MATERIAL AND METHODS

1. The mica technique

Isolated polytene chromosomes, NEs or nuclei were transferred by pipetting in storage buffer [0.09 M KCl, 0.06 M NaCl, 10 mM MgCl₂, 0.4 mM CaCl₂, 20 mM phosphate buffer (pH 5.2), 0.5% 2-mercaptoethanol, 0.5 mM phenylmethyl sulphonylfluoride (PMSF, Sigma), 0.08% Triton X-100, 0.13% Nonidet P40] to freshly cleaved mica pieces (about 5 x 10 mm), which were kept on depression slides placed on a cold stage (2-5^oC) of a preparation microscope. They were allowed to settle and attached in less than a minute. A few successful pilot experiments were also done on cover glasses freshly etched with 4% fluoric acid (HF), 10 min.

If the extraction and digestion were monitored by incident fluorescence microscopy, the material was stained with ethidium bromide 0.5-10 µg per ml (for 1-20 min), or for more specific DNA staining with DAPI (4“-6-diamidino-2-phenylindole: 0.25-0.5 µg per ml) in 2 M salt solution (see salt extraction) used for extraction, before the digestion steps.

2. Treatment procedures

2. 1. Salt extraction

2. 2. Enzyme digestions

The absence of protease activity in the hyaluronidase preparation was demonstrated by incubating it with ¹²⁵I labeled bovine serum albumin and analysing the incubation mixture by SDS-PAGE and autoradiography.

The absence of DNase activity was demonstrated by incubating 2.5 µg of plasmid pBR 322 DNA for 5, 10, 20, or 60 min at 20^oC in 5 mM MgCl₂, 10 mM Tris-HCl, pH 7.2, with 50 µg per ml of Hyalas and analysis on agarose gel electrophoresis.

The 2MS and other solutions contained, as a 0.5 mM solution, PMSF, which was added freshly from a 1 M stock solution in ethanol; yet no difference was noticed when PMSF was omitted.

2. 3. Washing, dehydration and staining

After detachment, the preparations were washed with water (transfered with the mica piece, plastic or wooden rod), and mounted on grids (75-100 mesh) with formvar and carbon support or (200 mesh grids) without it.

Mounting was done by floating selected, encircled replica pieces of the preparations on a drop in a wire loop, about 3 mm in diameter, slightly larger than a grid. After applying the loop over a grid, held in place by capillarity forces, the water was sucked off with a filter paper.

Stereo inspection of plates has been indispensable for the elucidation of relevant details of these bulky whole mounts. It was further found advisable to inspect the negatives directly in transmitted light with a good mirror stereoscope (Old Delft, x4.5), because with positive prints much of the details of the high-contrast plates is lost and the reversal of shades is unfavorable for interpretation. Inverted prints were made by rephotographing the original plates on Agfa Pan (25 or 100 ASA) film, from which final prints were made.

Cultures of both human and elk lymphocytes, as well as human COLO 320 colon carcinoma (containing double minute chromosomes) and Chinese hamster ovary (CHO) cell lines were used and fixed according to conventional cytogenetical methods. Briefly, after colchicine treatment for 1-9 hours the cells were collected, washed and treated with a hypotonic solution (75 mM KCl, at 37 ^oC or at room temperature, for 15-25 min) and fixed in methanol/acetic acid (3:1) and stored in a freezer (-20 ^oC).

Salivary glands from Drosophila melanogaster were isolated from third instar larvae in Ringer“s physiological solution (for insects) and either pretreated before spreading, with 45% acetic acid according to Burkholder (1976) or with 66% propionic acid, 66% citric acid (PA/CA) mixture according to Kalisch and Hägele (1981) or fixed in bulk with methanol/acetic acid before treatment and stored (-20^oC).

By this treatment, polytene chromosomes could not be released as individual chromosomes; only whole nuclei were found. They could, however, be released by spreading after the above pretreatment. Pretreatment was also necessary for spreading polytene chromosomes when salivary glands had been prefixed by methanol/acetic acid.

1) 4 M urea, 0.1 N HCl (U/HCl) (used according to Kalisch and Hägele, 1981, for polytene chromosomes).
2) Distilled water (used according to Burkholder, 1976, for polytene chromosomes).
3) 10-100 µg/ml of cytochrome c (Sigma) in TC buffer (10 mM Tris-HCl, pH 7.4, and 3 mM CaCl₂).

The spread material, floating on the surface, can be inspected under an inverted microscope (10x, 20x or 40x long distance objective). Selected material can be picked up by touching the surface with EM grids. The grids were either of Cu, Ni or Au: single hole, slot or 50-200 mesh coated with either Formvar and carbon or with plastic film made from pieces of polystyrene petri dishes (Nunc) dissolved to make a 0.2-0.3% solution in chloroform (Felluga and Martinucci, 1976) or better in 1,2-dichloroethane. After the material had been transfered to the grid, some of the following steps were taken as specified in each case.

a) Washing the grid in water (with or without addition of Photoflow (Kodak): 3 drops/50 ml) for about 10 s (3x) (as considered appropriate when salts are in the spreading solutions).
b) Washed in TC buffer 10 s (3x) or TM buffer, which is 10 mM Tris-HCl (pH 7.4), 3 mM MgCl2.
c) Washed in 60% acetic acid.
d) Washed in 50% acetic acid, at 100 oC, for 30-60 s (modified from Welter, Black and Hodge, 1984, for scanning electron microscopy of chromosomes).
e) Enzyme treatments: washed in 2MS solution (see Material and methods I. 2. 1, with or without detergent included), 10 s (3x), followed by TM buffer 10 s (3x), digested in DNase, 10 mg/ml (with or without RNase 10 mg/ml) in TM buffer for 30-60 min, room temperature, washed in TM buffer 10s (3x), washed in 2MS solution 10 s (3x), washed in TC buffer 10 s (3x).

5. Whole mounts of metaphase chromosomes

5. 1. Whole-mount centrifugation method (WMC)

5. 2. EM cytogenetic (EMC) method

A small amount (2-5 µl) of methanol/acetic acid-fixed cells (diluted to a suitable concentration as determined by pilot tests) was dropped onto prefrozen mica pieces (3x5 mm) (taken, immediately prior to dropping, out from dry ice in which the micas were laid on a slide). After application the preparations were left to dry for 1-2 minutes to let the cells attach firmly enough to the mica surface. This was followed by three washings in 60% acetic acid, 10 s each, and further washing in 50% acetic acid, at 100 ^oC, for 30-60 s (i.e. steps c and d).

Dehydration and staining were as above and then critical point-drying, followed by rotary Pt shadowing (7-8 degree angle) and an additional carbon evaporation were carried out. The replicas were then detached with warm (50-60^oC) 0.1 N NaOH, washed and mounted on grids (see Material and methods: I. 3).

6. DNA staining

DNA staining with the fluorescent dye DAPI (0.25-0.5 µg/ml in TC or TM buffer, 10-30 min) was performed either before or after the dehydration steps (without uranyl acetate staining). When necessary (particularly after steps c or d) step b was included and followed by embedding of the grids under a coverglass in 50-90% glycerol in a TM or TC buffer including antifading substances such as 10-20 mM DTT (dithiothreitol) or 2-mercaptoethanol with or without DAPI (0.25-0.5 µg/ml). When step (e) was performed this enabled prestaining in the 2MS solution with DAPI, which was used to follow the efficiency of digestion. After digestion the grids (or micas) were embedded in the glycerol solution with DAPI for final checking and photography.

7. EM stereoscopy and micrographs

Eukaryotic chromosome structure [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]