Microcontrolador

i-7v \/KA Chromosome

. Chromosome

^ ^ ^ L ^ b a n d no. (YM)

i7X YM

(YM)

7

6

5

If

h -

10

9

8

7

Well

studies presented in Section 3.4 of Chapter 3 indicated that bands 8, 9 and 10 of YM each contained single chromosomes (band 8, chromosome 12, band 9 chromosome 10, and band 10, chromosome 11). The 17X band which comigrated with YM band 10 may contain all of the three chromosomes within YM bands 8, 9 and 10. The most slowly migrating band of DNA in 17X, stained with an intensity which indicated that it was a doublet. In P. yoelii YM (and all strains of Plasmodia examined to date) this band contains chromosomes 13 and 14, and it seems likely that this was also the case in 17X. Because of the different distribution of the DNA bands of what was assumed to be an identical number of chromosomes in the two strains, it was

considered possible that the comigration of what appeared to be three chromosomes in strain 17X just in front of the most slowly migrating band of DNA in 17X could have affected the way this band migrated in 17X. However in less complete separations of the YM and 17X genomes when all of the bands which migrated in front of the slowest band co-migrated in both species, the largest 17X band still migrated less far than the most slowly migrating YM band indicating that there did seem to be a real difference in size. It is likely that the chromosome number will be fourteen in strain 17X also,

because this seems to be so in YM, and all other malaria parasites (see Chapter 3). Considering the size distribution of the chromosomes, although there are differences among some of the lager chromosomes, generally the karyotypes of P. yoelii YM and 17X are very conserved and are considerably larger than those of the other rodent malaria parasites (refer to Figure 3.2, which shows the resolved chromosomes of both P. yoelii strains and those of other rodent malaria parasites).

Thus it appears that although the karyotypes of the two parasites are generally very similar, on the basis of the comparison of the stained resolved DNAs of the two strains, at least four chromosomes (11, 12, 13 and 14) appear to have different electrophoretic mobilities in strain 17X, compared with strain YM.

The identical appearance of the small chromosomes, and the similar

appearance of the larger chromosomes supports the hypothesis that clones YM and 17X were very closely related.

The basis of the size differences cannot be determined from merely examining the resolved chromosomes of the two parasites, because without the identification of the individual chromosomes within bands, it is impossible to know which

chromosomes vary between the two strains. Hybridisation of the resolved 17X DNA with the specific DNA probes used for the identification of individual YM chromosomes is presented in Section 6.2.

6.2 Identification of P. yoelii 17X chromosomes using specific probes The DNA probes which were used to identify the smallest P. yoelii YM chromosomes hybridised to the corresponding bands in 17X indicating that the organisation of chromosomes 1 to 7 was identical in the two species. (Data not shown).

Please refer to Chapter 3 for sources for the probes used to identify individual chromosomes within the resolved PFGE DNA bands, following Southern hybridisation with each of the specific DNA probes.

Characterisation of the larger chromosomes of P. yoelii 17X with specific probes could not be completed because in addition to the fact that no probe

hybridised with chromosome 9 of YM, the chromosome 12 probe (SSUribo) did not hybridise to the only available blot containing well resolved 17X large chromosomes (probably because this blot had been repeatedly hybridised to other probes in the course of this work), and it was not possible to prepare another blot in the time remaining.

In work performed for Chapter 7, three independent chromosome 13/14 probes which had all hybridised with the most slowly migrating band of P. yoelii YM DNA were shown to also hybridise with the most slowly migrating band in 17X. See Figure 7.1, panels c, d, e, f. This result indicates that these two bands appear to contain equivalent chromosomes in the two strains. The chromosome 8 specific probe, MSP1, which hybridised to band 7 in YM also hybridised to a band which ran at almost the same position in 17X (See Figure 6.2, panels a and c). The DNA probe 9.11 which hybridised to band 9 (chromosome 10) in YM hybridised with the second slowest migrating band of 17X DNA which ran above the band 9 in YM (See Figure 6.2, panels d and e). The probe 9.2 which hybridised with band 10 (chromosome 11) in YM also hybridised with the second most slowly migrating band of 17X DNA, which on the blot used ran below the equivalent band of YM, but because the separation of the large chromosomes was not complete on the blot used this result was not very clear. From these results, it appeared that chromosome 8 in strains 17X and YM was of similar size. Chromosome 11 was smaller in strain 17X than in YM. Because there are no suitable chromosomal size markers to allow estimation of the sizes of the large chromosomes of malarial parasites, it is difficult to judge the magnitude of the

variations in chromosome size. In Chapter 3 the sizes of the YM chromosomal DNA bands were estimated to be; band 8, 1.75Mbp; band 9, 2.0Mbp; and band 10,

Figure 6.2

Southern blot of P. yoelii VIA and 17X resolved chromosomes,

hybridised with chromosome specific probes