Hormonal control of gene activity in polytene chromosomes

GENERAL

AND

COMPARATIVE

Hormonal

ENDOCRINOLOGY

Control

3, 159-167

SUPPLEMENT

of Gene

Activity

MARKUS Laboratoq

.for Developmental 8006

(1972)

in Polytene

Chromosomes”

LEZZI

Biology, Zwicla,

Swiss Federal Switzerland

Institute

oj

TechlLulog~q,

AND

Department

LAWRENCE of Biological Evanston,

I. GILBERT Xciences, Illinois

Northwestern 60201

University,

The most recent published work on the roles of ions and hormones on gene activation in Chironomus polytene chromosomes is discussed along v&h a series of as-yet-unpublished experiments. Studies utilizing ecdysone and juvenile hormone &a viva reveal the existence of specific chromosomal regions that. are sensitive to one or the other hormone. Similar effects can be elicited by changing the ionic milieu of the chromosomes including isolated chromosomes. The hormones do not appear to induce specific puffing patterns in isolated chromosomes; however, alternate possibilities are suggested for this lack of effect. Studies are reported on the normal sequence of puffing at the hormone-sensitive regions of the polytene chromosomes of both the salivary glands and the Malpighian tubules during development. The advantage of the latter structures is that, unlike the salivary glands, they remain intact during pupal life. Correlations are also drawn between the activity of an ecdysone-sensitive region and the appearance of an electrophoretically identifiable protein in the secretion of the salivary glands. These new data are consistent with our belief that insect hormones act indirectly to elicit gene activity by first causing alterations in the ionic constitution of the chromosomal milieu.

This presentation is in essence a progress report of studies in the field of hormonal control of insect gene activity utilizing as a baseline the recent review by one of us (Lezzi, 1970) ~ The reader is referred to this latter review for an in-depth treatment of the field. Since the problem is being investigated at several levels, we plan to discuss experiments in three research areas. In the area of molecular biology, one can ask which factors at the chromosomal level control gene activity? Does a relationship exist between these factors (ions) and the insect growth hormones (ecdysone and juveniie hormone)? At the level of endocrinology, we shall discuss the parameters ‘A portion gr:mL ,3&I-02818 Wealth.

of

this work was from the National

supported Institutes

involved in gene activation by juvenile hormone in z&o. In the field of celi physiology we intend to explore an example of cell function that appears to be controlled by genes previously activated by hormones, IONIC

[This section is based on data from Lezei and Gilbert (1970), Lezzi and Robert (1971)) and Robert (1971) .] There are two primary hypotheses concerning the means by which ecdysone administration results in gene activation The first is based on Karlson’s (1963) hypothesis that ecdysone acts directly at the level of the gene, and the second is that ecdysone elicits gene activation by indirect. means, perhaps by influencing the intranmclenr

by of 159

@ 1972 by

Academic

Press,

Inc.

INDUCTION OF DIFFERENTIAL GEXE ACTIVITY IN ISOIATED CHRQMOSONES

160

LEZZI

AND

ionic milieu (Kroeger, 1963). In the latter instance, changes in the ionic environment, would actually be the determining factors controlling gene activity. Both hypotheses are based on data derived from experiments concerning puffing at homologous regions of the salivary gland chromosomes of Chironomus (Keyl, 1968). These are regions I-18-C to I-19-B in Chironomus tentans and IIIdl to IIIdl.2 in Chironomus thummi. The most direct way to decide between the two hypotheses was to utilize isolated chromosomes, subject them to both varying concentrations of ecdysone and ions, and analyze their response in terms of puffing activity at specific loci. To obtain isolated polytene chromosomes, one can utilize microdissection of individual chromosomes from the salivary gland or isolate larger quantities by rupturing the envelopes of previously isolated nuclei. Both procedures are effective in yielding clean, intact chromosomes that respond essentially the same to changes in the ionic constitution of the incubation medium. The application of ecdysone or juvenile hormone to these isolated chromosomes does not influence the puffing pattern. However, it should be pointed out that these hormones may have to be bound or altered in some way before eliciting a responre. For example, we know that ecdysone-binding proteins exist in the salivary glands of Drosophila (Emmerich, 1970) and that specific proteins exist in the crustacean hepatopancreas that bind a yet unidentified metabolite of ecdysone (Gilbert et al. 1970) which may in fact be the active form of the hormone. Recently, juvenile hormone has been shown to be transported in the hemolymph of saturniids as a moiety of a specific lipoprotein (Whitmore and Gilbert, 1971) and may influence the insect’s internal structures only in this form. Notwithstanding these speculations, we know that inorganic ions do elicit large puff-like structures in isolated chromosomes within seconds of application. Theze ionitally induced puffed regions are sites at which DNA is preferentially transcribed when exogenous RNA polymerase and the four ribonucleoside triphosphates are pro-

GILBERT

vided. The activity pattern obtained under this regimen is not solely the result of amplification of a preexisting puffing pattern since the activity pattern also encompasses formerly inactive regions. The elicited pattern is highly reproducible and can be varied by altering the concentration of K+, Na+, and Mg”+, or Ca2+ in the incubation medium. Thus far, 8 chromosome regions have been identified that react differentially to different combinations of these 4 ions. Of particular interest are regions IIIdl (equivalent to I-18-C) which is activated in the presence of K+ rather than Na+, and IIIdl.l-1.2 (equivalent to I-19-AB1) which reacts to Na+ rather than K+. This strongly suggests that ions can activate genes in isolated chromosomes and that they do so in both a specific and differential manner without exogenous hormones or macromolecules. One may question whether the conditions utilized in the above experiments are physiological. The induction of the very large puff-like structures does require a low pH (4.3) and relatively high ionic strength (e.g., 550-650 miV Na’). However, similar results have been obtained using salt solutions at neutral pH and an ionic strength (e.g., 150-200 mM) that corresponds to the monovalent cation concentration of the nucleus (H. Kroeger, personal communication). Under these latter conditions it is again region I-18-C (equivalent to IIIdl) that responds to K+ rather than Na+ (150 d plus Mg’+ and Ca2+) and region I-19-AB (equivalent to IIIdl.l-1.2) that is affected by Na+ rather than by K+. The chromosome changes elicited by these ‘(more physiological” conditions are admittedly less striking than those obtained under conditions of low pH and high ionic strength, but are nevertheless induced in a differential manner even under these more moderate conditions. Theoretical considerations have led Robert (cf. Lezzi and Robert, 1971) to the conclusion that even minute variations in the relative concentra‘Region I-19-A contains 2 puffs. Since Clever (1962) designated these as 1-19-A, and I-19-B, we will term this region I-19-AB for simplicity sake.

HORMOKES

AND

GEWE

TABLE A

COMPARISON

OF THE

Chromosome 8. tentans

SPECIFICITY

161.

1

OF HORMONES

AND

IONS

IIIdl

Hormone

sensitivity

ecdysone ecdysone ecdysone juvenile juvenile

IIIdl.l-1.2 IIIal

BRb

hormone hormone ‘)

n Experimental material was the polytene chromosomes of the salivary Chironomus thum&. Homologous regions according to Key1 (1968). Tjata and Frigg (1967).

tions of K-, iYa+, Mg?+, and CaZ+ can suffice in eliciting highly specific alterations in the gene activity pattern. Whether these smail variations do indeed occur in the nucleus of the living cell is a matter of conjecture, but is presently being investigatecl in several laboratories (e.g., Lezzi and Kroeger) . Preliminary electrophysiological measurements do suggest however that ecdysone increases the nuclear K+/Na’ ratio (Kroeger, 1966) and that juvenile hormone decreases this ratio (Baumann! 1968). COMPdRISON

OF EFFECTS

IONIC ON

ON

PUFFING

ACTLVITYU

region C. thummi

1-18-C IV-2-B BKl J-l Y-AB

ACTIVITY

AND PUFFING

in vivo

Ion

sensitivit,y

in vitro

K+ Ki+ K’ Na” Na+ Mg2’ glands of Chironomus tentans and are from Lexzi (1967), and Lezei

and the effects of juvenile hormone ar,d Na+ on the other (Table 1). In an attempt to further clarify the respective roles of ions and hormones in eliciting puffs from the polytene chromosomes of Chironomus, the following requirements were established for further experimentation:

HORMONAL

It’ has been argued that gene activation by ions does not exhibit the same specificity as gene activation induced by hormones (Clever, 1968; Pelling, 1969; Swift, 1969). This criticism is based primarily on the data from in viva experiments with ecdysane (Clever, 1961), and juvenile hormone (Laufer and HoIt, 1970)) and the in. Z&TO experiments with ions (Lezzi, 1967). When some of the in viva hormone experiments were repeated (Lezzi and Gilbert, 1969; Lezzi and Frigg, 1971) and the results compared with puffing events during normal development, it was shown that the aforementioned criticism of the work with ions was based on what we consider a misinterpretation of the dat,a. Further analysis revealed a good correlation between the effects of ecdysone and K* on the one hand,

FIG. 1. Induction of a pilff at’ 1-19-A of Xalpighian tubule polyt,ene chromosomes from 6. tentalzs pupae by juvenile hormone. (a) control; received externally 0.5 ~1 DYISO and was killed 1.5 hr later. (b) experimental; received externally 3 pg of a mixture of juvenile hormone isomers dissolved in 0.5 ~1 DXISQ and was killed 1 hr later. (CJ experimental; received externally 3 pg of dichloromethyl farnesoat,e (juvenile hormone analog) dissolved in 0.5 pl DMSO and killed 2 hr later. r is unpuffed reference band used as standard for determinat,ion of the relative diameter of region I-19-A& 19 is puff I-19-A (outlined by horizontal lines); I8 is region I-18-C (arrows) in a puffed (a, b) or B&like stat,e, and vertical bar is 5 p. From C. Holdereggel (unpublished results).

162

LEZZI

AND

GILBERT

1. Study a chromosome region which is rapidly activated by ecdysone applied in viva. Region I-18-C meets this requirement (Clever and Karlson, 1960; Lezzi and Gilbert, 1969). 2. Study a chromosome region which is rapidly activated by juvenile hormone in vivo. Region I-19-AB meets this requirement (Lezzi and Gilbert, 1969; see also subsequent section and Figs. 1 and 2). 3. The chromosome regions in (1) and (2) above cannot be identical or even homologous. If they were, they could not be considered as specific for either hormone. 4. Insure that the chromosome regions of (1) and (2) above exhibit activity during normal development that reflects a reasonable approximation of the endogenous ecdysone and juvenile hormone titers. [Figs. 3 and 4 reveal that this is the case for regions I-18-C and I-19-AB. Compare with Shaaya and Karlson (1965), and Williams (1961) .] 5. If the requirements of (l)-(4) above are met, as they are for regions I-18-C and I-19-AB, then these two regions should be used as standards in studying the normal f 100

\

: : : I ( L

1

I 1

o

I 2

I

4h

2. Time and juvenile hormone dose depenof puff induction at region I-19-AB of Malpighian tubule polytene chromosomes from C. tentans pupae. Abscissa: time in hours after juvenile hormone administration. Ordinate: relative diameter of region I-IO-AB (see r in legend to Fig. 1) C is control animals receiving 0.5 ~1 DMSO. Indicated amomlts of juvenile hormone (mixture of isomers) dissolved in 0.5 ~1 DMSO were applied externally. 130 experimental and 130 control pupae were utilized in this graph. From C. Holderegger (unpublished results). FIG.

d ence

puffing behavior of other regions or the response of other regions to exogenous ecdysone and juvenile hormone. Our data have demonstrated that region I-19-AB is activated by juvenile hormone

\

\

\ \

’

I

I

I I /’ I I I I

\

\

Sal o-

\

‘\ \ \ \ \

I’ 50-

, 5’

‘\

I’

i

119AB

I-18-C

I-\ \ : I

d

: I

\--I

L M

Mpg

I

\ \

I

I , I PP

A

P M

M

FIG. 3. Puffing activity at region I-18-C of polytene chromosomes from C. tentuns during normal development. Abscissa: developmental stages. L = larva, PP = prepupa, P = pupa, A = adult, M = molt. Ordinate: f = frequency of puff development at region I-18-C of the salivary gland chromosomes. s = average size of the puff at region I-18-C of the Malpighian tubule polytene chromosomes as determined by morphological staging (1 = small puff; 2 = large puff or small BR). Sal denotes behavior in salivary glands as determined by Clever (1962, Figs. 4,8; 1963, Fig. 2). Unmarked vertical lines below abscissa indicate substages according to Clever. Mpg denotes behavior in Malpighian tubules as determined by C. Holderegger (unpublished results).

HORMONES

AND

GENE

163

ACTIVITY

I-19-AB

M

FIG.

4. Puffing activity at region I-IS-AB of polytene chromosomes of C. tentans during normai developd = relative diameter of region I-19-AB in the Malpighian tubule polytene chromosomes. f = frequency of puff development at 1-19-A and I-19-B of the salivary gland chromosomes. See legends to Figs. 2 and 3 for further explanation of values and abbreviations. A = Clever’s (1962, 1963) data for puff I-19-A, a:-id B = Clever’s (1962, 1963) data for puff 1-19-B of the salivary gland (Sal) chromosomes. AZ3 = Holderegger’s unpublished data for region I-IO-AB (puff 1-19-A and puff 1-19-B) of the Malpighian t,Libule (Mpg) chromosomes.

ment.

bllt not by ecdysone (Lezzi and Gilbert, l! 69). This is in contrast to Clever’s (1961) bl lief that, purr’ I-19-A is influenced by ec dysone because it reacts to prolonged ec zlysone treatment and is apparently enla :ged in the prepupa. However, it should

CORREL.~TIOM ECDYSONE

OF ACTIVITY AT REGIONS SD JUVENILE HORMONE Hormone

Chromosome region BRl

Ecdysone

sensitive Juvenile

hormone

I-19-AB IIIdl.l-1.2 IIIdl

BKb

IIIdl.l-1.2 IIIdl

TABLE 2 BRl, IIIal AND BRb WITH THE ACTIVITI’ OF ST~NDMSD SENSITIVE REGIONS DUR~KG NORMAL DFIVELOPMXWQ

region

1-18-C W-2-B

IIIal

be noted that the puff is drastically reduced in older prepupae (Clever, 1962). Table 2 shows clearly that, Balbiani ring 1 (BR1) is activated by ecdysone and its activi@ is positively correlated with two known ecdysone sensitive regions, I-18-C and IV-

Regression efficient +0.75* +0.50* -0.06 +0.70* -0.20 -0.60** +0.70*

co(b)

Number of animals 31 40 31 42 42 27 37

Response to hormone treatment in 26~0 ecdysone juvenile

(+ 34) hormone

( -- 36 j

juvenile

hormone

( + 27 j

juvenile

hormone

( - 6)

a Experimental material was the polytene chromosomes of the salivary glands of C. telztans (I-18-C: Ww 2-B, BRl, I-19-AB), and C. thummi (IIIal, IIIdl, IIIdl.l-1.2, BRb). The numbers in parentheses in the column denoting the response of a particular region to ecdysone or juvenile hormone signify the frequency difference (activity of experimental minus activity of control) and apply to the chromosome region in the first column. “Pb = 0<<0.001. +* Pb = 0 < 0.005. Data are from Lezzi and Gilbert (1969); Lezzi and Frigg (1971); Lezzi (unpublished results).

164

LEZZI

AND

FIG. 5. Effect of external application of juvenile hormone on the incorporation of 3H-uridine into puff IIIal of the salivary gland polytene chromosomes of’ prepupal C. thummi. (a) control; received 0.5 pl a.cetone:ol?ve oil (50: 1) and was killed 4 hr later. (b) experimental; received 3 pg juvenile hormone (mixture of isomers) dissolved in 0.5 ~1 acetone: olive oil (50: 1) and was killed 4 hr later. From Lezzi and Frigg (unpublished result)s).

2-B. In addition, Table 2 reveals that region IIIal is activated by juvenile hormone (see also Fig. 5) and that BRb cannot be considered to be a juvenile hormone-sensitive region. The results are in contrast to the findings of Laufer and Holt (1970)) and support the conclusions of Leexi and Gilbert (1969). EFFECTS OF JUVENILE HORMONE PUFFING OF PUPAL MAr,PIGHIAN TUBULE CHROMOSOMES

ON

[This section is based on the unpublished data of C. Holderegger.] As has been demonstrated for several other chromosome regions (e.g., Berendes, 1966), region I-19-AB exhibit,s the same general temporal activity in the polytene chromosomes of the Malpighian tubules as it does in the polytene chromosomes of the salivary glands (Fig. 4). Unfortunately, it is only during the end of the last larval instar that the polytene chromosomes of both structures can be compared since the salivary glands break down shortly after pupation. It is of interest that region I-19-AB is activated in the polytene chromosomes of both salivary glands and Malpighian tu-

GILBERT

bules after exogenous juvenile hormone treatment (Figs. 1 and 2; compare with Fig. 3, and Table 3 of Lezzi and Gilbert, 1969). The Malpighian tubule chromosomes seemed to be a more suitable system than the salivary gland chromosomes to analyze the effects of juvenile hormone on puffing for the following reasons. During the pupal stage, region I-19AB should not normally be active (Figs. 1 and 4) because of the presumed absence of juvenile hormone during this developmental period. As stated above, the salivary gland chromosomes cannot be studied during the pupal period. Pupae can be treated with juvenile hormone dissolved in DMSO rather than acetone, while prepupae cannot be so treated. The advantage of this solvent is that it is inert insofar as region I-19-AB is concerned. Finally, the diameter of the puffs of the polytene chromosomes of the Malpighian tubules can be measured with much greater accuracy than those of the salivary glands for structural reasons. With these advantages, the effect of juvenile hormone on puff induction in the polytene chromosomes of the Malpighian tubules was studied in a more detailed and quantitative manner than had been done previously. The most pertinent points of this study are compiled in Fig. 2 and reveal that the first statistically significant (P < 0.001) induction of a puff after juvenile hormone administration occurs in about 1 hr and that it is dose independent. The activity of region I-19-AB reaches a maximum and then declines. The greater the quantity of juvenile hormone applied, the earlier is the decline in activity after reaching the maximum (compare 30 ALg dose and 0.3-3 p,g dose). In all cases, the decline in activity is statistically significant (P < 0.001). Although the reason for the decline in puff activity is not known with certainty, it may be that the enzyme system that normally inactivates the juvenile hormone (Gilbert and Schneiderman, 1960) is stimulated in a dose-dependent manner by exogenously applied juvenile hormone. This idea finds support in the observation that a second application of juvenile hormone

HORMONES

AND

(3 pg) administered 1 hr after the first application causes a decline in the activity of region I-19-AB to the control level rather than amplifying the original stimulation. HORMONAL ACTIVITY GLAND

CONTROL OF AND SALIVARY FUNCTION

GENE

Among the regions of the salivary gland polytene chromosome that, are activated by ecdysone and inactivated by juvenile hormone is Balbiani ring 1 (Lezzi and Gilbert, 1969; Table 2) ~ This finding is of great interest since BRl can contribute up to 30% of the total nonnucleolar RNA synthesized in the nuclei of the cells of the salivary glands. In addition, the Balbiani rings appear to be import.ant in the primary function of the salivary gland, formation of a secretory product (cf. Grossbach, 1969). In 1969, Grossbach reported a method for fractionation of reduced and alkylated salivary gland secretions by microdisc electrophoresis on polyacrylamide gels. By a combination of this technique with a cytogenetic analysis of the chromosomal constitution of C. tentansC. pallidivittutus hybrids, he demonstrated that a single component (component 3) of the salivary gland secretion was absent from all specimens that had inactive BRl. Utilizing Grossbach’s procedure, an investigation was undertaken to determine whether component 3 is correlated with the activity of BRI during normal development. Preliminary results (W. Pankow, unpublished data) indicate that this is indeed the case. When t,he salivary glands of 4th instar larvae were analyzed, very little protein was detected with an electrophsretic mobility equivalent to Grossbath’s component 3. At the end of the 3rd instar, during the first day of the 4th instar and just prior to the molt to the pupa, however, one and possibly two protein bands appear with a mobility corresponding to component 3. It is of interest that the prepupal stage is characterized by clear visualization of this protein (component 3), relatively great activity of BRl and a high endogenous titer of molting hormone. This promises to be an excellent system

GENE

ACTIVITY

165

for further analysis of hormonal control of gene activity. Future work will be designed to answer the following questions, Can component 3 be induced in the salivary glands of young larvae by adminisiration of ecdysone? Dose juvenile hormone inhibit this process? Will K+ substitute for ecdysone in inducing synthesis of component 3 as it does in e!iciting activation of BRl ? This paper has sought to present the recent progress that has been made in L& fieId of hormonal contro1 of gene activity in the insect polytene chromosomes. As is true of many areas of the biological sciences, we find that. successful research begets additional unanswered questions. There is still no conclusive evidence that would lead one to unconditionally accept eit.her the hypothesis t.hat. insect hormones act directly on the genetic material or that they act via ionic intermediates. IIowever, the results of our studies on isolated chromosomes as well as the in vivo investigations with ecdysone and juvenile hormone fortify our conviction that the insect hormones elicit responses at, the chromosomal level by causing permeability changes that alter the ionic milieu of the chromosome. ACKNOWLEDGMEKTS We thank M. Robert (Saarbriicken), C. Holderegger (Ziirich), and W. Pankow (Ziirich) for allowing us to use their results prior to pub&+ tion. The juvenile hormone used in Nolderegger’s experiments was kindly provided by KofffmiznnLaRoche & Co.. Basel. We are grateful to Dr. F. E. Wiirgler and U. Graf for calculating the regression coefficients. REFERENCES BAUMANN, 6. (1968). J. Insect Phydol. 14, 14!%1476. BERENDES, H. D. (1966). 1. Eq. Zool. 162, 269218. CLEVER, U. (1961). Chlomosoma 12, 667-675. CLEVER, U. (1962). Chromosoma 13, 385-436. CLEVER, U. (1963). Chromosoma 14, 651-675. CLEVER, G. (1968). Annu. Rev. Genet. 2> 13-30. CLEVER, U., AND KARLSON, P. (1960). Ezp. Ccl% Res. 20, 623626. EMMERICH: H. (1970). Z. Vergl. PhysioL “68, 386402.

166

LEZZI

AND

GILBERT, L. I., AND SCHNEIDERMAN, H. A. (1960). Trans. Amer. Microsc. Sot. 79, 38-67. GILBERT, L. I., GORELL, T. A., AND CHINO, H. (1970). Eighth Int. Congr. Biochem. Lausanne, Switzerland. GROSSBACH, U. (1969). Chromosoma 28, 136-187. KARLSON, P. (1963). Perspect. Biol. Med. 6, 203214. KEYL, H.-G. (1968). Chromosoma 13, 486514. KROEGER, H. (1963). Nature 2,000, 12341235. KROEGER, H. (19%). Exp. Cell Res. 41, 64-80. LAUFER, H., AND HOLT, T. K. H. (1970). J. Exp. zooz. 173, 341-352. LEZZI, M. (1967). Chromosoma 21, 109-122. LEZZI, M. (1970). Int. Rev. Cytol. 29, 127-168. LEZZI, M., AND FRIGG, M. (1971). Mitt. Schweiz. Entomol. Ges. 44, (1) 163170.

GILBERT

LEZZI, M., AND GILBERT, L. I. (1969). Proc. Nat. Acad. Sci. U. S. 64, 498603. LEZZI, M., AND GILBERT, L. I. (1970). J. Cell. Xci. 6, 615-628. LEZZI, M., AND ROBERT, M. (1971). In “Results and Problems in Cell Differentiation.” (in press). PELLING, C. (1969). (I)) 239270.

Prog.

Biophys.

ROBERT, M. (1971). Chromosoma SHAAYA, E., AND KARLSON, P. Biol. 11, 424-432. SWIFT,

H.

WHITMORE, published WILLIAMS,

(1969).

Genetics,

(1961).

Biol.

Biol.

19

(in press). (1965). Develop.

Suppl.

E., AND GILBERT, observations. C. M.

Mot.

L. Bull.

61 (l), I.

439461.

(1971). 121,

DISCUSSION KING: Have you studied brain hormone-induced puffs described by Burdette and Kobayashi in Drosophila, and which ions can mimic them? Or does brain hormone have any effect at all on isolated chromosomes? Here is a third puffinducing hormone and a third opportunity to test your theory. LEZZI: We have not done any experiments with brain hormone. KARLSON: Most of the results described during the latter half of your presentation can be explained just as well by the assumption of a direct (not ion-mediated) action of hormones on the chromosomes. The main question is if the primary effect of hormone is only to change ion levels in the cell (or nucleus) and nothing else. This is the essence of the “ion hypothesis of gene activation.” In principle, then, it should be possible to induce the effects of ecdysone, e.g., in the Calliphora bioassay, by KCl. I understand that KC1 injection does not induce puparium formation. LEZZI: I must reemphasize that in general (maybe with the exception of the salivary glands of C. thummi) it is nonsense to apply ions to an intact cell or to a whole tissue because the cell protects itself against ionic changes in the external milieu. Our results are obtained exclusively with isolated chromosomes or nuclei. With intact salivary glands of C. tentans, for example, ions do not have a specific effect on puffing. KARLSON: How do you explain the results demonstrated by Sekeris that ecdysone stimulates RNA synthesis in isolated nuclei in the absence of K+ ions? LEZZI: It is probably not the nuclear membrane but the cell membrane where ecdysone causes the shift of K’. KARLSON: This is not the issue. In our system, we have isolated nuclei and ecdysone, with no ions in the incubation mixture, and nevertheless get stimulation of RNA synthesis. LEZZI: The ions do not need hormones, the hormones do not need ions, for exerting their respective effects with isolated nuclei. This suggests to me that the ions and the hormones act upon two different targets within the cell nuclei. KARLSON: Well, if you say that the hormone does not need ions to act, this is at variance with the original ion hypothesis. I can agree to this, but I would then say that the ion effects are highly reproducible artifacts. LEZZI: That is your concluding statement, Mister Chairman, not mine! CONTE: Isolated nuclei contain K’ despite being in ion-free media. Induct,ion may be mediated by redistribution of ions already within the nucleus at certain ion-sensitive gene loci which may or may not be coincident with hormone-sensitive gene loci. J. Ebert’s studies on animal virus production of eukaryotic tumor cells has shown that K’ is very important in the induction of specific viral proteins. In addition, the

Un-

572L585.

HORMONES

K+ effect virus. GILBERT:

appears Has

to be due anyone

to the

obtained

AND

GENE

activation effecix

ACTIVITY

of RNA of ecdysone

KARLSON: I think Emmerich did. LEZZI: As far as I know he did not, I mean,

polymerases on isolated

required

by

the

chromosomes?

with isolated polytene chromosomes. But we t.ried to do it using ecdysone or juvenile hormone, with a negative result (see Lezzi and Robert, 1971). DE KORT: Berendes (1961) did not succeed in inducing puffs with isolated lzilclei of salivary glands from D. Zydei. LEZZI: May I comment on this? Berendea wrote to me recently that-although he cannot observe a change in the puffing pattern-he still can measure a stimulation of the overall RNA synthesis when these nuclei are incubated in the presence of ecdysone. LAUFER: Critical to your hypothesis of antagonistic action of JH and ecdysone is repression of locus I-19-AB by ecdysone in Chironomus tentans in the prepupal stage, and a repression of Balbiani ring b in Chironomus thummi by ecdysone in the larva. Did you get such effects, and did you test for the effect of JN on Baibiani ring b in the prepupal stage as we did, getting positive enlargement? LEZZI: Concerning your first question: We did indeed find that eedysone causes regression of the puff in I-19-AB. This finding is in agreement with the observations by Clever and Karlson (1960) after short-term ecdysone application. Concerning your second question: We have not yet studied the effect of eedysone application on the activity of BRb (C. thummi).