Chapter 5 Flashcards
Methods of Creating Recombinant DNA
EcoRI-pSC101 Cloning Experiments
In the early 1970s, Herbert Boyer and Stanley Norman Cohen produced pSC101, the first plasmid vector for cloning purposes. Soon after successfully cloning two pSC101 plasmids together to create one large plasmid, they published the results describing the experiment, in 1973. The cloning of genes into plasmids occurred soon after. In 1980, pSC101 became the first patented commercial DNA cloning vector when patents were awarded to Boyer and Cohen. The “SC” stands for Stanley Cohen. Although the original pSC101 only contained tetracycline resistance and a restriction site for EcoRI, the commercially available pSC101 gained restriction sites for several enzymes, including HindIII, in addition to the EcoRI site. The publication of these experiments prompted a frenzy, as many scientists were concerned about the ethical implications of cloning and recombinant DNA, and thought that guidelines should be established before progress continued.
The Asilomar Recommendations
Published in 1975 by a group of >100 molecular biologists who gathered at Asilomar Conference Center in Monterey, California.
Proposed that DNA cloning should make use only of bacteria that had been genetically disabled so that they could not grow well outside of a test tube. Also proposed a ban on the cloning of viral oncogenes until further notice. (Remember that Peyton Rous had recently won the Nobel Prize in Physiology and Medicine in 1966 for viral oncogenesis)
The recommendations in this report later became codified in US law under the newly established Recombinant DNA Advisory Committee at the NIH, including both scientists and concerned non-scientists. The ban on cloning viral oncogenes was lifted in 1979 when restrictions loosened.
HindII
The first restriction enzyme characterized.
Discovered and characterized in 1970 by Hamilton Smith, isolated from the bacterium Haemophilus influenzae. The cut is as follows:
5’ - G T Py | Pu A C - 3’
3’ - C A Pu | Py T G - 5’
The SV40 Restriction Map
The first restriction map to be obtained.
Was completed in 1971 by Hamilton Smith’s colleague Daniel Nathans, who used HindII to cut the SV40 genome into 11 discrete fragments.
Discovery of the Origin of Replication
Determined by Daniel Nathans using his HindII restriction map of SV40.
By briefly radiolabeling bacteria undergoing viral replication and showing that the radiolabel always appeared first in one fragment and then spread around the map bidirectionally, Nathans demonstrated that there is a specific origin of replication at one point in the circular DNA, and that replication occurs on both strands.
