Protamine-like proteins: evidence for a novel chromatin structure.

Protamine-like (PL) proteins are DNA-condensing proteins that replace somatic-type histones during spermatogenesis. Their composition suggests a function intermediate to that of histones and protamines. Although these proteins have been well characterized at the chemical level in a large number of species, particularly in marine invertebrates, little is known about the specific structures arising from their interaction with DNA. Speculation concerning chromatin structure is complicated by the high degree of heterogeneity in both the number and size of these proteins, which can vary considerably even between closely related species. After careful examination and comparison of the protein sequences available to date for the PL proteins, we propose a model for a novel chromatin structure in the sperm of these organisms that is mediated by somatic-type histones, which are frequently found associated with these proteins. This structure supports the concept that the PL proteins may represent various evolutionary steps between a sperm-specific histone H1 precursor and true protamines. Potential post-translational modifications and the control of PL protein expression and deposition are also discussed.     

The Sperm Nuclear Basic Proteins (SNBPs) of the Sponge Neofibularia nolitangere: Implications for the Molecular Evolution of SNBPs

We have isolated and characterized for the first time, the SNBPs from an organism (Neofibularia nolitangere) of the phylum Porifera (Sponges). We have shown that these proteins consist of histones which, as expected, exhibit an amino acid composition very similar to that of other eukaryotic histones. The finding of histones in the sperm of these primitive organisms provides support to the notion that histones (SNBPs of the histone, H, type) were the proteins present at the onset of SNBP evolution. In contrast, a discrete number of alternative SNBP types (protamine-like, PL; protamine, P, types) seem to have appeared later on in the course of evolution and are found in both protostomes and deuterostomes, most likely as a result of processes of parallel evolution. Received: 5 March 1997 / Accepted: 6 March 1997     

Replacement of nucleosomal histones by histone H1-like proteins during spermiogenesis in Cnidaria: Evolutionary implications

We have analyzed the chromosomal protein composition of the sperm from several species belonging to three different classes (Hydrozoa, Scyphozoa, Anthozoa) of the phylum Cnidaria. In every instance, the sperm nuclear basic proteins (SNBPs) were found to consist of one to two major protein fractions that belong to the histone H1 family, as can be deduced from their amino acid composition and solubility in dilute perchloric acid, and the presence of a trypsin-resistant core. In those species where mature spawned sperm could be obtained, we were able to show that these proteins completely replace the somatic histones from the stem cells that are present at the onset of spermatogenesis. The presence of a highly specialized histone H1 molecule in the sperm of this phylum provides support for the idea that the protamine-like proteins (PL) from higher groups in the phylogenetic tree (and possibly protamines as well) may all have evolved from a primitive histone H1 ancestor.     

Basic nuclear protein pattern and chromatin condensation in the male germ cells of a tropical abalone, Haliotis asinina

The basic nuclear proteins (BNPs) in spermatozoa of a tropical abalone, Haliotis asinina, were composed of a majority of protamine-like (PL) protein and a small amount of histones H1 and H4. Abalone H1 and PL proteins exhibited strong immunological cross reactivities among themselves as well as with chick H5 and calf thymus H1. Thus, all these proteins may belong to the same family. Immunolocalization by indirect immunofluorescence and immunoelectron microscopy indicated that H1 and H4 were present in all steps of the male germ cells, however, with decreasing amount in late stage cells, particularly spermatids and spermatozoa. On the other hand, PL was present in middle step cells (secondary spermatocytes) with increasing amount in spermatids and spermatozoa when the chromatin became tightly packed. Thus, PL may be involved in the condensation of chromatin in the spermatozoa of this species.     

The structure of <Emphasis Type="Italic">Dreissena polymorpha</Emphasis> (Pallas) biocenosis and the role of mollusk in roach <Emphasis Type="Italic">Rutilus rutilus</Emphasis> (Linnaeus) feeding in Pleshcheevo Lake

As a result of studies on the macrozoobenthos composition of zebra mussel biocenosis in Lake Pleshcheevo carried out in 1996, a total of 44 species of bottom macroinvertebrates were found. The richest in terms of species composition were chironomids (17 species), oligochaetes (13 species), and mollusks and leeches (5 species each). After the introduction of zebra mussel Dreissena polymorpha (Pallas) into the lake, a natural biophilter was formed in the waterbody at depths of 4.5 to 9 m. This biophilter intercepts a substantial part of degrading macrophytes and allochtonous organic matter. Thus, the filter prevents the income of this organic matter in the lake’s profundal zone. Two roach Rutilus rutilus (Linnaeus) groups had inhabited Lake Pleshcheevo before dreissena introduction. Comparatively fast growing roach fed predominantly on mollusks of g. g. Valvata and Bithynia, while slowly growing roach fed mostly on zooplankton. After the introduction and mass development of D. polymorpha in the lake, roach started to consume these mollusks. This resulted in increases in the fish growth rate and maximum body size. Roach starts to feed on dreissena and other mollusks after reaching a body length of 15.0 cm. This relates to the third and the last change in the pharyngeal teeth shape, making possible for the fish to consume mollusks and to crush their shells.     

Histones are the major chromosomal protein components of the sperm of the nemerteans Cerebratulus californiensis and Cerebratulus lacteus

We have characterized for the first time the sperm nuclear basic proteins (SNBP) from two species of nemerteans: Cerebratulus californiensis and Cerebratulus lacteus. Gel electrophoretic and chromatographic (RP-HPLC) analysis of the nuclear sperm extracts indicate that histones are the major protein components which are present. The linker histones (histones of the H1 family) exhibit a rather unusual composition and some of them contain cysteine. Several histone H1 isoforms are present, one of which has a composition similar to that of other H1 histones found in the sperm chromatin of other groups of lower invertebrates.     

Engineering bacteriocin‐mediated resistance against the plant pathogen Pseudomonas syringae

The plant pathogen, Pseudomonas syringae (Ps), together with related Ps species, infects and attacks a wide range of agronomically important crops, including tomato, kiwifruit, pepper, olive and soybean, causing economic losses. Currently, chemicals and introduced resistance genes are used to protect plants against these pathogens but have limited success and may have adverse environmental impacts. Consequently, there is a pressing need to develop alternative strategies to combat bacterial disease in crops. One such strategy involves using narrow‐spectrum protein antibiotics (so‐called bacteriocins), which diverse bacteria use to compete against closely related species. Here, we demonstrate that one bacteriocin, putidacin L1 (PL1), can be expressed in an active form at high levels in Arabidopsis and in Nicotiana benthamiana in planta to provide effective resistance against diverse pathovars of Ps. Furthermore, we find that Ps strains that mutate to acquire tolerance to PL1 lose their O‐antigen, exhibit reduced motility and still cannot induce disease symptoms in PL1‐transgenic Arabidopsis. Our results provide proof‐of‐principle that the transgene‐mediated expression of a bacteriocin in planta can provide effective disease resistance to bacterial pathogens. Thus, the expression of bacteriocins in crops might offer an effective strategy for managing bacterial disease, in the same way that the genetic modification of crops to express insecticidal proteins has proven to be an extremely successful strategy for pest management. Crucially, nearly all genera of bacteria, including many plant pathogenic species, produce bacteriocins, providing an extensive source of these antimicrobial agents.     

Serotonergic cerebral cells control activity of cilia in the foregut of the pteropod mollusk <Emphasis Type="Italic">Clione limacina</Emphasis>

Bilaterally symmetrical pair of serotonergic cells, named C1 in Clione, has been described in the cerebral ganglia of all gastropod species. Here we describe a new role of C1 cells in gastropod mollusks: control of activity of ciliated epithelium in the foregut. Detailed morphological investigation of C1 neurons in the pteropod mollusk Clione limacina revealed that these cells among other destinations send their neurites into foregut where they produce intense arborization with large varicosities along the processes. Intracellular stimulation of a single C1 induced pronounced activation (often followed by inhibition) of cilia lining the foregut. This activation was substantially reduced by serotonin antagonist mianserin. Bath application of serotonin also induced transient increase in ciliary transport rate, followed by inhibition of ciliary activity up to its full cessation in some areas of isolated foregut. These data suggest that C1 in Clione may use serotonin to influence cilia in the foregut. Taking into account high homology of serotonergic cerebral cells across studied species we can speculate that these cells may be involved in the neural control of cilia in the foregut in other gastropod mollusks.     

Membrane phospholipid composition of hemocytes in the Pacific oyster Crassostrea gigas and the Manila clam Ruditapes philippinarum

The detailed sterol (free sterol proportions and compositions) and phospholipid (PL) compositions (relative proportions of PL classes and subclasses and their respective fatty acid (FA) compositions) of hemocyte membranes were investigated in two bivalve mollusks: the Pacific oyster Crassostrea gigas and the Manila clam Ruditapes philippinarum. Hemocyte membrane lipids of both species revealed similar general composition: i) their free sterol/PL ratio was above 0.4 and ii) their PL were predominated by the diacyl+alkyl forms of glycerophosphatidylcholine (PC), the plasmalogen form of glycerophosphatidylethanolamine (PE) and ceramide aminoethylphosphonate (CAEP). Free sterols were predominated by cholesterol in both species. Plasmalogen forms of PE and glycerophosphatidylserine (PS) represented 82-83% and 46-55% of total PE and PS, respectively. When compared to their respective diacyl+alkyl forms, plasmalogen forms of PE and PS were specifically enriched in non-methylene-interrupted (NMI) FA and 20:1n-11, suggesting a functional significance of these PL molecular species in bivalve hemocytes. Lysoglycerophosphatidylcholine (LysoPC) levels were found to be fairly high in hemocytes, accounting for about 8% of the PL. Some species-specific features were also found. LysoPC and glycerophosphatidylinositol (PI) FA compositions differed between Ruditapes philippinarum and Crassostrea gigas. CAEP proportion was higher in R. philippinarum than in C. gigas (14.5% and 27.9% of the PL, respectively). Hemolymph cell monolayer observations and flow-cytometric analyses revealed species-specific hemocyte morphology and sub-populations which could account for some of the observed species-specific membrane lipid compositions.     

Sperm Nuclear Basic Proteins (SNBPs) of Agnathans and Chondrichthyans: Variability and Evolution of Sperm Proteins in Fish

We have characterized for the first time SNBPs from the hagfish Eptatratus stouti (Myxini) and the lamprey Lampetra tridentatus (Cephalaspidomorphi) and have found that histones are the major protein components of the sperm of these agnathans. We have also conducted a systematic analysis of SNBPs from different groups of chondrichthyan fishes, including the skate Raja rhina and seven species of sharks. Together with our previous data showing the sporadic nature of SNBP evolution in bony fish (Saperas, N., Ausio, J., Lloris, D. and Chiva, M. [1994] J. Mol. Evol. 39: 282–295), the present study provides a unique insight into the overall evolutionary complexity and variability of the nuclear sperm proteins of fishes. It would appear that despite the discontinuous evolution of these proteins, the macroevolutionary pattern of histone (H type) → protamine-like (PL type) → protamine (P type) has been conserved in fish evolution, as it has in the evolution of other Deuterostomes. Received: 11 June 1996 / Accepted: 6 August 1996     

High mobility group-like proteins of the insect Plodia interpunctella

Nuclei from Plodia interpunctella larvae contain four major proteins, which are extracted by 5% perchloric acid and 0.35 M NaCl. The proteins have been designated PL1, PL2, PL3, and PL4. The amino acid analyses of these proteins show that they have high proportions of acidic and basic amino acid residues, a property characteristic of the high mobility group (HMG) proteins isolated from vertebrate tissues. Immunological characterization of these proteins clearly shows that PL1, PL2, and PL4 are more closely related to HMG1 dipteran proteins, while PL3 is more closely related to HMGI dipteran proteins. The possible relatedness of these proteins to HMG proteins is discussed.     

The Effect of Seasonal Shifts in Temperature on the Lipid Composition of Marine Macrophytes

The comparative study of lipid composition was carried out in four species of marine algae, Ahnfeltia tobuchiensis, Laminaria japonica, Sargassum pallidum, and Ulva fenestrata, as well as a higher plant grass wrack (Zostera marina). Plants were collected in the Japan Sea in spring at 2.9 and 5.5°C and in summer at 23°C. The main lipid components of membranes were determined, and the general patterns of the ratio of phospholipids (PL), glycolipids (GL), betaine (BL), and neutral (NL) lipids were discerned. The relative content of NL in all species (except A. tobuchiensis) was higher in summer. The level of triacylglycerols was as high as 18–37%. The content of individual classes of PL and GL varied between the spring and summer samples, the relative content of PL being higher in spring. In most species, the ratio of PL to GL decreased in summer. The content of free sterols did not depend on the season. The molar ratios of phosphatidylcholine and diacylglycerol-o-(hydroxymethyl)-(N,N,N-trimethyl)homoserine to free sterols varied from 0.9 to 1.7. The seasonal changes of lipid composition were apparently related to macrophyte adaptation to water temperature and to biology of their development.     

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.     

Complex chromatin condensation patterns and nuclear protein transitions during spermiogenesis: examples from mollusks

In this paper we review and analyze the chromatin condensation pattern during spermiogenesis in several species of mollusks. Previously, we had described the nuclear protein transitions during spermiogenesis in these species. The results of our study show two types of condensation pattern: simple patterns and complex patterns, with the following general characteristics: (a) When histones (always present in the early spermatid nucleus) are directly replaced by SNBP (sperm nuclear basic proteins) of the protamine type, the spermiogenic chromatin condensation pattern is simple. However, if the replacement is not direct but through intermediate proteins, the condensation pattern is complex. (b) The intermediate proteins found in mollusks are precursor molecules that are processed during spermiogenesis to the final protamine molecules. Some of these final protamines represent proteins with the highest basic amino acid content known to date, which results in the establishment of a very strong electrostatic interaction with DNA. (c) In some instances, the presence of complex patterns of chromatin condensation clearly correlates with the acquisition of specialized forms of the mature sperm nuclei. In contrast, simple condensation patterns always lead to rounded, oval or slightly cylindrical nuclei. (d) All known cases of complex spermiogenic chromatin condensation patterns are restricted to species with specialized sperm cells (introsperm). At the time of writing, we do not know of any report on complex condensation pattern in species with external fertilization and, therefore, with sperm cells of the primitive type (ect-aquasperm). (e) Some of the mollusk an spermiogenic chromatin condensation patterns of the complex type are very similar (almost identical) to those present in other groups of animals. Interestingly, the intermediate proteins involved in these cases can be very different.In this study, we discuss the biological significance of all these features and conclude that the appearance of precursor (intermediate) molecules facilitated the development of complex patterns of condensation and, as a consequence, a great diversity of forms in the sperm cell nuclei     

Heterogeneity of the histone-containing chromatin in sea cucumber spermatozoa : Distribution of the basic protein Φ0 and absence of non-histone proteins

Nuclei of spermatozoa of the sea cucumber Holothuria tubulosa contain the five somatic-type histones plus a sperm-specific histone H1 and a unique basic protein ₀, which is related to H1 in amino acid composition. No proteins of the High Mobility Group (HMG) type have been detected. The structure of this chromatin has been probed by nuclease digestion. Its behaviour is anomalous, since two distinct fractions of chromatin are recovered from these spermatozoa, which differ either in the presence or absence of the sperm-specific proteins H1 and ₀. This heterogeneous distribution is not found in conventional materials, such as calf thymus or chicken erythrocytes. Proteins H1 and ₀ are not uniformly distributed and may be localized in special regions of chromatin. Fragments containing long stretches of nucleosomes lacking both proteins can be recovered. At the same time, the chromatin fractions which contain these two proteins are shown to be less soluble. When an extensive digestion of chromatin is carried out yielding only nucleosomes and small oligomers, the H1 and ₀ proteins redistribute themselves on chromatin, the two proteins acting in a cooperative fashion in this process. Cross-linking experiments carried out in whole cells indicate a proximity of ₀ and H1, whereas no crosslinks have been detected between ₀ and any of the four nucleosomal histones. The ₀ protein may thus play a role similar to histone H1 and be only loosely associated with nucleosomal histones, but contribute to the structuration of chromatin during spermiogenesis.     

Purification and characterization of the protamines and related proteins from the sperm of a tunicate,Styela plicata

• 1.1. We have characterized for the first time the major sperm-specific nuclear proteins (X, P1 and P2) of the tunicate Styela plicata. Both P1 and P2 have an amino acid composition that allows us to classify them as protamine-like proteins.
• 2.2. The protein P1 of lower electrophoretic mobility has a trypsin-resistant core which is compositionally related to that of histones of the H1 family and to the PL-I protein found in the sperm of marine invertebrates. The evolutionary significance of this finding is discussed.
• 3.3. In addition to P1 and P2, the sperm nucleus of S. plicata contains a protein X component which is also compositionally related to PL-I proteins from bivalve molluscs.
• 4.4. Besides these sperm-specific proteins, a full complement of somatic-like histones, including a somatic-like histone H1, is also present. These histones represent only a small fraction of the total nuclear proteins of the sperm.

     

NUCLEAR HISTONE PROTEINS OF GAMETES IN AN OOGAMOUS AND TWO ISOGAMOUS BROWN ALGAE1

Nuclear basic proteins (histones) were studied in male and female gametes of the isogamous brown algae, Colpomenia bullosa (Saunders) Yamada and Analipus japonicus (Harvey) Wynne and sperm of the oogamous Cystoseira hakodatensis (Yendo) Fensholt by using SDS‐ and AUT‐PAGE. Four major core histones and several linker histone H1s were detected by electrophoresis. Each of the core histones was identified by amino acid sequence analysis and peptide mapping. Electrophoresis patterns of histones were the same in male and female gametes and quite similar between the two species. The composition of histone H1s in conspicuously condensed sperm nuclei of C. hakodatensis was different from that in isogamous gametes. Electrophoresis after micrococcal nuclease digestion of chromatin in male and female gamete nuclei of C. bullosa and A. japonicus and sperm of C. hakodatensis resulted in regular ladder patterns of DNA fragments (ca. 200 base pair). The chromatin of the brown algal gametes thus has the typical nucleosome structure. These results showed that chromatin condensation in sperm nuclei of C. hakodatensis was associated with a modification of linker histone H1 but not by change of core histones, replacement by other basic proteins, changes of repeating patterns, or disappearance of nucleosomes.     

Proteins associated with mitochondrial DNA protect it against the action of X-rays and hydrogen peroxide

It has been suggested in a number of investigations that the high vulnerability of mitochondrial DNA to reactive oxygen species and other damaging agents is due to the absence in mitochondria of histones complexed with DNA. In the present study it was shown that DNA-binding proteins of mitochondrial nucleoids were able to shield mitochondrial DNA from X-ray radiation and hydrogen peroxide, as nuclear histones did. Mitochondria, mitochondrial nucleoid proteins, and histones were isolated from mouse liver cells. The degree of damage to or protection of mitochondrial DNA was assessed from the yield of its PCR amplification product. The in vitro experiments demonstrated that mouse mitochondrial DNA, when in complex with mitochondrial nucleoids or nuclear histones, was damaged much less by radiation and/or hydrogen peroxide than in the absence of these proteins and histones. No significant difference between mitochondrial nucleoid proteins and nuclear histones was revealed in their efficiency to protect mitochondrial DNA from the damaging effect of radiation and hydrogen peroxide. It is likely that the nucleoid proteins in the mitochondria shield mitochondrial DNA against the attack of reactive oxygen species, thus significantly decreasing the level of the oxidative damage to mitochondrial DNA.     

Studies on the association of Metopa glacialis (Amphipoda, Crustacea) and Musculus discors (Mollusca, Mytilidae)

Associations between amphipods and mollusks are known from several examples, but the possible life-history strategies these amphipods utilize are often unknown. We present here the autecology of the stenothoid amphipod Metopa glacialis, an associate of the mytilid bivalve Musculus discors. Samples collected from northeast Spitsbergen of 279 M. discors show a prevalence of M. glacialis in 75%, with a variation between 1 and 41 amphipods within one mussel, the mean being 3.7. The associated amphipods have been analysed for several life-history traits such as size distributions, brood sizes and cohort compositions. Analyses of fatty acid composition of the associated species indicate different food sources for the amphipods and mollusks. Based on our data we propose two possible life-history strategies for M. glacialis: 3 or more seasons of single broods and parental care over more than 1 year after hatching, or multiple broods in one season and extended parental care. To our knowledge multiple broods in one season and extended parental care have never been reported in arctic amphipods.