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AssigneeAssociated Universities, Inc.
InventorsF. William Studier, Parichehre Davanloo, Alan H. Rosenberg, Barbara A. Moffatt, John J. Dunn
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This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the T7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.
A process for obtaining clones of a DNA sequence encoding an enzymatically active RNA polymerase of any T7-like bacteriophage, comprising the steps of:
Plasmid pAR1151, ATCC 39561, containing a DNA sequence encoding enzymatically active T7 RNA polymerase, said plasmid comprising base pairs 3145 to 5841 of T7 DNA inserted into the BamHI site of pBR322 by use of synthetic linkers.
A BamHI-ended DNA fragment containing a sequence encoding enzymatically active T7 RNA polymerase which is obtainable from plasmid pAR1151, ATCC 39561.
Plasmid pAR1173, ATCC 39562, consisting of plasmid pAR1151 modified to contain a single BglII site to the 5' side of the 5' terminus of the DNA sequence encoding enzymatically active T7 RNA polymerase and within the BamHI-ended DNA fragment containing said coding sequence.
A BamHI-ended DNA fragment, containing a sequence encoding enzymatically active T7 RNA polymerase and a BglII site to the 5' side of the 5' terminus of said coding sequence and which is obtainable from plasmid pAR1173, ATCC 39562.
Plasmid pAR1219, ATCC 39563, consisting of a DNA fragment containing the lacI gene and the lacUV5 promoter of E.coli inserted into the BglII site of pAR1173 in the orientation such that transcription from the lacUV5 promoter produces functional mRNA encoding enzymatically active T7 RNA polymerase.
A BamHI-ended DNA fragment containing the lacI gene, the lacUV5 promoter, and a sequence encoding enzymatically active T7 RNA polymerase, and which is obtainable from plasmid pAR1219, ATCC 39563.
A process for creating vectors in which a DNA sequence encoding enzymatically active T7 RNA polymerase can be transcribed from a promoter recognizable by a selected host cell or viral RNA polymerase, comprising the steps of:
A process for inserting the DNA sequence encoding enzymatically active T7 RNA polymerase into a bacterial chromosome, comprising the steps of:
Bacteriophage CE6, obtained from a genetic cross between DE2 and lambda cI857indlSam7, wherein said bacteriophage CE6 comprises a DNA sequence encoding enzymatically active T7 RNA polymerase and the cI857indlSam7 mutations.
Dependencies are inferred from the claim text.