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The transient expression of the luciferase reporter gene was used to detect the tissue-specific enhancer activity of the solitary extraviral long terminal repeat (LTR) of the human endogenous retrovirus K (HERV-K). The LTR was previously mapped to the 19q13.2 locus. It contains a number of potential regulatory elements including TATA box, binding sites for some nuclear factors, and a polyadenylation signal. However, an analysis of the genomic sequences close to the LTR did not reveal any known genes or the expressed sequences (EST), whose functioning could be regulated by this LTR. The enhancer activity can be preserved in the solitary LTR due to its involvement in the long-range control of genome functioning or by the absence of functional disruptive mutations within the human-specific LTR, because it is of a relatively young evolutionary age.

Reporter gene analysis of HERV-K solitary long terminal repeats (LTRs) showed that they retain detectable activity in human teratocarcinoma cells, and can direct the transcription in both orientations relative to the reporter gene. Deletion analysis demonstrated the possible existence of alternative promoters within the LTR as well as a silencer-like element in the U5 region. Our results indicate also that all-trans-retinoic acid is capable of modulating expression of the reporter gene directed by a HERV-K LTR in NT2/D1 cells.

Chimeric genes containing the genomic fragments of hepatitis B virus (HBV) coding for middle (M) and major/small (S) surface proteins were constructed and expressed in transgenic potato plants under the control of the cauliflower mosaic virus 35S promoter. Enzyme-linked immunosorbent assay (ELISA) revealed the presence of HBV surface antigen (HBsAg) determinants in extracts from transgenic plants carrying both constructions, the pre-S2 determinant was identified using the corresponding monoclonal antibodies (mAb) in a dot immunobinding assay. Polypeptides of the immunoaffinity-purified S and M proteins were analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. Two polypeptides of the M protein, probably representing different glycosylated forms, and the multimeric forms of HBsAg were observed.

When chromosomal proteins in chromatin or in mononucleosomes were extensively cross-linked with an imido ester, the H1-containing nonameric histone complex was revealed. In this complex, histone H1 is connected with the octamer of core histones. The cross-linking of H1 of the octamer is realized preferentially through H2a and H3 histones. Some HMG (high mortality group) proteins located presumably in the linker regions of a nucleosome fiber also take part in the formation of dimers, possibly with the histones of a nucleosomal core. The results suggest mutant interactions between some linker-associated proteins and intranucleosomal histones. Experiments involving extensive cross-linking of proteins in the purified mononucleosome subfractions demonstrated differences in the organization of core histones between 'complete' nucleosomes and nucleosomes lacking H1.

Earlier work in Felsenfeld’s laboratory showed that extensive digestion of chromatin with micrococcal nuclease results in a limit digestion set of discrete double-stranded DNA fragments of sizes ranging from about 130 base pairs (bp) to 45 bp [ 11. Alternatively, mild treatment of chromatin with nucleases produces a soluble mixture of mononucleosomes and their oligomers which can be separated from each other by sucrose gradient centrifugation [2,3] or by polyacrylamide gel electrophoresis [4-61. However, polyacrylamide gel electrophoresis of DNA . protein under nondenaturing conditions has proven to be a more successful method in studying the heterogeneity of nucleosomes and their composition. We have separated 4-5 main structural types of mononucleosomes: along with core particles (MNl), mononucleosomes were identified containing an additional segment of linker DNA and either Hl histone or non-histone protein HMG 14 or 17 [6-g]. We also discovered a set of small subnucleosomal particles which contained short DNA fragments and histones or non-histone proteins [3,6,7]. Two of them (SN2 and 3) comprising short DNA fragment and HMG protein originated apparently from transcriptionally active chromatin [8]. tion of nuclei from mouse L cells have been published in [8]. The isolated nuclei were suspended in buffer A [0.04 M NaCl, 2 mM MgC12, 10 mM triethanolamine (TEA)-HCl, pH 7.61 containing 1 mM CaClz at 5 X lo8 nuclei/ml. Then micrococcal nuclease (Worthington) was added to 75 units/ml and the digestion carried out at 37’C. The reaction was terminated by chilling in ice and the first supernatant (Sl) was obtained by centrifugation at 5000 X g. The digested nuclei were washed with buffer A and lyzed by addition of 2 mM Na-EDTA (pH 7.6). The second supernatant (S2) constituted of monoand oligonucleosomes was prepared. Both the supernatants were dialyzed against 1 mM Na-EDTA (pH 7.6) and clarified by centrifugation.