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J C Mottram

Publications and source records attributed to J C Mottram.

At least 55 records · Page 3Linked to original sources
PubMed1996

Gene disruptions indicate an essential function for the LmmCRK1 cdc2-related kinase of Leishmania mexicana.

The generation of homozygous null mutants for the crk1 Cdc2-Related Kinase of Leishmania mexicana was attempted using targeted gene disruption. Promastigote mutants heterozygous for crk1 were readily isolated with a hyg-targeting fragment, but attempts to create null mutants by second-round transfections with a bie-targeting fragment yielded two classes of mutant, neither of which was null. First, the transfected fragment formed an episome; second, the cloned transfectants were found to contain wild-type crk1 alleles as well as hyg and ble integrations. DNA-content analysis revealed that these mutants were triploid or tetraploid. Plasticity in chromosome number following targeting has been proposed as a means by which Leishmania avoids deletion of essential genes. These data support this theory and implicate crk1 as an essential gene, validating CRK1 as a potential drug target. L mexicana transfected with a Trypanosoma brucel homologue, tbcrk1, was shown to be viable in an immcrk1 null background, thus showing complementation of function between these trypanosomatid genes. The expression of crk1 was further manipulated by engineering a six-histidine tag at the C-terminus of the kinase, allowing purification of the active complex by affinity selection on Nl(2+)-nitriloacetic acid (NTA) agarose.

Alleles↗
PubMed1995

A family of trypanosome cdc2-related protein kinases.

The cyclin-dependent kinases, most notable of which is cdc2, are key regulators of the cell cycle, and are highly conserved in evolution. We have cloned and analysed three cdc2-related kinase-encoding genes (tbcrk1-3) from the protozoan parasite Trypanosoma brucei. tbcrk1 encodes a 34-kDa protein with 54% amino acid (aa) identity to the human cdc2, tbcrk2 a 39-kDa protein with 49% identity and tbcrk3 a 35-kDa protein with 54% identity. tbcrk1-3 have substitutions in the 16-aa sequence, the 'PSTAIRE' domain, that characterises the cdc2-related kinase family, to give PCTAIRE, PSTAVRE and PQTALRE motifs, respectively. The three kinases have conserved Tyr and Thr residues that are sites of phosphorylation in cdc2 and are important for regulating kinase activity. Southern blot analysis revealed that each tbcrk is a single copy gene. Pulse-field electrophoresis located the tbcrk genes to some of the largest of the trypanosome chromosomes at greater than 3 Mb. Western blots with anti-PSTAIRE polyclonal antibody detected proteins of 32, 43 and 65 kDa in all life-cycle stages and a 90-kDa protein in bloodstreams forms, implying the presence of a family of cdc2-related kinases. Trypanosomes have a remarkably large gene family of cdc2-related kinases for such a primitive organism. The crk genes may be involved in controlling aspects of the cell cycle which are linked to the differentiation of the parasite during its complex life cycle.

Amino Acid Sequence↗
PubMed1995

Antigen presentation by Leishmania mexicana-infected macrophages: activation of helper T cells specific for amastigote cysteine proteinases requires intracellular killing of the parasites.

Leishmania mexicana amastigotes proliferate in the phagolysosomes of mammalian macrophages. The parasites abundantly synthesize lysosomal cysteine proteinases, which are encoded by the lmcpb gene family. One of these genes was overexpressed in Escherichia coli, and the purified recombinant protein was used as an antigen to induce and establish a T helper 1 (Th1) cell line. The T cells recognize epitopes shared by the native cysteine proteinases and the recombinant protein. Infected bone marrow-derived macrophages induced to express major histocompatibility complex class II molecules by interferon (IFN)-gamma do not affect parasite viability. These macrophages fail to stimulate the proliferation of the T cell line. In contrast, strong T cell stimulation is observed after the parasites are killed by treatment with L-leucine methylester, or after activation of macrophages by IFN-gamma and tumor necrosis factor-alpha. It is concluded that infected macrophages efficiently present this lysosomal Leishmania antigen once the parasites are inactivated and degraded. This observation may be of considerable relevance for the outcome of Leishmania infections provided that it can be extended to other parasite antigens.

Animals↗
PubMed1994

Null mutants for the lmcpa cysteine proteinase gene in Leishmania mexicana.

The parasitic protozoon Leishmania mexicana possesses an abundance of developmentally regulated cathepsin L-like cysteine proteinases expressed at highest levels in amastigotes. We recently characterised lmcpa, a single-copy gene encoding one such proteinase, LmCPa, which differs from other homologues by possessing a 3-amino-acid insertion at the amino terminal of the predicted mature proteinase. To investigate the role of LmCPa in L. mexicana, we used gene-targeting of promastigotes with hygromycin- and phleomycin-resistance markers to generate null mutants by disrupting sequentially both alleles of lmcpa. The promastigote null mutants did not differ significantly from wild-type L. mexicana in growth rate or morphology and could differentiate to metacyclics and the amastigote-like form, both of which could infect the J774G8 macrophage-like cell line. The null mutant amastigote-like form obtained from the J774G8 cells could also establish rump lesions in CBA mice. By these criteria, therefore, LmCPa appears to be non-essential although there is the possibility that LmCPa could be required during development in the sandfly, a stage not analysed here. The apparent redundancy of LmCPa in amastigotes may be due to the presence of other cysteine proteinases and has implications for the choice of candidate targets for rationally designed anti-leishmanial drugs.

Alleles↗
PubMed1994

cdc2-related protein kinases and cell cycle control in trypanosomatids.

The molecular mechanisms that control the cell cycle have been studied extensively in yeast and higher eukaryotes. Investigations have centred on the cyclin-dependent kinase family of serine/threonine protein kinases, the best characterized of which is cdc2, a key regulatory element in the control of mitosis. Cell cycle control plays an important role in trypanosomes and Leishmania, not only in cellular proliferation, but also in the developmental system that controls the transfer of the parasite between hosts. In this review, Jeremy Mottram compares the family of trypanosome cdc2-related kinases with that of yeast and the higher eukaryotes.

Journal Article↗
PubMed1993

A novel CDC2-related protein kinase from Leishmania mexicana, LmmCRK1, is post-translationally regulated during the life cycle.

The p34CDC2 protein kinase is a key component in the regulation of the eukaryotic cell cycle. We have isolated from the protozoan parasite Leishmania mexicana mexicana a CDC2-related kinase gene (Lmmcrk1) encoding a 34-kDa protein kinase (lmmCRK1) which has 56% amino acid identity with the human CDC2 and contains a PCTAIR motif in place of the highly conserved PSTAIR box. lmmCRK1 was detected in all life cycle stages at comparable levels, yet its histone H1 kinase activity was detected in only the promastigote form, indicating that its activity is stage-regulated at a post-translational level. lmmCRK1 did not bind p13suc1 beads and Lmmcrk1 was unable to complement a fission yeast temperature-sensitive cdc2 mutant. These data suggest that Lmmcrk1 is unlikely to be the functional L. mexicana cdc2 homologue. A distinct histone H1 kinase activity that binds p13suc1 beads (SBCRK) was also detected, with activity that correlated with the division status of the developmental forms of the parasite, being present in the dividing stages of the parasite and absent in nondividing metacyclic forms. SBCRK is a candidate for the functional CDC2 homologue, but it does not react with an anti-PSTAIR monoclonal antibody on Western blots when eluted from p13suc1 beads, indicating a divergent PSTAIR box. These data suggest that a family of CDC2-related protein kinases are present in Leishmania. Some share sequence and biochemical properties with CDC2, but significant differences also exist, possibly reflecting the evolutionary distance between Leishmania and higher eukaryotes.

Amino Acid Sequence↗
PubMed1992

Characterization of a multi-copy gene for a major stage-specific cysteine proteinase of Leishmania mexicana.

lmcpb, a gene from Leishmania mexicana that encodes a major cysteine proteinase in the parasite, has been cloned and sequenced. LmCPb is related more to cysteine proteinases from Trypanosoma brucei and Trypanosoma cruzi than to a previously characterized cysteine proteinase, LmCPa, of L. mexicana. It contains a long C-terminal extension characteristic of similar enzymes of T. brucei and T. cruzi. The gene is multi-copy and tandemly arranged. lmcpb RNA levels are developmentally regulated with steady state levels being high in amastigotes, low in metacyclic promastigotes and undetectable in multiplicative promastigotes. This variation correlates with and may account for the stage-specific expression of LmCPb enzyme activity.

Amino Acid Sequence↗
PubMed1992

A developmentally regulated cysteine proteinase gene of Leishmania mexicana.

We have isolated a gene encoding a previously unreported class of trypanosomatid cysteine proteinase (CP) from the protozoan parasite Leishmania mexicana. The single-copy gene (lmcpa) [corrected]. has several unusual features that distinguish it from CP genes cloned from the related species Trypanosoma brucei and Trypanosoma cruzi. These include a shorter C-terminal extension of only 10 amino acids and a three-amino-acid insertion, GlyValMet, close to the predicted N-terminus of the mature protein. Northern blot analysis showed that the gene is expressed in all life-cycle stages but at higher levels in the amastigote stage in the mammal and in stationary phase promastigote cultures which contain the infective metacyclic form of the parasite. A precursor protein of 38 kDa was detected in amastigotes and stationary phase promastigotes with antisera specific to the LmCPa pro-region, but was barely detectable in early log-phase promastigotes. Anti-central domain antisera recognized the 38 kDa precursor and 24 and 27 kDa proteins. The major CPs of L. mexicana amastigotes, previously designated types A, B and C, were not detected with the antisera, suggesting that the gene codes for a previously uncharacterized CP in L. mexicana. The 24 kDa protein detected by the antiserum has no activity towards gelatin but apparently hydrolyses the peptide substrate BzPheValArgAMC. The relative levels of the 24 and 27 kDa proteins vary between the different life-cycle stages. The results indicate that expression of this CP is regulated at both the RNA and protein level.

Amino Acid Sequence↗
PubMed1991

tRNAs of Trypanosoma brucei. Unusual gene organization and mitochondrial importation.

In Trypanosoma brucei the U snRNA B gene (J. C. Mottram, K. L. Perry, P. M. Lizardi, R. Lührmann, N. Agabian, and R. G. Nelson (1989) Mol. Cell. Biol. 9, 1212-1223) is very tightly linked with other small RNA genes coding for tRNA(ACGArg), tRNA(CUULys), and a approximately 275-nucleotide RNA (RNA X) of unknown function. A similar genomic organization is found at the U6 snRNA locus, where the U6 gene is linked to tRNA(CGUThr) and tRNA(GUATyr) genes. The tRNA(Lys) and tRNA(Arg) genes are members of a multigene family, whereas the tRNA(Thr) and tRNA(Tyr) genes are single copy. Two additional tRNA(CUULys) genes and one tRNA(UUULys) gene were also isolated and sequenced and, together with a sequence previously published (D. A. Campbell (1989) Nucleic Acids Res. 17, 9479), appear to represent the entire gene family. Probes for tRNA(Lys), tRNA(Arg), tRNA(Thr), and tRNA(Tyr) were found to hybridize with mitochondrial and cytoplasmic tRNAs but not with mitochondrial DNA. This supports the hypothesis that mitochondrial tRNAs may be nuclear-encoded and imported from the cytosol into the mitochondrion.

Animals↗
PubMed1991

Conservation of cation-transporting ATPase genes in Leishmania.

DNA fragments isolated from Leishmania donovani ATPase genes were used to analyze the organization and expression of cation transporting ATPase genes in L. donovani, Leishmania tropica, Leishmania mexicana, Leishmania braziliensis, Trypanosoma brucei and Trypanosoma cruzi. The ATPase loci in all Leishmania species contained a tandem pair of ATPase genes arranged in head-to-tail orientation and separated by approximately 2 kb. No restriction site polymorphisms were detected in the internal portions of the Leishmania ATPase genes which contain domains conserved between the L. donovani and other eukaryotic plasma membrane ATPases. The ATPase locus of each of the four Leishmania species was mapped to a single small chromosome of approximately 750 kb. The ATPase locus of L. mexicana was differentially expressed. Promastigotes in exponential growth contained abundant transcripts from the upstream ATPase gene, while transcripts from the downstream gene were relatively scarce. Transcripts from the downstream ATPase gene increased in abundance in promastigotes allowed to reach the stationary phase of growth and were most abundant in amastigotes. The two trypanosome species were found to contain DNA fragments that hybridized strongly to the Leishmania ATPase gene.

Adenosine Triphosphatases↗
PubMed1990

Bloodstream and metacyclic variant surface glycoprotein gene expression sites of Trypanosoma brucei gambiense.

Trypanosoma brucei gambiense is the causative agent of chronic human sleeping sickness. Previous studies have indicated that T. b. gambiense isolates expressed the antigens U1 or L2 in both the metacyclic and early bloodstream form of the parasite life cycle. These studies suggested that L2 and U1 were likely to be metacyclic variant surface glycoproteins (mVSG). The basic copies of the genes encoding the VSGs L2 and U1 are present in single copy in non-expressing isolates of T. b. gambiense. Furthermore, they have been found to be maintained stably in a large number of stocks isolated from a wide geographic area over a 30-year period. The genomic DNA comprising the upstream 5' flanking regions of the U1 and L2 putative mVSG gene expression sites have been cloned from bloodstream forms of T. b. gambiense. The L2 expression site clone, containing 12.5 kb of sequences 5' to the VSG gene, was found to lack the 72/76-bp repeat unit generally found in the 'barren' region upstream of bloodstream form expression sites. The U1 expression site clone, containing 13.5 kb of the 5' flanking region, appeared to have the repeats, which were localized to 2 kb of DNA immediately 5' to the U1 mVSG gene. Neither the U1 nor the L2 clone was found to have ESAG2 or ESAG3 gene sequences, but both were found to have ESAG1 genes. The ESAG1 genes from the putative metacyclic expression sites and from the U1 and L2 bloodstream form expression sites (in the form of cDNA clones) were sequenced and compared to all other published ESAG1 sequences.

Amino Acid Sequence↗
PubMed1990

Cysteine proteinases of parasitic protozoa.

Proteinases are involved with many processes in living organisms. In recent years, there has been increasing interest in elucidating the functions the enzymes perform in parasites. These studies have revealed that one class of proteinases, the cysteine proteinases, predominates in many parasitic protozoa. In this article Mick North, Jeremy Mottram and Graham Coombs review what is known about the cysteine proteinases of parasitic protozoa and discuss the approaches being pursued in attempts to design antiparasite drugs based on inhibitors or substrates of these enzymes.

Journal Article↗
PubMed1989

A cysteine proteinase cDNA from Trypanosoma brucei predicts an enzyme with an unusual C-terminal extension.

A cDNA for a Trypanosoma brucei cysteine proteinase has been cloned and sequenced. The deduced protein can be divided into four domains, based on homologies with other cysteine proteinases: the pre-, pro- and central regions show considerable homology to the cathepsin L class of mammalian enzymes, whilst the long C-terminal extension distinguishes the trypanosome enzyme from all mammalian cysteine proteinases reported. This 108 amino acid extension, which includes 9 contiguous prolines near the junction with the central domain, appears likely to be processed in part to produce the mature enzyme, and may be involved in targeting the protein within the cell. The trypanosome genome contains more than 20 copies of the cysteine proteinase gene arranged in a long tandem array.

Amino Acid Sequence↗
PubMed1989

A transcriptional analysis of the Trypanosoma brucei hsp83 gene cluster.

Ten to twelve copies of the 83-kDa heat-shock protein gene (hsp83) from Trypanosoma brucei are arranged in a head-to-tail tandem array of 2.8-kb repeat units, which are transcribed to give 2.6-kb mature mRNAs. We have cloned and sequenced one of the repeat units. The gene encodes a putative protein of 81 kDa which is highly homologous to Hsp83 of Drosophila melanogaster (75%), Hsp90 of Saccharomyces cerevisiae (72%) and the C62.5 protein of Escherichia coli (61%). The 5' end of the mature mRNA was mapped by primer extension sequence analysis and shown to contain the spliced leader. The mapping of the 3' poly(A) addition sites by S1 analysis indicated that there is 218 nt of intergenic sequence linking the boundaries encoding the mature mRNA. Within this sequence are a number of elements conserved with the trypanosome hsp70 intergenic region, including a 14-nt sequence that also has homology to the Drosophila heat-shock consensus element.

Amino Acid Sequence↗