Identification of histone H1.z components in a Muscovy duck (Cairina moschata L.) population.

The general patterns of histone H1 proteins from erythrocyte nuclei of Muscovy duck individuals were similar to those of Pekin type ducks both in acetic acid-urea and 2D polyacrylamide gels. We show here that Muscovy duck histone H1.z in the tested population was represented by three different electromorphs, each presumably encoded by a distinct allelic gene. Accordingly, we have identified six phenotypes consisting of the homodimeric and heterodimeric combinations of the three isoforms. The frequency of the presumptive alleles ranged from 0.506 for the main allele z1 to 0.379 for allele z2 and only 0.113 for the rarest allele z3. In addition to a standard set of somatic H1 variants, an unusual protein X, absent in other avian species, was also revealed.

Natural allelic variation of duck erythrocyte histone H1b.

In our previous work (J. Palyga, Genetic polymorphisms of histone H1. b in duck erythrocytes. Hereditas 114, 85-89, 1991) we reported a genetic polymorphism of duck erythrocyte histone H1.b. Here, we screened H1 preparations in a two-dimensional polyacrylamide gel to refine the distribution of allelic forms of H1.b in fifteen duck populations. We have revealed that the frequency of H1.b allelic variants was significantly different among many conservative and breeding duck groups. While b(1) and b(3) were common in all populations screened, the allele b(2), with a slightly lower apparent molecular weight, was confined mainly to brown-feathered ducks (Khaki Campbell and Orpington) and descendent lines. The C- and N-terminal peptides released upon cleavage with N-bromosuccinimide and Staphylococcus aureus protease V8 from duck allelic histones H1. b2 and H1.b3, respectively, migrated differently in the gel, probably as a result of potential amino acid variation in a C-terminal domain.

Isolation and preliminary characterization of histone H1.b allelic variants from quail erythrocytes.

Our goal was to purify and characterize the allelic variants H1b1 and H1b2 of histone H1.b, one of the seven subtypes of this linker histone extracted from Japanese quail erythrocyte nuclei. These variants are revealed phenotypically as band H1.3 or part of band H1.4 by polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS). All H1 subtypes together were separated from H5 by gel-permeation chromatography through Bio-Gel P-150. H1 was then fractionated on a column of the cation-exchange resin Amberlite CG-50 by using a shallow guanidine hydrochloride gradient, which enriched subtype H1.b together with H1.z and overlapping with subtypes H1.a and H1.b. Alternatively purification of subtypes was achieved electrophoretically: total H1 fractions from quail with different H1 phenotypes were first resolved into sub-types by PAGE in acetic acid-urea; after staining, the appropriate H1.b bands from several parallel gel pieces were excised and the histone was concentrated by PAGE in SDS. After fragmentation of H1.b in the gel pieces with N-bromosuccinimide (NBS), PAGE in SDS indicated no difference between H1b1 and H1b2 in the C-terminal "half" of the polypeptides. In contrast, limited digestion with endoprotease V8 from Staphylococcus aureus has shown that differences, probably by a few residues in length, reside in the N-terminal part of the molecule, close to the amino-terminus.

Distribution of allelic forms of erythrocyte H1 histones in Japanese quail populations divergently selected for amount of weight loss after transient starvation.

Three polymorphic subtypes of erythrocyte histone H1 (H1.a, H1.b, and H1.z) were analyzed using a sodium dodecyl sulfate polyacrylamide gel in quail populations divergently selected for a high (line 1) or low (line 2) reduction in body mass following temporary food withdrawal. Both H1.b and H1.z histone alleles were found to be differently distributed in these populations during the selection period. The frequency of b1 in line 2 was approximately 1.9-2.8 times lower than in line 1 and approached the values in line 1 when the selection was suspended. Similarly, the frequency of allele z2 at locus H1.z increased significantly (about 1.6-2.3 times) in line 2 during selection and returned to the initial values when selection was stopped. On the other hand, allele a0 at locus H1.a was kept at relatively low levels (usually below 0.05) in both lines during selection. At that time its level was approximately three to four times lower than in a random mating control population. When selection was suspended, the frequency of a0 in line 1 increased significantly, approaching the values in the control line, and remained essentially unchanged in line 2. Thus, all three polymorphic histone H1 loci in quail responded through changes in allele frequencies to the breeding selection, which was directed at the amount of body weight loss upon transient starvation. It seems that either H1 histone locus could be linked to loci controlling the rate of body weight reduction following starvation or weight loss during fasting might be influenced by a panel of H1 histone alleles that can contribute to functional differences in avian chromatin.

Genes for polymorphic H1 histones are linked in the Japanese quail genome.

In a previous report (Palyga, J., Biochem. Genet. 29, 431-445, 1991), three subtypes of erythrocyte histone H1 were found to vary in a Japanese quail population. While H1.b and H1.z histones were each represented by two electromorphs differing in apparent molecular weights, a polymorphism of histone H1.a was connected with a lack of this protein in some birds. As a genetic basis for this variability was demonstrated only in H1.b, here genetic data are provided which indicate that both H1.a and H1.z are encoded by two codominant alleles at a locus. A linkage analysis of family data in 13 quail pedigrees has revealed a significant linkage between H1.a and H1.z and between H1.b and H1.z (lod scores about 12 and 5, respectively). Thus, a gene for histone H1.z is located between H1.a and H1.b in the quail genome.

Genetic variants of chicken erythrocyte histone H5.

Two allelic electromorphs a and b of chicken erythrocyte histone H5 have been detected in a sodium dodecyl sulfate polyacrylamide gel. In an acid-urea gel, however, each of the allelic variants was found to be accompanied by a slower migrating form. A comparison of alpha-chymotrypsin-digested products of H5.a and H5.b revealed that they differed in N-terminal domains. The H5 variants were distributed differently not only in various chicken races but also in distinct lines within a breed. Allele H5b was about 2.6-4.6 as abundant as its counterpart H5a in most chicken populations examined. These proportions were distorted in two Leghorn lines: the ratio of H5b to H5a was only 1.6 in line H22 and increased up to 32 in line G99.

The occurrence of a mutant dimerizable histone H5 in Japanese quail erythrocytes.

An allelic variant of linker histone H5 has been found in the erythrocytes of Japanese quail (Coturnix japonica) descended from a small group of feral birds captured on the island of Hawaii. This variant spontaneously forms protein dimers in vitro in the absence of reducing agents. That this depends upon the introduction of a sulfhydryl group (presumably because of a cysteine substitution) is indicated by its reaction with 2-nitro-5-thiocyanobenzoate and by its fluorescence after reaction with 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole. This is the first reported example of cysteine in a vertebrate linker histone and offers a specific reactive site for structural studies. A homozygous line for this form of H5 is being developed.

Linkage mapping and partial sequencing of 10 cDNA loci in the chicken.

Ten cDNA clones derived from chicken spleen cell mRNA have been partially sequenced and the genes which encode the mRNAs have been located within the linkage map of the chicken genome. The sequences of five of these clones show strong homology to known mammalian genes, the remainder show little homology to sequence present in the current databases. Interestingly, one of these clones appears to be the chicken homologue of the mammalian peptide transporter gene TAP2 and is located within the major histocompatibility complex. Two other clones are homologous to genes involved in protein synthesis and these are tightly linked in chickens, as in mice. These results suggest that partial sequencing and mapping of clones from selective cDNA libraries may be an efficient way of adding candidate genes to the chicken linkage map and that on a local scale there may be some conservation of grouping of genes between chickens and mammalian species.

Genetic polymorphism of histone H1.z in duck erythrocytes.

Three different phenotypes of erythrocyte histone H1.z were detected in several lines of duck by using two-dimensional PAGE. Electrophoresis and inheritance data have shown that two co-dominant alleles, z1 and z2, encode two proteins which differ slightly in their apparent molecular masses. Allele z1 was very abundant and was found at a frequency at least 0.94, while allele z2 was very rare and was present at a frequency of less than 0.06. Limited chemical and enzymic cleavages appeared to indicate that the allelic forms of histone H1.z differed in the C-terminal regions of their molecules.

A preliminary linkage map of the chicken genome.

We have used backcross progeny from a cross between two inbred lines of chickens to construct a linkage map of the chicken. The map currently consists of 100 loci, identified using either anonymous cloned fragments of genomic DNA or sequences corresponding to cloned genes. Parent birds were derived from two lines of White Leghorn chickens, which differ in susceptibility to a number of diseases. Restriction fragment length variants were identified by comparison of the DNA of these two parent birds using a panel of seven restriction enzyme digests and the segregation pattern observed in progeny of these two birds. Restriction fragment length variants were detected for approximately 41% of the clones tested, whether these were known genes or random genomic fragments. This high level of variability was also reflected in the presence of variation within the parental lines for some clones. The overall size of the linkage groups and the progressively higher incidence of linkage as further clones were added suggests that the map covers the majority of the genome, although it is unlikely that there are marker loci on all the microchromosomes. The present map will be of use in locating genes affecting disease resistance, but also illustrates the relative ease with which such maps for the chicken can be constructed.

Genetic polymorphism of erythrocyte histone H1 in Japanese quail.

Histone H1 from erythrocytes of Japanese quail was resolved in a sodium dodecyl sulfate (SDS)-polyacrylamide gel into five fractions differing in apparent molecular weights. A polymorphism of histone H1.1, H1.2, and H1.3 bands was detected among quail individuals. While some birds possessed either a high (phenotype .3+) or a low (phenotype .3+/.3-) level of H1.3, at least half of the quail population lacked this H1 band (phenotype .3-). Appropriate genetic crosses demonstrated that H1.3 behaved as though it was coded by a gene with two codominant alleles at an autosomal locus. Using two-dimensional polyacrylamide gel electrophoresis (acid-urea followed by SDS gels), it was found that birds .3+ contained polypeptides H1.b1 and H1.b'1; birds .3-, polypeptides H1.b2 and H1.b'2 with lower apparent molecular weights; and birds .3+/.3-, both types of polypeptides in equal proportions. The H1.b2 + H1.b'2 complement was not discernible in SDS gels, for it migrated together with H1.c' within band H1.4. It was found that a small number of birds lacking the H1.2 band in SDS gels failed to express histone H1.a. Since birds with phenotype .2- with a defective allele of the gene H1.a were simultaneously lacking the H1.3 band, it seems that the imperfect allele of the H1.a gene might be closely linked to the alleles producing H1.b2 + H1.b'2.

A comparison of the histone H1 complements of avian erythrocytes.

1. Histone H1 from chicken, turkey, duck and goose erythrocytes was resolved into six bands and that from quail into seven bands in an acetic acid-urea polyacrylamide gel. 2. A fast migrating minor subtype H1.e was detected in avian erythrocytes using two-dimensional polyacrylamide gel electrophoresis. 3. Although histone subtype H1.z from quail, turkey and duck was well separated in acid-urea gel, a similar protein in goose was found only in two-dimensional gel. This spot was absent in chicken. 4. Histone H1 spots .c, .c' and .d migrate in two-dimensional gel in a relatively constant manner forming a triangle-shaped pattern that facilitates comparison of H1 subtypes among various avian species.

Effect of retinyl acetate on nuclear proteins in rabbit liver.

1. Retinyl acetate injected intraperitoneally to adult rabbits fed on standard diet caused detectable changes in the polyacrylamide gel patterns of liver nucleoplasmic and 0.35 M NaCl-soluble chromatin proteins. 2. Both histones and non-histone proteins soluble in 5 M urea were not affected in vitamin A-treated animals. 3. It seems that variations in liver nuclear proteins from retinyl acetate-administered rabbits may reflect retinol-dependent alterations in structure and function of their chromatins.

Stability of liver nuclear proteins in inbred strains of mice.

1. A remarkable similarity in the gel patterns of liver nuclear proteins between four inbred strains of mice (A.CA, B10.A, CBA and DBA/2) was observed. 2. Only a very few quantitative differences were detected in the protein spot patterns of nucleoplasmic (spot of about 41 kDa) and chromatin (spot of about 37 kDa) non-histone proteins between those strains of mice. 3. Comparison of two-dimensional gel patterns of non-histone proteins from males and females revealed a few sex-linked spots. Nucleoplasmic protein with molecular weight of about 59 kDa and chromatin proteins with molecular weights of approximately 47 and 57 kDa were more abundant in liver nuclei of male mouse.

Effects of cadmium chloride and D-galactosamine on liver nuclear proteins of starved hens.

The effects of short term intraperitoneal administration of cadmium chloride and D-galactosamine on liver nuclear and chromatin levels of ribonucleic acid (RNA) and protein as well as the gel patterns of nuclear nonhistone proteins were examined in fed and starved chickens. It was shown that starvation, cadmium chloride and galactosamine caused a decrease of RNA and protein content in the liver nuclei and chromatin. Cadmium had no significant effect on the gel patterns of nuclear proteins whilst D-galactosamine and starvation produced quantitative changes of certain bands of nonhistone proteins. Although D-galactosamine caused an increase of the amount of histones among liver nucleoplasmic proteins both in fed and starved birds, cadmium brought about a similar effect in starved hens only.

Effect of alpha-amanitin on liver non-histone chromatin proteins of starved hens.

A diminution of RNA content in hen liver nuclei was observed after either prolonged starvation or short-term exposure to alpha-amanitin. Using polyacrylamide gel electrophoresis, it has been revealed a limited number of altered polypeptide bands in the gel patterns of 0.35 M NaCl- and 5 M urea-soluble non-histone proteins from liver chromatin of starved or alpha-amanitin-treated birds. The low-molecular-weight polypeptides were found to increase in the protein fractions from liver chromatin of alpha-amanitin-injected hens. Only two protein bands (48 and 79 kDa) in the gel patterns of 5 M urea-soluble chromatin fraction altered in similar manner both in starved and alpha-amanitin-treated animals. The amount of the 48-kDa protein decreased and that of the 79-kDa protein increased under these conditions. alpha-Amanitin seems to affect differently the non-histone chromatin proteins from starved and fed animals. The level of the 48-kDa urea-soluble protein was lower and that of the 64-kDa protein was higher in liver chromatin of starved animals receiving alpha-amanitin in comparison with the corresponding proteins from fed animals treated with this drug.

A disappearance of a 24-kDa acid-soluble protein from liver chromatin of normal and starved hens following D-galactosamine administration.

Normal and starved adult chickens were injected intraperitoneally with D-galactosamine hydrochloride (0.5 g/kg body weight) and 6 h later liver chromatin acid-soluble proteins were isolated. These proteins were resolved by a two-dimensional polyacrylamide gel electrophoresis in the presence of non-ionic detergent, Triton X-100, in the first dimension and anionic detergent, sodium dodecyl sulfate, in the second dimension. Although spotting patterns of acid-soluble chromatin proteins were remarkably similar between normal and starved control birds and those receiving D-galactosamine, a disappearance of a 24-kDa protein after administration of this agent was found. Moreover, it was shown that this protein was also completely absent in the chicken erythrocyte chromatin which was known to be inactive in RNA synthesis. It seems that the disappearance of the 24-kDa chromatin protein may be associated with inhibiting of transcription in hen liver after D-galactosamine administration and during hen erythrocyte maturation.

The composition of liver chromatin proteins from embryos, chicks and adult chickens.

The chemical composition of chromatin from the livers of 12-, 15- and 19-day-old embryos, of 1-day-old chicks and of adult chickens was analysed. The process of embryonic development is accompanied by an increase in non-histone chromatin proteins and chromatin RNA, as well as in the phosphorus content of chromatin phosphoproteins. The amount of these components decreases in the livers of 1-day-old chicks and adults. Two-dimensional polyacrylamide gel electrophoresis of acid-soluble chromatin proteins showed an increase in the amount of the H1 histone in 19-day-old embryos and adult chickens. Non-histone proteins of embryo liver chromatin showed a high content of the fraction of Mr of about 40 000; this was not the case for adult chickens. The non-histone protein fraction of Mr of about 120 000, characteristic of adult chicken liver proteins, was not found in the livers of 12- and 15-day-old embryos. Non-histone chromatin proteins isolated from the livers of animals of different age exhibited also quantitative differences.