STRUCTURE OF THE HYBRID TRANSFORMING UNIT
ROGER M. HERRIOTT
Department
of
Biochemistry, School of Hygiene and Public Health, The Johns Hopkins
University, Baltimore, Maryland
Received July 30, 1965
PECIFIC pairing of complementary strands was proposed by MARMUR
and
LANE(1960) and DOTY,
MARMUR,
EIGNERand SCHILDKRAUT
(1960) as the
mechanism of renaturation of DNA. Later, sedimentation studies of renatured
mixtures of isotopically light and heavy strands ( SCHILDKRAUT,
MARMUR
and
DOTY1961; ROWND1963), reenforced their case. In a test of this mechanism,
HERRIOTT(1961a) annealed a mixture of two DNAs from related strains of
Haemophilus influenme carrying different but linkable markers. It was thought
that on the basis of the proposed mechanism, hybrid units such as pictured in A
of Figure 1, should form and transform recipient cells to both markers. An increased number of doubly marked transformants was observed which was a
linear function of the annealed DNA concentration. A number of other models
of this unit were considered but none seemed more probable than model A. However, the low recovery of doubly marked units, and some recent results, suggest
that model A may not represent the doubly marked unit formed on renaturation.
Segregation, which was expected from the pictured hybrid unit, was not detected
in a clonal analysis (YAMASHITA
1964). Evidence on the mechanism of transcription (CHAMPEand BENZER1962; BAUTZ1963; GUILDand ROBISON1963;
MARMUR
and GREENSPAN
1963; HAYASHI,
HAYASHI
and SPIEGELMAN
1963)
A
B
5
+
IIIIIIlIlllllllII
+
Nb
f
IIIIII
IIIIIIII
FIGURE
1.-Models of the doubly marked units (heterozygotes) formed by annealing mixtures of singly marked denatured DNAs.
Genetics 52: 1235-1246 December 1965.
1236
R.
M. HERRIOTT
showed with remarkable uniformity that only one strand of DNA is transcribed
in uim. Model A with different markers on the opposite strands would therefore
not be expected to have both its markers transcribed. Yet it is possible as has
been suggested from studies by SANDERSON
(1965) on the polarity of operons in
Escherichia coli and Salmonella typhimurium, that transcription shifts in some
instances to the opposite strand after completion of an operon.
The reports of ZALESKA
and PAKULA
(1964) and BRESLER,KRENEVA,
KUSHEV
and MOSEVITSKI~
(1964a) that doubly marked transforming units were formed
by renaturating mixtures of B. subtilis DNAs did not resolve the above problem.
Decisive experiments were needed to indicate if any of the models discussed
1961a) and pictured in Figure 1 accounted for the properties
earlier (HERRIOTT
of the hybrid units (heterozygotes). The present report contains the results of
several new experiments designed primarily to determine if the hybrid unit
corresponds to model A. The results indicate that it does not. In fact, they virtually eliminate it as well as model C. Model B will account for the results. A
preliminary report of this work was presented at the Sixth International Biochemistry Congress in New York City, July, 1964.
MATERIALS A N D METHODS
The materials and methods used in the present work involving denaturation, renaturation and
biological assay of the Haemophilus transforming DNA have been described (HERRIOTT1963).
For the preparation of competent receptor cells and for other procedures of transformation see
GOODCAL
and HERRIOTT
(1961). Any deviations from these procedures or any new features will
be noted in the protocols for the individual experiments.
In general, DNAs purified from lysates of cell cultures having particular genetic markers
were diluted in 0.2 M NaCI-0.02 M Na, Citrate to 2 to 50 pg/ml. One ml aliquots in capped tubes
were denatured by heating in a boiling water bath for 5 minutes after which they were chilled
rapidly in ice water to prevent renaturation. Renaturation took place at 66 f 1" except when it
was convenient to let it run overnight, in which case the temperature was reduced to 56 to 57°C.
RESULTS
The very clear experiments of MARMUR
and GREENSPAN
1963; and HAYASHI,
HAYASHI
and SPIEGELMAN
1963, showing that only one strand of DNA is transcribed to messenger RNA in vim, made it necessary to reexamine the properties
of the heterozygote; for at first glance, Model A would not be expected to express
both its markers since they are on opposite strands. However, if the opposite
strands duplicate first and these two double stranded units then recombine yielding both markers on both strands, the apparent conflict would be resolved, for
then reading of one strand would include both markers. These added steps would
be expected to consume more time so it was decided to test the hypothesis by
comparing the time for expression of the heterozygote with that of a homozygous
unit in which both markers are on both strands. Included also was a hybrid made
by renaturing denatured doubly marked DNA with an excess of wild-type DNA.
In this case both markers are on but one strand. The results are shown in Figure 2.
1237
THE H Y B R I D T R A N S F O R M I N G UNIT
30
60
41
30
41
60
45
30
60
TIMElmin) between DNA UPTAKE AND EXPOSURE
TO BOTH ANTIBIOTICS
I. 1
EXPRESSION
TIME
TWO
SYMBOL
OF
ISO-TRANSFORMING
LINKED
NATURE
UNITS
Im
WITH
MARKERS
OF
IS0
UNIT
HOMOZYGOUS
0
HYBRID WITH
WILD TYPE
n
HETEROZYGOUS
CONFIGURATION
m
"
FIGURE
2.-Expression time of iso-transforming units with two linked markers. The time before challenge with two antibiotics us. percent of the double transformations found in 120 minutes.
Deta'ls of the experiments in Figure 2: Homozygous S-Nb D N A was isolated from cells resistant to 250 #g/ml streptoand HERRIOTT
1961). It was 60%
mycin ( S ) and 2.5 #g/ml of novobiocin (Nb) by the method described earlier (GOODGAL
linked.
Heterozygous S-Nb DNA was prepared by annealing at 65CZ'C for 4 hours a mixture of denatured S and of Nb DNAs
dissolved in 0.4 M NaC1-0.02 M Na, citrate. The fraction of double transformants to S transformants (S-Nb/S) obtained
with this DNA was about 0.01.
Hybrid S-Nb DNA was obtained by heating a mixture of 2 pg/ml of denatured homozygous S-Nb DNA and 8 flg/ml
of unmarked DNA 4 hours at 65rt2OC in 0.4 M salt solut~ou.
I n A and B (duplicate experiments), 3 flasks carrying 15 ml of Difco brain-heart infusion (BH) and 0.01 pg/ml of one
of the three DNAs noted above were brought to 37°C. 0.5 ml of 3 X l P / m l competent Rd Haemophilus was added with
stirring. After a 5 min uptake period DNAase and Mg++ were added to bring the final concentration to 5 pg/ml and
0.003 M respectively. While maintainiAg the agitation of the suspension at 37OC, samples were removed at the noted times
and plated with BH-agar containing the antibiotics. Since all points after 120 minutes followed a logarithmic progression,
it was concluded that the introduced markers were all duplicating by this time and so all curves were normalized to the
values obtained at 120 minutes. No cells resistant to both antibiotics were detectable prior to 30 minutes.
In C the concentrations of the various DNAs were adjusted so the number of transformants resistant to both antibiotics
in each instance was nearly the same. This meant a great difference in DNA concentrations. Thus the concentration of
DNA in the transformation mixture was 0.0001, 0.01 and 0.03 m / m l of homozygous, heterozygous and hybrid DNAs,
respectively. Otherwise they were treated as in A and B.
It may be seen that the two markers of the heterozygote were not expresszd
later than those of the homozygous unit. Although the two resistances of the
heterozygote developed consistently a few minutes earlier than those in the homozygote, this is not given special significance except to point out that this makes
the conclusion clearer than if the deviation were in the opposite direction.
Inhibition of DNA synthesis to delay heterozygote expression: It could be
argued that the time necessary for expression of antibiotic markers is so long that
small differences in time such as might be needed for duplication and recom-
1238
R . M. HERRIOTT
bination might not be recognized. As a further test of the hypothesis which includes the necessity for strand duplication, it was decided to determine if inhibitors of DNA synthesis delay the expression of heterozygotes.
5-fluorodeoxyuridine (FUDR) has been used extensively (BROCKMANand
ANDERSON
1963) for blocking DNA synthesis. It was reported (COHENet al.
1958) to inhibit by acting on the thymidilate synthetase in E . coli. Preliminary
studies showed that in submicrogram concentrations (0.9'5 p g / m l ) , this drug
inhibited DNA growth of H. influenzae (1 x 107/ml) for 2 hours. At levels of
1pg/ml and above the cells died.
In Figure 3 are shown the results of experiments in which the effect of FUDR
on the expression of hetero- and homozygous DNAs is compared. The fraction
of cells expressing is plotted against the time of challenge after uptake of DNA.
Another control containing FUDR and thymidine was also included. Two samples
were plated from each system at each time indicated. One of these plates contained
the antibiotics and hence measured those cells that had expressed. Addition of
the antibiotics to the second plate was delayed until the total time before exposure to the antibiotics was sufficient (a total of 2 hours after starting the experiment) to allow full expression by all transformed cells. A comparison of the
counts on the two plates provides the figure for the fraction of transformed cells
expressing at the time indicated.
The results seen graphically in Figure 3 show that there was no significant
difference in the effect of FUDR on the expression time of the hetero- and homozygous units. It seems doubtful therefore if the two types of DNA units differ
in their dependence on DNA synthesis for expression. This result does not lend
support to model A.
FUDR inhibition of normal expression: The literature contains little, if any,
evidence that DNA synthesis is necessary before introduced markers can express.
It was unexpected, therefore, to find FUDR inhibiting expression, especially of
the homozygous unit in Figure 3. FUDR may, of course, inhibit enzymes other
than thymidilate synthetase. I n H . influenzae conversion of citrulline to uracil
(HERRIOTT,
unpublished) is reduced by FLJDR; but as seen in Figure 3, whatever the nature of the inhibition of expression, it was completely reversed by
10 pg/ml of thymidine. This is tentatively taken to mean, therefore, that DNA
synthesis was needed before one or both of the markers could be expressed. A
similar delay was found in the development of erythromycin resistance by FUDR.
A study of other markers is needed to determine how widespread this effect is,
and HOTCHKISS
1960) gene expression was
but in at least one instance (LACKS
reported to begin immediately after DNA penetration, and so presumably it
would not require DNA synthesis. SUZUKI,YAMAGAMI
and SHIMAZU(1965)
have just reported some evidence suggesting that half the transformants need
DNA synthesis before expressing resistance to streptomycin.
A kinetic test of model A: The failure of the several tests in the previous
sections to lend support to model A led to a consideration of a more critical test
1963) that denatured wild-type DNA
of the model. It had been noted (HERRIOTT
renatured with genetically marked DNA as efficiently as did homologous DNA,
1239
THE HYBRID T R A N S F O R M I N G UNIT
A
A
= t FUDR
c
v)
C
?
I-
C
0
C
.c
U
e
U
60
90
135
180
T i m e (min.)
FIGURE3.-Effect of FUDR inhibition of DNA synthesis on the time a fraction of the cells
become resistant to two antibiotics following transformation with hetero- or homozygous DNAs.
Details of experiments in Figure 3: C e l h Frozen competent H. influenure cells were thawed, centrifuged, and resuspended in the initial volume of M-I a chemically defined growth medium ( R " N n 1965).
Heterozygous S-Nb DNA: This DNA was prepared by annealing for 4 hours at 65°C 1 ml of a 0.2 M NaCl solution of
two denatured DNAs in equal concentration, one carrying streptomycin resistance, the other novobiocin resistance. The
total DNA concentration was 10 fig/ml. After annealing the DNA was diluted 2 X 10*-foldand treated as indicated below.
Homozygous S-Nb DNA: This was the same as that described in the details for Figure 2. This DNA after dilution to
pg/ml in saline was mixed with cells in the various systems as noted below.
Procedure: (a) 0.5 ml of the above competent cells in M-I was mixed with 14 ml of M-I medium in a 125 ml flask
13.25 ml M-I medium
0.75 ml of 10 pg/ml FUDR
0.5 ml
and 0.5 ml diluted DNA was added. ( b j 0.5 ml cells
13.10 ml M-I medium
0.15 ml of loo0 pg/ml thymidine
0.75 ml FUDR
0.5
dilute DNA. (cj 0.5 ml cells
ml dilute DNA.
The above flasks were rotated at 3 7 O C and after an hour samples were taken a t intervals and were mixed on petri lates
with plain BH-agar or with BH-agar containing 50 fig/ml streptomycin and 2.5 pg/ml of novobiocin. After a totaf of 2
hours from the initiation of the experiment the first s e t of plates (containing no antibiotics) were overlaid with BH-agar
containing 100 s d m l streptomycin and 5 h. -.m l novobiocin. Viable-cell counts were Derformed at intervals during the
exposureto FUDR.
'I'he colonies appearing on the plates which had a 2 hour period before antibiotics were added served as a measure of
the trtal number of potential transformants at bme of sampling. The colonies on the plates which contained antibiotics
at &e lime of samplinp measured the cells which had developed resistance to both antibiotics. The latter number divided
by the former leads tv the fraction of transformants which have expressed at the time of sampling.
+
++
+
+
++
-
judging from the initial rate of recovery of either of two markers. If the heterozygote is represented by model A, then the rate of its formation would be expected
to be inhibited by the addition of denatured wild-type DNA during renaturation,
for the wild-type, but otherwise homologous, strands should compete for the
marked strands. Therefore, as a test of the model, the rate of formation of heterozygotes was examined in the presence of four times as much denatured wild-type
DNA, and compared to the rate when native or no wild-type DNA was present
to serve as a control. Since the strands of the native wild type do not separate at
the temperature of renaturation, the native DNA should have no influence on
the rate of heterozygote formation.
The results of this experiment, graphed in Figure 4, show that not only does
the denatured wild-type DNA fail to inhibit heterozygote formation, but in some
instances-ne
shown here-there was a slight increase. In other experiments
the increase was less, but in no case was there significant inhibition. In the
absence of an alternative explanation, this result virtually eliminates model A
of Figure 1 from further consideration.
Model C is also an unlikely candidate for the same reasons. The strands of
1240
R. M. H E R R I O T T
60
30
Time
at
90
56'C
120
(min.)
FIGURE4.-Initial
rate of formation of S-Nb heterozygotes in the presence and absence of
excess denatured wild-type DNA.
Details of experiments in Figure 4: Denaturation: 1 ml quantities of 10 pg/ml concentration of each of S, Nb and
wild-type DNAs dissolved in 0.2 M NaC1-0.02 M Na, citrate were heated in separate tubes in a water bath at 100°C for
5 min. They were then chilled quickly with agitation in an ice-water bath.
Renaturation. 1 ml of 0.2 M NaCl-0.2 M citrate containing 1 pg/ml of denatured S DNA 1 p /ml of denatured NbDNA and 8 pg/ml of denatured wild-type H. influenzae DNA vas placed in a water bath a; 5 6 - 8 . At intervals samples
were diluted 100-fold in saline-BH then 0.2/6 in BH growth medium containing 1 X lOs/ml competent H. influenzae.
After agitation for 30 minutes, 1 &l of this mixture was plated b mixing with 10 ml BH agar containing the usual
hemin and NAD. After incubating at 37°C for 90 minutes more x e plates were coated with 10 ml of double strength
antibiotic-BH-agar and incubated 24 to 48 hr at 37OC; the coloniek were then counted. A s a control, a tube simjlar to the
one above was prepared but 8 pg/ml native wild-type DNA replaced the denatured m l d type dming annealing.
denatured wild-type DNA would be expected to compete for the partially opened
units and inhibit formation of the proposed four-stranded unit.
Our failure to obtain biological evidence for hybrids of the type of model A
should not be construed as suggesting that such physical units are not present in
annealed mixtures. In similarly renatured systems of other DNAs ( SCHILDKRAUT
et al. 1961; ROWND1963) combined pycnographic and radioisotopic evidence of
their presence in expected proportions has been convincing. Such units are probably present in the renatured mixture of Haemophilus DNAs; but if, as noted
earlier, only one strand is used at some essential genetic step (SUZUKIet al. 1962;
HERRIOTT
1963; ROWND1963; BRESLERet al. 1964c; SUZUKIet al. 1965), then
for any given cell either, but not both, markers can be expressed.
Model B: With the elimination of models A and C the remaining model in
Figure 1 should be scrutinized in the light of all the evidence about the heterozygote. Model B, with both markers on one side, paired to a common strand,
provides an explanation for (a) the low recovery of heterozygotes, (b) the failure
to segregate, (c) the failure to distinguish between expression times of homo- and
heterozygotes, and (d) the effect of added denatured wild-type DNA on the
kinetics of heterozygote formation. To explain this last point, (d) , it is presumed
that in model B, added wild-type strands during renaturation would increase
the rate of formation of the proposed units by providing more common strands
THE HYBRID T R A N S F O R M I N G UNIT
1241
with which the two marked strands could pair; but by the same reasoning, unmarked strands should pair with the singly marked strands and therefore competitively inhibit the rate of formation of the doubly marked unit. This means
that the net effect would lie somewhere between the two extremes of enhancement and inhibition. Qualitatively, the results in Figure 4 support model B, for
the presence of four times as much denatured wild-type DNA had only a slight
effect on the rate of heterozygote formation.
Model B suggests that the order of the reaction might be greater than second
order. The writer (1963) has not observed values greater than 2 but LANYI
(1963) observed a figure of 2.55.
Model B and shearing forces: As a test of model B, it seemed reasonable to
expect it to show a weakness toward mechanical shearing forces since only one
of the strands has continuous covalent bonds. However, as seen in Figure 5, relative to homozygous DNA, the heterozygote was not weaker. No explanation can
be offered except that the prediction of heterozygote weakness was based on the
assumption that the loose unpaired ends of the two marked strands would not
lend stability against shearing forces to the unit.
Model B and a triply marked unit: If model B correctly represents the structure of the heterozygote, it would be expected that additional “linked” markers
might be introduced into the unit. It certainly would not fit model A. GOODGAL
pouog..
Thmugh #27
Ihpodormlo Woodlo
FIGURE
5.-Effect of shearing forces on the survival of hetero- and homozygous transforming
DNA.
Details of erperiments in Figure 5: Heterozygous S-Nb DNA: 1, ml of 5 ag/ml S DNA,+ 5 cLg/ml Nb DNA in 0.4 M
NaC1-0.02 M Na, citrate was heated at 100°C for 5 mm, then chllled quickly. This solutlon was then annealed at 57°C
overnight and m l e d to room temperature.
Homozygous DNA. Same as described in details for Figure 2.
Shearing: Both DNAs were diluted to 1 fia/ml in 0.15 M saline and 2 ml of each was reueatedly forced from a 2 ml
Luer-lock syringe through a 1 inch #27 gaGge needle at full manual pressure. After each-passage small aliquots were
removed to assay for residual transformations t o both markers.
R. M. HERRIOTT
TABLE 1
Formation of triply marked D N A b y renaturation of a mixture of
three different singly marked D N A s
Experiment 1
Marker
Dilution
Total
transf.
Titer/ml
transf. mix
Mixture of native DNAs
S
5 x 103 64,62
K
2 x 104 m,30
Nb
2 x 104 104,122
S-K-Nbf undiluted 50
3.1 x 105
6 x IO5
2.2 x 106
1.7
Renatured mixture of denatured
IO3 130,128
K
5 x 103 78,86
Nb
5 x 103 338,295
S-K-Nb-f undiluted 122
DNAs
1.2 x 105
4.1 x 105
1.6 x 106
4.3
s
No DNA
S-K-Nbt
...
.
1
0.03
Experiment 2
Calculated'
Dilution
Total
transf.
Titer/ml
transf. mix
4.5
2 x 103 iffi,18i
5 x 103 132,129
1 x IO4 296,307
undiluted 34
3.5 x 105
6.5 x IO5
3.0 x IO6
1.2
0.9
1 x 103 111,116 1.1 x 105
2 x 103 152,138 2.9 x IO5
5 x 103 384,345 1.8 x 106
undiluted 85
3.0
Calculated*
7
.
0.6
0.00
~~~~~~~~
* The calculated value is obtained by dividing the product of the titers of the individual markers by 9 X 10'". the square
and
of the cell concentration. This follows from the frequency relationship ( S / C ) X ( K / C ) X ( N b / C ) = (S-K-Nb)/C
assumes a random uptake of marker DNAs by eclls.
Fourteen plates of 2 ml each.
Details of experimenis shown in Table I: A tube containing 1 ml of 9 pg/ml of DNA from cells resistant to 250 pg/ml
of streptomycin and therefore designated S DNA was heated 5 minutes at 100°C then chilled in ice water. The solvent
for the DNA was 0.2 M NaC1-0.02 M Na, citrate throughout. A tube containing 6 pg/ml of DNA from cells resistant to
8 pg of kanamycin was similarly treated and finally a tube containing Nb DNA was similarly denatured.
0.5 ml of each of the denatured DNA solutions was mixed and the mixture was annealed at 63°C for 4 hours after
which the material was analyzed at the dilutions noted for individual (single) markers and triple resistances. A minimum of 48 hours was allowed for the S-K-Nb resistant colonies to form before colony counts were made.
+
(unpublished results) has observed that a kanamycin resistance gene ( K ) is
linked between the high streptomycin-resistance marker (SZ5O)and the novobiocin marker ( N P 5 ) .A mixture of the three separately prepared singly marked
DNAs was denatured and annealed in the usual manner and products analyzed
as shown in Table 1. Such annealed DNA produced nearly five times as many
triply marked transformants as calculated from random uptake of the individual
markers. In the control with a mixture of the three native DNAs, the observed
number of triply marked transformants was less than half of the expected number, so the difference between the control and experimental was at least tenfold.
Analyses of all the single and double markers after renaturation showed that the
observed triply marked transformants could have arisen from a double transformation by S-Nb and K DNAs. To decide if this possibility was a major source
of the triply marked transformants, the effect of the renatured DNA concentration
in the transformation mixture on the number of triply marked transformants
was studied. If all three markers are on a single unit of the renatured DNA, the
number of transformants should decrease directly with the DNA concentration,
yielding a slope of one. If double transformants are responsible for the results,
the effect of dilution should have a slope of two. The observed values of two
experiments plotted in Figure 6 fall on lines with a slope of one. It is concluded,
T H E HYBRID TRANSFORMING U N I T
1243
1
.06
'006
' '
:0001
DNA Cancentmtlon In tranrfomatlon mlxtun, pg/n
FIGURE
6.-Effect of dilution of annealed DNA on the number of triply marked transformants, a means of distinguishing among possible models.
Details of experiments in Figure 6: 1 nil samples of each of the 9 pg/ml of S, K and Nb DNAs were treated as
described in the details of Table 1 through the denaturation and annealing. 0.2 ml samples of this annealed DNA or
dilutions of it were added to 30 ml BH in a 125 ml flask containing 3XlOs/ml competent H . influenme. These mixtures
were rotated at 37°C for 30 minutes after which 2 ml was placed in each of 14 petri dishes and mixed with 10 ml of
plain BH-agar. After 2 hours these plates were overlaid with BH agar containing 100 pg/ml streptomycin, 15 pg/ml of
kanamycin and 5 pg/ml of novobiocin. The colonies were not counted until the plates had incubated at least 48 hours.
therefore, that triply marked units of DNA have been produced by renaturation.
They can be explained on the basis of model B.
DISCUSSION
BRESLERet al. (l964a) considered the transcription problem of a hybrid unit
made by annealing two B. subtilis DNAs. When they found no evidence of DNA
duplication during uptake and transformation, they concluded that messages
from both strands were transcribed. They did not consider the possibility that
model B would represent the heterozygote.
LANYI(1963) also reviewed the possible models in considering the nature of
the heterozygote. When an annealed mixture of sheared denatured wild-type
DNA and unsheared denatured doubly marked DNA failed to yield active doubly
marked units, LANYIconcluded that markers with a backbone discontinuity
would not transform to both markers; i.e., model B was unlikely.
1961b) on the formation of heterozygotes
An earlier experiment (HERRIOTT
from pairs of three different linkable markers led to the conclusion that the
farther apart the markers, the greater the proportion of hybrid units formed. At
the time it seemed that this result was best explained by model A and that
following strand-duplication the two double-stranded units could undergo recombination. Recombination frequency (Iinkage) of these markers in normal transforming DNA is known to increase with the distance between markers. Applied
1244
R.
M. HERRIOTT
to model B, the above facts suggest that the greater the map distance between
markers, the greater the chance that these markers will pair with a common
strand and both markers will become incorporated.
Several general features of the mechanism in genetic processes of bacterial
( 1962),
transformation are beginning to be clarified. Direct observations of LACKS
BODMER
and GANESAN
(1964), Fox and ALLEN (1964), and Buc (1965) that
only one strand of DNA is utilized by the cell are supported by finding that
hybrid units made by annealing of wild and marked strands are active in transformation (MARMUR
and LANE1960; SUZUKIet al. 1962; HERRIOTT
1963;
ROWND1963).
GUILDand ROBISON'S
(1963) evidence for separation of the individual strands
of novobiocin resistance marker by density centrifugation of the denatured DNA
suggests that both strands are functional in transformation, as expected from the
Watson-Crick model. That is, the lighter strand which induced antibiotic resistance 'was transcribed about 20 minutes sooner than the heavier one. It is presumed
that the heavier strand must generate a new complementary strand which is then
transcribed to RNA.
Renaturation of marked DNA in the presence of excess denatured wild-type
DNA produces twice the number of bifilar units carrying a marked strand, yet
the number of transformants is not higher than that obtained in the absence of
wild-type DNA (MARMUR
and LANE1960; HERRIOTT
1963). There must be a
compensatory reduction during incorporation. It was suggested (HERRIOTT
1963)
that perhaps either strand is utilized with equal probability, but not both strands.
Homozygous units would be expected to yield transformants almost twice as
et al. (1964b), and SUZUKI
frequently as the hybrid. ROWND(1963), BRESLER,
et al. (1965) have reached a similar conclusion.
The recent reports of BRESLER
et aZ. ( 1 9 6 4 ~ )and REBEYROTTE
(1965) on the
rescue of inactivated transforming markers by annealing them with denatured
wild-type DNA show that an intact strand, even when unmarked, is more effective than an inactivated marked strand in promoting incorporation of the marker
into a host genome.
The writer is glad to acknowledge the competent technical assistance of STELLA
N. WEATHERand ELEANOR
Y. MEYERin the performance of the experimental work. This investigation was
supported by Public Health Service research grant A1 01218, Public Health Service training
grant 5 T I GM 73, and Atomic Energy Commission Contract AT(30-1)-1371.
ALL
SUMMARY
Several properties of the hybrid transforming unit (heterozygote) had indicated that the generally assumed model with different markers on opposite
strands (model A) might not be correct. If transcription in general is restricted to
but one strand of the DNA, then duplication of the heterozygote strands, followed
by recombination which would put both markers on the same strand, would have
to precede transcription. On such a hypothesis expression of both markers of a
heterozygote would be expected to take longer than for the corresponding homozygous unit with both markers on both strands. When it was found that hetero-
T H E HYBRID TRANSFORMING U N I T
1245
and homozygous units needed the same time for expression, with or without
DNA synthesis being inhibited by FUDR, serious doubts developed for the
validity of the model. When the rate of formation of heterozygotes was not
sharply reduced by the addition of denatured wild-type DNA during annealing,
model A was abandoned. Model B, in which the two marked strands are partly
paired to a common strand, satisfied most of the known properties of the heterozygote. Support f o r this model was obtained from the formation of triply marked
units upon annealing a mixture of three singly marked DNAs.
L I T E R A T U R E CITED
BAUTZ,E. K. F., 1963 Physical properties of messenger RNA of bacteriophage T4. Proc. Natl.
Acad. Sci. U.S. 49: 68-74.
W. F., and A. T. GANESAN,
1964 Biochemical and genetic studies of integration and reBODMER,
combination in Bacillus subtilis transformation, Genetics 50 : 717-738.
BRESLER,
S. E., R. A. KRENEVA,
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