192 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 97221

T H E GEL E L E C T R O P H O R E S I S OF DNA

C. A A I J AND P. B O R S T
Department of Medical Enzymology*, Laboratory of Biochemistry, University of Amsterdam,
Amsterdam (The Netherlands)
(Received D e c e m b e r 3 i s t , 1971)

SUMMARY

I. We have compared the electrophoretic mobility of linear duplex DNAs

(bacteriophages T4, lambda, T 7 and q~29) and of circular duplex DNAs (replicative
form DNA of bacteriophage q~XI74, bacteriophage PM2 DNA and rat mtDNA) in
agarose gels. The method requires only o.5-1 #g DNA; the presence of the inter-
calating dye ethidium bromide in the electrophoresis buffer allowed the immediate
visualization of the fractionated DNA bands.
2. With all circular DNAs tested, the closed circular duplex form migrated
faster through the gel than the open circular duplex form. The migration rates of the
closed circular DNAs were inversely proportional to the logarithms of their molecular
weights (3.4" lO6, 6.0- lO6 and IO. lO 6, respectively).
3. In contrast to these small circular DNAs the linear DNAs (~b29, T7, lambda
and T4) with molecular weights from I I • lO 6 to IiO • lO 6, had similar electrophoretic
mobilities in 2.5 % agarose gels, whereas q529 DNA ran ahead in 0.6 % agarose gels.
4. At higher gel concentrations the migration rate of circular DNA decreased
to zero; the migration rate of the linear DNAs studied, however, decreased but re-
mained significantly above zero. Hence, T 4 DNA (Mr IiO. lO 6) migrates consider-
ably more rapidly through 4 % agarose than both ciI cular forms of q~X replicative
form DNA (Mr 3.4" lO6). Electrophoresis provides, therefore, a good method for the
separation of linear and open circular duplex DNA of the same molecular weight.

INTRODUCTION

Electrophoresis of macromolecules through porous gels 1 has proved useful for

the fractionation of polypeptides ~,~, RNAs 4,5 and ribosomes and their subunits 6,~.
For DNAs this technique has hardly been used. Thorne s,9 demonstrated that agar
gel electrophoresis could be used to separate the different molecular configurations
of polyoma viral DNA from each other and from host-cell DNA. More recently Taka-
hashi et al. 1° have shown that an inverse relation exists between the electrophoretic
mobility in agarose and the sedimentation coefficient of fragmented T2 DNA of
three different size classes.

* P o s t a l address: J a n S w a m m e r d a m Institute, Eerste Constantijn Huygensstraat 20,

Amsterdam, The Netherlands.

Biochim. Biophys. Acta, 269 (1972) 192-2oo

GEL ELECTROPHORESIS OF DNA 193

In this paper we present experiments on the electrophoretic behaviour of a

series of circular and linear duplex DNAs of various sizes in agarose gels. The results
show that electrophoresis provides a sensitive method for the separation of closed
and open circular DNAs and that it is uniquely suited for the separation of open
circular DNAs and linear DNAs of the same molecular weight.

METHODS

D N A preparations
T 4 phage particles were purified by differential centrifugation n and lambda
phage by (NH,)2SO4 precipitation TM. The purified viruses were incubated with I ~ug
deoxyribonuclease plus I #g ribonuclease/ml to degrade contaminating host-cell DNA
and RNA, if present. DNA was extracted from the purified viruses with 2 % sodium
dodecylsulphate followed b y phenol extraction.
T7 DNA was isolated from purified T 7 phages as described by Yamamoto et al. 13.
Bacillus subtilis phage q529 was grown and purified according to published
procedures x4'15. The phage DNA was extracted from the virus by pronase treatment
in o.I % sodium dodecylsulphate and phenol extraction.
Rat-liver mtDNA was prepared according to the method of Borst et al. 16.
Bacteriophage PM2 DNA was extracted from virus grown and purified as
described by Espejo and Canelo17; further purification was carried out according to
Espejo et al. TM.
Bacteriophage ~0XI74 was grown and purified according to a modified 19 pro-
cedure of Sinsheimer 2°'~1. The replicative form of q~xI74 DNA was isolated as de-
scribed by Jansz et al. 22.

Preparation o/gels
Agarose gels were prepared b y dissolving agarose (Behring or Seakem) in hot
water at twice the final gel concentration wanted. After cooling the homogeneous
solution to 4o°C an equal volume of 2 × F-buffer (see below) was added and the
mixed solution was poured into glass tubes (80 mm long; internal diameter, 8 mm).
Agarose gels containing ethidium were prepared in the same way, except that the
volume of 2 × F-buffer contained 200 #g or 20 #g ethidium bromide/ml, which re-
sulted in a final concentration of IOO #g or IO/zg ethidium bromide/ml in the gels,
as specified in the legends to figures.

Procedure o/electrophoresis
After polymerization of the gels, pieces of nylon stocking were fixed on the
bases of the tubes to prevent the gels from sliding out. The top few millimeters of
the gels were cut off with a razor blade to ensure a flat gel surface. In all experiments
the electrophoresis buffer F (0.04 M Tris-HCl-o.o2 M sodium acetate-2 mM diso-
dium EDTA adjusted with acetic acid to pH 7.7 at about 2o°C) was used. The time
between polymerization and starting the electrophoresis is not critical in the case
of the 1 % (or higher strength) agarose gels; however, a minimum period of ageing
of about 5 h at room temperature was used in the case of the 0.5-0.6 % gels. The
gels were pre-run in a vertical tube apparatus (Acrylophor, Pleuger) at the specified
Biochim. Biophys. Acta, 269 (1972) 192-2oo
I94 c. AAIJ, P. BORST

current for approximately 3o rain. The lower buffer (anode) contained IOO or IO #g
ethidium bromide/ml during pre-electrophoresis in the case of the ethidium-agarose
gels. Electrophoresis of ethidium-containing gels was carried out in a darkened
room. DNA samples (o.5-I.O #g, dissolved in o . o i - o . I ml of IO mM Tris-HCl-o.5 mM
disodium E D T A (final p H 7.6) containing at least 5 % (w/v) of sucrose) were layered
on the gels and electrophoresis was continued at room temperature. Time and current
of the electrophoretic runs are specified in the legends to the figures. After the run,
the pieces of nylon stocking were removed and the gels were gently blown out of
the tubes.

Visualization o~ the/ractionated D N A in the gel

The gels run without ethidium bromide were stained overnight in a buffer
containing ethidium bromide (50 mM sodium acetate/acetic acid (pH 4.5), I m M
E D T A and IOO #g ethidium bromide/ml). The ethidium not bound to DNA was
removed by washing the gels at least twice for 8 h in the same buffer without ethidium.
The gels, run in the presence of ethidium bromide, were destained immediately
omitting the staining procedure. Using a Universal ultraviolet lamp (CAMAC, type
TL-9oo at 350 nm) the clear orange bands could be observed in a darkened room and
photographed. Negative or positive prints of these photographs are reproduced. To
facilitate comparison, the results of one series of gels (Fig. 2) are presented schema-
tically.

Miscellaneous
The open and closed circular duplex forms of ~bXI74 replicative form DNA and
rat-liver m t D N A were separated by sucrose gradient centrifugation essentially as
described b y Borst et al. ~3.
For deoxyribonuclease treatment of the DNA samples, MgC12 was added to a
final concentration of 5 mM and incubation was carried out at 25 ° for the time in-
dicated with 0.02 ng pancreatic deoxyribonuclease (Worthington). The stock solu-
tion of the enzyme contained IO ng enzyme and IOO/~g bovine serum albmnin per
ml of IO mM Tris-HC1 (pH 7.4). The reaction was stopped b y adding E D T A (pH 7.5)
to a final concentration of 50 raM. The whole mixture was then layered on a gel for
analysis.

RESULTS

The three circular and four linear duplex DNAs, used in our experiments, are
listed in Table I. The integrity of each DNA was checked by analytical band sedi-
mentation through CsC1. The linear DNAs gave one sharp band, the circular DNAs
two bands corresponding to closed circles (Component I) and open circles (Compo-
nent II) (see refs 16, 23-25). In all cases the sedimentation coefficients were in reason-
able agreement with the values reported and no degraded DNA was detected.
Fig. I compares the migration rates of the four linear DNAs through two types
of gels, o.6 and 2.5 % agarose. In 2.5 % gels all DNAs migrate at approximately
the same rate even though their molecular weights v a r y from I I • lO 6 to IiO" lO 6.
In the o.6 % gels all DNAs run faster than expected, because of the increase in
Biochim. Biophys. Acta, 269 (1972) 192-2oo
GEL ELECTROPHORESIS OF D N A 195

TABLE I
SEDIMENTATION VALUES AND LITERATURE DATA. F R O M THE DNA PREPARATIONS USED IN THE
]~LECTROPHORETIC STUDIES

The sedimentation coefficients were determined by analytical band sedimentation in a Spinco

Model E centrifuge, as previously described 23.

DNA S~o,w Literature data Ref.

determined
S2o,~ M r × zo -e Configuration

~ X I 7 4 RF 21 + 17 21 + 17 3.4 circle 24
PYI2 27 + 21 28 + 21 6.0 circle 25
Mitochondrial 39+27 39+27 IO circle 16, 23
• 29 24 24. 5 II linear 14, 26
T7 3o 32 25 linear 27
Lambda 33 34 31 linear 27
T4 59 60-65 IiO linear 27, 28

r7

T4
Dda
lambda

9 ~'29

0 10 .20 (_ )
Distance migrated (ram) Distance migrated (ram)
Fig. I. Electrophoresis of various linear DNAs through agarose gels of different concentration.
Agarose gels were prepared as described in Methods. The gels in Series a consisted of 0.6 %,
those in Series b of 2. 5 % agarose. The gels were loaded with 0. 5 t*g of the DNA specified. Elec-
trophoresis for 3 h at 2.5 mA per gel. Note that the ~29 DNA band is leaning over because the
top of the gel was not properly aligned in this photograph.
i

p o r e size w i t h l o w e r gel c o n c e n t r a t i o n s , b u t t h e s m a l l e s t D N A (q529 D N A ) n o w m i -

g r a t e s a h e a d of t h e o t h e r t h r e e .
Fig. 2 c o m p a r e s t h e m i g r a t i o n of l i n e a r T 4 D N A to t h a t of t h e t h r e e c i r c u l a r
D N A s in I, 2 a n d 4 % a g a r o s e gels. I n t h e 1 % gel all c i r c u l a r D N A s are r e s o l v e d
i n t o t w o b a n d s , t h e closed a n d o p e n c i r c u l a r f o r m (see b e l o w ) , a n d b o t h f o r m s m i -
g r a t e f a s t e r t h a n T 4 D N A . W h e n t h e gel c o n c e n t r a t i o n is raised, t h e m i g r a t i o n r a t e
of t h e c i r c u l a r D N A s d e c r e a s e s m u c h m o r e t h a n t h a t of T 4 D N A w i t h t h e s t r i k i n g
c o n s e q u e n c e t h a t t h e l i n e a r T 4 D N A (Mr I i O • IO *) m o v e s m u c h f a s t e r t h r o u g h t h e
4 % gel t h a n t h e c i r c u l a r ~ X r e p l i c a t i v e f o r m D N A f o r m s (Mr 3.4 " IOe) •
T h e e l e c t r o p h o r e t i c b e h a v i o u r of t h e c i r c u l a r D N A s was s t u d i e d in m o r e d e t a i l
by a modified procedure. In this procedure the intercalating dye ethidium bromide
was a d d e d to t h e gel a n d t o t h e a n o d i c b u f f e r c o m p a r t m e n t t o k e e p t h e e t h i d i u m
c o n c e n t r a t i o n in t h e gel c o n s t a n t d u r i n g e l e c t r o p h o r e s i s . T h i s a l l o w e d t h e i m m e d i a t e

B i o c h i m . B i o p h y s . Aeta, 269 {1972) 192-2oo

196 c. AAIJ, P. BORSr

--- • ' ----- T4

@ - - l I --mr
---- • [] --- PM2
. . . . I I-- ~XRF
t I I I l i I

.... • ..... T4

E~ ---- I .... mt
. . . . . • • - - - PM2
....... I I - ~XRF
i I [ I I I I

• I T4

© II ~ - PM2
I --I ~XRF
I I
0 ll0 2Jo 310 410 5J0 60

Distonce migroted (mml

Fig. 2. Electrophoresis of various circu]ar DlXTAs and linear T# I)~TA t h r o u g h agarose gels. The
gels were loaded w i t h o . 5 - i . o / z g of the D N A indicated. Conditions of electrophoresis: Series a,
1% agarose, 2.5 h, 5 mA per gel; Series b, 2 % agarose, 5 h, 5 mA per gel; Series c, 4 % agarose,
5 h, 5 mA per gel.

v i s u a l i z a t i o n of the orange-coloured D N A b a n d s at the e n d of the r u n w i t h o u t time-

c o n s u m i n g s t a i n i n g a n d d e s t a i n i n g procedures. Contrast is improved, however, b y
d e s t a i n i n g a n d this was done in all e x p e r i m e n t s presented in this paper (see Methods).
Fig. 3 illustrates the effect of the presence of e t h i d i u m d u r i n g electrophoresis on the
m i g r a t i o n rates of various DNAs. The b i n d i n g of the positively charged dye to the
D N A led to a decrease in m i g r a t i o n rate. I n addition, the presence of e t h i d i u m led
to a reproducible s e p a r a t i o n of the closed a n d open circles of m t D N A (Fig. 3). Sepa-
r a t i o n was v a r i a b l e a n d sometimes a b s e n t in the absence of e t h i d i u m with m t D N A ,
b u t n o t with the other circular DNAs.
Since closed circles have a more compact configuration t h a n open circles it
could be a n t i c i p a t e d t h a t the closed circles would represent the faster of the two
b a n d s observed w i t h all circular DNAs. This was verified b y electrophoretic analysis
of closed a n d open circles first separated b y p r e p a r a t i v e b a n d s e d i m e n t a t i o n t h r o u g h
sucrose gradients a n d of open circles m a d e from closed circles b y a l i m i t e d deoxyri-

0 10 20 30 0 • t0 20
DiStance mlgrated(mm)

Fig. 3. The effect of the presence of ethidium during electrophoresis on the migration rate o
I)NA through agarose. Two series of o.6 % agarose gels were run in parallel; only the gels in Se
ries a contained IO/~g ethidium bromide/ml. The gels were loaded with o.5-1.o #g DNA, as speci-
fied. After the run the gels from Series b were stained with an ethidilm bromide-containingbuffer
(see Methods). Electrophoresis for 2 h at 2.5 mA per gel.
13iochim. Biophys. Acta, 269 (1972) 192-2oo
GEL ELECTROPHORESIS OF DNA 197

o lo zo 30 4o
Distance m i g r a t e d ( r a m )

®
®
®

D i s t a n c e mior'ated (ram)
Fig. 4, Comparative migration rates of the open and closed circular duplex forms of three cir-
cular D N A s . In all gels o.5 ~o agarose was used, containing ioo/~g ethidium bromide/ml. Series a:
gel I w a s loaded w i t h 0.25 fig u n f r a c t i o n a t e d mtDBIA; gel 2 w i t h 0. 5 fig m t D N A C o m p o n e n t I;
gel 3 w i t h o. 5 fig m t D N A C o m p o n e n t II. E l e c t r o p h o r e s i s for 3 h, 3.5 m A p e r gel. Series b: gel i
w a s loaded w i t h i/~g t o t a l PM2 DI~A; gels 2 a n d 3 w i t h I fig PM2 D N A , a f t e r t r e a t m e n t w i t h
d e o x y r i b o n u c l e a s e for 2 rain in gel 2 a n d for 3 ° rain in gel 3 (final v o l u m e , o.z ml). E l e c t r o p h o -
resis for 2 h 45 m i n a t 2. 5 m A p e r gel. Series c: gel I w a s loaded w i t h I fig u n f r a c t i o n a t e d # X
replicative f o r m D N A ; gel 2 w i t h 0.5 fig C o m p o n e n t I; gel 3 w i t h o.5/~g C o m p o n e n t II. E l e c t r o p h o -
resis for 3 h a t 2.5 m A p e r gel. C o m p o n e n t s I a n d II of m t D N A a n d ~ X replicative f o r m D ~ A
were s e p a r a t e d b y sucrose g r a d i e n t s e d i m e n t a t i o n (see M e t h o d s ) .

bonuelease treatment. The results of these experiments, presented in Fig. 4, clearly

show that with all three circular DNA preparations the closed circles migrate faster
than the open circles. Fig. 5 shows that the three closed circles are completely se-
parated; the rate of migration is inversely proportional to the log molecular weight
of the DNAs.
In some electropherograms of total circular DNA preparations, a third minor
band was observed, running just ahead of the open circles in 0.6 °/o agarose. This
band might represent linear molecules, arising b y the conversion of circular DNA
into linear DNA of the same length by the introduction of a double-stranded break.
Since linear molecules of this type are difficult to obtain free of circles, we have used
bacteriophage ~b29 DNA as a model for linear mtDNA. The molecular weight of ~b29

Fig. 5. Co-electrophoresis of closed circular d u p l e x D ~ A s of t h r e e m o l e c u l a r weights. O n o.5 %

a g a r o s e gels plus IOO/~g e t h i d i u m b r o m i d e / m l a m i x t u r e of t h e purified closed circles ~ X repli-
c a t i v e f o r m D ~ A (0. 5 #g), P M 2 DlxIA (0. 5 fig) a n d m t D ~ A (0.5/~g) w a s layered. T h e b a n d s
were identified b y electrophoresis of t h e s e p a r a t e c o m p o n e n t s on parallel gels in t h e s a m e r u n .

B$ochim. Biophys. Acta, 269 {1972) 192-2oo

198 c. AAIJ, P. BORST

DNA is only slightly higher than that of m t D N A (Table I). The molecule was re-
ported 2n to contain complementary single-stranded "sticky" ends, but this report
was not confirmed in this laboratory (P. Borst and G. J. C. M. Ruttenberg, unpub-
lished results) or elsewhere 14 and with our pronase-treated q)29 DNA preparations
artefactual circularization did not occur to a detectable extent. In 0.6 % agarose
gels q)29 DNA and open circular m t D N A have about the same mobility (Fig. 6a).
When the gel concentration was raised to 2 % agarose, however, q)29 DNA ran con-
siderablyfaster than both circular m t D N A components (Fig. 6b), as already antici-
pated from the results presented in Fig. 2. We infer from these results that the fast
minor component in m t D N A in Fig. 6b with the same electrophoretic mobility as
#29 DNA is either the linear duplex form of mtDNA or contaminating linear nuclear
DNA.

'" '" o ,o ,o
D i s t a n c e migrated(mm) D i s t a n c e migrated (ram)
Fig. 6. Electrophoresis of ~29 DNA and mtDNA through agarose gets of different concentrations.
The gels in Series a were o.6 % agarose and in Series b 2 % agarose; they contained IO/,g ethi-
dium bromide/ml. On gel I 0.25/~g #29 DN'A was loaded; on gel 2 o.5//g mtDNA. Conditions of
electrophoresis: Series a, 2.5 h at 2.5 mA per gel; Series b, 2.5 h at 5 mA per gel.

Finally, we note two practical points. Although electrophoretic mobilities of

various types of DNA through agarose were reproducible with most batches of aga-
rose, we have encountered batches that gave erratic results. Apparently the quality
of the agarose is important. Secondly, occasional DNA preparations failed completely
to enter the gel. We ascribe this to aggregation caused by contaminating protein, be-
cause it could be prevented by treatment of the DNA with pronase.

DISCUSSION
In 1967 Thorne 9 described the separation of closed and open circular forms of
polyoma viral DNA by electrophoresis through agar gels. We have extended his obser-
vations to other circular DNAs and improved the method by using 0.6 ° o agarose
instead of agar and by adding ethidium to the electrophoresis buffer. The latter al-
lows the immediate visualization of the bands at the end of the run, but it was pri-
marily added to improve the separation between closed and open circles. Ethidium
may be expected to have a differential effect both on the charge and the shape of
closed and open circles. The effect on charge is due to the restricted intercalation
of ethidium into closed circles at high ethidium concentrations 29. As a consequence
the mobility of closed circles should decrease less than that of open circles when ethi-
dium is added. The maximal difference in charge between open and closed circles
that could arise in this way is 7 % (calculated from ref. 29). The differential effect
Biochim. Biophys. Acta, 269 (1972) 192-2oo
GEL ELECTROPHORESIS OF D N A 199

of ethidium on shape is due to the fact that at high ethidium concentrations closed
circles are converted into extremely compact, tightly-twisted structures 3°-z2 that
should move faster through the agarose pores. Open circles merely increase in con-
tour length whereas their flexibility m a y even decrease somewhat 31. Rather dis-
appointingly, a clear increase in the ratio (mobility closed circle/mobility open circle)
was only observed with mtDNA. This indicates that the charge effect is too small to
be detected under our conditions.
W h y the expected effect of ethidium on shape does not show up in the smaller
DNAs is not clear. Two factors could be involved. The rod-like stiffness of highly-
twisted circles (see ref. 32) and the presence of (possibly rather inflexible) side
branches to these rod-like structures (see refs. 30 and 32). Both factors could impede
the rapid movement of the compact, ethidium-containing circles through the gel.
The fact t h a t ethidium resulted in increased separation of open and closed circles
in the case of m t D N A could be related to the number of twists in the DNA in the
absence of ethidium. The closed circular forms of PM2 DNA and ~bX replicative
form DNA contain about 60 °/o more twists per unit length in the absence of ethidium
than the corresponding form of m t D N A 33. The closed circles from the two bacterio-
phages might, therefore, be relatively compact in the absence of ethidium and as a
consequence the effect of ethidium would not show up.
The results presented in this paper show that electrophoresis provides a simple
and versatile method for the routine analysis of circular DNAs with molecular weights
up to IO • io 6. I t requires only o.5-1.o #g DNA and simple equipment and m a n y
samples can be analysed in one run. The method is insensitive to low-molecular-
weight contaminants that absorb in the ultraviolet and although this m a y be a dis-
advantage in some experiments, it makes the method uniquely suitable for moni-
toring the purification of circular DNAs from deproteinised lysates (C. Sol and C.
Aaij, unpublished experiments).
Although our experiments were started to study the electrophoretic behaviour
of circular DNAs, they have also provided new information on the behaviour of
linear DNAs in agarose gels. Previous reports had indicated a linear dependence of
the migration of small duplex DNAs 1° and RNAs 5 on the reciprocal of the log molec-
ular weight, but our results clearly show that this relation completely breaks down
at higher molecular weights. In addition, the dependence of migration rate on the
gel concentration is anomalous. Whereas the mobility of the circular DNAs decreases
to zero when the pore size of the gels is sufficiently decreased (Fig. 2), the effect
on the mobility of the linear DNAs is much less (Figs I and 2). We interpret these
effects as follows. When the size of the DNA is relatively small in relation to the pore
size, migration rate is strictly dependent on size. When the size of the DNA becomes
large in relation to the pore size, the circles are excluded from the gel but the linear
molecules are not, because they "crawl" like snakes head-on through the gels. A
circular molecule cannot do this because the stiffness of the helix prevents sharp
bending. On the basis of an entirely different approach Fisher and Dingman 34have
independently arrived at a similar conclusion for the migration of linear DNAs
through gels.
The different ways in which circular and linear DNAs move through agarose
gels, has a useful practical consequence. At present no good methods are available
to separate open circles and linear molecules of the same length (see ref. 35), but it
Biochim. ]3iophys. Acta, 269 (1972) 192-2oo
200 c. AAIJ, P. BORST

is clear from Figs 2 and 6 of this paper that electrophoresis solves this problem.
Adaptation of this technique to the preparative separation of circular and linear DNA
is under investigation.

ACKNOWLEDGEMENTS

We thank Mrs. I. Tiemersma-Meisner and Miss G. T. Noordhoek for expert

technical assistance. We are indebted to Dr S. 0. Warnaar and Dr E. M. Smit
for the q~XI74 replicative form DNA; to Mrs F. Fase-Fowler and Mr R. W. J. Thur-
ing for T 4 and lambda DNA; to Mr R. B. H.Schutgens for T 7 DNA; to Mrs N. van
Harten-Loosbroek for ~29 DNA and to Dr G. Woolfe of Boots Pure Drug Ltd.,
Nottingham, Great Britain, for a gift of ethidium bromide. This work was supported
(in part) by the Netherlands Foundation for Chemical Research (S.O.N.) with
financial aid from the Netherlands Organisation for the Advancement of Pure
Research (Z.W.O.) and by a grant from the Jane Coffin Childs Memorial Fund
for Medical Research.

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