Sergey Lavrov Jrme Djardin and Giacomo Cavalli 1 Introduction

Polytene chromosomes result from subsequent cycles of DNA replication that are not followed by nuclear division. In Drosophila, this occurs in the majority of larval tissues and is most prominent in salivary glands, where up to eleven rounds of replication events may occur. Because the replicated chromatids remain tightly aligned, this process leads to pairing of up to 2048 DNA strands, giving rise to a highly reproducible banding pattern (1). This exceptional structure allows cytological analysis of genes and their associated proteins with a relatively high resolution. Up to 5000 condensed bands separated by less condensed interband chromatin regions can be well resolved with electron microscopy, whereas conventional optical microscopy techniques allow about 1000 bands and interbands to be distinguished. Because the size of Drosophila genomic euchromatin is about 120 Mb, the level of resolution on a linear scale is in the order of 10-50 kilobases (kb), depending on the local degree of condensation of the chromosomal region of interest. Therefore, poly-tene chromosomes represent a formidable tool for cytological analysis of biological processes. Moreover, polytene chromosome assays can be readily implemented in most laboratories, because they require only standard transmission and fluorescence microscopy equipment and reagents and do not demand great technical expertise.

Polytene chromosomes can be used in many different experimental approaches. Early studies used them to investigate chromosome structure and organization. More recently, practical applications have often aimed at identifying the cyto-logical position of cloned genes and transgene insertions by in situ DNA

From: Methods in Molecular Biology, vol. 247: Drosophila Cytogenetics Protocols Edited by: D. S. Henderson © Humana Press Inc., Totowa, NJ

hybridization or at determining by immunostaining techniques whether proteins of interest are chromosomally associated. Double-labeling experiments allow one to study colocalization of chromosome-associated proteins (2,3). Polytene chromosome assays are also useful for mapping domains of proteinprotein interaction. This can be done by studying colocalization of interacting polypeptides in the presence of different types of mutation in each of them (4,5). Other applications involve dynamic studies of protein distribution in response to environmental stimuli such as heat shock (6,7), induction of gene expression by transcriptional activators (8,9) and treatment by agents like ribo-nucleases (10) or chemical inhibitors of transcription (11).

One common application is to determine whether a particular DNA sequence is associated with a protein of interest. Mapping the chromosomal location of transcriptional regulators can indicate if they are associated with loci containing putative target genes. A direct proof of binding to a regulatory element can be obtained by showing that a transgenic copy of the element induces an ectopic binding site on polytene chromosomes. Traditionally, this is achieved by two separate experiments. First, the transgene insertion is mapped by in situ DNA hybridization. Second, on separate preparations, immunostaining is performed in the transgenic line and compared to a wild-type background. Finally, the binding pattern is analyzed in order to determine if an additional binding site can be detected in the region of insertion of the transgene. This approach has been successfully used in many cases (12-14), but it has the major disadvantage that DNA hybridization and protein immunostaining are done on separate slides. Therefore, it can only be applied if the number of binding sites for the protein of interest is relatively small and, in particular, in the absence of endogenous binding sites in the cytological region of interest. Unfortunately, many chromosomal proteins display hundreds of binding sites in the genome (15,16). In this case, it is almost impossible to unambiguously determine if the transgene induces an ectopic binding site. We describe here a method that combines immunostaining of proteins and fluorescent in situ hybridization (immuno-FISH) for direct visualization of a specific DNA fragment and a protein of interest on the same chromosome. This method allows more refined mapping of protein binding sites compared to genes of interest and it can be widely used to map binding to both endogenous genes and transgenic insertions.

The method consists of two parts: In the first part, fluorescent protein staining of polytene chromosome preparations is performed. After immunostaining, a FISH protocol is applied in order to detect the DNA of interest with a different fluorochrome from the one used for immunostaining. Some protein epitopes and antibodies survive the FISH procedure and this allows image acquisition of DNA and protein staining on one slide using specific filter sets. When the protein staining does not survive FISH, it is still possible to do the experiment by sequentially performing immunostaining followed by acquisition of a series of images on slide positions that can be precisely monitored using the "xy" scale of the microscope stage or more accurate devices. After image acquisition, a FISH experiment is performed on the same slide and images are acquired at the same slide coordinates for analysis of in situ DNA labeling. Finally, the separately acquired images are accurately merged using imaging software. This somewhat complicated procedure has the advantage of allowing immuno-FISH experiments to be performed on any protein of interest, regardless of the stability of the antibody under the relatively harsh conditions used for FISH experiments.

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