volumes no.ii Novemberi976
Nucleic Acids Research
Repeating restriction fragments of human DNA.
Laura Manuelidis
Department of Pathology, Yale University School of Medicine, 310 Cedar Street,
New Haven,. CT 06510, USA
Received 17 August 1976
ABSTRACT
Human DNA digested with Hae III showed multiple repeats of a 170 base
pair fragment. The most prominent band was the 340 base pair dimer, estimated to be 0.8% of the entire genome. Eco Rj and Hha I yielded fragments
with similar electrophoretic mobility to the Hae III dimer. In each case
this band was markedly enriched in DNA reassociating at a C o t of £ 1.
Hybridization of the Hae III dimer to gels eluted on to filters demonstrated that the multiple Hae III fragments and Eco Ry fragments contained
compatible sequences. These sequences may comprise a distinct subclass of
DNA.
INTRODUCTION
Human DNA contains several satellites that are not easily resolved from
the bulk of the main band DNA. Each of these satellites is small in amount
(< 2% of the entire genome) and requires large scale preparative procedures using Cs.SO, with variable binding of silver and mercuric ions (1).
An alternate approach to the generation of specific fragments of the human
genome is made possible with the use of site-specific endonucleases, or restriction enzymes. Type II restriction enzymes cleave double stranded DNA
at sequence specific sites. They generally recognize sequences of 4-8
nucleotides long and in many cases their recognition sites and cleavage
points are known (2). Some restriction enzymes have in fact been used in
the analysis of whole bovine (3) and to a limited degree whole human DNA
(4). More recently they have become a useful tool in the characterization
of satellite DNAs from mouse (5) Drosophila (6) and other eucaryotes. Reassociation studies have suggested that a minimum of 9% of human DNA reassociates very rapidly (7) yet human satellites I, II and III together
have been recently reestimated to comprise <2% of the total genome (8).The
present study was undertaken In order to investigate the possibility of
other major repeated sequences in the human genome as well as to initiate
studies on defined fragments of DNA generated with this powerful technique.
In the present paper Hae III cleavage fragments consistent with a repeating
© Information Retrieval Limited 1 Falconberg Court London W1V5FG England
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unit are described and they are related to fragments generated by Eco R^
as well as to rapidly renaturing DNA.
MATERIALS AND METHODS
DNA Extraction. Human DNA was isolated from the following sources:
1) embryonic male brain, 2) adult brain, male and female, obtained within
8 hours of autopsy, 3) long term tissue culture lines of human intracranial
tumors (9) and A) male placenta within one hour of delivery. Tissues were
homogenized in isotonic sucrose
-
phosphate pH 7.2 and crude nuclei
pelleted after several washes and low speed centrifugation. Purified nuclei
labelled with 3H thymidine were made from tissue cultured cells as described (10). In each of these cases DNA was extracted from crude or
purified nuclear pellets after lysis and enzyme digestion with pronase
and RNAase (10) , followed by preparative equilibrium centrifugation in
CsCl. The entire peak of DNA was pooled, precipitated with 3 volumes of
70% ethanol at -20°C for 15 hours and after resuspension in a convenient
volume, dialyzed against 5mM Tris Cl pH 8.0 or water. DNA was kept
frozen in aliquots at -70°C.
Isolation of Heterochromatic DNA. Heterochromatic fractions of placental
nuclei were prepared and extracted essentially as described by Cosden and
Mitchell (11). Briefly this entailed homogenization of cells until >95% of
the cells were broken as monitored by phase contrast microscopy. Nuclei
were purified by centrifugation through 2.2 M sucrose, 1.5mM MgCl. at
72,000g in an SW 27 rotor for 1 hour; the nuclear pellet showed barely detectable cytoplasmic contamination. After washing the nuclei,they were resuspended in 45 ml of 0.25M sucrose and sonicated 2x15 seconds at 6.2 amps
on a W 140 D "Sonifier". Phase contrast revealed ^80% of the nuclei were
broken, and intact nucleoli with condensed chromatin remained. The heterochromatin was pelleted by centrifugation at lOOg for 5 minutes and extracted with phenol after lysis in sodium lauryl sulfate (SDS), and digestion with proteinase K (E.M. Biochemlcals, 200 ug/3 ml). This was
followed by ethanol precipitation, treatment with pancreatic RNAase (400ug/
ml), and phenol extraction. The phenol was removed by ether extraction.
The DNA was then pelleted with 2.5 volumes of ethanol in the presence of
0.3M Na Acetate overnight at -20°C and was stored in aliquots in 0.01 M
Na 2 SO 4 at -70°C.
Hydroxyapatite Fractionation. High molecular weight DNA (greater than
10
daltons, as judged by agarose gel electrophoretic mobility) or DNA
cleaved by the restriction enzyme Hae III (vide infra) were boiled in a
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siliconized tube for 6 minutes in water or 5mM Tris pH 8.0 to denature
the DNA. The tube was placed in a 63°C or 67°C water bath, hot 0.5 M Na
phosphate pH 6.8 was added to bring the salt concentration to 0.125 M,
and DNA was allowed to renature to a Cot«= 0.8. An equal volume of hot hydroxyapatite (1-2 ml) equilibrated in 0.05 M Na phosphate was added to the
DNA, mixed well and allowed to settle. The supernatant was withdrawn and
the slurry treated with 10-15 1 ml washes of 0.14 M Na phosphate to elute
single stranded DNA. Five washes with hot 0.5 M Na phosphate were then
used to elute double stranded DNA most of which eluted in the first two
washes. Recoveries of DNA were generally >98% as judged by radioactivity.
Restriction Enzyme Reactions. Hae III, Hind III and <|>x 174 used as a
molecular weight marker were the generous gift of G. Godson, and Hha I,
Hpa I and Hap II were the gift of J. Sklar. In later experiments active
enzymes were purchased from New England Bio Labs and Bethesda Research
Laboratories. Reactions were carried out at 37°C overnight in 7mM NaCl,
7mM MgCl., 5mM 8-mercaptoethanol and 5mM Tris Cl pH 7.5 for Hae III digests. Most of the other enzymes were reacted under similar conditions
except the NaCl was omitted; Eco Rj^ reactions contained 9.5mM MgCl,,, 9.5mM
Tris Cl pH 7.4, 13.5mM B-mercaptoethanol, 50mM NaCl and 100 yg of autoclaved gelatin. Small amounts of DNA (<1 yg) were digested with 1-2 units
of enzyme in a final volume of 50 yl. Digestion was considered complete if
further digestion(s) produced no change in band pattern. DNA incubated
under similar conditions without restriction enzymes was used in control
experiments and did not show any change in electrophoretic mobility. For
larger amounts of DNA (10-100 yg) ,enzyme concentrations were suitably scaled
up and reaction volumes were 200-500 pi. Reactions were terminated by
addition of 1/5 volume of 80% sucrose, 0.1 M EDTA, bromphenol blue and incubated at 37°C for 10 minutes.
Electrophoresis. 1% or 2% agarose gels (Seakem Agarose) containing
0.5 pg/ml of ethidium bromide were prepared as described (12) and were
poured in an E.C. 470 vertical slab gel apparatus. The gel thickness was
made to 1.5 or 3 mm and a 10 slot or 4 slot former was used. Polyacrylamide
slab gels (2.5%, 4%, 6%) were made according to Loenlng (13) in the same
apparatus. Gels were cooled to 10°C using circulating water and were prerun for a minimum of 45 minutes at 100 volts. Running buffer contained
Tris Borate 90mM pH 8.4 and 2.5mM EDTA.
Electrophoresis was started at £l0mA or 50 volts for 3/4 hour and then
increased to <20mA (or 100 volts) for 5-7 hours until the bromphenol blue
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tracking dye was 2-3 cm from the bottom. After electrophoresis gels were
laid on a U.V. light (U.V. Products, Inc.) and photographed with NP 55
Polaroid film using a Tiffin red 1 filter in series with a U.V. barrier
filter. For autoradiography gels were placed on a sheet of dialysis
membrane laid on a porous plate in a vacuum frame, sealed with Saran Wrap
and dried by suction overnight at 22°C. No radioactivity was lost when
dialysis membrane was used as the backing material. Dried gels were covered
with X-ray film and stored in the dark for 1 day-3 weeks prior to development.
Preparation of
P Labelled DNA. "Nick translation" of small aliquots of
32
P into selected samples of DNA obtained by
DNA (14) was used to introduce
gradient centrifugation, hydroxyapatite chromatography ,or eluted gel pieces.
32
? tri-
The reaction mixture contained a final concentration of 2-5mM a
phosphate (one or two) and 5mM each of the remaining unlabelled dNPTs,
50mM Tris Cl pH 8.0, lOmM B-mercaptoethanol, 6.7mM MgCl , 50 pg/ml of BSA
and 0.1-1 pg of DNA in a final volume of 50 pi. Pancreatic DNAase (stock =
1 pg/ml) was diluted on a sheet of parafilm 1:10,000 and 0.5 pi was added
followed by addition of 0.5 pi (1 unit) of DNA Polymerase I obtained from
Boehringer Mannheim. The reaction was allowed to proceed for 1 hour at
15-16°C and was terminated by addition of 5 pi of 0.2 M EDTA pH 7.5. After
addition of 100 pi of H.O the mixture was extracted with 100 ul of H.O
saturated phenol, centrifuged at 5000g for 8 minutes and the phenol phase
was back extracted with 100 pi of 50mM Tris Cl pH 7.4. The final volume
of the pooled aqueous phases was brought to 500 pi and the phenol removed
by ether extraction. E. coli tRNA was added to a final concentration of
20 pg/ml and Na Acetate to 0.3M and the DNA was precipitated with 2.5
volumes of 95% ethanol at -20°C. Resuspension and ethanol precipitation
were repeated two times and the final DNA pellet was stored at -70°C in
aliquots containing not more than 35,000 CPM/pl. Initial specific
activities were generally in the range of >5xlO
CPM/pg DNA.
Gel Elution. Specific bands were eluted from gels either by electrophoresis or by gel compression . For electrophoretic elution similar to that
described (15) gel fragments were placed on GFC Whatman paper tapped into
the nose of a 5 ml plastic pipette. Dialysis tubing was snuggly wrapped
over the nose, electrophoresis buffer was added and bubbles removed. The
pipettes were held in place in a Hoefer EF 301 cylindrical gel apparatus
and fragments eluted at 225 volts (3-4 mA/tube) for 4-5 hours "on ice. Recovery was >_ 85% for fragments of <500 base pairs. Alternatively frozen gel
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strips of agarose gels were homogenized or compressed (16) , filtered and
ethanol precipitated with tRNA carrier. In these elutions recoveries were
generally better than 50%.
Whole gels were denatured and eluted on to Millipore filters for filter
hybridization and autoradiography as initially described by Southern (17) ,
using the modification of Ketner and Kelly (18). Nick translated DNA was
reannealed to DNA immobilized on the filter after the filter was washed extensively in 2x SSC, and thereafter in the buffer described by Denhardt(19)
which consists of 0.02% Ficoll 4000, 0.02% BSA, 0.02% polyvinylpyrrolidine
(Sigma) in 3x SSC, in order to decrease the background in DNA-DNA hybrids.
Millipore strips were wrapped around a small plastic tube and inserted in a
larger tube so that 2 ml was sufficient to keep the entire strip wet. The
hybridization
hy
mixture contained 4x SSC with 0.1% SDS and 2-5x10 CPM of
32
P denatured DNA. DNA was denatured in the presence of 20 pg of M. lyso—
deicticus carrier DNA by adding 1/10 volume of IN NaOH for 15 minutes at
22eC. Immediately after neutralization with 1/10 volume of IN HC1 and 1/10
volume 0.5M Tris Cl pH 8.0 the DNA solution was added to the filter strips
soaking in 4x SSC-0.1% SDS; tubes were tightly capped, vortexed and incubated overnight at 62°C. Strips were washed extensively in 2x SSC at 22°C,
incubated in 500cc of Denhardt's buffer for 1 hour at 37"C to remove nonspecific bound DNA, washed again in 2x SSC and dried at 60°C on a glass
plate for autoradiography.
Ethidium bromide negatives or autoradlographs were scanned with a Joyce
Loebl
densitometer or with a Transidyne densitometer equipped with a laser
and attached to a Hewlett Packard 3380A computer, which calculated peak
distances in units of scan time.
RESULTS
Hae III Fragments of Human DNA. Human DNA from embryonic or adult brain,
brain tumors and placenta digested to completion with the enzyme Hae III
revealed a discrete series of bands on agarose-eth'idium bromide gel electrophoresis (Fig. 1, top). The most intensely fluorescent bands were present in DNA from both male and female sources and were present in a qualitatively equivalent pattern in purified nuclear DNA preparations and in
whole cell DNA. At least six of the bands were multiples of a single fragment length as suggested by their electrophoretic mobility (Fig. 1, bottom).
The dimer (band 2) was the most prominent fragment in all DNA preparations
examined. In addition, other bands with uneven multiples of this fragment
length were consistently observed; the strongest of these were a 1.3-mer,
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DISTANCE
U I G R A T C O
[ t i m i l
Figure 1. Top: densitometer
tracing of whole human DNA
from brain digested to completion with Hae III and
separated by electrophoresis
in 2% agarose; ethldlum bromide fluorescence. Center of
main peaks are computed in
densitometer minutes, s is
start of migration.
Bottom: Semilog plot of major
peaks mobility against multiples of fragment 1, numbered
bands 1-6 (•). In addition,
other somewhat weaker bands
show mobilities of a 1.3-mer
a 2.5-mer and a 3.5-mer (f ) .
a 2.5-mer and a 3.5-mer (Fig. 1).
In a series of experiments using restriction fragments <|>x 174 or G4
bacteriophage DNA whose sizes are known (20) the length of each of the
human bands was determined; a typical calibration is seen in Fig. 2; the
most prominent 2-mer was calculated to be 340 base pairs and estimation of
all bands is seen in Fig. 2. Control DNA not restricted with Hae III always
migrated as a single band j>2000 base pairs.
In order to estimate the amount of the entire genome formed by the
multiple fragments,densitometer tracings were cut and weighed and the
weight of the peaks (tangent skim from background DNA) compared to the
total fluorescence. Fluorescence was assumed to be proportional to concentration of DNA; this seemed reasonable when weights of bands with known
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-S
I20 0 - *
1050-*8 70-«6 0 0— »•
-1
15—
Figure 2. Calibration of molecular
weight of Hae III fragments.
Top: Left lane shows molecular
weight in base pairs of 4>x 174 (2ug)
digested with Hae III. At right is
Hae III digest of heterochromatic
male placental DNA. Of the multiple
fragments (numbered 1-6), the 2-mer
is the most prominent. Other faint
bands (-^1) and barely detectable
bands (<] ) as well as band resistant
to significant digestion (o)are seen.
s is start of migration, electrophoresis in 2.2% agarose, negative
image of ethidium bromide fluorescence.
Bottom: determination in base pairs
of human DNA bands using <(>xl74 fragments of known molecular weight for
calibration (—). Bands 1-6 are
multiples of 170 base pairs and very
faint bands ( V ) are consistent with
a 7,8 and 10-mer.
73-
to
S1000
40
210
CD
O
100
100
4
6
8
10
12
DISTANCE MIGRATED (cm.)
amounts of DNA were compared. Also in these studies gels were loaded with
exactly 5 or 10 yg of restricted DNA and yielded consistent estimates of
total DNA by weight. From these measurements the prominent 2-mer band was
calculated to comprise M ) . 8% of the genome and the weight of all the multiples (bands 1-6) together made up slightly less than 2% of the total
genome.
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Enrichment of Hae I I I Fragments In Rapidly Renaturing DNA. Hydroxya p a t l t e fractionation of unsheared DNA preparations (>_ 10
daltons) r e -
vealed considerable enrichment of the 1-mer and 2-mer species on r e s t r i c t i o n with Hae I I I (Fig. 3 , lane 2 ) . Although a l l hydroxyapatite
f r a c t i o n s were made to a Cot of <_ l f i n different experiments somewhat
v a r i a b l e percentages of t o t a l DNA were eluted i n the double stranded
f r a c t i o n s and were in the order of 7-13%. This v a r i a b i l i t y was considered
due in part to minor differences
in DNA preparations and in binding
capacity of d i f f e r e n t batches of hydroxyapatlte. In comparing a l l
32
rapidly reassociating fractions labelled with
different
P and digested to com-
pletion with Hae III it was noted that the lower the percent of DNA recovery from hydroxyapatite, the more enriched were the preparations in
fragments identified as multiples. In all these experiments unrestricted
DNA was seen at the origin. Hae III digests of whole DNA were also denatured and allowed to reassociate in order to purify fragments further
from contaminating single stranded tails. Although in these experiments
typical 2-mer fragments were observed they were not appreciably purer
than in preparations made from unsheared DNA.
Rapidly renaturing fragments should be enriched in heterochromatin.
Heterochromatin isolated from male placenta digested with Hae III as seen
in Fig. 2 showed some enrichment of identified bands in that the background DNA was decreased by 15-20!! and the 1-mer fragment weight was
"vl.6x the weight of the fragment seen in whole nuclear DNA digested at the
same time and electrophoresed with the same load of DNA. Enrichment of
these species in heterochromatin were not impressive when the band relatively resistant to Hae III digestion (o, Fig. 2) was compared. The
weight of this band in heterochronatlc preparations was "Wx that seen in
comparable whole nuclear digestions, which at the time of this report has
been identified as the Y satellite (2l).
Other Restriction Sites. Several other type II restriction enzymes
were tested on human DNA. "Nick translated" DNA was used to minimize the
amount of DNA tested as well as contaminants that might interfere with
enzyme activity. Enzymes were tested on whole nuclear and rapidly reassociating DNA, and some of these results are summarized in Table 1.
Eco R. yielded 3 discrete reproducible fragments consistent with a
molecular weight 340, 248 and 92 base pairs (b.p.). These bands were considerably enriched in rapidly renaturing DNA (Fig. 3, lane 3) as compared
to whole nuclear DNA (not shown) , where the shortest easily visible band
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Enzyme
Lots of Enzyme Tested
Result
1. Hind III
3
some digestion of total DNA;
most fragments >500 b.p.
2. Hlnf I
1
2 high M.W. fragments
3. Hha I
4
1
discrete bands (see text)
minimal digestion
6. Hae II
3
1
minimal digestion
7. Alu I
3
4. Hpa II
5. Eco R.
discrete bands (see text)
discrete bands, variable
results.
Table I: Other restriction enzymes tested on "nick translated" human whole
and rapidly renaturlng DNA.
was 340 b.p. In whole nuclear DNA most of the DNA was seen in the upper
1/3 of the gel. The 248 b.p. and 92 b.p. fragments together equal the
length of the 340 b.p. band. In several experiments with extensive Eco R^
redigestion the residual 340 b.p. fragment could not be further redigested.
These three bands thus are possibly related, and the 340 b.p. fragment may
lack one of the Eco R. sites.
Hha I restriction produced many fragments which were also enriched in
rapidly renaturing DNA as compared to whole nuclear DNA (Fig. 3, lanes 4
and 5). Again fragments with similar electrophoretic mobilities to those
seen in Hae III digests with molecular weights in the order of the 1-mer
and 2-mer (170 b.p. and 340 b.p.) were observed. Additional bands such as
those on either side of the 1-mer were calculated to be 188 b.p. and
148 b.p. respectively. Together these add up to almost 340 b.p. suggesting
that some of these fragments are related.
In order to test whether material in the Hae III 2-mer could be related
to or contain sequences similar' to the Hha I fragments,the 2-mer Hae III
band was eluted, "nick translated" and digested with Hha I (Fig. 3,lane 7).
Some of the fragments generated were similar to those seen on Hha I restrictions alone,and the remaining DNA was resistant to repeated Hha I digestion. Loss of some restriction sites (one Eco R, site and several Hha I
sites) may have occurred in some of the fragments. However, since the
Hae III 2-mer band had a significant background of other DNA more rigorous
experiments are needed to conclusively prove this hypothesis.
Filter Hybridization Studies. In order to find if the multiple Hae III
bands were In fact related to each other In sequence,the 2-tner band was
used to probe the entire spectrum of Hae III fragments immobilized on a
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Figure 3. Autoradiographs
of "nick translated"32p
labelled DNA. Restricted
DNA lanes 1-5 in 4% acrylamide electrophoresis on the
same slab gel. Hae III digestion shows considerable
enrichment of 1-mer and
2-mer fragments (—) in
rapidly renaturing DNA
(lane 2) as compared to
whole DNA preparation (lane
1). Eco R^ digestion (lane
3) shows a fragment of the
same electrophoretic mobility as the Hae III 2-mer
(o ); in addition note two
smaller fragments generated
by Eco Ri (*•) calculated to
be 248 and 92 base pairs
using Hae III 1-mer and 2tner bands for calibration.
Lanes 4 and 5 are Hha I
restrictions of rapidly re—
naturing and whole DNA respectively. Again note
fragments with similar mobilities to Hae III 1-mer
and 2-mer in both preparations (o)> these bands in the rapidly renaturing DNA are considerably enriched. Many other smaller bands are also noted in the Hha I digestions.
Lanes 6 and 7, another gel; eluted Hae III 2-mer band, nick translated control (lane 6) not restricted ,and lane 7, 2-mer band digested with Hha I.
I
- • —m
oi
-Mi
filter (see Methods). Eluted 2-mer material, forming a single 340 b.p.
band without other detectable species,
as seen in Fig. 3, lane 6, was
used as the sequence probe. Autoradiographs showed Hae III DNA fragments
capable of reannealing to this probe (Fig. 4, top). Autoradiographs carefully aligned on filter paper showed hybridization of material to
multiples 1-6 as well as to two additional fragments (7 and 8). Analysis
of autoradiographs confirmed that fragments able to hybridize were all related to even multiples 1-8 mer since they fell on a straight line when
the log of fragment size vs. distance migrated was plotted. Furthermore
there was apparent lack of hybridization to intermediate bands (the 2.5
and 3.5 mer) as well as to the very high molecular weight fragment (o).
Thus this experiment clearly indicated that the multiples are related to
each other in sequence yet the other bands can not as yet definitively be
assigned a similar sequence. It can also be seen that the 1-mer hybridized
less efficiently than would be expected from its amount on ethidium bro-
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Figure 4. 2-mer band eluted from agarose gel,nick translated and hybridized
with whole human DNA restricted with Hae III or Eco R^ subjected to electrophoresis and immobilized on filters. Densitometer tracings of fluorescence
(U.V.) is correlated with autoradiographic peaks (32p) in each case, s is
origin of migration. Top: Hae III autoradiographic peaks correspond with
bands of 1-8 mers. DNA peaks at o, 2.5-mer and 3.5-mer ( T ) do not show
significant hybridization as also demonstrated in aligned photographs at
right of 2.2% agarose gel and eluted gel autoradiography. Bottom: Hae III
2-mer band material also showed significant hybridization to major visible
ethidium bromide peaks ( V ) in Eco Ri digested DNA and a few minor bands
were also detected autoradiographically (f). At right is corresponding photograph of 1% agarose gel and autoradiograph
of eluted gel probed with the
Hae III 2-mer.
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mide gels. It is possible that the smaller size of the fragment limited its
efficiency of hybridization. Alternatively variation in other restriction
sites, such as Hha I sites, in these related molecules might decrease their
capacity to hybridize with a particular member. This latter concept could
also encompass the observation that the 7-mer band apparently hybridized
more efficiently than the 3-mer band.
Restriction with Eco R. suggested a relation between the 340 b.p.
fragment and the 2-mer of Hae III restriction. Filter hybridization of
the Hae III 2-mer to human nuclear DNA digested with Eco R. and separated
on an 1% agarose gel showed definitive hybridization of Eco R.. bands with
the Hae III 2-mer (Fig. 4, bottom). A few minor bands in addition to those
easily visualized with ethidium bromide fluorescence were brought out in
this hybridization.
DISCUSSION
In the present paper Hae III restriction fragments of human DNA have
been shown to consist of multiple repeating units containing a common and
specific sequence. These units share a common sequence with fragments produced by restriction with Eco R-^Evidence presented here also suggests a
similar sequence may be present in fragments produced by Hha I as judged
from analysis of rapidly renaturing DNA and digestion of the eluted Hae III
2-mer. The electrophoretic mobility of at least one fragment generated by
all three restriction enzymes displays a striking similarity. This fragment
was estimated to be in the order of 340 base pairs which is in reasonable
experimental agreement with a previous estimate of 360 base pairs for the
Eco R. band (4). One explanation for this common fragment could be clustering of these three restriction sequences at discrete loci in the repeating
sequence. Such a site would be made of some order of the sequences GCG'C
for Hha I (22) GG'CC for Hae III (23) and G'AATTC for Eco R. (24).
Clustering of restriction sites on repeating units have previously been
suggested for bovine DNA (3). Alternatively, specific regions of the repeating moiety in human DNA may have eliminated or inserted different restriction recognition sequences (5,6). It is of interest in the present
studies that the 2.5 mer and 3.5 mer did not show positive evidence for
sequences in common with the dimer and thus in human DNA do not appear to
support the idea of crossing over in "staggered" register of these uneven
multiples as proposed for mouse satellite (5).
The pattern of multiple Hae III fragments in human DNA is similar to
that seen in other purified eucaryotic DNAs (5,6). Its enrichment in
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rapidly renaturing fractions as demonstrated here is also consistent
with satellite DNA. However, fingerprints of this material purified in a
variety of ways shows a sequence of considerably higher complexity than
that of several simple human satellites purified in this laboratory (25).
The Hae III repeating fragment is estimated here to comprise ^27, of the
genome and thus is 100-fold more common than human ribosomal sequences (26)
which might be included in rapidly renaturing DNA fractions. Our preliminary results also indicate that these fragments do not significantly hybridize to known ribosomal sites on acrocentric human chromosomes (27).
The observation that these fragments are contained in approximately
equivalent or increased amounts in purified nuclei and heterochromatic
nuclear fractions as compared to whole cell DNA strongly argues against a
mitochondrial origin of these sequences. It is possible that the human
fragments described, although bearing certain physical similarities to
satellite DNA make up a somewhat distinct class of molecules and account
for the quantitative discrepancies noted between the amount of human
satellites (8) and rapidly renaturing DNA (7). The biological significance
of these fragments and their relation to other rapidly renaturing interspersed sequences or inverted sequences in human DNA need further
clarification.
ACKNOWLEDGEMENTS
The author gratefully acknowledges the excellent assistance of John Wu,
the valuable discussions with J. Sedat and generous advice of L. Sedat.
This work was supported by an NIH grant 5 R01 CA 15044-03. LM is the
recipient of an NIH Research Career Development Award 1 K04 NS 00101-02.
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