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Chapter 40 : Transposons and Their Applications

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Abstract:

This chapter focuses on the element Tn, which is the most extensively used transposon in . Numerous derivatives of Tn have been developed to facilitate the cloning and functional analysis of regulated genes identified by insertional mutations, and delivery vectors for obtaining Tn insertions function effectively in several other gram-positive species. This chapter also describes a new family of insertion elements based on derivatives of the transposon Tn that were specifically engineered for use in . The insertional mutagenesis systems now available for spp. are discussed briefly, primarily as a model for the establishment of such systems in gram-positive species in which barriers to the use of nonindigenous elements exist. Of particular importance for understanding the origins and properties of most of the Tn derivatives is the fact that the interval between and the nearest terminal inverted repeat consists of nonessential DNA that may be modified without interfering with transposition. Although transposons indigenous to other gram-positive bacteria, such as Tn and of , have been utilized effectively for insertional mutagenesis, only in the case of insertion elements have efforts been made to alter natural transposons to produce derivatives more useful for genetic analysis.

Citation: Youngman P. 1993. Transposons and Their Applications, p 585-596. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch40

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RNA Polymerase
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Streptomyces coelicolor
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Streptomyces lividans
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Figures

Image of Figure 1
Figure 1

Physical and functional map of Tn. Arrows indicate locations and orientations of transposon-associated genes: , gene encoding inducible erythromycin resistance; and , genes homologous to Tn3 genes with the same designations ( ). Hatched boxes at the ends represent 38-bp inverted repeats. The region labeled nonessential DNA consists of a 176-bp interval of DNA between the gene and the nearest inverted repeat that can be deleted or altered without interfering with transposition ( ). The nucleotide sequence of this 176-bp interval is given in reference . Hp, I restriction sites; N, I restriction sites. Other restriction sites are identified in footnote to Table 2 . The complete nucleotide sequence of 917 is given in reference , but it contains several errors, some of which (in the region) were corrected in a subsequent publication ( ).

Citation: Youngman P. 1993. Transposons and Their Applications, p 585-596. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch40
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Image of Figure 2
Figure 2

Genetic map of the chromosome showing locations of characterized Tn insertions relative to several commonly used chromosomal markers. Most of these insertions were not positioned by three-factor crosses, so except for insertional mutations that correspond to carefully mapped loci, locations are approximate and the relative positions of some closely spaced insertions are arbitrary. Additional information concerning specific insertions is given in Table 1 .

Citation: Youngman P. 1993. Transposons and Their Applications, p 585-596. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch40
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Figure 3

Delivery vector designed for selection of mini-Tnderivatives in ( ). Arrows with solid arrowheads represent locations and orientations of relevant genes: , chloramphenicol resistance gene of gram-positive origin and selectable in single copy in ; IS transposase, copy of the IS transposase gene provided with a ribosome-binding site appropriate for efficient translation in ; bla, lactamase gene conferring ampicillin resistance in ; , ribosome methyltransferase gene conferring resistance to erythromycin in . The segment marked Mini-Tn consists of the gene flanked by 307-bp inverted repeats that include the inside ends of IS ( ). ColE1, replication origin derived from pBR322 ( ); pE194Ts, replication functions derived from the temperature-sensitive gram-positive replicon pE194ts ( ). Most of the transposition-related elements of pHV1249 were constructed from pNK1250 ( ).

Citation: Youngman P. 1993. Transposons and Their Applications, p 585-596. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch40
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Tables

Generic image for table
Table 1

Catalog of Tn insertions (Continued)

Citation: Youngman P. 1993. Transposons and Their Applications, p 585-596. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch40
Generic image for table
Table 2

Tn Derivatives

Citation: Youngman P. 1993. Transposons and Their Applications, p 585-596. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch40
Generic image for table
Table 3

transposons

Citation: Youngman P. 1993. Transposons and Their Applications, p 585-596. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch40

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