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MICROCOPY RESOLUTION TEST CHART
NATIONAL BUREAU OF STANDARDS -1963
APR 7.469
.
-LEGAL NOTICE –
This report me proparod u an account of Government sponsored work. Noither tho Valtad
States, vor the Commissloo, aor any persoa sculos on behalf of the Commiuloa:
A. Yakas acy warranty or reprornudoa, expressed or impued, with respect to the accu-
racy, complotanou, or crafulness of tho Ilormation cor bed to this report, or that the wo
of way information, apparatu, motbod, or procos. daclosed la this report may not loirinco
I printely owned righto; or
B. Asnimos way liablues with respect to the wo of, or for damage resulting from the
lan of any information, apparatus, mothod, or procou disclosad in this report.
As und in the abcyo, “porsco acting on bedell of the Commission" Lacludes any om-
ploses or coatractor of the Commission, or amployee of such contractor, to do oxtent that
oocha omployee or coatructor of the Commission, or employce of much contractor properer,
desam pater, or provides accon to, noy tnformation purmant to his gaployment or contract
tid the Commission, or be omployment with such coatractor,
.
radores de
Stü
a
greement
:
under contract with the Union Carbide Corporation.
Research sponsored by the V. S. Atomic Energy Commission
Oak Ridge, Tennessee
Oak Ridge National Laboratory
Biology Division
J. E. Donnellan, Jr. and li. 3. Setlow
SPORES: NO THYMIVE DIMERS
PHOTOPRODUCTS IN IRRADIATED BACTERIAL
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CFSTI PRICES
IN NUCLEAR SCIENCE ABSTRACTS
RELEASED FOR ANNOUNCEMENTS
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Science


B. megeterium spores labeled with tritiated thymidine
.
were irradiated with monochromatic ultraviolet light and the
spore DNA analyzed for thymine containing photoproducts. No
thymine dimers were observed, but large amounts of new thymine
photoproducts were found. The unknown photoproduct was produced
in vitro by irradiation of DNA dried in the presence of various
Ultraviolet irraciation of vegetative cells of various
organisms and of DNA in solution forms dimers between adjacent
thymine residues in DNA strands. The formation of dimers
accuunts for a large fraction of the loss in the ability of
UV-irradiated DNA to prime DNA synthesis, and for the loss in
transforming activity of irradiated hemophilus influenzae DNA.3
Dimers have also been implicated as a major factor in the lethal
effects of Uv on cells.4
The resistance of bacterial spores to the deleterious
effects of such agents as uv, ionizing radiation, and heat has
often been attributed to a state of dryness within the spore.
Various mechanisms for achieving this state have been reviewed
and proposed by Lewis et al.5 If the DNA in a spore is "dry",
the resistance of spores to UV may arise from the fact that 1/10
as many dimers are produced by irradiation of dry DNA as are
made by irradiating wet dna.6
Spores of B. megaterium labeled with tritiated thymidine
were obtained by the following procedure. A liver extract medium'
(1% Wilson and co. "Liver Fraction B" in 10 mM potassium phosphate,
pH 6.5) containing methyl-43-thymidine was inoculated with a dilute.
suspension of heat shocked spores (60 C, 15 min). After shaking
for 36 hours at 30 C, the culture was harvested by centrifugation
and washed two times in 50 mm potassium phosphate, pH 4.5, followed
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14
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by at least three washes with water. The final pellet,
consisting of greater than 95% free spores, was suspended :
in distilled water at a concentration of 2 x 107 per ml and
irradiated with 2650 Å obtained from a large quartz prism
monochromator. The suspension contained 20,000-30,000 43
counts/min/ml. The average intensity was 2.7 x 103 ergs/mm2/min.
Following irradiation, the spores were collected by centrifugation.
They were broken in a "Wig-i-Bug" (Crescent Dental Manufacturing
Co., Chicago, Ill.) denta), ammalgamator using glass beads and
a glass ballº, and the DNA was extracted by a pheno) method.9
The extracted DNA was hydrolyzed in 97% Formic Acid at 175 C
ana radioactive products separated by descending chromatography
on Whatman #1 developed with n-butanol-acetic acid-1120
(30:4.5:11.2, v/v) Radioactivity was determined by cutting the
chromatograms into appropriate strips, eluting with water, and
counting in a liquid scintillation counter using a dioxane-
CO
napthalene-PPO-POPOP scintillator. Chromatograms of hydrolysates
of DNA from unirradiated spores contain less than 0.1% of the
total radioactivity, in regions other than thymine.
Figure 1 shows a chromatogram of the hydrolized DNA of
spores irradiated with 5 x 104 ex.es/mm2 together with a chromatogram
of the dimer region of hydrolized B. megaterium vegetative cells
irradiated with 2 x 103 ergs/mm2. Clearly the amount of thymine
diiner is negligible in the spores compared to vegetative cells. .
In small amount of radioactivity in the thymine dimer
rogion may be accounted for by the few percent vegetative cells
present in the spore preparation.
llowever, new and as yet unidentified photoproducts (a,b,c)
üre present in the hydrolized DNA of irradiated spores. At
5 X 104 ergs/mm2 photoproducts b + c involve some 30% of the
spore's thymine. Our measurements show that, at doses yielding
100% survival of colony forming ability (1000–2000 ergs/mm2)
the unknown photoproducts (b + c) are present to a total extent of
1-20 of the spore's thymine or some 50,000 thymine residues per
spore. We have succeeded in producing large amounts (12% of the
total thymine) of the photoproducts b + c in vitro by irradiating
E. coli DNA dried in the presence of various salts (Naci, Calcium
salt of 2,6-(dicarboxy)-pyridine). The photoproduct is not found
in . coli DNA dried from 0.02 M Tris-chloride buffer in the
absence of any other salts. Riklis reported a small amount of a
photoproduct with a similar Rp in irradiated-lyophilized DNA.6
The small amount of photoproduct (a) chromatographing in the
region of the um, dimer 10 has not been identified.
The absence of thymine dimers in the DNA of irradiated spores
is sufficient to explain their resistance to ultraviolet irradiation.
However, the appearance of large amounts of an unidentified photo-
product implies either that such a product does not interfere
with DNA synthesis or that the cells have a very efficient repair
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iñechanism for dealing with the photoproduct. In either case,
our data shows that during sporalation the physical state of .
the DNA within the cell is changed from that found in vegetative
cells and in solution.
References and Notes
1. A. Wacker. in Prog. Nucleic Acid Res., J. N. Davidson and W. E. Cohn,
D2. (Academic Press, New York) 1, 369 (1963); K. C. Smith, in
Photophysiology, A. C. Giese, Ed. (Academic Press, New York) 2, 329
(1964); J. K. Setlow and D. E. Duggan, Biochim. Biophys. Acta 87,
664 (1964)
J. E. Trosko, E. H. Y. Chu and W...Carrier, Radition Research in Press
(1965)
>
2. F. J. Bollum and R. B. Setlow, Biochim. Biophys. Acta 68, 599 (1963).
3. R. B. Setlow and J. K. Setlow, Proc. Natl. Acad. Scij. U.S. 48, 1250
(1962).
4. R. 3. Setlow, J. Cellular Comp. Physiol. 64 Suppl. 1, 51 (1964).
5. J. C. Levis, N. S. Snell, and H. K. Burr, Science 132, 544 (1960).
E. Riklis and E. Simson, Abst. Biophysical Soc. (1964); E. Riklis,
submitted to Can. J. Biochem. (1964).
7. H. S. Levinson and M. G. Sevag, J. Gen. Physiol. 36, 617 (1953),
8. L. I. Sacks, P. B. Percell, R. S. Thomas, and G. F. Bailey, J. Bacteriol.
87, 952 (1564).
9. J. E. Donnellan, Jr., E. H. Nags, and H. S. Levinson, in Spores III,
I. L. Campbell, Jr. and H. O. Halvorson, Ed. (Am. Soc. Microbiol.)
in press
10. R. B. Setlow, W. L. Carrier, and F. J. Bollum, Abst. Biophysical Soc.
(1965).
FIGURE LEGEND
FIGURE 1
Chromatograms of hydrolized DNA from B. megaterium
spores irradiated with 5 x 104 ergs/mm2 (solid
lines) and hydrolized B. megaterium vegetative
cells irradiated with 2 x 103 ergs/mm² (broken
lines). Wavelength of irradiating light was
2650 Å.
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DATE FILMED
5 / 20 / 66









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