Present in Dioscorea species, the alpha (a) class of carbonic anhydrase has been understood to demonstrate characteristics of a storage protein. The introns and exons of the carbonic anhydrase from Dioscorea macroura will be studied in order to accurately characterize the carbonic anhydrase (CAH) gene in the Dioscorea species and to deduce closer relationships between various Dioscorea species. The exons of the CAH sequence in D. macroura will be studied to notice any functional differences between the D. macroura species and the other sample species including Dioscorea cayensis, Dioscorea alata, Arabidopsis thaliana, Oryza sativa and Nicotiana langsdorffi. The introns of the genomic CAH sequence of D. macroura will be studied to make closer and more specific relationships between various Dioscorea species. It is hypothesized that the cDNA CAH sequence of D. macroura will share strong similarities with the Dioscorea cayensis and Dioscorea alata sequences found from the GenBank database. Specific active regions on the CAH sequence however, will vary from the corresponding regions in other species such as Arabidopsis and Oryza. Making deductions from the size of amplified CAH genes and from analysis of introns in sequences already on databases, it is expected that the introns will reveal information about closely related species and will better place D. macroura into the Dioscorea phylogenetic tree.

Prior to analysis, several necessary procedures were conducted. DNA was extracted from Dioscorea macroura and then amplified in order to isolate the genomic CAH gene. To receive a clean genomic sequence of the CAH gene, the DNA was cloned using an Invitrogen TA Cloning Kit. Samples of D. macroura cDNA were also used to amplify the CAH gene, which included exons only. Gel Electrophoresis was conducted with the amplified cDNA, which was then extracted from the gel. The cDNA of the D. macroura CAH gene was sent to the SUNY Stony Brook DNA Sequencing Facility and was sequenced in the forward and reverse directions. Gel Electrophoresis was conducted with the cloned DNA as well and will be sent to the Stony Brook Sequencing Facility for sequencing. As noticed from the gel electrophoresis, the cDNA CAH gene, containing only exons, ran from 750 to 850 base pairs, while the genomic DNA CAH gene, containing both introns and exons, ran from 1200 to 1500 base pairs. Once both sequences are obtained from the facility, they will be aligned and compared to each other to decipher the positions, lengths, and nucleotide sequences of the introns and exons of the D. macroura CAH gene. The genomic and mRNA sequences of the CAH gene found in the Arabidopsis thaliana, Oryza sativa and Nicotiana langsdorffi species were obtained from GenBank files and will be aligned and compared with the D. macroura sequences to derive differences and similarities in exon coding. This study is supported by the SIMONS Summer Fellowship Program and the Department of Ecology and Evolution at SUNY Stony Brook.