Nuclear changes and viability of exconjugants in conjugation between Chinese and American Paramecia

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Content NUCLEAR CHANGES AND VIABILITY OP EX-CONJUGANTS IN
CONJUGATION BETW EEN CHINESE AND AMERICAN PARAMECIA
A T h e s is
P r e s e n te d t o
t h e F a c u lt y o f th e D ep artm en t of Z o o lo g y
U n iv e r s i t y o f S o u tiie r n C a l i f o r n ia
In P a r t i a l F u l f il l m e n t
o f th e R eq u irem en ts f o r th e d e g r e e
M a ster o f S c ie n c e
b y
G hent G raves
F eb ru a ry 1950
UMI Number: EP67193
All rights reserved
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2 ^ ' 6 T y ^ n 0 y
This thesis, written by
....................... Ghent (^ayes ,Jr...........................
under the guidance of h..i3...Faculty Committee,
and approved by all its members, has been
presented to and accepted by the Council on
Graduate Study and Research in partial fulfill
ment of the requirements for the degree of
Master o f Science
Date. 9/ 21/50
Faculty Committee
Chairman
TABLE OP CONTENTS
PAGE
INTRODUCTION....................................................................................................... 1
H i s t o r i c a l r e v ie w ......................................................... 8
C o n j u g a t i o n ...................................................................................... 5
Genetics• • • • • • * • • • « 7
MATERIALS AND METHODS............................................................................. 10
M a t e r i a l s ....................................................................................................... 10
M ethods * . . 10
S t a in in g t e c h n iq u e .............................. 11
P e r c e n ta g e s t u d y ............................................................. 13
V i a b i l i t y s t u d i e s • • • • • • • .......................................... 14
OBSERVATIONS....................................................................................................... 15
V i a b i l i t y s t u d i e s . . 15
N u c le a r ch a n g es . . . . . . . . . . . 2 1
DISCUSSION. . ................................................................................. . . 2 4
SUm iARY................................................................................................................. 2 8
LITERATURE C I T E D ........................................................................................ 50
INTRODUCTION
Param eoium b u r a a r la h as b een d e s c r ib e d a s a ” s l i p p e r
shaped** a n im a l, a v e r a g in g b e tw e en 180 and 200 m icr o n s in
l e n g t h . I t h a s a g r e e n c o lo r w h ich i s due to th e p r e s e n c e
o f s y m b io t ic a l g a l c e l l s . T h is c h a r a c t e r i s t i c r e a d i l y
d i s t i n g u i s h e s i t from o th e r members o f t h e Genus P aram ecium ,
b u t a s J e n n in g s (1 9 3 9 ) p o in t e d o u t , a lth o u g h th e c o lo r may
be la c k in g due t o a more r a p id d i v i s i o n o f t h e o rgan ism
th a n t h a t o f t h e a l g a e . The a n t e r io r and i s u s u a l l y p o i n t
ed s l i ^ t l y downward, and in some c a s e s t h i s may b e p r o
n ou n ced s o a s t o g iv e a h o o k - lik e a p p e a r a n c e .
Param ecium b u r s a r i a . so f a r as i s known, c o n s i s t s
o f s i x v a r i e t i e s . (J e n n in g s 1 9 3 9 , J e n n in g s and O p itz 1 9 4 4 ,
Chen 1946a) • V a r i e t i e s I , I I , and I I I h a v e a l l b e e n fou n d
i n th e U n ite d S t a t e s , b u t V a r i e t i e s IV , V, and VI h a v e b e e n
i s o l a t e d o n ly i n E u ro p e. V a r i e t i e s IV , and V a r e o f R u ss ia n
o r i g i n , w h ile V a r ie t y VI i s from C z e c h o s lo v a k ia , E n glan d
and I r e l a n d . W ith in each o f th e s e s i x V a r i e t i e s are a
number o f m a tin g t y p e s . I t h a s b e e n d e te r m in e d t h a t a n im a ls
o f t h e same m a tin g ty p e do n o t n o m a l l y c o n ju g a te w h ile
in d iv i d u a l s o f d i f f e r e n t m a tin g t y p e s do so w ith a l a c r i t y .
The d e s i g n a t i o n s o f t h e s e m a tin g ty p e s a r e a l p h a b e t i c a l ,
th e r e b e in g f o u r i n V a r ie t y I , I I I , and V I, e i ^ t in
V a r ie t y I I , tw o in V a r ie t y IV , and one in V a r ie t y V.
2
Chen {u n p u b lish e d ) fo u n d t h a t c e r t a i n C h in e se c lo n e s
o f Param ecium b u r s a r ia m ate w ith A m erican c lo n e s o f P a ra
m ecium b u rs a r ia o f V a r ie t y I . The p u rp o se o f t h i s w ork
was to d e te r m in e w h e th e r or n o t t h e C h in ese a n im a ls w ere
a l s o o f V a r ie t y I , b y s t u d i e s o f (1 ) th e p e r c e n ta g e o f
m a tin g , (2) th e v i a b i l i t y o f th e e x - c o n j u g a n t s , and (3 )
th e n u c le a r c h a n g e s w hich ta k e p l a c e . The p ro b lèm es r e l a
t i o n t o c y t o lo g y i s a p p a r e n t, b u t i t s r e l a t i o n t o g e n e t i c s
i s som ewhat m ore o b s c u r e . The m a tin g r e a c t io n i s c o n t r o l
l e d by th e c e l l , and as th e r e a c t i o n s are u n ifo r m and c o n
s i s t e n t , d ie c o n t r o l l i n g m echanism may a r b i t r a r i l y be c o n
s ig n e d t o th e n u c le a r com ponents or t o t h e i r p r o d u c t s .
I n t h i s r e s p e c t t h e p rob lem i s r e l a t e d t o t h e f i e l d o f
g e n e t i c s .
H i s t o r i c a l R eview
Param ecium b u r s a r ia h a s b een e x t e n s i v e l y s t u d ie d
from a c y t o l o g i c a l p o in t o f v ie w , m ore s o i n some r e s p e c t s
th a n any o th e r s p e c i e s o f c i l l â t e s . Maupas (1 8 8 9 ) was
p erh a p s th e f i r s t t o g iv e a resum e o f t h e w ork on c i l i a t e s
t o t h a t d a t e . H is own w ork upon L eu con h yrs p a t u la and
th r e e o th e r o r g a n ism s i s o f p a r t i c u la r i n t e r e s t . He r e
c o rd ed o b s e r v a t io n s on c o n ju g a t io n o f t h i s and t h e o th e r
th r e e a n im a ls , w h ich are now ta k e n as i n d i c a t i n g t h e
3
e x i s t e n c e o f m a tin g t y p e s . He a l s o d e s c r ib e d c o n ju g a tio n
in Param ecium a u r e l i a . Param ecium oaudatum » and Param eoium
b u r s a r i a . H ow ever, w ith r e s p e c t t o t h i s l a t t e r s p e c i e s ,
h i s work was r a th e r s k e t c h y and in c o m p le t e .
A p p a r e n tly th e n e x t work upon t h e m a tin g ty p e s in
P a ra m ecia was t h a t o f Son n eb orn (1 9 3 7 ) , v h o w orked on
Param ecium a u r e l i a .
M a tin g t y p e s in Param ecium b u r s a r ia w ere d is c o v e r e d
b y J e n n in g s (1 9 3 8 ) s h o r t l y a f t e r t h e y w ere d is c o v e r e d in
Param ecium a u re l i a (S on n eb orn 1937) , and tw o new f i n d i n g s
w ere r e c o r d e d ; ( l ) s e x u a l i s o l a t i o n o f t h e v a r i e t i e s , and
(2 ) m u l t i p l e m a tin g t y p e s . The o n ly e x c e p t io n s t o c o m p le te
s e x u a l i s o l a t i o n o f v a r i e t i e s a r e t h e m a tin g r e a c t i o n s b e
tw e en m a tin g t y p e R o f V a r ie t y IV and m a tin g t y p e s B , K,
L , and M o f V a r ie t y I I . (S e e T a b le I ) . T h is i s o f l i t t l e
g e n e t i c a s s i s t a n c e th u s f a r , h o w e v e r , a s a l l o f t h e e x -
con ju g a n ts d ie i n su ch m a tin g s w ith o u t c o m p le tin g c o n ju g a
t i o n p r o c e s s e s . (Chen 1 9 4 6 c ) .
There ap p ear t o be s e v e r a l f a c t o r s v h io h a f f e c t th e
m a tin g r e a c t i o n . S e v e r a l o f t h e s e w ere en u m erated b y
J e n n in g s (J e n n in g s 1 9 3 9 ) . He fo u n d t h a t im m ature c lo n e s
e i t h e r had d i f f i c u l t y in m a tin g or d id n o t c o n ju g a te a t
a l l , and s u g g e s t e d t h a t th e o b s e r v e d tim e le n g th w h ic h
m u st e la p s e a f t e r a new g e n o ty p e i s form ed th r o u g h c o n ju
g a t io n r a n g e s from tw o w eeks t o s e v e r a l m onths or l o n g e r .
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5
The s h o r t e s t p e r io d o b s e r v e d f o r th e a tta in m e n t o f m a tu r ity
was t w e lv e d a y s* The n u t r i t i v e c o n d it io n i s a n o th e r im
p o r t a n t f a c t o r i n t h a t a n im a ls e i t h e r s t a r v e d or o v e r - f e d
do n o t g iv e th e s t r o n g e s t m a tin g r e a c t i o n s . R a th e r , t h e
m o st pronou nced c a s e s o f a g g l u t i n a t i o n and p a i r in g seem
t o r e s u l t tw o t o f o u r d ays a f t e r f e e d i n g . A l s o , m ix tu r e s
made from j u s t b e f o r e noon u n t i l ab ou t 4 o ’ c lo c k in th e
a fte r n o o n g iv e s t r o n g e r m a tin g r e a c t io n s th a n d o m ix tu r e s
made a t o t h e r tim e s o f th e d a y or n ig h t#
C o n ju g a tio n
The m ain f e a t u r e s o f c o n ju g a tio n o f Param ecium
b u r s a r ia w ere worked on b y sem e o f th e e a r l i e r i n v e s t i g a t o r s .
Hamburger (1 9 0 4 ) among them* H ow ever, s t u d i e s on tim ed
s t a g e s b y Chen have c o n t r ib u t e d much d e t a i l t o e a r l i e r
work*
A f t e r a g g l u t i n a t i o n and su b se q u e n t p a ir in g o c c u r s ,
th e f i r s t m a tu r a tio n d i v i s i o n ta k e s p l a c e . T h is i s a lo n g
and c o m p lic a te d d i v i s i o n , r e q u ir in g ab ou t h a l f o f t h e t o t a l
tim e o f c o n ju g a tio n f o r i t s c o m p le tio n . The d i v i s i o n d o e s
n o t o c c u r in th e m a c r o n u c le u s , b u t a f f e c t s o n ly th e m ic r o -
n u c le u s , w h ich d i v i d e s , one o f th e r e s u l t i n g p r o d u c ts d i s
i n t e g r a t i n g . The r e m a in in g m ic r o n u c le u s q u ic k ly u n d e r g o e s
a se c o n d d i v i s i o n , and a g a in one o f th e p r o d u c ts d i s i n t e
6
g r a t e s . The t h i r d p rogam ic d i v i s i o n r e s u l t s in two m ic r o
n u c l e i , one o f # 1 i c h i s s t a t i o n a r y , t h e o th e r m ig r a to r y *
T h ese a r e c a l l e d gam ete n u c l e i . The m ig r a to r y p r o n u c le u s
i n e a c h c o n ju g a n t p a s s e s i n t o th e o t h e r c o n ju g a n t th r o u g h
th e p o in t o f ju n c tu r e and f u s e s w ith th e s t a t i o n a r y n u c le u s
o f t h e o th e r a n im a l, fo r m in g w h at i s known a s t h e sy n c a r y o n
The sy n c a r y o n d i v i d e s , one o f t h e d a u g h te r n u c l e i d e g e n e r
a t e s , a n d one o f th e com p on en ts d i v i d e s t w i c e , tw o o f th e
p r o d u c ts fo r m in g ü ie m a c r o n u c le u s , w h ile th e r e m a in in g two
form th e m ic r o n u c le u s* The o ld m acro n u c le u s d i s i n t e g r a t e s
and i s a p p a r e n tly r e s o r b e d b y t h e c e l l *
Chen (1946b ) r e p o r t s a c o n s id e r a b le d i f f e r e n c e i n
th e le n g t h o f th e tim e o f c o n ju g a tio n r e q u ir e d b y d i f f e r
e n t v a r i e t i e s * V a r ie t y I h a s t h e s h o r t e s t m a tin g t im e ,
a p p r o x im a te ly tw e n ty -tw o t o t w e n t y - f o u r h o u r s , w h ile
V a r i e t i e s I I and IV n eed t h i r t y - t w o t o t h i r t y - s i x h o u rs
f o r c o m p le tio n o f c o n ju g a tio n *
No n u c le a r p r o c e s s e s a r e known in Param ecium b u r
s a r i a o th e r th a n f i s s i o n and c o n j u g a t io n . H ow ever, a u to
gamy d o e s o c c u r u n d er c e r t a i n abnorm al c o n d i t i o n s ,^ b u t
e n d o m ix is h a s n o t a s y e t b e e n d is c o v e r e d in Param ecium
b u r s a r i a . b y a n y o f th e i n v e s t i g a t o r s , t h o u ^ b o th o c c u r
r e n c e s a r e common in Param ecium au re l i a . (S o n n eb o rn , 1 9 3 9 ,
1 C o n ju g a tio n o f th r e e a n im a ls , in d u c t io n o f c o n ju
g a t io n in one m a tin g ty p e b y f l u i d from a n o th er m a tin g ty p e
(C h en , 1945 and 1 9 4 6 )•
7
W oodruff and Erdman 1 9 1 4 )•
T here Is e v id e n c e t o i n d i c a t e t h a t t h e r e o c c u r s
d u r in g c o n j u g a t io n a c e r t a i n am ount o f c y to p la s m ic e x c h a n g e .
H a r r is o n and G ow ler (1 9 4 5 ) r e p o r te d v i s i b l e p r o o f o f t h i s
c y to p la s m ic ex ch a n g e in c r o s s e s b e tw e e n a n im a ls vh ic h p o s
s e s s e d and th o s e w h ich la c k e d th e s y m b io t ic a lg a e * W hether
o r n o t th e s e m a t e r ia ls o th e r th a n th e n u c l e i , w h ic h a re
e x c h a n g e d , p la y any a p p r e c ia b le p a r t i n c a r r y in g h e r e d it a r y
f a c t o r s i s n o t d e f i n i t e l y know n.
G e n e tic s
Chen (1 9 4 0 a ) fo u n d common o c c u r r e n c e o f p o ly p lo id y
i n Param ecium b u r s a r i a . K im b a ll (1 9 4 3 ) s u g g e s t e d t h a t t h i s
m ig h t h ave m asked th e s i g n i f i c a n c e o f g e n e t i c s t u d i e s in
t h i s s p e c i e s . Chen p o in te d o u t (1 9 4 0 a ) t h a t c o n ju g a tio n
b e tw e e n a d i p l o i d or p o ly p lo id a n im a l and one w h ich i s
a m ic r e n u c le a te r e s u l t s in two e x - c o n j u g a n t s , each c o n t a in
in g h a l f o f th e chromosome com plem ent o f th e n orm al p a r e n t .
By a r e p e t i t i o n o f t h i s e x p e r im e n t, i t i s c o n c e iv a b le t h a t
th e d i p l o i d o r h a p lo id c o n d it io n c o u ld b e a t t a i n e d , th u s
s i m p l i f y in g th e p r o s p e c ts f o r f u r t h e r g e n e t i c s t u d y .
The in h e r it a n c e o f m a tin g t y p e , s e e m in g ly , h a s b e e n
th e m o st e x t e n s i v e l y s t u d ie d f e a t u r e o f th e g e n e t ic sy ste m
o f Param ecium b u r s a r ia . J e n n in g s (1 9 3 9 ) worked in t h e m ain
8
w ith V a r i e t i e s I and I I , and fo u n d t h a t in n e a r ly 100^ o f
th e c a s e s t h a t t h e two e x -c o n ju g a n ts of each p a ir w ere o f
th e same m a tin g t y p e # T h is w ould i t s e l f i n f e r t h a t t h e
m a tin g ty p e i s c o n t r o l l e d b y th e n u c le u s , and f u r t h e r ,
t h a t t h e t h i r d m a tu r a tio n d i v i s i o n i s e q u a t i o n a l. The
s m a ll p e r c e n ta g e o f e x -c o n ju g a n ts v h ic h do n o t e x h i b i t
t h i s s i m i l a r i t y o f m a tin g t y p e s a r e w e l l w it h in t h e l i m i t s
o f S ta n d a rd E r r o r , and m i ^ t be t h e r e s u l t o f a r e d u c t io n a l ,
r a t h e r th a n e q u a t io n a l t h ir d m a tu r a tio n d i v i s i o n . N orm al
l y , b o th e x -c o n ju g a n ts o f a b o u t h a l f o f t h e p a ir s a r e o f
th e same m a tin g ty p e as one o f th e p a r e n t s , w h i l e t h e r e
m ain d er a r e o f t h e same m a tin g ty p e a s t h e o th e r p a r e n t .
H ow ever, some o f t h e s e * * n orm al ex-con ju gan ts** d o n o t e x
h i b i t th e m a tin g ty p e o f e i t h e r p a r e n t , and t h i s may be
due to new r e c o m b in a tio n s o f th e n u c le u s .
M ost c lo n e s a re o f u n ifo r m m a tin g t y p e , b u t o c c a s -
3 i o n a l l y t h e y u n d erg o a p r o c e s s o f s e I f - d i f f e r e n t i a t i o n ,
th u s e n a b lin g th e members t o c o n j u g a t e . T h is s e l f - d i f f e r
e n t i a t i o n w h ich e n a b le s su c h a c lo n e t o p ro d u ce w i t h i n
i t s e l f m ore th a n one m a tin g t y p e , may be t h e r e s u l t o f
a u to g a m y , or e v e n e n d o m ix is . K im b a ll (1 9 4 3 ) t h in k s t h e s e
ch a n g es m i ^ t o c c u r i n t h e a b se n c e o f an y n u c le a r r e o r
g a n i z a t i o n s . H ow ever, as th e modus o p e r a n d i o f t h i s
phenom ena, and o f o t h e r s , h a s n o t b een b r o u ^ t t o l i ^ t
9
a s y e t , i t s u f f i c e s t o s a y t h a t th e y p r e s e n t problem s
w h ich a r e n o t o n ly i n t e r e s t i n g b u t v i t a l to t h e u n d e r
s t a n d in g o f t h i s one p h a se in t h e g e n e t i c s o f Param ecium
b u r s a r ia #
MATERIALS AND M ETHODS
M a te r ia ls
Two c lo n e s o f Param ecium b u r s a r ia were u s e d in th e
p r e s e n t w ork; G a8, w h ich was c o l l e c t e d n e a r Lake A rrow head,
C a l i f o r n i a , and P u 9 , w h ich w as o b ta in e d n e a r S h a n ^ a i ,
C hina* F or t h e s e c lo n e s I am in d e b te d t o D r. T . T . Chen*
M ethods
The m ethod o f c u lt u r e i s e s s e n t i a l l y t h a t d e s c r ib e d
b y J e n n in g s (1 9 3 9 ) w ith o n ly s l i ^ t m o d if ic a t io n s * I t
c o n s i s t s o f a l e t t u c e i n f u s i o n medium im p reg n a ted w ith
B a c i l l u s c lo a c a e . a s t r a i n o f b a c te r iu m v h ic h i s fo u n d i n
th e human a lim e n ta r y t r a c t , p a r t i c u l a r l y t h e c o lo n , in
la r g e num bers* F i r s t , l e t t u c e le a v e s are d e p r iv e d o f t h è i r
t h i c k v e i n l i k e p o r t i o n s , w ashed c a r e f u l l y i n r u n n in g ta p
w a te r and th e n d i s t i l l e d w a te r , and p la c e d upon f i l t e r
p a p er in a d r y in g o v e n . T hey a r e h e a te d to 1 5 6 ° C e n tig r a d e
f o r a p e r io d o f t h i r t y m in u te s t o a n h o u r * An e f f o r t m u st
be made t o s e e t h a t t h e le a v e s d o n o t r o a s t to o f a s t , and
t h a t th e b a k in g i s u n ifo r m w ith o u t b u r n in g . R e d i s t i l l e d
w a te r i s b r o u ^ t t o a b o i l i n g p o i n t , a llo w e d to s t o p bub
b l i n g and th e n m ixed w ith th e l e t t u c e pov/der a t a r a t i o o f
1 .5 grams t o 1000 c c . o f w a t e r . T h is i s a g a in b o i l e d f o r
5 m in u te s* In t h i s work 250 cc* E rlem ey er f l a s k s w ere u sed #
11
An e x c e s s o f c a lc iu m c a r b o n a te i s added b e f o r e a u t o c la v in g
f o r tw e n ty m in u te s a t tw e n ty pounds p r e s s u r e . The p u rp ose
o f t h i s c a lc iu m c a r b o n a te i s t o m a in ta in th e p r o p e r pH,
w h ich i s optimum a t 6 . 6 .
In o r d e r t o co m p lete t h e c u lt u r e f l u i d , th e b a c t e r i a
m u st be a d d e d , and a g a in t h e r e i s a d e f i n i t e p r o c e d u r e .
The b a c t e r i a a r e c u ltu r e d on s l a n t s o f a y e a s t a g a r medium
and th e n m ixed w ith 5 -1 0 c c . o f t h e p r e p a r e d l e t t u c e i n f u
s io n # When th e b a c t e r i a a re f u l l y and u n ifo r m ly d is p e r s e d
i n t h e medium 0 .5 cc# i s d rop p ed in t o a f l a s k c o n t a in in g
100 c c# o f l e t t u c e i n f u s i o n . T h is i s a llo w e d t o s ta n d
o v e r n ig h t , in o rd er t o a llo w th e b a c t e r i a t o m u l t i p l y ,
th e r e s u l t i n g c o n c e n tr a t io n b e in g th e optimum a c c o r d in g t o
J e n n in g s (1 9 3 9 ) # The c u lt u r e medium s h o u ld n o t be m ore
th a n two d a y s o ld #
In a l a t e r p a p e r , J e n n in g s (1 9 4 2 ) a d v is e d t h a t
o n ly o n e - h a lf o f th e c o n c e n tr a t io n o f l e t t u c e i n f u s i o n was
n eed ed and t h a t th e r e s u l t s w e re j u s t a s good# T h is h a l f
s t r e n g t h medium was t h a t u s e d in t h e s e s t u d i e s .
S ta in in p : te c h n iq u e # The s t a i n i n g te c h n iq u e em p loyed
was th a t d e s c r ib e d b y Chen ( 1 9 4 4 ) , and when f o llo w e d c o r
r e c t l y , in v a r ia b l y gave good r e s u l t s # The a n im a ls are
f i x e d in S c h a u d in n ’s f l u i d c o n t a in in g 6^ o f g l a c i a l a c e t i c
a c i d , and h e a te d to a te m p e r a tu r e o f 5 0 ° C e n tig r a d e #
12
Embryo l o g i c a l w atch g l a s s e s a r e p la c e d in a w a te r b a th
r e g u la t e d a t 6 2 ° C e n tig r a d e . A f t e r t h e c o n j u g a t in g p a ir s
become f i r m l y a t t a c h e d , th e y are f i x e d e v e r y h o u r , u n t i l
th e y s e p a r a t e , t o d e te r m in e t h e n u c le a r ch a n g es o c c u r in g
a t th e d i f f e r e n t s t a g e s * A f t e r 15 m in u t e s , th e S c h a u d in n ’ s
f l u i d i s r e p la c e d by 60% a l c o h o l . The 50% a l c o h o l i s r e
p la c e d a f t e r 10 m in u te s w ith 70% a l c o h o l , a n d th e s p e c i
mens are a llo w e d t o s ta n d from 10 m in u te s t o o v ern ig ja t*
A t t h i s p o in t th e p roced u re a llo w s t h e o p e r a to r t o p la c e
th e sp e c im e n s in an a i r t i g h t c o n ta in e r p r e v e n tin g e v a p o r a
t i o n o f th e a l c o h o l u n t i l h e i s r e a d y t o ru n the sp ec im en s
t h r o u ^ * The 70% a l c o h o l i s th e n r e p la c e d b y 83% a lc o h o l
a f t e r w h ich t h e y a re m o u n ted . A t h i n la y e r o f album en
i s sp r e a d on a c o v e r g l a s s and u s i n g a m i c r o p i p e t t e , th e
sp e c im e n s a r e p ic k e d ou t and d ropp ed o n to th e c e n t e r o f
th e c o v e r g la s s * The a l c o h o l and P a ra m ecia sp r e a d o v er th e
album en f i x a t i v e . P a r t i a l e v a p o r a tio n o f t h e a lc o h o l a l
low s th e a n im a ls t o make c o n t a c t w ith t h e alb u m en . A
d rop o r tw o o f 96% a l c o h o l i s th e n added t o f u r t h e r h a rd en
th e album en f i x a t i v e , a f t e r w h ich th e c o v e r g l a s s i s p la c e d
i n a p e t r i d is h c o n t a in in g 95% a l c o h o l and a llo w e d to sta n d
f o r 10 m in u te s* A d rop o f a 1.5% s o l u t i o n o f c o l l o d i o n
( d i s s o l v e d in e q u a l p a r t s o f a b s o lu t e a l c o h o l and e th e r )
i s u s e d , to form a l a y e r o v e r th e sp e c im e n s to p r e v e n t
t h e i r l o s s i n h a n d lin g *
13
The c o v e r g l a s s e s a re h y d r a te d th r o u g h 10% grad e
ch a n g es o f a l c o h o l downward t o w a t e r , and p la c e d in a 4%
ir o n alum m ordant s o l u t i o n f o r 24 h o u r s * A f t e r w a sh in g
i n r u n n in g w a te r f b r 2 t o 3 m in u te s th e sp e c im e n s are
p la c e d in a s l i ^ t l y a l k a l i n e s o l u t i o n o f *5% h e m a to x y lin
f o r a n o th e r 24 h o u r s *
Im m ersion in a s a t u r a t e d aq u eou s s o l u t i o n o f p i c r i c
a c id f o l l o w s a n o th e r b r i e f w a sh in g in r u n n in g w a ter* To
d e te r m in e when t h e d e s i r e d am ount o f d e s t a i n i n g h a s ta k e n
p l a c e , a c o v e r g l a s s may be rem o v ed , r in s e d in ta p w a t e r ,
exam in ed m i c r o s c o p i c a l l y , and p a s se d o v e r a c o n c e n tr a te d
s o l u t i o n o f ammonium h y d r o x id e * T h is s h o u ld be a p i l o t
p r e p a r a tio n , and i f i t i s r e tu r n e d t o th e p i c r i c a c id solu «
t i o n i t w i l l d e s t a i n a t a more r a p id r a t e th a n o t h e r s n o t
t r e a t e d in t h i s m anner *
The sm ears a re w ashed in r u n n in g w a te r f o r one
h o u r and b r o u ^ t t o a b s o lu t e a l c o h o l th r o u g h 10% c h a n g es
o f a l c o h o ls * T hey a r e c le a r e d f o r t e n m in u te s in c e d a r -
wood o i l , t e n m in u te s in x y l e n e , and m ounted w ith damar
o r perm ount*
P e r c e n ta g e o f p a i r i n g * I n t h e s t u d i e s made of p e r
c e n ta g e o f p a ir in g o n ly one p o s s i b l e a c c u r a te m ethod was
e x i s t e n t * A d e f i n i t e number o f a n im a ls o f one c lo n e was
p ic k e d o u t and m ixed w ith th o s e o f th e s e c o n d c lo n e , and
14
a f t e r m a tin g , th e p a ir s c o u n te d , by rem o v in g t h e p a ir s one
a t a t im e . H ow ever, th r e e m ix tu r e s b y volum e w ere m ade,
and th e p a ir s p ic k e d o u t . T h ese w ere made t o g e t an ap
p r o x im a te id e a o f t h e p e r c e n ta g e o f a n im a ls t h a t p a ir e d .
V i a b i l i t y s t u d i e s # In th e s t u d i e s o f t h e v i a b i l i t y
o f e x - c o n j u g a n t s , a f t e r th e p a ir s are f i r m l y a t t a c h e d ,
th e y w ere i s o l a t e d on d e p r e s s io n s l i d e s . Each p a ir was
p la c e d in a s e p a r a te d e p r e s s io n c o n t a in in g t h r e e d r o p s o f
day o ld c u lt u r e f l u i d . T h ese d e p r e s s io n s w ere num bered 1 ,
2 , 3 , e t c . A f t e r th e con ju g a n ts becam e s e p a r a t e d , each ex-
con ju g a n t was p la c e d in a s e p a r a te d e p r e s s io n la b e le d lA ,
IB , e t c . , r e s p e c t i v e l y . T h ese e x -c o n ju g a n ts were th e n
a llo w e d t o d i v i d e and grow f o r p e r io d s r a n g in g from t e n
d a y s t o f i f t e e n d a y s , w it h r e p la c e m e n t o f t h e o l d c u lt u r e
f l u i d w ith f r e s h medium e v e r y 2 or 3 d a y s .
OBSERVATIONS
The p u rp ose o f t h i s w ork was t o d e te r m in e w h eth er
th e C h in e s e P a ra m ecia b e lo n g to V a r ie t y I . S t u d ie s w ere
made on c o n ju g a tio n b e tw e en th e C h in e se c lo n e Pu9 and t h e
A m erican c lo n e Ca8 t o d e te r m in e (1 ) th e v i a b i l i t y o f t h e
e x - c o n j u g a n t s , (2 ) th e p e r c e n ta g e o f a n im a ls in a g iv e n
m ix t u r e , w h ich w o u ld c o n j u g a t e , and (3 ) th e n u c le a r c h a n g es
w h ich ta k e p la c e b e tw e en t h e two members o f a m a tin g p a ir
d u r in g c o n ju g a tio n #
V i a b i l i t y S t u d ie s
In t h i s p h ase o f th e s t u d y , an e f f o r t was made to
d e te r m in e th e p e r c e n ta g e s u r v i v a l x / y o f e x -c o n ju g a n ts in
m a tin g b etw een th e C h in ese c lo n e and t h e A m erican c lo n e #
T h is was done i n 2 grou p s o f 3 s t u c iie s e a c h , t h o u ^ t h e y
a r e l i s t e d in T ab le IV (P age 20) a s 6 c o n s e c u t iv e t e s t s f o r
c o n v e n ie n c e . In th e f i r s t 3 e x p e r im e n ts 9 , 1 7 , and 16
p a i r s w ere u s e d r e s p e c t i v e l y , a llo w e d t o s e p a r a t e , and
th e e x -c o n ju g a n t s c u lt u r e d . The e x -c o n ju g a n ts w h ich d id
n o t d i v i d e , as w e l l as t h o s e w h ich d ie d w ere c l a s s e d as
n o n - v ia b le # In th e f i r s t t e s t , 4 o f t h e 18 e x -c o n ju g a n ts
d i e d , g i v in g a s u r v i v a l p e r c e n ta g e o f 7 7 .7 , w h ile in th e
se c o n d t e s t 11 o f th e 34 e x -c o n ju g a n ts d ie d , sh o w in g a
67.6% s u r v i v a l r a t e . Of 32 e x -c o n ju g a n t s in t h e t h i r d
16
t e s t , 10 d ie d , a 6 8 .6 s u r v i v a l p e r c e n t a g e . Thus of a t o t a l
o f 84 e x -c o n ju g a n ts o b s e r v e d , th e s u r v i v a l p e r c e n ta g e a v e r
aged 71.1% .
The m a j o r it y o f th e a n im a ls w h ich p r o v e d n o n - v ia b le
i n t h is f i r s t group o f t e s t s e x h i b i t e d v/hat a p p ea r e d to b e
s m a ll a i r b u b b le s a d h e r in g t o th e s u r f a c e o f t h e c e l l .
A g a in , s u s p e c t in g t h a t e n v ir o n m e n ta l c o n d i t i o n s m i ^ t be
u n f a v o r a b le , a n o th e r room w it h c o n s t a n t l i ^ t and w h ich
c o n ta in e d no c h e m ic a ls was u s e d f o r t h e seco n d g ro u p , w ith
a n o t i c e a b le change in th e r e s u l t s . In th e f i r s t o f t h e s e ,
11 o f th e 100 e x -c o n ju g a n ts from 50 p a ir s d i e d , y i e l d i n g a
s u r v i v a l r a t e o f 89%. F o u r te e n o f 120 e x -c o n ju g a n ts d ie d
i n th e s e c o n d t e s t , th e r e b y sh o w in g a s u r v i v a l r a t e o f
88.4% . Two e x -c o n ju g a n ts w ere l o s t in h a n d lin g d u r in g th e
t h i r d o f t h e s e e x p e r im e n ts , o th e r w is e 15 were n o n - v i a b l e ,
s o t h a t th e 89.1% s u r v i v a l r a t e i t n o t e n t i r e l y a c c u r a t e .
H ow ever, t h e r e s u l t s a r e rem a rk a b ly c o n s i s t e n t , and th e
num bers s u f f i c i e n t t o b u f f e r an y d e v i a t i o n th e 2 l o s t a n i
m a ls m i ^ t h ave c a u s e d . Thus i n t h i s se c o n d group th e
a v e r a g e number o f v i a b l e a n im a ls o f 3 5 8 s t u d ie d was 88.8% .
The t o t a l number o f e x -c o n ju g a n t s s t u d ie d w as 4 4 2 ,
and th e a v e r a g e s u r v i v a l o f t h e 2 grou p s o f e x p e r im e n ts
com bined was 83.2% .
From March 2 2 , 1 9 5 0 , th e d a te on w h ic h th e a u th o r
17
r e c e i v e d th e two c lo n e s o f Param ecium b u r s a r ia c o n c e r n e d
i n t h i s w ork , f i f t y - t w o m ix tu r e s w ere m ade, to d e te r m in e
th e p e r c e n ta g e o f a n im a ls t h a t w o u ld c o n j u g a t e . T hree o f
t h e s e m ix tu r e s w ere made b y v o lu m e , th e p a ir s b e in g p ic k e d
o u t a s t h e y w ere fo r m e d . The r e m a in d e r of th e m ix tu r e s
w ere made b y m ix in g a d e f i n i t e number o f in d iv id u a ls from
e a c h c l o n e . Due t o d i f f i c u l t i e s , in t h e l a t e r group o f
t e s t s o n ly 10 m ix tu r e s gave c l e a r r e s u l t s .
I n T ab le I I {P age 19) i t w i l l b e s e e n t h a t a lth o u g ji
t h e r e w ere 25 t o 75 a n im a ls o f each ty p e p r e s e n t i n t h e
o r i g i n a l m ix t u r e , t h e f i n a l c o u n ts showed more o r g a n ism s to
b e p r e s e n t . T h is was due to m u l t i p l i c a t i o n b y f i s s i o n o f
th e a n im a ls in v o lv e d . The r e s u l t s in t h i s group o f e x p e r i
m e n ts , o v er p e r io d s r a n g in g from 2 t o 5 d a y s , showed an
a v e r a g e o f 48.9% p a i r i n g . No tem p o ra ry p a ir f o m a t i o n
a s r e p o r te d b y Chen (1 9 4 6 d ) was o b s e r v e d .
T a b le I I I , w h ich g i v e s th e p e r c e n t of p a i r in g o f
volum e m ix t u r e s , sh ow s a som ew hat h i ^ e r p e r c e n ta g e of
m a tin g th a n d id th e m ix tu r e s o f a d e te r m in e d number o f
a n im a ls . T hree m ix tu r e s o f 0 . 1 c c . , 0 .3 c c . , and 0 .5 o c . ,
p ro d u ced an a v e r a g e m a tin g p e r c e n ta g e o f 7 8 .8 . The a u th o r
o r i g i n a l l y in te n d e d t o in c lu d e p r o g r e s s iv e volum e m ix tu r e s
t o 3 .0 c c . , b u t th e i n a b i l i t y t o co u n t th e la r g e number o f
p a ir s w ith a n y d e g r e e o f a c c u r a c y p r e v e n te d t h i s . In th e
18
volum e m ix t u r e s , o o c a s s i o n a l l y , t h e r e ap p eared a s m a ll num
b e r o f * * threes** # T hey w ere a tta c h e d i n t h e same m anner as
ty p e s I and I I d e s c r ib e d b y Chan (1946b ) •
A lth o u g h a la r g e number o f m ix tu r e s was m ad e, a s
s t a t e d e a r l i e r , r e l a t i v e l y few o f them p rod u ced m ea su r a b le
r e s u l t s . T h is was due t o t h e f a c t t h a t , fo r som e u n d e te r
m in ed r e a s o n , t h e c lo n e s r e f u s e d t o m a te , or d id so w e a k ly
and o v e r su ch a p r o t r a c t e d p e r io d o f tim e t h a t i t was im
p o s s i b l e t o o b t a in a c c u r a te r e s u l t s .
B e li e v i n g t h a t t h e e n v ir o n m e n ta l c o n d i t i o n s m ig h t
be th e m ajor c o n t r ib u t in g f a c t o r t o t h i s w eak m a tin g r e a c
t i o n , th e a u th o r made th e t h r e e volum e m ix tu r e s in a n o th e r
room , and a n o t i c e a b le in c r e a s e in th e r e a c t i v i t y e n s u e d .
The seco n d l o c a t i o n h a d u n ifo r m l i f t i n g 2 4 h o u r s a d a y and
was f r e e from any p o s s i b l e c h e m ic a l c o n ta m in a tio n .
TA 6 L
1
E n
MIXTURE
Û R 1 0 J N A L
NO ANIMALS
OF EACH CLONE
NO. PA 1 R S
HO. SI N 6 LC
AN 1 M ALS
^ PAIRS Tl M E
(D a'< S )
X
2 5
|5 30 50 3
n
25
33 7 6 EST.
46.4 3
nr
2 5
> 7 58
36.9 Z
IS L
2 S
26 56 48.1
4
3C 5 0 47 39
70.6 6
XL
7 S 5 8 72 6 1.7 4
XU
75 46 46 45.7 4
x n t 50 26 65 44.4 4
Œ 5 0 32 79 4 4.7 4
z 25 1 9 56 40-4
4
TA#LE HI
MIXTURE
VOL. {c «.)U»CO
NO. A ^ im a w S NO. P A IR S NO. SIX aiE
ANJ M ALS
^ Pa ir s T I M E
(o*^VS)
I O. 1
156 6 4 ^8 8 2 . 0
2
31 0 .3 4 4 2 212 68 9 6 .5 2
n x 0 .5 72 \ E 3T . 2 .2 6 E ST 2 6 5 EST. 6 1.7 2
TABLE W
20
MIXTURE MUM&eR. OF
C X -C O N JÜGANT5
NUM&CR or
NON- v ia b l e s
SURVIVAL %
I l a 4 7 7 . 7
IL
34 II 6 7. C
n r Si. 10 6 8 . 6
m
lOO
M
87 o
X
ito
14 8 8 4
XI I3d
15
J
21
N u c le a r C hanges
T hree s e r i e s o f p r e p a r a tio n s w ere made o f the c o n
j u g a t io n b e tw e en t h e C h in e se and A m erican c lo n e s w ith w h ich
th e w ork was c o n c er n e d * In th e f i r s t 2 s e r i e s th e p a ir s
w ere f i x e d e v e r y 2 h o u r s , w h ile in th e l a s t s e r i e s th e
p a ir s w ere f i x e d e v e r y h ou r* S e v e r a l s l i d e s w ere n o t u s a b le
due t o a l o s s o f th e s p e c im e n s .
A s tu d y o f t h e s e s l i d e s r e v e a le d t h a t th e n u c le a r
ch an ges w h ich to o k p la c e d u r in g c o n j u g a t io n b e tw e e n th e
C h in ese and A m erican P a ra m ecia w ere n o r m a l* The f i r s t
p reg a m ic d i v i s i o n in v o lv e d t y p i c a l l y lo n g and c o m p lic a te d
p ro p h a se c h a n g e s , and r e q u ir e d r o u g h ly h a l f o f t h e t o t a l
tim e o f c o n ju g a tio n f o r i t s c o m p le tio n * S h o r t ly a f t e r t h e
o n s e t o f c o n j u g a t io n th e m ic r o n u c le u s s t a r t e d t o e n l a r g e ,
w h ile th e m a c r o n u c le u s w as s e e n t o f o l d a lo n g a d ia g o n a l
e u cis* (S e e P i g . 1 , p age 4 ) . At a s l i ^ t l y l a t e r s t a g e
o f t h e e a r l y p r o p h a se o f t h i s f i r s t m a tu r a tio n d i v i s i o n ,
th e m a cr o n u cle u s l o s t i t s e l l i p t i c a l sh a p e and b egan to
a p p ro x im a te a sp h e r e * M ea n w h ile, t h e m ic r o n u c le u s e lo n g a t e d
and became c r e s c e n t sh ap ed * (S e e F i g . 2 , page ) . Prom
t h i s p o in t u n t i l th e end o f c o n j u g a t io n , no n o t ic e a b le
ch a n g es w ere o b s e r v e d t o ta k e p la c e in th e m a c r o n u c le u s ,
th e ch a n g es b e in g c o n f in e d t o t h e m ic r o n u c le u s * I n t h e
l a t e p ro p h a se o f t h i s f i r s t d i v i s i o n , th e m ic r o n u c le u s
22
show ed e v e n more s w e l l i n g , and h ad a g a in a l t e r e d i t s form
from t h a t o f a c r e s c e n t , c h a r a c t e r iz e d h y r e a s o n a b ly w e l l
o r ie n t a t e d ch r o m a tin m a t e r i a l , t o a la r g e and som ew hat i r
r e g u la r s p h e r e w ith th e chrom osom es in no d e f i n i t e p a tte r n *
(S e e P i g . 3 , p a g e ) • H ow ever, s h o r t l y t h e r e a f t e r , th e
chrom osom es a rra n g ed th e m s e lv e s a lo n g a s p in d le and th e
n u c le u s d iv id e d , y i e l d i n g tw o m i c r o n u c l e i , on e o f w h ich
s l o w l y d i s i n t e g r a t e d , and was e v i d e n t l y r e s o r b e d b y t h e
c e l l * The r e m a in in g m ic r o n u c le u s u n d erw en t a se c o n d d i v i
s i o n much more r a p i d l y ; a s th e p ro p h a se was n o t as p r o t r a c t e d
a s in th e ca se o f th e f i r s t d i v i s i o n * A g a in d i s i n t e g r a
t i o n o f one o f th e p r o d u c ts o f th e d i v i s i o n o c c u r r e d * At
a b o u t 17 h o u r s a f t e r th e o n s e t o f c o n ju g a tio n th e t h i r d
m a tu r a tio n d i v i s i o n was c o m p le te d and ex ch a n g e o f p r o n u c le i
b e g a n t o ta k e p la c e * H ow ever, i n m ost i n s t a n c e s , b e f o r e
th e c o m p le tio n o f th e t h ir d d i v i s i o n , th e m ic r o n u c le a r
s p in d le had assum ed a p o s i t i o n n e a r th e o r a l r e g io n *
S e e m in g ly , th e gam ete n u c le u s , w h ich was n e a r e s t th e p o in t
o f ju n c tu r e in t h i s r e g i o n , m ig r a te d in t o th e o th e r c o n ju -
g a n t and u n it e d w ith i t s s t a t i o n a r y n u c le u s * (F ig u r e s
p a g e s ) * A f t e r th e ex ch a n g e o f p r o n u c le i and th e su b
s e q u e n t fo r m a tio n o f th e s y n c a r y o n was c o m p le te d , th e
sy n c a r y o n th e n d iv id e d * T h is w as th e f i r s t p o s t z y g o t i c
d i v i s i o n * Q u ite o f t e n , e v e n a t t h i s s t a g e , th e d i s i n t e
g r a t in g m ic r o n u c le u s o f th e t h i r d m a tu r a tio n d i v i s i o n was
23
s t i l l v i s i b l e . One o f t h e n u c l e i d e r iv e d from t h e f i r s t
p o s t z y g o t i c d i v i s i o n d e g e n e r a te d , w h il e th e o th e r d iv id e d
a g a in . The p r o d u c ts o f t h i s d i v i s i o n d id n o t d i s i n t e g r a t e
b u t ea ch u n d erw en t a f u r t h e r d i v i s i o n b e f o r e t h e a n im a ls
s e p a r a t e d . T h is s tu d y d id n o t in c lu d e oh a n g es su b se q u e n t
t o t h i s s t a g e , b u t a b o u t 15 h o u r s a f t e r th e consum ât io n o f
c o n j u g a tio n th e m a c r o n u c le u s i s r e so r b e d b y t h e c e l l , b e in g
r e p la c e d b y one form ed from two o f t h e m icro n u c l e i o f th e
t h i r d p o s t z y g o t i c d i v i s i o n #
D is c u s s io n
FaraBieoium b u r s a r ia i s a f a v o r a b le s p e c i e s f o r a
s t u d y o f t h i s t y p e b e c a u se o f t h e d eep s t a i n i n g c a p a c it y o f
th e m ic r o n u c le u s , a lth o u g h t h e r e i s q u it e o f t e n a v a r i a t i o n
o f t h i s c a p a c it y among t h e d i f f e r e n t s t r a i n s o f th e o r g a n
is m s . A l s o , as i t s **sex” r e a c t i o n s a r e t y p i c a l , an y m ajor
d e v i a t io n s a re r e a d i l y r e c o g n i z a b le .
C o n ju g a tio n b e tw e en a n im a ls o f d i f f e r e n t m a tin g
t y p e s from d i f f e r e n t l o c a l e s i s o f im p o r ta n c e i n sh e d d in g
l i g h t upon t h e r e l a t i o n s h i p s o f th e members o f t h a t m a tin g
t y p e , a s w e l l as e s t a b l i s h i n g t h e c y t o l o g i c a l p r o c e s s e s
i n v o l v e d . Much o f th e work w h ich h a s b een done on P a ra
m ecium b u r s a r ia h a s c o n cern ed i t s e l f w it h m a tin g s o f c lo n e s
from one c o u n tr y , or c r o s s e s o f c lo n e s o f d i f f e r e n t v a r i e
t i e s from d i f f e r e n t c o u n t r i e s . H ow ever, l i t t l e h a s b een
done upon c lo n e s o f th e same v a r i e t y from tw o su c h w i d e l y
s e p a r a t e l o c a l i t i e s a s C h in a and C a l i f o r n i a .
The r e s u l t s o f th e w ork on th e p e r c e n ta g e o f m a tin g
seem t o i n d i c a t e t h a t p h y s i c a l i n f l u e n c e s p la y an im p o r ta n t
r o l e in th e i n h i b i t i o n o f th e a g g l u t i n a t i v e r e a c t i o n . The
a v e r a g e p e r c e n ta g e o f p a i r in g was r o u ^ l y 20^ h ig h e r in
t h e volum e m ix tu r e s th a n in t h e m ix tu r e s o f a d e te r m in e d
number o f i n d i v i d u a l s . As th e r e w a s, c e r t a in ly ^ an e x c e s s
o f one t y p e , t h e s e f i g u r e s w ould seem o u t o f l i n e . H ow ever,
25
t h e r e a re f o u r p o s s i b l e e x p la n a t io n s o f t h i s a p p a r e n t d i s
c r e p a n c y ; (1 ) th e a g i t a t i o n r e s u l t i n g from t h e t r a n s f e r ,
(2 ) th e s m a ll number o f in d iv id u a ls p e r volum e o f f l u i d ,
(3 ) an e x c e s s o f f l u i d from one o f th e c l o n e s , and (4)
e x c e s s i v e or r e d u c e d f a c t o r s o f l i ^ t , h e a t , f o r e i g n e l e
m e n ts , e t c . in th e o r i g i n a l en v iro n m en t o f th e e x p e r im e n t s .
S in c e c a r e was ta k e n t o a v o id , as much a s p o s s i b l e , th e
f i r s t th r e e it e m s , i t c o u ld be assum ed t h a t som e su ch phy
s i c a l in f lu e n c e a s l i t e d in (4) m i ^ t b e r e s p o n s ib l e f o r
b o th th e s m a ll p e r c e n ta g e r e c o r d e d in T a b le I I and th e a b
s e n c e or d e la y o f t h e m a tin g r e a c t i o n i n t h e r em a in d er o f
th e m ix t u r e s . (S ee p a g e 1 5 , l i n e 13) . Chen (1 9 4 5 ) ob
s e r v e d t h a t f l u i d from one m a tin g ty p e was a b le t o p rod u ce
c o n j u g a t io n among th e members o f a c lo n e o f a n o th e r m a tin g
t y p e . T h is i n d i c a t e s p o s s i b l y t h a t th e medium o f a c lo n e
c o n t a in s a s u b s ta n c e w h ich in d u c e s a g g l u t i n a t i o n o f th e
a n im a ls in tr o d u c e d b y th e members o f th e c l o n e , and f u r t h e r ,
b e c a u s e o f n orm al a b s t in e n c e from m a tin g by members o f
d i f f e r e n t v a r i e t i e s , t h a t i t i s s p e c i f i c . The p r e s e n t
work show ed g a s b u b b le s on t h e s u r f a c e s o f m ost o f th e
n o n -a g g l u t i n â t in g c e l l s . H ow ever, in many m ix tu r e s , t h o u ^
c o n j u g a t io n , once s t a r t e d , p r o c e e d e d n o r m a lly , th e m a j o r it y
o f th e a n im a ls s t i l l w ere u n a b le t o a g g l u t i n a t e . The r e
s u l t s o f t h e s t u d y on t h e v i a b i l i t y o f t h e e x - c o n j u g a n t s ,
w h ich showed a s i m i la r d i f f e r e n c e in t h e tw o l o c a t i o n s
26
u s e d f o r th e w o rk , w o u ld seem t o b ea r o u t th e s u p p o s it io n
o f an u n d e te r m in e d f a c t o r , o r f a c t o r s , o f a p h y s i c a l n a t u r e ,
w h ich w ere r e s p o n s ib l e f o r t h e s e d i f f e r e n c e s . A p p a r e n tly
e i t h e r th e a g g l u t i n a t i v e s u b s ta n c e s e c r e t e d b y th e c e l l ,
was a l t e r e d or d e s t r o y e d , or th e c e l l i t s e l f w as im p a ir e d ,
t h e r e b y i n h i b i t i n g th e r e a c t i o n . The anim a l a a f f e c t e d
r e g a in th e a b i l i t y t o c o n ju g a te a f t e r a p e r io d o f t im e .
T h is f a c t , b e s id e th a t o f t h e c o n s i s t e n c y o f th e m a tin g
r e a c t i o n , te n d s t o e n tr e n c h m ore s o l i d l y , t h e chrom osom es
and g e n e s in t h e r o l e o f th e f i n a l c o n t r o l l i n g m echan ism s
o f t h e m a tin g r e a c t i o n s .
N o r m a lly , su c h a h i ^ v i a b i l i t y o f e x -c o n ju g a n ts a s
r e c o r d e d in T a b le IV i s th e r e s u l t o f a comp a ta b i l i t y e x
h i b i t e d o n ly b y a n im a ls b e lo n g in g t o th e same v a r i e t y .
Ga8 was t e s t e d and fo u n d t o b e lo n g t o V a r ie t y I , and t h e r e
f o r e , ju d g in g from th e r e s u l t s o b ta in e d i n t h e s e e x p e r i
m e n ts , t h e C h in e se c lo n e i s a l s o a member o f V a r ie t y I ,
th ou gh o f a d i f f e r e n t m a tin g t y p e . The h i g i p e r c e n ta g e
o f v i a b l e e x -c o n ju g a n ts p lu s th e n u c le a r ch a n g es n o r m a lly
e x p e c te d b e tw e e n two members o f a v a r i e t y , an d i n the
u s u a l le n g t h o f tim e e x p e c te d fo r c o n su m a tio n o f c o n ju g a
t i o n s t r o n g l y s u b s t a n t i a t e t h i s .
C o n s id e r in g t h e s e f a c t s ; ( l ) a f a i r l y h i ^ p e r c e n t
age o f m a tin g , (2 ) a low m o r t a l i t y among e x -c o n j u g a n t s , and
27
(3 ) n u c le a r ch a n g es n o r m a lly e x p e c te d in c o n j u g a t io n b e
tw e en a n im a ls w it h in t h e same v a r i e t y , i t i s a p p a re n t t h a t
members o f a s i n g l e v a r i e t y , t h o u ^ from d i f f e r e n t p a r t s
o f th e w o r ld , can and do m a in ta in s i m i l a r i t i e s o f g e n ic
m ake-up t o a llo w a t l e a s t c o m p a t a b ilit y in m a t in g . A l -
t h o u ^ th e g e o g r a p h ic a l b a r r ie r s im p osed up on t h e s e tw o
c lo n e s o f Param ecium b u r s a r ia seem a lm o st in s u p e r a b le ,
t h i s w ork adds e v id e n c e t h a t th e e x is t e n c e o f s p e c i f i c
m a tin g t y p e s i s u n i v e r s a l . In th e l i ^ t o f e v o lu t io n a r y
s t u d i e s upon o t h e r t y p e s o f o r g a n is m s , i t i s p o s s i b l e t h a t
t h e s e v a r i e t i e s and m a tin g ty p e s a r o s e from o n e o r i g i n a l
t y p e , and d iv e r g e d t h r o u ^ m u ta tio n or a c c u m u la tio n o f
m u ta tio n s when s e p a r a te d by some e f f e c t i v e form o f i s o l a
t i o n su ch a s g e o g r a p h ic a l i s o l a t i o n #
SUM M ARY
1 . The tw o c l o n e s , Pu9 and G a8, u s e d in t h i s w o rk ,
from C hina and C a l i f o r n i a , show ed no m arked m o r p h o lo g ic a l
d i f f e r e n c e s in e i t h e r th e n u c le a r or c y to p la s m ic com ponents#
2# Tim ed s t a g e s o f c o n ju g a tio n r e v e a le d n u c le a r
ch a n g es n o r m a lly e x p e c te d in a m a tin g b e tw e en tw o c lo n e s
o f d i f f e r e n t m a tin g ty p e s w it h in th e same v a r i e t y ; t h r e e
p r e garnie d i v i s i o n s , an ex ch a n g e o f p r o n u c le i w it h s u b s e
q u en t fo r m a tio n o f a s y n c a r y o n , and t h r e e p o s t z y g o t i c d i v i
s i o n s o f th e sy n ca ry o n #
3# A g g lu t in a t iv e t e n d e n c ie s i n some m ix tu r e s w ere
a b n o rm a l, p r o b a b ly due to t h e in f lu e n c e o f some d e t r im e n t a l
p h y s i c a l f a c t o r #
4# The l o s s , f o r a p e r io d o f t im e , and th e r e e s t a b
lis h m e n t o f th e a b i l i t y to c o n ju g a te p o s s i b l y i n d ic a t e t h a t
some s u b s ta n c e i n t h e medium o r th e c e l l i t s e l f was a l t e r e d ,
a s t h o s e a n im a ls w hich c o n ju g a te d e x h i b i t e d norm al n u c le a r
p r o c e s s e s # H ow ever, th e n u t r i t i v e c o n d it io n o f t h e c lo n e s
may h a v e b e e n th e m ain f a c t o r .
5# Of th e 2 ,6 3 6 a n im a ls in 13 m ix t u r e s , 1 ,6 4 6 w ere
p a i r e d , y i e l d i n g a t o t a l p e r c e n ta g e o f p a i r in g o f 6 1 .1 .
6# In th e v i a b i l i t y s t u d i e s 65 o f t h e 442 e x -
co n ju g a n ts d ie d # The o v e r - a l l s u r v i v a l p e r c e n ta g e was
8 7 . 6 .
29
7 . The good p e r c e n ta g e o f m a tin g in o t h e r m ix t u r e s ,
th e v i a b i l i t y o f th e e x -c o n j u g a n t s , and norm al n u c le a r p r o
c e s s e s are a l l i n d i c a t i o n s t h a t b o th a n im a ls a re o f t h e
same v a r i e t y .
LITERATURE CITED
C hen, T. T . 1 9 4 0 a P o ly p lo id y and i t s o r i g i n in PsLrameoium.
J o u r . H e r e d it y 31^ , 1 7 5 -1 8 4 (6 t e x t - f i g s . )
_______ 1940b A f u r t h e r s tu d y on p o ly p lo id y i n Param e
cium * J o u r . H e r e d it y , 51 ( 5 ) , 2 4 9 -2 5 1 (1 t e x t -
f i g J
1 9 4 0 c P o ly p lo id y in Param ecium b u r s a r i a . P r o c .
H a t. A ca d . S c i . , 26 ( ^ , 2 3 9 -2 4 0 ( l t e x t - f i g . )
_______ 1940d E v id e n c e s o f ex ch a n g e o f p r o n u c le i d u r in g
c o n ju g a tio n i n Param ecium b u r s a r i a . P r o c . H a t.
A c a d . S c i . , 2j6 , 2 4 1 - 2 4 3 .
_ _ _ _ 1940e C o n ju g a tio n o f th r e e a n im a ls in Param ecium
b u r s a r i a . P r o c . N a t. A cad . S c i . , 2 ^ , 2 3 1 -2 3 8
(1 3 t e x t - f i g s . )
________ 1 9 4 0 f C o n ju g a tio n i n Param ecium b u r s a r ia b e tw e e n
a n im a ls w it h v e r y d i f f e r e n t chromosome numbers and
b e tw e e n a n im a ls w ith and w ith o u t m i c r o n u c l e i .
P r o c . N a t. A cad . S c i . , 2 6 ( 4 ) , 2 4 3 - 2 4 6 .
1944 S t a i n in g n u c l e i and chrom osom es in p r o t o z o a .
S t a i n T e c h n o l., jL9 (3 j , 8 3 -9 0 (2 t e x t - f i g s . )
_______ 1 9 4 6 a V a r i e t i e s and m a tin g t y p e s in Param ecium
b u r s a r i a . I . New v a r i e t y and t y p e s from E n g la n d ,
I r e l a n d , and C z e c h o s lo v a k ia . P r o c . N a t. A cad .
S c i . , 32 ( 6 ) , 1 7 3 -1 8 1 (5 t e x t - f i g s . )
51
________ 1946b C o n ju g a tio n i n Param ecium b u r s a r ia # I#
C o n ju g a tio n o f t h r e e a n im a ls# Jou r# M orph#,
7 8 ( 5 ) , 3 5 3 -3 7 4 (2 1 p i s . , 1 t e x t - f i g . )
________ 1 9 4 6 c C o n ju g a tio n i n Param ecium b u r s a r i a . I I .
N u c le a r phenom ena i n l e t h a l c o n ju g a tio n b e tw e en
v a r i e t i e s . J o u r . M o rp h ., J79 ( 2 ) , 1 2 5 -2 0 8 (5 4 p i s . )
_______ 1946d T em porary p a i r fo r m a tio n i n Param ecium b u r
s a r i a . B i o l . B u l l . , 91 ( 1 ) , 1 1 2 - 1 1 7 .
D i l l e r , W. P . 1 9 3 6 . N u c le a r r e o r g a n iz a t io n p r o c e s s in
Param ecium a u r e l i a , w ith d e s c r i p t i o n s o f autogam y
and ”h e m ix is ” . J o u r . M orp h ., M ( 1 ^ ) , 1 1 - 6 7 .
_ _ _ _ _ _ 1940 N u c le a r v a r i a t i o n in Param ecium cau d atu m .
J o u r . M o rp h ., ^ (^) , 6 0 5 -6 2 9 (4 p i s . )
H am burger, C la r a 1 9 0 4 . D ie k o n ju g a tio n von param ecium
b u r s a r ia f o c k e . A r c h iv f u r P r o t is t e n k u n d e , 4 ( ^ ) ,
1 9 9 -2 3 9 (2 t e x t - f i g s . )
H a r r is o n , J . A . 1945 S e r e o l o g i c a l e v id e n c e o f c y to p la s m ic
ex ch a n g e d u r in g c o n ju g a tio n i n Param ecium b u r s a r ia .
S c i e n c e , 1 0 2 , 3 7 7 - 3 7 8 .
J e n n in g s , H . S . 1 9 3 8 . S e x r e a c t i o n t y p e s and t h e i r i n t e r
r e l a t i o n i n Param ecium b u r s a r i a . I . and I I .
C lo n e s c o l l e c t e d from n a t u r a l h a b i t a t s • P r o c .
N a t. A cad . S c i . , 2 4 ( 3 ) , 1 1 2 -1 1 7 (1 f i g . ) , 1 1 7 -
120 (2 f i g s . )
52
_ _ _ _ _ _ 1 9 3 9 G e n e tic s o f Param ecium b u r s a r i a . I . M atin g
ty p e s and g r o u p s , t h e i r i n t e r r e l a t i o n s and d i s
t r i b u t i o n ; m a tin g b e h a v io r an d s e l f s t e r i l i t y #
G e n e t i c s , 2 ^ ( 2 ) , 2 0 2 -2 3 3 (7 f i g s . )
_ _ _ _ _ _ 1941 G e n e tic s o f Param ecium b u r s a r i a . I I . S e l f
d i f f e r e n t i a t i o n and s e l f f e r t i l i z a t i o n o f c l o n e s .
P r o c . Am er. P h i l . S o c . , ^ (].) , 2 5 - 4 8 (3 f i g s . )
_ _ _ _ 1942 G e n e tic s o f Param ecium b u r s a r i a . I I I . I n
h e r it a n c e o f m a tin g ty p e i n c r o s s e s and c lo n a l
s e l f - f e r t i l i z a t i o n s . G e n e t i c s , 27 ( 2 ) , 1 9 3 - 2 1 1 .
_ _ _ _ _ _ 1944a Param ecium b u r s a r ia ; l i f e h i s t o r y . I .
Im m a tu r ity , m a t u r it y , and a g e . B i o l . B u l l . , 86
( 3 ) , 1 3 1 - 1 4 5 .
_ _ _ _ _ _ 1944b Param ecium b u r s a r ia ; l i f e h i s t o r y I I . Age
and d e a th o f c lo n e s in r e l a t i o n to th e r e s u l t s o f
c o n j u g a t io n . J o u r . E x p . Z o o l . , 96 (1 ,), 1 7 - 5 2 .
J e n n in g s , A . S . and P a u lin e O p itz 1944 G e n e t ic s o f P a ra
m ecium b u r s a r ia IV . A f o u r t h v a r i e t y from R u s s ia .
L e t h a l c r o s s e s w ith an A m erican v a r i e t y . G e n e t ic s ,
2 9 ( 6 ) , 5 7 6 - 5 8 3 .
K im b a ll, R . P . 1943 M atin g t y p e s i n t h e c i l i a t e d P r o t o z o a .
Q u a r t. R e v . B i o l . , 3^ (jL) , 3 0 - 4 5 .
M aupas, E . 1889 Le r a je u n is s e m e n t k aryogam iq u e c h e z l e s
C i l i é s . A r ch . Z o o l. e x p . e t g e n . , s e r . I I , 7^,
1 4 9 - 5 1 7 .
33
S o n n e b o r n , T . M. 1937 I n d u c tio n o f e n d o m ix is i n
Param ecium a u r e l i a . B i o l . B u l l . , 72 ( 2 ) , 1 9 6 -
202.
________ 1947 Param ecium and E u p lo te s A d vances in G e n e t i c s .
I . New Y ork: A cadem ic P r e s s I n c . , P u b li s h e r s ,
2 6 3 -3 5 8 .
34
.?■ /
Tr * '
)
F ig u r e 1
i n g l e a n im a l from th e GaS c lo n e of
Param ecium b u r s a r i a
F ig u r e 2
C o n ju g a tio n betw een c lo n e s Ca8 and Fu9
of Param ecium b u r s a r i a . About 7 h o u rs
a f t e r o n s e t of c o n j u g a t i o n .
3b
F ig u r e 3
P ro p h a se i n c o n j u g a t i o n o f two c lo n e s of P.
b u r s a r i a . About 8 h o u rs a f t e r o n s e t of con
j u g a t i o n .
37
F i g u r e 4
L ate p ro p h a se i n t h e second p regarnie
d i v i s i o n . About 12 h o u rs a f t e r o n s e t
o f c o n j u g a t i o n .
38
F ig u re
T e lo p h a se of t h i r d p regarnie d i v i s i o n ,
b e f o r e exchange of p r o n u c l e i . About
16 h o u rs a f t e r o n s e t of c o n j u g a t i o n .
39
i :
F i g u r e ô
Exchange of p r o n u el e i . About 17 h o u rs
a f t e r o n s e t of c o n j u g a t i o n .
40
F ig u r e 7
F o rm a tio n of s y n c a ry o n . About
17 h o u rs a f t e r o n s e t of c o n ju
g a t i o n .

Asset Metadata

Creator Graves, Ghent (author)

Core Title Nuclear changes and viability of exconjugants in conjugation between Chinese and American Paramecia

Contributor Digitized by ProQuest (provenance)

Degree Master of Science

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Biological Sciences,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c39-276645

Unique identifier UC11317207

Identifier EP67193.pdf (filename),usctheses-c39-276645 (legacy record id)

Legacy Identifier EP67193.pdf

Dmrecord 276645

Document Type Thesis

Format application/pdf (imt)

Rights Graves, Ghent

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

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University of Southern California Dissertations and Theses