Sequence-specific DNA uptake in Haemophilus transformation.

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RESUMO

Haemophilus cells efficiently take up Haemophilus DNA from the medium during transformation but do not take up other DNAs. To study the mechanism of this specificity we have cloned an 8.1-kilobase (kb) fragment of H. parainfluenzae DNA in the escherichia coli--pBR322 host--vector system and reisolated the DNA fragment for use as a defined probe. The 5'32P end-labeled 8.1-kb DNA is efficiently absorbed by competent Haemophilus cells whereas vector DNA present in the mixture is not, implying that the 8.1-kb DNA contains sequence-specific recognition sites that are needed for DNA uptake. Absorbed DNA can be recovered from cells as a 32P-labeled duplex of unaltered size for several minutes after uptake. We have determined the number and location of uptake sites in the 8.1-kb DNA by constructing a restriction endonuclease cleavage map and assaying fragments for uptake. Only two small fragments retain the ability to be absorbed. These fragments, 120 and 140 base pairs long, are 3.8 kb apart on the 8.1-kb fragment. We assume that each of these fragments contains a short common sequence, perhaps 8--12 base pairs long, that is the actual recognition site. We have shown by DNA competition assays, with the 8.1-kb DNA as a standard, that about 600 copies of the uptake sites are present in the Haemophilus genome.

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