Chromosomal assignment of gene encoding the largest subunit of RNA polymerase II in the mouse

The gene encoding the largest subunit of RNA polymerase II was mapped to mouse chromosome 11 by Southern blotting analysis of mouse-Chinese hamster somatic cell hybrids and by in situ hybridization. This assignment extends the previously defined homology between mouse chromosome 11 and human chromosome 17.     

Primary sequences of two small subunit ribosomal RNA genes from Plasmodium falciparum

We have determined the complete sequence of two structurally distinct 18S ribosomal RNA genes from the malarial parasite Plasmodium falciparum. S1 nuclease analyses demonstrate that only one of the genes is represented in stable rRNA populations isolated from blood-stage parasites. Comparisons of homologous rRNA genes from Plasmodium berghei and P. falciparum reveal that they are identical at 86% of their positions. From comparisons of the Plasmodium genes to that of humans, it was possible to design genus-specific as well as species-specific oligonucleotide probes that can be used to distinguish the parasite 18S ribosomal RNA from that of its host. The utilization of these probes as diagnostic reagents is discussed.     

Characterization and complete nucleotide sequence of a 5.8S ribosomal RNA gene from Plasmodium falciparum

The 5.8S and 5S rRNA components from the FCR-3/The Gambia strain of Plasmodium falciparum have been identified and the complete nucleotide sequence of a 5.8S ribosomal RNA gene determined. Unlike the 5S rRNA species, the 5.8S is a single homogeneous population of molecules of 157 nucleotides. Comparison of its nucleotide sequence with previously reported 5.8S rRNA sequences indicates that it is homologous to these molecules, but distantly related to them. The sequence of the 5.8S rRNA coding region from the pfrib-2 recombinant of the HG13 Gambian isolate of P. falciparum is identical.     

A genetic analysis of Plasmodium falciparum RNA polymerase II subunits in yeast

RNA polymerase II is an essential nuclear multi subunit enzyme that transcribes nearly the whole genome. Its inhibition by the alpha-amanitin toxin leads to cell death. The enzyme of Plasmodium falciparum remains poorly characterized. Using a complementation assay in yeast as a genetic test, we demonstrate that five Plasmodium putative RNA polymerase subunits are indeed functional in vivo. The active site of this enzyme is built from the two largest subunits. Using site directed mutagenesis we were able to modify the active site of the yeast RNA polymerase II so as to introduce Plasmodium or human structural motifs. The resulting strains allow the screening of chemical libraries for potential specific inhibitors.     

Citrus exocortis viroid RNA is associated with the largest subunit of RNA polymerase II in tomato in vivo

Summary.  An active replication complex of citrus exocortis viroid (CEV) was isolated as a chromatin-enriched fraction of infected tomato leaf with CEV RNA synthesis activity. This activity was solubilised from the chromatin with ammonium sulphate, but not with sarkosyl. Nucleoprotein complexes in the soluble fraction which bound to a monoclonal antibody to the carboxy terminal domain of the largest subunit of RNA polymerase II (8WG16) were affinity purified and contained plus- and minus-sense CEV RNA. The results support a role for RNA polymerase II in viroid replication and provide the first direct evidence of an association in vivo between host RNA polymerase II and CEV. Received March 28, 1999/Accepted July 21, 1999     

Mitochondrial RNA polymerase is an essential enzyme in erythrocytic stages of Plasmodium falciparum

Apicomplexan parasites are serious pathogens of animals and man that cause diseases including coccidiosis, malaria and toxoplasmosis. The importance of these parasites has prompted the establishment of genomic resources in support of developing effective control strategies. For the Eimeria species resources have developed most rapidly for the reference Eimeria tenella Houghton strain (http://www.genedb.org/Homepage/Etenella). The value of these resources can be enhanced by comparison with related parasites. The well characterised immunogenicity and genetic diversity associated with Eimeria maxima promote its use in genetics-led studies on coccidiosis and recommended its selection for sequencing. Using a combination of sequencing technologies a first draft assembly and annotation has been produced for an E. maxima Houghton strain-derived clone (EmaxDB; http://www.genomemalaysia.gov.my/emaxdb/). The assembly of a draft genome sequence for E. maxima provides a resource for comparative studies with Eimeria and related parasites as demonstrated here through the identification of genes predicted to encode microneme proteins in E. maxima.     

Lack of sequence diversity in the gene encoding merozoite surface protein 5 of Plasmodium falciparum.

The gene encoding merozoite surface protein 5 (MSP5) of Plasmodium falciparum is situated between the genes encoding MSP2 and MSP4 on chromosome 2. Both MSP4 and MSP5 encode proteins that contain hydrophobic signal and glycosylphosphatidylinositol (GPI) attachment signals and a single epidermal growth factor (EGF)-like domain at their carboxyl termini. The similar gene organization, location and similar structural features of the two genes suggest that they have arisen from a gene duplication event. In this study we provide further evidence for the merozoite surface location of MSP5 by demonstrating that MSP5 is present in isolated merozoites, partitions in the detergent-enriched phase following Triton X-114 fractionation and shows a staining pattern consistent with merozoite surface location by indirect immunofluorescence confocal microscopy. Analysis of antigenic diversity of MSP5 shows a lack of sequence variation between various isolates of P. falciparum from different geographical locations, a feature unusual for surface proteins of merozoites and one that may simplify vaccine formulation.     

A vaccinia virus isatin-beta-thiosemicarbazone resistance mutation maps in the viral gene encoding the 132-kDa subunit of RNA polymerase

The mutation in a vaccinia virus mutant resistant to inhibition by isatin-beta-thiosemicarbazone was mapped by marker rescue. DNA from the resistant mutant was cloned into cosmid and plasmid vectors and tested for its ability to convert wild-type vaccinia virus to IBT resistant virus in a helper-mediated marker rescue protocol. Resistance was mapped in this way to a 0.9-kb DNA fragment derived from the HindIII A fragment of vaccinia genome. Southern blot hybridization using this DNA as a probe demonstrated that the 0.9-kb fragment is contained within the DNA sequence encoding the second largest subunit of vaccinia RNA polymerase, rpo132. Thus, mutation of rpo132 can cause resistance to IBT in vaccinia virus.     

Inhibition of Plasmodium falciparum clag9 gene function by antisense RNA

We have previously shown by targeted gene disruption that the clag9 gene of Plasmodium falciparum is essential for cytoadherence to CD36. Here we report inhibition of the function of clag9 by the use of an antisense RNA vector as an alternative to targeted gene disruption. We transfected an antisense construct of clag9 into the P. falciparum clone 3D7 and when the resulting line was cultured in the presence of pyrimethamine it showed 15-fold lower cytoadherence to C32 melanoma cells than the control. Reversion to wildtype upon removal of the introduced plasmid provides direct evidence that the event responsible for the phenotypic change is not at an unrelated site and this approach provides a valuable new tool in malaria transfection technology.     

Inhibition of the growth of Plasmodium falciparum and Plasmodium berghei by the DNA polymerase inhibitor HPMPA.

The acyclic adenosine analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [HPMPA] belongs to a class of nucleoside analogues originally described as having potent activity against a broad spectrum of DNA viruses. We examined the effects of this class of drugs on the growth of cultured Plasmodium falciparum. Strong inhibition was observed by HPMPA (ID50 = 47 nM) at concentrations more than 1000-fold less than the cytotoxic dose for human cells. 3-deaza-HPMPA was even more strongly inhibitory (ID50 = 8 nM), whereas several other acyclic nucleosides were not effective. In mice infected with Plasmodium berghei, increase of parasitaemia can be blocked for 4-6 days by a single injection of HPMPA. Repeated drug administration blocks parasite growth for prolonged periods at doses that are clinically feasible. We also determined the inhibition of several purified Plasmodium DNA polymerases by diphosphorylated HPMPA (HPMPApp). DNA polymerase alpha-like enzymes of P. falciparum and P. berghei are inhibited with an IC50 = 40 microM and a gamma-like DNA polymerase from P. falciparum is even 40-fold more sensitive to the drug. The inhibition by HPMPApp is competitive with dATP, strongly suggesting that Plasmodium DNA polymerases are targets for this class of nucleotide analogue.     

Phat--a gene finding program for Plasmodium falciparum.

We describe and assess the performance of the gene finding program pretty handy annotation tool (Phat) on sequence from the malaria parasite Plasmodium falciparum. Phat is based on a generalized hidden Markov model (GHMM) similar to the models used in GENSCAN, Genie and HMMgene. In a test set of 44 confirmed gene structures Phat achieves nucleotide-level sensitivity and specificity of greater than 95%, performing as well as the other P. falciparum gene finding programs Hexamer and GlimmerM. Phat is particularly useful for P. falciparum and other eukaryotes for which there are few gene finding programs available as it is distributed with code for retraining it on new organisms. Moreover, the full source code is freely available under the GNU General Public License, allowing for users to further develop and customize it.     

Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase beta subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas.

Automated DNA sequencing was used to characterize mutations associated with rifampin resistance in a 69-bp region of the gene, rpoB, encoding the beta subunit of RNA polymerase in Mycobacterium tuberculosis. The data confirmed that greater than 90% of rifampin-resistant strains have sequence alterations in this region and showed that most are missense mutations. The analysis also identified several mutant rpoB alleles not previously associated with resistant organisms and one short region of rpoB that had an unusually high frequency of insertions and deletions. Although many strains with an identical IS6110 restriction fragment length polymorphism pattern have the same variant rpoB allele, some do not, a result that suggests the occurrence of evolutionary divergence at the clone level.     

Polymorphism in the gene encoding the Pfs48/45 antigen of Plasmodium falciparum. XI. Asembo Bay Cohort Project.

We have investigated the genetic diversity of the gene encoding the transmission-blocking vaccine antigen Pfs48/45 of Plasmodium falciparum parasites from western Kenya and compared it with parasite populations from Thailand, India, and Venezuela. We report 44 complete new sequences. Overall, the antigen is less polymorphic as compared with other pre-erythrocytic and blood stage antigens. Contrary to other P. falciparum antigens, the number of synonymous substitutions per synonymous site exceeds the number of non-synonymous substitutions per non-synonymous site. We have found that the Pfs48/45 gene of Kenyan parasites is more polymorphic than parasites from other geographic origins. Our analysis reveals that positive natural selection is involved in the maintenance of the observed polymorphism. No evidence of intragenic recombination was found. F(st) values reveal high levels of gene flow between India and Thailand, however, there are strong constraints in gene flow among Kenyan, Southeast Asian, and Venezuelan parasites. No alleles could be linked to a specific geographic region. The results of this study suggest that this gametocyte antigen, like other asexual blood stage antigens, is under selection pressure.     

Characterization of Plasmodium falciparum protein kinase 2

A sustained elevation of free Ca(2+) is observed on the rupture and release of merozoites of Plasmodium falciparum from the erythrocytes. The immunoelectron micrographs demonstrate that calmodulin is localized in merozoites. To elucidate the Ca(2+) signal of P. falciparum invasion, we attempted to characterize P. falciparum protein kinase 2 (PfPK2), which is homologous to human calcium calmodulin-dependent protein kinase (CaMK). PfPK2 was purified as a fusion protein that was labeled with [gamma-(32)P]ATP; this labeling was then eliminated by phosphatase. This phosphorylation was eliminated when the putative catalytic lysine residue of PfPK2 was replaced with alanine. PfPK2 phosphorylated histone II(AS) as a representative substrate in a Ca(2+)- and calmodulin-dependent manner. Calmodulin antagonists inhibited the phosphorylation of PfPK2 in vitro and markedly decreased the parasitemia of ring forms in an invasion assay, whereas CaMKII-specific inhibitors had no effect. PfPK2 was localized in the merozoites in the culture of P. falciparum. Thus, purified PfPK2 possesses protein kinase activity in a Ca(2+)- and calmodulin-dependent manner and the catalytic lysine of this protein was determined. These data suggest that PfPK2 is the Plasmodium protein kinase expressed in the merozoites during the invasion stage.