Cloning restriction enzyme

When compared with many cloned restriction enzymes, R-Fo I shares no apparent amino acid sequence similarities. It has no homology with the cognate M Fokl. 

The basic steps of gene cloning first involve cutting a precise DNA segment (gene) from a donor source DNA by use of a restriction enzyme (Figure 45.2). At the same time, a small looped... 

Workers in the early 1970s recognized that restriction enzymes provided tools not only for DNA mapping but also for constmction of new DNA species not found in nature. A collection of recombinant DNA species consisting of many passenger sequences joined to identical vector molecules is called a hbrary. Individual recombinant DNAs are isolated from single clones of the Hbrary for detailed analysis and manipulation. 

After a desired clone is obtained and mapped with restriction enzymes, further analysis usually depends on the deterrnination of its nucleotide sequence. The nucleotide sequence of a new gene often provides clues to its function and the stmcture of the gene product. Additionally, the DNA sequence of a gene provides a guidepost for further manipulation of the sequence, for example, lea ding to the production of a recombinant protein in bacteria. 

Type II restriction enzymes have received widespread application in the cloning and sequencing of DNA molecules. Their hydrolytic activity is not ATP-depen-dent, and they do not modify DNA by methylation or other means. Most importantly, they cut DNA within or near particular nucleotide sequences that they specifically recognize. These recognition sequences are typically four or six nucleotides in length and have a twofold axis of symmetry. For example, E. coU has a restriction enzyme, coRI, that recognizes the hexanucleotide sequence GAATTC ... 

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... 

The combination of restriction enzymes and various cloning vectors allows the entire genome of an organism to be packed into a vector. A collection of these dif-... 

The XE6 DNA was digested with several restriction enzymes and analysed via Southern blots using a probe derived fi-om the pgaW gene. A restriction map of the X.E6 clone revealed that the complete gene should be present on a 3.0 kb EcoRl fi agment (see Fig. 1). 

Each of the -6000 PCR products was then co-transformed into yeast along with the recipient vector that had been linearized using a restriction enzyme that digests the plasmid at the desired cloning site. The 70 bp of homologous flanking sequence on each end of the PCR products is sufficient for the yeast homologous recombination system to act upon and insert the PCR product into the vector (Hudson et al., 1997 Ma et al., 1987). 

We routinely use a mix of five mRNAs that are derived from the lys (ATCC no. 87482), trp (ATCC no. 87485), dap (ATCC no. 87486), thr (ATCC no. 87484), andphe (ATCC no. 87483) clones from the bacterium Bacillus subtilis cloned into a vector that contains a stretch of As. These RNAs are generated by in vitro transcription using a T3 in vitro transcription kit (e.g., MEGAscript from Ambion) of the linearized DNA template with the appropriate restriction enzyme. 

---