The sequence and organization of the core histone H3 and H4 genes in the early branching amitochondriate protistTrichomonas vaginalis

Among the unicellular protists, several of which are parasitic, some of the most divergent eukaryotic species are found. The evolutionary distances between protists are so large that even slowly evolving proteins like histones are strongly divergent. In this study we isolated cDNA and genomic histone H3 and H4 clones fromTrichomonas vaginalis. Two histone H3 and three histone H4 genes were detected on three genomic clones with one complete H3 and two complete H4 sequences. H3 and H4 genes were divergently transcribed with very short intergenic regions of only 194 bp, which containedT. vaginalis-specific as well as histone-specific putative promoter elements. Southern blot analysis showed that there may be several more histone gene pairs. The two complete histone H4 genes were different on the nucleotide level but encoded the same amino acid sequence. Comparison of the amino acid sequences of theT. vaginalis H3 and H4 histones with sequences from animals, fungi, and plants as well as other protists revealed a significant divergence not only from the sequences in multicellular organisms but especially from the sequences in other protists likeEntamoeba histolytica, Trypanosoma cruzi, andLeishmania infantum.     

Variation in Coding (NADH Dehydrogenase Subunits 2, 3, and 6) and Noncoding Intergenic Spacer Regions of the Mitochondrial Genome in Octocorallia (Cnidaria: Anthozoa)

Low rates of evolution in cnidarian mitochondrial genes such as COI and 16S rDNA have hindered molecular systematic studies in this important invertebrate group. We sequenced fragments of 3 mitochondrial protein-coding genes (NADH dehydrogenase subunits ND2, ND3 and ND6) as well as the COI-COII intergenic spacer, the longest noncoding region found in the octocoral mitochondrial genome, to determine if any of these regions contain levels of variation sufficient for reconstruction of phylogenetic relationships among genera of the anthozoan subclass Octocorallia. Within and between the soft coral families Alcyoniidae and Xeniidae, sequence divergence in the genes ND2 (539 bp), ND3 (102 bp), and ND6 (444 bp) ranged from 0.5% to 12%, with the greatest pairwise distances between the 2 families. The COI-COII intergenic spacer varied in length from 106 to 122 bp, and pairwise sequence divergence values ranged from 0% to 20.4%. Phylogenetic trees constructed using each region separately were poorly resolved. Better phylogenetic resolution was obtained in a combined analysis using all 3 protein-coding regions (1085 bp total). Although relationships among some pairs of species and genera were well supported in the combined analysis, the base of the alcyoniid family tree remained an unresolved polytomy. We conclude that variation in the NADH subunit coding regions is adequate to resolve phylogenetic relationships among families and some genera of Octocorallia, but insufficient for most species - or population-level studies. Although the COI-COII intergenic spacer exhibits greater variability than the protein-coding regions and may contain useful species-specific markers, its short length limits its phylogenetic utility.     

Phylogenetic relationships and unusual diversity in histone H4 proteins within the Tetrahymena pyriformis complex.

The 29 species of the Tetrahymena pyriformis complex are morphologically identical while being quite diverse at the molecular level. These species are also diverse relative to other eukaryotes. Phylogenetic relationships within the complex have been difficult to determine, because there are groups of closely related species, as well as individual species, that are highly divergent. We have sequenced portions of two highly conserved histone genes and the more rapidly evolving intergenic region between them. These sequences have been used to construct a phylogeny for the complex. A comparison of the amino-terminal portion of the histone H4 proteins from the species of the complex reveals a high degree of sequence diversity relative to that of other organisms. In contrast, the amino-terminal portions of the histone H3 proteins of the species in the complex are identical to each other. We also find that the pattern of nucleotide substitution in the intergenic region differs from that described for higher eukaryotes.     

Evolution of Histone H4 and H3 Genes in Different Ciliate Lineages

The histones H4 are known as highly conserved proteins. However, in ciliates a high degree of variation was found compared both to other eukaryotes and between the ciliate species. To date, only H4 histones of species belonging to two distantly related classes have been investigated. In order to obtain more detailed information on histone H4 variation in ciliates we undertook a comprehensive sequence analysis of PCR-amplified internal H4 fragments from 12 species belonging to seven out of the nine currently recognized ciliate classes. In addition, we used PCR primers to amplify longer fragments of H3 and H4 genes including the intergenic region. The encoded amino acid sequences reveal a high number of differences when compared with those of other eukaryotes and the ciliate species investigated. Furthermore, in some species H4 gene variants were detected, which result in amino acid differences. The greatest number of substitutions and insertions found was in the amino terminal region of the H4 histones. However, all sequences possess a conserved region corresponding to those of all other eukaryotic H4 histones. The histone gene variations were used to reconstruct phylogenetic relationships. The tree from our data matches perfectly with the ribosomal RNA data: The heterotrichs, which were considered as a late branching lineage, diverge at the base of the ciliate tree and groups formerly thought to represent ancestral lineages now appear as highly derived ciliates. Received: 4 April 1997 / Accepted: 1 August 1997     

Characterization of the promoter region of Tetrahymena genes.

The regions between adjacent histone H3 and H4 genes, as well as portions of the genes, from 22 species of Tetrahymena have been amplified using the polymerase chain reaction and sequenced. Both histone genes are transcribed divergently with initiation occurring within the intergenic region, thus 2 sets of 22 homologous Tetrahymena promoters can be compared. A sequence comparison of these regions reveals a single putative promoter element, with a consensus sequence TATCCAATTCARA, present in front of each gene. This sequence contains a 'CCAAT' box, which also occurs at 8 locations preceding other ciliate genes. No other putative promoter sequences are found in front of these sets of histone genes. Sequences searched for include 'TATA' boxes, 'GC' boxes and other sequences suggested as putative promoter elements for ciliate genes. The coding strand immediately preceding ciliate genes is very high in A content and the consensus sequence at the site of protein synthesis is AAAATGG.     

Structure of the intergenic spacer region from the ribosomal RNA gene family of white spruce (Picea glauca)

Five genomic clones containing ribosomal DNA repeats from the gymnosperm white spruce (Picea glauca) have been isolated and characterized by restriction enzyme analysis. No nucleotide variation or length variation was detected within the region encoding the ribosomal RNAs. Four clones which contained the intergenic spacer (IGS) region from different rDNA repeats were further characterized to reveal the sub-repeat structure within the IGS. The sub-repeats were unusually long, ranging from 540 to 990 bp but in all other respects the structure of the IGS was very similar to the organization of the IGS from wheat, Drosophila and Xenopus.     

An H3-H4 histone gene pair in the marine copepod Tigriopus californicus, contains an intergenic dyad symmetry element.

Histone genes are one of the most widely studied multigene families in eucaryotes. Over 200 histone genes have been sequenced, primarily in vertebrates, echinoderms, fungi and plants. We present here the structure and genomic orientation of an H3-H4 histone gene pair from the marine copepod, Tigriopus californicus. These histone gene sequences are the first to be determined for the class Crustacea and among the first to be determined for protostomes. The H4 and H3 genes in Tigriopus are shown to be adjacent, to have opposite polarity, and to contain a 26 bp region of dyad symmetry centrally located within the spacer region between the two genes. A similarly located dyad element has been found in yeast which contributes to the coordinated cell cycle control of the adjacent histone genes. The Tigriopus H3-H4 histone gene pair is clustered with one H2A and two H2B histone genes on a 15 kb genomic Bam H1 fragment. The H4 gene sequence predicts an H4 protein with an unusual serine to threonine substitution at the amino terminal residue. The H3 gene sequence predicts an H3 protein which is identical to the vertebrate H3.2 histone.     

Phylogenetic relationships amongTetrahymena species determined using the polymerase chain reaction

Summary The species of theTetrahymena pyriformis complex present a conundrum with regard to their highly conservative morphology and widely divergent molecular characteristics. We have investigated the phylogenetic relationships among these species using the nucleotide sequences from the histone H3II/H4II region of the genome. This region includes portions of the two histone coding sequences, as well as the intergenic region. The DNA sequences of these regions were amplified by the polymerase chain reaction (PCR) and the sequence of each was determined. Nucleotide substitutions and insertions/deletions within this set of sequences were compared to determine the phylogenetic relationships among the species of the complex. These data yield phylogenetic trees with identical topologies when different tree-building routines are used, indicating that the data are very robust.Glaucoma chattoni was used as an outgroup to root the trees for this analysis. The genome organization ofG. chattoni and the divergence of its histone H3II/H4II region sequence relative to those of the complex clearly indicate that this species has diverged considerably from the complex. These results show that PCR amplification analysis is feasible over considerable evolutionary distances. However, DNA-DNA hybridization may be more useful than sequence analysis in resolving the relationships among the closely related species in the complex.     

Phylogeography and conservation genetics of Hygrophila pogonocalyx (Acanthaceae) based on atpB–rbcL noncoding spacer cpDNA

Genetic variation in the atpB–rbcL intergenic spacer region of chloroplast DNA (cpDNA) was investigated in Hygrophila pogonocalyx Hayata (Acanthaceae), an endangered and endemic species in Taiwan. In this aquatic species, seed dispersal from capsules via elasticity is constrained by gravity and is thereby confined within populations, resulting in limited gene flow between populations. In this study, a total of 849 bp of the cpDNA atpB–rbcL spacer were sequenced from eight populations of H. pogonocalyx. Nucleotide diversity in the cpDNA is low (=0.00343±0.00041). The distribution of genetic variation among populations agrees with an isolation-by-distance model. Two geographically correlated groups, the western and eastern regions, were identified in a neighbor-joining tree and a minimum-spanning network. Phylogeographical analyses based on the cpDNA network suggest that the present-day differentiation between western and eastern groups of H. pogonocalyx resulted from past fragmentation. The differentiation between eastern and western populations may be ascribed to isolation since the formation of the Central Mountain Range about 5 million years ago, which is consistent with the rate estimates based on a molecular clock of cpDNA.Huang JC and Wang WK equally contributed to this work.     

Variability and inheritance of histone genes H3 and H4 in Vicia faba

Summary We have compared copy numbers and blothybridization patterns of histone genes (H3 plus H4) between and within individuals of broad bean (Vicia faba). Copy number differences among individuals in the population of 200 individuals were as great as 27 fold, and as much as 3.2 fold among separate leaves of the same plant. Among F₂ progeny from genetic crosses, up to a 5.4-fold range was seen (mean=3.5 fold), and among F₁ progeny of self-pollinated plants, up to a 5.9-fold range was observed (mean=2.3 fold). Histone gene blot-hybridization patterns for EcoRI and HindIII were also variable among individuals and indicated that the genes are probably clustered in only a few chromosomal loci. The degree of variation in histone gene copy number per haploid genome (2–55 copies, or 27 fold) was similar to that found previously for ribosomal RNA genes (230–22000, or 95 fold) of V. faba. However, the two gene families change independently, since individuals with a high or low copy number for one gene can have either a high or low copy number for the other. The mechanisms(s) for rapid gene copy number change may be similar for these gene families.     

Cloning and characterization of the major histone H2A genes completes the cloning and sequencing of known histone genes of Tetrahymena thermophila.

A truncated cDNA clone encoding Tetrahymena thermophila histone H2A2 was isolated using synthetic degenerate oligonucleotide probes derived from H2A protein sequences of Tetrahymena pyriformis. The cDNA clone was used as a homologous probe to isolate a truncated genomic clone encoding H2A1. The remaining regions of the genes for H2A1 (HTA1) and H2A2 (HTA2) were then isolated using inverse PCR on circularized genomic DNA fragments. These partial clones were assembled into intact HTA1 and HTA2 clones. Nucleotide sequences of the two genes were highly homologous within the coding region but not in the noncoding regions. Comparison of the deduced amino acid sequences with protein sequences of T. pyriformis H2As showed only two and three differences respectively, in a total of 137 amino acids for H2A1, and 132 amino acids for H2A2, indicating the two genes arose before the divergence of these two species. The HTA2 gene contains a TAA triplet within the coding region, encoding a glutamine residue. In contrast with the T. thermophila HHO and HTA3 genes, no introns were identified within the two genes. The 5'- and 3'-ends of the histone H2A mRNAs; were determined by RNase protection and by PCR mapping using RACE and RLM-RACE methods. Both genes encode polyadenylated mRNAs and are highly expressed in vegetatively growing cells but only weakly expressed in starved cultures. With the inclusion of these two genes, T. thermophila is the first organism whose entire complement of known core and linker histones, including replication-dependent and basal variants, has been cloned and sequenced.     

Characterization of two types of histone H2B genes from macronuclei of Tetrahymena thermophila.

Two histone H2B gene clones were isolated from macronuclei of Tetrahymena thermophila. Nucleotide sequences of the two clones were highly homologous within the coding region but not in the noncoding region. Comparison of the deduced amino acid sequences between the two clones showed three differences in a total of 121 amino acids. Each of the two clones contained a TAA triplet within the coding region, which appeared to code for a glutamine residue. To demonstrate the existence of histone mRNA containing UAA triplet, nuclease P1 protection mapping using total cellular RNA and nucleotide sequencing of primer extension products were carried out. The results clearly indicated that two cloned histone H2B genes were transcribed, giving rise to the major histone H2B mRNAs with a UAA triplet sequence in frame. The tentative 5'- and 3'-ends of histone H2B mRNAs were determined.     

Molecular identification of Tetrahymena species

Mitochondrial cox1 689 bp barcodes are routinely used for identification of Tetrahymena species. Here, we examine whether two shorter nuclear sequences, the 5.8S rRNA gene region and the intergenic region between H3 and H4 histone genes, might also be useful either singly or in combination with each other or cox1. We obtained sequences from ~300 wild isolates deposited at the Tetrahymena Stock Center and analyzed additional sequences obtained from GenBank. The 5.8S rRNA gene and portions of its transcribed flanks identify isolates as to their major clade and uniquely identify some, but not all, species. The ~330 bp H3/H4 intergenic region possesses low intraspecific variability and is unique for most species. However, it fails to distinguish between two pairs of common species and their rarer counterparts, and its use is complicated by the presence of duplicate genes in some species. The results show that while the cox1 sequence is the best single marker for Tetrahymena species identification, 5.8S rRNA, and the H3/H4 intergenic regions sequences are useful, singly or in combination, to confirm cox1 species assignments or as part of a preliminary survey of newly collected Tetrahymena. From our newly collected isolates, the results extend the biogeographical range of T. shanghaiensis and T. malaccensis and identify a new species, Tetrahymena arleneae n. sp. herein described.     

Cloning and structural analyses of partial nuclear rDNA fromBupleurum euphorbioides (Apiaceae)

For the cloning of nuclear ribosomal DNA (rDNA) fromBupleurum euphorbioides (Apiaceae), ten clones were screened by DNA-DNA hybridization method. Among them, two clones were strongly hybridized with a heterologous probe of rice rDNA and with an autologous probe of an internally-transcribed region ofB. euphorbioides amplified by PCR. We sequenced both ends of the two genomic clones aligned with a known sequence of rDNA. ITS2 sequences of the two clones showed 98% and 83% homology with the ITS2 sequence ofB. euphorbioides. Our clones showed 1 bp and 3 bp nucleotide substitutions in the 25S and intergenic spacer regions, respectively, and the ITS1 and 18S regions were both missing. Restriction enzyme sites and the orientation of both clones were analyzed for physical mapping purposes. Apart from the length difference between the two clones, we found restriction site variations in the 25S and intergenic spacer regions.