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Title: Methylotroph cloning vehicle

Abstract

A cloning vehicle comprising: a replication determinant effective for replicating the vehicle in a non-C[sub 1]-utilizing host and in a C[sub 1]-utilizing host; DNA effective to allow the vehicle to be mobilized from the non-C[sub 1]-utilizing host to the C[sub 1]-utilizing host; DNA providing resistance to two antibiotics to which the wild-type C[sub 1]-utilizing host is susceptible, each of the antibiotic resistance markers having a recognition site for a restriction endonuclease; a cos site; and a means for preventing replication in the C[sub 1]-utilizing host. The vehicle is used for complementation mapping as follows. DNA comprising a gene from the C[sub 1]-utilizing organism is inserted at the restriction nuclease recognition site, inactivating the antibiotic resistance marker at that site. The vehicle can then be used to form a cosmid structure to infect the non-C[sub 1]-utilizing (e.g., E. coli) host, and then conjugated with a selected C[sub 1]-utilizing mutant. Resistance to the other antibiotic by the mutant is a marker of the conjugation. Other phenotypical changes in the mutant, e.g., loss of an auxotrophic trait, is attributed to the C[sub 1] gene. The vector is also used to inactivate genes whose protein products catalyze side reactions that divert compounds from amore » biosynthetic pathway to a desired product, thereby producing an organism that makes the desired product in higher yields. 3 figs.« less

Inventors:
;
Issue Date:
OSTI Identifier:
7162964
Patent Number(s):
4824786 A
Application Number:
PPN: US 7-107244
Assignee:
Univ. of Minnesota, Minneapolis, MN (United States) PTO; EDB-94-118897
DOE Contract Number:  
AC02-82ER12029
Resource Type:
Patent
Resource Relation:
Patent File Date: 13 Nov 1987
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ANTIBIOTICS; BIOLOGICAL MARKERS; DNA-CLONING; DNA REPLICATION; ENDONUCLEASES; ANTI-INFECTIVE AGENTS; CLONING; DNA HYBRIDIZATION; DNA-ASE; DRUGS; ENZYMES; ESTERASES; HYBRIDIZATION; HYDROLASES; NUCLEIC ACID REPLICATION; ORGANIC COMPOUNDS; PHOSPHODIESTERASES; PROTEINS; 550200* - Biochemistry

Citation Formats

Hanson, R.S., and Allen, L.N. Methylotroph cloning vehicle. United States: N. p., 1989. Web.
Hanson, R.S., & Allen, L.N. Methylotroph cloning vehicle. United States.
Hanson, R.S., and Allen, L.N. Tue . "Methylotroph cloning vehicle". United States.
@article{osti_7162964,
title = {Methylotroph cloning vehicle},
author = {Hanson, R.S. and Allen, L.N.},
abstractNote = {A cloning vehicle comprising: a replication determinant effective for replicating the vehicle in a non-C[sub 1]-utilizing host and in a C[sub 1]-utilizing host; DNA effective to allow the vehicle to be mobilized from the non-C[sub 1]-utilizing host to the C[sub 1]-utilizing host; DNA providing resistance to two antibiotics to which the wild-type C[sub 1]-utilizing host is susceptible, each of the antibiotic resistance markers having a recognition site for a restriction endonuclease; a cos site; and a means for preventing replication in the C[sub 1]-utilizing host. The vehicle is used for complementation mapping as follows. DNA comprising a gene from the C[sub 1]-utilizing organism is inserted at the restriction nuclease recognition site, inactivating the antibiotic resistance marker at that site. The vehicle can then be used to form a cosmid structure to infect the non-C[sub 1]-utilizing (e.g., E. coli) host, and then conjugated with a selected C[sub 1]-utilizing mutant. Resistance to the other antibiotic by the mutant is a marker of the conjugation. Other phenotypical changes in the mutant, e.g., loss of an auxotrophic trait, is attributed to the C[sub 1] gene. The vector is also used to inactivate genes whose protein products catalyze side reactions that divert compounds from a biosynthetic pathway to a desired product, thereby producing an organism that makes the desired product in higher yields. 3 figs.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {1989},
month = {4}
}