Gene replacement by homologous recombination in the multicellular green alga Volvox carteri (original) (raw)

1997, Proceedings of the National Academy of Sciences of the United States of America

With only two different cell types, the haploid green alga Volvox represents the simplest multicellular model system. To facilitate genetic investigations in this organism, the occurrence of homologous recombination events was investigated with the intent of developing methods for gene replacement and gene disruption. First, homologous recombination between two plasmids was demonstrated by using overlapping nonfunctional fragments of a recombinant arylsulfatase gene (tubulin promoter͞arylsulfatase gene). After bombardment of Volvox reproductive cells with DNA-coated gold microprojectiles, transformants expressing arylsulfatase constitutively were recovered, indicating the presence of the machinery for homologous recombination in Volvox. Second, a well characterized loss-of-function mutation in the nuclear nitrate reductase gene (nitA) with a single G 3 A nucleotide exchange in a 5-splice site was chosen as a target for gene replacement. Gene replacement by homologous recombination was observed with a reasonably high frequency only if the replacement vector containing parts of the functional nitrate reductase gene contained only a few nucleotide exchanges. The ratio of homologous to random integration events ranged between 1:10 and 1:50, i.e., homologous recombination occurs frequently enough in Volvox to apply the powerful tool of gene disruption for functional studies of novel genes. Green algae of the order Volvocales range in complexity from unicellular Chlamydomonas through colonial genera to multicellular organism in the genus Volvox. The unicellular members of this order have proven to be excellent model systems for the biochemical and genetic analysis of cellular processes like photosynthesis, phototaxis and motility, and cell wall biogenesis. The more advanced members of this group, like Volvox have developed a multicellular form of organization with a complete division of labor between somatic and reproductive cells; in Volvox carteri, about 2,000 cells are somatic and only 16 cells are reproductive. Therefore, the Volvocales represent an ideal model system to study the prerequisite for the transition from unicellularity to multicellularity. Recently, the development of nuclear transformation (1, 2) and the introduction of reporter genes (3, 4) for the unicellular as well as the multicellular members of the Volvocales have made it possible to apply the powerful techniques of molecular genetics. However, the important genetic technique of gene replacement and gene disruption by homologous recombination has not yet been established in Volvox. Only a few successful reports on homologous recombination events exist for Chlamydomonas reinhardtii (5-7). Gene targeting by homologous recombination is a genetic tool that permits modification of cellular genes in a precise and predetermined fashion. This technique was initially used as a tool by yeast genetics because site-specific rather than random MATERIALS AND METHODS Recipient Strain. The Volvox strain 153-48, obtained from D. L. Kirk (Washington University, St. Louis), was used as the DNA recipient. Strain 153-48 is an F 1-female progeny of HB11A, a female strain of V. carteri f. nagariensis that has been described (17). This strain with wild-type morphology inherited from HB11A an allele that carries a loss-of-function mutation of nitA, the structural gene encoding nitrate reductase (18, 19). The spontaneous reversion rate of this allele is less than 3 ϫ 10 Ϫ9 per reproductive cell (D. L. Kirk, personal communication). Culture Conditions. Synchronous Volvox recipients were grown in Volvox medium (20) at 28°C in an 8-h dark͞16-h light (10,000 lx) cycle (21). The nonselective medium used was Volvox medium, supplemented with 1 mM NH 4 Cl; the selective medium was Volvox medium lacking NH 4 Cl and containing only nitrate as a nitrogen source. Construction of the Chimeric ␤-Tubulin͞Arylsulfatase Gene. The Volvox arylsulfatase gene was placed under the control of the Volvox ␤-tubulin promoter in Volvox by using genomic clones of Volvox ␤-tubulin (22) and Volvox arylsulfatase (23). Additional restriction sites were introduced by PCR to facilitate ligation of the parent DNAs. An EcoRV site was The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.