Cell, 1981 Dec, 27(3 Pt 2), 515 - 22 Regulation of SOS functions: purification of E . coli LexA protein and determination of its specific site cleaved by the RecA protein; Horii T et al.; The LexA protein of Escherichia coli was purified to more than 96% purity from cells harboring a recombinant plasmid carrying the lexA gene with the lacZ promoter sequence . The amino acid composition of the LexA protein and its amino-terminal sequence were analyzed . The results are in agreement with the prediction from the nucleotide sequence of the lexA gene . The LexA protein is cleaved into two polypeptides by E . coli RecA protein in the presence of ATP and single-stranded DNA . The site of the specific cleavage was determined by analyzing amino acid sequences of the cleaved products at the amino and carboxyl termini . The cleavage of the LexA protein by the RecA protein was found to occur at a single site between Ala84 and Gly85. Cell, 1981 Dec, 27(3 Pt 2), 523 - 31 Pausing and attenuation of in vitro transcription in the rrnB operon of E . coli; Kingston RE et al.; We have determined the effects of the nusA gene protein and the regulatory nucleotide guanosine tetraphosphate (ppGpp) on pausing and termination of transcription in the leader region of the rrnB operon in vitro . The leader region of rrnB contains several types of potential regulatory sequences that act at the level of RNA chain elongation and may be involved in control of bacterial growth . We have mapped a termination site, tL, located 260 bases from rrnB promoter P1 . Termination at tL is dependent on the nusA protein and is enhanced by ppGpp . The DNA sequence at tL shows striking homologies with trp t', a terminator also strongly affected in vitro by the nusA protein . These in vitro results suggest that rRNA transcription in vivo may be regulated in part through an attenuation mechanism that leads to termination of rrnB chains in the leader region . In addition to tL, elongation of transcription in the leader region is affected by several pause sites that are sensitive to the concentration of ppGpp and the presence of the nusA gene protein . The location and properties of these pause sites suggest that they may also play a role in regulation of rrnB transcription through a mechanism we have termed "turnstile" attenuation . One pause site, located 90 and 91 bases from P1, is unique in that it is not normally a site for transcriptional pausing, but is dependent on simultaneous binding of polymerase at rrnB promoter P2 . This leads to blockage of P1 transcripts at high RNA polymerase densities and may provide an additional locus for regulation of rrn transcription. Cell, 1981 Dec, 27(3 Pt 2), 507 - 14 Gene sequence of the lambda receptor, an outer membrane protein of E . coli K12; Clement JM et al.; The Escherichia coli K12 lambda receptor is a multifunctional outer membrane protein whose precursor, encoded in gene lamB, is cleaved during export . We present the DNA sequence of lamB and of the distal region that contains repetitive and palindromic sequences and could give rise to highly stable mRNA structures . The calculated molecular weight of the lambda receptor is 47,400 . Of the 421 amino acids, 89 are charged, mostly negatively . No region devoid of charged amino acids and long enough to serve as a transmembrane portion is detected . The distribution of charges presents special features that we comment upon in relation to the structure, functions and localization of the lambda receptor . Gene lamB is followed by molA, an unidentified reading frame corresponding to a 131-amino-acid peptide with the characteristics of an exported protein. Nucleic Acids Res, 1981 Nov 25, 9(22), 6103 - 114 The sequence of human serum albumin cDNA and its expression in E . coli; Lawn RM et al.; A recombinant plasmid has been constructed which contains the mature protein coding region of the human serum albumin (HSA) gene . Bacteria containing this plasmid synthesize HSA protein under control of the E . coli trp promoter-operator . The DNA sequence and predicted protein sequence of HSA were determined from the cDNA plasmid and are compared to existing data obtained from direct protein sequencing . The DNA sequence predicts a mature protein of 585 amino acids preceded by a 24 amino acid "prepro" peptide. Nucleic Acids Res, 1981 Nov 11, 9(21), 5689 - 96 Nucleotide sequence of the Aspergillus nidulans mitochondrial gene coding for the small ribosomal subunit RNA: homology to E . coli 16S rRNA; Kochel HG et al.; The complete primary structure of the 1437 bp gene coding for mitochondrial 15S rRNA and its flanking regions was determined by Maxam-Gilbert sequencing of cloned HindIII fragment H3 of A . nidulans mtDNA . The gene product reveals significant homology (59%) to E . coli 16S rRNA, and the potential secondary structures of both rRNA molecules are very similar, except that the hairpin structures 7, 8 and 30 of the Brimacombe 16S rRNA model are deleted, and that two sequences of 8 and 31 nucleotides are inserted in the mitochondrial species. Cell, 1981 Nov, 26(3 Pt 1), 333 - 43 Sensory transducers of E . coli are encoded by homologous genes; Boyd A et al.; The tsr and tar genes, which are widely separated in the E . coli genome, encode functionally analogous transducer proteins that focus and integrate two distinct classes of chemosensory information . Physical mapping of these genes was achieved by use of transposon Tn5 mutagenesis of cloned DNA fragments . The polar effects of Tn5 insertions in the tar-cheR-cheB-cheY-cheZ region indicated that these genes are cotranscribed from a promoter upstream of tar and revealed the existence of a new gene, tap (taxis-associated protein), lying between tar and cheR . DNA hybridization studies demonstrated that the tsr gene possesses sequence homologies with the tar and tap genes, suggesting that they have all evolved from a common ancestor . The tap gene encoded a polypeptide of apparent molecular weight of 65,000, which may constitute a transducer protein of unknown specificity. Cell, 1981 Nov, 26(3 Pt 1), 299 - 304 An E . coli gene coding for a protamine-like protein; Altman S et al.; Several open reading frames exist in the region of the tRNATyr/1 gene of the E . coli genome . One such sequence encodes a polypeptide 33 amino acids in length that, on the basis of size and amino acid sequence, bears a striking resemblance to the protamines found in trout sperm . DNA from the transducing phage phi 80 tRNATyr/1 su3+ directs in vitro synthesis of two small basic proteins that are not made when homologous DNA containing a deletion that overlaps both the su3+ gene and the gene for the putative protamine-like protein is used . DNA from phage that have regained the parental su3+ phenotype again direct in vitro synthesis of the basic proteins . Synthesis of basic protein is inhibited by the presence of ppGpp, as would be expected if the mRNA is part of a large RNA transcript that starts at the promoter for the tRNATyr/1 gene. Biokhimiia, 1981 Nov, 46(11), 2056 - 63 {Polypeptide synthesis dependent on kinetoplast DNA from Crithidia oncopelti in a cell-free system of E . coli}; Zaitseva GN et al.; The ability of a complex associate made up of maxi- and mini-circular molecules of kinetoplast DNA from C oncopelti to perform polypeptide synthesis in a cell-free system of conjugate transcription and translation of E . coli was demonstrated . The coding function of maxi-circular molecules and genetic inertness of mini-circular molecules of kinetoplast DNA during polypeptide synthesis in vitro was found . Total RNA from the kinetoplasts was shown to undergo translation in cell-free protein-synthesizing system of E . coli to form 9-10 polypeptides . The molecular weights of many polypeptides, whose synthesis in this system is performed by DNA and RNA from Kinetoplasts were found to be similar. Biofizika, 1981 Nov-Dec, 26(6), 1033 - 6 {Systems of H+-K+-exchange in E . coli . Interplay with ATPase complex F1.F0}; Martirosov SM et al.; A study has been made of the nature of H+-K+-exchanges in the strain E . coli AN 120 with mutation in gene uncA401 (the defect in Mg++-Ca++-ATPase) and in the strain E . coli AN 382 with mutation in gene uncB (F0 is insensitive to N,N1-dicyclohehylcarbodiimide, DCCD) . It was shown in E . coli AN 120 that no ATP-dependent and DCCD-sensitive TrkA system operates, exchanging 2H+ from a cell for K+ of the medium and actuating only in the presence of a difference of osmotic pressures between the cell and the medium . At the same time the strain E . coli AN 120 possesses An ATP-independent TrkF system exchanging H+ by K+ with variable stoichiometry and inhibited by DCCD . The TrkA and TrkF systems have lost the sensitivity to DCCD in E . coli AN 382 . Moreover the TrkA system in this mutant has turned insensitive to the variation of osmotic pressure in the medium, i . e . DCCD-insensitivity of the TrkA and TrkF systems as well as the osmotic sensitivity of the TrkA system are related to the F0 function . We do not exclude the chance of the ionic channel F0 being a portion of both systems of potassium accumulation. Gene, 1981 Nov, 15(2-3), 201 - 6 The identification and partial characterization of a plasmid containing the gene for the membrane-associated hydrogenase from E . coli; Glick BR et al.; Escherichia coli DNA was digested with restriction endonuclease PstI and ligated into the PstI site of plasmid pBR322 . Recombinant plasmids that were constructed in this manner were used to transform E . coli H61, a mutant with a decreased level of hydrogenase activity . Complementation of this hydrogenase mutation identified a bacterial clone carrying the gene for the membrane-associated E . coli hydrogenase in plasmid pBL101 . In E . coli minicells, the pBL101 DNA directed the synthesis of a protein of a size corresponding to that of the precursor of the E . coli membrane-associated hydrogenase, which appears to contain an uncleaved leader peptide . A restriction map of the cloned DNA was determined for 14 endonucleases. Int J Radiat Biol Relat Stud Phys Chem Med, 1981 Nov, 40(5), 507 - 24 Radiochemical cross-linking between proteins and RNA within 70 S ribosomal particles from E . coli MRE600; Giocanti N et al.; In the absence of oxygen, gamma-irradiation produces covalent links between some ribosomal proteins and 16 S RNA to 23 S RNA, within 70 S ribosomes from E . coli MRE600 . Under optimal conditions minimizing the structural modifications induced by radiations, in situ formed cross-links appear specific and reflect close RNA-protein contacts . In view of these results, the spatial organization of the 30 S, 50 S subunit interfaces is discussed . In addition, the gamma-irradiation technique reveals that subunit association induces modifications of some protein--RNA interactions. Biochim Biophys Acta, 1981 Oct 27, 655(3), 307 - 22 Polynucleotide synthetase of E . coli: an enzyme complex having polynucleotide phosphorylase as apoenzyme; Stavrianopoulos JG et al.; A previously described synthetase system of Escherichia coli that utilizes ribonucleoside triphosphates has been purified extensively and shown to consist of an apoenzyme and three protein factors . The apoenzyme itself was revealed to be polynucleotide phosphorylase . The conditions under which the latter - an enzyme incorporating nucleoside diphosphates - is converted to a system catalyzing the uptake of nucleoside triphosphates have been studied in detail with respect to primer requirements, the influence of triphosphates on diphosphate utilization and vice versa, and the possibly regulatory effect of the guanosine di- and triphosphates . The fully supplemented enzyme system (polynucleotide synthetase) incorporates GTP only in the presence of ATP, producing a polynucleotide with an A : G ratio near unity. Biochim Biophys Acta, 1981 Oct 27, 655(3), 446 - 8 Terminal strand-switching of E . coli RNA polymerase transcribing a truncated DNA fragment; Oostra BA et al.; When transcribing a restriction fragment containing the promoters and the first part of the rrnE operon of Escherichia coli, RNA polymerase holoenzyme starts exclusively on the promoters . Besides run-off transcripts, molecules longer than template-size are formed by terminal strand switch. Nucleic Acids Res, 1981 Oct 24, 9(20), 5483 - 92 Protein synthesis and competitive ESR binding studies with E . coli ribosomes and spin-labeled polynucleotides; Ozinskas AJ et al.; Enzymatically prepared spin labeled copolymers of (U)n were tested for their ability to direct polyphenylalanine synthesis in vitro using E . coli B enzymes and ribosomes . Spin labeling of the C5 position using (RUGT,U)n (1:100) or (RUTT,U)n (1:100) did not alter the amount of polyphenylalanine formed in comparison to (U)n . In contrast, the C4 spin labeled copolymer (ls4U,U)n (1:100) reduced phenylalanine incorporation by 70-75% of the (U)n control levels . ESR monitoring of competitive ribosome binding to equimolar mixtures of polynucleotides was demonstrated with the macromolecular probe (DUTT,dT)n (1:100), the DNA analogue of (RUTT,U)n . The ESR competition approach showed that the affinity of the ribosomes was essentially the same for (dT)n, (A,U,G)n, and (A,U,G)n + tRNArmet. Jpn J Antibiot, 1981 Oct, 34(10), 1410 - 5 {Study of cefamandole in neonatal purulent meningitis caused by E . coli (author's transl)}; Yamamoto T et al.; Cefamandole (CMD) was intravenously drip infusion administered at daily dose of 400 mg/kg to the neonate with purulent meningitis caused by E . coli which was resistant to ABPC . In clinical application, CMD was evaluated as effective, although 6 mg/kg/day of GM given concomitantly . No adverse effect and abnormal laboratory findings were observed . This study would support the clinical usefulness of CMD in severe neonatal infection especially like meningitis. Cell, 1981 Oct, 26(2 Pt 2), 205 - 11 Regulation of the S10 ribosomal protein operon in E . coli: nucleotide sequence at the start of the operon; Olins PO et al.; We have determined the DNA sequence of a 1250 base pair segment of the Escherichia coli chromosome that carries the promoter for the S10 ribosomal protein operon, the S10 gene and part of the L3 gene . A DNA fragment carrying the putative S10 promoter was cloned into the plasmid mini-Col E1, which contains a transcription termination signal close to the single Hind II site . Cells harboring the hybrid plasmid produced a relatively stable hybrid mRNA with the expected sequence, demonstrating that the promoter functions in vivo . Comparison of the mRNA sequence around the start of the S10 coding region, the presumed target site for L4 repressor protein, with the known binding site for L4 on 23S rRNA revealed the presence of sequence homologies . This supports the model of the translational feedback regulation of the S10 operon by L4. Poult Sci, 1981 Oct, 60(10), 2189 - 94 Rectal temperature and blood chemical responses of young chickens given E . coli endotoxin; Jones CA et al.; Two experiments were conducted to investigate the effect of intravenous injections of bacterial endotoxin in broiler chicks . In both experiments 5-weeks-old chicks were given a single intravenous injection of either distilled water (control) or 1 mg/kg Escherichia coli endotoxin (serotype 0.127:B8) in a volume of 1 mg/kg . In Experiment 1 rectal body temperatures were taken every hour for 24 hr postinjection . In Experiment 2, rectal temperatures and blood samples were taken at 3, 6, 9, 12, 15, and 24 hr following the administration of endotoxin . Hematocrits and plasma glucose, protein, sodium, potassium, magnesium, calcium, and inorganic phosphorus were measured at each time interval . Birds receiving endotoxin showed a significant increase in rectal temperature from 2 hr until 17 hr postinjection . The febrile response was biphasic with temperatures peaking at 3 to 5 hr and again at 9 to 12 hr . No significant changes in hematocrits occurred following endotoxin injection . Plasma protein, potassium, and calcium decreased significantly, while glucose increased significantly after endotoxin administration . No significant changes in plasma magnesium, sodium, or inorganic phosphorus were observed in endotoxin-treated chicks. J Biochem (Tokyo), 1981 Oct, 90(4), 1117 - 24 Selective photooxidation of histidine residues in polypeptide chain elongation factor Tu from E . coli; Nakamura S et al.; When EF-Tu was photooxidized for 20 min at 0 degrees C in the presence of 10 microM GDP and 5 microM rose bengal, the activity to promote the binding of {14C}Phe-tRNA to ribosomes was rapidly lost, while the activity to bind {3H}GDP remained intact . The activity of EF-Tu to interact with Phe-tRNA and ribosomes, as assessed by protection of {14C}Phe-tRNA against RNase A digestion and by methanol-induced uncoupled GTPase activity, respectively, was also inactivated under the above conditions . It was found, however, that these activities were fully protected in the presence of aminoacyl-tRNA and GTP, indicating that the active site(s) of EF-Tu for interaction with aminoacyl-tRNA and ribosomes could be protected against photooxidation in the ternary aminoacyl-tRNA . EF-Tu . GTP complex . Comparison of the amino acid composition of EF-Tu photooxidized in the form of EF-Tu . GDP with that of the intact EF-Tu revealed that only 1.4 residues of histidine were damaged . On the other hand, no histidine residue was lost when EF-Tu was oxidized in the presence of both aminoacyl-tRNA and GTP . The photooxidized EF-Tu . GDP was then partially degraded with trypsin and each of the resulting tryptic fragments, D, B, and C (Arai, Nakamura, Arai, Kawakita, and Kaziro (1976) J . Biochem . 79, 69-83), was analyzed for histidine content . The results indicated that fragments B, C, and D had lost 0.7, 0.5, and 0.2 residues of histidine, respectively . Since fragment B contains the cysteine residue which is essential for interaction with aminoacyl-tRNA and ribosomes, the above results suggest that a histidine residue in fragment B may also play an essential role in the interaction with aminoacyl-tRNA and ribosomes. Biokhimiia, 1981 Oct, 46(10), 1773 - 9 {Cobalamins and tRNA methyltransferase activity in E . coli cells}; Kal'nev VR et al.; The end of the logarithmic phase of growth of E . coli 113-3 cells in a mineral medium with methylcobalamin (MeCbl) occurs 60 minutes earlier than in the case of cells grown with cyancobalamin (CNCbl) . Chromatography on a Sephadex - DEAE cellulose column of the 105 000xg fractions of cell-free extracts of the cells grown in the presence of MeCbl showed both quantitative and qualitative differences . The ability of individual protein fractions to transfer the 14CH3-groups from the Adomet to submethylated tRNA of E . coli K12W6 in the cells grown with MeCbl increases 1.5 - 3-fold in comparison with those grown with CNCbl . A chromatographic analysis of methylated bases of tRNA did not show any qualitative differences . In the presence of MeCbl the incorporation of the CH3-groups into m7G is increased by 50%. Biochim Biophys Acta, 1981 Sep 29, 670(2), 294 - 7 Phosphorescence of alkaline phosphatase of E . coli in vitro and in situ; Horie T et al.; Escherichia coli K-12, which is rich in alkaline phosphatase, exhibits phosphorescence characteristic of tryptophan at room temperature . E coli mutants which do not have alkaline phosphatase do not show long-lived phosphorescence . The phosphorescence spectrum and lifetime of E . coli K-12 was similar to that of purified alkaline phosphatase from E . coli . These results indicate that the long-lived tryptophan phosphorescence in E . coli is likely to be derived from alkaline phosphatase in situ . The temperature dependence of tryptophan phosphorescence life-time of purified alkaline phosphatase and E . coli K-12 differ; this may imply that alkaline phosphatase in E . coli may be associated with the cell envelope and is therefore protected against structural changes in the protein which result in increased phosphorescence decay rates. Biochim Biophys Acta, 1981 Sep 28, 655(2), 243 - 50 Transformation of E . coli using homopolymer-linked plasmid chimeras; Peacock SL et al.; A number of parameters were explored to increase the transformation efficiency of E . coli with pBR322/eukaryotic DNA chimera, formed via d(A) . d(T) and d(G) . d(C) homopolymer tails . Of the E . coli strains analyzed, E . coli strain RR1 was the most efficient bacterial host . A clear optimum of nucleotide tail length existed for both types of homopolymer . The optimum hybridization temperature for chimera formation was found to be approx . 57 degrees C . In the case of d(A) . d(T)-linked chimeras, 30 min was sufficient for optimum chimera formation . In contrast, d(C) . d(G)-linked chimeras required up to 2 h to give the best yields (as measured by transformation efficiency) . Other minor factors affecting the transformation process are also explored and discussed. Nucleic Acids Res, 1981 Sep 25, 9(18), 4459 - 74 Identification of gene products programmed by restriction endonuclease DNA fragments using an E . coli in vitro system; Pratt JM et al.; DNA restriction enzyme fragments have been used to programme the synthesis of polypeptides in an in vitro system without apparent loss in fidelity compared with supercoiled templates . The system is extremely sensitive, less than 1 microgram of DNA can be used to direct the synthesis of 35S-labelled polypeptides of sufficiently high specific activity such that products can be identified by SDS-PAGE after a few hours autoradiography . The ability to analyse fragments can be used to readily assign specific proteins to small regions of the coding template, to identify cloned gene products distinct from those of the vector, and to identify cloned genes expressed from their own promoters . The in vitro system can be used successfully with bacterial DNA from other species and efficient extracts can be prepared from any E . coli K-12 strain, which should greatly facilitate the purification of factors controlling the expression of specific genes by complementation assay. Nucleic Acids Res, 1981 Sep 25, 9(18), 4669 - 76 Nucleotide sequence of the asnA gene coding for asparagine synthetase of E . coli K-12; Nakamura M et al.; We have subcloned the asnA gene of E . coli K-12, a gene coding for asparagine synthetase, from a previously cloned 6 mega-dalton segment of E . coli chromosome containing the DNA replication origin, ori, and asnA . The complete nucleotide sequence of the asnA gene was determined: the region of the structural gene extends 990 base-pairs at nucleotide positions 1434-2423 (see Fig . 3), which codes for a polypeptide of 330 amino-acid residues with a molecular weight of 36,688 daltons . The nucleotide sequences of the promoter and the ribosome-binding site of the gene are also assigned . We discuss the properties of its polypeptide. Genetics, 1981 Sep, 99(1), 1 - 23 Periodic selection, infectious gene exchange and the genetic structure of E . coli populations; Levin BR; As a consequence of sequential replacements by clones of higher fitness (periodic selection), bacterial populations would be continually purged of genetic variability, and the fate of selectively neutral alleles in very large populations of bacteria would be similar to that in demes of sexually reproducing organisms with small genetically effective population sizes . The significance of periodic selection in reducing genetic variability in these clonally reproducing species is dependent on the amount of genetic exchange between clones (recombination) . In an effort to determine the relationship between the rates of periodic selection, recombination and the genetically effective sizes of bacterial populations, a model for periodic selection and infectious gene exchange has been developed and its properties analyzed . It shows that, for a given periodic selection regime, genetically effective population size increases exponentially with the rate of recombination.--With the parameters of this model in the range anticipated for natural populations of E . coli, the purging effects of periodic selection on genetic variability are significant; individual populations or lineages of this bacterial species would have very small genetically effective population sizes.--Based on this result, some other a priori considerations and a review of the results of epidemiological and genetic variability studies, it is postulated that E . coli is composed of a relatively limited number of geographically widespread and genetically nearly isolated and monomorphic lineages . The implications of these considerations of the genetic structure of E . coli populations on the interpretation of protein variation and the neutral gene hypothesis are discussed. Biofizika, 1981 Sep-Oct, 26(5), 817 - 21 {Independent functioning of the H+-K+ ion exchange systems in E . coli}; Martirosov SM et al.; Two systems of potassium accumulation inhibited by N,N1-diciclohexilcarbodiimide (DCCD) can operate simultaneously and independently in the strain E . coli TK 1001 . These are the TrkA and TrkF systems . The strain E . coli TK 509 demonstrates only the TrkF system in the media with potassium concentrations higher than 1 meg . 1-1 . The onset of K+-uptake via the TrkF is observed much earlier than that in E . coli TK 1001 . Moreover the secretion of H+ increases considerably with the start of K+-uptake . DCCD-sensitive and counter-directed fluxes of H+ and K+ enhance proportionally both the increase of external potassium concentration and pH . Thus the E . coli cells possess at least two proton-potassium exchanging systems (TrkA and TrkF) sensitive to DCCD . The TrkA system operates a short time in response to an increase of the osmolarity in the medium and exchanges rapidly 2H+ for 1K+, whereas the TrkF maintains a slow exchange of H+ for K+ with the unstable ratio H+/K+ from 4 to 14. Sci Sin, 1981 Sep, 24(9), 1285 - 91 Tyrosine micro-region of E . coli L-asparaginase; Zhongxiao H et al.; The relationship between conformation change and activity of E . coli L-asparaginase has been studied with circular dichroism spectra and microcaloric methods . In many papers, it has been pointed out that the active site of L-asparaginase is closely related to tyrosyl residues . The present authors have studied the effects of L-cysteine on the activity and the conformation of L-asparaginase with UV difference spectra and kinetic methods . Moreover, we have studied the space arrangement of tyrosyl residues in the enzyme molecule . The results show that every enzyme molecule contains about 56 tyrosyl residues, 20 of which are in the hydrophobic core of the enzyme molecule, another 20 at the surface of the enzyme molecule, and the rest in the rifts and hollows of the enzyme molecule . Meanwhile, further study has also been made to determine the relationship between the changes of the enzyme activity and the ionization of tyrosyl residues as well as their chemical modification . By Zou Chenglu's graphical method we have proved that two tyrosyl residues at the surface of the enzyme molecule are the essential groups. Biokhimiia, 1981 Sep, 46(9), 1640 - 5 {Preparation of RNAase-free E . coli ribosomes active in initiation of protein biosynthesis}; Tsielens IE et al.; The level of contaminating RNAases in the main components of the protein biosynthesis initiation system, the initiation factors and ribosomes of E . coli, was studied . It was shown that the ribosomes are the major source of contaminating RNAases . A simple procedure for purification of ribosomes active in initiation including Sephadex G-200 gel-filtration of unwashed ribosomes in a 1.5 M NH4Cl-containing buffer was developed. Cell, 1981 Sep, 25(3), 765 - 72 E . coli mutant pleiotropically defective in the export of secreted proteins; Oliver DB et al.; A hybrid beta-galactosidase molecule containing a substantial portion of the amino-terminal sequence of the maltose-binding protein is inserted in the cytoplasmic membrane of E . coli; in this location, the protein has very low enzymatic activity . The strain producing it is, therefore, Lac- . Selection for derivatives of the fusion strain that are able to grow on lactose yields mutants in which the hybrid protein has become cytoplasmic, and thus has higher enzymatic activity . Among such derivatives, we have isolated a temperature-sensitive conditional lethal mutant that accumulates the precursor of the maltose-binding protein in the cytoplasm, and also accumulates precursors of alkaline phosphatase, lambda receptor protein and the ompF gene gene product . A number of periplasmic proteins are, however, properly localized at the nonpermissive temperature . The temperature-sensitive lesion has been genetically mapped to 2.5 min on the E . coli map, within or near a cluster of genes responsible for cell division and septation . The principle behind the genetic selection employed here should be useful in obtaining other secretion mutants to characterize the cell's secretion machinery. J Immunol, 1981 Sep, 127(3), 1152 - 6 How complement kills E . coli . II . The apparent two-hit nature of the lethal event; Wright SD et al.; We have studied the nature of complement (C) action on red blood cells and E . coli with respect to the number of "hits" required for membrane damage . Our method of analysis involves adding various amounts of purified C7 or C8 to serum preparations immunochemically depleted of C7 or C8, respectively, in order to construct dose-response curves for the action of C's terminal complex . The shape of the dose-response curves reflects the single or multiple-hit nature of C action . Our method confirms that C acts on red cells by a 1-hit mechanism, whether measured by lysis or by the permeation of a small molecule . In contrast, we find with E . coli that C-mediated outer membrane damage, inner membrane damage, and killing all appear to require more than 1 hit . We have also discovered a property of E . coli that displays a nonlethal 1-hit response to C that is particularly useful in the analysis of multiple-hit dose-response curves . Simultaneous measurements of this single-hit phenomenon and the multiple-hit killing of E . coli allow us to make direct comparisons of the amount of C needed for each response . On the basis of the midpoints of the single and multiple-hit curves, C-mediated membrane damage and killing of E . coli appear to be a 2-hit process. J Immunol, 1981 Sep, 127(3), 1146 - 51 How complement kills E . coli . I . Location of the lethal lesion; Wright SD et al.; We have studied the action of human complement (C) on E . coli membranes . We find, as have others, that C disrupts the outer membrane (OM), allowing the release of periplasmic proteins . In addition, we have found 1) that in the complete absence of lysozyme, C damages the inner membrane (IM), 2) IM damage is different from OM damage in that only small molecules traverse a damaged IM whereas macromolecules traverse damaged OM, 3) IM damage and OM damage occur with identical kinetics and dose response, suggesting that IM and OM damage are closely coupled events, and 4) upon the addition of purified C8 and C9 to the washed cellular intermediate, E . coli C 1-7, both IM and OM are damaged coordinately . These results, taken together, suggest that C damages E . coli membranes by acting at a site contiguous with both membranes . We speculate that C may simultaneously gain access to both membranes by acting at the junctions between IM and OM. Biochimie, 1981 Aug-Sep, 63(8-9), 699 - 707 Effects of partial deproteinization on the functional properties of 50S ribosomal subunits of E . coli; Guerin MF et al.; Sedimentation of E . coli 50S ribosomal subunits through sucrose gradients containing 10 mM Mg2+ and high concentrations of NaCl (0.1-1.0 M) leads to removal of proteins L16 and L25 . Analyses of the structural and functional properties of the protein depleted particles shows that removal of L16 and L25 from the 50S subunit causes loss of its ability to bind tRNA, to associate with the 30S subunit and to catalyze peptide bond formation . Reassociation of both L16 and L25 with core particles lacking these proteins is necessary for recovery of peptidyl transferase activity. Cell, 1981 Aug, 25(2), 507 - 16 Concerted strand exchange and formation of Holliday structures by E . coli RecA protein; DasGupta C et al.; RecA protein makes stable joint molecules from fully duplex DNA and molecules that are partially single-stranded; the latter may be either duplex molecules with an internal gap in one strand or molecules with single-stranded ends . Stable joint molecules form only when the end of at least one strand is in a homologous region . When RecA protein pairs linear duplex molecules and tailed molecules that share the same sequence end to end, the joints, which are located away from the single-stranded tails in most instances, have the electron microscopic appearance associated with the Holliday structure resulting from the reciprocal exchange of strands . The reaction leading to reciprocal strand exchange involves the concerted displacement of a strand from the end of the duplex molecule . These observations support the view that RecA protein makes stable joint molecules only by transferring strands and not by the side-by-side pairing of duplex regions. Cell, 1981 Aug, 25(2), 333 - 40 The methyl-accepting chemotaxis proteins of E . coli: a repellent-stimulated, covalent modification, distinct from methylation; Rollins C et al.; The methyl-accepting chemotaxis proteins (MCPs) of Escherichia coli are integral membrane proteins that have been shown to undergo reversible methylation in response to the addition of attractants . We have shown that a second, rapid modification of MCPI and MCPII occurs, which is repellent-stimulated . This modification, which is not methylation, was detected because it causes a decrease in mobility of the MCPs on 7.5% SDS-polyacrylamide gels with a high acrylamide to bisacrylamide ratio . We have designated this modification as the CheB-modification, as it is dependent on the CheB gene product . The CheB-modification causes a decrease in the isoelectric point of MCPII by one or two charge groups . The CheB-modification is not necessary for the methylation, nor does it preclude methylation of the MCPs . Both the CheB-modified form and the unmodified, unmethylated forms of the MCPs are stable to treatment with base, which results in the hydrolysis of the methylesters (demethylation) of the MCPs . The potential role of CheB-modification in chemotaxis is discussed. Atherosclerosis, 1981 Aug-Sep, 40(1), 65 - 73 Endothelial cell damage in piglet coronary artery after intravenous administration of E . coli endotoxin . A scanning and transmission electron-microscopic study; Pesonen E et al.; E . Coli endotoxin was administered to 6 piglets from a litter of 10 . Three days after the endotoxin stimulus 3 piglets showed definitive morphological evidence of endothelial damage to their left coronary artery . The proximal parts of the coronary artery were severely damaged . In scanning electron microscopy, the changes varied from disappearance of the microvilli to complete exfoliation of the endothelial cells . In cases of severe endothelial cell damage transmission electron microscopy revealed severe changes or even signs of cell death in the inner medial smooth muscle cells . Only of the piglets died prematurely . We are sufficiently encouraged to continue testing the theory that repeated endothelial cell damage initiates stenosing lesions in the coronary arteries. Biokhimiia, 1981 Aug, 46(8), 1488 - 98 {Properties and role of tryptophan residues in the polypeptide chain of elongation factor G from E . coli}; Kashparov IA et al.; Using chemical modification and spectrofluorimetry, it was shown that two tryptophane residues of the elongation factor G (EF-G) in positions 51 and 71 from the N-terminus are located on the surface of the EF-G molecule . The tryptophane residue in position 71 is effectively shielded against modification at binding of EF-G guanyl nucleotides . Modification of these tryptophane residues does not result in a loss of the nucleotide-binding activity but completely inhibits EF-G binding to the ribosome . Polarized fluorescence study showed that the relaxation properties of these exposed tryptophane residues essentially depend both on the presence of the C-terminal domain and on binding of nucleotides in the nucleotide-binding site located in the N-terminal part of EF-G . It was assumed that the C-terminal domain, the nucleotide-binding site and the site responsible for the EF-G binding to the ribosome are brought together in the three-dimensional structure of the elongation factor G. Nucleic Acids Res, 1981 Jul 24, 9(14), 3503 - 22 Studies on transfer ribonucleic acids and related compounds . XL . Synthesis of an eicosaribonucleotide corresponding to residues 35-54 of tRNAfMet from E . coli; Ohtsuka E et al.; An E . coli tRNAfMet fragment {C-A-U-A-A-C-C-C-G-A-A-G-G-U-C-G-U-C-G-G (bases 35-f54)} containing the anticodon triplet has been synthesized by the phosphotriester method involving protected oligonucleotide blocks . Di- or tri-nucleotide blocks were prepared by condensation of 2'-O-(o-nitrobenzyl) nucleotide derivatives and used for the synthesis of pentanucleotide blocks . The 5'-hydroxy, heterocyclic amino and internucleotide linkage were protected with monomethoxytrityl, acyl and p-chlorophenyl groups, respectively . The 3'-phosphates of the pentanucleotides, except for the GUCGG block where 2'-O-benzoyl 3'-O-(o-nitrobenzyl) N-isobutyrylguanosine was used, were protected with p-chlorophenyl and anilido groups . The anilido groups were removed by treatment with isoamyl nitrite and the 3'-phosphodiesters of resulting pentamers were activated with mesitylenesulfonyl nitrotriazolide to give protected decanucleotides in yields of 61-89% . The two decanucleotides were condensed similarly to yield the protected eicosanucleotide in a yield of 59% . The product was deblocked and purified by ion-exchange chromatography on DEAE-Sephadex A-25 and characterized by enzymatic hydrolysis after labelling the 5'-end by phosphorylation using polynucleotide kinase and {gamma-32P}ATP. Nucleic Acids Res, 1981 Jul 24, 9(14), 3491 - 501 Mechanism of ultraviolet-induced mutagenesis: the coding properties of ultraviolet-irradiated poly(dC) replicated by E . coli DNA polymerase I; Lecomte P et al.; We have identified three lesions rather than cyclobutane dimers which alter the properties of UV-irradiated poly(dC) as a template for E.coli DNA polymerase I, and have characterised these lesions with respect to their coding properties, rates of formation and decay, and their sensitivity to uracil DNA glycosylase . Our results lead us to conclude that these lesions are (1) cytosine hydrates, which code for cytosine and to a lesser extent thymine, (2) uracil hydrates, which code for adenine and are not sensitive to uracil DNA glycosylase, and (3) uracils, which code for adenine and are removed by uracil DNA glycosylase. Cell, 1981 Jul, 25(1), 259 - 67 Induction of SOS functions: regulation of proteolytic activity of E . coli RecA protein by interaction with DNA and nucleoside triphosphate; Phizicky EM et al.; Damage to cellular DNA or interruption of chromosomal DNA synthesis leads to induction of the SOS functions in E . coli . The immediate agent of induction is the RecA protein, which proteolytically cleaves and inactivates repressors, leading to induction of genes they control . RecA protein modified by tif mutations allows expression of SOS functions in the absence of inducing treatments . We show here that tif-mutant RecA protein is more efficient than wild-type RecA protein in interacting with DNA and nucleoside triphosphate . This result suggests that formation of a complex with DNA and nucleoside triphosphate is the critical event that activates RecA protein to destroy repressors after SOS-inducing treatments, and that damage to cellular DNA promotes this reaction by providing single-stranded DNA or active nucleoside triphosphate or both . Since dATP is the most effective nucleoside triphosphate in promoting repressor cleavage, we suggest that it is the natural cofactor of recA protein in vivo. Biokhimiia, 1981 Jul, 46(7), 1249 - 57 {Comparative study of properties of periplasmic and membrane-bound alkaline phosphatase of E . coli}; Krupianko VI et al.; The properties of three forms of periplasmic and one form of membrane-bound alkaline phosphatase of E . coli were studied . A practically complete agreement between the conditions for optimal activity of these enzymes (pHopt 9.5 +/- 0.1), effect of ionic strength and temperature, and accordance between the substrate specificities (the enzymes decompose all types of phosphate-containing ether bonds) were observed . A similarity was shown between the kinetic parameters (Km = 3.9 x 10(-5) + 4.3 x 10(-5) M) for the reaction of p-nitrophenylphosphate decomposition), type and constants of inhibition (Ki) of these enzymes by phosphate-containing compounds as well as between the values of energy (E = 5.95 + 6.2 kcal/mole) for activation of decomposition of this substrate . A structural similarity of active sites of these enzymes was assumed . It was also suggested that the membrane-bound form of the enzyme is a precursor of the periplasmic one. Biokhimiia, 1981 Jul, 46(7), 1267 - 76 {Dependence of threonine operon expression on the relA gene allelic state and the level of guanosine tetraphosphate in E . coli}; Perel'man BV et al.; The expression of threonine operon in Rel+ and Rel- E . coli cells was studied at the transcriptional level . The rel+ genotype and the lack of a specific amino acid--threonine--are necessary for the stimulation of threonine operon transcription . Under these conditions the RNA fraction, which is thr-mRNA, is increased 2-fold . The lack of arginine or histidine in Rel+ strain does not lead to derepression of the threonine operon . It is shown that in a cell-free system 0.1 mM ppGpp stimulate the synthesis of thr-mRNA on phage DNA lambda dthr and plasmid DNA PYN1107, containing total threonine operon 1.5--2-fold . It is assumed that ppGpp stimulates the initiation of transcription . Studies on the strain carrying spoT- mutation, which significantly lowers the rate of ppGpp degradation and results in suppression of rel- phenotype, revealed positive correlation between the intracellular level of ppGpp and the thr-mRNA fraction in the total transcript. Cell, 1981 Jul, 25(1), 251 - 8 Location of the tufB promoter of E . coli: cotranscription of tufB with four transfer RNA genes; Lee JS et al.; Previous nucleotide sequence studies have demonstrated that the structural genes for four transfer RNA species (thrU, tyrU, glyT and thrT) are positioned close to one another and near tufB, one of the structural genes for elongation factor Tu . We have carried out experiments to determine the position of the tufB promoter and thus to infer whether these five genes are in a single transcription unit . tufB cloned on plasmids was fused to Tc (in operon fusion) or to lacZ (in gene fusion) . These plasmids were then subjected to in vitro deletion to locate the promoter responsible for tufB transcription . In addition, the ability of wild-type tufB to complement kirromycin resistance was determined with deletion plasmids . The results indicate that the major promoter for tufB lies upstream from the four transfer RNA genes, and that there might be at least one weak internal promotor, possibly adjacent to tufB . Assuming that the four tRNA genes that lie between the major promoter and tufB are also transcribed from that promoter, we suggest that all five genes lie in a single transcription unit (thrUp-thrU-tyrU-glyT-thrT-tufB-tufBt) whose primary transcript is thus both a transfer RNA precursor and messenger RNA. Nucleic Acids Res, 1981 Jun 25, 9(12), 2853 - 69 The rRNA operon from Zea mays chloroplasts: nucleotide sequence of 23S rDNA and its homology with E.coli 23S rDNA; Edwards K et al.; The nucleotide sequence of 23S rDNA from Zea mays chloroplasts has been determined . Alignment with 23S rDNA from E.coli reveals 71 percent homology when maize 4.5S rDNA is included as an equivalent of the 3' end of E.coli 23S rDNA . Among the conserved sequences are sites for base modification . Chloramphenicol sensitivity and ribosomal subunit interaction . A proposal for the base pairs formed between 16S and 23S rRNAs during the 30S/50S subunit interaction is presented . The alignment of maize 23S rDNA with that of E.coli reveals three small insertion sequences of 25, 65 and 78 base pairs, whereas maize 16S rDNA shows only deletions when compared with the E.coli species. Nucleic Acids Res, 1981 Jun 25, 9(12), 2889 - 903 The nucleotide sequence of the cloned rpoD gene for the RNA polymerase sigma subunit from E coli K12; Burton Z et al.; We have determined the nucleotide sequence of the rpoD gene which codes for the sigma subunit of RNA polymerase from E . coli K12 . The gene, which we formerly cloned as a HindIII restriction fragment in the transducing phage, charon 25, was recloned into several plasmids . We have determined a 2600 base pair DNA sequence which includes the entire structural gene for sigma . The resulting amino acid sequence agrees with previous information obtained about sigma including the amino acid composition, partial sequence data for the N-terminus, the highly acidic nature of the polypeptide, and the cleavage pattern at cysteines . The molecular weight of 70,263 daltons calculated for the 613 amino acid polypeptides is significantly lower than had been determined previously by SDS polyacrylamide gel analysis. Gene, 1981 Jun-Jul, 14(1-2), 73 - 80 The cloning and analysis of the aroD gene of E . coli K-12; Kinghorn JR et al.; A 5.6-kb PstI fragment containing the structural gene (aroD) for 5-dehydroquinate hydrolyase (DHQase) of Escherichia coli K-12 has been cloned into recombinant plasmid pJKK12 . The bacterial fragment contains two Bg/II, one HpaII, one SalI and one XhoI site, but no EcoRI, HindIII or BamHI sites . the DHQase activity extracted from strains harboring pJKK12 had properties identical to those of the enzyme isolated from wild-type E . coli . The native protein appears to be a dimer composed of two 31 500 dalton subunits . aroD6 strains transformed with pJKK12 had an 11-fold and 34-fold increase in activity compared to untransformed wild-type controls grown on L broth and minimal medium, respectively . No increase of dehydroquinase activity was found in polynucleotide phosphorylase deficient strains of E . coli . At least four constitutively expressed genes are encoded on the fragment. Cell, 1981 Jun, 24(3), 707 - 17 Protein localization in E . coli: is there a common step in the secretion of periplasmic and outer-membrane proteins? Ito K, Bassford PJ Jr, Beckwith J. An E . coli strain carrying a fusion of the MalE and lacZ genes is induced for the synthesis of a hybrid protein, consisting of the N-terminal part of the maltose-binding protein and the enzymatically active C-terminal part of beta-galactosidase, by addition of maltose to cells . The secretion of the protein is initiated by the signal peptide attached to the N terminus of the maltose-binding protein sequence, but is not completed, presumably because the beta-galactosidase moiety of the hybrid protein interferes with the passage of the polypeptide through the cytoplasmic membrane . Thus the protein becomes stuck to the cytoplasmic membrane . Under such conditions, periplasmic proteins, including maltose-binding protein (encoded by the malE gene) and alkaline phosphatase, and the major outer-membrane proteins, including OmpF, OmpA and probably lipoprotein, are synthesized as precursor forms with unprocessed signal sequences . This effect is observed within 15 min after high levels of induction are achieved . The simplest explanation for these results and those of pulse-chase experiments is that specific sites in the cytoplasmic membrane become progressively occupied by the hybrid protein, resulting in an inhibition of normal localization and processing of periplasmic and outer-membrane proteins . These results suggest that most of the periplasmic and outer-membrane proteins share a common step in localization before the polypeptide becomes accessible to the processing enzyme . If this interpretation is correct, we can estimate that an E . coli cell has roughly 2 x 10(4) such sites in the cytoplasmic membrane . A system is described for detecting the precursor of any exported protein. Cell, 1981 Jun, 24(3), 695 - 706 Regulation of the synthesis of E . coli elongation factor Tu; Young FS et al.; Rapidly growing E . coli with two active genes (tufA, tufB) for elongation factor (EF) Tu contains three times as much EF-Tu (tuf) mRNA as EF-G (fus) mRNA on a molar basis, but about seven times as much EF-Tu as EF-G or ribosomes . The concentration and translational efficiency of fus (EF-G) mRNA is about that for ribosomal protein mRNAs . The high molar concentration of EF-Tu relative to EF-G or ribosomes is achieved in part by translating tuf mRNA more efficiently than these other mRNAs . In a tufA+ tufB-: : Mu strain, the tuf:fus mRNA ratio is 1, but the concentration of EF-Tu and tuf mRNA is the same as in the wild-type strain . Thus cells with only an active tufA gene increase the concentration but not the translational efficiency of tuf mRNA . In such cells the concentration of fus mRNA is almost three times that in the wild-type strain . Because the tufA gene is distal to but cotranscribed with the fus gene as part of the four gene str operon, the wild-type concentration of tuf mRNA in these tufB- cells must be produced by increasing the concentration of transcript corresponding to the entire str operon . Thus transcription of the tufA gene can only proceed from the str promoter . Extracts of the tufB- cells contain tuf transcripts that correspond not only in size to the entire 4.5 kb str operon, but also to the size (approximately 1 kb) of a tuf gene . Our evidence suggests that this 1 kb tuf transcript is derived by posttranscriptional modification of the primary str operon transcript and that this modification could in part explain the high translational efficiency of tuf mRNA. Nucleic Acids Res, 1981 May 25, 9(10), 2397 - 410 The properties of ATP-analogs in initiation of RNA synthesis catalyzed by RNA polymerase from E coli; Smagowicz WJ et al.; Various base and sugar modified derivatives of ATP and UTP were used as substrate analogs for the steady state initiation reaction ATP+UTP=pppApU and the single step addition reaction ApC+ATP=ApCpA . These reactions were carried out by E . coli RNA polymerase on T7 DNA in the presence of rifampicin . The steady state kinetic parameters of the analogs, either as substrates or inhibitors, were determined . On the basis of the obtained results it is concluded that purine NTP s in initiation require anti-conformation about the glycosidic bonds as well as gauche-gauche conformation of the C(4')-C(5') bonds . The latter conformation is also a prerequisite for substrates in elongation, whereas strict anti-conformation of glycosidic bonds is not. Nature, 1981 May 14, 291(5811), 117 - 21 Expression in E . coli of maize and wheat chloroplast genes for large subunit of ribulose bisphosphate carboxylase; Gatenby AA et al.; Synthesis of the large subunit polypeptide of ribulose bisphosphate carboxylase can be detected in Escherichia coli cells containing the chloroplast genes from maize and wheat . The chloroplast DNA sequences contain a 'promoter' that, in Escherichia coli, initiates transcription and translation of the genes . This synthesis of large subunits has been substantially increased using the powerful lambda promoter, PL, to drive transcription of the maize gene. Nucleic Acids Res, 1981 May 11, 9(9), 2223 - 37 Apparent association constants for E . coli ribosomal proteins S4, S7, S8, S15, S17 and S20 binding to 16S RNA; Schwarzbauer J et al.; We have previously reported the development of a technique utilizing nitrocellulose filters, which rapidly separates ribosomal protein-ribosomal RNA complexes from unbound protein . We have used this technique to obtain binding data for the association of proteins S4, S7, S8, S15, S17, and S20 with 16S RNA . With the exception of protein S17, the association behavior for each of these proteins exhibits a single binding site with a unique binding constant . The apparent association constants have been calculated and have been found to have a range from 1.6 x 10(6) M-1 for protein S7 to 7.1 x 10(7) M-1 for protein S17 . The Scatchard plot for the protein S17 binding data is biphasic, suggesting that within the RNA population two different binding sites exist, each with a different apparent association constant. Nucleic Acids Res, 1981 May 11, 9(9), 2153 - 72 Structural organization of the 16S ribosomal RNA from E . coli . Topography and secondary structure; Stiegler P et al.; Extensive studies in our laboratory using different ribonucleases resulted in valuable data on the topography of the E.coli 16S ribosomal RNA within the native 30S subunit, within partially unfolded 30S subunits, in the free state, and in association with individual ribosomal proteins . Such studies have precise details on the accessibility of certain residues and delineated highly accessible RNA regions . Furthermore, they provided evidence that the 16S rRNA is organized in its subunit into four distinct domains . A secondary structure model of the E.coli 16S rRNA has been derived from these topographical data . Additional information from comparative sequence analyses of the small ribosomal subunit RNAs from other species sequenced so far has been used. Biokhimiia, 1981 May, 46(5), 802 - 8 {Isolation and some properties of polynucleotide phosphorylase from E . coli}; Bagdonas AS et al.; A new method for isolation of polynucleotide phosphorylase from E . coli, including ion-exchange chromatography and gel-filtration has been developed . The method results in 300-fold purification of the enzyme, which being devoid of nuclease and phosphatase activities can further be utilized for oligonucleotide synthesis . It was shown that upon storage the enzyme loses the primer-independent activity and in the absence of NaCl can be used for further syntheses . An addition of NaCl stimulates the elongation of the oligonucleotide chain . Some advantages of polynucleotide phosphorylase from E . coli in comparison with the M . luteus enzyme are discussed. Cell, 1981 May, 24(2), 413 - 9 Identification of ribosomal protein S7 as a repressor of translation within the str operon of E . coli; Dean D et al.; A DNA-directed in vitro protein-synthesizing system was used to demonstrate that r protein S7 has the capacity to inhibit the translation of mRNA for the second and third gene products of the str operon (S7 and EF-G) but not for the first gene product (S12) . Translation of mRNA of the last gene product in the operon (EF-Tu) is also probably not inhibited by S7 . In addition, we localized the target site for S7 repressor action on the polycistronic str mRNA by examining the repressor activity of S7 in vitro using various template DNAs that contain the gene . The target site was found not to include a promoter-proximal portion of the mRNA for S12 . To test for regulatory properties of S7 in vivo, we inserted the S7 gene into a plasmid vector containing the ara regulatory elements such that S7 synthesis was placed under ara control . A specific increase in S7 synthesis caused by stimulation in transcription originating from the arabinose promoter decreased the synthetic rate for EF-G but had no effect on S12 or EF-Tu synthesis. Biull Eksp Biol Med, 1981 May, 91(5), 594 - 6 {Quantity of exogenous linear DNA absorbed by E . coli cells treated with Ca2+ cations}; Bukrinskii MI et al.; The amount of linear chromosomal DNA entering Ca2+-treated Escherichia coli K 12 cells was estimated from the degree of degradation of exogenous DNA by intracellular nucleases . At least 40% of DNA adsorbed by the cells in the nucleaso-resistant form (45 MD/cell) was discovered to be as intracellular . No significant difference was found between single- and double-stranded DNA. Mol Biol (Mosk), 1981 May-Jun, 15(3), 653 - 67 {Factors influencing the pulse character of RNA elongation in vitro by E . coli RNA polymerase}; Aivazashvili VA et al.; Pause location along primary structure of two RNA fragments each 200 nucleotide residues in the length synthesized from A1 promoters of T7 phage DNA and delta D111 T7 phage DNA was analyzed . No correlation between the location of pauses and GC-rich or self complementary regions of RNA were found . The location of pauses does not change upon the variation of the temperature or ionic strength . Concurrent variation of all four NTP concentrations also did not influence pausing pattern . However the distribution of pauses depends highly on the ratio of the individual substrate concentrations . Substitution of GTP by ITP changes the pausing pattern completely . Inorganic pyrophosphate (PPi) of inhibits RNA elongation preferentially in the regions: NAUN, CGUAG . The study of PPi action on RNA terminated with 3' OCH3-NMP suggest that the sequence-specific inhibition of RNA elongation may be a result of pyrophosphorolysis of terminal nucleotide residues of RNA . It was proposed that the pulse character of RNA elongation stems rather from differences in the kinetic constants of nucleotides attachment and pyrophosphorolysis from the 3'-termini of RNA than by termination signals encoded in the primary structure of DNA . The stable location of pauses in certain short oligonucleotides: AUG, AUU, AAU and some others is in favour of the hypothesis. Mol Cell Biochem, 1981 Apr 13, 36(1), 47 - 63 Chemistry and biology of E . coli ribosomal protein L12; Brot N et al.; E . coli ribosomal protein L12, because of its unique features, has been studied in more detail than perhaps any of the other ribosomal proteins . Unlike the other ribosomal proteins that are generally present in stoichiometric amounts, there are four copies of L12 per ribosome, some of which are acetylated on the N-terminal serine . The acetylated species, referred to as L7, has not been shown, as yet, to possess any different biological activity than L12 . A specific enzyme that acetylates L12 to form L7, using acetyl-CoA as the acetyl donor, has been purified from E . coli extracts . L12 is also unique in that it does not contain cysteine, tryptophan, histidine, or tyrosine, is very acidic (pI: 4.85) and has a high content of ordered secondary structure (approximately 50%) . The protein is normally found in solution as a dimer and also forms a tight complex with ribosomal protein L10 . There are three methionine residues in L12, located in the N-terminal region of the protein, one or more of which are essential for biological activity . Oxidation of the methionines to methionine sulfoxide prevents dimer formation and inactivates the protein . The four copies of L12 are located in the crest region(s) of the 50S ribosomal subunit . There is good evidence that the soluble factors, such as IF-2, EF-Tu, EF-G and RF, interact with L12 on the ribosome during the process of protein synthesis . This interaction is essential for the proper functioning of each of the factors and for GTP hydrolysis associated with the individual partial reactions of protein synthesis . The L12 gene is located on an operon that contains the genes for L10 and beta beta' subunits of RNA polymerase at about 88 min on the bacterial chromosome . DNA-directed in vitro systems have been used to study the unique regulation of the expression of these genes . Autogenous regulation, translational control, and transcription attenuation are regulatory mechanisms that function to control the synthesis of these proteins. Nature, 1981 Apr 2, 290(5805), 419 - 21 ATP-stimulated endoprotease is associated with the cell membrane of E . coli; Voellmy RW et al.; Despite knowledge of the physiological significance and regulation of protein degradation in bacteria, the pathway of proteolysis and the responsible enzymes are still not known . Degradation of cell proteins in bacterial and animal cells requires continuous ATP production, inhibition of which in Escherichia coli prevents the degradation of normal proteins in growing cells, accelerated breakdown of such proteins in starving cultures and the very rapid breakdown of abnormal proteins . Intracellular proteolysis proceeds by repeated endoproteolytic steps and ATP is required for the initial cleavages of the substrate . We have recently demonstrated ATP stimulation of proteolysis in extracts of bacterial and animal cells . These ATP-stimulated systems seem to be responsible for the rapid degradation of abnormal proteins in vivo, but they may also be involved in the catabolism of normal cell proteins, limited proteolysis, such as the processing of precursors for secreted or membrane proteins, and the selective inactivation of specific proteins, as occurs in the ATP-dependent cleavage of the lambda repressor by the recA protein . We report here that membrane fragments contain an ATP-stimulated protease that degrades cell proteins to large peptides (of molecular weight (MW) 71,500) which are then rapidly hydrolysed to amino acids by soluble ATP-independent enzymes. Nord Vet Med, 1981 Apr-May, 33(4-5), 218 - 23 Effect of pectin on secretion in pig jejunal loops challenged to enteropathogenic E . coli or enterotoxin (LT) . A preliminary report; Larsen JL; Perorally administered pectin in pigs could reduce the fluid accumulation in intestinal loops challenged to different dilutions of enteropathogenic E . coli strains, no effect was observed to enterotoxin (LT) preparations . Pectin seems to interact with the bacterial colonization . The neutralizing effect was most pronounced with low inoculation doses in the loops (10(3) and 10(5)), while high doses (10(9)) permitted the strains to exert their enteropathogenic effect (Table I) . Different batches gave different effects (Table II) and some preparations were extraordinarily effective (Table III) . Thus standardisation and testing is important in developing pectin preparations for diarrhoea prophylaxis. Biokhimiia, 1981 Apr, 46(4), 732 - 43 {Interaction of the membrane transport proteins in E . coli K12}; Kalachev IIa et al.; The inhibition kinetics of NO2PheGal transport by MeGlc in E . coli K12 were studied . The inhibitory effect was observed only at definite ratio of the corresponding transport proteins -- enzyme IIglc and beta-galactoside permease . It was shown that in this case, beside the repressive effect of MeGlc on beta-galactoside transport, beta-galactosides (GalSGal) can also inhibit the rate of MeGle accumulation . The data obtained suggest that in the region of maximal inhibitory effect the conformation of both membrane proteins are changed, which leads to an increase in the activity of enzyme IIglc and its affinity for MeGlc . It was assumed that the phenomenon observed is not unique and is in general conformity with the postulate that under certain conditions many bacterial membrane proteins can come into interaction, thus changing their activity. Biokhimiia, 1981 Apr, 46(4), 717 - 22 {Role of RNA polymerase in ensuring fidelity in copying the template during transcription in E . coli}; Kamzolova SG et al.; The heterogeneity of cell size of E . coli WU-36-10-11-12 and its four RNA-polymerase (rif-r) mutants with pleiotropic effect -- rpoB401, rpoB402, rpoB403 and rpoB409 was investigated for the purposeful choice of E . coli mutant with an altered fidelity of transcription . The stability of the phenotype of E . coli strains was shown to depend on the structural state of RNA polymerase . In vitro RNA-polymerase of the morphologically most unstable mutant rpoB402 incorporates non-complementary GMP or CMP on the poly {d(AT).d(AT)} template more frequently than the enzyme from the wild-type strain . The data obtained suggest that the beta-subunit of RNA-polymerase determines the fidelity of transcription and the selection of complementary nucleotides. Cell, 1981 Apr, 24(1), 243 - 9 Feedback regulation of ribosomal protein synthesis in E . coli: localization of the mRNA target sites for repressor action of ribosomal protein L1; Yates JL et al.; E . coli ribosomal protein L1 is a translational repressor of the synthesis in vitro of both proteins encoded in the L11 operon (L11 and L1) . L1 is shown to act at a single target site within the first 160 bases of the bicistronic mRNA, near (or at) the translation initiation site of the L11 cistron . Synthesis of L1 apparently requires translation of the preceding L11 cistron, allowing regulation of the synthesis of both proteins froma single mRNA target site . This observation suggests a sequential translation mechanism that results in the equimolar synthesis rates of the two proteins observed in vivo . It was found that the presence of 23S rRNA, but not 16S rRNA, relieves translational inhibition by L1 . L1 presumably recognizes structural features of the mRNA target site that are homologous to the L1-binding site of 23S rRNA . Although previous work indicated that translationally inhibited ribosomal protein mRNA is degraded in vivo, L1 repressor action in the present in vitro system was found not to involve mRNA degradation. J Microsc, 1981 Apr, 122(Pt 1), 15 - 22 A new approach to the study of the E . coli nucleoid; Haggis GH et al.; E . coli were examined by the freeze-fracture thaw-fix technique, embedded in thin fibrin gels . After glutaraldehyde fixation the bacterial nucleoid was found spread out over the surrounding fibrin . Addition of calcium and uranyl acetate to the fixative preserved the nucleoid in compact form . The spread nucleoid was then examined against a smooth mica background after freeze-thaw and osmotic lysis . These spreads were critical-point dried, rotary shadowed with platinum-carbon and viewed as stereo-pair micrographs . Structures seen are tentatively interpreted as clusters of polyribosomes, extended DNA, and supercoiled DNA complexed with proteins or polyamines . After osmotic lysis, glutaraldehyde alone preserves the nucleoid in compact form . Only where strands are broken, in freeze-fracture or freeze-thaw lysis, must uranyl acetate be added to the fixative to preserve a compact structure. Biokhimiia, 1981 Apr, 46(4), 699 - 707 {Phenylalanyl-tRNA synthetase from E . coli MRE-600 . Effect of chemical modification of lysine residues on the enzyme interaction with substrates}; Gorshkova II et al.; The effect of modification of Phe-RSase from E . coli MRE-600 by pyridoxal-5'-phosphate and 2', 3'-dialdehyde derivative of ATP and L-phenylalanynyl-5'-adenylate obtained by periodate oxidation on the enzyme interaction with substrates was investigated . It was shown that modification of Phe-RSase by pyridoxal-5'-phosphate and 2', 3'-dialdehyde derivative of ATP leads to a decrease of the aminoacylation rate without changing the rate of the ATP-{32P}-pyrophosphate exchange reaction . The substrate analogs L-phenylalanynol and L-phenyl-alanynyladenylate increase the degree of Phe-RSase inactivation in the aminoacylation reaction . tRNAphe strongly protects the enzyme against inactivation . ATP, both in the absence (in case of modification with pyridoxal-5'-phosphate) and in- the presence of Mg2+ and phenylalanine (in case of modification with o-ATP) exhibits a pronounced protective effect . L-Phe does not protect the enzyme against the inactivation by pyridoxal-5'-phosphate or o-ATP . The dissociation constant of the Phe-RSase{14C}-Phe-tRNAphe complex increases 2.5 -- 5-fold after the enzyme modification by pyridoxal-5'-phosphate, while the Km value for tRNAphe decreases approximately two times in the aminoacylation reaction . There are no changes in the Km values for amino acid and ATP and the Hill coefficients for all substrates tested . Modification of Phe-RSase by pyridoxal-5'-phosphate leads to a decrease of stability of the aminoacyladenylate -- enzyme complex . Oxidized L-phenylalanynyladenylate does not produce enzyme inactivation either by aminoacylation or in the isotropic ATP-PP iota exchange reaction . It is assumed that Phe-RSase from E . coli MRE-600 contains some lysine residues essential for binding and aminoacylation of tRNA, which do not occur in the ATP-binding subsite and aminoacyladenylate formation center. Cell, 1981 Apr, 24(1), 213 - 23 Homologous pairing and topological linkage of DNA molecules by combined action of E . coli RecA protein and topoisomerase I; Cunningham RP et al.; E . coli RecA protein and topoisomerase I, acting on superhelical DNA and circular single strands in the presence of ATP and Mg2+, topologically link single-stranded molecules to one another, and single-stranded molecules to duplex DNA . When superhelical DNA is relaxed by prior incubation with topoisomerase, it is a poor substrate for catenation . Extensive homology stimulates the catenation of circular single-stranded DNA and superhelical DNA, whereas little reaction occurs between these forms of the closely related DNAs of phages phi X174 and G4, indicating that, in conjunction with topoisomerase I, RecA protein can discriminate perfect or nearly perfect homology from a high degree of relatedness . Circular single-stranded G4 DNA reacts with superhelical DNA of chimeric phage, M13G ori 1, to form catenanes, at least half of which survive heating at 80 degrees C following restriction cleavage in the M13 region, but few of which survive following restriction cleavage in the G4 region . Electron microscopic examination of catenated molecules cleaved in the M13 region reveals that in most cases the single-stranded G4 DNA is joined to the linear duplex M13(G4) DNA in the homologous G4 region . The junction frequently has the appearance of a D loop, with an extent equivalent to 100 or more bp . We conclude that a significant fraction of catenanes were hemicatenanes, in which the single-stranded circle was topologically linked, probably by multiple turns, to its complementary strand in the duplex DNA . These observations support the previous conclusion that RecA protein can pair a single strand with its complementary strand in duplex DNA in a side-by-side fashion without a free end in any of the three strands. Eur J Immunol, 1981 Apr, 11(4), 286 - 90 Requirement of the immunogen (E . coli beta-galactosidase) for the response towards a determinant responsible for antibody-mediated enzyme activation, while antibodies binding some other site can be elicited by mitogen alone; Piccolella E et al.; Virgin mouse spleen cells cultured in vitro without antigen or mitogen produced a measurable amount of IgM binding E . coli beta-galactosidase (beta-gal) . Lipopolysaccharide (LPS) and LPS plus antigen enhance this response, which cannot be considered truly polyclonal since it does not include the production of antibodies directed towards a conformation-dependent determinant, responsible for the activation of a mutant beta-gal, and known otherwise to be highly immunogenic . By priming in vivo with alum-treated beta-gal (unable to elicit activating antibodies), an activating response is obtained in vitro by LPS plus antigen, but not by LPS alone . These results are compatible with a two-signal requirement for the activation of B cells and may be explained as follows: (a) the mitogen, in absence of immunogen, stimulates those clones which have received a specific signal from cross-reacting structures in the environment: (b) instead, no cross-reaction are available for the conformation-controlled structure of the "activating" determinant; thus, intact immunogen is required as well as mitogen . Because of this "uniqueness", the molecule of beta-gal offers a highly specific tool to probe carrier-hapten relationships in native proteins. Monatsschr Kinderheilkd, 1981 Apr, 129(4), 229 - 32 {Membranes properties of granulocytes in Job's-Syndrome with E . coli-septicemia (author's transl)}; Burdach SE et al.; Since the age of nine weeks a red haired girl suffered from purulent dermatitis and recurrent, systemic E . coli infections . She had an excessive hyperimmunoglobulinemia E, as well as impaired granulocyte adherence and chemotaxis . Though a sepsis was evident, the granulocytes exhibited a random FITC-Concanavalin A fluorescence . In spite of intensive treatment with various antibiotics and several granulocyte transfusions the child died at the age of 2 years and 11 months . As shown by the FITC-Concanavalin A distribution, the hyperimmunoglobulinemia E may have caused a decreased membrane fluidity causing the impaired adherence and chemotaxis . This could explain the pathophysiology of the Job's Syndrome. Nucleic Acids Res, 1981 Mar 25, 9(6), 1339 - 50 Electron microscopic analysis of transcription of a ribosomal RNA operon of E . coli; Hamming J et al.; Transcription in vitro of the E . coli ribosomal RNA operon, rrnE, was analysed by electron microscopy . The transcription initiation sites of the two rrnE promoters in tandem, P1 and P2, were mapped and the transcription from both sites was compared . The first and the second transcription initiation site are about equally used when all nucleotides are present at 200 microM . Lowering the concentration of the second promoter's start nucleotide CTP to 3 microM reduces the use of the P2 site sharply . At all CTP concentrations used the nascent RNA chains from P1 are in the average longer than those from P2 after a fixed transcription time . Most probably, this difference is caused by a longer average interval before formation of the productive complex with the second promoter. Nucleic Acids Res, 1981 Mar 11, 9(5), 1203 - 17 Attachment of protein affinity-labeling reagents of variable length and amino acid specificity to E . coli tRNAfMet; Schulman LH et al.; Transamination with bifunctional amines in the presence of bisulfite has been used to attach side chains of variable length to the N4-position of single stranded cytidine residues in E . coli tRNAfMet . Such side chains, terminating in reactive primary amino groups, have been coupled to a variety of N-hydroxysuccinimide esters . The resulting modified tRNAs carry protein affinity labeling groups capable of covalent reaction with a variety of amino acids. Vopr Med Khim, 1981 Mar-Apr, 27(2), 220 - 3 {Recognition sites of adenine DNA-methylases from cells of E . coli}; Lopatina NG et al.; DNA-methylases from a strain of E . coli CK were studied . Three adenine methylases were found in the strain studied, which were eluted by 0.16 M, 0.23 M and 0.7 M NaCl in phosphocellulose P-11 chromatography . According to this, the enzymes were designated as DM-A16, DM-A23 and MD-A70 . Indirect data on the presence of adenine specific methylases dissimilar in their recognition sites in cells of E . coli CK were obtained using the test of additional methylation modified by I . I . Nikol'skaya . These conclusions were confirmed, when the dinucleotide sequence was determined in the recognition site using DM-A23 and DM-A70 . The methylase DM-A23 was shown to recognize the dinucleotide sequence 5'...A-T...3' and DM-A70--the sequence 5'...A-G...3'. J Hered, 1981 Mar-Apr, 72(2), 125 - 6 The relationship between the dwarfing gene and E . coli infection in two populations of chickens; Mauldin JM et al.; Resistance to an E . coli challenge was studied in the 6th, 7th, and 8th backcross generations after the introduction of the sex-linked recessive dwarf gene (dw) into two populations of White Plymouth Rock chickens that had undergone bidirectional selection for juvenile body weight . In the B6 and B8 generations, the dwarf genotype from the HW line had significantly higher mortality and/or heart lesions than the heterozygotes while the homozygous normal chickens were intermediate . No association between the dwarf allele and the incidence of E . coli infection was observed in the HW line in any generation, but genotypes in the LW lines were influenced by social environment in susceptibility to the E . coli challenge . Under low social strife, the dwarf and heterozygote genotypes were more susceptible than the normal genotype to the E . coli challenge, while under a high social strife there were no differences among genotypes . Since the genotypes in the LW line responded differently to the disease challenge than those in the HW line, it was concluded that a line-dwarf genotype interaction was present. Int J Radiat Biol Relat Stud Phys Chem Med, 1981 Mar, 39(3), 265 - 71 Correlation of hyperthermic sensitivity and membrane microviscosity in E . coli K1060; Dennis WH et al.; We have demonstrated a positive correlation between membrane microviscosity and the temperature required to kill E . coli . Batches of cells with differing unsaturated fatty acid (u.f.a.) compositions were prepared from the u.f.a.-requiring E . coli K12 mutant K1060 . The membrane microviscosity of these cells is estimated from the extent of fluorescence polarization of the probe molecule 1,6-diphenyl-1,3-5,-hexatriene dissolved in the membrane . For the same growth temperature, cells grown in oleic acid (18:1) have a greater microviscosity and u.f.a . content than linolenic acid (18:3) grown cells . the rate of decrease in microviscosity with increasing temperature is correlated with the amount of u.f.a . present in the membrane . From survival curves determined at several hyperthermic exposures, one can interpolate the hyperthermic temperature required to kill 90 per cent of the cells in three hours . These equivalent kill temperatures are directly related to the cell microviscosity . These data support the hypothesis that cell membrane microviscosity plays a critical role in hyperthermic killing. Cell, 1981 Mar, 23(3), 689 - 97 Nucleotide sequence of the lexA gene of E . coli; Horii T et al.; Using a cloned fragment containing the lexA gene of E . coli, the entire nucleotide sequence of the lexA gene has been determined . The probable coding region of the lexA gene contains 606 nucleotide residues and encodes a single protein of 202 amino acids . The initiation site of in vitro transcription of the lexA messenger RNA has been determined by analysis of the 5' nucleotide sequence . Comparison of the DNA sequence of the promoter region of the lexA gene with that of the recA gene reveals the presence of sequences that are common to both . There is some similarity between the amino acid sequences of the lexA and the lambda repressor proteins. Immun Infekt, 1981 Mar, 9(1), 29 - 32 {Chemotherapy with fosfomycin, cefoxitin, and cefotaxime in experimental E . coli-pleuropneumonia (author's transl)}; Kruger C et al.; Two models of pneumonia--the experimental E . coli-pleuropneumonia and "intrapulmonary" E . coli-pneumonia--were employed in these studies . Only fosfomycin was effective in both models even at the low dosage of 100 mg/kg/d . The comparative drugs cefotaxime and cefoxitin, however, were not able to reduce the bacteria in both lungs even at very high dosages of 900 mg/kg and 300 mg/kg per day respectively over 6 days. Inflammation, 1981 Mar, 5(1), 55 - 60 Suppression of rabbit peritoneal macrophage migration by heat-labile E . coli toxin; Cushing AH et al.; Migration of rabbit peritoneal macrophages toward casein-serum was inhibited by preincubation of the cells with heat-labile toxin of Escherichia coli in direct relationship to the concentration of toxin in the incubation mixture . Cells preincubated with heated toxin or with toxin-antiserum migrated the same as those which had been incubated in toxin-free media . Toxin-preincubated cells had levels of cyclic AMP which were increased in direct relationship to the concentration of toxin in the preincubation mixture . Heated toxin failed to induce increased levels of cAMP in the cells at the highest concentration tested. Nature, 1981 Feb 26, 289(5800), 808 - 10 Expression of the E . coli uvrA gene is inducible; Kenyon CJ et al.; UvrA+-dependent excision repair is one of the most important systems in Escherichia coli for repairing UV-induced pyrimidine dimers and a variety of other forms of DNA damage . The uvrA protein acts in conjunction with the uvrB and uvrC gene products to introduce a nick at the of a DNA lesion and thus initiate the repair process . We have recently used the Mud(Ap, lac) operon fusion vector to identify a set of genes whose expression is induced by DNA damage . One Mud(Ap, lac) insertion mapped at the uvrA locus and made the cells sensitive to UV light . In this fusion strain, beta-galactosidase expression was induced by DNA-damaging agents in a recA+lexA+-dependent fashion . We were surprised by this result because uvrA+-dependent excision repair is observed both in cells in which protein synthesis has been inhibited and in recA- and lexA- cells, findings which have led to the conclusion that the uvrA gene product is constitutively expressed and not under the control of the complex recA+lexA+ regulatory circuitry (see below) . We have investigated this possibility further and describe here the generation and characterization of a set of fusions of the lac genes to the promoter of the uvrA gene . We confirm that the uvrA gene product is induced by DNA damage in a recA+lexA+-dependent fashion. Nature, 1981 Feb 19, 289(5799), 696 - 8 Molecular cloning of the K1 capsular polysaccharide genes of E . coli; Silver RP et al.; Epidemiological and immunological evidence indicates that the K1 capsular polysaccharide confers the property of virulence on Escherichia coli . E coli K1 is associated with invasive diseases in humans and in laboratory and domesticated animals . K1 isolates account for 80% of E . coli neonatal meningitis and comprise the majority of capsular types in neonatal septicaemia without meningitis and in childhood pyelonephritis . Passive administration of K1 antibodies prevented bacteraemia and meningitis in infant rats fed E . coli K1 . Nonencapsulated derivatives of these invasive K1 strains did not cause bacteraemia in infant rats, although intestinal colonization was similar to that of the parent strains (M . Achtman and R.P.S., unpublished results) . Several reports propose that the E . coli K1 capsular polysaccharide exerts an anti-phagocytic effect similar to that observed with other pathogenic encapsulated bacteria . One approach to studying whether the K1 antigen is sufficient to confer virulence of if other E . coli structures are necessary is to isolate the K1 genes for genetic and biochemical analysis . Recombinant DNA methodology provides a powerful tool for such an approach . Here, we report the molecular cloning of the E . coli K1 antigen genes . The cloned K1 genes synthesize a capsule in E . coli K12 indistinguishable chemically and immunologically from that of wild-type K1 strains. Nature, 1981 Feb 12, 289(5798), 555 - 9 Cloning of cDNA of major antigen of foot and mouth disease virus and expression in E . coli; Kupper H et al.; Double-stranded DNA copies of the single-stranded genomic RNA of foot and mouth disease virus have been cloned into the Escherichia coli plasmid pBR322 . A restriction map of the viral genome was established and aligned with the biochemical map of foot and mouth disease virus . The coding sequence for structural protein VP1, the major antigen of the virus, was identified and inserted into a plasmid vector where the expression of this sequence is under control of the phage lambda PL promoter . In an appropriate host the synthesis of antigenic polypeptide can be demonstrated by radioimmunoassay. Nucleic Acids Res, 1981 Feb 11, 9(3), 623 - 31 Identification of a protein coded by pR plasmid and involved in SOS repair in E . coli; Gigliani F et al.; The TP120 plasmid is known to determine enhanced UV survival in E . coli wild type an uvrB and PolA mutants but not in RecA mutant . In order to analyze the function involved in the SOS repair, we have constructed a new plasmid named pR derived by cleavage of TP120 with Hind III endonuclease . This new plasmid maintains the Ap and UV resistance . The insertion of Tn5 transposon in the plasmid allows to select several pR::Tn5 plasmids whose UV resistance was inactivated by the transposition . The comparison of the protein synthesis in the minicells of the pR and pR::Tn5 shows that the pR codes for a 22.000 M.W . dalton protein which is absent in protein pattern of pR::Tn5. Biull Eksp Biol Med, 1981 Feb, 91(2), 210 - 2 {Genetic features of the F-like pAP10-2 plasmid controlling synthesis of thermostable enterotoxin in E . coli cells}; Buianova NI et al.; A study was made of the genetic traits of F-like plasmid pAP10-2 that monitors the synthesis of thermostable enterotoxin after plasmid labelling with Tn9 transpozone . It has been shown that the test Ent-plasmid is a conjugative one, suppresses fertility functions of F plasmid and belongs to the F1 incompatibility group. Sci Sin, 1981 Feb, 24(2), 256 - 63 Expression of gene(s) in restricted fragment of lambda DNA in E . coli; Kuang DR et al.; Restriction endonucleases EcoR1 and BamH1 are used to produce fragments pBR322C (375 bp) and pBR322B (3987 bp) from pBR322, and to produce lambda F2A (65.6--71.3% of lambda DNA, 2679 bp) and lambda F2B (71.3--81% of lambda DNA, 4559 bp) from EcoR1 restriction fragment lambda F2 (65.6--81% of lambda DNA) of lambda cI857S7 DNA . By recombining pBR322B and lambda F2B in vitro, a new plasmid called pCB2 carrying promoters and structural genes cI and cro is constructed . The desired strain with pCB2 is selected from 338 transformants for its AprTcs and for its immunity to lambda infection . The length of the pCB2 DNA molecule is 2.66 +/- 0.33 micrometers and its MW is 5.51 +/- 0.68 x 10(6)d, as determined by electron microscope and agarose gel electrophoresis . The lengths of single strands and the double strands of the heteroduplex formed between lambda F2 and linear pCB2 (EcoR1 digested) agree well with the original design for its construction, From the above data, we come to the conclusion that pCB2 we constructed is a new plasmid with cI and/or cro gene expressed in E . coli. Nucleic Acids Res, 1981 Jan 24, 9(2), 339 - 47 Nucleotide sequence of the thrB gene of E . coli, and its two adjacent regions; the thrAB and thrBC junctions; Cossart P et al.; We have sequenced a DNA fragment containing the Escherichia coli thrA-thrB junction, the complete thrB gene and the thrB-thrC junction . The intergenic sequence thrA and thrB is only one base pair . The coding region for homoserine kinase is 927 base pairs long . It is followed by 114 base pair segment in an open reading frame predicting that thrC begins just after non-sense codon of thrB . The presence at the end of thrA and of thrB of sequences that can pair with the 3' end of the 16 S ribosomal RNA suggests that reinitiation of translation occurs at the end of the two genes . The deduced aminoacid sequence for homoserine kinase shows no striking homology with aspartokinase I homoserine dehydrogenase I. Nature, 1981 Jan 15, 289(5794), 194 - 5 Inducible repair of near-UV radiation lethal damage in E . coli; Peters J et al.; We present here the initial characterization of a new repair system which is induced in actively growing cultures of Escherichia coli and which repairs a major fraction of lethal damage produced by near-UV (310-400 nm) light . This system is different from the error-prone 'SOS' repair system for DNA, known to be induced in E . coli by treatments which damage DNA and/or inhibit DNA replication, such as irradiation by far-UV (254 nm) light or X rays, thymine starvation or treatment with naladixic acid or mitomycin C . The SOS response requires induction of an 'X' protein, the product of the recA gene, whereas the inducible repair system described here utilizes proteins distinct from the X protein. Acta Chir Scand, 1981, 147(7), 595 - 9 Intestinal hemodynamic effects of varying the route of infusion of live E . coli bacteria in the cat; Falk A et al.; The responses of the series-coupled vascular sections in the feline small intestine were studied in experimental sepsis induced following various routes of infusion of live E . coli bacteria . The intestinal hemodynamics were followed by means of plethysmography combined with direct recording of the intestinal venous outflow . After 2 hours of bacterial infusion the experiments were terminated . Infusion of E . coli in the inferior caval vein induced initially hypotension, decreased intestinal blood flow (Q) and increased intestinal vascular resistance (R) . Portal venous infusion induced, on the other hand, an initial arterial blood pressure increase, an increase of Q and a decrease of R . Aortal infusion evoked only minor initial changes . The early response was in all series followed by a progressive hypotension during which Q decreased and R increased gradually . There were no changes in intestinal tissue volume, indicating that there was no pooling of blood or extravasation of fluid, during the experiments . Intestinal mucosal lesions were equally distributed in the three series . Thus, depending on the route of infusion live E . coli induced intestinal vasoconstriction or vasodilatation . Regardless the route, there was no intestinal pooling of blood or fluid . Hypotension developed in most cats after 120 min, regardless the site of infusion and the initial vascular response. Acta Chir Scand, 1981, 147(7), 589 - 94 Central hemodynamic responses to venous, aortal or portal infusion of live E . coli bacteria in the cat; Falk A et al.; The central hemodynamic responses were studied in experimental sepsis in cats, following various routes of infusion of live E . coli bacteria . The aortic blood flow (ABF) was electromagnetically recorded . The pulmonary artery was cannulate for pressure recording . Platelet and white blood cell concentrations, PO2, PCO2, pH and oxygen saturation were measured at intervals . I.v . infusion of bacteria induced initially decreased ABF, systemic hypotension, pulmonary hypertension . Portal infusion evoked, on the other hand, increased ABF, but induced no significant change in systemic or pulmonary arterial blood pressures . Aortal infusion induced responses in between . The initial hemodynamic changes were followed by relative normalization after 5-10 min . Then, in all series, a progressive fall in ABF and systemic blood pressure were noticed . Within 5 min following bacterial infusion the platelet and white blood cell concentrations fell to 65 and 50%, respectively . In all series a moderate metabolic acidosis developed . Thus, the initial hemodynamic response following infusion of live E . coli was dependent on the route of infusion; intraportal infusion induced initially a more hyperdynamic state . The different initial central hemodynamic responses did not influence the subsequent development of a hypotensive shock state. Mol Biol (Mosk), 1981 Jan-Feb, 15(1), 103 - 14 {Membrane-bound precursor of E . coli periplasmic alkaline phosphatase: isolation and some properties}; Kolot MN et al.; Solubilization of membrane-bound alkaline phosphatase with 0.2% tritone X-100 and its purification to the homogenous state were performed . It was shown that the membrane-bound enzyme differed from the soluble enzyme by the N-terminal sequence, was more hydrophobic and was presented by one form of enzyme as compared to the three forms of the soluble one . By its substrate specificity this enzymes approximates the first form of the periplasmic enzyme, and does not differ from it by pH optimum, thermostability and the rate of inhibition by orthophosphate. Mol Gen Genet, 1981, 184(3), 562 - 3 N-Methyl-N'-nitro-N-nitrosoguanidine sensitivity of E . coli mutants deficient in DNA methylation and mismatch repair; Jones M et al.; E . coli mutants deficient in DNA methylation (dam) and mismatch repair (mut) have been characterized with respect to their sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . Dam bacteria are more sensitive than mutH, mutL, and mutS single mutant bacteria . Dam mutL and dam mutS double mutant bacteria are less sensitive than dam bacteria, whereas dam mutH double mutant bacteria are as sensitive as dam bacteria . This pattern of MNNG sensitivity may be a result of the specificity of the components of the E . coli mismatch repair system. J Med Virol, 1981, 8(4), 237 - 43 The conversion of hepatitis B core antigen synthesized in E coli into e antigen; MacKay P et al.; The antigen (HBeAg) of hepatitis B virus (HBV) is a polypeptide of 17-20,000 daltons closely associated with the core antigen (HBcAg) of Dane particles, from which it is released by a variety of disruptive procedures . HBeAg could be a unique component of HBV core particles or a derivative of HBcAg . To resolve this question immunodiffusion experiments were carried out with preparations of HBcAg synthesized in E coli carrying a recombinant plasmid from which the HBcAg, but no other HBV gene, was expressed . HBcAg was converted into HBeAg by proteolytic degradation under dissociating conditions, thus confirming at the molecular level that HBeAg is a component of HBcAg . This offers a new route to the detection of HBeAg and antibodies to the antigen. Mol Gen Genet, 1981, 183(1), 13 - 9 Transcription of the E . coli tufB gene: cotranscription with four tRNA genes and inhibition by guanosine-5'-diphosphate-3'-diphosphate; Miyajima A et al.; The transcription of the tufB gene by purified RNA polymerase holoenzyme was studied using the transducing phage lambda rifd 18 DNA and the hybrid plasmid pTUB1 DNA (Miyajima et al . 1979) as templates . The size of tufB mRNA synthesized in this system was about 1,700 nucleotides, and the same strand as for rrnB was transcribed . By electron microscopic examination of the R-loop formed between lambda fus3 DNA and tufB mRNA synthesized under the direction of pTUB1 DNA, it was found that the untranslated sequence of about 500 nucleotides is at the 5' end of tufB mRNA . The sequencing of the 5' region of tufB mRNA synthesized on the truncated template has revealed that the tufB gene is cotranscribed with its upstream genes for four tRNAs (thrU, tyrU, glyT, and thrT) . The synthesis of this mRNA molecule is completely abolished by low concentrations of ppGpp . Neither pppGpp, ppGp, nor pGpp was effective as inhibitor in this cell-free system. Nucleic Acids Symp Ser, 1981, (9), 229 - 33 Substrate specificity of CTP-synthetase from E . coli; Scheit KH et al.; The substrate specificity of CTP-synthetase from E . coli was investigated by means of UTP analogs . This study revealed that the three main structural elements of the UTP molecule were important for the substrate specificity of the enzyme . CTP-synthetase seems to possess an absolute requirement for the beta-D-ribose 5-triphosphate part in UTP . Substitutents in 5-position of UTP, exceeding the size of a tritium atom abolish substrate function. Nucleic Acids Symp Ser, 1981, (9), 207 - 9 Synthesis of unnatural P-N-bond catalyzed with E . coli ribosomes; Tarussova NB et al.; The model substances 2'(3')-O-{N-acetylmethionylaminomethylene-(P-methyl)phosphino} ester of adenosine-5'-phosphate, pA-(AcMetGlyP), and N-acetylmethionylaminomethylene-(P-methyl) phosphinoamide of phenylalanine, AcMetGly P PheOH, were synthesised . They were used for the ribosomal catalysis studying. Circ Shock, 1981, 8(4), 403 - 10 Effect of E coli endotoxin on the leakage of 14C-sucrose from phosphatidylcholine liposomes; Onji T et al.; The effect of E coli endotoxin on the leakage of 14C-sucrose from phosphatidylcholine liposomes in the absence or presence of Ca2+ was studied . Endotoxin decreased the leakage from liposomes from 27% to 4% in 5 hr when Ca2+ (1 mM) was incorporated into liposomes during sonication . The effect of endotoxin on the leakiness of liposomes was concentration dependent . Ca2+ alone increased the leakage of 14C-sucrose from liposomes . Mg2+ at concentrations higher than 5 mM exhibited an effect similar to that of Ca2+ . These findings suggest that endotoxin increases the molecular packing of phosphatidylcholine bilayers in the presence of Ca2+ or Mg2+ . A change in the physical state of membrane lipid bilayers induced by endotoxin may affect the function of biological membranes. Mol Gen Genet, 1981, 182(1), 7 - 11 Thermal resistance to photoreactivation of specific mutations potentiated in E . coli B/r ung by ultraviolet light; Fix D et al.; Mutagenesis by ultraviolet light was studied in a strain of E . coli ung, which lacks uracil-DNA glycosylase activity . Mutation potentiated by UV in cells already induced by nalidixic acid treatment was still photoreversible suggesting that pyrimidine dimers act directly as premutational photoproducts . Secondly, irradiated cells were held in buffer at 48 degrees C for 0 to 135 min to allow for deamination of cytosines in pyrimidine dimers . The mutation frequencies for class 2 de novo suppressor mutation, for class 2 converted suppressor mutation and for backmutation were individually determined, before and after photoreactivation, as a function of this thermal treatment . Backmutation remained sensitive to photoreactivation throughout the treatment but de novo and converted suppressor mutations rapidly developed resistance to photoreactivation . This resistance was not seen in an ung+ control . A model is proposed to account for the selective resistance based on the hypothesis that class 2 de novo and converted suppressor mutations normally result from UV by GC to AT transitions at T = C dimers . The model described deamination of the cytosine residues in these dimers to become uracil residues . In consequence, monomerization by photoreactivation in cells that can not repair uracils in DNA no longer reverse mutation and GC to AT transitions are established at the sites of uracils. Environ Mutagen, 1981, 3(4), 429 - 44 An E coli microsuspension assay for the detection of DNA damage induced by direct-acting agents and promutagens; McCarroll NE et al.; We have devised a microsuspension assay utilizing E coli indicator strains WP2, WP2 uvrA, WP67, CM611, WP100, W3110polA+, and p3478 pola- for the detection of chemically-induced preferential kill of repair-deficient strains . Data are presented from tests of 77 compounds representing a wide range of chemical classes which demonstrate the efficiency of the E coli microsuspension assay as both a qualitative and quantitative screen of DNA-modifying activity . Furthermore, the use of a battery of indicator strains lacking different repair systems offers the advantage of providing preliminary information concerning the mechanism of DNA damage induction by a test agent. Gastrointest Radiol, 1981, 6(2), 161 - 3 Massive gas embolism in E . coli septicemia; Jones B; The case of an elderly women with radiographic evidence of gas within her portal vein as well as the iliac and femoral arteries is presented . The underlying cause proved to be E . coli septicemia . Differential diagnosis and clinical significance of intravascular gas are reviewed. Gene, 1981 Jan-Feb, 13(1), 75 - 87 Variables affecting the selectivity and efficiency of retention of DNA fragments by E . coli RNA polymerase in the nitrocellulose-filter-binding assay; Strauss HS et al.; In this paper we characterize the effect of varying the solution conditions and filter-binding protocols on the extent and selectivity of DNA retention on nitrocellulose filters by DNA-binding proteins . These effects are illustrated by the binding interaction of Escherichia coli RNA polymerase with lambda and T7 phage DNA restriction fragments . We present procedures which will help enhance the selective retention of some DNA restriction fragments over others . These include increasing the pH and salt concentration, decreasing the enzyme-to-DNA ratio, and including an appropriate washing step . Selective binding is not dependent on the presence of Mg2+ . Although we only show data for RNA polymerase-DNA interactions, many of the principles discussed are likely to find practical applications in studying selective DNA-protein binding in general. Circ Shock, 1981, 8(1), 77 - 93 Mechanism of increased glucose uptake by skeletal muscle during E coli endotoxin shock in the dog; Raymond RM et al.; Carbohydrate metabolism of skeletal muscle was studied during 2 mg/kg E coli endotoxin shock in dogs . During natural (free-flow) conditions, glucose uptake by the muscle increased markedly during 6 hours of shock . Increased glucose uptake occurred concomitant with muscle ischemia and hypoxia . However, when muscle blood flow was held constant, thereby preventing local muscle ischemia and hypoxia, glucose uptake by the gracilis muscle did not change during shock . These results implicate local muscle ischemia and/or hypoxia as the mediator(s) of the increased muscle glucose uptake during shock . Further studies demonstrated that local muscle hypoxia was the stimulus for increased glucose uptake by skeletal muscle during endotoxin shock, and muscle ischemia per se did not alter muscle glucose uptake . Since approximately 50% of body mass is composed of skeletal muscle, the contribution of this organ system in the hypoglycemia of endotoxin shock in the dog may be substantial. Circ Shock, 1981, 8(1), 59 - 67 Does sodium pentobarbital anesthesia compromise clearance of bacteria or survival of dogs in lethal E coli shock? Archer LT, Beller-Todd B, Elmore O, White GL, Hinshaw LB. Recent reports have indicated that circulating leukocytes play a prominent role in promoting survival in endotoxin and live E coli shock . Anesthesia has been reported to compromise host defense by depressing phagocytic activity . This study was designed to determine whether sodium pentobarbital would adversely affect leukocyte concentrations, E coli clearance, blood glucose concentrations, and survival of dogs in lethal E coli shock . A leukocytosis was produced in some dogs using daily sublethal intravenous injections of E coli endotoxin for 3 days . Three groups of dogs were challenged with LD100 E coli: Group A (saline-pretreated, normal leukocyte count, unanesthetized); Group B (endotoxin-pretreated, leukocytotic, anesthetized); and Group C (endotoxin-pretreated, leukocytotic, unanesthetized) . Groups B and C reduced circulating E coli concentrations similarly but significantly more than Group A . All animals in Group A died, all in Group C lived, and 67% in Group B lived . The reduction in survival in Group B was probably due to the marked hemoconcentration in the anesthetized dogs which were unable to drink, rather than a compromise of their host defense . Inasmuch as phagocytic activity was similar between anesthetized and unanesthetized dogs, results suggest that barbiturate anesthesia is not deleterious to host defense. Int Arch Allergy Appl Immunol, 1981, 64(2), 167 - 70 A new technique for determination of immobilizing antibodies against E . coli using capillary tubes; Kaijser B; A new technique for determining immobilizing Escherichia coli antibodies is described . Capillary glass tubes were filled with semisolid, lactose-containing agar, supplemented with the antiserum to be tested . The highest dilution of antiserum still immobilizing the bacteria was registered. Mol Gen Genet, 1981, 184(2), 272 - 7 Rates of growth, ribosome synthesis and elongation factor synthesis in a tufA defective strain of E . coli; Gausing K; A tufA defective strain of E . coli was isolated which by a single deletion event acquired a tufA-lacZ fusion gene and lost the normal functional tufA gene (see accompanying paper) . A correlation between the growth rate of protein synthesis was decreased to about 50% in the tufA defective strain whereas the number of EF-Tu molecules per ribosome was about 80% compared to a normal strain . The results indicate that tufB gene expression was preferentially stimulated in the tufA defective strain but the increased EF-TuB synthesis was not sufficient to make up for the loss of normal EF-TuA synthesis . Introduction of a plasmid that carries a complete tufA gene and the preceeding fusA gene but not the str-promotor into the tufA defective strain did not alleviate the slow growth or low rate of EF-Tu synthesis showing that the high rate of EF-TuA synthesis compared to the other proteins in the str operon is not augmented by a strong second promotor for the tufA gene . The tufA-lacZ fusion which takes the place of the normal tufA gene was expressed at a high rate and the beta-galactosidase activity increased with the growth rate as expected. Mol Gen Genet, 1981, 181(3), 313 - 8 Selection for purine regulatory mutants in an E . coli hypoxanthine phosphoribosyl transferase-guanine phosphoribosyl transferase double mutant; Levine RA et al.; We have studied the relationship betwen purine salvage enzymes, 6-mercaptopurine resistance, and the purR phenotype in E . coli . Mutants resistant to 6-mercaptopurine were found to have defects in HPRT, the purR repressor, or in both . Analysis of these mutants led to the isolation of a hypoxanthine phosphoribosyl transferase-guanine phosphoribosyl transferase double mutant (hpt- gpt-) that is extremely sensitive to adenine . Two classes of adenine resistant mutants were isolated from this strain . The first class was deficient in APRT (apt-) while the second class represented purine regulatory mutants (purR-) . There is thus selection for the purR phenotype in a hpt- gpt- background. Adv Shock Res, 1981, 6, 15 - 26 Elevated plasma vasopressin concentrations during endotoxin and E . coli shock; Wilson MF et al.; Plasma vasopressin concentrations, measured by radioimmunoassay, were remarkably elevated during endotoxin and E . coli shock . The concentrations were often above 500 pg/ml in dogs and above 300 pg/ml in baboons; they reached 1200 pg/ml in two dogs and 1800 pg/ml in another . Plasma concentration in a quiet, hydrated subject is 4 pg/ml; osmoregulation is maximally effective at 20 pg/ml . Elevation of plasma vasopressin occurred by 15 minutes after beginning infusion of endotoxin or E . coli and reached concentrations of 200-350 pg/ml with a decrease in cardiac output but before hypotension, which suggests decreased thoracic blood volume and decompression of left atrial stretch receptors . Even higher vasopressin levels were associated with a reduction of arterial blood pressure . The typical pattern was an early peak elevation followed by a sustained plateau of plasma vasopressin concentration in dogs and baboons with endotoxin and/or E . coli-induced circulatory shock. Mol Gen Genet, 1981, 182(1), 143 - 7 A mutation affecting L-serine and energy metabolism in E . coli K12; Newman EB et al.; The effects of a pleiotropic mutation ssd are described . This mutation results in decreased efficiency in the use of glucose and fructose as carbon source, inability to use succinate or to grow anaerobically, an alteration in the activity of enzymes responsible for the synthesis and degradation of L-serine, increased resistance to certain antibiotics, and a deficiency in proline transport . This mutation resembles various previously described mutations thought to affect' energy coupling factor' and is located in the same region of the chromosome . While the gene product affected by this mutation is still unidentified, it is clear that L-serine metabolism cannot be understood merely in terms of providing L-serine and its derivatives. Mol Gen Genet, 1981, 184(3), 479 - 83 rho Mutations restore lamB expression in E . coli K12 strains with an inactive malB region; Colonna B et al.; lamB, the structural gene for lambda receptor, is the second gene of the malK-lamB operon in the malB region of the Escherichia coli K12 chromosome . lamB is essentially not expressed in the absence of an active malT gene product, the activator of the maltose regulon . A malT strain is resistant to phage lambda . We show that: (i) Introduction of rho mutations in malT mutants restores lamB expression to a level sufficient to render the strain sensitive to phage lambda; (ii) This restoration is not dependent on the main promoter of the malK lamB operon . It depends on the distal part of the malK gene . We propose that rho inactivation unmasks the activity of a promoter located near the distal end of malK . Experiments with Mu insertions in gene malK suggest that in the (-) orientation a Mu promoter is also able to allow lamB expression in a rho background. Mol Gen Genet, 1981, 184(2), 265 - 71 Construction and characterization of a tufA-lacZ fusion coding for an E . coli EF-Tu-beta-galactosidase chimeric protein; Gausing K; A new phage lambda cloning vector was constructed that has a single EcoRI site upstream from weakly expressed lacI-Z gene isolated by Muller-Hill and Kania (1974) . An EcoRI fragment containing the complete tufA gene of E . coli was cloned on the vector and the recombinant phage was crossed into the str operon that has tufA as its last gene . Subsequent selection gave rise to a tufA-lacZ fusion that codes for a chimeric peptide . The fused peptide has a molecular weight of 148,000 and contains 40% of the N-terminal of EF-Tu followed by part of the lac repressor-beta-galactosidase fusion . The specific activity of the fused peptide is about half of the activity of normal beta-galactosidase. Mol Gen Genet, 1981, 183(2), 341 - 7 DNA repair in E . coli strains deficient in single-strand DNA binding protein; Whittier RF et al.; Weigle reactivation and mutagenesis have been found to be defective in strains of E . coli deficient in single-strand DNA binding protein (SSB) . These defects parallel those previously found in prophage induction and amplification of recA protein synthesis in ssb- strains . Together, these results demonstrate a role for SSB in the induction of SOS responses . UV survival studies of ssb- recA- and ssb- uvr- strains are presented which also suggest a role for SSB in recombinational repair processes but not in excision repair . Studies of host cell reactivation support this latter conclusion. Mol Gen Genet, 1981, 183(2), 298 - 305 Cloning of E . coli pnp gene from an episome; Portier C et al.; Starting with an F' episome harboring a transposon inserted in the pnp gene (Portier 1980), we were able to identify an EcoRI restriction fragment carrying the pnp and argG genes . This fragment, from both wild-type and mutant episomes, was cloned ni pACYC184 . The presence of argG on the fragment allowed positive selection of the desired clones in an auxotrophic strain (argG) . A restriction map was established and a fragment of 3 megadaltons subcloned in the plasmid vector pBR322 . The pnp gene corresponds to about 50% of this subcloned segment and was roughly located by deletion mapping . The direction of transcription and locations of the promotor and gene extremities were determined by analyzing proteins synthesized in "maxi-cells" . In addition, the gene coding for a 10,000 dalton protein was found to reside adjacent to the beginning of the pnp structural gene . Strains carrying plasmids which express the pnp overproduce polynucleotide phosphorylase. Mol Gen Genet, 1981, 181(2), 241 - 7 Isolation of DNA fragments containing replicating growing forks from both E . coli and B . subtilis; Valenzuela MS et al.; By the use of a restriction enzyme digestion of gently lysed E . coli or B . subtilis cells, it is possible to isolate a minute fraction of the total DNA that has an unusually high sedimentation coefficient . Upon inspection of this DNA in the electron microscope, branched DNA fragments are observed . Single branched DNA fragments were analyzed by restriction enzyme and partial denaturation mapping techniques . The fragments appear to have the properties of growing forks excised from in vivo replicating intermediates . In B . subtilis, the minute fraction of DNA was also investigated by transformation assays and found to be greatly enriched for a marker near the origin and slightly enriched for a terminus marker. Circ Shock, 1981, 8(3), 263 - 71 In vitro effects of E coli endotoxin on K+-activated para-nitrophenylphosphatase activity and ouabain binding in dog hearts; Onji T et al.; The in vitro effect of E coli endotoxin on the activity of K+-activated paranitrophenylphosphatase (K+-PNPPase), an enzyme which represents the partial activity of (Na+ + K+)-ATPase enzyme system, was studied in isolated adult dog heart myocytes . The results were correlated with ouabain-binding studies . Endotoxin had an inhibitory effect on the Vmax for K+ activation as well as the Vmax for Mg++ and para-nitrophenylphosphate (PNPP) saturation . The inhibition was concentration-dependent and noncompetitive with K+, Mg++, and PNPP and furthermore, reversible . Endotoxin did not displace the bound 3H-ouabain from receptor sites nor did it affect the capacity of ouabain binding, indicating that the total enzyme concentration was not altered . From these findings, it is concluded that endotoxin in vitro affects the myocardial (Na+ + K+)-ATPase enzyme system by decreasing the turnover number of the enzyme molecule . The ability of endotoxin to modify myocyte membrane-associated enzyme activity may be responsible for altered heart metabolism and function in endotoxemia. J Interferon Res, 1981, 1(3), 381 - 90 Properties of a human alpha-interferon purified from E . coli extracts; Wetzel R et al.; A human alpha interferon, designated HuIFN-alpha A, produced in E . coli by direct expression of cloned cDNA {Goeddel et al., Nature 287, 411--416 (1980)} has been purified from bacterial extracts and characterized . The protein has a molecular weight (19,400 by SDS/PAGE) and amino acid composition consistent with the