Cloning and sequencing of the Leishmania major actin-encoding gene.

We report the cloning and characterization of the genomic sequence of the actin (Act)-encoding gene (act) from Leishmania major. Restriction maps of two genomic clones, as well as genomic Southern analysis strongly suggest that the act of L. major is a single-copy gene. A single 1.6-kb transcript is detected in Northern blots. The deduced amino-acid sequence shows 68-89% identity with Act sequences from other eukaryotes.

An actin footprint on villin. Single site substitutions in a cluster of basic residues inhibit the actin severing but not capping activity of villin.

Villin is a multidomain protein that severs, caps, and bundles actin filaments. We employed a chemical modification/cleavage strategy to identify residues whose chemical reactivities are reduced when villin is complexed with actin. We found that actin protects 3 methionine residues, Met125, Met379, and Met711 from oxidation by N-chlorosuccinimide. Because Met125 lies within the actin-severing domain of villin (44T), we probed this region for actin binding sites using a series of overlapping peptides each with an additional cysteine residue at their C terminus. Each peptide, as a disulfide-bonded dimer, was examined for actin cross-linking activity by electron microscopy and light scattering. Our results with M3R suggest this region contains an F-actin binding site and are consistent with proteolysis and deletion mutagenesis studies of gelsolin. Single substitution of the basic residues modulated actin severing but not capping activity of 44T. Circular dichroism and protease digestions did not detect alterations in secondary structure or conformational changes in the mutants, although some are cleaved more rapidly, thereby suggesting a change in the packing of the domains. Our results highlight that basic residues comprise part of the F-actin binding site that is involved in the actin severing activity of villin.

Fimbrin is a homologue of the cytoplasmic phosphoprotein plastin and has domains homologous with calmodulin and actin gelation proteins.

Fimbrin is an actin-bundling protein found in intestinal microvilli, hair cell stereocilia, and fibroblast filopodia. The complete protein sequence (630 residues) of chicken intestine fimbrin has been determined from two full-length cDNA clones. The sequence encodes a small amino-terminal domain (115 residues) that is homologous with two calcium-binding sites of calmodulin and a large carboxy-terminal domain (500 residues) consisting of a fourfold-repeated 125-residue sequence. This repeat is homologous with the actin-binding domain of alpha-actinin and the amino-terminal domains of dystrophin, actin-gelation protein, and beta-spectrin. The presence of this duplicated domain in fimbrin links actin bundling proteins and gelation proteins into a common family of actin cross-linking proteins. Fimbrin is also homologous in sequence with human L-plastin and T-plastin. L-plastin is found in only normal or transformed leukocytes where it becomes phosphorylated in response to IL 1 or phorbol myristate acetate. T-plastin is found in cells of solid tissues where it does not become phosphorylated. Neoplastic cells derived from solid tissues express both isoforms. The differences in expression, sequence, and phosphorylation suggest possible functional differences between fimbrin isoforms.

Sequence of the 24S alpha ribosomal RNA gene and characterization of a corresponding pseudogene from Trypanosoma cruzi.

Two genomic clones which cover a 30-kb region containing the ribosomal RNA cistron from Trypanosoma cruzi have been isolated. The location of the 18S, 24S alpha and 24S beta RNA species within the cistron was determined. The complete sequences of the genes corresponding to the 24S alpha RNA and to a small RNA (S1), as well as two internal transcribed spacers were obtained by sequencing a cDNA and a genomic fragment. A locus containing sequences related to the 24S alpha RNA has been determined. Sequence data and structural characterization of this locus strongly suggest that this region contains a 24S alpha RNA pseudogene.

Terminal beta-D-galactofuranosyl epitopes recognized by antibodies that inhibit Trypanosoma cruzi internalization into mammalian cells.

Polyclonal antisera to 80 - 90-kDa and to 50 - 60-kDa polypeptides of tissue-culture trypomastigotes which inhibit the interiorization of trypomastigotes of the Y strain (up to 70%) and of metacyclic trypomastigotes of the CL-14 clone (up to 50%) in cultured mammalian cells, were obtained. Both sera immunoprecipitate surface polypeptides of 90 kDa, 80 kDa, 72 kDa and 58 kDa in trypomastigotes and of 80 kDa, 77 kDa and 74 kDa in metacyclic trypomastigotes. These antigens are glycoproteins with affinity for concanavalin A. The antibodies (IgG class) of the inhibitory sera are mainly directed against carbohydrate epitopes, which were identified as being beta-D-galactofuranosyl units by radioimmunoinhibition assays. The direct involvement of the beta-D-galactofuranosyl unit in the process of parasite infection was verified using the synthetic disaccharide beta-D-galactofuranose (1----3)-alpha-D-mannopyranose which promoted, at 1 mg/ml concentration, 50% inhibition of internalization.