Cell, 1984 Apr, 36(4), 1073 - 80 Yeast DNA topoisomerase II is encoded by a single-copy, essential gene; Goto T et al.; The gene TOP2 encoding yeast topoisomerase II has been cloned by immunological screening of a yeast genomic library constructed in the phage lambda expression vector, lambda gt11 . The ends of the message encoded by the cloned DNA fragment were delimited by the Berk and Sharp procedure (S1 nuclease mapping) for the 5' end and mapping of the polyA tail portion of a cDNA fragment for the 3' end . The predicted size of the message agrees with the length of the message as determined by Northern blot hybridization analysis . The identity of the gene was confirmed by expressing the gene in E . coli from the E . coli promoter lac UV5 to give catalytically active yeast DNA topoisomerase II . Disruption of one copy of the gene in a diploid yeast creates a recessive lethal mutation, indicating that the single DNA topoisomerase II gene of yeast has an essential function. Gene, 1984 Apr, 28(1), 93 - 102 The rep mutation . VII . Cloning and analysis of the functional rep gene of Escherichia coli K-12; Bialkowska-Hobrzanska H et al.; The rep gene of Escherichia coli was isolated on a 6-kb PvuII fragment of plasmid pLC44-7 DNA from the Clarke-Carbon collection and cloned into pSC101 (to form pHBH8) and pBR322 (to form pHBH30) . The plasmids pHBH8 and pHBH30 were found to complement all rep mutations tested . The functional rep gene and its promoter were mapped to a 3.2-kb XhoI-BalI fragment on the basis of complementation data with deletion and insertion derivatives of the two plasmids; subcloning of various restriction fragments confirmed the assignment . EcoRI, HindIII, and HpaI restriction sites were found to reside within that region of the DNA required for expression of the rep function . A coupled in vitro transcription-translation system was used to show that only those plasmids containing a functional rep gene encoded a protein of about Mr 67 000 (the Mr of the rep protein) . No plasmids were found that complemented only the A or B classes of rep mutants (which differ in their ability to support the growth of P2 and M13 phages) . This result suggests that rep-A and rep-B are alleles of the same structural gene. Gene, 1984 Apr, 28(1), 29 - 35 Lambda phagemids and their transducing properties; Melnikov AA et al.; Two recombinant lambda DNAs, lambda gt::pMB9 and lambda NM::pBR322, containing, respectively, the pMB9 and pBR322 replicon were constructed and characterized . Both constructs (phagemid DNAs) transfect Escherichia coli cells, producing mature infectious phage progenies . Alternatively, drug-resistant colonies of transductants can be selected upon infection with these phages (phagemid particles) that maintain phagemid DNA in the cell in the form of covalently closed circular plasmids . The efficiency of transduction for nonlysogenic E . coli strains with lambda gt::pMB9 phage producing lambda repressor cIts ranges from 10(-7) to 10(-2) transductant colonies per input phage, depending on the temperature and strain used, while lambda NM::pBR322 phage carrying imm21 transduces with a frequency of up to 1 . This means that each lambda NM::pBR322 phagemid particle is capable of establishing itself in the cell as a nonlethal plasmid, permitting formation of a resistant bacterial colony . The maximal level of transduction with lambda gt::pMB9 was obtained when E . coli cells lysogenic for lambda were used . Thus, we believe that the efficiency of transduction is determined by the turn-on of the phage repressor in the transductant . In addition, we have found that all lambda gt::pMB9-containing transductants under certain conditions harbor precisely excised pMB9; excision of pBR322 from lambda NM::pBR322 has not been observed. Int J Radiat Biol Relat Stud Phys Chem Med, 1984 Apr, 45(4), 379 - 88 Repair of damage in double-stranded phi X174 (RF) DNA due to radiation-induced water radicals; Nabben FJ et al.; Experiments in which the yields of radiation-induced OH and H radicals were varied, showed that both types of water radicals inactivate phi X174 RF DNA to about the same extent as measured by transfection of the (irradiated) DNA to E . coli wild-type spheroplasts . On the other hand, using spheroplasts prepared from E . coli strains, deficient in one of the proteins involved in excision DNA repair (uvrA- or uvrC-) or in post-replication repair (recA-), clear differences between damage originating from OH or H radical attack were found . Part of the radiation damage due to H radicals appeared to be repairable by an uvrA-gene-dependent repair mechanism, whereas this repair pathway does not play an important role in the case of OH radical damage . The reverse applies to uvrC-gene-dependent repair, which only affects OH radical damage (obtained under anoxic conditions), but has no influence on damage due to H radicals . Irradiation of double-stranded phi X174 (RF) DNA in the presence of oxygen however, yields damage--due to OH radicals only--which appeared not to be sensitive to either uvrC- or uvrA-gene-dependent repair . Furthermore, post-replication repair (recA) has only very little effect on the amount of inactivation by H or OH radicals, when irradiation is carried out under anoxic conditions . We did not find significant inactivation due to hydrated electrons, whether the biological activity was determined by use of wild-type spheroplasts or of strains deficient in excision or post-replication repair proteins. Can J Microbiol, 1984 Apr, 30(4), 451 - 60 Morphological examination of cell surface structures of enterotoxigenic strains of Escherichia coli; Chan R et al.; The very fine sinuous K99 pili of enterotoxigenic strains of Escherichia coli can be visualized in shadowed and in negatively stained preparations, especially if the amorphous K30 glycocalyx is not produced, but these very delicate structures cannot be directly resolved in sectioned material . The K99 pili can, however, be thickened by the nonspecific accretion of K30 glycocalyx material, during its condensation as a result of dehydration, to the point where it can be resolved in sectioned material . This visualization is enhanced if the accreted and condensed glycocalyx is stained with ruthenium red . Alternatively and additionally, the K99 pilus can be thickened by the specific accretion of monoclonal antibodies so that it is made visible in sectioned material . The condensation of the hydrated K30 antigen glycocalyx of enterotoxigenic strains of Escherichia coli during dehydration can be prevented by stabilization using specific antibodies so that this capsular glycocalyx structure is identified in sectioned material and is seen in its correct distribution and dimensions . These methods allow the identification and visualization of bacterial surface structures, both in vitro and in vivo, and they provide a useful means of assessing the presence and distribution of these structures at all stages of the bacterial disease and a possible means of assessing their roles in the pathogenic process. Anal Biochem, 1984 Apr, 138(1), 66 - 7 An improved method for increasing the resolution and sensitivity of silver staining of nucleic acid bands in polyacrylamide gels; Kolodny GM; Staining of polyacrylamide gels with methylene blue prior to silver staining increases band resolution and sensitivity . This method permits resolution of multiple bands less than 1 mm apart, and is able to detect bands containing only 100 pg of RNA. Biochem J, 1984 Apr 1, 219(1), 205 - 10 Polyamine regulation of stringent control in a polyamine-auxotrophic strain of Escherichia coli; Goldemberg SH; Escherichia coli BGA8 , a mutant unable to synthesize putrescine, behaves as stringent or relaxed according to the presence or absence of polyamine, respectively, in the culture medium . The relaxed synthesis of RNA can be reverted back to stringent by addition of putrescine or spermidine . The stringent response depends on the concentration of the polyamine in the culture medium . The formation of guanosine 3'-diphosphate 5'-diphosphate elicited by amino acid starvation is stimulated at least 40-fold in putrescine-supplemented bacteria and only about 2-fold in putrescine-depleted cells. Proc Natl Acad Sci U S A, 1984 Apr, 81(8), 2295 - 7 Cloning and sequence analysis of cDNA for rat spleen thymosin beta 4; Wodnar-Filipowicz A et al.; Molecular cloning of a cDNA has established the sequence of the translated portion of the mRNA for rat spleen thymosin beta 4 . The presence of a methionyl initiator codon immediately preceding the codon for the first seryl residue of mature thymosin beta 4 is consistent with previous results indicating the absence of a signal peptide in the product translated in vitro from rat spleen mRNA . The cDNA sequence analysis also established the presence of two terminator codons immediately following the codon for the COOH-terminal seryl residue . Thymosin beta 4 is thus synthesized as a 5100-dalton peptide containing 44 amino acid residues . Removal of the initiator methionyl residue and acetylation of the NH2-terminal serine residue would yield mature thymosin beta 4 containing 43 amino acids . The absence of a signal peptide makes it unlikely that thymosin beta 4 is a secreted peptide. Proc Natl Acad Sci U S A, 1984 Apr, 81(7), 1966 - 70 A unique mechanism regulating gene expression: translational inhibition by a complementary RNA transcript (micRNA); Mizuno T et al.; The expression of the genes for the major outer membrane proteins OmpF and OmpC are osmoregulated . The ompC locus was found to be transcribed bidirectionally under conditions of high osmolarity and a 174-base transcript encoded upstream of ompC was found to inhibit the OmpF production and to substantially reduce the amount of the ompF mRNA . This RNA {mRNA-interfering complementary RNA (micRNA)} has a long sequence that is complementary to the 5' end region of the ompF mRNA . We propose that the micRNA inhibits the translation of the ompF mRNA by hybridizing with it . This RNA interaction may cause premature termination of the transcription of the ompF gene or destabilization of the ompF mRNA or both. J Bacteriol, 1984 Apr, 158(1), 69 - 72 Conversion of D-mannitol to D-ribose: a newly discovered pathway in Escherichia coli; Rosenberg H et al.; A mutant (mtlD) strain of Escherichia coli unable to oxidize mannitol-1-phosphate to fructose-6-phosphate was used to study the fate of mannitol-1-phosphate . D-{1-14C}mannitol entered the cells via the phosphotransferase system and was phosphorylated equally at carbon 1 or 6 . The label disappeared gradually from the mannitol-1-phosphate pool, and some 60% of the 14C was recovered in nucleic acids . Ribose was isolated from the purified RNA . The 14C label distribution in the isolated ribose precluded a simple hexose-to-pentose conversion by elimination of one terminal carbon from mannitol-1-phosphate . The 14C from mannitol-1-phosphate that did not enter macromolecules was found in CO2 and in some organic, non-phosphorylated compounds that were not identified . We suggest that the de novo synthesis of mannitol-1-phosphate in E . coli may be a reaction specifically dedicated to the biosynthesis of ribose. J Bacteriol, 1984 Apr, 158(1), 376 - 8 Mycoplasmas (Mollicutes) have a low number of rRNA genes; Amikam D et al.; DNA from Mycoplasma, Ureaplasma, Acholeplasma, and Spiroplasma species digested by restriction endonucleases was hybridized with probes consisting of portions of the rrnB rRNA operon of Escherichia coli and the rRNA operon of Mycoplasma capricolum . The results indicate the presence of only one or two sets of rRNA genes in the genome of Mollicutes linked in the procaryotic fashion, 16S-23S-5S. J Bacteriol, 1984 Apr, 158(1), 362 - 4 Genetic screen for cloned release factor genes; Weiss RB et al.; Nonsense suppression reflects competition between a nonsense suppressor tRNA and a translational release factor . This provides a simple way to screen for release factor genes cloned into a multicopy plasmid . We have confirmed that the expected competition occurs with the gene for release factor 2, cloned by C.T . Caskey et al . (C.T . Caskey, W.C . Forrester, W . Tate, and C.D . Ward, J . Bacteriol . 158:365-368, 1984), and used it to clone the gene for release factor 1. Arch Biochem Biophys, 1984 Apr, 230(1), 178 - 93 Relaxation time, interthiol distance, and mechanism of action of ribosomal protein S1; Odom OW et al.; The two sulfhydryl groups of ribosomal protein S1 from Escherichia coli have been labeled with fluorescent maleimides and the distance between them has been determined by nonradiative energy transfer . This distance was found to be approximately 27 A for both free S1 and S1 bound to 30 S subunits . This value probably represents an upper limit . The position of the fluorescence emission maximum indicates that both sulfhydryl groups are in a relatively hydrophobic environment . When poly(U) is added to labeled S1, either free or in 30 S subunits, the emission maximum shifts to the red by about 3 nm but without a detectable change in the interthiol distance . S1 labeled at one or both of its sulfhydryl groups retains most of its ability to enhance poly(U)-directed polyphenylalanine synthesis . About the same concentration of poly(U) is required to give the maximum shift in fluorescence as is required to give maximum polyphenylalanine synthesis, indicating that S1 binds poly(U) during translation . The peptide initiation inhibitor aurintricarboxylic acid almost completely quenches the fluorescence from either labeled sulfhydryl groups in S1 bound to ribosomes or free in solution . This quenching probably is due to energy transfer from the labeled sulfhydryls to bound aurintricarboxylic acid . Fluorescence anisotropy measurements indicated that the C-terminal domain of S1 is relatively rigid, but retains some independent movement when attached to ribosomes . The overall data are consistent with a model in which a region near the two sulfhydryl groups in the elongated C-terminal domain functions to sequester and bind mRNA to the ribosome during peptide synthesis. Proc Soc Exp Biol Med, 1984 Apr, 175(4), 458 - 67 Surface phenotype of LPS-binding murine lymphocytes; Jacobs DM et al.; We have characterized LPS+ murine lymphocytes by determining their surface phenotype using double-labeling immunofluorescence . In the spleen, 40% of cells were LPS+mu+ (B cells), 5% were LPS+Thy 1.2+ (T cells), and 5% were LPS+ cells bearing neither mu nor Thy 1.2 (null cells) . Peripheral lymph nodes contained 11% LPS+ B cells and 2% LPS+ T cells, and mesenteric lymph nodes contained 15% LPS+ B cells and 3% LPS+ T cells . In contrast, the 4% LPS+ cells in Peyer's patches were all B cells, and the thymus contained 4% LPS+ T cells . Only the spleen contained LPS+ null cells . Within each lymphoid organ examined, the fraction of total lymphocytes identified as LPS+mu+, LPS+Ia+, or LPS+ delta + was similar, indicating that LPS+ B cells possessed the surface phenotype mu+Ia+ delta +, characteristic of a mature B cell . This conclusion was supported by the absence of LPS+mu+Ia+ delta + cells in newborn spleens . The fraction of mu+Ia+ delta + cells which also binds LPS was highest in spleen and lowest in Peyer's patches . Assuming that cells are, on the average, more mature in the progression from spleen to lymph nodes to Peyer's patches, it would appear that LPS+ cells are a less mature fraction of the mu+Ia+ delta + pool, distinguished by the presence of an LPS binding site or receptor . These data illustrate selective binding of LPS predominantly to mature B cells, but also to small numbers of null cells and T cells . The relationship of this binding to cell activation is discussed by considering the characteristics of cells which can be activated by LPS to clonal growth or differentiation under appropriate conditions. J Pediatr, 1984 Apr, 104(4), 564 - 8 Appearance of secretory IgM and IgA antibodies to Escherichia coli in saliva during early infancy and childhood; Mellander L et al.; Unstimulated whole saliva was collected from 203 uninfected individuals at various ages from birth until adulthood . Levels of specific antibodies against Escherichia coli O antigens of secretory IgA, secretory IgM and IgG, as well as total amounts of SIgA, were determined using ELISA . Levels of SIgA antibodies found in adults were approached by the age of 12 months, but high levels could be attained earlier, presumably in response to antigenic exposure at the mucosal level . During the first few months of life, secretory IgM antibodies appeared in the saliva, possibly compensating for the relative lack of IgA. Exp Cell Res, 1984 Apr, 151(2), 314 - 21 Cyclosporin A is a differential inhibitor of eukaryotic RNA polymerases; Brack C et al.; In order to investigate the molecular mechanism of biological action of the drug cyclosporin A (CsA), we have studied its effect on eukaryotic RNA polymerase activities in two systems: (a) in vitro transcription; (b) injection into frog oocytes . The electron microscopic transcription R-loop method {1} has been used to assay for specific transcription of an immunoglobulin gene clone in HeLa cell lysates . In this system, CsA inhibits efficiently the transcription of immunoglobulin genes . Dilution experiments show that RNA polymerase II activity is inhibited by nanomolar concentrations (i.e . amounts comparable to the ones needed for the inhibition of lymphocyte stimulation), whereas polymerase III is inhibited at micromolar concentrations . In vitro transcription with E . coli RNA polymerase is not inhibited . Parallel experiments with the mushroom toxin alpha-amanitin have shown that the eukaryotic RNA polymerase II displays similar sensitivity towards the two cyclic peptides CsA and alpha-amanitin in vitro . When CsA is injected into frog oocytes, together with a cloned Xenopus laevis U1 small nuclear RNA gene, transcription of this gene by endogenous RNA polymerase II is inhibited by 60% . RNA polymerase III and RNA polymerase I transcription is not inhibited in the oocyte . Various possibilities for the selective action of CsA on stimulated lymphocytes are discussed. Cell, 1984 Apr, 36(4), 1089 - 95 Dispensable pieces of an aminoacyl tRNA synthetase which activate the catalytic site; Jasin M et al.; Recent data suggest that size polymorphism of aminoacyl tRNA synthetase is due to variable fusions of additional functional domains to a catalytic core so that, in a large synthetase, a substantial part of the polypeptide is dispensable for catalytic activity . We demonstrate here that a dispensable domain, joined to the catalytic core of a large synthetase, can activate the catalytic sites . This is shown by complementation of an activity-deficient mutant enzyme by protein fragments that contain internal deletions within the catalytic domain and are themselves devoid of activity . The complementation is dependent upon the presence of a defined segment of polypeptide that is remote in the sequence from the catalytic core . Substantial coupling has been established between dispensable and indispensable component pieces . This could be a mechanism to build efficiently large enzymes which integrate the catalytic sites with other previously shown functional roles. J Gen Microbiol, 1984 Apr, 130 ( Pt 4), 951 - 7 F and type 1 piliation of Escherichia coli; Biebricher CK et al.; F and type 1 piliation of various Escherichia coli K12 Hfr and F+ strains was re-examined by using a new visualization assay . In anaerobic cultures in rich media, bacteria were well F piliated throughout all growth phases . In aerobic cultures in rich media, F piliation reached a maximum in the mid-exponential phase . The yield of pili was up to 15 mg 1(-1) . In aerated cultures, F pili disappeared in the late exponential phase . In rich media F pili were on average 10-20 micron long; in synthetic media they were an order of magnitude shorter, and less numerous . Addition of metabolic poisons (NaCN, NaN3, Na3AsO4, phenylethyl alcohol) and starving the bacteria caused rapid disappearance of F pili under aerobic conditions, but had no influence on F piliation under anaerobic conditions . Type 1 piliation was not influenced by these drugs or by an alteration of growth conditions. J Gen Microbiol, 1984 Apr, 130 ( Pt 4), 941 - 9 Light-microscopic visualization of F and type 1 pili; Biebricher CK et al.; Methods for the direct visualization of F and type 1 pili of Escherichia coli in the light microscope are described . The method for visualizing F pili is based on the specific adsorption of fluorescent dye-labelled RNA phages to F pili . The best results were obtained with MS2 phages labelled with rhodamine B . Semi-quantitative determination of the amount of F pili is possible . Type 1 pili can be visualized rapidly and specifically by indirect immunofluorescence . Other structures on the cell surface are neither detected by, nor interfere with these assays . By using different fluorescent dyes the two methods can be combined and both F and type 1 pili can be determined in the same sample. Proc Natl Acad Sci U S A, 1984 Apr, 81(7), 2011 - 5 Oxidative inactivation of glutamine synthetase subunits; Nakamura K et al.; Escherichia coli glutamine synthetase (GS) was inactivated by a nonenzymic mixed-function oxidation system composed of ascorbate, O2, and Fe(III) . Partial inactivation of GS by this system leads to the formation of hybrid GS molecules (dodecamers) composed of both active and inactive subunits . Subunit interactions in these hybrid molecules are weaker than in the native enzyme, as indicated by the kinetics of subunit dissociation in the presence of 4 M urea . Heterologous subunit interactions in these hybrid molecules do not affect the affinity of active subunits for glutamate . Incubation of partially adenylylated GS preparations (n = 6.7) with the ascorbate system in the absence of substrates leads to preferential oxidative inactivation of unadenylylated subunits, whereas incubation in the presence of ATP and glutamate leads to preferential inactivation of adenylylated subunits. J Clin Microbiol, 1984 Apr, 19(4), 498 - 501 Monoclonal antibody enzyme-linked immunosorbent assay for identification of K99-positive Escherichia coli isolates from calves; Mills KW et al.; For Escherichia coli to produce diarrhea in animals it must possess the ability to attach to the epithelial cells of the intestine and to produce enterotoxins . Tests developed to differentiate pathogenic from nonpathogenic E . coli have relied on detection of adherence structures called pili or detection of the toxins . We utilized a monoclonal antibody to K99 pili in an enzyme-linked immunosorbent assay to detect the presence of K99 pili in E . coli isolated from calves . Twenty-three E . coli isolates that were known to be stable toxin positive were all shown to produce K99 pili . A 100% correlation also was shown between the presence of K99 antigen and production of stable toxin by E . coli isolates . Of the 251 isolates, 245 were negative by K99 enzyme-linked immunosorbent assay and stable toxin assay . The other six were positive on both tests . The enzyme-linked immunosorbent assay also was shown to be specific for K99 pili by antibody-blocking assays . The number of E . coli necessary for detecting K99 pili by enzyme-linked immunosorbent assay was determined to be 3.5 X 10(5) bacteria per ml. J Med Microbiol, 1984 Apr, 17(2), 141 - 50 The role of different K antigens of Escherichia coli in phagocytosis by polymorphonuclear leukocytes; Verweij-Van Vught AM et al.; The importance of K antigens from six strains of Escherichia coli for the interaction with polymorphonuclear leukocytes (PMNL) was studied . The major factor influencing this interaction was the ability of strains to activate complement by the classical route during opsonisation, this process being reduced for most K-positive strains . Interference of K antigens with the functioning of common pili as adhesions of eukaryotic cells was not observed nor a toxic effect of K antigens on PMNL. J Med Chem, 1984 Apr, 27(4), 429 - 32 Inhibitors of inosinic acid dehydrogenase . 2-Substituted inosinic acids; Wong CG et al.; A series of 2-substituted inosine monophosphate (IMP) and inosine derivatives were synthesized and tested for inhibitory activity against IMP dehydrogenase from Escherichia coli . All of the IMP analogues that possessed electron-withdrawing substituents on the phenyl ring of a benzylthio group placed at the 2-position of IMP showed strong inhibition, which was competitive with IMP . No evidence of hydrophobic interactions of the 2-substituent with the enzyme was observed. Bioorg Khim, 1984 Apr, 10(4), 567 - 9 {The nature of a covalent bond between the relaxation protein and ColE1-DNA in the relaxation complex}; Zhuklis KL et al.; O-{32P}phosphoserine was found to be the only phosphoamino acid in the acid hydrolysate of the {32P}ColE1 DNA-peptide produced by action of proteases on the ColE1 DNA relaxation complex . This finding suggests that the relaxation protein is bound to ColE1 DNA in the relaxation complex via a phosphodiester linkage between a serine hydroxyl of the protein and the 5'-phosphate of the terminal deoxycytidine residue of the DNA. J Biochem (Tokyo), 1984 Apr, 95(4), 909 - 16 The primary structure of phosphoenolpyruvate carboxylase of Escherichia coli . Nucleotide sequence of the ppc gene and deduced amino acid sequence; Fujita N et al.; The nucleotide sequence of the ppc gene, the structural gene for phosphoenolpyruvate carboxylase {EC 4.1.1.31}, of Escherichia coli K-12 was determined . The gene codes for a polypeptide comprising 883 amino acid residues with a calculated molecular weight of 99,061 . The amino acid sequence deduced from the nucleotide sequence was entirely consistent with the protein chemical data obtained with the purified enzyme, including the NH2- and COOH-terminal sequences and amino acid composition . The coding region is preceded by two putative ribosome binding sites, and is followed closely by a good representative of rho-independent terminator . The codon usage in the ppc gene suggests a moderate expression of the gene . The secondary structure of the enzyme was predicted from the deduced amino acid sequence. Lancet, 1984 Mar 31, 1(8379), 698 - 702 Human recombinant interleukin-2 partly reconstitutes deficient in-vitro immune responses of lymphocytes from patients with AIDS; Lifson JD et al.; The lymphokine interleukin-2 is required for the development of various cell-mediated immune functions that are known to be deficient in patients with acquired immunodeficiency syndrome (AIDS) . The effects of pure human recombinant interleukin-2 (rIL-2), produced by Escherichia coli containing the cloned human gene, on in-vitro immune responses were studied in 16 patients with AIDS and 10 age-matched healthy heterosexual men . Exposure of lymphocytes from most AIDS patients to 1-100 U/ml rIL-2, increased mitogen and alloantigen induced proliferation and augmented natural killer (NK) cell function in a dose-dependent manner . NK activity was the function most consistently improved, with deficient patient responses uniformly restored to normal after incubation of effector cells with rIL-2 . Patient responsiveness to rIL-2 did not appear to depend upon the primary manifestation of disease (opportunistic infection, Kaposi's sarcoma, or both) or other clinical variables . rIL-2 also augmented the responses of lymphocytes from health subjects, but to a lesser degree . Pure rIL-2 seems capable of at least partly reconstituting some in-vitro immunological defects characteristic of AIDS . The availability of highly purified rIL-2 makes in-vivo testing feasible. Biochem Biophys Res Commun, 1984 Mar 30, 119(3), 926 - 32 Trp holorepressor-trp operator interaction studied by protein distribution analysis; Haydock PV et al.; Trp repressor protein of Escherichia coli (Mr 24,700) undergoes a conformational change upon interaction with L-tryptophan that enables the resulting binary complex to bind with high specificity to several operator targets in double-stranded DNA . By protein distribution analysis it was shown that a significant fraction of Trp repressor is inert in operator binding . The equilibrium dissociation constant for Trp holorepressor-Trp operator interaction is 6.7 nM at 20 degrees in 0.05M NaCl, pH 7.4 . The Trp holorepressor-trp operator complex consists of one molecule of each of the participating species, even at high molar ratios of protein to DNA. Biochem Biophys Res Commun, 1984 Mar 30, 119(3), 860 - 7 Site-directed mutagenesis of cys148 in the lac carrier protein of Escherichia coli; Trumble WR et al.; The lac y gene of Escherichia coli which encodes the lac carrier protein has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys148 is converted to a glycine residue . Cells bearing the mutated lac y gene exhibit initial rates of lactose transport that are about 4-fold lower than cells bearing the wild type gene on a recombinant plasmid . Furthermore, transport activity is less sensitive to inactivation by N-ethylmaleimide, and strikingly, galactosyl 1-thio-beta-D-galactopyranoside affords no protection against inactivation . The findings suggest that although cys148 is essential for substrate protection against sulfhydryl inactivation, it is not obligatory for lactose: proton symport and that another sulfhydryl group elsewhere within the lac carrier protein may be required for full activity. Science, 1984 Mar 30, 223(4643), 1412 - 4 Biological activity of recombinant human interleukin-2 produced in Escherichia coli; Rosenberg SA et al.; The gene for interleukin-2 was isolated from the Jurkat cell line and from normal peripheral blood lymphocytes and, when inserted in Escherichia coli, was expressed at high concentrations . This interleukin-2 was purified to apparent homogeneity and tested for biological activity in a variety of assays in vitro and in vivo . The recombinant lymphokine supports the growth of murine and human interleukin-2 dependent cell lines, enhances the generation of murine and human cytolytic cells in vitro, and generates lymphokine activated killer cells from murine and human lymphocytes . It has a serum half-life of 2 to 3 minutes in the mouse and significantly enhances the generation of cytolytic cells in vivo after alloimmunization . No functional differences between native and the recombinant interleukin-2 molecules have been detected. Biochem Biophys Res Commun, 1984 Mar 30, 119(3), 1103 - 8 Inhibition of Escherichia coli fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate; Marcus F et al.; Fructose 2,6-bisphosphate, a potent inhibitor of fructose-1,6-bisphosphatases, was found to be an inhibitor of the Escherichia coli enzyme . The substrate saturation curves in the presence of inhibitor were sigmoidal and the inhibition was much stronger at low than at high substrate concentrations . At a substrate concentration of 20 microM, 50% inhibition was observed at 4.8 microM fructose 2,6-bisphosphate . Escherichia coli fructose-1,6-bisphosphatase was inhibited by AMP (Ki = 16 microM) and phosphoenolpyruvate caused release of AMP inhibition . However, neither AMP inhibition nor its release by phosphoenolpyruvate was affected by the presence of fructose 2,6-bisphosphate . The results obtained, together with previous observations, provide further evidence for the fructose 2,6-bisphosphate - fructose-1,6-bisphosphatase active site interaction. Biochemistry, 1984 Mar 27, 23(7), 1468 - 73 T4 RNA ligase mediated preparation of novel "chemically misacylated" tRNAPheS; Heckler TG et al.; T4 RNA ligase was employed for the condensation of Escherichia coli tRNAPhe missing cytidine-75 and adenosine-76 (tRNAPhe-COH; the acceptor "oligomer") with each of several chemically acylated derivatives of pCpA (the donor "oligomer") . The resulting "chemically misacylated " tRNAPheS were obtained in 20-65% yields following chromatographic workup on DEAE-cellulose and benzoylated DEAE-cellulose . Characterization of the chemically misacylated tRNAs was accomplished by (i) enzymatic reaminoacylation of chemically misacylated tRNAPhe with phenylalanine by E . coli phenylalanyl-tRNA synthetase following chemical deacylation of the "incorrect" amino acid, (ii) comparison of the hydrolytic effects of Cu2+ solutions on chemically and enzymatically prepared samples of N-acetyl-L-phenylalanyl- tRNAPheS , and (iii) measurement of the chromatographic behavior of the tRNA species derived from chemical misacylation . Biochemistry, 1984 Mar 27, 23(7), 1426 - 32 Defective proton ATPase of uncA mutants of Escherichia coli . 5'-Adenylyl imidodiphosphate binding and ATP hydrolysis; Wise JG et al.; The Escherichia coli uncA gene codes for the alpha-subunit of the F1 sector of the membrane proton ATPase . In this work purified soluble F1 enzymes from three mutant strains ( uncA401 , uncA447 , and uncA453 ) have been compared to F1 from a normal strain in respect to (a) binding of 5'-adenylyl imidodiphosphate (AMPPNP) to native enzyme in both the presence and absence of Mg, (b) high-affinity binding of MgATP to native enzyme, (c) total reloading of MgAMPPNP to nucleotide-depleted F1 preparations, (d, e) ability to hydrolyze MgATP at both high MgATP concentrations (d) (steady-state conditions) and low MgATP concentrations (e) where substrate hydrolysis occurs under nonsteady-state (" unisite ") conditions, and (f) sensitivity of steady-state ATPase activities to inhibitors of normal F1-ATPase activity . uncA mutant F1 showed normal stoichiometry of MgAMPPNP binding to both native (three sites per F1) and nucleotide-depleted preparations (six sites per F1) . Native uncA F1 preparations showed lower-than-normal affinity for MgAMPPNP and MgATP at the first site filled . Binding of AMPPNP in the absence of Mg was similar to normal, except that no increase in affinity for AMPPNP was induced by aurovertin . The uncA F1-ATPases had low but real steady-state rates of ATP hydrolysis, which were inhibited by aurovertin but relatively insensitive to inhibition by AMPPNP, efrapeptin, and sodium azide . Non-steady-state ( unisite ) ATP hydrolysis rates catalyzed at low substrate concentrations by uncA F1-ATPases were similar to normal.(ABSTRACT TRUNCATED AT 250 WORDS) Nucleic Acids Res, 1984 Mar 26, 12(6), 2745 - 58 Binding of Xenopus transcription factor A to 5S RNA and to single stranded DNA; Hanas JS et al.; Footprint competition assays are utilized to study the binding of Xenopus transcription factor A to a variety of single-stranded nucleic acids . The addition of Xenopus oocyte, yeast, or wheat germ 5S RNA as footprint competitors reveals that factor A binds these 5S RNAs with similar affinity . In contrast, factor A does not bind to E.coli 5S RNA or wheat germ tRNA in this assay . Factor A binding to single stranded DNA is also examined using footprint competition . Factor A binds preferentially to non-specific single stranded (M13) DNA versus double stranded (pBR322) DNA . Factor A binds equally well to single stranded DNA fragments containing either the coding or non-coding strands of the 5S RNA gene . Using single stranded M13 DNA as a competitor, the factor A-5S RNA gene complex is found to dissociate with a half-life of 5-6 min. Nucleic Acids Res, 1984 Mar 26, 12(6), 2641 - 8 DNA replication regulated by the priming promoter; Panayotatos N; ColE1 derivative plasmids were constructed in which the natural promoter that primes replication or, in addition, the region coding for the RNA I control element had been deleted . In all of these molecules priming of the origin was effected by read-through transcription from constitutive or inducible (lacUV5) promoters inserted farther upstream . In the latter case, regulation of lac repressor activity with IPTG resulted in controlled plasmid levels in vivo . These results indicated that, at least in the absence of other control elements, regulation of the priming promoter was sufficient to control DNA replication and determine plasmid copy number. Nucleic Acids Res, 1984 Mar 26, 12(6), 2969 - 85 The nucleotide and deduced amino acid sequences of the encephalomyocarditis viral polyprotein coding region; Palmenberg AC et al.; The nucleotide sequence of 7200 bases of encephalomyocarditis (EMC) viral RNA, including the complete polyprotein-coding region, was determined . The polyprotein is encoded within a unique translational reading frame, 6870 bases in length . Protein synthesis begins with the sequence Met-Ala-Thr, and ends with the sequence Leu-Phe-Trp, 126 bases from the 3' end of the RNA . Viral capsid and noncapsid proteins were aligned with the deduced amino acid sequence of the polyprotein . The proteolytic processing map follows the standard 4-3-4 picornaviral pattern except for a short leader peptide (8 kd), which precedes the capsid proteins . Identification of the proteolytic cleavage sites showed that EMC viral protease, p22, has cleavage specificity for gln-gly or gln-ser sequences with adjacent proline residues . The cleavage specificity of the host-coded protease(s) includes both tyr-pro and gln-gly sequences. Nucleic Acids Res, 1984 Mar 26, 12(6), 2901 - 16 An enhancer sequence from bovine papilloma virus DNA consists of two essential regions; Weiher H et al.; A comprehensive structural analysis of an enhancer sequence from bovine papilloma virus DNA is presented based on the construction and functional analysis of 20 mutant derivatives . The results, obtained in CV-1 tissue culture cells, show that this enhancer is a small genetic element--only 40 bp in length--that contains two essential regions . The two regions exhibit homology to each other and to DNA fragments from other viral genomes that also act as enhancers in the assay used . However, there is a certain latitude in the sequences that have enhancer activity in CV-1 cells, even within the critical regions . The results are discussed with respect to the model that enhancers are binding sites for tissue specific transcription factors . The formation of Z-DNA might be involved in the enhancement process . However, single base pair transitions in an 8 base pair stretch of alternating purines and pyrimidines within the BPV enhancer which conserve this pattern destroy enhancer function. Nucleic Acids Res, 1984 Mar 26, 12(6), 2851 - 9 The genes for the ribosomal proteins S12 and S7 are clustered with the gene for the EF-Tu protein on the chloroplast genome of Euglena gracilis; Montandon PE et al.; We characterize a DNA segment of the Euglena gracilis chloroplast DNA fragment Eco . N by nucleotide sequencing and S1 nuclease analysis . We show that this region, which is upstream of the previously sequenced tuf A gene, contains the genes for the ribosomal proteins S12 and S7 . The gene arrangement is 5'-rps 12-80 bp spacer-rps 7-174 bp spacer-tuf A, somewhat similar to the str operon of E . coli . The chloroplast S12 and S7 proteins contain 124 and 155 aminoacids, respectively, and are to 68% and 38% homologous with the corresponding E . coli proteins . The region is transcribed into a distronic mRNA of about 1.1 to 1.2 kb . The rps 12 and rps 7 genes, contrary to the tuf A gene, are not split. J Mol Biol, 1984 Mar 25, 174(1), 113 - 20 Identification of a development-specific promoter of Myxococcus xanthus; Inouye S; In the chromosome of Myxococcus xanthus, two homologous genes for protein S, a development-specific protein, are tandemly repeated with a 1.4 X 10(3) base-pair sequence between the two genes . Two synthetic oligodeoxyribonucleotides were used as specific probes for individual transcripts from the upstream gene 1 and the downstream gene 2, respectively . The gene 2 transcript was detected only during developmental growth, while the gene 1 transcript was not detected during developmental or vegetative growth . The major initiation site for the gene 2 transcription was determined to be 51 bases upstream of the initiation codon for gene 2 . The development-specific promoter of gene 2 was identified; it shows some homologies to the Escherichia coli promoter structures. J Biol Chem, 1984 Mar 25, 259(6), 3825 - 30 Prolipoprotein modification and processing enzymes in Escherichia coli; Tokunaga M et al.; Prolipoprotein signal peptidase, a unique endopeptidase which recognizes glycyl glyceride cysteine as a cleavage site, was characterized in an in vitro assay system using purified prolipoprotein as the substrate . This enzyme did not require phospholipids for its catalytic activity and was found to be localized in the inner cytoplasmic membrane of the Escherichia coli cell envelope . Globomycin inhibited this enzyme activity in vitro with a half-maximal inhibiting concentration of 0.76 nM . Nonionic detergent, such as Nikkol or Triton X-100, was required for the in vitro activity . The optimum pH and reaction temperature of prolipoprotein signal peptidase were pH 7.9 and 37-45 degrees C, respectively . Phosphatidylglycerol:prolipoprotein glyceryl transferase (glyceryl transferase) activity was measured using {2-3H}glycerol-labeled JE5505 cell envelope and {35S}cysteine-labeled MM18 cell envelope as the donor and acceptor of glyceryl moiety, respectively . 3H and 35S dual-labeled glyceryl cysteine was identified in the product of this enzymatic reaction . The optimal pH and reaction temperature for glyceryl transferase were pH 7.8 and 37 degrees C, respectively. J Biol Chem, 1984 Mar 25, 259(6), 3729 - 33 Effects of mutations at glycine residues in the hydrophobic region of the Escherichia coli prolipoprotein signal peptide on the secretion across the membrane; Inouye S et al.; Each of the 2 glycine residues in the hydrophobic region of the prolipoprotein signal peptide of Escherichia coli was systematically deleted or substituted with a valine residue by oligonucleotide-directed site-specific mutagenesis . Functional analysis of four such mutants as well as four double mutants, resulting from combinations of any two of the single mutations, revealed that (a) glycine residues at positions 9 and 14 could be replaced individually or at the same time with a valine residue without affecting the secretion of prolipoprotein; (b) the deletion of glycine at position 9 had no effect on the secretion of prolipoprotein whereas, when glycine at position 14 was deleted, the glyceride modification and the processing of the mutant prolipoprotein occurred at a much slower rate at 42 degrees C than those of the wild type prolipoprotein; and (c) the effects of deleting glycine at position 14 could be suppressed by the deletion of glycine at position 9, which resulted in shortening the hydrophobic region of the prolipoprotein signal peptide by 2 amino acid residues . These results indicate that the hydrophobic region of the prolipoprotein signal peptide has remarkable flexibility in terms of the relationship between its primary structure and function in protein secretion. J Biol Chem, 1984 Mar 25, 259(6), 3625 - 32 Differential scanning calorimetry of Cd(II) alkaline phosphatases; Roberts CH et al.; Differential scanning calorimetry has been employed to monitor structural alterations induced in the dimeric enzyme alkaline phosphatase on binding of Cd(II) (to the metal-free apoenzyme) and phosphate (Pi) (to the Cd(II) enzyme) . Cd(II) addition to the apoenzyme at pH 6.5 results in an increased transition temperature, suggesting a stabilizing effect of the bound metal ion . Two distinct structural forms of the protein are detected as discrete calorimetric transitions (Tm = 69-84 degrees C; 87-94 degrees C, respectively) . Distribution of the enzyme between these forms is found to depend on the exogenous Cd(II) concentration and the protocol of Cd(II) addition . These results indicate that conversion between the conformational forms is a slow process which appears to require specific levels of metal ion site occupancy . These studies, in which the exogenous Cd(II) concentration was varied from 10(-5) M to 10(-3) M suggest a structural basis for previously observed hysteretic phenomena observed on Cd(II) binding to the enzyme . Even at a minimum stoichiometry of Cd(II) (2 eq/mol of dimer) a single equivalent of Pi is sufficient to accelerate assumption of a stabilized form of the protein (Tm = 90 degrees C) . This is followed by a slow structural change paralleling the time course of formation of the functional 2 Cd(II) phosphoryl enzyme which displays two calorimetric transitions (Tm = 65 degrees C, 88 degrees C) . The low temperature transition does not appear if Pi is initially present at millimolar concentrations and is abolished on addition of Pi at concentrations in excess of 0.1 mM . These observations suggest the presence of a second, distinct Pi binding site on the 2 Cd(II) phosphoryl enzyme . This is supported by the changes observed in the 31P NMR chemical shift of Pi added to comparable enzyme samples . These data, including assessment of the effect of the presence of Mg(II), are discussed in terms of the mechanism of metal ion association to the enzyme and rearrangement of bound metal ions induced by Pi binding. Science, 1984 Mar 23, 223(4642), 1299 - 301 Total synthesis and cloning of a gene coding for the ribonuclease S protein; Nambiar KP et al.; A gene for ribonuclease S protein, has been chemically synthesized and cloned . The gene is designed to have 25 specific restriction endonuclease sites spaced at short intervals, permitting its structure to be rapidly modified . This flexibility facilitates tests of hypotheses relating the primary structure of the enzyme to its physical and catalytic behavior. Med J Aust, 1984 Mar 17, 140(6), 332 - 6 Acute obstructive suppurative cholangitis; Pillay SP et al.; Acute obstructive suppurative cholangitis is an uncommon condition which, if unrecognized, carries a high rate of mortality . This study of 12 patients highlights the clinical features as well as the poor outcome of delayed surgical decompression . Endoscopic and percutaneous drainage of the obstructed biliary tree in acutely ill patients may improve the results of subsequent surgery. Carbohydr Res, 1984 Mar 15, 126(2), 249 - 59 Structural studies of the O-specific side chain of the lipopolysaccharide from Escherichia coli O:7; L'vov VL et al.; The structure of the O-specific side-chain of the lipopolysaccharide from Escherichia coli O:7 has been investigated, using n.m.r . spectroscopy, methylation analysis, partial hydrolysis, and Smith degradation as the principal methods . It is concluded that the polysaccharide is constructed of repeating pentasaccharide units having the structure (formula; see text) where D-QuipNAc stands for 4-acetamido-4,6-dideoxy-D-glucopyranose . The 13C-n.m.r . spectrum of the polysaccharide has been interpreted completely. J Mol Biol, 1984 Mar 15, 173(4), 437 - 61 Multiple IS10 rearrangements in Escherichia coli; Raleigh EA et al.; We have investigated the occurrence of multiple transposon-promoted chromosomal rearrangements in Escherichia coli K12 strains containing transposon Tn10 . We show that a single Tn10 element, with its two closely spaced insertion sequence (IS10) elements, frequently gives rise to complex rearrangements that can be accounted for as the sum of two "classical" IS10 events . Using a strain containing differentially marked Tn10 elements at widely separated locations, we have investigated the possibility that IS10-promoted rearrangements occur in cell-wide "bursts", as expected if cells could occasionally undergo brief periods when all IS10 transposition events were activated, interspersed with longer periods of relative quiescence . We find no evidence for strong (greater than 60-fold), periodic cell-wide activation under our experimental conditions . The sensitivity of this experiment has been evaluated using an expression for the accumulation of double mutations in populations with heterogeneous, fluctuating mutation rates (see Appendix) . We discuss several mechanisms by which two closely linked IS10 elements could undergo coupled double events without cell-wide activation: local activation of small chromosomal regions, periodic bursts of synthesis of cis-acting transposase protein, and/or a propensity for elements that have actually engaged in one rearrangement event to initiate a second successive event immediately thereafter . We favor the last possibility. Eur J Biochem, 1984 Mar 15, 139(3), 541 - 5 Involvement of the 3' side of the anticodon loop of yeast tRNATyr in messenger-free binding to ribosomes . An electron-spin resonance study; Weygand-Durasevic I et al.; Electron-spin resonance (ESR) spectra of a nitroxide spin-label attached to residue i6A-37 of yeast tRNATyr were measured in complexes of deacylated tRNATyr with Escherichia coli ribosomes . A Scatchard plot, obtained in the absence of mRNA, indicated strong binding with an association constant of 1 X 10(7) l X mol-1, suggesting the P-site binding . The ESR spectrum of free tRNATyr, characteristic for a rapidly tumbling nitroxide, changes to a spectrum with extensively broadened lines in the ribosome-tRNA complex . The original spectrum can be restored upon long incubations of the complex with an excess of extraneous tRNA . ESR spectra suggest that the spin-label motion is drastically perturbed though not completely blocked in the ribosome-tRNATyr complex . Since ESR spectra of a spin-label attached to the opposite, i.e . 5', side of the anticodon loop are only slightly perturbed by the messenger-free binding to ribosomes {Rodriguez et al . (1980) J . Biol . Chem . 255, 8116-8120}, it is concluded that the two sides of the anticodon loop face entirely different environments when bound to the P site, the 3' side being oriented towards the surface of the ribosome, and the other side towards its environment or a large cavity. Eur J Biochem, 1984 Mar 15, 139(3), 547 - 52 Metabolism of tRNA in near-ultraviolet-illuminated Escherichia coli . The tRNA repair hypothesis; Blanchetot A et al.; The relA+-dependent stringent response is an important component of the mechanism of the near-ultraviolet-induced growth delay . However, the behaviour of the intracellular level of ppGpp is unexpected {Thomas et al . (1981) Eur . J . Biochem . 118, 381-387} and this led us to examine the metabolism of tRNAs during the illumination period and the growth lag that follows . Analysis of the gel electrophoresis migration profiles of tRNA molecules, synthesized prior to the illumination period, provides no evidence for tRNA degradation . Rather, it is suggestive of the rearrangement of some cross-linked tRNA species during the growth lag . By the same technique the neosynthesis of one or several tRNA species escaping the stringent response could be ruled out at the beginning of the growth lag . The behaviour of the cross-linked tRNAs was followed by a chromatographic procedure allowing the quantitative evaluation of the 8-13 link present in vivo . Upon illumination of growing cells, one observes an initial linear increase of the 8-13 link content . Unexpectedly this is followed during the illumination period by an abrupt decrease . The 8-13 link content then remains stable . The data above suggest that part of the 8-13 link (25-40%) is eliminated from tRNA without degradation of the molecules involved . A tRNA repair hypothesis is proposed: elimination of the 8-13 link would occur by scission of the N1-C1' glycosidic bonds at positions 8 and 13 of tRNA . It would be followed by reinsertion of uracil and cytosine in their respective positions. Biochemistry, 1984 Mar 13, 23(6), 1269 - 74 Inhibition of pyruvate dehydrogenase multienzyme complex from Escherichia coli with a radiolabeled bifunctional arsenoxide: evidence for an essential histidine residue at the active site of lipoamide dehydrogenase; Adamson SR et al.; Incubation of pyruvate dehydrogenase multienzyme complex (PD complex) from Escherichia coli with thiamin pyrophosphate, pyruvate, coenzyme A, Mg2+, and the radiolabeled bifunctional arsenoxide p-{(bromoacetyl)-amino}phenyl arsenoxide (BrCH214CONHPhAsO) led to the irreversible loss of lipoamide dehydrogenase (E3) activity . The mode of inactivation occurred by initial "anchoring" of the reagent via its -AsO group to reduced lipoyl residues on lipoate acetyltransferase (E2) (generated by substrates) followed by the delivery of the BrCH214CO- moiety into the active site of E3 where an irreversible alkylation ensued {Stevenson, K . J., Hale, G., & Perham, R . N . (1978) Biochemistry 17, 2189} . To account for nonspecific alkylations, not mediated by this delivery process, control experiments were conducted in which the radiolabeled bifunctional reagent was incubated with PD complex in the absence of substrates . E3 subunits were isolated from inhibited and control PD complexes by chromatography on hydroxylapatite in the presence of 8 M urea . Acid hydrolysis of the alkylated E3 and control E3 samples produced radiolabeled carboxymethylated amino acids that were identified and quantitated by high-voltage electrophoresis and amino acid/radiochemical analysis . The inhibited sample contained N3-(carboxymethyl)histidine and a small amount of S-(carboxymethyl)cysteine . These residues were not present in significant amounts in the controls . The loss of 81% of E3 activity correlated with the alkylation of about 0.7 residue of histidine and 0.1 residue of cysteine per mol of E3. Biochemistry, 1984 Mar 13, 23(6), 1047 - 51 Function of the repeating homologous sequences in nucleic acid binding domain of ribosomal protein S1; Suryanarayana T et al.; Ribosomal protein S1 contains in its RNA binding domain four repeating, homologous stretches of sequences . Its functionally active mutant form m1-S1 {Subramanian, A.R., & Mizushima, S . (1979) J . Biol . Chem . 254, 4309} contains only three repeating stretches . In order to assess the functional importance of this repeating sequence, we cleaved S1 at its reactive SH group on Cys-349 and isolated a fragment (S1-F4) that has lost two of the homologous stretches but retains all other essential elements . We find that ribosomes reconstituted with S1-F4 instead of S1 are functionally active in translating poly(U) and poly(A) but totally inactive in translating phage MS2 RNA . The significance of this result is discussed vis-a-vis the initiation step in translating natural mRNA, and a functional role for the tetrarepeat of S1 is suggested. Nucleic Acids Res, 1984 Mar 12, 12(5), 2243 - 58 The effect of nucleotide analogs on cell-free gene expression; Zimmer M et al.; The effects of the following pyrimidine nucleoside 5'-triphosphates: f5 UTP, br5 UTP, rTTP, s2 UTP, s4 UTP and s2 CTP on cell-free expression of the beta-galactosidase gene in lambda h80dlac DNA as well as the galactokinase gene in plasmid 01-14 were investigated . Only rTTP could substitute UTP in cell-free gene expression without restriction . Combinations of the other analogs with their respective natural congeners led to inhibition of gene expression . All analogs were found to inhibit transcription . Whereas br5 UTP and s4 UTP did not affect translation, mRNA containing s2 UMP or s2 CMP residues respectively was found to function poorly in translation . Only in the case of f5 UTP could ambiguitive behaviour be demonstrated . Whether mispairing of f5 UMP residues, responsible for this ambiguity takes place in transcription or in translation, could not be decided. Nucleic Acids Res, 1984 Mar 12, 12(5), 2499 - 508 The modelling of the decoding site of the Escherichia coli ribosome; Sarapuu T et al.; Individual ribosomal proteins S4, S9 and S13 were tested for their ability to interact with tRNA and synthetic polynucleotides . All three proteins bind to immobilized to Sepharose poly(A) and poly(U), while S4 and S13 form stoichiometric (1:1) complexes with tRNA in solution . We show that only the polynucleotide X S13 complexes are able to select their cognate tRNAs . In particular, the affinity of tRNAPhe to the binary poly(U) X S13 complex is about three orders of magnitude higher than that for poly(U) alone. FEBS Lett, 1984 Mar 12, 168(1), 166 - 8 Does the DNA methylase Eco dam pair nucleotide sequences to form site-specific duplexes? Buryanov YaI, Zinoviev VV, Vienozhinskis MT, Malygin EG, Nesterenko VF, Popov SG, Gorbunov YuA. The Eco dam methylase is active on denatured DNA and single-stranded synthetic oligonucleotides containing GATC sites . The results suggest that on interaction with single-stranded oligonucleotides the Eco dam methylase is able to form a duplex structure within the GATC site, and that this duplex site is a substrate for enzyme. Nucleic Acids Res, 1984 Mar 12, 12(5), 2461 - 72 The complete nucleotide sequence of the RNA coding for the primary translation product of foot and mouth disease virus; Carroll AR et al.; The complete nucleotide sequence of the coding region of foot and mouth disease virus RNA (strain A1061) is presented . The sequence extends from the primary initiation site, approximately 1200 nucleotide from the 5' end of the genome, in an open translational reading frame of 6,999 nucleotides to a termination codon 93 nucleotides from the 3' terminal poly (A) . Available amino acid sequence data correlates with that predicted from the nucleotide sequence . The amino acid sequence around cleavage sites in the polyprotein shows no consistency, although a number of the virus-coded protease cleavage sites are between glutamate and glycine residues. Nucleic Acids Res, 1984 Mar 12, 12(5), 2421 - 6 Physical mapping of the ribosomal RNA genes of Mycoplasma capricolum; Glaser G et al.; Physical mapping of the rRNA genes of Mycoplasma capricolum was done by digestion of the mycoplasmal DNA with EcoRI, PstI and BglII and hybridization with nick-translated probes consisting of defined portions of the rrnB ribosomal RNA operon of Escherichia coli . The results indicate that the rRNA genes in the chromosome of M . capricolum are arranged in two clusters, each organized in the order 5'-16S-23S-5S-3', resembling the order of the genes in the rrnB operon, with no large spacer regions separating the genes in each cluster. J Biol Chem, 1984 Mar 10, 259(5), 3368 - 74 Terminal oxidases of Escherichia coli aerobic respiratory chain . I . Purification and properties of cytochrome b562-o complex from cells in the early exponential phase of aerobic growth; Kita K et al.; Cytochrome b562-o complex, a terminal oxidase in the respiratory chain of aerobically grown Escherichia coli K12, was isolated in a highly purified form . The purified oxidase is composed of equimolar amounts of two polypeptides, with Mr = 33,000 and 55,000, determined by gel electrophoresis in the presence of sodium dodecyl sulfate . It contains 19.5 nmol of heme and 16.8 nmol of copper/mg of protein, but no detectable nonheme iron, phospholipid, ubiquinone, or menaquinone . In the difference spectrum at room temperature, the oxidase shows a single alpha absorption peak at 560 nm and at 77 K it shows two alpha absorption peaks at 555 and 562 nm . This oxidase combines with CO and the CO difference spectrum at room temperature has a peak at 416 nm and a trough at 430 nm in the Soret region . Its oxidation-reduction potential is estimated to be 125 mV (pH 7.4) and it is pH-dependent (-60 mV/pH) in medium of pH 6.0 to 7.4 . It catalyzes electron transport to oxygen via ubiquinol and ascorbate in the presence of phenazine methosulfate or N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride . This oxidase activity depends on phospholipids and is sensitive to respiratory inhibitors, such as 2-heptyl-4-hydroxyquinoline N-oxide, piericidin A, KCN and NaN3 . The divalent cations Zn2+, Cd2+, and Co2+ inhibit the oxidase activity extensively . The oxidase activity of the cytochrome b562-o complex was inhibited by photoinactivation with rose bengal, suggesting that the inhibition by zinc ion results from modification of a histidine residue of cytochrome o. J Biol Chem, 1984 Mar 10, 259(5), 2905 - 9 Escherichia coli pyruvate dehydrogenase complex . Thiamin pyrophosphate-dependent inactivation by 3-bromopyruvate; Apfel MA et al.; Inactivation of the pyruvate dehydrogenase complex by 3-bromopyruvate is thiamin pyrophosphate (TPP)-dependent . Inactivation with 2-14C- or 3-14C-labeled 3-bromopyruvate results in TPP-dependent covalent labeling of more than 60 sites in the complex, all of which are associated with the dihydrolipoyl transacetylase component . Inactivation by 3-bromo{1-14C}pyruvate labels up to 20 sites associated with dihydrolipoyl transacetylase, also with TPP dependence . Systemic chemical degradation of the complex inactivated by 3-bromo{2-14C}pyruvate under conditions that would convert lipoyl groups to S,S,-biscarboxymethyl dihydrolipoic acid produces S,S,-bis{14C}carboxymethyl dihydrolipoic acid . It is concluded that 3-bromopyruvate inactivates this complex by initially undergoing the first two steps of the usual catalytic pathway, TPP-dependent decarboxylation followed by reductive bromoacetylation of lipoyl moieties . The sulfhydryl groups of S-bromoacetyl dihydrolipoyl moieties generated by reductive bromoacetylation are then alkylated by 3-bromopyruvate as well as by bromoacetyl thioester groups associated with the complex. J Biol Chem, 1984 Mar 10, 259(5), 2798 - 802 The conserved 5 S rRNA complement to tRNA is not required for translation of natural mRNA; Zagorska L et al.; We have tested a putative base-paired interaction between the conserved GT psi C sequence of tRNA and the conserved GAAC47 sequence of 5 S ribosomal RNA by in vitro protein synthesis using ribosomes containing deletions in this region of 5 S rRNA . Ribosomes reconstituted with 5 S rRNA possessing a single break between residues 41 and 42, deletion of residues 42-46, or deletion of residues 42-52 were tested for their ability to translate phage MS2 RNA . Initiator tRNA binding, aminoacyl-tRNA binding, ppGpp synthesis, and miscoding were also tested . All of the measured functions could be carried out by ribosomes carrying the deleted 5 S rRNAs . The sizes and relative amounts of the polypeptides synthesized by MS2 RNA-programmed ribosomes were identical whether or not the 5 S RNA contained deletions . Aminoacyl-tRNA binding and miscoding were essentially unaffected . Significant reduction in ApUpG (but not poly(A,U,G) or MS2 RNA)-directed fMet-tRNA binding and ppGpp synthesis were observed, particularly in the case of the larger (residues 42-52) deletion . We conclude that if tRNA and 5 S rRNA interact in this fashion, it is not an obligatory step in protein synthesis. J Biol Chem, 1984 Mar 10, 259(5), 2734 - 41 Purification and characterization of succinyl-CoA: tetrahydrodipicolinate N-succinyltransferase from Escherichia coli; Simms SA et al.; Tetrahydrodipicolinate succinylase, an enzyme involved in the diaminopimelate-lysine pathway, was purified 1900-fold from crude extracts of Escherichia coli . The enzyme catalyzes the formation of CoA and N-succinyl-2-amino-6-keto-L-pimelate from succinyl-CoA and tetrahydrodipicolinate . The purified enzyme was shown to be homogeneous by polyacrylamide gel electrophoresis . The Stokes radius of the enzyme was determined from its elution volume on a Sephacryl S300 column and its sedimentation constant from sucrose density gradient centrifugation . These were 35 A and 4.7 (S20,w), respectively . The enzyme consists of two subunits each with a mass of 31,000 daltons, as determined using sodium dodecyl sulfate/polyacrylamide gel electrophoresis . Tetrahydrodipicolinate succinylase was shown to be a sulfhydryl enzyme . It has a pH optimum of 8.2 . The equilibrium lies predominantly in favor of product formation but the reverse reaction can be demonstrated in vitro. J Biol Chem, 1984 Mar 10, 259(5), 3375 - 81 Terminal oxidases of Escherichia coli aerobic respiratory chain . II . Purification and properties of cytochrome b558-d complex from cells grown with limited oxygen and evidence of branched electron-carrying systems; Kita K et al.; Cytochrome b558-d complex, a terminal oxidase in the respiratory chain of Escherichia coli K12 grown under the condition of limited oxygen, was purified to near homogeneity . The purified oxidase complex is composed of equimolar amounts of two polypeptides, with Mr = 26,000 and 51,000, determined with gel electrophoresis in the presence of sodium dodecyl sulfate . It contains 12.3 nmol of protoheme, 9.54 nmol of cytochrome d, and 26.6 nmol of iron/mg of protein . The enzyme is a "cytochrome bd-type oxidase," which shows absorption peaks at 558 and 624 nm in the difference spectrum at 77 K . This oxidase combines with CO, and the Soret region of the CO difference spectrum at room temperature has a peak at 420 nm and troughs at 430 and 442 nm . The oxidation-reduction potentials of cytochrome b558 and cytochrome d at pH 7.4 were estimated to be +10 mV and +240 mV, respectively . The cytochrome b558-d complex catalyzes the oxidation of ubiquinol-1, menadiol, and ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride . This oxidase activity was inhibited by the respiratory inhibitors piericidin A, KCN, and NaN3, but the cytochrome b558-d complex was less sensitive to the inhibitors than the cytochrome b562-o complex . The Km values for oxygen of purified cytochrome b558-d complex and cytochrome b562-o complex were determined to be 0.38 and 2.9 microM, respectively . Formation of a membrane potential by the reconstituted cytochrome b558-d complex in liposomes was observed with the fluorescent dye 3,3'-dipropylthiocarbocyanine iodide on addition of ubiquinol-1 . This is the first indication that there is a coupling site in the terminal oxidase, which contains cytochrome d . Steady state kinetics of cytochromes in the membrane showed that these oxidase complexes branch at the site of ubiquinone-8 in the respiratory chain . From these and previous results, branched electron-carrying systems of the E . coli respiratory chain are proposed. J Biol Chem, 1984 Mar 10, 259(5), 3202 - 9 Site specific deletions of regulatory sequences in a ribosomal protein-RNA polymerase operon in Escherichia coli . Effects on beta and beta' gene expression; Dennis PP; The 319 nucleotide long intergenic region between the rplL (L12) and the rpoB (beta) genes of the L10 operon contains a transcription attenuation sequence and a RNase III mRNA processing sequence . Four site specific deletions located within this intergenic space which remove either the transcription attenuation sequence or the RNase III mRNA processing sequence or both sequences have been isolated on recombinant DNA plasmids carrying this operon . Deletions of sequences surrounding the RNase III processing site result in a uniform 80-90% reduction in the translational efficiency of beta subunit mRNA . This reduction in translation efficiency appears not to be related to processing per se; transcription of the rpoB and rpoC genes and the translation efficiency of the respective mRNA sequences were indistinguishable in an RNase III processing defective mutant (rnc) and its isogenic parent (rnc+) . Deletions of the attenuator sequence result in a substantial increase in the downstream transcription of the beta subunit gene . The translational efficiency of RNase III processed beta subunit mRNA was found to be related in an inverse manner to the level of beta subunit synthesis . These result suggest that sequences on the mRNA in the vicinity of the RNase III processing site (i) are essential for efficient translation of beta subunit mRNA and (ii) are utilized for reducing the translational efficiency of the beta subunit mRNA when the beta subunit protein is produced in excess of that required for RNA polymerase assembly. J Biol Chem, 1984 Mar 10, 259(5), 2803 - 9 Vanadate inhibits the ATP-dependent degradation of proteins in reticulocytes without affecting ubiquitin conjugation; Tanaka K et al.; Reticulocytes contain a nonlysosomal, ATP-dependent system for degrading abnormal proteins and normal proteins during cell maturation . Vanadate, which inhibits several ATPases including the ATP-dependent proteases in Escherichia coli and liver mitochondria, also markedly reduced the ATP-dependent degradation of proteins in reticulocyte extracts . At low concentrations (K1 = 50 microM), vanadate inhibited the ATP-dependent hydrolysis of {3H}methylcasein and denatured 125I-labeled bovine serum albumin, but it did not reduce the low amount of proteolysis seen in the absence of ATP . This inhibition by vanadate was rapid in onset, reversed by dialysis, and was not mimicked by molybdate . Vanadate inhibits proteolysis at an ATP-stimulated step which is independent of the ATP requirement for ubiquitin conjugation to protein substrates . When the amino groups on casein and bovine serum albumin were covalently modified so as to prevent their conjugation to ubiquitin, the derivatized proteins were still degraded by an ATP-stimulated process that was inhibited by vanadate . In addition, vanadate did not reduce the ATP-dependent conjugation of 125I-ubiquitin to endogenous reticulocyte proteins, although it markedly inhibited their degradation . In intact reticulocytes vanadate also inhibited the degradation of endogenous proteins and of abnormal proteins containing amino acid analogs . This effect was rapid and reversible; however, vanadate also reduced protein synthesis and eventually lowered ATP levels in the intact cells . Vanadate (10 mM) has also been reported to decrease intralysosomal proteolysis in hepatocytes . However, in liver extracts this effect on lysosomal proteases required high concentrations of vanadate (K1 = 500 microM) and was also observed with molybdate, unlike the inhibition of ATP-dependent proteolysis in reticulocytes. J Biol Chem, 1984 Mar 10, 259(5), 3293 - 8 Dihydroorotase from Escherichia coli . Purification and characterization; Washabaugh MW et al.; Dihydroorotase (4,5-L-dihydroorotate amidohydrolase (EC 3.5.2.3}, which catalyzes the reversible cyclization of N-carbamyl-L-aspartate to dihydro-L-orotate, has been purified to homogeneity from an over-producing strain of Escherichia coli . Treatment of 70 g of frozen cell paste produces about 7 mg of pure enzyme, a yield of about 35% . The native molecular weight, determined by equilibrium sedimentation, is 80,900 +/- 4,300 . The subunit molecular weight, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 38,400 +/- 2,600, and by amino acid analysis is 41,000 . The enzyme is thus a dimer and contains 0.95 +/- 0.08 tightly bound zinc atoms per subunit when isolated by the described procedure, which would remove any loosely bound metal ions . Isoelectric focusing under native conditions yields a major species at isoelectric point 4.97 +/- 0.27 and a minor species at 5.26 +/- 0.27; dihydroorotase activity is proportionately associated with both bands . The enzyme has a partial specific volume of 0.737 ml/g calculated from the amino acid composition and a specific absorption at 278 nm of 0.638 for a 1 mg/ml solution . At 30 degrees C, the Michaelis constant and kcat for dihydro-DL-orotate (at pH 8.0) are 0.0756 mM and 127 s-1, respectively; for N-carbamyl-DL-aspartate (at pH 5.80), they are 1.07 mM and 195 s-1. Nature, 1984 Mar 8-14, 308(5955), 201 - 3 Mechanism of mutagenesis by O6-methylguanine; Eadie JS et al.; O6-methylguanine (O6meG) lesions of double-stranded DNA have been associated with mutation and neoplastic transformation . These lesions can, in principle, be produced by at least three different mechanisms: direct alkylation of G X C base pairs in double-stranded DNA; alkylation of guanine residues in single-stranded regions of DNA associated with replication forks; and alkylation of the DNA precursor pool followed by incorporation of O6-methyl deoxyguanosine triphosphate (O6-medGTP) during DNA replication . DNA biosynthesis subsequent to all three events will generate predominantly O6-meG X T base pairs as O6meG preferentially pairs with T . We show here that O6meG X T base pairs are mutagenic; that transalkylase repair has a direct role in the generation of mutations induced by alkylated pool nucleotides; and that the Escherichia coli mismatch repair system is capable of repairing mutagenic G X T intermediates. J Mol Biol, 1984 Mar 5, 173(3), 293 - 305 Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli host cells irradiated with ultraviolet light; Wood RD et al.; Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells . Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA . Non-irradiated phage grown in heavily irradiated uvr+ host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions . For non-targeted mutagenesis in heavily irradiated host cells, there were one to two mutant phage per mutant burst . From this and the pathways of lambda DNA synthesis, it can be argued that non-targeted mutagenesis involves a loss of fidelity in semiconservative DNA replication . A series of experiments with various mutant host cells showed a major pathway of non-targeted mutagenesis by ultraviolet light, which acts in addition to "SOS induction" (where cleavage of the LexA repressor by RecA protease leads to din gene induction): (1) the induction of mutants has the same dependence on irradiation for wild-type and for umuC host cells; (2) a strain in which the SOS pathway is constitutively induced requires irradiation to the same level as wild-type cells in order to fully activate non-targeted mutagenesis; (3) non-targeted mutagenesis occurs to some extent in irradiated recA recB cells . In cells with very low levels of PolI, the induction of non-targeted mutagenesis by ultraviolet light is enhanced . We propose that the major pathway for non-targeted mutagenesis in irradiated host cells involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and that the low polymerase activity leads to frameshift mutations during semiconservative DNA replication . The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage. Plasmid, 1984 Mar, 11(2), 166 - 77 A mathematical model for lambda dv plasmid replication: analysis of copy number mutants; Lee SB et al.; A mathematical model based on the molecular control mechanisms for lambda dv plasmid replication in a single Escherichia coli cell has been applied to simulate replication of mutant lambda dv plasmids . Model simulations of changes in repressor level and copy number resulting from mutations in the promoter-operator PROR region are consistent with experimental data . Calculated effects on lambda dv plasmid copy number of oligomer formation and of alternations in termination efficiency at tR1 also agree with experiment . The model has been employed to simulate the influence of cro mutants and of cro and tR1 double mutants on copy number and stable maintenance of lambda dv plasmid copy number . The genetic structure included in formulation of the replicon model provides a framework for relating changes in specific genetic loci on the plasmid with resulting alterations in host-plasmid system function. Plasmid, 1984 Mar, 11(2), 151 - 65 A mathematical model for lambda dv plasmid replication: analysis of wild-type plasmid; Lee SB et al.; A mathematical model for lambda dv plasmid replication in a growing single cell of Escherichia coli has been formulated and solved numerically . Quantitative description of the molecular control mechanism for initiation of lambda dv replication presumes regulatory functions of repressor and initiator proteins and transcriptional activation of the origin region . Random selection of a single plasmid for activation and replication is assumed, as is regular plasmid segregation to daughter cells . The model is capable of simulating the periodic changes in each regulatory element and the plasmid copy number during the cell cycle . The calculated average copy number, repressor concentration, and timing of plasmid replication agree well with experimental data . The simulated lambda dv plasmid replication rate is controlled primarily by transcription frequency . Initiation of plasmid replication is not related to variations in the levels of repressor or initiator proteins during the cell cycle . Simulation studies of perturbations in plasmid and repressor segregation indicate that replication regulation of the lambda dv plasmid compensates to readjust copy number to normal values in a few generations . Implications of these studies relative to the molecular mechanisms of replication control are discussed. Plasmid, 1984 Mar, 11(2), 141 - 50 Sequence analysis of the maize mitochondrial 26 S rRNA gene and flanking regions; Dale RM et al.; A single copy of the large ribosomal 26 S rRNA gene is found in the maize mitochondrial genome . The sequence of this gene and the flanking regions has been determined using the M13 dideoxy sequencing method . The maize mt 26 S rDNA shares a high degree of homology with the Escherichia coli 23 S rDNA, and the approximate 5' and 3' ends of the maize 26 S rDNA have been located by comparison with the E . coli sequence . The maize mt 26 S rDNA has also been compared with the sequences of the maize chloroplast 23 S rDNA, the human mitochondrial 16 S rDNA, part of the yeast mitochondrial 21 S rDNA, and the yeast cytoplasmic 25 S rDNA . In all cases, there are numerous regions of 70% or higher homology. Hoppe Seylers Z Physiol Chem, 1984 Mar, 365(3), 289 - 96 Purification and properties of phenylalanyl-tRNA synthetase from a higher plant (Phaseolus vulgaris); Rauhut R et al.; Phenylalanyl-tRNA synthetase from beans (Phaseolus vulgaris) was purified 2 800-fold to homogeneity with a 16% overall yield by salting-out chromatography, salting-out affinity chromatography, gel filtration and chromatography on DEAE-cellulose and hydroxylapatite . This combination minimizes potentially harmful effects of proteinases and products of the secondary metabolism of a green plant during the early steps . The molecular mass is 260 000 Da with a subunit structure of alpha 2 beta 2 (alpha = 59 000, beta = 70 000 Da) . Enzymatic activity was optimal with 20mM Mg2+ and 10mM KCl at pH 6.5 and pH 8.5, depending on the buffer substance . Kinetic measurements at low temperature and steady-state kinetics indicate that the esterification of tRNA or a step preceding it, but not the activation, are rate-determining at pH 7.65 . The cognate tRNAPhe is exclusively aminoacylated at the 2'-OH group . tRNAs from Escherichia coli and bean chloroplasts are not aminoacylated . No immunological relationship of the plant enzyme to other phenylalanyl-tRNA synthetases was revealed by immuno-diffusion and immunotitration with polyclonal antibodies raised against the enzymes from E . coli, yeast and hen liver . ATP analogs revealed a unique pattern of substrate properties with indication of conservation of ATP binding in the form of an ATP-Mg2+ complex in the anti-conformation with a coordination of the cation to the nitrogen in position 7 of the purine moiety. Cancer Res, 1984 Mar, 44(3), 1044 - 7 Augmentation of natural killer cell activity by lipopolysaccharide through separable effects on the binding of nonadherent lymphocytes to tumor targets and tumor killing; Salata RA et al.; A single-cell assay was utilized to study the augmentation by Escherichia coli lipopolysaccharide (LPS) of the cytotoxicity of human lymphocytes for the human myeloid tumor K562 . Preincubation with LPS at 20 micrograms/ml for 30 min at 37 degrees increased the binding of all nonadherent (NA) lymphocyte populations to K562 tumors {unseparated NA lymphocytes from 13.1 to 25.1%, immunoglobulin G Fc receptor-enriched lymphocytes from 27.6 to 42.9%, and immunoglobulin G Fc receptor-depleted lymphocytes from 14.0 to 23.7%, at p less than 0.001} . In contrast, interferon (IFN) at 10 units/ml had no effect on the overall binding of lymphocytes to K562 tumors . When lymphocyte-tumor conjugates were dispersed in agarose, cytotoxic activity of unseparated NA lymphocytes at 1 to 3 hr was markedly increased by preincubation with LPS (p less than 0.001) . However, LPS did not enhance cytotoxicity if conjugates were formed in its absence . IFN, likewise, increased cytotoxic activity in unseparated NA lymphocytes at 1 to 3 hr (p less than 0.001) . No synergistic cytotoxicity was seen with concurrent exposure to LPS and IFN . LPS increased cytotoxicity in the Fc receptor-enriched:tumor conjugates at 1 to 3 hr (p less than 0.001) and appeared to promote more efficient killing in individual conjugates over time . Cytotoxicity in the Fc receptor-depleted:tumor conjugates was not enhanced by LPS . Thus, LPS may enhance natural killer cell-like activity by increasing the binding of human lymphocytes to K562 tumors and by rearranging the population of binding cells to include more efficient killer cells . While the effects of LPS on binding appear independent of IFN, selective recruitment of more efficient killer cells could be through an IFN mechanism. Mikrobiologiia, 1984 Mar-Apr, 53(2), 285 - 9 {Expression of the resistance genes of plasmid RP4 in Escherichia coli cells grown by continuous cultivation}; Filonov AE et al.; The resistance to tetracycline decreased in Escherichia coli C600 cells containing plasmid RP4 and grown under the conditions of continuous cultivation . The population of cells containing plasmid RP4 is heterogeneous in the trait of tetracycline resistance, and most cells cannot grow in a selective medium with tetracycline at a concentration of 20 micrograms/ml . The decreased resistance to tetracycline was most pronounced for a glucose-limited chemostat culture and also in the presence of two plasmids, RP4 and pBS94 , in the cells . No decrease was found in the resistance to other drugs determined by plasmid genes. J Gen Microbiol, 1984 Mar, 130 ( Pt 3), 687 - 92 Genetic and structural evidence for the presence of propanediol oxidoreductase isoenzymes in Escherichia coli; Ros J et al.; The synthesis of propanediol oxidoreductase, an enzyme permitting the anaerobic metabolism of fucose and rhamnose, has been described as being controlled by the prd locus closely linked to the fuc locus in wild-type cells of Escherichia coli . However, strain AA-787, deleted in the fuc and prd loci, grew anaerobically on rhamnose, displaying propanediol oxidoreductase activity . From the deleted strain we derived a constitutive producer of propanediol oxidoreductase able to grow on 1,2-propanediol by oxidizing the diol to lactaldehyde which was further metabolized to lactate . Transduction experiments showed that this ability to use propanediol was closely linked to the rha locus . Peptide mapping of fucose- and rhamnose-induced propanediol oxidoreductase of wild-type cells established structural differences between the two enzymes, indicating two structural genes, one for each sugar metabolizing system. Can J Microbiol, 1984 Mar, 30(3), 411 - 5 Escherichia coli growth on lactose requires cycling of beta-galactosidase products into the medium; Huber RE et al.; Growth on lactose was found to be restricted in an Escherichia coli strain deficient in its ability to transport glucose and galactose . If the latter sugars were removed from the medium as they were being produced, a wild-type strain grew only poorly, while the transport-deficient strain did not grow at all . These results suggested that all of the products of beta-galactosidase action on lactose are released into the medium before being metabolized . This contention was strongly supported by the finding that the appearance of products in the medium was equal to lactose disappearance at three limiting lactose concentrations and by an experiment which showed that essentially all of the label from added lactose ( {1-14C}glucose) was found in the medium as glucose when chased with unlabelled lactose. Can J Microbiol, 1984 Mar, 30(3), 339 - 44 Involvement of outer membrane proteins in freeze--thaw resistance of Escherichia coli; Calcott PH et al.; Two families of Escherichia coli mutants with altered outer membrane protein components were examined for sensitivity to freezing and thawing and other stresses . A mutant unable to make the lipoprotein (lpo) was extremely sensitive to freezing and thawing in water or saline and to challenge with detergent, while the mutant unable to make the porin proteins (ompB) was more resistant than the isogenic wild type; strains unable to make the tsx and ompA proteins were slightly more sensitive to the stresses . Similarly, the lpo deficient strain exhibited more and the ompB less wall and membrane damage than the wild-type strains . Little difference in the extent of wall damage, but more membrane damage, was seen for the two tsx and the ompA strains when compared with the wild-type strain . The roles of the specific proteins in determining sensitivity to freeze-thaw are discussed. Vestn Khir Im I I Grek, 1984 Mar, 132(3), 95 - 6 {Use of CO2 laser for the prevention of postoperative wound suppuration}; Skobelkin OK et al.; The experiments with laboratory animals have convincingly shown that CO2-laser can be successfully used for prophylactics of suppurations of postoperative wounds in clinics. EMBO J, 1984 Mar, 3(3), 623 - 9 Mutagenesis of the three bases preceding the start codon of the beta-galactosidase mRNA and its effect on translation in Escherichia coli; Hui A et al.; The effect on the translation efficiency of various mutations in the three bases (the -1 triplet) that precede the AUG start codon of the beta-galactosidase mRNA in Escherichia coli was studied . Of the 39 mutants examined, the level of expression varies over a 20-fold range . The most favorable combinations of bases in the -1 triplet are UAU and CUU . The expression levels in the mutants with UUC, UCA or AGG as the -1 triplet are 20-fold lower than those with UAU or CUU . In general, a U residue immediately preceding the start codon is more favorable for expression than any other base; furthermore, an A residue at the -2 position enhances the translation efficiency in most instances . In both cases, however, the degree of enhancement depends on its context, i.e . the neighboring bases . Although the rules derived from this study are complex, the results show that mutations in any of the three bases preceding the start codon can strongly affect the translational efficiency of the beta-galactosidase mRNA. J Exp Med, 1984 Mar 1, 159(3), 828 - 43 Interrelationships of human interferon-gamma with lymphotoxin and monocyte cytotoxin; Stone-Wolff DS et al.; Crude preparations of interferon (IFN)-gamma derived from human peripheral blood leukocyte (PBL) cultures induced with 12-O-tetra-decanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA) were more cytotoxic to HeLa cells than partially purified nautral or highly purified recombinant human IFN-gamma preparations . Conditioned media from PBL cultures contained, in addition to IFN-gamma, a mixture of cytotoxins, including classic lymphocyte-derived lymphotoxin (LT), and a TPA-induced cytotoxic activity produced by the adherent cell population (presumably monocytes) . These two types of cytotoxins, indistinguishable in the mouse L929 cell LT assay, could be differentiated by an antiserum prepared against LT derived from the B lymphoblastoid cell line RPMI 1788 . This antiserum neutralized lymphocyte-derived classic LT but failed to neutralize the activity of the monocyte-derived cytotoxin . Processing of conditioned media by sequential chromatography on silicic acid, Con A-Sepharose, and DEAE-Sephacel failed to separate IFN-gamma from the LT activity . However, this procedure did remove the monocyte-derived cytotoxic activity present in the original starting material, leaving predominantly classic LT . This LT showed a slightly basic isoelectric point (pI 7.6) which partially overlapped the more basic pI range of IFN-gamma . The two lymphokine activities also could not be completely separated by fast protein liquid chromatography or molecular sieve chromatography . LT in these partially purified preparations was associated with a protein having an apparent molecular weight of 58,000 on gel filtration . This form dissociated partially into a 20,000 mol wt species after denaturation with 0.1% NaDodSO4 . IFN-gamma could be selectively removed from preparations containing both IFN-gamma and LT with the aid of monoclonal antibody to IFN-gamma . The addition of purified LT to purified E . coli-derived recombinant human IFN-gamma resulted in a marked synergistic enhancement of cytotoxicity for HeLa cells. J Bacteriol, 1984 Mar, 157(3), 828 - 32 Dual control of a common L-1,2-propanediol oxidoreductase by L-fucose and L-rhamnose in Escherichia coli; Chen YM et al.; Anaerobic growth of Escherichia coli on L-fucose or L-rhamnose as the sole source of carbon and energy depends on the regeneration of NAD from NADH by disposing the intermediate L-lactaldehyde as L-1,2-propanediol . The two parallel pathways, with their own permeases and enzymes encoded by two widely separated gene clusters, appear to share a single enzyme that catalyzes the formation of L-1,2-propanediol . Although this oxidoreductase is encoded by a gene at the fuc locus, the enzyme is inducible by both L-fucose and L-rhamnose . The inducibility by L-rhamnose is controlled by a gene at the rha locus with no other known functions, since the aerobic growth rate on L-rhamnose remains normal . L-1,2-Propanediol oxidoreductase activity is inducible only anaerobically, and the effect of the two methylpentoses operates at different levels: L-fucose exerts its influence post-transcriptionally; L-rhamnose exerts its influence transcriptionally. J Immunol, 1984 Mar, 132(3), 1300 - 4 Natural and recombinant Escherichia coli-derived interferon-gamma differ in their reactivity with monoclonal antibody; Le J et al.; Monoclonal antibody GIF-1 was found to neutralize human natural immune interferon (IFN-gamma), but not Escherichia coli-derived recombinant IFN-gamma . In addition, GIF-1 antibody failed to immunoprecipitate 125I-labeled recombinant IFN-gamma, whereas it precipitated natural IFN-gamma in a concentration-dependent manner . The lack of recognition of recombinant IFN-gamma by antibody GIF-1 may not be due to the absence of the oligosaccharide moiety in the molecules of recombinant IFN-gamma alone, because removal of carbohydrate from natural IFN-gamma by treatment with a mixture of glycosidases did not alter the selective binding of antibody, i.e., deglycosylated and untreated natural IFN-gamma were equally neutralized and immunoprecipitated by GIF-1 antibody . In addition, a minor monomeric component of natural IFN-gamma with the m.w . of 15,500, which apparently lacks carbohydrate, was also recognized by antibody GIF-1 . These results suggest that the discriminative recognition of natural and recombinant IFN-gamma by monoclonal antibody GIF-1 may be due to a conformational difference at or near the active regions of natural and recombinant human IFN-gamma molecules. Vaccine, 1984 Mar, 2(1), 93 - 9 Immunological activity of chitin and its derivatives; Nishimura K et al.; The effect of chitin and its derivatives on the activation of peritoneal macrophages in vivo, on the suppression of tumour growth in syngeneic mice and on the protection of the host against bacterial infection was examined . Thirty percent deacetylated chitin (30% DA-chitin), 70% DA-chitin and carboxymethyl-chitin (CM-chitin) induced cytotoxic macrophages most effectively . Chitosan, hydroxyethyl-chitin, dihydroxypropyl-chitin (DHP-chitin) and DHP-chitosan had moderate activities . Phosphorylated-, sulphonated- or acetyl-chitin, however, were less effective . Both 70% DA-chitin and DHP-chitosan were most active on the suppression of Meth-A tumour growth in BALB/c mice, and 30% DA-chitin had a moderate effect . For the stimulation of non-specific host resistance against Escherichia coli infection, 30% and 70% DA-chitin were effective. Aust Vet J, 1984 Mar, 61(3), 77 - 82 The effect of Escherichia coli endotoxin and culture filtrate on the lactating bovine mammary gland; Frost AJ et al.; The pathogenesis of coliform mastitis was studied by observing pathological changes in lactating glands after infusion of either endotoxin or the sterile culture filtrate (CCF) of the medium in which Escherichia coli strain B117 had been grown . Both infusions produced a rapid and intense inflammatory response by 4 h with a marked increase of serum proteins in the milk . Before dispersing into the milk, neutrophils were attached to the ductular epithelium; highest cell counts in the milk were recorded when the tissue reaction had waned . Oedema of the ductular epithelium occurred, particularly where neutrophils were actively migrating . The infusion of CCF produced, in addition to inflammation, degeneration and necrosis of ductular cells . The smallest lesions healed very rapidly . There was evidence of differing cell susceptibility to the necrotising toxin as well as uneven distribution over the epithelial surface . All changes observed were confined to the regions of the teat and lactiferous sinuses with little effect on the secreting tissue . The role of the necrotising toxin in the natural disease remains undetermined. Anal Biochem, 1984 Mar, 137(2), 351 - 9 Glycerol, sucrose, and other diol-containing reagents are not inert components in in vitro incubations containing aminoacyl-tRNA; Johnson AE et al.; The addition of glycerol, sucrose, or other diol-containing reagents to solutions of aminoacyl-tRNA (aa-tRNA) substantially increased the rate of hydrolysis of the aminoacyl ester bond . Glycerol at 4.9% (v/v) doubled the rate of deacylation for several aa-tRNAs and peptidyl-tRNAs, including fMet-tRNAMetf, while 1% (v/v) glycerol increased the deacylation rate by 20% . This effect was not caused by a nuclease contamination, and tRNA deacylated in the presence of glycerol could be fully recharged . The deacylation of aa-tRNA was accelerated by glycerol and sucrose even in the presence of EF-Tu X GTP . In addition, the extent of tRNA aminoacylation was reduced when glycerol was present at concentrations above 2% (v/v) . Thus, glycerol and sucrose are not necessarily inert or neutral additions to an in vitro incubation. Plasmid, 1984 Mar, 11(2), 178 - 81 Location of a function on RP1 that fertility inhibits Inc W plasmids; Yusoff K et al.; Two fertility inhibition (Fi+) functions which reduce R388 (Inc W) transfer were detected on RP1 (Inc P) . Neither function affected R388 -mediated surface exclusion but they could be distinguished by their effect on pilus production . One of the functions was located in the 6.5-kb Pst1 -C region of RP1, part or all of which also occurs on six Fi+ but not two Fi- Inc P plasmids studied. Med Tekh, 1984 Mar-Apr, (2), 12 - 6 {Automated biochemical analysis based on the use of electrosorption systems}; Andreev VS; An engineering method to separate biocomponents that is suitable for discrete and flow-type autoanalyzers is proposed . The principle is based on using electrosorption systems; it provides, to a high reproducibility, the separation of cells and biomacromolecules from another components of samples to be analysed and makes it possible to solve analytical problems which require the application of several currently available separation systems. J Biochem (Tokyo), 1984 Mar, 95(3), 729 - 36 Two autonomously replicating sequences near oli-1 gene of yeast mitochondrial DNA; Mabuchi T et al.; We cloned two autonomously replicating sequences from a short segment of mtDNA of an oligomycin-resistant petite yeast, O-111, into a vector pYleu 12 constructed from yeast LEU 2 gene and pBR 322 . These plasmids, pYmit 4 and pYmit 1, had frequencies of transformation of yeast as high as that of YEp 13, having a replicator of 2 mu DNA . They were maintained as plasmids in yeast under selective conditions and shuttled from yeast to E . coli . No evidence was obtained that these plasmids were incompatible with the wild-type mitochondrial genome . These sequences were located in intergenic regions. J Biochem (Tokyo), 1984 Mar, 95(3), 637 - 42 Phosphoenolpyruvate carboxylase of Escherichia coli . Specificity of some compounds as activators at the site for fructose 1,6-bisphosphate, one of the allosteric effectors; Kodaki T et al.; An investigation was performed to elucidate some unusual phenomena which had been observed with phosphoenolpyruvate (PEP) carboxylase {EC 4.1.1.31} of Escherichia coli . (i) Fructose 1,6-bisphosphate (Fru-1,6-P2) and GTP--the allosteric activators--were competitive with each other in the activation . (ii) Some analogs of PEP such as DL-2-phospholactate and 2-phosphoglycolate, which behaved as inhibitors in the presence of the activator (acetyl-CoA or dioxane), activated the enzyme to some extent in the absence of the activator . (iii) Ammonium sulfate deprived the enzyme of sensitivity to Fru-1,6-P2 or GTP but had no effect on the sensitivity to other effectors . It was found that the activation by the analogs was lost upon desensitization of the enzyme to Fru-1,6-P2 by reaction with 2,4,6-trinitrobenzene sulfonate . The activation by the analogs was not observed in the presence of 200 mM ammonium sulfate . In the presence of lower concentrations (0.1 mM) of PEP, ammonium sulfate activated the enzyme at concentrations less than 700 mM but had an inhibitory effect on the desensitized enzyme . These findings suggest that the unusual phenomena described above are a result of binding of the phosphate esters and sulfate ions with the Fru-1,6-P2 site of the enzyme or the active site depending on the reaction conditions. Res Vet Sci, 1984 Mar, 36(2), 187 - 93 Influence of diet on postweaning malabsorption and diarrhoea in the pig; Miller BG et al.; Five-day-old pigs challenged with 10(3) pathogenic Escherichia coli (nalidixic acid resistant) showed no clinical signs of disease until subsequently weaned at three weeks . Dietary manipulation was shown to influence xylose malabsorption, diarrhoea and bacterial proliferation after weaning . Brief, but not continuous, contact with the diet before weaning markedly increased the severity of subsequent disease after weaning . Immunogenicity of the weaning diet was critical for the development of the disease . Two diets, identical except that in one the protein source (casein) had previously been enzymatically hydrolysed, were compared . Pigs fed the predigested diet showed no clinical signs of post weaning diarrhoea whereas those fed the untreated casein all developed diarrhoea. Prikl Biokhim Mikrobiol, 1984 Mar-Apr, 20(2), 208 - 16 {Purification and properties of phenylalanyl-tRNA-synthetase from Escherichia coli MRE-600}; Ankilova VN et al.; A preparative scale method for isolation of highly purified phenylalanyl-tRNA synthetase from E . coli MRE-600 was developed . It consists of cell destroying, nucleic acid precipitation with streptomycine sulfate, fractionation with ammonium sulfate followed by chromatography on different carriers (Sephadex G-200, DEAE-cellulose, DEAE-Sephadex A-50, and hydroxyapatite) . The mode of cell destroying was found to affect the process of the further enzyme purification . The phenylalanyl-tRNA synthetase was purified 540-fold, with recovery being 20.6% and the specific activity - 540 units per mg protein . The enzyme content in the purified preparation was 80-90% judging by electrophoresis in PAAG . The molecular weights of the subunits determined by electrophoresis under denaturative conditions were found to be 102,000 +/- 4000 (beta) and 42,000 +/- 2000 (alpha) . The molecular weight of the native enzyme determined by gel filtration through Sephadex G-200 and electrophoresis at varied concentrations of polyacrylamide was found to be 340,000 +/- 20,000 . The Km values for tRNA, ATP and phenylalanine in the aminoacylation reaction are equal to 5.4 X 10(-7) M, 1,9 X 10(-4) M, and 3.7 X 10(-6) M, respectively. Mutat Res, 1984 Mar-Apr, 131(3-4), 97 - 100 UV irradiation alters deoxynucleoside triphosphate pools in Escherichia coli; Das SK et al.; UV irradiation of exponentially growing Escherichia coli increased intracellular concentration of dATP and dTTP without significantly changing the concentrations of dGTP and dCTP . These selective increases in dATP and dTTP pools are seen in wild-type E . coli K12 and AB1157, as well as in recA and umuC strains, and are proportional to UV dose . The possible significance of these findings with respect to induction of the SOS response and nontargeted mutagenesis are discussed. Mol Biol (Mosk), 1984 Mar-Apr, 18(2), 362 - 9 {Changes in the state of tryptophan residues in T4 phage lysozyme during binding to a competitive inhibitor}; Vedenkina NS et al.; The analysis of the intrinsic fluorescence parameters of T4 phage lysozyme in free state and in complex with inhibitor--disaccharide-tetrapeptide from the E . coli cell wall has been carried out . A comparison of the fluorescence changes with the results obtained by difference spectrophotometry and with the data of Elwell and Schellman on the intrinsic fluorescence of wild type WT and mutant eRI T4 phage lysozymes and a consideration of the three dimensional structure of the protein allows to represent the protein fluorescence parameters as a sum of contributions of the individual tryptophan residues . According to the proposed scheme Trp-126 does not emit neither in the free protein nor in the complex; the fluorescence parameters of Trp-158 (lambda m 332 nm, q = 0.27) are not affected by binding of the inhibitor, but all the fluorescence changes are due to the rise of the quantum yield (from 0.135 to 0.315) and the blue shift (from 332 to 328 nm) of the fluorescence of Trp-138. J Clin Microbiol, 1984 Mar, 19(3), 408 - 11 Colony incompatibility studies of enterotoxigenic Escherichia coli O126 isolated during one outbreak; Bettelheim KA; Strains of heat-stable enterotoxin-producing Escherichia coli belonging to O group O126 isolated during an outbreak of gastroenteritis were studied by the colony incompatibility phenomenon . They were compared with other isolates of O126 . All of the outbreak strains except one appeared related in these studies and were different from all other strains studied . The one different outbreak strain was isolated 6 weeks after all of the others and may represent some new factor . The study demonstrated the potential usefulness of monitoring strains during an outbreak for relatedness. Genetika, 1984 Mar, 20(3), 373 - 81 {Deletions of plasmid pRP3.1ts12 derived from RP1 leading to the suppression of the thermosensitive ts12 mutation and mucoid phenotype induction in Escherichia coli K-12}; Danilevich VN et al.; Properties of a temperature-sensitive in replication mutant pRP3.1ts12 derived from the broad host range RP1 plasmid have been studied . pRP3.1ts12 is a shortened variant of the temperature-sensitive RP1ts12 mutant carrying a deletion in a region from 2.3 to 7.6 MD . In contrast to RP1ts12, the plasmid pRP3.1ts12 is a leaky ts mutant and is characterized by an elevated frequency of reversions to the temperature-independent phenotype . Temperature-independent derivatives of pRP3.1ts12 were studied . Approx . 15% of these were found to induce mucoid growth of the host cells . As revealed from restriction endonuclease analysis, most of the latter derivatives contain deletions of small DNA segments in the region 0.56 to 2.3 MD of the RP1 map . The possible nature of the gene(s), whose deletions suppress the temperature-sensitive ts12 mutation and results in superproduction of Escherichia coli capsular poly-saccharide is discussed. EMBO J, 1984 Mar, 3(3), 631 - 5 A defined mutation in the protein export gene within the spc ribosomal protein operon of Escherichia coli: isolation and characterization of a new temperature-sensitive secY mutant; Shiba K et al.; We describe the properties of a temperature-sensitive mutant, ts24, of Escherichia coli . The mutant has a conditional defect in export of periplasmic and outer membrane proteins . At 42 degrees C, precursor forms of these proteins accumulate within the cell where they are protected from digestion by externally added trypsin . The accumulated precursors are secreted and processed very slowly at 42 degrees C . The mutation is complemented by expression of the wild-type secY (or prlA) gene, which has been cloned into a plasmid vector from the promoter-distal part of the spc ribosomal protein operon . The mutant has a single base change in the middle of the secY gene, which would result in the replacement of a glycine residue by aspartic acid in the protein product . These results demonstrate that the gene secY (prlA) is essential for protein translocation across the E . coli cytoplasmic membrane. EMBO J, 1984 Mar, 3(3), 545 - 50 Inducible repair of O-alkylated DNA pyrimidines in Escherichia coli; McCarthy TV et al.; The three miscoding alkylated pyrimidines O2-methylcytosine, O2-methylthymine and O4-methylthymine are specifically recognized by Escherichia coli DNA repair enzymes . The activities are induced as part of the adaptive response to alkylating agents . O2-Methylcytosine and O2-methylthymine are removed by a DNA glycosylase, the alkA+ gene product, which also acts on N3-methylated purines . O4-Methylthymine is repaired by a methyltransferase, previously known to correct O6-methylguanine by transfer of the methyl group to one of its own cysteine residues . It is proposed that certain common structural features of the various methylated bases allow each of the two inducible repair enzymes to recognize and remove several different kinds of lesions from alkylated DNA. Ann Immunol (Paris), 1984 Mar-Apr, 135C(2), 251 - 60 {Assay of rabbit anti-thermolabile enterotoxin antibodies from Escherichia coli by an immunoenzyme technic}; Germani Y et al.; The titration of rabbit anti-Escherichia coli heat-labile enterotoxin antibodies by an immunosorbent assay (ELISA) generally uses a GM1 coating step to lend specificity to the ELISA reaction at a time when the method is tempered by a lack of purified E . coli toxin . We have developed an adsorption method of the toxin to the polystyrene in order to simplify the technique . Three different immunosorbent preparations were tested to determine which of them yielded the most sensitive results. Am J Vet Res, 1984 Mar, 45(3), 586 - 91 Role of prostaglandins in pathogenesis of bovine mastitis induced by Escherichia coli endotoxin; Giri SN et al.; Four doses (5 to 100 micrograms, 1 dose/quarter) of Escherichia coli endotoxin were introduced into lactating mammary glands of 2 cows . There was no effect on milk prostaglandin (PG) E2 concentration, except that the concentration was increased from 200 pg/ml of milk to 1,060 pg/ml at post-treatment hour (PTH) 8 in cow 1 and from 75 to 420 pg/ml at PTH 4 in cow 2 after the highest dose 100 micrograms . Endotoxin caused a dose-dependent increase in milk PGF2 alpha concentrations in both cows . After the highest dose, PGF2 alpha was maximally increased from 200 to 3,500 pg/ml at PTH 4 in cow 1 and from 250 to 2,000 pg/ml in cow 2 at PTH 8 . The instillation of 50 micrograms of endotoxin in all 8 quarters of 2 more lactating cows caused no significant (P greater than 0.05) changes in milk PGE2 and thromboxane B2 concentrations, whereas milk PGF2 alpha was significantly increased from the base-line value of 642 to 2,683, 1,189, and 2,281 pg/ml at PTH 4, 8, and 12, respectively . The 6-keto-PGF1 alpha was also significantly increased from the base-line value of 305 to 871, 631, and 600 pg/ml at the corresponding times, respectively . A marked increase in vascular permeability, as judged by high concentrations of serum albumin in the whey, was observed as early as PTH 4 and peaked at PTH 12 followed by a gradual decline, although it remained significantly increased over the control for 48 hours after treatment.(ABSTRACT TRUNCATED AT 250 WORDS) Am J Trop Med Hyg, 1984 Mar, 33(2), 281 - 4 Outbreak of invasive Escherichia coli gastroenteritis on a cruise ship; Snyder JD et al.; An invasive strain of Escherichia coli (ONT:NM) was isolated from stool specimens from 7 of 10 ill passengers who developed diarrhea during a 5-day ocean cruise . The ill passengers had shared no common exposures off the ship before or during the cruise . Three of the persons whose stools were cultured were part of a tour group of 219 persons, and a food consumption and health history questionnaire was completed by 190 members (87%) of this tour group . Forty-seven (25%) had had diarrhea during the cruise; other symptoms among those with diarrhea included nausea (72%), abdominal cramps (68%), headache (68%), chills (60%), dizziness (53%), myalgias (43%), subjective fever (36%), and vomiting (26%) . The median duration of symptoms was 3 days . Eating at cold buffets on ship and eating potato salad, a buffet food item, were significantly associated with illness . No evidence of secondary spread of illness in household contacts of the ill person was found. Proc Natl Acad Sci U S A, 1984 Mar, 81(6), 1844 - 8 Molecular model for elongation of the murein sacculus of Escherichia coli; Burman LG et al.; Labeling experiments are presented that suggest that new (radioactive) strands of murein are initially inserted adjacent to old strands . After 8 min, new strands start to be inserted adjacent to the previously inserted radioactive strands . Analysis of these data suggests that, for Escherichia coli to double the length of the sacculus in each generation, about 90 separate membrane-bound enzyme complexes travel unidirectionally around the circumference of the cell . They travel at a constant rate, six times each generation, synthesizing, inserting, and crosslinking two strands of murein at a time, thereby doubling the length of the sacculus. Proc Natl Acad Sci U S A, 1984 Mar, 81(6), 1624 - 8 Possible ideal lac operator: Escherichia coli lac operator-like sequences from eukaryotic genomes lack the central G X C pair; Simons A et al.; Five DNA fragments have been cloned from yeast, chicken, and mouse DNA that titrate lac repressor in an Escherichia coli lac+ I+Z+ wild-type strain when on a multi-copy plasmid . The five repressor-binding sequences have been identified by DNA sequence determinations and DNase cleavage-inhibition patterns . They share the 14-base-pair symmetrical consensus sequence 5' T-G-T-G-A-G-C:G-C-T-C-A-C-A 3' (the colon represents the center of symmetry), which is an inverted repeat of 7 base pairs of the left half of the E . coli lac operator . A similar perfect palindromic DNA fragment--an 11-base-pair inverted repeat of the left half of the lac operator--was synthesized . The cloned synthetic DNA 5' G-A-A-T-T-G-T-G-A-G-C:G-C-T-C-A-C-A-A-T-T-C 3' binds lac repressor 8-fold more tightly than does wild-type E . coli lac operator DNA. Proc Natl Acad Sci U S A, 1984 Mar, 81(5), 1394 - 7 Contact points between transcription machinery and the fibroin gene promoter deduced by functional tests of single-base substitution mutants; Hirose S et al.; An efficient method for oligonucleotide-directed mutagenesis was developed to construct a set of site-specific mutations by using a mixture of oligodeoxyribonucleotides . With this method, as high as 40% of the tested clones turned out to be desired mutants . Seven single-point mutants were isolated in the "TATA" box region of the fibroin gene . In vitro transcription experiments showed that single-base transversions at the TATA box (A----T at po