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 se- quences 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).