Gene, 1984 Nov, 31(1-3), 247 - 50 Construction and properties of plasmid pKC30, a pBR322 derivative containing the pL-N region of phage lambda; Rao RN; The phage lambda N gene, contained on a HindIII-BamHI lambda DNA fragment (lambda coordinates 38 695-34 500), was inserted into plasmid pBR322 and cloned in Escherichia coli containing a defective lambda prophage . The plasmid is called pKC30 . lambda N-mediated pL transcription interfered with plasmid pKC30 maintenance . Plasmid pKC30 was stabilized (i) by repression of pL, (ii) by a nutL mutation, or (iii) by cloning an "N-unresponsive terminator" sequence downstream from N gene. Gene, 1984 Nov, 31(1-3), 205 - 11 Studies on deo operon regulation in Escherichia coli: cloning and expression of the deoR structural gene; Short SA et al.; Recombinant plasmid pBR322 derivatives containing the Escherichia coli deoR structural gene (coding for one repressor of the deo operon) and a mutant allele of the cmlA gene (chromosomally encoded chloramphenicol resistance) have been constructed and the positions of these genes on a 6.3-kb EcoRI fragment have been determined . Transformation of an E . coli deoR single mutant with any of the deoR+ plasmids resulted in complementation of the chromosomal deoR mutation . More importantly, however, transformation of a deoR cytR double mutant with the deoR+ plasmids also resulted in complete repression of Deo enzyme synthesis . Based on these data, we conclude that transcription of the deo operon initiating from both the cAMP/CRP-independent promoter-operator site, PO1, and the cAMP/CRP-dependent promoter-operator site, PO2, is negatively controlled by the deoR-encoded repressor, whereas the cytR-encoded repressor regulates deo operon expression only from the cAMP/CRP-dependent promoter-operator site, PO2. Gene, 1984 Nov, 31(1-3), 17 - 22 Electron microscopy can be used to measure DNA supertwisting; Sperrazza JM et al.; We have examined the use of electron microscopy (EM) to measure the degree of supertwisting of covalently closed circular fd and pBR322 DNAs . Band counting agarose gel electrophoresis was used to quantitate the number of twists in these DNAs . DNA species having moderate-to-low superhelical densities were prepared by treatment of the DNA with Drosophila topoisomerase 1 in the presence of ethidium bromide (EtBr) followed by isolation of discrete DNA topoisomers from preparative gels . These DNAs were then examined by EM using a direct mounting method employing a buffer containing spermidine . The crossovers in individual DNA molecules were counted and compared to the supertwist values obtained from gel studies . The results show that EM can provide a reliable estimate of the number of supertwists in DNAs having low-to-moderate superhelical densities. Gene, 1984 Nov, 31(1-3), 155 - 64 High-level expression in Escherichia coli of calcium-binding domains of an embryonic sea urchin protein; Muesing M et al.; A plasmid expression vector is described having features that facilitate high-level expression of eukaryotic DNA in Escherichia coli . The vector, designated pMAM17, carries the ColE1 rop gene under the control of the thermally inducible lambda PL promoter . The rop gene product is a negative regulator of ColE1 DNA replication, and its high-level expression is lethal to cells . However, cells harboring a plasmid with an insert in the rop gene grow normally under these conditions . pMAM17 has been used to investigate the properties of a family of proteins expressed in the dorsal ectoderm of sea urchin embryos . The coding sequences of these proteins (termed Spec proteins) have homology to the troponin C superfamily . Large amounts of the Rop-Spec fusion protein were produced at 42 degrees C in E . coli . Unfractionated E . coli extracts containing the fusion protein could be used to produce antibodies that were highly specific for Spec proteins present in crude extracts of sea urchin embryos . Analysis of the Rop-Spec fusion protein on SDS-polyacrylamide gels in the presence and absence of EGTA indicated that the fusion protein bound calcium ions in a manner characteristic of proteins of the troponin C superfamily . This behavior provides biochemical evidence that the Spec proteins are functionally homologous to other members of this superfamily. Gene, 1984 Nov, 31(1-3), 117 - 22 Temperature-dependent retention of a tryptophan-operon-bearing plasmid in Escherichia coli; Skogman SG et al.; Derivatives of plasmid pBR322 carrying the tryptophan operon are rapidly lost from a growing culture . The valS gene from Escherichia coli was combined with the tryptophan operon on a pBR322 derivative, pSGS21 . In a thermosensitive valS mutant of E . coli K-12 host, plasmid pSGS21 was stably inherited for over 200 generations at temperatures nonpermissive for the chromosomal mutation . At 30 degrees C, a temperature at which the host valSts protein is functional, the plasmid was lost at a rate of 1.2% per cell generation. Gene, 1984 Nov, 31(1-3), 109 - 16 Cloning of a third nitrate reductase gene from the cyanobacterium Anacystis nidulans R2 using a shuttle cosmid library; Kuhlemeier CJ et al.; A strategy for gene cloning in the cyanobacterium Anacystis nidulans R2 was developed which made use of a gene library constructed in a shuttle cosmid vector . The method involved phenotypic complementation of mutants with pooled cosmid DNA . The development of the procedure and its application to the cloning of a third gene involved in nitrate reduction are described. Biosci Rep, 1984 Nov, 4(11), 917 - 23 Nucleotide sequence of the thioredoxin gene from Escherichia coli; Hoog JO et al.; The nucleotide sequence of the thioredoxin gene from Escherichia coli was determined . The structural gene was identified on a cloned 3-kb PvuII fragment by hybridization with a synthetic oligodeoxyribonucleotide corresponding to a part of the amino acid sequence of thioredoxin . Restriction-enzyme fragments were used as templates in the dideoxy sequence method, directly and after subcloning into M13mp8 . A segment of 450 nucleotides was determined using both strands, alternatively, without extensive overlaps . The sequence contains the thioredoxin coding region, a potential ribosome-binding site, and a putative promotor region . The predicted amino acid sequence differs by two inversions from the previously given thioredoxin sequence . The revised sequence is presented and the results further show that thioredoxins from E . coli B and K12 are identical. Bioorg Khim, 1984 Nov, 10(11), 1535 - 43 {Synthesis of a full-length DNA copy of the hemagglutinin gene of the the influenza virus A H1N1 subtype, its cloning and primary structure}; Beklemishev AB et al.; Full-length DNA-copy of hemagglutinin gene RNA of influenza A virus strain A/Leningrad/54/1 was synthesized and cloned in E . coli plasmid pBR 327 . Its primary structure was determined by a modified Maxam - Gilbert procedure . The nucleotide sequence of this gene was compared with sequences of analogous genes of influenza strains A/USSR/90/77 and A/PR/8/34 . An addition of one nucleotide in non-translated region was found in the former. Mol Biol (Mosk), 1984 Nov-Dec, 18(6), 1445 - 60 {Co-translation folding, compartmentalization and modification of proteins}; Spirin AS; Transmembrane transport of polypeptide chains in the process of their synthesis on membrane-bound ribosomes and enzymic modification of the nascent polypeptides on membranes are reviewed . The possible role of ribosomes in protein folding and some other unsolved problems are discussed. Mol Cell Biochem, 1984 Nov, 65(1), 37 - 44 Effect of Escherichia coli lipopolysaccharide on the glucagon and insulin binding to isolated rat hepatocytes; Pagani R et al.; Number and affinity constant of low affinity binding sites of insulin and glucagon to isolated hepatocytes decreased when the cells were incubated with Escherichia coli 0111:B4 lipopolysaccharide . This effect agrees with a non-specific binding of lipopolysaccharide to hepatocytes, similar to the well-recognized non-specific binding of albumin . Also, binding of different lectins to their glycoprotein receptors did not affect the {14C}lipopolysaccharide interaction with the cell membrane surface . Endotoxin depresses gluconeogenesis from lactate when the precursor was incubated with the cells for short time intervals . The longer the preincubation interval with lipopolysaccharide, the higher the inhibition of gluconeogenesis in the absence and in the presence of glucagon . The effect of endotoxin was also studied on the glucagon-induced synthesis of cyclic AMP and the glucagon binding . Levels of cyclic AMP and hormone binding decreased with increasing both endotoxin concentrations and preincubation intervals at which cells were in contact with endotoxin. Mol Cell Biol, 1984 Nov, 4(11), 2266 - 72 High spontaneous mutation frequency in shuttle vector sequences recovered from mammalian cellular DNA; Ashman CR et al.; The recombinant shuttle vector pSV2gpt was introduced into V79 Chinese hamster cells, and stable transformants expressing the Escherichia coli gpt gene were selected . Two transformants carrying tandem duplications of the plasmid at a single site were identified and fused to simian COS-1 cells . Plasmid DNA recovered from the heterokaryons was used to transform a Gpt- derivative of E . coli HB101, and the relative frequency of plasmids carrying a mutation in the gpt gene was determined . The high frequency of Gpt- plasmids (ca . 1%) was similar to that observed when plasmid was recovered from COS-1 cells which had been transfected with pSV2gpt . Most of the mutant plasmids had rearrangements in the region containing the gpt gene. EMBO J, 1984 Nov, 3(11), 2605 - 11 A 145-base pair DNA sequence that positions itself precisely and asymmetrically on the nucleosome core; Ramsay N et al.; A 145-bp DNA sequence, cloned from Escherichia coli, was reconstituted into nucleosome core particles by a number of methods . The behaviour of the resulting complex upon sucrose gradient sedimentation and nucleoprotein gel electrophoresis closely resembled that of control bulk nucleosome core particles . DNase I digestion of the 32P-end-labelled complex revealed the 10-bp periodicity of cleavages expected for DNA bound on a histone surface . The narrow cleavage sites observed (1 bp wide) imply that the sequence occupies a single preferred position on the nucleosome core, accurate to the level of single base pairs . By relating the digestion pattern observed to the pattern of site protection found for random sequence nucleosomes, the DNA position was found to be offset by 17 bp from that in the normal core particle . A number of experiments argue against the involvement of length or end effects and suggest that it is some feature of the DNA sequence itself that determines this precise positioning of DNA on the nucleosome. EMBO J, 1984 Nov, 3(11), 2549 - 54 An assay for transient gene expression in transfected Drosophila cells, using {3H}guanine incorporation; Burke JF et al.; We have developed an assay for transient gene expression using a dominant-selectable marker previously employed to transform Drosophila cultured cells . Drosophila hydei cells transfected with a functional Escherichia coli xanthine guanine phosphoribosyl transferase gene (gpt), under the control of the long terminal repeats (LTRs) of the copia transposable element, rapidly incorporate guanine into acid-precipitable counts . Autoradiographic analysis in situ shows that approximately 20% of cells take up, and express, the gpt gene . This transient gpt expression depends on the Drosophila promoter sequences since vectors with the gpt gene in reverse orientation to the copia LTRs fail to incorporate guanine . Deletion analysis confirms that the LTRs are essential for gpt gene expression . Similarly, cells transfected with gpt controlled by the Drosophila 70 000 mol . wt . heat-shock (hsp 70) promoter show regulated guanine incorporation when heat shocked . The efficiency of the copia LTRs varies considerably between the cell lines we tested, whereas that of the hsp 70 promoter does not . The heterologous promoters of the Rous sarcoma virus (RSV) and simian virus 40 (SV40) function poorly in these cells. Proc Natl Acad Sci U S A, 1984 Nov, 81(22), 7207 - 11 A membrane protein encoded by Epstein-Barr virus in latent growth-transforming infection; Hennessy K et al.; The nucleotide sequence of an Epstein-Barr virus gene expressed in latently infected growth-transformed cells is known to include a long open reading frame containing a 33-base-pair repeat element . A bacterial fusion protein constructed from a portion of the reading frame and Escherichia coli beta-galactosidase was used to produce sera in rabbits against the previously unidentified gene product . The viral protein detected with these sera in latently infected cells varies in size with the number of copies of the DNA repeat element . Translation of the RNA in vitro yields a protein of similar size . As expected from its primary sequence, the protein is a membrane protein . Immunofluorescence studies with the rabbit antisera suggest that the protein is in the plasma membrane . Thus, this protein could be the lymphocyte-determined membrane antigen (LYDMA) responsible for the generation of T-cell immunity to latently infected cells. J Bacteriol, 1984 Nov, 160(2), 724 - 32 Transcriptional organization within an Escherichia coli cell division gene cluster: direction of transcription of the cell separation gene envA; Sullivan NF et al.; A cluster of at least 14 genes, each concerned with some aspect of cell envelope growth, morphogenesis, or function, is located at 2 min on the genetic map of Escherichia coli . We located the envA cell division gene and its promoter within the cluster and determined the direction of transcription of the gene by constructing fusions between its promoter and the galK coding sequence . In addition, we identified the promoter of a possible new gene lying between envA and the secA gene . We also present evidence from gene fusion studies which shows the direction of transcription of the ftsZ(sulB) division gene . The direction of transcription is the same for all three promoters and is the same as that of all other cluster genes for which this is known . We discuss the significance of this observation, together with the fact that every gene examined in sufficient detail within the cluster appears to have its own promoter and to be able to be expressed from isolated cloned fragments . Using a novel variable-copy plasmid vector, we demonstrate that the DNA fragment containing the envA gene is not stably maintained in multiple copies . The construction of two independent, nonoverlapping deletions allows us to conclude that the envA product itself is responsible for this effect. J Bacteriol, 1984 Nov, 160(2), 711 - 7 sn-Glycerol-3-phosphate auxotrophy of plsB strains of Escherichia coli: evidence that a second mutation, plsX, is required; Larson TJ et al.; sn-Glycerol-3-phosphate auxotrophs defective in phospholipid synthesis contain a Km-defective sn-glycerol-3-phosphate acyltransferase . Detailed genetic analysis revealed that two mutations were required for the auxotrophic phenotype . One mutation, in the previously described plsB locus (sn-glycerol-3-phosphate acyltransferase structural gene), mapped near min 92 on the Escherichia coli linkage map . Isolation of Tn10 insertions cotransducible with the auxotrophy in phage P1 crosses revealed that a second mutation was required with plsB26 to confer the sn-glycerol-3-phosphate auxotrophic phenotype . This second locus, plsX, mapped between pyrC and purB near min 24 on the E . coli linkage map . Tn10 insertions near plsX allowed detailed mapping of the genetic loci in this region . A clockwise gene order putA pyrC flbA flaL flaT plsX fabD ptsG thiK purB was inferred from results of two- and three-factor crosses . Strains harboring the four possible configurations of the mutant and wild-type plsB and plsX loci were constructed . Isogenic plsB+ plsX+, plsB+ plsX50, and plsB26 plsX+ strains grew equally well on glucose minimal medium without sn-glycerol-3-phosphate . In addition, plsX or plsX+ had no apparent effect on sn-glycerol-3-phosphate acyltransferase activity measured in membrane preparations . The molecular basis for the plsX requirement for conferral of sn-glycerol-3-phosphate auxotrophy in these strains remains to be established. J Bacteriol, 1984 Nov, 160(2), 706 - 10 Restoration of viability to an Escherichia coli mutant deficient in the 5'----3' exonuclease of DNA polymerase I; Boling M et al.; An Escherichia coli polA (Ex) mutant that is usually inviable at restrictive temperatures (43 degrees C) was found to grow normally at 43 degrees C when incubated in the presence of a membrane-containing fraction prepared from E . coli . This membrane fraction causes anaerobic conditions that are necessary but not sufficient for restoration of viability since some component present in the membrane fraction is also required for colony formation at 43 degrees C . The accumulation of small DNA fragments typical of aerobic growth of the polA(Ex) mutant was also seen under anaerobic conditions . The polA(Ex) strain was also much more sensitive than the isogenic wild-type strain to hydrogen peroxide. J Bacteriol, 1984 Nov, 160(2), 702 - 5 Two-component suppression of recF143 by recA441 in Escherichia coli K-12; Volkert MR et al.; Sensitivity to UV irradiation conferred by recF143 was partially suppressed by recA441 (also known as tif-1) . A temperature-conditional component depended on uvrA function and is thought to involve thermal induction of excision repair enzymes . In a uvrA6 mutant, a temperature-independent component of suppression was seen . This is thought to indicate that recA441 also caused temperature-independent changes in recA activity . Two hypotheses are offered to explain how recA441 produced both thermosensitive and thermoindependent effects. J Bacteriol, 1984 Nov, 160(2), 668 - 75 Genetic mapping of katF, a locus that with katE affects the synthesis of a second catalase species in Escherichia coli; Loewen PC et al.; A class of catalase-deficient mutants that was unlinked to katE was localized between mutS and cys at 59.0 min on the Escherichia coli genome . This locus was named katF . Transposon Tn10 insertions were isolated that mapped in both katE and katF loci . The catalase species present in katE+ and katF+ recombinants was found to be different from the main catalase activities, HPI and HPII, in several respects . It did not have an associated peroxidase activity; it was electrophoretically slower on native polyacrylamide gels; it eluted from DEAE-Sephadex A50 at a higher salt concentration; its Km for H2O2 was 30.9 mM as compared with 3.7 mM for HPI and HPII; its synthesis was not induced by ascorbate; and it did not cross react with HPI-HPII antisera . This new catalase was labeled HPIII. J Bacteriol, 1984 Nov, 160(2), 642 - 50 Spontaneous deletion of citrate-utilizing ability promoted by insertion sequences; Ishiguro N et al.; The citrate utilization (Cit+) transposon Tn3411 was shown to be flanked by directly repeated sequences (IS3411L and IS3411R) by restriction enzyme analysis and electron microscope observation . Cit- deletion mutants were frequently found to be generated in pBR322::Tn3411 by intramolecular recombination between the two copies of IS3411 . The flanking IS3411 elements of Tn3411 were shown to be functional insertion sequences by Tn3411-mediated direct and inverse transposition . Tn3411-mediated inverse transposition from pBR322::Tn3411 to the F-plasmid derivative pED100 occurred more efficiently than that of direct transposition of the Cit+ determinant . This was thought to be due to the differential transposability of IS3411L and IS3411R in the transposition process . The frequency of transposition of IS3411 marked with a chloramphenicol resistance determinant was much higher than IS3411-mediated cointegrate formation, suggesting that replicon fusions are not essential intermediates in the transposition process of Tn3411 or IS3411 . Spontaneous deletions occurred with high frequency in recA hosts . The spontaneous deletion promoted by homologous recombination between two IS3411 elements in Tn3411 was examined with deletion mutants. J Bacteriol, 1984 Nov, 160(2), 600 - 6 Construction and expression of hybrid plasmids containing the structural gene of the Escherichia coli K-12 3-deoxy-2-oxo-D-gluconate transport system; Mandrand-Berthelot MA et al.; The kdgT gene of Escherichia coli, which encodes for the 3-deoxy-2-oxo-D-gluconate transport system, was isolated as a ColE1-kdgT hybrid plasmid from the Clarke and Carbon bank . A restriction and genetic map of the min 88 region of the chromosome was established; by subcloning the restriction fragments into the plasmid vector pBR322, the kdgT gene was localized on a 1.4-megadalton PstI DNA fragment, and the direction of transcription of the gene was determined by making use of an in vitro gene fusion between kdgT and lacZ genes . Amplification of the gene product of kdgT was up to 14-fold the level found in a haploid strain . When plasmids bearing kdgT were expressed in an in vivo maxicell system, a specific polypeptide of 28,000 daltons appeared that was found to be associated with the membrane fraction. J Bacteriol, 1984 Nov, 160(2), 577 - 85 Identification of Escherichia coli region III flagellar gene products and description of two new flagellar genes; Bartlett DH et al.; Region III flagellar genes in Escherichia coli are involved with the assembly and rotation of the flagella, as well as taxis . We subcloned the flaB operon from a lambda fla transducing phage onto plasmid pMK2004 . Two additional genes were found at the flaB locus, and we subdivided the flaB gene into flaB1, flaBII, and flaBIII . The cheY suppressor mutations which have previously been mapped to flaB were further localized to flaB11 (Parkinson et al., J . Bacteriol . 155:265-274, 1983) . Until now, gene product identification has not been possible for these genes because of their low levels of gene expression . Overexpression of the flagellar genes was accomplished by placing the flaB operon under the control of the lacUV5 or tac promoters . Plasmid-encoded proteins were examined in a minicell expression system . By correlating various deletions and insertions in the flaB operon with the ability to complement specific flagellar mutants and code for polypeptides, we made the following gene product assignments: flaB 1, 60 kilodaltons; flaB 11, 38 kilodaltons; flaB111, 28 kilodaltons; flaC, 56 kilodaltons; fla0, 16 kilodaltons; and flaE, 54 kilodaltons. J Bacteriol, 1984 Nov, 160(2), 546 - 55 DNA sequence and transcriptional organization of essential cell division genes ftsQ and ftsA of Escherichia coli: evidence for overlapping transcriptional units; Robinson AC et al.; The DNA sequence of a cloned segment of the Escherichia coli chromosome containing ftsQ, ftsA, and part of the ftsZ gene was determined and interpreted for genetic complementation and promoter fusion data for the region . The contiguous genes ftsQ, ftsA, and ftsZ were transcribed in the same direction (clockwise on the genetic map) and each had at least one associated promoter which allowed it to be transcribed independently of neighboring genes . ftsA and ftsZ possessed promoters within the coding sequences of the juxtaposed upstream structural genes, and a promoter element for ftsA was surrounded by a region of twofold symmetry which corresponded closely to a symmetrical element in the region of a putative ftsZ promoter . The structural gene of ftsQ consisted of 838 nucleotides, encoding a 276-residue amino acid polypeptide of molecular weight 31,400; the structural gene of ftsA consisted of 1,260 nucleotides, encoding a 420-residue amino acid polypeptide of molecular weight 45,400 . The observation that the termination codon of ftsQ overlaps with a potential initiation codon for ftsA suggested that these two genes may be translationally coupled when transcription is initiated upstream of the ftsQ coding sequence. J Bacteriol, 1984 Nov, 160(2), 504 - 9 Identification of additional genes on transposon Tn10: tetC and tetD; Braus G et al.; Two genes (tetC and tetD) were identified and located on transposon Tn10 between gene tetA and insertion sequence IS10R . Genes tetC and tetD encode proteins of apparent subunit molecular weights of 23,000 and 18,000, respectively . The TetD protein was found to be membrane associated . Tetracycline resistance levels promoted by transposon Tn10 were found to be unaffected in Escherichia coli K-12 when mutants lacking tetC or tetC and tetD were tested . The nucleotide sequence of genes tetC and tetD is reported in the accompanying article (K . Schollmeier and W . Hillen, J . Bacteriol . 160:499-503, 1984). J Bacteriol, 1984 Nov, 160(2), 499 - 503 Transposon Tn10 contains two structural genes with opposite polarity between tetA and IS10R; Schollmeier K et al.; The nucleotide sequence of the central part of Tn10 has been determined from the rightmost HindIII site to IS10R . This sequence contains two open reading frames with opposite polarity . The in vivo transcription start points in this sequence have been determined by S1 mapping . These results define one minor and two major promoters . The transcription starts of the two major promoters are only 18 base pairs apart, and the transcripts show different polarity and overlap by 18 base pairs . The nucleotide sequence reveals two regions with palindromic symmetry which may serve as operators . Their possible involvement in the regulation of transcription of both genes is discussed . Taken together these results allow for a maximal coding capacity of 138 amino acids directed toward IS10R and 197 amino acids directed toward tetA . The possible function of these gene products is discussed . The accompanying article (Braus et al., J . Bacteriol . 160:504-509, 1984) presents evidence that these genes are expressed. Virology, 1984 Nov, 139(1), 11 - 21 Identification of the phage gene for host receptor specificity by analyzing hybrid phages of T5 and BF23; Heller KJ; The closely related coliphages T5 and BF23 differ in the receptor used for adsorption to cells . In order to identify phage genes encoding host receptor specificity, hybrid phages were isolated from crosses of T5 and BF23 . These hybrid phages were analyzed for their protein composition, kinetics of adsorption to various bacterial strains, and DNA restriction maps . Analyses of the protein compositions of purified tails suggested that only one tail protein was involved in the expression of host specificity . In T5 tails this protein was identified as a minor tail protein of an apparent molecular weight of 67K . A corresponding BF23 protein could not be detected . The DNA region encoding structural proteins was analyzed by digestion with the restriction enzymes BamHI, HindIII, and PstI . The distribution of restriction sites in these recombinants implied that the region affecting host specificity was located at ca . 90% of the T5 genome length . Evidence is presented that this gene is identical to the oad gene previously described (K.J . Heller and D . Bryniok (1984), J . Virol . 49, 20-25) . The location of other genes encoding tail proteins is discussed. J Cell Physiol, 1984 Nov, 121(2), 402 - 8 Effects of 2',3'-dideoxynucleosides on mammalian cells and viruses; Waqar MA et al.; Previous studies on the biological effects of the 2',3'-dideoxynucleosides (ddNs) have shown that while ddAdo is lethal to E . coli, ddThd has minimal effects on the growth of mammalian cell lines and that it inhibits retrovirus infection of some cell lines but not others . Previous studies have also shown that the 5'-triphosphate of ddThd, ddTTP, selectively inhibits cellular DNA polymerases beta and gamma and retroviral reverse transcriptases . Cellular DNA polymerase alpha is relatively resistant to ddTTP . We have extended these findings to show that the 5'-triphosphates of the other 3 ddNs (ddATP, ddCTP, and ddGTP) affect cellular DNA polymerases alpha, beta, and gamma in the same fashion as does ddTTP . We also show that all four ddNs in concentrations up to 100 microM have negligible effects on the growth of NIH Swiss 3T3 cells . These negligible effects may be due to inefficient intracellular phosphorylation of each nucleoside to the triphosphate . We have determined that, in several different cell lines, ddThd is phosphorylated only at a very slow rate to ddTTP, and in the one cell line tested (monkey CV-1 cells), ddAdo and ddGuo are also poorly phosphorylated . Both ddAdo and ddGuo, and probably ddThd, are converted by CV-1 cells to additional unknown compounds which may have biological activity . The four ddNs display effects of different magnitudes on certain virus infections . Although 30 microM ddThd inhibits herpes simplex I infection of CV-1 cells by 50%, 30 microM ddAdo has no effect . Infection of NIH Swiss 3T3 cells by 334C murine leukemia virus is inhibited 70-80% by ddAdo, ddCyd, and ddThd at 50 microM, but inhibition by 50 microM ddGuo is 100%. Cell, 1984 Nov, 39(1), 181 - 90 Transposase promotes double strand breaks and single strand joints at Tn10 termini in vivo; Morisato D et al.; We present evidence that Tn10 transposase promotes double strand breaks and single strand joints at Tn10 termini in vivo . Plasmids containing a shortened Tn10 element and a transposase overproducer fusion give rise, upon transposase induction, to new DNA species . The most prominent class is a circularized transposon molecule whose structure suggests that it arises from double strand breakage at the two transposon ends followed by covalent joining between the 3' and 5' ends of one of the two strands . We have used formation of the circularized transposon as a physical assay for the interaction between transposase and different mutant and wild-type termini . These experiments show that transposase protein interacts preferentially with the genetically most active termini in a way that suppresses productive interaction with weaker termini present on the same substrate molecule. Cell, 1984 Nov, 39(1), 173 - 80 Dual promoter control of the Escherichia coli lactose operon; Malan TP et al.; The control of transcription initiation at the lactose operon promoter was investigated in vitro . We found that an upstream promoter (termed lac P2) interfered with RNA polymerase binding at the principal promoter (termed lac P1) . The start site for lac P2 was located at base pair position -22 relative to the P1 start site . The addition of cAMP receptor protein and cAMP was shown to repress lac P2 and to activate lac P1 . Abortive initiation reactions for both promoters were used to investigate the coordinate repression-activation elicited by CRP-cAMP . The effects of lac promoter mutations (L8, Ps, and UV5) were consistent with an important RNA polymerase positioning role for CRP-cAMP in the activation of lac operon expression. Cell, 1984 Nov, 39(1), 163 - 71 Protein products of the bithorax complex in Drosophila; White RA et al.; A sequence from the Ubx 5' exon in the bithorax complex of Drosophila melanogaster was expressed as a fusion protein in bacteria . This protein was used to raise rabbit antisera and monoclonal antibodies . These antibodies detect antigens that, on protein blots and by immunofluorescence on whole mounts of imaginal discs, show the predicted segmental distribution of Ubx products . These products are predominantly, if not totally, localized in the cell nucleus . In the embryonic nervous system nuclei are labeled from the second thoracic segment to the eighth abdominal segment . There is no labeling in homozygous Df bxd100 embryos. Mol Biol (Mosk), 1984 Nov-Dec, 18(6), 1686 - 94 {Prediction of the ensembles of RNA secondary structures . Kinetic analysis of self-organization}; Mironov AA et al.; A new approach to the problem of prediction of secondary structures of RNA, which is based on the kinetic analysis of self-organising molecules is proposed . Structural reconstructions that take place during formation of secondary structures are described in terms of Markov process . A set of states and probability transition were defined . Monte-Carlo methods were used to describe this process . Probability distributions of various secondary structures depending on time are given . Examples of calculations for ensembles of secondary structures of some tRNAs are described . An effective method of steady-state ensemble research, which is based on a quick RESETTING of all possible variance of the secondary structures of RNAs is given . By ascribing to each of these structures the value of probabilities as a function of free energy it was possible to obtain the Boltzmann ensemble of secondary structures. Mol Biol (Mosk), 1984 Nov-Dec, 18(6), 1597 - 605 {Protein L16 of the Escherichia coli ribosome: possible role in protein biosynthesis}; Maimets TO et al.; We show that Escherichia coli 50S ribosomal subunits depleted of protein L16 can nevertheless catalyze the transfer of the peptide moiety from fMet-tRNA to puromycin, being, however, unable to use a fragment CACCA-Phe as an acceptor substrate . On the other hand, we found that protein L16 as well as its large fragment (amino acids 10-136) both interact with tRNA in solution (Kd approximately 10(-7) M) . Moreover, L16 interacts with CACCA-Phe in solution as well as protects 3' end of tRNA from the enzymatic degradation . We suggest that L16, although not being the peptidyl transferase as such, is involved in the binding of the 3' end cytidines of tRNA into the ribosomal A site. J Biochem Biophys Methods, 1984 Nov, 10(1-2), 1 - 12 The identification of tRNA isoacceptors fractionated by polyacrylamide gel electrophoresis; Igloi GL; A method is described by which specific tRNA isoacceptors may be identified in small amounts of bulk tRNA . The strategy relies on the retention of aminoacyl-tRNA by CNBr-Sepharose through covalent coupling of the alpha-NH2 group of the amino acid to the matrix . After removing unbound material by thorough washing, the bound specific isoacceptors are released by cleavage of the labile aminoacyl-tRNA ester bond through mild alkaline treatment . The product is analysed by two-dimensional gel electrophoresis and the spots obtained may be correlated with the pattern from bulk tRNA . Optimum sensitivity is achieved by combining the method with the recently introduced silver staining technique (Igloi, G.L . (1983) Anal . Biochem . 134, 184-188) for tRNA. Biochem Biophys Res Commun, 1984 Oct 30, 124(2), 625 - 8 The realm of the steady state in Escherichia coli; Cho S et al.; Only at cell concentrations of less than 10(7)/ml are E . coli in steady state as defined by three intrinsic properties: mean cell diameter, cellular specific gravity and the concentration of beta-galactosidase per cell . Such concentrations are below the range of turbidimetry. Eur J Pharmacol, 1984 Oct 30, 106(1), 113 - 9 Anomalous effects of anti-inflammatory corticosteroids in endotoxin-induced ocular inflammation; Williams RN et al.; The effect of anti-inflammatory corticosteroids upon leukocyte infiltration was investigated in vivo, using the eye as an experimental model . The inflammatory response was induced by injecting bacterial endotoxin into the vitreous body of the albino rabbit eye . Polymorphonuclear leukocyte (PMN) infiltration into the aqueous humor was quantified by counting cells directly, whereas accumulation of leukocytes in the ocular tissues was determined by measuring myeloperoxidase (MPO) activity . Systemically administered corticosteroids inhibited plasma protein leakage and PMN infiltration into the aqueous humor, but, anomalously, increased MPO activity and hence the number of PMNs in the ciliary body . These findings were confirmed qualitatively by histology . However, the clinical manifestations of the inflammatory responses were almost totally suppressed by the corticosteroids . We suggest that the inhibitory effect of corticosteroids on PMN infiltration into the aqueous humor, and possibly inflammatory exudates in general, represents the suppression of inflammatory cell activity in a manner which is unrelated to a direct effect on leukocyte movement. FEBS Lett, 1984 Oct 29, 176(2), 414 - 20 Sequence preferences in the binding to DNA of triostin A and TANDEM as reported by DNase I footprinting; Low CM et al.; Six or seven triostin-binding sites have been identified in a 160-base-pair DNA restriction fragment containing the tyr T promoter sequence . Each is centred round a CpG step, and the minimum binding site-size appears to be six base pairs . The sites are practically the same as those reported for echinomycin by DNase I digestion . Only two sites are protected by binding of TANDEM, the des-N-tetramethyl analogue of triostin A; they are centred around the sequences ATA or TAT. FEBS Lett, 1984 Oct 29, 176(2), 406 - 10 Eukaryotic elongation factor 2 loses its non-specific affinity for RNA and leaves polyribosomes as a result of ADP-ribosylation; Sitikov AS et al.; ADP-ribosylation of rabbit reticulocyte elongation factor 2 (EF-2) catalyzed by the A fragment of diphtheria toxin leads to a loss of its non-specific affinity for RNA . The removal of the ADP-ribose residue from EF-2 in the reverse reaction with nicotinamide restores its affinity for RNA . ADP-ribosylation of EF-2 is accompanied by its dissociation from the complexes with mono- and polyribosomes detected in the rabbit reticulocyte lysate at low ionic strength . The loss of the non-specific affinity of EF-2 for RNA as a result of ADP-ribosylation and, as a consequence, its decompartmentation from polyribosomes is assumed to be a reason for the diphtheria toxin-induced inactivation of the factor in eukaryotic cells. Nucleic Acids Res, 1984 Oct 25, 12(20), 7903 - 14 The photoreactivity of T-A sequences in oligodeoxyribonucleotides and DNA; Bose SN et al.; Photoaddition between adjacent adenine and thymine bases occurs, with a quantum yield of approximately 5 X 10(-4) mol einstein-1, when d(T-A), dT-A, d(pT-A), d(T-A-T), d(T-A-T-A) and poly(dA-dT) are irradiated, at 254 nm, in aqueous solution . The photoadduct thus formed is specifically degraded by acid to the fluorescent heterocyclic base 6-methylimidazo{4,5-b}pyridin-5-one (6-MIP) with retention of C(8) of adenine and the methyl group of thymine . This reaction, coupled with either spectrofluorimetric or radiochemical assay of 6-MIP isolated by high voltage paper electrophoresis, has been used to demonstrate formation of the adenine-thymine photoadduct on UV irradiation of poly(dA-dT).poly(dA-dT) and both native and denatured DNA from calf thymus and E . coli . Estimated quantum yields for this new type of photoreaction in DNA show that it is substantially quenched by base pairing . Possible biological implications of the photoreaction are discussed. J Mol Biol, 1984 Oct 25, 179(2), 171 - 83 Overinitiation of chromosome and plasmid replication in a dna Acos mutant of Escherichia coli K12 . Evidence for dnaA-dnaB interactions; Frey J et al.; The dnaAcos mutations are phenotypic suppressors of dnaAts46 that are co-transduced with dnaA, render the cell cold sensitive, and cause an excess of chromosome replication relative to cell mass when the cells are shifted from 42 degrees C to 32 degrees C . We have used pulse labelling and DNA-DNA hybridization to follow the effect of a temperature shift on the replication of the chromosome and of the plasmids pSC101, RTF-Tc, and lambda dv in such strains . After a shift of a dnaAcos strain from 42 degrees C to 32 degrees C (non-permissive temperature), initiation of the chromosome and replication of the plasmid pSC101 are stimulated, while the dnaA-independent plasmid RTF-Tc is not affected . The presence of pSC101 does not affect the level of overinitiation of the chromosome . The presence of lambda dv suppresses the cold sensitivity of dnaAcos mutants and allows the cells to grow at both 32 degrees C and 42 degrees C . The presence of lambda dv suppresses the overinitiation of chromosome and of pSC101 replication at 32 degrees C . Previous reports had shown that these suppressions involve an interaction between the dnaA product and the lambda P protein, which is also known to interact with dnaB . We show here that the mutant prophage P1 bac-crr, which produces high levels of a dnaB analogue, suppresses the dnaAcos phenotype, while wild type P1 does not . These results suggest that initiation involves interactions between the dnaA and dnaB products. Nucleic Acids Res, 1984 Oct 25, 12(20), 7741 - 52 Effect of superhelicity on the transcription from the tet promoter of pBR322 . Abortive initiation and unwinding experiments; Bertrand-Burggraf E et al.; Supercoiling of DNA is now known to have considerable effects on transcription in bacteria . By abortive initiation reaction (6) we have determined the binding constant KB and the forward rate of isomerization k2 as a function of temperature, pH and buffer for the tet promoter in a supercoiled plasmid . If the activation energy of isomerization is very similar to that obtained previously under the same conditions on a linearized plasmid (6) (respectively 21 +/- 5 kcal/mole and 13 +/- 5 kcal/mole) the supercoiling introduces very important and not well understood changes in the thermodynamic parameters of the association polymerase - promoter . Using the technique of superhelical DNA relaxation by eukaryotic topoisomerase I, we have determined the specific unwinding by RNA polymerase of the tet promoter of pBR322 (430 degrees) . This unwinding differs only slightly from the mean value (470 degrees) obtained for all the promoters of pBR322. J Biol Chem, 1984 Oct 25, 259(20), 12776 - 80 Biosynthesis and assembly of the polysialic acid capsule in Escherichia coli K1 . Activation of sialyl polymer synthesis in inactivate sialyltransferase complexes requires protein synthesis; Whitfield C et al.; The polysialosyl capsule in Escherichia coli K1 is not synthesized when cells are grown at 15 degrees C . Membranous sialyltransferase complexes isolated from cells grown at 15 degrees C spontaneously gain the ability to synthesize sialyl polymers when incubated at 33 degrees C for 2-4 h . Activation of the endogenous synthesis of polysialic acid is localized in a low-density vesicle fraction (Whitfield, C., Adams, D.A., and Troy, F.A . (1984) J . Biol . Chem . 259, 12769-12775) . The low density vesicles catalyzed protein synthesis, and spontaneous activation required protein synthesis . Immune blotting confirmed the presence of protein synthesis initiation factor, IF2, and ribosomal protein S6, in the low-density vesicle fraction . Temperature-upshift experiments identified four membrane proteins whose synthesis was temporally correlated with in vitro activation . These proteins have apparent molecular masses of 40, 21, 17.5, and 16 kDA . Although the function of these proteins is not known, they may relate to a sialyltransferase activity required to initiate sialyl polymer assembly, to an endogenous acceptor, or to a porin that may facilitate channeling of the polysialosyl chains through the outer membrane. J Biol Chem, 1984 Oct 25, 259(20), 12769 - 75 Biosynthesis and assembly of the polysialic acid capsule in Escherichia coli K1 . Role of a low-density vesicle fraction in activation of the endogenous synthesis of sialyl polymers; Whitfield C et al.; Escherichia coli K1 synthesizes a polysialic acid capsule when grown at 37 but not 15 degrees C . The derangement in sialyl polymer synthesis appears to result from the inability of 15 degrees C membranes to synthesize or assemble a functional endogenous acceptor (Troy, F.A., and McCloskey, M.A . (1979) J . Biol . Chem . 254, 7377-7387) . Membranes from cells grown at 15 degrees C spontaneously gained the ability to synthesize sialyl polymer after incubation at 33 degrees C for 2-4 h . The incubation-dependent activation of the endogenous synthesis of sialyl polymer in 15 degrees C membranes possessed two unusual features . First, the sialyltransferase was localized in a low density vesicle fraction (LDV; rho = 1.11 g/cm3) . Second, this fraction catalyzed protein synthesis, and protein synthesis was required for activation . A study of the LDV fraction showed: 1) their light density resulted from a 5- to 8-fold enrichment in lipid phosphate to protein ratio and their sialyltransferase activity was enriched 40-fold compared with unfractionated total membranes; 2) they contained proteins characteristic of inner and outer membranes including leader peptidase and lipoprotein; 3) they constituted 8% of the mass of unfractionated total membranes yet contained all of the endogenous sialyltransferase activity in 15 degrees C membranes . In contrast, LDV from 37 degrees C grown cells accounted for 4.8% of the membrane mass and only 12.5% of the endogenous sialyltransferase activity; 4) they were multilamellar and averaged 0.7 mu in diameter . Based on these results, we believe the LDV fraction is of physiological importance in sialyl polymer synthesis . Growth at 15 degrees C allowed identification and study of the LDV fraction possibly because of the altered thermotropic properties of the membrane phospholipids that occur when E . coli is grown at low temperature. J Biol Chem, 1984 Oct 25, 259(20), 12644 - 51 Biosynthesis of membrane-derived oligosaccharides . Novel glucosyltransferase system from Escherichia coli for the elongation of beta 1----2-linked polyglucose chains; Weissborn AC et al.; Membrane-derived oligosaccharides (MDO) of Escherichia coli are a family of substituted branched oligomers containing 8-12 residues of glucose that are joined by beta 1----2 and beta 1----6 linkages . MDO are localized in the periplasmic space of the cell, and their biosynthesis is regulated by the osmolarity of the medium (Kennedy, E . P . (1982) Proc . Natl . Acad . Sci . U . S . A . 79, 1092-1095) . We report here the initial characterization of a novel glucosyltransferase system that catalyzes the elongation of beta 1----2-linked polyglucose chains . The system requires: 1) a beta-D-glucoside such as the disaccharide sophorose (2-O-beta-D-glucosyl-glucose) or octyl beta-D-glucoside; 2) a trypsin-sensitive membrane fraction; 3) a heat-stable protein from the soluble fraction; 4) UDP-glucose; and 5) Mg2+ ions . Oligomers containing 6-10 glucose units (about the same size as MDO) that are joined by beta 1----2 linkages are major products of the enzyme system . Mutants in the recently mapped mdoA locus (Bohin, J . -P., and Kennedy, E . P . (1984) J . Bacteriol . 157, 956-957) are blocked in vivo at an early stage of MDO synthesis . It has now been found that mdoA mutants are defective in the membrane component, but not in the heat-stable protein that is required for the in vitro synthesis of beta 1----2-linked glucosyl oligomers . We conclude that the glucosyltransferase system described here has an essential function in the synthesis of MDO in vivo. Nucleic Acids Res, 1984 Oct 25, 12(20), 7859 - 75 The complete nucleotide sequence of a common cold virus: human rhinovirus 14; Stanway G et al.; The complete nucleotide sequence of the single-stranded RNA genome of human rhinovirus 14, one of the causative agents of the common cold, has been determined from cDNA cloned in E . coli . The genome is typical of the picornaviridae family, comprising a 5' non-coding region of 624 nucleotides, a long open reading frame of 6537 nucleotides (90.8% of the genome) and a 3' non-coding region of 47 nucleotides . Comparison of the nucleotide sequence and the predicted amino acid sequence with those of the polioviruses reveals a surprising degree of homology which may allow recognition of regions of antigenic importance and prediction of the virus polyprotein cleavage sites . The results presented here imply a closer genetic relationship between the rhinovirus and enterovirus genera than previously suspected. Nucleic Acids Res, 1984 Oct 25, 12(20), 7693 - 703 Nucleotide sequence of the repressor gene of the RA1 tetracycline resistance determinant: structural and functional comparison with three related Tet repressor genes; Unger B et al.; The tetracycline resistance determinant of RA1 was cloned . It consists of at least two genes oriented with opposite polarity, tetA for resistance and tetR for regulation . The transcriptional control sequence was identified and analyzed . It consists of overlapping promotors with divergent orientation and a tandem arrangement of operators . Nucleotide sequencing revealed two open reading frames . One codes for a protein which was identified as a Tet repressor by comparing its primary structure with those of other Tet repressors . The RA1 tetR gene codes for 218 amino acids with a calculated molecular weight of 24.4 kDa . In the primary sequence of the RA1-, pSC101-, Tn10-, and RP1/Tn1721-encoded Tet repressors, 36% of the amino acids are identical . This homology is clustered within the first 150 amino acids, 49% of which are identical among all four proteins . These results are discussed with respect to their structure and function in comparison to other DNA binding proteins. J Biol Chem, 1984 Oct 25, 259(20), 12826 - 30 Structure of the human gamma-fibrinogen gene . Alternate mRNA splicing near the 3' end of the gene produces gamma A and gamma B forms of gamma-fibrinogen; Fornace AJ Jr et al.; The gamma chain of human fibrinogen exists in 2 nonallelic forms, gamma A and gamma B, which differ only in their carboxyl termini . We have found that only one genomic locus exists for gamma-fibrinogen and that the gamma A and gamma B chains arise by alternate mRNA splicing near the 3' end of this gene . In contrast to the rat gamma B mRNA which is produced by alternate splicing with identical polyadenylation sites, human gamma B is produced when the eighth intervening sequence remains unspliced and a polyadenylation signal within this intron is used . The new carboxyl terminus is 16 amino acids longer than the gamma A protein, and although there is only minimal homology between the rat and human carboxyl termini they share a very high proportion of acidic amino acids. J Biol Chem, 1984 Oct 25, 259(20), 12678 - 84 Inhibition of superoxide dismutase by nitroprusside and electron spin resonance observations on the formation of a superoxide-mediated nitroprusside nitroxyl free radical; Misra HP; Nitroprusside appears to inhibit the known types of superoxide dismutases irrespective of their metal prosthetic group and regardless of the source from which the enzymes were isolated . Thus the copper-zinc enzyme from bovine erythrocyte or Neurospora crassa behaved identically as did the manganese enzymes from Escherichia coli or red alga and the iron enzyme from E . coli and a blue-green alga . The inhibition was dose dependent with a Ki = 2.5 X 10(-5) for nitroprusside . Nitroprusside does not bind to the copper moiety of copper-zinc enzyme and seems to compete with O2- for superoxide dismutase . These inhibitions by nitroprusside, which were elicited not only in purified enzymes but also in crude soluble extracts of biological samples, were rapidly reversible . Nitroprusside was found to react with O2- to form a paramagnetic species with three absorption lines of equal width with a separation AN = 15.0 G and a g value of 2.028 . The spin adduct appears to be a nitroxide radical and was stable for several minutes. Biochemistry, 1984 Oct 23, 23(22), 5095 - 102 Conformational transitions of thioredoxin in guanidine hydrochloride; Kelley RF et al.; Spectral and hydrodynamic measurements of thioredoxin from Escherichia coli indicate that the compact globular structure of the native protein is significantly unfolded in the presence of guanidine hydrochloride concentrations in excess of 3.3 M at neutral pH and 25 degrees C . This conformational transition having a midpoint at 2.5 M denaturant is quantitatively reversible and highly cooperative . Stopped-flow measurements of unfolding in 4 M denaturant, observed with tryptophan fluorescence as the spectral probe, reveal a single kinetic phase having a relaxation time of 7.1 +/- 0.2 s . Refolding measurements in 2 M denaturant reveal three kinetic phases having relaxation times of 0.54 +/- 0.23, 14 +/- 6, and 500 +/- 130 s, accounting for 12 +/- 2%, 10 +/- 1%, and 78 +/- 3% of the observed change in tryptophan fluorescence . The dominant slowest phase is generated in the denatured state with a relaxation time of 42 s observed in 4 M denaturant . Both the slowest phase observed in refolding and the generation of the slowest phase in the denatured state have an activation enthalpy of 22 +/- 1 kcal/mol . These features of the slowest phase are compatible with an obligatory peptide isomerization of proline-76 to its cis isomer prior to refolding. Biochim Biophys Acta, 1984 Oct 23, 790(2), 148 - 53 Effects of pH and detergent on the kinetic and electrochemical properties of the purified cytochrome d terminal oxidase complex of Escherichia coli; Lorence RM et al.; The cytochrome d terminal oxidase complex is one of the two terminal oxidases in the aerobic respiratory chain of Escherichia coli . In this paper, effects of pH and detergent on the electrochemical and kinetic properties of the enzyme are investigated . There are two significant conclusions . (1) The oxidation-reduction midpoint potential of the cytochrome b-558 component is sensitive to the detergent used to solubilize the complex . In particular, it is shown that octylglucoside and cholate cause a large decrease in the midpoint potential of cytochrome b-558, while they also result in the reversible inactivation of the oxidase . (2) The midpoint potentials of the cytochrome b-558, a1 and d components are sensitive to pH . More acidic solutions result in stabilizing the reduced forms of the redox-active groups, i.e., raising their midpoint potentials . This may be significant in view of the fact that it has been demonstrated that this enzyme catalyzes an electrogenic reaction and appears to function as a proton pump. FEBS Lett, 1984 Oct 15, 176(1), 176 - 8 The amino acid sequence of a small DNA binding protein from the archaebacterium Sulfolobus solfataricus; Kimura M et al.; The thermoacidophilic archaebacterium Sulfolobus solfataricus possesses several DNA binding proteins which may have a histone-like function . Two particularly dominant species have molecular masses of 7 and 10 kDa, respectively . We have purified one of the small proteins which occurs in relatively large amount and have determined its amino acid sequence . The protein is characterized by a high lysine content; in the N-terminal region the lysine residues occur in an alternating order: X-K-X-K-X-K-X-K . The amino acid sequence does not indicate any obvious homology to those DNA binding proteins whose sequences have been determined. Cell Immunol, 1984 Oct 15, 88(2), 393 - 400 Interferon enhances antibody-dependent cellular cytotoxicity when suboptimal concentrations of antibody are used; Basham TY et al.; Polymorphonuclear leukocytes (PMN) and killer (K) cells isolated from buffy coats from normal volunteers were tested for antibody-dependent cellular cytotoxicity (ADCC) against chicken erythrocytes (CRBC) with and without the addition of interferon (IFN) . Maximum enhancing activity was found when the anti-CRBC antibodies in the ADCC reaction were at suboptimal concentrations . All three species of pure recombinant Escherichia coli-derived interferon were compared for their ability to enhance ADCC in both effector systems . Recombinant IFN-gamma was found to be effective at lower doses than recombinant IFN-alpha A or recombinant IFN-beta, although maximum activity for all three species was similar in the PMN system . IFN-gamma also enhanced K-cell ADCC but to a lesser extent than in the PMN system . There appeared to be individual variation in response of the K-cell ADCC system to IFN-alpha A and IFN-beta at the doses tested. Biochem J, 1984 Oct 15, 223(2), 507 - 17 Nucleotide sequence encoding the iron-sulphur protein subunit of the succinate dehydrogenase of Escherichia coli; Darlison MG et al.; The nucleotide sequence of a 961 base-pair segment of DNA containing the sdhB gene, which encodes the iron-sulphur protein subunit of the E . coli succinate dehydrogenase, has been determined . The sdhB structural gene comprises 711 base pairs (237 codons, excluding the translational initiator and terminator) . It is separated by a 15 base-pair intergenic region from the preceding flavoprotein gene (sdhA) and is the distal gene of an operon that also includes genes (sdhC and D) encoding two hydrophobic subunits, sdhCDAB . The distal end of the sdh operon is linked to the 2-oxoglutarate dehydrogenase gene (sucA) by a complex region of dyad symmetry that is homologous with several potential intercistronic regulatory elements or transcriptional attenuators . The sdhB structural gene encodes a polypeptide of Mr26637 that is strikingly homologous with the iron-sulphur protein subunit of fumarate reductase (38% identity, increasing to 58% when conservative changes are included) . Both subunits contain 11 cysteine residues, 10 being conserved in three clusters resembling those found in ferredoxins . This work completes the sequence of a 9897 base-pair segment of DNA containing seven tricarboxylic acid cycle genes encoding three enzymes or enzyme complexes, citrate synthase (gltA), succinate dehydrogenase (sdh), and the 2-oxoglutarate dehydrogenase complex (suc), that are organized thus: gltA-sdhCDAB-sucAB. Virology, 1984 Oct 15, 138(1), 26 - 36 Detection of genomic-length soybean mosaic virus RNA on polyribosomes of infected soybean leaves; Vance VB et al.; Soybean mosaic virus (SMV)-related RNAs were examined in both polyribosomal and nonpolyribosomal fractions of systemically infected soybean leaves . Viral RNAs were detected by Northern blot hybridization analysis using two cloned SMV-cDNAs representing different regions of the viral genome as hybridization probes . Genomic length SMV-RNA (Mr of 3.3 X 10(6} was found in specific association with EDTA-sensitive polyribosomes of infected leaves, indicating that it functions as a messenger RNA in these cells . A smaller SMV-related RNA (Mr of 1.6 X 10(6} was sometimes detected in the polyribosomal fraction; however, reconstruction experiments indicate that this RNA is a breakdown product of the genomic-length RNA, generated during cell fractionation or RNA extraction . Two other SMV-related RNAs with Mr of 2.0 and 0.78 X 10(6) were sometimes detected in infected cells and were not generated from genomic SMV-RNA or intact virus particles in reconstruction experiments . However, these RNAs were exclusively associated with the EDTA-resistant, nonpolyribosomal fraction of infected cells . These data suggest that genomic-length SMV-RNA is the only viral RNA which is translated in these infected plants. Mech Ageing Dev, 1984 Oct 15, 27(2), 197 - 206 Expression of coliphage T7 in aging anucleate minicells of Escherichia coli; Libby RT et al.; Anucleate minicells produced by a mutated strain of Escherichia coli remain metabolically active for up to 48 h at 37 degrees C . Minicells of increasing age have been infected with the coliphage T7 . Infection results in the onset of transcription and translation producing T7 encoded polypeptides . Quantitative and qualitative changes in T7 gene expression result from infection of increasingly old minicells . There is no detectable increase in the frequency of error occurrence in the synthesis of T7 polypeptides in infected old minicells as compared to infected young minicells. Eur J Biochem, 1984 Oct 15, 144(2), 367 - 73 Nucleotide sequence of the respiratory D-lactate dehydrogenase gene of Escherichia coli; Campbell HD et al.; The structural gene for the respiratory D-lactate dehydrogenase of Escherichia coli, a membrane-bound flavoenzyme, has been subcloned from a 7 X 10(3)-base-pair chromosomal HindIII fragment containing the gene {Young, I . G., Jaworowski, A., and Poulis, M . (1982) Biochemistry 21, 2092-2095} . The complete nucleotide sequence of the 2340-base-pair PstI-SmaI subclone has been determined on both strands by the dideoxy chain termination method . A single large open reading frame is present in the nucleotide sequence . The reading frame is preceded by a good ribosome binding site and numerous possible promoter sequences, and is followed by a typical rho-independent termination sequence . The reading frame predicts that the D-lactate dehydrogenase polypeptide consists of 571 amino acids (including the initiating methionine residue) with Mr = 64613 . The protein does not have a low overall polarity, nor does it contain unusually hydrophobic stretches . It appears to contain a short repeat which is homologous with the well characterized L-lactate dehydrogenases in the vicinity of the 'essential' cysteine residue . Apart from this, homology with other proteins of known sequence has not been detected. Eur J Biochem, 1984 Oct 15, 144(2), 361 - 6 The properties of the large subunit of maize ribulose bisphosphate carboxylase/oxygenase synthesised in Escherichia coli; Gatenby AA; The maize chloroplast gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase has been expressed in Escherichia coli in vivo . This enables the properties of the native large-subunit polypeptide to be examined in the absence of small-subunit polypeptides, and avoids the use of denaturing agents . The product synthesised in bacteria is slightly larger (Mr 54300) than the form present in the chloroplast (Mr 53 300), suggesting the involvement of a precursor polypeptide . In addition several smaller polypeptides are synthesised, predominantly of molecular mass 41 and 30 kDa, but also some of 44 and 12-14 kDa . Pulse-chase experiments with {35S}methionine indicate that all the immunoprecipitable polypeptides are stable . The smaller products are probably the result of premature termination of translation . Virtually all of the large subunits are insoluble, whether synthesised at levels of 100-200 molecules per cell, or up to 60 000 molecules per cell . A small amount of the full-length polypeptide is soluble, but the major soluble product, as determined by sucrose gradient centrifugation, is a polypeptide of molecular mass 12-14 kDa . Ribulose bisphosphate carboxylase activity was undetectable in cell extracts, and binding of a mixture of the radiolabelled transition state analogues carboxyribitol 1,5-bisphosphate and carboxyarabinitol 1,5-bisphosphate could not be detected . It is proposed that other components are required to prevent the large subunit from adopting an inactive, insoluble conformation after, or during, synthesis. Eur J Biochem, 1984 Oct 15, 144(2), 295 - 303 Histidine residues in elongation factor EF-tu from Escherichia coli protected by aminoacyl-tRNA against photo-oxidation; Jonak J et al.; Complexes of Escherichia coli elongation factor EF-Tu with GTP or GTP and aminoacyl-tRNA were photo-oxidized by irradiation with visible light in the presence of rose bengal dye . EF-Tu was isolated, digested with trypsin, the resulting tryptic peptides were separated by high-performance liquid chromatography (HPLC), and the position of most of the peptides on the chromatogram was determined . Irradiation of complexes resulted in the inactivation of the factor (as tested by its capacity to interact with aminoacyl-tRNA) and was accompanied by the loss of its histidine residues (as revealed by amino acid analysis) and by the decrease in the amount of some tryptic peptides (as detected by HPLC) . Aminoacyl-tRNA, bound to EF-Tu during the irradiation, protected the protein from inactivation, from the loss of histidine residues and some of its peptides from photo-oxidative degradation . Comparison of quantities of individual tryptic peptides recovered from the irradiated EF-Tu X GTP X aminoacyl-tRNA complex with those from the irradiated EF-Tu X GTP complex revealed that histidine-containing peptides T12 and T15 as well as methionine-containing peptide T14 were in the ternary complex markedly protected against the photo-oxidative degradation . This finding suggests that their histidines, i.e . His-66 and His-118 respectively and at least one of the methionines (Met-91, 98 or 112) present in peptide T14 are located near to or at the binding site of EF-Tu for aminoacyl-tRNA and could be involved in the interaction between aminoacyl-tRNA and the factor. Biochem Biophys Res Commun, 1984 Oct 15, 124(1), 44 - 50 Molecular cloning of cDNA for rat glycine methyltransferase; Ogawa H et al.; Using a highly purified preparation of glycine methyltransferase mRNA, double-stranded cDNA was synthesized and inserted into the PstI site of pBR322 . The resulting recombinant DNA was used to transform E . coli X 1776 by conventional methods . Among tetracycline-resistant transformants, a number of colonies were found to contain cDNA sequence for glycine methyltransferase as examined by hybrid-selected translation . A restriction endonuclease cleavage map was constructed covering about 720 base pairs . With the cDNA as the probe, the content of the glycine methyltransferase mRNA was quantitated in various rat tissues and was found to be proportional to the specific enzyme activity. FEBS Lett, 1984 Oct 15, 176(1), 32 - 6 The L7/L12 proteins change their conformation upon interaction of EF-G with ribosomes; Gudkov AT et al.; The different functional complexes of ribosomes with elongation factor F (EF-G) were studied by digestion experiments with trypsin . It was found that upon interaction of EF-G with ribosomes the L7/L12 proteins are sensitive to trypsin and are trypsin resistant after dissociation of EF-G from ribosomes . The significance of conformational alterations in the L7/L12 and also in the other proteins in the translation process is discussed. J Immunol Methods, 1984 Oct 12, 73(1), 49 - 55 Immunoadsorption of histidinol dehydrogenase in the presence of urea; Andorn N et al.; Urea inhibits the activity of histidinol dehydrogenase from Escherichia coli B, prevents precipitation of the enzyme by specific antibodies and dissolves immunoprecipitates which were formed in the absence of urea . However, immunoadsorption of the enzyme to Sepharose-bound antibodies can take place in the presence of high concentrations (5-8 M) of urea . The immobilized antibody-bound enzyme exhibits almost full activity after removal of the urea and is inhibited by soluble specific antibodies in the presence of urea . The possibility of using immunoadsorption in the presence of urea for the study of insoluble proteins is discussed. Biochim Biophys Acta, 1984 Oct 12, 805(2), 137 - 42 Effect of dexamethasone, indomethacin, and lipopolysaccharide on the secretion of interstitial collagenase and type V collagenase by cultured macrophages; Mainardi CL; The rabbit alveolar macrophage secretes at least two collagenolytic metalloproteinases in vitro including an interstitial collagenase and a type V collagenase . Using assays previously shown to discriminate between these two activities, the secretion of these two enzyme activities was investigated . Both enzyme activities accumulated in culture over 11 days and the release of both were similarly inhibited by cycloheximide . Collagenolytic activity was negligible in cell lysates . The interstitial collagenase was found in a latent form but the type V collagenase activity was active in the culture medium . When cultured in the presence of dexamethasone, the secretion of both the enzymes were identically inhibited in a dose-dependent manner . Indomethacin was an effective inhibitor of secretion of both collagenases at a concentration of 10(-5) M but not at lower concentrations . Finally, bacterial lipopolysaccharide stimulated the secretion of both type V and interstitial collagenase by these cells . These studies indicate that, like the interstitial collagenase, the type V collagenase is released from the cell as synthesized and is not stored intracellularly . Protein synthesis is necessary for the release of both these collagenases . Furthermore, the release of type V collagenase responded to dexamethasone, indomethacin, and lipopolysaccharide in a manner identical to the secretion of the interstitial collagenase suggesting that synthesis and secretion of these two enzymes are regulated in a similar manner. Nucleic Acids Res, 1984 Oct 11, 12(19), 7389 - 400 Conformational alterations of transcription termination protein rho induced by ATP and by RNA; Engel D et al.; Transcription termination protein rho from Escherichia coli possesses an RNA-dependent ATP hydrolysis activity necessary for expression of its termination function . We have used the rate of trypsin-mediated inactivation of ATPase activity as a conformational probe to test for ligand binding-induced conformational changes in the rho polypeptide . When present in molar excess over rho polypeptide, trypsin inactivates rho ATPase by a first order process that correlates well with the loss of intact rho polypeptide . When rho protein binds poly(C) or poly(dC), its susceptible bonds become more accessible to trypsin action . On the other hand, when rho binds either ATP or ADP those bonds become less accessible . These results suggest that rho protein assumes an altered conformation when an RNA cofactor is bound and that is assumes a distinctly different conformation when a nucleotide substrate or product is bound . A special change in the accessibility of trypsin-susceptible bonds is also detected when rho in its complex with poly(C) is catalyzing the hydrolysis of ATP. J Biol Chem, 1984 Oct 10, 259(19), 12193 - 200 The synthesis of export-defective proteins can interfere with normal protein export in Escherichia coli; Bankaitis VA et al.; We have analyzed the kinetics of maturation for certain bacterial envelope proteins in Escherichia coli strains that are also concomitantly producing an export-defective protein . Our data indicate that proteins with defective signal peptides, rendered nonfunctional by either point mutation or deletion, interfere with the normal export of other envelope proteins . Expression of interference requires that the interfering protein: (i) exhibit a major export defect; (ii) be synthesized at a high rate; and (iii) be actively synthesized at the time interference is being measured . The latter data suggest that interference is a cotranslational process . Intragenic or extragenic suppression of the export defect exhibited by the interfering protein relieves interference in a manner that is directly related to strength of suppression . These and additional data suggest that interference occurs at a very early step in the secretory process . We interpret these results to indicate that proteins with defective signal peptides are still recognized as proteins destined for secretion and are, therefore, at least transiently incorporated into the cell's secretory pathway . The incorporation of an export-defective protein into the secretory pathway disrupts the normal protein traffic from the cytoplasm to the various extracellular compartments. J Biol Chem, 1984 Oct 10, 259(19), 11828 - 35 Stimulus-induced changes in methylesterase activity during chemotaxis in Escherichia coli; Kehry MR et al.; Responses of Escherichia coli to chemical stimuli are mediated by a family of sensory transducer proteins . These transmembrane proteins detect stimuli and convey sensory information to the flagella . Behavioral adaptation to environmental changes is correlated with the reversible methylation of the transducer proteins on several (4 to 6) specific glutamic acid residues to form methyl esters . We have assayed the activity of the chemotaxis-specific methylesterase, the product of the cheB gene, by measuring the product of the demethylation reaction, methanol, using a modification of a previous method (Toews, M.L., Goy, M.F., Springer, M.S., and Adler, J . (1979) Proc . Natl . Acad . Sci . U.S.A . 76, 5544-5548; Terwilliger, T.C., Bogonez, E., Wang, E.A., and Koshland, D . E., Jr . (1983) J . Biol . Chem . 258, 9608-9611) . In this study, we establish the use of a sensitive flow apparatus for measuring stimulus-induced changes in methylesterase activity of intact E . coli cells . Responses to positive and negative stimuli consist of transient decrease and increases in methylesterase activity, respectively . In addition, chase experiments demonstrated that all assayable methyl groups are nearly equivalent . The results are not consistent with the view that the methyl groups put on the transducer proteins as a result of a positive stimulus are the first to be removed when that stimulus is withdrawn . It seems more likely that recently added methyl groups become part of a pool of kinetically equivalent members, any of which can be removed when the stimulus is withdrawn . The flow measurements provide a powerful and simple biochemical assay that complements behavioral studies of chemotaxis. J Biol Chem, 1984 Oct 10, 259(19), 11804 - 11 Characterization of the structural and functional defect in the Escherichia coli single-stranded DNA binding protein encoded by the ssb-1 mutant gene . Expression of the ssb-1 gene under lambda pL regulation; Williams KR et al.; The ssb-1 gene encoding a mutant single-stranded DNA binding protein (SSB-1) has been cloned into a vector placing its expression under lambda pL regulation . This construction results in more than 100-fold increased expression of the mutant protein following temperature induction . Tryptic peptide analysis of the mutant protein by high-pressure liquid chromatography and solid-phase protein sequencing has shown that the ssb-1 mutation results in these substitution of tyrosine for histidine at residue 55 of SSB . This change could only occur in one step by a C----T transition in the DNA sequence which has been confirmed . Physicochemical studies of the homogeneous mutant protein have shown that in contrast to that of the wild-type SSB, the tetrameric structure of SSB-1 is unstable and gradually dissociates to monomer as the protein concentration is decreased from about 10 microM to less than 0.5 microM . The SSB-1 tetramer appears to be stable to elevated temperature (45 degrees C) but the monomer is not . We estimate the normal cellular concentration of SSB-1 (single chromosomal gene) to be 0.5-1 microM . Thus, there is a plausible physical explanation for our previous finding that increased expression of ssb-1 reverses the effects of a single gene (chromosomal) copy amount of SSB-1 (Chase, J.W., Murphy, J.B., Whittier, R.F., Lorensen, E., and Sninsky, J.J . (1983) J . Mol . Biol . 164, 193-211) . However, even though the in vivo effects of ssb-1 and most of the in vitro defects of SSB-1 protein are reversed simply by increasing SSB-1 protein concentration, the mutant protein is not as effective a helix-destabilizing protein as wild-type SSB as measured by its ability to lower the thermal melting transition of poly{d-(A-T)}. J Biol Chem, 1984 Oct 10, 259(19), 11756 - 62 Conformation-specific monoclonal antibodies to glutamine synthetase in Escherichia coli; Chung HK et al.; Glutamine synthetase from Escherichia coli is composed of 12 identical subunits and exists in various forms: unadenylylated, adenylylated, divalent cation bound (taut), and divalent cation free (relaxed) . The relaxed dodecamer readily dissociates into monomers upon exposure to 1 M urea or pH 8.0 . Glutamine synthetase can be inactivated irreversibly by oxidizing a particular histidine residue or by incubating with methionine sulfoximine and ATP . In order to establish hybridoma monoclones that produce antibodies capable of differentiating between different conformers of glutamine synthetase, homogeneous antibodies produced from 7 clones (10-76-1, 48-76-1, 68-2-1, 57-142-2, 72-104-1, 68-3-2, 57-8-1) were characterized for their binding specificity and effects on glutamine synthetase activity . Two antibodies (10-76-1, 48-76-1) bind only to the monomeric form, two antibodies (57-142-2, 68-3-2) bind only to the dodecameric forms (taut or relaxed) and the three others (68-2-1, 72-104-1, 57-8-1) bind to both forms . At a low antibody concentration, 68-3-2 binds preferentially to taut glutamine synthetase over oxidized glutamine synthetase . None of the 7 antibodies differentiates between unadenylylated and adenylylated form . Nevertheless, the gamma-glutamyltransferase activities of the resulting antibody-glutamine synthetase complexes were influenced variably depending upon the state of adenylylation and the divalent cation. Biochemistry, 1984 Oct 9, 23(21), 4934 - 9 Kinetics and subunit interaction of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system; Roossien FF et al.; Purified mannitol-specific enzyme II (EIImtl), in the presence of the detergent Lubrol, catalyzes the phosphorylation of mannitol from P-HPr via a classical ping-pong mechanism involving the participation of a phosphorylated EIImtl intermediate . This intermediate has been demonstrated by using radioactive phosphoenolpyruvate . Upon addition of mannitol, at least 80% of the enzyme-bound phosphoryl groups can be converted to mannitol 1-phosphate . The EIImtl concentration dependence of the exchange reaction indicates that self-association is a prerequisite for catalytic activity . The self-association can be achieved by increasing the EIImtl concentration or at low concentrations of EIImtl by adding HPr or bovine serum albumin . The equilibrium is shifted toward the dissociated form by mannitol 1-phosphate, resulting in a mannitol 1-phosphate induced inhibition . Mannitol does not affect the association state of the enzyme . Both mannitol and mannitol 1-phosphate also act as classical substrate inhibitors . The apparent Ki of each compound, however, is approximately equal to its apparent Km, suggesting that mannitol and mannitol 1-phosphate bind at the same site on EIImtl . Due to strong inhibition provided by mannitol and mannitol 1-phosphate in the exchange reaction, the kinetics of this reaction cannot be used to determine whether the reaction proceeds via a ping-pong or an ordered reaction mechanism. Biochemistry, 1984 Oct 9, 23(21), 4928 - 34 Energetic adaptation of ligand binding to subunit structure of tryptophan synthase from Escherichia coli; Wiesinger H et al.; The binding of indole and L-serine to the isolated alpha and beta 2 subunits and the native alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli was investigated by direct microcalorimetry to reveal the energetic adaptation of ligand binding to the subunit structure of a multienzyme complex . In contrast to the general finding that negative heat capacity changes are associated with ligand binding to proteins, complex formation of indole and the alpha subunit involves a small positive change in heat capacity . This unusual result was considered as being indicative of a loosening of the protein structure . Such an interpretation is in good agreement with results of chemical accessibility studies (Freedberg & Hardman, 1971) . Whereas the thermodynamic parameters of indole binding are not influenced by the subunit interaction, the large negative change in heat capacity of -6.5 kJ/(K X mol of beta 2) measured for the binding of L-serine to the isolated beta 2 subunit disappears completely when serine interacts with the tetrameric complex . These data demonstrate that the energy transduction pattern and therefore the functional roles of the substrates indole and L-serine vary strongly with the subunit structure of tryptophan synthase. Biochemistry, 1984 Oct 9, 23(21), 5069 - 76 Binding of S21 to the 50S subunit and the effect of the 50S subunit on nonradiative energy transfer between the 3' end of 16S RNA and S21; Odom OW et al.; Escherichia coli ribosomal protein S21 was labeled at its single cysteine group with a fluorescent probe . Labeled S21 showed full activity in supporting MS2 RNA-dependent binding of formylmethionyl-tRNAf to 30S ribosomal subunits . Fluorescence anisotropy measurements and direct analysis on glycerol gradients demonstrate conclusively that labeled S21 binds to 50S ribosomal subunits as well as to 30S and 70S particles . The relative binding affinities are in the order 70S greater than 30S greater than 50S . Other results presented appear to indicate that S21 is bound in the same position on either 50S subunits or 30S subunits as in 70S ribosomes, suggesting that the protein is bound simultaneously to both subunits in the latter . Addition of 50S subunits to 30S particles containing probes on S21 and at the 3' end of 16S RNA caused a decrease in the energy transfer between these points . The results correspond to an apparent change in distance from 51 to 61 A. Biochemistry, 1984 Oct 9, 23(21), 4955 - 61 X-ray analysis of the kinetics of Escherichia coli lipid and membrane structural transitions; Ranck JL et al.; Synchrotron radiation was used to follow the time course of the transitions, induced by temperature jump, in Escherichia coli membranes and their lipid extracts isolated from a fatty acid auxotroph grown with different fatty acids . We measured the relaxation times associated with the phase transitions as well as with the conformational transition of the hydrocarbon chains and observed different behavior as a function of chemical composition . Relaxation times of about 1-2 s were found at a hexagonal to lamellar phase transition and within a lamellar phase whose parameters display important variations with temperature when the conformational transition takes place . On the other hand, no delay was observed for a phase transition where large lipid or water diffusion was not needed . We have shown that phase transitions and conformational transitions are, to a large extent, uncoupled and that the relaxation times corresponding to the latter transition could be related to the size of the ordered domains . In all cases, the order to disorder conformational transition is more rapid than the disorder to order transition . Finally, the relaxation times of the disorder to order transition observed with the membranes and with their lipid extracts were found to be strongly correlated, indicating that the proteins do not play a role in this transition. Biochemistry, 1984 Oct 9, 23(21), 4921 - 8 Linkage of subunit interactions, structural changes, and energetics of coenzyme binding in tryptophan synthase; Wiesinger H et al.; The energetics of binding of the coenzyme pyridoxal 5'-phosphate (PLP) to both the apo beta 2 subunit and the apo alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli has been investigated as a function of pH and temperature by direct microcalorimetric methods . At 25 degrees C, pH 7.5, the binding process proceeds in the time range of minutes and shows a biphasic heat output which permits resolution of the overall reaction into different reaction steps . Binding studies on the coenzyme analogues pyridoxal (PAL), pyridoxine 5'-phosphate (PNP), and pyridoxine (POL) to the protein as well as a comparison of these results with data from studies on PLP binding to epsilon-aminocaproic acid have led to a deconvolution of the complex heat vs . time curves into fast endothermic contributions from electrostatic interaction and Schiff base formation and slow exothermic contributions from the interactions between PLP and the binding domain . The pH-independent, large negative change in heat capacity of about -9.1 kJ/(mol of beta 2 X K) when binding PLP to beta 2 is indicative of major structural changes resulting from complex formation . The much smaller value of delta Cp = -1.7 kJ/(mol of beta 2 X K) for binding of PLP to alpha 2 beta 2 clearly demonstrates the energetic linkage of protein-protein and protein-ligand interactions . Calorimetric titrations of the apo beta 2 subunit with PLP at 35 degrees C have shown that also at this temperature positive cooperativity between the two binding sites occurs . On the basis of these measurements a complete set of site-specific thermodynamic parameters has been established.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1984 Oct 9, 23(21), 4852 - 9 Solvent effects on allosteric equilibria: stabilization of T and R conformations of Escherichia coli aspartate transcarbamylase by organic solvents; Dreyfus M et al.; The activity of Escherichia coli aspartate transcarbamylase (ATCase) is markedly influenced by the addition of organic solvents to the assay medium . The cosolvents tested, which include simple aliphatic alcohols, amides, and ureas, as well as acetone and dioxane, fall into two different classes: the most polar ones (formamide, acetamide, N-methylformamide, and urea) stimulate the enzyme activity for all concentrations tested . In contrast, solvents that are less polar than water inhibit the enzyme at low concentrations but stimulate it at higher concentrations . No comparable effects are observed in the case of the isolated catalytic subunits, a non-regulated form of ATCase . Extensive kinetic studies on ATCase and on two of its Michaelian derivatives, 2-thioU-ATCase and carbamylated ATCase, indicate that solvents modulate the same allosteric transition that is responsible for homotropic interactions between the catalytic sites . The stabilization of the R state of ATCase by comparatively high concentrations of cosolvents is reminiscent of similar findings made on hemoglobin and glycogen phosphorylase, suggesting a common underlying mechanism . Addition of organic cosolvents to water is known to reduce hydrophobic interactions, and we suggest that this effect may preferentially stabilize the more "relaxed" conformations of allosteric proteins, because they have a larger surface exposed to solvent {Chothia, C . (1974) Nature (London) 248, 338-339} . On the other hand, we suggest that the stabilization of the T state by low concentrations of all but the most polar cosolvents simply reflects stronger electrostatic interactions in this conformation. Biochemistry, 1984 Oct 9, 23(21), 4837 - 43 Effect of template conversion from the B to the Z conformation on RNA polymerase activity; Butzow JJ et al.; Transition from the right-handed B to the left-handed Z conformation of DNA was studied by circular dichroism in parallel with the ability of the DNA to support RNA synthesis with Escherichia coli RNA polymerase . Since the B to Z transition is generally induced by a chemical agent, a definitive demonstration that a change in activity is due to the conformational change, and not to the agent itself or to other factors, requires the clear-cut correlation of template activity and conformation under a variety of conditions that result in conformational change . Such correlation was achieved by following the {Co(NH3)6}3+-induced transition of poly(dG-dC) X poly(dG-dC) and poly(dG-dm5C) X poly(dG-dm5C) and the Mg2+-induced transition of poly(dG-dm5C) X poly(dG-dm5C) . In addition, conditions were chosen to minimize possible aggregation . In each of these three systems, the B to Z conformational transition was accompanied by a substantial decrease in transcription activity . While the conversion from B to Z of poly(dG-dm5C) X poly(dG-dm5C) is induced by a 25-fold lower concentration of {Co(NH3)6}3+ than that required for the conversion of unmethylated polymer, in both cases the RNA polymerase activity is decreased at the same cation concentration as that producing the conformational transition . Neither {Co(NH3)6}3+ nor Mg2+ inhibits RNA synthesis with control templates that are not converted to Z under the same conditions, such as poly(dA-dT) X poly(dA-dT) or calf thymus DNA with {Co(NH3)6}3+ or poly(dG-dC) X poly(dG-dC) with Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1984 Oct 9, 23(21), 4998 - 5003 Specificity of the proton adenosinetriphosphatase of Escherichia coli for adenine, guanine, and inosine nucleotides in catalysis and binding; Perlin DS et al.; Specificity of the Escherichia coli proton ATPase for adenine, guanine, and inosine nucleotides in catalysis and binding was studied . MgADP, CaADP, MgGDP, and MgIDP were each good substrates for oxidative phosphorylation . The corresponding triphosphates were each substrates for hydrolysis and proton pumping . At 1 mM concentration, MgATP, MgGTP, and MgITP drove proton pumping with equal efficiency . At 0.1 mM concentration, MgATP was 4-fold more efficient than MgITP or MgGTP . Nucleotide-depleted soluble F1 could rebind to F1-depleted membranes and block proton conductivity through F0; rebound nucleotide-depleted F1 catalyzed pH gradient formation with MgATP, MgGTP, or MgITP . This showed that the nonexchangeable nucleotide sites on F1 need not be occupied by adenine nucleotide for proton pumping to occur . It was further shown that no nucleotide was tightly bound in the nonexchangeable sites of F1 during proton pumping driven by MgGTP in these reconstituted membranes, whereas adenine nucleotide was tightly bound when MgATP was the substrate . Nucleotide-depleted soluble F1 bound maximally 5.9 ATP, 3.2 GTP, and 3.6 ITP of which half the ATP and almost all of the GTP and ITP exchanged over a period of 30-240 min with medium ADP or ATP . Also, half of the bound ATP exchanged with medium GTP or ITP . These data showed that inosine and guanine nucleotides do not bind to soluble F1 in nonexchangeable fashion, in contrast to adenine nucleotides . Purified alpha-subunit from F1 bound ATP at a single site but showed no binding of GTP nor ITP, supporting previous suggestions that the non-exchangeable sites in intact F1 are on alpha-subunits. Biochim Biophys Acta, 1984 Oct 5, 783(1), 80 - 8 Isolation and characterization of two forms of Met-tRNAf deacylase from rabbit reticulocyte ribosomes; Kwan A et al.; We have separated and purified two forms of Met-tRNAf deacylase (or two separate enzymes), an activity that mediates in part the suppression of polypeptide chain initiation that occurs in heme deficiency or with double-stranded RNA, 1000-fold from the 0.5 M KCl wash of rabbit reticulocyte ribosomes . Deacylase I is a minor activity with an S20,w of 5.9, D20,w of 4.9 and Mr of 110 000, while deacylase II is the major activity with an S20,w of 3.3, D20,w of 7.1 and Mr of 43 000 . Both convert crude reticulocyte or pure yeast, wheat germ, and E . coli {35S}Met-tRNAf to {35S}methionine and tRNAfMet and have no effect on reticulocyte {35S}fMet-tRNAf, {3H}Ala-tRNA or {3H}Lys-tRNA . However, while deacylase I has similar activity throughout the pH range of 6.1-8.1, deacylase II has a sharp pH optimum at 7.9 and is almost completely inactive at 6.1 . In addition, deacylase II shows a much greater affinity for pure Met-tRNAf than deacylase I (Km of 1.5-3 nM vs . 100 nM), and, while deacylase II is selectively inhibited by tRNAfMet, deacylase I is inhibited similarly by any added tRNA. Biochim Biophys Acta, 1984 Oct 5, 783(1), 15 - 25 Synthesis and processing of 5 S rRNA from an rrnB minigene in a plasmid; Szeberenyi J et al.; A recombinant plasmid containing the promoters, terminators and only the intact 5 S rRNA gene of rrnB is expressed efficiently in Escherichia coli cells . In strains containing a thermolabile RNAase E (rne) full-length transcripts of the rrnB region from the plasmid and a partially processed intermediate product accumulate at non-permissive temperatures . Upon addition of chloramphenicol two additional plasmid-specific RNA molecules appear . They are shorter than the full-length transcripts . These species contain the 3'-end region of the full-length transcripts . Even though the 5' ends of these RNAs were most likely produced by degradative enzymes these 5' ends are not ragged . All these plasmid-specific RNAs are specific substrates for the two endonucleolytic RNA processing enzymes, RNAase E and RNAase III. J Mol Biol, 1984 Oct 5, 178(4), 941 - 8 Use of transposon-promoted deletions in DNA sequence analysis; Ahmed A; The usefulness of the dideoxy method for DNA sequencing can be greatly extended by the use of transposon-generated deletions . These deletions have the unique property of extending from a fixed nucleotide at the transposon terminus to various sites outside it . A plasmid (pAA3.7) carrying Tn9, which allows positive selection of such deletions as galactose-resistant colonies of Escherichia coli, is described . A cloned gene can thus be subdivided into a series of overlapping sequences, all of which are fused to a common sequence at the transposon terminus . Restriction fragments carrying the segments fused by deletions are cloned in M13, and sequenced using a primer complementary to the Tn9 terminus . Complete nucleotide sequence of the gene is assembled from sequence overlaps found in deletions with end-points approximately 350 base-pairs apart . The method is rapid, requires minimal in vitro manipulation, and is free from redundant information normally produced in shotgun sequencing. Biochim Biophys Acta, 1984 Oct 3, 776(2), 247 - 58 Localization of the galactoside binding site in the lactose carrier of Escherichia coli; Mitaku S et al.; The location of flurophores specifically bound to the lactose/H+ carrier of Escherichia coli was ascertained by the use of various collisional quenchers . The reporter groups were (1) the pyrenyl residue of N-(1-pyrenyl)maleimide attached to the essential cysteine residue 148, which is presumably at or near the galactoside binding site, and (2) the dansyl moieties of a series of fluorescent substrate molecules . The accessibility of these fluorophores from the lipid phase was assessed by nitroxyl-labelled fatty acids and phospholipids . By using a series of nitroxyl-labelled fatty acids carrying the quencher at different positions in the acyl chain, the position of a quenchable fluorophore with respect to the membrane normal can be determined . The accessibility of fluophores from the aqueous phase was assessed by using a water-soluble quencher, the N-methylpicolinium ion . The results of quenching studies suggest that the galactoside binding site is located within the carrier and that this binding site communicates with the aqueous phase through a pore. Acta Pathol Microbiol Immunol Scand {C}, 1984 Oct, 92(5), 331 - 3 Effects of methotrexate, ampicillin and gentamicin alone and in combination on the in vitro locomotion on human polymorphonuclear cells (PMN); Melby K et al.; The effect of ampicillin 25 mg/ml, gentamicin 5 mg/l and methotrexate 10 & 70 mg/l, alone and in combination, was tested for their influence on human polymorphonuclear cell-locomotion, using zymosan or a bacterial filtrate of E . coli, with or without 0.25 and 0.025 mg/l of ampicillin and gentamicin, respectively, as attractants . Methotrexate and gentamicin alone decreased the locomotion, using zymosan as attractant . No effect was observed using the pure bacterial filtrate . The filtrate containing antibiotics displayed a lower ability to induce locomotion when the leucocytes had been pre-treated with antibiotics alone or in combination with methotrexate but not with methotrexate alone . Aspects of these findings are discussed. Immunopharmacology, 1984 Oct, 8(2), 79 - 89 Effects of serum on C3b-stimulated release of prostaglandins and thromboxane B2 from human monocytes; Schenkein HA et al.; Previous studies have demonstrated that C3b stimulates the release of prostaglandin E and thromboxane B2 from human monocytes in serum-free cultures . We have examined the influence of serum on this phenomenon, and have found that addition of normal human serum or fetal calf serum to such cultures at concentrations of 0.1-5.0% results in enhanced release of both eicosanoids in C3b-stimulated cultures, while release in control cultures is unaffected or, in many cases, inhibited . Addition of human serum albumin or transferrin to serum-free cultures is not sufficient to mimic the serum effect . Fetal calf and normal human sera increase the efficacy of C3b in stimulating release of both prostaglandin E and thromboxane B2 at all but the lowest doses tested, but fail to significantly alter the kinetics or release or the acquired unresponsiveness of monocytes to C3b that occurs following culture periods of greater than 24 h . By preincubation of purified C3b in normal human serum, it can be shown that such serum degrades C3b stimulatory activity, but at a rate that is slow enough to permit expression of its activity when added at the beginning of the culture period . In contrast, the stimulatory activity of E . coli lipopolysaccharide is unaffected by pretreatment with serum . Thus, in contrast to the inhibitory effect of serum on the activity of C3 fragments in in vitro assays of certain mononuclear cell functions such as lymphocyte transformation and antibody production, serum enhances the ability of C3b to stimulate monocyte prostaglandin release. Appl Environ Microbiol, 1984 Oct, 48(4), 830 - 2 Isolation of Legionella species from drinking water; Hsu SC et al.; Three different species of Legionella were recovered from samples of water taken from chlorinated public water supplies where no coliform bacteria were simultaneously detected . Five of 856 samples yielded Legionella isolates . Three isolates were identified as Legionella pneumophila serogroup 1, the fourth was identified as Legionella dumoffii, and the fifth was identified as Legionella jordanis . Studies to determine the survival of L . pneumophila Flint 1 serogroup 1 in tap water at various temperatures and in tap water with added sodium hypochlorite were done . These organisms were found to survive for 299 days in tap water at 24 and 5 degrees C but not at 35 degrees C . A concentration of at least 0.2 mg of residual chlorine per ml was required to eliminate at least 90% of L . pneumophila and Escherichia coli inocula in 2 h. J Infect Dis, 1984 Oct, 150(4), 513 - 6 Plasmid transfer into members of the family Legionellaceae; Chen GC et al.; The plasmids RP4 and pPH1JI::Mu::Tn5 were transferred with moderate frequency from Escherichia coli to Legionella pneumophila (strain Bloomington 2) and Fluoribacter (Legionella) bozemanae (strain WIGA) . The frequency of transfer to Tatlockia (Legionella) micdadei (strain Tatlock) was much lower . In these transconjugant strains the plasmids were maintained following at least 10 nonselective passages . However, the introduced plasmids could not be transferred from these strains to several other strains of Legionella or Tatlockia with a detectable frequency, yet they could be transferred to a restriction-modification deficient (rm-) E . coli strain with moderate frequency . This suggests not only that the expression of transfer genes may be repressed but that the legionellae are poorer recipients than are the rm- E . coli. Arch Biochem Biophys, 1984 Oct, 234(1), 290 - 6 The primary structure of spinach acyl carrier protein; Kuo TM et al.; Acyl carrier protein (ACP) from spinach leaves has been purified to homogeneity by high-performance liquid chromatography with an anion-exchange column . The amino acid sequence of one major ACP in spinach leaves, ACP-I, has been determined by automated Edman degradation . It consists of the following 82 amino acids: (sequence in text) . Sequencing of the intact polypeptide provided data for the first 57 residues . Cleavage of the succinylated ACP with CNBr at Met-46, followed by sequencing of the fragment mixture, provided data for the final 36 residues . The C-terminal alanine was confirmed by carboxypeptidase Y digestion . The spinach ACP has 40, 70, and 25% homology with Escherichia coli, barley, and rabbit ACPs, respectively . The results not only provide the first complete sequence of a plant ACP, but also provide insight into the structural and evolutionary relationships among plant, animal, and bacterial ACPs. Microbiol Sci, 1984 Oct, 1(7), 168 - 75 The ins and outs of colicins . Part I: Production, and translocation across membranes; Pugsley AP; Most colicins are plasmid-encoded proteinaceous toxins which act on bacteria closely related to the producing strain (generally Escherichia coli), and which possess the almost unique ability to be translocated across up to four bilayer membranes, two each in the producing and target cells. Br J Pharmacol, 1984 Oct, 83(2), 433 - 42 A new canine model of endotoxin shock; Evans SF et al.; A new canine model of endotoxin shock has been developed in which spontaneous recovery of cardiovascular function is largely prevented, the haemodynamic effects of anaesthesia are minimized and intravascular volume replacement is given . This model has been evaluated using two groups of five adult mongrel dogs anaesthetized with alpha-chloralose and breathing spontaneously . Animals in one group were anaesthetized, instrumented and given Escherichia coli (E . coli) endotoxin intravenously, whilst those in the control group were subjected only to anaesthesia and instrumentation . E . coli endotoxin was given to dogs in the shock group as a bolus dose of 5 mg kg-1 followed by a continuous infusion at 2 mg kg-1 h-1 . This produced immediate, severe, cardiovascular depression, with precipitous falls in mean arterial pressure (MAP), cardiac index (CI), stroke index (SI) and left ventricular (LV) dp/dt max . There were associated increases in systemic and pulmonary vascular resistances . Arterio-venous oxygen content difference (C(a-v)O2) increased after induction of shock, and animals developed a progressive metabolic acidosis . Increasing haemoconcentration occurred, as evidenced by a rising haematocrit (PCV) . Hypovolaemia was reflected by a concurrent fall in pulmonary capillary wedge pressure (PCWP) . One hour after induction of shock, intravascular volume replacement was given in the form of a colloidal gelatin solution, as a bolus dose of 10 ml kg-1, followed by a continuous infusion at 10 ml kg-1 h-1 . Volume replacement reversed haemoconcentration, restored PCWP and produced some haemodynamic improvement, although in general, severe cardiovascular depression persisted throughout a three hour observation period . This severe endotoxin shock model has proved to be stable, reproducible and economical.(ABSTRACT TRUNCATED AT 250 WORDS) Arch Biochem Biophys, 1984 Oct, 234(1), 206 - 13 5-Azidoindole binding to the Escherichia coli tryptophan synthase alpha 2 beta 2 complex; Napier ML et al.; The tryptophan synthase alpha 2 beta 2 complex catalyzes tryptophan (Trp) biosynthesis from serine plus either indole (IN) or indole-3-glycerol phosphate (InGP) . The photoreactive 5-azido analog in IN (AzIN), itself a substrate in the dark, was utilized to examine the substrate binding sites on this enzyme . When irradiated with AzIN at concentrations approaching IN saturation for the IN----Trp activity (0.1 mM), in the absence of serine, the enzyme was increasingly inactivated (up to 70-80%) concomitant with the progressive binding of a net of 2 mol AzIN per alpha beta equivalent . Little or no cooperativity in the binding of the 2 mol AzIN was observed . In contrast, there was minimal effect on the IN----InGP activity . Under these conditions AzIN appeared to be incorporated equally into each subunit . No significant inactivation nor binding occurred in the presence of serine . A quantitatively similar inactivation of InGP----Trp activity was observed over the same AzIN concentration range, suggesting common IN sites for Trp biosynthesis from either indole substrate . At higher concentrations (0.1-0.7 mM), no further inactivation occurred, although there was extensive additional binding (up to 10 mol/alpha beta equivalent) . These data are consistent, although more clear-cut quantitatively, with the high- and low-affinity sites proposed from equilibrium dialysis studies . AzIN binding studies utilizing the isolated beta 2 subunit confirmed earlier reports suggesting the existence of many nonspecific IN binding sites on this subunit. Arch Biochem Biophys, 1984 Oct, 234(1), 151 - 60 Binding and reactivity at the "glucose" site of galactosyl-beta-galactosidase (Escherichia coli); Huber RE et al.; A large number of sugars and alcohols were tested to see how well they bound and how readily they reacted at the "glucose" site of the galactosyl form of beta-galactosidase . Two classes of compounds were found to bind well to the galactosyl form of the enzyme . One class contained sugars and alcohols similar in structure to D-glucose in its pyranose ring form, and the other class was composed of relatively hydrophobic sugars and alcohols . On the other hand, several factors seemed to control k4 . Large k4 values were found for straight-chain alcohols as compared to the values for the corresponding ring sugars . Also, if the acceptors had hydroxyl groups at the end of the molecule, the reactivity (k4) was greater than if hydroxyl groups were only in the middle of the molecule . In addition, if there was a hydroxyl at an asymmetric carbon next to a terminal hydroxymethyl group, it was necessary that it be in the same orientation as the D configuration of glucose; otherwise, the k4 was low . Overall, the results showed that it is the binding effect, more than the reactivity, which is responsible for the specificity at the "glucose" site . More specifically, these studies showed that the reason glucose is such an ideal molecule for transgalactosylation is that it leaves the galactosyl form of the enzyme very slowly, that is, k-a is relatively small . Thus, glucose remains attached to the galactosyl form of beta-galactosidase for a sufficient time to allow transgalactosylation to occur, while other acceptors, despite being as reactive (or more reactive) in terms of their k4 values, dissociate from the "glucose" site of the galactosyl form of the enzyme very readily and thus are poor acceptors. Proc Natl Acad Sci U S A, 1984 Oct, 81(19), 6154 - 8 Selective destruction of a host blood cell type by a parasitoid wasp; Rizki RM et al.; Foreign objects that enter the hemocoel of Drosophila melanogaster larvae are encapsulated by one type of blood cell, the lamellocyte, yet eggs of the parasitoid wasp Leptopilina heterotoma remain unencapsulated in D . melanogaster larval hosts that have many lamellocytes . Here we demonstrate that shortly after a female wasp oviposits in the hemocoel the lamellocytes undergo morphological changes and lose their adhesiveness . These affected blood cells are eventually destroyed as the parasitoid egg continues its development . The factor responsible for lamellocyte destruction, lamellolysin, is contained in an accessory gland of the female reproductive system and is injected along with the egg into the host hemocoel . Lamellolysin does not alter the morphology or the defense functions of the other types of blood cells in the host. J Bacteriol, 1984 Oct, 160(1), 161 - 8 Study of regulation and transport of hemolysin by using fusion of the beta-galactosidase gene (lacZ) to hemolysin genes; Juarez A et al.; Operon and gene fusions between lacZ and the hemolysin genes, hlyC and hlyA, were performed . These two genes are essential for the synthesis of active hemolysin and are transcribed from a common promoter (p1) . Whereas the amount of hemolysin produced in Escherichia coli is not changed by altering the hly gene dose, beta-galactosidase activity follows the gene dosage in both types of fusions when lacZ comes under the control of p1 . This indicates that hemolysin is not negatively regulated on the transcription or translation level . The products of the gene fusions hlyC::lacZ and hlyA::lacZ were identified in maxicells as stable proteins of 146,000 and 220,000 daltons, respectively . Both fusion proteins possess beta-galactosidase activity indicating that the performed fusions of lacZ to the hly genes do not destroy the reading frame of hlyC and hlyA . The fusion proteins HlyC-beta-gal and HlyA-beta-gal were predominantly detected in the cytoplasm, confirming previous data which suggested that the primary gene products of hlyC and hlyA are not transported across the cytoplasmic membrane. Infect Immun, 1984 Oct, 46(1), 74 - 80 Induction of immune and adjuvant immunoglobulin G responses in mice by Brucella lipopolysaccharide; Moreno E et al.; The immunogenic and adjuvant properties of Brucella abortus and Escherichia coli lipopolysaccharides (LPSs) were studied in endotoxin-responsive, athymic, and euthymic BALB/c mice and in responsive C3H/HeAu mice and congenic nonresponsive C3H/HeJ mice . Consistent with previous reports, E . coli LPS did not stimulate significant primary or secondary antibody responses in C3H/HeJ mice and induced the production of immunoglobulin M (IgM) and low levels of IgG in C3H/HeAu mice . In contrast, B . abortus smooth and rough LPS stimulated primary and secondary antibody responses and induced the production of IgM and high levels of IgG in both responsive and nonresponsive strains of C3H/He mice and in nude mice . When used as adjuvant, B . abortus LPS augmented the IgG plaque-forming-cell response of C3H/HeAu and BALB/c euthymic mice to the T-dependent antigen sheep erythrocytes . E . coli LPS augmented only the IgM plaque-forming-cell response in the same mouse strains . Neither B . abortus nor E . coli LPS was adjuvant for C3H/HeJ or nude mice . The dichotomy between the antibody and adjuvant responses of both C3H/HeJ mice and athymic mice to B . abortus LPS may be a function of the true thymus independence and dependence of these responses . In addition, the refractiveness of C3H/HeJ and nude mice to B . abortus LPS as adjuvant, but not as mitogen or polyclonal B cell activator, clearly dissociates these phenomena. Cancer Res, 1984 Oct, 44(10), 4465 - 9 Potent activation of mouse macrophages by recombinant interferon-gamma; Varesio L et al.; The ability of recombinant interferon-gamma (IFN-gamma) to activate mouse macrophages was investigated . The use of recombinant IFN-gamma has the advantage of being devoid of contaminating lymphokines . Two preparations of IFN-gamma were utilized, one which was not glycosylated and which was highly purified from Escherichia coli another which was glycosylated and which was from transfected COS-7 monkey cells . Both preparations of recombinant IFN-gamma activated murine macrophages to kill lymphoma and melanoma tumor targets, suggesting that glycosylation of the protein or the presence of other mammalian proteins is not essential for activation . Significant levels of cytolytic activity were induced from IFN-gamma (1 to 10 units/ml) . This activity was undiminished by treatment of the IFN-gamma preparations with polymixin B at doses which neutralized endotoxin (50 micrograms/ml) . Similarly, IFN-gamma, at low concentrations, induced an inhibition of migration by macrophages . Based on antiviral activity, IFN-gamma was shown to be 100 to 1000 times more potent than was IFN-beta as a macrophage-activating agent . Taken together, these results demonstrate that murine IFN-gamma is a macrophage-activating factor which is effective at physiological concentrations . Of particular interest is the observation that the nonglycosylated E . coli-derived IFN-gamma is active and therefore may be of value for therapeutic studies, since it can be easily produced in large amounts. J Biomol Struct Dyn, 1984 Oct, 2(2), 387 - 95 Periodic structurally similar oligomers are found on one side of the axes of symmetry in the lac, trp, and gal operators; Nussinov R et al.; Three well-defined E . coli operator regions were examined for recurring conformational deviation from a regular B-DNA helix . All three, the lac, trp, and gal, show repeats of the same set of neighboring helical twist angles . These angles recur with a periodicity equal to the helix periodicity on one side of the operator's axes of symmetry . The probability that their occurrence is random was found to be extremely small . Therefore, we propose that in addition to specific bases, repeating twist angles patterns are likely to be among the local parameters involved in repressor-operator recognition. J Biomol Struct Dyn, 1984 Oct, 2(2), 291 - 301 The absence of cruciform structures from pAO3 plasmid DNA in vivo; Lyamichev V et al.; We extracted pAO3 plasmid DNA from E . coli cells, having "frozen" the transition between cruciform and double-helical conformations in DNA . The characteristic feature of the DNA isola