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Modern Genetic Analysis Chapter 13. Transposable Genetic Elements
Insertion Sequences

Insertion sequences, or insertion-sequence (IS) elements, are now known to be segments of DNA that can move from one position on a chromosome to a different position on the same chromosome or on a different chromosome. When IS elements appear in the middle of genes, they interrupt the coding sequence and inactivate the expression of that gene. Owing to their size and in some cases the presence of transcription and translation termination signals, IS elements can also block the expression of other genes in the same operon if those genes are downstream from the promoter of the operon. IS elements were first found in E. coli in the gal operona set of three genes involved in the metabolism of the sugar galactose.

Physical Demonstration of DNA Insertion

Recall that phage l inserts next to the gal operon and that it is a simple matter to obtain ldgal phage particles that have picked up the gal region (page 288). When the IS mutations in gal are incorporated into ldgal phages and the buoyant density in a cesium chloride (CsCl) gradient of the phages is compared with that of the normal ldgal phages, it is evident that the DNA carrying the IS mutation is longer than the wild-type DNA. This experiment clearly demonstrates that the mutations are caused by the insertion of a significant amount of DNA into the gal operon. Figure 13-1 depicts this experiment in more detail.

Direct Visualization of Inserted DNA

When denatured ldgal DNA containing the insertion mutation is hybridized to denatured wild-type ldgal DNA, the extra piece of DNA actually can be located under the electron microscope. In such experiments, some of the DNA molecules that form in the mixture are not the parent duplexes but are heteroduplexes between one mutant and one wild-type strand. When point mutations are analyzed, the heteroduplexes are indistinguishable from the parental DNA molecules. However, in DNA containing the IS mutations, each heteroduplex shows a single-stranded buckle, or loop (Figure 13-2). This single-stranded buckle confirms the presence of an inserted sequence in the mutated DNA that has no complementary sequence in the wild-type DNA. The length of this single-stranded loop can be calibrated by including standardized marker DNA in the preparation. It proves to be approximately 800 nucleotides in length.

Identification of Discrete IS Elements

Are the segments of DNA that insert into genes merely random DNA fragments or are they distinct genetic entities? Hybridization experiments show that many different insertion mutations are caused by a small set of insertion sequences. In these experiments, the ldgal phages, which contain the gal gene, are isolated from the IS mutant bacteria, and their DNA is used to synthesize radioactive RNA in vitro. Certain fragments of this RNA are found to hybridize with the mutant DNA but not with wild-type DNA, indicative of the fact that the mutant contains an extra piece of DNA. These particular RNA fragments also hybridize to DNA from other IS mutants, showing that the same bit of DNA is inserted in different places in the different IS mutants.

On the basis of their patterns of cross-hybridization, the insertion mutants are placed into categories. The first sequence, the 800-bp segment identified in gal, is termed IS1. A second sequence, termed IS2, is 1350 bp long. Table 13-1 on the next page lists some of the insertion sequences and their sizes. The inverted repeats listed in Table 13-1 will be dealt with shortly under "Transposons."

SOURCE: M. P. Calos and J. H. Miller, Cell 20, 1980, 579595.

We now know that the genome of the standard wild-type E. coli is rich in IS elements: it contains eight copies of IS1, five copies of IS2, and copies of other less well studied IS types. It should be emphasized that the sudden appearance of an insertion sequence at any given locus under study means that these elements are truly mobile, with a capability for transposition throughout the genome. Presumably, they produce a mutation or some other detectable alteration of normal cell function only when they happen to end up in an "abnormal" position, such as the middle of a structural gene. Insertion sequences also are commonly observed in the F factor. Figure 13-3 on the following page shows an example of an F-lac episome.

MESSAGE
The bacterial genome contains segments of DNA, termed IS elements, that can move from one position on the chromosome to a different position on the same chromosome or on a different chromosome.

Orientation of IS Elements

Because of the base sequence, the two strands of ldgal+ DNA happen to have different buoyant densities. After DNA denaturation, they can be recovered separately in an ultracentrifuge. In some cases, the same strands (that is, parallel strands) from two different IS1 mutants form an unexpected hybrid with each other. Under the electron microscope, these hybrids have a peculiar appearance: each is a double-stranded region with four single-stranded tails (Figure 13-4). This observation is explained by assuming that the IS1 elements are inserted in opposite directions in the two mutants (Figure 13-5).

© 1999 by W. H. Freeman and Company. All rights reserved.