DNA Molecular mass marker= Sacc/7aromyces cerevisiae strain YNN295.
A
b
e
Sc YM
DNA Mr Mass
(kbp)
1600/2200 -
1140-
980.
Chromosome
band No.
Chromosomeband No.
1 7+
YM
Chromosome
band No.
Sc YM
11 10 9 81 7IDNA Mr Mass
(kbp)
2200
/1600
.1140-
Chromosome
band No.
have accurately determined the sizes of the chromosomes of each and have confirmed that the DNAs are of similar size and have equivalent mobilities.
3.1.2 Resolution of the smaller chromosomes using field inversion gel electrophoresis (FIGE)
Using a program designed to resolve DNA molecules of between 0.8 and I.SMbp (kindly suggested by D. Williamson, NIMR), the same seven bands produced using TAFE could be separated using FIGE (15s forward, 5s reverse, 19hrs, 200V, 100mA, 1% Geneline agarose) in less than half the time (see Figure 3.1.1, b). Although there was no improvement in the resolution of the genome, far greater quantities of DNA could be resolved in each lane of the FIGE gels as compared to the TAFE gels, a property which was found to be extremely useful for the work described in the following chapter. Under the conditions used the chromosomes migrated a distance which was a direct function of their size, and so the order of the
chromosomal bands was reversed, with the smaller bands ending the run closest to the well. The yeast size standards could not be used on these gels because the transition size (of approximately 0.4 - 0.5Mbp) fell within the range of their molecular sizes, with the result that at the end of the program, the medium sized chromosomes were closest to the well.
The genomic DNA bands resolved using TAFE and CHEF systems were numbered from one to seven in ascending order of size.
3.1.3 Contour-clamped homogeneous electric field (CHEF) electrophoresis Generally the separations produced using CHEFs were less reproducible than those produced by FIGE and TAFE, but when it worked well, this system could be used to separate the largest P. yoelii chromosomes. The program which resolved the genome most successfully (6min pulses, 72hr, 12min pulses, 24hrs, 100V, 100mA 1% Biorad Chromosomal Grade agarose) permitted the resolution of nine distinct bands (See Figure 3.1.1, panel c). Examination of CHEF and TAFE/FIGE separations and comparison with the yeast size-standards, allowed common bands to be identified. The smaller chromosomal bands were less well resolved by this CHEF protocol, and TAFE/FIGE bands 1 and 2 comigrated, as did bands 3 and 4. Band 5 ran separately, just distinct from band 6. Band 6 was not further resolved and remained refractory to separation when subjected to a range of different electrophoresis conditions and so the chromosomes within this band appear to have identical electrophoretic motilities.
Figure 3.1.2
Diagram o f the P. yoe//7 YM chrom osom al separations using the tw o differen t pulsed-field gel electrophoresis system s
Th e bands have been labelled In ascending ord er o f size 1) Separated using FIGE
2) Separated using CHEF
11 10 9 8 1) 7+ 2) 4,5 3 1,2
Band 7, the largest band seen on the TAFE/FIGE gels was resolved into five bands, which were numbered (from seven to eleven, in ascending order of size). These bands appeared to be of equal intensity, except for bands 9 and 11 which were approximately twice the intensity of the other large chromosomal DNA bands.
The largest S. ceævisiae (strain YP148) chromosome is 2.2Mbp, and this was the only marker DNA band which ran in the vicinity of the four largest P. yoelii
chromosomal DNA bands. PFGE gels do not tend to result in the distance migrated by a species being a direct function of its length. Different pulse times tend to open out different regions of a particular karyotype. For these reasons, the sizes of the large P. yoelii DNA bands could only be estimated. The bands were judged to be,
approximately, band 7 , 1.75Mbp; band 8, I.SOMbp; band 9, 2.0Mbp; and band 10, 2.1Mbp (see Figure 3.1.1, panels c and d). The size of band 11 could not be
determined because it ran clearly above all of the yeast markers indicating a size well in excess of 2.2Mbp. Accurate sizing of the larger chromosomes in other species has only been achieved by physical mapping, following restriction enzyme digestion.
The genetic material of P. yoelii is therefore distributed among at least eleven chromosomes, ranging in size from 0.8 to more than 2.2Mbp. Based on the density of staining of the chromosomes, bands 9 and 11 appear to be doublets, and band 6 a doublet, or possibly a triplet. This indicates that P. yoelii could have a chromosome number of 13 -15. For P. yoelii, and other rodent malarias in general, estimations of the chromosome number performed by physical means, such as the counting of the kinetochore bodies present in cells at metaphase, as Prensier and Slomianny published for P. falciparum (1986) have not been attempted. The bands seen following CHEF and TAFE/FIGE separations, and the numbering system used to identify each band, have been drawn out on a schematic (Figure 3.1.2).
3.2 Comparison of the chromosomes of P. yoelii with those of other Plasmodia
Samples of PFGE blocks containing the chromosomes of other Plasmodia, were obtained from colleagues in other laboratories, and run alongside P. yoelii chromosomes in a CHEF gel (see Figure 3.2).
The smallest chromosomes of P. yoelii appear to migrate at a significantly slower rate than the smallest chromosomes of other Plasmodia for which karyotypes have been published (e.g. P. falciparum', Kemp etal., 1992, P. chabaudf, Langsley et al., 1987, P. berghef, Janse etal., 1989), which are in all cases around O.GMbp. When