Birth-and-death evolution with strong purifying selection in the histone H1 multigene family and the origin of orphon H1 genes

Histones are small basic nuclear proteins with critical structural and functional roles in eukaryotic genomes. The H1 multigene family constitutes a very interesting histone class gathering the greatest number of isoforms, with many different arrangements in the genome, including clustered and solitary genes, and showing replication-dependent (RD) or replication-independent (RI) expression patterns. The evolution of H1 histones has been classically explained by concerted evolution through a rapid process of interlocus recombination or gene conversion. Given such intriguing features, we have analyzed the long-term evolutionary pattern of the H1 multigene family through the evaluation of the relative importance of gene conversion, point mutation, and selection in generating and maintaining the different H1 subtypes. We have found the presence of an extensive silent nucleotide divergence, both within and between species, which is always significantly greater than the nonsilent variation, indicating that purifying selection is the major factor maintaining H1 protein homogeneity. The results obtained from phylogenetic analysis reveal that different H1 subtypes are no more closely related within than between species, as they cluster by type in the topologies, and that both RD and RI H1 variants follow the same evolutionary pattern. These findings suggest that H1 histones have not been subject to any significant effect of interlocus recombination or concerted evolution. However, the diversification of the H1 isoforms seems to be enhanced primarily by mutation and selection, where genes are subject to birth-and-death evolution with strong purifying selection at the protein level. This model is able to explain not only the generation and diversification of RD H1 isoforms but also the origin and long-term persistence of orphon RI H1 subtypes in the genome, something that is still unclear, assuming concerted evolution.     

Cryopreservation and storage of mussel (Mytilus spp.) haemocytes for latent analysis by the Comet assay

Estuarine and coastal habitats are known to be polluted by a range of chemical contaminants from both industrial and domestic sources. Blue mussels (Mytilus spp.), which inhabit these areas, are widely used as bio-indicators in eco-toxicological studies, because of their sedentary nature and their ability to bio-accumulate contaminants. The analysis of DNA damage in mussel haemocytes is a valuable tool for biomonitoring but sampling issues related to storage, handling and transportation have often limited its application in large-scale monitoring programmes. This study uses a trial and error method to evaluate and validate a suitable protocol for cryopreservation of mussel haemocytes, thereby allowing material collected in the field to be analysed later under controlled laboratory conditions. Three different cell-culture media, i.e. Leibovitz-15, Hank's balanced salt solution and mussel physiological saline, along with four different cryoprotectants, i.e. dimethyl sulphoxide (10% and 20%), 1,2-propanediol (10%), ethylene glycol (10%) and glycerol (10%) were tested to assess their suitability for cryopreservation of mussel haemocytes for analysis in the comet assay. Experimental studies where mussel haemocytes were also exposed to UV radiation or benzo(a)pyrene were conducted in order to mimic environmental stresses and to verify the effectiveness of newly defined cryopreservation protocols. The comet assay was used to demonstrate that mussel haemocytes could be preserved at cryogenic temperatures for a month without altering levels of DNA damage, which could possibly be used for lab or field studies where time constraints or facilities do not allow instant analysis.     

Organization and expression of H1 histone and H1 replacement histone genes

The H1 family is the most divergent subgroup of the highly conserved class of histone proteins [Cole: Int J Pept Protein Res 30:433–449, 1987]. In several vertebrate species, the H1 complement comprises five or more subtypes, and tissue specific patterns of H1 histones have been described. The diversity of the H1 histone family raises questions about the functions of different H1 subtypes and about the differential control of expression of their genes. The expression of main type H1 genes is coordinated with DNA replication, whereas the regulation of synthesis of replacement H1 subtypes, such as H1° and H5, and the testis specific H1t appears to be more complex. The differential control of H1 gene expression is reflected in the chromosomal organization of the genes and in different promoter structures. This review concentrates on a comparison of the chromosomal organization of main type and replacement H1 histone genes and on the differential regulation of their expression. General structural and functional data, which apply to both H1 and core histone genes and which are covered by recent reviews, will not be discussed in detail.     

Expression of histone 1 (H1) and testis-specific histone 1 (H1t) genes during stallion spermatogenesis

In eukaryotic cells, the major protein constituents of the chromatin are histones, which can be divided into five classes, identified as H1, H2A, H2B, H3 and H4. During normal spermatogenesis, a testis-specific H1t is expressed in primary spermatocytes and believed to facilitate histone to protamine exchanges during spermiogenesis. In equine testes we detected the H1 protein at 22kDa by western blot analysis while H1t was detected at 29kDa. H1 protein was found to be expressed in all germ cells up to elongating spermatids (Sc) at stage IV. In peripubertal animals, there was a prolonged expression up to elongating spermatids (Sd1) at stage V. A fragment of the equine H1t gene was cloned (GenBank Accession No. AJ865320). The mRNA expression of H1t was found at the level in spermatogonia and in primary spermatocytes up to mid-pachytene at stage VIII/I, whereas H1t protein was found to be expressed up to round spermatides (Sa/Sb1) at stage VIII/I. In peripubertal animals, the H1t protein expression was detected up to elongating spermatids (Sb2) at stage II. Analysis of testes of different ages (< or =2 years) and (> or =3 years) by real-time RT-PCR revealed an increase of H1t mRNA expression, with a wide range of individual variety between 2 and 4 years old animals indicating a stable expression in animals older than 4 years old. This is the first study to show the testis-specific H1t in the stallion and gives evidence that the well-known peripubertal infertility in the stallion may be related to an insufficient histone to protamine exchange. The pattern of protamine gene expression, however, has still to be elucidated.     

Common phylogenetic origin of protamine-like (PL) proteins and histone H1: Evidence from bivalve PL genes

Sperm nuclear basic proteins (SNBPs) can be grouped into three main categories: histone (H) type, protamine (P) type, and protamine-like (PL) type. Protamine-like SNBPs represent the most structurally heterogeneous group, consisting of basic proteins which are rich in both lysine and arginine amino acids. The PL proteins replace most of the histones during spermiogenesis but to a lesser extent than the proteins of the P type. In most instances, PLs coexist in the mature sperm with a full histone complement. The replacement of histones by protamines in the mature sperm is a characteristic feature presented by those taxa located at the uppermost evolutionary branches of protostome and deuterostome evolution, while the histone type of SNBPs is predominantly found in the sperm of taxa which arose early in metazoan evolution; giving rise to the hypothesis that protamines may have evolved through a PL type intermediate from a primitive histone ancestor. The structural similarities observed between PL and H1 proteins, which were first described in bivalve molluscs, provide a unique insight into the evolutionary mechanisms underlying SNBP evolution. Although the evolution of SNBPs has been exhaustively analyzed in the last 10 years, the origin of PLs in relation to the evolution of the histone H1 family still remains obscure. In this work, we present the first complete gene sequence for two of these genes (PL-III and PL-II/PL-IV) in the mussel Mytilus and analyze the protein evolution of histone H1 and SNBPs, and we provide evidence that indicates that H1 histones and PLs are the direct descendants of an ancient group of "orphon" H1 replication-dependent histones which were excluded to solitary genomic regions as early in metazoan evolution as before the differentiation of bilaterians. While the replication-independent H1 lineage evolved following a birth-and-death process, the SNBP lineage has been subject to a purifying process that shifted toward adaptive selection at the time of the differentiation of arginine-rich Ps.     

Molecular cloning and expression of mRNAs encoding H1 histone and an H1 histone-like sequences in root tips of pea (Pisum sativum L.)

Two cDNA clones representing mRNAs, highly expressed in pea root tips, were isolated by mRNA differential display. Ribonuclease protection analyses showed different patterns of expression of these two messages in several pea tissues. Sequence analysis showed that the first clone, PsH1b-40, has 100% homology with a previously isolated H1 histone cDNA, PsH1b. However, it has an additional 30 nt at the 3 end which is absent in PsH1b, suggesting possible multiple polyadenylation sites in the same mRNA. The second clone, PsH1b-41, encodes a deduced 19.5 kDa protein of 185 amino acids with an isoelectric point of 11.5. The putative globular domain of the encoded protein showed 67–71% residue identity with globular domains of 28 kDa pea PsH1b H1 histone and Arabidopsis thaliana H1-1 H1 histone. It has 9 repeating motifs of (T/S)XXK. In the C-terminal domain, there are four lysine-rich repeating motifs of SXK(T/S)PXKKXK which may be involved in chromatin condensation and decondensation. Southern blot analysis of nuclear DNA shows that PsH1-41 belongs to a multigene family.     

Molecular evidence of hybridization in Florida's sheoak (Casuarina spp.) invasion

The presence of hybrids in plant invasions can indicate a potential for rapid adaptation and an added level of complexity in management of the invasion. Three Casuarina tree species, Casuarina glauca , Casuarina cunninghamiana and Casuarina equisetifolia , native to Australia, are naturalized in Florida, USA. Many Florida Casuarina trees are considered unidentifiable, presumably due to interspecific hybridization. We collected tissue from over 500 trees from Australia and Florida and genotyped these using amplified fragment length polymorphisms. Our goal was to determine the exact identity of the Florida species, including any putative hybrid combinations. In Australia, we found high assignment values to the three parental species, and no evidence of hybridization. In Florida, we found many trees with strong assignment to any one of the three species, as well as 49 trees with assignment values intermediate to C. glauca and C. equisetifolia , suggesting hybridization between these species. One population of 10 trees had assignment values intermediate to C. cunninghamiana and C. glauca , suggesting additional hybridization. For 69 of these putative hybrid and parental types, we sequenced a low-copy intron of nuclear G3pdh , and these sequences indicated that some Florida trees contain heterozygotic combinations of C. glauca and C. equisetifolia haplotypes. The presence of novel hybrids in the Florida invasion may enhance evolution of invasive traits in these species. Novel Casuarina hybrids in Florida have no coevolutionary history with any insects or diseases, which may be problematic for biological control efforts.     

Comparative analysis of gender-associated complete mitochondrial genomes in marine mussels (Mytilus spp.)

Marine mussels of the genus Mytilus have an unusual mode of mitochondrial DNA (mtDNA) transmission termed doubly uniparental inheritance (DUI). Female mussels are homoplasmic for the F mitotype, which is inherited maternally, while males are usually heteroplasmic, carrying a mixture of the maternal F mitotype and the paternally inherited M genome. Two classes of M genomes have been observed: "standard" M genomes and "recently masculinized" M genomes. The latter are more similar to F genomes at the sequence level but are transmitted paternally like standard M genomes. In this study we report the complete sequences of two standard male M. edulis and one recently masculinized male M. trossulus mitochondrial genome. A comparative analysis, including the previously sequenced M. edulis F and M. galloprovincialis F and M mtDNAs, reveals that these genomes are identical in gene order, but highly divergent in nucleotide and amino acid sequence. The large amount (>20%) of nucleotide substitutions that fall in coding regions implies that there are several amino acid replacements between the F and M genomes, which likely have an impact on the structural and functional properties of the mitochondrial proteome. Correlation of the divergence rate of different protein-coding genes indicates that mtDNA-encoded proteins of the M genome are still under selective constraints, although less highly than genes of the F genome. The mosaic F/M control region of the masculinized F genome provides evidence for lineage-specific sequences that may be responsible for the different mode of transmission genetics. This analysis shows the value of comparative genomics to better understand the mechanisms of maintenance and segregation of mtDNA sequence variants in mytilid mussels.     

Influence of genotype and geography on shell shape and morphometric trait variation among North Atlantic blue mussel (Mytilus spp.) populations

The influence of geography and genotype on shell shape (outline) and trait (morphometric) variation among North Atlantic blue mussels and their hybrids has been examined. Shape differences among reference taxa (Mytilus trossulus, Mytilus edulis and Mytilus galloprovincialis) were consistent with an association between taxon‐specific genes and shape genes. Newfoundland M. edulis × M. trossulus populations and northern Quebec M. trossulus populations exhibited an uncoupling of taxon‐specific genes from shape genes, whereas Nova Scotia M. trossulus populations and SW England M. edulis × M. galloprovincialis populations exhibited an association between taxon‐specific genes and shape genes. We found no evidence of a geographic effect (NE versus NW Atlantic) for shape variation, indicating that the genotype effect is stronger than any geographic effect at macrogeographic scales. Pronounced differences were observed in trait variability consistent with an association between taxon‐specific genes and trait genes in European populations, and trait divergence of New York M. edulis from all European mussels. Trait variability in mussels from Newfoundland, Nova Scotia and northern Quebec indicated an uncoupling of taxon genes from trait genes, whereas trait variability in SW England M. edulis × M. galloprovincialis populations was consistent with background genotype, indicating a strong association between taxon genes and trait genes. A pronounced macrogeographic split (NE versus NW Atlantic) regardless of taxonomy was observed, indicating that geography exerts a greater influence than genotype on trait variation at the macrogeographic scale. This is consistent with pronounced within‐taxon genetic divergence, indicative of different selection regimes or more likely of different evolutionary histories of mussels on either side of the North Atlantic. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96 , 875–897.     

Molecular evidence from ascidians for the evolutionary origin of vertebrate cranial sensory placodes

Cranial sensory placodes are specialised areas of the head ectoderm of vertebrate embryos that contribute to the formation of the cranial sense organs and associated ganglia. Placodes are often considered a vertebrate innovation, and their evolution has been hypothesised as one key adaptation underlying the evolution of active predation by primitive vertebrates. Here, we review recent molecular evidence pertinent to understanding the evolutionary origin of placodes. The development of vertebrate placodes is regulated by numerous genes, including members of the Pax, Six, Eya, Fox, Phox, Neurogenin and Pou gene families. In the sea squirt Ciona intestinalis (a basal chordate and close relative of the vertebrates), orthologues of these genes are deployed in the development of the oral and atrial siphons, structures used for filter feeding by the sessile adult. Our interpretation of these findings is that vertebrate placodes and sea squirt siphon primordia have evolved from the same patches of specialised ectoderm present in the common ancestor of the chordates.     

Molecular evidence from Ciona intestinalis for the evolutionary origin of vertebrate sensory placodes

Cranial sensory placodes are focused areas of the head ectoderm of vertebrates that contribute to the development of the cranial sense organs and their associated ganglia. Placodes have long been considered a key character of vertebrates, and their evolution is proposed to have been essential for the evolution of an active predatory lifestyle by early vertebrates. Despite their importance for understanding vertebrate origins, the evolutionary origin of placodes has remained obscure. Here, we use a panel of molecular markers from the Six, Eya, Pax, Dach, FoxI, COE and POUIV gene families to examine the tunicate Ciona intestinalis for evidence of structures homologous to vertebrate placodes. Our results identify two domains of Ciona ectoderm that are marked by the genetic cascade that regulates vertebrate placode formation. The first is just anterior to the brain, and we suggest this territory is equivalent to the olfactory/adenohypophyseal placodes of vertebrates. The second is a bilateral domain adjacent to the posterior brain and includes cells fated to form the atrium and atrial siphon of adult Ciona. We show this bares most similarity to placodes fated to form the vertebrate acoustico-lateralis system. We interpret these data as support for the hypothesis that sensory placodes did not arise de novo in vertebrates, but evolved from pre-existing specialised areas of ectoderm that contributed to sensory organs in the common ancestor of vertebrates and tunicates.     

Independent evolutionary origin of histone H3.3-like variants of animals and Tetrahymena.

All three genes encoding histone H3 proteins were cloned and sequenced from Tetrahymena thermophila. Two of these genes encode a major H3 protein identical to that of T. pyriformis and 87% identical to the major H3 of vertebrates. The third gene encodes hv2, a quantitatively minor replication independent (replacement) variant. The sequence of hv2 is only 85% identical to the animal replacement variant H3.3 and is the most divergent H3 replacement variant described. Phylogenetic analysis of 73 H3 protein sequences suggests that hv2, H3.3, and the plant replacement variant H3.III evolved independently, and that H3.3 is not the ancestral H3 gene, as was previously suggested (Wells, D., Bains, W., and Kedes, L. 1986, J. Mol. Evol., 23: 224-241). These results suggest it is the replication independence and not the particular protein sequence that is important in the function of H3 replacement variants.     

Proteome modifications of blue mussel (Mytilus edulis L.) gills as an effect of water pollution

The discharge of chemicals such as oil associated or not with derived products constitutes a real threat for the environment. We report here the differential expression of the blue mussel (Mytilus edulis) gill proteins corresponding to two contaminated environmental conditions: crude oil and offshore produced water. In order to evaluate and understand contaminants, effects and adaptive response of these organisms, we identified proteins using MS. The latter can be grouped into three main classes: proteins involved in the cellular structure, in metabolism, and in defence proteins.     

Leishmania (Viannia) panamensis: cloning of the histone H1 genes by representational difference analysis

We report the use of representational difference analysis to identify genes that have up-regulated expression in the amastigote life-cycle stage of Leishmania (Viannia) panamensis. This simultaneous process of selection and amplification allowed the cloning of several specific DNA fragments. One of them shows a high percentage of similarity with histone H1 genes from other Trypanosomatids and, as expected, is up-regulated in the amastigote life-cycle stage. The gene is present in two copies that are expressed at different levels in promastigotes and also in amastigotes, which seems to be a consequence of their different 3' untranslated regions.     

A new approach for the assessment of stochastic variation: analysis of behavioural response in blue mussel (Mytilus edulis L.)

One of the most effective techniques for evaluating stress is the analysis of developmental stability, measured by stochastic variation based particularly on fluctuating asymmetry, i.e. a variance in random deviations from perfect bilateral symmetry. However, the application of morphological methods is only possible when an organism lives under testing conditions during a significant part of its ontogenesis. Contrary to morphological characters, behavior can change very fast. Consequently, methods based on behavioural characters may have advantages over more traditional approaches. In this study we describe the technique of assessing stochastic variation, using not morphological, but behavioural characters. To measure stochastic variation of behavioural response, we assessed the stability of the isolation reaction of blue musselMytilus edulis at regular changes of salinity. With increasing temperature from +12°C to +20°C stochastic variation of the isolation reaction increased, which is a common response to change of environmental conditions. In this way, we have developed a method of assessing stochastic variation of behavioural response in molluscs. This method may find a great range of applications, because its usage does not require keeping animals in tested conditions for a long time.     

Morphological evidence for the evolutionary origin of Astigmata (Acari: Acariformes)

A century ago, Antonio Berlese first discussed the close phylogenetic relationship between the large mite groups Oribatida and Astigmata. Since then, information having phylogenetic value has greatly increased and the paradigms within which we interpret it have changed. Herein I refine the general hypothesis that Astigmata originated within oribatid mites and suggest Malaconothridae as a possible sister group. Among the 14 apomorphies used to support the origin of Astigmata within oribatid mites are possession of lateral opisthosomal glands, regression of hysterosomal setal pair f1, paired prelarval denticles, partially internalized chelicerae with incomplete adaxial walls, an atelobasic rutellum, pretarsal condylophores that articulate posteriorly with the tarsus, a dorsally fused palp tibia and tarsus and transdehiscent ecdysis. A further 13 apomorphies support the origin of Astigmata at some level within Malaconothroidea. These include absence of an oblique labiogenal articulation, presence of a distal rutellar lamella, shortening of the palp tarsus, larval regression of hysterosomal seta f2, loss of the bothridial seta in all instars, and several losses and modifications of leg setae. The hypothesis brings to light evolutionary questions that were previously obscured by incorrect or inappropriate classifications. The nomenclatural problems that arise from it are best solved by considering Astigmata as a subgroup within Oribatida.     

Characterization of AhMITE1 transposition and its association with the mutational and evolutionary origin of botanical types in peanut (Arachis spp.)

AhMITE1 is an active miniature inverted repeat transposable element (MITE) in peanut (Arachis hypogaea L). Its transpositional activity from a particular (FST1-linked) site within the peanut genome was checked using AhMITE1-specifc PCR, which used a forward primer annealing to the 5??-flanking sequence and a reverse primer binding to AhMITE1. It was found that transposition activation was induced by stresses such as ethyl methane sulfonate (EMS), gamma irradiation, environmental conditions, and tissue culture. Excision and insertion of AhMITE1 at this particular site among the mutants led to gross morphological changes resembling alternate subspecies or botanical types. Analysis of South American landraces revealed the presence of AhMITE1 at the site among most of the spp. fastigiata types, whereas the element was predominantly missing from spp. hypogaea types, indicating its strong association. Four accessions of the primitive allotetraploid, A. monticola were devoid of AhMITE1 at the site, indicating only recent activation of the element, possibly because of the ??genomic shock?? resulting from hybridization followed by allopolyploidization.     

Fatty acid profiles of Mytilus galloprovincialis (Lmk) mussel of subtidal and rocky shore origin

Fatty acid profiles of seeds of the mussel Mytilus galloprovincialis originating from two habitats (rocky shore and subtidal) were compared after transfer to the same habitat (subtidal). The objective was to study the initial levels of different fatty acids of metabolic importance and, furthermore, the variability of these fatty acids over the experimental period. The results show that of all fatty acids identified in both seed groups, the polyunsaturated fatty acids (PUFA) is the group with highest percentage. Within this group, the C20:5n-3 and C22:6n-3 fatty acids show the highest levels. Additionally, the mussels of subtidal origin presented higher initial levels than the rocky shore mussels with regard to fatty acids characterised by energetic-type functions, such as the C14:0, C16:0, and the C20:5n-3 fatty acids, among others. Fatty acids characterised by structural-type functions, e.g. C18:0, C22:6n-3 and non-methylene interrupted dienoic (NMID) with 20 and 22 carbons in rocky shore mussels presented higher levels than those of the subtidal mussels. However, it has not been ruled out that aside from influences relating to the functional aspects of the different fatty acids, the initial differences were also associated with the quantitative and qualitative differences of the available food in both habitats. Nevertheless, 22 days into the experiment (in the majority of cases) the initial differences disappear in the different fatty acids with metabolic importance. On the basis of these results, the influence that mussel origin could exercise on the variability of the fatty acid profiles of recognised metabolic importance is discussed.     

Isolation and characterization of two human H1 histone genes within clusters of core histone genes

Two human H1 histone genes, termed H1.3 and H1.4, were isolated from two cosmid clones. The H1.4 gene is associated with an H2B gene, whereas genes coding for all four core histones are located in the vicinity of the H1.3 gene. This cluster arrangement was found both in the two cosmid clones and on overlapping bacteriophage clones isolated from an EMBL3 library. In continuation of our previous analysis of two human H1 genes, this analysis raises the number of completely sequenced H1 histone genes within clusters of core histone genes to four.     

Immunological evidence for an H1° type of histone protein in chicken liver

We prepared monoclonal antibodies against chicken histone H5. These antibodies could be divided into two classes, and we present the results obtained with one representative antibody of each class. One class reacted exclusively with chicken H5, whereas the other additionally cross-reacted with rat H1(0) and with material present in adult but not embryonic chicken liver. The cross-reacting material in adult liver was identified by Western blotting as representing a minor band in histone preparations. The protein was not present in histone extracts from chicken erythrocytes. It is likely that this newly identified protein is a chicken H1(0) histone.