J. Cell Sci. 6, 615-627 (1970) 615

Printed in Great Britain

DIFFERENTIAL EFFECTS OF K+ AND Na+ ON

SPECIFIC BANDS OF ISOLATED POLYTENE

CHROMOSOMES OF CHIRONOMUS TENTANS

M. LEZZI AND L. I. GILBERTDepartment of Biological Sciences, Northwestern University, Evanston, Illinois 60201,U.S.A.

SUMMARY

Previous investigations on the effects of monovalent cations on the gene activities (formationof puffs) of polytene chromosomes utilized whole salivary glands or nuclei and suggested thatthe K+/Na+ ratio in the nuclear sap was of primary importance in influencing gene activities. Todetermine whether these effects of ions were direct or indirect, we have employed isolatedpolytene chromosomes incubated in salt solutions of varying ionic composition. Our observa-tions reveal differential effects of K+, Na+, Mg2+, Ca2+ and combinations thereof on specificchromosome regions. The band at region I-18-C that is specifically affected by K+ is the sameone that puffs when salivary gland nuclei are incubated in a K+-rich medium and this is believedto be an ecdysone-specific puff. Bands at region I-19-A are specifically affected by Na+and are thesame ones that form puffs when salivary gland nuclei are incubated in a Na+-rich medium. Thisregion is believed to be juvenile hormone-specific. Effects on the chromosome are also describedthat are the result of incubation in solutions containing Mg2+ and Ca2+. Analogies are drawnbetween the observed effects of ions on isolated chromosomes and their effects on chromatinisolated from mammalian cells. For example, when chromatin is exposed to solutions of increasingionic strength, hydration is followed by aggregation and histone dissociation. This may beanalogous to what is observed in the isolated chromosome under similar conditions; namely,expansion, contraction and expansion.

INTRODUCTION

The last several years have brought us a clearer understanding of the chemicalnature of those hormones controlling insect growth and development (see Gilbert,1969). However, the means by which these hormones exert their effects are still notwell understood. For the past decade, the giant polytene chromosomes of Dipterahave been utilized to investigate the mode of action of the insect moulting hormone(see Clever, 1968; Kroeger, 1968) due to the finding that application of moulting hormoneto Chironomus tentans results in the prompt appearance of specific puffs (Clever &Karlson, i960).

By incubating salivary glands of C. thummi in media of different K+/Na+ ratios,Kroeger (1963) could elicit the activation of a graded series of genetic loci. That is,different puffs were induced at specific sites of the polytene chromosomes. On thebasis of this and other findings, Kroeger (1963) postulated that the insect hormones,ecdysone and juvenile hormone, control gene activity by affecting the K+/Na+balance of the nuclear sap. Subsequent work supported this view in that ecdysone and

40 CEL 6

616 M. Lezzi and L. I. Gilbert

juvenile hormone appear to alter the nuclear K+/Na+ ratio (Kroeger, 1966; Baumann,1968) and K+ and Na+ do induce ecdysone- or juvenile hormone-specific puffs inisolated salivary gland nuclei (Lezzi, 1966, 1967c; compare with Lezzi & Gilbert,1969). The present paper examines the question of whether potassium and sodiumions act directly at the chromosome level.

MATERIALS AND METHODS

Isolation of chromosomes

Fourth instar C. tentans larvae (stage II-IV of Kroeger, 1964) were kept at 5 °C for at least2 h before use. Both salivary glands of an animal were excised. One was fixed and stained withorcein acetic acid and a squash was prepared (control I). The other gland was placed in a dropof cold isolation medium (200 mM sucrose, 10 mM MgCl2) 1 mM CaCl2, and 10 mni tris pH 7-6)on a piece of Parafilm that had been previously heat-sealed to a siliconized slide. The gland wasattached to the Parafilm by short fibres of coarse glass wool. A slit was made in the cytoplasm ofone of the salivary gland cells with a tungsten needle to form a passageway to the nucleus. Thenuclear membrane was torn open with a fine Pyrex glass needle. The chromosomes were thentransferred in the same drop of medium with the needle to a prepared hole (1 -2 mm diameter) inthe Parafilm layer. This hole served as an incubation chamber for the chromosomes. The chromo-somes were detached from the glass needle by pulling the needle through a small amount ofpolybutene (Lezzi & Kroeger, 1966). The chromosomes were then positioned on the glass bottomof the incubation chamber. After removing the remainder of the gland (which subsequently wasprepared as a squash to serve as control II), the chromosomes were washed by aspirating off allof the medium except that remaining in the incubation chamber and by applying a drop of saltsolution on top of the chamber. For each change of solution the above process was repeatedtwice. All salt solutions were buffered with 50 mM tris at pH 7-6. Further information on thecomposition of the test solutions is given in the text. All of the above manipulations were con-ducted under a Leitz dissecting microscope ( x 72). The isolation process took less than 5 min.

The isolated chromosomes were observed under the microscope by immersing the x 100 phaseobjective (Zeiss) directly into the isolation medium or salt solution. The appearance of thechromosome was recorded photographically (Wild Photoautomat) with a slightly closed con-denser diaphragm. The settings were constant during a series of observations on a single chro-mosome. Chromosomal diameters were measured only in non-puffed, non-constricted andnon-stretched chromosome regions and the measurements included at least 3 bands. A typicalseries of measurements was accomplished in about 15 min. In the cases of observations of specificbands, the chromosomes were usually fixed, after 10 min incubation, with orcein acetic acid,mounted in lactic acid, and compared with the chromosomes of controls I and II.

Incorporation of UTP-H3

After washing the isolated chromosome twice, 50 /tl of the appropriate salt solution con-taining 2 mM glutathione (for exact composition of solution see legend to Fig. 6) was applied tothe top of the incubation chamber. Then, 2 /tl of an RNA polymerase solution (see below) wereadded as were 2 fil of a solution containing 10 mM of each of the 4 ribonucleoside triphos-phates (UTP labelled with tritium (UTP-H3); specific activity, 1 Ci/mM; Schwarz BioResearchInc., Orangeburg, New York). The RNA polymerase (from Micrococcus lysodeikticus) waspurchased from Miles Laboratories, Elkhart, Indiana. It was concentrated by ammoniumsulphate precipitation and dissolved in storage medium (Bonner et al. 1968).

The chromosomes were then incubated at room temperature for 10 min while viewed underthe microscope. The chromosomes were fixed and stained with cold orcein acetic acid (pH 1 -6)for about 10 s, washed 3 times with ice-cold acetic acid (pH i-6) and rinsed 3 times with cold5 % formaldehyde solution. The latter usually had the same ionic composition as the saltsolution used for incubation except that the tris buffer had been replaced by triethanolaminebuffer (pH 7-6; triethanolamine from Calbiochem, Los Angeles, California). The slides re-

Ionic effects on isolated chromosomes 617

mained in formaldehyde solution for at least 4 h and in water overnight. Autoradiographs wereprepared by covering the slides with Kodak's NTB-3 photoemulsion (1:1 with water). Afteran exposure of approximately 2 months the autoradiographs were developed with Microdol-X(5 min), fixed with Rapid Fixer (3 min) and air-dried.

RESULTS

General effects of K+, Na+, Mg2+ and Ca2+ on chromosome structure

Chromosomes in isolation medium are condensed and their bands exhibit highcontrast. In 150 mM KC1 or NaCl solution the chromosomes are somewhat less con-tracted and the bands reveal less contrast. A decrease in the KC1 or NaCl concentra-tion results in the gradual expansion of the chromosomes (Fig. 1) and the bands become

250 r

5150 i-

100

100 200

Concentration (mM)

300

Fig. 1. Effect of various salt concentrations on the diameter of isolated chromosomes.Chromosome diameter is expressed as percent of diameter in isolation medium. Allpoints are averages of 2-5 measurements except that for 400 mM NaCl plus 12 mMMgCl2 which was a single determination. All solutions were buffered with 50 mM trisat pH 76. • , NaCl solution; O, KC1 solution; A, MgCl2 solution; A, NaCl solutioncontaining 12 mM MgCU; H, NaCl solution containing 12 mM MgCl2 and 19 mMCaCl..

less distinct. However, these changes are reversible. An increase in the KC1 or NaClconcentration above 150 mM also causes the chromosome to expand (to a greater degreewith NaCl; see Fig. 1 and Table 1) and the bands to fade (comparable to bands inFig. 2). If returned from concentrations between 200 and 400 mM to 150 mM, thechromosomes regain their distinct banding. For the most part, however, they have a

40-2

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9'5I I

11-11-5

11-11-5

12-5

I3-I3-51 0

1 0

12-5

13-515-516-5-17-5

% of original

1 0 0

116

116-121

116-121

1 3 1

137-142

105

i ° 5131142

163

174-184

618 M. Lezzi and L. I. Gilbert

smaller diameter than they first had in the 150 mM KCl or NaCl solution. Thisdecrease in diameter is more pronounced with a return to isolation medium. With asecond increase in KCl concentration the ultimate swelling of the chromosomes isgreater than with the first increase (Table 1).

Table 1. Effect of monovalent cations on the diameter of an isolated chromosome

Chromosome diameter

Incubation medium

A. Isolation Medium

B. 150 mM KCl250 mM KCl300 mM KCl350 mM KCl400 mM KCl

C. 150 mM KCl250 mM KCl300 mM KCl350 mM KCl400 mM KCl

D. 400 mM NaCl

A, 200 mM sucrose, 10 mM MgCL, 1 mM CaCL, 10 mM tris; pH 7-6.B, First treatment in increasing concentrations of KCl.C, Second treatment in increasing concentrations of KCl.D, First treatment in high concentration of NaCl.All salt solutions buffered with 50 mM tris, pH 7-6. The diameter was measured with an

ocular micrometer ( x 25). The original diameter is that measured in the isolation medium.

Once a concentration of 600 mM is reached, the bands remain faded and indistinctwhen returned to 150 mM KCl or NaCl although there is some slight chromosomalcontraction. It is only on being returned to the isolation medium or to 12 mM MgCl2solution that the banding reappears in these chromosomes and the chromosome dia-meter is reduced to 70% of the original. Staining with fast green at pH 8-2 (Alfert &Geschwind, 1953) reveals that chromosomes treated with 600 mM KCl or NaCl stillcontain histone.

When MgCl2 is used instead of KCl or NaCl, the same 3 phases of chromosomalexpansion, contraction and expansion are observed in solutions of increasing ionicstrength. However, condensation of the chromosomes is greater in Mg2+ and it occursat much lower concentrations than with K+ or Na+ (Fig. 1). A return of the chromo-somes from concentrations above 150 mM to 10 mM MgCl2 results in a reduction indiameter similar to that described for KCl and NaCl. CaCl2 yields essentially the samepattern as MgCl2 except that it causes less expansion than MgCl2 at concentrationsabove 100 mM.

Since Mg2+ and Ca2+ appear to be 4-10 times more effective than K+ or Na+ incausing chromosomal expansion, the addition of 12 mM MgCl2 to K

+- or Na+- contain-ing solutions should enhance the effect of the monovalent cations. This does occur

Ionic effects on isolated chromosomes 619

at KCl or NaCl concentrations above 300 mM or at concentrations above 350 mM withthe addition of 12 mM MgCl2 plus 1-9 mM CaCl2 (Fig. 1). These treated chromosomesalso show a reduction in diameter when returned to a K+- and Na+-free MgCl2solution. However, at KCl or NaCl concentrations below 300 or 350 mM, the presenceof 12 mM MgCl2 (with or without 1-9 mM CaCl2) appears to inhibit partially thechromosome expansion usually seen in KCl or NaCl solutions of above 150 mM(Fig. 1). In general, these chromosomes are more contracted and the bands moredense in such salt solutions than in simple KCl or NaCl solutions of the same ionicstrength (Fig. 1).

It should be stressed, however, that not all the bands remain condensed under theseconditions, but that there are single bands which fade and lose their compactness bothin the presence of Mg2+ (and Ca2+) and at a K+ or Na+ concentration as low as 150 mM(Figs. 4, 5). This fading of single bands can be detected especially well in stretchedchromosomes (Figs. 3, 5). By increasing the KCl or NaCl concentration, the numberof such fading bands increases (Fig. 3). This differential fading can also be observedwith simple KCl or NaCl solutions if the chromosomes have been exposed previouslyto a Mg2+- and Ca2+-containing medium (Fig. 3). This phenomenon is in contrast tothe more or less gradual fading of all the bands which occurs in chromosomes incubatedin KCl or NaCl solutions lacking divalent cations (i.e. solutions supplemented witho-i ITIM EDTA; Fig. 2).

Differential effects of K+- or Na+-containing salt solutions on chromosome regionsI-18-C and I-ig-A

Among the bands that fade in chromosomes exposed to KCl or NaCl solutionscontaining 12 mM MgCl2 (with or without 1-9 mM CaCl2) are a band at region

Table 2. Differential effects of monovalent cations on specific chromosomal regions

Cation

K+Na+K+

Na+

Chromosomeregion

I-18-CI-18-CI-19-AI-19-A

Number

Fading

13(2)

0

0(1)

6(4)

of bands

Unchanged

12(2)

30

Number ofpre-existing

puffs unchanged

35 (1)

9(0This table summarizes our total results. Salt solutions contained 150-200 mM KCl (or NaCl),

12 mM MgCl2 and 50 mM tris (pH y6). In most experiments the solutions also contained 2 mMglutathione and i-g mM CaCl2 while in some studies RNA polymerase and the 4 ribonucleosidetriphosphates were added. These added substances did not influence the differential effects ofK+ and Na+ (see text). Numbers in parentheses indicate number of equivocal results.

I-18-C and one or more bands at region I-19-A. In both regions the fading bands arepotential puff-forming sites. The reaction of the bands in these regions to K+ orNa+ is specific in that the band at I-18-C fades with salt solutions containing 150-200 mM KCl (Fig. 4) but not with solutions containing NaCl, whereas the converse

620 M. Lezzi and L. I. Gilbert

is true for the bands at I-19-A (Fig. 5 and Table 2). This is not true of all bands react-ing to K+- or Na+-containing solutions as several bands fade in solutions containingeither K+ or Na+ (e.g. the fading band in the lower part of Fig. 4).

Table 2 summarizes our observations of regions I-18-C and I-19-A on a total of39 chromosomes (19 utilized in experiments with K+; 20 utilized in experimentswith Na+). From this study of 78 chromosomal regions, the behaviour of 67 could beclearly described while the results obtained from the remaining 11 regions wereequivocal. Most of the chromosomes used were actually puffed at I-18-C or I-19-A orat both regions (2 cases). Only four chromosomes were not puffed in either region(one shown in Fig. 5). A puff present at one of the regions under study does notinterfere with the ability of the other region to respond specifically to one monovalentcation. A pre-existing puff does not appear to be drastically altered when exposed toeither KC1- or NaCl-containing salt solutions (Fig. 4).

Although the chromosomes seen in Figs. 4B and 5B were incubated in solutionssupplemented with RNA polymerase and the four ribonucleoside triphosphates, itshould be noted that none of these substances is essential for the difference in responseof the particular bands to K+ or Na+ although they as well as glutathione may act assynergists. That is, differential fading is initiated immediately after the addition ofsimple salt solutions of the proper composition. The presence of 1-9 inn CaCl2 in thesalt solution and in the K+- and Na+-free solutions used in some experiments forwashing the chromosomes after isolation seems to improve the specificity of fadingsince 8 of the 11 equivocal results (Table 2) were obtained with solutions lacking Ca2+.

After staining with orcein acetic acid, the bands which had faded during exposure to150-200 mM KC1- or NaCl-containing salt solutions appeared virtually identical tonormal puffs (compare Fig. 4c with fig. 3 A in Lezzi & Gilbert, 1969). However, incontrast to normal puffs, these expanded bands may not contain basic puff protein(see Lezzi (19676) for discussion of basic puff protein). Whether they contain theirfull complement of histone fractions could not be determined since Bloch's (1966)cytochemical method for histone differentiation yielded equivocal results although thechromosomes were fixed directly with neutral and isotonic formaldehyde solution.

Incorporation of UTP-H3 in chromosomes incubated in salt solutions of different ionicstrength

The following preliminary results were obtained from experiments originallyplanned to determine whether the single bands fading under the conditions describedabove are able to incorporate UTP-H3. Our results revealed that much higher resolu-tion autoradiographic techniques must be utilized to answer this question definitively.However, chromosomes, where most of the bands had expanded and faded duringincubation, showed homogeneous incorporation of UTP-H3 either over the entire lengthof the chromosome or over extended regions (Fig. 6). This observation is in contrast tothose made of chromosomes that maintained their 'normal' appearance duringincubation (i.e. chromosomes with primarily condensed bands) where incorporationof UTP-H3 occurred predominantly in the nucleoli, Balbiani rings and very largepuffs (Fig. 6). The latter incorporation pattern is also typical of totally expanded

Ionic effects on isolated chromosomes 621

chromosomes (and contracted chromosomes; Lezzi, 1967a) if, in contrast to the aboveexperiments, RNA polymerase is omitted from the incubation mixture.

The exact ionic strength of the final incubation mixture is not known with certaintysince evaporation occurs during the period of incubation. However, it is clear that inthose experiments where expansion and fading were recorded during incubation, thechanges were caused by high rather than by very low salt concentrations. The pre-sence of 12 mM MgCl2 in all of the salt solutions used for the incorporation experimentsis enough to ensure condensation of the chromosomes (Fig. 1). In two experiments inwhich fading and homogeneous incorporation occurred, the salt solution contained300 mM KC1 while in the other 5 cases it contained 200 mM or 150 mM KC1 or NaCl.In the experiments in which no expansion and no homogeneous incorporation wereobserved, the salt solution contained no KC1 or NaCl (2 cases), 125 mM NaCl (2 cases)or 150 mM KC1 or NaCl (12 cases). The overall degree of labelling varied in replicateexperiments and in 2 experiments there did not appear to be any incorporation of label.

DISCUSSION

The differential effects of K+, Na+, Mg2+, Ca2+ and several combinations of theseions were studied utilizing the isolated salivary gland chromosomes of Chironomustentans. The fact that the chromosomes were isolated by hand imposed limitations onthe numbers examined but the method has the virtue of yielding essentially 'naked'and normally appearing chromosomes. The finding that K+-containing salt solutionsand Na+-containing salt solutions differ in their effects on the structure of specificbands in these isolated chromosomes is of particular interest. The band at I-18-C thatis specifically affected by K+ is a band that forms a puff if isolated salivary gland nucleiare incubated in a K+-rich medium (Lezzi, 1966). The same puff is induced by treatinglarvae with the insect moulting hormone, ecdysone (Clever & Karlson, i960; Lezzi &Gilbert, 1969). Ecdysone in turn appears to increase the nuclear K+/Na+ ratio in thesalivary glands of Chironomus thummi (Kroeger, 1966). The bands at I-19-A whichspecifically fade in a Na+-containing salt mixture form puffs both in isolated salivarygland nuclei that are incubated in a Na+-rich medium (Lezzi, 1966) and when pre-pupae are treated with juvenile hormone (Lezzi & Gilbert, 1969). In contradistinc-tion to ecdysone, the juvenile hormone appears to decrease the nuclear K+/Na+ ratioin salivary glands (Galleria mellonella; Baumann, 1968). This may be a result of thejuvenile hormone interacting directly with the lipids of the membrane (Baumann, 1969).

The question of how change in the monovalent cation concentration of the im-mediate environment elicits puffing is not known. How K+ and Na+ induce fading ofspecific bands in the present experiments is also a matter for conjecture. However, wecan draw some analogies between our data and those available from experimentsutilizing isolated chromatin (for a detailed discussion see Lezzi, 1969 a). First,chromatin undergoes 3 alterations of state when exposed to solutions of increasingionic strength. These phases are hydration, aggregation, and histone dissociation andmay be analogous to the changes we observed in the isolated chromosomes; namely,expansion, contraction and expansion of the whole chromosome, or fading, condensa-

622 M. Lezzi and L. I. Gilbert

tion and fading of the individual band. (Compare with Gruzdev & Belaja's (1968)conclusion based on the results of similar experiments with the isolated salivary glandchromosomes of Chironomus dorsalis.) Secondly, treatment of chromatin with600 mM NaCl removes all of histone I and renders the chromatin soluble in saltsolutions in which untreated chromatin would aggregate (for example 150 mM NaCl).Polytene chromosomes treated with 600 mM KC1 or NaCl remain faded when incu-bated in 150 mM salt solutions whereas untreated chromosomes show distinct bandswith the latter solution. Thirdly, the dissociation of histone enhances the templateactivity of chromatin (measured as ribonucleotide incorporation into RNA in thepresence of excess RNA polymerase). Our data suggest that in isolated chromosomeswhere the bands expanded and faded as a result of incubation in solutions of highionic strength, template capability no longer appears restricted to the original activitysites. Finally, the presence of 12 mM MgCl2 and 1-9 mM CaCl2 is required for thedifferential effect of K+ or Na+ (both at 200 mM) on chromatin template activitywhich in turn is probably the direct result of the selective removal of histone I(Lezzi, 19696). The differential effects of K+ and Na+ (both at 150-200 mM) on thefading of specific bands in isolated polytene chromosomes also requires Mg2+ (12 ITIM)and possibly Ca2+.

This work, although to some extent preliminary, reveals the advantage of utilizingisolated chromosomes for studying gene activation. In this system, there is no questionof indirect action of added components via the cytoplasm, plasma membrane, nucleo-plasm or nuclear envelope. If, in contrast to assumptions made in connexion with thepresented studies, insect hormones do directly elicit chromosome puffs or Balbianiring enlargement, one should expect to see this gene activation as morphological changein the isolated chromosome. Such investigations are currently being conducted in thislaboratory.

This work was supported by grant AM-02818 to L. Gilbert and AI-06672 to H. Noll, bothfrom the National Institutes of Health. Dr. Lezzi's present address is: Zoologisches Institut derEidgenossischen Technischen Hochschule, Zurich, Switzerland.

REFERENCESALFERT, M. & GESCHWIND, I. I. (1953). A selective staining method for the basic proteins of

cell nuclei. Proc. natn. Acad. Sci. U.S.A. 39, 991-999.BAUMANN, G. (1968). Zur Wirkung des Juvenilhormones: Elektrophysiologische Messungen an

der Zellmembran der Speicheldriise von Galleria mellonella.J. Insect Physiol. 14, 1459-1476.BAUMANN, G. (1969). Juvenile hormone: Effect on bimolecular lipid membranes. Nature, Lond.

233, 316-317-BLOCK, D. P. (1966). Histone differentiation and nuclear activity. Ctiromosoma 19, 317-339.BONNER, J., CHALKLEY, G. R., DAHMUS, M., FAMBROUGH, D., FUJIMURA, F., HUANG, R. C. C,

HUBERMAN, J., JENSEN, R., MARUSHIGE, K., OHLENBUSCH, H., OLIVERA, B. & WIDHOLM, J.(1968). In Methods in Enzymology (Nucleic Acids), vol. 12 (ed. L. Grossman & K. Moldave),pp. 3-84. New York: Academic Press.

CLEVER, U. (1968). Regulation of chromosome function. A. Rev. Genet. 2, n-30.CLEVER, U. & KARLSON, P. (i960). Induktion von Puff-Veranderungen in den Speicheldriisen-

Chromosomen von Chironomus tentans. Expl Cell Res. 20, 623-626.GILBERT, L. I. (1969). The chemistry of insect hormones. Proc. Illrd int. Congr. Endocrin.,

Mexico City, pp. 304-310. Amsterdam: Excerpta Medica Foundation.

Ionic effects on isolated chromosomes 623

GRUZDEV, A. D. & BELAJA, A. N. (1968). The influence of the pH, tonicity and ionic strength ofsolutions on the size of polytene chromosomes (in Russian). Dokl. Akad. Nauk USSR(Cytologia), 10, 297-305.

KROEGER, H. (1963). Chemical nature of the system controlling gene activities in insect cells.Nature, Lond. 200, 1234-1235.

KROEGER, H. (1964). Zellphysiologische Mechanismen bei der Regulation von Genaktivitatenin den Riesenchromosomen von Chironomus thummi. Chromosoma 15, 36-70.

KROEGER, H. (1966). Potentialdifferenz und Puff-Muster. Elektrophysiologische und cytolo-gische Untersuchungen an den Speicheldriisen von Chironomus thummi. Expl Cell Res. 41,64-80.

KROEGER, H. (1968). Gene activities during insect metamorphosis and their control by hor-mones. In Metamorphosis: A Problem in Developmental Biology (ed. W. Etkin & L. I. Gilbert),pp. 185-219. New York; Appleton-Century-Crofts.

LEZZI, M. (1966). Induktion eines Ecdyson-aktivierbaren Puff in isolierten Zellkernen vonChironomus durch KC1. Expl Cell Res. 43, 571-577.

LEZZI, M. (1967a). RNS- und Protein-Synthese in Puffs isolierter Speicheldrusen-Chromo-somen von Chironomus. Chromosoma 21, 72—88.

LEZZI, M. (19676). Cytochemische Untersuchungen an Puffs isolierter Speicheldriisen-Chromosomen von Chironomus. Chromosoma 21, 89-108.

LEZZI, M. (1967c). Spezifische Aktivitatssteigerung eines Balbianiringes durch Mg2+ in iso-lierten Zellkernen von Chironomus. Chromosoma 21, 109-122.

LEZZI, M. (1969a). Differential gene activation in isolated chromosomes. Int. Rev. Cytol. (inPress).

LEZZI, M. (19696). Differential effects of sodium and potassium ions on the template activity ofrat liver chromatin. Physiol. Chem. Physics 1, 447-461.

LEZZI, M. & GILBERT, L. I. (1969). Control of gene activities in the polytene chromosomes ofChironomus tentans by ecdysone and juvenile hormone. Proc. natn. Acad. Sci. U.S.A. 64,498-503.

LEZZI, M. & KROEGER, H. (1966). Aufnahme von 22Na in die Zellkerne der Speicheldriisen vonChironomus thummi. Z. Naturf. 2,1b, 274-277.

(Received 13 October 1969)

624 M. Lezzi and L. I. Gilbert

Fig. 2. Effects of NaCl solution on the appearance of bands in an isolated chromosome.A, Isolation medium; B, 200 DIM NaCl plus o-i mM EDTA. This is a stretched regionof the chromosome which stretches even further during treatment in B. Verticalbar = 5 /im.Fig. 3. Effects of Mg2+ on the appearance of bands exposed to increasing concentra-tions of NaCl. A, Isolation medium (this is the same appearance as seen in 50-250 mMNaCl plus 12 mM MgCl2); B, 300 mM NaCl plus 12 mM MgCl2 (this is the sameappearance as seen in 300 mM NaCl); C, 400 mM NaCl. This stretched region of thechromosome had been exposed to the Mg2+- and Ca2+-containing isolation mediumfor 1 h before treatment with the NaCl solution. Vertical bar = 5 /tm.

Fig. 4. Effects of KC1 solution on the appearance of specific bands in an isolatedchromosome, A, Isolation medium; B, 150 mM KC1 containing 12 mM MgCl2 and2 mM glutathione; RNA polymerase and the 4 ribonucleoside triphosphates were alsoadded but differential fading was evident prior to these additions, c, Same chromo-some as in A and B but fixed and stained with orcein acetic acid. D, Homologouschromosome region from control I; fixed and stained. The fading band at regionI-18-C noted by dark line in upper portion of picture. Open circles delineate the pre-existing puffs at region I-19-A. Vertical bar = 5 /tm.

Ionic effects on isolated chromosomes

626 M. Lezzi and L. I. Gilbert

Fig. 5. Effects of NaCl solution on the appearance of specific bands in an isolatedchromosome, A, Isolation medium; B, 150 mM NaCl containing 12 mM MgCl2, i'9 HIMCaCl2 and 2 mM glutathione; RNA polymerase and the 4 ribonucleoside triphosphateswere also added but differential fading was evident prior to these additions. Note thatchromosome became stretched during treatment B. The fading bands of regionI-i9-A(/o) are noted by dark lines. Closed circle indicates the non-responding band atregion I-18-C (18). Vertical bar = 5 /tm.

Fig. 6. Autoradiographs of UTP-H3 incorporation into isolated chromosomes, A,Incubated in 150 mM NaCl (solution also contained 12 mM MgCl2, 1-9 mM CaCl2)2 mM glutathione, extra RNA polymerase, 4 ribonucleoside triphosphates with UTPlabelled). Note concentration of label at Balbiani rings (/, 2, 3) and nucleoli (no), B,Incubated in 300 mM KC1 (other constituents as in A). Note almost uniform labellingof the chromosome. Horizontal bar = 20 //rn.

Ionic effects on isolated chromosomes

6 A ••