Differential evolutionary rates of neuronal transcriptome in Aplysia kurodai and Aplysia californica as a tool for gene mining

Abstract

The marine mollusk Aplysia is a fascinating model organism for studying molecular mechanisms underlying learning and memory. However, evolutionary studies about Aplysia have been limited by the lack of its genomic information. Recently, large-scale expressed sequence tag (EST) databases have been acquired by sequencing cDNA libraries from A. californica and A. kurodai. The closeness between the two species allowed us to investigate rapidly evolving genes by comparing their orthologs. Using this method, we found that a subset of signal transduction genes in neurons showed rates of protein evolution higher than those of housekeeping genes. Moreover, we were also able to find several candidate genes that may be involved in learning and memory or synaptic plasticity among genes showing relatively higher K(a)/K(s) ratios. We also investigated the relationship between evolutionary rates and tissue distribution of Aplysia genes. They propose that the estimation of evolutionary rates cannot be a good marker to assess neuronal expression; however, it still can be an efficient way to narrow down the pool of candidate genes involved in neuronal functions for the further studies.

Figure 1.

Faster evolution rate of the mammalian homologs of signal transduction genes in Aplysia neurons (see text for details). (A) Evolutionary rates of signal transduction genes and housekeeping genes in Aplysia. (B) The K_a/K_s distribution of the same two subsets of brain derived genes in Aplysia.

Figure 2.

Differential expression levels of the genes showing the highest K_a/K_s values. (A) RT-PCR results on the 13 Aplysia genes showing highest evolutionary rates. Soluble acetylcholine receptor, hemocyanin, heart-type fatty acid–binding protein, and MIP were highly expressed in the central nervous system. One of these genes (MIP) was expressed only in CNS. K_a/K_s ratio for each gene is indicated in parenthesis (B) Expression levels of three genes (soluble acetylcholine receptor, hemocyanin, and heart-type fatty acid–binding protein) were confirmed by real-time PCR. Two genes (soluble acetylcholine receptor and heart-type fatty acid–binding protein) showed significantly higher expression levels in the CNS when compared to the other tissues (p < .05, ANOVA and Tukey’s multiple comparison test). Hemocyanin was also significantly enriched in the CNS except when compared to the OT (p > .05, ANOVA and Tukey’s multiple comparison test). CNS, central nervous system; BM, buccal mass; ST, stomach; GL, gill; OT, ovotestis; vATP, vacuolar ATP synthase subunit e; sAChR, soluble acetylcholine receptor; Haemo, hemocyanin; FABP, heart-type fatty acid–binding protein; CycloA, cyclophylin isoform; dhs14, dehydrogenase, short-chain family member; ATPsyn, ATP synthase, mitochondrial F1 cmplex alpha subunit; CREB2, ApCREB2; P4H, proline 4-hydroxylase; MIP, MIP-related protein precursor; Zn, zinc finger, HIT type 3; cct7, cct7-prov protein; TIF5, transcription initiation factor 5A.

Figure 3.

Differential expression levels of the genes showing the lowest K_a/K_s values. (A) RT-PCR results on the 14 Aplysia genes showing the lowest evolutionary rates. ENSANGP00000012700 and GTP-binding protein alpha-o subunit were highly expressed in the central nervous system. K_a/K_s ratio for each gene is indicated in parenthesis. (B) Expression levels of four genes (AGAP010957-PA, GTP-binding protein alpha-o subunit, guanine nucleotide regulatory protein beta subunit, and RHO_APLCA RAS-like GTP-binding protein RHO) were confirmed by real-time PCR. Two genes (AGAP010957-PA and GTP-binding protein alpha-o subunit) showed significantly higher expression levels in the CNS when compared to the other tissues (p < .05, ANOVA and Tukey’s multiple comparison test). GNRPb was also significantly enriched in the CNS compared to the ST and GL, and Rho showed significantly higher expression in CNS than in BM (p < .05, ANOVA and Tukey’s multiple comparison test). AGAP, AGAP010957-PA; GBPα, GTP-binding protein alpha-o subunit; Rho, RHO_APLCA RAS-like GTP-binding protein RHO; GNRPβ, guanine nucleotide regulatory protein beta subunit; S14, ribosomal protein S14; UnPP, unnamed protein product; S15, 40S ribosomal protein S15; L12, ribosomal protein L12; Cnot4, Cnot4-prov protein; APY1, YBOXH_APLCA Y-box factor homolog; S16, similar to Rps16 protein; Gluco, similar to glucocorticoid-induced gene 1.