Proc. Nat. Acad. Sci. USA Vol. 70, No. 1, pp. 80-83, January 1973
Release of Amplified Ribosomal DNA from the Chromomeres of Acheta (RNA cistrons/bybridization/oocytes/pachytene/meiosis) A. LIMA-DE-FARIA, HALINA JAWORSKA, AND T. GUSTAFSSON Institute of Molecular Cytogenetics, University of Lund, Lund, Sweden Communicated by Ake Gustafsson, October 20, 1972
ABSTRACT The major chromomeres of pachytene present in the satellite DNA (15, 16). (e) To check the ampli- chromosomes 6 and II in the oocytes of Acheta (Orthop- fication process further, hybridization at tera) are regions of amplification for ribosomal RNA cis- [3H]RNA-DNA trons (28 S and 18 S). During late prophase the amplified saturation was performed. The percentage of ribosomal RNA DNA copies are released from the chromomeres, which de- bound to DNA was nearly the same in testes and somatic crease in size as the amount ofDNA decreases. The number tissues but it was 4- to 5-times higher in ovaries (16). (f) of nucleotide pairs released from the major chromomere Further confirmation that the major chromomeres repre- of chromosome 11 is about 1.38 X 109. sent amplified regions of ribosomal DNA was obtained by The concept of chromomere-band-gene was determined by isolation of the DNA satellite on CsCl gradients and by cytological and genetic studies (1, 2) of polytene and preparation, with RNA polymerase, of complementary pachytene chromosomes. Recently, on the basis of new data [8H]RNA from satellite DNA. This complementary RNA from structural, genetic, and molecular analyses of chromo- was then hybridized in situ with the pachytene chromosomes meres, this concept is being expanded and revised. of Acheta oocytes, and it hybridized mainly with the major Structural analysis of pachytene and diplotene chromo- chromomeres (17). (g) The amounts of DNA in each pachy- somes established that the chromomeres consist of spiralized tene chromosome of the oocytes, in the pachytene chromo- segments of the chromosome (3, 4). Later, studies on the DNA somes of the spermatocytes, and in the major chromomeres of higher organisms disclosed that (a) there is no correlation were measured (18). between large amounts of DNA and obvious developmental This evidence indicates that the major chromomeres pre- complexity, (b) repeated DNA sequences occur in most sent in Acheta oocytes at early pachytene contain amplified eukaryotes (5), and (c) in certain bands of giant chromosomes ribosomal RNA cistrons. of insects the amount of DNA increases differentially (6-8). MATERIALS AND METHODS As a result of this evidence the chromomere was looked upon, The amount of DNA in Feulgen-stained pachytene chromo- not as a single genetic unit, but as a structure with multiple somes was measured by a Zeiss Scanning Microscope Photom- genetic functions consisting of repeated copies of given cistrons eter 05 connected to a Facit Tape Punch. The tapes were then or given DNA sequences (9-11). Recently, a more radical analyzed by a computer, the values being displayed as contour model has been proposed in which the chromomeres or bands maps of iso-brightness or as a volumetric display on an incre- would be the control elements, whereas the interbands or mental plotter. For the measurements, the chromomeres of fibrils between the chromomeres would be the coding se- every chromosome and all the chromosomes of a cell must lie quences of the chromosomal DNA (12). on a single plane. Human lymphocytes and rat-kidney nuclei Before presentation of the data on the release of DNA were used as standards. Details of the procedures used were copies from the chromomeres the principal data on gene described (18). amplification in Acheta should be mentioned. (a) At early pachytene there are five major chromomeres or DNA bodies RESULTS AND DISCUSSION in Acheta oocytes that are not present in the spermatocytes. In Acheta, every chromosome of the complement can be The major chromomeres are formed by extra DNA synthesis identified at pachytene, by its size and chromomere markers. occurring at the interphase and early prophase of meiosis This allows us to establish the number of amplification sites (13, 14). (b) Besides the main band, there is a DNA satellite, and to locate them in specific chromomeres (19). which has a calculated GC content of 56% (buoyant density The number of major chromomeres, their size from early 1.716 g/ml). (c) The DNA satellite of ovaries at the interphase to late pachytene, and their DNA content have been recorded. of meiosis, where the major chromomeres are largest, contains The amount of DNA in the five major oocyte chromomeres at 14% of the ovarian DNA, whereas the DNA satellite of testes, early pachytene is 4.18 pg, and 9.11 pg in the rest of the chro- where no major chromomeres are present, contains 0.8% of the mosomes. Three of the major chromomeres release their DNA total testes DNA (15). This result suggested that the large copies early in development, and they are hardly visible by DNA satellite seen in ovaries represents the DNA of the major middle pachytene. The two largest chromomeres are of about chromomeres. (d) To test this assumption Acheta DNA from the same size at early pachytene (Fig. 1), and at this stage ovaries (fractionated on CsCl gradients) and 28S and 188 contain, on the average, 2.00 pg and 1.48 pg. of DNA. The ribosomal RNA of Acheta labeled with 32p or 3H were hy- average amount of DNA per chromomere pair for the other bridized. The hybridization peak occurred at the position of chromomeres of the pachytene chromosomes of Acheta is 0.04 the DNA satellite. This shows that the ribosomal cistrons are pg. It can then be seen by middle-late pachytene that one of 80 Downloaded by guest on September 30, 2021 Proc. Nat. Acad. Sci. USA 70 (1978) DNA from Chromomeres of Acheta 81
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11 ~ ~ ~ - h* 6 1 11 5 11~~~~~~~~~~~~~~~~~~~~~1 2 6C/ ti i 64 4~~~~~ IL~~~~~~~~~~~~~~~~~~~~~~~~~~I 3 ) FIGS. 1-3. Feulgen-stained nuclei of Acheta at pachytene. At early pachytene (Fig. 1) the two major chromomeres, belonging to chromosomes 6 and 11, are about the same size. At late pachytene, stage 1 (Fig. 2), the major chromomere of chromosome 6 (which was separated from its chromosome by the squash) becomes a "puff" with the DNA copies separating from each other. The major chromomere of chromosome 11 has in the meantime released some of its DNA copies, becoming now only what could be called a large chromomere. Later, at late pachytene, stage 2 (Fig. 3), the major chromomere of chromosome 11 has released most of its copies and it is indistinguish- able from the smallest chromomeres of the chromosome complement, X 1,700. FIGS. 4-7. DNA measured in Feulgen-stained nuclei of Acheta at pachytene by the Zeiss Scanning Microscope Photometer (05. The extinction values were recorded on tape and processed by a computer. They are display'ed as a contour map of iso-brightness curves. The amount of DNA in the major chromomere of chromosome 6 remains practically constant, whereas that of the major chromomere of chromosome 1 1 decreases from early (Fig. 4) to middle (Fig. 5) to late, stage 1 (Fig. 6), and late, stage 2 (Fig. 7), pachytene. The amount of DNA released is 1.43 pg. Downloaded by guest on September 30, 2021 82 Genetics: Lima-De-Faria et al. Proc. Nat. Acad. Sci. USA 70 (1973) the amplified chromomeres is located at one end of chromo- [3H]uridine incorporation (13, 14). No appreciable number of some 6 and the other is located at one end of chromosome 11 copies is released from the amplified chromomere of chromo- (Figs. 2 and 3). At this stage, the DNA copies in the amplified some 6 throughout pachytene (Figs. 4-8). Only at late diplo- chromomere of chromosome 6 open out in the form of a "puff." tene do most copies leave this chromomere. On the other hand, The DNA copies are active in RNA synthesis, as checked by the amplified chromomere of chromosome 11 starts releasing EARLY MAJOR MAJOR CHROMOMERE CHROMOMERE CHROM. 6 CHROM. 11 DNA (PG) DNA (PG) 2.00O 1.48 2.23 2.16 8 FIG. 8. The same cells measured in Figs. 4-7 are shown in computer volumetric display. The major chromomere of chromosome 11 is indicated by an arrow. The average amount of DNA in pg is given on the left side for the major chromomere of chromosome 6 and on the right side for the major chromomere of chromosome 11. At late pachytene, stage 2 (bottom of figure) every chromosome could be identified separately. Downloaded by guest on September 30, 2021 Proc. Nat. Acad. Sci. USA 70 (1978) DNA from Chromomeres of Acheta 83 its copies during early pachytene. Its diameter decreases, from function? Chromomeres are present at every prophase but are early to late pachytene (stage 2), from 1.8Mm to 0.4 Mm. What particularly large and distinct at the first prophase of meiosis. was a DNA body or major chromomere at early prophase Is this connected with the more elaborate type of genetic becomes a large chromomere, then a chromomere of average information needed at this stage, in which more significant size, and finally a minute chromomere (see Figs. 4-7). During genetic events take place than in mitosis, and which precedes this process the DNA copies separate and the major chromo- fertilization? mere builds a smaller "puff" than that formed on chromosome 6. As the copies are released, the amount of DNA decreases NOTE ADDED IN PROOF from an average of 1.48 pg (early pachytene) to 0.27 pg Ribosomal DNA is the DNA that contains the segments (middle pachytene) to 0.11 pg (late pachytene, stage 1) and, (cistrons) coding for 28 5 and 18 5 ribosomal RNA together finally, to 0.05 pg (late pachytene, stage 2, Fig. 8). This means with sequences not homologous to this RNA ("spacer"). that as much as 1.4 pg of DNA is released from a single major chromomere during pachytene, transforming it into a minor Supported by research grants from the Swedish Natural Science chromomere. This amount of DNA corresponds to 1.38 X 109 Research Council, the Knut-and Alice Wallenberg Foundation, the Bank of Sweden Tercentenary Fund, and the Torsten and nucleotide pairs. Ragnar S6derberg Foundation. From the saturation experiments and the haploid DNA value of Acheta (2.00 pg) it has been calculated that in 1. McClintock, B. (1931) Proc. Nat. Acad. Sci. USA 17, 485- somatic tissues there are about 171 cistrons (single strand) 491. for ribosomal RNA (28 S and 18 S). Since most of these sites 2. Muller, H. J. (1935) Amer. Nat. 69, 405-411. located in the two major chromomeres of. chromosomes 6 3. Ris, H. (1957) in A Symposium on the Chemical Basis of He- are redity, eds. McElroy, W. D. & Glass, B. (The Johns Hopkins and 11, as shown by hybridization in situ with complemen- Press), pp. 23-62. tary RNA and with 28 S and 18 S Xenopus RNA (17, 20), the 4. Callan, H. G. (1955) in Symposium on the Fine Structure of major chromomeres of these chromosomes contain redundant, Cells (Leiden, 1954) (P. Noordhoff, Groningen, The Nether- tandemly duplicated ribosomal DNA sequences, as well as lands) Int. Union Biol. Sci. Ser. B 21, 89. DNA result from localized amplification of 5. Britten, R. J. & Kohne, D. E. (1969) in Handbook of Molecu- extra copies that lar Cytology, ed. Lima-de-Faria, A. (North-Holland Publ. cistrons coding for ribosomal RNA. Co., Amsterdam, London), pp. 21-36. What distinguishes the present data from previous informa- 6. Ficq, A. & Pavan, C. (1957) Nature 180, 983-984. tion on localized DNA increase and decrease is the following. 7. Rudkin, G. T. & Corlette, S. L. (1957) Proc. Nat. Acad. Sci. (a) Chromomeres are shown to contain (not necessarily to USA 43, 964-968. redundancy and gene amplification. (b) Chro- 8. Keyl, H.-G. (1965) Experientia 21 (191), 1-7. consist of) gene 9. Callan, H. G. (1967) J. Cell Sci. 2, 1-7. momeres are not only spiralized regions of the chromosome, as 10. Beermann, W. (1967) in Heritage from Mendel, ed. Brink, R. has been shown in several other species, but, at least for the A. (Univ. of Wisconsin Press), pp. 179-201. cases described here, extra DNA copies are also an integral 11. Edstrom, J. E. (1968) Nature 220, 1196-1198. part of their structure. (c) This redundancy and amplification 12. Crick, F. (1971) Nature 234, 25-27. that code for 28S 13. Heinonen, L. & Halkka, 0. (1967) Ann. Med. Exp. Fenn. 45, is for well-defined cistrons (the DNA sites 101-109. and 188 RNA), whereas in most previous cases it was not 14. Lima-de-Faria, A., Nilsson, B., Cave, D., Puga, A. & Jawor- known which genes were involved in DNA increase. (d) The ska, H. (1968) Chromosoma 25, 1-20. chromomeres studied here are not part of a somatic tissue, 15. Lima-de-Faria, A., Birnstiel, M. & Jaworska, H. (1969) Ge- such as the salivary glands, but are part of the germ line. (e) netics Supplement 61 (1), 145-159. to the 16. Pero, R., Lima-de-Faria, A., Stahle, U., Granstrom, H. & Not only the increase in DNA but also the decrease due Ghatnekar, R. (1973) Hereditas 73, in press. release of the copies could be measured in the very small 17. Ullman, J., Lima-de-Faria, A., Jaworska, H. & Bryngelsson, pachytene chromomere pairs, which are on the average about T. (1973) Hereditas 73, in press. 1/5 and 1/io of the diameter of the salivary gland chromosomes 18. Lima-de-Faria, A., Gustafsson, T. & Jaworska, H. (1973) Hereditas 73, in press. of Drosophila and Chironomus, respectively. 19. Lima-de-Faria, A., Daskaloff, S. & Enell, A. (1973) Heredi- Several questions arise. Do other chromomeres contain tas 73, in press. amplified copies of other genes? Once these copies are re- 20. Cave, M.D. (1971) Amer. Soc. Cell Biol. 11th Ann. Meeting leased are they destroyed at once? If not, do they still have a 1971, 48 abstr. Downloaded by guest on September 30, 2021