Recipient 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,
Plasmids pLV13-6, pLV131-3, and pLV131-4, which were used as unselected markers, were kindly provided by S. M. Miller (Washington University); each carries a different A DNA fragment plus a Volvox transposon (15) and will be described in detail elsewhere (S. M. Miller, personal communication). Cultivation Conditions. Recipient strains were grown in aerated flasks, as described (16). Standard Volvox medium (SVM) contained 0.5 mM urea plus 0.5 mM calcium nitrate (16), but selective medium (NSVM) contained only nitrate as a nitrogen source. Synchronous Gls/Reg cultures were obtained by inoculating 300 ml of SVM with 50 spheroids (selected when embryos were in early cleavage) and incubating for 3 days; this yielded =5 x 106 gonidia or embryos. Strains 153-40, -45, and -48 were cultured to a density of t10 spheroids per ml in flasks containing -1.5 liters of SVM; -2 x 105 gonidia or embryos could be recovered from such a culture.
Transformation Protocols. In initial successful experiments, mechanical disruption of cytoplasmic bridges linking embryonic cells (17) was used to provide cells access to exogenous DNA, as follows: Gls/Reg embryos (16- to 64-cell stage) were suspended in SVM containing nitA DNA and 50 mM sorbitol, broken into single cells in a Dounce homogenizer with a tight-fitting pestle (clearance, =2.5 Pm), and cultured in NSVM solidified with 0.375% SeaPlaque agarose (FMC). Plates were screened at intervals, and green, growing colonies were transferred to tubes containing NSVM. In all recent experiments, flowing helium (He) was used to bombard cells with gold particles (1-3 gam in diameter; Johnson Matthey GmbH, Karlsruhe, Germany, or Aldrich) that had been coated with nitA plasmid DNA by ethanol precipitation. Recipient spheroids were harvested by filtration and broken mechanically to release gonidia or embryos, which were then collected by mild centrifugation. For morphologically wild-type strains, 7% Percoll (Pharmacia) in SVM was used for the centrifugation step, so that the (floating) somatic cells could be removed from the (pelleted) gonidia. Target gonidia or embryos were resuspended in NSVM at a density of -4 x 105 per ml, cultured in the light for 15-120 min, and then bombarded by minor modifications of the methods of Takeuchi et al. (18): 10 y4 of DNA-coated gold particles in EtOH was placed on the screen of a Swinnex (Millipore) filter holder that was connected by tubing to a He tank; recipient cells were spotted on sterile filter paper in the bottom of a Petri plate and placed below the filter holder; a solenoid valve was then opened for -0. 1 sec to permit a rapid flow of He, which propelled the particles toward the cells. Parameter ranges tested extensively included: gun-to-target distance, 6-9 cm; He exit pressure, 4-6 bars (1 bar = 100 kPa); particle suspension, 125-250 ,g of gold and 125-750 ng of DNA; target, 104-2.5 x 105 mature gonidia or earlycleavage embryos. Controls included use of uncoated particles. Two selection protocols were tested: either the plate containing the bombarded filter was flooded with 30 ml of NSVM, covered, and returned to the culture chamber or cells were washed from the filter and cultured 2 days in 25 ml of NSVM and then aliquoted to wells of a multiwell plate. In both cases cultures were observed regularly, and each green, growing organism was transferred to a tube of NSVM. Putative transformants were tested for chlorate sensitivity by exposure to 8 mM potassium chlorate.
Preparation and Analysis of DNA. Plasmid DNAs were prepared by alkaline extraction (19). Genomic DNA was CsCl purified as described (4) or prepared by a newer miniprep method (15). Endonuclease digestion and electrophoresis of DNA (1-4 pg per lane on 0.8% agarose gels), preparation, hybridization, and autoradiography of Southern blots were all by previously described, conventional methods (4, 19), except that some Southern blots were probed with digoxigenin-labeled DNAs, according to directions provided by the vendor (Boehringer Mannheim). In all cases, final membrane washes were at 650C in 45 mM NaCl/4.5 mM sodium citrate/0.2% SDS/5 mM EDTA.