Escherichia coli

Drugs Exp Clin Res, 1985, 11(8), 469 - 78
Copper salicylate complex: thermoregulatory and biochemical effects; Hac EE et al.; The antipyretic properties of copper (II) salicylate and its effect on plasma copper, iron, zinc and ceruloplasmin concentrations was investigated in adult rabbits at an ambient temperature of 21.5 +/- 0.5 degrees C . The experiments indicated that copper salicylate (200 mg/kg/h i.v.) was a more potent antipyretic than sodium salicylate given in the same manner and doses . This pharmacological activity was found on a model of experimental fever induced by i.v . injection of lipopolysaccharide Escherichia coli at a dose of 1 microgram/kg . Furthermore, unlike sodium salicylate, this copper complex caused a decrease in core temperature in normothermic rabbits . At the same time copper salicylate activated heat dissipation much more efficiently than the parent drug, as manifested by decreases in vasomotor tone and reversal of postpyrogen inhibition of RF . As was expected, treatment with copper salicylate increased plasma copper and ceruloplasmin levels in both normothermic and febrile rabbits . These increases did not lead to any disturbances in iron and zinc concentrations . Neither salicylate affected postpyrogen falls in plasma iron concentrations . They both, however, delayed the appearance of zinc decreases in febrile rabbits . The results of this study suggest that copper modifies the thermoregulatory effects of salicylate . Moreover, the increased amounts of this metal do not seem to disturb seriously the ionic status of the blood.

Mol Gen Genet, 1985, 199(3), 381 - 7
Suppressors of temperature-sensitive mutations in a ribosomal protein gene, rpsL (S12), of Escherichia coli K12; Nashimoto H et al.; Temperature-sensitive (ts) mutations were isolated within a ribosomal protein gene (rpsL) of Escherichia coli K12 . Mutations were mapped by complementation using various transducing phages and plasmids carrying the rpsL gene, having either a normal or a defective promoter for the rpsL operon . One of these mutations, ts118, resulted in a mutant S12 protein which behaved differently from the wild-type S12 on CM-cellulose column chromatography . Suppressors of these ts mutations were isolated and characterized; one was found to be a mutation of a nonribosomal protein gene which was closely linked to the RNAase III gene on the E . coli chromosome . This suppressor, which was recessive to its wild-type allele, was cloned into a transducing phage and mapped finely . A series of cold-sensitive mutations, affecting the assembly of ribosomes at 20 degrees C, was isolated within the purL to nadB region of the E . coli chromosome and one group, named rbaA, mapped at the same locus as the suppressor mutation, showing close linkage to the RNAase III gene.

Gene, 1985, 35(1-2), 209 - 15
Cloning of the newt Pleurodeles waltlii chromosomal DNA; Muller JP et al.; Pleurodeles waltlii genomic DNA has been cloned using several phage lambda vectors . We have isolated approx . 600 000 clones, which correspond to about 20% of the total DNA sequences of this organism . This constitutes the first large gene library of a Urodele . The low yield of cloning was attributable to the abundance of highly repetitive sequences, since recombinations in the bacterial host could lead to the loss of clones . Indeed, the existence of highly repetitive sequences was directly demonstrated by hybridization between recombinants and the total genome, and some of the cloned DNA was found to be unstable . We suggest new methods for cloning the highly repetitive sequences.

Prog Pediatr Surg, 1985, 18, 139 - 45
Immunological consequences of splenectomy; Eibl M; A series of reports have dealt with the occurrence of overwhelming infections in splenectomized patients . Being the largest individual organ of the phagocytic apparatus, the spleen is responsible for the phagocytosis of insufficiently opsonized particles . These are taken up by macrophages, processed, and expressed, together with determinants of the HLA system, on the membrane of the macrophage . T cells recognize these structures and proliferate in response, thus inducing a series of immunoregulatory mechanisms . The anatomic design of the spleen allows for close contact between macrophages and T cells . Thus, splenectomy represents a major intervention into the immunologic system . Splenectomized patients have been shown to have low concentrations of IgM, decreased production of antibodies directed against pneumococci and Escherichia coli, and several defects in cellular immune function, including decreased numbers of T cells and a reduction in lymphocyte proliferative responses . Thus, the removal of the spleen affects certain immunological reactions, which are reflected by a number of clinical findings.

Mol Gen Genet, 1985, 198(3), 456 - 64
SOS induction by thermosensitive replication mutants of miniF plasmid; Sommer S et al.; MiniF, a 9.3 kb fragment of the dispensable F plasmid, carries genes necessary for its replication and partition as well as for the expression of an SOS signal . The arrest of replication of a thermo-sensitive miniFts at 42 degrees C induced SOS functions such as prophage lambda, sfiA expression, W-reactivation of UV-irradiated phage lambda . Two miniF ts9 and ts17 mutations were located within the KpnI fragment (43.6-46.9) in the minimal oriS replicon . Blocking miniF replication by incBC+ incompatibility genes situated in trans on a second plasmid also induced SOS functions . In contrast, if miniFts17 plasmid escaped the replication block at 42 degrees C by being inserted into pR325, there was no SOS induction . SOS induction by the arrest of miniF replication required the miniF lynA+ locus in cis, the host recA+ and lexA+ genes . We found that SOS induction was increased greatly near the stationary phase and that cell viability declined . During host cell exponential growth, miniFts9 and miniFts17 plasmids were lost rapidly, although SOS induction persisted for several cell generations . We postulate that lynA expresses a persistent product that may lead to the unwinding of chromosomal DNA.

Gene, 1985, 34(1), 47 - 54
Physical and genetic characterization of cloned enterobactin genomic sequences from Escherichia coli K-12; Fleming TP et al.; We have cloned genes responsible for enterobactin synthesis (entD) and transport (fepA,fes) from Escherichia coli K-12 . Relevant recombinant plasmids enabled EntD- and transport-defective mutants to grow on iron-limiting medium . Subcloning and deletion analysis demonstrated that the gene order is entD-fepA-fes . Protein synthesis studies in minicells suggest that FepA is first translated as an Mr 84 000 precursor, which is subsequently cleaved to the active Mr 81 000 receptor; the fes gene product is an Mr 44 000 protein; no polypeptide has been identified as the entD gene product.

Mol Gen Genet, 1985, 198(2), 336 - 47
Interaction of an antimutator gene with DNA repair pathways in Escherichia coli K-12; Lyons SM et al.; A mutation in the purB gene of E . coli K-12, isolated and partially characterized by Geiger and Speyer (1977), confers a temperature sensitive requirement for adenine and an antimutator phenotype at 30 degrees C . Several hypotheses about the mechanism of action of this mutation, named mud for mutation defective, were tested in the present work . The mud mutation has no effect upon the induction of the SOS response, so the antimutator phenotype is unlikely to be due to repression of mutagenic repair . Mud cells are resistant to the cytotoxic and mutagenic effects of alkylating agents such as MNNG, but this resistance is not due simply to derepression of the adaptive response . DNA isolated from mud cells is not undermethylated relative to DNA from purB+ cells, so the antimutator phenotype of mud cannot be due to reduced hotspot base-substitution mutation at methylated cytosine residues . Nor is there a longer lag in post-replicative DNA methylation, which indicates that there is no enhancement of mismatch repair resulting from an extended time window for strand discrimination . Measurement of nucleotide pool levels demonstrated an elevation of dCTP in mud cells and a reduction of all other nucleoside triphosphates.

Folia Microbiol (Praha), 1985, 30(1), 17 - 24
Protection of nonmodified phage lambda against EcoK restriction mediated by recA protein; Koukalova B et al.; A study was conducted to establish whether the EcoK-specific restriction, which is alleviated in E . coli cells after UV induction of the SOS response (Day 1977), is also alleviated under the influence of an increased level of recA protein without induction of other SOS functions . The host cells used were E . coli K-12, strain AB2497, and its derivatives; the nonmodified phage lambda was a mutant b2b5(vir) . An increase of the recA protein level was induced using the plasmid pX02, which is a recombinant of pBR322 carrying the recA gene of E . coli . AB2497(pX02) cells were found to exhibit a lower level of restriction than those without plasmid . The results indicate that the recA protein protects phage DNA during the process of restriction . A further factor affecting restriction is the growth phase of the culture of the restricting host: cells in the late stationary phase exhibit lower restriction than those in the exponential phase of growth . By a combination of these two factors (presence of plasmid pX02 and stationary growth phase) one can reduce the restriction of nonmodified phage about 300 times.

Genetika, 1985 Jan, 21(1), 46 - 53
{Integration of the related prophages lambda, phi 80 and their hybrid lambda att80 into the secondary chromosomal att sites of wild-type Escherichia coli}; Kholodii GIa et al.; The family of lambdoid phages displays a varying specificity of integration into the host chromosome . The lambda phage DNA failed to get inserted at the secondary attachment site(s) of the gal operon (frequency less than 2.6 X 10(-8)) in the presence of the primary (normal) one . By contrast, phi 80 and the lambda att80 hybrid integrated into wild-type Escherichia coli at least, at two secondary att sites of the btuB locus, the latter phage being also capable of integration in the vicinity of purE and purC (frequency 2 X 10(-3) to 10(-4)) . Integration of phi 80 and lambda att80 into btuB occurred with about the same frequency as in cells deleted for normal insertion site (0.7 divided by 4.0 X 10(-6)) . An analysis of the secondary lysogens with the prophage in btuB showed them to be polylysogens; the additional prophage(s) was found in the primary att site . We also failed to observe integration of phi 80 and lambda att80 with formation of secondary monolysogens into other foci (frequency less than 0.0035, if multiplicity of infection was 10(-3) or 10) . It is presumed that phi 80 and lambda att80 prophages get only integrated at secondary att sites in case the primary site is occupied.

Proc Natl Acad Sci U S A, 1985 Jan, 82(1), 88 - 92
Maximizing gene expression from plasmid vectors containing the lambda PL promoter: strategies for overproducing transcription termination factor rho; Mott JE et al.; We have constructed two plasmids in which transcription of the rho gene from Escherichia coli K-12 is under the control of the lambda phage PL promoter . In p31-356, the normal rho promoter is deleted, but the remainder of the rho leader region, including the ribosome binding site, is present . In p39-AS, the rho leader is completely absent, and the lambda cII ribosome binding site replaces that of rho . Under noninducing conditions, expression of rho protein from these plasmids is repressed by the lambda cI protein in hosts carrying lambda cryptic prophage . Induction using mitomycin C or nalidixic acid in a cryptic lysogen carrying the cI+ repressor resulted in the overproduction of rho protein to levels of 3%-5% of the total cellular protein with p31-356, and to levels of approximately equal to 40% with p39-AS . The overproduced protein is functionally indistinguishable from the rho protein isolated from the K-12 strain W3110, and it can be obtained from cells harboring p39-AS in yields of up to 25 mg of rho per g of cells . In contrast to chemical induction, heat induction in four cryptic lambda lysogens carrying the thermolabile cI857 repressor failed to yield the same high levels of rho protein (with either plasmid) . Our results show that chemical induction of PL-containing plasmid expression vectors can serve as a convenient and useful alternative to the commonly used method of heat induction.

J Bacteriol, 1985 Jan, 161(1), 96 - 104
Genetics of methyl-accepting chemotaxis proteins in Escherichia coli: cheD mutations affect the structure and function of the Tsr transducer; Callahan AM et al.; The tsr gene specifies a methyl-accepting membrane protein involved in chemotaxis to serine and several repellent compounds . We have characterized a special class of tsr mutations designated cheD which alter the signaling properties of the Tsr transducer . Unlike tsr null mutants, cheD strains are generally nonchemotactic, dominant in complementation tests, and exhibit a pronounced counterclockwise bias in flagellar rotation . Several lines of evidence showed that cheD mutations were alleles of the tsr gene . First, cheD mutations were mapped into the same deletion segments as conventional tsr mutations . Second, restriction site analysis of the transducing phage deletions used to construct the genetic map demonstrated that the endpoints of the deletion segments fell within the tsr coding sequence . Third, a number of the cheD mutants synthesized Tsr proteins with slight changes in electrophoretic mobility, consistent with alterations in Tsr primary structure . These mutant proteins were able to undergo posttranslational deamidation and methylation reactions in the same manner as wild-type Tsr protein; however, the steady-state level of Tsr methylation in cheD strains was very high . The methylation state of the Tar protein, another species of methyl-accepting protein in Escherichia coli, was also higher than normal in cheD strains, suggesting that the aberrant Tsr transducer in cheD mutants has a generalized effect on the sensory adaptation system of the cell . These properties are consistent with the notion that the Tsr protein of cheD mutants is locked in an excitatory signaling mode that both activates the sensory adaptation system and drowns out chemotactic signals generated by other transducer species . Further study of cheD mutations thus promises to reveal valuable information about the functional architecture of the Tsr protein and how this transducer controls flagellar behavior.

J Bacteriol, 1985 Jan, 161(1), 428 - 31
Locus affecting regulation of the colicin I receptor by iron; Worsham PL et al.; Using a strain containing a cir-lac operon fusion and a selective medium, we isolated a regulatory mutant of the colicin I receptor, which we have designated cirR . Cells carrying the cirR mutation were defective in the transcriptional regulation of cir by iron, but synthesis of other iron-regulated proteins was unaffected . cirR was found to be cis dominant . This is in contrast to previously described mutations in iron regulation which are pleiomorphic and trans dominant . Temperature regulation of colicin I receptor production was unaffected by cirR.

J Bacteriol, 1985 Jan, 161(1), 347 - 52
Temperature-sensitive catabolite activator protein in Escherichia coli BUG6; Benner D et al.; BUG6 is a temperature-sensitive cell division mutant which forms filaments at the nonpermissive temperature . Synthesis of the maltose- and galactose-binding protein-dependent transport systems is also temperature sensitive in BUG6 . Using operon and protein fusions of the maltose transport genes to lacZ, we observed that the temperature-sensitive control of the maltose transport system in BUG6 occurs at the transcriptional level . By P1-mediated transductions, we found that BUG6 contains two independent temperature-sensitive mutations . One maps between 2 and 3 min on the Escherichia coli linkage map, in close proximity to the fts-envA region . This mutation is responsible for temperature-sensitive cell division . The other mutation maps at 73 min in crp, the structural gene of the catabolite activator protein . The latter could be complemented by a hybrid plasmid carrying the wild-type crp as the only gene on a 0.9-kilobase HindIII-AluI restriction fragment . The mutation in crp alone was found to be responsible for the temperature-sensitive synthesis of the maltose transport system . Although it causes a complete block of transcription of the maltose transport genes at 41 degrees C, this mutation had only a marginal effect on the transcription of the lac operon.

J Bacteriol, 1985 Jan, 161(1), 207 - 11
Escherichia coli supH suppressor: temperature-sensitive missense suppression caused by an anticodon change in tRNASer2; Thorbjarnardottir S et al.; We describe the cloning and the DNA sequence of the Escherichia coli supH missense suppressor and of the supD60(Am) suppressor genes . supH is a mutant form of serU which codes for tRNASer2 . The supH coding sequence differs from the wild-type sequence by a single nucleotide change which corresponds to the middle position of the anticodon . The CGA anticodon of wild-type tRNA and CUA anticodon of supD tRNA is changed to CAA in supH tRNA, which is expected to recognize the UUG leucine codon . We propose that the supH suppressor causes the insertion of serine in response to this codon . The temperature sensitivity caused by supH may be due to a conformation of the CAA anticodon in the supH tRNASer that is slightly different than that in the corresponding tRNALeu species.

Am J Pathol, 1985 Jan, 118(1), 35 - 42
Complement and polymorphonuclear leukocytes do not determine the vascular permeability induced by intraocular LPS; Howes EL Jr et al.; The intravitreous injection of an endotoxin of Escherichia coli 055:B5 (LPS; 0.1-0.5 microgram/50 microliters of saline) induces ocular inflammation in rabbits that is maximal 20-24 hours later and disappears by 4 days . The inflammation is characterized by an alteration in ocular vascular permeability (OVP) measured by the ocular extravasation of 125I-albumin and an outpouring of leukocytes, most of which are polymorphonuclear leukocytes (PMNs), as determined by histopathologic study . Nitrogen mustard (mechlorethamine, 1.75 mg/kg) administered 3 days prior to LPS virtually eliminates PMNs in the circulation and those infiltrating ocular tissues 20 hours after intravitreous LPS, and yet the average increase in vascular permeability is not different from that of controls . Cobra venom factor (CVF; 300-400 units) 7 hours before intravitreous LPS produces a greater than 90% decrease in both hemolytic complement activity and zymosan-inducible serum chemotactic activity; yet 20 hours after LPS, the OVP is the same in CVF-treated rabbits and controls . For comparison, an ocular passive Arthus reaction (ovalbumin-anti-ovalbumin) was significantly affected by CVF pretreatment . Chemotactic activity in the aqueous humor is found in both CVF-treated and control rabbits 20 hours after intravitreous LPS . This activity attracts rabbit, but not human, PMNs, is partially heat-sensitive, and is not inhibited when PMNs are preincubated with C5a . These results indicate that neither PMNs nor circulating complement determine the OVP following intravitreous LPS, and that the chemotactic activity present in aqueous humor at the height of the inflammatory response is not primarily C5a.

Cancer Res, 1985 Jan, 45(1), 51 - 6
Interactions of benzo(a)pyrene diol-epoxides with linear and supercoiled DNA; MacLeod MC et al.; Previous spectroscopic studies of the major adduct formed by reaction of (+/-)-7r,8t-dihydroxy-9t, 10t-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE-I) with linear DNA have been interpreted to suggest that the adduct is not intercalated in the double helix . However, studies of the electrophoretic mobility of supercoiled DNA treated with BPDE-I suggest that the adduct is intercalated . To resolve these interpretations, we have studied the reaction of BPDE-I with supercoiled and linear DNA . The kinetics of DNA-catalyzed hydrolysis and of covalent binding are similar for the two DNAs; supercoiled DNA exhibits a 20% increase in the rate of hydrolysis of BPDE-I at low DNA concentration compared to linear DNA . Fluorescence excitation spectra and fluorescence quenching experiments provide no support for a model in which BPDE-I adducts are intercalated in supercoiled DNA . When deoxyribonucleoside adducts were analyzed by high-performance liquid chromatography, identical distributions of BPDE-I adducts were found for supercoiled and linear DNA . These data are consistent with a previously proposed model (Hogan, M . E., Dattagupta, N., and Whitlock, J.P., Jr . J . Biol . Chem., 256: 4504-4513, 1981; Taylor, E.R., Miller, K . J., and Bleyer, A . J . J . Biomol . Struct . Dyn., 1: 883-904, 1983), in which the major BPDE-I adduct in both linear and supercoiled DNA exists in a conformation which allows stacking with the neighboring base pair and introduces a "kink" into the path of the helical axis . Although this model provides an explanation for all available experimental data, there are undoubtedly other DNA adduct conformational models which are also consistent with the data.

Gene, 1985, 40(2-3), 353 - 7
Selection for the transfer of phenotypically nonselectable chromosomal mutations to recombinant plasmids through introduction of an altered restriction site; Chang YY et al.; We describe a simple method to select for transfer of mutant alleles from the Escherichia coli chromosome to a plasmid which formerly carried the wild-type (wt) allele . The wt allele on the plasmid is modified by introduction of a unique restriction site (e.g., XhoI) and transformed into a rec+ strain carrying the mutant allele on the chromosome . Upon homogenotization, the efficiency of which was increased by UV irradiation of the transforming plasmid {Chattoraj et al., Gene 27 (1982) 213-222}, plasmids carrying the mutant allele are formed which are resistant to XhoI . These plasmids are selected from the population by resistance to XhoI digestion coupled with the low transformation efficiency of linear DNA molecules in recA- strain . The method is efficient and rapid and has particular advantages in situations where the mutant allele is difficult to detect by its phenotype.

Gene, 1985, 40(2-3), 337 - 42
Molecular characterisation of the Stc- mutation of Escherichia coli K-12; Misra R et al.; The previously described Stc- (suppressor of TolC) mutation modifies the phenotype of tolC mutants from OmpF- to OmpF+ . Restriction mapping of chromosomal DNA from Stc+ and Stc- strains was performed to investigate the nature of the mutation which was shown to be a deletion, upstream of the ompC gene . DNA from the region of the deletion was cloned into pUC18 and a 650-bp PstI-EcoRI fragment was sequenced . The deletion started 49 bp upstream of the AUG start codon of the ompC gene, thus removing part of the ompC promoter and the whole of the micF gene . We suggest that the deletion of micF gives rise to the Stc- phenotype since the effect of micF expression is assumed to reduce ompF expression, and the Stc- phenotype involves increase in ompF expression.

Gene, 1985, 40(2-3), 259 - 66
Analysis of cosmids using linearization by phage lambda terminase; Rackwitz HR et al.; A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro . A plasmid capable of producing high levels of phage lambda terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised . After linearization, the cosmids were partially digested with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage lambda (Rackwitz et al., 1984) . High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.

Gene, 1985, 40(2-3), 217 - 29
New vectors for construction of recombinant high-copy-number yeast acentric-ring plasmids; Fagan MC et al.; Yeast acentric-ring plasmid 1 (YARp1), comprising 1453 bp of entirely yeast chromosomal DNA, is maintained in Saccharomyces cerevisiae as a high-copy, relatively stable plasmid . To determine the feasibility of using YARp1 as a yeast cloning vehicle, we subcloned the GAL1-10 promoter and the URA3 gene into YARp1 at different locations . To facilitate these constructions, a class of permuted YARp1 construction vectors was generated which enabled us to use various restriction sites in YARp1 as insertion points . Transformation frequencies, plasmid stabilities, and copy numbers of these YARp1 derivatives remained elevated, comparable to those of YARp1 itself . Also, when OMP decarboxylase was assayed using strains containing URA3-YARp's, specific activities of 100-300 times that of wild type were found . This evidence supports the use of YARp1 as a high-copy yeast-expression vector or for analyzing structural and regulatory DNA sequences.

Gene, 1985, 40(2-3), 175 - 82
Conversion of the FokI endonuclease to a universal restriction enzyme: cleavage of phage M13mp7 DNA at predetermined sites; Podhajska AJ et al.; Endonuclease FokI belongs to class IIS of restriction enzymes, for which the DNA cut points lie outside the enzyme-recognition sites . This permitted conferring new cleavage specificities by combining FokI with tailored oligodeoxynucleotide adapters . Such adapters carry a single-stranded (ss) target-recognition domain, complementary to the selected ss target DNA, and a double-stranded (ds) enzyme-recognition site . Neither enzyme nor adapter alone has endonucleolytic activity toward phage M13mp7 ss DNA, whereas the enzyme-adapter complex cleaves this ss target DNA at the particular sites foreordained by the sequence of the ss domain of the adapter . Two kinds of adapters (32 and 34 nucleotides long), with opposing orientations of the asymmetric FokI recognition site, were constructed and shown to direct specific cleavage under a variety of conditions . In addition to FokI, other class IIS enzymes, HphI, MboII and BbvI, which alone do not cleave ss DNA, are suitable for construction of tailored enzyme-adapter complexes with predictable cleavage specificities . This report provides a preliminary experimental confirmation for the proposal of Szybalski {Gene 40 (1985) 169-173} for the design of adapter-enzyme complexes with novel and predictable specificities . Theoretically, using this approach any sequence could be precisely cleaved at a predetermined point.

Gene, 1985, 40(1), 99 - 106
Expression of an Escherichia coli beta-galactosidase fusion gene in Aspergillus nidulans; van Gorcom RF et al.; We inserted in frame the Escherichia coli lacZ gene into the protein-coding region of the Aspergillus nidulans trpC gene and introduced the resultant fused gene into the A . nidulans genome . A functional beta Gal fusion protein was produced . Removal of the trpC transcription and translation initiation sequences from the fusion gene abolished production of the fusion protein, showing that expression is dependent on these sequences . Thus, lacZ fusions should be of use for estimating gene activity in a . nidulans.

Gene, 1985, 40(1), 93 - 8
Transient assay, by {3H}guanine incorporation of Escherichia coli xanthine-guanine phosphoribosyl transferase (GPT) in transfected human fibroblasts; Burke JF et al.; We have used {3H}guanine incorporation as a rapid and sensitive assay of xanthine-guanine phosphoribosyl transferase (GPT) activity in SV40 transformed human fibroblasts . The SV40 early promoter is more efficient than the Rous sarcoma virus long terminal repeat for transient expression of the gpt gene . The assay works well in a derivative of AT5BIVA which lacks hypoxanthine-guanine phosphoribosyl transferase (hprt-) and we show here how the assay has been adapted to work in the hprt+ AT5BIVA parent.

Gene, 1985, 40(1), 31 - 8
Nonrandom insertion of Tn5 into cloned human adenovirus DNA; McKinnon RD et al.; The bacterial transposable element Tn5 displays regional selectivity in target sites for transposition . To examine this integration specificity of Tn5, we have mapped 57 insertion events in a plasmid pXC1 containing a eukaryotic viral DNA fragment as a target for Tn5 insertional mutagenesis . We found a nonrandom distribution of integration sites in pXC1, suggesting preferred targets for transposition . However, DNA sequence analysis of seven mutants revealed no target site sequence specificity for Tn5 insertion . We demonstrated that the majority of these insertions mapped downstream from a fortuitous promoter sequence which was present and active in this cloned insert in pXC1 . Furthermore, when this promoter region was removed, Tn5 was able to transpose into previously unused upstream target sequences . Our data suggest that transcriptional activity may influence Tn5 transposition.

Gene, 1985, 40(1), 23 - 9
Expression of hepatitis B virus surface antigen P31 gene in Escherichia coli; Fujisawa Y et al.; A hepatitis B virus surface antigen (HBsAg) P31-coding DNA was constructed from a DNA fragment of the plasmid pHBr330 containing the entire hepatitis B virus (HBV) adr DNA and a chemically synthesized adaptor . The P31 gene was inserted into an expression vector, pTRP771, having an Escherichia coli tryptophan operon (trp) promoter to give a recombinant plasmid pTRP P31-R . The distance between the Shine-Dalgarno (SD) sequence and the initiation codon of P31 gene was adjusted to 9 bp . The expression level of HBsAg by E . coli 294{pTRP P31-R} was significantly elevated, in contrast to that of HBsAg by E . coli 294{pTRP SS-6} . Western blotting analysis has shown that E . coli{pTRP P31-R} synthesizes a specific polypeptide P31 of about 31 kDal, which reacts with anti-HBsAg antibody . The binding studies with polyalbumins from various species have also suggested that HBsAg P31 specifically binds to polymerized human serum albumin.

Gene, 1985, 40(1), 163 - 8
Control of cloned gene expression by promoter inversion in vivo: construction of the heat-pulse-activated att-nutL-p-att-N module; Podhajska AJ et al.; We have constructed a prototype of gene-expression plasmids with three novel properties: its "OFF phase" is absolute in all common hosts because the expression promoter is facing away from the studied gene and is blocked by a strong terminator; the "ON phase" is attained by the rapid and efficient inversion of the promoter; only a short heat pulse or exposure to other inducing agents is required to initiate this two-stage process . In the first stage, synthesis of the phage lambda Int protein is induced by the transient derepression of the properly engineered lambda xis- cIts857 prophage . In the immediately following second stage, Int causes inversion of a promoter cloned between the inverted ----P'OP phage att site and the normally oriented ----delta PO delta P' pseudo-bacterial att site . The inverted promoter can now control the expression of the studied gene and also of the lambda N gene cloned in tandem . The N product, in conjunction with the nutL site placed downstream of the promoter, permits efficient antitermination of any terminators present in the att sites, in the plasmid or in the cloned DNA, making this system efficient and of practical value . Employing the promoter-inverting plasmid, it was possible to obtain rapid onset and a high level of galactokinase synthesis from the cloned galK gene . Only a transient, 10-min induction at 42 degrees C was employed, permitting protein synthesis at 30 degrees C, which might be of importance for thermosensitive products . Furthermore, the entire promoter-inversion module can be transferred to any plasmid as a 1.3-kb AvaI-ClaI fragment (see Fig . 1).(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1985, 40(1), 15 - 22
Promoter activity and transcript mapping in the regulatory region for genes encoding ribosomal protein S15 and polynucleotide phosphorylase of Escherichia coli; Evans S et al.; The genes encoding ribosomal protein S15 (rpsO) and polynucleotide phosphorylase (pnp) occupy adjacent positions and are oriented in the same direction on the Escherichia coli chromosomes . The nucleotide sequence of the region controlling the expression of these two genes has been determined . Two in-phase gene fusions between pnp and lacZ were constructed . The fusions define the translational reading frame of the pnp gene and indicate that the expression of pnp is independent of the upstream rpsO gene . Transcript mapping with nuclease S1 demonstrated that the two genes are transcribed from separate promoters and that the rpsO-pnp intergenic space contains a strong transcriptional terminator . The transcriptional start points have been localized.

Gene, 1985, 40(1), 141 - 3
Cloning of the Escherichia coli K-12 guaC gene following its transposition into the RP4::Mu cointegrate; Moffat KG et al.; The guanosine 5'-monophosphate reductase gene, guaC, has been cloned into the multicopy vector pBR325 from the RP4::Mu cointegrate, pKGM62-1, and the gene product identified by in vitro transcription/translation as a protein of Mr 36 000 . Strains harbouring the recombinant plasmid had increased levels of GMP-reductase activity.

Gene, 1985, 39(2-3), 313 - 5
EcoK restriction during in vitro packaging of coliphage lambda DNA; Rosenberg SM; The K restriction system of Escherichia coli works in vitro {Meselson and Yuan, Nature 217 (1968) 1110-1114} . E . coli C lacks the K restriction system . I show that in vitro packaging in standard E . coli K-12-derived systems effects a loss of plaque-former output from K-unmodified lambda DNA relative to K-modified lambda DNA when compared with packaging in the E . coli C-derived system of Rosenberg et al . {Gene 38 (1985) 165-175} . I conclude that the EcoK restriction system is active in standard in vitro packaging systems . EcoK restriction during in vitro packaging could specifically depress recovery of some lambda and cosmid clones of eukaryotic DNA or any other DNA not modified for EcoK restriction.

Gene, 1985, 39(2-3), 305 - 10
A rapid procedure for DNA sequencing using transposon-promoted deletions in Escherichia coli; Ahmed A; A simple procedure has been developed for sequencing long fragments of DNA . The fragment (which can be several kb in length) is cloned in pAA3.7X, and subdivided into many overlapping segments by Tn9-promoted deletions . The deletions are isolated by positive selection for galactose resistance . A rapid plasmid preparation from several hundred galactose-resistant colonies is fractionated by agarose gel electrophoresis to pick a series of deletions terminating at approx . 200-bp intervals across the entire length of the fragment . Selected plasmids are purified by rapid alkaline extraction, and used directly for supercoil sequencing with a primer derived from IS1 . Sequences of adjacent deletions contain overlaps which are used to connect individual sequences to give the complete sequence.

Gene, 1985, 39(2-3), 263 - 7
Expression of a rat brain creatine kinase-beta-galactosidase fusion protein in Escherichia coli and derivation of the complete amino acid sequence of rat brain creatine kinase; Benfield PA et al.; Expression of a rat brain creatine kinase (CKB)/beta-galactosidase fusion protein in Escherichia coli has allowed isolation of a rat CKB cDNA clone by direct antibody screening of a rat brain gamma gt11 expression library . This clone is 1416 bp long and includes 202 bp of 3'-untranslated region and 29 bp of 5'-untranslated region . The coding sequence of this clone has enabled us to deduce the complete amino acid (aa) sequence of rat CKB protein.

Gene, 1985, 39(2-3), 173 - 80
Expression of tetracycline resistance in pBR322 derivatives reduces the reproductive fitness of plasmid-containing Escherichia coli; Lee SW et al.; Plasmid pBR322 and its numerous derivatives are used extensively for research and in biotechnology . The tetracycline-resistance (TcR) genes in these plasmids are expressed constitutively and cells carrying these plasmids are resistant to tetracycline . We have shown that expression of the TcR gene has an adverse effect on the reproductive fitness of plasmid-containing bacteria in both glucose-limited batch and chemostat cultures . If the TcR genes are inactivated at any one of three different restriction sites, mixed cultures of plasmid-free and plasmid-containing bacteria grow at the same rate.

Nucleic Acids Symp Ser, 1985, (16), 273 - 6
Efficient production of a human tumour necrosis factor in Escherichia coli; Fukui T et al.; To examine the effect of altering the nucleotide sequence of the Shine-Dalgarno (SD) region and the spacer sequence between the SD sequence and the AUG translation start signal, several plasmids were constructed which directed the synthesis of mature human tumour necrosis factor (TNF) under the control of the E . coli trp leader promoter . We found that the presence of the SD sequence, AAGGAGGT, which is complementary to the 3' end of 16S rRNA, gave the higher translational efficiency, and also, the presence of the spacer sequence which consists of only A and T residues raised the production of TNF 2-3 fold . The levels of expression of TNF were elevated over 20-fold by the alteration of the SD and spacer sequences and amounted to 20% of the total cellular proteins or approx . 5 X 10(6) molecules of TNF per cell.

Mol Gen Genet, 1985, 201(2), 360 - 2
Molecular cloning and nucleotide sequence of the HU-1 gene of Escherichia coli; Kano Y et al.; The Escherichia coli HU-1 was cloned by use of mixed synthetic oligonucleotides (17-mer) predicted from a portion of its amino acid sequence . The amino acid sequence of the HU-1 protein deduced from the nucleotide sequence is in good agreement with the published sequence . The nucleotide sequence has a possible promoter and a typical ribosomal binding site upstream from the translational initiation codon (GUG) of the HU-1 gene.

Mol Gen Genet, 1985, 201(2), 344 - 6
Effect of mutations in the RNA polymerase gene and that of the transcription termination factor rho on expression of the Escherichia coli galactose operon with an IS2 polar insertion; Yarulin VR et al.; We analysed the effects on the expression of the gal operon of six phenotypically different mutations in the Escherichia coli RNA polymerase genes in combination with wild-type, rho, and mutant, rho15, alleles of the gene for the transcription termination factor . RNA polymerase mutations can enhance (rpoB268) or reduce (rpoB255), rpoC3, rpoB265) termination by the rho15 factor at the IS2 terminator . The rpoC1 mutation enhances the transcription of the gal operon regardless of the IS2 insertion or the rho15 mutation . Thus RNA polymerase mutations can, independently, or in combination with the rho mutation, compensate for the IS-induced, specifically IS2-induced, termination, leading to a partial restoration of gene activity.

Mol Gen Genet, 1985, 201(2), 334 - 8
Multimer resolution systems of ColE1 and ColK: localisation of the crossover site; Summers D et al.; We have identified and characterised a stability function encoded by the high copy plasmid ColK . The function is analogous to ColE1 cer and maximises stability by maintaining plasmids in the monomeric state . In vivo recombination between cer and ckr (which share more than 90% homology at the DNA sequence level) produced a functional hybrid . Sequence analysis of hybrids indicates that recombination involving cer and ckr is site-specific and occurs within a 35 bp region of DNA which contains palindromic symmetry.

Mol Gen Genet, 1985, 201(2), 323 - 8
Regulation of the lkyB gene expression in Escherichia coli K-12 strains carrying an lkyB-lacZ gene fusion; Lazzaroni JC et al.; Phage MudII301 was used to isolate new periplasmic-leaky mutants of Escherichia coli K12 carrying an lkyB-lacZ gene fusion . The properties of strain JC2299 carrying the lkyB-2299 insertion mutation were identical to those of strain JC207 carrying the previously described lkyB-207 mutation . The LkyB-beta-galactosidase hybrid protein was partially extracellular and membrane bound . It was shown that both a nonsense (envZ-22) and a polar (ompR::Tn10) mutation in the ompB operon led to an increase of beta-galactosidase activity in the lkyB-lacZ fusion strain . On the other hand, mutations in the phoB, phoR, phoS, phoT, malT or envY genes had no effect on lkyB gene expression.

Mol Gen Genet, 1985, 201(2), 315 - 22
Isolation and characterization of Escherichia coli antimutators . A new strategy to study the nature and origin of spontaneous mutations; Quinones A et al.; To identify the nature and origin of spontaneous mutability we developed a screening procedure suitable to isolate antimutators showing a lower error rate than 10(-10) per base per replication . Among about 500,000 mutagenized colonies we found 20 mutants showing a reduced spontaneous mutability . These antimutators can be subdivided into three groups: (i) Mutants in which the level of spontaneous mutability is reduced due to an increase in efficiency of the error correcting mechanism (amu4) . (ii) Mutants which are deficient in several pathways of DNA repair . This finding supports the hypothesis that much spontaneous mutability is due to error-prone repair (amu59, amu47, amu50, amu62, amu43, amu38) . (iii) Mutants in which the antimutator effect seems to be the result of an auxotrophy such as Pur- (amu17), Thr- (amu1, amu28) and Ser- (amu31) . This finding might support the hypothesis that metabolically induced lesions are important in spontaneous mutagenesis . Eleven of these antimutators were mapped at ten bacterial loci in the following positions: amu31 (2 min); amu4 (4 min); amu62 (82 min); amu47 (85 min); amu59 (86 min); amu17 (89 min); amu50 (95 min); amu1/amu28 (100 min); amu38 (23-27 min) and amu43 (74-81 min).

Mol Gen Genet, 1985, 201(2), 301 - 7
The recN locus of Escherichia coli K12: molecular analysis and identification of the gene product; Picksley SM et al.; The recN gene which is necessary for inducible DNA repair and recombination in Escherichia coli has been cloned into the low copy plasmid vector pHSG415 . Analysis of the recombinant plasmid, pSP100, revealed a 5.6 Kb HindIII insert of chromosomal DNA . Transposon inactivation of recN function and analysis of a recN::Mu(Ap lac) fusion located the coding region to a 1.4 Kb region within a 2.1 Kb BglII-AvaI DNA fragment transcribed in a clockwise direction with respect to the chromosome map . The gene product was identified in maxicells as a 60,000 dalton protein . Synthesis of this protein was increased in cells lacking LexA activity or in strains carrying recN cloned into the multicopy vector pBR322 . Multiple copies of recN increase resistance to ionizing radiation in recN mutants but reduce the survival of a wild-type strain.

Mol Gen Genet, 1985, 201(2), 282 - 8
Genetical and functional organisation of the Escherichia coli haemolysin determinant 2001; Mackman N et al.; We have identified gene products corresponding to hlyC, hlyA and hlyD encoded by the Escherichia coli haemolytic determinant 2001 of human origin cloned into the recombinant plasmid pLG570 . The product of hlyC is required for the "activation" of the inactive 107K polypeptide encoded by the hlyA gene . The activated 107K protein constitutes the active haemolysin secreted into the medium . hlyB and hlyD are separate regions defined by complementation studies and encode functions essential for the export of haemolysin with hlyD encoding a 53K protein . Complementation studies using subclones and Tn5 insertions into pLG570 have revealed the presence of two major promoters upstream of hlyC and hlyD which transcribe the four hly genes in the same direction . Finally, we were able to reconstitute the complete haemolysin system from three different plasmids encoding hlyC, hlyA and hlyB + hlyD, respectively.

Mol Gen Genet, 1985, 201(2), 277 - 81
Rescue of transfected genes from mammalian cells by functional selection in Escherichia coli; Strauss M et al.; We have established procedures for reisolating a transfected gene from mammalian cells by selection in Escherichia coli for the function of the gene product using the Herpes simplex virus thymidine kinase gene as a model . Rescue of the gene is accomplished by three different methods . The tk gene is recloned into a plasmid in which it is hooked up by either the lac promoter or a lac/tk hybrid promoter, or the original plasmid is cut out of the host cell DNA . As the lac/tk hybrid gene can be expressed and selected both in the mammalian and E . coli cells, this type of gene rescue allows investigations on mutagenesis and methylation processes . Additionally, it offers a simple way of studying the integration of the transfected gene into the mammalian genome.

Mol Gen Genet, 1985, 201(2), 247 - 51
Role of translation in the UTP-modulated attenuation at the pyrBI operon of Escherichia coli; Clemmesen K et al.; A 273 bp DNA fragment containing the attenuator of the pyrBI operon was inserted into a synthetic cloning site early in the lacZ gene on a plasmid . By this operation the first few codons of lacZ were joined through a linker to the last 39 codons of the open reading frame for the putative pyrB leader peptide . In addition a gene fusion encoding a hybrid protein with beta-galactosidase activity was formed between the pyrB start and the rest of lacZ . This gene fusion is expressed from the lac promoter and the transcript is subject to facultative termination at the pyrBI attenuator . Different variants of the lacZ start were used that either contained a stop codon or directed the translation toward the attenuator in any of the alternative reading frames . The following results were obtained . No significant read-through of transcription over the pyrB attenuator was seen when the leader translation ended 49 nucleotide residues, or more, upstream of the attenuator symmetry, but a UTP-modulated attenuation was established if the leader translation was allowed to proceed across the attenuator as for the putative leader peptide or in a frame-shifted version . The regulation, however, was not as great as for the native pyrB gene . This is probably because the substitution of the normal start of the leader peptide by the start of lacZ alters the coupling between transcription and translation and thereby the attenuation frequency . It cannot, however, be ruled out that the pyrBI operon is regulated at the promoters in addition to the control by attenuation.

Mol Gen Genet, 1985, 201(2), 204 - 12
Identification of the genes and their polypeptide products responsible for aerobactin synthesis by pColV plasmids; Gross R et al.; Iron acquisition via aerobactin enhances the virulence of Escherichia coli . Genes that specify functions for aerobactin synthesis and iron(III)-aerobactin transport have been identified on several ColV plasmids . Previously, we cloned the locus for aerobactin synthesis from pColV-K311 and assigned to three loci termed aerA, aerB, and aerC the functions for hydroxylation of lysine, acetylation of the 6-amino group of 6-hydroxy-lysine and coupling of N-acetyl-N-hydroxy-lysine with citrate, respectively (Gross et al . 1984) . In this paper we show that aerA and aerB determine polypeptides with molecular weights of 50,000 and 35,000, respectively . We identified a fourth gene designated aerD that codes for a polypeptide with a molecular weight of 60,000, and which is required for the linkage of one residue of N-acetyl-N-hydroxy-lysine to citrate . The aerC gene product completes aerobactin synthesis by coupling the second N-acetyl-N-hydroxy-lysine to the monoacylated derivative citrate . The order of the genes in the operon was found to be aerD-aerB-aerC-aerA.

Mol Gen Genet, 1985, 201(2), 174 - 7
Reversion of a truncated gene for ampicillin resistance by genetic rearrangements in Escherichia coli K12; Iida S et al.; The composite transposon Tn2672 is a derivative of the Tn3-related transposon Tn902 whose bla gene providing ampicillin resistance had been inactivated by the insertion of the IS1-flanked multiple drug resistance transposon Tn2671 . Most ampicillin resistant revertants of Tn2672 are due to precise excision of Tn2671 . However, a rare Bla+ revertant which still retains all the previously acquired drug resistance markers was isolated . On this revertant, the 5' part of the split bla gene on Tn2672 has converted to an intact, active bla gene, and the entire Tn902 is structurally restored . In contrast, the adjacent IS1b element belonging to Tn2671 has its terminal 142 base pairs deleted . Despite of this rearrangement, the split 3' part of bla and its adjacent sequences have remained unchanged . Models are presented to explain the observed DNA rearrangements, and their similarity with gene conversion events is discussed.

Mol Gen Genet, 1985, 201(2), 151 - 7
Cloning of the Escherichia coli gene for the stringent starvation protein; Fukuda R et al.; In order to clone the Escherichia coli gene for the stringent starvation protein (SSP), we determined its N-terminal sequence as well as the sequence of two peptide fragments obtained by cyanogen bromide cleavage of the protein . We then chemically synthesized four sets of oligodeoxyribonucleotide mixtures that represented possible codon combinations for parts of these amino acid sequences . The synthetic oligonucleotides were labelled with 32P at their 5'-termini and used as hybridization probes to detect DNA fragments containing the complementary sequences . Genomic Southern hybridization of E . coli chromosomal DNA gave up to ten DNA fragments hybridizing with each probe but only a few hybridized with two or more of the probes . The latter fragments were cloned in pBR322 . By determining partial base sequences with a rapid method and examining proteins encoded by the DNA fragments, we were able to show that we had isolated a clone containing the complete SSP structural gene.

Folia Microbiol (Praha), 1985, 30(6), 474 - 8
Transformation of Streptomyces granaticolor with natural and recombinant plasmid vectors; Petricek M et al.; Protoplasts of Streptomyces granaticolor were found to be transformable by the broad-host-range plasmid pIJ350 but no transformants were detected when the narrow-host-range plasmid pIJ2 or the shuttle vector pPM66 (pIJ350--pBR322) isolated from E . coli cells were used . The onset of blue colour granaticin production by S . granaticolor cells was used as a marker to prepare protoplasts with a high transformation capacity . The presence of a restriction system is discussed.

Gene, 1985, 39(1), 89 - 93
A method for efficient gene isolation from phage lambda gt11 libraries: use of antisera to denatured, acetone-precipitated proteins; Timmins JG et al.; Experience with cloning pseudorabies virus (PRV) DNA in the lambda gt11 phage vector has shown that there are special requirements for the antisera used in screening the libraries, in addition to the requirement that the antisera recognize proteins on a Western blot . Initial screening of a lambda gt11 library of sheared PRV DNA fragments in Escherichia coli for expression of PRV antigens using PRV hyperimmune antisera was unsuccessful . It was only after screening the library with antisera raised against PRV proteins eluted from sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels that positive results were obtained . These "gel-slice" antisera (GSA) were equivalent in potency to hyperimmune antisera in standard immunoassays (including ELISA, immunoprecipitation, Western blots, and neutralization of virus), but only the GSA could recognize PRV fusion proteins expressed by recombinant lambda gt11 phage . This difference was seen despite the fact that hyperimmune antisera performed satisfactorily on Western blots of denatured PRV-infected cell extracts . These results show that the efficiency of screening expression libraries in E . coli can be improved if antibodies are raised against denatured proteins.

Gene, 1985, 39(1), 109 - 12
Identification of a mutation that relieves gamma-glutamyl kinase from allosteric feedback inhibition by proline; Rushlow KE et al.; A 1.75-kb DNA fragment containing the entire Escherichia coli proB+ gene has been sequenced . The proB locus encodes the structural gene for gamma-glutamyl kinase (GK), the enzyme responsible for the first step in proline biosynthesis, and the primary regulatory point of the pathway . We have previously reported the nucleotide (nt) sequence of a mutant proB gene isolated from an E . coli strain resistant to the toxic analog of proline, 3,4-dehydro-DL-proline (DHP) . This mutant gene encodes a GK which is refractory to allosteric feedback inhibition by proline (DHPR) . Comparison of the proB+ and DHPR proB sequences revealed a single base difference, an A-T to C-G transversion localized at nt position 428 within the amino acid (aa) coding region of proB . This mutation predicts an aa change from glutamic acid in the wild-type (wt) enzyme to alanine in the DHPR enzyme.

Gene, 1985, 36(3), 363 - 7
Restriction endonuclease EcoO109 from Escherichia coli H709c with heptanucleotide recognition site 5'-PuG/GNCCPy; Mise K et al.; A new restriction endonuclease, EcoO109, has been isolated from Escherichia coli H709c by polyethyleneimine (PEI) precipitation, DEAE-cellulose chromatography and heparin agarose chromatography . The yield was high, more than 3000 units/g of wet cells . The EcoO109 endonuclease recognizes and cleaves a nucleotide sequence of (formula: see text), in the presence of 10 mM Mg2+ . The enzyme will be useful for structural analysis and molecular cloning of DNA because of the stability, high yield and easy handling of the producer strain.

Gene, 1985, 36(3), 321 - 31
Development of a high-frequency transforming vector for Aspergillus nidulans; Ballance DJ et al.; The pyr4 gene of Neurospora crassa, which codes for orotidine-5'-phosphate decarboxylase, is capable of transforming an Aspergillus nidulans pyrG mutant by chromosomal integration, despite low homology between the transforming DNA and the recipient genome . Integration of pFB6, a plasmid carrying pyr4 and capable of replication in Escherichia coli, was not observed at the pyrG locus . The efficiency of transformation was considerably enhanced (50-100 fold) by inclusion in the transforming vector of a 3.5-kb A.nidulans chromosomal sequence, ans1 . Although this sequence was isolated on the basis of replicating activity in Saccharomyces cerevisiae, there was no evidence for such activity in A.nidulans . Part of the ans1 fragment appears to be reiterated in the A.nidulans genome, though it is not yet clear whether this is directly responsible for the high transformation frequency . The efficiency of transformation of A.nidulans by plasmids bearing ans1, using an improved protocol, was approx . 5 X 10(3) stable transformants per microgram of plasmid DNA.

Gene, 1985, 36(3), 241 - 7
The nucleotide sequence of the essential cell-division gene ftsZ of Escherichia coli; Yi QM et al.; The nucleotide sequence of a 1.8-kb fragment of Escherichia coli DNA containing the essential cell division gene ftsZ is reported . The FtsZ protein has an Mr of 40294 and has 23% charged residues with a calculated isoelectric point of 4.9 . The codon usage of the ftsZ gene reflects that of a highly expressed gene . Also located on this DNA fragment is the 3' end of the ftsA gene and the 5' end of the envA gene . These designations were confirmed by locating Tn5 insertions within the ends of these genes that inactivate each of these genes . A potential promoter for ftsZ overlapped the 3' end of the ftsA gene . A Tn5 insertion was located within the 3' end of the ftsA and within this potential promoter . No transcription terminators were evident between ftsA and ftsZ or between ftsZ and envA.

Gene, 1985, 36(3), 201 - 10
A rapid method of gene detection using DNA bound to Sephacryl; Langdale JA et al.; A rapid method of gene detection has been developed utilising DNA fragments immobilized on resins and a sandwich hybridization assay . This method permits the detection of restriction fragment length polymorphisms (RFLPs) without the need to immobilize sample DNA . The method is based on the use of two non-overlapping DNA restriction fragments, one of which is attached to a resin (fragment A) and the other 32P-labelled (fragment B) . Fragments A and B will not hybridize to each other unless there is a DNA or RNA fragment capable of hybridizing to both A and B present in the same reaction . Hybridization in this instance will result in the resin being radioactively labelled . The RFLP associated with the mutation causing sickle-cell anaemia was used as a model to develop the method . The resin Sephacryl S-500 appeared most suited to our method for two reasons: (i) DNA immobilization experiments using two coupling procedures and four resins indicated that Sephacryl S-500 bound the most DNA with very little non-covalent coupling . (ii) Hybridization experiments with DNA bound to a number of resins showed that DNA bound to Sephacryl S-500 hybridized most efficiently with a low level of nonspecific hybridization . Using optimum hybridization conditions 5 X 10(-18) mol of beta-globin DNA could be detected . The method has been used to distinguish between DNA from sickle, heterozygote and normal patients.

Adv Exp Med Biol, 1985, 185, 47 - 61
The structural proteins of the autonomous parvovirus feline panleukopenia virus; Carlson J et al.; Approximately 80% of the genome of feline panleukopenia virus was cloned into the plasmid pBR322 . The entire 3943 nucleotide sequence of the cloned portion of FPV was determined . This DNA includes the gene which codes for the structural proteins of the virus . Portions of this gene were expressed in E . coli as fusion proteins with bacterial proteins . Some of the fusion proteins were capable of raising neutralizing antibodies in guinea pigs . Through the use of deletion mapping, monoclonal antibodies, and synthetic peptides, attempts were made to localize the portion of the protein responsible for raising these antibodies.

Gene, 1985, 38(1-3), 57 - 64
Expression of the gag gene of human T-cell leukemia virus type I in Escherichia coli and its diagnostic use; Itamura S et al.; An expression plasmid, pHY202, was constructed which directs the synthesis of a fusion protein encoded by the gag sequence of human T-cell leukemia virus type I (HTLV-I) inserted into the lacZ' gene . Escherichia coli cells harboring pHY202 produced the 43-kDal LacZ'-Gag fusion protein with a yield of approx . 0.3% of total soluble proteins . The fusion protein is specifically recognized by monoclonal antibodies against the Gag proteins p19 and p24, and could be applicable for the diagnosis of HTLV-I infection, because almost all sera from HTLV-I carriers gave a positive response in the enzyme-linked immunosorbent assay (ELISA) employing the LacZ'-Gag hybrid protein purified by immunoaffinity column chromatography.

Gene, 1985, 38(1-3), 265 - 9
Nucleotide sequence of the phosphoenolpyruvate carboxylase gene of the cyanobacterium Anacystis nidulans; Katagiri F et al.; Nucleotide sequence of the open reading frame (ORF) for the phosphoenolpyruvate carboxylase gene (ppc) of the cyanobacterium Anacystis nidulans was determined . The ORF consists of 3159 bp and codes for 1053 amino acid (aa) residues . The codon usage of the ppc of A . nidulans is not so markedly different from that of the Escherichia coli ppc, yet, in A . nidulans the preferred codons are AAG for lysine and CCC for proline, whereas those are seldom used in the E . coli ppc.

Gene, 1985, 36(1-2), 87 - 95
One-step gene replacement in yeast by cotransformation; Rudolph H et al.; A general method to replace chromosomal DNA sequences of Saccharomyces cerevisiae by any in vitro modified DNA sequence has been developed and was applied to the PHO5 locus on chromosome II . A recipient strain was constructed in which part of the chromosomal PHO5 sequence was substituted by the URA3 gene . Replacement of this pho5-URA3 substitution by pho5 mutant alleles was achieved in one step by cotransformation with a pho5 DNA fragment and the self-replicating plasmid YEp13, which contains the LEU2 gene as a selectable marker . Leu+ transformants were selected, and the replacement events at the PHO5 locus were detected by their Ura- phenotype (1-4% of the Leu+ were Ura-) . In a similar way the PHO5 coding sequence was replaced by the sequence coding for human tissue-type plasminogen activator (t-PA).

Gene, 1985, 36(1-2), 79 - 86
The traM gene of the resistance plasmid R1: comparison with the corresponding sequence of the Escherichia coli F factor; Koronakis VE et al.; The 7.7-kb EcoRI fragment of the resistance plasmid R1 contains the gene for the TraM protein . The sequence was identified by the presence of an open reading frame (ORF) which is preceded upstream by two promoter sequences . Both these promoters were found to be active, although the more distant one predominates, as was judged by the relative abundance of mRNA of the expected length . The TraM protein could be synthesized in an in vitro DNA-dependent protein synthesis system if the DNA of the corresponding region was supplied as template . Comparison of the traM genes of R1 and the F factor showed a high degree of similarity, although a number of mutations, especially near the 5' terminus, introduce specific amino acid (aa) changes . Two-thirds of the 3' sequences differ mainly in silent mutations; hence the aa sequence of the corresponding carboxy-terminal portion of the protein is highly conserved . The 5' and 3' untranslated regions of the mRNAs show little homology . One of the promoter regions, the ribosome-binding sequences, and the transcription termination sites are located at comparable positions but differ in details.

Gene, 1985, 36(1-2), 37 - 44
Studies on deo operon regulation in Escherichia coli: cloning and expression of the cytR structural gene; Barbier CS et al.; The structural gene that encodes one repressor (the cytR-encoded repressor) of the Escherichia coli deo operon has been cloned from a lambda dmet transducing phage into the multicopy plasmid pBR322 by selecting for ApR, Lac- transformants of E . coli SS110(delta lac, cytR, tsx::lac) . Restriction maps for the cytR+ plasmids have been generated and the position of the cytR gene on the cloned insert of these plasmids has been determined through deletion analysis . Results from maxicell experiments employing pCB001 and its cytR- derivatives suggest that the cytR gene encodes a protein with a subunit Mr of 37 000 . In contrast to the complete repression of the deo operon obtained when deoR+ plasmids were introduced into E . coli SS201 (deoR, cytR), transformation of this DeoR-, CytR- strain with any of the cytR+ plasmids yields only clones which have phenotypes and Deo enzyme levels characteristic of a DeoR- single mutant . The data presented in this study are consistent with the interpretation that, in E . coli, the deoR-encoded repressor controls deo operon transcription initiating from both deo promoter-operator sites, PO1 and PO2 . In contrast, the cytR-encoded repressor regulates deo operon expression only through deo promoter-operator site PO2.

Gene, 1985, 36(1-2), 189 - 93
Evidence for autoregulation of the nusA-infB operon of Escherichia coli; Nakamura Y et al.; Analysis of three different nusA mutant strains suggests that the expression of the nusA-infB operon of Escherichia coli is regulated autogenously by the nusA gene product, a protein known to mediate transcription termination and antitermination . The cellular amounts of NusA and IF2 (infB) proteins are enhanced by a nusAts mutation which causes reduced transcription-termination activity . A nusAam mutant carrying the am ts suppressor, supFts6, overproduces the IF2 protein when the amount of NusA protein is reduced by the thermal inactivation of the supFts6 . A modified form of NusA with the cat protein of Mr of 24 000 attached to the C terminus of NusA is overproduced compared to the wild-type NusA and causes the overproduction of IF2.

Gene, 1985, 36(1-2), 113 - 22
Analysis of genes for 5S rRNA from the cricket, Acheta domesticus: two classes of repeating units; Benes H et al.; To examine the modulation of 5S rRNA gene activity during development in the cricket, Acheta domesticus, 5S X DNA was isolated from a lambda Charon 4 genomic library and characterized . Southern blot analysis of cloned A . domesticus genomic DNA revealed that restriction fragments of 3.0 and 2.1 kb represent two size classes of 5S X DNA repeating units; over 90% of the repeats measure 3.0 kb . Restriction analysis of two 5S X DNA clones suggests that the 2.1-kb repeats are not randomly interspersed within clusters of the larger 3.0-kb repeating units . Heteroduplex and restriction mapping of several clones indicate that the spacers of both repeating units account for their unusual length . The major difference between the two classes of repeats may lie in 0.9-kb spacer sequences to the 3.0-kb repeats.

Mol Gen Genet, 1985, 201(1), 76 - 81
Lethal mutations in the structural gene of an outer membrane protein (OmpA) of Escherichia coli K12; Freudl R et al.; The gene ompA encodes a major outer membrane protein of Escherichia coli . Localized mutagenesis of the part of the gene corresponding to the 21-residue signal sequence and the first 45 residues of the protein resulted in alterations which caused cell lysis when expressed . DNA sequence analyses revealed that in one mutant type the last CO2H-terminal residue of the signal sequence, alanine, was replaced by valine . The proteolytic removal of the signal peptide was much delayed and most of the unprocessed precursor protein was fractioned with the outer membrane . However, this precursor was completely soluble in sodium lauryl sarcosinate which does not solubilize the OmpA protein or fragments thereof present in the outer membrane . Synthesis of the mutant protein did not inhibit processing of the OmpA or OmpF proteins . In the other mutant type, multiple mutational alterations had occurred leading to four amino acid substitutions in the signal sequence and two affecting the first two residues of the mature protein . A reduced rate of processing could not be clearly demonstrated . Membrane fractionation suggested that small amounts of this precursor were associated with the plasma membrane but synthesis of this mutant protein also did not inhibit processing of the wild-type OmpA or OmpF proteins . Several lines of evidence left no doubt that the mature mutant protein is stably incorporated into the outer membrane . It is suggested that the presence, in the outer membrane, of the mutant precursor protein in the former case, or of the mutant protein in the latter case perturbs the membrane architecture enough to cause cell death.

Mol Gen Genet, 1985, 201(1), 51 - 5
The catabolite gene activator protein (CAP) is not required for indole-3-acetic acid to activate transcription of the araBAD operon of Escherichia coli K-12; Ebright RH et al.; Kline et al . (1980) have reported that indole-3-acetic acid (IAA) and four other indole derivatives are able to substitute for cAMP in activating expression of the ara regulon of E . coli . We have examined this phenomenon in detail, utilizing fusions between the structural gene for beta-galactosidase and the promoters for the araBAD, araE, and araFG operons . We confirm that IAA potently stimulates transcription from the araBAD promoter . The effect is highly specific to araBAD, as IAA has no, or only slight, effects on the araE and araFG operons . However, contrary to the results of Kline et al., we find that the action of IAA does not require CAP . Thus, IAA fully stimulates the transcription of araBAD in a strain which bears a complete deletion of the crp gene.

Mol Gen Genet, 1985, 201(1), 30 - 4
Untargeted mutagenesis induced by UV in the lacI gene of Escherichia coli; Christensen RB et al.; Using a nonselective method, we have estimated the proportion of untargeted mutations in the lacI gene of E . coli by transferring either irradiated or unirradiated F' pro lac plasmids from an excision deficient donor to an excision deficient pro lac deleted recipient that had been irradiated and allowed to induce recA dependent functions for 30 min . We find that about 10 percent of the mutations induced by either 3.5 Jm-2 or 7 Jm-2 UV are untargeted.

Folia Microbiol (Praha), 1985, 30(5), 407 - 13
New replication mutant pNH602 and its relationship to plasmid pAS3, another deletion derivative of plasmid R6K; Hochmannova J et al.; A stable deletion derivative pNH602 was obtained when the recently described higher-copy-number point mutant pNH601 of plasmid R6K was introduced to a minicells-producing strain of Escherichia coli . The size of plasmid pNH602 is 18.8 Mg/mol as determined by electron microscopy . The 7.2 Mg/mol fragment of R6K genome missing in pNH602 carries the Smr-determinant and the region finO and, according to the results of restriction analysis, it includes one EcoRI site . With its radioisotopically determined 33 copies of pNH602 per E . coli K-12 chromosome (npc), representing a 23% increase of the point mutant pNH601 and 150% enhancement of R6K npc, plasmid pNH602 differs from another closely related R6K deletion derivative pAS3 of the same size which exhibits only 20 npc . Both pNH602 and pAS3 plasmids are conjugative.

Folia Microbiol (Praha), 1985, 30(5), 401 - 6
Isolation and characterization of mutants of the RP4 plasmid coding for increased resistance to ampicillin; Cejka K et al.; E . coli strain J53(RP4) was mutagenized with ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine . Clones showing a two-to threefold increase in resistance to ampicillin were produced . This increase was not due to an increased number of RP4 copies per chromosome . The level of penicillinase activity was twice higher in comparison with the parental strain . No detectable changes were found in the region coding for the resistance to ampicillin on the plasmid by restriction analysis.

Gene, 1985, 37(1-3), 241 - 6
Solubility of the EcoRI restriction endonuclease and its purification from an over-producing strain; Luke PA et al.; The solubility of the EcoRI restriction endonuclease was measured in solutions of varying NaCl concentrations, at different temperatures and in the presence of DNA . The precipitation of the protein was enhanced by low NaCl concentrations, by elevated temperatures, and by the addition of DNA . These observations are discussed in relationship to the interaction of this protein with DNA . The purification of the EcoRI restriction enzyme from a strain of Escherichia coli that over-produces this enzyme was hampered by the insolubility of the protein, and hence the purification procedure was modified to optimize the recovery of active enzyme.

Gene, 1985, 37(1-3), 215 - 20
A powerful method for the preparation of cDNA libraries: isolation of cDNA encoding a 100-kDal nucleolar protein; Lapeyre B et al.; An easy and quick method to synthesize large cDNA molecules and to clone them with very high efficiency in the expression vector lambda gt11 is described . The technique employs RNase H and Escherichia coli DNA ligase treatment during second-strand synthesis, followed by repair of the ds cDNA extremities by S1 nuclease and PolIk (Klenow fragment) treatment . This treatment allows efficient addition of suitable linkers and results in a 100-fold increase in the yield of cloned cDNA, when compared with other published techniques . Using 75 ng of poly(A)+ RNA from CHO cells, we have prepared a library of 1.1 X 10(7) clones . This library was screened with polyclonal antibodies raised against a 100-kDal nucleolar protein of CHO cells . Five recombinants were isolated with inserts of 500-2500 bp . The average size of cDNA obtained by this method is considerable: the 2500-bp cDNA represents 90% of the mRNA coding for the 100-kDal protein.

Chromosoma, 1985, 92(5), 369 - 77
A cloned sequence, p82H, of the alphoid repeated DNA family found at the centromeres of all human chromosomes; Mitchell AR et al.; Clone p82H is a human DNA sequence which hybridises in situ exclusively to the centromeric regions of all human chromosomes . It is composed of approximately 14 tandemly repeated variants of a basic 172 bp sequence, and is related to the alphoid family . The organisation of the family of cross-hybridising sequences, detected by the clone p82H, is described both in the human genome and on certain chromosomes, and its relationship to known sequence families is discussed.

Mol Gen Genet, 1985, 200(3), 442 - 50
Autoregulation of the dnaA gene of Escherichia coli K12; Atlung T et al.; Regulation of the dnaA gene, which codes for an essential factor for the initiation of replication from the chromosomal origin, was studied in vivo using transcriptional and translational gene fusions . We found that the dnaA gene was autoregulated over a 30-fold range by the activity of dnaA protein . Expression from the dnaA promoter region of a dnaA"lacZ fusion was inhibited up to sevenfold by surplus dnaA protein and was stimulated up to fivefold upon thermoinactivation of the mutant protein in five different dnaA(Ts) strains . The autoregulation was found to be exerted at transcription from the major dnaA promoter and was eliminated by deletion of sequences around position -65 of this promoter where a 9-bp sequence, which is also found four times in the chromosomal origin, is located.

Mol Gen Genet, 1985, 200(3), 385 - 92
Analysis of the flanking regions from different haemolysin determinants of Escherichia coli; Knapp S et al.; The haemolysin (hly) determinant of the plasmid pHly152 contains an IS2 element at 469 bp upstream of the hlyC gene . The sequence at the other (right-hand) end (RS) also shows multiple hybridization with the plasmid pHly152 and the chromosome of some Escherichia coli strains but the nucleotide sequence of this region does not reveal the typical properties of an IS element . Similar arrangements in the regions flanking the hly determinant are also found on various Hly plasmids from uropathogenic E . coli strains . Chromosomal hly determinants lack both flanking sequences (IS2 and RS) in the immediate vicinity of the hly genes . The sequences immediately upstream of the hlyC gene have been determined from several chromosomal hly determinants and compared with the corresponding sequence of the hly determinant of the plasmid pHly152 . We show that these sequences, which contain one promoter (left promoter, phlyL) in all hly determinants tested, vary considerably although common sequence elements can still be identified . In contrast, only relatively few nucleotide exchanges have been detected in the adjacent structural hlyC genes . The A + T content of the 200 bp sequence upstream of hlyC is very high (72 mol% A + T) but even the structural hly genes show a considerably higher A + T content (about 60 mol%) than the E . coli chromosome on average (50 mol% A + T) suggesting that the hly determinant may not have originated in E . coli.

Mol Gen Genet, 1985, 200(3), 362 - 7
Autogenous regulation of synthesis of the replication protein in plasmid pSC101; Yamaguchi K et al.; A 1.3-kb segment of plasmid pSC101 includes the replication origin (ori) and the gene (rep) encoding the 37 kilodalton (K) protein required for autonomous replication of the plasmid . The present work describes the regulation of the rep gene expression . The gamma promoters PR and PL fail to promote rep gene expression when located upstream of a sequence with dyad symmetry overlapping the rep promoter, whereas elimination of this sequence allows expression and results in over-production of the rep protein . When expression of trpA'-lacZ is controlled under the rep promoter, beta-galactosidase is produced without the lac inducer . However, this enzyme synthesis is effectively reduced when the complete rep sequence is provided in trans . A partial disruption of the sequence with dyad symmetry relieves the repression . These results suggest that expression of the rep gene is negatively regulated by its own product and that the sequence with dyad symmetry plays the role of a receptor site for the rep protein.

Gene, 1985, 35(3), 223 - 35
The Streptomyces plasmid SCP2*: its functional analysis and development into useful cloning vectors; Lydiate DJ et al.; Detailed restriction maps of the plasmid SCP2* and its deletion derivative pSCP103 were constructed . DNA fragments carrying hygromycin (Hyg), thiostrepton (Thio) or viomycin-resistance (VioR) determinants were inserted into pSCP103, and various segments were deleted from the resulting plasmids . Changes in plasmid phenotypes associated with these insertions and deletions allowed the localisation and characterisation of plasmid replication, stability, transfer and fertility functions . Several useful cloning vectors were constructed . They are able to maintain large (greater than 30 kb) DNA inserts, with stable inheritance at a low copy number (1-2 per chromosome) and without structural rearrangements, in Streptomyces hosts . The vectors have a broad host range in the genus Streptomyces . One of them (pIJ903) is a shuttle vector for Streptomyces and Escherichia coli.

Dev Biol Stand, 1985, 60, 111 - 22
Cloning, expression and biological activity of a new variant of human interferon alpha identified in virus induced lymphoblastoid cells; Cohen S et al.; A synthetic oligonucleotide complementary to a highly conserved sequence in the IFN-alpha gene family, was used to screen a Namalva cDNA library . Among the cDNA clones having typical IFN-alpha traits, one was distinct from previously characterized IFN-alpha cDNAs . E . coli cells carrying this recombinant cDNA plasmid express an alpha-interferon activity . The sequence of this IFN-cDNA is extremely homologous (99.5%) to that of the IFN-alpha J gene and is designated IFN-alpha J1 . Several E . coli trp expression plasmids were constructed for efficient transcription and translation of the mature IFN-alpha J1 . The maximal level of expression (5 X 10(3) molecules/cells) was obtained from plasmid pJ1-4 . A synthetic consensus translation initiation sequence coupled to the trp p/o region (in pJ1-5) proved to be 10 times less effective in promoting metIFN production in bacteria, than the in-vitro mutated trpL initiation sequence carried on pJ1-4 . The bacterial IFN-alpha J1 was purified (to over 90% purity) to a specific activity of 1.3 X 10(8) units/mg . The antiviral activity of the purified IFN-alpha J1 was compared with other highly purified IFN-alpha species (bacterial IFN alpha A and alpha C, leukocyte IFN-alpha 1, leukocyte IFN mixture and Namalva IFN preparation) on a large panel of mammalian cell cultures . IFN-alpha J1 exhibits a distinct antiviral activity.

Dev Biol Stand, 1985, 60, 105 - 9
The immortalization of human lymphocytes by spheroplast fusion; Brzeski H et al.; A method for immortalizing animal cells based on the spheroplast fusion technique of Schaffner is being developed . Human lymphocytes have been fused with E . coli spheroplasts containing the plasmid pTSV3, which represents the entire SV40 genome cloned into the EcoRI restriction enzyme site of the plasmid pAT153 . The efficiency of transformation was examined using pTSV3 and derivatives which have deletions in parts of the late gene sequences . Although there was a marked increase in the survival time of the lymphocyte cultures after fusion compared with cells which had been mitogen-stimulated but not fused, the cells did not continue to proliferate indefinitely . Attempts are now being made to extend the survival time by culturing the transformed lymphocytes in the presence of feeder cells.

Mol Gen Genet, 1985, 200(2), 328 - 34
Molecular cloning and functional analysis of the cysG and nirB genes of Escherichia coli K12, two closely-linked genes required for NADH-dependent nitrite reductase activity; Macdonald H et al.; We have cloned two genes, nirB+ and cysG+ which are required for NADH-dependent nitrite reductase to be active, from the 74 min region of the Escherichia coli chromosome . Restriction mapping and complementation analysis establish the gene order crp-nirB-cysG-aroB . Both genes are trans-dominant in merodiploids and, under some conditions, can be expressed independently . The cysG+ gene can be expressed from both high and low copy number plasmids carrying a 3.6 kb PstI-EcoRI restriction fragment . Attempts to sub-clone the nirB+ gene into pBR322 on a 14.5 kb EcoRI fragment were unsuccessful, but this fragment was readily sub-cloned into and expressed from the low copy number plasmid pLG338 (Stoker et al . 1982) . Overproduction of the 88 kDa nitrite reductase apoprotein by strains carrying a functional nirB+ gene suggests that nirB is the structural gene for this enzyme.

Mol Gen Genet, 1985, 200(2), 266 - 71
The recQ gene of Escherichia coli K12: molecular cloning and isolation of insertion mutants; Nakayama K et al.; The recQ gene of Escherichia coli K12 was subcloned from plasmid pKO1 (Oeda et al . 1981) by monitoring the capacity of the resulting recombinant plasmids partially to reverse the increased ultraviolet (UV) sensitivity of a recF143 recQ1 double mutant . We were able to trace this complementation activity to a 3.4 kilobase (kb) SalI-PvuII fragment . Furthermore, analysis of the Tn3 insertion mutations that abolished the complementation revealed the exclusive localisation of such insertions in the same 3.4 kb segment . This segment was situated about 4 kb clockwise from corA on the chromosome, a result consistent with the transductional data previously reported . In addition, a comparison of our restriction endonuclease cleavage map with the published data has placed recQ between pldA and pldB . When relocated to the recQ site on the chromosome, the recQ::Tn3 mutations conferred partial resistance to thymineless death (TLD) or, in the case of a recBC sbcB background, recombination deficiency and increased UV sensitivity . This has provided the firm evidence that both the TLD resistance and the deficiency in the RecF recombination pathway result from loss of the functional recQ gene . We also identified the recQ gene product as a 74 kilodalton polypeptide by using the maxicell technique.

Mol Gen Genet, 1985, 200(1), 68 - 73
Identification of the gene appA for the acid phosphatase (pH optimum 2.5) of Escherichia coli; Dassa E et al.; A strain of Escherichia coli exhibiting reduced activity of the periplasmic enzyme acid phosphoanhydride phosphohydrolase (pH 2.5 acid phosphatase) was isolated . The mutation designated appA1 was located at 22.5 min on the E . coli genetic map . Acid phosphatase purified from an appA- transductant showed less than ten percent of the specific activity of an isogenic appA+ strain . The mutant enzyme was highly thermolabile and its Km for paranitrophenyl phosphate was increased about 20-fold . The mutant protein cross-reacted with antibody to the wild-type enzyme and had the same molecular weight and concentration in extracts as the wild-type enzyme . These findings strongly suggest that appA is the structural gene of the acid phosphatase.

Mol Gen Genet, 1985, 200(1), 21 - 6
Negative control of oriC plasmid replication by transcription of the oriC region; Tanaka M et al.; We have demonstrated that the replication of the oriC plasmid, carrying the replication origin of the Escherichia coli chromosome, is inhibited by transcriptional readthrough from an oriC flanking region of the plasmid . This was drawn from an examination of the replication of an oriC plasmid, pKZ4, which bears the lacOP segment at the right-hand side of oriC (the asnA side) in such an orientation that transcription from the lac promoter proceeds towards oriC . Replication of pKZ4 was found to be drastically inhibited by inducing transcription from the lac promoter with IPTG, an inducer of the lactose operon . When trp transcription attenuator termination sequences were inserted near the right-hand end of the oriC region of pKZ4, the replication of the plasmid became considerably insensitive to the inhibitory effect of IPTG . This indicates that the inhibition is due to the frequent leftward transcription, which initiates at the lac promoter and proceeds into the oriC region . Since IPTG inhibits the replication of pKZ4, but not that of another coexisting oriC plasmid which is devoid of the lacOP segment, the replication inhibition is judged to act only in cis . Transcription from the promoter of the chloramphenicol resistance gene also caused the inhibition of oriC plasmid replication.

Mol Gen Genet, 1985, 200(1), 176 - 81
Tn1721-encoded resolvase: structure of the tnpR gene and its in vitro functions; Rogowsky P et al.; A 760 base pair nucleotide sequence of transposon Tn1721 containing the resolvase (tnpR) gene has been determined . A 186 triplet open reading frame was assigned to tnpR, and the allocation of -35 and -10 promoter boxes was supported by mapping transcription initiation at 70 base pairs upstream of tnpR . Expression of tnpR under tac promoter control generated sufficient resolvase protein for enzyme purification and for in vitro studies . Purified Tn1721 resolvase requires supercoiling and two directly oriented resolution (res) sites . The enzyme resolves cointegrate substrates containing repeat copies of Tn1721 res, of Tn21 res, of Tn21 res/Tn1721 res, but not of Tn3 res.

Mol Gen Genet, 1985, 200(1), 169 - 75
The resolvase protein from the transposon Tn21; Halford SE et al.; The tac promoter was inserted into Tn21 upstream of the tnpR gene and the resultant plasmid was used to generate substantial amounts of resolvase . This protein was purified to homogeneity . The protein was characterized by amino acid sequence studies (which showed that an open-reading frame previously identified by DNA sequencing had been correctly assigned to the tnpR gene) and by molecular weight measurements (which demonstrated that the only active for of the protein in solution was dimeric) . Pure Tn21 resolvase catalysed site-specific recombinations between directly repeated res sites from Tn21 or Tn1721 but not from Tn3 nor on inverted res sites from Tn21.

Mol Gen Genet, 1985, 200(1), 14 - 20
Purification of the NusB gene product of Escherichia coli K12; Maekawa T et al.; The nucleotide sequence of the entire nusB gene of Escherichia coli has recently been determined and the amino acid sequence of its product deduced (Ishii et al . 1984; Swindle et al . 1984) . The NusB protein was purified by chromatography on Sephadex G-100, phosphocellulose and hydroxylapatite . Purification of the protein was monitored using 14C-labelled NusB protein, which was synthesized in a maxicell containing an nusB plasmid as a marker . The final product, which was at least 95% pure as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, had a molecular weight of about 16,000 and an isoelectric point of about 7.3 . Analytical data on the amino acid composition of the purified protein agreed with that deduced from the DNA sequence and indicated that this protein was indeed the product of the nusB gene.

Mol Gen Genet, 1985, 200(1), 138 - 44
Relationship between the proteins encoded by the exclusion determining locus of the IncI plasmid R144 and the cellular localization of these proteins in Escherichia coli K-12; Hartskeerl R et al.; A region of the IncI plasmid R144, determining and controlling exclusion (exc), codes for two proteins, designated 13K and 19K after their apparent molecular weights (respectively 13,000 and 19,000) . Both proteins were simultaneously affected by various mutations that resulted in exclusion deficiency . In this paper the relationship between these proteins as well as their cellular location is reported . We found no indications that the 19K protein is a precursor form of the 13K protein . Analysis of gene products of recombinant plasmids carrying exc as well as of several derivatives, however, provided a strong indication that the proteins result from overlapping genes . Besides, evidence was obtained that the 19K protein is essential for exclusion . Localization studies revealed that this protein exists in a membrane-bound form, associated at the periplasmic side of the inner membrane, and in a soluble form residing in the cytoplasm.

Mol Gen Genet, 1985, 200(1), 110 - 3
Nucleotide sequence of the iron regulatory gene fur; Schaffer S et al.; The fur gene of Escherichia coli is involved in all iron-regulated transcriptions hitherto studied . The nucleotide sequence of an 868 basepair fragment containing the fur gene was determined . There was only a single longer reading frame . The amino acid sequence derived from the nucleotide sequence comprised 148 amino acids that together made a polypeptide of 16,795 daltons . The amino acid sequence was confirmed by determination of the amino acid composition, the carboxy-terminal lysine residue and the internal Lys-Lys and Lys-Arg sequences of the isolated Fur protein . The nucleotide sequence contains typical initiation and termination sites for transcription and translation.

Mol Gen Genet, 1985, 199(3), 446 - 51
Cloning of the ARO cluster gene of Neurospora crassa and its expression in Escherichia coli; Catcheside DE et al.; We have constructed a phage, lambda Ncl, which comprises a 4.0 kb HindIII insert of Neurospora DNA into the immunity region of the vector lambda 598 . lambda Ncl complements the aroD6 mutation of E . coli, permitting the formation of galaxy plaques on medium lacking aromatic supplements, and transforms an aro-9 qa-2 Neurospora mutant to prototrophy at a low frequency . Low levels of 5-dehydroquinate hydrolyase (E.C.4.2.1.10.), with properties unlike those of the catabolic isoenzyme that is coded by qa-2, are present in E . coli aroD6 cell lysates following infection with lambda Ncl . lambda Ncl does not hybridize with qa-2 DNA and it is concluded that it contains at least the aro-9 region of the pentafunctional aro cluster gene.

Mol Gen Genet, 1985, 199(3), 388 - 95
Cointegration and resolution mediated by IS101 present in plasmid pSC101; Ishizaki K et al.; A certain class of cointegrate plasmids was found to occur between a pSC101 derivative and a second plasmid pBV320 in E . coli F- cells . Cleavage analysis and DNA sequencing showed that the cointegrate plasmid contained direct repeats of an insertion sequence IS101 at the recombination junctions, indicating that formation of cointegrates was mediated by IS101, which is a natural constituent of pSC101 . These cointegrates were formed only in cells which contained the transposon gamma-delta, suggesting that the gamma-delta sequence, which provides transposase, is responsible for cointegration . Whenever the cointegrate plasmids were present in cells containing gamma-delta or its related transposon Tn3, the cointegrates were dissolved to give pBV320::IS101 due to recombination at duplicated IS101 sequences in the cointegrates, suggesting that both gamma-delta and Tn3, which provide a resolvase, are responsible for the resolution of the cointegrates . Comparison between the nucleotide sequence of IS101 and those of gamma-delta and Tn3 shows a high degree of homology in the regions that have been shown to be the binding sites of resolvases, as well as in the terminal inverted repeats . However, there is no homology between IS101 and the other element, gamma-delta or Tn3, in the internal resolution site, at which the resolution event may occur.

Gene, 1985, 35(1-2), 71 - 82
Organisation and control of the Escherichia coli uvrC gene; Forster JW et al.; The Escherichia coli uvrC gene has been cloned into multicopy plasmids from the transducing phage lambda uvrC+ and the structural gene assigned to a 1.9-kb BglII fragment . Deletion of upstream sequences shows the presence of an in vivo uvrC promoter close to the start of the structural gene, as confirmed by subcloning the uvrC fragment into actively transcribed or 'promoter-free' restriction sites in various plasmid vectors . The control of uvrC transcription has been investigated using hybrid uvrC-cat operons . There are at least two promoters upstream of uvrC . Only the proximal promoter, some two orders of magnitude less effective than the cat promoter, is required for in vivo expression of the uvrC gene . We can find no evidence that expression of the uvrC gene on multicopy plasmids is either autogenously controlled or controlled by the product of the lexA gene.

J Membr Biol, 1985, 85(1), 87 - 92
Functional reconstitution of lens gap junction proteins into proteoliposomes; Nikaido H et al.; Membranes rich in junction complexes were prepared from bovine lens, and the fragments of the membranes were reconstituted into proteoliposomes with a large excess of phosphatidylcholine and dicetylphosphate . The osmotic swelling behavior of these liposomes showed that the lens junction membranes contributed protein components that produced channels with a nominal diameter of 1.4 nm . Most preparations of lens junctions produced rates of osmotic swelling much slower than those found in proteoliposomes containing equivalent amounts of Escherichia coli porin, and we discuss several possible explanations for this observation.

Folia Biol (Praha), 1985, 31(2), 115 - 20
Molecular cloning of integrated proviral DNA of bovine leukaemia virus from virus-producing foetal lamb kidney cells; Nyakatura G et al.; Over 90% of the total viral information together with the adjacent 5' cellular sequences were cloned in the lambdoid spi selection vector lambda-2558 by taking advantage of the unique EcoRI restriction site very close to the 3' long terminal repeat . Of the fifteen isolated recombinant phages with viral inserts, one has been propagated and its DNA isolated and subjected to preliminary restriction endonuclease analysis . The proviral insert has been found to be almost identical to the unintegrated proviral DNA reported by Kashmiri et al . (1984).

Basic Life Sci, 1985, 30, 397 - 414
DNA-protein interaction at the replication origins of plasmid chromosomes; Bastia D et al.; Novel techniques have been developed to purify replication initiator proteins of the plasmids R6K and pSC101 . The techniques consist of tagging the initiator cistrons at the C-terminus with beta-galactosidase-encoding DNA of Escherichia coli in the correct translational phase . The hybrid proteins are then rapidly purified by adsorption to and elution from a beta-galactosidase- specific affinity column . Two procedures have been devised to isolate the nonfused initiator proteins using the fused protein as a handle . The first procedure, called subunit association chromatography, exploits the association of a monomer of nontagged protein with that of beta-galactosidase-tagged protein in isolating both types of proteins by beta-galactosidase specific affinity column chromatography . The second procedure involves the fusion of the initiator protein to beta-galactosidase via a specific linker DNA . The linker DNA encodes a protein which is readily and specifically hydrolyzed by a sequence specific protease, thus releasing the initiator protein from beta-galactosidase . Using purified or partially purified initiator protein, we have demonstrated that the R6K encoded initiator protein (Pi protein) binds to a consensus 22 bp sequence at 2 regions of the plasmid chromosome . The pSC101-encoded initiator protein binds to sequences at or near the plasmid replication origin . At low concentrations the protein binds to a nucleation site and upon raising the concentrations of the protein binding is promoted at 4 adjacent sequences that have partial homologies with the nucleation sequence . Deletion of the binding site leads to a nonfunctional replication origin.

Basic Life Sci, 1985, 30, 335 - 54
Incompatibility and IncFII plasmid replication control; Rownd RH et al.; The DNA coding for replication control and incompatibility of the plasmid NR1 serves as a template in vivo and in vitro for RNA transcription in both directions . In the rightward direction, RNA synthesis begins from 2 different promoters, one of which is regulated and the other constitutive . In vivo, each of these transcripts is more than 1,000 nucleotides long, terminating near the estimated site for the origin of replication . These transcripts serve as messenger RNA for several proteins . One protein (repA1) is required for replication and another (repA2) serves as the repressor for the regulated rightward promoter . RNA synthesis in the leftward direction is constitutive and produces a single transcript of 91 nucleotides which is complementary in sequence to the rightward transcripts . This small transcript is the incompatibility product which regulates the replication of the plasmid . When the intracellular concentration of the small transcript is experimentally varied, the rate of translation of the rightward transcripts and the rate of initiation of replication (plasmid copy number) vary inversely to its concentration . The mode of action of this inhibitor RNA is likely to be formation of an RNA-RNA duplex with the rightward transcripts, thereby inhibiting the translation which would produce the required replication protein . The probability that a rightward transcript will escape interaction with the small RNA molecules and thus allow replication to initiate can be predicted from the laws of mass action based on base-stacking free energies for the likely sequences of initial contact . The estimated intracellular RNA concentrations, based on quantitative hybridization experiments, are agreement with those predicted from the calculated equilibrium constants for duplex formation.

Basic Life Sci, 1985, 30, 261 - 76
Regulation of replication and maintenance functions of broad host-range plasmid RK2; Thomas CM et al.; Replication of broad host-range plasmid RK2 depends on a cisacting vegatative replication origin oriVRK2 and the polypeptide product(s) of the trans-acting gene trfA as well as on host-specified products . The trfA gene is the second cistron in a polycistronic unit whose first cistron may be kilD, one of 4 known RK2-specified kil loci (kilA, B, C, and D) which are inhibitory for bacterial host or plasmid vector in the absence of kor functions which suppress in trans the effect of their respective kil genes . Transcription of the operon containing trfA is negatively regulated by the products of both the trfB locus (alias korD and korA) and korB . The loci, trfB and korB, are expressed from a single transcriptional unit which we propose to be negatively autoregulated by the products of both loci, although an additional, weaker and unregulated transcript may also express korB . While deletions in the oriVRK2 region have indicated the presence of copy number control elements adjacent to and possibly overlapping with the minimal oriVRK2 segment, the overriding control of copy number seems to reside in the trfB and korB loci which in conjunction appear to reduce expression of the trfA gene to levels limiting for replication . Coregulation of trfA with kil genes may indicate that kil genes play a role in plasmid maintenance other than replication.

Basic Life Sci, 1985, 30, 227 - 41
Genetic interactions of broad host-range plasmid RK2: evidence for a complex replication regulon; Figurski DH et al.; The kil and kor genes of RK2 are novel genetic determinants further that the kil and kor network constitutes a replication regulon, and that perhaps the function of this regulon is to ensure expression of trfA at appropriate levels . The complexity of this regulon may reflect an ability of the system to adapt to the intracellular environments of a variety of hosts . Indeed, there is tantalizing evidence that regions encoding kil or kor genes are important to host range (1,2,6,28; Schmidhauser and Helinski, pers . comm.) . We are therefore hopeful that the study of these genes and the eventual determination of the molecular basis of their actions will lead to a complete understanding of the replication control and broad host range capability of IncP plasmids.

Basic Life Sci, 1985, 30, 215 - 26
The partition functions of P1, P7, and F miniplasmids; Austin S et al.; The partition regions of P1, P7, and F miniplasmids are discrete DNA sequences of about 3 kb in length that will promote accurate partition of hybrid plasmids independent of the source of replication functions or the position or orientation of the elements . Each of the par regions seems to be very similarly organized, with open reading frames for essential proteins and a terminal site which appears to be analogous to the centromere of eukaryotic cells . When cloned, these terminal sites exert incompatibility against their respective parent plasmids presumably because they can compete with the parent plasmids as substrates for partition . We have determined the complete DNA sequence of the P1 par region . In addition to the open reading frame for the essential parA protein (42-44 kd), the region contains a second open reading frame which could encode a 38-kd protein . The 2 large open reading frames appear to form an operon that is negatively regulated from a site adjacent to the promoter and responds to the par gene products in trans . Both this site and the downstream "centromere" site, incB, contain blocks of extremely AT-rich sequences, which are postulated to be binding sites for par proteins . The incB and upstream AT-rich regions both contain 20-bp imperfect inverted repeats . Further downstream from the minimal incB sequence (172 bp) lies an additional region which is essential for partition . The further analysis of the P1 par region should be greatly facilitated by the finding that it can function in cis to stabilize pBR322 vectors under conditions where the copy number of pBR322 is reduced.

Basic Life Sci, 1985, 30, 151 - 72
Origin and initiation sites of lambda dv DNA replication in vitro; Tsurimoto T et al.; The sequence of lambda DNA essential for the unique initiation of replication was analyzed in an in vitro replication system . Fragments of lambda DNA were inserted into pBR322 and used as templates or were circularized in vitro in the absence of pBR322 and employed in the same tests . A 165-bp region left of the EcoRI site in the O gene of the lambda genome was defined as the functional origin . This region, which we defined as the ori region, carries, in order, the 4 19-bp repeat sequences where the O protein binds (ori-repeats), an A+T-rich stretch, and a region that constitutes part of a large palindromic structure . Regions that have long been suspected to participate in lambda DNA replication initiation, ice and oop were not required for the O, P-dependent lambda-specific replication initiation . The lambda dv and the "ori region plus" recombinant plasmids initiated DNA synthesis at or around this region, and the reaction depended on the presence of lambda-coded initiators, O and P proteins . Early replicative intermediates of lambda dv were prepared in an in vitro replication system in the presence of ddCTP, an inhibitor of DNA chain elongation . This system allows only that DNA synthesis that is a result of replication initiation . Using this system, the initiation site(s) of the DNA synthesis was finely analyzed by mapping the transition sites from primer RNA to DNA synthesis . Short-chain DNAs produced from regions near the ori region were purified from the intermediates . A fraction of the short-chain DNAs was covalently linked to primer RNA . The 5'-ends that had been linked to RNA (transition sites) were exposed by alkaline hydrolysis, labeled with 32P, and the transition sites were mapped along the nucleotide sequence of the genome . Two striking features emerged from this analysis: (i) The transition sites are located on both sides of the ori region, and no transition arose within the 165-bp ori region; (ii) The transition sites on both sides are not unique, but multiple, and are clustered in one of the 2 strands . Furthermore, their orientation demonstrates that the DNA synthesis in initiation of replication from the 2 sides of the ori region converge . The frequency of the "leftward" DNA synthesis is several times higher than that of "rightward" synthesis . These results reflect asymmetric bidirectional replication of lambda dv DNA, and may also reflect replication of lambda phage DNA.

Basic Life Sci, 1985, 30, 141 - 50
Initiation of replication of the Escherichia coli chromosomal origin reconstituted with purified enzymes; Kaguni JM et al.; A mixture of purified proteins has replaced a crude enzyme fraction capable of efficient replication of oriC-containing plasmids . The reconstituted enzyme system contains proteins which provide initiation, replication, and specificity functions required for dnaA-dependent replication specific for an oriC template . Replication can be separated into successive stages of RNA synthesis and DNA replication . Isolation of an intermediate no longer requiring RNA polymerase action requires the presence of dnaA protein, DNA gyrase, dnaB protein and dnaC protein . Intermediate formation likely involves binding of dnaA protein to a 9-bp sequence present 4 times as inverted repeats within the chromosomal origin sequence.

Prog Clin Biol Res, 1985, 178, 347 - 53
Immune response against the purified serotype specific antigen of bluetongue virus and initial attempts to clone the gene that codes for the synthesis of this protein; Huismans H et al.; Sheep were injected with different amounts of purified protein P2 of bluetongue (BT) virus (BTV) . About 3 X 50 mcg was required for the induction of neutralizing antibodies . Sheep injected with 3 X 10 mcg were, however, still largely protected when challenged with virulent virus . This has suggested the possibility of using P2 as a subunit vaccine and initiated an investigation of the possibility of synthesizing P2 by DNA-recombinant technology . In order to clone the gene that codes for the synthesis of P2 both the "shotgun" approach with unfractionated dsRNA and cloning of isolated segment 2 were investigated . The basic approach was to convert the dsRNA to DNA which was cloned into the Pst 1 site of E . coli plasmid pBR322 . The largest BTV-specific insert that was obtained in the initial experiments was just more than 2,000 base pairs long . The largest insert obtained when isolated segment 2 dsRNA was cloned was about 1,200 base pairs which represents about 1/3 of the P2 gene.

Prog Clin Biol Res, 1985, 177, 7 - 15
Molecular tools for the mapping of the human genome; Lehrach H et al.; The rapid progress in molecular cloning and DNA analysis techniques, together with the use of cloned DNA probes from specific chromosomes of the human genome might allow localisation and ultimately identification of genes defined by single mutations . The discrepancy between genetic dimensions expressed in centimorgans, each corresponding to millions of base pairs and distances easily accessible by molecular techniques amounting to at the most hundreds of kilobase pairs may be bridged with some new cloning techniques partially developed at the European Molecular Biology Laboratory . These techniques were designed to allow rapid cloning and analysis of large regions of mammalian genomes.

Mol Gen Genet, 1985, 198(3), 390 - 2
Transposon mutagenesis and genetic mapping of the rglA and rglB loci of Escherichia coli; Ravi RS et al.; The rglA and rglB genes code for two different proteins which cleave the hydroxymethylated cytosine residues of T-even phages . We isolated Tn10 and Tn5 insertion mutants of the above genes and of the genes in and around the rglA and rglB loci . These insertions were used to construct a detailed genetic map . Our results show that the rglA gene maps at 25.24 min and the rglB gene at 98.39 min on the standard Escherichia coli K12 genetic map.

J Basic Microbiol, 1985, 25(3), 197 - 201
Localization of a streptothricin acetyl transferase in cells of Escherichia coli K-12; Seltmann G et al.; Streptothricin acetyl transferase coded for by plasmids pIE636 and pIE637 in Escherichia coli K-12 was found to be located at the inner side of the cytoplasmic membrane.

Gene, 1985, 34(2-3), 197 - 206
Cointegrates carrying two copies of a Tn3 derivative in an inverted orientation; McCormick M et al.; We constructed a mutant of Tn3, Tn3 #2, that contains a 55-bp direct repeat of sequences near the amino-terminal coding region of the transposase, and an 8-bp EcoRI linker . This mutant transposase is functional . The plasmid carrying Tn3 #2, pMB8::Tn3 #2, recombines with the plasmid pHS1 at a frequency of 2.8 X 10(-7) recombinants per division cycle . This is similar to the recombination frequency of pHS1 and pMB8::Tn3+ (wild-type) which is 4.5 X 10(-6) recombinants per division cycle . One-third of the recombinants between pMB8::Tn3 #2 and pHS1 were approx . 22 kb in length . Restriction analysis and nucleotide sequencing showed that these large plasmids were Tn3 #2-mediated cointegrates formed by integration of pMB8::Tn3 #2 into pHS1 . However, unlike Tn3 tnpR- -mediated cointegrates that contain direct repeats of the incoming element, Tn3 #2-mediated cointegrates carry two copies of Tn3 #2 in the form of inverted repeats . Like the tnpR- repeats, the Tn3 #2 repeats occur at both junctions between the parental plasmids, and are associated with a 5-bp direct duplication of the pHS1 target site . Furthermore, these recombinants contain a small deletion starting precisely at the end of Tn3 #2 and extending into pMB8 sequences . We propose a model for the generation of Tn3 #2-mediated cointegrates.

Gene, 1985, 34(1), 87 - 93
A direct-selection vector derived from pColE3-CA38 and adapted for foreign gene expression; Vernet T et al.; The construction of a plasmid vector, pVT25, which allows an efficient and direct selection for transformed cells carrying recombinant plasmids is described . In this vector, the replicon and ApR gene from plasmid pBR327 are fused to the colE3 gene of pColE3-CA38, whereby positive selection is based on the inactivation of the lethal colicin E3 by the insertion of a foreign DNA fragment . However, pVT25 can be maintained within the Escherichia coli cells when complemented with another plasmid, pVT26, which expresses the colicin E3 immunity (imm) and the TcR phenotypes . Furthermore, pVT25 was used to regulate the expression of the synthetic human proinsulin gene fused to the colE3 gene at the single ClaI site . The production of the characteristic C-peptide of proinsulin, monitored by radioimmunoassay, was shown to be under the control of the inducible promoter of the colE3 gene.

Gene, 1985, 34(1), 17 - 26
IS50-mediated inverse transposition: specificity and precision; Nag DK et al.; The IS50 elements, which are present as inverted repeats in the kanamycin-resistance transposon, Tn5, can move in unison carrying with them any interstitial DNA segment . In consequence, DNA molecules such as a lambda::Tn5 phage genome are composed of two overlapping transposons - the kan segment bracketed by IS50 elements (Tn5), and lambda bracketed by IS50 elements . During direct transposition, mediated by IS50 "O" (outside) ends, the kan gene is moved and the lambda vector is left behind . During inverse transposition, mediated by the "I" (inside) ends of the IS50 elements, the lambda vector segment is moved and the kan gene is left behind . Direct transposition is several orders of magnitude more frequent than inverse transposition (Isberg and Syvanen, 1981; Sasakawa and Berg, 1982) . We assessed the specificity and precision of the rare events mediated by pairs of I ends by mapping and sequencing independent inverse transpositions from a lambda::Tn5 phage into the amp and tet genes of plasmid pBR322 . Using restriction analyses, 32 and 40 distinct sites of insertion were found among 46 and 72 independent inverse transpositions into the amp and tet genes, respectively . Eleven sites were used in two or more insertion events, and the two sites in tet used most frequently corresponded to major hotspots for the insertion of the Tn5 (by direct transposition) . The sequences of 22 sites of inverse transposition (including each of the sites used more than once) were determined, in eleven cases by analyzing both pBR322-IS50 junctions, and in eleven others by sequencing one junction . The sequence of the "I" end of IS50 was preserved and 9-bp target sequence duplications were present in every case analyzed . GC pairs were found at each end of the target sequence duplication in ten of the eleven sites used more than once, and also in seven of the other eleven sites . Our data indicate that transposition mediated by pairs of "I" ends is similar in its specificity and precision to the more frequent transposition mediated by IS50 "O" ends.

Gene, 1985, 33(3), 305 - 11
Cloning of the DNA repair genes mtcA, mtcB, uvsC, uvsD, uvsE and the leuB gene from Deinococcus radiodurans; Al-Bakri GH et al.; A gene library from Deinococcus radiodurans has been constructed in the cosmid pJBFH . A 51.5-kb hybrid cosmid, pUE40, that transduced Escherichia coli HB101 from leucine dependence to independence was selected, and a 6.9-kb fragment which carried the leuB gene from D . radiodurans was subcloned into the EcoRI site of pAT153 . The DNA repair genes mtcA, mtcB, uvsC, uvsD and uvsE, which code for two D . radiodurans UV endonucleases were identified by transforming appropriate repair-deficient mutants of D . radiodurans to repair proficiency with DNA derived from the gene library . Hybrid cosmid pUE50 (37.9 kb) containing an insert carrying both the mtcA and mtcB genes was selected and 5.6- and 2.7-kb DNA fragments carrying mtcA and mtcB, respectively, i.e., the genes that code for UV endonuclease alpha, were subcloned into the EcoRI site of pAT153 . The three genes uvsC, uvsD and uvsE, that code for UV endonuclease beta, were all present in the 46.0-kb hybrid cosmid pUE60 . The uvsE gene in a 12.2-kb fragment was subcloned into the HindIII site of pAT153 and the size of the insert reduced to 6.1 kb by deletion of a 6.7-kb fragment from the hybrid plasmid pUE62 . None of the uvs genes introduced into the UV-sensitive E . coli CSR603 (uvrA-) was able to complement its repair defect . The mtcA, uvsC, uvsD and uvsE genes were found in the 52.5-kb hybrid cosmid pUE70 . It is concluded that the DNA repair genes mtcA, mtcB, uvsC, uvsD and uvsE are located within an 83.0-kb fragment of the D . radiodurans genome.

Dev Biol Stand, 1985, 59, 11 - 22
Plasmid vectors for the regulated, high level expression of eukaryotic genes in Escherichia coli; Amann E; A series of plasmid vectors has been constructed for the regulated, high level expression of foreign genes in E . coli . The vectors express cloned genes under the control of the tac promoter, which is a hybrid of trp and lac promoter sequences . Some of our expression vectors carry in addition to the tac promoter, the efficient lacZ ribosome binding site followed by unique cloning sites . These vectors can be used to express cloned genes directly, i.e . in an unfused, mature form . A second type of vector provides, in addition to the above regulatory elements, a translation initiation codon (ATG) for the expression of genes which have been isolated in an incomplete form (for example: cDNA) . A third type of vector allow readily the construction of gene fusions to the E . coli beta-galactosidase gene, which may stabilize otherwise unstable eukaryotic proteins, and thus allows the production of high amounts of specific antigens in E . coli . With the above vectors, several eukaryotic and viral proteins, including SV40 small tumor antigen, human fibroblast interferon and herpes simplex glycoproteins have been expressed.

Ciba Found Symp, 1985, 112, 94 - 115
STb enterotoxin of Escherichia coli: cyclic nucleotide-independent secretion; Weikel CS et al.; Escherichia coli may produce a heat-labile enterotoxin (LT) or two heat-stable enterotoxins (STa, STb) . Experimentally, STb is consistently active only in 5 h-weaned pig intestinal loops (WPIL), an effect that is largely removable by rinsing . At least three mechanisms initiate small intestinal secretion: cyclic AMP (LT), cyclic GMP (STa) and calcium (A23187) . All three increase short-circuit current (SCC) in Ussing chambers by stimulating net Cl- secretion . STb significantly increases SCC within 2-5 minutes in Ussing chambers and is independent of cyclic AMP and cyclic GMP . When compared to crude culture filtrates of a non-toxigenic strain of E . coli, crude culture filtrates of STb did not alter Na+ or Cl- undirectional or net fluxes . However, the calculated residual ion flux (JRnet) increased significantly in STb-treated tissues and appeared to largely account for the STb-induced increase in SCC . Furosemide applied serosally (10(-3) M), the removal of extracellular calcium, and lanthanum chloride (10(-3) M) did not inhibit the effect of STb on SCC . Chlorpromazine (0.4 mM) completely inhibited STb-induced secretion in porcine loops . This inhibition was a non-specific reversal of the STb effect because in Ussing chambers, chlorpromazine simply induced an equal and opposite effect on SCC . These results indicate that STb initiates intestinal secretion in porcine jejunum in vitro by stimulating primarily non-chloride anion secretion in the absence of extracellular calcium . We postulate that STb causes bicarbonate secretion by a mechanism distinct from those of previously studied enterotoxins.

Ann Inst Pasteur Microbiol, 1985 Jan-Feb, 136A(1), 165 - 71
The role of the FtsZ protein (SfiB) in UV-induced division inhibition and in the normal Escherichia coli cell division cycle; Holland IB et al.; This paper describes some of the major characteristics of the SOS-dependent division arrest which occurs in Escherichia coli during repair of DNA damage following UV irradiation . We shall review the evidence that the inducible division inhibitor, SfiA, interacts directly with an essential division protein, FtsZ . On the basis of its pivotal role in division inhibition during the UV stress response and other properties of ftsZ, we propose that this is a key gene involved in the actual regulation of the division cycle in E . coli . We also propose that at least some components of the division machinery interact to form a specific complex which we designate as a "septalsome" . We shall discuss the possibility that a critical concentration of FtsZ is required to trigger the formation of an active septalsome prior to division . Alternatively, FtsZ might act to inhibit the formation of an active septalsome until a critical point in the cell cycle is reached.

Mol Gen Genet, 1985, 199(1), 117 - 22
Localization of the upstream regulatory sites of yeast iso2-cytochrome c gene; Iborra F et al.; In order to study the regulation of expression of the iso2-cytochrome c gene, we have constructed a fused gene between the 5'flanking region of the gene coding for the yeast iso2-cytochrome c and the coding region of the E . coli beta-galactosidase lacZ gene . When introduced in yeast cells this hybrid gene is expressed and regulated like the production of iso2-cytochrome c: it is under the control of the general catabolic repression and of the unlinked trans-acting CYP1 gene whose CYP1-18 allele causes an overproduction of iso2-cytochrome c . The expression of hybrid genes whose upstream region has been progressively shortened or altered by internal deletions was studied either in wild-type CYP1+ cells or in cells carrying the CYP1-18 allele grown either on glucose or on glycerol . It appears that the expression and the regulation of the iso2-cytochrome c gene is controlled by an upstream regulatory site composed of a positive and a negative element . This site is the target of regulation by the CYP1 gene product and, directly or through this gene, of the control by the general catabolic repression.

Biochimie, 1985 Jan, 67(1), 161 - 75
Specific binding of the cAMP receptor protein of Escherichia coli to the lactose operon promoter; Koop AH et al.; The nitrocellulose filter binding assay has been used to study effects of pH, temperature, ionic strength and magnesium ions on the specific binding of the cyclic adenosine 3',5'-monophosphate (cAMP) receptor protein (CAP) to the promoter of the lactose (lac) operon of Escherichia coli . The pH has a significant effect on binding with the greatest amount of specific binding appearing at pHs near 7 with a gradual decrease in binding as the pH is increased to 8 . Specific binding was observed at temperatures of 22 degrees C and 37 degrees C but not at 4 degrees C . The specific binding was also found to be a function of the concentration of magnesium acetate and potassium chloride, being dependent on the specific cation present, the total ionic strength, and the concentration of the CAP protein . All binding decreases as the ionic strength, increases, but this decrease occurs at a lower ionic strength in magnesium acetate than in potassium chloride . In a double label experiment the filter assay demonstrates that the cAMP-CAP complex preferentially binds to the wild-type lac promoter in the presence of a lac promoter mutated at the CAP binding site . Based on these results and comparisons with other experiments reported in the literature, buffer conditions that approximate the physiological state of a cell appear to be best for studying the interaction between CAP and the lactose promoter in vitro.

Biochimie, 1985 Jan, 67(1), 145 - 8
Indirect effects of the 3'-5' cyclic adenosine monophosphate binding protein (CAP) on the transcription of the malPQ operon in Escherichia coli; Gutierrez C et al.; Uninduced malPQ transcription, as followed by measuring beta-galactosidase expression in a strain carrying a malP-lacZ hybrid gene and grown in the absence of maltose, requires the presence of CAP . However this requirement is lost when the expression of malT, positive regulator gene of the maltose regulon, is rendered independent of CAP by a mutation in the malT promoter . This result suggests that the effect of CAP on uninduced malPQ expression is mediated through a modulation of MalT protein synthesis . The effect of CAP on the induced expression of malPQ is presumably mediated, in addition, through a modulation of the synthesis of the maltose transport system and, hence, of the entry of the inducer . Therefore the effect of CAP on malPQ expression seems to be merely indirect, and this is surprising since a CAP binding site is present at the malPQ promoter.

Biochemistry, 1985 Jan 1, 24(1), 51 - 8
Phosphoenolpyruvate synthetase and pyruvate, orthophosphate dikinase: stereochemical consequences at both the beta-phospho and gamma-phospho groups of ATP; Cook AG et al.; {(R)-16O,17O,18O}Phosphoenolpyruvate and adenosine 5'-O-{(gamma S)-beta gamma-17O,gamma-17O,18O}(3-thiotriphosphate) have been synthesized and used to determine the stereochemical course of the several displacements at phosphorus catalyzed by phosphoenolpyruvate synthetase and by pyruvate, orthophosphate dikinase, two enzymes that catalyze the formation of phosphoenolpyruvate from pyruvate and ATP . The catalytic mechanisms for each of these enzymes are believed to involve both phospho- and pyrophospho-enzyme intermediates . The stereochemical results are entirely in accord with these pathways: the beta-phospho group of ATP suffers overall retention of configuration that is presumably the consequence of two displacements with inversion, and the gamma-phospho group of ATP gamma S suffers inversion of configuration that is most probably the consequence of a single displacement at this center.

Plasmid, 1985 Jan, 13(1), 59 - 69
A two-stage molecular model for control of mini-F replication; Trawick JD et al.; It is known that mini-F replication requires production of a 29,000-Da protein, protein E, and origin of replication sequences mapping around 45 . kb . Further, control of replication is determined by two genes, copA and copB . In the present work a description is given of the cloning of an F restriction fragment containing the amino terminal portion of the protein E gene, repE, and associate promoter activity . It is shown that expression of this promoter is negatively regulated in trans by sequences taken from the F replication region of copA+ plasmids . However, the same sequences taken from six different copA- plasmids failed to repress expression of the promoter . Since prior studies have shown that copA+ determines a repressor of replication, it is now suggested that the above results are an accounting of where this repressor works . A hypothesis is also proposed to explain control of F replication by the copA and copB regulatory genes.

Can J Comp Med, 1985 Jan, 49(1), 1 - 9
A field trial to evaluate the efficacy of a combined rotavirus-coronavirus/Escherichia coli vaccine in dairy cattle; Waltner-Toews D et al.; A field trial was designed to determine the efficacy of a combination rotavirus-coronavirus/Escherichia coli vaccine on dairy farms in southwestern Ontario . In Part A of the trial, 321 cows on 15 farms were randomly assigned to either vaccination or placebo groups . On eight farms, 50% of the dams were vaccinated, while on the other seven farms, 80% of the dams were vaccinated . In Part B of the trial, 26 farms were randomly assigned to either a total vaccination program or to no vaccination program . Mortality, disease occurrence and weight gains were recorded on all calves for the first two weeks of life . In Part A, 23.5% of all calves were treated in the first two weeks of life, 20.9% were treated specifically for scours and 3.6% of live-born calves died . Enteropathogenic E . coli was identified on 13 of the 15 farms, rotavirus on 11 and coronavirus on ten . At least one of the three potential pathogens was found on every farm . There were no significant differences between calves from placebo-treated and vaccine-treated dams with regard to the proportion treated for all diseases, or for scours, or the proportion which died . Neither were there differences in days to first treatment for all diseases (seven days on average), days to first scour (6.7 days), duration of treatments (3.9 days for all diseases, 3.7 days for scours), or estimated weight gains (0.5 kg/day to 14 days) . These results were not altered when the presence or absence of enteropathogenic E . coli, rotavirus or coronavirus on the premises was accounted for.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Biol (Mosk), 1985 Jan-Feb, 19(1), 278 - 84
{A system of EcoRV restriction-modification: genes, enzymes and synthetic substrates}; Kraev AS et al.; The genes, encoding the restriction endonuclease and modification methylase EcoRV have been cloned from the natural plasmid pLG13 into pBR32 derivative vector pIL233 . A resultant clone, expressing both enzyme activities, was used as a source of DNA for sequencing these genes by a procedure, that employed construction of deletion derivatives used to locate borders (by means of a functional test) and to sequence ca . 300 bp near the deletion breakpoint . From the sequence data, we infer that the endonuclease, a 29 KDa protein, and the methylase, a 36 KDa protein, are transcribed from a 310 bp intergenic region in opposite directions . There is no apparent homology between the enzymes and genes of the EcoRI and the EcoRV systems . A synthetic decamer, containing the EcoRV endonuclease recognition sequence and a phosphoamide bond at the cleavage point, is not cleaved by the highly purified endonuclease; the unmodified synthetic decamer is cleaved at the same conditions, only that the cleavage occurs to produce a blunt end--GAT/ATC, and not in a place previously reported (GATAT/C).

Mol Gen Genet, 1985, 198(2), 358 - 9
Additional genes essential for replication of the mini-F plasmid from origin I; Tanimoto K et al.; We isolated a series of Tn5-insertional mutants from the mini-F plasmid, which has a deletion in the origin II region and replicates exclusively from origin I, and found that the mutants that had Tn5 in either the F4 or the F5 gene were defective in their replication . It is concluded that, in addition to the F3 gene on which we have reported previously, both the F4 and the F5 genes are essential for the replication from origin I.

Mol Gen Genet, 1985, 198(2), 323 - 8
Kinetoplast DNA segments function as promoters in Escherichia coli cells; Kuzmin EV et al.; By means of coupled transcription-translation in Escherichia coli cell-free system, an open reading frame was found in the Crithidia oncopelti maxi-circle kDNA segment cloned in the hybrid plasmid pCo1 . Subfragments from this region were tested for their ability to function as promoters in E . coli cells . For this purpose the vector pVE8 was constructed using the aminoglycoside phosphotransferase II (APTII) gene from the Tn5 transposon, and the pHC79 cosmid . After cloning of Sau3A fragments of pCo1 by insertion into pVE8 three types of plasmids containing promoter sequences were obtained . Two of these plasmids displayed promoter strength in E . coli cells greater than that of the normal promoter of the APTII gene . However the promoters found are not necessary for the coupled transcription-translation of kDNA in the E . coli cell-free system.

Mol Gen Genet, 1985, 198(2), 309 - 14
Activation of expression of a cloned archaebacterial gene in Escherichia coli by IS2, IS5, or deletions; Wood AG et al.; A DNA fragment from the methanogenic archaebacterium Methanococcus voltae, when cloned into the PstI site of the plasmid vector pBR322, complements the Escherichia coli argG mutation strongly or weakly depending on its orientation . Faster-growing variants derived from a strain containing the poorly expressed fragment were found to harbor plasmids which had undergone genetic rearrangements . Some of the plasmids were shown to have acquired an insertion element (IS2 or IS5), derived from the E . coli chromosome, close to the region essential for complementing activity . Other plasmids exhibited no homology with E . coli chromosomal DNA . These were found to represent multimeric forms of the parental plasmid in which 2-3 kb of DNA between the tet promoter and the argG-complementing region had been deleted . Growth rates of the variant strains in the absence of arginine varied significantly, suggesting differences in efficiency of activation of the cloned DNA.

Mol Gen Genet, 1985, 198(2), 243 - 54
Cloning and complementation analysis of the "Frizzy" genes of Myxococcus xanthus; Blackhart BD et al.; Fruiting-body formation in Myxococcus xanthus involves the aggregation of cells into raised mounds, where they sporulate . "Frizzy" mutants fail to aggregate into mounds, but rather aggregate into "frizzy" filaments (D.R . Zusman 1982) . The frizzy mutations (frz) were found to be genetically linked . The region of DNA carrying the frz genes was cloned in Escherichia coli by selecting for the kanamycin resistance element present on a transposon Tn5 insertion linked to the frz genes . Phage P1 mediated transduction of the cloned DNA into M . xanthus frizzy mutants showed that the cloned DNA could complement the frz mutations . The cloned DNA was analyzed by isolating and characterizing new Tn5 insertions at short intervals within the M . xanthus DNA and by constructing in vitro deletions . The mutated DNA was then transduced into M . xanthus where the cloned DNA became integrated into the bacterial chromosome as gene replacements or as merodiploids . The gene replacement strains allowed us to define the limits of the frz region, since Tn5 insertions in the frz genes resulted in the frizzy phenotype . The merodiploid strains allowed us to perform complementation analyses . Using appropriate crosses, we were able to identify 5-6 frz complementation groups on 7.5 kb of cloned DNA . One of the complementation groups was separated from the others by 1.4 kb of DNA, whereas the others were contiguous . The different frz loci behave as separate transcriptional groups although interactions between some of the gene products are indicated.

Mol Gen Genet, 1985, 198(2), 221 - 7
Genetic analysis of Tn7 transposition; Ouartsi A et al.; The purpose of this work was to localize the DNA regions necessary for the transposition of Tn7 . Several deletions of Tn7 were constructed by the excision of DNA fragments between restriction sites . The ability of these deleted Tn7s to transpose onto the recipient plasmid RP4 was examined . All the deleted Tn7s isolated in this work had lost their transposing capability . The possibility of complementing them was studied using plasmids containing all or part of Tn7 . Two deleted Tn7s could not be complemented by an entire Tn7 indicating that a DNA sequence greater than the 42 bp terminal sequence is needed for recognition of the transposon by a transposition function . Four other deleted Tn7s could be complemented by Tn7 . One of these was studied intensively in complementation experiments using different parts of Tn7 to obtain transposition . The results obtained allow us to propose that all genes needed for transposition of Tn7 onto plasmids are contained in a DNA segment of between 6.0 and 7.4 kb . Furthermore, one essential function must be contained in a DNA fragment longer than 2.5 kb on the right-hand end of Tn7 . The classification of Tn7 with regard to the other transposable elements is discussed.

Proc Natl Acad Sci U S A, 1985 Jan, 82(2), 474 - 8
Endonuclease III (nth) mutants of Escherichia coli; Cunningham RP et al.; Two strains that overproduce endonuclease III were found in a colony bank containing hybrid ColE1-Escherichia coli plasmids . The enzyme was identified in crude extracts by the degradation of partially depyrimidinated DNA in the presence of EDTA, by its sedimentation velocity, and by its associated thymine glycol-DNA glycosylase activity . An insertion mutation was produced by cloning the kanamycin-resistance gene of Tn5 into the plasmid copy of the nth gene . The mutation was then transferred to the chromosome in the following steps: (i) selection for chromosomal integration of the plasmid at 42 degrees C in a temperature-sensitive polA strain, (ii) curing via temperature shifts, and (iii) phage P1-mediated transduction of a new host . The insertion mutant, as well as a separately isolated deletion mutant, had no measurable glycosylase activity for DNA containing thymine glycol . Although such residues are common lesions in oxidized or irradiated DNA, the mutants were not unusually sensitive to H2O2 or gamma-rays . The insertion mutation had a mutator effect (4- to 22-fold enhancement) on one tested allele.

Proc Natl Acad Sci U S A, 1985 Jan, 82(2), 376 - 80
Ha-ras proteins exhibit GTPase activity: point mutations that activate Ha-ras gene products result in decreased GTPase activity; Manne V et al.; Several ras genes have been expressed at high levels in Escherichia coli and the resultant ras proteins were shown to be functional with respect to their well-known specific, high-affinity, GDP/GTP binding . We were able to detect a weak GTPase activity associated with the purified proteins . The normal cellular ras protein (p21N) exhibits approximately equal to 10 times higher GTPase activity than the "activated" proteins . Even though the turnover rate of the reaction is very low (0.02 mol of GTP hydrolyzed per mol of p21N protein per minute), the reaction appears to be catalytic; one molecule of p21N hydrolyzes more than one molecule of GTP . The GTPase and the GDP binding activities both have been recovered from a Mr 23,000 protein eluted following NaDodSO4/polyacrylamide gel electrophoresis, suggesting that these two activities are associated with the same protein . Mg2+ ions and dithiothreitol are required for GTPase activity and the optimal pH is between 7 and 8 . Guanidine X HCl, which is required for solubilizing bacterially expressed ras protein, is strongly inhibitory to GTPase activity at concentrations higher than 0.5 M.

Proc Natl Acad Sci U S A, 1985 Jan, 82(2), 297 - 301
On the mechanism of renaturation of complementary DNA strands by the recA protein of Escherichia coli; Bryant FR et al.; The renaturation of complementary DNA strands by the recA protein of Escherichia coli has been found to exhibit the following features . (i) Optimal renaturation occurs at recA protein levels below that required to saturate the DNA strands; saturating amounts of recA protein significantly reduce the rate of reaction . (ii) The reaction proceeds in the absence of a nucleotide cofactor but is markedly stimulated by ATP in the presence of 10 mM Mg2+ . A similar stimulation occurs in the absence of ATP when the Mg2+ concentration is increased from 10 mM to 30-40 mM . (iii) Both the ATP-stimulated and the Mg2+-stimulated reactions follow apparent first-order kinetics . These results, taken together with the known effects of ATP and Mg2+ on the state of aggregation of recA protein, suggest that the association of recA monomers may play an important role in recA protein-promoted DNA renaturation.

J Bacteriol, 1985 Jan, 161(1), 60 - 71
hisT is part of a multigene operon in Escherichia coli K-12; Marvel CC et al.; The Escherichia coli K-12 hisT gene has been cloned, and its organization and expression have been analyzed on multicopy plasmids . The hisT gene, which encodes tRNA pseudouridine synthase I (PSUI), was isolated on a Clarke-Carbon plasmid known to contain the purF gene . The presence of the hisT gene on this plasmid was suggested by its ability to restore both production of PSUI enzymatic activity and suppression of amber mutations in a hisT mutant strain . A 2.3-kilobase HindIII-ClaI restriction fragment containing the hisT gene was subcloned into plasmid pBR322, and the resulting plasmid (designated psi 300) was mapped with restriction enzymes . Complementation analysis with different kinds of hisT mutations and tRNA structural analysis confirmed that plasmid psi 300 contained the hisT structural gene . Enzyme assays showed that plasmid psi 300 overproduced PSUI activity by ca . 20-fold compared with the wild-type level . Subclones containing restriction fragments from plasmid psi 300 inserted downstream from the lac promoter established that the hisT gene is oriented from the HindIII site toward the ClaI site . Other subclones and derivatives of plasmid psi 300 containing insertion or deletion mutations were constructed and assayed for production of PSUI activity and production of proteins in minicells . These experiments showed that: (i) the proximal 1.3-kilobase HindIII-BssHII restriction fragment contains a promoter for the hisT gene and encodes a 45,000-dalton polypeptide that is not PSUI; (ii) the distal 1.0-kilobase BssHII-ClaI restriction fragment encodes the 31,000-dalton PSUI polypeptide; (iii) the 45,000-dalton polypeptide is synthesized in an approximately eightfold excess compared with PSUI; and (iv) synthesis of the two polypeptides is coupled, suggesting that the two genes are part of an operon . Insertion of mini-Mu d1 (lac Km) phage into plasmid psi 300 confirmed that the hisT gene is the downstream gene in the operon.

J Bacteriol, 1985 Jan, 161(1), 454 - 7
Regulation of expression of the crp gene of Escherichia coli K-12: in vivo study; Cossart P et al.; Expression of the crp gene was studied in vivo by use of a crp-lacZ gene fusion first constructed on a plasmid and then transferred onto the chromosome . Our in vivo data confirm the in vitro findings that crp is negatively autoregulated via the cyclic AMP-catabolite gene activator protein complex . We present evidence that gene crp is repressed by glucose.

J Bacteriol, 1985 Jan, 161(1), 361 - 7
Isolation and characterization of outer membrane permeability mutants in Escherichia coli K-12; Benson SA et al.; Escherichia coli normally requires the lamB gene for the uptake of maltodextrins . We have identified and characterized three independent mutations that allow E . coli to grow on maltodextrin in the absence of a functional lamB gene by allowing maltodextrins with a molecular weight greater than 1,000 to cross the outer membrane barrier . Two of the mutations map to the structural gene for the outer membrane porin OmpF, and the remaining mutation maps to the structural gene for the second major outer membrane porin, OmpC . These mutations increase the permeability of the outer membrane to small hydrophilic substances, antibiotics, and detergents . These mutations alter the electrophoretic mobility of the respective porin proteins.

J Bacteriol, 1985 Jan, 161(1), 292 - 8
Stable inheritance of plasmid R1 requires two different loci; Gerdes K et al.; The largest EcoRI fragment from plasmid R1 mediates a stability phenotype which is required to ensure the stable inheritance of this low-copy-number plasmid . When covalently linked to small, unstable R1 derivatives, this fragment makes the plasmids as stable as the wild-type R1 plasmid . A genetic analysis showed that two independently acting stabilization functions are encoded by this EcoRI fragment, both of which have the potential of partial stabilization of mini-R1 plasmids . The two loci are located at opposite ends of the fragment . Stabilization was also obtained by inserting these regions in unrelated, unstable plasmids from the p15 group . One of the two functions was very efficient in stabilizing such foreign replicons . Besides the stability phenotype, these genes exert incompatibility in an allele-specific manner . The stability functions do not seem to interfere seriously with the copy number of the plasmid.

J Bacteriol, 1985 Jan, 161(1), 133 - 40
Independence of cyclic AMP and relA gene stimulation of glycogen synthesis in intact Escherichia coli cells; Leckie MP et al.; Previous studies from our laboratory established that in Escherichia coli, glycogen synthesis is regulated by both the relA gene, which mediates the stringent response, and by cyclic AMP . However, those studies raised the question of whether this dual regulatory system functions in an independent or a dependent manner . We show here that this regulation is independent, i.e., each regulatory process can express its action in the absence of the other . Triggering the stringent response by amino acid starvation increased glycogen synthesis even in mutants lacking the ability to synthesize cyclic AMP or lacking cyclic AMP receptor protein; and cyclic AMP addition stimulated glycogen synthesis in relA mutant strains . We also show that physiological concentrations of GTP inhibit ADP-glucose synthetase (glucose-1-phosphate adenylyltransferase, EC 2.7.7.27), the rate-limiting enzyme of bacterial glycogen synthesis, in vitro . Because the stringent response is known to cause an abrupt decrease in the cellular level of GTP, modulation of ADP-glucose synthetase activity by this nucleotide could account for a substantial portion of the step-up in the cellular rate of glycogen synthesis observed when the stringent response is triggered.

J Bacteriol, 1985 Jan, 161(1), 123 - 7
Isolation and characterization of an Escherichia coli mutant lacking the cytochrome o terminal oxidase; Au DC et al.; A respiration-deficient mutant of Escherichia coli has been isolated which is unable to grow aerobically on nonfermentable substrates such as succinate and lactate . Spectroscopic and immunological studies showed that this mutant lacks the cytochrome o terminal oxidase of the high aeration branch of the aerobic electron transport chain . This strain carries a mutation in a gene designated cyo which is cotransducible with the acrA locus . Mutations in cyo were obtained by mutagenizing a strain that was cyd and, thus, was lacking the cytochrome d terminal oxidase . Strain RG99, which carries both the cyd- and cyo- alleles, grows normally under anaerobic conditions in the presence of nitrate . Introduction of the cyd+ allele into the strain restores the respiration function of the strain, indicating that the cytochrome o branch of the respiratory chain is dispensable under normal laboratory growth conditions.

Cell, 1985 Jan, 40(1), 171 - 81
Differential expression of photosynthesis genes in R . capsulata results from segmental differences in stability within the polycistronic rxcA transcript; Belasco JG et al.; We report that the light-harvesting and reaction center genes in the rxcA locus of R . capsulata are contained within a single operon and that their differential expression results predominantly from marked segmental differences in stability within the polycistronic rxcA transcript . The 3' portion of this transcript is rapidly degraded to give rise to either of two slowly decaying mRNA remnants, both of which encode only the light-harvesting polypeptides . The greater stability of these remnants accounts for nearly all of the difference between the concentrations of the light-harvesting and reaction center proteins . The unstable 3' portion of the transcript is delimited by two alternative stem-and-loop structures, which apparently act as barriers to 3' exoribonucleases and thereby protect the upstream RNA segment . When a DNA fragment containing the rxcA locus was fused to a plasmid promoter and transcribed in E . coli, the long precursor transcript was processed to two short messages of greater stability, as in R . capsulata.

Cell, 1985 Jan, 40(1), 147 - 58
Recombination site selection by Tn3 resolvase: topological tests of a tracking mechanism; Benjamin HW et al.; In vitro recombination by Tn3 resolvase of plasmids containing two directly repeated recombination (res) sites generates two singly interlinked catenated rings . This simple product catenane structure was maintained over a wide range of substrate supercoil densities and in a reaction mixture in which phage lambda Int-mediated recombination generated its characteristic multiply interlinked forms . Using substrates containing four res sites, we found that resolvase recombined neighboring res sites with high preference . This position effect implies that resolvase searches systematically along the DNA for a partner site . Intervening res sites in the opposite orientation did not prevent translocation . We analyzed the geometric arrangement of the interlocked rings after multiple recombination events in a four-site substrate and the pattern of segregation of nonspecific reporter rings catenated to the standard substrate . The results of these novel topological tests imply that the translocating enzyme may not make continuous contact with the DNA.

Cell, 1985 Jan, 40(1), 129 - 37
Transcriptional block caused by a negative supercoiling induced structural change in an alternating CG sequence; Peck LJ et al.; Using supercoiled plasmids containing a (CG)16 sequence downstream of a promoter, it is shown that purified E . coli RNA polymerase can transcribe through the sequence when it is in the B helical form . However, the polymerase together with its nascent transcript is blocked at the boundary of the CG sequence proximal to the promoter when the template is negatively supercoiled to flip the CG sequence to the left-handed Z-form . S1 nuclease mapping of in vivo transcripts from an E . coli gyrase temperature-sensitive mutant harboring the plasmids indicates that the bulk of the transcripts at either permissive or nonpermissive temperatures can proceed through the CG sequence, suggesting that the sequence is normally in the B helical form in vivo . The almost total blockage of transcription in vitro by the (CG)16 sequence in a highly negatively supercoiled DNA is not observed for a d(CA)21 X d(TG)21 insert.

Biull Eksp Biol Med, 1985 Jan, 99(1), 45 - 8
{Effect of corticotropin on the dehydrogenase activity of cells and tissues}; Korkach VI et al.; The authors studied the effect of native ACTH on dehydrogenase activity of isolated strips of rat diaphragm and suspension of E . coli cells, serotype O III:B4, grown on beef extract agar in a medium with different dehydrogenation substrates . ACTH activated dehydrogenase of rat diaphragm in a medium containing pyruvate, alpha-ketoglutarate, malate, beta-hydroxybutyrate, D-aspartic acid and did not alter it in a medium containing succinate . In contradistinction to rat diaphragm, ACTH activated dehydrogenase of E . coli cells whatever the substrates used (oxaloacetate, isocitrate, alpha-ketoglutarate, succinate, fumarate, malate, pyruvate, lactate, beta-hydroxybutyrate, glucose, D-aspartic acid . Synacthen (ACTH1-24) exerted a similar effect . It is suggested that the effects of ACTH are mediated via its influence on adenylate cyclase in the absence of receptors.

J Virol, 1985 Jan, 53(1), 19 - 24
Isolation and structural mapping of a human c-src gene homologous to the transforming gene (v-src) of Rous sarcoma virus; Gibbs CP et al.; We have utilized a lambda Charon 4A human genomic library to isolate recombinant clones harboring a highly conserved c-src locus containing nucleotide sequences homologous to the transforming gene of Rous sarcoma virus (v-src) . Four overlapping clones spanning 24 kilobases of cellular DNA were analyzed by restriction endonuclease mapping . Human c-src sequences homologous to the entire v-src region are present in a 20-kilobase region that contains 11 exons as determined by restriction mapping studies utilizing hybridization to labeled DNA probes representing various subregions of the v-src gene and by preliminary DNA sequencing analyses . A considerable degree of similarity exists between the organization of the human c-src gene and that of the corresponding chicken c-src gene with respect to exon size and number . However, the human c-src locus is larger than the corresponding chicken c-src locus, because many human c-src introns are larger than those of chicken c-src . alu family repetitive sequences are present within several human c-src introns . This locus represents a highly conserved human c-src locus that is detectable in human cellular DNAs from various sources including placenta, HeLa cells, and WI-38 cells.

Infect Immun, 1985 Jan, 47(1), 5 - 10
Construction of a conjugative plasmid with potential use in vaccines against heat-labile enterotoxin; Chen TM et al.; A conjugative plasmid with potential usefulness for vaccine strains was constructed . In the first step, a 5.9-kilobase DNA segment containing the two loci for the A and B subunits of heat-labile enterotoxin with a mutation in the gene for the A subunit was joined to the cloning vehicle pGA22, generating the nonconjugative plasmid pPMC4 with genes for resistance to tetracycline and chloramphenicol . In the second step, a segment of pPMC4 containing the genes for the A and B subunits, the gene for chloramphenicol resistance, and the replication genes of pGA22 was ligated to the genes for conjugal transfer of the F plasmid, generating the 54.9-kb plasmid pPMC5 . Eleven porcine Escherichia coli isolates were tested as recipients for pPMC4 and pPMC5 . For pPMC4, transformation and mobilization with a conjugative R plasmid were used to effect plasmid transfer . Only 1 of the 11 strains acted as a recipient in transformation . Mobilization with the R plasmid occurred with two strains, but the plasmids were altered during transfer . In contrast, pPMC5 was transferred with high frequency and unaltered to 9 of the 11 E . coli strains . Transconjugants from these nine matings produced high titers of the B subunit and no active heat-labile enterotoxin . Plasmid pPMC5 was stable in three porcine E . coli strains tested; plasmid pPMC4 was somewhat less stable in these strains . The method we describe for the construction of conjugative chimeric plasmids offers an opportunity for introducing genes with potential for immunization into bacterial strains that are suitable for colonizing the appropriate host sites.

Infect Immun, 1985 Jan, 47(1), 157 - 65
Cosmid cloning of Rickettsia prowazekii antigens in Escherichia coli K-12; Krause DC et al.; Rickettsia prowazekii DNA was partially digested with Sau3A or HindIII, ligated with the cosmid vector pHC79, packaged in vitro, and transduced into Escherichia coli HB101 . Cosmid cloning of Sau3A-digested rickettsial DNA yielded 1,288 ampicillin-resistant colonies; 798 cosmid clones resulted with HindIII-digested rickettsial DNA . Chimeric cosmid DNA was extracted from the latter gene bank, digested to completion with HindIII, and compared by agarose gel electrophoresis with a HindIII digest of rickettsial genomic DNA . The two digestion profiles were quite similar in their overall banding patterns, indicating that the clone bank was significantly representative of the rickettsial genome . When both clone banks were screened for expression of rickettsial antigens by enzyme-linked immunosorbent assay with goat anti-R . prowazekii serum, ca . 20% of the clones reacted positively . Two clones were randomly selected for more detailed analysis . Each contained a large chimeric plasmid (40.2 and 38.1 kilobases) which apparently yielded smaller deletion derivatives (13.6 and 12.6 kilobases) when transformed into an E . coli minicell strain . Each recombinant plasmid directed the synthesis of new protein species not observed in control minicells . One of the clones produced a 51,000-dalton protein in minicells, which comigrated with a protein reactive with anti-R . prowazekii serum . This protein was not present in negative controls . When antibodies to this protein were incubated with a Western blot of rickettsial total protein, they bound to a 52,000-dalton polypeptide . Hence, the cloned rickettsial gene product in E . coli corresponds to a protein of similar size in R . prowazekii . This study demonstrates the feasibility of cosmid cloning of rickettsial antigens in E . coli.

FEBS Lett, 1985 Jan 1, 179(1), 17 - 20
Inhibition of the activity of restriction endonucleases by spermidine and spermine; Kuosmanen M et al.; Physiological concentrations (0.5-2.0 mM) of spermidine and spermine were observed to inhibit the digestion in vitro of plasmid pJDB 207 by the restriction endonucleases BamHI (EC 3.1.23.6), EcoRI (EC 3.2.23.13), HindIII (EC 3.1.23.20), HpaI (EC 3.1.23.23) and PstI (EC 3.1.23.31) . The polyamines protected all the tested restriction sequences of DNA, since the activity of all endonucleases used was strongly inhibited . These results show the need for caution when using polyamines as experimental tools for recombinant DNA chemistry.

Curr Genet, 1985, 10(4), 313 - 20
Genetic analysis of transformation in a microconidiating strain of Neurospora crassa; Rossier C et al.; We have characterized Neurospora crassa transformants obtained with plasmid pDV1001 bearing the cloned catabolic dehydroquinase (qa-2+) gene (Hughes et al . 1983a) and fluffy 268 host strain producing only uninucleate microconidia allowing to isolate individual transformation products . The percentage of transformed nuclei in the mycelium and their stability were determined by genetic analysis of microconidia produced on selective or non-selective medium . About half of the transformants originating from mycelial spheroplasts were apparently homokaryotic . Catabolic dehydroquinase activity was in agreement with the proportion of transformed nuclei . The DNAs from four transformants analyzed by Southern hybridization showed restriction fragments expected for integration of pDV1001 into genomic DNA by non-homologous recombination . No plasmids could be rescued from the undigested DNAs of the transformants by transformation of E . coli . One transformant, fl268-6, was unstable and generated a high proportion of segregants . Plasmid pDV1001 sequences were absent in their DNA . Colonies originating from microconidia of strain fl268-6 on selective plates often lost the transformed character . These results suggest that instability in this transformant is due to the loss of integrated plasmid sequences during vegetative growth.

Curr Genet, 1985, 9(5), 383 - 8
Plasmid recovery from transformants and the isolation of chromosomal DNA segments improving plasmid replication in Neurospora crassa; Paietta J et al.; The efficient recovery of plasmid DNA from Neurospora crassa transformants is described . Lithium acetate-treated spores were transformed with plasmid DNA and grown in mass in liquid culture . The resulting mycelial growth was harvested and plasmid DNA was extracted and used to transform E . coli to ampicillin resistance . Although at low frequency, routine recovery of plasmid pSD3 which carries the Neurospora qa-2+ gene and pBR322 sequences has been demonstrated . About 10% of the recovered plasmids carried deletions and transformed Neurospora at a higher frequency . The liquid culture procedure was also used in attempts to isolate autonomously replicating sequences (ars) . In order to select for a stable vector which contains an ars sequence, a clone bank containing a selectable marker (qa-2+) and Neurospora chromosomal BamHI fragments was constructed and used to transform Neurospora . Several plasmids isolates resulting from a screening of the clone bank showed an improvement in the efficiency of recovery from Neurospora transformants . The properties of one such isolated plasmid, pJP102, suggest that it may contain an ars sequence . Some potential applications of these results for cloning in Neurospora and other filamentous fungi are discussed.

Microbios, 1985, 44(179-180), 169 - 84
Nicotinic acid transport in Escherichia coli; Rowe JJ et al.; The uptake of nicotinic acid by Escherichia coli is dependent on the presence of the enzyme nicotinic acid phosphoribosyl transferase and a source of energy . Glucose concentrations between 0.1 and 0.5%, a temperature of 46 degrees C and an external concentration of 2.5 X 10(-5) were optimal conditions for nicotinic acid uptake . Saturation kinetics occur with a Km of 1.75 microM and a Vmax of 0.116 nmoles/min/mg dry weight . The intracellular molarity of the accumulated pyridine compounds is 44-fold that of the initial concentration . Inhibitors of respiration and anaerobiosis do not significantly inhibit uptake rate . However, an inhibitor of glycolysis, uncouplers of ATP production and sodium arsenate reduce vitamin transport . A mutant defective in ATPase does not accumulate exogenously supplied nicotinic acid when lactate is used as an energy source, although L-proline, the transport of which is independent of ATP production, is accumulated.

Gene, 1985, 39(2-3), 231 - 8
Isolation and characterization of the Aspergillus niger trpC gene; Kos A et al.; The Aspergillus niger trpC gene was isolated by complementation experiments with an Escherichia coli trpC mutant . Plasmid DNA containing the A . niger trpC gene transforms an Aspergillus nidulans mutant strain, defective in all three enzymatic activities of the trpC gene, to Trp+, indicating the presence of a complete and functional trpC gene . Southern blot analysis of DNA from these Trp+ transformants showed that plasmid DNA was present but that this DNA was not integrated at the site of the chromosomal trpC locus . The A . niger trpC gene was localized on the cloned fragment by heterologous hybridization experiments and sequence analysis . These experiments suggest that the organization of the A . niger trpC gene is identical to that of the analogous A . nidulans trpC and the Neurospora crassa trp-1 genes.

Allerg Immunol (Leipz), 1985, 31(4), 245 - 57
{Determination of interleukin 1}; Friemel H et al.; Interleukin 1 is an essential factor of macrophage dependent T cell activation . As a method for determining IL 1 the estimation of its comitogenic activity is used with mouse thymocytes . For preparation IL 1 standards the macrophage cell line P388D1 or human leukocytes are stimulated with LPS . The mitogenic activity of the thymocytes is tested in five different mouse strains; LPS does not disturb the IL 1 determination in the concentration range between 10(2) micrograms ml-1 and 10(-3) micrograms ml-1 . Contrary to the Con A reactivity the susceptibility of the thymocytes on IL 1 is developing more slowly . The age of the animals for preparation thymocytes should not be under 8 to 10 weeks . IL 1 and IL 2 inhibitors must be considered in determining IL 1.

Mol Gen Genet, 1985, 201(2), 158 - 60
Effect of an umuC mutation on phage lambda induction; Jenek J et al.; A possible role of the umuC gene product in the induction of the SOS responses was examined . We compared the expression of a genetic fusion, in which gene lacZ, encoding beta-galactosidase in Escherichia coli is under the direct control of the cI repressor from prophage lambda, in a umuC+ strain and in an otherwise isogenic umuC- mutant . We found that two times higher UV doses were required to obtain a similar induction in the umuC+ strain as in the umuC mutant . In addition we showed that, at the same UV dose after a lag period, the specific activity of beta-galactosidase increased more rapidly in the umuC mutant . We suggest that the wild-type umuC gene product participates in the processing of the SOS inducing structures caused by UV irradiation or prevents formation of some of them . This is compatible with a role of the UmuC protein in DNA synthesis past a replication block.

Chromosoma, 1985, 93(2), 140 - 51
General recombination mechanisms in extracts of meiotic cells; Hotta Y et al.; RecA-like proteins have been purified from somatic and meiotic cells of mouse and lily . The rec proteins have been designated "s-rec" and "m-rec" to indicate their respective tissues of origin . The two proteins differ in molecular weight and in their response to temperature, the latter being consistent with the optimal temperature for physiological function of their tissues of origin . There is a major increase in m-rec protein with the entry of cells into meiosis, the peak of activity being early pachytene . Extracts of the cells and also those of yeast (Saccharomyces cerevisiae) have been prepared that have the capacity to catalyze homologous recombination . These extracts behave similarly to the m-rec proteins upon entry of cells into meiosis . Yeast transferred to sporulation medium displays a 100-fold increase in the recombination activity of the extract at about the time of entry into meiosis . The occurrence of peak levels of m-rec and recombination activity in extracts from cells in early pachytene points strongly to that stage as the time at which the enzymatic phase of recombination occurs.

Gene, 1985, 38(1-3), 165 - 75
Improved in vitro packaging of coliphage lambda DNA: a one-strain system free from endogenous phage; Rosenberg SM et al.; In previous systems for in vitro packaging of lambda DNA, phages are produced from the packaging components as well as from added DNA . We have developed a new genetic strategy for in vitro packaging that bypasses this endogenous phage problem . Our system employs a single bacterial strain whose lambda prophage codes for all of the packaging proteins but is deleted for cos, the packaging origin . Crude extracts of the single lysogen: (i) are virtually free from endogenous phages, (ii) package added lambda DNA efficiently and (iii) are easy to prepare . Using the cos- in vitro packaging system we show that packaging of lambda linear monomers is a second-order reaction, but that packaging from concatemers prepared by annealing or ligation is first order . We conclude that in our cos- system, linear monomers are a poor substrate for in vitro packaging but that packaging from concatemers works well.

J Interferon Res, 1985 Summer, 5(3), 471 - 6
Binding of unglycosylated and glycosylated human recombinant interferon-gamma to cellular receptors; Littman SJ et al.; Recombinant human interferons (IFNs), either unglycosylated produced in E . coli (rIFN-gamma) or glycosylated produced in CHO cells (g-rIFN-gamma), were labeled with 125I to similar specific activities to study their interaction with cell-surface receptors . When analyzed by gel electrophoresis, rIFN-gamma run as a single polypeptide of Mr 15,000-17,000, whereas g-rIFN-gamma separated into three components of Mr 20,000, 22,000, and 43,000, which corresponded to the known size of the two monomeric and one dimeric forms of glycosylated natural IFN-gamma . The binding of the two species of 125I-IFN-gamma was competed equally by rIFN-gamma in competition displacement experiments with Daudi cells, indicating that these IFNs bind with similar high affinity to the same receptors . KD values of 1.25 X 10(-10) and 2.5 X 10(-10) M were determined for g-rIFN-gamma and rIFN-gamma, respectively . This relatively small difference in KD does not apparently result in a detectable difference in biological activity, as measured by the increase in 2',5'-oligo(A) synthetase activity in IFN-treated HeLa and A549 cells . These results indicate that glycosylation of IFN-gamma does not play a significant role in its interaction with cellular receptors and in the induction of a biological response.

Circ Shock, 1985, 16(1), 19 - 28
Hemodynamic evaluation of endotoxic shock in anesthetized piglets: antagonism of endogenous vasoactive substances; Schrauwen E et al.; In anesthetized piglets the influence of an LD100 of Escherichia coli endotoxin (0.5 mg/kg IV, bolus injection) on several hemodynamic parameters and on survival time was studied . Endotoxin provoked a pronounced decrease in arterial pressure and cardiac output and an increase in portal venous and pulmonary arterial pressure and heart rate . Total peripheral and mesenteric vascular resistances displayed an initial increase followed by a sustained decrease, whereas pulmonary vascular resistance revealed a pronounced biphasic increase . All pigs died within 210 min following endotoxin administration . Pretreatment with the 5-HT2-antagonists R 41468 and R 50970 and with the prostanoid-synthesis inhibitor flurbiprofen induced a significant attenuation of the increase in pulmonary vascular resistance and beneficial effects on survival . Best survival results were obtained with prednisolone sodium succinate, although these results can only partly be ascribed to beneficial hemodynamic effects . The experiments with the opiate antagonists, however, point to detrimental effects.

Gene, 1985, 35(3), 333 - 42
Differential expression of influenza N protein and neuraminidase antigenic determinants in Escherichia coli; Jones IM et al.; Two influenza gene products of similar size and codon usage have been expressed in Escherichia coli under control of the phage lambda pR promoter . The influenza N protein (NP) was expressed in its entirety after fusion to a short (12 amino acid) segment of the lambda cro gene product and constituted about 1-2% of total soluble cell protein after induction . By contrast, constructions using the full length neuraminidase (NA) gene failed to give rise to detectable amounts of NA antigen after fusion to either the 12 amino acid Cro peptide or after fusion to bacterial beta-galactosidase (beta gal) . Rather, expression of NA antigenic determinants was only achieved after deletion of coding sequences at the 3' end of the beta gal-NA fusion construct such that the encoded protein precipitated within the cell.

Curr Genet, 1985, 9(6), 479 - 93
A fine restriction map of the linear mitochondrial DNA of Tetrahymena pyriformis: genome size, map locations of rRNA and tRNA genes, terminal inversion repeat, and restriction site polymorphism; Suyama Y et al.; A fine restriction map of the linear mitochondrial DNA of Tetrahymena pyriformis strain ST is presented . 1 . Based on agarose gel electrophoresis data together with limited nucleotide sequences available on some restriction fragments, we estimate the actual size of this genome to be about 55,000 base pairs . 2 . Seven tRNA gene locations have been assigned, which are scattered along the genome length . Six of these locations encode the genes for tRNA(phe), tRNA(his), tRNA(trp), and tRNA(glu), and the duplicate tRNA(tyr) genes which are located at the inverted terminal repeat segments . The tRNA gene(s) encoded in one location has not been identified . We have not yet found the tRNA(leu) and tRNA(met) genes, which were previously shown to be encoded in the genome (Chiu et al . 1974; Suyama 1982) . 3 . We have mapped the 14S rRNA gene by sequencing the 170 bp segment of EcoRI fragment 8 and by aligning its sequence with E . coli 16S rRNA . From our recent complete sequence data the gene size was found to be about 1,650 bp, which is unexpectedly large for the 14S rRNA which has an estimated size of 1,300 bp . The 14S rRNA is probably a cleavage product of the larger primary transcript of which 200-300 bases of the 5' end are missing . 4 . The duplicate copies of the 21S rRNA gene at the terminal duplication inversion segments were analyzed . ClaI fragment 7 (1,500 bp) corresponds in sequence from base position 850 to 2,390 of the 20S rRNA gene of Paramecium mitochondrial DNA (Seilhamer et al . 1984b) . The 21S gene is approximately 2,500 bp long . The presence of some restriction site polymorphism is apparent in this segment . 5 . Each of the 21S gene copies precedes the tRNA(tyr) gene, but the space flanking one tRNA(tyr) gene differs in size and restriction sites from the space flanking another tRNA(tyr) gene . Thus, this space corresponds to the segment of an imperfect match in the terminal duplication inversion of Goldbach et al . (1978a) . 6 . Saccharomyces cerevisiae mitochondrial probes including Cob, ATPase VI and IX, and cytochrome oxidase I gene sequences, 21S and 15S rRNAs, and mouse mitochondrial DNA showed no significant hybridization with any restriction fragments of Tetrahymena mitochondrial DNA . The results are in accordance with an extensive sequence divergence previously found in the Tetrahymena mitochondrial genome (Goldbach et al . 1977).

Trans R Soc Trop Med Hyg, 1985, 79(5), 569 - 76
Receptors and recognition mechanisms in intestinal infection; Farthing MJ; Commensal and pathogenic micro-organisms of the alimentary tract exhibit outstanding specificity with respect to host species, phenotype and age, and often to the type and location of epithelial cells within the gut . Specificity in attachment to the gut is achieved most commonly by ligand-receptor interactions at the pathogen-epithelial interface, commonly a bacterial lectin interacting with specific saccharides of the intestinal microvillus membrane . These bacterial lectins are usually associated with specialized attachment organelles known as pili or fimbriae . A variety of different bacterial pili have now been characterized with respect to their sugar specificities . Similar molecular events are important in the production of diarrhoea, notably the interaction of enterotoxins (cholera toxin, Escherichia coli toxins) with glycolipid receptors on the intestinal cell surface . In most instances, toxin-gut interactions exhibit less impressive specificity than is evident for attachment of micro-organisms to the intestinal epithelium . Identification and characterization of these processes has, and will continue to have, important implications for the biological control of enteric infection, particularly with respect to vaccine development.

Curr Top Cell Regul, 1985, 27, 221 - 32
The role of adenylyltransferase and uridylyltransferase in the regulation of glutamine synthetase in Escherichia coli; Rhee SG et al.; The regulation of GS activity involves two nucleotidylation cycles, the uridylylation cycle of PII and the adenylylation cycle of GS, which are catalyzed by two converter enzymes, uridylyltransferase and adenylyltransferase, respectively . The converter enzymes sense the fluctuation in the availability of nitrogen and accordingly regulate the activity of GS . On the other hand, the posttranslational modification of GS is tightly coupled to the transcriptional regulation of the glnA gene by unmodified PII protein acting as a repressor in the GS synthesis . Therefore, metabolic signals perceived by uridylyltransferase are transmitted through PII to two different levels of the regulation, namely, the posttranslational level and transcriptional level . In order to study the converter enzymes which exist in extremely low concentration, the glnD and glnE genes were cloned into a plasmid vector carrying the strong, regulatable lambda phage promoter . In this way, uridylyltransferase and adenylyltransferase were overproduced to the levels approaching 800- and 500-fold, respectively . The recombinant DNA technology also enabled us to examine the transcriptional regulation of the glnD and glnE genes . The expression of these genes was slightly repressed under nitrogen-excess conditions and the repressions were more pronounced under excess nitrogen plus carbon-limiting conditions . It was found that variations of the concentration of uridylyltransferase and adenylyltransferase also affect the rate of GS synthesis . Studies with strains harboring a multicopy plasmid, pglnD or pglnE, indicate that the elevated synthesis of the converter enzymes causes the enhancement of GS synthesis . In addition, the absence of one of the converter enzymes reduces the expression of the glnA gene . The parallel relationship between the converter enzymes and GS seems to derive from the binding capacity of the converter enzymes for the unbound PII, which is a repressor for the glnA gene . Therefore, it is believed that the metabolic regulation of the glnD and glnE genes is ultimately linked to the expression of the glnA operon.

Biol Cell, 1985, 54(3), 217 - 26
Subunit arrangement of Escherichia coli F1-ATPase studied by scanning transmission electron microscopy; Curgy JJ et al.; The shape and the arrangement of subunits in Escherichia coli F1-ATPase (ECF1) lacking the delta subunit have been explored with a high performance scanning transmission electron microscope . In tilting experiments, the ECF1 molecule appeared as a flat cylinder whose width (approx . 120 A) was about twice its height . The symmetry of front view projections of ECF1 has been investigated by computer analysis . In a population taken at random from the data bank, one third of the particles showed five-fold radial symmetry components, one third six-fold radial symmetry components and the last third no typical symmetry . The six-fold radial symmetry was consistent with a hexagonal arrangement of six large peripheric masses, which probably correspond to the three alpha and the three beta subunits of ECF1 . The five-fold radial symmetry was tentatively explained by a fusion of two juxtaposed peripheric subunits . Lateral projections showed a zig-zag organization of the large masses, suggesting that the large alpha and beta subunits are located on two levels, with some degree of intercalation between the subunits of the two levels.

Curr Top Cell Regul, 1985, 26, 207 - 19
Biophysical studies of Escherichia coli glutamine synthetase; Villafranca JJ et al.; The purpose of the EPR and NMR studies presented in this article was to determine the spatial relationship between the n1 and n2 metal ion sites of E . coli glutamine synthetase . Table I presents the distances between these two metal ion sites in various complexes using Mn(II) bound to each site . These studies also employed the transition-state analog, methionine sulfoximine, as an active-site probe as well as various nucleotide complexes . Two primary conclusions result from these data . The Mn(II)-Mn(II) distance changes from approximately 10 to approximately 8 A when nucleotides bind to the enzyme presumably as the result of a protein conformational change . Two Mn(II) ions can bind to the enzyme in the presence of the substitution-inert Co(III)-ATP complex, implying that the metal ion {Co(III)} coordinated to the beta-gamma phosphoryl groups in the complex is displaced from the normal n2 metal ion site . A model showing the probable spatial relationships among components of the active site is shown in Fig . 6 . This model comprises our current working hypothesis of the active site of glutamine synthetase . Further studies of distance relationships are presently underway in our laboratory and will be placed in the context of this model and the known kinetic mechanism.

Infection, 1985, 13 Suppl 2, S202 - 5
P-fimbriae studies on the diagnosis and prevention of acute pyelonephritis; Kallenius G et al.; Theoretically there are several ways to prevent pyelonephritis and renal scarring caused by P-fimbriated Escherichia coli . Screening for individuals at risk, e.g . those carrying P-fimbriated pyelonephritogenic E . coli or those with high receptor density on their uroepithelial cells, could perhaps define a population where prophylaxis with a receptor analogue or vaccination with P-fimbriae may be relevant . Epidemiological measures in neonatal and maternity wards may prevent the nosocomial spread of virulent bacteria and reduce the number of colonized infants . However, no such methods have so far had any proven clinical relevance, and today, the important concern is still to try by conventional means-as we have always done-to get an early diagnosis and to treat the patient without delay.

Gene, 1985, 37(1-3), 91 - 9
Nucleotide sequence of the glnA-glnL intercistronic region of Escherichia coli; Rocha M et al.; The nucleotide (nt) sequence of a 682-bp fragment containing the 3' end of the glnA gene, the region between the glnA and glnL genes, and the 5' end of the glnL gene from Escherichia coli was determined . This segment contains the region coding for the last 107 amino acids (aa) of glutamine synthetase, including the adenylylation site of this enzyme . The analysis of this sequence revealed two REP sequences, a Rho-independent terminator, the putative glnL promoter and the possible binding site for the glnG product, NRI.

Prog Clin Biol Res, 1985, 180, 317 - 28
Covalent modification of proteins by mixed-function oxidation: recognition by intracellular proteases; Rivett AJ et al.; Mixed-function oxidation of E . coli glutamine synthetase is a site-specific reaction involving covalent modification of specific amino acid residues . It causes loss of a specific histidine residue which is thought to be at one of the metal binding sites . The modified enzyme is catalytically inactive . Oxidative modification causes enhanced susceptibility to proteolytic attack and several different types of proteases recognize the oxidatively modified enzyme . This specific covalent modification increases the rate of degradation of glutamine synthetase to about the same extent as major structural modifications such as relaxation, subunit dissociation and denaturation . Moreover, the oxidative modification is one that is likely to occur in vivo . Adenylylation, which causes reversible inactivation of glutamine synthetase, has no effect on rate of proteolysis . We propose that the degradation of E . coli glutamine synthetase occurs by a two-step process . Control of the degradation is likely to be at the first step, which is inactivation by mixed-function oxidation of the enzyme . Metabolic control of the degradation process and the link with the nutritional state of the cell could be achieved by substrate protection against oxidative modification . At present, the apparent energy requirement of the degradation process is unexplained . Although most of our studies have involved E . coli glutamine synthetase, there is evidence that oxidative modification may be a general mechanism by which proteins are marked for degradation . Many enzymes are inactivated by oxidative modification which can be catalyzed by a variety of mixed-function oxidase systems . Several different types of intracellular proteases in E . coli and mammalian cells preferentially degrade the oxidized form of glutamine synthetase . Oxidative modification of proteins can occur in vivo and may be involved in intracellular protein turnover . It has also been implicated in host defense mechanisms and in aging.

Mol Gen Genet, 1985, 200(2), 322 - 7
Colonization factor antigen II (CFA/II) of enterotoxigenic Escherichia coli: molecular cloning of the CS3 determinant; Manning PA et al.; The genes for the cell surface associated antigen CS3, produced by CFA/II type enterotoxigenic Escherichia coli, have been cloned in the plasmid vector pBR322 to produce a family of recombinant plasmids . These plasmids contain a series of HindIII fragments of which a fragment of 4.6 kb is common to all those expressing CS3 . One of these plasmids, pPM474, has been subjected to mutagenesis with Tn1725 and deletions generated using Bal31 . This has defined a minimum region of 3.75 kb necessary for the production of CS3 on the cell surface and implying genetic complexity as has been observed with other fimbrial antigens . Analysis of the plasmid encoded proteins in E . coli K-12 minicells has confirmed this complexity.

Mol Gen Genet, 1985, 200(1), 60 - 4
Complementation and regulatory interaction between two cloned fimbrial gene clusters of Escherichia coli strain KS71; Rhen M et al.; Complementation experiments with cloned DNA fragments encoding either the KS71A, the KS71B or the KS71C fimbriae of the pyelonephritogenic Escherichia coli strain KS71 were used to localise the P-fimbrillin genes and to demonstrate regulatory interactions between the cloned genes . The structural genes of the KS71A and KS71B fimbriae were located within a common 1.1 kilobase pair ClaI-SmaI fragment, and it was shown that the gene clusters for these fimbriae could complement each other in trans . The gene cluster encoding the KS71C fimbriae did not complement for the other KS71 fimbriae . A DNA fragment, located near the KS71A fimbrillin gene, was found to enhance the production of the KS71B fimbriae in trans.

Mol Gen Genet, 1985, 199(3), 410 - 4
The fim genes responsible for synthesis of type 1 fimbriae in Escherichia coli, cloning and genetic organization; Klemm P et al.; The genes responsible for the expression of type 1 fimbriae, produced by the majority of E . coli strains, have been cloned from an E . coli K12 strain . The "passenger" DNA from an initial cosmid clone was reduced in size and subcloned in pACYC184 and pBR322 vectors . A DNA fragment of around 8 kbp was found to be required for the biosynthesis of type 1 fimbriae . This was further studied by transposon-mediated insertional inactivation and by BAL31-mediated deletions . Four genes, designated fimA, B, C, and D were found to be involved in the synthesis of the fimbriae . They encoded proteins that in their processed form appeared with apparent molecular weights of 16.5 kd, 23 kd, 26 kd, and 89 kd, the 16.6 kd polypeptide being the fimbrial subunit . The order to the genes was found to be: fimB, fimA, fimC, and fimD, organized in three transcriptional units.

Gene, 1985, 35(1-2), 45 - 54
Vectors for the direct selection of cDNA clones corresponding to mammalian cell mRNA of low abundance; Kowalski J et al.; We have constructed two cDNA cloning vectors which (1) carry the intergenic region of phage f1 and (2) permit efficient cloning (by the Okayama-Berg procedure) of full-length copies of mammalian mRNA in either orientation . Infection of cells harboring these vectors with f1 phage results in the encapsidation of single-stranded (ss) plasmid DNA carrying either the sense or the anti-sense sequence of the cDNA inserts . The complementary nature of the cDNA inserts in two such cDNA libraries facilitates preparative hybridization procedures . These vectors have general applicability to any eukaryotic system where changes in the abundance of mRNA transcripts are to be measured and the corresponding cDNA clones isolated.

Basic Life Sci, 1985, 30, 535 - 53
Genes and gene products involved in the synthesis of F-pili; Laine S et al.; Membrane fractions containing {35-S}methionine labeled proteins synthesized by Flac and Flac tra mutant strains or by lambda tra transducing phages expressed in such strains have been analyzed in order to investigate the pathway for synthesis of the F-pilin subunit and the gene products involved in synthesis of F-pili . Our data indicate that the synthesis of a mature F-pilin subunit requires the expression of at least 2 tra operon genes in addition to the structural gene for F-pilin, traA . In the absence of these activities, traA expression results primarily in the synthesis of a polypeptide, Ap14, with an apparent molecular weight of approximately 14,000 . We assume this polypeptide corresponds to the direct product of the traA gene . In the presence of traQ activity, the major detectable product of traA is a polypeptide, Ap7(Q), which migrates with an apparent molecular weight of 7,000, suggesting that traQ product may process or assist in the processing of Ap14 . Polypeptide Ap7(Q) is not, however, mature F-pilin, since it reacts poorly with anti-F-pilus-serum . Synthesis of a polypeptide which appears to be antigenically equivalent to F-pilin and which we assume requires a modification of the F-pilin N-terminus, is detected as synthesis of a polypeptide, Ap7* . This protein migrates slightly more slowly than Ap7(Q) on our polyacrylamide gels . Polypeptide Ap7*, can be efficiently precipitated with F-pilus antiserum, and can be detected in both inner membrane and outer membrane fractions under conditions where assembly of F-pili can occur . These data suggest that Ap7* is the mature F-pilin subunit and is assembled from an inner membrane pool . Synthesis of Ap7* appears to require traG activity, but may also be dependent upon additional tra activities.

Ann Rech Vet, 1985, 16(1), 41 - 6
{Anti-K99 vaccination and colostric protection of calves infected experimentally with Escherichia coli K99}; Contrepois M et al.; Colostral antibodies of cows vaccinated with E . coli B41 (O101: K99, F41) protect completely B41 experimentally infected calves . In order to know more precisely the role of K99, F41 antibodies in protection, calves receiving the colostrum of B41 vaccinated cows are infected with E . coli B44 (O9: K30: H-K99, F41) . Vaccination multiplies K99 antibodies in colostrum by seven . In B44 infected calves, specific K99, F41 antibodies protect only 3 out of 6 calves completely . Additive effect of antibodies against K99 and other surface antigens of enterotoxigenic E . coli seems desirable for a more complete protection.

Gene, 1985, 34(2-3), 187 - 96
Type 1C fimbriae of a uropathogenic Escherichia coli strain: cloning and characterization of the genes involved in the expression of the 1C antigen and nucleotide sequence of the subunit gene; van Die I et al.; The genes responsible for expression of type 1C fimbriae have been cloned from the uropathogenic Escherichia coli strain AD110 in the plasmid vector pACYC184 . Analysis of deletion mutants from these plasmids showed that a 7-kb DNA fragment was required for biosynthesis of 1C fimbriae . Further analysis of this DNA fragment showed that four genes are present encoding proteins of 16, 18.5, 21 and 89 kDal . A DNA fragment encoding the 16-kDal fimbrial subunit has been cloned . The nucleotide sequence of the structural gene and of the C- and N-terminal flanking regions was determined . The structural gene codes for a polypeptide of 181 amino acids, including a 24-residue N-terminal signal sequence . The nucleotide sequence and the deduced amino acid sequence of the 1C subunit gene were compared with the sequences of the fimA gene, encoding the type 1 fimbrial subunit of E . coli K-12 . The data show absolute homology at the N- and C-termini; there is less, but significant homology in the region between the N- and C-termini . Comparison of the amino acid compositions of the 1C and FimA subunit proteins with those of the F72 and PapA proteins (subunits for P-fimbriae) revealed that homology between these two sets of fimbrial subunits is also maximal at the N- and C-termini.

Gene, 1985, 33(3), 323 - 31
Cloning of aroG, the gene coding for phospho-2-keto-3-deoxy-heptonate aldolase(phe), in Escherichia coli K-12, and subcloning of the aroG promoter and operator in a promoter-detecting plasmid; Davies WD et al.; Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase{phe}), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG . A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG . The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis . A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector . Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.

J Biochem (Tokyo), 1985 Jan, 97(1), 205 - 11
Proton translocation coupled to nitrite reduction in anaerobically grown Escherichia coli; Sawada MT et al.; Proton translocation coupled to the reduction of nitrite was studied in anaerobically grown Escherichia coli . Extrusion of protons occurred by adding nitrite to an anaerobic suspension of wild-type cells . This extrusion was sensitive to a proton conductor, 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone . Dicyclohexylcarbodiimide, an inhibitor of H+-ATPase, prevented the proton extrusion linked to nitrite reduction, whereas this reagent had no effect on respiratory nitrate reduction to nitrite . Proton extrusion was undetectable when nitrite was added to a suspension of mutant cells defective in H+-ATPase . These results indicate that the proton extrusion associated with nitrite reduction to ammonia is not by redox pumps but by H+-ATPase . From the results obtained by the measurement of proton extrusion in nitrite reductase-deficient mutants, NADH-nitrite reductase system is suggested to involve the proton extrusion in whole cells of E . coli.

J Biochem (Tokyo), 1985 Jan, 97(1), 153 - 9
Cloning and expression of a novel variant of human interferon-gamma cDNA; Nishi T et al.; A cDNA library was prepared from the poly(A) mRNA isolated from human peripheral blood lymphocytes which were induced by combined treatment with phytohemagglutinin and a phorbol ester . Recombinant plasmids containing human interferon-gamma (HuIFN-gamma) cDNAs were identified by the oligonucleotide-hybridization method . Nucleotide sequence analysis showed that the nucleotide and amino-acid sequences of HuIFN-gamma cDNA in plasmid pIFN gamma-G4 differed from the published data at amino acid position 9 (CAA for glutamine versus AAA for lysine) . The cDNA in plasmid pIFN gamma-G4 was expressed under control of the simian virus 40 early promoter in monkey COS cells and a biologically active HuIFN-gamma was secreted from the cells . The cDNA was also inserted into an expression vector carrying an E . coli tryptophan promoter and was expressed in E . coli . The results suggest that the conversion from lysine to glutamine at amino acid position 9 might not affect the specific activity of HuIFN-gamma.

Comp Biochem Physiol A, 1985, 81(1), 165 - 9
Arylsulphatase A and acid phosphatase activities in plasma and leucocytes during LPS fever in the ox (Bos taurus); Kozak W et al.; Intravenous injection of E . coli LPS (0.5 micrograms/kg) produced the biphasic elevation of rectal temperature (TR) in conscious oxen . The fever was accompanied by a significant increase of the arylsulphatase A (AsA) activities in plasma and in mononuclear leucocytes . In polymorphonuclear cells a substantial decrease of the AsA activity after 1 hr fever was observed . After 3.5 hr of fever the polymorphonuclear activity of AsA restored to normal found before LPS administration . In contrast with AsA, the pyrogenic dose of LPS caused negligible changes of the acid phosphatase (AcP) activities in the sampled materials . Daily-repeated injections of pyrogen into the same oxen attenuated magnitudes of fever as well as AsA responses in plasma and granulocytes . Heat-induced hyperthermia provoked only minute changes of the AsA and AcP.

Virchows Arch B Cell Pathol Incl Mol Pathol, 1985, 48(3), 237 - 45
Localization of intravenously injected lipopolysaccharide (LPS) in the spleen of the mouse . An immunoperoxidase and histochemical study; Groeneveld PH et al.; In earlier studies we investigated the in vivo effects of lipopolysaccharide (LPS) on lymphoid and non-lymphoid cells in the mouse spleen . In order to find out whether LPS localizes in and/or on cells that are affected by this compound, the aim of the present study was to investigate the localization of intravenously injected LPS in the mouse spleen using an immunoperoxidase technique . At different time points after injection, the localization of LPS is demonstrated and LPS-containing cells are characterized . Most of the injected LPS has been taken up by marginal zone macrophages at 2 h after its administration whereas macrophages in the red pulp and at the periphery of the white pulp (marginal metallophils) have ingested less LPS . In the periarteriolar lymphocyte sheath, LPS is concentrated in a large number of acid phosphatase-negative, Ia-positive, large branched cells which were suggested to represent interdigitating cells . Moreover an extracellular dendritic localization pattern of LPS is demonstrated in the corona and central parts of the follicles at different time intervals after its injection . The significance of the localization pattern of LPS in the mouse spleen is discussed.

Plasmid, 1985 Jan, 13(1), 8 - 16
Expression of cloned plasmid regions encoding colonization factor antigen I (CFA/I) in Escherichia coli; Willshaw GA et al.; Molecular cloning from a plasmid encoding colonization factor antigen I (CFA/I) and heat-stable enterotoxin isolated two regions, 1 and 2, that are required for the production of CFA/I fimbriae . The level of CFA/I synthesis measured by ELISA was similar in an Escherichia coli K12 strain carrying regions 1 and 2 cloned separately on compatible plasmid vectors to that in the same strain containing the parental plasmid . The structural gene for the CFA/I fimbrial subunit was within region 1 . This region directed production in E . coli minicells of at least six independent polypeptides, of which the fimbrial subunit and at least three others appeared to be synthesized as precursor molecules that underwent processing . Cloned DNA containing CFA/I region 2 specified three polypeptides in minicells . Attempts to reduce the size of the cloned region 1 resulted in a derivative plasmid that carried the CFA/I structural gene but did not complement a region-2 recombinant plasmid to restore production of CFA/I fimbriae.

Comp Biochem Physiol C, 1985, 80(1), 99 - 104
The influence of hormones and inflammatory agents on C-reactive protein, cortisol and alanine aminotransferase in the plaice (Pleuronectes platessa L.); White A et al.; Endotoxin stimulates production of both C-reactive protein (CRP) and cortisol in the plaice within 24 hr . Cortisol alone (optimum dose i.p . 500 micrograms/300 g wt fish) also stimulates CRP production and the possibility that endotoxin acts through cortisol was examined . Dexamethasone suppresses cortisol production but elevates CRP . Cortisol levels are restored to normal within 24 hr of endotoxin injection . Turpentine and ACTH which stimulate cortisol do not affect CRP . Endotoxin and cortisol have no significant effect on alanine aminotransferase activity in the serum and liver although it is elevated in the serum within 24 hr of the administration of adrenalin or turpentine.

Comp Biochem Physiol C, 1985, 80(1), 161 - 5
The action of alpha-ketoglutarate dehydrogenase on 4-chloronitrosobenzene: evidence for species-dependent differences in active site properties; Doerge DR et al.; The reaction of bovine (Bos taurus) and porcine (Sus scrufa) cardiac alpha-ketoglutarate dehydrogenase complex (alpha-KGD) with 4-chloronitrosobenzene (I) was shown to produce a hydroxamic acid (IV) and a product due to a Bamberger rearrangement as previously shown for Escherichia coli alpha-KGD . The conversion of I into an active site-bound electrophile was general among the three alpha-KGD enzymes tested, but quantitative differences in products and kinetics were shown . The reaction of I was specific for the resolved alpha-ketoglutarate decarboxylase subunit.

Am J Vet Res, 1985 Jan, 46(1), 270 - 5
Expression of type 1 pili by Escherichia coli strains of high and low virulence in the intestinal tract of gnotobiotic turkeys; Dominick MA et al.; Highly virulent (strain 1) and weakly virulent (strain 3) Escherichia coli were examined using immunofluorescent and electron microscopic techniques to determine their ability to express type 1 pili in the intestinal tract of 3-week-old gnotobiotic turkeys . Turkeys were necropsied on postinoculation day (PID) 1, 2, 5, 8, and 12 . Nonpiliated forms of strains 1 and 3 were more numerous than piliated forms in cecal and colonic contents examined by negative staining electron microscopy . A piliated form of strain 1 was seen in intestinal contents on each PID and was more numerous in cecal contents than in colonic contents . The mucus blanket of the cecum and colon contained large numbers of bacteria, although organisms were rarely intimately associated with the intestinal epithelium . Immunofluorescent staining indicated large numbers of piliated forms of strains 1 and 3 within the mucus blanket of the cecum and colon on PID 2, 5, 8, and 12 . Piliated bacteria were infrequently seen in the ileal mucus blanket . Serum antibody titers to type 1 pili increased markedly by PID 5 and persisted in turkeys inoculated with strain 1 . In contrast, antibody titers in turkeys exposed to strain 3 increased gradually and varied markedly among birds at each PID . Type 1 pili may not be important for adherence of pathogenic E coli to intestinal epithelium of turkeys.

J Bacteriol, 1985 Jan, 161(1), 402 - 10
Conjugal transfer system of the IncN plasmid pKM101; Winans SC et al.; The conjugal transfer system of the broad-host range IncN plasmid pKM101 was analyzed genetically . Its organization differed significantly from that of the F plasmid . The tra genes are located in three regions, each between 3 and 4 kilobases in length . All of the genes in the first two regions are required for sensitivity to "donor-specific" phage which bind to the plasmid-mediated sex pilus, and these genes therefore are involved in the synthesis, and possibly retraction, of the sex pilus . The plasmid's origin of transfer was localized to a 1.2-kilobase region at an extreme end of the transfer region . Using two different methods, we have identified 11 complementation groups required for transfer . One of these, traC, is of special interest in that mutations at this locus can be partially suppressed if, prior to mating, cells carrying a traC mutant plasmid are incubated with cells which elaborate sex pili but are unable to transfer their plasmids . One possible explanation for this is that pilus-elaborating cells can donate traC gene product to a traC mutant in a form that can be reused.

Infect Immun, 1985 Jan, 47(1), 98 - 105
Purification and characterization of a receptor for the 987P pilus of Escherichia coli; Dean EA et al.; A receptor-containing fraction that caused visible aggregation of piliated Escherichia coli strain 987 bacteria was released into solution when brush borders from adult rabbit small intestines were stored . The receptor-containing fraction was separated into more than 50 bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 4 of which contained carbohydrate as demonstrated by staining with periodic acid-Schiff . Receptor activity in the receptor-containing fraction was associated with a low-molecular-weight (less than 14,000) glycoprotein as measured by Western blots . Extraction of brush borders with chloroform-methanol-water (4:8:3) resulted in increased pilus 987P receptor activity in the aqueous extract . Gel filtration chromatography of sodium dodecyl sulfate-solubilized aqueous extract yielded a fraction with 987P receptor activity that contained only the low-molecular-weight glycoprotein . The purified 987P receptor contained 81% carbohydrate and 19% amino acids by weight . Isoelectric focusing separated the 987P receptor receptor into 3 peaks corresponding to isoelectric points of 2.2, 3.8, and 4.1 . Each of the peaks contained 987P receptor activity . The 987P receptor activity was sensitive to periodate oxidation and to digestion with pronase . The strain 987-agglutinating activity of the receptor was inhibited by amino sugars and their N-acetylated derivatives, by compounds having a free amino group, by high concentrations of salt, and by lectins that recognize D-galactose or L-fucose.

Curr Genet, 1985, 9(7), 567 - 72
Yeast/herpes simplex virus thymidine kinase gene fusions yield fusion proteins with thymidine kinase activity; McNeil JB et al.; Expression in Saccharomyces cerevisiae of the Herpes Simplex Virus type-1 (HSV-1) thymidine kinase gene was accomplished by the construction of a gene fusion between the TK and a yeast gene . The fusion of yeast DNA sequences (which include a promoter and DNA that codes for the amino terminus end of the yeast gene product) with the TK gene resulted in a protein fusion with thymidine kinase activity.

Acta Physiol Hung, 1985, 65(1), 37 - 46
Preparation and characterization of myosin from cultured cells of Escherichia coli (02:K1 30156 strain); Fazekas S et al.; In this study the myosin preparations isolated from E . Coli cell cultures were analysed . The isolation of myosin from fresh cultures resulted in a substantially higher (approximately 10-fold) yield than from stored cells . The coli myosin, despite the two DEAE-cellulose treatments, contained more RNA and P-lipid than the myosin prepared from skeletal muscle . The RNA content can be removed gradually by acetone denaturation and lipid removal followed by subsequent washings . The fresh preparations contained protein-bound alkali-stable P; part of this was released by Cu ions . The coli myosin can be phosphorylated . The phosphate uptake depended on the concentrations of ATP and Mg2+ and on the time of incubation . The alkaline hydrolysate of the phosphorylated, washed lipid-free preparation was resolved into 8 P-containing peaks on ion-exchange chromatography . Of fractions P-Arg, Pi, P-Lys and 2 P-His conformer were identified by means of synthetic compounds, elution pattern and specific reactions . The remaining compounds could not be identified . The most abundant component was P-Arg suggesting that this compound might play an important role in the cytokinetic movements of E . coli.

Anat Rec, 1985 Jan, 211(1), 34 - 42
Alterations of immunoreactive somatostatin in thyroid C cells after induced hypercalcemia, hypocalcemia, and antithyroid drug treatment; Kameda Y et al.; In order to elucidate the functional significance of somatostatin in thyroid C cells, the alterations of immunoreactive somatostatin in the cells were investigated under various experimental conditions, i.e., hypercalcemia, hypocalcemia, and antithyroid drug treatment . Guinea pigs and rabbits, in which almost all C cells reveal the intense immunoreaction for somatostatin in addition to calcitonin, were used as experimental animals . After chronically induced hypercalcemia, somatostatin immunoreactivity conspicuously diminished coinciding with the decrease of calcitonin; somatostatin as well as calcitonin was responsive to induced hypercalcemia . After hypocalcemic tetany induced by injection of Escherichia coli L-asparaginase, C cells exhibited very intense immunoreactions for both calcitonin and somatostatin . After chronic treatment of ethylenethiourea, immunoreaction of somatostatin in C cells was the same as that of calcitonin . That is, when immunoreactivity for calcitonin remained unchanged, immunoreactivity for somatostatin was also intensive . However, when immunoreaction of calcitonin became very weak, the reaction of somatostatin was also weak . Thus, in all experimental conditions examined the alterations of immunoreactive somatostatin in C cells completely coincided with those of calcitonin . It seems likely that somatostatin in thyroid C cells exerts the synergistic effect on calcitonin action.

Mol Biol (Mosk), 1985 Jan-Feb, 19(1), 36 - 54
{The tRNA genes of pro- and eukaryotes . Organization, structure, transcription}; Venkstern TV; The up-to-date data on cytoplasmic tRNA genes, their number and structure, as well as organization in the genome and transcription are reviewed.

Mol Gen Genet, 1985, 198(2), 299 - 303
Intragenic suppression of the temperature-sensitivity caused by a mutation in a gene controlling transcription (fit) in Escherichia coli; Dass SB et al.; Starting from a transcription-defective strain harbouring a temperature-sensitive mutation in the fit gene, a rifampicin-resistant, temperature-insensitive derivative has been isolated . Genetic analysis of this derivative demonstrated the presence of a second temperature-sensitive mutation in the same gene . The two mutations mutually suppress each other's phenotype . From cotransduction experiments, the fit gene has been mapped 0.32 min and 0.16 min clockwise from the aroD and pps loci, respectively, at 37.5 min on the linkage map . The mutants harbouring either or both of the fit mutations are defective in RNA synthesis at the non-permissive temperature . The fit gene product is suggested to function as an accessory transcription factor.

J Dairy Sci, 1985 Jan, 68(1), 229 - 56
Enterotoxigenic Escherichia coli infections in newborn calves: a review; Acres SD; Diarrhea caused by enterotoxigenic Escherichia coli is an infectious bacterial disease of calves that occurs during the first few days of life . The Escherichia coli that cause the disease possess special attributes of virulence that allow them to colonize the small intestine and produce an enterotoxin that causes hypersecretion of fluid into the intestinal lumen . These enterotoxigenic Escherichia coli are shed into the environment by infected animals in the herd and are ingested by newborn calves soon after birth . There is some natural immunity to enterotoxigenic Escherichia coli; however, it often fails to protect calves born and raised under modern husbandry conditions . Hence, methods have been developed to stimulate protective immunity by vaccination of the dam . The protective antibodies are transferred passively to calves through the colostrum.

J Bacteriol, 1985 Jan, 161(1), 446 - 9
Cloning, sequence analysis, and expression of alteration of the mRNA stability gene (ams+) of Escherichia coli; Chanda PK et al.; The ams+ gene, which influences the stability of mRNA in Escherichia coli was cloned in pBR322 . The product of the gene, which is a 17,000-dalton protein, was expressed in expression vector pRC23, a derivative of pBR322 . The molecular weight is consistent with sequencing analysis which shows that the gene contains 595 nucleotides and has an open reading frame of 149 amino acids . We discussed the possible role(s) of the ams+ gene product in affecting mRNA stability.

J Bacteriol, 1985 Jan, 161(1), 212 - 8
Two control systems modulate the level of glutaminyl-tRNA synthetase in Escherichia coli; Cheung AY et al.; We studied the regulation of in vivo expression of Escherichia coli glutaminyl-tRNA synthetase at the transcriptional and translational level by analysis of glnS mRNA and glutaminyl-tRNA synthetase levels under a variety of growth conditions . In addition, strains carrying fusions of the beta-galactosidase structural gene and the glnS promoter were constructed and subsequently used for glnS regulatory studies . The level of glutaminyl-tRNA synthetase increases with the increasing growth rate, with a concomitant though much larger increase in glnS mRNA levels . Thus, transcriptional control appears to mediate metabolic regulation . It is known that glnR5, a regulatory mutation unlinked to glnS, causes overproduction of glutaminyl-tRNA synthetase . Here we showed that the glnR5 product enhances transcription of glnS 10- to 15-fold . The glnR5 mutation does not affect metabolic control . Thus, glnS appears to be regulated by two different control systems affecting transcription . Furthermore, our results suggest post-transcriptional regulation of glutaminyl-tRNA synthetase.

J Bacteriol, 1985 Jan, 161(1), 183 - 8
In vitro RNA polymerase interaction with a restriction fragment containing the Escherichia coli origin of replication; Greene RS et al.; The interaction of RNA polymerase with a restriction fragment containing the origin of Escherichia coli replication (oriC) was examined by methods used to investigate transcription promoter activities . Interactions of RNA polymerase with oriC were determined and characterized by agarose gel exclusion under conditions of polymerase binding and RNA synthesis initiation . These interactions were further demonstrated and defined by nitrocellulose retention experiments under various reaction conditions . The binding of RNA polymerase to the oriC fragment was compared to binding to the tetracycline promoter (tet), a known strong promoter of transcription . Specific localization of the RNA polymerase-oriC interaction was determined by restriction protection experiments . The binding of RNA polymerase was determined to be located near the HindIII site of oriC . These methods allowed the observation and characterization of a specific association of RNA polymerase with the origin of E . coli DNA replication.

Hybridoma, 1985 Spring, 4(1), 47 - 53
Establishment of hybridomas secreting monoclonal antibodies against C epsilon 2 and C epsilon 4 domains of human IgE; Ichimori Y et al.; Three kinds of hybridomas secreting monoclonal antibodies (MAbs) against the epsilon chain of human IgE were constructed by somatic cell hybridization between mouse myeloma P3U1 cells and spleen cells from BALB/c mice immunized with human IgE purified from the culture supernatant of U-266 cells . These MAbs were used effectively for the purification and determination of human IgE . The recognition site in the IgE molecule of each antibody was examined by using various epsilon chain fragment peptides produced in Escherichia coli . From these experiments, it was suggested that one recognized C epsilon 2 and the second C epsilon 4 . The third did not recognize the C epsilon 1-C epsilon 4 domains of the recombinant epsilon chain from E . coli, although it bound to the epsilon chain of natural human IgE.

Cancer Res, 1985 Jan, 45(1), 217 - 20
Prevention of methotrexate cytotoxicity by asparaginase inhibition of methotrexate polyglutamate formation; Jolivet J et al.; Escherichia coli asparaginase (Asnase) pretreatment of Asnase-sensitive L5178Y cells in vitro is thought to antagonize methotrexate (MTX) cytotoxicity through nonspecific inhibition of protein synthesis and MTX uptake . We have reexamined the mechanism of this interaction in view of recent data demonstrating the importance of MTX metabolism to polyglutamate derivatives (MTXPGs) in the cytotoxic effects of the antifolate . After a 3-hr exposure to 0.5 microM MTX, 67% of intracellular drug was in the form of MTXPGs containing a total of 2 to 5 glutamyl residues (MTX-Glu2-5), and cloning efficiency in drug-free medium was only 7% of untreated control . After a 3-hr pretreatment with E . coli Asnase (0.1 unit/ml), {3H}thymidine incorporation dropped by 29%, MTXPG formation during subsequent MTX exposure decreased by more than one-half (MTX-Glu2 unchanged; MTX-Glu3 and 4 decreased to 51.7 and 18.5% of levels achieved in cells not pretreated with Asnase; no MTX-Glu5 formed), and cloning efficiency increased to 71% of untreated control . This effect was not due to decreased MTX uptake into L5178Y cells or to decreased intracellular free L-glutamate or L-glutamine levels . A 3-hr exposure of L5178Y cells to media lacking L-isoleucine, an essential amino acid for cell growth, prior to MTX exposure inhibited {3H}thymidine incorporation by 37%, decreased subsequent MTXPG formation by 62%, and increased subsequent cloning in drug-free medium to control levels . Decreased MTXPG formation was responsible for the prevention of MTX cytotoxicity seen after both pretreatments . Unmetabolized MTX rapidly left L5178Y cells after removal of extracellular MTX . Consequently, lower levels of unbound intracellular drug, a prerequisite of drug activity, were maintained in pretreated than in control cells after passage in drug-free medium . Asnase pretreatment protects L5178Y cells from the cytotoxic effects of MTX, possibly through inhibition of cell growth which nonspecifically decreases MTXPG formation.

Orig Life Evol Biosph, 1985, 16(2), 97 - 116
RNA catalysis and the origin of life; Pace NR et al.; Until the discovery of catalytic RNAs, first the self-splicing intron in Tetrahymena and then the bacterial RNAse P, cellular enzymes had always seemed to be protein in nature . The recognition that RNA can catalytically make and break phosphodiester bonds simplifies some of the assumptions required of a rudimentary self-replicating entity . Available information on the chemistry of RNA-catalyzed reactions is reviewed, with particular attention to self-splicing introns and tRNA processing by RNase P . An explicit model for a self-replicating RNA is described . The model postulates a nucleotide binding/polymerization site in the RNA, and takes advantage of intrinsic fluidity in RNA higher order structure to dissociate parent and progeny complementary strands.

Dev Biol Stand, 1985, 62, 81 - 9
Potential vaccine antigens of the asexual blood-stages of Plasmodium falciparum; Anders RF et al.; We have constructed a cDNA clone library that contains many natural immunogens of the asexual blood-stages of Plasmodium falciparum . The corresponding parasite antigens have been identified with antisera raised against the antigens expressed in Escherichia coli or with monospecific human antibodies purified on adsorbents prepared from various clones . Sequencing studies on the clones have revealed that many malaria antigens contain extensive sequence repeats . These repeats encode antigenic epitopes that in several proteins have been shown to be immunodominant . One candidate vaccine molecule, the Ring-infected Erythrocyte Surface Antigen (RESA), which is transferred from inside merozoites to the erythrocyte surface at about the time of merozoite invasion, contains two blocks of antigenically cross-reactive repeats . The structures of other antigens containing extensive repeats are described and their possible significance to the host-parasite relationship is discussed.

Gene, 1985, 39(2-3), 239 - 45
Multiple-copy genes: production and modification of monomeric peptides from large multimeric fusion proteins; Kempe T et al.; A vector system has been designed for obtaining high yields of polypeptides synthesized in Escherichia coli . Multiple copies of a synthetic gene encoding the neuropeptide substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) have been linked and fused to the lacZ gene . Each copy of the SP gene was flanked by codons for methionine to create sites for cleavage by cyanogen bromide (CNBr) . The isolated multimeric SP fusion protein was converted to monomers of SP analog, each containing a carboxyl-terminal homoserine lactone (Hse-lactone) residue (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Hse-lactone), upon treatment with CNBr in formic acid . The Hse-lactone moiety was subjected to chemical modifications to produce an SP Hse amide . This method permits synthesis of peptide amide analogs and other peptide derivatives by combining recombinant DNA techniques and chemical methods.

Comp Immunol Microbiol Infect Dis, 1985, 8(3-4), 267 - 72
Intestinal and serum antibody response in gnotobiotic piglets to oral immunization with Escherichia coli; Dziaba KA et al.; The local and systemic immune response to a formolized E . coli oral vaccine was investigated in 13 gnotobiotic piglets . Beginning at ten days of age animals received a daily dose of 10(10) or 10(11) bacteria, on ten consecutive days . Intestinal loop tests with one animal of each group on day 26 showed protection which was more pronounced in the animal dosed 10(10) bacteria compared with the other immunized piglet . Immunoglobulin class-specific antibodies to O and K antigens were determined by ELISA technique . In serum no IgG or IgA antibodies were found, whereas IgM-anti O149 antibodies in both immunized groups reached their highest level at day 4 of dosing and decreased thereafter . IgM-anti K88 antibodies were first detected at day 10 of dosing . Both immunized groups had comparable serum levels at days 20 and 30 . Also in gut secretion the IgM antibody response was predominant, and higher levels were found in the 10(10) group than in the 10(11) group . IgG and IgA antibody response were also detected in secretion.

Acta Biochim Pol, 1985, 32(3), 187 - 97
Phosphotyrosine as a substrate of acid and alkaline phosphatases; Apostol I et al.; A new spectrophotometric method for following dephosphorylation of phosphotyrosine has been described . The absorption spectra of phosphotyrosine and tyrosine were plotted over the pH range from 3 to 9 . The change in absorbance accompanying the conversion of phosphotyrosine to tyrosine was the greatest at 286 nm . The difference absorption coefficients were calculated for several pH values . Dephosphorylation of phosphotyrosine by acid phosphatases from human prostate gland, from wheat germ and potatoes obeys the Michaelis-Menten equation, whereas alkaline phosphatases calf intestine and E . coli are inhibited by excess of substrate.

J Cell Biochem, 1985, 29(3), 225 - 37
The common alpha subunit of bovine glycoprotein hormones: limited formation of native structure by the totally nonglycosylated polypeptide chain; Strickland TW et al.; The folding of the bovine glycoprotein hormone alpha subunit, synthesized in bacteria following insertion of the nucleotide sequence coding for this polypeptide, has been studied to determine the effect that a complete lack of carbohydrate has on this process . The bacterially derived alpha polypeptide (bac-alpha), extracted from E . coli in the presence of reductant and denaturant, had an estimated 0.2% native structure as determined by a conformationally sensitive radioimmunoassay . Upon reduction of disulfide bonds and reoxidation in air, the amount of native structure increased about 18-fold . Approximately 2% of the refolded bac-alpha preparation combines with the beta subunit of human chorionic gonadotropin (hCG beta) to form a complex that binds to the gonadotropin receptor and elicits a biological response . Since the correct folding (by immunological criteria) of bac-alpha (ca 3%) is significantly greater than expected from a random formation of disulfide bonds (0.1%), it appears that correct folding of alpha subunit can occur in the complete absence of carbohydrate, though in very low yield . Native bovine lutropin alpha subunit (LH alpha) and chemically deglycosylated LH alpha (which retains two asparagine-linked N-acetyl glucosamine residues per alpha oligosaccharide) were subjected to the same reduction/reoxidation regimen as the bacterially produced alpha subunit . As has been reported previously {Giudice LC, Pierce, JG, J Biol Chem 251: 6392, 1976} intact LH alpha fully regained its native structure . The partially deglycosylated LH alpha also refolds to a native-like structure in high yield as assessed by immunological assays and by its ability to combine with HCG beta to form a biologically active complex . The data show that carbohydrate, while not obligatory for correct folding, greatly facilitates the formation of functional alpha subunit.

Adv Exp Med Biol, 1985, 185, 27 - 46
Foot-and-mouth disease and its antigens; Bachrach HL; Many factors combine to make foot-and-mouth disease (FMD) one of the most damaging and intractable disease of animals . These include its extreme contagion, wide geographic distribution, great multiplicity of both susceptible animal hosts and viral serotypes, a relatively short duration of immunity to a given serotype and a post-recovery carrier state of the virus in many animal species (e.g., cattle, sheep and goats) . Nevertheless, import restrictions and other actions of the U.S . Government have kept the United States free of FMD since 1929, even during outbreaks of the disease in Mexico and Canada in the early 1950's . Beginning in the late 1940's, the systematic vaccination of cattle has been practiced in several areas of the world with varying degrees of success . While this procedure has succeeded in Western Europe in greatly reducing the incidence of FMD, the presence of live virus in some batches of vaccine and the escape of virus from vaccine from manufacturing facilities are now responsible for a large proportion of the outbreaks that still occur there . A vaccine is needed that has no possibility of producing the disease . During the last few years, it has been demonstrated that capsid protein VP1, isolated from type A and C virions or biosynthesized in E . coli transformed with the gene for VP1, can be used to immunize livestock against FMD . Immunization of livestock has also been achieved with a 13 kd fragment (amino acid residues 55 through 179) cleaved with CNBr from the 213 amino acid long VP1 chain of type A virions . Immunogenic sites on intact virions, 12 S subunit particles and isolated VP1 chains have been studied by a combination of methods, including: assessment of the immunogenicity of VP1-specific fragments and synthetic peptides and mapping monoclonal antibodies (Mabs) generated with virus, VP1 and the 13 kd fragment to virus, 12 S subunits, VP1, VP1-specific fragments and a biosynthetic 32mer . Correlation of these results with sites having variant and serotype sequence variability indicates that the 136-179 region of type A12 VP1 possesses four putative neutralization-specific epitopes (ca . 137-143, 146-151, 152-157 and 170-175) . Of three neutralization-specific epitopes on type A12 virus, two are also present on 12 S subunits and isolated VP1 chains . Mabs to the three epitopes appear to neutralize virus by different mechanisms: by viral aggregation, by blocking the site on viral VP1 that binds to cell receptors or by interfering with a postreceptor attachment step, possibly penetration or uncoating.(ABSTRACT TRUNCATED AT 400 WORDS)

Gene, 1985, 38(1-3), 85 - 93
Physical mapping and nucleotide sequence of the rnpA gene that encodes the protein component of ribonuclease P in Escherichia coli; Hansen FG et al.; The rnpA gene, coding for the protein component of ribonuclease P (RNase P), was allocated to the dnaA region at 83 min of the E . coli K-12 map . This was accomplished through analysis of recombinant pBR322 plasmids, some of which complemented the temperature sensitivity of a strain carrying the rnpA 49 allele and restored the RNA processing activity . Although the temperature sensitivity of a strain carrying the rnp-241 allele could not be complemented by the rnpA+ plasmid, the RNA-processing activity was restored, suggesting that the rnp-241 mutation is allelic with rnpA 49 . In this analysis we also found two genes coding for proteins (60 and 50 kDal) of unknown function . The order of the genes located in this region is in the clockwise orientation: rpmH (5.4 kDal; ribosomal protein L34), rnpA (14 kDal; protein component of RNase P), a gene for a 60-kDal protein (inner membrane protein), a gene for a 50-kDal protein, and tnaA . All these genes are expressed in the clockwise orientation . From the DNA sequence of the rnpA gene region a very basic polypeptide with an Mr of 13773 could be deduced . We conclude that this polypeptide is the rnpA gene product, and is the protein component of RNase P . Comparison with previously published data on the transcription of rpmH suggests that the rnpA gene is the second gene in the rpmH operon.

J Interferon Res, 1985 Summer, 5(3), 521 - 6
On the relationship between human interferon alpha 1 and beta 1 genes; May LT et al.; The human interferon alpha 1 and beta 1 (IFN-alpha 1 and -beta 1) genes are approximately 56% identical in the nucleotide sequence across the regions coding for the mature proteins . An IFN-alpha 1 cDNA insert containing such sequences can be used to isolate 15 IFN-beta 1 cDNA colonies present in a 2600-strong library in Escherichia coli prepared from polyadenylated RNA extracted from human diploid fibroblasts (FS-4) induced with poly(I).poly(C) . Conversely, a clone containing an IFN-alpha 1 cDNA exhibited detectable hybridization with an IFN-beta 1 probe . One of the IFN-beta 1 cDNA clones isolated in these experiments had lost the internal PstI and PvuII sites due to a change at nucleotide 277 from a C to an A . As a consequence, this allele codes for Met-47 instead of Leu-47 . Suggestive evidence has been obtained for the presence of this allele in human genomic DNA (IFN-beta 1 Met-47).

J Interferon Res, 1985 Summer, 5(3), 455 - 63
Characterization of antibodies against recombinant HuIFN-gamma produced by hybridoma cells; Stefanos S et al.; Balbc/c mice were immunized with purified recombinant E . coli-derived human gamma-interferon (HuIFN-gamma) . Their spleen cells were fused with a mouse myeloma cell line (Sp2/0) . Hybridomas producing antibodies reacting with HuIFN-gamma were screened by a soluble-phase radioimmunoassay using pure 125I-labeled cloned IFN-gamma as antigen, and tested for their ability to neutralize the antiviral activity of IFN . Three hybridomas S1-1, S1-2, and S1-3, were cloned and subcloned and remained stable . Although the antibodies produced by clones S1-1 and S1-2 were both able to neutralize specifically the antiviral activity of natural and recombinant HuIFN-gamma, they appeared to recognize different epitopes on the HuIFN-gamma molecule . The antibodies produced by the S1-3 clone failed to neutralize the antiviral activity of either interferon . The antibodies from all three clones were characterized as IgG1 subclass . Their affinity constants were determined from competitive inhibition curves and ranged from 1 to 4.3 X 10(8) M-1.

J Interferon Res, 1985 Summer, 5(3), 445 - 53
Monoclonal antibodies as structural probes for oligomeric human interferon-gamma; Le J et al.; Monoclonal antibodies (MAb) B1 and B3, specific for human interferon-gamma (IFN-gamma) failed to immunoprecipitate heat-inactivated human IFN-gamma in solution . However, both MAb retained some reactivity with denatured IFN-gamma immobilized on vinyl plates . The two MAb have been employed in a sensitive immunoradiometric assay (IRMA) . In this IRMA one MAb was bound to polystyrene beads and used as immunoadsorbent . The second MAb, labeled with 125I, was used as the tracer to quantitate the amount of IFN-gamma bound to the immobilized MAb . Addition of unlabeled MAb B1 did not inhibit the binding of 125I-labeled MAb B3 (and vice versa), indicating that the two MAb react with two different and nonoverlapping epitopes . Yet, when the same MAb was used in IRMA as both immunoadsorbent and tracer, the amount of labeled MAb bound to a given concentration of natural or E . coli-derived recombinant human IFN-gamma was very similar as with two different MAb, indicating that a single IFN-gamma molecule must have two or more identical binding sites for each of the two MAb . These findings show that biologically active natural and recombinant human IFN-gamma exist in oligomeric form.

Int Arch Allergy Appl Immunol, 1985, 78(3), 318 - 25
Regulation of natural killer cell activity and interferon production in the rat lung following aerosol challenge; Farrell HE et al.; High levels of natural cytotoxicity were detected in vitro in cells from the lung interstitium of the rat following collagenase digestion of lung tissue . In contrast, alveolar cells exhibited negligible levels of natural cytotoxicity and furthermore suppressed the natural cytotoxicity of interstitial lung leukocytes . This suppression was alleviated following in vivo administration of indomethacin . The natural cytotoxicity of cells from both the lung and spleen was transiently suppressed following exposure of normal rats to aerosols of Escherichia coli lipopolysaccharide or of ovalbumin-sensitized rats to an ovalbumin aerosol . Parenchymal lung leukocytes, like those from the spleen and peripheral blood, showed enhanced cytotoxicity when treated with interferon in vitro . In addition interstitial leukocytes were capable of producing interferon when cocultured with P815 cells and, as was the case with the spleen, low density cells produced the most interferon . However, alveolar cells did not produce interferon in this system . These studies suggest that the lung is capable of self-regulation of the high levels of natural cytotoxicity present in interstitial tissue; alveolar cells or their products may suppress interstitial natural killer cells whilst interstitial leukocytes have the capacity to stimulate natural killer cells by producing interferon.

Gene, 1985, 37(1-3), 199 - 206
High-level expression of RNAs and proteins: the use of oligonucleotides for the precise fusion of coding-to-regulatory sequences; Sollazzo M et al.; We show that the fusion between regulatory sequences present on expression vectors and coding sequences can be efficiently achieved by oligonucleotide-directed mutagenesis . We have constructed single-stranded (ss) expression vectors that facilitate this process . These plasmids derive from vectors that have been used for the synthesis of quantities of proteins in Escherichia coli or RNAs in vitro . By inserting the origin of replication of the ss phage f1 into these plasmids it became possible to package their ss DNA into phage rods . Deletion of unwanted sequences or simple base changes can then be obtained by oligonucleotide-directed mutagenesis using the vector ss DNA as a template . We discuss the results of several experiments where this technique was applied to our expression vectors and we demonstrate the construction of a plasmid which efficiently synthesizes in vitro a regulatory RNA molecule that is involved in the control of plasmid copy number.

Ultramicroscopy, 1985, 16(3-4), 423 - 35
Comparative electron microscopic studies of single biomolecules negatively stained and freeze-dried metal-shadowed; Tesche B et al.; This paper deals with the development of physical fixation and contrasting procedures for the electron microscopic structure analysis of isolated biomolecules . It has been shown on isolated antibodies (IgG) that these preparation methods give better preservation of biomolecules than the commonly practiced chemical fixation and staining techniques . With regard to morphology and size, the data from the electron microscopic structure analysis of the tested antibodies (IgG) are in good agreement with those from X-ray structure analysis . With isolated ribosomal 40S subunits, the influence of chemical fixation and staining techniques on both the structure preservation and contrast medium distribution has been demonstrated.

CRC Crit Rev Biochem, 1985, 18(3), 239 - 80
The heat shock response; Craig EA; The response of cells to a heat shock or other stresses is the activation of a small number of genes which were previously inactive or transcribed at low levels . This response has been observed in a wide variety of bacterial, plant, and animal species . Evidence is accumulating that at least some of the proteins found in diverse species are similar, indicating a conservation of the response and the proteins in evolution . In a number of organisms a strong positive correlation has been found between the presence of heat shock proteins and ability of the organism to withstand thermal stress . This review attempts to assess the available data concerning the homology of proteins in different species, the localization of the proteins in cells, and the relationship between heat shock proteins and thermoresistance.

Acta Microbiol Pol, 1985, 34(2), 111 - 20
Mutagenic activity of radiosensitizers; Grzybowska E et al.; Seven radiosensitizers, six derivatives of nitroimidazole (coded P1 to P5 and one imidazole derivative--P6 were investigated for mutagenicity using 3 short-term tests: Ames test, prophage lambda induction and tryptophan reversion test . Out of seven investigated compounds five were not mutagenic . Only P1 derivative induces base pair substitutions . Another derivative of nitroimidazole: metronidazole induces base pair substitution and frameshift mutations . Its positive response in the prophage lambda induction test suggests that metronidazole provokes also epigenetic changes.

Mol Gen Genet, 1985, 199(3), 415 - 20
10 nm RecA protein filaments formed in the presence of Mg2+ and ATP gamma S may contain RNA; Register JC 3rd et al.; Filaments formed by the polymerization of RecA protein along DNA in the presence of Mg2+ and adenosine 5'-0-(3-thiotriphosphate) (ATP gamma S) are seen by electron microscopy to have a 10 nm diameter with a 9 nm helical repeat . When certain preparations of apparently pure RecA protein are incubated with Mg2+ and ATP gamma S in the absence of nucleic acid for extended times, very long filaments with the same 10 nm diameter and 9 nm axial repeat are seen . We show here that these long 10 nm filaments can contain RNA which is present as a contaminant of the RecA protein and poly(A) which is synthesized during the incubations by an activity that is apparently polynucleotide phosphorylase . RecA protein purified by a procedure developed in this laboratory did not contain RNA and did not form these very long 10 nm filaments . However, when exogenous RNA was added to this protein, 10 nm filament formation was observed.

Gene, 1985, 35(1-2), 199 - 207
Analysis of the ptsH-ptsI-crr region in Escherichia coli K-12: nucleotide sequence of the ptsH gene; De Reuse H et al.; The nucleotide sequence of an Escherichia coli DNA segment containing the ptsH gene and the first 162 nucleotides of the ptsI gene encoding, respectively, Hpr and enzyme I of the phosphoenolpyruvate-dependent glycose phosphotransferase system (PTS), was determined . The ptsH promoter was localized using the S1 mapping technique . A nucleotide sequence very similar to the consensus binding site for cAMP receptor protein was found in the -35 region of the ptsH promoter . The ptsH gene is transcribed in the same direction as the ptsI gene and the crr gene (encoding enzyme IIIGlc of the PTS) . Analysis of the nucleotide sequence substantiates the notion that the ptsH-ptsI-crr genes constitute a polycistronic operon.

Basic Life Sci, 1985, 30, 321 - 33
Construction of Co1E1 RNA1 mutants and analysis of their function in vivo; Polisky B et al.; We have carried out experiments designed to investigate the relationship between structure and function for the Co1E1 RNA1 species . RNA1 is a small RNA (108 nucleotides) that has been implicated in copy number control of the multicopy plasmid Co1E1 . In vitro, RNA1 inhibits the processing of the primer precursor required for initiation of DNA replication . The RNA1 gene is entirely complementary to the 5'-terminal region of the primer . We have functionally separated these 2 RNA species by cloning the RNA1 gene downstream from the S . marcescens trp promoter . When cloned in a Co1E1-compatible plasmid, a trp-RNA1 fusion has been shown to mediate Co1E1-type incompatibility in vivo . The construction scheme described here also generates mutant RNA1 species with altered sequences at the 5' terminus of RNA1 which have been assayed for function in vivo . These experiments have indicated that sequences at the 5' terminus play a critical role in RNA1 function.

Mol Gen Genet, 1985, 198(3), 503 - 8
Transcription and its regulation in the basic replicon region of plasmid R1; Light J et al.; The transcriptional units in the basic replicon of plasmid R1 were defined by means of gene fusions . It was found that in the wild-type plasmid there is one large mRNA encoding both the control factor copB and the positive replication factor repA . A second, internal transcription initiation site, the repA promoter, is usually repressed by the copB protein, and is therefore only of significance in the absence of this control factor . By induction of the repA promoter through gradual dilution of the copB repressor it was shown that translation of repA-mRNA, controlled by the copA-RNA, is significantly increased only when the rate of repA transcription is above a certain level . No indication was found for a possible interference from convergent copA transcription on repA transcription.

Gene, 1985, 34(2-3), 305 - 14
Use of primer-restriction-end adapters in a novel cDNA cloning strategy; Coleclough C et al.; We introduce a class of synthetic oligonucleotides, referred to as primer-restriction-end (PRE) adapters, which are bifunctional, one end serving as a primer for a polymerase reaction, while the other end can be ligated to restriction endonuclease digested DNA . Use of such adapters forms the basis of a new method for inserting single-stranded cDNA into cloning vectors, which involves very few separate biochemical modifications of the cDNA and so is appropriate when extensive fractionation of cDNA is desired prior to cloning . This novel methodology is highly efficient in producing full-length cDNA cloned in a predictable orientation within vectors, as we demonstrate by constructing and analysing clones of immunoglobulin lambda light chain cDNA in Escherichia coli.

Ann Inst Pasteur Microbiol, 1985 Jan-Feb, 136A(1), 105 - 10
Protein export in Escherichia coli; Pages JM et al.; Hyperproduction of phosphate-binding protein (PhoS) resulted in saturation of export sites, and pre-PhoS was accumulated both in the inner membrane and in the cytoplasm . The cytoplasmic pre-PhoS could not be exported post-translationally; only the membrane-associated precursor could be matured and exported . Preliminary evidence has been obtained for the existence of a translation stop . The pause site in pre-PhoS mRNA translation occurs when 70 to 80 amino acids have been assembled, and appears to be related to the coupling of synthesis and export.

J Immunol Methods, 1984 Dec 31, 75(2), 295 - 307
Development of a competitive enzyme-linked immunosorbent assay (ELISA) for Escherichia coli heat-stable enterotoxin (STa); Lockwood DE et al.; A sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the low molecular weight heat-stable enterotoxin (STa) in culture supernatant fluids of enterotoxigenic Escherichia coli (ETEC) . Competitive inhibition was observed between STa in solution and a glutaraldehyde-coupled STa-human serum albumin (HSA) conjugate bound to microtiter wells when antiserum raised against a glutaraldehyde-coupled STa-bovine serum albumin (BSA) conjugate was used as detecting antibody . No competition was observed with conjugates prepared using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or dimethyl suberimidate and antisera raised against each conjugate . A biotin/avidin system increased the sensitivity of the assay such that 133 pg/ml of purified STa can be detected in less than 4 h . The assay was used to detect and quantify STa in culture supernatant fluids from human, porcine, and bovine ETEC isolates . No cross-reactivity was observed with the heat-labile enterotoxin (LT) or the form of ST with biological activity only in piglets (STb) . Results from the quantitative STa ELISA showed good correlation (0.87) with the suckling mouse bioassay and a previously described radioimmunoassay . The quantitative assay was modified to reduce the total incubation time to less than 2 h . The qualitative STa ELISA provides a rapid and sensitive assay for clinical isolates of ETEC and should facilitate epidemiological studies on the incidence of STa-producing ETEC.

J Chromatogr, 1984 Dec 28, 317, 283 - 300
High-performance liquid chromatographic purification of deoxynucleoside 5'-triphosphates and their use in a sensitive electrophoretic assay of misincorporation during DNA synthesis; Revich GG et al.; This paper describes techniques and strategies for semi-preparative high-performance liquid chromatographic (HPLC) purification of 2'-deoxynucleoside 5'-triphosphates (dNTPs) . The procedure yields dNTPs that are sufficiently pure for use in a sensitive electrophoretic assay of misincorporation during DNA synthesis . Anion-exchange HPLC was used to purify the four normal dNTPs (dATP, dGTP, dCTP and dTTP), plus the chemically modified analogues, 5-BrdUTP, 5-IodUTP and 1,N6-etheno-dATP (epsilon dATP) . Baseline separations were achieved by isocratic elution of dNTPs with potassium dihydrogen phosphate mobile phase . In general, the resolution of dNTPs was highly dependent on pH, although the influence of mobile phase composition on separation of dNTPs was not the same for all three HPLC packing materials used . A Hewlett-Packard diode array detector was extremely valuable in the identification of contaminating peaks and in the development of optimal mobile phase conditions for dNTP purification . The pure dNTPs were used in the electrophoretic assay of misincorporation, yielding information about the mispairing potential of the modified dNTPs . BrdUMP and IodUMP were misincorporated in place of dCMP during chain elongation catalyzed by purified DNA polymerase I of Escherichia coli . epsilon dAMP was incorporated into DNA in place of dAMP, although at much lower efficiency than dAMP.

J Chromatogr, 1984 Dec 28, 317, 201 - 12
Application of high-performance liquid chromatography to the reconstitution of ribosomal subunits; Kerlavage AR et al.; We are currently utilizing reversed-phase high-performance liquid chromatography (RP-HPLC) in reconstitution experiments designed to study the structure and function of Escherichia coli ribosomes . The applications of RP-HPLC in these experiments include: (a) preparation of individual proteins or groups of proteins on a milligram scale for reconstitution pools, (b) analysis of the protein stoichiometry of reconstituted subunits, (c) determination of the extent and specificity of modification of proteins extracted from ribosomal subunits which have been subjected to chemical modification, and (d) resolution of modified forms of proteins S14 and L23 from the corresponding unmodified proteins . Proteins prepared by RP-HPLC from 30S and 50S ribosomal subunits were found to reconstitute into 30S and 50S subunits respectively, as well as into slower sedimenting particles . The reconstituted subunits contain a full complement of proteins and are active in ribosomal function assays, whereas the slower sedimenting particles lack several proteins and have little or no activity.

J Mol Biol, 1984 Dec 25, 180(4), 1023 - 51
Nucleotide sequence and transcription of the phenylalanine and tyrosine operons of Escherichia coli K12; Hudson GS et al.; A 4509 base-pair DNA fragment containing the phenylalanine and tyrosine operons of Escherichia coli K12 has been sequenced, and the pattern of transcription of these operons examined by S1 mapping, primer extension and galK fusion analyses . The phe operon consists of promoter, operator, leader region containing the phe attenuator and the pheA gene encoding chorismate mutase/prephenate dehydratase . The tyr operon consists of promoter, operator, a short leader region without an attenuator, and two structural genes aroF and tyrA encoding the tyrosine-sensitive isoenzyme of 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthetase and chorismate mutase/prephenate dehydrogenase, respectively . A bidirectional transcription terminator occurs between the two operons . The predicted amino acid sequences of chorismate mutase/prephenate dehydrogenase and chorismate mutase/prephenate dehydratase are homologous at their N termini, while the tyrosine-sensitive isoenzyme of DAHP synthetase is closely homologous to the phenylalanine-sensitive isoenzyme encoded by aroG.

J Biol Chem, 1984 Dec 25, 259(24), 15579 - 86
Morphology of proteoliposomes reconstituted with purified lac carrier protein from Escherichia coli; Costello MJ et al.; Proteoliposomes reconstituted with purified lac carrier protein from Escherichia coli were ultra-rapidly frozen and examined by freeze-fracture-etch electron microscopy . The proteoliposomes are greater than 95% unilamellar, and the majority are 30-150 nm in diameter . Fracture faces of proteoliposomes (at a protein:lipid molecular ratio of about 1:2500) display 7.0-nm diameter globular intramembrane particles uniformly distributed on convex and concave surfaces . Calculations of particle composition suggest that each intramembrane particle probably contains one or two molecules of the 46.5-kDa transmembranous lac carrier protein, depending on the correction factor for the thickness of the metal deposited to form the platinum/carbon replicas . Etched surfaces of the proteoliposomes are smooth . Incubation of the proteoliposomes with monoclonal antibody 4B1, which binds to an epitope in the lac carrier on the exterior of the proteoliposomes, dramatically alters the intramembrane particle distribution . After incubation with antibody, the convex (inner monolayer) fracture faces are nearly devoid of intramembrane particles, and an overall 4-fold reduction in the total number of intramembrane particles is observed.

J Biol Chem, 1984 Dec 25, 259(24), 15182 - 7
Kinetics of synthesis, processing, and membrane transport of heat-labile enterotoxin, a periplasmic protein in Escherichia coli; Hofstra H et al.; We report the detection in vivo of precursors to the A and the B subunits of the heat-labile enterotoxin (LT) in Escherichia coli . Both pre-LT A (Mr = 29,500) and pre-LT B (Mr = 13,500) are present in the spheroplast fraction of the bacteria after separation of the cells in spheroplasts and periplasm . Two smaller LT A related polypeptides (17 and 23 kDa) were also detected in the spheroplast fraction . Both were degraded with a half-time of about 40 s . Mature subunits (Mr = 27,500 for LT A, and 11,500 for LT B) are released from the spheroplasts soon after processing and occur freely in the periplasm not associated with the cytoplasmic or the outer membranes . Processing occurs mainly post-translationally for both the A and the B subunits . However, they show different kinetics of processing and subsequent segregation into the periplasm . Whereas pre-LT B is processed and released within seconds after chain termination, pre-LT A is processed and released more slowly, and a subfraction of mature LT A may reside in the cytoplasmic membrane for several minutes.

J Biol Chem, 1984 Dec 25, 259(24), 15025 - 7
Comparison of the basic Escherichia coli antizyme 1 and antizyme 2 with the ribosomal proteins S20/L26 and L34; Panagiotidis CA et al.; The two basic Escherichia coli proteins that inhibit ornithine and arginine decarboxylase and were named provisionally antizyme 1 and antizyme 2 (Heller, J.S., Rostomily, R., Kyriakidis, D.A., and Canellakis, E.S . (1983) Proc . Natl . Acad . Sci . U.S.A . 80, 5181-5184) are shown to have long identical sequences with the ribosomal proteins S20/L26 and L34, respectively . We have also isolated ribosomal proteins from purified E . coli ribosomes by established methodology and further purified them by our purification procedure for antizymes 1 and 2 . Of the various basic ribosomal proteins, two were found to have the same properties as antizyme 1 and 2 . These results indicate that these two basic E . coli antizymes are ribosomal proteins . The nature of the acidic antizyme remains to be elucidated.

J Biol Chem, 1984 Dec 25, 259(24), 15331 - 9
Interaction of aspartate and aspartate-derived antimetabolites with the enzymes of the threonine biosynthetic pathway of Escherichia coli; Shames SL et al.; The five enzymes responsible for the conversion of L-aspartate to L-threonine in Escherichia coli were purified to homogeneity and subsequently reconstituted in vitro in ratios approximating those found in vivo . 31P NMR was used to conveniently monitor the rates of consumption of the substrates ATP and NADPH, the accumulation of the intermediates beta-aspartyl phosphate and homoserine phosphate, and the formation of the products ADP, NADP+, and Pi in a single experiment . By this method, the flux of aspartic acid through the enzymes of the pathway was monitored in the absence and in the presence of several alternative substrates and inhibitors . Several known antimetabolites were found to be alternative substrates that ultimately became inhibitors of pathway flux . L-threo-3-Hydroxyaspartic acid was converted to 3-hydroxyhomoserine phosphate by the first four enzymes of the pathway . The antimetabolite L-threo-3-hydroxyhomoserine was found to bind to and inhibit aspartokinase-homoserine dehydrogenase I in a cooperative fashion (I 0.5 = 3 mM, nH = 2.5), similar to the action of the allosteric end product inhibitor L-threonine (I 0.5 = 0.36 mM, nH = 2.4) . In the presence of the remaining enzymes of the pathway, however, L-threo-3-hydroxyhomoserine was phosphorylated to the apparent ultimate antimetabolite L-threo-3-hydroxyhomoserine phosphate that was a potent inhibitor of threonine synthase and consequently of L-threonine biosynthesis . When aspartic acid alone was examined as a substrate of the enzymes of the pathway, no accumulation of the beta-aspartyl phosphate and homoserine phosphate intermediates was observed . However, in the presence of either 5 mM L-threo-3-hydroxyhomoserine or 5 mM L-threo-3-hydroxyhomoserine phosphate, homoserine phosphate was found to accumulate . In contrast to the homoserine phosphate and 3-hydroxyhomoserine phosphate intermediates, both of which were very stable, the acylphosphate intermediates beta-aspartyl phosphate and beta-3-hydroxyaspartyl phosphate were highly susceptible to hydrolysis, with first-order rate constants of 4.6 X 10(-3) min-1 and 4.5 X 10(-2) min-1 (pH 7.8, 25 degrees C), respectively.

J Mol Biol, 1984 Dec 25, 180(4), 881 - 909
Mechanism of CRP-cAMP activation of lac operon transcription initiation activation of the P1 promoter; Malan TP et al.; CRP-cAMP was shown to activate transcription initiation at the Escherichia coli lac promoter in vitro as a result of two separate effects . An indirect component of the activation resulted from an enhancement of the fraction of promoters productively bound by RNA polymerase . This effect was due largely to CRP-cAMP repression of RNA polymerase binding to an overlapping site (lac P2) within the promoter region . In addition, a direct enhancement of RNA polymerase binding at the principal lac promoter (lac P1) was found . The combination of indirect and direct activation by CRP-cAMP was suggested to be responsible for the large activation observed in vivo . Promoter strength parameters were also determined for the L8, UV5 and Ps promoters . The effect of CRP-cAMP on these mutant promoters was shown to be consistent with the activation mechanism deduced for the lac wild-type promoter . DNA supercoiling enhanced the promoter strength of the lac wild-type and UV5 promoters . The combination of supercoiling and CRP-cAMP was necessary for optimal promoter strength for the lac wild-type promoter.

J Mol Biol, 1984 Dec 25, 180(4), 1053 - 63
The nusA recognition site . Alteration in its sequence or position relative to upstream translation interferes with the action of the N antitermination function of phage lambda; Olson ER et al.; The phage lambda transcription antitermination protein, pN, acts with host factors, Nus, at sites on the phage genome, nut, to render RNA polymerase resistant to subsequent downstream termination signals . The NusA protein appears to recognize a seven to eight base-pair consensus sequence (5'Py-G-C-T-C-T-T(T)3') called boxA that is found in the promoter-proximal part of the nut region . Two types of change within or near the boxA sequence in the nutR region are shown to interfere with pN-mediated antitermination of transcription that has initiated at the upstream pR promoter . (1) A change of one base-pair (from G to T at the second position) in the boxA sequence significantly reduces pN action . (2) We prove that a frameshift mutation, cro delta 62, at the end of the gene promoter-proximal to the lambda nutR region, interferes with the pN antitermination reaction by allowing translation to proceed beyond cro into the nutR region . Using a series of plasmid constructions, we now show that the inhibition of antitermination caused by the cro delta 62 mutation can be suppressed when translation is terminated upstream from this mutation.

J Biol Chem, 1984 Dec 25, 259(24), 15373 - 6
Characterization of a sulfite reductase from Desulfovibrio vulgaris . Evidence for the presence of a low-spin siroheme and an exchange-coupled siroheme-{4Fe-4S} unit; Huynh BH et al.; We have studied a low-molecular-weight (Mr = 27,200) sulfite reductase from Desulfovibrio vulgaris (Hildenborough, NCIB 8303) with Mossbauer, EPR, and chemical techniques . This sulfite reductase was found to contain one siroheme and one {4Fe-4S} cluster . As purified, the siroheme is low-spin ferric (S = 1/2) which exhibits characteristic EPR resonances at g = 2.44, 2.36, and 1.77 . At 150 K, the observed Mossbauer parameters, delta EQ = 2.49 +/- 0.02 mm/s and delta = 0.31 +/- 0.02 mm/s, for the siroheme are typical for low-spin ferric complexes . The {4Fe-4S} cluster is in the 2+ state . The Mossbauer parameters, delta EQ = 0.95 +/- 0.02 mm/s and delta = 0.38 +/- 0.02 mm/s, for the cluster are almost identical to those observed for the {4Fe-4S}2+ cluster in the hemoprotein subunit of the sulfite reductase from Escherichia coli . Similar to the hemoprotein subunit of E . coli sulfite reductase, low-temperature Mossbauer spectra of D . vulgaris sulfite reductase recorded with weak and strong applied fields also show evidence for an exchange-coupled siroheme-{4Fe-4S} unit.

J Biol Chem, 1984 Dec 25, 259(24), 15000 - 2
Binding of rho factor to Escherichia coli RNA polymerase mediated by nusA protein; Schmidt MC et al.; The E . coli transcription termination factor rho binds specifically to purified nusA protein . Since nusA protein binds tightly to RNA polymerase, this provides a way of coupling rho to the elongating RNA polymerase complex through protein-protein interactions . These rho-nusA interactions may play a role in modulating rho action at certain terminators and could also be important in the action of antitermination factors such as lambda N protein.

Nucleic Acids Res, 1984 Dec 21, 12(24), 9237 - 48
The complete pattern of mutagenesis arising from the repair of site-specific psoralen crosslinks: analysis by oligonucleotide hybridization; Saffran WA et al.; Psoralen crosslinks were site-specifically placed in plasmid pBR322 near the BamHI site in the tet gene by enzymatically inserting mercurated nucleotides and reacting at the target site with a sulfhydryl-containing psoralen . The damaged plasmid was repaired in SOS-induced E . coli cells . Mutants were detected by colony hybridization to oligonucleotides in the target region, and their sequences were determined . The mutations are all base substitutions, 80% transitions and 20% transversions, similar to the mutations previously identified by the loss of tetracycline resistance . However, the mutation sites detected by a physical method, unconstrained by phenotypic changes, follow a broader distribution than those identified genetically . They occur primarily at favored psoralen crosslinking sites, where T-T and T-C interstrand crosslinks can be formed . A majority of these mutations are silent.

Nucleic Acids Res, 1984 Dec 21, 12(24), 9567 - 75
A method for measuring the non-random bias of a codon usage table; McLachlan AD et al.; We describe a new statistical method for measuring bias in the codon usage table of a gene . The test is based on the multinomial and Poisson distributions . The method is used to scan DNA sequences and measure the strength of codon preference . For E . Coli we show that the strength of codon preference is related to levels of gene expression . The method can also be used to compare base triplet frequencies with those expected from the base composition . This second type of codon bias test is useful for distinguishing coding from non-coding regions.

Nucleic Acids Res, 1984 Dec 21, 12(24), 9427 - 40
The complete nucleotide sequence of the adenylate cyclase gene of Escherichia coli; Aiba H et al.; The complete nucleotide sequence of the cya gene from E . coli was determined . The gene encodes a polypeptide consisting of 848 amino acid residues with a calculated molecular weight of 97,542 . The deduced protein structure reveals that cyclase is comprised of two domains, an amino-terminal region exhibiting catalytic activity and a carboxy-terminal region possibly carrying regulatory function . The frequent appearance of rare codons in the beginning of the gene as well as the sequence duplication in the promoter-initiator region suggest possible regulation(s) at the translational level . An unknown gene (cyaX) which seems to code for a very hydrophobic protein was found following the cya gene . Sequence analysis suggests that the cyax is a part of the cya operon.

Nucleic Acids Res, 1984 Dec 21, 12(24), 9271 - 85
DNA structural variations produced by actinomycin and distamycin as revealed by DNAase I footprinting; Fox KR et al.; The technique of DNAase I footprinting has been used to investigate preferred binding sites for actinomycin D and distamycin on a 160-base-pair DNA fragment from E . coli containing the tyr T promoter sequence . Only sites containing the dinucleotide step GpC are protected by binding of actinomycin, and all such sites are protected . Distamycin recognizes four major regions rich in A + T residues . Both antibiotics induce enhanced rates of cleavage at certain regions flanking their binding sites . These effects are not restricted to any particular base sequence since they are produced in runs of A and T by actinomycin and in GC-rich sequences by distamycin . The observed increases in susceptibility to nuclease attack are attributed to DNA structural variations induced in the vicinity of the ligand binding site, most probably involving changes in the width of the helical minor groove.

Nucleic Acids Res, 1984 Dec 21, 12(24), 9441 - 56
The gapped duplex DNA approach to oligonucleotide-directed mutation construction; Kramer W et al.; A simple and efficient method is described to introduce structurally pre-determined mutations into recombinant genomes of filamentous phage M13 . The method rests on gapped duplex DNA (gdDNA) molecules of the phage M13 genome as the key intermediate . In this gdDNA, the (+) and the (shorter) (-) strand carry different genetic markers in such a way, that a rigorous selection can be applied for phage carrying the markers of the (-) strand . For introduction of the mutation, a synthetic oligonucleotide with partial homology to a target site within the single stranded DNA region is annealed to the gdDNA . The oligonucleotide subsequently becomes part of the (-) strand by enzymatic DNA gap filling and sealing . This physical linkage is preserved at the genetic level after transfection of a recipient E.coli strain deficient in DNA mismatch correction, so that the synthetic marker can be selected from the phage progeny independent from its potential phenotype . It is demonstrated that by this method mutants can be constructed with marker yields in excess of 70%.

Nature, 1984 Dec 20-1985 Jan 2, 312(5996), 724 - 9
Human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin; Pennica D et al.; Human tumour necrosis factor has about 30% homology in its amino acid sequence with lymphotoxin, a lymphokine that has similar biological properties . Recombinant tumour necrosis factor can be obtained by expression of its complementary DNA in Escherichia coli and induces the haemorrhagic necrosis of transplanted methylcholanthrene-induced sarcomas in syngeneic mice.

Nature, 1984 Dec 20-1985 Jan 2, 312(5996), 721 - 4
Cloning and expression of cDNA for human lymphotoxin, a lymphokine with tumour necrosis activity; Gray PW et al.; A chemically-synthesized gene and natural complementary DNA coding for human lymphotoxin were isolated and engineered for expression in Escherichia coli . Purified recombinant lymphotoxin shows cytotoxic activity on murine and human tumour cell lines in vitro and causes necrosis of certain murine sarcomas in vivo.

EMBO J, 1984 Dec 20, 3(13), 3317 - 22
Protein fusions with the kanamycin resistance gene from transposon Tn5; Reiss B et al.; The gene for the neomycin phosphotransferase II (NPT II) from transposon Tn5 was fused at the amino or carboxy terminus to foreign DNA sequences coding for 3-300 amino acids and the properties of the fused proteins were investigated . All amino-terminal fusions examined conferred kanamycin resistance to their host cell, but profound differences in their enzymatic activity and stability were detected . Short additions to the amino terminus of the NPT II resulted in highly enzymatically active fusion proteins whereas long amino-terminal fusions often had to be proteolytically degraded to release active proteins . Fusions at the carboxy-terminal end of the NPT II protein did not always induce kanamycin resistance and their enzymatic activity depended more stringently on the nature of the junction sequence.

Nature, 1984 Dec 20-1985 Jan 2, 312(5996), 785 - 6
Virus- and cell-encoded tyrosine protein kinases inactivate DNA topoisomerases in vitro; Tse-Dinh YC et al.; Tyrosine protein kinase activity is associated with at least eight different retrovirus-encoded onc gene products and with cell receptors for epidermal growth factor, platelet-derived growth factor, tumour growth factor and insulin . Both the onc kinases and the growth factor receptors are membrane proteins whose enzymatic activity has been implicated in stimulation of growth . However, the mechanism by which a signal passes from the plasma membrane to the nucleus to initiate growth remains unknown . As DNA topoisomerases catalyse the interconversion of topological isomers of DNA and hence affect DNA replication, transcription and recombination, they may be involved also in stimulation of growth . Several DNA topoisomerases have been shown to form a covalent complex with DNA via a phosphotyrosine linkage . The DNA-protein complex is postulated to be an intermediate in breaking and rejoining of DNA . The aim of the present study was to determine whether tyrosine protein kinases modulate the activity of topoisomerases by phosphorylating the tyrosine residue involved in DNA binding . We report that incubation of Escherichia coli and calf thymus type I DNA topoisomerases with the Rous sarcoma virus transforming gene product, pp60src, and TPK75, a tyrosine protein kinase purified from normal rat liver, results in a 10-fold loss of topoisomerase activity.

Biochemistry, 1984 Dec 18, 23(26), 6522 - 9
Cooperative interactions in the system ribosomes-ribosomal protein S1-polynucleotide triplets; Goss DJ et al.; Association equilibria have been determined in the ternary system uridyl triplets (T)-ribosomal protein S1 (S)-ribosomes (Rb) depleted of S1 at 6 and 10 mM Mg2+ . For 1:1 stoichiometry of reactants, four thermodynamically independent equilibria characterize the ternary system . The binary interaction Rb + T was studied by following the fluorescence quenching of labeled ribosomes by added T . The Rb + T association constant for UpUpUp triplets was 10-20-fold greater than for ApUpG triplets . The interaction Rb + S was studied by following the changes in fluorescence anisotropy when labeled S1 reacted with ribosomes . The remaining two independent equilibrium constants (for S + T and RbT + S) were obtained from fits to observed anisotropy measurements when varying amounts of T were added to a solution of ribosomes and fluorescently labeled S1 . This indirect procedure allows one to measure S + T binding, an association that is difficult to determine directly . Over the concentration interval 5-10 mM Mg2+, the association constant for Rb + S increases with the sixth power of {Mg2+}, whereas the association constant for S + T decreases approximately 2-fold as Mg2+ is increased from 6 to 10 mM Mg2+ . T binds to Rb more tightly at 10 mM than at 6 mM Mg2+ . When S1 is bound to Rb, however, at 10 mM Mg2+ the binding constant for T is decreased 10-fold and the Mg2+ dependence is reversed . These interactions can be described in terms of coupling free energies . For the ternary complex, three linearly independent coupling free energies can be written.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1984 Dec 18, 23(26), 6484 - 91
Reaction of phenylglyoxal and (p-hydroxyphenyl) glyoxal with arginines and cysteines in the alpha subunit of tryptophan synthase; Eun HM et al.; The alpha subunit of tryptophan synthase from Escherichia coli is inactivated by phenylglyoxal and by (p-hydroxyphenyl)glyoxal . The use of these chemical modification reagents to determine the role of arginyl residues in the alpha subunit of tryptophan synthase has been complicated by our finding that these reagents react with sulfhydryl groups of the alpha subunit, as well as with arginyl residues . Analyses of the data for incorporation of phenyl{2-14C}glyoxal, for inactivation, and for sulfhydryl modification in the presence and absence of indole-3-glycerol phosphate indicate that two sulfhydryl groups and one arginine are essential for the activity . Our finding that the substrate protects the single essential arginyl residue but not the two sulfhydryl groups is consistent with the observed kinetics of partial protection by substrate or by a substrate analogue, indole-3-propanol phosphate . In contrast to phenylglyoxal, (p-hydroxyphenyl)glyoxal modifies two to three sulhydryl groups that are not protected by indole-3-glycerol phosphate and modifies none of the arginyl residues that are modified by phenylglyoxal.

Biochemistry, 1984 Dec 18, 23(26), 6369 - 77
Intrinsic zinc ion is essential for proper conformation of active Escherichia coli RNA polymerase; Solaiman D et al.; The DNA-dependent RNA polymerase (RPase) from Escherichia coli contained 2 mol of Zn/mol of holoenzyme (alpha 2 beta beta' sigma) . An in vitro protocol involving sequential denaturation of RPase in 8 M urea and low pH (2.2), in the presence of 10 mM ethylenediaminetetraacetic acid (EDTA), was developed to completely remove the two intrinsic Zn ions . Subsequent reconstitution of the denatured, Zn-free RPase in the absence and presence of 10(-5) approximately 10(-4) M ZnCl2 yielded respectively the inactive apoenzyme and active (50 +/- 10%) RPase containing one Zn ion (rec-Zn1-RPase) . Active rec-Cd1-RPase was similarly obtained when CdCl2 instead of ZnCl2 was used in the reconstitution . The use of 65Zn as a tracer in the two-step reconstitution procedure showed that the metal was incorporated into renatured enzyme only in the last step of reconstitution . The subunit location of the incorporated metal was identified to be in the beta subunit by the use of Affi-Gel Blue column chromatography of rec-Cd1-RPase . The analysis of apo- and rec-Zn1-RPases by sucrose density gradient sedimentation showed that the inactive apo-RPase appeared to be consisted of randomly folded protein species with S20,w values ranging from 5 to 18 S, while rec-Zn1-RPase contained a major, active 13S RPase species and a minor, inactive 7.9S species that could be separated by DNA-cellulose column chromatography . Both 13S and 7.9S RPase contained 1 mol of Zn and the five subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1984 Dec 18, 23(26), 6591 - 5
Evidence for a nucleotide binding site on the isolated beta subunit from Escherichia coli F1-ATPase . Interaction between nucleotide and aurovertin D binding sites; Issartel JP et al.; The nucleotide binding capacity and affinity of the isolated beta subunit from Escherichia coli F1-ATPase have been studied with radiolabeled ADP and ATP by an equilibrium dialysis technique . Each mole of beta subunit in the presence of EDTA bound 1 mol of ADP or ATP with Kd values of 25 microM and 50-100 microM, respectively . At a saturating concentration, aurovertin enhanced the affinity of ADP or ATP for the isolated beta subunit by 3-6-fold . The Kd values for the binding of ADP or ATP were also assessed through the enhancing effect of ADP on {14C}aurovertin binding (Issartel, J.-P., Klein, G., Satre, M., & Vignais, P.V . (1983) Biochemistry 22, 3485-3492); the Kd values determined by this approach were several times lower than in the absence of aurovertin, in agreement with results obtained by direct titration with radiolabeled ADP or ATP.

Biochemistry, 1984 Dec 18, 23(26), 6474 - 80
Purification and immunogenicity of fusion VP1 protein of foot and mouth disease virus; Shire SJ et al.; A procedure has been developed to purify foot and mouth disease virus (FMDV) VP1 surface antigens from recombinant Escherichia coli . The VP1 antigens are expressed as fusion proteins derived from the E . coli Trp operon and VP1 surface protein of FMDV . The procedure is capable of recovering greater than 96% of the desired product at a purity of greater than 96% . The resulting antigens induce significant levels of virus-neutralizing antibody in guinea pigs and cattle as determined by a mouse protection assay {Skinner, H.H . (1952) Proc . Int . Vet . Congr., 15th 1, 195} . E . coli contaminants have a deleterious effect on ion-exchange chromatography as well as immunogenicity of the expressed fusion VP1 antigens . The method presented removes significant E . coli contaminants, yielding fusion VP1 proteins which are immunogenically potent . In particular, virus neutralization titers at 100-micrograms dosage of the fusion VP1 proteins of the O1 and A24 serotypes are similar to that induced by the natural VP1 proteins isolated from FMD virions.

Biochemistry, 1984 Dec 18, 23(26), 6710 - 7
tRNA binding sites of ribosomes from Escherichia coli; Lill R et al.; 70S tight-couple ribosomes from Escherichia coli were studied with respect to activity and number of tRNA binding sites . The nitrocellulose filtration and puromycin assays were used both in a direct manner and in the form of a competition binding assay, the latter allowing an unambiguous determination of the fraction of ribosomes being active in tRNA binding . It was found that, in the presence of poly(U), the active ribosomes bound two molecules of N-AcPhe-tRNAPhe, one in the P and the other in the A site, at Mg2+ concentrations between 6 and 20 mM . A third binding site in addition to P and A sites was observed for deacylated tRNAPhe . At Mg2+ concentrations of 10 mM and below, the occupancy of the additional site was very low . Dissociation of tRNA from this site was found to be rather fast, as compared to both P and A sites . These results suggest that the additional site during translocation functions as an exit site, to which deacylated tRNA is transiently bound before leaving the ribosome . Since tRNA binding to this site did not require the presence of poly(U), a function of exit site bound tRNA in the fixation of the mRNA appears unlikely . Both the affinity and stability of binding to the additional site were found lower for the heterologous tRNAPhe from yeast as compared to the homologous one . This difference possibly indicates some specificity of the E . coli ribosome for tRNAs from the same organism.

Biochemistry, 1984 Dec 18, 23(26), 6614 - 8
Optically detected magnetic resonance of Escherichia coli glutamic acid specific transfer ribonucleic acid and its anticodon-anticodon complex with yeast phenylalanine-specific transfer ribonucleic acid; Taherian MR et al.; The low-temperature phosphorescence and the optically detected magnetic resonance (ODMR) spectra of Escherichia coli tRNA2Glu and its anticodon-anticodon complex with yeast tRNAPhe are reported . The ODMR signals are assigned to the modified base 5-{(methylamino)-methyl}-2-thiouracil (mnm5S2U) located at the "wobble" position of the anticodon . The zero-field splittings (zfs) are larger than those found for 1-methyl-2-thiouracil previously studied in a 1-methyluracil host system by ODMR . They are comparable, however, to those found for neat, polycrystalline 1-methyl-2-thiouracil and for the latter dissolved in ethyl acetate solvent . In the polycrystalline sample, five traps with widely varying zfs are assigned . A very large (15-25%) reduction in the D parameter of mnm5S2U is found to occur on formation of the anticodon-anticodon complex with yeast tRNAPhe . Additional ODMR signals found in the complex are assigned to the wybutine base of yeast tRNAPhe . The extreme sensitivity of the zfs parameters of S2U to environmental perturbations is ascribed to the variable involvement of the heavy atom containing chromophore, C = S, in the triplet-state wave function, which is largely localized on the C = C - C = O portion of the molecule.

Biochemistry, 1984 Dec 18, 23(26), 6327 - 34
Structure in solution of M1 RNA, the catalytic subunit of ribonuclease P from Escherichia coli; Guerrier-Takada C et al.; The structure of M1 RNA, the RNA component of Escherichia coli RNase P, has been probed by mild digestion with a variety of ribonucleases . The results have been used to generate a model for the two-dimensional structure of M1 RNA . This model is similar in many respects to an earlier model that was based entirely on theoretical considerations . M1 RNA was digested with RNase T1 in buffer containing 10 mM MgCl2 (in which M1 RNA, by itself, has no catalytic activity) and in buffer containing 60 mM MgCl2 (in which M1 RNA can cleave precursors to tRNA molecules) . Under these conditions, the main features of the secondary structure are similar, but several minor differences are apparent . Such subtle changes in structure are also observed when M1 RNA is present in a binary complex with a substrate molecule, the precursor to E . coli tRNATyr.

Biochem J, 1984 Dec 15, 224(3), 799 - 815
DNA sequence around the Escherichia coli unc operon . Completion of the sequence of a 17 kilobase segment containing asnA, oriC, unc, glmS and phoS; Walker JE et al.; The nucleotide sequence is described of a region of the Escherichia coli chromosome extending from oriC to phoS that also includes the loci gid, unc and glmS . Taken with known sequences for asnA and phoS this completes the sequence of a segment of about 17 kilobases or 0.4 min of the E . coli genome . Sequences that are probably transcriptional promoters for unc and phoS can be detected and the identity of the unc promoter has been confirmed by experiments in vitro with RNA polymerase . Upstream of the promoter sequence is an extensive region that appears to be non-coding . Conserved sequences are found that may serve to concentrate RNA polymerase in the vicinity of the unc promoter . Hairpin loop structures resembling known rho-independent transcription termination signals are evident following the unc operon and glmS . The glmS gene encoding the amidotransferase, glucosamine synthetase, has been identified by homology with glutamine 5-phosphoribosylpyrophosphate amidotransferase.

Experientia, 1984 Dec 15, 40(12), 1407 - 10
Chemotactic and random movement of cord-blood granulocytes; Marodi L et al.; Chemotactic responsiveness and random movement of cord-blood granulocytes were studied with a modified Boyden's method . Cord-blood granulocytes were less active chemotactically than granulocytes from healthy children and adults, whereas the random filter movement of the cells from all three sources was about the same . In cord sera, concentrations of cell directed chemotaxis inhibitors were equal to those in sera from other age groups . Compared with the situation in healthy children and adults, the generation of chemotactic factors in cord-blood sera was impaired . This impairment was not related to an increased activity of chemotactic factor inactivators . Measurement of the cyclic nucleotide levels in granulocytes from cord-blood and from children belonging to various age groups revealed that the cord granulocytes have significantly lower concentrations of cAMP and cGMP, which could have been responsible for the decreased chemotactic responsiveness.

J Mol Biol, 1984 Dec 15, 180(3), 549 - 76
Translation is a non-uniform process . Effect of tRNA availability on the rate of elongation of nascent polypeptide chains; Varenne S et al.; We reported elsewhere (Varenne et al., 1982) that, during synthesis of a number of colicins in Escherichia coli, intermediate nascent chains of discrete sizes accumulated, suggesting a variable rate of translation . In this paper, a detailed analysis provides arguments that this phenomenon, at least for the proteins under study, is not related to aspects of messenger RNA such as secondary structure . It is linked to the difference in transfer RNA availability for the various codons . Experimental analysis of translation of other proteins in E . coli confirms that the main origin for the discontinuous translation in the polypeptide elongation cycle is the following . For a given codon, the stochastic search of the cognate ternary complex (aminoacyl-tRNA-EF-Tu-GTP) is the rate-limiting step in the elongation cycle: transpeptidation and translocation steps are much faster . The degree of slackening in ribosome movement is almost proportional to the inverse of tRNA concentrations . The verification of this model and its possible physiological significance are discussed.

Biochem Biophys Res Commun, 1984 Dec 14, 125(2), 784 - 9
Sulfoxide configuration in sparsomycin determines time-dependent and competitive inhibition of peptidyl transferase; Ash RJ et al.; Sparsomycin, ScRs configuration, was the most potent of the four possible stereoisomers as a competitive inhibitor of peptide bond formation . In addition, the configuration of the two chiral centers dictated whether the compound exhibited time- and temperature-dependent inhibition of peptidyl transferase when incubated with polysomes prior to enzyme assay . The data corroborate the thesis that a peptidyl transferase-mediated acylation of the pivotal sulfoxide moiety and subsequent Pummerer rearrangement play a significant role in the inhibitory properties of sparsomycin.

Biochem Biophys Res Commun, 1984 Dec 14, 125(2), 600 - 5
Detection of proteins which recognize and bind oriC sequences; Weinberger M et al.; Extracts from E . coli capable of supporting in vitro oriC-dependent DNA replication have been examined with a protein blotting protocol to identify DNA-binding proteins . Four polypeptide chains with apparent affinity for oriC DNA were detected.

Nucleic Acids Res, 1984 Dec 11, 12(23), 9205 - 8
Partial methylation of two adjacent adenosines in ribosomes from Euglena gracilis chloroplasts suggests evolutionary loss of an intermediate stage in the methyl-transfer reaction; Van Buul CP et al.; Bacterial, cytoplasmic and organellar ribosomes from a wide phylogenetic spectrum of organisms have a characteristic m6(2)Am6(2)A structure near the 3' end of the RNA of the small ribosomal subunit (SSU) . We have studied one of the few exceptions to this extremely conserved post-transcriptionally modified sequence, i.e . dimethylation of only one of the two A's in chloroplasts from Euglena gracilis . It was established that only the A closest to the 5' end is dimethylated, the other one being unmodified . The methylation reaction was studied in vitro using ribosomes from a kasugamycin resistant mutant (ksgA) of Escherichia coli and purified methyl-transferase . Using limited amounts of the methyl donor S-adenosylmethionine (SAM) a partial level of methylation (50% of control) was attained . It is shown that in this case the 3' proximal A is dimethylated while the other is not . This suggests that dimethylation takes place in two successive stages . Apparently in E . gracilis chloroplasts the first stage of methylation does not occur.

Biochim Biophys Acta, 1984 Dec 11, 805(4), 337 - 44
Putrescine distribution in Escherichia coli studied in vivo by 13C nuclear magnetic resonance; Frydman B et al.; In order to study the intracellular polyamine distribution in Escherichia coli, 13C-NMR spectra of {1,4-13C}putrescine were obtained after addition of the latter to intact bacteria . The 13C-enriched methylene signal underwent line broadening . When the cells were centrifuged after 90 min the cell-bound putrescine peak had a linewidth of 23 Hz, while the supernatant liquid showed an unbound putrescine signal with a linewidth smaller than 1 Hz . By using 13C-enriched internal standards it could be shown that the linewidening was not due to the heterogeneity of the medium or to an in vivo paramagnetic effect . Cell-bound putrescine was liberated by addition of trichloroacetic acid and was therefore non-covalently linked to macromolecular cell structures . Cell-bound {13C}putrescine could be displaced by addition of an excess of {12C}putrescine . When samples of membranes, soluble protein, DNA, tRNA and ribosomes from E . coli were incubated with {1,4-13C}putrescine, strong binding was detected only in the ribosomal and membrane fractions . The ribosome-putrescine complex showed properties similar to those determined with the intact cells . By measuring the nuclear Overhauser enhancements eta, it was possible to estimate that only about 50% of the polyamine was linked to the macromolecules . Determination of the T1 values of free and ribosomal-bound putrescine allowed the calculation of a correlation time, tau c = 4 X 10(-7) s for the latter . T1 and tau c values found for the ribosome-putrescine complex were those expected for a motional regime of slowly tumbling molecules.

Nucleic Acids Res, 1984 Dec 11, 12(23), 8819 - 34
Structure of the spinach chloroplast genes for the D2 and 44 kd reaction-centre proteins of photosystem II and for tRNASer (UGA); Holschuh K et al.; We have determined the sequence of the spinach (Spinacia oleracea) chloroplast genes for the photosystem II proteins, D2 and the 44 kd reaction-centre, chlorophyll a-binding protein, and for tRNASer (UGA) . The 3' end of the D2 gene overlaps the first 50 bp of the 5' end of the gene for the 44 kd protein . Northern RNA hybridization analysis indicates the two genes are cotranscribed into a single 3.5 kb RNA . The predicted molecular weight of the 353-residue D2 protein is 39536 and that of the 473-residue 44 kd protein is 51816 . Both proteins are hydrophobic containing at least five possible membrane-spanning domains . D2 shows significant homology to the 32 kd herbicide-binding protein (Zurawski et al., (1982) Proc . Natl . Acad . Sci . USA 79, 7699-7703), and parts of the 44 kd protein show obvious similarities to parts of the 51 kd reaction-centre, chlorophyll a-binding protein of photosystem II (Morris and Herrmann (1984) Nucleic Acids Res . 12, 2837-2850) . The gene for tRNASer (UGA) which is on the opposite strand to and transcribed towards the photosystem II genes is 72% homologous with the corresponding Escherichia coli tRNASer.

Nucleic Acids Res, 1984 Dec 11, 12(23), 9083 - 93
Inhibition and enhancement of phleomycin-induced DNA breakdown by aromatic tricyclic compounds; Grigg GW et al.; Cationic aromatic tricyclic compounds including triphenylmethane dyes, phenazines, phenoxazines, acridines, phenothiazines, phenanthridinium compounds, anthracenes and xanthene dyes, which amplify cell killing in phleomycin-treated Escherichia coli B cells also modified phleomycin-induced breakdown of DNA to acid-soluble fragments . A plot of DNA breakdown as a function of concentration was bell-shaped for each of the active compounds, i.e . as the concentration increased, DNA breakdown was enhanced initially, but above a certain concentration, the proportion of DNA degraded declined, often to zero . One of the compounds, acriflavine, when tested also inhibited DNA breakdown following ultraviolet irradiation . A study, by sedimentation methods, of DNA single-strand breakage in phleomycin-treated E . coli cells, using 3 representative compounds, Crystal Violet, 3,6-diaminoacridine and Methylene Blue, revealed a consistent increase in DNA strand breaks as concentration of compound increased . In similar experiments with ethidium bromide the breakage yield/concentration curve exhibited a maximum . In general, however, it seems that the inhibition of DNA-breakdown observed at higher concentrations of these amplifying compounds is not explicable by an effect on the primary breakage event, but is due to suppression of exonucleolytic activity in the cells.

FEBS Lett, 1984 Dec 10, 178(2), 283 - 7
Stoichiometry of GTP hydrolysis in a poly(U)-dependent cell-free translation system . Determination of GTP/peptide bond ratios during codon-specific elongation and misreading; Gavrilova LP et al.; The stoichiometry of GTP hydrolysis during peptide elongation in the processes of codon-specific translation and misreading of polyuridylic acid was determined in a cell-free system in which all ribosomes were active in peptide synthesis . Ribosomes carrying oligophenylalanine presynthesized on poly(U) covalently bound to Sepharose were used . In the codon-specific translation of poly(Phe) on poly(U)-Sepharose at optimal Mg2+ concentration (6 mM MgCl2), the ratio of GTP cleaved to Phe polymerized was found to be about 2 (+/- 0.1) . Under the same conditions but during misreading (elongation of polyleucine on poly(U)-Sepharose) the GTP/Leu ratio increased 10 times (from 16 to 25 in different experiments).

FEBS Lett, 1984 Dec 10, 178(2), 223 - 7
The excision of AP sites by the 3'-5' exonuclease activity of the Klenow fragment of Escherichia coli DNA polymerase I; Bailly V et al.; The 3' AP endonucleases (class I) are said to hydrolyze the phosphodiester bond 3' to AP sites yielding 3'-OH and 5'-phosphate ends; on the other hand, the resulting 3' terminal AP site is not removed by the 3'-5' exonuclease activity of the Klenow fragment {1} . We show that AP sites in DNA are easily removed by the 3'-5' exonuclease activity of the Klenow fragment and that they are excised as deoxyribose-5-phosphate . It is suggested that the 3' AP endonucleases are perhaps not the hydrolases they are supposed to be.

J Biol Chem, 1984 Dec 10, 259(23), 14793 - 803
Mercurial-promoted Zn2+ release from Escherichia coli aspartate transcarbamoylase; Hunt JB et al.; The release of Zn2+ from aspartate transcarbamoylase (ATCase; c6r6) upon challenge by p-hydroxymercuriphenylsulfonate (PMPS) has been studied using the sensitive, high-affinity metallochromic indicator 4-(2-pyridylazo)resorcinol at pH 7.0 . When the--SH group of each catalytic (c) chain is protected, 1 Zn2+ is released for every 4 eq of PMPS added to ATCase during titration of the 24--SH groups of regulatory (r) chains . Moreover, the release of Zn2+ is a linear function of PMPS added, indicating that the rate-limiting step in Zn2+ release is mercurial attack on the 1st of the 4 r--SH groups bonded tetrahedrally to Zn2+ in an r chain near c:r contacts . Dissociation of ATCase is linked to Zn2+ release and mercaptide formation; e.g . upon addition of 4 eq of PMPS to ATCase in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer, 1/6th of ATCase is dissociated to c3 and r2 subunits at approximately 83% of the rate of Zn2+ release, with no accumulation of the c6r4 intermediate as is observed in KPO4 buffer . Adding less than or equal to 4 PMPS/ATCase, the release of Zn2+ is first-order in {PMPS} and is virtually independent of {ATCase} with an activation energy of 18 kcal/mol . With large excesses of PMPS, stopped-flow traces show a lag period followed by pseudo first-order release of Zn2+ from ATCase and the reaction order in {PMPS} = approximately 1.3 . Under these conditions, PMPS has a chaotropic effect on ATCase; the activation energy for Zn2+ release is much lower than that obtained with limiting PMPS and is increased by the presence of phosphate or active-site ligand from 6.6 to approximately 12 kcal/mol . A reasonable explanation of the observed kinetic data is that the organomercurial reagent binds reversibly to nitrogenous side chain groups in an ATCase molecule prior to the rate-limiting reaction with a sulfhydryl group.

J Biol Chem, 1984 Dec 10, 259(23), 14688 - 94
Asymmetric reconstitution of homogeneous Escherichia coli sn-glycerol-3-phosphate acyltransferase into phospholipid vesicles; Green PR et al.; The sn-glycerol-3-phosphate (glycerol-P) acyltransferase of Escherichia coli cytoplasmic membrane was purified in Triton X-100 (Green, P . R., Merrill, A . H., Jr., and Bell, R . M . (1981) J . Biol . Chem . 256, 11151-11159) and incorporated into mixed micelles containing Triton X-100, phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and beta-octyl glucoside . Enzyme activity was quantitatively reconstituted from the mixed micelle into single-walled phospholipid vesicles by chromatography over Sephadex G-50 . Activity coeluted with vesicles of 90-nm average diameter on columns of Sepharose CL-4B and Sephacryl S-1000 . These vesicles contained less than 2 Triton X-100 and 5 beta-octyl glucoside molecules/100 phospholipid molecules . Calculations suggested that up to eight 91,260-dalton glycerol-P acyltransferase polypeptides were incorporated per 90-nm vesicle . The pH dependence and apparent Km values for glycerol-P and palmitoyl-CoA of the glycerol-P acyltransferase reconstituted into vesicles were similar to those observed upon reconstitution by mixing of the enzyme in Triton X-100 with a 20-fold molar excess of sonicated phosphatidylethanolamine:phosphatidylglycerol:cardiolipin, 6:1:1 . The integrity of vesicles containing glycerol-P acyltransferase was established by trapping 5,5'-dithiobis-(2-nitrobenzoic acid) . Chymotrypsin inactivated greater than 95% of the glycerol-P acyltransferase in intact vesicles and cleaved the 91,260-dalton polypeptide into several vesicle-bound and several released peptides, indicating that critical domains of the enzyme are accessible in intact vesicles . Trinitrobenzene sulfonate and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene caused greater than 90% loss of glycerol-P acyltransferase in vesicles . Disruption of vesicles with Triton X-100 did not reveal significant latent activity . These data strongly suggest that the glycerol-P acyltransferase was reconstituted asymmetrically into the vesicles with its active site facing outward.

J Biol Chem, 1984 Dec 10, 259(23), 14824 - 8
Regulation of expression and nucleotide sequence of the Escherichia coli dapD gene; Richaud C et al.; Regulation of the Escherichia coli dapD gene involved in diaminopimelate and lysine biosynthesis was unknown as no convenient enzymatic assay was available until recently . This gene was cloned into pBR322 from a lambda transducing phage; its complete nucleotide sequence was established . This sequence shows that the dapD gene is composed of a single cistron encoding a 274-amino acid polypeptide, Mr 30,040 . Enzymatic activity measurements show that this gene encodes the tetrahydrodipicolinate N-succinyltransferase which catalyzes the third step of the specific lysine-diaminopimelate pathway . The transcriptional start of the dapD gene was localized; the identified promoter signals are weak compared to those from the E . coli promoter consensus sequence . The dapD gene-coding sequence is followed by a typical rho-independent transcriptional termination sequence . A study using an operon fusion constructed in vitro between the dapD promoter and the galK structural gene indicated that dapD gene expression is repressed by lysine; no attenuation-like sequence can be found to account for this regulation . At the present time, out of the 9 genes involved in diaminopimelate and lysine biosynthesis, 6 are known to be lysine regulated.

J Biol Chem, 1984 Dec 10, 259(23), 14505 - 7
The sequence of metK, the structural gene for S-adenosylmethionine synthetase in Escherichia coli; Markham GD et al.; The DNA sequence of the Escherichia coli metK gene has been determined . Protein sequence data for purified S-adenosylmethionine synthetase have also been obtained and confirm that metK is the structural gene for S-adenosylmethionine synthetase in E . coli . The sequence of the amino-terminal 35 residues of purified S-adenosylmethionine synthetase localizes the beginning of the coding region of the DNA . The open reading frame extends 1152 bases and codes for a 384-residue protein of Mr = 41,941 . The gene is transcribed clockwise on the E . coli chromosome . The DNA region 5' to the coding region was found to contain symmetrical sequences suggestive of operator structures and homologous to sequences upstream from other met genes sharing the same regulatory mechanism.

J Biol Chem, 1984 Dec 10, 259(23), 14472 - 80
The DNA dependence of the ATPase activity of DNA gyrase; Maxwell A et al.; We have studied the ATPase activity of DNA gyrase both in the absence and presence of DNA . In the absence of DNA we show that the gyrase B protein alone has a very low level of ATPase activity which can be increased many-fold by pretreatment of the B protein with heat or urea . When both the gyrase A protein and linear DNA are also present, the ATPase activity of the untreated B protein is greatly stimulated . We find that the extent of stimulation is dependent upon the length of the DNA but largely independent of DNA sequence . DNA molecules greater than 100 base pairs in length are much more effective in stimulating the gyrase ATPase than those of 70 base pairs or less, although short DNA molecules will stimulate the ATPase at high concentrations . The behavior of long and short DNA molecules with respect to ATPase stimulation is also reflected in their abilities to bind DNA gyrase . To account for these data we propose a model for the interaction of gyrase with ATP and DNA in which ATP hydrolysis requires the binding of DNA to two sites on the enzyme.

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