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Protamines, in the Footsteps of Linker Histone Evolution

2005; Elsevier BV; Volume: 281; Issue: 1 Linguagem: Inglês

10.1074/jbc.r500018200

1083-351X

José M. Eirín‐López, Lindsay J. Frehlick, Juan Ausió,

Animal Genetics and Reproduction

It was perhaps a lucky coincidence that the early attempts to establish the chemical composition of the cell nucleus were initially carried out on such diverse biological systems as salmon sperm heads (1Miescher F. Hoppe-Seyler's Med. Chem. Untersuchungen Ber. dtsch. Chem. Ges. 1874; 7: 376-379Crossref Scopus (46) Google Scholar) and geese and chicken erythrocytes (2Kossel A. Z. Physiol. Chem. 1884; 8: 511-515Google Scholar). Examination of sperm and erythrocyte systems, respectively, lead to the protamine and histone concepts (3Kossel A. The Protamines and Histones. Longmans Green and Co., London1928Google Scholar). We know with certainty that, with the exception of the male gametes, all somatic cells exclusively contain histones. Hence, in metazoans, protamines (4Felix K. Adv. Protein Chem. 1960; 15: 1-56Crossref PubMed Scopus (54) Google Scholar, 5Ando T. Yamasaki M. Suzuki K. Protamines: Isolation, Characterization and Function. Springer-Verlag, New York1973Crossref Google Scholar) are confined to the sperm nuclear chromatin, and even among sperm, protamines are not always present. Indeed, a large number of metazoans contain somatic-like histones in their sperm, and some crustaceans (order Decapoda) lack any chromosomal proteins in their sperm (6Bloch D.P. Histones of Sperm. Plenum Press, New York1976Crossref Google Scholar, 7Kasinsky H.E. Hnilica L.S. Stein G.S. Stein J.L. Histones and Other Basic Nuclear Proteins. CRC Press Inc., Boca Raton, FL1989: 73-163Google Scholar). Therefore, in contrast to the somatic nucleus, sperm chromatin may have a much more diverse protein composition. It was not until the first attempt of classification of the sperm nuclear basic proteins (SNBPs) 2The abbreviations used are: SNBPsperm nuclear basic proteinHhistonePprotaminePLprotamine-like proteinRIreplication-independent. by David Bloch (6Bloch D.P. Histones of Sperm. Plenum Press, New York1976Crossref Google Scholar, 8Bloch D.P. Genetics. 1969; 61: 93-111PubMed Google Scholar), an effort later on extended by Harold Kasinsky (7Kasinsky H.E. Hnilica L.S. Stein G.S. Stein J.L. Histones and Other Basic Nuclear Proteins. CRC Press Inc., Boca Raton, FL1989: 73-163Google Scholar), that a clearer picture started to emerge in this regard. sperm nuclear basic protein histone protamine protamine-like protein replication-independent. More recently, an enormous effort has been carried out in several laboratories, including our own (9Ausió J. Jamieson B.G.M Ausió J. Justine J.L. Advances in Spermatozoal Phylogeny and Taxonomy. 166. Mémoires du Muséum National d'Histoire Naturelle, Paris1995: 447-462Google Scholar, 10Chiva M. Saperas N. Caceres C. Ausió J. Jamieson B.G.M Ausió J. Justine J.L. Advances in Spermatozoal Taxonomy and Phylogeny. 166. Memories of the National Museum of Natural History, Paris1995: 501-514Google Scholar, 11Poccia D. Jamieson B.G.M Ausió J. Justine J.L. Advances in Spermatozoal Taxonomy and Phylogeny. 166. Memories of the National Museum of Natural History, Paris1995: 475-489Google Scholar, 12Oliva R. Dixon G.H. Prog. Nucleic Acids Res. Mol. Biol. 1991; 40: 25-94Crossref PubMed Scopus (362) Google Scholar, 13Lewis J. Song Y. de Jong M. Bagha S. Ausió J. Chromosoma (Berl.). 2003; 111: 473-482Crossref PubMed Scopus (140) Google Scholar, 14Hunt J.G. Kasinsky H.E. Elsey R.M. Wright C.L. Rice P. Bell J.E. Sharp D.J. Kiss A.J. Hunt D.F. Arnott D.P. Russ M.M. Shabanowitz J. Ausió J. J. Biol. Chem. 1996; 271: 23547-23557Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 15Wouters-Tyrou D. Martinage A. Chevaillier P. Sautiere P. Biochimie (Paris). 1998; 80: 117-128Crossref PubMed Scopus (144) Google Scholar), to extend this analysis to a large number of representative organisms from the different phylogenetic groups. With a broader perspective now available, SNBP heterogeneity can be restricted to three major groups or types: histone (H), protamine (P), and protamine-like (PL) (16Ausió J. J. Biol. Chem. 1999; 274: 31115-31118Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Histones consist of core histones (histones H2A, H2B, H3, and H4) and linker histones (histone H1 family). The names refer to the structural role of these proteins. Core histones are responsible for constraining DNA wrapped about a histone core to produce a nucleoprotein complex (chromatin subunit) known as a nucleosome core particle. Linker histones bind to the linker DNA regions connecting adjacent nucleosome core particles and assist in the folding of the chromatin fiber (17van Holde K.E. Chromatin. Springer-Verlag, New York1988Google Scholar). Protamines are a highly compositionally and structurally heterogeneous group of proteins (9Ausió J. Jamieson B.G.M Ausió J. Justine J.L. Advances in Spermatozoal Phylogeny and Taxonomy. 166. Mémoires du Muséum National d'Histoire Naturelle, Paris1995: 447-462Google Scholar). They exhibit a high charge density and a prevalence of arginine in their composition (13Lewis J. Song Y. de Jong M. Bagha S. Ausió J. Chromosoma (Berl.). 2003; 111: 473-482Crossref PubMed Scopus (140) Google Scholar), a fact that it is most likely related to the higher affinity with which this basic amino acid binds to DNA (18Ausió J. Greulich K.O. Haas E. Wachtel E. Biopolymers. 1984; 23: 2559-2571Crossref PubMed Scopus (20) Google Scholar). They lack any secondary structure in solution but may adopt a folded conformation upon interaction with DNA. Protamine-like proteins share compositional and structural similarities between histones and protamines (9Ausió J. Jamieson B.G.M Ausió J. Justine J.L. Advances in Spermatozoal Phylogeny and Taxonomy. 166. Mémoires du Muséum National d'Histoire Naturelle, Paris1995: 447-462Google Scholar, 16Ausió J. J. Biol. Chem. 1999; 274: 31115-31118Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Hence they represent a structurally intermediate group that will be discussed more extensively in the following sections of this review. To a certain extent, all three types of SNBP can be considered structurally analogous as all of them produce folded chromatin fibers of 30–50 nm (19Casas M.T. Ausió J. Subirana J.A. Exp. Cell Res. 1993; 204: 192-197Crossref PubMed Scopus (22) Google Scholar) regardless of the particular structure of the individual nucleoprotamine complexes. At the functional level, somatic histones bind to DNA in a highly dynamic way that not only helps in the folding of the genome but also has an important role in the epigenetic regulation of gene expression (20Ausió J. Abbott D.W. Zlatanova J. Leuga S.H. Chromatin Structure and Dynamics: State-of-the-Art. Elsevier, Amsterdam2004: 241-290Google Scholar, 21Fischle W. Wang Y. Allis C.D. Nature. 2003; 425: 475-479Crossref PubMed Scopus (547) Google Scholar). In contrast, protamine and protamine-like SNBPs of the spermatozoa bind very tightly to the genome to produce a maximal genome compaction, which completely abolishes the epigenetic information of the paternal histones (9Ausió J. Jamieson B.G.M Ausió J. Justine J.L. Advances in Spermatozoal Phylogeny and Taxonomy. 166. Mémoires du Muséum National d'Histoire Naturelle, Paris1995: 447-462Google Scholar) in this terminally differentiated system. This epigenetic silencing can be reverted only with the assistance of highly specialized protamine-removing proteins such as nucleoplasmin during the molecular events involved in nuclear metabolism during early fertilization (22Prado A. Ramos I. Frehlick L.J. Muga A. Ausio J. Biochem. Cell Biol. 2004; 82: 437-445Crossref PubMed Scopus (43) Google Scholar). The analogous structural DNA condensation potential of the three types of SNBPs raises a question as to the extent of structural homology between them. In the next sections we are going to discuss a series of recent papers that suggest that protamines and protamine-like proteins are evolutionarily related to linker histones. The overall extent of structural heterogeneity of the PL- and P-type SNBPs is better visualized when these proteins are compared within closely related phylogenetic groups. One such example can be found in mollusks. An early comparative study of SNBPs within this group (23Subirana J.A. Cozcolluel C. Palau J. Unzeta M. Biochim. Biophys. Acta. 1973; 317: 364-379Crossref PubMed Scopus (101) Google Scholar) revealed the presence, in some instances, of electrophoretically large proteins with lower mobility than histones and a composition rich in both arginine and lysine residues, such as those described in the clam Spisula solidissima. In other instances, such as in the cephalopods Octopus vulgaris and in Eledone cirrhosa, the SNBPs had higher electrophoretic mobility and a composition that ranged from arginine-rich, such as in the fish and bird protamines, to highly cysteine-rich, such as in mammalian protamines. The proteins of these three organisms have now all been sequenced (24Gimenez-Bonafe P. Ribes E. Sautiere P. Gonzalez A. Kasinsky H. Kouach M. Sautiere P.E. Ausió J. Chiva M. Eur. J. Cell Biol. 2002; 81: 341-349Crossref PubMed Scopus (27) Google Scholar, 25Gimenez-Bonafe P. Soler F.M. Buesa C. Sautiere P.E. Ausio J. Kouach M. Kasinsky H.E. Chiva M. Mol. Reprod. Dev. 2004; 68: 232-239Crossref PubMed Scopus (10) Google Scholar, 26Lewis J.D. McParland R. Ausió J. Biochemistry. 2004; 43: 7766-7775Crossref PubMed Scopus (14) Google Scholar). An initial structural characterization of the Spisula large SNBP component showed that the protein had a tripartite organization with a globular central core, which was found to bear a strong sequence similarity to the winged helix domain of histone H5 from bird erythrocytes, another terminally differentiated system (27Ausió J. Toumadje A. McParland R. Becker R.R. Johnson W.C. van Holde K.E. Biochemistry. 1987; 26: 975-982Crossref PubMed Scopus (41) Google Scholar). A SNBP protein with similar characteristics is extensively distributed throughout bivalve mollusks and has been called protamine-like protein I (PL-I) (28Ausió J. Mol. Cell. Biochem. 1992; 115: 163-172Crossref PubMed Scopus (38) Google Scholar, 29Ausió J. Comp. Biochem. Physiol. B. 1986; 85: 439-449Crossref Scopus (59) Google Scholar). The regions flanking this core are unstructured and are rich in both arginine and lysine (26Lewis J.D. McParland R. Ausió J. Biochemistry. 2004; 43: 7766-7775Crossref PubMed Scopus (14) Google Scholar). PL-I proteins have now been identified not only in mollusks but also in tunicates and in several fish (9Ausió J. Jamieson B.G.M Ausió J. Justine J.L. Advances in Spermatozoal Phylogeny and Taxonomy. 166. Mémoires du Muséum National d'Histoire Naturelle, Paris1995: 447-462Google Scholar, 30Lewis J.D. Saperas N. Song Y. Zamora M.J. Chiva M. Ausio J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4148-4152Crossref PubMed Scopus (75) Google Scholar, 31Watson C.E. Davies P.L. J. Biol. Chem. 1998; 273: 6157-6162Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 32Watson C.E. Gauthier S.Y. Davies P.L. Eur. J. Biochem. 1999; 262: 258-267Crossref PubMed Scopus (15) Google Scholar) where they represent the major SNBP component. In all instances (see Fig. 1A) they contain an internal folded domain that corresponds to the winged helix motif (33Graziano V. Gerchman S.E. Wonacott A.J. Sweet R.M. Wells J.R. White S.W. Ramakrishnan V. J. Mol. Biol. 1990; 212: 253-257Crossref PubMed Scopus (20) Google Scholar, 34Ramakrishnan V. Finch J.T. Graziano V. Lee P.L. Sweet R.M. Nature. 1993; 362: 219-223Crossref PubMed Scopus (659) Google Scholar), which is characteristic of the linker histones. Notably, in the cases of mollusks (35Carlos S. Hunt D.F. Rocchini C. Arnott D.P. Ausió J. J. Biol. Chem. 1993; 268: 195-199Abstract Full Text PDF PubMed Google Scholar) and tunicates (30Lewis J.D. Saperas N. Song Y. Zamora M.J. Chiva M. Ausio J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4148-4152Crossref PubMed Scopus (75) Google Scholar) the PL-I protein can undergo post-translational cleavage giving rise to a series of smaller PL proteins with increasingly higher arginine composition. In some instances, as in Mytilus (mussel), PL-III appear to have become independent genes. These phenomena have been taken as an indication that all PL proteins and possibly protamines are somehow related to a primitive linker histone precursor (16Ausió J. J. Biol. Chem. 1999; 274: 31115-31118Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) probably related to the replication-independent (RI) lineage that gave rise to the highly differentiated histone H5 from the nucleated vertebrate erythrocytes (36Eirin-Lopez J.M. Gonzalez-Tizon A.M. Martinez A. Mendez J. Mol. Biol. Evol. 2004; 21: 1992-2003Crossref PubMed Scopus (52) Google Scholar, 37Eirin-Lopez J.M. Ruiz M.F. Gonzalez-Tizon A.M. Martinez A. Ausio J. Sanchez L. Mendez J. J. Mol. Evol. 2005; 61: 398-407Crossref PubMed Scopus (9) Google Scholar). Interestingly, a potential structural relation between histone H5 and protamines has also been described in other invertebrates. Although there is still very little information about the protamines of insects (13Lewis J. Song Y. de Jong M. Bagha S. Ausió J. Chromosoma (Berl.). 2003; 111: 473-482Crossref PubMed Scopus (140) Google Scholar), a putative Drosophila protamine-like protein, which shares some extent of similarity to histone H5 and to the cysteine-rich protamines from mammals, has been identified in screens of transcripts expressed in the male germ line (38Russell S.R. Kaiser K. Genetics. 1993; 134: 293-308Crossref PubMed Google Scholar). One of the major conceptual stumbling blocks in trying to explain the transition from linker histones to protamines has been the difficulty in accounting for the evolutionary transition from the highly lysine-rich (25–30 mol %) composition, which is characteristic of histone H1 molecules (39Cole R.D. Anal. Biochem. 1984; 136: 24-30Crossref PubMed Scopus (118) Google Scholar), to the arginine-rich (≥30 mol %) composition of protamines. Although all PL proteins exhibit both a lysine- and arginine-rich composition (Arg + Lys = 35–50 mol %), they still have a distinct composition from the predominantly arginine-rich protamines. An important breakthrough in this direction came from a recent study of the PL proteins from two closely related tunicates: Styela monterreyensis and Ciona intestinalis (30Lewis J.D. Saperas N. Song Y. Zamora M.J. Chiva M. Ausio J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4148-4152Crossref PubMed Scopus (75) Google Scholar). The former contains an SNBP composition consisting of two PL-I-related proteins P1 and P2 (10Chiva M. Saperas N. Caceres C. Ausió J. Jamieson B.G.M Ausió J. Justine J.L. Advances in Spermatozoal Taxonomy and Phylogeny. 166. Memories of the National Museum of Natural History, Paris1995: 501-514Google Scholar, 40Saperas N.M. Chiva M. Ausió J. Comp. Biochem. Physiol. B. 1992; 103: 969-974Crossref Scopus (18) Google Scholar). Amino acid sequence analysis showed that these two proteins are indeed related, with the faster electrophoretic component corresponding to the C-terminal domain of the larger component (Fig. 1B). Furthermore, the faster PL component (P2) had an arginine-rich composition (58 mol %) (10Chiva M. Saperas N. Caceres C. Ausió J. Jamieson B.G.M Ausió J. Justine J.L. Advances in Spermatozoal Taxonomy and Phylogeny. 166. Memories of the National Museum of Natural History, Paris1995: 501-514Google Scholar), and more importantly, it consisted of repeated arginine clusters, which are characteristic of many invertebrate and vertebrate canonical protamines (13Lewis J. Song Y. de Jong M. Bagha S. Ausió J. Chromosoma (Berl.). 2003; 111: 473-482Crossref PubMed Scopus (140) Google Scholar). However, this SNBP composition appeared to be quite restricted to the genus Styela, as other tunicate species consisted only of the larger PL precursor (P1) molecule that apparently had not undergone the post-translational cleavage (10Chiva M. Saperas N. Caceres C. Ausió J. Jamieson B.G.M Ausió J. Justine J.L. Advances in Spermatozoal Taxonomy and Phylogeny. 166. Memories of the National Museum of Natural History, Paris1995: 501-514Google Scholar). In silico analysis based on the genome sequence available for the tunicate Ciona intestinalis revealed that the single unprocessed PL-I, which is present in this species, had an amino acid sequence that was strikingly similar to that of the Styela larger component except for the fact that its C-terminal region was lysine-rich (Fig. 1B). Careful analysis of the genomic nucleotide sequence encoding for Styela and Ciona PL-I show that the transition from lysine to arginine may have occurred as a result of a single frameshift mutation in the C-terminal domain of these molecules. In this way, a transition from a lysine-rich to an arginine-rich PL-I would have occurred very rapidly, most remarkably at the time when the arginine-rich PL-I underwent post-translational cleavage (Fig. 1B). There are, however, conserved residues within the C-terminal region of Styela and Ciona PL-I, which cannot be solely accounted for by a single frameshift mutation. This suggests that although the transition from Lys to Arg via frameshift would be the most efficient, other processes might have been involved. Work is currently in progress in our laboratory to identify these mechanisms. This rapid mechanism of evolution is in good agreement with the notion that the reproductive traits (including reproductive proteins) have evolved very quickly (41Wyckoff G.J. Wang W. Wu C.I. Nature. 2000; 403: 304-309Crossref PubMed Scopus (434) Google Scholar) and with the experimental evidence that indicates that, despite their rather simple amino acid composition and their high arginine contents, protamines are excellent molecular markers for evolution studies (13Lewis J. Song Y. de Jong M. Bagha S. Ausió J. Chromosoma (Berl.). 2003; 111: 473-482Crossref PubMed Scopus (140) Google Scholar). It is interesting to notice that in contrast to core histones, the evolutionary origins of which can now be traced back to archaebacteria (42Reeve J.N. Bailey K.A. Li W.T. Marc F. Sandman K. Soares D.J. Biochem. Soc. Trans. 2004; 32: 227-230Crossref PubMed Scopus (48) Google Scholar, 43Cubonova L. Sandman K. Hallam S.J. Delong E.F. Reeve J.N. J. Bacteriol. 2005; 187: 5482-5485Crossref PubMed Scopus (54) Google Scholar), the origin of histone H1 appears to have occurred earlier in eubacteria (44Kasinsky H.E. Lewis J.D. Dacks J.B. Ausió J. FASEB J. 2001; 15: 34-42Crossref PubMed Scopus (179) Google Scholar) (Fig. 2). Equally interesting is the fact that although archaeal histones contained a histone fold structure characteristic of core histones (45Arents G. Moudrianakis E.N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11170-11174Crossref PubMed Scopus (287) Google Scholar), they lacked the tails flanking this domain found in higher eukaryotes (42Reeve J.N. Bailey K.A. Li W.T. Marc F. Sandman K. Soares D.J. Biochem. Soc. Trans. 2004; 32: 227-230Crossref PubMed Scopus (48) Google Scholar). However, linker histones acquired the winged helix folded domain characteristic of higher eukaryotes in a reverse way. In other words, linker histones were initially composed only of a C-terminal region, and the acquisition of the core domain containing the winged helix motif occurred later in their evolution (Fig. 2) (44Kasinsky H.E. Lewis J.D. Dacks J.B. Ausió J. FASEB J. 2001; 15: 34-42Crossref PubMed Scopus (179) Google Scholar). Indeed, many protozoans contain a linker histone consisting only of the characteristic APK-rich C-terminal domain, which is critical for the stabilization of the folded chromatin structure (46Allan J. Mitchell T. Harborne N. Bohm L. Crane-Robinson C. J. Mol. Biol. 1986; 187: 591-601Crossref PubMed Scopus (263) Google Scholar). The long term evolution of the histone H1 family was recently shown to be best described by a birth-and-death process (36Eirin-Lopez J.M. Gonzalez-Tizon A.M. Martinez A. Mendez J. Mol. Biol. Evol. 2004; 21: 1992-2003Crossref PubMed Scopus (52) Google Scholar, 37Eirin-Lopez J.M. Ruiz M.F. Gonzalez-Tizon A.M. Martinez A. Ausio J. Sanchez L. Mendez J. J. Mol. Evol. 2005; 61: 398-407Crossref PubMed Scopus (9) Google Scholar, 47Nei M. Hughes A.L. Tsuji K. Aizawa M. Sasazuki T. Eleventh Histocompatibility Workshop and Conference. Oxford University Press, Oxford, UK1992: 2738Google Scholar), a mechanism based on recurrent gene duplication events under a strong purifying selection. This mechanism has favored the great diversification presented by the members of this family and was further enhanced by the presence of strong functional and structural constraints, which ultimately led the different H1 isoforms to the acquisition of specific functions (Fig. 2). Histone H1 diversification has been maintained throughout its evolutionary process in both plants and animals (48Kaczanowski S. Jerzmanowski A. J. Mol. Evol. 2001; 53: 19-30Crossref PubMed Scopus (11) Google Scholar), ranging from the high degree of heterogeneity observed in early protozoans, such as trypanosomes (49Gruter E. Betschart B. Parasitol. Res. 2001; 87: 977-984Crossref PubMed Scopus (9) Google Scholar), to extreme diversification and specialization in the case of mammals. To date, 11 different mammalian linker histones have been identified: 7 somatic variants, H1.1 to H1.5, H10 (20Ausió J. Abbott D.W. Zlatanova J. Leuga S.H. Chromatin Structure and Dynamics: State-of-the-Art. Elsevier, Amsterdam2004: 241-290Google Scholar, 50Parseghian M.H. Henschen A.H. Krieglstein K.G. Hamkalo B.A. Protein Sci. 1994; 3: 575-587Crossref PubMed Scopus (70) Google Scholar), and H1x (51Happel N. Schulze E. Doenecke D. Biol. Chem. 2005; 386: 541-551Crossref PubMed Scopus (57) Google Scholar); 3 sperm-specific variants, H1t (52Seyedin S.M. Kistler W.S. J. Biol. Chem. 1980; 255: 5949-5954Abstract Full Text PDF PubMed Google Scholar), H1t2 (53Martianov I. Brancorsini S. Catena R. Gansmuller A. Kotaja N. Parvinen M. Sassone-Corsi P. Davidson I. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2808-2813Crossref PubMed Scopus (171) Google Scholar), and HILS1 (54Yan W. Ma L. Burns K.H. Matzuk M.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10546-10551Crossref PubMed Scopus (139) Google Scholar, 55Iguchi N. Tanaka H. Yomogida K. Nishimune Y. Int. J. Androl. 2003; 26: 354-365Crossref PubMed Scopus (34) Google Scholar); and the oocyte-specific variant H1foo (56Tanaka M. Hennebold J.D. Macfarlane J. Adashi E.Y. Development. 2001; 128: 655-664Crossref PubMed Google Scholar). In addition, H1 evolution has also favored the differentiation of highly specialized isoforms such as histone H5 (57Neelin J.M. Callahan P.X. Lamb D.C. Murray K. Can. J. Biochem. Physiol. 1964; 42: 1743-1752Crossref PubMed Scopus (118) Google Scholar), an H1 replication-independent isoform restricted to terminally differentiated nucleated erythrocytes of birds, which also appears to be present in amphibians (58Khochbin S. Gene (Amst.). 2001; 271: 1-12Crossref PubMed Scopus (113) Google Scholar) and reptiles. The PL-I sperm-specific proteins, which appear at the end of spermiogenesis (yet another terminally differentiation process) in some vertebrate and invertebrate organisms (16Ausió J. J. Biol. Chem. 1999; 274: 31115-31118Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), would also belong to this classification. PL-I protein evolution and the possible link to protamine evolution is summarized in Fig. 2, where a hypothetical model involving the loss of the winged fold domain upon transition from lysine to arginine in precursor PL-I proteins is shown. Such a loss could be speculatively attributed to a gene duplication process, which has been common in both protamine evolution (59Black J.A. Dixon G.H. Nature. 1967; 216: 152-154Crossref PubMed Scopus (27) Google Scholar) and H1 evolution (36Eirin-Lopez J.M. Gonzalez-Tizon A.M. Martinez A. Mendez J. Mol. Biol. Evol. 2004; 21: 1992-2003Crossref PubMed Scopus (52) Google Scholar). Significantly, the PL genes in bivalve mollusks have been shown to occur in hyper-variable restriction fragment length polymorphism (RFLP) regions (60Heath D.D. Hilbish T.J. Genome. 1998; 41: 587-596Crossref PubMed Scopus (9) Google Scholar). The obvious changes in the structural properties of the proteins, derived from the lysine to arginine transition, determined the new mechanisms underlying protamine evolution. It is their high arginine content that allows protamines to tightly condense chromatin in the sperm nucleus. This feature represented a new (and the most important) constraint driving their evolution, which differed to that presented by somatic H1 proteins. In fact, both positive Darwinian (adaptive) selection (61Rooney A.P. Zhang J. Mol. Biol. Evol. 1999; 16: 706-710Crossref PubMed Scopus (93) Google Scholar) and an unusual form of purifying selection (62Rooney A.P. Zhang J. Nei M. Mol. Biol. Evol. 2000; 17: 278-283Crossref PubMed Scopus (62) Google Scholar) are the major mechanisms to which protamines are subject in their evolutionary process. The lysine to arginine transition and gene segregation may have taken place several times in the course of evolution. Whether this has involved a mechanism similar to that described in tunicates remains to be established. In this regard, it is interesting to note that the process of post-translational cleavage of PL-I precursors has occurred repeatedly in completely unrelated groups of organisms such as bivalve mollusks and ascidian tunicates. Remarkably, in both instances the next step in the evolution of these two groups, cephalopods (25Gimenez-Bonafe P. Soler F.M. Buesa C. Sautiere P.E. Ausio J. Kouach M. Kasinsky H.E. Chiva M. Mol. Reprod. Dev. 2004; 68: 232-239Crossref PubMed Scopus (10) Google Scholar, 63Lewis J.D. de Jong M.E. Bagha S.M. Tang A. Gilly W.F. Ausió J. J. Mol. Evol. 2004; 58: 673-680Crossref PubMed Scopus (8) Google Scholar) and cephalochordates (10Chiva M. Saperas N. Caceres C. Ausió J. Jamieson B.G.M Ausió J. Justine J.L. Advances in Spermatozoal Taxonomy and Phylogeny. 166. Memories of the National Museum of Natural History, Paris1995: 501-514Google Scholar), has involved the acquisition of an independent protamine gene encoding for a protein with characteristics almost identical to those of the PL-I arginine-rich fragments. In conclusion, if it can finally be proven that protamines with independent genes are related to linker histones through the process described above, this relationship would have resulted in the closure of an interesting evolutionary cycle in which the C-terminal domain of linker histones would have returned to the initial independent existence of its eubacterial/protozoan ancestry (see Fig. 2).