CHROMOSOMA

Chromosoma (Bed) (1989) 97:363-373

9 Springer-Verlag1989

Polytene chromosomes from ovarian pseudonurse cells of the Drosophila melanogaster otu mutant I. Photographic map of chromosome 3 Tapio I. Heino Department of Genetics, University of Helsinki, Arkadiankatu 7, SF-00100 Helsinki, Finland

Abstract. Certain mutant alleles of the otu locus in Drosophila melanogaster produce abnormal nurse cells in the ovaries. These cells are called pseudonurse cells (PNC), since they generate polytene chromosomes instead of endopolyploid ones and do not normally have an oocyte to nurse. The banding pattern of polytene chromosome 3 from the salivary glands (SG) and from PNCs of homozygous otu ~ females was compared and a detailed photomap of PNC chromosomes with different degrees of polyteny is presented. The banding pattern was found to be strikingly similiar in the two tissues. The puffing pattern of the PNC chromosomes was also studied and the function of the PNC chromosomes is discussed. No constrictions or breaks were found in the PNC chromosomes which seems to indicate that these sites, which are known to be underreplicated in the SG chromosomes, are equally replicated along with the rest of the chromosomes in the PNC nuclei.

Introduction The characteristic banding pattern seen in polytene chromosomes is of great importance in understanding the structure of eukaryotic interphase chromosomes. The banding pattern is with few exceptions always clearest in the polytene chromosomes found in the larval salivary glands (SG) of most species of Diptera, although polytene chromosomes exist in many other tissues too (see Ashburner 1970). Drosophila melanogaster is an ideal organism for comparing the banding pattern of polytene chromosomes, as long as only the SG are considered. Firstly, detailed SG chromosome maps made by C.B. and P.N. Bridges (see Lindsley and Grell 1968) are available. Secondly, a large and rapidly increasing number of exactly localized genes is known in this species. Therefore, possible differences in the banding pattern between the tissues studied can be examined at the chromomere or even at the gene level. In order to understand the functional significance of the polytene chromosome banding pattern, such comparisons should preferably be made between different developmental stages, e.g. larval and adult tissues and also between tissues whose metabolic functions differ radically. Unfortunately D. melanogaster does not normally have analyzable polytene chromosomes in adult tissues and, therefore, the larval SG chromosome banding pattern has only been compared with other larval tissues where polytene chromosomes exist, such as fat body (Richards 1980a), hindgut (Zhimulev et al.

1982; Hochstrasser 1987), midgut, and the prothoracic gland of normal (Hochstrasser 1987) and DTS-3 mutant larvae (Holden and Ashburner 1978). In all the above-mentioned cases the banding pattern has shown almost perfect homology between the compared tissues. Polytene chromosomes form naturally in the nurse cells of the adult ovary only in some species of Diptera (Stalker 1954). Redfern (1981) has studied polytene chromosomes from the nurse cells of the mosquito Anopheles stephensi and found that their banding pattern did not differ significantly from that of larval SG chromosomes. Quite the opposite result was obtained by Ribbert (1979) who, with combined inbreeding and selection, experimentally induced polytene chromosomes in normally endopolyploid nurse cells of Calliphora erythrocephala. Comparison of these chromosomes with polytene chromosomes from pupal bristle-forming cells did not reveal any homology between the two tissues. In D. melanogaster the nurse cells are normally endopolyploid so that the homologous chromatids remain separated, although they may be loosely conjoined, especially in ,the early stages of endomitosis (Painter and Reindorp 1939). Recently, H a m m o n d and Laird (1985) have show by in situ hybridization that some regions in the homologous chromatids, e.g. regions covering the 5S R N A and histone genes remain close to each other, even in the later stages of nurse cell development; however, on the whole the wild-type nurse cell nuclei of D. melanogaster are of little cytological value. Recently, some interesting female sterile mutations have been isolated in D. melanogaster which give rise to polytenization of chromosomes in the nurse cell nuclei instead of endomitosis (cf. King and Brining 1985). These cells are called pseudonurse cells (PNC), because they do not usually have an oocyte to nurse and because their nuclear morphology differs from that of the wild-type nurse cells (King et al. 1978). One of these mutations is the sex-linked otu ~ (for ovarian tumor, formerly calledfs(1)231). At present 17 different mutant alleles are known from the otu locus and the cytological anomalies they generate have been described by King and Riley (1982), Bishop and King (1984), and King et al. (1986). Up to now, the PNC chromosomes have proven to be difficult to analyze (King et al. 1981), because they are usually shorter than SG chromosomes and their banding is rarely clear. Recently Sinha et al. (1987) were able to compare the PNC chromosomes with SG chromosomes and showed that the banding pattern is in general similar, if not identical, in the two tissues. Here, we present a detailed

364 photomap of polytene chromosomes 3L and 3R from the PNCs of the otu ~ mutant. In addition to minor banding differences, some other structural differences were also found between the chromosomes from the two tissues. Results on banding similarities in the 3R chromosome in the two tissues have been presented by Heino (1985) as a preliminary report. Materials and methods

The stock. The otu 1 mutation is transmitted from generation to generation by matings between otu ~ males and otu~/ +females. The otu + allele is carried by the balancing chromosorhe F M 3 marked by the dominant mutation Bar. F M 3 also contains a series of paracentric inversions and two recessive lethal mutations (see Lindsley and Grell 1968). The X chromosome carrying the otu ~ mutation also carries mutations et" and I)24. In the otu ~ stock used, the second chromosome carries the dominant marker Cy combined with long paracentric inversions in both chromosome arms. Cy is lethal when homozygous. The other second chromosome in the stock carries a female sterile m u t a f i o n f e s with marker mutations en and bw and also a mutation Iv which lowers the preadult viability of homozygotes. The genotype of females and larvae studied was thus otu Ict" V24/otu I c t n V24; I n ( 2 L ) C y In(2R)Cy/fes cn lv bw. The flies were reared at 18 ~ C. Female larvae were separated from male larvae by the smaller size of their gonads. After squashing the SG of female larvae the otul/otu ~ homozygotes could be distinguished from the otu~/FM3 heterozygotes by the absence of inversion loops in the homozygotes. The heterozygosity of the second chromosome was determined by the presence of inversion loops in the Cy/fes cn 11)bw chromosome.

euchromatic arms of SG chromosomes are tightly attached to each other in this structure. In PNC chromosomes, no such chromocenter is found and the euchromatic chromosome arms are separated, but sometimes very thin and flexible heterochromatic fibers can be seen connecting the chromosome arms. Accordingly, five long chromosome arms and the tiny fourth chromosome can be seen in PNC nuclei. Different PNC nuclei contain chromosomes in various stages of polytenization (see below) so that, based on the morphology of the chromosomes, they have been classified as condensed, banded, or diffuse (King et al. 1981; Sinha et al. 1987). This study concentrates only on the banded chromosome class. In the stock we have used, the arms of the second chromosome can be readily distinquished from others in all of these morphological classes by the long inversion loops which result from the pairing of the cytologically normal second chromosome and the Curly chromosome which contains one long paracentric inversion in both chromosome arms. The cytologically normal X, 3L and 3R chromosomes can be identified in all these classes starting from the well-banded PNC chromosomes and proceeding gradually to shorter and more condensed, or diffuse, chromosomes. The comparison of the 3L and 3R chromosomes from the SG and well-banded PNC chromosomes is presented in Figures 1-5. Four PNC chromosomes were included in each of the figures and they were chosen to present different degrees of polyteny (see below). This was done to avoid preparative artifacts, when PNC chromosomes were compared with SG chromosomes. There is, however, only one representative of the SG chromosomes in each of Figures 1-5. The genotype of the larvae from which the SG squash preparations were made was the same as that of the flies studied (see Materials and methods). Estimates o f the polyteny levels o f P N C chromosomes

Cytological techniques. The PNC chromosomes are most clearly banded in 4 to 7 day old flies, but younger females (0-4 days) were also studied. The age of the flies was known with an accuracy of 24 h before preparation. The ovaries were dissected from etherized females in Ringer solution (Ephrussi and Beadle 1936). Ovaries were fixed with acetic: methanol (1:3) for 10rain to several hours and stained in lactic-acetic oreein (50 ml glacial acetic acid, 30 ml lactic acid, 20 ml distilled water and 2 g natural oreein) for 10-60 rain. Ovaries were then spread on a slide in 50% acetic acid with fine needles and squashed in the same solution. After squashing the coverslip was removed by pressing the slide on the surface of well-smoothed dry ice. Preparations were dehydrated in absolute ethanol (2 x 2 min) and mounted, after two xylene washes, in Entellan. The SG chromosomes were prepared by the same method as the PNC chromosomes with the exception of a shorter staining time (3-4 min). Preparations were observed with an Olympus BH-2 microscope and phase contrast illumination and photographed on Agfapan 25 film. Results

General characteristics o f P N C chromosomes

One of the typical features of polytene chromosomes of SG nuclei in Drosophila larvae is the chromocenter. The

The polyteny level of SG chromosomes is generally 1024C, but some nuclei reach a level of 2048C (for review see Richards 1985). Laird et al. (1980) have shown that, when the polyteny level doubles, the width of the chromosome increases by the square root of 2, i.e. with a coefficient of approximately 1.41. Based on this assumption the polyteny level of the PNC 3R chromosomes was estimated from Figures 3-5 by measuring from fixed distances the width of the SG chromosome and each of the PNC chromosomes (data not shown). In these calculations the length of the chromosomes was taken into account. The values fitted well to equal doublings of polyteny levels in the PNC chromosomes. The chromosomes in Figures 3b, 4b, c, and 5b were in the same polyteny class as the SG chromosome, i.e. in the estimated 1024C class. The chromosomes in Figures 3c, d, 4c, and 5c were in the 2048C class, that in Figure 4d was in the 4096C class and the chromosomes in Figures 3e, 4e and 5e fell into the 8192C polyteny class. Differences in the banding patterns o f the P N C and SG chromosomes

Comparison of the banding patterns of the PNC and SG chromosomes shows remarkable homology. Slight differences, however, were found and they are listed below. When differences were found between the SG and PNC chromosomes the banding pattern of the SG chromosome was in addition carefully compared with a set of photographs from

365

Fig. 1 a-e. Comparison of banding patterns in salivary gland (SG) and pseudonurse cell (PNC) chromosomes. Divisions 61-69 of the 3L chromosome in a SG, I~e PNC with different degrees of polyteny. Arrowheads mark differences in the banding or puffing patterns of the two tissues. Bar represents 10 gm

other genotypes and also with the photomaps of Lefevre (1976) and the new SG chromosome photomaps made by T.I. Heino published in Sorsa (1988). This was done to eliminate the possiblity of banding pecularities in the third SG chromosome of the otu 1 stock. The band designations

in the following comparison follow Bridges' (1941 a, b) revised maps. 6 2 C . This subdivision is always clearly shorter in the PNC chromosomes. The difference involves bands C3 and C4

366

Fig. 2a-e. Comparison of banding patterns in SG and PNC chromosomes. Divisions 70-80 of the 3L chromosome in a SG, b e PNC with different degrees of polyteny. Arrowheads mark differences in the banding or puffing patterns of the two tissues. Bar represents 10 gm

bringing bands C1 and C2 very close to the doublet D I - 2 in the P N C chromosomes (Fig. 1).

69ABC. The bands between A1 and C1 2 fuse extensively in the highly polytenized P N C chromosomes (Fig. I e).

64F. The SG chromosome band complex F1-3 is much

73A. The doublets A 1 - 2 and A3~4 are clearly separated in the P N C chromosomes (Fig. 2). In the P N C chromosomes there is possibly an additional band between the doublets not seen in the SG chromosomes (Fig. 2 c).

more prominent than the corresponding region in the P N C chromosomes. This difference may be due to puffing of the P N C chromosomes at least in the example in Figure 1 d and presumably also that in 1 e, but not in 1 c. 65E. In the PNC, the band complex E7-9 lies, at least in

some instances, closer to the complex E I ~ SG chromosomes (Fig. 1 b, c, e, but not 1 d).

than in the

68C. The prominent puff seen in the SG chromosomes can-

not be seen in the P N C chromosomes (Fig. 1).

75A. The doublet A8-9 can be seen more clearly in the P N C chromosomes and is occasionally also more prominent (Fig. 2 b, c). 75B. The puff in the SG chromosomes cannot be seen in the P N C chromosomes and the doublet B1-2 is very prominent in the P N C chromosomes (Fig. 2).

367

Fig. 3a-e. Comparison of banding patterns in SG and PNC chromosomes. Divisions 81-87 of the 3R chromosome in a SG, b-e PNC with different degrees of polyteny. Arrowheads mark differences in the banding or puffing patterns of the two tissues. Bar represents 10 gm

75C. The typical constriction in the SG chromosomes cannot be seen in the P N C chromosomes (Fig. 2).

strongly in the P N C chromosomes but not in the SG chromosomes (Fig. 3).

79-81E. The P N C chromosome arms are not connected to a c o m m o n chromocenter as in the SG nuclei (Figs. 2 and 3). The P N C chromosome is often clearly wider (Figs. 2c, d, e) in divisions 79 and 80 than the rest o f the chromosome forming a bulbous end to the proximal part of the 3L chromosome. Sometimes the presumed tiny fourth chromosome is connected to the proximal end of the 3L chromosome with heterochromatic fibers (Fig. 2b).

85F. The prominent puff formed by the bands in position

F1-5 cannol: be seen in the P N C chromosomes (Fig. 3). 86D. The constriction in the SG chromosomes cannot be

seen in the P N C chromosomes (Fig. 3). 87F. A more prominent band is found between doublets

8 7 F 1 ~ 1 3 and 88A1-2 in the P N C chromosomes than in the SG chromosomes (particularly Fig. 3 b and e).

84A. The bands of tight doublet A4-5 in SG are clearly

separated in the P N C chromosomes (Fig. 3 b-d).

88C. The band complex C1-4 is darker in the P N C chromo-

85D. The doublet D I - 2 and the bands in D10-12 puff

somes. In the SG chromosomes these bands are probably slightly puffed (Fig. 4).

368

Fig. 4a--e. Comparison of banding patterns in SG and PNC chromosomes. Divisions 88-93 of the 3R chromosome in a SG, b-e PNC with different degrees of polyteny. Arrowheads mark differences in the banding or puffing patterns of the two tissues. Bar represents 10 gm

881). The bands D5 and D6 are clearly darker in the PNC chromosomes. In the SG chromosomes these bands are probably slightly puffed (Fig. 4).

IOOA. A prominent puff is found in the PNC chromosomes, but not in the SG chromosomes. Bands A1-3 are involved in its formation (Fig. 5).

89E. The typical constriction seen in the SG chromosomes is not seen in the PNC chromosomes (Figs. 4 and 6).

The results presented above show that the banding pattern of the polytene chromosomes in PNC nuclei is remarkably similar to that of the SG chromosomes. This result is consistent with most of the studies in which polytene chromosomes from different tissues have been compared and only slight differences have been found in Drosophila (Holden and Ashburner 1978; Richards 1980a; Hochstrasser 1987) and other Diptera (earlier works cited in Beermann 1962; also Bedo 1976; Redfern 1981; Gupta and Singh 1983). As in these studies the banding pattern of the PNC chromosomes differs slightly when compared with that of the SG chromosome. In summary the slight differences between the PNC and SG chromosomes can be roughly divided into four groups: 1. Differences in the puffing patterns (64F, 68C, 75B, 85D, 85F, 91B, 93C, 94A and 100A) 2. Differences in the relative staining intensity of the bands (75A, 75B, 87F, 88C and 88D)

90E. The bands in the D1-2-E1-2 region form a tight band complex in the PNC chromosomes (Fig. 4 and also Fig. 6). 91B. The bands B5 and B6 form a puffin the PNC chromo-

somes but not in the SG chromosomes (Fig. 4c-d). 93C. A puff is seen in the PNC chromosomes but not in

the SG chromosomes. The doublet C4-5 is involved in its formation, but possibly also the more proximal bands C I - 3 (Fig. 4). 94A. The doublets A1-2 and A3~4 appear to be slightly puffed in the PNC chromosomes but not in the SG chromosome and no constriction occurs in this region in the PNC chromosomes (Fig. 5).

369

Fig. 5a-e. Comparison of banding patterns in SG and PNC chromosomes. Divisions 94 100 of the 3R chromosome in a SG, b-e PNC with different degrees of polyteny. Arrowheadsmark differences in the banding or puffing patterns of the two tissues. Bar represents 10 lain 3. Differences in the relative spacing between the bands (62C, 65E, 69ABC, 73A, 84A and 90DE) 4. The absence of constrictions in the PNC chromosomes (75C, 86D, 89E and 94A). Sinha et al. (1987) have coarsely mapped the 3R chromosome from PNC nuclei. Our mapping results differ from theirs particularly in the proximal parts of the chromosome. In the following we give the corrected positions based on our maps with the positions given by Sinha et al. (1987, Fig. 3) in parentheses. They are 84A (not 83D), 84D (84A), 84F (84D), 85D (85EF), 85F (86AB), 86D (86DE), 86E (87AB) and also in the distal part 92F (93A). They also report the presence of the 89E constriction in the PNC chromosomes which we do not see in clearly banded PNC chromosomes (Figs. 4 and 6). In the chromosome presented in their Figure 3 the 89E area is clearly narrower than the rest of the chromosome but this probably results from the tighter association of parallel chromatids in this position while the rest of the chromosome is slightly diffuse. At least in more prominently diffuse PNC chromosomes the

bands in the 89E position always form a tight region of synapsis while the parallel chromatids in the rest of the chromosome tend to separate from each other thus giving the chromosome a diffuse outlook (T. Heino, unpublished results). Sinha et al. (1987) have also reported the 10F-11A constriction as present in the PNC chromosomes and as a landmark of the X chromosome. In this case also our results differ from theirs since we have not seen this constriction in our preparations (T. Heino, unpublished resuits).

Discussion The banding pattern of the P N C chromosomes The banding pattern of the PNC chromosomes shows a remarkable similarity to that of the larval SG chromosomes. In fact, when one examines the published photomaps of the larval fat body polytene chromosomes (Richards 1980a), one can see that the fat body chromosomes

370

Fig. 6a-e. Comparison of banding patterns in PNC and SG chromosomes at a greater magnification. Divisions 89 and 90 in the 3R chromosome in a the Bridges (1941b) revised map, b in the PNC, e the SG from stock bw; st. Note the absence of a weak point in the PNC chromosome in 89E1-4 (arrows), the fusion of bands in 90DE (arrowheads) and the enormous size difference of the chromosomes. Bar represents 10 gm

differ from the SG chromosomes more than do the PNC chromosomes. This is rather surprising since the PNC chromosomes are from a developmental stage very different from that of the SG chromosomes. In addition, their function should differ radically from that of the SG chromosomes. If the PNC chromosome activities reflect the wildtype nurse cell chromosome activities (see below), the results presented here and by Sinha et al. (1987) would support more strongly the genetic rather than the epigenetic hypothesis (for review see Ashburner 1980; Richards 1985) for the basis of the polytene chromosome banding pattern. We should, however, remember the contradictory results obtained, e.g. by Ribbert (t979) with C. erythrocephaIa, and also recently by Bedo (1987) with Ceratitis capitata, where no homologies were found between the tissues studied. The conclusion drawn by Ribbert (1979) that the difference in the induced polytene chromosome banding pattern in Calliphora nurse cells is a result of the specialized function of germ line derived nurse cells has not gained support from Redfern (1981) with similiar studies in A. stephensi, or from our results. The PNC chromosomes are generated by a mutation in a gene and their presence in the ovaries is therefore abnormal as in the case of Calliphora (Ribbert 1979) where polyteny was experimentally induced in the nurse cell nuclei. The results with the PNC chromosomes also suggest that the unusual banding pattern in the nurse

cells of Calliphora does not result from their experimental induction. The remarkable similarity in the banding patterns of the PNC and SG chromosomes presented here and in Sinha et al. (1987) is contradictory to the results obtained by King et al. (1981) where banding differences were observed in the 2R chromosomes in these two tissues. The chromosome presented by King et al. (1981) was much shorter than those studied here and its alignment to SG chromosomes is indeed difficult without the help of longer PNC chromosomes. Closer examination of the " 2 R " chromosome in Figure 2 of King et al. (1981) has demonstrated that the chromosome is 3R instead of 2R so that what was taken as the telomere end is in fact the centromere end (not shown). The banding pattern of the 3R chromosome presented in Figure 2 of King et al. (1981) is essentially the same as the longer PNC 3R chromosomes studied here although it is difficult to interpret in certain locations because of the extensive fusion of the bands.

The puffing of the P N C chromosomes Most of the differences in the banding pattern between the PNC and SG chromosomes result from dissimilarities in the puffing pattern of the chromosomes. Tissue specific puffing patterns have been studied in detail in Dipteran

371 polytene chromosomes (for review see Richards 1985) and are well known to result from different gene activities in different tissues. Sinha et al. (1987) have reported the presence of 68C and 75B puffs in the banded PNC chromosomes as landmarks of the 3L chromosome which are also present in the SG chromosomes. We do not see any prominent signs of puffing in the 75B position in our preparations. The 68C region is widened in the PNC chromosomes but not puffed. The puffing of the 68C region is not expected in adult tissues since the puff is known to arise from the transcription of the Sgs genes whose expression is specific for salivary glands of the larva (Korge 1977). In this context it should be stressed that the SG landmark 2B puff which Sinha et al. (1987) claim to be present in the PNC chromosomes is not so located according to our observations; the puff in the PNC chromosomes is located at 3DE (T. Heino, unpublished results). The puffing of doublet 85D1 2 in PNC chromosomes is interesting because the same doublet puffs out in the SG chromosomes at the beginning of the prepupal stage (Ashburner 1972). This puff can also be induced with 20-OH-ecdysone (Biyasheva et al. 1985) like most of the puffs present during this developmental period (Richards 1980b). Although it is known that the adult female of D. melanogaster synthesizes ecdysone (Hodgetts et al. 1977), results on its production in the ovaries have been contradictory (compare Garen et al. 1977; Handler 1982). However it is very unlikely that the 85D1-2 puff is induced in the PNC chromosomes by ecdysone because none of the other ecdysone inducible puffs seen in the SG chromosomes at the prepupal and pupal stages are present in the PNC chromosomes. It is possible that other genes are transcribed from this position in the PNC chromosomes than those transcribed in the SG chromosomes during the prepupal period. M6ritz et al. (1984) have shown that puff formation at the same position in polytene chromosomes in two different tissues does not necessarily mean transcription of the same gene. In addition the puff in the PNC chromosomes seems to extend slightly more distally than the prepupal SG chromosome puff. It is therefore possible that genes located more distally are transcribed in the PNC chromosomes and the proximal chromatin containing the ecdysone inducible genes is passively involved in puff formation.

The function of the PNC chromosomes One of the most interesting questions concerning the PNC chromosomes is whether their gene activity reflects the activity of the wild-type nurse cells although they do not normally have an oocyte to nurse. This question is important since if the PNC chromosomes function like the wild-type nurse cell chromosomes, this would have important applications for studies on oogenesis and the effect of maternal genes on early embryogenesis in Drosophila. It has been shown that in the mature D. melanogaster oocyte there are about 6000 different maternal R N A sequences which have accumulated there during oogenesis (Hough-Evans et al. 1980). In addition it has been estimated that oogenesis requires the expression of approximately 70% of the genes that can be mutated to lethality (GarciaBellido and Robbins 1983). Most of these R N A species originate from the nurse cells. The majority of these are obviously produced in relatively small amounts, e.g.

m R N A s encoding proteins needed in the developmental pathways of the embryo. It is therefore expected that expression of such genes would not be seen as visible puffs in the PNC chromosomes. In contrast, one might expect to detect puff formation in chromosome areas known to harbor genes which are expected to be expressed strongly in nurse cells. Tubulin is a component of microtubules, needed in large amounts as components of the mitotic spindle during the cleavage divisions of the embryo. Tubulin-coding R N A components are poured into the oocyte from the nurse cells (Loyd et al. 1981; Gasch et al. 1988) as maternal products. In the mature egg cell the proportion of tubulins is 10% of non-yolk proteins (Loyd et al. 1981). The ~-tubulin genes located at chromosomal positions 67C4-6 and 84B3-6 (Kalfayan and Wensink 1982) are expressed as maternal RNAs during oogenesis. Also the shorter transcript of the fl2-tubulin gene at position 85D4-7 has been found to be abundantly present in 0-3 h embryos but not in later embryonic stages (Natzle and McCarthy 1984) suggesting a maternal origin for this R N A species. None of these chromosome sites shows any prominent sign of puff formation in the PNC chromosomes. It is however possible that these obviously large R N A amounts are produced slowly over a long period during oogenesis and therefore the puff is small or the parallel chromatids of the chromosome are not used simultaneously for transcription. It is known that mRNAs, coding for some of the heat shock proteins (Hsp83, Hsp28 and Hsp26), are produced in the ovaries of D. melanogaster without heat shock treatment (Zimmermann et al. 1983). These transcripts are produced by the nurse cells (Ambrosio and Schedl 1984), and at least the hsp26 (Cohen and Meselson 1985) and hsp28 genes (Hoffmann et al. 1987) have their own ovary specific promoters in addition to the promoters responding to heat shock. These hsp genes are transcribed with relatively high level of expression in the ovaries without heat shock (Zimmermann et al. 1983). The genes for the heat shock proteins in question lie in the 63BC and 67BC regions of the 3L chromosome. After heat shock giant puffs appear in these regions in the SG chromosomes of larvae. If it is assumed that the function of the PNC chromosomes is equivalent to the wild-type nurse cell chromosomes, we would expect some signs of puffing within these chromosome regions. However, we did not see any prominent puffing within these chromosome areas. It is probable that the appearance of giant puffs in the SG chromosomes after heat shock facilitates the rapid and vigorous transcription of these genes. If these m R N A s are produced by the PNC chromosomes they are obviously produced during a much longer period of time and the relaxation of chromomeres for transcription is stabilized. It is also possible that these heat shock genes are expressed during earlier stages of development, i.e. in less polytenized chromosomes than those studied here. Recently Storto and King (1987) have shown that certain heteroallelic combinations of otu alleles generate polytene chromosomes in their PNC nuclei and these flies are still fertile thus producing functional eggs. These results suggest that the PNC chromosomes are capable of generating appropriate transcripts which allow the egg to develop normally. It is however possible that there is a feedback mechanism from the egg cell to the pseudonurse or nurse cells at the time when the determination of the egg cell

372 is properly established. According to this hypothesis the egg cell would give the nurse cells a major signal to function normally. The developing oocyte has long been thought to be transcriptionally inactive until the cellular blastoderm stage (Zalokar 1976; Mahowald and Kambysellis 1980). Recently Haenlin et al. (1987) have shown at least one gene (KIO) affecting the dorsal-ventral pattern formation of the egg is transcribed by the egg cell nucleus as early as stage 6 of oogenesis (King 1970). It is therefore clear that transcription in very early oocyte development stages is not completely repressed and the hypothesis of production of such a feedback signal from the egg cell to the nurse cells is supported. What genes then lie in the locations of the prominent puffs seen in the P N C chromosomes? A search of the Drosophila literature did not reveal any genes in these positions whose massive expression is expected in the nurse cells. This finding might suggest that the function of the P N C chromosomes differs from that o f the wild-type nurse cell chromosomes but more detailed genome maps of Drosophila are necessary to answer this question.

The absence of weak points and ectopic pairing in the PNC chromosomes The euchromatic parts of D. melanogaster SG chromosomes are most probably equally replicated (Spierer et al. 1983; Spierer and Spierer 1984) the only exception being the constrictions seen regularly in certain polytene chromosome regions. The constriction covering bands 89E1-4 of the 3R chromosome has been shown to be a region of local underreplication (Spierer and Spierer 1984) in the SG chromosomes and the same probably holds for other constrictions. The chromosomes often tend to pair ectopically at constriction sites and SG chromosomes frequently break at these so-called weak spots. Such constrictions are observed in polytene chromosomes from other larval tissues at the same positions as in the SG chromosomes (Hochstrasser 1987). In the banded P N C chromosomes the regular SG constriction sites are not found, i.e. the widths of the bands in these regions do not differ from those of neighbouring bands. We therefore assume that they are equally replicated together with other euchromatic chromosome regions in the P N C chromosomes. Secondly we have never seen ectopic pairing between euchromatic chromosome regions in the P N C chromosomes. Moreover the P N C chromosomes have no tendency to break. Both features have been proposed to be signs of local underreplication (Zhimulev et al. 1982). It is therefore obvious that the euchromatic parts of the banded P N C chromosomes replicate equally throughout their total length. Ribbert (1979) also did not find ectopic pairing, breaks or constrictions in the nurse cell polytene chromosomes of Calliphora allthough these characteristics were present in the somatic trichogen cells. Therefore our results support his assumption that these features belong to the somatic polytene chromosomes but not to polytene chromosomes of germ line origin. It is also clear that local constrictions in polytene chromosomes and ectopic pairing are interdependent as Ashburner (1980) has already suggested. He has hypothesized that ectopic fibers are formed when chromosome regions containing similiar (heterochromatic) D N A sequences in the course of underreplication stick to each other when the replication forks are formed.

It is hoped that these maps of the 3L and 3R P N C chromosomes together with maps from other chromosomes (T. Heino, in preparation) will serve as reference maps for P N C chromosomes and will also encourage the use of these chromosomes for functional studies on the course of oogenesis in Drosophila. It should also be pointed out that the enormous size of certain P N C chromosomes compared with SG chromosomes (Fig. 6) facilitates the microdissection of desired chromosome bands (Schalenghe et al. 1981) for molecular cloning purposes.

Acknowledgement. While writing this manuscript the author was the recipient of a fellowship from the Eemil Aaltonen Foundation. References

Ambrosio L, Schedl P (1984) Gene expression during Drosophila melanogaster oogenesis: analysis by in situ hybridization to tissue sections. Dev Biol 105:80-92 Ashburner M (1970) Function and structure of polytene chromosomes during insect development. Adv Insect Physiol 7:1-95 Ashburner M (1972) Puffing patterns in Drosophila melanogaster and related species. In: Beermann W (ed) Results and problems in cell differentiation, vol 4. Springer, Berlin Heidelberg New York, pp 101-151 Ashburner M (1980) Some aspects of the structure and function of the polytene chromosomes of the Diptera. In: Blackmann RL, Hewitt GM, Ashburner M (eds) Insect cytogenetics. Symp R Entomol Soc London, vol 10. Blackwell Scientific, Oxford, pp 65-84 9 Bedo DG (1976) Polytene chromosomes in pupal and adult blackflies (Diptera: Simulidae). Chromosoma 57:387-396 Bedo DG (1987) Polytene chromosome mapping in Ceratitis capirata (Diptera: Tephritidae). Genome 29 : 598-611 Beermann W (1962) Riesenchromosomen. Protoplasmatologia VI/ D, Springer, Wien Bishop DL, King RC (1984) An ultrastructural study of ovarian development in the otu 7 mutant of Drosophila melanogaster. J Cell Sci 67:87-119 Biyasheva ZM, Belyaeva, ES, Zhimnlev IF (1985) Cytogenetic analysis of the X chromosome region 2B1-2-2B9-10 of Drosophila melanogaster. V. Puffing disturbances carried by lethal mutants of the gene dor are carried due to ecdysterone deficiency. Chromosoma 92 : 351-356 Bridges PN (1941 a) A revision of the left limb of the third chromosome of Drosophila melanogaster. J Hered 32: 64-65 Bridges PN (1941 b) A revision of the salivary gland 3R-chromosome map of Drosphila melanogaster. J Hered 32: 299 300 Cohen MS, Meselson M (1985) Separate regulatory elements for the heat-inducible and ovarian expression of the Drosophila hsp 26 gene. Cell 43:737-746 Ephrussi B, Beadle GW (1936) A technique of transplantation for Drosophila. Am Nat 70: 218-225 Garcia-Bellido A, Robbins LG (1983) Viability of female germ line cells homozygous for zygotic lethals in Drosophila melanogaster. Genetics 103:23~247 Garen A, Kauvar L, Lepesant JA (1977) Roles of ecdysone in Drosophila development. Proc Natl Acad Sci USA 74:5099-5103 Gasch A, Hinz U, Leiss D, Renkawitz-Pohl R (1988) The expression of ,81 and f13 tubulin genes of Drosophila melanogaster is spatially regulated during embryogenesis. Mol Gen Genet 211:8-16 Gupta JP, Singh OP (1983) Puffing patterns in polytene chromosomes of different tissues of Melanagromyza obtusa during development. J Hered 74:365-370 Haenlin M, Roos C, Cassab A, Mohier E (1987) Oocyte-specific transcription of fs(1)Kl0: a Drosophila gene affecting dorsalventral developmental polarity. EMBO J 6:801 807

373 Hammond MP, Laird CD (1985) Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanomelanogaster. Chromosoma 91:267-278 Handler AM (1982) Ecdysteroid titers during pupal and adult development in Drosophila melanogaster. Dev Biol 93 : 73-82 Heino TI (1985) The banding pattern of the 3R chromosome from the pseudonurse cells of D. melanogaster otu I mutant. Abstracts of the 9th European Drosophila Research Conference, Balatonszeplak, Hungary Hochstrasser M (1987) Chromosome structure in four wild-type polytene tissues of Drosophila melanogaster. The 87A and 87C heat shock loci are induced unequally in the midgut in a manner dependent on growth temperature. Chromosoma 95:197-208 Hodgetts RB, Saye B, O'Connor JD (1977) Ecdysterone titers during postembryonic development in Drosophila melanogaster. Dev Biol 60:310-317 Hoffmann EP, Gerring SL, Corces VG (1987) The ovarian, ecdysterone, and heat-shock-responsive promoters of the Drosophila melanogaster hsp 27 gene react very differently to perturbations of DNA sequence. Mol Cell Biol 7:937-981 Holden JJ, Ashburner M (1978) Patterns of puffing activity in the salivary gland chromosomes of Drosophila. IX. The salivary and prothoracic gland chromosomes of a dominant temperature sensitive lethal of D. melanogaster. Chromosoma 68 : 205-227 Hough-Evans BR, Jacobs-Lorena M, Cummings MR, Britten RJ, Davidson EH (1980) Complexity of RNA in eggs of Drosophila melanogaster and Musca domestica. Genetics 85:81-94 Kalfayan L, Wensink PC (1982) Developmental regulation of Drosophila ~-tubulin genes. Cell 29:91-98 King RC (1970) Ovarian development in Drosophila melanogaster. Academic Press, New York King RC, Brining J (1985) The origin and functioning of insect oocytes and nurse cells. In: Kerkut GA, Gilbert LI (eds) Comprehensive insect physiology, biochemistry and pharmacology, vol 1. Pergamon Press, Oxford, pp 37-82 King RC, Riley SF (1982) Ovarian pathologies generated by various alleles of the otu locus in Drosophila melanogaster. Dev Genet 3 : 69-89 King RC, Bahns M, Horowitz R, Larramendi P (1978) A mutation that affects female and male germ cells differentially in Drosophila melanogaster Meigen (Diptera: Drosophilidae). Int J Insect Morphol Embryol 7:359 375 King RC, Riley SF, Cassidy JD, White PE, Paik YK (1981) Giant polytene chromosomes from the ovaries of a Drosophila mutant. Science 212 :441443 King RC, Mohler D, Riley SF, Storto PD, Nicolazzo PS (1986) Complementation between alleles at the ovarian tumor locus of Drosophila melanogaster. Dev Genet 7 : 1-20 Korge G (1977) Larval saliva in Drosophila melanogaster: Production, composition and relationship to chromosome puffs. Dev Biol 58:339-355 Laird CD, Ashburner M, Wilkinson L (1980) Relationship between relative dry mass and average band width in the regions of polytene chromosomes of Drosophila. Chromosoma 76 : 175-189 Lefevre G Jr (1976) A photographic representation and interpretation of the polytene chromosomes of Drosophila melanogaster salivary glands. In: Ashburner M, Novitsky E (eds) Genetics and biology of Drosophila, vol 1a. Academic Press, London, pp 1-66 Lindsley DL, Grell EH (1968) Genetic variations of Drosophila melanogaster. Carnegie Inst Washington Publ 627

Loyd JE, Raft EC, Raft RA (1981) Site and timing of synthesis of tubulin and other proteins during oogenesis in Drosophila melanogaster. Dev Biol 86:272 284 Mahowald AP, Kambysellis MP (1980) Oogenesis. In: Ashburner M, Wright TRF (eds) Genetics and biology of Drosophila, vol 2d. Academic Press, London, pp 141-224 M6ritz T, Edstr6m JE, Pongs O (1984) Cloning of a gene localized and expressed at the ecdysteroid regulated puff 74EF in salivary glands of Drosophila larvae. EMBO J 3 : 289-295 Natzle JE, McCarthy BJ (1984) Regulation of Drosophila ~- and fl-tubulin genes during development. Dev Biol 104: 187-198 Painter TS, Reindorp EC (1939) Endomitosis in the nurse cells of the ovary of Drosophila melanogaster. Chromosoma 1 : 276-283 Redfer n CPF (1981) Homologous banding patterns in the polytene chromosomes from the larval salivary glands and ovarian nurse cells of Anopheles stephensi Liston (Culicidae). Chromosoma 83 : 221240 Ribbert D (1979) Chromomeres and puffing in experimentally induced polytene chromosomes of Calliphora erythrocephala. Chromosoma 74 :269-298 Richards G (1980a) The polytene chromosomes in the fat body nuclei of Drosophila melanogaster. Chromosoma 79 : 241-250 Richards G (1980b) Ecdysteroids and puffing in Drosophila melanogaster. In: Hoffman JA (ed) Progress in ecdysone research. Elsevier, New York, pp 363 378 Richards G (1985) Polytene chromosomes. In: Kerkut GA, Gilbert LI (eds) Comprehensive insect physiology, biochemistry and pharmacology, vol 2. Pergamon Press, Oxford, pp 255-300 Schalenghe F, Turco E, Edstr6m JE, Pirrotta V, Melli M (1981) Microdissection and cloning of DNA from a specific region of Drosophila melanogaster polytene chromosomes. Chromosoma 82: 205-216 Sinha P, Mishra A, Lakhotia SC (1987) Chromosomal organization of Drosophila tumours. I. Polytene chromosome organition and DNA synthesis in ovarian pseudonurse cells in otu mutants of D. melanogaster. Chromosoma 95:108-116 Sorsa V (1988) Chromosome maps of Drosophila, II. CRC Press, Boca Raton Spierer A, Spierer P (1984) Similiar level of polyteny in bands and interbands of Drosophila giant chromosomes. Nature 307:176-178 Spierer P, Spierer A, Bender W, Hogness DS (1983) Molecular mapping of genetic and chromomeric units in Drosophila melanogaster. J Mol Biol 168:35-50 Stalker HD (1954) Banded polytene chromosomes in the ovarian nurse cells of adult Diptera. J Hered 45 : 259-264 Storto PD, King RC (1987) Fertile heteroallelic combinations of mutant alleles of the otu locus of Drosophila melanogaster. Roux Arch Dev Biol 196:210-221 Zalokar M (1976) Autoradiographic study of protein and RNA formation during early development of Drosophila eggs. Dev Biol 49:425-437 Zhimulev IF, Semeshin VF, Kulikchov VA, Belyaeva ES (1982) Intercalary heterochromatin in Drosophila I. Localization and general characteristics. Chromosoma 87:197 228 Zimmermann JL, Petri W, Meselson M (1983) Accumulation of a specific subset of D. melanogaster heat shock mRNAs in normal development without heat shock. Cell 32:1161-1170 Received June 28, 1988 / in revised form August 21, 1988 Accepted by W. Beermann