Metaphase Chromosome Spreads Research Articles

Analyzing sister chromatid cohesion in mammalian cells.

The metaphase chromosome spread technique and subsequent analysis of sister chromatid cohesion is used for (clinical) diagnosis of genetic abnormalities that can cause aberrant sister chromatid cohesion. In addition, the technique can be used to assess the contribution of novel genes to the cohesion establishment and maintenance pathways. Cells are swelled in a hypotonic solution and fixed in Carnoy's solution. Samples are then dropped onto glass slides, and the spread chromosomes are stained and visualized by microscopy. Defects in sister chromatid cohesion can be easily assessed using this method, examples of which are given.

Chromosome Instability Underlies Stem Cell Dysfunction and Neoplasia In Widespread Hematologic Disease Induced By Deregulated Cyclin E

The Fbw7 ubiquitin ligase controls the expression of a number of oncoprotein substrates including cyclin E, Notch, c-Jun, and c-Myc. Using a knock-in mouse model (cyclin ET74AT393A), in which mutations were introduced into the cyclin E1 allele (Ccne1) to disrupt Fbw7-mediated ubiquitination specifically, we previously found that cyclin E dysregulation in vivo induces anemia with defects in erythroid differentiation and morphologic dysplasia of erythroid progenitors. We also found that cyclin ET74AT393A mice have fewer hematopoietic stem cells (HSCs) during steady-state hematopoiesis compared to wild-type counterparts. We performed serial transplantation experiments to assay comprehensively the self-renewal and multi-lineage reconstitution capacities of cyclin ET74AT393A HSCs. Contrary to our expectations, cyclin ET74AT393A HSC self-renewal appears normal after three rounds of serial transplantation; however, we identified defects in their multi-lineage reconstitution function. In cyclin ET74A T393A bone marrow erythroid cells, induction of a p53-dependent DNA damage response pathway appears to promote compensated erythropoiesis. In cyclin ET74A T393A HSCs, we similarly observed induced expression of canonical p53 target genes. We studied the effect of p53-loss on cyclin ET74A T393A HSCs and found that p53-null; cyclin ET74A T393A HSCs exhibit defects in both self-renewal and multi-lineage reconstitution. By enumerating chromosomes in metaphase spreads, we found p53-null; cyclin ET74A T393A hematopoietic stem and progenitor cells (HSPCs) demonstrate significant chromosomal instability (CIN). Importantly, we can recapitulate the self-renewal defects and CIN of cyclin ET74A T393A HSPCs with intact p53 by treating recipient animals with a single dose of 5-fluorouracil (5-FU). Thus, chromosomal stability is a key determinant for the maintenance of HSC self-renewal, and hematologic stress appears to unmask the potential for impaired Fbw7-dependent cyclin E ubiquitination to engender CIN in the presence of intact p53. Moreover, CIN is a characteristic feature of fatal T-cell malignancies that ultimately develop in recipients of cyclin ET74A T393A; p53-null HSCs. In pre-malignant thymocytes isolated from recipients of cyclin ET74A T393A; p53-null HSCs, aneuploidy is associated with the marked potentiation of cyclin E kinase activity in these cells by p53-loss. In malignant thymocytes, comparative genome hybridization analysis demonstrates clonal CIN associated with deregulated cyclin E expression combined with p53-loss. In toto, our data demonstrate the functional importance of cyclin E regulation by the Fbw7 ubiquitin ligase to the hematopoietic system and highlight CIN as a key mechanism underlying HSC dysfunction and malignancy induced by deregulated cyclin E in vivo. Disclosures:No relevant conflicts of interest to declare.

MetaSel: a metaphase selection tool using a Gaussian-based classification technique

BackgroundIdentification of good metaphase spreads is an important step in chromosome analysis for identifying individuals with genetic disorders. The process of finding suitable metaphase chromosomes for accurate clinical analysis is, however, very time consuming since they are selected manually. The selection of suitable metaphase chromosome spreads thus represents a major bottleneck for conventional cytogenetic analysis. Although many algorithms have been developed for karyotyping, none have adequately addressed the critical bottleneck of selecting suitable chromosome spreads. In this paper, we present a software tool that uses a simple rule-based system to efficiently identify metaphase spreads suitable for karyotyping.ResultsThe chromosome shapes can be classified by the software into four main classes. The first and the second classes refer to individual chromosomes with straight and skewed shapes, respectively. The third class is characterized as those chromosomes with overlapping bodies and the fourth class is for the non-chromosome objects. Good metaphase spreads should largely contain chromosomes of the first and the second classes, while the third class should be kept minimal. Several image parameters were examined and used for creating rule-based classification. The threshold value for each parameter is determined using a statistical model. We observed that the Gaussian model can represent the empirical probability density function of the parameters and, hence, the threshold value can be easily determined. The proposed rules can efficiently and accurately classify the individual chromosome with > 90% accuracy.ConclusionsThe software tool, termed MetaSel, was developed. Using the Gaussian-based rules, the tool can be used to quickly rank hundreds of chromosome spread images so as to assist cytogeneticists to perform karyotyping effectively. Furthermore, MetaSel offers an intuitive, yet comprehensive, workflow to assist karyotyping, including tools for editing chromosome (split, merge and fix) and a karyotyping editor (moving, rotating, and pairing homologous chromosomes). The program can be freely downloaded from "http://www4a.biotec.or.th/GI/tools/metasel".

Distribution of sex chromosomes (XY) in lymphocyte metaphase spreads of dairy bulls

Position of autosome and sex chromosomes in metaphase spreads is grate concerned of Cytogeneticians worldwide to understand cell biology. A few isolated studies have been conducted for the distribution of chromosomes in metaphase spread. Our studies reveal that most sex chromosomes (XY) remain on periphery and semi-periphery, 84.16% for X and 86.97% for Y respectively, in round metaphase spreads. The application of sex chromosome position in metaphase spreads is to easily find out sex chromosomes under microscope even without banding patterns. An another application is to identify or confirm sex chromosomes in unknown species on which cytogenetic studies have not been performed.

129 The molecular mechanism of breakage at fragile site FRA16D

Replication stress induces physical breakage at discrete loci in chromosomes, which can be visualized on a metaphase chromosome spread. These common fragile sites (CFS) are conserved across species and are hotspots for sister chromatid recombination, viral integration, rearrangements, translocations, and deletions (Glover et al 2005). Despite multiple theories, the molecular mechanisms of CFS expression and genomic instability are still not well understood. The fragile site FRA16D is of special interest because it is the second most highly expressed fragile site and is located within the WWOX tumor suppressor gene. Previous data identified a polymorphic AT repeat within a FRA16D subregion called F1 that causes chromosome fragility and replication fork stalling in a yeast model (Zhang and Freudenreich 2007). Recently, we have found that breakage increases in an AT repeat length-dependent manner. Our results suggest that the AT repeat in the context of F1 forms a secondary structure, making the region more vulnerable to breakage.

Cytogenetic characterization of Devario aequipinnatus (McClelland, 1839) and Devario yuensis (Arunkumar and Tombi, 1998) (Cypriniformes: Cyprinidae) from Manipur, northeast India

Cytogenetic characteristics of Devario aequipinnatus (McClelland, 1839) and Devario yuensis (Arunkumar and Tombi, 1998) from northeast India were studied by examining metaphase chromosome spreads from gill, epithelial, and kidney cells. The diploid chromosome numbers of both species were 2n = 50. The karyotypes consisted of 3 pairs of metacentric, 17 pairs of submetacentric, 3 pairs of subtelocentric, and 2 pairs of acrocentric chromosomes having the arm numbers NF = 96 in D. aequipinnatus; and 5 pairs of metacentric, 13 pairs of submetacentric, 6 pairs of subtelocentric, and 1 pair of acrocentric chromosomes having the fundamental arm numbers NF = 98 in D. yuensis. No sex chromosomes were detected cytologically in either of the species. The present study is the first report on the karyology of these 2 species from northeast India. Comparison of the chromosome numbers of these 2 fishes with those of other members of closely related genera of the subfamily Danioninae shows that 2n = 50 could be considered as a modal diploid chromosome number in this fish subfamily.

북방전복, Haliotis discus hannai 3배체의 세포유전학적 연구

북방전복, Haliotis discus hannai의 3배체를 제온자극으로 유도하고 부화유생인 trochophore를 이용하여 염색체 표본을 만들었고, 유도된 3배체와 정상 2배체 북방전복은 실내 유수식의 동일 환경에서 51개월 동안 사육되었으며 채취된 혈구를 채취하여 DNA 함량 측정에 사용되었다. 2배체 및 유도된 3배체의 염색체 수를 조사한 결과, 2배체 염색체 수는 2n = 36으로 나타났고, 3배체의 경우에는 3n = 54로 나타나 3배체는 2배체에 비해 1.5배의 염색체 수를 나타내었다. Flow cytometry로 인간의 백혈구를 control로 하여 북방전복의 DNA 함량을 측정한 결과, 북방전복의 DNA 함량은 1.743 pg/cell이었으며 3배체 북방전복의 DNA함량은 2배체 전복의 1.49배의 DNA 함량을 나타내어 3배체 특성인 모계 2n DNA 함량과 부계 n DNA함량을 나타내었다. In this study, we invesgated a cytogenetic analysis of the Pacific triploid abalone, Haliotis discus hannai induced by low temperature treatment. We got a lot of mitotic metaphase chromosome spreads from the triploid and diploid Pacific abalones' hatched larvae (trochophores). The chromosome number of diploid abalone was 2n = 36 and that of triploid abalone was 3n = 54, so the chromosome number of triploid abalone was 1.5 times higher than that of diploid abalone. We developed a modified flow cytometric method for Pacific abalone from the existing methods. We uesd 51 months aged triploid and diploid Pacific abalones' hemolymph to get the DNA contents by flow cytometry. The DNA content of diploid abalone was 1.743 pg/cell and the DNA content of triploid abalone was 1.49 times higher than that of diploid one. It proved that triploid abalone was consisted with two sets of maternal diploid and one set of paternal genome.

Folate deficiency is associated with the formation of complex nuclear anomalies in the cytokinesis‐block micronucleus cytome assay

Chromosomal instability (CIN) is an important hallmark to oncogenesis and can be diagnosed morphologically by the presence of nuclear anomalies such as micronuclei (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBuds). We have identified additional nuclear anomalies formed under folate-deficient conditions, defined as "fused" nuclei (FUS), "circular" nuclei (CIR), and "horse-shoe" nuclei (HS) and investigated their suitability for inclusion as additional CIN biomarkers in the lymphocyte cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. Although the morphological appearance of FUS, CIR, and HS suggested an origin from multiple NPB in the fusion region between the two nuclei, the very low frequency of dicentric chromosomes in metaphase spreads from these cultures did not support this model. Fluorescence in situ hybridization (FISH) analysis of cytokinesis-blocked binucleated (BN) cells with peptide nucleic acid probes for telomeres and centromeres (PNA-FISH) revealed a high proportion of fusion regions contained both centromeric and telomeric DNA. This suggests that folate deficiency may disrupt the process of sister chromatid separation and chromosome segregation during mitosis. It was concluded that the FUS, CIR, and HS morphologies represent promising biomarkers of CIN that are sensitive to folate deficiency, and further validation and investigation of the mechanisms responsible for their formation is warranted.

Karyotype analysis of mithun (Bos frontalis) and mithun bull x Brahman cow hybrids

We examined the cytogenetics of mithun (Bos frontalis), a domesticated version of the Asian gaur, and hybrids (F(1) generation) produced by artificial insemination of Brahman cows (Bos indicus) with mithun semen. Reproductive potential was also examined in the F(1) generation and a backcrossed heifer for utilization of heterosis. Metaphase chromosome spreads were examined by conventional staining and fluorescence in situ hybridization hybridized with the entire chromosome 1 of mithun as a specific probe. Chromosome 1 of mithun was found to be equivalent to Bos taurus chromosomes 2 and 28. The karyotype of the female mithun (N = 4) comprised 58 chromosomes, including 54 acrocentric and four large submetacentric chromosomes, without the four acrocentric chromosomes found in the domesticated species B. indicus. However, one of the four female mithuns with a normal mithun phenotype had an abnormal karyotype (2n = 59), indicating introgression from B. taurus or B. indicus. The F(1) karyotypes (N = 6, 3♂3♀) of the mithun bull × Brahman cow cross had 2n = 59, intermediate between their parents; they were consistent heterozygous carriers with a centric fusion involving rob(2;28), as expected. Two pronounced red signals were seen in the mithun karyotypes, three red signals in the mithun × Brahman hybrids, and four red signals in the Brahman cattle, in good agreement with centric fusion of bovine rob(2;28). The female backcross hybrid (N = 1) with 2n = 59 had a similar chromosome configuration to the F(1) karyotypes and had rob(2;28). Such female backcross hybrids normally reproduce; however, the F(1) bulls (N = 3) had not yet generated normal sperm at 24 months.

P3-01-01: Geminin Overexpression Prevents the Completion of Topoisomerase IIa Chromosome Decatenation Leading to Aneuploidy in Human Mammary Epithelial Cells.

Abstract Topoisomerase IIα (TopoIIα) cleaves DNA in a reversible manner, making it a valuable target for agents such as etoposide that trap the enzyme in a covalent bond with the DNA end it cleaves and prevents DNA re-ligation and triggers cell death in cancer cells. However, development of resistance to these agents limits their therapeutic use. In this study, we examined therapeutic targeting of geminin for improving the therapeutic potential of TopoIIa agents. Human mammary epithelial (HME) and breast cancer cell lines were used. Geminin, TopoIIα, Cdc7 silencing was done using specific siRNAs. Transit or stable inducible overexpression of these proteins and CKI∈ were also used, as well as several pharmacological inhibitors that target TopoIIα, Cdc7, or CKI∈. We manipulated HME cells expressing H2B-GFP, in order to detect chromosome bridges. Immunoprecipitation and direct western blot were used to detect interactions between these proteins and their total expression, respectively, whereas interactions on chromosomal arms were detected using the TARDIS assay. TopoIIα phosphorylation by Cdc7 or CKI∈ was done using in vitro kinase assay. The TopoGen decatenation kit was used to measure TopoIIα decatenation activity. Finally, comet assay and metaphase chromosome spread were used to detect chromosome breakages and changes in chromosome condensation or numbers, respectively. We found that geminin and TopoIIα interact in G2/M/early G1 cells on chromosomes, that geminin recruits TopoIIα to chromosomal decatenation sites or vice versa, and that geminin silencing in HME cells triggers the formation of chromosome bridges through suppressing TopoIIα access to chromosomal arms. CKIε kinase phosphorylates and positively regulates TopoIIα chromosome localization and function. CKIε kinase overexpression or Cdc7 kinase silencing (also phosphorylates TopoIIα in vitro), restored DNA decatenation and chromosome segregation in geminin-silenced cells before triggering cell death. In vivo, at normal concentration, geminin recruits the desumoylating enzymes SENP1 and SENP2 to desumoylate chromosomal bound TopoIIα and promote its release from chromosomes following completion of DNA decatenation. In cells overexpressing geminin, premature departure of TopoIIα from chromosomes is thought to be due to the fact that geminin recruits more of these desumoylating enzymes, or recruits them earlier, to chromosomal bound TopoIIα. This triggers premature release of TopoIIα from chromosomes, which we propose induces aneuploidy in HME cells, since chromosome breakages generated through were not sensed and/or repaired and the cell cycle was not arrested. TopoIIα recruitment and its chromosome decatenation function require normal level of geminin. Geminin silencing induces a cytokinetic checkpoint in which Cdc7 phosphorylates TopoIIα and inhibits its chromosomal recruitment and decatenation function. Geminin overexpression prematurely desumoylates TopoIIα, triggering its premature departure from chromosomes and leading to chromosomal abnormalities and the formation of aneuploid, drug resistant cancer cells. We propose that therapeutic targeting of geminin is essential for improving the therapeutic potential of TopoIIα agents. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-01.

Application of the novel and convenient IR/MAR gene amplification technology to the production of recombinant protein pharmaceuticals

Amplification of DHFR gene in CHO cells by selection of MTx has been widely applied to the establishment of stable cell lines that efficiently produce recombinant protein pharmaceuticals. However, the DHFR/MTx technology was highly time-and labor-consuming. On the other hand, we had found that a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR) initiates gene amplification in mammalian cells, and it quite efficiently generate the chromosomal HSR and/or the extrachromosomal DMs [1,2]. This is a completely original technology of gene amplification, and we have revealed the mechanism why and how such plasmid may mimic gene amplification [2,3]. Now, we aimed to adopt this technology to the industrial production of recombinant protein pharmaceuticals. We constructed plasmids with or without IR/MAR, with several promoters for drug resistant gene, antibody gene or Fc receptor gene, and with various orientations of these elements. The plasmid DNA with several physical structures were transfected to human COLO 320DM or hamster CHO DG44 cells, with or without another DNA. The transformed cells were selected by various conditions. The polyclonal transformants or the cloned cells were evaluated by the protein production (ELISA), as well as by the structure of amplified region (FISH). As a result, the usage of IR/MAR technology enabled us to obtain cells, in which the introduced genes were amplified to a few hundreds to thousands copies per cells as DMs or HSR of various size and shape (Figure ​(Figure1),1), which depended both on the vector constructs and the host cell lines. Such stable cells with amplified genes could be obtained within one month, and the protein production was increased more than a hundred-fold compared with the case without IR/MAR. A cell clone showed the specific production rate that reached almost the highest reported for antibody protein (45 pg/cell/day). Furthermore, we have found several novel ways that further improve the protein production level. For example, the combination of the IR/MAR and the DHFR/MTx technologies synergistically work and far more rapidly and easily generate the cells of higher production rate than previously. Figure 1 The IR/MAR plasmid can generates DMs (A), HSR (B), Ladder-HSR (C), and Fine ladder-HSR (D). Among the metaphase chromosome spread, we detected the plasmid sequence by FISH in green. Gene expression was generally higher from DMs than HSR. However, DMs ... In conclusion, the IR/MAR technology is a novel highly-competitive technology for use in recombinant protein production, and it further has potentials for improvement.

Chromosome analysis by spectral karyotyping of spermatozoa from an oligoasthenozoospermic carrier of a 10; 21 reciprocal translocation

Cytogenetic analysis of germ-line cells prior to intracytoplasmic sperm injection (ICSI) treatment is thought to be necessary for infertile males with an identified chromosomal abnormality. We analyzed the chromosomal karyotype of human spermatozoa from an oligoasthenozoospermic carrier of a reciprocal translocation t(10; 21). Cytogenetic analysis of 39 spermatozoa was performed by spectral karyotyping (SKY) and by ICSI into mouse oocytes. The motile morphologically normal spermatozoa were injected into mouse oocytes. Of these spermatozoa, 38 (97.4%) were activated. Twenty-one (53.8%) of the activated oocytes formed two pronuclei. Metaphase chromosome spreads from 13 spermatozoa were analyzed. Only one spermatozoon was normal and 2 spermatozoa exhibited balanced translocation. Nine and one spermatozoa showed abnormalities related and unrelated to the translocation, respectively. The numbers of normal/balanced spermatozoa were lower than those in previous reports analyzing reciprocal translocations using a previously described technique involving penetrated golden hamster oocytes. After genetic counseling with the carrier and his partner, ICSI treatment was performed. Healthy female and male infants were delivered at 37 weeks gestation via a Caesarean section. The female infant was a carrier of the reciprocal translocation and the male infant was confirmed normal on prenatal diagnosis at 16 weeks gestation. For genetic counseling prior to ICSI treatment, the incidence of unbalanced type spermatozoa after swim-up or Percoll gradient treatment should be investigated and discussed with couples having fertility problems related to oligozoospermia autosomal structural abnormalities.

Geminin overexpression prevents the completion of topoisomerase IIα chromosome decatenation, leading to aneuploidy in human mammary epithelial cells

IntroductionThe nuclear enzyme topoisomerase IIα (TopoIIα) is able to cleave DNA in a reversible manner, making it a valuable target for agents such as etoposide that trap the enzyme in a covalent bond with the 5′ DNA end to which it cleaves. This prevents DNA religation and triggers cell death in cancer cells. However, development of resistance to these agents limits their therapeutic use. In this study, we examined the therapeutic targeting of geminin for improving the therapeutic potential of TopoIIα agents.MethodsHuman mammary epithelial (HME) cells and several breast cancer cell lines were used in this study. Geminin, TopoIIα and cell division cycle 7 (Cdc7) silencing were done using specific small interfering RNA. Transit or stable inducible overexpression of these proteins and casein kinase Iε (CKIε) were also used, as well as several pharmacological inhibitors that target TopoIIα, Cdc7 or CKIε. We manipulated HME cells that expressed H2B-GFP, or did not, to detect chromosome bridges. Immunoprecipitation and direct Western blot analysis were used to detect interactions between these proteins and their total expression, respectively, whereas interactions on chromosomal arms were detected using a trapped in agarose DNA immunostaining assay. TopoIIα phosphorylation by Cdc7 or CKIε was done using an in vitro kinase assay. The TopoGen decatenation kit was used to measure TopoIIα decatenation activity. Finally, a comet assay and metaphase chromosome spread were used to detect chromosome breakage and changes in chromosome condensation or numbers, respectively.ResultsWe found that geminin and TopoIIα interact primarily in G2/M/early G1 cells on chromosomes, that geminin recruits TopoIIα to chromosomal decatenation sites or vice versa and that geminin silencing in HME cells triggers the formation of chromosome bridges by suppressing TopoIIα access to chromosomal arms. CKIε kinase phosphorylates and positively regulates TopoIIα chromosome localization and function. CKIε kinase overexpression or Cdc7 kinase silencing, which we show phosphorylates TopoIIα in vitro, restored DNA decatenation and chromosome segregation in geminin-silenced cells before triggering cell death. In vivo, at normal concentration, geminin recruits the deSUMOylating sentrin-specific proteases SENP1 and SENP2 enzymes to deSUMOylate chromosome-bound TopoIIα and promote its release from chromosomes following completion of DNA decatenation. In cells overexpressing geminin, premature departure of TopoIIα from chromosomes is thought to be due to the fact that geminin recruits more of these deSUMOylating enzymes, or recruits them earlier, to bound TopoIIα. This triggers premature release of TopoIIα from chromosomes, which we propose induces aneuploidy in HME cells, since chromosome breakage generated through this mechanism were not sensed and/or repaired and the cell cycle was not arrested. Expression of mitosis-inducing proteins such as cyclin A and cell division kinase 1 was also increased in these cells because of the overexpression of geminin.ConclusionsTopoIIα recruitment and its chromosome decatenation function require a normal level of geminin. Geminin silencing induces a cytokinetic checkpoint in which Cdc7 phosphorylates TopoIIα and inhibits its chromosomal recruitment and decatenation and/or segregation function. Geminin overexpression prematurely deSUMOylates TopoIIα, triggering its premature departure from chromosomes and leading to chromosomal abnormalities and the formation of aneuploid, drug-resistant cancer cells. On the basis of our findings, we propose that therapeutic targeting of geminin is essential for improving the therapeutic potential of TopoIIα agents.

Telomere and 45S rDNA sequences are structurally linked on the chromosomes in Chrysanthemum segetum L.

Some reports have shown that nucleolar organizer regions are located at the telomeric region and have a structural connection with telomeres at the cellular level in many organisms. In this study, we found that all 45S ribosomal DNA (rDNA) signals were located at telomeric regions on the chromosomes in Chrysanthemum segetum L., and the 45S rDNA showed distinct signal patterns on different metaphase chromosome spreads. The bicolor fluorescence in situ hybridization experiment on the extended fibers revealed that telomere repeats were structurally connected with or interspersed into rDNA sequences. The close cytological structure relation between rDNA and telomere sequences led us to use PCR with combinations of the telomere primer and the rDNA primer to obtain some fragments, which were flanked by different rDNA and telomere primer sequences. One representative clone CHS2 contains closely connected rDNA and telomere sequences, suggesting that the telomere sequence invaded into the conserved rDNA sequence. In addition, the sequences of some PCR clones were flanked by the single telomeric primer sequence or the rDNA primer sequence. These results suggested that homologous recombination occurred between tandem repeat units of rDNA sequences or telomere repeats at the chromosome terminus.

FISHprep: A Novel Integrated Device for Metaphase FISH Sample Preparation

We present a novel integrated device for preparing metaphase chromosomes spread slides (FISHprep). The quality of cytogenetic analysis from patient samples greatly relies on the efficiency of sample pre-treatment and/or slide preparation. In cytogenetic slide preparation, cell cultures are routinely used to process samples (for culture, arrest and fixation of cells) and/or to expand limited amount of samples (in case of prenatal diagnostics). Arguably, this expansion and other sample pretreatments form the longest part of the entire diagnostic protocols spanning over 3–4 days. We present here a novel device with an integrated expansion chamber to culture, arrest and fix metaphase cells followed by a subsequent splashing protocol leading to ample metaphase chromosome spreads on a glass slide for metaphase FISH analysis. The device provides an easy, disposable, low cost, integrated solution with minimal handling for metaphase FISH slide preparation.

Karyotype, NORs, and C-banding analysis of Pseudophoxinus firati Bogutskaya, Küçük & Atalay, 2007 (Actinopterygii, Cyprinidae) in the Euphrates River, Turkey

Pseudophoxinus firati Bogutskaya, Küçük & Atalay, 2007 (Actinopterygii, Cyprinidae) living in the Euphrates River system were analyzed in terms of their karyotype, C-banding, and nucleolus organizer region properties. Metaphase chromosome spreads were obtained from kidney cells. The diploid chromosome number was found to be 2n = 50, of which 19 pairs were meta-submetacentric and 6 pairs were subtelocentric, and the fundamental number was found to be 88. No sex chromosome differentiation was observed. C-banding revealed heterochromatin in the pericentromeric regions of 6 pairs of chromosomes. NORs were identified on the short arms of 2 pairs of medium-sized submeta-subtelocentric chromosomes. This study may contribute to other cytogenetic research.

An asymptomatic 61‐year‐old man with BCR‐ABL‐positive bone marrow following autologous transplantation for multiple myeloma

An asymptomatic 61‐year‐old man with <i>BCR‐ABL</i>‐positive bone marrow following autologous transplantation for multiple myeloma

Immunostaining of modified histones defines high-level features of the human metaphase epigenome

BackgroundImmunolabeling of metaphase chromosome spreads can map components of the human epigenome at the single cell level. Previously, there has been no systematic attempt to explore the potential of this approach for epigenomic mapping and thereby to complement approaches based on chromatin immunoprecipitation (ChIP) and sequencing technologies.ResultsBy immunostaining and immunofluorescence microscopy, we have defined the distribution of selected histone modifications across metaphase chromosomes from normal human lymphoblastoid cells and constructed immunostained karyotypes. Histone modifications H3K9ac, H3K27ac and H3K4me3 are all located in the same set of sharply defined immunofluorescent bands, corresponding to 10- to 50-Mb genomic segments. Primary fibroblasts gave broadly the same banding pattern. Bands co-localize with regions relatively rich in genes and CpG islands. Staining intensity usually correlates with gene/CpG island content, but occasional exceptions suggest that other factors, such as transcription or SINE density, also contribute. H3K27me3, a mark associated with gene silencing, defines a set of bands that only occasionally overlap with gene-rich regions. Comparison of metaphase bands with histone modification levels across the interphase genome (ENCODE, ChIP-seq) shows a close correspondence for H3K4me3 and H3K27ac, but major differences for H3K27me3.ConclusionsAt metaphase the human genome is packaged as chromatin in which combinations of histone modifications distinguish distinct regions along the euchromatic chromosome arms. These regions reflect the high-level interphase distributions of some histone modifications, and may be involved in heritability of epigenetic states, but we also find evidence for extensive remodeling of the epigenome at mitosis.

Development of fibroblast cell lines from the cow used to sequence the bovine genome

Source/methods: Two cell lines were initiated from 6-mm skin biopsies obtained from the Hereford cow whose DNA was used in sequencing the bovine genome. Biopsies were mechanically disrupted and digested in 0.25% trypsin to release individual cells. Immunohistochemistry was performed using a HistoMark Biotin Streptavidin Kit – AP System (KPL), as per the manufacturer s instructions. The primary antibodies used were mouse monoclonals against cytokeratin (1/5/10/14; Vector Laboratories, Inc.) and vimentin (Invitrogen), and the secondary antibody used was goat-anti-mouse IgG (KPL). The colorimetric portion of the analysis was performed using the HistoMark RED Phosphatase System (KPL). Metaphase chromosome spreads were prepared for cytogenetic analysis by the Cytogenetics Laboratory at the Corriel Institute for Medical Research, Camden, NJ, USA, as previously described. See Appendix S1 for more information.

A fluorescence in situ hybridization system for karyotyping soybean.

The development of a universal soybean (Glycine max [L.] Merr.) cytogenetic map that associates classical genetic linkage groups, molecular linkage groups, and a sequence-based physical map with the karyotype has been impeded due to the soybean chromosomes themselves, which are small and morphologically homogeneous. To overcome this obstacle, we screened soybean repetitive DNA to develop a cocktail of fluorescent in situ hybridization (FISH) probes that could differentially label mitotic chromosomes in root tip preparations. We used genetically anchored BAC clones both to identify individual chromosomes in metaphase spreads and to complete a FISH-based karyotyping cocktail that permitted simultaneous identification of all 20 chromosome pairs. We applied these karyotyping tools to wild soybean, G. soja Sieb. and Zucc., which represents a large gene pool of potentially agronomically valuable traits. These studies led to the identification and characterization of a reciprocal chromosome translocation between chromosomes 11 and 13 in two accessions of wild soybean. The data confirm that this translocation is widespread in G. soja accessions and likely accounts for the semi-sterility found in some G. soja by G. max crosses.