Biologically functional molecular chimeras

• US 4,468,464 A
• Filed: 11/09/1978
• Issued: 08/28/1984
• Est. Priority Date: 11/04/1974
• Status: Expired due to Term

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Cohen, Stanley N., Boyer, Herbert W.
• Primary Examiner(s): Tanenholtz, Alvin E.
• Application Number:
  US05/959,288
• Time in Patent Office: 2,119 Days
• Field of Search: 435/172, 435/68, 435/317, 435/91, 435/253
• US Class Current: 435/320.1
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: Y10S 435/82 Subcellular parts of microorganisms

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, which are bound to 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 are 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.

The invention was supported by generous grants of NIH, NSF and the American Cancer Society.

Claims

1. As a composition of matter, a biologically functional recombinant plasmid capable of selection and replication in a prokaryotic cell comprising:

a first DNA segment containing an intact replicon recognized by said cell derived by cleaving a virus or plasmid compatible with said cell at other than the replicon site, which segment is covalently joined in vitro at its ends to the complementary ends of a second DNA segment foreign to said cell and having at least one intact gene, said second DNA segment derived from a source which does not exchange genetic information with said cell.

2. As a composition of matter, a biologically functional recombinant plasmid having been cloned at least once and capable of selection and replication, said plasmid having first and second linear segments, wherein said first segment has an intact replicon recognized by a prokaryotic host and said second segment is a gene derived from a source which is foreign to and does not exchange genetic information with a prokaryotic host for said replicon.

3. A composition of matter according to claim 2, wherein said second segment is derived from eukaryotic source.

4. A composition of matter according to claim 2, wherein said first segment has a basis for selection.

5. A composition of matter according to claim 2, wherein said second segment has a basis for selection.

9. A composition of matter according to claim 2, wherein the basis for selection is resistance to a growth inhibiting substance.

11. A composition of matter according to claim 9, wherein said growth inhibiting substance is an antibiotic.

6. As a composition of matter, a biologically functional recombinant plasmid having been cloned at least once and capable of selection and replication, said plasmid having first and second linear segments, wherein said first segment has an intact replicon derived from a prokaryotic plasmid and said second segment is a gene derived from a source which is foreign to and does not exchange genetic information with a prokaryotic host for said prokaryotic plasmid.

7. A composition of matter according to claim 6, wherein said second segment is derived from a eukaryotic source.

8. A composition of matter according to claim 6, wherein the basis for selection is resistance to a growth inhibiting substance.

10. A composition of matter according to claim 8, wherein said growth inhibiting substance is an antibiotic.