Proc. Nati Acad. Sci. USAVol. 80, pp. 4770-4774, August 1983Genetics

Three organizations of human DNA(NaI centrifugation/repeated DNA/long repeats/short repeats/structural genes)

DAVID STRAYER*, N. HEINTZt, R. ROEDERt, AND D. GILLESPIE**Bary Ashbee Leukemia Research Laboratories, Department of Medical Oncology/Hematology, Hahnemann University Hospital, 230 North Broad Street,Philadelphia, Pennsylvania 19102; and tDepartment of Biological Chemistry, Division of Biology and Biomedical Sciences, Washington University, Schoolof Medicine, St. Louis, Missouri 63110

Communicated by Paul D. Boyer, March 29, 1983

ABSTRACT Human DNA was denatured, annealed to low Cot,and fractionated by centrifugation to equilibrium on gradients ofNaL. Three well-defined zones resulted. The topmost, least densezone contained satellite DNA. The bottom zone contained mole-cules bearing infrequent sequences and short repeated DNA. Themiddle zone contained molecules bearing infrequent sequences,short repeated DNA, and long repeated DNA. The Nal patternwas independent of single-strand DNA chain length from 5 to 30kdlobases. Structural genes were found in the organization bearinglong repeats.

About 30%o of the human genome consists of repeated se-quences (1), of which over half are repeated more than 1() timesper genome. Some of these repeated sequences are organizedin long, tandem arrays, while others are interspersed amongless frequently represented sequences including structural genes.Some of the major families of human interspersed repeated DNAsequences have been identified. The largest family consists of3 X 105 copies of related sequences, each about 300 nucleotideslong, with most members having a site that can be cleaved bythe restriction endonuclease Alu I (2). This group of sequencesis collectively called the Alu family and is represented by a clonedDNA named "Blur 8" (3). Other, less frequently represented,short, interspersed repeated sequences (SINES) have been re-ported more recently by Deininger et al (4) and Miesfeld et al.(5).Adams et at (6) reported a longer interspersed repeated DNA

sequence (LINES) that was 6,400 nucleotides long on the av-erage and was represented some 4 X 103 times per genome. Insix cloned representatives of the 6.4-kilobase (kb) repeat, theregion of apparent repetition ranged from 5 to 9 kb, some 20times longer than members of the Alu family. Maio et al (7) andGillespie et al (8) reported families of human LINES charac-terized by two Kpn I sites spaced 1,200, 1,500, 1,800, and 1,900nucleotides apart, respectively. Each of the four families of KpnLINES consists of about 5 x 103 members. The 6.4-kb repeatcontains Kpn 1,200-nucleotide LINES within it but carries nosequences closely related to Kpn 1,800-nucleotide LINES (9).Though each of the interspersed repeated DNA families is

assigned a length, the designation is a matter of convenienceand is not meant to define each member. Alu family members,for example, average 300 nucleotides, but lengths of individualmembers vary from <100 nucleotides to >350 nucleotides. Asmentioned above, the 6.4-kb repeat also varies in length.

Thus, the length of individual interspersed repeated DNAelements varies both among and within the different repeatedDNA families described above. Similarly, infrequent DNA se-quences vary in length. For these reasons we were surprisedto discover apparent severe limitations on the length ratio of

repeated sequences to adjacent infrequent sequences in humanDNA elements. Human DNA was denatured, annealed to lowCot so that only repeated DNA formed duplexes, then the hy-bridized complexes were fractionated according to the per-centage of double-strandedness by centrifugation to equilib-rium in Nal gradients. The three well-defined zones of DNAthat resulted conflicted with the expectation of a blur of valuesvarying around an average ratio of repeated DNA sequencelength to infrequent DNA sequence length. This communi-cation demonstrates that the three zones correspond to threespecific organizations that characterize human DNA. These arethe satellite DNA organizations, the short-repeat organization(10), and the long-repeat organization (11). Most human struc-tural genes studied belong to the long-repeat DNA organiza-tion.

METHODSCentrifugation in Nal. DNA was extracted as described (9).

The single-strand length of DNA was determined by electro-phoresis through agarose of DNA that had been denatured byheat and then incubated at 70'C for 10 min in 2 M formalde-hyde. All chain lengths in this paper refer to numbers of nu-cleotides contained in single DNA strands. A solution of DNAin NaI was made by mixing (i) 6.0 ml of saturated NaI stocksolution (see ref. 12), (ii) 0.5 ml of 0.1 M Tris chloride/0. 1 MEDTA, pH 8.0, (iii) 0.1 ml of ethidium bromide at 1 mg/ml,and (iv) 3.5 ml of denatured, self-hybridized DNA (0.4 mg) in0.15 M NaCi. The final refractive index was adjusted to 1.4390.Ten milliliters of solution and 2 ml of mineral oil was centri-fuged for 6 days at 36,000 rpm and 230C in a type 40 rotor.

Alternatively, the DNA solution in NaI was prepared by us-ing native DNA, held at 100'C for 15 min, and centrifuged for3 days at 36-380C and then for 6 days at 230C. Density gradientrelaxation was used to increase the separation of DNA speciesin several experiments (13).

After centrifugation, the tubes were held stationary withclamps, illuminated with ultraviolet light, and photographedwith Kodachrome 25 slide film and a 300-sec exposure. Thisprocedure was satisfactory for recording the position of bands.However, in the photographs, the DNA bands were broaderthan they appeared by visual inspection. After photography,==25 fractions were collected from the bottom of the tube. Eachfraction was mixed with 1 ml of H20 and stored in polypro-pylene tubes at 40C.

Preparation of Repeated DNA Probes. The Alu family ofinterspersed, repeated DNA was prepared from clone Blur 8obtained from P. Deininger. Plasmid was prepared as de-scribed by Birnboim and Doly (14), cut with BamHI endonu-

Abbreviations: LINES, long, interspersed repeated sequences; SINES,short, interspersed repeated sequences; kb, kilobase(s) or kdlobase pair(s);NaCl/Cit, 0.15 M NaCI/0.015 M sodium citrate, pH 7.0.

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Proc. Natl. Acad. Sci. USA 80 (1983) 4771

clease, fractionated on 3% agarose gels, and recovered fromagarose as described by Vogelstein and Gillespie (15). EcoRIand Xba I tandemly repeated DNAs were prepared by cuttinghuman DNA with EcoRI and Xba I endonuclease, respectively,fractionating the nuclease-treated DNA on 1% agarose gels, re-covering the 340-nucleotide fragment from agarose as above,then purifying each fragment by electrophoresis through 2%agarose gels. a-Satellite DNA clone (clone 6) was a generousgift from J. Maio. Fragments of LINES left by Kpn I were pu-rified as 1,200-, 1,500-, and 1,800-nucleotide fragments fromKpn I-treated DNA fractionated on 1% agarose gels and thenwere repurified on 1.5% agarose gels. LINES fragments showedno hybridization to Blur 8 DNA and vice versa.

Repeated DNA fragments were precipitated three times fromethanol, dissolved in 10 mM Tris chloride, and nick-translatedas described by Rigby et al. (16).

Molecular Hybridization. Aliquots of fractionated DNA werespotted on nitrocellulose in the following manner. Each samplewas incubated at 100'C for 10 min, one volume of 20X NaCl/Cit (1 x NaCl/Cit = 0.15 M NaCI/0.015 M sodium citrate, pH7.0) was added, and then the solution was passed through ni-trocellulose membranes (Schleicher & Schuell). The DNA-membranes were rinsed with 6x NaCl/Cit, air-dried, and bakedfor 2 hr at 80'C. Prior to hybridization, filters were washed asdescribed by Jeffreys and Flavell (17). Filters were examinedboth by radioautography and scintillation counting.

RESULTSNal Profiles of Denatured/Annealed Human DNA. The

results of centrifugation in Nal of denatured/annealed humanDNA are shown in Fig. 1. The length of this DNA averaged 30kb (single-strand length). The conditions of centrifugation re-sulted in separations that were determined almost totally by the

mid *main-*

FIG. 1. Annealing/banding of 30-kb human DNA. A solution ofDNA in NaI was made by mixing 6.0 ml of saturated Nal stock, 0.5 mlof 0.1MTris chloride/0.1 M EDTA (pH 8.0), 0.1 ml of ethidium bromide(1mg/mi), and 3.5 ml ofDNA (0.4 mg) in 0.015MNaCl. DNA was dena-tured at 100CC for 15 min and then cooled to400 in ice immediately priorto mixing. The average single-stranded length of the DNA was 30 kb.The refractive index of the solution was set to 1.4390, the solution waskept at 38TC for 3 days, and then centrifuged for 6 days at 23TC and 36,000rpm in a type 40 rotor. After centrifugation, the tube was photographedunder ultraviolet light using Kodachrome 25 slide film and a 300-secexposure. Bands are indicated. satt, Satellite.

percentage of double-strandedness in the DNA molecules ratherthan by base composition. The major features of the result werea prominent DNA band at a position corresponding to DNAthat was 25% double-stranded (designated main band), a fineband that was 80% double-stranded DNA (designated satelliteband), and a band between them that was 35% double-strandedDNA (designated midband). The main band contained about60% of the DNA, the satellite band contained 10%, and theremaining 30% was recovered as midband DNA. The bands werenot composed of DNA networks (see below).

This result was typical of human DNA from a variety of tis-sues from different individuals, as long as the single-strandedchain length was between 3 and 50 kb. Shorter DNA producedvery diffuse bands; longer DNA frequently produced mats thatinterfered with separation. Generally, however, the bands be-came sharper as the DNA length was increased. Random shear-ing from very high molecular weight (>100-kb average single-strand length) DNA to produce high molecular weight (10- to20-kb average single-strand length) segments produced sharpbands after centrifugation in NaL. A similar result was obtainedwith several DNA denaturing/annealing conditions. However,the sharpest bands were obtained by denaturing DNA in theNal solution and then centrifuging for 4 days at 370C, duringwhich period hybridization occurred, followed by a 7-day cen-trifugation at 230C. As the temperature of hybridization wasraised, the satellite band and midband were recovered in po-sitions corresponding to greater double-strandedness, but themain band remained essentially constant in position (Fig. 2). Inorder to visualize the satellite band more closely, these gra-dients were exposed longer during photography than in Fig. 1;consequently, the distinction between midband and main bandbecame blurred. Centrifugation of denatured human DNA at-5°C, a temperature known to prevent renaturation of DNAduring centrifugation (18), only produced main band.

The individual bands of DNA were recovered from severalNaI gradients, pooled, and reanalyzed. Each band ran true uponrecentrifugation (Fig. 3). Small amounts of DNA could be re-covered from between the major bands, and these, too, be-haved faithfully upon reanalysis (Fig. 3). Greater resolution wasoften obtained from recentrifugation of purified DNA bands.The top satellite DNA, which appeared as one faint band in thefirst centrifugation, was seen to be actually three distinct bandsupon recentrifugation. The major fraction of each isolated band,

F -~.

A B CFIG. 2. Annealing/banding of 5-kb human DNA. Human placen-

tal DNA with an average single-stranded length of 5 kb was subjectedto the same annealing/banding procedure described in the legend toFig. 1 with the following exceptions. Each centrifuge tube contained560 Ag of DNA, and the incubation temperature prior to centrifugationwas 42°C (tube A), 38°C (tube B), or 23°C (tube C). Bands are indicated.satt, Satellite.

Genetics: Straver et al.

Proc. Natl. Acad. Sci. USA 80 (1983)

satt -

mid -a-

1 Xa

a--- S 'at t

3 s a t!

20

0 5N

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FIG. 3. Recentrifugation of individual DNA bands. A solution (260ml) containing 360 ug ofthehuman placental DNA per ml (shown ana-lyzed in Fig. 2) was prepared as described in Fig. 1 and centrifuged at360C and 34,000 rpm in a Ti..60,rotor (8 tubes). After 3 days the tem-perature was lowered to 23TC, and centrifugation was continued for 6days. DNA from five separate regions (satt, satellite; B satt, below sat-ellite; mid, midand; main, main band; and below main band) was pooled,and each DNA region was rebanded in a second centrifugation for 6 daysat 230C and 36,00 rpm in a type 40 rotor. EachDNA region was againpooled, and aliquots (percentage of total recovered) from selected re-gions were combined and recentifuged under identical conditions intwo tubes along with double- or single-stranded DNA markers. TubeA contained satellite (20%), midband (2%), main-band (0.5%), and 4X174(80 W, single-standed marker) DNAs. Tube B contained satellite (2%),below-satellite (8%), main-band (0.5%), below-main-band (8%), and hu-man (60 u.g; double-stranded marker) DNAs.

when redenatured and rehybridized to itself, returned to itsoriginal position after centrifugation in Nal (Fig. 4).

Repeated Sequences in Main-Band, Midband, and Satel-lite-Band DNAs. These results suggested that limited anneal-ing of denatured human DNA produced complexes of definedclasses with regard to the weight ratio of double-stranded tosingle-stranded DNA regions. Because the duplexed regionswere likely to contain most of the highly and moderately re-peated elements, each band resulting from Nal centrifugationwas analyzed for specific repeated sequences. Repeated DNAsequence probes were prepared as described and hybridized tofractions collected from Nal gradients of denatured/annealed

mid

rIi r.

A D

FIG. 4. Recentrifugation after denaturation of midband and main-band DNAs. Twelve percent of the midband DNA (tube A) and 5% ofthe main-bandDNA (tube B) collected after the first centrifugation de-scribed in Fig. 3 were recentrifuged after denaturation (1000C for 10min) at 36,000 rpm and 230C in a type 40 rotor for 6 days.

5 10fraction number

FIG. 5. Hybridization of repeated DNA probes with 5-kb DNA re-covered from Nal gradients. Human placental DNA with an averagesingle-stranded length of 5 kb was subjected to the annealing/bandingprocedure described in the legend to Fig. 1. After centrifugation, frac-tions were collected from the bottom. Each fraction was held at 10000for 10 min, then 10 vol of 18x NaCl/Cit was added, and the sampleswere immediately filtered through nitrocellulose membranes. Molec-ular hybridization was as described. I, Main band, II, midband; mI, sat-ellite band; o, Blur 8 probe; e, Kpn 1,200-nucleotide probe; and F, Xba340-nucleotide probe.

5-kb DNA (Fig. 5). A probe prepared from a member of theAlu SINES family (Blur 8) hybridized primarily to main-bandDNA, whereas one prepared from the Kpn 1,200-nucleotideLINES family hybridized primarily to midband DNA. Thus,when short strands of DNA were renatured and fractionated onNal gradients, the bands that were formed were characterizedby the length of the repeated DNA element; the more densethe complexes, the shorter the repeated sequences.

a-Satellite DNA did not hybridize to main-band or midbandDNA under moderately stringent hybridization conditions (0.45M Na', 66QC) and washing conditions (0.02 M Na', 60'C).However, under a variety of relaxed conditions, cross-hybrid-ization to main-band DNA and midband DNA was observed,even when cloned a-satellite DNA was used (not shown). AnXba-cut satellite DNA probe (8) hybridized both to satellite-bandDNA and midband DNA but not to main-band DNA (Fig. 5).This probe preferentially cross-hybridized to a recently ampli-fied family of LINES (9). Quantitatively similar profiles wereobtained with limiting or excess probe, suggesting that the hy-bridization values in Fig. 5 do reflect the quantity of availablerepeated sequences in midband and main-band DNA.The same annealing/banding profile was given by 30-kb DNA

(Fig. 1) as by 5-kb DNA (Fig. 2, tube B). DNA fragments re-covered from the main band and midband were the same sizeas the starting DNA strands (not shown). With long DNA it wasnot always possible to quantitate the DNA in main band andmidband because of mat formation, especially in main-bandDNA. However, usually the distribution of bands from 30-kbDNA was the same as the distribution of bands from 5-kb DNA.Because similar NaI profiles of denatured/annealed DNA wereordinarily obtained from long and short DNA strands, and be-cause with short DNA the position of self-hybridized com-plexes in Nal correlated with the length of the repeated se-quence, we expected that the long repeat and short repeatorganizations of DNA would be essentially mutually exclusiveover long regions of DNA. Consequently, the hybridization ex-periments described above were repeated on denatured/an-nealed DNA of high chain length.LINES and SINES probes were hybridized to fractions col-

4772 Genetics: Strayer et d

Proc. Natl. Acad. Sci. USA 80 (1983) 4773

lected from 30-kb DNA centrifuged in Nal (Fig. 6). The hy-bridization pattern was more complex than had been obtainedwith 5-kb DNA. Alu SINES family probe hybridized to main-band DNA, to midband DNA, and to DNA located above mid-band in the Nal gradient. Kpn LINES probes hybridized tomidband DNA and to DNA located above midband but not sig-nificantly to main-band DNA.

Increased Resolution of Main-Band and Midband DNA. Themolecular hybridization results on 30-kb DNA suffer from thepotential problem of collection artifacts arising from networkformation, even in cases where networks are not apparent fromvisual inspection of the gradients or from examination of re-covered DNA. Furthermore, the assignment of particular re-peated sequences to main band or midband in NaI would bemore certain if the separation of the two bands were greater.The difference in band position in Nal of two denatured/an-

nealed complexes is dictated by the length ratio of double-stranded to single-stranded regions. Therefore, derivatizationsthat lower the buoyant density of duplex regions would be ex-pected to increase the separation between the two complexes.Intercalation of actinomycin D or distamycin A into double-stranded DNA decreases its buoyant density. Accordingly, weproduced complexes by redenaturing and reannealing 30-kb DNAand centrifuged them in Nal gradients containing actinomycinD and distamycin A. The dyes interfered with visual inspectionso that no photographs of these gradients can be presented;however, it was clear from hybridization of Alu SINES to DNAcentrifuged in the presence or absence of actinomycin D anddistamycin A that the buoyant density of the complexes wasreduced and that the band separation was increased (Fig. 7).In both cases LINES probes failed to hybridize to main-bandDNA, confirming the results of Fig. 6.

Therefore, midband and main-band DNA consist of two dis-tinct organizations. Main-band DNA carries infrequent se-quences and the Alu family of short repeated sequences butdoes not carry the Kpn families of long repeated sequences. Werefer to this as the short-repeat organization. Midband DNAconsists of both types of repeated DNA interspersed amonginfrequently represented (nonhybridized) regions. For con-venience, we call this the long-repeat organization, even thoughit contains short repeats as well.The Organization of Some Human Structural Genes. It is

obviously of interest to decide whether individual human struc-

20 v

0

N

l0 2

5

00

IO 20fraction number

FIG. 6. Hybridization of repeated DNA probes with 30-kb DNA re-covered from NaI gradients. The procedure was as described in the leg-end to Fig. 5, except that DNA with an average single-stranded lengthof 30 kb was used. I, Main band; II, midband; III, satellite band, o, Blur8 probe; and m and e, 1,200-nucleotide LINES probe.

I0s

5-C

N.0

n20-,_

10 I

5 l

-4

1O 20

fraction number

FIG. 7. Hybridization of repeated DNA probes with 30-kb DNAfractionated in Nal gradients containing actinomycin D plus dista-mycin A. The procedure was as described in Fig. 6 except that the Nalgradient was made to a refractive index of 1.438 and contained 20 ,ugof actinomycin D and 20 ,ug of distamycin A per ml. Centrifugationwas at 36,000 rpm and 36°C for 4 days then at 25,000 rpm and 23°C for7 days. Symbols are the same as in Fig. 6. (Upper) No drugs. (Lower)With actinomycin D and distamycin A.

tural genes lie within the midband or main-band type of DNAorganization. One way to determine this is to hybridize struc-tural gene probes to denatured/annealed DNA fractionated onNal. However, these probes will hybridize with pseudogenesin addition to structural genes. A second way to decide the or-ganization of a given structural gene is to assess DNA clones ofknown structural genes for the presence of LINES. If such clonescarrv both a structural gene and a nearby LINES sequence,

0

* S1A 5B SD 1D 48 40 7A 5F4A 4E 2A 1B 5E 7D u4A 5C

7C+D 4D 2B 1C 1 2 8 H

FIG. 8. Lines in human histone genes. Aliquots of 1 ,g of each DNAwere spotted on nitrocellulose membranes and hybridized to 32P-la-beled Kpn 1,200-nucleotide LINES. The first 20 samples representpBR322 subclones derived from histone genes that were originally clonedin A phage clones 1, 2, 4, 5, and 7 (see ref. 19). The subclones are ordered1C-lD-lA*-IB-lE, 2B-2A*, 4B*-4E-4D-4C*-4A, 5F-5E-5B-5D-5C*-5G-5A, and 7A*-7B*-7D-7C; * denotes subclones containing structural genes(8). Kpn LINES exist a maximum of 3 kb from each of two structuralgenes in A phage clone 1, 10 kb from a structural gene in A phage clone2, 2 kb from each of two structural genes in A phage clone 4, and 7 kbfrom a structural gene in A phage clone 5. Samples 21 and 22 represent1 ,tg of A phage clones html and htm2 (20). Sample 23 represents 1 ,gof Blur 8 clone DNA, and sample 24 represents 1 ,g ofhuman genomicDNA.

Genetics: Strayer et al.

Proc. Natl. Acad. Sci. USA 80 (1983)

then the structural gene must be constructed in the long-repeatDNA organization.

Fig. 8 shows a typical analysis of LINES in structural genes,specifically in human histone genes (19). A LINES probe washybridized to plasmid subclones containing 1-5 kb of DNA de-rived from larger A phage clones carrying human histone genes.From data such as that shown in Fig. 8, we conclude that LINESsequences are located within 5-10 kb of each structural histonegene. Therefore, most or all human histone genes utilize themidband organization. Similar results have been obtained withhuman tubulin genes (21), human albumin genes (22), humanU1 genes (23), and human A-chain immunoglobin pseudogenes,but not with active A-chain structural genes (24). These studieswill be reported separately; suffice it to say here that four of thefive human gene families studied contain LINES sequencesnearby, hence they must use the long-repeat organization.

DISCUSSION

Limited annealing of long, single strands of DNA producedstructures that formed duplexes over regions consisting of re-peated sequences. The duplex regions lowered the buoyantdensity of the DNA complex in direct proportion to the lengthratio of double-stranded to single-stranded elements. Thus, theposition of a DNA complex after the DNA was denatured, an-nealed, and centrifuged in a NaI gradient is a measure of thelength ratio of its repeated and infrequent sequences. The factthat three discrete, rather sharp bands were obtained uponcentrifugation of denatured/annealed human DNA suggestedthat repeated and infrequent sequences in the human genomeare organized in three preferred ways. This is not to say that anyselected repeated sequence must be spaced regularly in the ge-nome. However, it is to say that, in the aggregate, the lengthratio of repeated and infrequent sequences in a given genomeis a regular rather than a random feature. When each of thethree bands was purified, redenatured, reannealed, and re-centrifuged in NaI, most of the DNA returned to its originalbuoyant density. Therefore, each band in NaI corresponded toa preferred DNA organization structured around specific classesof repeated sequences.

The least dense band in NaL, the satellite band, was com-prised of tandemly repeated sequences, as reported by Maio(25). The two denser bands contained SINES and LINES.

The main band, the most dense band-i.e., the band withthe lowest double-stranded/single-stranded ratio-carried pri-marily, if not exclusively, SINES and lacked LINES as judgedby molecular hybridization with specific probes. The interac-tions leading to the duplex regions were primarily intramolecu-lar, short-range interactions because the main band was ob-served with DNA that was denatured, quick-cooled, andcentrifuged at -50C, a condition of centrifugation that pre-vents intermolecular rehybridization (18). Under this condi-tion, midband and satellite bands were not formed.The middle band in Nal contained both SINES and LINES

when the starting. DNA consisted of long strands but consistedprimarily of LINES when the starting DNA consisted of short(5-10 kb) DNA strands.

The results of this study reveal that the human genome is amixture of long- and short-repeat DNA organizations, each or-ganization continuing uninterrupted for over 50 kb. The short-repeat DNA organization may contain only short repeated se-quences, but the long-repeat DNA organization consists of bothlong and short repeated DNA elements. The SINES and LINESDNA organizations are probably equivalent to the "Xenopusorganization" (10) (SINES) and "Drosophila organization" (11)(LINES) discovered several years ago, with two distinctions.First, the Drosophila organization was considered to contain

LINES exclusively, whereas our results suggest that, in hu-mans, the LINES organization possesses both LINES andSINES. Second, human DNA was considered, to be arrangedin the Xenopus organization, whereas our results show that thehuman genome displays both the Xenopus-type and the Dro-sophila-type organizations. The human structural genes we haveexamined belong to the long-repeat organization.

The position of midband complexes in NaI gradients variesfrom one group of primates to another, suggesting that changesin long-repeat DNA organization have occurred recently duringevolution (8). It is now apparent that the changes involve changesin LINES, perhaps in the recent amplification of new LINESfamilies whose length or frequency is characteristic in each pri-mate group (8, 9). This is not meant to imply that the short-re-peat DNA organization is evolutionarily static; indeed, inter-spersion of Alu family members is known to occur frequentlyduring evolution. However, changes in the short-repeat DNAorganization may not produce detectable changes in the posi-tion of the main band in the Nal gradient assay of self-hybrid-ized complexes simply because in main-band complexes the ra-tio of repeats to infrequent sequences is so low.

We thank M. J. Caranfa for technical assistance and A. Porter and D.Valley for preparation of the manuscript. We are especially grateful toour collaborators, N. Cowan, A. Dugaiczyk, T. Manser, R. Gesteland,G. Hollis, P. Leder, J. Maio, and M. Zasloff for allowing us to discussunpublished results. This research was supported by Grants GM-27270and HD 13860 and from a donation by the Philadelphia Flyers.

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4774 Genetics: Strayer. et aL