Analysis Of Nuclear Organization Research Articles

Chromatin Conformation Capture-Based Analysis of Nuclear Architecture.

Nuclear organization and higher-order chromosome structure in interphase nuclei are thought to have important effects on fundamental biological processes, including chromosome condensation, replication, and transcription. Until recently, however, nuclear organization could only be analyzed microscopically. The development of chromatin conformation capture (3C)-based techniques now allows a detailed look at chromosomal architecture from the level of individual loci to the entire genome. Here we provide a robust Hi-C protocol, allowing the analysis of nuclear organization in nuclei from different wild-type and mutant plant tissues. This method is quantitative and provides a highly efficient and comprehensive way to study chromatin organization during plant development, in response to different environmental stimuli, and in mutants disrupting a variety of processes, including epigenetic pathways regulating gene expression.

Localizing DNA and RNA within nuclei and chromosomes by fluorescence in situ hybridization

The enormous potential of in situ hybridization derives from the unique ability of this approach to directly couple cytological and molecular information. In recent years, there has been a surge of success in powerful new applications, resulting from methodologic advances that bring the practical capabilities of this technology closer to its theoretical potential. A major advance has been improvements that enable, with a high degree of reproducibility and efficiency, precise visualization of single sequences within individual metaphase and interphase cells. This has implications for gene mapping, the analysis of nuclear organization, clinical cytogenetics, virology, and studies of gene expression. This article discusses the current state of the art of fluorescence in situ hybridization, with emphasis on applications to human genetics, but including brief discussions on studies of nuclear DNA and RNA organization, and on applications to clinical genetics and virology. Although a review of all of the literature in this field is not possible here, many of the major contributions are summarized along with recent work from our laboratory.

Interphase and Metaphase Resolution of Different Distances Within the Human Dystrophin Gene

Fluorescence in situ hybridization makes possible direct visualization of single sequences not only on chromosomes, but within decondensed interphase nuclei, providing a potentially powerful approach for high-resolution (1 Mb and below) gene mapping and the analysis of nuclear organization. Interphase mapping was able to extend the ability to resolve and order sequences up to two orders of magnitude beyond localization on banded or unbanded chromosomes. Sequences within the human dystrophin gene separated by less than 100 kb to 1 Mb were visually resolved at interphase by means of standard microscopy. In contrast, distances in the 1-Mb range could not be ordered on the metaphase chromosome length. Analysis of sequences 100 kb to 1 Mb apart indicates a strong correlation between interphase distance and linear DNA distance, which could facilitate a variety of gene-mapping efforts. Results estimate chromatin condensation up to 1 Mb and indicate a comparable condensation for different cell types prepared by different techniques.

Fluorescence in situ hybridization to interphase cell nuclei in suspension allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organization.

Fluorescence hybridization to interphase nuclei in liquid suspension allows quantification of chromosome-specific DNA sequences using flow cytometry and the analysis of the three-dimensional positions of these sequences in the nucleus using fluorescence microscopy. The three-dimensional structure of nuclei is substantially intact after fluorescence hybridization in suspension, permitting the study of nuclear organization by optical sectioning. Images of the distribution of probe and total DNA fluorescence within a nucleus are collected at several focal planes by quantitative fluorescence microscopy and image processing. These images can be used to reconstruct the three-dimensional organization of the target sequences in the nucleus. We demonstrate here the simultaneous localization of two human chromosomes in an interphase nucleus using two probe labeling schemes (AAF and biotin). Alternatively, dual-beam flow cytometry is used to quantify the amount of bound probe and total DNA content. We demonstrate that the intensity of probe-linked fluorescence following hybridization is proportional to the amount of target DNA over a 100-fold range in target content. This was shown using four human/hamster somatic cell hybrids carrying different numbers of human chromosomes and diploid and tetraploid human cell lines hybridized with human genomic DNA. We also show that populations of male, female, and XYY nuclei can be discriminated by measuring their fluorescence intensity following hybridization with a Y-chromosome-specific repetitive probe. The delay in the increase in Y-specific fluorescence until the end of S-phase in consistent with the results recorded in previous studies indicating that these sequences are among the last to replicate in the genome. A chromosome-17-specific repetitive probe is used to demonstrate that target sequences as small as one megabase (Mb) can be detected using fluorescence hybridization and flow cytometry.(ABSTRACT TRUNCATED AT 250 WORDS)