Plasmid for the overproduction of bacteriophage T3 RNA polymerase, transcription vectors that carry a promoter recognized by its polymerase, gene coding for T3 RNA polymerase and application of these plasmids

• US 5,037,745 A
• Filed: 04/01/1986
• Issued: 08/06/1991
• Est. Priority Date: 04/01/1986
• Status: Expired due to Term

• Current Assignee: University of Medicine and Dentistry of New Jersey (State of New Jersey)
• Original Assignee: University of Medicine and Dentistry of New Jersey (State of New Jersey)
• Inventors: McAllister, William T.
• Primary Examiner(s): Schwartz, Richard A.
• Assistant Examiner(s): NOLAN, SHARON
• Application Number:
  US06/846,931
• Time in Patent Office: 1,953 Days
• Field of Search: 435/172.3, 435/6, 435/91, 435/320, 435/194, 435/235, 536/27, 935/29, 935/14, 935/31, 935/41, 935/16, 935/72, 935/73
• US Class Current: 435/91.3
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli

Abstract

A dual phage RNA polymerase promoter having a polylinker with at opposite ends, a T3 phage RNA polymerase promoter and a phage RNA polymerase promoter, like T7; the promoters are linked to the polylinker in opposite orientation. A recombinant DNA vector containing a T3 phage RNA polymerase promoter and a different phage RNA polymerase promoter; the promoters are linked to a polylinker sequence in opposite orientation. A kit including a T3 promoter and containing appropriate components for synthesizing RNA transcripts that are complementary to either strands of a cloned DNA sequence, and for other applications. Gene coding for T3 RNA polymerase, a vector containing the gene and transformed microorganisms.

Claims

1. A dual phase RNA polymerase promoter vector having a polylinker with a multiple cloning site, which polylinker is linked at one of its ends to a T3 phage promoter which is recognized by and is specific for T3 RNA polymerase and on the other of its ends to a second phage promoter, which is a promoter other than a T3 RNA polymerase promoter, the two promoters being linked in opposite orientation with respect to the polylinker, wherein transcription from either of the phage promoters proceeds toward the other promoter and in a direction opposite from the direction of transcription of the other phage promoter and the phage polymerase capable of transcription from said promoters is specific and does not recognize the other phage promoter, and which second phage promoter is recognized by the other polymerase.

2. The dual polymerase promoter vector of claim 1 wherein the second promoter is selected from the group consisting of a promoter for T7 and SP6 RNA polymerases.

3. The dual polymerase promoter vector of claim 2 wherein the second promoter is a T7 polymerase promoter.

4. The dual RNA polymerase promoter vector of claim 2 wherein the second promoter is an SP6 promoter.

5. The dual phage RNA promoter vector of claim 1 wherein the cloning site contains HindIII and EcoRI restriction sites.

6. A recombinant DNA vector which comprises a polylinker sequence having linked to one of its ends a T3 phage promoter which is recognized by and is specific for T3 RNA polymerase and at the other of its ends, a second phage RNA polymerase promoter, the two promoters being linked in opposite orientation with respect to the polylinker, wherein said second promoter is a promoter other than a T3 phage RNA polymerase promoter and a DNA sequence inserted in the polylinker sequence having a multiple cloning site, the respective promoters being in opposite orientation and the recombinant DNA vector being capable of transcription of DNA to produce RNA strands complementary to either one of the strands of the inserted DNA sequence when there is supplied to the vector a phage promoter-specific polymerase, wherein transcription from either of the phage promoters proceeds toward the other promoter and in a direction opposite from the direction of transcription of the other phage promoter and the phage polymerase capable of transcription from said promoters is specific and does not recognize the other phage promoter, and which second phage promoter is recognized by the other polymerase.

7. The recombinant DNA vector of claim 6 wherein the second promoter is a T7 RNA polymerase promoter.

8. The recombinant DNA vector of claim 6 wherein the polylinker includes restriction sites XmnI and PvuI, positioned between the two promoter sequences.

9. The recombinant DNA vector of claim 6 wherein the T3 RNA polymerase promoter has a consensus sequence recognized by T3 polymerase.

10. The recombinant DNA vector of claim 9 wherein the consensus sequence comprises the following sequence TATTAACCCTCACTAAAGGGAGA.

11. The recombinant DNA vector of claim 6 wherein the cloning site contains HindIII and EcoRI restriction sites.

12. A recombinant DNA vector which comprises a polylinker sequence connected at one end to a SP6 phage RNA polymerase promoter and at the other end to a T3 phage promoter which is recognized by and is specific for T3 RNA polymerase, the two promoters being linked in opposite orientation with respect to the polylinker, wherein transcription from either of the phage promoters proceeds toward the other promoter and in a direction opposite from the direction of transcription of the other phage promoter and the phage polymerase capable of transcription from said promoters is specific and does not recognize the other phage promoter.

13. The recombinant DNA vector of claim 12 wherein the polylinker has an EcoRI restriction site at the end adjacent to the T3 promoter.

14. A recombinant DNA vector of claim 13 wherein the T3 RNA polymerase promoter has a consensus sequence recognized by a T3 polymerase.

15. A kit for genetic applications, especially for transcription of DNA to synthesize RNA transcripts complementary to either strand of a DNA sequence, which kit comprises in combination the following components:

a recombinantly produced T3 RNA polymerase, another recombinantly produced phage RNA polymerase and a dual promoter transcription vector having a T3 phage RNA promoter specific for said T3 RNA polymerase and another promoter specific for the other phage RNA polymerase, the respective promoters being linked at opposite ends of a polylinker having a multiple cloning site the two promoters being linked in opposite orientation with respect to the polylinker, wherein transcription from either of the phage promoters proceeds towards the other promoter and in a direction opposite from the direction of transcription of the other phage promoter, each phage promoter being recognized by the phage encoded RNA polymerase which is specific to that phage promoter and does not recognize the other phage promoter, and which second phage promoter is recognized by the other polymerase.

16. The kit of claim 15 wherein the other recombinantly produced phage RNA polymerase is from phage T7.

17. The kit of claim 15 wherein the promoter other than the T3 promoter is selected from the group of promoters consisting of T7 and SP6.

18. The kit of claim 15 wherein the promoter other than T3 is a T7 promoter.

19. The kit of claim 15 wherein the promoter other than T3 is a SP6 promoter.

20. The kit of claim 15 wherein the cloning site contains the restriction sites HindIII and EcoRI.

21. A method for producing in vitro RNA copies of a selected DNA sequence, which comprises the steps of inserting the selected DNA sequence in a polylinker of a recombinant DNA vector, mixing the vector with either one of a T3 phage RNA polymerase or another phage RNA polymerase and generating RNA copies of the inserted DNA sequence, the vector comprising a polylinker sequence containing a restriction site and having linked to one of its ends, a T3 RNA polymerase promoter and on the other of its ends to a second phage RNA polymerase promoter other than a T3 phage RNA polymerase promoter, and transcription proceeding from either of the phage promoters towards the other promoter and in a direction opposite from the direction of transcription of the other phage promoter, the T3 phage RNA polymerase recognizes the second phage RNA polymerase promoter and is specific to that promoter.

22. The method of claim 21 wherein the other RNA polymerase and specific promoter are from phage SP6.