Bacterial restriction enzymes

At least 1(X) bacterial restriction enzymes are known, and each catalyzes DNA cleavage in a specific and predictable way. These enzymes are the tools used to take DNA apart and reduce it to fragments of known size and nucleotide sequence. 

Bacterial plasmids are small, circular, duplex DNA molecules whose natural function is to confer antibiotic resistance to the host cell. Plasmids have several properties that make them extremely useful as cloning vectors. They exist as single or multiple copies within the bacterium and replicate independently from the bacterial DNA. The complete DNA sequence of many plasmids is known hence, the precise location of restriction enzyme... 

Most often proteins are the bacterial biopolymers studied using MALDI MS either from fractions or whole cells. They are not the only isolated cellular biopolymers studied by MALDI, nor the first. Very soon after the introduction of MALDI there were a few reports of the analysis of bacterial RNA or DNA from bacterial fractions. One of the first applications of MALDI to bacteria fractions involved analysis of RNA isolated from E. coli,4 Other studies included analysis of PCR-amplified DNA,5 6 DNA related to repair mechanisms7 and posttranscriptional modification of bacterial RNA.8 While most MALDI studies involve the use of UV lasers, IR MALDI has been reported for the analysis of double stranded DNA from restriction enzyme digested DNA plasmids, also isolated from E. coli.9... 

It is clear that both intact cell MALDI-TOF and PFGE have their limitations. PFGE analyses probes the chromosomal DNA of microorganisms for variations in the locations of specific restriction enzyme cleavage sites, while MALDI-TOF mass spectrometry of intact cells primarily examines abundant proteins such as ribosomal proteins35 and those associated with or near bacterial cell walls.58 In order for MALDI-TOF to detect a variation, a mutation must lead to noticeable changes in the expression of cell wall—associated... 

Cutting with restriction endonucleases is very useful for moving specific pieces of DNA around from place to place. It s also a useful way to name pieces of DNA. For example, a piece of DNA that is cut from a bigger piece of DNA is often named by size and given a surname that corresponds to the two restriction enzymes that did the cutting—the 0.3-kb EcoRI-BamHI fragment. Restriction enzymes themselves are named for the bacterial strains from which they were initially isolated. 

PUTTING YOUR DNA INTO A VECTOR Vectors are specialized pieces of DNA used to move other pieces of DNA around. Modern vectors are usually either bacterial plasmids or viral genomes. The act of isolating your DNA in the first place usually involves putting it into a vector and then selecting the vector that has your DNA in it. DNA pieces (called inserts when they are placed in a vector) are usually placed in vectors using restriction endonucleases. The vector is cut with two restriction enzymes of different specificity (Fig. 6-3). This removes a... 

Obtain samples of selected restriction enzymes and reaction buffer (supplied with enzymes). Also obtain a DNA sample, either a viral (lambda) or bacterial plasmid. 

Alkaliphilic Bacillus sp. AM-001 was aerobically grown to the early stationary phase at 37°C in alkaline medium (pH 9.0) containing 1% konjak mannan. Total chromosomal DNA obtained by the method of Saito and Miura( 14) was digested with Hindlll restriction enzyme. And 2 to 4 kbp DNA fraction of chromosomal DNA was collected by 1% agarose gel electrophoresis. The plasmid pUC19 was digested with Hindlll and then dephosphorylated with bacterial alkaline phosphatase. After the... 

The carboxyl-terminal domain of the mammalian enzymes is related to bacterial restriction methyltransferases. Journal of Molecular Biology, 203, 971-983. 

Restriction endonucleases are bacterial enzymes that cleave DNA at sequence specific sites. They were first discovered in 1970 [19]. Almost 2000 restriction enzymes have been identified since, and several hundred of these are commercially available [1]. Many mutations remove or create a particular restriction site in the DNA sequence. These mutations can be identified by PCR amplification, incubation of the product with the appropriate enzyme followed by visualisation of the fragments on an agarose gel. 

One of the major obstacles to molecular analysis of genomic DNA is the immense size of the molecules involved. The discovery of a special group of bacterial enzymes, called restriction endonucleases (restriction enzymes), which cleave double-stranded DNA into smaller, more manageable fragments, has opened the way for DNA analysis. Because each enzyme cleaves DNA at a specific nucleotide sequence, restriction enzymes are used experimentally to obtain precisely defined DNA segments called restriction fragments. 

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