra residues at the N- and the C-terminus of the plant enzyme, the C alpha atoms of the two enzymes can be superimposed with a root mean square deviation of only 1.52 A . Greater variations are found between the surfaces of the two molecules, as most of the identical residues between the two enzymes are either buried or clustered on the surface that lies adjacent to the ubiquitin-accepting cysteine . We suggest that this conserved surface functions in protein-protein binding during ubiquitin thiol ester formation.

J Theor Biol, 1993 Dec 21, 165(4), 659 - 72
An improved method for detection of words with unusual occurrence frequency in nucleotide sequences; Colosimo A et al.; A statistical analysis designed to deal with the problem of identifying rare or abundant "words" of arbitrary length in genomic fragments is presented . Our approach has the novelty of taking into account the statistical role of the presence of shorter words nested into longer ones and of introducing a Bayesian correction to minimize the effects of statistical fluctuations and of possible mistakes in genomic data . The method is successfully used in a thorough analysis of the abundance of short nucleotide sequences in the Escherichia coli genome.

J Mol Biol, 1993 Dec 20, 234(4), 965 - 74
Overproduction and purification of native and queuine-lacking Escherichia coli tRNA(Asp) . Role of the wobble base in tRNA(Asp) acylation; Martin F et al.; Escherichia coli tRNA(Asp) was overproduced in E . coli up to 15-fold from a synthetic tRNA(Asp) gene placed in a plasmid under the dependence of an isopropyl-beta,D-thiogalactopyranoside-inducible promoter . Purification to nearly homogeneity (95%) was achieved after two HPLC DEAE-cellulose columns . E . coli tRNA(Asp){G34} (having guanine instead of queuine at position 34) was obtained by the same procedure except that it was overproduced in a strain lacking the enzyme responsible for queuine modification . Nucleoside analysis showed that, except for the replacement of Q34 by G34 in mutant-derived tRNA(Asp), the base modification levels of both tRNAs are the same as those in wild-type E . coli tRNA(Asp) . Kinetic properties of tRNA(Asp){Q34} and {G34} with yeast AspRS compared to those in the homologous reactions in yeast and E . coli clearly indicate that the major identity elements are the same in both organisms: the conserved discriminant base and the anticodon triplet . In connection with this, we explored by site-directed mutagenesis the functional role of the interactions which, as revealed by the crystallographic structure, occur between the wobble base of yeast tRNA(Asp) and two residues of yeast AspRS . Their absence strongly affected aspartylation and the kd of tRNA(Asp) . Each contact individually restores almost completely the wild-type acylation properties of the enzyme; thus, wobble base recognition in yeast appears to be more protected against mutational events than in E . coli, where only one contact is thought to occur at position 34.

J Mol Biol, 1993 Dec 20, 234(4), 1270 - 3
Crystallization of the NADP(+)-dependent glutamate dehydrogenase from Escherichia coli; Korber FC et al.; The NADP(+)-dependent hexameric glutamate dehydrogenase from Escherichia coli has been crystallized as the apo-enzyme and also in the presence of its substrates 2-oxoglutarate, glutamate or NADP+, using either pulsed equilibrium microdialysis, or the hanging drop method of vapour diffusion . Three non-isomorphous, but related, crystal forms have been obtained, all of which belong to the orthorhombic system and are most likely to be in space group P2(1)2(1)2(1) . One crystal form is grown from ammonium sulphate, includes the apoenzyme and the binary complexes with 2-oxoglutarate or NADP+, and has cell dimensions a = 157.5 A, b = 212.5 A, c = 101.0 A with a hexamer in the asymmetric unit . Crystallizations using glutamate as the precipitant produced two further crystal forms, which show significant changes in the b and c cell dimensions with respect to the apo-enzyme crystals, with parameters a = 160.0 A, b = 217.5 A c = 92.4 A and a = 160.0 A, b = 223.0 A c = 92.4 A, respectively . X-ray diffraction photographs taken with synchrotron radiation show measurable reflections to beyond 3.0 A resolution.

J Mol Biol, 1993 Dec 20, 234(4), 1263 - 5
Crystallization and preliminary X-ray analysis of human tissue factor extracellular domain; Boys CW et al.; The extracellular domain (residues 1 to 220) of human tissue factor has been cloned and expressed in Escherichia coli and purified to isoelectric homogeneity . Single crystals suitable for X-ray analysis have been obtained by vapour diffusion . They belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with a = b = 45.2 A, c = 231.5 A, contain one molecule per asymmetric unit and diffract to 2.6 A resolution . Native and derivative data sets have been collected to 3.6 and 3.9 A, respectively.

J Mol Biol, 1993 Dec 20, 234(4), 1248 - 9
Crystallization and preliminary X-ray studies of L-aspartase from Escherichia coli; Shi W et al.; Single crystals of L-aspartate ammonia-lyase (L-aspartase) from Escherichia coli have been obtained by microdialysis at room temperature using polyethylene glycol 3350 and sodium acetate as co-precipitants . The crystals exhibit the symmetry of space group P2(1)2(1)2 with a = 156.5 A, b = 147.6 A, c = 102.5 A and diffract at least to 2.8 A.

J Mol Biol, 1993 Dec 20, 234(4), 1218 - 29
Role of an active site residue analyzed by combination of mutagenesis and coenzyme analog; Yano T et al.; Asp222 of aspartate aminotransferase is an active-site residue which interacts with the pyridine nitrogen of the coenzyme, pyridoxal 5'-phosphate (PLP) . The roles of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase have previously been explored by site-directed mutagenesis . These studies confirmed that a negatively charged residue at position 222 is essential for catalysis, but the reason for this remained speculative . In the present studies, the roles of Asp222 were clarified experimentally by analyzing the mutant D222A enzyme (Asp222 replaced by Ala) reconstituted with the coenzyme analog N(1)-methylated PLP (N-MePLP) . Spectroscopic and kinetic analyses showed that Asp222 stabilizes the protonated N(1) of PLP, raising the pKa value of N(1) by more than five units, in the active site of AspAT . The positive charge at N(1) accelerates abstraction of the alpha-proton from the amino acid substrate, stabilizing the transition state by 1.4 to 4.5 kcal.mol-1 in the reaction with aspartate . X-ray crystallographic (2.0 A resolution) and CD spectroscopic studies suggest that the coenzyme analog is not held in a proper orientation within the active site of D222A (N-MePLP) . This may account for the finding that the catalytic activity was recovered only partially by the reconstitution of D222A with N-MePLP . These results fully support the following postulated role of Asp222: the negative charge of Asp222 stabilizes the positive charge at N(1) of PLP and thereby enhances the function of PLP as an electron sink.

J Mol Biol, 1993 Dec 20, 234(4), 1197 - 206
Further examination of the intermediate state in the denaturation of the tryptophan synthase alpha subunit . Evidence that the equilibrium denaturation intermediate is a molten globule; Ogasahara K et al.; To further characterize the intermediate state in the denaturation of tryptophan synthase alpha subunit from Escherichia coli, we have carried out differential scanning calorimetry in various concentrations of urea near pH 8.5 . The heat capacity curve of the intermediate has no excess heat capacity nor any transition . This indicates that the intermediate is a thermodynamically denatured form . Although the intermediate retains significant CD signal in the far-uv region, the tertiary structure of the intermediate is disrupted as judged by the near-uv CD spectra and 1H NMR spectra in the aromatic region . This intermediate might be similar to a molten globule state . These results do not support our earlier proposal that the intermediate of the alpha subunit in the denaturation process retains an intact N-terminal domain, but that the C-terminal domain unfolds.

FEBS Lett, 1993 Dec 20, 336(1), 43 - 7
A mutation causing constitutive synthesis of the pyruvate dehydrogenase complex in Escherichia coli is located within the pdhR gene; Haydon DJ et al.; The aceE-aceF-lpd genes encoding the pyruvate dehydrogenase (PDH) complex of Escherichia coli are preceded by a gene encoding a putative transcriptional regulator, PdhR (formerly designated GenA) . Enzymological tests and studies with pdhR-lacZ and aceE-lacZ translational fusions have shown that a constitutive mutation (acec816), which increases PDH complex synthesis to the pyruvate-induced level in the absence of inducer, is recessive to the wild-type pdhR gene in trans . Sequence comparisons further showed that the acec816 mutation affects a single site in the pdhR gene leading to an Arg118 (CGU)-->Cys (UGU) substitution in the PdhR protein . The results support the view that synthesis of the PDH complex is regulated from the pdhR promoter of a pdhR-aceEF-lpd operon.

FEBS Lett, 1993 Dec 20, 336(1), 148 - 52
Crystallization and crystallographic investigations of ribonucleotide reductase protein R1 from Escherichia coli; Uhlin U et al.; Crystals of Escherichia coli ribonucleotide reductase protein R1 have been grown in complex with a synthetic peptide corresponding to the carboxyl end of protein R2 . Good quality crystals could only be obtained after improvement of the purification protocol and are of the space group R32 with hexagonal cell axes a = b = 226 A and c = 341 A . They contain 3 subunits per asymmetric unit and diffract to 2.5 A resolution in synchrotron radiation . A multiple isomorphous replacement map at 5.5A, improved by solvent flattening, shows that the dimeric molecules are elongated, about 110 A long . The dimer is thin in the middle around the molecular two-fold axis . The subunit is shaped like a bowl, probably with the active site in its center.

FEBS Lett, 1993 Dec 20, 336(1), 124 - 8
DnaK ATPase activity revisited; Palleros DR et al.; It has recently been reported that the ATPase activity of DnaK, a 70 kDa heat shock protein from E . coli, is autostimulated by increasing protein concentration {(1993) FEBS Lett . 322, 277-279}, suggesting that the DnaK dimer may be the enzymatically active species . In this paper we investigated the ATPase activity of different DnaK preparations; we found that the turnover number was very dependent on protein purification . With HPLC-purified DnaK we found a turnover number 20- to 50-fold lower than typical values previously published and no evidence of autostimulation, indicating that the monomer is the active species.

J Mol Biol, 1993 Dec 20, 234(4), 1259 - 62
Crystallization and preliminary X-ray analysis of gamma-glutamyltranspeptidase from Escherichia coli K-12; Kumagai H et al.; gamma-Glutamyltranspeptidase (EC 2.3.2.2) from Escherichia coli K-12 has been purified and crystallized by means of vapor diffusion in hanging drops . Two kinds of crystals on cell dimensions were found for X-ray diffraction analysis, one from ammonium sulfate and the other from polyethylene glycol 6000 as precipitants . The crystals of the orthorhombic form grown in the presence of 15% polyethylene glycol and 20 mM sodium acetate buffer were chosen for further analysis . The crystals belonged to space group P2(1)2(1)2(1), with cell dimensions of a = 128.1, b = 129.9 and c = 79.2 A, and two molecules constitute an asymmetric unit . These crystals diffracted to 2.0 A resolution and were suitable for X-ray crystallographic studies.

J Mol Biol, 1993 Dec 20, 234(4), 1140 - 57
Detecting frame shifts by amino acid sequence comparison; Claverie JM; Various amino acid substitution scoring matrices are used in conjunction with local alignments programs to detect regions of similarity and infer potential common ancestry between proteins . The usual scoring schemes derive from the implicit hypothesis that related proteins evolve from a common ancestor by the accumulation of point mutations and that amino acids tend to be progressively substituted by others with similar properties . However, other frequent single mutation events, like nucleotide insertion or deletion and gene inversion, change the translation reading frame and cause previously encoded amino acid sequences to become unrecognizable at once . Here, I derive five new types of scoring matrix, each capable of detecting a specific frame shift (deletion, insertion and inversion in 3 frames) and use them with a regular local alignments program to detect amino acid sequences that may have derived from alternative reading frames of the same nucleotide sequence . Frame shifts are inferred from the sole comparison of the protein sequences . The five scoring matrices were used with the BLASTP program to compare all the protein sequences in the Swissprot database . Surprisingly, the searches revealed hundreds of highly significant frame shift matches, of which many are likely to represent sequencing errors . Others provide some evidence that frame shift mutations might be used in protein evolution as a way to create new amino acid sequences from pre-existing coding regions.

FEBS Lett, 1993 Dec 20, 336(1), 19 - 22
The reaction of GroEL (cpn 60) with the ATP analogue 2',3' dialdehyde ATP; Thomson GJ et al.; The reaction of the E . coli chaperonin GroEL (cpn 60) with the ATP analogue 2',3' oxidised ATP (oATP) has been studied . Treatment with the reagent leads to loss of the ATPase activity of GroEL in a pseudo-first-order fashion; this can be prevented by inclusion of ATP in the reaction mixture . Measurements of the stoichiometry of the reaction indicate that the loss of activity corresponds to the incorporation of about one oATP per subunit of GroEL . From analysis of the sequences of modified peptides it is proposed that the reaction probably occurs with one or both of the two cysteines Cys-457 and Cys-518, although the instability of the adduct(s) makes a definite identification of the site(s) of reaction difficult . The involvement of Cys side chains in the reaction with oATP was confirmed by using Nbs2 (5,5'-dithiobis(2-nitrobenzoate)) to estimate thiol groups in both modified and unmodified GroEL.

J Mol Biol, 1993 Dec 20, 234(4), 998 - 1012
The role of RNA structure in determining RNase E-dependent cleavage sites in the mRNA for ribosomal protein S20 in vitro; Mackie GA et al.; An RNA encompassing the 3' 147 residues of the mRNA for ribosomal protein S20 in Escherichia coli constitutes a naturally occurring degradative intermediate whose formation depends on RNase E . We have investigated the role of internal stem-loop structures in the RNase E-dependent cleavage which generates this product from S20 mRNA in a partially fractionated processing system in vitro . Individual stem-loops have been removed by deletion or destabilized by point mutations . No single hairpin structure is absolutely required for RNase E-dependent cleavage at the site 147 residues from the 3' end of the RNA . Primary sequences or secondary structures 5' or 3' to this site exert only a modest influence on the specificity of cleavage but can strongly modify its rate . Moreover, mutations in the S20 mRNA which destabilize stems 5' or 3' to the prominent cleavage site also reveal several strong cryptic RNase E cleavage sites . These data greatly strengthen the hypothesis that RNase E is a single-strand specific endoribonuclease . Our data further demonstrate that stem-loop structures adjacent to the prominent cleavage site are unlikely to provide a site of recognition for RNase E . Rather, they appear to stabilize (or "anchor") the local secondary structure so that the cleavage site is single-stranded and to occlude alternative sites so that the initial products of cleavage resist further attack.

J Mol Biol, 1993 Dec 20, 234(4), 1021 - 37
An imbalance of HU synthesis induces mucoidy in Escherichia coli; Painbeni E et al.; Mutations in a number of loci, including the lon gene, dramatically increase the production of colanic acid capsular polysaccharide and render Escherichia coli K-12 mucoid . The lon gene, which encodes an ATP-dependent protease, is localized at ten minutes on the E . coli map and is very closely linked to the hupB gene coding for one of the two subunits of the histone-like protein HU . Surprisingly the introduction of a multi-copy plasmid carrying either the hupB or hupA gene into a wild-type E . coli strain, results in the overproduction of one of the HU subunits and repression of the synthesis of the other without changing the overall concentration of HU, also renders the cells mucoid . As in a lon strain, the transcription of the cps genes, the structural genes for the synthesis of colanic acid, is induced dramatically . Protease Lon negatively regulates cps genes by destabilizing RcsA, a positive regulator of capsule synthesis . Regulation of HU synthesis does not affect the steady state level of Lon, as judged by Western blotting . The UV sensitivity of the hup transformed lon+ bacteria is identical to the lon+ parental strain, suggesting that Lon activity for the degradation of SulA in these cells is normal . Using lac operon fusions to cps gene promoters and to the rcsA promoter we show that the deregulation of HU synthesis does not by pass the positive regulatory action of RcsA and RcsB for the expression of cps genes but functions by stimulating RcsA synthesis.

Science, 1993 Dec 17, 262(5141), 1886 - 9
Functional requirement of a site-specific ribose methylation in ribosomal RNA; Sirum-Connolly K et al.; The product of the PET56 nuclear gene of Saccharomyces cerevisiae was shown to be required for ribose methylation at a universally conserved nucleotide in the peptidyl transferase center of the mitochondrial large ribosomal RNA (21S rRNA) . Cells reduced in this activity were deficient in formation of functional large subunits of the mitochondrial ribosome . The purified Pet56 protein catalyzed the site-specific formation of 2'-O-methylguanosine on in vitro transcripts of both mitochondrial 21S rRNA and Escherichia coli 23S rRNA . These results provide evidence for an essential modified nucleotide in rRNA.

Cell, 1993 Dec 17, 75(6), 1061 - 70
Specific interactions between proteins implicated in splice site selection and regulated alternative splicing; Wu JY et al.; Specific recognition and pairing of the 5' and 3' splice sites are critical steps in pre-mRNA splicing . We report that the splicing factors SC35 and SF2/ASF specifically interact with both the integral U1 small nuclear ribonucleoprotein (snRNP U1-70K) and with the 35 kd subunit of the splicing factor U2AF (U2AF35) . Previous studies indicated that the U1 snRNP binds specifically to the 5' splice site, while U2AF35-U2AF65 heterodimer binds to the 3' splice site . Together, these observations suggest that SC35 and other members of the SR family of splicing factors may function in splice site selection by acting as a bridge between components bound to the 5' and 3' splice sites . Interestingly, SC35, SF2/ASF, and U2AF35 also interact with the Drosophila splicing regulators Transformer (Tra) and Transformer-2 (Tra2), suggesting that protein-protein interactions mediated by SR proteins may also play an important role in regulating alternative splicing.

Cell, 1993 Dec 17, 75(6), 1199 - 214
Disruption of Krox-20 results in alteration of rhombomeres 3 and 5 in the developing hindbrain; Schneider-Maunoury S et al.; The zinc finger gene Krox-20 is transcribed in two alternate segments (rhombomeres) of the developing hindbrain . To investigate its function, we have used homologous recombination to generate mice carrying an in-frame insertion of the E . coli lacZ gene within Krox-20 . Analysis of the beta-galactosidase pattern in heterozygous embryos confirmed the known profile with expression restricted to rhombomeres (r) 3 and 5 . Mice homozygous for the mutation die during the first two weeks after birth . Anatomical analysis of the hindbrain and of the cranial nerves during embryogenesis, combined with the determination of the expression patterns of rhombomere-specific genes, demonstrated that Krox-20 inactivation results in a marked reduction or elimination of r3 and r5 . We conclude that Krox-20, although not required for the initial delimitation of r3 and r5, plays an important role in the process of segmentation governing hindbrain development.

Nature, 1993 Dec 16, 366(6456), 690 - 4
A Drosophila homologue of human Sp1 is a head-specific segmentation gene; Wimmer EA et al.; Segmentation in Drosophila is based on a cascade of hierarchical gene interactions initiated by maternally deposited morphogens that define the spatially restricted domains of gap gene expression at blastoderm (reviewed in ref . 1) . Although segmentation of the embryonic head is morphologically obscured, the repeated patterns of expression of the segment polarity genes reflect the formation of seven head segments; two of these depend on the segmentation and homeotic genes used in the trunk, whereas the others form as a result of the activity of the head-specific genes orthodenticle (otd), empty spiracles (ems) and buttonhead (btd) . The genes ems and otd encode homeodomain proteins, suggesting that they may function as transcription factors . They are expressed in overlapping stripes in the early embryonic head of Drosophila, and their vertebrate homologues, otx and emx, are expressed in overlapping domains in the anterior central nervous system of the mouse embryo . We show here that btd is expressed in a stripe covering the head analgen of the segments affected in btd lack-of-function mutants and that btd encodes a zinc-finger-type transcription factor with sequence and functional similarity to the prototype mammalian transcription factor Sp1 (ref . 9) . When expressed in the spatial pattern of btd, a transgene providing Sp1 activity can support development of the mandibular segment in the head of btd mutant embryos . A ubiquitous transcription factor from humans can therefore replace an essential component of the genetic circuitry required to specify the development of a particular head segment in the fly.

Carbohydr Res, 1993 Dec 16, 250(1), 101 - 12
Suicide-substrate inactivation of beta-galactosidase by diazomethyl beta-D-galactopyranosyl ketone; BeMiller JN et al.; Diazomethyl beta-D-galactopyranosyl ketone (1) has been proven to be a mechanism-based, irreversible (suicide-substrate) inactivator of Aspergillus oryzae beta-D-galactosidase, but not an inactivator of E . coli lacZ beta-D-galactosidase . Compound 1 is stable in buffers of normal physiological pH . It is decomposed by H+, but not by nucleophiles . Inactivation of A . oryzae beta-D-galactopyranosyl ketone (2) nor diazomethyl alpha-D-galactopyranosyl ketone inactivated the enzyme and therefore inactivation is stereospecific, excess inhibitor could be separated from inactive enzyme without regain of activity and therefore it is bound irreversibly, and a second pulse of enzyme is inactivated at the same rate as enzyme inactivated to 95% activity by the first pulse . Diazomethyl beta-D-glucopyranosyl ketone (2) inhibited sweet almond beta-D-glucosidase.

Carbohydr Res, 1993 Dec 16, 250(1), 9 - 18
beta-Galactosidases of Escherichia coli with substitutions for Glu-461 can be activated by nucleophiles and can form beta-D-galactosyl adducts; Huber RE et al.; Nucleophiles activated the catalytic actions of beta-galactosidases with neutral or positively charged substitutions for Glu-461 . Aliphatic carboxylic acids increased the rate of hydrolysis of o-nitrophenyl beta-D-galactopyranoside if the pKa values of the carboxyl groups were > approximately 3.5 . Amino compounds activated if their pKa values were < approximately 8.5 . Imidazole, azide, and 2-mercaptoethanol also activated . Nucleophiles with high pKa values were able to activate the catalysis if the pH was high, and this showed that the lack of activation at pH 7.0 was because of protonation . Kinetic analysis showed that most of the nucleophiles that activated were bound to the active site, since the activation followed Michaelis-Menten type saturation kinetics . The binding seemed to be dependent upon the hydrophobicity; the longer the aliphatic chain, the stronger the binding . Gas-liquid chromatographic analysis showed that adducts of some type were formed during the reactions in the presence of many of the nucleophiles . Three of these adducts were purified and the nucleophiles were found beta-linked to D-galactose . This indicates that if an intermediate covalent bond is formed in the mechanism of beta-galactosidase action and if the nucleophile reacts to displace it, the intermediate covalent bond must have the alpha configuration and involve a group other than Glu-461.

Eur J Biochem, 1993 Dec 15, 218(3), 937 - 44
Formation of a functionally active sodium-translocating hybrid F1F0 ATPase in Escherichia coli by homologous recombination; Kaim G et al.; A deletion mutant of Escherichia coli lacking the genes for ATPase subunits a, c, b, delta and part of the alpha subunit was transformed with a plasmid containing the corresponding genes of the sodium-translocating ATPase of Propionigenium modestum . The respective DNA fragment of P . modestum was integrated into the genome of the E . coli deletion mutant by site-specific homologous recombination . The sites of this recombination event were identified by cloning and DNA sequencing . As a consequence of the recombination event, a functionally active hybrid ATPase was obtained by in vivo complementation . The biochemical characterization of this hybrid ATPase revealed high sensitivity to dicyclohexylcarbodiimide as well as strong activation by the addition of sodium ions . After reconstitution into liposomes, the hybrid ATPase catalysed the transport of Na+ upon ATP addition . In the absence of Na+, the ATPase hybrid was able to pump protons, as was shown by the ATP-dependent fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine.

Eur J Biochem, 1993 Dec 15, 218(3), 911 - 20
The actin/actin interactions involving the N-terminus of the DNase-I-binding loop are crucial for stabilization of the actin filament; Khaitlina SY et al.; Actin can be specifically cleaved between residues 42 and 43 with a novel protease from Escherichia coli A2 strain (ECP) {Khaitlina, S . Y., Collins, J . H., Kuznetsova, I.M., Pershina, V.P., Synakevich, I.G., Turoverov, K.K . & Usmanova, A.M . (1991) FEBS Lett . 279, 49-51} . The resulting C-terminal and N-terminal fragments remained associated to one another in the presence of either Ca2+ or Mg2+ . The protease-treated actin was, however, neither able to spontaneously assemble into filaments nor to copolymerize with intact actin unless its tightly bound Ca2+ was replaced with Mg2+ . Substitution of Mg2+ for the bound Ca2+ was also necessary to partially restore the ability of the protease-treated actin to inhibit the DNase I activity . The critical concentration for KCl-induced polymerization of ECP-treated ATP-Mg-G-actin, determined by measuring the fluorescence of pyrenyl label, was approximately threefold higher than that for actin cleaved between residues 47 and 48 using subtilisin, and 36-fold higher than the critical concentration for polymerization of intact actin under the same conditions . Morphologically, the filaments of ECP-treated actin were indistinguishable from those of intact actin . Comparison of the fluorescence spectra of pyrenyl-labelled actins and chemical cross-linking with N,N'-1,2-phenylenebismaleimide have, however, revealed structural differences between the filaments assembled from ECP-treated actin and those of intact as well as subtilisin-treated actin . Moreover, the filaments of ECP-treated actin were easily disrupted by centrifugal forces or shearing stress unless they were stabilized by phalloidin . The results are consistent with the direct participation of the region around residues 42 and 43 in the monomer/monomer interactions as predicted from the atomic model of F-actin {Holmes, K.C., Popp, D., Gebhard, W . & Kabsch, W . (1990) Nature 347, 44-49} and suggest that the interactions involving this region are of primary importance for stabilization of the actin filament . The mechanism of the regulation of actin polymerization by the tightly bound divalent cation is also discussed.

Eur J Biochem, 1993 Dec 15, 218(3), 1041 - 7
Conformational differences between complexes of elongation factor Tu studied 19F-NMR spectroscopy; Eccleston JF et al.; An analogue of elongation factor Tu (EF-Tu) from Escherichia coli was prepared by biosynthetic incorporation of 3-fluorotyrosine . The 19F-NMR spectra of the binary complexes of this protein with GDP, GTP and elongation factor Ts (EF-Ts) and the ternary complexes EF-Tu.GDP.aurodox and EF-Tu.GDP.EF-Ts were measured . EF-Tu contains ten tyrosine residues and all of the complexes studied gave complex 19F spectra with overlapping resonances . EF-Tu.GDP gave a spectrum in which two signals were markedly different from those shown by the other complexes, the two resonances being shifted downfield by at least 3.4 ppm and 0.9 ppm relative to their shifts in the other complexes . Such large downfield shifts can be explained by second-order electric field shielding effects resulting from these two tyrosine residues being in a sterically constrained environment in EF-Tu.GDP and with the steric restraints being released in all of the other complexes . The X-ray diffraction structure of EF-Tu.GDP shows that Tyr87 in the N-terminal domain (domain I) and Tyr309 in the C-terminal domain (domain III) are both buried within the protein and are close to each other: these residues are in regions of EF-Tu previously implicated in the structural changes between EF-Tu.GDP and EF-Tu.GTP by other workers . If these tyrosine residues correspond to the two downfield resonances of the spectra of EF-Tu.GDP, the results from the 19F-NMR would be consistent with these earlier indications that domain I interacts closely with domain III in EF-Tu.GDP and that the amino acids between Gly83 and Gly100 are an important part of this interaction . For all the other complexes studied, these tyrosines are in a less sterically crowded environment consistent with a weaker interaction between the two domains . The 19F-NMR spectrum of the trypsin-cleaved product of EF-Tu.GDP, from which the X-ray diffraction structural data have been obtained, shows no significant differences from the native protein so that trypsin cleavage causes no large changes in the protein's structure.

Gene, 1993 Dec 15, 135(1-2), 279 - 90
From the double-helix to novel approaches to the sequencing of large genomes; Szybalski W; Elucidation of the structure of DNA by Watson and Crick {Nature 171 (1953) 737-738} has led to many crucial molecular experiments, including studies on DNA replication, transcription, physical mapping, and most recently to serious attempts directed toward the sequencing of large genomes {Watson, Science 248 (1990) 44-49} . I am totally convinced of the great importance of the Human Genome Project, and toward achieving this goal I strongly favor 'top-down' approaches consisting of the physical mapping and preparation of contiguous 50-100-kb fragments directly from the genome, followed by their automated sequencing based on the rapid assembly of primers by hexamer ligation together with primer walking . Our 'top-down' procedures totally avoids conventional cloning, subcloning and random sequencing, which are the elements of the present 'bottom-up' procedures . Fragments of 50-100 kb are prepared in sufficient quantities either by in vitro excision with rare-cutting restriction systems (including Achilles' heel cleavage {AC} or the RecA-AC procedures of Koob et al . {Nucleic Acids Res . 20 (1992) 5831-5836}) or by in vivo excision and amplification using the yeast FRT/Flp system or the phage lambda att/Int system . Such fragments, when derived directly from the Escherichia coli genome, are arranged in consecutive order, so that 50 specially constructed strains of E . coli would supply 50 end-to-end arranged approx . 100-kb fragments, which will cover the entire approx . 5-Mb E . coli genome . For the 150-Mb Drosophila melanogaster genome, 1500 of such consecutive 100-kb fragments (supplied by 1500 strains) are required to cover the entire genome . The fragments will be sequenced by the SPEL-6 method involving hexamer ligation {Szybalski, Gene 90 (1990) 177-178; Fresenius J . Anal . Chem . 4 (1992) 343} and primer walking . The 18-mer primers are synthesized in only a few minutes from three contiguous hexamers annealed to the DNA strand to be sequenced when using an over 100-fold excess of hexamers and T4 DNA ligase at room temperature, preferably in the presence of the single-strand-binding (SSB) protein of E . coli . These 18-nt primers are immediately extended by the DNA polymerase, Sequenase 2.0, in the dideoxy sequencing reaction . Very high quality sequencing ladders are obtained for single-stranded DNA or denatured double-stranded approx . 50-kb fragments, as exemplified by phage lambda DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Experientia, 1993 Dec 15, 49(12), 1095 - 7
Anti-lipopolysaccharide activity of histatins, peptides from human saliva; Sugiyama K; Histatins are histidine-rich polypeptides secreted in human saliva . They were found to inhibit lipopolysaccharide (LPS)-mediated gelation of Limulus amoebocyte lysate, and to reverse the anti-complement action of LPS or lipid A . Histatins also gave precipitate bands in agarose gels with various LPS . The results indicate that histatins neutralized the activity of LPS by binding to the lipid A moiety of LPS.

Biochem Biophys Res Commun, 1993 Dec 15, 197(2), 792 - 7
Flavodoxin is required for the activation of the anaerobic ribonucleotide reductase; Bianchi V et al.; The inactive anaerobic ribonucleotide reductase from Escherichia coli is transformed by a multienzyme system and S-adenosylmethionine + NADPH into a radical protein that is enzymatically active . One of the activating enzyme components was earlier shown to be ferredoxin (flavodoxin):NADP+ reductase . Here we present evidence that flavodoxin, but not ferredoxin, also is a component of the system . Light reduced deazaflavin can substitute for the flavodoxin system . An additional unidentified low-molecular weight component further stimulates the reaction.

Biochem Biophys Res Commun, 1993 Dec 15, 197(2), 556 - 61
Characterization, cloning and expression of the 67-kDA annexin from chicken growth plate cartilage matrix vesicles; Cao X et al.; Analysis of a 67 kDa lipid-dependent Ca(2+)-binding protein from avian matrix vesicles revealed amino acid sequences homologous to mammalian annexin VI . PCR methods were used to identify a clone from an avian cDNA library that contained a full length copy of the 67-kDa annexin cDNA . This was restriction mapped, subcloned and sequenced . The cDNA sequence of the open reading frame showed 70 percent identity to that of murine annexin VI; the predicted amino acid sequence, 78 percent identity . There was no homology in the 5'- and 3'-untranslated regions . A plasmid was constructed that overexpresses the intact chicken 67-kDa matrix vesicle annexin in E . coli DH5 alpha in high yield; the physicochemical properties and the amino terminal sequence of the expressed protein exactly matched those of the native protein.

Proc Natl Acad Sci U S A, 1993 Dec 15, 90(24), 11980 - 4
Aspartate transcarbamoylase containing circularly permuted catalytic polypeptide chains; Yang YR et al.; Based on the demonstration that active enzyme is formed in vitro and in vivo from polypeptide fragments of the catalytic chains of aspartate transcarbamoylase (ATCase; EC 2.1.3.2) and the evidence that NH2 and COOH termini of wild-type chains are in close proximity, we constructed altered genes to determine whether circularly permuted catalytic chains could fold and assemble into active catalytic trimers . Two slightly different genetic constructs led to the expression in good yield of circularly permuted catalytic chains, which associated in vivo into active trimers . They, in turn, combined in vitro with wild-type regulatory dimers to form ATCase-like molecules . Both polypeptide chains began at residue 235 in a different domain from the NH2 terminus of wild type and had an overlapping sequence of eight residues at the COOH terminus . One had a six-amino-acid linker, and the other had a deletion of four residues . Enzymes containing rearranged chains were similar to their wild-type counterparts in physical properties . Whereas values of Vmax were close to those of wild-type trimers and ATCase, the Km values were more than 10-fold greater . Also the allosteric properties characteristic of wild-type ATCase were lacking in the enzymes containing permuted chains . Denaturation of trimers by urea was reversible, and recovery of activity in both rate and yield was comparable to that of wild-type trimers . The experiments demonstrate that folding of chains into clearly defined domains and the assembly of active, thermodynamically stable oligomers are not dependent on the positions of NH2 and COOH termini; the folded structures are a consequence of the final sequence and not the order of biosynthetic addition of amino acids.

Proc Natl Acad Sci U S A, 1993 Dec 15, 90(24), 11797 - 801
Methotrexate resistance in an in vivo mouse tumor due to a non-active-site dihydrofolate reductase mutation; Dicker AP et al.; A series of methotrexate (MTX)-resistant L1210 leukemia murine ascites tumors were developed in vivo and analyzed for drug resistance . Three of 20 tumors studied expressed an altered dihydrofolate reductase (DHFR) and each was identical, having a C to T base transition at nucleotide 46 in the DHFR gene as demonstrated by PCR and direct sequencing . This transition results in a Gly to Trp substitution at amino acid 15 of the enzyme . Purified altered enzyme displays significantly lower binding affinity for the antifolates MTX, trimetrexate, edatrexate, and trimethoprim with respective Ki values 165-, 76-, 30-, and 28-fold higher than values obtained for enzyme isolated from parental tumor (wild-type enzyme) . Substrate (dihydrofolate) and cofactor (NADPH) binding is also diminished for the mutant enzyme, although to a lesser extent (17.3- and 3.6-fold higher Km, respectively) . Gly-15 is highly conserved for all vertebrate species of DHFR but has no known interaction(s), either directly or indirectly, with bound cofactor, substrate, or inhibitor . Protein molecular modeling reveals that the affected residue is 9-12 A away from the enzyme active site and located in a region analogous to the mobile Met-20 loop domain characterized for Escherichia coli DHFR.

Proc Natl Acad Sci U S A, 1993 Dec 15, 90(24), 11753 - 6
Long-range structural effects in a second-site revertant of a mutant dihydrofolate reductase; Brown KA et al.; X-ray crystal structures have been determined for a second-site revertant (Asp-27-->Ser, Phe-137-->Ser; D27S/F137S) and both component single-site mutants of Escherichia coli dihydrofolate reductase . The primary D27S mutation, located in the substrate binding pocket, greatly reduces catalytic activity as compared to the wild-type enzyme . The additional F137S mutation, which partially restores catalytic activity, is located on the surface of the molecule, well outside of the catalytic center and approximately 15 A from residue 27 . Comparison of kinetic data for the single-site F137S mutant, specifically constructed as a control, and for the double-mutant enzymes indicates that the effects of the F137S and D27S mutations on catalysis are nonadditive . This result suggests that the second-site mutation might mediate its effects through a structural perturbation propagated along the polypeptide backbone . To investigate the mechanism by which the F137S substitution elevates the catalytic activity of D27S we have determined the structure of the D27S/F137S double mutant . We also present a rerefined structure for the original D27S mutant and a preliminary structural interpretation for the F137S single-site mutant . We find that while either single mutant shows little more than a simple side-chain substitution, the double mutant undergoes an extended structural perturbation, which is propagated between these two widely separated sites via the helix alpha B.

Proc Natl Acad Sci U S A, 1993 Dec 15, 90(24), 11678 - 82
Enhancer activity of light-responsive regulatory elements in the untranslated leader regions of cyanobacterial psbA genes; Li R et al.; Three psbA genes encoding the D1 protein of the photosystem II reaction center are differentially expressed under different light intensities in the cyanobacterium Synechococcus sp . strain PCC 7942 . Two of the three psbA genes, psbAII and psbAIII, are induced rapidly when light intensity is increased from 125 x 10(-6) mol.m-2.s-1 to 750 x 10(-6) mol.m-2.s-1 . A recombinational cloning vector that carries a transcriptional lacZ reporter gene was used to characterize the controlling elements responsible for light induction . At least three distinct cis elements are present in the regulatory regions of pbsAII and psbAIII: basal promoters, comparable to Escherichia coli sigma 70 promoters in position and sequence, confer constitutive expression of the genes under both low and high light intensities; negative elements upstream of the promoters down-regulate the expression of the corresponding gene; and sequences downstream of the promoters that correspond to the untranslated leader regions of the mRNAs (+1 to +41 in psbAII and +1 to +39 in psbAIII) are responsible for increased expression under high light . When these light-responsive elements were combined with an E . coli promoter (conII) in different positions and orientations, the expression of the lacZ gene was induced 4- to 11-fold . The induction of gene expression under high light by these enhancers was position independent but orientation dependent . When the elements were combined with the conII promoter in the correct orientation, they also conferred a small but reproducible level of light-responsive expression on this E . coli promoter.

Proc Natl Acad Sci U S A, 1993 Dec 15, 90(24), 11553 - 7
The relationship between synthetic and editing functions of the active site of an aminoacyl-tRNA synthetase; Kim HY et al.; We have analyzed, by site-directed mutagenesis, the molecular basis of the editing function and its relation to the synthetic function of Escherichia coli methionyl-tRNA synthetase . The data obtained fit a model of the active site that partitions an amino acid substrate between synthetic and editing pathways . Hydrophobic and hydrogen bonding interactions direct the cognate substrate methionine through the synthetic pathway and prevent it from entering the editing pathway . Two hydrophobic interactions are proposed: between the side chain of Trp-305 and a methyl group of methionine and between the benzene ring of Tyr-15 and the beta- and gamma-CH2 groups of the substrate . An essential hydrogen bond forms between the OH of Tyr-15 and an electron pair of the sulfur atom of methionine . Consistent with these functions, side chains of Trp-305 and Tyr-15 are localized on opposite sides of the cavity forming a putative methionine binding pocket that is observed in the three-dimensional crystallographic structure of methionyl-tRNA synthetase . Enzymes W305A, Y15A, and Y15F have diminished ability to discriminate against homocysteine in the synthetic reaction, compared to the wild-type enzyme . At the same time, mutant enzymes have lost the ability to discriminate against methionine in the editing reaction and edited Met-AMP to a similar extent as Hcy-AMP . Interactions of residues Arg-233 and Asp-52 of methionyl-tRNA synthetase with the carboxyl and amino groups, respectively, of the substrate, which are essential for the synthetic function, were also essential for the editing function of the enzyme . Deacylation of Met-tRNA to S-methylhomocysteine thiolactone catalyzed by W305A, Y15A, and Y15F mutant enzymes was only slightly impaired relative to the wild-type enzyme . However, enzymes R233Q, R233A, and D52A did not deacylate Met-tRNA . The model also explains why the noncognate homocysteine is edited by methionyl-tRNA synthetase.

EMBO J, 1993 Dec 15, 12(13), 5237 - 44
The beta 1 and beta 2 subunits of the AP complexes are the clathrin coat assembly components; Gallusser A et al.; The beta 1 and beta 2 subunits are the closely-related large chains of the trans-Golgi network AP-1 and the plasma membrane AP-2 clathrin-associated protein complexes, respectively . Recombinant beta 1 and beta 2 subunits have been generated in Escherichia coli . It was found that, in the absence of all the other AP subunits, beta 1 and beta 2 interact with clathrin and drive the efficient assembly of clathrin coats . In addition, beta 2 subunits and AP complexes compete for the same clathrin binding site . The appearance of the clathrin/beta coats is the same as the barrel-shaped structures formed with native AP complexes . It is proposed that the principal function of the beta subunits is to initiate coat formation, while the remaining subunits of the AP complexes have other roles in coated pit and coated vesicle function.

J Biol Chem, 1993 Dec 15, 268(35), 26738 - 44
Function of the zinc finger in Escherichia coli Fpg protein; Tchou J et al.; Fpg protein of Escherichia coli cleaves duplex DNA containing the oxidatively damaged base 8-oxo-7,8-dihydroguanine (Tchou, J., Kasai, H., Shibutani, S., Chung, M.-H., Laval, J., Grollman, A . P., and Nishimura, S . (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 4690-4694) . This DNA repair enzyme contains one zinc atom/protein molecule (Boiteux, S., O'Connor, T . R., Lederer, F., Gougette, A., and Laval, J . (1990) J . Biol . Chem . 265, 3916-3922); its N-glycosylase and apurinic/apyrimidinic lyase activities are physically associated . Amino acid sequence analysis reveals a putative single zinc finger motif of the CC/CC type located near the carboxyl terminus . A gel mobility shift assay was used to assay binding of Fpg protein to a noncleavable substrate analog, namely an oligodeoxynucleotide duplex containing a single tetrahydrofuran residue . High resolution hydroxyl radical DNA footprinting showed protection centered around the tetrahydrofuran residue . No footprint was observed on the complementary strand . To establish the role of COOH-terminal zinc finger in DNA binding and/or DNA cleavage, amino acid substitutions and an amber mutation were introduced at Cys-244 (C244S, C244H, C244A, and C244amber) . In addition, a double amino acid substitution was generated at Cys-244 and Cys-247 (C244S/C247S) . These mutant Fpg proteins lack DNA binding or cleavage activity, as tested in crude lysates of Escherichia coli . Wild type Fpg protein contains one zinc/protein molecule, whereas the mutant Fpg protein (C244S/C247S) lacks zinc, as measured by atomic absorption spectroscopy . This mutation did not significantly alter secondary structure, as assessed by circular dichroism spectroscopy . Our results suggest that Fpg protein utilizes its single COOH-terminal zinc finger motif in DNA binding.

J Biol Chem, 1993 Dec 15, 268(35), 26512 - 21
Calcium/calmodulin-dependent protein kinase I . cDNA cloning and identification of autophosphorylation site; Picciotto MR et al.; Ca2+/calmodulin-dependent protein kinase I (CaM kinase I) was previously purified from bovine brain (Nairn, A . C., and Greengard, P . (1987) J . Biol . Chem . 262, 7273-7281) based on its ability to phosphorylate the synaptic vesicle protein, synapsin I at site 1 . The cDNA for this protein kinase has now been cloned from both a rat and a bovine brain cDNA library and the complete amino acid sequence of rat CaM kinase I determined . The rat cDNA encoded a protein of 331 amino acids with a calculated M(r) of 37,545, and the encoded kinase was expressed in bacteria as a glutathione S-transferase fusion protein . The resulting fusion protein was purified by Sepharose-CaM affinity chromatography and shown to be totally dependent on Ca2+ and CaM for activity . Furthermore, the purified kinase phosphorylates synapsin I at the same site (site 1) as the endogenous brain enzyme . CaM kinase I is homologous to other known protein kinases and contains all nine invariant amino acids conserved in the catalytic domain of this class of enzymes . CaM kinase I was most identical to CaM kinase II both in the catalytic domain and in a short region at the COOH-terminal that is predicted to be the calmodulin-binding domain . CaM kinase I appeared to be encoded by a single gene . RNase protection assays detected the mRNA encoding CaM kinase I in all tissues examined . High concentrations of the kinase mRNA were found in all regions of the brain with frontal cortex showing the greatest level . CaM kinase I was autophosphorylated in a Ca2+/CaM-dependent manner at a threonyl residue (Thr-177) which is located at a position equivalent to that of the threonyl residue (Thr-197) autophosphorylated in cAMP-dependent protein kinase.

J Biol Chem, 1993 Dec 15, 268(35), 26476 - 81
Human X-linked phosphoribosylpyrophosphate synthetase superactivity is associated with distinct point mutations in the PRPS1 gene; Roessler BJ et al.; Superactivity of phosphoribosylpyrophosphate synthetase (PRS) is an X chromosome-linked disorder of purine metabolism, characterized by gout with uric acid overproduction and, in some families, neurodevelopmental impairment . Two highly homologous isoforms of PRS (PRS1 and PRS2), each encoded by a distinct X chromosome-linked locus, have been identified, and PRS1 and 2 cDNAs have been cloned . The entire 954-base pair translated regions of PRS1 and 2 cDNAs derived from cultured lymphoblasts and fibroblasts from two patients in whom purine nucleotide feedback resistance of PRS is associated with enzyme superactivity and neurodevelopmental defects were examined by direct sequencing after polymerase chain reaction amplification of PRS transcripts . Nucleotide sequences of PRS2 cDNAs from the patients and normal individuals were identical . In contrast, PRS1 cDNAs from the patients differ from normal PRS1 cDNA, each by a single base substitution . PRS1 cDNA from patient N . B . showed an A to G transition at nucleotide 341, corresponding to an asparagine to serine change at amino acid residue 113 of mature PRS1 . A G to C transversion at nucleotide 547, indicating an aspartic acid to histidine change at amino acid 182, was found for PRS1 cDNA from patient S . M . Point mutations at the sites identified in the PRS1 cDNAs of the two patients were confirmed by the results of RNase mapping analysis . Normal, N . B., and S . M . PRS1 cDNAs were introduced into Escherichia coli BL21 (DE3)/pLyS, and recombinant N . B . and S . M . PRS1s showed the purine nucleotide feedback resistance phenotypes characteristic of PRS from patients' cells.

J Biol Chem, 1993 Dec 15, 268(35), 26375 - 85
Escherichia coli-derived rat intestinal fatty acid binding protein with bound myristate at 1.5 A resolution and I-FABPArg106-->Gln with bound oleate at 1.74 A resolution; Eads J et al.; Rat intestinal fatty acid binding protein (I-FABP) is a 131-residue protein composed of two short alpha-helices (alpha I and alpha II) and 10 anti-parallel beta-strands organized into two nearly orthogonal beta-sheets . The structure of crystalline I-FABP with bound tetradecanoate (myristate) has been refined to a resolution of 1.5 A and compared to the 1.2 A structure of apo-I-FABP, the 1.9 A structure of I-FABP:hexadecanoate (palmitate) and the 1.75 A structure of I-FABP:9Z-octadecanoate (oleate) to determine how this model fatty acid receptor accommodates changes in the length of its fatty acid ligand . Myristate is located in the interior of the protein . A highly ordered, electrostatic network containing 7 hydrogen (H)-bonds links the OE1 and OE2 atoms of myristate's carboxylate group, the indole nitrogen of Trp82, NH1, and NH2 of Arg106, NE2, and OE1 of Gln115, and 2 interior ordered waters . The hydrocarbon chain of the bound fatty acid is slightly bent . Its convex face lies in a crevice, forming van der Waals contacts with the side chains of several hydrophobic and aromatic residues . Its concave face is exposed to an array of 8 interior ordered waters whose positions are stabilized by H-bond interactions with other waters, H-bond interactions with the side chains of polar/ionizable residues, and van der Waals contacts with the surface of the fatty acid . Addition of 2 or 4 methylenes to myristate produces remarkably little change in the positions of I-FABP's main chain and side chain atoms and interior ordered waters . The principal alterations are in the conformation of a surface opening (portal) connecting external and internal solvent and in the position of the benzene side chain of Phe55 . Changes in the conformation of the portal reflect movement of two of its components: the backbone of alpha II and a type I turn (Ala73, Asp74) connecting two beta-strands . The positions of the main chain atoms of Ala73 and Asp74 appear to be determined by their ability to form van der Waals contacts with the omega-terminus of the fatty acid . The side chain of Phe55 appears to function as an adjustable aromatic lid, located over the portal, whose position is dependent on an ability to form van der Waals contacts with a fatty acid's omega-terminus.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1993 Dec 15, 268(35), 26350 - 7
Human tumor necrosis factor alpha (TNF alpha) mutants with exclusive specificity for the 55-kDa or 75-kDa TNF receptors; Loetscher H et al.; To probe the ligand receptor interface, a number of point mutations were introduced in selected regions of human tumor necrosis factor (TNF) alpha by site-directed mutagenesis . The mutated proteins were expressed in Escherichia coli and analyzed for selective binding to recombinant 55- and 75-kDa TNF receptors in competition with radiolabeled wild-type TNF alpha . Generally, mutations in the loop from position 29 to 34 and at positions 86 and 146 preferentially impaired binding to the 75-kDa TNF receptor, whereas mutations in the region from 143 to 145 mainly affected binding to the 55-kDa TNF receptor . Mutation of the conserved Tyr87 resulted in a dramatic loss of binding activity to both receptors . The selectivity for one or the other receptor type was found to be enhanced by combining two or three point mutations, the effects of the single mutations with respect to receptor selectivity being at least additive . A combination of the mutations Arg32-->Trp and Ser86-->Thr yielded a double mutant (R32W-S86T) with wild-type binding to the 55 kDa, but no measurable binding to the 75-kDa TNF receptor . In contrast, combining the Asp143-->Asn and Ala145-->Arg mutations (D143N-A145R) resulted in a complete loss of binding to the 55-kDa TNF receptor, whereas binding to the 75-kDa TNF receptor was impaired by only 5-10-fold . In functional assays, selective activation of the 55-kDa TNF receptor by the R32W-S86T mutant elicited a full cytotoxic response in human KYM-1 cells and secretion of interleukin 6 and granulocyte-macrophate colony-stimulating factor in human umbilical vein endothelial cells . In contrast, stimulation of the 75-kDa TNF receptor with the D143N-A145R mutant as well as with agonistic antibodies failed to induce these responses.

J Biol Chem, 1993 Dec 15, 268(35), 26179 - 89
Enzymatic characterization of the individual mammalian primase subunits reveals a biphasic mechanism for initiation of DNA replication; Copeland WC et al.; The enzymatic mechanism of primase was investigated using Escherichia coli and baculoviral overexpressed mouse primase subunits, p49 and p58 . Neither of the singly purified primase subunits displayed primase activity alone, but the p49 subunit was able to extend a riboprimer, indicating that this subunit contains an RNA polymerase activity . The p58 subunit cooperated with the p49 subunit in binding the initiating purine to form the initial dinucleotide . After initiation, the p49 subunit alone was sufficient to extend the growing primer, but both the rate of p49 primer extension and its stability were influenced by the p58 subunit . The Km(ATP) in primer synthesis on poly(dT) of the p49-p58 heterodimeric primase complex was 10-fold higher than the Km(ATP) of the single p49 subunit in a ribo(A) primer extension assay . In addition, labeled ATP cross-linked to both of the individually purified subunits but with a striking difference in affinities; cross-linking was 11-fold more efficient to the p49 subunit . The interaction of the two primase subunits with polymerase alpha was also investigated . Immunoprecipitation experiments indicate that only the p58 subunit directly contacts the p180 subunit of DNA polymerase alpha . Competition experiments in the coupled primase-polymerase assay with a catalytically inactive mutant of DNA polymerase alpha and the Klenow fragment suggest that the DNA polymerase alpha-primase complex does not dissociate from the primer during the transition from RNA to DNA synthesis.

J Biol Chem, 1993 Dec 15, 268(35), 26162 - 70
DNA-induced conformational changes in RecA protein . Evidence for structural heterogeneity among nucleoprotein filaments and implications for homologous pairing; Kumar KA et al.; We have used circular dichroism as a probe to characterize the solution conformational changes in RecA protein upon binding to DNA . This approach revealed that RecA protein acquires significant amounts of alpha-helix upon interaction with DNA . These observations, consistent with the data from crystal structure (Story, R . M., Weber, I., and Steitz, T . (1992) Nature 355, 318-325), support the notion that some basic domains including the DNA binding motifs of RecA protein are unstructured and might contribute to the formation of alpha-helix . A comparison of nucleoprotein filaments comprised of RecA protein and a variety of DNA substrates revealed important structural heterogeneity . The most significant difference was observed with poly(dG) . poly(dC) and related polymers, rich in GC sequences, which induced minimal amounts of alpha-helix in RecA protein . The magnitude of induction of alpha-helix in RecA protein, which occurred concomitant with the production of ternary complexes, was 2-fold higher with homologous than heterologous duplex DNA . Most importantly, the stimulation of ATP hydrolysis by high salt coincided with that of the induction of alpha-helix in RecA protein . These conformational differences provide a basis for thinking about the biochemical and structural transitions that RecA protein experiences during the formal steps of presynapsis, recognition, and alignment of homologous sequences.

J Biol Chem, 1993 Dec 15, 268(35), 26041 - 4
In vitro heme O synthesis by the cyoE gene product from Escherichia coli; Saiki K et al.; The cytochrome bo complex is a heme-copper terminal quinol oxidase in the aerobic respiratory chain of Escherichia coli and contains low spin heme B, high spin heme O and CuB as the redox metal centers in subunit I . Based on site-directed mutagenesis studies on the cyoE gene in the cytochrome bo operon, we have postulated that the cyoE gene encodes a protoheme IX farnesyltransferase (heme O synthase) (Saiki, K., Mogi, T., and Anraku, Y . (1992) Biochem . Biophys . Res . Commun . 189, 1491-1497) . The present study demonstrates that the CyoE protein is localized in the cytoplasmic membrane and that the CyoE-overproduced membranes efficiently catalyze a conversion of exogenous ferrous protoheme IX and farnesyl diphosphate to heme O in the presence of divalent cations such as Mg2+ or Ca2+ . Thus, the cyoABCDE operon in E . coli encodes not only subunits of the cytochrome bo complex but also heme O synthase that is specifically required for functional expression of the bo-type quinol oxidase . Heme O seems to be an intermediate in heme A biosynthesis.

J Virol Methods, 1993 Dec 15, 45(2), 229 - 33
A liquid, colorimetric presence-absence coliphage detection method; Ijzerman MM et al.; A liquid, colorimetric presence-absence coliphage detection method based on the induction of beta-galactosidase by Escherichia coli is described . The release of beta-galactosidase in the medium due to lytic cell infections by coliphages permits the hydrolysis of a yellow chromogenic substrate that develops into a distinct red coliphage positive sample, while a coliphage negative sample remains yellow . This method has proven to be rapid, simpler to perform than an agar medium assay, easy to read and interpret, inexpensive, and highly sensitive.

J Virol Methods, 1993 Dec 15, 45(2), 161 - 7
Cytochemical analysis of human T cell leukaemia virus I LTR-regulated beta-galactosidase gene expression using a novel integrated cell system; Copeland KF et al.; To develop a reporter system to study the response of an integrated retroviral LTR and cellular and viral events which influence transcription, the 5' LTR of HTLV-1 was coupled to the Escherichia coli beta-galactosidase gene (lacZ) . This construct was assembled within a vector containing the neomycin resistance gene controlled by the SV40 promoter, and introduced into HeLa cells . Expression from the LTR in one clone was upregulated by positive regulators of HTLV-1 expression, including 12-O-tetradecanoylphorbol-13-acetate (TPA) and the HTLV-1 transregulatory protein (tax), as has been previously reported using transient transfection assays . This method proved to be a rapid and reproducible assay for the measurement of integrated viral LTR activation in a single cell system.

Biochem J, 1993 Dec 15, 296 ( Pt 3), 685 - 91
Arthrobacter D-xylose isomerase: chemical modification of carboxy groups and protein engineering of pH optimum; Siddiqui KS et al.; To try to lower the pH optimum, the carboxy groups of Arthrobacter D-xylose isomerase were coupled to glycinamide using a water-soluble carbodi-imide . In conditions that substituted all of the 59 carboxy groups in the denatured monomer, a maximum of 30 groups/monomer reacted in the native enzyme, whether in presence or absence of ligands, and the enzyme remained fully active and tetrameric throughout the coupling reaction . Purification by f.p.l.c . ion-exchange chromatography gave broad symmetrical peaks with increased pI, suggesting that the modified enzymes are essentially homogeneous . However, they are less stable than native enzyme in 8 M urea or on heating ('melting points' of 59 degrees versus 73 degrees C for the apoenzymes and 67 degrees versus 81.5 degrees C for the Mg(2+)-enzymes) . Kinetic studies of the D-fructose isomerase activity at 30 degrees C showed that the glycinamidylated enzyme had unaltered activation constant for Mg2+, and Km was also similar to that of the native enzyme at pH 7.3, but increased rapidly at higher pH rather than remaining constant . Vmax . was constant from pH 6.2 to 8.0, suggesting a reduced pKa for His-219, which controls Vmax . in the native enzyme (normally 6.0) . Three mutants were constructed by protein engineering with a view to reducing the pH optimum of enzyme activity . Two of these, Glu140-->Lys and Asp189-->Lys, could be detected in crude extracts of Escherichia coli by SDS/PAGE, but could not be purified, whereas mutant Trp136-->Glu was produced as a tetramer in amounts similar to the wild-type enzyme . However, it did not show any enzyme activity and was less stable in 0-9 M urea gradient PAGE.

Eur J Biochem, 1993 Dec 15, 218(3), 1071 - 82
Plasminogen-activator inhibitor type 2 (PAI-2) is a spontaneously polymerising SERPIN . Biochemical characterisation of the recombinant intracellular and extracellular forms; Mikus P et al.; Plasminogen-activator inhibitor type 2 (PAI-2) is a specific inhibitor of plasminogen activators (PA) that exists in an intracellular, low-molecular-mass form and a secreted, high-molecular-mass form that varies with respect to glycosylation . Here we have developed expression systems for both forms of PAI-2 and biochemically characterised the purified proteins . In order to obtain efficient secretion, we constructed an artificial signal sequence and fused it to the coding region of PAI-2 . With this construct, more than 90% of PAI-2 was secreted as a glycosylated, 60-kDa molecular-mass form, but the level of expression was low and unstable . To obtain higher expression of secreted PAI-2, a novel expression vector based on the Semliki-forest-virus replicon was used . Secreted PAI-2 was purified to homogeneity and N-terminal sequence analysis showed that the artificial signal peptide was correctly removed . The intracellular, non-glycosylated form of PAI-2 was expressed in Escherichia coli and purified to homogeneity . Both the secreted and the intracellular forms of PAI-2 were found to inhibit plasminogen activators by forming SDS-resistant complexes and the second-order rate constants were similar for both forms, ranging over 2.4-2.7 x 10(6) M-1s-1 for urokinase-type PA, 2.5-2.7 x 10(5) M-1s-1 for two-chain tissue-type PA and 0.8-1.2 x 10(4) M-1s-1 for single-chain tissue-type PA . None of the purified PAI-2 forms bound to vitronectin . Circular-dichroism spectral analysis revealed that PAI-2 has a CD spectrum that resembles ovalbumin more than PA-inhibitor type 1, confirming the greater similarity between these two members of the serine-protease inhibitor family . Similar to what has been described for the Z-form of alpha 1-antitrypsin, purified PAI-2 was found to spontaneously form polymers during incubation at room temperature . Attempts to convert PAI-2 to a stable locked conformation resembling the conformation of latent PAI-1 by treatment with diluted guanidinium chloride were unsuccessful . Instead, this treatment enhanced the formation of PAI-2 polymers, possibly by the loop-sheet polymerisation mechanism described for alpha 1-antitrypsin.

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