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Localization of repetitive and unique DNA sequences neighbouring the rabbit β-globin gene
Localization of Repetitive and Unique DNA Sequences Neighbouring the Rabbit /?-Globin Gene H.
.\.
X.
F~EIJMAKERS-VAS
DOMMELEX>
R. ii.
G.
C.
Section for Medical Enzymology and Molecular Laboratory of Biochemistry Uwiversity of Amsterdam Eerste Constant(jn Huygensstraat 20 Am.sterdam, The Netherlands
J.
E.
GROSVELD-1,
DE
BOER
FLAVELLt
M.
Biology
VARLEY
Department of Zoology University of Leicester University Road L&ester, England
A.
J.
JEFFREYS
Department of Genetics Unireraity of Leicester University Road L&x&r, England (Received
7 December
1979, and in revised form
17 March
1980)
DNA fragments containing the rabbit j-globin gene can be detected in restriction hybridizations with a labelled endonucleasc digests of rabbit DNX, usin, (I filtrr /?-globin cDNA plasmid as a probe for thr structural gene. -Analysis of the rea,mealing kinetics of these P-globin DNA fragments has been performed by denaturing rabbit DNA and renaturing t,o variolls C,t$ values, then detjermining by hydroxyapatite chromatography and subsequent filter hybridization whether a given P-globin DNA fragment, has anrlcnlcd bvith otller DKa via a rrpctitixrc DKLA tract contained in the fragmetlt, or instead has remained completely singlestratlded. This approach has revrxaled the presence of an intermrdiate repetitive DK.4 sc~~uonce about 0.6 to 2.4 kb from the 3’ end of the rabbit P-globin structural gone. The presence of t’his repetitive scquet~cc was confirmed by filter hybridization analysis of rabbit DNA4 with x,arious DNA fragments isolated from t Present,
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a cloned segment of rabbit genomic DNA containing the P-globin gene. A purified rabbit DXA4 fragment cont,aining sequences 0.9 to 2.1 kb from the 3’ end of the structtwal gene was found to hybridize to many DNA fragments in restriction endonttclease digests of rabbit DKA. 1~1 sit/l hybridizations t)o rabbit. metaphase chromosomes showed that copies of this repctitil-r sequence xere present on several chromosomes, and were preferentially localized in centromeric and telomeric regions. In contrast, the remaining sect,ion of the rabbit /%globin gent region examined, extending 1.4 kb from the 5’ end of the P-globin gene to 0.9 kb from the 3’ end of the gene and including the inter\-erring sequences withill the structural gene, was shown to consist of single copy DNS.
1. Introduction The pioneering work of Britben and his colleagues (Britten & Kohne, 1968) has demonstrated that eukaryotic genomes contain various classes of reiterated DNA in addition to DNA sequences present in only :a single copy per haploid genome. Highly repeat,ed sequences are found tandemly arranged (Southern, 1974) and commonly consist of simple DNA sequences (Sout hem, 1970). \vhich appear to be transcriptionally inactive (Walker, 1971) and restricted to heterochromatin (Jones, 1970; Pardue $ Gall, 1970). In addition, certain genes such an those coding for ribosomal RNAs (Birnstiel et al., 1971) and sea urchin histones (Birnstiel et al., 1974) are also found repeat,ed in tandem arrays. Much of the repetitive DNA with reiteration frequencies ranging from lo2 to lo5 appears t’o be spread throughout the eukaryotic genome, a,nd interspersed among single-copy DN’B sequences in a pattern that is apparently similar in many different organisms (Davidson et aZ., 1974). There is evidence that some of these repetit’ive sequences are linked to structural genes in the DNA of echinoderms (Davidson et al., 1975), a’nd evidence exist’s for a low frequency repetitive DNA segment in t)he vicinity of duck haemoglobin genes (Bishop & Freeman, 1974). Various authors have postulated that repetitive DNA sequences, linked to structural genes, might be involved in the control of gene expression (Britten & Davidson, 1969; Paul, 1972; Georgiev et al., 1974). To learn more about the possible role of repetitive DNA in the regulation of gene expression, it is essent’ial to know the types and locations of various repetitive DNA sequences which might be found near a given single-copy structural gene. In this paper we analyse the types of repetitive DNA that are found close bo the rabbit /3globin struct’ural gene. This approach has been made possible by the construction of a physical map of restriction endonuclease cleavage sites in and a,round this gene (Jeffreys & Flavell, 1977a;b) using a 32P-labelled P-globin cDNA plasmid (plasmid PpGl : Maniatis ft al., 1976) as a filt,er hybridization probe for rabbit DNA restriction fragments containing a /3-globin gene. The search for reiterated sequences has also been aided by the cloning of a fragment of rabbit’ DNA containing the /%globin gene by Van Den Berg et al. (1978). We have previously shown that a repetitive DNA segment is located to the 3’ side of the rabbit P-globin gene, and t,hat the DNA4 sequences next to the gene are present in less t’han 100 copies per haploid genome (Flavell et al., 1978a). We were interested in determining whether the DNA sequences flanking the jl-globin gene, and in particular on the 5’ side, are present in multiple copies per cell, as might be anticipated according to the gene regulation model proposed by Britten
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C%Davidson (1969). In addition, the rabbit ,%globin gene contains two intervening sequences (Jeffreys & Flavell, 1977b; Van Den Berg et al., 1978), and we were interested to learn whether these sequences are found only within the /3-globin gene or whet’her they are also present elsewhere in the genome. Our approach has shown that the 5’ extragenic region and the intervening sequences within t,he rabbit /3-globin gene consist of single copy DNA. In contrast, a region 0.9 to 2.1 kb-1 from the 3’ end of t#he structural gene contains a repet)itive DNA segment which is present in about 5000 copies per genomc.
2. Materials
and Methods
(a) Materials 1,ivt.r DSA was purified from an F1 hybrid between Alaska and Vienna White strains of rabbit, as described by Jeffreys & Flare11 (1977b). Purified PgGl DNA and Z-pCRI/ Rchr,6G-1 DNA (the latter being abbreriat,ed to R/?Gl in this paper) were provided by Professor C. \Veissmann, Inst,itut fur Molckularbiologie, Zurich. Restriction endonucleases EcoRI and PstI were purchased from the Medical Research Establishment, Porton Down, U.K. Endonucleases BumHI, HaeIII, HhaI and PvuII were purchased from New England BioLabs Inc. Endonuclease KpnI was prepared by t,hr method of Crawford & Robbins (1976). (b)
Filter
hybridization
analysis
of rabbit
P-glob&
USA
Restrictiolr endonuclease digestions of rabbit’ liver DNA, agarosc digest products, transfer of DNA t’o nitrocellulose filters by blotting, filt,ers wit’h 32P-labrlled PpGl DNA or 32P-labelled DNA fragments tion endonuclease digests of RPGl DNA \vere performed as described (1977w,b). (c)
Reassociation
analysis of P-glob& endonuclease digests
DLXA fragments of rabbit DSA
gel electrophoresis of and hybridization of isolated from restricby Jeffreys $ Flavrll
in restriction
Reassociation of digested DNA was performed at DNA concentrat,ions of 0.2 mp/ml to 10 mg/ml in 0.6 ar-sodium phosphate (pH 6.8), by heat denaturation (lOO”C, 5 min) followed by rapid cooling t,o 50°C and incubat,ion to the desired C,t value. Samples containing 30 pg DNA were then diluted to 0,12 >I-sodium phosphate and applied to a l-ml hydroxyapatite (Biorad HTP) column maint’ained at, 60°C. Completely single-st’randed DNA was elut,ed bj- washing with 10 column I-olumes of 0.12 >I-sodinm phosphate at 60°C. Duplex DNA was vluted by a wash witjh 5 column \-olumes of 0.4 ar-sodium phosphate at 60°C followed by 5 column volumes of 0.4 fir-sodium phosphate at 100°C to rccovor DNA present in networks. Column fract)ions were dialysed extensively against 10 mlsr-Tris .HCl (pH 7.5) for 3 days at) 4”C, concentrated by extraction of water with butan-2.ol, and DNA collected by precipitation with et,hanol. Samplrs were then denatured with alkali and fractionated by electrophoresis in agarose gels, DNA fragments were t,ransferred to nitrocellulose filters by blotting and P-glohin DNA-containing fragments detected by filt’er hybridization with 32P-labelled P/3Gl DN.1. All C,t values llave been corrected to t’he standard 0.12 ar-sodium phosphate. 6O’C condit,ions using the tables of Brieten et al. (1974). (d)
In situ
hybridization
Heparinized venous blood from an adult rabbit was l(PM1 1640 (Gibco Bio-Cult) medium supplemented strum and 2’;; (v/v) Bactophytohaemagglutinin M 0.4 &ml and the incubat’ion continued for 14 h. Cells -1 See r00tr10te
to ,,. 531.
cultured for 96 h at 37°C in 10 vol. with 10% (v/v) neonatal calf solution. Colchicine was added to were collected by centrifugation at
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100 g for 10 mm, suspended in 75 rnl\I-KCl, incubat,ed at 37°C for 20 min and recentrifugcd. The pellet was resuspended in a small volume of KC1 and fixed in 3 changes of ice cold 3: 1, methanol/acetic acid. Splash preparations were made onto glass microscope slides. Chromosome preparations were prepared for in situ hybridizations following the basic method of Gall & Pardue (1951). RNA was removed by incubating slides in 50 pg ribonuclease A/ml plus 10 units ribonuclease T,/ml in 2 x SSC at 37°C for 1 h (SSC is 0.15 Jr-NaCl, 15 mr\l-trisodium cit’rat,e, pH 7.0). Slides were then washed in 3 changes each of 2 x SSC, 700/, ethanol and 95% ethanol, arid air dried. C’hromosomal DNA was denatured in 70 rnsr-NaOH at room temperature for 3 mm, followed by washing in 700/, and 9576 ethanol and air drying. DNA samples to be used as probes were labelled with eH by nick t,rauslatioii using the method of Maniatis et al. (1975) as modified by Macgregor & Mizuno (1976). Each slide was liybridized under a cover slip for 12 h at, 37°C with 30 ~1 5Oe/, (v/x-) formamide, 4 :< SSC contaming 0.1 pp eH-laballed DNA ( IO6 to 2 x lo6 &s/mm per pg DNA), which had previously been denatured for 15 rnin in 0. I 31.NaOH and neutralized with HCl. Unbound labelled DNA was removed by washing with 3 changes of 2 x SSC at room temperature, 3 changes of 2 x SSC at 65°C. once in’2 x SSC plus 5oj (W/X-) t,richloroacetic acid for 5 min at 4”C, 3 times in each of 2 x SSC. 70% ethanol and 95% ethanol, and air dried. Slides were dipped in Kodak NTB-2 nuclear emulsion. dried and autoradiographed for 1 to 9 weeks. After developing and fixing. slides were stained with 294 Giemsa (Gurrs R-66) for 10 min and air dried.
3. Results (a) Reassociation analysis of P-glob& DNA fraqrnents: an intermediate repetitive sequence on the 3’ side of the gene JVe have described elsewhere how WC can specifically det,ect DnTA fragments, which contain the /3-globin (present in PPGl DKA). m rabbit DXA digested with various restriction endonucleases and how these fragments can be ordered into a physical map of cleavage sites within and around the P-globin structural gene (Jeffreys & Flavell, 1977a,b). This map (Fig. 1) shows the direction of transcription and the presence of two intervening sequences within the DNA sequences coding for /3-globin. Transcription
4
FIG. 1. A physical map of restriction endonuclease cleavage sites within and neighbouring the rabbit p-globin structural gene. Cleavage sites are shown for endonucleases LlnmHI (B), EcoRI (E), KpnI (K) and PatI (P). Regions coding for ,L-globin mRNA sequences are indicated by hatched boxes and intervening sequences by open boxes. The calibration scale in kb is contred on the int,ragenic RalnHI cleavage site. The derivation of this map is described elsewhere (Jeffrey5 & Flavell, 1977a,b; Van Den Berg at ccl., 1978). Although cleavage sites for a number of other restriction endonucleases have also been mapped, these are omitted here for the sake of clarity.
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To determine the presence of a repet’itive DNA sequence in a /Lglobin DNA4 fragment,, rabbit liver DNA digested with the appropriate restriction endonuclease was denatured and reassociated at 70°C in 0.6 M-sodium phosphate (pH 6.8), to the desired C,t value. Duplex DNA was then separated from completely single-stranded DNA by chromatography on hydroxyapatite at 60°C under the standard conditions described by Britten & Kohne (1968). The duplex and single-stranded DNA fractions were recovered, denatured with alkali and analysed for /?-globin DNA fragments by agarose gel electrophoresis, transfer of DNA fragments to a nitrocellulose filter by the method of Southern (1975) and filter hybridization with 32P-labelled Pj3Gl DNA (Flavell et al., 1978a). An estimate of repetition frequency of the sequences contained within a given restriction fragment can be determined by comparing its rate of annealing with that of single-copy DNA. In t’he following discussions all C,t values are corrected to the values for DNA annealing in 0.12 M-sodium phosphate (Britten et al.. 1974). When rabbit DNA digested with endonuclease EcoRI was denatured and reannealed, both the 2.5 kb and 0.9 kb /I-globin DNA fragments, which contain the 5’ and 3’ halves of the /?-globin gene, respectively (Fig. l), remained essentially single-stranded up to C,,t = 100 (Fig. 2). 0 n annealing to C,t = 1000, some of each fragment appeared in the duplex DNA fraction. As a control for the reassociation rat.t: of unique sequence DNA, we added to the rabbit DNA digests an amount, equivalent to a single gene copy, of a 1.5 kb plasmid vector PMB9 DNA fragment isolated from P/3Gl DNA which had been cleaved with EcoRI and BvaI (Jeffreys & Flavell, 1977a). This internal marker fragment reannealed with a C,t, of about 1000. (Fig. 2). The 3’ 0.9 kb EcoRI p-globin DNA fragment reannealed with similar kinetics, suggesting that this fragment of the rabbit genome contains only single-copy DNA. The reassociation rate of the 5’ 2.5 kb EcoRI /Lglobin DNA fragment was slightly higher than that of the 3’ EcoRI fragment and at C,t = 1000 it was mainly found in the duplex DNA fraction. This increased reannealing rate may be due to the length of this EcoRI fragment (2.5 kb compared with 1.5 kb for the single-copy marker; reannealing rates are proportional to the square root of DNA fragment length, see Wetmur & Davidson, 1968). The 3.6 kb fl-globin DNA fragment generated by cleavage of rabbit DNA with KpnI + PstI contains the entire @-globin gene (Fig. 1). In contrast to the EcoRI fragments, this component reannealed rapidly and almost completely entered the duplex DNA fraction on passing from C,t = 0.1 to C,t = 1 (Fig. 2). Hence this fragment must contain a repetitive DNA segment. Since neither of the EcoRI fragments contains this repetitive sequence, this places the intermediate repetitive element somewhere between the EcoRI and KpnI sites 0.6 to 2.4 kb on the 3’ side of the gene (Fig. 1). This result confirms and extends our previous observation that the 4.9 kb KpnI /3-globin DNA fragment, which also carries this region, was completely annealed at C,t = 60 (Flavell et al., 1978a). (b) Analysis
of rabbit
genomic
Analysis of the reannealing kinetics showed that a stretch of DNA from
Dh’d
sequences homologous DNA
to cloned R@l
of p-globin DNA fragment’s in total rabbit DNA 1.4 kb 5’ from the structural gene to 0.6 kb 3’
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FIG. 2. Reassociation analysis of P-globin DNA fragments grncrated by cleavage of rabbit DSA with endonucleases EC&I or KpnI + PstI. Rabbit liver DNA digests were heat-denatured and reannealcd to the indicated C,t values (corrected to the rate of annealing in 0.12 &I-sodium phosphate). Completely single-stranded DNA was separated from DNA containing double-strantletl regions by chromatography on hydroxyapatite. Single-stranded and double-stranded fractions derived from 30 pg of total DNA were alkali denatured, electrophoresed through a I.‘>“’i ,,, agarose go1 and transferred by blotting to a nit,rocellulose filter. ,t?-globin DNA fragments were detected by autoradiography after hybridization with 3ZP-labelled PgGl DNA (see Jeffreys & Flavoll, 1977n,b and Materials and Methods). in. = 30 pg unfractionatctl DNA digest: ,11 : marker DNA fragments (10 pg PpGl DNA x Hind111 (5.6 kb fragment) plus 25 pg P/3Gl DNA x EcoKI x dvcr1 x NindIII (2.3, 1.5, 1.2, 0.65 kb fragments; see Jeffreys & Flavell, 1977rr)). A single copy (15 pg) of a 1.5 kb PflGl DSA x EcoRI x i4cnI fragment, containing only plasmid vrctor PMB9 sequences was added to each 30 pg batch of rabbit liver DNA x EcoRI prior to t,hc, reassociaCon analysis.
from the gene and including the gene plus int’ervening sequences behaved as low repetitive or single-copy DKA. To confirm that these are indeed single-copy sequences, we have studied the hybridization bet’ween these sequences. isolated from a cloned fragment of rabbit DPU’A, and total rabbit, genomic DNA. A number of restriction endonuclease digest fragments of rabbit DNA were isolated from the recombinant plasmid R@Gl which contains the 4.9 kb KpnI ,!?globin DNA fragment shown in Figure 1 (Van Den Berg et al., 1978). The origin of t,hese purified fragments is shown in Figure 3. These fragments were then labelled with 32P by nick translation and used as probes in filter hybridizations with restriction endonuclease digests of total rabbit DNA. After hybridization, filters were given a high stringency wash in 0.1 x SSC at 65°C and remaining well-matched hybrids were detected by autoradiography (Fig. 4). Probes 1, 3, 4 and 5, which contained, respect-
BEPETITIVE
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Transcription
HhHM
M
5
6
FIG. 3. A physical map of a cloned segment of rabbit DKa containing the 8.globin gene, showing regions purified for use as probes for repetitive DNA sequences. The 4.9 kb Kpnl DNA fragment containing the 8.globin gene (see Fig. 1) has been purified by cloning into the EcoRI cleavage site of plasmid pCR1 to produce the recombinant plasmid RPGl (Van Den Berg et nZ., 1978). The map shows ,&globin structural gene sequences (hatched boxes), intervening sequences (open boxes) and pC’R1 sequences (solid boxes) flanking the cloned segment of rabbit DNA. Cleavage sites are shown for endonucleases HnmHI (B), EcoRI (E), Hat211 (H), JlspI (XI), P&I (P) and Pm11 (Pv). In addition, the positions of two HhnI (Hh) cleavage sites in pCR1 are shown; there are no HhaI cleavage sites in the rabbit DKA segment. The mapping of BnmHI, EeoRI and I’stI cleavage sites are described elsewhere (Jeffreys & Flavell, 1977a,b; Van Den Berg et nl., 1978). Cleavage sites for HueIII. MspI and PvuII in the cloned rabbit DSA were determined by th(, method of Smith Er Birnstiel (1976) (J. Van Den Berg, A. Van Ooycn and G. C. Grosveld, unpublished results). The origins of purified fragments of rabbit DNr\ used as probes for repetitive DX9 sequences am shown below the map. These probes were isolated from the rabbit DiYa4 fragment purified from a HhrT digest of RPGl DNA. Probes 1 and 3 t,o 6 Were purified from a HamHI -- HneIII double digest of this fragment by polyacrylamide gel elcct,rophoresis. using the procedure of Mazam & Gilbwt (1977). Probe 2 was similarly purified from a ~wrIT digest.
ively, 1.4 kb of 5’ extragenic sequences, coding sequences plus the small intervening sequence, coding sequences plus the large intervening sequence, and 0.9 kb of 3 ’ extragenic sequences, all detected only one 4.9 kb fragment in KpnI digests of rabbit DNA. Since this is the fragment cloned in RpGl, this suggests that probes 1 and 3 to 5 all consist of unique sequence DNA. The apparent lack of cross hybridization between these probes and additional rabbit DNA sequences is confirmed by analysis of PstI and EcoRI digests of rabbit genomic DNA (Fig. 4). As predicted, probes 1, 3 and 4 detect only the 2.5 kb EcoRI 5’ ,!I-globin DNB fragment, whereas probe 5 detects the 0.9 kb 3’ EcoRI fragment plus an addit’ional 1.8 kb fragment which presumably maps adjacent to the O-9 kb fragment. Similarly, probes 3, 4 and 5 all detect the single 6.7 kb Pstl /3-globin DNA fragment. In contrast, probe 1 maps to the 5’ side of this PstI fragment and detects instead a single new PstI fragment 13 kb in length. Finally, probe 2 detected the same DNA fragments as probe 1 (data not shown). The map positions of DNA fragments detected by probes 1 to 5 are shown in Figure 5. In all cases, these probes detect DNA fragments originating only from the /3-globin and surrounding sequences, demonstrating that these probes contain only single-copy DNA. 21
kb
15
Probe
:
AK
E
P
K
3 E
P
,2-----L* K
E
P
K
E
P
6 K
FIG. 4. Analysis of rabbit DNA fragments containing sequences homologous to DNA near the rabbit j3-globin gene. Rabbit DNA was digested with endonuclease PstI (P), KpnI (K) or EcoRI (E). 20 pg batches of DNA were alkali denatured, electrophoresed through 1% agarose slab gel and transferred by blotting to a nitrooellulose filter. Identical filters were then hybridized under non-stringent conditions (65°C in 3 x SSC for 2 days) with probes 1, 3, 4, 5 or 6 (Fig. 3) which had been labellcd with 32P by nick translation. After hybridization, unbound labelled DNA was washed from the filters, which were subsequently given a wash under stringent conditions (65°C in 0.1 x SSC). Labelled bands of DXA were detected by autoradiography. 411 hybridization and washing procedures were carried out as described by Jeffreys & Flavell(1977b).
P
E
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Transcription *
I kb I
I P
K E
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Probe
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5
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, P 6.7
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5. A physical fragments detected of DNA which have FIG.
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_I
Probe fragments
6
detected
map of DNA sequences near the rabbit p-globin gene, showing genomic DRA by probes isolated from sequences near the rabbit j%globin gene, plus regions been shown to contain unique or repetitive DNA sequences.
To investigate the possibility that the rabbit genome contains additional DNA sequences related to, but not identical with, sequences in probes 1 to 5, we hybridized these probes with filters containing restriction endonuclease digests of total rabbit DNA under conditions of low stringency, then washed the filters under increasingly stringent conditions by lowering the ionic strength of the washing solution. A typical result for probe 1 is shown in Figure 6. As the stringency is lowered, no additional labelled bands are detected until the lowest stringency (1 x SSC at 65°C) is reached, when the entire track of rabbit DNA digest shows a uniform labelling due to aspecific hybridization between probe and rabbit DNA (see Jeffreys $ Flavell, 1977a). Traces of a small number of additional discrete labelled components could be seen. Thus probe 1 contains essentially unique sequence DNA, although it is possible that part or all of this probe may consist of sequences present in a very limited number of 21*
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x K
E
kb 13 B-
Probe
I
Probe
6
4.9,
FIG. 6. Analysis of the stability of hybrids formed between total rabbit DNA and purified DNA sequences near the rabbit p-globin gene. 15 pg batches of rabbit DNA were digested with endonuclease PstI (P), KpnI (K) or EcoRI (E), electrophoresed through an agarose gel and transferred to a nitrocellulose filter. Identical filters probe 1 or probe 6 (Fig. 2). were hybridized in 3 x SSC at 65’C for 2 days with 32P-labellad Unbound label was removed and the filters were then given a more stringent wash in 1 x , 0.3 x or 0.1 x SSC at 65°C. Labelled bands were detected by autoradiography.
REPETITIVE
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additional diverged copies elsewhere in the genome. Similar results were obtained for probes 2 to 5 (data not shown). In contrast, probe 6 which contains sequences 0.9 to 2.1 kb from the 3’ end of the globin gene hybridizes at high stringency with many DNA fragments in restriction endonuclease digests of rabbit DNA, and labels the entire DNA track in filter hybridizations (Fig. 4). This DNA fragment must therefore contain a repetitive DNA sequence. This is consistent with the reassociation kinetics analysis which revealed a repetitive sequence somewhere within a region 0.6 to 2.4 kb from the 3 ’ end of the /3-globin gene. At lower stringencies, the labelling of rabbit DNA by probe 6 becomes considerably more intense (Fig. 6), and exceeds by orders of magnitude that seen with a single-copy probe. It is not known whether this is due to hybridization with related rather than identical repetitive sequences, or instead is due to artifactual loss of wellmatched hybrids in high stringency post-hybridization washes. (c) In situ hybridization of 3H-labelled R/&ICI DNA with rabbit metaphase chromosomes To identify the chromosome localization of the repetitive DNA sequences homologous to the sequence element on the 3’ side of the rabbit fl-globin gene, we hybridized rabbit lymphocyte metaphase chromosomes in situ with 3H-labelled R/?Gl DNA. Parallel hybridizations with 3H-labelled pCR1 vector DNA were performed to control for artifactual in situ hybridization between vector DNA and rabbit chromosomes. Similarly, 3H-labelled P/3Gl DNA containing rabbit ,Q-globin cDNA was used as a probe to control for hybridization to unique sequence DNA. After hybridization. unbound label was removed by a low stringency wash (2 x SSC at 65°C). As shown in Figure 7, 3H-labelled R/3Gl DNA labels a number of rabbit chromosomes, with a very marked preferential labelling of centromeric and telomeric regions. 89% of silver grains were found in these areas rather than in interstitial regions (Table 1). In contrast, neither pCR1 nor P/3Gl DNA label the metaphase preparation (data not shown), showing that the hybridization of chromosomes with RpGl DKA is due neither to cross-hybridization with vector nor to hybridization wit,h cloned rabbit single-copy sequences. The observed labelling pattern must therefore reflect, the chromosomal distribution of copies of the repetitive DNA element found on the 3’ side of the rabbit /?+globin gene.
4. Discussion Filter hybridization analysis of total genomic DNA and the cloning of specific nuclear DNA fragments have greatly aided the analysis of globin gene structure and the linkage arrangement of related globin genes (see, for example, Jeffreys $ Flavell, 19773; Tilghman et al.: 19783; Van Den Berg et al., 1978; Flavell et al., 19733; Lawn et al., 1978; Little et al., 1979). In the present paper, we show how both approaches can be used to analyse the interspersion of unique and repetitive DNA sequences at the fine structural level, and to investigate the linkage relationship between structural gene sequences and repetitive DNA elements. The region of the rabbit genome studied includes extragenic DNA sequences both 5’ and 3’ from the major adult p-globin gene, plus the coding and intervening sequences
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FIG. 7. 1n situ hybridization of rabbit metaphase chromosomes with sH-labelled RpGl DNA. Metaphase chromosomes were prepared from phytohaemagglutinin-stimulated rabbit lymphocytes, and hybridized with 3H-labelled RpGl DNA in 4 x SSC, 50”/: formamide at 37°C. Unbound label was removed by washing in 2 x SSC at 65”C, and label detected autoradiographically (9 weeks exposure). Chromosomes were subsequently stained with Giemsa. Typical labelled centromeres (c) and telomeres (t) are indicated by arrows; in some cases, it is not possible to distinguish between centromeric and telomeric labelling on acrocentric chromosomes (open arrow). Scale bar represents 10 pm.
within the structural gene. We initially used reassociation analysis of total rabbit DNA cleaved with restriction endonucleases to search for restriction fragments containing part or all of the /?-globin gene linked to a reiterated sequence. This approach showed that the 2.5 kb and O-9 kb EcoRI fragments, which contain, respectively, the 5’ and 3’ halves of the rabbit /3-globin gene plus extragenic sequences, reassociate very slowly with a Cot, of approximately 500 and 1000, respectively. A
REPETITIVE
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TABLE
b-GLOBIN
GENE
543
1
of silver grains over rabbit metaphusechromosomes hybridized in situ with 3H-labelEedR/K?1 DNA
Distribution
Number
Ko. of metaphases scored No. of silver grains scored Silver grains over: interstitial chromosome regions centromeres telomeres centromeres/telomerest Total number of grains over centromeres or telomeres
21 79 9 32 31 I 70
Percentage
(100, 11 41 39 9 89
Control in situ hybridizations with sH-labelled pCR1, PflGl and PMB9 DNA gave in each case 0.3 to 0.4 silver grain per metaphase, compared with 3.8 grains per metaphase with 3H-labelled RflGl DNA. This background labelling was distributed approximately equally between interstitial and (centromeric + telomeric) regions. t Where labelled chromosome is acrocentric and it is impossible to determine whether the centromere or telomere is labelled (see Fig. 7).
single copy of a 1.5 kb fragment of PMB9 DNA, the plasmid vector used in constructing PpGl (Maniatis et al., 1976), was added to restricted rabbit DNA prior to reassociation analysis to serve as a marker for the reassociation rate of unique sequence DNA. Since this marker fragment also reannealed with a C,t+ of about 1000 (Fig. 2), this suggests that both the 5’ and 3’ EcoRI p-globin DNA fragments contain DNA of a very low repetition frequency or unique sequence DNA. The measurement and interpretation of approximate C,t, values derived for /3globin DNA fragments is complicated by several factors. (1) The restriction endonucleases used generate large rabbit DNA fragments, many of which would be expected to form networks on reassociation by multimolecular annealing of long DNA fragments via reiterated sequences. Networks are difficult to recover from hydroxyapatite columns by salt elution alone (Hollenberg et al., 1972) and were recovered in the present study by a brief thermal denaturation at 100°C. Essentially complete recovery of DNA from hydroxyapatite was obtained and it is unlikely that network formation leads to selective loss of /I-globin DNA fragments in this experimental procedure. (2) These reassociation analyses cannot be extended to the very high C,t values required to obtain complete reannealing of unique sequence DNA, since prolonged reannealing leads to DNA degradation and loss of discrete /3-globin DNA bands in filter hybridizations. For example, annealing of rabbit DNA cleaved with EcoRI t,o C,t = 10,000 (10 mg/ml DNA, 70°C for 15 h) reduced the mean single-strand size of the DNA from about 5 kb to less than 1 kb (data not shown). As a result relatively inaccurate C,t+ values for unique or low reiterated sequences would have to be estimated from incomplete C,t curves.
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(3) The rate of reassociation of a DNA fragment is dependent on its length (Wetmur & Davidson, 1968), and since p-globin DNA fragments of different lengths were studied, the measured C,t, values would have to be corrected for the length of sequence over which hybridization is occurring ; this correction can only be applied to unique sequence DNA. These procedural complications make it very difficult to distinguish between unique sequence and very low repetit’ive sequence DNA using reassociation analysis of ,&globin DE’B fragments in total rabbit, DNA cleaved with a restriction endonuclease. However, an alternative more sensitive approach to analysing single-copy DNA is provided by the cloned rabbit /3-globin gene plus surrounding sequences (plasmid R/3Gl; made by Van Den Berg et al., 1978). Rabbit DNA fragments from this clone were used as filter hybridiza,tion probes in analyses of restricted rabbit DNA. If the purified fragment does not contain repetitive DNA sequences, then it’ should only detect its corresponding DXA fragment in total rabbit DNA. If instead low then a corresponding number of additional repetitive sequences are present, hybridizing fragments should be seen. A series of probes prepared from RPGl DNA and which covered 1.4 kb of 5’ extragenic sequence, 0.9 kb of 3’ extragenic sequence and most of the coding and intervening sequences, all behaved as unique sequence DNA by this criterion. We have previously shown that a probe containing as little as 45 bp of sequence perfectly complementary to a /3-globin DNA fragment is capable of detecting this fragment in filter hybridizations (Jeffrey3 $ Flavell, 19773). Thus any undetected reiterated sequences near the rabbit fl-globin gene must either be very short, or diverged in sequence from the corresponding cloned sequence, or be highly A + Trich and incapable of forming stable hybrids under the stringent conditions of hybridization used. Under relatively low stringencies (0.3 to I.0 x SSC at 65’C) rabbit /3-globin cDNA detects additional ,&related gene sequences (Jeffreys & Flavell, 1977b, and unpublished data), probably corresponding to three extra rabbit /3-globin genes described by Lacy et al. (1979). In contrast, extragenic probes, for example probe 1 in Figure 6, fails to detect sequences near these additional genes even after low stringency posthybridization washes. It therefore appears that sequences outside the p-globin genes are more diverged than those coding for p-globin. This phenomenon has been described in detail for the mouse pd-major and pd-minor globin genes (Konkel et al., 1979). It therefore appears that at least 1.4 kb of sequence on the 5’ side of the rabbit ,9-globin gene, covering all sequences except for 9 bp between the 5’ PvuII site and the beginning of the structural gene (see Fig. 3) are devoid of repetitive DNA elements. The significance of this finding in relation to possible roles of repetitive DNA sequences in eukaryotic genomes requires that the position of origin of transcription of the ,Y-globin gene be known. A 15 S precursor of mouse fl-globin mRNA has been described by several workers (Curtis $ Weissmann, 1976; Ross, 1976; Bastos & Aviv, 1977; Curtis et al., 1977a,b) and has been shown to be a continuous transcript of the /3-globin coding sequences plus both intervening sequences (Tilghman et al., 1978a,b; Schambijck et al., 1979). Furthermore, Weaver $ Weissmann (1979) have shown that the 5’ end of the 15 S mouse precursor is identical to that of mature mouse /3-globin
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mRNA, and they argue that the 5’ end of p-globin mRNA may well represent the point of initiation of transcription of the mouse /3-globin gene. Equally the rabbit /3-globin gene is also transcribed to give a 15 S precursor mRNA containing both intervening sequences (Flavell et al., 1979), and whose 5’ end is coincident with the 5’ end on the mRNA. Recent in vitro transcription experiments using RNA polymerase II and extracts of KB cells have shown that discrete transcripts with the same 5’ end as the 15 S pre mRNA and mRNA made in viwo can be obtained using RpGl DNA as template (P. Jat, R. I. Kamen and R. A. Flavell, unpublished results). The within the single-copy DNA sequences rabbit /3-globin promoter lies, therefore, characterized in this paper. Thus 1.4 kb of DNA upstream from the likely origin of transcription of the rabbit /3-globin gene appears to be devoid of reiterated DNA sequences. This is difficult to reconcile with the type of “receptor” genes, proposed by Britten & Davidson (1969), which were predicted to consist of intermediate repetitive DNA. From the arguments described above, we consider it unlikely that repetitive DNA elements are involved in the promotion of RNA synthesis. If such “receptor” genes exist, then they must act at a greater distance from the structural gene than hitherto expected. It is possible that the rabbit p-globin gene is exceptional, and for this reason it will be important to search for repetitive DNA elements on the 5’ side of other developmentally regulated genes. A similar comment may be made about the two intervening sequences within the rabbit /3-globin gene. Probes 3 and 4, which contain, respectively, the whole of the small intervening sequence and all except for the last 43 bp of the large intervening sequence (Fig. 3, see Van Den Berg et al., 1978), were devoid of reiterated sequences. These intervening sequences therefore appear to be unique sequence and are not found elsewhere in the genome, as might be expected if these sequences acted as common regulatory elements in many structural genes. The same result has been reported for the mouse /I-globin gene (Miller et al., 1978) and the chicken ovalbumin gene (Garapin et al., 1978). Both reassociation analysis of total rabbit DNA and the use of purified probes from the cloned rabbit /3-globin gene have revealed an intermediate repetitive sequence on the 3’ side of the structural gene somewhere within a segment of DNA located 0.9 to 2.1 kb from the end of the gene. This sequence reanneals with a Cat* of about 0.2, compared with a Cot, of about 1000 for unique sequence DNA (see Fig. 2). This suggests an approximate reiteration frequency of lOOO/O~X = 5000-fold for this repetitive DNA element. Probe 6 (Fig. 3) also contains this repetitive DNA element and hybridizes to many different fragments in restriction endonuclease digests of total rabbit DNA. There was no evidence for any discrete labelled components, as might be expected if most of these reiterated sequences are arranged in tandem within the rabbit genome. The heterogeneous pattern of hybridization to restricted rabbit DNA suggests instead that most or all of these sequences are arranged in an interspersed fashion within rabbit DNA. However, in situ hybridization of rabbit metaphase chromosomes with 3H-labelled plasmid RfiGl DNA, which occurs via this reiterated element, shows marked preferential labelling of centromeres and telomeres on a number of chromosomes. Thus the bulk of these sequences are located on more than one chromosome, and
546 appear
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The
chromosome localization of the rabbit /3-globin gene itself is not yet known. We do not know as yet whether the copy of the repetitive DNA sequence on the 3’ side of the rabbit p-globin gene is functionally linked with the expression of this gene, although this sequence is certainly not part of the 15 S precursor mRNA. In this respect, it is of interest that the human /I-globin gene also has a 3’ repetitive DNA element at the same position with respect to the structural gene (H. A. Hoeymakers, unpublished results). Insight into the function of the intermediate repetitive sequence and other sequences flanking the /3-globin gene will depend on suitable assay systems in which cloned globin genes can be expressed (Hamer & Leder, 1979 ; Mantei et al., 1979). This coupled with in vitro site-directed mutagenesis of sequences of interest in the cloned gene (Flavell et al., 1974; Mertz et aE., 1975) should ultimately make it possible to relate reiterated and other sequences to gene function in higher organisms. We thank Professor P. Borst, for helpful discussions. This work was supported in part by grants to P. Borst and one of t,he other authors (R. A. F.) from The Netherland Foundation for Chemical Research (SON) with financial aid from The Netherlands Organisation for the Advancement of Pure Research (ZWO) ; also by SRC grant no. GR/A 86466 awarded to Professor H. C. Macgregor and J. M. V ; A. J . J. was a postdoctoral fellow of the European Molecular Biolog)Organization during the early stages of this research.
REFERENCES Bastos, R. N. & Aviv. H. (1977). Cell, 11, 641-650. Birnstiel, M. L., Chipchase, M. & Spiers, J. (1971). Prog. NucZ. Acid Res. MoZ. BioZ. 11, 351-390. Birnstiel, M., Telford, J., Weinberg, E. & Stafford, D. (1974). Proc. Nat. Acud. Sci., U.S.A. 71, 29os2904. Bishop, J. D. & Freeman, K. B. (1974). Cold Spring Harbor Symp. Quant. Biol. 38, 707-716. Britten, R. J. & Kohne, D. E. (1968). Science, 161, 529-540. Britten, R. J. & Davidson, E. H. (1969). Science, 165, 349-357. Britten, R. J., Graham, D. E. & Neufeld, B. R. (1974). Methods Emzym,oZ. 29E, 363-418. Crawford, L. V. & Robbins, A. K. (1976). J. Gen. Viral. 31, 315-322. Curtis, P. J. & Weissmann, C. (1976). J. Mol. Biol. 106, 1061-1075. Curtis, P. J., Mantei, N., Van Den Berg, J. & Weissmann, C. (1977a). Proc. Nat. Acad. Sci., U.S.A. 74, 3184-3188. Curtis, P. J., Mantei, N. & Weissmann, C. (19776). Cold Spring Harbor Symp. Quant. Biol. 42, 971-984. Davidson, E. H., Graham, D. E., Neufeld, B. R., Chamberlin, M. E., Amenson, C. S., Hough, B. R. & Britten, R. J. (1974). Cold Spring Harbor Symp. Quunt. Biol. 38, 295-301. Davidson, E. H., Hough, B. R., Klein, W. H. & Britten, R. F. (1975). Cell, 4, 217-238. Flavell, R. A., Sabo, D. L., Bandle, E. F. & Weissmann, C. (1974). J. Mol. Biol. 89, 255-272. Flavell, R. A., Jeffreys, A. J. & Grosveld, G. C. (1978a). Cold Spring Harbor Symp. Quant. Biol. 42, 1003-1010. Flavell, R. A., Kooter, J. M., De Boer, E., Little, P. F. R. & Williamson, R. (1978b). Cell, 15. 25-41.
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Flavell,
R. A., Grosveld, G. C., Grosveld, F. G., Bernards, R., Kooter, J, M., De Boer, E. & Little, P. F. R. (1979). In From Gene to Protein, Miami Winter Symposium, in the press. Gall, J. G. & Pardue, M. L. (1971). Methods Enzymol. 21D, 470-480. Garapin, A. C., Lepennec, J. P., Roska,m, W., PBrrin, F., Cami, B., Krust, A., Breathnach, R., Chambon, P. & Kourilsky, P. (1978). Nature (London), 273, 349-354. Georgiev, G. P., Varshavsky, A. J., Ryskov, A. P. & Church, R. B. (1974). Cold Spring Harbor, Symp. Quant. Biol. 38, 869-884. Hamer, D. H. & Leder, P. (1979). Nature (London), 281, 35-40. Hollenberg, C. P., Borst, P., Flavell, R. A., Van Kreijl, C. F., Van Bruggen, E. F. J. & Arnberg, A. C. (1972). Biochim. Biophys. Acta, 277, 44-58. Jeffreys, A. J. & Flavell, R. A. (1977a). Cell, 12, 429-439. %Jeffreys, A. J. 8: Flavell, R. A. (1977b). Cell, 12, 1097-1108. Jones, K. W. (1970). Nature (London), 225, 912-915. Konkel, D. A., Maizel, J. V. & Leder, P. (1979). Cell, 18, 865-873. Lacy, E., Hardisolr, R. C., Quon, D. & Maniatis, T. (1979). Cell, 18, 1273-1283. Lawn, R. M., Frit,sch, E. F., Parker, R. C., Blake, G. & Maniatis, T. (1978). Cell, 15, 1157-I 174. Little, P. F. R., Flavell, R. A., Kooter, J. M., Annison, G. & Williamson, R. (1979). Nature (London), 278, 227-231. Macgregor, H. C. & Mizuno, S. (1976). Chromosoma (Berlin), 54, 15-25. Maniatis, T., Jeffrey, A. & Kleid, D. G. (1975). Proc. Nat. Acad.Sci., U.S.A. 72, 1184-1188. Maniatis, T., Kee, S. G., Efstratiadis, A. & Kafatos, F. C. (1976). Cell, 8, 163-182. Mantei, N., Boll, W. & Weissmann, C. (1979). Nature (London), 281, 40-46. Maxam, A. M. & Gilbert, W. (1977). Proc. Nat. Acad. Sci., U.S.A. 74, 560-564. Mertz, J. E., Carbon, J., Herzberg, M., Davis, R. W. & Berg, P. (1975). Cold Spring Harbor Symp. Quant. BioZ. 39, 69-84. Miller, H. I., Konkel, D. A. & Leder, P. (1978). Nature (London), 275, 772-774. Pardue, M. L. & Gall, J. (1970). Science, 168, 1356-1358. Paul, J. (1972). Nature (London), 238, 444-446. Ross, J. (1976). J. Mol. BioZ. 106, 403-420. SchambGck, A., Curtis, P., Boll, W. & Weissmann, C. (1979). Experientia, 35, 978. Smith, H. 0. & Birnstiel, M. L. (1976). NucZ. Acids Res. 3, 2387-2398. Southern, E. M. (1970). Nature (London), 227, 794-798. Southern. E. M. (1974). In Biochemistry of Nucleic Acids, Biochemistry Series One, vol. 6, pp. 101-139, MIP International Review of Science, Butterworths, London. Southern, E. M. (1975). J. Mol. BioZ. 98, 503-517. Tilghman, S. M., Curtis, P. J., Tiemeier, D. C., Leder, P. & Weissmann, C. (1978a). Proc. Nut. Acad. Sci., U.S.A. 75, 1309-1313. Tilghman, S. M., Tiemeier, D. C., Seidman, J. G., Peterlin, B. M., Sullivan, M., Maizel, J. V. & Leder, P. (1978b). Proc. Nat. Acad. Sci., U.S.A. 75, 725-729. Va,n Den Berg, J., Van Doyen, A., Mantei, N., SchambBck, A., Grosveld, G., Flavell, R. A. & Weissmann, C. (1978). Nature (London), 276, 37-44. Walker, P. M. B. (1971). Progr. Biophys. Mol. BioZ. 23, 145-160. Weaver, R. F. & \Veissmann, C. (1979). NucZ. Acids Res. 7, 1175-1193. Wetmur, J. R. & Davidson, N. (1968). J. Mol. BioZ. 31, 349-370.
.\.
X.
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Section for Medical Enzymology and Molecular Laboratory of Biochemistry Uwiversity of Amsterdam Eerste Constant(jn Huygensstraat 20 Am.sterdam, The Netherlands
J.
E.
GROSVELD-1,
DE
BOER
FLAVELLt
M.
Biology
VARLEY
Department of Zoology University of Leicester University Road L&ester, England
A.
J.
JEFFREYS
Department of Genetics Unireraity of Leicester University Road L&x&r, England (Received
7 December
1979, and in revised form
17 March
1980)
DNA fragments containing the rabbit j-globin gene can be detected in restriction hybridizations with a labelled endonucleasc digests of rabbit DNX, usin, (I filtrr /?-globin cDNA plasmid as a probe for thr structural gene. -Analysis of the rea,mealing kinetics of these P-globin DNA fragments has been performed by denaturing rabbit DNA and renaturing t,o variolls C,t$ values, then detjermining by hydroxyapatite chromatography and subsequent filter hybridization whether a given P-globin DNA fragment, has anrlcnlcd bvith otller DKa via a rrpctitixrc DKLA tract contained in the fragmetlt, or instead has remained completely singlestratlded. This approach has revrxaled the presence of an intermrdiate repetitive DK.4 sc~~uonce about 0.6 to 2.4 kb from the 3’ end of the rabbit P-globin structural gone. The presence of t’his repetitive scquet~cc was confirmed by filter hybridization analysis of rabbit DNA4 with x,arious DNA fragments isolated from t Present,
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a cloned segment of rabbit genomic DNA containing the P-globin gene. A purified rabbit DXA4 fragment cont,aining sequences 0.9 to 2.1 kb from the 3’ end of the structtwal gene was found to hybridize to many DNA fragments in restriction endonttclease digests of rabbit DKA. 1~1 sit/l hybridizations t)o rabbit. metaphase chromosomes showed that copies of this repctitil-r sequence xere present on several chromosomes, and were preferentially localized in centromeric and telomeric regions. In contrast, the remaining sect,ion of the rabbit /%globin gent region examined, extending 1.4 kb from the 5’ end of the P-globin gene to 0.9 kb from the 3’ end of the gene and including the inter\-erring sequences withill the structural gene, was shown to consist of single copy DNS.
1. Introduction The pioneering work of Britben and his colleagues (Britten & Kohne, 1968) has demonstrated that eukaryotic genomes contain various classes of reiterated DNA in addition to DNA sequences present in only :a single copy per haploid genome. Highly repeat,ed sequences are found tandemly arranged (Southern, 1974) and commonly consist of simple DNA sequences (Sout hem, 1970). \vhich appear to be transcriptionally inactive (Walker, 1971) and restricted to heterochromatin (Jones, 1970; Pardue $ Gall, 1970). In addition, certain genes such an those coding for ribosomal RNAs (Birnstiel et al., 1971) and sea urchin histones (Birnstiel et al., 1974) are also found repeat,ed in tandem arrays. Much of the repetitive DNA with reiteration frequencies ranging from lo2 to lo5 appears t’o be spread throughout the eukaryotic genome, a,nd interspersed among single-copy DN’B sequences in a pattern that is apparently similar in many different organisms (Davidson et aZ., 1974). There is evidence that some of these repetit’ive sequences are linked to structural genes in the DNA of echinoderms (Davidson et al., 1975), a’nd evidence exist’s for a low frequency repetitive DNA segment in t)he vicinity of duck haemoglobin genes (Bishop & Freeman, 1974). Various authors have postulated that repetitive DNA sequences, linked to structural genes, might be involved in the control of gene expression (Britten & Davidson, 1969; Paul, 1972; Georgiev et al., 1974). To learn more about the possible role of repetitive DNA in the regulation of gene expression, it is essent’ial to know the types and locations of various repetitive DNA sequences which might be found near a given single-copy structural gene. In this paper we analyse the types of repetitive DNA that are found close bo the rabbit /3globin struct’ural gene. This approach has been made possible by the construction of a physical map of restriction endonuclease cleavage sites in and a,round this gene (Jeffreys & Flavell, 1977a;b) using a 32P-labelled P-globin cDNA plasmid (plasmid PpGl : Maniatis ft al., 1976) as a filt,er hybridization probe for rabbit DNA restriction fragments containing a /3-globin gene. The search for reiterated sequences has also been aided by the cloning of a fragment of rabbit’ DNA containing the /%globin gene by Van Den Berg et al. (1978). We have previously shown that a repetitive DNA segment is located to the 3’ side of the rabbit P-globin gene, and t,hat the DNA4 sequences next to the gene are present in less t’han 100 copies per haploid genome (Flavell et al., 1978a). We were interested in determining whether the DNA sequences flanking the jl-globin gene, and in particular on the 5’ side, are present in multiple copies per cell, as might be anticipated according to the gene regulation model proposed by Britten
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C%Davidson (1969). In addition, the rabbit ,%globin gene contains two intervening sequences (Jeffreys & Flavell, 1977b; Van Den Berg et al., 1978), and we were interested to learn whether these sequences are found only within the /3-globin gene or whet’her they are also present elsewhere in the genome. Our approach has shown that the 5’ extragenic region and the intervening sequences within t,he rabbit /3-globin gene consist of single copy DNA. In contrast, a region 0.9 to 2.1 kb-1 from the 3’ end of t#he structural gene contains a repet)itive DNA segment which is present in about 5000 copies per genomc.
2. Materials
and Methods
(a) Materials 1,ivt.r DSA was purified from an F1 hybrid between Alaska and Vienna White strains of rabbit, as described by Jeffreys & Flare11 (1977b). Purified PgGl DNA and Z-pCRI/ Rchr,6G-1 DNA (the latter being abbreriat,ed to R/?Gl in this paper) were provided by Professor C. \Veissmann, Inst,itut fur Molckularbiologie, Zurich. Restriction endonucleases EcoRI and PstI were purchased from the Medical Research Establishment, Porton Down, U.K. Endonucleases BumHI, HaeIII, HhaI and PvuII were purchased from New England BioLabs Inc. Endonuclease KpnI was prepared by t,hr method of Crawford & Robbins (1976). (b)
Filter
hybridization
analysis
of rabbit
P-glob&
USA
Restrictiolr endonuclease digestions of rabbit’ liver DNA, agarosc digest products, transfer of DNA t’o nitrocellulose filters by blotting, filt,ers wit’h 32P-labrlled PpGl DNA or 32P-labelled DNA fragments tion endonuclease digests of RPGl DNA \vere performed as described (1977w,b). (c)
Reassociation
analysis of P-glob& endonuclease digests
DLXA fragments of rabbit DSA
gel electrophoresis of and hybridization of isolated from restricby Jeffreys $ Flavrll
in restriction
Reassociation of digested DNA was performed at DNA concentrat,ions of 0.2 mp/ml to 10 mg/ml in 0.6 ar-sodium phosphate (pH 6.8), by heat denaturation (lOO”C, 5 min) followed by rapid cooling t,o 50°C and incubat,ion to the desired C,t value. Samples containing 30 pg DNA were then diluted to 0,12 >I-sodium phosphate and applied to a l-ml hydroxyapatite (Biorad HTP) column maint’ained at, 60°C. Completely single-st’randed DNA was elut,ed bj- washing with 10 column I-olumes of 0.12 >I-sodinm phosphate at 60°C. Duplex DNA was vluted by a wash witjh 5 column \-olumes of 0.4 ar-sodium phosphate at 60°C followed by 5 column volumes of 0.4 fir-sodium phosphate at 100°C to rccovor DNA present in networks. Column fract)ions were dialysed extensively against 10 mlsr-Tris .HCl (pH 7.5) for 3 days at) 4”C, concentrated by extraction of water with butan-2.ol, and DNA collected by precipitation with et,hanol. Samplrs were then denatured with alkali and fractionated by electrophoresis in agarose gels, DNA fragments were t,ransferred to nitrocellulose filters by blotting and P-glohin DNA-containing fragments detected by filt’er hybridization with 32P-labelled P/3Gl DN.1. All C,t values llave been corrected to t’he standard 0.12 ar-sodium phosphate. 6O’C condit,ions using the tables of Brieten et al. (1974). (d)
In situ
hybridization
Heparinized venous blood from an adult rabbit was l(PM1 1640 (Gibco Bio-Cult) medium supplemented strum and 2’;; (v/v) Bactophytohaemagglutinin M 0.4 &ml and the incubat’ion continued for 14 h. Cells -1 See r00tr10te
to ,,. 531.
cultured for 96 h at 37°C in 10 vol. with 10% (v/v) neonatal calf solution. Colchicine was added to were collected by centrifugation at
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100 g for 10 mm, suspended in 75 rnl\I-KCl, incubat,ed at 37°C for 20 min and recentrifugcd. The pellet was resuspended in a small volume of KC1 and fixed in 3 changes of ice cold 3: 1, methanol/acetic acid. Splash preparations were made onto glass microscope slides. Chromosome preparations were prepared for in situ hybridizations following the basic method of Gall & Pardue (1951). RNA was removed by incubating slides in 50 pg ribonuclease A/ml plus 10 units ribonuclease T,/ml in 2 x SSC at 37°C for 1 h (SSC is 0.15 Jr-NaCl, 15 mr\l-trisodium cit’rat,e, pH 7.0). Slides were then washed in 3 changes each of 2 x SSC, 700/, ethanol and 95% ethanol, arid air dried. C’hromosomal DNA was denatured in 70 rnsr-NaOH at room temperature for 3 mm, followed by washing in 700/, and 9576 ethanol and air drying. DNA samples to be used as probes were labelled with eH by nick t,rauslatioii using the method of Maniatis et al. (1975) as modified by Macgregor & Mizuno (1976). Each slide was liybridized under a cover slip for 12 h at, 37°C with 30 ~1 5Oe/, (v/x-) formamide, 4 :< SSC contaming 0.1 pp eH-laballed DNA ( IO6 to 2 x lo6 &s/mm per pg DNA), which had previously been denatured for 15 rnin in 0. I 31.NaOH and neutralized with HCl. Unbound labelled DNA was removed by washing with 3 changes of 2 x SSC at room temperature, 3 changes of 2 x SSC at 65°C. once in’2 x SSC plus 5oj (W/X-) t,richloroacetic acid for 5 min at 4”C, 3 times in each of 2 x SSC. 70% ethanol and 95% ethanol, and air dried. Slides were dipped in Kodak NTB-2 nuclear emulsion. dried and autoradiographed for 1 to 9 weeks. After developing and fixing. slides were stained with 294 Giemsa (Gurrs R-66) for 10 min and air dried.
3. Results (a) Reassociation analysis of P-glob& DNA fraqrnents: an intermediate repetitive sequence on the 3’ side of the gene JVe have described elsewhere how WC can specifically det,ect DnTA fragments, which contain the /3-globin (present in PPGl DKA). m rabbit DXA digested with various restriction endonucleases and how these fragments can be ordered into a physical map of cleavage sites within and around the P-globin structural gene (Jeffreys & Flavell, 1977a,b). This map (Fig. 1) shows the direction of transcription and the presence of two intervening sequences within the DNA sequences coding for /3-globin. Transcription
4
FIG. 1. A physical map of restriction endonuclease cleavage sites within and neighbouring the rabbit p-globin structural gene. Cleavage sites are shown for endonucleases LlnmHI (B), EcoRI (E), KpnI (K) and PatI (P). Regions coding for ,L-globin mRNA sequences are indicated by hatched boxes and intervening sequences by open boxes. The calibration scale in kb is contred on the int,ragenic RalnHI cleavage site. The derivation of this map is described elsewhere (Jeffrey5 & Flavell, 1977a,b; Van Den Berg at ccl., 1978). Although cleavage sites for a number of other restriction endonucleases have also been mapped, these are omitted here for the sake of clarity.
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To determine the presence of a repet’itive DNA sequence in a /Lglobin DNA4 fragment,, rabbit liver DNA digested with the appropriate restriction endonuclease was denatured and reassociated at 70°C in 0.6 M-sodium phosphate (pH 6.8), to the desired C,t value. Duplex DNA was then separated from completely single-stranded DNA by chromatography on hydroxyapatite at 60°C under the standard conditions described by Britten & Kohne (1968). The duplex and single-stranded DNA fractions were recovered, denatured with alkali and analysed for /?-globin DNA fragments by agarose gel electrophoresis, transfer of DNA fragments to a nitrocellulose filter by the method of Southern (1975) and filter hybridization with 32P-labelled Pj3Gl DNA (Flavell et al., 1978a). An estimate of repetition frequency of the sequences contained within a given restriction fragment can be determined by comparing its rate of annealing with that of single-copy DNA. In t’he following discussions all C,t values are corrected to the values for DNA annealing in 0.12 M-sodium phosphate (Britten et al.. 1974). When rabbit DNA digested with endonuclease EcoRI was denatured and reannealed, both the 2.5 kb and 0.9 kb /I-globin DNA fragments, which contain the 5’ and 3’ halves of the /?-globin gene, respectively (Fig. l), remained essentially single-stranded up to C,,t = 100 (Fig. 2). 0 n annealing to C,t = 1000, some of each fragment appeared in the duplex DNA fraction. As a control for the reassociation rat.t: of unique sequence DNA, we added to the rabbit DNA digests an amount, equivalent to a single gene copy, of a 1.5 kb plasmid vector PMB9 DNA fragment isolated from P/3Gl DNA which had been cleaved with EcoRI and BvaI (Jeffreys & Flavell, 1977a). This internal marker fragment reannealed with a C,t, of about 1000. (Fig. 2). The 3’ 0.9 kb EcoRI p-globin DNA fragment reannealed with similar kinetics, suggesting that this fragment of the rabbit genome contains only single-copy DNA. The reassociation rate of the 5’ 2.5 kb EcoRI /Lglobin DNA fragment was slightly higher than that of the 3’ EcoRI fragment and at C,t = 1000 it was mainly found in the duplex DNA fraction. This increased reannealing rate may be due to the length of this EcoRI fragment (2.5 kb compared with 1.5 kb for the single-copy marker; reannealing rates are proportional to the square root of DNA fragment length, see Wetmur & Davidson, 1968). The 3.6 kb fl-globin DNA fragment generated by cleavage of rabbit DNA with KpnI + PstI contains the entire @-globin gene (Fig. 1). In contrast to the EcoRI fragments, this component reannealed rapidly and almost completely entered the duplex DNA fraction on passing from C,t = 0.1 to C,t = 1 (Fig. 2). Hence this fragment must contain a repetitive DNA segment. Since neither of the EcoRI fragments contains this repetitive sequence, this places the intermediate repetitive element somewhere between the EcoRI and KpnI sites 0.6 to 2.4 kb on the 3’ side of the gene (Fig. 1). This result confirms and extends our previous observation that the 4.9 kb KpnI /3-globin DNA fragment, which also carries this region, was completely annealed at C,t = 60 (Flavell et al., 1978a). (b) Analysis
of rabbit
genomic
Analysis of the reannealing kinetics showed that a stretch of DNA from
Dh’d
sequences homologous DNA
to cloned R@l
of p-globin DNA fragment’s in total rabbit DNA 1.4 kb 5’ from the structural gene to 0.6 kb 3’
536
H.
A.
M.
Log
Co’
:
M
in.
HOEIJMAKERB-VAS
-I -------------!A ss ds
DO;\lMELEN
ET
ilL.
0 ss
ds
ss
ds
ss
ds
4
ss
ds
in.
FIG. 2. Reassociation analysis of P-globin DNA fragments grncrated by cleavage of rabbit DSA with endonucleases EC&I or KpnI + PstI. Rabbit liver DNA digests were heat-denatured and reannealcd to the indicated C,t values (corrected to the rate of annealing in 0.12 &I-sodium phosphate). Completely single-stranded DNA was separated from DNA containing double-strantletl regions by chromatography on hydroxyapatite. Single-stranded and double-stranded fractions derived from 30 pg of total DNA were alkali denatured, electrophoresed through a I.‘>“’i ,,, agarose go1 and transferred by blotting to a nit,rocellulose filter. ,t?-globin DNA fragments were detected by autoradiography after hybridization with 3ZP-labelled PgGl DNA (see Jeffreys & Flavoll, 1977n,b and Materials and Methods). in. = 30 pg unfractionatctl DNA digest: ,11 : marker DNA fragments (10 pg PpGl DNA x Hind111 (5.6 kb fragment) plus 25 pg P/3Gl DNA x EcoKI x dvcr1 x NindIII (2.3, 1.5, 1.2, 0.65 kb fragments; see Jeffreys & Flavell, 1977rr)). A single copy (15 pg) of a 1.5 kb PflGl DSA x EcoRI x i4cnI fragment, containing only plasmid vrctor PMB9 sequences was added to each 30 pg batch of rabbit liver DNA x EcoRI prior to t,hc, reassociaCon analysis.
from the gene and including the gene plus int’ervening sequences behaved as low repetitive or single-copy DKA. To confirm that these are indeed single-copy sequences, we have studied the hybridization bet’ween these sequences. isolated from a cloned fragment of rabbit DPU’A, and total rabbit, genomic DNA. A number of restriction endonuclease digest fragments of rabbit DNA were isolated from the recombinant plasmid R@Gl which contains the 4.9 kb KpnI ,!?globin DNA fragment shown in Figure 1 (Van Den Berg et al., 1978). The origin of t,hese purified fragments is shown in Figure 3. These fragments were then labelled with 32P by nick translation and used as probes in filter hybridizations with restriction endonuclease digests of total rabbit DNA. After hybridization, filters were given a high stringency wash in 0.1 x SSC at 65°C and remaining well-matched hybrids were detected by autoradiography (Fig. 4). Probes 1, 3, 4 and 5, which contained, respect-
BEPETITIVE
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GENE
537
Transcription
HhHM
M
5
6
FIG. 3. A physical map of a cloned segment of rabbit DKa containing the 8.globin gene, showing regions purified for use as probes for repetitive DNA sequences. The 4.9 kb Kpnl DNA fragment containing the 8.globin gene (see Fig. 1) has been purified by cloning into the EcoRI cleavage site of plasmid pCR1 to produce the recombinant plasmid RPGl (Van Den Berg et nZ., 1978). The map shows ,&globin structural gene sequences (hatched boxes), intervening sequences (open boxes) and pC’R1 sequences (solid boxes) flanking the cloned segment of rabbit DNA. Cleavage sites are shown for endonucleases HnmHI (B), EcoRI (E), Hat211 (H), JlspI (XI), P&I (P) and Pm11 (Pv). In addition, the positions of two HhnI (Hh) cleavage sites in pCR1 are shown; there are no HhaI cleavage sites in the rabbit DKA segment. The mapping of BnmHI, EeoRI and I’stI cleavage sites are described elsewhere (Jeffreys & Flavell, 1977a,b; Van Den Berg et nl., 1978). Cleavage sites for HueIII. MspI and PvuII in the cloned rabbit DSA were determined by th(, method of Smith Er Birnstiel (1976) (J. Van Den Berg, A. Van Ooycn and G. C. Grosveld, unpublished results). The origins of purified fragments of rabbit DNr\ used as probes for repetitive DX9 sequences am shown below the map. These probes were isolated from the rabbit DiYa4 fragment purified from a HhrT digest of RPGl DNA. Probes 1 and 3 t,o 6 Were purified from a HamHI -- HneIII double digest of this fragment by polyacrylamide gel elcct,rophoresis. using the procedure of Mazam & Gilbwt (1977). Probe 2 was similarly purified from a ~wrIT digest.
ively, 1.4 kb of 5’ extragenic sequences, coding sequences plus the small intervening sequence, coding sequences plus the large intervening sequence, and 0.9 kb of 3 ’ extragenic sequences, all detected only one 4.9 kb fragment in KpnI digests of rabbit DNA. Since this is the fragment cloned in RpGl, this suggests that probes 1 and 3 to 5 all consist of unique sequence DNA. The apparent lack of cross hybridization between these probes and additional rabbit DNA sequences is confirmed by analysis of PstI and EcoRI digests of rabbit genomic DNA (Fig. 4). As predicted, probes 1, 3 and 4 detect only the 2.5 kb EcoRI 5’ ,!I-globin DNB fragment, whereas probe 5 detects the 0.9 kb 3’ EcoRI fragment plus an addit’ional 1.8 kb fragment which presumably maps adjacent to the O-9 kb fragment. Similarly, probes 3, 4 and 5 all detect the single 6.7 kb Pstl /3-globin DNA fragment. In contrast, probe 1 maps to the 5’ side of this PstI fragment and detects instead a single new PstI fragment 13 kb in length. Finally, probe 2 detected the same DNA fragments as probe 1 (data not shown). The map positions of DNA fragments detected by probes 1 to 5 are shown in Figure 5. In all cases, these probes detect DNA fragments originating only from the /3-globin and surrounding sequences, demonstrating that these probes contain only single-copy DNA. 21
kb
15
Probe
:
AK
E
P
K
3 E
P
,2-----L* K
E
P
K
E
P
6 K
FIG. 4. Analysis of rabbit DNA fragments containing sequences homologous to DNA near the rabbit j3-globin gene. Rabbit DNA was digested with endonuclease PstI (P), KpnI (K) or EcoRI (E). 20 pg batches of DNA were alkali denatured, electrophoresed through 1% agarose slab gel and transferred by blotting to a nitrooellulose filter. Identical filters were then hybridized under non-stringent conditions (65°C in 3 x SSC for 2 days) with probes 1, 3, 4, 5 or 6 (Fig. 3) which had been labellcd with 32P by nick translation. After hybridization, unbound labelled DNA was washed from the filters, which were subsequently given a wash under stringent conditions (65°C in 0.1 x SSC). Labelled bands of DXA were detected by autoradiography. 411 hybridization and washing procedures were carried out as described by Jeffreys & Flavell(1977b).
P
E
REPETITIVE
DNA
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,9-GLOBIN
539
GENE
Transcription *
I kb I
I P
K E
P
E
E K
E
P
m/A
..I Repetitive
+ h
Probe
I
\
IE 2.5 4 EK 2.5
+-7F
j ; b , ;
6 P 13 PK I.3
iK 4.9
I H I.6
im 1 I b
Probe
i-1 ,
+
2 I E 2.5 1 EK 2.5 iPK
I P 6-7 k-----H
/PK 3.6 jK 4.9
0.9
Fprobe ,
(faint)
Probes 3 8 4 E 2.5 1 EK 2.5
I 1 I
1.3,3.6
1
5
-j
LE
o.g,,.*
j EK 0.9,l.e
, P 6.7
iPK 3.6 H
H 1.2
d Many
5. A physical fragments detected of DNA which have FIG.
I”-
_I
Probe fragments
6
detected
map of DNA sequences near the rabbit p-globin gene, showing genomic DRA by probes isolated from sequences near the rabbit j%globin gene, plus regions been shown to contain unique or repetitive DNA sequences.
To investigate the possibility that the rabbit genome contains additional DNA sequences related to, but not identical with, sequences in probes 1 to 5, we hybridized these probes with filters containing restriction endonuclease digests of total rabbit DNA under conditions of low stringency, then washed the filters under increasingly stringent conditions by lowering the ionic strength of the washing solution. A typical result for probe 1 is shown in Figure 6. As the stringency is lowered, no additional labelled bands are detected until the lowest stringency (1 x SSC at 65°C) is reached, when the entire track of rabbit DNA digest shows a uniform labelling due to aspecific hybridization between probe and rabbit DNA (see Jeffreys $ Flavell, 1977a). Traces of a small number of additional discrete labelled components could be seen. Thus probe 1 contains essentially unique sequence DNA, although it is possible that part or all of this probe may consist of sequences present in a very limited number of 21*
540
H.
A.
M.
HOEIJMAKERS-VAN
DOMMELEN
o-3 P
17’
AL.
x K
E
kb 13 B-
Probe
I
Probe
6
4.9,
FIG. 6. Analysis of the stability of hybrids formed between total rabbit DNA and purified DNA sequences near the rabbit p-globin gene. 15 pg batches of rabbit DNA were digested with endonuclease PstI (P), KpnI (K) or EcoRI (E), electrophoresed through an agarose gel and transferred to a nitrocellulose filter. Identical filters probe 1 or probe 6 (Fig. 2). were hybridized in 3 x SSC at 65’C for 2 days with 32P-labellad Unbound label was removed and the filters were then given a more stringent wash in 1 x , 0.3 x or 0.1 x SSC at 65°C. Labelled bands were detected by autoradiography.
REPETITIVE
DNA
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RABBIT
/3-GLOBIN
GENE
541
additional diverged copies elsewhere in the genome. Similar results were obtained for probes 2 to 5 (data not shown). In contrast, probe 6 which contains sequences 0.9 to 2.1 kb from the 3’ end of the globin gene hybridizes at high stringency with many DNA fragments in restriction endonuclease digests of rabbit DNA, and labels the entire DNA track in filter hybridizations (Fig. 4). This DNA fragment must therefore contain a repetitive DNA sequence. This is consistent with the reassociation kinetics analysis which revealed a repetitive sequence somewhere within a region 0.6 to 2.4 kb from the 3 ’ end of the /3-globin gene. At lower stringencies, the labelling of rabbit DNA by probe 6 becomes considerably more intense (Fig. 6), and exceeds by orders of magnitude that seen with a single-copy probe. It is not known whether this is due to hybridization with related rather than identical repetitive sequences, or instead is due to artifactual loss of wellmatched hybrids in high stringency post-hybridization washes. (c) In situ hybridization of 3H-labelled R/&ICI DNA with rabbit metaphase chromosomes To identify the chromosome localization of the repetitive DNA sequences homologous to the sequence element on the 3’ side of the rabbit fl-globin gene, we hybridized rabbit lymphocyte metaphase chromosomes in situ with 3H-labelled R/?Gl DNA. Parallel hybridizations with 3H-labelled pCR1 vector DNA were performed to control for artifactual in situ hybridization between vector DNA and rabbit chromosomes. Similarly, 3H-labelled P/3Gl DNA containing rabbit ,Q-globin cDNA was used as a probe to control for hybridization to unique sequence DNA. After hybridization. unbound label was removed by a low stringency wash (2 x SSC at 65°C). As shown in Figure 7, 3H-labelled R/3Gl DNA labels a number of rabbit chromosomes, with a very marked preferential labelling of centromeric and telomeric regions. 89% of silver grains were found in these areas rather than in interstitial regions (Table 1). In contrast, neither pCR1 nor P/3Gl DNA label the metaphase preparation (data not shown), showing that the hybridization of chromosomes with RpGl DKA is due neither to cross-hybridization with vector nor to hybridization wit,h cloned rabbit single-copy sequences. The observed labelling pattern must therefore reflect, the chromosomal distribution of copies of the repetitive DNA element found on the 3’ side of the rabbit /?+globin gene.
4. Discussion Filter hybridization analysis of total genomic DNA and the cloning of specific nuclear DNA fragments have greatly aided the analysis of globin gene structure and the linkage arrangement of related globin genes (see, for example, Jeffreys $ Flavell, 19773; Tilghman et al.: 19783; Van Den Berg et al., 1978; Flavell et al., 19733; Lawn et al., 1978; Little et al., 1979). In the present paper, we show how both approaches can be used to analyse the interspersion of unique and repetitive DNA sequences at the fine structural level, and to investigate the linkage relationship between structural gene sequences and repetitive DNA elements. The region of the rabbit genome studied includes extragenic DNA sequences both 5’ and 3’ from the major adult p-globin gene, plus the coding and intervening sequences
542
H.
A.
M.
HO.EIJAMKERS-VAN
DOMMELEN
ET
AL.
FIG. 7. 1n situ hybridization of rabbit metaphase chromosomes with sH-labelled RpGl DNA. Metaphase chromosomes were prepared from phytohaemagglutinin-stimulated rabbit lymphocytes, and hybridized with 3H-labelled RpGl DNA in 4 x SSC, 50”/: formamide at 37°C. Unbound label was removed by washing in 2 x SSC at 65”C, and label detected autoradiographically (9 weeks exposure). Chromosomes were subsequently stained with Giemsa. Typical labelled centromeres (c) and telomeres (t) are indicated by arrows; in some cases, it is not possible to distinguish between centromeric and telomeric labelling on acrocentric chromosomes (open arrow). Scale bar represents 10 pm.
within the structural gene. We initially used reassociation analysis of total rabbit DNA cleaved with restriction endonucleases to search for restriction fragments containing part or all of the /?-globin gene linked to a reiterated sequence. This approach showed that the 2.5 kb and O-9 kb EcoRI fragments, which contain, respectively, the 5’ and 3’ halves of the rabbit /3-globin gene plus extragenic sequences, reassociate very slowly with a Cot, of approximately 500 and 1000, respectively. A
REPETITIVE
DNA
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TABLE
b-GLOBIN
GENE
543
1
of silver grains over rabbit metaphusechromosomes hybridized in situ with 3H-labelEedR/K?1 DNA
Distribution
Number
Ko. of metaphases scored No. of silver grains scored Silver grains over: interstitial chromosome regions centromeres telomeres centromeres/telomerest Total number of grains over centromeres or telomeres
21 79 9 32 31 I 70
Percentage
(100, 11 41 39 9 89
Control in situ hybridizations with sH-labelled pCR1, PflGl and PMB9 DNA gave in each case 0.3 to 0.4 silver grain per metaphase, compared with 3.8 grains per metaphase with 3H-labelled RflGl DNA. This background labelling was distributed approximately equally between interstitial and (centromeric + telomeric) regions. t Where labelled chromosome is acrocentric and it is impossible to determine whether the centromere or telomere is labelled (see Fig. 7).
single copy of a 1.5 kb fragment of PMB9 DNA, the plasmid vector used in constructing PpGl (Maniatis et al., 1976), was added to restricted rabbit DNA prior to reassociation analysis to serve as a marker for the reassociation rate of unique sequence DNA. Since this marker fragment also reannealed with a C,t+ of about 1000 (Fig. 2), this suggests that both the 5’ and 3’ EcoRI p-globin DNA fragments contain DNA of a very low repetition frequency or unique sequence DNA. The measurement and interpretation of approximate C,t, values derived for /3globin DNA fragments is complicated by several factors. (1) The restriction endonucleases used generate large rabbit DNA fragments, many of which would be expected to form networks on reassociation by multimolecular annealing of long DNA fragments via reiterated sequences. Networks are difficult to recover from hydroxyapatite columns by salt elution alone (Hollenberg et al., 1972) and were recovered in the present study by a brief thermal denaturation at 100°C. Essentially complete recovery of DNA from hydroxyapatite was obtained and it is unlikely that network formation leads to selective loss of /I-globin DNA fragments in this experimental procedure. (2) These reassociation analyses cannot be extended to the very high C,t values required to obtain complete reannealing of unique sequence DNA, since prolonged reannealing leads to DNA degradation and loss of discrete /3-globin DNA bands in filter hybridizations. For example, annealing of rabbit DNA cleaved with EcoRI t,o C,t = 10,000 (10 mg/ml DNA, 70°C for 15 h) reduced the mean single-strand size of the DNA from about 5 kb to less than 1 kb (data not shown). As a result relatively inaccurate C,t+ values for unique or low reiterated sequences would have to be estimated from incomplete C,t curves.
544
H.
A.
M.
HOEIJAMKERS-VAN
DOMMELEN
ET
AL.
(3) The rate of reassociation of a DNA fragment is dependent on its length (Wetmur & Davidson, 1968), and since p-globin DNA fragments of different lengths were studied, the measured C,t, values would have to be corrected for the length of sequence over which hybridization is occurring ; this correction can only be applied to unique sequence DNA. These procedural complications make it very difficult to distinguish between unique sequence and very low repetit’ive sequence DNA using reassociation analysis of ,&globin DE’B fragments in total rabbit, DNA cleaved with a restriction endonuclease. However, an alternative more sensitive approach to analysing single-copy DNA is provided by the cloned rabbit /3-globin gene plus surrounding sequences (plasmid R/3Gl; made by Van Den Berg et al., 1978). Rabbit DNA fragments from this clone were used as filter hybridiza,tion probes in analyses of restricted rabbit DNA. If the purified fragment does not contain repetitive DNA sequences, then it’ should only detect its corresponding DXA fragment in total rabbit DNA. If instead low then a corresponding number of additional repetitive sequences are present, hybridizing fragments should be seen. A series of probes prepared from RPGl DNA and which covered 1.4 kb of 5’ extragenic sequence, 0.9 kb of 3’ extragenic sequence and most of the coding and intervening sequences, all behaved as unique sequence DNA by this criterion. We have previously shown that a probe containing as little as 45 bp of sequence perfectly complementary to a /3-globin DNA fragment is capable of detecting this fragment in filter hybridizations (Jeffrey3 $ Flavell, 19773). Thus any undetected reiterated sequences near the rabbit fl-globin gene must either be very short, or diverged in sequence from the corresponding cloned sequence, or be highly A + Trich and incapable of forming stable hybrids under the stringent conditions of hybridization used. Under relatively low stringencies (0.3 to I.0 x SSC at 65’C) rabbit /3-globin cDNA detects additional ,&related gene sequences (Jeffreys & Flavell, 1977b, and unpublished data), probably corresponding to three extra rabbit /3-globin genes described by Lacy et al. (1979). In contrast, extragenic probes, for example probe 1 in Figure 6, fails to detect sequences near these additional genes even after low stringency posthybridization washes. It therefore appears that sequences outside the p-globin genes are more diverged than those coding for p-globin. This phenomenon has been described in detail for the mouse pd-major and pd-minor globin genes (Konkel et al., 1979). It therefore appears that at least 1.4 kb of sequence on the 5’ side of the rabbit ,9-globin gene, covering all sequences except for 9 bp between the 5’ PvuII site and the beginning of the structural gene (see Fig. 3) are devoid of repetitive DNA elements. The significance of this finding in relation to possible roles of repetitive DNA sequences in eukaryotic genomes requires that the position of origin of transcription of the ,Y-globin gene be known. A 15 S precursor of mouse fl-globin mRNA has been described by several workers (Curtis $ Weissmann, 1976; Ross, 1976; Bastos & Aviv, 1977; Curtis et al., 1977a,b) and has been shown to be a continuous transcript of the /3-globin coding sequences plus both intervening sequences (Tilghman et al., 1978a,b; Schambijck et al., 1979). Furthermore, Weaver $ Weissmann (1979) have shown that the 5’ end of the 15 S mouse precursor is identical to that of mature mouse /3-globin
REPETITIVE
DNA
NEAR
RABBIT
,6-GLOBIN
GENE
545
mRNA, and they argue that the 5’ end of p-globin mRNA may well represent the point of initiation of transcription of the mouse /3-globin gene. Equally the rabbit /3-globin gene is also transcribed to give a 15 S precursor mRNA containing both intervening sequences (Flavell et al., 1979), and whose 5’ end is coincident with the 5’ end on the mRNA. Recent in vitro transcription experiments using RNA polymerase II and extracts of KB cells have shown that discrete transcripts with the same 5’ end as the 15 S pre mRNA and mRNA made in viwo can be obtained using RpGl DNA as template (P. Jat, R. I. Kamen and R. A. Flavell, unpublished results). The within the single-copy DNA sequences rabbit /3-globin promoter lies, therefore, characterized in this paper. Thus 1.4 kb of DNA upstream from the likely origin of transcription of the rabbit /3-globin gene appears to be devoid of reiterated DNA sequences. This is difficult to reconcile with the type of “receptor” genes, proposed by Britten & Davidson (1969), which were predicted to consist of intermediate repetitive DNA. From the arguments described above, we consider it unlikely that repetitive DNA elements are involved in the promotion of RNA synthesis. If such “receptor” genes exist, then they must act at a greater distance from the structural gene than hitherto expected. It is possible that the rabbit p-globin gene is exceptional, and for this reason it will be important to search for repetitive DNA elements on the 5’ side of other developmentally regulated genes. A similar comment may be made about the two intervening sequences within the rabbit /3-globin gene. Probes 3 and 4, which contain, respectively, the whole of the small intervening sequence and all except for the last 43 bp of the large intervening sequence (Fig. 3, see Van Den Berg et al., 1978), were devoid of reiterated sequences. These intervening sequences therefore appear to be unique sequence and are not found elsewhere in the genome, as might be expected if these sequences acted as common regulatory elements in many structural genes. The same result has been reported for the mouse /I-globin gene (Miller et al., 1978) and the chicken ovalbumin gene (Garapin et al., 1978). Both reassociation analysis of total rabbit DNA and the use of purified probes from the cloned rabbit /3-globin gene have revealed an intermediate repetitive sequence on the 3’ side of the structural gene somewhere within a segment of DNA located 0.9 to 2.1 kb from the end of the gene. This sequence reanneals with a Cat* of about 0.2, compared with a Cot, of about 1000 for unique sequence DNA (see Fig. 2). This suggests an approximate reiteration frequency of lOOO/O~X = 5000-fold for this repetitive DNA element. Probe 6 (Fig. 3) also contains this repetitive DNA element and hybridizes to many different fragments in restriction endonuclease digests of total rabbit DNA. There was no evidence for any discrete labelled components, as might be expected if most of these reiterated sequences are arranged in tandem within the rabbit genome. The heterogeneous pattern of hybridization to restricted rabbit DNA suggests instead that most or all of these sequences are arranged in an interspersed fashion within rabbit DNA. However, in situ hybridization of rabbit metaphase chromosomes with 3H-labelled plasmid RfiGl DNA, which occurs via this reiterated element, shows marked preferential labelling of centromeres and telomeres on a number of chromosomes. Thus the bulk of these sequences are located on more than one chromosome, and
546 appear
H. to
A.
M.
be preferentially
HOEIJMAKERS-VAN localized
DOMMELEN within
centromeric
ET and
AL.
telomeric
regions.
The
chromosome localization of the rabbit /3-globin gene itself is not yet known. We do not know as yet whether the copy of the repetitive DNA sequence on the 3’ side of the rabbit p-globin gene is functionally linked with the expression of this gene, although this sequence is certainly not part of the 15 S precursor mRNA. In this respect, it is of interest that the human /I-globin gene also has a 3’ repetitive DNA element at the same position with respect to the structural gene (H. A. Hoeymakers, unpublished results). Insight into the function of the intermediate repetitive sequence and other sequences flanking the /3-globin gene will depend on suitable assay systems in which cloned globin genes can be expressed (Hamer & Leder, 1979 ; Mantei et al., 1979). This coupled with in vitro site-directed mutagenesis of sequences of interest in the cloned gene (Flavell et al., 1974; Mertz et aE., 1975) should ultimately make it possible to relate reiterated and other sequences to gene function in higher organisms. We thank Professor P. Borst, for helpful discussions. This work was supported in part by grants to P. Borst and one of t,he other authors (R. A. F.) from The Netherland Foundation for Chemical Research (SON) with financial aid from The Netherlands Organisation for the Advancement of Pure Research (ZWO) ; also by SRC grant no. GR/A 86466 awarded to Professor H. C. Macgregor and J. M. V ; A. J . J. was a postdoctoral fellow of the European Molecular Biolog)Organization during the early stages of this research.
REFERENCES Bastos, R. N. & Aviv. H. (1977). Cell, 11, 641-650. Birnstiel, M. L., Chipchase, M. & Spiers, J. (1971). Prog. NucZ. Acid Res. MoZ. BioZ. 11, 351-390. Birnstiel, M., Telford, J., Weinberg, E. & Stafford, D. (1974). Proc. Nat. Acud. Sci., U.S.A. 71, 29os2904. Bishop, J. D. & Freeman, K. B. (1974). Cold Spring Harbor Symp. Quant. Biol. 38, 707-716. Britten, R. J. & Kohne, D. E. (1968). Science, 161, 529-540. Britten, R. J. & Davidson, E. H. (1969). Science, 165, 349-357. Britten, R. J., Graham, D. E. & Neufeld, B. R. (1974). Methods Emzym,oZ. 29E, 363-418. Crawford, L. V. & Robbins, A. K. (1976). J. Gen. Viral. 31, 315-322. Curtis, P. J. & Weissmann, C. (1976). J. Mol. Biol. 106, 1061-1075. Curtis, P. J., Mantei, N., Van Den Berg, J. & Weissmann, C. (1977a). Proc. Nat. Acad. Sci., U.S.A. 74, 3184-3188. Curtis, P. J., Mantei, N. & Weissmann, C. (19776). Cold Spring Harbor Symp. Quant. Biol. 42, 971-984. Davidson, E. H., Graham, D. E., Neufeld, B. R., Chamberlin, M. E., Amenson, C. S., Hough, B. R. & Britten, R. J. (1974). Cold Spring Harbor Symp. Quunt. Biol. 38, 295-301. Davidson, E. H., Hough, B. R., Klein, W. H. & Britten, R. F. (1975). Cell, 4, 217-238. Flavell, R. A., Sabo, D. L., Bandle, E. F. & Weissmann, C. (1974). J. Mol. Biol. 89, 255-272. Flavell, R. A., Jeffreys, A. J. & Grosveld, G. C. (1978a). Cold Spring Harbor Symp. Quant. Biol. 42, 1003-1010. Flavell, R. A., Kooter, J. M., De Boer, E., Little, P. F. R. & Williamson, R. (1978b). Cell, 15. 25-41.
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