Emerin prevents BAF-mediated aggregation of lamin A on chromosomes in telophase to allow nuclear membrane expansion and nuclear lamina formation.

Several studies have suggested a role for the LEM-domain protein emerin and the DNA binding factor BAF in nuclear envelope reformation after mitosis, but the exact molecular mechanisms are not understood. Using HeLa cells deficient for emerin or both emerin and lamin A, we show that emerin deficiency induces abnormal aggregation of lamin A at the nuclear periphery in telophase. As a result, nuclear membrane expansion is impaired and BAF accumulates at the core region, the middle part of telophase nuclei. Aggregates do not form when lamin A carries the mutation R435C in the immunoglobulin fold known to prevent interaction of lamin A with BAF suggesting that aggregation is caused by a stabilized association of lamin A with BAF bound to chromosomal DNA. Reintroduction of emerin in the cells prevents formation of lamin A clusters and BAF accumulation at the core region. Therefore emerin is required for the expansion of the nuclear membrane at the core region to enclose the nucleus and for the rapid reformation of the nuclear lamina based on lamin A/C in telophase. Finally, we show that LEM-domain and lumenal domain are required for the targeting of emerin to exert its function at the core region.

Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability.

Histone H3 lysine 9 methylation has been proposed to provide a major "switch" for the functional organization of chromosomal subdomains. Here, we show that the murine Suv39h histone methyltransferases (HMTases) govern H3-K9 methylation at pericentric heterochromatin and induce a specialized histone methylation pattern that differs from the broad H3-K9 methylation present at other chromosomal regions. Suv39h-deficient mice display severely impaired viability and chromosomal instabilities that are associated with an increased tumor risk and perturbed chromosome interactions during male meiosis. These in vivo data assign a crucial role for pericentric H3-K9 methylation in protecting genome stability, and define the Suv39h HMTases as important epigenetic regulators for mammalian development.

The apical ectodermal ridge, fibroblast growth factors (FGF-2 and FGF-4) and insulin-like growth factor I (IGF-I) control the migration of epidermal melanoblasts in chicken wing buds.

The role of the apical ectodermal ridge and of fibroblast growth factors FGF-2 and FGF-4 and of the insulin-like growth factor I (IGF-I) in the control of the migration of epidermal melanoblasts was investigated using quail-chicken chimeras. Wing buds of a strain of unpigmented chicken were microsurgically modified in several ways (ablation, displacement or implantation of additional apical ectodermal ridges, implantation of grafts devoid of apical ectodermal ridges, ectopic application of growth factors) and received grafts containing quail neural crest cells. The distribution of the epidermal melanoblasts which had differentiated from the quail grafts revealed that both the apical ectodermal ridge and the growth factors invariably caused the migration of epidermal melanoblasts towards them. This leads to the conclusion that the presence of the apical ectodermal ridge is the sufficient condition to direct the migration of epidermal melanoblasts within the avian embryonic wing bud. Furthermore, FGF-2 and IGF-I and to a lesser extent FGF-4 play a decisive role in directing the migration of epidermal melanoblasts within chicken wing buds and are likely to be involved in the molecular cascade by means of which the apical ectodermal ridge controls the migration of epidermal melanoblasts.

Signal enhancement at the electron microscopic level using Nanogold and gold-based autometallography.

Immunoelectron microscopy using ultrasmall gold markers is a very sensitive method to detect molecules at high resolution. In order to discriminate the gold particles in the electron microscope, enlargement of gold particles is necessary. So far, mostly silver ions were used for deposition onto the surface of gold grains. In our study, we tested the selective deposition of gold instead of silver ions to enlarge gold particles. This was performed following immunogold detection of DNA at the surface of ultrathin sections embedded in the acrylic resin LR White (postembedding approach). Morphometric analysis of the distribution of DNA in human spermatocytes revealed that the method offers very good specificity and sensitivity and therefore is a good alternative to the use of silver for signal enhancement. This technique was also applied to the detection of ribosomal genes in human testis at the electron microscopic level by in situ hybridization. Ribosomal genes were localized in peri- and intranucleolar chromatin as well as in the dense fibrillar component of nucleoli.

Intranuclear anchoring of repetitive DNA sequences: centromeres, telomeres, and ribosomal DNA.

Centromeres, telomeres, and ribosomal gene clusters consist of repetitive DNA sequences. To assess their contributions to the spatial organization of the interphase genome, their interactions with the nucleoskeleton were examined in quiescent and activated human lymphocytes. The nucleoskeletons were prepared using "physiological" conditions. The resulting structures were probed for specific DNA sequences of centromeres, telomeres, and ribosomal genes by in situ hybridization; the electroeluted DNA fractions were examined by blot hybridization. In both nonstimulated and stimulated lymphocytes, centromeric alpha-satellite repeats were almost exclusively found in the eluted fraction, while telomeric sequences remained attached to the nucleoskeleton. Ribosomal genes showed a transcription-dependent attachment pattern: in unstimulated lymphocytes, transcriptionally inactive ribosomal genes located outside the nucleolus were eluted completely. When comparing transcription unit and intergenic spacer, significantly more of the intergenic spacer was removed. In activated lymphocytes, considerable but similar amounts of both rDNA fragments were eluted. The results demonstrate that: (a) the various repetitive DNA sequences differ significantly in their intranuclear anchoring, (b) telomeric rather than centromeric DNA sequences form stable attachments to the nucleoskeleton, and (c) different attachment mechanisms might be responsible for the interaction of ribosomal genes with the nucleoskeleton.

Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice.

It is commonly accepted, that regenerative capacity of striated muscle is confined to skeletal muscle by activation of satellite cells that normally reside quiescent between the plasmalemma and the basement membrane of muscle fibers. Muscular dystrophies are characterized by repetitive cycles of de- and regeneration of skeletal muscle fibers and by the frequent involvement of the cardiac muscle. Since during the longstanding course of muscular dystrophies there is a permanent demand of myogenic progenitors we hypothesized that this may necessitate a recruitment of additional myogenic precursors from an undifferentiated, permanently renewed cell pool, such as bone marrow (BM) cells. To this end normal and dystrophic (mdx) female mice received bone marrow transplantation (BMT) from normal congenic male donor mice. After 70 days, histological sections of skeletal and cardiac muscle from BMT mice were probed for the donor-derived Y chromosomes. In normal BMT recipients, no Y chromosome-containing myonuclei were detected, either in skeletal or in cardiac muscle. However, in all samples from dystrophic mdx skeletal muscles Y chromosome-specific signals were detected within muscle fiber nuclei, which additionally were found to express the myoregulatory proteins myogenin and myf-5. Moreover, in the hearts of BMT-mdx mice single cardiomyocytes with donor derived nuclei were identified, indicating, that even cardiac muscle cells are able to regenerate by recruitment of circulating BM-derived progenitors. Our findings suggest that further characterization and identification of the BM cells capable of undergoing myogenic differentiation may have an outstanding impact on therapeutic strategies for diseases of skeletal and cardiac muscle.

Autosomal-dominant, prelingual, nonprogressive sensorineural hearing loss: localization of the gene (DFNA8) to chromosome 11q by linkage in an Austrian family.

A four-generation family suffering from an autosomal-dominant, congenital, nonprogressive, nonsyndromic hearing loss was found in a rural region of Austria. The hearing loss was moderate to severe, a pure tone audiogram showing a U-shaped form with maximum loss at 2, 000 Hz. An initial genome search led to a lod score of 3.01 with markers on chromosome 15. This locus was registered as DFNA8 in the HUGO data base. Further sampling of the family, however, yielded data that reduced the maximal lod score with chromosome 15 markers to 1.81. The genome search was restarted using an ABITM genotyper, which eventually detected several positive two-point lod scores with markers from the long arm of chromosome 11. The highest value was 3. 6, which was seen with the marker D11S934. Haplotype analysis excluded the gene from the chromosomal region proximal from D11S898 and distal to D11S1309. These results place the gene in the region of the hearing loss gene DFNA12. Recent evidence suggests that the somewhat different phenotypes found in these two families are due to two different mutations in the human alpha-tectorine gene (Verhoeven et al., 1998).

Arrangement of individual human ribosomal DNA fragments on stretched DNA fibers.

We studied the arrangement of individual human ribosomal (r)DNA repeats by direct visualization of rDNA sequences. We used high resolution fluorescence in situ hybridization on preparations of DNA fibers released from interphase nuclei of HeLa cells. Probes from both the transcription unit and the intergenic spacer were used, and lengths of signals and of the gaps in between were measured and compared to molecular data. We could visualize the repetitive arrangement of individual rDNA sequences at the single gene level. No inversions or deletions were detected. The intergenic spacer was found to be shorter than expected, indicating a length polymorphism.

Signal amplification at the ultrastructural level using biotinylated tyramides and immunogold detection.

The tyramide amplification technique has recently been developed for signal enhancement in enzyme-linked immunosorbent assays and western blots. This method relies on using labelled tyramides as substrates for peroxidase, resulting in an immobilization of the labelled tyramide residues (tyramide reaction). We succeeded in establishing reliable protocols for the use of the tyramide reaction at the electron microscopic (EM) level. As model systems we chose the visualization of DNA in late spermatocytes, of actin in skeletal muscle, and the visualization of an rDNA probe after DNA-DNA in situ hybridization. We observed a significant increase in signal density after performing the tyramide reaction at the EM level. The tyramide amplification technique at the ultrastructural level therefore appears to be a useful tool to detect even a few epitopes present at the surface of a section as shown after in situ hybridization. It offers advantages over other amplification systems, such as the peroxidase-mediated deposition of diaminobenzidine, because of an increased spatial resolution, whereas specificity and sensitivity are comparable to the conventional immunogold detection method.

The transcription unit of ribosomal genes is attached to the nuclear skeleton.

The relationship between various loci of the ribosomal gene repeat and the nucleoskeleton was examined in agarose-embedded HeLa cells. The accessibility of intranucleolar structures to molecular probes was improved by dispersing the granular component of nucleoli, and unattached DNA was removed from permeabilized nuclei under "physiological" conditions by enzymatic digestion and subsequent electroelution. The cells were then hybridized in situ with various human rDNA probes for the transcription unit or for the intergenic spacer. A strong signal was detected with probes for the transcription unit but no signal was seen with probes for the intergenic spacer. These results show that only the transcription unit is strongly attached to the nucle(ol)ar skeleton and imply that rDNA is probably attached to the skeleton primarily via RNA polymerase complexes rather than via sequence-specific attachment sites. Nucleolar fibrillar centers, embedded into the nucle(ol)ar skeleton, provide structural support for these attachments.

Redistribution of ribosomal DNA after blocking of transcription induced by actinomycin D.

We report on the effect of different doses and times of incubation of the cytostatic drug actinomycin D (AMD) on nucleolar morphology, rRNA gene transcription and rDNA gene localization using in situ hybridization and the immunocytochemical detection of the human upstream binding factor (UBF) at the electron microscopic level in HeLa cells. Low doses of AMD (0.001 micrograms/ml, 30 min) selectively block rRNA gene transcription but alter neither nucleolar morphology nor the localization of rDNA with respect to the nucleolar components. Treatment with high doses of AMD (0.05 micrograms/ml, 1 h) resulted in a retraction of the rDNA out of the nucleolus in addition to the well-known blocking of rDNA transcription, total nuclear transcription and nucleolar segregation. Under these conditions accumulations of rDNA were found in patches of chromatin at the nucleolar periphery. We conclude that the blocking of rRNA gene transcription and the changes in nucleolar morphology, both induced by AMD at different doses, are independent phenomena.

The RNA polymerase I transcription factor UBF and rDNA are located at the same major sites in both interphase and mitotic pig embryonic kidney (PK) cells.

Indirect immunolabeling with anti-UBF antibodies, in situ hybridization with an rDNA probe, and confocal scanning laser microscopy were used to study nucleolar organizer regions (NORs) during the cell cycle in pig embryonic kidney (PK) cells. The chromosomal distribution of the polymerase I transcription factor UBF and rDNA was compared with the number of silver-stained NORs (Ag-NORs) present and nucleolar size. It was shown, both at interphase and mitosis, that the majority of UBF and rDNA signals were located at the same foci and that the amounts of UBF and rDNA at any given site were in a striking positive correlation. At mitosis, only the NORs were labeled; at interphase, the signals for both UBF and rDNA were arranged in necklace-like structures around the nucleoli. No chromosomal NORs without Ag-proteins or UBF were present, indicating that all NORs in PK cells are active at interphase. It was concluded that (1) UBF and rDNA co-localize throughout the cell cycle in PK cells; (2) their association with mitotic NORs is determined by the number of rDNA repeats, rather than by any differential ability of NORs to recruit the transcription factor; and (3) the amount of UBF can be correlated with the size and activity of the nucleoli at interphase.

Spatial distribution of sex chromosomes and ribosomal genes: a study on human lymphocytes and testicular cells.

The location of the sex chromosomes in relation to the rRNA genes in the nuclei of human lymphocytes and testicular cells was examined. Sex chromosomes were found to be located closer to ribosomal genes than would be expected assuming a random arrangement of these chromosomes with respect to rRNA genes. This proximity could be observed irrespective of the transcriptional activity of ribosomal genes indicating that the chromosomal material and not transcriptional activity is responsible for the intranuclear order of these chromosomes.

Human ribosomal RNA gene repeats are localized in the dense fibrillar component of nucleoli: light and electron microscopic in situ hybridization in human Sertoli cells.

The distribution of the human ribosomal gene repeat within human Sertoli cell nucleoli was investigated with the help of DNA-DNA in situ hybridization at the light and electron microscopic level. Probes from both the transcribed part of the gene repeat and the "non-transcribed" spacer were found to hybridize predominantly to the dense fibrillar component of nucleoli. It therefore can be concluded that the dense fibrillar component of nucleoli is the major site of the intranucleolar location of the ribosomal DNA. This holds true not only for the dense fibrillar component adjacent to fibrillar centers, but also for the dense fibrillar component remote from the fibrillar centers.

[DNA analysis and prenatal diagnosis in cystic fibrosis]. / DNA-Analyse und Pränataldiagnose bei cystischer Fibrose.

Cystic fibrosis (CF) is the most common autosomal recessive lethal genetic disorder in man, affecting 1 in 2,000 live births. The carrier frequency in Caucasians is 1 in 20. Approximately 70 per cent of the mutations correspond to a specific deletion on chromosome 7 (region 7q31). This delta F 508 mutation of the CF gene results in the loss of phenylalanine at position 508 in the gene product. About 50 per cent of the individuals with CF are homozygous for this deletion and suffer from pancreatic insufficiency. The remaining half are mostly compound heterozygotes between this deletion and another mutation. They exhibit less severe symptoms. Several simple polymerase chain reaction (PCR) tests are available to detect the delta F 508 mutation, allowing rapid prenatal diagnosis of the disease. In those families where other mutations of the CF gene are involved prenatal diagnosis is performed by means of tightly linked restriction fragment length polymorphisms (RFLPs). PCR tests are available for RFLP analysis too. Testing for the delta F 508 deletion, present in about 3 per cent of the normal population, may prove invaluable for extensive carrier screening. Thus, half of the couples at risk could be recognized before the first affected child is born.