A 27-bp synthetic DNA cassette was constructed which contains the restriction sites of the two rare-cutter enzymes NotI and SfiI and, in an overlapping arrangement, those of five enzymes with 6-bp recognition sequences: ApaI, BalI, NdeI, SacII, XmaIII. The protruding termini of the fragment allow its insertion into any EcoRI-cut DNA creating a new EcoRI site at one side of the cassette only. Th...

The Ty1 retrotransposon of Saccharomyces cerevisiae is bounded by long-terminal repeats (LTRs). We have constructed a variety of Ty1 elements in which the LTR length has been increased from the normal length of 334 bp to > 2 kb. Although small insertions in the LTR have minimal effects on transposition frequency, larger insertions dramatically reduce it. Nevertheless, elements with long LTRs ar...

Conjugative DNA transfer of IncI1 plasmid R64 is initiated by the introduction of a site- and strand-specific nick into the origin of transfer (oriT). In R64 oriT, 17-bp (repeat A and B) and 8-bp inverted-repeat sequences with mismatches are located 8 bp away from the nick site. The nicking is mediated by R64 NikA and NikB proteins. To analyze the functional organization of the R64 oriT region,...

Conformational transitions for a series of imperfect palindromes related to the dodecamer d(CGCGAATTCGCG) have been investigated. These sequences are: two isomeric 13-mers - d(CGCAGAATTCGCG) (13-merI) and d(CGCGAATTACGCG) (13-merII), 17-mer d(CGCGCGAATTACGCGCG) and 15-mer d(CGCGAAATTTACGCG). Insertion of a single adenine nucleotide prevents these sequences from being self-complementary. Analysi...

IS630 is a 1.15-kilobase sequence in Shigella sonnei that, unlike many mobile elements, seems not to mediate cointegration between different replicons. To assess its transposition, we constructed composite elements containing inverted copies of IS630 flanking a drug resistance gene. We found that these composite elements transposed to plasmid ColE1 in Escherichia coli. DNA sequencing showed tha...

SUMMARY Insertional mutagenesis is a powerful method for gene discovery. To identify the location of insertion sites in the genome linker based polymerase chain reaction (PCR) methods (such as splinkerette-PCR) may be employed. We have developed a web application called iMapper (Insertional Mutagenesis Mapping and Analysis Tool) for the efficient analysis of insertion site sequence reads agains...

Targeted transgenic mouse models, where an exogenous gene is inserted into a specified genomic locus to achieve its stable and reliable expression, have been widely used in biomedical research. However, the available methodologies for targeted insertion of sequences require many laborious steps that involve the use of embryonic stem (ES) cells. We recently developed Pronuclear Injection-based T...

We constructed insertion mutants of herpes simplex virus type 1 that contained a duplication of DNA sequences from the BamHI-L fragment (map units 0.706 to 0.744), which is located in the unique region of the L component (UL) of the herpes simplex virus type 1 genome. The second copy of the BamHI-L sequence was inserted in inverted orientation into the viral thymidine kinase gene (map units 0.3...

The IS6110 belongs to the family of insertion sequences (IS) of the IS3 category. This insertion sequence was reported to be specific for Mycobacterium tuberculosis complex and hence is extensively exploited for laboratory detection of the agent of tuberculosis and for epidemiological investigations based on polymerase chain reaction. IS6110 is 1361-bp long and within this sequence different re...