Page 59 - Genetics_From_Genes_to_Genomes_6th_FULL_Part2

218 Chapter 6 DNA Structure, Replication, and Recombination

mitotic recombination does not happen in every germ-line cells by injection. Why is the irrevers-
cell in the mouse? (This precaution is necessary ibility of the ΦC31 integrase–mediated reaction
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because a mouse which has many smc /smc valuable for placing the transgene into the
homozygous cells could die as an embryo, before it Drosophila genome?
reaches adulthood.) d. Bacteriophage ΦC31 must eventually reverse this
38. Suppose that you could inject a wild-type mouse zy- reaction. Why? How do you think the bacterio-
gote with a specific CRISPR RNA and the Cas9 en- phage can achieve this reversal?
zyme. The RNA directs the Cas9 enzyme to make a 40. Cre is a recombinase enzyme encoded by a gene in
double-strand break within a gene that you think may bacteriophage P1. The Cre enzyme promotes site-
be responsible for a heritable disease. Diagram in specific recombination between two copies of a 34 bp
rough form how you might inject at the same time an- long DNA sequence called loxP that is derived from
other nucleic acid molecule (here, a double-stranded the same bacteriophage.
DNA) to exploit homologous recombination so that Researchers use the Cre/loxP site-specific recom-
you could convert the wild-type allele of the gene to a bination system in order to make mice homozygous
specific mutant allele. for a deletion of any particular gene and only in a spe-
39. ΦC31 is a type of bacteriophage that infects cific tissue. You will see in Chapter 18 that scientists
Streptomyces bacteria. One gene in the bacteriophage first make mice where a pair of loxP sites are config-
genome specifies a recombinase called ΦC31 inte- ured in a particular way with respect to the gene to be
grase that works through a mechanism slightly differ- deleted.
ent from that of the recombinase shown in Fig. 6.30. a. Draw a diagram that shows the configuration of the
Most importantly, the two target DNA sequences are loxP sites that would enable deletion of a gene by
different from each other. One called attP is 39 base site-specific recombination.
pairs and is found on the circular bacteriophage chro- b. What else would the researchers need to introduce
mosome, while the other—attB—is 34 base pairs into the mouse genome in order to generate mice
long and is located on the much larger circular bacte- where only one tissue is homozygous for the gene
rial chromosome. Excepting two base pairs roughly deletion?
in the middle of both targets that are identical and c. Why do you think that scientists would want to
at which recombination takes place, the DNA generate mice like this?
sequences of attP and attB are completely different
from each other. d. Unlike the DNA rearrangements at attP and attB
a. Diagram the reaction that ΦC31 integrase per- sites catalyzed by ΦC31 integrase (described in
Problem 39), the DNA rearrangements caused by
forms. How could this reaction be important for Cre recombinase are reversible. Why?
the life cycle of the bacteriophage?
b. Using the diagram you just drew, explain why e. Why does the reversibility of Cre/loxP-mediated
recombination not interfere with the use of the
ΦC31 integrase cannot reverse the reaction. Cre/loxP system to generate mouse tissues with
c. Now consider how you might exploit this site- deletions?
specific recombination to place genes from another 41. Like Cre/loxP recombination, site-specific recombi-
species (a transgene) into the genome of an experi- nation mediated by the Flp/FRT system is reversible.
mental organism like Drosophila. Assume you can Why doesn’t this fact interfere with the experiment
make any DNA sequences you want and that you described in Problem 37?
can introduce these DNA sequences into fruit fly