Unique and repetitive DNA sequences in the genome of Chironomus tentans

Preliminary

Fig. 5. Light microscope preparation treated with 2 x SSC and Giemsa. Fig. 6. Replica of chromosomes, treated with 2 x SSC and Giemsa, showing ridging

plement results obtained by the more difficult and time consuming replica techniques. The results showed that the chromosomes collapsed after treatment with 2 x SSC which could be due to a rearrangement and/or a loss of chromosomal material. Cytoplasmic material appeared to be removed from the cell and collapse of the chromosomes may be a consequence of the loss of surrounding cytoplasmic material or more probably to a loss of material from the chrome. somes themselves. When chromosomes are stained with Giemsa after 2 x SSC treatment banding patterns are observed with the light microscope [5]. Replicas of these chromosomes showed a marked alteration of the chromosomal material leading to transverse ridging of the surface and to the appearance of fibrils. It is suggested from these results that the banding patterns obtained with Giemsa using light microscopy are due to the ridges absorbing more dye than the other parts of the chromosome. Work is currently in progress to determine whether these ridges on the chromosomes correspond to the band-

n ?tes

587

(arrowed).

ing observed by light microscopy using standard banding techniques. We are grateful to Dr A. T. Summer for discussion and assistance with the banding technique, to Mr N. Davidson for photographic assistance-and to Professor H. J. Evans for advice and encouragement. References 1. Caspersson, T, Lomakka, G & Zech, L, Hereditas 67 (1971) 89. 2. Christenhuss. R. Biichner. Th & Pfeiffer. R A. Nature 216 (1967) 379. ’ 3. Hunaerford. D A. Stain technol40 (1965) 333. 4. Net&&h, P’ W, Ampola, M G & ‘Vet&r, H G, Lancet ii (1967) 1366. 5. Sumner, AT, Evans, H J & Buckland, R A, Nature new biol 232 (1971) 31. Received April 27, 1972

Unique and repetitive DNA sequences in the genome of Chironomus tentans R. I. SACHS and U. CLEVER, Department of Biological Sciences, Purdue University, Lafayette, Ind. 47907, USA Summary It is estimated that 95.5 9/, of the DNA represents single-copy sequences. Sequences in the redundant Exptl Cell Res 74 (1972)

588 Preliminary notes sume the existence of long series of repetitive DNA units with similar or identical base sequences [2-4]. However, in some insects the fraction of DNA with reiterated base sequencesseemsto be relatively small and in addition, this fraction appears to be underreplicated during polytenization [5-71. No data are presently available about the composition of Chironomus DNA. Material and Methods

I

I

0.05

0.10

Fig. I. Abscissa: Cot values (mole x set/l); ordinate: inverse of fraction of single-stranded DNA (I/c). \-/ ,- l ,. larval DNA; o, embryo DNA. Renaturation kinetics of Chironomus (a, c) and E. coli (b) DNA. The dashed line in (a) is derived from the solid line by assuming that the slowly renaturing DNA fraction comprises 95.5 % of the genome. See text for further details.

fraction are repeated approx. 120 times. Heterodisperse high molecular weight RNA synthesized by salivary glands hybridizes in situ specifically to the Balbiani rings of salivary gland chromosomes. It is concluded that the DNA at these loci contains redundant sequences.

Late last instar larvae or egg masses, containing embryos of various developmental stages, of Chironomus tentans were homogenized in a lysing solution (8 M urea, 1 % SDS, and 0.01 M EDTA, 0.12 M PB (phosphate buffer, pH 7.4). The homogenate was extracted 3 times with an equal volume of chloroform and the aqueous phase passed through a hydroxyapatite column. The column was first washed with an 8 M urea-0.12 M PB solution and then with 0.024 M PB until the eluents had no measurable absorbance at 260 run. The selectively retained double-stranded DNA was eluted with 0.4 M PB. The eluent was dialysed against 0.1 x SSC, adjusted to 1 x SSC (0.15 M NaCI, 0.015 M sodium citrate), and the DNA precipitated with 2 vol of ethanol. The OD,,,,/,,, ratio was about 2 and the hyperchromicity 37.2%. The DNA, dissolved in 1 x SSC, was sheared according to Britten & Kohne 181to a strand length of about 400 nucleotides. The sheared DNA was denatured by heating to 100°C for 10 min and then quickly cooled to 60°C by the addition of 2!3 vol of ice-cold SSC. Fractions of DNA in double- or single-stranded form during renaturation were calculated from the chanae in hyuochromicity with time, measured by continuous 6D2,, recordina at 60°C in a Gilford 2000 suectroohotometer. u&g a 1 mm pathlength cuvette. * . Salivary glands were incubated for 2 h in a saline solution containing 3H-uridine and 3H-cytidine (250 &i/ml of each). RNA was extracted andsubjected to electrophoresis for 5 h on 2.2 % polvacrylamide gels as described previously [9]. The top gel slices, containing only heterodisperse large molecular weight RNA (> 4OS), were soaked overnight in 1 x SSC. The resulting solution contained 720 000 dpm/ml in RNA, but no measurable quantities of UV-absorbing material. The method of Pardue et al. [lo] was followed in the cytological hybridization experiments. Chromosomal DNA was denatured by boihng in 1 x SSC and quick cooling. After incubation with ‘H-RNA in 6 x SSC (25 ~1) at 65°C the squashes were treated with ribonuclease (20 peg/ml for 1 h) and covered with Kodak NTB-2 emulsion.

DNA stretches of up to 35,um length seem to be involved in the formation of some puffs in the polytene chromosomes of Chironomus tentans [I]. The RNA molecules synthesized may be 5 x lo6 D large and larger, and the size of RNA molecules extracted from individual puffs may vary with the size of these puffs [2]. Based upon these and related ob- Results and Discussion servations [3] several models to explain the In fig. 1, displaying the renaturation kinetics mechanism underlying puff-formation as- observed in our experiments, the inverse of Expti Cell Res 74 (197.2)

Preliminary notes 589 the fraction

of single-stranded DNA (C) is plotted versus C,,t, the product of the initial DNA concentration and time as defined by Britten & Kohne [8]. For DNA, or DNA fractions, homogeneous with regard to the multiplicity of base sequences this plot yields straight lines from which the Cot values at half renaturation (l/C ratio ==2) and the relative size of the homogeneous fractions (intersection with l/C axis) can be estimated. The experimental points for E. coli DNA (fig. 1b), which is essentially a homogeneous population of unique sequences [8], fall on a line intersecting with the ordinate at, or very close to, one. Half renaturation was obtained under our conditions at a C,t value of 5.75. Chironomus DNA showed fractions with different reassociation kinetics (fig. la). A major fraction renatured rather slowly. The experimental points fit onto a straight line, suggesting that this fraction is homogeneous. The extension of this line intersects with the I/C axis at a ratio of 1.047, corresponding to a fraction size of approx. 95.5% of total Chironomus DNA. To obtain the Coti of this fraction from the data of fig. la, both axes have to be renormalized accordingly. The derived line is included in fig. 1a and yields a Cot+value of 248, or approx. 43 times that of E. coli DNA. Since the Cot, values of homogeneous populations of DNA sequences are linearly related to the complexity of the DNA species compared [8, 1I], it follows that the slowly renaturing Chironomus DNA has a complexity of 1.23 x loll D (43 x 2.8 x log [12]). Since this represents only 95.5 9/, of the Chironomus DNA, the minimum total amount of DNA in the haploid genome as estimated from renaturation kinetics, i.e., the amount if the slowly renaturing sequences were present only once per haploid genome, is about 1.29 ,: 1011D, or 0.22 pg. According to microspectrometric measurements the hap-

Fig. 2. Autoradiograph of IVth chromosome of C/hnomus fentans hybridized with radioactive high molecular weight RNA synthesizedby salivary glands,The chromosome is unstained and photographed with phase contrast optics. (Left,) focused on chromosome to show position and size of Balbiani rings; (,right,) focused on photographic emulsion. Exposure was for 4 months. Approx. v 1 500.

loid amount of DNA in Chironomus tcwtans is 0.20 to 0.25 pg [13]. We conclude that the 95.5 “/b fraction of slowly renaturing DNA in Chironomus represents single copy sequences. It should be noted (fig. 1a) that the DNA extracted from embryos, containing essentially only diploid nuclei, showed the same renaturation kinetics, and thus the same fraction of unique DNA sequences, as the DNA extracted from larvae, their cell population being a mixture of diploid cells and cells of various degrees of polyteny (cf [6, 71). The initial renaturation reaction of Chironomus DNA is plotted on an extended scale in fig. I c, where corrections are made for the fact that only 4.50~ of the total DNA participates. The experimental points are more scattered than those of the slowly reacting Exptl Cell Res 74 (1972)

590 Preliminary notes

DNA fraction. This may be due to relatively larger experimental error, and/or to heterogeneity in the multiplicity of the sequences involved. For simplicity of calculation we will assume that we are dealing with a homogeneous class of DNA, renaturing according to the line included in fig. 1c. The Cot+ of this fraction would then be approx. 0.1, or 0.017 times the Coti of E. coli DNA. The complexity of DNA in this fraction would be 4.8 X 10’ D (0.017 X 2.8 X log). Toaccount for 4.5 % of the Chironomus genomeeach of these sequenceswould have to be repeated on the average about 120 times. This degree of sequence repetition is similar to that reported for other insect DNAs [5], but it is relatively low, when compared with the reported multiplicity of reiterated sequencesin the DNA of other organisms [8,11]. However, we have also failed to detect DNA duplexes formed at very low C,,t values, and thus representing a DNA fraction with a very high degree of sequence repetition, by:fractionation of 3H-methylated DNA on hydroxyapatite after incubation in 0.12 M PB at 60°C. The specific activity of the glandular RNA used for our in situ hybridization experiments was presumably considerably lower than that of the RNA synthesized under cellfree conditions which was used by other investigators for this type of experiments [7, IO]. When squashes of salivary glands were incubated with glandular RNA for 20 h, we obtained chromosome-associated silver grains in autoradiographs only after exposure for several months. In all our preparations, these silver grains were restricted to Balbiani rings 1 and 2 of the IVth chromosome (fig. 2). Control experiments with labelled E. coli RNA did not show silver grains at any chromosomal regions. This and the fact that the Balbiani ring-associated silver grains were resistant to ribonuclease treatment suggests that they represent specific DNA-RNA hyExptl Cell Res 14 (1972)

brids formed between DNA of the Balbiani ring loci and the heterodisperse, high molecular weight RNA synthesized by the glands (seeref. [IO] for a discussion of in situ hybridization). The restriction of hybridization to the Balbiani rings may be due to the fact that presumably most of the RNA used had been synthesized at these loci 12, 141. If correct, this would imply a relatively high degree of specificity of the reaction. The rate of DNA reassociation increases only slightly when small amounts of DNA in solution are incubated with increasingly large amounts of DNA bound to membrane filters [15]. Since the DNA at a specific chromosoma1 locus can be considered as immobilized DNA at high concentration, the hybridization conditions can be considered analogous, and the reaction rate should depend mostly on the concentration of the reactant in the solution. At the low RNA concentrations we used it is unlikely that hybridization of unique sequenceswould be detectable after 20 h of incubation (cf [16]). Therefore, the Balbiani rings appear to be sites of redundant DNA. The existence of redundant DNA at the Balbiani ring loci could be explained by assuming (i) that the cistrons at these sites are tandemly repeated [2, 31. However, since only 4.5 % of the total DNA is redundant, the Balbiani ring loci would be rather unique with this structure in the Chironomus genome. This implication could be avoided only by speculating, without any experimental basis at present, that the Balbiani ring DNA is amplified in the salivary glands. Alternatively, (ii) the single-copy sequencesof euchromatic DNA may generally be interspersed with short segments of redundant DNA sequences, with RNA being transcribed jointly from adjacent unique and redundant sequences. The alternation of unique and redundant sequences in euchromatic DNA has been inferred from renaturation kinetics

Preliminary

[l l] as well as from cytological hybridization experiments [7]. However, while we presently favor the second alternative, a decision cannot be made on the basis of our present data.

Note added in proof After this manuscrint was submitted a vauer by B Lambert, L Wieslander, B Daneholt, EmEsgyhazi & U Rineborcr fJ cell bio153 (1972) 407) anneared which also reports about in situ‘hybridization experiments in Ch. tentans. The apparently higher hybridization efficiencv achieved by these authors may have been due to their use of incubation media of very high radioactivity and their heat-denaturing of the RNA used to molecules of relatively small size, a step which was omitted by us. The results of these authors are in accord with our conclusions. This work was supported by NSF grant GB-14450 and a predoctoral fellowship from the NIH to R. I. S. We gratefully acknowledge the advice of Dr David Kohne and the technical assistance of Mrs Ingrid Storbeck.

References 1. Beermann, W & Bahr, G, Exptl cell res 6 (1954) 195. 2. Daneholt, B, Edstrom, J-E, Egyhlzi, E, Lambert, B & Ringborg, U, Chromosoma 28 (1969) 418. Daneholt, B, J mol biol 49 (1970) 381. :: Beermann, W, Cell differentiation and morphogenesis. North-Holland, Amsterdam (1966). 5. Laird, C & McCarthy, B, Genetics 63 (1969) 865. 6. Dickson, E, Boyd, J & Laird, C, J mol biol 61 (1971) 615. I. Gall, J, Cohen, E & Polan, M, Chromosoma 33 (1971) 319. 8. Britten, R & Kohne, D, Science 161 (1968) 529. 9. Rubinstein, L & Clever; U, Biochim biophys acta 246 (1971) 517. 10. Pardue, M L, Gerbi, S A, Eckardt, R A & Gall, J G, Chromosoma 29 (1970) 268. 11. Britten, R, RNA in development (ed E W Hanly). Universitv of Utah Press. Salt Lake Citv (1969). 12. Cairns, J,*Cold Spring Harbor symp quant‘biol28 (1963) 43. 13. Daneholt, B & Edstriim, J-E, Cytogenetics 6 (1967) 350. 14. Daneholt, B & Svedhem, L, Exptl cell res 67 (1971) 263. 15. McCarthy, B, Bact rev 31 (1967) 215. 16. Melli, M & Bishop, J 0, J mol biol40 (1969) 117. Received May 3, 1972 Revised version received June 12, 1972

notes

591

Isolation of bovine adrenal cortex mitochondria from the zona glomerulosa T. WAKABAYASHI, Department of Patho&,, Nagoya University School of Medicine, 65, Tsurumncho, Showa-ku, Nagoya, Japan Summary Isolation procedures for the bovine adrenal cortex mitochondria from the zona glomerulosa are described. Special care was taken to avoid contamination of the mitochondria from the zona fasciculata. Bovine adrenal cortex mitochondria in the zona glomerulosa in situ had a tubular or tubulo-vesicular form of cristae when they were examined in the electron microscope, whereas they showed a condensed form when isolated in sucrose:Tris or sucrose-TrisEDTA medium. BY contrast, mitochondria in the zona fasciculata and z&a reticularis, either in situ or isolated in a sucrose-Tris medium showed a vesicular form of cristae, whereas they showed a condensed form when isolated in a sucrose-Tris-EDTA medium. Well-coupled mitochondria were obtained from the zona glomerulosa when they were isolated in a sucroseTris-EDTA medium.

Correlation between adrenal cortex mitochondria and steroidogenesis has been extensively studied morphologically and biochemically. Adrenal cortex mitochondria both in the zona fasciculata and the zona reticularis have a vesicular form of the cristae which is characteristic of mitochondria in the steroid-producing organs. It is also known biochemically that adrenal cortex mitochondria have components for steroid hydroxylating pathways [l, 21. Recently isolated mitochondria from the zona fasciculata (plus the zona reticularis) of the bovine adrenal cortex have been extensively studied by Allmann et al. [3-51. However, there are no available data, up to now, on the isolated mitochondria from the zona glomerulosa although there have been some works on the zone using capsules stripped from bovine adrenals [6-83. Mitochondria in the zona glomerulosa have tubular or tubulo-vesicular cristae, and it is not known how they are correlated to steroidogenesis, especially in relation to the metabolism of aldosterone. For example, it is Exptl Ceil Res 74 (1972)