Reprinted from the PRocrepINGs OF THE NaTioNAL ACADEMY OF SCIENCES
Vol. 50, No. 4, pp. 664-672. October, 1963.

RESTRICTION OF IN VIVO GENETIC TRANSCRIPTION TO ONE OF
THE COMPLEMENTARY STRANDS OF DNA*

By M. Hayasur, M. N. Hayasui, anp 8. SPIEGELMAN

DEPARTMENT OF MICROBLOLOGY, UNIVERSITY OF ILLINOIS, URBANA

Communicated by H. E. Carter, July 1, 1963

Several recent investigations'—* demonstrate that when double-stranded DNA is
employed as a template in vitro, the DNA-dependent RNA polymerase mediates
the synthesis of RNA copies complementary to each of the two strands. The pri-
mary concern of the present paper is whether or not this situation obtams in the
intact cell.

Existent evidence suggests that zn vivo transcription may not mvolve both
strands. Thus, the base composition of unfractionated T2 complementary RNA‘ >
and the material collected by hybridization to DNA on columns*: 7 all have shown a
persistent and significant inequality of guanine to cytosine. This discrepancy
would be explained if only one strand is transcribed and if it has a bias toward a low
C:Gratio. Furthermore, it has been shown that sequences complementary to both
ribosomal®-" and soluble!!: 12 RNA exist in homologous DNA, implying that DNA
generates these molecular species. Nevertheless, G does not equal C nor does A
equal U in ribosomal RNA, suggesting’ that transcription in the corresponding re-
gion of the genome does not involve both strands. <A similar statement can be
made for the sRNA cistrons. The ability of 5-fluorouracil to restore® certain
mutants of bacteriophage T4 also leads to the conclusion that only one strand of the
r II region yields RNA messages.

None of these findings are decisive and they clearly do not define with certainty
the mechanism which reads the entire genome. The following three possibilities
can be entertained for genetic transcription: (1) all of both strands are transcribed
into complementary RNA; (2) both strands are employed but never in the same
regions; (3) only one of the two strands serves as a template for genetic messages.
We are ignoring the possibility of regions which are not transcribed at all in either
Vou. 50, 1963 BIOCHEMISTRY: HAYASHI ET AL. 665

strand. This would introduce three more possibilities, but they are not directly
relevant to the central issue here nor to the experiments to be described.

Examination of the possibilities mentioned suggests immediately that the hy-
bridization test of Hall and Spiegelman! can be employed to provide data useful
for deciding which of the three mechanisms operates in the cell. A successful res-
olution requires a source of radioactively labeled homologous complementary RNA
and the availability of at least one of the two strands of the relevant DNA in pure
form.

To provide the experimental requisites, the authors turned to the DNA virus,
#X174, which has been shown by Sinsheimer' to contain only one of the two com-
plementary strands. Nature has thus provided one of the necessary experimental
components in an easily accessible form. However, this useful feature can be
fruitfully exploited only if the complementary strand can also be obtained. It has
been found" that immediately after infection the complement to the injected strand
is synthesized, and a duplex results which has been called the ‘replicating form”
(RF). Asa preliminary to the experiments to be described here, it was therefore
necessary to devise a procedure for the isolation in pure state of the RF-DNA from
the infected cells. This was accomplished* by repeated chromatography on col-
umns of methylated albumin.

The availability of both RF-DNA and the single strand from the mature virus
particle made possible appropriate hybridizations with the RNA message fraction of
@X174. The hybridization tests and the base composition of the RNA hybridized
revealed the presence in infected cells of RNA complementary to only one of the two
strands in the RF-DNA. The data are consistent with the conclusion that one of
the two possible complements is either the principal or sole source of translatable
genetic information.

Materials and Methods.—Strains: The IDNA virus ¢X174 and its host £. col7, strain C, were
kindly provided by Dr. I. Tessman.

Media and buffers: Modified Penassay medium (MPM) was used as described by Nomura
et al. SCXD medium is composed of Tris-HCl] buffer 0.05 MW pH 7.3; 0.5% glycerol; 1073 M
phosphate; 1073 Af MgSO,; 0.5% casein hydrolysate and 1 pg/ml of FeCl. Adsorption medium
(AD) is Tris-HCl buffer 0.05 M pH 7.3; 10-8 M phosphate; 1073 AL MgSO.; 0.5% casein hy-
drolysate; 1 pg/ml of FeCl; 107-4 Af CaCh; 107? Wf NaCl; 10-2? W KCl. 3x) medium is
that described by Fraser and Jerrel;!® SSC" is 0.15 WM NaCl; 0.015 Af Na citrate; TM is 3 X
10-2 M Tris; 5 X 10-3. M Mg*t+; pH 7.4; TMS is3 x 10-2. V Tris; 5 x 107? M Mgt+; 0.01
M NaCl; HMP is 0.01 M PO,; pH 7.0.

Radioactive labeling: Pulse and uniform labeling carried out as described previously. P#
from the Oak Ridge National Laboratory was hydrolyzed to remove morganic pyrophosphate
prior to use. H*uridine (500 wc/112 wg), H*&-thymidine (2 we/yg), and C'-uridine (5.7 ue/mg)
were obtained from New England Nuclear Corp.

RNA and DNA purification: RNA was purified by the procedure of Hayashi and Spiegelman. ¥
Bacterial DNA was prepared by Marmur’s” method with the modification that, subsequent to
treatment with RNAase, aqueous phenol was used to remove protein. DNA from virus particles
was isolated according to Grossman ef al.'° The purification of ¢X174 replicating-form DNA
was carried out as detailed by Hayashi, Hayashi, and Spiegelman.*

Preparation of methylated-albumin-Kieselguhr (MAK) columns: The protocol of Mandell and
Hershey” was followed with one modification in the preparation of the methylated albumin.
The mixture of albumin, methanol, and HC! was incubated at 37°C for 5 days. In our hands this
resulted in preparations which yielded consistently superior separation of the DNA factors.

Conditions of infection: Log phase cells #. colt (strain C) were harvested at a density of 8-10 X
108/ml, washed with adsorption medium (AT) and resuspended in the same medium at 8-9

666 BIOCHEMISTRY: HAYASHI ET AL. Proc. N. A. 8.

10°/ml. Virus was added at a multiplicity of infection (m.o.i.) of about 20. The mixture was
kept at-20°C for 30 min and then diluted 10 times with prewarmed SCXD medium. This is
designated as O-time of infection. Under these conditions, uninfected cells amounted to less
than 0.5%. All experiments were carried out at 30°C.

Unlabeled and labeled virus: In general, the method of Hall et al.2! was followed. On comple-
tion of the lytic eycle, DNAase (5 ug/ml) was added and allowed to incubate. Following this,
CHCl, was added and cell debris centrifuged off. The supernatant was made 2.5 M with re-
spect to (NH4)2 SOx, the precipitate collected, resuspended, and dialyzed against 0.01 Af NHAC.
Tt was then loaded on a DEAE column which was washed with 0.01 Mf ammonium acetate, and
the virus was then eluted with 0.1 M ammonium acetate. Virus labeled with P** was purified in
the same way. Growth and infection were carried out in SCXD medium to which 10 uc/ml of P?
was added per ml one generation prior to introduction of virus at an m.o.i. of 0.1. Incubation
was continued to lysis. Labeling of viral DNA with H? was effected similarly with H®thymidine

at 5 pe/ml.
Infectious DNA: Infectivity of DNA was measured in protoplasts of H. cols C by the pro-

cedure of Guthrie and Sinsheimer.”*

Assay of radioactivity: Preparations of samples on millipore membranes and double channel
counting in a Packard scintillation spectrometer was carried out as described previously.'*

RNA base composition analysis: Bulk E. coli RNA was added as carrier to the P??-labeled
RNA being analyzed, and the mixture hydrolyzed with alkali (0.3 M NaOH) at 37°C for 15 br.
Chromatographic analysis of the resulting 2’-3’ nucleotides was performed using a Dowex-1-
formate column.’

Results. —@X174 message production in the infected complex: Investigation’ of
the ¢X174 E. coli C complex quickly revealed that the ¢X174 was incapable of shut-
ting off macromolecular synthesis specific to the host cell. Thus, it was found that
the RNA synthesized after infection was indistinguishable in over-all base composi-
tion from that observed in uninfected cells. Further, induced enzyme formation
could be instituted for considerable periods subsequent to infection. We were faced
therefore with the problem of detecting message production from $X174 in the
midst of the genetic messages generated by the host genome. This is, however,
readily achieved by use of the two label and co-chromatographie procedure intro~
duced and developed by Kano-Sueoka and Spiegelman.** The principle underlying
the device can be simply stated. Consider two labeled RNA preparations, one
identified by H? and the other by C.'* If a mixture is loaded on a column, the
elution profiles of the H? and C™ label should be identical if the two preparations
are the same, and should differ if one contains some component absent from the
other.

To detect message specific to the genome of ¢X174, pairs of infected and nou-
infected cultures were pulse-labeled with H?-uridine in one case and C4uridine in
the other. The RNA was purified from each, and a mixture loaded on an MAK
column. Figure 1 shows a series of the profiles obtained when such comparisons
were carried out during different periods of the infection. There is virtually no
discrepancy between the profiles of H* and C'* in Figure 1A suggesting that within
6.5 min little or no ¢X174 specific message has been produced in the infected cells.
However, examination of the profiles obtained for the 35-36.5 min interval (Fig.
1B) reveals discordancies unique to the infected complex. Finally (Fig. 1C), the
discrepancies between the H*® and C'4 become even more pronounced when the
examination is extended to a 50-51.5 period. The greatest discordancies occur in
the region to the right of the optical density peak corresponding to the 238 ribosomal
RNA. That these discrepancies are indeed referable to ¢X174 specific messages
VoL. 50, 1963 BIOCHEMISTRY: HAYASHI ET AL.

was tested by hybridizations along
the column using RF-DNA. A
pulse similar to that carried out in
Figure 1C and the pooled material
of the regions indicated in Figure 2
were hybridized to RF-DNA. The
complexed RNA is indicated by the
bar histogram. It is clear that
there is a peak of RNA hybridizable
to RF-DNA in the region corre-
sponding to 4.

From these and similar experi-
ments, one is led to conclude that
prior to five min virtually all of the
RNA messages found in the infected
complex originate from the host
genome. Viral specific messages
accumulate later in the infection.

Hybridization with RF-DNA and
with single-stranded mature DNA:
Because of the small size of @X-
DNA, some of the procedures which
have been useful in the past for de-
tecting and accumulating RNA-
DNA hybrids proved inconvenient.
Cesium chloride density centrifuga-
tions in swinging bucket rotors!*
yielded rather broad bands, partic-
ularly with heat-denatured mate-
rial. Further, neither the DNA-
agar columns of Bolton and Me-
Carthy’ nor the millipore filter tech-
nique of Nygaard and Hall?> were
found to be adequate for trapping
DNA of this size. Consequently, a
new procedure was devised?’ which
stemmed from the observation?
that single-stranded DNA of ¢X174
is very well separated from its
double-stranded counterpart on
MAK columns. RNA hybridized to
DNA chromatograms in about the
same position as the denatured DNA
to which it is complexed.

The outcome of chromatographic examinations of three different hybridizing ex-
periments are shown in Figure 3. To identify the hybrid with certainty the DNA
was labeled with P®? and the RNA with H?,

  
 

 

 

 

40

 

 

 

° 0 20 30 40 50 460 70
TUBE NUMBER

Fic. 1.—Detection of ¢X174 specific RNA
by two-label co-chromatography on MAK
column. The protocol and timing of each
experiment is diagrammed. Time ig meas-
ured from 0 min (see test). A noninfected
culture of E. coli was pulse-labeled with
C1+-uridine for 90 sec. Infected cells were
labeled with H*-uridine at the same time and
for the same period. On termination of in-
corporation, total RNA from each culture
was isolated and purified separately. A
mixture of the two samples was chromato-
graphed. The O.D. profile identifies pre-
existent cellular stable components (168 and
235 indicated by arrows).

% TOTAL
CPM

Tn all cases the H?-RNA employed

668 BIOCHEMISTRY: HAYASHI ET AL. Proc. N. A. 8.

Fig. 2.—Column identification of
$X174 specific RNA. $X174 in-
fected F. colt culture was pulse-

weaeo, labeled with H*-uridine for 90 sec,
COUNTS /6 MIN/O2 mi CPM 50 min after infection. The total
ie Waals RNA was isolated and chromato-

. L | 6000 graphed. O.D. profile identifies

pre-existing RNA. The pooled
samples in the regions indicated in
Hybridizoble the figure were concentrated. The
CPM same number of count from each
sample was hybridized with 207 of
RF-DNA which had been heat-
denatured in 1/10 SSC at 97-98°C
for 15 min. Hybridization was
carried out in 2 X SSC at 42.5°C for
18 hr. The reaction mixture was
[ 2000 chilled, and 307/ml of pancreatic
RNAase, free of contaminating
DNAase, was added. RNAase
i treatment was performed at 26°C

[ace a

 

  
    

2.0 +100,000

|
| Wa + 4000

 

   

SSSA NS ST

 

for 30 min. The reaction mixture
o- Bt patdrrrethomee + ++ 14 one was then loaded on an MAK column
0 zo 3000 BH O78 as described previously. Counts in
TUBE NUMBER the hybrid region were summed up
and are shown in the bar histo-
grams.
ONA RNA
Heat denatured = inf Buth (H3)
SEE RF (P32) > 80-515 min
[ . | 4
ONA 'N ’ Lo
Single (P32) X inf Bulk (M3) SO-5L.5 min '500 i Noct
h ©
n3 CPM N74
1000 @® iy
ase | NoCt UB
com Cone (M)} ©3 7600 “Ov
3000 | Lio sod 13 os
Noct 02 va 3 RNA
ia
t
1
;
ps2

 

   

 

 

 

 

res 10 20 30 4 $0. 60
1 TUBE NUMBER
ee . .
Fie. 3.—Hybrid separation by MAK
Heot denatured x inf "Sue (3) column. 20y of RF or $X single DNA,
Single (P32) 50-515 min both labeled with P*?, was hybridized with
bulk RNA derived by introducing H*
oo\cem WoC! lio uridine 50-51.5 | min after infection.
7 Hybridization, prior RNAase treatment,
o% L and column procedure are as detailed in
f S00 “xO Fig. 2. Note that the RNAase-resistant
02 ws ofepse core elutes earlier than the single-stranded
ana i \ OWA DNA and accompanying hybrid. By
P2560. jo} los washing with NaCl of suitable concentra-
a } \ tion, the core can be removed completely
/ Na from the column prior to the chromato-
X

 

graphic separation of the hybrid structure.

was bulk RNA obtained by introducing H*uridine 50-51.5 min after infection.
It is clear from Figures 34 and 3B that vegetative single-stranded DNA, whether
heated or not, cannot hybridize significantly to any of the H*-RNA included in
the reaction mixture. However, when heat-denatured RF-DNA is employed,
one observes an RNAase-resistant H*component in the region of the single-
stranded DNA and containing P®2. The faet that the radioactivity is alkali labile
Von, 50, 1963 BIOCHEMISTRY: HAYASHI ET AL. 669

and yields the expected 2’-3’, ribotides proves that it is RNA. These results sug-
gest that ¢X174-RNA message is incapable of complexing with the single-stranded
DNA but can hybridize with the RF-DNA.

Table 1 summarizes a series of hybridization experiments carried out with labeled
RNA derived from different periods of the infection. Comparatively little hybridi-

TABLE 1
HypripizaTions oF RNA wirH RF anp SIncLe-STrrRanpED DNA
RNA/DNA pulse-labeled between (min) REF Single-strand
5- 6.5 253 <60*
35-36.5 3,261 <160*
50-51.5 3,473 <160*

* These represent upper limits estimated from summation over all tubes in the hybrid regions of the
MAK column (cf. Fig. 3). The actual number is undoubtedly much lower.

20y of heat-denatured RF and 20y of single-stranded DNA were hybridized with bulk RNA pulse~
labeled with H*-uridine in the intervals indicated in the first column. The conditions of hybridiza-
tion, RNAase treatment, and subsequent isolation on an MAK column are as detailed in Fig. 2.

zation is observed between RF-DNA and RNA labeled between 5 and 6.5 min.
However, at later periods of infection the amount of hybridization observed with the
RF-DNA increases strikingly. In contrast, the single strand from the mature virus
exhibits virtually no capacity to hybridize with RNA from any interval. The
results in Table 1 agree with the profiles observed in Figure 1. Both indicate that
@X174 message is to be found in considerable amounts only late in the infectious
cycle.

Base composition of RNA hybridizable to RF-DNA: The results described indi-
cate that RNA messages produced in the F. coli-¢X174 complex hybridize ef-
fectively only with RF-DNA and not with single-stranded mature DNA of ¢X174.
This outcome offers strong support for the assertion that the component of the
RF-DNA which serves as a template for message production corresponds to the
complement of the DNA strand found in the vegetative particle. To complete the
proof it is necessary to demonstrate that the base composition of the RNA hy-
bridized to RF-DNA corresponds to that which this assertion predicts; the com-
plexed RNA should be complementary to the complement of the mature vegetative
strand. Asa consequence, it should possess a base composition which mimics that
of the vegetative DNA. The numerical situation is such as to make an experi-
mental test of this prediction easily attainable.

The necessary P*?-labeled RNA was prepared by 3-min labeling of the injected
complex at various times. As an added precaution a simple device, detailed in
Table 2, was used to make a preparation which would provide an adequate sample
of all ¢X174 messages. The labeled RNA thus obtained was hybridized to RF-
DNA and the hybrid mixture subjected to RNAase. The product was then chro-
matographed on MAK columns as in Figure 3. The hybrid region was collected,
hydrolyzed with alkali, and the base composition of the labeled nucleotides deter-
mined. The results are summarized in Table 2. The base composition of the RNA
hybridized coming from the 50-51.5 min period is in good agreement with that ob-
tained from the preparation expected to contain equivalent amounts of all messages
synthesized during the infection. Comparison of the three DNA base compositions
listed reveals that the hybridized RNA is similar to the vegetative single strand and

670 BIOCHEMISTRY: HAYASHI ET AL, Proc. N. A. 8

TABLE 2
Base Ratio oF HyBripizABLE RNA
Cc A U(T) G
RNA
all stage-pulsed 17.5 23.8 33.1 25.6
(0-54 min)
short pulse 17.5 25.5 34.0 23.0
(50-53 min)
DNA
@X174 single* 19 25 33 23
@X174 complementary f 23 33 25 19
oX174 RFT 21 29 29 21

* Taken from Sinsheimer.'4

+ Assumed complementary to the original strand.

{ Assumed double-stranded with single and complementary strands.

E, colt C in log phase was concentrated to 10%/ml in AD. 6@X174 was added at m.o.i. ~ 20. For the
complete message sample, adsorption of the phage was performed at 20°C for 30 min; 1 ml of this complex
was added every 3 min into prewarmed SCXD (150 ml) at 30°C under aeration. When the 18th complex
was added (51 min after 0 time), 20 me of P®? was pulsed for 3 min. The RNA was then isolated, purified,
and designated as ‘‘all stage-pulsed‘’ RNA. A short P32 pulse 50-53 min after infection was also performed
and RNA was isolated and purified.

6 mg of each bulk RNA was hybridized with 70y of heat-denatured RF; hybridization and RNAase
treatment are as detailed in Fig. 2. The hybrid was isolated on a MAK column and the base composition
determined as described in Methods.

is complementary to its complement. The prediction that the complement to the
mature strand generates genetic message would appear to be confirmed.

Discussion.—It will be recalled that analysis in cesium chloride gradients and
on MAK? columns have shown that the injected strand of ¢X174 is incorporated
into the RF duplex structure. The buoyant density and melting temperature®
of RF-DNA both agree that it is a double-stranded molecule containing mature
viral DNA as one component and its complement as the other. The fact that
@X174 message hybridizes to the RF-DNA and not to the mature single-stranded
DNA means that it is the complementary component of the RF-DNA which is in-
volved in the hybrid complex. The base composition of the hybridized RNA adds
further strong support to the validity of this conclusion. We obviously cannot
categorically state that the other strand does not produce any messages. However,
if it does, they represent less than 5 per cent of the population. There is also the
unlikely possibility that both are transcribed and that one of the transcriptions is
preferentially destroyed.

All of the results reported are consistent with the inference that one of the two
strands of the DNA duplex is predominantly, or solely, used to generate genetic mes-
sages. This conclusion does not deny the possibility” that effective transcription
of only one of the strands requires the presence of both strands.

It may perhaps be proposed that deductions derivable from the study of a single-
stranded DNA virus may not be generally applicable to organisms which normally
possess both complementary strands. However, it must be noted that message
production does not begin in this system until the double-stranded structure is con-
stituted. One is inclined therefore to believe that the situation being examined is
not so abnormal as to be completely unique. Nevertheless, general acceptance ob-
viously requires confirmation with other DNA systems.

The fact that only one strand is used possesses an interesting implication for the
problem of genetic inversions. Sequence inversion in a DNA molecule requires in
addition a 180° rotation of the inverted stretch in order to reconstitute the anti-
parallel 5’—3’-internucleotide linkage. This exchanges sequences between the
VoL. 50, 1963 BIOCHEMISTRY: HAYASHI ET AL. 671

strands. ‘Thus, inverted sequences will be lost to the transcription mechanism, so
that inversion necessarily results in a deletion. We predict therefore that tran-
seribable inversions resulting in nondeletion phenotypes will not be observed in
organisms which contain a continuous DNA duplex structure as the sole component
of their chromosomal apparatus. The corollary to this, for organisms which do
exhibit nondeletion inversions, is obvious.

We should like to conclude by noting briefly the discovery in uninfected host cells
of a strange genetic message of peculiar base composition, and complementary to a
restricted section of ¢X174 DNA. This RNA disappeared within 5 min after in-
fection of the host cell and thus did not complicate the experiments described here.
The significance of this unusual finding is at present under investigation.

Summary.—The experiments described were designed to determine whether in
vivo transcription of genetic information involves only one or both strands of the
DNA-duplex.

The experiments used DNA from the single-stranded virus ¢X174 and its purified
replicating form which contains the original strand and its complement. Ap-
propriate hybridization tests with these two DNA preparations and the RNA mes-
sage fraction were carried out. The results of the hybridization tests and of the base
composition of the RNA complex revealed the presence of RNA complementary to
only one of the two strands of the RF-duplex. The data are consistent with the con-
clusion that only one of the two complements in a DNA duplex is either the prin-
cipal or sole source of translatable genetic information. Among other conclusions,
these results imply that transcribable genetic inversions resulting in nondeletion
phenotypes are not to be expected within a continuous DNA duplex structure.

Note added in proof: The desirability of extending the conclusions reported here to DNA
which is normally double-stranded in the vegetative state has been met rather rapidly. C.
Greenspan and J. Marmur (personal communication) have separated the two strands of the SP-8
virus of B. megaterium and have shown that only one of them hybridized extensively with homolo-
gous message. G. P. Tocchini-Valentini, M. Stodolsky, A. Aurisicchio, F. Graziosi, M. Sarnat,
and E. P. Geiduschek (personal communication) have accomplished the same result with phage
alpha of B. subtilis and have obtained similar results.

* This investigation was aided by grants-in-aid from the U.S. Public Health Service and National
Science Foundation.

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