Nucleotide sequence of the kanamycin resistance transposon Tn903

The entire nucleotide sequence of the kanamycin resistance transposon Tn903 was determined by analyzing a mini-ColE1 derivative carrying Tn903. Tn903 was 3094 base-pairs in length and at both extremities possessed two identical inverted 1057 base-pair sequences. Furthermore, 18 bases at the ends of the 1057 base-pair sequence are themselves present in an invertedly repeated order as has been described for various insertion sequences. Analysis of initiation and termination codons in the Tn903 sequence indicated that Tn903 could possibly code for at least three high molecular weight polypeptides. One in the region between the two 1057 base-pair sequences is suggested to be the kanamycin resistance determinant (aminoglycoside 3′-phosphotransferase) from its location and size. The other polypeptides were located within the 1057 base-pair sequence and may be associated with transposition functions of Tn903.     

Organization of the Tn6-related kanamycin resistance transposon Tn2680 carrying two copies of IS26 and an IS903 variant, IS903. B.

35S incorporation studies showed that Candida tropicalis tRNA contained two thionucleosides, one of which was identified as 5-methyl-2-thiouridine. The other thionucleoside was alkali labile, and it appeared to be an ester. Pulse-chase experiments suggested that the two thionucleosides were structurally related. 5-Methyl-2-thiouridine was present in one of the lysine tRNAs. This is the first report of the presence of this nucleoside in a yeast tRNA.     

Construction of a correlated physical and genetic map of the Klebsiella pneumoniae hisDGO region using transposon Tn5 mutagenesis.

Multicopy plasmids containing the hisDG region of Klebsiella pneumoniae were mutagenized with transposon Tn5. The resulting plasmids were examined for their ability to complement hisD and hisG mutations in Escherichia coli. The physical location of Tn5 on each of the hisD::Tn5 and hisG::Tn5 plasmids was determined by restriction endonuclease analysis. By combining the two types of data, a precise correlated physical and genetic map of the K. pneumoniae hisDG region was constructed. Based on this analysis, the minimum sizes of the hisD and the hisG genes were calculated to be 1100 bp and 900 bp, respectively. The hisO(P) region was also identified. The insertional specificity of transposon Tn5 was shown to be very low. One unanticipated result was obtained: Tn5 insertions in the plasmid-borne hisG gene were not polar on hisD.     

Chromatin structure of transposon Tn903 cloned into a yeast plasmid

Transposon Tn903 contains the APH gene for kanamycin resistance, which is active in yeast [A. Jiménez and J. Davies (1980) Nature (London) 287, 869-871] and is flanked by two inverted repeats (IR) 1057 bp long. When plasmid pAJ50, carrying Tn903 and the 2-microns circle origin of replication, is cloned into Saccharomyces cerevisiae, nucleosomes are assembled in vivo on the prokaryotic DNA of the transposon. Indirect end labeling revealed that three nucleosomes are preferentially positioned on symmetrical sequences from both IRs. DNase I digestion also confirmed that the chromatin structure is symmetrical in both IRs. This suggests that sequence determinants are decisive for chromatin structure in these regions. We have calculated the rotational and translational fits [H. R. Drew and C. R. Calladine (1987) J. Mol. Biol. 195, 143-173] for the Tn903 sequence and the results indicate that the nucleosome positioning on the IRs is sequence-directed. Nucleosome deposition on the APH gene also occurs, but no clear positioning exists. Some sequence preference for positioning nucleosomes on the promoter can be predicted, especially from the translational fit. Experimental data indicate, however, that nucleosomes are absent from the promoter. Therefore, chromatin can be organized on prokaryotic DNA in a manner that resembles the typical eukaryotic chromatin structure.     

Transformation of Kluyveromyces lactis with the kanamycin (G418) resistance gene of Tn903

Direct selection of Kluyveromyces lactis resistant to the antibiotic G418 following transformation with the kanamycin resistance gene of Tn903 required the development of a procedure for producing high yields of viable spheroplasts and for the isolation of autonomous replication sequences (ARS). To obtain high yields of viable spheroplasts, cells were treated with (1) a thiol-reducing agent (L-cysteine), and (2) a high concentration of an osmotic stabilizer, 1.5 M sorbitol. Several ARS-containing plasmids were selected from a K. lactis recombinant DNA library in K. lactis and in Saccharomyces cerevisiae. Two of four ARS clones selected in K. lactis promoted transformation frequencies of 5-10 X 10(2) G418-resistant cells/micrograms of plasmid DNA. This frequency of transformation was at least twice as high as with ARS clones selected in S. cerevisiae. The stability of ARS-containing plasmids varied; after 20 generations of growth in the presence of G418, 16-38% of the cells remained resistant to the drug. In the absence of selection pressure less than 5% of the cells retained the drug-resistance phenotype. Plasmids containing the ARS1 or 2 mu replicon of S. cerevisiae failed to transform K. lactis for G418 resistance. Inclusion of S. cerevisiae centromere, CEN4, in a K. lactis ARS recombinant plasmid did not increase the stability of the plasmid in K. lactis, and marker genes on the vector segregated predominantly 4-:0+ through meiosis. We conclude that neither the ARS sequences or the centromere of S. cerevisiae was functioning in K. lactis.     

Tn5-rpsL: a new derivative of transposon Tn5 useful in plasmid curing

The rpsL gene of Escherichia coli was inserted into the BamHI site of transposon Tn5. This transposon was called Tn5-rpsL. Tn5-rpsL may be useful in microbiological studies when one wants to cure various bacterial genera of certain plasmid(s). A streptomycin-resistant (SmR) derivative of the host bacterial strain is first isolated. The plasmid(s) later to be cured are then labelled with Tn5-rpsL, which makes the cells Sm-sensitive. These cells can regain their resistance to Sm if they lose the Tn5-rpsL-tagged plasmid. Thus, plasmid-free bacteria are easily selected among SmR survivors. The frequency of occurrence of the plasmid-less variants of plasmid-containing wild-type Salmonella typhimurium measured by this method is given as an example.     

Detection and characterization of Tn2501, a transposon included within the lactose transposon Tn951

The DNA sequence spanning coordinates 9.9 to 16.4 kilobases of the lactose transposon Tn951 ( Cornelis et al., Mol. Gen. Genet. 160:215-224, 1978) constitutes a transposable element by itself. Unlike Tn951 ( Cornelis et al., Mol. Gen. Genet. 184:241-248, 1981), this element, called Tn2501 , transposes in the absence of any other transposon. Transposition of Tn2501 proceeds through transient cointegration and duplicates 5 base pairs of host DNA. Tn2501 is flanked by nearly perfect inverted repeats (44 of 48), related to the inverted repeats of Tn21 ( Zheng et al., Nucleic Acids Res. 9:6265-6278, 1982). Unlike Tn21 , Tn2501 does not confer mercury resistance.     

Restriction enzyme cleavage map of Tn10, a transposon which encodes tetracycline resistance.

A cleavage map of a recombinant plasmid carrying Tn10 was constructed for 13 different restriction enzymes. The Tn10 region of this plasmid contains cleavage sites for BamHI, AvaI, BglI, BglII, EcoRI, XbaI, HincII, HindIII, and HpaI. Restriction enzymes PstI, SmaI, KpnI, XhoI, SalI, and PvuI do not cleave within the Tn10 element. This map confirms the previously reported structure of this transposon; it is composed of a unique sequence (approximately6,400 base pairs long), which in part codes for the tetracycline resistance functions and is bounded by inverted repeats (approximately 1,450 base pairs long).     

Streptococcus faecalis R plasmid pJH1 contains an erythromycin resistance transposon (Tn3871) similar to transposon Tn917

The R plasmid pJH1 contains a 5.1-kilobase transposon ( Tn3871 ) that mediates inducible resistance to erythromycin. Three AvaI digestion fragments from this transposon are identical in size to and homologous with three AvaI-derived fragments from the previously described erythromycin resistance transposon Tn917 . These three DNA fragments account for greater than 90% of both transposons.     

Construction and characterization of versatile kanamycin-resistance cassettes derived from the Tn5 transposon

We have developed plasmids with the Tn5 kanamycin-resistance gene (kan) flanked either symmetrically or asymmetrically by several restriction sites. These can be used to provide a selectable genetic marker or to mobilize restriction sites and sense or nonsense codons into genes. The 1.3-kb kan cassette exhibits polarity effects in both directions.     

Rapid physical mapping by transposon Tn5 mutagenesis to localize the cloned yeast ILV2 gene

A rapid method for Tn5 mutagenesis of cloned genes on multicopy plasmids was used to map a yeast ILV2 mutant allele encoding a sulfometuron methyl-resistant acetolactate synthase. Twenty-one of 40 independent Tn5 insertions were within the 5.6-kilobase-pair cloned segment. Of these, seven adjacent transposition events inactivated the sulfometuron methyl resistance determinant, localizing the ILV2 gene to a minimum 1.4-kilobase-pair region.     

The kanamycin resistance transposon Tn2680 derived from the R plasmid Rts1 and carried by phage P1Km has flanking 0.8-kb-long direct repeats

Phage P1Km carries within the invertible DNA segment a 5-kb insertion with 0.8-kb terminal direct repeats flanking the kanamycin resistance determinant. The same structure was also found on the R plasmid Rts1, from which the Km resistance segment of P1Km was derived. Obviously, this Km resistance segment translocated as a unit to the P1 genome and it is therefore called Tn2680. Loss of the Km resistance determinant due to recombination between the flanking direct repeats occurs during vegetative growth of P1Km. Amplification of Tn2680 to tandem oligomers is documented and is thought to result from recombination between the flanking direct repeats. The flanking 0.8-kb repeats are different from known IS elements.     

G418 resistance in the yeast Saccharomyces cerevisiae: comparison of the neomycin resistance genes from Tn5 and Tn903

Summary The neo genes of Tn5 and Tn903 (Tn601) coding for amigoglycoside phosphotransferase type II and type I, respectively, were joined to the yeast adc 1 promoter and trp1 terminator and introduced into yeast (Saccharomyces cerevisiae) cells. Transformants were obtained by direct selection for G418 resistance. Plasmids containing the Tn5 neo gene induced antibiotic resistance only at low frequency, whereas colonies transformed with the Tn903 neo gene could be selected at high frequency (300–400 transformants/g plasmid DNA). The resistance threshold of transformed strains was increased to 30 mg G418/ml by both genes and high level expression of the bacterial genes in yeast could be shown using an in vitro phosphotransferase assay. The results indicate that this system can be used for high frequency transformation of wild-type strains and might in addition be used for the identification and isolation of promoter-active sequences.Abbreviations adc 1 alcoholdehydrogenase I gene - APH aminoglycoside-3-phosphotransferase - leu2 -isopropylmalate dehydrogenase gene - neo aminoglycoside-3-phosphotransferase gene - trp1 N-(5-phosphoribosyl)-anthranilate isomerase gene Dedicated to Prof. Dr. Dr. h. c. Karl Esser on the occasion of his 65th birthday     

Rapid physical mapping by transposon Tn5 mutagenesis to localize the cloned yeast ILV2 gene.

Techniques for in vivo cloning were used with the fast-growing nitrogen-fixing soybean microsymbiont R. fredii USDA 191. Selection for transfer of Tn5 insertions from R. fredii USDA 191 containing the gene-mobilizing plasmid pJB3JI provided recombinants at up to 400 times the background mutation level. These techniques may be useful for future genetic analysis of R. fredii.     

Identification of Tn2401, a transposon encoding multiresistance to aminoglycosides

A transposable element, Tn2401, was found in a clinical isolate of Pseudomonas aeruginosa. Tn2401 had a size of 7190 nucleotides and encoded aminoglycoside 3'-phosphotransferase and aminoglycoside 6'-N-acetyltransferase. The sequence encoding the former enzyme was homologous with that of Tn903. Pseudomonas aeruginosa strains harbouring this transposon were resistant to kanamycin, neomycin, lividomycin, ribostamycin, paromomycin, netilmycin, tobramycin, dibekacin, gentamicin, sisomicin, and butirosin.     

Properties of aminoglycoside-3'-phosphotransferases determined by kanamycin transposones Tn 601 and Tn 5

Aminoglycoside-3'-phosphotransferase I and II (APT-3'-I and APT-3'-II) has been purified to homogenity from the cells of E. coli containing the plasmids R6 and JR67, respectively. The purification procedure involved competitive affinity chromatography on neomycin-sepharose and gel-filtration on Sephadex G-100. The specific activity of APT-3'-I with the substrates--lividomycin A, neomycin B, paromycin, ribostamycin, kanamycins A and B--are 4.3, 2.8, 2.1, 1.6, 0.9 and 0.8 mol/min. mg protein, respectively. The specific activity of APT-3'-II with the substrates--ribostamycin, paromycin, kanamycins A and B, neomycin B--are 8.0, 7.2, 4.0, 4.5 and 3.6, respectively. Mg2+ is required for the activity of both enzymes. Co2+, Zn2+ and Mn2+ are active in case of APT-3'-I; however, these cations are less active than Mg2+. The pH-optimum of APT-3'-I and APT-3'-II is 7.0--7.5. High ionic strength is required for the activity of both enzymes. The molecular weights of APT-3'-I and APT-3'-II are about 36 000 and 26 000, respectively. The amino acid composition of APT-3'-I and APT-3'-II was determined. Both enzymes contain tryptophane residues whose fluorescence intensity decreased when ATP, but not amino-glycoside antibiotics, is added. The interrelationship between the molecular weights of these enzymes and the sizes of the loops of transposones Tn 601 and Tn 5, encoding APT-3'-I and APT-3'-II, is discussed.     

An alternative inverse PCR (IPCR) method to amplify DNA sequences flanking Tn5 transposon insertions

We have developed an alternative method to amplify DNA sequences flanking Tn5 transposon insertions. This method relies on the identical sequences of inverted terminal repeats, located at the 5' and 3' ends of Tn5, to determine the location and orientation of a transposon insertion within a restriction endonuclease fragment. From this information, PCR primers can be designed to selectively amplify by inverse PCR the DNA flanking one side of the transposon. This method avoids the problem of amplifying or cloning long sequences flanking Tn5. To demonstrate the applicability of this method, we generated Tn5 transposon mutants of Pseudomonas abietaniphila BKME-9 which no longer grew on dehydroabietic acid (DhA). The flanking sequence of one of the mutant (strain BKME-941) which accumulated 7-oxoDhA, was amplified.     

Spread and evolution of natural plasmids harboring transposon Tn5

Abstract: The presence of transposon Tn 5 was studied in 730 Enterobacteriaceae strains from clinical and sewage origin. From these strains, twenty-five conjugative plasmids harboring transposon Tn 5 were isolated. These plasmids were compared with pJR67 and pRYC119, the only previously studied plasmids harboring Tn 5 . A phylogenetic tree of the evolution of all different plasmids was proposed. Irrespective of their bacterial host and geographical place of isolation, some of the plasmids were shown to be identical. All of them can be included in only eight different prototypical plasmid species. Twenty-two plasmids (88%) carried an IncI1 incompatibility determinant as judged form DNA hybridization experiments. The presence of some other common resistance genes suggested that these plasmids are descendants of a common ancestor. These IncI1 plasmids could be grouped in six prototypical species. The results presented here suggest that Tn 5 spread in nature may be dependent on the conjugative ability of the IncI plasmids harboring the transposon, rather than on the efficiency of Tn 5 transposition between different replicons.