Towards many colors in FISH on 3D-preserved interphase nuclei

The article reviews the existing methods of multicolor FISH on nuclear targets, first of all, interphase chromosomes. FISH proper and image acquisition are considered as two related components of a single process. We discuss (1) M-FISH (combinatorial labeling + deconvolution + widefield microscopy); (2) multicolor labeling + SIM (structured illumination microscopy); (3) the standard approach to multicolor FISH + CLSM (confocal laser scanning microscopy; one fluorochrome - one color channel); (4) combinatorial labeling + CLSM; (5) non-combinatorial labeling + CLSM + linear unmixing. Two related issues, deconvolution of images acquired with CLSM and correction of data for chromatic Z-shift, are also discussed. All methods are illustrated with practical examples. Finally, several rules of thumb helping to choose an optimal labeling + microscopy combination for the planned experiment are suggested. Copyright (c) 2006 S. Karger AG, Basel

Chromosomal bar codes produced by multicolor fluorescence in situ hybridization with multiple YAC clones and whole chromosome painting probes

Colored chromosome staining patterns, termed chromosomal ‘bar codes’ (CBCs), were obtained on human chromosomes by fluorescence in situ hybridization (FISH) with pools of Alu-PCR products from YAC dones containing human DNA inserts ranging from 100 kbp to 1 Mbp. In contrast to conventional G- or R-bands, the chromosomal position, extent, Individual color and relative signal intensity of each ‘bar’ could be modified depending on probe selection and labeling procedures. Alu-PCR amplification products were generated from 31 YAC clones which mapped to 37 different chromosome bands. For multiple color FISH, Alu-PCR amplification products from various clones were either biotinylated or labeled with digoxigenin. Probes from up to twenty YAC clones were used simultaneously to produce CBCs on selected human chromosomes. Evaluation using a cooled CCD camera and digital image analysis confirmed the high reproducibility of the bars from one metaphase spread to another. Combinatorial FISH with mixtures of whole chromosome paint probes was applied to paint seven chromosomes simultaneously in different colors along with a set of YAC clones which map to these chromosomes. We discuss the potential to construct analytical chromosomal bar codes adapted to particular needs of cytogenetic investigations and automated image analysis

Chromosomal Rearrangements in Post-Chernobyl Papillary Thyroid Carcinomas: Evaluation by Spectral Karyotyping and Automated Interphase FISH

Structural genomic rearrangements are frequent findings in human cancers. Therefore, papillary thyroid carcinomas (PTCs) were investigated for chromosomal aberrations and rearrangements of the RET proto-oncogene. For this purpose, primary cultures from 23 PTC have been established and metaphase preparations were analysed by spectral karyotyping (SKY). In addition, interphase cell preparations of the same cases were investigated by fluorescence in situ hybridisation (FISH) for the presence of RET/PTC rearrangements using RET-specific DNA probes. SKY analysis of PTC revealed structural aberrations of chromosome 11 and several numerical aberrations with frequent loss of chromosomes 20, 21, and 22. FISH analysis for RET/PTC rearrangements showed prevalence of this rearrangement in 72% (16 out of 22) of cases. However, only subpopulations of tumour cells exhibited this rearrangement indicating genetic heterogeneity. The comparison of visual and automated scoring of FISH signals revealed concordant results in 19 out of 22 cases (87%) indicating reliable scoring results using the optimised scoring parameter for RET/PTC with the automated Metafer4 system. It can be concluded from this study that genomic rearrangements are frequent in PTC and therefore important events in thyroid carcinogenesis

Parasitic influences on the host genome using the molluscan model organism biomphalaria glabrata

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.The freshwater snail Biomphalaria glabrata is an intermediate host for Schistosoma mansoni parasites, causing one of the most prevalent parasitic infections in mammals, known as schistosomiasis (Bilharzia). Due to its importance in the spread of the disease B. glabrata has been selected for whole genome sequencing and is now a molluscan model organism. In order to aid the sequencing project and to understand the structure and organisation of B. glabrata’s genome at the chromosomal level, a G-banded karyotype has been established. Unlike in any other previous reports, two heteromorphic chromosomes have been identified in the genome of B. glabrata and for the first time snail ideograms have been produced. In addition to characterising the snail chromosomes, a methodology for mapping single copy B. glabrata genes onto these chromosomes has also been established, and 4 genes have successfully been mapped using fluorescence in situ hybridisation. In the relationship between a parasite and a host organism, it is of fundamental importance to understand the basic biology and interfere with the life cycle to reveal how the parasite controls and elicits host gene expression for its own benefit. This study is also directly addressing this aspect of host – parasite interactions by investigating the effects of schistosome infection on the genome and cell nuclei of the host snail B. glabrata. Upon infection with S. mansoni miracidia, genes known to be involved in the host response to the parasite are dramatically relocated within the interphase snail nuclei. These events are in conjunction with the up-regulation of gene expression, indicating a parasite induced nuclear event. Moreover, a differential response between the schistosome-resistant and schistosome-susceptible snails is also reported. This is the first time this has been described in a host – pathogen relationship. The precise organisation of the genome is critical for its correct functioning. The genome is non-randomly organised and this level of organisation is very much influenced by the nuclear architecture. Being a molluscan model organism with the availability of a unique cell line, B. glabrata is a remarkable organism for the studies of nuclear and genome biology. For this reason, in this thesis the snail nuclear architecture was also investigated. For the first time PML bodies, transcription factories, and nuclear myosin 1 beta have been visualised in the snail nuclei. A heat shock system was also developed to study the role of these structures in the snail. Upon heat stimuli gene loci were found to reposition and co-localise with transcription factories, which was in parallel with the up-regulation of gene expression. The mechanism of this genome reorganisation was explored by investigating nuclear motor structures in the snail. By using a motor inhibitor on snail cells, gene repositioning and subsequent expression after heat shock was blocked. This is the first time this has been shown in any organism. Thus, due to the ease of use of the snails with respect to maintenance, handling, and treatments, B. glabrata is making a very useful new model organism to study spatial genomic events