Transcription of Repetitive and Unique DNA Nucleotide Sequences in Pigeon Erythroid Cells with Different Degrees of Specialization

Transcription of Repetitive and Unique DNA Nucleotide Sequences in Pigeon Erythroid Cells with Different Degrees of Specialization K. G. GASARYAN, W. Z. TARANTUL and W. N. BARANOV I.V. Kurchatov Institute of Atomic Energy Moscow, U.S.S.R. Received June I915

HnRNA fractions with sedimentation coeficients > 45 S isolated from pigeon bone marrow as well as from the immature and mature erythroid cells of periferal blood were hybridised with a large excess of DNA fractionated on the basis of renaturation kinetics. 58-62% of the input RNAs were recovered as RNAase-resistant hybrids. About 1/3 (20%) of bone marrow > 45 S RNA found in the hybrids was hybridised with the repetitive and about 2/3 (40%) with the unique DNA sequences. In addition, a considerably smaller portion of > 45 S RNA from the LLreticufocytes’’ (13%) and “erythrocytes” (- 6%) was hybridised with the repetitive DNA.

Introduction

Methods

The systems where the cells acquire high degrees of specialisation are useful for studying relations between their reduced (predominantly cell-specific) mRNAs released into the cytoplasm and those DNA sequences of genome which are transcribed in the form of heterogenous nuclear RNA (HnRNA). Such studies may be conductive to the elucidation of the nature and functional role of DNA sequences localised in the transcriptional regions. This refers in particular to repeating sequences, some of which are thought to be involved in the regulation of gene activity [ l , 21. It has been shown by hybridisation of HnRNA with DNA excess, that 25-30% of this RNA are transcribed from the repeating and 70-75% from the single-copy nucleotide sequences [3-51. Such a proportion has always been found in actively metabolising cells of higher organisms irrespective of their species or tissue-specificity. At the same time, data appearing recently indicates that repeated DNA sequences represented in the HnRNA are reduced in the course of specialisation of muscle cells [61. In the present work we studied the representation and proportion of different nucleotide sequence families of pigeon genome in the > 45 S fraction of pulse-labelled RNA of erythroid cells with different degrees of specialisation.

Preparation and Labelling of Cells

Differentiation 6 , 41-46 (1976)

-

0 by Springer-Verlag 1976

Bone marrow cells (erythroblasts), immature cells of peripheral blood (reticulocytes), and mature erythrocytes of pigeon were used. The first two cell populations were obtained from pigeons anaemised by four daily injections of phenylhydrazine (15 mg/kg of body weight). The third cell population (erythrocytes) was obtained from non-anaemised pigeons. Anaemisation increases the proportion of erythroid forms in the bone marrow from 50%-80% and makes the erythroid population more homogeneous [71. On the 7th day after the first phenylhydrazine injection this cell population consists of polychromatic erythroblasts (20?6), basophylic erythroblasts (40%), orthochromatic erythroblasts (8%), proerythroblasts (7%) and erythrocytes (8%). It contains also about 18% of myeloid cells. The cells were obtained from the femoral and tibial bones and suspended in cold Parker’s medium 199. containing 8 units of heparin per ml and 30% of homologous serum. The cell suspension was filtered through 4 layers of sterile gauze and diluted by the same milieu to the final concentration of 3.5-4 x 10’ cells/ml. Population of reticulocytes was obtained from the same anaemic pigeons. The typical population contained about 75% reticulocytes, 15yo erythrocytes, 8% erythroblasts and 1.5% myeloid cells. Population of mature erythrocytes contained about 3.5% of cells other than erythrocytes. Blood of anaemic and normal pigeons was taken from the neck vessels of decapitated animals and diluted 4 times by the 199 medium containing 5 units of heparin per ml (the final cell concentration was 4-6 x lo8 cells/ml). All suspensions were incubated at 37’ C in slowly rotating flasks for 50 min with 100 uCi/ml of ’H-uridine (spec. activity 7.3 Ci/mM, U.S.S.R.). After the incubation, the suspensions were rapidly chilled in an ice bath, the cells were collected by centrifugation at 1000 g for 15 min, and used immediately or stored at -2OO C.

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K. G. Gasaryan et al.:

42 Isolation and Puripcation of RNA The total RNA was extracted by "hot-phenol-SDS" method [81 and fractionated in a sucrose gradient (5-20%). The fraction with the sedimentation coefficients higher than 45 S was precipitated by ethanol using a small amount of unlabelled E. coZi RNA as a carrier. RNA was precipitated by ethanol after dissolving in 0.05 M Tris-HC1,pH 7.0 - 0.05 M NaCl- 0.001EDTA - 0.4Ohsodium dodecyl sulphate (SDS) and purified by passing through the Sephadex G-25 column (3 x 5 cm). The specific activities of the total rapidly labelled RNA of erythroblasts, reticulocytes and erythrocytes were 8 x lo6, 5 x lo6 and 3 x lo6 counts/min/mg, respectively. Isolated > 45 S fractions contained approximately 25% of the total radioactivity of HnRNA (without taking into account the activity of the 4 S peak). They contained also some admixture of 28 S rRNA. According to rough estimates the specific radioactivity of the > 45 S fractions (from bone marrow cells, reticulocytes and erythrocytes) exceeded by an order of magnitude those of the corresponding total RNAs (see above).

Preparation of DNA DNA was isolated from pigeon reticulocytes by the phenol procedure followed by RNAase and pronase treatments. The DNA purified had the mw of 15-20 x lo6, RNA contamination - less than 1% protein - about 1%. It was fragmented by three passages through the French press at 120-130 kg/cm2. This resulted in a rather homogenous population of fragments (mw 300,000 according to sedimentation in analytical Spinco ultracentrifuge and 200,000 according to results of electron microscopic measurement). Fragmented DNA was denatured for 10 min in a boiling water bath and renatured at 62O C in 0.24 M of sodium phosphate buffer (containing equimolar amounts of Na,HPO, and NaH,PO,). Fractions with different renaturation kinetics (Table 1) were obtained by hydroxyapatite (HAP) column method [91. The purified fraction of unique sequences (fraction 3) was obtained after DNA renaturation to Cot = 1700 (this value is close to Cot,,, of renaturation of unique nucleotide sequences in the total pigeon reticulocyte nuclear DNA LlOl). Renatured DNA containing all the repeating and part of the unique sequences was denatured once more and then annealed to Cot = 1. It was separated into rapidly renaturing fraction (fraction 1) and the fraction renaturing at a moderate rate (fraction 2). DNA was desalted and concentrated by pressure ultrafiltration through the semi-permeable membrane.

-

-

RNA Hybridisation with Separated Fractions of DNA Hybridisation was conducted at 62O C in sealed ampules by annealing in 0.24 M Na-phosphate buffer. Experimental values of Cot are normalised to the conditions of 0.12 M Na-phosphate buffer using a correction coefficient 2.93 [ I l l . DNA concentration was 6 mg/ ml, the duration of annealing did not exceed 1I days. Approximately 2000 cpm of RNA (> 45 S) was added to the annealing mixtures. DNA-RNA mixtures were heated for 10 rnin in boiling water bath and then brought to the temperature of hybridisation. The amount of labelled RNA in hybrids was evaluated according to Melli and others 141. After annealing, the mixture was chilled to 4' C, diluted with 0.24 M phosphate buffer to a final DNA concentration of 50-100 pg/ml and treated with pancreatic RNAase (25 pg/ml, 25 rnin at 37" C). RNAase-resistant material was precipitated in the cold by 7% trichloroacetic acid (TCA), washed twice by

<

5% TCA and once by ethanol. The precipitates were dissolved in formic acid and counted in a liquid scintillation counter Mark 2 (USA.). To evaluate hybridisation background the control samples containing DNA fraction 1 were incubated at 2-4" C and those with DNA fractions 2 and 3, at 62O C to Cot values 0.1 and 10, respectively. The amount of radioactivity resistant to RNAase in the control incubations was 2-5% of the input radioactivity. This value was subtracted from the results obtained.

Results DNA Fractions Used for Hybridisation

Analysis of renaturation kinetics of the total fragmented DNA from pigeon erythroid cells using the calculation [ 10, 121 similar to that described by Wetmur and Davidson [ 131 revealed several classes of nucleotide sequences with different frequencies of repetition (Table 1). Three of them (Table 1b, c and d) were purified and characterised as kinetically homogeneous families [ 121. The fastest reassociating sequences (Table la) have not been subjected to detailed analysis except that the lowest frequency of repetitions presented in this fraction was roughly estimated [ 121. The hybridisation was conducted with the DNA initially separated into three fractions. The composition of these fractions is given in Table 1 which shows that only fraction 3 was homogeneous kinetically. RNA Usedfor Hybridisation and the Properties of Hybrids Fig. 1 presents sedimentation profiles of HnRNA isolated from the populations of erythroid cells. The lengths of RNA chains in the fractions > 45 S are greater by at least a factor than the length of the DNA fragments used. After heating RNA for 10 min in a boiling water bath, the sedimentation coefficient of such RNA decreased to about 14-16 S (Fig. 2). We did not conduct more drastic fragmentation of RNA before hybridisation in order to avoid RNA losses, as the prolonged annealing led to further fragmentation of RNA molecules (Fig. 2). Under our conditions after prolonged (10-12 days) annealing in the presence of 0.1% SDS more than 95% of RNA had a size of about 6 S. In other words, practically all RNA added to the annealing mixtures was able to participate in hybrid formation. However the sizes of the RNA chains changed during the annealing while the length of the DNA fragments remained constant. Fig. 3 shows that RNAase-resistant hybridisation product is eluted from HAP column at the same molarity of phosphate buffer which elutes native (fragmented)

43

Transcription of Repetitive DNA

'"I

28

s

18s

Fraction no. Fig. 1. Sucrose gradient sedimentation of total pulse-labelled RNA from erythroblasts (I), reticulocytes (2) and erythrocytes (3). Sam-

ples of RNA were centrifuged on a 5-2096 sucrose gradient in 0.05 M Tris-HC1 (pH 7.0) - 0.05 M NaCl M EDTA 0.4% SDS for I50 min at 40,000 rpm and 1 6 O C (RPS-40, Hitachi, PS-55 centrifuge). For the conditions of cell labelling see Methods

-7

28 s

I8 S

018 0.2L Phosphate buffer, M

Q12

( 10

Fig. 3. Salt elution of free RNA and RNAase-resistant hybrids from hydroxyapatite column. Samples of 6000 cpm of > 45 S erythroblast RNA were hybridised with 100 pg of DNA of the fraction 2 and with 500 pg of DNA of the fraction 3 up to Cot values about 1200 and 25,000, respectively (see Methods). After the treatment of hybrids with RNAase the concentration of phosphate buffer in the samples was brought up to 0.12 M, the hybrids were trapped on hydroxyapatite column at 60' C and eluted step-wise by the increasing concentrations of phosphate buffer

the > 45 S RNA with the repeating DNA sequences may be a reflection of the high (- 50%) GC-content of these DNA sequences in pigeon genome [lo, 121.

Hybridisation Kinetics of > 45 S HnRNA with the Repeating and Unique DNA Nucleotide Sequences

Fraction no. Fig. 2. Sucrose gradient sedimentation of erythroblast > 45 S RNA before annealing (1) after heating for 10 min at 100" C in 0.12 M phosphate buffer - 0.2%SDS (2) after annealing for 10 days in the same buffer at 62" C. Centrifugation was conducted as indicated in the legend of Fig. 1

DNA. Fig. 4 presents thermal elution profiles of hybrids. It is noteworthy that the hybrids obtained with the repeating nucleotide sequences are characterised by a markedly higher heat stability than those with the unique sequences. This difference was found in our experiments both when melting was conducted in solution [141 and on the hydroxyapatite column as shown in Fig. 5. The higher stability of hybrids which were formed by

Fig. 5 presents normalised kinetic curves of hybridisation of erythroblast RNA with the purified unique (fraction 3) and with the moderately repeating (fraction 2) DNA sequences. In the last case the kinetic curve is constructed after the subtraction of the hybrids formed with the single-copy sequences which are present in this fraction (see Table 1). The amount of such hybrids can be estimated from the kinetics of RNA hybridisation with purified single-copy DNA fraction 3. The Cot values for hybridisation with the repeating sequences of DNA fraction 2 (the points of Fig. 5 pertained to curve 1) were calculated, taking into account the concentration of the total DNA of fraction 2 in the annealing mixtures. In both cases of hybridisation (curves 1 and 2 of Fig. 5) experimental points can be fitted satisfactorily to

44

K. G. Gasaryan et al.: Table 1. The composition of DNA fractions used for hybridisation DNA fractions

1 2 3

% fraction in total DNA

20 45 35

Composition of fraction (96 total DNA)

a

b

c

13 -

5 3

12

_

-

-

d 2

-

-

30 35

-

a, b, c, d - nucleotide sequences of pigeon genome with repetition 1.8 x lo3, 70 and 1, respectively frequencies: 3.5 x lo’, [ 10, 121. The quantitative distribution of the sequence classes (a, b, c, d) in the separated DNA fractions is an average estimate based on (1) the analysis of the total kinetics of reassociation of the DNA [ 101 (2) the reassociation parameters obtained for purified sequence classes “b”,“c” and “d” [121, and (3) Cot values at which the fractions were obtained (see Methods) N

Temperature,

OC

Fig. 4. Melting of the hybrids by thermal elution from hydroxyapatite. 25,000 cpm of 3H-RNA were annealed with 1 mg of DNA of fraction 2 or with 5 mg of DNA of fraction 3 to Cot values 1000 and 50,000, respectively. RNAase-resistant hybrids (see Methods) were passed through the Sephadex G-200 column. Void volume was collected and the hybrids were adsorbed on the HAP column and washed with 0.12 M phosphate buffer containing 0.06% SDS. Then the temperature was raised stepwise and the material released from the column was eluted by 4 volumes of the same buffer. Absorbance at 260 nm and the radioactivity was determined in the eluted samples

‘I 0

1

3

2

L

5

are considered. For the purified unique sequences Cotl,, was approximately 5500. In hybridisation with the repeating sequences of DNA fraction 2 > 45 S RNA of reticulocytes and erythrocytes exhibited the Cot,,, values around 100 (data not presented), i.e. close to the value for the erythroblast RNA. As is seen from Fig. 5 , the kinetics of hybridisation with the repetitive DNA is not of a greater complexity than that observed in hybridisation of the same RNA with the unique sequences. This is an indication that sequences of a single moderately reassociating kinetic components of the DNA (see Table 1) participate in the hybridisation. An approximate calculation based ori comparison of Cot,,, values presented in the Fig. 5 indicates that DNA sequences which are repeated, on the average, 50 times per haploid genome, are involved in the reaction of hybridisation described by the data of curve 1.

lg Cot

Fig. 5 . Normalised kinetic curves of hybridisation of > 45 S erythroblast RNA with the moderately repeating (1) and with the unique (2) nucleotide sequences of DNA. Samples of 2000 cpm of the RNA were annealed with 500 ug of the DNA fraction 2 or with 2000 pg of the DNA fraction 3 as described in Methods. The amount of hybrids formed with the repetitive DNA sequences of fraction 2 was calculated for each Cot value by subtracting the hybrids formed in the same samples with unique sequences present in this DNA fraction using the data obtained in separate hybridisations with purified unique DNA

the theoretical curves of the second-order reaction kinetics with Cot,,, approximately 90 for the repeating sequences, which corresponds to Cot z 25 when only the repeating sequences presented in the DNA of fraction 2

The Content of Repetitive and Unique DNA Sequence Copies in > 45 S RNA of the Erythroid Cells Fig. 6 and Table 2 present the results in hybridisation of the studied RNAs with DNA in excess. RNA was annealed to Cot = about 5 with the repeating sequences of fraction 1. The level of hybridisation under such conditions did not exceed that observed at Cot = 1. Taking into account the kinetic data of Fig. 5 (curve 1) the hybridisation with DNA fraction 2 was conducted to Cot = about 1200. Under such conditions, the reaction with all the rarely repeating sequences must be completed. At this Cot value a certain number of hybrids are formed with the unique DNA sequences presented in this fraction. These hybrids we excluded from the

45

Transcription of Repetitive DNA

2

Table 2. Maximal values of hybridisation of > 45 S RNA from mature and immature erythroid cells with repeated and unique DNA sequences

a E r y t hroblosts

DNA fractions

Reticulocy tes

I

0

I

1

--4

Ret icu locytes

Erythroblasts

Reticulocytes

1 2

1.5 20.0"

13.0a

trace 6.0'

3

40.0

41.5

52.0

Total

61.5

61.0

58.0

0.5

Erythrocytes

The amount of the hybrids formed with the unique sequences subtracted.

I

200 300 LOO 100 DNA, ~g12000cprn3H-RNA

> 45 S RNA in RNAase-resistant hybrids, 5%

500

t

was RNAase-resistant after hybridisation with these DNA sequences. Thus, the relative proportion of repeated arid unique nucleotide sequences transcribed into the > 45 S RNA shifts in the course of maturation of erythroid cells in such a manner that the relative proportion of repetitions transcribed significantly decreases. Discussion

0

I

I

500

I

1000

I

1500

2000

DNA, pg/2000 cprn H-RNA

Fig. 6 a-c. Hybridisation of > 45 S RNA from erythroblasts or reticulocytes with excess of the DNA fractions 1 (a), 2 (b) and 3 (c). Samples of > 45 S RNA were hybridised with the indicated amounts of the DNA fractions 1, 2 or 3 to Cot values of about 5, 1200 and 5 x lo4, respectively, as indicated in Methods

values presented in Table 2. The annealing with purified unique DNA (fraction 3) was performed at Cot = about 5 x lo4. From the normalised curve 2 of Fig. 5 (assuming that the kinetics is of a second order) we conclude that at this Cot value the reaction with unique sequences proceeds approximately to 95%. Table 2 summarises the results on maximal levels of hybridisation of > 45 S RNA of erythroblasts, reticulocytes and erythrocytes with all DNA sequences obtained in these experiments. In each case 5 8 4 2 % of the RNA could be recovered as RNAase-resistant hybrids. From this, 1/3 of erythroblast RNA hybridised with the repetitive and 2/3 with the unique DNA sequences. In reticulocytes and erythrocytes, the proportion of RNA hybridising with the repeating sequences was found to be smaller by factors of 2 and 3, respectively. The hybridisation of the latter two RNAs with the sequences of DNA fraction 1 does not differ markedly from the background level, while about 1.5% of erythroblast RNA

Populations of immature and mature erythroid cells from pigeon bone marrow and from peripheral blood were used in the present work to reveal the possible changes in the pattern of transcription of reiterated and unique DNA sequences during specialisation of cellular functions. Man and Cole [61 reported recently that the proportion of RNAs copied from repeated sequences was markedly reduced during the conversion of proliferating myoblasts into the fused post-mitotic myotubes engaged in tissue-specific protein synthesis. Our experiments demonstrate essentially the same for the maturing nucleated erythroid cells. Three erythroid populations used in our work differed sharply in their ability to synthesise RNA. Total level of RNA synthesis in the bone marrow cells (erythroblasts) was nearly five times higher than that in the immature cells (reticulocytes) and about 10 times higher than in mature cells (erythrocytes) of peripheral blood, respectively [71. In the circulating erythroid cells the synthesis of rRNA is reduced to a negligible level, as compared to that in bone marrow [151. 70%-75% of HnRNA synthesised in the nuclei of circulating eythroid cells is unstable, and is decayed in 30-40 min [161. The data presented in this paper indicate that the proportion of the bone marrow > 45 S RNA which is hybridised with repetitive vs unique sequences of DNA in excess is nearly the same (- 1 : 3) as in other non-specialised cells [3-61. Considerably small-

K. G. Gasaryan et al.

46 er portion of the > 45 S RNA of the reticulocytes and erythrocytes is hybridised with the repetitive DNA sequences. The difference in hybridisation would be due to a more rapid loss of the repetitive part of the transcripts during the processing of HnRNA in the mature forms. However, this seems unlikely since in our experiments only the fractions of HnRNA larger than 45 S were used. These fractions appear to be minimally affected by processing [ 171 and consequently would largely reflect the properties of the genome regions from which they are transcribed. An interesting point concerns the frequency of repetitions which are found in the transcriptional sites and are represented in particular in the high molecular HnRNAs. In the pigeon erythroid cells these sites contain sequences repeating not more than 100 times per haploid genome (- 50 times by our estimations). This fact is worthy of note because a kinetic component with about 1800 repetitions per haploid genome is present in the moderately reassociating DNA of these cells. But this particular sequence family is not involved in the RNA synthesis (note that little or no hybrids were formed in hybridisation with DNA fraction 1 which includes a large portion of these sequences [Table 2]), while in other species 151 the sequences of comparable repetition frequency are actively transcribed into the high molecular RNA. The functional role of repetitions localised in transcriptional sites of eukaryotic genomes has not been cleared. At least some of these sequences, especially those which are transcribed into the high-molecular nuclear RNAs, may be the components postulated to participate in regulation of gene activity [ l , 21. If so, the

data presented above may suggest that for the regulation of genes which are functioning in highly specialised cells a smaller set of the repeating sequences is required.

References 1. Britten, R. J., Davidson, E. H.: Science 165, 349-357, 1969 2. Georgiev, G. P.: J. Theoret. Biol. 25, 473-490, 1969 3. Greenberg, J. R., Perry, R. P.: J. Cell Biol. 50, 774-786, 1971 4. Melli, M., Whitfield, C., Rao, R. V., Richardson, M., Bishop, J. 0.: Nature new Biol. 231, 8-12, 1971 5. Holmes, D. S., Bonner J.: Biochemistry 13, 841-848, 1974 6. Man, N. T., Cole, R. J.: Exp. Cell Res. 83, 328-334, 1974 7. Gasaryan, K. G., Kulminskaya, A. S., Ananjanz, T. G., Kiryanov, G. I.: Ontogenes SSSR 2, 263-275, 1971 8. Scherrer, K., Darnell, J. E.: Biochem. Biophys. Res. Cornmun. 7 , 486-490, 1962 9. Britten, R. J., Kohne, D. E.: Carnegie Znst. Wash. Yearbook 65, 78-106, 1967 10. Tarantul, W. Z., Baranov, Yu. N., Prima, V. I., Lipasova, V. A., Gasaryan, K. G.: Mol. Biol. SSSR 7 , 849-858, 1973 11. Britten, R. J.: In: Problems in Biology: RNA in Development. The Park City Int. Symp. on Problems in Biology (E.W. Hanly, ed.), pp. 187-217. University Utah Press 1969 12. Gasaryan, K. G., Kuznetsova, E. D., Tarantul, W. Z., Sivak, S.: (in preparation) 13. Wetmur, J. G., Davidson, N.: J. Mol. Biol. 31, 349-370, 1968 14. Gasaryan, K. G., Tarantul, W. Z., Baranov, Yu. N., Lipasova, V. A.: Mol. Biol. SSSR 8, 372-379, 1974 15. Tarantul, W. Z., Lipasova, W. A., Baranov, Yu. N., Gasaryan, K. G.: Biochemistry SSSR 38, 804-814, 1973 16. Gasaryan, K. G., Lipasova, V. A., Kirjanov, G. I., Ananjanz, T. G., Ermakova, N. G.: Mol. Biol. 5 , 680-689, 1971 17. Spohr, G., Imaizumi, T., Scherrer, K.: Proc. Nut. Acad. Sci. USA 71, 5009-5013, 1974