Interspecies conservation of gene order and intron-exon structure in a genomic locus of high gene density and complexity in Plasmodium.

A 13.6 kb contig of chromosome 5 of Plasmodium berghei, a rodent malaria parasite, has been sequenced and analysed for its coding potential. Assembly and comparison of this genomic locus with the orthologous locus on chromosome 10 of the human malaria Plasmodium falciparum revealed an unexpectedly high level of conservation of the gene organisation and complexity, only partially predicted by current gene-finder algorithms. Adjacent putative genes, transcribed from complementary strands, overlap in their untranslated regions, introns and exons, resulting in a tight clustering of both regulatory and coding sequences, which is unprecedented for genome organisation of PLASMODIUM: In total, six putative genes were identified, three of which are transcribed in gametocytes, the precursor cells of gametes. At least in the case of two multiple exon genes, alternative splicing and alternative transcription initiation sites contribute to a flexible use of the dense information content of this locus. The data of the small sample presented here indicate the value of a comparative approach for Plasmodium to elucidate structure, organisation and gene content of complex genomic loci and emphasise the need to integrate biological data of all Plasmodium species into the P.falciparum genome database and associated projects such as PlasmodB to further improve their annotation.

Targeted terminal deletions as a tool for functional genomics studies in Plasmodium.

We describe a transfection system that induces terminal deletions at specific chromosome ends in malaria parasites using a linear construct containing telomeric repeats at one end and plasmodial sequences able to drive homologous recombination at the other. A site-specific deletion was generated at one extremity of chromosome 5 of Plasmodium berghei, which was stably maintained in the parasite population selected after transfection. The telomeric repeat array introduced with the construct reached the average length observed in natural telomeres of Plasmodium, indicating that in vivo telomere addition occurred at the newly formed extremity. The expression of a mutant dhfr/ts gene conferring pyrimethamine resistance, used as a selectable marker, was not affected by the proximity to the telomeric sequences, either in the presence or absence of drug pressure. In addition, no transcriptional silencing was observed on insertion of the mutant dhfr/ts gene either in subtelomeric or internal positions that are transcriptionally silent in blood-stage parasites. This suggests that the activity of its promoter is not affected by the chromatin organization of the chromosomal context.

The putative gene for the first enzyme of glutathione biosynthesis in Plasmodium berghei and Plasmodium falciparum.

The putative gene for gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione biosynthesis, has been characterized both in Plasmodium berghei and Plasmodium falciparum. Protein sequence comparison between these two species reveals large conserved regions sharing more than 80% similarity, separated by less conserved portions. When the comparison is extended to known gamma-glutamylcysteine synthetases from other eukaryotes, a number of high similarity blocks are observed which may help in identifying sequence essential for protein function.

Genome plasticity and sexual differentiation in Plasmodium.

Spontaneous subtelomeric deletions of Plasmodium chromosomes have been observed both in natural infections and in laboratory maintained parasites. In the latter case, functions dispensable for asexual parasite multiplication and encoded at the extremities of the chromosomes are easily lost. In particular, spontaneous subtelomeric deletions have been characterised which affect gametocytogenesis both in Plasmodium berghei maintained in laboratory animals and in Plasmodium falciparum propagated in in vitro cultures. In order to identify these genetic determinants, and, potentially, other genes located subtelomerically, we designed a transfection system able to induce and select for controlled, site-specific subtelomeric deletions.

Developmental regulation of a Plasmodium gene involves the generation of stage-specific 5' untranslated sequences.

The B7 gene of Plasmodium berghei, highly conserved within the genus Plasmodium, encodes a nuclear protein most likely involved in chromatin assembly. In this study we describe the transcription pattern of B7 during asexual multiplication and sexual differentiation of the parasites in the blood of the vertebrate host. Two alternative transcripts have been identified: one, 1.4 kb in length is specific for asexual blood stages; the other, 1.8 kb in length is specific for sexually differentiated cells (gametocytes). The processed mRNAs are identical in their coding region and differ only in their 5' untranslated regions (5' UTRs). We show here that the differences in 5' UTRs are the result of two mechanisms: (1) the use of alternative transcription initiation sites mapped at least 1.4 kb apart, which imply the existence of separate, stage-specific promoters; (2) the splicing of a 765 bp gametocyte-specific intron at the 5' UTR of the 1.8 kb transcript.

A chromatin-associated protein is encoded in a genomic region highly conserved in the Plasmodium genus.

A single copy gene, pbB7, encoding a putative 26 kDa acidic protein has been isolated from Plasmodium berghei and appears to be part of a genomic region well conserved within the Plasmodium genus. The deduced amino acid sequence exhibits significant blocks of similarity with nucleosome assembly proteins from yeast and man. The nuclear localization of the natural protein and its close association with chromatin during the entire erythrocytic cycle of the parasite have been demonstrated using specific monoclonal antibodies against the pbB7 product expressed in Escherichia coli. These results suggest an involvement of this nuclear factor in the dynamics of chromatin packaging.

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.

Dynamics of telomere turnover in Plasmodium berghei.

Non-uniform composition in telomeric repeats at the extremities of Plasmodium 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 of Plasmodium 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.

Isolation of a distally located gene possibly correlated with gametocyte production ability.

Previous studies were focussed on the attempt to correlate observable variations in the size of Plasmodium berghei chromosomes with the loss of ability to produce viable gametocytes. A temporal coincidence between the appearance of a subtelomeric deletion on P. berghei chromosome 5 and the loss of the ability to produce viable gametocytes was observed in a clone (HPE) directly derived from the high gametocyte-producer clone 8417 during mechanical passages. Interestingly enough, three P. berghei sexual-specific genes have already been mapped on internal fragments of this chromosome. A novel gene, clone 150, isolated from a genomic library of clone 8417 using a probe enriched for sexual-specific transcripts, maps on chromosome 5 within 100kb from the telomere. Subtelomeric deletions of chromosome 5 affecting two non-producer clones involve part of the transcribed region of this gene.

Isolation of a distally located gene possibly correlated with gametocyte production ability

Previous studies were focussed on the attempt to correlate observable variations in the size of Plasmodium berghei chromosomes with the loss of ability to produce viable gametocytes. A temporal coincidence between the appearance of a subtelomeric deletion on P. berghei chromosome 5 and the loss of the ability to produce viable gametocytes was observed in a clone (HPE) directly derived from the high gametocyte-producer clone 8417 during mechanical passages. Interestingly enough, three P. berghei sexual-specific genes have already been mapped on internal fragments of this chromosome. A novel gene, clone 150, isolated from a genomic library of clone 8417 using a probe enriched for sexual-specific transcripts, maps on chromosome 5 within 100kb from the telomere. Subtelomeric deletions of chromosome 5 affecting two non-producer clones involve part of the transcribed region of this gene.

Chromosomal polymorphism and sexual differentiation in Plasmodium.

The correlation observed in several instances between the loss of ability to produce gametocytes and chromosomal rearrangements, prompted us to investigate in further detail the molecular bases of chromosomal polymorphism in Plasmodium. Generation of polymorphic karyotypes in Plasmodium involves important rearrangements, mostly occurring in subtelomeric position. Detailed analysis on the organisation of these regions have been carried out on the rodent malaria P. berghei and the human malaria P. falciparum. A 2.3kb sequence, tandemly organised in long clusters is shared by many P. berghei chromosomal ends. Variations in the copy number of this "module" account for most of the observed polymorphisms. In a P. falciparum cloned line (3D7) a common region spanning at least 40 kb, is present. It does not contain any repetitive structure other than the rep20 cluster, that appears to be completely contained within the common region. Notwithstanding the structural differences, human and rodent Plasmodia share the common feature of possessing long subtelomeric regions showing, thus, a homology between the different chromosomes.

A 40-kilobase subtelomeric region is common to most Plasmodium falciparum 3D7 chromosomes.

Starting from previous evidence indicating that some features are shared by several Plasmodium falciparum chromosomal extremities, a subtelomeric region present on most P. falciparum 3D7 chromosomes has been mapped. It was shown to occupy about 40 kb, and to include the proximal portion of pPftel. 1, the only telomeric clone described for P. falciparum [12], the complete 21-bp repetitive cluster and some conserved sites (PstI, EcoRI) proximally located with respect to this cluster.

Extensive turnover of telomeric DNA at a Plasmodium berghei chromosomal extremity marked by a rare recombinational event.

The dynamics of telomere turnover were studied in Plasmodium, whose telomeric structures consist of linear, recognisable sequences of two distinct repeats (TTTAGGG and TTCAGGG). Independent recombinant clones containing a well-defined chromosomal extremity of Plasmodium berghei, both before and after a rare insertion event took place, were obtained from clonal parasite populations and analysed. The insertion, which splits the original telomere and causes a significant reduction in the size of the telomeric structure, is shown to consist of an integer number of subtelomeric repeats typical of P.berghei, flanked on both sides by telomere-derived motifs. Analysis of the telomeric repeat sequence heterogeneity in the otherwise homogeneous populations examined, is compatible with a model in which diversification of a given telomere is driven by the occurrence of breakpoints whose frequency rapidly increases along the telomeric tract when moving in the outward direction. The breakpoints might be due either to terminal deletions followed by random serial addition of the two repeat versions, or to recombination events. The shortening/elongation mechanism is favoured against the recombination hypothesis because of the absence of higher-order patterns in the sequence of telomeric repeats.

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.

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.