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Electron microscopy of an induced “lampbrush stage” of the polytene chromosome
466 V. Sorsa et al. REFERENCES 1. Rose, G C, Texas rep biol med 12 (1954) 1074. 2. Paul, J, Quart j microscop sci 98 (1957) 279. 3. Sykes, J A & Moore, B E, Proc sot exptl biol med 100 (1959) 125. 4. Roberts, D C & Trevan, D J, J roy microscop sot 79 (1961) 361. 5. Ploem, J S, Thesis, p. 58. Amsterdam (1967). Received February 13, 1970
Electron microscopy of an induced 6‘lampbrush stage” of the polytene chromosome V. SORSA, K. PUSA, VIRPI VIRRANKOSKI and MARJA SORSA, Department of Genetics, University of Helsinki,
Helsinki
10, Finland
That the normal polytene structure of giant chromosomes could be transformed into a “lampbrush stage” by certain proteolytic
Fig. I. Electron micrograph of the normal banding pattern in an untreated X chromosome (subdivisions 2C2E) of Drosophila melanogaster. x 50 000. Fig. 2. Photomicrograph of a Drosophila polytene chromosome converted into a “lampbrush stage” by ureaalkali treatment. Light acetic haematoxylin stain. x 3 000. Exptl
Cell Res 60
Fig. 3. Low-power electron micrograph of an induced lampbrush stage of the giant salivary chromosome, revealing a distinct core (C) and band fibrils (II). x 15 000. Fig. 4. Electron micrograph of part of a cross-sectioned salivary chromosome in an induced lampbrush stage showing the core (C) with the band fibrils (B) protruding radially as whisks. x SO000. Jzxptl Cd Res 60
468
V. Soma et al.
Fig. 5. Electron micrograph of a longitudinal section of the polytene chromosome after mild proteolytic treatment. Thin fibrillar material (FM) is loosening from the chromosome. The orientation of the core fibrils (C) and protruding band fib& (B) is not yet clear. x 50 000. Fig. 6. Core region of an induced lampbrush chromosome after loosening of the thin fibrillar material. The multistranded nature of the fibrillar core (C) is obvious, although it is surrounded by band material (B), which still reveals coiling of the fibrils. x 85 000. Exptl
Cell Res 60
Electron microscopy of induced ~~rnpb~u~hch~amo~ome~ 46
treatments was known as much as thirty years ago [l, 2, 41 even though rarely cited in the recent literature. The light microscope, however, was able to reveal very little information on the essential structural changes taking place in the polytene chromosome as a result of alkali or urea-alkali treatments [3]. The squash-thin sectioning method developed for comparative light and electron microscopy of chromosomes [6, 71 facilitates a more thorough investigation of the structure of the lampbrush stage induced in polytene chromosomes by urea-alkali treatments. Material and Methods Salivary glands dissected from late third instar larvae of Drosophila melanogastm were immediately treated in vitro for 15 min in a 1: 1 mixture of a saturated aqueous solution of urea and 0.2 N NaOH, pH 13.0, at room temperature. After treatment, the glands were fixed for 2 h in acetic methanol (1 part glacial acetic acid : 3 parts acetone-free absolute methanol) on ice. Normal squash preparations were made on slides coated witb silicon. Well squashed lampbrush stages of the salivary chromosomes were photomicrographed by phase optics from fresh slides. The chromosomes were then “stained” with uranyl acetate during dehydration in series of absolute methanols, and prepared for electron microscopy according to.the prbcednre previously described by Sorsa [S]. The electron microscopic examination was carried out in The E!ectron Microscope Laboratory of the University of Helsinki, with a Philips EM 200 electron microscope, 80 kV voltage, using original magnifications from x 3 400 to x 10 500.
As a result of the treatment, various transition stages from rather mild induction to total dispersion of the banded polytene structure can be observed, obviously depending on the position of the cells affected. Individual chromomeres derived from the thicker bands seem to open up into masses of coiled and folded fibrils, ranging from 150 A to 500 A in thickness. During the opening phase, large amounts of very thin fibrillar material seem to be detached from the polytene chromosome. Longitudinally oriented strands, which we interpret as interband fibrils of the normal polytene chromosome, appear at first, in the early stages of proteolytic induction, as
coarse, tangled strands among the iffUSe mass situated all over the chromosome. In those cells which are obviously affected more strongly by the treatment, the fibres become more clearly parallel, forming a dense multistranded core to the chromosome, which then has a distinct ‘“lampbrush” appearance.Along this longitudinal core, at irregular intervals, darkly stained dotlike particles are visible, with dimensions in the range of 200 i%.In our opinion, these particles represent either crosssections of the transverse chromomeric fib&s or certain specific joining points between the longitudinal interband fibrils and the chromomerit fibrils. Preservation of the fibrillar core: in spite of treatment effective enough to remove at least most of the acidic proteins, suggeststhat the interchromomeric fibres form a relatively strong continuous framework within the polytene chromosome, to which the chromomer~c fibrils are connected at certain sites. Tile interpretation of these observations in terms of chromosome structure in general will be discussedin a forthcoming paper. Financial aid for this study was provided by- the National Research Council for Sciences.
REFERENCES 1. Calvin, M, Kodani, M & Goldschmidt, R, Proc natl acad sci US 26 (1940) 340. 2. Kodani, M, J hered 32 (1941) 146. 3. - Ibid 33 (1941) 115. 4. Painter, T S, Genes and chromosomes. Cold Spring Harbor symp on quantitative biology 9 (1941) 47. 5. Sorsa, M, Ann acad sci fenn A IV, Rio1 I46 (1969) 1. 6. Sorsa, M & Sorsa, V, Chromosoma 22 (1967) 32. 7. Sorsa, V & Sorsa, M, Ann acad sci fenn A IV, Rio1 105 (1967) 1. Received February 2, 1970
Electron microscopy of an induced 6‘lampbrush stage” of the polytene chromosome V. SORSA, K. PUSA, VIRPI VIRRANKOSKI and MARJA SORSA, Department of Genetics, University of Helsinki,
Helsinki
10, Finland
That the normal polytene structure of giant chromosomes could be transformed into a “lampbrush stage” by certain proteolytic
Fig. I. Electron micrograph of the normal banding pattern in an untreated X chromosome (subdivisions 2C2E) of Drosophila melanogaster. x 50 000. Fig. 2. Photomicrograph of a Drosophila polytene chromosome converted into a “lampbrush stage” by ureaalkali treatment. Light acetic haematoxylin stain. x 3 000. Exptl
Cell Res 60
Fig. 3. Low-power electron micrograph of an induced lampbrush stage of the giant salivary chromosome, revealing a distinct core (C) and band fibrils (II). x 15 000. Fig. 4. Electron micrograph of part of a cross-sectioned salivary chromosome in an induced lampbrush stage showing the core (C) with the band fibrils (B) protruding radially as whisks. x SO000. Jzxptl Cd Res 60
468
V. Soma et al.
Fig. 5. Electron micrograph of a longitudinal section of the polytene chromosome after mild proteolytic treatment. Thin fibrillar material (FM) is loosening from the chromosome. The orientation of the core fibrils (C) and protruding band fib& (B) is not yet clear. x 50 000. Fig. 6. Core region of an induced lampbrush chromosome after loosening of the thin fibrillar material. The multistranded nature of the fibrillar core (C) is obvious, although it is surrounded by band material (B), which still reveals coiling of the fibrils. x 85 000. Exptl
Cell Res 60
Electron microscopy of induced ~~rnpb~u~hch~amo~ome~ 46
treatments was known as much as thirty years ago [l, 2, 41 even though rarely cited in the recent literature. The light microscope, however, was able to reveal very little information on the essential structural changes taking place in the polytene chromosome as a result of alkali or urea-alkali treatments [3]. The squash-thin sectioning method developed for comparative light and electron microscopy of chromosomes [6, 71 facilitates a more thorough investigation of the structure of the lampbrush stage induced in polytene chromosomes by urea-alkali treatments. Material and Methods Salivary glands dissected from late third instar larvae of Drosophila melanogastm were immediately treated in vitro for 15 min in a 1: 1 mixture of a saturated aqueous solution of urea and 0.2 N NaOH, pH 13.0, at room temperature. After treatment, the glands were fixed for 2 h in acetic methanol (1 part glacial acetic acid : 3 parts acetone-free absolute methanol) on ice. Normal squash preparations were made on slides coated witb silicon. Well squashed lampbrush stages of the salivary chromosomes were photomicrographed by phase optics from fresh slides. The chromosomes were then “stained” with uranyl acetate during dehydration in series of absolute methanols, and prepared for electron microscopy according to.the prbcednre previously described by Sorsa [S]. The electron microscopic examination was carried out in The E!ectron Microscope Laboratory of the University of Helsinki, with a Philips EM 200 electron microscope, 80 kV voltage, using original magnifications from x 3 400 to x 10 500.
As a result of the treatment, various transition stages from rather mild induction to total dispersion of the banded polytene structure can be observed, obviously depending on the position of the cells affected. Individual chromomeres derived from the thicker bands seem to open up into masses of coiled and folded fibrils, ranging from 150 A to 500 A in thickness. During the opening phase, large amounts of very thin fibrillar material seem to be detached from the polytene chromosome. Longitudinally oriented strands, which we interpret as interband fibrils of the normal polytene chromosome, appear at first, in the early stages of proteolytic induction, as
coarse, tangled strands among the iffUSe mass situated all over the chromosome. In those cells which are obviously affected more strongly by the treatment, the fibres become more clearly parallel, forming a dense multistranded core to the chromosome, which then has a distinct ‘“lampbrush” appearance.Along this longitudinal core, at irregular intervals, darkly stained dotlike particles are visible, with dimensions in the range of 200 i%.In our opinion, these particles represent either crosssections of the transverse chromomeric fib&s or certain specific joining points between the longitudinal interband fibrils and the chromomerit fibrils. Preservation of the fibrillar core: in spite of treatment effective enough to remove at least most of the acidic proteins, suggeststhat the interchromomeric fibres form a relatively strong continuous framework within the polytene chromosome, to which the chromomer~c fibrils are connected at certain sites. Tile interpretation of these observations in terms of chromosome structure in general will be discussedin a forthcoming paper. Financial aid for this study was provided by- the National Research Council for Sciences.
REFERENCES 1. Calvin, M, Kodani, M & Goldschmidt, R, Proc natl acad sci US 26 (1940) 340. 2. Kodani, M, J hered 32 (1941) 146. 3. - Ibid 33 (1941) 115. 4. Painter, T S, Genes and chromosomes. Cold Spring Harbor symp on quantitative biology 9 (1941) 47. 5. Sorsa, M, Ann acad sci fenn A IV, Rio1 I46 (1969) 1. 6. Sorsa, M & Sorsa, V, Chromosoma 22 (1967) 32. 7. Sorsa, V & Sorsa, M, Ann acad sci fenn A IV, Rio1 105 (1967) 1. Received February 2, 1970