Transposon mutagenesis

[Paul N Hengen]

Ed (e.power at umds.ac.uk) wrote:

>  I forgot to include a subject line last time!
>>
>> Hi folks
>>
>> Can anyone recommend, and tell me where I can get hold of a transposon 
>> suitable for mutagenesis in E. coli. Ideally something on a Ts vector with 
>> antibiotic resistance markers and something like a lacZ structural gene to 
>> assess gene expression in the mutants.
>>
>> Cheers
>>
>> Ed

Ed, I replied to your post, but you might not see it without a
subject line. So, here it is again...


TITLE [TI]
    Construction of Tn5 lac, a transposon that fuses lacZ expression to
    exogenous promoters, and its introduction into Myxococcus xanthus.

AUTHOR [AU]
    Kroos L; Kaiser D

JOURNAL [JL]
    Proc Natl Acad Sci U S A 81: 5816-20 (1984)

ABSTRACT [AB]
    A promoterless trp-lac fusion fragment was inserted near one end of the
    bacterial transposon Tn5 in the correct orientation to fuse lacZ gene
    expression to promoters outside Tn5. The resulting transposon, Tn5 lac,
    retains the kanamycin-resistance gene of Tn5 and transposes in
    Escherichia coli at 6% the frequency of Tn5 to many different sites in a
    bacteriophage lambda target. Expression of beta-galactosidase, the
    product of the lacZ gene, from Tn5 lac insertions in phage lambda
    depends both on insertion into a transcription unit in the correct
    orientation and on the regulation of the promoter of the transcription
    unit, verifying that by transposition Tn5 lac can fuse lacZ expression
    to outside promoters. An insertion of Tn5 lac in bacteriophage P1 was
    isolated and used to introduce Tn5 lac into Myxococcus xanthus, a
    bacterium that undergoes multicellular development. Stable
    kanamycin-resistant transductants are obtained that contain no P1 DNA
    sequences but have Tn5 lac inserted at different sites in the Myxococcus
    chromosome. Individual transductants express different levels of
    beta-galactosidase. A chromogenic substrate of beta-galactosidase,
    5-bromo-4-chloro-3-indolyl beta-D-galactoside, is toxic in Myxococcus
    when cleaved in large amounts. In principle, Tn5 lac could be used to
    assay transcription in any bacterium in which Tn5 can transpose and
    beta-galactosidase can be measured.


TITLE [TI]
    A Tn3 lacZ transposon for the random generation of beta-galactosidase
    gene fusions: application to the analysis of gene expression in
    Agrobacterium.

AUTHOR [AU]
    Stachel SE; An G; Flores C; Nester EW

JOURNAL [JL]
    EMBO J 4: 891-8 (1985)

ABSTRACT [AB]
    The construction and use of a Tn3-lac transposon, Tn3-HoHo1, is
    described. Tn3-HoHo1 can serve as a transposon mutagen and provides a
    new and useful system for the random generation of both transcriptional
    and translational lacZ gene fusions. In these fusions the production of
    beta-galactosidase, the lacZ gene product, is placed under the control
    of the gene into which Tn3-HoHo1 has inserted. The expression of the
    gene can thus be analyzed by monitoring beta-galactosidase activity.
    Tn3-HoHo1 carries a non-functional transposase gene; consequently, it
    can transpose only if transposase activity is supplied in trans, and is
    stable in the absence of this activity. A system for the insertion of
    Tn3-HoHo1 into sequences specifically contained within plasmids is
    described. The applicability of Tn3-HoHo1 was demonstrated studying
    three functional regions of the Agrobacterium tumefaciens A6 Ti plasmid.
    These regions code for octopine catabolism, virulence and plant tumor
    phenotype. The regulated expression of genes contained within each of
    these regions was analyzed in Agrobacterium employing Tn3-HoHo1
    generated lac fusions.


TITLE [TI]
    Lambda placMu: a transposable derivative of bacteriophage lambda for
    creating lacZ protein fusions in a single step.

AUTHOR [AU]
    Bremer E; Silhavy TJ; Weisemann JM; Weinstock GM

JOURNAL [JL]
    J Bacteriol 158: 1084-93 (1984)

ABSTRACT [AB]
    We isolated a plaque-forming derivative of phage lambda, lambda placMu1
    , that contains sequences from bacteriophage Mu enabling it to
    integrate into the Escherichia coli chromosome by means of the Mu
    transposition system. The Mu DNA carried by this phage includes both
    attachment sites as well as the cI, ner (cII), and A genes. Lambda
    placMu1 also contains the lacZ gene, deleted for its transcription and
    translation initiation signals, and the lacY gene of E. coli, positioned
    next to the terminal 117 base pairs from the S end of Mu. Because this
    terminal Mu sequence is an open reading frame fused in frame to lacZ,
    the phage can create lacZ protein fusions in a single step when it
    integrates into a target gene in the proper orientation and reading
    frame. To demonstrate the use of this phage, we isolated lacZ fusions to
    the malB locus. These showed the phenotypes and regulation expected for
    malB fusions and could be used to isolate specialized transducing
    phages carrying the entire gene fusion as well as an adjacent gene
    (malE). They were found to be genetically stable and rarely (less than
    10(-7] gave rise to secondary Lac+ insertions. We also isolated
    insertions into high-copy-number plasmids. The physical structure of
    these phage-plasmid hybrids was that expected from a Mu-dependent
    insertion event, with the lambda placMu prophage flanked by the Mu
    attachment sites. Lac+ insertions into a cloned recA gene were found at
    numerous positions and produced hybrid proteins whose sizes were
    correlated with the position of the fusions in recA.


TITLE [TI]
    Transposable lambda placMu bacteriophages for creating lacZ operon
    fusions and kanamycin resistance insertions in Escherichia coli.

AUTHOR [AU]
    Bremer E; Silhavy TJ; Weinstock GM

JOURNAL [JL]
    J Bacteriol 162: 1092-9 (1985)

ABSTRACT [AB]
    We have constructed several derivatives of bacteriophage lambda that
    translocate by using the transposition machinery of phage Mu (lambda
    placMu phages). Each phage carries the c end of Mu, containing the Mu
    cIts62, ner (cII), and A genes, and the terminal sequences from the Mu S
    end (beta end). These sequences contain the Mu attachment sites, and
    their orientation allows the lambda genome to be inserted into other
    chromosomes, resulting in a lambda prophage flanked by the Mu c and S
    sequences. These phages provide a means to isolate cells containing
    fusions of the lac operon to other genes in vivo in a single step. In
    lambda placMu50, the lacZ and lacY genes, lacking a promoter, were
    located adjacent to the Mu S sequence. Insertion of lambda placMu50 into
    a gene in the proper orientation created an operon fusion in which lacZ
    and lacY were expressed from the promoter of the target gene. We also
    introduced a gene, kan, which confers kanamycin resistance, into lambda
    placMu50 and lambda placMu1, an analogous phage for constructing lacZ
    protein fusions (Bremer et al., J. Bacteriol. 158:1084-1093, 1984). The
    kan gene, located between the cIII and ssb genes of lambda, permitted
    cells containing insertions of these phages to be selected independently
    of their Lac phenotype.

--
*******************************************************************************
* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
* - - -  Methods FAQ list -> ftp://ftp.ncifcrf.gov/pub/methods/FAQlist - - -  *
* -  TIBS column archive -> http://www-lmmb.ncifcrf.gov/~pnh/readme.html - -  *
* - The BEST Molecular Biology HomePage -> http://www-lmmb.ncifcrf.gov/~pnh/  *
*******************************************************************************