Elisabetta Dore

Generation of chromosome size polymorphism during in vivo mitotic multiplication of Plasmodium berghei involves both loss and addition of subtelomeric repeat sequences.

Extensive chromosome size polymorphism arises in Plasmodium berghei during in vivo mitotic multiplication. Size differences between homologous chromosomes involve rearrangements occurring in the subtelomeric portions while internal chromosomal regions do not contribute significantly to chromosome size polymorphism. Differences in the copy number of a 2.3-kb subtelomeric repeated unit are shown to correlate with size variations, and in at least one case to account completely for the size difference between two variants of the same chromosome.

Organization of subtelomeric repeats in Plasmodium berghei.

Several (but not all) Plasmodium berghei chromosomes bear in the subtelomeric position a cluster of 2.3-kilobase (kb) tandem repeats. The 2.3-kb unit contains 160 base pairs of telomeric sequence. The resulting subtelomeric structure is one in which stretches of telomeric sequences are periodically spaced by a 2.1-kb reiterated sequence. This periodic organization of internal telomeric sequences might be related to chromosome-size polymorphisms involving the loss or addition of subtelomeric 2.3-kb units.

Telomeric motifs are present in a highly repetitive element in the Plasmodium berghei genome

Using as probes the subfragments of the telomeric sequence previously cloned by us from Plasmodium berghei DNA, we identified and cloned a 2.3 kb repeat, largely overlapping the original telomeric insert. Restriction mapping indicated that cloned inserts (2.3 kb in length) represented circularly permutated versions of a rather well conserved repeated element, at least in part organized in tandem. The 2.3 kb repeat family with a copy number of about 300 occupies about 4% of the whole genome. The copies are unevenly distributed among the chromosome-sized molecules revealed by pulsed field gradient electrophoresis. Complete sequence determination of the 2.3 kb element revealed that telomere-related motifs are present with a characteristic pattern in a set of tandem repeats, 27 bp long. The perfect conservation of these motifs as well as the pattern of chromosomal distribution suggest that we are dealing with a specialised structure subject to selective mechanisms of amplification and maintenance.

Further studies and electron microscopic characterization of Plasmodium berghei DNA.

The average length and the interspersion pattern of repetitive DNA sequences in the Plasmodium berghei genome have been studied by electron microscopy. Within the limitations posed by the relatively high genome complexity, analysis of partially renatured total DNA indicates that repetitive sequences do not occupy preferential positions along the genome, but are widely dispersed (one in approx. 8000 base pairs of unique DNA). Structures appearing as loops flanked by inverted repeats are present. Analysis of the repetitive fraction purified by hydroxyapatite chromatography shows that the average length of rapidly reassociating repetitive structures is around 800 base pairs with 90% of the length distribution between 400 and 1400 base pairs. Suitable extraction methods, preserving circularity of extrachromosomal DNA components, allow the detection of molecules which can be identified as mitochondrial DNA, 10.5 +/- 0.4 microns long.

Long insertions within telomeres contribute to chromosome size polymorphism in Plasmodium berghei.

During prolonged in vivo mitotic multiplication of a Plasmodium berghei ANKA clone (8417HP), parasites that contained an enlarged version of chromosome 4 were observed. Restriction mapping and hybridization results demonstrated that the extra DNA present in the enlarged chromosome consists of 2.3-kb tandem repeats, known to be normally located in subtelomeric position at several chromosomal ends but absent in the original chromosome. The inserted 2.3-kb units appeared to interrupt one of the original telomeres and to create an internal (approximately 1-kb-long) telomeric sequence.

Homologous telomeric sequences are present in different species of the genus Plasmodium

The telomeric sequence cloned from Plasmodium berghei (see M. Ponzi et al. (1985) EMBO J. 4, 2991-2995) was tested for species specificity. A telomeric and a subtelomeric fragment of the cloned insert served as separate, labelled probes on pulsed field gradient electrophoretical patterns and on genomic digests from the rodent malarias Plasmodium yoelii, Plasmodium chabaudi and from the human malaria Plasmodium falciparum. Results indicate that the subtelomeric fragment, abundantly represented in two chromosomes of P. berghei, is not present in the other DNA tested, while the telomeric fragment is present in every chromosome-sized molecule in all the species tested. The telomeric location in the other genomes of the sequences homologous to the P. berghei telomeric probe is confirmed by experiments with Bal 31 exonuclease. In all cases, the TaqI site appears to delimit the common telomeric portion.

Re-examination of earlier work on repetitive DNA and mosquito infectivity in rodent malaria

Previous results, relating mosquito infectivity to percentage of repetitive DNA in the genome of Plasmodia, are re-examined in the light of the finding that a parasite line used in the previous studies and classified as Plasmodium berghei NK65, was a mixed infection, where the major component appeared to be Plasmodium yoelii. This conclusion was reached through cloning and isoenzyme typing of different clones. Isoenzyme typing alone is not sufficiently sensitive to reveal contamination amounting to less than 20% in a mixture. Attention is drawn to the risk inherent in work with uncloned lines, where the proportions of species or sub-species present may vary according to line history and gametocyte viability.

Dynamics of telomere turnover inPlasmodium berghei

Non-uniform composition in telomeric repeats at the extremities ofPlasmodium chromosomes was exploited in order to obtain data on intraclonal diversification of telomeric sequences, relevant for the study of telomere regeneration dynamics. Families of sibling telomeric clones were obtained from several chromosomal ends ofPlasmodium berghei, and analysed so as to determine the exact points from which individual clones start to diverge. As much as 90% of the telomeric tract appears to be subject to events causing abrupt changes in the sequence of telomeric repeats. The results are compatible with the hypothesis that breakpoint probability is a continuously increasing function over the entire telomeric tract.

A site of intrinsic bending in a highly repeated element of Plasmodium berghei genome

The basic element of the 2.3 kb repetitive family, present in approximately 300 copies in the Plasmodium berghei genome, contains a bent DNA region. Indications of this given by anomalies in electrophoretic behaviour were confirmed by computational analysis of sequence data.

Presence of contaminating mitochondrial DNA from host reticulocytes in experimental infections of Plasmodium berghei

Superposition of two unrelated processes, namely terminal reticulocyte differentiation and synchronous plasmodial development, takes place in experimental infections of Plasmodium berghei. The first process is shown to be responsible for the appearance of some discrete restriction bands of host origin when DNA is extracted from leucocyte-free blood containing synchronous parasites at early stages of infection. These discrete DNA fragments cross-hybridize with host cell mitochondrial DNA. Purification steps are suggested to reduce this effect, which might be relevant also in the case of other plasmodial species exhibiting preference for reticulocytes as host cell.