RNA Polymerase II
Pol II refers to the protein coding gene which codes for the two largest subunits of the RNA Polymerase II enzyme. RNA polymerase II is involved in the synthesis of pre-mRNA (Shultz and Regier 2000, Liu et al. 1999). It is a single copy gene in fungi, though paralogous copies have been discovered in plants and trypanosomes. It is not entirely clear whether Pol II is a single copy or multiple copy gene in insects, though our lab has not encountered any evidence of paralogous copies in bees. Pol II is easy to amplify and align and contains several highly conserved regions which simplifies primer design (Liu et al. 1999).
The three-dimensional structure of Pol II has been described in Cramer et al. (2001), and there are already a substantial number of sequences available for this gene (Shultz & Regier 2000). The fragment that we have been able to amplify comes from the RPB2 gene, which encodes the largest of the Pol II subunits, and is involved in the catalyzation of the elongation reaction in mRNA transcription (Liu et al. 1999). The function of the RNA polymerases are common among all eukaryotic organisms (Sidow & Thomas 1994). Shultz and Regier (2000) demonstrated the utility of Pol II in resolving higher-level arthropod phylogeny, and Liu et al. (1999) showed that intron insertion/deletions occur within the Ascomycetes.
Using the recently published sequence of the honey bee genome, we located the Pol II homolog by blasting with partial insect Pol II sequences (from Shultz & Regier 2000) . Using this alignment we developed primers for amplifying and sequencing an approximately 2000 bp fragment of Pol II gene in bees (see pdf file below). Based on preliminary tests, these primers work on a broad survey of bees.
Pol II is probably the slowest single copy, protein-coding, nuclear gene that we have surveyed. Pol II evolves at approximately four times the rate of nuclear 18S ribosomal sequences (Sidow & Thomas 1994) but considerably slower than the other protein-coding genes described above. It is also more phylogenetically informative, recovering more lineages in Metazoa than 18S alone (Sidow & Thomas 1994). We are currently assessing the utility of Pol II for family level bee phylogeny using the primers listed on the attached pdf file.
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Gene Information: |
RNA Polymerase II primer information (.pdf file) |
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