Histone differentiation and nuclear activity

When fast green and eosin are used in combination to stain histones, nuclei display different affinities toward the dyes, some binding fast green exclusively, others binding eosin exclusively, and still others, both stains. In a given tissue, the frequencies of nuclei exhibiting the different colors remain fairly constant over a wide range of staining conditions. Nuclei of cells of the same type may stain differently, but when they are in the same stage of development or state of activity they tend to stain alike. Xenopus erythrocyte nuclei stain bright pink. Condensed mitotic and meiotic chromosomes stain purple. In the grasshopper spermatocyte, the main body of the interphase nucleus stains bright green, but the condensed chromosome stains purple. The mole crab sperm contains several distinct histone-like proteins, that differ in their amino acid compositions, within separate areas of the cell. In these sperms, the lysine-rich histones bind eosin, while the protamine-like protein and arginine-rich histone bind fast green. In general, the eosin and fast green bind preferentially to the lysine and arginine rich histones respectively, when the dyes are permitted to compete with one another. In several systems, including spermiogenesis and erythropoiesis, the aquisition of an eosinophilic component by the nuclei accompanies the slowing of RNA synthesis, and it is suggested that there may be a causal relationship between the two events, the eosinophilic histone effecting RNA synthesis within the nucleus as a whole.     

Model system studies of staining procedures for lysine and arginine residues.

Using polyacrylamide films containg poly-lysine, polyarginine and DNA as test models, a variety of reportedly specific staining procedures have been examine. Contrary to published observations, mixtures of fast green and eosin Y show no specific staining of either lysine or arginine. Both amino-acids bind eosin from the mixture more strongly than fast green. Arginine apparently has a greater affinity for this eosin than has lysine which contradicts previous reports that lysine will be stained by eosin arginine will stain with fast green, if proteins containing both amino-acids are stained with dye mixture. In films containing lysine and/or arginine picric acid is shown to bind specifically to the arginine. The picric acidarginine complex resists disruption in 0.004 M borate buffer which is a solvent used for subsequent staining of lysine residues with bromophenol blue. Picric acid may also be used as a hydrolysant and substitute for hydrocholoric acid in a Feulgen-like procedure which stains DNA to the same level as the classiclal hydrochloric acid based procedure while also staining arginine present.     

Model system studies of staining procedures for lysine and arginine residues

Summary Using polyacrylamide films containing poly-lysine, polyargine and DNA as test models, a variety of reportedly specific staining procedures have been examined. Contrary to published observations, mixtures of fast green and eosin Y show no specific staining of either lysine or arginine. Both amino-acids bind eosin from the mixture more strongly than fast green. Arginine apparently has a greater affinity for this eosin than has lysine which contradicts previous reports that lysine will be stained by eosin while arginine will stain with fast green, if proteins containing both amino-acids are stained with the dye mixture. In films containing lysine and/or arginine picric acid is shown to bind specifically to the arginine. The picric acid-arginine complex resists disruption in 0.004 M borate buffer which is a solvent used for subsequent staining of lysine residues with bromophenol blue. Picric acid may also be used as a hydrolysant and substitute for hydrochloric acid in a Feulgen-like procedure which stains DNA to the same level as the classical hydrochloric acid based procedure while also staining arginine present.     

Arginine-rich proteins in spherical inclusions of human locus coeruleus neurons demonstrated by benzil modification

Previous studies have shown that aminergic neurons in the normal human brain contain acidophilic cytoplasmic inclusions--called protein bodies (PBs)--that are reduced or absent in parkinsonism and disrupted in depression. The purpose of the present study was to elucidate the constitution of PBs in five formalin-fixed normal human brains using histochemical methods specific for histones, protamines, and the amino acid arginine. PBs were revealed with alkaline fast green and bromphenol blue, exhibiting a high content in histones and in protamines. They developed blue metachromasia with phosphotungstic acid-hematoxylin and green fluorescence with phenanthrenequinone, which established the presence of arginyl residues. Using benzil, which selectively modifies the guanido group of arginine, staining was blocked for each of the above two methods. The application of Mallory's trichrome procedure after benzil differentiated the PBs into an unstained core and a still fuchsinophilic rim. Since the fuchsinophilia of the rim was shown to persist after acetylation as well, we suggest that this rim probably contains acidic macromolecules that attach to the basic charges of the amphoteric acid fuchsin. We conclude that the PB are complex structures consisting of a core segregating arginine-rich proteins and a rim which probably contains macromolecules of an acidic nature.     

On the Diversity of Sperm Histones in the Vertebrates

Sperm histones display great variability in contrast to the conservation of most classes of somatic histones. To study this paradox, this series of papers examines the variation of histone patterns in the testis and sperm of vertebrates, particularly amphibians and reptiles, and attempts to relate such variation to genetically based sex determination as hypothesized by Bloch [Genetics Supplement 61, 93 (1969)]. In the present study we have investigated spermiogenesis in the newt Notophthalmus viridescens viridescens . Cytochemical experiments indicate that the basic nuclear proteins undergo progressive shifts from somatic type histone → very arginine-rich "stable protamine'in the later spermatids → protamine in the mature sperm. Electrophoresis of Notophthalmus histones extracted from chromatin reveals that the pattern of testes-specific basic proteins in the urodele is distinct from the pattern of testicular proteins in the anurans Bufo americanus and Xenopus laevis . Species within the class Amphibia therefore exhibit considerable diversification in their type of basic sperm proteins.     

CYTOCHEMICAL AND BIOCHEMICAL DETERMINATIONS OF CHANGES IN NUCLEAR HISTONE CONTENT DURING DIFFERENTIATION OF BARLEY ROOT CELLS

Changes in nuclear histone content in barley root cells have been studied by cytochemical methods for identification of histone subtypes and by conjunction with standard biochemical extraction procedure for various histone fractions and alkaline fast green stainability. The results obtained by the cytochemical methods indicate that the nuclear histones in cell nuclei found in their terminal stages of cellular differentiation or elongation contain histones rich in arginine, whereas the nuclei in meristematic cells contain histones rich in lysine. Cytochemicaly intermediate or transitional types of nuclear histones have been observed in cell nuclei which are undergoing differentiation or elongation and in chromosomes of mitotic nuclei. Information obtained from the conjunction of methods of biochemical extraction procedures for various histone fractions and alkaline fast green stainability indicate that the nuclei in well-differentiated cells contain predominantly histones rich in arginine (f3), whereas the nuclei of meristematic cells contain both very lysine-rich histones (f1) and slightly lysine-rich histones (f2). These results suggest the replacement of lysine-rich histones in the nuclei of meristematic cells by arginine-rich histones during cellular differentiation.     

Nuclear Basic Proteins of Xenopus laevis Sperm: Their Characterization and Synthesis during Spermatogenesis

Nuclear basic proteins from morphologically and functionally mature sperm of Xenopus laevis were analyzed by acid/urea/Triton X-100 polyacrylamide gel electrophoresis (AUT-PAGE). Six sperm-specific proteins (SP1-6) were identified in addition to somatic histones H3, H4 and smaller amount of H2A and H2B, but not H1. Of these, SP3–6 were unique in containing 33–41% arginine and having very low lysine/arginine ratios, while SP2 was more similar to H3 and H4 in having a lower arginine and higher lysine content. Fractionations of testicular cells at different spermatogenic stages by unit gravity sedimentation showed that primary spermatocytes and acrosomal vesicle spermatids possess typical somatic type histones but no SPs. Injection of [¹⁴C]-arginine into the testis and its tracing by fluorography on AUT-PAGE gels indicated that all somatic histones are synthesized during the stages between spermatogonia and primary spermatocytes, whereas SPs are synthesized at differentially regulated rates during the stages after acrosomal vesicle formation. In indirect immunofluorescence studies with anti-SP3-5 rabbit antiserum, a positive reaction was observed in the last step of spermiogenesis after the commencement of nuclear coiling.     

Basic Proteins of Plant Nuclei during Normal and Pathological Cell Growth

Histone proteins were studied by microphotometry of plant tissue sections stained with fast green at pH 8.1. For comparative purposes the Feulgen reaction was used for deoxyribose nuclei acid (DNA); the Sakaguchi reaction for arginine; and the Millon reaction for estimates of total protein. Analysis of Tradescantia tissues indicated that amounts of nuclear histone fell into approximate multiples of the gametic (egg or sperm) quantity except in dividing tissues, where amounts intermediate between multiples were found. In differentiated tissues of lily, corn, onion, and broad bean, histones occurred in constant amounts per nucleus, characteristic of the species, as was found also for DNA. Unlike the condition in several animal species, the basic proteins of sperm nuclei in these higher plants were of the histone type; no evidence of protamine was found. In a plant neoplasm, crown gall of broad bean, behavior of the basic nuclear proteins closely paralleled that of DNA. Thus, alterations of DNA levels in tumor tissues were accompanied by quantitatively similar changes in histone levels to maintain the same Feulgen/fast green ratios found in homologous normal tissues.     

3 types of sperm proteins in eukaryotes

Basic spermal proteins of various species of hydrobionts attributed to Pisces and Cephalopoda are studied. It is established that chromatin of nine species referring to two Cypriniformes families includes the somatic histones. Histone H1 of Cypriniformes is attributed to the lysine-rich type histones and contains 35% mol. of lysine and 0.7% mol. of tyrosine. Chromatin of 14 species of fish referring to nine families of the percoid fish superorder includes protamines similar to salmin, a typical protamine of salmon. The amino acidic analysis of protamine from the sandre sperma has shown that it contains 59% mol. of arginine and no tyrosine. Chromatin of three species from squid superorder referring to Cephalopoda includes gametones -- proteins differing from histones and protamines both in the electrophoretic mobility and amino acidic composition (75% mol. of arginine, 3% mol. of tyrosine).     

Characterization of the unusual basic proteins of cricket spermatid nuclei on the basis of their molecular weights and amino acid compositions

Typical somatic cell type histones are lost from the nucleus during late spermiogenesis in the house cricket; they are replaced by unusual basic proteins specific to the spermatid. We wish to characterize these proteins because they appear to determine the unusual chromatin structures of the spermatid. Molecular weights of the unusual basic proteins were estimated by chromatographing them on Bio-Gel A 0.5 M agarose columns eluted with 6 M guanidine hydrochloride. Two proteins named TH1 and TH2 have molecular weights in the range spanned by the somatic histones. The molecular weight of TH1 is 17 500 and that of TH2 is 15 500. Three additional spermatid proteins were also analyzed by molecular weight determination. They are called here protamines A, B and C, and they have molecular weights in the range typical of protamines. That of A is 6200, of B is 5500 and of C is 3800. They span the range from the large protamines typical of mammalian sperm to the small protamines of salmonid fish. The molecular weights of the TH proteins were also examined by electrophoresis on SDS-polyacrylamide gels. Amino acid compositions determined for TH1 and TH2 show that both are basic proteins rich in arginine relative to lysine. Their compositions are histone-like, but they appear to be distinct histone types rather than variant forms of the somatic histones.     

Schedule of Spermatogenesis in the Pulmonate Snail Helix aspersa, with Special Reference to Histone Transition

The schedule of spermatogenesis is determined from the times necessary for cells labeled with tritium thymidine during premeiotic DNA synthesis to pass through the successive spermatogenic stages. A transition from a typically somatic histone rich in lysine, to a histone rich in arginine is shown to occur during spermatid stages. A later shift to a protamine is observed in the maturing sperm. These changes are characterized by the use of in situ staining methods. The transition to an arginine-rich histone is accompanied by incorporation of tritium-labeled arginine, hence reflects synthesis of new protein. Comparison of the timing of arginine and thymidine incorporation, and independent measurements of DNA, show that in contrast to the case of premitotic chromosome duplication, the histone synthesis in the spermatid is unaccompanied by DNA synthesis. During the initial histone change, fine filaments are formed within the nucleus, which aggregate to form lamellae. This fine structure is lost during maturation of the sperm.     

Extraction, purification and characterization of the sperm protamines of the dog-fish Scylliorhinus caniculus

Dog-fish sperm nuclei contain four low molecular weight basic proteins called scylliorhinines. Protein Z3 is a typical arginine-rich protamine, whilst the three other components, Z1, Z2 and S4, are characterized by high arginine and cysteine contents. In contrast to protamine Z3, which can be directly solubilized by 0.25 M HCl, the three other protamines must be reduced and alkylated before acid extraction. They were further purified by ion-exchange chromatography on carboxymethyl-cellulose. The amino acid compositions and the N-terminal sequences reveal significant differences between scylliorhinines, particularly in their molecular size and amino acid diversity. Moreover, they show no common feature with other sperm-specific protamines previously described.     

The protamine family of sperm nuclear proteins

The protamines are a diverse family of small arginine-rich proteins that are synthesized in the late-stage spermatids of many animals and plants and bind to DNA, condensing the spermatid genome into a genetically inactive state. Vertebrates have from one to 15 protamine genes per haploid genome, which are clustered together on the same chromosome. Comparison of protamine gene and amino-acid sequences suggests that the family evolved from specialized histones through protamine-like proteins to the true protamines. Structural elements present in all true protamines are a series of arginine-rich DNA-anchoring domains (often containing a mixture of arginine and lysine residues in non-mammalian protamines) and multiple phosphorylation sites. The two protamines found in mammals, P1 and P2, are the most widely studied. P1 packages sperm DNA in all mammals, whereas protamine P2 is present only in the sperm of primates, many rodents and a subset of other placental mammals. P2, but not P1, is synthesized as a precursor that undergoes proteolytic processing after binding to DNA and also binds a zinc atom, the function of which is not known. P1 and P2 are phosphorylated soon after their synthesis, but after binding to DNA most of the phosphate groups are removed and cysteine residues are oxidized, forming disulfide bridges that link the protamines together. Both P1 and P2 have been shown to be required for normal sperm function in primates and many rodents.     

Detection of Sperm Histone Diversity Among Vertebrates by Alkaline Fast Green Staining

Testis sections from fifteen species from six classes of vertebrates were stained with alkaline fast green (AFG) to correlate staining differences with the known biochemical diversity of histones in the spermatozoa. After trichloroacetic acid (TCA) hydrolysis the sperm of some species known to contain sperm-specific histones did not stain. This correlation held if fixation in neutral buffered formalin was limited to 3 to 6 hr and hydrolysis was done at 90 C. The species whose sperm did stain after TCA hydrolysis could be divided into three groups. In some species the sperm no longer stained if, after TCA, the sections were treated with thioglycollic acid. These sperm contained basic proteins that were rich in cysteine. In turn, the group of species whose sperm continued to stain after TCA and thioglycollic acid treatments could be subdivided. The sperm of some were stained specifically without DNA hydrolysis if the AFG was made up with sodium chloride. These sperm contained sperm-specific histones. In other species the sperm did not stain under these conditions, and these sperm had a basic protein complement similar to that found in somatic cell nuclei. These correlations suggest that AFG staining can be used to detect sperm histone diversity in a wide range of organisms.     

Detection of sperm histone diversity among vertebrates by alkaline fast green staining

Testis sections from fifteen species from six classes of vertebrates were stained with alkaline fast green (AFG) to correlate staining differences with the known biochemical diversity of histones in the spermatozoa. After trichloroacetic acid (TCA) hydrolysis the sperm of some species known to contain sperm-specific histones did not stain. This correlation held if fixation in neutral buffered formalin was limited to 3 to 6 hr and hydrolysis was done at 90 C. The species whose sperm did stain after TCA hydrolysis could be divided into three groups. In some species the sperm no longer stained if, after TCA, the sections were treated with thioglycollic acid. These sperm contained basic proteins that were rich in cysteine. In turn, the group of species whose sperm continued to stain after TCA and thioglycollic acid treatments could be subdivided. The sperm of some were stained specifically without DNA hydrolysis if the AFG was made up with sodium chloride. These sperm contained sperm-specific histones. In other species the sperm did not stain under these conditions, and these sperm had a basic protein complement similar to that found in somatic cell nuclei. These correlations suggest that AFG staining can be used to detect sperm histone diversity in a wide range of organisms.     

The nuclear basic proteins of human testes and ejaculated spermatozoa

Human testicular nuclei were fractionated into two fractions according to their sedimentation in a sucrose density gradient. The nuclear basic proteins isolated from these two fractions were similar and also resembled electrophoretic mobilities and amino acid composition of the liver histones. Only quantitative differences among histone electrophoretic bands were observed. The nuclear basic proteins of ejaculated spermatozoa differed totally from those of the testes. The proteins could be divided into two categories on the basis of their electrophoretic mobilities, molecular weights and amino acid compositions. One group (SpH) was similar to testicular histones; another (HP) group was smaller, with nearly twice the electrophoretic mobility and a much higher arginine content. These proteins (HP) represent a new type of nuclear basic protein found in human tissues.     

Nature et évolution des protéines basiques au cours de la spermiogenèse chez Pleurodeles waltlii Michah., Amphibien urodèle

Résumé La spermatogenèse du Triton Pleurodeles waltlii a été étudiée au moyen de techniques cytochimiques et autoradiographiques. Les phénomènes spermiogénétiques ont été divisés en six stades caractéristiques en fonction de la forme du noyau et de l'évolution des structures extranucléaires: cou et filament de soutien de la queue.Les protéines basiques du noyau du spermatozoïde sont du type protamine, tandis que certaines structures extranucléaires se montrent riches en protéines basiques libres. Les transitions depuis les histones de type somatique présentes dans les jeunes spermatides I et II jusqu'aux protamines du spermatozoïde mûr par l'intermédiaire d'histones enrichies en arginine, montrent des phénomènes intéressants.La première transition depuis les histones de type somatique riches en lysine vers les histones enrichies en arginine est progressive. Elle se produit au cours des stades III et IV. Un gradient apparaît dans le noyau depuis la base vers le sommet. Au même moment, les protéines basiques libres apparaissent dans le cou.La perte des histones enrichies en arginine puis leur substitution par des protamines au niveau des noyaux des spermatides V est beaucoup plus brutale, elle intéresse tout le noyau. Ce dernier montre de même une disparition complète des acides aminés soufrés. Au même moment, les protéines basiques apparaissent dans la queue, de même que des acides aminés soufrés.Toutes ces observations plaident en faveur d'une origine nucléaire possible de certains composants de ces structures extranucléaires originales et riches en protéines basiques libres.

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Nature and development of the basic proteins during the spermiogenesis in Pleurodeles waltlii michah., urodele amphibian

Summary The spermiogenesis of the newt Pleurodeles waltlii has been studied with cytochemical and autoradiographical methods. The process of spermiogenesis has been divided into six characteristic steps in relation with the nuclear shape and the evolution of the extranuclear structures: the neck and the supporting filament of the tail.The nuclear basic proteins of the spermatozoon are of protamine type while the well developed extra-nuclear structures show a free basic proteins rich content. The transition from early spermatids somatic type histone to the mature sperm protamine via arginine-rich histone and the appearance of the free basic proteins in the tail show different interesting features. The first transition from the somatic rich lysine histone to the arginine rich histone which occurs at the spermatid III and IV steps, is gradual. A gradient appears in the nuclei from the base to the apex. Concurrently the free basic proteins appear in the neck. The loss of the arginine rich histone and its substitution by protamine in the spermatid V is more sudden and affects all the nuclei without a gradient, the sulfhydryl group content of the nuclei also disappear. At the same time, the basic protein of the tail appear as well as the sulfhydryl group.All these results are discussed in view of the hypothetical nuclear origin of these very unusual extranuclear basic proteins.

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Equipe de Recherche associée au C.N.R.S. Cytologie Ultrastructurale No. 129.

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Avec la collaboration technique de Melle F. de Sallier Dupin et de M. B. Morille.     

Characterization of protamines from four avian species

No data are available on the protamines of birds, with the exception of galline. We have characterized the protamines from four species of birds belonging to four different orders. All of them have very similar properties. They have been purified by carboxymethylcellulose chromatography and analyzed with respect to amino acid composition and electrophoretic behaviour. They are very arginine-rich proteins (63.4-67.3%) but do not contain lysine. Serine (12.0-18.2%), tyrosine (5.8-9.0%) and glycine (4.5-7.1%), along with arginine, make up the bulk of the amino acid residues in these molecules. The electrophoretic mobility of bird protamines in acetic acid-urea-polyacrylamide gels is intermediate between that of somatic histones and salmine. The molecular size, estimated from amino acid analysis and electrophoretic migration, is 65 +/- 5 amino acid residues.     

Cytochemical studies on cytoplasmic RNA-associated basic proteins in oocytes,somatic cells,and ribosomes

Summary Cytochemical studies on oocytes from representatives of several animal groups, of somatic tissues known to possess high levels of cytoplasmic RNA, and of isolated ribosomes indicated that basic proteins associated with the ribosomes may be stained by the methods currently in use for demonstrating histones. These proteins were resistent to weak acid extraction in fixed material and were labile to acetylation, but pretreatment with hot 5% TCA caused a measurable increase in staining with a modified Sakaguchi reaction. Such proteins were not confined to oocytes, but could be demonstrated with the TCA-alkaline fast green method in mouse pancreas and liver cells, and by the ammoniacal-silver method in all cells with high levels of cytoplasmic RNA. Microspectrophotometric studies onAscidia nigra andClavelina picta oocytes indicated that the highest concentration of cytoplasmic basic proteins was found in smaller oocytes, but the concentration of alkaline fast green stainable cytoplasmic RNA-associated basic proteins decreased in a pattern that differed significantly from that of stainable levels of cytoplasmic RNA. The implications of these findings to current concepts in developmental biology were discussed.Nucleoplasmic basic proteins with high levels of arginine which are not associated with nucleic acids were encountered in oocytes of all the echinoderm species studied and in the mouse.This work was supported by a U. S. Public Health Service grant HD-1499-04 and a Career Development Award 5-K3-6176-04.Contribution number 382 of the Bermuda Biological Station.     

ISOLATION AND CHARACTERIZATION OF CHROMATIN FROM NEUROSPORA CRASSA

Different preparations of chromatin isolated from mycelia of Neurospora crassa were analyzed for DNA-associated RNA and proteins. The UV absorption spectra, the ultrastructure of chromatin, and the amino acid composition of the acid-extractable proteins were studied. The protein:DNA ratios range from 1.5 to 2.8; the RNA:DNA ratios range from 0.5 to 1.24. UV absorption shows a macimum at 259 mµ and a minimum at 238–239 mµ. The E280/E260 ranges from 0.59 to 0.70. Electron microscopy reveals a fibrous structure with individual fibers of 120–150 A average diameter. Attempts were made to study the protein by polyacrylamide gel electrophoresis and amino acid analysis. The results indicate that Neurospora chromatin does not contain basic proteins comparable to calf thymus histone. The ratios of basic to acidic amino acids range from 0.93 to 1.19. On electrophoresis, no bands are seen whose positions correspond to those of histones. Staining for basic proteins with fast green or eosin Y at pH 8.2 also shows a negative reaction, suggesting the absence of histones.