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 oligodeoxyribonucleotide primers (10-mers) of arbitrary sequence are currently used in the polymerase chain reaction (PCR) to generate genomic fingerprints (RAPDs) for the characterisation and differentiation of organisms and for mapping loci of interest. Since the products of such reactions are generally less than 1 kb in size, the use of arbitrary primers on cDNA should generate RAPDs which are characteristic of expressed 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 non-dividing metacyclic forms of T. congolense differentiate to actively dividing bloodstream forms. In the technique herein, mRNA from each differentiated form was reverse transcribed into cDNA which was then used as the template in the PCR. The resultant products were examined by agarose-gel electrophoresis. As few as 10(3) trypanosomes were sufficient for the generation of a RAPD print after first amplifying the total cDNA through exploitation of the fixed 3' and 5' ends of trypanosome nuclear mRNAs. Differences in RAPD patterns between the differentiated forms examined were mainly due to differences in gene expression. The technique can rapidly identify genes expressed at very low levels and which are up- or down-regulated in the different forms examined. PCR products of interest are easily purified from the agarose gels for direct cloning and complete sequence determination due to their relatively small size (0.1-1 kb).
Murphy
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International Laboratory for Research on Animal Diseases (ILRAD), Nairobi, Kenya.
Pellé
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D000818:Animals; D001483:Base Sequence; D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis; D016054:DNA, Protozoan / Q000096:biosynthesis / Q000302:isolation & purification; D015870:Gene Expression / Q000235:genetics; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D009024:Morphogenesis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000096:biosynthesis; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000254:growth & development
D017931:DNA Primers; D018076:DNA, Complementary; D016054:DNA, Protozoan; D012333:RNA, Messenger