Biochem J, 1983 Jul 1, 213(1), 187 - 91 Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli; Lewendon A et al.; A procedure for the purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli is described . Homogeneous enzyme of specific activity 17.7 units/mg was obtained in 22% yield . The key purification step involves substrate elution of the enzyme from a cellulose phosphate column . The subunit Mr was estimated to be 49 000 by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate . The native Mr was estimated to be 55 000 by gel filtration, indicating that the enzyme is monomeric. Appl Environ Microbiol, 1983 Jul, 46(1), 37 - 43 Amplification of the aroA gene from Escherichia coli results in tolerance to the herbicide glyphosate; Rogers SG et al.; The predominant cellular target of the herbicide glyphosate is thought to be the enzyme 5-enolpyruvylshikimate-3-phosphoric acid synthase (EPSP synthase) . As a means of biologically testing this finding, we cloned a segment of DNA from Escherichia coli that encodes this enzyme . Clones carrying the gene for EPSP synthase were identified by genetic complementation . Cells that contain a multicopy plasmid carrying the EPSP synthase gene overproduce the enzyme 5- to 17-fold and exhibit at least an 8-fold increased tolerance to glyphosate . These experiments provide direct biological evidence that EPSP synthase is a major site of glyphosate action in E . coli and that, in an amplified form, it can serve as a selectable glyphosate resistance marker. Appl Environ Microbiol, 1983 Jul, 46(1), 291 - 2 Effect of pH on conjugal transfer at low temperatures; Singleton P et al.; The inhibitory effects of nonoptimal temperature and nonoptimal pH on F-type conjugal transfer were found to be synergistic . This finding is briefly discussed in an environmental context. J Clin Microbiol, 1983 Jul, 18(1), 23 - 8 Rapid test for identification of heat-labile enterotoxin-producing Escherichia coli colonies; Finkelstein RA et al.; A latex particle agglutination test is described which is suitable for the recognition of heat-labile enterotoxin-producing colonies of Escherichia coli immediately after primary culture on a variety of commonly used enteric diagnostic media . The test is simple and economical to perform, and the results are available in minutes. J Clin Microbiol, 1983 Jul, 18(1), 199 - 202 Genetic probes for enterotoxigenic Escherichia coli isolated from childhood diarrhea in the Central African Republic; Georges MC et al.; Escherichia coli strains were isolated from 778 children with diarrhea and 151 well children in the Central African Republic over a period of 1 year . These 929 strains were assayed for heat-labile and heat-stable enterotoxin production and were hybridized (probed) with structural genes for these enterotoxins . Twenty-four isolates from diarrheal patients and one isolate from a well child were found to be toxigenic by assay and probe . Minor discrepancies were encountered with both assays and probes during initial screening procedures, but the two methodologies were ultimately comparable. In Pract, 1983 Jul, 5(4), 135 - 46 Coliform mastitis; Jackson E et al.; The majority of coliform mastitis cases are relatively mild and self-limiting . Teat dipping and dry cow therapy will not prevent coliform problems, neither do they cause them . Bad housing and poor milking time hygiene lead to coliform mastitis . Efforts to control coliform mastitis must be directed at reducing exposure to the bacteria . In acute cases fluid therapy is indicated to counteract endotoxic shock. Drugs, 1983 Jul, 26(1), 80 - 90 Acute secretory diarrheas . Current concepts in pathogenesis and treatment; Hughes S; Acute secretory diarrheas constitute a major source of mortality and morbidity world-wide . Our current understanding of the underlying mechanisms involved is reviewed with particular reference to cholera and enterotoxigenic E . coli infections . From the physiological principles involved, a unified concept for the treatment of acute secretory diarrheas is presented . The importance of rehydration is highlighted and practical instructions for the use of oral glucose-electrolyte solutions in the treatment of acute secretory diarrhoeas are given, along with some discussion of the rationale behind their use and optimum composition . The important role of nutritional factors during acute diarrhoea is underlined and the place of various drugs, some established, some experimental, are briefly discussed. Radiat Res, 1983 Jul, 95(1), 158 - 64 The effects of radioprotectors on DNA polymerase I-directed repair synthesis and DNA strand breaks in toluene-treated and X-irradiated Escherichia coli; Billen D; In Escherichia coli made permeable to nucleotides by toluene treatment, a DNA polymerase I-directed repair synthesis is induced by exposure to X rays . This repair synthesis may be amplified and easily measured through inhibition of DNA ligase action . In an effort to learn more of the relationship between X-ray-induced strand breaks in cellular DNA and the extent of this repair synthesis, experiments designed to compare the influence of radioprotectors on both strand-break production and repair synthesis have been carried out . The results show that cysteamine, sodium formate, and glycerol not only protect against strand breaks but also reduce DNA polymerase I-directed repair synthesis . However, I-, an efficient hydroxyl radical scavenger, is not as effective a protective agent against strand breaks and does not measurably affect repair synthesis in our system. Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4446 - 9 Targeted mutation at cytosine-containing pyrimidine dimers: studies of Escherichia coli B/r with acetophenone and 313-nm light; Fix D et al.; We have tested the two-event model for UV mutagenesis producing class 2 suppressor mutations at glutamine tRNA genes in Escherichia coli . In the model used, the induction/indexing lesion is any type of pyrimidine dimer and the premutational photoproduct at the target site is a cytosine-containing dimer . Specific mutation-frequency responses were analyzed under conditions in which the ratio of thymine-thymine dimers to cytosine-containing dimers was modified by using 313-nm light and 0.0%, 0.1%, or 0.2% acetophenone . Changes observed in the production of class 2 suppressor mutations were consistent with the model and suggested that the G X C leads to A X T transitions responsible for class 2 suppressor mutations are targeted by cytosine-containing pyrimidine dimers at the mutational sites. Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4213 - 7 Testing an alternative model for the ribosomal peptide elongation cycle; Rheinberger HJ et al.; A kinetic analysis of poly(U)-dependent poly(Phe) synthesis with {14C}tRNAPhe and {3H}phenylalanine demonstrated that, in the course of efficient poly(Phe) synthesis, two tRNAs are present per 70S ribosome at all times, although at least 70% of the poly(Phe)-tRNAPhe is found at the peptidyl-tRNA (P) site . Together with our recent observation of a third tRNA-binding site on Escherichia coli ribosomes, these findings suggest a model for the peptide elongation cycle in which two tRNA molecules are present on the ribosome at both the pre- and the post-translocational state . This model predicts that deacylated tRNA is not released from the P site but translocated to the exit (E) site before release occurs . A series of translocation experiments with deacylated {14C}tRNAPhe at the P site and oligo {( 3H}Phe)-tRNA at the aminoacyl-tRNA (A) site proved that efficient elongation factor G-dependent translocation is not accompanied by a corresponding {14C}tRNAPhe release . However, significant {14C}tRNAPhe release was observed after translocation when an aminoacyl-tRNA was bound to the A site . Thus, deacylated tRNA is not released from the P site but is translocated to the E site, which therefore must be located "upstream" adjacent to the P site . Furthermore, the trigger for the release of deacylated tRNA from the E site is the binding of aminoacyl-tRNA to the A site. Proc Natl Acad Sci U S A, 1983 Jul, 80(13), 3884 - 8 Iron superoxide dismutase from Escherichia coli at 3.1-A resolution: a structure unlike that of copper/zinc protein at both monomer and dimer levels; Stallings WC et al.; The structure of iron superoxide dismutase (EC 1.15.1.1) from Escherichia coli has been determined at 3.1-A resolution . The dimeric molecule is constructed from identical subunits, which are two-domain polypeptides . The NH2-terminal domain is composed of two antiparallel crossing helices and the COOH-terminal domain is a three-layered structure characterized by mixed alpha/beta secondary structural features . The active center iron atoms, separated by 18 A and located near the monomer-monomer interface, are coordinated by two amino acid residues from each domain . Azide binding has been investigated by using difference Fourier techniques . Consistent with the notion of the independent evolution of the copper/zinc dismutase gene, the iron dismutase structure resembles the copper/zinc protein at neither the monomer nor the dimer level. J Virol, 1983 Jul, 47(1), 227 - 32 Construction of an infectious molecular clone of the autonomous parvovirus minute virus of mice; Merchlinsky MJ et al.; The linear single-stranded DNA genome of minute virus of mice, an autonomous parvovirus, was cloned in duplex form into the bacterial plasmid pBR322 . The recombinant clones of minute virus of mice were infectious when transfected into monolayers of human 324K cells and produced virus plaques with an efficiency of about 6% that obtained with duplex replicative-form DNA purified from cells infected with minute virus of mice . Southern blot analysis of transfected cells indicated that the cloned minute virus of mice genome requires both termini to be intact for excision and replication as a linear duplex molecule. J Med Chem, 1983 Jul, 26(7), 990 - 6 Theory and application of molecular potential energy fields in molecular shape analysis: a quantitative structure--activity relationship study of 2,4-diamino-5-benzylpyrimidines as dihydrofolate reductase inhibitors; Hopfinger AJ; A general formalism, based upon molecular mechanics pairwise potential functions, has been developed to compute the molecular potential energy fields inherent to a given molecule in a given conformation . Molecular descriptors are derived from the potential energy fields, which can be used in QSAR studies based upon molecular shape analysis . These descriptors have been computed for a set of 2,4-diamino-5-benzylpyrimidines that are dihydrofolate reductase (DHFR) inhibitors . A QSAR is derived in which DHFR inhibition activity can be explained in terms of molecular shape, as represented by differences in molecular potential energy fields between pairs of superimposed molecules, and the sum of the pi constants of substituents on the 3- and 4-position of the benzyl ring . This QSAR is superior to one developed earlier (Hopfinger, A . J . J . Med . Chem . 1981, 24, 818) in which molecular shape is described by common overlap steric volume . Ancillary information defining the "active" conformation and electrostatic nature of the binding site are realized in the construction of the QSAR. J Bacteriol, 1983 Jul, 155(1), 420 - 3 Inhibition of RNA synthesis by oxolinic acid is unrelated to average DNA supercoiling; Manes SH et al.; Oxolinic acid reduced RNA synthesis rates whether chromosome supercoiling decreased, increased, or remained unchanged . Thus, inhibition of RNA synthesis by oxolinic acid appears to involve factors other than average DNA supercoiling level . Coumermycin A1 caused RNA synthesis rates to increase or decrease roughly in parallel with DNA supercoiling. J Bacteriol, 1983 Jul, 155(1), 412 - 6 Expression of the Rickettsia prowazekii citrate synthase gene in Escherichia coli; Wood DO et al.; Recombinant DNA techniques were used to isolate the Rickettsia prowazekii citrate synthase gene on the plasmid vector pBR322 by functional complementation of a gltA mutation of Escherichia coli K-12 . Analysis of citrate synthase activity in crude extracts revealed that the enzyme expressed in E . coli retains the regulatory control mechanisms characteristic of the rickettsial enzyme. J Bacteriol, 1983 Jul, 155(1), 402 - 6 Involvement of a plasmid in Escherichia coli envelope alterations; Rosas SB et al.; A plasmid-containing wild-type Escherichia coli strain was treated with two plasmid-curing agents, sodium dodecyl sulfate and ethidium bromide . Plasmid elimination was accompanied by drastic changes in the morphology of the colonies . Analysis of the cured strain by scanning and transmission electron microscopy showed important alterations in size and morphology of the cells . Metabolic differences were also found between the wild-type and cured cells. J Bacteriol, 1983 Jul, 155(1), 391 - 7 Structure of fumarate reductase on the cytoplasmic membrane of Escherichia coli; Lemire BD et al.; The terminal electron transfer enzyme fumarate reductase has been shown to be composed of a membrane-extrinsic catalytic dimer of 69- and 27-kilodalton (kd) subunits and a membrane-intrinsic anchor portion of 15- and 13-kd subunits . We prepared inverted membrane vesicles from a strain carrying the frd operon on a multicopy plasmid . When grown anaerobically on fumarate-containing medium, the membranes of this strain are highly enriched in fumarate reductase . When negatively stained preparations of these vesicles were examined with an electron microscope, they appeared to be covered with knob-like structures about 4 nm in diameter attached to the membrane by short stalks . Treatment of the membranes with chymotrypsin destroyed the 69-kd subunit, leaving the 27-, 15-, and 13-kd subunits bound to the membrane; these membranes appeared to retain remnants of the structure . Treatment of the membranes with 6 M urea removed the 69- and 27-kd subunits, leaving the anchor polypeptides intact . These vesicles appeared smooth and structureless . A functional four-subunit enzyme and the knob-like structure could be reconstituted by the addition of soluble catalytic subunits to the urea-stripped membranes . In addition to the vesicular structures, we observed unusual tubular structures which were covered with a helical array of fumarate reductase knobs. J Bacteriol, 1983 Jul, 155(1), 275 - 80 Effects of inserting eight amino acid residues into the major lipoprotein on its assembly in the outer membrane of Escherichia coli; Inukai M et al.; A DNA sequence consisting of 24 base pairs was inserted into the structural gene (lpp) coding for the major lipoprotein of the Escherichia coli outer membrane which was carried on a high-copy-number plasmid in which expression was regulated through a lac promoter-operator region . This modification resulted in the insertion of eight amino acid residues, Glu-Glu-Phe-Leu-Glu-Glu-Phe-Leu, between the glutamine residue at position 9 and the leucine residue at position 10 of the wild-type lipoprotein sequence . When production of the mutant lipoprotein was induced by a lac inducer, the cells became swollen, showed unusual morphology, and eventually lysed . When the membrane fraction was analyzed after the induction, the mutant lipoprotein was found to have been normally secreted across the cytoplasmic membrane and assembled in the outer membrane . This lipoprotein was modified with glycerol and palmitic acid and even formed the bound form, which was linked covalently to peptidoglycan . The major difference between the membrane-associated mutant lipoprotein and the wild-type lipoprotein was that the mutant lipoprotein became sensitive to trypsin treatment . These results indicate that the substantial alteration in mutant lipoprotein structure near the amino-terminal end does not interfere with modification of the amino-terminal cysteine residue or cleavage of the signal peptide by the prolipoprotein-specific signal peptidase . However, this mutant lipoprotein assembled in the outer membrane appears to have deleterious effects with respect to envelope structure and cellular morphology and viability. J Bacteriol, 1983 Jul, 155(1), 228 - 37 Coordination of flagella on filamentous cells of Escherichia coli; Ishihara A et al.; Video techniques were used to study the coordination of different flagella on single filamentous cells of Escherichia coli . Filamentous, nonseptate cells were produced by introducing a cell division mutation into a strain that was polyhook but otherwise wild type for chemotaxis . Markers for its flagellar motors (ordinary polyhook cells that had been fixed with glutaraldehyde) were attached with antihook antibodies . The markers were driven alternately clockwise and counterclockwise, at angular velocities comparable to those observed when wild-type cells are tethered to glass . The directions of rotation of different markers on the same cell were not correlated; reversals of the flagellar motors occurred asynchronously . The bias of the motors (the fraction of time spent spinning counterclockwise) changed with time . Variations in bias were correlated, provided that the motors were within a few micrometers of one another . Thus, although the directions of rotation of flagellar motors are not controlled by a common intracellular signal, their biases are . This signal appears to have a limited range. J Bacteriol, 1983 Jul, 155(1), 113 - 21 Catabolism of phenylpropionic acid and its 3-hydroxy derivative by Escherichia coli; Burlingame R et al.; A number of laboratory strains and clinical isolates of Escherichia coli utilized several aromatic acids as sole sources of carbon for growth . E . coli K-12 used separate reactions to convert 3-phenylpropionic and 3-(3-hydroxyphenyl)propionic acids into 3-(2,3-dihydroxyphenyl)propionic acid which, after meta-fission of the benzene nucleus, gave succinate, pyruvate, and acetaldehyde as products . Enzyme assays and respirometry showed that all enzymes of this branched pathway were inducible and that syntheses of enzymes required to convert the two initial growth substrates into 3-(2,3-dihydroxyphenyl)propionate are under separate control . E . coli K-12 also grew with 3-hydroxycinnamic acid as sole source of carbon; the ability of cells to oxidize cinnamic and 3-phenylpropionic acids, and hydroxylated derivatives, was investigated . The lactone of 4-hydroxy-2-ketovaleric acid was isolated from enzymatic reaction mixtures and its properties, including optical activity, were recorded. Infect Immun, 1983 Jul, 41(1), 383 - 90 Evidence that a new enterotoxin of Escherichia coli which activates adenylate cyclase in eucaryotic target cells is not plasmid mediated; Green BA et al.; Escherichia coli SA53 produces a new enterotoxin that has a biological activity similar to that of E . coli heat-labile enterotoxin (LT) but is not neutralized by antiserum against LT or cholera enterotoxin . Strain SA53 contained two plasmids, pRB1 (69.2 +/- 4.3 megadaltons) and pRB2 (57.6 +/- 5.3 megadaltons) . Studies were undertaken to determine whether either plasmid was required for production of the LT-like toxin . We isolated a derivative of SA53 lacking both plasmids and confirmed that radioactively labeled pRB1 and pRB2 DNAs failed to hybridize to total DNA digests of the cured strain . The new enterotoxin was still produced by the cured strain, demonstrating that the gene(s) encoding the toxin was not located on pRB1 or pRB2 and was most likely on the bacterial chromosome . Although sonic extracts from SA53 contained no detectable LT antigen, plasmid pRB1 DNA did contain sequences with partial homology to the LT-A and LT-B genes . No sequence homology with LT genes was detected with pRB2 DNA . When the enterotoxin plasmid pCG86 was introduced into a rifampin-resistant derivative of SA53, LT was produced . Thus, plasmid-coded LT could be produced in the E . coli SA53 host, and the sequences homologous to LT in pRB1 were cryptic. Infect Immun, 1983 Jul, 41(1), 190 - 6 Physical and biological properties of U.S . standard endotoxin EC after exposure to ionizing radiation; Csako G et al.; Techniques that reduce the toxicity of bacterial endotoxins are useful for studying the relationship between structure and biological activity . We used ionizing radiation to detoxify a highly refined endotoxin preparation . U.S . standard endotoxin EC . Dose-dependent changes occurred by exposure to 60Co-radiation in the physical properties and biological activities of the endotoxin . Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis showed gradual loss of the polysaccharide components (O-side chain and R-core) from the endotoxin molecules . In contrast, although endotoxin revealed a complex absorption pattern in the UV range, radiation treatment failed to modify that pattern . Dose-related destruction of the primary toxic component, lipid A, was suggested by the results of activity tests: both the pyrogenicity and limulus reactivity of the endotoxin were destroyed by increasing doses of radiation . The results indicate that the detoxification is probably due to multiple effects of the ionizing radiation on bacterial lipopolysaccharides, and the action involves (i) the destruction of polysaccharide moieties and possibly (ii) the alteration of lipid A component of the endotoxin molecule. Infect Immun, 1983 Jul, 41(1), 137 - 44 Biphasic effect of pertussis vaccine on serum insulin in mice; Hewlett EL et al.; Administration of pertussis vaccine, consisting of whole, killed Bordetella pertussis organisms, causes hyperinsulinemia and enhanced secretion of insulin in response to a variety of secretagogues in rats and mice . In examining the time course and properties of this phenomenon, we discovered two distinct and separate effects of the bacteria on glucose and insulin levels in mice . First, a heat-stable (80 degrees C for 30 min) component causes a brief hyperinsulinemia which is +measureable by 1 h, maximal at 8 h, and ends in less than 48 h . This effect appears to be due to B . pertussis endotoxin, is mimicked by Escherichia coli endotoxin, and is associated with a transient, mild hypoglycemia . Second, there is a heat-labile component of the B . pertussis organism which induces a sustained (greater than 14 days), dose-dependent hyperinsulinemia which reaches a maximum at 5 to 7 days and has no associated hypoglycemia . The two effects are further distinguishable in that the early, endotoxin-induced hyperinsulinemia exhibits the normal suppressibility by exogenous epinephrine, whereas epinephrine markedly enhances the hyperinsulinemia occurring at 7 days . These two effects of B . pertussis appear to be mediated by different mechanisms and may be important in the well-recognized reactogenicity of pertussis vaccine in humans. Eur J Biochem, 1983 Jul 1, 133(3), 531 - 8 The accessibility of the active site and conformation states of the beta 2 subunit of tryptophan synthase studied by fluorescence quenching; Lane AN; The rate of quenching of the fluorescence of pyridoxal 5'-phosphate in the active site of the beta 2 subunit of tryptophan synthase from Escherichia coli was measured to estimate the accessibility of the coenzyme to the small molecules iodide and acrylamide . The alpha subunit and the substrate L-serine substantially reduced the quenching rate . For iodide, the order of decreasing quenching was: Schiff's base of N alpha-acetyl-lysine with pyridoxal 5'-phosphate greater than holo beta 2 subunit greater than holo alpha 2 beta 2 complex approximately equal to holo beta 2 subunit + L-serine greater than holo alpha 2 beta 2 complex + L-serine . The coenzyme in the beta 2 subunit is apparently freely accessible to both iodide and acrylamide (kappa approximately equal to 2 X 10(9) M-1 s-1), but the alpha subunit and L-serine decrease the rate by factors of 2-5 . Quenching of the fluorescence of the single tryptophan residue of the beta 2 subunit revealed that the apo and holo forms exist in different states, whereas the alpha subunit stabilizes a third conformation . As the alpha subunit binds to the beta 2 subunit, the tryptophan residue, which is within 2.2 nm of the active site of the beta 2 subunit, probably rotates with respect to the plane of the ring of the coenzyme, such that fluorescence energy transfer from tryptophan to pyridoxal phosphate is greatly reduced . The alpha subunit strongly protects the active-site ligand indole propanol phosphate from quenching with acrylamide, consistent with the active site being deep in a cleft in the protein . Iodide induces dissociation of the holo alpha 2 beta 2 complex {E . W . Miles & M . Moriguchi (1977) J . Biol . Chem . 252, 6594-6599} . The effect of iodide on the fluorescence properties of holo alpha 2 beta 2 complex allows us to estimate an upper limit for the dissociation constant for the alpha 2 beta 2 complex of 10(-8) M, in the absence of iodide. Eur J Biochem, 1983 Jul 1, 133(3), 481 - 9 The pyruvate dehydrogenase complex of Escherichia coli K12 . Nucleotide sequence encoding the dihydrolipoamide acetyltransferase component; Stephens PE et al.; The nucleotide sequence of the aceF gene, which encodes the dihydrolipoamide acetyltransferase component (E2) of the pyruvate dehydrogenase complex of Escherichia coli K12, has been determined using the dideoxy chain-termination method . The aceF gene comprises 1887 base pairs (629 codons excluding the initiation codon AUG); it is preceded by a short intercistronic segment of 14 base pairs containing a good ribosomal binding site, and it is followed closely by a potential rho-independent terminator . The results extend by 1980 base pairs the previously sequenced segment of 3780 base pairs containing the structural gene (aceE) of the pyruvate dehydrogenase component (E1) and they confirm that aceE and aceF are the proximal and distal genes of the ace operon . The amino terminus, carboxy-terminal sequence and amino acid composition of the acetyltransferase subunit predicted from the nucleotide sequence are in excellent agreement with previous studies with the purified protein . The predicted molecular weight (Mr = 65959) confirms experimental values derived from sedimentation equilibrium analysis and indicates that the higher values (78000-89000) that have been reported are due to unusual features of the protein that lead to anomalous mobilities during sodium dodecyl sulphate/polyacrylamide gel electrophoresis and in gel filtration . The primary structure fully supports conclusions, based on limited tryptic proteolysis, that the acetyltransferase subunit possesses two heterologous domains: the lipoyl domain and the subunit binding and catalytic domain . The lipoyl domain corresponds to the amino-terminal segment of the protein . It is acidic and contains three remarkably homologous repeating units of approximately 100 amino acids, each possessing a potential lipoyl binding site and a region that is characteristically rich in alanine and proline residues . The subunit binding and catalytic domain occupies most of the residual polypeptide in the carboxy-terminal segment. Arch Pathol Lab Med, 1983 Jul, 107(7), 361 - 3 Dissimilar manifestations of intrauterine infection with echovirus 11 in premature twins; Bose CL et al.; Echoviruses cause neonatal disease following intrauterine and intrapartum acquisition of the organism or by nosocomial infection . Dizygous twins apparently became infected following transplacental transmission of echovirus 11 . At 5 days of age, both twins experienced poor feeding, lethargy and hypothermia, and evidence of coagulopathy and hepatitis . During the sixth week of illness, the convalescence of twin A was complicated by peritonitis and sepsis, and the infant died . Pathologic findings included scattered foci of dystrophic myocardial calcification, distortion of hepatic architecture with fibrous connective tissue surrounding regenerative nodules and large foci of dystrophic calcification, and adrenal hemorrhagic necrosis and calcification . Twin B recovered without sequelae . The disease in twin A was unusual because of the extensive myocardial involvement . Also of interest was the variability of disease in twins who presumably had received a similar inoculum of organism by the same route. Science, 1983 Jul 1, 221(4605), 59 - 61 Requirement for signal peptide cleavage of Escherichia coli prolipoprotein; Inouye S et al.; Oligonucleotide-directed site-specific mutagenesis was applied to alter the cleavage site in the signal peptide of the major outer membrane lipoprotein of Escherichia coli . Replacing the glycine residue at the cleavage site with an alanine residue did not affect the processing of the signal peptide . However, when the same cleavage site was constructed by the deletion of the glycine residue, the signal peptide was no longer cleaved . These results indicate that stringent structural integrity at the cleavage site in the lipoprotein signal sequence is required for correct processing of prolipoprotein. Cancer Res, 1983 Jul, 43(7), 3247 - 52 O6-Methylguanine-DNA methyltransferase of human lymphoid cells: structural and kinetic properties and absence in repair-deficient cells; Harris AL et al.; Human lymphoid cell lines contain a DNA repair enzyme which removes the mutagenic alkylation lesion O6-methylguanine from DNA . The enzyme transfers the methyl group to a protein cysteine residue, generating S-methylcysteine, and is inactivated as a consequence of the reaction . Apparently the methylated enzyme represents a dead-end complex . The transfer reaction is very rapid and is completed in less than 1 min at 37 degrees, but methyl group transfer from single-stranded DNA or heavily damaged DNA is less efficient . The active methyltransferase and the methylated protein both have molecular weights of 21,000 to 22,000, as determined by gel filtration . Lymphoid cell lines proficient in repair of O6-methylguanine in vivo, Mex+, contain 10,000 to 25,000 molecules of the methyltransferase per cell . In contrast, repair-deficient cell lines, Mex-, do not contain detectable amounts of the enzyme . The latter point was verified by applying a partial purification procedure for the enzyme to cell-free extracts from two Mex- cell lines. J Gen Microbiol, 1983 Jul, 129 (Pt 7), 2277 - 83 Incompatibility properties of the IncFIII/FIV haemolytic plasmid pSU316 when integrated in the Escherichia coli chromosome; Navas J et al.; The haemolytic plasmid pSU316 is incompatible with members of the IncFIII and IncFIV incompatibility groups . Plasmid pSU307 (pSU316 hlyC::Tn5) was inserted by integrative suppression into the chromosome of JW112, a temperature-sensitive dnaA mutant of Escherichia coli . The incompatibility properties of this strain (SU51) were studied and it was found that: (1) plasmid pSU306 (pSU316 hlyA::Tn802) was rapidly lost from strain SU51 both at 30 degrees C and 42 degrees C; (2) the IncFIII plasmid pSU397 (ColB-K98::Tn802) was lost from strain SU51 and at 42 degrees C but not at 30 degrees C; and (3) the IncFIV plasmid R124 was stably maintained in strain SU51 at both temperatures . Revertants of pSU307 to the autonomous state could be obtained from SU51 . These revertants exerted incompatibility towards the prototype plasmids pSU306, pSU397 and R124 in the same way as pSU307 itself . Thus, strain SU51 provided a suitable method for distinguishing the three different incompatibility determinants of plasmid pSU316. Biochimie, 1983 Jul, 65(7), 437 - 40 {Specific association of the CRP-cyclic AMP complex with the control region of the lactose operon of Escherichia coli K 12: a direct fluorimetric study using DNA fragments of different lengths}; Baudras A et al.; Specific-site binding of the cAMP . CRP complex to the control region of the lactose operon of E . coli was measured directly . All of the protein molecules did bind specifically, and the binding constant for the major CRP site was not dependent on the length (62, 219 or 301 base pairs) of the DNA fragments used . Comparing the values of the binding constant measured for the major site and for the weaker "operator" CRP site, and referring to the published "consensus sequence" derived from the known CRP sites in a series of operons, we suggest that two sub-sites support additive contribution to the total binding free energy. Biochimie, 1983 Jul, 65(7), 417 - 25 CG sites and their nucleotide environment in the human gamma-delta-beta-globin gene cluster: distribution, frequency and possible frame for differential methylation; Poncet D et al.; Available restriction endonucleases including CG dinucleotides in their target sequences (most of them being unable to cut the DNA when the cytosine of the CG sequence is methylated) have been used to map cloned DNA covering the human gamma-delta-beta globin gene cluster . Since the human DNA fragments were cloned in Escherichia coli, only the internal cytosine in the sequence CCAT GG could be methylated . Thus, any recognized "CG enzyme" site can be detected since they are unmethylated . Results show that frequencies of "CG enzyme" sites regularly decrease from the gamma-globin region to the beta-globin region, the latter being very poor in "CG enzyme"' sites . The array of enzymes used here detects 4 times more CG sites than the classical MspI/HpaII system . Examination of previously sequenced parts of the gamma-delta-beta globin gene cluster shows that CG dinucleotides correspond to an average frequency of 1 out of 104 nucleotides in the gamma-globin region and 1 out of 138 nucleotides in the beta-globin region . In the gamma-globin region, 1 CG out of 4 or 5 may be detected by the enzymes used; the detected frequency is less than 1 out of 10 CG in the beta-region . Analysis of nucleotide environment around CG dinucleotides shows occurrence of local differences, the main sequences being CGG in the 5' side flanking the gamma genes and ACG in the corresponding area of the beta gene . The results presented introduce some new considerations about analysis of cytosine methylation which has been previously proposed as playing a role in the control of the activity of gamma, delta and beta genes respectively. Plasmid, 1983 Jul, 10(1), 31 - 44 Absence of cis-acting transposition immunity with Tn7; Hassan DM et al.; It is possible in two steps to insert into the plasmid RP4 two copies of the transposon Tn7 . This was demonstrated using a wild-type Tn7 in the first step, and a Tn7 derivative (carrying an additional marker), in the second step . The two successive transpositions occurred with the same polarity and frequency . The genetic structures of the resulting plasmids, predicted from the phenotypes of the bacterial host, were confirmed by direct analysis of the plasmid DNAs . Thus, the phenomenon of cis-acting transposition immunity, described with Tn1 or Tn3, does not take place in the case of Tn7. Plasmid, 1983 Jul, 10(1), 11 - 20 Cloning of an exclusion-determining fragment of the IncI plasmid, R144; Hartskeerl RA et al.; By cloning a distinct 8 MDa fragment of the IncI plasmid, R144, in the vector pACYC184, two recombinant plasmids were isolated . In these plasmids, pRAH303 and pRAH308, the inserted fragment was in opposite orientations . Both plasmids when present in a recipient strain caused a conjugation-specific exclusion in crosses with donor cells carrying the IncI plasmid R144 . Some derivatives of the recombinant plasmids in which parts were deleted, or in which Tn5 transposons were inserted, appeared to be exclusion negative . Analysis in minicells of the gene products of such plasmids together with those of the original recombinant plasmids revealed that the presence of two proteins, with apparent molecular weights of 13,000 and 19,000 Da could be correlated with the exclusion phenomenon. Mol Biol (Mosk), 1983 Jul-Aug, 17(4), 809 - 17 {Directed fragmentation of tobacco mosaic virus RNA: excision of 3' terminal sections, containing coat protein genes of tobacco mosaic virus}; Rodionova NP et al.; The present work demonstrated that RNA of different Tobacco mosaic virus strains (common; A14; Ni118, U2; wheat strain) are selectively cleaved by RNase H from E . coli in the presence of oligodeoxyribonucleotide d(TTTTTTTCCGG) complementary to the stretch of TMV RNA from the 842-nd to the 852-nd nucleotide from 3'-end, e . g . corresponding to the region adjacent to the 3'-terminus of origin of assemble of TMV . Such cleavage yields two main products . One of them--the short S-fragment--is the 3'-terminal part of TMV RNA . This conclusion is based on its ability to accept histidine and its molecular weight is about 0.28 X 10(6) . The other main product of TMV RNA cleavage by RNase H is the L-fragment . L-fragment is the 5'-terminal moiety of TMV RNA, as follows from its ability to direct the in vitro synthesis of p110-130, which is known to be encoded in the 5'-terminal TMV RNA region. Gene, 1983 Jul, 23(1), 35 - 40 A method for the generation of small pre-determined deletions in plasmid DNA: deletion analysis of the tetR region of vector pBR322; Heusterspreute M et al.; A general method is described that allows precise deletion of a chosen restriction fragment(s) from a plasmid having many cleavage sites for that restriction enzyme . The DNA to be deleted is first separated from the rest of the plasmid on a larger DNA fragment contained between two different unique restriction sites . This fragment is then subdigested by the restriction endonuclease of interest, which recognises two or more tetranucleotide (cohesive end or blunt end) sequences on the fragment, and is recloned between the two original unique restriction sites . The method is rapid, efficient, and the results are predictable . Examples are given in which predetermined HpaII (9 bp, 147 bp), TaqI (141 bp) and AluI (15 bp, 403 bp) fragments have been selectively removed from the tetR region of plasmid pBR322. Genetika, 1983 Jul, 19(7), 1210 - 2 {Localization of Kpnl restriction sites in the r-determinant of plasmid NR1}; Perebitiuk AN et al.; Restriction KpnI sites on a molecule of the plasmid NR1 were localized . The position of KpnA (58,0 MD) and KpnB (1,0 MD) fragments on a physical map of the NR1 molecule was shown using endonucleases EcoRI and SaII . Both restriction sites are situated on a r-determinant of the EcoJ fragment of the plasmid NR1. Biochem J, 1983 Jul 1, 213(1), 137 - 42 Studies by electron-paramagnetic-resonance spectroscopy of the molybdenum centre of spinach (Spinacia oleracea) nitrate reductase; Gutteridge S et al.; The molybdenum centre of spinach (Spinacia oleracea) nitrate reductase has been investigated by e.p.r . spectroscopy of molybdenum(V) in reduced forms of the enzyme . The resting enzyme gives no signals attributable to Mo(V) . However, on reduction with NADH, Mo(V) signals appeared at relatively short reaction times but decreased again on prolonged exposure to excess of the substrate as the enzyme was further reduced . On brief treatment of such samples with nitrate, Mo(V) signals reappeared but disappeared again on longer exposure to excess nitrate as the enzyme became fully reoxidized . Detailed investigation of the signals carried out in both 1H2O and 2H2O revealed the presence of two signal-giving species, referred to as 'signal A' and 'signal B', analogous to corresponding signals from nitrate reductase from Escherichia coli and from liver sulphite oxidase . Signal A has gav . 1.9767 and shows coupling to a single proton, exchangeable with the solvent, with A(1H)av . 1.3mT, whereas signal B shows no more than weak coupling to protons . Investigation of interconversion between the two species indicated that decreasing the pH from 8.0 to 6.7 had little effect, but that signal A was favoured by the presence of Cl- . This suggests, by analogy with recent work on sulphite oxidase by Bray, Gutteridge, Lamy & Wilkinson {Biochem . J . (1983) 211, 227-236} that Cl- is a ligand of molybdenum in the species giving signal A. Mol Cell Biol, 1983 Jul, 3(7), 1246 - 54 Differential activation of the mouse beta-globin promoter by enhancers; Berg PE et al.; A series of plasmids was constructed to study the effect of two enhancers, the simian virus 40 72-base-pair repeat and the Harvey sarcoma virus 73-base-pair repeat, on the mouse beta maj-globin promoter . These plasmids contain the mouse beta maj-globin promoter linked to the Escherichia coli galK gene, thus allowing galactokinase enzyme activity to be used as a measure of promoter function . In CV-1 (primate) cells, it was found that an enhancer is required for optimal promoter activity and that the simian virus 40 (primate) enhancer increases galactokinase fourfold more than the Harvey sarcoma virus (mouse) enhancer . In L (mouse) cells, however, the Harvey sarcoma virus enhancer is 1.3-fold stronger than the simian virus 40 enhancer . These data support the hypothesis that enhancer activity can be species specific . Furthermore, when both enhancers are present on the same plasmid, their effect is additive on the beta-globin promoter whether the plasmid is in CV-1 cells or L cells. Am J Trop Med Hyg, 1983 Jul, 32(4), 733 - 7 Migration of Entamoeba histolytica under agarose; Urban T et al.; A procedure for visualizing and quantifying motility of Entamoeba histolytica by migration under agarose is described . Agarose suspended in tissue culture medium 199 supplemented with bovine albumin was poured into plastic dishes and allowed to harden . Six pairs of wells were cut out in a circular configuration . To the inner wells a suspension of E . histolytica in Eagle's Medium (24 X 10(6) cells/ml) was added, and to the outer wells a chemoattractant or the control medium . After overnight incubation at 37 degrees C, the amebae were fixed and stained . The chemotactic and spontaneous migrations were measured in an enlarging projector . Escherichia coli filtrates, suspensions of intact and lysed erythrocytes, and the complement factor C5a acted as good chemoattractants . Both the random and chemotactic motility were correlated to the time of the incubation . Cytochalasin B effected a dose-related inhibition of both chemotactic and random migration, while colchicine caused a decrease of the chemotaxis only . The reproducibility of the method, measured by 10 intra-assay tests, was good . Thus, the described method can be useful for comparative determinations of the motility of different ameba populations . Furthermore, different factors affecting the motility of amebae can be studied. Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4432 - 6 Open reading frame expression vectors: a general method for antigen production in Escherichia coli using protein fusions to beta-galactosidase; Weinstock GM et al.; We have developed an Escherichia coli plasmid vector for the identification and expression of foreign DNA segments that are open reading frames (ORFs) . The 5' end of ompF, an E . coli gene encoding an abundant outer membrane protein, is used to provide a strong, regulated promoter, translation initiation site, and signal sequence for export from the cytoplasm . This sequence is coupled to the lacZ gene of E . coli so that expression of beta-galactosidase requires ompF transcription and translation signals . However, this hybrid gene is LacZ- because lacZ is out of frame with respect to ompF . Restriction enzyme recognition sites are located between ompF and lacZ to allow convenient insertion of DNA fragments . If an insert is an ORF of the correct length, ompF and lacZ become realigned in frame, resulting in a LacZ+ gene that produces a tribrid protein with the translation product of the insert sandwiched between OmpF and beta-galactosidase . The LacZ+ phenotype thus identifies clones containing an expressed ORF . To demonstrate the vector's utility we inserted a fragment from the herpes virus thymidine kinase gene and used the resulting tribrid protein to raise antibodies that precipitate thymidine kinase from herpes virus-infected cells . We also inserted a fragment from the E . coli lexA gene to produce a tribrid protein that is precipitated by antiserum raised with LexA protein . Thus, tribrid fusion proteins can be used to produce or detect antibodies and also to identify the product of a cloned gene. Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4422 - 6 F sex factor of Escherichia coli K-12 codes for a single-stranded DNA binding protein; Kolodkin AL et al.; In Escherichia coli K-12 strains that carry the mutation ssb-1 in the gene for single-stranded DNA binding protein, the presence of the F sex factor partially reverses the temperature-sensitive growth phenotype caused by the mutation . The region of F (EcoRI fragment 3) responsible for this compensation has been identified and subcloned onto pBR322 . A BamHI cleavage site has been found to intersect the essential coding region for this F function . By using this site, mutational blocks in the function have been constructed and used to identify a protein product (Mr approximately 22,000, slightly larger than the E . coli K-12 single-stranded DNA binding protein) which is correlated with the ssb-1-complementing activity . Labeled extracts from maxicells were used to show that this protein binds tightly to single-stranded DNA . The gene on F that codes for this protein is denoted ssf and is located at approximately 55.2 kilobases on the standard map of F, in the region transferred very early during bacterial conjugation. Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4403 - 7 Expression of a beta-galactosidase gene containing the ribosomal protein 51 intron is sensitive to the rna2 mutation of yeast; Teem JL et al.; The temperature-sensitive mutation rna2 causes the accumulation of higher molecular weight transcripts from the ribosomal protein 51 (rp51) gene of yeast and many other yeast ribosomal protein genes . We have determined the DNA sequence of the rp51 gene, confirming that it contains an intron and that the higher molecular weight transcript is an intron-containing precursor RNA . These data and other experiments suggest that the rna2 mutation affects mRNA processing (splicing) and that the presence of an intron is sufficient to render expression of a gene sensitive to the rna2 mutation . To test these hypotheses, we have inserted the rp51 intron into the coding region of a hybrid Escherichia coli beta-galactosidase gene, thereby interrupting the open reading frame subsequent to the initiating methionine codon . Despite the presence of the intron, the beta-galactosidase gene is expressed in yeast . Thus, the rp51 intron is properly excised from the normally intronless gene . The presence of the rp51 intron causes the beta-galactosidase activity to be sensitive to the rna2 mutation, consistent with the notion that this mutation affects gene expression at the level of splicing . The experiments suggest that an intron-containing beta-galactosidase gene can be used in a general way to study mRNA splicing. Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4228 - 32 Molecular cloning of a vitamin D-dependent calcium-binding protein mRNA sequence from chick intestine; Hunziker W et al.; We have constructed a recombinant cDNA library to facilitate study of the genomic actions of vitamin D3 and its hormonally active metabolite 1,25-dihydroxyvitamin D3 in initiation of the de novo biosynthesis of a 28,000-dalton vitamin D-dependent calcium binding protein (CaBP) present in chick intestine . The recombinant plasmids were prepared by the homopolymeric tailing and hybridization method using as a starting template poly(A)-enriched mRNA obtained from the intestinal mucosa of vitamin D3-replete (+D) chicks . Screening of 9,516 clones in this library was effected by using a comparative in situ colony hybridization technique with two {32P}cDNA probes; these probes were prepared from total poly(A)-RNA from chick intestinal mucosa of vitamin D-deficient (-D) chicks and a poly(A)-RNA specifically enriched for chick intestinal CaBP mRNA by immunoprecipitation of polysomes derived from vitamin D-replete (+D) chicks . We identified 26 clones that consistently displayed a significantly increased hybridization signal when comparing the -D vs . CaBP-enriched probe . Further evaluation of these clones by hybrid-selected translation showed the presence of CaBP-specific sequences . By "RNA gel" analysis of poly(A)-RNA, three independent mRNA species were found to hybridize to a CaBP clone; none of these RNA species were found in -D poly(A)-RNA . With this comparative colony hybridization procedure, we were able to identify CaBP-specific clones corresponding to a mRNA that is 0.1% of the total poly(A)-mRNA . The differential colony hybridization procedure using an enriched vs . a nonenriched probe should be of value in screening for other cDNA clones complementary to rare mRNA species. Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4223 - 7 The FLP protein of the yeast 2-microns plasmid: expression of a eukaryotic genetic recombination system in Escherichia coli; Cox MM; The FLP gene of the yeast 2-microns plasmid is involved in a site-specific recombination event that results in the inversion of a set of sequences within the plasmid . This gene has been cloned and expressed in Escherichia coli . Expression of the FLP gene results in efficient recombination within the bacterial cell, which is specific for plasmids containing at least one 2-microns plasmid recombination site . This work demonstrates that (i) FLP protein is actively involved in 2-microns plasmid recombination; (ii) no other factors specific to yeast are required for the reaction; (iii) FLP protein acts efficiently in trans; (iv) FLP protein will promote site-specific insertion and deletion reactions in addition to the inversion reaction; and (v) FLP-promoted recombination is not dependent upon any DNA structural features unique to yeast chromatin. J Bacteriol, 1983 Jul, 155(1), 69 - 73 Accumulation of cyclic GMP in filaments of Escherichia coli BUG6; Cook WR et al.; Experiments with Escherichia coli BUG6, a temperature-sensitive cell division mutant, have shown that at the restrictive temperature (42 degrees C) the loss of cell division potential (filamentation) was accompanied by an unusual increase in intracellular cyclic GMP (cGMP) . At the permissive temperature (30 degrees C), cell division proceeded normally, and cGMP did not accumulate . Increasing the osmotic strength of the medium with NaCl suppressed filamentation in BUG6 at 42 degrees C and also suppressed the temperature-sensitive accumulation of cGMP . The addition of nalidixic acid to BUG6 at 30 degrees C induced filamentation but failed to cause cGMP accumulation . A similar accumulation of cGMP has not been observed in other E . coli strains. J Bacteriol, 1983 Jul, 155(1), 407 - 11 Inhibition of secretion of a mutant lipoprotein across the cytoplasmic membrane by the wild-type lipoprotein of the Escherichia coli outer membrane; Lee N et al.; A globomycin-resistant mutant of Escherichia coli was found to produce a precursor of the major outer membrane lipoprotein (prolipoprotein), in which the glycine residue at position 14 within the signal peptide was replaced by an aspartic acid residue . The same mutation has been reported by Lin et al . (Proc . Natl . Acad . Sci . U.S.A . 175:4891-4895, 1978) . The structural gene of the mutant prolipoprotein was inserted into an inducible expression cloning vehicle . When the mutant prolipoprotein was produced in lipoprotein-minus host cells, 82% of the unprocessed protein was found in the membrane fraction, with the remaining 18% localized in the soluble fraction . However, when the production of the mutant prolipoprotein was induced in the wild-type lpp+ host cells, only 31% of the mutant prolipoprotein was found in the membrane fraction, leaving the remaining 69% in the soluble, cytoplasmic fraction . In addition, the assembly of the wild-type lipoprotein in these cells was not affected, whether the mutant prolipoprotein was produced or not . These results suggest that secretions of both mutant and wild-type prolipoproteins utilize the same component(s) responsible for the initial stages of secretion across the cytoplasmic membrane . However, it appears that the wild-type lipoprotein has a higher affinity for these components than does the mutant lipoprotein. J Bacteriol, 1983 Jul, 155(1), 398 - 401 Identification of a membrane protein induced concurrently with cell filamentation by cyclic AMP in an Escherichia coli K-12 fic mutant; Utsumi R et al.; A membrane protein with a molecular weight of 40,000 (40K protein) was induced concurrently with cell filamentation by cyclic AMP (cAMP) in a fic mutant . In the crp mutant and the wild-type strain, cell filamentation by cAMP was not observed, and the 40K protein was not induced . Induction of the 40K protein is regulated by the cAMP-cAMP receptor protein complex and is closely related to cell filamentation by cAMP in the fic mutant. J Bacteriol, 1983 Jul, 155(1), 317 - 29 Coliphage P1-mediated transduction of cloned DNA from Escherichia coli to Myxococcus xanthus: use for complementation and recombinational analyses; O'Connor KA et al.; We have found that coliphage P1 can be used to transduce cloned DNA from Escherichia coli to Myxococcus xanthus . Transduction occurred at a high efficiency, and no evidence for DNA restriction was observed . The analysis of the transductants showed that they fall into three general categories: (i) haploid cells which contain portions of the cloned DNA substituted for homologous chromosomal DNA; (ii) heterozygous merodiploids which contain the recombinant plasmid integrated into the chromosome at a region of homology; and (iii) homozygous merodiploids which contain two copies of a portion of the cloned DNA with the loss of the chromosomal copy of the genes . The merodiploids, once formed, are relatively stable . They were used to analyze two genes necessary for aggregation and thus fruiting body formation . P1 transduction also permits the reintroduction and substitution of mutated regions of cloned DNA into M . xanthus for the analysis of the role of the DNA in cellular physiology and development. J Bacteriol, 1983 Jul, 155(1), 265 - 74 Interactions between chemotaxis genes and flagellar genes in Escherichia coli; Parkinson JS et al.; Escherichia coli mutants defective in cheY and cheZ function are motile but generally nonchemotactic; cheY mutants have an extreme counterclockwise bias in flagellar rotation, whereas cheZ mutants have a clockwise rotational bias . Chemotactic pseudorevertants of cheY and cheZ mutants were isolated on semisolid agar and examined for second-site suppressors in other chemotaxis-related loci . Approximately 15% of the cheZ revertants and over 95% of the cheY revertants contained compensatory mutations in the flaA or flaB locus . When transferred to an otherwise wild-type background, most of these suppressor mutations resulted in a generally nonchemotactic phenotype: suppressors of cheY caused a clockwise rotational bias; suppressors of cheZ produced a counterclockwise rotational bias . Chemotactic double mutants containing a che and a fla mutation invariably exhibited flagellar rotation patterns in between the opposing extremes characteristic of the component mutations . This additive effect on flagellar rotation resulted in essentially wild-type swimming behavior and is probably the major basis of suppressor action . However, suppression effects were also allele specific, suggesting that the cheY and cheZ gene products interact directly with the flaA and flaB products . These interactions may be instrumental in establishing the unstimulated swimming pattern of E . coli. J Bacteriol, 1983 Jul, 155(1), 254 - 64 Cloning of the pif region of the F sex factor and identification of a pif protein product; Rotman GS et al.; This paper reports a detailed investigation of the pif region of the F factor responsible for inhibition of development of T7 and related "female-specific" phages . We have mapped a series of pif::Tn5 insertions to a region between 39.6 and 42.8 kilobases on the physical map of F . All pif::Tn5 insertions plated T7 at full efficiency; most were clustered in a 1.8-kilobase interval on both sides of the EcoRI site located at F coordinate 40.3 kilobases . A 5.2-kilobase Pst-I fragment with F coordinates 38.9 to 44.1 has been cloned into a pSC101 vector to create the Pif+ plasmid pGS103 . A series of Pif- deletion mutants and nonsense mutants were isolated from pGS103 . Using minicells carrying pGS103 or its derivatives, we have identified a 70,000-dalton pif protein. J Bacteriol, 1983 Jul, 155(1), 122 - 8 Regulation of expression of the colicin gene of I1 group plasmid TP110; Glazebrook JA et al.; The control of expression of the colicin Ib gene of the I1 group plasmid TP110 has been investigated . The colicin promoter was fused to the structural gene for beta-galactosidase, using the Mu d(Aprlac) phage, and the plasmid carrying this fusion was introduced into a variety of bacterial strains defective in genes involved in the "SOS" response . Colicin Ib belongs to that group of genes directly controlled by the repressor produced by the lexA gene, and expression was inducible by DNA-damaging agents . Mutations in uvrA, -B, and -C reduced the efficiency of induction by mitomycin C, as did mutations in recB . Mutations in recA and recF effectively prevented induction by mitomycin C, whereas mutations in lexA had contrasting effects, depending upon their effect on the properties of lexA protein . The spr-51 mutation (which inactivates lexA protein) led to constitutive expression, whereas the lexA3 mutation (which makes lexA protein refractory to cleavage by recA protein) completely inhibited inducible expression . In addition to lexA control, a TP110-coded function was identified which appeared able to inhibit colicin expression when the gene responsible was present in high copy number. Infect Immun, 1983 Jul, 41(1), 88 - 96 Acquired chemotactic inhibitors during infection with guinea pig cytomegalovirus; Tannous R et al.; Factors involved in neutrophil and monocyte migrations were serially studied in strain 2 guinea pigs undergoing initial cytomegalovirus infection and sham-inoculated controls . All studies remained unchanged in uninfected animals . Monocyte migrations and neutrophil spontaneous migration remained unchanged in infected animals . However, transient abnormalities occurred early in infection, comprising a decrease in neutrophil-directed migration towards C5-derived chemotactic fractions (C5-fr) and a decrease in the chemotactic activity of zymosan-activated plasma . Consequently, the presence of neutrophil- and chemotaxin-directed inhibitors in plasma was investigated . Normal neutrophils, C5-fr, Escherichia coli-derived bacterial factor, and the synthetic peptide F-met-leu-phe were first incubated with control or infected plasmas and then assayed for directed migration and lysosomal enzyme release . Results indicated the de novo appearance of both neutrophil- and chemotaxin-directed inhibitory activities in plasma during early infection . The neutrophil-directed inhibition was heat stable (56 degrees C for 120 min) and nonspecific (responses to all chemotaxins were inhibited) . The chemotaxin-directed inhibition was heat stable and C5-fr specific . The cytomegalovirus-induced inhibitors may be important in the enhanced susceptibility to concurrent opportunistic infections. Infect Immun, 1983 Jul, 41(1), 294 - 301 Escherichia coli lipopolysaccharides diminish and enhance cell function of human polymorphonuclear leukocytes; Henricks PA et al.; The effects of the lipopolysaccharide (LPS) of Escherichia coli J5 and 0111B4 on the function of human polymorphonuclear leukocytes (PMN) were tested . E . coli J5 is a UDP-galactose-4-epimerase-deficient mutant of E . coli 0111B4, and its LPS, therefore, contains mainly lipid A, as it lacks the polysaccharide side chains . PMN which had been incubated with J5 LPS showed decreased phagocytic, chemotactic, and metabolic activities as compared with control PMN . In contrast, incubation of PMN with 0111B4 LPS had no effect or even an enhancing effect on PMN function . When lipid A and the polysaccharide fraction were isolated from 0111B4 LPS, it was shown that lipid A had the same deleterious effect on PMN function as did J5 LPS and that the LPS fraction had no effect . When PMN were incubated with J5 LPS or lipid A, it could be shown that these structures were able to induce PMN to generate superoxide and chemiluminescence . 0111B4 LPS and the polysaccharide component were able to generate a metabolic burst by the PMN to a lesser extent . The induced defects in PMN function by J5 LPS could be prevented when polymyxin B or an oxygen-radical scavenger was present . We hypothesize that the lipid A portion of LPS is toxic for PMN due to the induction of toxic oxygen species by the PMN . These toxic oxygen species destroy the phagocytic, chemotactic, and metabolic activities of the PMN. Mikrobiologiia, 1983 Jul-Aug, 52(4), 563 - 8 {Metabolic limitation of DNA-polymerase I synthesis by Escherichia coli strain CM5199}; Soktoev SA et al.; The work was aimed at studying DNA polymerase I synthesis after induction of the vector prophage lambda polA (NM 964) in lysogenic Escherichia coli CM 5199 in the course of batch cultivation in different media and at various growth phases as well as upon "nutrient shifts" caused by adding organic compounds to the minimal medium . The enzyme activity was highest when the phage was induced at the exponential phase of growth and in media richer in their composition . The enzyme synthesis, the dynamics of protein and RNA content were studied after induction of the phage and enrichment of the medium; the studies have shown that synthesis of DNA polymerase I is influenced by limitation at two levels: (1) biosynthesis of amino acids and (2) biosynthesis of components of the protein-synthesizing apparatus . There is a direct correlation between DNA polymerase I biosynthesis under the control of vector DNA and the growth rate of the culture. J Antibiot (Tokyo), 1983 Jul, 36(7), 900 - 6 Aclacinomycin A-inhibition of phage phi X174 DNA synthesis in vitro; Tanaka A et al.; Aclacinomycin A inhibited the in vitro conversion of phage phi X174 single-stranded DNA to the replicative form DNA . DNA synthesis was inhibited by 50% in the presence of 15 microM aclacinomycin A . The inhibition was competitive with respect to template DNA (Ki = 13 microM) and was reversed by addition of Escherichia coli cell extracts . Short complementary strands approximately one-third of unit length molecule were synthesized in the presence of 15 microM aclacinomycin A . The data suggest that aclacinomycin A may inhibit the process of phi X174 DNA chain elongation by a direct interaction with the E . coli host enzymes. J Bacteriol, 1983 Jul, 155(1), 330 - 6 Two fep genes are required for ferrienterochelin uptake in Escherichia coli K-12; Pierce JR et al.; Escherichia coli mutants defective in the assimilation of iron from ferrienterochelin were isolated and characterized . One mutant was able to bind ferrienterochelin to its outer membrane but could not transport it into the cell . Complementation tests with lambda hybrid phage were employed to distinguish the defective gene, which we term fepB, from fepA, the structural gene for the outer membrane ferrienterochelin receptor protein . These same physiological and genetic tests were employed to tentatively classify several previously described fep mutants as carrying either fepA or fepB . The data demonstrate the existence of fepB and provide an explanation for previous difficulties in identifying fepB mutants. Bioorg Khim, 1983 Jul, 9(7), 900 - 5 {Modification of sulfhydryl groups in ribosomal proteins by a dinitrophenyl hapten}; Kozhukharova MS et al.; A dinitrophenyl hapten capable of protein SH-group modification was synthesized and the specificity of its reaction with SH-groups of the E . coli ribosomal proteins was studied . The possibility of incorporation of the Dnp-modified protein into ribosomal subunits by in vitro reconstitution was demonstrated with the ribosomal protein S12 . The Dnp-hapten attached to the protein S12 was found to be accessible for interaction with the Dnp-specific antibodies and therefore to be exposed on the surface of the reconstituted 30S subunit . Thus, the approach for incorporation of the antigenic groups into the protein components of supramolecular structures was proposed. J Gen Microbiol, 1983 Jul, 129 (Pt 7), 2079 - 89 Discriminated induction of SOS functions in Escherichia coli by alkylating agents; Barbe J et al.; Treatment of Escherichia coli with the alkylating agents diethyl sulphate, ethyl methane-sulphonate and N-methyl-N'-nitrosoguanidine produces a different pattern of expression of SOS functions . There is a full induction of recA-dependent inhibition of cell respiration, a slight induction of lambda prophage, and no inhibition of cellular division . In a comparative study with bleomycin, an agent which is able to induce these three SOS functions, we have also shown that the differences in expression of SOS functions are not due to any variation in the pattern of DNA synthesis, or DNA degradation after treatment with alkylating agents . These results suggest that the kind of damage induced in the DNA may be important in determining which SOS function is expressed. Fundam Appl Toxicol, 1983 Jul-Aug, 3(4), 209 - 14 Oxygen and redox-active drugs: shared toxicity sites; Brown OR et al.; Paraquat and nitrofurantoin can accept single electrons and, under appropriate conditions in tissues and cells, can pass these electrons to oxygen, thus participating in redox cycling . Similarities in the response of the target organ (the lung) and in subsequent pathology have also been observed among animals poisoned by oxygen and by these chemicals . We report evidence primarily obtained from Escherichia coli for common biochemical sites of toxicity for these agents . Common sites for oxygen and paraquat involve biosynthesis of specific amino acids, induction of genetic stringency via unloaded tRNAs resulting from amino acid deficiencies, decreased thiamin content, and impaired biosynthesis of pyridine nucleotide coenzyme biosynthesis for paraquat and oxygen . Inhibition of specific amino acid biosynthesis and induction of stringency also have been observed for nitrofurantoin . RNA and DNA biosynthesis are also impaired by oxygen; this has not been examined for paraquat or nitrofurantoin . There is a biochemical basis and preliminary data to support inhibition of NAD biosynthesis as a component of mammalian toxicity for these agents . Niacin may act to circumvent the consequences of the biochemical lesion at quinolinate phosphoribosyl transferase in NAD biosynthesis. Plasmid, 1983 Jul, 10(1), 66 - 72 Transcription of plasmid DNA in Escherichia coli minicells; Crooks JH et al.; Cellular RNA polymerase in association with plasmid DNA segregates into the minicells of minicell-producing strains . In general, one "plasmid equivalent" of RNA polymerase, reflecting the size of the segregating plasmid DNA and its efficiency of segregation, entered the minicell with the plasmid . The amount of RNA polymerase (measured as the amount of enzyme activity purified from minicells and the rate of RNA synthesis in plasmid-containing minicells), and not the DNA content, appeared to be rate-limiting in plasmid-mediated transcription in minicells . The purified minicell and cellular RNA polymerases showed the same sensitivity to rifampin and streptolydigin; both were associated with sigma factor, although the minicell enzyme appeared to have slightly less than the cellular enzyme . These studies demonstrate that transcription of plasmid DNA in minicells is a function of the efficiency of segregation and the amount of RNA polymerase which enters with the plasmid DNA . Because RNA polymerase is limiting, plasmids with relatively weak promoters for the vector genes should be used when attempting to identify products from inserted foreign DNA. Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4450 - 4 Identification of a precursor molecular for the RNA moiety of the processing enzyme RNase P; Gurevitz M et al.; A precursor molecule for 10Sb (M1) RNA, the RNA moiety of the RNA processing enzyme ribonuclease P (EC 3.1.26.5), is accumulated transiently in an Escherichia coli strain containing a plasmid that carries the 10Sb RNA gene . The same RNA precursor molecule is accumulated, in relatively large quantities, in a temperature-sensitive RNase E- mutant at the nonpermissive temperature . The RNA precursor includes 10Sb RNA and an extra 3' fragment that contains a termination stem and loop . It can be processed in vitro to a molecule the size of 10Sb RNA . None of the four endoribonucleases of E . coli--RNase III, RNase E, RNase F, or RNase P--takes part in this cleavage reaction . Therefore, we suggest that the processing of the precursor-10Sb RNA to 10Sb RNA is carried out by a thus-far unidentified endoribonuclease . The accumulation of a RNA molecule in a RNase E- mutant that does not contain a cleavage site for RNase E has been encountered previously and can be explained by assuming the existence of a RNA processing complex in the E . coli cell. Proc Natl Acad Sci U S A, 1983 Jul, 80(14), 4412 - 6 Selection of functional cDNAs by complementation in yeast; McKnight GL et al.; Yeast cDNA was prepared in a yeast expression plasmid to generate a cDNA plasmid pool composed of approximately 40,000 members . Several yeast mutants were transformed with the cDNA plasmid pool, and the cDNAs for ADC1, HIS3, URA3, and ASP5 were isolated by functional complementation . Restriction enzyme analysis confirmed the genetic identity of the ADC1, HIS3, and URA3 cDNAs and demonstrated that the URA3 cDNA contains 5' noncoding sequences . The relative abundance of the various cDNAs in the cDNA plasmid pool paralleled the abundance of the mRNAs in total poly(A)+ RNA, which ranged from approximately 0.01% to 1% . The utility of this approach to isolate rare cDNAs from higher eukaryotes is discussed. Cell, 1983 Jul, 33(3), 865 - 76 Expression of rRNA and tRNA genes in Escherichia coli: evidence for feedback regulation by products of rRNA operons; Jinks-Robertson S et al.; We have tested a model for global ribosome biosynthesis by examining the effects of increased gene dosage on the synthesis of rRNA . Increasing gene dosage does not lead to a significant increase in total rRNA transcription; i.e., rRNA synthesis from individual rRNA operons is reduced to keep total rRNA production unchanged . In contrast, when the plasmid-encoded rRNA operons used to increase gene dosage contain deletions within the rRNA coding region, rRNA transcription is gene-dosage-dependent; i.e., rRNA regulation is relieved . We find that the syntheses of most, if not all, tRNAs are also subject to the same controls as rRNA transcription . We conclude that the production of functional rRNA is monitored by the regulatory system that controls rRNA and tRNA transcription . We propose that rRNA and tRNA are negatively controlled by products of rRNA operons and discuss evidence suggesting that ribosomes are the key element involved in the postulated feedback regulation. Cancer Treat Rep, 1983 Jul-Aug, 67(7-8), 611 - 9 Leukocyte recovery with short-chain RNA fragments in cyclophosphamide-treated rabbits; Beljanski M et al.; Single-stranded short-chain RNA fragments, obtained by mild degradation of purified Escherichia coli ribosomal RNA(s) with pancreatic RNase A, exhibit particular biologic activities in vitro and in vivo . In vitro, these RNA fragments are used by DNA-dependent DNA polymerase I as primers to initiate the replication of DNA(s) isolated from rabbit bone marrow and spleen; they are inactive with DNA isolated from several normal tissues and cancerous cells . Administered iv, RNA fragments restore a normal level of circulating leukocytes in rabbits with high doses of cyclophosphamide (CP) . Granulocyte/lymphocyte balance, upset by daily CP administration, is also restored during the increase of both types of cells . No toxicity is observed, and numerous repeated doses of RNA fragments show no cumulative effect and do not lead to loss of leukopoietic stimulating activity . Tumor-bearing mice can be protected by RNA fragments against the toxic effect of CP without impeding the anticancer activity of this drug. Arch Biochem Biophys, 1983 Jul 1, 224(1), 196 - 205 Synthesis of polyadenylate-containing RNA in vitro in permeable cells of Escherichia coli B; Gopalakrishna Y et al.; As a starting point for the study of the biosynthesis of polyadenylated RNA in bacteria, the characteristics of RNA synthesis by cells of Escherichia coli B made permeable to small molecules by treatment with toluene were examined . Such cells mediated the incorporation of radiolabeled ribonucleoside triphosphates into RNA in a reaction that was sensitive to inhibitors of RNA polymerase and required the simultaneous presence of the four ribonucleoside triphosphates . Between 10 to 15% of the RNA synthesized under these conditions was polyadenylated as shown by affinity chromatography on oligo(dT)-cellulose . The presence of orthophosphate or dADP, inhibitors of polynucleotide phosphorylase, had no effect on the reaction and the rate of RNA synthesis was indistinguishable in the polynucleotide phosphorylase-deficient strain PR-7 and in its otherwise isogenic parent strain PR-100 . The poly(A) tracts associated with the newly synthesized RNA could be isolated after exhaustive digestion with pancreatic and T1 ribonucleases and accounted for 14% of the poly(A)-RNA . At least 74% of the poly(A) sequences were located at the 3' ends of RNA molecules and their weight-average length was 48 nucleotide residues . The size distribution of total RNA and poly(A)-RNA synthesized in the toluenized cell system was similar to that of the corresponding pulse-labeled fractions derived from growing cultures . The sequence complexity of poly(A)-RNA and unadenylated RNA synthesized in toluenized cells with {alpha-32P}CTP as the labeled substrate was analyzed by hybridization to fragments of Escherichia coli B DNA generated by digestion with EcoRI restriction endonuclease and immobilized on nitrocellulose sheets . Both RNA fractions hybridized with many DNA fractions, the hybridization patterns being similar with poly(A)-RNA and unadenylated RNA . This indicated that many different types of RNA transcripts synthesized in toluenized cells were subject to polyadenylation, but that polyadenylation was incomplete so that each transcript was present in both an adenylated and an unadenylated state. Arch Biochem Biophys, 1983 Jul 1, 224(1), 134 - 41 DNA sequesters endogenous mRNA during preparation of crude Escherichia coli extracts for protein synthesis; use of an S60 reduces the sequestered mRNA; Goldman E et al.; High backgrounds of endogenous incorporation of amino acids into protein in crude extracts of Escherichia coli are a consequence of endogenous messenger RNA . This RNA survives the preparation by virtue of protection or sequestering by endogenous DNA . Thus, DNase treatment in the crude extract leads to the elimination of this mRNA, while DNase treatment has no effect on the purified RNA . The endogenous mRNA also appears to be physically associated with DNA on CsCl gradients, and can be largely removed along with DNA by centrifugation of extracts at 60,000g . The top layer above a 60,000g centrifugation (S60) appears to be suitable for protein synthesis, and provides for lower background levels of endogenous messenger RNA. J Virol, 1983 Jul, 47(1), 202 - 16 Characterization of am404, an amber mutation in the simian virus 40 T antigen gene; Rawlins DR et al.; We analyzed the biological activity of an amber mutation, am404, at map position 0.27 in the T antigen gene of simian virus 40 . Immunoprecipitation of extracts from am404-infected cells demonstrated the presence of an amber protein fragment (am T antigen) of the expected molecular weight (67,000) . Differential immunoprecipitation with monoclonal antibody demonstrated that am T antigen was missing the carboxy-terminal antigenic determinants . The amber mutant was shown to be defective for most of the functions associated with wild-type T antigen . The mutant did not replicate autonomously, but this defect could be complemented by a helper virus (D . R . Rawlins and N . Muzyczka, J . Virol . 36:611-616, 1980) . The mutant failed to transform nonpermissive rodent cells and did not relieve the host range restriction of adenovirus 2 in monkey cells . However, stimulation of host cell DNA, whose functional region domain has been mapped within that portion of the protein synthesized by the mutant, could be demonstrated in am404-infected cells . A number of unexpected observations were made . First, the am T antigen was produced in unusually large amounts in a simian virus 40-transformed monkey cell line (COS-1), but overproduction was not seen in nontransformed monkey cells regardless of whether or not a helper virus was present . This feature of the mutant was presumably the result of the inability of am T antigen to autoregulate, the level of wild-type T antigen in COS-1 cells, and the unusually short half-life of am T antigen in vivo . Pulse-chase experiments indicated that am T antigen had an intracellular half-life of approximately 10 min . In addition, although the am T antigen retained the major phosphorylation site found in simian virus 40 T antigen, it was not phosphorylated . Thus, phosphorylation of simian virus 40 T antigen is not required for the stimulation of host cell DNA synthesis . Finally, fusion of am404-infected monkey cells with Escherichia coli protoplasts containing appropriate procaryotic suppressor tRNAs showed that am404 is a suppressible nonsense mutation. Immunology, 1983 Jul, 49(3), 519 - 28 Alpha-macroglobulin-induced release of anti-Ig-coated particles from a subpopulation of rabbit B lymphocytes; Mackin WM et al.; Approximately half of the rosettes formed by rabbit Ig+ lymphocytes (B cells) and anti-coated erythrocytes or glutaraldehyde-fixed bacteria are dissociated upon the addition of rabbit serum . Rabbit serum was fractionated and the rosette-dissociating activity was found in purified preparations of rabbit alpha 1- and alpha 2-macroglobulins . Studies designed to elucidate the mechanism of rosette dissociation suggested that the alpha-macroglobulins dissociated rosettes by causing the release or proteolytic cleavage of the membrane proteins complexed with the anti-Ig-coated particles . These data suggest that the alpha-macroglobulins may have a role in the interaction of B lymphocytes with particulate antigens. Zh Mikrobiol Epidemiol Immunobiol, 1983 Jul, (7), 59 - 62 {Electron microscopic study of the interaction of nephritogenic strains of Escherichia coli carrying D-mannose-resistant fimbriae with the cells of a human kidney line}; Gosteva VV et al.; The electron-microscopic study revealed that nephritogenic E . coli having L-mannose-resistant fimbriae contacted with subcultured human renal cells due to the interaction of fimbriae with microvilli and, less frequently, with the cytoplasmic membrane surrounding the main part of the cell, as well as with the electron-opaque fibrillar material in the intercellular space . The possibility of very close interaction was demonstrated; in some cases this interaction was so close that the outlines of bacterial and epitheloid cells followed each other, the invagination of the external membrane of the host cell being sometimes observed . The expediency of using ruthenium red for detecting fimbriae in morphological studies was shown . The cytopathogenic effect observed in this study and developing by the end of the 5-hour period from the moment of the inoculation of the monolayer with E . coli was manifested by the swelling of mitochondria accompanied by the partial ruptures of cristae, the widening of channels in the endoplasmic reticulum, the appearance of the secondary lysosomes and the increase of their number. Infect Immun, 1983 Jul, 41(1), 174 - 80 Reduction of the secretory response to Escherichia coli heat-stable enterotoxin by thiol and disulfide compounds; Greenberg RN et al.; We examined the effects of disulfide and thiol compounds on Escherichia coli heat-stable enterotoxin (ST) and cyclic GMP-induced secretion . Both cystamine and cystine (disulfide compounds) reduced the secretory responses to submaximal doses of ST in suckling mice (at 0.5 mumol per mouse) and reduced ST activation of guanylate cyclase (by 33 to 73% at 1 mM) . In higher doses, cystamine completely eradicated a maximally effective ST dose as well . In addition, the sulfhydryl (thiol) compounds cysteamine, cysteine, and acetylcysteine strikingly reduced the secretory response and the guanylate cyclase response to ST . Neither the disulfide nor the thiol compounds tested reduced cyclic GMP-induced secretion . These studies suggest that disulfide and thiol compounds both block ST-induced secretion before its activation of guanylate cyclase . Taken with the work of others, these findings suggest that disulfide compounds may alter the oxidation reduction state of a cell or act directly on the guanylate cyclase enzyme, whereas thiol compounds may inactivate ST itself by breaking its disulfide bridges, or it may alter guanylate cyclase activation by ST . Both families of compounds deserve further consideration among potential antisecretory agents for application in the control of ST-induced diarrhea. Eur J Biochem, 1983 Jul 1, 133(3), 499 - 507 Interaction between the different domains of aminoacyl-tRNA and the elongation-factor-Tu x kirromycin complex; Guesnet J et al.; In this work, we have studied the effect of aa-tRNA and derived 3' aminoacylated fragments on the EF-Tu GTPase in the presence of kirromycin, using two systems: without and with ribosomes . The aa-tRNA fragments were obtained by enzymatic digestion . Procedures for the enzymatic preparation of C-A-Val and Val-tRNA Val1 3' half molecule, as well as a purification method for short 3' aminoacylated fragments based on the amino group charge, were newly developed for this work . Aminoacyl-adenosine was found to be able to stimulate the EF-Tu x kirromycin GTPase, but only to a very small extent . Increasing the length of the aminoacylated fragments increased the stimulatory effect as follow: A-Val much less than C-A-Val less than C-C-A-Val less than 3' valyladenosine dodecanucleotide much less than Val-tRNA Val1 3' half molecule less than Val-tRNA Val1 . The presence of ribosomes did not affect the order of effectiveness, but increased the basic GTPase activity of EF-Tu x kirromycin and the stimulation by aa-tRNA, its 3' half molecule and even more by its 3' short fragments . The effect of aa-tRNA and derived 3' fragments in the absence of ribosomes was not influenced by MgCl2 concentrations of 5-30 mM whereas, in the presence of ribosomes, low concentrations of MgCl2 (5 mM) greatly reduced the stimulation of aa-tRNA and, to a lesser extent, also the effect of the C-C-A-aa as well as the basic activity of the EF-Tu x kirromycin GTPase . The extent of the stimulation by aa-tRNA, and even more by C-C-A-aa, depends on the nature of the amino acid . Among the aminoacyl side chains tested (Arg-, Phe-, Val-, Met-, Leu-, Lys-) arginine was found to be the most active and leucine the least . Our results show that (a) the 3' aminoacylated extremity is of prime importance for the stimulation of the EF-Tu GTPase, (b) in the 3' extremity there are critical sequences for the interaction with EF-Tu and (c) other domains of the aa-tRNA molecule are capable of influencing this reaction: one of the most important is the region including the T psi C loop and stem. Clin Chim Acta, 1983 Jun 30, 131(1-2), 75 - 85 Immunoassay of human muscle enolase subunit in serum: a novel marker antigen for muscle diseases; Kato K et al.; A sandwich enzyme immunoassay method for measurement of beta subunit of muscle enolase in human serum was developed by use of purified antibodies to enolase beta subunit and beta-D-galactosidase from Escherichia coli as label . The assay was specific to the beta subunit with no cross-reaction with the alpha and gamma subunits of human enolase . The measurable range was from 10 pg to 10 ng per assay tube or 1 to 1000 ng/ml serum . Coefficients of variation within-run and between-run for the assay of serum beta subunit were less than 14% . Normal adult sera contained about 6 ng/ml of the beta subunit, and the levels were significantly elevated in sera of patients with muscular dystrophy and those with myocardial infarction . Serum levels of the beta subunit correlated well with those of creatine phosphokinase, but poorly with those of myoglobin in the same samples . The specific distribution of beta subunit in skeletal muscle and heart was confirmed by measuring the levels in various tissue extracts. Nature, 1983 Jun 30, 303(5920), 770 - 4 Escherichia coli single-strand binding protein stabilizes specific denatured sites in superhelical DNA; Glikin GC et al.; Escherichia coli single-strand binding protein relaxes supercoiled DNA molecules containing the Drosophila melanogaster histone gene repeat unit, by stabilizing denaturation bubbles that map near the boundaries of the genes, at sites that in native chromatin have been shown to be hypersensitive to nucleases . A similar process may contribute to the propagation of such hypersensitive sites after their induction on the activation of gene expression. Biochim Biophys Acta, 1983 Jun 29, 745(3), 247 - 58 The pH-dependence of the binding of dihydrofolate and substrate analogues to dihydrofolate reductase from Escherichia coli; Stone SR et al.; The interaction of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli with dihydrofolate and folate analogues has been studied by means of binding and spectroscopic experiments . The aim of the investigation was to determine the number and identity of the binary complexes that can form, as well as pKa values for groups on the ligand and enzyme that are involved with complex formation . The results obtained by ultraviolet difference spectroscopy indicate that, when bound to the enzyme, methotrexate and 2,4-diamino-6,7-dimethylpteridine exist in their protonated forms and exhibit pKa values for their N-1 nitrogens of above 10.0 . These values are about five pH units higher than those for the compounds in free solution . The binding data suggest that both folate analogues interact with the enzyme to yield a protonated complex which may be formed by reaction of ionized enzyme with protonated ligand and/or protonated enzyme with unprotonated ligand . The protonated complex formed with 2,4-diamino-6,7-dimethylpteridine can undergo further protonation to form a protonated enzyme-protonated ligand complex, while that formed with methotrexate can ionize to give an unprotonated complex . A group on the enzyme with a pKa value of about 6.3 is involved with the interactions . However, the ionization state of this group has little effect on the binding of dihydrofolate to the enzyme . For the formation of an enzyme-dihydrofolate complex it is essential that the N-3/C-4 amide of the pteridine ring of the substrate be in its neutral form . It appears that dihydrofolate is not protonated in the binary complex. FEBS Lett, 1983 Jun 27, 157(1), 91 - 4 Quantitative study of interaction of deacylated tRNA with Escherichia coli ribosomes . Role of 50 S subunits in formation of the E site; Kirillov SV et al.; The 30 S subunit contains 2 sites for tRNA binding (Phe-tRNA, AcPhe-tRNA, tRNAPheOH) with the functional properties of D and A sites of the 70 S ribosome after attachment of 50 S subunit . The third (E) site specific for deacylated tRNA is introduced into 70 S ribosome by its 50 S subunit . The E-site binding of tRNAPheOH is not sensitive to either tetracycline and edeine, and practically codon-independent . The affinity constant of tRNAPheOH for the E site is 2-3 orders of magnitude lower than that for the D site. FEBS Lett, 1983 Jun 27, 157(1), 85 - 90 Physical properties of ribosomal proteins isolated under different conditions from the Escherichia coli 50 S subunit; Tumanova LG et al.; Physical properties of ribosomal proteins obtained with or without denaturating agents were compared . CD measurements and NMR studies have shown that proteins L2, L19, L24 and L30 isolated under denaturing conditions have the same properties as those prepared avoiding denaturating agents . CD and NMR spectra of proteins L1, L6, L11, L23, L25 and L29 obtained by us under denaturating conditions practically coincide with the data for the same proteins reported under 'mild' conditions . These findings suggest that the differences of reported physical properties can be due to different procedures of protein renaturation rather than to the methods of their isolation. J Mol Biol, 1983 Jun 25, 167(2), 259 - 74 Deletion mutagenesis of the Escherichia coli galactose operon promoter region; Busby S et al.; Using recombinant DNA technology we have created a series of progressively longer deletions both upstream and downstream from the Escherichia coli galactose operon regulatory region . The effects of these lesions on expression of the two overlapping galactose promoters have been quantitated after DNA fragments carrying these deletions were cloned in a plasmid vector, in which the beta-galactosidase gene could be expressed from the truncated galactose regulatory region . The results allow us to determine which sequences are necessary for the activity of the two promoters . Our results show that for the P1 promoter, which is controlled by the cyclic AMP-cyclic AMP receptor protein complex (cAMP-CRP), the sequence necessary for full activity starts 56 base-pairs upstream from the transcription initiation point . In contrast, for the P2 promoter, which functions in the absence of cAMP-CRP, the crucial sequence extends to only 39 base-pairs upstream from the transcription start . Deletions that cut into these sequences cause reductions in promoter strength, although some promoter activity is observed even when the "-35 region" of both P2 and P1 are deleted . Analysis of deletions originating downstream from the regulatory region shows that the elimination of the P1 and P2 Pribnow box sequences leads to loss of promoter activity . However, sequences downstream from the P1 start can be replaced without affecting the activity of either promoter . Finally examination of DNA fragments containing total deletions of both galactose promoters allows us to confirm that the flanking sequences contain no significant promoter activity and that the P1 and P2 promoters are principally responsible for galactose operon expression in vivo. J Biol Chem, 1983 Jun 25, 258(12), 7550 - 5 A novel proteolytic activity apparently initiating degradation of beta-galactosidase nonsense fragments in in vitro extracts of Escherichia coli; McKnight JL et al.; Our previous in vivo studies demonstrated that large premature fragments of beta-galactosidase are degraded in Escherichia coli by a common pathway, and the initial event appears to be a site-specific cleavage (McKnight, J . L., and Fried, V . A . (1981) J . Biol . Chem . 256, 9652-9661) . We now have developed a cell-free system that retains the specificity of this early cleavage event . Immunochemical techniques were used to isolate and quantitate the polypeptide substrate and products in pulse-chase experiments . The in vitro system has an activity that quantitatively converts the prematurely terminated A polypeptide of the lacZ non-sense mutant CSH-10 to the 90-kilodalton common B polypeptide intermediate observed in vivo . The activity is localized in the cytoplasm since the cleavage reaction is not affected by osmotic shock of whole cells or removal of the membrane fraction from cell-free extracts . The lon mutation capR9, which blocks this degradation pathway in vivo, does not affect the initial cleavage event in cell-free extracts of CSH-10 carrying this mutation . The in vitro cleavage event in extracts of lon+ CSH-10 or the isogenic lon- mutant is not stimulated by addition of ATP, not inhibited by depletion of ATP pools by hexokinase-2-deoxyglucose treatment, and not inhibited by EDTA or phenylmethylsulfonyl fluoride . These results suggest that the ATP-dependent proteolytic activity of the lon gene product does not directly catalyze this primary cleavage event. Nucleic Acids Res, 1983 Jun 25, 11(12), 3873 - 88 Determination of the promoter strength in the mixed transcription system . II . Promoters of ribosomal RNA, ribosomal protein S1 and recA protein operons from Escherichia coli; Kajitani M et al.; Using the in vitro mixed transcription system (Kajitani, M . and Ishihama, A . (1983) Nucleic Acids Res . 11, 671-686), we determined the two parameters of the promoter strength, i.e., the rate of open complex formation between RNA polymerase and promoter, and the saturation level of the open complex formation at equilibrium, for the promoters of ribosomal RNA (rrnE), ribosomal protein S1 (rpsA) and recA protein (recA) operons from Escherichia coli . Taken together with the previous determinations for lactose (lac(UV5)), tryptophan (trp) and ribosomal protein L10 (rp1J) operons, these studies revealed that the relative promoter strengths with respect to the kinetic parameter are 200, 70, 50, 40, 30, 20 and 2% of the reference promoter lacP(UV5) for recAp, rp1Jp, rpsAp3, trpP, rpsAp1, rrnEp1 and rrnEp2, respectively, under our standard reaction conditions (50 mM NaCl and 37 degrees C); and those with respect to the thermodynamic parameter are 70, 35, 20, 10, 10, 10 and 5% the level of lacP(UV5) for rrnEp2, trpP, rpsAp3, rp1Jp, rpsAp1, rrnEp1 and recAp, respectively . The order of the promoter strength, however, changes with variation of the salt concentration or reaction temperature. J Mol Biol, 1983 Jun 25, 167(2), 411 - 26 Topological arrangement of two transfer RNAs on the ribosome . Fluorescence energy transfer measurements between A and P site-bound tRNAphe; Paulsen H et al.; The relative arrangement of two tRNAPhe molecules bound to the A and P sites of poly(U)-programmed Escherichia coli ribosomes was determined from the spatial separation of various parts of the two molecules . Intermolecular distances were calculated from the fluorescence energy transfer between fluorophores in the anticodon and D loops of yeast tRNAPhe . The energy donors were the natural fluorescent base wybutine in the anticodon loop or proflavine in both anticodon (position 37) and D loops (positions 16 and 17) . The corresponding energy acceptors were proflavine or ethidium, respectively, at the same positions . Four distances were measured: anticodon loop-anticodon loop, 24(+/- 4) A; anticodon loop (A site)-D loop (P site), 46(+/- 12) A: anticodon loop (P site)-D loop (A site), 38(+/- 10) A: D loop-D loop, 35(+/- 9) A . Assuming that both tRNAs adopt the conformation present in the crystal and that the CCA ends are close to each other, the results are consistent with the two anticodons being bound to contiguous codons and suggest an asymmetric arrangement in which the planes of the two L-shaped molecules enclose an angle of 60 degrees +/- 30 degrees. J Biol Chem, 1983 Jun 25, 258(12), 7676 - 83 Cysteine starvation, isoleucyl-tRNAIle, and the regulation of the ilvGEDA operon of Escherichia coli; Harris CL et al.; The involvement of undermodified tRNA in the regulation of the ilvGEDA operon has been investigated using Escherichia coli C6, a relA-, Cys-, Met- mutant . This strain accumulates thionucleotide-deficient or methyl-deficient tRNA when starved for cysteine or methionine, respectively . The levels of threonine deaminase, the ilvA gene product, and transaminase B, the ilvE gene product, were both lower in cysteine-starved cells, as compared with either growing or methionine-starved cultures . When cysteine was added to cysteine-starved cells, growth ensued promptly and both enzyme activities returned to control levels . Treatment of recovering cultures with valine limited growth by isoleucine limitation, but did not cause a derepression of the ilvGEDA operon . Valine treatment of nonstarved or methionine-starved cells led to the expected increase in threonine deaminase and transaminase B activities . Cysteine-starved cells slowly regained the ability to derepress the operon after 3 h of recovery in complete medium . In contrast, the induction of the lac operon was normal in cysteine-starved cultures, even in the presence of valine . The loss of derepressibility of the ilvGEDA operon was correlated with the presence of a kinetically and chromatographically altered tRNAIle in cysteine-starved cells . No changes in tRNAIle were observed after methionine starvation . Using the periodate method, we found that the charging of tRNAIle increased from the normal level of 60 to 80% or greater after starvation for cysteine . Under conditions where the ilvGEDA operon was fully derepressed in nonstarved cells, the charging of tRNAIle fell to 27% . Unexpectedly, nearly identical results were obtained with cysteine-starved cells after an identical derepression test . These results suggest that factors other than the aminoacylation state of tRNAIle may be important in the regulation of this operon . In particular, modifications to tRNA which involve cysteine may be necessary for controlling the expression of the ilvGEDA operon in E . coli. J Biol Chem, 1983 Jun 25, 258(12), 7669 - 75 The cycling of Escherichia coli DNA polymerase III holoenzyme in replication; Burgers PM et al.; ATP-activated DNA polymerase III holoenzyme (holoenzyme) forms a stable initiation complex with primed DNA with concomitant hydrolysis of the ATP (Burgers, P . M . J., and Kornberg, A . (1982) J . Biol . Chem . 257, 11468-11478) . Upon replication of primed single-stranded circular DNA to a duplex circle with a small gap (RFII), the holoenzyme remains stably bound . Dissociation requires binding by ATP or the generally nonhydrolyzable analog, adenosine 5'-(3-thiotriphosphate) . Transfer of holoenzyme to another primed DNA absolutely requires ATP (or dATP) and takes about 2 min at 30 degrees C . The rate of cycling of holoenzyme is only slightly dependent on the concentration of primed DNA . However, the transfer time is reduced to only 2 to 5 s when it is intramolecular, as shown by movement to other primers on the same template chain . A rapid transfer of holoenzyme from a completed chain to another primer on the same template molecule is anticipated from the frequency of initiating nascent chains at the replicating fork of the cellular chromosome (about 1 per s at 37 degrees C) and the low cellular abundance of holoenzyme (about 10 to 20 molecules per cell). J Biol Chem, 1983 Jun 25, 258(12), 7379 - 85 Fatty acyl derivatives of glucosamine 1-phosphate in Escherichia coli and their relation to lipid A . Complete structure of A diacyl GlcN-1-P found in a phosphatidylglycerol-deficient mutant; Takayama K et al.; We have determined the complete structure of a glycolipid (designated lipid X) previously found to accumulate in certain Escherichia coli mutants defective in phosphatidylglycerol synthesis (Nishijima, M., and Raetz, C.R.H . (1979) J . Biol . Chem . 254, 7837-7844) . Based on fast atom bombardment mass spectrometry and proton nuclear magnetic resonance studies, this substance is an acylated metabolite of glucosamine 1-phosphate . Lipid X of E . coli has a Mr = 711.87 as the free acid (C34H66NO12P) and contains two beta-hydroxymyristate moieties, one attached as an amide at the 2 position and the other as an ester at the 3 position of the sugar . It has free hydroxyl groups at the 4 and 6 positions, and the anomeric configuration is alpha . The structure of lipid X from E . coli closely resembles the reducing end subunit of lipid A, and it might represent a very early precursor in the biosynthesis of lipid A . To our knowledge, fatty acyl derivatives of glucosamine 1-phosphate have not been reported previously. Nucleic Acids Res, 1983 Jun 25, 11(12), 4251 - 6 Blocked 5'-termini in the fragments of chromosomal DNA produced in cells exposed to the antitumor drug 4'-{(9-acridinyl)-amino}methanesulphon-m-anisidide (mAMSA); Marshall B et al.; Comparison of the sensitivity of DNA isolated from untreated and mAMSA-treated PY815 mouse mastocytoma cells to hydrolysis by E.coli 3'-exonuclease III and phage lambda or phage T7 5'-exonucleases show that the fragments of chromosomal DNA produced by mAMSA treatment have free 3'-OH termini and blocked 5'-termini. Nucleic Acids Res, 1983 Jun 25, 11(12), 4109 - 26 Site-dependent cleavage of pBR322 DNA by restriction endonuclease HinfI; Armstrong KA et al.; Cleavage of pBR322 DNA I by the restriction endonuclease HinfI is preferentially inhibited at specific HinfI cleavage sites . These sites in pBR322 DNA I have been identified and ordered with respect to the frequency with which they are cleaved . The HinfI site most resistant to cleavage in pBR322 DNA I is unique in that runs of G-C base pairs are immediately adjacent on both sites . Two differently permuted linear (DNA III) species were produced by cleavage with two different restriction endonucleases, PstI and AvaI . Only one of these linear molecules, that produced by PstI, exhibits the same preferential cleavage pattern as DNA I . The second linear species, that arising from AvaI digestion, shows pronounced relative inhibition of cleavage at the HinfI sites nearest the ends of the molecule (100 to 120 base pairs away, respectively) . This result suggest that proximity to the termini of a linear DNA molecule might also influence preferential cleavage . The possibility of formation of stem-loop structures does not appear to influence preferential cleavage by HinfI. J Mol Biol, 1983 Jun 25, 167(2), 227 - 43 Organization of the Escherichia coli chromosome around the genes for translation initiation factor IF2 (infB) and a transcription termination factor (nusA); Plumbridge JA et al.; The genes infB, for translational initiation factor IF2, and nusA for a protein involved in transcription termination are carried on a 4.8 X 10(3) base-pair DNA fragment . This fragment also carries promoters capable of expressing both genes . The order of these genes with respect to the surrounding genes is pnp, rpsO, infB, nusA, argG . Transcription of the two genes is anticlockwise on the standard Escherichia coli map, i.e . directed towards the genes rpsO and pnp . The presence of infB on multicopy plasmids enhances IF2 protein and messenger RNA levels only two to threefold compared to the normal haploid level. J Biol Chem, 1983 Jun 25, 258(12), 7840 - 6 Tandem promoters in the gene for ribosomal protein S20; Mackie GA et al.; Examination of the nucleotide sequence of the gene for ribosomal protein S20 (rpsT) of Escherichia coli suggested the presence of two promoters ("sites 1 and 2") separated by 90 base pairs (Mackie, G . A . (1981) J . Biol . Chem . 256, 8177-8182) . We have investigated the properties of purified or cloned DNA fragments containing one or other or both these sites for their ability to promote transcription in vivo and in vitro . In reactions in vitro containing DNA and purified RNA polymerase as the sole macromolecular components, both sites 1 and 2 act as promoters directing the synthesis of "runoff" transcripts . The 5' termini of such transcripts have been determined by direct sequencing or by identification of the 5' terminal nucleoside 5'-triphosphate, 3'-monophosphate . In site 1, the major transcript initiates with GTP at residue 141 in the DNA sequence . A minor start occurs at residue 142 and uses CTP as the initiating nucleotide . In site 2, the major transcript (approximately 55% of all initiations in site 2) initiates with CTP at residue 232 while minor transcripts, each comprising approximately 20% of the total, initiate at residues 231 and 233 with GTP and CTP, respectively . In four methods of assay which reflect to varying extents the usage of promoters in vivo, site 1 is responsible for 10-30% of the total transcription of the gene for S20 and site 2 the remainder . Sites 1 and 2 appear to act independently and additively in assays based on the rate of synthesis of S20 in a system for coupled transcription and translation . Together, the two promoters for S20 are from 10-25-fold more active than the fully induced lac operon promoter. J Biol Chem, 1983 Jun 25, 258(12), 7828 - 39 Carbodiimide inactivation of Escherichia coli RNA polymerase promoters on supercoiled simian virus 40 and ColE1 DNAs occurs by a one-hit process at salt concentrations in the physiological range; Hale P et al.; A previous study (Hale, P., Woodward, R . W., and Lebowitz, J . (1980) Nature 284, 640-644) showed that Escherichia coli RNA polymerase promoters on superhelical SV40 DNA are highly selective targets for chemical modification by the water-soluble carbodiimide, N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethyl carbodiimide (CMC) . To extend the inactivation analysis of supercoiled DNAs, we determined the number and location of RNA polymerase binding sites on the supercoiled and linear forms of ColE1 DNA . We also determined the site distribution of {3H} CMC on the superhelical form . This information, coupled with per cent inhibition of transcription versus CMC-bound curves, allowed a test of the specificity of the CMC inactivation by the Poisson equation . Curves were obtained for supercoiled SV40 DNA modified at 0 and 100 mM NaCl (2 mM NaPi, pH 7.0) and for supercoiled ColE1 DNA modified at 0, 100, and 320 mM NaCl . For supercoiled SV40 DNA, these data, coupled to our knowledge of the number of RNA polymerase binding sites from the study cited above, revealed an excellent fit to a one-hit inactivation by the Poisson equation for DNA modified at 100 mM NaCl . For ColE1 DNA, we obtained an excellent fit to a Poisson distribution when supercoiled DNA was modified at 320 mM NaCl . The Poisson distribution can be applied to {3H} CMC restriction fragment data with equivalent results . These results suggest that promoter sites can be forced into different structural conformations with variable degrees of unpairing. J Biol Chem, 1983 Jun 25, 258(12), 7469 - 75 Purification and characterization of Escherichia coli guanine-xanthine phosphoribosyltransferase produced by a high efficiency expression plasmid utilizing a lambda PL promoter and CI857 temperature-sensitive repressor; Liu SW et al.; The gene for Escherichia coli guanine-xanthine phosphoribosyltransferase was placed after the high efficiency lambda phage leftward promoter in plasmid pHEGPT also containing the lambda CI857 temperature-sensitive repressor . Guanine-xanthine phosphoribosyltransferase increases 780-fold when cells containing pHEGPT are shifted from 30 to 42 degrees C . Guanine-xanthine phosphoribosyltransferase represents approximately 5% of the protein in a crude extract of induced cells . Guanine-xanthine phosphoribosyltransferase may be purified to apparent homogeneity by ammonium sulfate fractionation, Sephadex G-100, and DEAE-cellulose column chromatography . The enzyme has a subunit molecular weight of 18,600 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behaves as a trimer during Sephadex G-100 column chromatography . Guanine-xanthine phosphoribosyltransferase is active from pH 7.5 to 10.5 with maximum activity at pH 9.5 . The enzyme is protected from heat inactivation by phosphoribosylpyrophosphate (PRPP) . At 65 degrees C, the enzyme has a half-life of 2 min in the absence of PRPP and 90 min in the presence of PRPP . The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for guanine, xanthine, hypoxanthine, and PRPP of 2.6, 39, 167, and 95 microM, respectively . The activity of the enzyme with guanine is 2-fold greater than that with xanthine and 3-fold greater than that with hypoxanthine. J Biol Chem, 1983 Jun 25, 258(12), 7395 - 401 The nucleotide sequence of the Mr = 28,500 flagellin gene of Caulobacter crescentus; Gill PR et al.; The DNA sequences which encode the Mr = 28,500 flagellin polypeptide of Caulobacter crescentus CB15 have been determined . The size of the protein, deduced from its DNA sequence (276 amino acids), is in agreement with its apparent molecular weight as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The distribution of arginine residues within the protein sequence encoded by the gene correlates with their relative location as predicted by peptide alignment analysis (Gill, P.R., and Agabian, N . (1982) J . Bacteriol . 150, 925-933) . DNA sequences 5' and 3' to the coding sequence were also determined . In the 5' region, DNA sequences homologous to consensus sequences associated with RNA polymerase recognition and transcription initiation sites in Escherichia coli (Pribnow box) are found . These are centered around 60, 90, and 120 base pairs upstream from the ATG codon at the beginning of the structural gene . Sequences 3' to the coding region were identified which might signal transcription termination . A typical E . coli 16 S ribosomal binding site (Shine-Dalgarno sequence) is located just 5' to the coding sequence, and for most of the amino acids there is a strong codon usage preference . Although this protein is exported from the cell (Gill, P.R., and Agabian, N . (1982) J . Bacteriol . 150, 925-933), the encoded NH2-terminal amino acid sequence is not different from the mature product. J Biol Chem, 1983 Jun 25, 258(12), 7345 - 51 Human beta-glucuronidase pinocytosis and binding to the immobilized phosphomannosyl receptor . Effects of treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase; Talkad V et al.; beta-D-Glucuronidase (EC 3.2.1.31) was purified to homogeneity from human spleen, and enzyme fractions from CM-Sephadex were examined for uptake by fibroblasts and retention by a column of immobilized phosphomannosyl receptor . Uptake and binding were enhanced by treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase, greatly reduced by prior treatment with alkaline phosphatase, and restored by subsequent treatment with alpha-N-acetylglucosaminyl phosphodiesterase . Immobilized phosphomannosyl receptor was used to separate high and low uptake enzyme forms . About 25% of the total beta-glucuronidase was retained by the receptor column and eluted with mannose 6-phosphate . The rate of uptake of retained enzyme was 2.5-3.0-fold greater than that of the enzyme applied to the receptor column . The fraction retained by the column was reduced to 5-10% by prior treatment of the enzyme with alkaline phosphatase . This phosphatase-resistant, receptor-retained fraction was taken up at only 24% the rate of non-phosphatase-treated, receptor-retained enzyme . However, its uptake was increased 7-fold by treatment with alpha-N-acetylglucosaminyl phosphodiesterase . The enhanced rate of pinocytosis conferred by treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase was destroyed by a subsequent treatment with alkaline phosphatase . These studies demonstrate that although most of the "high uptake" enzyme in beta-glucuronidase from human spleen binds to receptors through phosphomonoesters of mannose, a significant fraction can interact with immobilized phosphomannosyl receptor and be taken up by fibroblasts through interactions involving mannose 6-phosphate in diester linkage with N-acetyl-D-glucosamine. J Chromatogr, 1983 Jun 24, 262, 193 - 8 Characteristics of immobi