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Highly repeated DNA of the baboon: Organization of sequences homologous to highly repeated DNA of the African green monkey
J. Mol. Biol. (1979) 134, 835-842
LETTERS TO THE EDITOR
Highly Repeated DNA of the Baboon: Organization of Sequences Homologous to Highly Repeated DNA of the African Green Monkey In the African green monkey genome, 20% of the total DNA consists of a highly reiterated DNA sequence that occurs largely in long tandem arrays of a repeat unit that
is 172 base-pairs
in length.
The DNA of the baboon contains sequences in the baboon genome, these sequences comprise roughly 6% of the total DNA and alternate in a regular fashion with a DNA4 segment that may be distantly related to the monkey repeat unit. The sequences in the baboon that are homologous to the monkey repeat unit are contained within a 340 base-pair repeat unit of the highly repeated DNA fraction of the baboon. The extent of nucleotide divergence of the homologous repeated sequences between the two species is estimated to be about 10%.
homologous to this repeat unit. However,
Restriction enzyme analysis of genomic DNA from a variety of primates has demonstrabed the presence of highly reiterated DNA components whose repeat lengths are integral multiples of about 170 base-pairs (Griiss & Sauer, 1975; Fittler, 1977 ; Rosenberg et al., 1978; Donehower & Gillespie, 1979 ; Manuelides, 1978). Digestion of African green monkey (Cercopithecus aethiops) DNA with EndoR *Hind111 releases a set of DNA fragments of 172 base-pairs in length (AGMr(HindIII)-It); the nucleotide sequence specifying the most abundant residue at each position has been determined (Rosenberg et al., 1978). Sequences homologous to AGMr(HindIII)-1 represent about 20% of the African green monkey genomic DNA (Table 1) and occur both in long tandem arrays$ and interspersed with other sequences (Singer, 1979). The genome of the baboon Papio cynocephalus also contains tandem arrays of highly repeated DNA: partial digestion of baboon DNA with EndoR. BamHI releases a series of DNA fragments whose lengths are integral multiples of 340 basepairs (Donehower t Gillespie, 1979). Restriction enzyme analysis of the 340 base-pair reveals the presence of three EndoR. AluI sites within fragment (BABr(BamHI)-1) the monomer unit (Donehower & Gillespie, 1979; shown in Pig. 1). Therefore, digestion of total baboon DNA with EndoR. AluI releases the highly repeated DNA as a series t In this paper, we have adopted the nomenclature proposed by Rosenberg et al. (1978) for restriction enzyme fragments of African green monkey genomic DNA. The species of origin is designated by a 3-letter abbreviation, AGM for the monkey and BAB for the baboon, followed by I‘, to symbolize a reiterated DNA class; the enzyme generating the fragments is indicated in parenthesis; the integral multiple is indicated following a hyphen. Thus, the DNA fragments released following EndoR.HindIII digestion of African green monkey DNA are denoted as AGMr(HiadIII)-a; the monomer is then AGMr(HindIII)-1, the dimer AGMr(HindIII)-2. Similarly, the fragments produced by EndoR. BarnHI cleavage of baboon DNA are BABr( BanzHI)-n. $ Long tandem repeats of AGMr(HindIII)-1 have been separated as a satellite of main band DNA in buoyant density gradients and have been termed component a (Maio, 1971; Fittler, 1977). 836 0022-2836/79/320835-O-08
$02.00/O
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FIG. 1. Restriction map of the highly repeated DNA fraction of the baboon genome. The restriction map of the highly repeated DNA fraction of the baboon genome with respect to EndoR.BamHI (C ), EndoR.HindIII (C ) and EndoR. AZuI ( L ) is indicated in the central portion of the Figure. The indicated lengths (in base-pairs) of the DNA fragments were determined by electrophoretic mobilities relative to known standards (Donehower & Gillespie, 1979). The upper portion of the Figure shows the major products of a partial digestion of total baboon DNA witn EndoR.AZuI. The lower portion of the Figure shows the major products of a partial digestion of the BABr(BamHI)-1 monomer with EndoR.HindIII. Hatched regions of the various fragments correspond to regions of the repeat unit containing sequences homologous to AGMr(HindIII)-1 as determined by filter hybridization experiments shown in Figs 2 and 3.
of fragments whose lengths are multiples of 1’70 base-pairs. as well as othrr digest’iorl products (see Figs 1 and 3). In this study, it is demonstrated that sequences homologous to the dGMr(Hindlll)1 are present in baboon DNA within the BABr(BumHI)-1 and BABr(AZuI)-1 DNA fragments. The presence of sequences homologous t,o AGMr(HindIIT)-1 in baboon DNA was detected by hybridization of a 32P-labeled AGMr(Hin.dIII)-1 DNA probe to baboon DNA at 60°C (Table 1). Hybridization of the probe to baboon DNA occurred at a rate of 58.8 M-l s-l, or 6.5 times slower than to African green monkey DNA. The rate of the heterologous hybridizat,ion is retarded in proportion to the extent of divergence of the sequence between the two species (Bonner et al., 1973).
LETTERS
TO
THE
837
EDITOR
TABLE 1 Characteristics
of a DNA component homologous to AGMr(HindIII)-I in baboon and African green monkey genomes
1.7 x 10-Z 2.6x 1O-3
Baboon Monkey
58.8 382
3.0 (6.0) 19.4
74 83
Baboon DNA was prepared as described previously (Donehower et ccl., 1977). African green monkey DNA was prepared from BSC-1 cells, an established cell line derived from monkey kidney. Each DNA sample was sheared to a length of 250 to 450 base-pairs in a V&is 45 homogenizer. AGMr(HindIII)-1 DNA was isolated from BSC-I DNA by digestion with EndoR.HindIII DNA was preas described previously (Rosenberg et nl., 1978). 3ZP-labeled AGMr(HindIII)-1 pared by nick-translation followed by removal of fold-back sequences (Rigby et al., 1977). The specific radioactivity was 5.6 x lo6 cts/min per pg of DNA. Hybridization kinetics were measured in 0.12 x-phosphate buffer at 60°C. Driver DNA excess was 3 x IO3 to 3 x 105. Hybrid formation was assayed by hydroxyapatite column chromatography. The extents of hybridization were 86% in both cases. Under these condit,ions of hybridization, no self-reassociation of the tracer occurred; residual fold-back material constituted 6%. t Rate constant for the hybridization reaction. : The percentage of the total genome that is homologous to AGMr(H&dIII)-1 was determined from the rates of hybridization of monkey and baboon DNA relative to that of pure AGMrHin dII1) - 1 as follows : percentage
of genome
=
K(sample) K(AGMr(HindIII)-1)
x 100.
The
“ZP-labelled AGMr(HindIII)-1 tracer reannealed to pure AGMr(HindlII)-1 DNA with a of 505 x 10m4. The figure in parenthesis represents the percentage of the baboon genome when corrected for the effect of sequence divergence on the rate of reassociation. $ 32P-labelled AGMr(HindIII)-1 DNA was hybridized to either baboon DNA to a C,t value of buffer at 60°C. Following hybridization, 1 or monkey DNA to C,t value of 0.2 in 0.12 M-phosphate t,he double-stranded fraction (84% and 86.2% for baboon and monkey, respectively) was bound to hydroxyapatite in water-jacketed columns at 60°C. The temperature of the columns was then raised in 2 deg. C increments to 98°C. At each temperature, the columns were washed with 0.12 Mphosphate buffer to elute single-stranded DNA.
Cott value
Thermal chromatography of DNA duplexes formed from 32P-labeled AGMr(HindIII)1 and either monkey or baboon DNA demonstrated that the t, value of the heterologous duplex is 9 deg. C lower than that of the homologous duplex (Table 1). Therefore, the relative divergence of DNA sequences homologous to AGMr(HindIII)-1 between the two species is estimated to be about 10% based on the relative t, values (McCarthy & Church, 1970). Assuming that as a result of this divergence the rate of hybridization of 32P-labeled AGMr(HindIII)-1 to baboon DNA will be reduced twofold relative to the rate of the homologous reaction (Bonner et al., 1973), it can be calculated that roughly 6% of the baboon genome contains sequences homologous to AGMr(HindIII)-1 (Table 1). To examine the possibility that an additional large family of sequences, more distantly related to AGMr(HindIII)-1, is present in baboon DNA but not detected a,t 60°C. the extent of hybridization of 3ZP-labeled AGMr(HindIII)-1 to an excess of baboon DNA was measured as a function of the temperature of incubation at a constant C,tt value (Table 2). A reduction in temperature from 60°C to 50°C results tC,t,
DNA
concentration
x
time.
838
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Effect of temperature on the extent of hybridization of 32P-labeled AGMr(HindIII)-1 to baboon DNA Extent of hybridization? of [=P]AGMr( HindHI) (“4)
Temperature (“Cl
Baboon 66.4 (69.6): 64.0 (66.3) 57.1
50 66 60
_1
AMonkey 67.9 69.7 il.1
A 3ZP-labeled AGMr(HindIII)-1 tracer, prepared by nick-translation, was hybridized with each of the DNA preparations in 0.12 M-phosphate buffer at 60, 55 and 60°C. Hybrid formation was assayed by hydroxyapatite column chromatography as follows. Single-stranded DNA was eluted at the temperature of incubation, in 0.12 M-phosphate buffer. Double-stranded hybrids were eluted at 98’C, in 0.12 M-phosphate buffer. t To achieve comparable extents of hybridization of [“2P]AGMr(l-I i?ldIII)-1 to baboon and monkey DNA, hybridization was to C’,t 3 x 10m2 and 10e2 for baboon and monkey, respectively. $ The numbers in parentheses represent the corrected extent, of hybridizs.tion of [32P]AGMr(HindIII)-l to baboon DNA, which was calculated as : Extent Extent
of hybridization of hybridization
to monkey to monkey
DNA DNA
at t -:: 60°C at t 2 50, 55°C
Extent baboon
of hybridization t,o DNA at t = BOY, 55Y’, 1
in an increase in the observed extent of hybridization from 57.1*$ to 66+4q,. To correct for differences in the rate of hybridization as a function of temperature, the extent of hybridization of baboon DNA with AGMr(HindIII)-1 DNA at each temperature has been normalized to the extent of hybridization of monkey DNA with AGMr(HindIII)-1 at the same temperature. The corrected extent of hybridization of AGMr(HindIII)-1 DNA to baboon DNA at 50°C is 69.5”i’, (Table 2). Thus, at 50°C. the fraction of the baboon genomic DNA detected as homologous to AGMr(HindTII)-1 is about 13% as compared to 6% at 60°C. It is concluded that there are at least, t1v-o families of baboon sequences that are related to AGMr(HindIII)-I. The first,, a relatively closely related family, is detect’ed upon hybridization at 60°C (about, By;, of the total genome) and the second, a more distantly related family, is detected at) 50°C (about another 7% of the genome). It is possible that even more distantl) related sequences occur within the baboon genome which are not detected even at 50°C. The relationship between the highly repeated baboon DNA observed upon digestion of the total baboon DNA with the restriction enzymes EndoR. BamHI and EndoR. AZuI and the baboon sequence homologous to AGMr(HindIII)-1 was investigated. Total baboon DNA was treated with EndoR. BamHI, and the digestion products resolved on a polyacrylamide gel (Fig. 2(a)). The DNA fragments in the gel were transferred by the Southern technique (Southern, 1975) to nitrocellulose paper and hybridized to a 32P-labeled AGMr(HindIII)-1 probe (Fig. 2(b)). The results demonstrate that the monomer repeat unit of 340 base-pairs (BABr(BamHI)-1) contains sequences homologous to AGMr(HindIII)-1. To further localize the sequences homologous to AGMr(HindIII)-1 within the BABr(BamHI)-I DNA fragment, BABr(BamHI)-1 was isolated and further digested with EndoR.HindIII; previous studies have mapped two EndoR. Hind111 cleavage sites within the BABr( BamHI)- 1 fragment (Donehower & Gillespie, 1979). As shown in the lower part of Figure 1,
LETTERS
TO THE
340 -
- 340 - 285 340 - 225
170-
-
170
-
115
839
EDITOR
-
170 -
’ 340 .285 .225 .I70
- 55 (a)
(b)
Fro. 2. Sequences homologous to AGMr(HindITI)-1 in fragments of baboon and monkey DNA generated by restriction enzymes. Baboon DNA or monkey DNA were digested with EndoR.HindIII or EndoR*BalnHI for 4 h at 37°C. The digestion products were resolved by electrophoresis in a 5% polyacrylamide slab gel (25 V, 16 h) and stained with ethidium bromide (a). DNA fragments were then transferred to a nit,rocellulose filter (0.2 pm, Schleicher & Schuell) by a modification of the Southern technique (Southern, 1975; D. S. Singer, unpublished results). Hybridization of the 3aP-labelled AGMr(HindIII).1 probe to the filter followed the procedure of Botohan et al. (1976). A solution oontaining 0.02:/, Ficoll, 0.02% polyvinylpyrollidone, 0.02% bovine serum albumin, 100 pg Escherichin co& t,RNA/ml, 6 x SSC (SSC is 0.15 M-N&I, 0.015 M-sodium citrate, pH 7*0), 0.5% sodium tlodecyl sulfate and 3 x lo5 cts/min of 32P-labeled AGMr(HindII1).1 (spec. act. 8 x 10s cts/ min per pg) was incubated with the filter for 24 h at 66°C. Washing of the filters was as described by Botchan et al. (1976). The filter was exposed to Kodak X-omat film in the presence of intensifying screens for 3 days at ~ 70°C (b). Slot 1, monkey DNA (5 pg) digested with EndoR .HindIII (50 units) ; slot 2, AGMr(HindII1) -1 (5 pg) ; slot 3, baboon DNA (6 pg) digested with EndoR. BamHI (15 units) ; slot 4, baboon DNA (6 pg) digested with EndoR.HindIII (50 units); slot 5, BABr(BamHI)-1 (3 pg) digested with EndoR.HindIII (30 units). Fragment sizes (base-pairs) are indicated.
digestion of the BABr(BanzHI)-1 DNA fragment with EndoR*HindIII results in a series of five fragments (Fig. 2). The partial digestion products corresponding in length to 285, 225 and 170 base-pairs are observed to contain sequences homologous to AGMr(HindIII)-1 (Fig. 2(b)). H owever, under the present conditions of hybridization, DNA fragments of 115 and 5.5 base-pairs are not homologous to AGMr(HindIII)-1. These results indicate that, only a portion of the BABr(BamHI)-1 monomer contains sequences homologous to AGMr(HindIII)-1. These homologous sequences are contained within the 170 base-pair fragment released by EndoR *Hind111 digestion of the BABr(&mHI)-I monomer DNA. To further confirm this conclusion, the ability of the digestion products of baboon DNA DNA treated with EndoR. AZuI to hybridize to 32P-labeled AGMr(HindIII)-1 was investigated. Partial digestion of baboon DNA with EndoR. AluI generates a series of six fragments smaller than 340 base-pairs, in addition to the greater length multiples of the monomer (Fig. 3(a)). All of these fragments can be resolved on polyacrylamide gels (except for the two different fragments that are 170 base-pairs in
840
D.
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340
-290 -
220
-1770
-
I 70
120
120
-50
I
2
3
4
I
2
3
4
(b) Fro. 3. Sequences homologous to AGMr(Hi)zdIII)1 in fmyrnents of b&orm anti monlir~y I>Nd generated by restriction enzymes. Products of restriction enzyme digestion of baboon a.ntl monlwy I)SA \v<‘rr rewlwtl on polyacrylttmide slab gels, transferred to nit~rocellulosr paper and hybridizrtl to AGMr(HindIII)-1 as gel: (b) the autoradiogram of the filt~cr described in the legend to Fig. 2. (a) The cthidium-st,ainctl following hybridization. Slot 1, total baboon DNA (5 pg) digestetl with Etr~loK~Ni~r~ll II (20 units) ; ;ilot 2, t,otwl bitbo~m DNA (5 pg) digested with EndoR.HklIII (20 units) and EndoR. J~~vEHI (20 units): slot 3. purified BABr(HamHI)-1 (3 pg) digostcd \+iith EncloK~HintlIII (20 units): slot 4, total baboon DNA (5 pg) digested with EndoR .AZul (15 units). Fragment. sizes (base-pairs) are indicated.
length). The DNA fragments in the gel were transferred to nitrocellulose paper and hybridized to a 32P-labeled AGMr(HindIII)-1 DNA (Fig. 3(b)). The results again demonstrate that sequences homologous to AGMr(HindIJl)-1 art: contained within the highly repeated DNA of the baboon : baboon DKA fragments of 340,290, 170 and 120 base-pairs. generated by EndoR* Alul. hybridize with 32P-labeled AGMr(HindIII)-1 probe. However, baboon DNA fragments of 220 and 65 base-pairs that are also generated by parCal digestion of baboon DNA with EndoR.AZu.1. do not hybridize with the 32P-labeled AGMr(HindIIT)-1 probe. The observed lack of hybridization of the 220 base-pair fragment is unlikely to be due to inefficient transfer to or retention on the nitrocellulose filters, since the other baboon DNA fragments generat#ed in the same digestion by EndoR.AluI a,re observed to hybridize with the probe. Therefore, it is concluded that the DNA fragment of 220 base-pairs in length does not cont,ain sequences homologous to AGMr(HindIII)-1. Taken together, these results
LETTERS
TO
THE
EDITOR
841
demonstrate that in the highly reiterated DNA fraction of the baboon genome, sequences homologous to AGMr(HindIII)-1 alternate with a DNA fragment that is not homologous as defined by the hybridization conditions used here (see central portion of Fig. 1). The genomes of both baboon and African green monkey contain highly repeated DNA sequences that occur, at least in part, in long tandem arrays. In the monkey DNA, sequences homologous to AGMr(HindIII)-1 DNA represent about 20% of the total genome. Roughly three-quarters of these sequences occur in long tandem arrays (Rosenberg et al., 1978), while the remaining quarter is interspersed with unrelated sequences (Singer, 1979). Baboon DNA contains tandem arrays of a repeat unit of 340 base-pairs BABr(BamHI)-1. As assessed under the present conditions, sequences homologous to AGMr(HindIII)-1 are contained within a part of the rather than occurring in long tandem BABr(BamHI)-1 repeat unit. Therefore, repeats as in the monkey, the sequences homologous to AGMr(HindIII)-1 in the baboon alternate in a regular fashion with a DNA fragment that is sufficiently different that it no longer hybridizes under these conditions. It was noted that the to baboon DNA increases extent of hybridization of 32P-labeled AGMr(HindIII)-1 about twofold when the temperature of the hybridization reaction is reduced from 60°C to 50°C. This suggests the possibility that in the baboon the sequences homologous to AGMr(HindIII)-1 alternate with sequences more distantly related to AGMr(HindIII)-1. Recent nucleotide sequence analysis of the BABr(BamHI)-1 DNA fragment supports this notion (L. Donehower, unpublished results). The 170 base-pair fragment of BABr(BamHI)-1 released by EndoR. Hind111 has been partially sequenced and found to be very closely related to AGMr(HindIII)-1, whereas the other two fragments (115 and 55 base-pairs) appear to be related but not identical. Taken together, these data suggest that the mechanisms of sequence amplification and divergence lead to overall non-random sequence divergence. Since the divergence of the two species, the most striking changes in the highly repeated, complex sequence DNA fraction have been in the amount and organization of the DNA sequences closely related to AGMr(HindIII)-1. It, has recently been proposed (Fry & Salser, 1977) that simple sequence satellite DNA fractions are conserved between closely related rodent species. The present data suggest that highly repeated, complex sequence DNA fractions are also conserved between closely related primate species, but are represented in different frequencies and organization. i2’c gratefully tories this work
acknowledge Drs Maxine Singer and David Gillespie, in whose was done, for continued support and many helpful discussions.
Laboratory of Biochemistry, National Institutes of Health Bethesda, MD 20205, U.S.A.
National
Section on Molecular Hybridization Laboratory of Tumor Cell Biology, National Institutes of Health, Bethesda, MD 20205, U.S.A. Received
28 November
Institute
DINAH
SINGER?
LARRYDONEHOWERS
National
1978, and in revised
t Present atddrrss: Immunology 1 Present address: Laboratory U.S.A.
Cancer
labora-
Cancer
form
Institute
3 May
1979
Branch, NCI, NIH, Bethesda, MD 20205, U.S.A. of Tumor Virus Genetics, NCl, NIH, Bethesda,
MI)
20205,
842
D. SINGER
AND
L. DONEHOWER
REFERENCES Bonner, T. I., Brenner, D. J., Neufeld, B. R. & B&ten, R. J. (1973). J. Mol. Biol. 81, 123-135. Botchan, M., Topp, W. & Sambrook, J. (1976). Cell, 9, 269-287. Donehower, L. & Gillespie, D. (1979). J. Mol. BioZ. 134, 805-834. Donehower, L., Wong-Staal, F. t Gillespie, D. (1977). J. ViroZ. 21, 932-941. Fittler, F. (1977). Eur. J. Biochem. 74, 343-352. Fry, K. & Salser, W. (1977). Cell, 12, 1069-1084. Griiss, P. & Sauer, G. (1975). FEBS Letters, 60, 85-88. Maio, J. (1971). J. Mol. BioZ. 56, 579-595. Manuelides, L. (1978). Nature (London), 276, 92-94. McCarthy, B. & Church, R. (1970). Annu. Rev. Biochem. 39, 131-150. Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P. (1977). J. Mol. Bid. 113, 237-251. Rosenberg, H., Singer, M. & Rosenberg, M. (1978). Science, 200, 394-402. Singer, D. S. (1979). J. BioZ. Chem. 254, 5506-5514. Southern, E. M. (1975). J. Mol. BioZ. 98, 503-517.
LETTERS TO THE EDITOR
Highly Repeated DNA of the Baboon: Organization of Sequences Homologous to Highly Repeated DNA of the African Green Monkey In the African green monkey genome, 20% of the total DNA consists of a highly reiterated DNA sequence that occurs largely in long tandem arrays of a repeat unit that
is 172 base-pairs
in length.
The DNA of the baboon contains sequences in the baboon genome, these sequences comprise roughly 6% of the total DNA and alternate in a regular fashion with a DNA4 segment that may be distantly related to the monkey repeat unit. The sequences in the baboon that are homologous to the monkey repeat unit are contained within a 340 base-pair repeat unit of the highly repeated DNA fraction of the baboon. The extent of nucleotide divergence of the homologous repeated sequences between the two species is estimated to be about 10%.
homologous to this repeat unit. However,
Restriction enzyme analysis of genomic DNA from a variety of primates has demonstrabed the presence of highly reiterated DNA components whose repeat lengths are integral multiples of about 170 base-pairs (Griiss & Sauer, 1975; Fittler, 1977 ; Rosenberg et al., 1978; Donehower & Gillespie, 1979 ; Manuelides, 1978). Digestion of African green monkey (Cercopithecus aethiops) DNA with EndoR *Hind111 releases a set of DNA fragments of 172 base-pairs in length (AGMr(HindIII)-It); the nucleotide sequence specifying the most abundant residue at each position has been determined (Rosenberg et al., 1978). Sequences homologous to AGMr(HindIII)-1 represent about 20% of the African green monkey genomic DNA (Table 1) and occur both in long tandem arrays$ and interspersed with other sequences (Singer, 1979). The genome of the baboon Papio cynocephalus also contains tandem arrays of highly repeated DNA: partial digestion of baboon DNA with EndoR. BamHI releases a series of DNA fragments whose lengths are integral multiples of 340 basepairs (Donehower t Gillespie, 1979). Restriction enzyme analysis of the 340 base-pair reveals the presence of three EndoR. AluI sites within fragment (BABr(BamHI)-1) the monomer unit (Donehower & Gillespie, 1979; shown in Pig. 1). Therefore, digestion of total baboon DNA with EndoR. AluI releases the highly repeated DNA as a series t In this paper, we have adopted the nomenclature proposed by Rosenberg et al. (1978) for restriction enzyme fragments of African green monkey genomic DNA. The species of origin is designated by a 3-letter abbreviation, AGM for the monkey and BAB for the baboon, followed by I‘, to symbolize a reiterated DNA class; the enzyme generating the fragments is indicated in parenthesis; the integral multiple is indicated following a hyphen. Thus, the DNA fragments released following EndoR.HindIII digestion of African green monkey DNA are denoted as AGMr(HiadIII)-a; the monomer is then AGMr(HindIII)-1, the dimer AGMr(HindIII)-2. Similarly, the fragments produced by EndoR. BarnHI cleavage of baboon DNA are BABr( BanzHI)-n. $ Long tandem repeats of AGMr(HindIII)-1 have been separated as a satellite of main band DNA in buoyant density gradients and have been termed component a (Maio, 1971; Fittler, 1977). 836 0022-2836/79/320835-O-08
$02.00/O
0 1979 Academic Press Ina. (London) Ltd.
836
D.
SINGER
AND
1,. DOXEHOWER
FIG. 1. Restriction map of the highly repeated DNA fraction of the baboon genome. The restriction map of the highly repeated DNA fraction of the baboon genome with respect to EndoR.BamHI (C ), EndoR.HindIII (C ) and EndoR. AZuI ( L ) is indicated in the central portion of the Figure. The indicated lengths (in base-pairs) of the DNA fragments were determined by electrophoretic mobilities relative to known standards (Donehower & Gillespie, 1979). The upper portion of the Figure shows the major products of a partial digestion of total baboon DNA witn EndoR.AZuI. The lower portion of the Figure shows the major products of a partial digestion of the BABr(BamHI)-1 monomer with EndoR.HindIII. Hatched regions of the various fragments correspond to regions of the repeat unit containing sequences homologous to AGMr(HindIII)-1 as determined by filter hybridization experiments shown in Figs 2 and 3.
of fragments whose lengths are multiples of 1’70 base-pairs. as well as othrr digest’iorl products (see Figs 1 and 3). In this study, it is demonstrated that sequences homologous to the dGMr(Hindlll)1 are present in baboon DNA within the BABr(BumHI)-1 and BABr(AZuI)-1 DNA fragments. The presence of sequences homologous t,o AGMr(HindIIT)-1 in baboon DNA was detected by hybridization of a 32P-labeled AGMr(Hin.dIII)-1 DNA probe to baboon DNA at 60°C (Table 1). Hybridization of the probe to baboon DNA occurred at a rate of 58.8 M-l s-l, or 6.5 times slower than to African green monkey DNA. The rate of the heterologous hybridizat,ion is retarded in proportion to the extent of divergence of the sequence between the two species (Bonner et al., 1973).
LETTERS
TO
THE
837
EDITOR
TABLE 1 Characteristics
of a DNA component homologous to AGMr(HindIII)-I in baboon and African green monkey genomes
1.7 x 10-Z 2.6x 1O-3
Baboon Monkey
58.8 382
3.0 (6.0) 19.4
74 83
Baboon DNA was prepared as described previously (Donehower et ccl., 1977). African green monkey DNA was prepared from BSC-1 cells, an established cell line derived from monkey kidney. Each DNA sample was sheared to a length of 250 to 450 base-pairs in a V&is 45 homogenizer. AGMr(HindIII)-1 DNA was isolated from BSC-I DNA by digestion with EndoR.HindIII DNA was preas described previously (Rosenberg et nl., 1978). 3ZP-labeled AGMr(HindIII)-1 pared by nick-translation followed by removal of fold-back sequences (Rigby et al., 1977). The specific radioactivity was 5.6 x lo6 cts/min per pg of DNA. Hybridization kinetics were measured in 0.12 x-phosphate buffer at 60°C. Driver DNA excess was 3 x IO3 to 3 x 105. Hybrid formation was assayed by hydroxyapatite column chromatography. The extents of hybridization were 86% in both cases. Under these condit,ions of hybridization, no self-reassociation of the tracer occurred; residual fold-back material constituted 6%. t Rate constant for the hybridization reaction. : The percentage of the total genome that is homologous to AGMr(H&dIII)-1 was determined from the rates of hybridization of monkey and baboon DNA relative to that of pure AGMrHin dII1) - 1 as follows : percentage
of genome
=
K(sample) K(AGMr(HindIII)-1)
x 100.
The
“ZP-labelled AGMr(HindIII)-1 tracer reannealed to pure AGMr(HindlII)-1 DNA with a of 505 x 10m4. The figure in parenthesis represents the percentage of the baboon genome when corrected for the effect of sequence divergence on the rate of reassociation. $ 32P-labelled AGMr(HindIII)-1 DNA was hybridized to either baboon DNA to a C,t value of buffer at 60°C. Following hybridization, 1 or monkey DNA to C,t value of 0.2 in 0.12 M-phosphate t,he double-stranded fraction (84% and 86.2% for baboon and monkey, respectively) was bound to hydroxyapatite in water-jacketed columns at 60°C. The temperature of the columns was then raised in 2 deg. C increments to 98°C. At each temperature, the columns were washed with 0.12 Mphosphate buffer to elute single-stranded DNA.
Cott value
Thermal chromatography of DNA duplexes formed from 32P-labeled AGMr(HindIII)1 and either monkey or baboon DNA demonstrated that the t, value of the heterologous duplex is 9 deg. C lower than that of the homologous duplex (Table 1). Therefore, the relative divergence of DNA sequences homologous to AGMr(HindIII)-1 between the two species is estimated to be about 10% based on the relative t, values (McCarthy & Church, 1970). Assuming that as a result of this divergence the rate of hybridization of 32P-labeled AGMr(HindIII)-1 to baboon DNA will be reduced twofold relative to the rate of the homologous reaction (Bonner et al., 1973), it can be calculated that roughly 6% of the baboon genome contains sequences homologous to AGMr(HindIII)-1 (Table 1). To examine the possibility that an additional large family of sequences, more distantly related to AGMr(HindIII)-1, is present in baboon DNA but not detected a,t 60°C. the extent of hybridization of 3ZP-labeled AGMr(HindIII)-1 to an excess of baboon DNA was measured as a function of the temperature of incubation at a constant C,tt value (Table 2). A reduction in temperature from 60°C to 50°C results tC,t,
DNA
concentration
x
time.
838
D.
SINGER
AND
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TABLE
DONEHOWER 2
Effect of temperature on the extent of hybridization of 32P-labeled AGMr(HindIII)-1 to baboon DNA Extent of hybridization? of [=P]AGMr( HindHI) (“4)
Temperature (“Cl
Baboon 66.4 (69.6): 64.0 (66.3) 57.1
50 66 60
_1
AMonkey 67.9 69.7 il.1
A 3ZP-labeled AGMr(HindIII)-1 tracer, prepared by nick-translation, was hybridized with each of the DNA preparations in 0.12 M-phosphate buffer at 60, 55 and 60°C. Hybrid formation was assayed by hydroxyapatite column chromatography as follows. Single-stranded DNA was eluted at the temperature of incubation, in 0.12 M-phosphate buffer. Double-stranded hybrids were eluted at 98’C, in 0.12 M-phosphate buffer. t To achieve comparable extents of hybridization of [“2P]AGMr(l-I i?ldIII)-1 to baboon and monkey DNA, hybridization was to C’,t 3 x 10m2 and 10e2 for baboon and monkey, respectively. $ The numbers in parentheses represent the corrected extent, of hybridizs.tion of [32P]AGMr(HindIII)-l to baboon DNA, which was calculated as : Extent Extent
of hybridization of hybridization
to monkey to monkey
DNA DNA
at t -:: 60°C at t 2 50, 55°C
Extent baboon
of hybridization t,o DNA at t = BOY, 55Y’, 1
in an increase in the observed extent of hybridization from 57.1*$ to 66+4q,. To correct for differences in the rate of hybridization as a function of temperature, the extent of hybridization of baboon DNA with AGMr(HindIII)-1 DNA at each temperature has been normalized to the extent of hybridization of monkey DNA with AGMr(HindIII)-1 at the same temperature. The corrected extent of hybridization of AGMr(HindIII)-1 DNA to baboon DNA at 50°C is 69.5”i’, (Table 2). Thus, at 50°C. the fraction of the baboon genomic DNA detected as homologous to AGMr(HindTII)-1 is about 13% as compared to 6% at 60°C. It is concluded that there are at least, t1v-o families of baboon sequences that are related to AGMr(HindIII)-I. The first,, a relatively closely related family, is detect’ed upon hybridization at 60°C (about, By;, of the total genome) and the second, a more distantly related family, is detected at) 50°C (about another 7% of the genome). It is possible that even more distantl) related sequences occur within the baboon genome which are not detected even at 50°C. The relationship between the highly repeated baboon DNA observed upon digestion of the total baboon DNA with the restriction enzymes EndoR. BamHI and EndoR. AZuI and the baboon sequence homologous to AGMr(HindIII)-1 was investigated. Total baboon DNA was treated with EndoR. BamHI, and the digestion products resolved on a polyacrylamide gel (Fig. 2(a)). The DNA fragments in the gel were transferred by the Southern technique (Southern, 1975) to nitrocellulose paper and hybridized to a 32P-labeled AGMr(HindIII)-1 probe (Fig. 2(b)). The results demonstrate that the monomer repeat unit of 340 base-pairs (BABr(BamHI)-1) contains sequences homologous to AGMr(HindIII)-1. To further localize the sequences homologous to AGMr(HindIII)-1 within the BABr(BamHI)-I DNA fragment, BABr(BamHI)-1 was isolated and further digested with EndoR.HindIII; previous studies have mapped two EndoR. Hind111 cleavage sites within the BABr( BamHI)- 1 fragment (Donehower & Gillespie, 1979). As shown in the lower part of Figure 1,
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340 -
- 340 - 285 340 - 225
170-
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170
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115
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170 -
’ 340 .285 .225 .I70
- 55 (a)
(b)
Fro. 2. Sequences homologous to AGMr(HindITI)-1 in fragments of baboon and monkey DNA generated by restriction enzymes. Baboon DNA or monkey DNA were digested with EndoR.HindIII or EndoR*BalnHI for 4 h at 37°C. The digestion products were resolved by electrophoresis in a 5% polyacrylamide slab gel (25 V, 16 h) and stained with ethidium bromide (a). DNA fragments were then transferred to a nit,rocellulose filter (0.2 pm, Schleicher & Schuell) by a modification of the Southern technique (Southern, 1975; D. S. Singer, unpublished results). Hybridization of the 3aP-labelled AGMr(HindIII).1 probe to the filter followed the procedure of Botohan et al. (1976). A solution oontaining 0.02:/, Ficoll, 0.02% polyvinylpyrollidone, 0.02% bovine serum albumin, 100 pg Escherichin co& t,RNA/ml, 6 x SSC (SSC is 0.15 M-N&I, 0.015 M-sodium citrate, pH 7*0), 0.5% sodium tlodecyl sulfate and 3 x lo5 cts/min of 32P-labeled AGMr(HindII1).1 (spec. act. 8 x 10s cts/ min per pg) was incubated with the filter for 24 h at 66°C. Washing of the filters was as described by Botchan et al. (1976). The filter was exposed to Kodak X-omat film in the presence of intensifying screens for 3 days at ~ 70°C (b). Slot 1, monkey DNA (5 pg) digested with EndoR .HindIII (50 units) ; slot 2, AGMr(HindII1) -1 (5 pg) ; slot 3, baboon DNA (6 pg) digested with EndoR. BamHI (15 units) ; slot 4, baboon DNA (6 pg) digested with EndoR.HindIII (50 units); slot 5, BABr(BamHI)-1 (3 pg) digested with EndoR.HindIII (30 units). Fragment sizes (base-pairs) are indicated.
digestion of the BABr(BanzHI)-1 DNA fragment with EndoR*HindIII results in a series of five fragments (Fig. 2). The partial digestion products corresponding in length to 285, 225 and 170 base-pairs are observed to contain sequences homologous to AGMr(HindIII)-1 (Fig. 2(b)). H owever, under the present conditions of hybridization, DNA fragments of 115 and 5.5 base-pairs are not homologous to AGMr(HindIII)-1. These results indicate that, only a portion of the BABr(BamHI)-1 monomer contains sequences homologous to AGMr(HindIII)-1. These homologous sequences are contained within the 170 base-pair fragment released by EndoR *Hind111 digestion of the BABr(&mHI)-I monomer DNA. To further confirm this conclusion, the ability of the digestion products of baboon DNA DNA treated with EndoR. AZuI to hybridize to 32P-labeled AGMr(HindIII)-1 was investigated. Partial digestion of baboon DNA with EndoR. AluI generates a series of six fragments smaller than 340 base-pairs, in addition to the greater length multiples of the monomer (Fig. 3(a)). All of these fragments can be resolved on polyacrylamide gels (except for the two different fragments that are 170 base-pairs in
840
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340
-290 -
220
-1770
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I 70
120
120
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I
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3
4
(b) Fro. 3. Sequences homologous to AGMr(Hi)zdIII)1 in fmyrnents of b&orm anti monlir~y I>Nd generated by restriction enzymes. Products of restriction enzyme digestion of baboon a.ntl monlwy I)SA \v<‘rr rewlwtl on polyacrylttmide slab gels, transferred to nit~rocellulosr paper and hybridizrtl to AGMr(HindIII)-1 as gel: (b) the autoradiogram of the filt~cr described in the legend to Fig. 2. (a) The cthidium-st,ainctl following hybridization. Slot 1, total baboon DNA (5 pg) digestetl with Etr~loK~Ni~r~ll II (20 units) ; ;ilot 2, t,otwl bitbo~m DNA (5 pg) digested with EndoR.HklIII (20 units) and EndoR. J~~vEHI (20 units): slot 3. purified BABr(HamHI)-1 (3 pg) digostcd \+iith EncloK~HintlIII (20 units): slot 4, total baboon DNA (5 pg) digested with EndoR .AZul (15 units). Fragment. sizes (base-pairs) are indicated.
length). The DNA fragments in the gel were transferred to nitrocellulose paper and hybridized to a 32P-labeled AGMr(HindIII)-1 DNA (Fig. 3(b)). The results again demonstrate that sequences homologous to AGMr(HindIJl)-1 art: contained within the highly repeated DNA of the baboon : baboon DKA fragments of 340,290, 170 and 120 base-pairs. generated by EndoR* Alul. hybridize with 32P-labeled AGMr(HindIII)-1 probe. However, baboon DNA fragments of 220 and 65 base-pairs that are also generated by parCal digestion of baboon DNA with EndoR.AZu.1. do not hybridize with the 32P-labeled AGMr(HindIIT)-1 probe. The observed lack of hybridization of the 220 base-pair fragment is unlikely to be due to inefficient transfer to or retention on the nitrocellulose filters, since the other baboon DNA fragments generat#ed in the same digestion by EndoR.AluI a,re observed to hybridize with the probe. Therefore, it is concluded that the DNA fragment of 220 base-pairs in length does not cont,ain sequences homologous to AGMr(HindIII)-1. Taken together, these results
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demonstrate that in the highly reiterated DNA fraction of the baboon genome, sequences homologous to AGMr(HindIII)-1 alternate with a DNA fragment that is not homologous as defined by the hybridization conditions used here (see central portion of Fig. 1). The genomes of both baboon and African green monkey contain highly repeated DNA sequences that occur, at least in part, in long tandem arrays. In the monkey DNA, sequences homologous to AGMr(HindIII)-1 DNA represent about 20% of the total genome. Roughly three-quarters of these sequences occur in long tandem arrays (Rosenberg et al., 1978), while the remaining quarter is interspersed with unrelated sequences (Singer, 1979). Baboon DNA contains tandem arrays of a repeat unit of 340 base-pairs BABr(BamHI)-1. As assessed under the present conditions, sequences homologous to AGMr(HindIII)-1 are contained within a part of the rather than occurring in long tandem BABr(BamHI)-1 repeat unit. Therefore, repeats as in the monkey, the sequences homologous to AGMr(HindIII)-1 in the baboon alternate in a regular fashion with a DNA fragment that is sufficiently different that it no longer hybridizes under these conditions. It was noted that the to baboon DNA increases extent of hybridization of 32P-labeled AGMr(HindIII)-1 about twofold when the temperature of the hybridization reaction is reduced from 60°C to 50°C. This suggests the possibility that in the baboon the sequences homologous to AGMr(HindIII)-1 alternate with sequences more distantly related to AGMr(HindIII)-1. Recent nucleotide sequence analysis of the BABr(BamHI)-1 DNA fragment supports this notion (L. Donehower, unpublished results). The 170 base-pair fragment of BABr(BamHI)-1 released by EndoR. Hind111 has been partially sequenced and found to be very closely related to AGMr(HindIII)-1, whereas the other two fragments (115 and 55 base-pairs) appear to be related but not identical. Taken together, these data suggest that the mechanisms of sequence amplification and divergence lead to overall non-random sequence divergence. Since the divergence of the two species, the most striking changes in the highly repeated, complex sequence DNA fraction have been in the amount and organization of the DNA sequences closely related to AGMr(HindIII)-1. It, has recently been proposed (Fry & Salser, 1977) that simple sequence satellite DNA fractions are conserved between closely related rodent species. The present data suggest that highly repeated, complex sequence DNA fractions are also conserved between closely related primate species, but are represented in different frequencies and organization. i2’c gratefully tories this work
acknowledge Drs Maxine Singer and David Gillespie, in whose was done, for continued support and many helpful discussions.
Laboratory of Biochemistry, National Institutes of Health Bethesda, MD 20205, U.S.A.
National
Section on Molecular Hybridization Laboratory of Tumor Cell Biology, National Institutes of Health, Bethesda, MD 20205, U.S.A. Received
28 November
Institute
DINAH
SINGER?
LARRYDONEHOWERS
National
1978, and in revised
t Present atddrrss: Immunology 1 Present address: Laboratory U.S.A.
Cancer
labora-
Cancer
form
Institute
3 May
1979
Branch, NCI, NIH, Bethesda, MD 20205, U.S.A. of Tumor Virus Genetics, NCl, NIH, Bethesda,
MI)
20205,
842
D. SINGER
AND
L. DONEHOWER
REFERENCES Bonner, T. I., Brenner, D. J., Neufeld, B. R. & B&ten, R. J. (1973). J. Mol. Biol. 81, 123-135. Botchan, M., Topp, W. & Sambrook, J. (1976). Cell, 9, 269-287. Donehower, L. & Gillespie, D. (1979). J. Mol. BioZ. 134, 805-834. Donehower, L., Wong-Staal, F. t Gillespie, D. (1977). J. ViroZ. 21, 932-941. Fittler, F. (1977). Eur. J. Biochem. 74, 343-352. Fry, K. & Salser, W. (1977). Cell, 12, 1069-1084. Griiss, P. & Sauer, G. (1975). FEBS Letters, 60, 85-88. Maio, J. (1971). J. Mol. BioZ. 56, 579-595. Manuelides, L. (1978). Nature (London), 276, 92-94. McCarthy, B. & Church, R. (1970). Annu. Rev. Biochem. 39, 131-150. Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P. (1977). J. Mol. Bid. 113, 237-251. Rosenberg, H., Singer, M. & Rosenberg, M. (1978). Science, 200, 394-402. Singer, D. S. (1979). J. BioZ. Chem. 254, 5506-5514. Southern, E. M. (1975). J. Mol. BioZ. 98, 503-517.