APRT SELECTION SYSTEM IN PLANTS

Abstract

Most cells have an active turnover of many of their nucleic acids (particularly some types of RNA) which through degradative processes result in the release of adenine, guanine and hypoxanthine. These free purines are converted to their corresponding nucleotides through salvage pathways. Adenine is converted to its nucleotide form AMP by Adenine phosphoribosyltransferase (APRT) which is one of the enzymes associated with the purine salvage pathway. Since all organisms have a de novo pathway for the formation of AMP, APRT is classified as a `salvage enzyme'. The APRT enzyme, in general, does not show a high degree of specificity for the exact structure of adenine and can also act on cytokinins and adenine derivatives like 2,6-diaminopurine, 2-fluoroadenine and 6-methylpurine. The APRT enzyme can utilize adenine analogues as substrate and convert them into their nucleotide forms which are toxic. Plants that lack APRT activity (APRT-plants) survive in the presense of these analogues. The amount of adenine analogue used for selecting APRT-plants is such that it kills all APRT+ (wild type) plants. APRT+ plants survive when grown in the presense of azaserine and alanosine that block de novo synthesis of AMP. APRT-plants transformed with the wild type cloned gene can be selected from a mixture of transformed and non-transformed plants by selecting in the presense of adenine, azaserine and alanosine. The presense of APRT activity can be confirmed by assaying for the APRT enzyme. APRT activity has been detected in many plant species. The presense of a positive forward and backward selection system can thus allow the use of APRT as a selectable marker in plant gene transfer systems.