ultrastructure of polytene chromosomes of … · after this first-stage fixation, the preparation...

J. Cell Set. 45, 15-30 (1980)

Printed in Great Britain © Company of Biologists Limited

ULTRASTRUCTURE OF POLYTENE

CHROMOSOMES OF DROSOPHILA ISOLATED BY

MICRODISSECTION

MARGARET R. MOTT, E. J. BURNETT AND R. J. HILLCSIRO Genetics Research Laboratories, P.O. Box 184,North Ryde, NSW, 2113, Australia

SUMMARYDrosophila polytene chromosomes prepared by a new micromanipulative procedure, which

avoids acid squashing, have been examined at the ultrastrucrural level in the electron micro-scope. Puffs at 2B, 68C, 74EF, 75B and 85EF, have been examined in some detail, along withthe chromocentre and various interbands. The ultrastructure of these chromosomes, whichhave never been exposed to acid protein denaturants, compares favourably with that ofclassical acid-fixed specimens. Ribonucleoprotein particles in puffs are seen to be organized inlinear arrays and evidence is adduced for looped transcription units. Particles with charac-teristic sizes and morphologies are observed near the chromocentre, in puffs and in interbands.In interbands RNP particles and 'superbead'-like chromatin particles may be distinguished.Drosophila polytene chromosomes isolated by micro-manipulation should prove useful for thelocalization of native chromosomal proteins at an ulrrastructural level.

INTRODUCTION

The detailed morphology of Drosophila melanogaster salivary chromosomes survivesexposure to aqueous acetic acid, the solvent used since the 1930s in cytologicalpreparation of chromosomes in the classical squashing technique (Painter, 1934;Bridges, 1935). In fact this acid solvent and fixative concomitantly weakens the nuclearmembrane and confers mechanical strength on the chromosomes. However, in viewof the physical properties of aqueous acetic acid it is not surprising that it has somedeleterious effects at an ultrastructural level (Lakhotia & Jacob, 1974).

On return from aqueous acetic acid to simple aqueous solution, for example fortreatment with antisera, more detail is often lost. Furthermore, there is direct evidencethat acid fixatives can extract chromosomal proteins (Dick & Johns, 1968; Brody,1974; Pothier, Gallagher, Wright & Libby, 1975) and destroy protein antigenicity(Stenman, Rosenqvist, & Ringerts, 1975; Silver & Elgin, 1976). These factors raisedifficulties for biochemical studies of chromosome structure and function utilizingclassical acetic acid squash preparations.

Although there may be a number of solutions to this problem, perhaps the mostrigorous would be to isolate the polytene chromosomes by microsurgery and spreadthem by careful micromanipulation in saline, avoiding the use of aqueous acetic acidcompletely. In previous communications (Hill & Watt, 1977, 1978) it was demon-strated that this operation was possible and evidence was presented from light

M. R. Mott, E. J. Burnett and R. J. Hill

EM of microdissected polytene chromosomes 17

microscopy showing that even in the complete absence of acid fixation Drosophilasalivary chromosomes could be isolated in a state preserving fine structure thatcompares favourably with that in classical acid squashes. In what follows this analysisis extended to the electron-microscopic level. It will be shown that these chromosomes,in addition to being expected to contain native proteins (never having been exposed toacid protein denaturants), in fact combine the facility of mapping known chromosomeregions with preservation of ultrastructure comparing favourably with that previouslyseen only in sectioned material from glutaraldehyde-fixed whole glands (Skaer, 1977).They allow the resolution of structural elements not previously observed in Drosophilasalivary chromosomes.

MATERIALS AND METHODS

Isolation of salivary chromosomes by microdissection

This was performed essentially by the method of Hill & Watt (1977, 1978). Late third instarlarvae were selected for chromosome isolation from uncrowded, well-yeasted cultures. Theoperation was carried out in a room maintained at 19 CC.

Salivary glands were dissected out into a drop of 25 niM disodium glycerophosphate,10 mM KHjPO,,, 30 mM KC1, 10 mM MgClt, 162 mM sucrose, pH 6-8. Excess fat body wastrimmed away, taking care not to damage the glands. Subsequent microdissection steps werecarried out in salines based on that of Glancy (1946) - 90 mM KC1, 60 mM NaCl, 1 mM CaCla,5 mM sodium phosphate, pH 70, which shall be referred to as saline G. Glands were trans-ferred to 1 % Triton X-100, saline G for 20-30 s and then to 0025 % Triton X-100, saline Gfor 5-6 min. They were subsequently placed in a 3-/tl drop of o-oi % Triton X-100, saline Gsome distance from the well on a well slide. Several cells were torn open with a tungsten needleto release nuclei. The slide was flooded with 0-05 % formaldehyde, saline G. Clean nuclei, inthe optimal configuration for removal of the chromosomes by microsurgery (Hill & Watt,1978) were transferred on a blunt needle to the well.

A hole was torn in the nuclear membrane and the loosely adhering bundle of chromosomesremoved with fine glass needles. The chromosome complement was unravelled, flattened andallowed to attach to the glass using the glass needles. Once the chromosomes are attached theyremain so during the fixation steps for electron microscopy.

Preparation of spread chromosomes for electron microscopy

The method used was similar to that used by Mott & Callan (1975). The chromosomes weretreated with 3 % glutaraldehyde ino-i M phosphate buffer, pH 7-2 for 30 min, repeating withfresh glutaraldehyde in buffer for another 30 min. After this first-stage fixation, the preparationwas washed in several changes of buffer and then postfixed in 1 % osmium tetroxide, also inbuffer, for 30 min, repeating as before with fresh osmium tetroxide in buffer for another30 min. The preparation was again washed in several changes of buffer, a coverslip added, and

Fig. 1. Low-magnification phase-contrast micrograph of the complete chromosomespread isolated by microdissection from a salivary gland nucleus. Identified are:the X chromosome with the large puff at positions 2B; chromosome 3L present asa loop with the 2 ecdysone puffs at positions 74EF and 75B and attached micro-nucleolus {inn); and a small loop of chromosome 3R.Figs. 2, 3. Comparison of a phase-contrast micrograph (Fig. 2) with an electronmicrograph (Fig. 3) at the same magnification of the distal end of the X chromosome.Puffs 2B 5-6, 2B 13-17 are readily recognized. The arrow indicates a region ofasynapsis.

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the pieparation examined under phase-contrast. The fixed preparation was then dehydratedby passing through an ethanol series and the preparation in absolute ethanol was passed throughpropylene oxide to Epon embedding medium. The welled slide was placed on metal foil in aPetri dish and flooded with Epon, followed by fresh Epon and placing in a 60 °C oven toevaporate any remaining propylene oxide from, the preparation. Fresh Epon was again floodedinto the well and the preparation incubated overnight at 60 °C for polymerization. The slidewas again examined under phase-contrast and photographs of the chromosome group weretaken at various focal planes into the depth of the Epon film in the well, as the chromosomeswere not flattened along all their lengths to the coverslip. The phase-contrast micrographs wereused to monitor the trimming of the block and the cutting of the sections, and to identify thechromosomes and their landmarks.

The position of the chromosome group was marked on the underside of the coverslip andthen end-embedded under a Beem capsule of embedding medium and left to polymerize at60 °C for several days. The resulting peg of Epon with the end-embedded chromosomes wasthen split from the slide and bottom coverslip by placing in a slurry of solid COt and liquidnitrogen. The peg was filed down to about 1 cm length, the surface that had previously been incontact with the coverslip was examined under the dissecting binocular, and a small squarewith orientation marks was scratched on the surface so as to surround the barely visiblechromosome group, which in most cases occupied an area of about o-i mm1. Thereafter thissurface of the block was wetted with immersion oil and the block inverted on a slide. The filedother end of the block was now also wetted with immersion oil and a 13-mm coverslip laidacross it. The block was then examined under the phase-contrast microscope to check thechromosomes in the block with the photographs taken earlier and these photographs markedwith the appropriate orientation marks. These photographs were then used in monitoring thetrimming of the block with the dissecting microscope and LKB ultramicrotome. The blockwas finally trimmed using the LKB microtome with 550 glass knives to a block face of approxi-mately 02 m m x o i mm, just a little larger than the area of the chromosome group. 50-nmsilver sections were cut with a diamond knife (Friedrich Dehmer) on the LKB microtome,starting from the face of the block, and were picked up on carbon-coated Formvar grids.

The sections were stained with 4% aqueous uranyl acetate and bismuth subnitrate (Ains-worth & Karnovsky, 1972; Riva, 1974) and examined in a Siemens Elmiskop 1 at 80 and100 kV, a JEM 100S at 60 kV and a JEM iooCXat4o and 60 kV. Calibration of magnificationwas determined with a carbon-grating replica grid (Balzers Union).

RESULTS AND DISCUSSION

Known structures of the Drosophila salivary chromosomes isolated by micro-manipulation may be identified under the light microscope (Figs. 1, 2, 7, 8). They arethen readily located in the electron microscope and related to the classical chromosomemaps of Bridges (1935) arrived at from acid-squash preparations (Figs. 3, 9, 10).Most striking in the phase-contrast micrograph of the complete spread (Fig. 1) are theX chromosome with puffs in section 2 (compare Bridges, 1935), chromosome arm 3Lpresent as a loop with the 2 ecdysone-induced puffs at loci 74EF and 75B (compareAshburner, 1972) and an accompanying micronucleolus. Part of chromosome arm 3R

Figs. 4-6. Electron micrographs of the distal end of the X chromosome at highermagnifications.

Fig. 4. The tip of the X chromosome and the large puff at 2B 5-6, mn indicates amicronucleolar-like structure.

Figs, 5, 6. Parts of puff 2B 5-6 at higher magnification, showing linear arrays ofRNP. The arrows indicate possible stalks. Fig. 5 shows the looped configuration atthe periphery of the puff.

2 0 M. R. Mott, E. J. Burnett and R. J. Hill

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EM microdissected polytene chromosomes 21

is also identified. The thin sections of the end-embedded spread chromosomes at lowmagnifications (Figs. 3, 9, 10) revealed that the chromosomes are well preserved withwell defined bands, interbands, puffs and nucleolus. To gain a better indication of thestate of preservation of these chromosomes, particular structures on the X and 3rdchromosomes and at the chromocentre will now be examined in greater detail.

The X chromosome

A high-magnification phase-contrast micrograph of the distal region of the Xchromosome and the corresponding low-magnification electron micrograph arecompared in Figs. 2 and 3. Bands and puffs in general are easily correlated between the2 micrographs. Our electron micrographs of the X tip isolated in saline were comparedwith the published material on acid squash preparations, both by light microscopy(Ashburner, 1969) and electron microscopy (Berendes, 1970; Lossinsky & Lefever,1978). The 2 puffs in section 2 are most probably those identified by Ashburner at2B 5-6 and 2B 13-17. The present electron-microscope studies and those cited alldisplay fewer bands than Bridges' light-microscope map. In our preparation asynapsisis apparent at one point on the X chromosome (arrow in Fig. 3).

At higher magnifications (Figs. 4—6) the ultrastructural detail is seen to be excellentlypreserved despite the absence of acid fixation during the isolation procedure. It is atleast comparable to, and in some respects better than, that in the classical acid-squashpreparations (see for example, Berendes, 1970); moreover, there is no confusingbackground due to the network of nucleoplasmic fibrils which extends right throughthe chromosomes in sections through whole nuclei (Skaer, 1977; Skaer & Whytock,1977). The puff at 2B 5-6 and some, but not all, interbands contain putative ribo-nucleoprotein (RNP) granules of diameter 30-45 nm, consistent with transcriptionalactivity at these sites (Skaer, 1977) and according with the view of Crick (1971) thattranscription occurs in interbands and puffs. The ribonucleoprotein granules aresimilar to those found by Skaer on polytene chromosomes and identified as RNP byhim using the Bernhard staining technique. Vazquez-Nin & Bernhard (1971) alsoidentified analogous structures in Chironomus by autoradiography. The granules alsoappear similar morphologically to the RNP particles observed by Mott & Callan(1975) on the loops of amphibian lampbrush chromosomes. Chromatin threads areseen to run through the interbands (vide infra) and somewhat similar fibrils may beobserved within the puff.

Figs. 7-10. Micrographs of chromosome 3L present as a loop, and part of chromo-some 3R.

Figs. 7, 8. High-magnification phase-contrast micrographs at 2 different focalplanes. In Fig. 7 the puff on chromosome 3L at 68C is in focus. In Fig. 8 the 2 puffsat 74EF and 75B on chromosome 3L and a micronucleolus (mn) are in focus, as alsois part of chromosome 3R with the puff at 85EF.

Figs. 9, 10. Electron micrographs of part of the looped chromosome 3L sectionedat two different levels. Part of the chromocentre (cr), and the nucleolus (n) areindicated.


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The RNP particles in the puff can be seen, in more open regions, to be arranged inlinear arrays (Figs. 4, 6). Towards the periphery of the puff these structures displaylooped configurations (Figs. 4, 5). This is of interest since such loops have long beenobserved for the Balbiani rings of Chironomus (Beermann & Bahr, 1954; Lamb &Daneholt, 1979), but have never previously been observed in the puffs of Drosophila,despite extensive attempts to disclose them (Berendes et al. 1974). The particles appearto be clustered about an axial filament of diameter approximately 10 nm (Figs. 4-6).By analogy with the interpretation of such structures in Chironomus (Stevens & Swift,1966; Lamb & Daneholt, 1979), we provisionally interpret the arrays of particles inpuff 2B as nascent RNP attached to axes of transcriptionally active deoxyribonucleo-protein. Occasionally a short stalk may be seen apparently attaching the particles tothe axial filament (arrows in Figs. 5, 6). However, such 'stalks' are by no means asprominent in the present material as in the Balbiani rings (Beermann & Bahr, 1954);this may well reflect a difference in ease of unfolding of the RNP particles betweenDrosophila and Chironomus.

Even within the obviously active puff at 2B 5-6 there are some dense regions. Thesemay represent bands in the process of decondensing and/or clusters of RNP particles.Similar structures which may reflect either or both of these types of organization occurat many chromosomal loci, including puffs and interbands (vide infra). The use ofelectron-dense labelled antisera against histones and acidic proteins should proveinvaluable for distinguishing the nature of these structures.

Also in Fig. 4, a micronucleolar-like structure (rnn) can be seen. A number of theselarge globular particles with a fine granular texture similar to that of the nucleolus(Fig. 9) are seen adhering to the chromosomes. Many are visible in the light microscope(Hill & Watt, 1977). Most of these structures are apparently lost during acid fixation.Their precise chromosomal localization is presently under investigation in thislaboratory.

Chromosome 3

Chromosome 3L was identified by the ' ballet-skirt' puff at 68C and the 2 ' Chineselantern' puffs at 74EF and 75B. Chromosome 3L was seen to loop out from thechromocentre and return to it; the 2 phase-contrast micrographs (Figs. 7, 8) show the2 sides of the loop in slightly different focal planes. Fig. 7 shows the 'ballet skirt' at68C on one side of the loop to be in focus and the corresponding section is seen in

Figs. 11-13. Higher-magnification electron micrographs of the 2 large puffs at 74EFand 75B on chromosome 3L as depicted in Figs. 7-10.

Figs. 12, 13 are parts of puffs 74EF and 75B at higher magnification.Fig. 12 shows RNP particles arranged in loop configurations.Fig. 13 shows RNP particles clustered around an axis. The arrow indicates an

apparent stalk.Fig. 14. Higher-magnification electron micrograph of the puff at 68C on chromosome3L as depicted in Figs. 7-10.


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EM of microdissected polyUne chromosomes 25

Fig. 9 in the electron microscope. This electron micrograph depicts the puff at 68C,and the puff at 75B beginning to be sectioned. Fig. 8 shows the other side of the loopof 3L with the 'Chinese lanterns' at 74EF and 75B in focus and a micronucleolus(win), along with a segment of 3R containing the puff at 85EF. The electron micro-graph depicted in Fig. 10 shows 75B, and 74EF just beginning to be sectioned. Thenucleolus (n) has survived the isolation by microdissection, illustrating the generalhigh degree of preservation of structure and shows in Fig. 9 a uniform fine-granularappearance with vacuoles and granular inclusions. Part of the characteristic hetero-chromatic banding of the chromocentre (cr) is also present in Fig. 9 along with theproximal and distal ends of chromosome 3L adjacent to the nucleolus.

The puffs at 74EF, 75B and 85EF were examined at higher magnifications. Fig. 11shows the puffs at 74EF and 75B known to be produced during normal development inresponse to the increase in the titre of ecdysone at late third instar and prepupalstages. These puffs contain a large number of RNP particles of fairly uniform dia-meter of 20-35 run and as with the puff at 2B near the X tip the particles are oftenseen to be arranged in linear arrays sometimes forming loops (Fig. 12). Some of theparticles appear to be clustering around an axis and are possibly attached by shortconnecting stalks (arrow in Fig. 13). Again there are some dense masses of materialin the puffs which may represent clusters of RNP particles or fragments of bands.Separating the 2 'Chinese lantern' puffs is one intact band and fragments of severalother bands. Fine fibrils run into the puffs from adjacent chromomeres; these aregenerally oriented close to the chromosome axis and are probably deoxyribonucleo-protein fibres. They exhibit a range of beaded morphologies rather similar to that ofinterband chromatin fibres (vide infra)

Fig. 14 depicts the 'ballet skirt' puff at 68C with its characteristic morphology, thebands and fragments of bands forming a zig-zag pattern which produces the effect ofthe frill of the 'ballet skirt'. The RNP particles are much more sparse and range insize from 20 to 40 nm. They are rather obviously located in interbands between thezig-zag bands and fragmented bands, in contrast to the more active puff structuresat 2B, 74EF and 75B. This may well be due to the fact that the peak of transcriptionalactivity for 68C occurs earlier than that of 74EF and 75B (Ashburner, 1967) and thesparsely distributed particles at 68C may be a feature of declining transcription.Chromatin threads of variable diameter are obvious running through the 'balletskirt* puff.

In Fig. 15, the puff at 85EF on chromosome arm 3R can be seen to contain a highdensity of RNP particles of diameter 30—45 nm. Dense clumps of material can be seenwithin the puff and once again these may represent fragmented bands and/or clustersof RNP particles. This puff, judging by the criterion of density of RNP particles,

Fig. 15. Electron micrograph of part of chromosome 3R including the puff at 8sEF,indicated in the phase-contrast micrograph in Fig. 8.Fig. 16. Electron micrograph of the area around the chromocentre. Indicated are:convoluted banding (cb), circumferentially distributed granules (eg), large discreetgranules 50-60 nm (lg), thick fibrils (/) and a fine-granular structure (Jgs).

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seems quite active. Ashburner (1967) records region 85F as reaching its maximalactivity at puffing stage 8, just after the first peak of activity of 74EF and 75B, whichbegins to decline after puffing stage 7. As 2B 5-6 and 2B 13-17 are also representedby Ashburner (1969) as maintaining maximal states of development through puffingstages 2 to 9 and 5 to 9, respectively, the appearance of the major puffs described indetail in the present study is consistent with normally functioning chromosomesaround stage 7 to 8. In other words, major aspects of the puffing pattern appear quitenormal on the chromosomes isolated without acid fixation, with no suggestion ofpuffing artifacts.

The chromocentre

Unlike the considerable difficulty experienced by Lakhotia & Jacob (1974) in findingthe chromocentre when studying serial sections through whole salivary glands, thechromocentral region was exposed by microdissection and could be readily locatedusing the phase-contrast micrographs of the entire genome. Various structures wereidentified at the chromocentre (Fig. 16). Very convoluted and disorganized banding(cb), giving a sponge-like appearance and generally considered to be heterochromatin,is seen to have numerous discrete RNP granules (approx. 35 nm) in a characteristiccircumferential distribution (eg), as found by Lakhotia & Jacob (1974). From thisarea the various chromosome arms arise. Fibrils (/) when noted between the con-voluted bands were frequently rather thick, about 20 nm in diameter. In one part ofthis convoluted area significantly larger granules (Ig) are seen, of 50-60 nm diameteras noted also by Lakhotia & Jacob (1974) and Skaer (1977) in electron-microscopestudies of whole salivary gland cells. That these 2 types of granules at the chromo-centre are likely to be RNP was shown by Lakhotia & Jacob (1974), using auto-radiography.

Such transcriptional activity at the chromocentre is in contrast to the generalconcept of inactivity of heterochromatin. To the best of our knowledge these 50-60 nmparticles have never been observed in acid-fixed chromosome preparations. Thedense area of very fine granules (fgs) is possibly part of an embedded micronucleolusor a hitherto unidentified structure.

Interband structures

Several different classes of granules are apparent in interbands along the chromo-some. These include the large 50-60 nm granules in the chromocentral region and

Figs. 17-20. Interband granules.Fig. 17. Electron micrograph of a typical chromosome segment showing interband

granules.Fig. 18. Part of an interband (outlined by square in Fig. 17) at higher magnification.

The arrows indicate stalks attaching RNP granules to larger complexes.Figs. 19-21. High-magnification electron micrographs at the same magnification ofchromatin fibres in the interbands. s indicates a super-superbead or chromomerefragment apparently on one chromatid.

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28 M. R. Mott, E. J. Burnett and R. J. Hill

granules of 30-45 nm diameter in the interbands of the chromosome arms. These 2classes of particle are both thought to be RNP at the sites of transcription of activegenes. A fairly typical region of bands and interbands is shown in Fig. 17. NumerousRNP particles of 30-45 nm are seen in the interbands, some attached to fibrils andothers apparently free. As seen towards the right of Fig. 17, these particles can beattached to large complexes. Fig. 18 depicts part of this interband region at higherpower, with several RNP particles appearing to be on thin fibrillar stalks (arrows) ofapproximately 6 nm diameter. Somewhat similar clusters of RNP particles werefound by Berendes et al. (1974) and Derksen (1976) in Drosophila hydei.

A third class of interband granules can be distinguished on chromosomes isolatedby the present procedure. Chromatin interband fibres, probably representing theindividual chromatids, maybe observed running parallel to the axis of the chromosome.These fibres present a range of structures, some of which are shown in Figs. 19—21,and generally vary in diameter from 7 to 10 nm. Granules of varying sizes are oftenseen on the fibres. These particles appear rather irregular in shape, sometimes with along axis in the direction of the fibres; the short axis generally ranges from 20 to40 nm. Somewhat comparable structures have been noted by Sorsa & Sorsa (1967) onacid-squashed chromosomes. However, it is perhaps of interest that the range ofmorphologies observed in the present study bears a significantly closer resemblance tothe types of structure recorded by Olins (1978) for chromatin fibres prepared bylysing nuclei in saline without any exposure to acid. We provisionally interpret theirregular particles that we observe on the interband chromatin fibres as structuresanalogous to the nucleosome clusters of Olins or superbeads of Hozier, Renz &Nehls (1977). However, there may be no fixed upper size limit for these particles; inother words, some of them may even range up towards the size of the smallestchromomere fragments (s in Fig. 20).

The present study demonstrates that Drosophila polytene chromosomes have beenisolated by a micromanipulative procedure which avoids acid fixatives and shouldtherefore contain native protein molecules. The degree of preservation of ultra-structure in the absence of acid fixation has been demonstrated to be at least as good as,and in some respects better than, that of classical acid-fixed chromosomes. Forexample, ribonucleoprotein granules in puffs are seen to be organized in linear arraysand evidence is adduced for looped transcription units, as have been observed for sometime in the Balbiani rings of Chironomus, but which have not previously been seen inDrosophila. The puffing pattern observed for the X and third chromosomes isconsistent with a natural puffing stage; there was no evidence for puffing artifacts.

In fact, Drosophila polytene chromosomes prepared in this way should combine thepresence of native proteins with optimal preservation of ultrastructure, absence ofnucleoplasmic background, and ease of cytological mapping. They should provideuseful targets for the localization of chromosomal proteins at an ultrastructural levelby immunological and hormone-binding techniques.

We would like to thank Jeanette Gregory, Ralph Chapman and Penny Williams for assistancewith electron microscopy; Fujiko Watt and Doreen MacPherson for technical assistance; andGeoff. Grigg, James Rendel and Trevor Scott for support and encouragement.


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(Received 9 April 1980)

ultrastructure of polytene chromosomes of … · after this first-stage fixation, the preparation...

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