Ribosome degradation in ultraviolet light-damaged bacteria starved for an amino acid

lO6

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96141

RIBOSOME D E G R A D A T I O N IN U L T R A V I O L E T L I G H T - D A M A G E D BACTERIA STARVED FOR AN AMINO ACID

C. O. DOUDNEY

Research Laboratories, Department o/ Genetics, Albert Einstein Medical Center, Philadelphia, Pa. z9z4I (U.S.A.) (Received September 3oth, 1968)

SUMMARY

Degradation of RNA and of 3o-S and 5o-S ribosomes occurred when Escherichia coli H/r 3oR cells were exposed to 400 ergs/mm 2 of ultraviolet light and then incubated without arginine, a required amino acid. Under these conditions, the ribosomes remained stable for I h, but rapid breakdown began thereafter and eliminated the greater part of the ribosomes by 2 h. Arginine prevented most of the breakdown. Both the photoreversible pyrimidine dimer and non-photoreversible damage appeared to result in breakdown, but at this ultraviolet light dose most of the damage was not photoreversible. If 30 rain of postirradiation incubation with arginine was allowed prior to incubation without the amino acid, little degradation occurred on subsequent arginineless incubation, indicating that dark repair processes dependent on protein synthesis are capable of removing the cause of the breakdown, presumably through repair of damage to DNA resulting in recovery of capacity for DNA ieplication.

INTRODUCTION

During exponential growth of Escherichia coli, ribosomal RNA is stable (see a review b y NEIDHARDT1). However, when the bacteria are subjected to an unfavorable growth environment, such as stalvation for a specific essential metabolite, extensive breakdown of ribosomal RNA occurs 2. While certain conditions, such as the absence of Mg2+ from the medium, lead to fairly rapid ribosomal degradation, that which occurs in nitrogen-starved or amino acid-starved cells is slower (about 5 °/o per h) 2. Rapid degradation of 3o-S and 5o-S ribonucleoprotein particles has been found with bacteria starved for both thymine and a required amino acid 3. This breakdown began after 60 min of starvation and the 5o-S particles had almost disappeared by 12o rain. It did not occur when the bacteria were allowed to come to the end of the DNA replication cycle by means of incubation for I h with thymine but without the required amino acid before they were incubated without both thymine and the amino acid. This suggested that completion of the DNA replication cycle somehow prevents this sort of ribosome breakdown. The data are consistent with the hypothesis that interference with DNA replication (i.e., by thymineless incubation) somehow makes the riboBiochim. Biophys. Acta, 179 (1969) lO6-114

RIBOSOME DEGRADATION IN I R R A D I A T E D BACTERIA

107

somes susceptible to degradative processes which occur in the absence of a required amino acid. In this study, we have investigated the effect of ultraviolet light exposure on the stability of ribosomes and RNA in bacteria starved for a required amino acid. We found that this exposure, which results in a requirement for protein synthesis for recovery of capacity for DNA replication, also resulted in ribosome breakdown in amino acid-starved bacteria. If a period of time sufficient for "dark repair" and recovery of DNA replication were allowed before the amino acid starvation, however, the degradation of the ribosomes did not occur. The results support the general conclusion that interference with DNA replication (whether by thymineless incubation ol b y ultraviolet light damage) somehow creates intracellular conditions which render ribosomes unstable unless protein (or possibly RNA) synthesis occurs. MATERIALS AND METHODS

E. coli strains H/r 30 and H / r 3oR developed by Dr. EVELYN WITKIN4,5 were used in these studies. These strains require arginine; thus 4 °/~g/ml of L-arginine was added to the minimal growth medium. Culture growth procedures and ultraviolet light irradiation techniques have been described 6,v. Log-phase cultures were obtained by growth at 37 ° in minimal medium in large erlenmeyer flasks with vigorous aeration from a small inoculum to an absorbance of 0.255 (or approx. 3-IO s cell/ml), as measured by a Baush and Lomb "Spectronic 2o" spectrophotometer using a I.o-cm optical tube and at a wavelength of 660 nm. The doubling time of the bacteria in log phase was about 44 rain. The cells were harvested by collection on Millipore filters and rinsed on the filters with ice-cold minimal medium. They were then resuspended in an equal volume of ice-cold minimal medium. 25-ml samples were exposed in cold I4o-mm diameter petri dishes with magnetic stirring to ultraviolet light from a General Electric germicidal lamp (15 W), combined to the desired volume and held in an ice bath. The ultraviolet light dose below 28o0 ~, was 50 sec at the average rate of 8 ergs/mm ~ per sec as measured by a Hanovia model AV-971 photometer at the position of the suspension. This dose under the above conditions decreases survival (colony formation) to 23 °/o of the bacteria plated on minimal agar medium and results in a delay in initiation of net DNA synthesis of about 40 rain. After irradiation, 2oo-ml portions of bacterial suspension in 2-1 erlenmeyer flasks either were supplemented with L-arginine or left without it as indicated, warmed rapidly in hot water to 37 ° and incubated on a rotary shaker at 37 ° with sampling. In the studies of photoreversal of the ultraviolet light lesion, the ultraviolet light irradiated bacteria were exposed to high-intensity white light for 4 ° rain at 8 ° prior to the indicated supplementation of the medium and incubation at 37 ° . Controls were held at 8 ° in the dark for the same period of time. This period of photoreactivation was adequate to produce the maximum observable effect both in restoration of DNA replication and prevention of ribosomal breakdown. The techniques for photoreversal utilized have been described s. E. coli strain H/r 30 is a radiation-resistant strain requiting arginine and lacking the enzymatic capacity fer photoreversibility 4,5. Strain H/r 3oR is a revertant of H/r 30 which has regained capacity for photoreversibility 5. Tritium-labeled uridine (New England Nuclear Corp., Boston, Mass.) was added to the growth medium to a concentration of 15 pg and activity of 0.5/~C/ml at the Biochim. Biophys. Acta, 179 (1969) lO6-114

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C. O. DOUDNEY

start of culture growth. Io-ml samples were taken after various intervals of time for RNA, DNA and protein determinations and for the determination of incorporation of labeled uridine into RNA. The bacteria were removed from the labeled uridine with the filtration and resuspension in minimal medium prior to ultraviolet light exposure (see above) and incubation after ultraviolet light exposure was without labeled uridine. The cells were collected b y centrifugation at IO ooo × g, washed with cold o.5 M perchloric acid, and the nucleic acids hydrolyzed with hot acid (7o°). The techniques used for determination of quantities of nucleic acids and protein have been previously described 6,7. DNA was measured by the diphenylamine reaction. For determination of RNA. spectrophotometric measurements of nucleic acid hydrolysates were used, with correction for the optical absorption of the hydrolysate of the DNA present. The results were verified with the orcinol reaction. Protein was determined by the Folin method on the hot acid-insoluble cell fraction. Incorporation of labeled uridine into RNA was determined by plating appropriate samples of the bacterial suspension onto filter paper discs and treating them as described below. After incubation and sampling, as described above, the cells were centrifuged in the cold and washed twice with cold o.oi M Tris buffer supplemented with lO .4 M MgSO, (pH 7.4). The cells were then resuspended in the cold to a concentration of lO 1° cells/ml in the same buffer ccntaining IO #g/ml of deoxyribonuclease I (Worthington Biochemical Corp., Freehold, N.Y.) and lysed in a chilled French pressure cell at a controlled pressure of io ooo lb/inch ~. Incorporation of labeled uridine into RNA was determined by plating appropriate samples on filter paper discs and treating them as described below. The lysates were centrifuged in the cold at 20 ooo × g for 15 min. The highresolution density-gradient sedimentation analysis methods of BRITTEN AND ROBERTS9 were used for separating the ribonucleoprotein particles and the soluble RNA. Three 5 % to 20 °/o sucrose gradients with I ml of 4 °/o sucrose solution added to their surface were prepared (per centrifuge run). I ml of a supernatant was layered on the surface of a gradient and the tubes balanced with white mineral oil. The gradients were centrifuged for 16. 7 h at 27 272 x g in the Spinco model L ultracentrifuge in the cold using the SW 25.1 swinging bucket rotor. The gradients were collected by the punctured-tube drop method into a series of fractions, each about 0.5 ml in volume. Distribution of the RNA in the fractions was determined by optical absorption measurements with the Zeiss spectrophotometer at a wavelength of 260 nm, after proper dilution of the fractions with buffer. Radioactivity was determined by plating o.I ml of the fractions onto filter paper discs. The discs were dried with a heat lamp and put into beakers of cold 0. 5 M perehloric acid in ice to precipitate the nucleic acids. They were gently rinsed and neutralized with an alcohol ether-ammonium acetate mixture, heated for io rain at 46o and dried again. The [aH]uridine content expressed as counts/ min, was determined in the Packard scintillation counter. All operations were carried out under yellow room light from gold fluorescent lamps to avoid photoreactivation by the room light source. RESULTS

RNA degradation in ultraviolet light exposed bacteria during arginine starvation Radioactive uridine incorporated into the RNA of E. coli strain H/r 3oR cells Biochim. Biophys. Acta, 179 (1969) lO6-114

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RIBOSOME DEGRADATION IN IRRADIATED BACTERIA

during active growth was retained without appreciable diminution during subsequent incubation of 12o rain without arginine (Fig. I). When, however, the bacteria were exposed to 40o ergs/mm 2 ultraviolet light, and then stalved for arginine, a loss of label occurred after the first hour of incubation, so that after 12o min, only about 11

UttravioLet t r .~atment

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Fig. I. Degradation of R N A in a ultraviolet light exposed culture of E. coli strain H / r 3oR starved for arginine. The culture was grown for 16o min at 37 ° in minimal m e d i u m s u p p l e m e n t e d w i t h b o t h L-arginine and [3H]uridine (see MATERIALS AND METHODS). I t WaS t h e n filtered (vertical black line), w a s h e d w i t h ice-cold minimal m e d i u m and resuspended in ice-cold minimal m e d i u m (containing no arginine or labeled uridine). The culture was divided into two subcultures. One subculture (UV) was exposed to 4oo e r g s / m m * of ultraviolet light. The other control subculture (C) w a s held in the dark. B o t h s u b c u l t u r e s were w a r m e d rapidly to 37 ° and incubated on the r o t o r a r y shaker with sampling after the indicated periods of time h a d elapsed. The [3H]uridine c o n t e n t in the cold acid insoluble fraction of R N A obtained from these bacteria after the bacteria had been r e m o v e d from their g r o w t h m e d i u m b y s e d i m e n t a t i o n in the centrifuge at IO ooo × g w a s determined. Additional controls n o t s h o w n in the above figure were run. An unirradiated subculture incubated after filtration w i t h arginine showed iooo c o u n t s / m i n after 12o rain. An ultraviolet light exposed subculture incubated after filtration with arginine showed 92~ c o u n t s / m i n after 12o min. Fig. 2. Degradation of 3o-S and 5o-S ribosomes in an ultraviolet light exposed culture of E. coli strain H / r 3oR. The bacteria were handled as described in Fig. i. Samples were t a k e n immediately after filtration ( 0 - 0 ) and after 12o m i n of i n c u b a t i o n and subjected to sucrose d e n s i t y gradient analysis (see MATERIALS AND METHODS). One subculture was not irradiated and was incubated w i t h o u t arginine ( O - O ) . A second subculture was irradiated and incubated w i t h o u t arginine (V]-[]). A third subculture was irradiated and incubated w i t h arginine p r e s e n t ( , - . -m). The [~H]uridine c o n t e n t of the fractions of R N A so obtained was determined. The initial fraction a p p e a r s on the left and s u b s e q u e n t l y obtained fractions follow t o w a r d the right. T h u s the first large peak represents 5o-S ribonucleoprotein and the second smaller peak 3o-S ribonucleoprotein. The last fractions (after the 3o-S peak) contain soluble R N A of various classes and composition.

6o °/o of the label present at the start of incubation sedimented with the bacteria at io ooo ×g. This decrease in total labeled RNA was reflected to a greater extent in the loss of labeled ribosomes (Fig. 2). Thus while incubation of the unirradiated culture in the absence of arginine for 12o rain resulted in little change in the amount of label found in 5o-S and 3o-S fractions, incubation of the ultraviolet light exposed culture for an identical period resulted in a marked decrease in the 3o-S and especially in the 5o-S ribonucleoprotein particles, with an accumulation of slower-sedimenting soluble RNA. Incubation of the ultraviolet light exposed bacteria with arginine prevented a large part of the breakdown of the 3o-S and 5o-S ribonucleoprotein, though it did not prevent the accumulation of some slower-sedimenting RNA. One might imagine that the loss of sedimentable RNA and ribonucleoprotein particles observed with ultraviolet light exposed bacteria incubated without arginine Biochim. Biophys. dcta, 179 (1969) lO6-114

II0

C.O. D O U D N E Y

is due simply to cellular lysis. However, the 5o-S ribosomal particles were lost much more rapidly than total RNA. While some decrease in DNA occurred as would be expected in bacteria subjected to this dose of ultraviolet light and incubated under conditions where protein synthesis is not possible 1°, there was little decrease in sedimentable protein (Table I). An effort to compare numbers of bacteria present just TABLE

I

EFFECT OF INCUBATION WITHOUT ARGININE OF A ULTRAVIOLET LIGHT EXPOSED E. coli STRAIN H / r 3 o R ON D N A AND PROTEIN CONTENT

CULTURE OF

R e l a t i v e D N A a n d p r o t e i n c o n t e n t of t h e s e d i m e n t a b l e b a c t e r i a f r o m a u l t r a v i o l e t l i g h t e x p o s e d c u l t u r e of E. coli s t r a i n H / r 3 o R i n c u b a t e d i n t h e a b s e n c e of a r g i n i n e for 60 a n d 12o m i n a f t e r u l t r a v i o l e t l i g h t e x p o s u r e as c o m p a r e d t o c o n t e n t a t t i l e t i m e of i r r a d i a t i o n (i.e., I.O). F o r D N A , i.o = 5.8/*g/ml. For protein, i.o = 116.2/~g/ml.

T i m e o.i incubation (rain)

DNA Protein

6o

I2o

0.78 o.96

0.63 0.88

after ultraviolet light exposure with the numbers of the somewhat shrunken bacterial bodies found after 12o rain of incubation without arginine, utilizing the Petroff-Hauser chamber under the microscope, encountered difficulties due to unavoidable clumping but it was evident with each determination that these bodies represented not less than 75 % of the original number of bacteria present. Thus it seems unlikely that the loss in ribosomal particles is based on complete cell lysis and we suspect a more specific mechanism is involved in the ribosomal degradation observed. The delay of the degradation of the 5o-S ribonucleoprotein particles compared with that seen with total RNA (Fig. 3), which is to be expected, since these particles make up a considerable fraction of the total RNA. Thus the number of particles, based on optical absorption at 26o nm, remained constant for 60 rain and then began to break down. The 3o-S particles did not begin to decrease until after 9 ° min of incubation. The soluble RNA increased after 60 rain, presumably because of the accumulation of partially degraded 5o-S RNA. We have explored ribosomal degradation after ultraviolet light exposure in several other strains of E. coli requiring various other amino acids (methionine, tryptophan, tyrosine and leucine). The breakdown in 5o-S ribosomes in these strains seen after 12o rain of incubation without the required amino acid compared with that seen with H/r 3oR incubated without arginine. Thus we can conclude that the response is one general with amino acid starvation and not a special response to arginine starvation.

Dark repair o/the damage causing degradation At the dose of ultraviolet light given these bacteria (4oo ergs/mm 2) dark repair of capacity for DNA replication occurs and replication resumes after a lag period of some 4 ° min 7,s. This recovery involves repair-replication after excision of the pyrimidine dimers from the DNA n and also repair from some unknown non-photoreversible Biochim. Biophys. dcta, 179 (1969) l O 6 - 1 1 4

RIBOSOME DEGRADATION IN IRRADIATED BACTERIA

III

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Fig. 3- D e g r a d a t i o n of 3o-S a n d 5o-S r i b o s o m e s w i t h i n c u b a t i o n of a u l t r a v i o l e t light e x p o s e d c u l t u r e of H / r 3oR for i n c r e a s i n g t i m e periods. T h e c u l t u r e was g r o w n as described in Figs. I a n d 2. A f t e r filtration a n d u l t r a v i o l e t light e x p o s u r e t h e c u l t u r e w a s i n c u b a t e d in m i n i m a l m e d i u m lacki n g b o t h [nH]uridine a n d arginine for periods of t i m e ( O - a , o; O - - - O , 30; I - B , 6o; V]- - -[~, 90; A - & , i 2 o min) w h e n s a m p l e s were t a k e n a n d s u b j e c t e d to sucrose d e n s i t y g r a d i e n t analysis. I n this case t h e a b s o r b n c e at 260 n m (A260) of t h e v a r i o u s fractions is p r e s e n t e d in order to s h o w t h a t a l m o s t all t h e 5o-S r i b o s o m e s are s u b j e c t to b r e a k d o w n a f t e r this t r e a t m e n t a n d n o t a special h i g h l y labeled fraction. T h e r e s u l t s w h e n t h e r i b o s o m e s are m e a s u r e d b y [SH~uridine c o n t e n t are identical. Fig. 4. R e c o v e r y of E. coli H / r 3 o R f r o m u l t r a v i o l e t light d a m a g e responsible for r i b o s o m e deg r a d a t i o n w i t h i n c u b a t i o n w i t h o u t arginine. T h e b a c t e r i a were t r e a t e d as in Figs. 1- 3 e x c e p t t h a t i m m e d i a t e l y after u l t r a v i o l e t light e x p o s u r e L-arginine w a s a d d e d to t h e m i n i m a l g r o w t h m e d i u m , a n d after w a r m i n g t h e c u l t u r e to 37 °, i n c u b a t i o n was allowed for specific periods of t i m e ( X - x , o; A . . . & , IO; A - A , 2 0 ; N l . . . C ] , 3 ° , m - D , 4o a n d Q . . . O , 5 ° min) before a g a i n filtering, w a s h i n g r a p i d l y w i t h 37 ° m i n i m a l m e d i u m (without arginine) a n d r e s u s p e n d i n g in this m e d i u m at 37 °. I n c u b a t i o n w a s t h e n c o n t i n u e d for 12o m i n a n d s a m p l e s t a k e n for sucrose dens i t y g r a d i e n t analysis. T h e control culture ( O - O ) w a s n o t i n c u b a t e d a f t e r u l t r a v i o l e t light exposure; r a t h e r t h e s a m p l e for a n a l y s i s was t a k e n i m m e d i a t e l y . T h e slight decline in [nH]uridine c o n t e n t of t h e s a m p l e s t a k e n a f t e r 4 ° a n d 5o rain of i n c u b a t i o n w i t h arginine from t h a t of t h e 3 o - m i n s a m p l e p r o b a b l y r e p r e s e n t s a loss of label d u e to t h e increased i n c u b a t i o n period a n d n o t a reversal of recovery.

damage through processes involving RNA and protein synthesis s. Similarly, the damage resulting in ribosome degradation in the absence of arginine was found to be subject to dark repair, if the culture was allowed to incubate for a period of time with arginine before amino acid starvation (Fig. 4). When 30 rain of incubation with the amino acid was allowed before the starvation period, little degradation of 3o-S and 5o-S ribosomes was seen during the subsequent incubation.

Photoreversibility o~ the damage causing degradation As discussed above, both induction of the photoreversible pyrimidine dimer and some non-photoreversible damage is involved in the blocking of DNA replication by Biochim. J~iophys. ~4cta, 179 (1969) lO6-114

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this ultraviolet light dose 8. It was possible to explore the role of dimers in the effect on RNA by studying the effect of photoreversal treatment upon the response. With strain H/r 3oR a slight reduction in amount of 5o-S ribosome degradation was produced by previous light treatment (Table II). This could reflect the photoenzymatic elimination of pyrimidine dimers in that part of the bacterial population which has TABLE II EFFECT OF POSTIRRADIATION EXPOSURE TO PHOTOREVERSlNG LIGHT ON SUBSEQUENT DEGRADATION OF 5o-S RIBOSOMES OF E. coli STRAIN H / r 3 ° AND H / r 3oR. The b a c t e r i a were t r e a t e d as in Figs. 1- 3 e x c e p t t h a t one s u b c u l t u r e (UV + P R ) w a s e x p o s e d a f t e r i r r a d i a t i o n to i n t e n s e l i g h t for 4 ° m i n a t 8 ° while t h e o t h e r t w o s u b c u l t u r e s w e re h e l d a t 8 ° i n t h e d a r k d u r i n g t h i s period. A f t e r t h i s t r e a t m e n t one s u b c u l t u r e ( U V + A ) h a d a r g i n i n e a d d e d w h i l e two (UV, U V + P R ) d i d not. The c u l t u r e s were w a r m e d t o 37% i n c u b a t e d for 12o m i n arid t h e n s a m p l e s were s u b j e c t to sucrose d e n s i t y g r a d i e n t a n a l y s i s . The v a l u e s r e p r e s e n t t h e p e r c e n t of t h e t o t a l ~SH]uridine c o n t e n t of t h e 5o-S p e a k a t t h e t i m e of i r r a d i a t i o n l e ft a f t e r i 2 o rain.

Treatment

UV UV+PR UV+A

Relative counts/min (%) H/r 3 °

H/r 3oR

I2 II 79

11 24 83

not suffered non-photoreversible damage and therefore recovery of their capacity to make DNA. The larger part of the ribosomes degraded however even after photoreversal treatment. Thus we can suspect that the non-photoreversible damage blocking DNA replication ~ also results in ribosomal degradation. That no decrease in ribosomal degradation whatsoever is produced by light treatment of strain H/r 30 (which lacks the photoenzymatic capacity for splitting pyrimidine dimers) supports the contention that the decrease in ribosomal degradation produced by light in H/r 3oR, the revertant photoreversible strain, is due to photoenzymatic splitting of pyrimidine dimers and not some indirect effect of the light. DISCUSSION

BEN-HAMIDA AND SCHLESSINGER 12 have studied breakdown and synthesis of ribonucleic acid in E. coli starving for nitrogen and have presented evidence which suggested to them that the activation of nucleases responsible for the breakdown may be a direct consequence of the breakdown of polyribosomes when starvation begins. Degradation was found to increase as much as 5 fold when RNA synthesis is totally shut off suggesting that nucleases may be activated when RNA synthesis stops. Even so the rate of breakdown (5 % per h) observed under these conditions is far less than that demonstrated here with ultraviolet light damaged bacteria incubated without a required amino acid or previously shown with bacteria starved for required thymine and amino acids 3. While amino acid starvation would be expected to result in disruption of polyribosomes 13 and the blocking of RNA synthesis 14in the RNA stringent strains used in these studies resulting in the release of nucleases 12, the very rapid degradation of ribosomes observed cannot be accounted for on this basis alone. The Biochim. Biophys. dcta, i 7 9 (I969) I O 6 - I I 4

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results support the hypothesis that inhibition of DNA replication causes some cellular event to occur after 6o min of incubation under conditions of blocked RNA and protein formation which results in the rapid degradation cf ribosomes. This could result presumably either through the release cf additional nucleases or by the sensitization of the ribosomes to the nucleases already active in the cell. For the causative event to occur, it is clear that inhibition of active DNA synthesis (i.e., DNA synthesis in progress at the time of application of the inhibitory treatment) is necessary; thus when the bacteria were allowed to come to the end of their DNA replication cycle by incubation for I h without the required amino acid but with thymine in the medium, subsequent incubation without both a required amino acid and thymine did not lead to ribosomal degradation s . This is presumably because protein synthesis is required for the initiation of the new cycle of DNA replication 15 and this cannot occur because of the absence of the required amino acid in the subsequent incubation. The results make it evident that two types of photochemical lesions, both presumably blocking DNA replication, can produce degradation of ribosomes. With those bacteria presumably not suffering the non-photoreversible damage, the ribosomes do not degrade after photoreversing treatment with a strain of bacteria capable of photoreversal response but do degrade after treatment of a strain lacking the capacity for photoenzymatic splitting of pyrimidine dimers. Thus pyrimidine dimers produce this effect. Most of the damage producing the effect with the ultraviolet light dose given (400 ergs/mm z) however is not photoreversible. It has been shown previously that this ultraviolet light dose produces some non-photoreversible damage to most of the bacteria which blocks DNA replication (but has little effect on RNA and protein formation) 8. A postirradiation lag period of at least 30 rain is necessary for recovery of the capacity for DNA replication during which RNA and protein formation must occur 8. Similarly, if a recovery period of 30 rain is allowed with the required amino acid present, the subsequent breakdown of ribosomes with incubation without the amino acid is not observed. The mechanism responsible for the increased breakdown of the ribosomes under these conditions is not understood. A range of possibilities are conceivable to account for this phenomenon. At one extreme could lie extensive cellular disorganization which in a relatively nonspecific way either effects the release or activation of nucleases or alternatively sensitizes the ribosomes to nucleases already present and active. At the other extreme, the release or activation of nucleases or the sensitization of the ribosomes to their action could be a direct and specific effect of disruption of the DNA and RNA biosynthetic systems 3. This might cause an "unstabilization" of the ribosomes, making them subject to enzymatic attack 3. An intermediate possibility could involve a more or less complicated series of physiological interactions under these deleterious conditions which ultimately lead in a specific manner to the excessive release of nucleases or to the sensitization of the ribosomes to nuclease action.

ACKNOWLEDGEMENTS

The work was supported in part by A.E.C. contract AT (3o-1)3893. Some preliminary aspects of the work were executed in the Section of Genetics at the University of Texas, M.D. Anderson Hospital and Tumor Institute, Houston. The author wishes Biochim. Biophys. Acta, 179 (1969) lO6-114

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to thank Miss MARTHA J. FERGUSON and Miss BILLIE F. WHITE there and Miss CAROL M. THORNTON at his present institution for valuable technical assistance.

REFERENCES I 2 3 4 5 6 7 8 9 io ii 12 13 14 15

F. C. NEIDHARDT, Prog. Nucleic Acid Res. Mol. Biol., 3 (1964) 145. J. 1V[ANDELSTAM, Bacteriol. Rev., 24 (196o) 289. C. O. DOUDNEY, ][3. F. WHITE AND M. J. FERGUSON, Currents Mod. Biol., i (i967) 143. E. !V~.WITKIN, N. A. SICURELLA AND (3".M. BENNETT, Proc. Natl. Acad. Sci. U.S., 51 (1963) lO55. E. M. WITKIN, Mutation Res., I (1964) 22. C. O. DOUDNE¥, in F. SOBELS, Repair /rom Genetic Radiation Damage, Pergamon, Oxford, 1963 , p. 125. C. O. DOUDNEY AND C. S. YOUNG, Genetics, 47 (1962) 1125. C. O. DOUDNEY, Mutation Res., 3 (1966) 280. R. J. BRITTEN AND R. ][3. ROBERTS, Science, 131 (196o) 32. C. O. DOUDNEY, Biochem. Biophys Res. Commun., 4 (1961) 218. I{. ][3. SETLOW, P. SWENSON AND W. CARRIER, Science, 142 (1963) 1464. F. BEN-I-IAMIDA AND D. SCHLESSINGER, Biochim. Biophys. Acta, 119 (1966) 183. D. W. MORRIS AND J. A. DEMOSS, Proc. Natl. Acad. Sci. U.S., 56 (1966) 262. G. S. STENT AND S. BRBNNER, Proc. Natl. Acad. Sci. U.S., 47 (1961) 2005. P. HANAVCALT, O. MAALOE, I). CUMMINGS AND iV[. SCHAECTER, J. Mol. Biol., 3 (1961) 156.

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