Cloning of anthozoan fluorescent protein genes

Abstract

Many cnidarians display vivid fluorescence under proper lighting conditions. In general, these colors are due to the presence of fluorescent proteins similar to the green fluorescent protein (GFP) originally isolated from the hydrozoan medusa Aequorea victoria (Cnidaria: Hydrozoa). To optimize the search for new fluorescent proteins (FPs), a technique was developed that allows for the rapid cloning and screening of FP genes without the need for a prior knowledge of gene sequence. Using this method, four new FP genes were cloned, a green from Montastraea cavernosa (Anthozoa: Scleractinia: Faviidae), a cyan from Pocillopora damicornis (Anthozoa: Scleractinia: Pocilloporidae), a cyan from Discosoma striata (Anthozoa: Corallimorpharia), and a red from a second Discosoma species. Two additional green FPs were cloned, one from M. cavernosa and one from its congener Montastraea faveolata, from purified cDNA using PCR primers designed for the first M. cavernosa green FP. Each FP has recognizable amino acid sequence motifs that place them conclusively in the GFP protein family. Mutation of these products using a low-stringency PCR protocol followed by screening of large numbers of bacterial colonies allowed rapid creation of mutants with a variety of characteristics, including changes in color, maturation time, and brightness. An enhanced version of the new red FP, DspR1+, matures faster at 30 °C than the commercially available DsRed but matures slower than DsRed at 37 °C. One of the M. cavernosa green FPs, McaG2, is highly resistant to photobleaching and has a fluorescence quantum yield approximately twice that of EGFP-1.