No abstract

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][9][10][11][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.In bacterial transformation, cells acquire new genetic information by taking up and incorporating DNA directly from the medium. Not all bacteria are transformable; in fact, only a few species have evolved this mechanism for genetic transfer. Diplococcus pneumoniae, Bacillus subtilis, and Haemophilus influenzae are the most carefully studied examples. These strains become competent (i.e., develop the ability to take up DNA) only under certain conditions of growth, implying that competence is an inducible state. With H. influenzae, 100% of the cell population can be induced to competence by transferring logarithmic phase cells from a rich medium into a synthetic medium that allows continued protein synthesis without cell multiplication (1). As competence develops, the cells become efficient in transporting large DNA molecules into the cell so that such molecules are no longer susceptible to external DNases or to high-salt elution. In D. pneumoniae and B. subtilis, the transport mechanisms are nonspecific (2, 3)-i.e., DNA from any source may be taken up. In H. influenzae, on the other hand, uptake is highly specific for Haemophilus DNA; other DNAs are not taken up, nor do they compete with Haemophilus DNA for uptake. This specificity was first clearly demonstrated in a careful study by Scocca et al. (4). They suggested that discrimination occurred at the cell surface and involved recognition of either a characteristic modification pattern or a characteristic base sequence in Haemophilus DNA.To determine which of these alternatives is the basis for the recognition, we have cloned an 8.1-kilobase (kb) DNA fragment of H. parainfluenzae in Escherichia coli and then reisolated the fragment for transformati...

No abstract

No abstract

No abstract

The incorporation into a 7% polyacrylamide gel of a nucleic acid-specific photochemical reagent 4,5',8-trimethylpsoralen at a concentration of 1 mg/dl of acrylamide solution improves both the sensitivity and the efficiency of the transfer of plasmid DNA fragments from the gel onto nitrocellulose filters. This improvement allows detection by blot hybridization of DNA fragments as small as 57 bp long.

No abstract

No abstract