The proteoglycan bikunin has a defined sequence

Proteoglycans are complex glycoconjugates that regulate critical biological pathways in all higher organisms. Bikunin, the simplest proteoglycan, with a single glycosaminoglycan chain, is a serine protease inhibitor used to treat acute pancreatitis. Unlike nucleic acids and proteins, whose synthesis is template driven, Golgi-synthesized glycosaminoglycans are not believed to have predictable or deterministic sequences. Bikunin peptidoglycosaminoglycans were prepared and fractionated to obtain a collection of size-similar and charge-similar chains. Fourier transform mass spectral analysis identified a small number of parent molecular ions corresponding to monocompositional peptidoglycosaminoglycans. Fragmentation using collision-induced dissociation unexpectedly afforded a single sequence for each monocompositional parent ion, unequivocally demonstrating the presence of a defined sequence. The biosynthetic pathway common to all proteoglycans suggests that even more structurally complex proteoglycans, such as heparan sulfate, may have defined sequences, requiring a readjustment in the understanding of information storage in complex glycans.     

Fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei

An approach towards construction of two-dimensional (2D) and three-dimensional (3D) profiles of interphase chromatin architecture by quantification of fluorescence in situ hybridization (FISH) signal intensity is proposed. The technique was applied for analysis of signal intensity and distribution within interphase nuclei of somatic cells in different human tissues. Whole genomic DNA, fraction of repeated DNA sequences (Cot 1) and cloned satellite DNA were used as probes for FISH. The 2D and 3D fluorescence intensity profiles were able to depict FISH signal associations and somatic chromosome pairing. Furthermore, it allowed the detection of replicating signal patterns, the assessment of hybridization efficiency, and comparative analysis of DNA content variation of specific heterochromatic chromosomal regions. The 3D fluorescence intensity profiles allowed the analysis of intensity gradient within the signal volume. An approach was found applicable for determination of assembly of different types of DNA sequences, including classical satellite and alphoid DNA, gene-rich (G-negative bands) and gene-poor (G-positive bands) chromosomal regions as well as for assessment of chromatin architecture and targeted DNA sequence distribution within interphase nuclei. We conclude the approach to be a powerful additional tool for analysis of interphase genome architecture and chromosome behavior in the nucleus of human somatic cells. The text was submitted by the authors in English.     

Distribution of sugar-binding sites within interphase nuclei and mitotic chromosomes of a human cell line

Using gold labelled neoglycoproteins containing either alpha-D-glucose, N-acetyl-beta-D-glucosamine, alpha-D-mannose, 6-phospho-alpha-D-mannose, and alpha-L-fucose (BSA), we investigated their intranuclear binding sites in the TG human cell line. Although gold-labelled BSA did not give any noticeable labelling, the presence of 1% free BSA in the medium containing the gold labelled neoglycoproteins was revealed to be a key factor of the labelling. During interphase in the presence of free BSA most of the labelling was detected in the nucleoplasm. The border of the condensed chromatin, known to be the site of hnRNA synthesis as well as the interchromatin areas enriched in RNPs were labelled. Condensed chromatin also contained binding-sites. The nucleolus was seen to present low labelling in comparison with the labelling observed over the nucleoplasm. These nucleolar binding sites were located both in the dense fibrillar and granular components. No labelling could be detected over the fibrillar centers which are very conspicuous in this cell line. During mitosis sugar-binding sites were observed over the chromosomes. Data reported here show for the first time that lectin-like proteins and chromatin components are colocalized both during interphase and mitosis. In addition, within the nucleolus the presence of sugar-binding proteins was seen to be restricted to the dense fibrillar and granular components.     

The interphase distribution of satellite DNA-containing heterochromatin in mouse nuclei

The distribution of sites capable of binding mouse satellite-complementary RNA in the cytological hybridization reaction has been examined in mouse liver and testis interphase nuclei. The approach taken has been to combine hybridization with semi-thin sectioning and autoradiography in order to obtain a clear picture of the relationship of satellite DNA-containing structures to the rest of the interphase nucleus. In liver nuclei, hybridization occurs primarily with blocks of heterochromatin associated with the nuclear envelope. The most prominent of these, in terms both of size and intensity of hybridization, is the nucleolar stalk and the rest of the nucleolus-associated heterochromatin. The nucleolar body itself is not labeled, nor is much of the peripheral condensed chromatin ; in fact, a polarized distribution of satellite DNA is evident. In Sertoli and spematid nuclei, satellite DNA is found in a small number of large heterochromatin blocks with which the nucleolus is associated; some of this material bears a relationship to the nuclear envelope in these cells also.     

The fluorescence localization of mouse satellite DNA in interphase nuclei

Nuclei from various mouse tissues exhibit a pattern of fluorescence characteristic of the cell type when stained with the fluorescent compound Hoechst 33258. When such preparations are hybridized in situ with ³H-RNA complementary to the A-T rich satellite of mouse, it is clearly seen that only the fluorescent regions of the nuclei contain the satellite DNA. Thus Hoechst 33258 allows the precise localization of satellite DNA at all stages of the mouse cell cycle.     

The significance of telomeric aggregates in the interphase nuclei of tumor cells

Telomeres are TTAGGG repetitive motifs found at the ends of vertebrate chromosomes. In humans, telomeres are protected by shelterin, a complex of six proteins (de Lange [2005] Genes Dev. 19: 2100-2110). Since (Müller [1938] Collecting Net. 13: 181-198; McClintock [1941] Genetics 26: 234-282), their function in maintaining chromosome stability has been intensively studied. This interest, especially in cancer biology, stems from the fact that telomere dysfunction is linked to genomic instability and tumorigenesis (Gisselsson et al. [2001] Proc. Natl. Acad. Sci. USA 98: 12683-12688; Deng et al. [2003] Genes Chromosomes Cancer 37: 92-97; DePinho and Polyak [2004] Nat. Genetics 36: 932-934; Meeker et al. [2004] Clin. Cancer Res. 10: 3317-3326). In the present overview, we will discuss the role of telomeres in genome stability, recent findings on three-dimensional (3D) changes of telomeres in tumor interphase nuclei, and outline future avenues of research.     

The possible presence of aldehydes and carbohydrates in chromosomes and interphase nuclei

Summary A histochemical study of the mitotic chromosomes and interphase nuclei of rat regenerating liver and of root tips from Vicia faba and Trillium grandiflorum has been performed using the periodic acid-Schiff reaction. Carbohydrates and plasmalogen-like molecules were demonstrated on these cellular structures. The presence of such components was confirmed by biochemical studies upon nuclei isolated from rat liver.