ANALYSIS OF SEQUENCE HOMOGENIZATION IN RDNA ARRAYS OF HAEMONCHUS-CONTORTUS BY DENATURING GRADIENT GEL-ELECTROPHORESIS

Testing different theories of concerted evolution experimentally has b een hampered mainly due to the lack of appropriate model systems and t echnical limitations. In this study, we employed a denaturing gradient gel electrophoresis (DGGE) approach for the display and definition of nucleotide variations in the second internal transcribed spacer (ITS- 2) of ribosomal DNA (rDNA) of the parasitic nematode, Haemonchus conto rtus. The ITS-2 was amplified from individual adult nematodes by PCR a nd subjected to DGGE. Of the 94 individuals (representing nine differe nt populations) analysed, 13 different DGGE profiles were displayed. E ighteen bands representing those profiles were excised and sequenced. Sequencing defined 13 different types of ITS-2 with 12 nucleotide vari ations (4 transitions, 5 transversions, 1 insertion and 2 deletions) w hich could be related to particular positions of the predicted seconda ry structure for the ITS-2 pre-rRNA. The results showed that individua ls of interbreeding populations of H. contortus can have rDNA arrays t hat are partially or fully homogenised for different sequence variants (despite interindividual variation), suggesting that the homogenisati on process is driven mainly by intrachromosomal exchange. The findings also demonstrated the capacity of the DGGE-sequencing strategy to qua ntify the frequency of ITS-2 sequence types within individual nematode s from different populations without the need for cloning or Southern blot procedures. This has important implications for studying the mech anisms of sequence homogenisation in rDNA and pre-rRNA processing as w ell as for elucidating speciation events and population differentation at the molecular level.