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Immunofluorescent Staining of Polytene Chromosomes: Exploiting Genetic Tools

2003; Academic Press; Linguagem: Inglês

10.1016/s0076-6879(03)76025-x

1557-7988

Gena E. Stephens, Carolyn A. Craig, Yuhong Li, Lori L. Wallrath, Sarah C. R. Elgin,

Genetic and Clinical Aspects of Sex Determination and Chromosomal Abnormalities

This chapter describes a method for determining the in vivo distribution of chromosomal proteins on Drosophila polytene chromosomes. By combining genetic, biochemical, and molecular biology techniques with the cytological approach, a greater understanding of the molecular mechanisms of gene regulation can be gained. Few other systems offer the well-developed genetic tools in combination with the ability to perform cytological studies as Drosophila does. Immunostaining of Drosophila polytene chromosomes is a powerful tool for investigating the components of chromatin on a genome-wide scale. Many of the nuclei of Drosophila undergo multiple rounds of DNA replication without cell division during the larval stages of development, a process known as endoreduplication. While the euchromatic regions are copied ca. 10 times, the pericentric heterochromatin undergoes only a few rounds of replication, and centromeric satellite DNA and the Y chromosome are not amplified due to their heterochromatic nature. Immunological methods for studying the association of proteins with polytene chromosomes have been used to address a variety of biological questions. In wild-type flies, immunostaining has been used to determine the global distribution of one or more proteins; colocalization studies have been done to determine if a protein of interest might be in close association or part of a multiprotein complex with other proteins.