John Tonzetich 1 Introduction

Acetic orcein staining of polytene chromosomes was introduced in 1941 (1) shortly after the initial studies on aceto-carmine-stained chromosomes by Bridges (2) and has remained a standard method of preparation. Orcein dye can be purchased in both its natural form as extracted from two species of lichens, Rocella tinctoria and Lecanora parella, and a synthetic form. The mechanism of staining is not clearly understood because the stain itself is a variety of phenazones, which may interact at an acid pH with negatively charged groups or possibly interact hydrophobically with chromatin. Acetic acid fixation accommodates stretching of the chromosomes in the interband regions during a squash, thus providing for a higher resolution of the banding structure. The later addition of lactic acid to aceto-orcein (3) kept the glands softer in the fix and allowed for easier spreading of chromosomes. The method and its variations have appeared more recently in several publications (4,5).

Drosophila polytene chromosomes are found in a number of larval tissues, including the midgut, hindgut, and the fat body, but the largest chromosomes are found in the salivary glands of the third instar. They are referred to as interphase chromosomes and are structurally more comparable to highly amplified interphase chromatin than to mitotic chromosomes because the gland grows by endoreplication of DNA, thus increasing cell size rather than cell number. Each of the homologs is tightly synapsed in this somatic tissue and undergoes approx 10 rounds of endoreplication, producing 1024 chromatids closely associated in parallel arrays. The extent of polyteny varies depending on the position of cells (those in the narrower neck of the glands have a lesser

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

C value), the growth conditions, and the strain of fly (6). The average length of the chromosome arms is approx 200 ^m but varies with the amount of stretching (7). The mean width is about 3 ^m. All of the chromosome arms are associated at their centromeric regions, forming a centralized structure known as the chromocenter from which the chromosome arms radiate. The Y chromosome, which is heterochromatic and not highly endoreplicated, is completely contained within the chromocenter and is therefore not discernible. In males, the single X-chromosome consists of half the number of chromatids and typically stains visibly lighter than the paired X's in females. A nucleolus can often be seen associated with the X and Y chromosomes by a fine strand of chromatin. In Drosophila melanogaster the X chromosome is acrocentric, and the second and third chromosomes are metacentric, and the fourth chromosome is a very short, dotlike, acrocentric. The karyotypes of many Drosophila species can be traced to the rearrangements of six linkage groups designated A-F (8)

The polytene chromosomes of D. melanogaster contain approx 3000 bands. The distinctive banding pattern of each chromosome has been carefully delineated in cytogenetic maps both by light (9) and electron microscopy (10). The standard system of map nomenclature (2) divides the 4 chromosomes into 102 divisions, each of the chromosome arms containing 20 divisions (X: 1-20; 2L: 21-40; 2R: 41-60; 3L: 61-80; 3R: 81-100) excepting the small fourth chromosome, which is divided into divisions 101-102. Each numerical division is further subdivided into six lettered sections (A-F), each section containing approximately six consecutively numbed bands. The bands vary in size and clarity, the more evident bands typically delimiting map subdivisions. Similar but less detailed maps have been published for many Drosophila species (11).

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