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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
197 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 Gerrit­
sen, 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 temperatuurbehandeling, 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 (Beermann, 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 aktiviteit (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 octapeptide a-amanitine (Fiume en Wieland, 1970) Men neemt aan dat a-amamtine 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 puffvorming 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 interferentiemicroscopie 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 puffvorming 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 Chromosomenstruktur 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. Chromosoma (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
Untersuchungen anChironomus
Pettit, В J , Rasch, R W
Autoradiographische
tentons Chromosoma (Beri ) 15, 71-122 (1964)
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
Stedman, E , Stedman, E
Febs Letters 20, 148-152 (1972)
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
Teng, С , Hamilton, Τ Η
San Francisco-London-Amsterdam Holden Day Ine 1964
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 tempera­
ture 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 trypto­
phane. only a positive result «ith the staining reaction on the indole groups was
obtained lor induced puffs.—Injection of tntiated sodium acetate, methionine3
J
H -methyl, ethionine-H -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.
Introduction
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 c o n s i d e r a b l e q u a n t i t i e s of h i s t o n e s
a n d n o n - h i s t o n e p r o t e i n s ahsociated w i t h nucleic a c i d s . A p a r t from t h e i r
role a s 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 b e e n c o n s i d e r e d 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 g e n e t r a n s c r i p t i o n . B i o c h e m i c a l s t u d i e s on isolated
c h r o m a t i n i n d i c a t e d 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 i n t e r ­
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 t o n e s ( F r e n s t e r , 1 9 6 5 :
W a n g . 1 9 6 8 : T e n g a n d H a m i l t o n . 1969). ΐ η t h e s e in v i t r o s t u d i e s ,
h o w e v e r , a c i d i c 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 s o c i a t i n g h i s t o n e - D X A c o m p l e x e s , or r e v e r s i n g h i s t o n e 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 ( S p e l s b e r g a n d H n i l i c a , 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 l a r v a e , o n e oi t h e first
e v e n t s o c c u r r i n g u p o n g e n e a c t i v a t i o n s e e m s t o he a local a c c u m u l a t i o n
of n o n - h i s t o n e p i o t e i n s T h 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 i n d i n g capacity of c e i t a i n d y e s a t low p H (Swift, 1962
Beiendes
1967) T h e local i n c r e a s e in d y e b i n d i n g c a p a c i t y as o b s e i v e d w i t h light
g i e e n (Clcvei, 1961), fast g r e e n ( p H 2 4), m e r c u r i c b i o m p h e n o l b l u e
(Bei e n d e s u n p u b l i s h e d ) a n d n a p h t h o l \ e l l o w 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 t o reflect g e n e a c t i v i t y ( B e e i m a n n 1952
Polling, 1964) S i n c e 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
i n h i b i t i n g p i o t c i n s y n t h e s i s n e i t h e r a f f e c t e d t h e i n c i e a s e in d y e b i n d i n s ;
noi t h e f o i m a t i o n of puffs u p o n e x p e r i m e n t a l i n d u c t i o n it w a s s u g g e s t e d
t h a t t h e i n c i e a s e in d y e b i n d i n g l e s u l t s f i o m 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 r o m r/c novo s y n t h e s i s of p i o t e i n s ( B e r c n d e s 1968)
T h i s 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 h e p i c s e n c e ot t n t i a t e d a m i n o a c i d s n o specific labelling of t h e puff
sites ionici be olisci v e d (Sw itt 1964 P e t t i t a n d R a s c h 1966 Bei e n d e s
1967) So f a i , h o w c v c i o n l y R u d k i n (1962) h a s p i o v i d e d s o m e q u a n t i
t a t i v e e v i d e n c e foi a local i n c i e a s e in p i o t c i n c o n t e n t oí a c t i v e chi o m o
s o m e i egions
T h e a m i of t h e p i e s e n t s t u d v is t o q u a n t i f v local chan»c's m c h i
b i n d i n g c a p a o H d i n i n g s u b s e c | u c n t s t a g e s in t h e р ю с е ь ь ot puff f o r m a
t i o n 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 g i a p h i c 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 w h i c h 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 i n c r e a s e in d \ e b i n d i n g d u i m g
puff f oi m a t i o n
Material 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 37 0 C 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 (37 0 o) 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 tiyptophane 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 8 0 o 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)
1
b) For idcntilication of free a-ammo group ! the ninhydrin Sthitt reagent «as
used T h t staining procedure «asessentially as described b\ Yasuma and Ttchikawa
(1953) (see also Arnold. 19(58)
c) .Mercurv orange v a s 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 « a s 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 « a s 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
0
measurements «ere hydroljscd with X HCl toi 10 mm at (>0 C, 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
0
1 hour in a 0 25'Ό naphthol vello« S solution in 2 5 o acetic acid rinsed shoith in
0
l 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 t h 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 K e \ 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 X2
5X3
5K1
5N5
5 N,
Region 2-48 С
Band 2 - 4 7 B 2 a
IA430
ΙΑ 550
A 430
A 550
A 430/A 550
150
122
90
120
60
226
158
101
80
4«
96
79
91
107
103
57
29
44
61
44
20
17
38
36
60
110
65
87
73
96
80
47
92
71
107
1.27
1.75
1.90
1.80
2.83
1.24
1.05
1.25
1.50
1.34
Average
1.65 — 0.51
3o
30
09
80
77
IA430/TA550
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 « a s used. Unless stated
otherwise, the time of exposure was 7 days. After developing the films with
Kodak DI9h, fixation and thorough rinsing in t a p water, the slides were stained
through the film with 0.5 o o 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·
ï h . Κ. H. Holt:
68
3
out with L-ethionine-ethyl-H (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 (Calbiochem, 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 photo­
graphed 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 p H 2.4 and naphthol yellow S at low p H revealed an inten­
sive 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
Merrillic bromphonol blue on
basic and acidic pmteins
+ |
+
I
Fast green at pH 2 4 on free
cationit gtonps ot proteins
) |
-J-
-^
Лaphthoi u-llow S at pH 2.4 on
free cationic groups of proteins
f+
t-
I
Mercury orange on SH groups
-
—
—
-|-
-
τ
-¡- -*•
h
-
+ +
X (1 naphtln 1) etliv lene diamine
dihydrocliloiidc on indole groups
of tryptophane
Ninhydrm Schiff leagent on
free α-amino gioups
Fast green at pH H attei remoial
οί ш а і іс acids on basic proteins
(histones)
Fast green at pH 2 4 after
hot TCA extraction
I
—
—
+ -f
Fast green at ρ Η 2 4 after
pronase digestion
Fast green at ρ Η 2.4 after
RNase digestion
|-+
+4-
Intelbands
+
+
+
—
-
I
I
Xucleolus
+-^
4
f
I—
-ι­
+ —
+—
T h e d y e s i c a c t i n g with c a t i o n i c giuujjb oí p r o t e i n s sucli аь f a s t green
a n d n a p h t h o l y e l l o w S a t low p H were n o t influenced in t h e i r a f i i n i t y
t o i n d u c e d pufis by e x t r a c t i o n s of n u c l e i c a c i d s w i t h h o t TCA or d i g e s t i o n
of R K A with 1 m g / m l (4 h o u r s a t 3 7 4 ' )
Pronas,e d i g e s t i o n a f t e r s t a i n i n g w i t h f a s t green a n d n a p h t h o l y e l l o w S
d i d r e m o \ e t h e d y e s from t h e puffs a s well a s from t h e l e s t ot t h e c h r o m o s>ome a n d t h e n u c l e o l u s 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 w i t h
p r o n a s e no s t a i n i n g r e a c t i o n w i t h d y e s b i n d i n g t o c a t i o n i c g i o u p s w a s
o b s e r v e d (Fig 3)
F r o m t h e s e 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 i n d i n g c a p a c i t y as r e v e a l e d b y t h e n e w l y 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 c o n f i g u r a t i o n of
a l r e a d y p r e s e n t p r o t e i n s i n v o l v i n g t h e l i b e r a t i o n of c a t i o n i c g r o u p s a n d
i n d o l e 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 r e a c t i o n s u g g e s t s a
70
Th. К. H. Holt:
Ρ
Fig. За and b. Puff 4-81 В at 15 min after onset of a temperature treatment photo­
graphed 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
Local Protein Accumulation during Gene Activation. 1
71
relative ratio of absorption integrals
at ""/SSO η μ
region 2-1.8 С
i
n=10<j>
ψηΠΙ
H
n.-9
ό n= 9
n=9 9
n = 10
J
0
I
S
L
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 B 2 at one side and the bands 81 B 6 and 81 C I
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. Calcula­
tions 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 d a t a 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 t h a t 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
at ""/SSO mjj
5 L
region ί. 61Β i
¿η 9
η 10
in-10
άη 11
η IG Ο
¿ η 10
0
5 10
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 u l a t n e
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 i e a t m e n t For putt 4 81 В the
lelative incicase of the dye binding to catiomc gioups was 1 9 within
5 mm of t i e a t m e n t Also this puff revealed a maximum value ot the
quotient aftei 30 mm of t i e a t m e n t (Kig 5)
These icsults indicate t h a t the d j c binding capacitj of pioteins
piesent in each ol the two pufl regions studied, inereasts during puff
formation b\ about a factoi of 2 F u i t h e r m o i e these d a t a show t h a t 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 p a t t e r n ot mcoipoiation ot t u t i a t e d amino acids adimnisteied
at various times puoi to the onset of puff induction was investigated I t
is well established t h a t short pulses of t u t i a t e d 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 t o
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 -tryptophane (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
Incorpora tion in
Labeled
compound
Time of
administration
prior to puff
induction
J î H 3 L amino acids
40 hours
30 hours
20 hours
45 mm
15 mm
H 3 tryptophane
lb hours
15 mm
Methionine H 3 methyl
30 min
++
+
+
1
Band -Γ
λ 11
interband oleoli
C\to
plasm
_
_
_
_
=
±
+
^
+
+
Puffs
-i-
л-
—
-fc
_z:
h1
+
+
Etlnonint H cthjl
30 mm
-L
•Soditirn buarbonate C14
30 mm
-
-
Sodium acetate H J
30 mm
60 mm
+
+
+
+
1
±
+
_i_
^H
L
l· +
+-
++
+
+
+
1
1
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 d aminoacids 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-H 3 methyl (0 5 μΟ') at
30 mm before onset of a temperature tieatment 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-H 3 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 t n t i a t e d acetate (1 μC71aIva) or
C 14 -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 t n t i a t e d 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 carboxylation, 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 X Y S 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 nonpuffed 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.
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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. Auloiadiogiaphische Untersuchungen an СІпюпотич tenían^. Chromosoma (Beil.) IS. 71 122
(1904).
Pettit, B. J , Rasch, R. \Y : Tritiated histidme mcorpoiation into Оіочоріпіп sali­
vary 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, e d ) , 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
Тн К
hvdei
II H O L T
('ell ВіоІод\ Section. Department ol ZoolugN. L'mversitj of Nijmepen
Koeenecl Noxember 7. 1970
Ucepted N o \ c i n b e r l 4 . 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 t h a t of puff 4-81B, 210%. I t is concluded t h a t this increase in
dry mass is due almost entirely to a local accumulation of non-histone protein.
Introduction
I t is well e s t a b l i s h e d 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 display
an intensive
staining reaction with a variety
of
b i n d i n g a t low p H t o a v a i l a b l e c a t i o n i c g r o u p s of p r o t e i n s (Swift,
dyes
1962;
B e r e n d e s , 1 9 6 8 ; 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 y e l l o w S b i n d i n g a t successive s t a g e s of i n d u c e d puff g r o w t h i n
Drosophila
hydei r e v e a l e d a g r a d u a l i n c r e a s e 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 % i n t e m p e r a t u r e i n d u c e d puffs ( H o l t , 1970). T h i s
increase in d y e binding c a p a c i t y m a y result either from a n a l t e r a t i o n in
t h e c o n f i g u r a t i o n of p r o t e i n s p r e s e n t a t a puff f o r m i n g locus p r i o r 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 i n c r e a s e i n 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 a c i d s 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 , n o e v i d e n c e 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 w a s 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
an
i n t e r p r e t a t i o n of t h e i n c r e a s e d 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
change
in p r o t e i n
configuration
involving
the liberation
429
of
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 c o u l d n o t b e
catiomc
excluded
t h a t t h e failure t o o b s e r v e a definite i n c r e a s e of l a b e l e d m a t e r i a l i n t h e
i n d u c e d puffs w a s 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
i n v o l v e d 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 g e n e r a l l y c o n s i d e r e d t o b e a m o r p h o l o g i c a l
manifestation
of g e n e a c t i v i t y
S i n c e a c h a n g e i n local d y e
c a p a c i t y of a puff f o r m i n g r e g i o n o c c u r s a l m o s t i m m e d i a t e l y
binding
following
t h e o n s e t of puff i n d u c t i o n , i t w a s 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 g e n e a c t i v a t i o n
( B e r e n d e s , 1968). T h e r e f o r e
a
s t u d y o n t h e c h e m i c a l b a s i s of t h e i n c r e a s e 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 g e n e a c t i v a t i o n m
higher organisms.
I n o r d e r t o e s t a b l i s h w h e t h e r t h e i n c r e a s e i n 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 i n p r o t e i n c o n f i g u r a t i o n or t o a n i n c r e a s e i n p r o t e i n
m a s s , 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 w e i e
performed
after R N A digestion
Material and Methods
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 5 0 o 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 u i t h 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 prep­
arations of each gioup undergoing this procedure were stained with galloeyaninchromalum according to Emarson, 1951 after the К Nase treatment None of these
preparations revealed staining ot nucleoli, pufls and patches of cytoplasm in­
dicating 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 " D e l t a
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 K n o t t 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 chromo­
some 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 in­
crement 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 t h a t 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 4,81С 0 -82В 6 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:
rel. M
р
M puff
= —
—
M reference
A puff χ surface area
=
A reference χ surface area
Results
Relative
a n d 4-81B
values
for t h e t o t a l d r y m a s s of t w o puff r e g i o n s , 2 - 4 8 C
were established
a t 5, 10, 30, 60 a n d
120 m i n a f t e r
the
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 r v a e . T h e r e l a t i v e v a l u e s w e r e
also e s t a b l i s h e d for t h e s a m e c h r o m o s o m e r e g i o n s 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 stage which 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 ­
ment.
432
Th.Κ.H. Holt: Local Protein Arnimiilation during Gene Activation. TT
The selection of the regions 2^:8C and 4 - 8 I B 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 t h a n 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
of а Ь
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
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 t h a t 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 r e a t m e n t (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 - 8 I B . If the temperature treatment is
region 2 - 4 Θ С
θ
τ
-
ι
!
Ύ
η = 106
όη=9
η=10ί
Γ
-
Ι άη=10
L
"
¿η = 10
с>η=10
-
1
Ι
D
5 10
1
30
ι
ι
60
120
lime after onset of temperature treatment in mm
l'ig. 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
region t - B 1 В
о
lУ*
-
В
С) η = Β
ce
ϊ
τ
7
г
ra
E
Έ
с>η = 10
5
-
(»J
5
.4 —
ni
oc
30
ι
60
η=9 9
г
-
-
С>η = 10
-
3·'
(U
=
--
J
Ί
Ε
.
n = 1G0
Ο
Ο)
(Λ
ΙΌ
Οί
6 η = 10
τ
1 6
2
_
I
ι
ι
0
5 10
,
120
Time after onset of temperature treatment in mm
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 t h a t
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 measuicments 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 gallocyamnchiomalum 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 " o u t of p h a s e " DNA synthesis, it is likely
t h a t the increase in d i y mass value during puff formation is a consequence
Local Protein Accumulation during (.enc Activation. II
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
435
in a
p r e v i o u s p a p e r ( H o l t . 1070). t h e local i n c r e a s e in p r o t e i n c o n c e n t r a t i o n
is m o s t 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 equip­
ment as well as for the suggestions and introduction into the technique of inter­
ference 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 con­
tinuous 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 interferometry. In: General cytochemical methods (J. F. Danielli. ed.). p. ö S - l ö 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 aamanitin on chromosomal RNA synthesis in larval salivary glands of D hydei
was investigated at subsequent time intervals (90 min — 20 hrs) after in­
jection.
As revealed by autoradiography, 3H-uridine incorporation into the polytene 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 com­
parable 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 3 H-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 decondensed 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) sug­
gested that RNA synthesis is the crucial factor which determines the appear­
ance 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 argu­
ment in favor of his suggestion was the observation that loops of the am16
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
region i-BlB
region 2-32A
16
Τ
H
t
12
-
10
-
>
¿
ιι
08
ne
I
I
1
0
l
В
i
40
l
200
i
1000
β
10
200 1000
cone Іяд/mi;
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) con­
taining 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 inhib­
ition 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 treat­
ment 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 diam­
eter
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 treat­
ment The octapeptide is unable to inhibit puff formation completely. It
could be argued that the appearance oí the new puffs following a tempera19
relative puft size
regi
region 2-32 A
16
ί,-ΘΙΒ
(I
Η г
12
-
()
(>
<
10
>
.y/
OB
η e
1...
//
ι
—1
0
15
1
50
'i
1
20
^
!
1
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
3
H-Uridme incorporation in the puffs was estimated on the basis of grain
counts in autoradiographs obtained after 15 min of incorporation of 3 Huridine. 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 incuba­
tion medium was kept at 37 0 C. 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 37 0 C, were used as controls.
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 treat­
ment 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 in­
crease 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 -
48С
• ,jï
;
32А /
·*', .i·.·
!^»
^ ¿ .ν
, L^ ·,»
81 А
95 D-^-
^ ι г· *
V.
I ^
»
-' ^ f ^ збХ"
•.#••
®
-г
10/j
f
.1' ¿t^^k^·' t · . '
'я.·».· •
• ·
/02
/32AА
r
jr.
• · r-·
*
36A
28B
® ák
·
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
Untreated
15 min temperature
treatment in vitro
15 rmn temperature
treatment in vitro,
following a 5 hr
in vivo treatment
with α-amanitin * '
1 73±0 09
2.07±0 02
1.23±0 06
0 32±0 24
0 46±0 02
0.27±0 03
0 43±0 0 2 * 2
0 58+0 04
0.28±0 14
2-32A
2A&C
4-8 IB
* O-amamtin was also present in the incubation medium
*2 SD
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
Grain number ratios
puff/reference
Locus
2-28B
28C-30D
4-8 5 A
81C-84D
5-95D
99A-103D
Control
5 hrs treatment
with O-amarutin
0 60±0 07*
0 30+0 12
0 29+0 06
0 20±0 08
0 37±0 16
0 26+0 13
Absolute gram number
over reference regions
Control
5 hrs treatment
with Of-amamtin
31.0+10.5
50 1+21.8
59.3±19 9
47 8±22 5
48 3±18.7
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 chromo­
some 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
3
H-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 salivary 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 chromosomes. 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 chromosomes). In: Handbook of Molecular Cytology (A Lima-de-Fana E d ) , 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, 3 5 ^ 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 measurements on dye binding capacity at subsequent stages of puff formation in Drosophila 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 polymerase 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 polymerases 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, C E . 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
J.molec.Biol. 51, 327-340 (1970).
26
tentans salivary gland cells
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
2+
a-Amanitin specifically inhibits the action of Mn -dependent (chiomo1
2+
somal) RNA polymerase in vitro . The activity of Mg -dependent RNA
polymerase which, in the intact cell, resides in the nucleolus, is unaffected
by the toxin if tested in an in vitro system 2 3 4 .
However, in intact mammalian 5 6 as well as insect cells 7 the toxic octapeptide causes changes in nucleolar morphology and metabolism, fragmen­
tation of the nucleoli 7 8 9 and an impairment of the processing of nucleolar
RNA 7 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 midthird 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 DrosophilaR i n g e r 1 0 . At various time intervals, from 90 mm to 20 hrs after injection,
the incorporation of 3 H-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 3 H-uridine (spec.act. 24 Ci/mM) and 100 Mg/ml a-amanitin.
Autoradiographs were prepared as described previously 1 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 3 H-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 in­
creased as compared with the controls not submitted to an a-amanitin treat­
ment. 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 oiDrosophila 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 solution 1
instead of O-amanitin
Nucleoli
of a-amamtin
treated
n = 160
Nucleoli
of controls
Heavy labeling
(no grain counts possible)
89(5670
56 (29%)
Medium labeling
(more than 10 grains per square μ)
47(2970
64 (34%)
Weak labeling
(less than 10 grains per square μ)
24(15%)
71 (37%)
n= 191
A comparison of the radioactivity profiles of salivary gland RNA extract­
ed 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 RNA 1 4 , was
unaffected in the a-amanitin treated animals. On the basis of total radioac­
tivity in RNA extracted from salivary glands of larvae treated with a-amanitin 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, accumu­
lation 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-amanitm treated glands remains unknown. It is reasonable to assume unimpaired
7
transcription of 4S and 5S genes , 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
Fig.2. Radioactivity profile of
Ш
10
20
30
40
50
traction №
Η-uridine labeled RNA extracted from salivary glands
from mid-third instar larvae of Drosophila hydei which were injected with 0.5 μΟ/ΐ3Γν3
3
H-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 C E . 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 temperatuurbehandehng (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 ultramicrospectrophotometer m relatie tot het DNA-gehalte van bepaalde chromosoomgebieden 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 (Feulgenprocedure), 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 toe­
name kwam overeen met die welke gevonden was voor de toename in kleur­
stofbinding
Uit de totale groep van expenmenten kan geconcludeerd worden dat het
optreden van nieuwe puffs gepaard gaat met een lokale kwantitatieve toe­
name 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 chromo­
somale RNA-synthese werd onderdrukt
Een onderzoek van de werking van a-amamtme onder m vivoomstdndigheden (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 chromosomen-loci werden in hun RNA-synthese-aktiviteit geremd Autoradiografi­
sche 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 in­
corporatie 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 Gecon­
cludeerd 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 RNAaseremmer 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 kippeembryo 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 on­
waarschijnlijk
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 statusonderscheid
Th Κ Η Holt,
Nijmegen, 2 jum 1972