US4808537A
Expired / Lapsed
US
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Methanol inducible genes obtained from pichia and methods of use
Assignee: Research Corp Technologies Inc
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Filing: 1984-10-30
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Granted: 1989-02-28
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Expires: 2004-10-30
Abstract
Abstract A method for isolating and cloning methanol inducible genes from Pichia is taught. The regulatory regions are useful for the methanol regulated expression of heterologous genes.
Inventors
- 👤 David W. Stroman
- 👤 Paul F. Brust
- 👤 Steven B. Ellis
- 👤 Thomas R. Gingeras
- 👤 Michael M. Harpold
- 👤 Juerg F. Tschopp
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 22:32
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/80; C12N15/81; C12N15/815; C12N15/11; C12N15/52; C12N15/67; C12N15/68; C12N15/70; Y; Y10; Y10S; Y10S930/00; Y10S930/01; Y10S930/31
Claims Analysis
Total Claims: 34
Claim 1
Independent Claim
30 words
A method comprising: (a) culturing Escherichia coli NRRL B-15867 (LE392-pPG 6.0) in a nutrient medium; (b) disrupting the cultured cells; and (c) recovering plasmid pPG 6.0 from the disrupted cells.
Claim 2
Independent Claim
30 words
A method comprising: (a) culturing Escherichia coli NRRL B-15867 (LE392-pPG 6.0) in a nutrient medium; (b) disrupting the cultured cells; and (c) recovering plasmid pPG 6.0 from the disrupted cells.
Claim 3
Dependent Claim
38 words
A method in accordance with claim 1 further comprising: (d) digesting plasmid pPG 6.0 with the restriction enzymes HindIII and XhoI; and (e) recovering the DNA fragment characterized by the restriction map in FIG. 4a of the drawings.
Claim 4
Dependent Claim
38 words
A method in accordance with claim 1 further comprising: (d) digesting plasmid pPG 6.0 with the restriction enzymes HindIII and EcoRI; and (e) recovering the DNA fragment characterized by the restriction map in FIG. 4b of the drawings.
Claim 5
Dependent Claim
35 words
A method in accordance with claim 1 further comprising: (d) digesting plasmid pPG 6.0 with restriction enzyme EcoRI-HindIII; and (e) recovering the DNA fragment characterized by the restriction map in FIG. 7 of the drawings.
Claim 6
Dependent Claim
35 words
A method in accordance with claim 1 further comprising: (d) digesting plasmid pPG 6.0 with restriction enzyme HindIII; and (e) recovering the DNA fragment characterized by the restriction map in FIG. 1a of the drawings.
Claim 7
Independent Claim
29 words
A method comprising: (a) culturing Escherichia coli NRRL B-15868 (LE392-pPG 4.0) in a nutrient medium; (b) disrupting the cultured cells; (c) recovering plasmid pPG 4.0 from the disrupted cells.
Claim 8
Independent Claim
29 words
A method comprising: (a) culturing Escherichia coli NRRL B-15868 (LE392-pPG 4.0) in a nutrient medium; (b) disrupting the cultured cells; (c) recovering plasmid pPG 4.0 from the disrupted cells.
Claim 9
Dependent Claim
38 words
A method in accordance with claim 6 further comprising: (d) digesting plasmid pPG 4.0 with the restriction enzymes EcoRI and EcoRV; and (e) recovering the DNA fragment characterized by the restriction map in FIG. 5 of the drawings.
Claim 10
Dependent Claim
38 words
A method in accordance with claim 6 further comprising: (d) digesting plasmid pPG 4.0 with the restriction enzymes StuI and PvuII; and (e) recovering the DNA fragment characterized by the restriction map in FIG. 8 of the drawings.
Claim 11
Dependent Claim
38 words
A method in accordance with claim 6 further comprising: (d) digesting plasmid pPG 4.0 with the restriction enzymes EcoRI and PvuII; and (e) recovering the DNA fragment characterized by the restriction map in FIG. 2a of the drawings.
Claim 12
Independent Claim
29 words
A method comprising: (a) culturing Escherichia coli NRRL B-15969 (LE392-pPG 4.8) in a nutrient medium; (b) disrupting the cultured cells; (c) recovering plasmid pPG 4.8 from the disrupted cells.
Claim 13
Independent Claim
29 words
A method comprising: (a) culturing Escherichia coli NRRL B-15969 (LE392-pPG 4.8) in a nutrient medium; (b) disrupting the cultured cells; (c) recovering plasmid pPG 4.8 from the disrupted cells.
Claim 14
Dependent Claim
38 words
A method in accordance with claim 10 further comprising: (d) digesting plasmid pPG 4.8 with the restriction enzymes BamHI and SalI; and (e) recovering the DNA fragment characterized by the restriction map in FIG. 6 of the drawings.
Claim 15
Dependent Claim
38 words
A method in accordance with claim 10 further comprising: (d) digesting plasmid pPG 4.8 with the restriction enzymes SalI and EcoRI; and (e) recovering the DNA fragment characterized by the restriction map in FIG. 9 of the drawings.
Claim 16
Dependent Claim
38 words
A method in accordance with claim 10 further comprising: (d) digesting plasmid pPG 4.8 with the restriction enzymes BamHI and EcoRI; and (e) recovering the DNA fragment characterized by the restriction map in FIG. 3a of the drawings.
Claim 17
Independent Claim
30 words
A method comprising: (a) culturing Escherichia coli NRRL B-15870 (LE392-pPC 15.0) in a nutrient medium; (b) disrupting the cultured cells; and (c) recovering plasmid pPC 15.0 from the disrupted cells.
Claim 18
Independent Claim
30 words
A method comprising: (a) culturing Escherichia coli NRRL B-15870 (LE392-pPC 15.0) in a nutrient medium; (b) disrupting the cultured cells; and (c) recovering plasmid pPC 15.0 from the disrupted cells.
Claim 19
Dependent Claim
35 words
A method on accordance with claim 14 further comprising: (d) digesting plasmid pPC 15.0 with restriction enzyme PstI; and (e) recovering the DNA fragment characterized by the restriction map in FIG. 12 of the drawings.
Claim 20
Independent Claim
30 words
A method comprising: (a) culturing Escherichia coli NRRL B-15872 (LE392-pPC 6.7) in a nutrient medium; (b) disrupting the cultured cells; and (c) recovering plasmid pPC 6.7 from the disrupted cells.
Claim 21
Independent Claim
30 words
A method comprising: (a) culturing Escherichia coli NRRL B-15872 (LE392-pPC 6.7) in a nutrient medium; (b) disrupting the cultured cells; and (c) recovering plasmid pPC 6.7 from the disrupted cells.
Claim 22
Dependent Claim
35 words
A method in accordance with claim 16 further comprising: (d) digesting plasmid pPC 6.7 with restriction enzyme PstI; and (e) recovering the DNA fragment characterized by the restriction map in FIG. 14 of the drawings.
Claim 23
Independent Claim
30 words
A method comprising: (a) culturing Escherichia coli NRRL B-15871 (LE392-pPC 8.3) in a nutrient medium; (b) disrupting the cultured cells; and (c) recovering plasmid pPC 8.3 from the disrupted cells.
Claim 24
Independent Claim
30 words
A method comprising: (a) culturing Escherichia coli NRRL B-15871 (LE392-pPC 8.3) in a nutrient medium; (b) disrupting the cultured cells; and (c) recovering plasmid pPC 8.3 from the disrupted cells.
Claim 25
Dependent Claim
29 words
A method in accordance with claim 18 further comprising: (d) digesting plasmid pPC 8.3 with restriction enzyme PstI; and (e) recovering a DNA fragment of about 2100 base pairs.
Claim 26
Independent Claim
30 words
A method comprising: (a) culturing Escherichia coli NRRL B-15873 (MM294-pPC 8.0) in a nutrient medium; (b) disrupting the cultured cells; and (c) recovering plasmid pPC 8.0 from the disrupted cells.
Claim 27
Independent Claim
30 words
A method comprising: (a) culturing Escherichia coli NRRL B-15873 (MM294-pPC 8.0) in a nutrient medium; (b) disrupting the cultured cells; and (c) recovering plasmid pPC 8.0 from the disrupted cells.
Claim 28
Dependent Claim
29 words
A method in accordance with claim 20 further comprising: (d) digesting plasmid pPC 8.0 with restriction enzyme PstI; and (e) recovering a DNA fragment of about 750 base pairs.
Claim 29
Independent Claim
115 words
A method for isolating the p76 gene from a methanol assimilating yeast, said method comprising: (a) digesting total DNA with at least one restriction enzyme to give digested DNA; (b) size fractionating said digested DNA by agarose electrophoresis; (c) denaturating and binding the size fractionated DNA from step (b) to nitrocellulose filter to give bound DNA; (d) hybridizing said bound DNA with labeled p76 gene from Pichia pastoris; (e) identifying the unique size fragment of bound DNA which cross-hybridizes with the labeled p76 gene from P. pastoris; (f) size fractionating additional DNA which has been digested in accordance with step (a) to recover for cloning the unique size fragment of DNA identified in step (e).
Claim 30
Independent Claim
115 words
A method for isolating the p76 gene from a methanol assimilating yeast, said method comprising: (a) digesting total DNA with at least one restriction enzyme to give digested DNA; (b) size fractionating said digested DNA by agarose electrophoresis; (c) denaturating and binding the size fractionated DNA from step (b) to nitrocellulose filter to give bound DNA; (d) hybridizing said bound DNA with labeled p76 gene from Pichia pastoris; (e) identifying the unique size fragment of bound DNA which cross-hybridizes with the labeled p76 gene from P. pastoris; (f) size fractionating additional DNA which has been digested in accordance with step (a) to recover for cloning the unique size fragment of DNA identified in step (e).
Claim 31
Independent Claim
115 words
A method for isolating the p72 gene from a methanol assimilating yeast, said method comprising: (a) digesting total DNA with at least one restriction enzyme to give digested DNA; (b) size fractionating said digested DNA by agarose electrophoresis; (c) denaturing and binding the size fractionated DNA from step (b) to nitrocellulose filter to give bound DNA; (d) hybridizing said bound DNA with labeled p72 gene from Pichia pastoris; (e) identifying the unique size fragment of bound DNA which cross-hybridizes with the labeled p72 gene from P. pastoris; (f) size fractionating additional DNA which has been digested in accordance with step (a) to recover for cloning the unique size fragment of DNA identified in step (e).
Claim 32
Independent Claim
115 words
A method for isolating the p72 gene from a methanol assimilating yeast, said method comprising: (a) digesting total DNA with at least one restriction enzyme to give digested DNA; (b) size fractionating said digested DNA by agarose electrophoresis; (c) denaturing and binding the size fractionated DNA from step (b) to nitrocellulose filter to give bound DNA; (d) hybridizing said bound DNA with labeled p72 gene from Pichia pastoris; (e) identifying the unique size fragment of bound DNA which cross-hybridizes with the labeled p72 gene from P. pastoris; (f) size fractionating additional DNA which has been digested in accordance with step (a) to recover for cloning the unique size fragment of DNA identified in step (e).
Claim 33
Independent Claim
115 words
A method for isolating the p40 gene from a methanol assimilating yeast, said method comprising: (a) digesting total DNA with at least one restriction enzyme to give digested DNA; (b) size fractionating said digested DNA by agarose electrophoresis; (c) denaturing and binding the size fractionated DNA from step (b) to nitrocellulose filter to give bound DNA; (d) hybridizing said bound DNA with labeled p40 gene from Pichia pastoris; (e) identifying the unique size fragment of bound DNA which cross-hybridizes with the labeled p40 gene from P. pastoris; (f) size fractionating additional DNA which has been digested in accordance with step (a) to recover for cloning the unique size fragment of DNA identified in step (e).
Claim 34
Independent Claim
115 words
A method for isolating the p40 gene from a methanol assimilating yeast, said method comprising: (a) digesting total DNA with at least one restriction enzyme to give digested DNA; (b) size fractionating said digested DNA by agarose electrophoresis; (c) denaturing and binding the size fractionated DNA from step (b) to nitrocellulose filter to give bound DNA; (d) hybridizing said bound DNA with labeled p40 gene from Pichia pastoris; (e) identifying the unique size fragment of bound DNA which cross-hybridizes with the labeled p40 gene from P. pastoris; (f) size fractionating additional DNA which has been digested in accordance with step (a) to recover for cloning the unique size fragment of DNA identified in step (e).
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1992-02-28 | 1992-08-29 | 1993-02-28 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-02-28 | 1996-08-29 | 1997-02-28 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-02-28 | 2000-08-29 | 2001-02-28 | $7700.00 | Window Closed / Lapsed |