Enzymatic cleavage of a bacterial genome at a 10-base-pair recognition site.

Abstract

The circular genome of Staphylococcus aureus was cut into two fragments by a simple enzymatic method that cleaves a 10-base-pair site. The recognition sequence, A-T-C-G-mA J T-C-G-mA-T, was created by the combined use of the methylase MoCla I (A-T-C-G-mA-T) and the restriction endonucleaseDpn I (G-mA 4 T-C). This technique is insensitive to CpG methylation and in human DNA is predicted to produce fragments that, on average, are greater than five million base pairs. The ability to create such long pieces of DNA should facilitate mapping of large, complex chromosomes.Cutting chromosomes into a few, long fragments would markedly simplify the task of organizing and mapping complex genomic DNA. To reduce significantly the number of pieces that result from the cleavage of large genomes, it will be necessary to cut DNA into fragments that are greater than one million base pairs (1 Mb) and perhaps as large as 10 Mb. Recent work using pulsed-field electrophoresis (PFE) has resulted in the separation of intact, chromosomal DNA as large as 12 Mb (1, 2). However, present methods of cleaving DNA do not take advantage of this separation technology because they cut too frequently. It is generally true that the longer the recognition sequence of a DNA cleavage system, the less frequently it will cut and the larger the resulting fragments. To generate consistently pieces of DNA that are longer than 1 Mb requires a cutting technique with a recognition specificity of 10 base pairs (bp). The longest recognition sequence found in the case of type II restriction endonucleases is 8 bp (3), and on most genomes, the average size of fragments produced by these rare-cutting restriction enzymes is <1 Mb (4). A number of other enzymatic and chemical techniques that cut DNA less frequently than restriction endonucleases are still in the early stages of development (e.g., refs. 5 and 6).We previously described a method that uses adenine methylases to generate novel 8-and 10-bp recognition sequences for the methyladenine-dependent endonuclease Dpn I (G-mA I T-C; mA, N6-methyladenine) (7). It was possible with this technique to cleave a 10-bp recognition sequence on a specially constructed plasmid by using the adenine methylase M-Cla I (A-T-C-G-mA-T) with Dpn I. However, the technique had not been successfully applied to genomic DNA. We now demonstrate the improvement of the M-Cla I/Dpn I system and show that it can produce specific fragments of 1 Mb and greater in size by using the genome of the bacteria Staphylococcus aureus as a model substrate.MATERIALS AND METHODS DNA Preparation. Genomic DNA was prepared from S. aureus ISP8 that was provided by Peter Pattee (Iowa State University, Ames). The cells were grown to stationary phase, pelleted, and washed. Blocks or "inserts"of agarose containing S. aureus were made by first mixing SeaPlaque agarose (FMC BioProducts, Rockland, ME), final concentration of 1%, with 1010 cells per ml and allowing the mixture to solidify in 100-ul rectangular molds. Genomic DNA was prepared within the agaro...

Physical map of the chromosome of Neisseria gonorrhoeae FA1090 with locations of genetic markers, including opa and pil genes

“…Blocks were digested with 5 U of BglII (5' AGATCT 3'), NheI (5' GCTAGC 3'), or SpeI (5' ACTAGT 3') (Bethesda Research Laboratories [BRL]) in 120 ,ul of KGB (52) or with 5 U of PacI (5' TTAATTAA 3') (New England BioLabs) in NEBuffer 1 at 370C for 5 h. Digested blocks were melted at 65°C, loaded into a 1% agarose gel (15 by 15 cm) containing either individual wells or one large well extending the width of the gel, allowed to solidify, and subjected to pulsed-field gel electrophoresis.…”

Genotypic identification and phylogenetic analysis of the spotted fever group rickettsiae by pulsed-field gel electrophoresis

“…They studied rickettsiae genomes using randomly chosen DNA probes and RFLP analysis to determine genetic similarity on the basis of percent base pair mismatch. The principal disadvantage of these last two approaches is that only a small part of the genome is studied.Pulsed-field gel electrophoresis (PFGE) appears to be the best way to study the entire genome of SFG rickettsiae, since this has already been performed for other microorganisms (1,11,14,17,33,47,52,57). Several species of the family Rickettsiaceae have been studied by PFGE.…”

Pulsed-field gel electrophoresis studies of Rhizobiaceae

“…To show that the digests produced by these enzymes were complete, and to localize the nod and nif gene regions to fragments produced by rare-cutting restriction enzymes, we We treated the plugs with M.Clal followed by Ppnl (Weil and McClelland, 1989) because of the very large predicted size of the fragments (Table 3). Since Ppnl only is able to digest the sequence 5'-GATC-3' if N^-methyladenine is present, we first digested Rhizobiaceae PNA plugs with Ppnl ( Figure 2A).…”

“…Finally, we tested incubation of Rhizobiaceae DNA plugs with M.Clal alone, in the absence of Mg** ions (Weil and McClelland, 1989). Surprisingly, under these conditions, some digestion was observed for DNA in plugs from B. iaponicum strains and bacteroids, and from R. Flores et al, 1988;Romero et al, 1988;SoberonChaves and Najera, 1989).…”

“…after in vitro methylation with M.Clal. has been used to produce very large DNA fragments (Weil and McClelland, 1989). As can be seen from Table 3, the predicted fragment sizes for Rhizobiaceae genomic DNA treated with M.Clal/Dpnl is quite large.…”