Protamine precursors in human spermatozoa

Basic proteins isolated from human sperm nuclei are highly heterogeneous. Three groups of nuclear basic proteins have been characterized: somatic-type as well as testis-specific histones, protamines and basic proteins with an electrophoretic mobility which is intermediate between that of histones and that of protamines. Human protamines can be separated into 2 protein families with different amino acid composition and amino-acid sequence. Protamines HP1 differ in their degree of phosphorylation. Protamines HP2, 3 and 4 differ by their amino-terminal sequence. Intermediate basic proteins (HPI1, HPI2, HPS1, HPS2) share a common C-terminal sequence of 54 residues identical to the amino-acid sequence of protamine HP3; only their N-terminal regions are different. Taking into account these structural homologies, the intermediate basic protein HPI1 appears as a precursor of protamines HP2 and HP3.     

Histones of the fungi, Endomyces magnusii and Neurospora crassa]

Histones of Endomyces magnusii and Neurospora crassa were found to consist of four main fractions similar to calf thymus histones in their electrophoretic mobilities, molecular sizes and chromatographic behaviour on Akrilex P-60. Two of them are homologous to the most conservative histones H3 and H4. Other two fractions correspond to the histones H2A and H2B; however, they have some pecularities. A fraction of N. crassa histones corresponding to the H2B was isolated in a homogeneous state by means of gel filtration. It appeared to be very similar to calf thymus histone H2B in its amino acid composition.     

Basic nuclear proteins in testicular cells and ejaculated spermatozoa in man

Human testis was shown to contain a specific histone, TH2B, having the same electrophoretic mobility as rat TH2B. Testicular and ejaculated human sperm still possessed histones at 50% and 15% of the total basic nuclear proteins, respectively. Comparison of the electrophoretic patterns of histones from human testis, testicular sperm and ejaculated sperm implied that the histones may be removed in the order H2A and H1 before H3, H4 and H2B before TH2B. TH2B which is the major histone fraction in ejaculated sperm has no longer a strong affinity to DNA. TH2B in sperm nuclei could be separated from other basic nuclear proteins by Bio-Gel P-10 column chromatography and its amino acid composition is similar to that of rat TH2B, although no cysteine residue was found.     

Basic nuclear proteins in the aquatic fungus Achlya ambisexualis.

The complement of basic chromosomal proteins in the aquatic fungus Achlya ambisexualis has been characterized. Achlya nuclei contain proteins with electrophoretic mobilities on acetic acid/urea and dodecyl sulphate polyacrylamide gels which are comparable to rabbit kidney histones H3, H4 and H2A. In contrast, the behavior of putative H2B and H1 proteins from Achlya showed greater analogy on acid/urea gels to higher plant histones. A closely related water fungus Saprolegnia ferax contained basic nuclear proteins which were very similar to those of Achlya.     

Isolation and characterization of histones and other acid-soluble chromosomal proteins from Physarum polycephalum

Chromosomal basic proteins were isolated from amoebal and plasmodial stages of the acellular slime mold Physarum polycephalum. Polyacrylamide electrophoresis on high resolution acid-urea gels separated the five histone fractions in the sequence H1, H2A, H2B, H3, and H4. Under these electrophoretic conditions Physarum histones migrated more like plant (rye) than animal (calf) histones. Furthermore, Physarum histones H1, H2A, and H2B have higher molecular weights on sodium dodecyl sulfate (SDS) gels than the corresponding calf fractions. No differences were detected between amoebal and plasmodial histones on either acid-urea or SDS-polyacrylamide gel electrophoresis. Amoebal basic proteins were fractionated by exclusion chromatography. The five histone fractions plus another major acid-soluble chromosomal protein (AS) were isolated. The Physarum core histones had amino acid compositions more closely resembling those of the calf core histones than of rye, yeast, or Dictyostelium. Although generally similar in composition to the plant and animal H1 histones, the Physarum H1 had a lower lysine content. The AS protein was extracted with 5% perchloric acid or 0.5 M NaCl, migrated between histones H3 and H4 on acid-urea polyacrylamide gels, and had an apparent molecular weight of 15 900 on SDS gels. It may be related to a protein migrating near H1. Both somewhat resembled the high mobility group proteins in amino acid composition.     

Studies on Nuclei of Paramecium aurelia. II. Amino Acid Composition and Electrophoretic Properties of the Chromosomal Basic Proteins*

SYNOPSIS. The basic proteins of Paramecium aurelia nucleus were extracted from isolated nuclei and deoxyribonucleoprotein (DNP) of such nuclei. About 35–40% of the nuclear protein, predominantly a lysine-rich histone, is acid soluble. Five major components of the histone can be distinguished by polyacrylamide gel electrophoresis. Some components of Paramecium histone are similar to those of mammalian histones in their electrophoretic mobility, but they differ from the latter in the electrophoretic velocity and relative levels. The basic to acidic amino acid ratio of the histone from the ciliate is ～1.1–1.5, and its amino acid composition resembles closely that of yeast histone. Through the use of Sephadex G-200 gel filtration for purification of the histones extracted directly from isolated nuclei 2 basic proteins were resolved—component I, with an elution volume of 1.4, constitutes ～20% and component II, with an elution volume of 1.9, ～80%.     

Some Researches on Histones

The histone extracted from calf thymus glands is a complex system of proteins, which can be fractionated by chromatography on carboxymethyl cellulose columns into three principal fractions (1) very lysine-rich, (2) moderately lysine-rich, (3) arginine-rich. When examined by starch gel chromatography each of these gives more than one band. Methods have been devised for further separation of the components in some cases. The components show characteristic differences in end groups and certain amino acids as well as in their basic character. Histones extracted from various rat tissues can be separated into similar fractions, of which the amino acid analyses are similar to those derived from calf thymus, within the experimental error. To this extent, no species or tissue specificity of the fractionated histones was observed. Although all the histone fractions contain approximately one basic amino acid to three non-basic amino acids their structure is not regular, as Phillips has shown that in certain fractions the number of non-basic groups between two basic groups may vary from 0 to seven or more. The possible functions of histones are discussed.     

Trypanosoma cruzi histones. Further characterization and comparison with higher eukaryotes

Histones extracted from T. cruzi chromatin were analyzed in three electrophoretic systems. Our results show that a basic protein with some properties similar to those of histone H1 from higher eukaryotes is present in T. cruzi. However this protein presents different electrophoretic mobilities than H1 histone from higher eukaryotes in all three electrophoretic systems tested. Considering the marked differences observed in the electrophoretic mobilities of T. cruzi histones as compared with those from higher eukaryotes, it is proposed that histones are conservative proteins primarily with regard to their function.     

Histones from embryos of the sea urchin Arbacia lixula

Whole histones and histone fractions of the sea urchin, Arbacia lixula, embryos have been characterized by their appearance during development and by their amino acid composition. Comparison of electrophoretic mobility of the histone fractions from hatching blastula and gastrula stage embryos demonstrates the similarity of the basic proteins at these two stages. Histones F2a1 and F3 of hatching embryos are very similar to those of sperm, including the presence of cysteine in F2a1 from both sources. Both F2a1 and F3 display electrophoretic heterogeneity due to acetylation, not observed in the homologous sperm histones. F2a2 from embryos has different electrophoretic mobility than that from sperm, although their amino acid compositions are very similar. The relative proportion of F2a2 increases whereas that of F3 decreases during gastrulation. Slightly lysine-rich histone F2b could not be recovered from embryos by the standard methods of extraction. The very lysine-rich histone F1 of late embryos is partially phosphorylated and is remarkably different from that of sperm, notably by its higher electrophoretic mobility and lower content in arginine and proline. The significance of these results is discussed with regard to the structure and activity of chromatin.     

CHANGES IN NUCLEAR HISTONES DURING FERTILIZATION, AND EARLY EMBRYONIC DEVELOPMENT IN THE PULMONATE SNAIL, Helix aspersa

Calf thymus histories comprising two fractions, one rich in lysine, the other having roughly equal amounts of lysine and arginine, Loligo testes histones rich in arginine, and salmine, are compared with respect to their amino acid compositions, and their staining properties when the proteins are fixed on filter paper. The three types of basic proteins; somatic, arginine-rich spermatid histones, and protamine can be distinguished on the following basis. Somatic and testicular histones stain with fast green or bromphenol blue under the same conditions used for specific staining of histones in tissue preparations. The former histones lose most or all of their stainability after deamination or acetylation. Staining of the arginine-rich testicular histones remains relatively unaffected by this treatment. Protamines do not stain with fast green after treatment with hot trichloracetic acid, but are stained by bromphenol blue or eosin after treatment with picric acid. These methods provide a means for the characterization of nuclear basic proteins in situ. Their application to the early developmental stages of Helix aspersa show the following: After fertilization the protamine of the sperm is lost, and is replaced by faintly basic histones which differ from adult histones in their inability to bind fast green, and from protamines, by both their inability to bind eosin, and their weakly positive reaction with bromphenol blue. These "cleavage" histones are found in the male and female pronuclei, the early polar body chromosomes, and the nuclei of the cleaving egg and morula stages. During gastrulation, the histone complement reverts to a type as yet indistinguishable from that of adult somatic cells.     

Comparison of the amino acid sequences of human protamines HP2 and HP3 and of intermediate basic nuclear proteins HPS1 and HPS2. Structural evidence that HPS1 and HPS2 are pro-protamines

Two intermediate nuclear basic proteins HPS1 and HPS2 were isolated from human sperm. They were characterized by their electrophoretic mobility in acid-urea gels, their amino acid composition, and their peptide maps after digestion by endoproteinase Lys-C and by endoproteinase Glu-C. Their amino-terminal amino acid sequences have also been determined. The structural data thus obtained suggest that HPS1 and HPS2 are precursors of human protamines HP2 and HP3.     

An improved large scale fractionation of high mobility group non-histone chromatin proteins.

1. Methodology is presented for the large scale preparation and fractionation of high mobility group proteins from calf thymus chromatin. The total high mobility group protein from approx. 1 kg calf thymus tissue can be separated into five fractions by CM-Sephadex C25 ion-exchange chromatography. High mobility group proteins 1 and 2 comprise two fo the fractions. From a third fraction two more chromatin proteins, protein 3 and 17, can be isolated by trichloroacetic acid precipitation and CM-cellulose chromatography at pH 5.5. 2. The four proteins thus purified are lysine-rich proteins. Proteins 1 and 2 are additionally characterised by their high contents of acidic amino acids, as described previously (Goodwin, G. H. and Johns, E. W. (1973) Eur. J. Biochem. 40, 215-219). Proteins 3 and 17, having lower contents of acidic amino acids, are basic proteins similar to the histones. All four proteins exhibit single N-terminal amino acids; glycine is the N-terminal group of proteins 1, 2 and 3; protein 17 has a proline N-terminal amino acid. The proteins are not highly phosphorylated nor are they associated with appreciable quantities of nucleic acid.     

Histones of Neurospora crassa.

Neurospora crassa chromatin isolated by a rapid method minimizing proteolytic degradation contains approximately one weight of acid-extractable basic protein per weight of DNA. This basic protein consists of five major polypeptide species which are similar in size to the histone proteins of higher eukaryotes and are present in approximately the same molar ratios. These five polypeptides have been purified by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Their electrophoretic mobilities in polyacrylamide gels and their amino acid compositions indicate that they are histones homologous, although not identical, to the H1, H2A, H2B, H3, and H4 histones of mammals. The first 3 residues in the amino acid sequence of Neurospora H3 histone are identical to the first 3 residues in calf and pea H3; Neurospora H1, H2A, and H4 histones have blocked NH2 termini, like their mammalian counterparts. The finding of recognizable H1, H2A, H2B, H3, and H4 histones in Neurospora extends the range of eukaryotes now shown to contain a full complement of these strongly conserved chromosomal proteins, and supports the view that histones became involved in chromosome structure at a very early point in the evolution of eukaryotes.     

Chromatin from two classes of platyhelminthes display both protist H1 and higher eukaryote core histones

Histones from the parasitic platyhelminthes, Echinococcus granulosus and Fasciola hepatica, were systematically characterized. Core histones H2A, H2B, H3 and H4, which were identified on the basis of amino acid sequencing and mass spectrometry data, showed conserved electrophoretic patterns. Histones H1, identified on the basis of physicochemical properties, amino acid composition and amino acid sequencing, showed divergence, both in their number and electrophoretic mobilities, between the two species and among other organisms. According to these data, core histones but not H1 histones, would be stabilized during evolution at the level of platyhelminthes.     

Nuclear proteins. V. Studies of histone-binding proteins from mouse liver by affinity chromatography

We examined four extracts of mouse liver for histone-binding proteins using histone affinity chromatography and positively charged resins. The extracts used were cytoplasm and washes from isolated nuclei with buffers containing 0.05 M Tris, 0.15 M NaCl or 0.35 M NaCl. Proteins from the nuclear washes showed greater binding to the columns than proteins from the cytoplasm. The binding fractions were heterogeneous in gel electrophoresis systems. Proteins bound to affinity columns of individual histones were similar to those bound to columns of whole histone, polylysine and DEAE. A 25,000 dalton polypeptide (J2), found only in nuclear washes was a prominent histone-binding protein. It could be competitively eluted from DEAE with histones, suggesting polypeptide J2 may show a specific affinity for histones. Polypeptide J2 has an acidic to basic amino acid ratio of 1.58, and its amino acid composition is not similar to that of the high mobility group 1 protein. Polypeptide J2 binds to hydrophobic columns and may play a role in modifying histone-histone and histone-DNA interactions.     

Identification of the Pm and PmS human parotid salivary proteins as basic proline-rich proteins

Amino acid composition and electrophoretic mobility suggest that two polymorphic proteins in human parotid saliva, Pm and PmS, are basic proline-rich proteins. Comparison of two basic proline-rich proteins previously isolated by D. L. Kauffman and P. J. Keller (1979) [Arch. Oral. Biol. 24: 249], IB-6 and IB-9, with PmS and Pm demonstrated corresponding electrophoretic mobilities on cationic polyacrylamide slab gels. Further, the amino acid compositions of IB-9 and Pm were found to be similar. Although differences in amino acid composition and carbohydrate content were noted, such differences could be accounted for, suggesting that IB-9 and Pm are identical.     

Structure and histone composition of chromatin in Physarum polycephalum]

In Physarum polycephalum several degrees of organisation of deoxyribonucleoprotein fibres were found. The complexes of histones and the DNA duplex seem to "be packed" at first into a 100 A fibre and then into a 200 A fibre of DNP. In Ph. polycephalum the electrophoretic mobilities of histone fractions 4 and 6 are comparable to that of fractions f3/f2b and f2a1 of calf thymus, resp. Histone fractions 3 and 5 move a bit faster than fractions f1 and f2a2, resp. Thus, the myxomycete P. polycephalum is similar to higher eukaryotes as concerns the ultrastructure of chromatin and electrophoretic properties of histones.     

High mobility group (HMG) proteins in the tammar wallaby Macropus eugenii: quantitative variations between tissues and testis-specific co-extracted proteins

1. Tammar wallaby (Macropus eugenii, Marsupialia) proteins with similar electrophoretic mobilities to calf non-histone chromosomal proteins HMG 1, 2, 14 and 17 are perchloric acid extracted from whole tissues (liver, kidney, spleen, brain and testis) and purified liver nuclei (using PCA or 0.35 M NaCl). 2. Tammar and calf HMG 1 have similar amino acid compositions. 3. Two testis-specific basic proteins co-extracting with HMG-like proteins from both tammar and red kangaroo (Megaleia rufa) are found in whole testis, purified testis nuclei, but not epididymis. 4. Tammar HMG 2 separates into two components on both acid urea and SDS gels. The larger, more basic protein, HMG 2b, is relatively abundant in proliferating tissues (testis, spleen).     

The histones of some human tissues

1. Histones were examined from five human tissues, namely thymus, liver, placenta, bronchial tumour and peripheral leucocytes. 2. The four main histone fractions [F1, F2(b), F2(a), and F3] were isolated and characterized. 3. The amino acid analyses, N-terminal group analyses and electrophoretic patterns were very similar to those of the corresponding fractions of calf thymus. 4. The yields of fractions F1 and F2(b) were high in human thymus, in human bronchial tumour and in some preparations of normal human leucocytes. 5. It is concluded that the pattern of nuclear histones found in lower forms of life was conserved during the evolutionary process leading to man.     

Constancy of wheat histones during development

Summary Histones were extracted from leaves of winter- and spring-wheat seedlings, flowering shoots of spring wheat, shoots of vernalized and control winter wheat, and roots and shoots of winter wheat, and were compared by polyacrylamide-gel electrophoresis. No differences were found either in the electrophoretic mobility or relative quantity of the various fractions. Wheat histones contained fractions of the exact electrophoretic mobility as F2a1 and F3 of calf thymus and pea histones. Other major fractions of the wheat histones had electrophoretic mobilities similar to those of F1, F2b and F2a2 of peas.