Intact Chromosomes Research Articles

Chinese hamster ovary cell line DXB-11: chromosomal instability and karyotype heterogeneity

BackgroundChinese hamster ovary cell lines, also known as CHO cells, represent a large family of related, yet quite different, cell lines which are metabolic mutants derived from the original cell line, CHO-ori. Dihydrofolate reductase-deficient DXB-11 cell line, one of the first CHO derivatives, serves as the host cell line for the production of therapeutic proteins. It is generally assumed that DXB-11 is identical to DUKX or CHO-DUK cell lines, but, to our knowledge, DXB-11 karyotype has not been described yet.ResultsUsing differential staining approaches (G-, C-banding and Ag-staining), we presented DXB-11 karyotype and revealed that karyotypes of DXB-11 and CHO-DUK cells have a number of differences. Although the number of chromosomes is equal—20 in each cell line—DXB-11 has normal chromosomes of the 1st and 5th pairs as well as an intact chromosome 8. Besides, in DXB-11 line, chromosome der(Z9) includes the material of chromosomes X and 6, whereas in CHO-DUK it results from the translocation of chromosomes 1 and 6. Ag-positive nucleolar organizer regions were revealed in the long arms of chromosome del(4)(q11q12) and both chromosome 5 homologues, as well as in the short arms of chromosomes 8 and add(8)(q11). Only 19 from 112 (16.96%) DXB-11 cells display identical chromosome complement accepted as the main structural variant of karyotype. The karyotype heterogeneity of all the rest of cells (93, 83.04%) occurs due to clonal and nonclonal additional structural rearrangements of chromosomes. Estimation of the frequency of chromosome involvement in these rearrangements allowed us to reveal that chromosomes 9, der(X)t(X;3;4), del(2)(p21p23), del(2)(q11q22) /Z2, der(4) /Z7, add(6)(p11) /Z8 are the most stable, whereas mar2, probably der(10), is the most unstable chromosome. A comparative analysis of our own and literary data on CHO karyotypes allowed to designate conservative chromosomes, both normal and rearranged, that remain unchanged in different CHO cell lines, as well as variable chromosomes that determine the individuality of karyotypes of CHO derivatives.ConclusionDXB-11and CHO-DUK cell lines differ in karyotypes. The revealed differential instability of DXB-11 chromosomes is likely not incidental and results in karyotype heterogeneity of cell population.

Near-Chromosome-Level Genome Assembly of the Dark Septate Endophyte Laburnicola rhizohalophila: A Model for Investigating Root-Fungus Symbiosis.

The novel DSE Laburnicola rhizohalophila (Pleosporales, Ascomycota) is frequently found in the halophytic seepweed (Suaeda salsa). In this article, we report a near-chromosome-level hybrid assembly of this fungus using a combination of short-read Illumina data to polish assemblies generated from long-read Nanopore data. The reference genome for L. rhizohalophila was assembled into 26 scaffolds with a total length of 64.0 Mb and a N50 length of 3.15 Mb. Of them, 17 scaffolds approached the length of intact chromosomes, and 5 had telomeres at one end only. A total of 10,891 gene models were predicted. Intriguingly, 27.5 Mb of repeat sequences that accounted for 42.97% of the genome was identified, and long terminal repeat retrotransposons were the most frequent known transposable elements, indicating that transposable element proliferation contributes to its increased genome size. BUSCO analyses using the Fungi_odb10 data set showed that 95.0% of genes were complete. In addition, 292 carbohydrate active enzymes, 33 secondary metabolite clusters, and 84 putative effectors were identified in silico. The resulting high-quality assembly and genome features are not only an important resource for further research on understanding the mechanism of root-fungi symbiotic interactions but will also contribute to comparative analyses of genome biology and evolution within Pleosporalean species.

Kinetochore-independent mechanisms of sister chromosome separation.

Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore- independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.

Telomere length dynamics in response to DNA damage in malaria parasites

SummaryMalaria remains a major cause of morbidity and mortality in the developing world. Recent work has implicated chromosome end stability and the repair of DNA breaks through telomere healing as potent drivers of variant antigen diversification, thus associating basic mechanisms for maintaining genome integrity with aspects of host-parasite interactions. Here we applied long-read sequencing technology to precisely examine the dynamics of telomere addition and chromosome end stabilization in response to double-strand breaks within subtelomeric regions. We observed that the process of telomere healing induces the initial synthesis of telomere repeats well in excess of the minimal number required for end stability. However, once stabilized, these newly created telomeres appear to function normally, eventually returning to a length nearing that of intact chromosome ends. These results parallel recent observations in humans, suggesting an evolutionarily conserved mechanism for chromosome end repair.

In Situ Hybridisation-A Review

In situ hybridisation (ISH) depends on the integral blending of labelled DNA or RNA probes with typicalor irregular nucleic acid arrangements in intact chromosomes, cells or tissue segments. Compared withother molecular techniques appropriate to anatomical pathology, ISH appreciates better compatibility withhistopathologists in view of its closeness to immunohistochemistry. It has the exceptional preferred positionover other techniques to a great extent dependent on probe hybridization with nucleic acid removed fromhomogenized tissue probes - of permitting localization and representation of target nucleic acid sequencesinside morphologically recognizable cells or cell structures. In spite of the fact that ISH is to a great extentan examination apparatus, it is as of now making solid advances into demonstrative histopathology. Otherthan recognition of infective specialists, it likewise permits localization of destinations of contamination,explanation of systems of infection transmission and spread also, examination of the connection betweenviral specialists and malignant growth. Sex composing, localization of qualities on chromosomes andrecognition of basic and mathematical chromosomal changes in tumors are among different utilizations ofISH.

Linear, funnel, and multiple funnel schemes for stacking chromosomes that carry targeted recombinations in plants.

Up to five chromosomes that carry targeted recombinations can be stacked via a multiple funnel scheme, provided that the probabilities of inheriting intact chromosomes from donor parents are high. Targeted recombination involves inducing or selecting for recombination events at specific points in the genome to maximize genetic gain. Practical application of targeted recombination requires efficient breeding strategies to stack multiple chromosomes that carry such recombinations. Our objectives were to determine how many chromosomes with targeted recombinations can be feasibly stacked in a breeding program, and how the feasibility of stacking is affected by the crossing design, homozygosity versus heterozygosity of the donor lines, size of the chromosomal segment showing recombination, and probability of an intact chromosome being inherited. Based on a genetic model for maize (Zea mays L.) with 10 pairs of chromosomes, we examined different crossing schemes by simulation experiments and analytical studies with the goal of minimizing the number of generations and population sizes required for stacking. We found that targeted recombinations on up to five chromosomes can be stacked within practical constraints on time and resources. Linear and funnel schemes were less efficient than a multiple funnel scheme, which involved making all possible crosses in the first generation and stacking two additional chromosomes across multiple lines in subsequent generations. Homozygosity versus heterozygosity of the donor lines did not affect stacking efficiency. Population sizes and stacking efficiency were largely determined by the probability of intact chromosomal transfer from a donor parent to offspring. Such probability increased as the size of the chromosome segment from the donor decreased. When the probability of inheriting an intact chromosome was less than 0.15, population sizes needed for stacking became infeasibly large.

Three-dimensional super-resolution fluorescence imaging of DNA

Recent advances in fluorescence super-resolution microscopy are providing important insights into details of cellular structures. To acquire three dimensional (3D) super-resolution images of DNA, we combined binding activated localization microscopy (BALM) using fluorescent double-stranded DNA intercalators and optical astigmatism. We quantitatively establish the advantage of bis- over mono-intercalators before demonstrating the approach by visualizing single DNA molecules stretched between microspheres at various heights. Finally, the approach is applied to the more complex environment of intact and damaged metaphase chromosomes, unravelling their structural features.

Investigating SLX4‐Dependent Recruitment of a DNA Repair Protein to DNA Double Strand Breaks in Dividing Cells

Cancer is a disease that functions as a consequence of abnormal cell growth, generally caused by DNA damage that triggers abnormal cell function. One form of DNA damage is the double‐strand break (DSB) in which both strands of a chromosome are severed in near proximity. In normal cells, several forms of DSB repair, including single strand annealing (SSA) and synthesis dependent strand annealing (SDSA), fix the double strand break (DSB), restoring an intact chromosome, albeit sometimes with an altered DNA sequence. These DNA repair processes require the sequential action of numerous proteins that enable their many steps. In S. cerevisiae these include the Rad1‐Rad10 endonuclease complex, which cleaves nonhomologous DNA flaps in SSA and SDSA as one of the final steps.We have evidence that the Slx4 protein is partially required for the recruitment of Rad1‐Rad10 to DSB sites when cells are dividing (in S, G2 or M phases). Those experiments did not show the basis for the “partial” nature of the recruitment, but our hypothesis is that the Slx4‐dependency is tied to a specific phase of the cell cycle, possibly S phase. A plausible explanation is that when Slx4 exercises its checkpoint signal dampening role in S phase, which promotes homologous recombination, it indirectly recruits Rad1‐Rad10 to double‐strand break repair sites. Since, following DNA damage, checkpoint signal dampening is triggered (in part) by phosphorylation of Slx4 in S phase and the specific phosphorylation sites for this are known, we will test our hypothesis by creating mutant strains which lack these sites and repeating the earlier experiments using synchronized cell cultures. We began creating the mutant strains by cloning DNA plasmids containing the slx4‐7MUT gene, our focus, in which wild‐type SLX4 has been mutated to alanine at the seven phosphorylation sites activating checkpoint signal dampening. Candidate clones were screened by PCR and DNA sequencing. The successful candidate plasmids were transformed into a panel of W303 background yeast strains and integration of the mutant cassettes was confirmed by PCR and DNA sequencing screens. The strains were then crossed to strains bearing the fluorescence microscopy assay markers we will use in the fluorescence microscopy assay being employed. We have carried out preliminary fluorescence microscopy assays in which cultures of the progeny of these crosses were synchronized at the G1/S boundary and released back into the cell cycle that show acceptable arrest but the limited release back into the cell cycle that was observed requires further optimization. Using this separation‐of‐function mutant strain will allow us to compare recruitment of Rad1‐Rad10 in SLX4 wild‐type strains with Slx4 checkpoint signal dampening mutants. Understanding the process of Rad1‐Rad10 recruitment may aid in the design and development of a more effective means of cancer treatment.

Cell Cycle‐Dependent Cleaving of Holliday Junctions via Yen1 in S. cerevisiae

Double‐strand breaks (DSBs) in DNA can be harmful if left unrepaired. Eukaryotes use repair mechanisms, some of which involve intact chromosomes that template repair via an intertwined intermediate known as a Holliday Junction (HJ). In S. cerevisiae, recruitment of the endonuclease enzymes that cleave HJs back into individual chromosomes is not well understood. Yen1 resolves HJs but it’s unclear whether it localizes to anaphase bridges, the final intertwined regions during chromosomal separation. Yen1 appears in anaphase which occurs during a small time frame within the cell cycle, making it difficult to observe in an asynchronous cell population. Further, Yen1 is present in lower abundance relative to other proteins, therefore, microscopy experiments with fluorescently labeled Yen1 will suffer from low signal‐to‐noise. Our aim is to clone a yeast strain with a bright version of red fluorescent protein under the strong galactose (GAL1) promoter so that Yen1‐yEMRFP will be in higher abundance, thereby allowing us to track the position and timing Yen1 during HJ resolution. To construct this strain, we used adaptamer‐mediated Polymerase Chain Reaction (PCR), DNA digestions and ligations to prepare two DNA plasmids. Plasmid One contains a gene for yeast Enhanced Monomeric Red Fluorescent Protein (yEMRFP), a partial URA3 auxotrophic selection marker gene, and an upstream flanker sequence of the target yeast chromosome to direct integration. Plasmid Two contains the other half of the partial URA3 selection marker, the galactose promoter, and the downstream flanker sequence to target integration. Candidate clones were screened by PCR and DNA sequencing which showed that the candidate plasmids had the desired sequences and are ready to be integrated into the yeast chromosome. These DNA plasmids will allow cloning of a specialized yeast strain that will produce an improved signal‐to‐noise resolution to elucidate Yen1’s role at anaphase bridges.Support or Funding InformationThis research was funded by NIH grant, SC1GM127204.

Enhanced chromosome extraction from cells using a pinched flow microfluidic device.

Extraction and purification of intact chromosomes are critical sample preparation steps for transchromosomic research and other applications. The commonly used sample preparation methods lead to too few chromosomes with chromosome deactivation and degradation. In this paper, a "mild" chromosome extraction process that combines a chemical and mechanical lysis approach is introduced for the preparation of intact chromosomes that can readily be used for downstream processing. Metaphase cells are treated by chemical lysis buffer and pushed through a microfluidic pinched flow device. Cells are ruptured, and chromosomes are released by a combination of shear stress and chemical reagents. Chromosomes are released intact from the cell membrane into the solution. Simulations and experiments are performed to optimize the microfluidic device geometry and operation parameters. Cell rupture and chromosome release are found to be improved by the shear stress in the pinched flow device. Simulation results indicate that the maximum shear stress appears in the channel constriction region, and the narrow channel maintains constant shear stress. It is concluded that the constriction design, narrow channel width, and operation flow rate have a significate influence on chromosome release. Utilizing an optimized device, near-complete cell lysis is achieved and 4 times as many chromosomes are released (8% in control experiments to 25% in optimized pinched flow devices). Sample treatment time can also be reduced utilizing this combined chemical-mechanical chromosome release method.

Predicted genetic gains from introgressing chromosome segments from exotic germplasm into an elite soybean cultivar.

To improve an elite soybean line, introgress longer chromosome segments instead of QTL alleles from exotic germplasm. Broadening the diversity of cultivated soybean [Glycine max (L.) Merrill] through introgression of exotic germplasm has been difficult. Our objectives were to (1) determine if introgressing specific chromosome segments (instead of quantitative trait locus alleles) from exotic soybean germplasm has potential for improving an elite cultivar, and (2) identify strategies to introgress and pyramid exotic chromosome segments into an elite cultivar. We estimated genomewide marker effects for yield and other traits in seven crosses between the elite line IA3023 and seven soybean plant introductions (PIs). We then predicted genetic gains from having ≤ 2 targeted recombinations per linkage group. When introgression was modeled for yield while controlling maturity in the seven PI × IA3023 populations, the predicted yield was 8-25% over the yield of IA3023. Correlated changes in maturity, seed traits, lodging, and plant height were generally small but were in the favorable direction. In contrast, selecting the best recombinant inbred (without targeted recombination) in each of the PI × IA3023 populations led to negative or minimal yield gains over IA3023. In one PI × IA3023 population, introgressing and pyramiding only two linkage groups from recombinant inbreds into IA3023 was predicted to achieve an 8% yield gain over IA3023 without sacrificing the performance for other traits. The probability of inheriting intact chromosomes was high enough to allow introgression and pyramiding of chromosome segments in 5-6 generations. Overall, our study suggested that introgressing specific chromosome segments is an effective way to introduce exotic soybean germplasm into an elite cultivar.

Pulsed-Field Gel Electrophoresis Typing of Staphylococcus aureus Strains.

Pulsed-field gel electrophoresis (PFGE) is one of the most effective genotyping methods for the study of genetic relatedness of microorganisms as well as for epidemiologic investigation of outbreaks caused by pathogenic bacteria, including Staphylococcus aureus. The technique relies on the size patterns of large DNA fragments generated by the cleavage of intact bacterial chromosomes with a rare cutting restriction enzyme, and subsequently resolved by pulsed-field electrophoresis with periodic changes of the orientation of the electrical field across the gel. The high discriminatory power, improved reproducibility by standardization of experimental protocols and data analysis guidelines, and establishment of a national PFGE database of S. aureus profiles have made it a valuable means for global tracking of S. aureus infection sources and determination of genetic relatedness of the strains.

Fluorescence In Situ Hybridization (FISH) Analysis of the Locations of the Oligonucleotides 5S rDNA, (AGGGTTT)3, and (TTG)6 in Three Genera of Oleaceae and Their Phylogenetic Framework.

We report the cytogenetic map for a collection of species in the Oleaceae, and test similarities among the karyotypes relative to their known species phylogeny. The oligonucleotides 5S ribosomal DNA (rDNA), (AGGGTTT)3, and (TTG)6 were used as fluorescence in situ hybridization (FISH) probes to locate the corresponding chromosomes in three Oleaceae genera: Fraxinus pennsylvanica, Syringa oblata, Ligustrum lucidum, and Ligustrum × vicaryi. Forty-six small chromosomes were identified in four species. (AGGGTTT)3 signals were observed on almost all chromosome ends of four species, but (AGGGTTT)3 played no role in distinguishing the chromosomes but displayed intact chromosomes and could thus be used as a guide for finding chromosome counts. (TTG)6 and 5S rDNA signals discerned several chromosomes located at subterminal or central regions. Based on the similarity of the signal pattern (mainly in number and location and less in intensity) of the four species, the variations in the 5S rDNA and (TTG)6 distribution can be ordered as L. lucidum < L. × vicaryi < F. pennsylvanica < S. oblata. Variations have observed in the three genera. The molecular cytogenetic data presented here might serve as a starting point for further larger-scale elucidation of the structure of the Oleaceae genome, and comparison with the known phylogeny of Oleaceae family.

Development of a Fertility Restorer for inap CMS (Isatis indigotica) Brassica napus Through Genetic Introgression of One Alien Addition.

Novel Brassica napus cytoplasmic male sterility (CMS) with carpelloid stamens (inap CMS) was produced by intertribal somatic hybridization with Isatis indigotica (Chinese woad), but its RF (restorer of fertility) gene(s) existed in one particular woad chromosome that was carried by one fertile monosomic alien addition line (MAAL) of rapeseed. Herein, the selfed progenies of this MAAL were extensively selected and analyzed to screen the rapeseed-type plants (2n = 38) with good male fertility and to produce their doubled haploid (DH) lines by microspore culture. From the investigation of fertility restoration in the F1 hybrids with inap CMS, one DH line (RF 39) was identified to adequately restore male fertility and likely carried one dominant RF gene. Specifically, this restorer produced brown pollen grains, similar to the woad and the MAAL, suggesting that this trait is closely linked with the RF gene(s) and serves as one phenotypic marker for the restorer. This restorer contained 38 chromosomes of rapeseed and no intact chromosomes of woad, but some DNA fragments of woad origin were detected at low frequency. This restorer was much improved for pollen and seed fertility and for low glucosinolate content. The successful breeding of the restorer for inap CMS rendered this new pollination control system feasible for rapeseed hybrid production.

Direct Transfer of a Mycoplasma mycoides Genome to Yeast Is Enhanced by Removal of the Mycoides Glycerol Uptake Factor Gene glpF.

We previously discovered that intact bacterial chromosomes can be directly transferred to a yeast host cell where they can propagate as centromeric plasmids by fusing bacterial cells with S accharomyces cerevisiae spheroplasts. Inside the host any desired number of genetic changes can be introduced into the yeast centromeric plasmid to produce designer genomes that can be brought to life using a genome transplantation protocol. Earlier research demonstrated that the removal of restriction-systems from donor bacteria, such as Mycoplasma mycoides, Mycoplasma capricolum, or Haemophilus influenzae increased successful genome transfers. These findings suggested that other genetic factors might also impact the bacteria-to-yeast genome transfer process. In this study, we demonstrated that the removal of a particular genetic factor, the glycerol uptake facilitator protein gene glpF from M. mycoides, significantly increased direct genome transfer by up to 21-fold. Additionally, we showed that intact bacterial cells were endocytosed by yeast spheroplasts producing organelle-like structures within these yeast cells. These might lead to the possibility of creating novel synthetic organelles.

A microfluidic device for isolating intact chromosomes from single mammalian cells and probing their folding stability by controlling solution conditions

Chromatin folding shows spatio-temporal fluctuations in living undifferentiated cells, but fixed spatial heterogeneity in differentiated cells. However, little is known about variation in folding stability along the chromatin fibres during differentiation. In addition, effective methods to investigate folding stability at the single cell level are lacking. In the present study, we developed a microfluidic device that enables non-destructive isolation of chromosomes from single mammalian cells as well as real-time microscopic monitoring of the partial unfolding and stretching of individual chromosomes with increasing salt concentrations under a gentle flow. Using this device, we compared the folding stability of chromosomes between non-differentiated and differentiated cells and found that the salt concentration which induces the chromosome unfolding was lower (≤500 mM NaCl) for chromosomes derived from undifferentiated cells, suggesting that the chromatin folding stability of these cells is lower than that of differentiated cells. In addition, individual unfolded chromosomes, i.e., chromatin fibres, were stretched to 150–800 µm non-destructively under 750 mM NaCl and showed distributions of highly/less folded regions along the fibres. Thus, our technique can provide insights into the aspects of chromatin folding that influence the epigenetic control of cell differentiation.

Integration of Genetic and Cytogenetic Maps and Identification of Sex Chromosome in Garden Asparagus (Asparagus officinalis L.).

A genetic linkage map of dioecious garden asparagus (Asparagus officinalis L., 2n = 2x = 20) was constructed using F1 population, simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers. In total, 1376 SNPs and 27 SSRs were used for genetic mapping. Two resulting parental maps contained 907 and 678 markers spanning 1947 and 1814 cM, for female and male parent, respectively, over ten linkage groups representing ten haploid chromosomes of the species. With the aim to anchor the ten genetic linkage groups to individual chromosomes and develop a tool to facilitate genome analysis and gene cloning, we have optimized a protocol for flow cytometric chromosome analysis and sorting in asparagus. The analysis of DAPI-stained suspensions of intact mitotic chromosomes by flow cytometry resulted in histograms of relative fluorescence intensity (flow karyotypes) comprising eight major peaks. The analysis of chromosome morphology and localization of 5S and 45S rDNA by FISH on flow-sorted chromosomes, revealed that four chromosomes (IV, V, VI, VIII) could be discriminated and sorted. Seventy-two SSR markers were used to characterize chromosome content of individual peaks on the flow karyotype. Out of them, 27 were included in the genetic linkage map and anchored genetic linkage groups to chromosomes. The sex determining locus was located on LG5, which was associated with peak V representing a chromosome with 5S rDNA locus. The results obtained in this study will support asparagus improvement by facilitating targeted marker development and gene isolation using flow-sorted chromosomes.

Development of Large-scale Cross-linking Mass Spectrometry

Cross-linking mass spectrometry (CLMS) provides distance constraints to study the structure of proteins, multiprotein complexes and protein-protein interactions which are critical for the understanding of protein function. CLMS is an attractive technology to bridge the gap between high-resolution structural biology techniques and proteomic-based interactome studies. However, as outlined in this review there are still several bottlenecks associated with CLMS which limit its application on a proteome-wide level. Specifically, there is an unmet need for comprehensive software that can reliably identify cross-linked peptides from large data sets. In this review we provide supporting information to reason that targeted proteomics of cross-links may provide the required sensitivity to reliably detect and quantify cross-linked peptides and that a reporter ion signature for cross-linked peptides may become a useful approach to increase confidence in the identification process of cross-linked peptides. In addition, the review summarizes the recent advances in CLMS workflows using the analysis of condensin complex in intact chromosomes as a model complex.

Rapid emergence of independent \u201cchromosomal lineages\u201d in silvered-leaf monkey triggered by Y/autosome translocation

Sex/autosome translocations are rare events. The only known example in catarrhines is in the silvered-leaf monkey. Here the Y chromosome was reciprocally translocated with chromosome 1. The rearrangement produced an X1X2Y1Y2 sex chromosome system. At least three chromosomal variants of the intact chromosome 1 are known to exist. We characterized in high resolution the translocation products (Y1 and Y2) and the polymorphic forms of the intact chromosome 1 with a panel of more than 150 human BAC clones. We showed that the translocation products were extremely rearranged, in contrast to the high level of marker order conservation of the other silvered-leaf monkey chromosomes. Surprisingly, each translocation product appeared to form independent “chromosome lineages”; each having a myriad of distinct rearrangements. We reconstructed the evolutionary history of the translocation products by comparing the homologous chromosomes of two other colobine species: the African mantled guereza and the Indian langur. The results showed a massive reuse of breakpoints: only 12, out of the 40 breaks occurred in domains never reused in other rearrangements, while, strikingly, some domains were used up to four times. Such frequent breakpoint reuse if proved to be a general phenomenon has profound implications for mechanisms of chromosome evolution.

Novel Structural Abnormalities Involving Chromosomes 1, 17 and 2 Identified by Fluorescence In Situ Hybridization (FISH) and/or Cytogenetic Karyotyping in Kelly and SH-SY5Y Human Neuroblastoma Cell Lines, Respectively

Abstract: MYCN amplification and 1p36 deletion are important poor prognostic factors in neuroblastoma. 1p36 deletion and unbalanced translocations involving chromosomes 1, 17 and 2p were often reported in neuroblastoma cell lines. We aimed to investigate novel chromosomal abnormalities that are likely to affect neuroblastoma progression in Kelly and SH-SY5Y cell lines. Therefore, we analyzed the metaphase chromosomes using fluorescence in situ hybridization (FISH) method with probes specific to the chromosome bands 2p24 and 1p36 in SH-SY5Y and Kelly, respectively. Moreover, the rearrangements of chromosomes 1 and 17 from Kelly were re-examined by cytogenetic karyotyping. FISH analysis shows duplication of chromosome 2p24 on the long arm of a partner chromosome resulted from an unbalanced translocation der(?9)t(2;?9)(p24;q?34) in SH-SY5Y, suggesting that duplication of 2p24 locus containing MYCN gene may contribute to triggering MYCN amplification in neuroblastoma. On the other hand, FISH and karyotype analyses reveal three copies of chromosome 1 that consist of an intact chromosome 1, an extra derivative chromosome 1 with terminal and interstitial deletions (:p32→q25::q41→qter), and another including only interstitial deletion (pter→q25::q41→qter), leading to monosomy of the long arm segment 1q25-q41 in Kelly. These results suggest that chromosome 1q25-q41 may contain one or more tumour suppressor genes important for neuroblastoma progression. Together, FISH analysis shows that an additional 1p36 locus in Kelly is translocated to the short arm of extra chromosome 17, where the p53 tumour suppressor gene is located. Consequently, these novel structural abnormalities involving chromosomes 1, 17 and 2 could be contributed to the tumourigenicity of neuroblastoma cells.