Escherichia coli

J Bacteriol, 1990 Mar, 172(3), 1556 - 61
Cloning, nucleotide sequence, and expression of the Rhodobacter sphaeroides Y thioredoxin gene; Pille S et al.; Synthetic oligodeoxynucleotide probes based on the known amino acid sequence of Rhodobacter sphaeroides Y thioredoxin were used to identify, clone, and sequence the structural gene . The amino acid sequence derived from the DNA sequence of the R . sphaeroides gene was identical to the known amino acid sequence of R . sphaeroides thioredoxin . An NcoI site was created by directed mutagenesis at the beginning of the thioredoxin gene, inducing in the encoded protein the replacement of serine in position 2 by alanine . The 421-base-pair NcoI-PstI restriction fragment obtained was ligated in the pKK233-2 expression vector and the resulting hybrid plasmid was used to transform Escherichia coli strains lacking functional thioredoxin . Transformants that complemented mutations in the trxA gene were identified by increased colony size on rich medium, growth on minimal medium with methionine sulfoxide, and ability to support M13 growth and T7 replication; this latter phenotype implies correct interaction between R . sphaeroides thioredoxin and the product of T7 gene 5 . The presence of R . sphaeroides thioredoxin was further confirmed by enzyme assay.

Biophys J, 1990 Mar, 57(3), 555 - 66
Image analysis reveals that Escherichia coli RecA protein consists of two domains; Yu X et al.; The Escherichia coli RecA protein catalyzes homologous genetic recombination by forming helical polymers around DNA molecules . These polymers have an ATPase activity, which is essential for the movement of strands between two DNA molecules . One obstacle to structural studies of the RecA filament has been that the ATPase results in a dynamical polymer containing a mixture of states with respect to the bound ATP and its hydrolytic products . We have formed filaments which are trapped in the ADP-Pi state by substituting AIF4- for the Pi, and have used these stable filaments to generate a three-dimensional reconstruction from electron micrographs . The resolution of the reconstruction is sufficient to resolve the 38-k RecA subunit into two nearly equal domains . This reconstruction provides the most detailed view yet of the RecA protein, and serves as a framework within which existing biochemical data on RecA can be understood.

Aichi Gakuin Daigaku Shigakkai Shi, 1990 Mar, 28(1 Pt 2), 283 - 94
{Effect of lipopolysaccharide on biological properties and induction of alveolar bone resorption in rats}; Maki E; A study was made on the effects of lipopolysaccharides (LPS) from Escherichia coli and Actinobacillus actinomycetemcomitans on mitogenic response and the release of calcium from mouse calvaria . LPS exhibited significant mitogenic activity on mouse spleen cells, and increased the release of calcium from mouse calvaria in vitro . The effect of LPS from E . coli on alveolar bone resorption in Wistar male rats was also studied . LPS was infused by an Alzet miniosmotic pump that was implanted subcutaneously on the backs of the rats . A catheter connected to the pump was brought up to the maxillary right second molar using a nylon ligature . In this way the rats were continuously infused for 7 days with LPS . Alveolar bone loss was measured by an image-analyser (Nexus 6411) . Rats stimulated by LPS exhibited significant alveolar bone loss, but bone loss was not observed in rats stimulated by saline . Polymyxin B effectively inhibited the LPS-stimulated bone resorption.

Mikrobiol Zh, 1990 Mar-Apr, 52(2), 94 - 7
{The purification of beta-galactosidase by flow electrophoresis}; Andrienko VI et al.; A plant has been constructed for the free-flow electrophoresis . It permits carrying out electrophoretic separation both in a free flow and with the use of neutral grained fillers . Beta-galactosidase has been purified from the culture liquid of the Escherichia coli cell phage lysate . Its content in the purified material amounted to 81.2

Mol Gen Mikrobiol Virusol, 1990 Mar, (3), 27 - 9
{Localization in Escherichia coli of transcribed regions of the broad host range plasmid pBS222}; Dubeikovskii AN et al.; The set of insertions of the CmR-gene region devoid of the promoter into the broad host-range plasmid pBS222 regions transcribed in Escherichia coli cells has been obtained and characterized . Three transcribed regions of the plasmid that are dispensable for life functions of the plasmid have been identified by the insertions technique . The deleted plasmid derivatives have been isolated suitable for using as the broad host-range vectors.

Cell Differ Dev, 1990 Mar, 29(3), 187 - 94
Cell lineage analyses of epithelia and blood vessels in chimeric mouse embryos by use of an embryonic stem cell line expressing the beta-galactosidase gene; Kadokawa Y et al.; We have established an embryonic stem (ES) cell line, MS1-EL4, which has the potential to make various tissues in chimeric embryos and, at the same time, expresses the beta-galactosidase gene which was introduced as a good cell marker . To examine cell behavior and lineage during embryogenesis, we injected MS1-EL4 cells into host blastocysts and recovered chimeric embryos at various developmental stages . We examined the distribution of the MS1-EL4 cell derivatives by staining whole embryos with X-gal and by making serial paraffin sections . So far we have obtained the following results: (1) the MS1-EL4 cell line is useful for studying cell lineages because of its ubiquitous expression at least until the mid-gestation stage; (2) cells of the primitive ectoderm and its derivative epithelial tissues continue to intermingle with each other until the late primitive streak stage . Then, at early somite stages, cells of various epithelia stop intermingling and give rise to small coherent clones; (3) blood vessels of the yolk sac are formed by local aggregation of the ancestor cells and those of the embryo proper by proliferation and sprouting from fewer angiogenic cells.

Cell Differ Dev, 1990 Mar, 29(3), 181 - 6
A mouse embryonic stem cell line showing pluripotency of differentiation in early embryos and ubiquitous beta-galactosidase expression; Suemori H et al.; For analysis of chimeric mice made by injecting embryonic stem (ES) cells into host blastocysts, it is very desirable if the ES cells have a good cell marker that can distinguish them from host cells . It is ideal if the marker can be easily visualized in every type of cell and tissue throughout the embryogenesis . We tried to produce such ES cell lines by introducing an E . coli beta-galactosidase (beta-gal) gene construct by electroporation . One of the transformant lines (MS1-EL4) showed beta-gal activity in every undifferentiated stem cell . After being induced to differentiate in vitro, cells with various morphologies showed beta-gal activity . We also detected beta-gal activity in a wide variety of tissue elements in solid tumors made by injecting the MS1-EL4 cells into syngeneic mice . Then we produced chimeric embryos by injecting the MS1-EL4 cells into blastocysts and recovering the embryos at various developmental stages . We found that the MS1-EL4 cells contributed to various tissues and expressed beta-gal activity, including not only descendants of the inner cell mass but also the trophectoderm-derived extraembryonic ectoderm.

J Biochem Biophys Methods, 1990 Mar, 20(3), 181 - 8
Purification of recombinant ribonuclease T1 expressed in Escherichia coli; Shirley BA et al.; A protocol for the rapid purification of ribonuclease T1 expressed from a chemically synthesized gene cloned into Escherichia coli is described . QAE ion-exchange and Sephadex G-50 chromatography are used to give over 300 mg (88% yield) of pure ribonuclease T1 from 61 of liquid culture in 3 days . We also report a new absorption coefficient for RNase T1: E1%278 nm = 15.4.

Protein Eng, 1990 Mar, 3(4), 267 - 72
Efficient expression and Zn(II)-dependent structure of the DNA binding domain of the yeast GAL4 protein; Serikawa Y et al.; Three protein fragments of different sizes which contain the DNA binding domain of transcription factor GAL4 from Saccharomyces cerevisiae have been expressed in functional forms in Escherichia coli . DNase I footprinting and gel retardation assays showed that the purified proteins bound to the same DNA sequence on the gal1-gal10 promoter as intact GAL4 does . Denaturation--refolding experiments demonstrated that Zn(II) is necessary for maintenance of the conformation of the DNA binding domain of GAL4, as judged on UV-CD and 1H-NMR measurements, as well as for specific DNA binding.

J Photochem Photobiol B, 1990 Mar, 4(4), 371 - 8
Different lethal effects by enzyme-generated triplet indole-3-aldehyde in different Escherichia coli strains; Duran N et al.; Strains of Escherichia coli which lack 4-thiouridine (S4U) exhibit a higher survival rate than their wild-type parents which contain S4U after treatment with enzyme-generated triplet indole-3-aldehyde . In a similar manner to results obtained with monochromatic 334 nm UV light, the survival is related to single-strand breakage of DNA in E . coli containing the pBR 322 plasmid . The effects of the excited states generated by an enzymatic system suggest that S4U is an important chromophore in the lethal effects observed . The results also suggest that the energy transferred from triplet indole-3-aldehyde to S4U may also be passed from S4U of t-RNA to DNA, possibly through a singlet oxygen intermediate generated by excited S4U, resulting in a decrease in the survival rate of E . coli containing S4U . These results emphasize the importance of excited states in biological systems.

FEMS Microbiol Lett, 1990 Mar 1, 56(1-2), 201 - 5
Cloning and expression in Escherichia coli of tryptophan genes from Streptomyces griseus IMRU 3570; Rivero-Lezcano O et al.; Two Sau3A fragments of Streptomyces grisues IMRU 3570 were cloned in pBR322 as a vector . One of these clones contained the genetic information needed to complement trpA and trpB mutations in Escherichia coli . The other complements trpA, trpB and trpC mutations in E . coli . Both fragments originated in the same region of the chromosome but the latter is 1 kilobase (kb) longer in the region nearest the tetracycline promoter.

Br J Pharmacol, 1990 Mar, 99(3), 499 - 502
Relationships between tumour necrosis factor, eicosanoids and platelet-activating factor as mediators of endotoxin-induced shock in mice; Myers AK et al.; 1 . The toxicity of intravenous recombinant human tumour necrosis factor (rhTNF), a TNF fragment (TNF114-130), endotoxin and combinations of rhTNF or TNF114-130 were tested in mice . Neither rhTNF nor TNF114-130 was lethal alone, but when combined with a non-lethal dose of endotoxin, rhTNF provoked dose-dependent mortality, as did higher doses of endotoxin alone . 2 . Both the toxicity and the vasopermeability changes induced by endotoxin alone were blocked by the platelet-activating factor (PAF) antagonist BN52021, indomethacin or the dual cyclo-oxygenase/lipoxygenase inhibitor BW755C . 3 . The lethality of the combined low dose endotoxin/rhTNF challenge was unaffected by pretreatment with BN52021, indomethacin or BW755C, or by treatment at 6 h intervals with BN52021 or BW755C . 4 . The results of these studies suggest that TNF, a putative, early mediator of septic or endotoxin shock, cannot by itself mimic all of the effects of bacterial endotoxin in the model used in this study . Apparently, TNF works synergistically with other mediators whose release is stimulated by endotoxin . 5 . The results also suggest that the mechanism of shock production by the rhTNF/endotoxin combination in mice is not dependent on the early stimulation of eicosanoid or PAF synthesis by rhTNF.

Pharmacol Res, 1990 Mar-Apr, 22(2), 97 - 102
Inhibition of ornithine-decarboxylase produced by S(+) and R(-) ibuprofen in rats; Bruni G et al.; The differences between the S(+) and R(-) ibuprofen enantiomers in anti-inflammatory activity were assayed by measuring the release of 14CO2 in rats treated with labelled 14COOH-ornithine . Furthermore we investigated in vitro the inhibitory activity on ornithine-decarboxylase and the anti-inflammatory activity of R(-) and S(+) ibuprofen by using the carrageenin-induced paw oedema test in the rat.

J Clin Microbiol, 1990 Mar, 28(3), 591 - 5
Immunologic characterization of a 35-kilodalton recombinant antigen of Mycobacterium tuberculosis; Rumschlag HS et al.; A 35-kilodalton (kDa) recombinant antigen (35-kDa antigen) produced by Escherichia coli JM107 carrying DNA from Mycobacterium tuberculosis was purified and immunologically examined by in vivo and in vitro methods . A monoclonal antibody (2B2) was produced against the 35-kDa antigen . The protein was purified from the insoluble fraction of the recombinant E . coli strain by either affinity chromatography with the 2B2 monoclonal antibody or preparative isoelectric focusing . In enzyme-linked immunosorbent assay and Western blot (immunoblot) analyses, antibody to 2B2 reacted with whole-cell sonic extracts of M . tuberculosis and other slowly growing mycobacteria but not with two rapid growers, M . chelonae and M . fortuitum . An injection series totaling less than 1 mg of purified protein without adjuvant elicited a humoral response in guinea pigs . In one guinea pig, 10 micrograms of purified protein injected intradermally elicited both a humoral and a cell-mediated response . Results of these studies suggest that the 35-kDa antigen is a membrane-associated protein that stimulates both humoral and cell-mediated immune responses and should be evaluated as a vaccine candidate.

J Neurosci, 1990 Mar, 10(3), 1014 - 24
Immunolocalization of G protein alpha-subunits in the Drosophila CNS; Wolfgang WJ et al.; In order to uncover the role of G proteins in the integrative functioning and development of the nervous system, we have begun a multidisciplinary study of the G proteins present in the fruit fly, Drosophila melanogaster . In this report, we describe the distribution of 3 different G protein alpha-subunits in the adult Drosophila CNS as determined by immunocytochemical localization using affinity-purified antibodies generated to synthetic oligopeptide sequences unique to each alpha-subunit . Western blot analysis of membranes prepared from Drosophila heads indicates that antibodies specific for the Drosophila Go alpha and Gs alpha homologs recognize the appropriate protein species predicted by molecular cloning (Quan et al., 1989; Thambi et al., 1989) . The Gi alpha homolog could not be detected in head membranes by Western blotting, consistent with the negligible levels of expression observed for Gi alpha on Northern blots of head mRNA (Provost et al., 1988) . However, a Drosophila Gi alpha fusion protein could be detected by these antibodies following expression in E . coli . Immunolocalization studies revealed that the Go alpha and Gs alpha homologs are expressed at highest levels in neuropils and at intermediate levels in the cortex of all brain and thoracic ganglion areas . Only the lamina contained low levels of these alpha-subunits in the CNS . Additionally, Gs alpha appears to be associated with the cell membranes of neuronal cell bodies, while Go alpha has a more diffuse distribution, suggesting its presence in the cytoplasm as well as cell membranes . In contrast to the wide distribution of Go alpha and Gs alpha, Gi alpha has a surprisingly restricted distribution in the CNS . It is present at high levels only in photoreceptor cell terminations, glomerulae of the antennal lobes, and the ocellar retina . Little or no Gi alpha was detected in other brain regions or in the thoracic ganglion . Gi alpha, then, appears to be uniquely associated with some primary sensory afferents and their terminations, suggesting the presence of specific receptor and/or effector systems which mediate the transmission of primary sensory information in Drosophila.

J Infect, 1990 Mar, 20(2), 151 - 4
Detective work in continuous ambulatory peritoneal dialysis; al-Wali W et al.; We report five cases of continuous ambulatory peritoneal dialysis in which the mechanisms and sources of infection were established . We show how diligent enquiry and environmental investigation can explain the pathogenesis of infection and help in prevention by motivation of the patient.

Proc Natl Acad Sci U S A, 1990 Mar, 87(6), 2112 - 6
The trithorax gene, a trans-acting regulator of the bithorax complex in Drosophila, encodes a protein with zinc-binding domains; Mazo AM et al.; The trithorax (trx) gene functions in segment determination in Drosophila through interaction with genes of the bithorax complex and Antennapedia complex . Genetic evidence suggests that trx may be considered a positive regulator of homeotic genes . Sequencing of cDNAs corresponding to the entire trx transcription unit revealed the existence of an unusually long open reading frame encoding 3759 amino acids . The main features of the predicted trx protein are several cysteine-rich regions which can be folded into zinc finger-like domains . Cysteine-rich portions expressed from trx cDNAs in Escherichia coli are capable of zinc binding in vitro, suggesting a possible function for the trx product as a metal-dependent DNA-binding protein . Analysis of trx mutant embryos with antibody to the Ultrabithorax (Ubx) gene product showed decreased staining in parasegment 6 of the ventral nerve cord of late embryos . However, expression of Ubx was not affected in embryos carrying the lethal mutation trxE3, in which one of the putative zinc finger-like domains of the trx protein is deleted . This differential effect of the E3 mutation suggests that trx exhibits other function(s) besides those involved in the regulation of Ubx expression in the ventral nerve cord of the embryo.

Proc Natl Acad Sci U S A, 1990 Mar, 87(5), 1749 - 52
Hsp70 proteins, similar to Escherichia coli DnaK, in chloroplasts and mitochondria of Euglena gracilis; Amir-Shapira D et al.; The heat-shock response of Euglena gracilis was studied by pulse-labeling cells with {35S}sulfate at both the normal growth temperature (21 degrees C) and an elevated temperature (36 degrees C) . Analysis of the labeled proteins by polyacrylamide gel electrophoresis indicated that the rate of synthesis of at least 3 major and 15 minor polypeptides increased in cells grown at the higher temperature . Three of the proteins appear to be immunologically related to the ubiquitous approximately 70-kDa heat-shock protein (Hsp70) family . One protein of 68 kDa was found in the cytoplasm (P68cyt) and was the major heat-shock protein in Euglena gracilis . Two other proteins, 68 and 70 kDa, were localized in mitochondria (P68mit) and chloroplasts (P70chl), respectively, and they crossreacted with a polyclonal antibody raised against the Escherichia coli heat-shock protein DnaK . Like DnaK, P68mit and P70chl could be phosphorylated in vitro with {gamma-32P}ATP in a reaction that was stimulated by Ca2+ . A protein with characteristics similar to those of P70chl was also found in chloroplasts isolated from maize and spinach.

J Bacteriol, 1990 Mar, 172(3), 1392 - 9
Transcription of the ftsZ gene and cell division in Escherichia coli; Robin A et al.; The ftsZ gene of Escherichia coli, which lies in a cluster of cell division genes at 2 min on the genetic map, codes for a protein which is thought to play a key role in triggering cell division . Using an ftsZ::lacZ operon fusion, we have studied the transcription of the ftsZ gene under conditions in which cell division was either inhibited or synchronized in the bacterial population . In ftsZ, ftsA, ftsQ, and ftsI (or pbpB) mutants, there was no change in the differential rate of expression of the ftsZ gene in nonpermissive conditions, when cell division was completely blocked . Although the FtsZ protein is thought to be limiting for cell division, in synchronized cultures the ftsZ gene was expressed not only at the moment of septation initiation but throughout the cell cycle . Its expression, however, was not exponential but linear, with a rapid doubling in rate at a specific cell age; this age, about 20 min after division in a 60-min cycle, was different from the age at which the ftsZ::lacZ operon was duplicated . However, it was close to the age at which replication initiated and at which the rate of phospholipid synthesis doubled . During the transient division inhibition after a nutritional shift-up, ftsZ transcription again became linear, with two doublings in rate at intervals equal to the mass doubling time in the rich medium; it adopted the exponential rate typical of rich medium about 60 min after the shift-up, just before the bacterial population resumed cell division . The doubling in the rate of ftsZ transcription once per cycle in synchronized cultures and once per mass doubling time during the transition period after a nutritional shift-up reflects a new cell cycle event.

J Neurochem, 1990 Mar, 54(3), 762 - 70
A unique neurofilament from Torpedo electric lobe: sequence, expression, and localization analysis; Linial M et al.; A set of cDNA clones encoding a protein highly homologous to the mammalian middle-size class of neurofilaments (NF-M) was characterized . The amino acid similarity between the Torpedo and rat NF-M approaches 90% in the amino-terminal "rod-like" domain and is significantly lower in the carboxy-terminal tail . The Torpedo protein contains 13 tandem repeats of a unique six amino acid core, containing a Lys-Ser-Lys putative phosphorylation site . Surprisingly, the 3' untranslated region contains stretches of 80-90% nucleic acid homology with the mammalian, but not with the chicken sequences . This homology is greater than much of the coding region, suggesting that the 3' untranslated region of the message has an important functional role, perhaps governing RNA stability or localization . This Torpedo NF-M mRNA is expressed specifically in the electric lobe and was not detected in other tissues, including brain and spinal cord . A polyclonal antibody generated against a fusion protein synthesized in E . coli detects a 150-kDa protein in the electric lobe and organ, as well as a small amount of material in the brain . Cytochemical studies reveal immunoreactivity in electromotor neuron axons and terminals . Specific expression of neurofilament genes in subsets of central neurons may be important in determining the morphology and functional characteristics of specific neuronal subtypes.

Bioconjug Chem, 1990 Mar-Apr, 1(2), 123 - 31
Cleavage of DNA by electrochemically activated MnIII and FeIII complexes of meso-tetrakis (N-methyl-4-pyridiniumyl)porphine; Rodriguez M et al.; Electrochemical methods were used to activate MnIII and FeIII complexes of meso-tetrakis(N-methyl-4-pyridiniumyl)porphine (H2TMPyP) to cause cleavage of pBR322 DNA and to study their interaction with sonicated calf thymus DNA . Electrochemical reduction of MnIIITMPyP and FeIIITMPyP (at low concentrations) in the presence of O2 was required to activate these complexes . However, FeIIITMPyP at 1 x 10(-6) M produced DNA strand breakage without being electrochemically reduced . At low concentrations, FeIITMPyP was more efficient at cleaving DNA than MnIITMPyP . Reduction of O2 at a platinum electrode also produced some cleavage but to a much smaller extent . The oxidized form of MnIIITMPyP (charge 5+) has higher affinity for sonicated calf thymus (CT) DNA than the reduced form (charge 4+), as determined by the negative shift in E degrees' for the voltammetric wave in the presence of DNA . Both forms of FeIIITMPyP (charge 4+) interact with DNA to about the same extent . Differential pulse voltammetry was used to determine binding constants (K) and binding-site sizes (s) of the interaction of these metalloporphyrins with sonicated CT DNA . The data were analyzed assuming both mobile and static equilibria . MnIIITMPyP binds to DNA (5 mM Tris, 50 mM NaCl, pH 7) with K = 5 (+/- 2) x 10(6) M-1, s = 3 bp (mobile) or K = 3.6 (+/- 0.3) x 10(6) M-1, s = 4 bp (static) . FeIIITMPyP at that ionic strength caused DNA precipitation . At higher ionic strength (0.1 M Tris, 0.1 M NaCl, pH 7), FeIIITMPyP associates to DNA with K = 4.4 (+/- 0.2) x 10(4) M-1, s = 5 bp (mobile) or K = 1.9 (+/- 0.1) x 10(4) M-1, s = 6 bp (static).

Mol Microbiol, 1990 Mar, 4(3), 461 - 9
A soluble 60 kiloDalton antigen of Chlamydia spp . is a homologue of Escherichia coli GroEL; Bavoil P et al.; Two major 60 kD protein species can be separated by differential detergent extraction in Chlamydia spp . A Sarkosyl-soluble 60 kD protein is (i) structurally and antigenically distinct from the previously characterized 60 kD Omp2 outer membrane protein; and (ii) antigenically related to a bacterial common antigen of similar molecular weight which includes a 65 kD mycobacterial antigen and the GroEL heat-shock protein of Escherichia coli . Among GroEL homologues, the chlamydial protein (chl-GroEL) uniquely displays affinity towards immobilized thiol groups . The significance of this property is discussed with respect to the synthesis and assembly of the chlamydial disulphide-rich cell wall late in the growth cycle . Chl-GroEL is identical to the Triton X-100-soluble, ocular delayed-type hypersensitivity agent (Morrison et al., 1989), an essential component in the development of blinding trachoma . An autoimmune mechanism for chronic chlamydial diseases based on chl-GroEL homology to host proteins is hypothesized.

FEMS Microbiol Lett, 1990 Mar 1, 56(1-2), 183 - 8
Control of temperature-dependent synthesis of K99 fimbriae; van der Woude MW et al.; The influence of temperature on the production of K99 fimbriae by Escherichia coli was determined in cultures growing at constant specific growth rate in continuous cultures . In a wild type strain, in which the K99 operon is present on a low copy number plasmid, low cultivation temperature repressed the K99 production . This temperature-dependent production was not observed after introduction of multicopies of the regulatory region of the K99 operon into this strain, nor in E . coli K12 harbouring a recombinant, multicopy plasmid encoding the K99 operon . These results are in agreement with a regulation model in which a regulatory factor, most likely a repressor, inhibits expression of the K99 operon at low temperatures.

J Clin Microbiol, 1990 Mar, 28(3), 489 - 94
Characterization of Mycobacterium paratuberculosis and organisms of the Mycobacterium avium complex by restriction polymorphism of the rRNA gene region; Chiodini RJ; Nineteen Mycobacterium paratuberculosis strains, including strains of bovine, caprine, ovine, cervid, subhuman primate, and human origins, were compared with organisms of the M . avium complex by restriction fragment length polymorphism with a 5S rRNA gene probe as the reference DNA . Mycobacterial DNA was extracted, digested with several restriction enzymes, subjected to electrophoresis and Southern blotting, and then hybridized with a 5S rRNA gene probe from Escherichia coli . Hybridizing bands were visualized by autoradiography, and the sizes of the resulting rRNA fragments in kilobases were determined . Base substitutions were calculated on the basis of the number of shared fragments between species and strains . It was determined that M . paratuberculosis and the M . avium complex possess a single copy of the rRNA genes within their genomes and that the M . avium complex and M . paratuberculosis are a group of closely related organisms, likely with a common ancestral link . In proximity to the 5S rRNA gene exists a region or regions which display polymorphisms that are capable of species and subspecies differentiation . M . paratuberculosis strains isolated from humans, subhuman primates, and animals were found to be genetically identical to each other . M . paratuberculosis strains lacked the genetic heterogeneity (restriction fragment length polymorphisms) characteristic of most species, suggesting that this organism has unidirectional genetic selection . It is therefore assumed to be biologically isolated, occupying a unique and specific biological niche . This homogeneity was present in all strains, including those of animal and primate (subhuman and human) origin and strains isolated from different parts of the world.

Scand J Gastroenterol, 1990 Mar, 25(3), 203 - 9
Characteristics of immune-complex-induced chronic experimental colitis in rats with a therapeutic effect of sulphasalazine; Axelsson LG et al.; Experimental colitis was induced in rats by topical irritation of the colonic mucosa with 1 ml of 1% formalin followed by intravenous injection of 0.5 ml soluble immune complexes (IC) made in vitro in antigen excess and having characterized precipitation and complement activation profiles . The rats had been preimmunized with Escherichia coli O14:K7:H- to produce antibodies cross-reactive with colonic mucosa, thus aggravating the colitis to chronicity . Histologic evaluation of inflammation in the colon was performed on days 6, 12, and 18 by determining the number of phagocytic cells . The colitis was inhibited by sulphasalazine therapy given daily, 125.5 mumols (50 mg)/kg body weight, starting on the day when the inflammation was produced with IC and formalin . Sulphasalazine therapy significantly (p less than 0.05) decreased the number of phagocytic cells in the mucosa on days 12 and 18 but not on day 6 . The results may give a clue to the beneficial pharmacologic effects of sulphasalazine in the treatment of ulcerative colitis.

Proc Natl Acad Sci U S A, 1990 Mar, 87(6), 2289 - 93
Engrailed, a homeodomain protein, can repress in vitro transcription by competition with the TATA box-binding protein transcription factor IID; Ohkuma Y et al.; Engrailed (En) is a homeodomain protein that binds to a consensus sequence (NP) and plays an important role during Drosophila development . Purified En, which is produced in Escherichia coli, binds not only to this consensus sequence but also to the TATA box of the Drosophila Hsp70 promoter and of other eukaryotic promoters . Interestingly, En represses transcription of these promoters in an in vitro-reconstituted mammalian transcription system and footprint analyses show that En competes with the TATA box-binding protein transcription factor IID for binding to the TATA box . In contrast, a stable template-committed complex formed by preincubation of transcription factor IID with the promoter is not disrupted by addition of En, and in this case transcription is not repressed . These in vitro studies suggest a transcriptional repression mechanism, involving competition between En and transcription factor IID for TATA box binding, that may be involved in En-mediated repression in vivo.

J Infect Dis, 1990 Mar, 161(3), 518 - 24
Frequency and organization of pap homologous DNA in relation to clinical origin of uropathogenic Escherichia coli; Plos K et al.; The pap operon encodes the gal alpha 1-4gal beta specific adhesins of Escherichia coli . The presence and organization of pap homologous DNA was determined using two probes specific for pap in 217 uropathogenic E . coli samples by dot blot and Southern blot analysis . The frequency of pap homologous DNA was 76% in pyelonephritis, 69% in cystitis, and 52% in an asymptomatic bacteriuria group . Further, the gal alpha 1-4gal beta binding phenotype among the pap-positive strains was expressed more often in acute pyelonephritis (91%) than the cystitis (60%) or asymptomatic bacteriuria (52%) strains . This was explained in part by difference in organization of pap homologous DNA between the genotypically positive pyelonephritis and asymptomatic bacteriuria strains . The pyelonephritis isolates contained three copies of pap significantly more often than the asymptomatic bacteriuria strains, and the pyelonephritogenic O-antigen types had a general increase in pap copy number . The difference in expression of gal alpha 1-4gal beta adhesins between pyelonephritis and asymptomatic bacteriuria isolates was thus not only a function of the frequency of pap homologous DNA but also of phenotypic expression among genotypically pap-positive strains.

J Infect Dis, 1990 Mar, 161(3), 420 - 5
Peripheral blood CD4-mediated enhancement and CD8-mediated suppression in the presence of recombinant hepatitis B virus core antigen; Shirai M et al.; The proliferative response of peripheral blood T cells to hepatitis B core antigen (HBcAg) was studied in hepatitis B patients . CD4+ T cells from patients with chronic active hepatitis type B (CAH-B) exhibited a significant proliferative response to HBcAg, especially in hepatitis B envelope antigen (HBeAg)-positive patients . In contrast, there was no apparent T cell reaction to HBcAg in patients with CAH non-A, non-B, HBeAg-positive healthy carriers and in healthy volunteers . The proliferative response to CD4+ cells to bacterial extracts of Escherichia coli was always insignificant in all patients and healthy volunteers . The CD8+ cells did not proliferate in response to HBcAg in any subject, even in the presence of autologous irradiated CD4+ responder cells . The CD8+ cells, preactivated with HBcAg and HBcAg-reactive irradiated autologous CD4+ cells, suppressed the proliferative response to autologous CD4+ cells to HBcAg but not the response to phytohemagglutinin in HBcAg-responder CAH-B patients . CD4-mediated HBcAg-specific enhancement and CD8-mediated HBcAg-specific suppression in the peripheral blood compartments of HBcAg-responsive CAH-B patients are possible.

Proc Natl Acad Sci U S A, 1990 Mar, 87(5), 1845 - 9
Enhanced polymerization of recombinant human deoxyhemoglobin beta 6 Glu----Ile; Baudin-Chich V et al.; Polymerization of the deoxy form of sickle cell hemoglobin (Hb S; beta 6 Glu----Val) involves both hydrophobic and electrostatic intermolecular contacts . These interactions drive the mutated molecules into long fibrous rods composed of seven pairs of strands . X-ray crystallography of Hb S and electron microscopy image reconstruction of the fibers have revealed the remarkable complementarity between one of the beta 6 valines of each molecule (the donor site) and an acceptor site at the EF corner of a neighboring tetramer . This interaction constitutes the major lateral contact between the two strands in a pair . To estimate the relative importance of this key hydrophobic contact in polymer formation we have generated a polymerizing Hb with isoleucine at the beta 6 position (beta E6I) by site-directed mutagenesis . The mutated beta chains were produced in Escherichia coli and reassembled into functional tetramers with native alpha chains . Compared to native Hb S, the beta E6I mutant polymerizes faster and with a shortened delay time in 1.8 M phosphate buffer, indicating an increased stability of the nuclei preceding fiber growth . The solubility of the beta E6I mutant Hb is half that of native Hb S . Computer modeling of the donor-acceptor interaction shows that the presence of an isoleucine side chain at the donor site induces increased contacts with the receptor site and an increased buried surface area, in agreement with the higher hydrophobicity of the isoleucine residue . The agreement between the predicted and experimental differences in solubility suggests that the transfer of the beta 6 valine or isoleucine side chain from water to a hydrophobic environment is sufficient to explain the observations.

Infect Immun, 1990 Mar, 58(3), 801 - 7
The 987P gene cluster in enterotoxigenic Escherichia coli contains an STpa transposon that activates 987P expression; Klaasen P et al.; The genetic determinant for the production of 987P fimbriae has been cloned into pBR322 . Analysis of frequently occurring deletions in the resultant recombinant plasmid, pPK180, revealed that the 987P gene cluster contains a transposon that encodes the synthesis of heat-stable enterotoxin STpa and is flanked by inverted repeats of IS1 . Hybridization experiments with STpa- and 987P-specific probes demonstrated that a variety of STpa+ 987P+ wild-type Escherichia coli strains contained contiguous STpa-987P DNA, most likely on their chromosome . Transcription of the 987P gene cluster appeared to be activated by the adjacent IS1 element.

Infect Immun, 1990 Mar, 58(3), 794 - 800
Characterization of binding of Escherichia coli strains which are enteropathogens to small-bowel mucin; Wanke CA et al.; Before an enteropathogen binds to the small bowel, it must interact with the small-bowel mucus (SBM) layer . To determine whether this interaction involves specific binding of diarrheagenic Escherichia coli, we used a quantitative assay with labeled, purified rabbit SBM . Binding of SBM from an adult rabbit was significantly greater to strain 162, an agglutinating E . coli strain, than it was to RDEC-1, a rabbit pathogen, and was significantly greater to strain 2348/PMAR, an enteropathogenic E . coli strain, than it was to strains 1392+ and 1392-, which are enterotoxigenic E . coli strains with and without colonizing fimbriae, respectively . Binding of strains RDEC-1, 2348/PMAR, and 162-4 was significantly greater to SBM than to bovine serum albumin . Binding of all strains increased in a linear fashion with increasing amounts of SBM and was reproducible (r = 0.85) . Binding was significantly greater at pH 5.7 than at pH 7.4 or 8.0 for all five strains . Temperature did not alter the binding of any strain . Strains 162-4 and RDEC-1 bound significantly more to proximal SBM than to rabbit distal SBM, while strains 1392+ and 1392- bound significantly more to distal SBM . Oxidation of sugars from SBM significantly decreased the binding of all strains . Each pathogenic E . coli strain bound distinctively to SBM; the SBM sugars appeared to mediate this binding for all E . coli strains . Binding was also dependent on mucin characteristics, as binding varied by region of the gut (increased for proximal SBM for strains 162-4 and RDEC-1 and for distal SBM for strains 1392+ and 1392-) . The developmental age of the gut significantly affected binding only of the rabbit pathogen RDEC-1.

Infect Immun, 1990 Mar, 58(3), 695 - 702
Purification and characterization of the Dr hemagglutinins expressed by two uropathogenic Escherichia coli strains; Kist ML et al.; The fibrillar Dr hemagglutinins expressed by two uropathogenic Escherichia coli isolates were mechanically sheared from whole cells and subsequently purified by using anion-exchange high-pressure liquid chromatography . The isolated hemagglutinins were proteins with apparent subunit molecular masses of 14,500 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric points of 5.4 in denaturing isoelectric focusing gels . The two proteins were serologically related to each other but distinct from P fimbriae, as assessed by bacterial agglutination and immunoblotting . The amino acid compositions of the two hemagglutinins were highly similar both to each other and to other Dr hemagglutinins . N-terminal amino acid sequencing of the major hemagglutinin subunit proteins demonstrated homology with afimbrial E . coli adhesins.

Infect Immun, 1990 Mar, 58(3), 680 - 6
Polyclonal anti-idiotypic antibodies exhibit antigenic mimicry of limited type 1 fimbrial proteins of Escherichia coli; Paque RE et al.; Polyclonal anti-idiotypic antibodies (anti-Ids)(fim) developed against idiotypes on antibodies (Ab-1s) that specifically bind structural, organelle fimbrial proteins of Escherichia coli were able to modulate immune function in anti-Id(fim)-immunized mice . Proliferation or suppression of splenic lymphoid cell responses by polyclonal anti-Ids in tissue culture appeared to be dose dependent . Anti-Ids were able to induce a dose-dependent T-cell-mediated immunity specific for type 1 fimbrial antigen(s) in immunized animals when assessed in vitro, but they failed to elicit in vivo positive ear-swelling skin reactions . Anti-Ids were unable to induce protective immunity against an in vivo infectious challenge with E . coli in anti-Id-immunized adult animals, but they stimulated a specific, secondary antibody response in anti-Id-challenged mice . Anti-Ids stimulated the development of anti-anti-Ids (Ab-3s) specifically binding a fimbrial antigen(s) and revealed the presence of antibody idiotypes binding E . coli adhesin proteins in the 27- to 29-kilodalton range . Results suggest discrete, but subtle, immunomodulatory effects of the anti-Ids and potential vaccinoid properties capable of stimulating a specific humoral and cellular response in vivo.

Plant Cell, 1990 Mar, 2(3), 195 - 206
Acyl carrier protein (ACP) import into chloroplasts does not require the phosphopantetheine: evidence for a chloroplast holo-ACP synthase; Fernandez MD et al.; Import of the acyl carrier protein (ACP) precursor into the chloroplast resulted in two products of about 14 kilodalton (kD) and 18 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Time course experiments indicate that the latter is a modification derivative of the 14-kD peptide after the removal of the transit peptide . Substitution of serine 38 by alanine, eliminating the phosphopantetheine prosthetic group attachment site of ACP, produced a precursor mutant that gave rise to only the 14-kD peptide during import, showing that the modified form depends on the presence of serine 38 . Furthermore, these results demonstrate that the prosthetic group is not essential for ACP translocation across the envelope or proteolytic processing . Analysis of the products of import by nondenaturing, conformationally sensitive gels showed reversal of the relative mobility of the 14-kD peptide and the modified form, raising the possibility that the modification is the addition of the phosphopantetheine . Proteolytic processing and the modification reaction were reconstituted in an organelle-free assay . The addition of coenzyme A to the organelle-free assay completely converted the 14-kD peptide to the modified form at 10 micromolar, and this only occurred with the wild-type substrate . Reciprocally, treatment of the products of a modification reaction with Escherichia coli phosphodiesterase converted the modified ACP from back to the 14-kD peptide . These results strongly support the conclusion that there is a holo-ACP synthase in the soluble compartment of the chloroplast capable of transferring the phosphopantetheine of coenzyme A to ACP.

Res Microbiol, 1990 Mar-Apr, 141(3), 336 - 41
The plasmid-encoded arsenical resistance pump: an anion-translocating ATPase; Rosen BP; An anion-translocating ATPase has been identified as the product of the arsenical resistance operon of resistance plasmid R773 . When expressed in Escherichia coli this ATP-driven oxyanion pump catalyses extrusion of the oxyanions arsenite, antimonite and arsenate . Maintenance of a low intracellular concentration of oxyanion produces resistance to the toxic agents . The pump is composed of two polypeptides, the products of the arsA and arsB genes . This two-subunit enzyme produces resistance to arsenite and antimonite . A third gene, arsC, expands the substrate specificity to allow for arsenate pumping and resistance.

J Gen Microbiol, 1990 Mar, 136 ( Pt 3), 535 - 43
Characterization and comparison of mitochondrial DNAs and rRNAs from Penicillium urticae and P . chrysogenum; Sekiguchi J et al.; Mitochondrial DNA (mt DNA) from a patulin producer, Penicillium urticae (synonym P . griseofulvum), was 27.8 kb +/- 0.6 kb in size by electron microscopy and 27.2 kb by agarose gel electrophoresis . Restriction endonuclease maps for nine restriction enzymes were constructed, and eleven fragments which covered the total range of the mt DNA were cloned into the Escherichia coli plasmid vector pUC19 . Southern analysis of the native genomes of P . urticae and P . chrysogenum with six of the cloned fragments as probes indicated similar genome arrangements as well as similar restriction maps . Both the large and small rRNA genes of P . urticae and P . chrysogenum were located on these restriction maps using Southern hybridization, and the result also supported the similar arrangement . Agarose/formaldehyde gel electrophoresis indicated that the small rRNA was 1.5 kb in size in both species; but, surprisingly, the large rRNA was 4.2 kb in size for P . urticae and 3.5 kb for P . chrysogenum . These sizes were, respectively, 1.1 kb and 0.4 kb larger than those from the very closely related Aspergillus nidulans.

Mol Biol (Mosk), 1990 Mar-Apr, 24(2), 408 - 16
{Construction and expression in Escherichia coli of a gene of hybrid hemagglutinin H1-H3 of influenza virus}; Petrenko VA et al.; The hybrid gene of influenza virus hemagglutinin (HA) of the H1-subtype, carrying the sequence coding for the fragment of H3-subtype antigenic site B, was constructed . The product of expression of this gene in E . coli was obtained as a fusion protein with beta-galactosidase . The chimeric protein was shown to retain the antigenic properties of HA of H1-subtype and to interact specifically with antibodies against the synthetic peptide corresponding to the B site fragment of HA of the H3-subtype.

AIDS Res Hum Retroviruses, 1990 Mar, 6(3), 317 - 27
Epitope mapping of the HIV-1 gag region by analysis of gag gene deletion fragments expressed in Escherichia coli defines eight antigenic determinants; Marcus-Sekura CJ et al.; Immune response to HIV infection is generally characterized by appearance of antibodies to the gag protein p24 early in infection, and by apparent loss of p24 antibodies accompanied by increases in p24 antigen levels with disease progression . Precise definition of the immunodominant epitopes present in gag gene proteins has potential clinical significance . Seventeen anti-gag monoclonal antibodies (MAb) were used in enzyme-linked immunosorbent assays (ELISA) with antigens expressed by nine recombinant clones to define epitopes on HIV gag proteins which elicit an immune response . All of the MAbs tested, except two anti-p17, reacted with a clone which expresses the carboxyl terminal 13 amino acids of p17 and all of p24 and p15 . All anti-p24 MAbs reacted with clones containing all of p24 . MAbs reacted differentially with clones containing deleted regions depending on the antigenic portion expressed . Of thirteen potential identifiably different genomic regions which could be predicted from the genomic structure of the clones, eight different antigen epitopes were defined: two on p17, five on p24, and one on p15 (in the region corresponding to the carboxyl terminal protein p6) . Six regions did not appear to react with any of the monoclonal antibodies available . Identification of the epitopes present in the cloned antigens should allow their use to evaluate sera from HIV-infected donors at different clinical stages of progression to AIDS.

J Helminthol, 1990 Mar, 64(1), 1 - 8
Molecular analysis of the gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae; Sugane K et al.; The gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae was studied using recombinant DNA techniques . cDNA synthesized from poly(A)-rich mRNA from T . spiralis infective larvae was ligated into phage vector lambda gt11 DNA and packaged in vitro . The phages were propagated on Escherichia coli and a lambda gt11 expression library was constructed . A cDNA clone encoding a 46 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method . A clone containing nearly full-length cDNA for a 46 kDa protein was isolated . The gene encoding this 46 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the cDNA as a probe . The gene was transcribed to mRNA with approximately 1400 nucleotides and translated to 46 kDa polypeptide . The antigenic polypeptide was excreted/secreted as a 46 kDa native antigen . The antigenic beta-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera.

J Clin Microbiol, 1990 Mar, 28(3), 519 - 24
Expression in Escherichia coli of a dominant immunogen of Trypanosoma cruzi recognized by human chagasic sera; Cotrim PC et al.; A genomic clone expressing a Trypanosoma cruzi antigen in Escherichia coli was identified using human chagasic sera . Chagasic antibodies affinity purified on extracts of this clone recognized a high-molecular-weight protein expressed in all developmental stages of the parasite life cycle, as well as in various T . cruzi strains . The antigen is associated with the cytoskeleton of the parasite and localizes along the attachment region between the flagellum and the cell body . Antibodies to the recombinant antigen were detected in the sera of 115 chagasic patients from different endemic regions, but not in sera of patients with leishmaniasis, T . rangeli infection, or other parasitic diseases . Our data suggest that the presence of antibodies to this antigen may be specifically associated with Chagas' disease.

Arthritis Rheum, 1990 Mar, 33(3), 366 - 74
Lupus-inducing drugs alter the structure of supercoiled circular DNA domains; Zacharias W et al.; We analyzed the effects of procainamide (PROC), hydralazine (HYD), N-acetylprocainamide (NAPA), and L-canavanine (CAN) on circular supercoiled plasmids as models for chromosomal loop domains . The supercoil-dependent B-Z equilibrium in recombinant plasmids was used as an indicator of structural changes induced in circular DNA . Two-dimensional gel electrophoresis showed that PROC and HYD strongly inhibited supercoil-induced Z-DNA formation, whereas NAPA caused less pronounced changes in the B-Z equilibrium, and CAN had no effect . Gel retardation assays showed that the binding of a Z-DNA-specific autoimmune antibody to a Z-DNA-containing plasmid was strongly perturbed by HYD, but not influenced by CAN . Both PROC and NAPA showed moderate inhibition of antibody binding . Our results demonstrate the different potentials of these 4 drugs to interact with DNA and to alter the tertiary topology of DNA domains . It is conceivable that the in vivo capacity of PROC and HYD to induce antinuclear antibodies may be related to their ability to influence structural features in chromosomal DNA domains or nucleosomes, thus liberating antigenic structural epitopes in DNA and/or DNA-associated proteins.

Oncogene, 1990 Mar, 5(3), 397 - 403
Molecular genetic characterization of epitope-specific monoclonal antibodies against the myc family proteins; Ikegaki N et al.; The myc family proteins were used to produce monoclonal antibodies with defined specificities . The pattern of mosaic homology among the myc family proteins facilitated the efficient identification of monoclonal antibodies specific to myc homology box sequences . Sequential epitopes for pan-myc reactive monoclonal antibodies produced against N-myc/c-myc fusion protein were further defined by use of truncated myc proteins made in E . coli and synthetic oligopeptides corresponding to myc box sequences . One class of antibodies was found to be specific to the first myc box sequence, whereas the other was found to be reactive with the third myc box sequence . Further development of anti-myc monoclonal antibodies, especially those antibodies specific to each myc box sequence, would be likely to facilitate analysis of the possible biological functions of the myc proteins in vivo and in vitro.

Proc Natl Acad Sci U S A, 1990 Mar, 87(5), 1663 - 7
Amplified RNA synthesized from limited quantities of heterogeneous cDNA; Van Gelder RN et al.; The heterogeneity of neural gene expression and the spatially limited expression of many low-abundance messenger RNAs in the brain has made cloning and analysis of such messages difficult . To generate amounts of nucleic acids sufficient for use in standard cloning strategies, we have devised a method for producing amplified heterogeneous populations of RNA from limited quantities of cDNA . Whole cerebellar RNA was primed with a synthetic oligonucleotide containing the T7 RNA polymerase promoter sequence 5' to a polythymidylate region . After second-strand cDNA synthesis, T7 RNA polymerase was used to generate amplified antisense RNA (aRNA) . Up to 80-fold molar amplification has been achieved from nanogram quantities of cDNA . The amplified material is similar in size distribution to the parent cDNA and shows sequence heterogeneity as assessed by Southern and Northern blot analysis . Specific messages for moderate-abundance mRNAs for actin and guanine nucleotide-binding protein (G-protein) alpha subunits have been detected in the amplified material . By using in situ transcription to generate cDNA, sequences for cyclophilin have been detected in aRNA derived from single cerebellar tissue sections . cDNA derived from a single cerebellar Purkinje cell also has been amplified and yields material that hybridizes to cognate whole RNA and mRNA but not to Escherichia coli RNA.

Infect Immun, 1990 Mar, 58(3), 822 - 7
Characterization of monoclonal antibodies against the Escherichia coli hemolysin; Pellett S et al.; Twelve monoclonal antibodies (MAbs) produced against the Escherichia coli hemolysin (HlyA) encoded by the hemolysin recombinant plasmid pWAM04 were studied . HlyA derivatives from recombinant strains with different plasmids encoding HlyA amino-terminal and carboxy-terminal truncates, HlyA in-frame deletions, and HlyA frameshift mutations were used in immunoblots to localize the antigenic determinants for the anti-HlyA MAbs . The mapping of the MAb epitopes was also facilitated by immunoblotting analysis of HlyA polypeptide fragments derived by cyanogen bromide cleavage . The HlyA epitopes for 11 of the MAbs were mapped to relatively small linear regions of the cytolysin ranging from 28 to 160 amino acids . Five of the MAbs (C10, G8, E2, B7, and D12) neutralized HlyA hemolytic activity to varying degrees . The epitopes for these neutralizing MAbs were found to reside within the following HlyA regions: C10 and G8, amino acids 2 to 160; E2, amino acids 161 to 194; B7, amino acids 518 to 598; and D12, amino acids 626 to 726 . Hemolytically active HlyA was dependent on the action of the hlyC gene product . The D12 MAb recognized only HlyA produced by strains with an intact hlyC function . MAb A10 recognized an epitope within the HlyA region from amino acids 728 to 829 where a glycine-rich repeat domain exists; however, this MAb did not neutralize HlyA hemolytic activity . A HlyA domain map showing the anti-HlyA epitope location was constructed.

J Virol, 1990 Mar, 64(3), 1290 - 7
Virus-neutralizing monoclonal antibody to a conserved epitope on the duck hepatitis B virus pre-S protein; Lambert V et al.; In this study we used duck hepatitis B virus (DHBV)-infected Pekin ducks and heron hepatitis B virus (HHBV)-infected heron tissue to search for epitopes responsible for virus neutralization on pre-S proteins . Monoclonal antibodies were produced by immunizing mice with purified DHBV particles . Of 10 anti-DHBV specific hybridomas obtained, 1 was selected for this study . This monoclonal antibody recognized in both DHBV-infected livers and viremic sera a major (36-kilodalton) protein and several minor pre-S proteins in all seven virus strains used . In contrast, pre-S proteins of HHBV-infected tissue or viremic sera did not react . Thus, the monoclonal antibody recognizes a highly conserved DHBV pre-S epitope . For mapping of the epitope, polypeptides from different regions of the DHBV pre-S/S gene were expressed in Escherichia coli and used as the substrate for immunoblotting . The epitope was delimited to a sequence of approximately 23 amino acids within the pre-S region, which is highly conserved in four cloned DHBV isolates and coincides with the main antigenic domain as predicted by computer algorithms . In in vitro neutralization assays performed with primary duck hepatocyte cultures, the antibody reduced DHBV infectivity by approximately 75% . These data demonstrate a conserved epitope of the DHBV pre-S protein which is located on the surface of the viral envelope and is recognized by virus-neutralizing antibodies.

Agric Biol Chem, 1990 Mar, 54(3), 619 - 24
Effects of nutritional conditions on plasmid stability and production of tryptophan synthase by a recombinant Escherichia coli; Matsui T et al.; Effects of nutritional conditions and insertion direction of the tryptophan synthase (TSase) gene into a plasmid vector on the plasmid stability and the production of TSase in high cell concentration cultures were examined using recombinant Escherichia coli (E . coli K12 IFO 3301) harboring pBR322trpAB(1) (the TSase gene was inserted at the EcoRI site of pBR322 in the clockwise direction) and pBR322trpAB(2) (counterclockwise direction) . As to the effects of the insertion direction, the cells harboring pBR322trpAB(2) were slightly lower in the growth rate and the plasmid stability than those harboring pBR322trpAB(1) . However, the former was higher in the productivity of TSase and the final cell concentration attained than the latter . On the other hand, the addition of organic nutrients, especially yeast extract, to TK-25 medium was very effective to improve the plasmid stability . Among the components of yeast extract, L-glutamic acid was found to be effective to improve both the plasmid stability and the production of TSase . When 1 g/l of L-glutamic acid was added to TK-25 medium, a mineral synthetic medium developed for a high concentration culture, 115g (dry basis)/l of recombinant cells were obtained in 14 hr and the expression of TSase was maintained at 240-300 U/mg-protein during the cultivation.

Appl Microbiol Biotechnol, 1990 Mar, 32(6), 658 - 61
Synthesis of 9-(beta-D-arabinofuranosyl)guanine using whole cells of Escherichia coli; Zinchenko AI et al.; Synthesis of 9-(beta-D-arabinofuranosyl)guanine (ara-G) from 1-(beta-D-arabinofuranosyl)cytosine (ara-C) and guanine, guanosine or 2'-deoxyguanosine (dG) by glutaraldehyde-treated Escherichia coli BM-11 cells is described . It is shown that the concentration of phosphate ions, molar ratio of substrates and pH of the reaction medium are factors affecting product yield . Under optimum conditions ara-G was produced in the reaction mixture in a yield of 63%-65% based on dG as the best source of guanine base . The yield of isolated ara-G was 48%-53%.

Cytotechnology, 1990 Mar, 3(2), 133 - 40
Expression of human beta-interferon in Namalwa KJM-1 which was adapted to serum-free medium; Miyaji H et al.; A Namalwa cell line, KJM-1, which was adapted to serum-free medium is thought to be a very useful host cell line for recombinant DNA technology . Thus, the utility of Namalwa KJM-1 for expression of foreign genes was examined . As a model system human beta-interferon (beta-IFN) gene was engineered for expression in this cell line . For construction of the beta-IFN expression vector pSE1 beta 1-4, the expression vector pAGE107 was constructed and used . It contains simian virus 40 (SV40) early promoter, the rabbit beta-globin RNA processing signals for splicing and polyadenylation, and SV40 early RNA processing signal for polyadenylation . In addition to the above transcription unit, pAGE107 contains the ampicillin-resistance gene and G418-resistance gene . They can confer ampicillin resistance to Escherichia coli (E . coli) and G418 resistance to animal cells . To introduce plasmid DNA into cells, electroporation is a useful method (Wong, 1982; Potter, 1984) . We have established conditions for DNA-mediated transfection of Namalwa KJM-1 cell line by electroporation . Among pSE1 beta 1-4-introduced cells, clone 1-3 was further examined for the expression of beta-IFN in serum-free medium . The production level of beta-IFN was elevated with the increase of the cell density . The results indicated that the Namalwa KJM-1 cell line is useful for production of foreign gene products.

J Biotechnol, 1990 Mar, 13(4), 293 - 304
High-level expression of human insulin-like growth factor II in Escherichia coli; Rhee HJ et al.; A gene encoding mature human insulin-like growth factor II (IGF-II) was constructed from the modified IGF-II cDNA sequence and two double-stranded synthetic oligodeoxynucleotide linkers . It was fused to a truncated lacZ gene such that IGF-II was expressed as part of C-terminus of beta-galactosidase . This fused lacZ'-IGF-II gene was under the control of tac promoter and we overproduced the beta-galactosidase-IGF-II fusion protein in the Escherichia coli . The fusion protein formed inclusion bodies inside the cells . The fusion protein was purified from the isolated inclusion bodies and IGF-II protein was obtained from their fusion protein by CNBr cleavage . The released IGF-II was confirmed by its molecular weight as determined by SDS-PAGE and by its ability to bind anti-IGF antibody.

Biotechnol Prog, 1990 Mar-Apr, 6(2), 149 - 52
Effect of preinduction specific growth rate on recombinant alpha consensus interferon synthesis in Escherichia coli; Curless C et al.; The effect of preinduction specific growth rate on the yield of recombinant alpha consensus interferon in Escherichia coli K-12 was investigated . The cells used in the investigation contain a temperature-sensitive, walkaway plasmid bearing an insert that codes for alpha consensus interferon . Transcription of the recombinant gene is controlled by a lambda repressor/pL promoter system . The lambda promoter is regulated by the temperature-sensitive gene cI857 at 30 degrees C, but at 42 degrees C the promoter is derepressed . The cells were grown under glucose-limited conditions in a chemostat at pH 7 and 30 degrees C . Once steady state was achieved, the vessel temperature was raised to 42 degrees C and a fed-batch mode was initiated . Six dilution rates ranging from 0.025/h to 0.2/h were investigated . Cell dry weight, alpha consensus interferon content, glucose concentration, acetate concentration, and plasmid stability were measured . At each dilution rate, the expression level of alpha consensus interferon (g/g of cell dry wt) reached its maximum value approximately 3 h after induction . In addition, the expression level of alpha consensus interferon increases 4-fold as the dilution rate prior to induction is increased from 0.025/h to 0.2/h . Consequently, the expression of recombinant protein produced by E . coli is dependent on the preinduction specific growth rate.

Biochem Biophys Res Commun, 1990 Feb 28, 167(1), 301 - 9
Fission yeast cdc25 is a cell-cycle regulated protein; Ducommun B et al.; Fission yeast cell division is initiated by the cdc2/cdc13-cyclin protein kinase which in its catalytically active state comprises the mitotic inducer . During interphase the cdc2/cyclin complex is assembled in an inactive state that requires cdc25+ gene function for M-phase activation . The cdc25+ product, a 76 kd phosphoprotein, is shown to oscillate in abundance during the cell cycle, reaching a peak at G2/M, and to be sensitive to nitrogen starvation . The level of cdc25 is subject to feedback regulation involving both cdc25 and cdc2.

Biochemistry, 1990 Feb 27, 29(8), 2149 - 54
Role of diffusion in the folding of the alpha subunit of tryptophan synthase from Escherichia coli; Chrunyk BA et al.; The rate-limiting step in the folding of the alpha subunit of tryptophan synthase has been proposed to be the association of two folding units . To probe the role of diffusion in this rate-limiting step, the urea-induced unfolding and refolding of the protein was examined in the presence of a number of viscosity-enhancing agents . The analysis was simplified by studying the effect of these agents on folding unit dissociation, the rate-limiting unfolding reaction, and the reverse of the rate-limiting step in refolding . In the presence of ethylene glycol, the relaxation times for unfolding to the same final conditions increased with increasing concentration of the cosolvent . When the effects of the cosolvent on protein stability were taken into account, the rates were found to show a unitary linear dependence on the viscosity of the solution . Similar results were obtained with glycerol and low concentrations of glucose, demonstrating that the effect is general and not specific to any viscogenic agent . These results clearly demonstrate that the rate-limiting folding unit association/dissociation reaction in the alpha subunit of tryptophan synthase involves a diffusional process.

Biochemistry, 1990 Feb 27, 29(8), 2177 - 80
Site-directed mutagenesis of the hole-forming toxin aerolysin: studies on the roles of histidines in receptor binding and oligomerization of the monomer; Green MJ et al.; The six histidines of the channel-forming protein aerolysin have been replaced one at a time with asparagine by site-directed mutagenesis, and each of the modified proteins has been purified . Three proteins had the same hemolytic activity as native toxin, but the others, those changed at His107, His132, or His332, were less able to disrupt both human and rat erythrocytes . The largest reduction in activity, more than 100-fold, was observed with the His132 mutant protein . Studies with radioiodinated samples showed that it had approximately the same affinity as native aerolysin for the rat erythrocyte receptor . However, once bound to either rat or human erythrocytes, it was much less able to carry out the next essential step in hole formation, aggregation to form a stable oligomer . Aggregation was also reduced by replacing His107, but the contrast with native aerolysin and the effect on hemolytic activity were less pronounced . The protein modified at His332 behaved in a different way from those substituted at positions 107 and 132 . Its affinity for the rat erythrocyte receptor was considerably lower than the affinity of the wild-type protein, but when bound it appeared to aggregate normally . The results suggest that His132 and perhaps His107 are involved in the aggregation of aerolysin whereas His332 may be at or near the receptor binding site.

Biochemistry, 1990 Feb 27, 29(8), 2127 - 34
Human DNA topoisomerase II: evaluation of enzyme activity in normal and neoplastic tissues; Holden JA et al.; We have used both a quantitative filter binding assay and a decatenation assay to measure DNA topoisomerase II activity . The filter binding assay, which measures catenating activity, is able to detect topoisomerase II activity at 50-100-fold lower protein concentrations than the decatenation assay . Because of this remarkable sensitivity, we have been able to quantitate topoisomerase II activity in a variety of normal and neoplastic human tissues . The highest level of enzyme activity in normal tissues was found in the spleen and thymus . The highest level of enzyme activity in neoplasms was found in those that clinically behave in an aggressive manner and had a high proliferative status by flow cytometry . Surprisingly, these high topoisomerase II values in the neoplastic specimens are in the same range of values found in normal nonproliferating tissue . Since much previous data indicate that the enzyme is apparently a property of only proliferating cells, this finding might suggest that human tissues contain more than one form of the enzyme . The finding that 35-65% of the topoisomerase II activity in human tissues is resistant to teniposide suggests that more than one enzyme form exists.

Biochemistry, 1990 Feb 27, 29(8), 2075 - 80
Evidence for transition-state stabilization by serine-148 in the catalytic mechanism of chloramphenicol acetyltransferase; Lewendon A et al.; The function of conserved Ser-148 of chloramphenicol acetyltransferase (CAT) has been investigated by site-directed mutagenesis . Modeling studies (P . C . E . Moody and A . G . W . Leslie, unpublished results) suggested that the hydroxyl group of Ser-148 could be involved in transition-state stabilization via a hydrogen bond to the oxyanion of the putative tetrahedral intermediate . Replacement of serine by alanine results in a mutant enzyme (Ala-148 CAT) with kcat reduced 53-fold and only minor changes in Km values for chloramphenicol and acetyl-CoA . The Ser-148----Gly substitution gives rise to a mutant enzyme (Gly-148 CAT) with kcat reduced only 10-fold . A water molecule may partially replace the hydrogen-bonding potential of Ser-148 in Gly-148 CAT . The three-dimensional structure of Ala-148 CAT at 2.34-A resolution is isosteric with that of wild-type CAT with two exceptions: the absence of the Ser-148 hydroxyl group and the loss of one poorly ordered water molecule from the active site region . The results are consistent with a catalytic role for Ser-148 rather than a structural one and support the hypothesis that Ser-148 is involved in transition-state stabilization . Ser-148 has also been replaced with cysteine and asparagine; the Ser-148----Cys mutation results in a 705-fold decrease in kcat and the Ser-148----Asn substitution in a 214-fold reduction in kcat . Removing the hydrogen bond donor (Ser-148----Ala or Gly) is less deleterious than replacing Ser-148 with alternative possible hydrogen bond donors (Ser-148----Cys or Asn).

Biochim Biophys Acta, 1990 Feb 26, 1033(2), 207 - 9
Preparation of tritiated lipopolysaccharides from Escherichia coli K12; Peborde JP et al.; Tritiated lipopolysaccharide (LPS) from E . coli K12 was prepared by coupling {3H}ethanolamine to the LPS core residue ketodeoxyoctonate (KDO) via activation of its carboxylic function with N-hydroxysuccinimide or N-hydroxy-sulfosuccinimide . Specific activities of 1.5 microCi/mg and 9 microCi/mg were obtained, respectively . Experiments comparing the activity of native and derivatized LPS suggested that the preparation of the radiolabelled LPS did not alter the structural properties of E . coli K12 LPS . This probe will be useful for studying the interactions between LPS and proteins.

FEBS Lett, 1990 Feb 26, 261(2), 405 - 9
Escherichia coli of human origin binds to carcinoembryonic antigen (CEA) and non-specific crossreacting antigen (NCA); Leusch HG et al.; Immobilized carcinoembryonic antigen (CEA) and non-specific crossreacting antigen (NCA) bound 3 strains of E . coli of human origin . The binding was dose dependent, saturable, and of high avidity . Binding of the bacteria to CEA and NCA was completely abolished in the presence of 10 mM alpha-methyl D-mannopyranoside . Bacteria did not bind to concanavalin A . In addition, binding to deglycosylated CEA was either absent or significantly reduced . These findings indicate that the E . coli strains bind to D-mannosyl residues in CEA and NCA . Considering the tissue distribution of CEA (brush border of colonic epithelium) and NCA (granulocytes), these glycoproteins may be involved in the recognition of bacteria.

FEBS Lett, 1990 Feb 26, 261(2), 392 - 6
Structure-function analysis of epidermal growth factor: site directed mutagenesis and nuclear magnetic resonance; Dudgeon TJ et al.; The role of leucine-47 in determining the structure and activity of human epidermal growth factor was examined using site-directed mutagenesis . Wild type protein and four variants in which Leu47 was replaced by valine, glutamate, aspartate and alanine were produced from yeast . 1H NMR experiments demonstrated that substitution of Leu47 had little effect on the protein structure . The observed reduction in receptor binding affinity caused by the substitutions could thus be attributed to perturbation of a residue directly involved in receptor interactions.

FEBS Lett, 1990 Feb 26, 261(2), 358 - 60
Mapping of a dominant immunogenic region of synaptophysin, a major membrane protein of synaptic vesicles; Knaus P et al.; Synaptophysin is a major integral membrane protein of synaptic vesicles . Its transmembrane topology deduced from the cDNA sequence predicts 4 transmembrane regions and a carboxy-terminal cytoplasmic tail containing a characteristic pentapeptide repeat structure . The monoclonal antibody (mAb), SY38, binds to a cytoplasmic domain of synaptophysin . By using fusion proteins corresponding to truncated forms of the cytoplasmic tail, its epitope was located to a flexible segment in the center of the repeat structure . Four other mAbs (c7.1, c7.2, c7.3, c7.4) share the same epitope, which thus emerges as the major immunogenic region of this membrane protein.

J Biol Chem, 1990 Feb 25, 265(6), 3424 - 31
Biological significance of facilitated diffusion in protein-DNA interactions . Applications to T4 endonuclease V-initiated DNA repair; Dowd DR et al.; Facilitated diffusion along nontarget DNA is employed by numerous DNA-interactive proteins to locate specific targets . Until now, the biological significance of DNA scanning has remained elusive . T4 endonuclease V is a DNA repair enzyme which scans nontarget DNA and processively incises DNA at the site of pyrimidine dimers which are produced by exposure to ultraviolet (UV) light . In this study we tested the hypothesis that there exists a direct correlation between the degree of processivity of wild type and mutant endonuclease V molecules and the degree of enhanced UV resistance which is conferred to repair-deficient Eshcerichia coli . This was accomplished by first creating a series of endonuclease V mutants whose in vitro catalytic activities were shown to be very similar to that of the wild type enzyme . However, when the mechanisms by which these enzymes search nontarget DNA for its substrate were analyzed in vitro and in vivo, the mutants displayed varying degrees of nontarget DNA scanning ranging from being nearly as processive as wild type to randomly incising dimers within the DNA population . The ability of these altered endonuclease V molecules to enhance UV survival in DNA repair-deficient E . coli then was assessed . The degree of enhanced UV survival was directly correlated with the level of facilitated diffusion . This is the first conclusive evidence directly relating a reduction of in vivo facilitated diffusion with a change in an observed phenotype . These results support the assertion that the mechanisms which DNA-interactive proteins employ in locating their target sites are of biological significance.

J Biol Chem, 1990 Feb 25, 265(6), 3369 - 73
Cooperative biotin binding by streptavidin . Electrophoretic behavior and subunit association of streptavidin in the presence of 6 M urea; Sano T et al.; We describe the cooperativity in the biotin binding of streptavidin . We have developed an electrophoretic method which can separate streptavidin molecules with bound biotin from those without biotin . In 6 M urea, the electrophoretic mobility of streptavidin in polyacrylamide gels becomes significantly faster upon biotin binding . When streptavidin was titrated with biotin, only two major bands were observed on the gel, consisting of streptavidin molecules without bound biotin and those saturated with biotin . The change in mobility is due partly to the negative charge of the bound biotin, but it must reflect conformational changes of the protein molecule associated with biotin binding . Gel filtration chromatography showed that the streptavidin molecule dissociates into two subunit dimers in the presence of 6 M urea . These results suggest that the biotin binding by the streptavidin subunit dimer is cooperative and that some communication must exist between the two subunits.

J Biol Chem, 1990 Feb 25, 265(6), 3358 - 61
Expression in Escherichia coli and purification of a translocation-competent precursor of the chloroplast protein ferredoxin; Pilon M et al.; The precursor of the chloroplast protein ferredoxin from Silene pratensis was expressed in Escherichia coli . When a low copy number plasmid was used, the preferredoxin level was low, and the protein was soluble . The expression level was increased by using a high copy number plasmid . In protease-deficient cells transformed with the latter plasmid, the preferredoxin accumulated up to 1% of total protein, and it was found in insoluble aggregates . These aggregates were dissolved in 4 M urea, and the protein was purified to homogeneity . Amino-terminal sequencing confirmed the amino acid sequence as deduced from the copy DNA . However, the first methionine residue of the expected sequence was absent in E . coli . The purified precursor was readily imported by isolated chloroplasts and processed to the mature size.

J Biol Chem, 1990 Feb 25, 265(6), 3219 - 25
A new DNA binding mode for CAP; Hudson JM et al.; In the absence of cyclic AMP, the Escherichia coli cyclic AMP receptor protein (CAP) binds without detectable sequence specificity to restriction fragments containing lac and crp promoter sequences . Under standard conditions (10 mM Tris, 1 mM EDTA, pH 8.0), our estimates of the equilibrium constant and cooperativity parameter for complex formation are 114,000 +/- 1400 M-1 and 1.3 +/- 0.8, respectively . Thus, this interaction lacks the substantial cooperativity previously reported for CAP binding to genomic DNAs . Using the electrophoresis mobility shift assay, we find that complexes of increasing CAP content differ by a highly uniform mobility decrement . This result is most consistent with a binding mode in which little or no DNA bending occurs . The ability of CAP to distinguish between restriction fragments and genomic DNA, shown by the difference in binding cooperativity, suggests the existence of previously unsuspected DNA sequences or structures that modulate its binding cooperativity.

J Biol Chem, 1990 Feb 25, 265(6), 3183 - 8
Modifications of the active center of T4 thioredoxin by site-directed mutagenesis; Joelson T et al.; The active site sequence of T4 thioredoxin, Cys-Val-Tyr-Cys, has been modified in two positions to Cys-Gly-Pro-Cys to mimic that of Escherichia coli thioredoxin . The two point mutants Cys-Gly-Tyr-Cys and Cys-Val-Pro-Cys have also been constructed . The mutant proteins have similar reaction rates with T4 ribonucleotide reductase as has the wild-type T4 thioredoxin . Mutant T4 thioredoxins with Pro instead of Tyr at position 16 in the active site sequence have three to four times lower apparent KM with E . coli ribonucleotide reductase than wild-type T4 thioredoxin . The KM values for these mutant proteins which do not have Tyr in position 16 are thus closer to E . coli thioredoxin than to the wild-type T4 thioredoxin . The bulky tyrosine side chain probably prevents proper interactions to E . coli ribonucleotide reductase . Also the redox potentials of these two mutant thioredoxins are lower than that of the wild-type T4 thioredoxin and are thereby more similar to the redox potential of E . coli thioredoxin . Mutations in position 15 behave more or less like the wild-type protein . The kinetic parameters with E . coli thioredoxin reductase are similar for wild-type and mutant T4 thioredoxins except that the apparent kcat is lower for the mutant protein with Pro instead of Tyr in position 16 . The active site sequence of T4 thioredoxin has also been changed to Cys-Pro-Tyr-Cys to mimic that of glutaredoxins . This change does not markedly alter the reaction rate of the mutant protein with T4 ribonucleotide reductase or E . coli thioredoxin reductase, but the redox potential is lower for this mutant protein than for wild-type T4 thioredoxin.

J Biol Chem, 1990 Feb 25, 265(6), 3177 - 82
The primary structure of the 32-kDa subunit of human replication protein A; Erdile LF et al.; Replication protein A (RP-A) is a complex of three polypeptides of molecular mass 70, 32, and 14 kDa, which is absolutely required for simian virus 40 DNA replication in vitro . We have isolated a cDNA coding for the 32-kDa subunit of RP-A . An oligonucleotide probe was constructed based upon a tryptic peptide sequence derived from whole RP-A, and clones were isolated from a lambda gt11 library containing HeLa cDNA inserts . The amino acid sequence predicted from the cDNA contains the peptide sequence obtained from whole RP-A along with two sequences obtained from tryptic peptides derived from sodium dodecyl sulfate-polyacrylamide gel-purified 32-kDa subunit . The coding sequence predicts a protein of 29,228 daltons, in good agreement with the electrophoretically determined molecular mass of the 32-kDa subunit . No significant homology was found with any of the sequences in the GenBank data base . The protein predicted from the cDNA has an N-terminal region rich in glycine and serine along with two acidic and two basic segments . Monoclonal antibodies have been raised against the 70- and 32-kDa subunits of RP-A . The cloned cDNA has been overexpressed in bacteria using an inducible T7 expression system . The protein made in bacteria is recognized by a monoclonal antibody that is specific for the 32-kDa subunit of RP-A . This monoclonal antibody against the 32-kDa subunit inhibits DNA replication in vitro.

J Biol Chem, 1990 Feb 25, 265(6), 3111 - 5
The human growth hormone receptor . Secretion from Escherichia coli and disulfide bonding pattern of the extracellular binding domain; Fuh G et al.; A gene fragment encoding the extracellular domain of the human growth hormone (hGH) receptor from liver was cloned into a plasmid under control of the Escherichia coli alkaline phosphatase promoter and the heat-stable enterotoxin (StII) signal peptide sequence . Strains of E . coli expressing properly folded hGH binding protein were identified by blotting colonies with 125I-hGH . The E . coli strain capable of highest expression (KS330) secreted 10 to 20 mg/liter of culture of properly processed and folded hGH receptor fragment into the periplasmic space . The protein was purified to near homogeneity in 70 to 80% yield (in tens of milligram amounts) using ammonium sulfate precipitation, hGH affinity chromatography, and gel filtration . The unglycosylated extracellular domain of the hGH receptor has virtually identical binding properties compared to its natural glycosylated counterpart isolated from human serum, suggesting glycosylation is not important for binding of hGH . The extracellular binding domain codes for 7 cysteines, and we show that six of them form three disulfide bonds . Peptide mapping studies show these disulfides are paired sequentially to produce short loops (10-15 residues long) as follows: Cys38-Cys48, Cys83-Cys94, and Cys108-Cys122 . Cys241 is unpaired, and mutagenic analysis shows that the extreme carboxyl end of the receptor fragment (including Cys241) is not essential for folding or binding of the protein to hGH . High level expression of this receptor binding domain and its homologs in E . coli will greatly facilitate their detailed biophysical and structural analysis.

J Biol Chem, 1990 Feb 25, 265(6), 3489 - 96
DNA base composition determines the specificity of UvrABC endonuclease incision of a psoralen cross-link; Jones BK et al.; The sequences flanking a psoralen interstrand cross-link may determine how it is repaired . Our comparison of the Escherichia coli UvrABC endonuclease incision of a variety of specific cross-link sequences in a single natural DNA fragment showed that DNA base composition determines which of two cross-linked DNA strands will be incised . G/C enrichment of the region 6-12 bases 5' of the modified T on the furan-side strand results in preferential incision of the furan-side strand . When the G/C-rich region is on the 3' side, or on neither side, incisions occur on either strand . These effects of DNA base composition suggest that UvrAB can bind in two ways to a psoralen cross-link.

Nucleic Acids Res, 1990 Feb 25, 18(4), 891 - 4
Sequence-dependent structural variations of DNA revealed by DNase I; Brukner I et al.; Two global helix parameters important for DNA-DNase I interaction are the geometry of the minor groove and the DNA stiffness that resists bending toward major groove . Thus, local averaging of P-O3' bonds cutting frequencies (InP) reflects global helix parameters revealed by DNase I . Using the approximation that locally averaged InP values depend only on the type of the dinucleotide steps involved in the region of interaction, we calculated the collective contribution (sigma Dd) for ten different dinucleotide steps . Our results suggest that, at the first approximation, global varying helix parameters revealed by DNase I, might be predicted from sequence . Obtained sigma Dd function can be used as a sequence-dependent measure of protein-induced DNA flexure in the direction towards the major groove, which is usually connected to widening of the minor groove . In the course of analysis of Mg2+ and Mn2+ dependent DNase I digestions, no significant difference was found, in spite of the supposed differences in enzyme activity . These results suggest that if the second Mn2(+)-dependent active site exists, its activity is lower than that of the first one.

Carbohydr Res, 1990 Feb 25, 196, 101 - 9
Structure and serological properties of the capsular K11 antigen of Escherichia coli O13:K11:H11; Rodriguez ML et al.; The capsular K11 antigen of Escherichia coli contains glucose, fructose, and phosphate in the molar ratios 2:1:1, and a backbone of -4)-beta-D-glucopyranosyl-(1----4)-alpha-D-glucopyranosyl phosphate-(1----to which beta-D-fructofuranose is linked at position 3 of the beta-D-glucopyranosyl residue . The fructose, which is the immunodominant sugar of the K11 antigen, is released from the polysaccharide under mild acidic conditions (70 degrees, pH 5.0).

Nucleic Acids Res, 1990 Feb 25, 18(4), 901 - 11
DNA sequence analysis of five genes; tnsA, B, C, D and E, required for Tn7 transposition; Flores C et al.; A region of DNA sequence of the bacterial transposon Tn7, which is required for transposition, has been determined . This DNA sequence completes an 8351 base pair (bp) region containing five long open reading frames (ORF's) that correspond to the genetically defined genes, tnsA, B, C, D and E, required for Tn7 transposition . All of the ORF's are oriented in the same direction, ie . inward from the element's right end . The genes are in a very compact arrangement with the presumed initiation codons never more than two bases beyond the preceding termination codon . Domains with similarity to the helix-turn-helix genre of Cro-like, sequence specific DNA binding sites occur within the deduced amino acid (a.a.) sequence of the TnsA, TnsB, TnsD and TnsE proteins . Translation of the tnsC ORF reveals strong homology to a consensus sequence for nucleotide binding sites as well as a region of similarity to a transcriptional activator (MalT) . No striking a.a . sequence similarity to other DNA recombinases is observed . The possible roles of these proteins in Tn7 transposition is discussed in light of the analysis presented.

Nucleic Acids Res, 1990 Feb 25, 18(4), 895 - 900
TcA, the putative transposase of the C . elegans Tc1 transposon, has an N-terminal DNA binding domain; Schukkink RF et al.; Tc1 is a transposon present in several copies in the genome of all natural isolates of the nematode C.elegans; it is actively transposing in many strains . In those strains Tc1 insertion is the main cause of spontaneous mutations . The transposon contains one large ORF that we call TcA; we assume that the TcA protein is the transposase of Tc1 . We expressed TcA in E.coli, purified the protein and showed that it has a strong affinity for DNA (both single stranded and double stranded) . A fusion protein of beta-galactosidase and TcA also exhibits DNA binding; deletion derivatives of this fusion protein were tested for DNA binding . A deletion of 39 amino acids at the N-terminal region of TcA abolishes the DNA binding, whereas a deletion of 108 C-terminal amino acids does not affect DNA binding . This shows that the DNA binding domain of TcA is near the N-terminal region . The DNA binding capacity of TcA supports the assumption that TcA is a transposase of Tc1.

J Biol Chem, 1990 Feb 25, 265(6), 3447 - 54
Biochemical and physical characterization of exonuclease V from Escherichia coli . Comparison of the catalytic activities of the RecBC and RecBCD enzymes; Palas KM et al.; Biochemical evidence is presented that confirms exonuclease V of Escherichia coli consists of three distinct subunits encoded by the recB, recC, and recD genes . The recD gene encodes a Mr 60,000 polypeptide and physically maps 3' to the recB structural gene . The role of the recD subunit in exonuclease V function has been examined by comparing the catalytic activities of the purified RecBCD enzyme with the RecBC enzyme . The RecBC enzyme retains significant levels of DNA-dependent ATPase activity and DNA helicase activity . Endonucleolytic activity on single-stranded covalently closed DNA becomes ATP-dependent . Exonucleolytic activity on either single- and double-stranded DNA was not detected . Taken together with the phenotypic properties of recD null mutants, it appears that the exonucleolytic activities of the RecBCD enzyme are not required for genetic recombination and the repair of either UV-induced photoproducts or mitomycin C-generated DNA cross-links, but are essential for the repair of methyl methanesulfonate-induced methylation.

J Biol Chem, 1990 Feb 25, 265(6), 3189 - 92
Tetrahydrobiopterin biosynthetic activities in human macrophages, fibroblasts, THP-1, and T 24 cells . GTP-cyclohydrolase I is stimulated by interferon-gamma, and 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase are constitutively present; Werner ER et al.; Interferon-gamma induces tetrahydrobiopterin biosynthesis in human cells and cell lines . Macrophages are peculiar in the formation of large amounts of neopterin derivatives as compared to tetrahydrobiopterin (Werner, E . R., Werner-Felmayer, G., Fuchs, D., Hausen, A., Reibnegger, G., and Wachter, H . (1989) Biochem J . 262, 861-866) . Here we compare the impact of interferon-gamma treatment on activities of GTP-cyclohydrolase I (EC 3.5.4.16), 6-pyruvoyl tetrahydropterin synthase, and sepiapterin reductase (EC 1.1.1.153) in human peripheral blood-derived macrophages, normal dermal fibroblasts, THP-1 myelomonocytic cells, and the T 24 bladder transitional-cell carcinoma line . Upon interferon-gamma treatment, GTP-cyclohydrolase I activity is increased 7- to 40-fold, whereas 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase activities, which are constitutively present in all four investigated cells, remain unchanged . In fibroblasts and T 24 cells GTP cyclohydrolase I activity is the rate-limiting step of tetrahydrobiopterin biosynthesis . In macrophages and in THP-1 cells, however, the induced GTP cyclohydrolase I activity is higher than the 6-pyruvoyl tetrahydropterin synthase activity, leading to the accumulation of neopterin and neopterin phosphates.

Nucleic Acids Res, 1990 Feb 25, 18(4), 719 - 24
Structure, organization and evolution of the L1 equivalent ribosomal protein gene of the archaebacterium Methanococcus vannielii; Baier G et al.; The gene for ribosomal protein MvaL1 from the arachaebacterium Methanococcus vannielii was cloned and characterized . It is clustered together with the genes for MvaL10 and MvaL12, thus is organized in the same order as in E.coli and other archaebacteria . Unexpectedly, analysis of the sequence in front of the MvaL1 gene revealed an ORF of unknown identity, whereas in E.coli, Halobacterium and Sulfolobus solfataricus the gene for the L11 equivalent protein is located in this position . Northern blot analysis revealed a single tricistronic transcript encoding proteins MvaL1, MvaL10 and MvaL12 . The 5'-end of the MvaL1-L10-L12 transcript contains a region that has a sequence and structure almost identical to a region on the 23S rRNA which is the putative binding domain for MvaL1, and is highly similar to the E.coli L11-L1 mRNA leader sequence that has been implicated in autogenous translational regulation . Amino acid sequence comparison revealed that MvaL1 shares 30.5% identity with ribosomal protein L1 from E.coli and 41.5% and 33.3% identity with the L1-equivalent proteins from the archaebacteria H.cutirubrum and S.solfataricus respectively.

J Biol Chem, 1990 Feb 25, 265(6), 3153 - 60
Sensitivity of efflux-driven carrier turnover to external pH in mutants of the Escherichia coli lactose carrier that have tyrosine or phenylalanine substituted for histidine-322 . A comparison of lactose and melibiose; King SC et al.; Two Escherichia coli lactose carrier mutants (tyrosine or phenylalanine substituted for histidine 322) were studied under conditions of net efflux or equilibrium exchange . Net lactose efflux by either mutant was 10-20-fold slower than by the parent and was sensitive to extracellular pH (5.6-8.0) . The presence of extracellular lactose (equilibrium exchange) failed to accelerate loss of {14C}lactose, indicating that the step(s) rate limiting for exchange were also rate limiting for net lactose efflux . Net melibiose efflux by the Phe-322 mutant was comparable to the normal carrier, while that by the Tyr-322 mutant was 5-fold faster (pH 7.0) . Melibiose efflux by either mutant was sensitive to pH (5.6-8.0) . Melibiose in the extracellular medium significantly accelerated loss of {3H}melibiose from either mutant, showing that slow exchange is a sugar-specific phenomenon and not an intrinsic property of these mutants . The sugar-specific effect of these mutations could mean that the defect in these mutants is not on the path of the proton, although alternative explanations cannot as yet be eliminated . The modest effect of these mutations on the transport rate indicates that His-322 contributes a far smaller free energy increment to catalyzing of H+/galactoside cotransport than active site histidines contribute to catalyzing peptide bond hydrolysis in serine proteases . We interpret this to mean that in chemical terms the function of these catalytic histidine residues differ considerably.

Nucleic Acids Res, 1990 Feb 25, 18(4), 725 - 31
Derivation of clones from the choroideremia locus by preparative field inversion gel electrophoresis; van de Pol TJ et al.; By making use of preparative field inversion gel electrophoresis, we have constructed a lambda ZAP library that is highly enriched for sequences from the choroideremia locus . In vivo excision of pBluescript SK(-) constructs from lambda ZAP obviates the subcloning of DNA inserts and allows for rapid processing of several hundred recombinants . From a 625 kb Sfil fragment we isolated 7 clones that were physically mapped using microdeletions associated with the disease . One of these clones is located within, or just telomeric to, the choroideremia gene and detects two restriction fragment length polymorphisms (RFLPs) . Another clone detects a RFLP which maps centromeric to the disease locus . Together these probes should improve the reliability of linkage analysis in choroideremia families and should pave the way for the isolation of the choroideremia gene.

Lancet, 1990 Feb 24, 335(8687), 434 - 7
Development of antibodies to unprotected glycosylation sites on recombinant human GM-CSF; Gribben JG et al.; In 4 out of 16 patients receiving recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) in phase I/II studies antibodies developed to the recombinant protein . The antibodies react with sites on the native protein backbone which are normally protected by O-linked glycosylation but which are exposed in rhGM-CSF produced in yeast and Escherichia coli . Antigenicity of recombinant human proteins due to non glycosylation may have relevance to the choice of host system for production of factors for clinical use.

Cell, 1990 Feb 23, 60(4), 685 - 93
HIV-1 regulator of virion expression (Rev) protein binds to an RNA stem-loop structure located within the Rev response element region; Heaphy S et al.; HIV-1 Rev protein, purified from E . coli, binds specifically to an RNA transcript containing the 223 nucleotide long Rev response element (RRE) sequence . Rev binds to RRE in vitro with an apparent dissociation constant of 1 to 3 nM as determined by filter binding, gel mobility shift assays, or an immunoprecipitation assay using a monoclonal antibody specific for the Rev C-terminus . Antisense RRE sequences are bound by Rev with a 20-fold lower affinity than wild-type RRE sequences . The Rev-RRE complex forms even in the presence of a 10,000-fold molar excess of 16S rRNA, whereas formation of the low affinity antisense RRE-Rev complex is efficiently blocked by addition of excess 16S rRNA . A approximately 33 nucleotide fragment is protected from ribonuclease T1 digestion by the binding of Rev to RRE RNA, suggesting that Rev binds with high affinity to only a restricted region of the RRE . This protected fragment is unable to rebind Rev protein but has been mapped to a 71 nucleotide long Rev binding domain sequence that overlaps the protected fragment.

Eur J Biochem, 1990 Feb 22, 188(1), 61 - 6
Use of fragments of hirudin to investigate thrombin-hirudin interaction; Dennis S et al.; Site-directed mutagenesis was used to create hirudin in which Asn52 was replaced by methionine . Cyanogen bromide cleavage at this unique methionine resulted in two fragments . These fragments have been used to study the kinetic mechanism of the inhibition of thrombin by hirudin and to identify areas of the two molecules which interact with each other . The binding of the C-terminal fragment (residues 53-65) to thrombin resulted in a decrease in the Michaelis constant for the substrate D-phenylalanylpipecolylarginyl-p-nitroanilide (DPhe-Pip-Arg-NH-Ph) . The N-terminal fragment (residues 1-52) was a competitive inhibitor of thrombin . There was a small amount of cooperativity in the binding of the two fragments . Whereas hirudin and its C-terminal fragment protected alpha-thrombin against cleavage by trypsin, the N-terminal fragment did not . Hirudin and the N-terminal fragment completely prevented the cleavage of alpha-thrombin by pancreatic elastase while the C-terminal fragment afforded a lesser degree of protection . The results of these experiments with trypsin and elastase are discussed in terms of interaction areas on thrombin and hirudin.

Biochim Biophys Acta, 1990 Feb 22, 1015(3), 379 - 90
Subunit delta of H(+)-ATPases: at the interface between proton flow and ATP synthesis; Engelbrecht S et al.; The ATP synthases in photophosphorylation and respiration are of the F-type with a membrane-bound proton channel, F0, and an extrinsic catalytic portion, F1 . The properties of one particular subunit, delta (in chloroplasts and Escherichia coli) and OSCP (in mitochondria), are reviewed and the role of this subunit at the interface between F0 and F1 is discussed . Delta and OSCP from the three sources have in common the molecular mass (approximately 20 kDa), an elongated shape (axial ratio in solution about 3:1), one high-affinity binding site to F1 (Kd approximately 100 nM) plus probably one or two further low-affinity sites . When isolated delta is added to CF1-depleted thylakoid membranes, it can block proton flow through exposed CF0 channels, as do CF1 or CF1(-delta)+ delta . This identifies delta as part of the proton conductor or, alternatively, conformational energy transducer between F0 (proton flow) and F1 (ATP) . Hybrid constructs as CF1(-delta)+ E . coli delta and EF1(-delta)+ chloroplast delta diminish proton flow through CF0.CF1(-delta) + E . coli delta does the same on EF0 . Impairment of proton leaks either through CF0 or through EF0 causes "structural reconstitution' of ATP synthesis by remaining intact F0F1 . Functional reconstitution (ATP synthesis by fully reconstructed F0F1), however, is absolutely dependent on the presence of subunit delta and is therefore observed only with CF1 or CF1(-delta) + chloroplast delta on CF0 and EF1 or EF1(-delta) + E . coli delta on EF0 . The effect of hybrid constructs on F0 channels is surprising in view of the limited sequence homology between chloroplast and E . coli delta (36% conserved residues including conservative replacements) . An analysis of the distribution of the conserved residues at present does not allow us to discriminate between the postulated conformational or proton-conductive roles of subunit delta.

Biochemistry, 1990 Feb 20, 29(7), 1961 - 70
The LexA repressor and its isolated amino-terminal domain interact cooperatively with poly{d(A-T)}, a contiguous pseudo-operator, but not with random DNA: a circular dichroism study; Hurstel S et al.; The interaction of the entire LexA repressor and its amino-terminal DNA binding domain with poly{d(A-T)} and random DNA has been studied by circular dichroism . Binding of both protein species induces an about 2-fold increase of the positive circular dichroism band at about 270 nm of both polynucleotides, allowing a precise determination of the principal parameters as a function of mono- and divalent salt concentration and pH . Both proteins interact much more strongly (about 2000-fold) with poly{d(A-T)} than with random DNA as expected from the homology with the specific consensus binding site of LexA (CTGTATATATATACAG) . For both LexA and its DNA binding domain we find that the interaction with poly{d(A-T)} is cooperative with a cooperativity factor omega of about 50-70 for both proteins over a wide range of solvent conditions, suggesting that the carboxy-terminal domain of LexA is not involved in this type of cooperativity . On the contrary, no cooperativity could be detected for the interaction of the LexA DNA binding domain with a random DNA fragment . The overall binding constant K omega (or simply K in the case of random DNA) depends strongly on the salt concentration as observed for most protein-DNA interactions, but the behavior of LexA is unusual in that the steepness of this salt dependence (delta log K omega/delta log {NaCl}) is much more pronounced at slightly acidic pH values as compared to that at neutral or slightly alkaline pH . The behavior is not easily understood in terms of the current theories on the electrostatic contribution to protein-DNA interactions on the basis of polyelectrolyte theory . A comparison of the overall binding constant K omega of the entire LexA repressor and its DNA binding domain reveals that LexA binds only 20-50-fold stronger under a wide variety of salt and pH conditions . This result tends to demonstrate further that the additional energy due to the dimerization of LexA via the carboxy-terminal domain should be rather weak as expected from the small dimerization constant of LexA (2 X 10(-4) M-1).

Biochemistry, 1990 Feb 20, 29(7), 1907 - 13
Reversible dissociation and unfolding of aspartate aminotransferase from Escherichia coli: characterization of a monomeric intermediate; Herold M et al.; The unfolding and dissociation of the dimeric enzyme aspartate aminotransferase (D) from Escherichia coli by guanidine hydrochloride have been investigated at equilibrium . The overall process was reversible, as judged from almost complete recovery of enzymic activity after dialysis of 0.7 mg of denatured protein/mL against buffer . Unfolding and dissociation were monitored by circular dichroism and fluorescence spectroscopy and occurred in three separate phases: D in equilibrium 2M in equilibrium 2M* in equilibrium 2U . The first transition at about 0.5 M guanidine hydrochloride coincided with loss of enzyme activity . It was displaced toward higher denaturant concentrations by the presence of either pyridoxal 5'-phosphate or pyridoxamine 5'-phosphate and toward lower denaturant concentrations by decreasing the protein concentration . Therefore, bound coenzyme stabilizes the dimeric state, and the monomer (M) is inactive because the shared active sites are destroyed by dissociation of the dimer . M was converted to M* and then to the fully unfolded monomer (U) in two subsequent transitions . M* was stable between 0.9 and 1.1 M guanidine hydrochloride and had the hydrodynamic radius, circular dichroism, and fluorescence of a monomeric, compact "molten globule" state.

Biochemistry, 1990 Feb 20, 29(7), 1880 - 6
The transformed glucocorticoid receptor has a lower steroid-binding affinity than the nontransformed receptor; Nemoto T et al.; High-salt treatment of cytosolic glucocorticoid receptor (GR) preparations reduces the steroid-binding ability of the receptor and induces the conversion of the receptor from a nontransformed (non-DNA-binding) 9S form to a transformed (DNA-binding) 4S entity . Therefore, we decided to investigate the possible relationship between these two phenomena . Steroid-free GR was converted from a 9S to a 4S form by exposure to 0.4 M NaCl . The binding of {3H}triamcinolone acetonide {( 3H}TA) to the 9S form was almost saturated at a concentration of 20 nM, whereas {3H}TA was hardly bound to the 4S form at this concentration . The 4S form was efficiently labeled at 200 nM . Scatchard analysis of the GR exposed to 0.4 M NaCl in the presence of 10 mM molybdate showed the presence of two types of binding sites with apparent dissociation constants of 0.52 +/- 0.07 and 64.1 +/- 16.2 nM, respectively . In the absence of molybdate, the ratio of the lower affinity site was increased, but the total number of binding sites was not modified . The GR with the low {3H}TA-binding affinity bound to DNA-cellulose even in its unliganded state, whereas the form with the high affinity did not . Immunoblot analysis using anti-GR monoclonal antibody revealed no difference in molecular size (Mr 94000) between the high- and low-affinity entities . These results indicate that the transformed GR has a reduced {3H}TA-binding affinity as compared to the nontransformed GR.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1990 Feb 20, 29(7), 1744 - 9
Kinetics of binding of single-stranded DNA binding protein from Escherichia coli to single-stranded nuclei acids; Urbanke C et al.; The time course of the reaction of Escherichia coli single-stranded DNA binding protein (E . coli SSB) with poly(dT) and M13mp8 single-stranded DNA has been measured by fluorescence stopped-flow experiments . For poly(dT), the fluorescence traces follow simple bimolecular behavior up to 80% saturation of the polymer with E . coli SSB . A mechanistic explanation of this binding behavior can be given as follows: (1) E . coli SSB is able to translocate very rapidly on the polymer, forming cooperative clusters . (2) In the rate-limiting step of the association reaction, E . coli SSB is bound to the polymer only by one or two of its four contact sites . As compared to poly(dT), association to single-stranded M13mp8 phage DNA is slower by at least 2 orders of magnitude . We attribute this finding to the presence of secondary structure elements (double-stranded structures) in the natural single-stranded DNA . These structures cannot be broken by E . coli SSB in a fast reaction . In order to fulfill its physiological function in reasonable time, E . coli SSB must bind newly formed single-stranded DNA immediately . The protein can, however, bind to such pieces of the newly formed single-stranded DNA which are too short to cover all four binding sites of the E . coli SSB tetramer.

J Mol Biol, 1990 Feb 20, 211(4), 907 - 18
DNA-hybridization electron microscopy tertiary structure of 16 S rRNA; Oakes MI et al.; Seven regions of 16 S rRNA have been located on the surface of the 30 S ribosomal subunit by DNA-hybridization electron microscopy . This information has been incorporated into a model for the tertiary structure of 16 S rRNA, accounting for approximately 40% of the total 16 S rRNA . A structure labeled the platform ring is proposed for a region of rRNA within the central domain . This structure rings the edges of the platform and includes regions 655-751 and 769-810 . Another region, the recognition complex, consists of nucleotides 500 to 545, and occupies a region on the exterior surface of the subunit near the elongation factor Tu binding site . Ribosomal proteins that have been mapped by immunoelectron microscopy are superimposed onto the model in order to examine possible regions of interaction . Good correlation between the model locations of ribosomal proteins, and regions of rRNA protected by ribosomal proteins provide independent support for this model.

J Mol Biol, 1990 Feb 20, 211(4), 845 - 55
Biochemical properties of the Escherichia coli recA430 protein . Analysis of a mutation that affects the interaction of the ATP-recA protein complex with single-stranded DNA; Menetski JP et al.; The biochemical properties of the recA430 protein have been examined and compared to those of wild-type recA protein . We find that, while the recA430 protein possesses ssDNA-dependent rATP activity, this activity is inhibited by the Escherichia coli single-stranded DNA binding protein (SSB protein) under many conditions that enhance wild-type recA protein rATPase hydrolysis . Stimulation of rATPase activity by SSB protein is observed only at high concentrations of both rATP (greater than 1 mM) and recA430 protein (greater than 5 microM) . In contrast, stimulation of ssDNA-dependent dATPase activity by SSB protein is less sensitive to protein and nucleotide concentration . Consistent with the nucleotide hydrolysis data, recA430 protein can carry out DNA strand exchange in the presence of either rATP or dATP . However, in the presence of rATP, both the rate and the extent of DNA strand exchange by recA430 protein are greatly reduced compared to wild-type recA protein and are sensitive to recA430 protein concentration . This reduction is presumably due to the inability of recA430 protein to compete with SSB protein for ssDNA binding sites under these conditions . The cleavage of lexA repressor protein by recA430 protein is also sensitive to the nucleotide cofactor present and is completely inhibited by SSB protein when rATP is the cofactor but not when dATP is used . Finally, the steady-state affinity and the rate of association of the recA430 protein-ssDNA complex are reduced, suggesting that the mutation affects the interaction of the ATP-bound form of recA protein with ssDNA . This alteration is the likely molecular defect responsible for inhibition of recA430 protein rATP-dependent function by SSB protein . The biochemical properties observed in the presence of dATP and SSB protein, i.e . the reduced levels of both DNA strand exchange activity and cleavage of lexA repressor protein, are consistent with the phenotypic behavior of recA430 mutations.

J Mol Biol, 1990 Feb 20, 211(4), 739 - 49
How many EF-Tu molecules participate in aminoacyl-tRNA binding and peptide bond formation in Escherichia coli translation?
Ehrenberg M, Rojas AM, Weiser J, Kurland CG.
We have observed that two EF-Tu.GTP cycles are required to make one peptide bond during steady-state translation in an accurate and fast poly(U) translation system prepared from Escherichia coli . We have also found that there are two complexes of EF-Tu.GTP bound to one molecule of aminoacyl-tRNA under our experimental conditions . We suggest, on the basis of these data, that aminoacyl-tRNA enters the ribosomal A-site in a pentameric complex together with two EF-Tu and two GTP molecules . When the tRNA is delivered to the ribosome two GTP molecules are hydrolyzed . It is possible that the functional role of such an EF-Tu dimer is related to the function of the two L7/L12 dimers in the large ribosomal subunit.

J Mol Biol, 1990 Feb 20, 211(4), 689 - 90
Preliminary X-ray data for the periplasmic ribose receptor from Escherichia coli; Mahendroo M et al.; Crystals of the periplasmic ribose receptor for chemotaxis and transport in Escherichia coli have been examined by X-ray analysis . The crystals grow as elongated rectangular prisms with the symmetry of the orthorhombic space group P2(1)2(1)2(1) . The unit cell dimensions are a = 74.6 A, b = 88.8 A and c = 40.1 A . There is one molecule of molecular weight 28,500 per asymmetric unit.

Biochemistry, 1990 Feb 20, 29(7), 1777 - 91
CO recombination in cytochrome c peroxidase: effect of the local heme environment on CO binding explored through site-directed mutagenesis; Miller MA et al.; CO recombination to the cloned cytochrome c peroxidase {CCP(MI)} and mutants of CCP(MI) prepared by site-directed mutagenesis was examined as a function of pH by flash photolysis . The mutants examined included distal Arg 48----Leu, Lys; proximal Asp 235----Asn; and His 181----Gly . At alkaline pH, ferrous CCP(MI) was converted to a hexacoordinate form by a cooperative two-proton ionization, apparent pK(a) = 8.0 . This change was observed in all of the mutants, although in the His 181----Gly mutant, the conversion to the hexacoordinate form was the result of a single-proton ionization, implicating His 181 as one of the two residues deprotonated in this isomerization . The pH-dependent conversion of CO ferrous CCP(MI) from acidic to alkaline forms was also observed and was similar to that reported for cytochrome c peroxidase from bakers' yeast {Iizuka, T., Makino, R., Ishimura, Y., & Yonetani, T . (1985) J . Biol . Chem . 260, 1407-1412} . Photolysis of the acidic form of the CO complex of CCP(MI) produces a kinetic form of the ferrous enzyme (form A) which exhibits the slow rate of CO recombination (l1' approximately 10(3) M-1 s-1) characteristic of peroxidases, while photolysis of the alkaline form of the CO complex produces a second kinetic form (form B), which exhibits a much faster rate of recombination (l2' approximately 10(5) M-1 s-1) . Kinetic forms analogous to forms A and B were observed in all of the mutants examined . A third kinetic form (form B*) with a bimolecular rate constant l3' approximately 10(6) M-1 s-1 was also observed in the mutants at alkaline pH . Although the pH dependence for the conversion of form A to form B with increasing pH was altered by changes in the local heme environment, the rate of CO recombination by the respective forms was not dramatically altered in the mutants . Transient spectra of the reaction of CO with ferrous CCP(MI) after photolysis show that equilibrium between penta- and hexacoordinate ferrous enzyme is rapid relative to CO recombination . The presence of the internal sixth ligand has no discernible effect on the observed rate of recombination, however . The results presented indicate that in CCP(MI) the rate of ligand binding is determined primarily by isomerization of the protein from a closed conformation at acidic pH to an open conformation at alkaline pH and that polar effects of proximal Asp 235 and distal Arg 48 are of minor significance in the rate of CO recombination in both conformations.

J Mol Biol, 1990 Feb 20, 211(4), 897 - 906
DNA-hybridization electron microscopy . Localization of five regions of 16 S rRNA on the surface of 30 S ribosomal subunits; Oakes MI et al.; DNA-hybridization electron microscopy has been used to locate five regions of 16 S rRNA on the surface of 30 S ribosomal subunits . Biotinylated DNA probes that are complementary to selected regions of 16 S rRNA were hybridized to activated 30 S ribosomal subunits . These hybridized probes were reacted with avidin and localized by electron microscopy . The specificity of DNA binding was monitored with RNase H, which recognizes RNA-DNA hybrids and cleaves the RNA . Three of the five sequences examined were mapped on the platform . These sequences are 686-703, 714-733 and 787-803 . Region 1492-1505 is mapped in the cleft and region 518-533 is at the neck on the side opposite the platform, respectively.

Biochemistry, 1990 Feb 20, 29(7), 1757 - 63
Effects of nucleotide- and aurodox-induced changes in elongation factor Tu conformation upon its interactions with aminoacyl transfer RNA . A fluorescence study; Dell VA et al.; The effects of GDP and of aurodox (N-methylkirromycin) on the affinity of elongation factor Tu (EF-Tu) for aminoacyl-tRNA (aa-tRNA) have been quantified spectroscopically by using Phe-tRNA(Phe)-Fl8, a functionally active analogue of Phe-tRNA(Phe) with a fluorescein dye convalently attached to the s4U-8 base . The association of EF-Tu.GDP with Phe-tRNA(Phe)-Fl8 resulted in an average increase of 33% in fluorescein emission intensity . This spectral change was used to monitor the extent of ternary complex formation as a function of EF-Tu.GDP concentration, and hence to obtain a dissociation constant, directly and at equilibrium, for the EF-Tu.GDP-containing ternary complex . The Kd for the Phe-tRNA(Phe)-Fl8.EF-Tu.GDP complex was found to average 28.5 microM, more than 33,000-fold greater than the Kd of the Phe-tRNA(Phe)-Fl8.EF-Tu.GTP complex under the same conditions . In terms of free energy, the delta G degree for ternary complex formation at 6 degrees C was -11.5 kcal/mol with GTP and -5.8 kcal/mol with GDP . Thus, the hydrolysis of the ternary complex GTP results in a dramatic decrease in the affinity of EF-Tu for aa-tRNA, thereby facilitating the release of EF-Tu.GDP from the aa-tRNA on the ribosome . Aurodox (200 microM) decreased the Kd of the GDP complex by nearly 20-fold, to 1.46 microM, and increased the Kd of the GTP complex by at least 6-fold . The binding of aurodox to EF-Tu therefore both considerably strengthens EF-Tu.GDP affinity for aa-tRNA and also weakens EF-Tu.GTP affinity for aa-tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

J Immunol, 1990 Feb 15, 144(4), 1497 - 503
Specificity and inhibitory activity of antibodies to Plasmodium falciparum aldolase; Srivastava IK et al.; The multiplication of Plasmodium falciparum within RBC is energy-dependent and the glucose consumption of infected RBC is increased more than 50 times over the consumption of normal RBC . High levels of glycolytic enzymes such as fructose-1,6-diphosphate aldolase (p41) have been detected in infected RBC . Expression of the cloned aldolase gene of P . falciparum in Escherichia coli resulted in an enzymatically active polypeptide with a high sp . act . and the recombinant p41 aldolase was used for enzymatic and immunologic studies reported here . The presence of antibodies against p41 in the sera of human adults partially immune to malaria and immunization experiments in monkeys suggest that p41 is implicated in protective immune response against the parasite . Therefore, we analyzed the capacity of various antisera to inhibit P . falciparum aldolase activity . It was found that anti-p41 antibodies raised in mice, rabbits, and monkeys inhibited very efficiently aldolase activity in vitro up to dilutions higher than 10(-3) . In contrast none of the human sera with high levels of anti-p41 antibodies were able to inhibit parasite aldolase activity even at a dilution of 1/2 . The inability of human antisera to neutralize parasite aldolase is not related to antibody titers but is probably related to the specificity of the human antibodies . This finding is discussed in relation to homology of structure of P . falciparum and mammalian aldolase and to a possible mechanism of parasite adaptation and survival in its natural host.

J Biol Chem, 1990 Feb 15, 265(5), 2903 - 7
Olfactory-specific cytochrome P-450 (P-450olf1; IIG1) . Gene structure and developmental regulation; Nef P et al.; The olfactory neuroepithelium is the principal site of interaction for airborne molecules, mainly odorants, in the organism . The presence of an active cytochrome P-450-dependent oxidative metabolism in this tissue has not yet been studied as well as the hepatic cytochrome P-450-dependent oxidations . In this report, we describe cytochrome P-450olf1 (IIG1), a P-450 gene expressed at high levels uniquely in the olfactory epithelium . By Southern analysis and genomic DNA cloning, we demonstrate that a single copy of the P-450olf1 gene is present in the rat genome and contains 9 exons . We conclude that rat P-450IIG1 is a single gene subfamily . P-450olf1 gene expression was activated after birth in both male and female Sprague-Dawley rats and remained active in adult olfactory epithelium . A first maximum level of expression was reached around postnatal day 21 . The coincidence between the temporal gene activation of P-450olf1 and the postnatal increase in the sensitivity of olfactory response to odorants is consistent with a potential role of this enzyme in olfactory function.

J Biol Chem, 1990 Feb 15, 265(5), 2856 - 64
Regulation of manganese superoxide dismutase by lipopolysaccharide, interleukin-1, and tumor necrosis factor . Role in the acute inflammatory response; Visner GA et al.; We have demonstrated a dramatic induction of manganese superoxide dismutase (Mn-SOD) mRNA levels in response to lipopolysaccharide (LPS), interleukin-1, and tumor necrosis factor in pulmonary epithelial cells . These stimuli had no effect on the corresponding mRNA levels for the copper/zinc (Cu/Zn)-SOD . Identical treatments of pulmonary fibroblast cells with LPS showed only minor changes in the Mn-SOD mRNA levels demonstrating a cell type-specific effect for this acute inflammatory mediator . Furthermore, we have shown that hyperoxia has no effect within 24 h on Mn-or Cu/Zn-SOD mRNA levels in either fibroblasts or epithelial cells . The induction of Mn-SOD mRNA levels by LPS is completely inhibited by actinomycin . Treatment of cells with cycloheximide causes an induction equal to that for LPS, whereas co-treatment with cycloheximide and LPS resulted in a "super induction." This data is strongly suggestive of an important role for the Mn-SOD in the acute inflammatory response.

J Biol Chem, 1990 Feb 15, 265(5), 2450 - 5
Defective cation-coupling mutants of Escherichia coli Na+/proline symport carrier . Characterization and localization of mutations; Yamato I et al.; A major proline carrier in Escherichia coli encoded by the putP gene mediates proline/Na+ or Li+ symport . Proline carrier mutants with altered cation specificity were obtained by mutagenesis with nitrous acid in vitro of a plasmid carrying the wild-type putP gene . Two mutant strains harboring plasmid pMOP4135 and pMOP4141 could transport proline efficiently only in the presence of an increased concentration of sodium ion . Mutations of these plasmids, putP4135 and putP4141, caused reduction of affinity for Na+ of proline transport and binding, without remarkable change in the affinity for proline or in production of the carriers . Consistent with the lower affinity of the putP4141 carrier for Na+, the mutant carrier was supersensitive to N-ethylmaleimide inhibition . The pH dependence of proline binding was also changed in these mutant carriers . The lesions of putP4135 and putP4141 were located in the N-terminal part of the putP gene (ClaI-PvuII fragment) by in vitro recombination and subsequent examination of the phenotype of the transformants . DNA sequencing of these fragments revealed one base alteration of G to A at nucleotides 299 and 656 in pMOP4141 and pMOP4135, respectively, which corresponded to amino acid changes from Gly22 to glutamic acid and Cys141 to tyrosine, respectively.

Blood, 1990 Feb 15, 75(4), 976 - 83
Development, characterization, and subcellular location of DNAse activity in HL-60 cells and monocytes; Roberts PJ; The digestion of DNA from intact bacteria by human phagocytic cells was measured by the release of solubilized radiolabeled DNA . Two subclones from the human promyelocytic HL-60 cell line were unable to digest bacterial DNA unless they were previously induced to mature by incubation for several days with 1.25% dimethylsulfoxide (DMSO) . The maximal capacity of DMSO-induced HL-60 cells to digest DNA was similar to that of monocytes purified from peripheral blood (PB) and much greater than that of neutrophils . The increasing capacity to digest DNA during maturation was associated with the development of acid DNAse activity, measured in a cell-free system, and slightly preceded development of 12-O-tetradecanoyl phorbol 13-acetate-stimulated respiratory burst activity . The acid DNAse had a pH optimum of 5.0 and did not require the presence of calcium or 2-mercaptoethanol (2-ME) . A third subclone of HL-60 cells was able to digest DNA from intact bacteria without previous maturation, however, and this was associated with the presence of an alkaline DNAse which had a pH optimum between 7.0 and 8.0 and showed a dependence on calcium and 2-ME for maximal activity . The subcellular location of acid DNAse in DMSO-induced HL-60 cells was similar to that of monocytes in having a bimodal distribution on fractionated sucrose density gradients . The dense peak (mean density 1.195 g/mL) was located in the same region of the gradient as primary granule enzymes but the light peak (mean density 1.137 g/mL) did not codistribute with either plasma membrane, endoplasmic reticulum, or mitochondria, suggesting accumulation in a different organelle.

Biochem Pharmacol, 1990 Feb 15, 39(4), 737 - 44
Induction of mammalian topoisomerase II dependent DNA cleavage by nonintercalative flavonoids, genistein and orobol; Yamashita Y et al.; Two isoflavones, genistein (4',5,7-trihydroxyisoflavone) (1) and orobol (5,7,3',4'-tetrahydroxyisoflavone) (2) induced mammalian topoisomerase II dependent DNA cleavage in vitro . The cleavage activities of 1 and 2 were comparable to those of known antitumor agents with topoisomerase II dependent DNA cleavage activity such as 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and demethylepipodophyllotoxin ethylidene-beta-D-glucoside (VP-16) . Two flavones, fisetin (3,7,3',4'-tetrahydroxyflavone) (3) and quercetin (3,5,7,3',4'-pentahydroxyflavone) (4) showed topoisomerase II dependent DNA cleavage activity with similar potentials to that of Adriamycin . Addition of salt (0.5 M NaCl) to the reaction mixture containing genistein and topoisomerase II resulted in a great reduction of DNA cleavage, suggesting that the mechanism of the topoisomerase II dependent DNA cleavage induced by flavonoids is through the cleavable complex formation as seen with m-AMSA and VP-16 . DNA unwinding assay using mammalian topoisomerase I showed that both 1 and 2 did not intercalate into DNA but both 3 and 4 intercalated like m-AMSA . Other structurally related flavonoids could not induce topoisomerase II dependent DNA cleavage, indicating that the restricted structures of flavonoids were required for the cleavage activity.

J Biol Chem, 1990 Feb 15, 265(5), 2873 - 80
Polymeric sequences reveal a functional interrelationship between hydrophobicity and length of signal peptides; Chou MM et al.; We have examined the hydrophobicity component of signal peptide function using polymeric sequences in combination with cassette mutagenesis . Using homopolymeric units of either isoleucine, leucine, valine or alanine to replace the natural core segment of the Escherichia coli alkaline phosphatase signal peptide, the hydrophobicity requirements for export and processing were delineated . The transport properties of these mutants demonstrated that the net hydrophobicity determines the total extent of precursor processing, while a high mean hydrophobicity/residue is critical for complete, rapid processing and translocation . Moreover, alkaline phosphatase was converted from a periplasmic to an active membrane-anchored protein via a signal containing 20 leucine residues . This application of polymeric sequences allows systematic comparisons to be made, unambiguously revealing the hydrophobicity requirements governing specific steps in the transport process.

J Biol Chem, 1990 Feb 15, 265(5), 2707 - 12
Nucleoside diphosphate kinase from Myxococcus xanthus . II . Biochemical characterization; Munoz-Dorado J et al.; The gene that encodes the 16-kDa GTP-binding protein from Myxococcus xanthus has been cloned, and its DNA sequence has been determined . The gene has been expressed in Escherichia coli by using the lacZ promoter, and its gene product was overproduced (Munoz-Dorado, J., Inouye, M., and Inouye, S . (1990) J . Biol . Chem . 265, 2702-2706) . The gene product thus overproduced in E . coli was purified to homogeneity by a simple four-step procedure and crystallized . Gel filtration of the purified protein revealed that the protein forms a complex of an apparent molecular weight of 50,000, indicating that it exists as a trimer in the cell . It was found that the purified protein can bind not only GTP, but also equally well the other nucleoside diphosphates and triphosphates with no specificity for either the base or the sugar . Nucleoside monophosphates, Pi, and pyrophosphate do not bind to the protein . In the presence of Mg2+, the protein hydrolyzes nucleoside triphosphates to diphosphates and Pi . However, in the presence of EDTA, most of the phosphate remains bound to the protein . The phosphorylated protein can then transfer the phosphate group to a nucleoside diphosphate to form the corresponding nucleoside triphosphate in the presence of Mg2+ . The reaction is reversible, and it is considered to occur by a two-step ping-pong mechanism . These results unambiguously demonstrate that the M . xanthus 16-kDa GTP-binding protein is a nucleoside diphosphate kinase.

J Biol Chem, 1990 Feb 15, 265(5), 2702 - 6
Nucleoside diphosphate kinase from Myxococcus xanthus . I . Cloning and sequencing of the gene; Munoz-Dorado J et al.; By photoaffinity labeling with a photolysable analog of GTP, 8-N3GTP, we were able to find at least five distinct GTP-binding proteins in Myxococcus xanthus; two of them located in the membrane and the other three in the soluble fraction . The amino-terminal sequence of the 16-kDa GTP-binding protein from the soluble fraction was determined, and the gene that encodes this protein was isolated and cloned using degenerate oligonucleotides as a probe . The DNA sequence of the gene was determined, which did not show similarity with other known proteins . The gene product was overexpressed in Escherichia coli, by using the lacZ promoter, to a level of 13% of the soluble protein . Attempts to isolate deletion mutants were unsuccessful, although double crossing-over events leading to a deletion mutation of the gene were detected by Southern blot hybridization . This result indicates that this gene is essential for cell growth . In the following paper (Munoz-Dorado, J., Inouye, S., and Inouye, M . (1990) J . Biol . Chem . 265, 2707-2712), the gene product was biochemically characterized and identified to be a nucleoside diphosphate kinase.

J Biol Chem, 1990 Feb 15, 265(5), 2620 - 3
Substrate specificity and protonation state of Escherichia coli ornithine transcarbamoylase as determined by pH studies . Binding of carbamoyl phosphate; Zambidis I et al.; Binding of carbamoyl phosphate to Escherichia coli ornithine transcarbamoylase and its relation to turnover have been examined as a function of pH under steady-state conditions . The pH profile of the dissociation constant of carbamoyl phosphate (Kiacp) shows that the affinity of the substrate increases as pH decreases . Two ionizing groups are involved in carbamoyl phosphate binding . Protonation of an enzymic group with pKa 9.6 results in productive binding of the substrate with a moderate affinity of Kiacp approximately 30 microM . Protonation of a second group further enhances binding by roughly another order of magnitude . This ionization occurs with a pKa that shifts from less than 6 in the free enzyme to 7.3 in the binary complex . However, tighter binding of carbamoyl phosphate due to this ionization does not contribute to catalysis . The turnover rate (kcat) of the enzyme diminishes in the acidic pH range and is governed by an ionization with a pKa of 7.2 . Both the catalytic pKa of 7.2 and the productive binding pKa of 9.6 appear in the pH profile of kcat/KMcp . Together with earlier kinetic results (Kuo, L . C., Herzberg, W., and Lipscomb, W . N . (1985) Biochemistry 24, 4754-4761), these data suggest that the step which modulates kcat may occur prior to the binding of the second substrate L-ornithine.

Biochem J, 1990 Feb 15, 266(1), 227 - 34
Transport of the yeast ATP synthase beta-subunit into mitochondria . Effects of amino acid substitutions on targeting; Walker ME et al.; We have isolated the yeast ATP2 gene encoding the beta-subunit of mitochondrial ATP synthase and determined its nucleotide sequence . A fusion between the N-terminal 15 amino acid residues of beta-subunit and the mouse cytosolic protein dihydrofolate reductase (DHFR) was transcribed and translated in vitro and found to be transported into isolated yeast mitochondria . A fusion with the first 35 amino acid residues of beta-subunit attached to DHFR was not only transported but also proteolytically processed by a mitochondrial protease . Amino acid substitutions were introduced into the N-terminal presequence of the beta-subunit by bisulphite mutagenesis of the corresponding DNA . The effects of these mutations on mitochondrial targeting were assessed by transport experiments in vitro using DHFR fusion proteins . All of the mutants, harbourin from one to six amino acid substitutions in the first 14 residues of the presequence, were transported into mitochondria, though at least one of them (I8) was transported and proteolytically processed at a much reduced rate . The I8 mutant beta-subunit also exhibited poor transport and processing in vivo, and expression of this mutant polypeptide failed to complement the glycerol- phenotype of a yeast ATP2 mutant . More remarkably, the expression of I8 beta-subunit induced a more general growth defect in yeast, possibly due to interference with the transport of other, essential, mitochondrial proteins.

Biochem Pharmacol, 1990 Feb 15, 39(4), 781 - 6
Electrochemical studies and DNA damaging effects of the benzotriazine-N-oxides; Tocher JH et al.; The electrochemical behaviour of eight benzotriazine 1,4 di-N-oxides has been examined and compared with the mono- and zero-N-oxides . The di-N-oxides all show two reduction steps, an irreversible followed by a quasi-reversible response assigned to the 4 electron reduction of both N-oxide groups, followed by the 2 electron reduction of the benzotriazine ring . Mono- and zero-N-oxides show only a single, quasi-reversible reduction step, similar in character to the second reduction of the di-N-oxides . This has been assigned to reduction of the benzotriazine ring, with the available, redox-active, N-oxide group of the mono-N-oxide complex being reduced at less negative potentials, but only after ring reduction, hence only a single electrode response . The importance of reductive activation of the N-oxide group has been examined using a phi X174 double transfection technique which assays biologically relevant DNA damage . For the di-N-oxides, no effect on DNA was recorded under oxic conditions, however, DNA damage was marked under anoxic reduction conditions . The extent of DNA damage was found to increase with the acidity of the medium, suggesting the protonated form of the reduction product as being responsible for the cytotoxic action . The mono-N-oxide was shown to be biologically inactive under all conditions.

Anal Biochem, 1990 Feb 15, 185(1), 194 - 200
Site-directed mutagenesis by complementary-strand synthesis using a closing oligonucleotide and double-stranded DNA templates; Slilaty SN et al.; An approach for generating structures capable of directing full-length complementary-strand synthesis for double-stranded plasmid DNA is described . The structures are formed following heat denaturation and cooling of linearized plasmid DNA molecules in the presence of what is referred to as a "closing" oligonucleotide . Consisting of a sequence complementary to the free ends of one of the two plasmid strands, the closing oligonucleotide functions as an agent for recircularization of a DNA strand and generation of a primer-circular template structure suitable for polymerase-dependent full-length complementary-strand synthesis and ligation into a covalently closed heteroduplex molecule . When combined with a mutagenic oligonucleotide and uracil-substituted DNA templates, this approach allows site-directed mutagenesis to be performed directly on double-stranded DNA with a mutant formation efficiency of about 50%, a level amenable to rapid screening by DNA sequencing.

J Biol Chem, 1990 Feb 15, 265(5), 2888 - 95
Expression and characterization of RNase III and Era proteins . Products of the rnc operon of Escherichia coli; Chen SM et al.; The synthesis rates of ribonuclease III (RNase III) and Era proteins are relatively low, and expression of the era gene is translationally coupled with expression of the rnc gene . Expression of both genes is negatively controlled by RNase III itself . We have constructed plasmids that overproduce RNase III and/or Era proteins under the control of the lambda PL promoter . A plasmid with the rnc gene under PL control expresses RNase III at levels greater than 40% of total cellular protein . Another plasmid with the era gene under PL control and a modified translation-initiation signal produces up to 80% of total cell protein as Era . Each protein has been purified using simple and rapid procedures . Purified RNase III protein specifically processes mRNA transcripts containing known RNase III sites . The purified Era protein binds GDP and GTP and has GTPase activity . Kinetic analysis shows that one molecule of GTP or GDP is bound/Era peptide with a Kd of 5.5 microM for GTP binding and 1.0 microM for GDP binding . The Km of the Era GTPase is 9.0 microM, and the maximum catalyzed rate of GTP hydrolyzed/min/mol of Era protein at 37 degrees C is 9.8 mmol.

Anal Biochem, 1990 Feb 15, 185(1), 103 - 7
An electrophoretic method for the purification of RNA regions involved in protein crosslinking; Hajnsdorf E et al.; Direct information about structural interactions in ribonucleoprotein complexes can be obtained from crosslinking data . The purification of specific complexes, i.e., their separation from noncrosslinked proteins, from free RNA, and from other complexes, is essential for the identification of the bound proteins and the precise localization of their attachment sites in RNA . We describe a two-dimensional denaturing gel system which achieves this purification; in the first dimension basic proteins do not enter the gel and RNA--protein complexes are slowed down compared to protein free RNA, and in the second dimension sodium dodecyl sulfate improves the separation between the different complexes on the basis of their protein content.

Biochem Biophys Res Commun, 1990 Feb 14, 166(3), 1485 - 93
Characterization of a functional domain of human calpastatin; Uemori T et al.; Expression plasmids were constructed from the cDNA of human calpastatin to examine the contribution to the inhibition of calpain of highly conserved sequences in each of four repetitive domains . A series of deletion derivatives of domain 1 proteins, truncated at either the amino or carboxy terminus, were produced in E . coli . Deletion from the amino terminus past the amino terminal conserved sequence decreased the inhibition . When the middle conserved sequence, the M-sequence, was further deleted, no inhibition was detected, but deletion from the carboxy terminus past the carboxy terminal conserved sequence did not decrease the inhibition until the M-sequence was reached . Nuclear magnetic resonance and circular dichroism spectra showed that domain 1 has an unfolded structure . Peptides that contained the M-sequence and some neighboring sequences were synthesized to measure the minimum size of the inhibitory peptide, which was the M-sequence with the next six residues on the amino terminal side.

Biochem Biophys Res Commun, 1990 Feb 14, 166(3), 1163 - 70
Independent induction of interleukin 6 and prostaglandin E2 by interleukin 1 in human articular chondrocytes; Bunning RA et al.; Interleukin 6 is a cytokine with growth and differentiation activities on a number of cell types . Human articular chondrocytes produce interleukin 6 and this production appears to be constitutive but can be stimulated in a dose-dependent manner by interleukin 1 . Other stimulators of interleukin 6 production in chondrocytes include tumour necrosis factor-alpha, polyriboinosinic: polyribocytidylic acid and bacterial lipopolysaccharide . Interleukin 6 production is not inhibited by prostaglandin E2 but may be partially dependent on prostaglandin E2 production . Using an antiserum to interleukin 6 we have demonstrated that the production of prostaglandin E2 under basal conditions and in response to interleukin 1 is probably not mediated by interleukin 6.

Eur J Biochem, 1990 Feb 14, 187(3), 549 - 53
Purification and characterization of Escherichia coli RNase I . Comparisons with RNase M; Meador J 3rd et al.; The endoribonuclease, RNase I, was purified from the periplasm of Escherichia coli . Based on PAGE, it has molecular mass of approximately 27 kDa with a migration rate indistinguishable from that of the recently reported RNase M from E . coli . The amino acid sequence of the two enzymes must be very similar based on two-dimensional mapping of their tryptic peptides and suggests either a post-transcriptional modification to yield different proteins from the same gene or evolution of two genes by gene duplication . However, while RNase I could degrade each of the four ribonucleotide homopolymers, only poly(U) or poly(C) were good substrates for RNase M with possibly some hydrolysis of poly(A) . The reaction rate for poly(C) hydrolysis with RNase M was about ten times faster than for poly(U), while for RNase I the rates were about equal . Besides differences in specificity, RNase M was only located in the spheroplasts while RNase I found in the periplasm of growing cells . In terms of function, RNase I is known to cause degradation of rRNA during periods of stress or non-growth, whereas it has been proposed that RNase M is the endonuclease for mRNA degradation in growing cells.

Gene, 1990 Feb 14, 86(2), 291 - 5
Human recombinant interleukin-1 beta isolated from Escherichia coli by simple osmotic shock; Joseph-Liauzun E et al.; A synthetic gene coding for the C-terminal 153 amino acids of the human interleukin-1 beta (IL-1 beta) was used to produce large quantities of recombinant IL-1 beta in Escherichia coli . The expression of the synthetic gene was under the control of an inducible promoter . The recombinant protein was released from the cells by an osmotic shock . This procedure did not lyse the cells . The IL-1 beta that represented 90% of the total extracted protein was purified to homogeneity by a single chromatographic step . Sequence analysis revealed a heterogeneous N-terminal sequence resulting from the cleavage of the N-terminal methionine in 50% of the molecules and of both the N-terminal methionine and alanine in the other 50% . This recombinant IL-1 beta had a specific activity of 1.3 x 10(8) international units per mg.

Gene, 1990 Feb 14, 86(2), 185 - 92
Differential screening of murine ascites cDNA libraries by means of in vitro transcripts of cell-cycle-phase-specific cDNA and digital image processing; Lu X et al.; Cell-cycle-phase-specific cDNA libraries were prepared in the lambda gt10 vector and in the in vitro transcription vector, pBluescript . Plaques of the cDNA libraries prepared in the lambda gt10 vector were differentially screened with (a) in vitro transcripts of the cell-cycle-phase-specific cDNAs cloned in the transcription vector and (b) with first-strand cDNA of mRNA from phase-synchronous cells . The results suggest that first-strand cDNA can be replaced, at least in prescreening experiments, by in vitro transcripts of representative cDNA libraries prepared in in vitro transcription vectors . The fractions of differential clones detected with in vitro transcripts (1.2%) and with first-strand cDNA (1%) were in the same order . Individual clones selected by differential hybridization with in vitro transcripts could be verified by differential hybridization with cell-cycle-phase-specific first-strand cDNA . This indicates that the pattern of stage-specific prevalences of cDNA clones is essentially retained during careful amplifications of large cDNA libraries . The application of in vitro transcripts of stage-specific cDNA for differential screening experiments is of interest in cases where the amount of biological material is either limited or difficult to prepare . It also allows standardization of the probes in repeated screening experiments . Three clones reflecting cell-cycle-phase-specific mRNA prevalences were chosen and analyzed on the sequence level . Two sequences with S-phase prevalences were identified . They code for elongation factor EF1 alpha and for glyceraldehyde-3-phosphate dehydrogenase, respectively . The third sequence reflects the first cDNA of a mRNA with significant prevalence in G2-phase cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1990 Feb 14, 86(2), 137 - 44
Point mutations in a pBR322-based expression plasmid resulting in increased synthesis of bovine growth hormone in Escherichia coli; Watson N et al.; The bGH cDNA coding for bovine growth hormone (bGH) is expressed poorly in Escherichia coli using a pBR322-based expression plasmid . Random mutagenesis of the plasmid gave rise to two types of plasmid mutants which increased the expression of bGH . One class had single base changes in the first four codons of the bGH sequence . The second class had single base changes in regions of the plasmid involved in controlling plasmid replication but had little effect on plasmid copy number.

Biochem Biophys Res Commun, 1990 Feb 14, 166(3), 1284 - 92
Interaction of DnaK with ATP: binding, hydrolysis and Ca+2-stimulated autophosphorylation; Dalie BL et al.; The autophosphorylation of DnaK from Escherichia coli using ATP as phosphate donor is markedly stimulated by Ca+2 and to a lesser degree by Mn+2 . Mg+2 and other divalent ions are without effect in this reaction . Lanthanum, an agonist/antagonist of Ca+2, is also effective in stimulating the autophosphorylation . In contrast, Mg+2 but not Ca+2, markedly stimulates the hydrolysis of ATP catalyzed by DnaK . Also at 0 degrees, ATP forms a stable complex with DnaK without hydrolysis that is independent of cations . About 15% of the DnaK in E . coli is associated with membrane vesicles where it also can be phosphorylated in the presence of Ca+2.

Biochem Biophys Res Commun, 1990 Feb 14, 166(3), 1245 - 50
Increase in the catalytic rate of beta-galactosidase by selection in chemostats at changing dilution rates; Tsen SD; The experiments presented in this paper explore whether mutants with catalytically more active beta-galactosidase (E.C . 3.2.1.23) can be selected in lactose-limited chemostats . This experimental system has been chosen because lactose metabolism in Escherichia coli is well understood both from a biochemical and genetic point of view . In a lactose-limited chemostat with constant dilution rates, both beta-galactosidase and the lactose permease increased in quantity . The catalytic rate (kcat) of beta-galactosidase in populations showed no change . On the other hand, in chemostats with periodically changing dilution rates, the catalytic rate of beta-galactosidase increased dramatically . Therefore, the selection for beta-galactosidase with improved catalytic rate occurs in chemostats with fluctuating dilution rates but not in ones with constant dilution . These observations may be of value in the selection of other enzymes with enhanced catalytic rates.

Eur J Biochem, 1990 Feb 14, 187(3), 705 - 11
Expression of guinea-pig liver transglutaminase cDNA in Escherichia coli . Amino-terminal N alpha-acetyl group is not essential for catalytic function of transglutaminase; Ikura K et al.; Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl)lysine cross-links and the substitution of a variety of primary amines for the gamma-carboxamide groups of protein-bound glutamine residues . These enzymes are involved in many biological phenomena . Transglutaminase reactions also have been shown to be suitable for applied enzymology . In this study, as a first step of studies to elucidate the structure/function relationship of transglutaminase, we constructed an expression plasmid, pKTG1, containing a cDNA of guinea-pig liver transglutaminase between the NcoI and PstI sites of an expression vector, pKK233-2, and produced the liver transglutaminase as an unfused protein in Escherichia coli . The purified recombinant enzyme was indistinguishable from natural liver transglutaminase in some structural properties such as molecular mass, amino acid composition, and amino- and carboxyl-terminal sequences . However, the alpha-amino group of the amino-terminal alanine residue of the recombinant transglutaminase was not acetylated as was that of the natural enzyme . Comparison of the recombinant enzyme with the natural one did not indicate significant differences in specific activity and apparent Km values for substrates in the histamine incorporation into acetyl alpha s1-casein . The sensitivity to activation by Ca2+ and the rate of catalyzed protein cross-linking were also similar between recombinant and natural transglutaminases . These results indicated that the N alpha-acetyl group in natural liver transglutaminase has not a particular role in the catalytic function of this enzyme.

Biochemistry, 1990 Feb 13, 29(6), 1654 - 60
Expression of the amino-terminal half-molecule of human serum transferrin in cultured cells and characterization of the recombinant protein; Funk WD et al.; A human liver cDNA library was screened with a synthetic oligonucleotide, complementary to the 5' region of human transferrin mRNA, as a hybridization probe . The full-length human cDNA clone isolated from this screen contained part of the 5' untranslated region, the complete coding region for the signal peptide and the two lobes of transferrin, the 3' untranslated region, and a poly(A) tail . By use of oligonucleotide-directed mutagenesis in vitro, two translational stop codons and a HindIII site were introduced after the codon for Asp-337 . This fragment was inserted into two different expression vectors that were then introduced into Escherichia coli . As judged by NaDodSO4-polyacrylamide gel electrophoresis and Western blot analysis, however, recombinant hTF/2N was undetectable in bacteria transformed by these plasmids . Concurrently, we developed a plasmid vector for the expression of recombinant hTF/2N in eukaryotic cells . In this case, a DNA fragment coding for the natural signal sequence, the hTF/2N lobe, and the two stop codons was cloned into the expression vector pNUT, such that the expression of hTF/2N was controlled by the mouse metallothionein promoter and the human growth hormone termination sequences . Baby hamster kidney cells containing this hTF/2N-pNUT plasmid secreted up to 20 mg of recombinant hTF/2N per liter of tissue culture medium . Recombinant hTF/2N was purified from the medium by successive chromatography steps on DEAE-Sephacel, Sephadex G-75, and FPLC on Polyanion SI . The purified protein was characterized by NaDodSO4-PAGE, urea-PAGE, amino-terminal sequence analysis, UV-visible spectroscopy, iron-binding titration, and proton NMR.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1990 Feb 13, 29(6), 1624 - 32
cis-syn thymine dimers are not absolute blocks to replication by DNA polymerase I of Escherichia coli in vitro; Taylor JS et al.; Both Escherichia coli DNA polymerase I (pol I) and the large fragment of pol I (Klenow) were found to bypass a site-specific cis-syn thymine dimer, in vitro, under standard conditions . A template was constructed by ligating d(pCGTAT{c,s}TATGC), synthesized via a cis-syn thymine dimer phosphoramidite building block, to a 12-mer and 19-mer . The site and integrity of the dimer were verified by use of T4 denV endonuclease V . Extension of a 15-mer on the dimer-containing template by either pol I or Klenow led to dNTP and polymerase concentration dependent formation of termination and bypass products . At approximately 0.15 unit/microL and 1-10 microM in each dNTP, termination one prior to the 3'-T of the dimer predominated . At 100 microM in each dNTP termination opposite the 3'-T of the dimer predominated and bypass occurred . Bypass at 100 microM in each dNTP depended on polymerase concentration, reaching a maximum of 20% in 1 h at approximately 0.2 unit/microL, underscoring the importance of polymerase binding affinity for damaged primer-templates on bypass . Seven percent bypass in 1 h occurred under conditions of 100:10 microM dATP:dNTP bias, 1% under dTTP bias, and an undetectable amount under either dGTP or dCTP bias . At 100 microM in each dNTP, the ratio of pdA:pdG:pdC:pdT terminating opposite the 3'-T of the dimer was estimated to be 37:25:10:28 . Sequencing of the bypass product produced under these conditions demonstrated that greater than 95% pdA was incorporated opposite both Ts of the dimer and that little or no frame shifting took place.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1990 Feb 13, 29(6), 1539 - 45
Multiple replacements at position 211 in the alpha subunit of tryptophan synthase as a probe of the folding unit association reaction; Tweedy NB et al.; Equilibrium and kinetic studies on the folding of a series of amino acid replacements at position 211 in the alpha subunit of tryptophan synthase from Escherichia coli were performed in order to determine the role of this position in the rate-limiting step in folding . Previous studies {Beasty, A . M., Hurle, M . R., Manz, J . T., Stackhouse, T., Onuffer, J . J., & Matthews, C . R . (1986) Biochemistry 25, 2965-2974} have shown that the rate-limiting step corresponds to the association/dissociation of the amino (residues 1-188) and carboxy (residues 189-268) folding units . In terms of the secondary structure, the amino folding unit consists of the first six strands and five alpha helices of this alpha/beta barrel protein . The carboxy folding unit comprises the remaining two strands and three alpha helices; position 211 is in strand 7 . Replacement of the wild-type glycine at position 211 with serine, valine, and tryptophan at most alters the rate of dissociation of the folding units; association is not changed significantly . In contrast, glutamic acid and arginine dramatically decelerate and accelerate, respectively, both association and dissociation . The difference in effects is attributed to long-range electrostatic interactions for these charged side chains; steric effects and/or hydrogen bonding play lesser roles . When considered with previous data on replacements at other positions in the alpha subunit {Hurle, M . R., Tweedy, N . B., & Matthews, C . R . (1986) Biochemistry 25, 6356-6360}, it is clear that beta strands 6 (in the amino folding unit) and 7 (in the carboxy folding unit and containing position 211) dock late in the folding process.

Biochemistry, 1990 Feb 13, 29(6), 1444 - 51
Importance of domain closure for homotropic cooperativity in Escherichia coli aspartate transcarbamylase; Newton CJ et al.; The importance of the interdomain bridging interactions observed only in the R-state structure of Escherichia coli aspartate transcarbamylase between Glu-50 of the carbamoyl phosphate domain with both Arg-167 and Arg-234 of the aspartate domain has been investigated by using site-specific mutagenesis . Two mutant versions of aspartate transcarbamylase were constructed, one with alanine at position 50 (Glu-50----Ala) and the other with aspartic acid at position 50 (Glu-50----Asp) . The alanine substitution totally prevents the interdomain bridging interactions, while the aspartic acid substitution was expected to weaken these interactions . The Glu-50----Ala holoenzyme exhibits a 15-fold loss of activity, no substrate cooperativity, and a more than 6-fold increase in the aspartate concentration at half the maximal observed specific activity . The Glu-50----Asp holoenzyme exhibits a less than 3-fold loss of activity, reduced cooperativity for substrates, and a 2-fold increase in the aspartate concentration at half the maximal observed specific activity . Although the Glu-50----Ala enzyme exhibits no homotropic cooperativity, it is activated by N-(phosphonoacetyl)-L-aspartate (PALA) . As opposed to the wild-type enzyme, the Glu-50----Ala enzyme is activated by PALA at saturating concentrations of aspartate . At subsaturating concentrations of aspartate, both mutant enzymes are activated by ATP, but are inhibited less by CTP than is the wild-type enzyme . At saturating concentrations of aspartate, the Glu-50----Ala enzyme is activated by ATP and inhibited by CTP to an even greater extent than at subsaturating concentrations of aspartate.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1990 Feb 13, 29(6), 1436 - 43
Subcloning, characterization, and affinity labeling of Escherichia coli glycinamide ribonucleotide transformylase; Inglese J et al.; Glycinamide ribonucleotide transformylase (GAR TFase; EC 2.1.2.2) has been purified 70-fold to apparent homogeneity from Escherichia coli harboring an expression vector encoding the purN gene product, GAR TFase . The protein is a monomer of Mr 23,241 and catalyzes a single reaction . Steady-state kinetic parameters for the enzyme have been obtained . The structural requirements for cofactor utilization have been investigated and found to parallel those of the multifunctional avian enzyme . The enzyme was inactivated with the affinity label N10-(bromoacetyl)-5,8-dideazafolate in a stoichiometric and active-site-specific manner . The ionization state of the cofactor analogue in the enzyme-cofactor complex appears to require the dissociation of the proton at N3 of the pyrimidine within the complex.

Biochemistry, 1990 Feb 13, 29(6), 1425 - 35
Subcloning of the enterobactin biosynthetic gene entB: expression, purification, characterization, and substrate specificity of isochorismatase; Rusnak F et al.; The Escherichia coli entB gene, coding for the enterobactin biosynthetic enzyme isochorismatase, has been subcloned into the multicopy plasmid pKK223-3 under the control of the tac promoter . The resulting recombinant plasmid pFR1 expresses isochorismatase amounting to over 50% of the total cellular protein . The enzyme has been purified to homogeneity and a convenient assay developed . The enzyme has a Km for isochorismate of 14.7 microM and a turnover number of 600 min-1 . By use of 1H NMR spectroscopy, the progress of the reaction was followed with the expected formation of 2,3-dihydro-2,3-dihydroxybenzoate product . Several substrate analogues were also utilized by the enzyme including chorismic acid, the immediate precursor to isochorismic acid in the enterobactin biosynthetic pathway.

Biochemistry, 1990 Feb 13, 29(6), 1362 - 7
A comparison of strategies to stabilize immunoglobulin Fv-fragments; Glockshuber R et al.; Fv-Fragments of antibodies may dissociate at low protein concentrations and are too unstable for many applications at physiological temperatures . To stabilize Fv-fragments against dissociation, we have tested and compared three different strategies on the Fv-fragment of the well-characterized phosphocholine binding antibody McPC603 expressed and secreted in Escherichia coli: chemical cross-linking of the variable domains, introduction of an intermolecular disulfide bond, and construction of a peptide linker to produce a "single-chain" Fv-fragment . All the linked fragments show hapten affinities nearly identical with that of the whole antibody independent of protein concentration and are significantly (up to 60-fold) stabilized against irreversible thermal denaturation . All genetically engineered linked Fv-fragments can be obtained in native conformation in E . coli . The reported strategies for generating Fv-fragments with improved physicochemical properties may extend their usefulness in biotechnology as well as in therapeutic and diagnostic applications.

FEBS Lett, 1990 Feb 12, 261(1), 76 - 80
Enzymatic synthesis of dihydrosirohydrochlorin (precorrin-2) and of a novel pyrrocorphin by uroporphyrinogen III methylase; Warren MJ et al.; Uroporphyrinogen III methylase was purified from a recombinant hemB-strain of E . coli harbouring a plasmid containing the cysG gene . N-terminal analysis of this purified protein gave an amino acid sequence corresponding to that predicted from the genetic code . From the u.v./visible spectrum of the reaction catalysed by this SAM dependent methylase it was possible to observe the sequential appearance of the chromophores of a dipyrrocorphin and subsequently of a pyrrocorphin . Confirmation of this transformation was obtained from 13C-NMR studies when it was demonstrated, for the first time directly, that uroporphyrinogen is initially converted into dihydrosirohydrochlorin (precorrin-2) and then, by further methylation, into a novel trimethylpyrrocorphin.

FEBS Lett, 1990 Feb 12, 261(1), 28 - 30
Cluster of point mutations predetermined by a quasipalindromic nucleotide sequence in plasmid pBR322 DNA; Salganik RI et al.; Development of a cluster of point mutations due to the correction of an imperfect hairpin in plasmid DNA was investigated . Plasmid pBR322 DNA containing opposite double-strand DNA lesions in the region of a quasipalindrome was constructed . For this aim plasmid DNA was cleaved at the BamHI site, and cytosine residues of the sticky ends were modified by O-methylhydroxylamine . Modified linearized plasmid DNA was ligated and used for transformation of E.coli cells . Tetracycline-sensitive transformants were selected, and the mutants were characterized by restriction and sequencing analysis . One mutant contained a cluster of point mutations . The distribution of mutations was consistent with the cluster having arisen through correction of the imperfect hairpin formed by the quasipalindrome.

Nucleic Acids Res, 1990 Feb 11, 18(3), 619 - 24
Identification of the metalloregulatory element of the plasmid-encoded arsenical resistance operon; San Francisco MJ et al.; The regulatory region of the plasmid-encoded arsenical resistance (ars) operon was cloned as a 727-bp EcoRI-HindIII fragment . When cloned into a promoter probe vector this fragment conferred arsenite inducible tetracycline resistance in Escherichia coli, indicating that the fragment carried a regulatory gene, the arsR gene . A single region corresponding to -35 and -10 promoter recognition sites was identified . The transcriptional start site of the mRNA was determined by primer extension . The sequence has an open reading frame for a potential 13,179 Da polypeptide, termed the ArsR protein . The fragment was cloned into a temperature regulated expression vector . A protein with an apparent molecular mass of about 12 kDa was induced by either temperature or arsenite . This protein was purified and used to produce antibodies specific for the ArsR protein.

Nucleic Acids Res, 1990 Feb 11, 18(3), 589 - 97
The distribution of restriction enzyme sites in Escherichia coli; Churchill GA et al.; A statistical analysis of physical map data for eight restriction enzymes covering nearly the entire genome of E . coli is presented . The methods of analysis are based on a top-down modeling approach which requires no knowledge of the statistical properties of the base sequence . For most enzymes, the distribution of mapped sites is found to be fairly homogeneous . Some heterogeneity in the distribution of sites is observed for the enzymes Pstl and HindIII . In addition, BamHI sites are found to be more evenly dispersed than we would expect for random placement and we speculate on a possible mechanism . A consistent departure from a uniform distribution, observed for each of the eight enzymes, is found to be due to a lack of closely spaced sites . We conclude from our analysis that this departure can be accounted for by deficiencies in the physical map data rather than non-random placement of actual restriction sites . Estimates of the numbers of sites missing from the map are given, based both on the map data itself and on the site frequencies in a sample of sequenced E . coli DNA . We conclude that 5 to 15% of the mapped sites represent multiple sites in the DNA sequence.

Nucleic Acids Res, 1990 Feb 11, 18(3), 437 - 41
The influence of different modifications of elongation factor Tu from Escherichia coli on ternary complex formation investigated by fluorescence spectroscopy; Ott G et al.; A fluorescence titration assay was used to detect the effects of various modifications of E.coli elongation factor Tu on the formation of the ternary complex with aminoacyl-tRNAs . The treatment of EF-Tu.GDP with TPCK, an analogue of the 3'terminus of aminoacyl-tRNA, was found to have no influence on the conversion of EF-Tu.GDP to 'active' EF-Tu.GTP, but does decrease the affinity of the activated protein for yeast aminoacyl-tRNA by more than three orders of magnitude . Modification of the elongation factor by limited cleavage with trypsin, leading to the excision of amino acid residues 45-58, has only a minor influence on ternary complex formation . The equilibrium dissociation constant of the ternary complex with this trypsin-treated EF-Tu.GTP and E.coli Phe-tRNA(Phe) is only one order of magnitude higher than that of the ternary complex with native EF-Tu . Mutations in the amino acid residues 222 and 375 of EF-Tu also have little effect on ternary complex formation . Compared with TPCK-treated EF-Tu, the affinities of the two mutant species, designated EF-tuAR and EF-TuBO respectively, for {AEDANS-s2C}Tyr-tRNA(Tyr) are only slightly reduced and in the same range as trypsin-cleaved EF-Tu.

Nucleic Acids Res, 1990 Feb 11, 18(3), 605 - 11
Dam methyltransferase sites located within the loop region of the oligopurine-oligopyrimidine sequences capable of forming H-DNA are undermethylated in vivo; Parniewski P et al.; Several derivatives of pUC18 plasmid were constructed that contained oligopurine-oligopyrimidine (pur-pyr) motifs surrounded by Dam methylation sites . Inserts of two of the molecules (pPP1 and pPP2) were able to adopt the triple-stranded conformation in vitro and show in vivo a remarkable undermethylation of specific sites when grown in JM105 dam+ strain . Mapping experiments revealed that undermethylated GATC sequences were located exclusively within the single-stranded loop region of the sequence involved in H-DNA formation . Control molecules which either contained the pur-pyr tracts (pPPK and pKK42) or not (pUC18) and were not able to form the triple-stranded conformation were found to be normally methylated by the dam gene product in vivo . Location of GATC within the triplex forming sequence seems to be a prerequisite for achieving its in vivo undermethylation . E.coli host factors are involved in the observed phenomenon . This has been deduced from the fact that the undermethylated state of pPP1 and pPP2 does not depend on the phase of growth of host cells and is steadily maintained up to 50 hours, whereas the kinetics of Dam methylation in vitro of sites located within the triplex loop does not differ substantially from the kinetics of methylation of other sites on the vector . Full methylation can be readily achieved in vitro . Additional factor(s) that operate in vivo to control the undermethylated state are most likely proteins since the observed effect can be suppressed by chloramphenicol administration to the cell cultures.

Nucleic Acids Res, 1990 Feb 11, 18(3), 531 - 8
Codons 262 to 490 from the herpes simplex virus ICP4 gene are sufficient to encode a sequence-specific DNA binding protein; Wu CL et al.; The HSV-1 immediate early (IE) protein ICP4 (alpha 4, IE175, Vmw175) is an oligomeric molecule which activates transcription of viral early genes, represses transcription of viral IE genes, and binds to specific sequences in certain viral promoters . The extent to which these functions are interrelated has not been fully established . We have expressed truncated portions of the ICP4 gene in E . coli as trpE fusion proteins . DNA-binding studies with these hybrid proteins revealed that ICP4 residues 262 to 490 are sufficient for sequence-specific DNA-binding . DNA-binding was not detected with polypeptides extending from residue 262 to 464 or from residue 306 to 490 . Multiple bands of protein-DNA complexes observed in gel mobility shift assays indicate that residues 262 to 490 may also contribute to the oligomerization of ICP4.

Nucleic Acids Res, 1990 Feb 11, 18(3), 477 - 85
Localization of an oligodeoxynucleotide complementing 16S ribosomal RNA residues 520-531 on the small subunit of Escherichia coli ribosomes: electron microscopy of ribosome-cDNA-antibody complexes; Lasater LS et al.; The oligodeoxynucleotide dACCGCGGCTGCT, complementary to Escherichia coli small ribosomal subunit RNA residues 520-531, has been used to probe subunit conformation and to localize the sequence in the subunit . Conditions for binding of the cDNA to 30S subunits were optimized and specificity of the interaction was demonstrated by RNase H cleavage . Three kinds of terminal modification of this cDNA were used to allow its localization by immune electron microscopy . A solid phase support with 5'-dimethoxytrity-N6-delta 2-isopentenyl-adenosine linked to controlled pore glass was synthesized, and used to prepare oligomer with an added 3'-terminal residue of isopentenyl adenosine . cDNA with a 5' primary amine substituent was modified with 1-fluoro-2,4-dinitrobenzene to prepare 5'-dinitrophenyl oligonucleotide, and both modifications together gave doubly-derivatized probes . Immune electron microscopy with antibodies to dinitrophenol, isopentenyl adenosine, or both, was used to place the cDNA on 30S subunits . In each case the probe was placed at a single site at the junction of the head and body of the subunit, near the decoding site and the area in which elongation factor Tu is bound . It is proposed that this segment of ribosomal RNA functions in mRNA binding and orientation.

Science, 1990 Feb 9, 247(4943), 723 - 6
Growth factors induce phosphorylation of the Na+/H+ antiporter, glycoprotein of 110 kD; Sardet C et al.; The Na+/H+ antiporter, which regulates intracellular pH in virtually all cells, is one of the best examples of a mitogen- and oncogene-activated membrane target whose activity rapidly changes on stimulation . The activating mechanism is unknown . A Na+/H+ antiporter complementary DNA fragment was expressed in Escherichia coli as a beta-galactosidase fusion protein, and a specific antibody to the fusion protein was prepared . Use of this antibody revealed that the Na+/H+ antiporter is a 110-kilodalton glycoprotein that is phosphorylated in growing cells . Mitogenic activation of resting hamster fibroblasts and A431 human epidermoid cells with epidermal growth factor, thrombin, phorbol esters, or serum, stimulated phosphorylation of the Na+/H+ antiporter with a time course similar to that of the rise in intracellular pH.

Cell, 1990 Feb 9, 60(3), 487 - 94
Cyclin is a component of maturation-promoting factor from Xenopus; Gautier J et al.; Highly purified maturation-promoting factor (MPF) from Xenopus eggs contains both cyclin B1 and cyclin B2 as shown by Western blotting and immunoprecipitation using Xenopus anti-B-type cyclin antibodies . Immunoprecipitates with these antibodies display the histone H1 kinase activity characteristic of MPF, for which exogenously added B1 and B2 cyclins are both substrates . Protein kinase activity against cyclin oscillates in maturing oocytes and activated eggs with the same kinetics as p34cdc2 kinase activity . These data indicate that B-type cyclin is the other component of MPF besides p34cdc2.

Biochemistry, 1990 Feb 6, 29(5), 1271 - 5
Tyrosine-96 as a natural spectroscopic probe of the cytochrome P-450cam active site; Atkins WM et al.; The previously described correlation between the ferric spin equilibrium of cytochrome P-450cam and the environmental polarity of tyrosine residues (Fisher et al., 1986) has been further examined with the use of site-directed mutagenesis and active-site affinity reagents . Whereas the wild-type demonstrates an increase in environmental polarity of approximately one tyrosine residue, the mutant protein Y96F, in which Tyr-96 has been changed to Phe-96, demonstrates a lack of spin-state-dependent change in the second-derivative ultraviolet absorption spectrum . This suggests that the active-site Tyr-96 serves as a ultraviolet spectroscopic probe which can be utilized to determine the relative degree of water access to the active site for various substrate/protein complexes . The affinity reagent isobornyl mercaptan has been used to demonstrate the utility of this probe in determining the active-site polarity when substrate analogues are bound at the active site . In addition, the sensitivity of Tyr-96 to environmental polarity has been used to demonstrate that the product/enzyme complex, formed with 5-exo-hydroxycamphor, may be associated with increased water access to the heme iron . This may provide a means for turning off electron transfer when the product, instead of the substrate, is bound at the active site.

Biochemistry, 1990 Feb 6, 29(5), 1200 - 7
Structure-function analysis of mononucleotides and short oligonucleotides in the priming of enzymatic DNA synthesis; Nevinsky GA et al.; The reversed-phase chromatography technique was employed in the measurement of DNA synthesis at the primers d(pT)n, r(pU)n, d(pA)n, and r(pA)n (n = 1-16) in the presence of template poly(dA) or poly(dT) . DNA synthesis was catalyzed by Escherichia coli DNA polymerase I Klenow fragment, Physarum polycephalum DNA polymerase beta-like, P . polycephalum DNA polymerase alpha, and human placenta DNA polymerase alpha . Values of Km and Vmax were measured as functions of the primer chain lengths . It was found that all mononucleotides and small oligonucleotides served as primers of DNA synthesis . Values of the logarithm of both Km and Vmax increased linearly until primers had attained a chain length of 9-12 nucleotides, where a break was observed . The incremental as well as the absolute values of Km were interpreted in terms of free binding energies . These together with other data indicate that the 3'-ultimate nucleotide of the primer contributes a decisive amount of free energy of binding to DNA polymerase both from the nucleoside and from the phosphate moiety . The incremental increase is due to a complementary interaction between bases of primer and template buried in the binding cleft of the polymerase . It is also the ultimate nucleotide that determines whether the ribonucleotide or the deoxyribonucleotide is an efficient primer . It is of interest that the major results seem preserved for all four DNA polymerases . An energetic model for the binding of the template-primer was proposed and compared with available crystallographic data.

J Mol Biol, 1990 Feb 5, 211(3), 565 - 80
Secretion and membrane integration of a filamentous phage-encoded morphogenetic protein; Brissette JL et al.; The filamentous phage-encoded gene IV protein is required at high levels for virus assembly, although it is not a constituent of the virion . It is an integral membrane protein that does not contain an extended hydrophobic region of the kind often required for stable integration in the inner membrane . Rather, like a number of Escherichia coli outer membrane proteins, pIV is rich in charged amino acid residues and is predicted to consist of extensive beta-sheet structures . In phage-producing cells, pIV is primarily detected in the outer membrane, while in cells that produce it from the cloned gene, pIV is found in both the inner and outer membranes . The protein is synthesized as a precursor . Following cleavage of the signal sequence and translocation into the periplasm, the mature form is initially found as a soluble species . Soluble pIV then integrates into the membrane with a half-time of one to two minutes . Neither phage assembly nor other phage proteins are needed for this membrane integration, and phage assembly does not require the presence of the soluble form . The gene IV protein may be part of the structure through which the assembling phage is extruded.

J Biol Chem, 1990 Feb 5, 265(4), 2347 - 51
Human bone sialoprotein . Deduced protein sequence and chromosomal localization; Fisher LW et al.; A cDNA encoding the human bone sialoprotein was isolated from a lambda Zap expression library (made from cultured human bone cell poly(A)+ RNA) using radiolabeled rat bone sialoprotein cDNA (Oldberg, A., and Heinegard, D . (1988) J . Biol . Chem . 263, 19430-19432) as a probe . A 5' 1-kilobase EcoRI fragment of the purified 3-kilobase clone was sequenced and found to contain the entire protein-encoding region . The deduced protein sequence revealed a 317-amino acid protein (34,982 Da) containing a 16-amino acid hydrophobic signal sequence and a 33,352-Da protein destined to undergo extensive post-translational modifications before being secreted from the cell . A comparison of the human and rat protein sequences showed extensive (greater than 70%) amino acid identities including the Arg-Gly-Asp (RGD) tripeptide thought to confer the cell attachment activity observed previously for this protein . Also conserved were three regions rich in acidic amino acids and three regions rich in tyrosine . While all three tyrosine-rich regions appear to be composed of a nominal repeat structure, only the two carboxyl-terminal regions that flank the RGD sequence fit all three of the requirements for extensive tyrosine sulfation . Interestingly, human bone sialoprotein, whose final secreted product is approximately 50% carbohydrate, contains no cystines . Northern analysis showed that while bone cells are the major source of bone sialoprotein message production, other tissues may contain trace amounts of this message . Southern hybridization of DNA from human-rodent somatic cell hybrids that have segregated human chromosomes indicated that the gene is located on human chromosome 4 . The human bone sialoprotein gene is a single copy gene unlikely to exceed 11.1 kilobases in length . No restriction fragment length polymorphisms were observed with 12 different restriction enzymes in 10 normal individuals.

J Biol Chem, 1990 Feb 5, 265(4), 2124 - 31
Studies of the domain structure of mammalian DNA polymerase beta . Identification of a discrete template binding domain; Kumar A et al.; Characterization of the domain structure of DNA polymerase beta is reported . Large scale overproduction of the rat protein in Escherichia coli was achieved, and the purified recombinant protein was verified by sequencing tryptic peptides . This protein is both a single-stranded DNA binding protein and a DNA polymerase consisting of one polypeptide chain of 334 amino acids . As revealed by controlled proteolysis experiments, the protein is organized in two relatively protease-resistant segments linked by a short protease-sensitive region . One of these protease-resistant segments represents the NH2-terminal 20% of the protein . This NH2-terminal domain (of about 75 residues) has strong affinity for single-stranded nucleic acids . The other protease-resistant segment, representing the COOH-terminal domain of approximately 250 residues, does not bind to nucleic acids . Neither domain, tested as purified proteins, has substantial DNA polymerase activity . The results suggest that the NH2-terminal domain is principally responsible for the template binding activity of the intact protein.

J Biol Chem, 1990 Feb 5, 265(4), 2383 - 90
Gz, a guanine nucleotide-binding protein with unique biochemical properties; Casey PJ et al.; Cloning of a complementary DNA (cDNA) for Gz alpha, a newly appreciated member of the family of guanine nucleotide-binding regulatory proteins (G proteins), has allowed preparation of specific antisera to identify the protein in tissues and to assay it during purification from bovine brain . Additionally, expression of the cDNA in Escherichia coli has resulted in the production and purification of the recombinant protein . Purification of Gz from bovine brain is tedious, and only small quantities of protein have been obtained . The protein copurifies with the beta gamma subunit complex common to other G proteins; another 26-kDa GTP-binding protein is also present in these preparations . The purified protein could not serve as a substrate for NAD-dependent ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin . Purification of recombinant Gz alpha (rGz alpha) from E . coli is simple, and quantities of homogeneous protein sufficient for biochemical analysis are obtained . Purified rGz alpha has several properties that distinguish it from other G protein alpha subunit polypeptides . These include a very slow rate of guanine nucleotide exchange (k = 0.02 min-1), which is reduced greater than 20-fold in the presence of mM concentrations of Mg2+ . In addition, the rate of the intrinsic GTPase activity of Gz alpha is extremely slow . The hydrolysis rate (kcat) for rGz alpha at 30 degrees C is 0.05 min-1, or 200-fold slower than that determined for other G protein alpha subunits . rGz alpha can interact with bovine brain beta gamma but does not serve as a substrate for ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin . These studies suggest that Gz may play a role in signal transduction pathways that are mechanistically distinct from those controlled by the other members of the G protein family.

Biochim Biophys Acta, 1990 Feb 2, 1015(2), 264 - 8
Mutational analysis of the function of the a-subunit of the F0F1-APPase of Escherichia coli; Howitt SM et al.; In a model proposed for the structure of the a-subunit of the Escherichia coli F0F1-ATPase (Howitt, S.M., Gibson, F . and Cox, G.B . (1988) Biochim . Biophys . Acta 936, 74-80), a cluster of charged residues, including one arginine and four aspartic acid residues, lie on the periplasmic side of the membrane . On the cytoplasmic side, three pairs of lysine residues and an arginine residue are present . Site-directed mutagenesis was used to investigate the roles of these residues . It was found that none was directly involved in the proton pore . However, the substitutions of Asp-124 or Asp-44 by asparagine or Arg-140 by glutamine had similar effects in that the membranes from such mutants from which the F1-ATPase was removed were proton-impermeable . A combination of the Asp-44 mutation with either the Asp-124 or Arg-140 mutations in the same strain resulted in complete loss of oxidative phosphorylation . It was tentatively concluded that Asp-124 and Arg-140 form a salt bridge, as did Asp-44 with an unknown residue, and these salt bridges were concerned with the maintenance of correct a-subunit structure . Further support for this conclusion was obtained when second site revertants of a Glu-219 to histidine mutant were found to have either histidine or leucine replacing Arg-140 . Thus, the lack of the Asp-124/Arg-140 salt bridge might enable repositioning of the helices of the a-subunit such that His-219 becomes a functional component of the proton pore.

Biochim Biophys Acta, 1990 Feb 2, 1015(2), 216 - 22
Reaction of membrane-bound F1-adenosine triphosphatase of Escherichia coli with chemical ligands and the asymmetry of beta subunits; Bragg PD et al.; The three beta subunits of the isolated Escherichia coli F1-ATPase react independently with chemical reagents (Stan-Lotter, H . and Bragg, P.D . (1986) Arch . Biochem . Biophys . 284, 116-120) . Thus, one beta subunit is readily cross-linked to the epsilon subunit, Another reacts with N,N'-dicyclohexylcarbodiimide (DCCD), and the third one is modified on a lysine residue by 4-chloro-7-nitrobenzofurazan (NbfCl) . The binding site for the ATP analog, 2-azido-ATP, was not associated with a specific type of beta subunit (Bragg, P.D . and Hou, C . (1989) Biochim . Biophys . Acta 974, 24-29) . We now show that this binding site is a catalytic site as opposed to a noncatalytic nucleotide-binding site . NbfCl reacted with a tyrosine residue on the DCCD-reacting beta subunit in contrast to the different subunit location of the lysine residue labeled by the reagent . Thus, O to N transfer of the Nbf group in the free F1-ATPase involves transfer between subunits . The chemical labelling pattern of membrane-bound F1-ATPase differed from that of free F1 . The strict asymmetry of labeling of the free F1-ATPase was not observed . Thus, double labeling of beta subunits by several reagents was found . This suggests that the asymmetry was not induced by chemical modification, but is inherent in the structure of the ATPase.

Biochim Biophys Acta, 1990 Feb 2, 1015(2), 195 - 9
The chloroplast CF0I subunit can replace the b-subunit of the F0F1-ATPase in a mutant strain of Escherichia coli K12; Schmidt G et al.; The amino acid sequence of the CF0I subunit from the chloroplast F0F1-ATPase has only a low similarity to the amino acid sequence of the b-subunit of the E . coli F0F1-ATPase . However, secondary and tertiary structure predictions plus the distribution of hydrophobic and hydrophilic amino acids have indicated that these two subunits serve a similar function . This proposition was investigated directly . A cDNA clone for the chloroplast atpF gene, encoding the CF0I subunit, was altered by site-directed mutagensis such that the translation start site corresponded to the N-terminus of the mature protein . An E . coli mutant strain carrying a chain-terminating mutation in the uncF gene, encoding the b-subunit, was transformed with the plasmid carrying the altered atpF gene . The resultant transformant was able to grow on succinate and gave a growth yield similar to that of a wild-type control . Assays on membrane preparations from the transformant also clearly indicated that the mature CF0I subunit from spinach chloroplasts was able to replace the E . coli b-subunit in the E . coli F0F1-ATPase.

Scand J Immunol, 1990 Feb, 31(2), 225 - 35
Comparison of biological and immunological activities of human monocyte-derived interleukin 1 beta and human recombinant interleukin 1 beta; Molvig J et al.; Recombinant human interleukin 1 beta (rhIL-1 beta) and supernatants of Escherichia coli lipopolysaccharides-stimulated human monocyte (Mo) cultures, containing native human IL-1 beta (nhIL-1 beta), demonstrate significant differences when tested in the mouse co-stimulatory thymocyte (lymphocyte activating factor {LAF}) assay . The aims of the present study were to investigate this characteristic difference between rhIL-1 beta and Mo culture supernatants (Mo supernatants), and to compare the biological and the immunological activity of preparations of rhIL-1 beta and nhIL-1 beta during each step of an identical purification procedure . The biological activity of rhIL-1 beta/nhIL-1 beta preparations was characterized by the use of the LAF assay and the rat islet insulin release assay . An IL-1 beta enzyme-linked immunosorbent assay (ELISA) was established in order to compare the biological and immunological responses of the IL-1 beta preparations . We report that the significant difference between rhIL-1 beta and supernatants of Mo cultures, which was only demonstrable in the LAF assay, is due to the presence of interleukin 6 (IL-6) in the Mo supernatants . We describe a simple cation exchange chromatography separating nhIL-1 beta and IL-6 of Mo supernatants . The highly purified rhIL-1 beta possessing the correct amino-terminal sequence and nhIL-1 beta have identical biological and immunological activities demonstrating a specific biological activity (SBA) of 3 x 10(2) U/ng IL-1 beta . Thus, we have no indications of secondary or tertiary structural differences between rhIL-1 beta and purified nhIL-1 beta . In contrast, both in the LAF assay and in the rat islet insulin release assay the SBA of an amino-extended rhIL-1 beta form, Met-Glu-Ala-Glu-rhIL-1 beta, was only 1-2% of the SBA of rhIL-1 beta, suggesting that structural changes were introduced into the molecule by the amino-terminal extension . In the present study we have demonstrated that systematic combined testing of IL-1 beta preparations in two different biological assays and an immunological assay is useful for the characterization and comparison of the activity of recombinant and native IL-1 beta preparations purified by the use of exactly the same procedures.

Biochem J, 1990 Feb 1, 265(3), 899 - 902
Chorismate synthase . Pre-steady-state kinetics of phosphate release from 5-enolpyruvylshikimate 3-phosphate; Hawkes TR et al.; The pre-steady-state kinetics of phosphate formation from 5-enolpyruvylshikimate 3-phosphate catalysed by Escherichia coli chorismate synthase (EC 4.6.1.4) were studied by a rapid-acid-quench technique at 25 degrees C at pH 7.5 . No pre-steady-state 'burst' or 'lag' phase was observed, showing that phosphate is released concomitant with the rate-limiting step of the enzyme . The implications of this result for the mechanism of action of chorismate synthase are discussed.

Biochem J, 1990 Feb 1, 265(3), 725 - 9
The Escherichia coli cysG gene encodes S-adenosylmethionine-dependent uroporphyrinogen III methylase; Warren MJ et al.; The Escherichia coli cysG gene was successfully subcloned and over-expressed to produce a 52 kDa protein that was purified to homogeneity . This protein was shown to catalyse the S-adenosylmethionine-dependent methylation of uroporphyrinogen III to give a product identified as sirohydrochlorin on the basis of its absorption spectra, incorporation of 14C label from S-adenosyl{Me-14C}methionine and mass and 1H-n.m.r . spectra of its octamethyl ester . Further confirmation of the structure was obtained from a 14C-n.m.r . spectrum of the methyl ester produced by incubation of the methylase with uroporphyrinogen III, derived from {4.6-13C2}porphobilinogen, and S-adenosyl{Me-13C}methionine.

Arch Biochem Biophys, 1990 Feb 1, 276(2), 531 - 7
Isolation and structural characterization of three isoforms of recombinant consensus alpha interferon; Klein ML et al.; Recombinant DNA-derived consensus alpha interferon was expressed in Escherichia coli and purified . Isoelectric focusing of this purified protein indicated the presence of three isoelectric subforms of pI 6.1, 6.0, and 5.7 . These three subforms were preparatively separated by isoelectric focusing using Immobiline polyacrylamide gel and did not exhibit apparent differences in biological activity and tertiary structure . The pI 5.7 subform could also be separated from the pI 6.1 and 6.0 subforms by reverse-phase HPLC . Automated N-terminal amino acid sequence analysis of the pI 6.1 and 6.0 subforms yielded sequences corresponding to the methionyl and des-methionyl forms of the protein, respectively . Sequence analysis of the pI 5.7 subform indicated that its N terminus is blocked . To further determine the structure of the blocking moiety in the pI 5.7 subform, a blocked N-terminal tryptic peptide was isolated from HPLC peptide mapping of the S-carboxymethylated derivative . Results obtained from mass spectroscopic and amino acid analyses of this peptide suggest that it is blocked with an acetyl group at the N-terminal cysteine residue.

Arch Biochem Biophys, 1990 Feb 1, 276(2), 299 - 304
Visualization of ion-dependent conformational changes in Escherichia coli 23 S rRNA by scanning transmission electron microscopy; Mandiyan V et al.; Electron micrographs of Escherichia coli 23 S rRNA molecules obtained by scanning transmission electron microscopy, unstained and under nondenaturing conditions, reveal previously unresolved structural patterns . The complexity of the pattern is dependent upon the ambient ionic strength conditions . In water and in very low ionic strength buffer, the conformation of 23 S rRNA is characterized by an extended framework, with short side branches related to the secondary and tertiary structure of the molecule . The total length of this filamentous complex is approximately 2500 A, only about one-fourth of the length of 23 S rRNA when fully stretched under the denaturing conditions used for imaging by conventional electron microscopy . These data, supplemented by the determination of the linear density (M/L), suggest that in low ionic strength the backbone of 23 S rRNA is formed by a structure corresponding, on the average, to the mass of four nucleotide strands (M/L approximately equal to 480 Da/A) . With increasing ionic strength, 23 S rRNA coils into more compact forms . Molecules in these states can be characterized by apparent radii of gyration (RG), which can be calculated from the mass distribution within the digitized images of individual RNA molecules . The 23 S rRNA is in its most condensed form (RG = 115 A) in ribosomal reconstitution buffer; however, it still does not attain the compactness of the large subunit (RG = 69 A), nor does it show any resemblance to the native 50 S subunit . The net content of ordered secondary structure, as determined by circular dichroism spectroscopy, is not visibly affected by the changes of ionic strength conditions . These results imply that the observed conformational changes in 23 S rRNA are caused by intramolecular folding of the 23 S rRNA strands induced by the shielding effect of ambient charges.

Appl Environ Microbiol, 1990 Feb, 56(2), 551 - 4
Evidence that Escherichia coli accumulates glycine betaine from marine sediments; Ghoul M et al.; Escherichia coli grew faster in autoclaved marine sediment than in seawater alone . When E . coli was cultivated in sediment diluted with minimal medium M63 at 0.6 M NaCl, supplemented or not supplemented with glucose or with seawater, the osmoprotector glycine betaine was accumulated in the cells . The best growth occurred on glucose . Accumulation of glycine betaine was not observed with E . coli was grown in sterile seawater alone . The fact that E . coli grew better in the sediments than in seawater is attributed somewhat to the high content of organic matter in the sediment but mainly to the accumulation of glycine betaine . Thus, osmoprotection should be considered to be an additional factor in bacterial survival in estuarine sediments.

South Med J, 1990 Feb, 83(2), 253 - 4
Liver abscesses: successful treatment with choledochoduodenostomy; Fischer MJ; I have described two cases of hepatic abscess due to E coli after choledochoduodenostomy for choledocholithiasis, and have discussed the relationship between abscess formation and size of the anastomosis . I conclude that a small anastomosis may in some cases predispose to this problem . Treatment by percutaneous drainage of the abscess was successful in both cases.

Proc Natl Acad Sci U S A, 1990 Feb, 87(4), 1576 - 80
Induction and therapy of autoimmune diabetes in the non-obese diabetic (NOD/Lt) mouse by a 65-kDa heat shock protein; Elias D et al.; Insulin-dependent diabetes mellitus is caused by autoimmune destruction of the insulin-producing beta cells of the pancreas . The results described here indicate that a beta-cell target antigen in non-obese diabetic (NOD/Lt) mice is a molecule cross-reactive with the 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis . The onset of beta-cell destruction is associated with the spontaneous development of anti-hsp65 T lymphocytes . Subsequently hsp65 cross-reactive antigen becomes detectable in the sera of the prediabetic mice and some weeks later anti-hsp65 antibodies, anti-insulin antibodies, and anti-idiotypic antibodies to insulin antibodies become detectable . The hsp65-cross-reactive antigen, the autoantibodies, and the T-cell reactivity then decline with the development of overt insulin-dependent diabetes . The importance of hsp65 in the pathogenesis of insulin-dependent diabetes was confirmed by the ability of clones of anti-hsp65 T cells to cause insulitis and hyperglycemia in young NOD/Lt mice . Moreover, hsp65 antigen could be used either to induce diabetes or to vaccinate against diabetes, depending on the form of its administration to prediabetic NOD/Lt mice . Other antigens such as the 70-kDa heat shock protein (hsp70) had no effect on the development of diabetes.

Proc Natl Acad Sci U S A, 1990 Feb, 87(4), 1571 - 5
Identification of five putative yeast RNA helicase genes; Chang TH et al.; The RNA helicase gene family encodes a group of eight homologous proteins that share regions of sequence similarity . This group of evolutionarily conserved proteins presumably all utilize ATP (or some other nucleoside triphosphate) as an energy source for unwinding double-stranded RNA . Members of this family have been implicated in a variety of physiological functions in organisms ranging from Escherichia coli to human, such as translation initiation, mitochondrial mRNA splicing, ribosomal assembly, and germinal line cell differentiation . We have applied polymerase chain reaction technology to search for additional members of the RNA helicase family in the yeast Saccharomyces cerevisiae . Using degenerate oligonucleotide primers designed to amplify DNA fragments flanked by the highly conserved motifs V L D E A D and Y I H R I G, we have detected five putative RNA helicase genes . Northern and Southern blot analyses demonstrated that these genes are single copy and expressed in yeast . Several members of the RNA helicase family share sequence identity ranging from 49.2% to 67.2%, suggesting that they are functionally related . The discovery of such a multitude of putative RNA helicase genes in yeast suggests that RNA helicase activities are involved in a variety of fundamentally important biological processes.

Proc Natl Acad Sci U S A, 1990 Feb, 87(4), 1300 - 4
Generation of deletion derivatives by targeted transformation of human-derived yeast artificial chromosomes; Pavan WJ et al.; Mammalian DNA segments cloned as yeast artificial chromosomes (YACs) can be manipulated by DNA-mediated transformation when placed in an appropriate yeast genetic background . A "fragmenting vector" has been developed that can introduce a yeast telomere and selectable marker into human-derived YACs at specific sites by means of homologous recombination, deleting all sequences distal to the recombination site . A powerful application of the method uses a human Alu family repeat sequence to target recombination to multiple independent sites on a human-derived YAC . Sets of deletion derivatives generated by this procedure greatly facilitate restriction mapping of large genomic segments . Targeting recombination with single copy sequences, such as cDNAs, will have many additional applications . This approach establishes a paradigm for manipulation and characterization of mammalian DNA segments cloned as YACs.

Proc Natl Acad Sci U S A, 1990 Feb, 87(4), 1292 - 5
Are many Z-DNA binding proteins actually phospholipid-binding proteins?
Krishna P, Kennedy BP, Waisman DM, van de Sande JH, McGhee JD.
We used a Z-DNA affinity column to isolate a collection of Z-DNA binding proteins from a high salt extract of Escherichia coli . We identified one of the major Z-DNA binding proteins of this fraction, not as a protein involved in gene regulation or genetic recombination, but rather as an outer membrane porin protein . We then showed that several other known phospholipid-binding proteins (bovine lung annexins and human serum lipoproteins) also bind much more tightly to Z-DNA than to B-DNA . In all cases, this Z-DNA binding was strongly blocked by competition with acidic phospholipids, such as cardiolipin . Our results raise the question whether many of the Z-DNA binding proteins previously isolated are actually phospholipid-binding proteins.

Mutat Res, 1990 Feb, 243(2), 165 - 71
Induction of error-free DNA repair in Escherichia coli by thiamine deprivation; Sharma N et al.; Incubation of Escherichia coli AB1157 in a thiamine-deficient medium causes a large, time-dependent increase in resistance to UV-radiation (254 nm) and a fall in its UV-induced mutation frequency to histidine prototrophy which are abolished in its uvrA mutant, but only delayed in lon- and recA- cells . The response of the lexA3 mutant resembles that of the parental cells . These effects are very similar to those we have shown to be induced by heat shock and are clearly due to an error-free, DNA-excision repair-dependent process . They may represent a general response to non-mutagenic stress in these cells.

Clin Chem, 1990 Feb, 36(2), 198 - 200
Activity of phospholipase A2 in plasma increases in uremia; Costello J et al.; We measured phospholipase A2 (PLA2; EC 3.1.1.4) activity in normal and uremic plasma, using {1-14C}oleate-labeled autoclaved Escherichia coli as substrate . Hydrolysis of bacterial phospholipid by crude plasma from both groups was optimal at pH 5.5, was specific for the 2-acyl position of phospholipids, and had an absolute requirement for calcium . Activity was greatest in the presence of added Ca2+, 5 mmol/L, but this increase was inhibited by several divalent cations (Mg2+, Zn2+, Cu2+, Ba2+, Co2+, Pb2+, Fe2+) and by Fe3+ . PLA2 activity was also inhibited by heparin at acid and alkaline pH, normal plasma being more sensitive than uremic plasma to this inhibition . Enzyme activity in undiluted plasma was eightfold greater in uremic than in normal plasma . Dilution of plasma by two to fourfold increased the total activity of both normal and uremic plasma . However, the relative differences in total activity between the groups remained constant (eight- to 11-fold) . The cause and consequences of the increased PLA2 activity in uremia remain to be established.

Proc Soc Exp Biol Med, 1990 Feb, 193(2), 167 - 70
Endotoxin extends survival of adult mice in hyperoxia; Berg JT et al.; Research on endotoxin protection from oxygen toxicity is presently limited to the rat model since only rats have been protected by endotoxin . This study reports that endotoxin also extends survival of adult male mice in hyperoxia (greater than 99% oxygen at 1 ATA) . Initially, 4-month-old male mice were treated with Boivin-extracted E . coli endotoxin and placed in hyperoxia . Zymosan-primed mice receiving 2 or 10 micrograms endotoxin, and unprimed mice receiving 10-40 micrograms endotoxin, showed moderate protection against hyperoxia; 11/15 Boivin-treated mice survived 120 hours exposure to hyperoxia with time-of-death in hyperoxia = 126.7 +/- 4.4 hours (mean +/- SEM, n = 15) . This contrasts with untreated male mice; 0/4 survived 120 hours exposure to hyperoxia with mean survival = 103.5 +/- 3.5 hours . Mice receiving 20 or 60 micrograms Westphal-extracted endotoxin were not protected nor were older female mice receiving 20 micrograms Boivin-extracted endotoxin . This study suggests that age, sex, the extraction method used to obtain endotoxin, and possibly the time of year when endotoxin is administered, are important variables in allowing endotoxin to extend survival of mice in hyperoxia.

Proc Natl Acad Sci U S A, 1990 Feb, 87(3), 857 - 61
Expression cloning of a cDNA encoding the murine interleukin 4 receptor based on ligand binding; Harada N et al.; Interleukin 4 (IL-4) is a potent mediator of growth and differentiation for various lymphoid and myeloid cells . To isolate a cDNA encoding the murine IL-4 receptor, we have developed an expression cloning method that uses biotinylated ligand as a probe and that may be generally applicable to cloning of receptor genes . COS-7 cells transiently transfected with the cloned full-length cDNA bind murine IL-4 specifically with a Kd = 165 pM . Crosslinking of 125I-labeled IL-4 to COS-7 cells transfected with the cDNA reveals binding to proteins of 120-140 kDa . IL-4-responsive cells also express IL-4-binding proteins of 120-140 kDa but show additional bands at 60-70 kDa; the relationship of the smaller proteins to the larger ones is unclear . The nucleotide sequence indicates that the full-length cDNA encodes 810 amino acids including the signal sequence . While no consensus sequence for protein kinases is present in the cytoplasmic domain, a sequence comparison with the erythropoietin receptor, the IL-6 receptor, and the beta chain of the IL-2 receptor reveals a significant homology in the extracellular domain, indicating that the IL-4 receptor is a member of a cytokine receptor family.

Proc Natl Acad Sci U S A, 1990 Feb, 87(3), 1198 - 202
Specific interaction of the human immunodeficiency virus Rev protein with a structured region in the env mRNA; Cochrane AW et al.; A region of potential complex secondary structure within the human immunodeficiency virus env mRNA has been implicated in Rev-mediated export of viral structural mRNAs from the nucleus to the cytoplasm . By using an RNase protection gel-mobility-shift assay, we demonstrate that purified Rev protein forms a stable complex with this Rev-responsive RNA . RNAs with mutations designed to disrupt formation of a predicted stem structure no longer interact with Rev . However, Rev binding is restored upon annealing of the two complementary RNAs that make up the stem . These results suggest that direct interaction of Rev with the Rev-responsive element could facilitate transport of human immunodeficiency virus structural mRNAs from the nucleus to the cytoplasm.

Proc Natl Acad Sci U S A, 1990 Feb, 87(3), 1109 - 13
Site-specific DNA recombination system Min of plasmid p15B: a cluster of overlapping invertible DNA segments; Sandmeier H et al.; Plasmid p15B of Escherichia coli 15T- carries a 3.5-kilobase segment that undergoes frequent DNA inversion mediated by the DNA inversion enzyme Min, a member of the Din family of site-specific recombinases . While the previously described Din inversion systems invert a DNA segment between two crossover sites in inverted orientation, the Min system produces more complex DNA rearrangements . These have been physically characterized by electron microscopy and by restriction cleavage analysis . The results can best be explained by a model that involves six crossover sites (called mix) and predicts 240 isomeric forms of the invertible region . The model was confirmed by sequencing the six mix sites in plasmids that contain the invertible DNA segments in a frozen configuration . All mix sites fit the dix consensus sequence, and they are all good substrates for DNA inversion when carried in inverted orientation . Recombination between two mix sites in direct orientation was rare, in line with the notion that Din inversion systems are topologically biased to the inversion reaction . Another recently described multiple inversion system, the shufflon of the E . coli plasmid R64, is neither functionally nor structurally related to the Min system of p15B.

Proc Natl Acad Sci U S A, 1990 Feb, 87(3), 1042 - 6
Dominant lethal mutations in a conserved loop in 16S rRNA; Powers T et al.; The 530 stem-loop region in 16S rRNA is among the most phylogenetically conserved structural elements in all rRNAs, yet its role in protein synthesis remains mysterious . G-530 is protected from kethoxal attack when tRNA, or its 15-nucleotide anticodon stem-loop fragment, is bound to the ribosomal A site . Based on presently available evidence, however, this region is believed to be too remote from the decoding site for this protection to be the result of direct contact . In this study, we use a conditional rRNA expression system to demonstrate that plasmid-encoded 16S rRNA genes carrying A, C, and T point mutations at position G-530 confer a dominant lethal phenotype when expressed in Escherichia coli . Analysis of the distribution of plasmid-encoded 16S rRNA in ribosomal particles, following induction of the A-530 mutation, shows that mutant rRNA is present both in 30S subunits and in 70S ribosomes . Little mutant rRNA is found in polyribosomes, however, indicating that the mutant ribosomes are severely impaired at the stage of polysome formation and/or stability . Detailed chemical probing of mutant ribosomal particles reveals no evidence of structural perturbation within the 16S rRNA . Taken together, these results argue for the direct participation of G-530 in ribosomal function and, furthermore, suggest that the dominant lethal phenotype caused by these mutations is due primarily to the mutant ribosomes blocking a crucial step in protein synthesis after translational initiation.

Mol Cell Biol, 1990 Feb, 10(2), 785 - 93
The Z-DNA motif d(TG)30 promotes reception of information during gene conversion events while stimulating homologous recombination in human cells in culture; Wahls WP et al.; Tracts of the alternating dinucleotide polydeoxythymidylic-guanylic {d(TG)}.polydeoxyadenylic-cytidylic acid {d(AC)}, present throughout the human genome, are capable of readily forming left-handed Z-DNA in vitro . We have analyzed the effects of the Z-DNA motif d(TG)30 upon homologous recombination between two nonreplicating plasmid substrates cotransfected into human cells in culture . In this study, the sequence d(TG)30 is shown to stimulate homologous recombination up to 20-fold . Enhancement is specific to the Z-DNA motif; a control DNA fragment of similar size does not alter the recombination frequency . The stimulation of recombination is observed at a distance (237 to 1,269 base pairs away from the Z-DNA motif) and involves both gene conversion and reciprocal exchange events . Maximum stimulation is observed when the sequence is present in both substrates, but it is capable of stimulating when present in only one substrate . Analysis of recombination products indicates that the Z-DNA motif increases the frequency and alters the distribution of multiple, unselected recombination events . Specifically designed crosses indicate that the substrate containing the Z-DNA motif preferentially acts as the recipient of genetic information during gene conversion events . Models describing how left-handed Z-DNA sequences might promote the initiation of homologous recombination are presented.

J Bacteriol, 1990 Feb, 172(2), 977 - 85
Complementation of a reaction center-deficient Rhodobacter sphaeroides pufLMX deletion strain in trans with pufBALM does not restore the photosynthesis-positive phenotype; Farchaus JW et al.; The puf operon in Rhodobacter sphaeroides is composed of the genes for the photosynthetic reaction center L and M subunits, light-harvesting antenna complex I, and one other open reading frame termed pufX . Complementation of a reaction center-deficient, photosynthetically incompetent pufLMX deletion strain in trans with a fragment containing the entire puf operon, including pufX and an additional 1,100 base pairs of DNA downstream of pufX, restored the reaction center and the photosynthesis-positive phenotype . Complementation of the same strain with pufBALM restores the reaction center to the level seen with the entire puf operon but not the photosynthesis-positive phenotype . Northern (RNA) blot analysis revealed that oxygen regulated transcription was not blocked in the absence of pufX and the downstream region . Spectroscopic and protein analyses indicated that the pigment-binding protein complexes, including the reaction center, were expressed and showed normal absorption characteristics . A 20% reduction in the amount of light-harvesting antenna complex II and a corresponding increase in the amount of light-harvesting antenna complex I were observed in the deletion strain harboring the plasmid with the puf insert lacking the pufX gene and the downstream region compared with those complemented with the entire puf operon and an additional downstream 1,100 base pairs.

J Bacteriol, 1990 Feb, 172(2), 786 - 92
Effects of mutations in the repA gene of plasmid Rts1 on plasmid replication and autorepressor function; Terawaki Y et al.; We constructed a system in which wild-type RepA or RepAcop1 protein was supplied in trans in various amounts to coexisting mini-Rts1 plasmids by clones of the repA or repAcop1 gene under the control of the native promoter with or without its operator sequence . RepAcop1 protein which contains a single amino acid substitution (Arg-142 to Lys) within its 288 amino acids could initiate the replication of the mini-Rts1 plasmid efficiently at both 37 and 42 degrees C even if it was supplied in excess . In contrast, excess wild-type RepA inhibited plasmid replication at 37 degrees C but supported replication at 42 degrees C . Therefore, it appears that the initiator activity of RepA is not related to the incompatibility phenotype associated with an excess of RepA protein . An immunoblot analysis revealed that neither RepA nor RepAcop1 synthesis was temperature sensitive and that both were autogenously regulated to a similar extent because of the presence of an operator located immediately upstream of the promoter . Two mutant RepA proteins, each of which contains a 4-amino-acid insertion in the middle of the protein, maintained the autorepressor and incompatibility activities but lost the ori(Rts1)-activating function.

J Bacteriol, 1990 Feb, 172(2), 691 - 5
Effects of pH, glucose, and chelating agents on lethality of paraquat to Escherichia coli; Minakami H et al.; Retention of paraquat by Escherichia coli B was greatest after exposure at pH 9.0 and was progressively less after exposure at pH 7.0 and 5.0, respectively . This retained paraquat was capable of persistent growth inhibition . Uptake and retention of paraquat by E . coli B was dependent upon a carbon source, such as glucose . Under comparable conditions E . coli K-12 did not retain paraquat . The lethality of paraquat was seen in TSY medium but not in VB medium . The addition of Soytone, tryptone, or yeast extract, to the VB medium allowed the lethality of paraquat to be seen . A variety of chelating agents, including EDTA, 8-hydroxyquinoline, and o-phenanthroline, prevented the lethal effect of paraquat in TSY medium . Although EDTA protected against the lethality of paraquat, it did not protect against its bacteriostatic effect.

J Bacteriol, 1990 Feb, 172(2), 686 - 90
Effects of paraquat on Escherichia coli: differences between B and K-12 strains; Kitzler JW et al.; Escherichia coli B and K-12 are equally susceptible to the bacteriostatic effects of aerobic paraquat, but they differed strikingly when the lethality of paraquat was evaluated . E . coli B suffered an apparent loss of viability when briefly exposed to paraquat, whereas E . coli K-12 did not . This difference depended on the ability of the B strain, but not the K-12 strain, to retain internalized paraquat; the B strain was killed on aerobic tryptic soy-yeast extract plates during the incubation which preceded the counting of colonies . This difference in retention of paraquat between strains was demonstrated by delayed loss of viability, by growth inhibition, and by cyanide-resistant respiration after brief exposure to paraquat, washing, and testing in fresh medium . This difference was also shown by using {14C}paraquat . This previously unrecognized difference between E . coli B and K-12 has been the cause of apparently contradictory reports and should lead to some reevaluation of the pertinent literature.

J Bacteriol, 1990 Feb, 172(2), 653 - 7
Functional analysis of the Shiga toxin and Shiga-like toxin type II variant binding subunits by using site-directed mutagenesis; Jackson MP et al.; The B subunit of Shiga toxin and the Shiga-like toxins (SLTs) mediates receptor binding, cytotoxic specificity, and extracellular localization of the holotoxin . While the functional receptor for Shiga toxin, SLT type I (SLT-I), and SLT-II is the glycolipid designated Gb3, SLT-II variant (SLT-IIv) may use a different glycolipid receptor . To identify the domains responsible for receptor binding, localization in Escherichia coli, and recognition by neutralizing monoclonal antibodies, oligonucleotide-directed site-specific mutagenesis was used to alter amino acid residues in the B subunits of Shiga toxin and SLT-IIv . Mutagenesis of a well-conserved hydrophilic region near the amino terminus of the Shiga toxin B subunit rendered the molecule nontoxic but did not affect immunoreactivity or holotoxin assembly . In addition, elimination of one cysteine residue, as well as truncation of the B polypeptide by 5 amino acids, caused a total loss of activity . Changing a glutamate to a glutamine at the carboxyl terminus of the Shiga toxin B subunit resulted in the loss of receptor binding and immunoreactivity . However, the corresponding mutation in the SLT-IIv B subunit (glutamine to glutamate) did not reduce the levels of cytotoxicity but did affect extracellular localization of the holotoxin in E . coli.

J Bacteriol, 1990 Feb, 172(2), 648 - 52
Colicin cleavage by OmpT protease during both entry into and release from Escherichia coli cells; Cavard D et al.; Proteolysis of colicins A, E1, E2, and E3 was observed after they were added to whole cells carrying a functional ompT gene . Recombinant plasmid pML19 containing the ompT gene enabled two mutant strains to cleave the added colicins . On the other hand, two colicin A recombinants were split after release from the wild-type bacteria that produced them but not from ompT mutant cells.

J Bacteriol, 1990 Feb, 172(2), 515 - 8
Alkaline phosphatase fusions: sensors of subcellular location; Manoil C et al.; Alkaline phosphatase fusions allow genes to be identified solely on the basis of their protein products being exported from the cytoplasm . Thus, the use of such fusions helps render biological processes which involve cell envelope and secreted proteins accessible to a sophisticated genetic analysis . Furthermore, alkaline phosphatase fusions can be used to locate export signals . Specifying such signals is an important component of studies on the structure of individual cell envelope proteins . The basis of the alkaline phosphatase fusion approach is the finding that the activity of the enzyme responds differently to different environments . Thus, the activity of the fusion protein gives evidence as to its location . This general approach of using sensor proteins which vary in their function, depending on their environment, could be extended to the study of other sorts of problems . It may be that certain enzymes will provide an assay for localization to a particular subcellular compartment, if the environment of the compartment differs from that of others . For instance, the lysosome is more acidic than other intracellular organelles . A gene fusion system employing a reporter enzyme that could show activity only at the pH of the lysosome could allow the detection of signals determining lysosomal localization . Analogous types of enzymes may be used as probes for other subcellular compartments.

Infect Immun, 1990 Feb, 58(2), 550 - 6
Cloning and expression of the gene for the cross-reactive alpha antigen of Mycobacterium kansasii; Matsuo K et al.; The gene for the extracellular alpha antigen of Mycobacterium kansasii was cloned by using the alpha-antigen gene fragments of M . bovis BCG as probes . Gene analysis revealed that this gene encodes 325 amino acid residues, including 40 amino acids for the signal peptide, followed by 285 amino acids for the mature protein . A comparison of the nucleotide sequences of the genes isolated from these two mycobacterial species showed that the levels of DNA and amino acid homology were 84.8 and 89.1%, respectively . The hydropathy profiles were also compared, and two highly changed hydrophilic regions were observed, which might account for the antigenic diversity of this antigen or its acquirement of antigenic specificity.

Exp Cell Res, 1990 Feb, 186(2), 317 - 23
ATP-independent strand transfer protein from murine spermatocytes, spermatids, and spermatozoa; Higashitani A et al.; A protein-catalyzing D-loop formation is present in murine spermatocytes, spermatids, and spermatozoa, but is not found in somatic tissue or in premeiotic cells of the germline . Unlike the Escherichia coli RecA protein and the meiotic rec protein (m-rec) previously described, D-loop formation by this protein (referred to as "mAi-rec") does not require ATP . The meiotic profile of mAi-rec activity is only partly similar to that of m-rec . Like m-rec, it rises steeply during early prophase and reaches a peak at pachytene . Unlike m-rec, its activity remains high during the postmeiotic phase of spermatid development and is prominent in immunochemically stained spermatozoa . A polyclonal antibody to E . coli RecA reacts with mAi-rec and inhibits its activity . No such reaction occurs with m-rec protein . The extent of sequence homology between E . coli RecA and murine mAi-rec is highly limited; none of the several monoclonal antibodies tested reacted with mAi-rec.

J Biochem (Tokyo), 1990 Feb, 107(2), 228 - 35
Cloning of Xenopus RCC1 cDNA, a homolog of the human RCC1 gene: complementation of tsBN2 mutation and identification of the product; Nishitani H et al.; We isolated a cDNA clone from Xenopus cDNA libraries for the homolog of the human RCC1 gene, which is considered to be a regulator for the onset of chromosome condensation . The cloned Xenopus RCC1 cDNA encoded a protein of 424 amino acids which had the same seven homologous repeated domains of about 60 amino acids as found in human RCC1 cDNA . The overall identity of amino acid sequence between the human and Xenopus RCC1 protein was 76% . Specifically, in the repeated domain the amino acid sequence was highly conserved between both species . The identity of amino acids in this region was 82% . In the N-terminal region, albeit the overall identity was low (35%), some positively charged amino acids were conserved . The transcript for Xenopus RCC1 gene with the length of 2.2-kb was detected in both Xenopus oocytes and cultured somatic cells, A6 . The antibody prepared against Xenopus RCC1 protein produced in E . coli recognized 45 and 46 kDa proteins not only in Xenopus oocytes and A6 cells, but also in human and hamster cells . Xenopus RCC1 cDNA complemented the tsBN2 mutation, depending on the amount of its product in transformants . Thus, the RCC1 protein was suggested to regulate the onset of chromosome condensation in the eukaryote from amphibian to mammalian cells.

An Esp Pediatr, 1990 Feb, 32(2), 114 - 8
{Incidence of diarrhea among groups of children in the city of Seville}; Marquez Calderon S et al.; We studied prospectively the cases of diarrhoea that were produced during one year of follow-up in three groups of children (31 newborn, 62 of 1 year and 51 of 2 years) selected randomly from the Civil Register of Sevilla . The incidence of diarrhoea was 47 episodes for every 100 children per year, with a peak of higher incidence detected in July and August . Diarrhoea was more frequent in children of low socioeconomic level . The most common producers of diarrhoea were rotavirus (30%) and enteropathogenic E . coli (12%) . The most frequent accompanying symptoms of diarrhoea were anorexia (68%), fever (35%), abdominal pain (32%) and vomiting (30%).

Vet Immunol Immunopathol, 1990 Feb, 24(2), 169 - 75
The characterisation of equine interleukin-1; May SA et al.; Equine interleukin-1 has been produced from peripheral blood monocytes by stimulation with E . coli lipopolysaccharide . Sephacryl S200 gel filtration revealed a molecular weight of 17-18 kD . Chromatofocusing of the 17-18 kD peak identified four active fractions . Two major peaks were detected at pH 6.7 and pH 7, with smaller peaks at pH 6.3 and pH 5.9 . The pI 7 molecule is probably the equine form of IL-1 beta.

Inflammation, 1990 Feb, 14(1), 109 - 23
Chemotactic competence of neutrophils from neonatal calves . Functional comparison with neutrophils from adult cattle; Zwahlen RD et al.; Neonates demonstrate an increased susceptibility to infection . Defects in locomotory functions of newborn neutrophils may play a crucial role in this context . We therefore compared the migratory response of newborn (N-PMN) and adult (A-PMN) bovine neutrophils in a microwell filter assay . Stimulation with four different endotoxins (E . coli O128B:4 and O55B:5; S . abortus equi; S . typhimurium), with zymosan-activated plasma (ZAP) and with C5a induced dose-dependent migration of A-PMNs and N-PMNs . Migration of unstimulated cells and of cells stimulated with diluted ZAP or C5a was higher (P less than 0.05) in N-PMNs . Migration of A- and N-PMNs towards C5a was inhibited (P less than 0.001) by preincubation with either a steroidal (122 microM flumethasone) or nonsteroidal (3.3 microM phenylbutazone) antiinflammatory drug . Migratory responses of N-PMNs were inhibited less by SAIDs than were responses of A-PMNs (P less than 0.05); indeed dexamethasone slightly enhanced N-PMN responses towards C5a, and 510 microM flunixin meglumine enhanced C5a-induced migration in both age groups . Endotoxins from E . coli O55:B4, S . abortus equi, and S . typhimurium induced a higher rate of migration (P less than 0.05) in N-PMNs . In contrast to the above findings, measurement of the maximal distance of migration by the leading-front method did not reveal age-related differences . Migration speed of PMNs was lower after stimulation with C5a than with ZAP, but could be restored partly by adding human vitamin D-binding protein (Gc-globulin) . The demonstrated hyperirritability of bovine N-PMNs represents a major functional difference to neonatal neutrophils from other species, including man . It may additionally be related to altered PMN functions and neonatal disease susceptibility.

Arch Biochem Biophys, 1990 Feb 1, 276(2), 495 - 9
Reexamination of the 1H NMR assignments and solution conformations of L-carnitine and O-acetyl-L-carnitine using C-2 stereospecifically labeled monodeuterated isomers; Brewer F et al.; The two C-2 monodeuterated isomers of L-carnitine were synthesized by enzymatic hydration of crotonobetaine in D2O and by enzymatic proton exchange of L-{2-2H2}carnitine in H2O . These reactions, catalyzed by an induced Escherichia coli carnitine hydrolyase proceed stereospecifically . The two isomers of L-{2-2H}carnitine were examined by 1H NMR at 500 MHz, which allowed us to independently monitor the pD dependence and coupling constants of the H-2 protons . The results obtained indicate that there is little effect of the carboxyl charge on the conformational state(s) of L-carnitine about the C-2/C-3 bond . The NMR data obtained in this study do not support previous solution studies of the pH-dependent conformational changes for DL-carnitine nor the proposed conformation of O-acetyl-DL-carnitine in the crystalline state.

Proc Natl Acad Sci U S A, 1990 Feb, 87(4), 1615 - 9
Soluble antigen abrogates the appearance of anti-protein IgG1-forming cell precursors during primary immunization; Nossal GJ et al.; The anti-human serum albumin (HSA) B-cell repertoire of C57BL/6 mice was examined by culturing splenocytes at limiting dilution following polyclonal stimulation with Escherichia coli lipopolysaccharide and a lymphokine mixture . The frequency of anti-HSA precursors was determined before and after immunization with alum-precipitated HSA and 10(9) killed Bordetella pertussis organisms, by submitting clonal supernatants to an ELISA . Anti-HSA IgG1-forming precursors were rare in unimmunized spleens, representing approximately equal to 1 in 500,000 splenocytes or only approximately equal to 100 cells per spleen . Between day 5 and day 7 after immunization, this figure increased to approximately equal to 20,000 cells per spleen . Over the following 3 weeks, there was a progressive increase in the mean optical density generated in the clonal ELISA, presumably due to affinity maturation of the B-cell population . When freshly deaggregated HSA was injected before or even up to 4 days after challenge immunization, the appearance of anti-HSA IgG1-forming cell precursors was largely prevented . The effect was most marked with 5 mg or 1 mg of soluble HSA, but impressive partial effects could be seen with as little as 10 micrograms of HSA if administered before challenge immunization . Most of the few clones seen after the higher doses of the toleragen appeared to make antibody of low affinity . The capacity to influence the B-cell pool by soluble antigen administered just 1-2 days before the sudden appearance of IgG1 precursors argues against the totality of the effect being due to T-cell-mediated suppression and in favor of a direct effect on B cells.

Oral Surg Oral Med Oral Pathol, 1990 Feb, 69(2), 169 - 73
Prevalence of oral lesions among HIV-infected intravenous drug abusers and other risk groups; Barone R et al.; To study the prevalence of oral manifestations, we examined 217 patients infected with the human immunodeficiency virus (HIV) . Most of our patients were intravenous drug abusers (IVDAs) (65%) . Other risk categories were represented by IVDAs who were also male homosexuals or bisexuals (11%), male homosexuals and bisexuals (16%), sexual partners of HIV-infected patients (5%), and hemophilic persons and recipients of blood transfusions (3%) . Forty-six patients were women and 171 were men, with a median age of 27 years (range, 11 to 65 years) . At the time of first examination, 38% of patients had asymptomatic HIV infection, 36% had lymphadenopathy syndrome, 17% had AIDS-related complex, and 9% had AIDS . Oral manifestations were observed in 89 (41%) patients . Of these, 15 had asymptomatic infection, 23 had lymphadenopathy syndrome, 27 had AIDS-related complex, and 24 had AIDS . Increasing severity of disease was significantly associated with higher prevalence of oral lesions (p less than or equal to 0.0001) . Candidiasis was the most common oral lesion, followed by hairy leukoplakia . Kaposi's sarcoma, melanotic macules, herpes labialis, condyloma acuminatum, perioral molluscum contagiosum, and bacterial glossitis due to Escherichia coli infection were found in a small number of patients . Results of culture for fungi, available for 203 patients, revealed that 51% of patients with positive Candida cultures had clinical evidence of candidiasis . Our study demonstrates that oral lesions are also important signs of HIV infection among IVDAs . Early diagnosis of these manifestations is becoming increasingly significant in the practice of dentistry.

EMBO J, 1990 Feb, 9(2), 307 - 13
Transcriptional regulation and DNA methylation in plastids during transitional conversion of chloroplasts to chromoplasts; Kobayashi H et al.; During transitional conversion of chloroplasts to chromoplasts in ripening tomato (Lycopersicon esculentum) fruits, transcripts for several plastid genes for photosynthesis decreased to undetectable levels . Run-on transcription of plastids indicated that transcriptional regulation operated as a predominant factor . We found that most of the genes in chloroplasts were actively transcribed in vitro by Escherichia coli and soluble plastid RNA polymerases, but some genes in chromoplasts seemed to be silent when assayed by the in vitro systems . The regulatory step, therefore, was ascribed to DNA templates . The analysis of modified base composition revealed the presence of methylated bases in chromoplast DNA, in which 5-methylcytosine was most abundant . The presence of 5-methylcytosine detected by isoschizomeric endonucleases and Southern hybridization was correlated with the undetectable transcription activity of each gene in the run-on assay and in vitro transcription experiments . It is thus concluded that the suppression of transcription mediated by DNA methylation is one of the mechanisms governing gene expression in plastids converting from chloroplasts to chromoplasts.

J Neurochem, 1990 Feb, 54(2), 703 - 5
Rat brain glutamic acid decarboxylase sequence deduced from a cloned cDNA; Julien JF et al.; A cDNA clone complementary to the rat brain glutamic acid decarboxylase mRNA was isolated from a rat brain cDNA expression library using an antibody specific to the enzyme . The cDNA insert has been shown to direct the synthesis of an active protein in Escherichia coli . In this study, the nucleotide sequence of this clone, which includes the complete coding region, is presented . The predicted protein is 593 amino acids in length . The first 557 residues display a 95% identity when compared with the corresponding cat sequence . However, the deduced amino acid sequence of the carboxy-terminal end of the rat protein, downstream of residue 557, is totally different from the cat, whereas it agrees with a published partial peptidic sequence of the rat protein.

J Lab Clin Med, 1990 Feb, 115(2), 224 - 32
Recombinant human secretory leukocyte-protease inhibitor: in vitro properties, and amelioration of human neutrophil elastase-induced emphysema and secretory cell metaplasia in the hamster; Lucey EC et al.; Studies were undertaken to evaluate the in vitro properties of recombinant human secretory leukocyte-protease inhibitor (rSLPI) that had been made in Escherichia coli in an inactive form and refolded, and to determine whether emphysema and bronchial secretory cell metaplasia, induced in hamsters by intratracheal treatment with human neutrophil elastase (HNE), could be amelio-rated by prior intratracheal instillation of rSLPI . Chromatographic studies indicated that 3H-rSLPI formed a 1:1 complex with HNE . Blockage of the active site of HNE by a covalently bound tetrapeptide chloromethyl ketone reduced complex formation with 3H-rSLPI by more than 98% . Incubation of 3H-rSLPI-HNE complex with alpha 1-protease inhibitor for 3 hours at 37 degrees C decreased the amount of complex compared with incubation in the presence of bovine serum albumin (70% vs 27% dissociated) . The calculated dissociation rate constant was 1.1 x 10(-4) sec-1, indicating a 1.8 hour dissociation half-life . Dissociated 3H-rSLPI retained its ability to recombine with HNE . rSLPI was as effective at inhibiting HNE released from stimulated neutrophils as 3H-rSLPI was at inhibiting purified HNE . Intratracheal pretreatment of hamsters with 3000 micrograms of rSLPI as long as 8 hours before the intratracheal instillation of 250 micrograms of HNE, resulted in significant protection against induction of emphysema and secretory cell metaplasia . One and 4 hours after instillation of rSLPI, 59% and 44%, respectively, of the initial functional activity was recovered in lung lavage supernatant, indicating a half-life of approximately 2 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Anal Cell Pathol, 1990 Feb, 2(2), 115 - 23
Nuclear textural changes preceding endotoxin mediated enhancement of thromboplastin synthesis in human endothelial cells in vitro; Hubert I et al.; Human vascular endothelium plays a major role in hemostatic processes . Human venous endothelial cells (HEC) may promote coagulation by generation of thromboplastin . This tissue factor production is enhanced by bacterial lipopolysaccharide (LPS) . However, the mechanisms of this enhancement remain unclear . In order to quantify by image analysis the nuclear modifications induced by LPS on HEC, umbilical cord vein HEC were cultured in vitro with or without E . coli LPS (10 micrograms/ml) for 0 to 6 h . At the end of culture, tissue factor expression was evaluated by the ability of a cellular extract to shorten the coagulation time of human citrated plasma . Simultaneously, the morphology of LPS treated and control HEC was analysed using a SAMBA 200 cell image processor after Feulgen staining . This analysis indicates that LPS treatment induces nuclear modifications as early as 1 h after culture onset, before any tissue factor expression . This activity appears only between 2 and 4 h of culture with LPS . Our data show that image analysis permits the detection of very early nuclear events in HEC and that these events precede the expression of functional properties which may be implicated in thrombotic processes.

Zhonghua Jie He He Hu Xi Za Zhi, 1990 Feb, 13(1), 15 - 7, 61
{Thromboxane and prostacyclin in endotoxin-induced lung injury}; Hu S; Hypotension, respiratory failure and ARDS-like pulmonary morphological changes were induced by given continuous intravenous infusion of E . coli endotoxin (1-1.5 micrograms.kg/h) in goats . During endotoxin infusion, plasma TXB2 level rose markedly with peak at 0.5 h and then lowered, while 6-keto-PGF1 alpha elevated progressively with its highest level before death, and correlated with blood pressure and survival times negatively . So we suggested that prostacyclin may be one of the factors responsible for hypotension and death in late period of endotoxin induced shock and lung injury.

Indian J Med Sci, 1990 Feb, 44(2), 29 - 32
Sensitivity of the different tests of enterotoxigenicity of Escherichia coli and their correlation; Thawani G et al.; Enterotoxigenicity of E . coli isolates was tested in 136 cases of acute gastroenteritis . Heat labile toxin (LT) produced in-vitro was tested in rabbit ileal loop (RIL); vero cell line and Biktn plate . The results of live cultures were evaluated in RIL . The overall data of these four models were not statistically different . Elaboration of LT in these four models ranged from 14-21.4% . Out of the 20 LT producing strains 14 (70%) also revealed ST . Of the 6 positive reactors on vero cell line, appeared to produce vero toxin (VT) only . Out of 29 LT positive E . coli, 1 (3.45%) and 2 (6.89%) strains respectively revealed colonising factor antigen (CFA) I and II . The high incidence of ETEC showing both LT and ST has been highlighted in the age group 0-4 years, and its impact on nutritional status is discussed.

Genetika, 1990 Feb, 26(2), 197 - 205
{Transmission of amber mutants in phage T4 . V . Positive effect of heat shock proteins on the replication of amber mutants in gene 31}; Nivinskas RG et al.; The effect of growth of Escherichia coli BE, prior to infection, on multiplication of double amber mutant amN54-amNG71 in gene 31, mutant amN131-amNG114 in gene 26 and T4D wild-type at different temperatures has been studied . In the case of gene 31 mutant the increase in phage burst size, along with increase in growth temperature, was only observed . And this dependence seems to have the same character as the known dependence of growth temperature on cellular levels of heat shock proteins . Possibly, the product of gene 31 might be substituted to some extent by some heat shock protein . An antiserum against gene 31 protein immunoprecipitates heat shock protein, the molecular weight of which is close to the molecular weight of gene 31 protein . So, it seems likely that, in addition to supposed ability of this heat shock protein for functional substitution of gene 31 protein, these proteins might have some structural homology as well.

Biochem Cell Biol, 1990 Feb, 68(2), 559 - 66
Expression of mature pulmonary surfactant-associated protein B (SP-B) in Escherichia coli using truncated human SP-B cDNAs; Yao LJ et al.; The present communication documents attempts to produce the mature form of human surfactant-associated protein B (SP-B) by modification of the 5' and 3' regions of the cDNA and expression of the truncated cDNAs after insertion into the vector pKK223-3 . The 5' end of a cDNA for human SP-B (1407 base pairs) was reconstructed through the ligation of synthetic oligonucleotides to an internal PstI site in the 5' region . This construction coded for the initiation of protein synthesis at a Met codon adjacent to a codon for the N-terminal Phe of the mature polypeptide . Variable amounts of the 3' end of the human SP-B cDNA were deleted with mung bean nuclease and exonuclease III . The resulting blunt-ended 3' fragments were then ligated to a synthetic oligonucleotide linker designed to create a stop codon . The modified 5' and 3' ends were ligated to a short PstI-BamHI fragment isolated from the SP-B cDNA and inserted into the expression vector pKK223-3 . In vitro translation of sense mRNAs derived from the truncated SP-B cDNAs yielded oligopeptides of appropriate molecular weights, as indicated by urea - sodium dodecyl sulphate - polyacrylamide gel electrophoresis of either intact or immunoprecipitated reaction mixtures . Expression of SP-B in Escherichia coli was confirmed by Northern blot analysis for the mRNAs corresponding to the truncated cDNAs in appropriately transformed bacteria induced with the galactose analog isopropyl-beta-thiogalactoside . Western blot analysis using rabbit antisera prepared against bovine SP-B confirmed the presence of mature SP-B in lipid extracts of transformed E . coli, but the amounts were very small.(ABSTRACT TRUNCATED AT 250 WORDS)

Zh Mikrobiol Epidemiol Immunobiol, 1990 Feb, (2), 20 - 6
{The conjugational transfer of plasmids to Legionella strains}; Marakusha BI et al.; In experiments on conjugation the transfer of a number of R-plasmids having a wide range of hosts, such as plasmids RP1, R68.45, RP4, N3, RK2, S-a, those having a narrow range of hosts, such as plasmid R64, to strains of different Legionella species was shown . The frequency of transfer varied from 3.1 X 10(-3) to 9.4 X 10(-7) . The fact that the conjugation transfer was confirmed by the reverse transfer of plasmids from Legionella transconjugates to Escherichia coli strain K12, as well as by the detection of the DNA of the transferred plasmid by means of electrophoresis in agar gel . Plasmid RP1 showed different behavior in transconjugates of various Legionella species after several passages in a medium free of antibiotics . In the Legionella strain under study the unstable preservation of plasmid R64 was observed.

Mol Gen Genet, 1990 Feb, 220(3), 400 - 8
Measurement of in vivo expression of nrdA and nrdB genes of Escherichia coli by using lacZ gene fusions; Gibert I et al.; By using a promoter probe plasmid we investigated expression of the linked nrdA and nrdB genes coding for the two different subunits of the ribonucleoside diphosphate reductase enzyme of Escherichia coli . For this reason, nrdA-lacZ, nrdAB-lacZ and nrdB-lacZ fusions were constructed . Results obtained indicate that the nrdB gene has a promoter from which it may be transcribed independently of the nrdA gene . Furthermore, the nrdB gene may also be transcribed from the nrdA promoter . The expression of the nrdB gene is about 14-fold higher from the nrdA promoter than from its own promoter . The induction of both nrdA and nrdB genes by DNA-damaging agents in the wild-type strain as well as in several SOS mutants was also studied; nrdA gene expression was increased by these treatments in RecA+, RecA-, and LexAInd- strains, although in both RecA- and LexAInd- mutants the nrdA gene expression was considerably lower than that in RecA+ cells . nrdB gene expression was stimulated by DNA damage only when its transcription was from the nrdA promoter, but there was no effect when nrdB was transcribed from its own promoter . In addition, the basal level of nrdA-lacZ and nrdAB-lacZ fusions was reduced in strains containing either RecA- and LexAInd- mutations or a multicopy plasmid carrying the lexA+ gene, whereas the presence of a LexA51Def mutation increased the constitutive expression of both fusions . On the contrary, the basal level of the nrdB-lacZ fusion remained constant in all these strains . Together these results indicate that induction of the SOS response enhances expression of the nrd genes from the nrdA promoter.

Mol Gen Genet, 1990 Feb, 220(3), 366 - 72
Regulation of the phosphate regulon of Escherichia coli: properties of phoR deletion mutants and subcellular localization of PhoR protein; Yamada M et al.; The phoR gene is a bifunctional regulatory gene for the phosphate regulon of Escherichia coli . It acts as a negative regulator in the presence of excess phosphate and as a positive regulator with limited phosphate, through modification of PhoB protein . We constructed several phoR genes, with various deletions in the 5' regions, which were regulated by the trp-lac hybrid promoter . The PhoR1084 and PhoR1159 proteins that lack the 83 and 158 N-terminal amino acids, respectively, retained the positive function for the expression of phoA that codes for alkaline phosphatase, but lacked the negative function . The PhoR1263 protein that lacks the 262 N-terminal amino acids was deficient in both functions . An antiserum against PhoR1084 protein was prepared . Western blot analysis of the subcellular fractions obtained by differential centrifugation indicated that the intact PhoR and PhoR1084 proteins are located in the inner membrane and cytoplasmic fractions, respectively . The results suggest that PhoR protein is anchored to the cytoplasmic membrane by the amino-terminal region.

Mol Microbiol, 1990 Feb, 4(2), 315 - 9
Effect of positive redox potentials (greater than +400 mV) on the expression of anaerobic respiratory enzymes in Escherichia coli; Unden G et al.; The expression of fumarate reductase and other enzymes of anaerobic respiration in Escherichia coli was studied as a function of the redox potential (Eh) in the medium . Redox potentials up to +300 mV allowed full expression of fumarate reductase (frd) genes . Higher values resulted in decreased expression . The relationship between Eh and expression of frd could be approximated by the Nernst equation, assuming a redox couple with a midpoint potential Eo' = +400 mV to 440 mV . At Eh values greater than +510 mV (generated anaerobically by hexacyanoferrate(III} the degree of repression was the same as that obtained by O2 . Hexacyanoferrate(III) also caused decreased activities of dimethylsulphoxide (DMSO), nitrite and nitrate reductases . Since expression of these enzymes depends on FNR, the gene activator of anaerobic respiratory genes, it is suggested that the function of FNR is controlled by a redox couple of Eo' = +400 mV to 440 mV.

Mol Microbiol, 1990 Feb, 4(2), 265 - 73
Two precursors of the heat-stable enterotoxin of Escherichia coli: evidence of extracellular processing; Rasheed JK et al.; Expression of the gene of the methanol-soluble, heat-stable enterotoxin of Escherichia coli (STA) allowed the identification by SDS-PAGE of a cell-associated 7500 Dalton STA-related peptide; when similar experiments were performed with a phosphate buffer SDS-PAGE system, an additional Mr 9800 band became apparent . The 9800 Dalton form, pre-pro-STA, accumulated as an intracellular species when the experiments were performed in the presence of the proton ionophore CCCP (carbonylcyanide m-chlorophenylhydrazone); by pulse-chase experiments, it was shown that pre-pro-STA became a periplasmic Mr 7500 pro-STA and this form was chased to the culture supernatant; periplasmic and extracellular pro-STA showed the same electrophoretic mobility . A short time after the pulse, pro-STA was converted extracellularly to mature STA (Mr 4500) . It is proposed that STA is synthesized as pre-pro-STA, a 72-amino-acid peptide that is subsequently cleaved between amino acids 19 and 20 as it is translocated across the inner membrane . The resulting 53-amino-acid pro-STA is first detected in the periplasm and is then secreted to the culture supernatant . Pro-STA is cleaved extracellularly to yield mature STA (Mr 4500).

Mol Microbiol, 1990 Feb, 4(2), 253 - 64
Export and processing analysis of a fusion between the extracellular heat-stable enterotoxin and the periplasmic B subunit of the heat-labile enterotoxin in Escherichia coli; Guzman-Verduzco LM et al.; As an initial approach in the study of the mechanism of secretion of the extracellular heat-stable enterotoxin of Escherichia coli (STA), and in order to use this polypeptide as an extracellular carrier we previously constructed a fusion between the complete STA toxin (pre-pro-STA) and the mature B subunit of the periplasmic heat-labile enterotoxin (LTB); the resulting STA-LTB hybrid was not secreted to the extracellular environment, and cells expressing the hybrid lysed at temperatures above 35 degrees C . In this work we have established that the hybrid is initially detected as pre-pro-STA-LTB and converted to pro-STA-LTB, which lacks the 19 amino acids that share the properties of a signal peptide; the sequenced 17 amino-terminal residues of pro-STA-LTB defined the processing site of pre-pro-STA-LTB at pro-3phe-2ala-1 decreases gln+1 . This process was sensitive to an energy uncoupler (CCCP) and was correlated with translocation of pro-STA-LTB across the inner membrane . Additionally, we are able to show that although pre-pro-STA-LTB is processed at 37 degrees C and 29 degrees C, it is more efficiently processed at the latter temperature . At 37 degrees C, pro-STA-LTB was poorly released into the periplasm, resulting in accumulation of this protein, pre-pro-STA-LTB, and pre-beta-lactamase in the inner membrane, and in cell lysis . In contrast, at 29 degrees C pro-STA-LTB was localized in the periplasm and in the inner membrane, and pre-pro-STA-LTB and pre-beta-lactamase did not accumulate; however, translocation of periplasmic pro-STA-LTB across the outer membrane still did not occur, and a second processing step that would eliminate the pro segment from pro-STA-LTB was never observed . Thus, the fusion of pre-pro-STA and LTB resulted in a polypeptide that, while incompatible with secretion to the extracellular medium, is exported to the periplasm in a temperature-conditional fashion . This latter observation is consistent with an STA secretion pathway whereby pre-pro-STA is first processed to periplasmic pro-STA by the removal of a 19-amino-acid signal peptide.

Mol Microbiol, 1990 Feb, 4(2), 231 - 43
Nucleotide sequence and expression of an operon in Escherichia coli coding for formate hydrogenlyase components; Bohm R et al.; An 8kb segment of DNA from the 58/59 min region of the E . coli chromosome, which complements the defect of a mutant devoid of hydrogenase 3 activity, has been sequenced . Eight open reading frames were identified which are arranged in a transcriptional unit; all open reading frames were transcribed and translated in vivo in a T7 promoter/polymerase system . Analysis of the amino acid sequences derived from the nucleic acid sequences revealed that one of them, open reading frame 5 (ORF5), exhibits significant sequence similarity to conserved regions of the large subunit from Ni/Fe hydrogenases . Two of the open reading frames (orf2, orf6) code for proteins apparently carrying iron-sulphur clusters of the 4Fe/4S ferredoxin type . The product of one of the open reading frames, orf7, displays extensive sequence similarity with protein G from the chloroplast electron transport chain . ORF3 and ORF4, on the other hand, are extremely hydrophobic proteins with nine and six putative transmembrane helices, respectively . Over a limited hydrophilic sequence stretch, bordered by putative transmembrane areas, ORF3 and ORF4 exhibit homology with subunits 4 and 1 of mitochondrial and plastid NADH-ubiquinol oxidoreductases, respectively . The operon described, therefore, appears to comprise genes for redox carriers linking formate oxidation to proton reduction and for a hydrogenase of hitherto unique composition.

Mol Microbiol, 1990 Feb, 4(2), 169 - 87
Mapping of sequenced genes (700 kbp) in the restriction map of the Escherichia coli chromosome; Medigue C et al.; This paper describes software (written in Pascal and running on Macintosh computers) allowing localization of unknown DNA fragments from the Escherichia coli chromosome on the restriction map established by Kohara et al . (1987) . The program identifies the segment's map position using a restriction pattern analysis obtained with all, or some, of the eight enzymes used by Kohara et al . (1987) . Therefore, the sequenced genes available in the EMBL library may be localized on the E . coli chromosome restriction map . This allowed correction of the map (mainly by introducing missing sites in the published maps) at the corresponding positions . Analysis of the data indicates that there is only a very low level of polymorphism, at the nucleotide level, between the E . coli K12 strains used by the various laboratories involved in DNA sequencing . The program is versatile enough to be used with other genomes.

Genes Dev, 1990 Feb, 4(2), 277 - 86
Carboxy-terminal determinants of intracellular protein degradation; Parsell DA et al.; Using the amino-terminal domain of lambda repressor as a model system, we show that residues in an unstructured region at the extreme carboxyl terminus of the protein are important for determining its proteolytic susceptibility in Escherichia coli . Nonpolar amino acids are destabilizing when placed at the 5 carboxy-terminal residue positions, whereas charged and polar residues are stabilizing . The stabilizing effect of a single charged residue is greatest when it is at the terminal position and diminishes with increasing distance from the carboxyl terminus . The position of destabilizing sequences with respect to the free carboxyl terminus is important for their effect, but their distance from the folded portion of the protein is not important . Specific degradation of proteins with nonpolar carboxyl termini has been reconstituted in vitro using a partially pure, soluble fraction . This degradation is not ATP-dependent . Moreover, amino-terminal domain variants with nonpolar carboxy-terminal residues are still rapidly degraded in strains that are deficient in proteolysis of abnormal proteins . These data suggest that the degradation of amino-terminal domain variants with nonpolar carboxy-terminal residues involves proteolytic components distinct from those known to be important for the turnover of unfolded proteins in E . coli.

Chem Pharm Bull (Tokyo), 1990 Feb, 38(2), 474 - 6
Inhibitory effects of galloylglucose on nicotinamide adenine dinucleotide dehydrogenases of the aerobic respiratory chain of Escherichia coli; Konishi K et al.; The effects of pentagalloylglucose (1,2,3,4,6-penta-O-galloyl-beta-D-glucose) on the aerobic electron transport system of Escherichia coli were studied . The activity of nicotineamide adenine dinucleotide (NADH) reductase was inhibited by pentagalloylglucose, but the activities of succinate dehydrogenase, D-lactate dehydrogenase, and ubiquinol-1 (Q1H2) oxidase were not susceptible to the inhibitor . Because the presence of two kinds of NADH dehydrogenase in respiratory chain of Escherichia coli has been reported, we examined the effect of galloylglucose independently on both NADH dehydrogenases . Pentagalloylglucose is potent and specific inhibitor of both NADH dehydrogenases . One of the NADH dehydrogenases (NADH dh II) is more sensitive to the inhibitor than the other (NADH dh I).

Can J Microbiol, 1990 Feb, 36(2), 131 - 5
Purification of 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli strain 080; Prabha V et al.; Purification studies of 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) (EC 1.1.1.159) from Escherichia coli 080 showed that 1.59-fold purification could be achieved by heating (60 degrees C for 10 min) the ultracentrifuged enzyme preparation, and 6.46-fold purification was achieved by subsequent precipitation with ammonium sulfate . Further purification on Sephadex G-100 gel gave 10.1-fold purification . After pooling and concentrating the active fractions obtained from the Sephadex G-100 filtration, an 11.1-fold purification was achieved using DEAE-cellulose chromatography . The purified enzyme produced a single band on polyacrylamide gel electrophoresis and its molecular weight was determined to be 54,000 . The enzyme was immunogenic and showed immunoprecipitation with homologus antisera.

Trends Biochem Sci, 1990 Feb, 15(2), 53 - 9
Escherichia coli aspartate transcarbamoylase: the molecular basis for a concerted allosteric transition; Kantrowitz ER et al.; Aspartate transcarbamoylase from Escherichia coli has become a model system for the study of both homotropic and heterotropic interactions in proteins . Analysis of the X-ray structures of the enzyme in the absence and presence of substrates and substrate analogs has revealed sets of interactions that appear to stabilize either the 'T' or the 'R' states of the enzyme . Site-specific mutagenesis has been used to test which of these interactions are functionally important . By combining the structural data from X-ray crystallography, and the functional data from site-specific mutagenesis a model is proposed for homotropic cooperativity in aspartate transcarbamoylase that suggests that the allosteric transition occurs in a concerted fashion.

Mol Gen Mikrobiol Virusol, 1990 Feb, (2), 22 - 5
{Construction and analysis of the tularemia pathogen gene library in Escherichia coli}; Zakharenko VI et al.; The library of tularemia causative agent genes cloned on the pHC79 plasmid and the partial clonotek of these agents genes in Escherichia coli cells have been constructed . The immunochemical analysis has revealed seven clones of Escherichia coli harbouring the recombinant plasmids and expressing francisella antigens . The cloned sequences of francisella DNA as well as the recombinant plasmids containing them and coding for francisella antigens are capable of specific hybridization with the DNA from Francisella tularensis strains and Francisella novicida strain U112 . The cloned DNA sequences have the properties of the genetic radiospecific molecular DNA probe.

J Mol Endocrinol, 1990 Feb, 4(1), 61 - 9
Purification and properties of a recombinant DNA-derived ovine growth hormone analogue (oGH1) expressed in Escherichia coli; Wallis OC et al.; An Escherichia coli JM109 clone containing a plasmid, pOGHe101, based on pUC8 and the ovine GH (oGH) cDNA sequence, showed very high expression (up to 25% of total cell protein) of an oGH analogue (oGH1) after induction . oGH1 was found in the particulate fraction of induced bacteria, where electron-dense granules could be seen by electron microscopy . A simple method for the purification of oGH1 is described . The particulate fraction isolated from sonicated bacteria was dissolved in 6M guanidinium chloride containing dithiothreitol . After threefold dilution the proteins were reoxidized by gentle stirring overnight in air . Soluble renatured protein, recovered after dialysis, was further purified by ion-exchange and gel-filtration chromatography . Purified oGH1 had an Mr of 22,000, an isoelectric point of about 6.7 and an N-terminal sequence corresponding to that of oGH, with an extension of eight amino acids replacing the N-terminal alanine . oGH1 behaved similarly to authentic bovine GH in a radioimmunoassay, a radioreceptor assay and a weight-gain assay in hypophysectomized rats . Thus the renatured hormone appears to be correctly folded and the N-terminal extension has little or no effect on biological activity.

Aust Fam Physician, 1990 Feb, 19(2), 194 - 203
Travellers' diarrhoea; Looke DF; Travellers' diarrhoea may affect up to 50 per cent of travellers to developing countries . It is now known to be caused by a large number of different organisms with enterotoxigenic Escherichia coli as the major pathogen . By eating and drinking prudently, travellers can hope to reduce the chances of being affected . They should also be equipped with a strategy of rehydration and specific treatment so that the impact of the illness can be kept to a minimum.

Genet Res, 1990 Feb, 55(1), 1 - 6
A cya deletion mutant of Escherichia coli develops thermotolerance but does not exhibit a heat-shock response; Delaney JM; An adenyl cyclase deletion mutant (cya) of E . coli failed to exhibit a heat-shock response even after 30 min at 42 degrees C . Under these conditions, heat-shock protein synthesis was induced by 10 min in the wild-type strain . These results suggest that synthesis of heat-shock proteins in E . coli requires the cya gene . This hypothesis is supported by the finding that a presumptive cyclic AMP receptor protein (CRP) binding site exists within the promoter region of the E . coli htpR gene . In spite of the absence of heat-shock protein synthesis, when treated at 50 degrees C, the cya mutant is relatively more heat resistant than wild type . Furthermore, when heat shocked at 42 degrees C prior to exposure at 50 degrees C, the cya mutant developed thermotolerance . These results suggest that heat-shock protein synthesis is not essential for development of thermotolerance in E . coli.

Eur J Haematol, 1990 Feb, 44(2), 100 - 5
Effect of N-methionine-free, bacterially synthesized recombinant human granulocyte-macrophage colony-stimulating factor in a primate model; Akashi K et al.; We demonstrate the in vivo effects of bacterially synthesized, N-methionine-free recombinant human granulocyte-macrophage colony stimulating factor (rh GM-CSF) using a crab-eating monkey model . Monkeys were treated with cyclophosphamide (60 mg/kg) and administered with rh GM-CSF (30 micrograms/kg/d) subcutaneously (s.c.) for 7 days . Within 12 h, a transient increase of neutrophils (greater than 15.0 x 10(9)/l) was observed, and complete recovery of WBC counts was obtained by d 9 (d 16 in control monkeys) . Neutrophils and eosinophils were absolutely increased (greater than 8 x 10(9)/l) on d 10 . Readministration of rh GM-CSF (30 micrograms/kg/d, s.c.) for 3 d (including control monkeys) revealed absolute increases of neutrophils, eosinophils, monocytes and platelets . A two-fold increase of granulocyte/macrophage colony-forming units was also seen in the bone marrow, while the number of burst-forming units-erythroid was not affected . These data indicate that rh GM-CSF of this type stimulates granulopoiesis and thrombopoiesis in vivo.

Biophys J, 1990 Feb, 57(2), 381 - 3
Comparison of the dynamics of myoglobin in different crystal forms; Phillips GN Jr; Crystals have been grown of "sperm whale" myoglobin produced in Escherichia coli from a synthetic gene and the structure has been solved to 1.9 A resolution . Because of a remaining initiator methionine, this protein crystallizes in a different space group from native sperm whale myoglobin . The three-dimensional structure of the synthetic protein is essentially identical to the native sperm whale protein . However, the crystallographic B-factors for parts of the molecule are quite different in the two crystal forms, and provide a measure of the effect of different packing constraints on the flexibility of the protein . The effect of the packing forces is to reduce the mobility of the protein in the regions of contact and thereby introduce differences in mobilities between the two crystal forms . Discrepancies between mobilities calculated from molecular dynamics simulations and crystallography can be reduced by considering the data from both crystal forms.

Biotechniques, 1990 Feb, 8(2), 178 - 83
A rapid method for site-specific mutagenesis and directional subcloning by using the polymerase chain reaction to generate recombinant circles; Jones DH et al.; Site-specific mutagenesis and directional subcloning were accomplished by using the polymerase chain reaction to generate products that can recombine to form circular DNA . This DNA was transfected into E . coli without phosphorylation of primers, restriction enzyme digestion or ligation . Specifically, the polymerase chain reaction was used to generate products that when combined, denatured and reannealed, form double-stranded DNA with discrete, cohesive single-stranded ends . The generation of these cohesive ends of DNA permits the formation of precise, directional DNA joints without dependence on enzyme restriction sites . The primers were designed such that these cohesive single-stranded ends annealed to form circular DNA . The recombinant of interest was generated following only 14 amplification cycles . These recombinant circles of DNA were directly transfected into E . coli . In the mutagenesis protocol, the desired mutant was obtained at 83%-100% efficiency . Unwanted mutations were not detected, indicating a less than 0.025% nucleotide misincorporation frequency . In the directional subcloning protocol, inserts were positioned precisely in the recipient plasmid and were in the correct orientation . One unwanted mutation was detected after sequencing 900 bases, indicating a 0.11% nucleotide misincorporation frequency . Each manipulation, from setting up for the DNA amplification to transfection into E . coli . can easily be accomplished in one day.

Anticancer Drug Des, 1990 Feb, 5(1), 43 - 53
Recognition by the DNA repair system of DNA structural alterations induced by reversible drug-DNA interactions; Lambert B et al.; Ditercalinium (NSC 335153) was synthesized as a bifunctional DNA intercalator . It is made of two 7-H pyridocarbazole rings joined by a rigid bis-ethyl bispiperidine chain . It binds to DNA with high affinity and elicits anti-tumor activity on a variety of animal tumors . 1H n.m.r . studies of ditercalinium bis-intercalated into d(CpGpCpG)2 have shown that the intercalation process occurs from the large groove of the DNA helix while the two intercalated rings are separated by two base pairs . Because of the linking chain rigidity of ditercalinium, DNA conformation has to be altered to permit the intercalation of the two rings . DNA must be bent toward the minor groove . In E . coli, ditercalinium elicits a specific toxicity on polA strains which is suppressed by an additional uvrA mutation . In vitro, the purified UvrA and UvrB proteins bind to the DNA-ditercalinium complex in an ATP dependent manner . The UvrABC complex induces single-strand nicks, but only when ditercalinium is bound to negatively supercoiled DNA . The life-time of the UvrAB-DNA-ditercalinium complex is greater than 50 min when free ditercalinium concentration is maintained constant in the incubation medium . The cytotoxicity of ditercalinium in E . coli results from the induction of a futile and abortive DNA repair . The reversible ditercalinium-DNA complex mimics a bulky DNA lesion, yet the UvrABC endonuclease is unable to cope with a reversible lesion since it cannot eliminate the causative agent . The interaction of UvrA and UvrB proteins has also been studied with DNA and other DNA-binding drugs forming high-affinity complexes such as distamycin . The Uvr protein recognition process appears to be associated with specific DNA structural alterations . In eukaryotic cells, ditercalinium is concentrated in mitochondria . Mitochondrial DNA is rapidly and totally degraded . Mitochondrial DNA coded proteins being no longer synthesized, the respiratory chain is progressively inactivated . The stimulation of the glycolytic pathway allows the cells to continue growth for several generations . Dihydro-orotate dehydrogenase is located in the inner membrane of mitochondria and its activity is dependent on mitochondria energization . It becomes inactive after ditercalinium treatment . A drop of the pyrimidine pool is then observed . Complementation of treated cells with uridine decreases 10-fold the ditercalinium toxicity . The cellular delayed toxicity of ditercalinium results from the slow induction of a pyrimidineless state associated with the progressive inactivation of mitochondria . The results show that DNA structural alterations induced by reversible drug-DNA complexes can be recognized by DNA repair enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Mol Cell Probes, 1990 Feb, 4(1), 63 - 72
Monoclonal antibodies directed against the rev protein of human immunodeficiency virus type 1; Voll R et al.; The rev (art/trs) protein of human immunodeficiency virus type 1 (HIV-1), a phosphoprotein of 20 K apparent molecular weight, is essential to target the mRNA for virion polypeptides into the cytoplasm . The rev protein was expressed in Escherichia coli as a beta-galactosidase fusion protein with a cleavage site for proteinase factor Xa . The rev-specific fragment was isolated to immunize mice . Five stable hybridoma cell lines were obtained producing monoclonal antibodies that reacted with rev protein in Western blot and ELISA . Using the monoclonal antibodies in indirect immunofluorescence, the rev protein could be localized in the nucleus, mostly in the nucleoli, of Hela cells that were transfected with a eukaryotic rev expression plasmid.

J Microsc, 1990 Feb, 157 ( Pt 2), 187 - 203
Fuzzy sets-based classification of electron microscopy images of biological macromolecules with an application to ribosomal particles; Carazo JM et al.; Pattern recognition methods based on the theory of fuzzy sets are tested for their ability to classify electron microscopy images of biological specimens . The concept of fuzzy sets was chosen for its ability to represent classes of objects that are vaguely described from the measured data . A number of partitional clustering algorithms and an extensive set of cluster-validity functionals (some already reported and some newly developed) have been applied to a test-data set and to two real-data sets of images . One of the real-data sets corresponded to images of the Escherichia coli 50S ribosomal subunits depleted of proteins L7/L12 and the other set to images of the E . coli 70S monosome in the range of overlap views . These two latter sets had been previously studied by another clustering methodology . The new results obtained by the application of fuzzy clustering techniques will be compared to those previously obtained and some conclusions about the consistency of these classifications will be drawn from this comparison.

J Clin Microbiol, 1990 Feb, 28(2), 242 - 5
Molecular typing of nosocomial isolates of Legionella pneumophila serogroup 3; Tram C et al.; In Paris, France, an outbreak of pneumonia due to Legionella pneumophila serogroup 3 was observed in Necker (four cases) and Pitie (six cases) hospitals . Neither the 10 clinical isolates nor 5 tap water isolates from Necker Hospital harbored plasmids . Clinical and environmental serogroup 3 isolates and serogroup 3 reference strain Bloomington 2 were analyzed by chromosomal probe fingerprinting . rRNA, 16S and 23S from Escherichia coli and a randomly cloned 15-kilobase-pair nucleotide sequence from L . pneumophila serogroup 3 were used as probes . All strains tested showed a single pattern after HindIII digestion of DNA and hybridization with the 32P-end-labeled rRNA probe, whereas three patterns were obtained after hybridization with the 32P-labeled 15-kilobase-pair DNA probe . One pattern was given by all clinical and tap water isolates from Necker Hospital, another one was given by all clinical isolates from Pitie Hospital, and a last one was given by reference strain Bloomington 2 . Thus, molecular analysis showed that the two hospital outbreaks of legionellosis were unrelated and could link the outbreak in Necker Hospital to contaminated tap water.

Circ Shock, 1990 Feb, 30(2), 97 - 106
Effects of recombinant human superoxide dismutase on increased lung vascular permeability and respiratory disorder in endotoxemic rats; Schneider J et al.; A 4 hr intravenous infusion of Escherichia coli endotoxin in a total dose of 100 mg/kg produced significant morphological and functional pulmonary alterations in pentobarbitone anesthetized rats . Lung vascular permeability index was increased from 2.11 +/- 0.34 in normal rats to 4.82 +/- 0.65 in untreated endotoxemic rats . Treatment of endotoxemic rats with recombinant human superoxide dismutase (r-HSOD) in doses of 0.1, 0.215, and 0.464 mg/kg.min i.v., infused concomitantly with endotoxin, dose-dependently reduced the permeability index to 3.28 +/- 0.96, 2.83 +/- 0.55 (P less than 0.05), and 2.16 +/- 0.65 (P less than 0.05) . The wet lung weight was 523 +/- 15 and 664 +/- 46 mg/100 g bwt in normal and in untreated endotoxemic rats, respectively . r-HSOD dose-dependently inhibited the endotoxin-induced increase in wet lung weight to 617 +/- 40, 577 +/- 31, and 559 +/- 39 (P less than 0.05) mg/100 g bwt . r-HSOD (0.464 mg/kg.min) did not affect permeability index and wet lung weight in normal, nonintoxicated rats . Endotoxin infusion produced a significant increase in respiratory rate (max . +69%) and blood gas alterations, indicating a hyperventilatory hypocapnia in endotoxemic control rats . Infusion of r-HSOD (0.464 mg/kg.min) significantly inhibited the endotoxin-induced tachypnoe (max . +13%) and blunted the alterations in arterial hydrogen carbonate content and carbon dioxide tension . In conclusion, infusion of r-HSOD dose-dependently and significantly inhibited pulmonary edema formation and hyperventilatory dyspnoe in endotoxemic rats.

Circ Shock, 1990 Feb, 30(2), 81 - 95
Microvascular vasopressin effects during endotoxin shock in the rat; Baker CH et al.; We have demonstrated decreased microvascular sensitivity to norepinephrine during endotoxin shock possibly related to reduced sympathetic receptor activity (Baker et al.: Circ Shock 12:165-176, 1984) . The response to other vascular controls such as arginine vasopressin (AVP) may also be altered . Reactivity of the left cremaster muscle microvessels of pentobarbital anesthetized Wistar rats was studied using videomicroscopy and videodensitometry . Femoral arterial pressure (Pm), dose response curves of vessel diameters to topical arginine vasopressin (10(-15) to 10(-6) M), FITC-albumin mean transit times, and plasma velocities were obtained . Escherichia coli endotoxin (6 mg/kg i.v., LD100) was infused over a 1-hr period . Parameter measurements were repeated at 30 min and 90 min post-endotoxin . Both Pm and plasma velocities progressively decreased . Arteriolar constriction and the mean transit times of FITC-labeled albumin progressively increased . The threshold dose for AVP averaged 10(-9) M at control and decreased to 10(-14) M post-endotoxin . Venular diameters were not altered by AVP . The AVP antagonist did not alter the microvascular diameter response to endotoxin but did block the responses to topical and endogenous AVP since arterial pressure and flow velocity decreased at a significantly greater rate than in rats without antagonist . Plasma AVP levels were significantly increased by endotoxin . Reduced alpha adrenergic sensitivity may unmask the responsiveness to AVP or increase the sensitivity of AVP receptors . Increased endogenous AVP may require a smaller exogenous concentration of AVP for constriction.

Biotechnol Appl Biochem, 1990 Feb, 12(1), 28 - 33
Overproduction and crystallization of tryptophanase from recombinant cells of Escherichia coli; Tani S et al.; We have cloned the tryptophanase structural gene from Escherichia coli B/1t7-A into E . coli K-12 MD55 with a vector plasmid, pBR322 . The cloned cells produced a large amount of the enzyme corresponding to more than 30% of the total soluble protein . With the enzyme obtained by this overproduction system, we have prepared three different crystals of tryptophanase, apo-enzyme, holo-enzyme, and a complex of holo-enzyme and L-alanine, by using polyethylene glycol 4000 or potassium phosphate as a precipitant and the hanging drop method . These single crystals appeared to be suitable for X-ray diffraction analysis.

Am J Physiol, 1990 Feb, 258(2 Pt 2), R443 - 9
Centrally acting vasopressin contributes to endotoxin tolerance; Wilkinson MF et al.; Repeated daily intravenous injections of bacterial endotoxin induce a refractory state to their usual pyrogenic effects . The neuropeptide arginine vasopressin (AVP) has been implicated in natural fever suppression and may be involved in the process of pyrogenic tolerance to intravenous endotoxin . This study was conducted to test this hypothesis . Tolerance was induced by two successive daily intravenous injections of Escherichia coli endotoxin (50 micrograms/kg) into conscious unrestrained rats . This tolerance was maintained, unaltered, after a third or fourth subsequent injection . However, bilateral administration of an AVP V1-receptor antagonist (0.43-4.3 nmol) into the ventral septal area (VSA) of the rat brain markedly enhanced the thermoregulatory response to a third or fourth endotoxin challenge compared with saline controls . The effect of the V1 antagonist was dose related . In contrast, an AVP V2 antagonist (0.43 nmol) bilaterally injected into the VSA did not affect the tolerant reaction to endotoxin . Furthermore, neither saline nor the V1 antagonist significantly affected core temperature when administered within the VSA without subsequent endotoxin . These results are consistent with the hypothesis that AVP acts as an endogenous antipyretic within the VSA during fever . Moreover, the data suggest a possible role for centrally acting vasopressin during pyrogenic tolerance to E . coli endotoxin.

Virus Genes, 1990 Feb, 3(3), 213 - 20
Transient expression assay for qualitative assessment of gene expression by fowlpox virus; Dhawale S et al.; A transient expression assay for fowlpox virus (FPV) was developed to assess the feasibility of using heterologous promoters in FPV and to qualitatively determine relative promoter strength . A transient expression system for FPV has not been reported, and various methods used for transient expression in vaccinia-virus-infected cells produced negative results when used with FPV . Here a successful method for transient expression of E . coli beta-galactosidase in FPV-infected chick embryo fibroblasts is reported . This transient expression assay has been developed to qualitatively assess promoter recognition and gene expression by FPV . It should also prove useful in the identification of promoters from the FPV genomic library and in testing the accuracy of chimeric promoter-gene constructs.

Mol Gen Genet, 1990 Feb, 220(3), 419 - 24
Two different mechanisms for urea action at the LAC and TNA operons in Escherichia coli; Blazy B et al.; Urea, at concentrations which do not interfere with bacterial growth, specifically inhibits the expression of catabolite sensitive operons . To search for the target and the mechanism of urea action we measured lactose (lac) and tryptophanase (tna) specific mRNA synthesis in vivo and in vitro . We show that urea acts by two different mechanisms at these two catabolite sensitive operons, resembling the manner in which catabolite repression regulates lac and tna . At the lac promoter, urea abolishes transcription initiation or blocks an early step in mRNA elongation without interfering with the binding of RNA polymerase and catabolite gene activator protein (CAP) . At the tna promoter, urea does not abolish transcription initiation but could interfere with tnaC translation.

Mol Gen Genet, 1990 Feb, 220(3), 345 - 52
The DNA gyrase of Escherichia coli participates in the formation of a spontaneous deletion by recA-independent recombination in vivo; Miura-Masuda A et al.; A system for detecting a spontaneous deletion in Escherichia coli was developed and the role of DNA gyrase in deletion formation was studied . A derivative of lambda plac5, lambda AM36, was isolated in which whole pBR322 DNA was inserted in the lacZ gene and 227 bp of the lac gene duplicated at both sides of the pBR322 DNA . E . coli lac- strains lysogenized by lambda AM36 had a Lac- phenotype and segregated Lac+ revertants . Sequence analyses showed that the revertant was formed by a deletion that eliminated the inserted pBR322 DNA and one copy of the duplicated segments . The frequency of lac+ revertant formation was independent of recA function, was increased by oxolinic acid, an inhibitor of DNA gyrase, but was not increased in a lysogen of a nalidixic acid-resistant derivative . The reversion frequencies of temperature sensitive mutants of gyrA gene are 10 to 100 times lower than that of the wild-type strain . These results indicate that the DNA gyrase of E . coli participated in the in vivo deletion formation resulting from the direct repeats.

Mol Biol Med, 1990 Feb, 7(1), 59 - 72
Characterization of the humoral immune response to genital papillomaviruses; Galloway DA et al.; Human papillomaviruses (HPVs) have been detected in a wide range of proliferative lesions of squamous epithelium . A number of HPV types are associated with lesions of the genital tract . Some types (HPV types 6 and 11) are detected frequently in benign genital warts (condylomata acuminata); other types (HPV types 31, 33, 42 and others) are associated with dysplastic lesions of the uterine cervix; and certain HPV types (HPV types 16 and 18) are found in a high proportion of squamous cell carcinomas of the cervix and vulva . However, all of these HPV types have been detected also in normal epithelium . To date, investigators have relied primarily upon the detection of viral DNAs in clinical specimens as evidence of HPV infections . Such assays cannot determine whether past infections with HPVs have occurred which have subsequently resolved . Latent infections and current infections might also evade detection because of sampling errors or because of suboptimal sensitivity of DNA detection methods . Efforts to develop HPV serological assays have been hampered by the lack of appropriate viral antigens, since HPVs cannot be propagated in tissue culture and virions are not abundant in infected human tissues . Using HPV-encoded proteins expressed in Escherichia coli, we developed assays to measure human antibodies that react with HPV proteins . Human antibodies to late gene products (L1 and L2) of genital-type HPVs were more prevalent than antibodies to early gene products . However, approximately 15% of sera contained antibodies that reacted with the HPV16 E7-encoded protein, a gene product that has been implicated in HPV16-mediated cellular transformation . The human antibodies appeared to be type or "serotype" specific, because the antibodies did not cross-react with homologous proteins encoded by other HPV types . Antibodies to proteins encoded by HPV types 6 or 11 were detected in approximately 70% of adults, while antibodies to proteins encoded by HPV type 16 were found in approximately 50% of adults . Antibody prevalence was not associated with measures of sexual activity . There was also no significant difference between the prevalence of antibodies to HPV types 6 or 16 proteins in children when compared to the antibody prevalence in sexually active adults . These results suggest that infections by genital HPV types are widespread and frequently cause clinically inapparent infections . The viruses have a broad tropism for mucosal epithelium and are likely to be acquired by other modes, as well as by sexual transmission.

Arch Biochem Biophys, 1990 Feb 1, 276(2), 331 - 5
Role of the entC gene in enterobactin and menaquinone biosynthesis in Escherichia coli; Kaiser A et al.; Tn10 mutants of Escherichia coli MC4100 were screened for their inability to grow under iron deficiency and for their inability to grow under anaerobiosis in the presence of fumarate as an electron acceptor . A strain so obtained (E . coli PBB1) lacked the ability to convert chorismic acid to isochorismic acid . This shows that the gene (entC) encoding isochorismate synthase was mutated . E . coli PBB1 did not produce any detectable amounts of menaquinones (vitamin K2) or enterobactin . When supplemented with isochorismic acid this strain produced menaquinones, indicating that isochorismic acid is involved not only in enterobactin but also in menaquinone biosynthesis . The entC gene was isolated and was shown to be part of the enterobactin gene cluster: It was located on a DNA fragment (9 kb in length) which also carried the entA gene . The DNA fragment was identified by restriction site mapping and was compared to a previously published map of the enterobactin gene cluster . The entC gene on this fragment responds not only to conditions (iron deficiency) that stimulate enterobactin biosynthesis but also to anaerobiosis which results in increased isochorismic acid formation and increased menaquinone biosynthesis . We conclude that isochorismic acid, isochorismic synthase, and the gene (entC) encoding this enzyme are involved in catalytic events at a metabolic branch point from which both enterobactin and menaquinones originate.

Mutat Res, 1990 Feb, 243(2), 145 - 9
Non-phenotypic selection of N-methyl-N'-nitro-N-nitrosoguanidine-directed mutation at a predicted hotspot site; Gordon AJ et al.; The striking mutational specificity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) exhibited in the lacI gene in Escherichia coli allows comment on the phenotypic consequences of mutation at specific sequences that are not recovered after MNNG mutagenesis . We predict that the I+ phenotype is maintained when such silent positions are substituted by amino acids whose codons are generated by the MNNG-directed G:C----A:T transition . We chose the mutationally silent Gly200 codon (an MNNG hotspot motif sequence) to test this prediction . Through MNNG mutagenesis we have generated, identified and isolated a G:C----A:T transition at position 627 (5'-G-G-C-3') under non-selective conditions which creates the Gly200----Asp substitution . The I+ phenotype is retained for this altered repressor.

Proc Natl Acad Sci U S A, 1990 Feb, 87(3), 1149 - 53
Infection of cultured central nervous system neurons with a defective herpes simplex virus 1 vector results in stable expression of Escherichia coli beta-galactosidase; Geller AI et al.; We have developed a defective herpes simplex virus (HSV) vector system that permits the introduction of virtually any gene into mammalian central nervous system neurons . The prototype vector, pHSVlac, contains a transcription unit that places the Escherichia coli lacZ gene under the control of the HSV-1 immediate early 4/5 promoter . pHSVlac was propagated using the HSV-1 temperature-sensitive mutant ts K as helper virus . Infection of rat neurons in primary culture derived from various regions throughout the central nervous system, including spinal cord, cerebellum, thalamus, basal ganglia, hippocampus, occipital cortex, temporal cortex, and frontal cortex, resulted in stable expression of high levels of beta-galactosidase for at least 2 weeks, without cell damage . Since other genes can be expressed from pHSVlac, HSV-1 vectors may prove useful for delivery of genes into central nervous system neurons for studies on nervous system physiology or to perform gene therapy for neurological conditions.

Proc Natl Acad Sci U S A, 1990 Feb, 87(3), 1018 - 22
Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base; Otto H et al.; Photocycle and flash-induced proton release and uptake were investigated for bacteriorhodopsin mutants in which Asp-85 was replaced by Ala, Asn, or Glu; Asp-212 was replaced by Asn or Glu; Asp-115 was replaced by Ala, Asn, or Glu; Asp-96 was replaced by Ala, Asn, or Glu; and Arg-82 was replaced by Ala or Gln in dimyristoylphosphatidylcholine/3-{(3-cholamidopropyl)dimethylammonio}-1- propanesulfonate micelles at pH 7.3 . In the Asp-85----Ala and Asp-85----Asn mutants, the absence of the charged carboxyl group leads to a blue chromophore at 600 and 595 nm, respectively, and lowers the pK of the Schiff base deprotonation to 8.2 and 7, respectively, suggesting a role for Asp-85 as counterion to the Schiff base . The early part of the photocycles of the Asp-85----Ala and Asp-85----Asn mutants is strongly perturbed; the formation of a weak M-like intermediate is slowed down about 100-fold over wild type . In both mutants, proton release is also slower but clearly precedes the rise of M . The amplitude of the early (less than 0.2 microseconds) reversed photovoltage component in the Asp-85----Asn mutant is very large, and the net charge displacement is close to zero, indicating proton release and uptake on the cytoplasmic side of the membrane . The data suggest an obligatory role for Asp-85 in the efficient deprotonation of the Schiff base and in the proton release phase, probably as proton acceptor . In the Asp-212----Asn mutant, the rise of the absorbance change at 410 nm is slowed down to 220 microsecond, its amplitude is small, and the release of protons is delayed to 1.9 ms . The absorbance changes at 650 nm indicate perturbations in the early time range with a slow K intermediate . Thus Asp-212 also participates in the early events of charge translocation and deprotonation of the Schiff base . In the Arg-82----Gln mutant, no net transient proton release was observed, whereas, in the Arg-82----Ala mutant, uptake and release were reversed . The pK shift of the purple-to-blue transition in the Asp-85----Glu, Arg-82----Ala, and Arg-82----Gln mutants and the similarity in the photocycle and photoelectrical signals of the Asp-85----Ala, Asp-85----Asn, and Asp-212----Asn mutants suggest the interaction between Asp-85, Arg-82, Asp-212, and the Schiff base as essential for proton release.

Mol Cell Biol, 1990 Feb, 10(2), 625 - 33
Hormonal induction of transfected genes depends on DNA topology; Pina B et al.; Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thymidine kinase promoter of herpes simplex virus and the reporter gene chloramphenicol acetyltransferase of Escherichia coli respond to glucocorticoids and progestins when transfected into appropriate cells . In the human mammary tumor cell line T47D, the response to progestins, but not to glucocorticoids, is highly dependent on the topology of the transfected DNA . Although negatively supercoiled plasmids respond optimally to the synthetic progestin R5020, their linearized counterparts exhibit markedly reduced progestin inducibility . This is not due to changes in the efficiency of DNA transfection, since the amount of DNA incorporated into the cell nucleus is not significantly dependent on the initial topology of the plasmids . In contrast, cotransfection experiments with glucocorticoid receptor cDNA in the same cell line show no significant influence of DNA topology on induction by dexamethasone . A similar result was obtained with fibroblasts that contain endogenous glucocorticoid receptors . When the distance between receptor-binding sites or between the binding sites and the promoter was increased, the dependence of progestin induction on DNA topology was more pronounced . In contrast to the original plasmid, these constructs also revealed a similar topological dependence for induction by glucocorticoids . The differential influence of DNA topology is not due to differences in the affinity of the two hormone receptors for DNA of various topologies, but probably reflects an influence of DNA topology on the interaction between different DNA-bound receptor molecules and between receptors and other transcription factors.

J Bacteriol, 1990 Feb, 172(2), 802 - 7
Nucleotide sequence and transcriptional analysis of the Escherichia coli agp gene encoding periplasmic acid glucose-1-phosphatase; Pradel E et al.; The nucleotide sequence of the agp gene, which encodes a periplasmic glucose-1-phosphatase, was determined . The deduced amino acid sequence corresponds to a 413-amino-acid-residue polypeptide with a typical hydrophobic signal sequence of 22 amino acids . The mature protein lacks the N-terminal signal peptide and has a calculated Mr of 43,514 . Its promoter was defined by primer extension of the mRNA made in vivo . Like many genes under positive control, its -35 promoter region does not match the consensus . The agp gene is both preceded and followed by transcription termination signals, so it appears to be transcribed as a single unit.

J Bacteriol, 1990 Feb, 172(2), 538 - 47
Analysis and sequence of the speB gene encoding agmatine ureohydrolase, a putrescine biosynthetic enzyme in Escherichia coli; Szumanski MB et al.; The speB gene of Escherichia coli encodes the enzyme agmatine ureohydrolase (AUH) . AUH catalyzes the hydrolysis of agmatine to urea and putrescine in one of the two polyamine biosynthetic pathways in E . coli . Sequencing of a 2.97-kilobase-pair fragment of the E . coli chromosome containing speB revealed the presence of three intact open reading frames (ORFs), ORF1 and ORF2 on one strand and ORF3 on the opposite strand, as well as a truncated ORF, ORF4, which terminated 92 kilobase pairs upstream from ORF3 . ORF3 contained the coding sequence of the speB gene, as confirmed by complementation analysis . Two ORF3 transcripts were detected: a shorter transcript that included only ORF3 and a longer transcript that included both ORF3 and ORF4 . The short transcript was abundantly expressed when the ORF4 sequences were deleted, but when ORF4 and its upstream sequences were present, the polycistronic message predominated and the amount of the monocistronic message was drastically reduced . The promoter from which the shorter transcript was produced contained a TATACT sequence at position -12, but sequences upstream from the -12 position seemed to be irrelevant for promoter activity . The predicted amino acid sequence of AUH contained three regions of high homology to the arginases of yeasts, rats, and humans.

J Bacteriol, 1990 Feb, 172(2), 1035 - 42
Analysis of protein localization by use of gene fusions with complementary properties; Manoil C; This report describes a new transposon designed to facilitate the combined use of beta-galactosidase and alkaline phosphatase gene fusions in the analysis of protein localization . The transposon, called TnlacZ, is a Tn5 derivative that permits the generation of gene fusions encoding hybrid proteins carrying beta-galactosidase at their C termini . In tests with plasmids, TnlacZ insertions that led to high cellular beta-galactosidase activity were restricted to sequences encoding either cytoplasmic proteins or cytoplasmic segments of a membrane protein . The fusion characteristics of TnlacZ are thus complementary to those of TnphoA, a transposon able to generate alkaline phosphatase fusions whose high-activity insertion sites generally correspond to periplasmic sequences . The structure of TnlacZ allows the conversion of a TnlacZ fusion into the corresponding TnphoA fusion (and vice versa) through recombination or in vitro manipulation in a process called fusion switching . Fusion switching was used to generate the following two types of fusions with unusual properties: a low-specific-activity beta-galactosidase-alkaline phosphatase gene fusion and two toxic periplasmic-domain serine chemoreceptor-beta-galactosidase gene fusions . The generation of both beta-galactosidase and alkaline phosphatase fusions at exactly the same site in a protein permits a comparison of the two enzyme activities in evaluating the subcellular location of the site, such as in studies of membrane protein topology . In addition, fusion switching makes it possible to generate gene fusions whose properties should facilitate the isolation of mutants defective in the export or membrane anchoring of different cell envelope proteins.

Infect Immun, 1990 Feb, 58(2), 449 - 55
Binding of collagens to an enterotoxigenic strain of Escherichia coli; Visai L et al.; An enterotoxigenic strain of Escherichia coli, B34289c, has been shown to bind the N-terminal region of fibronectin with high affinity (G . Froman, L . M . Switalski, A . Faris, T . Wadstrom, and M . Hook, J . Biol . Chem . 259:14899-14905, 1984) . We now report that this strain also binds collagen . The binding of 125I-labeled type II collagen to bacteria was time dependent and reversible . Bacteria expressed a limited number of collagen receptors (2.2 x 10(4) per cell) and bound collagen with a Kd of 20 nM . All collagen types tested (I to V) as well as all tested cyanogen bromide-generated peptides {alpha 1(I)CB2, alpha 1(I)CB3, alpha 1(I)CB7, alpha 1(I)CB8, and alpha 2(I)CB4} were recognized by bacterial receptors, as demonstrated by the ability of these proteins to inhibit the binding of 125I-labeled collagen to bacteria . Of several unlabeled proteins tested in competition experiments, fibronectin and its N-terminal region strongly inhibited binding of the radiolabeled collagen to E . coli cells . Conversely, collagen competed with an 125I-labeled 28-kilodalton fibronectin fragment for bacterial binding . Collagen bound to bacteria could be displaced by excess amounts of either unlabeled fibronectin or its N-terminal fragment . Similarly, collagen could displace 125I-labeled N-terminal peptide of fibronectin bound to the bacterial cell surface . Bacteria grown at 41 degrees C or in the presence of glucose did not express collagen or fibronectin receptors . These results indicate the presence of specific binding sites for collagen on the surface of E . coli cells and furthermore that the collagen and fibronectin binding sites are located in close proximity, possibly on the same structure.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1990 Feb, 12(1), 19 - 24
{Synthesis, cloning and expression of human alpha-atrial natriodiuretic peptide gene in yeast system}; Lin H; Human alpha-ANP was synthesised using a yeast system . In order to obtain correct expression of the human alpha-ANP gene, a few bases on one strand were substituted by the corresponding yeast sequence, but those bases were not replaced in the complementary so a few unpaired bases existed . The gene was cloned into a shuttle vector (with alpha-factor) . The recombinants containing different human alpha-ANP DNA sequences were separated by BglII restriction analysis . We compared the expression level of human alpha-ANP from two kinds of genes and the results showed that the ANP expression level by the gene containing yeast sequences was lower (0.5-0.7 mg/L) than that expressed by the original gene (0.8-1.0 mg/L).

J Bioenerg Biomembr, 1990 Feb, 22(1), 27 - 38
Regulatory proteins of F1F0-ATPase: role of ATPase inhibitor; Hashimoto T et al.; An intrinsic ATPase inhibitor inhibits the ATP-hydrolyzing activity of mitochondrial F1F0-ATPase and is released from its binding site on the enzyme upon energization of mitochondrial membranes to allow phosphorylation of ADP . The mitochondrial activity to synthesize ATP is not influenced by the absence of the inhibitor protein . The enzyme activity to hydrolyze ATP is induced by dissipation of the membrane potential in the absence of the inhibitor . Thus, the inhibitor is not responsible for oxidative phosphorylation, but acts only to inhibit ATP hydrolysis by F1F0-ATPase upon deenergization of mitochondrial membranes . The inhibitor protein forms a regulatory complex with two stabilizing factors, 9K and 15K proteins, which facilitate the binding of the inhibitor to F1F0-ATPase and stabilize the resultant inactivated enzyme . The 9K protein, having a sequence very similar to the inhibitor, binds directly to F1 in a manner similar to the inhibitor . The 15K protein binds to the F0 part and holds the inhibitor and the 9K protein on F1F0-ATPase even when one of them is detached from the F1 part.

Proc Natl Acad Sci U S A, 1990 Feb, 87(3), 1129 - 33
Central role for the Escherichia coli minC gene product in two different cell division-inhibition systems; de Boer PA et al.; In Escherichia coli, selection of the proper division site at midcell requires the specific inhibition of septation at two other potential division sites, located at each of the cell poles . This site-specific inhibition of septation is mediated by the gene products of the minicell locus (the minB operon) that includes three genes, minC, minD, and minE . In this paper we show that one of the components of this division-inhibition system, the minC gene product, is also an essential component of another division-inhibition system, which is induced by derepression of the dicB gene and leads to inhibition of septation at all potential division sites . The two minC-dependent division-inhibition systems could be functionally distinguished by their different responses to the minE gene product . The results suggest a model in which a common mechanism, mediated by MinC, is responsible for the division block in a class of division-inhibition systems that can be independently activated by different proteins that determine the specific properties of these systems.

J Bacteriol, 1990 Feb, 172(2), 1077 - 84
Improved vector system for constructing transcriptional fusions that ensures independent translation of lacZ; Linn T et al.; An improved vector system has been developed for the in vitro construction of transcriptional fusions to lacZ . The principal feature is an RNaseIII cleavage site inserted between the polylinker cloning site and the promoterless lacZ gene . When these vectors are used to construct transcriptional fusions, the subsequent cleavage of the hybrid mRNA at the RNaseIII site generates an unchanging 5' end for the lacZ mRNA . In contrast to earlier vectors, this feature helps to ensure independent translation of the lacZ mRNA and, thus, the level of beta-galactosidase produced should accurately reflect the frequency of transcription of the upstream DNA sequences . Additional modifications of the vectors include removal of a weak transcriptional terminator between the cloning site and lacZ, insertion of a terminator downstream of lac, and alteration of restriction endonuclease cleavage sites to facilitate the in vitro construction of fusions . Both multicopy plasmid (pTL61T) and single-copy lambda (lambda TL61) vectors have been assembled . These vectors should be generally useful in scanning for transcriptional regulatory signals.

Plant Cell, 1990 Feb, 2(2), 153 - 61
Fungal toxins bind to the URF13 protein in maize mitochondria and Escherichia coli; Braun CJ et al.; Expression of the maize mitochondrial T-urf13 gene results in a sensitivity to a family of fungal pathotoxins and to methomyl, a structurally unrelated systemic insecticide . Similar effects of pathotoxins and methomyl are observed when T-urf13 is cloned and expressed in Escherichia coli . An interaction between these compounds and the membrane-bound URF13 protein permeabilizes the inner mitochondrial and bacterial plasma membranes . To understand the toxin-URF13 effects, we have investigated whether toxin specifically binds to the URF13 protein . Our studies indicate that toxin binds to the URF13 protein in maize mitochondria and in E . coli expressing URF13 . Binding analysis in E . coli reveals cooperative toxin binding . A low level of specific toxin binding is also demonstrated in cms-T and cms-T-restored mitochondria; however, binding does not appear to be cooperative in maize mitochondria . Competition and displacement studies in E . coli demonstrate that toxin binding is reversible and that the toxins and methomyl compete for the same, or for overlapping, binding sites . Two toxin-insensitive URF13 mutants display a diminished capability to bind toxin in E . coli, which identifies residues of URF13 important in toxin binding . A third toxin-insensitive URF13 mutant shows considerable toxin binding in E . coli, demonstrating that toxin binding can occur without causing membrane permeabilization . Our results indicate that toxin-mediated membrane permeabilization only occurs when toxin or methomyl is bound to URF13.

Sci China B, 1990 Feb, 33(2), 188 - 97
Selection of recombinant vaccinia viruses (Tian Tan strain) expressing hepatitis B virus surface antigen by using beta-galactosidase as a marker; Liu GQ et al.; We constructed a plasmid that contains a small piece of DNA with two vaccinia promoters running in opposite directions--a promoter from a late gene encoding an 11 K polypeptide (P11) and a promoter from an early gene encoding 25K (P25) . These promoters were isolated from the Tian Tan strain of vaccinia virus and were flanked by the thymidine kinase (TK) sequence of the same virus . Genes encoding the hepatitis B virus surface antigen (HBsAg) and the Escherichia coli beta-galactosidase (LacZ) were inserted downstream of the 11 K and 25 K promoters respectively so that coexpression plasmids were constructed . Recombinant vaccinia viruses were selected directly by picking blue plaques formed under overlaying agarose medium containing X-gal . HBsAg was expressed to high level by these recombinant viruses . These recombinant viruses showed reduced virulence on rabbit skin and induced anti-HBs after intradermal inoculation of rabbits.

Mol Microbiol, 1990 Feb, 4(2), 295 - 304
Pro-subtilisin E: purification and characterization of its autoprocessing to active subtilisin E in vitro; Ohta Y et al.; The formation of active subtilisin E from pro-subtilisin E requires the removal of the N-terminal pro-sequence of 77 residues . Pro-subtilisin E produced in Escherichia coli using a pINIII-ompA vector was first extracted with 6 M guanidine-HCl and 5 M urea and purified to homogeneity in the presence of 5 M urea . Upon drop dialysis against 0.2 M sodium phosphate buffer (pH 6.2), the purified pro-subtilisin in 5 M urea was processed to active subtilisin of which the N-terminal sequence and migration in SDS-polyacrylamide gel electrophoresis were identical to those of authentic active subtilisin E . This process was found to be very sensitive to the ionic strengths and anions used . Under the optimum conditions (dialysis against 0.5 M (NH4)2SO4 and 1 mM CaCl2 in 10 mM Tris-HCl buffer (pH 7.0) at 4 degrees C for 1 h), approximately 20% of pro-subtilisin E was converted to active subtilisin E . The activation process was not inhibited by Streptomyces subtilisin inhibitor, and pro-subtilisin E in which the active site was mutated (Asp32 to Asn) was unable to be processed under the optimum conditions . These results confirmed the previous hypothesis that the processing of pro-subtilisin occurs by an intramolecular, autoprocessing mechanism.

Proc Natl Acad Sci U S A, 1990 Feb, 87(4), 1511 - 5
Relationship between protein synthesis and concentrations of charged and uncharged tRNATrp in Escherichia coli; Rojiani MV et al.; We have continuously monitored Trp-tRNA(Trp) concentrations in vivo and, in the same cultures, measured rates of protein synthesis in isogenic stringent and relaxed strains . We have also manipulated cellular charged and uncharged {tRNA(Trp)} by two means: (i) the strain used contains a Trp-tRNA synthetase mutation that increases the Km for Trp; thus, varying exogenous Trp varies cellular Trp-tRNA(Trp); and (ii) we have introduced into the mutant strain a plasmid containing the tRNA(Trp) gene behind an inducible promoter; thus, total {tRNA(Trp)} also can be varied depending on length of induction . The use of these conditions, combined with a previously characterized assay system, has allowed us to demonstrate that (i) the rate of incorporation of Trp into protein is proportional to the fraction of tRNA(Trp) that is charged; for any given total {tRNA(Trp)}, this rate is also proportional to the {Trp-tRNA(Trp)}; (ii) uncharged tRNA(Trp) inhibits incorporation of Trp into protein; and (iii) rates of incorporation into protein of at least two other amino acids, Lys and Cys, are also sensitive to {Trp-tRNA(Trp)} and are inhibited by uncharged tRNA(Trp) . Our results are consistent with models of translational control that postulate modulating polypeptide chain elongation efficiency by varying concentrations of specific tRNAs.

Ann Surg, 1990 Feb, 211(2), 130 - 5
Tissue plasminogen activator reverses the deleterious effect of infection on colonic wound healing; Houston KA et al.; Fibrin deposition in response to bacterial peritonitis appears to predispose to residual infection in the peritoneal cavity . Our previous studies have demonstrated that intraperitoneal fibrinolysis using human recombinant tissue plasminogen activator (t-PA) prevented abscess formation in a rat intra-abdominal sepsis model . To investigate the potential adverse side effects of its use in the peritoneal cavity, the effect of t-PA on colonic anastomotic wound healing and on systemic coagulation parameters was examined in the rat . T-PA did not adversely affect colonic healing five and ten days after anastomosis . In animals infected intraperitoneally at the time of the anastomosis, t-PA reversed the inhibition of healing induced by perianastomotic abscesses at five days . This effect was mediated by the ability of t-PA to prevent perianastomotic abscess formation . After intraperitoneal administration, t-PA had no effect on prothrombin and partial thromboplastin times in either uninfected or infected animals and there was no evidence of clinical bleeding related to its use . These studies suggest that intraperitoneal fibrinolysis using t-PA may provide a safe, effective form of adjuvant therapy in the management of fibrinopurulent peritonitis.

Proc Natl Acad Sci U S A, 1990 Feb, 87(3), 862 - 6
Phage shock protein, a stress protein of Escherichia coli; Brissette JL et al.; Filamentous phage infection induces the synthesis of large amounts of an Escherichia coli protein, phage shock protein (Psp), the product of a previously undescribed gene . This induction is due to the phage gene IV protein, pIV, an integral membrane protein . The uninduced level of Psp is undetectable, but when induced by prolonged synthesis of pIV, it can become one of the most abundant proteins in the cell . Psp is also synthesized transiently in response to several stresses (heat, ethanol, and osmotic shock) . High-level synthesis occurs only after extreme treatment . Unlike the members of the heat shock regulon, Psp induction does not require the heat shock sigma factor, sigma 32; some stimuli that elicit sigma 32-dependent heat shock proteins do not induce Psp synthesis . The level of Psp induction after extreme stress is even higher in sigma 32 mutant cells, which are unable to mount a normal heat shock response, suggesting that these parallel stress responses are interrelated.

Proc Natl Acad Sci U S A, 1990 Feb, 87(3), 1037 - 41
In vivo properties of colicin A: channel activity is voltage dependent but translocation may be voltage independent; Bourdineaud JP et al.; The kinetics of K+ efflux caused by colicin A in Escherichia coli-sensitive cells have been investigated by using a K(+)-selective electrode . The order of magnitude of the rate of K+ efflux per colicin molecule was comparable to that of ion channels . The dependence of K+ efflux upon multiplicity, pH, temperature, and membrane potential (delta psi) was determined . The translocation of colicin A from the outer membrane receptor to the inner membrane and insertion into the inner membrane required a fluid membrane, but once inserted, the channel properties showed little dependence upon the state of the lipids . At a given multiplicity, the lag time before the onset of K+ efflux was found to reflect the time required for translocation and/or insertion of colicin into the cytoplasmic membrane . Opening of the channel only occurred above a threshold value of delta psi of 85 +/- 10 and 110 +/- 5 mV at pH 6.8 and 7.8, respectively . Conditions were designed for closing and reopening of the channel in vivo . These conditions allowed us to test separately the delta psi requirements for translocation and channel opening: translocation and/or insertion did not appear to require delta psi . The channel formed in vivo featured properties similar to that of the channel in lipid planar bilayers.

Proc Natl Acad Sci U S A, 1990 Feb, 87(3), 1032 - 6
Expression and purification of the leucine zipper and DNA-binding domains of Fos and Jun: both Fos and Jun contact DNA directly; Abate C et al.; The protein products of the fos and jun protooncogenes interact cooperatively in the form of a heterodimer with the activator protein 1 (AP-1) regulatory element . To characterize the properties of these proteins, we have expressed polypeptides comprised of the dimerization and DNA-binding domains of Fos and Jun in Escherichia coli . The mini-Fos (wbFos) and the mini-Jun (wbJun) proteins were purified to apparent homogeneity by using a nickel affinity chromatography procedure . Purified wbFos and wbJun associated rapidly in vitro and interacted cooperatively with the human metallothionein IIA AP-1-binding site . However, efficient DNA binding of wbJun and wbFos-wbJun complexes required an additional activity present in nuclear extracts . This activity was sensitive to alkylating agents and could be partially mimicked by the presence of reducing and stabilizing agents . DNase I footprinting experiments demonstrated that Jun homodimeric complexes and Fos-Jun heterodimeric complexes interacted with the same site on the human metallothionein IIA gene . Moreover, UV-crosslinking studies demonstrated that Fos and Jun contact DNA directly and that both proteins interacted equivalently with either strand of the AP-1-binding site.

Mol Cell Biol, 1990 Feb, 10(2), 689 - 95
DNA-mediated gene transfer into adult rat hepatocytes in primary culture; Rippe RA et al.; Proliferation-competent and differentiation-competent adult rat hepatocytes in primary culture were investigated for their ability to express reporter genes (firefly luciferase, bacterial chloramphenicol acetyltransferase, and bacterial beta-galactosidase) driven by tumor virus or eucaryotic promoters that vary in transcriptional efficiency and tissue specificity . Supercoiled plasmid DNA molecules were introduced into the cells by the calcium phosphate coprecipitation protocol of C . Chen and H . Okayama (Mol . Cell . Biol . 7:2745-2752, 1987) . Reporter gene expression was virtually restricted to hepatocytes and was efficient (2 to 20% of the cells) . The patterns and absolute levels of reporter gene expression depended on assay conditions employed (plasmid concentration {optimal at 2.4 micrograms of DNA per ml} and duration of exposure {optimal between 5 and 10 h}), culture growth cycle stages (lag, log, or stationary phase), properties and tissue specificity of the promoter(s) tested, and composition (and timing of fluid change) of the culture medium with or without the hepatocyte mitogen human transforming growth factor-alpha . Initial observations suggest that during hepatocellular growth transitions, human transforming growth factor-alpha differentially regulates exogenously introduced promoters associated with hepatocyte-specific function and proliferation . These findings provide a simple, fast, and powerful approach to analyzing the molecular and cellular biology of hepatocyte growth control.

Plant Mol Biol, 1990 Feb, 14(2), 147 - 61
Nucleotide sequence of Citrus limon 26S rRNA gene and secondary structure model of its RNA; Kolosha VO et al.; The complete nucleotide sequence of Citrus limon 26S rDNA has been determined . The sequence has been aligned with large ribosomal RNA (L-rRNA) sequences of Escherichia coli, Saccharomyces cerevisiae and Oryza sativa . Nine extensive expansion segments in dicot 26S rRNA relative to E . coli 23S rRNA have been identified and compared with analogous segments of monocot, yeast, amphibian and human L-rRNAs . A secondary structure model for lemon 26S rRNA has been derived based on the refined model of E . coli 23S rRNA . It has been compared with other eukaryotic L-rRNAs models in terms of location of functionally important regions . Origin and evolution of L-rRNA expansion segments are discussed.

Semin Cell Biol, 1990 Feb, 1(1), 37 - 45
A mitochondrial chaperonin: genetic, biochemical, and molecular characteristics; Hallberg RL; Mitochondria contain a matrix-localized protein complex composed of subunits homologous to the E.coli protein groEL . As with groEL in E.coli, the nuclear gene coding for the mitochondrial protein is essential for cell survival and the accumulation of the protein is elevated at heat shock-inducing temperatures . Biochemical analyses of wild type and mutant yeast strains have shown that this protein, hsp60, is required for the correct folding and assembly of newly imported, mitochondrially-targeted proteins . There is evidence suggesting a mandatory interaction between hsp60 and many imported, as well as mitochondrially-synthesized, proteins . The implications of these and other data are discussed.

Semin Cell Biol, 1990 Feb, 1(1), 1 - 9
The molecular chaperone concept; Ellis RJ; Molecular chaperones are a ubiquitous family of cellular proteins which mediate the correct folding of other polypeptides, and in some cases their assembly into oligomeric structures, but which are not components of those final structures . Known chaperones do not possess steric information for protein folding but inhibit unproductive folding and assembly pathways which would otherwise act as dead-end kinetic traps and produce incorrect structures . Chaperones function by binding specifically and non-covalently to interactive protein surfaces that are exposed transiently during cellular processes such as protein synthesis, protein transport across membranes, DNA synthesis, the recycling of clathrin cages, the assembly of organellar complexes from imported subunits, and stress responses . This binding is reversed under circumstances which favour correct interactions and in some cases ATP hydrolysis is involved in this reversal . Some chaperones bind specifically to a structural feature present in a wide range of unrelated proteins that is accessible only during the early stages of folding . The nature of this structural feature is unknown, but its identification is an important goal of current research . Knowledge of chaperone function may be important for the production of proteins for biotechnological purposes since in some cases chaperones may improve the yield of functional product . It is likely that chaperone diseases exist which result from the failure of certain proteins to fold correctly due to changes in chaperone structure.

New Biol, 1990 Feb, 2(2), 171 - 8
The DNA binding specificity of the Drosophila fushi tarazu protein: a possible role for DNA bending in homeodomain recognition; Nelson HB et al.; Segmentation in Drosophila melanogaster is controlled by a network of interacting genes, many of which encode a homeodomain that confers sequence-specific binding to DNA . One of these, fushi tarazu (ftz), is a transcription factor that regulates a number of segmentation and homeotic genes, including Antennapedia (Antp) . To determine the DNA binding specificity of the ftz homeodomain, we performed DNase I footprint analysis on ftz protein binding sites located near the two Antp promoters using a beta-galactosidase/ftz fusion protein synthesized in E . coli . A consensus sequence for the fusion protein's preferred binding site was derived from 19 sites . The consensus sequence contains an ATTA motif, as do the reported consensus sequences for the engrailed (en), even-skipped (eve), and bicoid (bcd) Drosophila homeodomain proteins . We propose DNA bending as an explanation for the presence of a shared motif between proteins with divergent recognition helices: according to this model, bases in ATTA would not directly contact amino acid side chains of the recognition helix but rather would be necessary for bending of the DNA around the homeodomain, perhaps facilitating important protein-DNA contacts.

Biochimie, 1990 Feb-Mar, 72(2-3), 157 - 67
The role of the mature part of secretory proteins in translocation across the plasma membrane and in regulation of their synthesis in Escherichia coli; MacIntyre S et al.; Presently available data are reviewed which concern the role of the mature parts of secretory precursor proteins in translocation across the plasma membrane of Escherichia coli . The following conclusions can be drawn; i) signals, acting in a positive fashion and required for translocation do not appear to exist in the mature polypeptides; ii) a number of features have been identified which either affect the efficiency of translocation or cause export incompatibility . These are: alpha) protein folding prior to translocation; beta) restrictions regarding the structure of N-terminus; gamma) presence of lipophilic anchors; delta) too low a size of the precursor . Efficiency of translocation is also enhanced by binding of chaperonins (SecB, trigger factor, GroEL) to precursors . Binding sites for chaperonins appear to exist within the mature parts of the precursors but the nature of these sites has remained rather mysterious . Mutant periplasmic proteins with a block in release from the plasma membrane have been described, the mechanism of this block is not known . The mature parts of secretory proteins can also be involved in the regulation of their synthesis . It appears that exported proteins are already recognized as such before they are channelled into the export pathway and that their synthesis can be feed-back inhibited at the translational level.

Microb Pathog, 1990 Feb, 8(2), 91 - 9
Expression of CFA/I fimbriae is positively regulated; Savelkoul PH et al.; Production of the plasmid-coded fimbrial antigen CFA/I of Escherichia coli requires both CFA/I region 1 and CFA/I region 2, which are separated by about 40 kb on the wildtype plasmid . The nucleotide sequence of region 2 was determined and contains an open reading frame (cfa d), encoding a protein of 265 amino acids . The protein has no signal sequence and upon sequence analysis appeared to be a DNA-binding protein . A plasmid was constituted, with a promoterless beta-galactosidase gene preceded by the promoter of region 1 . Introduction of a plasmid, carrying the cfa d gene, into a strain containing this construct enhanced expression of beta-galactosidase by at least five-fold indicating that the cfa d protein was enhancing expression from the promoter of region 1 . The cfa d gene sequence differed at 28 positions from the Rns gene, which encodes a protein that is a positive regulator of the expression of CS1 or CS2 fimbriae . It was shown that the cfa d gene and the Rns gene can functionally substitute each other in regulating fimbrial synthesis.

Clin Exp Immunol, 1990 Feb, 79(2), 209 - 14
Cloning and nucleotide sequence of cDNA for Ki antigen, a highly conserved nuclear protein detected with sera from patients with systemic lupus erythematosus; Nikaido T et al.; Patients with systemic lupus erythematosus (SLE) produce autoantibodies against a variety of nuclear antigens including Ki antigen . Although anti-Ki autoantibodies were found in a significant number of SLE patients, the nature of Ki antigen is poorly characterized . By using anti-Ki serum as a probe we have cloned a bovine cDNA directing the synthesis in Escherichia coli of a polypeptide immunologically indistinguishable from the authentic Ki antigen . A homologous human cDNA was also cloned and its nucleotide sequence predicted the entire primary structure of a novel nuclear protein with a molecular weight of 29 508 and with highly hydrophilic and weakly acidic character . The gene is highly conserved not only in the coding region but also in the 3'-untranslated region . The bacterially produced Ki antigen would be valuable for diagnosis of SLE.

Epidemiol Infect, 1990 Feb, 104(1), 127 - 33
Lack of association of Escherichia coli exhibiting both mannose-resistant haemagglutination and diffuse adherence to HEp-2 cells with acute diarrhoea in children; Cobeljic M et al.; Stool specimens from 631 children with acute diarrhoea and from 277 healthy controls were tested for the presence of non-enteropathogenic, non-enterotoxigenic Escherichia coli strains which mediated mannose-resistant haemagglutination of human erythrocytes (MRHA+) . Fifty-nine (34.9%) of 169 isolated MRHA+ strains but none of 210 MRHA- strains exhibited diffuse adherence (DA+) to HEp-2 cells . DA+ strains were found in 37 (5.9%) children with diarrhoea and in 22 (7.9%) controls . MRHA+/DA+ strains in comparison to MRHA+/DA- strains significantly less frequently expressed P fimbriae (10.7 vs . 73.6%), haemolysin production (12.5 vs . 63.2%), and MRHA of other species erythrocytes (21.4 vs . 84%) . These data demonstrate that E . coli which exhibit the diffuse pattern of adherence to HEp-2 cells also cause MRHA of human erythrocytes . Since these strains were found with similar frequencies in children with and without diarrhoea it seems that DA is not a marker of enteropathogenicity of E . coli.

Am J Vet Res, 1990 Feb, 51(2), 187 - 90
Prevalence of four enterotoxin (STaP, STaH, STb, and LT) and four adhesin subunit (K99, K88, 987P, and F41) genes among Escherichia coli isolates from cattle; Mainil JG et al.; Colony hybridizations with DNA probes for 3 heat-stable (STaP, STaH, and STb) enterotoxins and 1 heat-labile (LT) enterotoxin and for 4 adhesins (K99, F41, K88, 987P) were performed on 870 Escherichia coli isolates to determine pathotypes prevalent among enterotoxigenic E coli (ETEC) isolated from cattle in Belgium . One hundred thirty-two E coli isolates (15.2%) hybridized with probes STaP, K99, and/or F41 . The 5 other probes were not hybridized by E coli isolates . Therefore, only STaP enterotoxin and K99 and F41 adhesins were virulence factors of ETEC isolated from cattle . Two major pathotypes accounted for 95% of the ETEC: STaP+K99+F41+ (67.4%) and STaP+K99+ (27.3%) . The last 5% of probe-positive isolates had STaP+, STaP+F41+, or K99+F41+ minor pathotypes . Of 12 American ETEC isolates also assayed, 7 were positive with STb and/or 987P probes (pathotypes STaP+STb+, STaP+ 987P+, or STaP+STb+987P+) and may be porcine- rather than bovine-specific enteropathogens . The remaining 5 American ETEC isolates belonged to 3 minor pathotypes (STaP+, STaP+F41+, and K99+F41+) also found among Belgian E coli isolates . Such isolates may be derivatives of STaP+K99+F41+ or STaP+K99+ ETEC after in vivo or in vitro loss of virulence genes and/or non-ETEC isolates, which have acquired virulence genes by in vivo transfer.

J Infect Dis, 1990 Feb, 161(2), 343 - 7
Characterization of a new putative colonization factor (CS17) from a human enterotoxigenic Escherichia coli of serotype O114:H21 which produces only heat-labile enterotoxin; McConnell MM et al.; Enterotoxigenic Escherichia coli (ETEC) of serotype O114:H21, which produced only heat-labile enterotoxin (LT), gave mannose-resistant hemagglutination (MRHA) with bovine erythrocytes . One strain, E20738A, was shown to possess fimbriae of approximately 7.5 nm diameter . On SDS-PAGE two possible fimbrial polypeptides of molecular masses 17.5 and 15.5 kDa were seen; the 17.5-kDa band was the most prominent . Loss of LT and MRHA together from strain E20738A was associated with loss of a 100-MDa plasmid . An absorbed anti-strain E20738A serum reacted specifically with the 17.5- and 15.5-kDa polypeptides and bound to the intact fimbriae . This antiserum reacted positively in an ELISA with LT-positive E . coli strains of serogroups O8, O15, O48, O114, and O146 . The antiserum did not react with ETEC carrying known colonization factors . The term coli-surface-associated antigen (CS) 17 has been used to describe the fimbriae.

J Bacteriol, 1990 Feb, 172(2), 1114 - 20
F1C fimbriae of a uropathogenic Escherichia coli strain: genetic and functional organization of the foc gene cluster and identification of minor subunits; Riegman N et al.; The genetic organization of the foc gene cluster has been studied; six genes involved in the biogenesis of F1C fimbriae were identified . focA encodes the major fimbrial subunit, focC encodes a product that is indispensable for fimbria formation, focG and focH encode minor fimbrial subunits, and focI encodes a protein which shows similarities to the subunit protein FocA . Apart from the FocA major subunits, purified F1C fimbriae contain at least two minor subunits, FocG and FocH . Minor proteins of similar size were observed in purified S fimbriae . Remarkably, some mutations in the foc gene cluster result in an altered fimbrial morphology, i.e., rigid stubs or long, curly fimbriae.

Crit Care Med, 1990 Feb, 18(2), 190 - 7
Effect of leukotriene inhibitor LY-171883 on the pulmonary response to Escherichia coli endotoxemia; Gross D et al.; The effect of the leukotriene D4 (LTD4) receptor antagonist, LY-171883, on the respiratory and cardiovascular changes in endotoxemia was studied in 20 unanesthetized sheep . In group 1 (n = 2), 4 mg/kg LY-171883 was injected iv . In group 2 (n = 12), Escherichia coli endotoxin (1 micrograms/kg) was infused iv, and in group 3 (n = 6), 4 mg/kg LY-171883 was administered 15 min before and 30 min after the same dose of endotoxin . Infusion of LY-171883 in group 1 did not alter baseline ventilatory and cardiovascular measurements . A two-phase pulmonary response was observed in group 2: an early pulmonary hypertension phase in which pulmonary artery pressure (PAP) increased from 18.7 to 51.2 mm Hg (p less than .001), with a fall in cardiac index (CI) from 171 to 114 ml/min.kg (p less than .01) . The ratio of peak inspiratory/expiratory flow rate (PIF/PEF) increased from 1.08 to 1.35 (p less than .01) and the respiratory rate from 50 to 70 breath/min (p less than .005) 30 min postendotoxin . The flow rate measured at midexpiration time (V50) decreased from 81% to 25% of its peak expiration (p less than .001) and the airway resistance increased from 3.8 to 32.7 cm H2O/L.sec (p less than .001) . The second permeability phase was characterized by an increase in pulmonary lymph flow (QL) from 8.5 to 35.2 ml/h (p less than .01), a decrease in PaO2 from 76 to 61 torr (p less than .01), and an increase in pulmonary shunt ratio (Qsp/Qt) from 16% to 31% (p less than .005).(ABSTRACT TRUNCATED AT 250 WORDS)

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