Molecular cloning and characterization of horse DQA cDNA.

The research paper describes the successful cloning and sequencing of a specific antigen, horse Mhc class II DQA, using a lymphocyte-derived cDNA library. The aim was to compare the sequence similarity rate of this antigen with sequences from other species.

Methodology and Techniques Used

• The researchers began with the construction of a cDNA library sourced from a specific type of horse, the Eqca class I A2 homozygous stallion.
• The constructed library was cloned into a pcDNA I vector and screened following the methods laid out by Twomey and Krawetz in 1990.
• To screen the library, a probe was created by amplifying the second exon of the DQA gene, pulled from a heavily diluted portion of the cDNA library itself.
• The researchers designed primers based on conserved regions common to mammalian species. These specific sequences are listed in the paper.
• Clones from the library were then sequenced using vector primers, T7 and SP6.
• The end of these sequences presented the opportunity for the researchers to design and use new primers in a polymerase chain reaction.
• The resulting fragments were cloned into another system, the pT7Blue T-Vector, and both strands were sequenced once more, this time with vector primers T7 and U19.

Discoveries and Results

• The research team identified the Eqca-DQA cDNA clone to be 1115 basepairs in length.
• They predicted that the protein it encodes is made up of 232 amino acids, starting from the first residue of the cd domain.
• The sequence revealed that the signal peptide is composed of 23 residues and corresponds with those found in other mammalian species.
• The signal and mature peptides showed high sequence similarity when compared to identical sections in sequences from other species. They found 85% similarity with human sequences, 83% with sheep, 84% with pig, 80% with mouse, and 82% with cattle sequences.
• The sequence similarity with the horse DRA locus was much less, coming to only 51%.