Volume 7 Number 1 1979

Nucleic Acids Research

Restriction analysis of spacers in ribosomal DNA of Drosophila melanogaster

Eric O.Long and Igor B.Dawid

Department of Embryology, Carnegie Institution of Washington, Baltimore, MD 21210, and Laboratory of Biochemistry, National Cancer Institute, Bethesda, MD 20205, USA Received 5 July 1979 ABSTRACT The sequence arrangement of ribosomal DNA (rDNA) spacers in Drosophila melanogaster was analyzed with restriction endonucleases. Spacers, derived from cloned rDNA repeats and from uncloned purified rDNA, are internally repetitive, as demonstrated by the regular 250 base pairs interval between sites recognized by the enzyme Alu I. Length heterogeneity of spacers is due at least in part to varying numbers of repeated sequence elements. All spacers analyzed, whether derived from X or from Y chromosomal rDNA, have a very similar sequence organization. The distance separating the repeated nontranscribed spacer sequences from the 5' end of the transcribed region is conserved in all ten cloned fragments examined, and is probably less than 150 base pairs, as measured by electron microscopy. INTRODUCTION

Tandemly repeated genes are separated by nontranscribed sequences called spacers (1). Length heterogeneity in repeated gene units was observed in most cases studied. In the ribosomal genes (rDNA) of Xenopus laevis, this heterogeneity was shown to result from varying numbers of repeated sequences within the spacer (2). DNA sequencing studies have demonstrated tne presence of short repeated sequences in the spacers of the 5S RNA genes in X. laevis, X. borealis and Drosophila melanogaster (3-5), and in the spacer of ribosomal genes in X. laevis (6). However, length heterogeneity and internal sequence repetition are not general features of spacers, as demonstrated by the sequence uniformity of the minor oocyte 5S RNA genes in X. laevis (7) and of the ribosomal genes in

Bombyx mori (8). In D. melanogaster there are about 150 tandemly repeated genes coding for 18S and 28S rRNA (ribosomal genes) in each nucleolus organizer. One organizer is present on the X chromosome and one on the Y chromosome. Spacers in D. melanogaster rDNA are moderately heterogeneous in length (9) and are homologous on the two different chromosomes (10). We present here C Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England

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Nucleic Acids Research a restriction endonuclease analysis of the structure of uncloned and cloned rDNA spacers derived from both the X and Y chromosomes.

MATERIALS AND METHODS Preparation of DNA

All cloned rDNA fragments have been described previously (11) and were prepared from plasmids as described by Wellauer et al. (2). rDNA was partially purified from D. melanogaster (Oregon R) embryonic DNA by density equilibrium centrifugation in Hg - Cs2SO4 gradients as previously described

(9).

Restriction endonuclease digests of DNA were done according to the enzyme manufacturers (Bethesda Research Labs and New England Biolabs), and DNA fragments were separated by electrophoresis in agarose gels. DNA f ragments were extracted from agarose gels as described by Tabak and Flavell (12). Polyacrylamide gel electrophoresis and end-labeling of DNA fragments were performed according to Maxam and Gilbert (13). Mapping by partial restriction digests was done as described by Botchan et al. (14). Electron Microscopy Nuclear RNA larger than 28S, prepared from D. melanogaster embryos as described previously (11), was incubated with a restriction fragment containing rDNA spacer sequences for 3 hr at 42°C in 70% recrystallized formamide, U.3 M NaCl, 10 mM Tris-ECI (pH 8.5) and 1 mM El)TA. The sample was cooled to room temperature, diluted 40-fold to about I wg/ml DNA into 70% formamide, 3.5 M urea, 0.1 M Tris-tCl (pH 8.5), 10 mM EHTA and 50 9g/ml cytochrome C, and spread over a distilled water hypophase. fX174 and SV40 JNAs were included as size standards. All other procedures have been described previously (Z).

RESULTS AND DlISCUSSl0N

0. melanogaster ribosomal gene is shown in Figure 1. Maps of all the cloned rDNA repeats used in this study have been puolished (11). the various clones differ from one another in the lengths of their spacers, as will be discussed below, and with respect to the presence or absence of an insertion in the 28S gene region. The rDNA clones are designated umr (for Drosophila melanogaster ribosomal) followed by a letter and a number. The letter (a) represents clones derived from the X chlromosome, and the letter (Y) represents clones derived from the Y chromosume. other letters represent clones whose chromosomal origin is unknown. Wneli a cloned rDNA repeat is digested with the two restriction endonuA nap of one representative

206

Nucleic Acids Research 0 1 2 3 4 5 6 7 8 9 10 11 12 k NTS

ETS 18S, ITS

28S V

NTS

ETS 18S

Eco RI Fragmnt

Spam Fragmen

HindlI

Has m

Figure 1. Map of a ribosomal gene in Drosophila melanogaster. A ribosomal gene is shown, separated from its neighboring genes by nontranscribed spacer (NTS). Regions coding for the external and internal transcribed spacers (ETS and ITS, respectively) and for 18S and 28S rRNA are indicated. The single site recognized by the restriction enzyme Eco RI is indicated by a vertical bar. The site in the 28S rRNA coding sequence where insertions occur is indicated by a triangle. cleases Hind III and Rae III the largest resulting fragment corresponds to almost the entire spacer sequence (11). Rind III cuts at the very left end, and tae III close to the right end, of the spacer. We have determined that the Rae III site in the transcribed spacer is about 250 bp away from the 5' end of the 18S rRNA sequence (not shown). In this paper, a spacer fragment will be defined as the fragment resulting from double digestion of rDNA with Hind III and Rae III (Fig. 1). We chose our cloned rDNA repeat with the shortest spacer (DmreS4) and tested 22 different restriction enzymes on the spacer fragment (Table 1). Eighteen restriction enzymes did not cut this 3.6 kb fragment. The sites recognized by the 4 other restriction endonucleases were mapped in four different cloned spacers, Dmra55 and Dmra56, derived from the X chromosome, DmrY22, derived from the Y chromosome and Qmre54, whose chromosomal origin is not known. Dmra56 has the largest spacer fragment among our rDNA clones (5.4 Wb). The dind III fragment containing the spacer was prepared from these clones and was end-labeled at the 5' ends. The labeled fragments were then cleaved with Hae III to generate spacer fragments labeled only at the Hind III site. The sites recognized by Alu I, Taq I, Hinf I and

HIha I were mapped by partial digestion and agarose gel electrophoresis. Results obtained with Alu I are shown in Figure 2. Spacers are internally repetitive as evidenced by the regular interval between sites recognized by Alu I, and the number of this repeated sequence varies in different spacers. in order to obtain uncloned rDNA spacers partially purified rDNA was

207

Nucleic Acids Research Table 1. Dmre54

Restriction Endonucleases Tested on the Spacer Fragment from Clone

Enzyme a) Sma I Hae II Bam HI Pst I Pvu I Pvu II Sal I Xho I Hind II Bgl II Eco RI Hpa I Xba I Hind III Hinf I Hae III Hha I Hpa II Alu I Mbo I Taq I Bgl I

Recognition Sequence CCCGGG PuGCGCPy GGATCC CTGCAG CGATCG CAGCTG GTCGAC CTCGAG GTPyPuAC AGATCT GAATTC GTTAAC TCTAGA AAGCTT GANTC GGCC GCGC CCGG

AGCT GATC TCGA ?

Number of Cleavage Sites

0 0 0 0 0 0 0 0 0 0 0 0 0 0

1b)

o 1 0 7 0 1

0

a)Enzymes

are listed in decreasing order of (i) number of base pairs recognized, and (ii) G+C content of the recognition sequence.

b)Hae iII

cuts at the right end, and Hind III at the left end, of the spacer fragment.

digested with Hind III and Hae III and electrophoresed in a preparative agarose gel. Hae III does not cleave spacers but has many sites in most other DNA sequences. Therefore, much of the contaminating DNA and the ribosomal gene sequences were digested to small fragments. The remaining large fragments banded primarily at 4.1 and 4.4 kb, with other material between and immediately surrounding these bands. This size range corresponds to the size of spacers as determined previously by electron microscopy of uncloned rDNA (9). DNA fragments between 4 kb and 4.5 kb were extracted from the gel and analyzed by restriction endonuclease digestion in parallel witn ten different cloned spacer fragments (Fig. 3, Table 2). Every cloned spacer studied has a 1 kb Alu I-Hae III fragment at the right end, most of which corresponds to the external transcribed spacer. As expected, the same fragment is found in uncloned rDNA spacer. Therefore, 208

la c

";

Nucleic Acids Research

-o

054

a56

12 4 1 2 4

IcC

a55 Y22 1 2 4 1 2 4

a6 46

dli.

dewaa

d.

A..

C.

a

_ _.

w ^11

..

Figure 2. Restriction mapping of spacers from cloned rDNA repeats with the endonuclease Alu I. Four spacer fragments end-labeled at the Hind III site were subjected to partial restriction digestion with the endonuclease Alu I. The numbers 1, 2 and 4 represent arbitrary relative values for the product (enzyme units) x (time). The digests were electrophoresed in a 1.2% agarose gel.

divergence among different rDNA repeats transcribed/nontranscribed spacer, contrary to what was observed in X. laevis rDNA (14). Minor sequence divergence, not detected there seems to be no major sequence

at the boundary

209

Nucleic Acids Research

A

B

CM X Q

c

4

N

_

UL n n

(N Ur

n

'0

v

In

M

C Lnfi

e

CN

'

N

%-

Z

Q .

CN Z _- (N O CY i C CN C C co a e VI Le Lfu - I.N co -n f n R,, Atl-_

(l

r4

(N

_4

Figure 3. Restriction digests with endonuclease Alu I of cloned and uncloned rDNA spacers electrophoresed in a 2% agarose gel (A) and in 6% polyacrylamide gels (B and C). (C) is a partial digest and the arrow points to dimers of the repeated sequence. Size markers were X EcoRI-HindIII and pBR322 Alu I.

by this restriction enzyme analysis, may occur, but it is clear that the distance between the repetitive and the transcribed sequences has been conserved in most rONA repeats. As predicted from partial digestion mapping the enzymes Taq I, tlinf I and Hiha I cleave within the 1 kb Alu I- Hae III fragment and the sites for these three enzymes are conserved in the four clones studied (data not shown, see map in Fig. 4). The number of 250 bp repetitive elemeents within spacers varies from 5 to 12 in ten different cloned fragments. (The number of repetitive sequence elements is represented by the number of Alu I restriction sites and not by the number of fragments generated by Alu I.) We do not know whether shorter repetitive sequences occur within the 250 bp element. The Alu I fragment at the left of the spacer is variable in size. However a 1.9 kb fragment was found in 6 out of 10 clones and was also the 210

Nucleic Acids Research Table 2.

Sizes in bp of Alu I Fragments in Cloned and Uncloned rDNA Spacers

Alu I Fragments a) Spacer Fragment

Left

Middle (Sequence

Right b)

Repetition)

a5i c) a52 c)

a55d) b56C)

a56 d)

c52c) e52 c)

e54 d)

y15 C)

Y22 d) cDNA )

1900 1900 1900 1900 1900 1900

2650 1250 1875 1150 1900 1 850

250 250 250 250 250 250 250 250 250 250

(8) (7)

1000 1000 1000 1000 1000 1000 1000 1000 1000 1000

(7)

(12) (7) (5) (8) (7) (8) (9)

250

1000

1150

a) Sizes were determined from several agarose gels. AtHind III - Eco RI (17) and pBR322 Alu I (18).

b)The

right end of this fragment is the

Hae

Standards used were

III site.

c)These

spacers were analyzed by limit digestions. The sequence repetition was calculated from the sizes of Alu I fragments compared to the total size of the spacer fragment.

d) The position of all Alu I sites was determined by partial digests mapping

(Fig. 2).

most prominent fragment obtained from uncloned rDNA. All four clones derived from the X chromosome contain this 1.9 kb fragment suggesting that

it is present in most rDNA repeats on the X chromosome. Alu I fragments between 1 kb and 1.9 kb can be seen in uncloned rDNA spacer but we cannot be sure that they are derived from spacer sequences because the rDNA preparation was not pure. A detailed map of four cloned spacers is shown in Figure 4. The position of the restriction sites relative to the transcribed sequences (ETS and 18S rRNA) is only approximate because we know that there are discrepancies between measurements of spacer DNA by gel electrophoresis (used to measure restriction

fragments) and by electron microscopy (used to measure

coding sequences). 211

Nucleic Acids Research 0

2

1

28S

3

4

5

NTS

6 ETS 18S

TTTT}TTTTTTiT} l

_+

+rTTT iTT r

/a///v a56

vtt

f~~~~vt

_T

kb

X

TrT T T rTTTT

f//z

a55

///m

Y22

2

vYt

fT rT

l

w

/ZaX

e54

Figure 4. Maps of spacers f rom cloned rDNA. The sites in the spacer f ragment recognized by the following restriction endonucleases are shown: Hind III ( ); Alu I ( T); Taq I (V); Hinf I ( ?); Hha I ( t ) and Hae III ( ).

;

Alu I restriction digests of spacers were also electrophoresed in polyacrylamide gels (Fig. 3B). In 6% polyacrylamide the repetitive sequence element of 250 bp could be resolved into several variants.

in the repetitive

sequence

served in uncloned rDNA

into at least two, and can

exist in different

is not

spacer. up

a

This heterogeneity

cloning artefact because it is also ob-

The repetitive elements could be resolved Different variants

to four mobility variants.

spacers.

The sizes of DNA fragments could not be

determined in these polyacrylamide gels because the migration of sequenced DNA markers

was not a

linear function of the logarithm of their size.

It

is possible that the separation of repeated spacer fragments is due not

only to differences in size but also in base composition. When partial digests with Alu I were electrophoresed in 6% polyacrylamide gels, dimers of the repeated sequence elements also appeared heterogeneous (Fig. 3C). The pattern of these dimers confirmed that these heterogeneous repeated

elements spacers

can

be different in different spacers, in particular between

derived from the X and the Y chromosome.

Analysis in the electron microscope of heteroduplex molecules formed with cloned rDNA repeats from the X and the Y chromosome had shown that spacers on

the two chromosomes are homologous (10).

Deletion loops were

observed throughout the length of the nontranscribed spacer, suggesting

strongly an internal sequence repetition (15). Our restriction analysis nas demonstrated the presence of a repetitive sequence and a very conserved sequence

212

arrangement in different spacers, whether derived from X or from

Nucleic Acids Research Y chromosomal rDNA. The minor heterogeneity found in the repeated sequence elements is obviously too small to affect the formation of stable hetero-

duplexes between different spacers. We were able to determine the maximum distance separating the 5' end of the rRNA precursor and the repetitive spacer sequence by forming R-loops between rRNA precursor and a Hind III restriction fragment from cloned rDNA containing the spacer and part of the 18S rRNA coding sequence. Such Rlooped molecules had been used to determine the length of the external transcribed spacer (11). When spread in 70% formamide, spacer fragments often showed a pair of deletion loops. These loops must be the result of out-of-

Figure 5. R-loop between the rRNA precursor and the Hind III spacer f ragment from the cloned rDNA repeat Dmra56. The Hind III site at the right is within the 18S rRNA coding sequence (11). The rRNA precursor molecule is traced with a dotted line and DNA strands are traced with continuous lines. The arrow points to the region separating the 5' end of the RNA from the first loop in the spacer. 213

Nucleic Acids Research register pairing of repeated sequences, since a single cloned spacer f ragment was used. In some cases, deletion loops were observed very close to the 5' end of the precursor rRNA (Fig. 5). The distance between the proximal deletion loop and the 5' end of the RNA was measured, and the four shortest values were averaged at 130 ± 24 bp. This is a maximum estimate because the deletion loop can occur anywhere in the repeated sequence and not necessarily at the boundary between repeated and unique sequences.

Therefore, there are probably not more than 150 bp separating the repeated spacer sequence from the transcribed sequences. The corresponding region in X. laevis rDNA has recently been sequenced. It was found in a cloned rDNA repeat that 259 base pairs separate the repeated spacer sequence from the 5' end of the transcribed region (B. Sollner-Webb, personal communication). About half of the ribosomal genes in D. melanogaster are interrupted by insertions in the 28S rRNA coding region. We have shown that insertion transcripts are very rare in D. melanogaster (16), and it is therefore likely that most ribosomal genes with insertions are not transcribed. Nevertheless, the sequence arrangement at the boundary between repetitive and transcribed sequences, where control signals for initiation of transcription are likely to reside, is conserved in all cloned rDNA repeats examined. ACKNOWLEDGEMENTS We thank M. Rebbert for excellent technical assistance, D. Brown,

J. Ramirez, R. Reeder, H. Smith, B. Sollner-Webb and Y. Suzuki for gifts of various enzymes and P. Wellauer for communicating some of his unpublished experiments. E.O.L. was supported by a fellowship from the Swiss National Fund for Scientific Research.

REFERENCES

Fedoroff, N.V. (1979) Cell 16, 697-710. Wellauer, P.K., Dawid, I.B., Brown, D.D. and Reeder, R.H. (1976) J. Mol. Biol. 105, 461-486. 3. Fedoroff, N.V. and Brown, D.D. (1978) Cell 13, 701-716. 4. Korn, L.J. and Brown, D.D. (1978) Cell 15, 1145-1156. 5. Pirrotta, V. and Tschudi, C. (1978) In Genetic Engineering, Boyer, d. W. and Nicosia, S. Eds., pp. 127-134 Elsevier, North-Holland, Biomed. Press. 6. Boseley, P., Moss, T., Machler, M., Portmann, R. and Birnstiel, M. (1979) Cell 17, 19-31. 7. Brown, D.D., Carroll, D. and Brown, R.D. (1977) Cell 12, 1045-1056. 8. Manning, R.F., Samols, D.R. and Gage, L.P. (1978) Gene 4, 153-166. 9. Wellauer, P.K. and Dawid, I.B. (1977) Cell LU, 193-212. 1. 2.

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Nucleic Acids Research 10. 11. 12. 13. 14. 15. 16. 17.

18.

Wellauer, P.K., Dawid, I.B. and Tartof, K.D. (1978) Cell 14, 269-278. Dawid, I.B., Wellauer, P.K. and Long, E.O. (1978) J. Mol. Biol. 126, 749-768. Tabak, H.F. and Flavell, R.A. (1978) Nucleic Acids Res. 5, 2321-2332. Maxam, A.M. and Gilbert, W. (1977) Proc. Nat. Acad. Sci. USA 74, 560564. Botchan, P., Reeder, R.H. and Dawid, I.B. (1977) Cell 11, 599-607. Wellauer, P.K. and Dawid, I.B. (1978) J. Mol. Biol. 126, 769-782. Long, E.0. and Dawid, I.B. (1979) in Eucaryotic Gene Regulation, Axel, R. and Maniatis, T. Eds., Academic Press, New York, in press. Philippsen, P., Kramer, R.A. and Davis, R.W. (1978) J. Mol. Biol. 123, 371-386. Sutcliffe, J.G. (1978) Nucleic Acids Res. 5, 2721-2728.

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Restriction analysis of spacers in ribosomal DNA of Drosophila melanogaster.

Volume 7 Number 1 1979 Nucleic Acids Research Restriction analysis of spacers in ribosomal DNA of Drosophila melanogaster Eric O.Long and Igor B.Da...
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