* Unité d'Organisation Nucléaire et Oncogénèse, INSERM U579 and Unité de Rétrovirologie Moléculaire URA 1930, Institut Pasteur, Paris, France;
Service de Systématique Moléculaire, Institut de Systématique, Museum National d'Histoire Naturelle, Paris, France;
UMR 5143 CNRS, Paléobiodiversité, Museum National d'Histoire Naturelle, Paris, France; || Zoo de la Palmyre, Les Mathes, France; ¶ Services Vétérinaires, Zoo de Vincennes, Museum National d'Histoire Naturelle, Paris, France; and # Jardin Zoologique, Mulhouse, France
Correspondence: E-mail: ppineau{at}pasteur.fr.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: histone H4 PCR vertebrates
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Histones are structural and functional components of chromatin. Five classes of histones were originally characterized (H1, H2A, H2B, H3, and H4). The number of histone genes per haploid genome varies substantially from species to species from a few copies in mammals and birds up to approximately 500 copies organized in tandem clusters in certain sea urchin species. In mammals and birds, histone genes are usually mapping at a few genomic loci that contain clusters or individual genes. In humans, the largest cluster of histone genes, HIST1, is located on chromosome 6 (6p21-p22), and two smaller clusters, HIST2 (1q21) and HIST3 (1q41), are located on chromosome 1. In addition to the histone genes on these clusters, a histone H4 gene (HIST4) was identified on chromosome 12.
We focused on histone H4 genes considered as some of the most conserved genes in eukaryotes (DeLange and Smith 1971; Wells and Brown 1991; Thatcher and Gorovsky 1994). Each of the 14 human histone H4 genes encodes the same protein. The H4 genes in HIST2 and HIST4 on chromosomes 1 and 12 are, thus, very similar to the genes in HIST1 on chromosome 6, indicating that there is a strong selective pressure to preserve a similar nucleotide sequence in the coding region. Even so, histones H3 and H4 evolve on the order of 10 times more slowly than H2A and H2B (Thatcher and Gorovsky 1994). In addition, pseudogenes of histone H4 exist in the human genome sequence.
In the current report, we present a method for amplification of nuclear histone H4 genes that is suitable for DNA quality testing, as well as for species identification in the animal kingdom.
![]() |
Material and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Sequences Analysis
For all 43 species, amplified fragments were purified from agarose gels (Qiaex II kit, Qiagen, France) and ligated in the TOPO TA cloning vector (InVitrogen, France). Sequencing was performed using Thermosequenase (USB, Amersham) on an ABI373A sequencer (Applied Biosystems). Five clones were sequenced for each of these 43 species. In addition, between three and five clones from each mammoth specimens were sequenced. Sequences were aligned by using the ClustalW program (Thompson, Higgins, and Gibson 1994). Nucleotide distances were determined with DNAdist of the PHYLIP package version 3.5 (Felsenstein 1993). The tree was derived by neighbor-joining (NJ) analysis applied to pairwise distances calculated using the Kimura two-parameter method to generate unrooted trees. Robustness of the NJ phylogenetic tree was tested by boostrap analysis. Horizontal branch lengths are drawn to scale with the bar indicating 0.1 nucleotides replacements per site. The final output was generated with Treeview (Page 1996). The number at each node represents the percentages of bootstrap replicates (out of 100). Only bootstrap values of greater than 60 are given. The sequences can be found in GenBank under accession numbers AY675260 to AY675296.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amplification from Vertebrates, Nonvertebrates, and Clinical Samples
All vertebrate samples (n = 35) yielded amplification products either after a single round or a heminested amplification procedure (table 1). Those of the vertebrates samples yielding amplimers only after the second round of amplification originated in most cases (nine of 11 cases) from degraded tissue specimens obtained from animals experiencing severe acute hepatitis or from inappropriately stored samples (fig. 1A and B). In the remaining cases, (n = 25 of 36, or 67%), a single round of PCR cycles was sufficient to obtain a detectable amplimer. As expected, after 35 cycles, amplification of the smaller internal (181 bp) fragment was overall more efficient than that of the larger one (211 bp, 25 versus 20 positive samples). For invertebrates, the heminested procedure was often necessary (n = 3 of 9) to obtain a sufficient amplification product (table 1).
|
Relative Sensitivity of the Method
We next examined the amount of DNA required for HIST2H4 assay. An absolute comparison on different genomes is not possible because the exact genome sizes and composition with regard to the number of histone H4 genes are not known for most of the animal species. Broad differences in amplification efficiency attributable to the number of histone H4 genes per genome (sometimes in accordance with genome size) or to particular homology with degenerate primers may be detected by comparing different high-molecular-weight DNA samples (Bensasson et al. 2001; Ishaq et al. 1993). Six different samples were tested by serial 10-fold dilutions ranging from 100 ng to 0.1 fg. Species were chosen in each of the vertebrate classes (Bufo bufo, Mabuya quinquetanea, Melopsittacus undulatus, and Cercopithecus aethiops) and a nonvertebrate was also added (Locusta migratoria). First round of amplification yielded clear amplimeres within a range of 1 ng (Bufo bufo, Mabuya quinquetanea, Melopsittacus undulatus, and Cercopithecus aethiops) to 10 pg (Locusta migratoria), whereas the second round of PCR extended the detection range from 2 pg (Mabuya quinquetanea, and Cercopithecus aethiops) to 20 fg (Locusta migratoria, Bufo bufo, and Melopsittacus undulatus) (data not shown).
Degraded, Ancient, and Extinct DNA
Some samples, considered as difficult to explore (i.e., chimpanzee feces, ape teeth, and mammoth tissues) were then subjected to PCR for the HIST2H4 target gene. Amplification was successful in six out of 11 DNA samples extracted from chimpanzee feces (55%), and ape teeth yielded a product in seven out of nine cases (77%). The oldest DNA extracted from a tooth was dating from 1885 (G. gorilla). Finally, we were able to amplify three of three Siberian mammoth samples dating from 40,000 to 49,000 years before present (table 2). These data indicate that HIST2H4 amplification is robust enough to be performed on modern and old remains.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The strong conservation of histone H4 across evolution from slime molds and plants to mammals has been known since early studies were performed on proteins (DeLange and Smith 1971). In contrast, protists represent the only group displaying substantial variation within H4 sequences (Bernhard and Schlegel 1998; Van Den Bussche et al. 2000). Our data demonstrate the usefulness of HIST2H4 amplification in virtually all animal species (table 1). The assessment of nuclear DNA amplifiability (i.e., quality) in samples in which DNA is partially degraded is possible for any species when the nested procedure is used. With regard to quantitative aspects, HIST2H4 amplification may rival previously described methods aiming at rRNA or mtDNA. Ribosomal 18S/28S RNA (rRNA) genes are tandemly organized gene clusters on acrocentric arms of chromosomes (Zardoya and Meyer 1996). The number of rRNA genes per haploid genome varies greatly (50 to 800 copies) among species. A similar method is based on cytochrome b detection. This gene is encoded by mitochondrial genomes, which vary widely in number per cell (10 to 105) with tissue type, age, and pathology (Jansen 2000), thus, making them unsatisfactory for an accurate nuclear DNA quality assessment. The method developed in the current report is, however, also not strictly aimed at a single-copy nuclear gene. Indeed, in sea urchin (e.g., P. miliaris and S. Purpuratus) or in D. melanogaster, histones genes are organized into a single type of tandemly repeated (100 to 500 copies) cluster. The situation differs substantially in vertebrate species. In that case, the relatively low repetition (e.g., 14 H4 genes in man, four in mouse, and seven in chicken) has no consistent organization. In mammals and birds, clusters of histone genes may be composed of different repeating units, and sometimes no basic repeated entity is found in the cluster. Consequently, several nonallelic genes may be detected, with primers used primarily to detect HIST2H4 orthologs (Marzluff et al. 2002). Nevertheless, the relatively small number of paralogs of HIST2H4 makes this assay a rather faithful reflection of the status of a regular nuclear gene. HIST2H4 detection may, thus, represent an alternative or an additional target for characterization of samples from phylogenetically distant organisms (fig. 2). Similarly to previously published assays, the present procedure can be employed with a wide taxonomic utility (Meyer, Candrian, and Luthy 1994; Bataille et al. 1999). Because the mitochondrial genome is inherited maternally in most animals, the use of a nuclear DNA amplification method is essential when molecular data based on biparentally inherited sequences are needed (Palumbi and Cipriano 1998; Slade et al. 1998). In addition, recent analyses showed that phylogenetic results based on mitochondrial genomes often need to be confirmed with data from nuclear genes (Cotton and Page 2002). Similarly, several questions could not be resolved by rRNA sequence comparisons. Histone H4 may, thus, represent a truly independent marker for testing the robustness of rRNA or mtDNA phylogenies (Bernhard and Schlegel 1998). One peculiarity of histone H4 should, however, be kept in mind. Histone H4 is considered as virtually invariant at the protein level among vertebrates. Fortunately, the extent of overall nucleotide sequence divergence is higher than that of proteins (Piontkivska, Rooney, and Nei 2002). Most of the nucleotide sequence variation takes the form of synonymous substitutions, reaching apparently the saturation level in many species. It has been shown formerly that this situation may explain that distant species appear closely related on the phylogenetic tree (Piontkivska, Rooney, and Nei 2002).
|
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bataille, M., K. Crainic, M. Leterreux, M. Durigon, and P. de Mazancourt. 1999. Multiplex amplification of mitochondrial DNA for human and species identification in forensic evaluation. Forensic Sci. Int. 99:165170.[CrossRef][ISI][Medline]
Bellagamba, F., F. Valfre, S. Panseri, and V. Moretti. 2003. Polymerase chain reaction-based analysis to detect terrestrial animal protein in fish meal. J. Food. Prot. 66:682685.[ISI][Medline]
Bensasson, D., D. Petrov, D. Zhang, D. Hartl, and G. Hewitt. 2001. Genomic gigantism: DNA loss is slow in mountain grasshoppers. Mol. Biol. Evol. 18:246253.
Bernhard, D., and M. Schlegel. 1998. Evolution of histone H4 and H3 genes in different ciliate lineages. J. Mol. Evol. 46:344354.[ISI][Medline]
Cotton, J., and R. Page. 2002. Going nuclear: gene family evolution and vertebrate phylogeny reconciled. Proc. R Soc. Lond. B Biol. Sci. 269:15551561.[CrossRef][ISI][Medline]
Debruyne, R., V. Barriel, and P. Tassy. 2003. Mitochondrial cytochrome b of the Lyakhov mammoth (Proboscidea, Mammalia): new data and phylogenetic analyses of Elephantidae. Mol. Phylogenet. Evol. 26:421434.[CrossRef][ISI][Medline]
DeLange, R., and E. Smith. 1971. Histones: structure and function. Annu. Rev. Biochem. 40:279314.[CrossRef][ISI][Medline]
Felsenstein, J. 1993. PHYLIP (phylogeny inference package). Version 3.5. Distributed by the author, Department of Genetics, University of Washington, Seattle.
Hassanin, A., G. Lecointre, and S. Tillier. 1998. The 'evolutionary signal' of homoplasy in protein-coding gene sequences and its consequences for a priori weighting in phylogeny. C. R. Acad. Sci. III. 321:611620.[CrossRef][ISI][Medline]
Hecker, K. H., and K. H. Roux. 1996. High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR. Biotechniques 20:478485.[ISI][Medline]
Ishaq, A., S. Rizvi, D. Wells, and C. Tomlinson. 1993. A characterization of the H3 and H4 histone genes from the ascidian Styela plicata. Biochem. Biophys. Res. Commun. 194:775783.[CrossRef][ISI][Medline]
Jansen, R. 2000. Germline passage of mitochondria: quantitative considerations and possible embryological sequelae. Hum. Reprod. 15:112128.
Kocher, T., W. Thomas, A. Meyer, S. Edwards, S. Paabo, F. Villablanca, and A. Wilson. 1989. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc. Natl. Acad. Sci. USA 86:61966200.[Abstract]
Marzluff, W., P. Gongidi, K. Woods, J. Jin, and L. Maltais. 2002. The human and mouse replication-dependent histone genes. Genomics 80:487498.[CrossRef][ISI][Medline]
Meyer, R., U. Candrian, and J. Luthy. 1994. Detection of pork in heated meat products by the polymerase chain reaction. J. AOAC Int. 77:617622.[ISI][Medline]
Naito, E., K. Dewa, H. Ymanouchi, and R. Kominami. 1992. Ribosomal ribonucleic acid (rRNA) gene typing for species identification. J. Forensic Sci. 37:396403.[ISI][Medline]
Page, R. D. M. 1996. Treeview: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12:357358.[Medline]
Palumbi, S., and F. Cipriano. 1998. Species identification using genetic tools: the value of nuclear and mitochondrial gene sequences in whale conservation. J. Hered. 89:459464.
Piontkivska, H., A. Rooney, and M. Nei. 2002. Purifying selection and birth-and-death evolution in the histone H4 gene family. Mol. Biol. Evol. 19:689697.
Slade, R., C. Moritz, A. Hoelzel, and H. Burton. 1998. Molecular population genetics of the southern elephant seal Mirounga leonina. Genetics 149:19451957.
Sperling, F., G. Anderson, and D. Hickey. 1994. A DNA-based approach to the identification of insect species used for postmortem interval estimation. J. Forensic Sci. 39:418427.[ISI][Medline]
Thatcher, T., and M. Gorovsky. 1994. Phylogenetic analysis of the core histones H2A, H2B, H3, and H4. Nucleic Acids Res. 22:174179.[Abstract]
Thompson, J., D. Higgins, and T. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:46734680.[Abstract]
Vartanian, J., and S. Wain-Hobson. 2002. Analysis of a library of macaque nuclear mitochondrial sequences confirms macaque origin of divergent sequences from old oral polio vaccine samples. Proc. Natl. Acad. Sci. USA 99:75667569.
Van Den Bussche, R., S. Hoofer, C. Drew, and M. Ewing. 2000. Characterization of histone H3/H4 gene region and phylogenetic affinity of Ichthyophthirius multifiliis based on H4 DNA sequence variation. Mol. Phylogenet. Evol. 14:461468.[CrossRef][ISI][Medline]
Wallace, D. C., C. Stugard, D. Murdock, T. Schurr, and M. D. Brown. 1997. Ancient mtDNA sequences in the human nuclear genome: a potential source of errors in identifying pathogenic mutations. Proc. Natl. Acad. Sci. USA 94:1490014905.
Wan, Q., and S. Fang. 2003. Application of species-specific polymerase chain reaction in the forensic identification of tiger species. Forensic. Sci. Int. 131:7578.[CrossRef][ISI][Medline]
Wells, D., and D. Brown. 1991. Histone and histone gene compilation and alignment update. Nucleic Acids Res. 19:21732188.[ISI][Medline]
Zardoya, R., and A. Meyer. 1996. Evolutionary relationships of the coelacanth, lungfishes, and tetrapods based on the 28S ribosomal RNA gene. Proc. Natl. Acad. Sci. USA 9 3:54495454.[CrossRef]
|