The Wavelength Dependence of Ultraviolet Enhanced ... · of CV-l cells irradiated at 265 nm to...
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Western Kentucky UniversityTopSCHOLAR®
Masters Theses & Specialist Projects Graduate School
4-1978
The Wavelength Dependence of UltravioletEnhanced Reactivation in A Mammalian Cell-VirusSystemLeslie C. James
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Recommended CitationJames, Leslie C., "The Wavelength Dependence of Ultraviolet Enhanced Reactivation in A Mammalian Cell-Virus System" (1978).Masters Theses & Specialist Projects. Paper 1705.http://digitalcommons.wku.edu/theses/1705
THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET ENHANCED
REACTIVATION IN A MAMMALIAN CELL-VIRUS SYSTEM
A Thesis
Presented to
the Faculty of the Department of Biology
Western Kentucky University
Bowling Green, Kentucky
In Partial Fulfillment
of the Requfrements f or the Degree
Master of Science
by
Leslie C. James
April, 1978
AUTHORIZATION FOR USE OF THESIS
ermission is hereby
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O reserved to the author for the making of any copies of this the sis except for brief sections for re search or scholarly purposes .
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-"~J l . ..
Dean Of the
THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET ENHANCED REACTIVATION IN A MAMMALIAN CELL-VIRUS SYSTEM
Graduat . COllege
Recommended --_-f:0::!-0C7'/_7~ / 7,...::d,--/ __ (Date)
~~ 7?~tf2 - Director of Thesis _
ACKNm~LEDGMENTS
I would like to t hank Dr. Fernando Morgado and Dr.
Larry Elliott for serving on my committee and for their
gu idance during my graduate program.
I would a lso like to t hank the excellent student
worker staff for their competence and cheerfulness in
performing the routine tasks which were necessary for the
completion of this project .
Special thanks go to my fellow workers in the Bio
physics - Virology lab . Mr. Bobby Cobb, Mr. Richard Detsch
and Mr . Ed Ryan for always being available for irradiations,
and Mr . Daniel Knaue r and Mr. Dennis Fry for assisting in
experiments all deserve special consideration .
Very special thanks go to Ms. Sharon Moore for her
daily assistance in this research pro j ect , and also r
the typing of this manuscript .
Lastly, I would like to thank Dr . Thomas P. Coohill
for t eaching me how to do research , and for encouraging
and supporting me during al l of my research and academic
endeavors.
iii
ACKNOWLEDGMENTS
LIST OF TABLES .
LIST OF FIGURES
TABLE OF CONTENTS
INTRODUCTION AND LITERATURE REVIEW
MATE RIALS AND METHODS
RESULTS
DISCUSSION
BIBLIOGRAPHY
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page
iii
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vi
1
5
10
21
2 4
LIST OF TABLES
Table 1 . Fluence rate ranges for monochromatic expOsures to cells and the slope of the averages RER responses .. . ..
v
page
9
Figur e 1 .
Figur e 2.
Figure 3 .
Figu re 4.
Figure 5.
Figure 6 .
LIST OF FIGURES
The effect of 265 nm radiation on the capacity of CV-l cells to SUpport the growth of either unirradiated ((), ~ ) or irradiated (500 J m- 2 ) ( .. , A ) HSV- l . . . . . . . . . . . . .
The effect of 235 nm radiation on cell capaci ty for unirrad~ated ( () or irr ad i ated (500 J m- ) ( .. ) virus . .
The effect of 302 nm radiation On cell capaci ty for unirradiated ( () or irradiated (500 J m- 2 ) virus .. .. .
The reactivation curves for the ability of CV- l cells irradiated at 265 nm to reactivate irradiated (500 J m-2) HSV-l
The virus reactivation curves for cells irradiated at 235 nm . . . . . . .
The virus reactivation curves for cells irradiated at 302 nm . . . . . . . . .
Figure 7 - 11. The average reactivation factors for RER of HSV- l by CV-l cells at different wavelengths . . . .
Figure 12. An action Spectrum for RER plotted as the slope (quantum corrected) of the lines from Figures 7 t h rough 11. An absorption spectrum for DNA is included (Jagger , 1967) ...... . . . ... .
vi
page
11
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20
THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET ENHANCED
REACTIVATION IN A ~~LIAN CELL-VIRUS SYSTEM
Leslie C. James April , 1978 2 7 pages
Di r ected by: Dr . Thomas P . Coohill, Dr . Larry Elliott , Dr . Fernando Morgado
Department of Biology Western Kentucky University
The effect of ultraviolet (UV) radiation in t he wavelength
region 230 nm to 30 2 nm on the ability of an irradiated
mammalian cell to reactivate UV-irradiated mammalian virus
was tested . An action spectrum for radiation enh anced reacti-
po i nts to a combined nucleic acid-protein target for UV
vation (RER) is presented . The shape of the action spectrum
biological events involved in RER does not a llow the identi-
of the results of others involving the biochemical and photo-
radiation effects on this cellular parameter. An analysis
to this effect . Studies involving an analogous phenomenon
fication of the macromolecule which is the major contL b uto r
involve similar mechanisms .
in bacteria (Weigle reactivation) imply that RER and NR may
INTRODUCTION AND LITERATURE REVIEW
Today our environment contains many artificial light
sources. Increasingly, interest has focused on t h e effects
of these sources on biological cells and mol~cules . The wave
length s which impart the most damage to cells are those in
the ultraviolet (UV) region. Therefore, UV-light is an
important environmental agent , and can be used as an analytical
tool to modify selective biological events. Several authors
have reported on the effects of ultraviolet light on several
mammalian cell parameters, for example : DNA replication
(I-Ihitfield, et al., 1961; Powell, 1962; Cleaver, 1965;
Rasmussen and Painter, 1966; Cleaver, 1967), mutation (Rao
and Puck , 1970; Trosko and Chu, 1971), chromosomal damage
(Chu, 1965) and cell death (Todd, et a l , 1968) . If the
effect under investigation is studied at a sufficient number
of wavelengths, an action spectrum of e f fect as a function of
wavelength can be plotted . A comparison of this spectrum a nd
the absorption spectra of important biological molecules can
help identify the target molecu le (or molecules) responsible
for the studied response (Jagger, 1967).
An action spectrum has recently been published for the
effect of UV-light on mammalian host cell capacity (Coohill,
et al ., 1977). Cellular capacity is defined by Benzer and
Jacob (1953) as the ability of a cell to support the growth
of a particular virus. This cellular function is sensitive
to UV-radiation, irradiation of the cell Usually decreasing
its capacity to suppo rt viral growth (Anderson , 1948; Powell,
1 959; Bockstahler and Lytle, 1970; COppey, 1971). If the
virus was also UV- damaged and then allowed to infect UV-
irradiated cells, the expected result would be a furthe r
decrease in capacity , but this is not always the case.
I~hile investigating other phenomena in the Escherichia
coli-phage l a mbda system, Weigle (1953) made a surprising -observation: the survival of UV- irradiated phage lambda
increased if the host cells (or phage - bacterium complexes)
were also irradiated . If the bacteria were irradiated before
phage adsorption , the viral reactivating ability of the cells
was retained for several days . Also , there was a higher
proportion of mutants among reactiva ted phages than among non -
reactivated phages . Among these mutants , no reversions to
Dulbecco, 195 3) . the parent type were observed (Weigle, 1953; I~eigle and
Since the discovery of Weigle reacti vation I~R has been
demonstrated in several phage - bacterial systems (Rupert and
Harm , 1966). an analogous phenomenon has been more reCently
reported in mammalian cells (Bock stahler and Lytle, 1970;
Hellman , ~ ~., 1974; Lytle, ~ al. , 1974; Lytle and Benane ,
1975; BOCkstahler, ~ al ., 1976; Hellman , ~ al . , 1976; Lytle,
~ al . , 1976; Lytle, 1977) involving the reactivation of UV-
irradiated mammalian viruses grown in irradiated mammalian
cells . The term "radiation enhanced reactivation " (RER) has
2
been recommended to describe this phenomenon in mammalian
cells since the molecular eVents that produce this reaction
may not be identical to those that produce Weigle reactivation in bacteria (Lytle, 1977).
3
No single theory has been accepted to eXplain the mechanisms
of WR and HER, but many researChers are in agreement that a Uv
inducible repair process is responSible for both phenomena
(Devoret, 1973; Sedgewick, 1975; Lytle, 1977). This inducible
repair process was termed SOS repa ir by Radman (1974), the
term "SOS" implYing that DNA damage initiates its expression.
This mechanism is conSidered to be error-prone because its
inducement generates a large number of mutants. The induci_
bility of SOS repair indicates that protein synthesis is
required for its presence. Lytle (1972, 1977) has Shown that
HER is absent in mammalian cells treated With . ' otein syntheSis
inhibitors, implYin g the role of a protein in e nhanced reacti_
Vation. In add~tion , a delay of several days between cell
irt"adiatjon and vira l inoculation USually increases the
amount of HER observed in many mrunrnalian cells (BoCkstahler,
~ ~., 1976). These data indicated that the mec hanism of
enhanced reactivation is ind ucible and long-lived.
WR (dnu possibly also HER) has been interpreted by Lytle,
~ al. (1976) to be the result of the cell's response to DNA
d a mage which remains after eXcision repair, or to that damag",
Which remains in the absence of eXcision repair. Excision
repair is the mechanism which is responsible for the remova l
of Pyrimidine dimers . Pyrimidine dimers are the major cause
of damage to cells due to UV-light. RER and ~IR are both
independent of excision repair. The UVR genes controllinq
excision repair in E. coli are not required for expression of
WR (Witkin, 1974). A similar finding has been made in the
exci sion repair deficient cells of xeroderma pigmentosum
patients in that the absence of excision repair does not in
hibit the cells' expression of RER (Lytle, et al., 1976).
4
Finally , RER is present only for nuclear replicating
viruses . This suggests that the c e llular mechanisms responsible
for reactivation are located in the cell nucleus (Bockstahler
and Lytle , 1977).
The implications of the phenomena RER and IvR to the
study of mutation, virus activation , and carcinogenesis have
been recently stated by Devoret, et al . (1976) and Witkin
(1976). In fact, they have suggested that all three phenomena
are controlled by the same, or a closely related, mechanism
which is induced by UV-light .
In an attempt to elucidate the mech anism involved in
radiation enhanced reactivation in mammalian cells , experiments
were conducted involving the UV-wavelength (230 nm to 302 nm)
dependence of the ability of irradiated monkey kidney cells
to enhance the survival of UV-irradiated herpes simplex virus.
MATERIALS AND METHODS
Cell Cultures
A permanent line of African green monkey kidney cells
(CV- l) was obtained from Dr . L . E . Bockstahler of the Bureau
of Radiological Health, Rockville, Maryland 20852. These
cells were maintained in a growth medium consisting of lX
Minimum Essential Medium (Eagle's) with the following
components added per liter: 10 % fetal calf serum (Reheis
Chemical Co., Kankakee, Illinois 60901) 0 . 25 g L-glutamine,
16 ml of SOX amino acids, 8 ml of 100 X vitamins, 80,000 units
penicillin, 0 . 08 g streptomycin (International Scientific
Industries , Cary , Illinois 60013), 100,000 units mycostatin
(E . R. Squibb, Princeton , New Jersey 08450) and buffered to
pH 7.5 with 0.3 g sodium bicarbonate. Cel ls were incubated
in closed flasks or in petri dishu i n clo sed ( a pproximately
5 % CO2 ) chambers at 37oC.
Virus Assay: Plaque Forming Ability (pfa)
A macroplaque strain of HSV-l ( Herpes hominus) was
obtained from Dr. C. D. Lytle of he Bureau of Radiological
Health , Rockville , Marylan d 20852. The virus was inoculated
into confluent monolayers of CV-l cells, harvested , and kept
in vials at -40o C until use . For virus assay freshly con
flue~t monolayers of cells were inoculated with an appropriate
viral titre (approximately 100 plaque forming units/plate)
6
in a maintenance medium consisting of IX MEM supplemented with
0.26 gil L-glutamine and 2% fe tal calf serum and kept at 370
c
for 90 min. The inoculum was then removed and 4 ml o f growth
medium Containing 0.25 % immune serum globulin (Armour Pharm
aceutica l, Kankakee , Illinois 60901) was added to prevent non
cellular viral transfer . After inoculation with virus, cell
monolayers were incubated for two days in the case of unirrad
iated virus and three days in the c a se of irradiated virus to
allow for visible plaque formation (Ross, ~ al. , 1971) .
Following incubation, cells were stained by adding to the
growth medium one drop of a 3% aqueous solution of crystal
violet in 20% ethyl alcohol and 0 . 8% ammonium Oxalate (Carolina
Biological Co., Burlington, North Carolina 27215). The resulting
solution was poured off after 10 min and plaques were counted.
Monochromatic Exposures
To obtain sUfficient exposure rates , a 2 . 5 kW high
presSure mercury - xenon lamp was used (929B Hanovia Lamp, Newark,
New Jersey 07105). This source was air cooled to remove ozone,
and infrared radiation was partially removed from the beam by
means of a circulating water filter. Spectral separation was
achieved by means of two 25 cm grating rnonochromators coupled
in tandem (GM 250, Schoeffel Inst. , Westwood , New Jersey 07675) .
Half band widths were measured at several wavelengths and varied
from 1 . 5 to 6.0 nm depending on the slit width. The Spectral
purity of the monochromator was further checked by plaCing a
7
series of cut-off filters into the exit beam . In this study,
only wavelengths corresponding to individual lines of the
mercury spectrum were used, and these were fOund to be Spectral_
ly pure. EXPOsure rates were measured at different times
during an experiment by plaCing a calibrated UV-sensitive
photodiode (Cal- Uv , United Detector Technology, (UDT) , Inc. ,
Santa Monica , California 90405) in the sample position and
measuring the photocurrent produced by the radiation beam with
a Keithley 610B electrometer (Keithley Inst., Cleveland, Ohio
44101). Cells or Viruses were irradiated in horizontally
oriented petri diShes. The UV beam was deflected downWard
onto the diShes by means of a 45 - degree front - surfaced mirror .
DiShes were rotated at 0.5 rps to compensate for POSsible
inhomogeneities in the radiation beam . EXPosure times Varied from 1 to 120 sec .
2 EXPOsure rates varied from 0.08 to 24 W/m .
Before exposure to radiation , the growth medium was
removed from confluent monolayers of cells i p lastic petri
diShes by Suction . The mono layers we re then rinsed twicp in
Dulbecco ' s phOSphate buffered saline (PBS) (Dulbecco and Vogt ,
1954) to remove UV-absorbing components in the medium. The cell
monolayers were kept mOist during irradiation by the presence
of 2 ml of PBS . Virus was also irradiated, as a SUspenSion,
in PBS . Virus was irradiated at 40 c and cells were irradiated
at room temperature (22 ! IOC) . During any experiment ten
monolayers were assayed at each fluence , and experiments were
r 2peate6 at least three times for each wavelength. Additional
B
experiments were run at peak wavelengths to ins u re the accuracy
of t he peak response level .
Immediately following irradiation of the virus , the viral
suspension was diluted into maintenance medium and inoculat ed
onto the cells . Cells were incubated and stained as described
above.
All viral irradiations were at 265 nm at a fluence of
500 J/m2 . This fluence lowered the pfa of the virus to approx
imately 1% of a control (unirradiated) virus , but this survival
varie d som~what from expe riment to experiment. Cells were
irradiated a t different wavele ngths as shown in t he results
(Table 1) .
TABLE 1
FLUENCE RATE RANGES FOR MONOCHROMATIC EXPOSURES TO CELLS AND THE SLOPE OF THE AVERAGED RER RESPONSES
Fluence Wavelength rate re~ge RER RER Slope (in mn) (1'1 m ) Slope (Ouantum corrected)
230 0 .08 - 0 . 30 0.041 0.053 235 0.50 - 2.00 0.055 0.071 240 1.10 - 3.90 0.071 0 . 089 245 1. 30 - 5 .10 0.074 0.091 248 1.25 - 5.40 0 . 114 0.139 254 0 . 45 - 1.45 0.146 0.174 260 0.05 - 0.20 0.210 0.244 265 0.30 - 1.10 0.206 0.235 270 0.60 - 2 .00 0 . 220 0.246 275 0.75 - 2.60 0.190 0 .209 280 0 . 90 - 3.70 0 .152 0 . 164 289 1.05 - 3.50 0.060 0.063 297 6 . 00 - 19.00 0.011 0 . 011 302 8 . 00 - 24 .00 0.004 0.004
9
RESULTS
Figure 1 represents the data obtained from two separate
experiments involving the effect of UV-radiation at 265 nm
on cellular capacity for the growth of unirradiated and
irradiated virus. The abscissa indicates the fluence of
radiation; the ordinate is the fractional survival of cellular
capacity compared with unirradiated c ontrols. Since each mono
layer contained 20-100 plaques, each data point corresponds
to 200 to 1000 plaques. The upper curves obtained from ir
radiating cells but not viruses show a loss of capacity as
fluence is increased. The lower curves obtained from irrad
iating both cells and viruses show an increase in pfa at low
fluences fOllowed by a decrease at higher fluences. Figures
2 and 3 represent data obtained from experiments conducted
at two additional wavelengths (235 nm an r J 2 nm , respective ly).
In figure 2 are indicated results for a wa vel e ngth (235 nm)
which incurs less damage than 265 nm. At 235 nm, no decrease
i n the capacity to host unirradiated virus (upper curve ) is
seen at the fluence which at 265 nm (Figure 1 ) decreases the
capacity to 10 % of a control. Correspondingly , the reactivation
sh0wn at 235 nm (lower curve) is less than that shown at 265
nm. Figure 3 represents a wavelength (302 nm) which is also less
damaging to the cells than is 265 nm, a larger fluence is
required to lower cellular capacity to support virus growth .
11
Figure 1. 'I'he effect of 265 nm radiation On the capacity of CV-I cells to support the growth of either_
2 unirradiated (O' ~ ) or irradiated ( 500 J m ) (. ,A) HSV-1.
265 I'Im
>- 1.0 ~
(.) 0.5 ~
~ (.)
C) Z
0.1 Z -~ .05 ~ w a::
lL... 0 .01 Z 0 .005 ~ 0 ~ a:: lL...
0 10 20 30 40
FLUEI\CE IN ~l
12
Fiqure 2. Tb effect of 235 na radiation on c II capact J tor un1rradiated ( 0 ) or irr d1 ted (~OO J .- ) ( . ) virus.
>-...... 0 235nm
~ ~I.O (') 0.5 z z -<l ~ W a:: 0.1 LL
° .05 z 0 t-O <l .01 c:: LL
-
0 10 20 30 40 50 60 FLUENCE IN J/m2
13
Figure 3. The effect of 302 ~ radi for un rradiatad ( 0 ) or ( . ) viru •.
tion 0 cell capac ty irr dta ad (500 J .-2)
>-..... 302" ... 0 <l: 1.0 a.. <l: 0 0.5 C) Z
Z
<l: 0.1 ~ W cr .05 lJ... 0
Z 0
.0' ..... 0 <1 .005 0::: lJ...
o 25 t() 5 1 125 FLUENCE It, J/ ,.,.2
14
The l ower cur ve repr esents th radiation ennanc d reactivat on
da ta for this wavelength. As in figure 2, the reactivation
apparent for l02 nm i. las. than that tor 265 n..
Experiments were co plated at an additional eleven wave
length. and yielded siailar data. Por purpo ... of .i~licity,
only thr e important wavelength. are illuatra ed, one at the
peak response level and one at each end ot th wavelength.
tested. The shape of these r diatlon enhanced r ct~Yat 01'1
curves can vary from experi8ent to exper t.
To ttempt to arrive at an action _peet or thb effect with such a variat on in re.ponse uld be difficult. Howeva , it analys • • lia1ted !nth foll ng ay.,
tha probl m of choo.ing a c rta1n lev 1 of r c ivat.ion i.
alleviated:
1. The pta of rr d at d virus
divided by the pfa of unirr diat v rla on cell. tha the a tlu no. Thi. ratio i. ref rc toa.thee
factor (Bockat. hler, !! al., 1916, Caille _p
1972 / Radl!lan and Devor t, U7l). •
2. Only that port~on of the re.,pclnl,. that 1nYOl
e
t.1Yat.~
Det .,
increaaa in th abov ra 10 ia us
of r activation. to d t rain th a·:>uftt
l. Each RER experi t. as done on the • d Y capacity ex rl nt for unirr diat. d v ru p ss g nu r c U •.
Onc t a l1naita re illpO d, th n a 1 n on 8 mi-log paper w • oPt ined or e cn a r n . '" in,
three important wavelength. were chosen to illustrate data.
Piqure 4 represents typical data analyz d a. above for
several experiments at 265 nm. Each line in f qure • rep
resent. data from one experiment at 265 n.. Slope. re
calculated for each of these line. and an average slope value
was obtained for the wavelength. The lines n figure 5 rep
resent experiments at 235 nm, and the 1 nes n figure 6 rep
resent experiments at 302 nm. Calculated slopes are a1ao
presented for these wavelengths.
In order to obtain one repre ntative line for ea
wavelength, the data point. re then nOrrMlized to t cor-
responding data point. on th 1 1 ne (~.i. line E of
figure .). Th s valu ., calculat, d for all a lengt • were th n replotted in figures 7 tbrouq,h 11. The val of
the slope of this final line was eons d red to
value for th wav length.
In fi9ures 7 through 11 are ah nth effecta 0 uv-
radi tion of 14 different avel "9thS on r dtat on • ..la4n
reactivation. The alop of th re ctiv tion f ctor
for each w velength and 18 found lhted in T le l.
since photochemical ch nges occurring n bioloq c 1
Ho_vee,
are du to photon ab.orption, not j ust to the en rqy of x
posure, th slope valu s w re quantum corr cted ( ) (J r,
1967). The OC values re also l i at d 1n Tab1 1.
15
In figure 12 is sho the corrected ct on SDec-.:naa
for RER plott d a alope veraua w n9th. An orpt on
apectrUlll for A is includ d for CO/IIDalr1.0n (J 99 r, 1 61) .
16
Figure 4. The reactivation curv • tor the 1 ty of CV-l cell. irr,diat d at 2 5 na to re ctiv t. rr dia (500 J. I HSV-1.
-
265nm
01
~ .06
::! c
z 0 f-
~ .01 -f-~ .006 w ..... Q:
o 5 10 15 FUDa:
17
Figur 5. The viru. react vation curvea for cella read ted at 235 nJII.
z o I-
~
.04
.01
~ .001
o
o a
10 20 .30 Fl.JJEta II
•
~QPfS ...... . ~ . . • • II - -•
18
igure 6. The virus r act vat ion curve. to e.U. lrrad a at 302 nJD.
a: o 0 .1 I-
~ LL .05
z o I<l > t5 .01 <l W a:
.005
302"fI'\
8
o 500 1000 1500 2000 2500 FLUE NC IN J"/ rtlt
19
Figures 7 - 11. The averaged r activation factor. for KEX of HSV-1 by CV-l cell. at different av 1 Dgtb ••
-
~ .05
~ u.:
z 0
DI -~ t- ~ 'ci W Q:
o
f,;0PEs HO~7.& • •
10 20 30
FlLEta
, , Uo
40 50
J/rtf
2.54
240
~
60
Q: .05
E u II
z 0 .01 -
260 I-
~ -I-U <f W
- ·220 a · .20 a::
• t1'O - .217
o 5 10 15
FLL£NCE:
cr f2 ~ z 0 ~ <l > ~ U <l W cr
0.1
.01
. 005
280
SL~ . 2,..-. • - .1iD e •
o 10 20 JO 40 50
FLUEf\CE I. ~'
cr .05 ~ ()
i1.
z 0 I- .01 et >
&OfEs -I- .005 Si w cr
o 250 500 750 1000 &SQ)
FU.£ E ~.
20
Figure 12. An ct on apectrua tor • plotted •• .10 (quantWII corrected) of the 1 n • fro. I'i ure 7 throug 11. An orptiQn. for • included (3 99 r. 1967).
.3
.2
~ 0 ~
.I
IlCTION SPECTRLM
-DNA~I
/
I /
/ /
/'
230 240 250 260 270 200 290 300
A IN nm
OISCUSSIO
An analysis of the action spectrua pre. nt.ed in figure
12 leads to several conclusions. Kajor protions of the •
action spectrum are similar in shape to the published action
spectrum tor cell capacity to support unirradiat.d virus 9th
in the s cell-virus syste (coon!1l,!! al., 1917 , . Since
mammalian cell c pacity is considered to be a A depend nt
function (Lytle and Benane, 1975 ; Coppey and Nocentini, 1 7',
Coohill, !! al., 1977) it s re sanAble to conclude that
is, a least partially, also 0 A dependent. ver, the ~.
action spectrUlll has its peal< n the ran fro. 2'0 to 275 llA.
Although a pea at 2 0 na indicates the lnyolv ll t ot ftucleic
acid ( A or AI, a peak &bov this va l ue ( 2 na) lip! es
protein involv nt (Ja9g r, 19 7). Th r fore, the ct Oft
spectrum pres nted her s 0 th 1nvol nt ot be n 0 1 c
oid and protein. This interpretation s able
one modification. Proteins also in 0
w v.lengths below 250 na , but this a c ion
rb _r trua
show an incr ase in aD orption in thiS r gion. In
the action .pectrua below 2 0 n .
rapidly than doe. the abaorption
s em to indicate th invol nl of
• i n val
etr or
not
molecules) in the RE r spons at thes berter a ths .
Whether this effect is dir ct (an in ract on) or t
(shielding of the t rg t molecule)
tion.
ai s fur r e r
In bacteria, WR is clos ly aSSOCiated with prOphage
induction and mutagenesis. Witkin (1976) and Devoret (1976)
have Suggested that a COImIOn inducible regulatory aignal aay
Control all three pheno na. That all of the proc sa a re
quire poa -irradiation protein syntheaia is corollary evi
22
of induoibility (Witkin, 1976). n (1974, 197 ) hal e 1-
Oped thi a concept further into a ·805· 1ndue ble eeror-prone
repair hypothesis. He haa also lhown that SOS-induc1ng treat
ments allow DNA replication of the bacteriophag .XI7C to
prOceed p st (or around) pyriJUdine , penanal
communication). In addition, HR i. photoreact Vahle in e
teria (Heigle , 1953) POinting to pyriJlidine d1 ra al a lor
cause of this r aponae. WR in bacteria ia not pendent Oft
exciSion r p ir (Radman and Devoret, 1'711. Pinally, sin
WR is vident in Single str ad aa 11 a do Ie r'ande'd
phages (Ono nd Shiaazu, 19 6/ T s n d Oz 1, 1 '0) •
not likely that a reco in conal type 0 r pair ia
for its e xpression.
Results of similar ex ri n a using .... ~l n cella
be n done. Lytle,!!!!. (1 16 ) have I
ia not necessary for RER. Thi, r ault,
relults of Hellman, !!!l. (197) ho
tha xc I on r
th
t t s decreased in the pr enc of eaf in, could be int rpret
luggesting that post-replication ~ pair aay be n
RER (Nilsson and Leh n , 1'75). Th r suIt, of Lytle (1 71)
with Kilh cat virul (a I ngl st vi) r ail r
to thoa ntioned &bov for I nqle b et r1opftag.1
r
2 3
This resul t may mean t h at an inducible error-prone .override.
repai r sys tem ( Radman , 1974 and 1976) is common to both systems.
Hence , the results inVOlvinq RER in m 11an cells agree
in principle with the results Obtained with WR in bacteria with
o ne e xception; Lytle and Benane (1975) have reported that
Photoreactivating light treatment of potoroo cells has I ttle
Or no effect on delayed RER. This latter reSUlt L.plies th t
py r imidine dimers are not essential for RER, at least in marSupial cells.
In summary, th actiOn spectrum for RER in aa-=a11an cells
can be interpreted as evidence for involv t of both nUCleic
acid and protein in this pheno non. Corollary data of others
on the b iochemical events involved in this response 0 not all
one to distinquish betw neither DOlecuie as the .. ,or t r t
for UV-radiation effects on RER.
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