tetracycline and Chromosome-Deletion

Shigella flexneri SFL124, with a deletion encompassing all, or nearly all, of the coding sequence of gene aroD was obtained after selection on a fusaric acid medium supplemented with 2,3-dihydroxybenzoic acid for tetracycline-sensitive mutants of S. flexneri SFL114 which is an aroD::Tn10 transductant. Two of 20 tetracycline-sensitive mutants tested in colony hybridization with a 32P-labelled DNA probe of approximately 1400 base pairs (comprising all except the 75 N-terminal base pairs of the coding region of gene aroD) did not hybridize. The selected mutant SFL124 is Congo-red positive, invades and shows a limited multiplication in HeLa cells and does not cause keratoconjunctivitis in guinea-pigs. It is well tolerated by Macaca fascicularis monkeys, is excreted for up to 4 days, elicits a slight inflammatory reaction in the colonic mucosa, stimulates significant secretory IgA responses in the intestine and serum IgA and IgG responses against the S. flexneri cell envelope lipopolysaccharide. The immune response conferred a complete protection against challenge with 1 x 10(11) (equivalent to a 100 LD50 dose) live S. flexneri SFL1.

Topics: Administration, Oral; Animals; Antibodies; Bacterial Vaccines; Chromosome Deletion; Congo Red; Dysentery, Bacillary; Female; Guinea Pigs; HeLa Cells; Humans; Immunoenzyme Techniques; Immunoglobulin A; Immunoglobulin G; Intestine, Large; Macaca fascicularis; Mutation; Sensitivity and Specificity; Shigella flexneri; Tetracycline; Vaccination

Two plasmids which express either nearly intact or truncated filamentous hemagglutinin (FHA) from Bordetella pertussis and which are marked with a tetracycline resistance (Tcr) gene were transformed into Salmonella dublin SL1438, an aroA deletion mutant intended for use as an attenuated oral vaccine against salmonellosis. These S. dublin recombinants, when fed to mice, induced serum immunoglobulin, immunoglobulin M (IgM), and sometimes IgA antibody responses to FHA and S. dublin. In addition, IgA antibodies against FHA were found in gut wash fluids. S. dublin carrying pDB2300, a multicopy plasmid encoding truncated FHA protein, induced a better antibody response than did S. dublin carrying pDB2000, a low-copy-number plasmid encoding full-sized FHA. Administration of tetracycline to mice enhanced the stability of recombinant plasmids, and tetracycline-treated mice developed higher anti-FHA titers. Although neither strain examined is suitable for use in a human oral vaccine, these data demonstrated that an immune response against B. pertussis FHA could be induced by oral administration of live attenuated recombinant strains of S. dublin and suggested that development of a live oral attenuated vaccine against pertussis may be possible.

Topics: Animals; Antibodies, Bacterial; Bacterial Vaccines; Bordetella pertussis; Chromosome Deletion; Female; Gene Expression; Hemagglutinins; Male; Mice; Mice, Inbred BALB C; Plasmids; Salmonella; Salmonella Infections, Animal; Tetracycline; Vaccines; Vaccines, Attenuated; Vaccines, Synthetic

The ability to clone a variety of sequences with varying capabilities of adopting non-B structures (left-handed Z-DNA, cruciforms or triplexes) into three loci of pBR322 was investigated. In general, the inserts were stable (non-deleted) in the EcoRI site (an untranslated region) of pBR322. However, sequences most likely to adopt left-handed Z-DNA or triplexes in vivo suffered deletions when cloned into the BamHI site, which is located in the tetracycline resistance structural gene (tet). Conversely, when the promoter for the tet gene was altered by filling-in the unique HindIII or ClaI sites, the inserts in the BamHI site were not deleted. Concomitantly, the negative linking differences of the plasmids were reduced. Also, inserts with a high potential to adopt Z-DNA conformations were substantially deleted in the PvuII site of pBR322 (near the replication origin and the copy number control region), but were less deleted if the tet promoter was insertion-mutated. The deletion phenomena are due to the capacity of these sequences to adopt left-handed Z-DNA or triplexes in vivo since shorter inserts, less prone to form non-B DNA structures, or random sequences, did not exhibit this behavior. Sequences with the potential to adopt cruciforms were stable in all sites under all conditions. These results reveal a complex interrelationship between insert deletions (apparently the result of genetic recombination), negative supercoiling, and the formation of non-B DNA structures in living Escherichia coli cells.

Topics: Chromosome Deletion; DNA Transposable Elements; DNA, Bacterial; Escherichia coli; Genes; Genes, Bacterial; Mutation; Plasmids; Promoter Regions, Genetic; Recombination, Genetic; Regulatory Sequences, Nucleic Acid; Tetracycline

Tn7 transposes from the chromosome of Pseudomonas aeruginosa into the plasmid R68.45 with tandem IS21, at up to 400 times the frequency that it transposes into R68, which has only one copy of IS21. While R68::TN7 derivatives are stable, R68.45::Tn7 isolates undergo frequent deletions. Instability of R68.45 occurs whether Tn7 is inserted into the plasmid (cis configuration) or into the bacterial chromosome (trans configuration). The deletions of R68.45 start at the junction between the tandem IS21 copies and proceed clockwise, ending in the region of oriT. It appears that Tn7 and IS21 can mutually stimulate transposition of each other.

Topics: Chromosome Deletion; Chromosome Mapping; DNA Restriction Enzymes; DNA Transposable Elements; DNA, Bacterial; Gene Expression Regulation; Kanamycin; Pseudomonas aeruginosa; R Factors; Tetracycline

We have investigated by Southern blot hybridization the rate of IS10 transposition and other Tn10/IS10-promoted rearrangements in Escherichia coli and Salmonella strains bearing single chromosomal insertions of Tn10 or a related Tn10 derivative. We present evidence for three primary conclusions. First, the rate of IS10 transposition is approximately 10(-4) per cell per bacterial generation when overnight cultures are grown and plated on minimal media and is at least ten times more frequent than any other Tn10/IS10-promoted DNA alteration. Second, all of the chromosomal rearrangements observed can be accounted for by two previously characterized Tn10-promoted rearrangements: deletion/inversions and deletions. Together these rearrangements occur at about 10% the rate of IS10 transposition. Third, the data suggest that intramolecular Tn10-promoted rearrangements preferentially use nearby target sites, while the target sites for IS10 transposition events are scattered randomly around the chromosome.

Topics: Chromosome Deletion; Chromosome Inversion; Culture Media; DNA Transposable Elements; Drug Resistance, Microbial; Escherichia coli; Galactose; Histidine; Recombination, Genetic; Salmonella; Selection, Genetic; Tetracycline

Plasmids isolated from two different clinical isolates of Salmonella typhimurium, both resistant to the antibiotics ampicillin, tetracycline, streptomycin and chloramphenicol, were used to transform Escherichia coli. Segregation of antibiotic-resistance determinants occurred in both cases. Analysis of plasmids from one set of segregants by DNA-DNA hybridisation indicated that the segregation was due to precise deletions in the transforming plasmid.

Topics: Ampicillin; Anti-Bacterial Agents; Chloramphenicol; Chromosome Deletion; Escherichia coli; Phenotype; R Factors; Salmonella typhimurium; Streptomycin; Tetracycline; Transformation, Bacterial

Reducing sugars such as glucose or glucose 6-phosphate (Glc-6-P) have been shown previously to modify the amino groups of nucleotides and single-stranded DNA. We have examined the mutagenic effect of Glc-6-P-induced lesions in the double-stranded DNA plasmid pBR322. Seventeen mutants of the Ampr Tets phenotype were isolated from plasmid preparations whose transforming capacity had been decreased by incubation with Glc-6-P. A number of the mutant plasmids were found to have undergone gross DNA alterations, including insertions and deletions, as well as the development of multiple species originating from a single cell. The ability of an endogenous reducing sugar to induce extensive DNA rearrangements suggests that these lesions may be significant contributors to cellular mutation.

Topics: Chromosome Deletion; DNA Replication; DNA, Bacterial; Fusaric Acid; Glucose-6-Phosphate; Glucosephosphates; Mutation; Oxidation-Reduction; Penicillin Resistance; Plasmids; Tetracycline; Transformation, Genetic

A chromosomal tetracycline resistance (Tcr) determinant previously cloned from Streptococcus mutans into Streptococcus sanguis (Tobian and Macrina, J. Bacteriol. 152:215-222, 1982) was characterized by using restriction endonuclease mapping, deletion analysis, and Southern blot hybridization. Deletion analysis allowed localization of the Tcr determinant to a 2.8-kilobase region of the originally cloned 10.4-kilobase sequence. This cloned determinant hybridized to a representative of the tetM class of streptococcal Tcr determinants but not to representatives of the tetL and tetN classes and, like other tetM determinants, mediated high-level resistance to tetracycline and low-level resistance to minocycline. A portion (approximately 3 kilobases) of the isolated streptococcal fragment was subcloned into Escherichia coli, where it conferred resistance to tetracycline and minocycline. Two proteins with apparent molecular weights of 33,000 and 35,000, encoded by the S. mutans DNA, were synthesized in E. coli minicells. Insertion of DNA into a unique SstI site of the cloned S. mutans fragment resulted in inactivation of Tcr expression in E. coli and S. sanguis, as well as loss of production of both the 33,000- and 35,000-dalton proteins in E. coli minicells. Incubation of minicells in subinhibitory concentrations of tetracycline did not result in changes in the levels of synthesis of either protein. Our data suggest that at least one of these proteins is involved in the expression of Tcr.

Topics: Chromosome Deletion; Chromosomes, Bacterial; Cloning, Molecular; DNA Restriction Enzymes; Drug Resistance, Microbial; Escherichia coli; Nucleic Acid Hybridization; Phenotype; R Factors; Streptococcus mutans; Tetracycline

The structural gene region for tetracycline resistance on Tn10 consists of two complementation groups, tetA and tetB (M. S. Curiale and S. B. Levy, J. Bacteriol. 151:209-215, 1982). Using a series of deletion mutants, we have determined that the tetA region is 450 to 600 base pairs long and that the tetB region, which is adjacent to tetA, is 600 to 750 base pairs long. Point mutations in either tetA or tetB affected the amount and size of the inducible inner-membrane Tet protein synthesized in Escherichia coli maxicells. Moreover, deletions in these regions led to the synthesis of an appropriately smaller Tet protein. A single tetracycline-inducible RNA of about 1,200 bases was detected that was homologous with the tetracycline resistance structural gene region. These results indicate that the tetA and tetB complementation regions represent two parts of a single gene encoding two domains of the tetracycline resistance protein Tet.

Topics: Bacterial Proteins; Chromosome Deletion; DNA Transposable Elements; Drug Resistance, Microbial; Escherichia coli; Genes; Genes, Bacterial; Genetic Complementation Test; Membrane Proteins; Mutation; Plasmids; Tetracycline

Broad host-range plasmid RK2 is able to replicate in a controlled manner in most Gram negative bacterial species. To analyze the elements of its control mechanism, we have measured the copy number in Escherichia coli of mini-RK2 replicons isogenic except for defined deletions in regions adjacent to the vegetative replication origin, oriVRK2, which have previously been implicated in copy number control because of their expression of plasmid incompatibility. The results indicate that while the previously defined 700-bp HaeII oriVRK2 fragment carries one copy control element (copA), a second (copB) lies at least partly outside this fragment towards the tetracycline resistance genes of RK2. Deletions affecting both these regions give a mini replicon with a copy number of 35-40 compared with 4-7 for parental RK2. Further incompatibility experiments indicate that targets for both incA (copA) and incB (copB) lie within the 700-bp HaeII oriVRK2 fragment.

Topics: Base Sequence; Cell Division; Chromosome Deletion; DNA Restriction Enzymes; Drug Resistance, Microbial; Escherichia coli; Genes, Bacterial; Plasmids; Tetracycline

pT181 is a fully sequenced 4.4-kb 20 copy Tcr plasmid from Staphylococcus aureus. Its replication system involves a unique unidirectional origin embedded in the coding sequence for a plasmid-determined protein, RepC, that is required for initiation. When joined to a 55 copy carrier plasmid, pE194, pT181 excludes autonomous isologous replicons by inhibiting their replication. Two types of spontaneous pT181 copy mutants have been isolated, one that eliminates sensitivity to this inhibition and another that does not. A spontaneous 180-bp deletion, delta 144, eliminates both the inhibitory activity and sensitivity to it. This deletion increases copy number by 50-fold and RepC production by at least 10-fold. It is located directly upstream from the repC coding sequence and the deletion-bearing plasmid supports the replication of inhibitor-sensitive plasmids in cells containing active inhibitor. This effect is probably due to the overproduction of RepC by the delta 144 plasmid. On the basis of these results, it is suggested that RepC synthesis is negatively controlled by an inhibitor that is encoded directly upstream from the repC coding sequence and acts as a tareget set in the same region. It is likely, therefore, that pT181 replication rate is determined by the level of RepC.

Topics: Bacterial Proteins; Chromosome Deletion; DNA Replication; DNA, Bacterial; Drug Resistance, Microbial; Plasmids; Staphylococcus aureus; Tetracycline

The transposon Tn10-encoded TET repressor controls the expression of tetracycline resistance as well as its own synthesis. The antibiotic tetracycline functions as an inducer for both genes, which are transcribed in divergent directions from a common start area. The interaction of the TET repressor with the regulatory sequence of the tetracycline resistance operon is investigated by equilibrium and kinetic methods. The wild-type control sequence contains two nearly identical operators separated by only ten base-pairs. A deletion mutant lacking one of the operators is constructed by controlled digestion with exonuclease Bal31. It serves to prove that the two TET operators are each occupied by a TET repressor dimer in the wild-type tet operon regulatory sequence. The association constants are approximately identical for both operators between 10(12) and 10(13) M-1 as derived from kinetic data. The half-life of the TET repressor--tet operator complex is 12 minutes when competed with tet operator DNA and two minutes when competed with the inducer tetracycline. The dissociation of the repressor--operator complex has no apparent activation enthalpy but has an activation entropy of -320 J/mol K, indicating the involvement of solvent or counterion condensation. The dissociation rate constant of the tetracycline--TET repressor complex depends strongly on temperature. The activation enthalpy is 160 kJ/mol, indicating extremely strong binding of the drug. This result is discussed with respect to the necessary sensitivity of a regulated resistance gene. The native structure of the TET repressor is a dimer, as demonstrated by molecular exclusion chromatography. The elution behavior of the TET repressor--tetracycline complex indicates clearly that the repressor--inducer complex remains a dimer. The results are discussed with respect to the regulatory functions of the components.

Topics: Base Sequence; Chromosome Deletion; DNA Transposable Elements; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Gene Expression Regulation; Kinetics; Macromolecular Substances; Molecular Weight; Operon; Repressor Proteins; Tetracycline; Transcription Factors

A high-copy plasmid, pGA217, which carries a deletion (lacking the carboxy-terminal 20 amino acids) of the structural gene for ribosomal protein L10 (rplJ) is lethal to the cell in the absence of the gene (rplL) for r-proteins L7/L12, but only if the upstream operon for r-proteins L11 (rplK) and L1 (rplA) is present on the same plasmid. Measurements of beta-galactosidase activity of a hybrid protein expressed by a rplL-lacZ fusion indicated that the L10 fragment peptide which lacks the carboxy-terminal 20 amino acids is capable of exerting feedback regulation. Double transformation experiments with two compatible plasmids showed that the detrimental effect of the rplJ deletion on pGA217 can be reversed by the addition of a second plasmid which carries a functional gene for L7/L12. These two pieces of evidence suggest that the lethal effect of pGA217 is due to its property of feeding back on L7/L12 production from the chromosomal rplK gene. The upstream rplKA operon was inferred to have a cis-acting, stimulating effect on rplJ expression from the following evidence: (1) donor plasmids carrying the genes for L11 and/or L1 fail to exert a trans-acting effect, (2) deletion mutants which removed portions of rplK and/or rplA, but maintained the rplKA promoter, rplKp, still retained a severe growth-inhibiting effect. We suggest that these results can be explained by assuming that there is transcription from the rplKA promoter through rplJ and perhaps beyond.

Topics: Chromosome Deletion; Drug Resistance, Microbial; Escherichia coli; Genes; Genes, Bacterial; Mutation; Operon; Plasmids; Ribosomal Proteins; Tetracycline; Transcription, Genetic

Palindromic arrangements of 2 X 400 to 2 X 1517 bp of nucleotide sequences were created by in vitro manipulation of Escherichia coli plasmids. As a consequence of its method of formation, each palindrome possessed at its center the recognition site for a particular restriction endonuclease. Eight out of eight palindromes, having at their centers the recognition sites for BamHI, BglI, BglII, HindIII, PstI, SalI, XhoI, and Xma III, were shown to be inviable when transformed into E. coli. The smallest of these palindromes had a half-length of approx. 400 bp. The lethality of palindromic sequences for their carrier plasmids was circumvented at low frequencies by spontaneous in vivo deletion events which removed the centers of symmetry of the palindromes. The frequencies of such deletions were less than 1% in most cases, but varied significantly both with the palindromic sequence in question and with the surrounding nonpalindromic sequences of the carrier plasmid. We confirmed the viability of a plasmid with a 147-bp palindrome [Bergsma et al., Gene 20 (1982) 157-167] and found that this palindrome (derived from SV40) does not confer viability on the plasmids with long palindromes.

Topics: Base Sequence; Chromosome Deletion; DNA Replication; DNA, Bacterial; Escherichia coli; Genes, Lethal; Plasmids; Tetracycline

Transposon Tn10 is 9300 bp in length, with 1400 bp inverted repeats at its ends. The inverted repeats are structurally intact IS-like sequences (Ross et al., 1979). Analysis of deletion mutants and structural variants of Tn10, reported below, shows that the two IS10 segments contain all of the Tn10-encoded genetic determinants, both sites and functions, that are required for transposition. Furthermore, the two repeats (IS10-Right and IS10-Left) are not functionally equivalent: IS10-Right is fully functional and is capable by itself of promoting normal levels of Tn10 transposition; IS10-Left functions only poorly by itself, promoting transposition at a very low level when IS10-Right is inactivated. Complementation analysis shows that IS10-Right encodes at least one function, required for Tn10 transposition, which can act in trans and which works at the ends of the element. Also, all of the sites specifically required for normal Tn10 transposition have been localized to the outermost 70 bp at each end of the element; there is no evidence that specific sites internal to the element play an essential role. Finally, Tn10 modulates its own transposition in such a way that transposition-defective point mutants, unlike deletion mutants, are not complemented by functions provided in trans; and wild-type Tn10, unlike deletion mutants, is not affected by functions provided in trans from a "high hopper" Tn10 element.

Topics: Base Sequence; Chromosome Deletion; DNA Transposable Elements; Drug Resistance, Microbial; Genetic Complementation Test; Mutation; Tetracycline

Topics: Base Sequence; Chromosome Deletion; Chromosome Inversion; DNA Transposable Elements; Escherichia coli; Genetic Complementation Test; Mutation; Recombination, Genetic; Salmonella; Tetracycline

Topics: Bacterial Proteins; Base Sequence; beta-Lactamases; Chromosome Deletion; DNA Transposable Elements; DNA, Bacterial; Escherichia coli; R Factors; Rec A Recombinases; Temperature; Tetracycline; Transformation, Genetic

Topics: Chromosome Deletion; Drug Resistance, Microbial; Escherichia coli; Genes; Mutation; Phenotype; Plasmids; Recombination, Genetic; Staphylococcus aureus; Tetracycline

Closed circular DNA interacts with complementary sequences of single-stranded DNA to form displacement loop (D loop) structures in vitro. The site of D-loop formation can be directed by using single-stranded DNA derived from a selected restriction fragment. Circular DNA containing a D loop can then be linearized by cleavage with endonuclease S1. This cleavage appears to remove a limited number of nucleotides from each strand of the circular DNA substrate. Incubation with polynucleotide ligase followed by propagation in vivo leads to circular DNA molecules that bear small, single deletions in the region of the single-stranded DNA sequence chosen for the formation of the D loops. We have utilized these manipulations of DNA to construct tetracycline-sensitive deletion mutants of plasmid pBR322. The level of mutagenesis obtained by the procedure is sufficiently high that selective growth and screening procedures are not necessary for the isolation or identification of mutants. The frequency, variety, and small size of the deletions obtained within the selected target regions present considerable advantage for genetic and biochemical analysis. The method is quite general in rationale and should be immediately applicable to phage and viruses having infectious circular DNA genomes or recombinant DNA species propagated in circular plasmid vectors.

Topics: Chromosome Deletion; Chromosome Mapping; Cloning, Molecular; DNA, Circular; DNA, Single-Stranded; DNA, Superhelical; Drug Resistance, Microbial; Endonucleases; Mutation; Plasmids; Polynucleotide Ligases; Tetracycline

Using a set of overlapping deletion mutants in the tetracycline-resistance transposon Tn10, it has been established that certain regions of the Tn10 genome exert a powerful inhibition on translocation of an intact Tn10 element into the bacterial genome. Such inhibition is strongly temperature dependent: at 37 degrees C translocation is inhibited by at least a factor of 100; no inhibition of translocation is detected at 30 degrees C.

Topics: Chromosome Deletion; Chromosomes, Bacterial; DNA Transposable Elements; Drug Resistance, Microbial; Mutation; Recombination, Genetic; Salmonella typhimurium; Temperature; Tetracycline

A simple technique has been devised that allows direct plate selection of tetracycline-sensitive clones from a predominantly tetracycline-resistant population. The technique is especially useful in genetic methodologies based on the use of tetracycline resistance transposons, such as Tn10. Potential uses of the method include selection of deletion mutants, fine-structure mapping, generalized mapping, construction of multiply marked strains, elimination of tetracycline resistance transposons and plasmids and cloning. The technique is based on our finding that tetracycline-resistant cells are hypersensitive to lipophilic chelating agents, such as fusaric acid. This finding supports the contention that certain metal ions critically facilitate tetracycline uptake and leads us to suggest possible molecular mechanisms for tetracycline resistance.

Topics: Carboxylic Acids; Cations; Chelating Agents; Chromosome Deletion; Chromosome Inversion; DNA Transposable Elements; Drug Resistance, Microbial; Fusaric Acid; Picolinic Acids; Quinolines; Salmonella typhimurium; Selection, Genetic; Tetracycline

5 antibiotics, 4 of which inhibit protein synthesis in different ways, and 1 of which inhibits bacterial cell-wall synthesis, were tested in a battery of microbial assays for possible genetic effects. All the antibiotics, chloramphenicol, tetracycline, gentamicin, oleandomycin and phosphonomycin induced forward mutation to L-azetidine-2-carboxylic acid resistance in Escherichia coli WP2. This response was closely correlated with the toxic effects and was inferred to be deletion mutation. In addition, chloramphenicol was weakly active in reversion of the frame-shift mutation in Salmonella typhimurium TA98, gentamicin caused petite induction in S. cerevisiae at pH 4.4--4.7 and tetracycline gave a significant reponse with gene conversion and petite induction also in S. cerevisiae but at pH 7.2. The results, particularly those with E. coli, cast doubts on the validity of testing specifically designed antibacterial agents in bacteria, and raise serious problems in the evaluation of such data in terms to human populations.

Topics: Animals; Anti-Bacterial Agents; Chloramphenicol; Chromosome Deletion; Drug Resistance, Microbial; Escherichia coli; Fosfomycin; Gene Conversion; Gentamicins; Microsomes, Liver; Mutagenicity Tests; Oleandomycin; Rats; Saccharomyces cerevisiae; Salmonella typhimurium; Species Specificity; Tetracycline

Integration of the tetracycline resistance transposon Tn10 into lacI of a lacI-lacZ gene fusion permits the isolation of deletions that excise DNA from one end of Tn10 and fuse Tn10 genes with lacZ in such a manner that chimeric proteins with beta-galactosidase activity are produced. The synthesis of the chimeric proteins is under the same control as the transposon genes. Thus, regulation of expression of Tn10 genes can be investigated by measuring beta-galactosidase activity. Analysis of Tn10-lacZ fusions revealed different deletion endpoints within Tn10; lacZ has been fused to at least three different Tn10 genes or operons. Two of these genes are under the control of a tetracycline repressor.

Topics: beta-Galactosidase; Chromosome Deletion; Drug Resistance, Microbial; Escherichia coli; Genes; Genes, Regulator; Hot Temperature; Lac Operon; Mutation; Salmonella typhimurium; Tetracycline; Translocation, Genetic

Insertions of Tn10 are characterized by the presence of a direct repetition of a 9-bp target gene sequence on either side the insertion. The occurrence of these repetitions undoubtedly reflects an important feature of the way in which DNA molecules are broken and joined during translocation. Our experiments further suggest, however, that these 9-bp sequences are probably not responsible for Tn10-insertion specificity and that their presence is not required for normal Tn10 translocation elsewhere. We therefore suggest that the genetic information which controls the quality and quantity of Tn10 translocation actually resides somewhere other than these 9-bp sequences. We presume that much of this information lies within the ends of Tn10 itself and that signals on the target DNA which guide Tn10 to preferred positions must occur near, but not actually at, the eventual physical site of the integration event. Consideration of Tn10-promoted deletions and inversions reemphasizes the role of these ends in Tn10-promoted recombination events. Since Tn10-promoted events almost always consist in joining the physical end of one of the putative IS sequences of Tn10 to some other DNA segment, one comes again to the conclusion that these ends must contain important genetic information governing recombination events.

Topics: Bacteriophage lambda; Base Sequence; Chromosome Deletion; Chromosome Inversion; DNA Transposable Elements; DNA, Bacterial; DNA, Viral; Drug Resistance, Microbial; Recombination, Genetic; Tetracycline; Translocation, Genetic

Spontaneous tetracycline-sensitive, transfer-deficient mutants of R100-1 were selected and analysed by genetic complementation tests and with the restriction endonuclease EcoR1. While some of the Tets Tra- mutants were caused by a single deletion event which removed the Tetr genes and extended into the neighbouring transfer genes, other mutants were the result of the deletion of the Tetr genes within Tn10 which was accompanied by an inversion of adjacent DNA sequences. A clustering of deletion and inversion endpoints occurred in the traA gene. Some of the transfer genes of R100-1 were assigned to EcoR1 fragments.

Topics: Chromosome Deletion; Chromosome Inversion; Conjugation, Genetic; DNA Restriction Enzymes; DNA Transposable Elements; Escherichia coli; Genes; Genetic Complementation Test; Mutation; R Factors; Tetracycline

Topics: Chromosome Aberrations; Chromosome Deletion; Chromosome Inversion; Chromosomes, Bacterial; Drug Resistance, Microbial; Histidine; Operon; Recombination, Genetic; Salmonella typhimurium; Tetracycline

Deletion mutants have been derived from a plasmid-cloned repeating unit of Xenopus laevis oocyte 5S DNA by introducing the transposable chloramphenicol-resistance element Tn9 into the AT-rich spacer sequence near the 5' terminus of the X. laevis 5S rRNA gene in a recombinant plasmid and then selecting plasmids which had lost the transposable element. Plasmids lacking the entire transposable element and various portions of the AT-rich spacer sequence flanking the original site of Tn9 integration have been obtained, and their ability to support transcription of the remaining X. laevis 5S rRNA gene has been tested in X. laevis oocyte nuclei. The deletion mutants analyzed in the present study retain the 49 nucleotide nonrepetitive sequence immediately adjacent to the 5' terminus of the gene, but lack as much as 80% of the repetitive AT-rich spacer sequence (Fedoroff and Brown, 1978). Such deletion mutants are fully active templates for 5S rRNA synthesis. This implies that the AT-rich spacer, which comprises half or more of each repeating unit in X. laevis oocyte 5S DNA, is relatively unimportant for correct initiation of transcription, and that if there are extragenic sequences with promoter function, they are likely to reside in the short nonrepetitive region immediately adjacent to the gene.

Topics: Animals; Base Sequence; Chloramphenicol; Chromosome Deletion; Chromosome Mapping; DNA, Recombinant; Drug Resistance; Mutation; Plasmids; RNA, Ribosomal; Tetracycline; Transcription, Genetic; Translocation, Genetic; Xenopus

We report here the physical structures of deletions and inversions promoted by the translocatable tetracycline-resistance element Tn10. DNA/DNA heteroduplex and restriction enzyme analyses of alterations in the genome of bacteriophage lambda suggest that both types of DNA alterations almost always originate at the internal termini of the 1400 bp terminal inverted repetitions of Tn10. Tn10-promoted deletions remove a single contiguous DNA segment beginning at one such terminus; Tn10-promoted inversions are more complex, and involve both an inversion and a specific deletion of Tn10 DNA.

Topics: Chromosome Deletion; Chromosome Inversion; Coliphages; DNA, Viral; Nucleic Acid Hybridization; R Factors; Recombination, Genetic; Tetracycline; Translocation, Genetic

Topics: Chromosome Deletion; Chromosome Mapping; Chromosomes, Bacterial; Drug Resistance, Microbial; Escherichia coli; Genetic Engineering; Mutation; Phenotype; Plasmids; Salmonella Phages; Salmonella typhimurium; Tetracycline; Translocation, Genetic

Strains of Escherichia coli K12 heterozygous for the R100-1 tetracycline resistance region were constructed. They carried the wild-type Tetr genes in the chromosome and single site Tets mutations on plasmids. Some heterozygotes could not express tetracycline resistance fully after induction. The mutant tet allele was thus partially dominant. When heterozygotes carrying the dominant tet mutant were plated on agar containing 20 mg/ml tetracycline, mutants which grew normally occurred at a frequency of 1-4 X 10(-4). Analysis of these dominance relief mutants showed that in 53/56 isolates the dominant tet allele was lost forming either Tra+ or Tra- deletion mutants of the plasmid. The mutation frequency was not affected either by the host chromosomal recA mutation or by the temperature of growth of the culture.

Topics: Chromosome Deletion; Chromosomes, Bacterial; Drug Resistance, Microbial; Escherichia coli; Genes, Dominant; Heterozygote; Mutation; R Factors; Tetracycline

The majority of tetracycline-sensitive (Tets) mutants of R100 and R100-1 are multisite (deletion) mutants. About 50% of these are also transfer-deficient, indicating that the Tetr locus is closely linked to the transfer genes. Tet(s) mutants with single-site lesions are also described.

Topics: Chromosome Deletion; Chromosomes, Bacterial; Conjugation, Genetic; Drug Resistance, Microbial; Escherichia coli; F Factor; Mutation; R Factors; Tetracycline