Clara Frontali

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.

Divergence of Noncoding Sequences and of Insertions Encoding Nonglobular Domains at a Genomic Region Well Conserved in Plasmodia

Abstract. To identify conserved features in the rapidly diverging portions of a well-conserved locus, completely sequenced in Plasmodium falciparum and Plasmodium berghei, a computational method based on recurrence analysis was exploited. At the level of the genomic sequence, in both species, introns and intergenic sequences—though subject to rapid diversification—do not drift without constraints, but rather coevolve, in the sense that they maintain not only an AT-rich base composition, but also a consistent use of recurring (AT)n tracts. One of the two genes present in the conserved locus encodes a protein that exhibits blocks of high similarity to the first enzyme in glutathione biosynthesis (γ-glutamylcysteine synthetase) but bears long low-complexity insertions, absent in other organisms. From an analysis of the aminoacid sequence, different constraints appear to act on the borders and on the central part of the insertions. Albeit maintaining a strong bias toward hydrophylic residues, central portions diverge more rapidly than borders, through point mutation and differential presence of entire tracts.

Detection of latent sequence periodicities.

A method is proposed for the automatic detection of serial periodicities in a linear sequence. Its application to DNA subtelomeric sequences from two lower eukaryotes, P.falciparum and S.cerevisiae, reveals ordered patterns organised in hierarchical periodicities, not easily recognizable by other methods. The possible implications concerning the evolution of tandemly repetitive arrays are discussed in light of a model which involves, as successive steps, random repeat modification, the fusion of differently modified repeat versions into longer units, and the amplification of (and/or homogenization to) the more recent repeat units.

GENOME PLASTICITY IN PLASMODIUM

Extensive genome plasticity inPlasmodium involves frequent loss of dispensable functions under non-selective conditions, polymorphisms in subtelomeric repetitive regions, as well as rapid and apparently concerted variation in the intra-genic repetitive arrays that are typical of plasmodial antigen genes. As an example of the latter type of variation, the region of the merozoite surface antigen gene MSA-1 ofPlasmodium falciparum, which encodes a tri-peptide repeat, is analysed in detail. The example illustrates how evasion of the immune defenses of the vertebrate host can be achieved through repeat homogenization mechanisms, acting at the DNA level, and leading to rapid fixation of variant epitopes. The remarkable ability of Plasmodia to utilize mechanisms which operate on its own nuclear DNA in the course of mitotic multiplication is discussed against the need of life cycle closure as a haploid unicellular. The possibility is suggested that active genomic diversification in a (clonal) multicellular population evolved as an adaptive tool.

Structure and superstructure of Plasmodium falciparum subtelomeric regions

Walking and jumping procedures were employed to obtain a consensus map of the 35-40 kb subtelomeric region shared by many chromosomal extremities in Plasmodium falciparum strain 3D7, and to characterise the portions flanking the rep20 tract, which is known to contain tandemly repeated, apparently degenerate, 21-bp repeats. The borders of rep20 were shown to harbour short (possibly locally homogenised) patterns of non-degenerate 12-, 17-, 23- and 28-bp repeats. The central repetitious portion of the consensus map was estimated to be about 18 kb in length, and to be separated from the telomere by approx. 11 kb of non-repetitious sequence, maintained with high fidelity at different chromosomal ends. Several kilobases of similarly conserved, non-repetitious sequence flank rep20 on its proximal side. Computer analysis of the rep20 sequence suggested that a peculiar superhelical winding originates from the conservation of identical nucleotide groups in phase with the pitch of the double helix, overcoming the effect of repeat degeneration in in other positions of the 21-bp unit.

SIMILARITY IN OLIGONUCLEOTIDE USAGE IN INTRONS AND INTERGENIC REGIONS CONTRIBUTES TO LONG-RANGE CORRELATION IN THE CAENORHABDITIS ELEGANS GENOME

A method is presented which allows detection of a sequence correlation effect not related to patchiness in base composition or to preferences in codon usage. Recurrence plots providing local views of oligonucleotide recurrence regimen show that introns and intergenic regions are often characterised by a highly recurrent use of oligonucleotides. By window analysis it is possible to score a long sequence for the recurrence of a given subset of oligos while filtering away the effects of short-range correlations. Long-range exploration of chromosome III from Caenorhabditis elegans reveals that consistent use of recurrent oligonucleotides in introns and intergenic regions generates a correlation effect that extends over several megabases.