ISOLATION OF PHASE‐SPECIFIC COMPLE‐MENTARY DNAS FROM CARPOSPOROPHYTES OF GRACILARIOPSIS LEMA‐NEIFORMIS USING MAGNETIC BEADS AND PCR

Kamiya, M. 1 , Moon, D. A. 2 , Kawai, H. 1 & Goff, L. J. 21 Kobe University Research Center for Inland Seas, 2746 Iwaya, Awaji‐cho 656‐2401 Japan; 2 Department of Biology, University of California, Santa Cruz, CA 95064 USA Although the morphology and developmental patterns of carposporophyte stage have been investigated well, there are few molecular, genetic, or biochemical data about this stage. The greatest obstacle to this research has been that the conventional methods to isolate tissue‐specific genes require a lot of tissues, but the carposporophyte is very tiny and mostly embedded in female gametophyte tissues. Recent advanced techniques have allowed the subtractive cloning of differentially expressed genes from small amounts of tissue or cells. We applied the subtractive hybridization method using magnetic beads and PCR to the analysis of phase‐specific cDNAs from carpo‐sporophytes of Gracilariopsis lemaneiformis (Gracilariales, Rhodophyta). A hundred cystocarps were dissected to isolate gonimoblast tissues, and total RNAs were extracted from the gonimoblast tissues and the female gametophyte branches, respectively. Messenger RNAs were captured on paramagnetic oligo‐dT beads, followed by first‐strand cDNA synthesis on the beads. Three rounds of subtractive hybridization between the amplified second‐strand carposporophyte cDNA in solution and the first‐strand gametophyte cDNA attached to magnetic beads were sufficient to remove common genes present in both gametophyte and carposporophyte stages. A specific PCR product from the nuclear GAPDH gene was readily amplified from gametophyte and carposporophyte cDNA, but no amplification was observed using the subtracted carposporophyte cDNA as template. This control PCR product demonstrates that the hybridization steps successfully removed the common GAPDH cDNA, which is found in both stages, giving confidence that the remaining genes cloned from the subtracted carposporophyte cDNA library are stage‐specific.