Chromosoma (Berl.) 62, 217--242 (1977)

CHROMOSOMA 9 by Springer-Verlag 1977

RNA Synthesis in Isolated Polytene Nuclei from Chironomus tentans Horst Hameister Max-Planck-Institut fiir Biologie, Abteilung Beermann, Tfibingen; present address: Institut f/Jr Humangenetik und Anthropologie, Albertstral3e 11, 7800 Freiburg, Federal Republic of Germany

Abstract. An RNA synthesizing system with isolated polytene nuclei from Chironomus tentans is described. This system allows one to monitor the effect of salt concentration on chromosome structure and to assign in vitro R N A synthesis to structural modifications of the chromosome (i.e. nucleoli, Balbiani rings and puffs).-At a salt concentration of 0.15 M monovalent cations (standard salt medium=SSM) chromosomal structure appears to be best preserved during in vitro incubation. At low and high ionic strength the bands decondense and the microscopically visible chromosomal structure is lost completely. These three states of condensation and decondensation are distinguished with respect to RNA synthesis: (1) in low salt overall R N A synthesis is depressed, (2) in SSM ribosomal RNA synthesis predominates and continues for 30 min, (3) in high salt R N A synthesis is stimulated 3-4 fold again. This stimulation is due solely to chromosomal, non-ribosomal R N A synthesis, which proceeds in high salt for more than 10 h, though new initiation of RNA chains is prevented. Molecular weight determinations of the RNA synthesized demonstrate a time dependent increase in size of the newly synthesized molecules under these conditions.-Autoradiographs of nuclei incubated in SSM reveal prominent label in nucleoli, significant label in Balbiani rings and rather reduced activity at other sites. Addition of various exogenous RNA polymerases does not markedly alter this pattern. Autoradiographs of nuclei incubated in high salt exhibit extensive R N A synthesis spread over the chromosomes. Preparations of autoradiographs from isolated chromosomes show that the high salt induced label is localized in single bands. Though the majority of bands is still unlabelled, the actual number of bands exhibiting incorporation in high salt is higher than in any individual functional state in vivo. These results are discussed in terms of activated and preactivated genes.

Introduction

The giant polytene chromosomes of the dipteran Chironomus tentans have been the subject of many investigations on the cytogenetic and molecular basis of

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cell differentiation. U s i n g this material B e e r m a n n , 1952, i n t e r p r e t e d p u f f formation as expression of differential gene activation. This p i o n e e r i n g o b s e r v a t i o n was followed by further detailed descriptions of developmental-specific puffing (Mechelke, 1953; Becker, 1959) a n d characteristic changes of the p u f f p a t t e r n after ecdysone a d m i n i s t r a t i o n (Clever, 1961). T h e c o n c e p t o f p u f f f o r m a t i o n as the m a n i f e s t a t i o n of gene activity was greatly s u p p o r t e d by the direct d e m o n s t r a t i o n that puffs are the sites of active R N A synthesis (Pelling, 1964). The c h a r a c t e r i s a t i o n of some m a j o r t r a n s c r i p t i o n a l ( D a n e h o l t , 1972; L a m b e r t , 1972) a n d t r a n s l a t i o n a l p r o d u c t s (Grossbach, 1969) has also been described (for review see: D a n e h o l t , 1975). W e have m a d e use of these c h r o m o s o m e s to study R N A synthesis in a cell free system. T o this end, p o l y t e n e nuclei or c h r o m o s o m e s have b e e n isolated by a m e t h o d a d a p t e d f r o m that originally described by R o b e r t , 1971. The characteristics o f in vitro t r a n s c r i p t i o n u n d e r different salt c o n d i t i o n s by template b o u n d , e n d o g e n o u s R N A polymerases a n d v a r i o u s exogenous polymerases will be described in r e l a t i o n to the p a t t e r n o n R N A synthesis in vivo a.

Materials and Methods Materials

Larvae of Chironomus tentans, population P16n, were raised at 18~ according to Beermann (1952). Heparin (sodium salt) and Triton-X-100 were obtained from Serva, Heidelberg. ATP, CTP, GTP and UTP (trisodium salts) were from Boehringer, Mannheim, (5-3H)UTP was purchased from Amersham Buchler, Braunschweig; c~-amanitinwas a generours gift from Prof. Dr. Th. Wieland, Heidelberg. All other chemicals were analytical grade.

Methods 1. Isolation of Nuclei. The nuclei were isolated by the method of Robert (1971) with the following modifications. The Chironomus Ringer (CR-buffer) contained: 0.087 M NaC1, 0.0032 M KC1, 1.3 mM CaC12, 4 mM MgC12, 10 mM Tris-Hcl pH 7.3. The nuclear isolation buffer (IS-buffer) contained the same salts as cited for the CR-buffer. To transform the viscous salivary gland secretions to a soIid rubbery material, which can be easily removed, the pH was adjusted to 5.8 with Trismaleate and 0.2% Triton-X-100 was used instead of digitonin. Chironomus tentans possesses about 40 nuclei in each of the two salivary glands. Approximately 1000 nuclei can be isolated within 3 h by a single person. Ease of isolation is dependent on the size of the glands. A good isolation yields 80% of the nuclei free of visible adherent cytoplasrn. 2. Isolation of Chromosomes 2. Up to 10 glands were incubated at 0~

in IS-buffer for 45 rain. Then they were transferred to a glass Dounce type homogenizer and disrupted by 8 strokes of the loose fitting pestle. The homogenate was strained through a layer of cheese-cloth and centrifuged for 1 min at 500 rpm. The supernatant was discarded and the pellet taken up in 100 I~l CR-buffer. The chromosomes were collected out of the droplet under the dissection microscope. 3. Incubation Conditions for in vitro RNA Synthesis. Whole nuclei or isolated chromosomes were

used immediately after isolation. If not otherwise stated incubation for RNA synthesis was carried out at 25~ for 30 min in 10 ~tl final volume. The standard salt medium (SSM) contained: 0.09 M 1 2

This report was presented in parts at the FEBS Meetings in Budapest, 1974, and Paris, 1975 This method was kindly made available by Dr. C.P. Hollenberg

R N A Synthesis in Isolated Polytene Nuclei of Chironomus

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KC1, 0.06 M NaC1, 0.025 M Tris-HC1 pH 7.3, 5 mM MEA, 0.1 mM EDTA, 2 m M MnC12, 0.2 m M each of ATP, CTP, GTP and 2 g C i of 3H-UTP (spec. act.: 11.1 Ci/mmole or as indicated). In high salt medium all constituents are identical except that the sodium concentration was raised to 0.51 M NaC13.

4. Autoradiography. The nuclei or chromosomes were collected out of the incubation medium, washed in SSM and pipetted on to a gelatinized slide. Nuclei and chromosomes incubated in high salt are decondensed and as such not easily visible in the incubation medium. Nuclei are recognized by their nuclear membrane, chromosomes are detectable only under oblique illumination by their faint outline. Such nuclei or chromosomes can be transferred to SSM and allowed to reconstitute. The material is then settled onto a gelatinized slide, fixed in ethanol:acetic acid (3: 1), washed in cold 5% TCA and covered with Kodak A R 10 autoradiographic stripping film and processed according to Pelling (1964). Exposure time was usually 14 d.

5. Quantitative Determination of RNA Synthesis. In order to measure total UTP incorporated by whole nuclei it is necessary to disrupt the nuclei by addition of 1.0 ml of a solution of 2% SDS and 0.5 mg/ml Pronase in 0.1 M NaC1 and further incubation of the whole mix for at least 1.5 h, usually 3 h. This treatment increases the efficiency of counting about ten times, After adding 100 gg of yeast R N A as carrier the acid insoluble material was precipitated by 0.5 ml of ice-cold 4% TCA containing 5% (v/v) of saturated Na~PzO7-solution. The precipitate was collected on Millipore filters, washed 5-times with the TCA solution, dried and radioactivity was determined in a toluene based scintillator. R N A synthesis is completely dependent on the presence of all four nucleoside triphosphates and the product is RNase sensitive (results not shown).

6. Test with Exogenous Added RNA Polymerases. E. coli R N A polymerase was from Boehringer, Mannheim, calf thymus RNA polymerase A was kindly provided by Dr. Chambon, Strasbourg, and calf thymus R N A polymerase B by Drs. Schmincke and Hausen, Tttbingen. The nuclei were preincubated in S S M + 0 . 2 m M each of ATP, CTP, GTP and UTP in a final volume of 50 gl to exhaust the endogenous, template bound R N A polymerase activity. The nuclei were collected, washed extensively in SSM to separate unlabelled triphosphates and used for a further incubation under the same conditions with 2 gCi 3H-UTP instead of cold UTP in the presence of the respective exogenous R N A polymerase and c~-amanitin where required.

7. Sedimentation Analysis of RNA. A defined number o f nuclei was incubated for R N A synthesis in 20 gl high salt medium to yield 10,000 cpm -FCA precipitable radioactivity in RNA. Reactions were stopped by addition of 1.0 ml of ice-cold S S M + 1 mg/ml heparin. The mixture was quickly stirred, centrifuged and the supernatant containing the free nucleoside triphosphates was discarded. 10 gl of Drosophila hydei r-RNA, prepared by a modified Kirby method as described by Diez (1973), was added to the pellet. Lysis of the nuclei was carried out for 3 h at 37~ by stirring in 100 gl of 50 m M sodiumacetate pH 5.0, 10 m M EDTA, 0.5% disodium naphthalene-l,5-disulphonate, 1.0 % sodium tri-isopropyl-naphthalenesulphonate, 6 % sodium-4-aminosalicylate and 0.5 % Sarkosyl. Then the mixture was extracted twice with freshly distilled p h e n o l + 0 . 5 % 8-hydroxychinoline to which after some shaking an equal volume of chloroform was added. The aqueous pkase was made 0.24 M in ammonium acetate and precipitated with ethanol. The pellet was dissolved in 100 gl of centrifugation buffer (10 m M Tris-HC1 pH 7.3, 50 m M NaC1, 1 m M EDTA, 0.5% SDS) and layered on a 11.6 ml gradient of 5-20% sucrose in centrifugation buffer, then centrifuged at 40,000 rpm for 5 h at 12~ 30 fractions were collected and TCA precipitated on Millipore filters before counting. 3 The following abbreviations were used: EDTA, ethylene-diaminetetraacetate; MEA, 2-mercaptoethanol; SDS, sodiumdodecyl sulfate; SSM, standard salt medium (0.09 M KC1, 0.06 M NaCI, 0.025 M Tris-HC1 p H 7.3, 2 m M MnClz, 5 mM MEA, 0.1 m M EDTA); TCA, trichloroacetic acid

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H. Hameister

Fig. 1. Isolated salivary gland nucleus of Chironomus tentans. Note the nuclear envelope which is preserved completely. Roman numerals indicate chromosomes. BR, Balbiani ring; N, nucleolus. Bar = 20 gm

Results L RNA Transcription in Standard Salt Medium

The salt combination of the SSM was chosen according to the following criteria: (1) preservation of the banding structure of the chromosomes during incubation, (2) maximal R N A synthesis compatible with the above precondition. In accordance with the data of Glancy, 1946, it was found that a combination of 0.09 M KC1 and 0.06 M NaC1 was most effective for maintaining chromosome structure. A cytological squash-preparation of a nucleus incubated 30 min in SSM is shown in Figure 1.

1. Dependence of R N A Synthesis on Nuclear Concentration Figure 2 demonstrates the dependence of R N A synthesis on the number of nuclei incubated. The relation is shown to be linear over a wide range of nuclear concentration. Variation of DNA content per nucleus due to variation of the degree ofpolytenisation (in full grown larvae between 21~ chromatids) did not effect the results on R N A synthesis noticeably, and it is possible to carry out reproducible tests with 40 nuclei each. After 12 rounds of replication the mean D N A content of a salivary gland nucleus in our preparation was determined to be ca. 2 mgg (Daneholt and Edstr6m, 1967). With our nuclear isolation procedure it was impossible to determine routinely the DNA content of each batch of nuclei. However from

R N A Synthesis in Isolated Polytene Nuclei of Chironornus

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1200

~o~I000 800 L

o

Fig. 2. Dependence on nuclear concentration. The indicated number of nuclei was incubated in the SSM synthesis mix 30 rain at 25~ as described in Methods

600

-m

4oo

i

200

oJ " I

0 10 20

I

I

I

40

80

120

Nuclei

E

'~'400

Fig. 3. Time-course of R N A synthesis. Nuclei were incubated in the SSM synthesis mix for the indicated periods, c~-amanitin was added to the mix at a concentration of 1 gg/ml. 9 9 without c~-amanitin, 9 . . . . 9 with c~-amanitin

300

8c 200 i

9

//

100 I

I

10

20

I

I

30 40 Time [rain]

I

t

50

60

the parameters mentioned we estimate that each test with 40 nuclei contains between 40 and 80 mgg DNA.

2. Time-Course of R N A Synthesis The time course of R N A synthesis is shown in Figure 3. Only a minor fraction of total R N A synthesis is sensitive to ~-amanitin. It amounts to 5-15% in different preparations of nuclei. This suggests that under our conditions some 90% of R N A synthesis is due to synthesis of r-RNA and t-RNA.

3. Stability of Synthesis Products When nuclei were incubated for periods longer than 30 min the acid precipitable radioactivity did not further increase. In order to ascertain, whether this result is due to true cessation of synthesis, to attainment of an equilibrium between synthesis and degradation or to release of R N A from the nuclei, the following experiment was carried out (Table 1). After normal incubation with labelled U T P for 30 min nuclei were separated from the labelled medium and chased

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Table 1. Stability of synthesis products. All nuclei were incubated together for 30 min in 25 ~tl of the SSM R N A synthesis mix as cited in Materials and Methods. After incubation the T C A insoluble radioactivity was determined at once or the nuclei were further incubated under various conditions at 25~ At the end of the second incubation period radioactivity was determined again. T C A precipitable radioactivity in the supernatant, due to possible release of the RNA, is also noted Incubation

3H-UTP incorporated (cpm)

30 rain supernatant I 30 r a i n + 3 0 min 30 m i n + 6 0 min 30 m i n + 3 0 min 30 min + 60 min supernatant II

398 57 360 330 347 392 19

"

chase chase chase chase

in in in in

SSM SSM SSM+4 NTP" SSM + 4 N T P a

0.2 m M each of ATP, CTP, G T P and U T P

E

(Mn2§ /

800 '~ 600

o

6O0

/

8 c 400

40O

,,.~,-,~.,~,,--~ ~o

200

I

& a

,.,.o

I

0,0

0,1

I

I

0,2 0,3 KC1 IN]

(Mg2+)

8O0

I

I

0,4

0,5

~~

200q

0,0 b

I

P

I

1

I

0,1

0,2

0,3

0,4

0,5

KCl N]

Fig. 4a and b. Salt dependence of R N A synthesis. Nuclei were incubated for R N A synthesis 30 rain at 25~ in 10 gl of the following mix: 25 m M Tris-HC1 p H 7.3, 5 m M MEA, 0.1 m M E D T A , 0.2 m M each of ATP, CTP and G T P and 2 pCi 3H-UTP. 2 m M MnC12 (a), 5 m M MgC12 (b) and KC1 were added as indicated, e - - e without ~-amanitin, o - - o with c~-amanitin to 1 gg/ml

in SSM with or without cold nucleotides. The comparison of the recovered radioactivities clearly shows that there is little release from the nuclei or degradation of RNA to acid soluble products during the chase period. The constancy in amount of radioactivity seen after 30 min incubation is therefore due to a cessation of RNA synthesis.

4. Salt Dependence of RNA Synthesis The salt dependence of RNA synthesis in the presence of either Mn 2+ or Mg 2+ is shown in Figure 4. A prominent stimulation of RNA synthesis with

RNA Synthesis in Isolated Polytene Nuclei of Chironomus

223

increasing ionic strength is found only in the presence of M n 2 +. This stimulation is solely due to R N A polymerase B activity as demonstrated by its inhibition with 1 gg/ml c~-amanitin. The R N A polymerase A and perhaps C activity is either unaffected or even rather depressed in high salt, as seen by its behaviour in the presence of Mg 2§ The stimulation of R N A synthesis with increasing ionic strength is correlated with a progressive decondensation of the chromosome which is essentialy complete in 0.45 M NaC1 (Robert, 1971; own observation). Very low salt ( < 0.05 M NaC1), too, causes a decondensation of the chromosome banding structure (Glancy, 1946; Beermann, 1962). In contrast to high salt very low salt induced decondensation does not result in significant stimulation of R N A synthesis (Fig. 4).

H. R N A Transcription in High Salt As shown before R N A synthesis can be enhanced by raising the ionic strength in the incubation medium. This enhancement applies only to ~-amanitin-sensitive R N A polymerase B activity at the puff sites. To the best of our knowledge a high salt medium, i.e. 0.6 M monovalent cations, is a non-physiological ionic environment for chromosomes. The chromosomes are disintegrated and are no longer visible as structural entities in the light microscope. Nevertheless we decided to further study the characteristics of this stimulation.

1. Time-Course of R N A Synthesis in High Salt The kinetic characteristics of R N A synthesis in high salt are shown in Figure 5. Whereas :~-amanitin-insensitive polymerase A and C activity is rate stimulated to reach a plateau after only 10 min, the total a m o u n t of R N A synthesis at this time point, i.e. total 3H-UTP incorporated, is similar to that in SSM (Fig. 3). On the other hand, plymerase B activity is enhanced both in synthesis rate, proceeding at a decreasing rate for more than 1 h, and in the maximal

3000

~ ' 2 500

& -~ 2ooo

Fig. ft. Short time-course of RNA synthesis in high salt. The assays were carried out in triplicate and 25 nuclei each were incubated for the cited periods in SSM to which NaC1 had been added to a final concentration of 0.51 M (high salt). 9 - - e without ~-amanitin, 9 .... 9 with 1 gg/rnl ~-amanitin

'1500 c

=,

1000

500

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~ .z>...o_ _ _ . . 6 _

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20

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30 40 50 Time [min]

I

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70

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extent o f R N A synthesized, w h i c h after 1 h surpasses r i b o s o m a l R N A synthesis by a f a c t o r o f 5. A s seen in F i g u r e 3, in S S M c h r o m o s o m a l R N A synthesis a c c o u n t s for o n l y 5 - 1 5 % o f t o t a l synthesis. F i g u r e 5 shows t h a t in high salt R N A synthesis t a k e s p l a c e at a high rate linearly o n l y d u r i n g the first 10 min. L a t e r o n the rate declines a n d seems to be e x h a u s t e d after one hour. But when synthesis was f o l l o w e d for longer times a small b u t c o n t i n u o u s increase o f 3 H - u r i d i n e i n c o r p o r a t i o n was observed. F i g u r e 6 shows a t i m e - c o u r s e where synthesis was m e a s u r e d over a p e r i o d o f 18 h. This l o n g t i m e - c o u r s e d e m o n s t r a t e s a b i p h a s i c n a t u r e o f R N A synthesis in high salt. T h e first h o u r o f relatively active R N A synthesis c o r r e s p o n d s to the short t i m e - c o u r s e o f F i g u r e 5. This increase is f o l l o w e d b y a linear but slower rate for 12 h b e f o r e a p l a t e a u is reached. H i g h salt s t i m u l a t e d R N A synthesis is due o n l y to p o l y m e r a s e B activity at n o n - r i b o s o m a l c h r o m o s o m a l sites a n d since new- or r e i n i t i a t i o n o f R N A p o l y m e r a s e s is p r e v e n t e d in high salt ( F u c h s et al., 1967; H y m a n a n d D a v i d s o n , 1970), it c a n o n l y be a t t r i b u t e d to e l o n g a t i o n by p r e b o u n d R N A p o l y m e r a s e s a l o n g the t e m p l a t e . D u r i n g the first h o u r n e a r l y 1/3 o f the t o t a l activity, d e t e r m i n e d after 16-18 h, is i n c o r p o rated. T o c o n f i r m t h a t synthesis does in fact level off at this time, several experim e n t s were p e r f o r m e d , one o f which is s h o w n in the inset o f F i g u r e 6. Nuclei were i n c u b a t e d in the presence o f c o l d U T P for 9.5 h b e f o r e t r i t i a t e d U T P

6000

5000

/,

Z u

4000

cpm 1200 1000

.

~u- 3 0 0 0

800 600

.E

400 200

2000 f

10

12

10

12

1000

14

16

18 o

o

2

4

6

8

14 16 18 Time [hours]

20

Fig. 6. Long time-course of RNA synthesis in high salt. The assays were carried out as cited in the legend to Figure 5. The arrow indicates the addition of further 2 gCi 3H-UTP (spec. activity: 48.2 Ci/mmole) and ATP, CTP and GTP to 0.2 mM in the appropriate buffer. | indicates values, obtained without a second addition of triphosphates, o indicates RNA synthesis in the presence of 1 gg/ml c~-amanitin. - Inset: The same experiment was carried out in the presence of 1 mg/ml heparin. The nuclei were incubated for 9.5 h in high salt as cited in the legend to Figure 5, except that 3H-UTP was replaced by the same amount (0.004 mM) of unlabelled UTP. After 9.5 h observation of the reaction was started by the addition of 2 pCi 3H-UTP (spec. activity: 48.2 Ci/mmole, i.e. 0.004 mM UTP) and ATP, CTP, GTP to 0.2 mM in the appropriate buffer. Note the identical time scale

R N A Synthesis in Isolated Polytene Nuclei of Chironomus

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of high specific activity was added and synthesis followed up to 19.5 h. During this test heparin was added to 1 mg/ml according to Cox (1973) to inhibit possible RNAse activities. Under these conditions the plateau reached after 12-16 h remains stable and has to be taken as real. The plateau is not due to depletion of nucleoside triphosphates, as shown by the fact, that the plot is uninfluenced by omission of a second addition of substrate after 9.5 h. Also shown in Figure 6 are two determinations of c~-amanitinresistant activity which again exhibits a similar timecourse as in standard salt due to early termination of nucleolar synthesis.

2. Size Determination of the R N A Synthesized in High Salt As outlined above the kinetics of synthesis in high salt are considered to be due to continuous growth of R N A chains. To prove this, several attempts have been made to isolate R N A and determine its length distribution. It emerged that under high salt conditions the length of the R N A synthesized remained constant irrespective of the incubation time. In Figure 7a the size distribution of two R N A preparations from nuclei incubated for 30min or 150rain is shown. Both samples contain R N A of similar size (5-8 S). During longer incubation periods only a broadening of the peak is observed. Though this broadening cannot be a consequence of RNAse activity alone, in a further experiment heparin was added to 1 mg/ml. The long time-course is uninfluenced by the presence of heparin (cf. Fig. 6), but size determination

/

T% 30 40

15' 30' 150' cpm 3000

T~ 40

cpm 1800

50

1600

60

1200

70

50 2000

60

i /it lO0O

800

6,I 1 top

.&l

80

9o

400

70

LA

A

.........

"k,.,.,.,-',,.,

90

.'.

Fraction

bottom O0

0

b

top

Fraction

I 100 bottom

Fig. 7a and b. Size determination of R N A synthesized in high salt. a Nuclei were incubated in high salt R N A synthesis mix for 30 min e - - e or 150 min 9 . . . . o. The R N A was purified as indicated in Methods, layered onto a 5-20% sucrose gradient, centrifuged 5 h, 12~ at 40,000 rpm, collected into 30 fractions with automatic absorbance reading and TCA precipitated before counting. 260 nm absorbance of Drosophila hydei 18 S and 28 S r-RNA, run in the same tube. b Nuclei were incubated 25 min z~. . . . zx, 30 min e - - e , and 150 min o . . . . o, in the high salt R N A synthesis mix with addition of 1 mg/ml heparin. Further processing was the same as cited above

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H. Hameister

o f the p r o d u c t s reveals a striking change. The R N A chain grows and attains a m o d a l size o f 18 S after 150 min (Fig. 7b). This result is consistent with a c o n t i n u o u s p r o p a g a t i o n o f the R N A polymerase along the template. With increasing time a broadening o f the peak also takes place in the presence of heparin, which makes it difficult to determine the size o f R N A f r o m nuclei, which had been incubated for periods longer than 150 min. Similar attempts to isolate R N A o f distinct size out o f SSM incubated nuclei were unsuccessful.

3. Effect o f the High Salt Treatment The stimulation o f R N A synthesis in c h r o m a t i n by high salt is a general p h e n o m e n o n and has been observed by m a n y authors (Widnell and Tata, 1966; Novello and Stirpe, 1969; Butterworth et al., 1971; Cox, 1973). It has usually been attributed either to dissociation o f inhibitory proteins at high ionic strength (Butterworth et al., 1971 ; B r a d b u r y et al., 1973) or to a m o r e general systematic effect on template c o n f o r m a t i o n (de Pomerai et al., 1974). In an a t t e m p t to distinguish between these two alternatives the experiment detailed in Table 2 was performed. Nuclei were isolated and divided into three aliquots. One aliquot was preincubated 2 h at 0 ~ in SSM, another for 2 h at 0 ~ C in high salt, the third was tested without preincubation. All three examples were diluted and tested for R N A synthesis in SSM and high salt. I f the release o f c h r o m o s o m a l proteins in high salt is responsible for the stimulation and reassociation is prevented by rapid dilution into SSM, the characteristics o f the stimulated state should be retained. The results clearly show that this is

Table 2. Effect of high salt treatment The nuclei were isolated and divided into three samples. The nuclei of the first sample were incubated for RNA synthesis 30 min at 25~ in 50 gi of the standard or high salt synthesis mix. The nuclei of the second and third sample were preincubated 2 h at 0~ IN 10 gl standard or high salt medium, then each sample diluted with an adjusted salt solution to give 50 ~tl of standard or high salt synthesis mix and further incubated for RNA synthesis 30 min at 25~ Determination of RNA synthesis was as mentioned in methods Preincubation

Incubation for RNA Synthesis 30 min, 25~

(3H-UTP) incorporated (cpm)

standard salt 0.6 M salt

624 1045

___----, standard salt 0.6 M salt

576 1054

120 min, 0~ -

standard salt 0.6 M salt

~_~---~ ~ ~

standard salt 0.6 M salt

540 1097

RNA Synthesis in Isolated Polytene Nuclei of Chironomus

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not the case. Loss of c h r o m o s o m a l proteins alone can therefore probably not account for high salt stimulation.

III. Autoradiography of S S M Incubated Nuclei 1. Transcription by Endogenous R N A Polymerase Figure 8 shows a typical autoradiograph of giant chromosomes recovered from a nucleus incubated in SSM for 30 min at 25~ immediately after isolation. The incorporation pattern is characterized by heavy labelling of the two nucleoli situated in c h r o m o s o m e 2 and 3. Nucleolar incorporation is spread evenly over the entire area of the nucleolar mass. Even in nuclei incubated for shorter times this general pattern is observed. A restriction of nucleolar labelling to a c h r o m o s o m a l centre, due to condensation of the nucleolus organizer (Pelling, 1964) and often seen in vivo, has never been observed under our in vitro conditions. Incorporation in the chromosomes differs from the in vivo pattern in that only the Balbiani rings exhibit the intense incorporation known from in vivo studies, while distinctive puff activity is restricted to few sites and the remainder of the chromosomes is only diffusely labelled. C h r o m o s o m a l activity is often found to be even lower than shown in Figure 8. All nuclei, even if isolated from animals raised under very different conditions show this predominant nucleolar incorporation and low puff activity. This is

Fig. 8. Autoradiograph of SSM incubated nuclei. Nuclei were isolated and incubated for 30 min at 25~ in the SSM RNA synthesis mix. For further details see Methods. Symbols as in Figure 1. Bar = 20 gm

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Hameister

in accordance with the low e-amantin-sensitivity of R N A synthesis shown in Figure 3.

2. Transcription by Heterologous, Exogenous R N A Polymerases In an attempt to enhance chromosomal activity during in vitro transcription, experiments were performed with various exogenous R N A polymerases. Since R N A polymerase from C. tentans was not available we decided to employ well defined R N A polymerase preparations from other sources although we are aware of the possible restrictions on interpretation of data imposed by the use of such heterologous systems. Addition of polymerase B to freshly prepared nuclei leads t o nearly 30% Stimulation of incorporation in SSM (results not shown). To assign incorporation of label to synthesis by exogenous polymerases it is necessary to avoid incorporation by the endogenous template associated R N A polymerase. This can be achieved by preincubation of the nuclei in the presence of unlabelled triphosphates. After 30 min the endogenous R N A polymerase activity is exhausted (Fig. 9). The requirement for triphosphates indicates that the decrease in R N A synthesis is due to active polymerisation and not to progressive inactivation of the enzyme. Figure 10b shows an autoradiograph of a nucleus pretreated as described and then incubated in the presence of labelled UTP. No label comparable to the labelling induced by endogenous enzyme is detectable over the nucleoli nor over Balbiani rings or puffs.

a) Transcription with Calf Thymus RNA Polymerase A and B. A nucleus, to which the R N A plymerase A was added during the second incubation, is shown in Figure 10c. Polymerase A induces incorporation of triphosphates in the two nucleoli while incorporation in Balbiani rings and puffs is low. Figure 10d on the other hand presents an autoradiograph of a nucleus to which the nucleoplasmic form B polymerase has been added. Surprisingly this incorporation pattern is the same as that obtained with R N A polymerase A, i.e. marked induction of R N A synthesis within the two nucleoli. The extent of label within Balbiani rings and puffs, where one would expect incorporation with R N A polymerase B, again is low.

% %'100,

80

. . . . . . ..o___.o

2

\\\\\\\%

L .~8_40 20 i

E

I

I

10

20

I

I

I

30 40 50 Time [mini

I

I

60

70

Fig. 9. Exhaustion of RNA synthesis capacity. Nuclei were isolated and preincubated at 25~ for the cited periods 9 9 in standard salt +0.2 mM of all four triphosphates, o .... o in standard salt. Then they were washed free of cold triphosphates 3 times in SSM and further incubated in the SSM RNA synthesis mix for 30 rain as detailed in Methods

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Fig. 10a-f. RNA synthesis mediated by different exogenous RNA polymerases, a Nuclei were incubated for RNA synthesis immediately after isolation (see also Fig. 8). The other nuclei were preincubated in a cold RNA synthesis mix as detailed in Methods and washed free of triphosphates before further incubation, b Second incubation without added exogenous enzyme (control), e with l gl RNA polymerase A from calf thymus, d with 2 ~tl RNA polymerase B from calf thymus, e with 2 pl RNA polymerase B from calf thymus and 1 gg/ml ~-amanitin, f with l ~tl RNA polymerase from E. coli (" holo-enzyme "). Bar=20

The two R N A p o l y m e r a s e s differ n o t o n l y in their salt r e q u i r e m e n t s for m a x i m a l R N A synthesis b u t also in their sensitivity to ~ - a m a n i t i n , with form B being sensitive to this toxin. T h e R N A synthesis c a p a c i t y o f p o l y m e r a s e B was t h e r e f o r e tested in the presence o f 1 g g / m l ~ - a m a n i t i n . P r e c a u t i o n s were t a k e n to first s a t u r a t e the p o l y m e r a s e with ~ - a m a n t i n b e f o r e a d d i n g the nuclei by p r e i n c u b a t i n g the p o l y m e r a s e a n d c~-amanitin for 5 m i n at 0 ~ (CochetM e i l h a c a n d C h a m b o n , 1974). A s s h o w n in F i g u r e 10e the i n c o r p o r a t i o n p a t t e r n is the s a m e as in the a b s e n c e o f e - a m a n i t i n .

b) Transcription with E. coli R N A Polymerase. F i g u r e 10f shows a n a u t o r a d i o g r a p h o f a nucleus l a b e l l e d in the presence o f E. coli R N A p o l y m e r a s e . T h e

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i n c o r p o r a t i o n p a t t e r n differs c o m p l e t e l y f r o m t h o s e p r e s e n t e d before. Silver grains are d i s t r i b u t e d u n i f o r m l y t h r o u g h o u t the c h r o m o s o m e s w i t h o u t a n y specific localisation.

IV. Autoradiography in High Salt 1. H i g h Salt I n c u b a t e d Nuclei A s p o i n t e d o u t before nuclei lose their s t r u c t u r a l i n t e g r i t y w h e n i n c u b a t e d in high salt. T h e y c a n nevertheless be i n d u c e d to r e g a i n their distinct b a n d i n g p a t t e r n s if d e c o n d e n s a t i o n in high salt is r e s t r i c t e d to a l i m i t e d time, before r e t u r n i n g t h e m to S S M , where c o m p l e t e r e c o n d e n s a t i o n t a k e s place. L o n g e r i n c u b a t i o n times in high salt l e a d to irreversible a l t e r a t i o n o f the structure, until after p e r i o d s o f r e c o n d e n s a t i o n l o n g e r t h a n 3 h, r e c o n d e n s a t i o n to m i c r o scopically visible structures is impossible. W e have t a k e n a d v a n t a g e o f this ionic s t r e n g t h d e p e n d e n t reversibility o f c h r o m o s o m e structure to s t u d y R N A

Fig. l l a - d . High salt incubated nuclei. Nuclei were isolated, incubated 5 rain at 25~ in the high salt RNA synthesis mix, recondensed in standard salt and prepared for squash preparations or autoradiography as detailed in Methods. a Squash preparation stained with carmine-orcein-acetic acid; b same as a, but fixed before complete recondensation was achieved. Note the nuclear membrane, e Autoradiograph of a high salt incubated nucleus; d survey of several nuclei, isolated from different animals and collected on the same slide. Bar = 20 gm

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synthesis by autoradiography in chromosomes that had been incubated in high salt. As the nuclear envelope remains visible in high salt, it is possible to collect the decondensed nuclei out of the medium and to transfer them to SSM. In Figure 11 a are presented some representative examples of squash preparations of nuclei treated as described. The chromosomes do recondense in SSM to entities smaller than before, which makes it difficult to identify them accurately, moreover since the nuclear envelope turns out to be very stable and does not allow separation of chromosomes during squashing. The nucleus shown in Figure l l b was fixed and squashed before complete recondensation was achieved in SSM. Such a preparation gave rise to clear separated chromosomal bands, but identification of single chromosomes was even worse. Figure 11 c shows a representative example of an autoradiograph of a nucleus which had been incubated in high salt for 5 min. The incorporation pattern has changed completely compared to that of nuclei incubated in SSM. Label is now found predominantly in the non-nucleolar regions and seems to be evenly distributed. Detailed examination, however, reveals a banded structure of the grain distribution, i.e. activity appears to be restricted to individual bands. Incorporation in the Balbiani rings is prominent and occurs to the same extent as observed in SSM. Nucleolar R N A synthesis is rather reduced in relation to chromosomal R N A synthesis. As shown in Figure 11 c both nucleoli exhibit slight incorporation. This is in accordance with the high ~-amanitinsensitivity of in vitro R N A synthesis under these conditions (Fig. 5). It should be pointed out that nuclei even if collected from different animals always show the same intense labelling pattern when incubated for 5 min in high salt. No significant variation in the degree of incorporation was found, as is often seen after in vivo labelling. This is demonstrated in a selection of different nuclei from one slide (Fig. 11 d).

2. High Salt Incubated Individual Chromosomes In the autoradiograph shown in Figure l l c there are distinguishable sites of evenly distributed grains, others with grains arranged in a banded structure and yet others showing decreased incorporation, especially on chromosome 4. But it is not possible to assign label on these autoradiographs to single bands or puffs, since the chromosomes overcondense on being returned to SSM after the high salt treatment. But the condition shown in the nucleus depicted in Figure 11 b raised the possibility that this difficulty could be circumvented by isolating single chromosomes, incubating them as described for nuclei in high salt and then recondense them only partially in SSM. When pipetted into the high salt medium, single chromosomes lose their banded structure as was shown for whole nuclei. The chromosomes swell in high salt, but under oblique illumination they can be identified by a faint outline for the first minutes. This enables one to collect the decondensed chromosomes out of the medium and reconstitute them in SSM. Figure 12 shows some chromosomes which were fixed and squashed just as they began to recondense visibly in the low salt medium. They are thus more extended than in vivo and show

Fig. 12a-d. High salt incubated chromosomes. The chromosomes were isolated, incubated 5 min at 25~ in the high salt R N A synthesis mix and processed for autoradiography just as they became visible during recondensation in SSM. a Partially recondensed chromosome 2; b chromosome 1 with some puffs indicated (cf. Fig. 13 a); e chromosome 2 completely recondensed; d chromosome 3 with some puffs indicated (cf. Fig. 13b). N, nucleolus. B a r = 2 0 lain

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Fig. 13a and b. Constant incorporation pattern in high salt. Representative examples of chromosomal sections from a the right arm of chromosome 1; b the right arm of chromosome 3, compared with the respective section of the chromosome map (Pelling, 1964). Each bracket in the chromosome map indicates a puff observed in vivo. Bar=20 gm

234 Table 3. Countings of puffs on the left arm of c h r o m o s o m e 3 expressed in high salt C h r o m o s o m e s were isolated, incubated 5 min at 25~ in the high salt R N A synthesis mix and prepared for autoradiography as detailed in Methods. The examples listed originate from different animals. The puffs expressed were counted under the light-microscope. Since the identity of some puffs remains questionable. a m i n i m u m and a m a x i m u m n u m b e r of puffs is given for every example

H. Hameister

Sample

N u m b e r of puffs Minimum

I II III IV V VI

48 50 47 50 48 49

VII

48

Maximum 54 54 52 56 50 54 54

clear separation of labelled bands. Figure 12a shows a single chromosome 2. Good recondensation into single bands was only achieved for the left part, including 1A-5A. The rest of the chromosome, including the nucleolar region, has not undergone sufficient recondensation to allow puff identification. In Figure 12b-d typical examples of chromosome 1, 2 and 3, respectively, are shown which were fixed after recondensation. In chromosomes of this quality it is apparent that incorporation is restricted to individual bands and not evenly spread over the whole chromosome, as would be suspected from the results with nuclei (Fig. 1l c). Estimates of the number of labelled sites indicate that most of the bands do not exhibit RNA synthesis under the conditions employed. Further analysis reveals that the position and number of bands labelled on chromosomes incubated in high salt is reproducible and constant. In Figure 13a and b this is demonstrated for two selected chromosome segments, the right arm of chromosome 1, section 14-17, and the right arm of chromosome 3, section 12-13. The number of puffs expressed in high salt was counted in identifiable sections of the three long chromosomes 1, 2 and 3 and Table 3 summarizes the data from the left arm of chromosome 3 which turned out to be best represented among our chromosome preparations. A maximum and minimum number of puffs is listed because of the uncertainties involved in such counts. On the average 51 puffs were expressed in high salt. This corresponds to nearly 20% of all the bands listed in this section and is slightly in excess of the number of active sites which Pelling, 1964, compiled in an elaborate analysis of RNA synthesizing sites in the salivary gland of C. tentans. In other words 80% of the bands remain inactive under these conditions (see also Discussion).

V. Nucleolar Spreading Preparations In some autoradiographs of nuclear preparations incubated in SSM silver grains were not evenly distributed over the nucleoli but tended to be concentrated over numerous carrotshaped subunits, often widely separated from each other. On close inspection an ordered arrangement of these subunits reveals itself (Fig. 14).

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Fig. 14a-e. Nucleolar spreading preparations. Nuclei were isolated and incubated 30 min at 25 ~ C in the SSM R N A synthesis mix (see also Fig. 8). Arrow indicates the inner wheel-like structure. NI and N2 are nucleolar masses originating from different nuclei. Note the reduced c h r o m o s o m a l activity. B a r = 2 0 ixm

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It appears that (1) the subunits are arranged in tandem, (2) several of these sets of subunits lie in parallel. These characteristics suggest that the subunits are ribosomal cistrons. It is known from the EM-pictures of Miller and Beatty, 1969, that the ribosomal cistrons are arranged in tandem separated by the non-transcribed spacer. As we are dealing with polytene chromosomes several of these strings of spaced subunits are ordered side by side. This arrangement of the ribosomal cistrons does not hold true for the inner of the nucleolus which is built up by wheellike ordered cistrons as depicted in Figure 14b and c. The assumption that these nucleolar subunits represent ribosomal cistrons is not in conflict with estimates of their size and R N A synthesizing capacity as calculated from grain counts. In spite of the limitations of the technique a crude estimate of the number of nucleolar subunits is often feasible. Thus the nucteolus shown in Figure 14a appears to be built up of ca. 200 subunits. The following observations may also be significant. Within the same nucleus the number of expressed ribosomal cistrons is similar in the two nucleoli of chromosome 2 and 3. But there is great variation in the number of expressed ribosomal cistrons between nucleoli from different nuclei with a comparable level of polyteny (Fig. 14c).

Discussion

The giant chromosomes of Dipterans offer obvious advantages as a system for the in vitro study of transcription. They represent a form of interphase chromatin which is, in contrast to the chromatin preparations usually studied, structurally highly defined and amenable to microscopic analysis. For our in vitro system this well known in vivo structure of the giant chromosomes was the point of departure in judging the salt concentration employed. An optimal salt concentration was arrived at from cytological experiments with isolated chromosomes (Glancy, 1946; Lezzi, 1967; Robert, 1971). A concentration in the range of 0.15 M monovalent cations (0.09 M K C I + 0 . 0 6 M NaC1) (Glancy, 1946) was found to be most effective. This is in accordance with determinations of the activity of monovalent cations in rat liver nuclei (Siebert and Langendorf, 1970). Though of the divalent cations Ca 2 + is most effective in maintenance of chromosome structure, it was replaced by 2 m M Mn 2§ , since Ca 2+ acts inhibitory to R N A synthesis in vitro.

L RNA Synthesis in the Standard Salt Medium Under the salt conditons defined above R N A synthesis proceeds linearly for 30 min at 25~ Edstr6m and Beermann, 1962, determined the R N A content of Chironomus tentans chromosomes to be 1/7-1/8 of the D N A by weight. This R N A turns over within 30 rain (Egyhazi, 1974) leading to in vivo transcriptional activity of 0.004 gg R N A • min 1 x gg 1 DNA, similar to nuclear R N A turnover calculated elsewhere (Brandhorst and McConkey, 1974). R N A synthesis in vitro on the other hand is more than two orders of magnitude less

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than that derived from in vivo calculations (Fig. 3) and this is borne out again by the autoradiographic data showing disproportionately low activity at puff sites in vitro (Fig. 8), In an attempt to stimulate the activity of the in vitro system exogenous heterologous polymerases were added. They were shown to stimulate incorporation by some 30%. Polymerase A induces an incorporation pattern which is in accordance with the template specificity of this enzyme (Fig. 10c). This does not hold true for polymerase B, which also leads to marked induction of synthesis in the nucleoli. This may be a consequence of the fact that the DNA within the nucleoli is more despiralized than at other chromosomal sites and thus more available for binding and elongation by polymerases (Laird et al., 1976). Since addition of exogenous eukaryote enzymes gives rise in every case to the endogenous incorporation pattern and the incorporation mediated by polymerase B in the nucleolus turned out to be ~-amanitin-resistant, the question arises whether this label is due to elongation by the added enzymes themselves or to mere activation of endogenous polymerases. Such activation would circumvent difficulties in the initiation of the endogenous enzymes on a double stranded template (Dez~l& et al., 1974). It should be emphasized that the E. coli "holoenzyme" is unable to induce a specific incorporation pattern (Fig. 10t). The nature of our template enables the spatial specificities of added enzymes to be investigated. The results presented make it clear that precautions have to be taken in the interpretation of chromatin transcription by exogenous polymerases.

IL RNA Synthesis in the High Salt Medium Severe deviations from the standard salt concentration to hypotonic (Glancy, 1946; Beermann, 1962) and to hypertonic conditions (Robert, 1971) cause a reversible disappearance of the banding structure. Decondensation in low salt coincides with a decrease in RNA synthesis capacity while decondensation in high salt results in an increase in RNA synthesis (Fig. 4; Marushige and Bonner, 1966; Cox, 1973; Widnell and Tara, 1964; Reeder and Roeder, 1972; Leake et al., 1972). High salt decondensation was shown to be a differential process beginning at selective gene sites and countinuing with further increase of salt concentration at other bands (Robert, 1971). At 0.45 M monovalent cations the banding structure is lost completely. However there is no stringent correlation between high salt decondensation and RNA synthesis. Thus, the left arm of chromosome 3 contains about 250 bands of which on the average only 51 are expressed as sites of elevated transcriptional activity in high salt. In other words, 80% of all the bands remain inactive under conditions of stimulated transcription (Table 3). The increase in synthetic capacity in high salt is specific for nucleoplasmic, Mn 2§ dependent RNA polymerase B (Fig. 4; Widnell and Tate, 1964; Novello and Stirpe, 1969; Cox, 1973; Zylber and Penman, 1971), implying some fundamental differences in enzyme or, more likely, template characteristics between nucleolar and chromosomal sites. Within the ribosomal cistrons the DNA is

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normally unfolded (Miller and Beatty, 1969; Meyer and Hennig, 1974). It is suggested, that at the other active chromosomal sites the DNA remains in a more condensed state, which is released in high salt. The increase in synthetic activity may be a result of (1) dissociation of inhibitory proteins (Lindigkeit et al., 1974) and or (2) general transitions in chromatin conformation (de Pomerai et al., 1974). The results presented in Table 2 make it unlikely that (1) alone can account for the 30-50-fold stimulation in polymerase B activity. Although several doubts can be raised to whether reassociation of proteins in standard salt has been effectively inhibited in this experiment, the result seems to favour the second alternative. This idea is further supported by the observation that the presence of heparin in SSM also leads to much enhanced puff activity and concomitant decondensation (unpublished observation). It has been suggested that at least part of the stimulatory effect of this polyanion is due to an undefined gross change in chromatin conformation (Groner et al., 1975). Both heparin and high salt decondense the chromosomes and stimulate RNA synthesis, however both are also capable of completely inhibiting reinitiation of RNA polymerases (Fuchs et al., 1967; Walter et al., 1967; Hyman and Davidson, 1970). Thus it is clear that the RNA synthesis observed under these conditions must be attributed to prebound RNA polymerases.

III. Autoradiography in High Salt Further evidence in support of this notion comes from a comparison of the incorporation pattern observed in vitro in high salt and those found in vivo. In vivo one is confronted with great fluctuation in the extent of RNA synthesis, which can amount to a factor of 1000 between salivary gland nuclei of different animals (Pelling, 1964). Furthermore there exists stage specific variation in the puff pattern during development (Beermann, 1952; Becker, 1959; Clever, 1961). One can distinguish between developmentally non specific puffs, which are expressed always, and developmentally specific puffs, which are expressed only at particular points during the lifetime of the salivary gland. Pelling (1964) has made a detailed investigation of puffs expressed in vivo and compiled them in a puff map. Some 300 bands were found to be capable of puffing in the differentiated salivary gland. In chromosome 1 it was possible to identify a maximum of 97 puffs. However, the number observed on individual first chromosomes varied between 37 and 61, i.e. at any one time only about 50% of the potential puffs are found to be expressed simultaneously. In vitro, in the presence of high salt, we find quite a different picture. No variation is observed in the extent of overall synthesis, i.e. all nuclei or chromosomes from different animals exhibit equally strong incorporation. Furthermore, the same set of puffs is always expressed under these conditions. A detailed puff analysis was done for the left arm of chromosome 3 incubated in high salt. Pelling, 1964, listed 43 puffs in this section with 4-5 additional ones of uncertain localisation. In vitro, with high salt 51 puffs are expressed on the average (Table 3) and these 51 puffs are active in RNA synthesis in

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every case observed. This observation is interpreted as indicating the presence of RNA polymerases at all gene sites which become active at any time in this differentiation state. These gene sites are distinguished from the 80% of bands which remain untranscribed in this tissue by the presence of bound RNA polymerases in quantities leading to detectable RNA synthetic capacity. One then has to distinguish two functional states of these sites: (1) activated sites, where polymerases are currently synthesizing RNA, (2) preactivated sites, where the enzymes are bound in an arrested state. Fine regulation of transcription leading to full read-out of the gene is then required to occur downstream of the binding site of the polymerase. Such a gate could also control the number of bound polymerases allowed to read further into the gene. In high salt the function of this second control-site is abolished, and the bound RNA polymerases read through this site. A similar second regulation site downstream of the binding site and just before the beginning of the structural part of the cistron has been demonstrated in the tryptophan operon of E. coli (Bertrand et al., 1975). Several other authors have reported the unmasking of RNA polymerases in chromatin by different treatments. High salt was used to decouple RNA polymerases in resting mouse kidney epithelial cells (Moore et al., 1974), polyanions to unmask them in the facultative heterochromatic male chromosomes in the testis of mealy bugs (Miller et al., 1971) and Sarkosyl to demonstrate the occurence of two classes of RNA polymerases B in chromatin, one actively transcribing and a second class of resting polymerases, derepressed by Sarkosyl treatment (Green et al., 1975). Moreover these authors reported RNA polymerase B activity in mitotic cells, which is comparable to the activity in interphase cells, when released by Sarkosyl (Gariglio et al., 1974).

IV. Time-Course in High Salt

The stimulation of the Mn 2§ dependent RNA polymerase B becomes prominent with time (Fig. 5). Whereas nucleolar RNA synthesis levels off after 10-15 rain, chromosomal RNA synthesis continues at a high but decreasing rate for 12-16 h (Fig. 6). As high salt inhibits reinitiation of polymerases (Fuchs et al., 1967; Hyman and Davidson, 1970), synthesis for up to 16 h has to be attributed to elongation by prebound enzymes. In this case the RNA molecules isolated ought to increase in length with time as a consequence of uninterrupted polymerisation. In high salt alone only RNA molecules of a constant size of 5-8 S were isolated. In the presence of heparin, itself an inhibitor of initiation, it is possible to demonstrate chain elongation with time. It is important to note that heparin does not alter the main features of the long time-course. These results are in accordance with those obtained by other authors (Cox, 1973; Groner et al., 1975) and have been interpreted as direct effect of heparin on the chromatin, perhaps leading to some unwinding of the DNA. Since the long time-course is accompanied by continuous chain growth it is of interest to ask how long the transcriptional unit might be to account for such synthetic behaviour. The rate of in vitro transcription of chromatin

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has been determined to be ca. 1 nucleotide/sec (Cedar and Felsenfeld, 1973; Cox, 1973). This would enable the whole o f an average c h r o m o m e r e o f 40 kb to be transcribed in ca. 10 h.

V. Structural Organisation of Ribosomal Cistrons in the Nucleolus If one c o m p a r e s the EM-pictures of ribosomal cistrons (Miller and Beatty, 1969; Meyer and Hennig, 1974) with the a u t o r a d i o g r a p h s o f nucleoli shown in Figure 14, a n d considers that we are dealing with polytene c h r o m o s o m e s , the conclusion appears inescapable that the grain assemblies or nucleolar subunits observed represent active ribosomal cistrons. A s s u m i n g that each subunit corresponds to one cistron, the n u m b e r o f cistrons active in R N A synthesis under in vitro conditions is rather low. The salivary c h r o m o s o m e s o f late 4th instar Chironomus tentans are built up by at least 2000 chromatids a n d within each c h r o m a t i d the ribosomal cistrons are reiterated 43 times according to hybridisation data (Hollenberg, 1976). T h u s the expected n u m b e r o f ribosomal cistrons would be in the range o f 100,000 whereas our estimates o f active ribosomal cistrons are in the range o f 200 with vast variation (Fig. 14a). The observed regulation o f R N A synthesis involves b o t h nucleoli o f c h r o m o some 2 and 3 to the same extent. G r o s s differences in the n u m b e r o f subunits between the two nucleoli o f the same nucleus were never observed. Whether this regulation principle applies to all nuclei o f the same gland c a n n o t be definitely decided, since the nuclei c o m b i n e d on the same slide originate f r o m different animals. The b r o a d distribution o f nucleolar size in different nuclei tends to refute the possibility o f such global nucleolar regulation m e c h a n i s m in the gland.

Acknowledgements. I am obliged to Dr. C. Pelling for his suggesting this investigation and for continuous interest and support. The skilful help by Ms. F. Hofmann is gratefully acknowledged. P.A. Hardy aided in preparing the manuscript. This research was supported by the Deutsche Forschungsgemeinschaft.

References Becker, H.J.: Die Puffs der Speicheldrfisenchromosomen von Drosophila melanogaster. Chromosoma (Berl.) 10, 654~678 (1959) Beermann, W.: Chromosomenkonstanz und spezifische Modifikationen der Chromosomenstruktur in der Entwicklung und Organdifferenzierung yon Chironomus tentans. Chromosoma (Berl.) 5, 139-198 (1952) Beermann, W. : Riesenchromosomen, Protoplasmatologia, VI/D, 1-161. Wien: Springer 1962 Bertrand, K., Korn, L., Lee, F., Platte, T., Squires, C.L., Squires, C., Yanofsky, C. : New features of the regulation of the tryptophan operon. Science 189, 22-26 (1975) Bradbury, E.M., Carpenter, B.G., Rattle, H.W.E. : Magnetic resonance studies of desoxyribonukleoprotein. Nature (Lond.) 241, 123-126 (1973) Brandhorst, B,P., McConkey, E.H.: Stability of nuclear RNA in mammalian cells. J. molec. Biol. 85, 451-463 (1974) Butterworth, P.H.W., Cox, R.F., Chesterton, C.J.: Transcription of mammalian chromatin by mammalian DNA-dependent RNA-polymerase. Europ. J.Biochem. 23, 229-241 (1971)

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Received December 6, 1976 / Accepted February 10, 1977 by W. Beermann Ready for press March 1, 1977