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I Solovei

Publications and source records attributed to I Solovei.

18 recordsLinked to original sources
PubMed2006

Towards many colors in FISH on 3D-preserved interphase nuclei.

The article reviews the existing methods of multicolor FISH on nuclear targets, first of all, interphase chromosomes. FISH proper and image acquisition are considered as two related components of a single process. We discuss (1) M-FISH (combinatorial labeling + deconvolution + wide-field microscopy); (2) multicolor labeling + SIM (structured illumination microscopy); (3) the standard approach to multicolor FISH + CLSM (confocal laser scanning microscopy; one fluorochrome - one color channel); (4) combinatorial labeling + CLSM; (5) non-combinatorial labeling + CLSM + linear unmixing. Two related issues, deconvolution of images acquired with CLSM and correction of data for chromatic Z-shift, are also discussed. All methods are illustrated with practical examples. Finally, several rules of thumb helping to choose an optimal labeling + microscopy combination for the planned experiment are suggested.

Cell Nucleus↗
PubMed2001

Transcripts of the MHM region on the chicken Z chromosome accumulate as non-coding RNA in the nucleus of female cells adjacent to the DMRT1 locus.

The male hypermethylated (MHM) region, located near the middle of the short arm of the Z chromosome of chickens, consists of approximately 210 tandem repeats of a BamHI 2.2-kb sequence unit. Cytosines of the CpG dinucleotides of this region are extensively methylated on the two Z chromosomes in the male but much less methylated on the single Z chromosome in the female. The state of methylation of the MHM region is established after fertilization by about the 1-day embryonic stage. The MHM region is transcribed only in the female from the particular strand into heterogeneous, high molecular-mass, non-coding RNA, which is accumulated at the site of transcription, adjacent to the DMRT1 locus, in the nucleus. The transcriptional silence of the MHM region in the male is most likely caused by the CpG methylation, since treatment of the male embryonic fibroblasts with 5-azacytidine results in hypo-methylation and active transcription of this region. In ZZW triploid chickens, MHM regions are hypomethylated and transcribed on the two Z chromosomes, whereas MHM regions are hypermethylated and transcriptionally inactive on the three Z chromosomes in ZZZ triploid chickens, suggesting a possible role of the W chromosome on the state of the MHM region.

Animals↗
PubMed2001

Arrangements of macro- and microchromosomes in chicken cells.

Arrangements of chromosome territories in nuclei of chicken fibroblasts and neurons were analysed employing multicolour chromosome painting, laser confocal scanning microscopy and three-dimensional (3D) reconstruction. The chicken karyotype consists of 9 pairs of macrochromosomes and 30 pairs of microchromosomes. Although the latter represent only 23% of the chicken genome they containalmost 50% of its genes. We show that territories of microchromosomes in fibroblasts and neurons were clustered within the centre of the nucleus, while territories of the macrochromosomes were preferentially located towards the nuclear periphery. In contrast to these highly consistent radial arrangements, the relative arrangements of macrochromosome territories with respect to each other (side-by-side arrangements) were variable. A stringent radial arrangement of macro- and microchromosomes was found in mitotic cells. Replication labelling studies revealed a pattern of DNA replication similar to mammalian cell nuclei: gene dense, early replicating chromatin mostly represented by microchromosomes, was located within the nuclear interior, surrounded by a rim of late replicating chromatin. These results support the evolutionary conservation of several features of higher-order chromatin organization between mammals and birds despite the differences in their karyotypes.

Animals↗
PubMed2001

Non-random radial higher-order chromatin arrangements in nuclei of diploid human cells.

A quantitative comparison of higher-order chromatin arrangements was performed in human cell types with three-dimensionally (3D) preserved, differently shaped nuclei. These cell types included flat-ellipsoid nuclei of diploid amniotic fluid cells and fibroblasts and spherical nuclei of B and T lymphocytes from peripheral human blood. Fluorescence in-situ hybridization (FISH) was performed with chromosome paint probes for large (#1-5) and small (#17-20) autosomes, and for the two sex chromosomes. Other probes delineated heterochromatin blocks of numerous larger and smaller human chromosomes. Shape differences correlated with distinct differences in higher order chromatin arrangements: in the spherically shaped lymphocyte nuclei we noted the preferential positioning of the small, gene dense #17, 19 and 20 chromosome territories (CTs) in the 3D nuclear interior--typically without any apparent connection to the nuclear envelope. In contrast, CTs of the gene-poor small chromosomes #18 and Y were apparently attached at the nuclear envelope. CTs of large chromosomes were also preferentially located towards the nuclear periphery. In the ellipsoid nuclei of amniotic fluid cells and fibroblasts, all tested CTs showed attachments to the upper and/or lower part of the nuclear envelope: CTs of small chromosomes, including #18 and Y, were located towards the centre of the nuclear projection (CNP), while the large chromosomes were positioned towards the 2D nuclear rim. In contrast to these highly reproducible radial arrangements, 2D distances measured between heterochromatin blocks of homologous and heterologous CTs were strikingly variable. These results as well as CT painting let us conclude that nuclear functions in the studied cell types may not require reproducible side-by-side arrangements of specific homologous or non-homologous CTs. 3D-modelling of statistical arrangements of 46 human CTs in spherical nuclei was performed under the assumption of a linear correlation between DNA content of each chromosome and its CT volume. In a set of modelled nuclei, we noted the preferential localization of smaller CTs towards the 3D periphery and of larger CTs towards the 3D centre. This distribution is in clear contrast to the experimentally observed distribution in lymphocyte nuclei. We conclude that presently unknown factors (other than topological constraints) may play a decisive role to enforce the different radial arrangements of large and small CTs observed in ellipsoid and spherical human cell nuclei.

Amniotic Fluid↗
PubMed2000

Topology of double minutes (dmins) and homogeneously staining regions (HSRs) in nuclei of human neuroblastoma cell lines.

Amplification of the MYCN gene is a characteristic feature of many neuroblastomas and is correlated with aggressive tumor growth. Amplicons containing this gene form either double minutes (dmins) or homogeneously staining regions (HSRs). To study the nuclear topology of these tumor-specific and transcriptionally active chromatin structures in comparison to chromosome territories, we performed fluorescence in situ hybridization with a MYCN probe and various chromosome paint probes, confocal laser scanning microscopy, and quantitative three-dimensional image analysis. The dmins formed dot-like structures in interphase nuclei and were typically located at the periphery of complexly folded chromosome territories; dmins noted in the chromosome territory interior were often detected within an invagination of the territory surface. Interphase HSRs typically formed extremely expanded structures, which we have never observed for chromosome territories of normal and tumor cell nuclei. Stretches of HSR-chromatin often extended throughout a large part of the cell nucleus, but appeared well separated from neighboring chromosome territories. We hypothesize that dmins are located within the interchromosomal domain (ICD) space and that stretches of HSR-chromatin align along this space. Such a topology could facilitate access of amplified genes to transcription and splicing complexes that are assumed to localize in the ICD space.

Cell Nucleus↗
PubMed2000

Chromosome territories, interchromatin domain compartment, and nuclear matrix: an integrated view of the functional nuclear architecture.

Advances in the specific fluorescent labeling of chromatin in fixed and living human cells in combination with three-dimensional (3D) and 4D (space plus time) fluorescence microscopy and image analysis have opened the way for detailed studies of the dynamic, higher-order architecture of chromatin in the human cell nucleus and its potential role in gene regulation. Several features of this architecture are now well established: 1. Chromosomes occupy distinct territories in the cell nucleus with preferred nuclear locations, although there is no evidence of a rigid suprachromosomal order. 2. Chromosome territories (CTs) in turn contain distinct chromosome arm domains and smaller chromatin foci or domains with diameters of some 300 to 800 nm and a DNA content in the order of 1 Mbp. 3. Gene-dense, early-replicating and gene-poor, middle-to-late-replicating chromatin domains exhibit different higher-order nuclear patterns that persist through all stages of interphase. In mitotic chromosomes early replicating chromatin domains give rise to Giemsa light bands, whereas middle-to-late-replicating domains form Giemsa dark bands and C-bands. In an attempt to integrate these experimental data into a unified view of the functional nuclear architecture, we present a model of a modular and dynamic chromosome territory (CT) organization. We propose that basically three nuclear compartments exist, an "open" higher-order chromatin compartment with chromatin domains containing active genes, a "closed" chromatin compartment comprising inactive genes, and an interchromatin domain (ICD) compartment (Cremer et al., 1993; Zirbel et al., 1993) that contains macromolecular complexes for transcription, splicing, DNA replication, and repair. Genes in "open," but not in "closed" higher-order chromatin compartments have access to transcription and splicing complexes located in the ICD compartment. Chromatin domains that build the "open" chromatin compartment are organized in a way that allows the direct contact of genes and nascent RNA to transcription and splicing complexes, respectively, preformed in the ICD compartment. In contrast, chromatin domains that belong to the "closed" compartment are topologically arranged and compacted in a way that precludes the accessibility of genes to transcription complexes. We argue that the content of the ICD compartment is highly enriched in DNA depleted biochemical matrix preparations. The ICD compartment may be considered as the structural and functional equivalent of the in vivo nuclear matrix. A matrix in this functional sense is compatible with but does not necessitate the concept of a 3D nuclear skeleton existing of long, extensively arborized filaments. In the absence of unequivocal evidence for such a structural matrix in the nucleus of living cells we keep an agnostic attitude about its existence and possible properties in maintaining the higher-order nuclear architecture. Quantitative modeling of the 3D and 4D human genome architecture in situ shows that such an assumption is not necessary to explain presently known aspects of the higher-order nuclear architecture. We expect that the interplay of quantitative modeling and experimental tests will result in a better understanding of the compartmentalized nuclear architecture and its functional consequences.

Animals↗
PubMed1999

Identification of the gene-richest bands in human prometaphase chromosomes.

The human genome is a mosaic of long, compositionally homogeneous DNA segments, the isochores, that can be partitioned into five families, two GC-poor families (L1 and L2), representing 63% of the genome, and three GC-rich families (H1, H2 and H3), representing 24%, 7.5% and 4-5% of the genome, respectively. Gene concentration increases with increasing GC levels, reaching a level 20-fold higher in H3 compared with L isochores. In-situ hybridization of DNA from different isochore families provides, therefore, information on the chromosomal distribution of genes. Using this approach, three subsets of reverse or Giemsa-negative bands, H3+, H3* and H3-, containing large, moderate, and no detectable amounts, respectively, of the gene-richest H3 isochores were identified at a resolution of 400 bands. H3+ bands largely coincide with the most heat-denaturation-resistant bands, the chromomycin-A3-positive, DAPI-negative bands, the bands with the highest CpG island concentrations, and the earliest replicating bands. Here, we have defined the H3+ bands at a 850-band resolution, and have thus identified the human genome regions, having an average size of 4 Mb, that are endowed with the highest gene density.

Base Composition↗
PubMed1998

Specific chromomeres on the chicken W lampbrush chromosome contain specific repetitive DNA sequence families.

Chromomeres 1 and 3 of the chicken W lampbrush chromosome contain most of the EcoRI and XhoI repeat sequence families respectively. These chromomeres were stained with DAPI and their sizes relative to other W chromomeres were observed. Their relative contents of EcoRI and XhoI repeats were determined using fluorescence in situ hybridization with genomic probes for each of the two repeat families. There were two types of W chromosome in the chickens (White Leghorn and Rhode Island Red) used in this study with respect to the amount of EcoRI repeat. A high-copy-number type has about 4000 copies of the 1.2-kb repeat per genome and shows a large fluorescence signal on W chromomere 1. A low-copy-number type has about 700 copies per genome and does not have a detectable chromomere 1 on W chromosome, nor does it show FISH labelling in the region normally occupied by chromomere 1. The genome of Fayoumi chickens has about one-sixth the amount of the XhoI sequence family of White Leghorns. W lampbrush chromomere 3 is much smaller and its FISH labelling with the XhoI probe is much weaker in Fayoumis than in White Leghorns. These results demonstrate that in the chicken W chromosome, specific chomomeres are occupied by specific DNA repeat sequence families.

Animals↗
PubMed1997

Nuclear pore complex structure in birds.

The nuclear envelope consists of two parallel membranes enclosing an aqueous lumen. In places there are pores in both membranes at which the two membranes are joined. Within these pores reside the nuclear pore complexes. The current structural models of the nuclear pore complex have been derived from a number of studies using different electron microscopical techniques. Recently, using surface imaging techniques such as field emission in-lens scanning electron microscopy, novel structures have been identified, particularly at the periphery of the structure, most notably the nucleoplasmic basket. One limitation of the current models is that they are based almost entirely on nuclear envelopes isolated from amphibian oocytes and a pressing question is whether this structure is the same in other organisms and tissues. Here we have studied the structure of nuclear envelopes isolated from bird oocytes. We show that the overall structure is remarkably conserved. In particular, recently discovered peripheral structures appear very similar. We see variations in basket conformation but believe that this is related to the functional states of individual pore complexes.

Animals↗
PubMed1997

Molecular characterization and cytological mapping of a non-repetitive DNA sequence region from the W chromosome of chicken and its use as a universal probe for sexing carinatae birds.

A non-repetitive genomic DNA region of about 25 kb was cloned from the W chromosome of chicken using a genomic library prepared from a single W chromosome of the chicken. This region was mapped by fluorescence in situ hybridization (FISH) with mitotic and lampbrush chromosomes to a position between the major EcoRI family and the pericentromeric Xhol family on the W chromosome. A 0.6-kb EcoRI fragment (EE0.6) subcloned from this region consists of a sequence that can be obtained by the exon-trapping procedure and flanking sequences. Sequences, which are closely similar to that of EE0.6, are widely conserved on the W chromosomes of Carinatae birds, as revealed by Southern blot hybridization to HindIII-digested female and male genomic DNAs from 18 species of birds belonging to eight different taxonomic orders. The female sex of those birds can be determined by the presence of an unambiguous female-specific band. For many species of birds, the female sex can also be determined by polymerase chain reaction (PCR) using a set of primers from the flanking sequences in the chicken EE0.6.

Animals↗
PubMed1997

Nuclear Pore Complex Structure in Birds

The nuclear envelope consists of two parallel membranes enclosing an aqueous lumen. In places there are pores in both membranes at which the two membranes are joined. Within these pores reside the nuclear pore complexes. The current structural models of the nuclear pore complex have been derived from a number of studies using different electron microscopical techniques. Recently, using surface imaging techniques such as field emission in-lens scanning electron microscopy, novel structures have been identified, particularly at the periphery of the structure, most notably the nucleoplasmic basket. One limitation of the current models is that they are based almost entirely on nuclear envelopes isolated from amphibian oocytes and a pressing question is whether this structure is the same in other organisms and tissues. Here we have studied the structure of nuclear envelopes isolated from bird oocytes. We show that the overall structure is remarkably conserved. In particular, recently discovered peripheral structures appear very similar. We see variations in basket conformation but believe that this is related to the functional states of individual pore complexes.

Journal Article↗
PubMed1996

Characterization of DNA sequences constituting the terminal heterochromatin of the chicken Z chromosome.

Two clones, pCZTH5-8 and pCZTH12-8, were isolated from a female chicken genomic library by screening with sequences obtained from genomic libraries which had been constructed from a terminal region of a single Z chromosome of chicken utilizing laser microbeam irradiation and PCR amplification. Fluorescence in situ hybridization to the mitotic Z chromosome and the lampbrush ZW bivalent of chicken demonstrated that both the cloned sequences are located in the heterochromatic region of the Z chromosome at the end opposite to the pairing region with the W chromosome. The sequences pCZTH-8 and pCZTH12-8 are distributed widely on both the telomeric bow-like loops (TBL) and the region I (short loops region) of the Z lampbrush chromosome. These clones, pCZTH12-8 particularly notably, hybridized also to the TBLs of lampbrush bivalents 1-4 of chicken. Both sequences are transcribed in the lampbrush stage oocytes on the Z chromosome and on other macrobivalents. The subfragment of pCZTH5-8 which hybridizes to the TBLs and the insert of pCZTH12-8 contain regions that are closely similar in sequence. The pCZTH-8 sequence has no internal repeats and may be part of the 24-kb macrosatellite repeating unit that is evident after Nhel digestion of the genomic DNA. A cloned 24-kb unit, pFN-1, does not show significant DNA curvature, but cytosines of its CpG dinucleotides may be highly methylated in vivo. This contrasts with the repeat sequences of the W heterochromatin which not only have highly methylated CpG but are also strongly curved. The 24-kb unit is repeated about 830 times in the diploid genome of a female chicken, suggesting that nearly the entire terminal heterochromatin on the Z chromosome consists of this macrosatellite family. Sequences of the greater part of the pCZTH-8 are restricted to the genus Gallus but the sequence of one subregion which hybridizes to TBLs is present in the genomes of the order Galliformes.

Animals↗
PubMed1995

Single stranded nucleic acid binding structures on chicken lampbrush chromosomes.

In chicken oocytes, proteins of the K/J family or their analogs, such as are known to be involved in mRNA processing in humans, are closely associated with nascent C-rich RNA transcripts on the loops of lampbrush chromosomes. Using labelled single stranded nucleotide probes and an antibody to protein K, these C-rich transcripts have been mapped to six different pairs of lampbrush loops situated on 3 macrochromosomes, the sex bivalent (ZW) and certain microchromosomes. Each of these loop pairs has a distinctive morphology. The observations represent cytological evidence of the connection between K-proteins and C-rich RNA transcripts. Another structure, the spaghetti marker of macrochromosome II, also preferentially binds C-rich homonucleotides. This spaghetti marker has a highly distinctive fine structural organization that is quite unlike that of lampbrush loops. Its proteins are not recognised by antibodies to protein K. Homonucleotide binding loops are recommended as potentially extremely valuable as markers on physical maps of chicken chromosomes.

Animals↗
PubMed1994

The arrangement and transcription of telomere DNA sequences at the ends of lampbrush chromosomes of birds.

The arrangement of loops and chromomeres at the ends of lampbrush chromosomes in four species of bird is described with reference to chromomeres, loops and transcription units. Unlike the situation described in lampbrush chromosomes of amphibians, the lampbrush chromosomes of birds end in a terminal chromosome with conspicuous loops emerging from it. The fine-scale morphology of the ribonuclear protein matrix of these terminal loops is different from that of the majority of loops elsewhere on the chromosomes. In many cases the loops associated with the terminal chromomere are open ended, emerging from the chromomere but not returning to it at the other end. The distal ends of terminal open-ended loops therefore represent the true ends of the chromatids that make up a lampbrush half-bivalent. The pattern of binding of three telomeric DNA sequence probes to the terminal regions of bird lampbrush chromosomes, under conditions of DNA/DNA and DNA/RNA transcript in situ hybridization has been investigated by fluorescence in situ hybridization. All three probes gave the same results. With DNA/DNA and DNA/RNA transcript hybridization, three classes of structure were labelled: the terminal chromomere, a small number of interstitial chromomeres and the terminal transcription unit on telomere loops. Labelling of telomere loops, but not of terminal or interstitial chromomeres, was eliminated by ribonuclease treatment before in situ hybridization. The labelled regions of telomere loops were spaced away from the labelled terminal chromomere by an unlabelled sub telomeric transcription unit. After DNA/DNA in situ hybridization, no labelled loops were seen. DNA/RNA transcript in situ hybridization with single-stranded hexamers of each strand of telomeric DNA showed that the terminal transcription unit on telomere loops represents transcription exclusively from the C-rich strand of the repeat outwards towards the end of the chromosome. It is concluded that transcription specifically of the C-rich strand of strictly terminal clusters of telomere repeats is an obligatory event on the lampbrush chromosomes of birds and is unlikely to represent indiscriminate readthrough from proximally located gene elements.

Animals↗
PubMed1993

Avian sex chromosomes in the lampbrush form: the ZW lampbrush bivalents from six species of bird.

The ZW bivalent has been identified and characterized in detail in its lampbrush form in oocytes of chicken, quail, turkey, pigeon, chaffinch and sparrow. The sex bivalent in all six species looks like a single highly asymmetrical chromosome. Most of it has the typical lampbrush organization. The terminal one-fifth is relatively thick and condensed and bears only a few pairs of lateral loops: this condensed terminal region is the W chromosome; the part with normal lampbrush morphology is the Z. The two are connected by a single near terminal chiasma. The fine scale morphology and arrangement of loops and markers on Z and W chromosomes are described for each species and lampbrush maps have been constructed. The identification of the lampbrush sex bivalent is based on the following criteria. The asymmetrical chromosome has two centromere regions. In the interstitial region of the asymmetrical chromosome where the junction between Z and W chromosomes is supposed to be, there are telomere-specific loops and telomeric DNA sequences and there is good morphological evidence for the presence of a chiasma. There are W chromosome specific DNA sequences in the region of the asymmetrical lampbrush chromosome that is thought to represent the W. Breed-specific variations in the morphology of the chicken W chromosome with respect to the sizes, numbers and arrangements of axial chromomeres and distributions of specified repeated DNA sequence families have been identified, offering one of the first examples of definitive correlation between a repeat family and a single chromomere. The lampbrush chromosomes of all the birds examined, except quail, terminate in distinctive free hanging loops. These are a novel feature in the sense that at the end of each chromatid there is a large transcription unit terminating in a cluster of telomeric DNA sequences.

Animals↗
PubMed1992

A novel structure associated with a lampbrush chromosome in the chicken, Gallus domesticus.

At a site near the end of the short arms of lampbrush bivalent 2 in the chicken (Gallus domesticus) there is always a marker structure that appears in the phase-contrast light microscope as a solid object with diffuse edges measuring about 4 microns across. When examined by transmission electron microscopy in thin section, this object appears as a loose bundle of fibres. In some preparations individual fibres appear 15-16 nm thick, smooth in outline and solid in cross-section. In other preparations they are 32-38 nm thick, rougher in outline and ring-like in cross-section. High-resolution scanning electron micrographs of the chromosome 2 marker show it to be a loose bundle of spaghetti-like fibres that is quite unlike anything previously seen on a lampbrush chromosome of any organism. As with the sectioned material, fibres in some preparations were smooth and 15-16 nm in diameter, whereas those in others were more knobbly and about 35 nm thick. The fibres appear to branch and in some cases it is clear that the daughter strands of a branch have the same dimensions as the parent strand. Free ends are rare. Total length of fibre material present at one marker locus is estimated to be between 500 and 2000 microns. Similar structures are not present on the lampbrush chromosomes of quail, wood pigeon or chaffinch. The nature of this fibrous marker, referred to in this paper as the "spaghetti marker", is discussed in relation to lampbrush chromosome function and to events that take place during the lampbrush phase of oogenesis in chicken. Evidence is discussed in relation to the possibility that the chromosome 2 marker represents a novel form of nuclear RNP or the specific association of some structural protein with one chromosome locus.

Animals↗
PubMedDate not supplied

Crossing over in chicken oogenesis: cytological and chiasma-based genetic maps of the chicken lampbrush chromosome 1.

Chiasmata in diplotene bivalents are located at the points of physical exchange (crossing-over) between homologous chromosomes. We have studied chiasma distribution within chicken lampbrush chromosome 1 to estimate the crossing-over frequency between chromosome landmarks. The position of the centromere and chromosome region 1q3.3-1q3.6 on lampbrush chromosome 1 were determined by comparative physical mapping of the TTAGGG repeats in the chicken mitotic and lampbrush chromosomes. The comparison of the chiasma (=crossing over)-based genetic distances on chicken chromosome 1 with the genetic linkage map obtained in genetic experiments showed that current genetic distances estimated by the high-resolution genetic mapping of the East Lansing, Compton, and Wageningen chicken reference populations are 1.2-1.9 times longer than those based on chiasma counts. Conceivable reasons for this discrepancy are discussed.

Animals↗