THE USE OF ARBITRARY PRIMERS AND THE RADES METHOD FOR THE RAPID IDENTIFICATION OF DEVELOPMENTALLY-REGULATED GENES IN TRYPANOSOMES

Biological processes, such as the cell-division cycle, differentiation and development, are driven by changes in gene expression. Short olig odeoxyribonucleotide primers (10-mers) of arbitrary sequence are curre ntly used in the polymerase chain reaction (PCR) to generate genomic f ingerprints (RAPDs) for the characterisation and differentiation of or ganisms and for mapping loci of interest. Since the products of such r eactions are generally less than I kb in size, the use of arbitrary pr imers on cDNA should generate RAPDs which are characteristic of expres sed genes. To assess this possibility, two model systems were employed ; one in which actively dividing Trypanosoma brucei brucei bloodstream forms differentiate to non-dividing forms, and the second in which no n-dividing metacyclic forms of T. congolense differentiate to actively dividing bloodstream forms. In the technique herein, mRNA from each d ifferentiated form was reverse transcribed into cDNA which was then us ed as the template in the PCR. The resultant products were examined by agarose-gel electrophoresis. As few as 10(3) trypanosomes were suffic ient for the generation of a RAPD print after first amplifying the tot al cDNA through exploitation of the fixed 3' and 5' ends of trypanosom e nuclear mRNAs. Differences in RAPD patterns between the differentiat ed forms examined were mainly due to differences in gene expression. T he technique can rapidly identify genes expressed at very low levels a nd which are up- or down-regulated in the different forms examined. PC R products of interest are easily purified from the agarose gels for d irect cloning and complete sequence determination due to their relativ ely small size (0.1-1 kb).