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Interband fibrils of polytene chromosomes
Cell Differentiation, 12 (1983) 137-142
137
Elsevier Scientific Publishers Ireland, Ltd.
Interband fibrils of polytene chromosomes V e i k k o Sorsa Deoartment of Genetics, University of Helsinki, Salornonkatu 17A, SF ~00100 Helsinki 10, Finland
(Accepted 16 July 1982)
Polytenization of chromosome complement is characteristic to the differentiation of salivary gland cells of Dipteran insects. Ultrastrueture of polytenized chromosomes was studied in Drosophila cells by means of thin section EM. According to the high-voltage EM of whole-mountedpolytenechromosomesthe interband fibers are 20-30 nm wide helices of nucleosomefibril, which implies that the DNA content of interband fibres is ca. 20-30 times their axial length in chromosomes. To test this concept the ultrastructural organization of interband fibrils was studied from the salivary gland chromosomes of Drosophila melanogaster after different fixations and differential stretching of identified interbands. The results do not support the existence of nucleosomehelices in the interbands. The thickness of individual interband fibrils is only about 5 nm in the thin sections of unstretched chromosomes and the length of fibrils can be increased only ca. 2 times by the stretching, which indicates that their DNA is already in relatively extended stage. polytene chromosomes
interbandfibrils
thin section EM
1. Introduction Repeated replication of chromatids in the interphase nuclei is one of the most characteristic events during the differentiation of salivary gland cells in the larval development of Dipteran insects. The structural differentiation of individual chromatids into the chromomeres and interchromomeres appears as a regular banding pattern in the polytenized interphase chromosomes. About 40 years ago C.B. and P.N. Bridges identified and mapped ca. 5060 bands and interbands in the salivary gland chromosomes of Drosophila melanogaster (cf. Lindsley and Grell, 1968). The electron microscopic analyses of the banding pattern in thin-sectioned salivary gland chromosomes have shown that the number of bands and interbands is even higher than the one depicted in the revised camera lucida maps of Bridges (cf. Saura and Sorsa, 1979a, b; Saura, 1980; Sorsa and Saura, 1980a, b). Alanen (1981) has shown that Bridges' bands can be detected also in the whole-mount EM, if the acidfixed polytene chromosomes are spread instead of squashing (cf. Ris, 1976).
chromatidstructure
Drosophilamelanogaster
Although Steffensen (1963) failed to show the continuity of thymidine labeling through the interbands, the presence of D N A has been indisputably proved by the DNase treatments (Lezzi, 1965) and by the acridine orange staining (Wolstenholme, 1966). Correspondingly, the results of DNase digestion (Sorsa and Sorsa, 1969) and thymidine autoradiography (Virrankoski and Sorsa, 1969; Sorsa and Virrankoski-Castrodeza, 1972) indicate that the central axis of induced lampbrush stage of polytene chromosomes contains DNA. Beermann (1972) estimated that the D N A content of interbands is about 5% of the total D N A content of polytene chromosomes. The localizations of R N A polymerase B (cf. Jamrich et al., 1977), of uridine labeling (cf. Semeshin et al., 1979) and of transcription products (cf. Skaer, 1977; Mott et al., 1980) into the interband regions have suggested that the D N A content of interbands is > 5%. The thickness of interband fibers observed in the whole-mounts of squased chromosomes (Ris, 1976) as well as the densitometry of EM negatives of similarly prepared polytene chromosome also indicate that the
0045-6039/83/0000-0000/$03.00 © 1983 Elsevier Scientific Publishers Ireland, Ltd.
138 proportion of interband DNA may be much higher than proposed by Beermann (1972) (cf. Laird et al., 1980; Laird, 1980; Laird et al., 1981). According to the densitometric results of Laird (1980) > 26% of the total DNA is located in the interband regions of polytene chromosomes. Accordingly, several arguments have been presented in favor of the hypothesis that the interbands of polytene chromosomes may represent active genes (cf. Crick, 1971; Speiser, 1974; Zhimulev and Belyaeva, 1975). On the other hand, the recent localization of Z-DNA into the interbands of salivary gland chromosomes of Drosophila (Nordheim et al., 1981) supports the view that the interbands are involved in the regulation of genes which are located in bands (cf. Paul, 1972; Sorsa, 1976). Earlier results of the EM of interbands have been quite contradictory depending on the methods used for the preparation of polytene chromosomes (cf. Swift, 1962; Sorsa and Sorsa, 1967; Ris, 1976). In particular, the observations concerning the thickness of individual interband fibers have yielded controversial results (cf. Ris and Korenberg, 1979). This is manifest also in the recent densitometric results of EM negatives of whole-mounted and thin-sectioned polytene chromosomes (cf. Laird, 1980; Sorsa, 1982). As the fine structure and DNA content of interbands are of great importance for understanding the structural and functional organization of polytene chromosomes we evidently need more exact data of the dimensions of individual chromatin fibrils in the interbands identified according to the reference maps of Bridges. To test the structural hypothesis revealed by the whole-mount EM that the interband fibers are composed of ca. 30 nm wide chromatosome helices (Laird et al., 1980) the ultrastructure of Bridges' interbands was studied from thin-sectioned polytene chromosomes after differential stretching.
2. Material and methods
Salivary glands of the third instar larvae of
Drosophila melanogaster were fixed within 3-5 s
after dissection in cold FAR-solution (4% formaldehyde in insect-Ringer) or with cold AMsolution (glacial acetic acid and absolute methanol 1:3) ca. 15 min in about + 4 ° C at a refrigerator. After a rapid squashing phase in 45% acetic acid between the silicon-coated slide and coverslip and removal of the coverslip in 50% ethanol the polytene chromosomes were dehydrated in a series of ethanols (70-99.5%). After staining in cold (UAM (2% uranyl acetate in absolute methanol) the preparations were rinsed in 2-3 absolute ethanols, moved into the 1 : 1 propylene oxide-ethanol and finally embedded from propylene oxide into Durcupan ACM (Fluka). After polymerization of embedding material on slides the chromosomes were chosen with a phase contrast microscope, photographed and marked on the surface of Durcupan layer for exact setting the capsules. BEEM-capsules were filled with a slightly softer mixture of Durcupan and put upside down on the marked salivary gland chromosomes. After polymerization the blocks were removed from slides with the polytene chromosomes, trimmed and thin-sectioned for the EM. The contrast was increased in FAR-fixed chromosomes by a short post-staining with Pb-citrate. The polytene chromosomes were studied with a Philips EM 200 electron microscope at the Department of Electron Microscopy of the University of Helsinki using 80 KeV. The electron micrographs were taken on 35 mm fine grane film by using original magnifications ×5000-18000. The salivary gland chromosome regions were identified according to the revised reference maps of Bridges (Lindsley and Grell, 1968) (see Figs. 1 and 2). Regions of polytene chromosomes, which are easy to recognize in all kinds of preparations were chosen for studying the structure of Bridges' interbands from the high resolution micrographs. The region 56F 10-17 is located between the prominent band complexes of 56F 1-9 (known as 5S RNA locus) and 57A 1-2 in chromosome 2R. The other regions 71F, 26B and 31D can be seen also in the low magnification micrographs of wholemounted polytene chromosomes (cf. Laird, 1980; Laird et al., 1980).
139
Fig. 1. Electron micrographs showing the interbands and bands in the distal part of Bridges' subdivusion 56F in differentially fixed and stretched salivary gland 2R chromosomes of Drosophila melanogaster. The identification of bands follows the revised reference maps of Bridges (cf. Lindsley and Grell, 1968). New bands found in the EM are marked with+signs. Scale bars, 100 nm. (a) A relatively unstretched region between the distal heavy bands 56F 6 - 9 of the 5S RNA gene locus (cf. Sorsa, 1973) and the next prominent doublet band 57A 1-2 in a 2R chromosome fixed with F A R and stained in U A M + Pb citrate. (b, c) The region 56F 10-17 in two moderately stretched chromosomes fixed with F A R (b) and with AM (c) and stained in UAM. (d) The region 56F 10-14 in a more stretched 2R fixed with F A R and stained in U A M + Pb citrate. The axial fibrils between the bands are quite extended already in the unstretched 2R (a), and the thickness of individual interband fibrils (thin arrows) is only about 5 nm, while the thicker fibers (thick arrows) obviously represent pairs or bundles of the 5 nm fibrils.
140
Fig. 2. Electron micrographs of interbands and bands in Bridges' subdivisions 71F of 3L, 26B and 31D of 2L (a, b and c), and fine structure of interbands after a stronger stretching of polytene chromosomes after fixation in F A R (d-e) and in A M (f). Individual
141
3. Results To preclude the possibility that the 20-30 nm wide nucleosome helices, which are assumed to exist in the interbands of whole-mounted polytene chromosomes, may be distroyed by overstretching (cf. Ris and Korenberg, 1979), the same interbands are compared in unstretched and stretched stage. Figs. 1 and 2 show the fine structure of stained chromatin in thin-sectioned salivary gland chromosomes after different fixations and differential stretching. The length of interbands can not be stretched more than ca. 1.5-2 times without destroying the faintest bands. The thickness of individual interband fibriles (ca. 5 nm) is not remarkably reduced by the stretching. Higher orders of helical structures or even nucleosomes cannot be recognized in the fibrils connecting Bridges' bands. The cross section area of a 5 nm thick cylindrical fiber is ca. 20 nm 2. According to the unineme concept of chromatids each one of those ca. 5 nm thick interband fibrils represents a chromatid in the polytenized interphase chromosome.
4. Discussion and Conclusions According to the results of Laird et al. (1980) and Laird (1980) the cross-section area of chromatin of a single chromatid is ca. 2500 nm 2 in an average band and ca. 650 nm 2 in interbands. The cross-section area of interband chromatin (650 nm 2) is thus about 26% of the cross-section area of band chromatin (2500 nm 2) per chromatid. This estimate of Laird (1980) is in good accordance with the direct observations of the thickness of interband fibers in the whole-mounted squashes of polytene chromosomes studied with the high-voltage EM (cf. Ris, 1976; Laird, 1980).
However, a comparison of the cross-sectional value of ca. 20 nm 2 estimated for the individual chromatids in the interbands of thin-sectioned salivary gland chromosomes with the figures reported by Laird et al. (1980) shows an obvious contradiction. The cross-section of individual chromatids in interbands of thin-sectioned chromosomes (20 nm 2) is < 0.8% of the cross-section (2500 nm 2) of average chromatid in bands and ca. 3% of the cross-section of interband fiber (ca. 650 nm 2) in whole-mounts. The result of the first comparison (0.8%) is quite understandable, because the average cross-sectional value per chromatid (2500 nm 2) is obviously based on measurements of rather prominent bands. The result of the latter comparison (ca. 3%) is more difficult to explain. There seems to be a difference of more than 30 times between the cross-sectional values of interbands fibers in the thin-sectioned and wholemounted polytene chromosomes. If we accept as a fact that on average ca. 120 nm long and only ca. 5 nm thick, already rather extended interchromomeric fibrils are not capable of forming equally long or even longer nucleosome helices with a diameter of ca. 30 nm, the following questions have to be answered. What is the real number, length and fine structure of those ca. 30 nm thick interband fibers which appear when the acid-fixed and squashed polytene chromosomes are prepared for the EM by using the whole-mount method? What is their relation to the interbands depicted in the revised reference maps of Bridges? The EMs of thin-sectioned salivary gland chromosomes of Drosophila seem to suggest that those 30 nm thick fibers in the interbands may not represent individual chromatids between two Bridges' bands. More likely they should be interpreted as bundles of parallel chromatids. This hypothesis may be roughly checked by estimating the number of interband fibers in a clear fully
interband fibrils are ca. 5 nm thick or even less in the more stretched regions. The regularly coiled fibrils in the neighborhood of stretched bands (thick arrows in d) may represent the uncoiling process of nucleosome fibers. Stretching of polytene chromosomes causes the breakage of many bands and thus the border lines of interbands and bands become unclear. Small chromomeres (thick arrows in e) may disappear completely. This, together with the shifting of individual chromomeresout of bands into the interband areas makes it difficult to get reliable densitometric results from interbands. The overlappingof d and e is marked with white stars on the micrographs. (f) A higher magnification electron micrograph of an interband region in a thicker section of AM-fixed and UAM-stained salivary gland chromosome.Scale bars, 100 nm.
142 p o l y t e n i z e d i n t e r b a n d . O n the o t h e r hand, the high cross-sectional m a s s a n d D N A c o n t e n t o b t a i n e d for the i n t e r b a n d s in the d e n s i t o m e t r i c studies of E M negatives of w h o l e - m o u n t e d p o l y t e n e chrom o s o m e s indicate, as suspected b y L a i r d (1980), that u n d e t e c t e d Bridges' b a n d s were i n c l u d e d in the a s s u m e d i n t e r b a n d s . This possibility can be easily checked b y a c o m p a r i s o n of the s c a n n e d i n t e r b a n d s to the revised reference m a p s of Bridges. T h e p r o p o s e d e x p l a n a t i o n for the high D N A c o n t e n t of i n t e r b a n d s is also s u p p o r t e d b y the d e n s i t o m e t r i c results of the E M negatives of t h i n - s e c t i o n e d salivary gland c h r o m o s o m e s 2L (Sorsa, 1982). In this study, which also i n c l u d e d analyses o f very n a r r o w b a n d s , the average crosssection area of b a n d c h r o m a t i n was e s t i m a t e d to b e o n l y ca. 806 n m 2 p e r c h r o m a t i d . This corres p o n d s to a single cylindrical fiber having a d i a m eter of ca. 32 nm. A c c o r d i n g l y , b o t h the cross-section area (ca. 650 n m 2) a n d the c o r r e s p o n d i n g d i a m e t e r (ca. 29 nm) of the a s s u m e d i n t e r b a n d fibers in the w h o l e - m o u n t e d p o l y t e n e chrom o s o m e s are close to the cross-sectional a r e a a n d d i a m e t e r e s t i m a t e d for the average c h r o m o m e r e s in thin-sectioned p o l y t e n e c h r o m o s o m e s , . T h e results of thin-section E M of salivary g l a n d c h r o m o s o m e s suggest that the m a i n reason for the structural p a r a d o x of p o l y t e n e c h r o m o s o m e s as it was i n t r o d u c e d b y L a i r d (1980) o b v i o u s l y is the different i n t e r p r e t a t i o n of n u m b e r , length a n d thickness of i n d i v i d u a l c h r o m a t i d s in the int e r b a n d s a n d the d e f i n i t i o n of i n t e r b a n d regions in the w h o l e - m o u n t e d a n d in the t h i n - s e c t i o n e d pol y t e n e c h r o m o s o m e s . T h e d i m e n s i o n s of i n d i v i d u a l fibrils in Bridges' i n t e r b a n d s of t h i n - s e c t i o n e d salivary g l a n d c h r o m o s o m e s suggest that the observations m a d e b y m e a n s of the high voltage E M f r o m the w h o l e - m o u n t e d squashes are in need of re-checking with higher r e s o l u t i o n a n d with the exact c o m p a r i s o n to the b a n d i n g p a t t e r n d e p i c t e d in the revised reference m a p s of Bridges.
Acknowledgments I wish to t h a n k Ms. Virpi V i r r a n k o s k i C a s t r o d e z a , M.Sc., A n j a O. Saura Ph.Lic. a n d M s R i i k k a S a n t a l a h t i for skilful thin-sectioning of the salivary g l a n d c h r o m o s o m e s . T h e s t u d y was finan-
cially s u p p o r t e d b y the N a t i o n a l Research Council of Sciences of F i n l a n d .
References Alanen, M.: Hereditas 95, 295-321 (1981). Beermann, W,: In: Results and Problems in Cell Differentiation, 4., ed. W Beermann (Springer-Verlag, New York) pp. 1-33 (1972). Crick, F.: Nature (London) 234, 25-27 (1971). Jamrich, M., A.L. Greenleaf and E.K.F. Bautz: Proc. Natl. Acad. Sci. U.S.A. 74, 2079-2083 (1977). Laird, C.D.: Cell 22, 869-874 (1980). Laird, C.D., M. Ashburner and L. Wilkinson: Chromosoma 76, 175-189 (1980). Laird, C.D., L. Wilkinson, D. Johnson and C. Sandstr~m: In: Chromosomes Today, 7. eds. M. Bennett, M. Bobrov and G. Hewitt (George, Allen & Unwin, Hemel Hempstead) pp. 74-83 (1981). Lezzi, M.: Exp.Cell Res. 39, 289-292 (1965). Lindsley, D.L. and E.H. Grell: Genetic variation of Drosophila melanogaster (Carnegie Institution of Washington) Publ. 627 (1968). Mott, M.R., E.J. Burnett and R.J. Hill: J. Cell Sci. 45, 15-30 (1980). Nordheim, A., M.L. Pardue, E.M. Lafer, A. M/311er, B.D. Stollar and A. Rich: Nature (London) 294, 417-422 (1981). Paul, J.: Nature (London) 238, 444-446 (1972). Ris, H.: Sixth Eur. Conf. Electron Microscopy, Jerusalem 2, 21-25 (1976). Ris, H. and J. Korenberg: In: Cell Biology 2nd ed. D.M. Prescott and L. Goldstein (Academic Press, New York) pp. 267-361 (1979). Saura, A.O.: Hereditas 93, 295-309 (1980). Saura, A.O. and V. Sorsa: Hereditas 90, 39-49 (1979a). Saura, A.O. and V. Sorsa: Hereditas 90, 257-267 (1979b). Semeshin, V.F., I.F. Zhimulev and E.S. Belyaeva: Chromosoma 73, 163-177 (1979). Skaer, R.J.: J. Cell Sci. 26, 251-266 (1977). Sorsa, M. and V. Sorsa: Chromosoma 22, 32-41 (1967). Sorsa, V.: Hereditas 74, 297-301 (1973). Sorsa, V.: Hereditas 82, 63-68 (1976). Sorsa, V.: Hereditas 97, 103-113 (1982). Sorsa, V. and A.O. Saura: Hereditas 92, 73-83 (1980a). Sorsa, V. and A.O. Saura: Hereditas 92, 341-351 (1980b). Sorsa, V. and M. Sorsa: Eur. Dros. Res. Conf. Abstr., Den Haag, The Netherlands (1969). Sorsa, V. and V. Virrankoski-Castrodeza: Hereditas 71: 139-144 (1972). Speiser, C.: Theor. Appl. Genet. 44, 97-99 (1974). Steffensen, D.M.: Genetics 48, 1289-1301 (1963). Swift, H.: In: Molecular Control of Cellular Activity, ed. J.M. Allen (McGraw-Hill, New York) pp. 73-125 (1962). Virrankoski, V. and V. Sorsa: Eur. Dros. Res. Conf. Abstr., Den Haag, The Netherlands (1969). Wolstenholme, D.: Genetics 53, 357-360 (1966). Zhimulev, I.F. and E.S. Belyaeva: Theor. Appl. Genet. 45, 335-340 (1975).
137
Elsevier Scientific Publishers Ireland, Ltd.
Interband fibrils of polytene chromosomes V e i k k o Sorsa Deoartment of Genetics, University of Helsinki, Salornonkatu 17A, SF ~00100 Helsinki 10, Finland
(Accepted 16 July 1982)
Polytenization of chromosome complement is characteristic to the differentiation of salivary gland cells of Dipteran insects. Ultrastrueture of polytenized chromosomes was studied in Drosophila cells by means of thin section EM. According to the high-voltage EM of whole-mountedpolytenechromosomesthe interband fibers are 20-30 nm wide helices of nucleosomefibril, which implies that the DNA content of interband fibres is ca. 20-30 times their axial length in chromosomes. To test this concept the ultrastructural organization of interband fibrils was studied from the salivary gland chromosomes of Drosophila melanogaster after different fixations and differential stretching of identified interbands. The results do not support the existence of nucleosomehelices in the interbands. The thickness of individual interband fibrils is only about 5 nm in the thin sections of unstretched chromosomes and the length of fibrils can be increased only ca. 2 times by the stretching, which indicates that their DNA is already in relatively extended stage. polytene chromosomes
interbandfibrils
thin section EM
1. Introduction Repeated replication of chromatids in the interphase nuclei is one of the most characteristic events during the differentiation of salivary gland cells in the larval development of Dipteran insects. The structural differentiation of individual chromatids into the chromomeres and interchromomeres appears as a regular banding pattern in the polytenized interphase chromosomes. About 40 years ago C.B. and P.N. Bridges identified and mapped ca. 5060 bands and interbands in the salivary gland chromosomes of Drosophila melanogaster (cf. Lindsley and Grell, 1968). The electron microscopic analyses of the banding pattern in thin-sectioned salivary gland chromosomes have shown that the number of bands and interbands is even higher than the one depicted in the revised camera lucida maps of Bridges (cf. Saura and Sorsa, 1979a, b; Saura, 1980; Sorsa and Saura, 1980a, b). Alanen (1981) has shown that Bridges' bands can be detected also in the whole-mount EM, if the acidfixed polytene chromosomes are spread instead of squashing (cf. Ris, 1976).
chromatidstructure
Drosophilamelanogaster
Although Steffensen (1963) failed to show the continuity of thymidine labeling through the interbands, the presence of D N A has been indisputably proved by the DNase treatments (Lezzi, 1965) and by the acridine orange staining (Wolstenholme, 1966). Correspondingly, the results of DNase digestion (Sorsa and Sorsa, 1969) and thymidine autoradiography (Virrankoski and Sorsa, 1969; Sorsa and Virrankoski-Castrodeza, 1972) indicate that the central axis of induced lampbrush stage of polytene chromosomes contains DNA. Beermann (1972) estimated that the D N A content of interbands is about 5% of the total D N A content of polytene chromosomes. The localizations of R N A polymerase B (cf. Jamrich et al., 1977), of uridine labeling (cf. Semeshin et al., 1979) and of transcription products (cf. Skaer, 1977; Mott et al., 1980) into the interband regions have suggested that the D N A content of interbands is > 5%. The thickness of interband fibers observed in the whole-mounts of squased chromosomes (Ris, 1976) as well as the densitometry of EM negatives of similarly prepared polytene chromosome also indicate that the
0045-6039/83/0000-0000/$03.00 © 1983 Elsevier Scientific Publishers Ireland, Ltd.
138 proportion of interband DNA may be much higher than proposed by Beermann (1972) (cf. Laird et al., 1980; Laird, 1980; Laird et al., 1981). According to the densitometric results of Laird (1980) > 26% of the total DNA is located in the interband regions of polytene chromosomes. Accordingly, several arguments have been presented in favor of the hypothesis that the interbands of polytene chromosomes may represent active genes (cf. Crick, 1971; Speiser, 1974; Zhimulev and Belyaeva, 1975). On the other hand, the recent localization of Z-DNA into the interbands of salivary gland chromosomes of Drosophila (Nordheim et al., 1981) supports the view that the interbands are involved in the regulation of genes which are located in bands (cf. Paul, 1972; Sorsa, 1976). Earlier results of the EM of interbands have been quite contradictory depending on the methods used for the preparation of polytene chromosomes (cf. Swift, 1962; Sorsa and Sorsa, 1967; Ris, 1976). In particular, the observations concerning the thickness of individual interband fibers have yielded controversial results (cf. Ris and Korenberg, 1979). This is manifest also in the recent densitometric results of EM negatives of whole-mounted and thin-sectioned polytene chromosomes (cf. Laird, 1980; Sorsa, 1982). As the fine structure and DNA content of interbands are of great importance for understanding the structural and functional organization of polytene chromosomes we evidently need more exact data of the dimensions of individual chromatin fibrils in the interbands identified according to the reference maps of Bridges. To test the structural hypothesis revealed by the whole-mount EM that the interband fibers are composed of ca. 30 nm wide chromatosome helices (Laird et al., 1980) the ultrastructure of Bridges' interbands was studied from thin-sectioned polytene chromosomes after differential stretching.
2. Material and methods
Salivary glands of the third instar larvae of
Drosophila melanogaster were fixed within 3-5 s
after dissection in cold FAR-solution (4% formaldehyde in insect-Ringer) or with cold AMsolution (glacial acetic acid and absolute methanol 1:3) ca. 15 min in about + 4 ° C at a refrigerator. After a rapid squashing phase in 45% acetic acid between the silicon-coated slide and coverslip and removal of the coverslip in 50% ethanol the polytene chromosomes were dehydrated in a series of ethanols (70-99.5%). After staining in cold (UAM (2% uranyl acetate in absolute methanol) the preparations were rinsed in 2-3 absolute ethanols, moved into the 1 : 1 propylene oxide-ethanol and finally embedded from propylene oxide into Durcupan ACM (Fluka). After polymerization of embedding material on slides the chromosomes were chosen with a phase contrast microscope, photographed and marked on the surface of Durcupan layer for exact setting the capsules. BEEM-capsules were filled with a slightly softer mixture of Durcupan and put upside down on the marked salivary gland chromosomes. After polymerization the blocks were removed from slides with the polytene chromosomes, trimmed and thin-sectioned for the EM. The contrast was increased in FAR-fixed chromosomes by a short post-staining with Pb-citrate. The polytene chromosomes were studied with a Philips EM 200 electron microscope at the Department of Electron Microscopy of the University of Helsinki using 80 KeV. The electron micrographs were taken on 35 mm fine grane film by using original magnifications ×5000-18000. The salivary gland chromosome regions were identified according to the revised reference maps of Bridges (Lindsley and Grell, 1968) (see Figs. 1 and 2). Regions of polytene chromosomes, which are easy to recognize in all kinds of preparations were chosen for studying the structure of Bridges' interbands from the high resolution micrographs. The region 56F 10-17 is located between the prominent band complexes of 56F 1-9 (known as 5S RNA locus) and 57A 1-2 in chromosome 2R. The other regions 71F, 26B and 31D can be seen also in the low magnification micrographs of wholemounted polytene chromosomes (cf. Laird, 1980; Laird et al., 1980).
139
Fig. 1. Electron micrographs showing the interbands and bands in the distal part of Bridges' subdivusion 56F in differentially fixed and stretched salivary gland 2R chromosomes of Drosophila melanogaster. The identification of bands follows the revised reference maps of Bridges (cf. Lindsley and Grell, 1968). New bands found in the EM are marked with+signs. Scale bars, 100 nm. (a) A relatively unstretched region between the distal heavy bands 56F 6 - 9 of the 5S RNA gene locus (cf. Sorsa, 1973) and the next prominent doublet band 57A 1-2 in a 2R chromosome fixed with F A R and stained in U A M + Pb citrate. (b, c) The region 56F 10-17 in two moderately stretched chromosomes fixed with F A R (b) and with AM (c) and stained in UAM. (d) The region 56F 10-14 in a more stretched 2R fixed with F A R and stained in U A M + Pb citrate. The axial fibrils between the bands are quite extended already in the unstretched 2R (a), and the thickness of individual interband fibrils (thin arrows) is only about 5 nm, while the thicker fibers (thick arrows) obviously represent pairs or bundles of the 5 nm fibrils.
140
Fig. 2. Electron micrographs of interbands and bands in Bridges' subdivisions 71F of 3L, 26B and 31D of 2L (a, b and c), and fine structure of interbands after a stronger stretching of polytene chromosomes after fixation in F A R (d-e) and in A M (f). Individual
141
3. Results To preclude the possibility that the 20-30 nm wide nucleosome helices, which are assumed to exist in the interbands of whole-mounted polytene chromosomes, may be distroyed by overstretching (cf. Ris and Korenberg, 1979), the same interbands are compared in unstretched and stretched stage. Figs. 1 and 2 show the fine structure of stained chromatin in thin-sectioned salivary gland chromosomes after different fixations and differential stretching. The length of interbands can not be stretched more than ca. 1.5-2 times without destroying the faintest bands. The thickness of individual interband fibriles (ca. 5 nm) is not remarkably reduced by the stretching. Higher orders of helical structures or even nucleosomes cannot be recognized in the fibrils connecting Bridges' bands. The cross section area of a 5 nm thick cylindrical fiber is ca. 20 nm 2. According to the unineme concept of chromatids each one of those ca. 5 nm thick interband fibrils represents a chromatid in the polytenized interphase chromosome.
4. Discussion and Conclusions According to the results of Laird et al. (1980) and Laird (1980) the cross-section area of chromatin of a single chromatid is ca. 2500 nm 2 in an average band and ca. 650 nm 2 in interbands. The cross-section area of interband chromatin (650 nm 2) is thus about 26% of the cross-section area of band chromatin (2500 nm 2) per chromatid. This estimate of Laird (1980) is in good accordance with the direct observations of the thickness of interband fibers in the whole-mounted squashes of polytene chromosomes studied with the high-voltage EM (cf. Ris, 1976; Laird, 1980).
However, a comparison of the cross-sectional value of ca. 20 nm 2 estimated for the individual chromatids in the interbands of thin-sectioned salivary gland chromosomes with the figures reported by Laird et al. (1980) shows an obvious contradiction. The cross-section of individual chromatids in interbands of thin-sectioned chromosomes (20 nm 2) is < 0.8% of the cross-section (2500 nm 2) of average chromatid in bands and ca. 3% of the cross-section of interband fiber (ca. 650 nm 2) in whole-mounts. The result of the first comparison (0.8%) is quite understandable, because the average cross-sectional value per chromatid (2500 nm 2) is obviously based on measurements of rather prominent bands. The result of the latter comparison (ca. 3%) is more difficult to explain. There seems to be a difference of more than 30 times between the cross-sectional values of interbands fibers in the thin-sectioned and wholemounted polytene chromosomes. If we accept as a fact that on average ca. 120 nm long and only ca. 5 nm thick, already rather extended interchromomeric fibrils are not capable of forming equally long or even longer nucleosome helices with a diameter of ca. 30 nm, the following questions have to be answered. What is the real number, length and fine structure of those ca. 30 nm thick interband fibers which appear when the acid-fixed and squashed polytene chromosomes are prepared for the EM by using the whole-mount method? What is their relation to the interbands depicted in the revised reference maps of Bridges? The EMs of thin-sectioned salivary gland chromosomes of Drosophila seem to suggest that those 30 nm thick fibers in the interbands may not represent individual chromatids between two Bridges' bands. More likely they should be interpreted as bundles of parallel chromatids. This hypothesis may be roughly checked by estimating the number of interband fibers in a clear fully
interband fibrils are ca. 5 nm thick or even less in the more stretched regions. The regularly coiled fibrils in the neighborhood of stretched bands (thick arrows in d) may represent the uncoiling process of nucleosome fibers. Stretching of polytene chromosomes causes the breakage of many bands and thus the border lines of interbands and bands become unclear. Small chromomeres (thick arrows in e) may disappear completely. This, together with the shifting of individual chromomeresout of bands into the interband areas makes it difficult to get reliable densitometric results from interbands. The overlappingof d and e is marked with white stars on the micrographs. (f) A higher magnification electron micrograph of an interband region in a thicker section of AM-fixed and UAM-stained salivary gland chromosome.Scale bars, 100 nm.
142 p o l y t e n i z e d i n t e r b a n d . O n the o t h e r hand, the high cross-sectional m a s s a n d D N A c o n t e n t o b t a i n e d for the i n t e r b a n d s in the d e n s i t o m e t r i c studies of E M negatives of w h o l e - m o u n t e d p o l y t e n e chrom o s o m e s indicate, as suspected b y L a i r d (1980), that u n d e t e c t e d Bridges' b a n d s were i n c l u d e d in the a s s u m e d i n t e r b a n d s . This possibility can be easily checked b y a c o m p a r i s o n of the s c a n n e d i n t e r b a n d s to the revised reference m a p s of Bridges. T h e p r o p o s e d e x p l a n a t i o n for the high D N A c o n t e n t of i n t e r b a n d s is also s u p p o r t e d b y the d e n s i t o m e t r i c results of the E M negatives of t h i n - s e c t i o n e d salivary gland c h r o m o s o m e s 2L (Sorsa, 1982). In this study, which also i n c l u d e d analyses o f very n a r r o w b a n d s , the average crosssection area of b a n d c h r o m a t i n was e s t i m a t e d to b e o n l y ca. 806 n m 2 p e r c h r o m a t i d . This corres p o n d s to a single cylindrical fiber having a d i a m eter of ca. 32 nm. A c c o r d i n g l y , b o t h the cross-section area (ca. 650 n m 2) a n d the c o r r e s p o n d i n g d i a m e t e r (ca. 29 nm) of the a s s u m e d i n t e r b a n d fibers in the w h o l e - m o u n t e d p o l y t e n e chrom o s o m e s are close to the cross-sectional a r e a a n d d i a m e t e r e s t i m a t e d for the average c h r o m o m e r e s in thin-sectioned p o l y t e n e c h r o m o s o m e s , . T h e results of thin-section E M of salivary g l a n d c h r o m o s o m e s suggest that the m a i n reason for the structural p a r a d o x of p o l y t e n e c h r o m o s o m e s as it was i n t r o d u c e d b y L a i r d (1980) o b v i o u s l y is the different i n t e r p r e t a t i o n of n u m b e r , length a n d thickness of i n d i v i d u a l c h r o m a t i d s in the int e r b a n d s a n d the d e f i n i t i o n of i n t e r b a n d regions in the w h o l e - m o u n t e d a n d in the t h i n - s e c t i o n e d pol y t e n e c h r o m o s o m e s . T h e d i m e n s i o n s of i n d i v i d u a l fibrils in Bridges' i n t e r b a n d s of t h i n - s e c t i o n e d salivary g l a n d c h r o m o s o m e s suggest that the observations m a d e b y m e a n s of the high voltage E M f r o m the w h o l e - m o u n t e d squashes are in need of re-checking with higher r e s o l u t i o n a n d with the exact c o m p a r i s o n to the b a n d i n g p a t t e r n d e p i c t e d in the revised reference m a p s of Bridges.
Acknowledgments I wish to t h a n k Ms. Virpi V i r r a n k o s k i C a s t r o d e z a , M.Sc., A n j a O. Saura Ph.Lic. a n d M s R i i k k a S a n t a l a h t i for skilful thin-sectioning of the salivary g l a n d c h r o m o s o m e s . T h e s t u d y was finan-
cially s u p p o r t e d b y the N a t i o n a l Research Council of Sciences of F i n l a n d .
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