Gene coding for a protein having T3 polymerase activity

• US 5,102,802 A
• Filed: 01/02/1991
• Issued: 04/07/1992
• Est. Priority Date: 04/01/1986
• Status: Expired due to Fees

• 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:
  US07/636,686
• Time in Patent Office: 461 Days
• Field of Search: 435/252.3, 435/320.1, 435/252.33, 435/256, 435/252.35, 435/254, 435/252.31, 536/27
• US Class Current: 435/252.33
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: Y10S 435/94 Saccharomyces

Abstract

A cloned DNA sequence encoding T3 RNA polymerase is provided. This DNA can be inserted into an expression vector for recombinant expression of the peptide. The peptide can be used with dual promotor vectors containing T3 specific promoters.

Claims

1. A cloned DNA sequence having the following nucleotide sequence:

ATGAACATCATCGAAAACATCGAAAAGAATGACTTCTCAGAAATCGAACTGGCTGCTATCCCGTTCAACAC ACTGGCTGACCACTACGGAAGCGCCTTGGCTAAAGAGCAGTTGGCTTTAGAACATGAGTCTTATGAGCTAG GCGAGCGCCGCTTCCTCAAGATGCTTGAGCGTCAAGCGAAAGCTGGTGAGATTGCAGACAACGCAGCCGCT AAGCCGTTACTCGCTACGCTTCTCCCTAAGTTAACCACACGTATCGTCGAGTRGGCTCGAAGAGTACGCATC GAAGAAAGGCCGCAAGCCTAGCGCATACGCACCGCTCCAGTTACTCAAGCCGGAGGCCTCCGCGTTTATCA CCCTGAAAGTTATCCTTGCGTCACTAACCAGTACGAACATGACAACCATTCAGGCCGCTGCTGGTATGCTG GGGAAAGCCATTGAGGACGAGGCACGATTTGGGCGCATCCGTGACCTAGAAGCGAAGCACTTCAAGAAGCA CGTTGAGGAACAGCTAACAAGCGCCACGGGCAAGTCTACAAGAAAGCATTATGCAGGTGGTCGAGGCCGA TATGATTGGTCGAGGTCTGCTTGGTGGCGAGGCGTGGTCTAGCTGGGATAAAGAAACCACGATGCACGTAG GGATTCGCCTGATTGAAATGCTGATTGAATCCACGGGTCTGGTGGAATTACAGCGCCACAACGCAGGTAAC GCAGGCTCTGACCATGAGGCACTGCAACTGGCCCAAGAGTACGTGGACGTATTAGCGAAGCGTGCAGGCGC CTGGCGGGTATCTCTCCGATGTTCCAGCCGTGTGRTCGTACCGCCGAAACCTTGGGTAGCAATCACAGGGGG CGGCTATTGGGCTAACGGTCGCAGACCTTTGGCACTCGTTCGCACTCACTCTAAGAAGGGCTTGATGCGCT ACGAAGACGTTTACATGCCAGAAGTCTACAAGGCTGTGAACCTCGCGCAAAACACCGCATGGAAAATCAAC AAGAAAGTTCTTGCTGTTGTCAATGAGATTGTTAACTGGAAGAATTGCCCGGTAGCAGACATTCCATCGCT GGAGCGCCAAGAGTTACCGCCTAAGCCTGACGACATTGACACCAACGAGGCAGCGCTCAAGGAGTGGAAGA AAGCCGCTGCTGGTATCTATCGCTTGGACAAGGCACGAGTGTCTCGCCGTATCAGCTTAGAGTTCATGCTG GAGCAGGCCAACAAGTTCGCAAGTAAGAAAGCAATCTGGTTCCCTTACAACATGGACTGGCGCGGTCGTGT GTACGCTGTGCCGATGTTCAACCCGCAAGGCAACGACACTGACGAAAGGTCTGCTGACCCTTGCTAAAGGCA AGCCAATCGGTGAGGAAGGTTTCTACTGGCTGAAAATCCACGGTGCGAACTGTGCGGGTGTTGATAAGGTT GGTTCCATTCCCGGAGCGCATCGCGTTCATTGAGAAGCACGTAGACGACATTCTGGCTTGCGCTAAAGACC CAATCAATAACACTTGGTGGGCTGAGCAGGATTCACCGTTCTGTTTCCTCGCGTTTTGCTTCGAGTATGCA GGCGTTACGCACCACGGTCTGAGCTACAATTGCTCTCTGCCGCTGGCGTTCGACGGGTCTTGCTCTGGTAT CCAGCACTTCTCCGCGATGCTCCGCGATGAGGTAGGCGGTCGTGCGGTTAACCTGCTGCCAAGCGAAACCG TGCAGGACATTTACGGCATCGTTGCACAGAAAGTAAACGAGATTCTCAAACAGGATGCAATCAACGGCACG CCTAACGAGATGATTACCGTGACCGACAAGGACACCGGGGAAATCTCAGAGAAGCTCAAACTTGGAACCTC AACGCTGGCGCAACAGTGGCTGGCATATGGTGTAACCCGTAGCGTAACTAAACGTTCGGTCATGACGCTGG CTTACGGTTCCAAGGAGTTCGGCTTTCGTCAACAGGTATTGGATGACACCATTCAGCCTGCAATTGACAGC GGTAAGGGCTTGATGTTCACCCAACCGAACCAAGCGGCTGGCTATATGGCTAAGCTGATTTGGGATGCGGT AAGCGTGACCGTAGTTGCAGCGGTTGAGGCGATGAACTGGCTCAAATCTGCCGCTAAGCTGCTGGCTGCTG AGGTCAAGGACAAGAAGACCAAGGAGATTCTGCGCCACCGTTGCGCGGTTCACTGGACTACGCCGGACGGC TTCCCGGTCTGGCAGGAATACCGCAAGCCACTCCAGAAGCGTCTCGATATGATTTTCTTAGGGCAATTCCG TCTGCAACCGACGATTAATACCCTCAAGGATTCAGGCATTGACGCACACAAGCAGGAGTCTGGCATCGCTC CTAACTTTGTTCACTCACAGGACGGTAGCCACCTCCGCATGACAGTCGTTTATGCTCACGAGAAGTATGGC ATTGAGCCTTTGCGCTCATCCATGACAGCTTTGGGACTATCCCGGCAGACGCTGGTAAGCTCTTTAAGGCT GTGCGTGAAACGATGGTTATCACCTATGAGAACAACGATGTGCTGGCAGACTTCTACTCTCAGTTTGCCGA CCAGCTACACGAGACCCAACTGGACAAGATGCCTCCGCTTCCGAAGAAAGGAAACCTGAACCTGCAAGACA TTCTCAAGTCTGACTTTGCCTTTGCATAA, which nucleotide sequence encodes a polypeptide having T3 RNA polymerase activity or a nucleotide sequence encoding a functionally equivalent polypeptide.

2. A recombinant plasmid adapted for transformation of a microbial host and subsequent replication therein comprising an expression vector which carries the nucleotide sequence of claim 1 encoding a polypeptide having T3 RNA polymerase activity or the functional equivalent thereof.

3. A transformed microorganism which includes the recombinant plasmid of claim 2.

4. The transformed microorganism of claim 3 wherein the microorganism is selected from the group consisting of the genera Escherichia, Saccharomyces, Bacillus, Neurospora and Streptomyces.

5. The transformed microorganism of claim 4 wherein the microorganism is E. coli.

6. The transformed of claim 5 wherein the microorganism is E. coli BL21.

7. The transformed microorganism of claim 4 which is free of proteolytic enzyme activity, thereby avoiding cleavage of the polymerase.

8. The transformed E. coli of claim 5 which carries expression vector pCM56.

9. A cloned DNA sequence having the following nucleotide sequence:

ATGAACATCATCGAAAACATCGAAAAGAATGACTTCTCAGAAATCGAACTGGCTGCTATCCCGTTCAACAC ACTGGCTGACCACTACGGAAGCGCCTTGGCTAAAGAGCAGTTGGCTTTAGAACATGAGTCTTATGAGCTAG GCGAGCGCCGCTTCCTCAAGATGCTTGAGCGTCAAGCGAAAGCTGGTGAGATTGCAGACAACGCAGCCGCT AAGCCGTTACTCGCTACGCTTCTCCCTAAGTTAACCACACGTATCGTCGAGTGGCTCGAAGAGTACGCATC GAAGAAAGGCCGCAAGCCTAGCGCATACGCACCGCTCCAGTTACTCAAGCCGGAGGCCTCCGCGTTTATCA CCCTGAAAGTTATCCTGCGTCACTAACCAGTACGAACATGACAACCATTCAGGCCGCTGCTGGTATGCTG GGGAAAGCCATTGAGGACGAGGCACGATTTGGGCGCATCCGTGACCTAGAAGCGAAGCACTTCAAGAAGCA CGTTGAGGAACAGCTTAACAAGCGCCACGGGCAAGTCTACAAGAAAGCATTATGCAGGTGGTCGAGGCCGA TATGATTGGTCGAGGTCTGCTTGGTGGCGAGGCGTGGTCTAGCTGGGATAAAGAAACCACGATGCACGTAG GGATTCGCCTGATTGAAATGCTGATTGAATCCACGGGTCTGGTGGAATTACAGCGCCACAACGCAGGTAAC GCAGGCTCTGACCATGAGGCACTGCAACTGGCCCAAGAGTACGTGGACGTATTAGCGAAGCGTGCAGGCGC CTGGCGGGTATCTCTCCGATGTTCCAGCCGTGTGTCGTACCGCCGAAACCTTGGGTAGCAATCACAGGGGG CGGCTATTGGGCTAACGGTCGCAGACCTTTGGCACTCGTTCGCACTCACTCTAAGAAGGGCTTGATGCGCT ACGAAGACGTTTACATGCCAGAAGTCTACAAGGCTGTGAACCTCGCGCAAAACACCGCATGGAAAATCAAC AAGAAAGTTCTTGCTGTTGTCAATGAGATTGTTAACTGGAAGAATTGCCCGGTAGCAGACATTCCATCGCT GGAGCGCCAAGAGTTACCGCCTAAGCCTGACGACATTGACACCAACGAGGCAGCGCTCAAGGAGTGGAAGA AAGCCGCTGCTGGTATCTATCGCTTGGACAAGGCACGAGTGTCTCGCCGTATCAGCTTAGAGTTCATGCTG GAGCAGGCCAACAAGTTCGCAAGTAAGAAAGCAATCTGGTTCCCTTACAACATGGACTGGCGCGGTCGTGT GTACGCTGTGCCGATGTTCAACCCGCAAGGCAACGACATGACGAAAGGTCTGCTGACCCTTGCTAAAGGCA AGCCAATCGGTGAGGAAGGTTTCTACTGGCTGAAAATCCACGGTGCGAACTGTGCGGGTGTTGATAAGGTT GGTTCCATTCCCGGAGCGCATCGCGTTCATTGAGAAGCACGTAGACGACATTCTGGCTTGCGCTAAAGACC CAATCAATAACACTTGGTGGGCTGAGCAGGATTCACCGTTCTGTTTCCTCGCGTTTTGCTTCGAGTATGCA GGCGTTACGCACCACGGTCTGAGCTACAATTGCTCTCTGCCGCTGGCGTTCGACGGGTCTTGCTCTGGTAT CCAGCACTTCTCCGCGATGCTCCGCGATGAGGTAGGCGGTCGTGCGGTTAACCTGCTGCCAAGCGAAACCG TGCAGGACATTTACGGCATCGTTGCACAGAAAGTAAACGAGATTCTCAAACAGGATGCAATCAACGGCACG CCTAACGAGATGATTACCGTGACCGACAAGGACACCGGGGAAATCTCAGAGAAGCTCAAACTTGGAACCTC AACGCTGGCGCAACAGTGGCTGGCATATGGTGTAACCCGTAGCGTAACTAAACGTTCGGTCATGACGCTGG CTTACGGTTCCAAGGAGTTCGGCTTTCGTCAACAGGTATTGGATGACACCATTCAGCCTGCAATTGACAGC GGTAAGGGCTTGATGTTCACCCAACCGAACCAAGCGGCTGGCTATATGGCTAAGCTGATTTGGGATGCGGT AAGCGTGACCGTAGTTGCAGCGGTTGAGGCGATGAACTGGCTCAAATCTGCCGTAAGCTGCTGGCTGCTG AGGTCAAGGACAAGAAGACCAAGGAGATTCTGCGCCACCGTTGCGCGGTTCACTGGACTACGCCGGACGGC TTCCCGGTCTGGCAGGAATACCGCAAGCCACTCCAGAAGCGTCTCGATATGATTTTCTTAGGGCAATTCCG TCTGCAACCGACGATTAATACCCTCAAGGATTCAGGCATTGACGCACACAAGCAGGAGTCTGGCATCGCTC CTAACTTTGTTCACTCACAGGACGGTAGCCACCTCCGCATGACAGTCGTTTATGCTCACGAGAAGTATGGC ATTGAGCCTTTGCGCTCATCCATGACAGCTTTGGGACTATCCCGGCAGACGCTGGTAAGCTCTTTAAGGCT GTGCGTGAAACGATGGTTATCACCTATGAGAACAACGATGTGCTGGCAGACTTCTACTCTCAGTTTGCCGA CCAGCTACACGAGACCCAACTGGACAAGATGCCTCCGCTTCCGAAGAAAGGAAACCTGAACCTGCAAGACA TTCTCAAGTCTGACTTTGCCTTTGCATAA, which nucleotide sequence encodes a polypeptide having T3 RNA polymerase activity.