Mutation of the killer plasmagene in Paramecium aurelia

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Mutation of the killer plasmagene in Paramecium aurelia

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Mutation of the Killer HLasmagene In Paramecium aurelia

by

Euih f, Dippell

/if* /W

SuSsaitted to the Faculty of the Graduate School in partial fulfillment of the requirement® for the degree Doctor of Philosophy in the Department of Zoology Indiana University 1949

ProQuest Number: 10295204

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is d e p e n d e n t u p o n th e quality o f th e c o p y subm itted. In th e unlikely e v e n t th a t th e au th o r did n o t s e n d a c o m p le te m anuscript a n d th e re a re missing p a g e s , th e s e will b e n o te d . Also, if m aterial h a d to b e re m o v e d , a n o te will in d icate th e deletion.

uest, ProQ uest 10295204 Published by ProQ uest LLC (2016). Copyright o f th e Dissertation is held by th e Author. All rights reserved. This work is p ro te c te d a g ain st unauthorized copying u n d er Title 17, United States C o d e Microform Edition © ProQ uest LLC. ProQ uest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346

Th« author wishes to acknowledge with sincere gratitude the invaluable sug~ gestions of Dr# T.M. Sonnebom given throw-out the cours© of this investi­ gation and to express appreciation for his advice* interest and unlimited patience during th© writing of this mnuscripb*

Th© author is also indebt­

ed to Hr. Pao Kuo Chao for technical aid received during the eytological part of this study and to Hiss Martha Dippell, Miss Joann© Whallon, Mr* David K&nnoy and Dr* Mary L* Austin for aid in th© final preparation of this rniiu* script*

TABLa m C0STW3

Pag® X* Introduction*.*......... •.••••*..••#••••*•........

1

XX. The mutant killer stocks********..... •••*#.*,••.....

3

A* Detection and maintenance.

««•••.••*••**••••••••

3

B* Origin of the mutant stocks.....*•*•••*•••••••••••*••

6

C* The tm general types of killing and resistance shown fey the mutant stocks...

*.....

&

D. The phenotype of the mtant stocks*»*..«•.•••••*••••• 10 XIX* The inheritance of the mutant killer characters**. A* Materials and methods*

....

17 17

B. the genic constitution of the mutant killers......

29

C. Discussion of the genetic analysis ....

65 70

17* The kappae of the mutant stocks....... A. MLdenee for their existence and mutation. ... 1* Genetic evidence*

.... .••*••••••••*••••*..•« 70

2* Gytological evidence*

...

B* Conclusions**... V* General dlscuasion

70

80 93

....

71. Summary...******.,*...... VXI. Bibliography*....... *........

98 106 110

QytoplMf&a control of the killer trait in certain stocks of Paramecium aurella has been demonstrated to depend upon the presence of self-siultipXying, visible bodies in the cell cytoplasm (Freer, 1946, 1946a3 3ormbomP 1947)* These bodies, referred to as ttkappa,t particles by Sonnebom, bear a striking resemblance to the nuclear genes In their ability to control a hereditary trait and in their power of self-duplication* However, one of the naosfe fundamental and important properties exhibited by nuclear genes, that of mutability, was until recently demonstrated for cytoplasmic determinants of heredity only in the case of the plastlds of green plants (Renner, 1934)*

In 1946, the writer published a preliminary

report of mutations of kappa*

(Sonnebom in 1946 and Freer in 1946

reported what they believed to be indications of a kappa mutation but the cultures in question m m never analysed*) The present paper presents in detail the genetic analysis demonstrating that mutation of kappa and not of the K gens pro­ vides the physical basis for five observed changes or mutations of the killer character. Four of these mutations arose in stock 51 and the fifth in stock 47, both stocks belonging to variety 4

#?hla investigation was supported by Indiana University and by Rockefeller Foundation grants-in-aid of research on Protozoan Genetics at Indiana University* The author was m A*A*U*W, Fellow during part of the period over which this investigation extended*

2 of P# corolla* A brief account 1® Included of the origins of tho mutant stocks, the diverse typo or typos of killing they smnifost and the nature of the kappas present in the mutants*

II. THE m A.

KHASt STOCKS

Sataction ana aadntananca.

1* The kUUaa jw U onof aoarco Btoctta 51 ami 47 tram

Sonnebom (1939. 19A3) has shewn that the paraiaecln produced by killer stocks causes sensitive animals to undergo a series of changes leading to death of the affected animals*

In

stock 51, the typical pre-leihal stages in this scries have been described and diagramed (Sonnebom, 1946) and are represented here by photomicrograph® (Plate 1). When killer and sensitive animals are grown at an average rate of one fission per day at 27° C* (see Freer, 1948* for method of controlling fission rate in test tube culture) and animals from these two cultures are mixed at the same temperature,, stage k (Plate 1) can he recognised about two hour® after exposure of sensitives to 51 paramecin* the re­ maining stages (Plate 1, SUE) follow in succession, B being reached approximately 12 hours after exposure and disintegration of the corpses occurring some time after 24 hours from initial exposure* However, the time at which the first morphological change® become apparent and the duration of the various stages are dependent upon a nustoer of factor©, e.g., temperature and nutritive conditions of both killer and sensitive animals. Sonnebom (1939, 1943) has further shown that different killer stocks may evoke specific and characteristically different responses from the same sensitive stock* Thus, the second killer

stock mentioned above, stock 47 of variety 4* produced a para* msoin causing sensitive animals to develop a large vacuole in the posterior part of the body (Plate 2)* After a period of several years during which the stock m s no longer employed for ©xperimental work, a teat for kHXing revealed that stock 47 had lost its killer character and had become sensitive to the paramecin produced by other killer stocks*

2* Mutant killer defined* Changes in the killer diameter of stocks 51 and 47 were regarded as mutations if (1) the variants differed from the parent stocks in type or strength of killing as Measured by the mssaher of sensitive animals killed in a mixture with a given number of k&lero, and (2) if such differences rmmined persaanenb* ly heritable throughout vegetative and sexual reproduction*

At­

tention was not directed toward discovery or investigation of mutations involving changes from killing to ssenaliivity*

3* Method of detection* Mutations of the killer character were discovered by mixing animals of a killer stock with those t r m a sensitive stock and observing that the response of the sensitive animals to the paramecin in ih& Medium did not follow the expected characteristic series of changes brought about by 51 or 47 paramecin*

Five dif­

ferent sensitive stock® wore employed initially in mixtures with killer© but the most sensitive stock, stock 31 of variety

was

5 selected as the standard to be used in subsequent tests, 4* Method of isolating ours mutant stocks, A series of single anln&l. isolations was made fro® any culture displaying a mutant type of killing when mixed with sensitives* One-half of the total number of animals isolated was grown at maximal rate, while the remaining half was grown at an average rate of one fission per day*

(The necessity for maintain­

ing the isolations at different rates of growth is pointed out cm p* 22)* When each single animal isolation had given rise, by re­ peated fission, to a culture containing approximately 1,000 animals, a sample of each ©lone was tested for its ability to kill and for its type of killing* 5* Maintenance of the mutant stocks* Several of the m t m t killer clones obtained by the method outlined in the preceding paragraph were transferred from depressions slides to test tubes and were carried as stock cultures, being given each day enough standard culture medium to permit an average growth rat© of one fission per day*

At approximately ten-

day intervals throughout the period in which these culture® were used for experimental purposes, new subcultures were started from established clones, either as single animal isolations in the manner ^S;

described in (4) above or m mas® subcultures in which on® 6© two thousand animal® of a given culture were transferred to & fresh, sterile test tub© for continued culture*

Sach new subculture

was routinely checked for the presence of the mutant killer character*

6

6* Designations of the mutant stocks, the following system of r^msneXaiurft has boon adopted according to which the designation of any klllor mutant stock consist® of three parts#

(1) the designation of the parent (non-

imitated) stock free# which the mutant was derived, (2) the letter %** identifying the culture as a saxtant type, and ($) a number designation to distinguish among the various mutant cultures*

Thus,

killer mutants derived from stock 51 are referred to as 51ml, 51®2, etc*

(In an earlier paper, Dippdl (1943) discussed four mutant

stock® v&tixout reference to specific designations*

According to

the above extern, these stocks can. now be classified as follows# the Rfirst mutant,1* 51m23 the "second mutant," Slnal* the "third mutant,w 51m3 and the "fourth, mutant," 51m4«) B*

Origin of the mutant stocks* Of the five mutation® analysed in this study, nowa was

obtained throng the deliberate use of any mutagenic agent* All. arose, Insofar as is known, by spontaneous mutation* 1* Mutant shocks derived frost stock 51* a*

Origin of 51ml and 51m2* IS#© stock bottle cul­

tures of killer stock 51, mating type 911, were the sources from Which 51ml and 51®2 were obtained*

Stock cultures serve as reserve

supplies and are maintained on boiled hay infusion inoculated with Aerobacter aeroaenes (Sonnebom, 1950)* Of the two stock cultures of the 51 killer referred to above, one was subcultured from the other in 1946, and the following year one of these culture® was

7 found to contain 51ml, the other, 51is2. Whether or not the entire culture in each ease m e of a mutant type was not determined, tut the possibility m e suggested by the fact that of sixty single animal isolations made from each of the two stock cultures (repre­ senting

approximately 2*5%to 5$ of the total number of animals In

each culture), all were mutant type killers• b.

Origin of 51mj3« A third mutation was discovered

in a mass subculture of the 51killer stock, mating type YIX, pos­ sessed by Dr* J»A* Harrison ofTemple University and Obtained originally from Dr* Soimsbortio e*

51&4* mentioned in an earlier paper (Dlppell,

194S) proved upon further investigation to be other than a true mutation and 1© not considered in the present paper* d*

Origin of 51mJ^ A single animal isolation from

a 1 1weak killer" culture gave rise to a mutant type clone desig­ nated 51m5* 'ML® ssutant proved to be essentially a non-killer but resistant to 51 paramecin*

It1® therefor® designated as the 51®5

or E ("resistant®) mutant*

The "weak

killer” clone originated, in

turn, from a single animal isolated from a teat tube culture of killer stock 51, mating type VII. 2* Origin of the mutant stock 47ml derived from stock 47* During that period in which stock 47 was a killer, a mass subculture was sent to Father J*A* Frisch of Canisius College* In 1946, a subculture of this stock was returned to Sonncbom’s laboratory and, when first tested, was found to possess a type of killing Identical with that of stock SL* Since Father Frisch did not possess stock 51, however, there was no possibility for con-

a fusion of this stock with the isutant. the fact that 47&L was de­ rived from stock 47 and not from 51 was further confirmed by breed­ ing analysis (p. *°). C.

the two general types of killing and resistance manifested by the mutant stocks. Among the five mutant killers only two general types of killing

have thus far been found. The first type appears to be identical with that produced by 51 paramecin and will be referred to through­ out this paper as 51 killing. The second type, discovered in 51ml* differs strikingly fro® 51 and partially resales the killing action of stock G (variety 2} described by Sonnebom (1939) and ©ore ex­ tensively by Freer (1946). This type of killing will be referred to as "spinner1* or "Sp" killing action.

Sensitive animals exposed to

51ml paramecin reverse their normal direction of rotation and sub­ sequently increase the speed of this rotation* resulting in violent spinning of the sensitives on their longitudinal axes with little or no forward movement.

Up to this stag®, the pro-lethal effects

aro Identical with those produced by stock G paramecin* but in later stages the two differ. Sensitives exposed to 51®1 (Sp) killing, un­ like those exposed to Q type of killing described by Freer (1946), frequently develop one or two large vacuoles within the body or from a huge blister extending along the afeoral body surface* into which the body contents subsequently empty.

Frequently the animals settle

to the bottom and become almost completely paralysed before death.

9 (See Plate 3 for photomicrographs of the most characteristic prelethal stages following the early wspinning” stage of sensitives*) This type of killing progresses to completion sore rapidly than that of 51 or G. The Sp killer further differs from the original 51 killer in conditions required for maintenance of the killer trait* Unlike 51 killers v&ioh can retain their killing capacity when grown at maximal rate, animals producing Sp killing action must be grown at a rate of less than thr®« fissions per day to maintain their killer character. Of the five mutants thus far analysed, one (51ml) is pure for the Sp type of killing and one (51m5) is pur® for the 51 type of killing, although this mutant kills very weakly and inconsistently* The remaining mutant stock®, 5&m2, $lm$ and 47ml are mixed with respect; to the two killer types? cultures derived from single animal® of these mutant stocks kill some sensitives in one way and other animal® of the same sensitive stock in a second way* It has previously hm n shown that killer animal® of any on® stock are resistant to their o m paramecin* This same relationship was demonstrated for the mutant killers among which two general types of resistance w@r© found to exist, correlated with the two types of killing described above* D.

The nhenotyp® of the mutant stocks* It ha® been shown by several investigators that a killer stock,

resistant to its own paramecin, can he sensitive to & different type of paramecin produced by another stock.

In addition therefore, to

10 the initial tests (p. 5} determining the capacity of the mutant stocks to Jdll sensitive stock 31 or to b© killed by killer stock

51* this relation of resistance or sensitivity of a particular mutant to paramecin* liberated by other killer stocks was investi­ gated in order to obtain a more complete picture of the phenotype of each mutant stock. The most direct and conclusive results were obtained from testa in which living animals of one stock wore exposed to the paramecin of a second stock in the absence of living animals of the second stock. This animal-free "paramecin brei1 1was obtained by drawing into a hypodemie syringe a concentrated culture of the animals whose killing action was to be studied and forcing b h m repeatedly through a 25 gauge hypodermic needle until direct microscopic observation proved that no intact paranoia were any longer present (donnebom,

1950), living animals to be tested for resistance or sensitivity to this killer brei were then added to a known volume of the brei. Standardisation of all stocks used in this and the second experi­ ment reported was a&eoagtftehedl by growing each stock in test tubes at m approximate rate of one fission per day for five days prior to testing. Only cultures in which more than 90$ of the animals were in vegetative condition (i.e., cultures in which less than 10$ of the animals were undergoing nuclear reorganization) were used. Mixtures of brei and sensitive animals were observed after 1, 3* 6, 12, 24 and 4® hours.

Results are summarised in Table I.

The brei

u type of teat yields simple and clear cut result* free from the possible complication of mutual killing effects when two kinds of killers are mixed together* In the second type of test* living animls of each mutant kil­ ler stock, the nonnmitated 51 killer stock and the standard sensi­ tive stock 31 were mixed in all possible combinations of two each* In conducting the tests, approximately equal numbers of animal®

(100 to 200) from each culture were mixed and observed after 2, 5, 3, 24 sad 43 hours*

Hesults are presented in fable II,

Although

difficulty was encountered in an attempt to relate a particular type of killing to o m or the other of the stool® present in mix­ tures containing living animals of the two killer stock®, the results obtained were in complete agreement with those of the brei tests*

546715

12

TABLE 1* The result® obtained when living animals of stock 51# the mutant killer stocks and sensitive stock 31 arc mixed with brei obtained from these msm cultures* (-) Indicates no killing, (51) indicates 51 type kil­ ling, (Sp) Indicates spinner type of killing and (-(51) ) refers to extremely weak 51 type killing* Each record represents a composite of the results of three separate tests conducted within a four-month period*

actant killer stocks giwa nia ' j a r f t B T

sensitive stock

13

TABUS II •

llpauXts obtained when living animals of stock 5t# #10 mutant killer stocks and sensitive stock 31 are mixed together in all possible oorabinatlons of two each* (of* Table X legend for es^lanation of term®.) Each record represents a composite result of six separate tests carried out within a six-month period*

living animals original ' killer stock ........51... 51 yiii 51ua 51m3 51m5 m 31

SiA Sp Sp am Sp 51

sensitive Mutant killer stocks stock 51ml 51m2 51=0. 5ta5.Jtftol _ .31. ... n 5lf3p Sp m am am 51 Sp 3p mm 51 Sp Sp

Sp 51 «Sp * 51,Sp

- Sp 51 Sp Sp 51 Sp Sp Sp «• 5l,Sp - 3p -(51) Sp « 51,Sp *(51) 51,3p| -

u From the above data, one can determine for each stock its typo of killing and its resistance or sensitivity to the p&ramocins produced fey other stocks» Those throe aspects of the killer character, when combined, comprise the killer phenotype of a stock and are susemrlaed in Table III for each of the mutant stocks as well m

for the original 51 stock*

The killer phenotype of’each mutant stock has remined con­ stant, with two exceptions, over the entire course of this investi­ gation* One of these exceptions, the 51m5 or B mutant, exhibited two slight but significant changes following several months of culture* it first a non-killer and resistant to 51 paramecin, it later occasionally displayed extremely weak killing action of the 51 type, affecting only o m ©r two sensitives in a test In which hundreds would have been affected by paramecin liberated from an equal number of stock 51 killers. Further, when grown at maximal, rate, It displayed slight sensitivity to 51 paramecin. Tests conducted six months after the above changes were observed indicated no further changes in killing action but slight increase la the number of animals sensitive to 51 paramecin*

The nature

of this change has not been investigated* The second exception, 47ml, initially produced only one type of killing which was Identical with that of stock 51#

It was a

somewhat stronger killer than 51, however, as evidenced by the fact that sensitive animals exposed to 4*ftal paramecin passed through

15

TABi& XII* The killer phenotype of the mutants arsd original 51 killer stock* This table shows the type or types of killing action on sensitive stock 31* the strength of the killing reaction is indicated by the number of (4) symbols * Also shown is the resistance (E) or sensitivity (S) of a given stock to the two general types of paramecin found among the mutant killers, via*, 51 paramecin associated with 51 type of killing and 51ml paramecin as« sociated with Sp type of killing*

Stocks

Type of killing action on sensitive stock 31 51 ..3p........

Reaction to narameclns 511 M L

Mutantss m l

51m2

ee

51h3

f + 4-

53L»5

4m

» to

& or K~S a

3 or 3-S

14

16

I II Totals

7

4

&

7

13

The segregation ratios are unmistakably close to 1:1.

this

is of special interest, since, In Experiment I, the ratio of 7*4 for clone 5b might have aroused suspicion in view of the fact that this clone was th® mate of 5a which gave th® aberrant 14*1 result.

Th© additional data indicate that 5b was, as ex­

pected, a hstero^ygot® Kk regardless of the cause of the dis­ crepancy in the F2 from 5a. A third experiment carried out as a check against the first two reported, involved a repetition of th© original cross (Experiment I) in which 51ml m s again mated with stock 186 and an F2 generation was again obtained by autogamy.

(The particular

pair selected to give rise to the F2 generation bears by coin­ cidence, the same number designation as that shown in Experiment I but does pot imply any relationship to the latter.) The F2 data from Fl 2b are close to the expected 1*1 ratios. The F2 data from the two members of Fl pair #5 are not so close, but

60 they do not differ significantly from 1:1, either separately or together* Tables XVI and XVII show the F2 results of crossing 51m2 x 186 and $1th3 x 186*

In both cases the rssiilts arc an

close agreement with a Isl ratio ( pp* 61-62)• In the cross of the 4?ml killer x stock 186 the first experiment yielded an F2 group deviating significantly from th© expected 1:1 ratio*

A second experiment was therefore run as

a check on the results obtained from Experiment I* Both sets of data are recorded in Table XVIII, p* 63* As previously indicated, this cross Involves the mating of two diverse stocks, unlike th© four crosses previously re­ ported above in which a stock highly isogenic with killer stock 51 Is mated with mutants derived from stock 51* The results ob­ tained in th© two eases, however, are comparable, as indicated la th® discussion following Table VIII, p* 40* Furthermore, any of the three variety 4 stocks (29, 32 or 186) which are homozygous kk can be used in crosses to stock 51 or 47 in order to investigate genic control of the 51 killer character* (It will also be recalled from the discussion accompany­ ing Table VIII that the marked discrepancy between the number of F2 clones obtained and the number tested in the above cross is due to fZ mortality normally resulting from crosses between two divers® stocks*)

61

TABLE XVI* Cro®8 of 51®2 killer x stock 186.

exconj* #..

Fl

la

S

b

K

2a

X

b

X

# clones obtained

F2 ' result of test: theoretical observed rfK 3 ..... $TT.._3

15* -

15

5 10

7ir.

7i_

15

7

6



6ft

a

7

7h

7k

7

7

. 15....

K

15

7

7

b

K

-.15.....

9

6

6a

K

15

8

7

7i

b

K

7

8

_.„.7i

8a

K

15

8

6

b totals

X

0 120

>

_..^15______

59 57’

.....

.. 7ft....7i .... 7i ._7L_____

7

7

58

58

62

TABLE XVII.

©x~ conj. t ..... FX

Cross of 51hi3 killer x stock 1.36.

# clones obtained

2a

K

30

b

S

30

4a

K

b

T2 result of test* observed theoretical 1' r~ g ■^ 13

17

15

15

30

18

12

15

15

K

30

16

13

14*

Ui

6a

K

28

16

12

14

14

b Totals

K

0 23

54

s

us

r

w

63

TABUS XVIXI# Cross of 47&L killer x stock 186, Experiment 1 exconj, # , la b 2a b 6a b Totals

PI 3 K S K K K

# clones obtained 60* 60 0 60 60 „,60 240

P2 result of teste theoretical observed It s m 3

26 20 17 11 w ~

9

17$

17i

19 13 11 n

19$ 15 11 63

19i 15 11 &3

Ifocperiraent 11

exconj. # la b 3& b Totals'

Fl

"# clones obtained

3 K 5 K

0 60 0 ,,69

120

P2 result of test theoretical observed & 3 M rS

29

26

26

2k.. a 54 50

26 52

26 52

23

6.4 In ixperisaent I, Table XVIII, three of the four Fl killer olonee gave rise to F2 generations displaying a clearly defined lsl segregation into killer and sensitive clones*

The

fourth clone, lb, however, showed a significant deviation from the expected lsl ratio* Here, as in the case of the aberrant ratio reported In the 51ml x 186 cross (Table XV), no satisfactory ex­ planation can be given, and again since the Fl was discarded after obtaining the F2 isolations and before these clones could be tested, the cross could not be repeated with the same material* Supporting evidence for the result® obtained in th© first experiment was forthcoming in a cross of one of the 47tel F2 kil­ lers from Fl 2b (Experiment I, Table XVIII) to stock 186*

The auto­

gamous P2 clones derived from the Fl generation of this cross yield­ ed a distribution of 34 killer cultures to 50 sensitive cultures, a close approximation to a 1*1 ratio (Experiment II). (c)

Discussion.

Prom results obtained in crosses of

the mutant killers to stock 186, it must be concluded that each mutant stock differs from 186 by only one, or effectively one, gene with respect to genetic control of the killer character. The same situation was found to hold in crosses of original stock 51 to stock 186 (Sonnebom, 1947)* Since th® mutant killers were found to be effectively identical with stock 51 in genes con­ trolling th© killer character (pp. 30-50), it follows that the dif­ ference between the mutants and stock 186 is at effectively the

65

same locus, the K locus*

Cross III, moreover, brings out one im­

portant point not shorn by Crosses I and H .

Th© first two crosses

give no information as to whether the K gene is essential for the maintenance of th© mutant killer traits because this gen© was present in homozygous condition in all th® materials employed*

While it was,

of course, to b© anticipated that th© K gem is essential for the mutant as well aa for the original killers, this remained an assumption without the data from Cross III. These results make it clear that the substitution of homozygous k for the K gem in the mutants results in th© irreversible loss of th© killer trait.

Th© latter fact, to­

gether with information obtained from Crosses I air* II, provides th© final evidence that the mutants and the original killer are not only identical in gems involved in the killer trait, but that the gone K function© apparently in the same way in th© mutant© as in th® originals, namely, by controlling th© maintenance of the killer cytoplasmic factors. C. Discussion of the genetic analysis

;

Th® general interrelations between gene, cytoplasmic factor and trait in determination of the killer character of stock 51 have already been pointed out (Sonneborn, 1943, 1945, 1947)*

It will be recalled that the

trait, production of paramecin, depends upon the presence of kappa. The latter, injtum, can b® maintained only in the presence of gene K. Thus, a change in either one of these component parts of the killer system or of others like them could result in a change in th© type of killing

66

manifested by the cell*

For example, this change in character could

conceivably b® due to (a) a imitation of th® killer gene, K, (b) a mutation of some other gene influencing the killer character, (c) a mutation of kappa or perhaps of some other cytoplasmic element. It will be seen from the results of the genetic analysis reported that the consequences which must necessarily follow if either (a) or (b) is true, stand in complete disagreement with the observed facts, as shown in the following discussion* In (a) above, the change in killer character is explained by assuming a mutation of the original K gene to &*• As a consequence, we would expect that replacement of the K gen® in a 51 cell by Its mutant counterpart, K % should result in transformation of the original killer into a mutant type killer, as must have occurred at the origin of the mutation on this hypothesis*

That such a transformation does

not occur, however, is shown by the fact that the assumed K» gene con­ tinue® to support th® original 51 killer character in precisely th© same manner as the K gene which it replaced (pp* 45*-4?)# Further, if mutation of the & gene is responsible for pro­ duction of th© mutant character, then &, when substituted for K® in a cell, should result in transformation of th© mutant into a 51 type killer (or, more unlikely, into a sensitive)*

On the contrary, the

K gen© from stock 51 maintains the mutant character apparently as ef­ ficiently m the presumed mutant gene, &f (pp*3t-4o). Whether or not the #K gene mutation” hypothesis can be com­

67

pletely discounted on the strength of this evidence depends, in part, upon our understanding of th® functions of K and kappa*

Precisely

what the interrelations are between these two have not yet been es­ tablished* However, fro® present knowledge of the chemical nature and function of genes and gene-like bodies and from the studies of Sonnebom demonstrating the maintenance of kappa to depend upon the presence of gene K, it appears reasonable to assume that (l) K might produce a product directly or indirectly utilizable by kappa and (2) watation of K to K* would most likely result in a change in gene pro­ duct from that produced by K.

If we accept for the present these as­

sumptions, then several alternative possibilities become immediately apparent v&th respect to the functional relationship between K* and kappa. On the one hand, the kappa present in a $1 killer animal may be incapable of utilising the product of K*• Since kappa depends for its maintenance upon the gen© (or gene product), its inability to utilise this product should result in its loss from the cell.

Thus,

stock 51 animals receiving the Kf gen® should be transformed into sensi­ tives.

On the contrary, the data show (pp. 45"-#*) that the 51 killer

character is not lost but 1® maintained In those animals homozygous for the killer gen® fro© th® mutant anti lacking th® gene K from stock 51. On the other hand, if we assume kappa to be capable of utiliz­ ing the K* product, at least two further alternatives need to be con­ sidered} (1) kappa may b© able to utilize K1 product equally as well as

63

K product*

In this event* however* no change In type of killing

should occur and* by definition* a imitation would be excluded*

(2)

The K* product utilized by kappa could modify th® latter in such a way as to result in change in type of killing by the cell*

(Th©

relationship existing between kappa and paramecin is not yet under­ stood but it is known that kappa is necessary for production of para­ mecin*) Under such conditions we would expect that replacement of the 51 & gen® is a 51 killer cell by th© K* gene would result in transformation of this animal, into a mutant-type killer*

On the con­

trary, the data show that the K* gene supports th© original 51-type killing possessed by the 51 killer cell*

It is possible, of course,

that modifications induced in kappa as a result of utilizing Kf pro­ duct could persist in th® absence of K h

This condition, however,

would represent in effect a ttdoubltn mutation? change of K to K* and & change or mutation of kappa to a modified form* A third and equally untenable alternative designed to explain the killer character mutations on the basis of a change in the killer gene postulates the d© novo origin of kappa from or by means of the K* gene*

Several sources of evidence exist to disprove this possibility,

the most critical of which is th© fact that the assumed mutant gene, present in horaoaygous condition in a cell lacking kappa, does not initiate production of kappas the cell and its progeny remain sensitive* Finally, additional and decisive evidence against the K gene mutation hypothesis was obtained when the kappa in the mutant killers was

69

diluted out of the aniiaals (p, 7< ), Under these conditions it could be shown that the mutant killer character also was irreversibly lost* The second of three hypotheses set forth to account for the killer character imitations assumes a change in a gene (or genes) other than K which Influences expression of the killer character. At least one such gene is already known (gene S: bomiebom, 1947) and it is probable that more exist*

It is not inconceivable, there­

fore, that the mutation of one of these Ms©condary" $etei$ainors could result in a change in type of killing*

While this result could be

distinguished by proper crosses from that of the K1 result above, it would be excluded by the same considerations set forth in regal'd to th© K 1 gene* It thus becomes necessary to abandon the hypothesis which seeks to explain the killer character mutations on the basis of changes in genes controlling th© killer trait*

The further pos­

sibility that s cm® intranuclear factor other than the gene® might be responsible for the mutations is not indicated as would be expected from breeding experiments already discussed*

We are therefore forced

to consider the remaining alternative which would locate the physical basis of th© killer imitations in th© cytoplasm of th© killer cell, Recalling th© important rol© played by kappa in the determination of the 51 killer character (Soimobom, 1943)* the search for a cytoplasmic constituent responsible for the killer mutations was first of all directed toward investigating th© presence and possible mutation of killer cytoplasmic factors in th© mutant stocks.

(Section IV)*

70 IV. THE KAPPAS OF THE MUTANT STOCKS Th© fact that kappa is necessary for the detemination of th® killer trait in stock 51 (flonnebom* 1943) and that 4 of the $ mutant killers were derived from killer stock 51* indicated early in th® course of study that th® mutant killers probably also con* tained kappa.

If true* the possibility that th® kappa in th© orig­

inal stocks could mutate and subsequently give rise to the mutant killer phenotypes would appear not unlikely in view of its gene-like nature (flonnebom, 1943) (Freer, 1948a and in press).

Indeed* the

results of the crosses described in flection III* while discounting gene mutations as the prime factor responsible for changes in the killer characters of stocks 51 and 47* supported* on th© other hand* the hypothesis accounting for mutations of th© killer characters on the basis of changes in kappa.

Th© present section* therefor©* takes into account th© evidence for th® existence of kappa in th© mutant stocks and the origin of th® new killer phenotypes as a result of kappa mutation. Pertinent data from th© preceding sections as well as new genetic and eytological evidence are presented in the form of a series of experiments, Th© general discussion and conclusions are reserved for Section V.

A. Evidence for the existence and mutation of kappa in the mutant rtPBla..

71 1*

Genetic evidence. Experiments I-X.

Crosses of th© mutant killers with th© and stock 1&6 (pp. 5"V-6j ) indicated

51 sensitive stock

that mutant types of kappa might be involved in the determination of the killer phenotypes*

Mhen cytoplasm was not exchanged between

th® mates in these crosses* the Fl killer member of a pair invariably remained a killer of the mutant type and the Fl sensitive member invariably remained a sensitive and gave rise only to sensitive progeny*

When cytoplasm was exchanged between mates* on the other

hand* th© sensitive member of the pair was transformed into a killer of the mutant type with which it mated,

the data from one of the

two type® of crosses (mutant killer® x 51S) is cited in Table XIX, p* 72* The results of the second type of cross (mutant killers x stock 1&6) are comparable with those reported in Table XIX* Both show that the killer character of the mutant stocks, like that of stock 51, follows the cytoplasm in inheritance*

Hence, killer cyto­

plasmic factors must be present also In the mutant killer animals* Experiment XX*

Additional information extending and con­

firming th© result© reported in Experiment® I-X were obtained from a cross of two killersj th© original 51 killer anti the 51ml mutant* The latter, It will be recalled, produces a type of killing action (Sp) very different from that manifested by stock 51 animals (p.8 ). From a mating between these killers, ten pairs were ob­ tained for study.

Five of these pairs showed no visible delay in

TABLE XIX, The effect of transfer of cytoplasm between mates in crosses of mutant KK killers x stock 51 &K sensitives. Column 2 gives the d eaignation of the ^airs used in the experiment* Column 3 indicates whether separation at time of conjugation was rapid (ft), resulting in no cytoplasmic exchange or delayed (D), producing cytoplasmic exchange between mates* Column 4 shows whether the axconjugants of each pair produced sensitive (S) or killer (K) cultures* The phenotype of the culture from the original killer member of the pair Is shown on the lefthand side, that from the original sensitive member, on the right-hand side* In every case, the type of killing corresponds to that of the killer parent used in the cross* — .. Gross

Pair #

3 or Q separation

...... ..-~.