A gene family encoding heterogeneous histone H1 proteins in Trypanosoma cruzi.

A gene family encoding a set of histone H1 proteins in Trypanosoma cruzi is described. The sequence of 3 genomic and 4 cDNA clones revealed the presence of several motifs characteristic of histone H1, although heterogeneity at the polypeptide level was evident. The clones encode histone H1 proteins of an unusually small size (74-97 amino acids), which lack the globular domain found in histone H1 of higher eukaryotes. All histone H1 mRNAs from T. cruzi are polyadenylated, although no typical polyadenylation signal was found. Furthermore, the genes encoding the histone H1 proteins in T. cruzi are found in a tandem array containing 15-20 gene copies per haploid genome. This tandem array is located on a large chromosome of 2.2 Mb.

H 1 histone and histone variants in Trypanosoma cruzi.

Trypanosoma cruzi chromatin is not condensed in chromosomes during mitosis. In previous studies a characteristic H 1 was not found in SDS or in acid-urea-PAGE. Consequently, it was proposed that the particular behavior of T. cruzi chromatin in dividing cells was due to the absence of an H 1 histone. In the present work, histones from this parasite were systematically characterized by spectrofluorometric analysis, amino acid composition, PAGE in one and in two dimensions, differential extraction with PCA and TCA, immunological cross-reactivity with antisera, and immunoblotting. We conclude that T. cruzi contains all five histones, H 1 presenting solubility and immunological properties similar to those in other species, but with a particular electrophoretic mobility in Triton-PAGE. Thus an explanation other than the absence of H 1 should be offered in order to understand the behavior of T. cruzi chromatin during mitosis. Moreover, histone variants were described by two-dimensional PAGE. The presence of histone variants suggests that they may participate in the regulation of cell proliferation and differentiation of this parasite, as it has been postulated for higher eukaryotes.

Trypanosoma cruzi histones. Further characterization and comparison with higher eukaryotes.

Histones extracted from T. cruzi chromatin were analyzed in three electrophoretic systems. Our results show that a basic protein with some properties similar to those of histone H1 from higher eukaryotes is present in T. cruzi. However this protein presents different electrophoretic mobilities than H1 histone from higher eukaryotes in all three electrophoretic systems tested. Considering the marked differences observed in the electrophoretic mobilities of T. cruzi histones as compared with those from higher eukaryotes, it is proposed that histones are conservative proteins primarily with regard to their function.

Histone synthesis in Trypanosoma cruzi.

Trypanosoma cruzi is an ancient, parasitic eukaryote which does not undergo chromatin condensation during cell division. This behavior may be explained if one considers the strong amino acid sequence divergence of Trypanosoma histones compared to higher eukaryotes. In the latter organisms histone synthesis is coupled to DNA replication. Considering the nonconserved amino acid sequence of T. cruzi histones, as well as the absence of chromatin condensation in this organism, we have studied histone synthesis in relation to DNA replication in this parasite. We have found that core histones and a fraction of histone H1 are synthesized concomitantly to DNA replication. However, another fraction of histone H1 is constitutively synthesized.

Presence of histone H2B in Trypanosoma cruzi chromatin.

The organization of chromatin in protists presents some characteristic features. In Trypanosoma cruzi, no condensation of chromatin into chromosomes is observed during cell division. A systematic characterization of histones should provide information on this peculiar behaviour. Histone H2B from this parasite was characterized by selective dissociation from chromatin in 0.8 M NaCl, by its elution pattern in narrow-bore reversed phase high performance liquid chromatography, by polyacrylamide gel electrophoresis and by partial sequencing of its amino terminal domain. This chromosomal protein differs from histone H2B of other species. The first 12 amino acids are missing which explains its lower molecular weight when compared to human histone H2B. Correspondingly, the amino terminal domain of T. cruzi histone H2B is 25-30% shorter than other histones H2B. Moreover, three out of four acetylation sites present in human histone H2B are missing in T. cruzi histone H2B. The differences in size and in acceptor sites for acetylation of T. cruzi histone H2B when compared to human histone H2B may represent a functional feature to consider for the understanding of the chromatin cycle of condensation in this parasite.

Unambiguous identification of histone H1 in Trypanosoma cruzi.

The existence of histone H1 has been questioned in Trypanosomatids. We report here the presence of a histone H1 in the chromatin of Trypanosoma cruzi. This protein was purified by narrow-bore reversed phase HPLC and its amino acid composition analyzed and compared with histones H1 from other species. Furthermore, the purified chromosomal protein was digested with proteases and the amino acid sequences of the resulting peptides were analyzed by the automated Edman degradation. The sequences obtained were found to present a high degree of homology when compared to the carboxy terminal domain of other known histones H1.

Histone H1 and core histones in Leishmania and Crithidia: comparison with Trypanosoma.

The Trypanosomatidae family is characterized by flagellated protozoa presenting a kinetoplast. Several genera of this family contain species that are pathogenic to man and domestic animals. Their chromatin is not condensed into chromosomes during cell division. As a contribution to the understanding of basic aspects of their genome organization, we present a systematic characterization of the histones from three genera of the Trypanosomatidae family. Crithidia fasciculata and Leishmania mexicana show core nucleosomal histones with electrophoretic mobilities both similar to and different from those of Trypanosoma cruzi and higher eukaryotes. Another protein is extracted from the chromatin of these organisms by procedures designed to purify histone H1. This protein presents elution profiles by HPLC and amino acid composition of histone H1. Considering these data and the high mobility of this protein in Triton-acetic acid-urea-polyacrylamide gel electrophoresis, as well as its position relative to the nucleosomal core histones in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we postulate that Crithidia and Leishmania possess a histone H1 shorter than that of higher eukaryotes as we have previously shown to be the case for T. cruzi. The possible presence of a shorter histone H1 in these trypanosomatids may explain the absence of chromatin condensation during cell division in these flagellates.

Extremely divergent histone H4 sequence from Trypanosoma cruzi: evolutionary implications.

Trypanosoma cruzi presents six histones electrophoretically resolved in three gel systems. Indirect evidence shows that one of these histones, named e, corresponds to H4 in other species. We present evidence that histone e is H4 by sequencing its amino terminal end. The amino terminal of T. cruzi histone H4, unlike that of other H4s examined thus far is not blocked. Moreover, this protein presents two variants. This partial amino acid sequence of T. cruzi histone H4 differs greatly from homologous sequences of human, yeast, or Tetrahymena. Since the conservatism of the core histones (H2A, H2B, H3, and H4) is clearly illustrated by comparative sequence analyses, the data shown here demonstrates that T. cruzi histone H4 is the most divergent reported. Quantitative analysis of the data suggests that the rate of substitutions in the histone H4 amino terminal sequence varies among different lineages. We postulate a slow-down in the evolutionary rate of histone H4 amino terminal domain in the metazoa branch related perhaps to the appearance of a novel function for this domain.

Histone genes in trypanosomatids.

Histone genes in Trypanosomatids are of considerable interest because these flagellates do not condense their chromatin during mitosis. In contrast to higher eukaryotes, histone genes in Trypanosomatids are found on separate chromosomes, and their transcripts are polyadenylated. Sequence similarity of Trypanosomatid core histones with those of higher eukaryotes is found predominantly in the globular region; the N-terminal is highly divergent. Finally, in general, Trypanosomatid histones H1 are of low molecular weight, bearing closest homology to the C-terminal region of the higher eukaryote histones H1. These features constitute interesting targets for a rational approach to the study of these protozoa, as discussed here by Norbel Galanti and colleagues.

Chromatin and histones from Giardia lamblia: a new puzzle in primitive eukaryotes.

The three deepest eukaryote lineages in small subunit ribosomal RNA phylogenies are the amitochondriate Microsporidia, Metamonada, and Parabasalia. They are followed by either the Euglenozoa (e.g., Euglena and Trypanosoma) or the Percolozoa as the first mitochondria-containing eukaryotes. Considering the great divergence of histone proteins in protozoa we have extended our studies of histones from Trypanosomes (Trypanosoma cruzi, Crithidia fasciculata and Leishmania mexicana) to the Metamonada Giardia lamblia, since Giardia is thought to be one of the most primitive eukaryotes. In the present work, the structure of G. lamblia chromatin and the histone content of the soluble chromatin were investigated and compared with that of higher eukaryotes, represented by calf thymus. The chromatin is present as nucleosome filaments which resemble the calf thymus array in that they show a more regular arrangement than those described for Trypanosoma. SDS-polyacrylamide gel electrophoresis and protein characterization revealed that the four core histones described in Giardia are in the same range of divergence with the histones from other lower eukaryotes. In addition, G. lamblia presented an H1 histone with electrophoretic mobility resembling the H1 of higher eukaryotes, in spite of the fact that H1 has a different molecular mass in calf thymus. Giardia also presents a basic protein which was identified as an HU-like DNA-binding protein usually present in eubacteria, indicating a chimaeric composition for the DNA-binding protein set in this species. Finally, the phylogenetic analysis of selected core histone protein sequences place Giardia divergence before Trypanosoma, despite the fact that Trypanosoma branch shows an acceleration in the evolutionary rate pointing to an unusual evolutionary behavior in this lineage.