| RRC ID |
33514
|
| 著者 |
Gray MR, Colot HV, Guarente L, Rosbash M.
|
| タイトル |
Open reading frame cloning: identification, cloning, and expression of open reading frame DNA.
|
| ジャーナル |
Proc Natl Acad Sci U S A
|
| Abstract |
A plasmid was constructed that facilitates the cloning and expression of open reading frame DNA. A DNA fragment containing a bacterial promoter and the amino terminus of the cI gene of bacteriophage lambda was fused to an amino-terminally deleted version of the lacZ gene. An appropriate cloning site was inserted between these two fragments such that a frameshift mutation was introduced upstream of the lacZ-encoding DNA. This cloning vehicle produces a relatively low level of beta-galactosidase activity when introduced into Escherichia coli. The insertion of foreign DNA at the cloning site can reverse the frameshift mutation and generate plasmids that produce a relatively high level of beta-galactosidase activity. A large fraction of these plasmids produce a fusion protein that has a portion of the lambda cI protein at the amino terminus, the foreign protein segment in the middle, and the lacZ polypeptide at the carboxyl terminus. The production of a high level of beta-galactosidase and a large fusion polypeptide guarantees the cloning of a DNA fragment with at least one open reading frame that traverses the entirety of the fragment. Hence, the method can identify, clone, and express (as part of a larger fusion polypeptide) open reading frame DNA from among a large collection of DNA fragments.
|
| 巻・号 |
79(21)
|
| ページ |
6598-602
|
| 公開日 |
1982-11-1
|
| DOI |
10.1073/pnas.79.21.6598
|
| PMID |
6815653
|
| PMC |
PMC347175
|
| MeSH |
Base Sequence
Cloning, Molecular*
Galactosidases / genetics*
Genes
Genetic Engineering / methods*
Lac Operon
Mutation
Operon
beta-Galactosidase / genetics*
|
| IF |
9.412
|
| 引用数 |
60
|
|
WOS 分野
|
BIOLOGY
|
| リソース情報 |
| 原核生物(大腸菌) |
pMR100 |
