Date of Award
Master of Arts (MA)
Dr. A. Thomas Weber
Previous studies in Dictyostelium mucoroides have sought to identify genes specific for macrocyst development. A gene named A-11, previously isolated as a cDNA, appears to be one of these genes. The cDNA of A-11 was made from macrocyst (8h) mRNA, and Northern analysis did not detect any A-11 transcript during sorocarp formation. The cDNA sequence for A-11 has been obtained. To determine if the cDNA of A-11 is complete, primer extension and RNA sequencing were performed using an end-labeled oligonucleotide complementary to positions 1221 to 1239 of A-11 cDNA. The two longest extension products were 102 bases in 101 bases. As there were only 84 bases from the 19-mer oligonucleotide primer to the 5’-end of A-11 cDNA, this indicated that the A-11 cDNA was not a complete reverse transcript of A-11 mRNA. It was short of full length by 18 nucleotides. RNA sequencing with dideoxynucleoside triphosphates showed the nucleotides missing from the cDNA were 5’NNTNNAACAAATAAATAA3’. There are several AUG potential translation start sites in the 5’-end of A-11. To determine which AUG was the initiation codon, hybrid-selection of A-11 mRNA from my macrocyst (4h) total mRNA and in vitro translation of A-11 mRNA were performed. The resulting translation product had a molecular weight of 25 kDa which approximated the size of the prediction protein initiated at the first AUG codon of A-11 mRNA.
Ma, Weilie, "Determination of the transcriptional start site and in vitro translation of the A-11 mRNA in Dictyostelium mucoroides." (1994). Student Work. 3305.