voiu,-m1o number 21932
`
`Nucleic Acids Research
`
`Simple repeated sequences in human satellite DNA
`
`M.Frommer, J.Prosser, D.Tkachuk, A.H.Reisner. and P.C.Vincent
`
`liancruatsu Memorial Institute, Sydney Hospital, Macquarie Street, Sydney, N.S.W. 2000, and
`CSIRO, Molecular and Cellular Biology Unit, P.O. Box 184, North Ryde, N.S.W. 21 13, Australia
`
`Received 4 November 1981; Accepted 4 December 1981
`
`433%
`
`In an extensive analysis, using a range of restriction endonucleases,
`HinfI and TaqI were found to differentiate satellites I, II and III 5 IV.
`Satellite I is resistant to digestion by TaqI, but is cleaved by HinfI to
`yield three major fragmnts of approximate size 770, 850 and 950bp,
`associated in a single length of Dun.
`The 770bp fragment contains
`recognition sites for a number of other enzymes, whereas the 850 and 950bp
`fragments are "silent" by restriction enzym analysis. Satellite II is
`digested by HinfI into a large number of very sall (10—80bp)
`fragments,
`many of which also contain TaqI sites.
`A proportion of the HinfI sites in
`satellite II have the sequence 5'GA(g)TC.
`The Hinfl digestion products of
`satellites III and IV form a complete ladder, stretching from 15bp or less
`to more than 25obp, with adjacent multimers separated by an increment of
`Sbp. The ladder fragments do not contain TaqI sites and all HinfI sites
`have the sequence 5'GA(A)TC. Three fragments from th Hinfl ladder of
`satellite III have beenTsequenced, and all consist of a tandemly repeated
`Sbp sequence, 5'TTCCA, with a non-repeated, G+C rich sequence, 9bp in
`length, at the 3' end.
`
`INTRODUCTION
`
`in part, be isolated as four cryptic
`Human highly repeated DNA can,
`satellites, each of which is separated from main band DNA by isopycnic
`
`centrifugation in an appropriate density gradient (1-4). Satellites I, II,
`III and IV are A+T rich compared to main band DNA (4) and constitute about
`
`5% of the human genom (5).
`Satellites II, III and IV have been characterised in terms of the
`
`products of digestion with restriction endonucleases EcoRI and HaeIII,
`
`whereas satellite I is resistant to digestion by these two enzymes. Several
`
`fractions of satellites II, III and IV have been identified by digestion with
`
`a "ladder" of fragments which are
`(i)
`EcoRI and HaeIII (6). These include:
`exact multiples in length of a 170 base pair (hp) monmer, (ii)
`various
`
`fragments which do not bear any obvious size relationship to the ladder
`sequences
`fragments, (iii)
`a 3400bp male specific fragment (7, 8), and (iv)
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`that are resistant to digestion by either EcoRI or HaeIII or both enzymes.
`Satellites III and IV have been found to be identical by a range of
`
`criteria,
`
`including restriction enzyme analyses. Satellite II is similar to
`
`satellites III and IV in the size classes of products of digestion with
`EcoRI and HaeIII, but has been distinguished fro satellite III by the
`
`observation that different sequence variants of the l70bp ladder material
`
`are incorporated into satellite II.
`
`The repeated sequences which make up the 170bp ladder in satellites II
`
`and III, particularly the 340bp and 680bp coponents, are present in large
`
`quantities in unfractionated human DN (9).
`
`A 340bp fragmnt and part of a
`
`680bp fragment, fro an EcoRI digest of total B, have been directly
`
`to provide consensus sequences for a portion of the
`isolated and sequenced,
`170bp ladder material (10). This work has yielded no evidence for any
`
`internal repeat within the l70bp monomer.
`
`There is evidence that shorter repeated sequences are also present in
`
`the human DNA satellites. The sequence determined for part of a single
`cloned 1770bp fragment,
`isolated fro an EcoRI digest of satellite III,
`
`shows an obvious, thoug irregular, 4-9bp internal repeat and numerous
`
`HinfI and TaqI sites (ll). Evidence for relatively simle repeated
`
`sequences in satellites I and II has been obtained from DNA fingerprinting
`studies (12).
`
`We report here that preparations of satellites II, III and IV may vary
`
`considerably in their content of the EcoRI or HaeIII 170bp ladder components,
`
`but that all preparations of satellites III and IV contain, as a major
`
`component, a conserved repeated sequence, Sbp in length. This sequence
`
`in HinfI digests of satellites
`appears as a ladder, with a 5bp periodicity,
`III and IV. Satellite II,
`like satellite III, consists mainly of sequences
`
`which are identified by the presence of frequent HinfI sites, but the size
`
`distribution of fragmnts obtained by digestion with HinfI is entirely
`
`In satellite II, unlike satellite III, sequences which contain
`different.
`HinfI sites also contain nuerous TaqI sites. Satellite I is characterised
`
`by a relative lack of Hinfl sites. This satellite contains a sequence which
`consists of sun resistant to restriction enzyme digestion, associated with a
`
`sequence containing clustered recognition sites for a nuber of enzymes.
`
`METHODS
`
`DNA extraction and preparation of satellite Dan
`from male placentae and
`DNA was extracted by the methd of Harmr (13),
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`fro preparations of leukaemic cells obtained fro male patients (with
`
`acute myeloblastic or chronic granulocytic leukaemia) who were undergoing
`leucepheresis for therapeutic purposes.
`Isolation of human satellites I,
`
`II, III and IV, using Ag+ or H9++/CB2 50» density gradients, was carried
`out as previously described (5).
`
`Restriction endonuclease digestion
`EcoRII endonuclease was a gift of Dr. Keith Brown. All other
`
`restriction endonucleases were purchased fro New England Biolabs.
`
`Digestions were carried out under the following conditions:-
`Aval, AvaII, Bg1II, DdeI, HgaI, HindIII, HinfI, Mbol, 5au3AI, Sau96I:
`
`6mM Mqclz,
`6mH Tris-Hcl, pH 7.5, 50mM Nacl,
`0.lmg/ml bovine serum albumin (BSA).
`
`6mM B-mercaptoethanol,
`
`BamHI, SalI, XbaI, X1101:
`
`6m.H Tris-I-IC1, pH 7.9,
`
`150'm.M NaCl,
`
`6111M M9012,
`
`O.lmg/ml BSA.
`
`Fnu4HI, HaeIII, TaqI:
`
`6mM Tris—HCl, pH 7.4, 6mH NaCl,
`
`6mM MgCl2,
`
`6mM B-nnrcaptoethanol, 0.lmg/ml BSA.
`EcoRII, Mspl:
`10mM Tris—BCl, pH 7.5,
`
`6mM KC1,
`
`lOmH Mgclz,
`
`6mM B-mercaptoethanol, 0.lmg/ml BSA.
`ECORI: 100mM Tris-HCI, pH 7.5,
`501135! NaC1, Smfl Hgclz, 0.1mg/ml BSA.
`
`Digestions were carried out for 2 hours at 37°, except for TaqI digestions,
`
`which were carried out at 65°. Reaction mixtures, of total volume 10ul,
`
`contained 0.05-3ug DNA and sufficient enzyme to ensure complete digestion
`
`within two hours. Reactions were terminated by heating to 65° for 5 min,
`followed by rapid cooling to 0°.
`
`Labelling of restriction fragments and gel electrophoresis
`with the exception of HaeIII, all the restriction enzyme used in this
`
`study cleaved double stranded DNA,
`
`to produce single stranded 5' ends which
`
`were then filled in by the action of reverse transcriptase, using one or two
`
`P-nucleoside triphosphates (14). When digests contained a
`appropriate 32
`large number of small fragments,
`the incubation with labelled nucleoside
`
`triphosphate was followed by incubation with an excess (0.1-0.2m) of all
`
`four unlabelled nucleoside triphosphates. The reaction was terminated by
`
`heating to 65°, followed by rapid cooling to 0°.
`
`Restriction fragments were separated by electrophoresis in vertical
`
`gels of 4% or 12% polyacrylamide (O.5m thick) or 1.5% agarose
`
`“P-labelled fragments were visualised by
`(0.8n'.sn or 3m thick).
`autoradiography. HaeIII digestion products were stained with ethidiwm
`
`bromide an visualised under ultraviolet light.
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`Various restriction endnuclease digests of the_plasmid PBR322 were
`
`used as standards (15). when calculating sizes of small, end-labelled
`
`fragmnts, it was necessary to take into account differences between
`
`restriction enzymes in the length of the 5'+ 3' staggered cut and thus in
`the number of nucleoside triphosphate units added by reverse transcriptase.
`
`D Egguencing
`For sequencing, HinfI fragents of satellite III, containing [0'32P]ATP
`at the 3' end of each strand, were eluted from 12% polyacrylamide gels. The
`fragments were denatured,
`the strands were separated by electrophoresis in St
`
`polyacrylamide gels at low ionic strength, and the single stranded DA was
`
`sequenced according to Maxam and Gilbert (16).
`
`In each case,
`
`the more
`
`slowly moving strand was eluted from the denaturing gel and sequenced.
`
`Southern transfers and filter hybridisation
`DA fragents were transferred from 1.5% agarose gels to nitrocellulose
`filters by the method of Southern (17). Hybridisation was carried out
`
`using a ma probe labelled with [a-“ppm: by nick translation (13) .
`
`RESULTS
`
`Satellites I, II and III are extensively cleaved by HinfI
`Satellites I, II and III can be effectively differentiated by comparison
`
`of th products of digestion with HinfI (Figure 1). Satellite I is broken
`down into a number of high molecular weight fragments (Figure la). The gel
`
`shows three prominent bands of approximate size 770bp, 8S0bp and 950bp,
`a small amount of undigested material at the origin and a large band at
`
`about 3500bp, which, on further analysis in 1.5! agarose gels, proved to be
`
`a smear of high molecular weight material.
`
`satellites II and III are
`
`(Figure 1, b and c). There is very little
`almost entirely digested by HinfI
`undigested material, either at the origin or at the 3500bp position. The
`
`low molecular weight fragments in Hinfl digests of satellites II and III
`
`have been further resolved by electrophoresis in 12% polyacrylamide gels
`
`(Figure 2). Satellite II is cleaved by HinfI into a large nuber of very
`
`small fragments (Figure 2b);
`
`the sallest fragments are less than 10bp in
`
`length, and there are very few fragments of size greater than Bobp. The
`
`Hinfl digestion products of satellite III form a complete ladder stretching
`
`fro 15bp or less to more than 250bp (Figure Zj). Adjacent components of
`the ladder are separated fro each other by an increment of Sbp.
`
`Although the Hinfl digests shwn in Figure 2 wre obtained uner
`
`55°
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`"4’ <‘v— ‘'
`
`’-'
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`4*! polyacrylamide
`Figure l.
`gels of [cl-32P]ATP—la.be-lled
`fragments produced by digestion
`of satellites I, II and III
`with HinfI.
`a: Hinfl digested satellite I.
`Fragment sizes,
`in base pairs,
`are marked on the left side.
`b: Hi.nfI digested satellite II.
`c: Hinfl digested satellite III.
`d:
`[<1-32P]C'I'P-labelled
`fragments from digestion of
`PBR322 with MspI.
`Fragment
`sizes,
`in base pairs, are
`marked on the right side;
`these have been adjusted to
`make them comparable in size
`with end—labelled Hi_nfI
`fragments, and give an
`indication of sizes of
`fragments in lanes b and c.
`
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`
`b
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`
`conditions which normally result in a complete reaction,
`
`the satellite III
`
`HinfI ladder has the appearance of a partial digest. However, when samples
`of both satellites II and III were incubated with increased concentrations of
`
`there was no change in the amount of any of the
`HinfI or for longer times,
`satellite III ladder components or the satellite II fragments.
`In addition,
`
`individual HinfI ladder components, eluted from a gel, were not further
`digested by reincubation with an excess of Hinfl. We conclude,
`therefore,
`
`that the digestions shown in Figures 1 and 2 were coplete, and that the
`
`age
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`12% polyacrylamide gels of HinfI and TaqI digestion products of
`Figure 2.
`satellites II and III.
`a and h:
`[u-3zP]CTP-labelled fragmnts fra
`digestion of PBR322 with Hsyl.
`Fragment sizes,
`in base pairs, are marked.
`b: Einfl digest of satellite II, fragmnts labelled with [a-32P]ATP.
`c: HinfI/TaqI double digest of satellite II, fragments labelled with
`[u-32P]AT? and [a-32P]CTP.
`d: Taql digest of satellite II, fragmnts
`labelled with [a—”p]crp.
`e: Hinfl/Taql double digest of satellite :1,
`fragments labelled with [a.-32P]C'I'P.
`f: HinfI digest of satellite II,
`fragments labelled with [cc-"P]C'I'P.
`g and 1:
`[a—32P]CTP-labelled fragments
`from digestion of PBR322 with Sau96I.
`i: HinfI digest of satellite III,
`fragmnts labelled with [a-32P]CTP.
`j: HinfI digest of satellite III,
`fragments labelled with [a-”p]A'm>.
`1;: Taql digest of satellite 111,
`fragments labelled with [a-3zP]CTP.
`
`552
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`fragmnt sizes and concentrations accurately reflect the distribution of
`HinfI sites in satellites II and III.
`
`HinfI, with recognition site 5'G‘ANTC, will cleave four possible
`sequences, and it was of interest to determine whether satellites II and III
`
`could be further differentiated by analysis of the sequence of HinfI sites.
`
`HinfI digests were incubated either with [a—32P]ATP, which will label all
`
`fragments, or with [a-32P]CTP, which will label only fragments produced by
`
`cleaving the sequences 5'GA(g)TC. Satellite III contains virtually no HinfI
`sites with a central
`(g) pair (Figure 2i). Short term autoradiographs
`(15-60 minute exposure), as used in all other HinfI analyses, failed to
`
`reveal any bands in the gels of la-32P]CTP-labelled HinfI digests of satellite
`III. Exposure time of 15 hours or more were required to reveal the HinfI
`
`Sbp ladder in these gels,
`
`indicating that very few of the HinfI sites in all
`
`By contrast,
`size classes of the ladder have the base composition 5'GA(g)TC.
`a considerable proportion of the HinfI sites in satellite II contain a central
`
`in autoradiographs
`(g) pair (Figure 2f). Comparisons of band intensities,
`with increasing exposure times,
`showed that gels of satellite II HinfI
`
`digests contain a few bands which are essentially unlabelled by [a-32P]CTP,
`some bands which incorporate a large amount of the CTP label and a majority of
`
`bands which are labelled to an intermediate extent by [a-32P]CP.
`
`Tgql digestion products of satellites II and III
`DNA sequences which are digested into small fragments by HinfI might be
`
`expected also to contain recognition sites for TaqI(5'T+cGA), since part of
`two adjacent HinfI sites will form a TaqI site. Products of TaqI digestion
`of satellites II and III are shown in Figure 2
`(d and k). Satellite II is
`
`extensively cleaved by TaqI,
`
`to yield a large nuber of small fragmnts,
`
`the
`
`majority of which range from 35bp to l90bp. The double digests of
`satellite II with TaqI and HinfI (Figure 2, c and e) contain very few
`
`indicating
`fragmnts of the sam size as those in the TaqI single digest,
`that most of the sequences which contain TaqI recognition sites also
`
`contain HinfI recognition sites.
`
`A nuber of new bands appear in the gels
`
`including a prominent band of approximately
`of HinfI/TaqI double digests,
`5bp. Satellite III, on the other hand, is almost entirely resistant to
`
`In autoradiographs exposed for 15 hours, a
`(Figure 2k).
`digestion by TaqI
`faint Sbp ladder could be seen in the Taql digest of satellite III, with
`
`fraqmnts of the same size as those in the HinfI ladder. The amount of
`
`material cleaved by TaqI could not be increased by increasing the enzyme
`concentration or the digestion time. Thus, it appears that the resistance
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`of satellite III to digestion by TaqI indicates a real lack of Taql sites
`in this DN.
`
`Cggparison of satellite preparations by digestion with EcoRI, HaeIII and
`HinfI
`
`Figure 3A shows an autoradiograph of a 1.5$ agarose gel in which
`
`[a-32P]ATP—labelled fragments from EcoRI digests of a number of
`satellite II, III and IV preparations have been separated. Although the
`
`170bp ladder can be identified in all preparations,
`
`there is considerable
`
`variation between preparations in the concentrations of the 170bp ladder
`components.
`
`Satellites III and IV:
`
`Seven preparations of satellites III and IV have
`
`been assayed. Three preparations of satellite III, digested with EcoRI,
`yield only faint bands at 340bp and 680bp (Figure 3A, c and i, one
`preparation is not illustrated). One preparation of satellite IV
`
`f) yields a complete and prominent 170bp ladder, with the
`(Figure 3A,
`monomer just visible, and the 2x, 3X, 4x, 5x, 6x,
`7X and BM multimers
`
`present in the gel, along with 2200bp and 3400bp fragments. One
`
`preparation of satellite IV and two preparations of satellite III fall into
`
`an intermediate class, where a nuber of the ladder components can be
`
`detected in low concentrations (Figure 3A, g and h; Figure 4).
`
`A number of othr experimental procedures were used in attempts to
`detect more of the 170bp ladder material in all satellite III and IV
`
`preparations:
`
`(i) Digestion of satellite preparations with EcoRI and
`
`HaeIII, electrophoresis in 4% polyacrylamide and 1.5% agarose gels, and
`
`Southern transfer of fragments,
`(ii)
`staining with ethidium broide.
`1.5! agarose gels of EcoRI and HaeIII digests of satellites III and IV,
`
`from
`
`to nitrocellulose strips,
`
`followed by hybridisation with an
`
`la-32P]ATP-labelled, nick translated satellite IV probe, known to contain
`large amounts of 170bp ladder components.
`(iii) Direct digestion of
`
`[u-3zP]ATP-labelled, nick translated DNA with EcoRI and HaeIII. None of
`thse procedures yielded any evidence of greater concentrations of 170bp
`
`ladder components than those found in the original, end—label1ed, EcoRI
`
`digests shown in Figure 3A. We conclude that different amounts of 170bp
`ladder material are contained in different preparations of the same
`satellite, soetimes from the same individual,
`isolated to the same
`
`standards of ultracentrifugal purity.
`
`Figure 3B shows an autoradiograph of a 4% polyacrylamide gel,
`
`containing [a—32P]ATP-labelled fragmnts from Hinfl digests of the sam five
`
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`H
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`Individual and DNA prep. no:
`X2
`Y
`2
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`
`X1
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`IV
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`XI 2 X?
`
`Figure 3. Variability between satellite preparations.
`A: 1.5% agarose gel of EcoRI digestion products of satellites II, III and IV.
`E:
`4% polyacrylamide gel of HinfI digestion products of satellites III and IV.
`The satellite type is noted below each lane. The individual,
`from whose DNA
`the satellite was prepared,
`is identified by a letter, X, Y or z. Where two
`DA preparations were made from one individual,
`the preparation number is
`identified by a subscript. Fragment sizes,
`in base pairs, are marked at
`either side of the autoradiographs. Lanes d, e and 1 are standard digests
`of PBR322 with EcoRI/PstI, HinfI and Mspl, respectively. All digests were
`end-labelled with la-3zP]ATP except the Mspl standard digest (lane 1),
`which was labelled with [a-3 1>1cr1>.
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`Fragments
`
`Smear
`_
`re none
`
`;;;;’<—-Oflgin
`
`4i polyacrylamide gel of
`Figure 4.
`EcoRI digestion products of satellite
`111,
`labelled with [a—3zP]AT'P.
`Arrows show bands and smear regions
`from which DNA was eluted and
`redigested with HinfI.
`
`J4-
`
`_‘_
`
`L‘
`
`1330—>
`
`350-»
`680—>
`
`340—> T
`
`satellite III and IV preparations, as illustrated in Figure 33 after
`
`digestion with EcoRI. The Sbp HinfI ladder is identical in all five
`A preparations, both in number and relative intensity of bands.
`The
`
`satellite IV preparation which contains high concentrations of both HaeIII
`
`and EcoRI
`
`l70bp ladder material,
`
`shows additional digestion products with
`
`including prominent l70bp and 340bp bands. The other preparations
`HinfI,
`of satellite III and IV show faint 340bp bands and sometimes 170bp bands,
`
`the amount of each reflecting the amount of material digested by EcoRI.
`
`Satellite II:
`
`Two preparations of satellite II, fro different
`
`individuals, also differ in the concentration of repeated sequences cleaved
`
`by EcoRI (Figure 3A, a and b). The sam preparations yielded virtually
`
`identical fragment patterns when digested with Hinfl, with perhaps sam
`small differences in the concentration of three or four larger Hinfl
`
`bands (70-100bp).
`
`Satellite I: Three preparations of satellite I from different individuals
`
`were all resistant to digestion by EcoRI, and showed identical patterns for
`the major products of Hinfl digestion.
`
`Fate of the Hinfl Sbp ladder in an EcoRI digest of satellite III
`EcoRI digestion products of satellite III, separated in a 4%
`
`polyacrylamide gel, are illustrated in Figure 4.
`
`In addition to the bands
`
`detected in 1.5% agarose gels, a concentrated smear of material at around
`
`the 3500bp position and a more diffuse smear of material behind all the
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`To determine the fate of the HinfI Sbp
`larger EcoRI bands can be seen.
`ladder in an EcoRI digest of satellite III, DNA fram each band and smar
`
`region, as marked in Figure 4, was eluted and redigested with Hinfl. The
`
`to yield a
`origin and 3500bp smear were almost entirely digested by HinfI,
`pattern of fragments indistinguishable from that seen in Hinfl digests of
`
`total satellite III.
`
`The Hinfl ladder was also detected in redigested
`
`smar regions from either side of the 1360bp and B50bp EcoRI bands. Table 1
`
`lists the series of bands obtained by digestion of eluted EcoRI fragments
`with HinfI. The redigested 1360bp and 850bp EcoRI fragments contained a
`
`trace of the HinfI 5bp ladder, similar in concentration to that seen in
`
`adjacent smear regions. The relative intensities of smear regions and
`
`bands,
`
`in the EcoRI digest shown in Figure 4, give an indication of the
`
`relative proportions of Sbp HinfI ladder material and 170bp EcoRI ladder
`material in this satellite III preparation.
`
`Repeated ggggence gggpgnents of satellite I
`Preparations of satellites I, II, III and IV were analysed with a
`
`range of restriction enzymes, as listed in the Hthods section. Of the
`
`enzymes tested, only Hinfl cleaved all preparations of satellites III and Iv
`
`to a major extent, and only HinfI and TaqI cleaved both preparations of
`
`satellite II to a major extent.
`
`satellite I was cleaved extensively by
`
`AvaII, DdeI, EcoRII, Fnu4HI, Hinfl and Mbol.
`
`Fragment sizes for
`
`32P-nucleoside triphosphate labelled digests of satellite I with these
`
`Table l: Hinfl digestion products of eluted fragmnts
`fro EcoRI digest of satellite III.
`
`Eluted fragment
`size (bp)
`
`Major products of
`digestion with HinfI
`(hp)
`
`171,169
`
`340
`171,169
`195,l00,50
`
`340
`195,l00,50
`
`Hinfl Sbp ladder
`
`Minor products of
`digestion with HinfI
`(bp)
`
`340
`295,50
`95. 80
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`enzyims are displayed in Figure 5. Double digests were carried out using all
`possible pairs of the five enzymes 1-\vaII, EcoRII, Fnu4HI, HinfI and Mbol.
`
`including undigested origins and 3500bp smear
`In addition, all bands,
`regions, were cut out of 4% polyacrylamide gels of satellite I digested
`
`with Fnu4HI and HinfI. The DEA eluted from these bands was redigested with
`a number of enzymes. These mapping experiments have indicated that the five
`
`enzymes AvaII, EcoRII, Fnu4HI, HinfI and Mbol all cleave the same component
`
`EcoR ll
`
`Sau96 I
`(Ava ll)
`, _0rigin ;_Oriuin V
`
`Fnu4H I
`
`Hinf I
`
`Mbo I
`
`Dds I
`
`" origin
`
`-7 -Origin.
`
`g_'_Origin
`
`;_Origin
`
`---20v:-u:»
`-~2"‘U"=
`
`— «—2m;r.)
`z«I'7'-)0
`
`<
`
`«.«'J«,:
`
`1 <
`
`-ew
`525 j <
`"V"
`
`be
`
`
`
`J?lr)(>
`
`-/—/mm: I
`
`‘ «"?’!<fl'-
`.
`
`_4._,
`
`1 OF
`
`_ r
`
`4% polyacrylamide gels of restriction endonuclease digestion
`Figure 5.
`products of satellite I. Fragments were end-labelled with appropriate
`32P—nuc1eoside triphosphates. Fragent sizes are given in base pairs.
`Sizes of large fragmnts (1000-3000bp) were determined by electrophoresis of
`satellite I digests in 1.5% agaross. All gels also contain an approximately
`3500bp band, which indicates an accumulation of undigested material.
`Fragments which have been located in the mapped segment of satellite I are
`marked with arros. The location of the remainder of the fragments is not
`known.
`
`- 5
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`of the satellite. This component appears to consist of a region which is
`
`"noisy" by restriction enzyme analysis, associated with a "silent" region.
`The "noisy" region is bounded by two Hinfl recognition sites, 770bp apart,
`and contains recognition sites for the four other enzyme studied.
`A
`
`preliminary map of the Hinfl fragments of satellite I is provided in
`
`Figure 6; moredetailedsequence analysis of satellite I will be provided
`in a later publication.
`
`Base sequence of Hinfl restriction fragments of satellite III
`Base sequences were determined for three fragmnts, of approximate
`
`length 4Sbp, 50bp and 100bp,
`
`from the Hinfl Sbp ladder of satellite III.
`
`The base sequences are presented fro the 3' end, for the first 42 bases of
`
`the 45bp fragment,
`
`the first 47 bases of the 50bp fragment and the first
`
`91 bases of the l0Obp fragent (Figure 7). The exact sequences at the 5' end
`
`of the fragments were not determined and therefore the exact size of each
`fragment is not known. All three sequences consist of a tandemly repeated
`
`unit, 5'TTCCA, with an apparently identical, G+C rich sequence,
`9bp in length, at the 3' end of each fragment.
`The 100bp fragment is
`clearly a dimer, consisting of two nearly identical subunits, one of which
`
`is 49bp in length. The 9bp G+C rich sequence is present twice. The
`
`525
`
`,es ,
`
`5’cG('%)cc
`Ava ll
`Sau96 u 5'GGNCC
`*—x EcoR u
`s'cc($)Gc.
` — Fnu4H I 5’GCNGC
`
`—-—-—:"°jj—=-—- Hinf I
`
`5'GANTC
`
`-——+——:o59o——— Mbol
`
`5'GATC
`
`A
`
`B
`
` —-
`\_____________,___.___————J
`
`Fnu4H I --2000bp fragment
`contains Hinfl
`I50 & 950bp fragments
`
`Figure 6. Restriction site map of satellite I.
`A: Recognition sites and fragment sizes for individual enzymes.
`B: Restriction site map of satellite I for AvaII/Sau96I, EcoRII, Fnu4HI,
`Hinfl and MboI.
`
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`Fragment
`size
`
`45bp
`
`50bp
`
`w
`5
`w
`5*
`mi
`5*
`m
`5
`3'G-—GGG--CIACCTTIACCTT|ACCT-IACCTTIACETTIACCTTIACC
`
`5
`10
`Z)
`25
`30
`5
`so
`#5
`3'GccGGGcTc|AcgTg|@ccTT|AccTT|AccTT|AccTT|Ac§TT|AccTT|Acc
`
`5
`
`10
`
`1.5
`
`Z0
`
`Z5
`
`30
`
`5
`
`H0
`
`'05
`
`100bp
`
`3'G--GGG--CIACQTIACCTTIACCT-IACCTTIACCTIACCTTIACCTTIACCTT
`5D
`55
`60
`E5
`70
`75
`BO
`5
`93
`
`GG—GGG---IACCTTIACCTTIACCTT|ACCTT|ACCTT[A-—TT|ACC
`
`Figure 7. Base sequences of the 45bp, 50bp and 100bp Hinfl fragmnts of
`satellite III. The Sbp monomers, 5'TTCCA, are marked and positions where
`the consensus sequence has clearly diverged fro the monmr sequence are
`underlined. Positions where the consensus sequence could not be easily
`read are left blank. Positions in the 45bp sequence where a single base
`change would generate a HinfI and TaqI site are marked with arrows.
`The 100bp fragmnt is presented as two tandem repeats of 49bp each.
`
`sequence at the junction between the subunits is 5'éETCéKi 50 the
`absence of an A betwen positions 49 and 50 explains the lack of a Hinfl
`site at the centre of the dimer.
`
`DISCUSSION
`
`Criteria of purity of satellite preparations
`The data presented here confirm that the human satellites I, II and III
`are distinct DNA fractions. All satellite preparations will contain a
`
`certain proportion of contaminating repeated sequences; for instance,
`satellite I contains a very small amount of the satellite III HinfI ladder,
`
`and EcoRII digests of all satellites may contain very small amounts of a
`
`series of fragmnts which appear as prominent repeated sequence components
`
`In addition, variable amounts of the
`in EcoRII digests of total DA.
`repeated sequences which form the EcoRI and HaeIII 170bp ladders are
`
`isolated in satellites II, III and IV. The l70bp ladder forms a major part
`of the repeated sequences in total human DNA, and appears to consist of a
`collection of related sequences, variants of which have different
`chranosoal locations (19) and may be isolated in different satellite
`
`preparations (6).
`
`We have observed that sequence variants, which differ in
`
`nuber and position of Hinfl sites (Table 1), may be isolated in sate111te 111,
`The 170hp ladder material cannot be termed a contaminant of satellite
`
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`preparations, since the relative amount of the ladder fragments in BcoRI
`digests does not correlate with the relative purity of satellite
`
`preparations, as determined by buoyant density analysis, with subsequent
`curve analysis of the resultant DNA peak. Preparation of satellites
`
`enriched for these sequences has proved to be a useful method for studying
`
`them (6,19). However, we locate the 170bp ladder material as a relatively
`
`minor component in most satellite II, III and IV preparations, and its
`
`presence cannot be used as a criterion of purity for any satellite
`preparation.
`
`Restriction enzym analysis of satellite I
`No data from restriction enzyme analysis of human D satellite I have
`
`been published to date. The restriction site data for satellite I,
`
`presented here, allow a coarison of satellite Iwithsatellites II and III.
`Satellite I has been classed as one of a “family of A+T rich“ (FAT)
`
`satellites, with simple sequence characteristics,
`
`in human DA (10,12).
`
`The lack of restriction enzyme sites in large portions of the satellite
`
`supports the DNA fingerprinting data (12), that satellite I consists
`
`of a simple sequence or a set of simple sequences. Yet this A+T rich
`
`satellite contains a region with frequent sites for a number of
`
`restriction enzymes with related, G+C rich, recognition sequences -
`
`The sequence
`5'GCNGC.
`AvaII: 5'GG(;)CC, EcoRII: 5'CC($)GG, Fnu4HI:
`relationships between the "noisy" region and the adjacent "silent"
`
`regions of satellite I remain to be established.
`
`Or anisation of s
`
`le s
`
`ences in satellites II and III
`
`Satellite III has been characterised in terms of the short
`
`repeated sequence, 5'TTCCA, which is a major component of all
`satellite III and IV preparations.
`The sequence can be isolated from
`
`Hinfl digests of the satellite, where it appears as a ladder of
`
`fragments, with adjacent fragments separated by an increment of Sbp.
`
`The presence of HinfI sites within the Sbp repeated sequence is not
`
`surprising; only one base substitution is required for each HinfI site,
`
`5'TTCCATTCCA+5'TTCGATTCCA. However,
`
`the lack of TaqI sites in the
`
`satellite III Hinfl fragmnts is unexpected, since such single base
`
`substitutions must yield one Taql site (5'TCGA) for every HinfI site.
`
`The lack of TaqI sites presumably indicates that any sequence change
`
`leading to the generation of a HinfI site in the Sbp repeated sequence
`
`has occurred as part of a larger change in the region immediately 5'
`
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`to the newly generated Hinfl site.
`
`In the three fragments sequenced,
`
`this change has involved the insertion of an 8 or 9bp, G+C rich sequence
`into the Sbp repeated sequence,
`immediately 5'
`to the HinfI site.
`
`Further studies will establish the distribution of the 9bp sequence
`
`in all fragent size classes of the HinfI ladder, and thus yield
`information about the origin and relationships of the ladder
`
`compo