nuclease activity associated with the ustilago maydis virus induced

volume 6 Number 12 1979 Nucleic Ac ids Research

Nuclease activity associated with the Ustilago maydis virus induced killer proteins

Ronit Levine, Y.Koltin and Judith Kandel

Department of Microbiology, Faculty of Ufe Sciences, Tel Aviv University, Ramat Aviv, Israel

Received 20 July 1979

ABSTRACT

An ̂ n vitro nuclease activity was found to be associated with the puri-fied killer proteins of ustilaRQ maydis. The proteins are effective againstsingle stranded ENA, single and double stranded UNA. Endonucleolytic activitywas confirmed by cleavage of circular molecules of 0x174 and PM2. Doublestranded ENA did not appear to serve as a substrate.

INTRODUCTION

During the last decade the existence of viruses in fungi was confirmed

by the isolation and characterization of these particles.1!2 In only a few

species, the presence of viruses is expressed by the excretion of a substance

toxic to sensitive cells of the same species and closely related species.3'1*

The toxic substance was shown to be a protein.1* >5f6 >7 In UstilaRQ maydis

the excretion of the killer toxin Is always associated with the presence of

virus particles with a segmented genome of dsENA.® Three different killer

proteins (KP1, KP4 and KP6) have been identified thus far and each killer

type is associated with a different but related virus. Cells carrying a

specific virus type are sensitive to the two other killer toxins. Each

killer strain is immune to the killer toxin i t produces.

A unique pattern of dsHNA is typical to each of the 3 viruses associa-

ted with the 3 killer specifici t ies.9 '1 0 The synthesis of the toxins is11,12,13

directly coded for or Induced by specific segments of the viral genome.

The 3 toxins are proteins with a molecular weight of ca. 10000. The 3 pro-

teins can be distinguished, however, by various criteria such as thermola-

bil i ty, pH dependence, killing kinetics and migration in polyacrylamide gels.

Furthermore, resistance to the 3 toxins is conferred by 3 independent reces-

sive genes.10!11* The mode of action and the site of action of the toxins is

unknown. Exposure of sensitive cells to the toxins leads to cell death and

the prolongation of the generation time among survivors.

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Nucleic Acids Research

Recent genetic findings suggest a role for the killer protein that may

link a distinct biological function in the etiology of the virus with the

killer effect. The assumptions are based on attempts to combine killer

specificities by the formation of viral hybrids.

Some of the dsKNA segments of the viral genome are unique to each of

the 3 viruses. Hybrids can be recognired by the reaasortment of the mole-

cules and by the combination of killer specificities as determined in a bio-

assay on lavns of sensitive cells. However, all the attempts to combine

specificities in crosses between different killer strains failed.10 The out-

come of such crosses indicated specific exclusion relations between the dif-

ferent viruses. The introduction of 2 related but different viruses into a

single cell led to the exclusion of one or both viruses. The relations be-

tween the 3 viruses were defined and are constant. The phenomenon involves

the removal of at least some segments of the dsRKA by degradation or by the

inhibition of its replication. The exclusion relations are an inherent pro-

perty of the viruses and the host has no effect on the outcome. In crosses

in which one of the parents is a mutant lacking the dsRNA segment associated

with the killer expression, the restrictions on hybrid formation are removed.

New hybrids were formed by this procedure containing the dsRNA segments from

both parents.13 Thus, the genetic data linking the exclusion phenomenon with

the killer activity suggest that the killer protein acts in infected cells

as a nudease preventing the superinfection of cells. The killing function

exerted by the extracellular protein may result from its ability to penetrate

specific cells.

The reported study examines the association of nuclease activity with

the purified killer toxin and determines its substrate specificity in vitro.

MATERIALS AMD METHODS

Strains - The strains of £. maydls used in the study were No. 77 (con-

taining P4 virus) , No. 75 (containing P6 virus) and strain No. 18 sensitive

to both killer proteins of type 4 (KP4) and of type 6 (KP6) . All the strains

are currently in the collection at Tel Aviv University and were obtained from

Dr. P.R. Day, Genetics Department at the Connecticut Agricultural Experiment

Station, New Haven, Conn.

Purification of the toxin - The toxin was recovered from the supernat-

ant of liquid cultures of the killer strains. The cells were grown for 48-

7 2 hours in a rotary shaker incubator at 25 C in Ustllafto complete medium.

The volume of the supernatant was decreased by lyophylization to 1/20 its

3718



original volume and the proteins were precipitated with 3 volumes of cold

acetone (-20 C). The acetone was evaporated and the precipitate was

disolved in 1/100 the original volume in 5 mM Tris-HCl (pH 7.1). The sample

was applied to a column of Cellex-D (Bio-Rad) (4 x the sample volume) prev-

iously equilibrated with the same buffer. Toxin activity was eluted with the

buffer wash, whereas, most of the polysaccharides remained on the column.

The killer activity was followed by a diffusion zone method in which the

sensitive strain served as an indicator for the activity. The indicator

strain was plated over a base layer of agar. Sterile absorbant discs (Sch-

leicher and Schull, No. 2668) were placed on the indicator lawn and 15 yl

from each fraction were applied. The lawns were incubated at 25 C for 24 hrs

and activity was identified as an inhibition zone surrounding the discs. The

fractions containing activity were pooled, concentrated by lyophilization and

dialysed for 24 hrs in the cold against 5 mM Tris-HCl buffer (pH 7.1).

Further purification was obtained by gel filtration on Bio-Gel P30

(80 x 3.0 cm) and P60 (75 x 2.0 cm) followed by filtration through P10

(27 x 1.0 cm) that has an exclusion limit of 12000 MW. The degree of purity

was examined by gel electrophoresis in 12.5Z polyacrylamide disc gels con-

taining SDS according to Weber and Osborn.-15 The position of the toxin in

the gels was identified by the elution of the activity from 2 mm slices of

the gel and the protein was examined in a replicate gel that was stained

with 0.21 Coomassie Blue. After P10 the killer proteins were apparently

homogenous (Fig. 1). In addition, the purity of the proteins was confirmed

by isoelectric focusing (IEF) in polyacrylamide gels according to Wrig-

ley16 using amphollnes with a pH range from 10.0 to 3.0. A single band was

discernible after staining with Coomassie Blue. The pH gradient was deter-

mined in 1.5 mm slices from duplicate unstained gels and the range attained

was from 9.0 to 2.5. Killer activity was eluted from the sliced sections

corresponding to the protein band. Some of the reactions described below

were repeated with the proteins eluted from the IEF gels.

Cell-free protein synthesizing system - The effect of the killer pro-

teins on iji vitro translation was determined with the reticulocyte system

described by Pelham and Jacobson17 using as exogenous mRNA either globin

mRNA or parotid mRNA (provided by Dr. M. Gorecki, Weizmann Institute of

Science). The incorporation of methlonine 35S into proteins was followed.

Killer protein concentrations were determined by the method of Lowry, Rose-

brough, Farr and Randall.18

Nuclease activity - (a) Polysome profile: The effect of the killer pro-

3719



Fig. 1 SDS polyacrylamlde gels ofKP6. Samples of toxin followingchromatography on P30 (left) and P10(right) run in 12.5Z gels.

iteins on the polysomes in a reticulocyte cell-free protein synthesizing

system containing endogenous globin mRNA was determined according to

Wreschner, Melloul and Herzberg.^9 The reticulocyte system was incubated

for 7.5 mln at 37 C with the killer proteins. The polysomes profile was

determined in 15-401 exponential sucrose gradients prepared in 10 mM Tris-

HC1 (pH 7.4) containing 75 mM KC1, 5 mM MgCl . After centrifugation (in

SW56 rotor at 48000 rpm for 60 tain) , the gradients were scanned at 260 run

with an ISCO continuous flow spectrophotometer.

(b) Ribosomal RNA: Reticulocyte ribosomal RNA was extracted with

phenol as described by Wreschner, Melloul and Herzberg.19 Incubation

(20 Pg rRNA) with killer proteins was performed in 10 mM Tris-HCl (pH 7.4)

containing 50 mM KC1 and 4 mM MgCl2 for 30 mln at 37 C. After incubation

the profile of the rRNA was determined in 2.5Z polyacrylamide gels contain-

ing SDS by scanning the gels at 260 ran.

(c) Ribohomopolymers: Specificities of the killer proteins were tested

with homopolymers of RNA and compared with RNaseA and Tl. The method used

was described by Zimmerman and Sandeen.20 All the polymers were purchased

from Sigma (St. Louis, Mo.). Since the degradation of the polymers is de-

termined by the amount of acid soluble material, the homopolymers were init-

ially dialysed for 18 hours against 200 volumes of 20 mM Tris-HCl buffer

(pH 7.5) containing 1M NaCl and 1 mM EDTA. The dialysis was continued for an

additional 30 hours with 5 mM Tris-HCl (pH 7.5). All dialyses were performed

3720



In the cold. After dialysis the samples were maintained at -15 C. The re-

action was performed with 0.16 uM of each polymer in 10 uM Tris-HCl buffer

(pH 7.4) with an incubation period of 20 min at 37 C. The reaction was ter-

minated with cold perchloric acid (2.4Z v/v) and bovine serum albumin (1 mg/

ml) was added as a carrier. After 5 min on ice, the samples were centrifuged

for 10 min at llOOOg and the optical density in the supernatant was deter-

mined at 260 nm.

(d) Double stranded RNA: Substrates for the killer proteins included

phenol extracted dsRNA from the Ustllago viruses (PI, P4, P 6 ) 1 0 and from

reovirus (type III) provided by Dr. W. Joklik, Duke University. Incubation

of dsRNA with the proteins was performed in 40 mM Tris-Acetate (pH 7.4) in

the presence of 1 mM EDTA. The effect of the killer proteins was determined

in 5Z polyacrylamide gel electrophoresis as described by Shatkin, Sipe and

Loh.21 A synthetic dsRNA of poly I-poly C (P.R. Biochem) was also used.

The method for the detection of the activity of the killer proteins on this

substrate was according to Zimmerman and Sandeen.2" The synthetic dsRNA

was pretreated with RNaseA (10 ug/ml) and Tl (40yg/ml) to remove single

stranded stretches.

(e) ssDNA and dsDNA: Bacterlophage (Jxl74 DNA (single stranded) that

was extracted according to Razin, Sedat and Sinsheimer22 was provided by Dr.

A. Livne, Heizmann Institute of Science. Bacteriophage PM2 DNA (double

stranded) was extracted according to Espejo, Canelo and Sinsheimer.23 Since

both phages DNAs are circular, endonucleolytic activity was examined by the

conversion of circular molecules to linear forms. Nuclease activity was de-

termined at 37 C In 10 mM Tris-HCl buffer (pH 7.4) containing 50 mM KC1 and

4 mM MgCl using 0.4 pg 0x174 or 2 ug PM2 DNA. The reaction was stopped by

the addition of 20 mM EDTA. The effect on the DNA was followed by the mi-

gration of the molecules in 1.2Z agarose slab gel electrophoresis (90 mM

Tris-borate buffer, pH 8.3, with 2 mM EDTA). The gels were stained with

ethidium bromide (1 vg/ml).21* Control experiments to identify the position

of the linear molecule of PM2 were performed with Hpall (Bio Labs, Mass.)

that has one restriction site on the PM2 DNA.25

Phosphodiesterase activity - Non specific phosphodiesterase activity was

determined according to Laakowski26 using as a substrate Ca-bis-nitrophenyl

phosphate and p-nitrophenyl-thymidine 5' phosphate. The final substrate

concentration was 1 mM in 10 mM Tris-HCl (pH 7.4). The reaction mixture was

incubated at 37 C and the optical density change, determined by the release

of p-nitrophenol, was measured at 400 nm in a Gilford Spectrophotometer.

3721



The reaction was tested and performed simultaneously with snake venom pro-

vided by Dr. A. Bdullah, Tel Aviv University.

RESULTS

Inhibition of in vitro translation - The killer proteins KPA and KP6

were tested for their effect on in vitro translation using either exogenous

globin or parotid mRNA in a reticulocyte system. The protein samples in-

cluded KP4 eluted following IEF gel electrophoresia. The results (Table 1)

show that both KP4 and KP6 effectively inhibit in vitro translation in the

reticulocyte system with either mRNA. The degree of inhibition after 1 hr

incubation Increased with toxin concentration and reached a level greater

than 90X. It is unclear, however, from this experiment which of the com-

ponents of the translation system are affected. Therefore, the effect of

the killer proteins on the polysomes was tested.

Polysomes derived from a reticulocyte system containing endogenous

globin mRNA were incubated with concentrations of killer proteins that in-

hibit 50Z of the translation in the system. Alterations were observed In

the polysome profile in sucrose gradients (Fig. 2). The majority of the

polysomes were converted to monosomes. Thus, the killer proteins may in-

Table 1 Inhibition of in vitro translation by the Ustilago killer proteins

Killer Proteins yg

0.0

4 2.8

5.6

7.0

8.0

16.0

20.0

4 I*

II

6 2.8

5.6

7.0

Globin35S met

67083

52247

28174

-

14986

2613

-

46194

7781

9594

4024

2692

mRNA

X Inhibition

0

22

58

-

78

96

-

31

88

86

94

96

Parotid35S met Z

18343

8203

-

6553

3607

-

1765

14150

3078

3727

-

1711

mRNA

Inhibition

0

55

-

64

80

-

90

23

83

55

-

91

* Killer activity detected from the isoelectric focusing gel corresponding

to the single band. I and II refer to two consecutive slices from the gel.

3722



TOP

Fig. 2 Sucrose density gradient centrifugation of polysomes.From left to right: Arrows indicate the position of tetrasomes, trisomes,disomes and monosomes. A. Incubated for 7.5 min., no additions;B. Incubated for 7.5 min with KP4; C. Incubated for 7.5 min with KP6.

hibit translation through a dissociation of ribosomes and mRNA or from the

endonucleolytic degradation of mRNA. If the latter were correct one would

anticipate that the killer proteins would possess the ability to degrade

ssRNA. Ribosomal RKA, as a single stranded RNA substrate, was incubated

with the killer proteins for 30 min at 37 C. The concentration of the pro-

teins used was sufficient to inhibit in vitro translation by 30Z. The ribo-

somal RNA profile detected in acrylamide gels after the incubation clearly

indicated the degradation of the 28S and 18S rRHA (Fig. 3). The resulting

products do not include any discrete fragments and appear to be a result of

multiple cuts. Therefore, although other alternatives for the inhibition

of protein synthesis in the ̂ n vitro system cannot be ruled out, it is clear

that the killer proteins possess a nuclease activity capable of degrading

ssRNA and probably sufficient for the dissociation of polysomes to monosomes.

The substrate specificity of the ribonuclease activity was tested with

various ribohomopolymers and compared with known ribonucleases. The killer

proteins are quite distinct from RNaseA and Tl (Fig. 4) and differ from

each other. The affinity for a specific nucleotide in the killer protein is

not clear as with RNaseA and Tl. KP4 displays the highest affinity for

poly(I) followed by poly(C). Homopolymers of poly(G), poly(A) and poly(U)

are degraded to some extent. KP6 does not display such clear preference for

any of the 5 homopolymers. The affinity of KP4 for poly (I) is similar to

RNase Tl, but the latter displays almost no activity on poly (C), poly (A)

3723



- M I G R A T I O N *

Fig. 3. Digestion of rRNA by killer proteins. Ribosomal RNA was incubatedin the presence or absence (control) of KP4 or KP6 for 30 min. The entirereaction mixture was electrophoresed in 2.5Z SDS polyacrylamide gels.Gels were scanned at 260 tun.

Fift. 4 Digestion of ribohomopolymersby killer proteins and known RNases.

ILc c « u I

3724



and poly (U) whereas KP4 degrades well poly (C).

The activity on the homopolymers displayed by the killer proteins was

obtained with protein concentrations sufficient to inhibit 90Z of the in

vitro translation. The concentration was 4 times greater than needed to

obtain a maximal zone of inhibition on lawns of sensitive cells. The level

of activity on the homopolymers suggests that these molecules are not ideal

substrates for the killer proteins. Thus, the results indicate that the

proteins can act on the homopolymers, but optimal activity may require

unique nucleotide sequences rather than a repeating nucleotide sequence.

An indication that the proteins do not merely act as nonspecific phos-

phodiesterases was obtained in tests using Ca-bis-p-nitrophenylphosphate

and p-nitrophenyl-thymidine 5' phosphate. The use of the latter substrate27

could specifically identify cleavage at the 5' position'and could indicate

if a single pyrimidine is sufficient for the enzymatic cleavage. Tests were

run with killer proteins at various stages of purity and no phosphodiester-

ase activity was detected on either substrate using protein concentrations

sufficient to inhibit 90Z of the jji vitro translation and after prolonged

incubations over 24 hours. The lack of activity on p-nitrophenyl-thymidine

5' phosphate suggests that the killer proteins require a polynucleotlde of

a minimal sire or a specific sequence upon which to act.

To test if the killer activity displayed by the purified killer pro-

teins is a general characteristic of ribonucleases of low molecular weight

which may enable such molecules to permeate cell membranes, ribonucleases

were applied on lawns of sensitive cells in a manner similar to the test of

killer proteins.6 Any killing effect should have been discernible by a

clear zone of inhibition on the sensitive lawns, but none were detected.

The ribonucleases tested were RNaseA (200 ug), RNaseB (12.5 yg), RNaseTl

(0.87 vig) , and EHaseN (0.6 ug) . Approximately 5 ug of the killer proteins

produce a clearly defined maximal zone of inhibition.

The effect on dsRNA - Genetic data indicated a relation between the

killer proteins and the exclusion phenomenon, possibly through the degrada-

tion of specific dsRNA molecules. If the killer proteins act as dsRNA nu-

cleases certain predictions can be drawn based on the specific interactions

defined in genetic crosses. For example, crosses of P6 and PI result in

progeny containing only a partial virus genome. Therefore, incubation of

PI dsRHA with the KP6 should result in the degradation of those segments

of dsRNA specific for PI.

DsRNA from each virus PI, P4 and P6 was incubated with the proteins

3725



KP4 or KP6 for 30 min at 25 C in 40 mM Tris-Acetate at pH 7.4. DsRNA from

reo virus (Type 3) was also incubated with both KP4 and KP6. Following

the incubation, the samples were loaded on 5Z polyacrylamide gels and

electrophoresed according to the standard conditions for the dsRKA of

Ustilago.u The results with reo dsRNA and PI dsKNA incubated with KP4 and

KP6 are shown in Fig. 5. Although some dsRNA molecules are below the level

of resolution, most bands in the treated material can be traced to corres-

ponding diffuse bands that migrate at a slower rate than normal. The de-

crease in the rate of migration of the dsRNA molecules, especially notice-

able with the higher molecular weight species of reo virus, may suggest

some interaction between the proteins and the dsRNA molecules. However,

repeated deproteinizations with phenol and SDS did not restore the normal

pattern of migration of the dsRNA after incubation of the dsRNA with the

KPs. Additional deproteinization with proetase K, prior to the extraction

with phenol and SDS, also did not affect the pattern of migration of the

treated dsRNA and the migration was retarded when compared with the migra-

tion of untreated dsRNA from the same virus.

The effect of the killer proteins on dsRNA does not seem to involve

nicking or digestion. Tests with Poly I • Poly C, similar to those con-

m

...4A ' M<1

Fig. 5. Polyacrylamide gel electrophoresis of dsRNA following incubationwith killer proteins. A. Reovirus dsRNA (left to right) untreated, 40 ugKP4, 80 ug KP4. B. PI dsRNA (left to right) untreated, 52 ug KP4, 78 ugKP4, 40 ug KP6, 80 pg KP6, 120 tig KP6. All gels were 5Z polyacrylamide.

3726



ducted with the ribohomopolymers, did not reveal acid soluble material and

the sensitivity of Poly I • Poly C was not appreciably increased by joint in-

cubation of both the killer proteins and RNaseA or Tl.

The results suggest some conformational change in the dsRHA by the

killer proteins, similar to the effect of UV irradiation on reo virus

dsRNA.28 The interactions between KP4 and KP6 with the dsRNA of all 3

viruses does not correspond to the exclusion relations defined In genetic

crosses. The results, however, do not exclude the involvement of the killer

proteins in the exclusion mechanism by means other than degradation of

dsRNA.

The effect on DNA - The results of incubation of the killer proteins

with DNA substrates are shown in Figures 6 and 7.

The digestion of 0x174 ssDNA as a function of time is shown in Fig. 6.

The untreated DNA (position 1) contains the circular molecules (upper band)

and linear molecules (lower band). The conversion of the circular molecule

to the linear form increased with time of incubation (position 2-5) and the

linear form was digested to nondiscrete products (position 5). Similar

results were obtained following incubations with increasing protein con-

centrations for a single time period.

The effect of incubation of the killer proteins KP4 or KP6 with dsDNA

is shown in Fig. 7. The untreated molecules of PM2 (position 4) display

1 3 4 gig 6. Agarose slab gel of 0x174ssEHA incubated with KP4. 1. Untreated,the major band corresponds to the cir-cular form. 2-4. Incubation with 20 ygKP4 for 15, 30 and 45 min in the absenceof Mg and KC1. 5. Incubation with 20 ugKP4 for 30 min in the presence of 4 mMMgCl, and 50 mM KC1. The same resultswere obtained with KP6.

3727



Fig. 7. Agarose slab gel ofPM2 dsDNA following Incubationwith killer proteins. Position1, 2, 3 Incubation with KP6 for45, 30 and 15 min. Position 5,6,7, incubation with KP4 for 45, 30and 15 min. Incubations were at37 C.

2 bands, the upper band corresponding to the relaxed form of the super-

coiled circular molecule that is seen in the lower band. The lowest faint

band in the untreated DNA corresponds to the linear form of the PM2 DNA.21*

Confirmation of the position of the linear form was obtained in control ex-

periments with the restriction enzyme Hpall. Incubation of the PM2 DNA with

KP4 (position 5-7) and KP6 (position 1-3) resulted in an increased intensity

of the band corresponding to the linear form of the molecule and a decreased

intensity of the bands corresponding to the relaxed form and the super-

coiled molecules. If the reaction is performed as a function of time, the

sequence of events leading to the degradation of PM2 DNA are suggested.

To the left of the untreated material the results of incubation of PM2 DNA

with a constant concentration of KP6 for periods of 15, 30 and 45 min (from

right to left) is shown. Likewise, to the right of the untreated material,

the results of incubation of PM2 with one concentration of KP4 for varying

time periods is shown. In both cases the sequence that can be suggested

is the initial conversion of the supercoiled molecule to a relaxed form

followed by the conversion of the relaxed form to a linear molecule. As

shown in Fig. 7, linear form of the molecules accumulate within the dura-

tion of these experiments. Identical results were obtained with KP's

eluted from IEF gels. Also, the gradual conversion of DK\ of SV40 form I

to form II and finally to form III, was detected after incubation with KP4

and KP6 for varying durations.

DISCUSSIOM

The search for nuclease activity associated with the purified killer

3728



proteins was stimulated by the genetic studies that suggested the involve-

ment of these proteins in the exclusion of dsBNA molecules. The results

of in vitro experiments indicate that the proteins act as nucleases. How-

ever, results of experiments with EHA and DNA substrates indicate no

activity with dsBNA and suggest no preference for double stranded DNA mole-

cules. The effective degradation of rRNA and of 0x174 DMA is clearly a nu-

cleolytic activity on single stranded molecules. The nuclease activity on

PK2 dsDNA does not necessarily imply the cleavage of dsDNA since the super-

coiled molecules contain single stranded regions. »30 PM2 supercoiled

molecules can be cleaved by enzymes specific for single stranded DMA.

The nature of the cuts introduced by the killer proteins is currently under

investigation, but the course of events suggested by results obtained with

the 0x174 DNA and the relaxed form of PM2 is already suggestive of activity

on single strands by endonucieolytic cuts.

In addition, killer proteins possess a unique property of cell recog-

nition. Nucleases of a similar sire, tested in the reported study, did

not affect the cells. The small killer proteins may be internally differ-

entiated into a catalytic region and a recognition region in a structure

similar to that proposed for some of the bacterial colicins, some of which

also act as nucleases.32 Mutants of a killer protein of Ustilago dis-

playing such differentiation have been isolated.12

The inability to demonstrate the activity of the killer proteins in the

exclusion of dsRNA molecules does not conclusively rule out their activity

intracellularly as dsRNA nucleases. Earlier studies with deletion mutants

of the virus P6 led to the conclusion that a part of the viral genome is

essential for the processing of the killer protein.6 Such a processing

during excretion may alter substrate specificity. A simple modification by

crosslinking of a single strand specific nuclease such as EHaseA was

recently demonstrated by Wang and Moore.33 The modoficatlon led to an

increased affinity for dsKNA molecules. Similarly, it can be viewed that

an enzyme specific for dsRNA may be modified as it is excreted. Experi-

mental modifications of the killer proteins and ln-vitro translation to

obtain the preprocessed protein may serve to test the validity of these

assumptions.

These results do not indicate the site of action of the killer pro-

teins. A study of their effect on nucleic acid metabolism and protein

synthesis in vivo is a basic corollary to the reported results.

3729



ACKNOWLEDGMENTS

The a u t h o r s w i s h t o thank D. M e l l o u l and D r s . M. H e r z b e r g and M.

Gorecki for their assistance in material and advice. The study was

supported in part by the Branch for Basic Research, Israel Academy of

Sciences.

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SDS, sodium dodecylsulphate; ssDNA, s i n g l e stranded EN A; dsDNA,double stranded DMA; IEF, i s o e l e c t r i c focus ing; BNase, r ibonuclease;rRNA, ribosomal RNA; mRNA, messenger RNA; met, methiooine.

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