Can J Microbiol, 1992 Jul, 38(7), 699 - 704 Escherichia coli serotyping and disease in man and animals; Orskov F et al.; Serotyping of Escherichia coli is useful, but complex, with 173 O antigens, 80 K antigens, and 56 H antigens, which can all be subdivided into partial antigens . The O, K, and H antigens can be found in nature in many of the possible combinations . The final number of E . coli serotypes is very high, 50,000-100,000 or more . The number of frequent pathogenic serotypes is, however, limited . Two main groups of such frequent serotypes are (i) serotypes from diarrhoeal disease and (ii) serotypes from extraintestinal disease . Serotypes from diarrhoeal diseases are mostly species specific, and could at present be used as epidemiological markers for bacterial clones equipped with special virulence markers, such as toxins and adhesins . Their O-antigen lipopolysaccharides may be regarded as virulence factors . These strains are not inhabitants of the normal intestine . Serotypes from extraintestinal diseases constitute a different set of clones, which are good colonizers of the intestinal tract, that under certain conditions succeed in invading host tissues . They are characterized by virulence factors different from those found in strains from diarrhoeal disease . Thus, the two groups of pathogenic E . coli are both composed of a limited number of clones for which the O:K:H serotypes are excellent, although not faultless, markers. Agents Actions, 1992 Jul, 36(3-4), 312 - 6 Lobenzarit disodium (CCA) inhibits the proliferation of human endothelial cells and the activity of DNA polymerase alpha; Takeuchi F et al.; N-(2)-Carboxyphenyl-4-chloroanthranilic acid disodium {Lobenzarit disodium (CCA)} is widely used for the treatment of patients with RA in Japan; however, the pharmacological mechanism of the compound is still unclear . In this report, the effect of CCA on the proliferation and DNA synthesis of endothelial cells was examined . CCA inhibited DNA synthesis in endothelial cells at a rather lower concentration than that in fibroblasts and HeLa cells . The DNA polymerase alpha activity was inhibited by CCA at a lower concentration than E . coli DNA polymerase I and avian myeloblastosis virus reverse transcriptase . Thus, CCA is a potent inhibitor of DNA polymerase alpha and this inhibitory effect could cause the inhibition of endothelial cell proliferation, which may be related to the therapeutic and pharmacological mechanisms of CCA. FEMS Microbiol Lett, 1992 Jul 1, 73(1-2), 31 - 6 Synthesis of unusual thick pili by Escherichia coli of EPEC serogroup O119; Bradley DE et al.; Unique very thick pili were found in varying numbers on cells of five out of 11 clinical isolates of enteropathogenic Escherichia coli belonging to the classic EPEC serogroup O119 . They were approximately 12.5 nm in diameter, which is thicker than any other known E . coli pilus type . Analysis of the plasmid profiles of the O119 isolates showed that one strain was plasmid-free while the remainder contained numerous plasmids with a wide range of sizes . The thick pilus determinants were located on a 140-kb non-transferrable plasmid . They were not associated with adherence or a putative 90-kb enteroadherence factor plasmid. FEMS Microbiol Lett, 1992 Jul 1, 73(1-2), 149 - 53 Bovine Escherichia coli of serotypes O55:H4 and O55:H21 which produce CNF2 express P fimbriae and Vir surface antigen, respectively; Blanco J et al.; We have characterized the toxic and adhesive properties of Escherichia coli strains producing the second type of cytotoxic necrotizing factor (CNF2) and belonging to the classic enteropathogenic serogroup O55 . Bovine CNF2 strains of serotype O55:H4 express P fimbriae as do pyelonephritic Escherichia coli that cause infections in humans . In contrast, strains of serotype O55:H21 which produce CNF2 from bovine origin possess the Vir surface antigen . One human strain of serotype O55:H- was positive for production of CNF2, but was negative for the two adhesive factors and for mannose-resistant haemagglutination. Biol Chem Hoppe Seyler, 1992 Jul, 373(7), 547 - 54 Flow cytometric analysis of protease activities in vital cells; Rothe G et al.; The analysis of lysosomal proteases in cell lysates is complicated by pH-dependent and oxidative changes of their activity and complex formation with cytosolic inhibitors . Therefore, new flow cytometric methods were developed for the intracellular measurement of protease activities in viable cells . Intracellular cleavage of substrates such as Z-Arg-Arg-4-trifluoromethylcoumarinyl-7-amide to green fluorescent 7-amino-4-trifluoromethylcoumarin (AFC) in viable neutrophils and monocytes was only detected following phagocytosis of Escherichia coli . A measurement of the cysteine or serine proteinase activities in resting human leukocytes was, however, not possible with AFC derivatives as the overlapping blue fluorescence of the substrates reduces sensitivity . Nonfluorescent bis-substituted peptide derivatives of rhodamine 110 (R110), which are intracellularly cleaved to green fluorescent mono-substituted R110 and free R110 proved to be more sensitive substrates . The activity of the lysosomal cysteine proteinases of human monocytes or rat macrophages, i.e . cathepsin B and L, was specifically measured with (Z-Arg-Arg)2-R110, (Z-Phe-Arg)2-R110, or (Z-Ala-Arg-Arg)2-R110 . Fluorescence generation was completely inhibited by Z-Phe-Ala-diazomethane or E-64 . The serine proteinases of human neutrophils were analyzed with Elastase-substrates such as (Z-Ala-Ala)2-R110 or (Z-Ala-Ala-Ala)2-R110 . Specificity was shown by inhibition with diisopropylfluorophosphate. Anal Biochem, 1992 Jul, 204(1), 90 - 5 Application of 3-{3-(3-(trifluoromethyl)diazirin-3-yl)phenyl}-2,3- dihydroxypropionic acid, carbene-generating, cleavable cross-linking reagent for photoaffinity labeling; Bochkariov DE et al.; N-Hydroxysuccinimide ester of 3-{3-(3-(trifluoromethyl)diazirin-3-yl)phenyl}-2,3-dihydroxypro pionic acid was successfully tested in a ribosomal tRNA binding system . It is an originally designed trifluoromethyl-diazirine-based cleavable cross-linking reagent with a very short distance between the active points (about 8.5 A) . The reagent was coupled to the amino acid amino group of Phe-tRNAPhe to obtain a photoactivatable analog of peptidyl-tRNA . This analog was bound to ribosomes and the complex was irradiated with uv light . After isolation, the cross-linked product was cleft by periodate treatment to reveal the properties of the new reagent. Antimicrob Agents Chemother, 1992 Jul, 36(7), 1441 - 6 Identification of the human immunodeficiency virus reverse transcriptase residues that contribute to the activity of diverse nonnucleoside inhibitors; Condra JH et al.; The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is potently inhibited by a structurally diverse group of nonnucleoside compounds . These include pyridinone derivatives, tetrahydroimadazo{4,5,1-j,k}{1,4}-benzodiazepin-2(1H)-one and -thione, and BI-RG-587 (nevirapine) . The compounds act noncompetitively, by an unknown mechanism, with respect to template-primer and nucleotide substrates . Despite a high degree of similarity between the HIV-1 and HIV-2 RTs, the HIV-2 enzyme is totally insensitive to these inhibitors . Using a novel method for joining DNA sequences, we have exploited this difference between the two enzymes to identify the regions of the RT that contribute to the compounds' inhibitory activities . The relative in vitro sensitivities of HIV-1/HIV-2 chimeric and site-specific mutant enzymes were determined . Sensitivity to inhibition was largely, though not exclusively, dependent upon the RT region defined by amino acid residues 176 to 190, with specific contributions by residues 181 and 188 . The region defined by residues 101 to 106 was found to functionally interact with the domain from 155 to 217 . In addition, the functional equivalence of the three inhibitor groups was shown. Mol Gen Genet, 1992 Jul, 234(1), 121 - 8 Localised mutagenesis of the fts YEX operon: conditionally lethal missense substitutions in the FtsE cell division protein of Escherichia coli are similar to those found in the cystic fibrosis transmembrane conductance regulator protein (CFTR) of human patients; Gibbs TW et al.; After localised mutagenesis of the 76 min region of the Escherichia coli chromosome, we isolated a number of conditionally lethal mutants . Some of these mutants had a filamentation temperature sensitive (fts) phenotype and were assigned to the cell division genes ftsE of ftsX, whereas others were defective in the heat shock regulator gene rpoH . Both missense and amber mutant alleles of these genes were produced . The missense mutant ftsE alleles were cloned and sequenced to determine whether or not the respective mutations mapped to the region of the gene encoding the putative nucleotide binding site . Surprisingly, most of these mutant FtsE proteins had missense substitutions in a different domain of the protein . This region of the FtsE protein is highly conserved in a large family of proteins involved in diverse transport processes in all living cells, from bacteria to man . One of the proteins in this large family of homologues is the human cystic fibrosis transmembrane conductance regulator (CFTR), and the FtsE substitutions were found to be in very closely linked, or identical, amino acid residues to those which are frequently altered in the CFTR of human patients . These results confirm the structural importance of this highly conserved region of FtsE and CFTR and add weight to the current structural model for the human protein. Proc Natl Acad Sci U S A, 1992 Jul 1, 89(13), 5779 - 83 Identification of Tyr-185 as the site of tyrosine autophosphorylation of recombinant mitogen-activated protein kinase p42mapk; Rossomando AJ et al.; Tyrosine phosphorylation of 42-kDa mitogen-activated protein kinase (p42mapk) occurs during expression of the recombinant protein in Escherichia coli, as well as during in vitro phosphorylation of the protein purified from this source . Structural analyses were performed to identify the site(s) of tyrosine phosphorylation of recombinant p42mapk, both during expression of the protein in E . coli and during in vitro incubations with ATP/Mg2+/Mn2+ . Mass spectrometry and phosphopeptide mapping showed that tyrosine phosphorylation of recombinant p42mapk occurs on Tyr-185, the site of regulatory tyrosine phosphorylation that occurs in mitogen-stimulated mammalian cells. Mutat Res, 1992 Jul, 282(3), 203 - 7 Inhibition of recA induction by the radioprotector 2-mercaptoethylamine; Naslund M et al.; Our earlier finding that the radioprotective action of 2-mercaptoethylamine (MEA) is counteracted by ascorbate suggests a biochemical mechanism of action, which is supported by observations that MEA is not radioprotective in Rec- E . coli strains . In this study we show that MEA inhibits the induction of the recA gene by UV- or gamma-irradiation or by nalidixic acid in Escherichia coli strain GE94, which contains a recA-lacZ fusion . This effect, which may be counteracted by cysteine, indicates that in general MEA inhibits the induction of SOS functions. Mutat Res, 1992 Jul, 282(3), 183 - 90 Mutagenic specificity in DNA base sequence by irradiation of health lamp light (UV-B) in Escherichia coli; Okaichi K et al.; A shuttle vector, pZ189, carrying a bacterial suppressor tRNA marker gene, was irradiated with health lamp (HL) light containing UV-B . Plasmid mutations were scored by transforming an indicator strain of Escherichia coli carrying a suppressive blue amber mutation in the beta-galactosidase gene . Plasmid survival was also measured by transforming activity of the indicator strain . The majority of mutations induced by HL light were GC-AT transitions (69%) and the rest were transversions (31%) . Some hot-spots in the mutations were observed by sequencing the suppressor gene . Mutagenic specificity in DNA base sequences induced by HL in E . coli agrees well with previous reports about 254-nm or 313-nm light effects on mammalian cells . This agreement may depend on the substitution of the inserted base instead of a G residue at the opposite site of a damaged C residue from conformational change of DNA structure in both bacterial and mammalian cells. Eur J Biochem, 1992 Jul 1, 207(1), 305 - 13 Molecular cloning, stage-specific expression and cellular distribution of a putative protein kinase from Plasmodium falciparum; Zhao Y et al.; A putative protein kinase gene (PfPK2) has been isolated from the human parasite Plasmodium falciparum by using a mixed oligonucleotide pool which corresponds to a highly conserved region of serine/threonine protein kinases . The complete nucleotide sequence of 5 kb suggests the existence of a second transcriptional unit besides that of the PfPK2 gene, separated by a highly (A+T)-rich region and transcribed in a different orientation . No intron sequence exists in PfPK2 . The predicted amino acid sequence of PfPK2 contains features characteristic of eukaryotic serine/threonine protein kinases . Within its putative catalytic domain it shares 33%, 30%, and 28% amino acid identities with rat calcium-calmodulin-dependent protein kinase, human protein kinase C, and bovine cAMP-dependent protein kinase, respectively . Outside the catalytic domain, however, PfPK2 has no homology with regulatory domains of other protein kinases, indicating PfPK2 might be modulated by signals different from those of higher eukaryotes or might be associated with other regulatory subunits . Using a specific antiserum raised in rabbits against a recombinant fragment of the protein expressed in Escherichia coli, PfPK2 was found to be expressed in a stage-specific fashion and mainly localized in the parasitic membrane. EMBO J, 1992 Jul, 11(7), 2675 - 83 Replication control of plasmid R1: RepA synthesis is regulated by CopA RNA through inhibition of leader peptide translation; Blomberg P et al.; The replication frequency of plasmid R1 is post-transcriptionally controlled by an antisense RNA, CopA, that binds to the leader region in the RepA mRNA, CopT, and ultimately inhibits the synthesis of the replication initiator protein RepA . We present results demonstrating that CopA controls RepA synthesis indirectly . A reading frame for a 24 amino acid leader peptide (Tap, translational activator peptide) is located in the region between the copA and repA genes . A translational fusion between the tap and lacZ genes was used to demonstrate that tap is translated and controlled by CopA . Stop codons (UAA, UAG and UGA) introduced at three different positions within the tap gene led to a severe decrease in repA expression . Specific suppression of the stop codons reversed the effect . This indicates that tap translation is required for RepA synthesis . Phylogenetic comparisons between IncFII-like plasmids, together with previous in vitro and in vivo results (Ohman and Wagner, 1989, 1991), suggest that a stable RNA stem-loop structure sequesters the repA ribosome binding site irrespective of CopA-CopT duplex formation . The results presented here show that ribosomes translating the tap reading frame have to terminate close to the start codon of repA to permit reinitiation (direct translational coupling), and that transient disruption of the inhibitory RNA stem-loop is insufficient for activation of repA translation . The possibility that direct translational coupling is required because of a suboptimal repA RBS cannot be excluded.(ABSTRACT TRUNCATED AT 250 WORDS) EMBO J, 1992 Jul, 11(7), 2627 - 32 The positive regulator CfaD overcomes the repression mediated by histone-like protein H-NS (H1) in the CFA/I fimbrial operon of Escherichia coli; Jordi BJ et al.; CFA/I fimbriae of enterotoxigenic Escherichia coli are expressed at 37 degrees C and not at 20 degrees C . Expression of CFA/I fimbriae requires two DNA regions (regions 1 and 2) which are separated by 40 kb on the wild type plasmid . Region 2 encodes a protein (CfaD) which activates the promoter in region 1 . We investigated whether the histone-like protein H-NS (H1) of E.coli is involved in the temperature regulated expression of CFA/I fimbriae . As demonstrated recently with other temperature regulated genes, a mutation in the gene coding for this nucleoid-associated H-NS (H1) protein resulted in derepression of CFA/I expression . CFA/I fimbriae were now expressed both at 20 degrees C and 37 degrees C . More strinkingly, the positive regulator CfaD was not needed for CFA/I expression in an H-NS- strain . This indicates that CfaD diminishes an inhibitory effect of the H-NS nucleoid-associated protein . We also showed that in the H-NS- strain the CfaD protein still has a positive effect on the transcription of CFA/I. EMBO J, 1992 Jul, 11(7), 2457 - 63 The aerolysin membrane channel is formed by heptamerization of the monomer; Wilmsen HU et al.; The cytolytic toxin aerolysin has been found to form heptameric oligomers by SDS-PAGE electrophoresis, STEM mass measurements of single oligomers and image analysis of two-dimensional membrane crystals . Two types of crystal, flat sheets and long regular tubes, have been obtained by reconstitution of purified protein and Escherichia coli phospholipids . A noise-filtered image of the best crystalline sheets reveals a structure with 7-fold symmetry containing a central strongly stain-excluding ring that encircles a dark stain-filled channel 17 A in diameter . The ring is surrounded by seven arms each made up of two unequal sized domains . By combining projected views and side-views, a simplified model of the aerolysin channel complex has been constructed . The relevance of this structure to the mode of action of aerolysin is discussed. Mutat Res, 1992 Jul, 268(1), 59 - 64 Analysis of phage M13mp2 mutants produced from transfection of phage DNA having N4-aminocytosines at defined sequence positions; Matsumoto K et al.; N4-Aminocytidine is mutagenic in various organisms . In the cell, this cytidine analog is metabolized into N4-aminodeoxycytidine 5'-triphosphate, which will then be incorporated into DNA and mutation will result during the replication of the DNA . To prove that the N4-aminocytosine residue in DNA is indeed the site of mutagenesis, we prepared a series of phage M13mp2 DNA samples that bear N4-aminocytosine residues at a few defined positions in the lacZ alpha region, by carrying out in vitro limited extension of primed phage DNA . We then transfected the DNAs to Escherichia coli and examined the progeny phages for the forward mutations . The M13mp2 DNAs bearing N4-aminocytosines produced mutant phages at high frequencies . Furthermore, DNA sequencing of the resulting mutants demonstrated that both AT-to-GC and GC-to-AT mutations took place at those positions where N4-aminocytosine residues were originally present. J Bacteriol, 1992 Jul, 174(14), 4558 - 75 TnphoA and TnphoA' elements for making and switching fusions for study of transcription, translation, and cell surface localization; Wilmes-Riesenberg MR et al.; We describe a set of elements based on the transposon TnphoA for making transcriptional fusions to the lacZ gene and for making translational fusions to the phoA or lacZ structural gene . Each element can be switched, one for another, by homologous recombination, thereby allowing testing for transcription, translation, or cell surface localization determinants at the same site within a gene . We describe three kinds of elements for making each fusion type . Two kinds are transposition proficient (Tnp+): one encodes kanamycin resistance, and the other encodes tetracycline resistance . The third kind is transposition defective (Tnp-) and encodes kanamycin resistance . In addition, we describe one Tnp- element that has no reporter gene and encodes chloramphenicol resistance; this element is used primarily as a tool to aid in switching fusions . Switching is efficient because each element has in common 254 bp of DNA at the phoA end and 187 bp (or more) of DNA at the IS50R end of TnphoA, and switching is straightforward because individual elements encode different drug resistances . Thus, switched recombinants can be selected as drug-resistant transductants, and they can be recognized as ones that have lost the parental drug resistance and fusion phenotype . Further, switching Tnp+ elements to Tnp- elements reduces problems due to transposition that can arise in P1 crosses or cloning experiments . Some TnphoA and TnphoA' elements cause polar mutations, while others provide an outward promoter for downstream transcription . This feature is especially useful in the determination of operon structures . Strategies for the use of TnphoA and TnphoA' elements in gene analysis are also described. Biotechnology (N Y), 1992 Jul, 10(7), 795 - 8 Single-step purification on DEAE-sephacel of recombinant polypeptides produced in Escherichia coli; Ortega S et al.; We describe a method for the purification of recombinant proteins based upon the selective interaction of the choline-binding domain of the pneumococcal murein hydrolase and tertiary amines . Proteins of interest, fused to the binding domain by a peptide linker, containing the cleaving sequence recognized by blood coagulation factor Xa, can either be assayed for biological activities in vitro and in vivo or have the binding moiety removed to yield a totally unmodified form, suitable for clinical and functional studies . The method can also be applied to the production of low molecular mass peptides . The principle of the technique is illustrated with acidic fibroblast growth factor and with a neuropeptide-like fragment of ten amino acids contained within its sequence. Blood, 1992 Jul 1, 80(1), 170 - 8 Infection of hematopoietic progenitor cells by human cytomegalovirus; Maciejewski JP et al.; The susceptibility of hematopoietic progenitor cells to infection by human cytomegalovirus (HCMV) was investigated using several strains of HCMV, including the recombinant strain RC256 . RC256 is derived from the laboratory strain Towne and contains the Escherichia coli LacZ gene coding for beta-galactosidase (beta-gal) regulated by an early HCMV promoter . Expression of LacZ allowed the detection of HCMV in individual hematopoietic cells . Clonogeneic bone marrow (BM) progenitors, including CD34+ cells, could be infected with HCMV and would then form normal hematopoietic colonies . By polymerase chain reaction (PCR) amplification of DNA, HCMV could be detected in both erythroid and myeloid colonies . LacZ activity was observed predominantly in cells of myelomonocytic lineage . When cells derived from HCMV-infected progenitors were cocultivated with permissive human fibroblasts, infectious virus expressing LacZ was recovered . Although no characteristic HCMV cytopathology was observed in BM colonies, high virus to cell ratios resulted in a moderate inhibition of colony formation . Since infected hematopoietic progenitors can harbor HCMV for weeks and through several differentiation steps in culture, we postulate that in vivo these cells may serve as a reservoir of latent virus and contribute to HCMV dissemination. J Immunol, 1992 Jul 1, 149(1), 113 - 9 Granulocyte colony-stimulating factor (G-CSF) gene transduction in murine adenocarcinoma drives neutrophil-mediated tumor inhibition in vivo . Neutrophils discriminate between G-CSF-producing and G-CSF-nonproducing tumor cells; Colombo MP et al.; We have previously demonstrated that the murine colon adenocarcinoma C-26 cell line transduced with the human gene for the granulocyte CSF (G-CSF) loses tumorigenic activity through a mechanism that involved massive targeting of neutrophils at the site of tumor injection . The suppression of tumorigenicity by G-CSF was limited to the G-CSF-producing cells and was not transferred to nonproducing C-26 cells in a mixed tumor transplantation assay . We present direct evidence that neutrophils are involved in this phenomenon . We firstly examined, by electron microscopy (EM), the morphology of tumor infiltrates obtained 2, 5, and 10 days after s.c . injection of a mixture of G-CSF-producing and -nonproducing C-26 cells into syngeneic BALB/c mice . The EM analysis showed at 5, but not at 2 or 10 days, the presence of neutrophils in intimate contact with tumor cells . We then investigated whether neutrophils discriminate between G-CSF-producing and -nonproducing C-26 cells . To this aim, C-26 cells were transduced, via retroviral vector, with the Escherichia coli LacZ gene and mixed tumor transplantation assays were performed by injecting a mixture of G-CSF-producing beta-gal- and G-CSF-nonproducing beta-gal+ C-26 cells at different ratios . Histologic and EM analysis of the tumors growing at the site of injection were carried out . Five days after injection, treatment with x-gal revealed, at the histochemical level, the presence of neutrophils around G-CSF producing beta-gal- cells; cell-cell contacts and fusion of cell membranes were detected by EM only between neutrophils and G-CSF-producing cells . In vitro experiments, performed in Boyden chambers, confirmed that the G-CSF produced by C-26 cells was a chemoattractant for neutrophils . In addition, a colorimetric, cytostatic assay revealed that neutrophils were able to inhibit the growth of G-CSF-producing but not of G-CSF-nonproducing C-26 cells . Thus the tumor take after injection of G-CSF-producing C-26 cells seems to be controlled in situ through two major mechanisms namely neutrophil chemotaxis and neutrophil-mediated tumor inhibition . The results indicate that neutrophils can discriminate between G-CSF-producing and -nonproducing tumor cells and that neutrophils infiltrate the tumor mixture as long as G-CSF-producing cells are present. J Biotechnol, 1992 Jul, 24(3), 235 - 51 Plasmid instabilities of single and three-plasmid systems in Escherichia coli during continuous cultivation; Maschke HE et al.; Plasmid instabilities in E . coli JM103 carrying three plasmids (pRK248cI, pMTC48, pEcoR4) and a single plasmid system (pTG206) for the production of fusion EcoRI (SPA::EcoRI) and catechol 2,3-dioxygenase, respectively, were investigated in continuous cultures under selective and non-selective conditions . In a three-plasmid system, pRK248cI was lost gradually together with pMTC48 from the host under non-selective conditions . The selective pressure against pRK248cI stabilized the pMTC48 . This indicates that the loss of pMTC48 under non-selective conditions was caused by the loss of cI857 gene (coded by pRK248cI) which resulted in the overproduction of the toxic gene product (coded by pMTC48) . In the case of single plasmid (pTG206) system, the plasmid lost from the host under non-selective conditions . This plasmid was stabilized in the host growing under selective conditions . During this period we obtained some ampicillin resistant colonies which gave low levels of enzyme activities compared to the normal plasmid bearing cells . Plasmid analysis from the above cells showed that the plasmid has undergone structural instability . Further, restriction analysis of this plasmid exhibited an additional PvuII site in a 0.9 kbp fragment that was integrated near the tet promoter which controls the expression of the xyl E gene, thereby resulting low levels of enzyme activities . Our results indicate that some of the IS elements which are present in the host chromosome were responsible for such instabilities to turn off the synthesis by inserting into the tet promoter region to lower the protein formation during the bioprocess. Biosci Biotechnol Biochem, 1992 Jul, 56(7), 1012 - 6 Syntheses of recombinant yellowtail and flounder growth hormones in Escherichia coli; Watahiki M et al.; For syntheses of recombinant yellowtail and flounder growth hormones (r-yGH and r-fGH) in E . coli, expression plasmids were constructed . The expression level of r-yGH and r-fGH in the host cells were very high, reaching 15 and 8% of the total protein, respectively . These product proteins were accumulated in inclusion bodies in the cells . The recombinant hormones were isolated from the pellets ina glutathione reduction/oxidation buffer . The refolded hormones were further purified by DEAE-Toyopearl 650M chromatography to homogeneity . The purified r-yGH and r-fGH were composed of 188 and 174 amino acid residues, respectively, having amino-terminal sequences starting with methionine . The recombinant hormones had potent growth-promoting activities on juvenile rainbow trout Salmo gairdneri in a dose-dependent manner. Biotechnology (N Y), 1992 Jul, 10(7), 790 - 4 Synthesis of a functional anti-phytochrome single-chain Fv protein in transgenic tobacco; Owen M et al.; We have expressed a synthetic gene that encodes an antigen-binding single-chain FV protein (scFV) in transgenic tobacco plants . The scFV gene was created by polymerase chain reaction (PCR) amplification of the variable domain coding regions from a mouse monoclonal hybridoma cell line . The monoclonal cell line secretes an IgG1 antibody that binds to the plant regulatory photoreceptor protein, phytochrome . The cloned scFV gene was expressed initially in Escherichia coli and shown to produce a 28 kD, phytochrome-binding binding scFV protein . Transgenic tobacco plants expressing the scFV gene were also found to produce a functional scFV protein, and seeds from transgenic R1 progeny displayed aberrant phytochrome-dependent germination . The scFV from transgenic tobacco could be isolated, to near homogeneity, by a single phytochrome-Sepharose affinity chromatography step. Res Immunol, 1992 Jul-Aug, 143(6), 601 - 10 TNF alpha-mediated tissue damage in mouse footpads primed with mycobacterial preparations; al Attiyah R et al.; Tissue sites involved in certain types of inflammation become sensitive to the destructive effect of a subsequent injection of tumour necrosis factor alpha (TNF alpha) . To try to further delineate the cascade of effector and regulatory events controlling this activity, a new model is described and its main properties characterized . C57BL/GrFa mice received mycobacterial products subcutaneously in the footpads . Recombinant TNF alpha was injected 24 h later into the same sites . To assess the tissue-destructive effect of TNF alpha in these "primed" footpads, swelling and haemoglobin content of injected footpads were measured, 16 h and 20 h respectively after the injection of TNF alpha . When loaded with either Escherichia coli LPS (10 micrograms) or Mycobacterium vaccae soluble sonicate (17 micrograms), footpads were reactive to the subsequent injection of 1 microgram recombinant TNF alpha, as assessed by both swelling and haemoglobin content . When C57BL/GrFa mice received 10(9) autoclaved M . vaccae subcutaneously in the back 10 days before the footpad was "primed" with soluble M . vaccae sonicate, the destructive effect of TNF alpha was significantly enhanced, becoming 5-10-fold greater than that seen in sites "primed" with an optimal dose of LPS . This higher reactivity was abrogated by a single dose of anti-CD4 given just before the injection of TNF alpha . This local reactivity to TNF alpha of skin sites loaded with mycobacterial products is compared to the local LPS-dependent Shwartzman reaction, and the relevance of this assay as a model with which to delineate the mechanisms of tissue damage in tuberculosis is discussed. J Gen Microbiol, 1992 Jul, 138 ( Pt 7), 1495 - 502 Molecular cloning of a determinant coding for fimbrial antigen F165(1), a Prs-like fimbrial antigen from porcine septicaemic Escherichia coli; Harel J et al.; The genetic determinant coding for F165(1) fimbriae was cloned from the chromosome of the porcine Escherichia coli wild-type strain 4787 (O115:K-:H51:F165) . The fimbrial determinant was further subcloned into the BamHI site of pACYC184 and a restriction map was established . On Southern hybridization, identity between the chromosomally encoded prs-like determinant of strain 4787 and its cloned counterparts was demonstrated . The cloned F165(1) fimbriae and those of the wild-type strain possessed a major protein subunit of molecular mass 18.5 kDa . Strains expressing F165(1) fimbriae were detected using an F165-specific polyclonal antiserum and caused mannose-resistant haemagglutination and agglutination of Forssman latex beads . Antiserum against the cloned F165(1) fimbriae recognized a 18.5 kDa band in the parent strain 4787. Mol Microbiol, 1992 Jul, 6(14), 1995 - 2007 Mycobacteria contain two groEL genes: the second Mycobacterium leprae groEL gene is arranged in an operon with groES; Rinke de Wit TF et al.; In contrast to other bacterial species, mycobacteria were thus far considered to contain groEL and groES genes that are present on separate loci on their chromosomes, Here, by screening a Mycobacterium leprae lambda gt11 expression library with serum from an Ethiopian lepromatous leprosy patient, two DNA clones were isolated that contain a groEL gene arranged in an operon with a groES gene . The complete DNA sequence of this groESL operon was determined . The predicted amino acid sequences of the GroES and GroEL proteins encoded by this operon are 85-90% and 59-61% homologous to the sequences from previously characterized mycobacterial GroES and GroEL proteins . Southern blotting analyses with M . leprae groES- and groEL-specific probes demonstrate that similar groESL homologous DNA is present in the genomes of other mycobacteria, including Mycobacterium tuberculosis . This strongly suggests that mycobacteria contain a groESL operon in addition to a separately arranged second groEL gene . Using five T-cell clones from two leprosy patients as probes, expression of the M . leprae GroES protein in Escherichia coli after heat shock was demonstrated . Four of these clones recognized the same M . leprae-specific GroES-derived peptide in a DR2-restricted fashion . No expression of the groEL gene from this operon was detected in E . coli after heat shock, as tested with a panel of T-cell clones and monoclonal antibodies reactive to previously described GroEL proteins of mycobacteria. Int J Radiat Biol, 1992 Jul, 62(1), 21 - 32 Repair of ionizing radiation damage in primate alpha DNA transfected into rat cells; Bases R et al.; The time-course of repair of irradiated primate alpha DNA was studied after transfection and recovery from rat NRK cells . Rat cells were chosen for transfection because they have no alpha DNA . Plasmid pBUC4 alpha 10, containing 10 tandem 172 bp alpha DNA subunits in its 5 kbp DNA, was irradiated and introduced into the rat cells by electroporation . The transfected alpha DNA was then recovered from NRK nuclei free of extraneous rat DNA, permitting study of the fate of the transfected alpha DNA in time-course experiments . alpha DNA continuously entered nuclei for processing in the first 2.5 h after transfection . The pool of damaged bases in alpha DNA in NRK nuclei was detectable 2.5 h after transfection . Radiation-induced alpha DNA fragments of electrophoretic mobility intermediate between those of unit nucleotide length were prominent in sequencing gel analyses of alpha DNA for 5-150 min after transfection . These intermediate mobility fragments initially disappeared with T 1/2 of 6-20 min . The alpha DNAs of intermediate mobility are presumed to be intermediates in DNA repair . Residual DNA base damage which had not been processed in the transfected cells could later be unmasked in vitro by conversion to strand breaks by beta-elimination using heat and piperidine or endonuclease III of E . coli . Irradiation of the recipient NRK cells with 5 Gy 4 hours before transfection prolonged the time during which intermediate mobility species could be found, consistent with the increased frequency of intermediate mobility species observed in DNA of monkey CV-1 cells pretreated with small doses of radiation before 300 Gy (Bases et al . 1990). J Bacteriol, 1992 Jul, 174(14), 4538 - 48 Characterization of Escherichia coli glnL mutations affecting nitrogen regulation; Atkinson MR et al.; Nitrogen regulator II (NRII), the product of the Escherichia coli glnL (ntrB) gene, regulates the activation of transcription of glnA and the Ntr regulon by catalyzing the phosphorylation and dephosphorylation of the transcription factor NRI . Previous results have indicated that under conditions of nitrogen excess, transcriptional activation is prevented by an NRI-phosphate phosphatase activity that is observed when NRII and another signal transduction protein known as PII (the glnB product) interact . The availability of PII for this interaction is controlled by a uridylytransferase/uridylyl-removing enzyme, encoded by glnD, that reversibly modifies PII in response to intracellular signals of nitrogen availability . Here we describe the isolation and characterization of missense mutations in glnL that suppress the Ntr- phenotype resulting from a leaky glnD mutation . The regulation of glnA expression in the pseudorevertants was found to vary from complete insensitivity to ammonia in some strains (GlnC phenotype) to nearly normal regulation by ammonia in other strains . Sequence analysis indicated that in 16 instances suppression was due to point mutations at 14 different sites; 10 different mutations resulting in a variety of phenotypes were identified in a cluster extending from codons 111 to 154 flanking the site of NRII autophosphorylation at His-139 . Complementation experiments with multicopy plasmids encoding NRII or PII showed that suppression by GlnC glnL alleles was eliminated upon introduction of the plasmid encoding NRII but was not affected by introduction of the plasmid encoding PII . Conversely, suppression by certain glnL alleles that resulted in regulated expression of glnA was eliminated upon introduction of either the plasmid encoding NRII or that encoding PII . We hypothesize that mutants of the latter type result in a subtle perturbation of the NRII-PII interaction and suggest two possible mechanisms for their effects. Infect Immun, 1992 Jul, 60(7), 2828 - 34 Passive protection of suckling infant mice against F41-positive enterotoxigenic Escherichia coli strains by intravenous inoculation of the dams with monoclonal antibodies against F41; Duchet-Suchaux M et al.; Ten monoclonal antibodies (MAbs) against five different epitope clusters of adhesion factor F41 (two MAbs per cluster) were tested for protection of infant mice against an oral challenge with F41-positive enterotoxigenic Escherichia coli (ETEC) B2C and B41M . Infant mice suckling dams intravenously inoculated with MAbs were orally challenged, and the survival rates were measured for 12 days after inoculation and challenge . Irrespective of their epitope specificity, all F41 MAbs given in a single dose of 4 mg per dam had a protective effect against both ETEC strains . In contrast, one K99 MAb of the same isotype and given in the same dose as the F41 MAbs did not protect infant mice at all . A reduction in the dose of F41 MAbs to 0.032 mg per dam resulted in a decrease in protection . Two different MAbs against the same epitope cluster were not necessarily equally protective . Combining MAbs two by two, whether the MAbs recognized the same epitope cluster or not, resulted in protective activity essentially similar to that obtained with each MAb separately, without any improvement . Therefore, one MAb against any epitope may be sufficient for protection . Enzyme-linked immunosorbent assay (ELISA) titers of MAbs in the serum of dams were similar, irrespective of the epitope specificity of the MAbs, and gradually decreased from day 1 to day 12 after inoculation . We found a good correlation between colostrum and milk ELISA titers of MAbs and serum ELISA titers of MAbs . Colostrum and milk MAb titers were 10-fold lower than corresponding serum MAb titers and stayed high until day 5 after inoculation . The most protective MAb had the highest ELISA titers in colostrum and milk for the first 5 days after inoculation . ETEC strain B2C colonized the intestines of infant mice suckling MAb-inoculated mothers until day 12 after challenge . Intestinal levels of the challenge strain were high on day 2 but never reached the very high numbers (10(9) to 10(10)) described previously in a diarrheic infant mouse model . MAbs did not eliminate the challenge ETEC strain from the intestines of infant mice. Infect Immun, 1992 Jul, 60(7), 2648 - 56 pap-and pil-related DNA sequences and other virulence determinants associated with Escherichia coli isolated from septicemic chickens and turkeys; Dozois CM et al.; Escherichia coli isolates from septicemic or healthy chickens and turkeys from Quebec were serotyped, examined genotypically by using DNA probes specific for the pil and pap fimbrial systems and the aerobactin siderophore system, and examined phenotypically for lethality in day-old chicks, hemagglutination, serum resistance, and aerobactin production . Serogroups O78 and O1 were most common in septicemic chickens and turkeys . pap+ isolates from chickens were associated with septicemia, and pap+ isolates from turkeys were associated with lethality in day-old chicks . Four of nine pap+ isolates from septicemic turkeys expressed P adhesin, whereas all pap+ isolates from septicemic chickens were negative for P adhesin . The pil+ genotype was associated with septicemia in chickens and with serum resistance in isolates from turkeys . Mannose-sensitive hemagglutination of guinea pig erythrocytes was associated with septicemia in chickens and turkeys, although this phenotype was not associated with pil+ isolates from turkeys . Serum resistance was associated with isolates from septicemic turkeys and with lethality in isolates from chickens . The aerobactin system was associated with isolates from septicemic chickens and turkeys . Overall, results indicated that (i) genotypic examination may reveal virulence-associated traits which differ from the typically expected phenotype and/or are not readily expressed in vitro, and (ii) certain phenotypic and genotypic traits associated with E . coli causing extraintestinal disease in humans and animals are also associated with E . coli causing avian septicemia. Plant J, 1992 Jul, 2(4), 549 - 57 Potato virus X as a vector for gene expression in plants; Chapman S et al.; The suitability of potato virus X (PVX) as a gene vector in plants was tested by analysis of two viral constructs . In the first, the GUS gene of Escherichia coli was substituted for the viral coat protein gene . In the second, GUS was added into the viral genome coupled to a duplicated copy of the viral promoter for the coat protein mRNA . The viral construct with the substituted coat protein gene accumulated poorly in inoculated protoplasts and failed to spread from the site of infection in plants . These results suggest a role for the viral coat protein in key stages of the viral infection cycle and show that gene replacement constructs are not suitable for the production of PVX-based gene vector . The construct with GUS coupled to the duplicated promoter for coat protein mRNA also accumulated less well in protoplasts than the unmodified PVX, but did infect systemically and directed high level synthesis of GUS in inoculated and systemically infected tissue . Although there was some genome instability in the PVX construct, much of the viral RNA in the systemically infected tissue had retained the foreign gene insertion, especially in infected Nicotiana clevelandii plants . These data point to a general utility of PVX as a vector for unregulated gene expression in plants. Arch Biochem Biophys, 1992 Jul, 296(1), 122 - 8 Cloning, expression, and nucleotide sequence of glgC gene from an allosteric mutant of Escherichia coli B; Ghosh P et al.; The Escherichia coli B mutant strain CL1136 accumulates glycogen at a 3.4- to 4-fold greater rate than the parent E . coli B strain and contains an ADPglucose synthetase with altered kinetic and allosteric properties . The enzyme from CL1136 is less dependent on the allosteric activator, fructose 1,6-bisphosphate, for activity and less sensitive to inhibition by AMP than the parent strain enzyme . The structural gene, glgC, for the allosteric mutant enzyme was selected by colony hybridization and cloned into the bacterial plasmid pBR322 by insertion of the chromosomal DNA at the PstI site . One recombinant plasmid, designated pKG3, was isolated from the genomic library of CL1136 containing glgC . The cloned ADPglucose synthetase from the mutant CL1136 was expressed and characterized with respect to kinetic and allosteric properties and found to be identical to the enzyme purified from the CL1136 strain . The mutant glgC was then subcloned into pUC118/119 for dideoxy sequencing of both strands . The mutant glgC sequence was found to differ from the wild-type at the deduced amino acid residue 67 where a single point mutation resulted in a change from arginine to cysteine. Protein Sci, 1992 Jul, 1(7), 910 - 6 Cis proline mutants of ribonuclease A . I . Thermal stability; Schultz DA et al.; A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol . 185, 60-89) . The expressed protein, which contains an additional N-terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A . The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification . Site-directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made . In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing . Thermal unfolding experiments on four single mutants give delta Tm approximately equal to 10 degrees C and delta delta G0 (apparent) = 2-3 kcal/mol . The reason is that either the substituted amino acid goes in cis, and cis<==>trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild-type conformation, that allows the substituted amino acid to form a trans peptide bond. Biull Eksp Biol Med, 1992 Jul, 114(8), 204 - 5 {The molecular cloning of a genetic region determining the surface exclusion system of the F-like plasmid pAP42}; Krivskaia KS et al.; Molecular cloning of genetic region of F-like plasmid pAP42, coding its surface exclusion system (system Six V) was performed . Restriction and genetic analysis of recombinant plasmids showed that six V locus is situated in Sal I-fragment f5 (4.2 MD) of this plasmid. J Biochem (Tokyo), 1992 Jul, 112(1), 57 - 62 Expression and purification of growth hormone-releasing factor with the aid of dihydrofolate reductase handle; Iwakura M et al.; Expression of a fusion protein composed of dihydrofolate reductase and a derivative of growth hormone-releasing factor resulted in the formation of inclusion bodies in Escherichia coli at 37 degrees C . Among various chemicals, such as detergents, protein denaturants, and acetic acid, tested for the ability to dissolve the inclusion bodies, acetic acid, Brij-35, deoxycholic acid sodium salts, guanidine-HCl, and urea showed a strong solubilizing effect without damaging the DHFR activity . Acetic acid was useful in terms of preparing GRF derivatives, since it could be easily removed by lyophilization, and this made it easy to perform the succeeding BrCN treatment for cutting out the GRF derivative from the fusion protein . The GRF derivative was purified by reversed phase HPLC from the BrCN digest of the acetic acid extract, and its growth hormone-releasing activity was demonstrated . However, for obtaining a highly purified fusion protein itself, solubilization of inclusion bodies by urea was preferred because urea was the only agent which did not cause serious precipitation of the regenerated fusion protein after 10-fold dilution of the extracted inclusion bodies with buffer . The fusion protein was highly purified by means of a methotrexate affinity chromatography. J Biochem (Tokyo), 1992 Jul, 112(1), 1 - 6 Activation of the osmoregulated ompF and ompC genes by the OmpR protein in Escherichia coli: a study involving chimeric promoters; Takayanagi K et al.; The expression of each of the Escherichia coli ompF and ompC genes is activated by the common positive regulator, OmpR, in response to the medium osmolarity . The promoters for these genes consist of canonical -35 and -10 regions, and upstream OmpR-binding sites . In this study, we constructed a functional ompF-ompC chimeric promoter consisting of the OmpR-binding site of ompF, and the -35 and -10 regions of ompC, which was fused to the lacZ gene on a low-copy-number plasmid . It is known that the ompF and ompC genes are expressed in a different manner in cells with mutations in the ompR gene . In this respect, it was found that the ompF-ompC chimeric promoter behaves just like ompF promoter with respect to an ompR mutation (ompR472) . It was revealed that, in this chimeric promoter, the OmpR-binding site must be located stereospecifically with respect to the -35 and -10 regions for OmpR-dependent transcription of the ompF-ompC chimeric promoter to occur . These results are discussed in relation to the structures and functions of the ompF and ompC promoters, the occurrence of a DNA curvature in the promoter regions being implied, which may be an important parameter for the transcription activation by the OmpR protein. Development, 1992 Jul, 115(3), 729 - 35 Spatial and temporal expression of the I factor during oogenesis in Drosophila melanogaster; Lachaume P et al.; The I factor is a functional non-viral retrotransposon, or LINE, from Drosophila melanogaster . Its mobility is associated with the I-R hybrid dysgenesis . In order to study the expression pattern of this LINE in vivo, a translational fusion between the first ORF of the I factor and the lacZ gene of Escherichia coli has been carried out and introduced in the genome of reactive (R) flies . Homozygous transgenic Drosophila lines have been established and analysed . ORF1 expression is limited to germ-line cells (nurse cells and oocyte) between stage 2 and 10 of oogenesis . No somatic expression is found . Position effects may limit the level of expression of a given transgene but do not modify its basic pattern of expression during the development of the fly . This reproducible control demonstrates both that I factor is driven by its own promoter, probably the internal one suggested by Mizrokhi et al . (Mizrokhi, L.J., Georgevia, S.G . and Ilying, Y.V . (1988) . Cell 54, 685-691), and that tissue-specific regulatory sequences are present in the 5' untranslated part of the I factor . The nuclear localization of the fusion protein reveals the presence of nuclear localization signals (NLS) in the ORF1-encoded protein correlating with the possible structural and/or regulatory role of this protein . This expression is restricted to dysgenic and reactive females, and is similar in the two conditions . All the results obtained in this work suggest that I factor transposition occurs as a meiotic event, between stage 2 and 10 of the oogenesis and is regulated at the transcriptional level . It also appears that our transgene is an efficient marker to follow I factor expression. J Pediatr Gastroenterol Nutr, 1992 Jul, 15(1), 63 - 72 Field trial of an infant formula containing anti-rotavirus and anti-Escherichia coli milk antibodies from hyperimmunized cows; Brunser O et al.; Two groups of 124 and 108 children, respectively, living in urban Santiago, Chile in low socioeconomic conditions were prospectively followed for 6 months for their incidence of diarrhea . Each cohort was divided into two subgroups receiving either a commercial milk formula or the same formula containing 1% (wt/wt) bovine milk immunoglobulin concentrate from cows hyperimmunized with human rotaviruses and the major enteropathogenic Escherichia coli (EPEC) serogroups . Neither group differed with respect to incidence of diarrhea (98 episodes in 117 treated children versus 95 episodes in 115 control children), duration and clinical symptoms of diarrhea, and weight gain . Furthermore, neither group differed with respect to isolation of rotavirus (14 and 13 isolates in treatment and control groups, respectively) and isolation of enteropathogenic E . coli (14 and 15 isolates in treatment and control groups, respectively) . The treatment but not the control formula contained neutralizing antibody against all human rotavirus serotypes . Titers were comparable to human breast milk samples . All isolated EPEC serogroups were included in the vaccine used for the immunization of the cows . The treatment, but not the control formula, protected mice against a lethal challenge with an EPEC strain . In conclusion, feeding an antibody-supplemented formula had no positive effect on diarrheal diseases under the conditions of a fairly well-controlled small-scale field trial. Biokhimiia, 1992 Jul, 57(7), 1057 - 64 {Insertional mutagenesis of protease 2A of the poliomyelitis virus and its substrate, simultaneously expressed in Escherichia coli cells}; Slobodskaia OR et al.; In order to clarify the structural features of protein substrates which determine their sensitivity towards poliovirus 2A protease, a high-efficiency bacterial expression system for cDNA of the poliovirus genome fragment has been developed . The expressed protein encompasses the C-end half of the VP1 capsid protein . 2A protease, and a large portion of the 2B protein . Virus-specific products were found in the insoluble fraction of bacterial cell lysates which were mainly represented by two proteins . These proteins appeared to be produced via the cleavage of the expressed protein by 2A protease at the Tyr-Gly site located between the VP1 protein and the 2A protease proper . The accumulation of two viral proteins can be prevented by a four amino acid insertion into the 2A gene locus in close vicinity of the putative catalytic His residue . The determinants of specificity toward the 2A action are located within the region flanking the cleavable Tyr-Gly dipeptide from its N-side. FEMS Microbiol Lett, 1992 Jul 1, 73(1-2), 1 - 6 Carbon monoxide-binding properties of the cytochrome bo quinol oxidase complex in Escherichia coli are changed by copper deficiency in continuous culture; Ciccognani DT et al.; A strain of Escherichia coli having elevated levels of cytochrome bo and lacking the cytochrome bd quinol oxidase was grown in chemostat culture at low copper levels . Such cells had lowered levels of copper and of total cytochrome b . Cytochrome o concentration was unchanged when assayed by conventional CO difference spectroscopy, but apparently diminished by 80% in copper-deficient cells as determined by photodissociation of bound CO at 193 K . This is attributed to depletion of copper in the oxidase of copper-deficient cells, causing rapid recombination of photodissociated CO to haem O . CO recombination was also more sensitive to low intensities of actinic light in copper-depleted oxidase . The results illustrate a further similarity between the active sites of o- and aa3-type terminal oxidases. Plasmid, 1992 Jul, 28(1), 80 - 5 Overproduction of four functionally active proteins, TnsA, B, C, and D, required for Tn7 transposition to its attachment site, attTn7; Flores CC et al.; The bacterial transposon Tn7 encodes five trans-acting transposition genes, tnsA, B, C, D, and E . Tn7 requires four of these genes, tnsA, B, C, and D, for a novel transposition pathway: high-efficiency site-specific transposition to a chromosomal attachment site, attTn7 . Plasmids that individually allow inducible overexpression of proteins from the first initiation codon of four of these genes were constructed . Escherichia coli strains carrying these plasmids were used to overexpress the TnsA, B, C, and D proteins . The abundance and the apparent relative molecular mass of these proteins were examined and the latter was compared to those predicted from wild-type Tn7 . The functionality of these proteins, encoded by an overexpression construct, was demonstrated by the fact that they could efficiently trans-complement a defective mini-Tn7 carrying only the cis-essential Tn7 termini in an in vivo assay for transposition to attTn7. Jpn J Cancer Res, 1992 Jul, 83(7), 705 - 13 Identification of antibodies against human papillomavirus type 16 E6 and E7 proteins in sera of patients with cervical neoplasias; Sasagawa T et al.; We have developed a sensitive and specific ELISA method using the s10-fusion proteins of human papillomavirus (HPV) 16 E6 and E7, expressed in E . coli . Sera from 97 women (30 patients with invasive cervical cancers, 26 patients with cervical intraepithelial neoplasia III (CIN III) and 38 healthy women) were tested for the presence of antibodies to E6 and E7 proteins . Eight (27%) of the 30 cervical cancer sera, five (19%) of the 26 CIN sera and none of the 38 normal sera were reactive with E6 proteins (cut-off point: absorbance (A) = 0.59, x +3SD) . Ten (33%) of the 30 cervical cancer sera, two (8%) of the 26 CIN III sera and none of the 38 normal sera were reactive with E7 proteins (cut-off point: A = 0.40, x +3SD) . The mean absorbance for anti-E7 antibody in positive cases was higher in cancer patients than in CIN III patients, while that for E6 did not differ between these two groups . Interestingly, six (50%) of 12 cancer sera which reacted with either E6 or E7 proteins were reactive for both proteins, whereas none of the sera from the CIN III patients reacted with both proteins . The high prevalence rates and high absorbance values for HPV 16 E6 and E7 antibodies in association with malignant transformation suggest that detection of these antibodies may be a useful diagnostic aid for cervical cancer-associated HPV 16. Biol Chem Hoppe Seyler, 1992 Jul, 373(7), 523 - 8 Using proteinase trapping to detect revertants of inactive rhinoviral 2A proteinase mutants; Luderer M et al.; The 2A proteinase of human rhinovirus 2 cleaves itself off the growing polyprotein at its own N terminus during translation; this property was used to develop an in vivo screening system with the lacZ gene fragment of M13mp18 . The fusion of an active 2A proteinase to the C-terminus of the alpha-fragment did not affect alpha-complementation, as the proteinase cleaved itself off the alpha-fragment . However, an inactive 2A proteinase remained fused to the alpha-fragment hindering alpha-complementation . Random mutations were then introduced into the 2A gene site by PCR amplification . Mutants defective in alpha-complementation (thus containing an inactive 2A proteinase) were obtained at an efficiency of 5%, mutants showing reduced 2A activity at an efficiency of 1% . Mutants showing reduced or no 2A activity were then subjected to PCR mutagenesis . Three mutants reactivating an inactive 2A proteinase were examined and the compensatory changes determined. Mol Microbiol, 1992 Jul, 6(14), 1877 - 86 Identification and molecular analysis of glgS, a novel growth-phase-regulated and rpoS-dependent gene involved in glycogen synthesis in Escherichia coli; Hengge-Aronis R et al.; The putative stationary-phase sigma factor (sigma S) encoded by rpoS is essential for glycogen synthesis, but is not required for the transcription of glgC and glgA, which encode ADP-glucose-pyrophosphorylase and glycogen synthase, respectively . Using a mini-Mu random chromosomal library and a screen for glycogen overproduction, we identified a novel gene (glgS) involved in glycogen synthesis . glgS maps at 66.6 min (3247 kb) on the chromosome and constitutes a monocistronic operon . It encodes a hydrophilic and highly charged small protein, with a molecular weight of 7886, which is strongly expressed in minicells . Experiments with single-copy chromosomal glgS::lacZ gene fusions indicated that glgS expression is controlled by sigma S as well as by cAMP . Two transcriptional start sites were mapped in the upstream regulatory region of glgS . The glgSp1 transcript was absent in a cya mutant, whereas an rpoS mutant did not synthesize the glgSp2 transcript . Although glycogen synthesis is strongly stimulated by overproduction of GlgS and is inhibited by a glgS null mutation, glgS does not affect the expression of the glgCAP operon . Its potential role in the metabolic control of glycogen synthesis is discussed . Also, evidence is presented to show that the amount of glycogen accumulated in vivo in early stationary-phase cells is mainly determined by sigma S-controlled gene expression and allosteric activation of GlgC, whereas the absolute levels of expression of glgCAP as well as the intracellular concentration of cAMP are of minor importance. J Virol Methods, 1992 Jul, 38(1), 81 - 92 Characterization of nonradioactive hybridization probes for detecting infectious bursal disease virus; Lee LH; Reverse transcription followed by the polymerase chain reaction was used to amplify a fragment of infectious bursal disease virus (IBDV) strain P3009 genome . The amplified DNA fragment was annealed into the plasmid pUC18 and used to transform Escherichia coli strain JM109 . A clone that contained IBDV-specific nucleotide sequences was selected and designated pC23 . The DNA fragment within pC23 was 320 base pairs in length and designated C23 . Radiolabeled probes prepared from C23 hybridized to genome segment A of strain P3009 by a northern-blot hybridization assay . Biotin-labeled probes prepared from C23 and pC23 either by using nick translation (designated C23/NT and pC23/NT, respectively) or by direct introduction of biotin molecules into C23 and pC32 (designated C23/BH and pC23/BH, respectively) were used in the dot blot hybridization assay for detecting IBDV strains . All four biotinylated probes detected three serotype 1 viruses and one serotype 2 IBDV . However, they did not cross-react with nucleic acids extracted from mock-infected cells or from seven unrelated avian viruses . Probe pC23/BH detected as little as 0.04 ng of IBDV RNA, while the other three probes were less sensitive and detected approximately 1 ng of IBDV RNA . In addition, the probe pC23/BH detected IBDV RNA in bursa tissues from commercial broiler raising farms following the dot blot hybridization. Biochem J, 1992 Jul 1, 285 ( Pt 1), 91 - 7 Binding of the cyclic AMP receptor protein of Escherichia coli and DNA bending at the P4 promoter of pBR322; Brierley I et al.; The binding of the Escherichia coli cyclic AMP receptor protein (CRP) to its specific site on the P4 promoter of pBR322 has been studied by gel electrophoresis . Binding to the P4 site was about 40-50-fold weaker than to the principal CRP site on the lactose promoter at both low (0.01 M) and high (0.1 M) ionic strengths . CRP-induced bending at the P4 site was investigated from the mobilities of CRP bound to circularly permuted P4 fragments . The estimated bending angle, based on comparison with Zinkel & Crothers {(1990) Biopolymers 29, 29-38} A-tract bending standards, was found to be approximately 96 degrees, similar to that found for binding to the lac site . These observations suggest that there is not a simple relationship between strength of CRP binding and the extent of induced bending for different CRP sites . The apparent centre of bending in P4 is displaced about 6-8 bp away from the conserved TGTGA sequence and the P4 transcription start site. Mol Microbiol, 1992 Jul, 6(13), 1829 - 39 PhoA gene fusions in Legionella pneumophila generated in vivo using a new transposon, MudphoA; Albano MA et al.; To enable effective use of phoA gene fusions in Legionella pneumophila, we constructed MudphoA, a derivative of the mini-Mu phage Mu dII4041, which is capable of generating gene fusions to the Escherichia coli alkaline phosphatase gene (EC 3.1.3.1) . Although an existing fusion-generating transposon, TnphoA, has been a useful tool for studying secreted proteins in other bacteria, this transposon and other Tn5 derivatives transpose inefficiently in Legionella pneumophila, necessitating the construction of a more effective vector for use in this pathogen . Using MudphoA we generated fusions to an E . coli gene encoding a periplasmic protein and to an L . pneumophila gene encoding an outer membrane protein; both sets of fusions resulted in alkaline phosphatase activity . We have begun to use MudphoA to mutate secreted proteins of L . pneumophila specifically, since this subset of bacterial proteins is most likely to be involved in host-bacterial interactions . This modified transposon may be useful for studies of other bacteria that support transposition of Mu, but not Tn5, derivatives. Mol Microbiol, 1992 Jul, 6(13), 1739 - 45 Differential regulation of two genes encoding lysyl-tRNA synthetases in Escherichia coli: lysU-constitutive mutations compensate for a lysS null mutation; Kawakami K et al.; Lysyl-tRNA synthetases are synthesized in Escherichia coli from two distinct genes, lysS and lysU, which are regulated differentially . A strain which is null for lysS, the constitutive gene, was created by gene disruption (lysS1) and exhibited cold-sensitive lethality . Hence, lysS is dispensable at high temperatures . This cold sensitivity was suppressed by a multi-copy plasmid carrying lysU, the inducible gene . These data are interpreted as indicating that lysS is functionally replaceable by lysU for cell growth, and that the cold sensitivity of lysS1 is caused by insufficient expression of lysU at low temperatures . To investigate the mechanism of lysU expression, cold-resistant bypass mutations were isolated from lysS1, and named als (for abandonment of lysS) . Two als mutations which were linked to lysU contain IS2 insertions upstream of the lysU promoter . They caused a 16-19-fold increase in the lysU-mRNA level . Furthermore, deletion mutations created immediately upstream of the lysU promoter restored growth of lysS1 . These results suggest that transcription of lysU is negatively controlled by a cis-element located upstream of the promoter. J Cell Biol, 1992 Jul, 118(2), 227 - 44 Disulfide bond formation during the folding of influenza virus hemagglutinin; Segal MS et al.; To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues . Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues . We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds. Eur J Biochem, 1992 Jul 1, 207(1), 61 - 8 EPR and redox characterization of iron-sulfur centers in nitrate reductases A and Z from Escherichia coli . Evidence for a high-potential and a low-potential class and their relevance in the electron-transfer mechanism; Guigliarelli B et al.; The redox properties of the iron-sulfur centers of the two nitrate reductases from Escherichia coli have been investigated by EPR spectroscopy . A detailed study of nitrate reductase A performed in the range +200 mV to -500 mV shows that the four iron-sulfur centers of the enzyme belong to two classes with markedly different redox potentials . The high-potential group comprises a {3Fe-4S} and a {4Fe-4S} cluster whose midpoint potentials are +60 mV and +80 mV, respectively . Although these centers are magnetically isolated, they are coupled by a significant anticooperative redox interaction of about 50 mV . The {4Fe-4S}1+ center occurs in two different conformations as shown by its composite EPR spectrum . The low-potential group contains two {4Fe-4S} clusters with more typical redox potentials (-200 mV and -400 mV) . In the fully reduced state, the three {4Fe-4S}1+ centers are magnetically coupled, leading to a broad featureless spectrum . The redox behaviour of the high-pH EPR signal given by the molybdenum cofactor was also studied . The iron-sulfur centers of the second nitrate reductase of E . coli, nitrate reductase Z, exhibit essentially the same characteristics than those of nitrate reductase A, except that the midpoint potentials of the high-potential centers appear negatively shifted by about 100 mV . From the comparison between the redox centers of nitrate reductase and of dimethylsulfoxide reductase, a correspondence between the high-potential iron-sulfur clusters of the two enzymes can be proposed. Eur J Biochem, 1992 Jul 1, 207(1), 195 - 200 Enhanced cell-free transcription of the ribosomal protein L32 gene by the polyoma virus enhancer PEA3 DNA-binding protein; Yoganathan T et al.; The mouse-ribosomal-protein-L32-gene promoter contains a 12-bp sequence motif within the 5'-upstream region termed the beta element which shows significant similarity with the consensus sequence of the polyoma-virus-enhancer PEA3 . A cloned PEA3 DNA-binding protein, expressed in Escherichia coli and purified, activates the expression of the ribosomal-protein-L32 gene in a cell-free system . Moreover, the PEA3 protein participates in the formation of the ribosomal-protein-L32-promoter-preinitiation-transcription complex . The preinitiation complex formed with PEA3 is resistant to competition by oligonucleotides containing the beta element . In addition anti-PEA3 serum interacts with a factor in mouse L1210 nuclear extract that binds to the beta element, causing a supershift in a mobility-shift assay . Our study demonstrates for the first time that the PEA3 protein can transactivate a cellular gene in a cell-free transcription system. Eur J Biochem, 1992 Jul 1, 207(1), 177 - 83 A hybrid protein of urokinase growth-factor domain and plasminogen-activator inhibitor type 2 inhibits urokinase activity and binds to the urokinase receptor; Ballance DJ et al.; The binding of urokinase-type plasminogen activator (uPA) to its specific cell-surface receptor (uPAR) localises the proteolytic cascade initiated by uPA to the pericellular environment . Inhibition of uPA activity or prevention of uPA binding to uPAR might have a beneficial effect on disease states wherein this activity is deregulated, e.g . cancer and some inflammatory diseases . To this end, a bifunctional hybrid molecule consisting of the uPAR-binding growth-factor domain of uPA (amino acids 1-47; GFuPA) at the N-terminus of plasminogen-activator inhibitor type 2 (PAI-2) was produced in Saccharomyces cerevisiae . The purified protein inhibited uPA with kinetics similar to placental or recombinant PAI-2 and was also found to bind to U937 cells and to FL amnion cells . GFuPA-PAI-2 competed with uPA, the N-terminal fragment of uPA and a proteolytic fragment of uPA (amino acids 4-43) in cell binding experiments, indicating that the molecule bound to the cells via uPAR . Hence, both the uPA-inhibitory and uPAR-binding domains of the hybrid molecule were functional, demonstrating the feasibility of the novel concept of introducing an unrelated, functional domain onto a member of the serine-protease-inhibitor superfamily. Am Rev Respir Dis, 1992 Jul, 146(1), 32 - 8 Dibutyryl cyclic AMP attenuates lung responses induced by endotoxin in conscious sheep; Koyama S et al.; Dibutyryl cyclic AMP (DBcAMP) could inhibit the production of prostanoids and modulate the pulmonary vascular responses induced by endotoxin . Diffuse lung injury after endotoxemia in sheep is accompanied by the production of prostanoids and an increase in endothelial permeability . To determine whether exogenous DBcAMP could prevent the endotoxin responses, we measured pulmonary hemodynamics, gas exchange, and lung lymph responses to an intravenous infusion of Escherichia coli endotoxin (1.0 micrograms/kg over 30 min) in unanesthetized sheep in the presence and absence of DBcAMP (30 micrograms/kg/min) infused intravenously for 6 h beginning 1 h before endotoxin infusion or for 4.5 h after 30 min of treatment with endotoxin infusion . We also measured circulating leukocytes and lung lymph and plasma concentrations of thromboxane B2 (TXB2) and prostacyclin (6-keto-PGF1 alpha) metabolites by radioimmunoassay . DBcAMP infusion before endotoxin infusion decreased endotoxin-induced pulmonary hypertension and hypoxemia and markedly attenuated the increased lung lymph flow and lymph protein clearance . DBcAMP after endotoxin only attenuated the increased lung lymph flow and lymph protein clearance . DBcAMP treatment both before and after endotoxin infusion blocked endotoxin-induced increases in lung lymph and plasma TXB2 and 6-keto-PGF1 alpha . DBcAMP did not affect the number of circulating leukocytes . Although DBcAMP alone did not affect the pulmonary and systemic hemodynamics and lung lymph balance, the potential that DBcAMP directly modulates the pulmonary vascular responses to endotoxin as a vasodilator could be expected . We conclude that DBcAMP infusion attenuates lung dysfunction caused by endotoxemia, possibly by preventing prostanoid release and modulating the pulmonary vascular responses. J Bacteriol, 1992 Jul, 174(14), 4856 - 9 Isolation and characterization of Escherichia coli mutants blocked in production of membrane-derived oligosaccharides; Weissborn AC et al.; We report a new procedure for the facile selection of mutants of Escherichia coli that are blocked in the production of membrane-derived oligosaccharides . Four phenotypic classes were identified, including two with a novel array of characteristics . The mutations mapped to two genetic loci . Mutations in the mdoA region near 23 min are in two distinct genes, only one of which is needed for the membrane-localized glucosyltransferase that catalyzes the synthesis of the beta-1,2-glucan backbone of membrane-derived oligosaccharides . Another set of mutations mapped near 27 min closely linked to osmZ; these appear to be in the galU gene. J Bacteriol, 1992 Jul, 174(14), 4530 - 7 Fis plays a role in Tn5 and IS50 transposition; Weinreich MD et al.; The Fis (factor for inversion stimulation) protein of Escherichia coli was found to influence the frequency of transposon Tn5 and insertion sequence IS50 transposition . Fis stimulated both Tn5 and IS50 transposition events and also inhibited IS50 transposition in Dam-bacteria . This influence was not due to regulation by Fis of the expression of the Tn5 transposition proteins . We localized, by DNase I footprinting, one Fis site overlapping the inside end of IS50 and give evidence to strongly suggest that when Fis binds to this site, IS50 transposition is inhibited . The Fis site at the inside end overlaps three Dam GATC sites, and Fis bound efficiently only to the unmethylated substrate . Using a mobility shift assay, we also identified another potential Fis site within IS50 . Given the growth phase-dependent expression of Fis and its differential effect on Tn5 versus IS50 transposition in Dam-bacteria, we propose that the high levels of Fis present during exponential growth stimulate transposition events and might bias those events toward Tn5 and away from IS50 transposition. J Bacteriol, 1992 Jul, 174(13), 4509 - 12 Cloning, expression, and nucleotide sequence of a mutant glgC gene from Escherichia coli B; Meyer CR et al.; A mutant glgC gene contained in a 10.9-kb PstI fragment was cloned from the Escherichia coli B strain SG5 via colony hybridization by using a wild-type glgC probe . The altered allosteric properties of the expressed ADPglucose synthetase were found to result from the conversion of proline to serine at amino acid residue 295. J Bacteriol, 1992 Jul, 174(13), 4212 - 7 Construction of isogenic urease-negative mutants of Helicobacter pylori by allelic exchange; Ferrero RL et al.; Isogenic urease-negative mutants of Helicobacter pylori were constructed by allelic replacement . A region of cloned H . pylori DNA containing the structural urease genes (ureA and ureB) was disrupted by insertion of a mini-Tn3-Km transposon . Electrotransformation of H . pylori cells with kanamycin-ureB-disrupted derivative plasmids resulted in isolation of kanamycin-resistant H . pylori transformants . Competence for electrotransformation appeared to be restricted to certain wild-type H . pylori isolates; only 1 isolate (of 10 tested) was consistently transformed . Two of the kanamycin-resistant H . pylori transformants were further studied and shown to be urease negative . Southern hybridization analyses demonstrated that the urease-negative mutants had been constructed by allelic exchange involving simultaneous replacement of the ureB gene with the kanamycin-ureB-disrupted copy and loss of the vector . Immunoblot studies of whole-cell extracts of the isogenic ureB mutants with anti-H . pylori sera indicated the absence of a polypeptide with an apparent molecular mass of 61 kDa; thus, the mutants no longer synthesized the UreB product . Generation of stable, genetically engineered urease mutants of H . pylori will be useful for addressing the role of urease in the pathogenesis of H . pylori infection. Cancer Res, 1992 Jul 1, 52(13), 3760 - 7 Oncogene complementation in fetal brain transplants; Wiestler OD et al.; Using a neural transplantation model and retrovirus-mediated gene transfer, we have introduced the oncogenes v-Ha-ras and v-myc into the developing rat brain . Upon insertion of a construct encoding v-Ha-ras and the Escherichia coli beta-galactosidase marker gene, the retroviral vector was found to be expressed in neurons, astrocytes, and endothelial cells of the graft . After latency periods of several months, fascicular neoplasms with expression of S-100 protein were observed in 50% of the transplants . The foreign genes were shown to be highly expressed in the tumors and in intact donor cells, by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside histochemistry, indicating that an activated Ha-ras oncogene has the potential to initiate neoplastic transformation of glial cells . Introduction of the v-myc oncogene into 15 grafts resulted in only a single primitive neuroectodermal tumor . However, simultaneous expression of the v-Ha-ras and v-myc genes yielded highly malignant, polyclonal neoplasms in all recipient animals, as early as 13 days after transplantation, from which cell lines could be easily derived . In addition, neoplastic transformation was also observed in vitro following introduction of ras and myc into embryonic forebrain cultures and into newborn cerebellar cultures . These data indicate a powerful complementary transforming effect of ras and myc on neural progenitors in vivo and in vitro . Coexpression of ras and myc may, therefore, provide a highly efficient tool for transforming neural precursor cells in distinct segments of the central nervous system at different stages of development. Infect Immun, 1992 Jul, 60(7), 2870 - 3 Binding of class II Escherichia coli enterotoxins to mouse Y1 and intestinal cells; Donta ST et al.; The binding of class II Escherichia coli heat-labile enterotoxins (LT) to Y1 tissue-cultured cells and mouse intestinal cells was studied and compared with that of class I toxins, including cholera enterotoxin . All radioiodinated (125I) toxins retained their biological activities in both model systems, but only LTIIb could be shown to bind specifically to target cells . LTIIa could inhibit the binding of both class I and LTIIb toxins, a finding which correlates with its ability to bind to multiple gangliosides . LTIIb could not inhibit the binding of the other enterotoxins . The binding and activity of class II toxins could not be modulated by prior exposure of target cells to the B subunit of LTI. Infect Immun, 1992 Jul, 60(7), 2572 - 80 Expression of receptors for enterotoxigenic Escherichia coli during enterocytic differentiation of human polarized intestinal epithelial cells in culture; Kerneis S et al.; To study the expression of human intestinal receptors for enterotoxigenic Escherichia coli (ETEC), the human polarized intestinal epithelial cell line Caco-2 in culture and several subpopulations of HT-29 cells in culture--parental (mainly undifferentiated) HT-29 cells (HT-29 Std), an enterocytelike subpopulation obtained by selection through glucose deprivation (HT-29 Glc-), and an enterocytelike subpopulation obtained by selection through glucose deprivation which maintains its differentiation characteristics when switched back to standard glucose-containing medium (HT-29 Glc-/+)--were used . Since Caco-2 spontaneously differentiated in culture under standard culture conditions (in the presence of glucose) and HT-29 cells were undifferentiated when cultured under standard conditions (HT-29 Std) and differentiated when grown in a glucose-free medium (HT-29 Glc-), we studied the expression of the receptors for colonization factor antigens (CFA) I, II, and III and the 2230 antigen of ETEC in relation to enterocytic differentiation . We provide evidence that expression of ETEC CFA receptors develops in parallel with other differentiation functions of the cultured cells . The expression of ETEC-specific brush border receptors was studied by indirect immunofluorescence using antibodies raised against purified ETEC CFA . No ETEC receptors were detected in HT-29 Std or short-term-cultured Caco-2 cells . However, among the population of HT-29 Std cells, 2 to 4% of the cells were found to bind ETEC, and these cells expressed positive carcinoembryonic antigen immunoreactivity . This indicated that among the population of undifferentiated HT-29 cells, clusters of differentiated cells were present . ETEC CFA receptors were expressed in the apical and basolateral domains of differentiated HT-29 cells, whereas in differentiated Caco-2 cells only apical expression was observed . Both in HT-29 cells (HT-29 Glc-/+) and in Caco-2 cells cultured under standard conditions, ETEC CFA receptors develop as a function of day in culture . This indicated that the expression of the ETEC CFA receptors was a growth-related event . Indeed, ETEC CFA receptors developed in step with the apical expression of differentiation-associated proteins. Curr Genet, 1992 Jul, 22(1), 41 - 5 Genetic transformation of the symbiotic basidiomycete fungus Hebeloma cylindrosporum; Marmeisse R et al.; The pAN7.1 plasmid containing the E . coli hygromycin B phosphotransferase gene was used to transform protoplasts of the ectomycorrhizal fungus Hebeloma cylindrosporum . Hygromycin-resistant transformants were selected at a frequency of one to five per micrograms of transforming DNA . Southern blot analyses revealed multiple copy integration of the transforming plasmid into the genome . The selection system was used to introduce other genes of interest by co-transformation . Two plasmids, one containing tryptophan biosynthesis genes and the other the NADP-glutamate dehydrogenase gene from the saprophytic basidiomycete Coprinus cinereus, were successfully introduced into the H . cylindrosporum genome with up to 70% efficiency of co-transformation . The hygromycin resistance phenotype was stably maintained during growth of transformants on hygromycin-free medium . All transformants retained their ability to form mycorrhizae with the habitual host plant Pinus pinaster, making them suitable for future physiological studies. Arch Biochem Biophys, 1992 Jul, 296(1), 337 - 46 Exogenous quinones directly inhibit the respiratory NADH dehydrogenase in Escherichia coli; Imlay J et al.; The ability of naphthoquinones to generate reactive oxygen species has been widely exploited in studies of oxidative stress . However, excess superoxide dismutase and catalase failed to protect Escherichia coli in rich medium against growth inhibition by plumbagin, indicating that its toxic effect was not due to the production of partially reduced oxygen species . Respiration failed immediately upon the addition of growth-inhibitory levels of plumbagin . Studies in vitro showed that plumbagin and other redox-active quinones intercept electrons from NADH dehydrogenase, the primary respiratory dehydrogenase in glucose-containing media . An excess of oxidative substrate, such as plumbagin, inactivates this enzyme, which appears to be redox-regulated . The resultant respiratory arrest is a cautionary example of metabolic dysfunction from redox-cycling drugs that cannot be attributed to superoxide or hydrogen peroxide. J Virol, 1992 Jul, 66(7), 4591 - 6 Activation of simian virus 40 transcription in vitro by T antigen; Coulombe J et al.; Simian virus 40 is repressed when the viral early gene product large tumor antigen (TAg) binds to specific sites within the viral origin and DNA replication ensues . Late transcription is activated by TAg, even in the absence of viral DNA replication . We show here that TAg produced in human 293 cells can selectively activate Simian virus 40 transcription in a cell-free system . In the absence of DNA binding by TAg, early and late transcription are both activated, as they are in vivo, suggesting that the effect might be mediated by a cellular component(s) utilized by both the early and late promoters . When TAg binds to the viral origin of replication, early transcription is repressed but the late promoter activation is unaffected . Various preparations of TAg differed in their activities, with some able both to bind DNA and to activate transcription and others able to do only one or the other . Since these variations might be explained by variable amounts of associated factors that copurified with TAg, we asked whether a bacterially derived protein could regulate transcription . An NH2-terminal 272-amino-acid fragment of TAg, produced in Escherichia coli as a glutathione S-transferase fusion protein, retains the ability to activate transcription in vitro, similar to that of the full-length protein . Structural features of this region that might be important are discussed. J Virol, 1992 Jul, 66(7), 4390 - 8 Expression of the Marek's disease virus homolog of herpes simplex virus glycoprotein B in Escherichia coli and its identification as B antigen; Chen X et al.; Marek's disease (MD) is an oncogenic disease of chickens caused by MD virus (MDV) . Among the major glycoproteins found in MDV-infected cells are gp100, gp60, and gp49, detected by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with antisera previously shown to be reactive with B antigen in immunodiffusion analysis . Following treatment with tunicamycin (TM), an inhibitor of N-linked glycosylation, the same sera were reported to detect two molecules called pr88 and pr44 . However, the gene encoding B antigen was not unequivocally identified . Recently, an MDV homolog of the gene encoding herpes simplex virus glycoprotein B (gB) was identified and sequenced (L . J . N . Ross, M . Sanderson, S . D . Scott, M . M . Binns, T . Doel, and B . Milne, J . Gen . Virol . 70:1789-1804, 1989) . To determine whether the MDV gB homolog gene might encode the B antigen, antisera against trpE fusion proteins of the MDV gB homolog (trpE-MDV-gB) were prepared . These antisera immunoprecipitated gp100, gp60, gp49, and a 92-kDa precursor polypeptide (pr88, now designated 92-kDa pr88, in the presence of TM) from MDV-infected cell lysates . On the basis of size comparison, trpE-MDV-gB competition and blocking assays, and the fact that gp100, gp60, gp49, and 92-kDa pr88 could be detected in MDV-infected cells with antisera specific to both MDV B antigen and the gB homolog, it was concluded that (i) the MDV gB homolog gene encodes MDV B antigen and (ii) 92-kDa pr88 is the primary precursor polypeptide . The antisera against trpE-MDV-gB also contained antibody reactive with the herpesvirus of turkey gB homolog, consistent with the known antigenic relatedness between the MDV and herpesvirus of turkey B antigens . TM inhibition data and results from pulse-chase analysis with MDV-infected cells show that MDV gB homolog processing involves cotranslational glycosylation of 92-kDa pr88 to form gp100, which is then cleaved to form gp60 and gp49, the N- and C-terminal halves, respectively, of gp100 . This processing pathway is consistent with those of other gB homologs, further supporting the gene identification described above . The conclusions of this study will facilitate future research on the immunobiology of MD, especially studies on the mechanism of immunoprotection. Rev Roum Physiol, 1992 Jul-Dec, 29(3-4), 57 - 62 Phagocytic response in rats following chemical sympathectomy with 6-hydroxydopamine; Derevenco P et al.; Wistar rats were injected i.p . at 2, 4 and 7 days after birth with 6-OHDA (50 mg/kg) . At maturity the phagocytic response of neutrophils was elicited by Escherichia coli (EC) lipopolysaccharide (LPS) . Before and at 3 and 24 hours after the injection of LPS (1 mg/kg i.v.) the leucocyte (L) and neutrophil (N) counts and the phagocytic activity of N in blood against EC have been tested . In controls (6 female and 7 male rats) the number of N increases significantly after 3 h . The L and N responses were similar in controls and in treated (SyX) animals . In controls the percentage of phagocytic active N(PA) and the number of bacteria incorporated by 100 n(IB) shows significant rises at 3 h . In the SyX groups (7 females and 7 males) PA does not increase; the IB decreases significantly in females at 3 and 24 h . In conclusion chemical sympathectomy depresses the PR to LPS in rats; this suggests a stimulatory action of the sympathetic system on the phagocytic immune reaction . An immunological sexual dimorphism exists. Protein Sci, 1992 Jul, 1(7), 850 - 60 Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho . I . Binding of ATP; Geiselmann J et al.; Escherichia coli transcription termination factor rho is an RNA-dependent ATPase, and ATPase activity is required for all its functions . We have characterized the binding of ATP to the physiologically relevant hexameric association state of rho in the absence of RNA and have shown that there are six ATP binding sites per rho hexamer . This stoichiometry has been verified by a number of different techniques, including ultracentrifugation, ultrafiltration, and fluorescence titration studies . We have also shown that ATP can bind to isolated monomers of rho when the hexamer is dissociated with the mild denaturant myristyltrimethylammonium bromide, demonstrating that each promoter of rho carries an ATP binding site . The six binding sites that we observe in the rho hexamer are not equivalent; the hexamer contains three strong (Ka approximately 3 x 10(6) M-1) and three weak (Ka approximately 10(5) M-1) binding sites for ATP . The binding constant of the weak binding site is just the reciprocal of the enzymatic Km for ATP as a substrate; thus these weak sites, as well as the strong sites, can, in principle, take part in the catalytic cycle . The asymmetry induced (or manifested) by ATP binding reduces the symmetry of the rho hexamer from a D3 to a pseudo-D3 state . This "breakage" of symmetry has implications for the molecular mechanism of rho, because an asymmetric structure can lead to directional helicase activity by invoking directionally distinct RNA binding and release reactions (see Geiselmann, J., Yager, T.D., & von Hippel, P.H., 1992c, Protein Sci . 1, 861-873). Protein Sci, 1992 Jul, 1(7), 843 - 9 Structural homology between rbs repressor and ribose binding protein implies functional similarity; Mauzy CA et al.; The deduced amino acid sequence of the rbs repressor, RbsR, of Escherichia coli is homologous over its C-terminal 272 residues to the entire sequence of the periplasmic ribose binding protein . RbsR is also homologous to a family of bacterial repressor proteins including LacI . This implies that the structure of the repressor consists of a two-domain binding protein portion attached to a DNA-binding domain having the four-helix structure of the LacI headpiece . The implications of these relationships to the mechanism of this class of repressors are discussed. Protein Sci, 1992 Jul, 1(7), 831 - 42 Structural and functional analyses of the repressor, RbsR, of the ribose operon of Escherichia coli; Mauzy CA et al.; The DNA sequence encoding the rbs repressor protein, RbsR, has been determined . Amino acid sequence analyses of the product of an rbsR-lacZ fusion and of affinity-purified RbsR demonstrate that translation begins at an unusual codon, TTG, and that the initial amino acid is removed during maturation of the protein . DNA-binding assays indicate that RbsR binds to a region of perfect dyad symmetry spanning the rbs operon transcriptional start site and that the affinity for the rbs operator is reduced by addition of ribose, consistent with ribose being the inducer of the operon . RbsR is a member of a family of homologous repressor proteins having very similar DNA-binding sites and binding to similar operator sequences. J Postgrad Med, 1992 Jul-Sep, 38(3), 112 - 4, 111 Hyperbaric oxygen therapy in diabetic foot; Doctor N et al.; To study the effect of hyperbaric oxygen therapy in chronic diabetic foot lesions, a prospective controlled study was undertaken . Thirty diabetics with chronic foot lesions were randomised to study group (conventional management and 4 sessions of hyperbaric oxygen therapy) and control group (conventional management) . The patients were assessed for average hospital stay, control of infection and wound healing . The control of infection spread was quicker . Positive cultures decreased from initial 19 to 3 in study group as against from 16 to 12 in the control group . (p < 0.05) . This difference was most pronounced for Escherichia coli . Also, the need for major amputation was significantly less in the study group (n = 2) as against the control group (n = 7) (p < 0.05) . The average hospital stay was not affected . We conclude that hyperbaric oxygen therapy can be safely used and is beneficial as an adjuvant therapy in chronic diabetic foot lesions. Fiziol Zh, 1992 Jul-Aug, 38(4), 56 - 62 {The ultrastructural changes in the glomeruli and juxtaglomerular apparatus at different periods of endotoxic shock}; Kharlanova NG et al.; It is shown that after endotoxin injection the ultrastructural changes in the glomeruli can favour development of the acute renal insufficiency . In the initial and intermediate periods of the endotoxin shock the granular and agranular forms of juxtaglomerular cells hyperfunction, respectively, are revealed as well as an increase of renin activity in plasma . At the stage of the late endotoxemia ultrastructural alterations are stabilized . The juxtaglomerular cells synthesize and accumulate secretory granules, but renin activity in plasma decreases almost to the initial level. J Struct Biol, 1992 Jul-Aug, 109(1), 70 - 7 Configuration of interdomain linkers in pyruvate dehydrogenase complex of Escherichia coli as determined by cryoelectron microscopy; Wagenknecht T et al.; The dihydrolipoyl transacetylase (E2p) component of the pyruvate dehydrogenase complex (PDC) of Escherichia coli is a multidomain polypeptide comprising a catalytic domain, a domain that binds dihydrolipoyl dehydrogenase (E3-binding domain), and three domains containing lipoic acid (lipoyl domains) . In PDC 24 subunits of E2p associate by means of interactions involving the catalytic domains to form the structural core of PDC . From cryoelectron microscopy and computer image analysis of frozen-hydrated isolated E2p cores it appears that the lipoyl domains are located peripherally about the core complex and do not assume fixed positions . To further test this interpretation the visibility of the lipoyl domains in electron micrographs was enhanced by specifically biotinylating the lipoic acids and labeling them with streptavidin . In agreement with the studies of native, unlabeled E2p cores, cryoelectron microscopy of the streptavidin-labeled E2p cores showed that the lipoic acid moieties are capable of extending approximately 13 nm from the surface of the core . Localization of the E3-binding domains was accomplished by cryoelectron microscopy of E2p-E3 subcomplexes prepared by reconstitution in vitro . Frequently an apparent gap of several nanometers separated the bound E3 from the surface of the core . The third component of PDC, pyruvate dehydrogenase (E1p), appeared to bind to the E2p core in a manner similar to that observed for E3 . These results support a structural model of the E2p core in which the catalytic, E3-binding, and three lipoyl domains are interconnected by linker sequences that assume extended and flexible conformations. Protein Sci, 1992 Jul, 1(7), 861 - 73 Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho . II . Binding of RNA; Geiselmann J et al.; The rho protein of Escherichia coli interacts with the nascent RNA transcript while RNA polymerase is paused at specific rho-dependent termination sites on the DNA template, and (in a series of steps that are still largely undefined) brings about transcript termination at these sites . In this paper we characterize the interactions of rho with RNA and relate these interactions to the quaternary structure of the functional form of rho . We use CD spectroscopy and analytical ultracentrifugation to determine the binding interactions of rho with RNA ligands of defined length ({rC}n where n > or = 6) . Rho binds to long RNA chains as a hexamer characterized by D3 symmetry . Each hexamer binds approximately 70 residues of RNA . We show by ultracentrifugation and dynamic laser light scattering that, in the presence of RNA ligands less than 22 nucleotide residues in length, rho changes its quaternary structure and becomes a homogeneous dodecamer . The dodecamer contains six strong binding sites for short RNA ligands: i.e., one site for every two rho protomers . The measured association constant of these short RNAs to rho increases with increasing (rC)n length, up to n = 9, suggesting that the binding site of each rho protomer interacts with 9 RNA nucleotide residues . Oligo (rC) ligands bound to the strong RNA binding sites on the rho dodecamer do not significantly stimulate the RNA-dependent ATPase activity of rho . Based on these features of the rho-RNA interaction and other experimental data we propose a molecular model of the interaction of rho with its cofactors. J Neurobiol, 1992 Jul, 23(5), 481 - 90 Preparation and biological properties of native and recombinant ciliary neurotrophic factor; Gupta SK et al.; CNTF (ciliary neurotrophic factor), purified from rabbit sciatic nerves by a relatively simple procedure, is bioactive in tissue culture at low picomolar concentration and appears as a doublet on polyacrylamide gel electrophoresis (PAGE) . In these nerves, CNTF accounts for more than one-half of the survival-promoting activity on ciliary neurons . The concentration of CNTF in rabbit sciatic nerves is estimated to be 5 nmol/kg, more than 1000 times higher than would seem to be required to support neurons if the neurotrophic factor were homogeneously distributed . With recombinant DNA technology, rat CNTF has been synthesized in Escherichia coli, purified without denaturating agents, and found to be bioactive at a slightly lower concentration than CNTF extracted from rabbit sciatic nerves . After radioiodination, CNTF retains biological activity but is not specifically internalized and retrogradely transported in motor and sensory axons . In peripheral nerves, ciliary neurotrophic factor differs biologically from nerve growth factor (NGF) by its much higher tissue concentration and apparent lack of internalization by peripheral nerve axons. Biochem Biophys Res Commun, 1992 Jun 30, 185(3), 987 - 92 Identification of two types of homologous DNA pairing activity in mouse cells; Mishina Y et al.; We have identified two types of homologous DNA pairing activity in mouse cell extracts by a strand-transfer assay . Both activities are separated from each other by anion-exchange chromatography; neither of them needs ATP . One requires magnesium ion and is stimulated by Escherichia coli single-stranded DNA binding protein, whereas the other does not require the ion and shows a higher affinity for a left-handed Z-DNA. Biochem Biophys Res Commun, 1992 Jun 30, 185(3), 874 - 80 The N-terminal region of HIV-1 integrase is required for integration activity, but not for DNA-binding; Schauer M et al.; HIV-1 integrase binds to both double- and single-stranded DNA with Kd-values of around 20 nM, irrespective of sequence similarities with the termini of the viral LTR . For integration activity, however, the correct LTR sequence of the substrate is required . The putative zinc-binding site present at the N-terminus of the protein is not essential for DNA binding, since deletion mutants of the protein lacking this sequence show similar affinity towards DNA as the wild-type; however, these mutants are not capable of performing the LTR-cleavage and integration reactions . Thus, it appears that the N-terminal part of the integrase is essential for catalytic activity. Biochem Biophys Res Commun, 1992 Jun 30, 185(3), 1133 - 40 Inhibition of DNA polymerases by tripeptide derivative protease inhibitors; Taguchi T et al.; Benzyloxycarbonyl(Z)-Leu-Leu-Leu-al and dansyl(Dns)-Leu-Leu-Leu-CH2Cl, well known as protease inhibitors, effectively inhibit the activities of DNA polymerases alpha, beta and gamma from rat liver and pol I from Escherichia coli, but the ability of these inhibitors to inhibit terminal deoxynucleotidyl transferase (TdT) is weak . The mode of inhibition by these tripeptide analogues is non-competitive with dNTP . The Ki values for Z-Leu-Leu-Leu-al and Dns-Leu-Leu-Leu-CH2Cl are 6.25 x 10(-5) M and 6.56 x 10(-5) M, respectively. Biochem Biophys Res Commun, 1992 Jun 30, 185(3), 1000 - 4 Proteolytic processing of amyloid beta protein precursor (APP) by thrombin; Igarashi K et al.; Search for proteases responsible for an altered processing of APP which generates intermediates containing beta/A4 peptide is preceding to understand the formation of beta amyloid deposits characteristic of Alzheimer's disease, since many studies reveal that APP is ordinarily processed so as not to generate beta amyloid . Here, we have examined the action of thrombin, a serine protease in the blood clotting, in APP processing . Thrombin cleaved the mouse recombinant APP695 in vitro, resulting in the accumulation of 28 kDa fragment . The immunoblot analysis showed that the fragment is derived from the carboxy-terminal side of the recombinant APP695 . Further, amino acid sequencing exhibited that the fragment is generated by the cleavage at Arg 510-Ile 511 and therefore includes entire beta/A4 peptide . We consider that the 28 kDa fragment is a possible intermediate for beta/A4 peptide . Thus thrombin may be involved in the altered processing of APP. Biochemistry, 1992 Jun 30, 31(25), 5937 - 44 The mRNA binding track in the Escherichia coli ribosome for mRNAs of different sequences; Bhangu R et al.; Interactions between mRNA and rRNA on the 30S ribosomal subunit or 70S ribosome have been determined by photochemical cross-linking experiments using synthetic mRNA analogs substituted with 4-thiouridine . A set of RNA molecules containing different sequences has been used to determine the extent to which binding contacts are sequence dependent . The 16S rRNA and 23S rRNA nucleotides that form a part of the binding site have been identified by reverse transcription . The nucleotides are U1381, G1338, G1300, G1156, A845, U723, G693, A532, G497, U420, G413/A412, and G436 of 16S rRNA and U887 of 23S rRNA . Several additional nucleotides (U1065 of 23S rRNA and A1227, G818, G524, and G423 of 16S rRNA) are seen for some, but not all, of the mRNAs . Results obtained with two mRNAs containing the Shine-Dalgarno sequence were similar to those obtained with mRNAs lacking the Shine-Dalgarno sequence . Eight of these cross-linking sites were also seen when a mixture of RNA was used in which there are 12 random nucleotides preceding and seven random nucleotides succeeding an AUG codon . These results indicate that to a large extent placement of the mRNA in the ribosome does not depend upon its primary sequence. Biochemistry, 1992 Jun 30, 31(25), 5878 - 87 Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate; Yano T et al.; Asp222 is an invariant residue in all known sequences of aspartate aminotransferases from a variety of sources and is located within a distance of strong ionic interaction with N(1) of the coenzyme, pyridoxal 5'-phosphate (PLP), or pyridoxamine 5'-phosphate (PMP) . This residue of Escherichia coli aspartate aminotransferase was replaced by Ala, Asn, or Glu by site-directed mutagenesis . The PLP form of the mutant enzyme D222E showed pH-dependent spectral changes with a pKa value of 6.44 for the protonation of the internal aldimine bond, slightly lower than that (6.7) for the wild-type enzyme . In contrast, the internal aldimine bond in the D222A or D222N enzyme did not titrate over the pH range 5.3-9.5, and a 430-nm band attributed to the protonated aldimine persisted even at high pH . The binding affinity of the D222A and D222N enzymes for PMP decreased by 3 orders of magnitude as compared to that of the wild-type enzyme . Pre-steady-state half-transamination reactions of all the mutant enzymes with substrates exhibited anomalous progress curves comprising multiphasic exponential processes, which were accounted for by postulating several kinetically different enzyme species for both the PLP and PMP forms of each mutant enzyme . While the replacement of Asp222 by Glu yielded fairly active enzyme species, the replacement by Ala and Asn resulted in 8600- and 20,000-fold decreases, respectively, in the catalytic efficiency (kmax/Kd value for the most active species of each mutant enzyme) in the reactions of the PLP form with aspartate . In contrast, the catalytic efficiency of the PMP form of the D222A or D222N enzyme with 2-oxoglutarate was still retained at a level as high as 2-10% of that of the wild-type enzyme . The presteady-state reactions of these two mutant enzymes with {2-2H}aspartate revealed a deuterium isotope effect (kH/kD = 6.0) greater than that {kH/kD = 2.2; Kuramitsu, S., Hiromi, K., Hayashi, H., Morino, Y., & Kagamiyama, H . (1990) Biochemistry 29, 5469-5476} for the wild-type enzyme . These findings indicate that the presence of a negatively charged residue at position 222 is particularly critical for the withdrawal of the alpha-proton of the amino acid substrate and accelerates this rate-determining step by about 5 kcal.mol-1 . Thus it is concluded that Asp222 serves as a protein ligand tethering the coenzyme in a productive mode within the active site and stabilizes the protonated N(1) of the coenzyme to strengthen the electron-withdrawing capacity of the coenzyme. Biochemistry, 1992 Jun 30, 31(25), 5861 - 8 Mechanism of action of sparsomycin in protein synthesis; Theocharis DA et al.; Before CI isomerizes to C*I, we detect a competitive phase of inhibition (Ki = k5/k4 = 0.05 microM) which eventually, by increasing the concentration of I, becomes linear mixed noncompetitive and involves C*I in place of CI . The equilibration of C and I according to reaction 2 is much slower than the equilibration between C and S in reaction 1 (time-dependent inhibition) . The inactivation plots obey reaction 2 and allow us to estimate k6 as equal to 2.2 min-1 . The isomerized C*I, free of excess I, can be studied as a mixture with complex C . From the kinetics of the regeneration of C from C*I, in the presence of puromycin, we can estimate k7 to be between 0.22 min-1 and 0.06 min-1 . Although the isomerized C*I survives after adsorption on cellulose nitrate filter disks, it does not survive after gel chromatography on a Sepharose CL-4B column but is converted quantitatively to complex C containing D of unchanged reactivity . This result does not support the proposed {Flynn, G . A., & Ash, R . J., (1990) Biochem . Biophys . Res . Commun . 166, 673-680} chemical reaction between D and I toward new products . The isomerized C*I can be obtained not only from the already-made complex C but also de novo from D, R, and M . In the latter case, the reactions which lead to C are represented by the following hypothetical scheme: D + R + M in equilibrium with DRM or C (binding reaction) . When C*I is formed de novo, this reaction is coupled to reaction 2 and the ultimate product is a mixture of C and C*I.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1992 Jun 30, 31(25), 5841 - 8 Biochemical characterization of the Oct-2 POU domain with implications for bipartite DNA recognition; Botfield MC et al.; B-cell specific regulation of immunoglobulin gene expression provides a model for the interaction of promoter and enhancer elements with eukaryotic sequence-specific DNA binding proteins . A critical element of this system, the octamer site (5'-ATGCAAAT-3'), is recognized by the B-cell transcription factor Oct-2 . Octamer recognition is mediated by the POU domain, a conserved structural motif which--like the zinc finger and leucine zipper--defines a family of related transcription factors . Homologies among POU sequences suggest a bipartite structure, consisting of an N-terminal POU-specific subdomain and C-terminal variant homeodomain connected by a linker of variable length and sequence . As a first step toward a molecular understanding of the Oct-2 POU domain and its mechanism of DNA recognition, we have overexpressed in Escherichia coli the intact POU domain and subdomains as thrombin-cleavable fusion proteins and have purified these fragments to homogeneity following digestion with thrombin . Biochemical and biophysical characterization yields the following results . (i) The intact POU domain (166 residues) is monomeric and exhibits high-affinity octamer-specific DNA-binding activity . (ii) Limited proteolytic digestion demonstrates that the POU domain contains two proteolytically stable subdomains (the POU-specific subdomain and the variant homeodomain) connected by a proteolytically sensitive linker . (iii) The isolated subdomains are each monomeric and do not interact to form noncovalent heterodimers.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1992 Jun 30, 31(25), 5687 - 91 Intramolecular catalysis of a proline isomerization reaction in the folding of dihydrofolate reductase; Texter FL et al.; The cis/trans isomerization of the peptide bond preceding proline residues in proteins can limit the rate at which a protein folds to its native conformation . Mutagenic analyses of dihydrofolate reductase (DHFR) from Escherichia coli show that this isomerization reaction can be intramolecularly catalyzed by a side chain from an amino acid which is distant in sequence but adjacent in the native conformation . The guanidinium NH2 nitrogen of Arg 44 forms one hydrogen bond to the imide nitrogen and a second to the carbonyl oxygen of Pro 66 in wild-type DHFR . Replacement of Arg 44 with Leu results in a change of the nature of the two slow steps in refolding from being limited by the acquisition of secondary and/or tertiary structure to being limited by isomerization . The simultaneous replacement of Pro 66 with Ala (i.e., the Leu 44/Ala 66 double mutant) eliminates this isomerization reaction and once again makes protein folding the limiting process . Apparently, one or both of the hydrogen bonds between Arg 44 and Pro 66 accelerate the isomerization of the Gln 65-Pro 66 peptide bond . The replacement of Arg 44 with Leu affects the kinetics of the slow folding reactions in a fashion which indicates that the crucial hydrogen bonds form in the transition states for the rate-limiting steps in folding.