EP0077650A2
Expired / Lapsed
EP
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Cloning vectors
Assignee: Eli Lilly and Co
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Filing: 1982-10-14
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Granted: —
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Expires: 2002-10-14
Abstract
Abstract The present invention discloses selectable pairs of recombinant DNA cloning vectors for use in Streptomyces and related organisms. A pair of recombinant DNA cloning vectors comprises:a) plasmid pEL7, andb) a second plasmid that is functionally dependent upon plasmid pEL7, said second plasmid comprising a restriction fragment of plasmid pEL7 and one or more different DNA segments that convey resistance to at least one antibiotic when transformed into a sensitive host cell, said host cell being susceptible to transformation, cell division, and culture.
Inventors
- 👤 Jeffrey Tobin Fayerman
- 👤 James Albert Mabe
- 👤 Walter Mitsuo Nakatsukasa
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-06 14:01
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/74; C12N15/76; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/822; Y10S435/886
Claims Analysis
Total Claims: 40
Claim 1
Independent Claim
68 words
A pair of recombinant DNA cloning vectors, said pair comprising: a) plasmid pEL7, and b) a second plasmid that is functionally dependent upon plasmid pEL7, said second plasmid comprising a restriction fragment of plasmid pEL7 and one or more different DNA segments that convey resistance to at least one antibiotic when transformed into a sensitive host cell, said host cell being susceptible to transformation, cell division, and culture.
Claim 2
Dependent Claim
68 words
A pair of recombinant DNA cloning vectors, said pair comprising: a) plasmid pEL7, and b) a second plasmid that is functionally dependent upon plasmid pEL7, said second plasmid comprising a restriction fragment of plasmid pEL7 and one or more different DNA segments that convey resistance to at least one antibiotic when transformed into a sensitive host cell, said host cell being susceptible to transformation, cell division, and culture.
Claim 3
Dependent Claim
30 words
The pair of claim 1 wherein the one or more DNA segments in the second plasmid are segments that convey resistance to either or both of antibiotics thiostrepton and neomycin.
Claim 4
Dependent Claim
9 words
The recombinant DNA cloning vector which is plasmid pEL7.
Claim 5
Dependent Claim
9 words
The recombinant DNA cloning vector which is plasmid pEL7.
Claim 6
Dependent Claim
60 words
A recombinant DNA cloning vector which is a plasmid that is functionally dependent upon plasmid p E L7, said plasmid comprising a restriction fragment of plasmid pEL7 and one or more different DNA segments that convey resistance to at least one antibiotic when transformed into a sensitive host cell, said host cell being susceptible to transformation, cell division, and culture.
Claim 7
Dependent Claim
60 words
A recombinant DNA cloning vector which is a plasmid that is functionally dependent upon plasmid p E L7, said plasmid comprising a restriction fragment of plasmid pEL7 and one or more different DNA segments that convey resistance to at least one antibiotic when transformed into a sensitive host cell, said host cell being susceptible to transformation, cell division, and culture.
Claim 8
Dependent Claim
17 words
The vector of claim 4 which is plasmid pEL7.I, pEL7.2, pEL7.3, pEL7.4, pEL7.5, pEL7.6, pEL7.7, or pEL7.8.
Claim 9
Dependent Claim
12 words
A recombinant DNA cloning vector which is plasmid pLRl, pLR2, or pLR4.
Claim 10
Dependent Claim
12 words
A recombinant DNA cloning vector which is plasmid pLRl, pLR2, or pLR4.
Claim 11
Dependent Claim
61 words
A recombinant DNA cloning vector that is functional in E. coli and is functionally dependent upon plasmid pEL7 when transformed into Streptomyces comprising: a) a functional E. coli replicon containing an antibiotic resistance conferring restriction fragment of an E. coli plasmid, and b) a restriction fragment of a member of the pair of recombinant DNA cloning vectors recited in Claim 1.
Claim 12
Dependent Claim
14 words
The vector of claim 7 which is plasmid pLR5, pLR6, pLR7, pLR9 or pLRll.
Claim 13
Dependent Claim
11 words
A transformed restrictionless host cell comprising the vector of claim 3.
Claim 14
Dependent Claim
20 words
The cell of claim 9 which comprises a pair of the recombinant DNA cloning vectors of claim 1 or 2.
Claim 15
Dependent Claim
14 words
The cell of claim 10 which comprises a vector of claim 4 or 5.
Claim 16
Dependent Claim
22 words
The cell of claim 9, 10 or 11 which is a restrictionless cell of Streptomyces, Streptosporangium, Actinoplanes, Nocardia, Micromonospora, Bacillus, or Staphylococcus.
Claim 17
Dependent Claim
16 words
A transformed restrictionless host cell comprising the recombinant DNA cloning vector of claim 7 or 8.
Claim 18
Dependent Claim
9 words
The cell of claim 13 which is E. coli.
Claim 19
Dependent Claim
11 words
The cell of claim 13 which is Stre p to- myces.
Claim 20
Dependent Claim
17 words
The cell of claim 12 or 15 which is Streptomyces of species fradiae, aureofaciens, coelicolor, or ambofaciens.
Claim 21
Dependent Claim
9 words
The cell of claim 16 which is Streptomyces ambofaciens.
Claim 22
Dependent Claim
79 words
A process for isolating plasmid pEL7 which comprises cultivating Streptomyces ambofaciens/pEL7 NRRL 12523 under submerged aerobic conditions in a nutrient medium containing assimilable sources of carbon, nitrogen and minerals at a pH in the range of 5 to 9 and at a temperature in the range of 15° to 45°C., harvesting the cells thus formed, lysing the harvested cells, solvent extracting the lysate supernatant, precipitating the plasmid DNA from the extract, and separating the plasmid pEL7 from the precipitate.
Claim 23
Dependent Claim
79 words
A process for isolating plasmid pEL7 which comprises cultivating Streptomyces ambofaciens/pEL7 NRRL 12523 under submerged aerobic conditions in a nutrient medium containing assimilable sources of carbon, nitrogen and minerals at a pH in the range of 5 to 9 and at a temperature in the range of 15° to 45°C., harvesting the cells thus formed, lysing the harvested cells, solvent extracting the lysate supernatant, precipitating the plasmid DNA from the extract, and separating the plasmid pEL7 from the precipitate.
Claim 24
Dependent Claim
18 words
A process for preparing plasmid pLRl which comprises ligating HindIII treated plasmid pIJ2 to HindIII treated plasmid pBR322.
Claim 25
Dependent Claim
18 words
A process for preparing plasmid pLRl which comprises ligating HindIII treated plasmid pIJ2 to HindIII treated plasmid pBR322.
Claim 26
Dependent Claim
18 words
A process for preparing plasmid pLR2 which comprises ligating HindIII treated plasmid pIJ6 to HindIII treated plasmid pBR322.
Claim 27
Dependent Claim
18 words
A process for preparing plasmid pLR2 which comprises ligating HindIII treated plasmid pIJ6 to HindIII treated plasmid pBR322.
Claim 28
Dependent Claim
18 words
A process for preparing plasmid pLR4 which comprises ligating BamHI treated plasmid pLR2 to BamHI treated plasmid pBR322.
Claim 29
Dependent Claim
18 words
A process for preparing plasmid pLR4 which comprises ligating BamHI treated plasmid pLR2 to BamHI treated plasmid pBR322.
Claim 30
Dependent Claim
57 words
A process for preparing a plasmid that is functionally dependent upon plasmid pEL7 which comprises ligating one or more different DNA segments that convey resistance to at least one antibiotic when transformed into a sensitive host cell onto a restriction fragment of plasmid pEL7, and self ligating the resulting recombinant DNA to produce said functionally dependent plasmid.
Claim 31
Dependent Claim
57 words
A process for preparing a plasmid that is functionally dependent upon plasmid pEL7 which comprises ligating one or more different DNA segments that convey resistance to at least one antibiotic when transformed into a sensitive host cell onto a restriction fragment of plasmid pEL7, and self ligating the resulting recombinant DNA to produce said functionally dependent plasmid.
Claim 32
Dependent Claim
14 words
The process of claim 22 wherein the DNA segment conveys resistance to antibiotic thiostrepton.
Claim 33
Dependent Claim
24 words
The process of claim 23 for preparing plasmids pEL7.1 and pEL7.2 which comprises ligating BamHI treated plasmid pLR2 to BamHI treated plasmid p EL7.
Claim 34
Dependent Claim
15 words
The process of claim 22 wherein the DNA segment conveys resistance to the antibiotic neomycin.
Claim 35
Dependent Claim
23 words
The process of claim 25 for preparing plasmids pEL7.3 and pEL7.4 which comprises ligating Pstl treated plasmid pLRl to Pstl treated plasmid pEL7.
Claim 36
Dependent Claim
17 words
The process of claim 22 where the DNA segment conveys resistance to the antibiotics thiostrepton and neomycin.
Claim 37
Dependent Claim
30 words
The process of claim 27 for preparing plasmids pEL7.5 and pEL7.6 which comprises ligating a mixture of Pstl treated plasmid pEL7, Pstl treated plasmid pEL7.l, and Pstl treated plasmid pLRl.
Claim 38
Dependent Claim
30 words
The process of claim 27 for preparing plasmids pEL7.7 and pEL7.8 which comprises ligating a mixture of Pstl treated plasmid pEL7, Pstl treated plasmid pEL7.2, and Pstl treated plasmid pLRl.
Claim 39
Dependent Claim
64 words
A process for preparing a plasmid that is functional in E. coli and is functionally dependent on plasmid pEL7 when transformed into Streptomyces which comprises ligating a) a functional E. coli replicon containing an antibiotic resistance conferring restriction fragment of an E. coli plasmid, and b) a restriction fragment of a member of the pair of recombinant DNA cloning vectors recited in Claim 1.
Claim 40
Dependent Claim
25 words
The process of claim 30 for preparing plasmids p L R5 and pLR6 which comprises ligating BamHI treated plasmid pEL7 to BamHI treated plasmid pBR322.
References Cited (Backward Citations)
No citation records indexed.
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