Filamentous fungi functional replicating extrachromosomal element

• US 4,486,533 A
• Filed: 01/24/1983
• Issued: 12/04/1984
• Est. Priority Date: 09/02/1982
• Status: Expired due to Fees

• Current Assignee: Alan Lambowitz
• Original Assignee: St. Louis University
• Inventors: Lambowitz, Alan M.
• Primary Examiner(s): Wiseman, Thomas
• Assistant Examiner(s): Martinell, James
• Application Number:
  US06/460,684
• Time in Patent Office: 680 Days
• Field of Search: 435/68, 435/70, 435/91, 435/172, 435/254, 435/317, 536/27
• US Class Current: 435/6.13
• 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

Abstract

A functional extrachromosomal element capable of replication in filamentous fungi is provided. The extrachromosomal element employs (1) a mitochondrial replicating element or (2) a lower organism replication sequence recognized by the fungus, in combination with foreign DNA to provide replication, transcription, and translation of foreign regulatory elements and genes. The extrachromosomal element is exemplified by a mitochondrial replicating system from Neurospora.

The cell strain E. coli HB101 containing the plasmid pALS-1-1 has been deposited at the A.T.C.C. on July 13, 1982, for patent purposes and given the designation ATCC 39157.

The cell strain E. coli HB101 containing the plasmid pALS-2 has been deposited at the A.T.C.C. on July 13, 1982, for patent purposes and given the designation ATCC 39158.

Claims

1. A method for producing autonomously replicating vectors for use in Neurospora which comprises:

isolating mitochondria having autonomously replicating plasmid DNAs from Neurospora;

linearizing said plasmid DNAs and inserting said plasmid DNAs into a cloning vector to produce a hybrid cloning plasmid and introducing said hybrid cloning plasmid into an amplifying host to provide a modified host;

growing said modified host for said cloning vector to clone said mitochondrial plasmid DNA and isolating said hybrid cloning plasmids;

introducing at least a portion of said cloning plasmid containing at least a portion of said linearized mitochondrial plasmid into a Neurospora and selecting for a Neurospora in which said portion is stably maintained outside of the mitochondria.

2. A method according to claim 1, wherein said portion of said linearized mitochondrial plasmid is joined to at least one marker recognized by a Neurospora.

3. An extrachromosomal hybrid DNA derived at least in part from mitochondrial plasmid DNA from a Neurospora said extrachromosomal hybrid DNA being capable of stable extrachromosomal maintenance outside of the mitochondria in a Neurospora host.

4. Hybrid DNA comprising a plasmid having a prokaryotic replication sequence and a replication sequence derived from Neurospora mitochondrial plasmid DNA, wherein said hybrid DNA is capable of stable maintenance outside of the mitochondria as an extrachromosomal element in Neurospora.

5. Hybrid DNA according to claim 4 including a marker capable of selection in a Neurospora.

6. Hybrid DNA according to claim 4 including a gene expressing a protein foreign to a Neurospora.

7. A Neurospora containing a hybrid DNA according to claim 4 and capable of stable replication as an extrachromosomal element in said Neurospora.

8. A host according to claim 7, wherein said prokaryotic replication sequence is from E. coli.

9. A host according to either of claims 7, or 10, wherein said plasmid includes a marker capable of selection in said Neurospora and a structural gene foreign to said Neurospora.