Ewa Górnicka-Michalska

Allelic Polymorphism of Histone H1.a in Duck Erythrocytes

In each eukaryotic cell ® ve classes of histones are found. Four of them (H2A, H2B, H3, and H4) play a key role in the organization of the nucleosome core. These proteins are evolutionarily conserved, in contrast to histone H1, which has been reported to vary in its structure within cells, tissues, individuals, and species (Køyszejko-Stefanowicz et al., 1989). Allelic polymorphism of histone H1 has been found in erythrocytes of some bird species [quail and goose (Paøyga, 1991c, 1990a)], rabbit liver (Paøyga, 1990b), and mouse spermatocytes (Zweidler, 1984). In the present work, polymorphic forms of histone H1.a, a nonallelic subtype of histone H1 of duck erythrocytes, have been identi® ed.

Sequence variants of chicken linker histone H1.a

Two allelic isoforms (H1.a1 and H1.a2) of histone H1.a were identified within two conservative flocks (R11 and R55) of Rhode Island Red chickens. These proteins form three phenotypes: a1, a2 and a1a2. Birds with phenotype a1 were most common (frequency 0.825–0.980) while the a1a2 chickens appeared relatively rarely (0.017–0.175). The third phenotype a2, not detected in the tested populations, has only been revealed in progeny of the purpose‐mated a1a2 birds. The polymorphism of histone H1.a was observed in all examined chicken tissues, so that the H1 preparations isolated from the lung, spleen, kidney and testis from the same individual exhibited identical phenotypes (a1, a2, or a1a2). This finding, together with inheritance data, supports the genetic nature of the H1.a polymorphism. As indicated by cleavages with α‐chymotrypsin and protease V8, the H1.a1 and H1.a2 are two highly related proteins which differ within N‐terminal part of their C‐terminal tails. Only a single nonconservative amino acid substitution between both H1.a allelic isoforms was detected by Edman degradation: glutamic acid present at position 117 in histone H1.a1 was replaced by lysine in histone H1.a2. Furthermore, using microsequencing techniques we have found a sequence homology between the N‐ and C‐terminal parts of an unknown minor protein H1.y, present in the phenotype a2, and similar regions of histone H1.b.

Two polymorphic linker histone loci in Guinea fowl erythrocytes

A variable migration of linker histone H1.b and H1.c spots in two-dimensional polyacrylamide gel patterns of total erythrocyte histone H1 has been detected during population screening in two differently plumaged Guinea fowl strains. Alloforms, H1.b1 and H1.b2 as well as H1.c1 and H1.c2, differing in apparent molecular weights tended to form only phenotypes b1 and b2 or c1 and c2 in a white-feathered strain while all phenotypes (b1, b2 and b1b2 or c1, c2 and c1c2, respectively) were present in a black-feathered population. Accordingly, the white-feathered population significantly deviated from the Hardy-Weinberg principle (chi-square test, d.f=1, p<<0.001) due to a lack of heterozygotes while the black-feathered population conformed to the Hardy-Weinberg equilibrium (p>0.05) at both H1.b and H1.c loci. Differential electrophoretic mobilities of the C-peptides from a partial chemical cleavage (N-bromosuccinimide) or limited enzymatic digestion (α-chymotrypsin and protease V8) of the histone H1.b and H1.c alloforms seem to indicate that altered amino acid sequence segments might be located either at the C-terminal end of globular domain or in the C-terminal domain itself.

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.

Phenotypic variation of erythrocyte linker histone H1.c in a pheasant (Phasianus colchicus L.) population

Our goal was to characterize a phenotypic variation of the pheasant erythrocyte linker histone subtype H1.c. By using two-dimensional polyacrylamide gel electrophoresis three histone H1.c phenotypes were identified. The differently migrating allelic variants H1.c1 and H1.c2 formed either two homozygous phenotypes, c1 and c2, or a single heterozygous phenotype, c1c2. In the pheasant population screened, birds with phenotype c2 were the most common (frequency 0.761) while individuals with phenotype c1 were rare (frequency 0.043).

Genetic variants of chicken erythrocyte histone H5

Two allelic electromorphs a and b of chicken eryhrocyte 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 α‐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.

Allelic isoforms of the chicken and duck histone H1.a

Two isoforms of the erythrocyte histone H1.a were identified in two conservative flocks of Rhode Island Red chickens and six conservative flocks of ducks. The H1.a1 and H1.a2 isoforms formed three phenotypes (a1, a2 and a1a2) and were electrophoretically similar in the two species. The frequency of phenotype and histone H1.a allele occurrence varied within the genetic groups of birds, but the relatively rare allele a2 was only detected in chicken and duck strains with colored feathers. Using mass spectrometry, we established that the difference between the measured masses of the duck H1.a isoforms was 156 Da. Since this value corresponds to the mass of the arginine residue alone or to the combined mass of the valine and glycine residues, we believe that the polymorphism of duck histone H1.a might have originated from sequence variation. A mass difference of 1 Da observed between chicken H1.a isoforms corresponded well to the previously detected Glu/Lys substitution (0.9414 Da) at position 117.

Linker Histone H1.b is Polymorphic in Grey Partridge (Perdix perdix)

This study was aimed at characterizing allelic variations of erythrocyte histone H1.b by comparing the electrophoretic patterns of histone H1.b from individuals of grey partridge (Perdix perdix) population. As two alloforms, H1.b1 and H1.b2, were discerned in the screening gels, the histone H1.b was regarded to be a polymorphic protein encoded by a gene with two codominant alleles, b1 and b2, at a locus. The tested population was found to be at Hardy-Weinberg equilibrium (χ2 = 0.834, p = 0.361), with only a minor heterozygote deficiency (fixation index F = 0.136). Since the histone H1.b alloforms were identified in a two-dimensional gel containing sodium dodecyl sulfate, with no significant differences in their migration pattern in an one-dimensional acetic acid polyacrylamide gel, we assumed that the H1.b alloforms possessed a similar net charge and differed in their apparent molecular weights. A comparison of N-bromosuccinimide-cleaved and α-chymotrypsin-digested products of histone H1.b alloforms revealed slight differences in the velocity of C-terminal peptides and a similarity in migration of their N-terminal peptides in one-dimensional sodium dodecyl sulfate-polyacrylamide gel. Therefore, it seemed that the histone H1.b alloforms might differ in this amino acid sequence in a protein segment between N-bromosuccinimide cleavage site and the very C-terminus.

Linker histone-like proteins in Muscovy duck (Cairina moschata L) erythrocyte chromatin.

Linker Histone‐Like proteins (LHL1 and LHL2) were identified within a linker histone complement of Muscovy duck erythrocyte chromatin. Polyacrylamide gel electrophoretic patterns of N‐bromosuccinimide‐cleaved LHL products as well as liquid chromatography‐electrospray‐ion trap mass spectrometry analyses of trypsin‐digested LHL peptides revealed structural similarity of LHL1 to histone H5 and between LHL2 and histone H1 subtypes.