DISEÑO DE PLANTAS INDUSTRIALES

No.

No.

^

No.

(PyY)

(Pb)

(PyY)

9, 10. 11

1 0

12

13. 14

13. 14

9 .1 0 ,1 1 I

8

5 .6

1.2

^

##

13. 14

1 0.

12

5.6

of not only the sequence of the rhoptry protein genes, (because the hybridisations were carried out under the normal conditions of high stringency), but in their organisation, and perhaps even their high copy number.

A locus of the gene family is present on the doublet containing chromosomes 13 and 14 in P. berghei, which is also the case in the ‘wild-type’ P. yoelii strain, 17X. P. berghei is a reticulocyte-restricted parasite which has not undergone any radical phenotypic alterations like that observed for P. yoelii by Yoeli et al., (1975). Since its adaptation into laboratory mice, P berghei has been observed to cause gradually rising parasitaemia over a period of a few weeks as the host reticulocyte population increases in response to parasitisation. P. berghei infections are lethal to the mice if untreated (Landau and Boulard, 1978). P. berghei has never been reported to extensively invade mature erythrocytes. It was postulated that the rhoptry protein gene locus on chromosomes 13/14 might in some way mediate or be associated with reticulocyte-restriction.

The indication that there may be a connection between the reticulocyte- restriction and the presence of rhoptry protein gene sequence(s) on the chromosome 13/14 doublet was extremely interesting. It was decided to test for the presence of an association between the progeny of a genetic cross between P. yoelii strains YM and 17X. Because it was anticipated that a certain amount of time would be required to examine the products of the cross, work was begun on this project, before

characterisation of the gene family in 17X was completed. This work is presented in Chapter 7.

6.4 Hybridisation of two-dimensional FIGE Rsa\ Southern blot of 17X to E8 When the attempts to produce two-dimensional Southern blots using FIGE- resolved YM chromosomal DNA were finally successful they revealed a great deal of information concerning the chromosomal location and copy number of the rhoptry protein gene family in this strain. It was anticipated that the use of the same technique would also reveal a similar amount of information about strain 17X and allow a more complete comparison of the distribution of the gene family in the two parasites. However there were technical problems to be overcome in the production of 2D Southern blots with 17X. Because it was necessary to resolve the two largest bands of 17X DNA in order to examine the rhoptry protein gene locus within each separately, it was necessary first to separate the chromosomes using a CHEF system, before they were digested. As in Chapter 3, when a CHEF 2D blot was made for YM in order to examine the distribution of larger chromosomes, problems were encountered due

to the lack of reproducibility of CHEF separations. When suitable gels were produced, the amount of DNA that could be digested was very low because CHEF gels do not resolve well heavily loaded samples. Gel strips wider than 5mm could not be digested because wider strips did not allow adequate penetration of the restriction enzyme and resulted in partially digested products being present on the final Southern blots.

Because there was much less DNA present on a typical 2D blot produced using CHEF in the first dimension rather than FIGE, the signals seen on these Southern blots were correspondingly lower, times of exposure to film increased, and as a result, it was much more difficult to detect weaker signals above background. Because of these technical constraints only one successful 2D Southem blot was produced using 17X DNA (using the restriction enzyme Rsal). Other blots which were attempted using the enzymes EcoRI and Apa\, in order to allow a fuller comparative study of the

organisation of the gene family in the two parasites, were not successful, and the work could not be completed during the time-frame of this project.

An autoradiograph of this CHEF-Rsal 2D Southern blot of strain 17X, hybridised with E8 is shown in Figure 6.4. Although the signal was comparatively weak and many of the less intense bands were not seen, all of the major bands detected on the FIGE-2D Southern blots of YM DNA could be detected on this blot. In addition to the major spots recognised from the YM blot, three additional spots of similar intensity could also be distinguished. These spots have been marked with arrows on the photograph of the autoradiograph. Two of these spots (of 1.2 and 3.5kbp), were derived from the most slowly migrating band of DNA (containing chromosomes 13 and 14), and one (of 3.0kbp) was from the second most slowly migrating band of DNA (containing chromosomes 9,10, 11, and 12). The two novel fragments derived from the most slowly migrating band of DNA could represent two different regions of the same gene, or could represent shorter, independent, rhoptry protein related sequences, perhaps present on different chromosomes. The 3.0kbp novel spot derived from the second slowest migrating band of 17X DNA may

represent sequence from an incomplete gene copy, or may be associated with other sequences which comigrate with other bands, and so cannot be distinguished. Similar sized spots to these novel species could not be identified at other locations in YM. The spots of hybridisation derived from 17X DNA band 6 (which contains two

chromosomes 5, and 6), did not all align directly above one another, but showed the same distribution into either the front or the rear of the original doublet band, as in YM,

Figure 6.4