BamHI restriction site

Figure 9 Construction of plasmid pKE4-9SH. The promoter region and the clostridial hydrogenase structure gene were amplified by PCR. Both fragments were ligated at their BamHI restriction sites and cloned into the shuttle vector pKE4.

Figure 3.117 Oiganization of the gene cluster for pimaricin biosynthesis. The transcriptional direction and tiie relative sizes of the predicted orfs are indicated by pointed boxes, orfs corresponding to the PKS are indicated in color. Additional orfs of putative pimaricin tailoring, regulation, and resistance functions are indicated in gray. The 3P end of an unidentified orf (orfX) is indicated in white. Only BamHI restriction sites (B) are indicated. With permission from Aparicio et al. (2000). Copyright 2000, Elsevier.

The primers were also designed to flank the sequence with Nco and BamHI restriction sites, suitable for insertion into a pET-11d vector, without altering the amino acid sequence of the encoded BKR protein. 

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. 

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.

Figure 3. Characterization of plasmids carrying chimeric beta-tubulin genes. The top line represents the restriction map of the N. crassa beta-tubulin gene. Restriction sites are abbreviated as follows H, Hindlll Sa, Sail E, EcoRI Nc, Ncol Pv, PvuII B, BamHI. The black lines represent the portion from the plasmid pSV50 and white lines represent the portion from the plasmid pEFSO. The plus and minus indicate whether the transformants were able to grow on medium containing the chemicals.

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.

Fig. 1. Schematic diagram of pCR with the sphaeroides operon as a donor. Three of the classes of recombinants that arose from the chimera rescue experiments are also shown. The hatched boxes represent sphaeroides sequences the open boxes r resent capsulatus sequences. Only the Pstl sites that define the su lone of pJWl (3) used in this experiment are shown. Restriction sites B=BamHI E = coRI H = //i/iDIII P = Pstl S = Sad.

Hindm EcoT22T BamHI PslI PstI Ximl StuI Ncol AfUI (restriction sites)... 

Figure 24.2. pGaTB. To construct promoter-GAL4 fusions, the GAL4-encoding BamHI/Notl fragment of pGaTB is subcloned downstream from the appropriate promoter sequence in a P-element vector. Unique restriction sites are presented in boldface type. 

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