PDF hosted at the Radboud Repository of the Radboud University

Nijmegen

The following full text is a publisher's version.

For additional information about this publication click this link.

http://hdl.handle.net/2066/147529

Please be advised that this information was generated on 2017-12-05 and may be subject to

change.

LOCAL GENOME ACTIVITIES

IN DROSOPHILA HYDEI

GIANT CHROMOSOMES

Th.K.H. HOLT

LOCAL GENOME ACTIVITIES IN DROSOPHILA HYDbl GIANT CHROMOSOMES

PROMOTOR PROF DR H D. BERENDES

2

LOCAL GENOME ACTIVITIES

IN DROSOPHILA HYDEI GIANT CHROMOSOMES

PROEFSCHRIFT

TER VERKRIJGING VAN DE GRAAD VAN DOCTOR

IN DE WISKUNDE EN NATUURWETENSCHAPPEN

AAN DE KATHOLIEKE UNIVERSITEIT Tfc NIJMEGEN,

OP GEZAG VAN DE RECTOR MAGNIFICUS,

DR G. BRENNINKMEIJER,

HOOGLERAAR IN DE FACULTEIT

DER SOCIALE WETENSCHAPPEN,

VOLGENS BESLUIT VAN DE SENAAT

IN HFT OPt NBAAR TE VERDEDIGEN

OP VRIJDAG 2 JUNI 1972

DES NAMIDDAGS Tb 2 UUR PRECIES

DOOR

THOMAS KAREL HENDRIK HOLT

GEBOREN TE HAARLEM

1 9 7 2

THOBEN OFFSET NIJMEGEN

Gaarne wil ik mijn dank uitspreken aan hen, die hebben bijgedragen aan het tot stand komen van dit proefschrift Veel dank ben ik verschuldigd aan allen van de afdeling Genetica, in het bijzonder mijn collega's Dr. Ρ J. Helmsing, Dr Η J. Leenders, Drs С L M Poels, С Alonso Md en Drs J. Derksen Tot slot wil ik bedanken Prof Dr G Τ Rudkin voor correctie van de Engelse tekst en het begeleidend commentaar. Mevrouw I van Heteren-Van Kol en Mevrouw G В Lammers-Jacobs voor het vele typewerk en de Heer J Gerrit­sen, die met 7ijn stai de illustraties verzorgde

4

Aan mijn uouw Aan mijn ouders

6

INHOUDSOPGAVE

I Inleiding

II Referenties

HI Local protein accumulation during gene activation I Quantitative measurements on dye binding capacity at subsequent stages of puff formation in Drosophila hydei Reprinted from Chromosoma (Beri ) 32, 64-78 (1970)

IV Local protein accumulation during gene activation II Interferometnc measurements of the amount of solid material in temperature induced puffs of Drosophila hydei Reprinted from Chromosoma (Beri ) 32,428435 (1971)

V Induction of chromosome puffs in Drosophila hydei salivary glands

after inhibition of RNA synthesis by α-amanitin Preprint accepted

by Chromosoma (Bed )

VI Effects of a-amanitin on nucleolar structure and metabolism in Drosophila hydei Preprint submitted to Expenentia (Basel)

VII Samenvatting

INLEIDING

Er is nog slechts weinig bekend over de mechanismen die een rol spelen bij de derepressie van genen in het eukaryoten genoom

Verschillende macromoleculaire substanties, waaronder RNA (Frenster, 1965, Britten en Davidson, 1969, Mayfield en Bonner, 1971), histonen (Stedman en Stedman, 1950) en niel-histon eiwitten (Teng en Hamilton, 1969, Helmsing en Berendes, 1971) zouden een rol in de genregulatie kun­nen spelen De met-histon eiwitten, ook wel als zure eiwitten gedefinieerd, worden wel beschouwd als de beste kandidaten voor een regulatorische rol bij de genaktivenng en -inaktivermg (Wang, 1971, O'Malley, Spelsberg, Schrader Chytil en Steggels, 1972)

De belangrijkste argumenten ten gunste van met-histon eiwitten als speci­fieke derepressoren werden verkregen uit experimenten, waarbij gebruik gemaakt werd van steroid-hormonen, die in staat zijn specifieke genen te aktiveren Het vervellingshormoon ecdyson kan in Dipteren larven bepaalde genen in hun aktiviteit beïnvloeden (Clever en Karlson, 1960, Berendes, 1967,Poels, 1970)

In de speekselklieren van Dipteren larven is een aktief gen, of een groep van aktieve genen, morphologisch te herkennen als lokale verdikking(en) in de reuzechromosomen, de 'puffs' (Beermann, 1952)

Een onderzoek naar het gedrag van deze zogenaamde puffs onder natuur­lijke, als wel onder bepaalde experimentele omstandigheden, kan een inzicht geven in de factoren die de aktiviteit van bepaalde genloci beïnvloeden Niet alleen hormonen, ook andere agentia, bijvoorbeeld een temperatuurbehan-deling, kan op specifieke plaatsen in het reuzechromosoom een puff Te voorschijn roepen (Ritossa, 1962, Berendes, Van Breugel en Holt, 1965, Ashburner, 1970)

Een onderzoek naar de macromoleculaire samenstelling in puffs met behulp van speciale kleurmethodes toonde aan, dat puffs geen veranderde histon-hoeveelheid bevatten in vergelijking tot hetzelfde chromosoomgebied in met-aktieve toestand (Swift, 1964, Gorovsky en Woodard, 1967) Er waren echter aanwijzingen dat zure eiwitten in de puffs werden geaccumu­leerd tijdens de vorming van de puffs (Clever, 1961, Berendes, 1967) Kwan­titatieve en kwalitatieve eigenschappen van de zure eiwitten in de puffs, waren echter niet onderzocht Bovendien werd accumulatie van eiwit tijdens

ч

puffinduktie in twijfel getrokken Immers de tot zover verkregen gegevens zouden evengoed op een toename van bindmgsplaatsen voor kleurstof kun­nen berusten

Pogingen om een synthese van zure eiwitten in de puffs aan te tonen met behulp van radioaktieve aminozuren waren met succesvol gebleken (Beer-mann, 1964, Pettit en Rasch, 1966, Berendes, 1967) Ook kon niet gecon­cludeerd worden dat er een strukturele modificatie van eiwitten optrad (Benjamin en Goodman, 1969, Clever en Ellgaard, 1970)

ben zeer belangrijk kenmerk van de puffs is de hoge RNA-synthese aktivi-teit (Pelling, 1964) Het was bekend dat de RNA in de puffs werd geas­socieerd tot nbonucleoproteine partikels, in welke vorm het door de puff voorgebrachte product het chromosoom verliet De accumulatie van eiwitten zou dus kunnen dienen ter verpakking van het nieuw gesynthetiseerde RNA

Anderzijds kon niet worden uitgesloten dat de zure eiwitten tijdens de initiatie van de genaktiviteit een belangrijke funktie zouden vervullen Deze laatste suggestie werd ondersteund door het feit, dat ondanks de remming van de RNA-synthese met behulp van dctinomycine D de eiwitaccumulatie optreedt (Berendes, 1968) Actinomycine D bindt zich aan het DNA en verhindert de transcriptie (RNA synthese) (Reich, Franklin, Shatkin en Tatum, 1961)

Een andere remstof, die invloed heeft op de RNA synthese, is het octa-peptide a-amanitine (Fiume en Wieland, 1970) Men neemt aan dat a-amam-tine een complex vormt met een der RNA-polymerasen, het polymerase-B (Jacob, Sajdel en Munro, 1970, Kedinger, Gniazdowski, Mandel, Gissinger en Chambón, 1970) en op deze wijze specifiek de synthese van hoogmoleculaire RNA inhibeert (Serflmg, Wobus en Pamtz, 1972)

Met behulp van deze remstof zou men dus eveneens kunnen nagaan of bij het ontstaan van een puff de accumulatie van zure eiwitten en het initiëren van de RNA-synthese-aktiviteit gekoppelde processen zijn Een ander pro­bleem, dat men met behulp van het gebruik van deze remstof zou kunnen onderzoeken, is de vraag of voor de initiatie van RNA-synthese-aktiviteit eventueel een reeds aanwezige RNA benodigd is

Tijdens het onderzoek, waarvan de resultaten in dit proefschrift zijn samengevat, werd getracht een inzicht te verkrijgen in de rol die de zure eiwitten en de initiatie van RNA-synthese spelen bij het proces van puff-vorming Als eerste onderdeel van het onderzoek werd de kinetiek van de kleurstofbinding aan eiwitten in de bepaalde puff-gebieden tijdens het ont­staan van de puffs nagegaan en werd een poging ondernomen om de eiwitten met specifieke cytochemische methoden te karakteriseren Ook werd gebruik gemaakt van radioaktieve voorlopers van eiwitten en reaktieve groepen van eiwitten

10

Het bleek noodzakelijk gebruik te maken van de methodiek van inter-ferentiemicroscopie om de kwantiteit droge stof in de puffs te bepalen, aangezien de eerder gebruikte cytochemische methodieken onvoldoende bleken om te kunnen onderscheiden tussen de accumulatie van eiwit en een eventuele conformatie-verandermg van reeds aanwezige eiwitten.

Door gebruik te maken van het octapeptide a-amanitine werd geprobeerd de relatie tussen de accumulatie van het zure eiwit en de initiatie van de RNA-synthese te bepalen. In eerste instantie werd hiertoe de remmende werking van a-amanitine op de chromosomale RNA-synthese en de puff-vorming in detail onderzocht.

Toen uit deze onderzoekingen bleek, dat het a-amanitine in vivo of in vitro toegediend een duidelijk ander werkingsspectrum bleek te bezitten dan bij reakties met de gezuiverde Polymerasen, werd als zijlijn van het bovenge­noemde projekt, een studie gemaakt van de ribosomale RNA-synthese. Deze bleek onder invloed van de remstof gemodificeerd te worden.

12

REFERENTIES

Ashburner, M Patterns of puffing activity m the salivary gland chromosomes of

Drosophila. V. Responses to environmental treatments. Chromosoma (Beri ) 31,

356-376(1970)

Beermann, W Chromomerenkonstanz und spezifische Modifikationen der Chromoso­

menstruktur in der Entwicklung und Organdifferenzierung von Chironomus tentans.

Chromosoma (Beri.) 5,139-198 (1952).

- Control of differentiation at the chromosomal level. J.exp.Zool. 157, 49-62 (1964).

Berendes, H.D The hormone ecdysone as effector of specific changes in the pattern of

gene activities of Drosophila hydei Chromosoma (Bed.) 22, 274-293 (1967).

- Aminoacid incorporation into giant chromosomes of D hydei. Dros Inf.Serv. 42,

102-103 (1967).

- Factors involved in the expression of gene activity in polytene chromosomes. Chromo­

soma (Bed.) 24, 418-437 (1968).

- Breugel, F.M.A. van, Holt, Th.K.H. Experimental puffs in Drosophila hydei salivary

gland chromosomes Chromosoma (Bed.) 16, 35-47 (1965).

Benjamin, W.B., Goodman, R.M. Phosphorylation of dipteran chromosomes and rat liver

nuclei. Science 166, 629-630 (1969).

Bntten, R J , Davidson, E.H.. Gene regulation for higher cells. A theory. Science 165,

349-357(1969).

Clever, U. Genaktivitaten in den Riesenchromosomen von Chironomus tentans und ihre

Beziehungen zur Entwicklung I. Genaktivierungen durch Ecdyson. Chromosoma

(Bed.) 12,607-675 (1961).

- Control of chromosome puffing. In The control of nuclear activity. (L Goldstein,

ed.), p.161-186 New York Prentice-Hall Inc. 1967.

- EUgaard, E.G. Puffing and histone acetylation in polytene chromosomes. Science 169,

373-374(1970).

- Karlson, P. Induktion von Puffanderungen in den Speicheldrüsenchromosomen von

Chironomus tentans durch ecdyson. Exp.Cell Res. 20, 623-627 (1960).

Fiume, L., Wieland, Th. Amamtins Chemistry and action. Febs Letters 8, 1-5 (1970).

Frenster, J.H A model of specific de-repression within interphase chromatm. Nature

(Lond ) 206, 1269-1270(1965)

Gorovsky, M.A., Woodard, J. Histone content of chromosomal loci active and inactive in

RNA synthesis J Cell Biol 33,723-728(1967).

Helmsing, P.J , Berendes, H D Induced accumulation of nonhistone proteins in polytene

13

nuclei of Drosophila hydei J Cell Biol 50, 893-896 (1971)

Jacob, S Τ , Sajdel, Ь M , Munro, Η N Different responses of soluble whole nuclear RNA

polymerase and soluble nucleolar RNA polymerase to divalent cations and to inhibi­

tion by O-amamtm Biochem biophys Res Commun 38,765-770(1970)

Kedinger, С , Gniazdowky, M , Mandel, J L , Gissinger, F , Chambón, Ρ Oí-Amanitm a

specific inhibitor of two DNA-dependent RNA polymerase activities from Calf

thymus Biochem biophys Res Commun 38, 165-171 (1970)

Mayfield, J E , Bonner, J Tissue differences in rat chromosomal RNA Proc nat Acad

Sci (Wash ) 68, 2652-2655(1971)

O'Malley, В W , Spelsberg, Th С , Schrader, W Τ , Chytil, F , Steggels, A W Mechanisms

of interaction of a hormone-receptor complex with the genome of an eukaryotic target

cell Nature (Lond ) 235, 141-144(1972)

Peiling, С Ribonukleinsäure-Synthese der Riesenchromosomen Autoradiographische

Untersuchungen anChironomus tentons Chromosoma (Beri ) 15, 71-122 (1964)

Pettit, В J , Rasch, R W Tntiated-histidine incorporation into Drosophila salivary

chromosomes J cell comp Physiol 68,325 334(1966)

Poels, С L M Time sequence in the expression of vanous developmental characters

induced by ecdysterone m Drosophila hydei Develop Biol 23, 210-225 (1970)

Reich, E , Franklin, R M Shatkin, A J , Tatum, E L Effect of actinomycin D on cellu­

lar nucleic acid synthesis and virus production Science 134, 556-557 (1961)

Ritossa, F A new puffing pattern induced by temperature shock and DNP in Drosophila

Expenentia (Basel) 18, 571-572 (1962)

Serfling, E , Wobus, U , Pamtz, R Effect of a-amanitin on chromosomal and nucleolar

RNA synthesis in Chironomus thummi Febs Letters 20, 148-152 (1972)

Stedman, E , Stedman, E Cell specificity of histones Nature (Lond ) 166, 780-781

(1950)

Swift, H The histones of polytene chromosomes In The nucleohistones (J Bonner and

Ρ Τ s'o, eds ), ρ 169-183 San Francisco-London-Amsterdam Holden Day Ine 1964

Teng, С , Hamilton, Τ Η Role of chromatin in estrogen action in the uterus II Ног

mone induced synthesis of non-histone acidic protems which restore histone inhibited

DNA dependent RNA synthesis Proc nat Acad Sci (Wash ) 63, 465^72 (1969)

Wang, Τ Y Tissue specificity of non-histone chromosomal proteins Exp Cell Res 69,

217-219(1971)

14

Chromosoma (Beri.) 32, 64—78 (1970) (( by Springer-Verlag 1970

Local Protein Accumulation during Gene Activation

I. (Quantitative Measurement* on Dye Binding Capacity

at Subsequent Stages of Puff Formation in Drosophila hydei

'ΓΗ. Κ Η. HOLT

(VU Biologv Section, Department of Zoology university of Nijmegen

Received July lb, 1970 < Accepted July 2Γ>, 1970

Abstract. MeasuromciUs of the integrated absorbaney of naphthol yellow S binding to protein (430 nra) and Feulgen-stamed DNA (530 nm) of two puff regions in Drotophih hydei polvtene chromosomes revealed u significant inerease in the naphthol vellow S binding capacity during the first З min of puff induction. The ratio of integrated absorption values at 430 and 330 nm of two chromosome regions. 2-48 Г and 4-81 В «ere determined relative to the ratio of absorption values at 430 and 330 am of a reference band. These determinations «ere earned out in a non-puffed state and at 3. 10, 30, 60 and 120 mm after onset of a tempera­ture treatment inducing puffs in these regions. The quotient of the absorption ratio of the puff region and the ratio of the reference band provides a relative measure tor naphthol vellow .S binding to protein. The staining reaction was absent after pronase treatment —The relative increase in naphthol yellow S binding was most obvious during the first 5 miti after onset of puff induction. The binding of naphthol yellow S was increased by a factor 1.7 for puff 2-48C, and a factor 1.9 loi putf 4-81 B. The maximum value, indicating a relative increase by a factor 1.8 in puft 2-48 С and a factor 2.2 in pull 4-81 В «a ï attained in both puffs at 30 mm after onset of puff induction.—Among staining procedures performed on sulphydryl groups, free α-ammo acids and indole groups of trypto­phane. only a positive result «ith the staining reaction on the indole groups was obtained lor induced puffs.—Injection of tntiated sodium acetate, methionine-H3-methyl, ethionine-HJ-ethyl, (/"-sodium bicarbonate, α mixture of 13 H'-labelled L-amino acids and H-'-tryptophane at various time intervals prior to puff induction failed to result m a specific incorporation of any of these radioactive substances into newly induced pulls.

I n t r o d u c t i o n

E u k a r y o t i c o h r o m o s o m e s c o n t a i n cons iderab le q u a n t i t i e s of h i s tones

a n d n o n - h i s t o n e prote ins ahsociated wi th nucleic ac ids . A p a r t from t h e i r

role as s t r u c t u r a l p r o t e i n s , b o t h t y p e s h a v e been cons idered as c a n d i d a t e s

for t h e r e g u l a t i o n ot gene t r a n s c r i p t i o n . Biochemica l s t u d i e s on isolated

c h r o m a t i n ind ica ted t h a t t h e r a t e of t r a n s c r i p t i o n is r e g u l a t e d b y inter­

a c t i o n of n o n - h i s t o n e (acidic) p r o t e i n s a n d h i s tones ( F r e n s t e r , 1965:

W a n g . 1968: T e n g a n d H a m i l t o n . 1969). ΐ η these in v i t r o s t u d i e s ,

however , ac idic p r o t e i n s a n d o t h e r p o l y a n i o n ^ a p p e a r e d t o be i n c a p a b l e

of d i s soc ia t ing h i s t o n e - D X A complexes , or revers ing h i s tone i n h i b i t e d

D X A - d e p e n d e n t R X A s y n t h e s i s (Spelsberg a n d Hni l ica , 1969).

Local Protem Amimnlation during Gene Actuation Τ fió

I n t h e p o l i t e n e c h r o m o s o m e s of d i p t e r a n la rvae , one oi t h e first

even t s occurr ing upon gene a c t i v a t i o n seems to he a local a c c u m u l a t i o n

of non-h i s tone p i o t e i n s Th i s e v e n t is d e m o n s t i a t e d b \ a c h a n g e in

b ind ing capacity of c e i t a i n d y e s a t low p H (Swift, 1962 B e i e n d e s

1967) T h e local increase in d y e b ind ing c a p a c i t y as obse ived wi th l ight

g i een (Clcvei, 1961), fast green ( p H 2 4), mercu r i c b i o m p h e n o l b lue

(Bei endes unpub l i shed ) a n d n a p h t h o l \e l low S pai allels t h e f o i m a t i o n

of puffs, which a t e t h o u g h t to reflect gene a c t i v i t y ( B e e i m a n n 1952

Polling, 1964) Since p i e t t e a t m e n t of t h e l a i v a c with a n t i b i o t i c s

inh ib i t ing p io tc in syn thes i s ne i the r affected t h e inc iease in d y e bindins;

noi the fo imat ion of puffs upon e x p e r i m e n t a l i nduc t i on it was sugges ted

t h a t t he inc iease in d y e b ind ing l e su l t s f iom a c c u m u l a t i o n of p i e e x i s t m g

p i o t e i n s l a t h e i t h a n f rom r/c novo syn thes i s of p i o t e i n s (Bercndes 1968)

This i n t e i p r e t a t i o n is s u p p o i t e d b j t h e fact t h a t d i n i n g puft f o i m a t i o n

in t he p icsence ot t n t i a t e d a m i n o ac ids no specific labell ing of t h e puff

s i tes ionici be olisci ved (Sw itt 1964 P e t t i t a n d Rasch 1966 Bei e n d e s

1967) So fa i , howcvci on ly R u d k i n (1962) h a s p iov ided some q u a n t i

t a t i v e evidence foi a local inc iease in p io tc in c o n t e n t oí ac t i ve chi o m o

some i egions

The ami of t he p i e s e n t s tudv is t o quant i fv local chan»c's m c h i

b ind ing c a p a o H d i n i n g subsec |ucnt s t ages in t h e рюсеьь ot puff f o r m a

t ion i n d u c e d b> l e m p c i a t u i c t i e a t m e n t s F u r t h c i m o r e s o m e a u t o r a d i o -

giaphic e \ p e i i i n e n t s will be l e p o i t e d t h e i c s u l t s of which m a y bo

i c l c v a n t foi t h e i n t e i p i e t a t i o n of t h e increase in d \ e b i n d i n g d u i m g

puff f oi m a t i o n

Mater ia l and Methods

VII L\[xrimcnts «eie carried out with late third inst.ir larvae ot ι wild tvpe stock of ¡Jio^oplalii lii/dei reaied under standaid conditions as described prev louslv (Berendes, 190>5)

The induetion of specific puffs wa1· performed by tiansterring the Ігіі ле trom 2"> to 37 V After lö nun at 370C a number of new puffs were іесоігшчесі in the salivarv gland chiomosomesj (Bciendcs, van Bieugel and Holt 19b5) bor investiga tions of these puffs the following standaid piocedure «us applied

.Sahvarv glands were dissected, fixed tor 2 mm in 7 TO ncuti.il (butfeicd) formaldclnde (pH 7 2) and squashed in ΊΤΌ acetic acid Immediatelv after squashing, the slides were frozen on drv ice. the coverslip removed and post fixed foi I hour in a solution composed ot 1 part foiiiialdehvclc (370o) and 9 parts 950(> ethanol ¡subsequentlv the slides were passed through an ethanol >-nes and thoroughly iinsed in distilled water

Staining Piocedutei

Ли, following btaining piocedures «ere applied a) For detection ot indole gioups of t iyptophane the slides were innnersed for

1.3 mm in a solution of 2°.) N-(I-naphthv 1) ethylene diamine dihvdiochloiidc· dissolved in a mixture of equal parts of 95"n ethanol and N HCl lollowing a pre treatment of 10 mm with a freshh prepared reigent containing 80o sodium nitrite

о Сііпіпюыип» (ßi il ) ltd M

66 Th Κ H Holt

m β X HCl according to Breummer, Carver and Thomas (1957) (see also Breummer and Thomas, 1958)

b) For idcntilication of free a-ammo group1! the ninhydrin Sthitt reagent «as used Tht staining procedure «asessentially as described b\ Yasuma and Ttchikawa (1953) (see also Arnold. 19(58)

c) .Mercurv orange vas used foi detection of sulplndrvl gioujis The staining procedure described by K a v a n n u a (1900) was applied (see also λ\ led. ]9()h)

d) A saturated aqueous men uric bromphenol blue solution «as applied to identif\ basic and acidic proteins The method of Ma7ia. Bre«ei and Altcit (195S) was followed

e) Staining of fiec cationic pioups of piotems «as performed either with fast green at low pH (2 4) according to Alfert and (.eschwmd (1953). or with naphthol yellow S according to Deitch (1955) As counterstain aceto carmine «as used in fast gieen stained preparations and the reuigen leaction in naphthol yellow S staining pioeedures

The combined naphthol jellow S-Feulgen procedure (Deitch, 1955) «as used for qiiantiti\e measurements on protein and DNA Preparations used foi these measurements «ere hydroljscd with X HCl toi 10 mm at (>00C, stained with Schiffs reagent for 90 mm followed by repeated rinsing in a frtshlv made sulfurons acid bleach After rinsing in distilled water the preparations «ere stained tot 1 hour in a 0 25'Ό naphthol vello« S solution in 2 50o acetic acid rinsed shoith in l 0 o acetic acid, dclndrated m thiee changes of teitiarv buU 1 alcohol and mounted in Caeda\ (r ι 1 55)

After this staining procedure, induced temperature puffs show a strong naphthol yello« S staining reaction (Fig 1). I t was observed that on some preparations the staining intensitv of the DNA and pioteins varied « i th the location of the ( bromo some sets within the preparation Chromosomes situated in the centei ot the preparation were more intensely stained with both reagents than chromosomes at the edges of the preparation

Cytophotomett y

Although most of the nuclei of the third instar salivai \ glands are m.icti\c with respect to DNA svnthesis. the le\el of poh tern among the population of nuclei \aries For this reason, measurements on naphthol yellow S binding л\еіе performed relative to DNA. For comparison of the absorbancy measurements on puff regions, the same procedure was applied to a distinct band in the vitmitv of the puff region. Absorption measuicments on naphthol yellow S binding and the Feulgen reaction «ere carried out with the Universal Alikro Spectral-Photometer (UMSP, Zeiss) at two wave lengths, 430 nm and 550 nm iespecti\eU. ac coi ding to the scanning principle.

Absorption curves of the Vwo dyes used are sutliciently separated to avoid an overlap at their absorption maxima (Deitch, 19(i(i) For possible systematical errors involved in the staining and measuring procedure applied, the analysis of Deitch (19ÒG) and Ke\ l (1905) maj be referred to

All chromosome legions, whether puffed oi non puffed on «Inch quantitative measurements were performed, were free of contamination from cytoplasmic constituents Quantitative measurements on local naphthol vello« 8 binding relative to DXA were performed bv establishing the ratio ot the integpeted absorp tion values at 430 nm and at 550 nm in a puff region over the integrated absorption values at the same wavelengths in a particular band m the neighbourhood of the puff region This measurement was made for two temperature puffs, 2-48 С and 4 81 В respectively Measurements on non-pufied band regions in the same chi omo some revealed an almost constant ratio of integrated absorption values (1A) at

Local Protein Accumulation during (iene Activation. I <i7

430 η m and 550 nm. However, this ratio varied considerably if compared in different preparations. It was found that the quotient of the 430/550 1A ratio of a particular band generally gives a value of the same order of magnitude (Table 1).

Table 1. Exam ¡ile of the result« of absorbanci/ nieasureiiieiits at 430 and ¡iöO um of reyioii 2-4SC in a non-puffed situation and on the reference band :і-{7 Пг 3

ТА = Integrated absorption resulting from various scanning lines over the puff region.— A - Absorption resulting from one scanning line over the band.

Nucleus

26 Nj 26 N u

26 X, 26 X, 26 X,

5 X 2

5 X 3

5K1

5 N 5

5 N,

Region

IA430

150 122 90

120 60 3o 30 09 80 77

2-48 С

ΙΑ 550

226 158 101

80 4« 96 79 91

107 103

Band 2

A 430

57 29 44 61 44 20 17 38 36 60

- 4 7 B 2 a

A 550

110 65 87 73 96 80 47 92 71

107

Average

IA430/TA550

A 430/A 550

1.27 1.75 1.90 1.80 2.83 1.24 1.05 1.25 1.50 1.34

1.65 — 0.51

If the DNA content of the puff forming region remains constant during puft formation, this quotient may be used as a relativi; measure for comparison of naphthol yellow S binding to the puff regions at subsequent stages after induction. So far, there is no indication for a change in DNA content of a region undergoing puff formation in Drosojiliiiti (Swift, 1962).

All dyes wore commercially obtained, fast green I'CF and prosanilin Iron) Chroma Gesellschaft; mercury orange from Serva; naphthol yellow S and N-(l-naphthyl)-ethylene diamine dihydrochloride from Fhika A.O. and bromphenolblue from Merck.

A utoradioijrnph ι/

For autoradiography Kodak ARIO stripping film «as used. Unless stated otherwise, the time of exposure was 7 days. After developing the films with Kodak DI9h, fixation and thorough rinsing in tap water, the slides were stained through the film with 0.5oo toluidine blue in distilled water. The staining was bleached in 7()0o ethanol, the preparations were dehydrated and mounted in Euparal or, after passing a xylene series, in Caedax.

The incorporation of amino acids into experimentally induced puffs was studied by injection of an L-aminoacid mixture containing ISHMabeled amino acids (act. 1 mC/ml) at various times prior to the onset of puff induction. Similar experiments were performed with HMryptophane (Spec. act. 200 mC/mM). The amino acids were administered by injection of 1 μΐ/larva.

In order to test whether or not acetylation of histoncs is involved in gene activation in polytene chromosomes of Drosophila hydei, larvae were injected a t 60 and 15 min prior to puff induction with tritiatcd sodium-acetate (spec. act. 100 mC/mM) (Pogo, Allfrey and Mirsky, 1966). Similar experiments were carried

5·

68 ï h . Κ. H. Holt:

out with L-ethionine-ethyl-H3 (spec. act. 42.2 mC/mM) and with neutralized L-ethionine-methyl-H3 (spec. act. 134 mC/mM). In order to test whether ethylation or methylation is involved in gene activation (Friedman, Shull and Färber, 1909; Gershey. Haslett and Allfrey. 1969). Carboxylation was studied by using XaHC140 (act. 5 mC/ml). All radioactive substances were obtained from XKX.

RNA and DXA were extracted by treating the preparations with 50o TCA at 90° С (20 min). Chromosomal proteins were digested with 1 mg/ml pronase (Calbio-chem, В grade) at pH 8.0. In order to retain an intact chromosome image, pronaso digestion was carried out for 10 min at 37° C.

Fig. 1 Fig. 2

Fig. la and b. Puff 2-48C at 15 min after onset of a temperature treatment photo­graphed with an interference filter at 550 nm (a) and 430 nm (b) after staining with

naphthol yellow S-Feulgen

Fig. 2. Puff 2-48C at 15 min after onset of a temperature treatment photographed with an interference filter setting at 020 nm after staining for indole groups of

tryptophane

Results

Staining Reactions

Among the staining procedures applied, mercuric bromophenol blue, fast green at pH 2.4 and naphthol yellow S at low pH revealed an inten­sive staining reaction with induced temperature puffs. As compared with puffs already present at the time of onset of the temperature treatment the dye binding in the newly induced puffs revealed a particular strong staining reaction. From the other procedures used, only the reaction on indole groups of tryptophane gave a positive reaction on induced puffs (Fig. 2). The ninhydrin-Schiff reagent and mercury orange did not show a specific effect on induced puffs. A full account of the results of all staining procedures, including the reactions of band and interband regions and the nucleolus is given in Table 2.

Lot<il Protem Aociirmilation dining CJene Activation. 1 09

Tabic 2. titutiiuH/ lenctuum of temjietntuie induced ¡шЦя. bttiids. tiitnbands and nucleolus ajlci fonnuldehyde fixation

A strong reaction is indicated as I - . a reaction as —, and no icaition as —.

btainmg piocedme I'ufls Bands Intel- Xucle-bands olus

Merrillic bromphonol blue on + | + I + - ^ basic and acidic pmteins

Fast green at pH 2 4 on free ) | -J- -^ 4 f cationit gtonps ot proteins

Лaphthoi u-llow S at pH 2.4 on f + t- I I — free cationic groups of proteins

Mercury orange on SH groups - — — -|-

X (1 naphtln 1) etliv lene diamine I - τ -¡- -*• dihydrocliloiidc on indole groups of tryptophane

Ninhydrm Schiff leagent on — h - + + free α-amino gioups

Fast green at pH H attei remoial — + 4 - + -ι­οί ш а і іс acids on basic proteins (histones)

Fast green at pH 2 4 after + -f + + + — hot TCA extraction

Fast green at ρ Η 2 4 after — -pronase digestion

Fast green at ρ Η 2.4 after | - + I I + — RNase digestion

T h e dyes i c a c t i n g with ca t ionic giuujjb oí p ro te ins sucli аь fas t green

a n d n a p h t h o l yel low S a t low p H were n o t inf luenced in t h e i r a f i in i ty

t o i n d u c e d pufis by e x t r a c t i o n s of nucle ic ac ids wi th h o t TCA or d iges t ion

of R K A with 1 mg/ml (4 h o u r s a t 3 7 4 ' )

Pronas,e d iges t ion a f ter s t a i n i n g wi th fast green a n d n a p h t h o l yel low S

did r e m o \ e t h e d y e s from t h e puffs a s well a s f rom t h e lest ot t h e c h r o m o -

s>ome a n d t h e nuc leo lus U p o n p i e - t i e a t m e n t of t h e p i e p a r a t i o n s wi th

p r o n a s e no s t a i n i n g r e a c t i o n wi th d y e s b i n d i n g t o cat ionic g i o u p s was

observed (Fig 3)

F r o m these d a t a it m a y be c o n c l u d e d t h a t t h e local i n e i e a s e in d3'e

b inding c a p a c i t y as revea led b y t h e newly i n d u c e d puffs is d u e t o e i t h e r

a n іпсгеаье i n p r o t e i n m a s s , or t o a c h a n g e in t h e conf igura t ion of

a l r e a d y p r e s e n t p r o t e i n s involv ing t h e l ibera t ion of ca t ionic g r o u p s a n d

indole g r o u p s of t i y p t o p h a n e T h e l a t t e i s t a m i n g reac t ion sugges t s a

70

Ρ

Fig. За and b. Puff 4-81 В at 15 min after onset of a temperature treatment photo­graphed with an interference filter setting at 580 r m after staining with fast green at pH 2.4 (a), b following pretreatment with 1 mg/ml preñase for 10 min at 37°С

role of non-basic proteins (non-histones). This suggestion is supported by the virtual absence in the same regions of fast green binding at high p H after removal of nucleic acids (see also Swift, 1964, and Gorovsky and Woodard, 1966, 1967).

M irrospectrophotometric Measurements

Absorption measurements were made at 430 and 550 nm on two temperature induced puffs, 2-480 and 4-81В respectively. The results of these measurements were compared with those on the same regions in control animals. The preparations used for these measurements generally were passed together through the dehydration and staining procedures in order to avoid as far as possible variations in the staining procedure.

Integrated absorption values were established by scanning the puff regions at the two wave lengths including at one side of region 2-48C band 4 8 B 4 a n d at the other side band 48C2. As reference the ratio of absorption values at 430 and 550 nm of band 47 В2,3 was used. For puff 4-81B, the boundaries of the region measured were taken between

Th. К. H. Holt:

Local Protein Accumulation during Gene Activation. 1 71

relative ratio of absorption integrals

at ""/SSO η μ

region 2-1.8 С

n=10<j> ψ η Π Ι

H

i n.-9

ό n = 9 n = 9 9

n = 10

J I L 0 S 10 60 120

time after onset temperature treatment in mm

Fig. 4. Ratios of absorption integrals at 430 and 550 nm of region 2-48C relative to the reference band 2-4715:м at a non-puffed state (0) and at various time

intervals after onset of puff induction

the bands 81 В1 and 81 B2 at one side and the bands 81 B6 and 81 CI at the other side. Band 80C1, 2 was used as reference. The puff region and the reference band were always measured in the same chromosome. The poiyteny level of the two chromosome regions is identical, provided that the nucleus is inactive in DNA synthesis. The measurements on the two puff regions were performed at an unpuffed btate and at ,), 10, 30, 60 and 120 min after onset of the temperature treatment. Calcula­tions of the quotient of the ratios of integrated absorption values at 430 and 050 nm of the puff region and the reference band provided a relative measure for the change in naphthol yellow S binding during puff formation. At each of the times indicated 9-11 measurements were made. The variation in the data obtained from these measurements (Table 1) may be partly due to a variation resulting from the staining procedure although it cannot be excluded t h a t they are caused by true differences in dye binding to different nuclei of the same gland.

It was found in the control animals that the quotient of the two puff regions over the reference band was, on the average, 1.65. After the onset of puff formation, the quotient revealed an evident increase (Figs. 4 and 5). From Fig. 4 it may be concluded t h a t naphthol yellow S binding to puff 2-48C increased by a factor 1.7 within 5 min after onset of the temperature treatment. A maximum value of the quotient is attained

72 Th Κ H Holt

relative ratio ot absorption integrals

5 L at ""/SSO mjj

η 10

¿η 9

άη 11

¿η 10

0 5 10

region ί. 61Β i

in-10

η IG Ο

60 120 time after onset o< temperature treatment in min

Fig Ί Ratios ol absoiption inteprals at 43(1 and 550 nm o\ region 4 81 В ulatne to the reieiime band 4 SOtj _ a t a non pufiecl state (()) and at valions time

intervals after onset of putt induction

at 30 mm alter onset of the tempeiature t ieatment For putt 4 81 В the lelative incicase of the dye binding to catiomc gioups was 1 9 within 5 mm of t ieatment Also this puff revealed a maximum value ot the quotient aftei 30 mm of t ieatment (Kig 5)

These icsults indicate that the d jc binding capacitj of pioteins piesent in each ol the two pufl regions studied, inereasts during puff formation b\ about a factoi of 2 Fuithermoie these data show that in contiast to the suggestion of Bciendes (1968), the level ot thi dye binding does not deci ease after the puffs have attained their maximum diametei

A utoradiography

The pattern ot mcoipoiation ot tu t ia ted amino acids adimnisteied at various times puoi to the onset of puff induction was investigated I t is well established that short pulses of tut ia ted ammo acids if gi\tn by injection into the laivae shortly before the onset of puff induction (within 1 houi), do not result in a specific incorporation in newly foimcd putts (Pettit and Rasch, 1966 Beiendes, 1967) Thest, findings wcie confiimed b^ expenments in which 1 μΐ pei lai\ae of a mixtuie of

15 H 3 labeled amino acids (1 μΟ/μ!) or H 3 tryptophane (1 μϋ/μΐ) was

injected 15 min betore onbet of temperatuie treatment

Local Protein Accumulation during Gene Activation. I 73

Fig. 6. Autoradiograplis of the puffs 2-48C and 4-81В induced by a 20 min temperature treatment at 20 h after administration of a mixture of 15 tritiated

L-amino acids

The temperature puffs did not show a speeifie strong incorporation as compared with non-puffed chromosome regions.

Longer intervals between the administration of radioactive amino acids and the onset of a temperature treatment were chosen in order to determine whether or not the synthesis of proteins t h a t might be involved in puff formation occurs at a relatively low rate throughout development, or a t specific periods of development. If pre-existing proteins become incorporated during puff formation, these proteins should become labeled if the radioactive precursors are available over a long period of development and become expressed in newly induced puffs by a specific increase in grain density in the autoradiographs. The tritiated amino acid mixture (1 μΐ containing 1 μC/larva) was injected

40 hrs, 30 hrs, 20 hrs, and 6 hrs respectively before onset of puff induction.

In the autoradiographs of the salivary gland chromosomes no specific

high label density was observed over the newly induced puffs (Fig. 6),

although in each of the experiments cytoplasm, chromosomes and nucleo­

lus were labeled. Similar experiments were carried out with H 3 -trypto-

phane (1 μΐ/larva containing 1 μ0).

74 Th Κ H Holt

Table 3 hicot¡ют!ion of labeled compounds IH bund and inlerbands. induced puffs, nucleoli and cytoplasm

+ hlndicateb hcaw labelling, + labelling, z i v e r v weak labelling, —no labelling

Labeled compound

J î H3 L amino acids

H 3 tryptophane

Methionine H 3 methyl

Etlnonint H1 cthjl

•Soditirn buarbonate C14

Sodium acetate H J

Time of administration prior to puff induction

40 hours 30 hours 20 hours 45 mm 15 mm

lb hours 15 mm

30 min

30 mm

30 mm

30 mm 60 mm

Incorpora

Puffs

_

= -i-

л-— + + + + - L

-

+ +

tion in

Band -Γ interband

_

± + -fc _z:

h 1

+ + 1

-

+ +

λ 11 oleoli

_

^ ± +

l· + 1

+ + + + + 1

C\to plasm

_

+ + _i_

^H

+ -L

+ 1

-

+ + +

Albo thebe expenmentb failed to show a ьрееіпс incoi poi ation of this amino acid into the newly induced puffs (Table 3)

Kxpenments in which larvae weie fed with a mixture of H damino-acids by supplying the larvae every day during a 4 day period with 30 μ( ' also failed to leveal spécifie incoipoiation of labeled piotcins into newh induced puffs

Injection of 1 μΙ of neutralized L-methiomne-H3 methyl (0 5 μΟ') at

30 mm before onset of a temperature t ieatment gave no indication for

mcth^lation to be involved in the process of puff formation The chromo­

somes -rtcrc randomly labeled Only the nucleolus appealed to show a

specific high incorporation of labeled methyl gioups A ыгшіаі lesult was obtained after injection of 1 μΐ L-ethionine-H3 ethyl (0 5μ(71αι\α)

Although the chromosomes and the nucleolus weie labeled no specific

high incorporation into newly induced tempeiatuie puffs could be

obsei\ed Also upon administration of tnt ia ted acetate (1 μC71aIva) or

C14-sodium bicarbonate prior to puff induction no specific incoipoiation

into newly induced puffs was found Clnomosomes, nucleolus and cyto­

plasm were labeled The results obtained with tnt iated acetate agí ees well with earlier observations on Droiophila hydei (Betendes, unpub­lished) Droòophila melanogaster (Ellgaaid 1967) and Chironomus (Clcvet, 1967)

Local Protein Acciiiniilation during Gene Activation. 1 70

From the autoradiographic experiments reporter!, no evidence was obtained for an accumulation of pre-existing proteins, nor for carboxyla-tion, acetylation, methylation or ethylation of substances during puff formation.

Discussion Absorption measurements on dyes bound to rationie groups of

proteins in relation to Feulgen-stained DXA indicated an evident increase in protein-dye binding capacity of regions becoming active in transcription. During the first 5 to 10 min of puff formation, the binding capacity for naphthol yellow S (NYS) is about a factor 2 higher when compared to the same region in a non-puffed situation. This factor was established by comparison of the quotient of the integrated absorption ratio at 430 nm/.550 nm of the puff region, and the absorption ratio of a reference band with a similar quotient obtained from the region in a non-puffed situation. Although providing only relative values, these quotients can be used as a relative measure for XYS binding. It was established that the ratio of the absorption values at 430 and 550 nm obtained by one scanning line through bands of the same chromosome provided a constant value. The same holds true for interband regions. Other chromosomes on the same slide or on a different slide may give a different ratio. It may be supposed however, that within a small area of the same chromosome the dye binding is proportional to the amount of free cationic groups of proteins and DXA respectively, whether a band or a putt is measured. By calculating the quotient variations between different chromosomes are hardly reflecterl anymore.

This quotient should, for a certain region, be identical in different chromosomes, provided that a) the binding of the XYS to the puff region is always proportional to the binding in the reference band and b) that the level of polyteny of the band(s) included in the puff region is identical with that of the reference band.

Failure with respect to the latter requirement may be responsible for deviations from the average quotient recorded in Table 1. Smaller deviations may result from true variations in binding capacity of the XYS in the different regions used, due to differences ¡n the degree of stretching (a very weak activity of the regions cannot be excluded). The comparison of quotients of the same region at subsequent stages of puff formation revealed an obvious increase. An increase as such can be brought about cither by an increase in the DNA content of the reference band without a simultaneous increase in cationic groups of protein, or by an increase in NYS-binding to protein during puff formation. On account of the constant protein/DXA ratio in the bands as shown by the absorption measurements, it is likely that the increase in the quotient value during puff formation is due to a raise in XYS binding in the puff region.

70 Th. Κ. H. Holt:

Tilo increase in ΝΥΝ bindinjí during puff formation may be due to either a loeal change in the configuration or composition of chiOmosomal proteins involving an increase in the number of eationie groups, or to a local increase in the quantity of non-histone proteins. With respect to the first possibility it could be assumed that acetylation, methylation or ethylation of histones is involved in puff formation. If so. the binding capacity of the histones for XYS at low pH could become increased. This, at the same time, would decrease the number of binding sites of the histones for fast green at a high pH.

So fai' there is no evidence for a change in the staining properties of histones in puff regions as compared with the same region in a non-puffed condition. Swift (1964) and Gorovsky and W'oodard (1966: 1967) performed densitometric measurements on chromosomes of DroKophila virilis. stained with alkaline fast green. They found no difference in histone quantity in the same region, whether puffed of non-puffed. In addition, no indication was obtained from the autoradiographic experi­ments reported here that acetylation. methylation or ethylation is involved in puff formation. Moreover, changes in histone configuration as such, would not account for the positive reaction obtained after staining for indole groups of tryptophane. I t is therefore, more likely that acidic proteins provide the binding sites for the dyes attaching to eationie groups.

From the autoradiographic experiments it seems unlikely that carboxylation of these proteins does occur during puff formation. Similarly, phosphorylation may be excluded as a factor involved (Benjamin and Goodman. 1969).

Administration of labeled amino acids, including tryptophane at various intervals prior to the onset of puff induction failed to give any indication for a specific local increase in protein content during gene activation. These results agree with those obtained for Drowphila viriiis (Pettit and Rasch, 1966).

On the other hand, preliminary data obtained from interference microscope measurements revealed that in induced puffs the quantity of solid material is increased in comparison with the non-puffed state even after extraction of RNA. These data suggest that some substance, e.g. protein, accumulates during puff formation. If this interpretation is correct the failure in obtaining evidence for protein accumulation by autoradiographic analysis of the amino acid incorporation patterns might be due to a very low turnover of the proteins involved.

Aektunrlediiemenis. 1 am indebted to Professor Dr. H. D. Berendes for his continuous interest in the work, and his stimulating discussions. Professor Dr. H. G. Keyl is acknowledged for his interest in this study and tor providing me with

Local Protein Accumulation during Gene Activation. 1 77

the possibility of using the L'MSI'. Some measurements were carried out at the Institute for Anthropology ol the L'nivcrMty of Amsterdam. Thanks are due to Drs>. J . B. Bijlsma and V. Koperdraad for providing me with the opportunity of using the L'.M.SP.

Deferences

Alfert. M., Geschwind, J . .).· A selective staining method for the basic proteins of cell nuclei. Proc. nat. Acad. S( ι (Wash.) 39, 991-999 (1953).

Arnold, M.· Histochcmie. Herlm-Heidelberg-iVew York: .Spimger 19()H. Becrmann, W.: Chromomerenkonstanz und spezifische Moditikationen der ('hromo-

somenstriiktur in der Entwicklung und Organdiffcrcnzierung von Chiiuiwiiius tentans. Chromosoma (Bed.) ,'ι, i:)9-198 (1952).

Benjamin, \Y. В., (ioodman. Ι? ЛГ. : Phosphorylation of dipteran clnomo.soincs and rat liver nuclei. Science 1««. (Й9 030 (19G9).

Berendes, Η. Ό.: Salnar\ gland iunction and ( hromosomal puffing patterns m Diowplnla hydeì. Chromosoma (Beri.) I". 35-77 (1965).

— The hormone ecdysone as effector of specific changes in the patlcrn ot sienc activities ol T)ioso¡>hilei hi/dri. ('hiOinosoma (Beri.) 22. 274-293 (I9(i7).

— Factors involved in the expression ot gene activity m polvtene ι liromosomcs. Chromosoma (Beil.) 24. 418-437 (19(i8).

— Ammo acid incoiporation into mant chromosomes ol Ιίιυ^υρΙιιΙα ln/ilit. Dros. Ini. Sei4. 42. 102 (197(1).

— Breugel, F Μ. Λ. van. Holt, Th. К. H..: Experimental pufts m Drosophila In dei salivary gland chromosomes. Chromosoma (Beri.) IG, 35 47 (19()5).

Brcummcr, X. ('., Carver. M. .Г., Tilomas, IJ. E.: A tryptophane liistcx licniical method. J . Histochem. Cytochem. .», 140-144 (1957).

— Thomas,L.E. : Tryptophane lustücheiiiical method. J . Histochem. Cvtochcm ti, 75 (1958).

Clever, (J.: Genaklivitatcn in den Biesenchromosomen von Chiroimum* lenltm* und ihre Bezielumgen zur Entwicklung. I. Genaktiv icningen durch Ecdyson. Chromosoma (Beri.) 12. 007 075 (1901).

— Control of chromosome pulting. In: The control of nuclear aclivitv (I,. Gold­stein, ed.), p. 101-180. Neu York: Prentice-Hall Inc. 1907.

Deitch. A. U. : Cytophotometry of proteins. In · Introduction to quantitative cvto-chemistry (G. L. Wied. ed.). p.451 408. New York-London: Academic Picss 1900.

— Microspectrophotometru .study ot the binding of the anionic dye. naphthol vellow S by tissue sections and bv purified proteins. Lab. Invest 4. 324 351 (1955).

Ellgaard. E G.. Gene activation without histone acetvlation m Ihowplula weliina-(/ачіеі: Science 157. 1070 1072 (1907).

Frcnster, J . H . : Nuclear polyamons as de-repressors of synthesis of ribonucleic acid. Nature (bond.) 2П<>, 080-083 (1905).

Friedman, M., Shull, K. H., Färber, li.: Highly selective in rno ethylation ol tat liver nuclear protein bv etlnonine. Βίοι hem. biophys. Kes. ('onimun. 34, 857-804 (1909).

Gershey, E. L., Haslett, G. W., Vidah, (ί., Allfrey, Y. G.: Chemical studies on histone methvlation. Evidence for the occurence of 3-methvl histidme m avian erythrocyte histone tractions. J . biol. ("hem. 244. 4871 -4877 (1909).

Gorovsky, Μ. Α.. Woodard. J . : Histone-DNA ratios in five regions of a polvtene chromosome ol Drosojihih lirilit. J . Cell Biol. 31, 41 A (1960).

— — Histone content of chromosomal loci active and inactive in RNA synthesis. J . Cell Biol. 33, 723-728 (1907).

78 Th. К. H Holt Local Protein Acdimulutioii durinp Gene Activation. I

Kawamiira. >,.. Cytochemual and quantitative stucU ol proteia-boimd siil[h\di\l and disulphide groups in eggs of Aibnrtn during the first cleavage E.\p. (VII Res 20, 127^128 (1900).

Keyl. H. G. Duphkationen von Untereinheiten der chromosomalen DXS wahrend der Evolution von Chiionumui Ihuntnii. Chromosonni (Beil ) 17. 139 180 (196,")).

Mazia, D.. Brewer. Ρ. Α.. Alfert. M The eytoehemieal staining and measuiement of protein with mercuric bromphenol blue Biol Bull (Woods Hole) 104. 57-07 (1953).

Peiling. С : Ribonukleinsäure Synthese der Rie.senehromosomen. Auloiadiogiaphi-sche Untersuchungen an СІпюпотич tenían^. Chromosoma (Beil.) IS. 71 122 (1904).

Pettit , B. J , Rasch, R. \Y : Tritiated histidme mcorpoiation into Оіочоріпіп sali­vary chromosomes. J . cell, comp Physiol. (»8. 325-334 (1900).

Pogo, B. G. T.. Allfrcv. V'. G.. Mirsky. A E. RNA synthesis and histone acetvla tion during the course of gene activation in Ivmphocytes. Proc nat Acad. Sci (Wash.) 55, 805-812 (1900).

Rudkm, G. T. : Nucleic acid metabolism in giant с Іігопюмтіеь ot Drow/i/iilii mehinogaslei Ann. Histochim , Suppl. 2, 77-34 (1902).

Spelsberg, Th С , Hnihca. L S · The effects of acidic proteins and RNA on the histone inhibition of the DNA-dependent RNA synthesis in vitio Biochim biophys. Acta (Amst ) 195, 03-75 (1909)

Swift, H. : Nucleic acids and cell morphology m dipteran salivary glands In The molecular control ot cellulai activity (J.Allen, ed) , ρ 73-125. New York. McGraw-Hill 1902.

— The histones of polytene chromosomes Tn: The iiiicleohiHtone.s (J. Bonne ι and Ρ Ts'o, eds.), p. 109-183. San Francisco-London-Amsterdam : Holden Day Inc. 1904.

Teng, C, Hamilton, Τ Η.: Role of chromatin m estrogen action m the uteius. 11 Hormone induced synthesis of non-histone acidic proteins wine h restore histone inhibited DNA-dependent RNA synthesis. Proc. nat. Acad. Sci. (Wash ) 03, 405-472 (1909).

Wang, T. Y.· Restoration of histone-mhibited DNA dependent RNA synthesis by acidic chromatin proteins. Exp. Cell Res. 53, 288-291 (1908).

Wied, G. L. : Introduction to quantitative cytochemistry New York London : Academic Press 1906.

Yasuma, Α., Itchikawa, Τ Nmhydrin-Schitt and alloxane Schift stammg. A new histochemical staining method for protein. J Lab chim Med. 41. 296-29!) (1953).

Drs. Th. Κ. H. Holt Zoologisch Laboratorium Afdeling Celbiologie Drieluuzenveg 200 Nijmegen, Toernooiveld Nederland

Chromosoma (Beri ) .·)2, 42Я 4.4.5 (1971) ( b\ Springer Verlag 1971

Local Protein Xtrumulalion during Gene ^ctivalion

II. Interfcrometnr Measurements of the Amount of Solid Material

in Temperature Induced Pufls of Drosophiln hvdei

Тн К II H O L T

('ell ВіоІод\ Section. Department ol ZoolugN. L'mversitj of Nijmepen

Koeenecl Noxember 7. 1970 Ucepted No\cinberl4. 197(1

Abstract. Relative values for the dry mass in puff-forming regions of Drosophila hydei salivary gland chromosomes were established with a Leitz double beam interference microscope. All measurements were made after RNA digestion.

Optical path differences per unit area of the induced puffs 2 ^ 8 0 and 4-81В (temperature induced) and a cytoplasm-free background were recorded. In each of the nuclei used for these measurements, the same procedure was applied to two reference regions in the vicinity of the puff, region 2, 4 7 A ^ 8 B and region 4. 81C-82C respectively. For comparison of the dry mass values of a puff region at various time intervals after the onset of puff induction, ratios of the optical path differences of the puff region over that of the reference region were calculated.

These ratios were established at 5, 10, 30, 60 and 120 mm after the onset of a temperature treatment and compared with the ratio m non-treated animals. From the data it can be concluded that the dry mass in the induced puffs increases gradually up to 30 min. At this time the dry mass ratio for puff 2^480 has reached a value of 150% and that of puff 4-81B, 210%. I t is concluded that this increase in dry mass is due almost entirely to a local accumulation of non-histone protein.

Introduction

I t is well e s tab l i shed t h a t t h e puffs in D i p t e r a n p o l y t e n e c h r o m o ­

somes d i sp lay a n i n t e n s i v e s t a i n i n g r e a c t i o n w i t h a v a r i e t y of d y e s

b i n d i n g a t low p H t o ava i lab le cat ionic g r o u p s of p r o t e i n s (Swift, 1962;

B e r e n d e s , 1968; H o l t , 1970). R e c e n t l y , p h o t o m e t r i c m e a s u r e m e n t s of

n a p h t h o l yel low S b i n d i n g a t successive s tages of i n d u c e d puff g r o w t h in

Drosophila hydei r e v e a l e d a g r a d u a l increase in d y e b i n d i n g c a p a c i t y u p t o

a p p r o x i m a t e l y 2 0 0 % in t e m p e r a t u r e i n d u c e d puffs (Holt , 1970). T h i s

increase i n d y e b i n d i n g c a p a c i t y m a y resu l t e i t h e r from a n a l t e r a t i o n in

t h e conf igura t ion of p r o t e i n s p r e s e n t a t a puff forming locus pr ior t o t h e

o n s e t of puff f o r m a t i o n or t o a n a c t u a l local increase in p r o t e i n con­

c e n t r a t i o n . A u t o r a d i o g r a p h i c a l s t u d i e s w i t h r a d i o a c t i v e a m i n o ac ids so far

failed t o i n d i c a t e d e n o v o p r o t e i n s y n t h e s i s in i n d u c e d puffs ( P e t t i t a n d

R a s c h , 1966). F u r t h e r m o r e , no ev idence for a n a c c u m u l a t i o n of p r e l a b e l e d

p r o t e i n s i n t o t h e puff regions was o b t a i n e d . T h e s e d a t a f a v o u r e d a n

i n t e r p r e t a t i o n of t h e increased p r o t e i n d y e b i n d i n g c a p a c i t y in t e r m s of a

Local Protein Accumulation during Gene Activation. TT 429

c h a n g e in p r o t e i n conf igurat ion invo lv ing t h e l i b e r a t i o n of c a t i o m c

g r o u p s for d y e b i n d i n g . On t h e o t h e r h a n d , i t could n o t be e x c l u d e d

t h a t t h e fai lure t o observe a definite increase of labeled m a t e r i a l i n t h e

i n d u c e d puffs was a c o n s e q u e n c e of a v e r y low t u r n o v e r r a t e of p r o t e i n s

invo lved in puff f o r m a t i o n .

T h e puffing p h e n o m e n o n іь genera l ly cons idered t o be a m o r p h o l o g i c a l

m a n i f e s t a t i o n of gene a c t i v i t y Since a c h a n g e in local d y e b i n d i n g

c a p a c i t y of a puff f o r m i n g region occurs a l m o s t i m m e d i a t e l y following

t h e o n s e t of puff i n d u c t i o n , i t was a s s u m e d t h a t t h i s f e a t u r e is o n e of

t h e p r i m a r y e v e n t s in gene a c t i v a t i o n (Berendes , 1968). T h e r e f o r e a

s t u d y o n t h e chemica l bas is of t h e increase in d y e b i n d i n g m i g h t con­

t r i b u t e t o a b e t t e r u n d e r s t a n d i n g of t h e m e c h a n i s m of gene a c t i v a t i o n m

higher o r g a n i s m s .

I n o r d e r t o e s tab l i sh w h e t h e r t h e increase in d y e b i n d i n g c a p a c i t y is

d u e t o a c h a n g e in p r o t e i n conf igura t ion or t o a n increase in p r o t e i n

mass , i n t e r f e r o m c t r i c m e a s u i e m e n t s o n i n d u c e d puffs weie p e r f o r m e d

af ter R N A d i g e s t i o n

M a t e r i a l a n d M e t h o d s

All expenment.s were performed with late third instar larvae of a wild tyjie hto< к of Drosophila hydei reared under standard conditions.

Interference measurements were earned out on two puffs, 2 ^ 8 0 and 4-81B induced by a temperature treatment. The induction of these and some other puffs is brought about by transferring larvae from 2.5° Ut 37° С (Beiendes. van Breugel and TTolt, 1965). Measurements on these chromosome regions weie made on salivary gland squashes of non treated animals and of animals submitted for 5, 10, 30, 60 or 120 mm to a teinperature treatment.

After dissection, the salivaiy glands were fixed for 2 mm in 7 50o neutral buffered formaldehyde (pH 7 2) and squashed in 40% acetic acid Tmmediately after squashing the slides were fro7en on dry ice and the siliconized covers!ip was removed. Subsequently, the preparations were postfixed dining 1 hour m a 1:9 formaldehyde (37%)-ethanol (95%) mixture, followed by thorough rinsing in distilled water All preparations were submitted lo a HNase treatment (1 mg/ml, Type I-A, Sigma) loi 2 hours at 37° After tins tieatment the prcpaiations were rinsed ui th distilled water and passed through three glycerol dianges over a period of 12 hours (glycerol, Meuk. analyt. grade, 87% aq sol w/w ; n = 1453). The preparations were covered with fresh glyceiol and a cover glass Some prep­arations of each gioup undergoing this procedure were stained with galloeyanin-chromalum according to Emarson, 1951 after the К Nase treatment None of these preparations revealed staining ot nucleoli, pufls and patches of cytoplasm in­dicating that the !{>> V digestion had been successlul

For the determination of the relative dry mass quantity of the puff regions at successive stages of puff formation a Leit/. double beam mtciference microscope with a tungsten light souice was applied The emission was stabilized by a "Del ta elektiomca" powei supply. All measurements were performed under conditions of interference contrast (even field) at 546 nm with a 50 χ (10/85) objective and a lOx eye piece The light intensities were recorded with a Knott photomultiplier combined with a Knott power supply and a Digitec voltmeter A Leitz photo-

430 ï h . Κ . H . Holt:

¿432 Ößi

Fig. 1. a Phase contrast photograph of a part of chromosome 4 of Drosophila hydei including region 4-81B (A) and the region used as a reference (JB). a and b are background areas with a surface identical to Л and В respecitvely. These areas were used for the determination of the optical path difference, b The same chromo­some part as in (a) is shown after 30 min of temperature treatment of the larva.

At region 4-81B a puff has developed

multiplier housing containing a variable diaphragm and a viewing prism were used for adjustment.

Using interference contrast, the difference in light intensity between a definite area of the background and an identical area of a biological object is linearly related to the dry mass of the object. By measuring optical path differences

A area (o.p.d.), the dry mass can be calculated from the equation M a = ,„„ in which

M = dry mass, z l = t h e o.p.d. of the area measured and a = the refractive in­crement of the object in water. This equation can be used only if certain conditions have been fulfilled. The immersion fluid should penetrate the object completely. By comparing the light intensities from equally sized areas of the background and selected parts of the object to be measured, integrated values of optical path differences can be obtained (Fig. 1). The differences in light intensity values are expressed relative to the difference between minimum and maximum intensity of the background (Fig. 2). This method of measuring the o.p.d. can only be applied if the maximum o.p.d. of the biological object does not exceed a value of 0.3 λ. Only under this condition, the relationship between light intensity and o.p.d. is linear (Davies, 1958). In order to meet this condition, it is necessary to immerse the polytene chromosomes in a medium with a refractive index higher than that of water. Embedding in glycerol (re= 1.453) serves this purpose. (See: Grampp et al., 1960; Galjaard and Szirmai, 1965).

The dry mass of chromosome regions after RNA digestion is composed of DNA and proteins. I t is well established that the DNA/protein ratio in inactive chromosome regions is fairly constant (Paul and Mateyko, 1970; Holt, 1970). However, if chromosome regions of different cells in the same preparation or in different preparations should be compared, the absolute quantities of DNA plus protein may vary due to differences in the level of polyteny of the chromosomes. As discussed in a previous paper (Holt, 1970) this feature which may cause incorrect

Local Protein Accumulation during Gene Activation. I I 431

object

background

Fig. 2. Schematic representation of the wave pattein in the fringe field during a measurement on a chromosome region. The value between maximum and minimum during a setting of the instrument is compared with the optical path difference between the object A and the background a. The o.p.d. by means of photo-electric

Α-α λ readings in even field interference contrast, J =

max — mm π

values if the quantities are directly compared, can be overcome by comparing ratios of quantities in puff regions олгег that of non-puffed reference regions in the same chromosome. In this study region 2,47A^8B 1 was chosen аь a reference for puff 2-480 and region 4,81С0-82В6 was chosen as reference for puff 4-81B. Since the integrated o.p.d. of a puff region and the reference region is measured over areas with a different surface which always is delimited by the same chromosome bands., the calculation of the relative values should involve a correction for the difference in surface area measured. I t is evident that sui face areas of puffs as well as reference regions may vary due to variations in degree of stretching of the chromosomes and to variations in the structural organization of the chromosomal sites (despirahzation of the nucleohistone in puffs).

It these factors are taken into account the following equation can be applied for the calculation of a relative value for the total dry mass of a puff region:

M puff A puff χ surface area rel. M = — — =

р M reference A reference χ surface area

Results

R e l a t i v e va lues for t h e t o t a l d r y m a s s of t w o puff regions, 2-48C

a n d 4 - 8 1 B were e s t a b l i s h e d a t 5, 10, 30, 60 a n d 120 m i n af ter t h e

o n s e t of a t e m p e r a t u r e t r e a t m e n t of t h e l a rvae . T h e re la t ive va lues were

also es tabl i shed for t h e s a m e c h r o m o s o m e regions in l a r v a e of t h e s a m e

d e v e l o p m e n t a l s t a g e w h i c h were n o t s u b m i t t e d t o a t e m p e r a t u r e t r e a t ­

m e n t .

432 Th.Κ.H. Holt: Local Protein Arnimiilation during Gene Activation. TT

The selection of the regions 2^:8C and 4-8IB from a total of 5 regions

responding with puff formation to a temperature treatment was made

because these regions are located in chromosome sections which appear

to be well stretched in a high percentage of the nuclei. Furthermore, the

region which is involved in a completely expanded puff at these loci

can be delimited more precisely than for the other temperature sensitive

loci.

In temperature treated larvae and in the controls, 8 to 11 sets of in­

tensity measurements were made on the regions mentioned at any of the

time intervals after the onset of the temperature treatment As shown in

the Table, the relative values for the total dry mass of the puff regions

vary considerably in different nuclei. However, despite this individual

Table. Relative values of total dry mass m region 2-48 С (rel. М'2—IS С)

Nucleus Unpuffed 5 min after the onset no.

1 2

3 4 5 6 7 8 9

10

0.395 0.435 0.456 0.460 0.495 0.596 0.588 0.472 0.360 0.680

of а Ь

0.808 0.633 0.740 0.740 0.534 0.830 0.640 0.930 0.735 0.564

Average relative value 0.494±0.101 0.715±0.123

variation it became evident that the average values calculated on the basis of relative values from individual regions in a puffed condition differed significantly ( p < 0.001) from the average value of the same region in non-puffed state. Irrespective of the time of temperature treatment, the average relative values for total dry mass of puff regions were higher than those of the same regions in non-puffed condition. I t appeared, from a comparison of the average values of the puft regions at successive stages after the onset of puff induction, that the average relative value for the dry mass gradually increases until 30 min after the onset of the temperature t reatment (Figs. 3 and 4). At this moment the average value has increased by a factor of 1.5 for region 2-48C and by a factor of 2.1 for region 4-8IB. If the temperature treatment is

θ τ Ύ

-

η=10ί

Ι

- с

Γ "

ό η = 9

ά η = 1 0

L

>η=10

-

-

1 Ι 1

¿η = 10

ι

region 2 - 4 Θ С

ι !

η = 106

ι

l'ig.

D 5 10 30 60 120

lime after onset of temperature treatment in mm

3. Average iclativc аіись for the dry mass in region 2-48C in untreated animals and at suoresaive stages after puff induction by a temperature treatment

С

о

l -

У*

- В

ce

ϊ 7 ra

1 6

E

Έ 5

Ο

Ε .4 Ο) (Λ ΙΌ Οί -»- 3 ( J ·'

5 (U

= 2 n i

oc

С

τ

с

n = 1G0

-Ί

—

С

г -

г

>η = 10

J-

>η = 10

_

I ι ι

)η = Β

.

6 η = 10

--

ι

region t - B 1 В

τ

η = 9 9

, 0 5 10 30 60

Time after onset of temperature treatment in mm

120

Fig. 4. Average relative values for the dry mass in region 4-81B in untreated

animals and at successive stages after puff induction by a temperature treatment

434 Th Κ H Holt

extended (nei я period, lonjiei than 30 min no mei ease of the a\eiao;o ielati\e л alno toi di\ mass m the puff legions oeeuis Puft 2-480' іс\са1ь a lowoi \aluo at GO mm than at 30 mm whereas at 120 mm the d\eragt di\ mass \A\UL IS M Í J similar to that at 30 mm (Fig 3) The dry mass values for puff 4-81B at 60 and 120 mm art lower than that at 30 mm (Fig 4)

Discussion

The data presented clearly demonstrate that the formation of two puffs which ait experimentally induced by a temperature treatment is accompanieel by an actual inciease in dry mass pei unit aioa of the chromosome I he inciease in relative values of the diy mass in the puff regions at successne stages of puff formation and maintenance of the puffed condition is verj similar to the increase in dye binding to pioteins at low pH (Holt 1970)

A comparison of the photometirc data on dye binding to proteins with the rnteiferometne data obtamed rn the present rmestrgation suggests that the increase in d \ e brnding capacrtj of chmrnosome regions under going puff formation is due to an inciease in piotein mass rathei than to a change in the configuration of ahead} piesent proteins

This suggestion seems likely because the inteifeiomctiie measuic-ments were made on chromosome piepaiations which were subrmttcd to RNA digestion by RXase

Although some of the RXA in the completely expanded puffs may be present in association with protein in the form of the characteristic R N P particles (ßerendes, 1969), RXase treatments appeared to be effective in the degradatrorr of RNA from these structures after frxatron wrth foimaldehyde (Stevens and Swift, 1966) The effectiveness of the RNA degiadation was fuithei indicated by application of the gallo-cyamnchiomalum staining piocedurc which failed to demonstrate the presence of RNA in the induced puffs after treatment with RXase RNA, theicforc may be evcluded as a factor contributing to the diy mass per unit area of the chromosome in the measurements reported Wrth regaid to the acid dye binding capacity of the puff regions the presence or absence of RXA appeared to be without conseeiuenees Neither cxtiaction of RXA with TCA, nor digestion with RXase affected the acid dye binding capacity of the puff legions (Holt, 1970)

Undei the condrtions used, DNA and proteins seem to be the mam components contributing to the dry mass quantity in the chromosomes Since in Droiophila hydei, like in other Drosophila species, puff foimation does not involve a local "ou t of phase" DNA synthesis, it is likely tha t the increase in diy mass value during puff formation is a consequence

Local Protein Accumulation during (.enc Activation. II 435

of a local incroa&c in p r o t e i n c o n c e n t r a t i o n . As was discussed i n a

prev ious p a p e r ( H o l t . 1070). t h e local increase in p r o t e i n c o n c e n t r a t i o n

is most likely t o be d u e t o a n a c c u m u l a t i o n of n o n - h i s t o n e p r o t e i n .

Acknowlnlgniicnts. The interference measurements «ere earned oui at the Dept. ot Cell Biology of the Medical Faculty a t Kottordam The author is very indebted to Prot. Dr. H. Caljaard for providing him with the opportunity to use the equip­ment as well as for the suggestions and introduction into the technique of inter­ference microscopy. Also the help of Mr. J de .losselm de Jong is gratefully acknowledged. Thanks are further due to I'rof Dr. H . I ) . Rerendes lor his con­tinuous support and interest

ІІСІРГСПРСЧ

Berendes. H. D. : Factors involved in the expression of gene activity in polytene chromosomes. Chromo.soma (Beri.) ¿4. 418-4.47 (ІЖІН).

- Activités des chromosomes polyténiques et des chromosomes plumcux. Induction and control of puffing. Ann. d'Embryol Morphogenèse. Suppl. 1. l.").4-lß4 (1969).

— Breugel. F. Μ. Α. van. Holt, Th. Κ. H.: experimental puffs in Drosnphila hyde> salivary gland chromosomes. Chromosoma (Beri.) Itt. :t.V47 (ΙΘΙΜ).

Davies. H. C : The determination of mass concentration by microscope inter-ferometry. In: General cytochemical methods (J. F. Danielli. ed.). p. öS- lö l . Хеи York: Academic Press 1958.

Einarson, L.: On the theory of gallocyanin chromalum staining anil lis application for quantitative estimation of basophilia A selective staining of evquisite progressa it_\ . Acta path, murobiol. se.ind. '28. 82-102 (19.Ή)

(¡aljaard, H.. Szirmai. J. Α.. Determination of the dry miss of tissue sections 1>\ interference microscopy. .). roy. micr. Soc 8t. 27 42 (19(10).

Grampp. W.. Hallen. О.. Rosengren. В.: Mass determination b\- mterterenee- and X ray microscopy. Exp. Cell Res. 19. 4:!7-442 (I9fi(i).

Holt. Th. Κ. Η : Local protein accumulation during gene acti\,ition. i. Quantitative measurements on dje binding capacitv at subsequent stages of putt lormation in Diowphilii lu/rlci. Chromo.soma (Beri.) :12. ()4-78 (1970).

Paul. J . S., Malevko, С. ΛΙ : Quantitative interference nucroscopv of jxilytene chromosomes 11. f'vtochemicnl studies vu iiltramicrospectrophotometrv. E\p. Cell Res. 59. 2:ì7-24:ì (1970).

Pettit. B. .1.. Rasch. R.W'. T n t n t e d hislidme imorporatmn into Itmsoplnln salivary chromosomes. .1 cell. comp. Phvsiol. l>8. .42.") .'t:t4 (19()(>).

¡Stevens, В Л.. Svwlt. Η.: ΗΝ Λ transport Irom nucleus to cvtoplasm in ('hitonnmus salivary glands. J (VII Biol. SI. Ô.V77 (19(i(i).

Swift. H.: Xucleic acids and cell mnrphologv in diptei'an salivary glands. In. The molecular control of cellular activitv (J. Allen, ed ) ρ TU 125. New York. McGraw-Hill 1902.

Drs. Th K. H. Holt Zoologisch Laboratorium Afdclmsi Celbiologie Driehiii/eivveg 2·Ν· .Nijmegen. Tocrnooivekl Nederland

INDUCTION OF CHROMOSOME PUFFS IN DROSOPHILA HYDE1 SALIVARY GLANDS

AFTER INHIBITION OF RNA SYNTHESIS BY α-ΑΜΑΝ1ΤΙΝ

Th.K.H. Holt and A.M.C. Kuijpers

Dept. of Genetics, University of Nijmegen, The Netherlands

15

ABSTRACT

The effect of injection of various concentrations (4 ng-0.5 д£/]аг а) of a-amanitin on chromosomal RNA synthesis in larval salivary glands of D hydei was investigated at subsequent time intervals (90 min — 20 hrs) after in­jection.

As revealed by autoradiography, 3H-uridine incorporation into the poly-tene chromosomes was greatly reduced as compared with that in control larvae. In α-amanitin injected larvae, new chromosome puffs could be induced by a temperature treatment. These puffs never attained a size com­parable to that in the controls. The newly induced puffs did not show specific incorporation of 3 H-undine.

Puffs which were active before the toxin was jpplied undergo a reduction in size and incorporation of 3H-undine.

INTRODUCTION

Some'of the most prominent features of puffs in the polytene chromosomes of Diptera are: a strong capacity for binding protein-staining dyes, a decon-densed state of the nucleoprotein and transcriptional activity (Berendes and Beermann, 1969). It was suggested that each of these features provides an essential contribution to the morphological manifestation of a puff (Berendes, 1968). The strong dye binding capacity results from accumulated non-histone proteins (Holt, 1970; 1971). The transcriptional activity results in new RNA molecules which become associated with protein within the puff. These complexes appear as RNP-particles (Beermann and Bahr, 1954; Swift, 1965; Stevens and Swift, 1966; Vasquez-Nin and Bernhard, 1971, Berendes, 1972).

In contrast to the idea that the accumulating protein, the despirahsation of nucleoprotein fibrils and the de novo synthesis of RNA are all processes which are involved in the determination of puff size, Beermann (1966) sug­gested that RNA synthesis is the crucial factor which determines the appear­ance of a puff. Arguments for this suggestion come from studies on the effect of various inhibitors of RNA synthesis on the persistence of puffs and Balbiani rings in Chironomus salivary gland chromosomes. A further argu­ment in favor of his suggestion was the observation that loops of the am-

16

phibian lampbrush chromosomes disappear if RNA synthesis is inhibited by actinomycin D (Gall and Callan, 1963) or by a-amamtin (Mancino, Nardi, Corvaja, Fiume and Mannozzi, 1971)

In Drosophda hydei, injection of high concentrations of actinomycin D failed to produce a complete disappearance of the puffs Moreover, the antibiotic did not inhibit de novo formation of puffs upon experimental induction (Bercndes, 1968) Recently, these íesults were confirmed for putts induced by the steroid ecdysterone (Poels, 1972) It was argued (Clever, 1968), that the results obtained indicate that the antibiotic did not inhibit de novo RNA synthesis completely in spite of the fact that it was undetec­table by autoradiography (see also Ellgaard and Clever, 1971) The iailuie ol the puffs to regress in the presence of the antibiotic could be due to a rigid configuration of the DNA resulting from its interaction with actino my cm

In order to find an answer to the question whether or not de novo RNA synthesis is the only factor determining the morphological manifestation of a puft, the ellect of α-amanitin was investigated. It is well established that this toxic octapeptide inhibits primarily chromosomal RNA synthesis by binding to the Mn2+-dependent RNA polymerase Bi and Вц (II) (Jacob, Sajdel and Munro, 1970 Kedinger, Gniazdowski, Mandel, Gissinger and Chambón, 1970) Theretore, if a-amanitm should be effective in complete inhibition of chromosomal RNA synthesis as well as in the suppression of the appearance of new puffs upon their experimental induction, it could be concluded that RNA synthesis is essential tor the formation of a puff

MATERIALS AND METHODS

All experiments were performed in vivo with mid third instar larvae of a wild type laboratory stock of Drosophila hydei reared under standardized condi­tions

Induction of new puffs was carried out by transferring the larvae from 25° to 370C After 15 mm at 37°C the salivary glands were dissected, fixed with alcohol-acetic acid (3 1), stained with aceto-orcein and squashed in 45'/ acetic acid The changes in the puffing pattern occurring as a consequence ot the temperature treatment were described previously (Berendes, Van Breugel and Holt, 1965) The relative size of the puffs was measured as described by Berendes (1968)

Protein staining was performed with Fast green (FCF) at pH 2 4 (Alfert and Geschwind, 1953) RNA synthesis was studied by autoradiography as previously described (Berendes, 1968) Exposure time was 7 days All auto

17

relative puft size

IB

16

H

12

10

08

ne

Τ

t

-

>

-

region 2-32A

¿

ι

I

ι

I 1 l i l i

region i-BlB

0 В 40 200 1000 β 10 200 1000 cone І я д / m i ;

Fig.l. Relative size of the puffs 2-32A and 4-81В following a 15 mm temperature treat­

ment at the end of a 5 hr pretreatment with vanous Oi-amanitin concentrations Each

point represents average values of 15 measurements on puff size in animals injected with

0 5 pi of a solution containing either none, 8, 40, 200 or 1000 Mg/ml Oi-amamtm. Δ,

control value in animals which did not receive an injection of Oi-amanitm nor a tempera­

ture treatment

radiographs were post-stained with 0.57' toluidine blue. 3H-Uridine (spec.act. 24 mCi/mM) was applied in vitro by incubation of the salivary glands during 15 min in Drosophila Ringer according to Becker (1959) con­taining 0.05 mCi/ml 3H-uridine.

The evaluation of the incorporation of the 3H-undine was based on grain counts. The number of grains over the temperature-induced puffs and that over a reference region were established. For the puffs 2-32A and 2-48Ç, region 468, - 47C, was used as a reference. For the puff 4-81 Β,, region TlAj — 75A4 was used. Grain counts were also performed on regularly active puffs, 2-28B, 4-85A and 5-95D. For these puffs, the regions

18

28C-30D, 81С 84D and 99A- 103D were used as reference regions In all instances, the results are expressed as ratios of grain numbers over the putted and the reference regions which do not contain obvious puffs

α AmaniUn was purchased from Boehrmger

RESULTS

a Puff induction in the presence of a-amamtm In order to establish the concentration oí α-amanitin which is most effec­

tive m the inhibition of chromosomal activity, groups of 15 larvae were injected with 0 5 μΐ/larva of a solution of either 0, 8 Mg/ml, 40 Mg/ml, 200 pg/l or 1000 Mg/ml α-amanitin. Five hours after injection the larvae were submitted to a temperature treatment (15 nun) and the relative size of the temperature-induced puffs, 2-32A and 4-8IB was measured As shown in Fig 1, none of the concentrations applied was effective in a complete inhib­ition of puff formation However, the response of both puffs measured, became very small if a concentration of 0 5 μg α-amamtin per larva was applied When a lower concentration of the octapeptide was applied (0 1 μg/laгva) for 5 hours, the response of puft 4-81В to the temperature treat­ment was almost the same as after injection of 0 5 μg of a-amanitin, whereas puff 2-32A under the same conditions attained a significantly larger diam­eter

In order to establish the time required to obtain effective inhibition of de novo putt formation, groups of 20 larvae were injected with 0 5 μΐ of a α-amanitin solution containing 500 μg/ml A temperature treatment was applied either directly, at 90 min, at 5 hours or at 20 hours after injection Measurements on the size of the puffs 2-32A and 4-8IB revealed that even at 20 hours after injection of the a-amamtin, small puffs did develop as a consequence of the temperature treatment (Fig. 2).

Apart from the appearance of new puffs in the presence of α-amamlin, it was observed that already present pulfs did not regress completely, though their diameters were smaller than in control glands.

The small pulfs which did arise alter a temperature treatment in the presence of α-amanitin revealed a strong staining reaction with Fast green at a low pH, indicating that accumulation of proteins within these puffs (see Holt, 1970) took place (Fig 3)

These results show that a-amarutin is effective insofar as it suppresses the development of tuli size puffs lollowing the onset of a puff inducing treat­ment The octapeptide is unable to inhibit puff formation completely. It could be argued that the appearance oí the new puffs following a tempera-

19

relative puft size

ί,-ΘΙΒ

. y / 1 . . .

^ ! 1 0 15 50 20 0 1.5 50 20

time after treatment with α-smanitin in hours

Fig.2. Relative size of the puffs 2-32A and 4-8IB following a 15 mm temperature treat­

ment immediately after injection, 90 nun, 5 hrs or 20 hrs after injection of

0 25 yug/larve Of-amamtin. Each point represents an average of 20 measurements Δ, con­

trol value (see Fig. 1).

ture treatment does occur because under the conditions applied, de novo RNA synthesis in the puff regions is not inhibited completely.

b. Undine Incorporation in the Presence of a-armnitin 3H-Uridme incorporation in the puffs was estimated on the basis of grain

counts in autoradiographs obtained after 15 min of incorporation of 3H-uridine. Larvae were injected with 0.5 ¿ig oamanitin and five hours later the salivary glands were dissected and incubated for 15 mm in Ringer's medium containing 100 ßg/m\ α-amanitin and 0.05 mCi/ml 3H-undine. The incuba­tion medium was kept at 370C. Salivary glands dissected from larvae 5 hours after injection of the solvent and incubated thereafter for 15 mm in Ringer's medium with 3H-uridine at 370C, were used as controls.

16

Η -

12

10

OB

η e

-

(

-

I

(

ι —

region 2-32 A

г

)

( > <

//

1 1 'i 1

>

regi

20

Fig.3. Puff 4-81B. a and a , region 4-81B of a salivary gland chromosome from an un­

treated larva, b and b ' , the same region after 15 min temperature treatment in vivo, с and c 1 , the same region following a 15 min temperature treatment after 5 hrs pretreatment

with 0.5 μg O-amanitin in vivo.

a, b and с photographed with red filter (590 nm); a 1 , b 1 and с , photopgraphed with

green filter (530 nm). The chromosomes were stained with aceto-orcein and fast green at

pH 2.4.

A comparison of autoradiographs of salivary gland chromosomes from a-amanltin treated animals with those of the controls revealed that in all instances the pattern of chromosomal incorporation in the α-amanitin treat­ment glands was different from that in the controls. Whereas in the controls the chromosomes display a typical irregular grain distribution, high numbers of grains over puffed regions and very low numbers over non puffed areas, the α-amanitin treated glands revealed a more or less homogeneous grain distribution over the whole chromosomes (Fig. 4). In the most, but not all nuclei of α-amanitin treated animals, the labeling density was obviously less than in those of untreated controls. The labeling density of nuclei within one gland as well as in different glands varied greatly. Grain numbers over newly induced temperature puffs in the chromosomes 2, 4 and 5 as well as over reference areas in the same chromosome were established. From table 1 it may be concluded that none of the newly appearing puffs revealed an in­crease in grain number relative to the reference region after induction in the presence of α-amanitin. The ratio of grain numbers over the already present puffs 2-28B, 4-85A and 5-95D and their reference regions was lower in the α-amanitin treated than in control larvae (Table 2). However, a comparison of the absolute grain numbers over the reference regions in α-amanitin treated and control glands showed for two of the reference regions chosen an increase in grain numbers. This increase may be a consequence of an overall nonspecific labeling of the chromosomes which is regularly observed in the α-amanitin treated glands. However, whereas the puffs recorded in Table 2 could easily be identified in the controls because of their high grain density

21

А К , . У28В

- чса -

• ,jï 32А / 48С

!^»

; · * ' , .i·.·

^ ¿ .ν , L^ ·,» 81 А 95 D-^-

^ ι г· * V. I ^ »

-' ^ f ^ збХ" •.#••

® -г f 10/j

. 1 ' ¿ t ^ ^k^ · ' t · . ' /32A

• · r-·

® ák

/02 А

' я . · » . · • • · r

36A

j r . · *

28B dOvj

Fig.4.

a. H-uridine incorporation during 30 min temperature treatment in vitro. Puffs and

their local incorporation are evident at the loci 2-28B, 2-32A, 2-36A, 2-48C, 4-8IB,

5-95D.

b. Same experiment as in a. Glands were obtained from larvae injected with 0.5 jUg

tt-amanitin 5 hours before.

22

Table 1 Ratios of grain numbers over induced puffs and reference areas in the same

chromosome Grain numbers were established over the puffs 2-32A and 2-48C and over a

reference area 2-46B! to 2-47Ci , the puff 4-81 В and the reference area 71 A! to 75A4

The values presented are averages of 10 determinations

Treatment applied

Puff locus

2-32A

2A&C

4-8 IB

Untreated

0 43±0 0 2 * 2

0 58+0 04

0.28±0 14

15 min temperature

treatment in vitro

1 73±0 09 2.07±0 02

1.23±0 06

15 rmn temperature

treatment in vitro,

following a 5 hr

in vivo treatment

with α-amanitin * '

0 32±0 24

0 46±0 02

0.27±0 03

* O-amamtin was also present in the incubation medium

* 2 S D

Table 2 Ratios of grain numbers of the regularly present puffs 2-28B, 4-85 A and 5-95 D

The grain number over the puff areas and over reference areas, 2-28C to 30D for puff

2-28B 4-81C to 84D for puff 4-85A and 5-99A to 103D for puff 95D was established

Each value recorded is an average of 10 determinations * S D

Locus

2-28B

28C-30D

4-8 5 A

81C-84D

5-95D

99A-103D

Grain number ratios

puff/reference

Control

0 60±0 07*

0 29+0 06

0 37±0 16

5 hrs treatment

with O-amarutin

0 30+0 12

0 20±0 08

0 26+0 13

Absolute gram number

over reference regions

Control

31.0+10.5

59.3±19 9

48 3±18.7

5 hrs treatment

with Of-amamtin

50 1+21.8

47 8±22 5

71.5±51 8

relative to the chromosomal background, the same regions did not show an obviously higher grain density if compared with neighbouring nonputfed regions in the a-amamtm treated glands It, therefore, may be suggested that despite the increase in absolute grain numbers in the reference areas, the undine incorporation in the puff regions is reduced in the α-amamtin treated glands

23

So far it is unknown whether the increased labeling over entire chromo­some sites in some of the a-amanitin treated glands results from an over all transcription or from defects in the processing of RNA transcribed by polymerase(s) which are not affected by the toxin It was established that in D hydei as well as in other Dipteran species the processing ot nucleolar RNA was impaired In many nuclei the nucleolus is fragmented and labeled mate rial is dispersed over the entire nucleus or at least over a substantial part of it (Holt, 1972 Serfhng, Wobus and Pamtz, 1972)

DISCUSSION

The toxic octapeptide α-amanitin is widely used as a specific inhibitor of chromosomal RNA polymerase activity (Fiume and Wieland, 1970, Jacob, Muecke, Sajdel and Munro, 1970, Kedinger, Gmazdowski, Mandel, Gissinger and Chambón, 1970 Lindell, Weinberg, Morris, Roeder and Rutter, 1970 Kedinger, Naret and Chambón, 1971) It was recently reported that the toxin not only impairs chromosomal RNA synthesis in the polytene chromo­somes of various Diptera (Beermann, 1971, Wobus, Pamtz and Serfhng, 1972 Ashburner, 1972), but also affects the processing of nucleolar RNA (Serfhng Wobus and Pamtz, 1972 Holt, 1972) An effect of the toxin at the nucleolar level was also reported for mammalian cells (Jacob, Sajdel and Munro, 1970,Niessing, Schmeders, Kunz, Seifart and Sekens, 1970)

Although the toxin generally reduces or inhibits the incorporation of 3H-uridine in already present puffs, the chromosomes remain labeled and display in many nuclei an uniform grain distribution Despite this uniform background the normal transcription products of the puffs (see Daneholt, bdstrom Egyhazi Lambert and Ringborg, 1970) are not produced in the presence of a-amanitin The characteristic high molecular weight RNA is not synthesized in glands treated with a-amanitin (Serfhng, Wobus and Pamtz, 1972)

The present study indicates that a temperature treatment is effective in the induction of the specific puffs if the glands have been submitted for five hours to the action of the toxin Although these puffs did not reveal any evidence for an increased labeling as compared to their inactive state, it is difficult to exclude that RNA synthesis was initiated in these loci In partic­ular, the random increase in grain number over the chromosomes which is found in many nuclei of the α-amanitin treated animals make it hard to distinguish as to whether a particular locus is actually transcribed or not At least some loci developed a puffed appearance and did show a specific high level of precursor incorporation in the presence of α-amamtin (Beermann,

"M

1971). On account of these observations, it may be concluded that the toxin

has a differential effect on chromosomal transcription. This could reflect the

activity of different polymerases, some of which are impaired by the toxin,

whereas others are not. Because of the observed differential action of the

a-amanitin on chromosomal transcription a definitive conclusion as to

whether or not transcription is a prerequisite for the development of a

morphological puff cannot be given.

ACKNOWLEDGEMENTS

The authors are indebted to Prof. Dr. H.D. Berendes and Prof. Dr. G Τ

Rudkm for many stimulating discussions.

REFERENCES

Ashbumer, M Ecdysone induction of puffing in polythene chromosomes of Drosophila

melanogüster Effects of inhibitors of RNA synthesis. Exp.Cell Res 71, 433-440

(1972).

Alfert, M., Geschwind, J.J. A selective staining method for the basic proteins of cell

nuclei Proc.nat Acad.Sci. (Wash ) 39, 991-999 (1953).

Becker, H.J. Die Puffs der Speicheldrusenchromosomen von Drosophila melanogaster 1.

Mitt Beobachtungen zum Verhalten des Puffmusters im Normalstamm und bei zwei

Mutanten, Giant and Lethal-Giant-Larvae Chromosoma (Beri ) 10, 654-678 (1959)

Beermann, W.. Differentiation at the level of the chromosomes. In Cell Differentiation

and morphogenesis, pp.24-54. Amsterdam-London North-Holland Publ.Cy (1966)

- Effect of Oamanitine on puffing and intranuclear RNA synthesis in Cluronomus sali­

vary glands Chromosoma (Beri.) 34, 152-167 (1971).

- Bahr, F. The submicroscopic structure of the Balbiam rmg. Exp.Cell Res. 6, 195-201

(1954)

Berendes, H.D. Factors involved in the expression of gene activity in polytene chromo­

somes. Chromosoma (Beri.) 24,418-437 (1968).

- The control of puffing in Drosophila hydei In Developmental studies with giant

chromosomes (W. Beermann, ed.). Berlin-Heidelberg-New York. Springer-Verlag 1972

(in press).

Beermann, W. Biochemical activity of interphase chromosomes (polytene chromo­

somes). In: Handbook of Molecular Cytology (A Lima-de-Fana Ed) , p.500-519.

Amsterdam-London North-Holland Publ.Cy. (1969).

Breugel, F M.A. van, Holt, Th.K H : Experimental puffs in Drosophila hydei salivary

gland chromosomes Chromosoma (Beri ) 16, 35^7 (1965)

?5

Clever, U Regulation of chromosome function. Ann.Rev Genet 2, 11-30 (1968).

Daneholt, В , Edstrom, Η.Ε., Egyházy, E., Lambert, В., Rmgborg, U. RNA synthesis in a

Balbiam ring in Chironomus tentons Cold Spr Harb.Symp.quant Biol. 35, 513-519

(1970).

Ellgaard, E.G , Clever, U RNA metabolism during puff mduction. Chromosoma (Beri.)

36,60-78(1971).

Fiume, L , Wieland, Th. Amanitins. Chemistry and action. Febs Letters 8, 1-5 (1970)

Gall, J.G., Callan, H.G. Structure and function of lampbrush chromosomes. Proc.lóth

Int.Congr Zool. 4, 280-282 (1963).

Holt, Th.K.H.· Local protein accumulation during gene activation. I. Quantitative meas­

urements on dye binding capacity at subsequent stages of puff formation in Droso-

phila hydei. Chromosoma (Beri.) 32, 64-78 (1970).

- Local protein accumulation during gene activation. II. Interferometnc measurements

of the amount of solid material in temperature induced puffs of ürosophila hydei.

Chromosoma (Beri ) 32, 428-435 (1971).

- Effects of O-amamtin on nucleolar structure and metabolism in Drosophila hydei

Expenentia (Basel) submitted for publication (1972).

Jacob, S.T., Muecke, W , Sajdel, E.M , Munro, H N. Evidence for extranucleolar control

of RNA synthesis in the nucleolus. Biochem biophys.Res.Commun. 40, 334-342

(1970).

- Sajdel, E.M., Munro, H N. Different responses of soluble whole nuclear RNA poly­

merase and soluble nuclear RNA polymerase to divalent actions and to inhibition by

ttamamtin. Biochem biophys.Res.Commun. 38, 765-770 (1970).

Kedinger, C, Gmazdowski, M., Mandel, J.L., Gissinger, F., Chambón, P.: OAmamtin. a

specific inhibitor of two DNA-dependent RNA polymerase activities from calf thymus

Biochem biophys Res.Commun. 38, 165-171 (1970)

- Naret, P., Chambón, P. Structural evidence for two Ctamamtm sensitive RNA poly­

merases m calf thymus. Eebs Letters 15, 169-174 (1971).

Lindell, Τ J., Weinberg, F., Morris, P.W., Roeder, R.G., Rutter, W.J. Specific inhibition

of nulear RNA polymerase II by O-amanitine. Science 170,447-449(1970).

Mancino, G., Nardi, I., Corvaja, N , Fiume, L., Mannozzi, V. Effects of Oamamtin on

Tntums lampbrush chromosomes. Exp.Cell Res. 64, 237-239 (1971)

Niessing, J., Schmeders, В., Kunz, W., Seifart, К H., Sekens, CE. Inhibition of RNA

synthesis by O-amarutin in vivo. Z.Naturforsch. 25b, 11 19-1125 (1970).

Peiling, С : Puff RNA in polytene chromosomes Cold Spr.Harb.Symp quant Biol 35,

521-531 (1970).

Poels, C.L.M.· Mucopolysaccharide secretion from Drosophila salivary gland cells as a

consequence of hormone induced gene activity. Cell Diff. 1, in press (1972).

Ringborg, U , Deneholt, В., Edstrom, J.E., Egeyházi, E., Lambert, В.' Electrophoretic

characterization of nucleolar RNA from Chironomus tentans salivary gland cells

J.molec.Biol. 51, 327-340 (1970).

26

Serfling, I-., Wobus, U., Paratz, R. Effect of (ï-amanitin on chromosomal and nucleolar

RNA synthesis in Chironomus thummi polytene chromosomes Febs Letters 20,

148-152(1972).

Stevens, В J., Swift, H.· RNA transport from nucleus to cytoplasm m Chironomus sali­

vary glands. J.Cell Biol. 31, 55-77 (1966).

Swift, H. Molecular morphology of the chromosome. In: The Chromosome. In Vitro

(C J. Dawe ed.) 1, 2 6 4 9 . Baltimore. Williams, Wilkins, Cy. (1965).

Vasquez-Nin, G., Bernhard, W. Comparative ultrastructural study of penchromatm- and

balbiam ring granules. J.Ultrastruct.Res. 36, 842-860 (1971).

Wobus, U , Panitz, R., Serfling, E. CV-Amamtin. its effect on RNA synthesis in polytene

chromosomes. Expenentia (Basel) 27, 1202-1203(1971).

27

28

EFFECTS OF α-ΑΜΑΝΙΤΙΝ ON NUCLEOLAR STRUCTURE

AND METABOLISM IN DROSOPHJLA HYDFJ

Th.K.H. Holland A.M.C. Kuijpeis

Address Depl. of Genetics, University of Nijmegen

Dnehuizerweg 200, Nijmegen, The Netherlands

29

a-Amanitin specifically inhibits the action of Mn2+-dependent (chiomo-

somal) RNA polymerase in vitro1. The activity of Mg2+-dependent RNA

polymerase which, in the intact cell, resides in the nucleolus, is unaffected

by the toxin if tested in an in vitro system2 3 4 .

However, in intact mammalian 5 6 as well as insect cells7 the toxic octa-

peptide causes changes in nucleolar morphology and metabolism, fragmen­

tation of the nucleoli7 8 9 and an impairment of the processing of nucleolar

RNA7 8 were recently reported. The present paper describes the effects of

a-amanitin on nucleolar morphology and RNA metabolism in poly tene sali­

vary gland nuclei of Drosopliila hydei after injection of the toxin into mid-

third instar larvae.

METHODS

Mid-third instar larvae of a laboratory stock of Drosopliila hydei were inject­

ed with various concentrations of a-amamtin (Boehrmger) in Drosophila-

Ringer 1 0 . At various time intervals, from 90 mm to 20 hrs after injection,

the incorporation of 3H-uridine into chromosomes and nucleoli of explanted

glands was investigated autoradiographically (see below). These experiments

revealed that at 5 hrs after injection of 0.5 μg a-amanitin per larva, a maxi­

mal reduction of chromosomal incorporation was achieved 1 1 . All observa­

tions to be reported here were obtained following the application of 0.5 μg

a-amanitin per larva. At 5 hrs after injection of the toxin, salivary glands

were dissected and incubated for 15 min in Ringer's solution 1 0 containing

0.05 mCi/ml 3H-uridine (spec.act. 24 Ci/mM) and 100 Mg/ml a-amanitin.

Autoradiographs were prepared as described previously1 2 and exposed for 7

days.

The diameters of compact nucleoli were measured with an ocular microm­

eter and labeling density was established on the basis of gram numbers per

surface area.

The incorporation of 3 Η-uridine into newly synthesized RNA was assessed

by sucrose gradient centnfugation of RNA extracted from 100 salivary

glands according to the method of Pelling 1 3 . The isotope was incorporated

in vivo during 30 min, 5 hrs after the injection of 0.5 μg/larva a-amanitin.

Larvae were injected with 0.5 μΓι 3H-uridine. Carrier RNA was extracted

with SDS-phenol from 0.5 g frozen-powdered larvae. Radioactivity was

measured with a Philips scintillation spectrometer and calculated after

quench correction as distintegrations per min.

30

RESULTS AND DISCUSSION

Among 30 pairs of glands prepared for autoradiography, 18 revealed varying degrees of fragmentation or dispersion of the nucleolus in 20 to 90r/r of the nuclei (Fig.la.b). The diameters of compact nucleoli were significantly in­creased as compared with the controls not submitted to an a-amanitin treat­ment. The average diameter was 37.4+6.0 μ (n = 27) in a-amanitin treated animals compared to 22.1 ±8.2 μ (η = 26) in the controls. Moreover, the nucleoli of a-amanitin treated salivary gland cells were labeled more densely than those in the controls (table 1), suggesting a defect in the mechanism(s) involved in the release of RNA from the nucleolus.

Fig. 1. H-uridine incorpo­

ration in nucleoli oiDroso-

phila hydei salivary glands.

a and b, Nucleoli from

larvae treated for 5 hours

with 0.5 pg a-amanitin (in­

jected) and subsequent in­

cubation of the salivary

glands ( 15 min) in Ringer's

solution containing 0.05

mCi/ml 3 Η-uridine and 100

pg/ml a a ma nit in. c, Nu­

cleolus of a control animal

injected with Ringer's solu­

tion, 5 hours before dissec­

tion of the glands and 15

min incubation with 0.05

mCi/ml H-uridine.

3]

Table 1· Grain density of H-undine labeling in salivary gland nucleoli of a-amanitin

treated larvae of D. hydei and in nucleoli of control animals which were injected with

Ringer's solution1 instead of O-amanitin

Heavy labeling

(no grain counts possible)

Medium labeling

(more than 10 grains per square μ)

Weak labeling

(less than 10 grains per square μ)

Nucleoli

of a-amamtin

treated

n = 160

89(5670

47(2970

24(15%)

Nucleoli

of controls

n= 191

56 (29%)

64 (34%)

71 (37%)

A comparison of the radioactivity profiles of salivary gland RNA extract­ed from a-amanitin treated and control animals (Fig.2) revealed obvious differences in the relative amounts of 28S, 18S and low molecular weight (3-7S) RNA.

The relative quantity of a fraction with a molecular weight greater than 28S, which should include the 38S precursor of ribosomal RNA1 4, was unaffected in the a-amanitin treated animals. On the basis of total radioac­tivity in RNA extracted from salivary glands of larvae treated with a-ama-nitin in comparison with that of the controls, it seems that transcription of nucleolar RNA is not quantitatively impaired by the toxin. This suggestion finds support from the autoradiographical data. On the other hand, accumu­lation of the radioactivity in the nucleolus and the decrease in 18 and 28S RNA species indicate that the processing of the ribosomal precursor RNA may be impaired following the application of the toxin in vivo. The similar indications obtained in studies on the nucleolar RNA of Chironomus'1 15 in which the toxin was applied to isolated salivary glands are probably not artifacts of the in vitro conditions. Because, in the experiments described, whole salivary glands were extracted, the origin of the strongly increased relative quantity of low molecular weight RNA in the 3-7S region in a-ama-nitm treated glands remains unknown. It is reasonable to assume unimpaired transcription of 4S and 5S genes7, to which has been added small molecules from other sources, possibly breakdown products of ribosomal RNA of which the normal processing has been impaired.

32

OD 260

10 20 30 Ш 10 20 30 40 50 traction №

Fig.2. Radioactivity profile of Η-uridine labeled RNA extracted from salivary glands

from mid-third instar larvae of Drosophila hydei which were injected with 0.5 μΟ/ΐ3Γν3 3H-uridine at 5 hours after injection of 0.5 β% O-amaratm/larva. The glands were dissected

30 mm after injection of the isotope. In each of three replicate experiments 100 pairs of

salivary glands were used for RNA extraction as described by Peiling' 3 . a, Profile of RNA

from control glands obtained from animals which received an injection of Ringer's solution

instead of ûi-amamtin. b, Profile of the RNA of m vivo Oamamtin treated salivary glands.

33

ZUSAMMENFASSUNG

Fünf Stunden nach Injektion von 0.5 pg a-Amanitin in Larven des III. Stadiums von Drosophila hydei zeigen viele Nukleolen der Speicheldrüsen­zellen eine Fragmentierung und die meisten eine erhöhte Markierung mit 3 H-Uridin.

In Tieren, die mit a-Amanitin behandelt wurden, ist der Grad des 3 H-Uri­din Einbaus in 28S- und 18S-RNS geringer, während der Einbau in nieder­molekulare RNS (3-7S) im Vergleich mit Kontroll-Tieren stark zugenommen hat.

REFERENCES

1. L. Fiume and Th. Wieland, Febs Lett. 8 (1970).

2. S.T. Jacob, E.M. Sajdel and H.N. Munro, Biochem.Biophys.Res.Commun. 38, 765

(1970).

3. С. Kedinger, M. Gniazdowski, J.L. Mandel, F. Gissinger and P. Chambón, Biochem.

Biophys.Res.Commun. 38, 165 (1970).

4. T.J. Lindeil, F. Weinberg, P.W. Morris, R.G. Roeder and W.J. Rutter, Science 170,

447(1970).

5. S.T. Jacob, W. Muecke, E.M. Sajdel and H.N. Munro, Biochem Biophys.Res.

Commun 40, 334(1970).

6. J. Niessing, B. Schmeders, W. Kunz, K.H. Seifart and CE. Sekens, Z.Naturforech

256, 1119(1970).

7. E. Serfling, U. Wobus and R. Panits, Febs Lett. 20, 148 (1972).

8. V. Marmozzi and L. Fiume, Expl.Cell Research 67, 311 (1971).

9. F. Meyer-Schultz and A. Porte, Cytobiol. 3, 387 (1971).

10. H.J. Becker, Chromosoma 10, 654 (1959).

11. Th.K.H. Holt, in preparation.

12. H.D. Berendes, Chromosoma 24, 418 (1968).

13. С Pelling, Cold Spring Harbor Symp.Quant.Biol. 35, 521 (1970). 14.

14. U. Ringborg, B. Daneholt, J.E. Edström, E. Egyházi and E. Lambert, J.mol.Biol. 51,

327(1970).

15. U. Wobus, R. Pamtz and E. Serfling, Experientia 27, 1202 (1971).

34

SAMENVATTING

Het onderzoek van de veranderingen in lokale macromoleculaire samenstel­ling welke optreedt tijdens de aktivering van een chromosoom-locus werd uitgevoerd aan puffs, welke werden geïnduceerd door een temperatuurbehan-dehng (Berendes, Van Breugel en Holt, 1965) Door overbrengen van larven van 25CC naar 370C ontstaan m de polytene speekselklier chromosomen van Drosophila hydei 6 nieuwe puffs

In eerste instantie werd de kleurstofbinding van Naphthol Yellow S in twee van de opkomende puffs gemeten met behulp van een Zeiss ultramicro-spectrophotometer m relatie tot het DNA-gehalte van bepaalde chromo-soomgebieden De metingen werden uitgevoerd na kleuring van kneusprepa­raten met een Feulgen-kleunng en een Naphthol Yellow S kleuring (pH 2 4) Het DNA reageert na hydrolyse specifiek met de basische fuchsine (Feulgen-procedure), terwijl eiwitten reageren met N Y.S Op verschillende tijdstip­pen na begin van de temperatuurbehandeling werden metingen verricht De nieuwe puffs bereiken hun maximum-afmeting tussen 10 en 20 minuten na het begin van de behandeling Tijdens deze periode neemt de N Y S -binding aan eiwit toe tot ongeveer 200% van de oorspronkelijke waarde De kleur­stofbinding kan worden verwijderd door een behandeling met het proteolyti­sche enzym pronase

Speciale kleunngsmethoden werden toegepast om de aanwezigheid van tryptofaan aan te tonen Deze methoden gaven aanwijzingen voor de aanwe­zigheid van tryptofaan in het 'puff-eiwit Deze gegevens maken het waar­schijnlijk dat zich een met-histon eiwit in de puffs bevindt

Autoradiografische experimenten waarbij de inbouw van radioaktieve aminozuren in de puffs werd onderzocht leverden een negatief resultaat De aminozuren werden in de larven geinjiceerd voordat de puff-induktie plaats­vond en wel op verschillende tijdstippen tussen 15 min en 40 uur voor het begin van de puff-induktie

De inbouw van acetaat, methionine en ethiomne werd eveneens onder­zocht Geen van deze groepen werd specifiek in de puffs ingebouwd tijdens het ontstaan van de puffs

Metingen van de hoeveelheid droge stof in de individuele puffs op verschil­lende momenten van hun ontwikkeling werden verricht met behulp van een Leitz dubbelstraal interferentie-microscoop Bij de preparatie van de te

35

meten chromosoomgebieden werd zorggedragen voor volledige extractie van nieuw gesynthetiseerde RNA uit de puffs. Deze metingen gaven een toename in droge stof tijdens het onstaan van de puff aan. De kinetiek van de toe­name kwam overeen met die welke gevonden was voor de toename in kleur­stofbinding

Uit de totale groep van expenmenten kan geconcludeerd worden dat het optreden van nieuwe puffs gepaard gaat met een lokale kwantitatieve toe­name in met-histon eiwit Dit eiwit wordt niet de novo gesynthetiseerd

De accumulatie van het eiwit bleek onafhankelijk van de initiatie van RNA-synthese in de puffs op te treden Remming van de RNA-synthese met actmomycine D verhinderde de accumulatie van het eiwit niet Ditzelfde effect werd gevonden indien met een andere remstof, het a-amamtine welke specifiek de Mn2+-afhankelijke RNA-polymerase В inaktiveert, de chromo­somale RNA-synthese werd onderdrukt

Een onderzoek van de werking van a-amamtme onder m vivoomstdn-digheden (na injectie in de larve) wees uit dat remming van de chromosomale RNA-synthese eerst met zeer hoge concentratie (5 Mg/larve) en na lange incubatie (minimaal 5 uur) effectief geschiedde Echter, met alle chromo-somen-loci werden in hun RNA-synthese-aktiviteit geremd Autoradiografi­sche experimenten wezen uit, dat RNA-synthese in nieuw geïnduceerde puffs met werd geïnitieerd Bepaalde reeds aktieve puffs bleken echter RNA-syn­these, hoewel op een lager niveau, te continueren

Opmerkelijk was de observatie dat in α-amanitine behandelde larven ook de nucleolaire RNA-synthese een modificatie scheen te ondergaan De in­corporatie van 3H-uridme leek aanmerkelijk verhoogd in vergelijking met de onbehandelde kontrolelarven Bovendien werd in vele cellen fragmentatie van de nucleolus waargenomen

Een studie van de nucleolaire RNA wees uit, dat de synthese van de nbosomale voorloper niet door het a-amamtine geremd was De ombouw van deze voorloper tot 18,28S RNA was echter duidelijk gereduceerd. Ook werd een enorme toename van laagmoleculaire RNA (3-7S) waargenomen Gecon­cludeerd mag worden dat a-amamtine onder in vzvo-omstandigheden de ombouw van de nbosomale RNA beïnvloedt, terwijl op het niveau van de chromosomale polymerase-aktiviteit de remming onvolledig is, dan wel bepaalde specifieke RNA-polymerase-aktiviteiten onaangetast laat

36

STELLINGEN

I Het opkomen van een puff gaat gepaard met een accumulatie van met-histon eiwitten

Dit proefschrift

II De hypothese van Ellgaard en Clever, dat puff-vorming mede het gevolg is van RNA-degradatie, wordt met ondersteund door de resultaten van hun expenmenten

Ellgaard, E G , Clever, U , Chromosoma (Beri ) 36, 60-78, 1971

III De proefomstandigheden, waaronder Clark en Felsenfeld hun experimenten deden, rechtvaardigen met de door deze auteurs getrokken conclusie, dat de DNA in chromatine voor de helft bedekt is met eiwitten

Clark, R J , Felsenfeld, G , Nature New Biol 229, 101-106, 1971

Musky, A E , Proc nat Acad Sci (Wash ) 68, 2945-2948, 1971

Itzhaki, R F , Biochem J 125, 221-224, 1971

IV Het is te betwijfelen of het gebruik van diethylpyrocarbonaat als RNAase-remmer bij de isolatie van polysomen, zoals voorgesteld door Schrauwen en Lmskens, als algemene methodiek aan te bevelen is

Schrauwen, J , Lmskens, Η F , Planta (Bed ) 102, 227-285, 1972

V Een experimenteel bewijs, dat de primordiale kiemcellen van het kippe-embryo actief migreren van de 'germinal crescent' naar de gonade, is met geleverd

Dubois, R J , J Exp Morph 21 225-270, 1969

VI De veronderstelling van Truden en Franklin, dat tijdens hun experimenten de 'coat'-eiwitten van virussen geassocieerd blijven met nbosomen, is zeer on­waarschijnlijk

Truden, J L , Franklin, R M , J Virol 9, 75-84, 1972

VII De hypothese van Ray, White en Brock, dat het moleculaire mechanisme van thermophihe bij bacteriën berust op verandenngen in de celmembraan, is aanvechtbaar

Ray, Ρ Η , White, D С , Brock, Τ D , J Bact 108, 227-235, 1971

Vili Het is niet juist om, bij een gedragsgenetische analyse, de waarde van de bestudering van de effekten van afzonderlijke genen te zeer te benadrukken, met verwaarlozing van de multifactonele (polygene) bepaling van het gedrag

Thiessen, D D , Psychol Bull 75, 103-105, 1971

Wücock, J , Psychol Bull 72, 1-29, 1969

IX In tegenstelling tot de gangbare opvatting, kan de verhoging van het fosfaat­gehalte van het oppervlaktewater slechts voor een gering deel toegeschreven worden aan het gebruik van fosfaatmeststoffen in de landbouw

Landbouwkundig Tijdschrift 82, 185-186, 1970

De Margriet, 6 juni 1970

X Het aanwezig zijn van persoongebonden parkeerplaatsen is niet rationeel, het is te beschouwen als een naïeve poging tot het creëren van een statusonder-scheid

Th Κ Η Holt,

Nijmegen, 2 jum 1972