Nuclear transformation of Volvox carteri.

Abstract

Stable nuclear transformation of Volvox carten was achieved using the cloned V. carteni itAr gene (which encodes nitrate reductase) to complement a nitA mutation. Following bombardment of mutant cells with plasmid-coated gold particles, putative transformants able to utilize nitrate as a nitrogen source were recovered with an efficiency of -2.5 x 10-5. DNA analysis indicated that the plasmid integrated into the genome, often in multiple copies, at sites other than the nitA locus. Cotransformants were recovered with a frequency of 40-80% when cells were cobombarded with a selected and an unselected marker. Thus, V. cartie becomes one ofthe simplest multicellular organisms that Is accessible to detailed molecular studies of genes regulating cellular differentiation and morphogenesis.Volvox carteri is a multicellular organism with a complete division of labor between somatic and reproductive cells (1, 2). Genetic analysis (1-4) has led to the hypothesis that a small number of loci act to cause differentiation of these two cell types (5), and patterns of cell-type-specific gene expression in wild-type and mutant embryos are consistent with that hypothesis (6). However, detailed molecular analysis ofthese putative regulatory loci has awaited a method for transforming the organism with exogenous DNA.We repeatedly tried to transform V. carteri with various bacterial or plant selectable markers that were introduced by microinjection, electroporation, particle bombardment (7), UV-laser microbeam irradiation (8), agitation with glass beads (9), etc. As with the related unicellular alga, Chlamydomonas reinhardtii, reproducible transformation with heterologous selectable markers was not achieved, possibly because of an inability of these algae to express heterologous genes. Again in parallel with C. reinhardtii (9-12), success in transforming V. carteri has come with the availability of a homologous selectable marker: here we report use of the recently cloned nitrate reductase-encoding gene of V. carteri, nitA (13), to complement a nitA mutation. MATERIALS AND METHODSRecipient Strains. Strains used as DNA recipients were F1 female progeny of HB11A, a previously described, multiply marked strain of V. carteri f. nagariensis (4). All of these strains inherited from HB11A a stable mutant allele (reversion rate, <10-8) that confers resistance to chlorate, abolishes the ability to utilize nitrate as a nitrogen source, maps to the nitA locus, and is therefore inferred to be a stable loss-of-function mutation of nitA, the gene encoding nitrate reductase (13). Strain 153-81 was given the mnemonic Gls/ Reg ("gonidialess/regenerator") because it inherited from HB11A a regA mutation that causes somatic cells to redifferentiate as gonidia (asexual reproductive cells) and also has a spontaneous mutation at the gis ("gonidialess") locus that results in an absence of any "true" gonidia (6). GIs/Reg was used in initial studies because it has only one type of cells, all of which can reproduce; thus it provides a homogeneous populati...

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