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 or