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1.10 CLONING RESTRICTION MODIFICATION SYSTEMS

1.10.1 INTRODUCTION

Sixteen years ago, the first paper appeared describing the cloning of a complete restriction modification system in E. coli (Mann et al , 1978). The cloned restriction modification systems provide material for investigating the biology of DNA modification and

restriction and the biochemistry of DNA-protein interactions. They also constitute new, often more convenient or prolific sources from which restriction and modification enzymes can be purified. An example of over-expression of a restriction endonuclease is the Kpnl restriction-modification system, cloned and expressed in E. coli (Hammond et a l, 1990). The genes for restriction and méthylation were isolated sequentially (Section 1.10.3). Using a two step method, the strain was constructed with two compatible plasmids, an inducible plasmid with the Kpnl restriction gene which is activated at elevated temperatures and a pBR322 derivative expressing the methylase. The new strain produced 10 million units of Kpnl restriction endonuclease activity/g of wet weight cells. This was several 1000 fold higher than the level of Kpnl produced by the source bacteria

K pneumoniae (Hammond er a/., 1990).

There are many different methods of cloning restriction modification systems. The cloning methods use the functional properties of the restriction modification system, i.e., the ability to restrict foreign DNA and the ability to methylate DNA to isolate the clones.

1.10.2 PLASMIDS CODING FOR RESTRICTION MODIFICATION SYSTEMS

Natural plasmids can be subcloned if the restriction modification systems are coded within the plasmid. Purification of the plasmid, i.e., removal of chromosomal DNA prior to cutting and ligation, reduces the complexity of the DNA. The resulting fragments can

then be characterised. This is preferable to preparing genomic libraries and screening for restriction modification systems. EcdRl, EcoRR and EcoRV, PaeR ll and PvmII are all found on naturally occurring plasmids and have all been cloned in this manner (Wilson

1988).

The ^joll RM system was also cloned in this method (Karyagina et a l, 1990). Shigella sonnet strain 47 contains a complex array of plasmids. The S, sonnet 47 plasmids were transferred to E. colt by both transformation and conjugation. The plasmids were selected by susceptibility to phage infection (Section 1.10.3). The genes for the

endonuclease and the methylase are located on two small plasmids (designated -P6 and

P4 respectively) of S. sonnet 47.

1.10.3 SELECTION BASED ON SUSCEPTIBILITY TO PHAGE INFECTION

Some of the earliest restriction modification systems were cloned on the principle that cells that restrict are less susceptible to phage infection. Clones containing the HhaR

(Mann et a l, 1978) and Pst\ genes (Walder et a l, 1984) were isolated by their ability to survive infection. For the Pstl cloning experiments, transformants carrying hybrid plasmids of P. stuartü 164 chromosomal DNA and pBR322 were screened using bacteriophage X. Survivors were assayed for restriction activity. However, this method has been found to be of limited value as cloned restriction modification genes do not always manifest sufficient phage resistance to confer selective survival.

1.10.4 SELECTION BASED ON MODIFICATION

A more popular method of cloning restriction modification genes is based on selection using DNA modification. Methylase selection involves screening for methylase clones by exposing DNA from transformed hosts with the corresponding restriction endonuclease.

Survival indicates the presence of the methylase gene, since the DNA of the host is modified and insensitive to attack by the restriction endonuclease. Where restriction and modification are closely linked, both genes can be cloned simultaneously.

The Banl restriction modification system was cloned in a single step method. The chromosomal DNA from B. aneurinolyticus was partially digested with Sau2>Pd. The DNA was ligated to BamYil digested pBR322 and used for transformation of E. coli.

Plasmid DNA was isolated from approximately 10 000 ampiciUin resistant transformants. Plasmid DNA was digested with Banl and reintroduced into E. coli. The resultant

transformants were assayed for restriction activity. The genes for both restriction and modification were isolated on a 3.1kb fragment (Maekawa et a l, 1990).

Methylase selection does not necessarily yield a complete restriction system. Even attempts to clone larger regions of DNA failed to produce an active endonuclease gene in the BamHl and Ddel systems (Howard et a l, 1986; Brooks et a l, 1989). In these cases the failure stemmed from the introduction of the endonuclease gene into a host which is not adequately protected by méthylation. These restriction modification systems were cloned by a multi-stage procedure;

(i) The methylase gene was cloned by in vitro selection.

(ii) The genes were manipulated to achieve full méthylation of the host.

(iii) The modified cells were used as recipients of the restriction gene in a separate round of cloning.

The Ddel restriction modification system (Howard et a l, 1986) was cloned in this two step procedure as were the BamHl (Brooks et a l, 1989) and the StyLTl systems (De-Backer and Colson, 1991).

The genes for the ^ I restriction-modification system were isolated and expressed via a different method. The restriction endonuclease was purified to homogeneity, the N terminal sequence determined and used to make an oligonucleotide probe which was used to screen the Sfil modified libraries (VanCott, 1990).

A potential obstacle to cloning RM genes lies in the discovery that some strains of E. coli

react adversely to cytosine or adenine modification. The méthylation of adenine induces the SOS repair response (Heitman and Model, 1987; Raleigh e t al , 1988). Adenine methylated DNA in certain sequences is recognised by the product of a locus named mrr . It has been suggested that mrr encodes an endonuclease that cleaves adenine methylated DNA and that DNA scission induces SOS. Cytosine methylases foreign to E. coli also induce restriction. Restriction is due to two genetically distinct systems mcrB and mcrA

(modified cytosine restriction) that differ in their sequence specificities (Table 1.3). When cloning RM systems it is therefore advisable to use mutant strain of E. coli mcrAr,

mcrB', mrr'), in which these systems are defective.

Table 1.3. Méthylation systems in E. coli

System

Sequence Recognised

m rr

G AC where A i$ methylated