Deoxyribonucleic acid instability in the presence of chloramphenicol in ultraviolet-light-exposed bacteria

Vol. 4, No. 3,1961

BIOCHEMICAL

AND RIOPHYSICAL RESEARCH COMMUNICATIONS

DEOXYRIBONUCLEICACID INSTABILITY IN THE PRESENCEOF CHLORAMPHENICOLIN ULTRAVIOLET-LIGHT-EXPOSED BACTERIA* C. 0. Doudney Section of Genetics, Department of Biology, The University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, Texas Received February 8, 1961 It has been demonstrated

that the ultraviolet

induced lag in deoxyribonucleic (Kelner, Drakulic

acid (DNA) synthesis

1953) is overcome through

acid (RNA) and protein and Errera,

chloramphenicol synthesis

(chl)

(Harold

the synthesis

and Ziporin, In studies

1959).

cultures

(UV)

in bacteria

of ribonucleic

1958; Doudney, 1959;

of the effects

on induced mutation

in "synchronized"

light

of

and on nucleic

of Escherichia

coli

posed to UV, Doudney and Haas (196Oa; 1960b) demonstrated a relation

exists

synthesized

at the time of chl addition

of DNA synthesis DNA synthesis

between the amount of postirradiation

that rate

The maximum rate of

in the presence of chl occurs when the amount of in the culture

of the relation

at the time of chl addition.

of RNA formation

have been extended to log phase cultures where similar

exRNA

and the relative

in the presence of chl.

RNA has just doubled These studies

acid

results

are observed

of E.

(Doudney, unpublished

In the course of these studies

it was observed

is added immediately

UV-exposure,

that,

data).

when chl

in the amount of DNA takes place with

a marked decline This incubation in chl,

chl-promoted

UV-exposure appears not

instability

following

to DNA synthesis

of DNA after

*This work was supported in part by U. S. Atomic Energy Commission contract no. AT-(40~l)-2139. 218

BIOCHEMICAL

Vol. 4, No. 3,1961

to be related however

it

nucleic

acid

presence

directly

AND BIOPHYSICAL

to the

UV-induced

may be related

to

precursor

synthesis

of

chl

of UV-exposed

(Drakulic

lag

in DNA synthesis;

such phenomena

as the

in UV-exposed

failure

cells

of

in the

1961) and chl-promoted

et al.,

bacteria

RESEARCH COMMUNICATIONS

(Okagaki,

death

1960).

RESULTS If

chl

is

progressive Burton

immediately

decrease

reaction)

in the

creased

by eighty

is blocked protein

by the is

that

minutes

percent

the

cells

incubation

amount

the

cubation

following

for

exposure after If

in

is

UV-exposure,

added

initiated

of

indicaafter

15

a decline

in

incubation after

no decrease

of DNA is

amount

of chl

15 minutes

chl

in-

synthesis

150 minutes,

produces

by the

of DNA is de-

protein

Addition

a

15 minutes

amount

no decrease

intact.

begins

synthesis

after

Though

1).

incubation

same level.

is observed;

begins

(Table

following

of DNA, which

to about

which

the

remain

exposure,

of DNA (as measured

of chl,

with

radiation

incubation,

addition

observed

after

amount

is observed,

At 150 minutes

cubation.

ting

added

in

chl,

30 minutes

in amount after

in-

of DNA

60 minutes

incubation. RNA synthesis exposed

cells

in the

lowing

UV-exposure

minutes

and then

RNA in the is

added

after

15 minutes

this

period

place

presence (Table

ceases;

culture

next

synthesis

takes

with

15 minutes in not

of incubation eventually

but

at a reduced

of chl

1).

incubation

incubation, appreciably the

ceases.

proceeds

is no decrease

subsequent

rate

affected;

of

chl

for

45 of

If

chl

of RNA in the however,

of synthesis

fol-

in amount in chl.

synthesis

No effect

219

in UV-

added:immediately

RNA synthesis

there

rate

declines

after and

on RNA synthesis

BIOCHEMICAL

Vol. 4, No. 3,196J

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE I Effect of chloramphenicol on the amount of RNA and DNA in UVexposed cultures of Escherichia coli strain B/r, with postirradiation incumon.

Time of chloramphenicol addition** 0

Min.

15

30

0.92

15

postirradiation

incubation*

45

60

75

0.79

0.72

0.58

0.45

p.20

0.18

1.02

1.02

0.88

0.78

0.65

0.40

0.22

30

1.02

1.04

1.04

1.01

1.18 1.48 amount RNA***

1.73

0

1.12

1.22

1.35

1.31

1.33

1.35

1.35

15

1.25

1.45

1.60

1.66

1.66

1.61

1.60

30

1.28

1.48

1.77

2.03

2.41

-

-

Relative

Relative

amount

105

DNA***

150

*Minimal (salts-glucose) medium is inoculated with a 7 hr liquid culture of Escherichia coli strain B/r to an optical density of 0.08 at-s= in optical tubes with a 1.8 cm lightpath in a Bausch and Lomb type 33-29-40 calorimeter. The culture is incubated at 37°C with vigorous aeration until an optical density of 0.43 is reached. The culture is chilled in an ice bath. Twenty ml samples in large petri dishes, are exposed with mechanical stirring, to 30 set UV light at 30 cm from a Gates "Raymaster" mercury vapor lamp. The culture is rapidly rewarmed to 37°C and incubation, with aeration, continued. **After the indicated minutes of incubation following UV-exposure chloramphenicol is added to a concentration of 20 -"#*DNA: ug per ml. 1=37 ug per 5 ml sample of cell suspension based on a purified salmon sperm DNA standard. RNA: 1=180 ug per 5 ml sample of cell suspension based on a purified yeast RNA standard. Five ml culture samples, taken at appropriate intervals during the postirradiation incubation period for analysis of RNA, DNA and protein, are chilled, spun down in the cold and resuspended in cold 0.5 N perchloric acid. The suspension is spun again to obtain a precipitate containing the nucleic acids and protein. Nucleic acids are hydrolyzed by incubation in hot erchloric acid for 50 minutes at 7O*C for DNA is that of Burton [$-u-)yd Rosen, 1950 P l . Analysis For RNA determination the ultraviolet adsorption at 260 ml.l and 290 mn is determined (Visser and Chargaff, 1950) and the amount of DNA, as determined by the Burton analysis of the same sample, is then subtracted with correction for extinction coefficients. Protein is determined by the Folin method.

is observed

for

added

30 minutes

after

at

least

90 minutes, following

when the

UV-exposure. 220

antibiotic

is

BtOCHEMlCAL

Vol. 4, No. 3,1961

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

DISCUSSION The effect chl-promoted

of chl decline

basis,

the

on the

functional

cellular

may

synthesis cells

be explained

sitivity

of the

(Doudney,

presence

on this

basis.

cellular

effect

of chl

lethality to the are

lowing

DNA to chl

to chl

UV-exposure of time

that

period

following

1961)

syntheses

to the

sen-

effect

of

material

chl

parallels

chl-promoted

1960) may be related Cells

approximately

of &. 20-30

unpublished

DNA remains

in

W-exposure the

(Okagaki, chl.

material

of these

time

of

et al.,

Similarly,

DNA to

(Doudney,

Failure

(Drakulic

260 mu absorbing

for

may depend

DNA.

Furthermore,

soluble

of the

sensitive

same period

a comparable

UV-exposure

sensitivity

precursors)

of chl

to the On this

260 mu absorbing

on RNA synthesis.

following

cell.

Sensitivity

data).

of acid

its cellular

soluble

in the

unpublished

on synthesis

of the

during

may be related

of DNA in the

RNA (or

of acid

is observed

B/r

to form integrity

to chl

the

in amount

capacity

UV-exposed

on RNA synthesis

strain

minutes

data), sensitive

coli

which

folis the

to chl.

ACKNOWLEDGMENT The author technical

wishes

to thank

Miss

Janet

Allison

for

capable

assistance. REFERENCES

Burton,

K.,

Biochem.

J. 62,

315 (1956). (1959).

Doudney,

C. O.,

Nature

l&t,

Doudney,

C. O.,

and Haas,

F. L.,

Biochim.

Biophys.

Acta 40,

and Haas,

F. L.,

Genetics

42,

(1960b).

375 (l%Ca).

Doudney, Drakulic, 495

C. O., M., (1959

and Errera, 1

M.,

Biochim.

l

221

Biophys.

1481

!Acta 2,

BIOCHEMICAL

Vol. 4, No. 3,196l

Drakulic,

M., Smit, S., and Stavric,

Acta &, Harold,

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

M.,

Biochim.

Biophys.

77 (1961).

F. M., and Ziporin,

Z. Z.,

Biochim.

Biophys.

Acta 2,

439 (1958). Kelner, Okagaki,

A., H.,

J. Bacterial. J. Bacterial.

a,

259 (1953).

a,

277 (1960).

Ogur, M., and Rosen, G.,

Arch Biochem. 3,

Visser,

E.,

E., and Chargaff,

J. Biol.

222

262 (1950).

Chem. a,

703 (1948).