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Olfactory receptor (OR) genes constitute the molecular basis for the sense of …

Biology Articles » Zoology » Primatology » Loss of Olfactory Receptor Genes Coincides with the Acquisition of Full Trichromatic Vision in Primates » Materials and Methods

Materials and Methods
- Loss of Olfactory Receptor Genes Coincides with the Acquisition of Full Trichromatic Vision in Primates

Design and test of degenerate primers. OR genes have a coding region that is approximately 1 kb long and contains no introns. In order to test the performance of degenerate primers, we sequenced 30 genes amplified with each primer pair in human and mouse and compared the composition of the different OR families in the sample to that of the full OR gene repertoire of these two species (Glusman et al. 2001; Zhang and Firestein 2002). We also compared the sample estimates of the proportion of pseudogenes to the proportion in the entire OR repertoire of human and mouse. Since the degenerate primers amplify only 670 bp of the approximately 1 kb coding region of the OR gene, a subset of the coding region disruptions will fall in segments of OR genes not amplified by our primers. As a result, the true fraction of OR genes carrying coding region disruptions will be underestimated by our approach. We therefore determined the proportion of OR genes with at least one disruption within the corresponding 670 bp in the entire human and mouse OR gene repertoires (47.7% and 16.3% in humans and mouse, respectively).

We first tested an existing set of primers, used by Rouquier et al. (2000), but found significant deviations from the family composition of the full OR repertoire in both species. As an illustration, among the 60 OR genes obtained in humans, 36.6% were of the subfamily 7E (all pseudogenes), significantly more than expected given the true proportion of the 7E subfamily in the full human OR gene repertoire (12.4%, p = 2 × 10−6, assessed by FET). As a consequence of these biases, estimates of the proportion of pseudogenes in human and mouse obtained with these primers (Rouquier et al. 2000) differ significantly from the true value (p < 0.01, assessed by FET).

We proceeded by designing new pairs of degenerate primers for the OR gene family by using the program HYDEN (Fuchs et al. 2002; Linhart and Shamir 2002). The first primer pair, PC1 (PC1–5′: CTSCAYSARCCCATGTWYHWYTTBCT, PC1–3′: GTYYTSAYDCHRTARAYRAYRGGGTT), was designed based on class 1 human OR sequences only. The second primer pair, PC2 (PC2–5′: YTNCAYWCHCCHATGTAYTTYTTBCT, PC2–3′: TTYCTNARGSTRTAGATNANDGGRTT), was designed based on solely class 2 human OR sequences, excluding all genes that belong to subfamily 7E. Both primer pairs were designed to amplify a 670-bp product that approximately covers the region from transmembrane domains 2–7 of the OR protein. As a first step, we used each primer pair to amplify and sequence (see below) 30 genes from human genomic DNA. We found that PC1 primer pairs amplify OR class 1 and OR class 2 genes in roughly equal proportions. PC2 primer pairs amplified only OR class 2 genes, including members of the 7E OR subfamily. Based on the OR family composition that we observed for the 60 genes, we estimated that if we constructed a sample containing 25% of genes amplified with PC1 and 75% of genes amplified with PC2, we would obtain an unbiased representation of the familial composition of the human OR gene repertoire. This approach was validated by amplifying and examining 100 genes collected in the same way from human as well as from mouse.

PCR and DNA sequencing Each primer pair was used to amplify a set of eight reactions in each species using a temperature-gradient PCR. The use of several annealing temperatures for each species yielded a greater diversity of amplified OR genes. PCR was performed in a total volume of 25 μl, containing 0.2 μM of each deoxynucleotide (Promega, Madison, Wisconsin, United States), 50 pmol of each primer, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris (pH 8.3), 2 U of Taq DNA polymerase, and 50 ng of genomic DNA. Conditions for the PCR amplification from all species were as follows: 35 cycles of denaturation at 94°C, annealing at a gradient temperature of 48°C to 60°C, and extension at 72°C, each step for 1 min. The first step of denaturation and the last step of extension were 3 min each. The PCR products were separated and visualized in a 1% agarose gel. From each amplification set (a given primer pair in a given species), all successful products were mixed and subjected to cloning using a TA cloning kit (Boehringer, Mannheim, Germany). Cloning was followed by a touchdown PCR using the vector primers for amplifications from isolated bacterial colonies. Products were purified using the High Pure PCR Product Purification Kit (Boehringer). Sequencing reactions were performed in both directions on PCR products, using the vector primers and the dye-terminator cycle sequencing kit (Perkin Elmer, Wellesley, Massachusetts, United States) on an ABI 3700 automated sequencer (Perkin Elmer).

Sequence analysis After base calling with the ABI Analysis Software (version 3.0), the data were edited and assembled using the Sequencher program, version 4.0 (GeneCodes Corporation, Ann Arbor, Michigan, United States). Assembly of the clones was done using a similarity cutoff of 98%. This cutoff ensures that Taq-generated mutations that may have been sequenced in individual clones are not counted as independent genes. Clones that were collapsed to the same contig by the assembly process were counted as one gene. Once 25 and 75 genes (independent contigs) were identified from PC1 and PC2 primer pairs, respectively, a majority consensus was generated for each gene. In order to confirm that only OR genes were amplified from all the species, we used the consensus sequences of all genes from all species as queries in a BLAT search against the human genome sequence (http://genome.ucsc.edu/). In every case, the best hit was a human OR gene. This analysis was also used to insure that none of the genes were an artifact of (“jumping”) PCR fusion. Finally, each consensus sequence was searched for an uninterrupted open reading frame (ORF) in all six possible frames. If an uninterrupted ORF was found, the gene was annotated as intact. If no ORF was identified, the gene was annotated as a pseudogene. This approach probably results in an underestimate of the proportion of pseudogenes, as not all OR genes with an intact coding region are functional. Mutations in promoter or control regions of OR genes may lead to reduced or no expression. Similarly, radical missense mutations in highly conserved positions of the OR protein may result in dysfunction (Menashe et al. 2003). Although it is known that there are several highly conserved positions among OR genes, it is not always straightforward to ascertain which, if any, of these positions is necessary to retain function. We therefore chose the most straightforward definition of a pseudogene: a gene without a full ORF.

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