Department of Biology, Center for Advanced Research in Environmental Genomics (CAREG), University of Ottawa, 30 Marie Curie Street, Ontario, Canada K1N 6N5
Gamma-aminobutyric acid (GABA) and glutamate mediate fast synaptic inhibitory and excitatory neurotransmission in the CNS, respectively. GABA is synthesized from glutamate in a single enzymatic step by glutamic acid decarboxylase (GAD) (Martin and Rimvall 1993
). In addition to its role as a neurotransmitter in higher brain centers, GABA is involved in the regulation of neuroendocrine function by acting on the hypothalamo-pituitary axis (Trudeau et al. 2000a
). At least two isoforms of GAD exist in mammals, which have molecular weights of 65 kDa (GAD65) and 67 kDa (GAD67). Both GAD65 and GAD67 are coexpressed in GABAergic neurons, but they differ in cellular distribution, in regulation, and in their interaction with the cofactor pyridoxal 5'-phosphate, PLP (Erlander et al. 1991a;
Erlander and Tobin 1991b;
Bu et al. 1992
; Sheikh, S. B. Martin, and D. L. Martin 1999
).
The genes coding for GAD65 and GAD67 show highly similar deduced amino acid sequences. The similarity of their intron-exon organization in humans led Bu and Tobin (1994)
to suggest that these genes derive from a common ancestral GAD gene, yet GAD65 and GAD67 are encoded by two distinct genes located on human chromosomes 10 and 2, respectively (Erlander et al. 1991a;
Bu et al. 1992
; Edelhoff et al. 1993
). They are thought to have arisen as a result of a gene duplication event early in vertebrate evolution, after the branching off of the ascidians but before the divergence of the teleosts from the main vertebrate lineage (Bosma et al. 1999
).
A third form of GAD, designated GAD3, was recently cloned from the armed grenadier (Coryphaenoides [Nematonurus] armatus), a deep-sea fish (Bosma et al. 1999
). It shows only 64% identity to either GAD65 or GAD67 at the amino acid level, whereas grenadier GAD65 and GAD67 are 73% identical to each other at the amino acid level. This novel GAD was shown to group separately from the other GAD isoforms in phylogenetic analyses; however, its evolutionary relationship with GAD65 and GAD67 could not be resolved (Bosma et al. 1999
). It was suggested that the ancestral GAD may have undergone two phases of gene duplication events to produce the three expressed forms of GAD (Bosma et al. 1999
).
This study set out to examine some of the important events occurring in GAD evolution by cloning and sequencing several new vertebrate cDNA fragments. We first examined the evolution of GAD around the putative time of divergence of GAD65 and GAD67 by studying the amphioxus, three representative agnathans, and four species from the Chondrichthyes. Single, divergent forms of the GAD gene were identified from the amphioxus and each agnathan species, whereas two forms with high sequence similarity to GAD65 and GAD67 were cloned from two Chondrichthyan species. This shows that GAD65 and GAD67 genes originated after the divergence of agnathans and gnathostomes but before the divergence of Chondrichthyes, more than 450 MYA.
Amphioxus (Branchiostoma lanceolatum) was provided by Dr. John Bishop (Plymouth Marine Laboratory, England). Hagfish (Myxine glutinosa) and hedgehog skate (Raja erinacea) were provided by Dr. Jim Ballantyne (University of Guelph). Lampreys were collected by Dr. Claude Renaud (Canadian Museum of Nature, Ottawa). Greenland sleeper shark (Somniosus microcephalus) brain was collected in Cumberland Sound, Baffin Island, by Dr. Aaron Fisk (Canadian Wildlife Services, Hull). Ratfish (Hydrolagus colliei) were obtained from the Bamfield Marine Station, British Columbia, and brains were collected by Dr. S. Perry (University of Ottawa). Dogfish (Squalus acanthias) were collected by V.L.T. near Aberdeen, Scotland. Eels (Conger conger) were collected by V.L.T. near Scalloway, Shetland, and I. Napier (NAFC) provided trawling and aquarium facilities. Electric fish (Apternotus leptorhynchus) were provided by W. Ellis and L. Maler (University of Ottawa). Lungfish (Lepidosiren paradoxa) were caught courtesy of Dr. G. Somoza (Universidad de San Martin, Argentina). Alligator (Alligator mississippiensis) brain was provided by Dr. L. Guillette (University of Florida). Axolotls (Ambystoma mexicanum) were provided Dr. Bob Johnson (Toronto Zoo). Brains were rapidly dissected, pooled on dry ice, and stored at -80°C until RNA extraction.
Total RNA, and in some cases mRNA, was isolated from the frozen tissue samples using the acid guanidinium thiocyanate method (Chomczynski and Sacchi 1987
), followed by either the Oligotex mRNA kit (Qiagen) or the Straight A's kit (Novagen) for certain samples. First-strand cDNA was synthesized from mRNA or total RNA using Oligo (dT) primer and Superscript II reverse transcriptase (GIBCO BRL). Using the protocol described by Bosma et al. (1999)
, annealing temperatures ranging from 43°C to 51°C and a standard MgCl2 concentration of 1.5 mM, partial GAD sequences of GAD65 and GAD67 were amplified. Goldfish GAD3 was amplified using the primers gGAD3 f/r2 (f: TGT GGG ATG GTG CGA GGA AGA G; r2: GTC TGC TAC AAG AGT GCA ACC AGC TG), with an annealing temperature of 63°C. Desired products of approximately 550 bp were extracted from 1% agarose gel using the QIAquick gel extraction kit (Qiagen) and ligated into the pCRII-TOPO cloning vector, which was then transformed into One Shot competent cells (Invitrogen, San Diego, Calif.). Plasmids were isolated using the Wizard Miniprep kit (Promega). Inserts were sequenced by the Canadian Molecular Research Services (Ottawa); both strands of three to six inserts were sequenced for each GAD consensus sequence obtained. All the nucleotide consensus sequences obtained were translated to predicted amino acid sequences. Accession numbers of previously published sequences are described in Bosma et al. (1999)
and Trudeau et al. (2000a)
.
Maximum-likelihood trees of predicted partial amino acid sequences were calculated using the TREE-PUZZLE version 5.0 program (Strimmer and von Haeseler 1996
). The JTT model of amino acid substitutions (Jones, Taylor, and Thornton 1992
), and one fixed and eight gamma-distributed rates of heterogeneity were used. The tree was rooted with Ciona intestinalis GAD, an urochordate known to be an out-group to all the other species analyzed. Alternative amino acid tree topologies were compared using user-defined trees, the JTT model of substitutions (Jones, Taylor, and Thornton 1992
), and eight gamma-distributed rates of heterogeneity. The RRTree version 1.1.10 program was used to perform relative rate tests on the GAD amino acid sequences (Robinson et al. 1998
; Robinson-Rechavi and Huchon 2000
).
A few mammalian GAD gene sequences have long been available. However, GADs from species representing the other major groups of vertebrates have been sequenced only recently (Bosma et al. 1999
; Trudeau et al. 2000a
). We obtained 21 new partial GAD sequences 541547 bp in length from 14 different species. These sequences represent the central one-third of the coding sequence of the GAD genes. Two forms were obtained from alligator, axolotl, lungfish, electricfish, eel, ratfish, and skate. Single forms were cloned from dogfish shark, greenland shark, both lamprey species, hagfish, and amphioxus. In addition, a GAD3 sequence 588 bp in length was obtained from goldfish brain. This sequence is 86% identical, both at the nucleotide and at the amino acid level, to grenadier GAD3.
Phylogenetic analysis shows that GAD genes cluster in five groups: the protochordate out-group, the agnathans, GAD3, GAD65, and GAD67 (fig. 1 ). These groupings of GAD were all found to evolve at similar rates (results not shown). The GAD65 and GAD67 sequences, which include most of the known vertebrate GADs, form two well-defined groups within the tree, each of these groups following the general model of vertebrate evolution. Alternative tree topologies were tested to determine whether the inferred phylogenetic position of the GAD3 and agnathan GAD genes was significant. None of the alternative topologies (table 1 ) we tested were significantly different from the topology shown in figure 1 . A similar tree was also obtained using the PROML maximum-likelihood method of the PHYLIP package (Felsenstein 1989). The main relationships of the tree obtained using this method were identical to those shown in figure 1 , except for GAD3 which was shown to branch as a weakly supported out-group to all other vertebrate GAD genes (results not shown).
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We sequenced GAD from the amphioxus, which is considered to be the direct ancestor of all modern vertebrates (Holland and Chen 2001
). We found that the amphioxus GAD groups separately from the sequences for both GAD65/GAD67 and agnathan GADs but directly adjacent to Ciona GAD, a protochordate sequence. Bosma et al. (1999)
had previously suggested that the GAD expressed by Ciona intestinalis is most likely a derivative of the archetypal chordate form. The single form we cloned from amphioxus is probably derived from the same ancestral gene. To further elucidate the time of divergence, we cloned a single GAD form from a parasitic lamprey, a nonparasitic lamprey, and a hagfish. Our data support a hagfish-lamprey clade because all three species grouped together. However, the sequence data available are not sufficient to resolve the phylogenetic position of the agnathans with the GAD65, GAD67, or GAD3 groups (table 1
). Although studies on mitochondrial DNA have yielded phylogenies grouping lampreys with tetrapods, with hagfishes grouping separately (Rasmussen, Janke, and Arnason 1998
), more recent studies on ribosomal rDNA sequences have shown moderate to strong support for the monophyly of these three cyclostomes (Mallatt and Sullivan 1998
). Among the lamprey GADs, only minor DNA sequence differences were observed, which did not result in any amino acid differences (results not shown). This demonstrates a remarkable conservation of the GAD gene between these two species despite their very different life histories. Our research seems to suggest that the agnathan form of GAD evolved separately from GAD65 and GAD67 and may represent a descendant of an ancestral form of the gene. Furthermore, the two isoforms of GAD found in mammals and other vertebrates were identified in the two extant subclasses of the Chondrichthyes: the holocephalans (ratfish) and the elasmobranchs (skate). These two GADs clearly group with GAD65 and GAD67 in this study.
Lungfish GAD sequences consistently group with tetrapods for both GAD65 and GAD67, supporting the proposal that terrestrial vertebrates have evolved from lobe-finned fishes (subclass Sarcopterygii; Zardoya and Meyer 1997
; Longhurst and Joss 1999
). Only six living species of lungfish and the coelacanth, a single lobe-finned fish, now represent this subclass. The South American lungfish is the first of these species from which GAD has been cloned. Some recent controversial studies have shown lungfish as the most ancestral of the fishes, with Chondrichthyes occupying a terminal position in the piscine tree (Rasmussen and Arnason 1999a,
1999b
). Our study does not support this conclusion but shows trees placing Sarcopterygians as the sister group to tetrapods as being more likely.
We also sequenced the alligator GAD65 and GAD67 genes. Both were shown to group with known turtle and bird sequences. Using the available sequence information, it was not possible to group together anapsids (e.g., turtles) and diapsids (e.g., crocodilians and birds); however, the common ancestry of the GAD genes from these two groups is clear. We obtained two different forms of GAD65 from the brain of a conger eel that differ by 16 amino acids in the sequence portion obtained. These likely represent the result of a recent duplication in this species, a hypothesis which requires further investigation.
Previous phylogenetic studies identified a third form of GAD from a deep-sea fish, the armed grenadier (Bosma et al. 1999
). In our study, we were able to clone this form of GAD from goldfish, thus demonstrating its expression in at least two divergent teleost species. GAD3-deduced amino acid sequences are found to be most similar to GAD65 and GAD67 sequences when compared with the human genome using a BLAST search (results not shown). The additional sequences added to our phylogenetic analysis are yet to resolve the origin of GAD3 because trees grouping GAD3 alone or groupings with either of the other GAD groups are not statistically different (table 1
). Furthermore, relative rate tests show that the different GAD genes evolve at similar rates (results not shown). Although two comparisons (GAD3 vs. GAD67 and GAD3 vs. agnathan GADs) are significantly different at the 5% level, neither of these comparisons is significant at the 0.5% level when the alpha level is corrected for the 10 comparisons conducted (Rice 1989
). These results are consistent with previous relative rate test analyses on a smaller GAD sequence data set (Bosma et al. 1999
). The position of this novel GAD on the phylogenetic tree is suggestive of an early duplication event. Had GAD participated in both genome duplication events as described earlier, four GADs would have been expected. With the presence of three distinct GADs in teleosts, we hypothesize that the putative fourth form could be silent in many vertebrate classes. Moreover, the GAD3 form may have gone silent in some classes, but this has yet to be rigorously tested. A novel function may be reasonably assumed for GAD3 because this gene likely would have degenerated into a pseudogene had it not had a separate function (Shimeld 1999
). Furthermore, differential distribution for GAD3 has been established in the brain of the deep-sea grenadier. It has been found to be more highly expressed in the cerebellum than in the other brain regions, a pattern not consistent with the distribution of GAD65 or GAD67, which are both more abundant in telencephalon (Trudeau et al. 2000b
). This differential distribution also implies a novel function for GAD3.
In conclusion, we have presented evidence of the existence of multiple forms of GAD in species that diverged after the agnathans. Although our results cannot resolve the precise relationship of the lamprey and hagfish sequences relative to the three GAD isoforms found in higher vertebrates, the presence of both GAD65 and GAD67 in Chondrichthyes demonstrates that these two GAD isoforms originated before the divergence of the cartilaginous fishes about 450 Myr ago.
Supplementary Material
The new sequences reported here were submitted to GenBank. Accession numbers are B. lanceolatum GAD: AF432147; R. erinacea GAD65 and GAD67: AF432148 and AF432149; S. microcephalus GAD67: AF432150; Ambystoma mexicanum GAD65 and GAD67: AF432151 and AF432152; L. paradoxa GAD65 and GAD67: AF432153 and AF432154; Carassius auratus GAD3: AF432155; M. glutinosa GAD: AF432156; Petromyzon marinus GAD: AF432157; Ichthyomyzon unicuspis GAD: AF432158; H. colliei GAD65 and GAD67: AF432159 and AF432160; Alligator mississippiensis GAD65 and GAD67: AF432161 and AF432162; C. conger GAD65A and GAD65B: AF432164 and AF432163; Squalus acanthias GAD67: AF432165; A. leptorhynchus GAD65 and GAD67: AF432166 and AF432167.
Acknowledgements
Discussions with Dr. Peter Bosma and tissue collections by many colleagues are acknowledged with appreciation. This research was supported by scholarships and funds from NSERC (Canada) to K.L., L.M., V.G., G.D. and V.L.T.
Footnotes
Keywords: glutamic acid decaboxylase
GAD
gamma-aminobutyric acid
neurotransmitter
molecular phylogeny
chordate evolution
amphioxus
agnatha
cartilaginous fishes
Address for correspondence and reprints: V. L. Trudeau, Department of Biology, Center for Advanced Research in Environmental Genomics (CAREG), University of Ottawa, 30 Marie Curie Street, Ottawa, Ontario, Canada K1N 6N5. E-mail: vtrudeau{at}science.uottawa.ca
.
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