Accardo Maria Carmela, Dimitri Patrizio
Dipartimento di Genetica e Biologia Molecolare and Istituto Pasteur-Fondazione Cenci-Bolognetti, Università "La Sapienza", Rome, Italy.
Methods Mol Biol. 2010;659:389-400. doi: 10.1007/978-1-60761-789-1_30.
The organization of eukaryotic chromosomes into euchromatin and heterochromatin represents an enigmatic aspect of genome evolution. Constitutive heterochromatin is a basic, yet still poorly understood component of eukaryotic genomes and its molecular characterization by means of standard genomic approaches is intrinsically difficult. Drosophila melanogaster polytene chromosomes do not seem to be particularly useful to map heterochromatin sequences because the typical features of heterochromatin, organized as it is into a chromocenter, limit cytogenetic analysis. In contrast, constitutive heterochromatin has been well-defined at the cytological level in mitotic chromosomes of neuroblasts and has been subdivided into several bands with differential staining properties. Fluorescence in situ hybridization (FISH) using Bacterial Artificial Chromosomes (BAC) probes that carry large genomic portions defined by sequence annotation has yielded a "revolution" in the field of cytogenetics because it has allowed the mapping of multiple genes at once, thus rendering constitutive heterochromatin amenable to easy and fast cytogenetics analyses. Indeed, BAC-based FISH approaches on Drosophila mitotic chromosomes have made it possible to correlate genomic sequences to their cytogenetic location, aiming to build an integrated map of the pericentric heterochromatin. This chapter presents our standard protocols for BAC-based FISH, aimed at mapping large chromosomal regions of mitotic heterochromatin in Drosophila melanogaster.
真核生物染色体组织成常染色质和异染色质是基因组进化中一个神秘的方面。组成型异染色质是真核生物基因组的一个基本组成部分，但人们对其了解仍然很少，通过标准基因组方法对其进行分子表征本质上是困难的。果蝇多线染色体似乎对绘制异染色质序列不是特别有用，因为异染色质组织成染色中心的典型特征限制了细胞遗传学分析。相比之下，组成型异染色质在神经母细胞有丝分裂染色体的细胞学水平上已经得到了很好的定义，并被细分为具有不同染色特性的几个带。使用携带由序列注释定义的大片段基因组的细菌人工染色体（BAC）探针进行荧光原位杂交（FISH）在细胞遗传学领域产生了一场“革命”，因为它允许一次定位多个基因，从而使组成型异染色质易于进行快速的细胞遗传学分析。事实上，基于BAC的FISH方法应用于果蝇有丝分裂染色体，使得将基因组序列与其细胞遗传学位置相关联成为可能，目的是构建一个着丝粒周围异染色质的综合图谱。本章介绍了我们基于BAC的FISH标准方案，旨在绘制果蝇有丝分裂异染色质的大染色体区域。
