Structural alterations of chromosome 2 in Leishmania major as evidence for diploidy, including spontaneous amplification of the mini-exon array

We have utilized pulsed field electrophoresis to characterize several karyotypic alterations in Leishmania major. Promastigotes of the LT252 line contain three small chromosomes, of 300, 350 and 385 kb. Quantitative densitometry of ethidium bromide-stained gels suggest that these chromosomes are present in equal levels (2:2:2). Two derivatives of this line, one appearing spontaneously (LT252 delta) and one obtained following selection with methotrexate (11-MTXR20), exhibit altered levels of these chromosomes, in the ratio of 2:1:3, respectively. The variant pattern in both lines is due to an increase in size of chromosome 2, yielding a new chromosome similar in size to chromosome 3. The enlarged chromosome 2 of the LT252 delta line is a result of amplification of the mini-exon gene array normally located on this chromosome, which increases from about 93 to 150 copies of the 0.44-kb mini-exon tandem repeat, as shown by quantitative hybridization and sizing of the mini-exon array. In contrast, the increased size of chromosome 2 within the methotrexate-resistant mutant 11-MTXR20 is not due to mini-exon amplification. In both variant lines, there are equal levels of the wild-type and enlarged chromosome 2, and the wild-type chromosome 2 is now present at 50% of the level of chromosome 1. These and other data suggest that Leishmania is diploid for chromosomes bearing housekeeping genes such as the mini-exon locus.