Long-term memory in Drosophila is separable into two components: consolidated, anesthesia-resistant memory and long-lasting, protein-synthesis-dependent memory. The Drosophila memory mutant radish is specifically deficient in anesthesia-resistant memory and so represents the only molecular avenue to understanding this memory component. The radish gene was identified by positional cloning and comparative sequencing, a mutant stop codon was found in in gene CG15720 from the Drosophila Genome Project. Induction of a wild-type CG15720 transgene in adult flies acutely rescues the mutant's memory defect. The phospholipase A2 gene, previously identified as radish [Chiang, 2004), maps 95 kb outside the behaviorally determined deletion interval and is unlikely to be radish. The Radish protein is highly expressed in the mushroom bodies, centers of olfactory memory. It encodes a protein with 23 predicted cyclic-AMP-dependent protein kinase (PKA) phosphorylation sequences. The Radish protein has recently been reported to bind to Rac1 (Formstecher, 2005), a small GTPase that regulates cytoskeletal rearrangement and influences neuronal and synaptic morphology (Folkers, 2006).

The specific defect of the radish mutant, diminished ARM, normal long-lasting protein-synthesis-dependent memory, provides a key to obtaining mechanistic information about ARM. The radish mutant was isolated in a screen for learning-defective flies after chemical mutagenesis. Genetic recombination and deletion mapping of the memory defect placed the gene in a 140-kb stretch in region 11D of the X chromosome. a transcript analysis and information from the Drosophila genome project indicated 17 genes in this interval. 105 kb of the genomic 11D region DNA was PCR-amplified from radish flies and from the parental wild-type Canton-S (C-S) flies, the products were sequenced, and the sequenceswere compared. The sequences analyzed included the ORFs of all identified genes in or near the interval. In this comparison, only a 1-nt difference was detected between mutant and parent strains, a C-to-T transition in the ORF of the CG15720 predicted gene. This mutation converts a glutamine codon in the wild-type ORF into an amber stop codon in the mutant radish DNA (Folkers, 2006).

Previous mapping of the memory phenotype revealed that the radish mutation is uncovered by the Df (1)105 X-chromosome deficiency (Folkers, 1993). However, the CG15720 ORF lies proximal of the Df (1)105 breakpoint, but close enough so that the deletion could plausibly eliminate its transcription. To verify that this was the case, RT-PCR was used to isolate the CG15720 transcript from C-S/Df (1)105, radish/Df (1)105, and radish/C-S flies. The region of the mutation was further amplified and the products were sequenced. The results were all consistent with Df (1)105 cis-acting elimination of radish gene transcription, C-S/Df (1)105 gave only wild-type sequence; radish/Df (1)105 gave only mutant sequence; radish/C-S gave 50:50 wild-type and mutant sequence (Folkers, 2006).

The CG15720 gene is expressed in the fly head. Northern blot analysis revealed a single 6-kb transcript. This transcript was amplified by RT-PCR, its ORF was sequenced, and it was found to span five exons. The radish mutation is at the 3' end of the fourth exon, which should truncate the protein in radish mutant flies. The inferred mutant protein lacks the C-terminal 64 aa (Folkers, 2006).

To ascertain whether CG15720 is, in fact, the radish gene, wild-type CG15720 gene expression was reestablished in radish mutant flies and ARM was assayed. The wild-type CG15720 ORF DNA was subcloned into the germ-line transformation vector hs-CaSpeR under the control of the inducible promoter for heat-shock protein 70. The hs-CG15720 construct was injected into mutant radish embryos and two resulting transformant flies were bred into homozygous populations (hs-rsh-1, hs-rsh-2). The hs-CG15720 transgene was induced with a 25-min heat shock (37°C), transgene expression required a wait of 1h, and the flies were trained in a odor-discrimination paradigm. Two hours later, the flies were anesthetized by cooling on ice for 2 min, they were allowed to recover, and memory was tested 1 h thereafter (Folkers, 2006).

Expression of the hs-CG15720 transgene in mutant radish flies restored normal ARM. Without heat-shock induction of the hs-CG15720 transgene, the flies remembered as poorly as mutant radish flies. Western blot analysis indicated that the transgene was expressed after heat shock and not in its absence. (Transgenic rescue of memory was also obtained without anesthesia). These results strongly indicate that the CG15720 gene is radish (Folkers, 2006).

Induction of this transgene in radish⁺ flies elicited no additional ARM, indicating that this transgene specifically rescues the radish memory defect. When ARM was assayed after heat shock, rsh⁺ scores were 0.15 ± 0.05; scores for transformed w, rsh⁺[hs-rsh-1] flies were 0.15 ± 0.03. Without heat-shock treatment, scores for w, rsh⁺ were 0.18 ± 0.04; scores for transformed w, rsh⁺[hs-rsh-1] flies were 0.19 ± 0.04 (Folkers, 2006).

Chiang (2004) have reported that the radish gene encodes a phospholipase A2 gene (PLA2) with the designation CG4346. However, this gene was found to be far outside the deletion, Df(1)N105, that defines the proximal end of the radish interval. PCR-amplification of Df(1)N105 DNA indicates that the proximal breakpoint of this deletion is 95 kb distal of the PLA2 gene. Recently Dubnau and colleagues have done additional experiments, including repeating their complementation crosses with reciprocal parental genotypes. Their recent results indicate that PLA2 and radish are different genes (J. Dubnau, personal communication to Folkers, 2006) (Folkers, 2006).

It is concluded that the Radish protein functions acutely in the adult fly to engender ARM, because its expression 1 h before training is sufficient to rescue memory in mutant radish flies. PKA-dependent phosphorylation of the Radish protein, as suggested by numerous PKA target sites, would logically link ARM to the cAMP pathway that functions in short-term memory in Drosophila. The idea is inconsistent with the findings that rutabaga flies have substantial ARM, although the reduction in cAMP levels caused by the mutation is partial (Folkers, 2006).

The high Arg/Ser content of the inferred amino acid sequence and the observed homology to Arg/Ser-rich splicing factors both suggest a role in RNA processing for the Radish protein. This notion appears to be contradicted by the finding that the translation inhibitor cycloheximide does not affect ARM. However, protein synthesis in these experiments was reduced by only 50% (Folkers, 2006).

Finding the Radish protein in the MBs is consistent with the known importance of the MBs in olfactory memory, the expression of many other memory-relevant genes (including cAMP cascade components) there, and the observation that blocking MB output abolishes ARM. Staining in the MB calyx and lobes indicates the presence of the Radish protein in neurites. Nuclear localization is not apparent, in current immunostains, but it may be present under other conditions, e.g., on activation of PKA (Folkers, 2006).

Long-term memory in Aplysia and mice is correlated with changes in synaptic morphology. One last property of the Radish protein argues for its role in synaptic morphology. Formstecher (2005) carried out an extensive high-throughput yeast two-hybrid screen for interacting Drosophila gene products using 102 proteins as bait. Intriguingly, this analysis identified an interaction between CG15720 (Radish) and Rac1. Rac1 is a small GTPase of the Rho family that regulates cytoskeletal assembly to influence neuronal and synaptic morphology in Drosophila and mammals. Furthermore, Rac1 lies in the same cell-signaling pathways as Pak1 and fragile-X protein (FMRP), both of which have documented effects on synaptic and behavioral plasticity. Strikingly, amino acids 538–579 of the Radish protein are sufficient for its binding to Rac1, and these residues are all deleted in the mutant Radish protein. Therefore, it is plausible that the critical function of Radish in ARM is to change synaptic morphology through Rac1 interaction (Folkers, 2006).

The Radish protein is similar to a predicted protein from the mosquito Anopheles gambiae (66% identity) and to one from the honey bee Apis mellifera (45% identity). It has no striking homology to proteins with known function. Sequence comparison with mammalian proteins reveals low homology (25%) to Arg/Ser-rich splicing factors. 29% of amino acids in the Radish protein are either arginines (14%) or serines (15%) (Folkers, 2006).

The inferred Radish amino acid sequence has 23 potential PKA and 14 protein PKC phosphorylation sites. The Radish sequence also has five bipartite nuclear localization signals (NLSs); and each NLS overlaps with a PKA target site. The truncated protein in the radish mutant lacks two PKA target sequences and two NLSs (Folkers, 2006).