BamHI, restriction enzyme

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. 

By contrast, other restriction enzymes cut at different portions of a DNA molecule, forming an "offset" break with "sticky" ends. For example, the restriction enzyme known as BamHI cuts between two GG nitrogen bases, but at different parts of the DNA molecule, forming "overhangs or sticky ends. These sticky ends provide locations at which new nitrogen base sequences can be inserted. 

Other combinations of restriction enzymes may be used to excise the cloned fragment form the plasmid, each cleaving at a different side of the cloning site. However, avoid using BamHI, as the restriction site may not have been reconstructed in the recombinant fragments after ligation. 

A couple of important points exist about the consensus. First, not all bases in the consensus are conserved to the same amount. The bases marked with bold type and underlined are more conserved than the others, and the -10 region is more conserved overall than is the -35 region. Secondly, the promoter sequence is asymmetrical that is, it reads differently in one direction than in the other. (Compare this to the recognition sequence for the restriction enzyme BamHI, GGATCC.) This asymmetry means that RNA polymerase gets directional information from the promoter in addition to information about the starting point for transcription. 

EXAMPLE 8.17 Among the most important enzymes of recombinant DNA technology are the restriction enzymes. These are endonucleases that cleave DNA only at specific sequences of bases (called restriction sites). Typically, restriction sites are palindromic, in other words, the sequences are the same in the 5 — 3 and 3 — 5 strands. Restriction enzymes are produced by bacteria as an antiviral defense, and they cleave the DNA of viruses (bacteriophages) that infect them. However, they do not cleave host bacterial DNA. Fig. 8-17 shows the restriction sites of three common restriction enzymes, BamHI, EcoRI, and PvuII. Because BamHI and EcoRI cleave their restriction site asymmetrically, they produce overhangs in the cleaved DNA, called sticky ends. Conversely, PvuII cleaves symmetrically, producing blunt ends. 

Fig. 8-17 Restriction sites of the BamHI, EcoRI, and PvuII restriction enzymes. The cieavage positions are indicated by arrows.

BamHI and other restriction endonucleases are dimeric enzymes that bind to a DNA palindrome and cut both strands at equivalent positions. The cut leaves two ends with complementary overhangs that will... 

Arrows indicate the phosphodiester bonds cleaved by each restriction endonuclease, Asterisks indicate bases that are methylated by the corresponding methylase (where known), N denotes any base. Note that the name of each enzyme consists of a three-letter abbreviation (in italics) of the bacterial species from which it is derived, sometimes followed by a strain designation and Roman numerals to distinguish different restriction endonucleases isolated from the same bacterial species, Thus BamHI is the first (I) restriction endonuclease characterized from Bacillus amyloliquefaciens, strain H. 

Figure 39-4 Restriction map of the igH chain locus.The sites of the enzyme restriction sites determine the sizes of the fragments that are visualized using the igHJ6 probe as illustrated (e.g., BamHI, Xbai, and BglH enzymes yield 16 kb, 6.2 kb, and 3.8 kb fragments, respectively). Restriction patterns different from the germline patterns are indicative of a novel rearrangement event.

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