Organization of a family of highly repetitive sequences within the human genome

J. Mol.

Viol.

(1982) 154, 51-63

Organization

of a Family of Highly Repetitive Within the Human Genome

S. M. I)ARI,IXG,

Sequences

J. &‘I. CRAMPTON AND R. WILLIAMSON

Ikportment of Biochemistry. St Mary’s Hospital Medical School liniversity of London, London W2 IPC, E’ngla.?td (Rrcel;ved 20 August

1981)

Recombinarl t. clones cont.airling the highly repetitive human DNA sequence approximat.cly 340 base-pairs in length obt,ained after EcoRI digestion (xRI-DNA) were c~loned in plasmid pAT153. Two clones contained a single copy of the nRIDKA sequence, and the third had an insert with two copies of the sequence in tandem. LVhen radioactive recombinant DNA was hybridized to total human DNA partially digested with EcoRI. a series of multiple bands was obtained up to 22 rc>prats in length, demonstrating that the nRI-DNA sequencbes occur in tandem arrays in tbr gencmic DNA. A reassociation analysis using isolated insert DSA from one of the recombinant clones showed that, the family of sequences is repeated 28.000 times in the human genome. Clones containing the %RI-DNA sequence were also isolakd from a library of human genomic DNA in bacteriophage /\. Using these clones it was shown that, in at least some cases, the repetitive element is bounded by Dh’A less abundant, than the &I sequence.

1. Introduction In humans. as in all eukaryotes, DNA seyuences are present at a variety of multiplicities (Britten & Kohne. 1968: Davidson & Written, 1979). The majority of sequences coding for proteins appear to exist in one or a few (sopies per haploid genomc. However. other elements are present in higher copy numbers, up to several hundred thousand per genome. Among the highly repetit,ive element,s in human DNA are seyuences that separat,r from the bulk of the DNA on isopycnic gradient, cent,rifugation, and arc known as sntellites ((‘orneo et al.. 1!167). Other highly repetitive elements have the same buoyant density as main band DNA, and either type of repeat may be cut specifically by a particular restricbion endonuclease (Cooke, 1976: Manuelidis, 197%). For example, total digestion of human DNA with the enzyme EcoRI gives rise t,o prominent bands of approximately 340 bpf and 680 hp. The 340 bl) sequencae. known as &RI-DNA, comprises approximately 07~59;~ of the tot,al human DSA (Manuelidis Rr Wu, 1978). Th. IS &-DNA repeat unit has been partialI? sequenced and is homologous to sitnilar DNA sequences in the African green monkey (Manuelidis $ Wu. 1978). The sequencr contains stop codons in all reading frames and thus is unlikely t’o code for a polypeptide. t Abbreviations used: bp, base-pair; C,t. is the product of concentration x time (8). and C,lt value of C,t at which a reaction is 50% complete: kb. IO3 bases or base-pairs, 0022-2836/82/0100~51-13

$02.00/O

51

Q 1982 Academic Press Inc. (London)

is the

Ltd.

~~anuelidis & Wu (197X) h ave shown. using pattially purified %Rl- UNA, that the majority of this sequence family occurs in t~andern arrays. Manuelidis (19786) has also shown hy in .sifrl hybridization t,hat t,hr *RI-UNA sequences are clustered wound t’he wntIromeres of some human chromosomes. However. it is possihle that this sequence is also interspersed throughout) nowcentromeric regions of the chromosomes. In contrast, other families of repetitive elements such as the .-IL/l family (Houvk ~1 ~1.. 1979) are int,erspersed with single copy 1)X,4 and not found in tandem arrays (Jelinek et nl., 1980). repetitive sequences in eukaryotic I)SA It has 1~~1 suggested that’ moderately havr roles in gene regulation (L)avidson & Hritten, 1973) or are replic:at,ion origins (.Jriinrk rl rtl.. 1980). More recently. proposals have kwen put. forward that. repetit,ive I)NAs have no function except t.hat. of’ “sur~~ival” within the genome (L)oolitt)le & Sapienza. 1980; Orgel & Crick, 1980). l\‘e describe here the cloning of representatives of the uRI-USA family and their subsequent use in the study of the organization of the family in total human DNA and in large. cloned segments of human I)NA.

2. Materials

and Methods

High molec*ular weight Dh’A was prepared fi-orn minced normal placenta as described by *Jeffrevs 8: Flavell (1979). Plasmid DPiA was prepared f’rom 1-I cultures by the detergent Iysate method of Clewell 8 Helinski (1969). The DNA was separated from R,S,4 by (pH 7%). cerlt,rifugation through a 15’$, to 30”,, sucrow gradient in 10 m&f-Tris, I miv-EDTA Purified

clones selected f’rom the genomic library

descaribetl by Loenen

& Hrammar

wrr

grown ant1 the DIL’A isolatrtl

as

(1980).

All restriction according to

rndonuc~leases were purchased from Bet,hesda Research Labs, and were used DNA samples were digested at a the manufacturer’s specifications. conrentration of50 pg/ml. Complete digestion of DNA was obtained by incubating 2 m&s of enzyme/pg of DNA for at least 6 h at 37°C in the specified buffer. Partial digestions were obtained by incubating 1 unit of enzyme/pg of DNA at 37”C, and stopping the reaction at appropriate time int,rrvals by adding OS! vol. 05 wEDTA.

RestrictSion endonuclease-digested DNA samples the products analysed on 1.5O;, (w/v) agarose or was carried out for approximately 16 h at 35 1’ in EDTA (pH 7.7) with ethidium bromide added to

were made to 49, in Ficoll/Orange G and gels. Electrophoresis 5Y, p 01y acrylamide 10 rnM-Tris, 20 mw-sodium acetate, 2 mMa rotlcentrat~ion of 0.25 mg/ml.

EcoRI Human genomic DNA was fractionated 011 a l.Fi”>, agarose gel after complete digestion. After visualization wit,h short-wave u.v. light the 340 bp band was excised from the gel and the DNA isolated by the freeze-squeeze method of Thuring et al. (1975) using the modifiration of Arrand (1978). The DNA was extracted onre with chloroform/isoamyl alcohol (24 : 1 v/v) and precaipitated overnight at - 20°C by the addition of 2 vol. ethanol. The DNA was recovered by centrifugation at 40,000 revs/min, 20°C for 1 h and redissolved in 10 rnhI-Tris (pH 7%).

(e) Puri&ation

of inser/ D,VA from

recombinant

plasmids

The recombinant plasmids pRHR-3, pRHR-5 and pRHR-6 were digested to completion with EcoRI and the products of digestion were run on a 571~ polyacrylamide gel in 40 mm Tris, 20 mrv-sodium acetate, 2 mM-EDTA (pH 7.7) with no ethidium bromide. Ultraviolet, shadowing was used for visualization of the DNA fragments as U.V. irradiation of ethidium bromide-stained DNA may result in nicking of the DNA. The gel was layered onto a U.V. fluorescent thinlayer chromatography plate (Kieselgel 60 F 254, Merck) and illuminated with a short-wave U.V. lamp. Slices containing insert DNA were excised from the gel. diced and put, into heat-sealed 1 ml disposable pipette tips plugged with siliconized glass-wool (Maxam & Gilbert. 1977); 0.6 ml of elution solution (0.5 M-ammonium acetate, O+W sodium dodecyl sulphate, I mM-EDTA, and tRNA at 5 pg/ml) was added and the tops of the tips were sealed with Parafilm. The tips were heat,ed for 16 h at 37°C. The Parafilm was removed arId t.he Cps of t.he tubes cut off. The elut,ed solution was drained out and collected irl 15 ml plastic centrifuge tubes. The pofyacrylamide fragments left in the tips were re-washed wit.h OC?ml of elution solution. Preclpltation of the DNA with ethanol was as described in the previous section. The pellet was dissolved in 200 ~1 water and spun for 30 s in a microfuge to remove any residual polyacrylamide fragments. (f) Radioactivr

labelling

of DA\‘-4 fragments

DNA fragments (approx. 0.5 pg) were labelled using 20&i of [32P]thymidinr triphosphate (obtained from the Radiochemical Centre. Amersham) by nick-translation as described by Rigby et al. (1977). After 1.5 h the reaction was terminated by adding 01 vol. W5 wEDTA and the radioactively labrlled DNA was separated from unincorporated nuclroside triphosphates by chromatography on Sephadex G-50 using 3 x SW as elutiorr buffer (1 x SSC is 0.15 M-NaCl. O-015 M-sodium ritrate, pH 7.6). (g) Hybridizxtion

to tJ,z’.t immobilized

onto nitrocelfukw

Rest,ricted DN,A in agarose gels was denatured and neutralized in situ and transferred by blotting at 4°C’ onto nitrocellulose paper using the t’echnique of Southern (1975). The filters were baked for at least 2 h at, 80°C. ,411 washes and hybridizations were at 65°C. Filters were prewashed for hybridization in 3 x SSC for 30 min, transferred for 2 h t,o 3 x SW. IO x Denhardt’s solution (Denhardt, 1966). and finally washed in hybridization buffer of 3 x SW. 10 x Denhardt’s, @l(& sodium dodecyl sulphate. 50 pg sonicated, denatured herring sperm DNA/ml. 10 pg poly(A)/ml for 16 h. The 32P-labelled probe. in 3 x SSC, was denatured by heating for 5 min at 100°C and was added to 10 ml of hybridization buffer. The filters were incubated with t,he probe for 16 h. Post-hybridization washes were carried out in either 3 x WC. 0.1 ()<> sodium dodecyl sulphatr or 0.1 x SW. Cl?6 so&urn doderyl sulphat,r tlepending on t,he levels of stringency required. The filters were dried and exposed to Fuji Sray film with an intensifying screen at -70°C. (h) Uon,ing Purified plasmid pAT153 was digested t,o completion with EcoRI and dephosphorylated using bacterial alkaline phosphatase (Bethesda Research Labs) according to the supplier’s instructions. The efficiency of dephosphorplation was test,ed by assaying for re-ligation of t,hc plasmid ends. Partially pure ,&I-DNA, isolated from a gel. was ligated into the plasmid vector pATI. in 66 mu-Tris. 6.6 mM-MgCl,, 10 mmdithiothreitol, 0.4 PM-ATP (pH 7.6) at 3 : 1 molar ratio of plasmid : insert for 2 h at room temperature. The ligated material was diluted 2O+fold wit,h TCM buffer (10 mM-Tris, 10 mWI!aC’l,, 10 mM-MgCl,, pH 7.5) and added to cells of E,scherichia coli K12 strain HBlOl made competent for transformation by the method described by Wensink et al. (1974). Transformation was carried out as described by Woods et al. (1980). Transformants were picked into microtitre wells and grown up in L broth and ampicillin (50 p&ml). Celfs were transferred onto 9 cm nitroceflulose filters using a replicator block and

54

S. M. I)AKI,IN(:

El’ .4L.

growu overnight at 37’(’ ou agar supplemented with ampicillin (50 &ml). The plasmids were amplified by transferring t,he filters to agar plates containing 17Og of cxhloramphenic-ol/mI for a further 16 h at 37°C”. The DNA was immobilized and sealrd onto the filter paper (Grunstrin & Hogness, 1975), except that the filters were not treated wit)h proteinase K nor washed with chloroform. The filters \vere then washed aud hybridized as detailed in the previous section, using a 32P-labelled. partially pure preparation of oiRI-DXA isolated from a gel as a probe. All cloning experiments were carried out, under cbontainmrnt conditions as recommended by the GMA(G guidelines for the I1.K on recombinant rcxsearcah.

Sir@-c:op.v human DNA was prepared from highly sheared DNA. which was soniuated t.o approximately 600 bp iu 1eugt.h. Thr preparative hybridization was carried out in @14 Mphosphate buffer and initially t,he DNA was boiled for .‘, min. Renaturation was at 6.5”C to a f’,f of 50 and the reaction was stoppd by placing in a (‘OJethanol bath. The samples were allowed to thaw and were made t,o a volume of 1 ml using 0.12 M-phosphate buffer. Fractionation on hydroxylapat,ite was used to separate single-strandrd DSA (eluted with 0.14 M-phosphate buffer) from double-stranded DXA (eluted with 0.48 M-phosphatr buffer). The single-stranded eluate was denaturrd and rehyhridizrd to a C’,t of 50 and again passed through hydroxylapatitr. The eluted single-st,randcd fraction is termed single-copy DNA and was used in the kinetic reaction discussed. Insert DNA from the recombinant pRHR-3 was hybridized at 65°C to human DSA sheared to 500 to 1000 bp in length aft,er boiling for 5 min. Hybridizations were carried out at a c*oncent,ration of 0.3 Av-XaCl and samples were t,akcu at appropriate time intervals and put ou ice. The extent of hybridization was assayed by percsentage resistance to S, nurlease (Getz CJ/al., 1975). The kinetics of hybridization for single-cop\ 1)X4 werp analysed under idrntical vondit.ions t,o provide a kinetic* standard for the react,ion. The f!,/+ values in thr t.ext. are from the. analyst+ iu W3 M-NaU.

The human genomica library was obtained from Dr Tom Maniatis (Lawn r/ nl., 1978). This library contains 15.000 to 20,000 bases inserts of human DNA in bacteriophage X (Iharon 4A (Blattner uf (~1..1977) and was screened for clones containing IKI-DNA sequences using thr in s&c plaque hybridization technique of Henton & Davis (1977). Plaques werr picked into 200 ~1 of phage buffer (21.5 mM-KH2f’0,. 49 rnM-Na,HPO,, 85 m&l-NaCl. I m&1i&+0,. 0.1 mW.?aCl,, O~oO1°~,, (w/v) gelatin, pH 7.15) and rescreened to ensure clone purity. The phagc werr amplified using plate lysat,es as desc~ribetl by Rlattner it ~1. (1977).

3. Results Human genomic DNA. totally digested with the restric6on endonuclease EcoRI and separated according to molecular weight by electrophoresis on an agarose gel. reveals two bands when stained with ethidium bromide (Fig. l(a)). one 340 bp and one 680 bp in length. The prominence of these bands against the background of the rest of the DNA shows that there is a family or families of DNA sequences defined by EcoRI restriction sites which is highly repetitive in the human genome. The and this paper analyses this family of 340 bp monomer unit is termed nRI-DSA repeats. Initial studies were carried out to investigate the distribution of &RI-DNA within the human genome. aRI-DNA. estimated at 7Y& purity (Mauuelidis. 1978a), was isolated from an agarose gel. nick-translated with [3zPldNTPs. and

HI-MAS

REI’ETITIVE

SEQUENCE

55

ORGASIZATION

hybridized to Southern blots of partial and total EcoRI digests of human genomiv DXA. A “ladder” of radioactive bands is seen (Fig. 1) in the tracks containing DNA partially digested with k:coRI. The bands of higher molecular weight gradually disappear as the digests tend to completion. the only two bands

(b)

0.66

Lb

(cl

(dl

(Cl

(f)

(9)

(h)

(il

Ij)

(k)

-

0*34kb-

VI<:. I. (a) Hums I)NA totally digested with ~‘coR1. electrophoresed on a 1596 agarose gel and stained wit.)] et,hidium bromide to show the 340 bp (nRI-I)NA) and 680 bp bands. (b) to (k) .;\utoradil)pr3n1 of hylwidization of nick-translated. partially pure mRl-USA to Southern blots of partial and total &‘coKI digests of human IJSA: left to right. digests tend to completion with tracks (j) and (k) containing tot,;cl digests. The final post-h~bridi~atiol, wash was in @I x SW. 1 kb = 1O3hp.

S. M. I)ARLING

56

ET AL.

remaining after total digestion being the 340 bp and 680 bp bands. The size of the largest. identifiable band in the partial digest, is 4.1 kb. a dodecamer of the 340 bp monomer. Considering t.he limits of resolution of the gel system and the fact, that a partially pure probe was used, it can be concluded that the nRI-DNA family occurs in tandem arrays of at least 12 units in lengt,h. Members of the &RI-DNA family were cloned so that) purified probes would be available for furt)her investigations. The plasmid pAT153 was chosen as a cloning vect,or since it has only one E~coRl restriction site and since most repeating units of ,xRI-I)IVA cont.ain a single EcoRI restriction sit,e. Three out of ten transformant,s chosen hybridized to part,ially pure, labelled &RIDNA probe. and these putative recornbinants were called pRHR-3. -5 and -6 (Eco_KI, I(igh Bepeat). When the rec.ombinant pla,smidn were restricted with EcoRI a 340 bp insert, was cut from each plasmid, in addition to t,he linearized pATI : pRHR-6 gave a 340 bp insert of t.wioe the intensity of ethidium bromide st,aining as t,he other two clones and it seems probable that two 340 bp inserts had been ligated into the plasmid vector in tandem. In order t’o test whether the insert, in each of the three clones is nRI-DNA, the recombinant plasmids were nick-translated and hybridized with Southern blots of total F:coRI digests of human DNA. These were compared wit,h equivalent blots hybridized with part.iall,v pure rwRI-DNA. As can be seen from Figure 2(c) the clone pRHR-3 hybridizes strongly at the 340 bp and 680 bp positions. as did pRHR-5 and pKHR-6 (data not shown). The hybridizat,ion patterns are identical for each recombinant. Therefore all the clones contain rwRI-DNA. However. other faint bands can be seen when genomic DNA digested to complet,ion is hybridized with ~loncd fiRI-DXA probes. which cannot, be seen in equivalent tracks hybridized witch partially pure zRI-DNA. These bands are of higher molecular weight : only some are mult.imers of the 340 bp monomer. It would seem that the cloned 340 bp sequence is able to detect) #&I-DNAc*ontaining penomic fragments that were not previously identified using a partially pure preparat,ion of ;RI-DNA. The recombinant, plasmid pRHR,-3 was also hybridized to South ern blots of li:coRI partial digests of human DNA (Fig. S(a) and (I))): multimers of up t.o 22 of the 340 bp units are seen. Once again the higher sensitivity obtained wit,h the cloned sequence shows larger multimeric forms of 8~RI-I)SA than were seen when partially pure nRI-DNA was used as a probe, although this may also be due to a slightly more partial digestion. A microdensit,ometer tracing of track (c) shows that there are prominent bands out, of phase w&h the 340 bp unit. These bands are sized at 076 kb and 1.45 kb, and ~iv/” of the total also do not represent “half-unit,a” ; each (.onstitutes approximately hybridization as estimated from the area, under the tracing. (b) Reitemtion

frequency

and sequence hetwogen,eity

of the c\RI seqwnee

To estimate the reiteration frequency of the rRI-DNA family within the human genome. the k&et.& of hybridizat,ion of the cloned sequence to human DNA were studied. The renaturation of human single-copy DNA was followed under identical condit,ions as a kinetic standard, and the result,ing Cot curves are shown in Figure 3. From the C,tt values of 0.0457 for &T-DNA and 1000 for single-copy DNA t.he

HVMAN

REPETITIVK

SEQI’EXCE

ORC:ASI%ATIOX

57

number of copies per haploid genome caZu be estimated at 22,000 for aRI-DNA. Sequence heterogeneity withiu the &RI-DNA family was studied by mapping pRHR-3. pRHR-5 and pRHR-6 using restriction enzymes. Enzymes that did not cut any of the cloned inserts were XbnI, HaeII, P&I, HpaII, Sau96a, BarnHI, RgZII and KpnI. Figure 4 shows that pRHR-6, as already suggested from the iuitial analysis of the clones, coutaius two 340 bp inserts. Despite the possible different orientations of the inserts, the restriction sites vary considerably between the four cloned 340 bp sequerices. All contain one MhoII site, two coutain a Fnu4HI site. each at a different positiori, and oue contaius a Hind111 site. The restriction sites cover 26 bp; at least five of which show a base change. Therefore, at the restriction sites st.udied. approximately ZOqi,, of the nucleotides show variation between the four ~loued sequences. The sequence variability of the xRI-DNA family was also investigat,ed by hybridizing the recombinarit pRHR-3 to Southeru blot,s of human genome DN.4 (a)

(b)

(cl

1.45 kb

“pi,

-

0.76

kb

Q-

0.34

Lb

FIG. 2. Autoradiogram of a Southern blot of partial ((a) and (b)) and total (c) KcoRI digests of human DNA hybridized with the recombinant plasmid. pRHR-3. DNA in (a) was digested for 15 mirl and DNA in (b) was digested for 30 min. The final post-hybridization was in @I x SSC.

S. $121.I)ARI,lS(:

58

p

60-

ET

.4L

A/

; 1; I” 40-

s

A 20%

/

FIG:. 3. Kinetic analysis of the hybridization of 321’-labelled aRI-l)SA tu excess sheared human 1IS.A (---A--). For comparison. a kinetic analysis of single-copy human I)NA was carried out under identical conditions (-A-A-).

ECORI pRHR-3

u

pRHR-5

u

Em RI

Mboll

Fnu4HI

Mbo II

EcoRI

Fnu4HI

0 I

100 bp I

Eco RI

EcoRI

MboII

HmdIlI

EmRI

Mbo II

Eco RI

pRHR-6 200

bp I

NC:. 4. Restriction maps of the 4 cloned 340 bp mcm~~nwrs. Kestrirtion fractionated on 5’35,polyacrylarnide gels.

digests of the inserts only were

digested to completion wit)h restriction er~do~~uc~leases other than EcoRI (Fig. 5). The enzymes were chosen on t,he basis of t,he data obtained from the restriction mapping of the cloned inserts. D1L’A digested with Mb011 (Fig. 5(a)) gives a ladder when hybridized with rRI-DNA. The lowest hand has a molecular weight of approximately 340 bp and most of the bands, some of which are more prominent than others, differ by 340 bp. This shows t)hat there is one Mb011 site within the majority of the 340 bp units. Figure 5 (b) and (d). containing DNA digested with Hind111 and BgZII. respect.ively, show that, t.hese rest~riction endonucleases only cut.

(a)

0.68

(b)

(cl

(d)

(0)

hb

FIG;. 5. Autoradiogram of hxbridizatiorr of the nick-translated recombinant pRHR-3 blots of huncul gencmic I)NA dipsted with wrri~ms restriction enzymes: (a) M6oII: (c) X/MI : (d) RglTI : (e) Ah/I. The final post-h? bridization wash was in 0.1 x SW.

to Southern (b) Hind111

the &I-DZ;A rarely, if at all. as only higher molecular weight DNA hybridizes to the recombinant, plasmid. Xbnl and il/~l restriction sit,es would seem to be present in t,hr &RI-DNA wit,h a frequencay between these two ext,remes (Fig. 5(c) and (e)).

In order to study the distribution and organization of this highly repetitive DNA family at a finer level, clones from a phage library of human genomic DNA were analysed. The genomic library was given by Dr T. Maniat.is (Lawn et al., 1978) and it is important that the method of c4oning does not result in loss of organizatjion of &I-DNA. Human foetal liver was partially digested with the restriction rndonucleases HrreIIT and AZuI and large fragments (15 to 20 kb) were ligated t,o

S. M. DARLING

ET

ilL

FIG. 6. Screening of the human genomic library : autoradiogram of the distribution hybridizing to aRI-DNA. The final post-hybridization wash was in 3 x SW.

of clones

Clone I RRRRRRRRRRRRRRRR

Clone 2 R

0 I

R

I kb I

R

HR

RHR

R

HR

R

2kb I

FIG. 7. Restriction maps of clones from the genomic library which hybridize to &RI-DSA. (0) Shows the location of aRI-l)NA within each clone derived from hybridization data not shown. Digested DNA was fractionated on 1.5’:;, agarose gels.

the vector h Charon 1A using Ec.oRI linkers. The resulting library has a >990/; probability of containing any DNA sequence present in the genome. The library was screened for clones hybridizing to the insert of pRHR-3 (Benton & Davis. 1977). The final post-hybridization wash was carried out to a low stringency of 3 x SSC so that all possible members of the family would hybridize to the insert, despite the sequence heterogeneity observed. Figure 6 shows the results of the initial screening of the library. Positively hybridizing plaques give varying intensities of signal, showing that clones in the library contain differing amounts of &RI-DNA sequence. Individual clones which differed in hybridization signal were picked in order to study the organization of otRI-DNA.

Restriction maps were constructed by analysis of the fragments obtained after digestion of genomic clones with the enzymes HindTII and EcoRI. For the six cblones picked. two different classes of strurt)ural organization of rRI-I)KA were seen. (%mes of one class, an example of which is clone I. gave a strong hybridization signal and were found to consist entirely of &RI-DNA : clone I has 15 310 bp units in t>andem array. Clone 2. an example of the second class. gave a weak hybridization signal and contains *RI-DNA interspersed with non-1R1-1)XA in a complex manner (Fig. 7). The location of %RI-DNA within each clone is derived from hybridization data not shown. To ascertain the repetitive nature of the ~oI~-IRI-T>SA. nick-translated t’otal human I)XA was hybridized to Soutjhern blots of restriction digests of clone 2. Some I)NA fragments that were seen in t,he gel did not, hybridize to the t,otal human I)NA probe and therefore are not highly repeated (Fig. 7). The nature of these sc~quen~s associated with the #cxRI repeat are currently under investigation.

4. Discussion

Four uRI-1)SA sequences in three plasmid clones were isolated. and b> comparison of restriction endonuclease sites. approximately 20(:,, sequel1c.e variation is found. However, this figure is derived from looking at only 26 bp of the IRI-l)?;X sequence and the remaining nucleotides may or may not) show this level pure preparation of of variation. Manuelidis & Wu (1978) sequenced a partially xRI-1)SA and found no ambiguity in the sequence family. However. this met,hod of analysis would not detect a nucleotide variation that was trot, present in at1 important proport,ion of nRI-DNA copies at a given position. By comparison. the method of restriction mapping of individual cloned members of the rR1 farnil? gives a direcat indicat,ion of sequence variation within the family. This amount of’ heterogeneity may need to be considered when reviewing t,he kinetic analysis of the ~RI-I)SL4 family. The number of copies expected for this sequence can be calculat,ed from the figure of 0.75”” which Manuelidis (1978) reported as t)he proport’ion of rRT-I)SA in the genorne. This gives a value of 55.000 copies per haploid genome (OW)75 x 2.5 x lo9 bp haploid genome/340 bp insert). while the observed C’,f+ value gives a figure of 22,000 copies. The tnost likely factor causing this approximateI> twofold difference between the observed and expec%ed reiteration frequencies is that due t)o mismatching between the renatured duplex molecules. Rritjtrn & Kohne (I 968) found that reassociated repet,itive 1)X*4 may melt gradually over a \vide temperature range showing that there must be considerable mispairing in the renatured duplex molecules. They suggest,ed the concept that repetitive, components consist of families of related sequences. Thus, the repetitive fractions observed to retrature at a given C’,f value have a higher repetition frequency than implied 1,. the observed C,tt value. Our data from the restriction mapping of’ thus c~loned -\RI-I)SA would suggest that there is likely t,o be mismatching betwee reassociatjrd sequences.

(b) Orywnizntion

of nRI-DNA

within

the human yenome

The cloned aR1 probes were able to detert, longer end-to-end repeats of the 340 bp unit tha,n were observed when a partially purified sequence isolated from agarose gels is hybridized to equivalent filters. When the clone pRHR-3 was hybridized to partial EcoRI digests of total genomic DNA, the longest fragment resolved was a 22-mer, which shows that the periodicity in the sequence extends over long st,retches. When the probe was hybridized to DNA digested to completion with E’coRI, many of the higher molecular weight bands disappeared, as occurred in the hybridization using the partially pure sequence. However, new bands were seen. which are not visible when the partially pure probe is hybridized t,o total &oRI digests of human DNA. These bands. of Q76 kb and 1.45 kb each, constitute approximately Fi’:, of the total hybridizing rRI-DNA as shown by microdensitometer tracing of the autoradiogram. This demonstrates t,hat there are structural arrangements for the nRI-DNA family within the human genome other than t’andem arrays of a 340 bp repeating unit. If the distribution of the 340 bp sequence is random throughout the genome then of the t,otal number of genomic clones in bhe library would give a positive 440, signal when hybridized with aRI-DNA. This figure is derived from the predicted value of 55.000 copies of &RI-DNA per haploid genome and the number of clones. on average 20 kb in size, required to represent every DNA sequence present in the human genome at least once (55,000/2.5 x 109/! x 104). A t,otal of 69, of the clones were found to hybridize wit.11 cloned rRI-DNA. further in t,he library of the family wit.hin the human demonstrat.ing the non-random distribution genome. When six clones containing &RI-DNA were isolated from a genomic library and analysed, two different st’ructural arrangements were seen. One class (three clones) consisted entirely of otRI-DNA sequences in tandem. The second (also of three clones) containing rRI-DNA sequences interspersed in a complex manner with DNA that was not highly repetitive. Clone 2, a member of this second class. gives 1.45 kb fragments on digestion with /CcoKl, and bands of this size are prominent, in the complete EcoRI digest of genomic DNA when hybridized wit,h cloned &RI-DNA: sequences of DNA of 1.45 kb are flanked on one or both sides by xRI-DNA such that the EcoRI sit,e is iu the middle of the 340 bp unit. Thus two different, structural organizations of the rRl-DNA family within t,he human genome have been found. In one arrangement the DNA occurs in tandem arrays. some of which are of at least 22 repeat units in length. The second interspersed with other DNA arrangement contains sequences of this family sequences that are not highly repetitive in nature. We thank Dr Tom Maniatis for the generous gift of his human genomic library, and Dr Peter Little for helpful discussions. This research is funded by the U.K. Medical Research Council. REFERENCES Arrand, J. E. (1978). J. Oen. Viral. 41. 573-586. Benton,

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196. 180- 18%.

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