Discrete-Length Repeated Sequences in Eukaryotic Genomes (DNA Homology/Nuclease SI/Silk Moth/Sea Urchin/Transposable Element) WILLIAM R

Proc. Nati Acad. Sci. USA Vol. 78, No. 7, pp. 4016-4020, July 1981 Biochemistry

Discrete-length repeated sequences in eukaryotic genomes (DNA homology/nuclease SI/silk moth/sea urchin/transposable element) WILLIAM R. PEARSON AND JOHN F. MORROW Department of Microbiology, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205 Communicated by James F. Bonner, January 12, 1981

ABSTRACT Two of the four repeated DNA sequences near the 5' end ofthe silk fibroin gene (15) and one in the sea urchin the 5' end of the silk fibroin gene hybridize with discrete-length Strongylocentrotus purpuratus (16, 17). families of repeated DNA. These two families comprise 0.5% of the animal's genome. Arepeated sequencewith aconserved length MATERIALS AND METHODS has also been-found in the short class of moderately repeated sequences in the sea urchin. The discrete length, interspersion, and DNA was prepared from frozen silk moth pupae or frozen sea sequence fidelityofthese moderately repeated sequences suggests urchin sperm (15). Unsheared DNA [its single strands were that each has been multiplied as a discrete unit. Thus, transpo- >100 kilobases (kb) long] was digested with 2 units ofrestriction sition mechanisms may be responsible for the multiplication and enzyme (Bethesda Research Laboratories, Rockville, MD) per dispersion of a large class of repeated sequences in phylogeneti- ,Ag of DNA (1 unit digests 1 pg of A DNA in 1 hr) for at least cally diverse eukaryotic genomes. The repeat we have studied in 1 hr and then with an additional 2 units for a second hour. most detail differs from previously described eukaryotic trans- Alternatively, silk moth DNA was sheared to 6-10 kb (single- posable elements: it is much shorter (1300 base pairs) and does not stranded length) and sea urchin DNA was sheared to 1.2-2.0 have terminal repetitions detectable by DNAhybridization. A sim- kb or 4-6 kb. A typical DNA sample (1 ml at 200 ,ug/ml) in 150 ple technique for identifying such discrete-length repeated se- mM NaCl was then denatured by addition of25 A1l of3 M NaOH quences is described, at 40C and neutralized by successive addition of 3 M NaOAc Repeated DNA sequences comprise 20-50% of most animal (pH 6.8) to 75 mM, 200 mM 1,4-piperazinediethanesulfonic genomes (1) and >50% of many plant genomes (2). Detailed acid (Pipes, pH 6.8) to 20 mM, and 3 M HOAc to 75 mM. Al- information has become available on the organization, distri- kaline denaturation was used to ensure complete strand sepa- bution, and evolution ofmany satellite and repeated structural ration of the longer DNA fragments. The denatured silk moth gene DNAs, but few details are known about the arrangement DNA was incubated to Cot 1 [Cot is the product of total con- of the bulk of the moderately repeated, interspersed DNA se- centration ofnucleotide residues (in mol/liter) and time ofhy- quences present in most eukaryotes. Recent work that links bridization (in sec); 170 Ag/ml for 30 min at 600C] and chilled genetic and structural studies on.repeated sequences in yeast on ice; sea urchin DNA was incubated to Cot 10. The DNA was (3, 4) and Drosophila (5-7) indicates that some eukaryotic re- then incubated with nuclease S1 at 5-20 units per Ag of DNA peated sequences are transposable elements analogous to those (Boehringer Mannheim; 1 unit digests 1 Ag ofdenatured DNA found in prokaryotes. These eukaryotic transposable elements in 30 min at 370C) in 150 mM NaCV200 mM NaOAc, pH 4.3/ are >5000 nucleotides (Nt) long, much longer than the short 0.2 mM ZnSOJ4 mM dithiothreitol for 1 hr at 370C. The re- repeated sequences found throughout the metazoa (1). action was stopped with EDTA (20 mM) and the mixture was In most eukaryotes, and in silk moths and sea urchins in par- extracted with phenol twice. The DNA duplexes were dialyzed ticular, repeated sequences that average -400 Nt are inter- overnight against 1 mM Tris base/0.05 mM EDTA, pH 8, con- spersedwith single-copy DNA averaging 1000-3000 Nt. In con- centrated 10- to 20-fold by 2-butanol extraction, and subjected seem to have few to electrophoresis (15). trast, some animals, including Drosophila, Electrophoresis of DNA fragments, filter transfer, and hy- short repeated sequences interspersed with nonrepeated DNA bridization are described (15). Nitrocellulose sheets with 0.2- (8-10). Our studies have been concerned with repeated se- when reten- quence organization in genomes that have a repeat intersper- ,m pores (Schleicher & Schuell, BA83) were used sion typical of most animals (1, 11-13). tion of fragments shorter than 250 Nt was critical. The length ofa repeated sequencerefers to the duplex region Restriction fragment probes were prepared from Hae III, formed when a repeated sequence renatures. The unreacted BamHI, or EcoRI digests of plasmids pBF41 (15) and pFbl9 single-strand tails mark the ends. These single-strand tails have (18). Plasmid DNA restriction fragments were end labeled with- been visualized with the electron microscope (14) or degraded out denaturation (15). The labeled fragments were separated on with nuclease S1 and the remaining duplexes were fractionated 4% polyacrylamide gels, and individual fragments were cut out, on the basis of length (13). We have used Southern transfer eluted, and used for hybridization. hybridization of populations of nuclease Sl-trimmed repeated sequences to examine the distribution of lengths of individual RESULTS members of various repeated-sequence families. We find that Length of Repeated Sequences in Silk Moth DNA. Frag- some repeated-sequence families consist ofmembers that have ments from the plasmid pBF41, which contains the silk fibroin the same length, while other families have members that differ gene and 5700 Nt 5' to the gene, were hybridized with restric- in length (15). Three discrete-length repeated sequences are tion fragments oftotal Bombyx DNA or a population ofnuclease described, two in the DNA of the silk moth Bombyx moti near Abbreviations: Cot, product oftotal concentration ofnucleotide residues The publication costs ofthis article were defrayed in part by page charge (in mol/liter) and time ofhybridization (in sec); kb, kilobase(s); Nt, nu- payment. This article must therefore be hereby marked "advertise- cleotides or nucleotide pairs of single-stranded or double-stranded nu- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. cleic acid, respectively. 4016 Downloaded by guest on September 27, 2021 Biochemistry: Pearson and Morrow Proc. Nati Acad. Sci. USA 78 (1981) 4017

Sl-trimmed repeated-sequence DNA duplexes. Four distinct bands can be seen in the ethidium fluorescence of the total repeated sequences were found (15). Two of the sequences, a Bombyx repeated DNA population. The repeated-sequence 1.3-kb sequence in the Bam C fragment and a 5-kb sequence families homologous to the Hae B and Bam C fragments appear hybridizing with the Hae B/Bam A fragment (Fig. 1), are note- sufficiently abundant to produce these fluorescent bands. Ad- worthy because oftheir discrete length and conserved internal ditional families of discrete-length sequences can be seen in sequence. A member of the 1.3-kb repeated-sequence family nuclease SI-trimmed repeated-sequence duplexes from the is present 5000 Nt beyond the 5' end of the silk fibroin gene. Bombyx genome (Fig. 1). There are 1000 members of this repeated family distributed Distribution ofthe Discrete-Length Sequences Throughout throughout the Bombyx genome, accounting for a total of 1300 Bombyx DNA. Hybridization of repeated DNA probes to nu- kb, 0.3% of the Bombyx genome. clease Sl-trimmed repeated-sequence duplexes provides in- Part of the 5-kb discrete-length repeated sequence is found sight into both the length and the distribution of members of in the Hae B/Bam A fragment, the restriction fragment prox- a repeated-sequence family. The discrete distribution of the imal to the start ofthe silk fibroin mRNA (Fig. 1). This repeated fragments that react with the 1.3-kb repeated sequence is due sequence starts at least 600 Nt upstream from the 5' end ofthe to sequences adjacent to each 1.3-kb repeat that do not renature silk gene and continues away from the gene for up to 1000 Nt by Cot 1. The adjacent sequences remain single stranded be- (15). It is bounded by a single copy sequence proximal to the cause they are different for different copies ofthe 1.3-kb repeat. silk fibroin gene and by a different repeated sequence up- As the nuclease S1 concentration is increased from 5 to 20 units/ stream. A complete member of the 5-kb family is not present 1Lg of DNA,.single-strand tails are more completely trimmed near the 5' end of the fibroin gene; a 600-1000 Nt homologous and the 1.3-kb and several shorter repeated-sequence families region is apparently present. The 5-kb repeated-sequence fam- become visible above a background oftotal DNA duplexes (Fig. ily is found about 200 times in the Bombyx genome and accounts 1). The length distribution of the fragments hybridizing to the for 1000 kb (0.2%) of the total DNA (15). Faint 5-kb and 1.3-kb 1.3-kb sequence probe (Bam C) shifts from one trailing above 1.3 kb to a sharp 1.3-kb band. The absence of bands above 1.3 kb at the mild nuclease S1 - = - - < digestion criterion shows that most ofthe 1.3-kb sequences are Nuclease SI )C$ . X Nuclease not repeated in tandem. This is confirmed by digestion of total X 5 20 5) 20 "-' = S1 X Bombyx DNA with a variety of restriction enzymes that cut outside the sequence (BamrHI, HindIII) or within the sequence 23.8 -m only a few times (EcoRI). The hybridizing fragments in 9.5 -i these 6.6 digests have the broad length distribution expected for a se- 4.36 quence randomly interspersed throughout the genome (Fig. 1). EcoRI would be expected to generate 1000-Nt and 300-Nt bands 2.27 iftandem copies were present. The sequence in the Hae B fragment that reacts with the 5.0-kb family also hybridizes to a broad 9l range of restriction fragments (Fig. 1). Conservation of Internal Restriction Sites in the Discrete- 0.54 Length Repeated Family. The hybridization of the 1.3-kb repeated sequence with specific Hae III fragments of total Bom- byx DNA is due to conservation of restriction sites within the repeated-sequence family (Figs. 1 and 2). The Hae III fragments Barn C Hae B Bam A of Bombyx DNA that react with different parts of the 1.3-kb repeated sequence are the same length as the corresponding Hae III fragments of plasmids pBF41 and pFbl9. Thus, the cloned 1.3-kb repeated sequence near the silk fibroin gene is FIG. 1. Discrete-length repeated sequences near the 5' end of the representative of the silk fibroin gene. The restriction map shows the 5'-end region of this repeated-sequence family. gene (right) and 5700 Nt of 5' flanking sequence. The thickened line The conservation of restriction sites in the various copies of represents DNA that encodes fibroin mRNA (and delimits an inter- the 1.3-kb repeated sequence can be used to map its ends. Re- vening sequence containing a repeated element). The open boxed reaction of pBF41 fragmeoc Hae I with a Bombyx Hae III digest gions denote the four sequences repeated elsewhere in the genome (15). shows a predominant band at the same Mr as Hae I, 170 Nt (Fig. The locations of the Hae HI (6), BamHI (O), and EcoRl ( ) sites are shown. The ethidium fluorescence lanes (extreme left) show the dis- 2). Consequently, the Hae I fragment is identified as totally tribution of repeated-sequence lengths. Bands were visible at 1300, within the 1.3-kb repeated-sequence family. Similarly, the pre- 460, 280, and 160 Nt. The autoradiographs show the lengths of nu- dominant Hae III fragments of Bombyx DNA that hybridize clease Si-trimmed repeated sequences or restriction fragments of with pBF41 Bam C have the same Mr as Hae G and Hae Y, 560 Bombyx DNA that react with the two 32P-labeled fragments indicated from plasmid pBF41. The nuclease Si-trimmed repeated sequences Nt and 350 Nt, respectively (Figs. 2 and 3). were formed from shearedBombyx DNA (50 ,ug per lane) that had been The fragments identified as within the repeated sequence by denatured, renatured to Cot 1, and incubated with 5 or 20 units of nu- the above criteria, Hae G, I, and Y, account for 1070 ofthe 1300 clease S1 per pg of DNA (Bam C, left) or 10 units/;kg (Hae B/Bam A). Nt in the repeated sequence, leaving about 230 Nt to be dis- The 5 units/pg repeated fraction contains 27% of total Bombyx DNA; the 20 tributed at the ends in Hae H and Hae X. The 350-Nt pBF41 units/jg duplexes contain 22%. The restriction fragments rep- Hae H resent the entire Bombyx genome (10 ug per lane, Bam C; 20 gg per fragment reacts with the 1.3-kb nuclease S-trimmed lane, Hae B/Bam A). The fragments were subjected to electrophoresis duplex but not with a Bombyx Hae III fragment 350 Nt long in an agarose gel, transferred to filters, and tested for homology to the (not shown), locating one end of the repeat within the Hae H 32P-labeled plasmid fragment indicated (15). Ten copy equivalents (3.5 fragment. The other end of the repeat must be located in Hae ng) of a Bam digest of BF41 are also shown (1OX). The Hae III frag- X (Fig. 2), as ments of Bombyx DNA that react with Bam C are estimated as 910, the Eco C fragment does not react with a Hae X- 730,560, and 350 Nt. Marks at extreme left indicate 6.6,2.27,0.54, and length fragment (550 Nt) in a Bombyx Hae III digest. 0.18 kb. The 480-Nt band of total Bombyx DNA that hybridizes with Downloaded by guest on September 27, 2021 4018 Biochemistry: Pearson and MoiITOW Proc. NatL Acad. Sci. USA 78 (1981)

r jna 0 0 . ). qrO .q m gq¢ m - ; m Q~~~CEI--rII= -r~~~~~~~ ~w _raN

_ _m

500_ Eco.C -I |Y |||I*_ 300 - -_ _ 150

Eco C Eco B Hoe J Hoe I Bam C Eco RI X1 B I A ( BamC ) pFbl9 Hoe II x Y I G II H

1 500 NT G 1 I I H X pBF41

FIG. 2. Conservation of restriction sites within a discrete-length repeated sequence. Restriction fragments from plasmids pFbl9 (18) and pBF41 (15) and total Bombyx DNA were separated on 2% agarose gels, blotted, and probed with different cloned fragments around the 1.3-kb repeated sequence. The samples contained equimolar quantities of the 1.3-kb sequence [1 ,ug of pFbl9 digested with EcoRI (R19) or Hae m (H19), 500 ng of pBF41 Hae Im fragments (H41), and 10 ,ug ofBombyx DNAHae Im fragments (HB)]. The filters were probed with the Eco B andEco C fragments of pFbl9, which are not present in pBF41, and Hae J, Hae I, and Bam C from pBF41. The reactions were carried out at 550C in 1 M NaCl, and the products were washed at 3700 in 50 mM NaCl, in which the melting temperature (Tm) of 30% G + C DNA is 720C. The map of theEcoRl ( I ),BamHI (F), and Hae m (6) sites was deduced from the overlap of fragments Bam C and Hae J with fragment Hae Y. Hae Y reacts primarily with Eco B, which orders Eco B and Eco C and Hae Y and Hae X away from BF41. The minor Bombyx Hae digest fragments that hybridize with Bam C are 730 and 910 Nt long. They are identified as Hae G + I and Hae G + Y on the basis of their lengths and hybridizations with Eco B, Hae J, and Hae I. They may reflect partial digestion or rare DNA sites resistant to Hae m digestion. Multiple bands in the Hae I hybridization appear to be due to partial digest products Hae H + I and Hae G + I. The binding of the short fragments is variable. Although hybridization to 170-Nt (Hae I) and 150-Nt (Eco C) fragments is often seen, at other times (32P-labeled Bam C hybridization), the short fragments are not found. Eco C shows some crossreaction with sequences in Eco B, and part of Eco B reacts with a sequence at the 5' end of Eco A (seen in reactions ofHae J and Bam C with Eco B).

the Hae B/Bam A fragment indicates common Hae III sites in gence of the silk fibroin genes of the different species is about many of the 100-200 full-length members of the 5.0-kb family 220C between B. monri and A. polyphemus and 12'C between (Fig. 3). A. polyphemus and S. cynthia. The divergence ofthe total sin- Absence of Large Terminal Redundancies in the 1.3-kb gle-copy DNAs ofB. mori and A. polyphemus is more than 240C Repeated-Sequence Family. We have examined the ends ofthe (unpublished results). Repeated sequences diverge less than 1.3-kb sequence for crosshomology. With 230 Nt at the ends single-copy DNA in echinoderms and vertebrates (17). ofthe sequence to be distributed between fragments Hae X (550 Distribution of Two Repeated Sequences in Sea Urchin Nt) and Hae H (350 Nt), terminal repetitions larger than 40 Nt DNA. Repeated-sequence clones from the sea urchin S. pur- should produce crosshybridization ofHae H with Hae X and Eco puratus (16, 17) have also been used to probe nuclease S1- C. No crossreaction has been identified between fragments in trimmed repeated sequence duplexes. CS2101 contains a 320- pBF41, which contains only one end ofthe repeated sequence, Nt fragment ofDNA that is repeated 700 times in the sea urchin and pFbl9 fragments, which contain the other end. [See, for genome. CS2101 has been shown to react predominantly with example, the absence of hybridization of pFbl9 Eco C with the class of short repeated sequences in sea urchin DNA and pBF41 Hae H (350 Nt; Fig. 2).] We should be able to detect with a broad distribution of EcoRI fragments from either S. a perfect match of 20 Nt or a 300-Nt homology with 30% mis- purpuratus or S. franciscanus DNA (19). CS2101 reacts with match under the conditions used. The strong hybridization of a discrete band of nuclease Sl-trimmed sea urchin repeated the 40-Nt Hae J fragment shows the sensitivity of the hybrid- sequence duplexes (Fig. 4). ization reaction (Fig. 2). The discrete band ofCS2101 hybridization contrasts sharply The 1.3- and 5.0-kb Repeated Sequences Are Not Found in with the ladder of trimmed duplexes that react with CS2133 Related Silk Moths. The two discrete-length families of re- DNA, which is included for comparison. CS2133 contains a 310- peated sequences near the fibroin gene together contain 0.5% Nt fragment repeated 2100 times and a second 310-Nt fragment oftotal Bombyx DNA (1000 copies and 200 copies). No examples repeated only 60 times (16). EcoRI digests of total sea urchin ofeither family have been found in distantly related Antheraea DNA suggest that CS2133 may be tandemly clustered (19). The polyphernus or Samia cynthia silk moth DNAs. The search for ladder of nuclease Sl-trimmed duplexes one, two, three, or related sequences (Fig. 3) was carried out at a very low criterion, more multiples of the 2133 repeated-sequence length is the 350C below the melting temperature of 30% G + C DNA. As result ofregularly spaced nuclease S1-sensitive sites. Such sites few as five DNA fragments with as much as 30% mismatch may be due to localized sequence diversity or unusual base should have been detected after long exposures. The diver- composition. Downloaded by guest on September 27, 2021 Biochemistry: Pearson and Morrow Proc. Natl. Acad. Sci. USA 78 (1981) 4019

x A B A B 0 S 2000-

*.. _ - 1700 1150- .:!:!;.I!! a- 1400 910- 1* p1300 - 3400 730- 2600 560- 580- _ - 400- -1660 350- * _- 610 - 480 -830

CS2101 CS2133 Born C Hoe B/ Bam A FIG. 4. Discrete-length interspersed and tandemly repeated sequences in sea urchin DNA. Sea urchin DNA (S. purpuratus) was FIG. 3. The 1.3-kb and 5.0-kb repeated sequences in three silk sheared to 1.6 kb (A) or 5.0 kb (B), denatured, and annealed to Cot 10. moth genomes. Hae III fragments from 5 pg of Bombyx mori (B), An- The renatured DNA was trimmed with nuclease S1, and the duplexes theraeapolyphemus (A), and Samia cynthia (S) DNAs were separated were separated on 2.0% (CS2101) or 0.7% (CS2133) agarose gels and on 2% agarose gels, blotted, and probed with BF41 Bam C (1.3-kb re- blotted to nitrocellulose. The filters were probed with the cloned sea peat) or Hae B/Bam A (5.0-kb repeat). The Hae B/Bam A lanes also urchin repeated sequences at 700C in 1 M NaCl and washed at 550C included 50 copy equivalents of a Pst I digest of BF41 and 15 copy in 50 mM NaCl. The minor band found in C52101 lane A is probably equivalents of aHae Il/Bam HI digest of BF41 (50X). The filters were due to some digestion of the repeated sequence and is the same length incubated at 450C in 1 M NaCl and washed at 370C in 50 mM NaCl. as the cloned repeated-sequence probe. The 480-Nt Hae III fragment of B. mori DNA, which reacts with the Hae B/Bam A probe, is part of the 5.0-kb repeated sequence. lion years ago. However, S. purpuratus has 20 times more copies of the CS2101 sequence (17). Discrete-length repeated sequences must be multiplied and DISCUSSION dispersed by different mechanisms from scrambled or tandemly repeated sequences. Recombination mechanisms may exploit Hybridization of repeated DNA probes with nuclease S1- sequence homologies between repeated elements in tandem or trimmed repeated-sequence duplexes can divide repeated-se- scrambled sequences. For example, unequal crossover may be quence families into three distinct groups. First, some families used to multiply or correct populations of tandem sequences contain tandemly repeated sequences such as ribosomal RNA (22, 23). Homologous but nonreciprocal recombination mech- and histone genes. Members of the sea urchin CS2133 re- anisms have also been proposed for transposition, inversion, or peated-sequence family are also often found in tandem arrays. deletion ofrepeated sequences (8, 24). On the otherhand, these Second, members ofdiscrete-length repeated-sequence fam- would not serve to distribute many copies of a sequence among ilies are usually found interspersed among different sequences. different nonrepeated sequences. For instance, the copia se- Then, the nuclease S1 Southern analysis described presents a quences in Drosophila do not display detectable homology with discrete band, as for the 1.3- and 5-kb repeats ofsilk moth DNA a host site (25). and the sea urchin clone CS2101. The transposable repeated Precisely copied discrete-length repeated-sequence families sequences in Drosophila, copia, and 412 are also flanked by are probably generated by a mechanism that distributes an in- unique sequences (6) and display a discrete nuclease S1 South- dividual repeated sequence as a unit, without involving its tan- ern band with a radioactive probe (19). One copy of the Tyl dem duplication at any site. If, on the contrary, the 1.3-kb family transposable element ofyeast (3) is also found at a given location. had arisen by tandem duplication to a form -a, Y, G, I, b, a, Y, Third, some dispersed repeated-sequence families have a G, I, b... in a linear array, even discrete-length parts of such diffuse range of repeat lengths. The intervening sequence in an array could have the form -a, Y, G, I, b- and also -I, b, a, Y, the silk gene and one of the repeated sequences beyond the 5' G- or -G, I, b, a, Y-. The pattern ofbands ofHae III fragments end ofthe gene react with repeated duplexes from 250 to several of Bombyx DNA that react with the 1.3-kb sequence suggests thousand nucleotides long that have no apparent pattern (15). that -a and b- are always on the end and that Y, G, and I are Similar scrambled repeated-sequence families have been found always in the middle in the same order. in Drosophila and chicken DNAs (8, 21). The 8 repeat at the The 1.3-kb sequence has evidently been distributed as a unit. ends ofthe Tyl and other transposable sequences in yeast would Two broad classes of mechanisms may be envisioned, one in- react with a variety of repeated-sequence lengths (3, 4). volving existence of the element free of the host genome (for The discrete length and tandemly reiterated types of re- instance, as a virus) and the other without a free state (trans- peated sequences display a high degree of sequence homology position) (26). The fact that the 1.3-kb sequence is much smaller within a family. The conservation of the Hae III sites in almost than all of the numerous animal virus genomes known makes every copy of the 1.3-kb repeated sequence has allowed map- a viral origin unlikely. ping ofits ends in the same way that isolation ofmultiple copies The 1.3-kb repeated sequence and other members of dis- of the sequence would. And there are at least 100 copies of a crete-length sequence families may be transposable elements 480-Nt Hae III fragment ofthe 5.0-kb repeated sequence else- similar to those found in prokaryotes and other eukaryotes (26). where in the Bombyx genome. The CS2101 clone melts only Such elements are duplicated as a unit throughout chromosomal 1°C lower after hybridizing with S .franciscanus DNA than with DNA and are sometimes associated with rapidly reverting ge- S. purpuratus DNA, though these species diverged 10-20 mil- netic loci (4). Moderately repeated DNA sequences have been Downloaded by guest on September 27, 2021 4020 Biochemistry: Pearson and Morrow Proc. Nad Acad. Sci. USA 78 (1981)

found to move over short time scales in yeast (3, 4) and occupy 4. Roeder, G. S., Farabaugh, P. J., Chaleff, D. T. & Fink, G. R. different chromosomal locations in closely related strains of (1980) Science 209, 1375-1380. Drosophila (6, 27). In the absence ofgenetic markers, it is dif- 5. Finnegan, D. J., Rubin, G. M., Young, M. W. & Hogness, D. S. (1978) Cold Spring Harbor Symp. Quant. Bowl 42, 1053-1058. ficult to provide direct evidence oftransposition in other DNAs, 6. Potter, S. S., Brorein, W. J., Dunsmuir, P. & Rubin, G. M. but the identification of precisely duplicated discrete-length (1979) Cell 17, 415-427. sequences suggests that a similar mechanism may be respon- 7. Gehring, W. J. & Paro, R. (1980) Cell 19, 897-904. sible for the multiplication of many moderately repeated se- 8. Wensink, P. C., Tabuta, S. & Pachl, C. (1979) Cell 18, 1231- quences in eukaryotic genomes. 1246. We have not been able to detect any redundancy at the ends 9. Manning, J. E., Schmid, C. W. & Davidson, N. (1975) Cell 4, 141-155. ofthe 1.3-kb sequence. The results rule out terminal repetitions 10. Crain, W. R., Eden, F. C., Pearson, W. R., Davidson, E. H. >50 base pairs long. The absence of long terminal repetitions & Britten, R. J. (1976) Chromosome 56, 309-326. and the size ofthe 1.3-kb element distinguish it from previously 11. Efstratiadis, A., Crain, W. R., Britten, R. J., Davidson, E. H. described transposable elements in eukaryotes. Those of yeast & Kafatos, F. C. (1976) Proc. Natl. Acad. Sci. USA 73, 2289-2293. and Drosophila, and the analogous integrated proviruses of 12. Gage, L. P., Friedlander, E. & Manning, R. F. (1976) in Molec- RNA tumor viruses, are 5000 base pairs or larger and are ular Mechanisms in the Control of Gene Expression, eds. Nier- lich, D. P., Rutter, W. J. & Fox, C. F. (Academic, New York), bounded by direct repeats of 300-1200 base pairs (28). All pp. 593-598. known transposable elements have terminal repetitions. How- 13. Eden, F. C., Graham, D. E., Davidson, E. H. & Britten, R. J. ever, in many prokaryotic insertion sequences and transposons, (1977) Nucleic Acids Res. 4, 1553-1567. these are very short. IS4, for example, has only 18-base inverted 14. Chamberlin, M. E., Britten, R. J. & Davidson, E. H. (1975)1. repeats (with two internal mismatches) at its ends (26). Such Mol. Biol 96, 317-333. terminal bounding the 1.3-kb sequence could 15. Pearson, W. R., Mukai, T. & Morrow, J. F. (1981)J. Biol Chem. short repetitions 256, 4033-4041. not be detected by the experiments described here. 16. Klein, W. H., Thomas, T. L., Lai, C., Scheller, R. H., Britten, The function ofthis class ofrepeated sequences is unknown. R. J. & Davidson, E. H. (1978) Cell 14, 889-900. There is ample evidence that transposable loci can affect gene 17. Moore, G. P., Scheller, R. H., Davidson, E. H. & Britten, R. J. expression (4, 7, 29), Other sequences may be relics of earlier (1978) Cell 15, 649-660. insertions that have lost critical sequences for transposition. 18. Ohshima, Y. & Suzuki, Y. (1977) Proc. Nati Acad. Sci. USA 74, a mechanism for rearranging re- 5363-5367. Such sequences could provide 19. Moore, G. P., Pearson, W. R., Davidson, E. H. & Britten, R. J. gions of neighboring single-copy DNA. It has also been sug- (1981) Chromosoma, in press. gested that the transposable elements in eukaryotes serve no 20. Rubin, G. M., Brorein, W. J., Dunsmuir, P., Flavell, A. J., purpose in the organism (8, 30, 31). Levis, R., Strobel, E., Toole, J. J. & Young, E. (1981) Cold Spring Harbor Symp. Quant. Biol 45, in press. We thank P. Bingham for describing the nuclease S1 Southern blot 21. Eden, F. C., Burns, A. T. H. & Goldberger, R. F. (1980)J. Biol M. Rubin for of and Chem. 255, 4843-4853. technique, G. discussions transposable elements, 22. Smith, G. P. (1974) Cold Spring Harbor Symp. Quant. Biol 38, M. Kahler for manuscript preparation. The unpublished results cited 507-513. above were obtained by N. T. Chang and J. F. Morrow. The studies 23. Petes, T. D. (1980) Cell 19, 765-774. on sea urchin repeated sequences were carried out in collaboration with 24. Scherer, S. & Davis, R. W. (1980) Science 209, 1380-1384. R. J. Britten at the Kerckhoff Marine Laboratory and were supported 25. Dunsmuir, P.,.Brorein, W. J., Simon, M. A. & Rubin, G. M. by a Carnegie Institution fellowship to W.R. P. This research was sup- (1980) Cell 21, 575-579. ported by Grants GM26557 and T32 CA 09139 from the National In- 26. Calos, M. P. & Miller, J. H. (1980) Cell 20, 579-595. stitutes of Health. 27. Strobel, E., Dunsmuir, P. & Rubin, G. M. (1979) Cell 17, 429- 439. 1. Davidson, E. H., Galau, G. A., Angerer, R. C. & Britten, R. J. 28. Levis, R., Dunsmuir, P. & Rubin, G. M. (1980) Cell 21, 581-588. (1975) Chromosoma 51, 253-269. 29. McClintock, B. (1956) Cold Spring Harbor Symp. Quant. Biol. 2. 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