| Conventional genetic analysis of retroviruses is extremely labor-intensive, because each viral mutant must be maintained as an individual cell line. This thesis presents the development of genetic footprinting, a novel technique for high-throughput analysis of in-frame insertion or block substitution mutants in retroviruses or any gene for which a genetic or physical selection can be devised. Genetic footprinting has been applied to three model systems: the supF tRNA, the TEM-1 beta-lactamase protein, and the first 1000 nucleotides of the HIV-1 genome. In each case, the density of mutants analyzed has greatly exceeded that found in previous studies of that DNA segment. These structure-function analyses have confirmed previously-known features, and allowed the identification of novel determinants within these genes.; Genetic footprinting involves creating libraries of mutants, bearing in-frame insertions at diverse positions, by a modified in vitro transposition reaction. To allow conversion of insertion mutants to block substitution mutants, two type IIS restriction endonuclease recognition sites are incorporated into the inserted sequence. Cleavage at the type IIS sites removes the inserted sequence as well as a fixed amount of flanking wild-type sequence; the cleaved mutant is ligated to a double-stranded oligonucleotide that precisely replaces the deleted sequence, generating a block substitution mutant.; The phenotypic analysis of mutants by genetic footprinting is accomplished by subjecting libraries of mutants to en masse functional selections, and isolating DNA from the mutant populations before and after selection. The representation of each mutant within the pre- and post-selected populations is determined retrospectively by a genetic footprinting polymerase chain reaction (PCR), in which each mutant gives rise to a PCR product of unique electrophoretic mobility. This thesis shows that genetic footprinting can be used to perform parallel, quantitative phenotypic analysis on a large number of mutants without the need for cloning and sequencing of individual mutants. |
