Daicel Arbor Biosciences present on the design and synthesis of custom FISH probes for use in various cytogenetic applications.

Protocol for preparing metaphase chromosome spreads in human cells

Polycomb group (PcG) proteins silence gene expression by chemically and physically modifying chromatin. A subset of PcG target loci are compacted and cluster in the nucleus; a conformation that is thought to contribute to gene silencing. However, how these interactions influence gross nuclear organization and their relationship with transcription remains poorly understood. Here we examine the role of Polycomb-repressive complex 1 (PRC1) in shaping 3D genome organization in mouse embryonic stem cells (mESCs). Using a combination of imaging and Hi-C analyses, we show that PRC1-mediated long-range interactions are independent of CTCF and can bridge sites at a megabase scale. Impairment of PRC1 enzymatic activity does not directly disrupt these interactions. We demonstrate that PcG targets coalesce in vivo, and that developmentally induced expression of one of the target loci disrupts this spatial arrangement. Finally, we show that transcriptional activation and the loss of PRC1-mediated interactions are separable events. These findings provide important insights into the function of PRC1, while highlighting the complexity of this regulatory system.

Chromosome painting is a useful technique for distinguishing specific chromosomes (fragments), elucidating the genetic relationships of different genomes or chromosomes, and identifying chromosomal rearrangements. The development of chromosome- or genome-specific probes is fundamental for chromosome painting. The possibility for developing such probes specifically painting homoeologous chromosomes in allopolyploid species has been questioned since that chromosomes belonging to the same homoeologous group share highly conserved sequences. In the present study, we attempted to construct a wheat chromosome 4D-specific oligo probe library by selecting 4D-specific sequences in reference genome of common wheat cv. Chinese Spring (CS, 2n = 6x = 42, AABBDD). The synthesized library contains 27,392 oligos. Oligo painting using the probe library confirmed its specificity, shown by that only chromosome 4D could be painted in three wheat genotypes and CS nulli-tetrasomic line N4AT4D. Oligo painting was successfully used to define the 4D breakpoints in CS deletion lines involving 4D and two wheat-Haynaldia villosa 4D–4V translocation lines. Thirteen wheat relatives and a Triticum durum-H. villosa amphiploid were used for oligo painting. Except the 4D in two Aegilops tauschii accessions, the 4M in Ae. comosa and 4U in Ae. umbellulata could be painted. In tetraploid Ae. ventricosa, both 4D and 4M could be painted; however, the signal intensity of 4M was less compared with 4D. No painted chromosome was observed for the other alien species. This indicated that the relationship among D/M/U was closer than that among D/A/B as well as D with genomes H/R/Ss/Sc/Y/P/N/J. Our successful development of 4D-specific oligo probe library may serve as a model for developing oligo probes specific for other homoeologous chromosomes.

Acute promyeloid leukemia (APL) is characterized by the oncogenic fusion protein PML-RARα, a major etiological agent in APL. However, the molecular mechanisms underlying the role of PML-RARα in leukemogenesis remain largely unknown.

Abstract Drosophila males have evolved a unique system of chromosome segregation in meiosis that lacks recombination. Chromosomes pair at selected sequences suggesting that early steps of meiosis may also differ in this organism… Diploid germline cells must undergo two consecutive meiotic divisions before differentiating as haploid sex cells. During meiosis I, homologs pair and remain conjoined until segregation at anaphase. Drosophila melanogaster spermatocytes are unique in that the canonical events of meiosis I including synaptonemal complex formation, double-strand DNA breaks, and chiasmata are absent. Sex chromosomes pair at intergenic spacer sequences within the ribosomal DNA (rDNA). Autosomes pair at numerous euchromatic homologies, but not at heterochromatin, suggesting that pairing may be limited to specific sequences. However, previous work generated from genetic segregation assays or observations of late prophase I/prometaphase I chromosome associations fail to differentiate pairing from maintenance of pairing (conjunction). Here, we separately examined the capability of X euchromatin to pair and conjoin using an rDNA-deficient X and a series of Dp(1;Y) chromosomes. Genetic assays showed that duplicated X euchromatin can substitute for endogenous rDNA pairing sites. Segregation was not proportional to homology length, and pairing could be mapped to nonoverlapping sequences within a single Dp(1;Y). Using fluorescence in situ hybridization to early prophase I spermatocytes, we showed that pairing occurred with high fidelity at all homologies tested. Pairing was unaffected by the presence of X rDNA, nor could it be explained by rDNA magnification. By comparing genetic and cytological data, we determined that centromere proximal pairings were best at segregation. Segregation was dependent on the conjunction protein Stromalin in Meiosis, while the autosomal-specific Teflon was dispensable. Overall, our results suggest that pairing may occur at all homologies, but there may be sequence or positional requirements for conjunction.

Spatial organization of chromosome territories and interactions between interphase chromosomes themselves, as well as with the nuclear periphery, play important roles in epigenetic regulation of the genome function. However, the interplay between inter-chromosomal contacts and chromosome-nuclear envelope attachments in an organism’s development is not well-understood. To address this question, we conducted microscopic analyses of the three-dimensional chromosome organization in malaria mosquitoes. We employed multi-colored oligonucleotide painting probes, spaced 1 Mb apart along the euchromatin, to quantitatively study chromosome territories in larval salivary gland cells and adult ovarian nurse cells of Anopheles gambiae, An. coluzzii, and An. merus. We found that the X chromosome territory has a significantly smaller volume and is more compact than the autosomal arm territories. The number of inter-chromosomal, and the percentage of the chromosome–nuclear envelope, contacts were conserved among the species within the same cell type. However, the percentage of chromosome regions located at the nuclear periphery was typically higher, while the number of inter-chromosomal contacts was lower, in salivary gland cells than in ovarian nurse cells. The inverse correlation was considerably stronger for the autosomes. Consistent with previous theoretical arguments, our data indicate that, at the genome-wide level, there is an inverse relationship between chromosome-nuclear envelope attachments and chromosome–chromosome interactions, which is a key feature of the cell type-specific nuclear architecture.

A novel tetraploid S. spontaneum with basic chromosome x = 10 was discovered, providing us insights in the origin and evolution in Saccharum species.

Fluorescence in situ hybridization using probes based on oligonucleotides (oligo-FISH) is a useful tool for chromosome identification and karyotype analysis. Here we developed two oligo-FISH probes that allow the identification of each of the 12 pairs of chromosomes in rice (Oryza sativa). These two probes comprised 25 717 (green) and 25 215 (red) oligos (45 nucleotides), respectively, and generated 26 distinct FISH signals that can be used as a barcode to uniquely label each of the 12 pairs of rice chromosomes. Standard karyotypes of rice were established using this system on both mitotic and meiotic chromosomes. Moreover, dual-color oligo-FISH was used to characterize diverse chromosomal abnormalities. Oligo-FISH analyses using these probes in various wild Oryza species revealed that chromosomes from the AA, BB or CC genomes generated specific and intense signals similar to those in rice, while chromosomes with the EE genome generated less specific signals and the FF genome gave no signal. Together, the oligo-FISH probes we established will be a powerful tool for studying chromosome variations and evolution in the genus Oryza.