The size of the transcription unit in balbiani ring 2 of Chironomus tentans as derived from analysis of the primary transcript and 75 S RNA

J. Mol. Bid. (1978) 124,223-241

The Size of the Transcription Unit in Balbiani Ring 2 of Chironomus tentans as Derived from Analysis of the Primary Transcript and 75 S RNA STEVEN T. CAsEt AND BERTIL DANEHOLT$

Department of Histology Karolinska Institutet S-104 01 Stockholm 60 Sweden (Received 12 December 1977) The size of the transcription unit for 75 S RNA in Balbiani ring 2, a giant puff in the salivary glands of Chironornua tentans, has been derived from determinations of the size of salivary gland 75 S RNA as well as of the size of the primary transcript in BR2 5. Salivary gland 75 S RNA, predominantly located in the cytoplasm, was analyzed by electrophoresis in formaldehyde-containing agarose gels. The molecular weight was shown to be at least 12 x lo6 but probably not exceeding 13 to 14 x 106. The size was also determined by contour length measurements in the electron microscope, which gave a molecular weight of 12.3 x 106. Since this value fell within the size range established by the electrophoretic method, it was accepted as an estimate of the size of 75 S RNA. The biochemical and electron microscopic evidence for considering 75 S RNA as a single, covalently linked polynucleotide is discussed. Balbiani ring 2 RNA, containing the finished primary transcript as well as growing RNA molecules, was analyzed in agarose/formaldehyde gels with salivary gland 75 S RNA as an internal size marker. From the distribution of the nascent RNA it was concluded that the finished primary transcript essentially migrated coincident with 75 S RNA. Thus, the molecular weight value of 12.3 x lo6 was also adopted for the primary transcript. This would imply that the corresponding transcription unit in BR2 comprises about 37 x lo3 base-pairs of DNA. The predicted size of the transcription unit in BR2 was related to the information available on the BR2 chromomere and the adjacent interchromomeres. It was concluded that only a minor part of the unit (less than 5 x 103 base-pairs) can be accommodated in an interchromomere adjacent to the BR2 chromomere. Most, if not all, of the unit has to be present in the BR2 chromomere itself. On the other hand, there is more DNA in a BR2 chromomere (> 100 x lo3 base-pairs) than can be accounted for by one single 7.5 S RNA transcription unit.

1. Introduction In contemporary studies of the eukaryotic genome it is important to define and characterize individual non-ribosomal transcription units. Transcriptionally active t Present address: Kline Biology Tower, Yale University, New Haven, Conn. 06520, U.S.A. $ Author to whom correspondence should be addressed. # Abbrevietion used: BR2, Balbiani ring 2. 223 0022-2836/78/260223+ 19 $02.00/O

0 1978 Academic Press Inc. (London) Ltd.

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units can be visualized in the electron microscope which makes it feasible to st,udy the range as well as the frequency of transcription unit sizes (Foe et al., 1976; Laird et al., 1976; McKnight & Miller, 1976). Only occasionally, however, e.g. in the case of the fibroin gene (McKnight et al., 1976), has it been possible to identify specifically the active genes which have been seen in the electron microscope. Information on t,he sizes of the transcription units can also be derived from biochemical ana.lyses of pulse-labelled heterogeneous nuclear RNA (Derman et aZ., 1976: Perry et al., 1976) and from investigations on the effects of ultraviolet irradiation on its synthesis (Giorno & Sauerbier, 1976; Goldberg et al., 1977). Again, only in a few cases have defined primary transcripts been studied (Daneholt, 1972 ; Egyhszi, 1975 ; Lizardi, 197&J). Usually, due to the complexity of newly synthesized RNA as well as to its rapid processing, it ha,s been difficult to isolate and study specific non-ribosomal primary transcripts in order to obtain information about their corresponding transcription units (for a review, see e.g., Lewin, 1975). The salivary glands of Chironomw tentans offer one of the few systems available for the direct examination of defined primary transcripts. Because of the exceptional size of their giant chromosomes, it has been feasible t.o isolate specific chromosome segments (Edstrom & Beermann, 1962 ; Lambert & Daneholt, 1975) and to characterize RNA synthesis in these regions (for a review, see Daneholt, 1975). Most attention has been focused on the products made at one giant, tissue-specific puff, called Balbiani ring 2 located on the IVth chromosome. The finished transcript, 75 S RNA, as well as the growing molecules can be studied. Furthermore, of particular interest for the analysis of the primary transcript is the fact that 75 S RNA from BR2t is transported into the cytoplasm (Daneholt & Hosick, 1973) and further into polysomes (Daneholt et al., 1977a) wnhout a major reduction in size. Along with RNA from BRl (and perhaps also from other puffs), BR2 RNA constitutes a major cytoplasmic 75 S RNA species (for a review, see Case $ Daneholt, 1977). In the present study we have derived the size of the transcription unit in BR2 by determining the size of both salivary gland 75 S RNA and the primary transcript in BR2. The size of the unit was then related to the information available on the band-interband structure of the BR2 region. We concluded that only a minor part of the unit can be accommodated in interchromomeric DNA. Therefore, the transcription unit for 75 S RNA in BR2 has to be primarily or completely located within the BR2 chromomere.

2. Materials and Methods (a) Cultiwation of animals larvae were cultured in plastic tubs containing saline (0.04% (w/v) NaCl) and enough pieces of torn cellulose paper initially to cover the bottom of the tubs. Cultures at were provided with moderate aeration and kept on a 16/8-h cycle of daylight/darkness C. ten&as

18°C. Animals were fed twice weekly with nettle powder dissolved in saline in an amount not in excess of what the animals in a culture could clear from the bottom of the tubs prior to the subsequent feeding. Under the above conditions larvae from most cultures

pupate and develop into midges. The average time to develop from eggs to pupae was about 7 weeks. For our studies, only early to mid 4th instar larvae with a length of about’ 14 to 16 mm were selected t See footnote

to p. 223.

from

rapidly

growing

cultures.

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conditions

When larvae were to be incubated with isotopic precursors in vizro, as many as 25 animals were placed into a small cup containing 20 ml of saline and a few pieces of torn cellulose paper. The following isotopes (Amersham) were used: [3H]cytidine (spec. act. > 25 Ci/mmol) and [3H]uridine (> 40 Ci/mmol) added to yield a final concn of 5 rCi/ml c>ach; alternatively [‘%]cytidine and [14C]uridine (both > 450 mCi/mmol) each at a final concn of 0.5 &i/ml. Animals were bathed in isotopic saline for 3 days at 18’C. Labeling of explanted glands in vitro was performed in 25 ~1 of Cannon’s medium containing 100 &i of both r3H]cytidine and [3H]uridinr, as previously described h> in vitro were performed for 45 min at 18’C’. Lambert, & Daneholt (1975). All incubations whicll is in excess of the time needed to saturate the BRs wit,11 isotope, (Danellolt et al.. 1969a; Egyhitzi, 1975). Subsequently, glands were fixed in 707; ethanol for 1 h at 4°C ant1 then placed in a solution of ethanol/glycerol (1 : 1) for at least 1 II prior to rnicrodissect,iol1. Microdissection of various cellular components was accomplished with a dr Fonhrunc~ micromanipulator. as recently described by Lambert bt Daneholt (1975). (c) Extraction

of

RhrA

Tot,al salivary gland nucleic acids were extracted according to C-0 & Daneholt ( 1976) with some modifications. As many as 10 glands were simultaneously extracted for 10 mirl in 400 ~1 of extraction medium kept at 2 to 4°C on a thermostatically controlled cold plat,c mounted on the stage of a dissecting microscope. The extraction medium was a solution (TES) of 20 mw-Tris *HCl (pH 7.4), 10 mM-Na,EDTA and 0.576 sodium dodecgl sulfat,e to wllich 1 mp of Pronase/ml (Calbiochem, RNAase-free) was added. The medium was predigested at 37°C for 30 min prior to use. We have tested and found TES medium to bcs superior to our previous extraction and electrophoretic buffers with respect to protectiorl against RNAase. Nucleic acids could be precipitated from the soluble portion of the extract by the addition of 0.1 vol. 1 M-NaCl and 2.5 vol. absolute ethanol or, alternatively, thus soluble extract could be extracted with phenol prior to precipitation with ethanol. Wllil,. electrophoret,ic profiles on non-denaturing gels were essentially the same with or withollt phenol extractions, this step was found to be imperative for analyses on formaldehyderont’aining gels in order to avoid tailing. Extractions utilizing freshly distilled phenol saturated with TES medium at pH 7.4 or pH 9.0 yielded identical recoveries of 75 S RNA. 13H]RNA was obtained from fixed glands or microdissected cellular components using cbxactly the, same procedures except that the TES medium cont’ained only 2 rnbl-Na,EDTA (rl) Electrophoresis

in n,on-denaturing

and

denatwin,g

agarose

gels

Elcctrophoresis under non-denaturing conditions was performed in horizontad 0.75”,, et al. (1969b). The gels werp, however. agarose slab gels essentially according to Daneholt prepared in TES medium (pH 8.0) and were placed on a fresh piece of Parafilm whiclr covered an ultraviolet fluorescent screen. The use of a hydrophobic surface under t,he pcbl eliminated trailing artefacts of nucleic acid bands which could be observed if the gel was cut into longitudinal sections. Ethanol pellets were dissolved in 30 ~1 of TES medium anti apphcld t’o sample troughs. Electrophoresis was carried out, at room temperature using a \-oltage gradient of 3 V/cm (measured across the surface of the gel) and using recircldating TES medium as the electrophoretic buffer. After the initial 15 rnirl, the sllrfaco of tllc Gaul was lnoistened with TES medium and covered with Parafilm t,o prrvent evaporation. Near tjhe end of the run the top layer of Parafilm was removed and thr position of 4 S 01’ rihosomal RNA bands briefly visualized under a u.v. lamp and marked on t,hr gels. Sca,nning of gels were made with a Vitatron spectrophot,omcter. The percentage of t,hch RNA mass represented by 75 S RNA was determined by comparing the integrated areas under the 4 main RNAase-sensitive peaks in unstained gels scanned at 254 nm. The total amount of C. tentans RNA in a gel was determined by comparing the summat,ion of tt~es~~ areas to tracings of a dilution series of known amounts of Escherichia coli transfer RX.4 (measured as 41 fig/A260 unit) simultaneously elect,rophnrrnt~tl ill parallrl t,rougtlx on t hr. same gel. When radioactivity measurements were to be made, gels were cut, int,o 1.1.mm slices and placed in a toluene-based scintillation fluid containing Soluene 100 (Daneholt &

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Hosick, 1973) at room temperature overnight or at 60°C for 2 h prior to cooling and counting in a Packard Tricarb liquid scintillation spectrometer. The slab gel system was also applied for electrophoresis under denaturing conditions. Formaldehyde-containing agarose gels were prepared according to the procedure of Lehrach et al. (1977). The electrophoretic and gel buffer was, however, modified according to Lizardi et al. (1975) and contained 2.2 M-formaldehyde, 10 mm-NazEDTA and 40 mMtriethanolamine-HCl. Slab gels of higher agarose concentrations (0.5% and 075%) were moulded as described earlier (Daneholt et al., 19693), while 0.3% gels were made according to McDonell et al. (1977). Samples were dissolved in the electrophoretic buffer containing 50% (v/v) formamide and heated to 60°C for 5 min (Lehrach el al., 1977). After cooling to room temperature, the samples were applied to gels which, during electrophoresis were sandwiched between Parafilm as described for the non-denaturing gels. However, of the run), the before the top layer of Parafilm was added (15 min after the initiation denaturation buffer was removed from the application trough and replaced with gel buffer. Electrophoresis was performed at room temperature with recirculating gel buffer. The voltage gradient was 1.5 V/cm or 3 V/cm as indicated in the text. In order to evaluate the effects of electro-osmosis in formaldehyde-containing agarose gels, 3 batches of Dextran (Pharmacia) (molecular weights of 0.5, 5.2 and 15 x 108, respectively) were analyzed in these gels. The migration of the 3 fractions proved to be size-dependent : the apparent mobilities of the Dextran fractions amounted to about 10% of that found for RNA of corresponding sizes (Dextran and RNA moved in opposite directions). Since in the present study only the relative mobilities of the various RNA fractions were of interest, it was not necessary to correct the recorded apparent mobilities for the minor and proportional effects of the electro-osmotic flow. (e) Purification

of 75 S

RNA

A number of extractions of salivary gland RNA were carried out and the RNA was collected. About 250 to 300 pg of RNA were dissolved in 200 ~1 of TES medium and applied to a preparative 0.75% agarose slab gel. Electrophoresis was performed at room temperature under non-denaturing conditions as described above. When the separation was completed, the 75 S RNA band was visualized under a U.V. lamp, cut from the gel, and prepared for micro-electrophoretic elution (Case & Daneholt, 1976). This latter procedure was modified on 2 points: TES (pH 8.0) was used as buffer, and the elution took place at room temperature. Purified 75 S RNA was ethanol precipitated from TES buffer for subsequent electrophoretic or electron microscopic studies. Our average yield after elution and ethanol precipitation was 20 to 25% of the input RNA. (f) Electron microscopy To obtain strongly denaturing conditions prior to and during spreading, the hyperphase was made from a stock of 5.9 M-urea/74% formamide prepared according to Kung et aZ. (1975) using recrystallized formamide (Robberson et al., 1971). Aqueous electrolytes (5%, v/v) were added to the urea/formamide solution to yield a final concn of 10 mM-Tris (pH 85), 1 mM-Na,EDTA. Ethanol-precipitated RNA to be spread for microscopy was then dissolved directly in the buffered urea/formamide, and cytochrome c was added to a concn of 30 pg/ml just prior to spreading. The final hyperphase was 95% by vol. (5.9 Murea/74% formamide). 50-~1 samples were spread on a hypophase of distilled water at a flow-rate of 100 to 120 pl/min as regulated by a syringe pump. Samples were adsorbed to 200-mesh copper grids covered with carbon-coated 1.5% parlodion films. Grids were dehydrated in 90% (v/v) ethanol, stained in alcoholic 1O-4 M-uranyl acetate, rinsed in ethanol and dried. The grids were then rotary-shadowed with 2.5 cm of platinum/palladium (SO/ZO) wire at an angle of 5 to 8”. Samples were visualized and photographed in a Philips EM300. Extended molecules were photographed at random and their negatives projected to a final enlargement of 100,000 x . Molecules were traced and their contour lengths measured manually using a micrometer which was converted into a map-measuring device. Magnification calibrations were obtained by a carbon replica of a diffraction grating containing 2160 lines/mm. Internal or external length standards, HeLa cell 28 S rRNA and E. coZi 23 S rRNA,

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BALBIASI

RING

9

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respectively, were included in each experiment. The standard rRNA species were purified from agarose gels and analyzed by electron microscopy as described above for 7.5 S RNX

3. Results (a) Amount

and intracellular

distributio~a of 75 S RNA

The secretory cells of C. tentans salivary glands contain at least four prominent size classes of RNA when analyzed by gel electrophoresis. Figure l(a) shows that when an extract of total salivary gland nucleic acids is electrophoresed in 0.75% agarose gels. four major RNAase-sensitive peaks are observed: a slowly migrating 75 S RNA, the two ribosomal RNA species (18 S and 28 S RNA) and 4 S RNA. From a series of densitometric tracings (see Materials and Methods) we have determined that an average gland contains 2.6 ( f 0.4) pg of RNA, which is in good agreement wit,h earl&I reports (Rubinstein & Clever, 1972 ; LGnn & Ed&rem, 1977). The 75 S peak represents about 3.5% (IL y 9, with a range of 2.7 to 64%) of the mass of total RNA. Our present value of 75 S RNA being equal to 3.5% of the t,otal cellular RNA content i;; more than twofold higher than earlier results (Daneholt & Hosick, 1973) and probably reflects t,he increased efficiency with which we can ext)ract undegraded 75 S RX.4 from unfixed glands. Our results imply an average of about 91 ng of 75 8 RNA pel gland, or 2.6 ng per salivary gland cell (there are typically 30 t’o 40 secretory cells pt’r gland). An electrophoretic analysis of RNAlabeled in viva for t,hree days is presented in Figure l(c). The RNAase-sensitive radioactive profile closely resembles the densitometric tracing in Figure l(a) and indicates that under the conditions utilized, the abundant cellular RNA species are labeled. Figure l(b) and (d) indicates that, both the optical density and radioactivity of the 75 S peaks would be indicative of a single electrophoret’ic species under the present electrophoretic conditions. It has recently been shown that about 95%, of the salivary gland 75 8 RNA is in long-term (7 days) present in the cytoplasm (EdstrGm et al., 1978). Furthermore, labeling experiments (Daneholt & Hosick, 1973), it leas established that more than 95%, of the labeled 75 S RNA resides in the cytoplasm. In t,he present, study t,ht: salivary glands incorporated isotopic precursor for t’hree days in viva. From ele&rophoretic profiles of microdissected nuclear and cytoplasmic fractions (data not shown). we could estimate that after three days of labeling ill viwo: at least 83% of the radioactive 75 S RNA was located in the cytoplasm. We can t,herefore conclude that total as well as long-term labeled 75 S RNA (Fig. l(a) and (c), respectively) is predominantly located in the cytoplasm. (b) Determination

of the size of 75 S RNA

in denaturing

gels

Cellular 75 S RNA was subjected to electrophoresis under denaturing condibions in formaldehyde-containing agarose gels as follows. Radioactive salivary gland RNA containing amounts with measureable optical density of rRNA was precipitated in ethanol, dissolved in 2.2 M-formaldehyde, 40 mM-triethanolamine (pH 8.0), 10 m&INa,EDTA, 50% (v/v) formamide, and denatured for five minutes at 60°C prior to its application to the gel. Identically treated samples of E. coli RNA as well as pulsc~labeled HeLa cell nuclear RNA containing amounts with measureable optical densit!, of HeLa cell rRNA were applied in parallel troughs on the same gel. Thus each run of 75 S RNA had an overlapping combination of internal and ext’ernal molecular

228

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1234567123 Distance

migrated

(cm)

(T I

I

I

7

d)

IO00

4

IO

20

30

40

50

60

IO

20

500

30

Slice no FIG. 1. Electrophoresis of C. tentans total salivary gland RNA in 0.75% agarose gels, (a) About 26 pg of RNA were electrophoresed in a 0.76% agarose gel made in TES medium: 20 m&r-Tris (pH %O), 10 mM-Na,EDTA, 0.5% sodium dodecyl sulfate (see Materials and Methods). Spectrophotometric tracings were made at 254 nm prior to ( ) or after (. . . . .) treating the gel with RNA&se. To accomplish this treatment, nucleic acids were fixed and sodium dodecyl sulfate removed as follows. The gel was placed in a large volume of 5% trichloroacetic acid (CC1,COOH) at room temperature, then placed at 4°C and stirred vigorously for 2 h, a change of cold acid was added for 1 h, then extensive rinsing was carried out in running water overnight. During this procedure the 4 S band will diffuse out of the gel. Next, the gel was treated with RNA&se A at 60 pg/ml in 20 mlvr-Tris (pH 7.4), 20 mivf-N&j, 2 mM-Na,EDTA at 30°C for 12 h. When purified DNAs were electrophoresed into similar gels and subjected to the same treetment, they were uneffected. (b) A spectrophotometric tracing at 646 nm of part of a parallel gel containing a 75 S RNA peak which has been enhanced by staining with 0.1 oh Toluidine blue in 20 mM-sodium acetate (pH 6.2) followed by extensive rinsing in the same buffer. Nucleic acids were fixed and the dodecyl sulfate removed from the gel prior to staining, as described in (a). (c) A radioactive profile of one half of a 30 rg sample of [3H]RNA obtained from glands in half of the sample was treated Th e remaining animals labeled for 3 days in viva (-@-a--). with RNAese A at 50 pg/ml in 20 miw-Tris (pH 7.4), 10 mrd-NaZEDTA for 1 h at 37°C. Sodium dodecyl sulfate was added to 0.6% along with 20 pg of E. coli RNA and the sample subjected to electrophoresis in a parallel gel (-O-O-). (d) The 76 S RNA region in (c) replotted on an expanded scale.

TRANSCRIPTION

UNIT

SIZE

IN

BALBIANI

RING

2

2”‘) -.

weight standards. Figure 2(a) is an example of the results obtained in such an experiment and shows the position of 75 S RNA relative to various standards. As is seen more clearly in Figure 2(b), 75 S RNA withstands this denaturing treatment and still migrat,es as a single symmetrical peak. On the other hand, C. tentans 28 S rRNA is sensitive to denaturation and migrates on these gels coincidently with 18 S rRIL’A (Fig. 2(a)). Similar observations have been made previously for the large rRNA of a number of insectIs (Applebaum et al., 1966 ; Greenberg, 1969 ; Rubinstein & Clever, 1971).

Slm “0 FIQ. 2 Electrophoresis of salivary gland [sH]RNA in a denaturing agarose ge1. (a) Salivary gland RNA labeled for 3 days in viva, was extracted, phenol-treated and ethanolprecipitated. The RNA pellet (20 rg) was dissolved in 2.2 M-formaldehyde, 40 mM-triethanolamineHCl (pH 8.0), 10 mM-Na,EDTA containing 50% (v/v) formamide and denatured at 60°C for 5 min. The RNA was electrophoresed in a formaldehyde-containing 0.3% agarose gel at 3 V/cm (see Materials and Methods). The positions of HeLa cell 45 8 and 28 S RNA and E. coli 23 S RNA were obtained from parallel runs. (b) The 75 S RNA peak from (a) replotted on an expanded scale.

We proceeded to use the above approach to estimate the molecular weight, of 75 S RNA by calculating its mobility relative to reference RNA molecules. During t’he initial phase of this work we observed a mobility phenomenon which has been reported recently for large single-stranded or duplex DNA (McDonell et al., 1977) and RNA (Lehrach et aE., 1977) molecules. Such molecules can exhibit erroneously high relative mobilities when electrophoresed in aqueous or denaturing gels which have channels too small to accommodate the radius of these molecules in their equilibrium conformation. These authors suggest that in a moderately limiting gel mesh and a sufficiently high voltage gradient these macromolecules can uncoil and “snake” through the gel end-on, thus migrating relatively more rapidly than smaller reference molecules which are being sieved in their unperturbed conformations. Because of this information, we have analyzed 75 S RNA in formaldehyde-containing gels of varying agarose concentrations and low voltage gradients, as suggested by Lehrach et al. (1977). The mobility of 75 S RNA was determined in formaldehyde-containing gels of 0.3,

S. T. CASE

230

AND

B. DaNEHOLT

0.5 and 0.75% agarose at voltage gradients series of experiments can be seen in Figure can be seen that by applying either 1.5 V/cm lated the apparent molecular weight of 75 12.1 x 106, respectively). By increasing the

of 1.5 and 3.0 V/cm. The results of this 3. By comparing Figure 3(a) and (b), it or 3 V/cm to 0.3% agarose gels we calcuS RNA to be about 12 x lo6 (11.9 and gel concentration to 0.5% agarose, the 0.3 % 3.0 V/cm

(b) 75 s

\ \

0 45 S HeLa \

\

\

. 28 S HeLo \

\

.

. 23 S \ Ecoh

\.

45 S Hello

45 S HelLn \

\

\

28 S H&o

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\

l

28 S HeLo ;\23

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HeLa

0

28 S HeLa \

Mobility

(arbitrary

S E COll

0 23 S E. coli \

units)

FIQ. 3. The effect of gel concentration and voltage gradient on the mobility of 76 S RNA in denaturing agarose gels. The mobilities of 76 S RNA and various reference molecules were obtained at the gel concentrations and voltage gradients indicated in the panels and were expressed in arbitrary units (migration distance (no. of slices) x 103/potential gradient (V/cm) x time (min)). The mobilities have been plotted as a function of the logarithm of molecular weight in order to estimate values for 75 S RNA. The molecular weights of reference molecules are those obtained by electron microscopy (Wellauer & Dawid, 1973a,b). The points in each panel are the average of at least 2 determinations from independent experiments.

TRANSCRIPTION

UNIT

SIZE

Ih’

BALBIAXI

RING

2

L’31

use of either voltage gradient results in a similar apparent molecular weight determinat,ion but, as indicated in Figure 3(c) and (d), the values have now decreased to 10.4 and 10.3 x 106, respectively. Results with 0.75% agarose gels were more sensitive to variations in the voltage gradient. Figure 3(e) indicates an apparent molecular weight of 7.9 x 106 at 1.5 V/cm while Figure 3(f) indicates a value of 7.5 x IO6 at 3.0 V/cm. The electrophoretic data are summarized in Figure 4 where the apparent molecular weights obtained at, a given voltage gradient are plotted as a function of agarose concentration. The obvious decline in apparent molecular weight with increasing gel concentration. and at) some point increasing voltage gradient, is typical of the molecular weight-independent, migration described above. In theory, once the gel mesh is large enough to allow a molecule t,o enter in its equilibrium conformation, t#hen identical apparent, molecular weights will result upon decreasing the gel concentration and voltage gradient, at least over a range of values where t,he molecule of int,erest and appropriate standards are still being sieved. This is demonstrated in Figure 4 where we have entered values for HeLa cell 45 S precursor rRNA based on its mobilitIy relative to 28 S RN4 as well as to E. coli 23 S RNA (cf. Fig. 3). Since it, would appear that the curve shown for 75 S RNA has not reached a similar plateau, we predict that the values obtained in the lowest gel concentration are nearest to the actual values. The lowest concentration formaldehyde-containing gel in which WY have obtained highly reproducible results is 0.3% agarose. Thus our best estimate obtained by denaturing gel elect,rophoresis is that the molecular weight of cellular 75 S RNA is 12 x IO’. However, since we were not’ able to test significantly lowrr gel concentrations. we cannot rule out that 75 S RNA could be as much as 13 to 14 x 106, as judged 1)~ extrapolat’ing the curve in Figure 4 to Oo/, a g arose. Thus we have used electron microscopy as an alt’ernative method to estimate the size of 75 S RXA.

-.-

FIG. 4. A summary of apparent molecular weight values obtained for 76 S RNA under various clectrophoretic conditions in denaturing agarose gels. The apparent, molecular weights for 75 S RNA were estimated from the mobilities of 75 S RNA relative to t,he size markers (see .Fig. 3) and are plotted here as a function of agarove gel concentration at each voltage gradient: (i)) 1.5 V/cm; (0) 3.0 V/cm. The effect of t,hexe same changes in electrophoretic parameters on HeLa cell 45 S RNA is also illuxt,rated. In this case, the data points for 45 S RNA were obtained from its mobility compared to smaller RNA markers: (A) 1.6 V/cm; (A) 3.0 V/cm.

232

S. T. CASE

AND

(c) Electron microscopic

B. DANEHOLT analysis

of 75 X RNA

75 S RNA suitable for electron microscopy was purified and characterized as follows. Each 250 to 300 pg of 3H-labeled salivary gland RNA was separated on a preparative agarose gel which had an electrophoretic profile essentially like the one shown in Figure l(c). After separation was complete, the 75 S band was briefly visualized under ultraviolet light, cut out from the gel, and electrophoretically eluted as previously described (Case & Daneholt, 1976). Prior to spreading for electron microscopy, samples were checked for their integrity by gel electrophoresis. In a rerun in the agarose gel (Fig. 5(a)), the purified 75 S RNA migrated as a defined RNA species with the

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(a)

300 75 s 23Sl6S

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200

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100

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Fm. 5. Electrophoresis of purified 76 S RNA in non-denaturing and denaturing agarose gels. (a) One portion of a sample of purified 3H-labeled 75 S RNA was electrophoresed in a 0.76% (see Materials and Methods). Another portion of agarose gel made in TES medium (-e-e-) the sample was pretreated with RNAase A at 2 pg/ml in 1 x SSC (0.16 M-N&I, 16 mMsodium citrate) for 6 min on ice prior to mixing with an equal volume of 2 x TES medium for electrophoresis (-O-O-). A third portion was preincubated in 0.2 M-NaOH at 37°C for 16 h (-x-x -) prior to neutralization with 2 M-Tris (pH 8.0) and mixing with an equal vol. of 2 x TES medium. E. coli RNA was added to all samples just prior to electrophoresis. The results of these parallel gels are plotted together since their RNA markers migrated to identical positions. (b) Purified 3H-labeled 76 S RNA was denatured in formaldehyde/formamide as described in the legend to Fig. 2 and electrophoresed in a formaldehyde-containing 0.6% agarose gel (see Materials and Methods) until it reached approximately the same position as the 76 S peak shown in (a). Positions of 1. co& RNA markers am indicated.

TRANSCRIPTION

UNIT

STZE IN R.ilLBTANI

RING

2

-‘:!:I

same mobility as the 75 S peak of total gland RNA (cf. Fig. l(b)), which excludes any major degradation of 75 S RNA during its isolation. Figure 5(a) also demonstrates that purified 75 S RNA is RNAase-sensitive and alkali-labile. Another t’est for integrity is presented in Figure 5(b). The purified 75 S RNA was run in a denaturing agarose gel and again behaved as a single species migrating to the same po&ion in the gel as the 75 S peak of total gland RNA (cf. Fig. 3(d)), which indicates that, me arc’ :!ealing with a single species and that a minimum number of single-&and breaks harcl occurred during the preparative stages. Preparations exhibiting denaturing gel profiles such as bhe one shown in Figure 5(b) were used for electron microscopy. After purificabion, 75 S RNA was ethanol precipit)ated and dissolved directly in the spreading solution which, with the addition of aqueous electrolytes, was 95% (v/v) (5.9 M-urea/74% formamide) (Kung et al., 1975) as described in Materials and Methods. Since initial spreads indicated that 75 S RNA lacked secondary structure under these conditions, purified HeLa cell 28 S rRNA was included as an internal length standard as well as indicator of stable secondary structure (Wellauer & Dawid, 1973a). An example taken from one such spread is shown in Figure 6. While more than 90% of the HeLa cell 28 S RNA molecules displayed thrii characteristic pattern of hairpin loops, at least 97% of all 75 S RNA molecules examined were virtually free of such structures. Well extended molecules were photographed at random for contour length measurcments. Approximately 38% of the 75 S RNA molecules measured fall into the peak area indicated on t,he histogram in Figure 7(a) and have a mean contour 1engt)h are likely to ,: L?, = 7.75 (&O-7) pm. The smaller molecules of the distribution represent’ degradation products, most of them probably arising as a result of shearing, when the 75 S RNA molecules are spread for electron microscopy. It should, however, also be realized that the minor amount of degraded RNA present in Figure 5(b) will seem more prominent in Figure 7(a) because of the different type of plotting method used (mass in Fig. 5(b) wersus no. of molecules in Fig. 7(a)) (cf. Fig. 3 in Lizardi et al.. 1975). Length determinations were also made for HeLa cell 28 S RNA as an internal lengt,h standard (( Lj, == 1.11 (& 0.6) pm) and simultaneous parallel spreads of E. coli L’3 S RNA as an external length standard ((L), = 0.68 (A 0.09) pm). From their respective molecular weights of 1.76 x lo6 (Wellauer & Dawid, 1973a) and 1.07 Y 1O6 (Stanley & Bock, 1965), we calculate that, under the conditions described, we obt,ain a linear density of 1.58 x lo6 daltons/pm. Thus from F‘igure 7(a) we calculate that t,ln molecular weight of 75 S RNA is 12.3 & 1.1 x 106. Samples of 75 S RNA in urea/formamide (without HeLa cell 28 S RNA) wert’ heated 30 to 60 seconds at temperatures ranging up to 80°C prior to cooling, the addition of cytochrome c and spreading at room temperature. As shown in Figure 7(b). while the mean contour length decreased to 6.99 (jO.7) pm, 380/b of the molecules still remained distributed in that portion of the histogram in Figure 7(a) on which we based our molecular weight calculation. As we will discuss below, since most of the initially full-length 75 S RNA molecules resisted this extreme denaturabion step. \\‘t~ conclude t)hat 75 S RNA is a single covalently linked polynucleotidc. Final1.v. we wanted to establish a correlation between the RNAase-sensitive elect.rophoretic profile of purified 75 S RNA shown in Figure 5(a) and t,he molecules w(~ observed in the electron microscope. Therefore a sample of 75 S RKA was given a similarly mild RNAase treatment and mounted for microscopy. Figure 7(c) clear]?. indicates t’he RNAase-sensitivity of the 75 S RNA molecules that \ve have observed.

FIG.

A mixture of purified C. tenlnns 75 S RNA and HeLa (pH 8.5), 1 mw-Na,EDTA, and 30 pg cytochrome c/ml. loops (Wellauer & Dawid, 1973n). The bar equals 1 pm.

6. 4n electron micrograph of C. tentclns 75 S RNA. cell 28 S rRNA was spread from a hyperphase Arrows indicate HeLa cell 28 S RNA molecules

of 95% (5.9 M-~ea/74’$!~ formamide), displaying their characteristic pattern

10 m.n-Tris of hairpin

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ib) 30 25 20 -

I

I

I

I

I

I

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20 - 15 IO 5I

2

hlr-l 3 4

5

I 6

I 7

I 8

I 9

I IO

Length (pm) PIG. 7. Contour length measurements of C. tentrrns 75 S RNA. (a) Contour length measurements were obtained for a mixture of C. tentrrns 75 S RNA and HeLa cell 28 S rRNA molecules spread from the hyperphase described in the legend to Fig. 6. This histogram shows the distribution obtained by measuring 250 molecules of 75 S RNA and 110 molecules of 28 S RNA (indicated by the filled area). The cross-hatched area of the histogram indicates those molecules from which we calculate a mean length
236

S. T. CASE

(d) Electrophoretic

analysis

AND

B. DANEHOLT

of the primary

transcript

in BR2

In order to determine the size of the primary transcript in BR2 by electrophoresis, salivary glands were labeled in vitro for 45 minutes in the presence of [3H]RNA precursors. Subsequently, the glands were fixed, and BR2s were isolated by microdissection. To obtain cellular 75 S RNA as an internal size marker, salivary glands were also labeled in vivo for three days in the presence of 14C-labeled precursors and afterwards fixed. The 3H-labeled BR2 RNA and the 14C-labeled salivary gland RNA were then co-extracted and analyzed in a formaldehyde-containing 0.3% agarose gel. Figure 8 depicts the result of the electrophoretic analysis. 3H-labeled BR2 RNA shows a sharp increase in radioactivity towards the high molecular weight side of the distribution, while the activity towards the low molecular weight side is gradually falling. This asymmetric profile confirms earlier results obtained in non-denaturing 1% agarose gels (Daneholt, 1972) and is likely to represent a spectrum of growing RNA molecules, the largest ones being the finished, or almost finished primary transcripts (see Discussion). The peak of the BR2 RNA is essentially at the position of the internal size marker, 14C-labeled salivary gland 75 S RNA (Fig. 8). However, there

Slice no.

FIG. 8. Electrophoresis of a mixture of BR2 RNA and cellular 76 S RNA in a denaturing agarose gel. and total [l%]RNA from 10 in viva [3H]RNA from 140 in vitro labeled BR2s (-a-@-) labeled glands (-O-O-) were co-extracted, denatured, and electrophoresed in a formaldehydecontaining 0.3% agarose gel (see Materials and Methods). For the simplicity of presentation, only the 75 S peak of the 14C-labeled RNA has been plotted.

seems to be a minor difference, the peak of the BR2 RNA being one slice to the right of the salivary gland 75 S RNA. This would mean that the primary transcript could be slightly smaller than the cellular 75 S RNA. On the other hand, due to the nature of the nascent profile (see Discussion), the primary transcript is somewhat larger than is indicated from the position of the peak of the nascent distribution. The difference between the primary transcript and the cellular 75 S RNA is therefore probably less than one slice, and we conclude that the primary transcript is of about the same size as cytoplasmic 75 S RNA. We therefore also accept the determination of the size of 75 S RNA as a reasonable estimate of the size of the primary transcript in BR2.

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4. Discussion (a) Xize am! integrity

qf 75

X RNA

We have det,ermined the size of salivary gland 75 S RNA by gel electrophoresis and electron microscopy under denat,uring conditions. From the relativch mobility of 75 S RNA in formaldehyde-containing 0.3% agarose gels we e&mate a molecular weight of approximately 12 x lo6 as shown in Figure 3(a) and (b). However, since Figures 3 and 4 demonstrate that, even under these conditions we cannot rule out t’hc possibility t)hat our results are still being affected by migrational arbefacts, we simultaneously determined the molecular weight of 75 S RNA via electron microscopy. From the rat,io of contour lengths of 75 S RNA compared to an internal length standard, as shown in Figure 7(a), we calculate that the molecular weight of 75 S RNA is 12.3 + 1.1 x 106. Thus from these two independent met,hods wc conclude that’ a 75 S RNA molecule is 37.000 nucleotides in lengt,h. Implicit in t,he above conclusion is evidence indicating that 75 S RNA is a single RN,4 chain rat&her than two or more molecules held toget)her by overlapping regions of intermolecular hydrogen-bonding. During the course of our observations in thts elect,ron microscope we have never seen any duplex structures with tails indicative ot intermolecular associations at internal sites along molecules, as have been recently visualized in HeLa cell heterogeneousnuclear RNA (Federoffetal., 1977). Furthermore. we have not observed any internal hairpin structures such as those detected on several RNA viruses which have been shown to represent end-to-end overlaps of t,wo cont’iguous molecules by a particularly stable region of hydrogen-bonding (Kung et al.. 1975,1976). Yet’, if such overlapping regions existed in 75 S RNA and were composed of a simple linear duplex of less than 100 base-pairs. it, is unlikely that, we would havct been able to observe them. Further evidence against the existence of intermolecular associations in 75 8 RNA can be derived from the experiment in which 75 S RNA was dissolved in urea: formamide and heated to 80°C prior to spreading (Fig. 7(b)), since it can be argued that hydrogen-bonding of even a pure rG.rC homopolymer should have been eliminated in t,hat particular experiment. The t, of an rG.rC duplex is 97.6”C in 1-3 mMNat (Chamberlin, 1965). In thermal melting studies of DNA, Kung et al. (1976) ham, observed that a given percentage volume of (5.9 M-urea/740/, formamide) depressed the t, to the same extent as the same percentage volume of pure forma,midc. It has been reported t,hat the t, values of various RNA duplexes can be reduced b>an average of 0.4 deg. C/l% f ormamide (Bockstahler, 1967; Kielland-Hrandt’ 8 Nilsson-Tillgren, 1973). Thus we would estimate that in our spreading solution for microscopy. which is 95% (v/v) (5.9 M-urea/74’/” formamide) the t, of’ an RSA duplex would be decrea#sed by 38 deg. C. On the ot’her hand, t,he aqueous portion of the hyperphase contributes a monovalent cation concentration of about’ ti rn>l (Kung et ab.. 1975) which would increase the t, of an RNA duplex. Compared to a concentration of 1.3 mM, this increase would be about 9.5 deg. C. based on an average 12 cleg. C linear increase in t, with the logarithm of ionic strength (Mont,agnier & Sanders, 1963; Kaerner & Hoffmann-Berling, 1964; Nayak & Baluda, 1968: KiellandBrandt & Nilsson-Tillgren, 1973). Therefore the predicted net t,, of an rG. rC duplex in our hyperphase would be around 69’C (97.6 - 38 + 9.5), i.e. well below t,hc tcmperat’ure used in our melting experiment. The possibility of intermolecular reassociations taking place after the melting at

238

S. T. CASE

AND

B. DANEHOLT

high temperature (during subsequent cooling and spreading) seems unlikely in view of the following observation. The hairpin structures of HeLa cell 28 S rRNA are probably 80% G+C (Wellauer & Dawid, 1973b) based on the base composition of RNAaseresistant fragments obtained from other vertebrate 28 S rRNAs (Wikman et al., 1969; Schibler et al., 1975). While more than 90% of HeLa cell 28 S RNA molecules displayed characteristic secondary structures when spread directly at room t,emperature, we have observed that only 22% display such structures (19% were small terminal loops) if heated to 80°C prior to spreading at room temperature. Thus even the majority of these highly stable regions of intramolecular hydrogen-bonding could not reform. Since the distribution of 75 S RNA contour lengths after such a heat treatment essentially reflects the distribution obtained without heating, we conclude that 75 S RNA consists of a single covalently linked polynucleotide chain. (b) Xize of the primary

transcript

in BR2

In the present investigation the size of the primary transcript in BR2 was compared to cellular 75 S RNA in formaldehyde-containing 0.3% agarose gels. Since the size determination of cellular 75 S RNA in a 0.3% agarose gel agreed with that found by electron microscopy, such a gel should be suitable for analysis of molecules at least as large as 75 S RNA. In formaldehyde/0.3% agarose gels 3H-labeled BR2 RNA showed an asymmetric profile with a peak and a gradually declining slope towards a symmetric the low molecular weight side, while 14C-labeled 75 S RNA exhibited peak (Fig. 8). Several lines of evidence have attributed the asymmetry of the BR2 profile to the presence of a spectrum of growing RNA molecules (for discussion, see Daneholt, 1975). For example, it has been demonst#rat)ed by hybridization in situ that BR2 RNA is synthesized within BR2 (Lambert et aZ., 1972). Moreover, the predominent ultrastructural components in BR2 are lampbrush-like loops (Beermann & Bahr, 1954; Stevens & Swift, 1966), which are likely to constitute transcription complexes (for discussion, see Case & Daneholt, 1977). Finally, experiments with known to block the initiation of 5,6-dichloro-l-/3-n-ribofuranosyl-benzimidazole, transcription (Egyhizi, 1974), provide further strong support for the idea that BR2 RNA represents growing RNA molecules (Egyh&zi, 1975). If the nascent hypothesis is accepted, the peak of the BR2 RNA distribution should represent the finished, or almost finished primary transcripts. Figure 8 shows that the peak of the distribution was almost coincident with the cellular 75 S RNA fraction, though the peak of the BR2 profile migrated one slice further into the gel than the cellular 75 S RNA. This could mean that the BR2 transcript is slightly smaller than the cytoplasmic 75 S RNA. There is, however, another explanation for this minor difference: the peak of a nascent distribution (such as in Fig. 8) is likely to be located somewhat to the right of the position of the finished primary transcript because activity from unfinished, smaller molecules will be superimposed predominantly on the right side of the activity distribution of the finished branscripts. Therefore the difference in position in the gel between the completed primary transcript and 75 S RNA would be less than one slice, and we conclude that the primary transcript in BR2 has approximately the same molecular size as the salivary gland 75 S RNA. The agreement in size between the primary transcript in BR2 and the predominantly cytoplasmic cellular 75 S RNA (see Results), is of considerable interest, since cytoplasmic 75 S RNA is known t,o contain BR2 sequences (Lambert & EdstrGm, 1974). Furthermore, as a result of studies of polysomal RNA by electrophoresis

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(Daneholt et al., 1977a) and hybridization in situ (Wieslander & Daneholt. 1977). it was possible to conclude that BR2 75 S RNA enters polysomes. In fact, most of thcl cytoplasmic 75 S RNA resides in polysomes (Daneholt et al., 1977a). The similarity in size of the primary t)ranscript and cytoplasmic 75 S RNA therefore suggests that there is no major size change during the t)ransfer of this branscript from the gene to the polysomes, although a minor size change might have gone undetected due to the* exceptional size of 75 S RNA. Our data show that in a eukaryotic cell all giant nuclear RN.4 molecules are not necessarily bound t#o be cleaved into considerably smaller molecules (for discussion on processing, see e.g. Darnell, 1976), but some might servtl as precursors to giant RNA in the cytoplasm. Another example of this latt,er possibility is known from studies on the synthesis of fibroin messenger RNA in t’he silk gland of Bomhyx mob. The precursor to the fibroin mRNA is similar in size (Lizardi. 1976a), or maybe slightly larger (Lizardi, 19766) than the high molecular weight fibroin mRNA (5.8 x 106, according to Lizardi et al. (1975)). (c) The transcription

unit in BR2 und its relatiorr to rhromonwric

lJX.4

the present study the size of the transcription unit in BR2 (37 x 103 base-pairs). is derived from analysis of the primary transcript and 75 S RNB. It’ should. therefore. first be emphasized that in this paper we use the definit,ion of a transcription unit as t,he DNA segment corresponding to the primary transcript, which does not necessarily have to be identical to the segment of DNA between the initiation and termination sites for the RNA polymerase. Keeping t(his qualifying statement in mind, we want to discuss the available information on the transcription unit in BR2 in relation to the structure of the giant chromosome. A recent investigation was performed in order to elucidat’e the locat’ion of the 75 S RNA4 transcription unit(s) in the BR2 region of chromosome IV. Comparing the structure of the BR2 region in salivary glands nit,11 that, in Malpighian tubules, Daneholt et al. (19776) concluded that only one band, designated t,he BR2 band, in region IV-3B is involved in the puffing process in BR2. though it was not, excluded that adjacent interbands could also participate. The following considerations suggest, however, that only a minor part of a 75 S RN,4 transcription unit can be located in interband DNA. According to Beermann (1976) interband DNA constitutes less than 5% of total DNA in C. tuataw. Since an average chromomere contains nearly 100 x lo3 base-pairs of DNA (Daneholt’ & Edst,riirn, 1967). this implies that there is less than 5 x lo3 base-pairs in an average interchromomere. Since the interbands adjacent to the BR2 band are not exceptionally large (Daneholt et al.. 19773), it is possible to conclude that an interchromomere adjacent’ to the BR:! chromomere can only accommodate a minor part of a 75 S RNA transcription unit. Most of the unit, perhaps even all, is likely to be present wit,hin the BR2 chromomerc itself. On the other hand the BR2 band is a rather broad band (Daneholt et ul.. 1977h). and thus each BR2 chromomere probably contains considerably more DNA than t#he 100 ti 103 base-pairs of an average-sized chromomere (Daneholt $ Edstriim. 1967). Consequently, one would have to postulate multiple copies of a 75 S RNA transcription unit, 37 X lo3 base-pairs in size, in order to account for all the DNA in a BRI chromomere. However, a major alternative to this idea would be that one 75 S RNA t’ranscription unit could be sitting among an excess of nucleotide sequences of unknown function. This latter possibility would be in agreement with a study of Sorsa et al. (1973) on the white locus in Drosophila melunogader. Based upon a combined In

240

S. T. CASE AND

genetic and cytological analysis, the band DNA has non-essential

B. DANEHOLT

they concluded that at this particular or at least ill-defined functions.

locus most of

Expert technical support has been provided throughout this study by Eva Martensson, Jeanette Nilsson and Sigrid Sahlen. One of us (S. T. C.) thanks R. Weber (University of Bern, Switzerland) for providing facilities in which Toni Wyler gave instructions for spreading RNA in urea/formamide. U. Pettersson (University of Uppsala, Sweden) has kindly provided us with samples of HeLa cell nuclear and cytoplasmic RNAs and K. Granath (Pharmacia, Uppsala, Sweden) with Dextran fractions of different molecular sizes. Finally we thank Hannele Jansson for typing this manuscript. Our research was supported by grants from the Swedish Cancer Society, Magnus Bergvalls Stiftelse and Karolinska Institutet (Reservationsanslaget). One author (S. T. C.) has been the recipient of a National Institutes of Health National Research Award from the National Institute of General Medical Sciences (U.S.A.).

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