Chris J. Delves

The multifunctional folic acid sythesis fas gene of Pneumocystis carinii appears to encode dihydropteroate synthase and hydroxymethyldihydropterin pyrophosphokinase

We describe the cloning of a multifunctional folic acid synthesis (fas) gene from Pneumocystis carinii. The nucleotide sequence contains an open reading frame interrupted by three introns, that encodes a protein of 740 amino acids with an Mr of 97,278. The predicted Fas protein has homology to two enzyme domains, dihydropteroate synthase and 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase, both of which are involved in folate synthesis, and at least one other region of unknown function.

The tubulin genes of the human malaria parasite Plasmodium falciparum, their chromosomal location and sequence analysis of the α-tubulin II gene

We report the isolation and sequencing of genomic clones encompassing the entire alpha-tubulin II gene from the human malaria parasite Plasmodium falciparum. This gene is closely related to, but significant different from the alpha-tubulin I gene that we have described previously. These two genes represent the entire complement of alpha-tubulin sequences in this organism and are expressed in a stage-specific manner. The alpha-II gene is present as a single copy and encodes a tubulin molecule with a predicted length of 450 amino acid residues (49.7 kDa). Like the alpha-I gene, it contains two introns, which are in identical positions to those of alpha-I, but are about one-third smaller. The deduced alpha-II protein is very similar to alpha-tubulin I (94.2% amino acid identity), except for notable differences across residues 40-45. In addition, unlike the great majority of alpha-tubulin genes (including alpha-I), alpha-II does not encode a terminal tyrosine residue. Using pulsed field gel electrophoresis we demonstrate that the two alpha-tubulin genes, together with the single beta-tubulin gene, are unlinked, all residing on different chromosomes. We assign alpha-I to chromosome 9, alpha-II to chromosome 4 and beta-tubulin to chromosome 10.

Isolation and molecular characterization of the bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase gene from Toxoplasma gondii

Toxoplasma gondii is an important cause of AIDS-related opportunistic infection, manifest as toxoplasmic encephalitis. The clinical treatment of choice is the synergistic combination of antifolate agents, pyrimethamine and sulphadiazine, of which the latter targets the parasites dihydropteroate synthase (DHPS) activity. Here, we describe the isolation of the gene encoding this activity in T. gondii. The nucleotide sequence contains an open reading frame interrupted by five introns, which encodes a protein of 664 amino acids with an M(r) of 72991. Sequence analysis revealed that, in addition to DHPS, the predicted protein contains a second enzyme function, hydroxymethyldihydropterin pyrophosphokinase (PPPK). This enzyme immediately precedes DHPS in the folate biosynthetic pathway. The bifunctional arrangement of the T. gondii pppk-dhps gene is the same as that observed in the related protozoan parasite, Plasmodium falciparum, and confirms previous biochemical data that these activities were inseparable. Recently, specific mutations within conserved motifs of the DHPS gene of P. falciparum have been identified which give rise to sulphonamide drug resistance. Analysis of seven clinical isolates of T. gondii did not reveal any similar mutations in this limited sample of organisms that had been subjected to drug pressure.

Expression of α and β tubulin genes during the asexual and sexual blood stages of Plasmodium falciparum

Abstract Malaria parasites switch to sexual development after a period of vegetative growth in the hosts erythrocytes. This switch, vital for parasite transmission to mosquitoes, is little understood at the genetic level. Likely candidates for developmental control are the α- and β-tubulin subunits required for microtubule assembly. We report here that the transcription of the α- and β-tubulin genes in Plasmodium falciparum show a radically different pattern of transcription in the asexual and sexual phases of parasite growth. Our studies lead to the conclusion that three transcripts of the β-tubulin gene differ by sequences in their 5′- or 3′-untranslated regions.

Cloning of a β-tubulin gene from Plasmodium falciparum

We describe the isolation and characterization of a gene for β‐tubulin from the malaria parasite, Plasmodium falciparum. This organism appears to contain a single gene encoding β‐tubulin. A single transcript from this gene can be detected in the total RNA of the parasites asexual blood stages. The complete sequence for the gene has been elucidated. It has two introns, one of which has a position identical to that of a related parasite, Toxoplasma gondii. The gene shows the usual preference for codons with A or T in the third position. The predicted amino acid sequence is compared with that of T. gondii and the human host. Further comparisons between these and fungal sequences of β‐tubulins resistant to benomyl, a drug binding this protein, highlight differences that could be exploited in the development of parasite‐specific anti‐tubulin drugs.

Isolation of α‐tubulin genes from the human malaria parasite, Plasmodium falciparum: sequence analysis of α‐tubulin

As a step towards indentifying exploitable differences between host and parasite at the molecular level, we have isolated and sequenced genomic clones encompassing an entire α‐tubulin gene (designated α‐tubulin I) from the human malaria parasite, Plasmodium falciparum. The gene, which contains two introns, encodes a product with a predicted length of 453 amino acid residues (50.3 kD). The protein sequence shows a high degree of homology to other α‐tubulins, particularly that of the coccidian parasite, Toxoplasma gondii (94%), whose gene carries introns in identical positions. Only one copy of the α‐tubulin I gene itself was found, although a second gene designated α‐II was also identified which is closely related but which differs at both the nucleotide and amino acid sequence levels. The α‐I and β‐tubulin genes were found to reside on different chromosomes.

Identification of Plasmodium falciparum-infected mosquitoes using a probe containing repetitive DNA

A cloned repetitive DNA sequence (rep20) was evaluated as a diagnostic probe specific for Plasmodium falciparum sporozoites using experimentally infected mosquitoes squashed directly on nylon filters. Head/thorax portions of mosquitoes, killed 14-16 days after ingesting P. falciparum-infected blood, gave positive signals when examined for the presence of P. falciparum sporozoite DNA by hybridisation. This correlated with the number of oocysts found in a sample of the same batch of mosquitoes examined by dissection. No positive signals were obtained with 50 Plasmodium berghei-infected mosquitoes probed with the rep20 sequence. The results indicate that a probe containing rep20 may be useful in the rapid and specific incrimination of vectors carrying P. falciparum sporozoites. The value of repetitive DNA in the diagnosis of malaria is discussed.

Cloning and sequence of a β-tubulin cDNA from Pneumocystis carinii: possible implications for drug therapy

This work describes the isolation and characterization of a full‐length cDNA clone encoding β‐tubulin from the pathogen Pneumocystis carinii, P. carinii contains a single gene encoding β‐tubulin. The complete sequence of this cDNA has been determined and its inferred amino acid sequence compared with the β‐tubulins from other organisms. This analysis augments the data indicating that P. carinii should be classified as a fungal organism. Further comparisons between the P. cariniiβ‐tubulin and those of fungal β‐tubulins resistant to benomyl, a β‐tubulin‐binding drug, indicate a difference which may be exploited in the development of a new drug therapy for P. carinii pneumonitis. These results suggest that, theoretically, a drug presently administered for treatment of nematode worm infections may be an effective agent against P. carinii, without being toxic to the mammalian host. This possibility is currently being investigated.

Two domains with amino-acid sequence similarity are required for dihydroneopterin aldolase function in the multifunctional folic acid synthesis Fas protein of Pneumocystis carinii.

The folic acid synthesized gene (fas) of Pneumocystis carinii (Pc) codes for a multifunctional enzyme (Fas) known to catalyse three consecutive steps leading to the production of dihydropteroate in the de novo folate synthesis pathway. Previously, we predicted that a domain, designated FasB (amino acids (aa) 161-280), of the 740-aa multifunctional protein contains the first of the three enzyme activities in the pathway, namely dihydroneopterin aldolase (DHNA), since it shares 23% aa identity with the DHNA of Streptococcus pneumoniae (Sp). We now extend these findings to show that a second domain, FasA (aa 39-160), whose function was previously unknown, shares 27% sequence identity with the adjacent FasB domain, indicative of functional similarity. FasA is also 18% identical with the DHNA from Sp. Recombinant baculoviruses were constructed which directed the production of either FasA, FasB or FasAB polypeptide species in cultured Spodoptera frugiperda (SF9) insect cells. No DHNA activity is associated with either fasA or fasB when produced as single domains in the insect-baculovirus system. However, DHNA activity was detected in SF9 extracts containing the overproduced FasAB polypeptide. The results of aa sequence alignments and expression studies suggest that FasA and FasB may be two subunits of the DHNA enzyme moiety within the multifunctional Fas protein of Pc. An alternative interpretation of the results is also discussed.