A new type of IS1-mediated deletion

Summary Genetical tests and DNA sequence analysis revealed that the mechanism of formation of IS1-induced type I and type II deletions differs.IS1-mediated type II deletions occur at the termini of the integrated element and do not remove the element. This process is independent of the cellular recA system and does not involve DNA sequence homology.Conversely, the formation of IS1-induced type I deletions differs substantially. They require recA gene product, small DNA sequence duplications and a topological arrangement of the DNA molecule to allow alignment of duplications.     

IL-6 is an intermediate in IL-1-induced thymocyte proliferation

Both IL-1 and IL-6 have been shown to be comitogenic for lectin-stimulated thymocytes. Thymocytes cultured in the presence of IL-1 produce IL-6 themselves. This IL-6 production is caused by a cell population with low buoyant density. After removal of these cells, IL-6 or IL-2 are still co-mitogenic for thymocytes whereas IL-1 is not. Addition of IL-1 to such thymocytes renders them about 100-fold more sensitive to IL-6. At all conditions proliferation is inhibitable with antibodies to IL-2 and to the IL-2R. Our experiments show that IL-1-driven proliferation of thymocytes is dependent on endogenous IL-6 production and that in the classical thymocyte assay IL-1 has a dual role: it induces IL-6 production and it greatly increases the sensitivity for IL-6.     

Characteristics of the deletion mutant of plasmid R6K]

Deletion plasmide R6Kdelta with the mol wt of 17.2.10(6) dalton isolated from the E. coli chi 925 (R6K) is described. This plasmide expresses no resistance to streptomycin, is replicated in the E. coli K12 under relaxed control and is resistant to the treatment with the eliminating agents. Analysis of plasmide DNA with the aid of electrophoresis in agarose gel demonstrated that R6K delta has one site attacked by restriction endonucleases Eco. RI and Bam HI. These data were confirmed by the determination of the transforming activity of the corresponding DNA restrictors. It is supposed that the isolated plasmide was identical with plasmide RSF1040. A possibility of using R6K delta as a genetic vector for obtaining recombination DNA molecules in vitro is discussed.     

Tn1721-induced mutation in an isoprenol degrading plasmid fromPseudomonas putida

Summary Transformation experiments using pRO1825 containing the tetracycline resistance transposonTn1721 were conducted to induce mutations in plasmid pSRQ50, which coded for acyclic isoprenol degradation. Transformants that were tetracycline resistant were examined for their ability to utilize geraniol. Strains unable to utilize geraniol were observed to have a 0.6 kb addition on the C fragment of pSRQ50.     

The F plasmid carries an IS3 insertion within finO

DNA-DNA hybridization studies support the conclusion that coding sequences of the fertility inhibition gene, finO, are present on the F plasmid and map downstream of the transfer region, entirely within 99.19-2.0 F. In addition, the results indicate that the IS3a element at 100/0-1.26 F is inserted within the F finO coding region. This insertion may have inactivated the finO gene on the F plasmid.     

PCR-mediated deletion of plasmid DNA

The PCR-mediated plasmid DNA deletion method is a simple approach to delete DNA sequences from plasmids using only one round of PCR, with two primers, and without ligation or purification prior to in vivo recombination. By using only PCR, the method is sequence independent and, as shown in this study, is applicable to various sizes of plasmids and deletions using minimal primer design.     

Isolation and preliminary characterization of R6K plasmid deletion mutants.

Several deletion mutants of R6K have been isolated by mutagen treatment of bacterial host carrying wild type of the plasmid and search for clones that lost ampicillin or streptomycin resistance. The molecular weight of the mutants as estimated by agarose gel electrophoresis was 15 times 10(6) minus 23 times 10(6) compared to 26 times 10(6) for the parental plasmid. The mutants were characterized in respect of the level of resistance to ampicillin and frequency of conjugational transfer. Some of the mutants were found to differ in Eco RI digestion pattern from the wild type.     

Phenotypic reversion of an IS1-mediated deletion mutation: a combined role for point mutations and deletions in transposon evolution

We have physically characterised a deletion mutant of the R plasmid R100 which has lost all of the antibiotic resistances, including chloramphenicol resistance (Cmr), coded by its IS1-flanked r-determinant. The deletion was mediated by one of the flanking IS1 elements and terminates within the carboxyl terminus of the Cmr gene. DNA sequence analysis showed that the mutated gene would produce a protein 20 amino acids longer than the wild-type due to fusion with an open reading frame in the IS element. Surprisingly for a deletion mutation, rare, spontaneous Cmr revertants could be recovered. Two of the four revertants studied had frame shifts due to the insertion of a single AT base pair at the same position; the revertants would code for a protein five amino acids shorter than the wild-type. The other two revertants had acquired duplications of the 34-bp inverted terminal repeat sequences of the IS1 element and would direct the synthesis of a protein six amino acids longer than the wild-type. The reverted Cmr markers were still capable of transposition. These observations suggest a role for point mutations and small DNA rearrangements in the formation of new gene organisations produced by mobile genetic elements.     

Mapping and characterization of an E. coli mutant defective in IS1-mediated deletion formation

Summary A mutant of E. coli has been isolated in which the frequency of IS1-mediated deletion formation is reduced as much as 100 fold. The mutation causing this reduction, designated del, was mapped to a position near 61 min, in the vicinity of lysA and galR. Strains carrying a deletion of the del gene were constructed, and these exhibit a significant reduction in the frequency of IS1 excision in addition to impairment of deletion formation. A bacteriophage capable of transducing lysA and del was also isolated.     

Characterization of deletion derivatives of an autonomously replicating Neurospora plasmid.

We previously described two plasmids that replicate autonomously in both Neurospora and E. coli (Stohl and Lambowitz, Proc. Natl. Acad. Sci., U.S.A. 80, 1058-1062, 1983). One plasmid, pALS1, consists of the Neurospora ga-2+ gene (3 kb Hind III-fragment), the mitochondrial plasmid from N. intermedia strain P405-Labelle, and E. coli plasmid pBR325. The other, pALS2, is a putative deletion derivative of pALS1 that lacks most or all of the Labelle insert and that was repeatedly recovered from Neurospora transformants. We have now sequenced the region encompassing the deletion in five pALS2 plasmids isolated independently in two different laboratories. All five plasmids are identical in this region and completely lack the Labelle insert. We have also characterized an additional deletion derivative that retains a small (approximately 0.5 kb) segment of the Labelle insert. The results for pALS2 suggest that pBR325 plus the ga-2+ segment constitute a Neurospora replicon.     

The site-specific deletion in plasmid pBR322

The formation of a deletion derivative of plasmid pBR322, designated pBR322 delta 1, was observed during cloning of various eukaryotic DNAs, when the BamHI site of the plasmid vector was used for construction of the recombinant molecules. The restriction analysis of six independently isolated pBR322 delta 1 plasmids allowed establishment of their complete identity. Similar deletion derivatives were also formed as a result of transformation of Escherichia coli cells by the linear form of vector pBR322 produced by BamHI cleavage, but not by SalI or HindIII. The endpoints of the deletion in one of the pBR322 delta 1 plasmids occurred at positions 375 and 16666 bp from the EcoRI site, as determined by sequence analysis. Formation of pBR322 delta 1 is most probably due to site-specific recombination between the sequence in the 1666-1670 bp region and the BamHI end of the linear pBR322 molecule. THe deletion was not controlled by the recA system of the host bacteria.     

Functional organization of the ends of IS 1: specific binding site for an IS1-encoded protein

The IS 1-encoded protein InsA binds specifically to both ends of IS1, and acts as a repressor of IS1 gene expression and may be a direct inhibitor of the transposition process. We show here, using DNasel 'foot-printing' and gel retardation, that the InsA binding sites are located within the 24/25 bp minimal active ends of IS1 and that InsA induces DNA bending upon binding. Conformational modification of the ends of IS1 as a result of binding of the host protein integration host factor (IHF) to its site within the minimal ends has been previously observed. Using a collection of synthetic mutant ends we have mapped some of the nucleotide sequence requirements for InsA binding and for transposition activity. We show that sequences necessary for InsA binding are also essential for transposition activity. We demonstrate that InsA and IHF binding sites overlap since some sequence determinants are shared by both InsA and IHF. The data suggest that these ends contain two functional domains: one for binding of InsA and IHF, and the other for transposition activity. A third region, when present, may enhance transposition activity with an intact right end. This 'architecture' of the ends of IS1 is remarkably similar to that of IS elements IS10, IS50 and IS903.     

Role of replication in IS102-mediated deletion formation

Summary Intramolecular transposition produces replicon dissociation in a bireplicon; this reaction is homologous to the well-characterized IS-associated deletions in the case of a monoreplicon. However the frequencies at which these two reactions occur differ by a factor of more than 10² in favor of deletion formation. This raises the question of how these deletions occur. We show that the presence of a productive replication on the fragment to be deleted interferes with deletion formation. Our results also suggest that the deleted fragment is not degraded during deletion formation.     

Convenience of plasmid R6K higher-copy-number deletion derivative for cloning of larger DNA fragments

Vector properties of plasmid pNH602, a higher-copy-number deletion mutant of plasmid R6K, were tested by cloning the 6.5 Mg/molBam HI pSa fragment carrying determinants of resistance to four antibiotics in the uniqueBam HI site of pNH602. The resultingin vitro constructed recombinant plasmid pNH606 was found to be stable, conjugative, multicopy (20 copies of pNH606 perE. coli chromosome were estimated) and to ensure the increased expression of different genes responsible for the antibiotic resistance. The pSa fragment inserted in theBam HI site of plasmid pNH602 (located in Tn2660) was proved to be transposable to other replicons. Recombinant plasmid pNH606 was analyzed using restriction enzymesBam HI andEco RI and its physical and genetic map was constructed.     

Molecular cloning and mapping of a deletion derivative of the plasmid Rts 1

The plasmid pTW20 is a deletion derivative of the kanamycin resistance plasmid Rts1. By digesting pTW20 DNA with EcoRI endonuclease six fragments were generated, and each was cloned in the vector plasmid pACYC184. These cloned EcoRI fragments were further digested with various endonucleases, and the cleavage map of pTW20 was constructed. A SalI fragment (1.5 Md) in E1 (the largest EcoRI fragment; 11.5 Md) contained the genes kan (kanamycin resistance) and puv (uv sensitization of host). An electron microscopy study of a BamHI fragment containing kan revealed the presence of a transposon-like structure in the fragment. The smallest EcoRI fragment E6 (2.0 Md) was capable of autonomous replication in a polA host, indicating that E6 contained replication genes of pTW20. These genes were found to be located on a 1.1-Md HindIII fragment in E6. Two incompatibility genes were identified on the pTW20 genome, one located on each of the fragments E6 and E5 (3.5 Md), and expressed T incompatibility independently. The nature of the temperature sensitivity of pTW20 was discussed.     

Conservation of IS66 homologue of octopine Ti plasmid DNA in Rhizobium fredii plasmid DNA

Summary DNA sequences homologous to the T-DNA region of the octopine Ti plasmid from Agrobacterium tumefaciens are found in various fast-growing Rhizobium fredii strains. The largest fragment (BamHI fragment 2) at the right-boundary region of the core T-DNA hybridizes to more than one plasmid present in R. fredii. However, one smaller fragment (EcoRI fragment 19a) adjacent to the core T-DNA shows homology only with the plasmid carrying the symbiotic nitrogen-fixation genes (pSym). Hybridization data obtained with digested R. fredii USDA193 pSym DNA suggests that the homology is mainly with two HindIII fragments, 1.7 kb and 8.8 kb in size, of the plasmid. The 1.7 kb HindIII fragment also hybridizes to two regions of the virulence plasmid of A. tumefaciens, pAL1819, a deletion plasmid derived from the octopine Ti plasmid, pTiAch5. Hybridization studies with an insertion element IS66 from A. tumefaciens indicate that the 1.7 kb HindIII fragment of R. fredii plasmid, homologous to the T-DNA and the virulence region of Ti plasmid, is itself an IS66 homologue.     

New replication mutant pnh602 and its relationship to plasmid pAS3, another deletion derivative of plasmid R6K

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.