[Cloning of tobacco DNA fragment with promoter properties in a transgenic plant].
A selection system for isolating DNA sequences with transcription-promoting activity by their functioning in bacterial cells has been proposed. Tobacco nuclear DNA fragments were inserted in front of the promoterless neomycin 3'-phosphotransferase II (NPT-II) gene and promoter-like sequences were identified by their ability to restore NTP-II activity in E. coli cells. One of these recombinant plasmids was introduced in tobacco protoplasts by direct gene transfer and transformed calli were isolated by kanamycin selection. The NTP-II expression in regenerated transgenic plants were highest in root, slightly lower in stem and were practically absent in leaf. Sequence analysis of cloned segment showed the presence of conserved sequences essential for promoter activity in eukaryotic cells. A transcription start site was observed by S1 mapping. The size of protected fragments corresponds to the initiation of transcription 176 and 179 base pairs upstream the initiation codon in tobacco and 75 base pairs in E. coli.
Domanskiĭ
N N
NN
Gening
L V
LV
Kovalenko
P G
PG
Medvedeva
T V
TV
Galkin
A P
AP
Gazarian
K G
KG
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering; D019868:Kanamycin Kinase; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000235:genetics; D010947:Plants, Toxic; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*
D004247:DNA; D010770:Phosphotransferases; D019868:Kanamycin Kinase
Klonirovanie fragmenta DNK tabaka, obladaiushchego svoĭstvami promotora v transgennom rastenii.