Abstract
The chicken H2B gene family comprises eight members (H2B-I to H2B-VIII), which are all located in two major histone gene clusters. All of them have been shown to encode four different protein variants (classes I to IV). In the DT40 chicken B cell line, the H2B-V gene, encoding the class III H2B variant, constituted about 10% of the total intracellular mRNA from all the H2B genes. To study the nature of this particular variant in vivo, we generated heterozygous (H2B-V, +/-) and homozygous (H2B-V, -/-) DT40 mutants by targeted integration. The remaining H2B genes were shown to be expressed more in these mutants than in the wild-type cell lines. The growth rate of DT40 cells was unchanged in the absence of the H2B-V gene. Two-dimensional polyacrylamide gel electrophoresis showed that the protein patterns were, on the whole, similar between the wild-type and homozygous cell lines. However, within this constant background, some cellular proteins disappeared or decreased quantitatively in the homozygous mutants, and several other proteins increased or newly appeared. These results suggest that the class III H2B variant participates negatively or positively in regulation of the expression of particular genes that encode the proteins that vary in DT40 cells. This type of regulation is possibly mediated through alterations in nucleosome structure over the restricted regions involving the putative genes of the DT40 genome.
Highlights
Chickens have fewer copies of the histone genes, ranging from six copies of the H1 gene to about ten copies of the core genes (H2A, H2B, H3, and H4) [1,2,3] than most higher eukaryotes, which possess large numbers of the genes of each histone subtype, ranging from several dozen to hundreds (4 –7)
We have further demonstrated that the intracellular mRNA levels from H2B-V in the oviduct and lung of chickens were at most one-half those in the kidney, but the total mRNA level from all the H2B genes was roughly equal in these three tissues [30]
To determine the intracellular levels of H2B mRNAs in DT40 cells, we applied the RNase protection method using probe H2B-V, which consisted of the antisense RNA fragment derived from the 128-bp 5Ј-flanking region involving the 5Ј-untranslated sequence plus the 224-bp 5Ј-coding region of H2B-V, in addition to the 78-bp flanking sequence of the plasmid vector (Fig. 2B)
Summary
Chickens have fewer copies of the histone genes, ranging from six copies of the H1 gene to about ten copies of the core genes (H2A, H2B, H3, and H4) [1,2,3] than most higher eukaryotes, which possess large numbers of the genes of each histone subtype, ranging from several dozen to hundreds (4 –7). In a Saccharomyces cerevisiae mutant with disruption of one of two H2A/H2B gene pairs encoding two different variants H2A and H2B, the arrangement of nucleosomes over CYH2 and UBI4 and the centromere of chromosome III was dramatically disrupted, but nucleosomes over HIS4 and GAL1 and the telomeres appeared essentially normal. In this mutant, HIS4 was constitutively expressed and GAL1 repression was unaffected by the mutation. Results obtained in in vivo and in vitro experiments showed that H1 histone acted as a general repressor of transcription in several higher eukaryotic systems [32,33,34,35,36,37], not in yeast
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