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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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rying DNA from human chromosome 20 (those clones light up autoradiographically). This technique, hybridization of cloned DNA without an electrophoreticseparation step, is referred to as dot blotting. These techniques can also be carried out without the grid arrangement of colonies by using replica plating as described in chapter 7. Thus, a speci c gene can be located after shotgun cloning.
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An entirely different method used to locate particular cloned genes utilizes the actual expression of the cloned genes in the plasmid-containing cells. If a eukaryotic gene is cloned in an E. coli plasmid downstream from an active promoter, that gene may be expressed (transcribed and translated into protein). Plasmids that allow the expression of their foreign DNA are termed expression vectors. There are many problems with this technique because bacteria normally would not express eukaryotic genes. However, special vectors have been developed in which the cloning site is just downstream from a promoter.The eukaryotic gene thus becomes part of the prokaryotic gene, producing a fusion protein, usually with only a few amino acids from the prokaryote. Of course, the eukaryotic gene must be in the correct orientation and in the correct codon reading frame for appropriate translation, meaning that the success rate of this technique is relatively low. A particular protein product can be located by western blotting, a method completely analogous to either
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Southern blotting or dot blotting. In this technique, probing is done with antibodies speci c for a particular protein, rather than using a radioactive oligonucleotide probe. A second antibody, speci c to the rst and labeled with a marker, usually uorescent, locates the rst antibody. For example, assume we are looking for the expression of a particular protein in the clones of gure 13.14. The clones would be transferred to a nitrocellulose membrane, where they would be lysed (e.g., with chloroform vapor).Then an antibody, speci c for the particular protein, would be applied. A second antibody, speci c for the rst antibody and labeled with a uorescent marker, would be applied to the lters. Fluorescence of the second antibody would locate the presence of the rst antibodies and thus indicate which of the clones is expressing the particular gene ( g. 13.15).
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Eukaryotic Vectors
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The work we have described so far involves introducing chimeric plasmids into bacteria, primarily E. coli. However, there are several reasons why we want to extend these techniques to eukaryotic cells (box 13.1). First, a prokaryote like E. coli is not capable of fully expressing some eukaryotic genes since it lacks the enzyme systems necessary for some posttranscriptional and posttranslational modi cations such as intron removal and some protein modi cation. Second, we also wish to study the organization and expression of the eukaryotic genome in
Nitrocellulose filter with twelve clones carrying pBR313 plasmids with inserts
Lyse cells, apply antibodies
Dot blot autoradiograph of 288 clones of DNA from a mouse-human hybrid cell line. After lysing samples of each clone on a nitrocellulose lter, the investigator hybridized the clones with radioactive probes for human-speci c sequences. The two dark spots indicate clones carrying human DNA. The slight background radiation in most other spots provides the spot pattern needed to orient the investigator.
Ultraviolet light reveals clone expressing the probed-for gene Figure 13.15 Western blot technique is used to locate an expressed protein from among many clones. Clones that may carry the expressed protein are lysed. Tagged antibodies are applied to locate the protein; a second antibody locates the rst antibody either through a uorescent color marker, as shown here, or with autoradiography.
(Source: Courtesy of Nick O. Bukanov.)
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
BOX 13.1
Ethics and Genetics
The Recombinant DNA Dispute
Paul Berg (1926 ).
(Courtesy of Dr. Paul Berg.)
aul Berg shared the 1980 Nobel Prize in chemistry for creating the first cloned DNA molecule, a hybrid phage that contained the genome of the simian tumor virus, SV40. The fact that he could do this work was worrisome to many people, himself included. The recombinant DNA dispute was underway. Berg voluntarily stopped inserting tumor virus genes into
phages that attack the common intestinal virus E. coli. People continue to worry about the dangers of working with recombinant DNA. One immediate and obvious concern is that cancer or toxin genes will escape from the laboratory. In other words, recombinant DNA technology could create a bacterium or plasmid that contained toxin or tumor genes. The modi ed bacterium or plasmid could then accidentally infect people. A 1974 report by the National Academy of Sciences led to a February 1975 meeting, which took place at the Asilomar Conference Center south of San Francisco. Berg convened this meeting, which over one hundred molecular biologists attended. The recommendations of the Asilomar Committee later formed the basis for
of cial guidelines developed by the National Institutes of Health (NIH). In essence, NIH established guidelines of containment. Containment means erecting physical and biological barriers to the escape of dangerous organisms. The NIH guidelines de ned four levels of risk, from minimal to high, and four levels of physical containment for them (called P1 through P4). The most hazardous experiments, dealing with the manipulation of tumor viruses and toxin genes, require extreme care, which included negativepressure air locks to the laboratory and experiments done in laminar ow hoods, with ltered or incinerated exhaust air. Biological containment means developing host cells and manipulated vectors that are incapable of successful reproduction outside the lab, even if they escape. High-risk work was done with host cells or vectors that were modi ed. For example, a bacterium of the E. coli strain EK2 cannot survive in the human gut because it has mutations that do not permit it to synthesize thymine or diaminopimelate.The lack of thymine-
vivo (in the living system), something we can only accomplish by working directly with eukaryotic cells. Finally, we wish to learn how to manipulate the genomes of eukaryotes for medical as well as economic reasons.To these ends, we discuss eukaryotic plasmids and the direct manipulation of eukaryotic genomes in vivo.
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