Fusion reporter gene for bacterial luciferase

• US 5,196,524 A
• Filed: 01/06/1989
• Issued: 03/23/1993
• Est. Priority Date: 01/06/1989
• Status: Expired due to Term

• Current Assignee: Novartis Ag
• Original Assignee: Eli Lilly and Company
• Inventors: Roberts, Jean L., Kirchner, Gretchen, Gustafson, Gary D., Ingolia, Thomas D.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Nisbet, T.
• Application Number:
  US07/294,170
• Time in Patent Office: 1,537 Days
• Field of Search: 424/94.4, 435/69.7, 435/8, 435/189, 935/78, 935/47
• US Class Current: 536/23.2
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/80 for fungi
• CPC Class Codes: C12N 15/8209 Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 2830/42 being an intron or intervening sequence for splicing and/or stability of RNA
• CPC Class Codes: C12N 2830/70 from fungi
• CPC Class Codes: C12N 2830/702 yeast
• CPC Class Codes: C12N 2840/20 translation of more than one cistron
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12Y 114/14003 Alkanal monooxygenase FMN (1.14.14.3), i.e. bacterial-luciferase

Abstract

Novel fusion reporter genes, fusion reporter proteins, and an improved reporter system for measuring the relative activity of a promoter sequence. A luxAB fusion gene of the present invention is particularly useful as a reporter gene and is derived from the fusion of a luxA gene and a luxB gene from Vibrio harveyi. The gene products of the luxA and luxB genes are the α- and β-subunits, respectively, of a bacterial luciferase. A fusion protein encoded by a luxAB fusion gene is a single active protein and is particularly useful as a reporter protein having luciferase activity. An advantage of such a reporter system to assay gene expression in many cells which contain FMNH₂, such as bacterial and yeast cells, is that an immediate and quantitative assessment of gene expression may be made from real-time light measurements using intact cells.

Claims

1. A recombinant DNA encoding a fusion protein having bacterial luciferase activity comprising operable linked in the 5'"'"' to 3'"'"' direction, the coding region of the luxA gene, a DNA linker, and the luxB gene, wherein the coding region of the luxA and the luxB genes are isolated from Vibrio harveyi and the DNA linker consists essentially of TCTAGA.

22. A DNA cassette of claim 1 which comprising the following elements 5'"'"' to 3'"'"', operably linked:

a promoter DNA segment, a fusion reporter gene of claim 1.

23. A plasmid vector comprising the fusion reporter gene of claim 22.

24. A host cell transformed with the plasmid vector of claim 23.

2. A fusion reporter gene that is luxAB.

3. A DNA cassette comprising a promoter operably linked to a fusion reporter gene of claim 2.

4. A plasmid vector comprising a fusion reporter gene of claim 2.

11. A host cell transformed or transfected by the plasmid vector of claim 4.

5. A plasmid vector that is pIT20.

12. A host cell transformed or transfected by the plasmid vector of claim 5.

6. A plasmid vector that is pIT24.

13. A host cell transformed or transfected by the plasmid vector of claim 6.

7. A plasmid vector that is pIT21.

14. A host cell transformed or transfected by the plasmid vector of claim 7.

8. A plasmid vector that is pGAR117.

15. A host cell transformed or transfected by the plasmid vector of claim 8.

9. A plasmid vector that is pSV2lux.

16. A host cell transformed or transfected by the plasmid vector of claim 9.

10. A plasmid vector that is pIT22.

17. A host cell transformed or transfected by the plasmid vector of claim 10.

18. A transformant that is E. coli K12 JA221/pIT20 (NRRL B-18301).

19. A transformant that is E. coli K12 JA221/pIT24.

20. A transformant that is Sacchraromyces cerevisiae DBY746/pIT21.

21. A transformant that is Nicotiana plubmaginifolia/pGAR117.