Volume 3

Gene expression / Benjamin Lewin.

  • Lewin, Benjamin.
Date:
1974-1977
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Gene expression / Benjamin Lewin. Source: Wellcome Collection.

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    series of experiments. Schmidt, Besemer and Starlinger (1976) took ad vantage of phages carrying IS1 or IS2 in opposite polarities to isolate the insertion sequences as the sole regions able to form duplex DNA when the denatured phage genomes are annealed. Gel electrophoresis and elec tron microscopy of the isolated IS DNA showed that IS1 is 820-850 base pairs long and IS2 has a length of 1300-1350 base pairs. Ohtsubo and Ohtsubo (1976) developed a method for the isolation of inverted repeat sequences, essentially relying upon their ability to renature very much more rapidly than other sequences. Applied to the insertion sequences, this identified lengths for the duplex stems of the isolated IS DNAs of 710-730 base pairs for IS 1, 1220-1270 base pairs for IS2, and 1290-1340 base pairs for IS3. The availability of isolated DNA molecules corre sponding to the insertion sequences may provide a useful probe for locating them readily in genetic elements and also may make possible the determination of their sequences by direct means. The isolation of insertion sequences by virtue of their polar effects in the gal operon implies that they must carry signals that influence gene expression. In at least one case polarity has been shown to result from the premature termination of transcription and it is possible that this applies to all instances of this phenomenon (see Adhya, Gottesman and De Crombrugghe, 1974). This premature termination appears to take place at sites subject to the action of the rho termination factor; rho-dependent termination is suppressed by certain bacterial mutations and a study of the effect of IS sequences in such mutant hosts may lead to definition of the types of control signal that they carry. For example, Reyes, Gottes man and Adhya (1976) have shown that certain bacterial mutations able to suppress polarity are effective upon IS1 but not IS2 or IS3; and the polarity of an insertion probably of the IS2 type is suppressed by the N function of phage lambda, which is an antiterminator (see Chapter 4). Further experiments of this type may resolve the relationship between the control signals carried on the various insertion sequences and their con nection with the signals utilized in the bacterial cell. What is the source of these insertion sequences? In a search for small Plasmids carrying just these sequences, Saedler and Heiss (1973) were unable to find any covalently closed circles of appropriate size in E.coli. But when denatured bacterial DNA was annealed with the DNA of \gal phages carrying either IS1 or IS2, the extent of hybridization was greater than that shown with control phages lacking the IS sequences. The increase corresponds to the presence of about 6-9 copies of IS1 and 4-6 copies of IS2 in the chromosomes of the E.coli K12 strains that were used. This demonstrates that these sequences are present in bacterial DNA, although we do not yet know how much variation there may be in