Specific Centromere Research Articles

The haplotype-resolved telomere-to-telomere carnation (Dianthus caryophyllus) genome reveals the correlation between genome architecture and gene expression.

Carnation (Dianthus caryophyllus) is one of the most valuable commercial flowers, due to its richness of color and form, and its excellent storage and vase life. The diverse demands of the market require faster breeding in carnations. A full understanding of carnations is therefore required to guide the direction of breeding. Hence, we assembled the haplotype-resolved gap-free carnation genome of the variety 'Baltico', which is the most common white standard variety worldwide. Based on high-depth HiFi, ultra-long nanopore, and Hi-C sequencing data, we assembled the telomere-to-telomere (T2T) genome to be 564 479 117 and 568 266 215bp for the two haplotypes Hap1 and Hap2, respectively. This T2T genome exhibited great improvement in genome assembly and annotation results compared with the former version. The improvements were seen when different approaches to evaluation were used. Our T2T genome first informs the analysis of the telomere and centromere region, enabling us to speculate about specific centromere characteristics that cannot be identified by high-order repeats in carnations. We analyzed allele-specific expression in three tissues and the relationship between genome architecture and gene expression in the haplotypes. This demonstrated that the length of the genes, coding sequences, and introns, the exon numbers and the transposable element insertions correlate with gene expression ratios and levels. The insertions of transposable elements repress expression in gene regulatory networks in carnation. This gap-free finished T2T carnation genome provides a valuable resource to illustrate the genome characteristics and for functional genomics analysis in further studies and molecular breeding.

Atypical low-copy number plasmid segregation systems, all in one?

Plasmid families harbor different maintenances functions, depending on their size and copy number. Low copy number plasmids rely on active partition systems, organizing a partition complex at specific centromere sites that is actively positioned using NTPase proteins. Some low copy number plasmids lack an active partition system, but carry atypical intracellular positioning systems using a single protein that binds to the centromere site but without an associated NTPase. These systems have been studied in the case of the Escherichia coli R388 and of the Staphylococcus aureus pSK1 plasmids. Here we review these two systems, which appear to be unrelated but share common features, such as their distribution on plasmids of medium size and copy number, certain activities of their centromere-binding proteins, StbA and Par, respectively, as well as their mode of action, which may involve dynamic interactions with the nucleoid-packed chromosome of their hosts.

Epigenetic rewriting at centromeric DNA repeats leads to increased chromatin accessibility and chromosomal instability

BackgroundCentromeric regions of human chromosomes contain large numbers of tandemly repeated α-satellite sequences. These sequences are covered with constitutive heterochromatin which is enriched in trimethylation of histone H3 on lysine 9 (H3K9me3). Although well studied using artificial chromosomes and global perturbations, the contribution of this epigenetic mark to chromatin structure and genome stability remains poorly known in a more natural context.ResultsUsing transcriptional activator-like effectors (TALEs) fused to a histone lysine demethylase (KDM4B), we were able to reduce the level of H3K9me3 on the α-satellites repeats of human chromosome 7. We show that the removal of H3K9me3 affects chromatin structure by increasing the accessibility of DNA repeats to the TALE protein. Tethering TALE-demethylase to centromeric repeats impairs the recruitment of HP1α and proteins of Chromosomal Passenger Complex (CPC) on this specific centromere without affecting CENP-A loading. Finally, the epigenetic re-writing by the TALE-KDM4B affects specifically the stability of chromosome 7 upon mitosis, highlighting the importance of H3K9me3 in centromere integrity and chromosome stability, mediated by the recruitment of HP1α and the CPC.ConclusionOur cellular model allows to demonstrate the direct role of pericentromeric H3K9me3 epigenetic mark on centromere integrity and function in a natural context and opens interesting possibilities for further studies regarding the role of the H3K9me3 mark.

PLK1 facilitates chromosome biorientation by suppressing centromere disintegration driven by BLM-mediated unwinding and spindle pulling

Centromeres provide a pivotal function for faithful chromosome segregation. They serve as a foundation for the assembly of the kinetochore complex and spindle connection, which is essential for chromosome biorientation. Cells lacking Polo-like kinase 1 (PLK1) activity suffer severe chromosome alignment defects, which is believed primarily due to unstable kinetochore-microtubule attachment. Here, we reveal a previously undescribed mechanism named ‘centromere disintegration’ that drives chromosome misalignment in PLK1-inactivated cells. We find that PLK1 inhibition does not necessarily compromise metaphase establishment, but instead its maintenance. We demonstrate that this is caused by unlawful unwinding of DNA by BLM helicase at a specific centromere domain underneath kinetochores. Under bipolar spindle pulling, the distorted centromeres are promptly decompacted into DNA threadlike molecules, leading to centromere rupture and whole-chromosome arm splitting. Consequently, chromosome alignment collapses. Our study unveils an unexpected role of PLK1 as a chromosome guardian to maintain centromere integrity for chromosome biorientation.

Movember GAP1 PDX project: An international collection of serially transplantable prostate cancer patient-derived xenograft (PDX) models.

While it has been challenging to establish prostate cancer patient-derived xenografts (PDXs), with a take rate of 10-40% and long latency time, multiple groups throughout the world have developed methods for the successful establishment of serially transplantable human prostate cancer PDXs using a variety of immune deficient mice. In 2014, the Movember Foundation launched a Global Action Plan 1 (GAP1) project to support an international collaborative prostate cancer PDX program involving eleven groups. Between these Movember consortium members, a total of 98 authenticated human prostate cancer PDXs were available for characterization. Eighty three of these were derived directly from patient material, and 15 were derived as variants of patient-derived material via serial passage in androgen deprived hosts. A major goal of the Movember GAP1 PDX project was to provide the prostate cancer research community with a summary of both the basic characteristics of the 98 available authenticated serially transplantable human prostate cancer PDX models and the appropriate contact information for collaborations. Herein, we report a summary of these PDX models. PDX models were established in immunocompromised mice via subcutaneous or subrenal-capsule implantation. Dual-label species (ie, human vs mouse) specific centromere and telomere Fluorescence In Situ Hybridization (FISH) and immuno-histochemical (IHC) staining of tissue microarrays (TMAs) containing replicates of the PDX models were used for characterization of expression of a number of phenotypic markers important for prostate cancer including AR (assessed by IHC and FISH), Ki67, vimentin, RB1, P-Akt, chromogranin A (CgA), p53, ERG, PTEN, PSMA, and epithelial cytokeratins. Within this series of PDX models, the full spectrum of clinical disease stages is represented, including androgen-sensitive and castration-resistant primary and metastatic prostate adenocarcinomas as well as prostate carcinomas with neuroendocrine differentiation. The annotated clinical characteristics of these PDXs were correlated with their marker expression profile. Our results demonstrate the clinical relevance of this series of PDXs as a platform for both basic science studies and therapeutic discovery/drug development. The present report provides the prostate cancer community with a summary of the basic characteristics and a contact information for collaborations using these models.

Non-random Mis-segregation of Human Chromosomes

SummaryA common assumption is that human chromosomes carry equal chances of mis-segregation during compromised cell division. Human chromosomes vary in multiple parameters that might generate bias, but technological limitations have precluded a comprehensive analysis of chromosome-specific aneuploidy. Here, by imaging specific centromeres coupled with high-throughput single-cell analysis as well as single-cell sequencing, we show that aneuploidy occurs non-randomly following common treatments to elevate chromosome mis-segregation. Temporary spindle disruption leads to elevated mis-segregation and aneuploidy of a subset of chromosomes, particularly affecting chromosomes 1 and 2. Unexpectedly, we find that a period of mitotic delay weakens centromeric cohesion and promotes chromosome mis-segregation and that chromosomes 1 and 2 are particularly prone to suffer cohesion fatigue. Our findings demonstrate that inherent properties of individual chromosomes can bias chromosome mis-segregation and aneuploidy rates, with implications for studies on aneuploidy in human disease.

Structure and Distribution of Centromeric Retrotransposons at Diploid and Allotetraploid Coffea Centromeric and Pericentromeric Regions.

Centromeric regions of plants are generally composed of large array of satellites from a specific lineage of Gypsy LTR-retrotransposons, called Centromeric Retrotransposons. Repeated sequences interact with a specific H3 histone, playing a crucial function on kinetochore formation. To study the structure and composition of centromeric regions in the genus Coffea, we annotated and classified Centromeric Retrotransposons sequences from the allotetraploid C. arabica genome and its two diploid ancestors: Coffea canephora and C. eugenioides. Ten distinct CRC (Centromeric Retrotransposons in Coffea) families were found. The sequence mapping and FISH experiments of CRC Reverse Transcriptase domains in C. canephora, C. eugenioides, and C. arabica clearly indicate a strong and specific targeting mainly onto proximal chromosome regions, which can be associated also with heterochromatin. PacBio genome sequence analyses of putative centromeric regions on C. arabica and C. canephora chromosomes showed an exceptional density of one family of CRC elements, and the complete absence of satellite arrays, contrasting with usual structure of plant centromeres. Altogether, our data suggest a specific centromere organization in Coffea, contrasting with other plant genomes.

Rapid molecular assays to study human centromere genomics.

The centromere is the structural unit responsible for the faithful segregation of chromosomes. Although regulation of centromeric function by epigenetic factors has been well-studied, the contributions of the underlying DNA sequences have been much less well defined, and existing methodologies for studying centromere genomics in biology are laborious. We have identified specific markers in the centromere of 23 of the 24 human chromosomes that allow for rapid PCR assays capable of capturing the genomic landscape of human centromeres at a given time. Use of this genetic strategy can also delineate which specific centromere arrays in each chromosome drive the recruitment of epigenetic modulators. We further show that, surprisingly, loss and rearrangement of DNA in centromere 21 is associated with trisomy 21. This new approach can thus be used to rapidly take a snapshot of the genetics and epigenetics of each specific human centromere in nondisjunction disorders and other biological settings.

Unfavorable Prognostic Impact of MYC Increased Copy Number (ICN) in Patients with Diffuse Large B-Cell and High-Grade Lymphoma Treated with Immuno-Chemotherapy (ICT)

Background: MYC rearrangements occur approximately in 5-15% of diffuse large B-cell lymphoma (DLBCL), and are known to unfavorably impact on patients (pts) survival, especially if associated to BCL2 or/and BCL6 rearrangements (double/triple-hit). Previous studies have shown that also a MYC gene ICN may confer a worse prognosis to these pts (Yoon, Histopathology 2008), but only few groups have transferred this observation into the clinical practice. However, this finding may account for those cases that, despite the absence of MYC rearrangements, show a similar aggressive clinical course. For this reason, a careful study of all aberrations involving MYC geneshould be widely applied. We report a single center experience on the clinical outcome of pts with MYC ICN in the setting of aggressive B-cell lymphoma.Methods: In the study period (Aug 2011-Aug 2015) we performed FISH analysis in all consecutive pts with de novo or transformed DLBCL or B-cell lymphoma, unclassifiable (BCLU) that displayed Ki67 >80% or intense bcl2 protein expression. Interphase FISH has been performed on 23 μm thick sections of formalin fixed paraffin embedded tissues, using splitsignal DNA probes (Dako) specific for the following loci: 8q24 (MYC), 18q21 (BCL2) and 3q27 (BCL6). For each sample, 60 evaluable nuclei with complete FISH signals were scored. ICN was considered when 3 or more copies of the gene studied were identified.Almost all pts with a MYC translocation have been treated according to Burkitt lymphoma (BL)-like regimens as first line therapy (FLT); pts with MYC ICN have been treated with BL-like regimens from 2013, whereas standard ICT (R-CHOP or R-CHOPlike) has been used for these pts diagnosed before 2013. Overall survival (OS) was evaluated by Kaplan Meier method.Results: In the 4-year study period, 317 consecutive pts were diagnosed with de novo or transformed DLBCL or BCLU. Twenty-one (7%) showed a translocation of MYC, in single (14%), double (67%) or triple hit (19%), with respect to BCL2 and BCL6 rearrangements. Interestingly, 8 (2.5%) pts with no MYC translocation showed MYC ICN, ranging from 3 up to 10 gene copies per cell. This aberrant gene copy number was observed in > 80% of the analyzed nuclei in 7 cases. The characteristics of the pts with MYC gene rearrangement or ICN are summarized in Table 1.Of the 8 pts with MYC ICN, 5 had de novo DLBCL, 2 had transformed DLBCL and 1 had BCLU. Seven pts also showed BCL2 and/or BCL6 rearrangement/ICN. Three pts were treated with standard ICT and 5 with a BLlike regimen. Two pts (25%) responded to the FLT, showing one complete and one partial remission (CR and PR, respectively); both received BLlike regimen. The patient with PR progressed after 7 months, and received a second line treatment likewise the 6 nonresponders to FLT. None of them had a response and eventually died, with a median OS of 10.5 months. Cause of death was lymphoma in all cases. The patient who obtained a CR was still in remission at 29 months from the end of the FLT. The survival of these pts, compared to pts with MYC rearrangement, is shown in Figure 1 (respectively 12% vs 57% at 2 years, p 0.04).Conclusion: These preliminary data show a dismal prognosis of pts with aggressive B cell lymphoma and MYC ICN, significantly worse than pts harboring a MYC translocation, independently of the treatment received. To better understand the genetic defect underlying this FISH pattern, further analysis using a specific centromere probe for chromosome 8 is ongoing on these and new cases. A previous study distinguished gain from amplification of MYC based on the number of gene copies, identifying 2 groups with different prognosis (Valera, Hematologica 2013). In our hands, there was a wider range of gene copy number in each patient, and the outcome was homogeneously poor. Overall, the biological consequence of this alteration is still to be fully explained, and only a few studies have addressed this point so far (Stasik, Hematologica 2010). Further investigation on larger cohort groups is necessary to confirm the unfavorable prognostic role of MYC ICN in aggressive B cell lymphoma. Such insights would help practitioners to determine more accurate therapeutic approaches, in order to improve the outcome of this specific subset of pts. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Regulatory Effect of INMAP on Centromere Proteins: Antisense INMAP Induces CENP-B Variation and Centromeric Halo

CENP-B is a highly conserved protein that facilitates the assembly of specific centromere structures both in interphase nuclei and on mitotic chromosomes. INMAP is a conserved protein that localizes at nucleus in interphase cells and at mitotic apparatus in mitotic cells. Our previous results showed that INMAP over-expression leads to spindle defects, mitotic arrest and formation of polycentrosomal and multinuclear cells, indicating that INMAP may modulate the function of (a) key protein(s) in mitotic apparatus. In this study, we demonstrate that INMAP interacts with CENP-B and promotes cleavage of the N-terminal DNA binding domain from CENP-B. The cleaved CENP-B cannot associate with centromeres and thus lose its centromere-related functions. Consistent with these results, CENP-B in INMAP knockdown cells becomes more diffused around kinetochores. Although INMAP knockdown cells do not exhibit gross defects in mitotic spindle formation, these cells go through mitosis, especially prophase and metaphase, with different relative timing, indicating subtle abnormality. These results identify INMAP as a model regulator of CENP-B and support the notion that INMAP regulates mitosis through modulating CENP-B-mediated centromere organization.

Induction of a whole chromosome loss by colcemid in human cells elucidated by discrimination between FISH signal overlap and chromosome loss

Aneuploidy is a change in the number of chromosomes and an essential component in tumorigenesis. Therefore, accurate and sensitive detection of aneuploidy is important in screening for carcinogens. In vitro micronucleus (MN) assay has been adopted in the recently revised International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) S2 guideline and can be employed to predict both clastogenic and aneugenic chromosomal aberrations in interphase cells. However, distinguishing clastogens and aneugens is not possible using this assay. The Organization for Economic Co-operation and Development (OECD) guideline TG487 therefore recommends the use of centromere/kinetochore staining in micronuclei to differentiate clastogens from aneugens. Here, we analyzed numerical changes of a specific chromosome in cytokinesis-blocked binucleated cells by fluorescence in situ hybridization (FISH) using the specific centromere probe in human lymphoblastoid TK6 cells treated with aneugens (colcemid and vincristine) or clastogens (methyl methanesulfonate [MMS] and 4-nitroquinoline-1-oxide [4-NQO]). Colcemid and vincristine significantly increased the frequencies of nondisjunction and loss of FISH signals, while MMS and 4-NQO slightly increased only the frequency of loss of FISH signals. The loss of FISH signals of a specific chromosome from two to one per nucleus implies either a loss of a whole chromosome or an overlap of two signals. To distinguish a chromosome loss from signal overlap, we investigated the number of FISH signals and the fluorescent intensity of each signal per nucleus using a probe specific for whole chromosome 2 in binucleated TK6 cells and primary human lymphocytes treated with colcemid and MMS. By discriminating between chromosome loss and FISH signal overlap, we revealed that colcemid, but not MMS, induced a loss of a whole chromosome in primary lymphocytes and TK6 cells.

The CENP-A nucleosome: a dynamic structure and role at the centromere

The centromere is a specialized locus that directs the formation of the kinetochore protein complex for correct chromosome segregation. The specific centromere histone H3 variant CENP-A has been described as the epigenetic mark of this chromatin region. Several laboratories have explored its properties, its partners, and its role in centromere formation. Specifically, two types of CENP-A nucleosomes have been described, suggesting there may be more complexity involved in centromere structure than previously thought. Recent work adds to this paradox by questioning the role of CENP-A as a unique centromeric mark and highlighting the assembly of a functional kinetochore in the absence of CENP-A. In this review, we discuss recent literature on the CENP-A nucleosomes and the debate on its role in kinetochore formation and centromere identity.

A Coordinated Interdependent Protein Circuitry Stabilizes the Kinetochore Ensemble to Protect CENP-A in the Human Pathogenic Yeast Candida albicans

Unlike most eukaryotes, a kinetochore is fully assembled early in the cell cycle in budding yeasts Saccharomyces cerevisiae and Candida albicans. These kinetochores are clustered together throughout the cell cycle. Kinetochore assembly on point centromeres of S. cerevisiae is considered to be a step-wise process that initiates with binding of inner kinetochore proteins on specific centromere DNA sequence motifs. In contrast, kinetochore formation in C. albicans, that carries regional centromeres of 3–5 kb long, has been shown to be a sequence independent but an epigenetically regulated event. In this study, we investigated the process of kinetochore assembly/disassembly in C. albicans. Localization dependence of various kinetochore proteins studied by confocal microscopy and chromatin immunoprecipitation (ChIP) assays revealed that assembly of a kinetochore is a highly coordinated and interdependent event. Partial depletion of an essential kinetochore protein affects integrity of the kinetochore cluster. Further protein depletion results in complete collapse of the kinetochore architecture. In addition, GFP-tagged kinetochore proteins confirmed similar time-dependent disintegration upon gradual depletion of an outer kinetochore protein (Dam1). The loss of integrity of a kinetochore formed on centromeric chromatin was demonstrated by reduced binding of CENP-A and CENP-C at the centromeres. Most strikingly, Western blot analysis revealed that gradual depletion of any of these essential kinetochore proteins results in concomitant reduction in cellular protein levels of CENP-A. We further demonstrated that centromere bound CENP-A is protected from the proteosomal mediated degradation. Based on these results, we propose that a coordinated interdependent circuitry of several evolutionarily conserved essential kinetochore proteins ensures integrity of a kinetochore formed on the foundation of CENP-A containing centromeric chromatin.

Mitotic Defects Lead to Pervasive Aneuploidy and Accompany Loss of RB1 Activity in Mouse LmnaDhe Dermal Fibroblasts

BackgroundLamin A (LMNA) is a component of the nuclear lamina and is mutated in several human diseases, including Emery-Dreifuss muscular dystrophy (EDMD; OMIM ID# 181350) and the premature aging syndrome Hutchinson-Gilford progeria syndrome (HGPS; OMIM ID# 176670). Cells from progeria patients exhibit cell cycle defects in both interphase and mitosis. Mouse models with loss of LMNA function have reduced Retinoblastoma protein (RB1) activity, leading to aberrant cell cycle control in interphase, but how mitosis is affected by LMNA is not well understood.ResultsWe examined the cell cycle and structural phenotypes of cells from mice with the Lmna allele, Disheveled hair and ears (LmnaDhe). We found that dermal fibroblasts from heterozygous LmnaDhe (LmnaDhe/+) mice exhibit many phenotypes of human laminopathy cells. These include severe perturbations to the nuclear shape and lamina, increased DNA damage, and slow growth rates due to mitotic delay. Interestingly, LmnaDhe/+ fibroblasts also had reduced levels of hypophosphorylated RB1 and the non-SMC condensin II-subunit D3 (NCAP-D3), a mitosis specific centromere condensin subunit that depends on RB1 activity. Mitotic check point control by mitotic arrest deficient-like 1 (MAD2L1) also was perturbed in LmnaDhe /+ cells. LmnaDhe /+ fibroblasts were consistently aneuploid and had higher levels of micronuclei and anaphase bridges than normal fibroblasts, consistent with chromosome segregation defects.ConclusionsThese data indicate that RB1 may be a key regulator of cellular phenotype in laminopathy-related cells, and suggest that the effects of LMNA on RB1 include both interphase and mitotic cell cycle control.

Cytogenetic studies in seven individuals with an i(Xq) karyotype

Seven individuals with stigmata of the Turner syndrome, large Barr bodies and a late replicating, structurally abnormal X-chromosome, have been reinvestigated with different banding techniques to elucidate the mechanisms leading to formation of isochromosomes. Dicentric chromosomes With separated C-blocks were found in five of these individuals. The two remaining patients had a slightly larger and a nearly double sized block of centromeric heterochromatin. Using the centromeric model proposed by Lima-de-Faria in 1956, the size of these blocks can be related to different locations of break points within the centromere or in the short arm. The presence of a 45, X cell line in all cases is probably due to anaphase lag secondary to instability of an abnormal isochromosome centromere. The mechanisms behind centromere inactivation in dicentrics are still obscure and not likely to be disclosed before the ultrastructure of these abnormal centromeres is known in more detail, The necessity of a more specific centromere nomenclature is emphasized. Additional rare or unexpected findings were normal menstruation in one individual, bipartite Barr bodies in two cases and one pericentric inversion of chromosome 9.

A tale of two centromeres—diversity of structure but conservation of function in plants and animals

The structural and functional aspects of two specific centromeres, one drawn from the animal kingdom (Drosophila) and the other from the plant kingdom (maize), are compared. Both cases illustrate an epigenetic component to centromere specification. The observations of neocentromeres in Drosophila and inactive centromeres in maize constitute one line of evidence for this hypothesis. Another common feature is the divisibility of centromere function with reduced stability as the size decreases. The systems differ in that Drosophila has no common sequence repeat at all centromeres, whereas maize has a 150-bp unit present in tandem arrays together with a centromere-specific transposon, centromere retrotransposon maize, present at all primary constrictions. Aspects of centromere structure known only from one or the other system might be common to both, namely, the presence of centromere RNAs in the kinetochore as found in maize and the organization of the centromeric histone 3 in tetrameric nucleosomes.

Dynamics of inner kinetochore assembly and maintenance in living cells

To investigate the dynamics of centromere organization, we have assessed the exchange rates of inner centromere proteins (CENPs) by quantitative microscopy throughout the cell cycle in human cells. CENP-A and CENP-I are stable centromere components that are incorporated into centromeres via a “loading-only” mechanism in G1 and S phase, respectively. A subfraction of CENP-H also stays stably bound to centromeres. In contrast, CENP-B, CENP-C, and some CENP-H and hMis12 exhibit distinct and cell cycle–specific centromere binding stabilities, with residence times ranging from seconds to hours. CENP-C and CENP-H are immobilized at centromeres specifically during replication. In mitosis, all inner CENPs become completely immobilized. CENPs are highly mobile throughout bulk chromatin, which is consistent with a binding-diffusion behavior as the mechanism to scan for vacant high-affinity binding sites at centromeres. Our data reveal a wide range of cell cycle–specific assembly plasticity of the centromere that provides both stability through sustained binding of some components and flexibility through dynamic exchange of other components.

Probing plasmid partition with centromere‐based incompatibility

Low-copy number plasmids of bacteria rely on specific centromeres for regular partition into daughter cells. When also present on a second plasmid, the centromere can render the two plasmids incompatible, disrupting partition and causing plasmid loss. We have investigated the basis of incompatibility exerted by the F plasmid centromere, sopC, to probe the mechanism of partition. Measurements of the effects of sopC at various gene dosages on destabilization of mini-F, on repression of the sopAB operon and on occupancy of mini-F DNA by the centromere-binding protein, SopB, revealed that among mechanisms previously proposed, no single one fully explained incompatibility. sopC on multicopy plasmids depleted SopB by titration and by contributing to repression. The resulting SopB deficit is proposed to delay partition complex formation and facilitate pairing between mini-F and the centromere vector, thereby increasing randomization of segregation. Unexpectedly, sopC on mini-P1 exerted strong incompatibility if the P1 parABS locus was absent. A mutation preventing the P1 replication initiation protein from pairing (handcuffing) reduced this strong incompatibility to the level expected for random segregation. The results indicate the importance of kinetic considerations and suggest that mini-F handcuffing promotes pairing of SopB-sopC complexes that can subsequently segregate as intact aggregates.

Maize centromeres: organization and functional adaptation in the genetic background of oat.

Centromeric DNA sequences in multicellular eukaryotes are often highly repetitive and are not unique to a specific centromere or to centromeres at all. Thus, it is a major challenge to study the fine structure of individual plant centromeres. We used a DNA fiber-fluorescence in situ hybridization approach to study individual maize (Zea mays) centromeres using oat (Avena sativa)-maize chromosome addition lines. The maize centromere-specific satellite repeat CentC in the addition lines allowed us to delineate the size and organization of centromeric DNA of individual maize chromosomes. We demonstrate that the cores of maize centromeres contain mainly CentC arrays and clusters of a centromere-specific retrotransposon, CRM. CentC and CRM sequences are highly intermingled. The amount of CentC/CRM sequence varies from approximately 300 to >2800 kb among different centromeres. The association of CentC and CRM with centromeric histone H3 (CENH3) was visualized by a sequential detection procedure on stretched centromeres. The analysis revealed that CENH3 is always associated with CentC and CRM but that not all CentC or CRM sequences are associated with CENH3. We further demonstrate that in the chromosomal addition lines in which two CenH3 genes were present, one from oat and one from maize, the oat CENH3 was consistently incorporated by the maize centromeres.

Detection of trisomy 18 on formalin-fixed and paraffin-embedded material by fluorescence in situ hybridization.

Formalin-fixed and paraffin-embedded autopsy material from 10 fetuses and infants with unknown karyotype and anomalies suggestive of trisomy 18 were subjected to fluorescence in situ hybridization (FISH). Nuclei were extracted from the tissues and hybridized with a chromosome 18-specific centromere probe. The hybridization was successful in 9 of 10 cases. Two cases showed three hybridization signals in most of the nuclei (74% and 85%). These had anomalies frequently occurring with trisomy 18 (congenital heart defect, omphalocele, and horseshoe kidney). Two cases showed a mixture of two and three signals (47%/49% and 59%/36%), suggesting the possibility of mosaicism. One of these cases had anomalies consistent with a trisomy 18 phenotype. In the other case intrauterine growth retardation and syndactylies suggested triploidy. Hybridization with a chromosome 8-specific probe gave a distribution of two and three signals (34% and 62%, respectively). This result strengthened the suspicion of a possible triploid mosaicism. In five of the cases most of the nuclei showed two signals (85% to 88%). However, as only one type of tissue was examined for enumeration of chromosome 18, the possibility of organ mosaicism or other chromosome aberrations cannot be excluded. The FISH technique is applicable on macerated and autolysed formalin-fixed tissue, making it possible to retrospectively analyze autopsy material from aborted and stillborn fetuses and infants. This analysis contributes to a better quality of perinatal autopsies and is helpful in parental counseling.