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U Canosi

Publications and source records attributed to U Canosi.

At least 19 recordsLinked to original sources
PubMed1996

A highly precise reporter gene bioassay for type I interferon.

We describe the setting up and validation of a reporter gene assay for type I IFN based on monkey Vero cells transfected with pMx-Luc, a plasmid carrying the luciferase gene under the control of the type I IFN inducible mouse Mx1 promoter. Vero cells were stably transfected with pMx-Luc and clone 3-143/5 was selected on the basis of luciferase inducibility by IFN-beta. A linear dose-response relationship was found between 1 and 16 IU/ml IFN-beta. The assay was shown to be specific for IFN-alpha and -beta as no effect by a number of other cytokines including IFN-gamma could be detected. In order to render the IFN-beta reporter gene assay protocol more suitable for routine assays, a 3 x 3 balanced parallel line assay design was applied using a 96-well luminometer for luminescence measurement. The assay was shown to be precise with a coefficient of variation of less than 9%. This assay is characterized by high precision coupled to high efficiency, as reflected by a very short assay duration (1 day), when compared to the classical cytopathic effect assays for IFNs and the previously published IFN reporter gene assay based on growth hormone measurement (Lleonart, R., Näf, D., Browning, H. and Weissmann, C. (1990) A novel, quantitative bioassay for type 1 interferon using a recombinant indicator cell line. Biotechnology 8, 1263-1267).

Animals↗
PubMed1994

Determination of haptoglobin expression in IL-6 treated HepG2 cells by ELISA and by RNA hybridization--evaluation of a quantitative method to measure IL-6.

Interleukin-6 (IL-6) is known to be an important modulator of acute phase (AP) protein expression in hepatocytes both in vivo and in vitro. In the present study the inducing activity of IL-6 on the expression of the AP protein haptoglobin (HP) by the human hepatoma cell line HepG2, has been evaluated. HP mRNA inducibility was analysed by Northern and slot-blot hybridization, while HP protein was detected by means of an ELISA procedure. A dose-response relationship from 0.3 to 4.8 ng/ml of a human recombinant IL-6 preparation derived from a Chinese hamster ovary (CHO) cell line was observed after 48 h of treatment. Comparable results were obtained by analysing both HP mRNA expression and HP protein secretion. Detectable induction of HP protein secretion was observed with as little as 25 pg/ml of IL-6. The effect of IL-6 was potentiated by dexamethasone, while an inhibition on HP mRNA inducibility could be prevented by lowering the foetal calf serum (FCS) concentration to 1%. Preliminary data indicate that neither IL-1 beta nor TNF-alpha were able to induce significantly HP mRNA expression and protein secretion. The activity ratio between two IL-6 preparations (from CHO and E. coli cells) obtained with a conventional IL-6 bioassay (i.e., T1165 cell growth assay) was comparable to that obtained in the induction of HP expression. The nominal specific activity of the CHO-derived IL-6 was two to three times higher with both responses.

Animals↗
PubMed1989

Cyanogen bromide cleavage at methionine residues of polypeptides containing disulfide bonds.

A method for cleaving polypeptides at their methionine residues without affecting intramolecular disulfide bonds is described. This method may be applied for cleaving recombinant heterologous hybrid polypeptides with release of the interesting peptide. The method may also be applied to assign the correct positions of disulfide bonds in protein molecules.

Chromatography, High Pressure Liquid↗
PubMed1988

Expression in Escherichia coli and characterization of human growth-hormone-releasing factor.

A chemically synthesized DNA sequence, coding for the 44 amino acid residues of human growth-hormone-releasing factor (GRF) preceded by a tryptophan codon, was cloned in frame with Escherichia coli trpE gene within a pBR322-derived plasmid. GRF was expressed in E. coli as a fused polypeptide chain (TrpE-GRF) and then the GRF amino acid sequence was released from the fused protein by specific chemical cleavage at the tryptophan residue using o-iodosobenzoic acid. The thioether group of the methionine residue of GRF was converted in the sulfonium salt derivative, in order to prevent irreversible oxidation of methionine to the sulfone derivative by the o-iodosobenzoic acid reagent. GRF was purified by HPLC and characterized in terms of amino acid composition after acid hydrolysis, protein sequencing and gel electrophoretic behaviour. These data clearly established that the biosynthetic GRF was identical to the natural one, except for the lack of amidation at the carboxyl-terminal amino acid. Far-ultraviolet circular dichroism measurements established that both biosynthetic and natural GRF are devoid of secondary structure in aqueous solution at neutral pH, whereas both peptide samples achieve a high percentage of helical structure in the presence of trifluoroethanol.

Chromatography, High Pressure Liquid↗
PubMed1985

The genome of Bacillus subtilis phage SPP1: structure of an early promoter.

The strongest of five 'early' promoters of Bacillus subtilis phage SPP1 was localized in a DNA restriction fragment by analysis of RNA polymerase binding and R-loop formation. The nucleotide sequence of the promoter region was established. The signal structures identified were similar to those recognized by the sigma 55 RNA polymerase of B. subtilis. The promoter precedes an open reading frame with 51 codons. A protein with the Mr predicted from the nucleotide sequence was identified in minicells.

Autoradiography↗
PubMed1982

SPP1-mediated plasmid transduction.

The virulent Bacillus subtilis phage SPP1 transduces plasmid DNA. Plasmid-transducing phages contain only plasmid DNA. Such DNA represents a concatemer of monomeric plasmid molecules with the molecular weight of mature SPP1 DNA. Biological parameters of plasmid transduction are described.

Bacillus subtilis↗
PubMed1981

Plasmid transformation in Bacillus subtilis. The significance of partial homology between plasmid and recipient cell DNAs.

A series of hybrid plasmids consisting of pC194 or pUB112 and B. subtilis DNA were constructed. In contrast to plasmid pC194, purified monomeric forms of such plasmids were active in transformation, provided the recipient cells were recombination proficient. Similarly the monomers of pC194 derived plasmids, containing bacteriophage phi 105 DNA were able to transform phi 105 lysogenic but not nonlysogenic cells. From the results it is concluded that the presence of DNA/DNA homology between chromosomal DNA of the recipient cell and part of the hybrid plasmids used is a sufficient condition to endow monomeric plasmids with transforming activity.

Bacillus subtilis↗
PubMed1981

Plasmid transformation in Bacillus subtilis. Alterations introduced into the recipient-homologous DNA of hybrid plasmids can be corrected in transformation.

Various alterations (deletions, additions, inversions) were introduced into portions of pC194/B. subtilis or pC194/phi 105 hybrid plasmid molecules which are homologous to the DNA of recipients in transformation. These plasmids are stably maintained in transformations of recombination deficient cells. In transformations of recombination proficient cells, they can be corrected to those plasmid forms into which the alterations were originally introduced. This correction is most pronounced when transformations are performed with monomeric ccc forms of hybrid plasmids. It is suggested that correction is a consequence of mismatch repair occurring in the synapsis of homologous portions of plasmid and resident DNAs.

Bacillus subtilis↗
PubMed1981

Plasmid transformation in Bacillus subtilis: fate of plasmid DNA.

Only multimeric, and not monomeric forms of B. subtilis plasmids can transform B. subtilis cells (Canosi et al. 1978). This finding prompted us to study the physico-chemical fate of plasmid DNA in transformation. Competent cells of B. subtilis were exposed to either unfractionated preparations or to preparations of multimeric plasmid DNA. Plasmid DNA was re-extracted from such cells and then analyzed by sedimentation and isopycnic centrifugation and also defined by its sensitivity to nuclease S1 degradation. No double-stranded plasmid DNA could be recovered from cells transformed with unfractionated plasmid preparations which contained predominantly monomeric covalently closed circular (CCC) DNA. Re-extracted plasmid DNA was single-stranded, had a molecular weight considerably smaller than monomer length DNA and had been subject to degradation to acid soluble products. However, when transformations were performed with multimeric DNA (constructed by in vitro ligation of linearized pC194 DNA), both double-stranded and partially double-stranded DNA could be recovered in addition to single-stranded DNA. We assume that plasmid DNA is converted to a single-stranded form in transformation, irrespective of its molecular structure. Double-stranded and partially double-stranded DNAs found in transformation with multimeric DNA would be the products of intramolecular annealing.

Bacillus subtilis↗
PubMed1981

Plasmid transformation in Bacillus subtilis: effects of insertion of Bacillus subtilis DNA into plasmid pC194.

We have constructed a hybrid plasmid pBC1, which consists of plasmid pC194 with an insert of B. subtilis DNA as its HindIII restriction site. This plasmid is stably maintained in B. subtilis. In contrast with pC194, monomeric ccc forms of pBC1 are active in transformation. Transformations with these monomeric molecules of pBC1 have a stringent requirement for recombination proficiency, as defined by recE in the recipient cell. The extent of dependence of the transforming activity of oligomeric pBC1 DNA on the recombination proficiency of the recipient cell decreases with increasing oligomer size. A model of DNA processing during plasmid transformation of B. subtilis is presented.

Bacillus subtilis↗
PubMed1980

The effect of trimethylpsoralen--crosslinks on entry of donor DNA in transformation and transfection of Bacillus subtilis.

Transforming chromosomal DNA, irradiated with long-wave UV light in the presence of 4,5',8-trimethylpsoralen (TMP) binds to competent B. subtilis cells as effectively as non-treated DNA, but its transforming activity is strongly reduced. Uptake studies show that the entry of transforming DNA, after some stimulation by short periods of irradiation in the presence of TMP, decreases proportionally with the dose of irradiation. Crosslinking was quantitated by electron microscopy. Since the number of crosslinks increases proportionally with the dose of irradiation, it is suggested that entry of donor DNA is prevented by crosslinks. The inhibition of entry of DNA is paralleled both by decreased breakdown of crosslinked DNA interacting with competent cells, and decreased breakdown by nuclease activity liberated during protoplasting of competent cultures. These data support the model of Lacks et al. (1976) which postulates that a membrane-bound deoxyribonuclease is engaged in the entry of donor DNA into the competent cell. The transforming activity of the chloramphenicol-resistance carrying plasmid pC194, originally obtained from Staphylococcus aureus, is also destroyed by TMP crosslinks. Contrary to chromosomal DNA, its association with the cells is stimulated by long-wave UV irradiation in the presence of TMP, but experiments are presented suggesting that the DNA is still vulnerable to the action of exogenous pancreatic deoxyribonuclease. Transfecting SPP1 DNA is also inactivated by TMP crosslinks. Marker rescue of transfecting DNA containing crosslinks occurs; the extent of rescue of one marker is considerably in excess of that of linked markers.

Bacillus subtilis↗
PubMed1980

Restriction and modification in Bacillus subtilis: identification of a gene in the temperate phage SP beta coding for a BsuR specific modification methyltransferase.

A gene coding for a modifying DNA-methyltransferase which methylates the central C in the BsuR recognition sequence 5'GGCC was identified in the genome of the temperature Bacillus subtilis phage SP beta. This gene is expressed only after induction of the prophage by either mitomycin C or UV. The presence of active methyltransferase in induced cells leads to modification of BsuR recognition sites in SP beta DNA as well as in heterologous DNA.

Bacillus subtilis↗
PubMed1979

Different specific activities of the monomeric and oligomeric forms of plasmid DNA in transformation of B. subtilis and E. coli.

(1) The low residual transforming activity in preparations of monomeric, supercoiled, circular (CCC) forms of the plasmids pC194 and pHV14 could be attributed to the presence in such isolates of a small number of contaminating multimeric molecules. (2) E. coli derived preparations of pHV14, as in vitro recombinant plasmid capable of replication in both E. coli and B. subtilis, contain oligomeric forms of plasmid DNA in addition to the prevalent monomeric CCC form. The specific transforming activity of pHV14 DNA for E. coli is independent of the degree of oligomerization, whereas in transformation of B. subtilis the specific activity of the purified monomeric CCC molecules is at least four orders of magnitude less than that of the unfractionated preparation. (3) Oligomerization of linearized pHV14 DNA by T4 ligase results in a substantial increase of specific transforming activity when assayed with B. subtilis and causes a decrease when used to transform E. coli.

Bacillus subtilis↗
PubMed1979

Effect of 6-(p-hydroxyphenylazo)-uracil on the homologous and heterologous transduction processes in Bacillus subtilis.

We have studied the effect of 6-(p-hydroxyphenylazo)-uracil on the recombination processes that operate in the homologous and heterologous transduction mediated by PBS1 and SP10 phages of Bacillus subtilis. The results obtained demonstrate that the process of heterologous genetic exchange is sensitive to this compound, whereas the homologous process is not. The present data, along with those of our previous work (U. Canosi, A. G. Siccardi, A. Falaschi, and G. Mazza, J. Bacteriol. 126:108--121, 1976), suggest that the DNA polymerase III is involved in the recombination process that operates in transformation and heterologous transduction, whereas homologous transduction follows a partially independent pathway not involving this protein.

Bacillus subtilis↗
PubMed1978

The relationship between molecular structure and transformation efficiency of some S. aureus plasmids isolated from B. subtilis.

DNA preparations of the chloramphenicol resistance determining S. aureus plasmids pC194, pC223, and PUB112 can be fractionated by gel electrophoresis into various bands. Electronmicroscopic investigations of these various molecular species obtained with pC194 indicated that, depending on the preparations, 70 to 80% of the molecules were monomers, while the rest consisted of various classes of concatemeric and/or interlocked multimers. Measurements of the specific transforming activity of the various molecular classes indicated that the monomers had less than one thousandth the activity of the multimeric plasmic DNA. pC194 DNA of high specific transforming activity could also be obtained by ligation of HindIII generated monomers into concatemeric DNA.

Bacillus subtilis↗
PubMed1978

Studies on transduction process by SPP1 phage.

The conditions for optimal transduction efficiency of the Bacillus subtilis phage SPP1 have been investigated. By irradiating transducing lysates with u.v. light we have been able to obtain a fivefold increase in the number of transductants and to reduce strongly the interference caused by infective particles. Any dependence of SPP1 transduction on PBSX induction has been ruled out by the use of xin mutants, which are unable to induce the defective phage. SPP1 mediated transduction is susceptible to the restriction and modification system of B. subtilis. The rec functions involved in the recombination of the SPP1 transduced DNA fragment are probably identical to those required in DNA transformation and heterologous PBS1 transduction.

Bacillus subtilis↗
PubMed1977

Host cell reactivation of Bacillus subtilis bacteriophages.

Host cell reactivation of ultraviolet-irradiated phage can be used as a probe of the bacterial repair system and to determine phage and cellular contributions to the repair process. Using the Bacillus subtilis phages SPP1, SP01, phie, and phi29, we found that the uvr-1 and polA functions are involved in the host cell reactivation of the four phages. SPP1 was the only phage whose reactivation was also decreased in recA, recD, and recF mutant cells. We studied variations of host cell reactivation for SPP1 during spore outgrowth; at high ultraviolet doses the activity of a spore repair system requiring deoxyribonucleic acid polymerase I became evident. The spore repair system was completely replaced by the vegetative one by 120 min of outgrowth.

Bacillus subtilis↗