The University of Georgia Research Foundation patent family

University of Georgia Research Foundation, Inc. has a granted patent in the United States on methods and materials for selecting transgenic cells based on arabitol or ribitol as positive selectable markers.  Patent application was also filed in Australia.

The invention relates to isolated polynucleotide molecules coding for a protein possessing arabitol/ribitol dehydrogenase enzymatic activity and a protein possessing arabitol/ribitol kinase enzymatic activity.  It also relates to a positive selection system that involves conferring to transformed cells (includes protoplasts, as well as cells of plants, animals and microorganisms) the ability to metabolize arabitol, ribitol and/or mannitol and selecting the transformed cells.

Technology overview

D-arabitol and ribitol are two of the four possible pentitols (five-carbon sugar alcohol, C5H12O5) formed by the reduction of ribose.  In 2001, LaFayette and Parrott reported that E. coli strains B and K-12 cannot metabolize these pentitols.  However, E. coli strain C originally isolated in the Lister Institute, London, in 1920 (NCTC 1983) can metabolize both D-arabitol and ribitol and thus grow on these pentitols when they are the sole carbon source.  This ability is due to the presence of two genes coding for arabitol deydrogenase and ribitol dehydrogenase that convert arabitol and ribitol into xylulose and ribulose, respectively.

The structure of a pentitol

Plants are not able to metabolize arabitol and ribitol. The positive selection strategy in this aspect is to engineer plant cells by introducing an arabitol or ribitol dehydrogenase gene so that the transformed cells can utilise arabitol or ribitol as carbon source. For example, when a gene coding for a bacterial arabitol dehydrogenase is transferred into and expressed in a plant cell, the cell will be able to grow in a medium containing D-arabitol by converting arabitol into the plant-metabolizable xylulose,  whereas an untransformed plant cell will not proliferate.

Specific patent information

Patent/Application Number Title, Independent Claims and Summary Assignee
US 7005561 B2

  • Earliest priority – 8 Mar 2000
  • Filed – 3 Mar 2001
  • Granted – 28 Feb 2006
  • Expected expiry – 8 Aug 2021 (see disclaimer in the patent)
Title – Arabitol or ribitol as positive selectable markers

Claim 1
An isolated polynucleotide molecule comprising at least one gene of interest and at least one selectable marker gene, wherein said at least one selectable marker gene comprises a nucleotide sequence which selectively hybridizes under high stringency conditions to the complement of a nucleotide sequence shown in SEQ ID NO: 2, or a plant optimized version thereof, wherein said nucleotide sequence encodes for a protein possessing ribitol dehydrogenase enzymatic activity and a protein possessing ribitol kinase enzymatic activity.
Claim 6
A method of selecting transformed cells from a population of cells comprising;a) introducing into the genome of a cell a gene of interest and a selectable marker gene;
b) obtaining transformed cells;
c) supplying to the population of cells a marker compound wherein said transformed cells have a selective advantage over non-transformed cells due to expression or transcription of the the selectable marker gene in the presence of the marker compound; and
d) selecting said transformed cells from the population of cells; wherein said selectable marker gene comprises a nucleotide sequence which selectively hybridizes under high stringency conditions to the complement of a nucleotide sequence shown in SEQ ID NO: 2, or a plant optimized version thereof, wherein said nucleotide sequence encodes a protein that possesses ribitol dehydrogenase enzymatic activity and a protein that possesses ribitol kinase enzymatic activity; and said marker compound comprises arabitol, ribitol, or mannitol.
Claim 12
An isolated polynucleotide molecule comprising a nucleotide sequence which selectively hybridizes under high stringency conditions to the complement of a plant optimized version of the nucleotide sequences shown in SEQ ID NO: 2, and wherein said nucleotide sequence encodes for a protein possessing ribitol dehydrogenase activity and a protein possessing ribitol kinase activity.
Claim 13
An isolated polynucleotide molecule comprising at least one gene of interest, and at least one selectable marker gene, wherein said at least one selectable marker gene comprises a nucleotide sequence encoding SEQ ID NOS.: 3 and 4.
Claim 14
An isolated polynucleotide molecule comprising at least one gene of interest, and at least one selectable marker gene, wherein said at least one selectable marker gene comprises a nucleotide sequence which selectively hybridizes under high stringency conditions to the complement of a nucleotide sequence shown in SEQ ID NO: 1, or a plant optimized version thereof, wherein said at least one selectable marker gene encodes for a protein possessing arabitol dehydrogenase enzymatic activity.
Claim 15
A method of selecting transformed cells from a population of cells comprisinga) introducing into the genome of a cell a gene of interest and a selectable marker gene;
b) obtaining transformed cells;
c) supplying to the population of cells a marker compound wherein said transformed cells have a selective advantage over non-transformed cells due to expression or transcription of the selectable marker gene in the presence of the marker compound; and
d) selecting said transformed cells from the population of cells; wherein said selectable marker gene comprises a nucleotide sequence which selectively hybridizes under high stringency conditions to the complement of a nucleotide sequence shown in SEQ ID NO: 1, or a plant optimized version thereof, and encodes a protein having arabitol dehydrogenase enzymatic activity; and wherein said marker compound is arabitol.
Claim 16
A method of selecting transformed cells from a population of cells comprisinga) introducing into the genome of a cell a gene of interest and a selectable marker gene;
b) obtaining transformed cells;
c) supplying to the population of cells a marker compound wherein said transformed cells have a selective advantage over non-transformed cells due to expression or transcription of the selectable marker gene in the presence of the marker compound; and
d) selecting said transformed cells from the population of cells; wherein said selectable marker gene comprises a nucleotide sequence encoding SEQ ID NO.: 3, and a nucleotide sequence encoding SEQ ID NO.: 4; and wherein said marker compound is ribitol.

University of Georgia Research Foundation

Remarks

The related patent application in Australia (AU 200140117) has lapsed. A PCT application was also filed (WO 2001/66779).

Search strategy

Search details
Date of search 31/05/2006
Database searched Patent Lens
Type of search Simple, stemming on
Collections searched AU-B, US-A, US-B, EP-B, WO
Search terms “Positive AND selection AND transformation AND cell” in abstract
Results 117 hits
Comments This search results in patents from several patent families that related to the positive selection topic:

1. the Syngenta family represented by WO 1993/05163

2. the “University of Georgia Research Foundation, Inc.” family  represented by US 7005561

titled “Arabitol or ribitol as positive selectable markers”.