Transposons as Portable Regions of Homology


The sefA gene encodes a type of pilus that is unique to Salmonella enteritidis. Given a sefA+ strain, a sefA::Kan mutant, and a P22 generalized transducing lysate grown on a random pool of Tn10 (tetracycline resistant) transposon insertions:

  1. How could you isolate a strain with a Tn10 insertion near the sefA::Kan mutation? Draw a diagram showing the donor and recipient DNA with any transposition or recombination events, and indicate the medium you would use for each selection or screen.

    ANSWER:

  2. Given a P22 phage lysate grown on a strain with a Tn10 insertion linked to the sefA+ gene, how could you isolate point mutations in the sefA gene? Draw a figure indicating the donor and recipient DNA and describe any selections you would use. [You do not need to describe how to screen for sefA point mutants.]

    ANSWER:


Transposon insertion mutations in the sefA gene decrease the virulence of Salmonella enteritidis in mice. Does this result prove that the sefA gene product is involved in virulence? Briefly explain your answer.

ANSWER: No. Recall that transposons are usually strongly polar of expression of downstream genes. Thus, an insertion in sefA would prevent expression of the downstream genes sefB, sefC, and sefD. It is possible that sefA itself plays no role in virulence but one of these other genes is required for virulence. [How would you test this possibility?]


The srl operon is required for growth on sorbitol as a sole carbon source.

  1. Given a lysate of phage P1 grown on a random pool of random Tn10 insertions into the wild-type Shigella chromosome and a Srl- recipient, how could you isolate a Tn10 insertion linked to the srl operon? [Show the crosses you would do and indicate the phenotype of each possible class of recombinant obtained. Be sure to indicate how you would confirm that the Tn10 insertion is linked to the srl operon.]

    ANSWER:

  2. How could you use this Tn10 insertion linked to the srl+ operon to isolate point mutations in srl? [Draw a figure indicating the donor and recipient and any selection or screen you would use.]

    ANSWER:


Transposons that carry antibiotic resistance genes can be used as a convenient genetic markers: it is possible to require inheritance of the transposon by selecting for antibiotic resistance, and it is possible to test for loss of the transposon by screening for antibiotic sensitivity. For example, transposons can be used for genetic mapping as shown in the following example. P22 lysates grown on two transposon insertion mutants that are linked to the put operon (zcc::Tn10) were used to transduce putP and putA mutants as shown in the table below.

Draw a map showing the relative cotransduction frequencies and locations of zcc-4::Tn10, zcc-6::Tn10, putP, and putA.

ANSWER:


Using the transposon insertions in the putA gene shown below, how could you isolate a deletion mutation that removes the material between putA2096::Tn10 and putA2097::Tn10? [Draw a diagram showing the genotype of the donor and recipient, the recombination events, the phenotype selected, and the genetic map of the final deletion mutant.]

ANSWER: Two ways of constructing the desired deletion using Tn10 as a portable region of homology are shown in the boxes below. Note that you need a way to select for any strains used -- it is easy to select for a strain that carries both a Tn10 (TetR)insertion and a MudJ (KanR) insertion because the two transposons encode different antibiotic resistances, but it is not trivial to construct a strain with two copies of Tn10 in the same gene.

It is also possible to obtain TetR Lac- colonies by simply transducing out the MudJ insertion without resulting in a deletion. Therefore, if you used the second approach for constructing the deletion it would be essential to do backcrosses to test for the deletion.


It is possible to isolate E. coli mutants with IS1 inserted at many different sites in the galE gene. When inserted in one orientation the insertions are polar on the galT and galK genes, but when inserted in the opposite orientation the insertions are not polar. Suggest a likely explanation for this observation.

ANSWER:


The put genes are required for growth on proline as a carbon or nitrogen source. By transduction between P22 grown on a donor strain with a Tn10 insertion to the right of the put operon, and a recipient strain with a Tn10 insertion to the left of the put operon, Muro-Pastor and Maloy (1995. J. Biol. Chem. 270: 9819-9827) isolated a tandem chromosomal duplication of the put operon. The donor and recipient strains are shown below (abc and xyz simply represent genes on the left or right hand side of the put operon shown for orientation):

  1. Draw a diagram showing how you could select for the chromosomal duplication. Indicate what phenotype(s) you would select for.

    ANSWER:

  2. How could you demonstrate genetically that you had actually constructed the duplication. ANSWER: This could be done in several ways. For example, the duplication strain could be transduced with phage carrying a putA::Cam mutation with selection for TetR, KanR, and CamR. The resulting colonies should be Put-, indicating that the second (wild-type) copy of putA is has been disrupted in the transductants.


Given a S. typhimurium pyr::MudJ mutant (i.e., a pyrimidine auxotroph) and phage P22 grown on a random pool of Tn10 insertions in the S. typhimurium chromosome, how could you isolate a Tn10 insertion near (i.e. "linked to") the pyr mutation?

ANSWER: Transduce a pyr- recipient with the pool of Tn10 insertions, selecting for TetR transductants. Replica print the TetR transductants onto minimal plates without pyrimidines and with tetracycline. Colonies that grow are potential transductants that brought in a copy of pyr+ with a closely located ("linked") Tn10 insertion.


After transduction of a pyr mutant with a random pool of Tn10 insertions, TetR Pyr+ transductants were obtained. How could you determine the linkage between the Tn10 insertion and the pyr mutation? [Draw a picture of the crosses you would do and label the donor and recipient phenotypes in each cross.]

ANSWER: A "backcross" of the TetR Pyr+ transductants with the Tets Pyr- parent will confirm whether the Tn10 is actually linked to the pyr locus and, if it is linked, this cross will also indicate how closely linked the two loci are (i.e., the cotransduction frequency).


phoA67 is a missense mutation in the E. coli alkaline phosphatase gene that has a dominant-negative phenotype -- that is, strains that have a tandem duplication with one copy of the phoA67 gene and one copy of the phoA+ gene are phenotypically PhoA-. [The tandem duplication with Tn10 at the join-point is shown below.]

In the absence of tetracycline, the phoA67 / phoA+ duplication segregrated in 10% of the cells. Two classes of segregrants were obtained. Draw a picture showing how the segregration occurs and indicate the phenotypes of the two types of segregrants.

Answer: see diagram in the box below.


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Last modified October 30, 2003