2 MARCO TEORICO
2.1 DIRECCIONAMIENTO ESTRATÉGICO
compared with other malaria chromosomes, P. yoelii showed a much less even distribution of chromosome migrations, with all chromosomes migrating more slowly than the chromosomes of the other species (See Figure 3.2). The only exception is the largest band of DNA which migrates to approximately the same position as those of the other rodent malarias. In P. chabaudi, P. berghei, and P. vinckei, this band is known to be a doublet containing chromosomes 13 and 14 (Sheppard et a/., 1989). Judging by the density of ethidium bromide staining, this band is probably also a doublet in P. yoelii. Despite the use of altemate PFGE protocols designed to open up specific areas of the karyotype, resolution of additional bands was not possible, indicating that some chromosomes had identical electrophoretic motilities. The
chromosome 13/14 doublet has been estimated to be 3.5Mbp in other malaria species (Foote and Kemp, 1989), which gives an estimation of the size of this band in P. yoelii, although because of the condensation of the karyotype observed over this region, there could be differences between the sizes of the bands which are not apparent on these gels.
As stated previously, the rate at which DNA species migrate through pulsed- Ifield gels is dependent on their size and base composition. DNA with a high G + C
content migrates more rapidly than DNA with a high A + T content. In the only study in which the base composition of P. yoelii DNA was analysed, it was found to be 24% G + C, the same as the other rodent malarias (Chance et al., 1972). As stated in
j Chapter 1, these estimations have been subsequently shown to be erroneously high, and the G + C content of the DNA in these parasites appears to be around 18% (McCutchan et al., 1984). P. yoelii was not included in this later study, but the fact that it was found to be the same as the other rodent malarias by Chance et al., and also on the basis of comparison of the sequence of known genes, indicates that all rodent malarias have a similar base composition. In this case the different mobilities of the chromosomes of P. yoelii, as compared with those of the other rodent malarias is probably a result of their being larger.
All of the malarias which have been examined to date have fourteen
chromosomes (Janse et al., 1989, Kemp et al., 1992). This may also be the case for P. yoelii, but alternatively, because most chromosomes appear to be significantly larger than those of the other species, while the DNA contents of the nuclei of each species appear to be similar (Weber, 1988) the chromosome number for P. yoelii, may be less than fourteen. Until a comprehensive study examining the number of genetic linkage groups is published for this species, or a kinetochore count is performed, or the chromosome number is determined by other means, it cannot be
assumed that the chromosome number for P. yoelii, will be the same as for the other Plasmodia.
P. yoelii is believed to be a close relative of P. berghei, and was originally classified as a subspecies of this species (Landau and Chabaud, 1965), but it can be seen that the karyotypes of these parasites are quite different. Whether this is an indication that the evolutionary distance between these two parasites is greater than has been previously believed remains to be investigated.
3.3 Chromosome quantitation
One method of quantifying the chromosome number of an organism is to restrict genomic DNA and examine the number of fragments containing telomere repeat sequences. The reiterated telomere repeat sequence is refractory to digestion by all known restriction enzymes, and so each chromosome will give rise to two telomere-containing sequences following restriction. This technique was first applied to P. falciparum (Biggs et al., 1989), by using the restriction enzyme EcoRI to digest separated chromosomes, which were then electrophoresed in standard submarine gels, transferred to membranes and hybridised with a telomeric repeat probe. This two-dimensional approach was developed to avoid the superimposition of similarly sized fragments arising from different chromosomes. For P. falciparum chromosomes the telomeres are bounded by repetitive regions refractory to restriction, with adjacent subtelomeric regions containing interspersed recognition sites for EcoRI and other enzymes (Dore etal., 1986). Two differently sized fragments, both under ISkbp, were observed for all P. falciparum chromosomes, giving a maximal size for the telomeres. Dore et al., (1990) compared the DNAs of P. falciparum, P. chabaudi and P. yoelii digested with three different restriction enzymes (Haelll, H/ndlll, and EcoRI) and concluded that the number of telomere repeats added to each chromosome end was much more variable for P. yoelii than for the other species, so much so that when the P. yoelii DNA was restricted with EcoRI as described by Biggs et al., (1986), only smears resulted on the telomeric probe autoradiographs. In an attempt to circumvent this problem, infrequently cutting enzymes were selected in this study.
3.3.1 Hybridisation of telomeric probe P.tell to Apa\ restricted genomic DNA In one-dimensional TAFE test blots, Apal (recognition site GGGCCC) produced telomere-containing fragments of SOkbp - I.SMbp. Under the PFGE
conditions used to separate DNAs in this range, fragments differing by several tens of kilobase pairs comigrate, the effect of large variations in telomere length is minimised,
and the telomere fragments form discrete bands. When a Southern blot of P. yoelii genomic DNA that had been restricted with Apal and resolved using TAFE, was hybridised with the telomeric probe P.teH (for the origin of this probe see Table 3.1), a series of discrete fragments, ranging in size from 10 to 1 SOkbp could be observed (data not shown). The presence of these bands indicated that if the same technique were to be applied to resolved chromosomes, then two discrete telomere-containing fragments might be distinguished from each chromosome.
3.3.2 Hybridisation of P.tell to Apal-digested FIGE-resolved P. yoelii YM chromosome fragments, resolved in the second dimension by CHEF
A strip of a FIGE gel in which the six smallest P. yoeiii DNA bands were well resolved, was restricted with Apal and then set across the top of a second (CHEF) gel, which was used to resolve the Apal restriction fragments from each band. A CHEF gel was used for the second dimension because TAFE gels were not wide enough to permit inclusion of all of the resolved chromosomes at once. After transfer of this gel to nitrocellulose, the blot was hybridised with the telomeric probe. Following autoradiography, two clear telomere signals could be seen from each of bands 1-5, indicating that each must have contained a single chromosome (see Figure 3.3.2). Four spots could be distinguished from band 6 (which must therefore contain at least two chromosomes). Seven clear bands were derived from band 7+ (indicating a minimum of four chromosomes). One of the spots derived from bands 7+ was much more intense, which was probably the result of superimposition of more than one band. When the chromosomes within band 7+ had been resolved using CHEF, five bands were seen (see above), and so this result was not surprising. Thus P. yoelii must have a minimum of twelve chromosomes. Weaker hybridisation signals arising from each band may be due to the presence of telomere-related sequences present internally within the chromosomes. Such sequences have been implicated in
homologous recombination events, one of the mechanisms used by Plasmodia for the generation of genetic diversity (Janse, 1993).