GB2328209A

GB2328209A - Vectors which express a coloured protein for detection of integration into a cell

Info

Publication number: GB2328209A
Application number: GB9717341A
Authority: GB
Inventors: Stefan Andreas Oehler; David Christopher Gubb
Original assignee: Individual
Current assignee: Individual
Priority date: 1997-08-16
Filing date: 1997-08-16
Publication date: 1999-02-17
Also published as: GB9717341D0

Abstract

A process for the detection of integration of transformation vector DNA into a target cell comprises the insertion, into said DNA, of a DNA sequence which either encodes a coloured protein or encodes a protein which develops colour on exposure to an appropriate co-factor. The said sequence may be used in the production of a reporter construct by operatively linking thereto a regulatory element from a gene, especially for the transformation of plants and animals. The preferred protein is either a cytochrome (in which the co-factor is an iron ion), especially cytochrome ba3 of Thermus thermophilus (in which the cofactor may be a copper ion), or a carotenoid binding protein (in which the co-factor is the appropriate carotenoid).

Description

Title DNA vectors capable of expressing a coloured protein In order to genetically engineer strains of micro organisms, animals and plants with useful commercial applications it is necessary to develop transformation vectors based on mobile genetic elements. Direct identification of those treated cells in which the transformation vectors have become incorporated provides a difficult problem and a number of solutions have been proposed. Some early approaches to this problem included incorporating into the transformation vector DNA capable of expressing a novel enzyme, cells carrying the transformation vector integrated into their DNA were identified by supplying enzyme substrates giving a pigmented product. This method requires addition of purified enzyme substrate molecules which are expensive.

Alternative approaches are to confer antibiotic or insecticide resistance to a cell using a vector containing a gene coding for a antibiotic protein or for metabolising insecticidal molecules. Transformed cells can then be selected on antibiotic or insecticide containing medium. This works well, but there are serious concerns about releasing genetic strains carrying antibiotic or insecticide resistance into the natural environment.

A recent modification has been to use a DNA vector that encodes a gene expressing a 'green fluorescent' protein such as aequorin (see for example US patent Number 5422266 Cormier et. al.). Whilst this provides a direct method of identification this invention has the significant disadvantage that the marker protein is visible only under ultraviolet illumination. Thus the technique is limited to those applications in which analysis may be carried out under ultraviolet illumination and for example cannot be simply applied to those tissue which are naturally fluorescent, for example any chlorophyll containing materials.

In addition it has been suggested that such vectors may also used as reporter constructs where the regulatory element from a gene is operatively linked to the DNA sequence encoding for a 'green fluorescent' protein (see for example US patent Number 5291084, Chalfie et. al.) In this application the expression of the marker fluorescent protein may be used to select cells expressing a protein of interest or by being linked to promotors for genes which are activated in response to infection may be used to investigate infection processes within plants and animals or in horticulture may be employed in the efficient application of herbicides etc. An alternative approach is when a reported gene with a minimal enhancer sequence is integrated at random sites in the host DNA. These "enhancer trap" vectors adopt the pattern of expression of the host genes adjacent to their site of integration. Potential applications are the production of variegated flower patterns with the coloured protein or specific patterns in the body of insect pests, for monitoring the populations of released organisms with the Sterile Insect Technique (SIT). Again this technique is limited as the marker protein is visible only under ultraviolet illumination.

We have devised methods for the detection of transcription vectors and for reporter constructs whereby the problems associated with the existing technology are mitigated or overcome.

Statement of Invention According to the first aspect of this invention there is provided a method of detection of integration of transformation vector DNA into a target cell which comprises insertion of the DNA sequence encoding for a coloured protein into the transformation vector, upon integration and expression of the transformation vector the protein will be produced and colour will be developed in the presence of an appropriate co-factor.

In a preferred embodiment of this invention the protein is a cytochrome protein and in a preferred embodiment the protein is cytochrome ba3 from Therm us thermophilus. In this embodiment a purple intracellular pigmentation will be apparent when the protein is expressed in the presence of a physiological level of the copper ion. In a further embodiment of the invention the copper binding site of the natural protein is modified to accommodate other heavy metals. This would allow the pigmentation to be developed only when the cell was provided the specific heavy metal.

In a further embodiment of the invention the protein is a cytcochrome that binds an iron ion.

In a further embodiment of the invention the protein is a carotenoid binding protein and pigmentation is developed in the presence of an appropriate carotenoid. This carotenoid may be physiologically present within the cell or may be supplied exogenously.

According to the second aspect of the invention there is provided a reporter construct here the regulatory element from a gene is operatively linked to the DNA sequence encoding for a coloured protein. In this application the expression of the marker pigmented protein may be used to select cells expressing a protein of interest or by being linked to promotors for genes which are activated in response to infection may be used Particular applications of reporter constructs include, but are not restricted to envisaged are as disease indicators of crop plants. We intend to drive coloured protein expression under promotors for genes activated during bacterial and tungal expressions. We intend to engineer a strain of tomatoes that expresses a coloured protein when infected with a tungal infection.

Claims

Claims

I. A method of detection of integration of transformation vector DNA into a target cell which comprises insertion into the transformation vector the DNA sequence encoding for a coloured protein.
2. A method as claimed in claim 1 in which the protein is a cytochrome protein.
3. A method as claimed in claim 2in which the protein is a cytochrome ba3 from Therm us thermophilus.
4. A method as claimed in claim 2 in which the cytochrome protein binds an iron ion
5. A method as claimed in claims 2 to 4 in which the metal ion binding sites of natural cytochromes are modified to accommodate other metal ions.
6. A method as claimed in claim 1 in which the protein is a carotenoid binding protein.

7. A method of producing a reporter construct where the regulatory element from a gene is operatively linked to the DNA sequence encoding for a coloured protein as claimed in claims 1 to 6.
7. A method of producing an enhancer trap using the DNA sequence encoding for a coloured protein as claimed in claims 1 to 6.
8. Any plants and animal strains derived from the method of claims.