Structural and functional studies of insertion element IS200

Abstract

The nucleotide sequence of the insertion element IS200 has been determined partially, including the junctions between the element and the host chromosome at the insertion site. At most, two bases (A-A) are found repeated at the junctions and could be duplications of host sequences generated by the insertion of the element. No obvious sequence repeats, either direct or inverted, have been detected between the sequences just within the two ends of the element. The element is an extremely strong block to host transcription across the insertion site. A sequence similar to known transcription termination signals was found just within the element near the right end. Removal of less than 50 base-pairs at the right end of the element abolishes the transcription block. The putative terminator sequence is located within this 50 base-pair region. Genetic studies suggest that the element contains a promoter located more than 93 base-pairs from its left end. The proposed promoter and terminator are in proper orientation to form an internal transcription unit.

Distribution of insertion sequence IS200 in Salmonella and Shigella

“…This problem is usually overcome by the isolation and labelling of an internal IS200 fragment (i.e. the 0.3 kb EcoRI-Hind111 fragment: see Lam & Roth, 1986). However, the existence of an additional EcoRI site in the hybrid phage DNA (Lam & Roth, 1986;Casades6s & Roth, 1989) makes the isolation tedious.…”

“…the 0.3 kb EcoRI-Hind111 fragment: see Lam & Roth, 1986). However, the existence of an additional EcoRI site in the hybrid phage DNA (Lam & Roth, 1986;Casades6s & Roth, 1989) makes the isolation tedious. Handling of M13Ho176 in genetic experiments also has specific limitations, since it can only infect F-containing strains and has no selectable markers.…”

“…Radioactive labelling of the 0.3 kb EcoRI-Hind111 fragment of pIZ45 provides an excellent substrate for both Southern blotting and colony hybridization. If genomic DNA is digested with a restriction enzyme that does not cut within IS200, such as PvuII or AvaI (Casadesus & Roth, 1989;Lam & Roth, 1986), the number of IS200 copies present in the DNA tested can be read directly as the number of hybridization bands observed in the autoradiography. With non-radioactive labelling procedures, clear-cut Southern patterns were only obtained when the number of hybridization bands was low (< 10).…”

“…dysenteriae, suggesting the existence of a related sequence. To ascertain whether any of the hybridization bands observed was due to the presence of IS630, a Shigella IS element that shows homology to one IS200 end (Matsutani et al, 1987), we carried out Southern hybridization analysis using a 5'-32P-labelled 32-mer homologous to the left end of both IS630 and IS200 (Lam & Roth, 1986;Matsutani et al, 1987). Genomic DNAs were digested with AuaI, an endonuclease that does not cut IS200 or IS630 (Casadesiis & Roth, 1989;Matsutani et al, 1987).…”

“…The chromosome of the standard, wild-type strain S. typhimurium LT2 contains six copies of IS200; moreover, one insertion mutation caused by IS200 is known: his-0984 : : IS200 (Lam & Roth, 1983a, b). Molecular analysis of this mutation unveiled several interesting features of the element: ( 1 ) it does not contain inverted repeats; (2) upon insertion, it does not generate duplication of host DNA sequences; (3) it is absolutely polar, at least in one orientation, because of the presence of a strong, rhoindependent terminator (Lam & Roth, 1986). Despite intensive efforts, hunts for new insertion mutants in Salmonella have been unsucessful and insertion elements other than IS200 have not been found (Casadesus & Roth, 1989).…”