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Studies of deoxyribonucleic acids in spermatocytes of Cancer productus Randall Astell, Caroline Ruth 1966

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STUDIES OF DEOXYRIBONUCLEIC ACIDS IN SPERMATOCYTES OF CANCER PRODUCTUS RANDALL by CAROLINE RUTH ASTELL B.Sc,  U n i v e r s i t y of B r i t i s h Columbia, 1964  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of ZOOLOGY  We accept t h i s t h e s i s as conforming r e q u i r e d standard  THE  to the  UNIVERSITY OF BRITISH COLUMBIA May, 1966  In  presenting  requirements Columbia, for  agree  reference  extensive granted It  I  for  is  by  and  copying the  Department  gain  thesis  in  partial  advanced  degree  at  the  that  the  Library  study.  I  of  thesis  Head  understood  financial  an  this  this of  that  shall  my  further for  Department  copying not  be  or  University  make  agree  allowed  Columbia  it  that  scholarly or  by  his  publication  of  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  shall  fulfilment  without  of  of  the  British  freely  available  permission purposes  of  for  may  be  representatives. this  thesis  my w r i t t e n  for  permission  TABLE OF CONTENTS Page I.  SPERMATOGENESIS IN CANCER PRODUCTUS Abstract L i s t o f Tables L i s t of Figures  i i a iiia  Introduction  1  M a t e r i a l s and Methods 1. T i s s u e s e c t i o n s 2. T i s s u e squashes 3. D e t e r m i n a t i o n o f DNA r e p l i c a t i o n by i n c o r p o r a t i o n o f H^-thymidine, in vitro  3 3 3  Results 1. S e c t i o n s o f the t e s t e s and vasa deferentia 2. Squashes o f t e s t i c u l a r t i s s u e 3. In v i t r o i n c o r p o r a t i o n of H^thymidine i n t o crab t e s t i c u l a r  5  tissue  II.  ia  4  5 11  16  Discussion  17  Summary  19  Literature Cited  20  INTRACELLULAR LOCALIZATION OF AN ANOMALOUS DNA IN CANCER PRODUCTUS Abstract L i s t o f Tables L i s t of F i g u r e s Introduction  ib iib iiib 22  Page M a t e r i a l s and Methods 1. I s o l a t i o n of n u c l e i 2. In v i t r o i n c o r p o r a t i o n of H^thymidine and autoradiography (i) I n c o r p o r a t i o n of H^thymidine, i n v i t r o (ii) Autoradiography  24 27  Results 1. I s o l a t i o n of n u c l e i 2. In v i t r o i n c o r p o r a t i o n of H^thymidine i n t o crab DNA and autoradiography (i) In v i t r o i n c o r p o r a t i o n of H -thymidine . (ii) Autoradiography  31 31  Discussion  46  Summary  54  Literature Cited  55  3  27 27 28  35 35 38  ACKNOWLEDGMENT I would l i k e to express my a p p r e c i a t i o n t o Drs. D.T.  Suzuki  and M. Smith f o r suggesting  the problem and  t h e i r d i r e c t i o n d u r i n g the course of t h i s  study.  I would a l s o l i k e t o thank Dr. R.P. K l e t t f o r the work he has done i s o l a t i n g and c h a r a c t e r i z i n g the DNA. Without the i n f o r m a t i o n he has provided,  the r e s u l t s of  the n u c l e a r i s o l a t i o n and a u t o r a d i o g r a p h i c  s t u d i e s could  not have been i n t e r p r e t e d c r i t i c a l l y . I am g r a t e f u l to Drs. L.K. P i t e r n i c k and C.V. Finnegan f o r t h e i r c r i t i c a l r e a d i n g of t h i s  thesis.  SPERMATOGENESIS IN CANCER PRODUCTUS  ABSTRACT Spermatogenesis s t u d i e d t o determine  i n Cancer productus R a n d a l l was  the a v a i l a b i l i t y of c e l l s and chrom-  osomes s u i t a b l e f o r the l o c a l i z a t i o n of crab dAT.  I t was  found that throughout most of the year the r e p r o d u c t i v e t r a c t contained r e s t i n g spermatogdnial c e l l s and a few r e s i d u a l spermatophores.  Between A p r i l and J u l y there was  a b u r s t of m e i o t i c a c t i v i t y observed both c y t o l o g i c a l l y and by the i n c o r p o r a t i o n of a r a d i o a c t i v e DNA p r e c u r s o r , tritiated-thymidine. The mean h a p l o i d chromosome number  f o r C.  produc-  tus was found to be 47.0 + 4.1, lower than the p r e v i o u s l y r e p o r t e d number of 58.  iia  LIST OF TABLES Table  Page I.  Chromosome counts on primary spermatocytes o f C. productus  15  iiia  LIST OF FIGURES Figure 1.  Page D o r s a l view o f the haemocele o f a Cancer productus male (carapace '. '• "  7  2a. S e c t i o n of t e s t e s i n r e g i o n I I  8  removedT"!  b. S e c t i o n o f t e s t e s i n r e g i o n I I I  8  c. S e c t i o n of t e s t e s i n lower r e g i o n III  8  3.  S e c t i o n of the vas deferens ( r e g i o n IV)  9  4a,b, and c. S e c t i o n i n r e g i o n II of the t e s t e s showing the synchrony of the c e l l c y c l e at any one p o i n t . . . . . . . .  10  5a, and b. Squashes of primary spermat o c y t e s i n l a t e prophase  13  6a, and b. P o l a r view of metaphase i n squashes of primary spermatocytes  14  1 INTRODUCTION An  anomalous DNA  Sueoka (1961) was  subsequently  a l t e r n a t i n g adenine and percent  guanine and Although  the chromatin are a few  d i s c o v e r e d i n the genus Cancer by shown to c o n s i s t p r i m a r i l y of  thymine r e s i d u e s with l e s s than  c y t o s i n e (Swartz et a l . , 1961). c e l l u l a r DNA  of the nucleus  exceptions  i s usually associated with  (de R o b e r t i s ejt alL. , 1960) , there  such as the s m a l l amounts of DNA  i n c h l o r o p l a s t s (Edelman <et jal. , 1965) and Reich, 1965).  1964;  two  and mitochondria  Rabinowitz e t a l . , 1965;  E x t r a n u c l e a r DNA  found  Suyama and  Preer,  u s u a l l y c o n s t i t u t e s l e s s than  percent of the t o t a l c e l l u l a r DNA,  (Luck  one  although Corneo et a l .  (1966) r e p o r t t h a t m i t o c h o n d r i a l DNA  from yeast  (Saccharomyces  c e r e v i s i a e and Saccharomyces c a r l s b e r g e n s i s ) makes up ten cent of the c e l l u l a r DNA. r i c h i n adenine and  T h i s l a t t e r DNA  thymine (75%).  l i n g the DNA  location.  shown to be  In order to d e f i n e the  b i o l o g i c a l s i g n i f i c a n c e of crab dAT, mine i t s i n t r a c e l l u l a r  was  per-  i t i s d e s i r a b l e to d e t e r -  T h i s has been done by  w i t h a r a d i o a c t i v e i s o t o p e and  locating  label-  the  l a b e l by the a u t o r a d i o g r a p h i c methods d e s c r i b e d i n S e c t i o n II (page 2 2 ) . The necessary  data r e p o r t e d i n S e c t i o n I were provided  as the  background i n f o r m a t i o n f o r the l o c a l i z a t i o n s t u d i e s .  P r e l i m i n a r y work i n d i c a t e d t h a t t e s t i c u l a r t i s s u e i s most s u i t a b l e f o r o b s e r v a t i o n of d i v i d i n g c e l l s necessary  f o r the  d e t e r m i n a t i o n of i n c o r p o r a t i o n of r a d i o a c t i v e p r e c u r s o r s  2  d u r i n g DNA s y n t h e s i s . observed  D i s t i n c t chromosomes can a l s o be best  i n d i v i d i n g c e l l s o f the t e s t e s .  T h e r e f o r e a study  of spermatogenesis i n the crab Cancer productus  R a n d a l l was  undertaken to determine the a v a i l a b i l i t y o f s u i t a b l e for  cells  the a u t o r a d i o g r a p h i c l o c a t i o n of crab dAT d u r i n g the  year.  3 MATERIALS AND Cancer  productus was  METHODS  chosen f o r t h i s study  i t has been shown (Smith, 1963)  because  that t h i s s p e c i e s c o n t a i n s  t h i r t y percent crab dAT whereas another west coast s p e c i e s , Cancer magister Dana, c o n t a i n s o n l y ten percent crab Mature Cancer  productus males were c o l l e c t e d  and V i c t o r i a , B.C.  from May  l o g i c a l examination,  1964  dAT.  at Vancouver  to March 1966.  For c y t o -  the t e s t e s and vasa d e f e r e n t i a were  d i s s e c t e d from l i v e animals and immediately  fixed i n ethyl  alcohol: acetic acid  then passed  (3:1).  The t i s s u e was  through two changes of e t h y l a l c o h o l to 95% a l c o h o l and  then  s t o r e d i n 70% a l c o h o l at 4°C. 1.  Tissue Sections For t i s s u e s e c t i o n s , s m a l l p i e c e s of f i x e d  were dehydrated  to e t h y l a l c o h o l ,  embedded i n p a r a f f i n .  Sections 5^  tissue  c l e a r e d i n benzene, and t h i c k were prepared  s t a i n e d "either by the Feulgen procedure or w i t h methyl pyronin Y ( C u l l i n g ,  1963).  and green-  The s l i d e s were then dehydrated  to xylene and mounted i n permount. 2.  T i s s u e Squashes The f i x e d  tissue  (stored  i n 70% a l c o h o l at 4 C) G  was  g r a d u a l l y hydrated to d i s t i l l e d water and s t a i n e d by the Feulgen procedure.  A f t e r s t a i n i n g , the t i s s u e was  f r e e z i n g i n 45% a c e t i c a c i d .  stored  by  (The best s t a i n i n g r e s u l t s w i t h  4 Feulgen f o r crab t i s s u e were obtained IN HC1  f o r 20 minutes at 60°C.)  c y t o l o g i c a l examination by of 45%  a c e t i c a c i d and  squashing.  The  t i s s u e was  prepared f o r  t e a s i n g a small piece i n a drop  to be prepared, the squash was c o v e r s l i p removed.  The  by a c i d h y d r o l y s i s i n  When permanent s l i d e s were  f r o z e n on dry i c e and  s l i d e was  graded a l c o h o l s e r i e s (70%  the  then passed through a  to e t h y l a l c o h o l ) to xylene  and  then mounted i n permount. 3.  D e t e r m i n a t i o n of DNA Tritiated-Thymidine, The  R e p l i c a t i o n by I n c o r p o r a t i o n in vitro  i n v i t r o i n c o r p o r a t i o n of t r i t i a t e d - t h y m i d i n e  i n t o the t e s t e s was  studied using  the procedure d e s c r i b e d  Methods and M a t e r i a l s , S e c t i o n I I (page 27). were taken throughout the year and  Tissue  cytologically.  Following  thymidine, the DNA  the i n c o r p o r a t i o n of  from the t i s s u e was  (This was  was  examined  tritiated-  i s o l a t e d and  c a r r i e d out by Dr. R.P.  charac-  K l e t t of  the  F i s h e r i e s Research Board of Canada.  See  f o r the d e t a i l s of the i s o l a t i o n and  characterization.)  amount of i n c o r p o r a t i o n was  measured by  K l e t t ejt a_l. (1966)  i s o l a t i n g the  and measuring the r a d i o a c t i v i t y of the t o t a l DNA DNA  and  crab  dAT)  was  estimated from the UV  unit i s equivalent  i n CPM  to 50  per  ^g  DNA.  (The DNA  absorbance at 260 /^.g DNA  in  samples  p a r t of each sample  used f o r an i n c o r p o r a t i o n study w h i l e the r e s t was  terized.  of  (Sueoka and  m/A  The DNA  (both main concentration .  An  ODr,™  Cheng, 1962) .)  5  RESULTS The r e p r o d u c t i v e t r a c t of Cancer c o n s i s t s of two haemocoele.  productus males  lobes l y i n g d o r s a l and a n t e r i o r i n the  The organ i s a s o f t , h i g h l y t u b u l a r s t r u c t u r e  p a r t i a l l y embedded i n the hepatopancreas.  There i s an  i n c r e a s e i n the s i z e of the t u b u l e s towards the c e n t r e of the haemocoele where they lead i n t o the vasa d e f e r e n t i a on each s i d e ) .  Although i t i s d i f f i c u l t  to determine  (one  from  the gross morphology where the t e s t e s end and the vasa d e f e r e n t i a . begin, S p a l d i n g (1942) observed a r a t h e r sharp s i o n l i n e i n C a r c i n u s maenus m i c r o s c o p i c a l l y .  divi-  He r e p o r t e d  t h a t the e p i t h e l i a l c e l l s l i n i n g the t u b u l e s change suddenly from simple squamous e p i t h e l i a l c e l l s to columnar cells.  This transition i n e p i t h e l i a l c e l l  observed i n Cancer  productus and was  epithelial  types was  also  used as the d i v i s i o n  l i n e between the t e s t i s and vas d e f e r e n s .  The vasa  deferentia  open to the o u t s i d e j u s t p o s t e r i o r to the bases of the l a s t p a i r of w a l k i n g l e g s . 1.  S e c t i o n s of the Testes and Vasa D e f e r e n t i a S e c t i o n s of the t e s t e s and vasa d e f e r e n t i a  during  the s p r i n g showed that the r e p r o d u c t i v e system i n the male could be d i v i d e d i n t o approximately four r e g i o n s a c c o r d i n g to the c e l l types found w i t h i n the t u b u l e s . are the upper  testis  testis  and the vas deferens ( I V ) .  (III),  These four r e g i o n s  ( I ) , the middle t e s t i s  ( I I ) , the lower  (See F i g u r e 1 f o r  6  the approximate  extent of these f o u r r e g i o n s . )  Region  found to c o n t a i n mostly spermatogonial c e l l s and primary spermatocytes. spermatogonial c e l l s ,  Region II c o n t a i n s a narrow band of a l a r g e r e g i o n of spermatocytes,  and  ( F i g u r e 2a).  I I I c o n s i s t s of a l a r g e area of f r e e spermatids  spermatozoa w i t h bands of d i v i d i n g spermatocytes b).  was  resting  a s m a l l band of spermatids undergoing maturation Region  I  and  (Figure 2  The band of spermatogonial c e l l s c h a r a c t e r i s t i c of  r e g i o n s I and I I was  absent i n r e g i o n I I I .  In the  lower  part of r e g i o n I I I the t r a n s i t i o n from simple squamous e p i t h e l i a l to columnar e p i t h e l i a l c e l l s i s observed 3b).  T h i s d i v i s i o n of a tubule i n t o areas of c e l l  corresponds to the r e g i o n s d e s c r i b e d by B i n f o r d the A t l a n t i c crab Menippe mercenaria (1918) i n Cancer  magister.  (Figure types  (1913) i n  (Say) and by Fasten  Region IV, the vas deferens  packed w i t h spermatophores ( F i g u r e 3 ) .  was  The w a l l s of the  t u b u l e s are made up of a s i n g l e l a y e r of columnar  epithelial  cells. Thus, d u r i n g the s p r i n g , Cancer  the r e p r o d u c t i v e t r a c t of  productus males r e p r e s e n t s a wave of m e i o t i c a c t i v i t y  showing a p r o g r e s s i o n of stages b e g i n n i n g w i t h r e s t i n g spermatogonial c e l l s i n the upper t e s t i s  (Region I) and end-  i n g w i t h mature spermatozoa enclosed i n spermatophores i n the vas was  deferens. striking;  The synchrony  of the c e l l s w i t h i n a tubule  a l l c e l l s of one type w i t h i n a t u b u l e were at  the same stage of c e l l d i v i s i o n  (see F i g u r e 4a, b and c ) .  7a  F i g u r e 1.  D o r s a l view of the haemocoele o f a Cancer p r o d u c t u s male (carapace  removed)  T - t e s t e s ; HP - hepatopancreas; V - vas d e f e r e n s ; H - h e a r t ; I , I I , I I I , and IV - r e g i o n s o f the r e p r o d u c t i v e t r a c t (see t e x t f o r d e t a i l s ) .  HP T  V H  8a  Figure  2.  a - S e c t i o n of t e s t i s i n r e g i o n I I . Magnification  120X  b - S e c t i o n of t e s t i s i n r e g i o n I I I . Magnification  120X  c - S e c t i o n of t e s t i s i n lower r e g i o n I I I . M a g n i f i c a t i o n 120X SG - spermatogonia! c e l l s ; SM - spermatocytes; SPTD - spermatids; SZ - spermatozoa; CE - columnar e p i t h e l i a l  cells.  D  9a  F i g u r e 3.  Section GE  of the vas deferens ( r e g i o n I V ) . Magnification 120X  - columnar e p i t h e l i a l  tophores.  cells;  SP  - sperma  9  10a  F i g u r e 4.  S e c t i o n s i n r e g i o n I I of the t e s t i s showing the synchrony of the c e l l c y c l e at any one p o i n t , a - magnification  120X  b - m a g n i f i c a t i o n 455X c - magnification  1200X.  10  c  11 Once the r e l a t i v e l o c a t i o n of the v a r i o u s c e l l types was  determined,  f u r t h e r c y t o l o g i c a l study was  almost e x c l u s i v e l y on squashed of complete  t i s s u e which permits the study  c e l l s i n each stage of  spermatogenesis.  A survey of t e s t i c u l a r t i s s u e throughout showed that m e i o t i c a c t i v i t y as determined of d i v i d i n g c e l l s was and J u l y .  performed  the year  by the  presence  l i m i t e d to the i n t e r v a l between A p r i l  A f t e r that time, the s i z e of the t e s t e s decreased  and the number of spermatocytes  diminished.  During the r e s t  of the year the only c e l l s observed were spermatogonial i n the t e s t i s proper. diameter, but s t i l l  cells  The vas deferens a l s o decreased i n  contained a few spermatophores,  presumably  a r e s i d u e from the b u r s t of m e i o t i c a c t i v i t y d u r i n g the previous s p r i n g . The m e i o t i c c e l l s of C. productus are very s m a l l ; i t was  estimated t h a t the diameters of primary and  spermatocytes were approximately 6 - 8 ^ tively.  in size  i n diameter.  obtained.  2.  respec-  were a p p r o x i -  Spermatophores were q u i t e v a r i a b l e  (Figure 3) depending  they c o n t a i n e d .  phore  and 4 - 6 A ,  The spermatids and mature, spermatozoa  mately 3/A  secondary  on the number of  spermatozoa  E s t i m a t e s v a r y i n g from 20 - 60ju.  were  S p a l d i n g (1942) estimated the s i z e of a spermato-  from C a r c i n u s maenus to be 4 5 ^  by 35yu .  Squashes of T e s t i c u l a r T i s s u e Much of the work was  concentrated on the primary  12 spermatocytes  to p r o v i d e i n f o r m a t i o n necessary f o r p o s s i b l e  s t u d i e s on chromosomes. to be p r o t r a c t e d .  Interphase i n these c e l l s  appears  When prophase (begins, the chromatin o f the  nucleus condenses i n t o a Feulgen p o s i t i v e mass.  At l a t e  prophase,  (distilled  a f t e r a 20 minute hypotonic treatment  water) f o l l o w e d by standard f i x a t i o n and s t a i n i n g procedures, the chromosomes are c l e a r l y v i s i b l e i n squashed p r e p a r a t i o n s . In many cases, p a i r e d homologues could be seen, c o n f i r m i n g that the c e l l s b e i n g observed were primary spermatocytes (see F i g u r e 5a and b ) . g r e a t l y condensed structures  By e a r l y metaphase the chromosomes were and appear- d as s m a l l F e u l g e n - p o s i t i v e  ( F i g u r e 6a and b ) .  The s i z e o f these " g r a n u l a r "  chromosomes was estimated to be 0.5 - 0 . 7 ^ The  i n diameter.  l a r g e number o f chromosomes i n C. productus i s  c o n s i s t e n t w i t h the numbers found i n c r u s t a c e a n s i n g e n e r a l (see Makino, 1951).  F a s t e n (1924) has r e p o r t e d that the  h a p l o i d number i n C. productus i s 58.  H i s counts were made  on p o l a r views o f metaphase i n primary spermatocytes smears.  However chromosome counts o f the same c e l l  i n tissue types i n  the present study r e s u l t e d i n a mean h a p l o i d number of 47.0 i  4.1 (Table I ) .  I t can be seen that t h e r e i s c o n s i d e r a b l e  v a r i a t i o n although none of the numbers observed are as h i g h as the h a p l o i d number r e p o r t e d by F a s t e n (1924). counts on c e l l s i n l a t e prophase and b) were a l s o l e s s than 58.  Chromosome  (such as those i n F i g u r e 5a In these p r e p a r a t i o n s , how-  ever, the chromosomes were spread out over a f a i r l y wide area by the squashing procedure, so that d i s t i n c t i o n between  13a  F i g u r e 5.  a and b - Squashes of primary spermatocytes i n l a t e prophase.  PH - p a i r e d  homologues.  M a g n i f i c a t i o n 1920X  13  b  14a  F i g u r e 6.  a and b - P o l a r view of metaphase i n squashes of primary spermatocytes.  M a g n i f i c a t i o n 1920X  1°SM - primary spermatocytes; 2°SM - secondary  spermatocytes.  14  b  15  Table I.  Chromosome counts on p r i m a r y s p e r m a t o c y t e s of C. p r o d u c t u s . (Counted i n p o l a r metaphase views.)  45  42  45  49  47  55  46  47  43  46  46  49  49  50  44  45  49  47  45  55  48  46  47  57  38  51  40  45  x = 47.0  +  4.1  16 chromosomes b e l o n g i n g to adjacent c e l l s c o u l d not always be made w i t h c e r t a i n t y . The mature sperm when s t a i n e d by the Feulgen procedure  showed that the DNA of the c e l l i s contained i n  the p e r i p h e r a l r i n g i n c l u d i n g the r a y s . was F e u l g e n - n e g a t i v e .  The c e n t r a l r e g i o n  T h i s i s i n agreement w i t h Fasten's  (1918) d e t a i l e d study o f spermiogenesis  i n C. magister i n which  he t r a c e d the f a t e of the nucleus and concluded radial  that the  arms or r a y s of the spermatozoon " o r i g i n a t e as o u t -  growths from the n u c l e a r - m i t o c h o n d r i a l cup".  Andrews (1904)  s t a t e d that i n the c r a y f i s h Cambarus a f f i n i s the arms o f the sperm a r i s e from the n u c l e i of the spermatid, although i n two other s p e c i e s of c r a y f i s h , Cambarus v i r i l i s  and Cambarus  immunis, F a s t e n (1914) s t a t e d that the arms of the spermatozoan are c y t o p l a s m i c i n o r i g i n . ^*  In v i t r o I n c o r p o r a t i o n o f T r i t i a t e d - T h y m i d i n e i n t o T e s t i c u l a r Tissue The  Crab  amount o f i n c o r p o r a t i o n o f t r i t i a t e d - t h y m i d i n e  i n t o DNA i n crab t e s t i c u l a r t i s s u e c o r r o b o r a t e s the c y t o l o g i c a l evidence that m e i o s i s o c c u r s d u r i n g A p r i l to e a r l y J u l y .  The  r a d i o a c t i v i t y of the DNA i s o l a t e d from the t i s s u e was measured i n CPM per JUL g DNA. are l i s t e d  Values obtained from seven  below. December 6 March 30 May 15 May 26 June 14 August 18 September 22  55 350 600 460 400 < 20 < 20  experiments  DISCUSSION T h i s study of spermatogenesis  i n Cancer  productus  has shown i t to be very s i m i l a r to that of other c r u s t a c e a n s , p a r t i c u l a r l y crabs and c r a y f i s h  (Menippe mercenaria,  ( B i n f o r d , 1913); C a r c j n i d e s maenus (Broekhuysen); magister  (Fasten, 1918); Cancer  and C. productus  oregonensis,  and Cambarus i r r o r a t u s  (Andrews,  (Fasten, 1914.)  a l l of these s p e c i e s the p e r i o d of m e i o t i c  activity i s brief. are reduced  C  (Fasten, 1924); Cambarus a f f i n i s  1904); Cambarus v i r i l i s In  gracilis,  Cancer  During the r e s t of the year the t e s t e s  i n s i z e and c o n t a i n a few spermatogonial  The vasa d e f e r e n t i a are a l s o reduced  cells.  i n s i z e and c o n t a i n o n l y  a few spermatophores which are i n t e r p r e t e d i n t h i s study to be remnants of the preceeding b u r s t of m e i o t i c a c t i v i t y . M e i o t i c a c t i v i t y and p r o d u c t i o n of mature spermatozoa, however, do not seem to be necessary p r e r e q u i s i t e s to mating a l l s p e c i e s of decapods. , (Broekhuysen  in :  (1936) s t a t e s t h a t once  the crab C a r c i n i d e s maenus had reached m a t u r i t y , although there was  v a r i a t i o n i n the amount of contents of the v a s a  d e f e r e n t i a , mating could occur a l l the year round.  Fasten  (1914), found that f o l l o w i n g mating of the c r a y f i s h C. in  September and October,  were g r e a t l y reduced  the t e s t e s and v a s a d e f e r e n t i a  i n s i z e and the t u b u l e s were e i t h e r  empty or contained a few spermatogonial c e l l s and ual  virilis  spermatozoa u n t i l ; ,the f o l l o w i n g June.  e a r l y s p r i n g ( A p r i l and May)  resid-  He s t a t e s that i n  these animals may  before there i s m e i o t i c a c t i v i t y i n the t e s t e s .  again mate  18 The c o r r e l a t i o n between the c y t o l o g i c a l  determin-  a t i o n of m e i o t i c a c t i v i t y and the b i o c h e m i c a l d e t e r m i n a t i o n of thymidine i n c o r p o r a t i o n as assays of spermatogenesis good, and i t i s suggested  was  that e i t h e r method may be used as  a measure of m e i o t i c a c t i v i t y . The major d i s c r e p a n c y between t h i s study and p r e v i ous r e p o r t s i s w i t h the estimate of chromosome number f o r Cancer  productus.  I t was e x p e r i m e n t a l l y found to be 47.0 i  4.1 ( h a p l o i d number) i n these s t u d i e s whereas the p r e v i o u s count was r e p o r t e d to be 58 (Fasten, 1924). were made on p o l a r views of metaphase I.  Both e s t i m a t e s  The o n l y major  refinement i n techniques was the use of hypotonic treatment of the t i s s u e as a means of producing w e l l d e f i n e d chromosomes. The  l a r g e v a r i a t i o n i n chromosome number i s l i k e l y  due to the t e c h n i c a l d i f f i c u l t i e s encountered chromosomes. is  i n c o u n t i n g the  In any event, f u r t h e r study of chromosome  numbers  suggested. T h i s study of spermatogenesis  i n C. productus has  r e v e a l e d that the c e l l types best s u i t e d f o r chromosome are the primary spermatocytes.  I t has a l s o d i s c l o s e d  studies  that  c e l l s at t h i s stage are a v a i l a b l e f o r the s h o r t p e r i o d of A p r i l to e a r l y J u l y .  Attempts  to induce spermatogenesis  d u r i n g the  r e f r a c t o r y p e r i o d by m a i n t a i n i n g male crabs f o r s i x to e i g h t weeks at 10 - 12°C i n c i r c u l a t i n g sea water w i t h a 16 hour photoperiod i n d i c a t e d that there was some p o s i t i v e These experiments were not pursued.  response.  19 SUMMARY An i n v e s t i g a t i o n of spermatogenesis productus R a n d a l l was  i n Cancer  c a r r i e d out i n order to e s t a b l i s h a  c e l l u l a r system s u i t a b l e f o r a u t o r a d i o g r a p h i c l o c a l i z a t i o n of t r i t i u m - l a b e l l e d crab dAT w i t h i n the c e l l . i t was  Cytologically,  found that throughout most of the year the t e s t e s are  reduced  i n s i z e and c o n t a i n r e s t i n g spermatogonial  cells,  w h i l e the vasa d e f e r e n t i a c o n t a i n a few r e s i d u a l spermatophores.  Beginning i n A p r i l  there was  c y t o l o g i c a l and  chemical evidence of m e i o t i c a c t i v i t y i n the t e s t e s . activity i s short-lived, early  bioThis  l a s t i n g u n t i l the end of June or  July. Chromosome counts on primary spermatocytes  from  p o l a r views of metaphase I gave a mean h a p l o i d number of 47.0  + 4.1.  T h i s i s c o n s i d e r a b l y lower than the p r e v i o u s l y  r e p o r t e d number of 58  (Fasten, 1924).  20 LITERATURE CITED Andrews, E.A., 1904. Breeding h a b i t s of c r a y f i s h . N a t u r a l . 38:165-206.  Amer.  B i n f o r d , R., 1913. The germ c e l l s and the process of f e r t i l i z a t i o n i n the crab, Menippe mercenaria. J . Morph. 24:147-201. Broekhuysen, G.J., 1936. On development, growth and d i s t r i b u t i o n of C a r c i n i d e s maenus ( L . ) . Arch, neere. Z o o l . 2:257-399. Corneo, G., C. Moore, D. Rao Sandi, L . I . Grossman, and J . Marmur, 1966. M i t o c h o n d r i a l DNA i n yeast and some mammalian s p e c i e s . S c i e n c e 151:687-689. C u l l i n g , C.F.A., 1963. Handbook of h i s t o p a t h o l o g i c a l techniques, 2nd e d i t i o n . Butterworths, London. de R o b e r t i s , TE.D.P. , W.W. Nowinski, and F.A. Saez, 1960. General Cytology, t h i r d e d i t i o n . W.B. Saunders Company, P h i l a d e l p h i a and London. Edelman, M., J.A. S c h i f f , and H.T. E p s t e i n , 1965. S t u d i e s of development i n Euglena. XII Two types of s a t e l l i t e DNA. J . Mol. B i o l . 11 :T6"9-774. Fasten, N. , 1914.. Spermatogenesis of the American c r a y f i s h Cambarus v i r i l i s and Cambarus immunis (?) w i t h s p e c i a l r e f e r e n c e to s y n a p s i s and the chromatoid bodies. J . of Morph. 25:587-649. , 1918. Spermatogenesis of the P a c i f i c coast e d i b l e crab Cancer magister Dana. B i o l . B u l l . 34:277-307. , 1921. The e x p l o s i o n of the spermatozoa of the crab Lophopanopeus b e l l u s (Stimpson) Rathbun. B i o l . B u l l . " T H 288-300. of  , 1924. Comparative stages i n the spermatogenesis v a r i o u s Cancer c r a b s . J . of Morph. 39:47-61.  K l e t t , R.P., M. Smith, C R . A s t e l l , D.T. Suzuki, and I.H. Goldberg, 1966. D e o x y r i b o n u c l e i c a c i d s i n crabs of the s u p e r f a m i l y brachyrhyncha. Submitted to J . Mol. B i o l . Luck, D.L., and E. Reich, 1964. DNA i n m i t o c h o n d r i a of Neurospora c r a s s a . Proc. N a t l . Acad. S c i . U.S. 52:931938. Makino, S., 1951. An a t l a s of the chromosome numbers i n animals. The Iowa S t a t e C o l l e g e Press, Ames, Iowa.  21 Rabinowitz, M., J . S i n c l a i r , L. de S a l l e , R. Haselkorn, and H.H. S w i f t , 1965. I s o l a t i o n of DNA from mitochondria of c h i c k embryo h e a r t and l i v e r . Smith, M. 1963. D e o x y r i b o n u c l e i c a c i d s i n crabs of the genus Cancer. Biochem. Biophys. Res. Comm. 10:67-72. S p a l d i n g , J.F., 1942. The nature and formation o f the spermatophore and sperm plug i n C a r c i n u s maenus. Quart. J . M i c r o s . S c i . 83:399-422. Sueoka, N., 1961. V a r i a t i o n and h e t e r o g e n e i t y of base comp o s i t i o n of d e o x y r i b o n u c l e i c a c i d s : A c o m p i l a t i o n o f o l d and new data. J . Mol. B i o l . 3^:31-40. , and T.-Y. Cheng, 1962. F r a c t i o n a t i o n o f n u c l e i c a c i d s w i t h the methylated albumin column. J . Mol. B i o l . _4:161-172. Suyama, Y., and J.R. Preer, 1965. M i t o c h o n d r i a l DNA protozoa. G e n e t i c s 52:1051-1058.  from  Swartz, M.N., T.A. Trautner, and A. Kornberg, 1962. Enzymatic s y n t h e s i s of d e o x y r i b o n u c l e i c a c i d s . XI F u r t h e r s t u d i e s i n nearest neighbour base sequence i n d e o x y r i b o n u c l e i c a c i d s . J . B i o l . Chem. 237:1961-1967.  INTRACELLULAR LOCALIZATION OF AN ANOMALOUS DNA IN CANCER PRODUCTUS  ib ABSTRACT i  Spermatocyte n u c l e i were i s o l a t e d from Cancer productus R a n d a l l by sedimentation of a nonidet P.40 t r e a t e d t i s s u e homogenate i n a l i n e a r sucrose g r a d i e n t . cent of the DNA  Thirty  per-  i n the p r e p a r a t i o n of i n t a c t n u c l e i was crab  dAT. Attempts to l a b e l the crab dAT w i t h  tritiated-  thymidine w h i l e r e p l i c a t i o n of main DNA was i n h i b i t e d by actinomycin D, were u n s u c c e s s f u l . g r a p h i c examination  However, i n an a u t o r a d i o -  of c e l l s i n which both types of DNA were  l a b e l l e d , n i n e t y percent of a l l the g r a i n s developed nucleus.  over the  These o b s e r v a t i o n s i n d i c a t e d that the c e l l u l a r  site  of crab dAT i s i n the nucleus. Prolonged  i n c u b a t i o n s of t i s s u e  ( i n v i v o and i n  v i t r o ) i n order to o b t a i n l a b e l l e d metaphase chromosomes were u n s u c c e s s f u l .  Thus i t i s suggested  that d i s c r i m i n a t i o n  of main DNA  and crab dAT must be accomplished  are f i x e d .  Proposed  heat denatured  a f t e r the c e l l s  methods are a c r i d i n e orange s t a i n i n g of  chromosomes, DNA/DNA h y b r i d i z a t i o n of  t r i t i a t e d - c r a b dAT, or r e p l i c a t i o n of crab dAT r e g i o n s , i n situ.  iib LIST OF TABLES Table  Page I.  II.  III.  IV.  V.  E f f e c t of actinomycin D on the i n c o r p o r a t i o n o f H^-thymidine i n t o crab DNA  37  Number of g r a i n s above the nucleus and cytoplasm i n samples p r e s t a i n e d with Feulgen or p o s t - s t a i n e d w i t h methyl green-pyronin Y  41  The r a t i o of mean number o f n u c l e a r g r a i n s to c y t o p l a s m i c g r a i n s f o r t i s s u e s t a i n e d w i t h Feulgen and methyl green-pyronin Y  42  Number o f g r a i n s per c e l l f o r t i s sue incubated w i t h and without actinomycin D  44  Adjusted mean number of g r a i n s over the nucleus and cytoplasm f o r p r e and p o s t - s t a i n e d t i s s u e (see t e x t for details)  49  iiib LIST OF FIGURES Figure  Page  1.  Procedure f o r i s o l a t i o n of spermatocyte n u c l e i  2.  D i s t r i b u t i o n of d i f f e r e n t c e l l  26  types  and n u c l e i i n the sucrose g r a d i e n t s  32  3.  Isolated  33  4.  Spermatocyte l y s i s i n nonidet P.40  34  5. 6.  I s o l a t e d spermatocyte n u c l e i I n c o r p o r a t i o n of H^-thymidine i n t o main DNA and crab dAT a. c o n t r o l (without actinomycin D) b. 5y/g/ml actinomycin D  36  39  Autoradiographs led tissue  40  7.  spermatocytes  of H^-thymidine l a b e l -  22  INTRODUCTION Two types of DNA i n the crabs Cancer b o r e a l i s and Cancer i r r o r a t u s were demonstrated by Sueoka cesium c h l o r i d e d e n s i t y c e n t r i f u g a t i o n .  (1961) u s i n g  The minor component  was found to have a buoyant d e n s i t y c o r r e s p o n d i n g to a DNA r i c h i n adenine and thymine.  S t u d i e s on the minor DNA com-  ponent of Cancer b o r e a l i s by Swartz et a l . (1961) r e v e a l e d that t h i s DNA was, i n f a c t ,  s i m i l a r to the dAT-copolymer  s y n t h e s i z e d de novo by Schachman e t a l . (1960), and hence i t i s r e f e r r e d to as crab dAT.  L i k e s y n t h e t i c dAT, crab dAT has  been found t o be a double-stranded molecule composed primari l y o f an a l t e r n a t i n g sequence of adenine and thymine  (Swartz  et al., 1961) . Crab dAT has s i n c e been found i n f i v e other s p e c i e s of the genus Cancer, and may c o n s t i t u t e approximately 10 or 30% of the t o t a l c e l l u l a r DNA depending of the s p e c i e s (Smith 1963,  1964).  Crab dAT does not appear to be l i m i t e d t o  c e r t a i n t i s s u e s , having been found i n the same p r o p o r t i o n i n somatic as w e l l as g o n i a l c e l l s Smith, p e r s o n a l  (Cheng and Sueoka, 1964;  communication).  The h i g h p r o p o r t i o n o f crab dAT i n c e l l s and i t s unusual base sequence are of i n t e r e s t from the s t a n d p o i n t o f i t s function. cellular  One o f the f i r s t  problems to be answered  i s the  l o c a t i o n o f the crab dAT.  F r a c t i o n a t i o n o f crude c e l l homogenates has i n d i c a t e d  23 that crab dAT i s found Smith,  unpublished).  i n the n u c l e a r p o r t i o n  Two methods which may be u s e f u l i n  determining the n u c l e a r l o c a t i o n 1.  demonstration  location  of crab dAT a r e :  of i t s e x i s t e n c e i n i s o l a t e d  or n u c l e a r chromatin, 2.  intact  nuclei  and  of t r i t i u m - l a b e l l e d  autoradiographic  ( K l e t t and  dAT w i t h i n c e l l s by  procedures.  Both of these methods were employed i n t h i s  study.  24 MATERIALS AND METHODS Male crabs of the s p e c i e s Cancer productus which c o n t a i n s 30% dAT (Smith, the experiment.  Randall,  1963) were used throughout  Animals c o l l e c t e d  i n Burrard  I n l e t and at  V i c t o r i a , B.C. were used almost e x c l u s i v e l y d u r i n g A p r i l to J u l y , when m e i o t i c a c t i v i t y and i n c o r p o r a t i o n of t r i t i a t e d thymidine 1.  i n t o c e l l s of the t e s t e s were found  I s o l a t i o n of N u c l e i The  productus The  t o be maximal.  t e s t e s were d i s s e c t e d from a mature Cancer  and placed i n 10 ml. of f i l t e r e d  sea water at 4°C.  t i s s u e was d i s r u p t e d w i t h a loose f i t t i n g g l a s s - T e f l o n  homogenizer and the suspension mesh.  filtered  through a f i n e  nylon  The f i l t r a t e was spun at 2500 rpm f o r 5 minutes (0-  3°C), and the supernatant  discarded.  pended i n 1.5 ml. of sea water (4°C), three l i n e a r sucrose to 1.00 M s u c r o s e )  1  gradients  The p e l l e t was  resus-  c a r e f u l l y l a y e r e d onto  (0.5 ml. each) (0.25 M,  sucrose  and spun at 4000 rpm f o r 30 minutes (4°C)  i n a spinco model L u l t r a c e n t r i f u g e u s i n g a SW 39 r o t o r .  The  l a y e r o f spermatocytes (see F i g u r e 2) was c o l l e c t e d u s i n g a pasteur  p i p e t t e , washed w i t h sea water, c e n t r i f u g e d , and  resuspended i n 1.5 ml. of 1.00 M sucrose c o n t a i n i n g nonidet . P.40 at a c o n c e n t r a t i o n of 0.1% a c c o r d i n g to the method of  1 A l l s o l u t i o n s of sucrose used were 0.003 M i n CaCl2 and 0.003 M i n MgCl2« The 0.25 M sucrose s o l u t i o n a l s o contained 0.25 M NaCl. The c e n t r i f u g e tubes were 5 ml. c a p a c i t y c e l l u l o s e n i t r a t e tubes.  25 O'Brien (1964a) i n order to l y s e the c e l l membrane. nonidet P.40 England.)  was  a gift  of the S h e l l Chemical Co. L t d . ,  The s o l u t i o n was  shaken g e n t l y f o r 15 minutes,  l a y e r e d onto a second l i n e a r sucrose g r a d i e n t to  (The  (1.05 M sucrose  2.5 M sucrose) and spun at 15,000 rpm f o r 30 minutes.  n u c l e i were c o l l e c t e d from the bottom of the tube.  The  This  procedure f o r the i s o l a t i o n of n u c l e i i s o u t l i n e d i n F i g u r e 1. At  each step, the degree of s e p a r a t i o n of  spermatophores, or n u c l e i was t r a s t photomicroscope.  assessed u s i n g a Wild phase con-  Some of the i s o l a t e d n u c l e i were a i r  d r i e d , or squashed and f r o z e n onto s l i d e s , s t a i n e d f o r DNA  and c h a r a c t e r i z a t i o n of DNA  (see  and f i x e d  by the Feulgen procedure ( C u l l i n g ,  r e s t of the n u c l e i were used f o r DNA  by Dr. R.P.  cells,  isolation.  from these n u c l e i was  and  1963).  The  The  isolation  c a r r i e d out  K l e t t of the F i s h e r i e s Research Board of Canada  K l e t t e t a l . (1966) f o r complete d e t a i l s of the p r o c e d u r e ) . Briefly,  dodecylsulfate, detergent.  the n u c l e i were l y s e d w i t h 25% sodium  and the DNA  The crude DNA  components i n a at  44,770 rpm  at  22°C.  of  DNA  separated from the p r o t e i n and  was  then separated i n t o i t s two  cesium c h l o r i d e d e n s i t y g r a d i e n t by s p i n n i n g  (spinco model E u l t r a c e n t r i f u g e ) f o r 16 hours  A UV photograph was  and crab dAT was  t r a c i n g of the UV  taken and the r e l a t i v e amount  determined from a microdensitometer  photograph.  26a  F i g u r e 1.  Procedure f o r i s o l a t i o n of spermatocyte n u c l e  DISSECT  TESTES  I N T O S E A WATER  (4 C )  DISRUPT TISSUE, CONNECTIVE TISSUE VARIOUS  CELL  I  T Y P E S AND  SPERMATOPHORES  CENTRIFUGE PELLET  OF V A R I O U S  CELL  FILTER  1  TYPES,  2 5 0 0 RPM,  30  MIN.  SPERMATOPHORES  R E S U S P E N D I N S E A WATER, L A Y E R ONTO G R A D I E N T S  25  M  SUCROSE  0 M  SUCROSE  C E N T R I F U G E , 4 0 0 0 RPM, 3 0 MIN., C O L L E C T S P E R M A T O C Y T E S , WASH, / CENTRIFUGE SPERMATOCYTES S U S P E N D I N 1.00 M S U C R O S E C O N T A I N I N G 0 . 1 % N P . 4 0 , SHAKE G E N T L Y , 15 MIN. L A Y E R ONTO G R A D I E N T  1.05 M  SUCROSE.  4  t 2.5 M  SUCROSE  CENTRIFUGE,  1 5 0 0 0 RPM,  30 i  COLLECT NUCLEI  MIN.  27 2.  In v i t r o I n c o r p o r a t i o n o f T r i t i a t e d - T h y m i d i n e and Autoradiography (i)  I n c o r p o r a t i o n of t r i t i a t e d - t h y m i d i n e - i n The  with s c i s s o r s ,  vitro  t e s t e s were removed from a l i v e crab, minced and placed i n a t i s s u e c u l t u r e medium c o n t a i n -  i n g 5.0 ml. E a g l e s ' minimal medium w i t h a supplement of 10% c a l f serum, 1 umole n o n - e s s e n t i a l amino a c i d s , 10 jumoles Lglutamine,  0.5 ^umoles 5 - f l u o r o d e o x y u r i d i n e  u n i t s each of p e n i c i l l i n and streptomycin, NaCl. 5  (FUDR), 250 and 2.75 mmoles  When t i s s u e s were to be incubated w i t h actinomycin D,  /*-g/ml ( f i n a l c o n c e n t r a t i o n ) was added to the medium.  t i s s u e was preincubated tritiated-thymidine  The  at 20°C f o r one hour a f t e r which the  (50 juc,  6.7 c/mole) was added.  The i n c u -  b a t i o n was c a r r i e d out f o r three hours at 20°C, and was f o l lowed by s i x washings o f the t i s s u e , each w i t h 5.0 ml o f the t i s s u e c u l t u r e medium (without FUDR or actinomycin D) c o n t a i n i n g an excess o f u n l a b e l l e d thymidine  (100 umoles).  t i s s u e to be used f o r autoradiography  was f i x e d f o r 1/2 hour  i n a s o l u t i o n c o n t a i n i n g e t h y l a l c o h o l and g l a c i a l acid  (3:1) and then s t o r e d i n 70% a l c o h o l at 4°C.  The  acetic The  remainder o f the t i s s u e was f r o z e n and used f o r the i s o l a t i o n and c h a r a c t e r i z a t i o n o f the DNA ( K l e t t e t a l . , 1966). The  s p e c i f i c a c t i v i t y o f the i s o l a t e d DNA  (includ-  i n g main DNA and crab dAT) was measured and expressed as counts  per minute per jug DNA.  (The amount of DNA was e s t i m -  ated by measuring the absorbance at 260 nyx  and u s i n g the  28 conversion factor (Sueoka  50 jug DNA i s e q u i v a l e n t t o one OD 60  u  n  i  t  2  and Cheng, 1962).)  The main DNA and crab dAT were  then separated i n a cesium s u l f a t e d e n s i t y g r a d i e n t i n the presence o f H g al.  (1965).  + +  a c c o r d i n g to the procedure o f Davidson e_t  F i f t y to s i x t y f r a c t i o n s were c o l l e c t e d  the bottom of the c e n t r i f u g e tube.  The absorbance  from  at 260 ny*.  and the CPM per ml. were measured f o r each f r a c t i o n . (ii)  Autoradiography The t i s s u e t o be used f o r autoradiography was  e i t h e r p r e s t a i n e d by the Feulgen procedure or l e f t unstained u n t i l a f t e r the autoradiographs had been developed.  I f the  l a t t e r method were f o l l o w e d , the s t a i n used was methyl pyronin Y ( C u l l i n g , The  green-  1963).  autoradiographs were prepared a c c o r d i n g to the  procedure o f Dr. J . Naylor ( p e r s o n a l communication). t i s s u e was squashed  The  i n 45% a c e t i c a c i d onto subbed s l i d e s ,  f r o z e n on dry i c e , the c o v e r s l i p removed, and the s l i d e placed i n e t h y l a l c o h o l .  (Subbed  s l i d e s were prepared by  d i p p i n g a c i d - c l e a n e d s l i d e s i n t o a subbing s o l u t i o n c o n t a i n i n g 0.5% g e l a t i n and 0.1% chromium potassium s u l f a t e , and placing  them i n dust proof s l i d e boxes to a i r dry.)  s l i d e s were p l a c e d i n two changes of e t h y l a l c o h o l , each, and then coated w i t h melted  (40°C) Kodak NTB-3  The 5 minutes liquid  emulsion u s i n g a s t a i n l e s s s t e e l spreader under a Wratten series 2 safelight.  The coated s l i d e s were a i r d r i e d f o r  29 2-4 ing  hours, then s e a l e d i n b l a c k p l a s t i c s l i d e boxes c o n t a i n -  c a l c i u m c h l o r i d e and s t o r e d at 4°C.  p e r i o d was from 2 - 1 0  The u s u a l exposure  days.  The autoradiographs were developed f o r 10 minutes under a Wratten diluted  s e r i e s 2 s a f e l i g h t u s i n g Kodak D-19 developer  1:1 w i t h d i s t i l l e d water.  w i t h water,  f i x e d f o r 15 minutes  The s l i d e s were r i n s e d i n Kodak hardening f i x e r , and  then washed f o r one hour i n c o l d running water.  The e n t i r e  d e v e l o p i n g procedure was c a r r i e d out at 4°C. I f the t i s s u e had been p r e s t a i n e d by the Feulgen procedure,  the autoradiographs were a i r d r i e d and mounted  w i t h permount.  I f the t i s s u e was not p r e s t a i n e d , the s l i d e s  were placed i n 5% c h l o r o f o r m i n e t h y l a l c o h o l chloroform i n e t h y l alcohol  (two changes,  (5 min.), 50%  5 min. each), and  then c l e a r e d i n a c l e a r i n g s o l u t i o n c o n t a i n i n g 2 volumes methyl s a l i c y l a t e : 1 volume e t h y l a l c o h o l : 1 volume xylene, for  1/2 hour.  The unstained autoradiographs were then  a l l y hydrated down to d i s t i l l e d water, green-pyronin Y f o r 45 to 60 minutes, i n acetone,  gradu-  s t a i n e d w i t h methyl differentiated  quickly  acetone: xylene (1:1), c l e a r e d i n xylene, and  mounted w i t h permount. G r a i n counts on autoradiographs o f Feulgen s t a i n e d tissue  (not incubated w i t h actinomycin D) were made u s i n g a  Z e i s s phase c o n t r a s t microscope.  The number of g r a i n s over  the nucleus and over the cytoplasm were counted f o r t h i r t y  30  cells. methyl  G r a i n c o u n t s w e r e a l s o made o n t i s s u e s t a i n e d w i t h green-pyronin  Y.  For both  groups,  t h e mean number o f  n u c l e a r g r a i n s ( x ) and t h e mean number o f c y t o p l a s m i c (y) w e r e c a l c u l a t e d .  The r a t i o o f mean number n u c l e a r  mean number o f c y t o p l a s m i c g r a i n s (—) was d e t e r m i n e d , y 95% c o n f i d e n c e  grains over and t h e  l i m i t s on t h i s r a t i o c a l c u l a t e d ( G o l d s t e i n ,  1965). A c o m p a r i s o n o f t h e number o f g r a i n s l y i n g stained  over  n u c l e i was made f o r t i s s u e i n c u b a t e d w i t h o r w i t h o u t  5 JA. g/ml a c t i n o m y c i n D. was c a l c u l a t e d  The mean number o f g r a i n s p e r c e l l  f o r t i s s u e w i t h a c t i n o m y c i n D ( x ^ ) and w i t h o u t  a c t i n o m y c i n D ( x g ) . The h y p o t h e s i s using a t test.  t h a t x± =fc X2 was t e s t e d  31 RESULTS 1.  I s o l a t i o n of N u c l e i Homogenization  Cancer  and f i l t r a t i o n of the t e s t e s of a  productus r e s u l t e d i n a s e p a r a t i o n of the c o n n e c t i v e  t i s s u e from the v a r i o u s c e l l first  types and spermatophores.  l i n e a r sucrose g r a d i e n t (0.25 M to 1.00  found to separate the primary and secondary the f r e e spermatozoa and spermatids, i n t o three d i s t i n c t l a y e r s spermatocytes The Cancer  (see F i g u r e 2 ) .  spermatocytes,  The  isolated  are shown i n F i g u r e 3. e f f e c t of nonidet P.40  on t e s t i c u l a r c e l l s i n  i n 1.00  (1964a,b).  s o l u t i o n of  M sucrose, the c e l l s underwent a s e r i e s  m o r p h o l o g i c a l changes, p r i m a r i l y a darkening of the nucleus  followed by a darkening of the cytoplasm to  was  and the spermatophores  When the spermatocytes were placed i n a 0.1%  of  M sucrose)  c o r r o b o r a t e s the o b s e r v a t i o n s of O'Brien  nonidet P.40  The  (Figure 4).  In one  f i v e minutes the c e l l membrane disappeared f r e e i n g the  nucleus.  Although O'Brien  (1964a) observed  c y t o l y s i s and fragmentation of amphibian  that n u c l e a r  e r y t h r o c y t e and  l i v e r n u c l e i began 10 to 20 minutes a f t e r the a d d i t i o n of 0.1%  nonidet P.40,  spermatocyte  unchanged f o r at l e a s t 30  n u c l e i of crabs remained,  minutes.  R e c e n t r i f u g a t i o n of the detergent t r e a t e d t o c y t e s i n a second  sperma-  l i n e a r sucrose g r a d i e n t (1.05 M - 2.5  sucrose) e f f e c t i v e l y separated n u c l e i from the c e l l  M  debris.  32a  F i g u r e 2.  D i s t r i b u t i o n of d i f f e r e n t c e l l types and i n the s u c r o s e g r a d i e n t s . a)  0.25 , ,., .  b)  1.05  " > ^  1.00  M gradient  2.5 M g r a d i e n t  nuclei  I N T E R F A C E  '  I  25M  S P E R M A T I D S , S P E R M A T O Z O A  S P E R M A T O C Y T E S  I  LOOM  S P E R M A T O P H O R E S  . I N T E R F A C E  1.05 M  C E L L  A  2.5 M  S^-^L  N U C L E I  D E B R I S  33a  F i g u r e 3.  I s o l a t e d spermatocytes.  Phase c o n t r a s t ,  M a g n i f i c a t i o n 2360X  34a  F i g u r e 4.  Spermatocyte l y s i s i n n o n i d e t  P.40  M a g n i f i c a t i o n a p p r o x i m a t e l y 2000X.  34  35 The  isolated nuclei  bottom o f the tube. normal.  ( F i g u r e 5a and b) c o l l e c t e d from the These n u c l e i were m o r p h o l o g i c a l l y  I t was observed  that treatment  of c e l l s i n meta-  phase w i t h nonidet P.40 permitted the i s o l a t i o n o f n u c l e a r chromatin  ( F i g u r e 4 and F i g u r e 5b). The i s o l a t e d  were found  to s t a i n p o s i t i v e l y w i t h  Feulgen.  A UV a b s o r p t i o n photograph of the DNA from the n u c l e i and spun i n a cesium  nuclei  isolated  chloride density gradi-  ent by Dr. R.P. K l e t t i n d i c a t e d t h a t both main DNA and crab dAT were present.  Microdensitometer  t r a c i n g s o f the  UV photograph showed that the r e l a t i v e p r o p o r t i o n o f main DNA and crab dAT were c h a r a c t e r i s t i c of Cancer  productus  (70% main DNA, 30% crab dAT). 2.  In v i t r o I n c o r p o r a t i o n o f T r i t i a t e d - T h y m i d i n e Into Crab DNA and~Autoradiography (i)  In v i t r o i n c o r p o r a t i o n of t r i t i a t e d - t h y m i d i n e The  i n c o r p o r a t i o n of t r i t i a t e d - t h y m i d i n e i n t o  total  c e l l DNA i n three separate t e s t s w i t h actinomycin D i s given i n Table I .  These r e s u l t s c l e a r l y show t h a t actinomycin D  does i n h i b i t the i n c o r p o r a t i o n of t r i t i a t e d - t h y m i d i n e crab DNA,  the degree of i n h i b i t i o n v a r y i n g from  into  approximately  one f i f t h to one h a l f the c o n t r o l v a l u e . When the t o t a l c e l l DNA was c e n t r i f u g e d i n a  CSSO4  d e n s i t y g r a d i e n t i n the presence o f H g , the crab dAT and + +  main DNA were separated i n t o two bands.  The f r a c t i o n s  l e c t e d from the tubes showed two DNA peaks measured  col-  36a  F i g u r e 5.  I s o l a t e d spermatocyte n u c l e i .  Phase c o n t r a s t .  CHR - i s o l a t e d n u c l e a r c h r o m a t i n , a - m a g n i f i c a t i o n 2360X b - m a g n i f i c a t i o n 1920X  b  Table I.  E f f e c t o f actinomycin D on the i n c o r p o r a t i o n o f H^-thymidine i n t o crab DNA.  FXPVRTMFNT  EXPERIMENT  CONTROL CPM/jx g DNA  TEST ( 5 g / m l AD) CPM/,* g DNA  „ % INHIBITION  A  May  15, 1965  640  390  39  May  26, 1965  460  244  48  June 14, 1965  400  315  22  38 s p e c t r o p h o t o m e t r i c a l l y at a wavelength  o f 260 ra.jx. .  The  s p e c i f i c a c t i v i t y o f each f r a c t i o n was a l s o determined.  The  o p t i c a l d e n s i t y and s p e c i f i c a c t i v i t y o f each f r a c t i o n are shown i n F i g u r e 6a and b f o r one t y p i c a l experiment.  In the  i n c u b a t i o n without actinomycin D (Figure 6a), both the main DNA and crab dAT were h e a v i l y l a b e l l e d w i t h thymidine. of  tritiated-  In the actinomycin D - t r e a t e d t i s s u e  incorporation  t r i t i a t e d - t h y m i d i n e was e q u a l l y depressed i n both DNA  components ( F i g u r e 6b). These r e s u l t s were not expected, and their  i m p l i c a t i o n s w i l l be d i s c u s s e d . (ii)  Autoradiography Under low m a g n i f i c a t i o n the autoradiographs showed  a remarkable  l o c a l i z a t i o n of g r a i n s over the nucleus (see  F i g u r e 7a, b and c ) .  G r a i n counts made w i t h both Feulgen  and methyl green-pyronin Y s t a i n e d t i s s u e are given i n Table II..  F o r t i s s u e p r e s t a i n e d w i t h Feulgen and exposed f o r 4  days,  the mean number o f g r a i n s per c e l l above the nucleus  (x) was 41.20 w i t h a standard e r r o r o f 1 3.10. ber o f g r a i n s above the cytoplasm  (y) was 2.53 ± .42. The  r a t i o i i was c a l c u l a t e d to be 16.29. y  The 95% c o n f i d e n c e  i n t e r v a l on t h i s r a t i o i s 14.34 t o 18.68. same experiment,  The mean num-  exposed t h r e e days,  T i s s u e from the  and p o s t - s t a i n e d w i t h  methyl green-pyronin Y gave an x of 33.1 ± 2.71, y o f 1.4 ± .21,  and a r a t i o £ of 24.36. y  t h i s r a t i o i s lb.21 results  t o 62.39.  The 95% c o n f i d e n c e i n t e r v a l on Table I I I summarizes the  f o r the g r a i n counts f o r both Feulgen and methyl  39a  Figure 6.  I n c o r p o r a t i o n of crab  dAT  a - control b - 5  - t h y m i d i n e i n t o main DNA  g/ml.  (without actinomycin actinomycin  D.  D)  and  •41 _C0  CD J O  p.  IH  in  o  00  hoo  1/  .AT-  o X  o  CD CM  <  Jtf  idAT  20 FRACTION  40  FIGURE 6a  AT  20 FRACTION  40  FIGURE 6b  40a  F i g u r e 7.  Autoradiographs of H^-thymidine . l a b e l l e d a - Low power.  Magnification.150X.  b - Phase c o n t r a s t .  Magnification  768X.  c - Phase c o n t r a s t .  Magnification  2360X.  tissue,  40  41  Table I I .  Number o f g r a i n s above the nucleus and cytoplasm i n samples o f c e l l s p r e - s t a i n e d w i t h Feulgen o r p o s t - s t a i n e d w i t h methyl green-pyronin Y.  Feulgen (exposed 4 days) ;lear Cytoplasmic 50 29 44 42 29 60 23 44 41 45 40 76 76 67 28 55 39 49 50 40 20 34 29 32 25 20 30 42 37 35  1 1 2 1 2 4 3 2 2 2 3 4 5 4 2 5 3 2 4 2 1 3 3 3 2 1 2 2 3 2  y - 2.53  41.20 Sx  Methyl green.-pyronin Y (exposed 3 days) Nuclear Cytoplasmii  s y ± . 4 2  =±3.10  '  32 42 21 22 8 24 27 22 33 21 19 22 17 24 52 29 33 23 47 33 37 24 42 30 26 30 47 76 56 25  1 2 1 2 0 1 1 2 0 1 2 1 0 3 1 2 1  i I 0 2 1 1 3 2 1 2 3 2 1  x = 33.13 Sx  y - 1.36  sy = ± . 2 i  =±2.71  X  =  y  16.29  •-  24.36  Table  III.  The r a t i o of mean number of nuclear g r a i n s to cytoplasmic sue s t a i n e d w i t h Feulgen and methyl green-pyronin Y.  grains f o r t i s -  Lower 95% R a t i o x/- converted Upper 95% CL Lower 95% CL Staining R a t i o XA- Upper 95% Procedure CL on r a t i o CL on r a t i o %N %C %N %C %N %C  Feulgen  16.29  18.68  14.38  94.16  5.78  94.92 5.08  93.48 6.52  MG-Y  24.36  62.39  15.27  95.97  3.94  98.42 1.58  93.85 6.15  43 green-pyronin  Y stained tissue.  The  ratio  may  be  converted  y to percent n u c l e a r g r a i n s and percent c y t o p l a s m i c g r a i n s by the f o l l o w i n g method. percent cytoplasmic g r a i n s = (.- + y percent n u c l e a r g r a i n s = 100  1)  -  100 +  1)  y Table I I I i n c l u d e s the percent nuclear and  cytoplasmic grains  f o r the r a t i o 4r and a l s o f o r the upper and y l i m i t s on these r a t i o s .  low 95-%  The  demonstration  t h a t both main DNA  and crab  were l a b e l l e d w i t h t r i t i a t e d - t h y m i d i n e ( F i g u r e 6a) almost a l l the l a b e l was crab dAT  nuclear  must be l o c a t e d i n the  (Table I I I ) ,  confidence  and  preincubated without  nucleus. tissue  The mean number of g r a i n s f o r t i s s u e  actinomycin D was  126.9  ± 31.8,  the mean number f o r t i s s u e incubated w i t h 5 yUg/ml. The  while actinomy-  c i n D was  60.8  was  to be h i g h l y s i g n i f i c a n t u s i n g a t t e s t to compare  found  + 20.0.  that  i n d i c a t e s that  G r a i n counts made on actinomycin D-treated are shown i n Table IV.  dAT  d i f f e r e n c e between the two  values  means. The  f a i l u r e to s p e c i f i c a l l y  to an attempt to l a b e l the main DNA  l a b e l the crab dAT l e d  p r e f e r e n t i a l l y with  t r i t i a t e d - d e o x y c y t i d i n e , s i n c e crab dAT would remain r e l a t i v e l y  Table IV.  Number of g r a i n s per c e l l f o r t i s s u e incubated w i t h and without actinomycin D. Without AD  With 5 ^ g / m l  135  77  141  46  175  74  122  77  173  58  116  37  88  80  193  50  105  43  107  69  118  85  84  61  101  31  131  63  125  61  %=  126.9  62  S%=  31.8  81 63 63 65 x  2  =  60.8  Sx  2  =  20.0  AD  45  u n l a b e l l e d due percent).  Thus crab dAT  of the c e l l The  c y t i d i n e content should  method would not be  ( l e s s than  correspond to the  that i s (are) F e u l g e n - p o s i t i v e  l i n g crab dAT cell  to i t s low  as c r i t i c a l  and  region(s)  unlabelled.  as s p e c i f i c a l l y  label-  because the Feulgen p o s i t i v e r e g i o n ( s )  that i s (are) u n l a b e l l e d may  j u s t be main DNA  not r e p l i c a t i n g at the time of i n c o r p o r a t i o n .  two  of  the  that  was  However,  an  i n v i t r o i n c o r p o r a t i o n experiment u s i n g t r i t i a t e d - d e o x y c y t i dine was  c a r r i e d out  according  to the procedure f o r i n v i t r o  t r i t i a t e d - t h y m i d i n e i n c o r p o r a t i o n experiments previously. The  FUDR was  omitted from the i n c u b a t i o n  s p e c i f i c a c t i v i t y of the DNA  background  (R.P.  (described  was  only s l i g h t l y  K l e t t , unpublished d a t a ) .  l a c k of i n c o r p o r a t i o n was  a l s o observed  The  mixture.) above  almost  total  autoradiographically.  46 DISCUSSION DNA  e x t r a c t e d from i s o l a t e d ,  n u c l e a r chromatin  of Cancer productus  t h i r t y percent crab dAT.  i n t a c t n u c l e i or spermatocytes  When both DNA  led with t r i t i a t e d - t h y m i d i n e ,  components are  t i o n of crab  label-  a u t o r a d i o g r a p h i c evidence  i n d i c a t e s t h a t the bulk of t h i s l a b e l i s n u c l e a r . experiments  contains  These  provide c r i t i c a l evidence f o r the n u c l e a r  loca-  dAT.  Reich (1964) has shown t h a t at h i g h c o n c e n t r a t i o n s the a n t i b i o t i c actinomycin D(AD) of DNA  synthesis.  He proposed  i s an e f f e c t i v e that AD  a c t s by a s p e c i f i c  b i n d i n g to the guanine r e s i d u e s of the DNA.  I t has been  demonstrated t h a t the amount of AD bound to DNA to the guanine content of the DNA, s h i p was  not found  Support  i n h i b i t i o n of RNA content of the DNA  i s related  although t h i s  relation-  to be d i r e c t l y p r o p o r t i o n a l to the amount  of guanine i n the molecule et a l . , 1964).  inhibitor  (Goldberg ert stl. , 1962;  Goldberg  of Reich's model i s the f i n d i n g t h a t  s y n t h e s i s a l s o i n c r e a s e s as the guanine increases.  Goldberg  e_t aJ. (1964) found  t h a t the i n c o r p o r a t i o n of a l a b e l l e d p r e c u r s o r i n t o s y n t h e s i z e d on the primer s y n t h e t i c dAT was  u n a f f e c t e d by 100  than two  percent guanine) was  i n the presence  of AD.  (no guanine r e s i d u e s )  /^g/ml of actinomycin Ci  ever, i n c o r p o r a t i o n i n t o RNA  RNA  (AD).  u s i n g the primer crab dAT i n h i b i t e d by some 24  How(less  percent  These o b s e r v a t i o n s i n d i c a t e t h a t AD  might s e l e c t i v e l y i n h i b i t s y n t h e s i s of main crab DNA,  while  47  crab dAT would be much l e s s a f f e c t e d , and  thus permit i t s  l o c a l i z a t i o n a u t o r a d i o g r a p h i c a l l y . However, the i n c o r p o r a t i o n of t r i t i a t e d - t h y m i d i n e was the same extent i n both DNA  i n h i b i t e d to  types.  There are s e v e r a l pos-  s i b l e e x p l a n a t i o n s f o r these r e s u l t s . of a l a r g e r r e p l i c a t i n g u n i t ted throughout crab dAT if  I f crab dAT  ( f o r example i t may  i s part  be  distribu-  a l l the chromosomes), the r e p l i c a t i o n of the  might be dependent on r e p l i c a t i o n of main DNA.  s y n t h e s i s of main DNA  dAT.  approximately  i s inhibited,  Thus,  so i s that of crab  A second p o s s i b i l i t y c o u l d be the e x i s t e n c e of a mech-  anism of feedback i n h i b i t i o n which, responds to h i g h concent r a t i o n s of dGTP and  dCTP and  shuts o f f a l l DNA  In t h i s case, AD bound to main DNA c a t i o n and cause an accumulation suggestion and  . i s t h a t crab DNA  synthesis.  would prevent  of dGTP and  polymerase may  i n h i b i t i o n of the p r o d u c t i o n of i t s RNA  would r a p i d l y r e s u l t i n a c e s s a t i o n of DNA f i n a l p o s s i b i l i t y r e q u i r e s the messenger RNA merase to be produced on the main crab  its repli-  dCTP. be  A  short-lived,  message by replication. f o r DNA  i n two  the other p o s t - s t a i n e d w i t h methyl green-pyronin  (Table I I ) .  poly-  Feulgen,  Y,  the mean  higher i n the pre-  However, t h i s t i s s u e was  f o r f o u r days whereas the p o s t - s t a i n e d t i s s u e was three.  This  samples of the  same t i s s u e , one of which had been p r e s t a i n e d w i t h  stained tissue  AD  DNA.  In comparing the g r a i n counts  number of g r a i n s over the nucleus was  third  exposed  exposed f o r  When the d i f f e r e n c e i n exposure time i s c o r r e c t e d f o r  48 by m u l t i p l y i n g the mean number of g r a i n s i n p r e s t a i n e d t i s sue by three f o u r t h s , the adjusted mean v a l u e s are x = 31.15 and y = 1.90  (Table V ) .  T h i s c o r r e c t i o n f o r exposure time  g i v e s a mean n u c l e a r g r a i n count s i m i l a r to the value i n the p o s t - s t a i n e d sample. 30 percent  However, c y t o p l a s m i c counts d i f f e r by  (Table V ) .  T h i s apparent  d i f f e r e n c e may be  e x p l a i n e d by the method of c o u n t i n g g r a i n s . Feulgen s t a i n e d t i s s u e were performed microscope  i n order to determine  G r a i n counts on  u s i n g a phase c o n t r a s t  the extent of the cytoplasm.  Rupture of the c e l l membranes i n the squashing r e s u l t s i n an e x t r u s i o n of the cytoplasm. ably overlapped  procedure  T h i s leakage  prob-  a c e r t a i n number of random background g r a i n s  that happened to be a s s o c i a t e d w i t h c o n n e c t i v e t i s s u e and were i n c l u d e d i n the counts. of  On the other hand, g r a i n  counts  methyl green-pyronin Y s t a i n e d t i s s u e were made u s i n g a  l i g h t microscope. was determined  In t h i s case the extent of the cytoplasm  by the appearance of RNA p o s i t i v e r e g i o n s ,  thereby m i n i m i z i n g the e x t e n t o f the cytoplasm. Demonstration  of the n u c l e a r l o c a l i z a t i o n of crab  dAT i n v i t e s f u r t h e r attempts of  t h i s component.  to determine  Short term i n c o r p o r a t i o n experiments  ( d e s c r i b e d on page 27) produced only.  Prolonged  the n u c l e a r s i t e ( s )  labelled  interphase n u c l e i  i n c o r p o r a t i o n time o n l y r e s u l t e d i n c e l l  death and c y t o l y s i s a f t e r 8 - 1 2  hours (at 20°C),  without  producing l a b e l l e d chromosomes.  I n c u b a t i o n of the c e l l s at  5°C r e s u l t e d i n 85 percent s u r v i v a l a f t e r s i x days but no  Table V.  Adjusted mean number of g r a i n s over the nucleus and cytoplasm f o r p r e - and p o s t - s t a i n e d t i s s u e (see t e x t f o r d e t a i l s ) .  Staining Procedure  Mean No. Grains Over Nucleus  Mean No. Grains Over Cytoplasm  prestained  41.20  2.53  ( p r e s t a i n e d ) X 3/4  31.15  1.98  post-stained  33.13  1.36  CD  50  detectable  cell divisions.  under these c o n d i t i o n s tained,  and  Therefore  i t was  presumed  the c e l l s were merely being  not c u l t u r e d .  An  that the p e r i o d between DNA  main-  alternate explanation  r e p l i c a t i o n and  the  that  may  be  appearance  of d i s t i n c t chromosomes i s p r o t r a c t e d . Attempts to l a b e l chromosomes i n v i v o , r e s u l t e d i n no s i g n i f i c a n t i n c o r p o r a t i o n , w h i l e a l l attempts to l a b e l of the f r a c t i o n s of DNA  one  or to l a b e l chromosomes were unsuc-  cessful . A l t e r n a t i v e experiments might d i s t i n g u i s h between the DNA  components i n f i x e d c e l l s .  ures that might be  Three p o s s i b l e proced-  f e a s i b l e i n t h i s respect  are  fluorescent  s t a i n i n g of heated chromosomes w i t h the dye  a c r i d i n e orange,  h y b r i d i z a t i o n of l a b e l l e d crab dAT  regions  chromosomes, and  w i t h dAT  s p e c i f i c r e p l i c a t i o n of dAT  of  the  r e g i o n s of  the  chromosomes, i n s i t u . Nash and s a l i v a r y gland  Plaut  (1964) reported  c e l l s of D r o s o p h i l a  orange r e s u l t e d i n b r i g h t yellow flame red f l u o r e s c e n c e of the DNA, orange.  in situ,  t h a t s t a i n i n g of  melanogaster w i t h a c r i d i n e  fluorescence  of cytoplasmic  RNA.  of DNA  Heat  gave flame red f l u o r e s c e n c e  and  denaturation with acridine  Thus the c o l o r of a c r i d i n e orange permits a v i s u a l  d i s c r i m i n a t i o n between double- and  single-stranded  nucleic  acids. The  r e l a t i v e guanine and  c y t o s i n e content of  DNA  51 determines i t s thermal d e n a t u r a t i o n and Doty, 1959). percent dAT  (^3  (Smith,  GC)  has  GC)  has  a Tm  of approximately  and  crab  66.4°C  T h i s d i f f e r e n c e i n Tm v a l u e s might  t i o n by a c r i d i n e orange s t a i n i n g .  (42  83.2°C, w h i l e  the s e l e c t i v e d e n a t u r a t i o n of crab dAT,  of t h i s nature  (Marmur  productus main crab DNA  a Tm of approximately  percent 1963).  Thus i n C.  temperature (Tm)  permit  thus i t s detec-  P r e l i m i n a r y experiments  i n d i c a t e t h a t h e a t i n g to 75°C does r e s u l t i n  a d e t e c t a b l e change i n the c o l o r of DNA  fluorescence  although  no d i s c r i m i n a t i o n between chromosomes or chromosome r e g i o n s was  possible. The  d i s c o v e r y by S c h i l d k r a u t et a l . (1961) t h a t  s i n g l e - s t r a n d e d DNA t e r i a could anneal  isolated  from two  d i f f e r e n t r e l a t e d bac-  to form double-stranded  h y b r i d DNA  was  followed by the demonstration t h a t the r e l a t i v e amount of h y b r i d i z a t i o n of b a c t e r i a l messenger RNA DNA  fragments of a d i f f e r e n t s t r a i n may  to s i n g l e - s t r a n d e d be used as a measure  of g e n e t i c r e l a t e d n e s s between them (McCarthy and 1963) .  Bolton,  Hoyer e_t a l . (1964) f u r t h e r showed t h a t i n v e r t e b r a t e s  p a r t i c u l a r l y mammals, the amount of DNA-DNA b i n d i n g could a l s o be used as an estimate these  of g e n e t i c r e l a t e d n e s s .  s t u d i e s have been c a r r i e d out on i s o l a t e d DNA  molecules.  Undoubtedly DNA  tightly coiled  and  However, and  i n metaphase chromosomes i s  bound by p r o t e i n , f a c t o r s which would  decrease the e f f i c i e n c y of h y b r i d i z a t i o n .  RNA  52 Recently, Wolff  and Trosko  (1965) r e p o r t e d t h a t  t r y p s i n d i g e s t i o n of chromosomes i s o l a t e d from V i c i a removes much of the chromosomal p r o t e i n w i t h a  faba  concomitant  i n c r e a s e i n chromosome l e n g t h by a f a c t o r of at l e a s t t h r e e . Thus, i t i s proposed that c e l l - f r e e p r e p a r a t i o n s of crab chromosomes might be t r e a t e d w i t h t r y p s i n , heated presence  of l a b e l l e d crab dAT  then s l o w l y c o p i e d .  Any  at 100°C f o r 10 minutes and  h y b r i d i z a t i o n of chromosomal  w i t h the l a b e l l e d crab dAT graphically.  i n the  could then be detected  DNA  autoradio-  S i m i l a r experiments done on non-crab dAT-con-  t a i n i n g animals  such as C a r c i n u s maenus (Sueoka, 1961)  would  provide a c o n t r o l f o r n o n - s p e c i f i c b i n d i n g . P r e s c o t t , et a l . (1966) have r e p o r t e d a method of o b t a i n i n g DNA  synthesis, i n s i t u ,  as the template.  u s i n g depolymerized  They have shown that a c i d treatment  squashed p r e p a r a t i o n s f o l l o w e d by i n c u b a t i o n w i t h DNA  p r e c u r s o r s and c a l f thymus DNA  of  labelled  polymerase, r e s u l t e d i n  i n c o r p o r a t i o n of the l a b e l i n the c e l l s . cells,  DNA  Therefore, i n crab  i t might be p o s s i b l e to incubate squashed p r e p a r a t i o n s  of primary  spermatocytes  t r i t i a t e d - t h y m i d i n e and  ( p r e t r e a t e d w i t h acid) w i t h o n l y t r i t i a t e d - d e o x y a d e n o s i n e , which should  s e l e c t i v e l y permit the r e p l i c a t i o n of crab dAT  regions.  These r e g i o n s could then be detected a u t o r a d i o g r a p h i c a l l y . The crab dAT somes .  now  demonstration  of the nuclear l o c a l i z a t i o n of  demand i t s f u r t h e r l o c a l i z a t i o n on the chromo-  The establishment of a random or non-random  53  d i s t r i b u t i o n of the s a t e l l i t e DNA w i l l have a g r e a t e r on p o s s i b l e f u n c t i o n s of t h i s  component.  bearing  54 SUMMARY Spermatocyte n u c l e i of Cancer productus were i s o l a t e d by treatment  of a t i s s u e homogenate w i t h 0.1% nonidet  P.40 i n sucrose f o l l o w e d by c e n t r i f u g a t i o n i n a l i n e a r sucrose g r a d i e n t . n u c l e i was found  T h i r t y percent of the DNA to be crab dAT.  dence, autoradiography  i n these  Corroborating t h i s  intact evi-  on c e l l s i n which both DNA components  were l a b e l l e d w i t h t r i t i a t e d - t h y m i d i n e i n d i c a t e s the n u c l e a r l o c a t i o n of both DNA  types.  I n c u b a t i o n of t i s s u e i n actinomycin D f a i l e d to p r e f e r e n t i a l l y i n h i b i t main DNA r e p l i c a t i o n and depressed the r e p l i c a t i o n of both DNA components e q u a l l y .  The i m p l i -  c a t i o n s of t h i s o b s e r v a t i o n are d i s c u s s e d . L a b e l l e d chromosomes were never obtained  after  i n c u b a t i o n of t e s t e s w i t h the r a d i o a c t i v e p r e c u r s o r s . v i v o attempts  to l a b e l c e l l s a l s o f a i l e d .  Ta  Three methods f o r  the chromosomal l o c a l i z a t i o n of crab dAT u s i n g f i x e d chromosome p r e p a r a t i o n s are suggested.  55 LITERATURE CITED Cheng, T.-Y., and N. Sueoka, 1964. A polymer s i m i l a r to p o l y a d e n y l a t e - thymidylate i n v a r i o u s t i s s u e s of a marine crab. Science 143:1442-1443. C u l l i n g , C.F.A., 1963. Handbook of h i s t o p a t h o l o g i c a l techniques, 2nd e d i t i o n . Butterworths, London. Davidson, N., J . Widholm, U.S. Nandi, R. Jensen, B.M. O l i v e r a , and J.C. Wang, 1965. P r e p a r a t i o n and propert i e s of n a t i v e crab dAT. Proc. N a t l . Acad. S c i . U.S. 53:111-118. Goldberg, I.H., M. Rabinowitz, and E. Reich, 1962. B a s i s of actinomycin a c t i o n . I. DNA b i n d i n g and i n h i b i t i o n of RNA polymerase s y n t h e t i c r e a c t i o n s by actinomycin. Proc. N a t l . Acad. S c i . U.S. 48:2094-2101. , and E. Reich, 1964. Actinomycin i n h i b i t i o n of RNA s y n t h e s i s d i r e c t e d by DNA. F e d e r a t i o n Proc. 23: 958-964. G o l d s t e i n , A., 1965. New York.  Biostatistics.  The MacMillan  Co.,  Hoyer, B.H., B.J. McCarthy, and E.T. B o l t o n , 1964. A molecul a r approach to the s y s t e m a t i c s of higher organisms. Science 144:959-967. K l e t t , R.P., M. Smith, C R . A s t e l l , D.T. Suzuki, and I.H. Goldberg, 1966. D e o x y r i b o n u c l e i c a c i d s i n crabs of the s u p e r f a m i l y brachyrhyncha. Submitted t o the J . of Mol. Biol. McCarthy, B.J., and E.T. B o l t o n , 1963. An approach to the measurement of g e n e t i c r e l a t e d n e s s among organisms. Proc. N a t l . Acad. S c i . U.S. 50:156-164. Marmur, J . , and P. Doty,, 1959. Heterogeneity i n deoxyribonuc l e i c a c i d s . I. Dependance on composition of the conf i g u r a t i o n a l s t a b i l i t y of d e o x y r i b o n u c l e i c a c i d s . Nature 183:1427-1429. Nash, D., and W. P l a u t , 1964. On the d e n a t u r a t i o n of chromosomal DNA i n s i t u . Proc. N a t l . Acad. S c i . U.S. 51: 731-735. O'Brien, B.R.A., 1964a. The p a r t i a l c y t o l y s i s of the amphibi a n e r y t h r o c y t e and l i v e r parenchyma c e l l by a nonionogenic s u r f a c e a c t i v e agent. J . C e l l . B i o l . 20:521525.  56 O'Brien, B.R.A., 1964b. A r a p i d method f o r the i s o l a t i o n and c o l l e c t i o n of n u c l e i from whole c e l l suspensions. J. C e l l . B i o l . 20:525-529. Reich, E., 1964. Actinomycin: C o r r e l a t i o n of s t r u c t u r e and f u n c t i o n of i t s complexes w i t h purines and DNA. Science 143:684-689. S c h i l d k r a u t , C.L., J . Marmur, and P. Doty, 1961. The format i o n of h y b r i d DNA molecules and t h e i r use i n s t u d i e s on DNA homologies. J . Mol. B i o l . 3_:595-617. Schachman, H.K., J . Adler, CM. Radding, I.R. Lehman, and A. Kornberg, 1960. Enzymatic s y n t h e s i s of d e o x y r i b o n u c l e i c acid. VII. S y n t h e s i s of a polymer of deoxyadenylate and deoxythymidylate. J . B i o l . Chem. 235:3242-3249. Smith, M., 1963. D e o x y r i b o n u c l e i c a c i d s i n crabs of the genus Cancer. Biochem. Biophys. Res. Comm. 10:67-72. , 1964. Deoxyribonucleic Mol. B i o l . 9^17-23.  a c i d s of C r u s t a c e a .  J.  Sueoka, N., 1961. V a r i a t i o n and h e t e r o g e n e i t y of base comp o s i t i o n of d e o x y r i b o n u c l e i c a c i d s : A c o m p i l a t i o n of o l d and new data. J . Mol. B i o l . 3_:31-40. , and T.-Y. Cheng, 1962. F r a c t i o n a t i o n of n u c l e i c a c i d s w i t h the methylated albumin column. J . Mol. B i o l . 4:161-172. Swartz, M.N., T.A. Trautner, and A. Kornberg, 1962. Enzymatic s y n t h e s i s of d e o x y r i b o n u c l e i c a c i d . XI. F u r t h e r s t u d i e s on nearest neighbour base sequence i n d e o x y r i b o n u c l e i c a c i d s . J . B i o l . Chem. 237:1961-1967. Trosko, J.E., and S. Wolff, 1965. Strandedness of V i c i a faba chromosomes as r e v e a l e d by enzyme d i g e s t i o n s t u d i e s . J . C e l l . B i o l . 26:125-135. Von B o r s t e l , R.C., D.M. P r e s c o t t , and F . J . Bollum, 1966. I n c o r p o r a t i o n of n u c l e o t i d e s i n t o n u c l e i of f i x e d c e l l s by DNA polymerase. Submitted to J . C e l l . B i o l .  

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