EM Mapping and Sources of Errors

In ultrathin sections of polytene chromosomes, large thick bands can usually be seen easily. Visualization of thin faint bands depends mainly on two factors: (1) the method of fixation and treatment prior to embedding and (2) the thickness of the sections analyzed.

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

Fig. 1. The 9A-10B region of the X chromosome. The chromosome was fixed in 3 : 1 ethanol/acetic acid. Scale bar: 1 |im.

1.1.1. Chromosome Fixation

In our experience (5-7), fixation of salivary glands in 3% glutaraldehyde produces contracted chromosomes in which it is very difficult to discern many thin bands (see also ref. 8). Fixation in 3% formaldehyde preserves the visibility of thin bands; however, it makes squashing of the glands difficult and results in poorly spread chromosomes. This is also the case for glutaraldehyde fixation. Salivary gland treatment with 45% acetic acid, the fixative used by Bridges, allows the detection of thin bands but results in the formation of doublets of thick bands. Treatment with 60% lactic acid significantly reduces the number of observable thin bands. Long-term storage of preparations in 96% ethanol (9) results in a drastic contraction of the bands. Only fixation in a 3 : 1 mixture of alcohol (ethanol or methanol) and acid (acetic or propionic) gives the desired result, allowing both good chromosome spreading and visualization of the greatest number of the bands. The polytene chromosome in Fig. 1 is an example. Comparison of this electron micrograph with the revised map of Bridges (1) reveals a characteristic difference: Most of the bands indicated as doublets by Bridges appear as singlets in Fig. 1. After analyzing many other chromosome regions, we concluded that practically all heavy bands are single and that fixation in 45% acetic acid or in a hydrated 3 : 1 mixture of alcohol : acetic acid leads to the formation of double vacuolated bands. We consider such vacuolization to be an artifact of fixation. It should be noted that the best

Fig. 2. Banding pattern of polytene chromosome regions in serial sections of different thickness: (a) 50-70 nm and (b) 120-150 nm. Scale bar: 1 |im.

way to detect thin bands, like those in Fig. 1, is through analysis of serial sections of many chromosomes.

1.1.2. Thickness of Sections

Accuracy of polytene chromosome mapping as well as interpretation of the observed images depends both on the DNA content and the degree of DNA decompaction in a given structure. Therefore, section thickness plays an important role in the analysis of banding pattern (10). Figure 2 shows thin (50-70 nm) and thick (120-150 nm) sections in two regions, namely 9-10 and 48-49 of the D. melanogaster X and 2R chromosomes, respectively, made from the same Araldite block. In the 50- to 70-nm ultrathin section (see Fig. 2A) it is difficult to identify very thin bands and to interpret the structure of large bands. In the thick section, two bands positioned close to each other may look like a single band (see Fig. 2B). Although neither image furnishes a detailed picture of the banding pattern, the comparison nevertheless demonstrates a clear advantage of the thick section over the thin one. In the thin section, many of the large bands exhibit a reticular, fine flaky structure. Faint bands are tightly juxtaposed to large ones and it is quite impossible to determine the exact number of bands, as they look like an indivisible network of chromatin. In the 120- to 150-nm sections, faint bands cover a larger cross section, which facilitates their detection (compare the 9E-10A region in Figs. 1 and 2). The same conclusion was made earlier by Berendes (8), who proposed to use even thicker sections (150-200 nm). However, large neighboring bands in such sections often look fused.

Finally, the detectability of bands depends on the extent of stretching of the polytene chromosome. Therefore, the quality of chromosome structure should be monitored using the light microscope before sectioning is done with an ultratome.

1.1.3. Mapping Rules

The diversity of band morphology of polytene chromosomes requires that certain mapping rules be followed for correct interpretation of the data obtained. Such rules were formulated earlier (11) and can be summarized as follows:

1. The banding pattern of a region has to be reproducible in serial sections of at least two or three chromosomes. Single dense structures of very small size (approx 1/20 of chromosome diameter) with approximately equal length and width are not regarded as bands.

2. The best fixative is a 3 : 1 alcohol : acid mixture. Vacuole like formation in large bands increases with increasing fixative hydration, which gives rise to doublet artifacts.

3. In estimating the number of bands taking part in puff development, the beginning and end stages of the process should be included in the analysis and the chromosomes of larvae at different developmental stages should be studied.

4. The optimum thickness of sections used for mapping is 120-150 nm.

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