Methods 40 (2006) 360–364 www.elsevier.com/locate/ymeth 1046-2023/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2006.09.001 Functional genomics of histone modiWcation and non-histone chromosomal proteins using the polytene chromosomes of Drosophila Joel C. Eissenberg ¤ Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104, USA Accepted 16 July 2006 Abstract Knowing the genomic distribution of chromosomal proteins and of histone modiWcations provides essential insight into function. The giant polytene chromosomes of the Drosophila larval salivary glands provide a high-resolution genomic map with a resolution exceeded only by chromatin immunoprecipitation. ImmunoXuorescence localization of chromosomal proteins and speciWc post-translational mod- iWcations of histones is a simple and rapid tool for the functional genomics of chromosomal proteins.  2006 Elsevier Inc. All rights reserved. Keywords: Drosophila; Polytene chromosomes; ImmunoXuorescence 1. Introduction In his standard reference “A photographic representa- tion and interpretation of the polytene chromosomes of Drosophila melanogaster salivary glands,” [16] begins with the following encomium: “Cytogeneticists owe an enduring debt to C.B. Bridges (and to his son, P.N. Bridges) whose maps of the polytene chromosomes of D. melanogaster salivary glands ƒ have long served as the standards of reference for recording chromosome breakpoints, gene and puV locations, and all other features of cytogenetic interest.” More than 70 years after the Wrst maps were published [6], the salivary gland polytene chromosomes continue to be an invaluable tool in dissecting the molecular interac- tions that underlie gene expression and other aspects of nuclear metabolism. Nearly complete ampliWcation of chromosomal material and nearly perfect alignment of all homologous strands cooperate to generate a detailed and stereotypical pattern of bands, constrictions and puVs that provide the landmarks for detailed mapping using a com- pound light microscope. The sequenced Drosophila genome has been mapped to speciWc bands and interbands. The average DNA content per band is ca. 25–30 kb [27,5], providing a degree of cytological resolution without paral- lel in any other organism. However, other than the diVeren- tial ampliWcation of euchromatin over heterochromatin, polytene chromatin diVers in no measurable respect from the chromatin of any other kind of interphase cell. In the age of chromatin immunoprecipitation (ChIP) and gene chip microarrays, it has become possible to map the distribution of speciWc chromosomal proteins in virtually any cell type to a resolution, in some cases, of 250– 500 bp. These experiments are expensive and time-consum- ing, the more so if comparisons between two or more proteins are required. In contrast, immunolocalization of proteins on Drosophila polytene chromosomes aVords a rapid assay for genome-wide distribution and co-localiza- tion of chromosomal proteins at a resolution only an order of magnitude less than ChIP. In certain cases, the resolution can approach that of ChIP (see below). Since Drosophila expresses homologs of nearly all of the chromosomal pro- teins found in other higher eukaryotes, as well as most or all covalent histone modiWcations, immunoXuorescence * Fax: +1 314 977 9205. E-mail address: eissenjc@slu.edu