To examine whether mating can occur within as well as between clones of Trypanosoma brucei, we transformed three T. brucei subspecies stocks with heterologous genes conferring resistance to either hygromycin or Geneticin and carried out a series of inter-and intraclone matings in all possible double drug combinations. Double drug-resistant hybrids were recovered from three of the six out-crosses, but not from any of the three intraclone matings. However, further analysis of cloned progeny trypanosomes from one of the out-crosses using RFLP markers, molecular karyotyping and RAPD (random amplification of polymorphic DNA) produced unequivocal evidence that intraas well as interclone mating had occurred. The progeny of interclone mating were double drug-resistant and heterozygous at 9 of 13 loci examined. In contrast, the progeny of intraclone mating had no demonstrable input of genetic material from the hygromycin-resistant parent and were similar to the Geneticin-resistant parent for most markers, except for five loci which were heterozygous in the Geneticin-resistant parent but homozygous in these clones (aldolase, THTl glucose transporter, procyclin, tubulin and cDNA 23). In addition, PFGE showed considerable karyotypic rearrangements in these clones and loss of genetic material was evident from RAPD and VSG (variant surface glycoprotein) gene fingerprint analysis. We conclude that intraclone mating can occur in trypanosomes, but only during out-crossing, suggesting that meiosis and/or fusion are triggered by a diffusible factor.

In the order Kinetoplastida, genetic exchange has been demonstrated only in the genus Trypanosoma. Analysis of kinetoplast DNA (kDNA) in genetic crosses of T. brucei has shown that whereas maxicircles are inherited uniparentally, minicircles are inherited from both parents. This result was confirmed for a new cross of T. b. brucei and T. b. rhodesiense by restriction enzyme digestion and Southern analysis of purified kDNA. By hybridisation with small minicircle-derived probes, we could demonstrate the presence of particular parental minicircles in the kDNA of hybrid progeny clones. All hybrid clones had inherited two minicircles from one parent despite two of the four clones having maxicircles from the other parent. The results suggest that rather than small-scale exchange of minicircles between parental networks, gross breakdown and reassembly of the minicircle network occurs during genetic exchange.