US4237224A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Process for producing biologically functional molecular chimeras
Assignee: Leland Stanford Junior University
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Filing: 1979-01-04
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Granted: 1980-12-02
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Expires: 1999-01-04
Abstract
Abstract Method and compositions are provided for replication and expression of exogenous genes in microorganisms. Plasmids or virus DNA are cleaved to provide linear DNA having ligatable termini to which is inserted a gene having complementary termini, to provide a biologically functional replicon with a desired phenotypical property. The replicon is inserted into a microorganism cell by transformation. Isolation of the transformants provides cells for replication and expression of the DNA molecules present in the modified plasmid. The method provides a convenient and efficient way to introduce genetic capability into microorganisms for the production of nucleic acids and proteins, such as medically or commercially useful enzymes, which may have direct usefulness, or may find expression in the production of drugs, such as hormones, antibiotics, or the like, fixation of nitrogen, fermentation, utilization of specific feedstocks, or the like.
Inventors
- 👤 Stanley N. Cohen
- 👤 Herbert W. Boyer
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 18:07
CPC Classifications
C; C12; C12N; C12N15/00; Y; Y10; Y10S; Y10S435/00; Y10S435/82; Y10S435/8215; Y10S435/822; Y10S435/848; Y10S435/849; Y10S530/00; Y10S530/808
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
174 words
A method for replicating a biologically functional DNA, which comprises: transforming under transforming conditions compatible unicellular organisms with biologically functional DNA to form transformants; said biologically functional DNA prepared in vitro by the method of: (a) cleaving a viral or circular plasmid DNA compatible with said unicellular organism to provide a first linear segment having an intact replicon and termini of a predetermined character; (b) combining said first linear segment with a second linear DNA segment, having at least one intact gene and foreign to said unicellular organism and having termini ligatable to said termini of said first linear segment, wherein at least one of said first and second linear DNA segments has a gene for a phenotypical trait, under joining conditions where the termini of said first and second segments join to provide a functional DNA capable of replication and transcription in said unicellular organism; growing said unicellular organisms under appropriate nutrient conditions; and isolating said transformants from parent unicellular organisms by means of said phenotypical trait imparted by said biologically functional DNA.
Claim 2
Independent Claim
174 words
A method for replicating a biologically functional DNA, which comprises: transforming under transforming conditions compatible unicellular organisms with biologically functional DNA to form transformants; said biologically functional DNA prepared in vitro by the method of: (a) cleaving a viral or circular plasmid DNA compatible with said unicellular organism to provide a first linear segment having an intact replicon and termini of a predetermined character; (b) combining said first linear segment with a second linear DNA segment, having at least one intact gene and foreign to said unicellular organism and having termini ligatable to said termini of said first linear segment, wherein at least one of said first and second linear DNA segments has a gene for a phenotypical trait, under joining conditions where the termini of said first and second segments join to provide a functional DNA capable of replication and transcription in said unicellular organism; growing said unicellular organisms under appropriate nutrient conditions; and isolating said transformants from parent unicellular organisms by means of said phenotypical trait imparted by said biologically functional DNA.
Claim 3
Dependent Claim
12 words
A method according to claim 1, wherein said unicellular organisms are bacteria.
Claim 4
Dependent Claim
18 words
A method according to claim 2, wherein said transformation is carried out in the presence of calcium chloride.
Claim 5
Dependent Claim
50 words
A method according to claim 3, wherein said phenotypical trait is resistance to growth inhibiting substance, and said growth is carried out in the presence of a sufficient amount of said growth inhibiting substance to inhibit the growth of parent unicellular organisms, but insufficient to inhibit the growth of transformants.
Claim 6
Dependent Claim
13 words
A method according to claim 1, wherein said unicellular organism is E. coli.
Claim 7
Dependent Claim
14 words
A method according to claim 1, wherein said predetermined termini are staggered and cohesive.
Claim 8
Dependent Claim
13 words
A method according to claim 6, wherein said joining conditions includes enzymatic ligation.
Claim 9
Dependent Claim
33 words
A method according to claim 6, wherein said cohesive ends are formed by staggered cleavage of said viral or circular plasmid DNA and a source of said second segment with a restriction enzyme.
Claim 10
Dependent Claim
16 words
A method acording to claim 6 wherein said cohesive termini are formed by addition of nucleotides.
Claim 11
Dependent Claim
20 words
A method according to claim 1, wherein said predetermined termini are blunt end and said joining conditions include enzymatic ligation.
Claim 12
Dependent Claim
83 words
A method for replicating a biologically functional DNA comprising a replicon compatible with a host unicellular organism joined to a gene derived from a source which does not exchange genetic information with said host organism, said method comprising: isolating said biologically functional DNA from transformants prepared in accordance with claim 1; transforming unicellular microorganisms with which said replicon is compatible with said isolated DNA to provide second transformants; and growing said second transformants under appropriate nutrient conditions to replicate said biologically functional DNA.
Claim 13
Independent Claim
69 words
A method for producing a protein foreign to a unicellular organism by means of expression of a gene by said unicellular organism, wherein said gene is derived from a source which does not exchange genetic information with said organism, said method comprising: growing transformants prepared in accordance with any of claims 1 and 11 under appropriate nutrient conditions, whereby said organism expresses said foreign gene and produces said protein.
Claim 14
Dependent Claim
12 words
A method according to claim 12, wherein said protein is an enzyme.
Claim 15
Dependent Claim
33 words
A method according to claim 11, wherein said method is repeated substituting said biologically functional DNA from transformants prepared in accordance with claim 1 with second or subsequent transformants to produce additional transformants.
References Cited (Backward Citations)
US3813316A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1983-12-02 | 1984-06-02 | 1984-12-02 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1987-12-02 | 1988-06-02 | 1988-12-02 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1991-12-02 | 1992-06-02 | 1992-12-02 | $7700.00 | Window Closed / Lapsed |