No abstract

SummaryThe calpain family is named for the calcium dependence of the papain-like, thiol protease activity of the well-studied ubiquitous vertebrate enzymes calpain-1 (µ-calpain) and calpain-2 (m-calpain).Proteins showing sequence relatedness to the catalytic core domains of these enzymes are included in this ancient and diverse eukaryotic protein family. Calpains are examples of highly modular organization, with several varieties of amino-terminal or carboxy-terminal modules flanking a conserved core. Acquisition of the penta-EF-hand module involved in calcium binding (and the formation of heterodimers for some calpains) seems to be a relatively late event in calpain evolution. Several alternative mechanisms for binding calcium and associating with membranes/phospholipids are found throughout the family. The gene family is expanded in mammals, trypanosomes and ciliates, with up to 26 members in Tetrahymena, for example; in striking contrast to this, only a single calpain gene is present in many other protozoa and in plants. The many isoforms of calpain and their multiple splice variants complicate the discussion and analysis of the family, and challenge researchers to ascertain the relationships between calpain gene sequences, protein isoforms and their distinct or overlapping functions. In mammals and plants it is clear that a calpain plays an essential role in development. There is increasing evidence that ubiquitous calpains participate in a variety of signal transduction pathways and function in important cellular processes of life and death. In contrast to relatively promiscuous degradative proteases, calpains cleave only a restricted set of protein substrates and use complex substraterecognition mechanisms, involving primary and secondary structural features of target proteins. The detailed physiological significance of both proteolytically active calpains and those lacking key catalytic residues requires further study. Gene organization and evolutionary historyThis review focuses on the eukaryotic calpains, although genome databases reveal bacteria, but no archaea, with sequences related to the catalytic core domains (domains dI and dII) of the classical calpains, the criterion used for designating a protein as a calpain. Only single copies of calpain-coding genes are found in the small number of sequenced or partially sequenced protozoan genomes, such as those of the apicomplexan parasites Plasmodium falciparum, Theileria annulata and Cryptosporidium parvum [1][2][3], and of the amitochondrial parasite Entamoeba histolytica [4]. No calpain-like sequences were identified in the human pathogen Giardia lamblia, a diplomonad often considered to be the most basal eukaryotic organism [5]. Protozoan calpains lack a domain containing EF-hand-type Ca 2+ -binding sites, as also do plant and fungal calpains, and thus it seems likely that the proposed cysteine proteasecalmodulin gene fusion leading to the classical calpain structure (for earlier reviews see [6][7][8]) occurred exclusively within the animal lineage....

Abstract. The sequence of tubulin-tyrosine ligase (TTL), the enzyme catalyzing the ATP-dependent posttranslational addition of a tyrosine to the carboxyterminal end of detyrosinated ot-tubulin, has been determined. TTL from bovine and porcine brain was purified by immunoaffinity chromatography and extensively characterized by protein sequencing. Oligonucleotides derived from the protein sequence were synthesized and partial eDNA sequences were obtained using reversed transcribed brain mRNA in polymerase chain reactions. Polymerase chain reaction fragments were used to isolate a full-length eDNA clone from a randomly primed hgtl0 eDNA library obtained from embryonic porcine brain mRNA. Porcine TTL is encoded by 1,137 nucleotides corresponding to 379 amino acid residues. It has a molecular weight of 43,425 and a calculated isoelectric point of 6.51. Northern blot analysis revealed a surprisingly long mRNA (~ 6 kb in embryonic porcine brain). The protein sequence of TTL shares no extended homology with the sequences in the data banks. TTL contains a potential serine phosphorylation site for cAMP-dependent protein kinase (RKAS at positions 73 to 76). Residues 244 to 258 lie at the surface of the molecule. A rabbit antibody raised against a synthetic peptide corresponding to this sequence binds to native TTL. The same sequence contains the cleavage site for endoproteinase Glu-C (residue 248) previously shown to convert TTL into a nicked derivative in which the two fragments still form a tight complex but don't display enzymatic activity.

The structural basis of mitosis, spindle organisation and chromosome segregation, in the unicellular parasite Trypanosoma brucei is poorly understood. Here, using immunocytochemistry, fluorescent in situ hybridisation and electron microscopy, we provide a detailed analysis of mitosis in this parasite. We describe the organisation of the mitotic spindle during different stages of mitosis, the complex ultrastructure of kinetochores and the identification of a potential spindle-organising centre in the mitotic nucleus. We investigate the dynamics of chromosome segregation using telomeric and chromosome-specific probes. We also discuss the problems involved in chromosome segregation in the light of the fact that the T. brucei karyotype has 22 chromosomes in the apparent presence of only eight ultrastructurally defined kinetochores.

African trypanosomes show monoallelic expression of one of about 20 telomeric variant surface glycoprotein (VSG) gene-expression sites (ESs) while multiplying in the mammalian bloodstream. We screened for genes involved in ES silencing using flow cytometry and RNA interference (RNAi). We show that a novel member of the ISWI family of SWI2/SNF2-related chromatin-remodelling proteins (TbISWI) is involved in ES downregulation in Trypanosoma brucei. TbISWI has an atypical protein architecture for an ISWI, as it lacks characteristic SANT domains. Depletion of TbISWI by RNAi leads to 30-60-fold derepression of ESs in bloodstream-form T. brucei, and 10-17-fold derepression in insect form T. brucei. We show that although blocking synthesis of TbISWI leads to derepression of silent VSG ES promoters, this does not lead to fully processive transcription of silent ESs, or an increase in ES-activation rates. VSG ES activation in African trypanosomes therefore appears to be a multistep process, whereby an increase in transcription from a silent ES promoter is necessary but not sufficient for full ES activation.

No abstract

The Trypanosoma brucei nuclear genome contains about 100 minichromosomes of between 50 to 150 kilobases and about 20 chromosomes of 0.2 to 6 megabase pairs. Minichromosomes contain nontranscribed copies of variant surface glycoprotein (VSG) genes and are thought to expand the VSG gene pool. Varying VSG expression allows the parasite to avoid elimination by the host immune system. The mechanism of inheritance of T. brucei chromosomes was investigated by in situ hybridization in combination with immunofluorescence. The minichromosome population segregated with precision, by association with the central intranuclear mitotic spindle. However, their positional dynamics differed from that of the large chromosomes, which were partitioned by kinetochore microtubules.

Most eukaryotic genomes contain large regions of satellite DNA. These arrays are often associated with essential chromosomal functions, but remain largely absent from genome projects because of difficulties in cloning and sequence assembly. The numerous small chromosomes of the parasite Trypanosoma brucei fall into this category, yet are critical to understanding the genome because of their role in antigenic variation. Their relatively small size, however, makes them particularly amenable to physical mapping. We have produced fine-resolution maps of 17 complete minichromosomes and partial maps of two larger intermediate-sized chromosomes. This revealed a canonical structure shared by both chromosomal classes based around a large central core of 177-bp repeats. Around the core are variable-length genic regions, the lengths of which define chromosomal class. We show the core region to be a repetitive palindrome with a single inversion point common to all the chromosomes of both classes, suggesting a mechanism of genesis for these chromosomes. Moreover, palindromy appears to be a feature of (peri)centromeres in other species that can be easily overlooked. We propose that sequence inversion is one of the higher-order sequence motifs that confer chromosomal stability.[Supplemental material is available online at www.genome.org. The following people kindly provided reagents, samples, or unpublished information as indicated in the paper: S. Melville and P. Borst.]African trypanosomes of the species Trypanosoma brucei are extracellular protozoan parasites of the mammalian bloodstream. They survive for long periods in the host bloodstream through a process of antigenic variation involving periodic switching of the major cell surface protein, variable surface glycoprotein (VSG; see Barry and McCulloch 2001). To facilitate this important survival mechanism, there exists in T. brucei a highly specialized gene organization that includes a large number of small chromosomes-classified on the basis of electrophoretic mobility as either minichromosomes (MCs) or intermediate-sized chromosomes (ICs), as shown in Figure 1 (see also El-Sayed et al. 2000). The MCs are small (30-150 kb), linear, and very numerous. A population of ∼100 MCs, comprising ∼10% of the nuclear DNA, is maintained by T. brucei as a means of expanding the number of available telomeric VSG genes (VSGs). Loci from this repertoire of MC VSGs, despite being fewer in number than those on other chromosomes, are preferred genes for antigenic switching events early in parasitemia (Robinson et al. 1999). Such switching events proceed by duplicative recombination of VSGs of minichromosomal origin into an active VSG expression site on a larger chromosome, the MCs themselves being transcriptionally silent.Central to the role of MCs in T. brucei pathogenesis is the diversity of VSGs they carry. During nuclear division, MCs are segregated via interactions with the mitotic spindle (Ersfeld and Gull 1997), and this segregation proceeds with considerable fidelity (Wickstead et ...