Structure of allelic variants of subtype 5 of histone H1 in pea Pisum sativum L.

The pea genome contains seven histone H1 genes encoding different subtypes. Previously, the DNA sequence of only one gene, His1, coding for the subtype H1-1, had been identified. We isolated a histone H1 allele from a pea genomic DNA library. Data from the electrophoretic mobility of the pea H1 subtypes and their N-bromosuccinimide cleavage products indicated that the newly isolated gene corresponded to the H1-5 subtype encoded by His5. We confirmed this result by sequencing the gene from three pea lines with H1-5 allelic variants of altered electrophoretic mobility. The allele of the slow H1-5 variant differed from the standard allele by a nucleotide substitution that caused the replacement of the positively charged lysine with asparagine in the DNA-interacting domain of the histone molecule. A temperature-related occurrence had previously been demonstrated for this H1-5 variant in a study on a worldwide collection of pea germplasm. The variant tended to occur at higher frequencies in geographic regions with a cold climate. The fast allelic variant of H1-5 displayed a deletion resulting in the loss of a duplicated pentapeptide in the C-terminal domain.

A mutation, tl2, in pea (Pisum sativum L.) affects leaf development only in the heterozygous state.

After gamma irradiation of pea seeds, a mutation designated as tendril-less2 (tl2) was induced. In the heterozygous state, it transforms tendrils into very narrow leaflets that resemble the heterozygote phenotype of the classic tl mutation. The tendrils of the double heterozygote tl2/+, tl/+ are converted into oval leaflets. Unlike tl, the novel mutation in the homozygous state does not affect tendrils. The leaf phenotype of homozygotes tl2/tl2 and Tl2/Tl2 do not differ in the tl/+ background. However, the anthocyanin pigmentation is strongly suppressed in petals of tl2/tl2 plants. Some hypotheses to explain the unusual phenotypic manifestation of tl2 are suggested.

Tertiary trisomics in the garden pea as a model of B chromosome evolution in plants.

It is hypothesized that, in plants, genetically empty B chromosomes may originate from the extra chromosome (E) of tertiary trisomics if (i) the region of basic chromosomes homologous to the E (H-region) harbors a sporophytic lethal covered by the wild-type allele in E, and (ii) crossing-over between E and the H-region is suppressed. Under these conditions, most loss-of-function mutations occurring in the H-region are deleterious for haploid gametophytes, whereas those occurring in E are neutral or advantageous for hyperploid (n+1) gametophytes. As a result, natural selection at the gametophyte level can lead to the degeneration of E, leaving the H-region intact. Using Hammarlund translocation T(3-6)a, we synthesized two trisomic lines of the garden pea (Pisum sativum L.), where E was composed of the short arms of chromosomes 3 and 6 and the H-region carried recessive markers. In the trisomic line TRIS, we found few crossovers between E and the H-region. In the trisomic line TRUST, obtained after a change of basic chromosome constitution, recombination in this region was completely suppressed. After induction in the H-region of TRUST of a recessive sporophytic mutation rmv, two 15-chromosome lines of stable trisomics were established. One of them passed 11 generations, having produced more than 6000 individuals, all of them trisomic, and E remained present as a single element with no pairing partners. No tetrasomics were detected in these lines. If such trisomics occurred in nature, their extra chromosomes are likely to become a B chromosome.

Mortality of pollen grains may result from errors of meiosis: study of pollen tetrads in Typha latifolia L.

In the cattail Typha latifolia the four haploid products of meiosis remain attached and form the flat tetrad of pollen grains. Gametophytic lethals arisen de novo in diploid cells of sporophyte must manifest themselves as pollen tetrads with two dead grains. This could allow to estimate the rate of recessive lethals arresting pollen grain development. We studied pollen samples collected from 44 sprouts in two populations in the vicinity of Novosibirsk. The anomalous tetrads T1, T2, T3, and T4 carrying one, two, three, and four dead grains, respectively, were detected in each sampled individual. The mean frequency of all anomalous tetrads in the two populations was 3.4% and 8.7%. The frequencies of tetrad classes varied widely among the individuals with correlation coefficient up to 0.94, but their ratios remained nearly constant. The majority of anomalous tetrads were presented by T1 and T2 classes (their sum comprising 72.7 and 74.0% in two populations), T1 being a little more abundant. The observed pattern of frequencies of tetrads with dead grains can be explained by errors of male meiosis such as chromosome non-disjunction in both meiotic divisions. The tetrads with two dead pollen grains may result mostly from non-disjunction in anaphase I, and those with one pollen grain from non-disjunction in anaphase II, thus making tetrad analysis ineffective for estimating the rate of gametophytic lethals.

[Structural characteristics of lysine-rich histone from the sperm of a mollusk Anodonta piscinalis]. / Osobennosti stroeniia bogatogo lizinom gistona spermy bezzubki Anodonta piscinalis.

Sperm of freshwater bivalve mollusk Anodonta piscinalis was found to contain two fractions of lysine-rich histone: somatic histone H1 and sperm-specific protamine-like histone, named Hp. A detailed analysis of H1 and Hp structure was carried out by means of N-bromosuccinimide, chymotrypsin and pepsin cleavage followed by determination of the lysine residue number, positive charge and molecular length of obtained fragments by the method of incomplete succinylation. It has been shown, that Anodonta histone H1, like the avian histone H5, contains 3 tyrosine residues in the central hydrophobic domain of the molecule. Histone Hp contains 5 tyrosine residues, 3 of which are localized in the hydrophobic domain, while the rest two--in the COOH-terminal part of the molecule, characterized by a strong positive charge. Such unusual disposition of tyrosine residues in the lysine-rich histone has been found for the first time. All the regions of histone Hp molecule contain a great number of arginine residues. The only phenylalanine residue is localised approximately in the middle of the polypeptide chain for both H1 and Hp molecules. On the basis of structure homology between histones H1 and Hp the origin of Hp from H1 in the course of evolution is proposed.

[Evolutionary interrelationship of histones H1 and H5 in vertebrates]. / Evoliutsionnye vzaimootnosheniia gistonov H1 i H5 u pozvonochnykh.

Lysine-rich histones of some amphibians and fishes were studied. Electrophoretically purified subfractions were cleaved at residues of tyrosine, methionine, aspartic acid and phenylalanine. The fragments thus obtained were investigated by the method of incomplete succinylation which permitted us to determine the number of lysine residues, positive charge in acid conditions and molecular lengths of polypeptides. It was found that in anura and shark the fraction H1a resembled the histone 5 of birds in its N-terminal half part of the molecule. However this fraction proved to be non-tissue-specific. Other histone 1 fractions characteristic for vertebrates were represented in the present study by molecular variant H1s which was different from H1a fraction by the number and position of tyrosine, methionine and aspartic acid residues. The erythrocyte-specific fraction of the lysine rich histone was found in the following families of fishes: Salmonidae, Percidae and Cyprinidae. A high degree of homology in the structure of N-terminal half of H1s and histone 5 of fishes has been observed. On the basis of these results we propose a hypothesis of the independent origin of the avian and fish H5 from different fractions of H1.

[Determination of lysine residue number, positive charge and molecular lengths of histone H1 and H5 by a method of incomplete succinylation]. / Opredelenie chisla ostatkov lizina, polozhitel'nogo zariada i molekuliarnoi dliny gistonov H1 in H5 metodom nepolnogo suktsinilirovaniia.

A simple method for determination of lysine residue number and positive charge of histones H1 and H5 in the acetic acid -- urea system has been developed. The method is based on incomplete succinylation of lysine residues and allows an accurate determination of protein molecular length. The accuracy of the method has been demonstrated by determination of goose and chicken histones H5 and their fragments. The calibrating curve for determination of molecular lengths of histones H1 and H5 has been plotted. Using the incomplete succinylation method, a detailed analysis of Lymantria dispar histone H1 structure has been carried out. The method under discussion can be used for determining lysine residue number, positive charge and molecular length of practically any protein.

[Histone H1 of reptiles, homologous to histone H5 of birds]. / Giston H1 presmykaiushchikhsia, gomologichnyi gistonu H5 ptits.

Lysine-rich histone H1 of animals from three reptilian orders was studied. Electrophoretically pure H1 histone subfractions were cleaved at residues of tyrosine, methionine, aspartic acid and phenylalanine. The fragments obtained were studied by modified method of incomplete succinylation which permitted to determine the number of lysine residues, the positive charge and molecular length of polypeptides. The structural homology between the fastest reptilia H1 subfraction and avian H5 histone has been shown.

[Molecular variants of histone H5 of linnet (Acanthis flammea)]. / Molekuliarnye varianty gistona H5 chechetki (Acanthis flammea).

Five electrophoretic variants of H5 histone were detected in the population of linnet (Acanthis flammea). Using the method of incomplete succinilation the number of lysine residues, electrophoretic positive charge and molecular length were determined for some variants of H5 histone and for their fragments, obtained after treatment with n-bromosuccinimide and chymotrypsin. The difference in structure of H5 variants was found to be connected with the region, confined by the phenylalanine residue and the C-end of the molecule. The minimal difference in molecular length of fragments carrying the variable region was found to be 6 amino acids, two of them are basic. A series of "regular" fragments was detected after mild treatment with trypsin. The number of these fragments increased in parallel with the increase of histone length. In accordance with the scheme proposed, the difference in structure of linnet H5 variants is caused by insertion of the regular region consisting of tandem repeats (from 3 to 7 times) of the elementary hexapeptide.

[Histones of Salmonidae fishes. Comparative study of histones from different species and intraspecies variability of Oncorhynchus nerka H1]. / Sravnenie gistonov raznykh vidov lososevykh ryb i vnutrividovaia izmenchivost gistona H1 Oncorhynchus nerka.

The comparative electrophoretic properties of erythrocyte histones from 7 Salmonidae species were investigated. Using Na-SDS gel electrophoresis, it was shown that all the species studied possess the erythrocyte-specific fraction of histone H5. High resolution gel electrophoresis in acetic acid--urea gels demonstrated differences in the subfractional composition of histone H1 from erythrocytes and liver of O. nerka. The analysis of the sample of 40 individuals from the same population revealed the existence of intraspecies polymorphism in histone H1 subfractional composition.

[Dansylated amino acid separation by polyacrylamide gel electrophoresis]. / Razdelenie dansilirovannykh aminokislot metodom élektroforeza v poliakrilamidnom gele.

The paper describes a method for separation of dansylated amino acids by polyacrylamide gel electrophoresis. The methods allows a simultaneous analysis of 20-30 samples. The sensitivity of the method is 1 x 10(-9)-1 x 10(-10) M amino acid. The method permits separation of all amino acids formed during acid hydrolysis of proteins except for two pairs: Ile, Phe and Val, Asp.

[Microelectrophoretic analysis of histones from single chromosomes and nucleoli of mosquito (Chironomis plumosus) larvae]. / Mikroelektroforeticheskii analiz gistonov iz individual'nykh khromosom i iadryshek lichinki komara (Chironomus plumosus).

A simple method of microelectrophoresis for histone analysis from single fragments of polythenic chromosomes of chironomid salivary glands has been developed. The chromosomes or their fragments obtained micrurgically were dissolved in a microdroplet of 8 M urea and 1 N sulfuric acid solution. Electrophoresis was performed in 30--60 m diameter polyacrylamide gel cylinders under a layer of vaseline oil. The histones were separated into 6 electrophoretic bands which correspond to fractions H4, H2b, H2a+H3, H3-dimer, and two subfractions of histone H1. Comparison of histone electrophoregrams of the I, II, and III chromosomes showed their almost absolute identity. The ratio of fractions H4, H3, H2A, H2B as well as the ratio of histone H1 subfractions in the nucleus and in the rest of the IV chromosome cannot be visually distinguished.

[Study of evolutionary changes in subfraction composition of histone H1 in birds]. / Issledovanie evoliutsionnykh izmenenii v subfraktsionnom sostave gistona H1 ptits.

Analysis of electrophoretic mobility of histone H1 subfractions from liver, brain and erythrocytes of 41 bird species was carried out. Subfractions of erythrocyte H1 histones from each species were compared with those of thrush Turdus musicus. The majority of species proved to possess a set of electrophoretically similar subfractions. Interspecific differences in histone H1 were mainly due to the differences in the ratio of those subfractions. The identity of the electrophoretic mobility and similar contribution to H1 histone from the respective tissues in different species permits to consider certain subfractions as homologous ones. The conservation of electrophoretic mobility for homologous subfractions of the birds of different orders shows a high evolutionary conservatism of the corresponding genes. It seems that in the course of evolution only a change in the expression of some of those genes occurs.

Study of the relationship between the subfraction composition of histone F1 and the type of tissue in birds.

The subfraction composition of lysine-rich histone has been studied with the aid of polyacrylamide gel electrophoresis. The subfraction compositions of the histone F1 of several tissues from the chicken, pigeon, and titmouse have been compared. The histone F1 from the tissues investigated consists of four or five subfractions of similar number and electrophoretic mobility (1, 1a, 2, 3, and 4). In the different avian species each subfraction varied its mobility independently of the others. The chicken tissues investigated can be divided into two classes, depending on the relative concentration of subfractions 2 and 3 (A and B): Class A (subfraction 2 is smaller than 3) includes the brain, liver, skeletal muscle, heart, muscular layer of the stomach, and pancreas, and class B (subfraction 2 is larger than 3) includes the intestinal mucosa, thymus, and testes, as well as the liver, heart, and pancreas from a 21-day embryo. Such a division of the tissues corresponds to the varying rate of their cellular renewal. In a parallel examination of the relative concentrations of the individual subfractions in the same tissues from the three avian species it has been found that the relative concentration of subfractions 3 and 2 is increased in the skeletal muscles, heart, brain, and liver, that subfraction 2 is increased in the intestinal mucosa, that subfractions 4 and 3 are increased in the pancreas, and that subfractions 1, 1a, and 4 are increased in the erythrocytes. The results obtained may be interpreted as a consequence of some relationship between the subfraction composition of histone F1 and the type of tissue of the source.