Escherichia coli

J Biochem (Tokyo), 1988 Jul, 104(1), 30 - 4
High-level expression of human BSF-2/IL-6 cDNA in Escherichia coli using a new type of expression-preparation system; Tonouchi N et al.; BSF-2 (B cell stimulatory factor-2/IL-6) is a member of the lymphokine family and responsible for B cell differentiation . Expression plasmids of human BSF-2 cDNA were constructed using a trp promotor/operator and a trpA terminator . In an extract of Escherichia coli HB101 holding "direct" expression plasmid pBSF-2D, activity of BSF-2 was detected, but overproduction was not observed . A "fused" expression system was therefore developed to prepare the recombinant protein . In this system, cDNA was expressed as a fused protein with human IL-2 N-terminal peptide . In the case of the fused BSF-2 expression plasmid, pBSF-2F, inclusion bodies were observed and overproduction of the protein occurred . As this fused protein had a Phe-Arg-Ala sequence at the junction of hIL-2 and BSF-2, it was possible to process mature BSF-2 from the fused BSF-2 by treatment with kallikrein and aminopeptidase P . From 1 liter of E . coli culture, 45 mg of mature BSF-2 was purified; it had a relative biological activity equal to that of natural BSF-2 purified from T cells.

Can J Microbiol, 1988 Jul, 34(7), 905 - 7
Thymineless death in Escherichia coli mutants deficient in the RecF recombination pathway; Nakayama K et al.; Like recF and recQ mutants studied earlier, two other classes of Escherichia coli mutants defective in the RecF conjugal recombination pathway, recJ and recO, were found to be partially resistant to thymineless death . In contrast, a recN mutant, also belonging to the pathway, was indistinguishable from the wild type with respect to thymineless death.

Infection, 1988 Jul-Aug, 16(4), 215 - 21
Polymorphonuclear leucocyte dysfunction during short term metabolic changes from normo- to hyperglycemia in type 1 (insulin dependent) diabetic patients; Kjersem H et al.; Polymorphonuclear leucocyte (PMN) ingestion of particles coated with lipopolysaccharide (LPS) from Escherichia coli was compared to other PMN functions in seven patients with insulin dependent diabetes mellitus (IDDM) during short-term controlled metabolic changes from normo- to hyperglycemia without ketoacidosis . Factors known to interfere with PMN functions were excluded . PMN ingestion of particles coated with both LPS and bovine serum albumin became reduced from normo- to hyperglycemia . PMN motility was impaired in IDDM, but did not seem to be affected by short-term changes in metabolic control . PMN metabolism did not change from normo-to hyperglycemia . Particle-uptake by diabetic PMN is impaired after short term hyperglycemia in the range normally occurring in diabetics in every-day life.

Mol Microbiol, 1988 Jul, 2(4), 531 - 8
The structure of a plasmid of Chlamydia trachomatis believed to be required for growth within mammalian cells; Comanducci M et al.; Sequence analysis of a 7.5 kb DNA plasmid isolated from Chlamydia trachomatis shows 8 open reading frames (ORFs) regularly spaced along most of the sequence . One of these ORFs encodes a 451-amino-acid polypeptide highly homologous to the DnaB protein of Escherichia coli . A region between ORFs 6 and 7 contains a cluster of alternating ATs and a 22 bp sequence tandemly repeated 4 times, suggesting a replication control region . Several ORFs correspond to plasmid-specific polypeptides that have been described . Codons ending with A or T are more frequent, as might be expected from the high A/T content (64%) of the plasmid, and codon usage is similar to that of the C . trachomatis chromosomal gene, omp1L2.

Mol Microbiol, 1988 Jul, 2(4), 497 - 505
The integral membrane protein from a virulent isolate of transmissible gastroenteritis virus: molecular characterization, sequence and expression in Escherichia coli; Britton P et al.; Subgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA clones . Part of a new clone and a previously reported clone were sequenced and used to construct the viral gene for integral membrane protein . A single open reading frame (ORF) encoding a polypeptide of 262 amino acids, relative molecular mass (Mr) 29,459, was identified . The positive identification of the polypeptide as the integral membrane protein was demonstrated by the production in E . coli of a chimaeric protein comprising most of the ORF encoding the Mr 29,459 polypeptide and beta-galactosidase . The chimaeric protein reacted with a specific monoclonal antibody to viral integral membrane protein and antibodies raised against the chimaeric protein immune precipitated the viral protein . Comparison with the sequence of an avirulent isolate indicates amino acid residues that may be important in pathogenicity.

Mol Microbiol, 1988 Jul, 2(4), 473 - 9
Molecular characterization of malQ, the structural gene for the Escherichia coli enzyme amylomaltase; Pugsley AP et al.; The structural gene for the Escherichia coli enzyme amylomaltase, malQ, is the second gene in the malPQ operon . The nucleotide sequence of malQ shows that the gene encodes an Mr 78360 protein close to the experimentally determined Mr of purified amylomaltase (72000-74000) . The malQ initiation codon was identified by sequence analysis of clustered deletions around the 5' end of the gene . One of these deletions removed the first 5 bases from the malQ coding sequence . Strains carrying a plasmid with this truncated malQ gene under lacZ promoter control and out-of-frame with the first four codons of lacZ were Mal- . The Mal+ phenotype could be restored by inserting small, random fragments of E . coli chromosomal DNA into the unique EcoRI site . Nucleotide sequencing showed that the inserts either joined the lacZ and malQ sequences in frame, or contained a new translation start signal and coding sequence in frame with malQ . These results indicate that amylomaltase could be useful as a reporter protein in gene fusion studies.

J Med Virol, 1988 Jul, 25(3), 329 - 37
Low incidence and high titers of antibodies to hepatitis B virus X-protein in sera of Chinese patients with hepatocellular carcinoma; Liang XH et al.; Sera of patients from China with hepatocellular carcinoma (HCC) were tested for the presence of HBc/HBe- and HBx antibodies by immunoblotting using recombinant MS2 or beta gal fusion proteins as substrate . Antibodies against HBx were detected in four out of 68 HBsAg positive and in one out of three HBsAg negative sera, antibodies against HBc/HBe in 52 and two serum samples, respectively . Competition experiments in which sera were preincubated with individual viral proteins synthesized in E . coli were carried out to demonstrate the specificity of signals obtained in immunoblot analyses . In the five anti-HBx positive sera, the antibody titer against X fusion protein was higher than against core fusion protein and in one of these sera anti-x activity could be demonstrated even at a serum dilution of 1:50,000 . These data indicate that X antibodies occur rarely in Chinese patients and are not serodiagnostic for HCC . The high titer of X antibodies in some patients shows that the X protein can be highly immunogenic in vivo . Induction of antibody formation may be triggered by X protein expressed from integrated viral DNA.

Circ Shock, 1988 Jul, 25(3), 197 - 207
Contractile function of aortic smooth muscle in guinea pig endotoxin shock; Kutsky P et al.; Contractile responsiveness of aortic rings from control and endotoxin-shocked guinea pigs was examined in vitro . Aortae were removed 16 h following intraperitoneal administration of 4 mg/kg of Escherichia coli endotoxin or sterile saline . Although no shift in the dose-response curve or maximal contraction to norepinephrine (10(-9)-10(-4) M) was observed, there was an enhanced contractile response to KCl in shock rings . Calcium dose-response curves in both 60 mM and normal (5.4 mM) KCl were shifted upward in shock rings . This upward shift was abolished in normal KCl in the presence of the Ca2+ blocker D 600 (50 microM), suggesting enhanced Ca2+ influx via voltage-dependent Ca2+ channels in shock vascular smooth muscle . A deleterious effect of high cytoplasmic Ca2+ on vascular smooth muscle mitochondrial function may play a role in endotoxin shock.

Mol Cell Biol, 1988 Jul, 8(7), 2837 - 47
Epstein-Barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells; Margolskee RF et al.; Efficient transfection and expression of cDNA libraries in human cells has been achieved with an Epstein-Barr virus-based subcloning vector (EBO-pcD) . The plasmid vector contains a resistance marker for hygromycin B to permit selection for transformed cells . The Epstein-Barr virus origin for plasmid replication (oriP) and the Epstein-Barr virus nuclear antigen gene have also been incorporated into the vector to ensure that the plasmids are maintained stably and extrachromosomally . Human lymphoblastoid cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10(6) to 10(7) independent transformants . Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells . Furthermore, since EBO-pcD plasmids are maintained as episomes at two to eight copies per cell, intact cDNA clones can be readily isolated from transformants and recovered by propagation in Escherichia coli . By using such vectors, human cells have been stably transformed with EBO-pcD-hprt to express hypoxanthine-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2 (CD8) . Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10(6) total clones or one clone of EBO-pcD-Leu-2 per 2 x 10(4) total clones can be recovered intact from the transformed cells . The ability to directly select for expression of very rare EBO-pcD clones and to then recover these episomes should make it possible to clone certain genes where hybridization and immunological screening methods are not applicable but where a phenotype can be scored or selected in human cell lines.

Proc Natl Acad Sci U S A, 1988 Jul, 85(14), 5269 - 73
Alterations of amino acid repeats in the Escherichia coli hemolysin affect cytolytic activity and secretion; Felmlee T et al.; The primary structure of the Escherichia coli hemolysin polypeptide (HlyA) is used to predict intramolecular structures involved in the secretion and cytolytic activity of the molecule . The C-terminal region of HlyA contains a repeated, 8-amino acid chain represented by the consensus sequence Leu-Xaa-Gly-Gly-Xaa-Gly-Asn-Asp . Three in vitro derived mutations of hlyA are described that encode molecules missing various portions of the C-terminal region, including the repeat region . The wild-type and mutated HlyA molecules were analyzed for the ability to be secreted and to lyse erythrocytes . Hemolytic activity absolutely requires the presence of the repeats . The ability of the mutated HlyA molecules to initiate membrane translocation and be secreted required the presence of the C terminus and, to a degree, the repeated amino acid octets.

Proc Natl Acad Sci U S A, 1988 Jul, 85(14), 5146 - 50
Human p53 oncogene contains one promoter upstream of exon 1 and a second, stronger promoter within intron 1; Reisman D et al.; To gain insight into how transcription of the human p53 oncogene is controlled, we characterized the regulatory regions of the gene . A 3.8-kilobase-pair (kbp) EcoRI restriction fragment encompassing the 5' end of the human p53 gene, as well as subfragments generated by restriction digests, was cloned upstream of the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and CAT activity was assayed in extracts of transfected cells . Two types of CAT vectors were used: Epstein-Barr virus oriP-derived constructs that were stably introduced into the human cell lines K562, Raji, and HL-60, and pSV0-CAT-derived constructs that were transiently introduced into the monkey cell line COS . By this approach we have identified two promoters for the human p53 gene . One promoter, p53P1, is located 100-250 bp upstream of the 218-bp noncoding first exon; a second, stronger promoter, p53P2, maps within the first intron . CAT activity and expression of CAT RNA indicate that p53P2 functions up to 50-fold more efficiently than p53P1 . We conclude that the expression of the human p53 gene may be controlled by two promoters and that differential regulation of these promoters may play an important role in the altered expression of the gene in both normal and transformed cells.

Virology, 1988 Jul, 165(1), 262 - 7
Identification of human papillomavirus type 11 E4 gene products in human tissue implants from athymic mice; Brown DR et al.; A new method has recently been described for the growth of human papillomavirus type 11 (HPV11), an agent associated with genital warts, in human tissue xenografts implanted under the renal capsules of athymic nude mice (J . W . Kreider et al., 1986, J . Virol . 59, 369-376; 1987, J . Virol . 61, 590-593) . With this model it is now possible to study productive HPV11 infection under controlled laboratory conditions . To identify proteins encoded by the HPV11 E4 open reading frame in infected implants, we have cloned an HPV11 E4 genomic DNA fragment representing all of the E4 region thought to be expressed in vivo, as evidenced by cDNA cloning and R loop mapping . The cloned HPV11 fragment was expressed in Escherichia coli as a cro-beta-galactosidase E4 fusion protein (Gal-E4 fusion) . Rabbit antibodies raised against the Gal-E4 fusion protein were affinity purified using an HPV11 E1 E4 fusion protein . The E1--E4 protein was synthesized independently by expressing an HPV11 E1--E4 cDNA in E . coli using a second expression vector . Affinity-purified anti-E4 antibodies identified putative E4 proteins of 10 and 11 kDa in both the condylomatous cyst walls and in the desquamated cells in the cavities of HPV11-infected human skin implants from athymic mice . Similar proteins were not detected in uninfected controls . Implications for use of the athymic mouse system are discussed.

Virology, 1988 Jul, 165(1), 200 - 8
Proteins encoded by bovine viral diarrhea virus: the genomic organization of a pestivirus; Collett MS et al.; The genome of bovine viral diarrhea virus (BVDV) contains a single large open reading frame capable of encoding 449 kDa of protein . Short segments from along the length of the molecularly cloned BVDV genome were engineered so as to be expressed as bacterial fusion polypeptides in Escherichia coli . These BVDV analog fusion proteins were used as immunogens to generate a panel of sequence-specific antisera . These antiserum reagents were in turn employed in immunoprecipitation analyses to identify the authentic BVDV protein to which they were directed . The results allowed for the identification and positioning along the genome of BVDV gene products accounting for approximately 83% of the coding capacity of the virus . A preliminary map of the genetic organization of BVDV is presented and discussed.

Proc Natl Acad Sci U S A, 1988 Jul, 85(13), 4799 - 803
Isolation of gene fusions (soi::lacZ) inducible by oxidative stress in Escherichia coli; Kogoma T et al.; Mu dX phage was used to isolate three gene fusions to the lacZ gene (soi::lacZ; soi for superoxide radical inducible) that were induced by treatment with superoxide radical anion generators such as paraquat and plumbagin . The induction of beta-galactosidase in these fusion strains with the superoxide radical generating agents required aerobic metabolism . Hyperoxygenation (i.e., bubbling of cultures with oxygen gas) also induced the fusions . On the other hand, hydrogen peroxide did not induce the fusions at concentrations that are known to invoke an adaptive response . Introduction of oxyR, htpR, or recA mutations did not affect the induction . Two of the fusion strains exhibited increased sensitivity to paraquat but not to hydrogen peroxide . The third fusion strain showed no increased sensitivity to either agent . All three fusions were located in the 45- to 61-min region of the Escherichia coli chromosome.

Proc Natl Acad Sci U S A, 1988 Jul, 85(13), 4677 - 81
Model for how type I restriction enzymes select cleavage sites in DNA; Studier FW et al.; Under appropriate conditions, digestion of phage T7 DNA by the type I restriction enzyme EcoK produces an orderly progression of discrete DNA fragments . All details of the fragmentation pattern can be explained on the basis of the known properties of type I enzymes, together with two further assumptions: (i) in the ATP-stimulated translocation reaction, the enzyme bound at the recognition sequence translocates DNA toward itself from both directions simultaneously; and (ii) when translocation causes neighboring enzymes to meet, they cut the DNA between them . The kinetics of digestion at 37 degrees C indicates that the rate of translocation of DNA from each side of a bound enzyme is about 200 base pairs per second, and the cuts are completed within 15-25 sec of the time neighboring enzymes meet . The resulting DNA fragments each contain a single recognition site with an enzyme (or subunit) remaining bound to it . At high enzyme concentrations, such fragments can be further degraded, apparently by cooperation between the specifically bound and excess enzymes . This model is consistent with a substantial body of previous work on the nuclease activity of EcoB and EcoK, and it explains in a simple way how cleavage sites are selected.

J Bacteriol, 1988 Jul, 170(7), 3281 - 2
Regulation of the Escherichia coli secA gene by protein secretion defects: analysis of secA, secB, secD, and secY mutants; Rollo EE et al.; SecA protein synthesis levels were elevated 10- to 20-fold when protein secretion was blocked in secA, secD, and secY mutants or in a malE-lacZ fusion-containing strain but not in a secB null mutant . An active secB gene product was not required to derepress secA, since SecA levels were elevated during protein export blocks in secB secY and secB malE-lacZ double mutants.

J Bacteriol, 1988 Jul, 170(7), 3262 - 8
Promoter of the Mycoplasma pneumoniae rRNA operon; Hyman HC et al.; RNA transcripts starting from the 5' end of the single Mycoplasma pneumoniae rRNA operon were analyzed by several methods . By primer extension analysis a start site was found 62 nucleotides upstream from the start site of the 16S rRNA . This site was preceded by a putative Pribnow box; however, a defined -35 recognition region was absent . The cloned rRNA operon was transcribed in vitro by using purified RNA polymerase of Escherichia coli . A single start site could be demonstrated within a few nucleotides of the start site found by primer extension analysis of M . pneumoniae transcripts . When fragments from the cloned operon were used as hybridization probes, S1 nuclease mapping yielded a single transcript extending approximately 193 nucleotides upstream from the 16S rRNA start site . The region surrounding this endpoint did not resemble any known promoter sequence . Dot blot hybridization of M . pneumoniae RNA to three oligonucleotides consisting of nucleotides -5 to -21, -38 to -54, and -112 to -132 (from the start of the 16S rRNA gene) indicated that most rRNA transcripts were processed at the stem site preceding the 16S rRNA gene . The majority of the longer precursor transcripts, extending beyond this point, did not extend further upstream to an oligonucleotide consisting of nucleotides -112 to -132 . It was concluded that transcription of the rRNA operon of M . pneumoniae is initiated by a single promoter . The nucleotide sequence of the region is presented.

J Bacteriol, 1988 Jul, 170(7), 3094 - 101
Minicell-forming mutants of Escherichia coli: production of minicells and anucleate rods; Jaffe A et al.; The Escherichia coli minB mutant originally isolated is known to septate at cell poles to form spherical anucleate minicells . Three new minicell-producing mutants were isolated during a screening by autoradiography for chromosome partition mutants giving rise spontaneously to normal-sized anucleate cells . These min mutants were affected close to or in the minB locus . Autoradiography analysis as well as fluorescent staining of DNA showed that in addition to minicells, these strains and the original minB mutant also spontaneously produced anucleate rods of normal size and had an abnormal DNA distribution in filaments . These aberrations were not associated with spontaneous induction of the SOS response . Inhibition of DNA synthesis in these mutants gave rise to anucleate cells whose size was longer than unit cell length, suggesting that the min defect allows septation to take place at normally forbidden sites not only at cell poles but also far from poles . Abnormal DNA distribution and production of anucleate rods suggest that the Min product(s) could be involved in DNA distribution.

J Bacteriol, 1988 Jul, 170(7), 3040 - 5
Construction of a dihydrofolate reductase-deficient mutant of Escherichia coli by gene replacement; Howell EE et al.; The dihydrofolate reductase (fol) gene in Escherichia coli has been deleted and replaced by a selectable marker . Verification of the delta fol::kan strain has been accomplished using genetic and biochemical criteria, including Southern analysis of the chromosomal DNA . The delta fol::kan mutation is stable in E . coli K549 {thyA polA12 (Ts)} and can be successfully transduced to other E . coli strains providing they have mutations in their thymidylate synthetase (thyA) genes . A preliminary investigation of the relationship between fol and thyA gene expression suggests that a Fol- cell (i.e., a dihydrofolate reductase deficiency phenotype) is not viable unless thymidylate synthetase activity is concurrently eliminated . This observation indicates that either the nonproductive accumulation of dihydrofolate or the depletion of tetrahydrofolate cofactor pools is lethal in a Fol- ThyA+ strain . Strains containing the thyA delta fol::kan lesions require the presence of Fol end products for growth, and these lesions typically increase the doubling time of the strain by a factor of 2.5 in rich medium.

J Virol, 1988 Jul, 62(7), 2474 - 82
The product of the bovine papillomavirus type 1 modulator gene (M) is a phosphoprotein; Thorner L et al.; The M gene of bovine papillomavirus type 1 has been genetically defined as encoding a trans-acting product which negatively regulates bovine papillomavirus type 1 replication and is important for establishment of stable plasmids in transformed cells . The gene for this regulatory protein has been mapped in part to the 5' portion of the largest open reading frame (E1) in the virus . We constructed a trpE-E1 fusion gene and expressed this gene in Escherichia coli . Rabbits were immunized with purified fusion protein, and antisera directed against the product were used to identify the M gene product in virus-transformed cells . In this way a polypeptide with an apparent molecular mass of 23 kilodaltons was detected . The virus-encoded product is phosphorylated and can be readily detected by immunoprecipitation assays from cells transformed by the virus . Cells that harbor viral DNA without M as integrated copies do not produce this protein, whereas cells that harbor integrated viral genomes which are defective for another E1 viral gene important for plasmid replication, R, do produce this protein . The protein has an anomalously low electrophoretic mobility . An in vitro translation product of an SP6 RNA product of a sequenced cDNA predicts a molecular mass of 16 kilodaltons for the protein, and this in vitro translation product has an electrophoretic mobility identical to that of the in vivo immunoprecipitated protein . The results of these studies confirm our previous genetic studies which indicated that part of the E1 open reading frame defined a discrete gene product distinct from other putative products which may be encoded by this open reading frame.

J Virol, 1988 Jul, 62(7), 2358 - 65
Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli; Terry R et al.; The gag-pol precursor protein of the avian sarcoma-leukosis virus is processed into three known pol-encoded mature polypeptides; the 95- and 63-kilodalton (kDa) beta and alpha subunits, respectively, of reverse transcriptase and the 32-kDa pp32 protein . The pp32 protein possesses DNA endonuclease activity and is produced from the precursor by two proteolytic cleavage events, one of which removes 4.1 kDa of protein from the C terminus . A 36-kDa protein (p36pol) which retains this C-terminal segment is detectable in small quantities in virions . We have constructed Escherichia coli plasmid clones that express the C-terminal domains of pol corresponding to pp32 and p36 . These proteins have been purified by column chromatographic methods to near homogeneity . No significant differences could be detected in the enzymatic properties of the bacterially produced p32pol and p36pol proteins . Both possess DNA endonuclease activity and, like the pp32 protein isolated from virions, can cleave near the junction of two tandem avian sarcoma-leukosis virus long terminal repeats in double-stranded supercoiled DNA substrates . In the presence of Mg2+, both p32pol and viral pp32 cleave either strand of DNA 2 nucleotides 5' to the junction.

J Virol, 1988 Jul, 62(7), 2243 - 50
Identification and procaryotic expression of the gene coding for the highly immunogenic 28-kilodalton structural phosphoprotein (pp28) of human cytomegalovirus; Meyer H et al.; Human cytomegalovirus contains a structural polypeptide that is 28 kilodaltons in apparent molecular size and is reactive in Western blot (immunoblot) analysis with the majority of human sera . The gene coding for this polypeptide was mapped on the genome of human cytomegalovirus strain AD169 . A monoclonal antibody specific for the 28-kilodalton polypeptide was used to screen a cDNA library constructed from poly(A)+ RNA of human cytomegalovirus-infected cells in the procaryotic expression vector lambda gt11 . Hybridization of cDNA with cosmid and plasmid clones mapped the gene to the HindIII R fragment . The gene was transcribed into a late 1.3-kilobase RNA . The nucleotide sequence of the coding region was determined . Parts of the 28-kilodalton polypeptide were expressed in Escherichia coli as hybrid proteins fused to beta-galactosidase . In Western blots these proteins were recognized by human sera . Antibodies raised against the hybrid proteins reacted specifically with the viral antigen in immunoprecipitations and Western blots . In vitro phosphorylation of HCMV virions and immunoprecipitation showed that the 28-kilodalton polypeptide was phosphorylated.

FEMS Microbiol Immunol, 1988 Jul, 1(2), 109 - 14
A new method for induction of specific polyclonal antibodies using immunostained proteins on nitrocellulose membrane, and HIV 1 core antigen and Escherichia coli verotoxin as examples; Sandkamp O et al.; Two polyclonal antibodies against verotoxin 1 and the core protein p24 of HIV 1 were raised in mice by a new immunization procedure . Both proteins were transferred to nitrocellulose, reacted with polyspecific antisera and the antigen-antibody complexes were then visualized by immunostaining . For preparation of antisera the stained protein bands were cut from the nitrocellulose sheets and implanted subcutaneously into the backs of BALB/c mice, without any adjuvant . A single booster was given 4 weeks later by implanting a second strip . All mice produced high titers of antibody directed against the antigen used for immunization . Thus, antibodies of high specificity can be elicited against protein bands in stained immunoblots.

J Gen Microbiol, 1988 Jul, 134 ( Pt 7), 1795 - 1805
Stability of plasmid sequences in an acute Q-fever strain of Coxiella burnetii; Samuel JE et al.; The rickettsial pathogen Coxiella burnetii undergoes a variation in which virulent isolates (phase 1) become avirulent (phase 2) after repeated passage in a non-immunologically competent host . Biochemically, this variation is associated with a lipopolysaccharide modification and possibly other factors . Genetically, the regions of DNA responsible for phase variation have not been identified . We have sought to determine whether the plasmid identified in acute disease isolates, QpH1, which represents approximately 5% of the coding capacity of this organism is involved in phase variation . Plasmids from phase 1 and phase 2 variants (designated QpH1 and QpH2, respectively) were compared by restriction endonuclease digestion and Southern blot hybridization to determine whether sequence changes in the phase 2 plasmid might account for changes in the virulence of phase 2 organisms compared with that of phase 1 cells . Using over 20 different restriction enzymes, no changes in DNA restriction fragment patterns were detected regardless of whether the phase change occurred during egg or tissue culture passage . The plasmid-specific mRNAs produced from metabolically active, purified cells were identical for each phase type . Using QpH1 or QpH2 DNA as a template, the mRNA produced by an E . coli extract was also identical . Finally, the proteins encoded by either plasmid in an in vitro transcription/translation reaction were identical . These data indicate that within the limits of our analysis, the plasmid DNA from C . burnetii phase variants is structurally and functionally the same and is therefore unlikely to be involved in phase variation.

J Gen Microbiol, 1988 Jul, 134 ( Pt 7), 1747 - 53
Serotype-specific monoclonal antibodies against the H12 flagellar antigen of Escherichia coli; Whitfield C et al.; The flagellar filaments of morphotype E isolates of Escherichia coli characteristically possess an apparent helically arranged sheath structure, surrounding the central core of the filament . Re-examination of the type strains of H-serotypes belonging to morphotype E showed that all but serotype H34 possessed the expected morphology . Heterogeneity was observed in both the diameter of filaments from individual morphotype E strains and in the Mr of individual flagellins . There was no apparent correlation between these two features . Monoclonal antibodies (MAbs) of the IgM class were raised against serotype H12 flagella . In Western immunoblotting and agglutination tests, the MAbs recognized the H12 antigen of six isolates with different O:K antigen combinations . The MAbs were H-serotype-specific, with no significant reaction with the H-antigens of other morphotype E strains . The location of the serotype-specific H12 epitope(s) was studied by immunolabelling with colloidal gold markers . The epitope was surface-exposed and appeared to be helically arranged on the flagellar filament . The pattern of colloidal gold labelling was consistent with the possibility that the H12 serotype-specific epitope resides in the apparent sheath structure.

J Chromatogr, 1988 Jul 1, 444, 47 - 65
High-resolution chromatography of nucleic acids on the Gen-Pak FAX column; Stowers DJ et al.; High-performance liquid chromatography (HPLC) on a Gen-Pak FAX column has been used to separate and purify microgram amounts of single- and double-stranded DNA and RNA molecules . HPLC of mixtures of DNA restriction fragments showed that fragments within the size range 0.125-23.1 kilobase were easily resolved . Supercoiled (form I) plasmid DNA molecules were readily separated from single-stranded circular DNA of the same length and from various DNA conformational isomers including nicked (form II) and linear (form III) species . Topological isomers generated from supercoiled plasmid DNA molecules by DNA topoisomerase I exhibited different retention times than supercoiled molecules . Supercoiled (form I) DNA molecules were resolved from fully relaxed (form IV) molecules . Synthetic oligonucleotides of 74 and 128 nucleotides in length were separated from failure sequences, as well as from other contaminating synthesis products . Single-stranded circular M13mp18 DNA molecules sufficiently pure for use in automated DNA sequencing systems were prepared by HPLC on a Gen-Pak FAX column . HPLC was also used to fractionate linear double-stranded porcine rotavirus genomic RNA fragments into size classes between 0.3 and 3 kilobase . Finally, HPLC of unfractionated Escherichia coli tRNA molecules resolved multiple species . In all cases, HPLC on Gen-Pak FAX was carried out in phosphate or Tris buffers at neutral pH in the presence of sodium chloride . Columns were not damaged by repeated exposure to impure samples, provided they were cleaned frequently with sodium hydroxide and acetic acid . Although procedures for resolution of the various size ranges for each class of DNA and RNA molecules require further optimization, our preliminary data on the separations obtained, the moderate salt concentrations employed, and the durability of the matrix suggest that this column merits further study.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1988 Jul, 269(1), 7 - 14
Modes of action of colicins E4-E7: rates of basic biosyntheses inhibition; Smarda J et al.; The progress of the inhibition of prominent biopolymer syntheses (namely of DNA, RNA and protein synthesis) in cultures of susceptible bacteria Escherichia coli treated with colicins E4, E5, E6 or E7 was followed . The method used was the measurement of incorporation of specific radioactive precursors into an instantly precipitable fraction . All the three syntheses being inhibited successively, progressively and with different urgency during unfolding of the lethal effect of each colicin, emphasis was put on a comparative mathematical analysis of the development of the biosyntheses inhibition rates . In bacteria treated with colicins E4, E5 or E6, protein synthesis was blocked preferentially and most heavily, followed by cessation of DNA synthesis and finally also by a rather slowly proceeding impairment of RNA synthesis . (In E6-treated cells, damage to DNA synthesis started prior to that to protein synthesis.) Thus, the effect of colicins E4, E5 and E6 follows the general pattern of colicin E3 action . Bacteria treated with colicin E7 developed an immediate block of DNA synthesis, soon followed by protein and (again after a significant delay) by RNA synthesis switch-off . Thus the action of colicin E7 is closely related to that of colicin E2 . The presumptive direct targets of these colicins remain to be elucidated.

Cytometry, 1988 Jul, 9(4), 316 - 24
Phagocytosis, intracellular pH, and cell volume in the multifunctional analysis of granulocytes by flow cytometry; Rothe G et al.; Phagocytosis of Escherichia coli K12 strain bacteria was used to measure by flow cytometry the functional activities of human granulocytes in whole blood or buffy coat preparations . In a first measurement, the increase in electric cell volume and acridine orange (AO) green and red fluorescence were used to quantify the degree of phagocytosis . In a second measurement, the intracellular pH and esterase activity of each cell were determined with 1,4-diacetoxy-2,3-dicyanobenzene to obtain information on the metabolic activities during phagocytosis and degradation of bacteria . The DNA of dead cells was simultaneously counterstained with propidium iodide in both assays . The volume, the AO green and red fluorescence, the internal pH, and esterase activity were automatically averaged for all granulocytes or lymphocytes of a measurement . The calculated mean values were transferred into the self-learning database of the DIAGNOS1-program system . The functional granulocyte parameters of normal healthy individuals can be used as reference values for the automated diagnosis of abnormal granulocytes in various infectious disease states . The assays require 1 ml of heparinized whole blood and the results are available within 1 hour.

Mol Biochem Parasitol, 1988 Jul, 30(1), 19 - 26
Schistosoma mansoni: localisation of antigenic regions on the 31 kilodalton diagnostic protein; Felleisen R et al.; Antigenic sites on the 31 kDa diagnostic protein of Schistosoma mansoni (Sm31) were identified using the cDNA fragment H3 cloned in the expression vector pEx34b . This fragment encodes approximately two-thirds of the polypeptide . A set of deletion mutants was generated by the exonuclease Bal31 and the resulting shortened proteins synthesised in Escherichia coli as fusions to MS2 polymerase were tested in Western blots with human schistosomiasis patient sera . Three antigenic regions were identified, one of which was narrowed down by appropriate restriction sites to a sequence specifying only 27 amino acid residues . Examination of a longer MS2 fusion product extending into the N-terminus of the protein, corresponding to the nearly full length Sm31 sequence, revealed that its reactivity in immunoblots is comparable with that of the H3 clone . This suggests that additional antigenic sites, which might be more reactive than those already identified, are absent from the remaining part of the protein.

Eur J Biochem, 1988 Jul 1, 174(4), 585 - 92
Cloning and expression of cDNA for human vascular anticoagulant, a Ca2+-dependent phospholipid-binding protein; Maurer-Fogy I et al.; Based on sequence information from tryptic peptides an almost full-size cDNA coding for the human vascular anticoagulant was isolated from a placental cDNA library and sequenced . The coding region was cloned into an Escherichia coli expression vector and the protein expressed at high levels . The recombinant protein was purified and found to be indistinguishable from its natural counterpart in several biological assays.

Science, 1988 Jul 1, 241(4861), 74 - 9
Aminoacylation of synthetic DNAs corresponding to Escherichia coli phenylalanine and lysine tRNAs; Khan AS et al.; Synthetic DNA oligomers (tDNAs) corresponding to Escherichia coli tRNA(Phe) or tRNA(Lys) have been synthesized with either deoxythymidine (dT) or deoxyuridine (dU) substituted in the positions occupied by ribouridine or its derivatives . The tDNAs inhibited the aminoacylation of their respective tRNAs with their cognate amino acids, but not the aminoacylation of tRNA(Leu) with Leu . In the presence of aminoacyl-tRNA synthetase, species of both a tDNA(Phe) synthesized with a 3' terminal riboadenosine and a tDNA(Lys) containing only deoxynucleotides could be aminoacylated with the appropriate amino acids, although the Michaelis constant Km and observed maximal rate Vmax values for aminoacylation were increased by three- to fourfold and decreased by two- to threefold, respectively . The aminoacylation of synthetic tDNAs demonstrates that the ribose backbone of a tRNA is not absolutely required for tRNA aminoacylation.

Proc Natl Acad Sci U S A, 1988 Jul, 85(13), 4710 - 4
RNase PH: an Escherichia coli phosphate-dependent nuclease distinct from polynucleotide phosphorylase; Deutscher MP et al.; Final trimming of the 3' terminus of tRNA precursors in Escherichia coli is thought to proceed by an exonucleolytic mechanism . However, mutant strains lacking as many as four exoribonucleases known to act on tRNA still grow normally and process tRNA normally . Extracts from such a multiple-RNase-deficient strain accurately mature tRNA precursors exonucleolytically in vitro in a reaction that requires inorganic phosphate . Here we show that this reaction is not due to polynucleotide phosphorylase (PNPase) but, rather, that it is mediated by a phosphate-requiring exonuclease that we have named RNase PH . Purified PNPase is incapable of completely processing tRNA precursors, and extracts from a PNPase- strain retain full activity for phosphorolytic processing . Although both PNPase and RNase PH act in a phosphorolytic manner, they differ substantially in size and substrate specificity . RNase PH has a molecular mass of 45-50 kDa and favors tRNA precursors as substrates . The possible physiological role of RNase PH and the advantages of phosphorolytic processing are discussed.

Proc Natl Acad Sci U S A, 1988 Jul, 85(13), 4667 - 71
Identification of a region in the S1 subunit of pertussis toxin that is required for enzymatic activity and that contributes to the formation of a neutralizing antigenic determinant; Cieplak W et al.; The S1 subunit of pertussis toxin possesses two regions (homology boxes), each spanning 8 residues, that are nearly identical in sequence to similarly located regions in the enzymatically active A fragments of two other ADP-ribosylating toxins: cholera toxin and Escherichia coli heat-labile toxin . This observation suggests a functional role for one or both of these regions in enzymatic activity . We have examined the role of one of these regions, located near the amino terminus of the S1 subunit, by using a high-level recombinant expression system and progressive truncation of the gene sequence encoding the amino terminus of the molecule . A series of six truncated, recombinant proteins were produced at high levels in E . coli and examined for their enzymatic and antigenic properties . The three molecules that lacked most or all of the homology box delimited by amino acid residues 8 and 15 lacked detectable enzymatic activity . All of the three molecules in which the box was retained exhibited detectable activity . Only those recombinant molecules that possessed the homology box reacted with a neutralizing and passively protective monoclonal anti-S1 antibody . These findings identify the region of homology located near the amino terminus of S1 as an apparent enzymatic subsite and a potentially important antigenic determinant.

J Bacteriol, 1988 Jul, 170(7), 3110 - 4
Cyclic AMP-cyclic AMP receptor protein as a repressor of transcription of the spf gene of Escherichia coli; Polayes DA et al.; The spf gene of Escherichia coli encodes an unstable 109-nucleotide RNA, spot 42 RNA; the level of this RNA was reduced three- to fivefold when cells were grown in the presence of 3',5'-cyclic AMP (cAMP) . We show that this regulation occurs through reduction in transcription and depends on both cAMP and the cAMP receptor protein (CRP) but is independent of the de novo protein synthesis . Through deletion analysis of the spf gene promoter, we have identified sequences that are important in the synthesis of spot 42 RNA . Deletion of sequences upstream of -77 completely eliminated the negative control of cAMP-CRP and resulted in high constitutive levels of transcription . This region contained a sequence that both conformed to the consensus binding site for cAMP-CRP in positively regulated promoters and acted as a cAMP-CRP binding site in a gel retardation assay . Deletion of sequences between positions -77 and -60 greatly reduced the level of transcription in the presence or absence of cAMP-CRP, indicating that at least part of this region is a binding site for a positive-acting transcription factor (or RNA polymerase itself) . We propose that the proximity of the two sites defined here allows for the negative control of spf gene transcription by cAMP-CRP . In particular, if only one site at a time can be occupied, the binding of cAMP-CRP would interfere with the binding of a transcription factor.

J Virol, 1988 Jul, 62(7), 2525 - 9
A single 66-kilodalton polypeptide processed from the human immunodeficiency virus type 2 pol polyprotein in Escherichia coli displays reverse transcriptase activity; Le Grice SF et al.; We have cloned the entire pol gene of human immunodeficiency virus type 2 into a high-level Escherichia coli expression system . Induction of cultures containing the recombinant plasmid, p2RTL1, leads to rapid accumulation of polypeptides of 66, 54, and 34 kilodaltons . We have designated the larger polypeptides reverse transcriptase, and we have designated the smaller polypeptide endonuclease . Purification of reverse transcriptase via ion-exchange and affinity chromatography yields the 66-kilodalton polypeptide, with which reverse transcriptase activity is associated . Purified enzyme furthermore displays a higher apparent molecular weight than its counterpart from human immunodeficiency virus type 1.

Nature, 1988 Jun 30, 333(6176), 869 - 71
The structure of trp pseudorepressor at 1.65A shows why indole propionate acts as a trp 'inducer'; Lawson CL et al.; The trp repressor is a small dimeric regulatory protein which controls the expression of three operons in Escherichia coli . The inactive aporepressor protein must bind two molecules of L-tryptophan to form the active repressor . If desamino analogues of L-tryptophan such as indole propionate (IPA) are substituted for L-tryptophan, an inactive pseudorepressor is formed . Because the desamino analogues thus cause derepression of operons under control of the trp repressor, they appear to be 'inducers' . We have determined the crystal structure of the pseudorepressor and refined it to 1.65 A . The molecular structure was compared to that of the nearly isomorphous orthorhombic form of the repressor . Surprisingly, the indole ring of IPA is in the same position as the indole ring of L-tryptophan in the repressor, but is 'flipped over' . As a result, the carboxyl group of IPA is oriented toward the DNA-binding surface of the protein and is in a position where it sterically and electrostatically repels the phosphate backbone of both operator and non-operator DNA . This explains why IPA acts as an apparent trp inducer.

Gene, 1988 Jun 30, 66(2), 295 - 300
Expression and excretion of human pancreatic secretory trypsin inhibitor in lipoprotein-deletion mutant of Escherichia coli; Kanamori T et al.; We constructed a gene coding for the 56-amino acid human pancreatic secretory trypsin inhibitor (PSTI), and ligated it on a plasmid downstream from the trp promoter and the signal peptide sequence of alkaline phosphatase . The resulting plasmid was transfected into a lipoprotein deletion mutant (Escherichia coli JE5505) and the plasmid-carrying cells were induced with 3-indoleacrylic acid . A considerable amount (50 micrograms/ml culture) of the mature PSTI protein was detected in the culture supernatant . The excreted PSTI was identical to the natural PSTI protein with respect to the trypsin-inhibiting activity, the N-terminal and the C-terminal amino acid sequences and the amino acid composition.

Gene, 1988 Jun 30, 66(2), 269 - 78
Highly efficient positive selection of recombinant plasmids using a novel rglB-based Escherichia coli K-12 vector system; Noyer-Weidner M et al.; We have developed pBR328-derived vectors which allow highly efficient positive selection of recombinant plasmids . The system is based on the rglB-coded restriction activity of Escherichia coli K-12 directed against 5-methylcytosine (5mC)-containing DNA . The vectors code for cytosine-specific, temperature-sensitive DNA methyltransferases (ts-Mtases), whose specificity elicits RglB restriction . 5mC-free vector DNA - a prerequisite to allow establishment of such plasmids in cells expressing the RglB nuclease activity - can be prepared from cultures grown at 42 degrees C . At 30 degrees C the vector plasmids are vulnerable to RglB restriction due to the expression of suicidal Mtase activity . Cloning a DNA fragment into the ts-Mtase-coding gene disrupts the lethal methylation and thus permits selection of such recombinant plasmids at 30 degrees C . The standard vector used, pBN73, contains unique recognition sites for nine restriction enzymes within the ts-Mtase-coding gene, which can be used independently or in combination for the construction of recombinant plasmids selectable by the rglB-coded activity . Plasmid pBN74, which carries the determinants for both the ts-Mtase and the RglB nuclease, contains seven unique sites within the ts-Mtase-coding gene . While selection of recombinant plasmids derived from pBN73 obligatorily requires the employment of rglB+ strains, selection of pBN74 derivatives can be performed independent of the E . coli-host genotype . It remains to be elucidated whether positive selection of pBN74-derived recombinant plasmids can also be achieved in hosts other than E . coli . Plasmids pBN73, pBN74 and the recombinants are structurally stable . Generally applicable procedures, as developed during the establishment of this vector system, are described; they allow the isolation of ts-Mtases and facilitate the cloning of genes coding for nucleases directed against 5mC-containing DNA.

Gene, 1988 Jun 30, 66(2), 259 - 68
Nucleotide sequence and transcriptional analysis of a third function (Flm) involved in F-plasmid maintenance; Loh SM et al.; The leading region of the conjugative F plasmid encodes for a function, Flm, capable of extending the maintenance of normally unstable plasmids . Nucleotide sequencing and functional studies of flm locus have shown that it consists of at least two genes, flmA and flmB, which are physically and functionally homologous to hok and sok of parB in plasmid R1 . The 52-amino acid flmA-coded polypeptide is almost identical to the hok product which has been shown to be a membrane-associated lethal protein {Gerdes et al., EMBO J . 5 (1986) 2023-2029} . Gene flmB codes for a 100 nucleotide, non-translated, complementary RNA which overlaps the 5' leader sequence of the flmA RNA . The flmA RNA also encodes an open reading frame (ORF70) which overlaps the flmA-coding sequence and may be a third gene involved in the Flm function . S1 analysis and functional studies suggest that the antisense flmB RNA binds to the flmA RNA and suppresses the expression of the lethal product, presumably by blocking coupled translation of ORF70 and flmA . Secondary structure analysis predicts that the flmA RNA is extremely stable compared to the regulatory flmB RNA . We suggest that when these RNA species are retained by cells which have lost the F plasmid, the more stable flmA RNA will eventually be translated thus leading to cell death . This phenomenon provides a third mechanism, additional to ParFIA and Ccd functions, to ensure maintenance of the F plasmid in a growing bacterial population.

Gene, 1988 Jun 30, 66(2), 245 - 58
The nucleotide sequence of a plasmid determinant for resistance to tellurium anions; Jobling MG et al.; The plasmid pMJ606 contains a 5-kb insert specifying resistance to tellurium salts (TeR) which was cloned from the large conjugative plasmid pMER610 . Nucleotide sequence analysis of this insert has identified five open reading frames (ORFs) . ORFs 1, 2, 4 and 5 correspond in terms of their predicted polypeptide products to the 41, 15.5, 22 and 23-kDa polypeptides, respectively, which are synthesised by the resistance determinant in maxicells . An additional ORF, ORF3, whose product has not been identified is predicted by the sequence data . The sequence of the presumptive polypeptide specified by ORF3 indicates a membrane location . The nucleotide and predicted amino acid sequences of ORF4 and ORF5 show considerable homology which is consistent with the duplication and minor divergence of an ancestral gene . ORFs 4 and 5 appear to retain the same function and while each can function separately and contribute to the resistance mechanism, the full level of wild-type resistance requires both genes to function . The transcriptional signals for the TeR determinant may be located beyond and 5' of the sequences cloned in pMJ606.

Gene, 1988 Jun 30, 66(2), 279 - 94
Total chemical synthesis of a gene for hepatitis B virus core protein and its functional characterization; Nassal M; We have chemically synthesized a DNA duplex of 560 nucleotides that codes for the hepatitis B virus (HBV) core protein . The synthetic gene contains 27 unique internal restriction sites . Thereby, it can easily be mutagenized by replacement of rather short restriction fragments . A number of restriction recognition sequences are in common between the synthetic and the authentic gene, thus allowing for the transfer of synthetic segments into the cloned viral genome . Several unexpected mutations in the synthetic gene were readily corrected utilizing the multiple unique restriction sites . In Escherichia coli, the expression level of the synthetic gene product amounts to about 4% of the total soluble protein . It forms particles closely resembling native HBV cores . After transfer of the synthetic gene into the viral genome, transient expression in a hepatoma cell line yields proteins indistinguishable from the native gene products . The synthetic gene thus provides a useful tool for studies on the structure and function of the isolated HBV core protein as well as the gene and its various products in the viral life-cycle.

Gene, 1988 Jun 30, 66(2), 163 - 81
High-level transient expression of influenza virus proteins from a series of SV40 late and early replacement vectors; Huylebroeck D et al.; We have constructed a collection of simian virus 40 (SV40) plasmid vectors useful for transient or constitutive expression of cDNA or genomic DNA in animal cells . Most vectors contain several unique restriction sites downstream from the SV40 late or early promoter, and are available with or without the virus-specific splicing signals . The use of these vectors for transient expression in monkey cells of X47 (H3N2) influenza hemagglutinin (HA) and matrix protein (M1) was demonstrated . Membrane-bound (HAm) as well as secreted forms of the HA glycoprotein lacking the sequence of the C-terminal anchor (HA-) have been obtained . Depending on the insert, the type of vector and the amount of transfected DNA, HA levels in COS cells {Gething and Sambrook, Nature 293 (1981) 620-625} transfected with late replacement SV40 vectors vary from 10(9) (HAm) to 10(8) (HA-) molecules per transfected cell . The maximum expression levels with early replacement vectors in COS cells are at least 50 times lower . In addition to the optimalization and the characterization of the expression of each vector-coded influenza protein, cotransfections, including vectors expressing HAm, neuraminidase (NA) and M1, were undertaken . The latter experiments did not result in a measureable amount of HAm or NA in the cell culture medium, suggesting that expression of these three structural viral proteins does not result in budding of (empty) influenza particles from the cell surface.

Biochem Biophys Res Commun, 1988 Jun 30, 153(3), 1244 - 50
Site-directed mutagenesis of the cAMP-binding sites of the recombinant type I regulatory subunit of cAMP-dependent protein kinase; Kuno T et al.; The type I regulatory subunit (R-I) of rat brain cAMP-dependent protein kinase was expressed in E . coli and site-directed mutagenesis was used to substitute amino acids in the putative cAMP-binding sites . The wild-type recombinant R-I bound 2 mol of cAMP/mol subunit, while two mutant R-Is with a single amino acid substitution in one of the two intrachain cAMP-binding sites (clone N153:a glutamate for Gly-200, and clone C254:an aspartate for Gly-324) bound 1 mol of cAMP/mol subunit . When these two substitutions were made in one mutant, cAMP did not bind to this mutant, indicating that binding of cAMP to N153 or C254 was to their nonmutated sites . Competition experiments with site-selective analogs and dissociation of bound cAMP from mutant R-Is provided evidence for strong intrachain interactions between the two classes of cAMP-binding sites in R-I.

Nature, 1988 Jun 30, 333(6176), 824 - 9
DNA sequence determinants of CAP-induced bending and protein binding affinity; Gartenberg MR et al.; The sites of DNA bending induced by binding catabolite activator protein are identified and shown to coincide with positions where DNA grooves face the protein . The bendability of DNA with different sequences at these bend centres parallels the bending preference of the sequences in nucleosomal DNA . Anisotropic DNA bendability significantly affects the structure and strength of regulatory protein-DNA complexes.

Gene, 1988 Jun 30, 66(2), 319 - 23
Expression in Escherichia coli of a Moloney murine leukemia virus reverse transcriptase whose structure closely resembles the viral enzyme; Hizi A et al.; We have constructed an expression plasmid containing the portion of the Moloney murine leukemia virus genome encoding the reverse transcriptase (RT) . When introduced into Escherichia coli this plasmid induces the synthesis of a 70-kDa protein . The RT made in E . coli differs from the viral protein only in that there are two new amino acids, methionine and glycine, substituted for the threonine found at the N terminus of the viral enzyme . Approximately half of the E . coli synthesized RT enzyme is soluble in cell extracts . This protein is active in an RT assay, and like the enzyme purified from virions, is more active in the presence of Mn2+ than Mg2+ . We have also constructed a plasmid that induces the synthesis of an RT-integration protein fusion.

J Chromatogr, 1988 Jun 29, 443, 381 - 97
Separation of proteins by reversed-phase high-performance liquid chromatography . II . Optimizing sample pretreatment and mobile phase conditions; Nugent KD et al.; The effects of separation variables such as temperature, pH and composition of the mobile phase (including additives such as chaotropes, ion-pairing agents and surfactants), sample size and sample pretreatment for reversed-phase high-performance liquid chromatography (RP-HPLC) of proteins is examined . Experimental optimization of these parameters using the preferred instrumental and column conditions described previously lead to well behaved chromatographic performance for most proteins . This allowed us to achieve the required level of performance for the first dimension (RP-HPLC) separation of most protein samples by the chromatophoresis process.

J Immunol Methods, 1988 Jun 28, 111(1), 1 - 9
Erythropoietin-beta-D-galactosidase . The generation, purification and use of a fusion protein; Nielsen OJ et al.; A human erythropoietin (Epo) cDNA fragment encoding the complete erythropoietin peptide sequence was fused to the 3'-end of the lacZ gene in the polylinker region of the high expression vector, pUR 278 . Escherichia coli bacteria were transformed with the recombinant plasmid harboring the hybrid Epo-beta-D-galactosidase gene . After induction with isopropyl-thiogalactoside large amounts of the fusion protein, Epo-beta-D-galactosidase were synthesized in the transformed bacteria . The fusion protein was partially purified and shown to exhibit intact galactosidase enzymatic activity . Although no biological activity of the Epo counterpart of the fusion protein was detected both in an in vivo and in an in vitro bioassay, the fusion protein served as an effective antigen for the production of anti-erythropoietin antibodies . Antifusion protein antibodies raised in rabbits were shown to react with the intact human Epo molecule from erythropoietin producing culture supernatants . The affinity of these anti-fusion protein antibodies was sufficiently high to permit the development of a sensitive radioimmunoassay for human Epo . This fusion protein approach is a relatively straightforward and rapid method of generating antibodies with specificity for any protein encoded by a cloned eukaryotic gene.

Biochemistry, 1988 Jun 28, 27(13), 4952 - 6
Direct photoaffinity labeling of ribonucleotide reductase from Escherichia coli using dTTP: characterization of the photoproducts; Kierdaszuk B et al.; Subunit B1 of Escherichia coli ribonucleotide reductase contains one type of allosteric binding site that controls the substrate specificity of the enzyme . This site binds the allosteric effector dTTP as well as other nucleoside triphosphates . Cross-linking of dTTP to protein B1 by direct photoaffinity labeling, as well as the isolation and sequence determination of the labeled tryptic peptide, has recently been reported {Eriksson, S., Sjoberg, B.-M., Jornwall, H., & Carlquist, M . (1986) J . Biol . Chem . 261, 1878-1882} . In this study, we have further purified the dTTP-labeled peptide and characterized it using UV spectroscopy . Two types of dTTP-cross-linked peptide were found: one having an absorbance maximum at 261 nm typical for a dTTP spectrum, i.e., containing an intact 5,6 double bond, and one minor form with low absorbance at 261 nm . In both cases, the same amino acid composition was found, corresponding to the peptide Ser291-X-Ser-Gln-Gly-Gly-Val-Arg299 in the B1 sequence with X being Cys-292 cross-linked to dTTP . Isotope labeling experiments revealed that one proton in the 5-methyl group of thymine was lost during photoincorporation . Therefore, the cross-linking occurs via the 5-methyl group to Cys-292 in a majority of incorporated dTTPs, but a second, possibly 5,6-saturated form of incorporated nucleotide was also detected . The reasons for the high stereospecificity of the reaction and the possible structure of the allosteric site of protein B1 are discussed.

Biochemistry, 1988 Jun 28, 27(13), 4800 - 4
Ligand-induced structural constraints in human dihydrofolate reductase revealed by peptide-specific antibodies; Ratnam M et al.; Peptides from human dihydrofolate reductase (DHFR) generated by cyanogen bromide cleavage and corresponding to residues 15-52, 53-111, 112-125, and 140-186 (carboxyl terminus) were purified and used to immunize rats . Titration of the immune sera against denatured human DHFR by solid-phase immunoassay showed that peptides 15-52 and 140-186 were relatively highly immunogenic, unlike the native enzyme which is most immunogenic in the sequence 53-111 . The antisera were specific for the corresponding peptides used for immunization . Antibodies to peptides 15-52, 53-111, and 140-186 cross-reacted with native human DHFR in solution in competition assays . However, the binding of nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and the inhibitors folate and methotrexate, both in binary and in ternary complexes with the enzyme, caused a striking reduction in binding of antibody . Using a sensitive radioactive assay, it was found that antisera to peptides 15-52 and 140-186, both of which exhibited a high antibody titer, caused significant inhibition of DHFR . Because peptide 140-186 does not include any active-site residues, it is concluded that at least in this case all the antibodies bound to regions outside the active site . Since comparison of the X-ray structures of the chicken liver DHFR holoenzyme with the apoenzyme reveals no changes in secondary structural elements (alpha-helices and beta-sheets), the reduction in antibody binding to DHFR-ligand complexes must not involve epitopes within these structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1988 Jun 28, 27(13), 4777 - 80
Stereochemistry of phospho group transfer catalyzed by a mutant alkaline phosphatase; Butler-Ransohoff JE et al.; The stereochemical course of the phospho group transfer catalyzed by mutant (S102C) alkaline phosphatase from Escherichia coli was investigated by using 31P nuclear magnetic resonance spectroscopy . Transphosphorylation from 4-nitrophenyl (Rp)-{16O, 17O, 18O}phosphate to (S)-propane-1,2-diol occurs with overall retention of configuration at phosphorus . This result is consistent with the view that the hydrolysis of substrates by this mutant enzyme proceeds by way of a covalent phosphoenzyme intermediate in the same manner as the wild-type alkaline phosphatase.

Biochemistry, 1988 Jun 28, 27(13), 4735 - 40
Spectroscopic and hydrodynamic studies reveal structural differences in normal and transforming H-ras gene products; Pingoud A et al.; We have recorded the circular dichroism spectra of the cellular and the viral H-ras gene products both in the absence and in the presence of guanine nucleotides and analyzed these spectra in terms of the secondary structure composition of these proteins . It is shown that the GTP complex of the ras proteins has a different secondary structure composition than the GDP complex and, furthermore, that there are differences in the secondary structure of the viral ras protein and the cellular ras protein . We have also recorded and analyzed the circular dichroism spectrum of the isolated guanine nucleotide binding domain of the Escherichia coli elongation factor Tu (EF-Tu), which has been considered as a model for the tertiary structure of the ras proteins {McCormick, F., Clark, B . F . C., LaCour, T . F . M., Kjeldgaard, M., Norskov-Lauritsen, L., & Nyborg, J . (1985) Science (Washington, D.C.) 230, 78-82} . Our data show that the guanine nucleotide binding domain of EF-Tu (30% alpha-helix and 16% beta-pleated sheet for the GDP complex) has quite a different secondary structure composition than the ras proteins (e.g., the cellular ras protein has 47% alpha-helix and 22% beta-pleated sheet for the GDP complex), indicating that the protein core comprising the guanine nucleotide binding site might be similar but that major structural differences must exist at the portion outside this core . Normal and transforming ras proteins also differ slightly in their hydrodynamic properties as shown by sedimentation velocity runs in the analytical ultracentrifuge.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1988 Jun 28, 27(13), 4687 - 95
Complementary oligodeoxynucleotide probes of RNA conformation within the Escherichia coli small ribosomal subunit; Lasater LS et al.; The large RNA molecule within each ribosomal subunit is folded in a specific and compact form . The availability of specific 16S RNA sequences on the surface of the small ribosomal subunit has been probed by using complementary oligodeoxynucleotides . The hybridization of 8-15-nucleotide-long oligomers to their RNA complements within the subunit was quantitated by using a nitrocellulose membrane filter binding assay . The probes have been grouped into classes on the basis of sequence-specific binding ability under different conditions of ionic environment, incubation temperature, and subunit activation state {as defined by the ability to bind phenylalanyl-tRNA in response to a poly(uridylic acid) message} . Oligodeoxynucleotides complementary to nucleotides flanking 7-methylguanosine residue 527 and to the 3'-terminal sequence bound 30S subunits regardless of the activation state . Oligodeoxynucleotides that complement 16S ribosomal RNA residues 1-16, 60-70, 685-696, and 1330-1339 and the sequence adjacent to the colicin E3 cleavage site at residue 1502 all bound efficiently only to subunits in an inactivated conformation . Probes complementary to residues 1-11 and 446-455 bound only inactivated subunits, and then with low efficiency . Sequences complementary to nucleotides 6-16, 99-109, 1273-1281, and 1373-1383 bound 30S subunits poorly regardless of the activation state . With one exception, each probe was bound by native or heat-denatured 16S ribosomal RNA (as determined by size-exclusion chromatography) . We conclude that complementary oligodeoxynucleotide binding efficiency is a sensitive measure of the availability of specific RNA sequences under easily definable conditions.

Biochemistry, 1988 Jun 28, 27(13), 4680 - 6
Isolation, nucleotide sequence, and expression of a cDNA encoding pig citrate synthase; Evans CT et al.; Citrate synthase is a key enzyme of the Krebs tricarboxylic acid cycle and catalyzes the stereospecific synthesis of citrate from acetyl coenzyme A and oxalacetate . The amino acid sequence and three-dimensional structure of pig citrate synthase dimers are known, and regions of the enzyme involved in substrate binding and catalysis have been identified . A cloned complementary DNA sequence encoding pig citrate synthase has been isolated from a pig kidney lambda gt11 cDNA library after screening with a synthetic oligonucleotide probe . The complete nucleotide sequence of the 1.5-kilobase cDNA was determined . The coding region consists of 1395 base pairs and confirms the amino acid sequence of purified pig citrate synthase . The derived amino acid sequence of pig citrate synthase predicts the presence of a 27 amino acid N-terminal leader peptide whose sequence is consistent with the sequences of other mitochondrial signal peptides . A conserved amino acid sequence in the mitochondrial leader peptides of pig citrate synthase and yeast mitochondrial citrate synthase was identified . To express the pig citrate synthase cDNA in Escherichia coli, we employed the inducible T7 RNA polymerase/promoter double plasmid expression vectors pGP1-2 and pT7-7 {Tabor, S., & Richardson, C . C . (1985) Proc . Natl . Acad . Sci . U.S.A . 82, 1074-1078} . The pig citrate synthase cDNA was modified to delete the N-terminal leader sequence; then by use of a synthetic oligonucleotide linker, the modified cDNA was cloned into pT7-7 immediately following the initiator Met . A glutamate-requiring (citrate synthase deficient), recA- E . coli mutant, DEK15, was transformed with pGP1-2 and then pT7-7PCS . pT7-7PCS complemented the E . coli gltA mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1988 Jun 28, 27(13), 4712 - 20
Mutations in the nucleotide binding loop of adenylate kinase of Escherichia coli; Reinstein J et al.; The adk gene of Escherichia coli has been used to overexpress the adenylate kinase protein in two ways: (1) by cloning the adk gene with its own promoter into pEMBL plasmids, which have an increased copy number, and (2) by deleting the adk promoter and cloning the gene behind the regulatable tac promoter . Adenylate kinase comprises up to 40% of the soluble cellular extracts from E . coli strains containing these plasmids . Mutations have been introduced into the gene by site-directed mutagenesis to exchange amino acids in the nucleotide binding loop, which is highly conserved in many mononucleotide binding proteins . The mutation of Lys13----Gln is nearly inactive, whereas the Pro9----Leu and the Gly10----Val mutant proteins have an increased Km for both substrates and a Vmax that is similar to wild type . Proton NMR measurements of the proteins show that a major structural change seems to have taken place for the Pro9----Leu and Gly10----Val mutants . The results are discussed in the light of the kinetic mechanism for adenylate kinase and the three-dimensional structure of the protein.

Biochemistry, 1988 Jun 28, 27(13), 4698 - 705
Functional consequences of the arabinosylcytosine structural lesion in DNA; Mikita T et al.; Cytosine arabinoside (araC) is a potent antileukemic agent that is misincorporated into DNA in the course of its action . We have developed a chemical synthetic method that allows site-specific introduction of araC into synthetic DNA oligomers . We describe here the utilization of these oligomers as primer/template substrates for in vitro DNA synthesis reactions and as fragments for DNA ligation . These studies were undertaken to investigate the manner in which sites of araC misincorporation constitute sites of DNA dysfunction . AraCMP at the primer terminus dramatically reduced the rate of next nucleotide addition for Escherichia coli polymerase I (Klenow fragment) (Pol I), T4 polymerase, HeLa cell polymerase alpha 2 (Pol alpha 2), and AMV reverse transcriptase . Polymerases with associated 3'-5' exonuclease activity preferentially excised araCMP from the primer terminus prior to chain elongation . AraCMP-terminated fragments were ligated more slowly than control fragments by T4 DNA ligase . AraCMP located at an internucleotide site in the template markedly slowed replicative bypass for Pol I, T4 polymerase, and Pol alpha 2, but not for reverse transcriptase . Synthesis was partially arrested after insertion of the correct nucleotide opposite the lesion site . These results suggest a complex mechanism for the inhibition of DNA replication by araC when it is misincorporated into DNA.

J Biol Chem, 1988 Jun 25, 263(18), 9015 - 9
Sequence of thioredoxin reductase from Escherichia coli . Relationship to other flavoprotein disulfide oxidoreductases; Russel M et al.; The DNA sequence of the Escherichia coli gene encoding thioredoxin reductase has been determined . The predicted protein sequence agrees with an earlier determination of the 17 amino-terminal amino acids and with a fragment of the protein containing the redox-active half-cystines . Similarity between E . coli thioredoxin reductase and other flavoprotein disulfide oxidoreductases is quite limited, but three short segments, two of which are probably involved in FAD and NADPH binding, are highly conserved between thioredoxin reductase, glutathione reductase, dihydrolipoamide dehydrogenase, and mercuric reductase.

J Biol Chem, 1988 Jun 25, 263(18), 8953 - 7
Studies on the mutator gene, mutT of Escherichia coli . Molecular cloning of the gene, purification of the gene product, and identification of a novel nucleoside triphosphatase; Bhatnagar SK et al.; The mutator gene, mutT, has been cloned into an expression vector and overproduced in Escherichia coli . The gene product has been purified to over 90% homogeneity as judged by gel electrophoresis and amino acid analysis . The amino acid composition of the protein and the sequence of the 20 amino acids of the N-terminal region agree well with the nucleotide sequence of the gene reported by Akiyama et al . (Akiyama, M., Horiuchi, T., and Sekiguchi, M . (1987) Mol . Gen . Genet . 206, 9-16) and indicate that the first of the potential initiation codons (position 164) of the open reading frame in the PvuII fragment carrying the mutT gene is the site of initiation of translation of the 15,000-Da polypeptide . A novel nucleoside triphosphatase activity which has a preference for dGTP is associated with the purified protein, and preliminary experiments are consistent with the notion that the mutT gene product is the enzyme responsible for this activity.

J Biol Chem, 1988 Jun 25, 263(18), 8735 - 9
The RNA N-glycosidase activity of ricin A-chain . The characteristics of the enzymatic activity of ricin A-chain with ribosomes and with rRNA; Endo Y et al.; Ricin A-chain cleaves the N-glycosidic bond at A-4324 in 28 S rRNA when intact rat ribosomes are the substrate . Cleavage occurs at a concentration of the toxin of 1 X 10(-10) M, and specificity for this single residue is retained when the concentration is as high as 3 X 10(-7) M . The apparent Michaelis constant (Km) for the reaction is 2.6 microM, and the turnover number (Kcat) is 1777 min-1 . The same N-glycosidic bond is cleaved by ricin A-chain in naked 28 S rRNA, but at a greatly reduced rate . The Km value for this reaction is 5.8 microM . The results suggest that the A-chain has a similar affinity for 28 S rRNA in ribosomes and in the absence of ribosomal proteins . Ricin A-chain has no effect on 23 S rRNA in Escherichia coli ribosomes, however, the N-glycosidic bond at A-2600 in naked 23 S rRNA is cleaved by the toxin; this corresponds to the ricin site in eukaryotic 28 S rRNA . Since the Km value (3.3 microM) for the reaction with E . coli 23 S rRNA approximates that obtained with rat liver ribosomes, it is possible that E . coli ribosomal protein(s) protect this site against ricin attack in intact ribosomes . Ricin A-chain also acted on naked 16 S rRNA cleaving the N-glycosidic bond of adenine at position 1014 . The results suggest that ricin A-chain recognizes a specific structure in rRNA, perhaps a loop and stem having the sequence GAGA in the loop.

J Biol Chem, 1988 Jun 25, 263(18), 8710 - 5
The expression in Escherichia coli of recombinant human platelet factor 4, a protein with immunoregulatory activity; Barone AD et al.; In order to establish more firmly the immunoregulatory effect of platelet factor 4 (PF4) and develop a means to provide material for possible clinical use, the nucleotide sequence for PF4 was synthesized utilizing a ligation strategy of six duplexes ranging from 27 to 43 base pairs in length . The individual oligodeoxynucleotides were synthesized on an automated system . The resultant gene segment (226 base pairs), which incorporated convenient HindIII and BamHI overhangs at the 5' and 3' ends, respectively, was cloned into the pIN-III-ompA-2-expression vector in Escherichia coli, affording a fusion protein of Mr = 8900 with 7 additional amino acids at the amino terminus and 4 at the carboxyl terminus and with aspartic acid rather than asparagine in position 47 . The recombinant PF4 (rPF4) was purified by heparin-agarose affinity chromatography and reverse-phase high performance liquid chromatography . It reacted with a monoclonal mouse anti-human PF4 antibody on a Western blot and in an enzyme-linked immunosorbent assay . The rPF4 protein exhibited an immunoregulatory effect like that of human PF4 in its ability to reverse concanavalin A-induced immunosuppression in BALB/c mice.

J Biol Chem, 1988 Jun 25, 263(18), 8666 - 70
Identification of the reactive sulfhydryl groups of S-adenosylmethionine synthetase; Markham GD et al.; S-Adenosylmethionine synthetase from Escherichia coli is rapidly inactivated by N-ethylmaleimide . In the presence of excess N-ethylmaleimide inactivation follows pseudo first-order kinetics, and loss of enzyme activity correlates with the incorporation of 2 eq of N-{ethyl-2-3H}maleimide/subunit . Preincubation of the enzyme with methionine and the ATP analog adenylylimidodiphosphate reduced the rate of N-ethylmaleimide incorporation more than 30-fold . Two N-{ethyl-2-3H}maleimide-labeled tryptic peptides were purified from the modified enzyme by reverse phase high performance liquid chromatography . The modified residues were identified as cysteine 90 and cysteine 240 by comparison of the amino acid compositions of these peptides with the protein sequence . These are the first residues to be implicated in the activity and/or structure of the enzyme . N-Ethylmaleimide-modified S-adenosylmethionine synthetase exists mainly as a dimer in conditions where the native enzyme is a tetramer . Accumulation of the dimer parallels the loss of the enzyme activity . When an enzyme sample was partially inactivated, separation of tetrameric and dimeric enzyme forms by gel filtration revealed that the residual enzyme activity was solely present in the tetramer and N-{ethyl-2-3H} maleimide was present predominantly in the dimer . Gel filtration studies of the tetramer-dimer equilibrium for the native enzyme indicated that the dissociation constant between the tetramer and dimers is less than 6 x 10(-11) M . Similar studies for the N-ethylmaleimide-modified protein indicated that the dissociation constant of the tetramer is approximately 4 x 10(-4) M . Upon modification the strength of dimer-dimer interactions is diminished by at least 9 kcal/mol.

J Biol Chem, 1988 Jun 25, 263(18), 8727 - 34
Protease Ti, a new ATP-dependent protease in Escherichia coli, contains protein-activated ATPase and proteolytic functions in distinct subunits; Hwang BJ et al.; In addition to protease La (the lon gene product), Escherichia coli contains another ATP-dependent protease, Ti . This enzyme (approximately 340 kDa) is composed of two components, both of which are required for proteolysis . Both have been purified to homogeneity by conventional procedures using {3H}casein as the substrate . The ATP-stabilized component, A, has a subunit molecular weight of 80,000 upon gel electrophoresis in the presence of sodium dodecyl sulfate, but it behaves as a dimer (140 kDa) upon gel filtration . Component P, which is relatively heat stable, is inactivated by diisopropyl fluorophosphate and can be labeled with {3H} diisopropyl fluorophosphate . It has a subunit size of 23 kDa, but the isolated component behaves as a complex (260 kDa) of 10-12 subunits . The isoelectric point of component A is 7.0 and that of P is 8.2, and their amino acid compositions differ considerably . The purified enzyme has an ATPase activity that is stimulated 2-4-fold by casein and other protein substrates but not by nonhydrolyzed proteins . Component A also shows ATPase activity which can be stimulated by casein . Addition of component P (which lacks ATPase activity) inhibits basal ATP hydrolysis by A and makes this ATPase more responsive to casein . Although component P contains the serine active site for proteolysis, it shows no proteolytic activity in the absence of component A, Mg2+, and ATP or dATP . Other nucleoside triphosphates are not hydrolyzed and do not support proteolysis . Protease Ti has a Km for ATP of 210 microM for hydrolysis of both casein and ATP . Casein increases the Vmax for ATP without affecting the Km . A Mg2+ concentration of 5 mM is necessary for half-maximal rates of ATP and casein hydrolysis . Ca2+ and Mn2+ partially support these activities . Thus, protease Ti shares many unusual properties with protease La (e.g . coupled ATP and protein hydrolysis and protein-activated ATPase), but these functions in protease Ti are associated with distinct subunits that modify each other's activities.

J Biol Chem, 1988 Jun 25, 263(18), 8716 - 23
Construction of a recombinase-deficient mutant recA protein that retains single-stranded DNA-dependent ATPase activity; Bryant FR; The recA1 mutation is a single point mutation that replaces glycine 160 of the recA polypeptide with an aspartic acid residue . The mutant recA1 protein has a greatly reduced single-stranded DNA-dependent ATPase activity at pH 7.5 compared to the wild-type protein . Interestingly, the recA1 protein does exhibit a vigorous ATPase activity at pH 6.2 . To explore the molecular basis of this pH effect, we used site-directed mutagenesis to replace aspartic acid 160 of the recA1 polypeptide with an isosteric, but nonionizing, asparagine residue . The new {Asn160}recA protein catalyzes ATP hydrolysis at pH 7.5 with the same turnover number as the wild-type protein . This result suggests that the activation of the recA1 protein ATPase activity that occurs at pH 6.2 may be due, in part, to neutralization of the negatively charged aspartic acid 160 side chain . Although it is an active single-stranded DNA-dependent ATPase, the {Asn160}recA protein is unable to complement a recA deletion in vivo and is unable to carry out the three-strand exchange reaction in vitro . Further examination of ATP hydrolysis (under strand exchange conditions) revealed that the ATPase activity of the {Asn160}recA protein is strongly suppressed in the presence of Escherichia coli single-stranded DNA-binding protein (a component of the strand exchange assay), whereas the ATPase activity of the wild-type recA protein is stimulated by the E . coli protein . To account for these results, we speculate that ATP may induce specific conformational changes in the wild-type recA protein that are essential to the DNA pairing process and that these conformational changes may not occur with the {Asn160}recA protein.

J Biol Chem, 1988 Jun 25, 263(18), 8872 - 8
Acetyl-coenzyme A:polysialic acid O-acetyltransferase from K1-positive Escherichia coli . The enzyme responsible for the O-acetyl plus phenotype and for O-acetyl form variation; Higa HH et al.; The capsular polysaccharide of Escherichia coli K1 is a linear polymer of N-acetylneuraminic acid in alpha-2,8 linkage . Certain substrains of E . coli K1 (designated OAc+) modify the polysaccharide by O-acetylation of the sialic acids . We demonstrate here an acetyl-coenzyme A: polysialosyl O-acetyltransferase activity that is found only in E . coli K1 OAc+ substrains . When form variation between the O-acetyl-positive and -negative states occurred in strain D698:K1, the fluctuations were accompanied by appropriate changes in the expression of enzyme activity . Thus, expression of this enzyme can account for the OAc+ phenotype and for the form variation between OAc+ and OAc- . The enzyme was solubilized in nonionic detergent and freed of endogenous acceptor activity by DEAE-cellulose chromatography, and its general properties were determined . Analysis of the reaction product showed a highly preferential acetylation reaction that was confined to polysialosyl units of greater than 14 residues . Acetyl groups were shown to be transferred to both the 7- and the 9-positions of the sialic acid residues . The partially purified enzyme was stable even after prolonged incubation at 57 degrees C . In contrast, any further purification resulted in loss of activity, even at 4 degrees C . Treatment of the stable enzyme with a polysialic acid-specific endoneuraminidase caused a similar loss of enzyme stability . This effect of the endoneuraminidase could be protected against by the addition of exogenous polysialic acid . This indicates that the partially purified enzyme contains traces of endogenous polysialic acid substrate that are required for the stability of the enzyme . Finally, the enzyme can O-acetylate the polysialic acid chains on the eucaryotic protein neural cell adhesion molecule, suggesting that enzymatic recognition of the substrate requires only the polysialic acid sequence.

J Biol Chem, 1988 Jun 25, 263(18), 8765 - 70
A homologous sequence between H+-ATPase (F0F1) and cation-transporting ATPases . Thr-285----Asp replacement in the beta subunit of Escherichia coli F1 changes its catalytic properties; Noumi T et al.; A sequence of 10 amino acids (I-C-S-D-K-T-G-T-L-T) of ion motive ATPases such as Na+/K+-ATPase is similar to the sequence of the beta subunit of H+-ATPases, including that of Escherichia coli (I-T-S-T-K-T-G-S-I-T) (residues 282-291) . The Asp (D) residue phosphorylated in ion motive ATPase corresponds to Thr (T) of the beta subunit . This substitution may be reasonable because there is no phosphoenzyme intermediate in the catalytic cycle of F1-ATPase . We replaced Thr-285 of the beta subunit by an Asp residue by in vitro mutagenesis and reconstituted the alpha beta gamma complex from the mutant (or wild-type) beta and wild-type alpha and gamma subunits . The uni- and multisite ATPase activities of the alpha beta gamma complex with mutant beta subunits were about 20 and 30% of those with the wild-type subunit . The rate of ATP binding (k1) of the mutant complex under uni-site conditions was about 10-fold less than that of the wild-type complex . These results suggest that Thr-285, or the region in its vicinity, is essential for normal catalysis of the H+-ATPase . The mutant complex could not form a phosphoenzyme under the conditions where the H+/K+-ATPase is phosphorylated, suggesting that another residue(s) may also be involved in formation of the intermediate in ion motive ATPase . The wild-type alpha beta gamma complex had slightly different kinetic properties from the wild-type F1, possibly because it did not contain the epsilon subunit.

J Biol Chem, 1988 Jun 25, 263(18), 8611 - 4
Evidence that glutamic acid 49 of tryptophan synthase alpha subunit is a catalytic residue . Inactive mutant proteins substituted at position 49 bind ligands and transmit ligand-dependent to the beta subunit; Miles EW et al.; Glutamic acid 49 of the alpha subunit of tryptophan synthase from Escherichia coli is an essential residue since 19 mutant proteins substituted at position 49 were found previously to be inactive . Our present findings that five mutants of the alpha subunit, substituted with Asp, Lys, Ala, Phe, or Gly at position 49, bind a substrate analog normally are further evidence that glutamic acid 49 is a catalytic base . Ligands of the alpha subunit also have similar effects on site-site interactions between the beta subunit and the wild type or mutant alpha subunits . These effects include inhibition of the activity of the beta subunit, reduction of the dissociation constant for D-tryptophan, and increase of the equilibrium concentration of a quinonoid intermediate formed with L-tryptophan.

J Biol Chem, 1988 Jun 25, 263(18), 8943 - 52
Synthesis, antiviral activity, and conformational characterization of mouse-human alpha-interferon hybrids; Raj NB et al.; Reciprocal hybrids were constructed between human and mouse interferons (IFNs), and their antiviral activity was examined on different target cells and compared to the activity of the parental molecules . In addition, we used a number of predictive algorithms on a data base of the available alpha-interferon sequences to propose a working model for the overall conformation of the alpha-interferon molecule that is consistent with the structural predictions . Remarkable conservation within the predicted alpha-helical segments of the interferon molecule was observed . We propose that the observed changes in the activity and specificity of the hybrids obtained are largely due to the sequences present in the loops at the ends of the major helical structures; these are less conserved, contain beta-bends, and are generally hydrophilic and flexible . The data on the constructed mouse-human hybrids have shown that the activity on human cells is contributed by determinants present in the N-terminal 122 amino acids of human IFN, thus implicating one or more loops within this region (e.g . loops 1-12, 25-38, 70-74, and 103-113) . The activity on bovine cells appears to be localized mainly in sequence 60-121, implicating the role of loops 70-74 and/or 103-113 of the human IFN molecule . The specificity of mouse IFN for mouse cells is in some or all of the loops (70-74, 103-113, 134-139, and 163-166) in the C-terminal sequence . The proposed working model should provide guidelines for the study of the specificity of action in molecular terms.

Nucleic Acids Res, 1988 Jun 24, 16(12), 5391 - 406
The core region of human glutaminyl-tRNA synthetase homologies with the Escherichia coli and yeast enzymes; Thommes P et al.; We have isolated from a Lambda-gt 11 library a human cDNA clone with one open reading frame of about 2400 bases . A stretch of about 350 amino acids in the deduced amino acid sequence is up to 40 percent identical with parts of the known amino acid sequences of E . coli and yeast glutaminyl (Gln)-tRNA synthetase . The isolated cDNA sequence corresponds to an internal section of a 5500 bases long mRNA that codes for a 170 kDa polypeptide associated with Gln-tRNA synthetase . Thus, the human enzyme is about three times larger than the E . coli and two times larger than the yeast Gln-tRNA synthetase . The three enzymes share an evolutionarily conserved core but differ in amino acid sequences linked to the N-terminal and C-terminal side of the core.

Nucleic Acids Res, 1988 Jun 24, 16(12), 5305 - 22
Evolution and mutagenesis of the mammalian excision repair gene ERCC-1; van Duin M et al.; The human DNA excision repair protein ERCC-1 exhibits homology to the yeast RAD10 repair protein and its longer C-terminus displays similarity to parts of the E . coli repair proteins uvrA and uvrC . To study the evolution of this 'mosaic' ERCC-1 gene we have isolated the mouse homologue . Mouse ERCC-1 harbors the same pattern of homology with RAD10 and has a comparable C-terminal extension as its human equivalent . Mutation studies show that the strongly conserved C-terminus is essential in contrast to the less conserved N-terminus which is even dispensible . The mouse ERCC-1 amino acid sequence is compatible with a previously postulated nuclear location signal and DNA-binding domain . The ERCC-1 promoter harbors a region which is highly conserved in mouse and man . Since the ERCC-1 promoter is devoid of all classical promoter elements this region may be responsible for the low constitutive level of expression in all mouse tissues and stages of embryogenesis examined.

Nucleic Acids Res, 1988 Jun 24, 16(12), 5557 - 68
Expression of glutathione peroxidase I gene in selenium-deficient rats; Reddy AP et al.; We have characterized a cDNA pGPX1211 encoding rat glutathione peroxidase I . The selenocysteine in the protein corresponded to a TGA codon in the coding region of the cDNA, similar to earlier findings in mouse and human genes, and a gene encoding the formate dehydrogenase from E . coli, another selenoenzyme . The rat GSH peroxidase I has a calculated subunit molecular weight of 22,155 daltons and shares 95% and 86% sequence homology with the mouse and human subunits, respectively . The 3'-noncoding sequence (greater than 930 bp) in pGPX1211 is much longer than that of the human sequences . We found that glutathione peroxidase I mRNA, but not the polypeptide, was expressed under nutritional stress of selenium deficiency where no glutathione peroxidase I activity can be detected . The failure of detecting any apoprotein for the glutathione peroxidase I under selenium deficiency and results published from other laboratories supports the proposal that selenium may be incorporated into the glutathione peroxidase I co-translationally.

Nucleic Acids Res, 1988 Jun 24, 16(12), 5439 - 58
Upstream and downstream transcriptional control signals in the yeast retrotransposon, TY; Fulton AM et al.; The yeast retrotransposon, Ty, shares many structural and functional features with retroviral proviruses . These include production of a terminally redundant major transcript . There are also two less abundant transcripts of 5.0 kb and 2.2 kb . Ty transcription is regulated by cell-type, that is it is reduced 5-20 fold in a/alpha diploids as compared to haploids . However control of expression of Ty is not well understood . By deletion analysis we have identified regions of the element which are involved in the activation and regulation of transcription . These signals are found both upstream and downstream of the mRNA start site . The downstream signals are within the region encoding the major Ty proteins . This organisation of transcriptional control signals is discussed with reference to the organisation of control signals in other yeast genes and in retroviral proviruses and other retro-elements.

Nucleic Acids Res, 1988 Jun 24, 16(12), 5345 - 59
Gene regulation on broad host range plasmid RK2: identification of three novel operons whose transcription is repressed by both KorA and KorC; Thomas CM et al.; The product of the korA gene of broad host range plasmid RK2 is a key transcriptional repressor which regulates not only the expression of the essential replication gene trfA but also its own expression and that of the kilA operon . It has previously been proposed that korA also encodes a positive activator of transcription of the korC gene, which may act as a transcriptional antiterminator . Here we show that the action of korA in relation to korC can be explained entirely through the korA protein's property as a transcriptional repressor . The limited ability of the previously cloned korC gene to suppress kilC on its own is shown to be due to the fact that korC in RK2 is transcribed from the bla promoter of Tn1 which was deleted in the original korC clones . We demonstrate that korA is a second repressor along with korC of three operons, one of which encodes kilC, the other two not having been described previously and serving an as yet unknown function . We have designated these operons kcrA, B and C for KorC-regulated . Putative kilC is designated kcrC . The homology between the expression signals of these operons suggests that they have arisen by duplication . This is confirmed in the case of kcrA and B by the existence of considerable homology between the products of the first ORFs in each of these operons.

Nucleic Acids Res, 1988 Jun 24, 16(12), 5277 - 90
A second RNA-polymerase can bind specifically to the bla promoter of Tn3, repressing transcription initiation; Duval-Valentin G et al.; We showed earlier that the region of the bla promoter of Tn3 protected by the RNA-polymerase (RNAP), has the normal size (about 60bp) at RNAP/promoter molar ratio r less than or equal to 2, but rises to about twice this extent as r increases . We confirm here that the species corresponding to normal and extended footprint distinguish by their electrophoretic mobilities . Furthermore, inspection of the complexes by electron microscopy confirms that at r greater than 2, the bla promoter can bind specifically a second RNAP particle, as compared to the 1:1 complex observed at r less than or equal to 2 . At r greater than 2, the ability of the bla promoter to initiate transcription in vitro is repressed when compared to the complex 1:1 obtained at r less than or equal to 2 . The unexpected decrease in initiation efficiency as the concentration of RNAP particles is increased, together with the striking sequence homology of the bla promoter with promoters of stable RNA, suggest that in vivo, this promoter could be regulated by growth rate.

FEBS Lett, 1988 Jun 20, 233(2), 432 - 6
Automated Sanger dideoxy sequencing reaction protocol; Zimmermann J et al.; The protocol for Sanger dideoxy chain termination reactions in DNA sequencing is tedious and prone to errors due to the repetitive character of the pipetting steps . An industrial robot, with the addition of a few simple parts, was programmed to automate the dideoxy sequencing reactions . The system is set up in a short time for routine operation and it is faster and more reliable than a human operator . It is flexible and allows variations and optimization of the standard procedure . Disposable microtiter plates at a controlled temperature are used . In one reaction cycle (about 50 min) up to 48 templates are processed . Up to 450 bases were resolved in automated DNA sequencing on samples prepared by the robot . The protocol is applicable to fluorescent as well as to radioactive labeling.

FEBS Lett, 1988 Jun 20, 233(2), 367 - 70
Degradation of epidermal growth factor receptors by cathepsin L-like protease: inhibition of the degradation by c-Ha-ras gene products; Hiwasa T et al.; Extract of NIH3T3 mouse fibroblasts contains a protease which can cleave epidermal growth factor receptor (EGF receptor) . This protease was tentatively named cathepsin X and purified to near homogeneity . The characteristics of cathepsin X were similar to those of cathepsin L and the proteolytic activity of cathepsin X was inhibited by c-Ha-ras gene products.

FEBS Lett, 1988 Jun 20, 233(2), 326 - 30
Protein NMR resonance assignment by isotropic mixing experiments on random fractionally deuterated samples; LeMaster DM; The 108-residue protein E . coli thioredoxin has been uniformly enriched to 50% with deuterium at all carbon-bound hydrogen positions . Isotropic mixing (i.e . TOCSY) experiments have been conducted for both the deuterated and natural-abundance samples . Using a 54 ms mixing time correlation peaks can be seen for all four protons on the benzenoid ring of tryptophan in both samples . The deuteration results in an average decrease in cross-sectional area of a factor of 2-3 for the TOCSY cross-peaks . The cross-peak intensities for the deuterated sample systematically decrease as a function of the number of protons involved in the transfer process thus overcoming a common ambiguity in the TOCSY experiment.

FEBS Lett, 1988 Jun 20, 233(2), 347 - 51
Identification of alpha-subunit Lys201 and beta-subunit Lys155 at the ATP-binding sites in Escherichia coli F1-ATPase; Tagaya M et al.; Binding of about 1 mol of adenosine triphosphopyridoxal to Escherichia coli F1-ATPase resulted in the nearly complete inactivation of the enzyme {(1987) J . Biol . Chem . 262, 7686-7692} . About two thirds of the label was bound to the alpha-subunit, and the rest to the beta-subunit . The present study revealed that Lys201 in the alpha-subunit and Lys155 in the glycine-rich region of the beta-subunit are the major sites labeled with this reagent . Thus, these two residues might be located close to the gamma-phosphate of the bound ATP.

J Mol Biol, 1988 Jun 20, 201(4), 697 - 716
Probing the assembly of the 3' major domain of 16 S rRNA . Interactions involving ribosomal proteins S2, S3, S10, S13 and S14; Powers T et al.; We have used rapid probing methods to follow the changes in reactivity of residues in 16 S rRNA to chemical and enzymatic probes as ribosomal proteins S2, S3, S10, S13 and S14 are assembled into 30 S subunits . Effects observed are confined to the 3' major domain of the RNA and comprise three general classes . (1) Monospecific effects, which are attributable to a single protein . Proteins S13 and S14 each affect the reactivities of different residues which are adjacent to regions previously found protected by S19 . S10 effects are located in two separate regions of the domain, the 1120/1150 stem and the 1280 loop; both of these regions are near nucleotides previously found protected by S9 . Both S2 and S3 protect different nucleotides between positions 1070 and 1112 . In addition, S2 protects residues in the 1160/1170 stem-loop . (2) Co-operative effects, which include residues dependent on the simultaneous presence of both proteins S2 and S3 for their reactivities to appear similar to those observed in native 30 S subunits . (3) Polyspecific effects, where proteins S3 and S2 independently afford the same protection and enhancement pattern in three distal regions of the domain: the 960 stem-loop, the 1050/1200 stem and in the upper part of the domain (nucleotides 1070 to 1190) . Proteins S14 and S10 also weakly affect the reactivities of several residues in these regions . We believe that several of the protected residues of the first class are likely sites for protein-RNA contact while the third class is indicative of conformational rearrangement in the RNA during assembly . These results, in combination with the results from our previous study of proteins S7, S9 and S19, are discussed in terms of the assembly, topography and involvement in ribosomal function of the 3' major domain.

Biochem Biophys Res Commun, 1988 Jun 16, 153(2), 875 - 80
In vivo phosphorylation of isocitrate lyase from Escherichia coli D5H3G7; Hoyt JC et al.; This report describes the in vivo phosphorylation of isocitrate lyase and examines the possible consequences to the control of the Kreb's cycle and glyoxylate bypass . NADP-specific isocitrate dehydrogenase from E . coli was the first bacterial protein whose enzymic activity was shown to be modulated by reversible phosphorylation . This enzyme has been thought to be solely responsible for the partitioning of isocitrate between the Kreb's cycle and glyoxylate bypass . No studies to date have examined the possible role of isocitrate lyase in controlling this flux.

Biochem Biophys Res Commun, 1988 Jun 16, 153(2), 767 - 72
L-leucine and its analogue: specific inhibitors for S-benzyl-L-cysteine-p-nitroanilide-hydrolyzing enzyme in Escherichia coli B; Murata K et al.; An enzyme that catalyzes hydrolysis of S-benzyl-L-cysteine-p-nitroanilide was purified from E . coli B . The enzyme was a monomer with a molecular weight of 82,000 . In addition to L-cysteinylglycine, the enzyme hydrolyzed various glycine-containing dipeptides most efficiently at pH 7.0 . The enzyme required no metal ions for activity and was specifically inhibited by L-leucine and its analogue with free carboxyl group at the physiological concentrations.

Biochem Biophys Res Commun, 1988 Jun 16, 153(2), 753 - 9
Mutant isolation and cloning of the gene encoding protease VII from Escherichia coli; Sugimura K; A mutant of Escherichia coli lacking protease VII, the outer membrane-associated protease which specifically cleaves paired basic residues (1), was isolated by using N-methyl-N'-nitro-N-nitrosoguanidine treatment . The mutant exhibited no significant change as for its growth rate and microscopic feature compared with wild cells . The gene encoding protease VII was cloned by using complementation analysis of protease VII (-) mutation . The minicell experiment showed that the gene encoded a putative precursor protein of 38,000 Mr which was processed into a protein of 36,000 Mr suggesting the presence of a signal peptide on the putative precursor.

Thromb Haemost, 1988 Jun 16, 59(3), 378 - 82
Endotoxin-induced platelet activation in human whole blood in vitro; Csako G et al.; The effect of purified bacterial endotoxin was studied on human platelets in vitro . In adding up to 1 microgram/mL of a highly purified endotoxin, we found neither aggregation nor ATP release in heparinized or citrated human platelet-rich plasma . On the other hand; endotoxin at concentrations as low as a few ng/mL (as may be found in septic patients) caused platelet aggregation in both heparinized and citrated human whole blood, as monitored by change in impedance, free platelet count, and size . Unlike collagen, the platelet aggregation with endotoxin occurred after a long lag phase, developed slowly, and was rarely coupled with measurable release of ATP . The platelet aggregating effect of endotoxin was dose-dependent and modified by exposure of the endotoxin to ionizing radiation . Thus, the activation of human platelets by "solubilized" endotoxin in plasma requires the presence of other blood cells . We propose that the platelet effect is mediated by monocytes and/or neutrophils stimulated by endotoxin.

Biochem Biophys Res Commun, 1988 Jun 16, 153(2), 760 - 6
Mechanism of the EPSP synthase catalyzed reaction: evidence for the lack of a covalent carboxyvinyl intermediate in catalysis; Wibbenmeyer J et al.; In order to detect covalent reaction intermediates in the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase reaction, we have investigated the interaction of EPSP synthase with the reaction product EPSP . An exchange of EPSP-methylene protons could be demonstrated by incubating EPSPS with EPSP in D2O . Since trace amounts of contaminating Pi would lead to reversal of EPSPS reaction and hence methylene proton exchange, we added pyruvate kinase, ADP, Mg++ and K+ . Under these conditions, any contaminating Pi that is converted to PEP is trapped as ATP . No exchange of EPSP protons with those of the solvent could be detected in the presence of this trap system, suggesting that enzyme-bound EPSP is unable to form a covalent tetrahedral complex . Incorporation of {14C} from {14C}-S3P and {14C}-PEP into EPSP could be detected, but only in the absence of a PEP (or Pi) trap system . This indicates that for the exchange reaction, Pi is required, and also indicates the absence of a covalent intermediate, unless the carboxyvinyl-enzyme-bound S3P is completely restricted from exchange.

Biochem J, 1988 Jun 15, 252(3), 909 - 12
Evidence that the pyrromethane cofactor of hydroxymethylbilane synthase (porphobilinogen deaminase) is bound through the sulphur atom of a cysteine residue; Hart GJ et al.; Hydroxymethylbilane synthase (porphobilinogen deaminase) from Escherichia coli uses a novel pyrromethane cofactor to bind the growing pyrrolic chain for hydroxymethylbilane biosynthesis {Hart, Miller, Leeper & Battersby (1987) J . Chem . Soc . Chem . Commun . 1762-1765} . We show that this cofactor is bound to the protein through the sulphur atom of a cysteine residue.

J Biol Chem, 1988 Jun 15, 263(17), 7929 - 32
2-Deoxy-2-fluoro-D-glycosyl fluorides . A new class of specific mechanism-based glycosidase inhibitors; Withers SG et al.; Mechanism-based glycosidase inhibitors are of considerable use in studies of enzyme mechanism, in studies of glycoprotein processing, and possibly therapeutically in control of sugar uptake . This paper describes a new general approach to mechanism-based inactivation of glycosidases which involves trapping a covalent glycosyl enzyme intermediate . This is achieved by use of 2-deoxy-2-fluoro-D-glycosyl fluorides, for which the rate of hydrolysis of the fluoroglycosyl enzyme intermediate is extremely slow, resulting in accumulation of the intermediate . Eleven different glycosidases were tested with their corresponding 2-deoxy-2-fluoro-D-glycosyl fluorides . Eight of the eleven were inactivated, four of them according to pseudo first-order kinetics and four according to a more complex kinetic scheme . The specificity of these inhibitors was investigated by assaying for inhibition of one enzyme with four different 2-deoxy-2-fluoro-D-glycosyl fluorides . Large differences in inactivation rate were observed which paralleled previously observed substrate specificities.

J Biol Chem, 1988 Jun 15, 263(17), 8282 - 7
Chemical and immunological characterization of the 21-kDa ADP-ribosylation factor of adenylate cyclase; Kahn RA et al.; The ADP-ribosylation factor (ARF) is a 21-kDa GTP-binding protein cofactor in the cholera toxin-catalyzed ADP-ribosylation of the stimulatory regulatory subunit of adenylate cyclase . Purified bovine brain ARF was digested with cyanogen bromide, and peptides were purified and sequenced . Approximately 25-30% of the protein was sequenced in this manner . Peptides contained consensus sequences for GTP-binding proteins but were distinct from any of the previously published GTP-binding proteins . Antibodies were raised in rabbits against both protein and synthetic peptide fragments of ARF . Specific ARF immunoreactivity was detected in every eukaryotic tissue or cell examined, including yeast, slime mold, and man . No ARF immunoreactivity was observed when Escherichia coli proteins were tested . Immunoblotting revealed the majority of ARF to be present in the 100,000 x g supernatant . Immunological cross-reactivity with the cytosolic factor indicate that it and ARF are likely to be the same protein . ARF is shown to be myristylated at the amino terminus . The potential role of myristylation in cellular localization is discussed.

Gene, 1988 Jun 15, 66(1), 77 - 85
cDNA cloning and sequence analysis of a chicken gene expressed during the gonadal development and homologous to mammalian cytochrome P-450c17; Ono H et al.; A cDNA clone, pLOA0511, was isolated from the cDNA library prepared from the left ovaries of one- to three-day-old chickens . The transcript corresponding to this cDNA clone is approx . 1.9 kb in length and is present in steroidogenic tissues (ovary, testis and adrenal gland) but is undetectable in non-steroidogenic tissues . Relative abundance of the transcript in the ovary and testis is high and developmentally regulated but is much lower in the adrenal gland . Relative levels of this transcript in the developing left ovary and the regressing right ovary of the one- to three-day-old chicken are similar . The nucleotide sequence of this cDNA clone shows significant homology to some mammalian cytochromes P-450 . Out of the 508 amino acids (aa) coded, 244 and 243 aa residues are identical with those of the bovine and human cytochrome P-450c17 (steroid 17 alpha-hydroxylase/17,20 lyase), respectively . There are three highly conserved regions among the bovine, human and chicken sequences, one of which is unique to P-450c17 . These data suggest strongly that this cDNA corresponds to the mRNA of a chicken cytochrome P-450c17.

Gene, 1988 Jun 15, 66(1), 97 - 106
Structure and characterisation of a duplicated human alpha 1 acid glycoprotein gene; Merritt CM et al.; Human alpha 1-acid glycoprotein (AGP), also known as orosomucoid, is a major acute-phase plasma protein . The amino acid sequence of AGP, which was determined by sequencing from protein isolated from pooled plasma, contained amino acid substitutions in 21 different positions . Genomic and cDNA clones which correspond to one of the possible amino acid sequences have been previously reported . In this paper we present the complete nucleotide sequence of a second gene, AGP2 which is located approx . 3.3 kb downstream from AGP1 . The derived amino acid sequence of AGP2 contains 19 of the possible alternative amino acid substitutions as well as two additional differences . It is clear from the results presented here that the AGP in human plasma is the product of two separate gene loci.

Gene, 1988 Jun 15, 66(1), 87 - 96
Isolation and characterization of the Bos taurus beta-casein gene; Gorodetsky SI et al.; The expression of casein genes in the mammary cells is regulated by peptide and steroid hormones . To investigate the controlling mechanisms we have isolated and characterized the bovine beta-casein gene . The gene has the size of 8.6 kb, which is 7.8 times longer than the corresponding mRNA composed of nine exons . The genomic clones include additional 8.5-kb and 4.5-kb sequences of the 5'- and 3'-flanking regions . We have determined the sequences of the 5' and 3' ends of the gene and compared them with the respective sequences of the rat beta-casein gene . Conserved sequences are identical or homologous to the potential binding sites for nuclear factors and for glucocorticoid and progesterone receptors . The regulatory region contains two different TATA signals and a repeat sequence between them.

J Biol Chem, 1988 Jun 15, 263(17), 8037 - 43
Cloning and expression of cDNA for human endonexin II, a Ca2+ and phospholipid binding protein; Kaplan R et al.; Endonexin II is a member of the family of Ca2+-dependent phospholipid binding proteins known as annexins . We cloned human endonexin II cDNA and expressed it in Escherichia coli . The apparent size and Ca2+-dependent phospholipid binding properties of purified recombinant endonexin II were indistinguishable from those of the placental protein . A single mRNA of approximately 1.6 kilobase pairs was found to be expressed in human cell lines and placenta and was in close agreement with the length of the cDNA clone (1.59 kilobase pairs) . The cDNA predicted a 320-amino acid protein with a sequence that was in agreement with the previously determined partial amino acid sequence of endonexin II isolated from placenta . Endonexin II contained 58, 46, and 43% sequence identity to protein II, calpactin I (p36, protein I), and lipocortin I (p35), respectively . The partial sequence of bovine endonexin I was aligned with the sequence of endonexin II to give 63% sequence identity . Like these other proteins, endonexin II had a 4-fold internal repeat of approximately 70 residues preceded by an amino-terminal domain lacking similarity to the repeated region . It also had significant sequence identity with 67-kDa calelectrin (p68), a protein with an 8-fold internal repeat . Comparing the amino-terminal domains of these four proteins of known sequence revealed that, in general, only endonexin II and protein II had significant sequence identity (29%) . Endonexin II was not phosphorylated by Ca2+/phospholipid-dependent enzyme (protein kinase C) even though it contained a threonine at a position analogous to the protein kinase C phosphorylation sites of lipocortin I, calpactin I, and protein II.

J Biol Chem, 1988 Jun 15, 263(17), 8204 - 9
Conservative replacement of methionine by norleucine in Escherichia coli adenylate kinase; Gilles AM et al.; Escherichia coli grown in limited methionine and excess norleucine media accumulate cyanogen bromide-resistant species of proteins after the methionine supply is exhausted . Bacteria, transformed by recombinant plasmid pIPD37 carrying the adk gene and grown under limiting methionine and excess norleucine, synthesize 16-20% of adenylate kinase molecules having all 6 methionine residues replaced by norleucine . Species showing only partial replacement of methionine residues by norleucine are identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after cyanogen bromide treatment of pure enzyme . Norleucine-substituted adenylate kinase shows structural and catalytic properties similar to the wild-type protein as indicated by circular dichroism spectroscopy and kinetic experiments but exhibits a much higher resistance to hydrogen peroxide inactivation under denaturing conditions.

J Biol Chem, 1988 Jun 15, 263(17), 8072 - 7
Arginine substituted for leucine at position 195 produces a cyclic AMP-independent form of the Escherichia coli cyclic AMP receptor protein; Harman JG et al.; Mutant forms (CRP*) of the Escherichia coli cAMP receptor protein (CRP) that activate CRP-dependent promoters in the absence of the normal allosteric effector (cAMP) have been described . A previous report (Harman, J . G., McKenney, K., and Peterkofsky, A . (1986) J . Biol . Chem . 261, 16332-16339) detailed the properties of three CRP* mutant proteins . One protein, 220 CRP, has amino acid substitutions at positions 127 and 170 and low CRP* activity in vivo . A second protein, 222 CRP, has the amino acid substitutions present in 220 CRP and a third substitution (arginine for leucine) at position 195 . 222 CRP has high CRP* activity in vivo and high apparent affinity for lacP DNA relative to the 220 CRP in vitro . In this report, we evaluate the effect of a single amino acid substitution at position 195 (leucine to arginine) on CRP activity both in vivo and in vitro . Cells (cya delta crp delta/pJH8crpR195) containing R195 CRP were found to exhibit a CRP* phenotype, expressing a variety of CRP-dependent genes in the absence of added cAMP . R195 CRP exhibited both CRP* activity in vitro and increased apparent affinity for cAMP relative to wild-type CRP . CRP titration experiments performed using an in vitro lac transcription system suggest that the isolated substitution of arginine at position 195 does not confer on CRP the high lacP affinity that distinguishes the 220 and 222 forms of CRP . These findings lead us to the conclusion that the effects of multiple mutations in CRP can be both cumulative and interactive.

J Biol Chem, 1988 Jun 15, 263(17), 8003 - 10
The cloning, DNA sequence, and overexpression of the gene araE coding for arabinose-proton symport in Escherichia coli K12; Maiden MC et al.; A lambda placMu1 insertion was made into araE, the gene for arabinose-proton symport in Escherichia coli . A phage containing an araE'-'lacZ fusion was recovered from the lysogen and its restriction map compared with that of the 61-min region of the E . coli genome to establish the gene order thyA araE orf lysR lysA galR; araE was transcribed toward orf . A 4.8-kilobase SalI-EcoRI DNA fragment containing araE was subcloned from the phage lambda d(lysA+ galR+ araE+) into the plasmid vector pBR322 . From this plasmid a 2.8-kilobase HincII-PvuII DNA fragment including araE was sequenced and also subcloned into the expression vector pAD284 . The araE gene was 1416-base pairs long, encoding a hydrophobic protein of 472 amino acids with a calculated Mr of 51,683 . The amino acid sequence was homologous with the xylose-proton symporter of E . coli and the glucose transporters from a human hepatoma HepG2 cell line, human erythrocytes, and rat brain . The overexpressed araE gene product was identified in Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of cell membranes as a protein of apparent Mr 35,000 +/- 1,150 . Arabinose protected this protein against reaction with N-ethylmaleimide.

J Biol Chem, 1988 Jun 15, 263(17), 8066 - 71
Homogeneous Escherichia coli endonuclease IV . Characterization of an enzyme that recognizes oxidative damage in DNA; Levin JD et al.; Agents that act via oxygen-derived free radicals form DNA strand breaks with fragmented sugar residues that block DNA repair synthesis . Using a synthetic DNA substrate with a single type of sugar fragment, 3'-phosphoglycolaldehyde esters, we show that in Escherichia coli extracts the only EDTA-resistant diesterase for these damages depends on the bacterial nfo (endonuclease IV) gene . Endonuclease IV was purified to physical homogeneity (Mr = 31,000) from an E . coli strain carrying the cloned nfo gene and in which the enzyme had been induced with paraquat . Although heat-stable and routinely assayed in the presence of EDTA, endonuclease IV was inactivated in the absence of substrate at 23-50 degrees C by either EDTA or 1,10-phenanthroline, suggesting the presence of an essential metal tightly bound to the protein . Purified endonuclease IV released phosphoglycolaldehyde, phosphate, and intact deoxyribose 5-phosphate from the 3'-end of DNA, all with apparent Km of 5-10 nM . The optimal KCl or NaCl concentration for 3'-phosphoglycolaldehyde release was 50-100 mM . The purified enzyme had endonuclease activity against partially depurinated DNA but lacked significant nonspecific nuclease activities . Endonuclease IV also activated H2O2-damaged DNA for repair synthesis by DNA polymerase I . Thus, endonuclease IV can act on a variety of oxidative damages in DNA, consistent with a role for the enzyme in combating free-radical toxicity.

Biochemistry, 1988 Jun 14, 27(12), 4325 - 31
Carbon-13 and deuterium isotope effects on the catalytic reactions of biotin carboxylase; Tipton PA et al.; 13C and 2H kinetic isotope effects have been used to investigate the mechanism of enzymic biotin carboxylation . D(V/K) is 0.50 in 80% D2O at pD 8.0 for the forward reaction and 0.57 at pD 8.5 for the phosphorylation of ADP by carbamoyl phosphate . These values approach the theoretical maximum limit for a reaction in which a proton is transferred from a sulfhydryl to a nitrogen or oxygen base . Therefore, it appears that this portion of the reaction is at or near equilibrium . 13(V/K) at pH 8 is 1.007; the small magnitude of this number suggests that the reaction is almost fully committed by the time the carbon-sensitive steps are reached . There does not appear to be a reverse commitment to the reaction under the conditions in which 13(V/K) was determined . A large forward commitment is consistent with the failure to observe positional isotope exchange from the beta gamma-bridge position to the beta-nonbridge position in {18O4}ATP or washout of 18O from the gamma-nonbridge positions . Transfer of 18O from bicarbonate to inorganic phosphate in the forward reaction was clearly observed, however . These observations suggest that biotin carboxylase exists in two distinct forms which differ in the protonation states of the two active-site bases, one of which is a sulfhydryl . Only when the sulfhydryl is ionized and the second base protonated can catalysis take place . Carboxylation of biotin is postulated to occur via a pathway in which carboxyphosphate in formed by nucleophilic attack of bicarbonate on ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1988 Jun 14, 27(12), 4222 - 6
Identification of a pterin derivative in Escherichia coli DNA photolyase; Wang BY et al.; DNA photolyase from Escherichia coli contains reduced flavin adenine dinucleotide plus a second chromophore, partially characterized in previous studies . Both chromophores function as sensitizers in catalysis . The second chromophore has been identified as a 6-substituted pterin derivative . The compound is oxidized with permanganate to yield 6-carboxypterin or reduced with sodium cyanoborohydride to yield a 5,6,7,8-tetrahydropterin derivative . The second chromophore exhibits spectral properties (lambda max = 360, 255 nm, pH 2) similar to that observed for 7,8-dihydropterin cations . The compound does not exhibit a spectrally detectable pKa around 4 but is converted to a dication (lambda max = 346, 255 nm) in strong acid (pKa approximately 1) . Similar ionization behavior is observed with 7,8-dihydropterin derivatives that are alkylated at N(5) . The instability of the second chromophore in weakly alkaline solution is due to a fully reversible conversion to a labile bleached form . As compared with other pterin derivatives, the hydrolytic instability is unusual but is very similar to that observed for 5,6-dialkyl-7,8-dihydropterinium salts . It is proposed that the second chromophore is a 7,8-dihydropterin with substituents at positions 5 and 6 . The discovery that a pterin derivative functions as a photosensitizer in DNA repair is apparently the first example of a photobiological function for pterins.

Biochemistry, 1988 Jun 14, 27(12), 4317 - 25
Catalytic mechanism of biotin carboxylase: steady-state kinetic investigations; Tipton PA et al.; Biotin carboxylase was purified from Escherichia coli by a new procedure, and its steady-state kinetic parameters were examined . MgATP and bicarbonate add to the enzyme randomly, followed by addition of biotin . Both bicarbonate and MgATP add in rapid equilibrium . A catalytic base with a pK of 6.6 is observed in V/K profiles . Inactivation studies also revealed a sulfhydryl group in the active site that is essential for catalysis . It is proposed that the acid-base catalysts are necessary for the tautomerization of biotin, which presumably enhances its nucleophilicity toward the carboxyl group donor . A second enzymic group with a pK of 6.6, whose role is unknown, is seen in Vmax profiles . The pH profiles for the biotin carboxylase catalyzed phosphorylation of ADP by carbamoyl phosphate have the same shape as the profiles for the forward reaction, which demonstrates that the enzymic bases assume the same protonation states for catalysis of transphosphorylation in either direction . The lack of reactivity of thionucleotide analogues of ATP when Mg is used as the divalent metal ion suggests that both metal ions required for reaction coordinate to the nucleotide . The second metal ion appears to be absolutely required for reaction and not merely an activator of the reaction . Characterization of a bicabonate-dependent biotin-independent ATPase activity strongly suggests that carboxylation proceeds via a carboxyphosphate intermediate.

Biochemistry, 1988 Jun 14, 27(12), 4391 - 5
Normal cellular Ha ras p21 protein causes local disruption of bilayer phospholipid; Montgomery GW et al.; We have investigated the interactions of the p21 protein of c-Ha ras with its phospholipid environment . Gel filtration of detergent-"solubilized" p21 revealed that this preparation consisted of a mixture of multimolecular aggregates of protein and phospholipid and also a population of individual p21 molecules . Addition of 8 M urea to p21 preparations increased the solubility of the molecule in detergent solutions upon the removal of this denaturant . The progressive addition of the detergent cholate appeared to increase the efficiency of p21 preparations to bind GTP . This affinity for GTP was not removed even at high detergent concentrations, when delipification of the p21 was presumably effected . Modification of the composition of the phospholipid species surrounding the protein did not appear to alter its affinity for GTP . Electron spin resonance studies with membrane spin-labels indicated a perturbation of the bilayer extending to between 44 and 100 phospholipids surrounding the molecule . However, no evidence was found for any population of intimately bound phospholipid, which is seen as an annulus of about 30 lipids in transmembrane proteins such as Ca2+-ATPase . From these results we propose that the Ha ras p21 protein has the ability to associate directly with the membrane in a manner clearly discernible from that of a transmembrane protein.

Nucleic Acids Res, 1988 Jun 10, 16(11), 5089 - 105
Selection of DNA binding sites by regulatory proteins: the LexA protein and the arginine repressor use different strategies for functional specificity; Berg OG; The DNA sequences in the operator sites of the arginine regulon and of the SOS regulon have been subject to a statistical analysis . A quantitative correlation is found between the statistics of sequence choice and the activity at individual operator sites in both systems, as expected from theoretical considerations {Berg & von Hippel, J.Mol.Biol . (1987) 193, 723-750} . Based on these correlations it is possible to predict the effect of various sequence mutations . There is a significant difference in the slopes of the correlation lines between sequence and activity for the two systems . From this difference it can be expected that individual point mutations in the ARG boxes will have a much smaller effect on activity than similar changes in the SOS boxes . This difference may be related to a strong cooperative activity at tandem ARG boxes while the binding at SOS boxes appears to be mostly noncooperative.

Nucleic Acids Res, 1988 Jun 10, 16(11), 5067 - 73
The oriC unwinding by dam methylation in Escherichia coli; Yamaki H et al.; It has been shown that dam methylation is important in the regulation of initiation of DNA replication in E.coli . The question then arises as to whether dam methylation in the oriC region mediates any structural changes in DNA involved in the regulation of initiation of DNA replication . We demonstrate that the thermal melting temperature of the oriC region is lowered by adenine methylation at GATC sites . The regulation of initiation of DNA replication by dam methylation may be attributed to the ease of unwinding at GATC sites in oriC.

Nucleic Acids Res, 1988 Jun 10, 16(11), 4875 - 90
d(GATC) sequences influence Escherichia coli mismatch repair in a distance-dependent manner from positions both upstream and downstream of the mismatch; Bruni R et al.; The role of d(GATC) sites in determining the efficiency of methyl-directed mismatch repair in Escherichia coli was investigated . Transfection of host bacteria, both proficient and deficient in mismatch repair, with a series of artificially constructed M13 heteroduplexes showed that a decrease in the total number of d(GATC) sequences within these vectors lowered the efficiency of repair in vivo . Single hemimethylated d(GATC) sequences were still able to direct the correction event to the unmethylated strand, providing that the mismatch to d(GATC) site distance was shorter than approximately 1 kb . In excess of this distance, the effect of hemimethylated d(GATC) sites on mismatch correction was almost unnoticeable . The directionality of the repair event could be dictated by d(GATC) sequences situated both upstream and downstream of the mispair, suggesting that this important antimutagenic pathway can proceed bidirectionally.

Nucleic Acids Res, 1988 Jun 10, 16(11), 4841 - 51
Circular DNA of 3T6R50 double minute chromosomes; van der Bliek AM et al.; In pulsed field gradient gel electrophoresis (PFGE) the intact deproteinized circular DNA of Mycoplasma (800 kb) and Escherichia coli (4700 kb) remains trapped in the slot . We show here that gamma-irradiation of the DNA in agarose plugs is a convenient method to partially convert these circles into full-length linears, migrating with the expected mobility in PFGE . We have used this method to study the structure of Double Minute chromosomes (DMs) from the methotrexate (MTX)-resistant mouse cell line 3T6R50 . Intact deproteinized DM DNA is immobile in these gels, but is converted into a single band of about 2500 kb by either gamma-irradiation, DNaseI in the presence of Mn2+, or restriction enzymes . We conclude that the DM DNA in 3T6R50 cells consists of a homogeneous population of 2500-kb circles.

Nucleic Acids Res, 1988 Jun 10, 16(11), 5075 - 88
Search for the optimal sequence of the ribosome binding site by random oligonucleotide-directed mutagenesis; Min KT et al.; Synthetic DNA duplexes corresponding to the ribosome binding site (RBS) were synthesized through the phosphite method on solid support . The synthetic RBS DNA with partial random sequences was inserted into an appropriate site between the lpp-lac promoter and the beta-galactosidase structural gene in plasmid pMKT2 . The level of beta-galactosidase expression was correlated with the color intensity of the recombinant colonies on X-gal plates . The bluest colonies were isolated and characterized with respect to beta-galactosidase enzyme activity and RBS sequence . There was good correlation between color intensity and the level of the enzyme activity, and this provided a reliable phenotypic screening method in the search for the optimal regulatory sequences . Novel RBS sequences obtained here show not only the unique nucleotide distribution, but also strong complemetarity to the 3' end region of 16S rRNA, from which could be deduced a generalized RBS sequence, the position of the SD region, and the 16S rRNA position mediated during translation initiation.

Nucleic Acids Res, 1988 Jun 10, 16(11), 5031 - 8
The relative importance of Escherichia coli exonuclease III and endonuclease IV for the hydrolysis of 3'-phosphoglycolate ends in polydeoxynucleotides; Siwek B et al.; In vitro, in the presence of Mg++, the 3'-phosphoglycolatase activity of endonuclease IV is about 4-times smaller than that of exonuclease III for the same AP endonuclease activity . It thus seems that endonuclease IV has only a minor role in the repair of strand breaks limited by 3'-phosphoglycolate ends in Escherichia coli even after the amount of enzyme has been increased by induction with O2 -generating agents.

N Engl J Med, 1988 Jun 9, 318(23), 1481 - 6
Detection of circulating tumor necrosis factor after endotoxin administration; Michie HR et al.; Cytokines, products of stimulated macrophages, are thought to mediate many host responses to bacterial infection, but increased circulating cytokine concentrations have not been detected consistently in infected patients . We measured plasma concentrations of circulating tumor necrosis factor alpha (cachectin), interleukin-1 beta, and gamma interferon, together with physiologic and hormonal responses, in 13 healthy men after intravenous administration of Escherichia coli endotoxin (4 ng per kilogram of body weight) and during a control period of saline administration . Eight additional subjects received ibuprofen before receiving endotoxin or saline . Plasma levels of tumor necrosis factor were generally less than 35 pg per milliliter throughout the control period, but increased 90 to 180 minutes after endotoxin administration to mean peak concentrations of 240 +/- 70 pg per milliliter, as compared with 35 +/- 5 pg per milliliter after saline administration . Host responses were temporally associated with the increase in circulating tumor necrosis factor at 90 minutes, and the extent of symptoms, changes in white-cell count, and production of ACTH were temporally related to the peak concentration of tumor necrosis factor . Ibuprofen pretreatment did not prevent the rise in circulating tumor necrosis factor (mean peak plasma level, 170 +/- 70 pg per milliliter) but greatly attenuated the symptoms and other responses after endotoxin administration . Concentrations of circulating interleukin-1 beta and gamma interferon did not change after endotoxin administration . We conclude that the response to endotoxin is associated with a brief pulse of circulating tumor necrosis factor and that the resultant responses are effected through the cyclooxygenase pathway.

FEBS Lett, 1988 Jun 6, 233(1), 95 - 9
Is translation inhibited by noncognate ternary complexes?
Bilgin N, Ehrenberg M, Kurland C.
We studied the influence of an error-prone isoacceptor (tRNALeu4), as well as an intermediate (tRNALeu2) and a weak (tRNAVal) competitor of tRNAPhe on the poly(Phe) synthesis rate . Even at very high excess concentrations of these noncognate ternary complexes there was no significant effect on the translation rate . Our result argues against the assertion that in vivo translation is slowed down by noncognate tRNA and favours the hypothesis that the incorrect ternary complex concentrations are too low to saturate the ribosomes in vivo.

J Biol Chem, 1988 Jun 5, 263(16), 7830 - 7
Synthesis and expression of a gene coding for the calcium-modulated protein S100 beta and designed for cassette-based, site-directed mutagenesis; Van Eldik LJ et al.; As an initial step in studies aimed at addressing the question of what common and unique features of the S100 family of proteins are related to their specific functions and localizations, a gene coding for one of the S100 proteins, S100 beta, has been prepared by ligation of 12 overlapping, synthetic oligonucleotides . Automated DNA sequence analysis demonstrated that the final construct has the expected structure . The gene was inserted into a plasmid vector that contains a tac promoter and ampicillin-resistance gene, thus allowing both amplification and direct expression cloning in Escherichia coli . The gene was designed to allow rapid, efficient changes of single or multiple amino acids by using cassette-based mutagenesis while the gene is resident in the vector . The expressed protein (VUSB-1) is indistinguishable from bovine brain S100 beta in terms of electrophoretic mobility, reactivity with antibodies to S100 beta, amino acid composition, and partial amino acid sequence analysis . Preparations of expressed protein are also functionally similar to bovine brain S100 beta as determined by aldolase activator activity and neurite extension factor activity, supporting the concept that these activities are a property of the S100 beta polypeptide.

J Biol Chem, 1988 Jun 5, 263(16), 7655 - 9
The NH2-terminal 21 amino acid residues are not essential for the papain-inhibitory activity of oryzacystatin, a member of the cystatin superfamily . Expression of oryzacystatin cDNA and its truncated fragments in Escherichia coli; Abe K et al.; Oryzacystatin, a proteinaceous cysteine proteinase inhibitor (cystatin) in rice, is comprised of 102 residues (Met1-Ala102) (Abe, K., Emori, Y., Kondo, H., Suzuki, K., and Arai, S . (1987) J . Biol . Chem . 262, 16793-16797) . We constructed an expression plasmid containing a full length oryzacystatin cDNA at the multi-cloning site of pUC18 and produced a lacZ'-oryzacystatin fusion protein in Escherichia coli . The partially purified expressed protein efficiently inhibits papain activity assayed using N-benzoyl-DL-arginine-2-naphthylamide as a substrate . We also constructed expression plasmids lacking the 5'- and 3'-regions of cDNAs that encode NH2- and COOH-terminally truncated oryzacystatins . An N-truncated oryzacystatin lacking Gly5 and retaining Gln53-Val54-Val55-Ala56-Gly57 inhibited papain as efficiently as the full length oryzacystatin, although both Gly5 and Gln53-Gly57 (oryzacystatin numbering) are conserved among members of most cystatin superfamilies . However, another N-truncated oryzacystatin lacking the NH2-terminal 38 residues was almost completely inactive . On the other hand, a COOH-terminally truncated oryzacystatin lacking the COOH-terminal 11 residues possesses potent papain-inhibitory activity, whereas another COOH-terminally truncated oryzacystatin lacking 35 residues shows much less inhibitory activity, although it retains the two well conserved features Gly5 and Gln53-Gly57 . These results indicate that the NH2-terminal 21 residues containing Gly5 and the COOH-terminal 11 residues are not essential, suggesting that a portion of the polypeptide segment containing Gln53-Gly57 is necessary for oryzacystatin to elicite its papain-inhibitory activity efficiently.

J Biol Chem, 1988 Jun 5, 263(16), 7620 - 7
Properties of a defined mutant of Escherichia coli thymidylate synthase; Maley GF et al.; A mutant of Escherichia coli thymidylate synthase (F3-TS), resulting from the replacement of a tyrosine for a cysteine 50 amino acids from the amino-terminal end, has been purified to homogeneity and found to contain less than 0.2% of the activity of the native enzyme (thyA-TS) . Although this protein formed a ternary complex with 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) and 5,10-methylenetetrahydrofolate, like the native enzyme, the extent of complex formation was significantly impaired as determined by equilibrium dialysis and circular dichroism . Thus, unlike the native enzyme, where 2 mol of FdUMP were present in each mole of ternary complex, F3-TS contained less than 1 mol of FdUMP/mol of ternary complex . Similarly, the binding of dUMP by F3-TS was greatly diminished relative to thyA-TS, but its binding as well as that of FdUMP could be improved by the presence of either the folate substrate or a tight binding folate analogue, 10-propargyl-5,8-dideazafolate (PDDF) . However, despite the fact that PDDF enhanced the binding of FdUMP and dUMP to F3-TS, the binding of PDDF to the mutant enzyme was also greatly impaired . This contrasts with the native enzyme, which, under the same conditions, bound about 2 mol of PDDF/mol of enzyme in the presence or absence of either FdUMP or dUMP . Circular dichroism analyses with PDDF in the presence of dUMP or FdUMP yielded analogous results, but the effects were less dramatic than those obtained by equilibrium dialysis . Evidence in support of a structural difference between thyA-TS and F3-TS was obtained by demonstrating that the latter protein was 15-fold slower in forming a ternary complex with dUMP and PDDF than the former and that the mutant enzyme was less stable than the native enzyme.

J Biol Chem, 1988 Jun 5, 263(16), 7639 - 45
Structure and expression of the puf operon messenger RNA in rhodospirillum rubrum; Belanger G et al.; In Rhodospirillum rubrum, the genes coding for the alpha and beta polypeptides of the B880 antenna (pufA,B) and the L and M polypeptides of the photoreaction center (pufL,M) are clustered on operon puf . In oxygen-limited cells, the puf mRNA is present as species of 2561, 640, and 617 nucleotides . Aerated cells contain only traces of these mRNAs . The large mRNA encodes the alpha,beta, L, and M polypeptides, whereas the small mRNAs encode only alpha and beta . S1 nuclease protection mapping showed these transcripts to have a common 5' end, immediately downstream of a region of dyad symmetry and at 166 nucleotides upstream of the initiation codon of pufB . The 3' termini of the small transcripts are located in the intercistronic region between pufA and pufL, downstream of another region of dyad symmetry . This region is highly conserved in Rhodospirillum rubrum, Rhodobacter capsulatus, and Rhodobacter sphaeroides and shares 61% sequence similarity with the repetitive extragenic palindromic sequences of Escherichia coli . The slightly heterogeneous 3' termini of the large transcript are downstream of a region of dyad symmetry characteristic of rho-independent transcription termination . Following a shift from oxygen-limited to aerated conditions, the pufL,M and the pufA,B mRNAs decayed with respective half-lives of 9 and 20 min . These high relative stabilities, attributed to secondary structure, are in accord with the mole ratio (2:1) of the pufA,B/pufL,M messages . While the differential expression of alpha,beta/L,M congruent to 15 is thought to be due, in part, to this relative stability, the main factor may be a more efficient translation initiation for pufA,B than for pufL,M.

J Mol Biol, 1988 Jun 5, 201(3), 497 - 506
Effect of point mutations on the in-vitro pore properties of maltoporin, a protein of Escherichia coli outer membrane; Dargent B et al.; Maltoporin (LamB protein), a protein of Escherichia coli outer membrane forms ionic channels with a selectivity for maltose and maltodextrins (Dargent et al., 1987) . The effect of different point mutations on maltoporin pore properties was investigated in vitro with planar bilayers . The mutations belong to three classes in terms of selective maltose transport in vivo: class A (substitution at positions 259 and 382) does not affect maltose transport, class B (position 163 and 245) decreases maltose transport down to 20 to 30%, and class C (position 18) almost completely abolishes selective maltose transport . This in-vitro study reveals that class A does not affect the pore properties in contrast to class B substitutions . The class B maltoporins are still able to form channels but display some specific features and altered specificity for maltose and maltodextrins . The substitution (Gly18----Val) alters trimer stability and impedes pore function (class C mutant) . Thus, there is a good correlation between the specific transport properties of the mutated maltoporins in vivo and their behavior in vitro . These data, in combination with the asymmetric orientation of the protein within the bilayer and topological considerations, indicate that residues 245 and 163 do not belong to the selectivity filter . Mutations at these sites cause hindrance at the mouth of the pore on the outer domain of maltoporin.

J Biol Chem, 1988 Jun 5, 263(16), 7753 - 9
Targeting of porin to the outer membrane of Escherichia coli . Rate of trimer assembly and identification of a dimer intermediate; Reid J et al.; Porin, a transmembrane protein in the outer membrane of Escherichia coli, exists in a trimeric structure which is not dissociated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 25 degrees C . This unusual stability was utilized in the study of the conformational changes which accompany the targeting of porin to the outer membrane . A delay of 16-44 s between completion of synthesis of a monomer and its assembly into a trimer was found from the ratio of monomers to trimers found in exponentially growing cells . Pulse-chase experiments showed that rapid processing of precursor OmpF molecules was followed by assembly into sodium dodecyl sulfate-resistant oligomers with a half-time of 20 s at 30 degrees C . An intermediate in assembly was isolated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis below 10 degrees C and was identified as a metastable dimer.

Science, 1988 Jun 3, 240(4857), 1302 - 9
DNA damage and oxygen radical toxicity; Imlay JA et al.; A major portion of the toxicity of hydrogen peroxide in Escherichia coli is attributed to DNA damage mediated by a Fenton reaction that generates active forms of hydroxyl radicals from hydrogen peroxide, DNA-bound iron, and a constant source of reducing equivalents . Kinetic peculiarities of DNA damage production by hydrogen peroxide in vivo can be reproduced by including DNA in an in vitro Fenton reaction system in which iron catalyzes the univalent reduction of hydrogen peroxide by the reduced form of nicotinamide adenine dinucleotide (NADH) . To minimize the toxicity of oxygen radicals, the cell utilizes scavengers of these radicals and DNA repair enzymes . On the basis of observations with the model system, it is proposed that the cell may also decrease such toxicity by diminishing available NAD(P)H and by utilizing oxygen itself to scavenge active free radicals into superoxide, which is then destroyed by superoxide dismutase.

Cell, 1988 Jun 3, 53(5), 781 - 93
Myogenic lineage determination and differentiation: evidence for a regulatory gene pathway; Pinney DF et al.; Stable myogenic cell lines have been derived at a high frequency by transfection of a cloned multipotential mouse embryo cell line, C3H 10T1/2, with cloned human DNA linked to a selectable neomycin resistance gene . The myogenic phenotype remains linked to neomycin resistance during secondary transfections . Although proliferative in growth conditions, these cell lines maintain the ability to differentiate and express muscle-specific proteins . We conclude that there is a simple genetic basis for myogenic determination and that a single gene, myd, converts 10T1/2 cells to a myoblast lineage . Southern blot analysis demonstrates nonidentity of myd and the MyoD1 gene . Northern blot analysis shows that myd-transfected myogenic lineages express MyoD1 mRNA while parental 10T1/2 cells do not . These results suggest that a dependent regulatory gene pathway mediates myogenic determination and differentiation.

Cell, 1988 Jun 3, 53(5), 713 - 22
Zeste encodes a sequence-specific transcription factor that activates the Ultrabithorax promoter in vitro; Biggin MD et al.; Zeste is a Drosophila regulatory gene that is required for transvection at the bithorax complex . Here we find that purified zeste protein binds to multiple sites just 5' of the initiation site of Ubx RNA . Zeste protein purified from Drosophila cells or from E . coli expressing the zeste gene activates Ubx transcription in vitro . This activation is dependent on the presence of zeste protein binding sites, as it is not observed with a Ubx promoter lacking these sites or with an Adh promoter . These results suggest that transvection involves regulatory elements that act at the level of transcriptional initiation and may be mechanistically similar to activation of transcription by enhancer elements, except that transvection occurs across paired chromosomes . These findings are consistent with the hypothesis that zeste may play a more important role in the normal regulation of Ubx and its other target genes than current genetic evidence implies.

Nature, 1988 Jun 2, 333(6172), 470 - 3
Molecular cloning and expression of the major protein kinase C substrate of platelets; Tyers M et al.; In platelets, agonists that stimulate phosphoinositide turnover cause the rapid phosphorylation of a protein of apparent relative molecular mass (Mr) 40-47,000, called P47, by protein kinase C (PKC) . Diverse identities have been ascribed to P47 including lipocortin, inositol 1,4,5-trisphosphate 5-phosphomonoesterase, pyruvate dehydrogenase alpha subunit and an actin regulatory protein . We have isolated human P47 clones by immunological screening of a lambda gt11 complementary DNA library from HL-60 cells, a human promyelocytic leukaemia cell line . P47 recombinants thus identified hybridized to a 3.0 kilobase (kb) messenger RNA in mature white blood cell lines; the same mRNA was induced in HL-60 cells during differentiation . A 1,050 base pair (bp) open reading frame that could encode a protein of Mr40,087 was confirmed by comparison with peptide sequences from platelet P47, and by expression of the putative recombinant P47 in E . coli and in vitro . The P47 sequence appears to have been conserved throughout vertebrate evolution, and is not similar to any other known sequence including human lipocortin and the alpha subunit of pyruvate dehydrogenase . The P47 protein contains a potential Ca2+-binding 'EF-hand' structure and a region that strongly resembles known PKC phosphorylation sites.

Nature, 1988 Jun 2, 333(6172), 473 - 5
An in vivo recombinant RNA capable of autocatalytic synthesis by Q beta replicase; Munishkin AV et al.; A variety of small RNAs ranging from tens to hundreds of nucleotides in length grow autocatalytically in a Q beta replicase (Q beta phage RNA-dependent RNA polymerase) reaction in the absence of added template, and similar RNAs are found in Q beta phage-infected Escherichia coli cells . Three such RNAs have been sequenced . One of them that is 221 nucleotides (nt) long ('MDV-1' RNA) has been found to be partially homologous to Q beta phage RNA 8, which might be considered as an indication of its origination from by-products of the Q beta RNA replication . To gain further insight into the origin and function of these RNAs, we have sequenced a new RNA, 120 nt long, isolated from the products of spontaneous synthesis by the nominally RNA-free Q beta replicase preparation . The minus strand of this RNA appeared to be a recombinant RNA, composed of the internal fragment of Q beta RNA (approximately 80 nt long) and the 33-nt-long 3'-terminal fragment of E . coli tRNA(1Asp) . This seems to be the first strong indication of RNA recombination in bacterial cells . The various implications of this finding are discussed.

J Appl Physiol, 1988 Jun, 64(6), 2675 - 83
Effects of endotoxemia on the sheep lung microvascular membrane: a two-pore theory; Bradley JD et al.; We analyzed the effects of Escherichia coli endotoxin infusion on pulmonary microvessels in sheep by using a two-pore mathematical model of the microvascular barrier . Five sheep were prepared with lung lymph fistulas and instrumented to measure pulmonary arterial and left atrial pressures . Multiple indicator-dilution curves (with 125I-labeled albumin, 51Cr-labeled erythrocytes, {14C}urea, and 3H2O) were measured at base line and during phases 1 and 2 of the endotoxin response . Alterations in the membrane integrity in response to endotoxin infusion were quantified by using a two-pore theory of the microvascular barrier that incorporated lymph, protein, pressure, and multiple indicator measurements . The modeling results showed a slight change in the size of the pores during phase 1 but a 56% decrease in the number of small pores and a twofold increase in the number of large pores with respect to base-line values . During phase 2 the large pore size increased by 40%, and the total number of pores returned to base-line values . The analysis showed that endotoxin effects on fluid and protein exchange in the lung cannot be explained by hemodynamic and surface area changes alone . An apparent increase in lung microvascular permeability occurs during phases 1 and 2 of the endotoxin reaction, with a substantial decrease in perfused microvascular surface area during phase 1.

J Appl Physiol, 1988 Jun, 64(6), 2463 - 7
Fluid filtration coefficient of isolated goat lungs was unchanged by endotoxin; Winn R et al.; The Starling fluid filtration coefficient (Kf) of blood-perfused excised goat lungs was examined before and after infusion of Escherichia coli endotoxin . Kf was calculated from rate of weight gain as described by Drake et al . {Am . J . Physiol . 234 (Heart Circ . Physiol . 3): H266-H274, 1978} . These calculations were made twice during base line and then at hourly intervals for 5 h after infusion of 5 mg (approximately 250 micrograms/kg) of E . coli endotoxin or after injection of oleic acid (47 microliter/kg) . All lungs were perfused at constant arterial and venous pressure under zone 3 conditions . Base-line Kf averaged 27 +/- 10 and 20 +/- 4 (SD) microliter.min-1.cmH2O-1.g dry wt-1 for endotoxin and oleic acid groups, respectively . It was unchanged in the endotoxin group throughout the experiment but approximately doubled in the oleic acid lungs . Pulmonary arterial and venous pressures were not changed significantly during the course of these experiments in either group . Lung wet-to-dry weight ratios of these lungs were 5.6 +/- 0.6 and 6.1 +/- 0.5 ml/g for the endotoxin and oleic acid groups, respectively . This compares with 4.6 +/- 0.5 ml/g for normal, freshly excised but not perfused goat lungs . The small change in lung water and unchanged pulmonary pressures after both endotoxin and oleic acid suggest that lung injury was minimal . We conclude that 1) endotoxin does not cause a direct injury to the endothelium of isolated lungs during the first 5 h of perfusion, and 2) neutrophils are not sufficient to cause increased Kf after endotoxin infusion in this preparation.

Br J Pharmacol, 1988 Jun, 94(2), 282 - 4
Attenuation of arterial blood pressure fall in endotoxin shock in the rat using the competitive bradykinin antagonist Lys-Lys-{Hyp2, Thi5,8, DPhe7}-Bk (B4148); Weipert J et al.; The selective competitive bradykinin (Bk) antagonist, B4148 (Lys-Lys-{Hyp2, Thi5,8, DPhe7}-Bk) infused at 100 micrograms kg-1 min-1 into rats produced a significant inhibition of the hypotensive effect of Bk and had no effect against acetylcholine-induced responses . In a rat model of endotoxin shock, the fall in mean arterial blood pressure in response to an intravenous injection of lipopolysaccharide from E . coli was significantly attenuated by the same infusion of B4148 compared to controls . These findings suggest that kinins are involved in the hypotensive response to endotoxin shock in rats . The development of potent Bk antagonists offers a new experimental approach for evaluating the role of kinins in this and other disease states and potential therapy in such disorders.

Proc Natl Acad Sci U S A, 1988 Jun, 85(12), 4280 - 4
Probing the salt bridge in the dihydrofolate reductase-methotrexate complex by using the coordinate-coupled free-energy perturbation method; Singh UC; The importance of the ionic interaction due to the formation of the salt bridge between the Asp-27 and the pteridine ring in Escherichia coli dihydrofolate reductase-methotrexate complex has been studied by using the free-energy perturbation method . The calculation suggests that the ion-pair contribution to the binding energy is insignificant, as the enzyme surroundings do not stabilize the salt bridge to the extent of the desolvation of the charged groups . The activation barrier for the proton exchange between the pteridine ring and the Asp-27 is calculated to be 20.1 kcal/mol (1 cal = 4.184 J) by using the coordinate-coupled perturbation method, implying that this may be a channel to the proton exchange from the pteridine ring to the solvent . The Gibbs-energy difference of binding between the Asn-27 and Ser-27 is calculated to be 3.2 kcal/mol and is mainly due to the electrostatic interactions.

Mutat Res, 1988 Jun, 208(2), 101 - 4
Induction of chromatid aberrations by TEM and maleic hydrazide is differently affected by pretreatment of Vicia faba root-tip meristems with methyl iodide; Rieger R et al.; Treatment of Vicia faba main root meristems with methyl iodide (MeI) 2 h before challenge treatment with triethylene melamine (TEM) significantly reduced the yield of metaphases with chromatid aberrations, i.e., resulted in clastogenic adaptation . Combined treatment with MeI and TEM increased the aberration yield; MeI treatment alone (10(-3) M, 0.5 h) was without clastogenic effect . No protective effects were observed after MeI pretreatment and challenge treatment by maleic hydrazide (MH) . The data obtained in V . faba are compared to those previously reported for E . coli.

Circ Shock, 1988 Jun, 25(2), 75 - 83
Amrinone administration in endotoxin shock; Vincent JL et al.; This study explored the hemodynamic effects of amrinone, a phosphodiesterase inhibitor, in association with intravenous fluids, in the treatment of endotoxin shock . Mongrel dogs were anesthetized with pentobarbital and mechanically ventilated with room air . Treatment was started 30 min after slow intravenous administration of 3 mg/kg of E . coli endotoxin . In the first part of the study, ten dogs were resuscitated for 30 min with intravenous saline alone (10 ml/kg) and for the next 3 h by saline (10 ml/kg/h) and amrinone 40 micrograms/kg/min . During this latter period, arterial pressure remained stable while cardiac output significantly increased from 3.1 +/- 0.5 to 5.2 +/- 0.7 l/min (P less than 0.01), and oxygen delivery increased from 616 +/- 92 to 983 +/- 156 ml/min (P less than 0.01) . Comparison with control animals revealed that amrinone infusion prevented the decrease in left ventricular stroke work and markedly increased oxygen delivery . In the second part of the study, 18 dogs were treated by saline infusion titrated to maintain pulmonary artery balloon-occluded pressure at baseline level . In ten dogs, amrinone was added 60 min after endotoxin administration at a dose of 40 micrograms/kg/min . Total amount of fluids infused averaged 87 +/- 14 ml in the amrinone-treated dogs and 64 +/- 15 ml in the control dogs (differences nonsignificant) . Oxygen delivery and oxygen consumption increased significantly in the amrinone-treated dogs (from 541 +/- 36 to 1063 +/- 176 ml/min, P less than 0.01, and from 145 +/- 23 to 202 +/- 38 ml/min, P less than 0.01, respectively) but not in the control dogs . The amrinone-treated dogs had lower PaO2 and higher venous admixture than the control dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Circ Shock, 1988 Jun, 25(2), 61 - 74
Cardiopulmonary changes with intermittent endotoxin administration in sheep; Godsoe A et al.; Chronic sepsis was induced by administering endotoxin (lipopolysaccharide--LPS) at 12-hr intervals to sheep . The animals (n = 7) responded to the first dose of LPS with increased pulmonary arterial pressure (PAP), systemic vascular resistance, plasma and lymph thromboxane B2 (TxB2) concentrations, and lung lymph flow rate concurrent with a reduction in the cardiac index (CI) . Subsequent doses of LPS produced an elevation of PAP and TxB2 which was progressively attenuated and eventually disappeared . With LPS the lung lymph flow was markedly elevated and CI increased . This latter was transient and associated with a reduction in systemic vascular resistance . Concomitant with the cardiopulmonary changes prekallikrein levels were not diminished, but there was a statistically significant reduction in C1-esterase inhibitor . The administration of LPS was discontinued after 5 days and the cardiopulmonary variables rapidly returned to baseline levels . Chronic endotoxemia appears to be associated with an elevated pulmonary microvascular permeability and a tendency toward a hyperdynamic circulation but with an appreciable degree of refractoriness associated with regional hemodynamics and eicosanoid biosynthesis.

Circ Shock, 1988 Jun, 25(2), 103 - 9
Effects of chronic endotoxaemia on oxygen consumption at different ambient temperatures in the unanaesthetised rat; Goran MI et al.; Oxygen consumption has been measured at different ambient temperatures at intervals during the intravenous infusion of endotoxin (1 mg/kg.day-1) from a subcutaneously implanted osmotic minipump in unanaesthetised rats . On day 1 of the infusion oxygen consumption was elevated at ambient temperatures of 10, 28, and 31 degrees C but not at 20 degrees C, compared with pair-fed saline-infused controls . There was a significant negative correlation between oxygen consumption on days 1 and 3 and environmental temperature (10, 20, and 28 degrees C) in both groups, but the regression line describing the relation in endotoxin-infused rats was displaced above that for the saline-infused control without a change in slope . The "minimal observed" oxygen consumption, which is taken as an estimate of basal metabolic rate, was elevated by the infusion of endotoxin . The endotoxin-induced increase in "minimal observed" oxygen consumption was removed by indomethacin (5 mg/kg.sc) on day 1 of the infusion but was ineffective on days 3 and 7.

Biull Eksp Biol Med, 1988 Jun, 105(6), 730 - 3
{Ultrastructural mechanisms of the effect of myelopeptides on brain lesions in rats receiving endotoxin and the role of receptor-mediated endocytosis}; Bardakhch'ian EA et al.; It has been demonstrated that myelopeptide is capable of preventing some ultrastructural alterations that take place in the brain in response to intravenous lipopolysaccharide injection by neuronal receptor blockade.

J Cell Biol, 1988 Jun, 106(6), 2011 - 22
Generation of antisera that discriminate among mammalian alpha-tubulins: introduction of specialized isotypes into cultured cells results in their coassembly without disruption of normal microtubule function; Gu W et al.; To assay the functional significance of the multiple but closely related alpha-tubulin polypeptides that are expressed in mammalian cells, we generated three specific immune sera, each of which uniquely recognizes a distinct alpha-tubulin isotype . All three isotypes are expressed in a tissue-restricted manner: one (M alpha 3/7) only in mature testis, one (M alpha 4) mainly in muscle and brain, and the third (M alpha 6) in several tissues at a very low level . A fourth specific antiserum was also generated that distinguishes between the tyrosinated and nontyrosinated form of a single alpha-tubulin isotype . Because individual tubulin isotypes cannot be purified biochemically, these sera were raised using cloned fusion proteins purified from host Escherichia coli cells . To suppress the immune response to shared epitopes, animals were first rendered tolerant to fusion proteins encoding all but one of the known mammalian alpha-tubulin isotypes . Subsequent challenge with the remaining fusion protein then resulted in the elicitation of an immune response to unique epitopes . Three criteria were used to establish the specificity of the resulting sera: (a) their ability to discriminate among cloned fusion proteins representing all the known mammalian alpha-tubulin isotypes; (b) their ability to uniquely detect alpha-tubulin in whole extracts of tissues; and (c) their capacity to stain microtubules in fixed preparations of cells transfected with sequences encoding the corresponding isotype . The transfection experiments served to demonstrate (a) the coassembly of M alpha 3/7, M alpha 4, and M alpha 6 into both interphase and spindle microtubules in HeLa cells and NIH 3T3 cells, and (b) that the M alpha 4 isotype, which is unique among mammalian alpha-tubulins in that it lacks an encoded carboxy-terminal tyrosine residue, behaves like other alpha-tubulin isotypes with respect to the cycle of tyrosination/detyrosination that occurs in most cultured cells.

Proc Natl Acad Sci U S A, 1988 Jun, 85(12), 4406 - 10
DNA base sequence changes and sequence specificity of bromodeoxyuridine-induced mutations in mammalian cells; Davidson RL et al.; By using a shuttle vector system developed in our laboratory, we have carried out studies on the molecular mechanism by which 5-bromodeoxyuridine (BrdUrd) induces mutations in mammalian cells . The target for mutagenesis in these studies was the Escherichia coli gpt gene that was contained within a retroviral shuttle vector and integrated into chromosomal DNA in mouse A9 cells . Shuttle vector-transformed cells expressing the gpt gene were mutagenized with BrdUrd and cells with mutations in the gpt gene were selected . Shuttle vector sequences were recovered from the mutant cells, and the base sequence of the mutant gpt genes was determined . The great majority of the BrdUrd-induced mutations involving single-base changes were found to be G.C----A.T transitions . We have shown that mutagenesis by BrdUrd depends upon perturbation of deoxycytidine metabolism . Thus, the current results suggest that BrdUrd mutagenesis involves mispairing and misincorporation of BrdUrd opposite guanine in DNA, driven by nucleotide pool perturbation caused by BrdUrd and the resulting imbalanced supply of triphosphates available for DNA synthesis . The results also revealed a very high degree of sequence specificity for the BrdUrd mutagenesis . BrdUrd-induced G.C----A.T transitions occurred almost exclusively in sequences with two adjacent guanine residues . Furthermore, in approximately equal to 90% of the cases, the guanine residue involved in mutation was the one in the more 3' position.

Proc Natl Acad Sci U S A, 1988 Jun, 85(12), 4276 - 8
Use of site-directed mutagenesis to elucidate the role of arginine-166 in the catalytic mechanism of alkaline phosphatase; Butler-Ransohoff JE et al.; The guanidinium group of arginine-166 has been postulated to act as an electrophilic species during phosphorylation of alkaline phosphatase . Its role could be either to stabilize the developing negative charge on the oxygen of the leaving group or the pentacoordinate transition state or to help bind the -PO2-3 group . We have produced via site-directed mutagenesis two Escherichia coli alkaline phosphatase mutants (lysine-166 and glutamine-166) to test whether the guanidinium group plays a critical role in catalysis . Comparative kinetic characterization of the lysine-166 and glutamine-166 mutants indicates that the charge at residue 166 is not required for the hydrolysis of phosphate monoesters . Small decreases in kcat are observed for both the lysine and glutamine mutants, relative to the wild-type enzyme, but the value for the uncharged glutamine mutant is only one-third that of lysine . Thus, the stabilizing effect of the positively charged guanidinium group does not appear to play a major role in the rate-limiting step for substrate hydrolysis . A significant effect on the Km value is seen only for the glutamine mutant.

Proc Natl Acad Sci U S A, 1988 Jun, 85(12), 4242 - 6
Codon choice and gene expression: synonymous codons differ in their ability to direct aminoacylated-transfer RNA binding to ribosomes in vitro; Thomas LK et al.; Phe-tRNA (anticodon GAA)--polypeptide-chain elongation factor Tu-GTP ternary complexes react faster with ribosomes programmed with UUC codons than with ribosomes programmed with UUU codons . A similar preference is shown by Leu-tRNA2 (anticodon GAG) complexes, which react faster with ribosomes programmed with CUC than with those programmed with CUU . The difference is seen in the rate of ternary-complex binding to the ribosome; no differences are seen in peptide-bond formation . Highly expressed mRNAs in Escherichia coli favor codons terminating in cytosine rather than uracil when both codons are read by a single tRNA with an anticodon beginning with guanine . The results suggest that intrinsic differences between the efficiencies of synonymous codons play an important role in modulating gene expression in E . coli.

Proc Natl Acad Sci U S A, 1988 Jun, 85(12), 4148 - 52
Aspartic acid substitutions affect proton translocation by bacteriorhodopsin; Mogi T et al.; We have substituted each of the aspartic acid residues in bacteriorhodopsin to determine their possible role in proton translocation by this protein . The aspartic acid residues were replaced by asparagines; in addition, Asp-85, -96, -115, and -112 were changed to glutamic acid and Asp-212 was also replaced by alanine . The mutant bacteriorhodopsin genes were expressed in Escherichia coli and the proteins were purified . The mutant proteins all regenerated bacteriorhodopsin-like chromophores when treated with a detergent-phospholipid mixture and retinal . However, the rates of regeneration of the chromophores and their lambda max varied widely . No support was obtained for the external point charge model for the opsin shift . The Asp-85----Asn mutant showed not detectable proton pumping, the Asp-96----Asn and Asp-212----Glu mutants showed less than 10% and the Asp-115----Glu mutant showed approximately equal to 30% of the normal proton pumping . The implications of these findings for possible mechanisms of proton translocation by bacteriorhodopsin are discussed.

Radiat Res, 1988 Jun, 114(3), 550 - 5
Mechanisms of the radioprotective effect of cysteamine in Escherichia coli; Korystov YuN et al.; The values of the oxygen effect (m) and the maximal protective effect of cysteamine (DMF*) were estimated for four Escherichia coli strains: AB1157 (wild type), AB1886 (uvrA), AB2463 (recA), and p3478 (polA) . A correlation made between DMF* and m as well as the kinetics of the increase of DMF with oxygen depletion showed that the protective effect of cysteamine is realized by three mechanisms: (i) anoxia achieved by oxygen reduction, with the DMF varying from 2.2 to 4.2 for different E . coli strains (this protection is the major contribution to the entire mechanism); (ii) lowering of the indirect radiation effect; i.e., for 50 mM cysteamine DMF does not exceed 1.1; and (iii) increase of the efficiency of enzymatic repair . The latter effect of cysteamine is registered only with the wild-type E . coli, the DMF being not less than 1.4.

J Urol, 1988 Jun, 139(6), 1323 - 4
Successful transurethral drainage of bilateral seminal vesicle abscesses; Frye K et al.; We report the successful management of bilateral seminal vesicle abscesses with transurethral unroofing and drainage of the abscess cavities . The diagnosis was confirmed by computerized tomography . Transurethral drainage became necessary after percutaneous drainage had proved to be inadequate.

J Bacteriol, 1988 Jun, 170(6), 2886 - 9
Complete nucleotide sequence of the 16S rRNA gene of Mycobacterium bovis BCG; Suzuki Y et al.; The complete nucleotide sequence of the 16S rRNA gene of Mycobacterium bovis BCG was determined . Its coding region was estimated to be 1,536 base pairs long . The nucleotide sequence of the gene in M . bovis BCG has homologies of 75 and 89% with those of Escherichia coli and Streptomyces lividans, respectively.

J Bacteriol, 1988 Jun, 170(6), 2639 - 45
Truncated forms of Escherichia coli lactose permease: models for study of biosynthesis and membrane insertion; Stochaj U et al.; Using in vitro DNA manipulations, we constructed different lacY alleles encoding mutant proteins of the Escherichia coli lactose carrier . With respect to structural models developed for lactose permease, the truncated polypeptides represent model systems containing approximately one, two, four, and five of the N-terminal membrane-spanning alpha-helices . In addition, a protein carrying a deletion of predicted helices 3 and 4 was obtained . The different proteins were radiolabeled in plasmid-bearing E . coli minicells and were found to be stably integrated into the lipid bilayer . The truncated polypeptides of 50, 71, 143, and 174 N-terminal amino acid residues resembled the wild-type protein in their solubilization characteristics, whereas the mutant protein carrying an internal deletion of amino acid residues 72 to 142 of the lactose carrier behaved differently . Minicell membrane vesicles containing truncated proteins comprising amino acid residues 1 to 143 or 1 to 174 were subjected to limited proteolysis . Upon digestion with proteases of different specificities, the same characteristic fragment that was also produced from the membrane-associated wild-type protein was found to accumulate under these conditions . It has previously been shown to contain the intact N terminus of lactose permease . This supports the idea of an independent folding and membrane insertion of this segment even in the absence of the C-terminal part of the molecule . The results suggest that the N-terminal region of the lactose permease represents a well-defined structural domain.

J Bacteriol, 1988 Jun, 170(6), 2555 - 9
Different effects of recJ and recN mutations on the postreplication repair of UV-damaged DNA in Escherichia coli K-12; Wang TC et al.; Two mutations known to affect recombination in a recB recC sbsBC strain, recJ284::Tn10 and recN262, were examined for their effects on the postreplication repair of UV-damaged DNA . The recJ mutation did not affect the UV radiation sensitivity of uvrB and uvrB recF cells, but it increased the sensitivity of uvrB recN (approximately 3-fold) and uvrB recB (approximately 8-fold) cells . On the other hand, the recN mutation did not affect the UV sensitivity of uvrB recB cells, but it increased the sensitivity of uvrB (approximately 1.5-fold) and uvrB recF (approximately 4-fold) cells . DNA repair studies indicated that the recN mutation produced a partial deficiency in the postreplication repair of DNA double-strand breaks that arise from unrepaired daughter strand gaps, while the recJ mutation produced a deficiency in the repair of daughter strand gaps in uvrB recB cells (but not in uvrB cells) and a deficiency in the repair of both daughter strand gaps and double-strand breaks in uvrA recB recC shcBC cells . Together, these results indicate that the recJ and recN genes are involved in different aspects of postreplication repair.

J Bacteriol, 1988 Jun, 170(6), 2549 - 54
Perturbed chromosomal replication in recA mutants of Escherichia coli; Skarstad K et al.; When initiation of DNA replication is inhibited in wild-type Escherichia coli cells by rifampin or chloramphenicol, completion of ongoing rounds of replication (runout of replication) leads to cells containing two, four, or eight fully replicated chromosomes, as measured by flow cytometry . In recombination-deficient recA strains, a high frequency of cells with three, five, six, or seven fully replicated chromosomes was observed in addition to cells with two, four, or eight chromosomes . recA mutants affected only in the protease-stimulating function behaved like wild-type cells . Thus, in the absence of the recombinase function of RecA protein, the frequency of productive initiations was significantly reduced compared with that in its presence . DNA degradation during runout of replication in the presence of rifampin was about 15% . The DNA degradation necessary to account for the whole effect described above was in this range or even lower . However, a model involving selective and complete degradation of partially replicated chromosomes is considered unlikely . It is suggested that the lack of RecA protein causes initiations or newly formed replication forks to stall but remain reactivatable for a period of time by functional RecA protein.

J Bacteriol, 1988 Jun, 170(6), 2543 - 8
Evidence that the fadB gene of the fadAB operon of Escherichia coli encodes 3-hydroxyacyl-coenzyme A (CoA) epimerase, delta 3-cis-delta 2-trans-enoyl-CoA isomerase, and enoyl-CoA hydratase in addition to 3-hydroxyacyl-CoA dehydrogenase; Yang SY et al.; Genetic complementation of a mutant defective in fatty acid oxidation (fadAB) with plasmids containing DNA inserts from the fadAB region of the Escherichia coli genome was studied . The mutant containing the hybrid plasmid with a 5.2-kilobase (kb) PstI-SalI fragment was found to overproduce 3-hydroxyacyl-coenzyme A (CoA) epimerase and delta 3-cis-delta 2-trans-enoyl-CoA isomerase as well as three other beta-oxidation enzymes by 16- to 18-fold compared with the wild-type parental strain LE392 . The purification of a fully functional multienzyme complex of fatty acid oxidation from the transformant ultimately established that the 5.2-kb DNA fragment contained an entire fadAB operon . Since immunotitration of cell extracts with antibodies against the fatty acid oxidation complex proved that all 3-hydroxyacyl-CoA epimerase and delta 3-cis-delta 2-trans-enoyl-CoA isomerase activities were associated with the complex, no genetic loci other than the fadAB operon encoded these two enzymes . Moreover, the binding of antibodies caused parallel inhibition of four component enzymes, whereas 3-ketoacyl-CoA thiolase activity was slightly increased . These findings support the suggestion that the epimerase and isomerase as well as enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase are located on the same polypeptide . The results of this study, together with published data (S.-Y . Yang and H . Schulz, J . Biol . Chem . 258:9780-9785, 1983), lead to the conclusion that 3-hydroxyacyl-CoA epimerase, delta 3-cis-delta 2-trans-enoyl-CoA isomerase, and enoyl-CoA hydratase in addition to 3-hydroxyacyl-CoA dehydrogenase are encoded by the fadB gene.

J Bacteriol, 1988 Jun, 170(6), 2521 - 6
Cloning of the C-terminal cytoplasmic fragment of the tar protein and effects of the fragment on chemotaxis of Escherichia coli; Oosawa K et al.; A gene encoding only the C-terminal portion of the receptor-transducer protein Tar of Escherichia coli was constructed . The gene product was detected and localized in the cytoplasmic fraction of the cell by immunoblotting with anti-Tar antibodies . The C-terminal fragments from wild-type and mutant tar genes were characterized in vivo . The C-terminal fragment generated from tar-526, a mutation that results in a dominant "tumble" phenotype, was found to be deamidated and methylated by the CheB and CheR proteins, respectively . The C-terminal fragment derived from a wild-type gene was poorly deamidated, and the C-terminal fragment derived from tar-529, a dominant mutant with a "smooth swimming" phenotype, was not apparently modified . Cells carrying the C-terminal fragment with the tar-526 mutation as the sole receptor-transducer protein showed a high frequency of tumbling and chemotaxis responses to changes in intracellular pH . These results suggest that the cytoplasmic C-terminal fragment of Tar retains some of the functions of the whole protein in vivo.

J Bacteriol, 1988 Jun, 170(6), 2485 - 92
Mutations in the leader sequence and initiation codon of the gene for ribosomal protein S20 (rpsT) affect both translational efficiency and autoregulation; Parsons GD et al.; We have transferred the complete structural gene and part of the leader for ribosomal protein S20 of Escherichia coli to a controllable expression vector and have used oligonucleotide-directed mutagenesis to create mutations in the untranslated leader of the plasmid-borne gene . We have assayed for posttranscriptional regulation of the synthesis of S20 after inducing transcription of the mutant S20 mRNA from the expression vector . We found that two mutations lead to loss of feedback control of S20 synthesis: (i) a change of the initiation codon from UUG to AUG and (ii) a replacement of part of the S20 leader with a nonhomologous sequence including an AUG initiation codon . These mutations also lead to increases in both the intrinsic translational efficiency of the plasmid-encoded S20 mRNA in vitro and its half-life in vivo . A double mutation (GA to CT) at residues -3 and -4 relative to the initiation codon does not result in overproduction of S20 . Rather, it reduces translational efficiency in vitro and mRNA stability in vivo . Our results demonstrate the fundamental importance of the UUG initiation codon in mediating autogenous repression of S20 synthesis.

J Bacteriol, 1988 Jun, 170(6), 2457 - 61
Osmotic regulation of biosynthesis of membrane-derived oligosaccharides in Escherichia coli; Kennedy EP et al.; The osmotic regulation of the biosynthesis of membrane-derived oligosaccharides (MDO) in strains UB1005 and DC2 of Escherichia coli K-12 was examined; this regulation was previously reported by Clark (J . Bacteriol . 161:1049-1053, 1985) to be different from that observed by Kennedy for other strains of E . coli (Proc . Natl . Acad . Sci . USA 79:1092-1095, 1982) . Osmotic regulation of the synthesis of MDO in UB1005 and DC2 is in fact indistinguishable from that previously reported for other strains of E . coli, with maximum production of MDO occurring in the medium of lowest osmolarity . The report of Clark to the contrary was apparently based on the inadequate methods for the measurement of MDO employed in that study . MDO are localized in the periplasm of wild-type E . coli cells . However, strain DC2, selected for hypersensitivity to a range of antibiotics, released most of its MDO into the medium, apparently as a result of greater outer membrane permeability.

J Bacteriol, 1988 Jun, 170(6), 2448 - 56
Nucleotide sequence and gene-polypeptide relationships of the glpABC operon encoding the anaerobic sn-glycerol-3-phosphate dehydrogenase of Escherichia coli K-12; Cole ST et al.; The nucleotide sequence of a 4.8-kilobase SacII-PstI fragment encoding the anaerobic glycerol-3-phosphate dehydrogenase operon of Escherichia coli has been determined . The operon consists of three open reading frames, glpABC, encoding polypeptides of molecular weight 62,000, 43,000, and 44,000, respectively . The 62,000- and 43,000-dalton subunits corresponded to the catalytic GlpAB dimer . The larger GlpA subunit contained a putative flavin adenine dinucleotide-binding site, and the smaller GlpB subunit contained a possible flavin mononucleotide-binding domain . The GlpC subunit contained two cysteine clusters typical of iron-sulfur-binding domains . This subunit was tightly associated with the envelope fraction and may function as the membrane anchor for the GlpAB dimer . Analysis of the GlpC primary structure indicated that the protein lacked extended hydrophobic sequences with the potential to form alpha-helices but did contain several long segments capable of forming transmembrane amphipathic helices.

Int J Radiat Biol Relat Stud Phys Chem Med, 1988 Jun, 53(6), 977 - 82
In vivo adenylate cyclase activity in ultraviolet- and gamma-irradiated Escherichia coli; Chatterjee A et al.; The incorporation of {14C}adenine into the cyclic AMP fraction by whole cells of Escherichia coli B/r was taken as a measure of the in vivo adenylate cyclase activity . This activity was significantly inhibited by irradiation of the cells either with 60Co gamma-rays or with UV light from a germicidal lamp, suggesting inhibition of cyclic AMP synthesis . The incubation of cells after irradiation with lower doses (50-100 Gy) of gamma-rays produced a significant increase of in vivo adenylate cyclase activity, whereas there was no significant change after higher doses (150 Gy and above) . Dark incubation of cells after irradiation with UV light (54 J m-2) led to recovery of enzyme activity to the level measured in unirradiated cells . Thus it appears that the catabolite repression of L-arabinose isomerase induced by UV light, as well as gamma-irradiation, is due to reduced cyclic AMP synthesis in irradiated cells.

Infect Immun, 1988 Jun, 56(6), 1513 - 7
Molecular analysis of enterotoxin plasmids of enterotoxigenic Escherichia coli of 14 different O serotypes; Danbara H et al.; A total of 104 isolates of enterotoxigenic Escherichia coli derived from diarrheal patients from more than 10 countries were examined for serotype and toxigenicity . The transferability and molecular structure of the enterotoxin plasmids from each isolate were also examined . Enterotoxin plasmids from serotypes such as O6, O25, O27, O126, O128, and O159, which are frequently associated with E . coli diarrhea (classical strains) generally did not transfer by conjugation from clinical isolates, whereas those from serotypes such as O7, O17, O80, O98, O139, O150, and O153, which are rarely associated with diarrhea (rare strains) transferred almost always from the clinical isolates by conjugation . Analyses of enterotoxin plasmids by restriction endonucleases and DNA-DNA hybridization with the enterotoxin probes revealed that the strains with the same O serotype and toxigenicity carry closely related enterotoxin plasmids . These results suggest that classical strains resulted from the dissemination of ancestral clones which received enterotoxin plasmids long ago, while the rare strains acquired the enterotoxin plasmids recently by conjugation and have not yet been spread to the same degree as the ancestral clones.

Arch Surg, 1988 Jun, 123(6), 714 - 7
Moxalactam vs tobramycin-clindamycin . A randomized trial in secondary peritonitis; Stellato TA et al.; One hundred five patients with peritonitis were randomized to receive either tobramycin sulfate plus clindamycin phosphate or moxalactam alone before surgical intervention . Fifty-nine patients were evaluable . A mean of 3.1 (moxalactam) and 3.5 (tobramycin-clindamycin) pathogens per patient were identified . Overall success rate was 85% (tobramycin-clindamycin, 24/30; moxalactam, 26/29) . When patients with appendicitis were excluded, there was an observed but not statistically significant advantage of moxalactam over tobramycin-clindamycin (85% vs 67%) . There were five deaths (tobramycin-clindamycin, four; moxalactam, one) . Other complications included hypoprothrombinemia (tobramycin-clindamycin, five; moxalactam, five), renal dysfunction (tobramycin-clindamycin, three; moxalactam, one), and superinfection (tobramycin-clindamycin, nine; moxalactam, six) . More wound infections were noted in the group given tobramycin-clindamycin . These data suggest that moxalactam is as safe and efficacious as tobramycin plus clindamycin . The observed benefits of this agent warrant study in a larger sample to verify advantages of moxalactam over combination therapy.

Cancer Res, 1988 Jun 1, 48(11), 2969 - 74
Identification of the human papillomavirus type 18 E6 and E6 proteins in nuclear protein fractions from human cervical carcinoma cells grown in the nude mouse or in vitro; Schneider-Gadicke A et al.; We recently reported the transcription patterns of human papillomavirus (HPV) type 18 sequences in human cervical carcinoma cell lines . The open reading frames (ORFs) E6* and E6 represent the 5'-terminal cistrons in HPV18 mRNAs . ORF E6* was assumed to be specific for HPV types associated with genital carcinomas . To identify the predicted gene product, ORF E6* from a HeLa cDNA clone was expressed as an MS2 fusion protein in Escherichia coli . The C-terminal 23 amino acid residues were chemically synthesized . A panel of monoclonal antibodies was generated, recognizing E6* and E6* plus E6, respectively . In human cervical carcinoma cell lines grown in vitro these monoclonal antibodies specifically immunoprecipitate the putative Mr 17,000 and 18,000 HPV18 E6 proteins in nuclear protein fractions . In a HPV18 DNA containing human cervical carcinoma established in nude mice, these monoclonal antibodies specifically immunoprecipitate a polypeptide with a molecular weight of 6500 as predicted for the HPV18 ORF E6* gene product in a nuclear protein fraction.

AIDS Res Hum Retroviruses, 1988 Jun, 4(3), 199 - 210
B and T cell reactivities after immunization of macaques with HIV subcomponents; Wahren B et al.; A model system was established for studies of humoral and cellular immunity to human immunodeficiency virus (HIV) antigens after vaccination . Macaques (Macaca fascicularis) were immunized with purified HIV, an infected cell extract rich in gp120 or polypeptides of cloned genes representing parts of p24, gp41, and gp120 . Western blot analysis best showed the appearance of antibodies to nucleocapsid proteins, and antibodies to higher molecular weight envelope glycoproteins were better demonstrated by radioimmunoprecipitation . With whole HIV, antibodies to p24 appeared first, and sometimes were the only ones to be demonstrable . Several immunizations with HIV or recombinant polypeptides were required to obtain antibodies to gp120, and the responses were weak . Although the envelope-specific response was weak, this was the only component that mediated neutralizing capacity . Escherichia coli-derived viral transmembrane polypeptide (g)p41 also had a poor immunizing effect . IgG synthesis from B cells in vitro was demonstrable to antigens and generally paralleled the antibody titers of sera after multiple immunizations . The HIV-specific lymphocyte proliferation response as measured by DNA synthesis was best seen with polypeptide p24-15, followed by the other antigens.

Scand J Immunol, 1988 Jun, 27(6), 705 - 16
Endotoxin-stimulated human monocyte secretion of interleukin 1, tumour necrosis factor alpha, and prostaglandin E2 shows stable interindividual differences; Molvig J et al.; The secretions of interleukin 1 (IL-1), tumour necrosis factor alpha (TNF), and prostaglandin E2 (PGE2) of low-dose E . coli lipopolysaccharide (LPS)-stimulated human monocytes (M phi) were investigated in an endotoxin (ET)-free milieu (less than 1.6 pg LPS/ml) . Human M phi cultures from nine healthy men were stimulated with 0, 12.5-500, and 250,000 pg LPS/ml as measured by a very sensitive Limulus test . The IL-1 activity was tested by the mouse costimulatory thymocyte (LAF) assay, which was thoroughly standardized and characterized (interassay variation 22-24%, intra-assay variation 3-7%) . Spontaneous M phi secretions of IL-1, TNF, and PGE2 were negligible, but 12.5 pg LPS/ml significantly stimulated the secretions of these M phi products and the monokine responses to 500 and 250,000 pg LPS/ml were almost in the same range . It was demonstrated that the secretions of IL-1-TNF and TNF-PGE2 were strongly correlated . Pronounced interindividual differences in LPS responsiveness were demonstrated, and two low-responders, one of whom was HLA-DR1,2-positive, were identified . Three first-degree relatives of the DR1,2-positive low-responder had similar low responses . Furthermore, M phi cultures were prepared weekly for 4 weeks from four HLA-DR different men and the only DR2,2 homozygous individual had low monokine responses . In conclusion, stable interindividual differences in in vitro monokine and PGE2 secretions of LPS-stimulated M phi were demonstrated . It is suggested that HLA-DR2-positive individuals may be low responders.

Eur J Immunol, 1988 Jun, 18(6), 957 - 9
Interleukin 6 is involved in interleukin 1-induced activities; Helle M et al.; Human monocytes produce a number of soluble mediators involved in regulation of inflammation and lymphocyte growth and differentiation such as interleukin 1 (IL 1) and tumor necrosis factor . Recently, the cDNA of another monocyte-derived factor, interleukin 6 (IL 6), was cloned . Herein we show that purified E . coli-derived recombinant IL 6 (rIL 6) is as active as IL 1 in the thymocyte assay . In addition, IL 1 and IL 6 synergize strongly in stimulating thymocyte proliferation . Another property shared by IL 1 and IL 6 is their pyrogenicity . Human rIL 6 induces a monophasic fever after i.v . injection into rabbits . Together with the observation that IL 1 induces IL 6 in a variety of cells including thymocytes, our data suggest that IL 6 is involved in many of the pleiotropic effects of IL 1.

Mol Immunol, 1988 Jun, 25(6), 535 - 43
IgM anti-erythrocyte autoantibodies specific for buried and neo-antigens using cellular-ELISA assays; Jonusys AM et al.; The aim was to develop a cellular-ELISA assay to detect natural autoantibodies specific for bromelain-treated mouse red blood cells (BrMRBC) . High, unexpected IgM titres against normal mouse red blood cells (NMRBC) were detected in day 7-14 sera of CBA mice treated with E . coli lipopolysaccharide (LPS) . These "autoantibodies" bound to normal mouse red blood cells in the presence or absence of commonly used c-ELISA adhering agents . Such high reactivity to NMRBC was never detected using complement dependent haemolytic assays in earlier work in this system . The question whether these IgM alpha-NMRBC molecules were binding nonspecifically (via Fc) or specifically (via Fab) was answered indirectly by comparing the binding titres of LPS-stimulated serum and several purified IgM antibody preparations (alpha-PC, alpha-KLH, MOPC 104E) on the same antigen coated plates . The observed binding ratios (titre on antigen X: titre on NMRBC) varied widely between different antibody sources, indicative of specific binding . In addition no significant unequivocal binding against NMRBC could be detected in vivo (LPS-stimulated mice) nor could bound IgM antibody be detected in a suspension-c-ELISA assay (high binding titres to BrMRBC could be detected in the latter test system) . In conventional c-ELISA assays, modification of normal erythrocyte by adhesion to plastic microtitre plates appears to expose or create "neoantigens" on NMRBC which are not encountered in suspension-type c-ELISA, nor in lytic or agglutination assays where the erythrocyte targets are in suspension at physiological pH and isotonicity.

Can J Microbiol, 1988 Jun, 34(6), 822 - 5
Effect of ascorbate on oxygen uptake and growth of Escherichia coli B; Richter HE et al.; The addition of ascorbate to aerobically growing cultures of Escherichia coli B caused only a short pause in growth and no subsequent change in the rate or extent of growth . The effect of ascorbate on oxygen uptake varied from inhibition in minimal medium to stimulation in rich medium . Cyanide-resistant growth and oxygen uptake were stimulated by ascorbate . Both the rate and extent of anaerobic growth were stimulated in proportion to the amount of ascorbate added when fumarate was the terminal electron acceptor . Ascorbate had no effect on any aspect of anaerobic growth in the absence of a terminal electron acceptor or in the presence of nitrate.

J Biochem (Tokyo), 1988 Jun, 103(6), 950 - 3
Immunoassay for the beta gamma subunits of GTP-binding proteins and their regional distribution in bovine brain; Asano T et al.; Antibodies were raised in rabbits against the beta gamma subunits of bovine brain GTP-binding proteins, and were purified with a beta gamma-coupled Sepharose column . Purified antibodies reacted strongly with 36,000-dalton beta subunit and slightly with 35,000-dalton beta and gamma subunits, but not with other proteins in an immunoblot assay . Using these purified antibodies, a sensitive enzyme immunoassay method for the quantification of brain beta gamma was developed . The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli . The minimum detection limit of the assay was 3 fmol, or 130 pg . Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of beta gamma were determined . The beta gamma were detected in all the regions, and the highest concentrations were observed in the cerebral cortex and nucleus caudatus . The concentrations of beta gamma were higher than those of alpha subunit of GTP-binding protein, Go, in all the regions.

Biochimie, 1988 Jun, 70(6), 773 - 82
Genetic engineering of methionyl-tRNA synthetase: in vitro regeneration of an active synthetase by proteolytic cleavage of a methionyl-tRNA synthetase--beta-galactosidase chimeric protein; Hirel PH et al.; The construction of a family of plasmids carrying derivatives of metG, the gene for E . coli methionyl-tRNA synthetase, is described . These plasmids allow expression of native or truncated forms of the enzyme and easy purification of the products . To facilitate the characterization of modified enzymes with very low catalytic activity, a specialized vector was constructed, in which metG was fused in frame with lacZ, the gene for beta-galactosidase . This plasmid expresses a methionyl-tRNA synthetase-beta-galactosidase chimeric protein, which is shown to carry the activities of both enzymes . This hybrid can be purified in a single step of affinity chromatography for beta-galactosidase . The methionyl-tRNA synthetase moiety can be regenerated by mild proteolysis, thus providing a simple method for purifying and studying mutated proteins.

Biochimie, 1988 Jun, 70(6), 727 - 33
Improving the stability of a foreign protein in the periplasmic space of Escherichia coli; Anba J et al.; An efficient expression/export vector comprising the entire phoS (phosphate binding protein) gene fused to a synthetic gene encoding the human growth hormone releasing factor (mhGRF) has recently been constructed {1} . The hybrid protein (PhoS-mhGRF) was exported to the periplasmic space . However, in this location proteolytic degradation occurred at the C-terminal region . Phenylmethylsulfonyl fluoride (PMSF) increased the stability of the hybrid protein indicating that a serine protease may be involved in the proteolytic cleavage . The correct export and subsequent degradation of the recombinant protein in the periplasmic space were demonstrated in situ using double immunogold labeling on ultrathin sections . Using a phoS-based expression/export vector in the presence of PMSF, 2-4 mg of hybrid protein per liter of culture could be obtained.

J Appl Physiol, 1988 Jun, 64(6), 2410 - 9
Pathological supply dependence of systemic and intestinal O2 uptake during endotoxemia; Nelson DP et al.; When systemic delivery of oxygen (QO2 = blood flow X arterial O2 content) is reduced, the systemic O2 extraction ratio {(CaO2 - CVO2)/CaO2; where CaO2 is arterial O2 content and CVO2 is venous O2 content} increases until a critical limit is reached below which O2 uptake (VO2) becomes limited by delivery . Patients with adult respiratory distress syndrome and sepsis exhibit supply dependence of VO2 even at high levels of QO2, which suggests that a peripheral O2 extraction defect may be present . We tested the hypothesis that endotoxemia might produce a similar defect in the efficacy of tissue O2 extraction by determining the whole-body critical systemic QO2 (QO2 c) and critical extraction ratio in a control group of dogs and a group receiving a 5-mg/kg dose of Escherichia coli endotoxin . QO2 c was determined in each group by measuring VO2 as QO2 was gradually reduced by bleeding . The VO2 and QO2 of an isolated segment of small intestine were also measured to determine whether O2 extraction was impaired within a local region of tissue . The dogs were anesthetized, paralyzed, and ventilated with room air . Systemic QO2 was reduced in stages by hemorrhage as hematocrit was maintained . The systemic and intestinal critical points were determined from a plot of VO2 vs . QO2 . The mean systemic QO2 c and critical O2 extraction ratio of the endotoxemic group (12.8 +/- 2.0 and 0.54 +/- 0.11 ml.min-1.kg-1) were significantly different from control (6.8 +/- 1.2 and 0.78 +/- 0.04) (P less than 0.001), indicating that endotoxin administration impaired systemic extraction of O2 . Endotoxin also increased base-line systemic VO2 {6.1 +/- 0.7 (before) to 7.4 +/- 0.1 (after)} (P less than 0.001) . The critical and maximal intestinal O2 extraction ratios of the endotoxemic group (0.47 +/- 0.10 and 0.71 +/- 0.04) were significantly less than control (0.69 +/- 0.06 and 0.83 +/- 0.05) (P less than 0.001) . In addition, intestinal reactive hyperemia disappeared in six of seven endotoxemic dogs, whereas it remained intact in all control dogs . Thus endotoxin reduced the ability of tissues to extract O2 from a limited supply at the whole body level as well as within a 40- to 50-g segment of small intestine . These results could be explained by a defect in microvascular regulation of blood flow that interfered with the optimal distribution of a limited QO2 in accordance with tissue O2 needs.

J Gen Virol, 1988 Jun, 69 ( Pt 6), 1241 - 6
Reduction of yellow fever virus mouse neurovirulence by immunization with a bacterially synthesized non-structural protein (NS1) fragment; Cane PA et al.; Part of a yellow fever virus-specified non-structural protein (NS1) was expressed in Escherichia coli as a fusion protein with beta-galactosidase . Immunization of mice with this partially purified NS1-beta-galactosidase fusion protein induced yellow fever virus-specific antibodies and provided some protection against intracerebral challenge with the virus.

J Exp Med, 1988 Jun 1, 167(6), 1951 - 6
Regulation of the acute phase and immune responses in viral disease . Enhanced expression of the beta 2-interferon/hepatocyte-stimulating factor/interleukin 6 gene in virus-infected human fibroblasts; Sehgal PB et al.; We have defined the expression of the mRNA for, and secretion of, IFN-beta 2/hepatocyte-stimulating factor/IL-6 (IFN-beta 2/IL-6) in human diploid fibroblasts (FS-4 strain) infected with different RNA- and DNA-containing viruses . RNA blot-hybridization analyses carried out 6-8 h after the beginning of infection showed that the RNA-containing Sendai virus (paramyxoviridae) enhanced IFN-beta 2/IL-6 mRNA levels 10-fold, followed, in decreasing order, by encephalomyocarditis (EMC, picornaviridae), vesicular stomatitis (VSV, rhabdoviridae), Newcastle disease virus (NDV, paramyxoviridae), and influenza A (Flu, myxoviridae) viruses . The DNA-containing pseudorabies virus (PR, herpesviridae) enhanced IFN-beta 2/IL-6 mRNA levels sixfold, while the effect of adenovirus type 5 (Ad5, adenoviridae) was considerably less and comparable with that of NDV or Flu . A rabbit antiserum raised against E . coli-derived human IFN-beta 2/IL-6 was used in immunoprecipitation experiments to monitor the secretion of 35S-methionine-pulse-labeled IFN-beta 2/IL-6 proteins by fibroblasts up to 7 h after the beginning of infection . Enhanced levels of secretion of IFN-beta 2/IL-6 (2-14-fold) were observed in every instance evaluated (Sendai, EMC, VSV, Flu, PR, Ad5 viruses) . A biological consequence of enhanced secretion of IFN-beta 2/IL-6 was the ability of media from infected FS-4 cell cultures to enhance by 8-15-fold the synthesis and secretion of a typical acute phase plasma protein (alpha 1-antichymotrypsin) by human hepatoma Hep3B2 cells . These observations make it likely that IFN-beta 2/IL-6 mediates, in part, the host response to acute virus infections.

Proc Natl Acad Sci U S A, 1988 Jun, 85(11), 3980 - 4
Interleukin 2-diphtheria toxin fusion protein can abolish cell-mediated immunity in vivo; Kelley VE et al.; De novo expression of the interleukin 2 receptor (IL-2R) is a critical and pivotal event in initiation of an immune response . Targeting the low-affinity IL-2-binding p55 subunit of the high-affinity IL-2R with the rat anti-mouse IgM monoclonal antibody M7/20 suppresses a variety of T-cell-mediated reactions, including transplant rejection, autoimmunity, and delayed-type hypersensitivity (DTH) . A hybrid IL-2-toxin gene was constructed from the diphtheria toxin gene by replacing the DNA encoding the diphtheria toxin receptor-binding domain with the DNA encoding the receptor-binding domain of IL-2, and the fusion protein encoded by the hybrid gene was expressed in Escherichia coli {Williams, D.P., Parker, K., Bacha, P., Bishai, W., Borowski, M., Genbauffe, F., Strom, T.B . & Murphy, J.R . (1987) Protein Eng . 1, 493-498} . We examined the action of the chimeric IL-2-toxin fusion protein on an in vivo T-cell mediated response, DTH . The IL-2-toxin fusion protein was found to be a potent immunosuppressive agent . Treatment of mice with the IL-2-toxin blocks DTH and prevents expansion of IL-2R+ T cells . Indeed, IL-2-toxin treatment targets IL-2R+ T cells in vivo and is shown to selectively eliminate their appearance in draining lymph nodes . DTH suppression was observed even in mice possessing high titers of antibodies to diphtheria toxoid.

J Bacteriol, 1988 Jun, 170(6), 2841 - 9
Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli; Giaever HM et al.; It has been shown previously that Escherichia coli accumulates endogenously synthesized trehalose under osmotic stress . We report here that E . coli contained an osmotically regulated trehalose-phosphate synthase which utilized UDP-glucose and glucose 6-phosphate as substrates . In the wild type, the synthase was induced by growth in glucose-mineral medium of elevated osmotic strength and the synthase itself was strongly stimulated by K+ and other monovalent cations . A laboratory strain which expressed the synthase at a high constitutive level was found . GalU mutants, defective in synthesis of UDP-glucose, did not accumulate trehalose . Two genes governing the synthase were identified and named otsA and otsB (osmoregulatory trehalose synthesis) . They mapped near 42 min in the flbB-uvrC region . Mutants with an otsA-lacZ or otsB-lacZ operon fusion displayed osmotically inducible beta-galactosidase activity; i.e., the activity was increased fivefold by growth in medium of elevated osmotic strength . Mutants unable to synthesize trehalose (galU, otsA, and otsB) were osmotically sensitive in glucose-mineral medium . But an osmotically tolerant phenotype was restored in the presence of glycine betaine, which also partially repressed the synthesis of synthase in the wild type and of beta-galactosidase in ots-lacZ fusion mutants.

J Bacteriol, 1988 Jun, 170(6), 2511 - 20
Transcriptional and posttranscriptional regulation of manganese superoxide dismutase biosynthesis in Escherichia coli, studied with operon and protein fusions; Touati D; Protein and operon fusions between the manganese superoxide dismutase (MnSOD) gene, sodA, and genes of the lactose operon were constructed in an attempt to explore the effects of various factors on MnSOD expression and the level at which they operate . In sodA-lacZ protein fusions, induction of beta-galactosidase perfectly mimicked MnSOD induction (i.e., beta-galactosidase was not expressed in anaerobiosis and was induced by oxygen, redox-cycling compounds in aerobiosis, and iron chelators in anaerobiosis) . In tac-sodA operon fusions, MnSOD induction was monitored only by the lactose operon inducer isopropyl-beta-D-thiogalactopyranoside . Various plasmids carrying part or all of the sodA regulatory and structural region inhibited aerobic beta-galactosidase induction in sodA-lacZ fusions . This included plasmids carrying only the transcription start and upstream region and also plasmids which did not contain this region and in which MnSOD was under foreign transcriptional control . The role of metal ions was also investigated . Addition of Mn(II) enhanced MnSOD activity but did not affect induction . The anaerobic expression of MnSOD from the oxygen-insensitive tac promoter was enhanced threefold by iron-chelating agents, implying a posttranscriptional or most likely a posttranslational modulation of enzyme activity via metal ions . To accommodate all these data, multiregulation of MnSOD is proposed.

J Virol, 1988 Jun, 62(6), 1849 - 54
Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors; Falkner FG et al.; Mycophenolic acid, an inhibitor of purine metabolism, was shown to block the replication of vaccinia virus in normal cell lines . This observation led to the development of a dominant one-step plaque selection system, based on expression of the Escherichia coli gpt gene, for the isolation of recombinant vaccinia viruses . Synthesis of xanthine-guanine phosphoribosyltransferase enabled only the recombinant viruses to form large plaques in a selective medium containing mycophenolic acid, xanthine, and hypoxanthine . To utilize the selection system efficiently, we constructed a series of plasmids that contain the E . coli gpt gene and allow insertion of foreign genes into multiple unique restriction endonuclease sites in all three reading frames between the translation initiation codon of a strong late promoter and synthetic translation termination sequences . The selection-expression cassette is flanked by vaccinia virus DNA that directs homologous recombination into the virus genome . The new vectors allow high-level expression of complete or partial open reading frames and rapid construction of recombinant viruses by facilitating the cloning steps and by simplifying their isolation . The system was tested by cloning the E . coli beta-galactosidase gene; in 24 h, this enzyme accounted for approximately 3.5% of the total infected-cell protein.

J Gen Microbiol, 1988 Jun, 134 ( Pt 6), 1717 - 21
The rate and topography of cell wall synthesis during the division cycle of Escherichia coli using N-acetylglucosamine as a peptidoglycan label; Cooper S et al.; The rates of synthesis of peptidoglycan and protein during the division cycle of Escherichia coli were measured by the membrane elution technique using cells differentially labelled with N-acetylglucosamine and leucine . During the first part of the division cycle the ratio of the rates of protein and peptidoglycan synthesis was constant . The rate of peptidoglycan synthesis, relative to the rate of protein synthesis, increased during the latter part of the division cycle . These results support a simple, bipartite model of cell surface increase in rod-shaped cells . Prior to the start of constriction the cell surface increases only by lateral wall extension . After cell constriction starts, the cell surface increases by both lateral wall and pole growth . The increase in surface area is partitioned between the lateral wall and the pole so that the volume of the cell increases exponentially . No variation in cell density occurs, because the increase in surface allows a continuous exponential increase in cell volume that accommodates the exponential increase in cell mass . The results are consistent with the constant density of the growing cell and the surface stress model for the regulation of cell surface synthesis . In addition, the elution pattern suggests that the membrane elution method does work by having the cells effectively bound to the membrane by their poles.

J Gen Microbiol, 1988 Jun, 134 ( Pt 6), 1549 - 59
Iron-regulated outer-membrane proteins of Escherichia coli strains associated with enteric or extraintestinal diseases of man and animals; Chart H et al.; The SDS-PAGE patterns of the iron-regulated outer-membrane proteins from 70 strains of Escherichia coli isolated from various human and animal infections were analysed and the nature of the siderophores produced was examined . Iron-regulated 81 kDa and 74 kDa protein bands seen in SDS-PAGE gels were characterized further by immunoblotting using anti-81 kDa and anti-74 kDa (Cir) sera . The results showed considerable differences between the patterns of the iron-regulated outer-membrane proteins exhibited by the different strains . Nevertheless, three distinct and characteristic profiles, based on the most prominent bands expressed, could be identified, although not all strains produced patterns which matched with one of these . These results suggest the possibility of using the pattern of iron-regulated outer-membrane proteins expressed, as well as siderophores produced, as a new set of markers to characterize groups of pathogenic E . coli.

J Gen Microbiol, 1988 Jun, 134 ( Pt 6), 1499 - 507
A haemoprotein is not involved in the control by oxygen of enteric nitrogenase synthesis; Smith A et al.; Strains of Escherichia coli containing the Nif+ plasmid pRD1 were used to investigate the possibility that haem proteins are involved in the regulation by O2 of nif expression . Strains lacking 5-aminolaevulinate synthase (HemA-), and hence normally unable to synthesize haem proteins, showed an identical response to O2 in the presence or absence of added aminolaevulinate (and hence of haem proteins) . It was concluded that the regulatory protein NifL is not a haem protein.

Am Rev Respir Dis, 1988 Jun, 137(6), 1441 - 8
Endotoxin-induced inflammation and injury of the guinea pig respiratory airways cause bronchial hyporeactivity; Folkerts G et al.; It was investigated whether an endotoxin-induced airway inflammation and injury correlated with the induction of bronchial hyperreactivity . Guinea pigs were treated with an endotoxin aerosol, and 4 and 24 h later lung lavages were performed and a differential cell count was made . The number of neutrophils and monocytes was significantly increased (p less than 0.005) at these times . After 24 h, the number of eosinophils and lymphocytes was also increased (p less than 0.005) . The number of alveolar macrophages remained unchanged . Histologic examination revealed increased intraluminal mucus and an influx of erythrocytes and neutrophils in the tracheal and bronchial lumen at both time points after the endotoxin aerosol . The epithelium was morphologically changed and contained many neutrophils . Focal matting and/or loss of cilia also occurred . Airway smooth muscle responsiveness was measured in vitro on isolated guinea pig tracheal smooth muscle preparations 4 and 24 h after endotoxin aerosol . No differences in the maximal responses or slope factors of the dose-response curves for carbachol, histamine, or isoprenaline were detected between the control and endotoxin-exposed groups . The EC50 value of the histamine dose-response curve 4 h after endotoxin nebulization was slightly but significantly (p less than 0.05) increased, indicating decreased sensitivity . Responsiveness in vivo was measured in anesthetized spontaneously breathing guinea pigs 24 h after the endotoxin aerosol . The histamine-induced increase in pulmonary resistance was reduced by about 35% in the endotoxin-nebulized group (p less than 0.01) . It can be concluded that an influx of inflammatory cells accompanied by injury of the airways induces hyporeactivity of the guinea pig respiratory tract.

Pathol Biol (Paris), 1988 Jun, 36(5 Pt 2), 731 - 4
{Effect of fosfomycin on cellular immune reactions as affected by cyclophosphamide or corticosteroids}; Gillissen G et al.; The effect of fosfomycin (FM) has been examined on the "footpad swelling reaction" (FPSR) as a model for cellular immunity and on the course of experimental infection with FM resistant E . coli using normal and immunocompromised mice . FM given the day of immunization improved FPSR response progressively up to a dose of 30 mg/kg, higher doses had no effect . If FM was injected 1 or 3 days after immunization doses of 30 mg/kg induced a considerable stimulation, whereas higher doses were inhibitory . In contrast to the high dose of 50 mg of FM per kg, 30 mg/kg increased the stimulatory effect induced by small doses of CY and reduced the inhibitory effect of high CY doses . The depressing effect of hydrocortisone (HC) was also reduced by 30 mg/kg of FM, but not by the higher one . In experimental infection with E . coli, FM in doses of 30 mg/kg was more effective than the higher ones in reducing mortality rate . Comparable results were obtained with CY pretreated animals.

Biochem Int, 1988 Jun, 16(6), 1083 - 93
Effect of various oxygen free radical scavengers in preventing tissue injury caused by Escherichia coli in pyelonephritic mice; Kaur A et al.; Reactive oxygen species have been found to be responsible for the tissue injury caused in experimental pyelonephritis in mice . The extent of lipid peroxidation (as assayed by malondialdehyde formation) was found to be increased significantly (p less than .001) in the infected group as compared to the normal mice . Superoxide dismutase and catalase (oxygen free radical scavengers) showed a significant decrease (p less than .001) in the extent of lipid peroxidation even in the presence of infection . Dimethyl sulfoxide, a hydroxyl ion scavenger, was however found to be effective only at 4 and 7 days postinfection (p less than .001) . Allopurinol, an inhibitor of xanthine oxidase, did not significantly (p greater than .05) inhibit the formation of lipid peroxides, even upto 7 days postinfection . There was a significant decrease (p less than .05) in the activities of renal brush border membrane enzymes used as markers of renal tissue damage (i.e . alkaline phosphatase, leucine amino-peptidase and gamma-glutamyl transpeptidase) in the infected group as compared to the normal group . In the presence of superoxide dismutase, dimethylsulfoxide and catalase except allopurinol, the activities of all the enzymes but maltase were found to be increased significantly (p less than .05) as compared to the infected group . There was a significant increase (p less than .01) in the bacterial count in the presence of superoxide dismutase and DMSO in infected mice as compared to the infected control mice . However, no significant difference was observed in the catalase and allopurinol treated groups.

Vet Microbiol, 1988 Jun, 17(2), 159 - 69
Effect of experimentally-induced villus atrophy on adhesion of K88ac-positive Escherichia coli in just-weaned piglets; Cox E et al.; Three- to four-week-old, just-weaned piglets were infected with transmissible gastroenteritis (TGE) virus and the next day with K88ac+ enterotoxigenic Escherichia coli (ETEC) . Histological examination of caudal jejunum and ileum of piglets killed 2-3 days after virus challenge (1-2 days after ETEC infection) revealed severe villus atrophy especially in the jejunum compared with controls (P less than 0.05) . Four-5 days after TGE virus infection villus length increased and after 7 days it was near normal . Villi scraped from jejunal and ileal mucosa of the piglets were incubated in vitro with K88ac+ E . coli and the number of bacteria adhering to 250 micron villus brush border was counted . Attachment of bacteria to villi of piglets killed 2-3 days after TGE virus infection was significantly decreased in comparison with adhesion to villi of non-infected piglets or of piglets killed 7 days after the virus infection . Correlation between in vitro adhesion and villus height was 0.6649 (P less than 0.001) . The results suggest that the experimentally-induced villus atrophy was attended with a temporarily diminished susceptibility of villus enterocytes to adhesion of K88ac+ E . coli.

Mol Gen Mikrobiol Virusol, 1988 Jun, (6), 23 - 30
{Structural rearrangements of the plasmid R68,45 in cells of Brucella suis}; Gorelov VN et al.; The mobilizing activity of the plasmid R68,45 in Escherichia coli and Brucella abortus has been studied . The plasmid R68,45 has been found to lose its ability to mobilize the chromosome in Brucella suis cells . The experiments on the conjugational transfer of R68,45 were confirmed by restriction analysis of the plasmid DNA . R68,45 has been shown to lose Cma in brucella cells via deletion . The molecular mechanisms of deletion process in brucella and other bacteria are discussed.

Mol Cell Probes, 1988 Jun, 2(2), 125 - 30
DNA hybridization with oligodeoxyribonucleotide probes for identifying enterotoxin-producing Escherichia coli; Cravioto A et al.; The DNA hybridization method using oligodeoxyribonucleotide probes was compared with the GM, ELISA and the infant mouse assay for determining production of LT and ST, respectively, by 2000 strains of Escherichia coli . Sensitivity and specificity by DNA hybridization for LT were 98.7 and 99.8%, and for ST were 78.8 and 99.6%.

J Appl Bacteriol, 1988 Jun, 64(6), 487 - 95
Production of antibody to lipopolysaccharide (LPS) after immunization with a LPS-polymyxin B-agarose immunogen; Ryan LK et al.; A method was devised to produce antibodies to lipopolysaccharide (LPS) in guinea-pigs following a single immunization . The antigen was prepared by mixing polymyxin B-agarose with LPS from Escherichia coli O55:B5 . Use of the agarose support allowed purification of the complex by simple washing procedures . Twenty-nine days after a single injection of the immunogen mixed with Freund complete adjuvant all animals demonstrated antibody to the LPS portion of the complex . No antibodies were detected to the polymyxin B component . Typical titres of LPS as measured by ELISA were 2(11) . After, a booster immunization, titres of LPS antibody were further increased and a greater avidity was noted . In contrast to other methods which have been employed for production of antibody to LPS, use of the polymyxin B-agarose complex has the following advantages: ease of antigen preparation, ready purification of the complex, potent immunostimulation, and under the conditions employed here, LPS-specific antibody production, without accompanying antibody to polymyxin B.

EMBO J, 1988 Jun, 7(6), 1881 - 8
Recognition of the P1 plasmid centromere analog involves binding of the ParB protein and is modified by a specific host factor; Davis MA et al.; The P1 plasmid partition system is responsible for segregation of daughter plasmids during division of the Escherichia coli host cell . The P1-encoded elements consist of two essential proteins, ParA and ParB, and the cis-acting incB region . The incB region determines partition-mediated incompatibility and contains the centromere-like site parS . We have isolated and purified the two proteins . ParB binds specifically to the incB region in vitro . DNase I footprinting assays place a strong binding site over the 35-bp parS sequence previously shown to be sufficient for partition when the Par proteins are supplied in trans . A weaker site lies within the incB region in sequences that are important for specifying incompatibility, but are not essential for partition . Gel band retardation assays show that a host factor binds specifically to the incB sequence . The factor strongly stimulates binding of ParB . Cutting the region at a site between the two ParB binding sites yields two fragments that can bind ParB but not host factor . Thus, information for host-factor binding lies in the region determining the specificity of plasmid incompatibility . The roles of parB and the host factor in partition and the specificity of plasmid incompatibility are discussed.

EMBO J, 1988 Jun, 7(6), 1871 - 9
Correlation between the conformation of Escherichia coli -10 hexamer sequences and promoter strength: use of orthophenanthroline cuprous complex as a structural index; Spassky A et al.; The lac and gal control regions contain two functional overlapping promoters P1 and P2 . Point mutations can shift transcription from P1 to P2 and vice versa . We show that the reactivity of DNA fragments towards nucleolytic attack with orthophenanthroline cuprous complex can be used to predict which promoter competes more efficiently for RNA polymerase binding . Furthermore, similar changes in reactivity are observed as closed complexes isomerize to form the final open complexes, provided that the functional start is taken as a reference . We found a correlation between the reactivity pattern of -10 regions in uncomplexed DNA and the rate of open complex formation.

EMBO J, 1988 Jun, 7(6), 1831 - 5
ProOmpA spontaneously folds in a membrane assembly competent state which trigger factor stabilizes; Crooke E et al.; The precursor protein proOmpA can translocate across purified Escherichia coli inner membrane vesicles in the absence of any other soluble proteins . ProOmpA, purified 2000-fold in the presence of 8 M urea, is competent for translocation following rapid renaturation via dilution . ATP, the transmembrane electrochemical potential, and functional secY protein are essential for the translocation of proOmpA renatured by dilution . The kinetics of its translocation and the level of translocation at each concentration of ATP are indistinguishable from that of proOmpA renatured by dialysis with trigger factor . After dilution, the proOmpA rapidly loses its competence for membrane assembly . However, this competence is stabilized by trigger factor . Assembly-competent proOmpA is in a protease-sensitive conformation, whereas proOmpA which has lost this competence is more resistant to degradation . This suggests that the primary role for trigger factor in in vitro protein translocation is to maintain precursor proteins in a translocation-competent conformation . We propose that a properly folded precursor protein and ATP are the only soluble components which are essential for bacterial protein translocation.

EMBO J, 1988 Jun, 7(6), 1785 - 91
Partial purification and substrate analysis of bacterially expressed HIV protease by means of monoclonal antibody; Hansen J et al.; Retroviruses code for a specific protease which is essential for polyprotein precursor processing and viral infectivity . The HIV-specific protease has been predicted to be an aspartic protease which is located at the amino terminus of the pol gene . We have prepared several constructs for bacterial expression of the protease . Two of them span the whole protease region and result in its autocatalytic activation . Analysis of the dynamics of this activation indicates a two-step process which starts at the carboxy terminus and ends at the amino terminus of the protease . The activated protease is a molecule of 9 kd as evidenced by monoclonal antibody in immunoblot analysis . A construct in which the carboxy terminus of the protease is deleted results in a stable, enzymatically inactive 27-kd protein which proved useful as substrate since it contains one of the predicted cleavage sites . The stability of this protein indicates that the carboxy-terminal sequences of the protease are essential for its activity and its autocatalytic activation . The protease which is very hydrophobic was solubilized by acetone treatment and passaged over ultrogel and propylagarose columns for partial purification . It elutes as a dimer and tends to aggregate . It is inhibited by pepstatin A in agreement with its expected active site and its theoretical classification as aspartic protease . Cleavage of the gag precursor results in the mature capsid protein, p17 . The protease does not, however, cleave the denatured 27-kd substrate or the denatured gag precursor . Therefore its specificity appears to be not solely sequence- but also conformation-dependent . This property needs to be taken into account for the development of protease inhibitors for therapy of AIDS.

Clin Exp Immunol, 1988 Jun, 72(3), 510 - 5
Two calcium-binding proteins associated with specific stages of myeloid cell differentiation are expressed by subsets of macrophages in inflammatory tissues; Zwadlo G et al.; Using a monoclonal antibody to macrophage migration inhibition factor (MIF), two proteins were isolated from supernatants of Concanavalin A-stimulated human peripheral blood mononuclear cells which seem to have complexed to a third component carrying the MIF activity . They are therefore designated MIF-related proteins or MRP-8 and MRP-14 according to their apparent molecular weights . Partial amino acid sequences have been determined and their cDNA have been cloned and expressed in Escherichia coli . Both are calcium-binding proteins and MRP-8 seems to be largely homologous to the cystic fibrosis antigen (Dorin et al., 1987) . Antisera were raised in the rabbit against the recombinant proteins and their expression in cells and tissues studied using immunohistological techniques . The proteins are only found in blood granulocytes and monocytes . In culture the number of positive monocytes sharply increased and then declined with time, suggesting that their expression is associated with early stages of monocyte/macrophage differentiation and absent from resident macrophages in all tested tissues . In acute inflammatory reactions, e.g . gingivitis, MRP-8 is never seen in the tissue, whereas MRP-14 is expressed by intravascular monocytes and perivascular macrophages . In contrast, in chronic inflammation, e.g . rheumatoid arthritis, MRP-8 is also expressed by macrophages in the tissue . From this it is concluded that MRP-8 and MRP-14 are expressed sequentially at defined stages of monocyte/macrophage differentiation and that dysregulation of this process in chronic inflammation is mirrored by the presence of MRP-8-positive macrophages in the tissue.

Vaccine, 1988 Jun, 6(3), 269 - 77
Adjuvant activity of Escherichia coli heat-labile enterotoxin and effect on the induction of oral tolerance in mice to unrelated protein antigens; Clements JD et al.; The ability of Escherichia coli heat-labile enterotoxin (LT) to influence the induction and maintenance of tolerance was examined in animals primed orally with a soluble protein antigen, ovalbumin (OVA), or in animals primed orally with two unrelated protein antigens administered simultaneously, OVA and bovine serum albumin (BSA) . LT is immunologically and structurally related to the cholera enterotoxin (CT), which has been shown to be capable of abrogating oral tolerance to protein antigens when delivered simultaneously with the antigens . In this study, simultaneous administration of LT with OVA was shown to prevent the induction of tolerance to OVA and to increase the serum anti-OVA IgG response 30- to 90-fold over OVA-primed and PBS-primed animals, respectively . This effect was determined to be a function of the enzymatically active A subunit of the toxin since the B (binding) subunit alone was unable to influence tolerance induction . Animals fed LT with OVA after the initial OVA prime developed a significantly lower serum IgG and mucosal IgA anti-OVA response than those fed LT with OVA in the initial immunization, indicating that prior exposure to the antigen reduces the effectiveness of LT to influence tolerance and its ability to act as an adjuvant . LT was not able to abrogate tolerance once it had been established . Serum IgG and mucosal IgA responses in animals receiving LT on only a single occasion, that being upon first exposure to antigen, were equivalent to responses after three OVA/LT primes, indicating that commitment to responsiveness occurs early and upon first exposure to antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Endocrinol, 1988 Jun, 2(6), 579 - 86
Characterization of proliferin-related protein; Colosi P et al.; Proliferin-related protein (mPRP) is a member of the PRL/GH family in the mouse . We have generated an antiserum against mPRP expressed as a bacterial fusion protein; this antiserum detects mPRP in the conditioned media of placental tissue cultures as a heterogeneous population of glycoproteins . We have also expressed mPRP in mammalian tissue culture cells and purified the secreted protein . N-terminal sequence analysis of the purified protein reveals that it is secreted as a 214 amino acid protein after removal of a 30 amino acid signal polypeptide . An antiserum raised against the purified protein detects high levels of mPRP in maternal serum during gestation . The site of synthesis of this protein has been localized by in situ hybridization to the basal zone of the day-10 mouse placenta, which is distinct from the site of synthesis of other placental proteins in this family.

Mol Gen Genet, 1988 Jun, 212(3), 543 - 7
Inhibition of gene expression of T7-related phages by prophage P1; Hausmann R et al.; The gene expression of nine phages of the T7 group was compared after infection of Escherichia coli B(P1) . With the exception of phage 13a which grew normally, all of them infected E . coli B(P1) abortively . Differences were found in the efficiency of host killing which ranged from 100% for phage 13a to 37% for phage A1122 . Infection by T7 prevented colony formation by about 70% of the cells but they showed filamentous growth until about 2 h after infection . It was shown by SDS-polyacrylamide gel electrophoresis and autoradiography of {35S}methionine-labelled phage-coded proteins that all phages except for 13a showed measurable expression only of the early genes . No correlation was observed between killing capacity and the pattern of gene expression, and the ability to hydrolyse S-adenosyl-methionine (SAM, a cofactor for the P1 restriction endonuclease) by means of a phage-coded SAMase . Mixed infection of E . coli B(P1) with 13a and T7 yielded mixed progeny indistinguishable from that observed after mixed infection of the normal host E . coli B . Genetic crosses with amber mutants of 13a and T7 showed that the 13a marker opo+ (overcomes P one), required for growth on B(P1), is located in the early region, to the left of gene 1 (RNA polymerase gene).

Int J Lepr Other Mycobact Dis, 1988 Jun, 56(2), 265 - 73
Identification of T-cell-activating recombinant antigens shared among three candidate antileprosy vaccines, killed M . leprae, M . bovis BCG, and mycobacterium w; Mustafa AS; Antigenic crossreactivity among three candidate antileprosy vaccines, killed Mycobacterium leprae, BCG, and Mycobacterium w, was studied using T-cell lines and clones raised from BCG- and killed-M . leprae-vaccinated subjects . To identify the crossreactive antigens, the T-cell lines and clones were tested against Escherichia coli lysates containing 65-, 36-, 28-, 18-, and 14-kilodalton (kDa) and 13B3 M . leprae antigens and 65-, 19-, and 12-kDa M . tuberculosis antigens . The short-term T-cell lines, which compared to T-cell clones are easy to raise and maintain, were equally effective in identifying the T-cell-activating recombinant antigens . The reactivity pattern of the T-cell lines and the clones suggested that 65-kDa M . leprae and M . tuberculosis antigens are present in M . leprae, BCG, and Mycobacterium w; 18-kDa M . leprae antigen is shared between M . leprae and Mycobacterium w, 13B3 M . leprae antigen is possessed by M . leprae and BCG . These and other unidentified T-cell-activating antigens shared among candidate leprosy vaccines may be the basis for induction of in vivo sensitization to M . leprae antigens after vaccination with BCG or Mycobacterium w.

Br Poult Sci, 1988 Jun, 29(2), 371 - 8
Effect of Escherichia coli endotoxin on tissue lipoprotein lipase activities in chickens; Griffin HD et al.; 1 . Four-week-old broiler chickens were injected intravenously with from 0.01 to 1 mg of E . coli endotoxin/kg body weight or with saline . 2 . At all doses used endotoxin markedly depressed food intake and lipoprotein lipase activities in muscle and adipose tissue within 8 h . Heart lipoprotein lipase activity was significantly depressed only at doses of 0.1 mg endotoxin/kg body weight or greater . 3 . Treatment of birds with 0.3 mg endotoxin/kg body weight reduced post-heparin lipoprotein lipase activity to 0.13 of that in control birds in 8 h . 4 . Endotoxin generally depressed plasma very-low-density lipoprotein concentration . Plasma non-esterified fatty acid concentration was significantly elevated only in birds given 1 mg endotoxin/kg body weight . 5 . Fatty acid synthetase activity in the liver of endotoxin-treated birds was significantly lower than in control birds 16 h after administration of endoxin, but not after 8 h . 6 . These results show that tissue lipoprotein lipase activity in birds is very responsive to E . coli endotoxin, as in mammals . Hypertriglyceridaemia occurs only occasionally in endotoxin-treated chickens, most probably because of the particularly close relationship between food intake and hepatic lipoprotein synthesis in birds.

Eur J Epidemiol, 1988 Jun, 4(2), 135 - 43
The hemolysin of Escherichia coli; Bhakdi S et al.; Many strains of E . coli elaborate a hemolysin which is responsible for the zone of beta-hemolysis surrounding bacterial colonies on blood agar . The significance of this cytolysin as a determinant of bacterial pathogenicity has been established in animal models with the use of genetically engineered, isogenic bacterial strains . An analogous role in human infections has been inferred from the high association of hemolysin production with disease . Studies at a molecular genetical level have defined 4 genes that are required for the synthesis, post-translational modification and secretion of the hemolysin . The structural gene hlyA encodes for a 107-110,000 polypeptide, which must be modified in an unknown manner to its active form by the product of the neighboring hlyC gene . Genes hlyB and hlyD encode for proteins that export the molecule to the extracellular medium . The signal for secretion is contained in the C-terminal portion of the toxin molecule . The secreted hemolysin attacks plasma membranes of target mammalian cells by inserting as a monomer into the bilayer and generating hydrophilic transmembrane pores of approximately 2 nm effective diameter . The pores display a marked selectivity for cations over anions and pore-opening is dependent on the presence of a correct transmembrane potential . Binding to a membrane target does not require the presence of a specific receptor, and pores may be generated in planar lipid membranes consisting solely of phosphatidylcholine . Pore formation in nucleated cells can trigger secondary reactions such as stimulation of arachidonate metabolism with release of lipid mediators, probably initiated by passive influx of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, 1988 Jun, 7(5), 329 - 36
Identification and characterization of cryptic polyadenylation sites in the 3' region of a pea ribulose-1,5-bisphosphate carboxylase small subunit gene; Hunt AG; The polyadenylation signal of a pea ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (rbcS) gene has been studied using in vitro mutagenesis and Ti plasmid-mediated transformation of tobacco . Analysis of a mutant that is lacking sequences upstream from -6 (relative to the "normal" site of polyadenylation of RNAs from the rbcS-E9 gene) reveals a number of alternate polyadenylation sites located downstream from the normal poly(A) site . Transcripts carrying these sequences end at any one of at least seven sites between 20 and 300 bases downstream from the normal site . These sites are seen in populations of transgenic plant cells, and also in independent transgenic plants.

Am J Vet Res, 1988 Jun, 49(6), 743 - 6
Strains of Escherichia coli associated with urogenital disease in dogs and cats; Wilson RA et al.; Selected strains of Escherichia coli associated with urogenital disease in dogs and cats were evaluated for 3 virulence factors associated with human uropathogenic strains . Urogenital strains of E coli from dogs and cats had high prevalence of alpha hemolysin and were clustered in 5 to 10 somatic serogroups, attributes also shared by human uropathogenic strains . However, the canine and feline urogenital strains failed to have increased prevalence of mannose-resistant hemagglutination, as has been reported for human uropathogenic strains.

J Am Vet Med Assoc, 1988 Jun 1, 192(11), 1581 - 4
Immune-mediated polysynovitis in four foals; Madison JB et al.; The deposition of immune complexes in the synovial membrane resulted in polysynovitis in 4 foals . All 4 foals had an infection at a site other than the joints . The polysynovitis was characterized by marked effusions of affected joints and joint stiffness . Bacterial and mycoplasmal cultures of the joints did not yield growth . Staining of synovial membrane biopsy specimens with fluorescein-labeled anti-equine IgG revealed immune complexes in the synovial membrane . Immune-mediated polysynovitis might develop in foals with bacterial infections . We propose that deposition of immunoglobulin in the synovial membrane of the affected foals was caused by an increase in circulating immune complexes formed as a result of the primary disease processes.

Eur J Biochem, 1988 Jun 1, 174(2), 411 - 6
Expression of atrial natriuretic factor as a cleavable fusion protein with chloramphenicol acetyltransferase in Escherichia coli; Dykes CW et al.; Recombinant fusion proteins containing human atrial natriuretic factor, ANF(1-28) joined to chloramphenicol acetyltransferase (CAT) via cleavable linker sequences have been produced in Escherichia coli . The linker sequences were designed to allow the release of authentic ANF(1-28) following proteolytic cleavage by enterokinase or thrombin, or chemical cleavage with 2-(2-nitrophenylsulphenyl)-3-methyl-3'-bromoindolenine . Proteins, containing ANF(1-28) fused to the carboxyl-terminal region of CAT (using the ScaI restriction site in the cat gene), were largely soluble in E . coli and were obtained in higher yield than analogues containing ANF(1-28) linked to shorter CAT sequences . The longer derivatives also retained CAT activity allowing subsequent purification by affinity chromatography.

Eur J Biochem, 1988 Jun 1, 174(2), 405 - 10
The isolation and characterisation of human atrial natriuretic factor produced as a fusion protein in Escherichia coli; Knott JA et al.; Human atrial natriuretic factor {ANF(1-28)} has been isolated from a fusion protein produced in Escherichia coli . ANF(1-28) was linked to a naturally occurring E . coli protein, chloramphenicol acetyltransferase, via unique cleavage sequences susceptible to either human thrombin digestion, or the chemical action of 2-(2-nitrophenylsulphenyl)-3-methyl-3'-bromoindolenine (BNPS-skatole) . The linker sequences were Gly-Val-Arg-Gly-Pro-Arg and Trp respectively . The liberated ANF was purified by reversed-phase HPLC . Optimised cleavage conditions released 5-10% (by mass) of the maximal yield of ANF(1-28) from the fusion protein with the thrombin-susceptible linker, whilst a 2-5% (by mass) yield was observed from the fusion protein with the tryptophan linker after BNPS-skatole treatment . The purified cleavage products were biologically active and shown to comprise intact ANF(1-28) . Fast-atom-bombardment mass spectrometry confirmed {MH}+ of 3079 m/z, consistent with ANF(1-28).

J Immunol, 1988 Jun 1, 140(11), 3838 - 43
Characterization of murine IL-1 beta . Isolation, expression, and purification; Huang JJ et al.; One cDNA clone encoding a truncated murine IL-1 beta (M IL-1 beta) sequence was isolated from a murine macrophage cDNA library . We reconstituted the coding sequence of the 152-residue mature protein and expressed it in Escherichia coli . rM IL-1 beta was purified to homogeneity and characterized by oligonucleotide and NH2-terminal sequence analysis . Purified rM IL-1 beta exhibited biologic activity equivalent to 7.8 x 10(7) units/mg in the murine thymocyte proliferation assay and 9.9 x 10(3) units/mg in the human gingival fibroblast PGE2 production assay, indicative of species specificity . The isoelectric point of rM IL-1 was found to be 8.85 . The circular dichroism spectrum revealed that the secondary structure of M IL-1 is indistinguishable from that of the human protein . Receptor binding studies indicated the rM IL-1 bound to murine EL-4.1 thymoma cells in a specific and dose-dependent fashion with an affinity of 32 pM . Competition binding data suggested that murine and human IL-1 compete for a single class of receptor . Antisera were generated in rabbits against both murine and human IL-1 . Results of ELISA binding and antisera neutralization assays indicated that there are common antigenic sites between the two IL-1 beta molecules . These domains are of functional importance because they are capable of mediating the neutralization of biologic activity.

Mol Gen Genet, 1988 Jun, 212(3), 412 - 7
Functional and structural homology among regulatory cistrons of pili-adhesin determinants in Escherichia coli; Goransson M et al.; Expression of the digalactoside-binding Pap pili involves two trans-acting regulatory genes, papB and papI . Using pap-lac operon fusions and DNA hybridization probes derived from pap DNA we tested whether or not other pili-adhesin determinants from different Escherichia coli strains encode homologs to the pap regulatory genes . Digalactoside-specific clones of serotypes F72 and F11 complemented papB and papI mutants of the Pap (serotype F13) clone and DNA hybridization analysis showed that the clones are homologous in the DNA sequences encoding the two regulatory genes . Similar results were obtained with an S-pili determinant which mediates binding to sialic acid-containing receptors and the findings suggest that the regulatory regions may be more conserved than other genes in different pili-adhesin gene clusters . Determinants for type 1-pili (mannose-specific binding) and for pili associated with enterotoxigenic E . coli (K88, K99, CFAI, CFAII) did not appear to contain DNA sequences homologous to papB or papI . E . coli strain J96, which was the origin of the pap DNA, was found to carry two additional copies of papB-papI homologous sequences in the chromosome . In strains expressing more than one kind of pili the trans-active gene products thereby may allow for regulatory interaction between separate pili-adhesin gene systems.

Curr Genet, 1988 Jun, 13(6), 487 - 94
Isolation of a DNA fragment which complements glutamine synthetase deficient strains of S . pombe; Barel I et al.; From a gene bank of S . pombe DNA, a 5.6 kb clone was isolated which complemented mutants defective in glutamine synthetase (GS) activity . Sub-cloning fragments of this 5.6 kb clone showed that the complementing activity was localised in a 1.6 kb HindIII-AvaI fragment and a partial DNA sequence revealed an open reading frame preceded by TATA sequences and a TGACTA sequence . Plasmid constructs carrying up to 3.4 kb of DNA used to transform gln- strains gave transformants which showed a wide range of GS activity, in some cases 100 times the wild-type level . These constructs identify DNA sequences lying downstream from the putative coding sequence which have effects on the total amount of enzyme activity, but do not affect the control imposed by the nitrogen source on which the cells are grown.

Proc Natl Acad Sci U S A, 1988 Jun, 85(11), 3767 - 71
Suppression of the Escherichia coli ssb-1 mutation by an allele of groEL; Ruben SM et al.; A series of spontaneous suppressors to the temperature-sensitive phenotype of the single-stranded DNA-binding protein mutation ssb-1 were isolated . A genomic library of EcoRI fragments from one of these suppressor strains was prepared by using pBR325 as the cloning vector . A 10.0-kilobase class of inserts was identified as carrying the ssb-1 gene itself . A second class of 8.3-kilobase inserts was shown to contain the groE region by (i) restriction analysis, (ii) Southern hybridization of the 8.3-kilobase insert to groE+ DNA, and (iii) identification of the gene products by similar migration on polyacrylamide gels . Subcloning demonstrated that an intact mutant groEL gene was necessary for suppression and that plasmids carrying the 8.3-kilobase insert could suppress mutants carrying groES- but not groEL- genes for phage lambda growth . The suppressor, designated as groEL411, was specific for the ssb-1 allele . In ssb-1 groEL411 cells, DNA synthesis stopped after a shift to 42.5 degrees C but rapidly recovered within minutes . The data suggest a direct interaction between the single-stranded DNA-binding protein and GroEL proteins in DNA replication.

Infect Immun, 1988 Jun, 56(6), 1475 - 84
Isolation and nucleotide sequence of the F17-A gene encoding the structural protein of the F17 fimbriae in bovine enterotoxigenic Escherichia coli; Lintermans P et al.; The genetic determinant for production of the fimbrial F17 adhesive antigen was isolated from a bovine enterotoxigenic Escherichia coli strain . The F17-A gene, coding for the structural component of the F17 fimbrial adhesin, was cloned and sequenced . An open reading frame of 540 base pairs encoding a polypeptide of 180 amino acids, of which the NH2-terminal 21 residues are characteristic of a signal sequence, has been characterized . The mature protein lacks histidine, methionine, and tryptophan . A possible promoter and ribosome binding site as well as a possible site for termination of transcription are proposed . An important homology of the F17-A protein with fimA and papA fimbrial proteins was found . The N-terminal sequence of the mature F17-A pilin is extremely similar to the N-terminal sequence of the G fimbriae identified on human pyelonephritogenic E . coli strains.

Diagn Microbiol Infect Dis, 1988 Jun, 10(2), 93 - 101
Papillomaviruses in human skin warts and their incidence in an Argentine population; Corley E et al.; Human papillomavirus genomic types present in human warts of an Argentine population were studied . HPV DNA from single warts was obtained using an alkaline extraction procedure that resulted in a clean DNA preparation, which could be analyzed with several endonucleases . This method was used to isolate and insert the HPV DNAs of two genomic types into the Bam HI site of the pBR322 plasmid . Restriction maps of both HPV DNAs were constructed . According to these maps, one of the genomic variations was identical to HPV1a and the other to HPV2a . The incidence of HPV2 and of HPV1 in different types of skin warts was studied by a dot blot hybridization assay . Twenty-two out of 28 common warts were positive for HPV2 and negative for HPV1; four were positive for HPV1 and negative for HPV2 and two were negative for both . Five out of six plantar warts were positive for HPV1, and one was negative for both . Three out of seven filiform warts were positive for HPV2, three were positive for both probes, and one was negative for both . Southern blot analysis of HPV2 positive samples indicated that 80% were HPV2a and 20% another subtype not yet characterized . All plantar warts contained HPV1a . Msp I/Hpa II restriction analysis confirms previous results indicating that HPV1a DNA is partially methylated, while no evidence of methylation was found for HPV2a DNA.

EMBO J, 1988 Jun, 7(6), 1897 - 905
Isolation and characterization of the Tn3 resolvase synaptic intermediate; Benjamin HW et al.; We have isolated in quantitative yield the synaptic intermediate formed during site-specific recombination by Tn3 resolvase and characterized it by restriction endonuclease mapping, electron microscopy and topological methods . The intermediate accumulates at low reaction temperatures and is stabilized by crosslinking of the resolvase protomers with glutaraldehyde . The DNA-resolvase complex that maintains the structure of the intermediate (the synaptosome) is approximately 100 A in diameter, forms specifically at resolution (res) sites, and requires two res sites in a supercoiled DNA molecule . Resolvase bound to individual res sites protects approximately -0.5 supercoil per site from relaxation by a topoisomerase, whereas the formation of the synaptosome protects -3 supercoils and condenses the associated DNA to a supercoil density 2.5 times that of the non-complexed substrate . Although recombination requires two directly repeated res sites, both direct and inverted sites form synaptosomes . We conclude that the specificity of recombination is achieved by a three-stage recognition system: binding of resolvase to separate sites, formation of the synaptosome and determination of site orientation from within the complex.

EMBO J, 1988 Jun, 7(6), 1889 - 95
The partition locus of plasmid pSC101 is a specific binding site for DNA gyrase; Wahle E et al.; A protein in extracts of Escherichia coli that specifically binds the stabilizing par sequence of pSC101 was identified as DNA gyrase . The purified enzyme protects par against digestion by DNase I and exonuclease III . Competition assays demonstrate that gyrase has a 40-fold higher affinity for the 100-bp par sequence than for nonspecific DNA and that par is the major gyrase-binding site in pSC101 derivatives used in this and other studies . Within par, AT-rich sequences occur with a pronounced 10-bp periodicity that is shifted by 5 bp from a similar periodicity of GC-rich sequences . As judged by DNase I digestion, the GC sequences are exposed on the outside of the DNA wrapped around gyrase . The data suggest that the site-specificity of DNA gyrase may be partly determined by the bendability of the DNA . A 4-bp deletion that interferes with Par function in vivo also reduces the affinity for gyrase in vitro . However, a deletion of par causes little reduction in superhelical density in vivo . We conclude that DNA gyrase, while involved in the Par function, may not affect plasmid stability through its supercoiling activity or by an influence on DNA replication.

Mol Gen Genet, 1988 Jun, 212(3), 393 - 404
Apparent gene conversion in an Escherichia coli rec+ strain is explained by multiple rounds of reciprocal crossing-over; Yamamoto K et al.; Gene conversion, the non-reciprocal transfer of sequence information between homologous DNA sequences, has been reported in lower eukaryotes, mammals and in Escherichia coli . In an E . coli rec+ strain, we established a plasmid carrying two different deleted neo genes (neoDL and neoDR) in an inverted orientation and then selected for homologous recombination events that had reconstructed an intact neo+ gene . We found some plasmids that had apparently experienced intramolecular gene conversion . Further evidence, however, suggests that they are products of multiple rounds of reciprocal crossing-over, apparently involving two plasmid molecules . First, most of the Neo+ clones contained multiple types of Neo+ plasmids, although the frequency of producing the neo+ clones was low . Second, all the neo+ clones also contained, as a minority, one particular form of dimer, which can be formed by reciprocal crossing-over between neoDL of one plasmid molecule and neoDR of another plasmid molecule . Third, in reconstruction experiments, we cloned and purified this dimer and transferred it back into the rec+ cells . The dimer gave rise to clones containing multiple types of neo+ recombinant monomers, including those apparent gene conversion types, and containing only few molecules of this dimer plasmid.

Biochem J, 1988 Jun 1, 252(2), 563 - 9
Expression of a gene encoding a novel ferredoxin in the cyanobacterium Synechococcus 6301; Cozens AL et al.; A gene was discovered in the cyanobacterium Synechococcus 6301 that encodes a protein highly related to members of the {2Fe-2S} ferredoxin family found in chloroplasts and cyanobacteria . It follows a cluster of seven genes encoding subunits of the cyanobacterial ATP synthase complex . It is transcribed as a monocistronic mRNA of 408 nucleotide residues . Transcription starts at a site 55 bp upstream of the initiator methionine codon . Transcriptional initiation and termination signals with sequences similar to those found in Escherichia coli are not present . Comparison of the predicted sequence of the ferredoxin protein with those of other cyanobacterial and plant ferredoxins shows an average sequences identity of about 40% . Twelve amino acid residues are invariant, including the four cysteine residues that provide ligands for the {2Fe-2S} cluster . The deduced Synechococcus ferredoxin protein sequence has a C-terminal extension of eight amino acid residues relative to most other 2Fe-2S ferredoxins except for those from halobacteria, which also have a C-terminal extension . The sequence of the Synechococcus protein is most closely related to ferredoxins from the two complex cyanobacteria Chlorogloeopsis fritschii and Mastigocladus laminosus . The deduced protein sequence is not that of the major soluble ferredoxin that has been isolated from Synechococcus 6301 and is reported in the accompanying paper {Wada, Masui, Matsubara & Rogers (1988) Biochem . J . 252, 571-575} . So it appears to be a novel {2Fe-2S} ferredoxin and Synechococcus 6301 contains at least two {2Fe-2S} ferredoxins, which may have different roles in vivo.

Virus Res, 1988 Jun, 10(4), 343 - 56
Construction of recombinant fowlpox viruses as vectors for poultry vaccines; Boyle DB et al.; Plasmid vectors have been constructed which allow the construction of infectious fowlpox virus (FPV) recombinants expressing foreign genes . The foreign genes were inserted within the thymidine kinase (TK) gene of FPV contained in these vectors . To facilitate the selection of recombinants the Escherichia coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene was developed as a dominant selectable marker . This marker operates in a wide variety of cell types and obviates the need for TK- cell lines for selection of TK- recombinants when foreign genes have been inserted within the TK gene of FPV . The general approach adopted was to construct plasmid vectors in which the FPV TK was interrupted by the Ecogpt gene under the control of a poxvirus promoter in tandem with a gene of interest under the control of another poxvirus promoter . Selection of viruses expressing the Ecogpt gene simultaneously selects for recombinants carrying both the Ecogpt gene and the gene of interest . Using this approach a series of plasmid vectors was constructed in which the FPV TK gene was interrupted by the Ecogpt gene under the control of the P7.5 vaccinia virus promoter in tandem with the A/PR/8/34 haemagglutinin gene under the control of the PL11 vaccinia virus promoter . A recombinant FPV constructed using these plasmids had the expected genome arrangement, expressed influenza haemagglutinin, and induced haemagglutination-inhibiting antibodies when inoculated into chickens . These techniques should allow the construction of a variety of recombinant FPVs expressing poultry vaccine antigens . Such recombinants should be a very cost-effective means of delivering vaccines to poultry.

J Appl Physiol, 1988 Jun, 64(6), 2568 - 74
Prostaglandin E2 attenuation of sheep lung responses to endotoxin; Brigham KL et al.; Prostaglandin (PG) E2 can inhibit inflammatory responses of neutrophils and lymphocytes, including eicosanoid release . Diffuse lung injury after endotoxemia in sheep is accompanied by sequestration of neutrophils and lymphocytes in the lungs, and eicosanoids mediate some of the pathophysiology of the response . To determine whether exogenous PGE2 could prevent the endotoxin response, we measured pulmonary hemodynamics, gas exchange, and lung lymph responses to infusion of Escherichia coli endotoxin (0.5 micrograms/kg iv over 30 min) in unanesthetized sheep in the presence and absence of PGE2 (0.5 micrograms.kg-1.min-1) infused intravenously for 4 h beginning 0.5 h before endotoxin infusion . We also measured lung lymph concentrations of thromboxane B2 (TxB2) and prostacyclin metabolite, 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), by radioimmunoassay and leukotriene B4 (LTB4) by gas chromatography-mass spectrometry . PGE2 decreased endotoxin-induced pulmonary hypertension and hypoxemia and markedly attenuated the lymph flow and lymph protein clearance responses . PGE2 also attenuated endotoxin-induced increases in lung lymph TxB2 and 6-keto-PGF1 alpha and decreased lymph LTB4 flow after endotoxin without decreasing lymph LTB4 concentrations . We conclude that PGE2 infusion attenuates lung dysfunction caused by endotoxemia, possibly by preventing endogenous release of other eicosanoids.

Oncogene, 1988 Jun, 2(6), 539 - 44
The Escherichia coli Ras-like protein (Era) has GTPase activity and is essential for cell growth; March PE et al.; The era gene of Escherichia coli encodes a protein (Era) which is similar to the eucaryotic RAS family of proteins . We report here that purified Era possesses both GTP-binding and GTPase activities . Era is also shown to be loosely associated with the inner membrane of E . coli . Overproduction of Era to 5% of the total cellular protein does not apparently alter either cell growth or cAMP levels . Disruption of the era gene by insertional inactivation is shown to be lethal by construction of a conditional lethal era mutant strain.

Eur J Biochem, 1988 Jun 1, 174(2), 387 - 9
The stereochemical course of D-glyceraldehyde-induced ATPase activity of glycerokinase from Escherichia coli; Bethell RC et al.; D-Glyceraldehyde-induced hydrolysis of adenosine (R)-5'-{gamma-17O,18O,thio}triphosphate catalysed by glycerokinase from Escherichia coli gives inorganic {16O,17O,18O}thiophosphate with the (S)-configuration, showing that the reaction proceeds with inversion of configuration at phosphorus . This result provides powerful support for the chemically most plausible mechanism, namely, that the hydrate of D-glyceraldehyde is the effective substrate which after phosphorylation or thiophosphorylation eliminates inorganic phosphate or inorganic thiophosphate, respectively, with regeneration of D-glyceraldehyde.

Proc Natl Acad Sci U S A, 1988 Jun, 85(12), 4237 - 41
Escherichia coli host factor for site-specific DNA inversion: cloning and characterization of the fis gene; Koch C et al.; The Escherichia coli (Es . coli) protein Fis (factor for inversion stimulation) stimulates site-specific DNA inversion of the G segment in phage Mu by binding to a recombinational enhancer . By using synthetic oligonucleotides deduced from the amino-terminal amino acid sequence, we have cloned the gene (termed fis) encoding this specific DNA-binding protein . The DNA sequence shows that the Fis protein is basic and contains 98 amino acids . A helix-turn-helix sequence motif characteristic of many DNA-binding proteins is located at the carboxyl-terminal end of the protein . By marker exchange, we have constructed an insertion mutation of fis . Fis is nonessential for Es . coli growth; however, inversion of the G segment of a Mu prophage was not detected in the fis mutant . The fis gene is located between 71 and 72 min on the Es . coli genetic map.

Proc Natl Acad Sci U S A, 1988 Jun, 85(12), 4138 - 42
Cooperative DNA binding of heterologous proteins: evidence for contact between the cyclic AMP receptor protein and RNA polymerase; Ren YL et al.; Four cAMP-independent receptor protein mutants (designated CRP* mutants) isolated previously are able to activate in vivo gene transcription in the absence of cAMP and their activity can be enhanced by cAMP or cGMP . One of the four mutant proteins, CRP*598 (Arg-142 to His, Ala-144 to Thr), has been characterized with regard to its conformational properties and ability to bind to and support abortive initiation from the lac promoter . In the absence of cGMP, CRP*598 shows a more open conformation than CRP, as indicated by its sensitivity to proteolytic attack and 5,5'-dithiobis(2-nitrobenzoic acid)-mediated subunit crosslinking . Binding of wild-type CRP to its site on the lac promoter and activation of abortive initiation by RNA polymerase on this promoter are effected by cAMP but not by cGMP . CRP*598 can activate lacP+-directed abortive initiation in the presence of cAMP and less efficiently in the presence of cGMP or in the absence of cyclic nucleotide . DNase I protection ("foot-printing") indicates that cAMP-CRP* binds to its site on the lac promoter whereas unliganded CRP* and cGMP-CRP* form a stable complex with the {32P}lacP+ fragment only in the presence of RNA polymerase, showing cooperative binding of two heterologous proteins . This cooperative binding provides strong evidence for a contact between CRP and RNA polymerase for activation of transcription . Although cGMP binds to CRP, it cannot replace cAMP in effecting the requisite conformational transition necessary for site-specific promoter binding . In contrast, the weakly active unliganded CRP*598 can be shifted to a functional state not only by cAMP but also by cGMP and RNA polymerase.

Proc Natl Acad Sci U S A, 1988 Jun, 85(11), 3723 - 7
General method for quantifying base adducts in specific mammalian genes; Thomas DC et al.; A general method has been developed to measure the formation and removal of DNA adducts in defined sequences of mammalian genomes . Adducted genomic DNA is digested with an appropriate restriction enzyme, treated with Escherichia coli UvrABC excision nuclease (ABC excinuclease), subjected to alkaline gel electrophoresis, and probed for specific sequences by Southern hybridization . The ABC excinuclease incises DNA containing bulky adducts and thus reduces the intensity of the full-length fragments in Southern hybridization in proportion to the number of adducts present in the probed sequence . This method is similar to that developed by Bohr et al . {Bohr, V . A., Smith, C . A., Okumoto, D . S . & Hanawalt, P . C . (1985) Cell 40, 359-369} for quantifying pyrimidine dimers by using T4 endonuclease V . Because of the wide substrate range of ABC exinuclease, however, our method can be used to quantify a large variety of DNA adducts in specific genomic sequences.

J Infect Dis, 1988 Jun, 157(6), 1203 - 11
Antigenic and immunogenic properties of a hepatitis A virus capsid protein expressed in Escherichia coli; Johnston JM et al.; We have constructed a recombinant plasmid producing, in bacteria, a hepatitis A virus (HAV) capsid protein that may be useful as a subunit vaccine . HAV VP1 coding sequences were fused in-frame to NH2-terminal Escherichia coli TrpE coding sequences under the control of the tryptophan promoter . In the absence of exogenous tryptophan, E . coli containing this recombinant plasmid produced high levels of an 88-kilodalton fusion protein that was recognized in immunoblots by antibodies to TrpE and HAV . The TrpE/HAV VP1 protein was gel-purified and used to immunize rabbits . The resulting antiserum reacted with denatured HAV VP1 in immunoblots but did not react with intact virus . However, subsequent inoculation of an immunized animal with a subimmunogenic dose of inactivated, whole HAV resulted in the rapid appearance of a stable, virus-neutralizing antibody response.

J Bacteriol, 1988 Jun, 170(6), 2832 - 40
Recognition of Escherichia coli attTn7 by transposon Tn7: lack of specific sequence requirements at the point of Tn7 insertion; Gringauz E et al.; Transposon Tn7 inserts at high frequency into a specific site in the Escherichia coli chromosome called attTn7 . We show that the point of Tn7 insertion in attTn7 lies within the transcriptional terminator of the bacterial glmS gene . We have exploited the glmS transcription terminator to isolate mutants with altered sequences at the point of Tn7 insertion and have used these mutants to show that the nucleotide sequence at the point of Tn7 insertion is irrelevant to attTn7 target activity . Thus, the nucleotides which provide attTn7 target activity are distinct from the point of Tn7 insertion . We have also examined the effect of transcription on the capacity of attTn7 to act as a target for Tn7 transposition . Our results suggest that transcription of attTn7 does not modulate its Tn7 target activity.

J Bacteriol, 1988 Jun, 170(6), 2763 - 9
Nucleotide sequence of aceK, the gene encoding isocitrate dehydrogenase kinase/phosphatase; Klumpp DJ et al.; In Escherichia coli, the phosphorylation and dephosphorylation of isocitrate dehydrogenase (IDH) are catalyzed by a bifunctional protein kinase/phosphatase . We have determined the nucleotide sequence of aceK, the gene encoding IDH kinase/phosphatase . This gene consists of a single open reading frame of 1,734 base pairs preceded by a Shine-Dalgarno ribosome-binding site . Examination of the deduced amino acid sequence of IDH kinase/phosphatase revealed sequences which are similar to the consensus sequence for ATP-binding sites . This protein did not, however, exhibit the extensive sequence homologies which are typical of other protein kinases . Multiple copies of the REP family of repetitive extragenic elements were found within the intergenic region between aceA (encoding isocitrate lyase) and aceK . These elements have the potential for combining to form an exceptionally stable stem-loop structure (delta G = -54 kcal/mol {ca . -226 kJ/mol}) in the mRNA . This structure, which masks the ribosome-binding site and start codon for aceK, may contribute to the downshift in expression observed between aceA and aceK . Another potential stem-loop structure (delta G = -29 kcal/mol {ca . 121 kJ/mol}), unrelated to the REP sequences, was found within aceK.

J Bacteriol, 1988 Jun, 170(6), 2716 - 24
Genetics of the iron dicitrate transport system of Escherichia coli; Pressler U et al.; Escherichia coli B and K-12 express a citrate-dependent iron(III) transport system for which three structural genes and their arrangement and products have been determined . The fecA gene of E . coli B consists of 2,322 nucleotides and encodes a polypeptide containing a signal sequence of 33 amino acids . The cleavage site was determined by amino acid sequence analysis of the unprocessed protein and the mature protein . For the processed form a length of 741 amino acids was calculated . The mature FecA protein in the outer membrane contains at the N terminus the "TonB box," a pentapeptide, which has hitherto been found in all receptors and colicins which functionally require the TonB protein . In addition, the dyad repeat sequence GAAAATAATTCTTATTTCG is proposed to serve as the binding site of the Fur iron repressor protein . The fecB gene was mapped downstream of fecA and encodes a protein with an apparent molecular weight of 30,000 . It was synthesized as a precursor, and the mature form was found in the periplasm . The fecD gene follows fecB and was related to a membrane-bound protein with an apparent molecular weight of 28,000 . In Mu d1 insertion mutants upstream of fecA, the fec genes were not inducible by iron limitation and citrate, indicating a regulatory region, termed fecI, which controls fec gene expression.

J Bacteriol, 1988 Jun, 170(6), 2599 - 611
Fine-structure mapping and identification of two regulators of capsule synthesis in Escherichia coli K-12; Brill JA et al.; Positive and negative regulatory elements involved in the synthesis of colanic acid, the capsular polysaccharide of Escherichia coli K-12, have been identified previously . RcsB, a positive regulator for transcription of the structural genes of colanic acid synthesis (cps), is a protein of about 26 kilodaltons which probably acts as a multimer, rcsC, which maps close to rcsB at 48 min on the E . coli chromosome, exerts a negative effect on expression of the structural genes and codes for a protein of about 100 kilodaltons . The two genes appear to be transcribed in opposite directions, with the C-terminal ends of the genes being less than 0.3 kilobases apart . Multicopy expression of rcsB is lethal in rcsC mutants which carry cps-lac fusions, probably owing to accumulation of intermediates in the capsule synthesis pathway in these cells . Examination of double mutants and cells carrying multicopy rcsB+ plasmids reveal an rcsA-independent pathway for capsule synthesis . We hypothesize that RcsC may act as an environmental sensor, transmitting information to the RcsB positive regulator.

J Bacteriol, 1988 Jun, 170(6), 2568 - 74
A new pleiotropic mutation causing defective carbohydrate uptake in Escherichia coli K-12: isolation, mapping, and preliminary characterization; Mahajan SK et al.; A new pleiotropic mutation, designated cup-1 (for carbohydrate uptake), which impairs the ability of Escherichia coli cells to grow on a large number of phosphotransferase system (PTS) and non-PTS carbohydrates by blocking their entry into the cells, has been isolated, partially characterized, and mapped . The mutants grew poorly even on rich and glucose minimal media . Fast-growing revertants rapidly accumulated in cultures grown on either of the above two media and made stable maintenance of the mutation difficult . Several extragenic suppressor mutations that permitted cup cells to grow on specific single sugars or groups of sugars have been isolated . One such suppressor, which enabled cup cells to grow as well on glycerol minimal medium as their wild-type parent, has been helpful in stably maintaining these cells in this medium . cup-1 has been mapped to 97 min on the standard E . coli map . It cotransduced with a transposon Tn10 inserted clockwise to it and (very weakly) with uxuA . Surprisingly, it failed to cotransduce with pyrB, argI, or valS, three markers located nearby but counterclockwise to it . In F' merodiploids, cup-1 was dominant over its cup+ allele . Cyclic AMP permitted growth of cup-1 cells on some sugars but not all . Apparently, reduced cyclic AMP level and therefore noninduction of several sugar operons is one but not the only effect of cup.

Arch Surg, 1988 Jun, 123(6), 752 - 5
Abnormal rabbit heterophil chemotaxis following thermal injury . An in vivo model of an abnormality of the chemoattractant receptor for f-met-leu-phe; Davis JM et al.; Previous studies have shown that the decreased neutrophil migratory responsiveness seen in burned patients correlates with the extent of thermal injury and the extent of the neutrophil-specific granule deficiency . To understand better the relationship between the neutrophil dysfunction, degranulation, and thermal injury, a rabbit model was studied . Eighteen rabbits were burned over 20% of their surface area . Assay of peripheral blood heterophils disclosed decreased migratory activity compared with preburn levels and decreased lysozyme content vs preburn levels, but no change in the beta-glucuronidase content . The specific binding of tritiated formyl-methionyl-leucyl-phenylalanine to peripheral blood heterophils was increased fivefold over that of control cells . These studies indicate that, following thermal injury, there is a selective decrease of specific granule contents and an increase in chemoattractant binding to the cell and also suggest an abnormality in chemoattractant receptor processing . The rabbit provides a convenient model for the study of compromised host defenses following thermal injury.

Virology, 1988 Jun, 164(2), 450 - 7
Use of vaccinia virus vectors to study the synthesis, intracellular localization, and action of the human immunodeficiency virus trans-activator protein; Falkner FG et al.; A recombinant vaccinia virus that expresses the human immunodeficiency virus (HIV) trans-activator (tat) gene was constructed . The tat polypeptide migrated anomalously with an apparent molecular mass of 14 kDa on a sodium dodecyl sulfate-polyacrylamide gel and reacted with polyclonal anti-tat serum . The tat protein was localized predominantly in the cell nucleus despite the absence of other HIV proteins or intranuclear HIV DNA . Additional recombinant vaccinia viruses that contain the Escherichia coli chloramphenicol acetyltransferase (CAT) gene under control of an early vaccinia promoter were constructed . Insertion of the HIV trans-activator-responsive (tar) sequence at the precise start of the CAT mRNA decreased CAT expression slightly . Trans-activation of vaccinia virus-encoded tarCAT failed to occur when CV-1 or HeLa cells were coinfected with the recombinant vaccinia virus expressing tat or when a HeLa cell line containing stably integrated copies of tat was used for infection, indicating the absence of transcriptional or translational effects under these conditions.

Virology, 1988 Jun, 164(2), 301 - 8
Enzymatic activity of poliovirus RNA polymerase synthesized in Escherichia coli from viral cDNA; Rothstein MA et al.; Plasmids have been constructed that contain DNA sequences that direct the expression of the poliovirus RNA-dependent RNA polymerase, in the form of recombinant fusion proteins . Inclusion of an additional gene for the poliovirus protease results in cleavage of the fusion protein to yield a 52-kDa, enzymatically active, polymerase protein, apparently identical to the functional enzyme isolated from virus-infected HeLa cells . A large amount of polymerase protein accumulates as particulate or insoluble material in bacteria, and this protein has little or no activity . However, significant amounts of soluble, active enzyme are recovered, such that the resulting specific activity of crude bacterial extracts is greater than that obtained from virus-infected HeLa cells . Purification of the enzyme from Escherichia coli is readily accomplished, and yields a preparation that will copy poliovirion RNA as template, in the presence of oligo(U) primer . The availability of cloned DNA sequences encoding catalytically active RNA polymerase will allow genetic manipulations to initiate structure-function studies of this enzyme.

J Virol, 1988 Jun, 62(6), 2115 - 23
Identification of immunoreactive antigens of human papillomavirus type 6b by using Escherichia coli-expressed fusion proteins; Jenison SA et al.; Human papillomavirus (HPVs) infect the genital epithelium and are found in proliferative lesions ranging from benign condylomata to invasive carcinomas . The immunological response to these infections is poorly understood because of the lack of purified viral antigens . In this study, bacterially derived fusion proteins expressing segments of all the major open reading frames (ORFs) of HPV type 6b (HPV-6b) have been used in Western blot (immunoblot) assays to detect antibodies directed against HPV-encoded proteins . The most striking reactivities present in sera from patients with genital warts were to the HPV-6b L1 ORF protein and, to a lesser extent, to the HPV-6b L2 ORF protein . Two cases of reactivity to HPV-6b E2 ORF were observed, but no reactivities were seen with other HPV-6b constructs . Two sera reacted with the HPV-16 L2 fusion protein, and two sera reacted with the HPV-16 E4 protein . The antibodies directed against the HPV-6b fusion proteins showed no cross-reactivity with comparable regions of the HPV-16 ORFs . This assay provides a useful approach for further studies of HPV serology.

J Virol, 1988 Jun, 62(6), 1999 - 2006
Expression of simian virus 40 T antigen in Escherichia coli: localization of T-antigen origin DNA-binding domain to within 129 amino acids; Arthur AK et al.; The genomic coding sequence of the large T antigen of simian virus 40 (SV40) was cloned into an Escherichia coli expression vector by joining new restriction sites, BglII and BamHI, introduced at the intron boundaries of the gene . Full-length large T antigen, as well as deletion and amino acid substitution mutants, were inducibly expressed from the lac promoter of pUC9, albeit with different efficiencies and protein stabilities . Specific interaction with SV40 origin DNA was detected for full-length T antigen and certain mutants . Deletion mutants lacking T-antigen residues 1 to 130 and 260 to 708 retained specific origin-binding activity, demonstrating that the region between residues 131 and 259 must carry the essential binding domain for DNA-binding sites I and II . A sequence between residues 302 and 320 homologous to a metal-binding "finger" motif is therefore not required for origin-specific binding . However, substitution of serine for either of two cysteine residues in this motif caused a dramatic decrease in origin DNA-binding activity . This region, as well as other regions of the full-length protein, may thus be involved in stabilizing the DNA-binding domain and altering its preference for binding to site I or site II DNA.

J Virol, 1988 Jun, 62(6), 1925 - 31
Interaction of the bovine papillomavirus type 1 E2 transcriptional control protein with the viral enhancer: purification of the DNA-binding domain and analysis of its contact points with DNA; Moskaluk CA et al.; The E2 gene of bovine papillomavirus type 1 positively and negatively regulates the transcriptional enhancer located in the long control region of the viral genome . The DNA-binding domain of the E2 gene product was suspected to interact with the DNA sequence motif ACCN6GGT . We have shown that the carboxy-terminal 126 amino acids of the E2 protein constitute the DNA-binding domain . In this paper we described the expression of the E2 carboxy terminus in Escherichia coli and its subsequent purification . We provide definitive evidence that the protein recognizes the ACCN6GGT motifs in the viral enhancer . We show by methylation protection, methylation interference, and ethylation interference that the E2 protein contacts the DNA at the GG residues of the consensus sequence on both DNA strands . A gel retardation-DNase I footprint assay has revealed that the E2 DNA-binding domain exhibits different affinities for different ACCN6GGT motifs, indicating that nucleotides other than the conserved ACC and GGT sequences probably modulate the affinity of the DNA sequence for the E2 protein.

J Biomol Struct Dyn, 1988 Jun, 5(6), 1259 - 66
Effect of uridine dethiolation in the anticodon triplet of tRNA(Glu) on its association with tRNA(Phe); Houssier C et al.; The effect of U(34) dethiolation on the anticodon-anticodon association between E . coli tRNA(Glu) and yeast tRNA(Phe) has been studied by the temperature jump relaxation technique . An important destabilization upon replacement of the thioketo group of s2U(34) by a keto group, was revealed by a lowering of melting temperature of about 20 degrees C . The measured kinetic parameters indicated that this destabilization effect was originated in an increase of dissociation and a decrease of association rate constants by a factor of 4 to 5 . Modifications in both stacking interactions and flexibility in the anticodon loop would be responsible for this effect.

Mol Immunol, 1988 Jun, 25(6), 555 - 63
Immunological evidence for differences in the exposed regions of OmpF porins from Escherichia coli B and K-12; Pages JM et al.; Nine monoclonal antibodies (MoF 0-8) directed against the native form (trimeric) of outer membrane protein OmpF of Escherichia coli B were obtained and characterized . All these antibodies bind to OmpF porin in intact E . coli B cells but not OmpF from E . coli K-12 cells which only differ at positions 66, 117 and 262 in the sequence . These antibodies exhibit a specificity to the native form, failing to recognize the denatured form in a liquid immunorecognition assay . Four tested antibodies are able to protect against colicin A, a bacteriotoxin using OmpF as receptor . One monoclonal antibody (MoF 0) is specific to the external topology of native porin in the outer membrane and three antibodies could recognize epitopes present in each conformation of subunits of trimer form . It is concluded that the region around the 66th and more probably around the 262nd amino acids are involved in cell-surface exposed epitopes . Moreover, these results support the assumption that the conformation of protruding regions of OmpF from E . coli B and K-12 are different.

Anal Biochem, 1988 Jun, 171(2), 271 - 6
Inorganic pyrophosphatase as a label in heterogeneous enzyme immunoassay; Baykov AA et al.; Inorganic pyrophosphatase from Escherichia coli has been employed as a label in heterogeneous enzyme immunoassays . Enzyme-antibody conjugates were prepared with the use of glutaraldehyde and purified by gel permeation chromatography . Enzyme activity was measured by means of a sensitive one-step color reaction between phosphate, molybdate, and malachite green . The sensitivity in terms of absorbance readings was four to eight times higher than that of peroxidase-based assays . The color change (yellow to greenish blue) inherent in the use of pyrophosphatase as the labeling agent is highly suitable for visual analysis . Other merits of pyrophosphatase include the remarkable stability of the enzyme and its substrate, its compatibility with bacteriostatic agents, and its low Michaelis constant . Examples of the use of phosphatase in the assay of human alpha-fetoprotein and immunoglobulin G are presented.

Eur J Immunol, 1988 Jun, 18(6), 849 - 54
T cell epitopes on the 36K and 65K Mycobacterium leprae antigens defined by human T cell clones; Van Schooten WC et al.; To identify the molecular localization and specificity of Mycobacterium leprae antigenic determinants inducing T cell activation, we studied the reactivity of M . leprae-reactive T cell clones from two tuberculoid leprosy patients towards a battery of different mycobacterial strains and purified mycobacterial antigens . Of the 38 T cell clones tested 8 appeared to be M . leprae specific (specificity A), another 8 were cross-reactive with at least one of the three mycobacterial strains, M . lepraemurium, M . vaccae and M . scrofulaceum (specificity B), 5 were reactive with most but not all strains (specificity C) and the remaining 18 were reactive with all 17 mycobacterial strains tested (specificity D), but not with nonmycobacterial antigens . All T cell clones were tested with the 36K and 65K antigen isolated from M . leprae, and with the M . leprae and M . bovis BCG 65K proteins produced in E . coli by recombinant DNA . Four T cell clones appeared to recognize epitopes on the 36K antigen, nine T cell clones recognized the 65K antigen . These 2 M . leprae antigens, 36K and 65K, thus seem to contain major T cell epitopes . At least 3 different epitopes could be defined on the 36K antigen of which one is M . leprae specific, one of specificity B and one of specificity C . Two distinct epitopes were discerned on the 65K antigen of which one is M . leprae specific and one of specificity D . The M . leprae-specific epitopes on the 36K and 65K antigen may help in the development of a specific serodiagnostic and skin test.

Proc Natl Acad Sci U S A, 1988 Jun, 85(11), 3918 - 22
Mutagenesis by the autoxidation of iron with isolated DNA; Loeb LA et al.; Oxygen free radicals are highly reactive species generated by many cellular oxidation-reduction processes . These radicals damage cellular constituents and have been causally implicated in the pathogenesis of many human diseases . We report here that oxygen free radicals generated by Fe2+ in aqueous solution are mutagenic . Aerobic incubation of luminal diameter X174 am3 (amber 3 mutation) DNA with Fe2+ results in decreased phage survival when the treated DNA is transfected into Escherichia coli spheroplasts . Transfection of the treated DNA into SOS-induced spheroplasts results in an increase in mutagenesis as great as 50-fold . Both killing and mutagenesis can be prevented by binding of Fe2+ with deferoxamine or by the addition of catalase or mannitol . These results suggest that DNA damage and mutagenesis brought about by Fe2+ are likely to occur by a Fenton-type mechanism that involves the generation of (i) hydrogen peroxide by the autoxidation of iron and (ii) hydroxyl radicals by the interaction of the hydrogen peroxide with Fe2+ . DNA sequence analysis of the Fe2+-induced mutants indicates that reversion of the phage phenotype to wild type occurs largely by a transversion type of mutation involving substitution of deoxyadenosine for thymidine opposite a template deoxyadenosine . Mutagenesis is not abolished by incubation of Fe2+-treated luminal diameter X174 am3 DNA with an apurinic endonuclease and only partially abolished by incubation with alkali, suggesting that a large fraction of the mutagenesis by oxygen free radicals is not caused by formation of apurinic sites but instead involves an as-yet-to-be-defined alteration in deoxyadenosine . These findings raise the possibility that free iron localized in cellular DNA may cause mutations by the generation of oxygen free radicals.

Infect Immun, 1988 Jun, 56(6), 1633 - 40
Use of recombinant antigens expressed in Escherichia coli K-12 to map B-cell and T-cell epitopes on the immunodominant 65-kilodalton protein of Mycobacterium bovis BCG; Thole JE et al.; In gene libraries of Mycobacterium bovis BCG, Mycobacterium tuberculosis, and Mycobacterium leprae, recombinants were frequently encountered that expressed an immunodominant 65-kilodalton (kDa) protein antigen that was shown to react with a high proportion of mycobacterium-reactive human and murine T cells and murine monoclonal antibodies . In this study, recombinant antigens were used to map T-cell and B-cell epitopes on the M . bovis BCG 65-kDa protein that was previously designated MbaA . Four different T-cell-epitope-containing regions (amino acid residues 1 through 16, 17 through 61, 85 through 108, and 235 through 279) were defined that were recognized by seven T-cell clones from patients with tuberculoid leprosy . These regions are distinct from two previously described T-cell epitopes recognized by T cells from a tuberculosis patient . As T-cell clones restricted by different class II determinants were shown to be specific for different regions on the 65-kDa protein, the presented data suggested that the products of different human leukocyte antigen class II loci and alleles present different parts of MbaA to the immune system . B-cell epitopes recognized by 20 monoclonal antibodies were assigned to eight different regions of MbaA . Using 15 of these antibodies, we previously showed that MbaA was antigenically related to a common antigen present in many bacterial species . The dispersed localization of the involved epitopes defined here shows that various different parts of MbaA are indeed conserved . These results show that well-defined recombinant antigens are useful tools for the localization of both B- and T-cell-epitope-containing regions of a protein . Peptides synthesized from the sequences of such regions may then exactly define the epitopes relevant for the development of specific diagnostic tests or of vaccines against mycobacteria.

Biochemistry, 1988 May 31, 27(11), 3966 - 74
Processivity in early stages of transcription by T7 RNA polymerase; Martin CT et al.; Immediately following initiation of transcription, T7 RNA polymerase enters a phase in which dissociation of the enzyme-DNA-RNA ternary complex significantly competes with elongation, a process referred to in the Escherichia coli enzyme as abortive cycling {Carpousis, A.J., & Gralla, J.D . (1980) Biochemistry 19, 3245-3253} . Characterization of this process in the T7 RNA polymerase system under various reaction conditions and on templates with differing message sequences reveals that conversion to a highly processive ternary complex occurs after incorporation of eight bases and that the relative competition between dissociation and elongation up to this point is influenced by several different forces . In particular, the sequence dependence of abortive falloff suggests that dissociation is favored immediately following incorporation of UMP and is less likely following incorporation of GMP into the RNA message . Abortive cycling is unchanged in transcription from a synthetic oligonucleotide template which is double-stranded in the promoter region but single-stranded throughout the entire message region . This result proves that melting and reannealing of the DNA duplex in the coding region do not contribute to abortive cycling . Furthermore, weakening of promoter binding by an order of magnitude affects abortive cycling only slightly, suggesting that strong interactions with the promoter are not the major cause of abortive cycling . Kinetic analyses show that conversion to a highly processive ternary complex after the incorporation of eight bases may reflect a large decrease in the unimolecular rate of dissociation of the complex due to increased contacts between the nascent RNA and the DNA template and between RNA and enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1988 May 31, 153(1), 334 - 9
Nonhistone protein HMG1 removes the transcriptional block caused by left-handed Z-form segment in a supercoiled DNA; Waga S et al.; The effect of HMG1 on the transcriptional block caused by left-handed Z-form in a highly negatively supercoiled DNA was examined using a supercoiled plasmid containing a (CG)10 sequence downstream of promoters . The transcription by E . coli RNA polymerase was blocked at the boundary of alternating CG sequence in the Z-form . In the presence of HMG1, RNA polymerase could transcribe through the CG sequence resulting in the chain elongation of transcripts . The addition of HMG1 allowed the stalled RNA polymerase at the CG block to resume transcription . These suggest that HMG1 may remove the Z-block by flipping it back into the B-form.

Biochem Biophys Res Commun, 1988 May 31, 153(1), 359 - 64
Amino acid sequence of S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum as deduced from the cDNA sequence; Kasir J et al.; S-Adenosyl-L-homocysteine hydrolase has been cloned from a lambda gt11 cDNA library prepared from Dictyostelium discoideum that had been starved for 3 hours . The sequence of the cloned cDNA was determined and the deduced amino acid sequence was compared to the amino acid sequence of rat AdoHcy hydrolase . When the sequences from the two species were aligned, 74% of the amino acids were in identical positions . If conservative changes were taken into account the homology was 84% . Because differences have been reported in the binding characteristics of NAD+ to the D . discoideum and rat AdoHcy hydrolases, changes in the amino acids of the putative NAD+-binding site were of particular interest . Six changes were observed in this region but the changes appeared to be in regions that are not critical to the three dimensional folding of the NAD+-binding site.

Biochemistry, 1988 May 31, 27(11), 3983 - 90
{3H}-p-azidopuromycin photoaffinity labeling of Escherichia coli ribosomes: evidence for site-specific interaction at U-2504 and G-2502 in domain V of 23S ribosomal RNA; Hall CC et al.; Previously we (1) showed that {3H}-p-azidopuromycin was a functional puromycin analogue that, on photolysis in the presence of 70S ribosomes from Escherichia coli, photoincorporated site specifically into proteins L23, L18/22, and L15 {Nicholson, A.W., Hall, C.C., Strycharz, W.A., & Cooperman, B.S . (1982) Biochemistry 21, 3809-3817} and (2) used immunoelectron microscopy to localize the principal sites of p-azidopuromycin photoincorporation within the 50S subunit {Olson, H.M., Nicholson, A.W., Cooperman, B.S., & Glitz, D.G . (1985) J . Biol . Chem . 260, 10326-10331} . These studies are here continued by identification of the principal sites of {3H}-p-azidopuromycin photoincorporation into ribosomal RNA . The major portion of such photoincorporation, 72%, takes place into 23S rRNA . Analysis by hybridization of the photoaffinity-labeled rRNA to restriction enzyme fragments of plasmid pKK3535, which contains rrnB DNA, using a refinement of a recently developed methodology {Hall, C.C., Smith, J.E., & Cooperman, B.S . (1985) Biochemistry 24, 5702-5711}, shows that the most prominent {3H}-p-azidopuromycin photoincorporation occurs within bases 2445-2668 in domain V {Noller, H.F . (1984) Annu . Rev . Biochem . 53, 119-162} of 23S rRNA . Photoincorporation into this region is site specific, as demonstrated by the decrease in photoincorporation of radioactivity when unlabeled puromycin is included in the photolysis solution . Significant site-specific photoincorporation also occurs within bases 489-681 in domain II of 23S RNA . Further localization, by the method of reverse transcriptase primer extension {Barta, A., Steiner, G., Brosius, J., Noller, H.F., & Kuechler, E . (1984) Proc . Natl . Acad . Sci . U.S.A . 81, 3607-3611}, provides evidence that U-2504 and G-2502 are the principal sites of p-azidopuromycin interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1988 May 31, 27(11), 3900 - 6
Physical properties of DNA in vivo as probed by the length dependence of the lac operator looping process; Bellomy GR et al.; Plasmid constructs containing a wild-type (O+) lac operator upstream of an operator-constitutive (Oc) lac control element exhibit a length-dependent, oscillatory pattern of repression of expression of the regulated gene as interoperator spacing is varied from 115 to 177 base pairs (bp) . Both the length dependence and the periodicity of repression are consistent with a thermodynamic model involving a stable looped complex in which bidentate lac repressor interacts simultaneously with both O+ and Oc operators . The oscillatory pattern of repression with distance occurs with a period approximating the helical repeat of DNA and presumably reflects the necessity for proper alignment of interacting operators along the helical face of the DNA . In the length regime examined, the presence of the upstream operator enhances repression between 6-fold and 50-fold depending upon phasing . This reflects a torsional rigidity of DNA in vivo that is consistent with in vitro measurements . The oscillatory pattern of repression is best fit with a period of either 9.0 or 11.7 bp/cycle but not 10.5 bp/cycle . This periodicity is interpreted as reflecting the average helical repeat of the 40-bp interoperator region of plasmid DNA in vivo, suggesting that the local helical repeat of DNA in vivo may differ significantly from 10.5 bp/turn . The apparent persistence length needed to fit the data (aapp) is only one-fifth the standard in vitro value . This low value of aapp may be due in part to DNA bending induced by catabolite activator protein (CAP) bound to its site between the interacting operators.(ABSTRACT TRUNCATED AT 250 WORDS)

Philos Trans R Soc Lond B Biol Sci, 1988 May 31, 319(1193), 121 - 6
Import of proteins into mitochondria; Eilers M et al.; A mounting body of evidence suggests that cytoplasmically synthesized proteins destined to be imported into the mitochondrial interior must at least partly unfold to penetrate across the mitochondrial membranes . During post-translational import, this unfolding process appears to be a major rate-limiting step . It can be blocked by ligands that stabilize the protein's native conformation and appears to be accompanied by the cleavage of ATP outside the mitochondrial inner membrane.

Biochem Biophys Res Commun, 1988 May 31, 153(1), 328 - 33
Molecular cloning, sequence analysis, and expression of a human liver cDNA coding for fructose-6-P,2-kinase:fructose-2,6-bisphosphatase; Algaier J et al.; A cDNA coding for 378 amino acids from the C-terminus of the human liver bifunctional enzyme, Fructose-6-phosphate,2-kinase:Fructose-2,6-bisphosphatase was isolated, sequenced, and expressed in E . coli K38 . The expressed protein, identified by specific immunoassay, showed Fru 2,6-bisphosphatase activity but no Fru 6-P,2-kinase activity, demonstrating directly that the Fru 2,6-bisphosphatase activity resides in the C-terminal region . The Km for Fru 2,6-P2 was 4.3 microM . Fru 6-P was a noncompetitive inhibitor (Ki = 2.9 microM), and formed a phosphorylated intermediate when incubated with Fru 2,6{2-32P}P2 . The subunit Mr of the enzyme was 36,600, and the active enzyme showed Mr = 37,000 by gel filtration.

Gene, 1988 May 30, 65(2), 285 - 92
Nucleotide sequence and expression in Escherichia coli of cDNA of swine pepsinogen: involvement of the amino-terminal portion of the activation peptide segment in restoration of the functional protein; Tsukagoshi N et al.; A clone, pSPcA2, which carries the full-length swine pepsinogen cDNA was isolated . The coding sequence comprised the signal peptide {15 amino acids (aa)}, the activation peptide segment (44 aa) and mature pepsin (327 aa) . The deduced amino acid sequence agrees with the published sequence with two exceptions . Asparagine instead of aspartate is present at aa positions 19 and 308 . Two types of plasmids, pAS and pUCtacSPc series, were constructed for expressing swine pepsinogen cDNA . These plasmids directed the synthesis of polypeptides which were detected by employing an antibody to swine pepsinogen . However, all the polypeptides formed aggregates and showed no acid protease activity . Only the protein directed by pAS5 regained the acid protease activity after renaturation procedures . The activity was completely inhibited by pepstatin . Furthermore, the renatured pAS5 protein was spontaneously converted to pepsin under acidic conditions . The presence of Arg-8 in the activation peptide segment appears important for the stabilization of the pepsinogen molecule.

Gene, 1988 May 30, 65(2), 195 - 202
Acquisition of new metabolic capabilities: multicopy suppression by cloned transaminase genes in Escherichia coli K-12; Berg CM et al.; The four general transaminases of Escherichia coli K-12 have overlapping, but discrete, substrate specificities and participate in the final step in the synthesis of at least seven different amino acids . Through the use of strains that have mutations in one or more transaminase genes and carry a different wild-type (wt) gene on a multicopy plasmid, it was possible to detect instances in which an amplified wt gene suppressed nonallelic mutations . In these cases, overproduction of the enzyme permitted a broader range of substrates to be used at physiologically significant levels, either because a low catalytic efficiency (in the case analyzed here) or a low affinity of the enzyme towards the substrate prevented its effective utilization under normal conditions . Consequently, by compensating for a low catalytic reaction rate, enzyme overproduction circumvents the original lesion and restores biosynthetic activity to the mutant strain . The suppression of a mutation in one gene by amplified copies of a different wt gene is termed 'multicopy suppression' . This phenomenon is useful for detecting poorly expressed genes, for detecting duplicate genes, for identifying secondary functions of the products of known genes, and for elucidating the metabolic role of the product of the suppressed gene.

Gene, 1988 May 30, 65(2), 167 - 77
Overproduction, purification and crystallization of TaqI restriction endonuclease; Barany F; Under phoA promoter control, TaqI endonuclease was overproduced to 5% of Escherichia coli cellular proteins . This was achieved by fusing the endonuclease gene to the first four codons of the alkaline phosphatase signal sequence . For maximal overproduction (30% of cellular proteins), a putative 14-bp hairpin within the endonuclease coding sequence was replaced with degenerate codons . In addition, TaqI methylase was required to protect host DNA . The endonuclease was purified in sufficient amounts for crystallization.

Gene, 1988 May 30, 65(2), 149 - 65
The TaqI 'star' reaction: strand preferences reveal hydrogen-bond donor and acceptor sites in canonical sequence recognition; Barany F; TaqI endonuclease recognizes and cleaves its canonical sequence, TCGA, with complete fidelity under standard conditions . In the presence of some organic solvents, TaqI endonuclease introduced additional single-strand and double-strand cuts at sequences termed TaqI 'star' sites . Using 'middle-labeled' DNA, the relative rates of cleavage of each strand were simultaneously determined for several star sites . These star recognition sequences differed from the canonical sequence by a single base, and all potential star sites were either nicked or cleaved . Star sites within the middle labeled substrate represented ten of the twelve possible star sequences for each strand . For each group of identical star sites, one strand was consistently preferred for cleavage . Based on these preferences, a model for TaqI recognition of the TCGA sequence is proposed . According to this model, sequence discrimination is mediated by eight hydrogen bonds formed between TaqI and the cognate nucleotides within the major groove.

Nucleic Acids Res, 1988 May 25, 16(10), 4539 - 54
Modifications of guanine bases during oligonucleotide synthesis; Yeung AT et al.; Guanine bases are sensitive to modification during automated DNA synthesis and processing reactions . Methods for the detection of two types of guanine modifications are described . The first method uses the higher reactivity of the modified G base to KMn04 oxidation than T bases, and thus allows detection by chemical DNA sequencing . The second method makes use of the Escherichia coli nucleotide excision repair enzyme UvrABC endonuclease which can detect "bulky" base modifications at each nucleotide in the synthetic DNA . Though the chemical structures of the two modifications are not known, they may be related . Both types of G modifications are often found in oligonucleotides synthesized by the methoxy-diisopropyl-phosphoramidite (MEDP) chemistry but non-detectable in the products of the beta-cyanoethyl-diisopropyl-phosphoramidite (CEDP) chemistry . The Rubin and Schmid pyrimidine-specific chemical DNA sequencing procedure (Rubin, C.M., and Schmid, C.W . (1980) Nucleic Acids Res . 8, 4613-4619) was found to be applicable to oligonucleotides synthesized by the CEDP chemistry, and to oligonucleotides synthesized by the MEDP chemistry if precautionary measures are taken to destroy the signals produced by the highly KMnO4 sensitive modified guanine bases . We also show how chemical DNA sequencing might be useful for diagnosing other chemical modifications in synthetic oligonucleotides.

Nucleic Acids Res, 1988 May 25, 16(10), 4287 - 98
Synthesis of a gene for the HIV transactivator protein TAT by a novel single stranded approach involving in vivo gap repair; Adams SE et al.; The synthesis of a gene for the HIV TAT protein is described using a novel approach that capitalises on the ability to synthesise oligonucleotides of greater than 100 bp in length . It involves the synthesis of large oligomers covering one strand of the desired gene in its entirety and the use of small complementary bridging and adapter oligonucleotides to direct the assembly and cloning of the large oligomers . After ligation to the cloning vector the partially single stranded intermediate is transformed directly into the recipient bacterial host where the plasmid is repaired . The synthetic tat gene has been expressed in HeLa cells and is shown to trans-activate TAR+ but not TAR- HIV LTR-CAT constructs.

J Biol Chem, 1988 May 25, 263(15), 7378 - 85
Characteristics of Z-DNA helices formed by imperfect (purine-pyrimidine) sequences in plasmids; McLean MJ et al.; The capacities of three synthetic sequences to adopt left-handed helices were evaluated in recombinant plasmids . The sequences consisted of very short runs of (CG)n (n = 2-4) interspersed with runs of alternating A.T base pairs and/or with regions of non-alternating base pairs . The plasmids were studied by two-dimensional gel electrophoresis to determine the natures of the conformational transitions and their free energies of formation . These results coupled with analyses with chemical (diethyl pyrocarbonate, osmium tetroxide, and bromoacetaldehyde) and enzymatic (S1 nuclease, T7 gene 3 product, and MHhaI) probes indicated that the entire sequence was adopting a left-handed helix in each case . In one of these sequences, Z-DNA formation necessitated the retention of the anti conformation of one of the guanines in a region of non-alternation . In a sequence which contains out-of-phase regions of alternation, our results indicate the formation of a separate left-handed helix in the central (CG)2 region, thus forming two Z-Z junctions . In summary, we conclude that only very short regions of alternating CG are necessary to effect the B to Z transition and that this conformational change can be transmitted through non-alternating regions . A set of empirical rules governing the characteristics of the B to Z transition and the types of left-handed helices in supercoiled plasmids was derived from studies on a systematic series of 17 plasmids.

J Biol Chem, 1988 May 25, 263(15), 7261 - 5
A functional decaisoleucine-containing signal sequence . Construction by cassette mutagenesis; Kendall DA et al.; An alkaline phosphatase signal sequence optimized for formation of a hydrophobic alpha-helix functions very efficiently in the transport process . This mutant contained a core region comprised of 9 consecutive leucine residues (Kendall, D . A., Bock, S . C., and Kaiser, E . T . (1986) Nature 321, 706-708) . We have now constructed a second mutant containing a decaisoleucine core region . Isoleucine was chosen because it is an isomer of leucine with comparable hydrophobicity but in synthetic peptides isoleucine favors beta-sheet formation . Surprisingly, this mutant precursor was also processed efficiently, and mature alkaline phosphatase was correctly targeted to the Escherichia coli periplasm . Since the effective length of a beta-strand is extended relative to an alpha-helix, conformational differences should be mirrored by the relative effectiveness of shortened polyisoleucine and polyleucine core regions . However, analysis of two additional mutants containing truncated segments of either polyleucine or polyisoleucine did not reveal any differences and both accumulate as precursors . We conclude that these mutants do not adopt critically different structures . This comparative analysis was facilitated by construction of a new plasmid, CASS3 . This plasmid contains unique restriction sites flanking the DNA region coding for the signal sequence hydrophobic core segment . Consequently, the wild type core-encoding region can be readily replaced with synthetic oligonucleotides coding for new structural units and multiple amino acid substitutions can be made without the need for step-wise mutagenesis.

J Biol Chem, 1988 May 25, 263(15), 7211 - 5
Chelating peptide-immobilized metal ion affinity chromatography . A new concept in affinity chromatography for recombinant proteins; Smith MC et al.; We report our experimental results supporting the hypothesis that a specific metal-chelating peptide (CP) on the NH2 terminus of a protein can be used to purify that protein using immobilized metal ion affinity chromatography (IMAC) . The potential utility of this approach resides with recombinant proteins since the nucleotide sequence that codes for the protein can be extended to include codons for the chelating peptide and thereby generate the gene for a chimeric CP-protein that can be cloned, expressed, and affinity-purified with immobilized metal ions . The chelating peptide purification handle could then be removed chemically or enzymatically after purification has been achieved to generate a protein with the natural amino acid sequence . The feasibility of using a chelating peptide as a purification handle has been demonstrated using a leuteinizing hormone-releasing hormone (LHRH) analog, 2-10 LHRH, which contains the previously identified chelating peptide, His-Trp, on the NH2 terminus . 2-10 LHRH had a high affinity for a Ni(II) IMAC column due to the NH2-terminal dipeptide sequence His-Trp, forming a coordination complex with Ni(II), whereas the controls, 3-10 LHRH and 4-10 LHRH, lacking the CP sequence, did not bind . Furthermore, 2-10 LHRH could be purified from a mixture of histidine-containing peptides on a Ni(II) IMAC column in one step . His-Trp proinsulin was used as a model of a recombinant CP-protein . The S-sulfonates of His-Trp-proinsulin and proinsulin were isolated from Escherichia coli engineered to overproduce these proteins as trpLE' fusion proteins . His-Trp-proinsulin(SSO3-)6 had a higher affinity for immobilized Ni(II) than proinsulin (SSO3-)6 . Both proteins were eluted by decreasing the pH or by introducing a displacing ligand into the buffer . Ni(II) eluted from the column with much higher concentrations of displacing ligand than the proteins.

J Biol Chem, 1988 May 25, 263(15), 7181 - 5
Purification and reconstitution of Escherichia coli proline carrier using a site specifically cleavable fusion protein; Hanada K et al.; We previously constructed a bifunctionally active membrane-bound fusion protein, in which Escherichia coli proline carrier (the product of the putP gene) was linked with beta-galactosidase (the product of the lacZ gene) through a collagen linker (Hanada, K., Yamato, I., and Anraku, Y . (1987) J . Biol . Chem . 262, 14100-14104) . The proline carrier was purified from this site specifically cleavable fusion protein . Cytoplasmic membranes overproducing the fusion protein were solubilized with dodecylmaltoside, and the solubilized fraction was subjected to anti-beta-galactosidase IgG-Sepharose chromatography . The fusion protein was specifically adsorbed to the immunoaffinity resin and then treated with collagenase for splitting the proline carrier moiety of the fusion protein from the beta-galactosidase moiety . The collagenase used for the collagenolysis was then removed by anti-collagenase IgG-Sepharose chromatography . In this way, the proline carrier was purified to more than 95% homogeneity of the protein . Proline transport in proteoliposomes reconstituted with the purified carrier was dependent on the membrane potential and the chemical gradient of Na+ across the membrane with apparent Michaelis constants for proline and for Na+ stimulation of 3.6 microM and 31 microM, respectively . These results indicated that the proline carrier mediates electrogenic Na+/proline symport.

Nucleic Acids Res, 1988 May 25, 16(10), 4465 - 82
RglB facilitated cloning of highly methylated eukaryotic DNA: the human L1 transposon, plant DNA, and DNA methylated in vitro with human DNA methyltransferase; Woodcock DM et al.; In vitro methylation of Bluescribe plasmid DNA (pBS) with human placental DNA methyltransferase to 6% 5-methylcytosine (mC) reduced transformation efficiencies in rglB+ host strains C600 and DS410 by almost 2 orders of magnitude . By contrast, the rglB- derivative of DS410 showed no reduction in transformation efficiency with methylation while the rglB- derivative of C600 was partially tolerant to methylation . Further, we show that the 1.8 kilobase (kb) and 1.2 kb KpnI fragments derived from the human L1 repeat have respectively 18.3% and 2.3% mC in vivo . Using these hyper- and hypo-methylated genomic segments ligated into the pBS plasmid, transformants with the highly methylated 1.8 kb L1 insert were recovered at 17 to 40 fold higher frequency with the rglB- host strains than with the rglB+ hosts . In addition, recombinant phage (lambda 2001) containing inserts of plant genomic DNA with 26.7% mC (from Petunia hybrida) when plated on rglB- hosts gave titres up to 222 times higher than on the rglB+ strains.

Nucleic Acids Res, 1988 May 25, 16(10), 4447 - 63
Cloning and characterization of a photolyase gene from the cyanobacterium Anacystis nidulans; Yasui A et al.; A 2 kb fragment was isolated from an Anacystis nidulans genomic DNA library by hybridization with synthetic oligonucleotide probes derived from the N-terminal amino acid sequence of Anacystis photolyase . This fragment contains a 1452 bp-long open reading frame encoding a polypeptide of 484 amino acids (Mr 54475) . Antibodies raised against purified Anacystis photolyase reacted with extracts of cells harboring fused genes between lacZ of Escherichia coli and this gene . A 40.7% similarity was found between the deduced amino acid sequences of Anacystis and E . coli photolyases, notwithstanding the difference in chromophore structure.

Nucleic Acids Res, 1988 May 25, 16(10), 4353 - 67
Prediction and demonstration of a novel Epstein-Barr virus nuclear antigen; Allday MJ et al.; The protein sequence predicted by the Epstein Barr virus (EBV) BERF4 open reading frame includes a tetrapeptide, Lys-Arg-Pro-Arg (KRPR), shown for other proteins to be a component of a signal for rapid nuclear localization . A subgenomic fragment of EBV DNA containing BERF4 has been incorporated into an expression vector, transfected onto primate cells and the nuclear distribution of the resulting protein established by immunofluorescence using EBV positive human sera . These sera contained high titres of antibodies to a fusion protein, produced in E . coli, consisting of beta-galactosidase and the C-terminal 167 amino acids of BERF4 . Immunoaffinity purified antibodies reactive with the EBV component of the fusion show the molecular weight of this antigen in EBV immortalized B-cell lines to be about 160 kD . The demonstration that BERF4 contains an exon encoding a nuclear protein identifies a new EBNA gene (EBNA-6) and suggests that KRPR is a signal sequence common to a number of viral and cellular nuclear polypeptides which bind to nucleic acids and may therefore be of predictive value in identifying karyophilic proteins.

Nucleic Acids Res, 1988 May 25, 16(10), 4341 - 52
Site-specific degradation of Streptomyces lividans DNA during electrophoresis in buffers contaminated with ferrous iron; Zhou X et al.; Streptomyces lividans DNA contains a modification which makes it susceptible to double-strand cleavage during electrophoresis in buffers contaminated with ferrous iron (which may be present in some batches of EDTA) . The cleavage of the DNA is site-specific and the average fragment size resulting from limit digestion of total S . lividans DNA is about 6kb . DNA from Streptomyces coelicolor A3(2) and several other Streptomyces strains, and from E . coli, is not cleaved under the same conditions . A S . lividans mutant has been isolated which lacks the DNA modification . We suspect that many reports of "poor" preparations of S . lividans plasmids may be due to the above effect.

Nucleic Acids Res, 1988 May 25, 16(10), 4315 - 29
Intra-RNA cross-linking in Escherichia coli 30S ribosomal subunits: selective isolation of cross-linked products by hybridization to specific cDNA fragments; Stiege W et al.; M13 clones were constructed with cDNA inserts corresponding to specific regions of E . coli ribosomal RNA . The DNA from the clones was immobilized by coupling to diazobenzyloxymethyl cellulose, and was used for the selective isolation by hybridization of cross-linked RNA complexes containing the complementary sequences . Immobilized DNA samples with inserts complementary to four different regions covering bases 735-1384 of the 16S RNA were hybridized with a mixture of 16S RNA fragments generated by partial digestion of 30S subunits that had been cross-linked by ultraviolet irradiation in vivo . After dehybridization, the individual RNA fragments and cross-linked complexes were separated by gel electrophoresis and analysed by our usual procedures . Nine cross-links are described; four of these are hitherto unobserved "secondary structural" cross-links, and one is a new "tertiary structural" cross-link between positions 243-247 and 891-894 of the 16S RNA.

J Biol Chem, 1988 May 25, 263(15), 7431 - 6
RecA-mediated strand exchange reactions between duplex DNA molecules containing damaged bases, deletions, and insertions; Hahn TR et al.; RecA protein from Escherichia coli promotes homologous pairing and strand exchange between duplex DNA molecules if one is partially single-stranded . Using linear duplexes and circles with a single-stranded gap as the substrates, this reaction generates nicked circular heteroduplex DNA and linear molecules with single-stranded ends . The completion of strand exchange can be demonstrated by the production of nicked circular heteroduplex DNA detected by gel electrophoresis and autoradiography using radiolabeled linear molecules . When the effect of ultraviolet damage to the substrate DNA was tested, strand exchange was found to pass 30 or more pyrimidine dimers in each duplex . In contrast, exchanges were blocked or severely slowed by interstrand cross-links and monoadducts produced by psoralen and 360 nm light . Deletions and insertions of from 4 to 38 base pairs in the DNA substrates had little effect on the production of nicked circular heteroduplex DNA . However, those of 120 base pairs, or greater, reduced the product yield to a level below the threshold of detection . These results contrast with those obtained in related three-stranded reactions (Bianchi, M . E., and Radding, C . M . (1984) Cell 35, 511-520), in which stable heteroduplex products with 500 or 1300 unpaired bases were obtained when the insert was located within a single-stranded circular substrate.

J Biol Chem, 1988 May 25, 263(15), 7386 - 96
Influence of DNA sequence on the formation of non-B right-handed helices in oligopurine.oligopyrimidine inserts in plasmids; Hanvey JC et al.; A systematic study was conducted on seven recombinant plasmids harboring synthetic inserts which had all purines on one strand and all pyrimidines on the complementary strand (Pur.Pyr) . The inserts ranged in G+C content from 100% {G19.C19} to 0% {A20.T20} with intermediate contents at 66% {(TCC)8.(GGA)8}, 50% {(CT)12.(AG)12 and (TTCC)6.(GGAA)6}, 33% {(TTC)8.(GAA)8}, and 25% {(GAAA)6.(TTTC)6} . The specific reactions at the base pair level of these inserts with enzymatic (S1 and P1 nucleases) and chemical (bromoacetaldehyde, OsO4, diethyl pyrocarbonate, and dimethyl sulfate) probes were evaluated as influenced by pH, negative supercoiling, and ionic strength (NaCl) . Supercoil-induced relaxation studies using two-dimensional gels also provided important conformational information . We conclude that the five inserts with 66-25% G+C adopt a non-B right-handed conformation which is stabilized by negative supercoiling . Low pH (pH values 4.5-5.0) tends to stabilize this structure but is not essential for its formation . Surprisingly, an end bias of reactivity from the center toward the 5'-end of the purine strand of these inserts was generally found for the enzymatic and chemical probes which was irrespective of the orientation of the insert in the pRW790 vector . An intramolecular triple-stranded model for the unusual structure of the insert accounts most favorably for these observations . Unexpectedly, the A20.T20 insert seems to remain in an orthodox right-handed B-conformation under all conditions tested . The G19.C19 insert does adopt a non-B right-handed structure as for the five inserts with 66-25% G+C, but the pattern of reactivities and hence its conformation is different.

J Biol Chem, 1988 May 25, 263(15), 7370 - 7
The role of DNA sequence in the formation of Z-DNA versus cruciforms in plasmids; McLean MJ et al.; The capacities of four synthetic sequences containing runs of perfectly alternating purine-pyrimidine base pairs (bp) to adopt left-handed structures were evaluated in a homologous family of recombinant plasmids . All the sequences had the same G+C content (50%) and consisted of simple tetranucleotide repeat units but differed in the relative orientations of these units . For some of the sequences, several alternate secondary structures were theoretically possible; a variety of probes (S1 nuclease, bromoacetaldehyde, OsO4, T7 gene 3 endonuclease, supercoil-induced gel relaxation studies) under a wide range of reaction conditions was used to determine which structures were adopted as a function of superhelical stress . The precise positions at the bp level of reactions with these chemical and enzymatic probes were determined . We conclude that for short (20-24 bp) sequences containing runs of alternating (T-G) and (C-A), the cruciform state is preferred over the similarly allowable left-handed form provided that symmetry constraints allow . However, these sequences can be induced to form a left-handed helix under appropriate conditions . This is the first demonstration of plasmid inserts which will adopt more than one unusual DNA structure in response to negative superhelical stress . The structural properties of a molecule containing a Z-Z junction were studied, and we conclude that the disruption caused by this feature extends over only a few bp although it requires a high energetic penalty.

J Biol Chem, 1988 May 25, 263(15), 7136 - 40
The dnaA protein of Escherichia coli . Abundance, improved purification, and membrane binding; Sekimizu K et al.; Immunoassays of dnaA protein in extracts from five strains showed a rather constant abundance relative to cell mass, with a variation of 800-2100 molecules/cell; overproducing cells contained 100-fold that number . About half of the dnaA protein in wild type cells was solubilized by a lysis procedure . Within the insoluble fractions, dnaA protein was identified by its characteristic high-affinity binding of ATP . An improved, rapid procedure for purifying dnaA protein from overproducing cells appears to depend on its coprecipitation with phospholipids and depends on solubilization by guanidine HCl . The procedure, with a 5-fold increased yield, also eliminates a potent ATPase contaminant . Purified dnaA protein, unlike dnaB and dnaC proteins, binds to phospholipid vesicles as judged by analysis on sucrose gradient centrifugation.

J Biol Chem, 1988 May 25, 263(15), 7131 - 5
Cardiolipin activation of dnaA protein, the initiation protein of replication in Escherichia coli; Sekimizu K et al.; ATP binding to dnaA protein is essential for its action in initiating the replication of plasmids that bear the unique origin of the Escherichia coli chromosome (oriC) . ADP bound to that site renders dnaA protein inactive for replication . Diphosphatidylglycerol (cardiolipin), a diacidic membrane phospholipid, displaces the bound nucleotide, and in the presence of components that reconstitute replication, fully reactivates the inert ADP form of dnaA protein . The monacidic phosphatidylglycerol is one-tenth as active as cardiolipin, whereas the neutral phosphatidylethanolamine, the principal E . coli phospholipid, is inactive . Fluphenazine, a tranquilizer drug, blocks cardiolipin activation of dnaA protein, in keeping with the inhibitory action of such agents on phospholipid-dependent enzymes . With the use of this drug to terminate cardiolipin action, dependence of the activation on time, elevated temperature, and high levels of ATP was demonstrated . Cardiolipin binding of nucleotide-free dnaA protein prevents binding of ATP and initiation of oriC replication . Removal of a fatty acid from cardiolipin by phospholipase A reverses this inhibitory effect . The strong and specific interaction of cardiolipin, a cell membrane component, with an essential nucleotide-binding site of dnaA protein, the protein essential for the initiation of chromosome replication, may be an important element in regulating the cell cycle.

J Biol Chem, 1988 May 25, 263(15), 7124 - 30
Sequential early stages in the in vitro initiation of replication at the origin of the Escherichia coli chromosome; Sekimizu K et al.; Complexes previously identified in the reconstitution of stages in the initiation of replication of plasmids (oriC) bearing the origin of the Escherichia coli chromosome have been examined further . These are: (i) an ATP complex of dnaA protein, (ii) an initial complex of ATP.dnaA protein with oriC DNA, (iii) an open complex in which a portion of the oriC duplex has been opened by dnaA protein action, (iv) a prepriming complex of the open complex with dnaB, dnaC, and HU proteins, and (v) a complex with a small bubble opened at oriC by dnaB helicase action and by coating with single strand-binding protein (SSB) . Helicase and gyrase actions can enlarge the bubble; coupling to priming and replication propagates bidirectional fork movement . Formation and stability of these complexes are profoundly affected by ATP, Mg2+, and temperature, as well as the levels of the participating proteins, including HU and SSB . As examples, the open complex is stable to isolation at a temperature near 38 degrees C but not at 24 degrees C; the prepriming complex requires an elevated temperature and high ATP levels for its formation, but is maintained at a low temperature and is destabilized by Mg2+ . These successive steps, subject to a variety of controls, are designed to open the supercoiled duplex for priming and bidirectional replication.

Cell, 1988 May 20, 53(4), 649 - 57
Site-directed mutagenesis of the cell-binding domain of human fibronectin: separable, synergistic sites mediate adhesive function; Obara M et al.; Polypeptide sequences required for function of the cell-binding domain of human fibronectin were analyzed by site-directed mutagenesis . Site-specific deletion of the putative recognition sequence Arg-Gly-Asp-Ser or an Asp-to-Glu mutation decreased the adhesive activity of fibronectin fusion proteins expressed in E . coli by greater than or equal to 97% . A second functional site over 0.5 kb away was identified by deletion mutagenesis . These mutants also showed a greater than or equal to 96% loss of activity, indicating cooperativity between sites . The two classes of mutant protein displayed synergism of activity in a trans complementation assay . Effective actin microfilament bundle organization was also dependent on the combined function of both sites . Thus, fibroblast adhesion and intracellular response to the fibronectin cell-binding domain involve two synergistic sites, each of major quantitative importance.

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