Conserved epitopes on plant H1 histones recognized by monoclonal antibodies

Abstract

A series of monoclonal antibodies specific for distinct regions of HI histone from the plant Nicotiana tabacum were obtained from fusion experiments with spleen cells of mice immunized with tobacco nuclear extracts. These monoclonal antibodies were characterized and the evolutionary conservation of the epitopes in higher plants and animals studied by immunoblotting and enzyme-lfnked immunosorbent assay (ELISA). Whereas some epitopes appear restricted to the Solanaceae plant family, others are common to all higher eukaryotes tested and even detectable on nuclear proteins of yeast. ELISA experiments performed with isolated tobacco chromatin give some indications of the differential accessibility of the epitopes after interaction of H1 histone with the nucleosome.Inside the chromatin of eukaryotic cells the first order of DNA packing is reached by wrapping the DNA around a protein octamer composed of two molecules of histones 2A, 2B, 3, and 4 [I]. This protein-DNA complex, or nucleosome, has been found in all eukaryotic organisms studied up to now and shows in each case highly conserved structural characteristics [2]. In addition to the four nucleosomal histones, a fifth group exists which is characterized by a high lysine and arginine content. These H1 histones associate with the DNA at a point where it enters and leaves the nucleosome [3]. Furthermore, H 1 histone, once bound, aids in condensation of the nucleosome chains and in the formation and maintenance of higher-order structures in the chromatin [4, 51.Studies on the evolutionary conservation of histone proteins have shown that histones involved in nucleosome assembly are among the most conserved proteins found in eukaryotes [6 -111.In contrast to the nucleosomal histones, H1 histones show considerable sequence variation [9]. Even in a single tissue several subtypes of H1 coexist [6]. Furthermore, tissue-specific as well as development-specific HI-histone variants have been characterized [12, 131. Some variants show different abilities to condense DNA and chromatin [14 -161 which may play a role in the control of gene expression [12].Despite the significant variations in molecular mass and amino acid sequence existing among H1 histones, studies in animals revealed that the general organization of the molecule is preserved, and consists of a central hydrophobic globular domain flanked by one highly variable N-terminal domain and a hydrophilic C-terminal region [12].Only two reports on the amino acid sequence of plant H1 histones have been published until now [17, 181. The data indicate that the general structure of H1 histones has been maintained in plants. However, the amino acid sequence data remain too fragmentary to draw conclusions on the extent and modalities of evolutionary conservation of H1 histones in plants.An alternative to amino acid sequence comparisons to determine the conservation of specific structures in a protein is the use of antibodies that recognize discrete epitopes. Such an approach has been widely used in the structural characteriza...

Existence of yeast genomic fragments hybridizing with the sea urchin histone H1 gene.

“…Elsewhere we presented evidence that a yeast analogue of histone H 1 could be detected immunologically as a contaminant in some HMG preparations (12). Positive immunochemical identification of H1-like proteins in yeast has also been reported by others (6,8). As a continuation of this work (12), we attempted the identification in the yeast genome of nucleotide sequences homologous to the H 1 gene from sea urchin.…”

A bacterial peptide acting as a plant nuclear targeting signal: the amino-terminal portion of Agrobacterium VirD2 protein directs a beta-galactosidase fusion protein into tobacco nuclei.

“…Nuclei were isolated according to Mazzolini et al (17). Samples were treated with 2% glutaraldehyde prior to the assay.…”

Production and characterisation of cell- and tissue-specific monoclonal antibodies for the flatworm Macrostomum sp.

“…Monoclonal antibodies are available for all developmental model organisms such as, for example, C. elegans (Goetinck and Waterston 1994), Drosophila (Patel 1994), Xenopus (Itoh et al 1994) or mouse (Kubota et al 1996). Antibody libraries were raised to dissect, for example, nuclear structure in animals (Saumweber 1991) and plants (Mazzolini et al 1989).…”