Biomedical subjects

P F Ambros

Publications and source records attributed to P F Ambros.

At least 19 recordsLinked to original sources

PubMed2007

Induction of senescence in MYCN amplified neuroblastoma cell lines by hydroxyurea.

Recently, it was shown that MYCN amplified cells spontaneously expulse extrachromosomally amplified gene copies by micronuclei formation. Furthermore, it was shown that these cells lose their malignant phenotype and start to age. We tested whether it is possible to encourage neuroblastoma tumor cells to enter the senescence pathway by low concentrations of the micronuclei-inducing drug hydroxyurea (HU). We studied the effect of HU on 12 neuroblastoma cell lines with extra- or intrachromosomally amplified MYCN copies and without amplification. Two extrachromosomally amplified neuroblastoma cell lines (with double minutes) were investigated in detail. Already after 3 weeks of HU treatment, the BrdU uptake dropped to 25% of the starting cells. After 4 weeks, enlarged and flattened cells (F-cells) and increased granularity in the majority of cells were observed. A drastic reduction of the MYCN copy number-down to one copy per cell-associated with CD44 and MHCI upregulation in up to 100% of the HU treated neuroblastoma cells was found after 5-8 weeks. Telomere length was reduced to half the length within 8 weeks of HU treatment, and telomerase activity was not detectable at this time, while being strongly expressed at the beginning. All these features and the expression of senescence-associated-beta-galactosidase (SA-beta-GAL) in up to 100% of the cells support the hypothesis that these cells entered the senescence pathway. Thus, low-dose HU is a potent senescence elicitor for tumor cells with gene amplification, possibly representing an attractive additional strategy for treatment of this subset of tumors.

Antineoplastic Agents↗

PubMed2006

FKHRL1-mediated expression of Noxa and Bim induces apoptosis via the mitochondria in neuroblastoma cells.

Protein kinase-B (PKB) and its target, the forkhead transcription factor like 1 (FKHRL1)/FoxO3a, have been suggested as regulators of neurotrophin-mediated cell survival in neuronal cells. We analyzed human neuroblastoma cells and found that FKHRL1 was phosphorylated, suggesting its inactivation. To study FKHRL1 function, we infected SH-EP and NB15 cells with a 4OH-tamoxifen-regulated FKHRL1(A3)ER(tm) transgene. Activation of FKHRL1 promoted cytochrome-c release and caspase-dependent apoptosis. FKHRL1 induced TRAIL and the BH3-only proteins Noxa and Bim, implicating both extrinsic and intrinsic death pathways. However, expression of dnFADD did not inhibit FKHRL1-induced cell death, whereas Bcl2 protected against apoptosis. This excluded the death-receptor pathway and suggested that cell death decision is regulated by Bcl2-rheostat. Importantly, RNAi knockdown of Noxa or Bim decreased apoptosis, indicating that Noxa and Bim cooperate to mediate FKHRL1-induced cell death. We conclude that Noxa and Bim establish a connection between FKHRL1 and mitochondria, and that both BH3-only proteins are critically involved in FKHRL1-induced apoptosis in neuroblastoma.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2005

DEC1 expression in 1p-aberrant oligodendroglial neoplasms.

BACKGROUND: Expression of hypoxia-related tissue factors in 1p-aberrant oligodendroglial neoplasms diminishes patient outcome. Differentiated embryo-chondrocyte expressed gene 1 (DEC1) has been described as novel hypoxia-related tissue factor. In our study, we assessed the expression of DEC1 in 1p aberrant oligodendroglial neoplasms and its association with necrosis and expression of hypoxia-inducible factor 1alpha (HIF-1alpha), carbonic anhydrase-9 (CA9), and vascular endothelial growth factor-mRNA (VEGF). MATERIALS AND METHODS: 44 primary and 16 recurrent oligodendroglial neoplasms with 1p-aberrations were investigated immunohistochemically for the expression of DEC1, HIF-1alpha, and CA9. Expression of VEGF was investigated using in situ hybridization. DEC1 expression was correlated with necrosis and with expression of HIF-1alpha, CA9, and VEGF. RESULTS: DEC1 was expressed in tumor cell nuclei, and occasionally in nuclei of endothelial cells, and glial and neuronal cells of surrounding brain tissue. High expression (>10% of tumor cells immunolabeled) of DEC1 was found in 56 cases, low expression (<10% of tumor cells immunolabeled) was found in 3 cases. In 1 case no expression of DEC1 was evident. DEC1 expression showed no topographical association with necrosis or expression of HIF-1alpha, CA9, or VEGF. CONCLUSION: DEC1 expression is found in the majority of 1p-aberrant oligodendroglial neoplasms and does not correlate with necrosis or expression of HIF-1alpha, CA9, VEGF. Thus, immunohistochemical analysis of DEC1 expression is in our hands not suitable for detection of tissue hypoxia in this type of primary brain tumor.

Adult↗

PubMed2004

Automatic quantification of gene amplification in clinical samples by IQ-FISH.

BACKGROUND: The reliable detection and quantification of gene amplifications is crucial to clinical practice. Although there are different detection techniques, the fluorescence in situ hybridization (FISH) method has become highly accepted over past years because it is a reliable, robust, and quick method. Unfortunately, automatic quantification of gene amplification based on fluorescence intensities has not been possible thus far. Because current spot counting methods are reliable only when analyzing low amplification rates, we attempted to establish another method, i.e., to quantify the intensity of different FISH signals using an automatic fluorescence microscopical device on interphase nuclei: interphase quantitative FISH (IQ-FISH). METHODS: We quantified the fluorescence intensities of the differently labeled FISH probes (MYCN and D2Z) hybridized to three different neuroblastoma cell lines, six peripheral blood (PB) samples, 10 spiked PB samples, and nine neuroblastoma samples using the Metafer4 system (MetaSystems, Altlussheim, Germany). To obtain the MYCN copy number per cell, the ratio between the fluorescence intensities of the MYCN gene and reference sequence (D2Z) was calculated. For automatic analysis of the HER-2/neu status in tumor cells, labeled FISH probes specific for HER-2/neu and a chromosome 17-specific probe were hybridized to peripheral blood and tumor specimens and analyzed using the automatic device. RESULTS: When measuring the fluorescence intensity per cell for both probe pairs (MYCN/D2Z and HER-2/17p), amplified and non-amplified cells, showed distinct peaks with only little overlap. Whereas normal cells showed a fluorescence ratio peak for MYCN/D2Z between 200 and 800, cells with MYCN amplification clearly exceeded this ratio value (1000 to 25,000). When mixing a varying number of MYCN amplified cells (range 9-91%) to normal PB, the spiked tumor cells could be identified. Even one neuroblastoma tumor cell in 1000 mononucleated cells could reliably be detected using our device. In neuroblastoma patient samples, non-amplified cells were distinguished from amplified cells. Automatically and manually counted signals gave matching results in amplified and non-amplified samples. HER-2/neu-amplified cells were automatically detected in the breast cancer samples analyzed. CONCLUSION: The automatic measurement of fluorescence signal intensities not only allows a reliable discrimination between non-amplified and amplified cells but also exact quantification of amplified sequences. This is the prerequisite for the following applications: detection of amplified cells in the bone marrow and second-look specimens; comparison between primary and relapse or pre- and post-chemotherapeutic specimens; detection of tumors with focal gene amplification; and quantification of elimination of amplified gene sequences.

Automation↗

PubMed2003

Quality assessment of genetic markers used for therapy stratification.

PURPOSE: Therapy stratification based on genetic markers is becoming increasingly important, which makes commitment to the highest possible reliability of the involved markers mandatory. In neuroblastic tumors, amplification of the MYCN gene is an unequivocal marker that indicates aggressive tumor behavior and is consequently used for therapy stratification. To guarantee reliable and standardized quality of genetic features, a quality-assessment study was initiated by the European Neuroblastoma Quality Assessment (ENQUA; connected to International Society of Pediatric Oncology) Group. MATERIALS AND METHODS: One hundred thirty-seven coded specimens from 17 tumors were analyzed in 11 European national/regional reference laboratories using molecular techniques, in situ hybridization, and flow and image cytometry. Tumor samples with divergent results were re-evaluated. RESULTS: Three hundred fifty-two investigations were performed, which resulted in 23 divergent findings, 17 of which were judged as errors after re-evaluation. MYCN analyses determined by Southern blot and in situ hybridization led to 3.7% and 4% of errors, respectively. Tumor cell content was not indicated in 32% of the samples, and 11% of seemingly correct MYCN results were based on the investigation of normal cells (eg, Schwann cells). Thirty-eight investigations were considered nonassessable. CONCLUSION: This study demonstrated the importance of revealing the difficulties and limitations for each technique and problems in interpreting results, which are crucial for therapeutic decisions. Moreover, it led to the formulation of guidelines that are applicable to all kinds of tumors and that contain the standardization of techniques, including the exact determination of the tumor cell content. Finally, the group has developed a common terminology for molecular-genetic results.

Biomarkers, Tumor↗

PubMed2002

Prognostic impact of chromosomal aberrations in Ewing tumours.

Although greater than 50% of Ewing tumours contain non-random cytogenetic aberrations in addition to the pathognomonic 22q12 rearrangements, little is known about their prognostic significance. To address this question, tumour samples from 134 Ewing tumour patients were analysed using a combination of classical cytogenetics, comparative genomic and fluorescence in situ hybridisation. The evaluation of the compiled data revealed that gain of chromosome 8 occurred in 52% of Ewing tumours but was not a predictive factor for outcome. Gain of 1q was associated with adverse overall survival and event-free survival in all patients, irrespective of whether the tumour was localised or disseminated (overall survival: P=0.002 and P=0.029; event-free survival: P=0.018 and P=0.010). Loss of 16q was a significant predictive factor for adverse overall survival in all patients (P=0.008) and was associated with disseminated disease at diagnosis (P=0.039). Gain of chromosome 12 was associated with adverse event-free survival (P=0.009) in patients with localised disease. These results indicate that in addition to a 22q12 rearrangement confirmation in Ewing tumours it is important to assess the copy number of 1q and 16q to identify patients with a higher probability of adverse outcome.

Adult↗

PubMed2002

[Detection, quantification and characterization of disseminated tumor cells].

There are different reasons why the detection of disseminated tumor cells (DTCs) in the hematopoetic system is important. On the one hand the detection of disseminated tumor cells can provide vital information about a tumor's tendency to develop metastases. In some localized epithelial but also in embryonic tumors, for example a correlation between disseminated tumor cells and unfavorable outcome was observed (6, 14). These studies are based on the assumption that those tumor cells which appear in the hematopoetic system at a very early stage are responsible for the development of metastases. Another important aspect is the monitoring of the disease response to cytotoxic drugs by quantifying DTCs. During and after therapy there is no other possibility (except for an operation) to either directly analyze the effects the therapy has on the tumor cells or to determine their biological characteristics. The dissemination in the hematopoetic system, however, reveals the response to therapy and the biological features of the tumor cells. The prerequisites for such bone-marrow diagnosis, however, is the unequivocal identification of disseminated tumor cells. So in order to avoid false positive results (which are a risk in bone-marrow diagnostics), a system was developed to distinguish tumor cells from non-neoplastic cells and to facilitate insights into the biological make-up of tumor cells (2, 11).

Antineoplastic Agents↗

PubMed2002

[Cell proliferation and cell death in disseminated tumor cells].

The occurrence of occult metastases of solid tumors at initial diagnosis or during follow-up is of crucial therapeutical importance. The sensitive detection of such cells in hematological samples depends on tissue specific cellular markers. The demonstration of minimally disseminated tumor cells at a given timepoint is, however, only a snapshot, which does not give any information about the potential and dynamics of the cells in question. Functional differences may fundamentally influence the impact of a positive finding. The analysis of cell proliferation and cell death (apoptosis) in disseminated tumor cells, for instance, defines, whether the dissemination process is progressive or regressive. With a newly developed automatic image analysis station the investigation of functional parameters in isolated cells from clinical samples became possible. The studies presented here demonstrate, that such techniques allow an improved identification of isolated tumor cells with clinical importance.

Cell Death↗

PubMed2001

The EWS protein is dispensable for Ewing tumor growth.

EWS encodes a ubiquitously expressed RNA binding protein with largely unknown function. In Ewing sarcoma family tumors (EFT), one allele is rearranged with an ETS gene. This is the first description of an EFT with a complete EWS deficiency in the presence of two copies of a rearranged chromosome 22 carrying an interstitial EWS-FLI1 translocation. Absence of EWS protein suggested that it is dispensable for EFT growth. By sequencing of EWS cDNA from unrelated EFTs, we excluded inactivation of EWS as a general mechanism in EFT pathogenesis. Rather, EWS was found to be uniformly expressed in two splicing variants of similar abundancy, EWSalpha and EWSbeta, which differ in a single amino acid. Three EWS negative cell lines were established, which will serve as valuable models to study normal and aberrant EWS function upon reintroduction into the tumor cells.

Alleles↗

PubMed2001

Identification of a novel exon in apolipoprotein E receptor 2 leading to alternatively spliced mRNAs found in cells of the vascular wall but not in neuronal tissue.

Novel members of the low density lipoprotein receptor family were identified in human endothelial and vascular smooth muscle cells utilizing a homology-cloning strategy. Four novel mRNA transcripts could be identified as isoforms of the apolipoprotein E receptor 2 (apoEr2): one form lacking three ligand binding repeats (nucleotides 497-883) but containing a novel ligand binding repeat adjacent to a unique cysteine-rich domain preceding the epidermal growth factor precursor domain of apoEr2, forms lacking the O-linked sugar domain, and forms containing a 59-amino acid deletion within the cytoplasmic tail. By fluorescence in situ hybridization for chromosome mapping, we could confirm that the novel alternative forms of apoEr2 are splice variants of transcripts from a single copy gene on chromosome 1p34. To analyze whether the different splice variants of apoEr2 mRNA are expressed in a splice variant-specific pattern, we concentrated on the central nervous system, where high expression of apoEr2 has been described originally. By means of splice variant-specific in situ hybridization, we could confirm that apoEr2 mRNA is abundantly expressed in brain tissue and, with exception of the newly identified ligand binding domain, all mRNA splice variants exhibited a similar expression pattern. The mRNA of the newly identified ligand binding domain, however, was expressed in brain only in cells of the vascular wall, confirming data from Northern blotting, where the mRNA of the newly identified ligand binding domain was found in several tissues but was absent in brain tissue.

Alternative Splicing↗

PubMed2001

Neuroblastoma cells provoke Schwann cell proliferation in vitro.

BACKGROUND: A subset of human neuroblastomas (NBs) has the capacity to mature completely, imitating sympathetic ganglia. Previously, we showed that the neuronal population in spontaneously maturing NBs usually has a near-triploid DNA content without 1p deletions, and we concluded that the constantly diploid Schwann cells (SCs) do not belong to the neoplastic component of these tumours. We therefore hypothesised that NB cells are able to stimulate SC proliferation, and that SCs trigger NB differentiation. PROCEDURE: We performed in vitro experiments to test this model and to test whether SCs can also influence the growth of aggressive NBs. Human SCs were co-cultivated with NB tumours and cell lines, and were harvested after defined time intervals. Proliferative activity of the SCs and the NB cells was determined by visualisation of 5-bromo-2'-deoxyuridine (BrdU) incorporation or Ki-67 staining. Neurite outgrowth and neurofilament (NF) expression were analysed immunocytochemically and apoptotic rate was determined by a terminal deoxynucleotidyl transferase-mediated dUTP-X fluorescein nick end labelling (TUNEL) assay. RESULTS: Human NB tumours or cell lines unequivocally increased the proliferation of SCs in vitro. In cocultivated NB cells, the proliferative activity was not altered in the first days of cocultivation, although neurite outgrowth and NF expression were enhanced. However, after 10 days, the mitotic rate of neuroblastic cells decreased and the apoptotic rate showed a marked increase. CONCLUSIONS: The results of the cocultivation experiments provide an experimental hint that the in vivo growth of SCs in NBs is caused by the neoplastic neuroblasts, and they also indicate that cells from peripheral nerves can influence the growth of aggressive NB cells if cocultivated.

Antigens, Neoplasm↗

PubMed2001

Intratumoural heterogeneity of 1p deletions and MYCN amplification in neuroblastomas.

BACKGROUND: At least three genetic hallmarks identify aggressive tumour behaviour in neuroblastomas; amplification of the oncogene MYCN; deletion (loss of heterozygosity [LOH]) at the short arm of chromosome 1 (del1p36), seen in approximately 28% of the cases; and di-tetraploidy. The MYCN oncogene is amplified in approximately 23% of all neuroblastomas and becomes important for the stratification of therapy in localised and 4s tumours. Up to now, it has been believed that the genetic constellation of neuroblastic tumours is stable and does not alter during tumour evolution or during tumour progression. PROCEDURE: Using fluorescence in situ hybridisation techniques (FISH) to investigate different tumour areas on touch preparations and histological sections, we show that genetic heterogeneity can be detected in neuroblastomas, especially in tumours detected by urinary mass screening. CONCLUSION: The identification of such cell clones is important, because the MYCN amplification and/or the deletion at 1p36 appear to be responsible for aggressive local growth and development of metastases.

Chromosomes, Human, Pair 1↗

PubMed2001

Automatic detection and genetic profiling of disseminated neuroblastoma cells.

BACKGROUND: Rare tumor cells circulating in the hematopoietic system can escape identification. On the other hand, the nature of these cells, positive for an immunologiCal tumor marker, cannot be determined without any genetic information. PROCEDURE: To overcome these problems a novel computer assisted scanning system for automatic cell search, analysis, and sequential repositioning was developed. This system allows an exact quantitative analysis of rare tumor cells in the bone marrow and peripheral blood by sequential immunological and molecular cytogenetic characterization. RESULTS AND CONCLUSIONS: In that virtually all tumor cells in a mixing experiment could be recovered unambiguously, we can conclude that the sensitivity of this approach is set by the number of cells available for analysis. Sequential FISH analyses of immunologically positive cells improve both the specificity and the sensitivity of the microscopic minimal residual disease detection.

Biomarkers, Tumor↗

PubMed2001

Absence of somatostatin receptor expression in vivo is correlated to di- or tetraploid 1p36-deleted neuroblastomas.

BACKGROUND: Poor prognosis in childhood neuroblastoma is associated with deletions of chromosome region 1p36 and di/tetraploid DNA content. PROCEDURE: Forty-six patients with histopathologically proven neuroblastoma were investigated for in vivo expression of somatostatin receptors (SR) by 111In-pentetreotide scintigraphy. All tumors were analyzed for cytometric DNA content and chromosome 1p36 integrity. RESULTS: SR expression was detected in 28 tumors (61%) and correlated with young age, localized clinical stage, and favorable outcome. Fourteen tumors showed deletion at chromosome 1p36, thirteen of which did not show SR expression (P< 0.001). A triploid DNA content was correlated with the presence of SR (23 of 25, P< 0.001). No tumor with deletion of chromosome 1p36 and di/tetra DNA content showed SR expression (chi2 = 29.88, d.o.f. = 2, P < 0.001). CONCLUSIONS: We conclude that SR expression is related to genetic features of prognostic significance. This may be assessed with a minimally invasive scintigraphic method.

Adolescent↗

PubMed2001

Fine mapping of a tumour suppressor candidate gene region in 1p36.2-3, commonly deleted in neuroblastomas and germ cell tumours.

BACKGROUND: A common genetic feature of neuroblastomas, which is also an important prognostic factor, is deletion of chromosome region 1p. The deletion of 1p often involves a deletion of varying size, with a consensus region within the most distal bands 1p36.2-3. The neuroblastoma SRO (shortest region of overlap of (deletions) presented earlier by our group was defined distally by the cluster of loci D1S80/ D1Z2/CDC2L1 and proximally by loci D1S244, i.e., approximately 25 cM. The 1p deletions are, however, not restricted to neuroblastoma tumours. In fact, a large spectrum of tumour types display deletions to varying degrees of 1p. PROCEDURE: We have exploited the possibility of using deletions of other tumour types, preferentially that of germ cell tumours, and combining the deletions with that of the neuroblastoma SRO. Also in germ cell tumours, distal 1p-deletions have been shown to have prognostic significance. RESULTS: We found in our germ cell tumours a SRO ranging from D1S508 to D1S200. Interestingly, this region only partially overlapped (approximately 5 cm) with our neuroblastoma SRO in region D1S508 to D1S244. We have thus focused on analysing this smaller region in the search for genes involved in the genesis of different cancers. We have performed radiation hybrid mapping of a large number of markers, STSs, ESTs, and others known to reside in 1p. We have also initiated the development of a BAC contig of the region. FISH, and fibre-FISH mapping of BACs were also performed. CONCLUSIONS: The data presented here constitute an ongoing work with the aim of identifying and cloning gene(s) important for development of germ cell tumours, neuroblastomas, and possibly other tumours.

Chromosome Mapping↗

PubMed2001

Prognostic significance of DNA di-tetraploidy in neuroblastoma.

BACKGROUND: Identification of biological factors may provide tools to discriminate poor risk neuroblastoma patients of diagnosis, to ultimately offer risk adapted treatment intensity. PROCEDURES: Tumour cell DNA content, MYCN amplification (NMA), deletion of the short arm of chromosome 1 (del 1p) as well as three serological markers were assessed in 179 children with neuroblastoma. RESULTS: Localised regional disease (stage 1 to 3) was diagnosed in 98 patients, and disseminated disease in 81 patients (65 with stage 4, 16 with stage 4s). Median age at diagnosis was 12 months and the median observation time 4 years. Sixty-seven of 179 patients had near di-tetraploid tumours (37%), with a significantly worse prognosis of 44% overall survival at 4 years in comparison with 88% in near triploid tumours (P < .001). The near di-tetraploid group showed a significant correlation with additional adverse biological factors (NMA, del 1p: P < 0.001), age over 1 year (P< 0.001), clinical stage 4 (P< 0.001), elevated ferritin (P = 0.023), and elevated LDH (P< 0.001). Multivariate analysis based on the overall (OS) and event free survival (EFS) estimations revealed that near di-tetraploidy was the most powerful biological factor, with a P-value of <0.001 for EFS and OS, followed by NMA (P = 0.015) for OS and del 1p (P= 0.047) for EFS. CONCLUSIONS: This analysis underlines the important influence of near di-tetraploidy on prognosis, and suggests that more efforts should be undertaken to implement this factor in future studies.

Adolescent↗

PubMed2001

Pathology and biology guidelines for resectable and unresectable neuroblastic tumors and bone marrow examination guidelines.

The recommendations concerning tumor and bone marrow handling for the evaluation of molecular-biologic and molecular-genetic and immunologic markers presented in this paper were developed by the SIOP Europe Neuroblastoma Pathology and Biology and Bone Marrow Group. Although the Guidelines were developed for neuroblastic tumors (neuroblastoma, ganglioneuroblastoma and ganglioneuroma), they are applicable to all other tumor entities as well. The paper is subdivided in three main parts. The Pathology Guidelines give an overview about the handling, sectioning and securing of tumor material in case of resectable and non-resectable neuroblastic tumors. The Guidelines encompass open biopsies, tru cut biopsies, fine needle aspirations, and bone marrow aspiration. The importance of the pathologic evaluation for the interpretation of the molecular-genetic and molecular-biologic results, which also includes the exact determination of the tumor cell content is stressed. Besides this, recommendations concerning tumor material obtained after cytotoxic therapy, immunohistologic and immuno-cytologic issues and lymph node examination are addressed. In the Biology Guidelines, the different methods for MYCN, chromosome 1p36 investigations and DNA content measurements are discussed and DNA probes are recommended. Furthermore, specified definitions and a common terminology already used in the SIOP Europe Neuroblastoma Group are presented. In the Bone Marrow Guidelines, recommendations concerning the methods to be employed are given and the most important pitfalls are demonstrated. Both the use of standardized methods and the application of a common language will, it is hoped, contribute to the quality and reliability of collected data and thus to a better comparability between and among research reports. These improvements should prove to be of great value for the affected patients.

Blotting, Southern↗

PubMed2001

Characterisation of pericentrometric and sticky intercalary heterochromatin in Ornithogalum longibracteatum (Hyacinthaceae).

The hexaploid liliaceous plant Ornithogalum longibracteatum (2n=6x=54) has a heterochromatin-rich bimodal karyotype with large (L) and small (S) chromosomes. The composition and subgenomic distribution of heterochromatin was studied using molecular and cytological methods. The major component of centromeric heterochromatin in all chromosomes is Satl, an abundant satellite DNA with a basic repeat unit of 155 bp and an average A+T content (54%). The major component of the large blocks of intercalary heterochromatin in L chromosomes is Sat2, an abundant satellite DNA with a basic repeat unit of 115 bp and a high A+T content (76%). Additionally, traces of Sat2 can be detected at the centromeric regions of S chromosomes, while minor amounts of Satl are discernible in intercalary heterochromatin of L chromosomes. The chromosomal localisation pattern of Sat2 is consistent with the fluorescent staining pattern obtained with the A+T-specific DNA ligand 4'-6-diamidino-2-phenylindole (DAPI). A+T-rich intercalary heterochromatin is sticky and tends to associate ectopically during mitosis. Sister chromatid exchange clustering was found at the junctions between euchromatin and heterochromatin and at the centromeres. The pattern of mitosis-specific phosphorylation of histone H3 was not uniform along the length of the chromosomes. In all L and S chromosomes, from early prophase to ana-/telophase, there is hyperphosphorylation of histone H3 in the pericentromeric chromatin and a slightly elevated phosphorylated histone H3 level at the intercalary heterochromatin of L chromosomes. Consequently, the overall phosphorylated histone H3 metaphase labelling resembles the distribution of Satl in the karyotype of O. longibracteatum.

Base Sequence↗