Chapter 10

14.  Ligase is an essential enzyme within all cells that seals breaks in the sugar–phosphate backbone of DNA. In DNA replication, ligase joins Okazaki fragments to create a continuous strand, and, in cloning, it is used to join the various DNA fragments with the vector. If it were not added, the vector and cloned DNA would simply fall apart.

15.  Each cycle takes 5 minutes and doubles the DNA. In 1 hour, there would be 12 cycles; so the DNA would be amplified 212 = 4096-fold.

18.  You could isolate DNA from the suspected transgenic plant and probe for the presence of the transgene by Southern hybridization.

22. 

  1. The transformed phenotype will map to the same locus. If gene replacement was due to double crossing over, the transformed cells will not contain vector DNA. If a single crossing over took place, the entire vector will now be part of the linear Neurospora chromosome.

  2. The transformed phenotype will map to a different locus from that of the auxotroph if the transforming gene was inserted ectopically (i.e., at another location). Ecotopic incorporation could also be inferred by reverse PCR.

23.  Size, translocations between known chromosomes, and hybridization to probes of known location can all be useful in identifying which band on a pulsed-field gel corresponds to a particular chromosome.

33.  The region of DNA that encodes tyrosinase in “normal” mouse genomic DNA contains two EcoRI sites. Thus, after EcoRI digestion, three different-size fragments hybridize to the cDNA clone. When genomic DNA from certain albino mice is subjected to similar analysis, no DNA fragments contain complementary sequences to the same cDNA. This result indicates that these mice lack the ability to produce tyrosinase because the DNA that encodes the enzyme must have been deleted.

36.  The promoter and control regions of the plant gene of interest must be cloned and joined in the correct orientation with the glucuronidase gene, which places the reporter gene under the same transcriptional control as the gene of interest. The text describes the methodology used to create transgenic plants. Transform plant cells with the reporter gene construct, and, as discussed in the text, grow them into transgenic plants. The glucuronidase gene will now be expressed in the same developmental pattern as that of the gene of interest, and its expression can be easily monitored by bathing the plant in an X-Gluc solution and assaying for the blue reaction product.