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An Integrated Description of Polytene Chromosome Structure

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An Integrated Description of Polytene Chromosome Structure Hrrrditas 95: 2Y5-321 An integrated description of polytene chromosome structure M. ALANEN Dc,purtmiwt of Genetics, Uniwrsity of Helsinki, Finlund* ALANEN, M. 1981. An integrated description of polytene chromosome structure. - Hereditas 95: 295-321. Lund, Sweden. ISSN 0018-0661. Received April 28, 1981. An integrated description from nucleohistone fiber level to light microscopic level of polytene chromo- somes is presented. The integration is based on the results obtained by different new applications of electron microscopic techniques and on the analysis of earlier data of chromatin structure. A model for the organization of polytene chromosomes in vivo is extrapolated from the EM-data by approximating the effects of the physico-chemical parameters changing during preparative procedures. The nucleosome unit fiber forms loops, which are organized into about 500A diameter coils with 24 nucleosomes per turn of the double fiber. leading to an overall packing ratio of about 1:78. The coils are packed differentially into structural units forming the polytene bands, which are separated from each other by interbands of unit fibers with no higher order coiling evident. For the general higher order structure of chromatin a model capable of explaining former observations in other eukaryotic chromosome systems is proposed. A whole mount method suitable for nucleosome fiber examination in polytene chromosomes is intro- duced. A sectioning method preserving the nucleosomal structures and a quick method for whole mounting polytene chromosomes retaining their light microscopic morphology are also described. M. Alanen. Recombinant DNA Laboratory, University of Helsinki, Haartmaninkatu 3, SF40290 Helsinki 29, Finland Electron microscopic study of eukaryotic chro- soon amply confirmed (e.g., OLINSet al. 1975; mosomes is a prerequisite for integrating bio- RATTNERet al. 1975; LANGMOREand WOOLEY1975). chemical and genetic data into a coherent descrip- Since then the nucleosomes have become the most tion of the structure of chromatin. The fibrous extensively studied structural features of chroma- network of chromatin threads underlying the light tin (e.g., CHAMBON1978). Conventional electron microscopic structures has long been studied by microscopy has not been very successful in un- electron microscopic whole mount methods (e.g., raveling the internal structure of isolated nucleo- WOLF^ 1965; Du PRAW1965; GALL1966; SOLARI somes (POONand SELIGY1978, 1980) but, when 1968). Variation in the observed fiber diameters, combined with electron scattering and crystallo- mainly effected by different preparative condi- graphic techniques, electron microscopy has been tions (cf. SOLARI1968; BORNKAMMand SONNENBICH-of central importance also in this respect (LANG- L~R1973; BRASCH1976) combined with the lack of a MORE and WOOLEY1975; FINCHet al. 1977). higher order reference structure, limited, how- In attempts to describe the different orders of ever, the use of electron microscopic methods in chromatin fibers with the nucleosome as a refer- interpreting the organization of the DNA double ence structure electron microscopic investigation helix into higher order structures. has been the main approach. Superhelical coiling The situation changed considerably when the of the nucleosome unit fiber as represented by the subunit structure of chromatin (OLINSand WRIGHT solenoid model (BRAMet al. 1975; FINCHand KLUG 1973: OLINS~~~OLINS1974) was demonstrated and 1976; WORCELand BENYAJATI1977) and the group- a general model of the chromatin unit fiber as a ing of nucleosomes into similar-sized clusters as beaded string was presented (KORNBERG1974). The represented by the superbead structure (HOZIER existence and the general morphology of round et al. 1977; STRATLINGet al. 1978; SCHEERand particles with relatively constant diameter, i.e. ZENTGRAF1978) have both been indicated as the nucleosomes (OUDETet al. 1975) on the chromatin basis of the next higher order of organization fiber under certain preparative conditions was above the nucleosome unit fiber. Nucleosome clusters of variable size as well as multimers of * Present address: Rwombinant DNA Laboratory. University of such clusters (OLINS1978; PRUITTand GRAINGER Hdsinki. Haartmaninkatu 3, SF-00290 Helsinki 29, Finland 1980) have also been reported repeatedly, but it is 296 M.ALANEN Hrrrdirrrs Y5 (IYBI) possible that all the above-mentioned structures the general higher order organization of eukaryo- are only more or less transitional conformations of tic chromatin, at least in this respect. collapsing chromatin fibers in vitro (RATTNERand The aim of the present study is to probe the HAMKALO1978 a, b, 1979: MiLLERet al. 1978: BASU structure of Dipteran salivary gland chromosomes 1979). The systems thus investigated include with different electron microscopic techniques in meiotic chromosomes (e.g., SCHEERand ZENTCRAForder to aquire an integrated description of 1978: WEITHand TRAUT1980; RAITNERet al. 1980) chromosome structure from nucleohistone fibers and somatic cells in metaphase (RATTNERand to light microscopic structures. The nucleosome HAMKALO1975, 1978a, b: LEPAULTet al. 1980) as fiber structure will be made visible by methods, well as in interphase (e.g., WOODCOCKet al. 1976: that have proved successful in other systems (cf. RATTNERand HAMKALO1979). HoZlER et al. 1977). The higher order band-inter- Unfortunately, polytene Chromosomes lack, so band structure will be investigated by whole- far, a nucleosomal fiber description. Nucleosomes mounting identifiable, acid fixed polytene have been reported to occur in Dipteran polytene chromosomes (ALANENand SORSA1978) and by Chromatin (WOODCOCKet al. 1976: SORSA1976) but thin sectioning aldehyde fixed material. it has not been possible to see interpretable or clearly resolved structures, mainly because of methodological difficulties in applying whole- mount electron microscopic techniques to poly- Experimental procedures tene material. So, the description of polytene Nucleosome sprcuds chromosome fiber structure rests mainly on sec- tioning data (SORSAand SORSA1967a, b, 1968a, b; Fourth instar larvae of Chironomu~tentans were KIKNADZEet al. 1976) and on whole mount data of used. Salivary glands excised in the hemolymph severely treated Chromosomes (e.g., SORSA1973a; were transferred immediately on their release into SORSA and VIRRANKOSKI-CASTRODEZA 1976). The a drop of incubation buffer modified from D’AN- elucidation of the organization of the nucleosome CELO (1946). The buffer contained YO mM KC 1, 60 fiber in polytene chromosomes is essential be- mM NaCI, 5 mM EDTA, 0.5 mM dithiothreitol. cause of the wealth of higher order structures that 4.1 mM KH,PO,, 5.9. mN Na,HPO,(pH 6.2). The these systems offer for research in contrast to buffer was made I I;i of Nonidet P40 and 0.2 % of most other eukaryotic chromosome systems. As a Triton X-100. A gland was incubated in the de- first approximation polytene chromosomes can be tergent buffer (DB) for 5 min and then washed in regarded as multistranded cables in which each the buffer without detergents (B) twice for 2 min strand represents an individual, extended inter- and left in the third drop for an additional 20 min. phase chromatid in perfect register with other All the foregoing preparative steps were executed identical chromatids. The most conspicuous mor- at 04°C. Half a gland was introduced onto a phological feature resulting from this exact pairing hypophase of bidistilled water, with alcohol of multiple chromatids is undoubtedly the forma- cleaned forceps. Chromosomes were picked up tion of alternating high density bands and low with formvar coated hydrophilic grids. The mate- density interbands along the whole length of poly- rial was stained with 2 57 uranyl acetate in 75 V tene Chromosomes. Most of the research on the methanol for 30 s and rinsed thereafter in absolute electron microscopic organization of these methanol for 5-10 s before air drying. The speci- structures has been done in this laboratory by mens were rotary shadowed with platinum-palla- Marja and Veikko Sorsa (e.g., SORSAand SORSA dium (80:20) wire wound around a tungsten wire. 1967a, b: 1968a. b: M. SORSA1969: V. SORSA1974. Shadowing angle of 8” was used. 1976). In spite of the drawbacks of the methods used (e.g., DERKSENand SORSA1972: SORSA1973e) a Thin srctions basically loop-like organization of polytene chromatin has been clearly indicated (e.g., SORSA Fourth instar larvae of Chironomits p(illi(li~,itt~itii~s 1973b. 1976). As Chromatin loops have been con- were used. Salivary glands were incubated in sistently reported in other chromosome systems 3.7 % formaldehyde in 0.1 M Sflrensen’s phos- as well (e.g., CALLAN1963: So~s~andSORSA 1967b: phate buffer (pH 6.2) for 7 min. The foregoing MARSDENand LAEMMLI1979: BENYAJATIand WORCEL preparative steps were executed at 0-4°C. The 1976) the elucidation of polytene Chromosome material was further fixed at room temperature for structure should give valuable informatoin also on an additional 8 min and placed into a drop of 45 % Hrrrditus Y5 (1981) EUKARYOTICCHROMOSOME STRUCTURL 297 acetic acid for 3 min. The material was squashed Results between well siliconized cover slip and object slide. After removal of the cover slip in liquid Nucleosome spreads nitrogen the material was dehydrated and stained Salivary glands from Chironomus tentans were with 2 9f uranyl acetate in alcohol series. The incubated in a buffered salt solution containing material was embedded in Epon via propylene detergents
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