studies on the sal locus in drosophila pseudoobscura . iii. the molecular pattern of dna in active...

STUDIES ON THE SAL LOCUS IN DROSOPHILA PSEUDOOBSCURA. 111. THE MOLECULAR PATTERN OF DNA

IN ACTIVE AND INACTIVE CHROMOSOME REGIONS1

Laboratories of Anatomy, Department o f Animal Biology, School of Veterinary Medicine, University of Pennsylvania,

Philadelphia, Pennsylvania

The banding pattern at the tip of salivary gland chromosome 3 in Drosophila pseudoobscura is determined by a nearby locus, sal (salivary). A number of cytological changes in the neighborhood of the sal locus have been obtained. Their effects on the appearance of bands are interpreted in terms of the h e structure of the chromosome and the arrangement of the DNA double helix. Bands as viewed in the light microscope may become dispersed, as in puffs, or compacted, as in position effects involving their transposition to heterochromatic regions. Changes of bands are interpreted in terms of dispersion or compaction of DNA.

Secondary puffing is induced in a paired sal chromosome which does not puff when unpaired, by a sal' homologue which puffs when unpaired. This is explained as being due to osmotic effects caused by added protein and gene products (RNAs) diffusing from the sal+ to the sal chromosome, when they are paired, through submicroscopic interconnecting spaces within the chromosomes.

I n t r o d u c t i o n

In the phenomenon of variegated position effect following transposition of a locus to a heterochromatic chromosome region, the phenotypic character con- trolled by the locus is manifested variably from to cell to cell of a tissue. In experiments with actinomycin, Perez-Davila and Baker ( 1967) obtained suggestive indications that the effect of heterochromatic regions in modifying the phenotype might be primarily within the chromosome; i.e., replication or transcription of the locus might be affected in the first place, resulting in modification of the phenotype.

The ideal material for a study of position effect would be a gene which determined a phenotypic characteristic of the salivary gland cell (such as the cytoplasmic granules described by Beermann in Chironornus pallidivittatus, 196'1) and whose activity (or most intense activity) was associated with p u a g at a site identified cytogenetically as the location of the gene. It should then be pos- sible to correlate the activity of the gene from cell to cell, as indicated by the degree of puffing (or the state of the band) with the degree of expression of the phenotypic character in each cell.

The mutant salivary (sal) in Drosophila pseudoobscura has been shown with fair certainty (Levine and Van Valen, 1962; Gersh, 1972b) to be located at the tip of chromosome 3 and to inhibit puffing in a region a few bands proximal to the srpl locus. Although this mutant falls short of the requirements just stated, it proved of great interest to review a detailed study of this locus in light of information on the fine structure of the salivary gland chromosomes of Drosophila rnelarwgaster (Gersh and Gersh, 1973). Problems dealt with in this review are concerned with (1) autonomy and non-autonomy of puffing; and (2) changes in activity of genes in the polytene chromosome in relation to the molecular arrangement of DNA.

'Aided by a research grant (5R01-GM14607) from the U.S. Public Health Service. Manuscr~pt received March 22, 1973.

Can. J. Genet. CytoL 15: 497-507. 1973.

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498 EILEEN SUTTON GERSH

Most of the facts on the sal locus underlying the discussion of these problems are presented elsewhere in two separate articles. In the first (Gersh, 1972a) it was shown that a change in banding pattern induced by the sal allele could be interpreted as a compaction of DNA into bands in contrast to the partially puffed condition in the wild type ( s a p ) chromosome. It was also shown that when unpaired in a sal+/sal heterozygote, each chromosome shows the banding pattern characteristic of its own genotype, though when paired, both show the sal' or an intermediate pattern. In the second article (Gersh, 1972b) evidence was presented that sal is a regulator locus separated by a few bands from the bands it controls; that mutation of the sal locus or transfer of bands to a heterochromatic region affects them similarly; and that the change probably consists of a compaction of DNA and concomitant inactivation of genetic loci whose function is undetermined.

The interpretation in terms of fine structure is based on a description of the distribution at the submicroscopic level of nucleic acids in the salivary gland nuclei of Drosophila melanogaster larvae. No one would doubt, I believe, that the polytene chromosomes are fundamentally similar in the three genera in which they are most commonly studied: Drosophila, Chironomus, and Scima. It seems perfectly legitimate, therefore, to use a study of one Drosophila species to interpret data obtained from another species of the same genus.

Autonomy of Puffing in Heterozygotes

As mentioned, in saZ+/sal heterozygotes, both chromosomes show the puffing in region 81 (according to the maps of Tan, 1937) characteristic of sal' when they are paired in this region. Nevertheless, when the chromosomes are unpaired they behave autonomously, each showing the banding pattern characteristic of its own genotype.

This is not a new phenomenon. Reports of similar behavior of puffed regions have been published (Ashburner, 1967, 1970; Mechelke, 1960). In larvae heterozygous for a puff, while some puffs cause puffing of the homologue when paired, others do not (Ashburner, 1970; Beermann, 1952; Panitz, 1965; Hsu and Liu, 1948). Ashburner has suggested two alternative explanations for the induction of puffing in the homologue. One alternative is that the puffing is due to diffusion of mRNA from the puffed chromosome to its non-puffed homologue. Such diffusion would not occur to any extent, of course, unless the homologues were paired. The second alternative involves a more complex interaction between regulating and structural genes in the region of the puff.

The study of the fine structure of the salivary chromosome and examination of the illustrations of induced puffs in the homologues of heterozygotes both lend some support to an interpretation similar to the first.

Electronmicrographs of salivary chromosomes (Gersh and Gersh, 1973) show that larger bands are divided into sub-bands or DNA-rich lamellae, alternating with RNA-rich larnellae. The many DNA double helices exist as second order helices (with diameter and period of about 400A) which are closely packed in bands or sub-bands, but are separated where they course through RNA-rich lamellae, interbands and puffs. It is assumed that histones (coiling proteins) are responsible for binding to the DNA double helix and maintaining it in the form of higher order coils (Zubay, 1964). In the compact masses of DNA of bands and sub-bands, the second order helices are folded or coiled and may be assumed to be cross-linked by other histones (linking proteins or interhelix proteins). C

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STUDIES ON THE SAL LOCUS IN DROSOPHILA 499

There is some evidence that acidic proteins, perhaps in cooperation with "chromosomal RNA" (Huang and Bonner, 1965) play a part in gene activation by releasing specific DNA sequences from bound histones (Paul, 1970). Accom- panying this process a swelling commonly occurs, affecting the whole nucleus or localized parts of it (Gurdon, 1968; Auer et al., 1970). This is presumably due to the intake of water by osmosis caused by the increased protein content.

It has been shown that acidic protein is present in puffs but not in bands of the salivary chromosome (Swift, 1962; Rudkin, 1964; Schultze et d., 1965). Some of it is presumably bound to histone that holds the DNA in a compact form at other stages. Free molecules of this acidic protein must exist in equilibrium with the bound form. It is very likely that the presence of this additional protein has an osmotic effect that results in the initial swelling of the puff, which may later be enhanced by the RNA molecules produced by transcription from the DNA template.

In the salivary chromosomes there are submicroscopic spaces which are continuous with those in the nucleoplasm like the interconnecting spaces of a sponge. They are present wherever the DNA is not densely packed and are continuous from one chromosome to another of a synapsed pair (Gersh and Gersh, 1973). Thus the activating acidic protein molecules accumulated at the site of an active gene in one homologue can move laterally through these submicroscopic spaces into the other homologue and initiate swelling there even though the corresponding allele may not be activated. It is hardly necessary to assume, as does Ashburner (1967), that the transcribed RNA is bound within the chromosome at the site of the puff, or that it would be unable to diffuse through the nucleoplasm outside the chromosome and eventually to reach an unpaired homologue. The high con- centration of acidic protein molecules at and around the polytene transcription sites of the homologue favor the rapid passage of this protein into the other homologue when it is synapsed. When unsynapsed, this homologue is exposed only to the nucleoplasm, where the acidic protein is present in greater dilution. Ash- burner comments that the degree of puffing of the 64C region in Drosophila melano- gmter in a synapsed heterozygote is always less than in the parent homozygous for the puff, as would be expected if the products of a single chromosome were distri- buted between the two chromosomes of the heterozygote. According to Mechelke (1960), in a hybrid between a strain that has a Balbiani ring and one that lacks it, the paired chromosomes form a small puff which extends across both of them. The reduced puffing can be interpreted as a sharing of substance accumulated by one homologue between the two of them. Inspection of the illustrations of puff heterozygotes with one homologue that does not puff when synapsed shows that this homologue is often slightly puffed and the other much more so (e.g., Beermann, 1952, Fig. 37; Panitz, 1965, Figs. 4,5,7,13). According to the present interpretation this would be likely to happen to the homologue which puffs only secondarily, as the result of diffusion of substances from the other chromosome. Only in the illustrations of Hsu and Liu (1948, Figs. 3,8) and Ashburner (1970, Figs. 2,3,6) are the homologues completely unpuffed, and in these cases the puffs involve bands which are normally moderately to highly compacted. In these cases failure to pilff may be due to the impermeability of the compacted DNA to the activating protein.molecules (Mirsky et al., 1972). Another possibility is that the mutant, non-pufEng region is inactive precisely because this allele has a modification of the specific receptor region so that the activating protein cannot affect it. In C

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Panitz's (1965) examples also, there are dense bands opposite to the puffed regions, and these may remain dense while the neighboring light bands disappear in the (secondarily induced) puff (Panitz's Figs. 6,14). In Panitz's Fig. 5, a dense band is broken up, as if partially puffed, at the side where it abuts on its puffed homologue. Following this interpretation in the instance studied here, the sap banding pattern is due to a degree of puffing caused by the activation of certain genes in section 81. This genetic activity may b'e presumed to follow the release of DNA, mediated by acidic protein, from the compact banded state to a relatively extended condition in which the primary DNA double helix is organized in secondary coils with a diameter and pitch of about 400A. The compact banded state, in which both coiling protein and interhelix protein are involved to keep these genes inactive or relatively inactive, is seen in the sal banding pattern. In the heterozygote, each condition is autonomous when the homologues are unpaired, but when they are paired, acidic protein and perhaps mRNA diffuse from the sd+ to the sal chromosome, disrupting its banding pattern and causing secondary puffing. It is of some interest that the s d locus itself is apparently distal to the bands in which the puffing occurs, suggesting that it may be a regulator gene whose product initiates activity of other genes not far away in the same chromosome (Gersh, 1972b).

Effect of Heteroohromatin on the Banding at the Tip of Chromosome 3 In T (Y; 3; 4) 190cvm', the tip of chromosome 3 is translocated to the Y

chromosome (Gersh, 1972b). It shows quite variable banding. Extreme examples are shown in Figs. 1A,B. The number of individual bands in this fragment of chromosome 3 is at least 15, counting a doublet as one band. The bands which distinguish the sal pattern are bands 10 to 14. The fragments in Fig. 1 have fewer than 15 clear bands. In some nuclei the fragment of chromosome 3 could not be found at all, at the chromocenter or elsewhere. It may be noted that whenever more than four of five bands are visible, the most proximal are heavily stained and tend to resemble those in the sal banding pattern. In some fragments in which the sequence of bands is incomplete and the dark bands not visible, the most proximal bands merge into diffuse heterochromatic material (Fig. 1B). The proximal heterochromatic regions in Drosophila pseudmbscura are less extensive than in D . nzelanogaster (see Figs. 2,3), and the heterochromatic material in question is not dense enough to obscure heavy bands, even if it were to overlay them. Frequently, moreover (Figs. 3,4), the amount of heterochromatic material is further reduced by the separation of the heterochromatic part of the X from the chromocenter in which the other chromosomes are joined.

The interpretation of this variability, commonly referred to as "heterochromatization" of euchromatic banded regions, can be pursued at different levels. First

Fig. 1. D. pseudoobscura, T190cvmfsal+/sal. A. Part of a salivary gland squash pre- paration. Chromosomes labeled according to Tan's maps (1937): P, proximal part, D, distal part of chromosome; T3D, translocated distal tip of chromosome 3 at chromocenter next to heterochromatic material (het); arrow shows dark proximal bands of the translocated fragment. Inset, drawing of area enclosed in lines which includes translocated tip of 3 and small chromosome 5. B. Part of another nucleus. At upper left, translocated tip of 3 (number of visible bands much reduced) adjacent to heterochromatic material, and tip of normal 4. At lower right, tip of normal 3 (3D) and translocated tip of 4 (T4D) attached to proximal part of 3 (3P). Inset, drawing of region enclosed in lines, which includes translocated tip of 3 and tip of normal 4 (X 1,680). C

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Fig. 2. D. melanogaster, chromocenter. A. Part of nucleus showing (chrc) aggregation of proximal heterochromatic regions of salivary chromosomes, which are labeled according to Bridges' maps (1935). The unlabeled chromosome at lower right intrudes from an adjacent squashed nucleus (X 1,320). B. Enlargement of chromocenter region (X 2,800). C. Drawing of chromocenter region; arrows indicate the limit of heterochromatic material in each chromosome arm (X 1,320). Compare with Fig. 3. C

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Fig. 3. D. pseudoobscura, chromocenter. A. Part of nucleus showing (chrc) chromocenter formed by autosomes, the heterochromatic region of the X chromosome being ex- cluded; D, distal part, P, proximal part of chromosome. There is a translocation between chromosomes 2 and 3 at the point marked T, distal to which each chromosome 3 is unpaired (3D) (X 1,320). B. Enlargement of chromocenter region (X 2,800). C. Drawing of chromocenter region; arrows indicate limits of heterochromatic material in each chromosome arm (X 1,320). Compare with Fig. 2. C

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Fig. 4. D. pseudoobscura, T190cvABJ. The tip of chromosome 4 was originally translocated to chromosome 3, as in T190cv7"' (Fig. lB, lower right). Subsequently this translocated chromosome was broken in the part of chromosome 3 proximal to the dark bands ( b l ) and puff (p) of section 80 and to a few bands (b2) of section 79. The distal fragment (T4D) was reattached to heterochromatic material at the chromocenter (het). A. Part of nucleus showing relationship of chromosomes. The heterochromatic part of the X chromosome (X het) is separated from that of the autosomes (X 1,320). B. Enlargement of region enclosed in lines, showing bands of chromosome 3 distal ( b l ) and proximal (b2) to the puff (p), which remains fully puffed in the translocated fragment (T4D) (X 2,800). C. Drawing of region enclosed in lines (X 1,320). C

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it is worth while to review as a whole, and interpret at the level of the light microscope, the information given above about the variations in banding pattern. Fol- lowing this an interpretation will be made in terms of fine structure.

Znterpretation of banding patterns at the light microscope level: A mutation from sal' to sal results in a change of banding pattern which may be interpreted as a consolidation of diffuse material including DNA in the proximal part of section 81 (Tan's 1937 map) into heavy bands, arranged in three groups. The same change is brought about by a deficiency of bands in the distal part of section 81, presumably including the sal locus. Conversely, one might say that the substitution of a fully active sal' allele for a partially or totally inactive allele results in a puffing of the proximal part of section 81 and a separation of material from the bands, so that the heavy bands become slender and diffuse.

Transfer of the tip of chromosome 3, with the sal' allele, to the heterochromatic Y chromosome has the same effect as mutation to sal. Although there is not necessarily any change in the distal region of 81 where sal is presumed to be located, the proximal bands of section 8 1 become consolidated. Sometimes there is a more extreme change: the proximal bands are not visible in any form, though the most distal bands can still be identified. An even more extreme state is found in some nuclei where the banded tip of chromosome 3 cannot be seen at all. At this stage the bands which are presumed to include the sal locus are also affected, of course, and indeed they may also appear more dense than usual in the intermediate states where few bands are visible.

The interpretation adopted here is, briefly, that sal is a regulatory locus that normally switches on a group of genes in the proximal part of 81. Inactivation of the sol locus has, as a consequence, the inactivation of this group of genes. In position effect, these genes may be inactivated by "heterochromatization," regardless of whether the sap locus is likewise inactivated or not.

Interpretation of banding patterns in terms of fine structure: Turning to the submicroscopic level, the first point to be considered is that in the puffing process, part of the substance of relatively compact bands is dispersed throughout the puff. In fact it has been found, as stated above, that the DNA in puffs is relatively loosely organized, being arranged in separated second order helices of approximately 400A in diameter and period (Gersh and Gersh, 1973). Also the DNA which is sur- rounded by the nucleolus is in the form of thin strands (small aggregates of second order coils). Thus, although the nucleolus-organizing region is adjacent to neighboring dense heterochromatic regions, the nucleolus-organizing DNA itself is more dispersed. By contrast, the DNA in the proximal heterochromatic regions generally is massively packed. These findings are in agreement with the view, recognized for several years (see for instance, Littau et d., 19684), that active chromatin is finely dispersed and compacted chromatin is inactive. It has been concluded (Beermann, 1962) that in the salivary gland chromosome the puffs and nucleolus-organizing region are actively transcribing RNA, while heterochromatic regions are inactive. This conclusion is borne out by the electron microscope results, which show, on the one hand, an abundance of RNA-containing granules in and around puffs, as well as around the nucleolus-organizing DNA; and on the other, a paucity of RNA in heterochromatic regions. Considered in these terms, the s d locus seems to control a group of genes, by releasing their DNA, which C

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would otherwise be bound into compact, dense bands and kept inactive. This release can be thought of as a process such as acetylation or phosphorylation (AU- frey, 1970; Shepherd et al., 1971a,b), or of competition by acidic protein molecules produced as a result of sal' activity, which loosens the bonds between DNA and the coiling and linking proteins which keep the DNA coils in the compact state. The released DNA, in the form of second order helices coursing through the puffed regions, can then become available as a template for transcription of mRNA. In the absence of sal' activity, however, these genes remain bound in the compact bands.

The DNA of the chromocentral heterochromatic regions of the chromosomes also is kept in a highly condensed state, perhaps by cross-linking proteins, and when the tip of chromosome 3 is transposed to such a region, the effect of these binding proteins may extend to the DNA of the transposed tip, compacting the proximal bands of region 81 even though the sal' allele may remain unchanged. The well-known polarized "spreading effect," in which loci progressively further from the breakpoint and from the heterochromatic material are affected, but in diminishing degree (Lewis, 1950), may be due to a relative rigidity or density of the chromocenter region. If the extended interband DNA is pulled in toward and bound to this dense or rigid center, and if this binding occurs progressively to interband regions further along the chromosome but to varying extents from cell to cell, then the closest loci will be affected most frequently, and those further along the chromosome will only be affected when the more proximal loci are already inactivated. Thus, in T190cv70f the bands in the proximal part of section 81 are compacted because they are immediately adjacent to the break point and to heterochromatic material. In some nuclei the proximal bands seem to be completely disorganized and indistinguishable from the heterochromatic regions, in which case the next most proximal bands appear unusually compact, as described by Schultz (1939) in another instance of position effect. Incidentally, I have obtained another translocation in which the tip of chromosome 3 has been transferred to a heterochromatic region. In this rearrangement (T190cvm') the puff in section 80, though translocated to a proximal heterochromatic region, remains puffed (Fig. 4), presumably because it is at some distance from the break point, from which it is separated by bands of section 79 (Tan's 1937 map).

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studies on the sal locus in drosophila pseudoobscura . iii. the molecular pattern of dna in active...

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