| RRC ID |
33446
|
| 著者 |
Nam HG, Loechel S, Fried HM.
|
| タイトル |
Plasmids allowing transcription of cloned DNA by Salmonella typhimurium phage SP6 RNA polymerase to produce RNAs with authentic 5'-terminal sequences.
|
| ジャーナル |
Gene
|
| Abstract |
We wished to determine whether there is any specific sequence downstream of the start point of the SP6 promoter which is required for its function in the plasmid pSP64 (Melton et al., 1984). Lack of such specificity would permit in vitro synthesis of an RNA molecule having a 5'-terminal sequence identical to its wild-type in vivo counterpart. To test its requirement, we replaced all of the SP6 sequence downstream of the transcription start point with heterologous nucleotides (nt) and found that any sequence will suffice to permit efficient and accurate transcription. These results permitted construction of plasmids for synthesis of 'authentic' transcripts from cloned DNA. In one case, by an oligodeoxynucleotide-mediated site-specific deletion, we placed the start point of yeast gene TCM1 at nt + 2 of the SP6 promoter and produced in vitro TCM1 mRNA with a wild-type 5'-terminal sequence. We also constructed a vector, pSP64 delta 1, in which the SalI/AccI/HincII recognition sites of pSP64 reside at nt + 2 through + 7. Plasmids such as pSP64 delta 1 may be more useful in some cases as insertion of any DNA fragment at one of these three sites will yield a transcript in which only two to four nt are derived from the vector.
|
| 巻・号 |
46(1)
|
| ページ |
57-64
|
| 公開日 |
1986-1-1
|
| DOI |
10.1016/0378-1119(86)90166-6
|
| PII |
0378-1119(86)90166-6
|
| PMID |
3026927
|
| MeSH |
Base Sequence
Chromosome Deletion
Cloning, Molecular
DNA / metabolism*
DNA Restriction Enzymes
DNA-Directed RNA Polymerases / metabolism*
Nucleic Acid Conformation
Operon
Plasmids*
Protein Biosynthesis
Salmonella Phages / enzymology
Salmonella Phages / genetics*
Salmonella typhimurium / enzymology
Salmonella typhimurium / genetics*
Species Specificity
Transcription, Genetic*
|
| IF |
2.984
|
| 引用数 |
5
|
|
WOS 分野
|
GENETICS & HEREDITY
|
| リソース情報 |
| 原核生物(大腸菌) |
pSP64Delta1 |
