Positive selection using glucosamine-6-phosphate

A selection method for selecting genetically transformed plant cells from a population of plant cells, comprising:

    transforming a population of plant cells with an expressible nucleotide sequence having at least 75% identity to SEQ ID NO: 1 and which encodes an enzyme having glucosamine-6-phosphate deaminase activity that converts a component or a metabolic derivative thereof when present in a high concentration in a medium into a nutrient, thereby producing a population of genetically transformed and non-transformed cells;

    introducing said population of plant cells to a medium comprising said component or metabolic derivative thereof, wherein the component or the metabolic derivative thereof is present in said medium in a high concentration and is toxic to said non-transformed plant cells and is a source of both carbohydrate and nitrogen for the selectable, genetically transformed plant cells or if a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatized substrate, then that derivatized substrate provides an allosteric effect on the gene product; and

    culturing said plant cells thereby selecting at least a portion of said transformed plant cells over said non-transformed plant cells; and, wherein said enzyme having glucosamine-6-phosphate deaminase activity converts glucosamine-6-phosphate to fructose-6-phosphate.

A composition comprising a population of transformed and non-transformed plant cells, said transformed plant cells being transformed with an expressible nucleotide sequence having at least 75% identity to SEQ ID NO: 1 and which encodes an enzyme having glucosamine-6-phosphate deaminase activity which converts a component or a metabolic derivative thereof into a nutrient, and a medium comprising said component or metabolic derivative thereof, wherein the component or the metabolic derivative thereof is present in said medium in a high concentration and is toxic to said non-transformed plant cells; and, wherein said enzyme having glucosamine-6-phosphate deaminase activity converts glucosamine-6-phosphate to fructose-6-phosphate.

A construct for genetically transforming a non-transformed plant cell to produce a selectable genetically transformed plant cell, said construct comprising: a plant promoter operably linked to an expressible nucleotide sequence encoding a glucosamine-6-phosphate deaminase gene product and having at least 75% identity to SEQ ID NO: 1; and, wherein said glucosamine-6-phosphate deaminase gene product converts glucosamine-6-phosphate to fructose-6-phosphate.

A transgenic plant produced from a cell transformed with an expressible nucleotide sequence having at least 75% identity to SEQ ID NO: 1 and encoding an enzyme having glucosamine-6-phosphate deaminase activity.

An E. coli strain selected from the group consisting of NCIMB 40852, NCIMB 40853, and NCIMB 40854.

Claim 1

A selection method for selecting from a population of cells one or more selectable genetically transformed cells;
    wherein the population of cells comprises selectable genetically transformed cells and possible non-transformed cells;
    wherein each of the selectable genetically transformed cells comprises a first expressable nucleotide sequence and optionally a second expressable nucleotide sequence;
    wherein a component or a metabolic derivative thereof when present in a low concentration in a medium is a nutrient for both the selectable genetically transformed cells and the non-transformed cells;
    wherein the component or the metabolic derivative thereof when present in a high concentration in a medium is toxic to the non-transformed cells;
    wherein the first nucleotide sequence codes for a gene product capable of converting the component or the metabolic derivative thereof when present in a high concentration in a medium to a nutrient for the selectable genetically transformed cells;
    the method comprising the step of introducing the population of cells to a medium;
    wherein the medium optionally comprises a high concentration of the component or the metabolic derivative thereof; and
    either wherein the component or the metabolic derivative thereof is a source of both carbohydrate and nitrogen for the selectable genetically transformed cells;
    or wherein if a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatised substrate, then that derivatised substrate is capable of providing an allosteric effect on the gene product.

Use of glucosanmine-6-phosphate deaminase as a selection means for selecting a genetically transformed cell over a non-transformed cell.

Claim 22

Use of a gene coding for glucosamine-6-phosphate deaminase for providing a selection means for selecting a genetically transformed cell over a non-transformed cell.

Claim 23

Use of a gene coding for glucosamine-6-phosphate deaminase obtained from NCIMvB 40852 or NCIMB 40853 or NCIMB 40854 for providing a selection means for selecting a genetically transformed cell over a non-transformed cell.