Insertional mutagenesis using a synthetic lac operator - PubMed (original) (raw)
Insertional mutagenesis using a synthetic lac operator
D S Bautista et al. Gene. 1989.
Erratum in
- Gene 1990 Mar 1;87(1):155
Abstract
We have developed a novel cassette for generating insertion mutants in multi-copy bacterial plasmids. The cassette consists of synthetic oligodeoxyribonucleotides (oligos) which form a DNA duplex following reconstitution in vitro, due to sequence complementarity. It contains a 21-bp segment of the lac operator (lacZo), to provide a readily detectable phenotypic marker. Bacterial colonies harboring plasmids with insertions of this cassette are blue due to constitutive expression of the lac operon resulting from titration of lac repressor molecules by plasmid-borne lacZo sequences. Synthetic oligos containing a desire sequence may be added to the cassette by complementary ends for targeted insertion into plasmids. Sequencing of the resulting insertion mutants is facilitated by using oligos within the cassette as primers for bidirectional sequencing. This allows a complete characterization of each insertion in terms of location, structure of flanking sequences, and orientation of the inserted oligo. We have used this system to construct a series of mutants in early region 1a genes of human adenovirus type 5. For this purpose we designed a cassette which had all three possible translational reading frames open when inserted in one orientation, and all reading frames closed in the other orientation. The cassette also had BamHI restriction sites at each end which could be used to 'collapse' mutants, reducing the size of each insert to 6 bp.
Similar articles
- A synthetic translation-terminator gene. A tool for dissecting the translation direction of a gene.
Maruyama IN, Horikoshi K, Nagase Y, Soma M, Nobuhara M, Yasuda S, Hirota Y. Maruyama IN, et al. Gene Anal Tech. 1989 May-Jun;6(3):57-61. doi: 10.1016/0735-0651(89)90011-3. Gene Anal Tech. 1989. PMID: 2656460 - Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids.
Williams SG, Cranenburgh RM, Weiss AM, Wrighton CJ, Sherratt DJ, Hanak JA. Williams SG, et al. Nucleic Acids Res. 1998 May 1;26(9):2120-4. doi: 10.1093/nar/26.9.2120. Nucleic Acids Res. 1998. PMID: 9547269 Free PMC article. - The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers.
Vieira J, Messing J. Vieira J, et al. Gene. 1982 Oct;19(3):259-68. doi: 10.1016/0378-1119(82)90015-4. Gene. 1982. PMID: 6295879 - Mutation accumulation in vivo and the importance of genome stability in aging and cancer.
Dollé ME, Giese H, van Steeg H, Vijg J. Dollé ME, et al. Results Probl Cell Differ. 2000;29:165-80. doi: 10.1007/978-3-540-48003-7_9. Results Probl Cell Differ. 2000. PMID: 10838700 Review. No abstract available.
Cited by
- Phosphorylation at the carboxy terminus of the 55-kilodalton adenovirus type 5 E1B protein regulates transforming activity.
Teodoro JG, Halliday T, Whalen SG, Takayesu D, Graham FL, Branton PE. Teodoro JG, et al. J Virol. 1994 Feb;68(2):776-86. doi: 10.1128/JVI.68.2.776-786.1994. J Virol. 1994. PMID: 8289381 Free PMC article. - An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3.
Bett AJ, Haddara W, Prevec L, Graham FL. Bett AJ, et al. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8802-6. doi: 10.1073/pnas.91.19.8802. Proc Natl Acad Sci U S A. 1994. PMID: 8090727 Free PMC article. - Anti-adenovirus type 5 cytotoxic T lymphocytes: immunodominant epitopes are encoded by the E1A gene.
Routes JM, Bellgrau D, McGrory WJ, Bautista DS, Graham FL, Cook JL. Routes JM, et al. J Virol. 1991 Mar;65(3):1450-7. doi: 10.1128/JVI.65.3.1450-1457.1991. J Virol. 1991. PMID: 1704928 Free PMC article. - Determination of the origin-specific DNA-binding domain of polyomavirus large T antigen.
Sunstrom NA, Acheson NH, Hassell JA. Sunstrom NA, et al. J Virol. 1991 Dec;65(12):6998-7003. doi: 10.1128/JVI.65.12.6998-7003.1991. J Virol. 1991. PMID: 1658390 Free PMC article.
Publication types
MeSH terms
LinkOut - more resources
Full Text Sources
Other Literature Sources