Positioning of a nucleosome on mouse satellite DNA inserted into a yeast plasmid is determined by its DNA sequence and an adjacent nucleosome.

It has earlier been shown that multiple positioning of nucleosomes on mouse satellite DNA is determined by its nucleotide sequence. To clarify whether other factors, such as boundary ones, can affect the positionings, we modified the environment of satellite DNA monomer by inserting it into a yeast plasmid between inducible GalCyc promoter and a structural region of the yeast FLP gene. We have revealed that the positions of nucleosomes on satellite DNA are identical to those detected upon reconstruction in vitro. The positioning signal (GAAAAA sequence) of satellite DNA governs nucleosome location at the adjacent nucleotide sequence as well. Upon promoter induction the nucleosome, translationally positioned on the GalCyc promoter, transfers to the satellite DNA and its location follows the positioning signal of the latter. Thus, the alternatives of positioning of a nucleosome on satellite DNA are controlled by its nucleotide sequence, though the choice of one of them is determined by the adjacent nucleosome.

Nucleosome positioning on yeast plasmids is determined only by the internal signal of DNA sequence occupied by the nucleosome.

The possible role of border factors in determining the nucleosome positioning on a DNA sequence was investigated. To this end a family of recombinant plasmids based on Gal10Cyc1 promoter and neomycin phosphotransferase gene NPTII were created. A DNA sequence adjoining the GalCyc promoter was varied in these plasmids. Three nearly equally represented nucleosome positions on the GalCyc promoter were found. In the basal plasmid an FRT sequence adjoins the GalCyc promoter at the right. It contains an internal signal of multiple positioning. Its replacement with different DNA sequences does not affect nucleosome positioning on the GalCyc promoter. The nucleosome positioning on the GalCyc promoter does not depend on nucleosome positioning (or its absence) on adjoining sequences. The same is true for nucleosome positioning on FRT sequence. It was found also that nucleosomes' positioning on the NPTII gene and their mutual disposition, namely the spacing between neighboring nucleosomes (linker length) are determined by the location of positioning signals only. Generally the nucleosome positioning in our experimental model is determined solely by internal DNA sequence occupied by nucleosome. On the other hand, the action of this internal positioning signal does not extend to neighboring DNA sequences.

Segment selection during 'phase variation'-type regulation of gene replacement mediated by FLP recombinase in the yeast Saccharomyces cerevisiae.

FLP recombinase has recently been used as a tool to direct the exchange between invertible DNA segments, called 'Phase variation'-type regulation of gene replacement in eukaryotic cells. Using an appropriate selective medium, positive segment selection was shown to be efficient during the regulation of gene replacement. The efficiency was determined from the copy number ratio of invertible segments with the use of the neomycinphosphotransferase II (NPTII) gene bearing invertible segments located on the episomal yeast plasmid, and the resident 2-microns circle. Without the selection the segments copy number ratio was retained in growing cells. The results obtained are an evidence for the efficiency of positive segment selection during the 'Phase variation'-type regulation of gene replacement in eukaryotic cells.

Nucleomeric organization of chromatin.

In the nuclei fixed in situ, as well as in nuclei in low-ionic-strength solutions containing magnesium ions, chromatin is represented by globular nucleomeric fibrils 20-25 nm in diameter. Staphylococcal or endogenous nucleases cleave chromatin fibrils to nucleomers and multinucleomers. On removal of firmly bound magnesium, the nucleomers unfold into chains of four, six or eight nucleosomes. Mild staphylococcal nuclease digestion of nuclear chromatin releases mononucleomers, dinucleomers and trinucleomers that sediment in the sucrose density gradient in the presence of EDTA as 37-S, 47-S and 55-S particles, respectively. The mononucleomers in the sucrose density gradient with MgCl2 sediment as 45-S particles. The determination of the length of staphylococcal-nuclease-digested DNAs contained in the chromatin fragments showed that a nucleomer is composed of 8, and a dimer and trimer of 14-16 and 21-24 nucleosomes, respectively. When deprived of Mg2+ ions, the monomers lose their compactness (45 S) and become loose particles (37 S). This transition is completely reversible if nucleomers contain histone H1. Removal of this histone or dialysis of the nucleomer against EDTA at low ionic strength results in the complete unfolding of the nucleomer into a chain of nucleosomes. A structural model of a nucleomer fibril is suggested where the helicity of the nucleosome chain in a nucleomer (two turns of four nucleosomes each) is periodically discontinued. Such an organization of chromatin apparently provides additional hindrances for site-specific recognition of DNA in chromatin but permits local changes (within a single nucleomer) in chromatin when a hindrance is abolished.

Intragenome distribution of 5-methylcytosine in DNA of healthy and wilt-infected cotton plants (Gossypium hirsutum L.).

Fractionation of DNA of healthy and wilt-infected cotton plants has been carried out according to the reassociation kinetics and the content of GC and 5-methylcytosine in the resulting fractions has been studied. The genome of cotton plant was found to be methylated quite unevenly. The GC rich (GC equals 64.7 mole%) fraction of highly reiterated sequences (Cot equals 0-3.7 times 10- minus 2) has a high content of 5-methylcytosine (5.8 mole%), whereas the methylation degree of the fraction of unique sequences (Cot larger than or equal to 487) is very low (the 5-methylcytosine content is about 0.5 mole%). In plants being infected with wilt, the 5-methylcytosine content in DNA or cotton leaves decreases two-fold; no changes in the structure and molecular population of DNA has been found. The sharp change in the 5-methylcytosine content in DNA of infected plants takes place at the expense of the decrease in the 5-methylcytosine content in fractions of highly reiterated sequences. The methylation degree of unique sequences (structural genes) remains unchanged.