Complex transcription from the extrachromosomal DNA encoding mitochondrial functions of Plasmodium yoelii.

Abstract

AU tested members of genus Plasmodium contain tandemly arrayed, transcribed, extrachromosomal DNA with a unit length of 6.0 kb. This DNA contains two open reading frames with potential to encode cytochrome c oxidase subunit I (coxl) and cytochrome b (cob) as well as fragments of rRNA genes scattered on both strands. At least 10 discrete RNA molecules transcribed during erythrocytic stages of a rodent malarial parasite, Plasmodium yoelii, were recognized by the 6.0-kb DNA probes. The RNA molecules of 1.4 and 1.1 kb were identified as encoding coxi and cob, respectively. Primer extension and RNA sequencing were used to locate and characterize 5' ends of these two RNAs, showing that an identical 12-nucleotide sequence, 5'-TATTTTT TGTTT-3', was present at these positions. This sequence may act as a promoter or as an RNA processing signal. A stem-loop structure signifying a possible transcription termination was present at the end of the coxi open reading frame. At least six discrete RNA molecules of less than 250 nucleotides were recognized by different fractions of the 6.0-kb DNA. The largest of these, 200 nucleotides, was also characterized by primer extension and RNA sequencing. This molecule had a high homology to portions of the large-subunit rRNA domains IV and V. Other, small RNA molecules were recognized by regions of the 6.0-kb DNA that had homology to the highly conserved peptidyltransferase domain of large-subunit rRNA. These results show that tlte unusual compactly organized mitochondrionlike DNA of malarial parasites is transcribed in a complex attern.Malarial parasites, members of genus Plasmodium, belong to phylum Apicomplexa of kingdom Protista. Organisms within this phylum include many important pathogens, such as species of the genera Toxoplasma, Babesia, and Eimeria, in addition to Plasmodium species, all of which are intracellular parasites. They possess a complex array of organelles, some of which may assist in invasion of host cells; others have unknown functions. Among the organelles of Plasmodium species are mitochondria, whose contributions to the parasite physiology are not entirely clear. Because mammalian Plasmodium species rely mainly on glycolysis for energy generation and lack many citric acid cycle enzymes (22), mitochondria have been thought to act primarily as electron sink for dihydro-orotate dehydrogenase during pyrimidine biosynthesis (17). And yet, observations with rhodamine 123 staining (11,24) and studies with mitochondrial inhibitors (4, 16) give a hint of organelles that may play a wider role in parasite biochemistry.Until recently, not much was known about the organelle DNA of malarial parasites. A circular DNA molecule of about 35 kb was observed by electron microscopy and assumed to be the mitochondrial genome (12,18,28). This assumption appeared to be substantiated when genes encoding rRNA sequences resembling those of procaryotes and mitochondria were found within the 35-kb circular DNA of Plasmodium falciparum (15). However, through DNA sequencing of a 6.0-kb tandemly...

The evolution of primate malaria parasites based on the gene encoding cytochromebfrom the linear mitochondrial genome

“…The gene encoding cytochrome b of Plasmodium has a high A ϩ T content in all of the lineages studied, which is in agreement with previous reports (23,27,38). The average A ϩ T content is 73.35%, with a 95% confidence interval around the mean of 72.98-73.73%, calculated using only one sequence from P. falciparum.…”

“…We obtained five sequences of P. falciparum, one from a field isolate from western Kenya and four laboratory adapted strains: ''Santa Lucia'' from Central America, ''3D7'' from West Africa, ''Malaysian-4'' from Malaysia, and one from China. The Plasmodium yoelii sequence was obtained from the literature (23).…”

Extrachromosomal DNA in the Apicomplexa

“…Even though detailed knowledge of the mechanism of transcription is lacking, the finding of transcripts underpins a general belief that the malarial organelle is functional in the erythrocytic stages. The three protein-coding genes (cyt b, cox I, and cox III) give rise to transcripts commensurate with their size (102), consistent with evidence suggesting that their polypeptide products are present and functional (26). It is not clear if these transcripts are processed from a larger precursor, although 5-kb transcripts of both strands were found in P.…”

Unidirectional dominance of cytoplasmic inheritance in two genetic crosses of Plasmodium falciparum.

“…This was important because in Toxoplasma gondii, sequences related to the 6-kb DNA molecules have been found on chromosomal DNA in multiple copies (25), and a similar situation in P. falciparum would have complicated analysis of inheritance. The 6-kb probe consisted of a plasmid clone containing 5.8 kb of the P. falciparum 6-kb DNA molecule (35,39), whereas the probe for the 35-kb DNA consisted of a 0.7-kb region of the molecule (13) amplified by PCR. Figure 1 shows an ethidium bromide-stained PFG gel in which the 14 nuclear chromosomes were size fractionated.…”

“…Support for a possible algal relationship of apicomplexans is offered by the observations that rRNA genes in mitochondrial genomes of Plasmodium spp. (10,19,35,37,39,40) and Chlamydomonas reinhardtii (2) are both fragmented and scrambled, although the fragments differ in size, order, and arrangements. Detailed studies of dinoflagellate organelle genomes have not been reported.…”