Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA.

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres.

Antigenic variation at the surface of infected erythrocytes in plasmodium falciparum

Os membros da família dos genes var de Plasmodium falciparum codificam para receptores que desempenham um papel importante na patogenicidade da malária. O mecanismo responsável pela seleçäo da expressäo dos diferentes membros da família dos genes var ("switching") tem sido estdado utilizando populações de parasitas clonados, selecionados por suas características adesivas. O parasita expressa um único gene var o estágio de trofozoíto do seu ciclo de vida. Análises dos sítios de expressäo, ativos ou inativos, dos genes var demonstraram que o controle da expressäo ocorre durante a transcriçäo e a ativaçäo destes genes ocorre "in situ". Observamos que näo há sobreposiçäo no repertório dos genes var para diferentes isolados de laboratório, sugerindo desta maneira a existência de mecanismos para a geraçäo de diversidade desta família gênica. Experimentos de "fluorescence in situ hybridization" (FISH) mostraram que as extremidades dos cromossomos de P. falciparum estäo fisicamente associados e que esta formaçäo é importante para a geraçäo da diversidade dos genes var.

Identification of the telomere in Trypanosoma cruzi reveals highly heterogeneous telomere lengths in different parasite strains.

Here we describe the cloning and characterisation of the Trypanosoma cruzi telomere. In the Y strain, it is formed by typical GGGTTA repeats with a mean size of approximately 500 bp. Adjacent to the telomere repeats we found a DNA sequence with significant homology to the T.cruzi 85 kDa surface antigen (gp85). Examination of the telomere in nine T.cruzi strains reveals differences in the organisation of chromosome ends. In one group of strains the size of the telomere repeat is relatively homogeneous and short (0.5-1.5 kb) as in the Y strain, while in the other, the length of the repeat is very heterogeneous and significantly longer, ranging in size from 1 to >10 kb. These different strains can be grouped similarly to previously existing classifications based on isoenzyme loci, rRNA genes, mini-exon gene sequences, randomly amplified polymorphic DNA and rRNA promoter sequences, suggesting that differential control of telomere length and organisation appeared as an early event in T. cruzi evolution. Two-dimensional pulsed field gel electrophoresis analysis shows that some chromosomes carry telomeres which are significantly larger than the mean telomere length. Importantly, the T.cruzi telomeres are organised in nucleosomal and non-nucleosomal chromatin.

Expression of trans-sialidase and 85-kDa glycoprotein genes in Trypanosoma cruzi is differentially regulated at the post-transcriptional level by labile protein factors.

To adapt to different environments, Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, expresses a different set of proteins during development. To begin to understand the mechanism that controls this differential gene expression, we have analyzed the levels of amastin and trans-sialidase mRNAs and the mRNAs encoding members of the 85-kDa glycoprotein gene family, which are differentially expressed in the T. cruzi stages found in the mammalian host. Amastin mRNA is expressed predominantly in intracellular and proliferative amastigotes. trans-Sialidase mRNAs are found mostly in forms undergoing transformation from amastigotes to trypomastigotes inside infected cells, whereas mRNAs encoding the 85-kDa glycoproteins appear only in the infective trypomastigotes released from the cells. The genes coding for these mRNA species are constitutively transcribed in all stages of T. cruzi cells, suggesting that expression is controlled post-transcriptionally during differentiation. Inhibition of transcription by actinomycin D revealed that each mRNA species has a relatively long half-life in stages where it accumulates. In the case of the trans-sialidase and 85-kDa glycoprotein genes, mRNA accumulation was induced by treatment with the protein synthesis inhibitor cycloheximide at the stages that preceded the normal accumulation. Therefore, mRNA stabilization may account for mRNA accumulation. mRNA degradation could be promoted by proteins with high turnover, or stabilization could be promoted by forming a complex with the translational machinery at defined times in development. Identification of the factors that induce mRNA degradation or stabilization is essential to the understanding of control of gene expression in these organisms.

Two distinct groups of mucin-like genes are differentially expressed in the developmental stages of Trypanosoma cruzi.

Sialic acid acceptors of Trypanosoma cruzi are abundant mucin-like glycoproteins linked to the parasite membrane by a glycosylphosphatidyl inositol (GPI) anchor. They are heterogeneous and variable in different parasite stages. The protein portion of these mucins contains many threonine residues, and is thought to be encoded by a heterogeneous gene family. To investigate whether the high degree of heterogeneity in the mucin gene family is responsible for the diversity of mucins expressed on the parasite surface, we have studied the expression of mucin genes in several developmental stages of T. cruzi. We have found that mucins are expressed in all parasite stages. By using conserved sequences at 3' end of translated sequences of the gene family and the splice leader sequence, we have isolated 120 mucin-like cDNAs by RT-PCR from epimastigote and trypomastigote mRNAs. All transcribed genes contain conserved 5' and 3' regions, which code for the signal peptide, the sequence for GPI anchor addition, and a conserved domain rich in threonine residues. The internal portions of these genes are highly variable in size and sequence, and can be grouped in two major categories. One group contains KP(1-2)T(6-8) repeats, a motif found in mammalian mucins in the central region. This group is expressed preferentially in the trypomastigote forms ready to be released from the infected mammalian cell. The other has highly variable sequences in the central portion, and is expressed in all parasite stages. Because the number of synonymous substitutions is equivalent to the non-synonymous substitutions in the second group, they are probably evolving neutrally. On the other hand, the KP(1-2)T(6-8) containing genes have more synonymous substitutions and are most likely under a strong selective pressure. We propose that the group of KP(1-2)T(6-8) motif corresponds to the highly glycosylated mucins of the trypomastigote stages. In the other group proteolysis may remove the central domain yielding small mucins, such as the mucins found in insect derived stages of T. cruzi.

Organization of trans-sialidase genes in Trypanosoma cruzi.

Trypanosoma cruzi trans-sialidase is encoded by a family of genes containing a conserved region, which corresponds to the catalytic and amino-terminal domain of the enzyme. Most, but not all genes, also encode a variable region formed by 12 amino acid repeats at the carboxy-terminus of the protein that are not required for enzymatic activity. To design gene knock-out strategies and understand how trans-sialidase expression is regulated, we have studied the genome organization of trans-sialidase genes. We show here that the different types of trans-sialidase genes are distributed in more than one chromosomal band with sizes ranging from 0.8 to 1.5 Mb pairs in several T. cruzi strains. In the Y-strain, all repeat-containing genes are localized in one chromosomal band of 1.1 Mb, while the repeat-minus genes are in two chromosomes of 0.82 and 0.79 Mb. The repeat-containing genes have similar catalytic and intergenic regions, but variable lengths of the repeated region. The trans-sialidase genes with the repeats are in tandem of up to 12 genes in at least four different clusters. Each cluster contains genes with different numbers of repeats, according to the physical maps of eight independent cosmids, and in the same cluster there are genes that code for active and inactive trans-sialidases. There are 80 +/- 30 copies of the repeat-containing genes grouped in two NotI fragments of 120 and 180 Kb. Therefore, in the Y-strain, the trans-sialidase genes containing repeats might be arranged in three to four clusters in two homologous chromosomes, each cluster having up to 12 genes with different repeat numbers.