RACE PCR on the ccNR1.1 subunit proved to be difficult, particularly in its 3’ end. To obtain functional assays, it was therefore necessary to optimize different reaction parameters, like Taq polymerases, PCR programs and primers.
In the 3’ end this implied the use of a different Taq polymerase, since the Platinum® Taq seemed to be operating close to its limits. The Advantage 2 polymerase mix was used, as this polymerase mix is supposed to be particularly useful for difficult templates. In addition to containing the modified and more robust BD TITANIUM™ Taq polymerase, it contains a proof reading enzyme, and is reported by the manufacturer to amplify DNA templates as large as 20 kb.
The fact that a different Taq polymerase had to be used to achieve successful 3’ RACE of ccNR1.1 might have to do with the quality of the primers. In the GeneRacer™ protocol the linker-specific primers (GeneRacer™ 5’ and 3’ primers) were listed as having melting temperatures between 72 and 78 °C. When this was calculated using the Primer3 software, these primers were assigned melting temperatures ranging from 65 and 69 °C. Further, GSP RACE primers were designed using Primer3, and they were given Primer3 calculated melting temperatures ranging from 70 to 72 °C (Table 2). Thus, there was a large discrepancy in melting temperatures between GeneRacer™ primers and GSPs. This was not discovered until late in course of the project, and brought along large variations
Discussion in melting temperature (~4.4°C) within every single primer pair. The basis of touchdown
PCR is to start out with annealing temperatures similar to those of primer melting temperatures, and to gradually lower it to get a more efficient amplification as PCR products are accumulating (Don et al., 1991). Primers with different melting temperatures will require different annealing temperatures, and the closer the melting temperatures of a primer pair, the larger their potential of success. Thus, the primers used for 3’ RACE of ccNR1.1 were far from ideal.
4.2 NMDAR Subunit Sequences
There are greater sequence similarities in between ccNR2 subunits than between ccNR2 and ccNR1.1 (Figure 9) or ccNR3A. All ccNR2 ESTs were obtained in the 5’ end (N- terminal) of the gene. This was done to be able to distinguish between them in PCR reactions and to enable phylogenetic analyses.
4.2.1 Primer Design
Primers designed for EST cloning of ccNR1.1, resulted in an amplicon containing the ligand binding loop S1, the pore element P and the transmembrane segment TM3 (Figure 2). The primers where placed in highly conserved regions. These turned out to represent β-plate and α-helix structures (data not shown). These structures are considered important for protein function and tend to be conserved across species (Sitbon and Pietrokovski, 2007). Positions for primers were Primers for EST cloning of ccNR3A were aimed toward the membrane pore element P, the transmembrane TM3 and part of the ligand binding loop S2. These ccNR3A elements are highly conserved between species, as well as between ccNR3 subunits, especially on the nucleotide level (see Figure 2 and Figure 5, section 3). Thus, the ccNR3A identity could not be verified via nucleotide BLAST, and could only be determined via amino acid BLAST. Further, the highly conserved sequence excluded accurate assessment of NR3 phylogenetics. This would have been possible, had the ccNR3A EST been retrieved from the less conserved pre-transmembrane region (as was done for ccNR2A.1-D.1).
Discussion
4.2.2 Crucian Carp Paralogs
The two paralogs found for ccNR1.1, ccNR2A.1 and ccNR2D.1 displayed DNA mutation rates of 3.3 %, 8.3 % and 8.1 %, respectively. These mutations affected the amino acid sequence with a rate (functional mutation rate) of 0 %, 36 % and 14.3 %. The sequences investigated were located to analogous regions of the 5’ ends (ccNR1.1.1 amino acid area 238-381, Figure 5). The DNA mutation rates tell us that the ccNR2A.1 and ccNR2B.1 genes are mutating twice as fast as the ccNR1.1 gene. Speculatively, mutations in the ccNR1 gene may produce dysfunctional proteins that are selected against. The functional mutation rates reflect this possibility, as no amino acid changes are seen in the ccNR1.1 protein. This indicates the need for uniform and stable NR1 function. In the light of the dose-model proposed by Force et al. (1999), both ccNR1 paralogs might be needed to cover the demand for NR1 subunits. Thus, both copies must retain their functionality. Further, the high amino acid mutation rates of NR2A.1 and NR2B.1 indicate that functional changes in this region of ccNR2 proteins are not detrimental for their function. Indeed, for ccNR2A every third mutation ends in changes in amino acid composition. The high amino acid mutation rate may be due to two factors. Firstly, the mutations may not affect protein function. Secondly, the mutations may occur in a part of the protein where functional changes are allowed.
In accordance with the DDC model by Force et al. (1999) the paralogs of ccNR2A.1 and ccNR2D.1 might have become subfunctionalized or neofunctionalized. The wider variety of NMDAR subunits in crucian carp could reflect a need for seasonal change in subunit composition. Indeed, in another cyprinid, the zebrafish, the expression of NMDAR subunit paralogs have been proven to vary during the course of embryonic CNS development. While the zebrafish paralog NR1.1 mRNA is found in the brain, retina and spinal cord 24 hours postfertilization (hpf), the NR1.2 is expressed only in the brain and detected 48 hpf. The NR2 paralogs displayed a much more diversified expression: both NR2A paralogs were expressed in the brain 48 hpf, no NR2B or NR2C paralog could be detected embryonically and while the NR2D.1 was expressed in the forebrain, retina and spinal cord 24 hpf, the NR2D.2 was only found in the retina (Cox et al., 2005). The regional expression of the ccNR2A.1 and ccNR2D.1 paralogs in crucian carp remains to
Discussion be assessed, along with the confirmation of the existence and expression of paralogs for
ccNR2B and ccNR2C.
Another analysis performed of the two ccNR1.1 paralogs revealed 48 nucleotide mutations within the 5’ region of the gene (amino acid 60-545 in Figure 5). These mutations resulted in 2 differences in amino acid composition (amino acid 96 and 433). In general, the amino acids are conserved in these two local regions of the gene (Figure 5). This implies a functionally important segment where amino acid transitions would be undesired to preserve protein function. However, interestingly, the two residues also differ between the other species compared in Figure 5. Taken together, the transitions between the two crucian carp ccNR1.1 paralogs would not likely alter the paralogs’ function.