Genetic engineering of the multicellular green alga Volvox: a modified andmultiplied bacterial antibiotic resistance gene as a dominant selectable marker

The green alga Volvox represents the simplest multicellular organism: Volvo x is composed of only two cell types, somatic and reproductive. Volvox, the refore, is an attractive model system for studying various aspects of multi cellularity. With the biolistic nuclear transformation of Volvox carteri, t he powerful molecular genetic manipulation of this organism has been establ ished, but applications have been restricted to an auxotrophic mutant servi ng as the DNA recipient. Therefore, a dominant selectable marker working in all strains and mutants of this organism is required. Among several gene c onstructs tested, the most advantageous results were obtained with a chimer ic gene composed of the coding sequence of the bacterial ble gene, conferri ng resistance to the antibiotic zeocin, modified with insertions of two end ogenous introns from the Volvox arylsulfatase gene and fused to 5' and 3' u ntranslated regions from the Volvox beta 2-tubulin gene. In the most suitab le plasmid used, the gene dosage was increased 16-fold by a technique that allows exponential multiplication of a DNA fragment. Go-transformation of t his plasmid and a non-selectable plasmid allowed the identification of zeoc in resistant transformants with nuclear integration of both selectable and non-selectable plasmids. Stable expression of the ble gene and of genes fro m several non-selectable plasmids is demonstrated. The modified ble gene pr ovides the first dominant marker for transformation of both wild-type and m utant strains of Volvox.