J. Cell Set. 26, 251-266 (1977) 251

Printed in Great Britain

INTERBAND TRANSCRIPTION IN DROSOPHILA

R. J. SKAERDepartment of Haematological Medicine, Hills Road, Cambridge, England

SUMMARYMost puffs contain perichromatin ribonucleoprotein granules 30-40 nra in diameter; other

puffs contain ribonucleoprotein granules 25 nra in diameter or mixtures of these and peri-chromatin granules. All puffs contain fragments of band material possibly from several bands.By examining progressively smaller puffs the transcriptionally active region is shown to liewithin an interband. Some transcriptionally active interbands are so small that there can be nosignificant contribution of decondensed band material to the interband. Up to 33 % of allinterbands contain significant evidence of transcription. These findings are discussed inrelation to the use of the terms heterochromatin and euchromatin to describe the bandingpattern of polytene chromosomes.

INTRODUCTION

Polytene chromosomes show a very clear-cut subdivision into bands and interbands.While there appears to be, in parts of the X chromosome of Drosophila at least,a reasonably accurate correspondence between the number of bands and the numberof complementation groups (Judd & Young, 1974; Beermann, 1972) the functionalreason for the clear-cut alternation of condensed and dispersed chromatin is not clear.Transcriptionally active chromatin is dispersed and since bands disperse and becomeinvisible in the light microscope when puffs or Balbiani rings are formed at sites ofgene expression, it is customary to equate the bands rather than the interbands withthe sites of genes. Since 95 % of the DNA is in the bands (Beermann, 1972), thissupposition would seem reasonable. Crick (1971), however, has pointed out that thereis probably sufficient DNA in the interbands of Drosophila to code for the range ofproteins it is likely to make. Keyl (1975) compared the lampbrush chromosomes inthe spermatocytes of Chironomus with polytene chromosomes from the salivaryglands of the same animal and has suggested that there is a numerical correspondencebetween the loops on the lampbrush chromosomes and the bands of the polytenechromosomes. Such an equation between dispersed loops and condensed bands wasmade plausible by the absence of condensed chromomeres from the lampbrushchromosomes - the loops apparently arise directly from the axial fibres. Keyl suggeststhe interbands of polytene chromosomes correspond to the transcriptionally inactive,axial fibres of lampbrush chromosomes. Such a correspondence would make it veryunlikely that interbands, though they are made up of dispersed chromatin, could everbe transcriptionally active.

Sites of gene expression contain ribonucleoprotein (RNP) granules calledperichromatin granules. If a brief pulse of tritiated uridine is administered to a

252 R. J. Skaer

transcriptionally active cell the radioactive label is found first in perichromatin fibrils(Nash, Puvion & Bernhard, 1975; Fakan, Puvion & Spohr, 1976); the label is thenfound in regions rich in perichromatin granules (Vazquez-Nin & Bernhard, 1971;Lakhotia & Jacob, 1974). Perichromatin granules have a size range of 30-45 nm andhave been shown to contain RNA by the Bernhard staining technique. They are foundin puffs (Stevens & Swift, 1966), Balbiani rings (Vazquez-Nin & Bernhard, 1971;Daneholt, 1975), transcriptionally active regions of chromatin in liver cells (Puvion &Bernhard, 1975; Fakan et al. 1976) and lampbrush loops of amphibian oocytes (Mott& Callan, 1975; Angelier & Lacroix, 1975). There is thus firm evidence that they areassociated with transcription.

I have found perichromatin and other RNP granules to be present in largeamounts not only in puffs but also in many interbands. Some are found in interbandsso thin that it is difficult to believe that the band has, by decondensation, contributedany material to the transcriptionally active interband. Thus transcription originatesin, and may be confined to, the interbands. The contribution made to transcriptionallyactive sites by band material remains to be determined.

MATERIALS AND METHODS

Salivary glands from 3rd instar larvae of the Canton-S strain of Drosophila melanogaster werefixed by placing the living larvae in the fixative and immediately cutting them across half-waydown the body, just behind the tip of the salivary glands (which can be seen through the ventralbody wall). If the posterior half of the body is completely severed with scissors, muscularcontraction of the anterior part of the body ejects the salivary glands with their attached fatbody into the fixative. In good dissections the 2 glands stick out into the fixative like a pair ofrabbit's ears and if the anterior half of the larva is moved gently through the fixative immedi-ately after dissection the fixative has very good access to the cells. The fat body is not removed;any attempt to do this damages the cells of the salivary gland by stretching them, and anartifactual network is produced in the nuclear sap (Skaer & Whytock, 1977).

The fixative was 3 % glutaraldehyde (redistilled as described in Skaer & Whytock, 1976, orbought from Polysciences as unbuffered 8 % glutaraldehyde) which was buffered to pH 6 7 with005 M HEPES and contained 025 M sucrose and 125 mM calcium chloride. The calciumconcentration was occasionally raised to 4 mM. A fixative temperature of 10 °C gave bestfixation; on the one hand the material was free from traces of network in the nuclear sap thattended to appear at higher temperatures (Skaer & Whytock, 1977), on the other hand thebands did not become diffuse as occurs during fixation at o °C. Osmium tetroxide was not usedas it did not improve the fixation of the nuclei and prevented histochemistry.

Glands were dehydrated in a cold ethanol series, treated with propylene oxide and embeddedin Spurr or Araldite resin. Sections were stained with uranyl acetate and lead citrate andexamined in an AEI EM6B electron microscope. The Bernhard staining technique was carriedout as described in Bernhard (1969). All pictures are of unsquashed chromosomes in nuclei.

RESULTS AND DISCUSSION

Morphology of puffs

Puffs are recognized as slightly enlarged regions of chromosome, containingaggregates of darkly staining granules in the size range 25-45 nm (FiSs- *> 2> 4)- Mostpuffs contain almost exclusively large granules 35-40 nm - perichromatin granules(Figs. 1, 8, 13). These granules stain with the Bernhard staining method for RNA(Fig. 14); although the technique is empirical its results seem fairly reliable, for it is

Interband transcription 253

immediately obvious when successful bleaching of the DNA has occurred. Underthese circumstances ribosomes and other structures known to contain RNA arestained. A few puffs contain almost exclusively small granules 20—25 n m m diameter -just slightly larger than ribosomes (Fig. 2). Many puffs contain both small and peri-chromatin granules (Fig. 4). Very occasionally puffs near the chromocentre containvery large granules 45-50 nm in diameter (Fig. 3). Lakhotia & Jacob (1974) suggestpuffs with these granules are located at the base of the X chromosome.

The relationship between the small granules found in puffs and the perichromatingranules is not clear. Yamamoto (1970) has found that Balbiani rings in Chironomuscontain granules with an apparent particle diameter of 40-50 nm, after heat shock andregression of the Balbiani rings there is regeneration of granules which 1 h after heatshock have a range of apparent particle diameters that reach peaks at both 22-5 nmand 38 nm. Six hours after the heat shock there is an enhanced ring size and manygranules are present whose diameters are in the range 40-50 nm. This might suggestthat the 22-5-nm granules are a precursor of the 40-50 nm granules. In this caseFig. 2 might be a developing puff. Alternatively granules of this size may be the normalfinal transcription product of that particular puff, although the presence of a very fewlarger granules militates against this idea. In the heat shock puff at locus 2-48 BC ofDrosophila hydei the puff product is large — up to 400 nm in diameter, and complex,with a large RNA-free core surrounded by a cortex of 30-nm particles that containRNA (Derksen, 1976). Here, too, it appears that particles less than 30 nm are formedand these form the final puff product by aggregation and remodelling. Two sizes ofgranule — small, approx. 20 nm diameter and large, 30—38 nm diameter - are alsofound on some loops in lampbrush chromosomes of oocytes of Pleurodeles (Angelier &Lacroix, 1975). One category of small (25 nm) ribonucleoprotein particle found inchromatin, however, does not appear to be a precursor of perichromatin granules.Thus interchromatin granules are slightly or not significantly labelled with tritiateduridine even after prolonged labelling (Fakan &Bernhard, 1973; Fakan etal. 1976). Itis thus not clear what function the small granules perform - nor whether all RNPgranules of this size in the nucleus should be included in the same category - recog-nized morphologically as interchromatin granules. In certain situations some may beprecursors of the larger perichromatin granules.

Puffs also commonly contain many fragments of band material (Figs. 1, 2, 4, 13).Whether these bands are fragmented by transcription or whether they are naturallydotted can only be settled by studying known bands under different physiologicalstates. These fragments are often not in lateral register with each other (Fig. 4) so itis not clear whether or not they are all fragments of the same band dispersed bytranscription. In smaller puffs, however, the fragments of band material retaina greater degree of lateral register and in these it appears that several bands withina puff are surrounded by perichromatin granules. The progressive loss of lateralregister of band material in a puff will contribute to the loss of visibility in the lightmicroscope of band material when puffs develop. Many puffs appear to contain frag-ments of several bands (Fig. 1). This applies to small puffs for only in these can pieces

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254 R- J- SkaeT

of band material be allocated to their band. It seems unlikely that several adjacentbands should simultaneously become transcriptionally active to make up each puffseen in the light microscope. It seems more likely that perichromatin granules spreadalong the chromosome away from their original site of synthesis and pass betweenbands where these are dotted, until the granules are contained by an unbroken bandabove and below the puff (Fig. i).

The arrays of perichromatin granules in Fig. 13 (arrow) may possibly be movingthrough gaps in the dotted band and may be orientated by the longitudinal orientationof interband fibres rather than actually synthesized in these positions. Fig. 11 alsosuggests that perichromatin granules may move longitudinally in the chromosomeaway from their site of synthesis and may pass through gaps in bands.

The extent of band decondensation in a puff is difficult to assess. Fragments ofbands that may not necessarily be transcriptionally active may be included in the puff.Moreover the accumulation of transcription products causes the puffing region toincrease in volume so that fragments of band material become increasingly separatedfrom each other. Only extensive serial sectioning will reveal the true volume of bandmaterial in a puff. If the puff shown in Fig. 4 were produced from a single band ofaverage size (o-i fim) it would have increased in length by some 25 times and perhapsdoubled in width. These figures are not entirely reliable for although it is a fairlyaccurately longitudinal section, chromosomes in nuclei tend to kink at a large puffperhaps through the greater activity of one allele on the pair of homologues that makeup the chromosome. Moreover the possibility of longitudinal spread of transcriptionproducts makes the estimate of volume increase of a puff unreliable. Berendes (1971)claims his pictures show complete decondensation of a band, so that the band structuredisappears in the puffed region 2-48 BC of D. hydei.

Interband transcription

If one studies smaller and smaller puffs the transcriptionally active region is seen tolie within an interband (Figs. 5, 6, 8—11). Perichromatin granules occur often as a singlerow down the centre of an interband (Fig. 5) that is bounded by unbroken bands oneither side. Fig. 5 shows an interband of average thickness (o-i /tm); Figs. 6 and 8 showvery small interbands (0-07 /im) so band decondensation is unlikely to have con-tributed significantly to the interband. Although perichromatin granules are oftenevenly spread across the width of the chromosome in the interband (Figs. 5, 9, 11),they are sometimes grouped in patches. This agrees with the findings of Scheer,

Fig. 1. Typical puff containing abundant darkly staining perichromatin granules(diameter approximately 35 nm). Smaller granules also present. The sea of granulessurrounds numerous fragments of band material. The longitudinal limits of the puff areset by substantial unbroken bands above and below the puff. At the sides the peri-chromatin granules can be seen extending beyond the limits of the puff into thenuclear sap. x 46000, scale bar 1 fiva.

Fig. 2. Puff containing only small granules (diameter approximately 20 nm) - verymuch the same size as ribosomes (bottom right hand corner of figure), x 46000, scalebar 1 /im.

Interband transcription 255

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R. J. Skaer

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Interband transcription 257

Trendelenburg & Franke (1976) that in maturing oocyte nuclei, as transcriptionbecomes less active some ribosomal RNA matrix units are fully active, some partiallyactive with sparse RNA, and some completely inactive, all in the same nucleus.

Sometimes in Drosophila, groups of perichromatin granules occur opposite breaksin an adjacent band (Fig. 10). This might be interpreted as evidence that transcriptionoccurs at sites of band decondensation. The appearance is sufficiently uncommon forthis interpretation not to be generally true. In Fig. 11, a transcriptionally fairly activeinterband is bounded by broken bands with transcriptionally inactive interbandsbeyond them. Perichromatin granules from the active interband appear to be passingbetween breaks in the bands and entering the neighbouring, transcriptionally inactiveinterbands. In the light of the demonstration that transcription can occur in interbandsbounded by unbroken bands, this seems a more plausible explanation for the effectin Fig. 11 than that the band has decondensed at the gap containing the perichromatingranules, and that these granules have spread into the neighbouring interband.

Some interbands contain arrays of small granules that are slightly larger thanribosomes (Fig. 7). As in the case of puffs with this size of granule (Fig. 2) it is notclear if the interband or the puff is developing (or regressing) or is producing its finalsize of granule.

It is interesting that Alonso & Berendes (1975), searching for the location of 5 Sribosomal RNA genes in D. hydei should find RNP particles in the interband next toband 2-2 3 B 1, 2 and conclude that 'the data presented in this paper do not excludethe possibility that only a small number of the 5 S RNA cistrons, which could be situatedin the interband adjacent to band 2-2 3 B 1, 2 are transcribed. The interband doesshow incorporation of [3H]uridine and contains RNP-like particles. The diameter ofthese particles (~ 30 nm) is very similar to that of RNP particles observed in variouschromosome puffs'. If these are indeed ribosomal RNA it is interesting that it ispackaged in much the same way as perichromatin granules, some of which may makeup messenger RNA (Stevens & Swift, 1966; Egyhazi, 1976).

Number of transcriptionally active interbands

Glands vary somewhat in the extent of their transcriptional activity, but as many asone-third of all interbands contain perichromatin granules (Fig. 12). This figure isapparently due to the very large number of small active interbands rather than to theextensive longitudinal spread of perichromatin granules to many inactive interbandsfrom an active puff. Most active interbands in Fig. 12 have inactive neighbours.A possible further source of error would be the infiltration of interbands by peri-chromatin granules that are present in the nuclear sap. If this occurred to a significantextent one would expect to find large numbers of interbands with a single granule per

Fig. 3. Puff containing large (40-45 nm diameter) granules. This puff is situated veryclose to the /? heterochromatin of the chromocentre. x 50000, scale bar 0-5 /tm.

Fig. 4. Large puff containing both perichromatin and smaller RNP granules. Bandmaterial is present. The nuclear sap (top right) contains a network, for the fixativecontained 4 mM calcium chloride. Perichromatin granules are visible in the network,x 54000, scale bar 0 5 /'m.

R. J. Skaer

Interband transcription 259

section. In fact, interbands containing so few granules are relatively rare - onecommonly sees interbands free of granules or with 3 or more per section.

Since there are over 5000 bands in Drosophila, and if one-third of the interbands areactive, more than 1500 sites of transcription must be active at any one time. This ismuch higher than the number assessed autoradiographically (10% of the bands;Pelling, 1964). However, it agrees with recent observations on the very wide distributionof Drosophila RNA polymerase B in polytene chromosomes, as measured by immuno-fluorescence (Plagens, Greenleaf & Bautz, 1976). From this number of transcription-ally active sites it is clear that most interbands that contain perichromatin granules aretranscribing at a low, steady rate - otherwise at a slightly later stage these would be1500 puffs and this is never seen. Only a few active interbands can be early stages inthe development of larger puffs - indeed, many fewer than the number of large puffsat any one stage, since some puffs exist for substantial periods of time. It is thereforepossible that these relatively rare developing puffs have special transcriptionalfeatures - such as a particular category of granule.

Implications for euchromatin and heterochromatin

The morphological demonstration of transcriptional activity in the interbands,places the interbands, together with the puffs, firmly as euchromatin; and, byimplication, the bands, as regions of condensed transcriptionally inactive (unless oruntil they decondense to contribute to a puff) heterochromatin. Bands also share withother forms of heterochromatin the property of late replication — Kalisch & Hagele(1976) found that puffs label early in the replication cycle of polytene chromosomes;they also replicate quickly. Prominent bands, on the other hand, label later in thereplication cycle and have a long labelling period. This view of interbands and puffsas euchromatin and bands and centromeric regions as heterochromatin is in conformitywith the original subdivision of chromatin on the basis of compactness of packing(Heitz, 1928).

Geneticists, however, are accustomed to regard both the bands and interbands ofpolytene chromosomes of Drosophila as euchromatin, and for these chromosomes theyrestrict the term 'heterochromatin' to the chromocentre, some telomeres, and a few

Fig. 5. Interband (100 nm thick) containing a row of perichromatin granules acrossthe centre of the interband (arrow), x 50000, scale bar 0 5 /»m.

Fig. 6. Small interband containing perichromatin granules (arrowed), x 50000, scalebar 05 fim.

Fig. 7. Interband containing numerous ribosome sized granules. At top right areribosomes in the cytoplasm, x 56000, scale bar 0 5 /im.

Fig. 8. Length of chromosome containing a puff with perichromatin granules (top)and a small interband with 3 perichromatin granules (arrow). The next interbanddown is also transcriptionally active. The nuclear sap contains a network as thefixative contained 4 mM calcium chloride, x 50000, scale bar 0 5 fim.

Fig. 9. Interband containing perichromatin granules. At the bottom is part of thenucleolus. x 50000, scale bar O'5 fim.

260 R. J. Skaer

Interband transcription 261

intercalary regions on the chromosome arms (Dobzhansky, 1944). The reason for therestriction of the term ' heterochromatin' to these regions seems to be 2-fold. (1) Theseregions apparently contribute very little to the phenotype - they are genetically almostsilent. (2) Apparently similar regions of early mitotic chromosomes are highlycondensed.

Early in prophase in Drosophila the satellite-rich a heterochromatin together withwhat may be /? heterochromatin are condensed as a dense proximal mass in eachchromosome. This condensed region makes up 30-50% of the length of the Xchromosome in early prophase (Cooper, 1959), and a very large proportion of it is thesatellite-rich DNA of the region around the centromere (Gall, Cohen & Polan, 1971).The latter is scarcely or not represented in the polytene chromosomes (Gall et al.1971).

The mitotic pattern of heterochromatin, however, is not a particularly good guideto the regions that are made up of heterochromatin in interphase. Thus the entirecategory of facultative heterochromatin (Brown, 1966) is not represented in the mitoticpattern of bands. Moreover, a precise correspondence between interphase hetero-chromatin, and the bands of constitutive heterochromatin at mitosis is not to beexpected, since the metaphase pattern is produced progressively by differential fusionof small regions of heterochromatin. These by a complex pattern of accretion ofheterochromatin make up the relatively small number of bands that comprise themetaphase pattern (Rohme, 1974). Rohme found, indeed that, in the prematurely con-densed chromosomes of the Indian muntjac, this progressive fusion occurs particularlyin the proximal regions of chromosomes - the main sites of a and /? heterochromatin inDrosophila. The pattern of bands in polytene chromosomes, on the other hand, isconstant with time and is altered only by the development and regression of puffs.

The features by which heterochromatin, in the sense used by geneticists, is recog-nized in the polytene chromosomes is by its nebulous, fuzzy appearance, with noclear-cut banded structure; it usually, though by no means always, stains strongly(Dobzhansky, 1944). It is not clear that these features, particularly in the intercalaryregions are sufficiently diagnostic to separate the material with these properties fromthe material of the bands, in the range of whose structure these features can beaccommodated.

The /? heterochromatin at the chromocentre is clearly a region of the chromosomeswith very special properties, both cytological in the staggered and contorted nature ofits bands (Lakhotia & Jacob, 1974) and genetic, in its apparent genetic inertness. The

Fig. 10. Interband with perichromatin granules localized opposite breaks in theadjacent band, x 50000, scale bar 0 5 /im.

Fig. 11. Transcriptionally active interband containing numerous perichromatingranules that appear to have passed through a break in the adjacent band into theinterband above, x 72000, scale bar 0 5 fim.

Fig. 12. Low-power view of a length of chromosome showing a puff (p). Similarperichromatin granules can be seen in the arrowed interbands. Approximatelyone-third of the interbands contain perichromatin granules. Cytoplasm (c) showsribosomes. x 25000, scale bar 0-5 /tm.

afisj R. J. Skaer

w >•

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Interband transcription 263

restriction of the term 'heterochromatin' as applied to polytene chromosomes, almostexclusively to the region of the chromocentre is particularly unfortunate since (apartfrom a tiny region of a heterochromatin in some species) the staggered interbands inthis region are continuously and highly active transcriptionally. This was firstdemonstrated by Lakhotia & Jacob (1974) by means of autoradiography and electronmicroscopy; I have confirmed their findings. Such a demonstration demands a reassess-ment of the mechanism of position effect variegation, for it is not immediately obviouswhy genes transposed into the chromocentre by a break next to the gene in questionand a break in the /? heterochromatin, should be genetically inactivated (Rudkin,1965; Baker, 1968). The problem remains of why a region that is so active transcrip-tionally and to which many transcribed sequences will bind (Spradling, Penman &Pardue, 1975) should be genetically relatively inert (Hilliker & Holm, 1975).

Thus it does not appear that the restriction of the term 'heterochromatin' to theseregions of polytene chromosomes is justified. Use of the term 'a heterochromatin'(Heitz, 1934) for that region around the centromere of mitotic cells that is rich insatellite DNA, and the term '/? heterochromatin' for the staggered bands of thechromocentre does not exclude the possibility of describing the remainder of the bandsas, say, 'y heterochromatin'. It is interesting that Lefevre (1976) has also come to theconclusion that ' " euchromatic" bands that are compact, stained, and visible must,paradoxically, be in a heterochromatic state. Only puffed regions and interbands aretruly in a euchromatic state'. He reaches this conclusion for largely different reasonsthan have been given above.

Interphase chromosomes increase in length progressively through Gx to S, andthen progressively shorten through G2 to mitosis (Rao & Wilson, 1976; Hittelman,Rao & Rao, 1976). These changes in length are associated with complex patterns ofdecondensation on the one hand and differential condensation and fusion on the other.The chromomere pattern thus changes throughout the cell cycle. The fact that thebanding pattern in the polytene chromosomes is the same in all tissues of D.melanogaster (Beermann, 1972) presumably means that polytenization occurs only atone, very precise part of the cell cycle. Since DNA replication occurs at this time, thepattern possibly represents a particular, early 5-phase banding pattern. There is noparticular reason to assume this pattern is any more functionally fundamental than,say, a G2 pattern, which would presumably be different. This may account for thefact that there are some 5000 bands in Drosophila but only some 2000 bands inChironomus. There is no particular reason to assume the numbers of genes in these2 flies are very different. It may be coincidence that in Drosophila the number of

Fig. 13. Small puff containing perichromarin granules. The granules surround piecesof band material. The granules appear to have passed between the fragments of thedotted band (arrow) as far as the thick unbroken band at the top. x 50000, scale bar0-5 /tm.

Fig. 14. Section of length of chromosome and cytoplasm stained with the BernhardEDTA bleaching technique to show RNA. Ribosomes are visible in the cytoplasm atright. A puff (p) contains darkly staining perichromatin granules (arrows). These arealso visible between the bleached bands b. x 54000, scale bar 0-5 /im.

264 R. J. Skaer

genes has, where it has been tested, corresponded to the number of bands or inter-bands. It may be that relatively frequent decondensed regions are required in a chromo-some for easy access of DNA polymerases and RNA polymerases. These regionswould, of course, be part of a complex, reproducible pattern. The remainder of thechromosome would be relatively tightly packed heterochromatin.

There are two possible ways in which a puff could develop from a transcriptionallyactive interband: (1) The bands might not decondense at all but be broken and thepieces forced apart laterally by the accumulation of transcription products so thechromosome becomes locally fatter. Transcription to differing extents across theinterband might cause the fragments of band to lose their lateral register with eachother, so that, as in the chromocentre, the banded structure disappears in the lightmicroscope. An argument against this view would be that there might be insufficientDNA just in an interband to stretch across a large puff or Balbiani ring of Chironomuswithout the band contributing some DNA. It is not known, however, how long theDNA fibres are in puffs or Balbiani rings. The long loops to which the stalkedperichromatin granules of Balbiani rings are attached are assumed by Daneholt (1975)to be DNA on the basis of some histochemistry performed by Stevens & Swift (1966).The histochemical pictures are very difficult to interpret; perichromatin granules,moreover, do not normally attach directly to DNA. In lampbrush chromosomes theperichromatin granules clearly attach to transcribed RNA fibrils (Mott & Callan,1975). If an interband becomes a puff by an active interband increasing its activitywithout the adjacent band decondensing to some extent and contributing to the puff,one would not expect the DNA sequence of the transcription product to change onpurring. The interbands would presumably have to be located precisely in relation tosequences that are needed for transcription.

(2) The transcriptionally active interband might enlarge into a puff not only by theaccumulation of transcription products but also by recruitment of decondensed DNAfrom an adjacent band. If this occurred the DNA sequences transcribed might changeas the puff develops. On such a view the precise placing of the interband in relationto DNA sequences needed for transcription would not necessarily be of crucialimportance. If the puff enlarged sufficiently by decondensation of the band the DNAsequences necessary to specify an appropriate protein would eventually be reached.

These possibilities are being investigated with known developing puffs, but thesituation is complicated by the possible incorporation into the puff of adjacentinactive dotted bands. Moreover, since 95 % of the DNA is in the bands a slight errorin measuring the volume of band material in a puff would make a great deal of differ-ence to the result.

I am most grateful to Miss S. Whytock and Mr J. P. Emmines for expert technical assistance.I thank Dr Peter Lawrence and Professor H. G. Callan for very helpful comments on thiswork. Dr H. le B. Skaer read and criticized the manuscript. This work was supported by theLeukaemia Research Fund.

Interband transcription 265

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(Received 20 January 1977)