Abstract: Genome sequencing projects generate a wealth of information; however, the ultimate goal of such projects is to accelerate the identification of the biological function of genes. This creates a need for comprehensive studies to fill the gap between sequence and function. Here we report the results of a functional genomic screen to identify genes required for cell division in Caenorhabditis elegans. We inhibited the expression of approximately 96% of the approximately 2,300 predicted open reading frames on chromosome III using RNA-mediated interference (RNAi). By using an in vivo time-lapse differential interference contrast microscopy assay, we identified 133 genes (approximately 6%) necessary for distinct cellular processes in early embryos. Our results indicate that these genes represent most of the genes on chromosome III that are required for proper cell division in C. elegans embryos. The complete data set, including sample time-lapse recordings, has been deposited in an open access database. We found that approximately 47% of the genes associated with a differential interference contrast phenotype have clear orthologues in other eukaryotes, indicating that this screen provides putative gene functions for other species as well.

Abstract: To help understand mechanisms of vertebrate genome evolution, we have compared zebrafish and tetrapod gene maps. It has been suggested that translocations are fixed more frequently than inversions in mammals. Gene maps showed that blocks of conserved syntenies between zebrafish and humans were large, but gene orders were frequently inverted and transposed. This shows that intrachromosomal rearrangements have been fixed more frequently than translocations. Duplicated chromosome segments suggest that a genome duplication occurred in ray-fin phylogeny, and comparative studies suggest that this event happened deep in the ancestry of teleost fish. Consideration of duplicate chromosome segments shows that at least 20% of duplicated gene pairs may be retained from this event. Despite genome duplication, zebrafish and humans have about the same number of chromosomes, and zebrafish chromosomes are mosaically orthologous to several human chromosomes. Is this because of an excess of chromosome fissions in the human lineage or an excess of chromosome fusions in the zebrafish lineage? Comparative analysis suggests that an excess of chromosome fissions in the tetrapod lineage may account for chromosome numbers and provides histories for several human chromosomes.

Abstract: Allopolyploid hybridization serves as a major pathway for plant evolution, but in its early stages it is associated with phenotypic and genomic instabilities that are poorly understood. We have investigated allopolyploidization between Arabidopsis thaliana (2 n = 2 x = 10; n , gametic chromosome number; x , haploid chromosome number) and Cardaminopsis arenosa (2 n = 4 x = 32). The variable phenotype of the allotetraploids could not be explained by cytological abnormalities. However, we found suppression of 20 of the 700 genes examined by amplified fragment length polymorphism of cDNA. Independent reverse transcription–polymerase chain reaction analyses of 10 of these 20 genes confirmed silencing in three of them, suggesting that ∼0.4% of the genes in the allotetraploids are silenced. These three silenced genes were characterized. One, called K7, is repeated and similar to transposons. Another is RAP2.1 , a member of the large APETALA2 (AP2) gene family, and has a repeated element upstream of its 5′ end. The last, L6, is an unknown gene close to ALCOHOL DEHYDROGENASE on chromosome 1. CNG DNA methylation of K7 was less in the allotetraploids than in the parents, and the element varied in copy number. That K7 could be reactivated suggests epigenetic regulation. L6 was methylated in the C. arenosa genome. The present evidence that gene silencing accompanies allopolyploidization opens new avenues to this area of research.

Abstract: Expression profiling using DNA microarrays holds great promise for a variety of research applications, including the systematic characterization of genes discovered by sequencing projects. To demonstrate the general usefulness of this approach, we recently obtained expression profiles for nearly 300 Saccharomyces cerevisiae deletion mutants. Approximately 8% of the mutants profiled exhibited chromosome-wide expression biases, leading to spurious correlations among profiles. Competitive hybridization of genomic DNA from the mutant strains and their isogenic parental wild-type strains showed they were aneuploid for whole chromosomes or chromosomal segments. Expression profile data published by several other laboratories also suggest the use of aneuploid strains. In five separate cases, the extra chromosome harboured a close homologue of the deleted gene; in two cases, a clear growth advantage for cells acquiring the extra chromosome was demonstrated. Our results have implications for interpreting whole-genome expression data, particularly from cells known to suffer genomic instability, such as malignant or immortalized cells.

Abstract: The large-scale chromatin organization of the major histocompatibility complex and other regions of chromosome 6 was studied by three-dimensional image analysis in human cell types with major differences in transcriptional activity. Entire gene clusters were visualized by fluorescence in situ hybridization with multiple locus-specific probes. Individual genomic regions showed distinct configurations in relation to the chromosome 6 terrritory. Large chromatin loops containing several megabases of DNA were observed extending outwards from the surface of the domain defined by the specific chromosome 6 paint. The frequency with which a genomic region was observed on an external chromatin loop was cell type dependent and appeared to be related to the number of active genes in that region. Transcriptional up-regulation of genes in the major histocompatibility complex by interferon-gamma led to an increase in the frequency with which this large gene cluster was found on an external chromatin loop. Our data are consistent with an association between large-scale chromatin organization of specific genomic regions and their transcriptional status.

Abstract: During interphase in the budding yeast, Saccharomyces cerevisiae, centromeres are clustered near one pole of the nucleus as a rosette with the spindle pole body at its hub. Opposite to the centromeric pole is the nucleolus. Chromosome arms extend outwards from the centromeric pole and are preferentially directed towards the opposite pole. Centromere clustering is reduced by the ndc10 mutation, which affects a kinetochore protein, and by the microtubule poison nocodazole. This suggests that clustering is actively maintained or enforced by the association of centromeres with microtubules throughout interphase. Unlike the Rabl-orientation known from many higher eukaryotes, centromere clustering in yeast is not only a relic of anaphase chromosome polarization, because it can be reconstituted without the passage of cells through anaphase. Within the rosette, homologous centromeres are not arranged in a particular order that would suggest somatic pairing or genome separation.

Abstract: Chromosomal rearrangements are important resources for genetic studies. Recently, a Cre-loxP-based method to introduce defined chromosomal rearrangements (deletions, duplications, and inversions) into the mouse genome (chromosome engineering) has been established. To explore the limits of this technology systematically, we have evaluated this strategy on mouse chromosome 11. Although the efficiency of Cre-loxP-mediated recombination decreases with increasing genetic distance when the two endpoints are on the same chromosome, the efficiency is not limiting even when the genetic distance is maximized. Rearrangements encompassing up to three quarters of chromosome 11 have been constructed in mouse embryonic stem (ES) cells. While larger deletions may lead to ES cell lethality, smaller deletions can be produced very efficiently both in ES cells and in vivo in a tissue- or cell-type-specific manner. We conclude that any chromosomal rearrangement can be made in ES cells with the Cre-loxP strategy provided that it does not affect cell viability. In vivo chromosome engineering can be potentially used to achieve somatic losses of heterozygosity in creating mouse models of human cancers.

Abstract: Arabidopsis thaliana is an important model system for plant biologists. In 1996 an international collaboration (the Arabidopsis Genome Initiative) was formed to sequence the whole genome of Arabidopsis and in 1999 the sequence of the first two chromosomes was reported. The sequence of the last three chromosomes and an analysis of the whole genome are reported in this issue. Here we present the sequence of chromosome 3, organized into four sequence segments (contigs). The two largest (13.5 and 9.2 Mb) correspond to the top (long) and the bottom (short) arms of chromosome 3, and the two small contigs are located in the genetically defined centromere. This chromosome encodes 5,220 of the roughly 25,500 predicted protein-coding genes in the genome. About 20% of the predicted proteins have significant homology to proteins in eukaryotic genomes for which the complete sequence is available, pointing to important conserved cellular functions among eukaryotes.

Abstract: The C-band distribution patterns of 105 angiosperm species were compared to identify general patterns or preferential sites for heterochromatin. The base-specific fluorochrome reaction of heterochromatin for 58 of these species and the role played by the average chromosome size in band distribution were also considered. The results showed that heterochromatin was preferentially located in similar chromosome regions, regardless of the distance from the centromere. This trend results in generalized bands, with heterochromatin distribution being identical in most chromosomes of a karyotype. Such bands very often displayed the same fluorochrome reaction, suggesting possible repeat transfer between non-homologous sites. Chromosome size may also play a role in heterochromatin location, since proximal bands were much more common in small-sized chromosomes.

Abstract: The aim of this study was to develop an improved procedure for preparation of chromosome suspensions, and to evaluate the potential of flow cytometry for chromosome sorting in wheat. Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity (flow karyotypes) obtained after the analysis of DAPI-stained chromosomes were characterized and the chromosome content of all peaks on wheat flow karyotype was determined for the first time. Only chromosome 3B could be discriminated on flow karyotypes of wheat lines with standard karyotype. Remaining chromosomes formed three composite peaks and could be sorted only as groups. Chromosome 3B could be sorted at purity >95% as determined by microscopic evaluation of sorted fractions that were labeled using C-PRINS with primers for GAA microsatellites and for Afa repeats, respectively. Chromosome 5BL/7BL could be sorted in two wheat cultivars at similar purity, indicating a potential of various wheat stocks for sorting of other chromosome types. PCR with chromosome-specific primers confirmed the identity of sorted fractions and suitability of flow-sorted chromosomes for physical mapping and for construction of small-insert DNA libraries. Sorted chromosomes were also found suitable for the preparation of high-molecular-weight DNA. On the basis of these results, it seems realistic to propose construction of large-insert chromosome-specific DNA libraries in wheat. The availability of such libraries would greatly simplify the analysis of the complex wheat genome.

Abstract: For introducing regions of human chromosomes greater than a megabase into cells or animals, we have developed a chromosome-cloning system in which defined regions of human chromosomes can be cloned into a stable human minichromosome vector in homologous recombination-proficient chicken DT40 cells. The stable minichromosome vector allowed a 10 Mb-sized region of the mitotically unstable human chromosome 22 to be stably maintained in mouse embryonic stem (ES) cells, and in mice. Furthermore, we demonstrated functional expression of human genes from the HAC in mice. This study describes a stable cloning and expression system for greater than megabase-sized regions of human chromosomes.

Abstract: The mutation rates of cancer cells to drug and multidrug resistance are paradoxically high, i.e., 10−3 to 10−6, compared with those altering phenotypes of recessive genes in normal diploid cells of about 10−12. Here the hypothesis was investigated that these mutations are due to chromosome reassortments that are catalyzed by aneuploidy. Aneuploidy, an abnormal number of chromosomes, is the most common genetic abnormality of cancer cells and is known to change phenotypes (e.g., Down's syndrome). Moreover, we have shown recently that aneuploidy autocatalyzes reassortments of up to 2% per chromosome per mitosis because it unbalances spindle proteins, even centrosome numbers, via gene dosage. The hypothesis predicts that a selected phenotype is associated with multiple unselected ones, because chromosome reassortments unbalance simultaneously thousands of regulatory and structural genes. It also predicts variants of a selected phenotype based on variant reassortments. To test our hypothesis we have investigated in parallel the mutation rates of highly aneuploid and of normal diploid Chinese hamster cells to resistance against puromycin, cytosine arabinoside, colcemid, and methotrexate. The mutation rates of aneuploid cells ranged from 10−4 to 10−6, but no drug-resistant mutants were obtained from diploid cells in our conditions. Further selection increased drug resistance at similar mutation rates. Mutants selected from cloned cells for resistance against one drug displayed different unselected phenotypes, e.g., polygonal or fusiform cellular morphology, flat or three-dimensional colonies, and resistances against other unrelated drugs. Thus our hypothesis offers a unifying explanation for the high mutation rates of aneuploid cancer cells and for the association of selected with unselected phenotypes, e.g., multidrug resistance. It also predicts drug-specific chromosome combinations that could become a basis for selecting alternative chemotherapy against drug-resistant cancer.

Abstract: The small fourth chromosome of Drosophila melanogaster (3.5% of the genome) presents a puzzle. Cytological analysis suggests that the bulk of the fourth, including the portion that appears banded in the polytene chromosomes, is heterochromatic; the banded region includes blocks of middle repetitious DNA associated with heterochromatin protein 1 (HP1). However, genetic screens indicate 50–75 genes in this region, a density similar to that in other euchromatic portions of the genome. Using a P element containing an hsp70-white gene and a copy of hsp26 (marked with a fragment of plant DNA designated pt), we have identified domains that allow for full expression of the white marker (R domains), and others that induce a variegating phenotype (V domains). In the former case, the hsp26-pt gene shows an accessibility and heat-shock-inducible activity similar to that seen in euchromatin, whereas in the latter case, accessibility and inducible expression are reduced to levels typical of heterochromatin. Mapping by in situ hybridization and by hybridization of flanking DNA sequences to a collection of cosmid and bacterial artificial chromosome clones shows that the R domains (euchromatin-like) and V domains (heterochromatin-like) are interspersed. Examination of the effect of genetic modifiers on the variegating transgenes shows some differences among these domains. The results suggest that heterochromatic and euchromatic domains are interspersed and closely associated within this 1.2-megabase region of the genome.

Abstract: In cultivated banana, Musa spp., there are four known genomes, A, B, S, and T. These correspond to the genetic constitutions of wild Eumusa species M. acuminata, M. balbisiana, M. schizocarpa and the Australimusa species, respectively. Most cultivated clones are triploid or diploid, they have been classified into genomic groups according to chromosome numbers and morphological traits. Genomic in situ hybridization (GISH) enabled us to differentiate the chromosomes of these four genomes; however, a distal portion of the chromosomes remained unlabelled. GISH was used to determine the exact genome structure of interspecific cultivated clones.In most cases the results were consistent with the chromosome constitution estimated by means of phenotypic descriptors.The one notable exception, the clone ’Pelipita’, has the 8 A and 25 B chromosomes instead of the predicted 11 A and 22 B. GISH also enabled us to determine the chromosome complement of a few clones that could not be classified only on the basis of phenotypic descriptors and chromosome counts. The rDNA sites were located in Musa species. They appeared to be often associated with satellites, which can be separated from the chromosomes, representing a potential source of error for chromosome counting using classical techniques.

Abstract: Segregation of DNA in bacterial cells is an efficient process that assures that every daughter cell receives a copy of genomic and plasmid DNA. In this review, we focus primarily on observations in recent years, including the visualization of DNA and proteins at the subcellular level, that have begun to define the events that separate DNA molecules. Unlike the process of chromosome segregation in higher cells, segregation of the bacterial chromosome is a continuous process in which chromosomes are separated as they are replicated. Essential to separation is the initial movement of sister origins to opposite ends of the cell. Subsequent replication and controlled condensation of DNA are the driving forces that move sister chromosomes toward their respective origins, which establishes the polarity required for segregation. Final steps in the resolution and separation of sister chromosomes occur at the replication terminus, which is localized at the cell center. In contrast to the chromosome, segregation of low-copy plasmids, such as Escherichia coli F, P1, and R1, is by mechanisms that resemble those used in eukaryotic cells. Each plasmid has a centromere-like site to which plasmid-specified partition proteins bind to promote segregation. Replication of plasmid DNA, which occurs at the cell center, is followed by rapid partition protein-mediated separation of sister plasmids, which become localized at distinct sites on either side of the division plane. The fundamental similarity between chromosome and plasmid segregation-placement of DNA to specific cell sites-implies an underlying cellular architecture to which both DNA and proteins refer.

Abstract: We examined the structure, intranuclear distribution and activity of ribosomal DNA (rDNA) in Nicotiana sylvestris (2n = 2x = 24) and N. tomentosiformis (2n = 2x = 24) and compared these with patterns in N. tabacum (tobacco, 2n = 4x = 48). We also examined a long-established N. tabacum culture, TBY-2. Nicotiana tabacum is an allotetraploid thought to be derived from ancestors of N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). Nicotiana sylvestris has three rDNA loci, one locus each on chromosomes 10, 11, and 12. In root-tip meristematic interphase cells, the site on chromosome 12 remains condensed and inactive, while the sites on chromosomes 10 and 11 show activity at the proximal end of the locus only. Nicotiana tomentosiformis has one major locus on chromosome 3 showing activity and a minor, inactive locus on chromosome 11. In N. tabacum cv. 095-55, there are four rDNA loci on T3, S10, S11/t and S12 (S11/t carries a small T-genome translocation). The locus on S12 remains condensed and inactive in root-tip meristematic cells while the others show activity, including decondensation at interphase and secondary constrictions at metaphase. Nicotiana tabacum DNA digested with methylcytosine-sensitive enzymes revealed a hybridisation pattern for rDNA that resembled that of N. tomentosiformis and not N. sylvestris. The data indicate that active, undermethylated genes are of the N. tomentosiformis type. Since S-genome chromosomes of N. tabacum show rDNA expression, the result indicates rDNA gene conversion of the active rDNA units on these chromosomes. Gene conversion in N. tabacum is consistent with the results of previous work. However, using primers specific for the S-genome rDNA intergenic sequences (IGS) in the polymerase chain reaction (PCR) show that rDNA gene conversion has not gone to completion in N. tabacum. Furthermore, using methylation-insensitive restriction enzymes we demonstrate that about 8% of the rDNA units remain of the N. sylvestris type (from ca. 75% based on the sum of the rDNA copy numbers in the parents). Since the active genes are likely to be of an N. tomentosiformis type, the N. sylvestris type units are presumably contained within inactive loci (i.e. on chromosome S12). Nicotiana sylvestris has approximately three times as much rDNA as the other two species, resulting in much condensed rDNA at interphase. This species also has three classes of IGS, indicating gene conversion has not homogenised repeat length in this species. The results suggest that methylation and/or DNA condensation has reduced or prevented gene conversion from occurring at inactive genes at rDNA loci. Alternatively, active undermethylated units may be vulnerable to gene conversion, perhaps because they are decondensed and located in close proximity within the nucleolus at interphase. In TBY-2, restriction enzymes showed hybridisation patterns that were similar to, but different from, those of N. tabacum. In addition, TBY-2 has elevated rDNA copy number and variable numbers of rDNA loci, all indicating rDNA evolution in culture.

Abstract: Cytogenetic studies were carried out in the fish, Hoplias malabaricus, from the Parque Florestal do Rio Doce (Brazil). This population is characterized by 2n = 42 chromosomes for both males and females and an XX/XY sex chromosome system, confirmed through several banding methods. Females show 24 metacentric, 16 submetacentric and 2 subtelocentric chromosomes. Males show 24 metacentric, 17 submetacentric and 1 subtelocentric chromosomes. While the X chromosome is easily recognized (the only subtelocentric element), the Y chromosome is somewhat difficult to identify but appears to correspond to the smallest submetacentric in the male karyotype. In-situ hybridization with an 18S rDNA probe showed 10 well-labeled chromosomes, including the X chromosome. The 5S rDNA is interstitially located in a single metacentric pair independent of the 18S rDNA sites. The NOR on the X chromosome is always active and occurs adjacent to a heterochromatic distal segment on the long arm. Variations in size of the NORs and/or heterochromatic segment correspond to a polymorphic size condition observed in the X chromosome. The present results confirm the XX/XY sex chromosome system in the population analyzed as well as a new cytotype in the Hoplias malabaricus group.

Abstract: Mutations in the forkhead-like 7 (FKHL7) gene have been recently shown to cause juvenile glaucoma and anterior segment anomalies. We report on a three-generation family with Axenfeld-Rieger syndrome (ARS), harboring an alteration in the FKHL7 gene. Genetic linkage analyses excluded the ARS phenotype from chromosomes 4q25 and 13q14, the locations of the PITX2 and RIEG2 loci, respectively. Evidence of linkage was observed with markers at 6p25, near the FKHL7 gene. Direct sequencing of FKHL7 detected a C67T mutation that segregated with the ARS phenotype in this family, but was not detected in over 80 control chromosomes. This mutation is predicted to cause a nonsense mutation of the FKHL7 protein (Gln23Stop) upstream of the forkhead DNA-binding domain, and thus to generate a truncated FKHL7 protein product. This discovery broadly implicates FKHL7 in ocular, craniofacial, dental, and umbilical development.

Abstract: Domestic cats and dogs are important companion animals and model animals in biomedical research. The cat has a highly conserved karyotype, closely resembling the ancestral karyotype of mammals, while the dog has one of the most extensively rearranged mammalian karyotypes investigated so far. We have constructed the first detailed comparative chromosome map of the domestic dog and cat by reciprocal chromosome painting. Dog paints specific for the 38 autosomes and the X chromosomes delineated 68 conserved chromosomal segments in the cat, while reverse painting of cat probes onto red fox and dog chromosomes revealed 65 conserved segments. Most conserved segments on cat chromosomes also show a high degree of conservation in G-banding patterns compared with their canine counterparts. At least 47 chromosomal fissions (breaks), 25 fusions and one inversion are needed to convert the cat karyotype to that of the dog, confirming that extensive chromosome rearrangements differentiate the karyotypes of the cat and dog. Comparative analysis of the distribution patterns of conserved segments defined by dog paints on cat and human chromosomes has refined the human/cat comparative genome map and, most importantly, has revealed 15 cryptic inversions in seven large chromosomal regions of conserved synteny between humans and cats.

Abstract: Although cold acclimation in cereals involves the expression of many cold-regulated genes, genetic studies have shown that only very few chromosomal regions carry loci that play an important role in frost tolerance. To investigate the genetic relationship between frost tolerance and the expression of cold-regulated genes, the expression and regulation of the wheat homolog of the barley cold-regulated gene cor14b was studied at various temperatures in frost-sensitive and frost-tolerant wheat genotypes. At 18/15 degrees C (day/night temperatures) frost-tolerant plants accumulated cor14b mRNAs and expressed COR14b proteins, whereas the sensitive plants did not. This result indicates that the threshold temperature for induction of the wheat cor14b homolog is higher in frost-resistant plants, and allowed us to use this polymorphism in a mapping approach. Studies made with chromosome substitution lines showed that the polymorphism for the threshold induction temperature of the wheat cor14b homolog is controlled by a locus(i) located on chromosome 5A of wheat, while the cor14b gene was mapped in Triticum monococcum on the long arm of chromosome 2Am. The analysis of single chromosome recombinant lines derived from a cross between Chinese Spring/Triticum spelta 5A and Chinese Spring/Cheyenne 5A identified two loci with additive effects that are involved in the genetic control of cor14b mRNA accumulation. The first locus was tightly linked to the marker psr911, while the second one was located between the marker Xpsr2021 and Frost resistance 1 (Fr1).

Abstract: Despite several loss of heterozygosity studies, a comprehensive genomic survey of pheochromocytomas is still lacking. To identify DNA copy number changes which might be important in tumor development and progression and which may have diagnostic utility, we evaluated genetic aberrations in 29 sporadic adrenal and extra-adrenal pheochromocytomas (19 clinically benign tumors and 10 malignant lesions). Comparative genomic hybridization was performed using directly fluorochrome-conjugated DNA extracted from frozen (16) and paraffin-embedded (13) tumor tissues. The most frequently observed changes were losses of chromosomes 1p11-p32 (86%), 3q (52%), 6q (34%), 3p, 17p (31% each), 11q (28%), and gains of chromosomes 9q (38%) and 17q (31%). No amplification was identified and no difference between adrenal and extra-adrenal tumors was detected. Progression to malignant tumors was strongly associated with deletions of chromosome 6q (60% versus 21% in clinically benign lesions, P = 0.0368) and 17p (50% versus 21%). Fluorescence in situ hybridization confirmed the comparative genomic hybridization data of chromosomes 1p, 3q, and 6q, and revealed aneuploidy in some tumors. Our results suggest that the development of pheochromocytomas is associated with specific genomic aberrations, such as losses of 1p, 3q, and 6q and gains of 9q and 17q. In particular, tumor suppressor genes on chromosomes 1p and 3q may be involved in early tumorigenesis, and deletions of chromosomes 6q and 17p in progression to malignancy.

Abstract: We have generated transgenic mice by pronuclear microinjection of a murine satellite DNA-based artificial chromosome (SATAC). As 50% of the founder progeny were SATAC-positive, this demonstrates that SATAC transmission through the germline had occurred. FISH analyses of metaphase chromosomes from mitogen-activated peripheral blood lymphocytes from both the founder and progeny revealed that the SATAC was maintained as a discrete chromosome and that it had not integrated into an endogenous chromosome. To our knowledge, this is the first report of the germline transmission of a genetically engineered mammalian artificial chromosome within transgenic animals generated through pronuclear microinjection. We have also shown that murine SATACs can be similarly introduced into bovine embryos. The use of embryo microinjection to generate transgenic mammals carrying genetically engineered chromosomes provides a novel method by which the unique advantages of chromosome-based gene delivery systems can be exploited.

Abstract: Multicolour fluorescence in situ hybridisation (FISH) analysis of interphase nuclei in cleavage stage human embryos has highlighted a high incidence of postzygotic chromosomal mosaicism, including both aneuploid and ploidy mosaicism. Indeed, some embryos appear to have a chaotic chromosomal complement in a majority of nuclei, suggesting that cell cycle checkpoints may not operate in early cleavage. Most of these studies, however, have only analysed a limited number of chromosomes (3-5), making it difficult to distinguish FISH artefacts from true aneuploidy. We now report analysis of 11 chromosomes in five sequential hybridisations with standard combinations of two or three probes and minimal loss of hybridisation efficiency. Analysis of a series of arrested human embryos revealed a generally consistent pattern of hybridisation on which was superimposed frequent deletion of one or both chromosomes of a specific pair in two or more nuclei indicating a clonal origin and continued cleavage following chromosome loss. With a binucleate cell in a predominantly triploid XXX embryo, the two nuclei remained attached during preparation and the chaotic diploid/triphoid status of every chromosome analysed was the same for each nucleus. Furthermore, in each hybridisation the signals were distributed as a mirror-image about the plane of attachment, indicating premature decondensation during anaphase consistent with a lack of checkpoint control.

Abstract: Very little is known about genetic abnormalities involved in the development of pilocytic astrocytoma, the most frequently occurring brain tumour of childhood We have analysed 48 pilocytic astrocytoma specimens using comparative genomic hybridization Only five of 41 tumours from children showed abnormalities detectable by comparative genomic hybridization, and in each case this represented gain of a single chromosome Interestingly, two of seven tumours from adults showed abnormalities, which were multiple and relatively complex Six of the seven tumours showing abnormalities were from female patients (two adults and four children) The most frequently detectable abnormality was gain of 9q341-qter, which was present in three cases (two adult and one paediatric) © 2000 Cancer Research Campaign