Nucleotide sequences of HS-α satellite DNA from kangaroo rat dipodomys ordii and characterization...

Cell, Vol. 12, 1069-1084. December 1977, CopyrIght 0 1977 by MIT

Nucleotide Sequences of HS-a Satellite DNA from Kangaroo Rat Dipodomys ordii and Characterization of Similar Sequences in Other Rodents

Kirk Fry* and Winston Salser Molecular Biology Institute and Department of Biology University of California Los Angeles, California 90024

Summary

The most common repeated nucleotide sequences of the highly repetitive satellite HS-r~ fraction from kangaroo rat Dipodomys ordii was determined using ribosubstitution methods. This sequence was 6 nucleotides long and represented about 25% of the total HS-cu satellite DNA, while the remaining DNA was composed of sequence variants related to the most common sequence. The sequences of the commonest of these variants are reported. Furthermore, the most common repeated sequence was identical to that reported for the cy satellite of guinea pig Cavia porcellus. The (Y satellites of guinea pig, Cavia porcellus, pocket gopher, Thomomys bottae and antelope ground squirrel, Ammospermo- philus leucurus, are shown to have sequences in common with the kangaroo rat. This implies that the simplest repeated sequences of mammalian satellite DNAs may persist over much longer evolutionary times than previously thought.

Attempts to explain the very rapid quantitative changes in satellites whose sequence is strongly conserved have led us to consider that they might have a role in sympatric speciation. Among the novel features of the model presented is that fluctuations in satellites could be due to “speciation genes.” Such genes would confer a strong selective advantage in certain situations, and could explain the many puzzling instances in which large numbers of new related species have appeared over a short evolutionary span.

Introduction

Highly repeated DNA sequences appear to be a common feature of all eucaryotic genomes. Be- cause their base compositions are significantly different from those of less highly repeated DNA, large arrays of highly repeated sequences frequently can be separated in buoyant density gradients. The genome of the kangaroo rat, Dipodo- mys ordii, has been shown by Hatch and Mazrimas (1970, 1974) to represent one of the most extreme examples, with > 50% of its DNA in three major classes of satellite DNA.

The role of these sequences in the genome has

* Present address: Department of Biochemistry, Stanford Univer- sity School of Medicine, Stanford, California 94305.

not been determined. Evidence indicates that satellite DNAs do not have coding or regulatory roles. The technique of in situ hybridization has located the large arrays of tandemly repeated satellite DNA in the genetically inactive aggregated pericentric heterochromatin (Jones, 1970; Pardue and Gall, 1970; Yasmineh and Yunis, 1970; Prescott et al., 1973; Bostock and Christie, 1975). Analysis of the basic repeated sequences indicate extremely simple sequences for which coding functions would be improbable (Southern, 1970, 1975; Fry et al., 1973; Peacock et al., 1973; Gall and Atherton, 1974; Biro et al., 1975). In the case of kangaroo rats, great variation in the amounts of particular satellite DNAs has been found in several closely related species. For instance, the amount of HS satellite in D. ordii is 30% of the total DNA observed in density gradients, whereas in D. deserti, no HS satellite is observed (Mazrimas and Hatch, 1972). It is therefore improbable that a large percentage of any particular satellite must be conserved for a presumed regulatory function.

The available evidence suggests that the function of these highly repeated sequences, if any, is most probably in “chromosomal behavior:” folding, recombination, meiotic pairing or disjunction in the germ line tissue. Large deletions of the X (or Y) heterochromatin have little effect on the viability of Drosophila melanogaster, but do affect fertility and disjunction in male meiosis (Gershenson, 1933; Cooper, 1959; Lindsley and Grell, 1967; Pea- cock et al., 1973). Miklos and Nankivell (1976) and Yamamoto and Miklos (1977) argue that heterochromatin, which contains satellite DNA, regulates recombination.

In this paper, we report inter- and intraspecies relationships of possible functional significance, refuting the original proposal that the most common repeat units of satellites arise de novo in a very short evolutionary time (Southern, 1970; Hen- nig and Walker, 1970). Initial attempts to detect homology between satellites from different species failed (Flamm, Walker and McCallum, 1969a, 1969b; Hennig and Walker, 1970), but recent experiments have demonstrated cases where similarities between highly repeated sequences of closely related species of mice (Sutton and McCallum, 1972), Crustacea (Graham and Skinner, 1973), chimpanzee and human (Prosser et al., 1973), and Drosoph- ila (Gall and Atherton, 1974) are suggested by RNA-DNA hybridization experiments or direct sequence determinations. We report here the most common repeated sequence of the HS-a satellite of Dipodomys ordii (kangaroo rat) and show sequence relationships with the (Y satellite (p = 1.706 g/ml) of Cavia porcellus (guinea pig), CY satellite @ = 1.713 g/ml) of Thomomys bottae (pocket gopher)

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and (Y satellite @ = 1.708 g/ml) of Ammospermo- philus leucurus (antelope ground squirrel). These data suggest sequence “conservation” in these mammals over much longer evolutionary time spans than previously shown.

Results

The most commonly iterated sequence of HS-a satellite DNA from kangaroo rat, Dipodomys ordii, has been determined using ribosubstitution methods to produce base-specific cleavages at rA, rG and rC residues in the separated strands of the satellite DNA. The data presented below allow us to deduce the sequences of the major fragments from each cleavage. These fragments can then be assembled in a unique way to establish the most common sequence. Evidence also exists for a number of sequence variants. Since the most common iterated sequence is identical to the sequence proposed for guinea pig CY satellite (Southern, 1970), and since both pocket gophers and antelope ground squirrels have satellites banding at similar density in the denatured state (Mazrimas and Hatch, 1977), we have also compared fingerprints of the satellites from these three specres.

Fragments from the Heavy Strand The basic repeated sequence of the HS-(r satellite was determined from data derived independently from each strand of the satellite DNA. We obtained labeled and ribosubstituted heavy strand sequences by copying the light strand of HS-a with rGTP substituted for dGTP. Either a-32P-dATP, (Y- 32P-dCTP or a-3ZP-rGTP served as a radioactive precursor in separate experiments. Figure 1 is an autoradiograph of the fingerprint resulting when the ribo G-substituted DNA complementary to the light strand is cleaved at rG nucleotides by T2 ribonuclease and electrophoresed in two dimensions. Inspection reveals a relatively simple fingerprint containing only three or four fragments of significant molar yield. Table 1 outlines the evidence used to deduce the sequences of the radioactive fragments in Figure 1. We obtained the evidence listed in this table from analysis of fragments from the fingerprint in Figure 1 and from two other fingerprints of similar experiments in which label was introduced as Lu-32P-dATP or (Y- =P-TTP.

As an example, we will briefly explain the deduc- tion of the sequence T-T-A*rGp for the spot desig- nated G-2 as it is summarized in Table 1. (In

general, the IUPAC-IUB Commission on Biochemi- cal Nomenclature rules have been followed with the following exceptions: all nucleotides are considered deoxynucleotides unless preceded by a small “r” to denote a ribonucleotide; 32P-labeled phosphates are denoted by “*“; slashes indicate where the sequence is enzymatically cleaved). First, as indicated by column 1 of Table 1, the position of this fragment in the fingerprint (Figure 1) suggested a base composition of two Ts, one or two As and one rG residue according to relationships summarized by Barrel! (1971) and Salser et al. (1972). When the fragment was synthesized using &*P-TTP as precursor and digested to 3’ mononucleotides by spleen phosphodiesterase, label was recovered in T,* (95%) and rG,* (5%) (Table 1, column 4). Transfer of label from T to T establishes the existence of the dinucleotide sequence T-T, while the fact that neither A nor C is labeled shows that the sequences T-T must be at the 5’ end of the original fragment. The appearance of 5% label in rGp shows that rGp is followed only occasionally by T. In confirmation of these results, micrococcal nuclease digestion of the fragment released 80% of the label in T’T, 10% in TMP and 10% in A-rG,* (Table 1, column 5). Digestion with DNAase I gave products cut from the 5’ end of the fragment with mobilities consistent with T’T and T’T-A (Table 1, column 6). In an experiment using (w-32P-dATP as the radioactive precursor, nearest- neighbor analysis indicated that T is always the 5’ neighbor to A, ruling out a sequence of A-A (Table 1, column 4). Nearest-neighbor analysis using (Y- 32P-rGTP indicated that A is the 5’ neighbor of rG, and that the 3’ neighbor is G 66% of the time (Table 1, column 4). The only sequence which can account for the above data is G/T-T-A-rGp/.66G. (Note that because the (Y satellite sequences contain substantial quantities of sequence variants, individual nearest-neighbors are frequently present in substantially less than molar yield as is here indicated by the .66G, which shows that the sequence G-T-T-A-rG is followed by G 66% of the time.) This sequence was confirmed by the use of partial exonuclease digestions to produce inter- mediate fragments which were analyzed as described in Experimental Procedures (Table 1, columns 8 and 9). The sequence assignments of the other fragments depend upon similar reasoning from the data found in Table 1.

Fragments from the Light Strand To establish sequences present in the light strand, rC-substituted DNA was synthesized in vitro from a

Figure 1. Autoradiograph of Fingerprints Showing T2 RNAase Digest of rG-Substituted Heavy Strand of the HS-a Satellite DNA

The radioactive precursor was &*P-rGTP. Fractionation was by the standard fingerprinting technique as described in Experimental Procedures.

Nucleotide Sequences of HS-a Satellite DNA 1071

-CELLULOSE ACETATE pH 35

P “0 a

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Table 1. Fragwnts Produced by Cleavage at G in the Heavy Strand of HS-alpha Satellite

1. Spot Nmber 2. Ettlmated Base

Corpotitlon Spleen 3. Proposed Radloactlve Molar Digestfon

Sequence Precursor Vleld Products

G-l rGTP 1.59 rGP dATP .16 rGp T-TP 1.71 rGp

5’ Terminal Mlcrococcal Products of Two Partial Digestion

Partial DNAse I Dimensional

Products Spleen Venom

Digestion System #l Product(s) Product(s)

rGp rGp rGD

rGTP 1.00 5% Tp

$11 zp

t-G/T-T-A&p

dATP

l-TP

1.00 WI% Tp [;;r{ ;,TP T-T-A 9% rGP

T-A-rGp T-T-A

1% Ap 1.00

(5;) A-rGp 95% Tp

5% rG I

'f$; ;,TP ;::-A

T-A-rGp T-T-A T-T

10%) A-rGp

G-3 lA,lrG

WA-r6p

rGTP

ZP

.8D 5: Tp 36% rGp 50% b

.15 rG nona

A-rGp

G-4 rGTP

lT.lrG dATP

ffi/ThP TTP

.a3 95% Tp T-rGp 5% rGp

.57 2% Tp T-rGp

T-rGp

G-5

3T.l-2A.lrG

rG/T-T-T-A-rGp

rGTP dATP TTP

.37 n.d.

.34 n.d.

.3D 95% Tp I;;;{ ;,TP T-T T-T-A-rGp T-T-T-A 2% ffip T-T-T T-T-T 3% Ap

[!:I ?rGp T-T

G-6 rGTP .13 40% A 52% rG

lT.2A.lrG dATP T-A A-A-rG

G/T-A-A-rGp TTP none

Column 1: Spot number refers to the number assigned to the fragments on the fingerprints in the figures. The base composition is estimated by the position of the fragment on the standard two-dimensional electrophoresis systems described in Experimental Procedures. The proposed sequence IS deduced from the data entered in the table. Column 2: Radioactive precursor refers to the labeled &lP-triphosphate used to label the fragment and digestion products in the horizontal row across from the entry. Since the a-:‘“P is transferred from the 5’ position to the 3’ position of the digestion products, the precursor is not always present in the fragment. For example, T-rGp IS labeled by a-,“P-dATP whenever the following sequence is present: rG-T-rG’Ap Column 3: The molar yield is the amount of the fragment present relative to the sequence T-T-A-rG in each fingerpnnt. This was determined by adjusting the amount of radioactivity observed in the fragment for the number of radioactive phosphates as determined by the sequence of the fragment and then comparing this number with the number similarly determined for T-T-A-rGp. For example, when A- rGp was labeled with &‘“P-rGTP, it was determined by total spleen digestion that the 3’ terminal phosphate was labeled 61% as often as the internal phosphate. The total radioactivity observed in the fragment was therefore divided by 1.61 before comparing it to the number determined for T-T-A-rGp. Column 4: List of spleen digestion products and relative amounts. Numbers in parentheses indicate visual estimates from X-ray film density; others were quantitated in a low background gas flow geiger counter. Column 5: List of products of micrococcal digestion and relative amounts as determined on the two-dimensional chromatography system 1 as described in Experimental Procedures and by Whitcome et al., (1974). Column 6: Products resulting from DNAase I digestion of the fragments and two-dimensional chromatography as described in Experimental Procedures. These represent the sequences found on the 5’ ends of the fragments. Column 7: Lists the sequence for an undigested fragment determined by position on the two-dimensional chromatography system used to analyze micrococcal digestion products (Experimental Procedures). Columns 8 and 9: Products of partial digestion with spleen and venom phosphodiesterase. - .-

heavy strand template, introducing the radioactive precursors &2P-dATP, &*P-TTP or &*P-rCTP in separate experiments. The results of cleavage at rC-substituted DNA are summarized in Table 2.

The most common fragments from the light strand were T-A-A-rCp from the sequence rC/T-A-A-rCp/ .75rC, and rC from the sequences rC/rC/rC and rC/rC/T. As discussed above, the most common

Nucleotide Sequences of W-U Satellite DNA 1073

fragments from the heavy strand were T-T-A-rGp from the sequence G/T-T-A-rGp/.66G, and rGp from the sequences rG/rG/T and rG/rG/rG. These sequences can be assembled to give a repeating unit of T-T-A-G-G-G :

(Y-3’) G/G/G/ G/G/T

G/T-T-A-G/G . .

(3’-5’) Cl C- A-A-T/C c/c/c

T/C/C

Ribosubstitution and Cleavage at rA While there is at least a two-nucleotide overlap at

every point in the sequence assembled above, the numerical data do not unequivocally establish the length of the run of C-G pairs, nor do they ade- quately define the sequence variations present. To answer these questions, we carried out experiments with substitution and cleavage at rA nucleotides in each strand. If the hypothesized sequence is correct, we would expect to obtain the whole repeated sequence rA/G-G-G-T-T-rA/G (a permu- tation of the sequence T-T-A-G-G-G mentioned above) in one fragment from the heavy strand, with other fragments representing sequence variants. Figure 2 shows a representative two-dimensional homochromatographic fingerprint of fragments produced by rA cleavage of ribosubstituted DNA synthesized in vitro from the light strand

Table 2. Fragnents Produced by Cleavage at C in the Light Strand of HS-alpha Satellite

1 2:

Spot Number Estimated Base 5' Terminal Coqmition Spleen Micrococcal Products of Two Partial Partial

3. Prqosed Radioactive Molar Digestion Digestion DNase I Dimensional Spleen Venom Seouence Precursor Yield Products Products Digestion system I1 Product(s) Product(s)

C-l rCTP 2.28 100% rCp 1rC dATP

rCP none

WrCP TTP .75 rCP

c-2 rCTP 1.44 95% Tp lT.lrC

T-rCp 5% rCp

dATP .51 100% rCp rC/T-rC TTP .oa T-rCp

c-3 rCTP 1.00 43% rCp 100% A-rCp 57% Ap

lT.2A.lrCp dATP 1.00 48% Ap (GO%) T-Ap T-A A-A-rCp T-A-A 47% Tp (20%) A-rCp T-A-A T-A

5% cp C/T-A-A-rCp TTP none

c-4 rCTP .a0 42% rC A-rCp 58% Ap

lA.lrCp dATP .09 (100%) rC A-rCp WA-rCp TTP .21 A-rCp

c-5 rCTP .2a 43% Tp 40% rCp

lT.lA.lrC 17% Ap mixture dATP .07 38% Tp A-rCp rC/A-T-rCp 56% rCp T-rCp

and 6% *C/T-A-rCp TTP none

C-6 lT.3A.rCp

rC/T-A-A-A-rCp

rCTP

dATP

TTP

.24 47% rCp 53% Ap

.23 35% Tp 40% Ap A-A-A-rC T-A-A-A 61% Ap 40% T-Ap A-A-rC T-A-A

3% rCp 3% A-Ap A-rC T-A 1% Gp 3% rCp

3% Tp 1% A-Gp

none

c-7

2T.rCp

rC/T-T-rCp

rCTP

dATP TTP

.12 71% Tp 100% T-Tp 15% rCo

7% Gp' 7% Ap

.05 100% rCp (60%) T-Tp

.12 86% Tp (2’3%) TP 14% rCp (20%) rCp

Sequence data of fragments from the light strand of HS-ry satellite produced by cleavage at rC nucleotides.Table headingsareas inTable 1,and the molar yields are relativetothe fragment T-A-A-rCp.

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(SiF

E?

-AH 11

-AH 8

-AH 5 =-AH12

AH 13 -AH 3

P I

\ AH 10

CELLULOSE ACETATE pH 3.5 Figure 2. Autoradiograph of T2 RNAase Digest of rA-Substituted Heavy Strand of HS-a Satellite DNA

The radioactivity was introduced with &*P-rATP. Electrophoresis in the first dimension was on cellulose acetate, while chromatography in the second dimension was on a 40 x 36 cm DEAE-cellulose thin-layer plate developed with homomixture adjusted to pH 4.2 with acetic acid. “B” and “P” mark the positions of the blue and pink dye markers.


satellite template. Since the radioactive precursor was a-32P-rATP, all fragments present will be labeled (except for 3’ rAMP, where it is not followed by rA) in direct relation to their quantitative amounts in the satellite. Inspection of this fingerprint reveals a number of sequences in addition to the major sequence predicted above. Data used to establish the sequences of these fragments are presented in Table 3. All the sequences are derived easily from the most common sequence G-G-G-T- T-rA: for example. G-G-T-T-T-rA involves a change of a G to T, while the two fragments of G-G*rA and T-T’rA would be produced by a change of a G to A.

Table 4 summarizes the data from two-dimensional fingerprints of radioactive fragments obtained from rA cleavage of ribosubstituted DNA synthesized in vitro from the HS-a heavy strand DNA. The two fragments of highest yield are rAMP and C-C-C-T-rAp as predicted by our original hy- pothesis. Other fragments are complementary to the sequence variants established by the data from the material synthesized from the opposite strand (Table 3).

Reconstruction of the Basic Sequence It is possible to construct the repeating sequence (Y-3’) G-G-G/T-T-A-G-G-G/T-T-A by matching the overlapping sequences of the fragments G/T-T-A-rG/Gp and rA/G-G-G-T-T-rA/G from the heavy strand of the HS-cu satellite of D. ordii. This gives three nucleotide overlaps at every point. The sequence is supported by the independent data from the light strand where the major fragments were found to be rA/C-C-C-T-rAp/rA and rC/T-A-A-rCp/rC. These fragments support the repeating sequence (5’-3’) . T-A-A/C-C-C-T-A-Al C-C-C. The two reconstructed sequences are complementary and are shown in Figure 3 with the various fragments fitted together.

Variants to the Basic Sequence As pointed out above, all the fingerprints reveal fragments not present In the most common iterated sequence. Table 5 lists the fragments for which sequence data are available and their relative molar yields. Table 6 presents eleven sequence variants suggesting how the observed fragments are related to the most common sequence. Since about 20% of the label in each fingerprint is in minor fragments whose sequences have not been determined due to the small amount of radioactivity present, there are other minor sequence variants remaining to be characterized. Of the 80% of characterized material, about 25% is devoted to the most common repeat, with the remaining 75% in sequences related to the most common sequence by one or two base substitutions.

Interspecies Sequence Comparisons As previously mentioned, the most common repeated sequence of kangaroo rat HS-(Y satellite is identical to that published by Southern (1970) for the guinea pig (Y satellite. Subsequently, similar heavy satellites were found in the pocket gopher, Thomomys bottae, and the antelope ground squirrel, Ammospermophilus leucurus (Mazrimas and Hatch, 1977). Figure4 compares fingerprints of the fragments from the heavy strand of each of these satellites made in vitro while ribosubstituting with rGTP and introducing label as &*P-dTTP. The fingerprints show only very small drfferences in the sequences present. Segments of nucleic acid which give identical fingerprints do not necessarily have identical sequences. In this case, however, the fingerprint pattern is unusually simple and highly distinctive, so that the data shown give a strong indication that the four satellites tested have identical major sequences as well as srmilar sequence variants. The relative quantities of each oligonucleotide fragment are given In Table 7. The amounts of the minor fragments provide a measure of the relative amounts of each sequence variant present.

Discussion

lntraspecies satellite sequence relationships as found in Drosophila virilis (Gall and Atherton, 1974) or the p and y satellites of guinea pigs (Southern, 1974a) have suggested a common evolutionary origin for different satellites found in the same organism. This appears not to be the case in Dipodomys ordii. From work presented here and other studies carried out in our laboratory on the HS-p and MS satellites found in D. ordii, we have determined the simplest repeated sequences present in the three major satellite classes (Fry et al., 1973; Salser et al., 1976). The most common repeated sequences of the three satellites found in kangaroo rats are not related to each other in any simple way. This observation argues against any evolutionary model necessitating that the three kangaroo rat satellites (or multiple satellites in other eucaryotes) be derived from a single ancestral satellite.

Persistence of Satellite Sequences over Long Evolutionary Time Spans In earlier studies of satellite DNAs, it was found that among several closely related rodent species, the amount of satellite DNA varied greatly and showed different densities in CsCl gradients (Hen- nig and Walker, 1970; Walker, 1971). Due to these observations and the additional observation that satellites from these species cross-hybridized poorly, if at all, it was hypothesized that satellite

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Table 3. FracJmts Produced by Cleavage at A in the Heavy Strand of HS-alpha Satellite

1. spotNuber 5' Terminal 2. Estlrted Base Spleen Micrococcal Products of Two

coqmsition Radioactive Molar Digestion Digestion DNase I Dimensional 3. PropoSed sequence Precursor Yield Products Products Digestion System Yl

AH-1 rATP 1.20

31.2G.rA

mixture of d/G-f;ldT-T-T-rAp

M/G-T-G-T-T-rAp

.86

.56

77% Tp 13% Gp 10% rAp

26% Tp 36% Gp 38% rAp

47% Tp 39% Gp

3% rAp

i2”dj

TP T-rAp T-Tp (G,T)p GP I-A Und. rAp T-rAp (T.G)P G-Gp TP G-Gp Und. T-Tp r Ap (G.T)p(l ~%)GP

G-G G-G-T

G-T-G

G-T G-T-T(t G-T-G and/or G-G-T

.race)

AN-2 rATP 1.00

2T.3G.rA

rA/G-G-G-T-T-rAp

dGTP

TTP

1.00

1.00

67% Tp 25% Gp

8% rAp

14% Tp 51% Gp 29% rAp 35% Tp 47% Gp

4% rAp 14% Und.

(40%) TP (35%) T-rAp ';;'-'j A;-TIP

(35%) Und. (35%) rAp mg' h;;F'P

30% Tp 7% rAp 2% G-Gp 2% (G.T)P 2% T-Tp 1% G-Cp

G-G G-G-G

none observed

AH-3

G.ZT.rAp

rA/G-T-T-rAp

rATP .67

dGTP .39

(1O'M TP

::: ;; 40% Und.

TTP .83 51% Tp 49% Gp

(80%) Trinuclaottde

Trinuclwtlde rAp (T.G)p(l%)T-TP C-T) T-rAp Trlnucleotlde TP (G.T)P

T;;F GP

observed

G-T see note #l at end of table

An-4

4TT.G.rA

rAfG.3T)-T-rAp

rATP .53

dGTP not eluted dTTP .27

50% Und. 50% Tp

59% Tp 41% Gp

30% Und. 40% Tp 20% (T,G)p 10% T-Tp

G-T G-T-T G-T-G

AH-5

PT.rA rA/T-T-rAp

rATP

dGTP TTP

d

.49

.37

.81

91% Tp 9% rAp

100% rAp 91% Tp

9% rAp ('357:) T-Tp

(5%) HAP T-T-rA* 'after alkaline

ohosohatase

AH-6

4G.T.rAp

rA/G-G-G-T-G-rAp

An-7

PG.rAp

d/G-G-rAp

rATP

dGTP

TTP

.45

.41

not counted

24% Tp 66% Gp 10% rAp 20% Tp 54% Gp 26% rAp

I::;; (254 Und.

G-G G-G-G

rATP dGTP

TTP

.43

.26

.67

[ ; ;;; ,;TP

7%.Gp 93% rAp

95% G-Gp 5% rAp

100% rAp

AH-8 rATP .37

G.rA dGTP .26 d/G-rAp TTP none

89% Gp 11% rAp

100% rAp 100% rAp

G-rAp

G-rAp G-rAp

Nucleotide Sequences of I-IS-o Satellite DNA 1077

Table 3. Continued

:: 3.

AH-9

Spot N&r Estimted Base Caposition Pnmosed Saauence

3G.T.rA

26&A

W(G.G.G.T)rAp

Radioactive Precursor

rATP

dGTP

Molar Yield

.19

.ll

Spleetl Digestion Products

51% TP 21% Go 27% rip

17% Tp 47% Gp 24% rAp 12% Und.

Micrococcal Digestion Products

n.d.

5’ Terminal Products of DNase I Digestion

Two Dimensional System Rl

see note #2 at end of table

ITTP .73 n.d. n.d.

AH-10 4G.rAp

3G:f.rAp rA/G-G-G-G-rAp

and/or rA/(3G.T;-rAp

rATP

dGTP

dTTP

.18

.lO

not eluted

(303 Tp (40;.3 Gp (30f$ rAp n.d.

n.d.

(4tT ) rAp (30:‘) (G,T)p (30:‘) G-Gp

see note $2 at end of table

AH-II

1.A

VA/T-rA$l

AH-12

rATP

dGTP dTTP

rATP

.23

n.d. n.d.

.14

67’ T ll- G 22’: rAp

100:. rAo 100:’ rAb

92% Tp n.d.

T-rAp

T-rAo T-rA;

T,G.rA

rA/T-G-rAp

AH-13

26.2T.rA and/or

G,3T,rA

rA/(LG.T)-T-rAp and/or

rA/(G.ZT)-T-rAp

dGTP

dTTP

.I7

none

8f’ rAp 54:’ Tp 46: rAp

rATP .ll

dGTP .21

TTP 1.50

587 Tp 42:’ rAp 22:’ Tp 20% Gp 405 rAp 17’y Und. 36X Tp 44:’ Gp 202 Und.

7U: G-Gp 20’ (T.G)p lo? Und.

see note 42 at end of table

Note Yl: Partial digestion with spleen exonoclease yielded the product T-T-rAp. Note 12: The Position of the spots on the fingerprint establishes the fragment length as 5 and the relative ratio of G to T. The

separation of these spots was poor making it difficult to determine if AH-10 contained none or one T. AH-9 has one more T and one less G than AH-10 and AH-13 has one more T and one less G than AH-IO.

Sequence data of fragments from the heavy strand of HS-a satellite produced by cleavage at rA nucleotides. Table headings are as in Table l.and the molar vields are relativetothe fraqment G-G-G-T-T-rAp.

DNAs in the closely related rodents studied were not evolutionarily related and had appeared relatively recently, having arisen since speciation had occurred. Southern (1970) proposed that these satellites originated de novo independently in each species by saltation of portions of nonsatellite sequence.

Later studies showed that a number of satellite DNAs from different but closely related species have sequence relationships indicating that they are derived from common ancestral sequences. Prosser et al. (1973) demonstrated a satellite common to chimpanzee and man; Gall and Atherton (1974) found a satellite common to D. virilis and D. americana; and Graham and Skinner (1973) demonstrated sequence conservation in Crustacea. Sutton and McCallum (1972) showed by cross-hybridization studies that closely related Mus species have related satellites. Using Sutton and Mc- Callurn’s (1972) data of the lowered melting tem-

peratures of the heteroduplexes, Southern (1974a) calculated that the rate of divergence was 1% of the bases changed per lo6 years for rodents.

The results above showed that the 1970 Southern model could not be strictly true, but left open the possibility that it was basically correct in picturing satellite sequences as relatively poorly conserved, drifting rapidly once created. Smith (1976) subsequently proposed a model which postulated that satellite sequences would be created inevitably even if they had no function. According to this model, highly repetitive satellite-like sequences would be produced as a trivial consequence of repeated unequal crossover events. The same unequal crossover events would also keep the satellite sequences homogeneous by randomly ampli- fying some repeats and deleting others.

The data we present in this paper indicate the persistence of particular satellite sequences over much longer evolutionary times than has previ-

Cell 1078

Table 4. Fragments Produced by Cleavage at A in the Light Strand of HS-alpha Satellite

1. Spot Nuber 2. Estiuted Base

Caposition 3. Proposed

Sequmce Radioactive PreWrSpr

lblar Yield

Spleen Digestion Products

Micrococcal Digestion Products

5’ Terminal Products of DNase I Diqestion

TWO Dimensional System al

Partial Spleen Product(s)

M-l rATP

3C. T. rAp

35%rAJ(F!C,T)C-rAp

bS%rA/C-C-C-T-rAp

dCTP

TTP

1.54

1.54

1.54

23:: cp 43’: Tp 34% Ap 60% cp 26" Tp 14. Ap 92: cp

5^' Ap 3: Und

44% Ap 44’ Tp 12;: cp

'p:';'; cA;P "' r

(1"') TP 95.' cp

4: rAp I.' c-cp

c-c c-c-c

N.O. C-C-T-rAp C-T-rAp

AL-2 rATP

ZC.ZT,rA and

3C.ZT.rA W~J$~T)~AP

rAfX.ZTlrAa

dCTP dTTP

.64

.88

.80

17;; cp 39:. Tp 33’ rAp 11': Und. 4 spots 4 spots

Attempts to repurify the spots revealed a mixture of at least 4 spots. Correlations could not be made.

M-3 rATP .75 100' rAp TAP rb dCTP 5.38 100 rAp HAP rNrAp dTTP .51 1001 rAp HAP

AL-4

2C.T.rAp

WC-C-T-W

AL-5

rATP

dCTP

TTP

rATP

.36 5: cp 51; Tp

y&q ;;

44 rAp (lOi) cp .38 71:’ cp (75%) c-cp c-c

13’ Tp (204) rAp 15% rAp (57) T-Cp

.29 957, (80’) c-cp N.O. C-T-rAp 5” (lD?) C-Tp

(10:') rA - __-

.39 12’1 cp

G.T.EC,rAp and

G.T,3C,rAp dCTP

28> Tp 21" rAp 4W Und.

60 I1 j9l Cp ril (75") c-cp G-C 21.: Tp (25%) ;-CP G-C-C 407 Gp

x2 271 cp X2 (307) G-Cp 12 G-C 18X To (307) rAo N-N-N's 20” Gp

9% rAp 26”: Und.

(15:) T-Cp (159) c-cp

(5%) CD i5%j Tb

rA/(G,T.PC)rAp

rA/fG.:;C)rAp

AL-6 C.rAP rNC-rAp

N-7

dTTP

rATP dCTP dTTP

rATP

.21 I1 90% cp #l (65%) Cp Xl C-T (faint) 10% Gp (35%) Und. C-T-C (faint)

X2 8iX Cp X2 (60%) Cp X2 N.O. 12% Gp 7% rAp

y;,’ u;;. P

.40 86% cp N.O.

C-rAp 14% rAp

N.O.

.23 8% Cp 48% Tp

C.T.rAp

WC-T-rAp

dCTP

dTTP

.Ol

.16

44% rAp 51% Tp 49% rkp 93% cp

7% rAp 100% C-Tp

rATP .24 59% Tp 414 ..an

T-rAp

M-9 1c. T. rAp

mixture of rNC-C-C-C-T-rAp

and

rATP

dCTP

dTTP .36

.35

.52

38% cp 34% Tp 28% rAp

60% Cp 26% Tp 14% rAp 93% cp

7% I-AD

(40%) rAp (20%) 1c.T)~ (20%) TP

c-c c-c-c

100% cp N.O.

Sequence data of fragments from the light strand of HS satellite produced by cleavage at rA nucleotides.Table headings are as in Table 1,andthe molar yields are relativetothe fragment C-C-C-T-rAp.

Nucleotide Sequences of HS-u Satellite DNA 1079

ously been demonstrated. This persistence is so great as to rule out Smith’s (1976) model in its simplest form. Our data show that four species from the three major rodent suborders defined by Romer (1966) have a satellite with the same repeated sequences. Romer (1966) and Colbert (1969) cite evidence that these three suborders of rodents separated from each other at about the same time, during the Eocene period 40-50 million years ago. Accordrng to Southern’s (1974a) estr- mate for the rate of divergence of base sequence determined for mouse satellites, one would estr- mate 40-50% of the bases to have changed from the orrgrnal ancestral sequence of the (Y satellites. This clearly is not the case. It therefore appears that the simplest repeated sequences of satellite DNAs may normally persist over much longer evolutionary times than previously thought.

Alternatively, it might be proposed that this is a case of convergent evolution, with independently arising satellites only chancing to have the same major repeat sequence. This may possibly explain the similarity noted by Skinner et al. (1974) between the repeating sequence T-A-G-G found in hermit crabs and the sequence T-T-A-G-G-G found in rodents. We believe that the correlations which we have observed in rodents are not due to convergent evolution, since not only the major sequence (in its entirety) but also most of the sequence variants are common to all three rodent suborders. This conservation of the minor sequence variants is

G/T-T-A-G/G A/ G-G-G-T-T-A/G

il 2 3 4 5 6; HEAVY STRAND (5' - 3') A-G-G-G; T-T-A-G-G-G iT-T-A-G

. . . . .,. . .

LIGHT STRAND (3' - 5') T-C -C-C i A-A-T-C-C-C !A-A-T-C

A/A-T-C-C-C/A

C/A/A C/C-A-A-T/C T/C/C

ClClC

A-G-G-G-T-T-A-G

t -t -a -4 -lJ -g-t G-T-T-A-G-G

g -g -t -t -a -g

A-G-G-G-T-T-A-G

Figure 3. Major Repeated Sequence of HS-u Satellite DNA Based on Data from Ribosubstitution and Cleavage at rG and rA in the Heavy Strand and rC and rA in the Light Strand

In the upper part of the figure, the repeated sequence is presented with the sequences deduced from the various experiments aligned over or under the positions where they fit in that sequence. The lower portion of the figure shows how the longest sequences deduced can be arranged to provide at least 5 nucleotide overlaps along each of the sequences. Those sequences in lowercase type are complementary to sequences deduced from the light strand.

also in contradiction to the model proposed by Smith (1976). According to his computer simula- tions, any mutation which arises has an equal chance of being increased in number and perhaps of even becoming the dominant sequence. In such a case, even a very short period of evolutionary divergence should have sufficed to give different “variant” sequences. The conservation of the mi- nor sequences suggests that they may have an important role in the function of this satellite DNA.

Libraries of Satellite Sequences To explain the conservation of satellite sequences over long evolutionary periods during which they seem to appear and disappear many times, we have suggested a new model (Salser et al., 1976) in which it is proposed that the rodents (and perhaps other mammalia) share a common library of satellite sequences. In each species, certain members of this library may be amplified and appear as major satellite peaks, while other satellite sequences are present at low levels undetecta- ble in the analytical ultracentrifuge. According to this model, the rapid evolutionary changes under- gone by satellite DNAs would be for the most part quantitative. Appearances of new satellites would usually represent amplification of one of the satellites already present at low level in the “library”

spot x

AH-5 T-T-r& 0.4 dP/T 0.5 AH-6 G-G-G-T-G-r& 0.6 AL-4 t-C-T-r& 0.3 AH-7 G-6.rAp AL-5 (G,2-3C,r-?Ap 0.4 AH-8 G-r& i.: AL-6 C-rlip AH-9 (2-1G,i-*T)~Ap AL-7 I\“-10 (3-4G,O-lT,rAp z.: AL-8 K”’ i.: 0.2 AH-I, T-r& 0.2 AL-9 t-C-G(T,C,rl\p 0.4 AH-,2 T-G-r/ip AH-13 (l-ZG.1-ZT~T-rAp t.1 Molar yields were originally determined (Tables 1-4) relative to an arbitrarily chosen fragment produced by a particular cleavage, making it impossible to compare directly the relative amount of one sequence determined in Table 1 to another in Table 3. The molar yields were renormalized by the following procedure, so they are directly comparable. The relative molar yields determined from the different radioactive precursors in each table were averaged for each fragment. The sequences determined from the G cleavage of the heavy strand should contain all the sequences in the A cleavage of the heavy strand, and the summation of yield times length of all sequences in G-l through G-6 should equal a similar computation with AH-1 through AH-13, and, similarly, C-l through C-7 and Al-l through Al-g. Since molar yields in each group, however, were originally relative to an arbitrarily chosen sequence in each group, they are not equal. The relative molar yield in each group was therefore proportionally varied so that the summation of yield times length of all sequences in each orouo was eaual

Cell 1080

123156 1. . ..T-T-A-G-G-G WJne 62,AW,Ai-2.u I.0

2. I-T-A-G-A-G &.a 5 4”.8.G.Z.IH-Z,AL-2.C.3 .5

3. -.,-T-R-G-G-T G-T 6 RN-1 .G-SAL-IL-6 .5

1. T-G-A-G-G-G. T-G * A&6,11-9 .5

5. I-T-A-t-6-A ., G.+ 6 A”-IAH-IA-Z .3

6 . ..l-T-A-t-T-t t-T 5 G-Z,M-IAL-I,C-3 .3

I .I-A-A-G-G-G. T 4 2 I\&9.G.L,AL-2 .*

sequencpr ‘or WhlCh prt,a, evidence e*>rtr.

8. .T-T-A-G-T-T. &.T 5 and 6 AH-4.CI~4A.TWT .2

9. . ..6-A-A-G-G-G.. L4,r-a 1 and 2 AH-IO,G,A-A-‘ .2

IO. . ..T-T-A-G-G-C... l&c 6 AL-5 .*

1,. . . ..-G-A-G-G-A... T-G.&.+ 2 and 6 I\“-12,AH.I.AL-Z 1

12. .T-I-A-A-G-G.. &.a I A”.1J.CZ 1

Primary and variant sequences present in HS-a satellite of D. ordii. Column 1 is a list of the sequences. Columns 2 and 3 describe how the sequence is related to the most common sequence. The sequences represented by the numbers under “supporting sequences” can be assembled to derive the particular variant. The relative molar yield of each sequence was estimated from the yields of the sequences in Table 5. In some cases, a fragment is used in more than one variant. In these cases, the amount was divided among the different sequences. The sequences for which partial evidence exists are offered only as oossibilities.

rather than appearance de novo as in the original Southern (1970) model.

We propose that the essential step in the evolution of a specific satellite DNA may be its acquisi- tion of a biological function so that it will be maintained in the library of satellite sequences over long evolutionary periods. According to this view, “candidate” satellite sequences are created de novo frequently, either by an unspecified salta- tory replication or by unequal homology-depend- ent crossing over at the replication fork or as sister chromatid exchange. Only infrequently, however, would one of these “candidate” satellites acquire a biological function which would cause it to be maintained over long evolutionary periods. Many of the possible roles proposed for satellite DNAs would necessitate that they interact specifically with recognition proteins. The library of satellite sequences would be accompanied by a cognate library of genes for the recognition proteins, and the need to maintain the specific recognition might explain the evolutionary selection pressure respon- sible for conservation of the satellite sequences.

Quantitative Variations in Satellite DNA-a Possible Role in Speciation Our original (Salser et al., 1976) proposal of the concept of a “library” of satellite sequences has

Figure 4. Comparative Autoradiographs of Fingerprints Showing T2 RNAase Digests of rG-Substituted Heavy Strand of the a Satellite DNAs from Four Species

(a) Dipodomys ordii, (b) Cavia porcellus, (c) Thomomys bottae and (d) Ammospermophilus leucurus. The radioactive label was introduced with w~‘P-TTP.

been supported by our own subsequent work and by that of Peacock et al. (1977). These investigators have used in situ hybridization to polytene chromosomes to show that species which appear to have lost satellites usually retain detectable numbers of copies. Thus the sequence remains present in the library and may again be amplified at some future time. Our model as described above is in- complete in that it fails to explain why there are enormous rearrangements and changes in the amounts of individual satellite DNAs during very short evolution times. There is, for instance, great variability in the relative amounts of satellites present in closely related species of Dipodomys. The extreme case is D. deserti, in which isopycnic banding in the analytical ultracentrifuge detects none of the three satellites which make up 52% of the genome of D. ordii.

Although such variations fitted well with the previous models of drifting satellites, they pose a paradox when considered in the light of a library of highly conserved sequences. How can such radical fluctuations in amount over short evolutionary periods be consistent with functional impor- tance sufficient to guarantee conservation of sequence over long evolutionary periods? Do such rearrangements serve a purpose? Is there special “machinery” to perform such rearrangements?

From the first discovery of satellite DNAs, one of the popular hypotheses has been that they have a role in meiotic pairing. When we considered this role in the light of the questions posed above, it was obvious that a sudden change in the amount or localization of the satellite sequences in the progeny of a single individual could provide a very crude “species” barrier in which matings within the group were fertile, but matings with the general population were more or less infertile. Such events would normally be disadvantageous unless they occurred in individuals carrying a fortunate juxta- position of alleles which permitted them to exploit a new ecological niche. In this case, the lack of competition in the new niche might compensate for the gametic wastage due to infertile matings with the original population, and such events could serve as a first crucial step toward sympatric speciation.

This has led us to a model which proposes that the rapid evolutionary changes in satellites and other heterochromatin are due to the activity of gene systems whose major role is to foster sympatric speciation. In the conventional view (for example, see Mayr, 1963), new species can arise only as

Nucleotide Sequences of l-6-a Satellite DNA 1081

+ + CELLULOSE ACETATE pH 3.5

n

$ I! r

* i

0. e

2 t

CM- G4 2

, i

4

b

Cell 1082

~GP

T-T-R-rGp

T-T-T-A-rGp

(Te.Ap) rGP

T-&p

(T,,A,C) rGP

(C,T) rGP

R-rGp

Tg-dP

(1*-C) rGP

(A.1) ~GP

T-T-IQ

46

23

13

3.9

2.9

2.8

2.3

1.6

1.0

0.8

0.6

0.6

33

25

13

5.8

5.7

2.4

2.8

2.6

2.2

1.9

1.9

2.2

31

27

24

3.7

4.2

3.0

1.0

1.2

1.4

0.7

0.5

29

23

26

5.8

5.1

1.7

0.5

1.8

2.3

0.8

0.4

1.3

C-rGp 0.5 0.3 0.2 0.4

(CAT) rCP 0.5 1.1 0.5 0.7

(T.A.,) ~GP 0.3 0.7 1.0 0.8

Comparative yields in percentage of :‘2P in fragments seen in Figure 4 produced by RNAase T2 digestion of the rG-substituted heavy strand of the four a satellites. All the radioactive spots in the fingerprints were cut out and quantitated in a liquid scintilla- tion counter. The percentages obtained are not proportional to the molar yield of each fragment, since no correction was made for the different amounts of radioactivity transferred from the nearest-neiahbors to each fraament.

a consequence of evolution in separate popula- tions during their long genetic isolation by some geographic barrier (allopatric speciation). Unfortu- nately, many instances have been reported where it is difficult to imagine what geographical barriers could have intervened to cause the complete genetic isolation demanded by the allopatric model. Any species carrying a gene system fostering sympatric speciation would have a great evolutionary advantage in any situation where large numbers of empty niches suddenly become available (as when there is a widespread climatic change or invasion of a new continent). Such a mechanism might therefore explain some discontinuous aspects of evolution such as the very rapid radiations of new species over a brief evolutionary time span. The details of the model will be presented elsewhere (W. Salser, manuscript in preparation).

Experimental Procedures

Templates Separated strands of HS-a satellite of kangaroo rat (Dipodomys ordii), (Y satellite of guinea pig (Cavia porcellus), and heavy satellite of pocket gopher (Thomomys bottae) and antelope ground squirrel (Ammospermophilus leucurus) were gifts from F. Hatch and J. Mazrimas. They were prepared from liver tissue of several animals of each species by published methods (Hatch and Mazrimas, 1974) and dialyzed against 0.001 M Tris-Cl, 0.0001 M EDTA (pH 6.0) with storage at 49) until use.

Synthesis of Ribosubstituted Satellite DNA DNA polymerase I was used to copy the separated strands of the satellite under conditions allowing one deoxyribonucleotide to be totally replaced with the corresponding ribonucleotide. In

each experiment, label was introduced as a single &2P-dNTP or rNTP to obtain nearest-neighbor information (Berg, Fancher and Chamberlin. 1963; Fryet al., 1973).The incubation conditions were as follows: 67 mM Tris-Cl (pH 7.4); 0.67 mM MnCI,; 66 PM dNTPs; 330 PM rNTP (rA, rC or rG); 16-50 pg/ml satellite template; 33 PM &*P-dNTP, 70-120 Ci/mM; 5.5 pg/ml oligonucleotide primers IO-20 bases long, prepared by fractionation of a DNAase I digest of calf thymus DNA (Fry, 1974); and 30 wg/ml DNA polymerase I. Maximal syntheses, as monitored by acid- precipitable counts, occurred by about 90 min. In early experiments, reactions were terminated by heating at 100°C for 5 min. Later experiments indicated that phenol extraction was prefera- ble, since native product was less likely to be lost by binding to surfaces during subsequent steps.

As with all indirect methods of sequencing DNA, the fidelity of copying is crucial to an accurate determination of sequence. Two papers have questioned the fidelity of copying while substi- tuting with nbonucleotides (Van de Sande, Loewen and Khorana, 1972; Wu et al., 1972). Our conditions are substantially different that those used in these studies and have been fully discussed (Salser, 1974).

Purification and Digestion of Ribosubstituted DNA After the reaction was terminated, ribosubstituted DNA was separated from small molecular weight reaction components by gel filtration on a Bio-Gel P-60 (100-200 mesh; Bio-Rad) column (0.5 cm x 15 cm) equilibrated with 0.1 M sodium acetate and 0.001 M EDTA. The peak of radioactive ribosubstituted DNA in the exclu- sion volume was mixed with 2 parts absolute ethanol at 0°C. This solution was placed in a 15 ml corex centrifuge tube previously treated with a 5% solution (v/v) of dichlorodimethylsilane in chloroform. After several minutes, the precipitated, ribosubstituted DNA was pelleted by centrifugation at 0°C (15.000 g x 20 min). and the supernatant was poured off. Residual sodium acetate was removed by gently washing the walls of the centrifuge tube with 1 ml of cold absolute ethanol. The precipitated, ribosubstituted DNA was resuspended in 0.02 ml of 0.1 M KOH using a capillary tube to wash the surface of the tube at least 3 times. Recoveries, as roughly monitored using a hand geiger counter with identical sample geometry, were usually better than 95% and never ~80%. To prevent cleavage of the alkali-sensitive bonds at !he ribose bases, the solution was immediately desalted by chromatography over a 1 mm x 20 mm pyridinium Dowex-50 column poured in the tip of a disposable Pasteur pipette. The 0.01 ml (1 drop fractions) which contained the radioactive ribosubstituted DNA were combined (4 or 5 fractions) and dried on a plastic polyethylene sheet coated with dichlorodimethylsilane in preparation for further enzymatic digestion.

Two-Dimensional Fingerprinting Technique The fingerprints presented here were prepared by the standard electrophoretic methods (Barrel, 1971). with the second dimen- fion transfer carried out using an improved method (Southern, 1974b). Homochromatography solvents were prepared by two methods. The first was a minor variation of homomix C described by Brownlee and Sanger (1969). The second was prepared the same way, except that after addition of urea, the solution was adjusted to pH 4.2 with acetic acid (Kleid. Agarwal and Khorana, 1975). We found that this variation provides superior resolution and separates fragments of identical length on the basis of purine to pyrimidine ratios.

Method of Analysis Ribosubstituted DNA synthesized using only one radioactive triphosphate was digested with T2 ribonuclease (if ribosubstituted with A or G) or pancreatic ribonuclease A (if ribosubstituted with C) and fingerprinted either by homochromatography on thin-layer plates or by the standard two-dimensional method on DEAE paper. In some cases, samples were analyzed by both techniques. Radioactive fragments were recovered by standard methods de-


scribed by Barrel (1971) repurified, subdivided and subjected to a number of analyses, as described in detail below. To determine the nearest-neighbor 5’ to the originally labeled base, complete digestion with spleen phosphodiesterase was carried out, followed with thin-layer chromatography to identify the products. Di- and trinucleotide sequences contained in the fragments were determined by micrococcal nuclease digestion and subsequent thin-layer chromatography as previously described by Whitcome, Fry and Salser (1974). As described below, DNAase I digestion

with thin-layer chromatography was used to determine the di- and trinucleotide sequences from the 5’ end fragments. Finally, in some cases, standard methods of partial digestion with spleen or snake venom phosphodiesterase were carried out with the resulting fragments analyzed either by one-dimensional electrophoresis (Brownlee and Sanger. 1969) or by two-dimensional methods of electrophoresis and homochromatography (Sanger et al., 1973).

Radioactive material eluted from the fingerprints was frequently found to include more than one fragment. For those fragments which migrate slowly in the second dimension on DEAE paper, homochromatography in one dimension was carried out on DEAE-cellulose thin-layer plates to obtain further purification. Suspected mixtures eluted after separation by homochromatography were electrophoresed in one dimension on DEAE paper in 7% formic acid.

Total digestion of fragments to 3’ mononucleotides for nearest- neighbor analysis was carried out in 0.01 ml with spleen phosphodiesterase (Worthington, code SPH with no additional purification) at 6 mg/ml in 0.01 M ammonium acetate (pH 5.7) 0.002 M EDTA, 0.05% Tween 80 and 0.01 M ammonium tartrate. No carrier RNA was added to material eluted from homochromatographs, while 10 pg of carrier RNA were added to fragments eluted from DEAE paper. Samples were digested overnight at 37”C, and the products were chromatographed on thin-layer plates prepared with polyethyleneimine-impregnated cellulose (Brinkman. Cellulosepulver MN 300 PEI). These were developed in a solution of 1200 g (NHdSO,, 26.8 g Na,HPOI,7HI0. 12.8 g NaH,PO,.H,O, 40 ml n-propanol and water to a final volume of 2680 ml.

The use of micrococcal nuclease digestion to characterize oligonucleotides has previously been described (Whitcome et al., 1974). The enzyme conditions have been modified for digestion of material eluted from homochromatograms. Fragments were digested overnight at 37°C with 0.01 ml of 20 fig/ml micrococcal nuclease (nuclease. Staphylococcus aureus, Worthington code NFCP) dissolved in 0.1 M Tris-Cl (pH 8.9) 0.01 M MgClr; 10 pg of carrier RNA were added to material eluted from DEAE paper. In some cases, it was desirable to use different amounts of digestion, since for the conditions given, C-rich fragments may be digested almost completely to mononucleotides, but G-rich fragments are sometimes left undigested.

DNAase I attacks olrgonucleotides by endonucleolytrc cleavage leaving 5’ phosphate groups. The final digestion products from ribosubstituted fragments are of the forms N-N and N-N-N from the 5’ end, pN-N and pN-N-N from the internal sections, and prNp, pN-rNp and pN-N-rNp from the 3’ end (Vanecko and Laskowski, 1961; Laskowski. 1966; Matsuda and Ogashi, 1966). The di- and trinucleotides from the 5’ end, lacking terminal phosphate groups, have less total charge and can be clearly separated from the fragments of the interior and 3’ ends of the molecule of PEI-cellulose thin-layer plates developed in two dimensions (Fry, 1974; 8. E. Wallace and W. Salser, manuscript in preparation). From the position of the radioactive dinucleotide monophosphates compared to ultraviolet-absorbing markers, the identity of the two nucleotides on the 5’ end of the fragment may be unequivocally determined. It is usually possible to determine the third nucleotide from the 5’ end from the position of the trinucleoside diphosphate and the knowledge of the two nucleotides on the 5’ end. Since some of the relevant trinucleoside diphosphates chromatograph close together, however, it is occa-

sionallydifficult to distinguish the third nucleotide without further analysis.

The DNAase I digestions of fragments eluted from either homochromatography or DEAE paper were carried out at 37°C for 24 hr with 0.01 ml of a solution of DNAase I (Worthington, code DPFF) at a concentration of 1 .O-2.5 mg/ml in 0.1 M Tris-Cl. 0.02 M MgCI, (pH 7.0). After digestion, markers were added to the solution, and chromatography was carried out on PEI thin- layer plates. The first dimension solvent was 0.25 M formic acid adjusted to pH 3.4 with NH,OH; the plate was subsequently dried, washed in methanol and dried again before second-dimension development in 0.2 M LiCI.

Analysis of the partial exonuclease digestion products of an oligonucleotide provides a powerful method of sequence determination. The methods used here involve minor technical modifi- cations of published methods reviewed by Barrel (1971) and by Sanger et al. (1973). Partial spleen phosphodiesterase digestion of material from homochromatographs (Worthington SPH) was carried out in a 0.01 ml solution at an enzyme concentration of 2 mg/ml in 0.1 M ammonium acetate (pH 5.7). 0.002 M EDTA. 0.05% Tween 80 and 0.01 M ammonium tartrate. Samples were taken for electrophoresis at 0, 15. 30 and 60 min. For fragments eluted from DEAE paper, the enzyme concentration was reduced to 0.4 mg/ml.

Snake venom phosphodiesterase (Worthington, code VPH) was repurified as described by Sulkowski and Laskowski (1971). Digestion conditions were previously described by Barrel (1971). except that the venom phosphodiesterase concentration used for material eluted from homochromatograms was 0.5 mg/ml, and for material from DEAE paper 0.05 mg/ml. Samples were taken at 7.5. 15 and 30 min.

Acknowledgments

We would like to thank Joseph Mazrimas and Fred Hatch for generous supplies of purified and characterized satellite DNAs. without which this work would have been impossible, and for their enthusiasm and encouragement throughout. This research has been supported by grants from the NSF as well as by grants from the USPHS. W. S. was the recipient of a USPHS Career Development Award.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 21, 1977; revised August 17, 1977

References

Barrel, B. G. (1971). Fractionation and sequence analysis of radioactive nucleotrdes. In Procedures in Nucleic Acid Research, 2. G. L. Cantoni and D. R. Davies, eds. (New York: Harper and Row), pp. 751-779.

Berg, P., Fancher, H. and Chamberlin, M. (1963). The synthesis of mixed polynucleotides containing ribo- and deoxyribonucleo- tides by purified preparations of DNA polymerase from Esche- richia colr. In Informational Macromolecules, H. J. Vogel, V. Bryson and J. 0. Lampen, eds. (New York: Academic Press), pp. 467-483.

Brro, P. A.. Carr-Brown, A., Southern, E. M. and Walker, P. M. B. (1975). Partial sequence analysis of mouse satellite DNA: evidence for short range periodicities. J. Mol. Biol. 94, 71-86.

Bostock, C. J. and Christie, S. (1975). Chromosomes of a cell lme of Dipodomys panamintinus (kangaroo rat), a banding and autoradiographic study. Chromosoma51, 25-34.

Brownlee. G. G. and Sanger, F. (1969). Chromatography of 3zP- labelled oligonucleotides on thin layers of DEAE-cellulose. Eur. J. Biochem. 71, 395-399.

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Nucleotide sequences of HS-α satellite DNA from kangaroo rat dipodomys ordii and characterization...

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