We have used this vector to clone some of the extremely toxic genes in sequenced genomes and describe here the analysis of the putative Type II restriction endonuclease genes in J99 and the functional characterization of their companion DNA methylase genes using novel antibodies. R-M systems, which are believed to be a primitive bacterial immune system, by alternatively turning on/off a subset of numerous R-M systems. INTRODUCTION Bacterial restrictionCmodification (R-M) systems are traditionally divided into three major types: I, II and III. The designations are based on enzyme subunit composition, co-factor requirements, DNA specificity characteristics and reaction products (1). The Type II R-M systems Metoprolol are the simplest. They usually have two impartial polypeptides: a restriction endonuclease which cleaves DNA and a corresponding DNA methyltransferase (methylase), which protects endogenous DNA from endonuclease digestion by methylating the endonuclease recognition sequence (2). Type II restriction endonucleases recognize specific sequences and cleave precisely either within or very close to their recognition sequences. They are key enzymes in biotechnology and molecular biology, and new specificities remain desirable (3,4). The traditional approach to screen for restriction endonucleases was to grow small cultures of individual strains, prepare cell extracts and then test the crude cell extracts for their ability to produce specific fragments on small DNA molecules (5). Using this approach, about 12 000 strains have been screened worldwide to yield the current harvest of more than 3000 restriction endonucleases (6). Roughly, one in four of all strains examined, using a biochemical approach, shows the presence of a Type II restriction enzyme. This is much lower than the average number of putative R-M systems predicted from a computer analysis of sequenced microbial genomes (Table ?(Table11). Table 1. Potential DNA R-M systems in sequenced microbial genomes based on the computational Metoprolol identification of putative methylase genes and the presence of adjacent ORFs of unknown function AR391.23?no candidates???0266951.6631423?22J991.6431623?23species3.57?1?315would be an example. The column labeled mR contains genes that share similarity with methylation-dependent restriction systems. More than two dozen bacterial and archaeal genomes have been completely sequenced within the last 5 years (7; Table ?Table1).1). The complete sequences of these genomes have revealed a remarkable fact: >80% of the genomes appear to have at least one DNA R-M system and 75% of these genomes appear to contain multiple R-M systems, Metoprolol most of which have never been assayed biochemically (Table Metoprolol ?(Table1).1). The extreme case is usually J99 which contains almost two dozen R-M systems, of which 16 appear to be Type II (8,9). The availability of these complete genome sequences has opened new avenues to screen for Type II restriction endonucleases. First, bioinformatics methods can be used to identify methyltransferase genes and nearby open reading frames (ORFs) are candidate endonuclease genes. Those candidate genes can then be cloned and their gene products tested biochemically. However, characterizing restriction endonuclease genes by cloning and expression can be very challenging, because the gene products are cytotoxic when expressed unless the endogenous genomic DNA is completely guarded by methylation (10). This means that usually both Metoprolol R and M genes must be cloned together or, more reliably, the M gene must be cloned and expressed first to provide a safe recipient. In theory an R gene could be safely expressed under the control of an inducible promoter such as the T7 promoter in the absence of protective methylation. However, while expression can be repressed >100-fold in the T7 expression system (11), the cloning and expression of extremely toxic genes, such as those for restriction endonucleases, often cannot be achieved presumably because of low level expression from read-through transcripts (H.Kong and R.J.Roberts, unpublished observations). While the idea of using opposing promoters to modulate gene expression has been described previously (12,13), it has not been successfully employed as a method to clone a toxic gene. One system, which relies upon conditional expression of a gene encoding spectinomycin resistance, has proved to be a useful genetic selection for genes encoding proteins capable of exhibiting transcriptional repressor-like activity (14C16). These studies showed that transcriptional inactivation of a gene can be achieved with an antisense promoter. Here we describe a new cloning vector, pLT7K, which combines repression and an antisense promoter as dual control elements to repress basal expression but nevertheless permits TSPAN9 inducible expression of a cloned gene. We have used this vector to clone some of the extremely toxic genes in sequenced genomes and describe here the analysis of the putative Type II restriction endonuclease genes in J99 and the functional characterization of their companion DNA methylase genes using novel antibodies. Finally we discuss the biological significance of the numerous R-M systems in J99 genomic.