Basic Nuclear Proteins Research Articles

Protamine-like sperm nuclear basic proteins in the primitive frogAscaphus trueiand histone reversions among more advanced frogs

Sperm nuclear basic proteins (SNBPs) that condense chromatin are very diverse. In animals, evolution of SNBPs has proceeded from lysine-rich histone H type in sponges to more arginine-rich protamine-like PL and protamine P types. Yet sporadic PL/P to H reversions are known to occur in both protostomes and deuterostomes. To determine why this is the case, we have examined SNBPs in eleven anuran species. We find that sperm of the primitive, internally fertilizing archeobatrachian frog A. truei (family Ascaphidae) has PL/P type (42 mol % arginine), with an electrophoretic profile similar to SNBPs in another archeobatrachian, externally fertilizing Leiopelma hochstetteri (family Leiopelmatidae). Cytochemistry of sperm nuclei in the advanced, externally fertilizing neobatrachian frogs Crinia signifera and C. deserticola (family Myobatrachidae) indicates that they have reverted to H type SNBPs. This is also known to be the case in externally fertilizing Rana (family Ranidae) and Silurana, a subgenus of Xenopus (family Pipidae). Such a trend, from PL/P type SNBPs in two archeobatrachians to sporadic reversions to H type in more advanced frogs, parallels the ultrastructural simplification from complex A. truei introsperm to neobatrachian aquasperm that Jamieson et al. (1993. Herpetologica 49:52-65) attribute as a secondary reversion to external fertilization.

Two murine and human homologs of mab-21, a cell fate determination gene involved in Caenorhabditis elegans neural development.

We report the cloning and genetic characterization of one human and two murine homologs of the mab-21 cell fate specification gene. mab-21 participates in the formation of sensory organs in the male nematode tail, and is essential for other developmental functions elsewhere in the Caenorhabditis elegans embryo. The expanding mab-21 gene family, which is strikingly conserved in evolution, includes two putative Drosophila members. The two mammalian genes, encoding 41 kDa nuclear basic proteins, are expressed in partially overlapping territories in the embryonic brain, eye and limbs, as well as in neural crest derivatives. Recent genetic data implicating mab-21 as a downstream target of TGF-beta signaling, together with the distribution of mab-21 transcripts in the mouse embryo, propose these novel genes as relevant factors in various aspects of vertebrate neural development.

Protamine‐like sperm nuclear basic proteins in the primitive frog Ascaphus truei and histone reversions among more advanced frogs

Sperm nuclear basic proteins (SNBPs) that condense chromatin are very diverse. In animals, evolution of SNBPs has proceeded from lysine-rich histone H type in sponges to more arginine-rich protamine-like PL and protamine P types. Yet sporadic PL/P to H reversions are known to occur in both protostomes and deuterostomes. To determine why this is the case, we have examined SNBPs in eleven anuran species. We find that sperm of the primitive, internally fertilizing archeobatrachian frog A. truei (family Ascaphidae) has PL/P type (42 mol % arginine), with an electrophoretic profile similar to SNBPs in another archeobatrachian, externally fertilizing Leiopelma hochstetteri (family Leiopelmatidae). Cytochemistry of sperm nuclei in the advanced, externally fertilizing neobatrachian frogs Crinia signifera and C. deserticola (family Myobatrachidae) indicates that they have reverted to H type SNBPs. This is also known to be the case in externally fertilizing Rana (family Ranidae) and Silurana, a subgenus of Xenopus (family Pipidae). Such a trend, from PL/P type SNBPs in two archeobatrachians to sporadic reversions to H type in more advanced frogs, parallels the ultrastructural simplification from complex A. truei introsperm to neobatrachian aquasperm that Jamieson et al. (1993. Herpetologica 49:52–65) attribute as a secondary reversion to external fertilization. J. Exp. Zool. 284:717–728, 1999. © 1999 Wiley-Liss, Inc.

Analysis of hamster protamines: primary sequence and species distribution.

Basic nuclear proteins were isolated from the sperm of the Syrian hamster Mesocricetus auratus and characterized by gel electrophoresis, amino acid analysis, and sequencing. Analyses of the proteins by gel electrophoresis show that sperm of this species contain both protamines 1 and 2. The two proteins were purified by HPLC and the complete primary sequence of hamster protamine 1 was determined by automated amino acid sequence analysis. The protein sequence was subsequently confirmed by sequencing the PCR-amplified protamine 1 gene. The first forty-two residues of the hamster protamine 2 sequence were obtained by amino acid sequence analysis of the isolated protein, and this sequence was also confirmed and extended by sequencing the gene. Total basic nuclear protein was also isolated from sperm of six other species of hamsters, the protamines were identified by HPLC and amino acid analysis, and the proportion of protamines 1 and 2 in each species was determined. Marked differences in the protamine 2 content of sperm were observed among the different species of hamster. This variation and the high level of sequence similarity between mouse and hamster protamines provide insight into how the two protamines may be organized in sperm chromatin. Mol. Reprod. Dev. 54:273-282, 1999. Published 1999 Wiley-Liss, Inc.

Sperm Nuclei of Discoglossus pictus (Anuran Amphibian) Contain a Unique, Histidine-rich Basic Protein

Fractionation with reverse-phase HPLC and electrophoretic analyses revealed that the nuclear basic proteins in sperm of an anuran amphibian, Discoglossus pictus, consisted of weakly basic proteins without histones. Amino acid analyses indicated that none of these proteins were histones or protamines because of the low amounts of Lys and Arg. The predominant protein among those fractionated possessed an unusually high content of His and extremely low amounts of Arg and Lys, indicating that this is a unique nuclear basic protein not reported previously. The induction of a highly decondensed state of sperm nuclei upon their incubation in egg extract was accompanied by the removal of most, if not all, of this His-rich protein from the nuclei.

Structure and expression of the highly repetitive histone H1-related sperm chromatin proteins from winter flounder.

In the late stages of spermatogenesis, winter flounder produce a family of high molecular mass (80-200 kDa) basic nuclear proteins (HMrBNPs) that combine with the normal complement of histones to produce condensed sperm chromatin with an increased nucleosomal repeat length. The HMrBNPs have a biased amino-acid composition in which Arg, Ser, Lys and Pro are abundant because of their presence in many simple peptide repeats. The organization of these repeats was deduced by cDNA cloning. The predominant repeating units are related 26- and 30-amino-acid sequences that in turn are linked by 6-amino-acid spacers to form 58- and 62-amino-acid repeats. Subsets of these repeats are also present, such as a dispersed 20-amino-acid repeat and a tandem array of nine heptapeptides at the C-terminus. The HMrBNPs appear to have evolved from an extreme H1 variant that has an N-terminal tail of HMrBNP-like sequence linked to an H1 globular region. Based on sequences of the most abundant HMrBNP cDNAs, and the lack of hybridization between HMrBNP mRNAs and a DNA probe for the H1 globular region, the latter domain appears to have been lost during expansion and amplification of the HMrBNP-like repeats. Transcripts of the HMrBNP and H1 variant genes are present in testis RNAs only during the mid-spermatid stage of spermatogenesis, at the same time that HMrBNPs in their highly phosphorylated form first appear in the nucleus. Judging by the lack of a lag between HMrBNP mRNA synthesis and translation, the mRNAs for these highly basic proteins are not stored for any length of time. Instead, the deposition of HMrBNPs onto DNA, which coincides with the major reorganization and silencing of the chromatin, may be controlled by dephosphorylation.

Transcriptional and translational regulation of gene expression in haploid spermatids.

During spermiogenesis, round spermatids undergo complex morphological, biochemical, and physiological modifications resulting in the formation of mature spermatozoa. While in round spermatids histones and non-histone proteins are replaced by transition proteins, in elongating spermatids, transition proteins are removed from the condensing chromatin and are replaced by protamines, which are the principal basic nuclear proteins of mature spermatozoa. The tightly packed DNA-protamine complexes cease transcription several days before the completion of spermiogenesis. Thus, major modifications in both nuclear and cytoplasmic structures continue throughout spermiogenesis, stringent temporal and stage-specific gene expression is a prerequisite for the correct differentiation of round spermatids into mature spermatozoa. The genes for transition proteins and protamines are transcribed in round and elongating spermatids. Transcription is regulated via methylation and trans-acting factors that bind to the TATA-box, the CRE-box, or other specific DNA sequences in the promoter region. The transcripts are stored as ribonucleoprotein particles in a translationally repressed state for several days and are translated in elongating and elongated spermatids. It has been demonstrated that, in haploid spermatids, essentially every mRNA exhibits evidence of translational repression. Translational regulation involves protein repressors that bind to the poly-A tail or specific RNA sequences located in the 3'-UTR.

Recent and rapid amplification of the sperm basic nuclear protein genes in winter flounder

The high molecular weight basic nuclear proteins (HMrBNPs), which are tightly bound to sperm chromatin in winter flounder, are made up of imperfect reiterations of simple peptide sequences that contain phosphorylatable DNA-binding motifs. Genomic Southern blots hybridized with probes to the coding and non-coding regions of HMrBNP mRNA showed that HMrBNP sequences form a complex multi-gene family. Previously, one gene (2B) was used to establish an evolutionary link between histone H1 and the HMrBNPs. Further examination of this complex, multi-gene family has now revealed that the majority of the HMrBNP genes are linked as 4.5 kb direct tandem repeats that each contain a 2.8 kb coding region and a 1.7 kb intergenic region (IR). These findings, combined with the cloning of the IR, established that the tandemly repeated genes lack introns and code for the abundant 3 kb HMrBNP mRNAs that produce the prominent 110 kDa HMrBNP. Southern blotting of DNAs from other righteye flounder species showed that HMrBNP multi-gene families were present in closely related species, though with substantial differences in restriction patterns and band intensities, but were not detected in more distantly related flounders. These observations are consistent with recent and rapid elaboration of the HMrBNP gene family.

Variability of sperm nuclear basic proteins in the three-spined stickleback and related species of Gasterosteoidei

In the present study we have examined the sperm nuclear basic proteins (SNBPs) from mature testes of the three-spined stickleback Gasterosteus aculeatus and three related teleosts in the suborder Gasterosteoidei. Acid/urea polyacrylamide gel electrophoresis (PAGE) of SNBPs from Gasterosteus wheatlandi and Pungitius pungitius (family Gasterosteidae), as well as Aulorhynchus flavidus (family Aulorhynchidae), shows the largest variability of SNBPS in any suborder of teleosts thus far examined. Amino acid analysis reveals a new type of protamine-like SNBP with a high level of arginine (37 mol% in G. aculeatus) along with the presence of histidine and lysine, in agreement with cytochemical results. A PAGE developmental profile shows that the SNBP from freshwater G. aculeatus may undergo post-translational modification during spermiogenesis or, alternatively, may be produced from a higher molecular weight precursor. SNBP variability within the G. aculeatus species complex is much more limited. Although small differences exist between SNBPs from anadromous and freshwater G. aculeatus in lysine/arginine ratios and in the number of multiple electrophoretic bands, small or no differences are detected among SNBPs from different geographically distinct freshwater populations. An exception is in sticklebacks from Paxton Lake and Enos Lake, BC, where relatively recent divergent populations of benthic and limnetic populations are found. Here, G. aculeatus benthic populations show higher levels of residual somatic histones in testes along with the SNBPs, suggesting that chromatin condensation in these mature sperm may differ from that in limnetics.

The amino acid sequence of the ram spermatidal protein 3--a transition protein TP3 or TP4?

As in other mammals, several nuclear basic proteins replace histones during the differentiation of germinal cells into spermatozoa in the ram. These proteins called transition proteins (TP) are later replaced by protamines. The amino acid sequence of the ram spermatidal protein 3 has been established by Edman degradation of the protein and of its fragments generated from digestion with endoproteinase Lys-C and pepsin and from the coding sequence of the gene and of the cDNA. The ram protein 3 is a basic protein of 109 residues (calculated Mr 13,200) with arginine and lysine residues uniformly distributed along the polypeptide chain. Of the 13 serine and threonine residues, 9 are located in structural motifs where they could be phosphorylated and, thus, modulate the binding of the protein to DNA. The tyrosine residues at position 33 and position 93, located in a basic environment, and the tryptophan residue at position 29 could be involved in the interactions of the protein with DNA through the stacking of their aromatic ring between the nucleotide bases. The ram protein 3 differs completely from the two well-defined transition protein families TP1 and TP2, which are also synthesised transiently during mammal spermiogenesis. In contrast with the rat TP3, ram protein 3 does not correspond to a precursor of a protamine. However, it shares structural similarities with both transition proteins TP3 and TP4 of the boar. The ram protein 3 and the boar transition proteins TP3 and TP4 probably belong to the same transition-protein group and would play similar functions in the chromatin remodelling during spermiogenesis. As protamine and transition-protein TP1 and TP2 genes from mammals, the coding sequence of the gene of ram protein 3 is interrupted by one intron but its organisation is different.

Architectural DNA-Binding Properties of the Spermatidal Transition Proteins 1 and 2

Mammalian spermiogenesis is characterized by replacement of somatic histones by a set of basic nuclear transition proteins thought to be actively involved in the chromatin remodeling process. The two major transition proteins of the elongating spermatids, namely TP1 and TP2, were expressed and purified using a bacterial expression system. Both topoisomerase and ligase-mediated supercoiling assays demonstrated that TP1, as well as TP2, did not produce detectable changes in the twist and/or writhe of DNA molecules upon binding. Ligase-mediated circularization assay further demonstrated that neither of the transition proteins under study produced bends in linear DNA but that they both have the capacity to stimulate oligomerization of linear DNA fragments. We further established that the transition proteins arein vitrosubstrates for the Ca+2-phospholipid-dependent protein kinase (PKC) as well as the cAMP-dependent protein kinase (PKA). PKC phosphorylation was found to strongly weaken the DNA-condensing ability of TP2. These results suggest that the major transition proteins represent architectural factors able to stabilize DNA in a nonsupercoiled state, thereby promoting DNA condensation.

Expression of mRNA and protein of nucleoproteins during human spermiogenesis.

The most important event determining the nuclear status of sperm cells is the replacement of histones by protamines, which are the basic nuclear proteins of mature spermatozoa. A first step in this exchange is the displacement of histones by transition proteins (TP). Our study demonstrates, for the first time, the sequential expression of the testis-specific histone (H1t) and the transition proteins (TP1 and TP2) during normal human spermatogenesis. H1t mRNA could only be detected in the cytoplasm of mid and late pachytene spermatocytes. Concomitant with the onset of H1t transcription, the H1t protein appeared in the nuclei of pachytene spermatocytes and remain as a nuclear protein constituent up to step 5 spermatids. While transition protein 1 gene TNP-1 mRNA was present in spermatids from step 2 to early step 4, the TP1 protein occured, with temporal delay, in the nuclei of step 3 and step 4 spermatids. The TP2 protein was observed in the nuclei of spermatids from step 1 to step 5. The transition protein 2 gene TNP-2 mRNA was only detected by reverse transcription-polymerase chain reaction, but not on paraffin sections. These data demonstrate a strong temporal association between H1t gene transcription and synthesis of the H1t protein. Since the TP1 protein appeared with temporal delay we can assume that the corresponding TNP-1 mRNA is translationally delayed.

The High Molecular Weight Chromatin Proteins of Winter Flounder Sperm Are Related to an Extreme Histone H1 Variant

Unlike mammals, birds, and most other fishes, winter flounder completes spermatogenesis without replacing its germ cell histones with protamines. Instead, during spermiogenesis, these fish produce a family of high molecular weight (80,000-200,000) basic nuclear proteins (HMrBNPs) that bind to sperm chromatin containing the normal complement of histones. These large, basic proteins are built up of tandem iterations of oligopeptide repeats that contain phosphorylatable DNA-binding motifs. Although the HMrBNPs have no obvious homology to histones, protamines, or other sperm-specific chromatin proteins, we report here the isolation of a clone (2B) from a winter flounder genomic DNA library that establishes a link between the HMrBNPs and histone H1. The 2B sequence contains an open reading frame, which, when conceptually translated, encodes a 265-residue protein. At its N terminus the translation product contains numerous simple repeats that match the oligopeptides contained within the HMrBNPs. Unexpectedly, the C terminus of the putative protein shows 66% identity and 76% conservation to the histone H1 globular domain. This connection suggests that the HMrBNPs may have originated from the extended N-terminal tail region of a testis-specific, H1-like linker histone.

Nuclear basic proteins in spermiogenesis

In animal species, spermiogenesis, the late stage of spermatogenesis is characterized by a dramatic remodelling of chromatin which involves morphological changes and various modifications in the nature of the nuclear basic proteins. According to the evolution of species, three situations can be observed: a) persistence of somatic histones or appearance of sperm-specific histones: b) direct replacement of histones by generally smaller and more basic proteins called protamines: and c) occurrence of a double nuclear basic protein transition: histones are not directly replaced by protamines but by intermediate basic proteins which are themselves replaced by one or several protamines. However, in some species, two kinds of intermediate basic proteins can be distinguished in spermatid nuclei: transition proteins and protamine precursors. Whereas transition proteins are not structurally related either to histones or to protamines, protamine precursors are further processed at the end of spermiogenesis to give rise to the mature protamine. The molecular characteristics of the protamines as well as number of prutamine types present in the spermatozoon vary from species to species. In some cases, protamine-encoding genes, although present, are not expressed to a significant level. The diversity and the precise function of intermediate basic proteins remain open to discussion. Some of them are the precursors of protamines but the mechanism, sequential or not, as well as the enzyme(s) involved in the proteolytic processing, remain to be discovered.

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.

Histone H1 variants as sperm‐specific nuclear proteins of Rana catesbeiana, and their role in maintaining a unique condensed state of sperm chromatin

Amino acid analyses of nuclear basic proteins of an anuran amphibian, Rana catesbeiana, revealed that they are comprised of a full set of core histones and three types of lysine-rich, sperm-specific proteins. On the basis of their amino-acid compositions and partial amino-acid sequences of their trypsin-resistant cores, the sperm-specific proteins could be defined as members of the histone H1 family. Both micrococcal nuclease digestion and electron microscopy indicated that sperm chromatin consists of nucleosomal and fibrillar DNA structures which are irregularly interspersed with each other. When sperm nuclei were incubated with nucleoplasmin, nuclei decondensed to some extent, and the sperm-specific H1s were removed, but not completely. The residual sperm-specific histone H1 variants were also found in reconstituted male pronuclear chromatin, comprising regularly spaced nucleosomes. We conclude that sperm-specific histone H1 variants are essential for chromatin condensation in the sperm nuclei, but that their complete removal is not necessary for the remodeling into somatic chromatin that takes place after fertilization. Mol. Reprod. Dev. 47:181–190, 1997. © 1997 Wiley-Liss, Inc.

ANTIBODIES TO HISTONES IN AUTOIMMUNE HEPATITIS OF CHILDHOOD

Antibodies to histones (AHA) are directed against basic nuclear proteins which are complexed with DNA to form nucleosomes that are important in cell replication. These antibodies may be one of the reactivities of antinuclear (ANA) positivity in autoimmune disorders. Serum samples from 18 patients (median age 6 years 7 months) who satisfied criteria for autoimmune hepatitis were studied at onset of the disease: 14 were ANA negative by indirect immunofluoresce on rat liver: 9 of them were anti- liver/kidney microsome antibody (LKM) positive, 3 anti-smooth muscle antibody (SMA) positive, 1 liver anti-cytosol positive, and 1 patient was negative for all reactivities tested. Four patients were ANA seropositive by at a titer of at least 1:100 and 2 of them were also SMA positive. Twenty-seven healthy subjects, comparable for age were included in the study. Serum levels of IgG antibodies to total histones, dsDNA and nucleosomes were assessed by ELISA: sera presenting antibody levels greather than mean + 2 SD of controls were considered positive. Results expressed as number of positive subject/ number of subject tested are shown in the table: Moreover among the 7 ANA negative patients reactive to histones: 5 were anti-LKM positive, I anti-SMA and I anti-cytosol positive. Nine anti-histone positive patients were also tested at irst biochemical remission: in none of them anti-histones antibody levels were higher than mean + 2SD of controls while 5/9 had still anti-LKM or SMA reactivity. In conclusion, antibodies to histones: i) are common in children with autoimmune hepatitis; ii) are associated with ANA reactivity although they can also be found in sera of ANA negative patients;: iii) may represent an early marker of disease remission.

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.

Isolation of the major basic nuclear protein and its localization on chromosomes of the dinoflagellate, Oxyrrhis marina

Basic nuclear proteins were extracted from isolated nuclei of Oxyrrhis marina. The HPLC pattern of the extract showed a single major peak, which consisted of a major band with an apparent molecular mass of 23 kDa on an SDS-PAGE gel. We designated this protein as Np23 because of its apparent molecular mass. The amino acid composition of this protein revealed its extremely basic nature with a high lysine content. Polyclonal antibodies were raised against Np23. Immunofluorescence microscopy showed that Np23 was localized within the nucleus of dividing and non-dividing cells as well, and immuno-electron microscopy showed that the protein was localized only on chromosomes. These data established that Np23 is the major basic chromosome protein of Oxyrrhis marina.

Primary structures of sperm‐specific basic nuclear proteins and gene expression in Japanese newt, Cynops pyrrhogaster

Electrophoretic analyses of acid extracts from mature sperm of newt, Cynops pyrrhogaster, on acid/urea/Triton X-100 polyacrylamide gel showed the exclusive occurrence of sperm-specific nuclear basic proteins (SBPs), which moved faster than somatic histones on the gel. These SBPs were eluted separately by reversed phase-high-performance liquid chromatography as two large peaks and a few small peaks. Of these, only the small peaks disappeared with treatment of the acid extracts with alkaline phosphatase before they were injected into the column, so that there were only two distinct components: NP1 and NP2. Determination of amino acid sequences by the Edman method as well as by sequencing of cDNA for both components indicated that each protein consisted of 43 (NP1) or 48 (NP2) amino acid residues, rich in arginine residues (53.5% in NP1; 47.9% in NP2), forming the clusters. They had molecular masses of 5,386 Da (NP1) and 5,748 Da (NP2), respectively. Northern blot analysis using cDNAs as probes indicated that mRNAs for both NP1 and NP2 occurred not in primary spermatocytes but in round spermatids. In situ hybridization analyses using antisense RNA for NP1 as a probe clearly showed the first appearance of NP1 mRNA at the late stage of round spermatid. Mol. Reprod. Dev. 46:243–251, 1997. © 1997 Wiley-Liss, Inc.