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

Abstract

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.

Figure 1

(A) Tiling path of the genomic DNA clones used to assemble the B9 locus in addition to marker B9 (16). (B) Molecular probes used in northern blot analysis relative to their position in the B9 locus. Letters in alphabetic order represent names of the genomic probes and RT numbers refer to RT–PCR probes. (C) Gene organisation in the B9 locus, confirmed by cDNA clone, RT–PCR and 5′-RACE analyses as described. Black and grey boxes represent coding and UTR regions, respectively, and lines the introns. Arrows indicate the direction of transcription and asterisks mark the gametocyte-specific genes. Models (a) and (b) for PbORF2 and PbORF3 represent the two most diverse cDNA clones and 5′-RACE models, respectively. The coding region in model (a) of PbORF2 remains undetermined due to a frameshift and/or internal stop codons as a result of alternative splicing. (D) Compilation of results after northern blot analysis with the probes from (B). T, trophozoite stage; G, gametocyte stage; S, schizont stage. Probe b shows the expression of PbOMP decarboxylase, PbORF1 and PbORF2 relative to each other. Probe b shows a very faint band in the region of 1.6 kb in the G lane compared to the T lane due to unequal amounts of parasite RNA. Probe RT-2 shows three clear bands in the region of 1.6 kb, with equal amounts of parasite RNA loaded in the G and T lanes. The pattern of multiple bands for this probe is most likely a representation of the different alternative splice transcripts.

Figure 2

(A) A visual representation of GlimmerM gene prediction in the B9 locus of P.berghei in relation to the organisation of the coding regions as it became apparent during this study. The predictions with the highest probability are shown in grey; the predictions with lower probability in white. AsteriskS indicate gametocyte-specific genes. No experimental evidence or any significant sequence homology was found for the gene predicted between ORF4 and ORF5 in P.berghei. (B) The same annotation applies for P.falciparum. For the second exon of ORF5 in P.falciparum the ORF continues from the end of our sequence for another 1059 nt (contig c10m345, TIGR database). Without this additional sequence GlimmerM is unable to predict the exon accurately.