[Chromatin folding in human spermatozoa. I. Dynamics of chromatin remodelling in differentiating human spermatids].

Changes in chromatin structure at different stages of differentiation of human spermatids were studied. It was shown that, in nuclei of early spermatids, chromatin is loosely packed and its structural element is an 8-nm fiber. This "elementary" fiber is predominant at the initial stages of differentiation; in the course of maturation, it is replaced by globular elements approximately 60 nm in diameter. In intermediate spermatids, these globules start to condense into fibrillar aggregates and reduce their diameter to 30-40 nm. At all stages of spermatid maturation, except the final stages, these globules are convergence centers for elementary fibers. This remodelling process is vectored and directed from the apical (acrosomal) to the basal pole of the nucleus. In mature spermatids, the elementary 8-nm fibers are almost absent and the major components are 40-nm fibrillar aggregates. The nuclei of mature spermatids are structurally identical with the nuclei of spermatozoa with the so-called "immature chromatin," which are commonly found in a low proportion in sperm samples from healthy donors and may prevail over the normal cells in spermiogenetic disorders. The cause of this differentiation blockade remains unknown. Possibly, the formation of intermolecular bonds between protamines, which are required for the final stages of chromatin condensation, is blocked in a part of spermatids. The results of this study are discussed in comparison with the known models of nucleoprotamine chromatin organization in human spermatozoa.

[Nucleosome positioning within neomycinphosphotransferase gene (NPTII) on yeast plasmid in repressed and active state].

Yeast recombinant plasmid containing FRT-sequence flanked by hybrid GAL-CYC promoter and NPTII gene was developed. GAL-CYC promoter contains four UAS sequences and two closely associated TATA-boxes in CYC part. This construct provides galactose-inducible synthesis of neomycinphosphotransferase from NPTII gene, and, thus, resistance of transformed cells to G418 antibiotic. Nucleosome positioning within NPTII gene in repressed and active states was studied. Under repressive conditions (growth on glucose) stable positioning of three nucleosomes was detected. Two nucleosomes are localized in CYC-part. One of them encompasses both TATA-boxes. The third nucleosome overlaps FRT sequence and start of NPTII gene coding sequence. All three nucleosomes show multiple positioning. It suggests possibility of nucleosome sliding along DNA. After induction of NPTII expression by galactose sliding of two nucleosomes is detected. Sliding leads to exposure of TATA-box and long promoter segment. Sliding results in stable repositioning of nucleosomes at new sites. 5'-distal nucleosome moves closer to UAS-sequences. As a results UAS becomes spatially closer to TATA-box. This proximity facilitates assembly of preinitiation complex. Nucleosomes slides independently from each other. The second nucleosome moves towards FRT-sequence and repositions at its nucleosome positioning signal. Galactose-induced expression does not affect nucleosome positioning with coding region of NPTII gene. Unidirectional sliding and repositioning are detected without induction after deacetylase inhibition with trichostatine A. Basal expression of NPTII gene was shown without activation of GAL-CYC promoter and after spatial uncoupling of coding sequence and promoter by gene inversion. In these cases it seems that expression is driven by TATA-like element in FRT-sequence. This element is located in permanently exposed area (in vivo data).

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.

[The dependence of the 3-dimensional organization and length of the nucleolonema in the hepatocyte nucleoli of normal and regenerating rat livers on the nucleosome structure of the intranucleolar chromatin]. / Zavisimost' trekhmernoi organizatsii i dliny nukleolonemy iadryshek gepatotsitov normal'noi i regeneriruiushchei pecheni krysy ot nukleosomnoi struktury vnutriiadryshkovogo khromatina.

A biochemical and ultrastructural stereo-morphological analysis, with special reference to spatial organization and length of nucleonema and Ag-positive zones, was performed for various modifications of nucleolonemal type nucleoli in normal and regenerating (6 and 22 hours after partial hepatectomy) rat hepatocytes. To determine possible disorders on nucleosomal and supranucleosomal levels, chromatin DNA degradation was carried out during micrococcal nuclease hydrolysis, followed by analysis of electrophoretically separated particles. Functional characterization of intranucleolar chromatin was performed by testing the rate of DNA degradation after DNAase I treatment as well as by detection of free G-C pairs during titration with actinomycin D. Transcriptional activity of nucleoli was determined according to the intensity of [14C]-UTP uptake with isolated nucleoli. It is shown that the total chromatin from control nucleoli contains nucleosomal fibrils, although deprived of high compactization level. Nucleosomes themselves are strongly destabilized. In activated nucleoli structural differences of chromatin are more perceptible. In 6 hour preparations the bulk of chromatin fibrils (about 70%) undergo a further relaxation and lose the nucleosomal structure. Therefore at this point of experiment, the maximum length of nucleolonema and Ag-positive zones was registered in addition to the highest quantity of free G-C pairs, and sensibility to DNAase I transcriptional activity of isolated nucleoli. 22 hours after hepatectomy, the transcriptional activity and functional parameters of intranucleolar chromatin markedly decreased compared to the 6 hour period. Simultaneously, the share of chromatin restituting the nucleosomal structure increased, while the length of nucleolonema was shorter than in nucleoli 6 hours after hepatectomy. The main results could be resumed in the following way: the general composition of nucleolonemal type nucleolus variations described in our experimental conditions is in close relation with the with the compactization grade of ribosomal DNP-fibrils.