Passenger DNA

Plasmid Vectors for Facile Introduction of Passenger DNA and Selection of Recombinants. The map of a commonly used plasmid vector, pUC19 (7), is shown in Figure 2. Three parts of the vector are key to its utility. The origin sequence, oh, allows the repHcation of plasmid DNA in high copy number relative to the chromosome. A gene, amp, encoding the enzyme beta-lactamase, which hydrolyzes penicillin compounds, allows... 

If an antibody to the protein of interest is available, it is sometimes possible to use vector sequences, eg, the beta-galactosidase promoter sequence, to direct the transcription of the passenger DNA into messenger RNA and the translation of that mRNA into protein which can be recognized by the antibody. Although this method is somewhat less reHable than the use of nucleic acid probes, specialized vectors are available for this purpose. 

Plasmids. See also Plasmid vectors expression, 12 474 mobilizable, 12 471 in salicylic acid manufacture, 22 8 Plasmid vectors, for passenger DNA introduction and recombinant selection, 12 501 Plasmin, 5 175... 

The piece of DNA to be cloned is tailed with poly(dT). After annealing and ligation recombinant plasmids carrying the passenger DNA will be formed. 

On this medium the desired recombinants do not grow because the Tet1 gene has been inactivated. This allows selection of colonies containing passenger DNA (Fig. 26-9). A further selection procedure is required to establish that the piece of DNA inserted into the recombinant plasmid is one that is desired. 

Of the 50 genes present in native X, only about half are necessary for replication in the lytic cycle. Thus, it is possible to delete about 1 / 3 of the genome to make room for more passenger DNA. However, to form mature phage particles the length of the DNA must be at least 75% of the native length. No more than 110% of the native amount may be present. The total DNA must fall between 38 and 53 kb in length. 

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