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 exhibit