size and distribution of polyadenylic acid sequences in drosophila polytene dna and rna

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Biochimica et Biophysica Acta, 479 (1977) 235--245 © Elsevier/North-Holland Biomedical Press

BBA 99036

SIZE AND DISTRIBUTION OF POLYADENYLIC ACID SEQUENCES IN DROSOPHILA POLYTENE DNA AND RNA

CARLOS ALONSO, MONTSERRAT PAGES and M. LUISA GARCIA

Centro de Biolog(a Molecular, Universidad AutSnoma Madrid, Facultad de Ciencias, Canto Blanco, Madrid-34 (Spain)

(Received February 28th, 1977) (Revised manuscript received July 14th, 1977)

Summary

[3H]Poly(U) hybridizes very rapidly to polytene DNA from Drosophila hydei. When hybridization is performed at 30°C in 2 X SSC to a large excess of DNA, 95% of the poly(U) becomes ribonuclease resistant. Also, complementary RNA transcribed in vitro from polytene DNA hybridizes to poly(U). 023-- 0.25% of the DNA is composed of (dA)-rich sequences and 0.23--0.31% of cRNA hybridizes to [3H]poly(U). The length of the (dA)-rich sequences on the DNA and cRNA is 40 nucleotides. The T m value of these hybrids formed between DNA or cRNA • poly(U) is 45 ° C.

The poly(A) fragments from cytoplasmic RNA ranged from 80 to 170 nucleotides in lenght, and migrated in polyacrilamide gels as a broad peak.

The average sizes of the poly(A) fragments from the poly(A)-containing RNA transcribed by nuclei isolated from salivary glands in vivo or in vitro were 40, 70, 170 and 70 nucleotides, respectively. Hybridization in situ of [3H]- poly(U) to chromosome squashes indicated that the (dA)-rich sequences are randomly distributed over the whole genome.

Introduct ion

It seems that the understanding of poly(A) metabolism is of interest in the study of gene expression. Sequences of polyadenylic acid residues have been detected in mRNA and HnRNA [1]. Most of the cytoplasmic m R N A molecules appear to be transcribed from unique DNA sequences [2,3] and it has been proposed that the addition of poly(A) to the 3' end of nuclear precursors may be an important step in mRNA maturation and transport [4,5]. There is how-

Abbreviations: SSC, standard saline-citrate solution (0.15 M NaCI/0.15 M trisodium citrate, pH 7.0): cRNA, complentary RNA.

236

ever growing evidence that a substantial amount of the cytoplasmic mRNA is not polyadenylated [6,7 ]. Because it is most likely that the polyadenylation of RNA molecules is post-transcriptional [8] and as indicated by several authors [5,9] the addition of adenylate is made to a previous polyadenylated RNA and not to poly(A-)-RNA, the possibility is raised that the regulatory control mech- anism by which only some of the mRNA molecules are polyadenylated may be encoded in the DNA itself. Nuclear precursors of the mRNA can be transcribed from DNA fragments containing and lacking poly(dT) stretches which could serve as primers for the lengthening of the poly(A) segments in the RNA.

In order to determine the proportion of the poly(A) sequences in the DNA and cRNA and to measure their size, the poly(A) sequences have been identified by hybridizing them to 3H-labeled poly(U). The size of nuclear and cytoplasmic poly(A)-containing RNA was estimated by polyacrilamide gel electrophoresis.

It has been reported by Bishop [10] that poly(A) sequences in duck DNA that react with poly(U) are interspersed with non-reactive sequences. Therefore we have hybridized radioactive poly(U) to polytene chromosomes 'in situ' to detect possible specific or random distribution of poly(A) in the genome. These experiments indicate that there are no fragments in the DNA of a size similar to those found in mRNA and that there is a widespread distribution of the poly(A) fragments over the whole genome.

Materials and Methods

Throughout this study early third instar larvae of a laboratory stock of Drosophila hydei were used.

DNA preparation DNA was prepared from polytene nuclei as previously described [11]. After

the last purification step the DNA was dissolved in 0.01 M phosphate buffer and applied to a hydroxylapati te column equilibrated with 0.01 M pphosphate buffer. The hydroxylapat i te column was washed twice with 5 bed volumes of 0.12 M phosphate buffer at 50°C. Then the DNA was eluted from the column with 0.4 M phosphate buffer. The solution was passed through a 23 gauge needle, dialyzed against 0.1 × SSC, freeze dried and dissolved in the annealing buffer. The hyperchromicity of the DNA was 39.%.

Synthesis of cRNA and [3H]poly(U) Complementary non-labeled RNA was prepared by 'in vitro' transcription of

8 pg nuclear DNA obtained as indicated above. The DNA was transcribed with 5 units of Escherichia coli RNA polymerase each, for two periods of 45 min in the presence of 0.4 mM of each of the four non-labeled nucleotides. The RNA was extracted according to Perry et al. [12]. The aqueous phase was passed through a column of Sephadex SP 50 and the RNA precipitated with 2 vols. of ethanol. [3H]Poly(U) was synthesized as described by Bishop et al. [ 10] with a specific activity of 8.4 • 10 s dpm/pg.

Hybridization to [3H]poly(U) Sheared DNA or cRNA were annealed in filters or in solution to an excess of

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[3H]poly(U) in 2 × SSC at 30°C for 3.5 h. P o l y ( U ) w a s present in a 10-fold excess over the poly(U) binding capacity of the DNA of cRNA. Although in poly(U) excess the hybridization rate between poly(A) and poly(U) is indepen- dent of the poly(A) concentration in the reaction mixture [13], reacting conditions similar to the 'in situ' hybridization experiments (see further) were want- ed. For that reason minifilters of an area equal to that occupied by the squashed chromosomes were prepared following in part the method of Lambert [14]. From a large nitrocellulose filter washed several times with 2 × SSC at 60°C, 9 mm: small filters were cut and placed on a siliconized slide. Then 1/~1 of 0.1 XSSC containing 0.012 #g of either native or denatured DNA was put on the filter and dried in vacuum at 4°C for 2 h. Control experiments containing equal amounts of labeled Drosophila hydei DNA indicated that the DNA remained in the filter even after 7 h of incubation in 2 × SSC at 45°C. 0.024 ng of labeled poly(U) in 5 pl of 2 × SSC were placed over the filters and covered with liquid paraffin. Hybridization in solution was also carried out on siliconized slides in 5 #l containing 0.012 #g DNA or cRNA. After the incubation period the samples were treated for 20 min at 0°C with pancreatic ribonuclease (20 pg/ml in 2 × SSC). Bovine serum albumin (250 #g) and 10% trichloroacetic acid were added to each sample. The precipitate was collected on GF/C filters, dried and counted in a Beckman scintillator counter with an efficiency of 25%.

Preparation of 3H-labeled nuclear and cytoplasmic poly(A )-containing RNA Labeled nuclear and cytoplasmic RNA were extracted from third instar lar-

vae injected with 25 pCi of [3H]adenine (spec. act. 27 Ci/mM). After 2 h the larvae were homogenized at 0°C for 5 min in a buffer containing 100 mM NaCl, 10 mM CaC12, 30 mM Tris • HC1 (pH 7.3), 25/~g/ml polyvinyl sulfate, 35 ~zg/ml spermine, 1% diethyl pirocarbonate and 0.5% NP40. The nuclei were pelleted at 4000 rev./min for 5 min. Labeled nuclear RNA was also obtained from polytene nuclei, isolated from salivary glands wi thout the use of detergents, as previously described [15].

The RNA was extracted at 4°C from either the cytoplasmic or nuclear fraction with phenol/chloroform. The RNA was precipitated from the final aqueous phase by the addition of 2 vols. of ethanol. After 8 h at --30°C the precipitate was collected by centrifugation at 10 000 rev./min, dissolved in buffer containing 0.7 M NaC1, 50 mM Tris • HC1 pH 7.5; amd EDTA 10 mM in 25% Forma- mide and applied to a poly(U)-Sepharose column (Pharmacia). The column wa washed with 3 bed volumes of this buffer and once with buffer wi thout formamide. The poly(A)-containing RNA eluted with water at 50°C. The solution was lyophilized and the RNA dissolved in 2 × SSC.

Measurement of poly(A ) size The poly(A)-containing RNA samples eluted from the poly(U)-Sepharose

column were treated for 30 min at 37°C with 2/~g/ml pancreatic RNAase and 100 units of T1 RNAase/ml (Sigma) (both previously heated at 80°C for 15 min). After addition of SDS (final concentration 0.5%) and digestion with 20 pg/ml pronase for 15 min, the RNA sample was extracted with phenochloro- form and the poly(A) fragments precipitated with 2 vols. ethanol in the presence of 4 S and 5 S E. coli RNA. The Mr of the poly(A) or poly(U) was calcu-

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lated considering that 5 S and 4 S RNA behave electrophoretically as poly(A) molecules of 115 and 58 nucleotides long, respectively [16--17]. Electrophor- esis was carried out on 10% polyacrylamide gels at 5 mA/gel [18]. 1 mm gel slices were digested overnight at room temperature with NH3. The radioactivity was counted with 20 ml of butyl PBD-ethoxy ethanol toluene scintillation liquid.

Size measurements of the poly(A) segments in the DNA and cRNA were carried out as described in the Results section.

Hybridization in situ The in situ hybridization experiments were carried out as previously described

[19]. Previous to the hybridization reaction the slides were treated at 37°C for 1.5 h with 400 pg/ml pancreatic RNAase and 100 units/ml of RNAase T1. 5 pl of the hybridization solution (2 × SSC) containing 1.2 ng [3H]poly(U) were placed over denatured or non-denatured chromosomes. The incubation was done at 30°C for 3.5 h under liquid paraffin. The slides were treated with 20 /~g/ml pancreatic RNAase at 0°C in 2 × SSC for 30 min. The exposure time varied from 2 to 3 months.

Results

Amount of polyadenylic acid in DNA and cRNA As indicated by Shenkin and Burdon [20] [3H]poly(U) hybridizes very read-

ily to denatured DNA. The Cot 1/2 for poly(U) is 5.9 • 10 -4 mol • 1 -~ [21]. Un- der our conditions 95% of the poly(U) hybridized to a large excess of Droso- phila DNA becomes ribonuclease resistant. Complementary RNA transcribed in vitro from polytene DNA also hybridizes to poly(U). The opt imum temperature of the reaction for both DNA and cRNA was 30°C. Table I shows the saturation of denatured and native DNA and cRNA by [3H]poly(U) at a ratio of 1 : 10. Hybridization saturation experiments in which large amounts of denatured or native polytene DNA (10 pg/ml) were annealed to [3H]poly(U) (0.4 pg/ml) indicated that after 1.5 h at 30°C in 2 × SSC a plateau is reached and 11.2 and 6.3% of the input [3H]poly(U), respectively, becomes ribonuclease resistant. This shows that the amount of [3H]poly(U) (0.24 ng) that was amealed with 0.012 pg DNA (Table I) is about 10 times the poly(U) binding capacity

T A B L E I

S A T U R A T I O N O F DROSOPHILA H Y D E I D N A A N D c R N A BY P O L Y U R I D I L I C A C I D

0 . 0 1 2 p g o f d e n a t u r e d , n a t i v e D N A o r c R N A w e r e i n c u b a t e d w i t h 0 . 2 4 n g [ 3 H ] p o l y ( U ) in 2 X SSC f o r 3 .5 h a t 3 0 ° C . H y b r i d s were e s t i m a t e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . T h e n u m b e r s i n d i c a t e aver-

age v a l u e s o f f o u r d i f f e r e n t e x p e r i m e n t s . C o r r e c t i o n s h a v e b e e n m a d e f o r b a c k g r o u n d w i t h n o D N A ( l e s s

t h a n 0 . 2 % o f t h e r a d i o a c t i v i t y was r e t a i n e d w i t h D N A o r c R N A ) .

P e r c e n t r e a c t i o n P e r c e n t r e a c t i o n U r i d i l i c a c id r e s i d u e s ( D N A f i x / p o l y ( U ) ) ( D N A s o l / p O l y ( U ) ) b o u n d p e r g e n o m e * (X 1 0 6 )

D N A d e n a t u r e d 0 . 4 3 - - 0 . 4 8 0 . 3 7 1 .4

D N A n a t i v e 0 . 2 3 ~ 0 . 2 5 0 .20- - -0 .25 0 .S c R N A - - 0 .23-- -0 .31 - -

D N A f i x , D N A f i x e d t o f i l t e r s ; D N A s o l , D N A i n l i q u i d p h a s e . * H a p l o i d D N A c o n t e n t o f Drosophila hydei p o l y t e n e c h r o m o s o m e s = 1.7 - 108 B.P. = 1 . 0 2 • 1011

d a l t o n s .

239

of the nucleic acids. At saturation, 0.43--0.48% of denatured DNA fixed to nitrocellulose filters and 0 .23 -0 .25% of native DNA reacts with [3H]poly(U). However, when the hybridization reaction was carried out in solution, only 0.37% of denatured DNA and 0 .20 -0 .21% of native DNA formed complexes with poly(U). This is expected if three-stranded complexes are formed with denatured DNA [22] and if the poly(dT)-DNA sequences compete with poly- (U) for poly(dA) in solution. In such a case 0.23--0.25% should be taken as the amount of Drosophila hydei DNA which can be saturated with poly(U). In order to know the number of poly(U) residues bound per haploid genome, the DNA content of polytene nuclei was measured by ultraviolet microcytopho- tomet ry [23]. We know that each late third instar salivary gland nucleus has 355 -+ 18 pg DNA at the polytene level and therefore 0.173 pg on the average at the haploid level, taking 2048 as the polytenization number. Thus the haploid genome has 1 . 7 3 - 1 0 8 base pairs, equivalent to 1 .02 . I 0 '~ dalton. Since 0.25% of the DNA can be saturated with poly(U), the number of poly(U) residues bound per haploid genome is approx 8 • l 0 s. Saturation experiments between cRNA and poly(U) indicated that 0 .23 -0 .31% (0.23, 0.27, 0.25, 0.31%) of the cRNA, in four experiments with different batches of cRNA, re- acted with poly(U). The complexes between poly(A) and po ly (U)a re three- stranded, although after ribonuclease treatment half of the poly(U) is sensitive to ribonuclease [10].

Size of polyadenylic acid sequences in DNA and RNA Previous communications have shown that approx. 3.5% of the mRNA and

5.5% of HnRNA from salivary glands contain poly(A) sequences [15]. Follow- ing digestion of labeled poly(A)-containing RNA, as described in Materials and Methods, the size of the poly(A) fragments was analyzed in 10% acrylamide gels. The poly(A) fragments from cytoplasmic RNA migrated as a broad peak implying a significant heterogeneity in size. This ranged from 80 to 170 nucleotides. On the other hand, the poly(A} fragments of nuclear RNA transcribed in vivo did not show such heterogeneity, migrating as discrete peaks correspond- ing to fragments averaging 40, 70 and 150 nucleotides in lengths. The average size of the poly(A} fragments of the poly(A)-containing RNA transcribed from isolated nuclei is 70 residues (Fig. 1). In order to know whether or not significant amounts of sequences containing elements other than adenine residues were present in the putative poly(rA) fragments, these were applied to a poly- (U) Sepharose column equilibrated with NaC1/Tris. HC1/EDTA/formamide buffer. The column was washed with 5 bed volumes of Tris buffer CSB. 95-- 100% of the label was retained in the column and only eluted with water at 50 ° C. The homogenei ty of the poly(rA) fragments was also estimated from the Tm value of the hybrids formed between the poly(rA) fragments from mRNA or nuclear RNA and poly(U), melted under the conditions described in the legend to Fig. 2. The Tm value of the hybrids was 66 and 64°C, respectively. We presume therefore that less than 5% heterogeneity as to base composit ion must be in the poly(rA) fragments [24--25].

Fig. 1 also indicates the size of the poly(A) fragments in DNA and cRNA. Following incubation of the DNA or cRNA with an excess of poly(U), the re- maining non-hybridized poly(U) was digested with pancreatic RNAase. After-

u

Q..

wards the nucleic acids were precipitated with 2 vols. of ethanol in the presence of a 500-fold excess of cold poly(U) over the [3H]poly(U) used for the hybridization reaction and 4 S and 5 S E. coli RNA. The precipitate was dissolved in E buffer (0.4 M Tris. HC1 pH 7.2/0 .06 sodium acetate/0.003 M EDTA) and heated in a water bath at 100°C for 3 min to melt the hybrids. Electrophoresis was carried out in 10% polyacrilamide gels.

Control experiments indicated that less than 5% of the [3H]poly(U) which was in a hybrid form remained ribonuclease resistant under conditions in which free poly(U) is acid-soluble. Therefore after heating in the presence of a 500-fold excess of cold poly(U), the [3H]poly(U) was single stranded and did not form complexes with poly(dA} or poly(rA). Therefore the location on the gels of the

6 0 0

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4 5 0

3 0 0

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10

10 20

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5S 4S BLUE MARKER

240

p

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Slice no.

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5 S 4S BLUE MAKER

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Sl ice no.

Fig. 1. Po l yac r i l am ide gel e lec t rophores is o f p o l y ( A ) f ragments f r o m cy top lasm ic p o l y ( A ) - c o n t a i n i n g R N A , nuc lea r R N A , R N A f rom isola ted nucle i , nuc lea r DNA and c R N A . Po ly (A) s egmen t s were ob ta ined f rom c y t o p l a s m i c p o l y ( A ) con ta in ing R N A or nuc lea r R N A . These samples were t r e a t ed for 30 rain at 37°C wi th 2 /~g/ml pancrea t i c RNAase and 100 uni t s of T 1 RNAase , and e x t r a c t e d wi th p h e n o l / c h l o r o - f o r m as descr ibed in Materials and Methods . The isolat ion of the p o l y ( A ) f r a g m e n t s f r o m DNA or c R N A was descr ibed in the Resul ts sec t ion . E lec t rophores i s o f the p o l y ( A ) f r a g m e n t s was car r ied ou t on 10% p o l y a c r y l a m i d e gels using 4 S and 5 S as marke r s . A. Po ly (A) f r ag men t s f r o m cy to p l a smic po ly (A) -con - ta i rdng R N A (¢ =). B. Po ly (A) f r a gme n t s f r o m nuc lea r R N A (e e ) and R N A f r o m isolated nuclei (o o). C. Po ly (A) f r a gme n t s f r o m nuc lea r DNA (e -') and c R N A (o . . . . . . o).

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0 0

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80 ~; ~! 70 ~

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Temlx 1. C )

Fig. 2. Melt ing curve of the c o m p l e x e s f o r m e d b e t w e e n d e n a t u r e d D N A (~- --), c R N A (~ ~), p o l y ( r A ) (= ¢) and p o l y ( d A ) (o . . . . . -o) wi th [ 3 H ] p o l y ( U ) . D e n a t u r e d D N A (0 .012 /~g)or c R N A (0 .012 /~g) were hyb r id i zed wi th 0 .24 ng [ 3 H ] p o l y ( U ) fo r 3.5 h in 2 X SSC a t 30°C. Fo l lowing incu- ba t ion , several samples [10 ] were m i x e d and d i lu ted to 1 ml w i th 2 × SSC con ta in ing 20 ~g cold po ly- (U). The t e m p e r a t u r e was raised a f te r 5 rain a t the t e m p e r a t u r e ind ica ted 0.1 ml po r t i ons were r e m o v e d , t r ans fe r red to an ice b a t h and d i lu ted wi th 1 ml i ce /bov ine s e r u m a l b u m i n and t r i ch lo roace t i c acid were added to each tube . The prec ip i ta te was co l lec ted on G F / C filters, dr ied and c o u n t e d . P o l y ( d A ) - [ 3 H ] - p o l y ( U ) and po ly ( rA) • [ 3 H ] p o l y ( U ) were p r e p a r e d b y annea l ing 4 /~g/ml o f p o l y ( d A ) or p o l y ( r A ) w i th an equal a m o u n t of [ 3 H ] p o l y ( U ) in 2 × SSC for 30 m i n a t 55°C.

2 4 2

[3H]poly(U) fragments was not in any way influenced by the size of the DNA or cRNA molecules to which they have hybridized. As expected, poly(A) fragments of the same size were found in both cases (DNA or cRNA) migrating in the gels as segments with an average length of 40 nucleotides. We may calculate then that there are 20 000 poly(A) fragments of length approx. 40 nucleotides in Drosophila hydei DNA. In order to test the specificity of the hybrids formed, these were melted in the presence of a large excess of cold poly(U) as described in the legend to Fig. 2. The Tm value of the hybrids was 45°C. The low Tm and the broad transition are probably due to short poly(A)sequences containing some internal heterogeneity in base composition [24--25]. It is worth knowing that the poly(A) sequences found in HnRNA and mRNA are not totally ho- mogenous in base composition [2,27,28].

In situ hybridization Whether or not the polyadenylic acid residues in the DNA are all clustered or

interspersed with non-reactive sequences throughout the whole genome was checked by in situ hybridization experiments. It is possible to observe in Fig. 2 that grains of radioactivity are found randomly distributed all over the chromosomes. The centromere and the region of the six chromosome rich in highly repetitive sequences [29] did not accumulate any significant number of grains.

From grain counting, time of exposure and the specific activity of [3H]poly- (U) the amount of [3H]poly(U) retained in the preparation was estimated. Among 20 chromosome sets scored, taken from 3 slides, it was observed that 530 -+ 30 radioactive grains accumulate over each polytene nucleus. Assuming that the autoradiographic detection of 3H is 5--10% in cytological preparations [30] and knowing that the specific activity of [3H]poly(U) used for in situ

Fig. 3. D i s t r i b u t i o n o f [3H]polF(U)in salivary gland c h r o m o s o m e s o f Drosophila hydei. H y b r i d i z a t i o n in s i tu w a s p e r f o r m e d at 3 0 ° C f o r 3 . 5 h . A f t e r t h e i n c u b a t i o n r e a c t i o n , t h e s l i d e s w e r e t r e a t e d w i t h 20 ~ g / m l pancrea t i c R N A a s e at 0°C in 2 X SSC for 30 m i n . T h e e x p o s u r e t i m e w a s 3 m o n t h s . X 2000 .

243

hybridization is 8.4 • 10 s dpm per pg, 1 pg of [3H]poly(U) would produce 7.7 • 103 grains in 3 months. We have calculated therefore that 530 + 30 grains accu- mulated over 3 months correspond to 0.065--0.071 pg [3H]poly(U). Since there are 355 + 18 pg of DNA per polytene nucleus and 0.23% of the native DNA can be saturated with poly(U) in filters, the amount of [3H]poly(U) detected over a chromosome set represents 8--9% of the expected value. It has been estimated that the hybridization efficiency for 5 S RNA in situ is approx. 10% [31]. Therefore in situ hybridization experiments in which poly(U) is used as a probe, can provide a quantitative estimate of the amount of poly(A) on chromosome squashes. Whether or not the same efficiency will hold for in situ hybridization with mitotic chromosomes should be further tested.

Discussion

There seems to be no doubt at the present time that the addition of polyadenylic acid to mRNA is post-transcriptional and, if it is likely that the addition is made to a previously polyadenylated RNA and not to transcribed poly- (A)-RNA [5,9], poly(A) sequences should be found in segments of the DNA which encode mRNA precursors. It is then expected that more poly(A) sequences will be found in organisms with larger numbers of genes. It is known, however, that not all of the poly(A) fragments in the HnRNA are used for the lengthening of the poly(rA) attached to the 3' end of mRNA since poly(A) sequences internal to the HnRNA or mRNA molecules do exist [22,27] in some cases.

There are reports that both prokaryotic and eukaryotic DNA contain poly(A) sequences [18,10]. The difference between the highest prokaryote and the lowest eukaryote is more than three orders of magnitude when the number of uridylic acid residues is related to the analytical complexity of the DNA. It remains to be seen whether the differences observed can be related to the number of genes of each of the organisms. It is known however that even though the analytical complexity of the DNA (and the number of genes) from E. coli and M. Lysodeikticus is greater than that from Phage ~ and Simian Virus 40 the poly(A) content varies significantly [20]. There are not enough compara- tive data to be able to make any correlation between DNA complexity, number of genes and amount of poly(A). From this point of view it would be of interest to know if the poly(A) fragments are evenly distributed over the chromosomes or clustered in some regions as are most of the satellites. In Drosophila hydei in situ hybridization of [3H]poly(U) results in labeling of all the chromosomes without any significant difference between subregions. We therefore conclude that poly(A) is distributed along the DNA interspersed with non-poly(A) sequences. Whether or not these poly(A) sequences form part of or serve as primers for the poly(A) found in HnRNA and mRNA is not yet obvious.

From the work on Dictyostelium mRNA and mRNA precursors it is known that cellular mRNA yields approximately equimolar amounts of poly(A)100 and poly(A)2s and that similar segments are also found in mRNA precursors isolated from labeled cells. In this case therefore one poly(A) chain at least is common to both HnRNA and mRNA. We have not detected in Drosophila hydei any mRNA-associated poly(A) fragments similar to the ones found in

2 4 4

DNA. We may conclude that the longer (poly(A)80_170) sequences originate post-transcriptionally. Recent work from this laboratory has shown that polyadenylation of some mRNA molecules takes place outside the chromosomal region from which they were transcribed (unpublished). There are, however, poly(A) sequences on HnRNA from nuclei labeled in vivo or in vitro, of a length similar to those on the DNA, suggesting that these sequences are transcribed from DNA. The data do not, however, allow us a conclusion with regard to the loss of these sequences during HnRNA processing or their identity with the long ones found in mRNA. The different size of poly(A) fragments from HnRNA, mRNA and DNA seems to support Bishop's conclusion that the mRNA-associated poly(A) fragments are not the components found in DNA [10]. Since, however, this conclusion comes only from size measurements and Tm values, we cannot exclude the possibility that the entire DNA poly(A) frag- ment or a fraction of it forms part of the poly(A)-mRNA.

Further experiments are needed to determine whether or not they are at the 3' end of coding sequences in nuclear pre-mRNA and to test the proportion of the coding sequences which may or may not have such a sequences of poly(A). This will, no doubt, shed considerable light on the relationship between poly(A) metabolism and gene expression.

Acknowledgements

This work was supported by research grants from FundaciSn Juan March and ComisiSn Asesora. C.A. is a F.J.M. fellow.

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