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Tamarin: Principles of Genetics, Seventh Edition
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III. Molecular Genetics
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13. Genomics, Biotechnology, and Recombinant DNA
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The McGraw Hill Companies, 2001
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Restriction Mapping
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Figure 13.24 Expression of green uorescent protein in root
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cells of the plant Arabidopsis thaliana under uorescent light. Chromosomes are visible in a cell undergoing mitosis and chromatin is visible as circles of green in interphase nuclei. The green uorescent protein gene was fused to the carboxy terminus of the gene for the transcription factor Cry2, controlling genes for the phototrophic response (bending toward light).
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(Copyright Sean Cutler, Stanford University Plant Biology Department.)
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Figure 13.23 Luminescent transgenic tobacco plant containing
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the re y luciferase gene. The plant was watered with luciferin, resulting in a re y glow. ( Science VU/Keith V. Wood/Visuals
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Unlimited.)
the Ti plasmids of Agrobacterium tumefaciens, as alluded to earlier. When a plant is infected with A. tumefaciens containing the Ti plasmid, a crown gall tumor is induced when the Ti plasmid transfects the host plant, transferring the T-DNA region. Those cells transfected with the T-DNA are induced to grow as well as to produce opines that the bacteria feed on. Much recent research has concentrated on engineering Ti plasmids to contain other genes that are also transferred to the host plants during infection, creating transgenic plants. One series of experiments has been especially fascinating. Tobacco plants have been transfected by Ti plasmids containing the luciferase gene from re ies. The product of this gene catalyzes the ATP-dependent oxidation of luciferin, which emits light. When a transfected plant is watered with luciferin, it glows like a re y ( g. 13.23). The value of these experiments is not the production of glow-
ing plants, but rather the use of the glow to report the action of speci c genes. In further experiments, the promoters and enhancers of certain genes were attached to the luciferase gene. As a result, luciferase would only be produced when these promoters were activated; thus, the glowing areas of the plant show where the transfected gene is active. One of the more recent reporter systems developed uses a gene from jelly sh that produces a green uorescent protein. The value of this system is that it reports when ultraviolet light shines on it, rather than requiring an addition, as in the luciferase system. The gene for the green uorescent protein is recombined with a gene in question, and then the transfection is performed. If the gene in question has transferred successfully, carrying the gene for the green uorescent protein, the uorescent protein will report it when activated by ultraviolet light ( g. 13.24).
RESTRICTION MAPPING
The number of cuts that a restriction enzyme makes in a segment of double-stranded DNA depends on the size of that DNA, its sequence, and the number of base pairs in the recognition sequence of the particular enzyme. That is, a restriction enzyme with only three base pairs in its recognition sequence will cut more times than one with six base pairs in its sequence, since the probability of a sequence occurring by chance is a function of the length of that sequence. A sequence of three bases occurs more 1/64 base pairs) than a seoften by chance (1/43 quence of six bases (1/46 1/4,096 base pairs). HindII,
Tamarin: Principles of Genetics, Seventh Edition
III. Molecular Genetics
13. Genomics, Biotechnology, and Recombinant DNA
The McGraw Hill Companies, 2001
Thirteen
Genomics, Biotechnology, and Recombinant DNA
for example, cuts the circular DNA of the tumor virus SV40 into eleven pieces; some restriction enzymes can cut E. coli DNA into hundreds of pieces. The product of the action of a restriction enzyme on a DNA sample is called a restriction digest. Using electrophoresis, we can separate the fragments of a restriction digest by size. With techniques to be described later, we can locate the restriction sites on the original gene or piece of DNA. That is, we can construct a map of the restriction recognition sites that will give us the physical distance between sites, in base pairs ( g. 13.25). This restriction map is extremely valuable for several reasons. For example, when the radioactive nucleotide tritiated thymidine was added for a very short period of time during the beginning of DNA replication in SV40 viruses, the radioactivity always appeared in only one restriction fragment. This demonstrated that SV40 replication started from a single, unique point; that point
was localized to a particular segment of the SV40 chromosome. In addition, a restriction map often allows researchers to correlate the genetic map and the physical map of a chromosome. Certain physical changes in the DNA, such as deletions, insertions, or nucleotide changes at restriction sites, can be localized on the genetic map. These changes can be seen as changes in size, or in the total absence, of certain restriction fragments when compared with wild-type DNA. This information allows us to see changes in the DNA; it also gives us information about the evolution of species (see chapter 21). The differences in fragment sizes are called restriction fragment length polymorphisms (RFLPs) and have proven valuable in pinpointing the exact location of genes and determining the identity or relatedness of individuals. A restriction digest is also useful for isolating short segments of DNA that can be easily sequenced.
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