Cloning and molecular characterization of CnTEF1 which encodes translation elongation factor 1alpha in Cryptococcus neoformans.

Degenerate PCR primers were synthesized based upon known translation factor 1alpha (TEF1) sequences. Touchdown PCR with these primers utilizing Cryptococcus neoformans strain M1-106 genomic DNA as template produced a DNA fragment containing a portion of CnTEF1. This DNA fragment was used as a hybridization probe to clone a cDNA version of CnTEF1 from C. neoformans strain B3501. Comparison of the genomic and cDNA nucleotide sequences revealed the presence of six introns in CnTEF1. The nucleotide sequence of CnTEF1 from these two strains of C. neoformans were 98% identical. Codon bias for most amino acids encoded by CnTEF1 was similar to that observed in Saccharomyces cerevisiae for highly expressed genes. This codon bias was also observed in the C. neoformans ACT gene. CnTEF1 encoded a protein (CnEF-1alpha) consisting of 459 amino acids with a calculated MW of 50.3 kDa from C. neoformans strain B3501. CnTEF1 from strain M1-106 encoded a protein with one additional aa. Both C. neoformans proteins possessed a high degree of identity throughout their length to fungal, human, and plant EF-1alpha proteins.