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Study of extracellular ribonuclease activity in ustilago hordei Bech-Hansen, Nils Torben 1970

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A  STUDY  OF  EXTRACELLULAR RIBOMJCLEASE IN  HORDE  USTILAGO  ACTIVITY  I  by  Nils B.Sc,  Torben Bech-hiansen  University  A THESIS THE  of  SUBMITTED  British  IN  REUU I K t M b N I S  PARTIAL H)K  f HI-  MASTER  OF  in  Department  the  Columbia,  1968  FULFILMENT I1RGRFF  OF  SCIENCE  of Botany  We a c c e p t  this  thesis  required  THE  as  conforming  to  the  standard  UNIVERSITY  OF  January,  BRITISH 1970  COLUMBIA  OF  In  presenting  this  an a d v a n c e d  degree  the  shall  I  Library  f u r t h e r agree  for  scholarly  by  his  of  this  written  thesis at  the U n i v e r s i t y  make  tha  it  purposes  for  may  financial  is  of  gain  Columbia  February 20, 1970  of  Columbia,  British  by  for  shall  the  that  not  requirements I  agree  r e f e r e n c e and copying of  t h e Head o f  understood  Botany  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, C a n a d a  of  for extensive  be g r a n t e d  It  fulfilment  available  permission.  Department  D a t e  freely  permission  representatives. thesis  in p a r t i a l  or  that  study.  this  thesis  my D e p a r t m e n t  copying  for  or  publication  be a l l o w e d w i t h o u t  my  if  ABSTRACT  Extracellular sporldial  r i b o n u c l e a s e a c t i v i t y was d e t e c t e d In c u l t u r e media of  and m y c e l i a l  was maximal  a t pH 5.0, 6.5 and 8.0.  was a f u n c t i o n o f was c o i n c i d e n t . the I n i t i a l  cultures of UstIlago horde!.  the c e l l  density.  Enrichment o f  The s e c r e t i o n o f Release of pH k.S  the RNase a c t i v i t y and pH 7.5  activity  the simple g l u c o s e and s a l t s medium delayed  s e c r e t i o n of a c t i v i t y .  not enhance the amount o f a c t i v i t y  The presence o f RNA In the medium d i d released.  o f RNA in the medium was not r e q u i r e d f o r Into the medium, i t  The RNase a c t i v i t y  Furthermore, s i n c e the presence  the r e l e a s e of the RNase a c t i v i t y  i s suggested the s y n t h e s i s  and s e c r e t i o n  is a  constitutive  function. N-methyl-N'-nitro-N-nltrosoguanidlne strains.  S e l e c t i o n methods  c e l l u l a r RNase a c t i v i t y a r e  was used to produce  f o r the d e t e c t i o n o f s t r a i n s discussed.  auxotrophic  deficient  in e x t r a -  in  TABLE OF CONTENTS  Page INTRODUCTION  1  MATERIALS AND METHODS  8  1.  Biological Material  8  2.  Culturtng  8  3.  C u l t u r e Media and S e l e c t i o n P l a t e s  9  k.  Assays  9  5.  Chemical M u t a g e n e s i s , D e t e c t i o n and S c r e e n i n g o f Mutants.  6.  The P r e p a r a t i o n o f CMC-RNA  . . .  11 12  RESULTS  13  1.  E v i d e n c e f o r E x t r a c e l l u l a r R t b o n u c l e a s e s In U. horde I  13  2.  Release o f Ribonuclease A c t i v i t y .  15  3.  RNA a s a Carbon Source f o r U_. h o r d e i  20  k.  Chemical M u t a g e n e s i s , Mutants D e t e c t e d and Screened  22  5.  CMC-p-Toluenesulfonate  22  M o d i f i e d RNA  DISCUSSION  29  SUMMARY AND CONCLUSIONS  39  BIBLIOGRAPHY  *1  APPENDIX A APPENDIX B  .  *»5 *6  iv  LIST OF TABLES Page Table Table  I.  of U. horde!  23  II. Preliminary screening of possible mutant Isolates of U_. horde I  2k  Table  III.  Table  IV.  NG treatments of s t r a i n E^  Selection method f o r detecting e x t r a c e l l u l a r RNase-defIcient mutants using CMC modified RNA  $k  Selection scheme f o r the detection of three classes of e x t r a c e l l u l a r RNase-deficient mutants using model substrates  36  LIST OF FIGURES Page Figure 1. Figure 2.  Figure 3.  Figure 4.  Figure 5. Figure 6.  RNase a c t i v i t y of c e l l - f r e e minimal medium from E« stationary culture, measured across pH range 3.0 to 9.2  14  Growth o f strains E j and 1^ cultured In e i t h e r minimal or complete medium and the release of RHase a c t i v i t y (pH 7-5)  16  Growth of s t r a i n Ej In minimal and complete medium and the release o f RNase a c t i v i t y (pH 4.5 and pH 7.5)  17  Growth o f a stable mycelial s t r a i n in complete medium and the release of RNase a c t i v i t y (pH 4.5 and pH 7.5)  18  The release of RNase a c t i v i t y (pH 4.5 and pH 7.5) by s t r a i n E^~ grown on minimal medium plus RNA.  19  Dilution experiment  21  ACKNOWLEDGEMENTS  The author wishes to extend his sincere appreciation to Professor Clayton Person for his suggestions and c r i t i c i s m s during the Investigation and especially In the preparation of this thesis; Gratitude i s a l s o expressed  to Or. Michael Smith and Or. Michael Shaw,  co-supervisors, for their encouragement and helpful discussions.  A special  word o f thanks to Or. Smith who generously allowed me to use space and equipment In his lab, and who provided many useful c r i t i c i s m s during the preparation of this thesis. To Or. Peter Gilham, many thanks f o r I n i t i a t i n g my Interest in this study and o f f e r i n g helpful discussions. The discussions and constructive c r i t i c i s m s of my fellow-colleagues In the Botany and Biochemistry  Departments during the research and preparation  of this thesis are g r a t e f u l l y acknowledged and appreciated. My thanks to Margaret Shand for the technical assistance.  To Barbara  Szuts, Cathy Davison, Rita Rosbergen and Bruce Stewart for their assistance In the preparation of this thesis, thank you.  1  INTRODUCTION  Enzymes o f t e n o c c u r In the c u l t u r e f l u i d s o f b a c t e r i a and  fungi.  Sometimes t h e t r p r e s e n c e Is a r e s u l t o f the d e a t h o r l y s i s o f a f r a c t i o n o f the c e l l s  In the c u l t u r e but  c e l l u l a r (Lampen, 1965)*  In o t h e r c a s e s the enzymes a r e t r u l y e x t r a -  An e x t r a c e l l u l a r enzyme o r exoenzyme I s , by  d e f i n i t i o n , one w h i c h can be produced and  r e l e a s e d by the c e l l w i t h o u t  a l t e r a t i o n t o c e l l s t r u c t u r e g r e a t e r than t h a t c o m p a t i b l e w i t h the normal p r o c e s s e s o f growth and  reproduction  any  cells'  ( P o l l o c k , 1963)•  S t u d i e s o f e x t r a c e l l u l a r enzymes produced by microbes have been reviewed e x t e n s i v e l y by D a v l e s (1963), by P o l l o c k (1963) and general  by Lampen (1965).  Some  f e a t u r e s a r e r e v e a l e d from a comparison o f t h o s e e x t r a c e l l u l a r enzymes  t h a t have been s t u d i e d  ( P o l l o c k , 1963):  I) they a r e s m a l l II)  In s i z e - l e s s than 80,000  M.W.;  they have l i t t l e o r no c y s t e i n e i n t h e i r p r i m a r y s t r u c t u r e ;  III)  they o f t e n need c a l c i u m  ions f o r a c t i v a t i o n and  Iv) they o c c u r most f r e q u e n t l y In g r a m - p o s i t i v e  stabilization;  b a c t e r i a and  and  fungi.  g e n e r a l i z a t i o n s a r e based m a i n l y on s t u d i e s o f b a c t e r i a s i n c e r e l a t i v e l y  Such few  s t u d i e s have been c a r r i e d out w i t h f u n g i . E x t r a c e l l u l a r enzymes a r e g e n e r a l l y c o n s i d e r e d c e l l ' s r e g u l a r p r o t e i n s y n t h e s i z i n g system.  t o be s y n t h e s i z e d  the  S t u d i e s o f an e x t r a c e l l u l a r  p e n i c i l l i n a s e produced by B a c i l l u s s u b t i l Is (Kushner and P o l l o c h ,  1961)  showed t h a t the l i b e r a t i o n o f the enzyme r e q u i r e s membrane s y n t h e s i s . ( 1 9 6 8 ) , who  by  Beaton  s t u d i e d S t a p h y l o c o c c u s a u r e u s , suggested t h a t r e l e a s e o f  p e n i c i l l i n a s e I n v o l v e s a l t e r a t i o n o f membranous s t r u c t u r e s . In h i s h y p o t h e s i s  v i s u a l i z e s the f o r m a t i o n  and  s e c r e t i o n of  as b e i n g a s s o c i a t e d s p e c i f i c a l l y w i t h the mesosomes.  Lampen (1965) penicillinase  This proposal,  based  2 on studies i n prokaryotes, may of s e c r e t i o n In eukaryotes. e x t r a c e l l u l a r proteases  not be t o t a l l y compatible with the phenomena In Neurospora crassa It has been suggested that  located i n membrane-bound v e s i c l e s are released  e x t r a c e l l u l a r l y when they "cross the plasma membrane as i n t a c t p a r t i c l e s by means of Invaginations o f the plasmalemma" (Nat l i e «Jt a L , Electron micrographs have recently been obtained  19^5).  ( S t e i n , 1970) which a l s o  support the Idea that I n t r a c e l l u l a r v e s i c l e s are Involved i n the secretton of c e l l products by U. horde! (Pers.) Lagerh.  Probably the process of c e l l  secretton In eukaryotes has been best studied i n higher organisms, f o r example wtth pancreatic c e l l s (production and release of zymogen).  In t h i s example  zymogen i s synthesized on the rIbosomes of the endoplasmic reticulum moves into the I n t e r c l s t e r n a l c a v i t i e s of the E.R. the Golgi complex by t r a n s i e n t connections.  (E.R.),  and then to v e s i c l e s of  These v e s i c l e s then move to the  c e l l surface where they fuse with the plasma membrane to release the zymogen e x t r a c e l l u l a r l y (Oe R o b e r t l s , 1965)•  In U. hordeI where there Is no Golgl  complex ( S t e i n , 1970), the production and release of e x t r a c e l l u l a r products cannot f o l l o w t h i s sequence of events.  Although I t Is apparent  that there Is no s i n g l e or standard mechanism of s e c r e t i o n used by a l l eukaryotlc c e l l s the process appears In a l l cases to involve movement, v i a v e s i c l e s , of proteins from t h e i r s i t e s of synthesis to the plasma membrane. M i c r o b i a l ribonucleases, both i n t r a - and e x t r a - c e l l u l a r , have been reviewed i n d e t a i l by Egaml and Nakamura (1969)*  These authors noted t h a t ,  In general, I n t r a c e l l u l a r RNases have no n u c l e i c a c i d s p e c i f i c i t y , have a s i z e of 30,000 - 40,000 M.W.  u n i t s , are h e a t - l a b i l e and are exonuclease,  tn contrast to e x t r a c e l l u l a r RNases which have base s p e c i f i c i t y , a s i z e of 11,000 - 13,000 M.W.  u n i t s , are heat-stable and are endonucleases.  Nlshlmura and Nomura (1959)* who  Investigated the mode of formation of  e x t r a c e l l u l a r RNase In 6. subtl1 Is ( s t r a i n H), found that the RNase a c t i v i t y  3 in the medium Increased markedly when growth entered the s t a t i o n a r y phase and then continued to increase at a constant rate during the s t a t i o n a r y phase.  In a l a t e r study they found that the e x t r a c e l l u l a r RNase of IB. s u b t l l i s  d i f f e r e d from the i n t r a c e l l u l a r RNase i n such c h a r a c t e r i s t i c s as optimum pH, heat s t a b i l i t y and ion requirement (Nlshlmura and Haruo, I 9 6 0 ) . This contrasts with the f i n d i n g that one of the I n t r a c e l l u l a r RNases of Neurospora crassa Is very s i m i l a r to the e x t r a c e l l u l a r RNase (Takai ejt a K ,  1967)*  The amount of e x t r a c e l l u l a r RNase In the medium can be a f f e c t e d by the conditions of c u l t u r e ; i t has been shown by Yanaglda et aj_. (1964), who worked with U. area, that where RNA or poly U (which was not a substrate f o r the RNase) was supplied as the s o l e source of carbon, the production of an e x t r a c e l l u l a r guanyloribonuclease  was enhanced.  G l i t z and Dekker (1964) a l s o found that  an e x t r a c e l l u l a r guanyloribonuclease  accumulated In the c u l t u r e medium of  U. sphaerogena when RNA was added as the sole carbon source. considered these to be Inducible enzymes.  They therefore  In a subsequent study (Artma et a l . ,  1968) four RNases were p u r i f i e d from the growth medium of U_. sphaerogena: RNase U j , a guanyloribonuclease;  RNases Uj, and U j , both of them puryloribonucleases;  and RNase U^, which was not b a s e - s p e c i f i c . These workers reported a s t r i k i n g increase In the release of RNases Uj and U^, together with a small increase i n RNases  and My  f o l l o w i n g the a d d i t i o n of RNA as the s o l e source of phosphorus  In the c u l t u r e medium.  In contrast with the r e s u l t s obtained  In studies of  JJ. zea and U. sphaerogena, RNA was reported not to enhance the formation or release of e x t r a c e l l u l a r RNases by H. crassa (Takai ejt al_., 1967). The  Importance of RNA-degrading enzymes i n growing c e l l s i s discussed  In d e t a i l by Egaml and Nakaraura (1969), who summarize t h e i r probable p h y s i o l o g i c a l r o l e s as f o l l o w s : 1) metabolism of RNAs; t l ) p r o t e c t i o n against penetration by phage  RNA;  i l l ) supply of n u t r i e n t s by degrading e x t r a c e l l u l a r  RNA;  k  i v ) a c t i v a t i o n o f D N A - s p e c i f l c endonuclease  I by removing  Inhibitory  RNA. E x t r a c e l l u l a r RNases o f t e n have base s p e c i f i c i t i e s and  i n c o n s i d e r i n g the  r o l e o f such RNases i t i s known t h a t g u a n l n e - r i c h n u c l e o t i d e s tend t o form a g g r e g a t e s and  i t Is t h e r e f o r e suggested  a c i d phosphodtester  t h a t RNases s p e c i f i c f o r g u a n y l f c  bonds would y i e l d d i g e s t i o n p r o d u c t s which may  through the c e l l membrane (Egami and Nakamura, 1969)*  diffuse  On the w h o l e , t h e  m i c r o b i a l e x t r a c e l l u l a r r t b o n u c l e a s e s , because o f t h e i r ease o f p u r i f i c a t i o n and t h e i r s t a b i l i t y , have found g r e a t f a v o u r w i t h the b i o c h e m i s t w h i l e t h e general b i o l o g i c a l  r o l e o f such e x t r a c e l l u l a r p r o d u c t s remains, f o r t h e  most p a r t , u n r e s o l v e d . S p e c i e s o f t h e genus U s t l l a g o a r e u s u a l l y c o n s i d e r e d t o be o b l i g a t e p a r a s i t e s because they a r e e n t i r e l y dependent on t h e i r h o s t s p e c i e s d u r i n g a t l e a s t p a r t o f the l i f e c y c l e .  The a s s o c i a t i o n between U s t l l a g o s p e c i e s and  t h e i r h o s t s a r e u s u a l l y h i g h l y s p e c i f i c and, a l t h o u g h e x t r a c e l l u l a r enzymes o f s e v e r a l U s t l l a g o s p e c i e s have been s t u d i e d , t h e r e i s as y e t no  evidence  t o i n d i c a t e t h a t the p r o d u c t i o n o f e x t r a c e l l u l a r enzymes c o n t r i b u t e s i n any p a r t i c u l a r way  t o the s u c c e s s o f t h e s e s p e c i e s d u r i n g t h e i r  pathogenic  phase. One approach  c o n s i d e r e d u s e f u l f o r e v a l u a t i n g the importance o f e x t r a -  c e l l u l a r enzymes o f p a r a s i t i c fungt  I n v o l v e s the p r o d u c t i o n o f mutants d e f i c i e n t  In the a b i l i t y t o r e l e a s e a s p e c i f i c a c t i v i t y and the study o f the a b t l i t y o f such mutants t o p a r a s i t i z e the h o s t . d a t e been produced  o n l y by UV  Irradiation  Mutants tn U s t l l a g o hordet have t o (Hood, 1966), but u s e f u l  o f t h i s method o f m u t a t i o n r e q u i r e d t h e treatment o f s y n c h r o n o u s l y cultures.  The c h e m i c a l mutagen N - m e t h y l - N ' - n t t r o - N - n t t r o s o g u a n t d t n e  o r n t t r o s o g u a n l d i n e ) was used R N a s e - d e f I c i e n t mutants.  tn the p r e s e n t study In a t t e m p t s  H o l l l d a y and H a l l l w e l l  application growing (NG  t o produce  (1968) have p r e v i o u s l y  5 used NG with Ustilago maydls to produce e x t r a c e l l u l a r DNase-defIclent mutants. Mandell and Greenberg  {1961) f i r s t reported the mutagenic action of NG  In bacteria and t h i s compound has subsequently been shown to be highly Adelberg et al_. ( 1 9 6 5 ) studied the action of NG In f j c h e j j c h t a  mutagenic.  c o l i and found that a high y i e l d of mutations was obtained in conditions which gave over 50 percent survival rate.  Mutagenic action of NG has  s i m i l a r l y been studied In Salmonella typhlmurium Schlzosaccharomyces  (Elsenstark et a l . ,  1965),  pombe (Loprleno and Clarke, 1965) and Arabldopsls thai Iana  (Muller and Gichner, 1 9 6 4 ) .  Loprieno and Clarke ( 1 9 6 5 )  in their study reported  the following order for decreasing r a t i o s of mutagenesis to l e t h a l i t y : NG > nitrosomethylurethane > u l t r a v i o l e t l i g h t > nitrous a c i d . acts on DNA and RNA  to produce 7-methylguanine  In v i t r o , NG  (Craddock, 1 9 6 8 ; Lawley, 1 9 6 8 ;  McCalla, 1967) and a small amount of 3-methyladenine (Lawley, 1 9 6 8 ) . The  in vivo action of NG on DNA  i s s p e c i f i c for the r e p l i c a t i n g region of  the bacterial genome (Cerda"-01medb e£ a1_., In fungi.  1 9 6 8 ) , but this has yet to be shown  In another in vivo study, Baker and Tessman ( 1 9 6 8 ) found d i f f e r e n t  mutagenic s p e c i f i c i t i e s in phages SI3 and T 4 following treatment with  NG.  With S13 (as in Salmonella typhlmurium) both transitions of GC to AT and of AT to GC were Induced predominated of the DNA  In about equal frequences, whereas GC to AT transitions  In phage T 4 .  The molecular environment  of the r e p l i c a t i o n region  (which should depend on the s p e c i f i c mechanism of r e p l i c a t i o n ,  the nature of the DNA  polymerase and the base composition of the DNA)  suggested to be responsible f o r the differences.  Is  The authors point out that  care should be taken in making generalizations about the s p e c i f i c i t y of a p a r t i c u l a r mutagen (e.g. beyond saying that NG The macromolecular  Is an a l k y l a t i n g agent).  action of NG In vivo is not r e s t r i c t e d to DNA  (Cerda-Olmedo and Hanawalt, 1 9 6 7 ) .  Besides causing a l t e r a t i o n of DNA which  can be recognized and repaired by the dark repair mechanism, NG can Inactivate  6 proteins and Inhibit protein synthesis. Inhibit RNA  To a lesser extent HG can a l s o  synthesis, resulting In a reduction In the rate of synthesis of  proteins and the production of small amounts of non-functional protein. HG may by  a l s o cause misreading of the genetic code, which can be suppressed  streptomycin. Whether HG or diazoraethane  unanswered question.  Is the reactive species tn vivo i s s t i l l  The former view Is supported  an  by Mandell and Greenberg  (I960) and McCalla (1967) and the l a t t e r by Cerda-Olmedo and Hanawalt (1968), As well as having an e f f i c i e n t procedure for Inducing mutations, one must also have a s e l e c t i o n method for detecting those mutants which are of s p e c i f i c Interest.  For a detailed consideration of the problems Involved  see the Discussion. In the present study, one approach that was considered for the s e l e c t i o n of RNase-defIcient  mutants Involved th® use of a d e r i v a t i v e of RNA which altered  the attack of base-spectfIc RNases.  The modification of  rtbonuclease  degradation products by the addltton, tn this case, of a water-soluble car bodIImlde to a dinucleotlde substrate was  f i r s t reported by Gilham (1962).  N-cyclohexy1-N'-B-(^-raethylmorpholln!um)ethyIcarbodlImlde p-toluene-sulfonate (CMC-p-toluenesulfonate) was  shown to react s p e c i f i c a l l y with the bases  guanine, u r a c i l and thymidine (Ko and Gilham, 1967). Pyrlmldine-speclfIc RNases are r e s t r i c t e d to degradation at cytosine when the RNA sulfonate.  substrate has previously been reacted with CMC-p-tolueneSimilarly It should be feasible to l i m i t the action of RNases of  other base s p e c i f i c i t i e s (P.T. Gtlham, personal communication). The p r a c t i c a l application of this method of limiting RNase action was demonstrated by Lee ejt aj_. (1965) for pancreatic RNase in the preparation of trinucleotides containing a terminal cyttdlne, by Naylor e£ aJL  (1965) f o r  RNase on CMC-modified polynucleotides, and by Sanger et a l . (1968) tn the  7 r e s t r i c t i o n of RNase A on modified  5s RNA f o r p a r t i a l hydrolyses to  f a c i l i t a t e base sequence analysis. After establishing the presence of ribonuclease a c t i v i t y In the culture medium of the barley-sraut fungus (Ustilago horde1 (Pers.) Lagerh.), this thesis was undertaken to gain Information about the possible role of the enzymes responsible f o r RNase a c t i v i t y . were also detected two parts:  (Protease and amylase a c t i v i t i e s  but these were not studied.)  The study ts divided into  one, the study of the RNase a c t i v i t y in the non-pathogenic  phase of the l i f e cycle (nature of a c t i v i t y , secretion pattern, control of secretton); and two, chemical mutagenesis of U. hordet In attempts to produce mutants d e f i c i e n t In RNase a c t i v i t y which would be useful In considering the Importance of e x t r a c e l l u l a r RNases In the host parasite relationship of U. hordeI and i t s host, c u l t i v a t e d barley (Hordeum vulgare L . ) .  8  MATERIALS AND METHODS  1.  BIOLOGICAL MATERIAL  The two standard monosporldlal ( I . e . haplotd) l i n e s o f U s t l l a g o hordeI (Pers.) Lagerh. used In t h i s study were developed by Hood (1966) and designated by him as E^ and 1^ . Both these l i n e s were used i n enzyme studies while E j only was used tn mutagenesis experiments. A m y c e l i a l c u l t u r e derived from a complementation t e s t (Dlnoor and Person, 1969) o f two s p o r l d l a l l i n e s derived from a s i n g l e t e l l o s p o r e , Met  Pan Arg A and Met Pan Arg a (Drs. Jean Mayo and C O . Person,  personal communication), was studied f o r production o f e x t r a c e l l u l a r RNase.  2.  CULTURING  L i q u i d c u l t u r e s were grown In a New Brunswick psycrotherm incubator a t 22° C on a shaking table (100 RPM).  Agar plates were a l s o incubated a t 22° C.  In order to measure c e l l density o f c u l t u r e s a t d i f f e r e n t stages o f growth, c u l t u r e s were grown i n l i q u i d c u l t u r e i n 125 ml Erlenmeyer f l a s k s f i t t e d with Klett-Summerson c o l o r ( m e t r i c tubes (side-arm f l a s k s ) . the  By turning  f l a s k s sideways, the side-arm f i l l e d with c u l t u r e medium whose t u r b i d i t y  was then measured on a Klett-Summerson p h o t o e l e c t r i c colorimeter f i t t e d w i t h a red f i l t e r .  This allowed one t o f o l l o w a c u l t u r e from a c e l l density of  10^ c e l l s / m l (0-5 K.U.) through t o s t a t i o n a r y phase which occurred a t a c e l l  8  density near 2 x 10 c e l l s / m l (*»00-/»50 K.U.).  9  3.  CULTURE MEDIA AND SELECTION PLATES  Culture media, minimal and complete, were prepared according t o the procedures outltned by Hood (1966).  Minimal medium contained  20 ml Vogel's  s a l t s o l u t i o n (see Appendix A) and 10 g glucose per one l i t e r o f d i s t i l l e d water.  Complete medium was a minimal medium enriched with 5 g D l f c o yeast  e x t r a c t , 5 g s a l t - f r e e casein hydrolysate (N.B. Co.), 50 mg tryptophan and 10 ml vitamin s o l u t i o n (see Appendix A) per one l i t e r o f minimal medium. For s o l i d medium 2.0$ Difco bacto agar was added. Supplemented minimal medium was prepared according to HoiIIday (1961). The i n d i v i d u a l growth f a c t o r s were added to minimal medium as required: amino a c i d s , 100 mg; purines and pyrlmldlnes, 10rag;and vitamins, 1 mg per liter. I n i t i a l screening o f auxotrophs was c a r r i e d out on agar plates of minimal medium supplemented with yeast e x t r a c t , vitamins, o r casein hydrolysate. In amounts normally used f o r complete medium. Auxotrophs were stored on agar s l a n t s (complete medium plus 2.5% agar) at 4° C and transferred every four weeks. RNA plates containing 0.5% yeast RNA were prepared by adding MI 11(pored 10% RNA s o l u t i o n t o autoclaved regular minimal medium containing one percent agar.  4.  ASSAYS  (a) Rlbonuclease a c t i v i t y was determined q u a n t i t a t i v e l y by measuring the absorbance of a c i d - s o l u b l e degradation products according to the assay f o r RNase T., described by Takahashi (1961) and modified by Arima e t a l . (1968).  10  The reaction mixture contained 0.1 ml of enzyme s o l u t i o n , 0.25 ml of 0.2 H T r l s buffer, pH 7.5, o r 0.2 M sodium acetate buffer, pH k.$, 0.1ralof -2 2 x 10  M E0TA, 0.3 ml of d i s t t l l e d water and 0.25 ml of yeast sodium  ribonucleate (Schwarz), 10 mg/ml freshly prepared before use. The hydrolysis was allowed to proceed f o r 30 min at 37° C a f t e r the additton o f RNA solution and was stopped with 0.25 ml of 0.75$ uranyl acetate In 254 p e r c h l o r i c a c i d . The reaction mixture was centrifuged and 0.2 ml of supernatant s o l u t i o n was diluted In 4.8 ml of d i s t i l l e d water and the absorbance at 260 nm was read on a Unicam SP 800 spectrophotometer f i t t e d with s i l i c a c e l l s of one centimeter l i g h t path.  The amount o f enzyme that under the standard assay conditions and  t h i r t y minutes of hydrolysis would produce an increase tn absorbance o f one at 260 nm was defined as one enzyme unit (Takahashl, 1961). An a l t e r n a t i v e , q u a l i t a t i v e assay f o r RNase a c t i v i t y s i m i l a r to that used by Hoi Itday and Hall(well  (1968) f o r ONase and e a r l i e r by J e f f r i e s et aJL (1957)  was employed In assaying f o r a c t i v i t y released from colonies of U. horde!. Since factors such as agar concentration, per cent RNA and depth of agar influenced this plate assay, the assay was c a r r i e d out under standard optimal conditions, using plates containing 1% agar, 0.5$ RNA and 8 ml of minimal medium per 8.5 cm diameter Petri plate.  These RNA plates were spotted or spread  with sportdta and grown f o r about f i v e days at 22° C or they were spotted with culture medium and the plates were floated tn a 37° C water bath f o r t h i r t y minutes.  To stop the hydrolysis, the plates were flooded with 10% t r i c h l o r o -  a c e t i c acid  (TCA).  To test the v a l i d i t y of this assay, ten m i c r o l i t e r s o f  p u r i f i e d pancreatic RNase (100-0.01 mtcrograms/ml, approximately 20,000 units/ml; Worthington) were spotted on such RNA plates.  Where there was no RNase, a white  p r e c i p i t a t e formed while In areas of RNase spotting cleared areas were v i s i b l e . This assay proved to be a rapid and s e n s i t i v e method f o r detecting a c t f v t t y that may be due to RNases.  11 (b) Phosphodiesterase (PDE) I and II (both described by R a z z e l l , 1967) were assayed f o r by the method of Razzell (H. Smith, personal communication). For PDE I the stock s o l u t i o n contained 0.1 ml M T r l s , pH 9.3, 0.05 ml 0.2 H MgClg, 0.10 ml para-nltrophenyl thymtdlne-5' phosphate, 5 mlcromoles per ml (I.e. 8.3 O.D./ml measured a t 272 nm In 0.01 M HC1), pH 9.3, and 0.75 ml water. To 0.1 ml of stock warmed 2 minutes a t 37° C about 20 m i c r o l i t e r of enzyme s o l u t i o n was added and incubated f o r one hour.  The reaction was stopped with  0.25 R>1 0.3 H NaOH and the contents o f the tube mixed by Inverting several times, made up to 1 ml with water and absorbance a t 400 nm was determined. For phosphodiesterase II the stock s o l u t i o n contained 0.25 ml H ammonium a c e t a t e , pH 5*9, 0.05 ml 0.02 M sodium E0TA, 0.1 ml 2,4-dlnltrophenyl thymidine-^' phosphate, 10 mlcromoles per m l , and 0.55 ml water.  The assay  procedure was as f o r P0E I. The use of 2,4-dlnltrophenyl thymldlne-3* phosphate, a m o d i f i c a t i o n o f Von TigerStrom and Smith (1969). allows d i r e c t q u a n t i t a t i v e measurement at pH 5.9 and 360 run, since the 2,4-dlnltrophenoxlde anion has maximum absorbancy near the assay pH. This Is an improvement over the use o f p-nltrophenyl thyraidine-3' phosphate as a substrate, since p-nltrophenoxide has l i t t l e absorbance a t pK 5.9. (c) Phosphomonoesterase,  a c i d and a l k a l i n e , were measured by the method  of Artma e t a l . (1968).  5.  CHEMICAL MUTAGENESIS, DETECTION AND SCREENING OF MUTANTS  To Induce mutations, w i l d type, log phase s p o r i d l a ( E ^ ) grown In l i q u i d minimal medium were p e l l e t e d and resuspended a t a concentration of about 10  c e l l s per ml In c i t r a t e b u f f e r at pH 5.0 or 5.7 i n 40 ml Nalgene tubes.  S u f f i c i e n t NG stock s o l u t i o n was added to give a f i n a l concentration o f 0.1 mg NG per ml o f medium. 22-24  C.  Standard NG treatment lasted f o r f i f t e e n minutes a t  Stock s o l u t i o n of 2 mg per ml o f NG was made fresh with each  12 treatment. Treated c e l l s were Immediately washed with c i t r a t e buffer and then with complete medium before being suspended  In the treatment volume of complete  medium and transferred to SO ml Erlenmeyer flasks to be Incubated f o r about ten hours before p l a t i n g . Samples of cultures treated tn this way were d i l u t e d to give 50 to 100 colonies per plate when spread on complete medium.  Colonies of these plates  were r e p l i c a t e d , using the method o f Lederberg and Lederberg (1952), as follows;  to minimal plates to determine auxotrophs, to RNA plates to  detect RNase-deftctent colonies and, as a f t n a l step, to complete plates to confirm that transfer of each colony had been made onto a l l previous plates. After f i v e to ten days, each plate was examined and the auxotrophs were tentatively Identified;  these were further defined by their a b t l t t y to respond  to casein hydrolysate, to vitamin solution or to yeast extract.  Final  c l a s s i f i c a t i o n o f an auxotroph was based on a p o s i t i v e response to a stngl® compound added to minimal plates. Arlma et ej[, (1968) reported four e x t r a c e l l u l a r RNases In Ustilago sphaerogena, two with sharp optima at pH 4.5 and two others with a broader optima at pH 7.5. study.  These observations were taken as the basis f o r beginning thts  RNA plates were made with a pH of 6.0 or greater In order to assay f o r  deficiency o f the RNases with the broad optima (I.e. Uj and U^).  6.  THE PREPARATION OF CMC-RNA  The water-soluble carbodlImlde derivative of RNA was prepared by the method of Ho et aj_. (1967).  The reactants, CMC-p-toluenesulfonate  (Aldrlch)  and yeast sodium ribonucleate (Schwarz), were reacted f o r 26-30 hours.  13 RESULTS  1.  EVIDENCE FOR EXTRACELLULAR RIBONUCLEASES IN U. HORDEI  (a) I n i t i a l q u a l i t a t i v e and quantitative assays f o r RNase a c t i v i t y in the medium of a stationary phase E^ and 7*5*  culture gave p o s i t i v e r e s u l t s , both at pH 4.5  Assaying across pH range 3.0 to 9.2 demonstrated two pH maxima,  one at pH 5.0 and another at pH 8.0 (Fig. t ) . was obtained at pH 6.5;  in addition, a third maximum  t h i s maximum has not been reported  previously.  Some preliminary attempts at i d e n t i f y i n g the number of RNases released by U. horde1 were made (see Discussion). (b) Evidence that the RNase a c t i v i t y was due to the release of extrac e l l u l a r RNases came from observation of culture samples under the l i g h t microscope.  In samples taken during the period of maximal Increase in RNase  a c t i v i t y no rupture c e l l s or c e l l fragments were seen.  U. hordeI character-  I s t l c a l l y does not lyse but rather, becomes mycelial towards the end of i t s log  phase of growth. (c) A s l i g h t amount of a c t i v i t y  (0.1 mM/ml/hr) due to phosphodiesterase I  was found to be associated with the c e l l surface, which, on centrlfuging out the c e l l s and assaying the c e l l - f r e e culture medium was not detectable.  Phospho-  diesterase II a c t i v i t y was not detected on the c e l l surface or In the culture medium. (d) A ten day o l d l i q u i d culture of s t r a i n E j  in minimal medium, that had  been in the stationary phase of growth f o r f i v e days, was assayed f o r phosphomonoesterase (PME) a c t i v i t y .  A c t i v i t y was detected only under acid conditions  (pH 5.5) and primarily in c e l l s containing culture medium (0.54 mM/hr/ml); a small amount was found in c e l l free medium (0.04 mM/hr/ml), possibly due to c e l l death or lyses.  Figure 1:  The RNase a c t i v i t y of c e l l - f r e e minimal c u l t u r e medium,In which s t r a i n  had been grown to s t a t i o n a r y phase,  measured across the pH range 3*0 to 9.2.  FIGURE I  pH  UNITS  2.  RELEASE OF RlBONUCLEASE ACTIVITY  (a) Figure 2 presents RNase a c t i v i t y for  and  representative graphs to show the release of 1^* grown in complete and  A sharp increase In RNase a c t i v i t y occurred stationary phase maximal release was  In minimal medium.  in late log phase and by early  achieved.  The release of e x t r a c e l l u l a r  enzymes during this phase of growth has been observed not only with RNases but also with many other e x t r a c e l l u l a r enzymes, and may  be regarded as a  rather general c h a r a c t e r i s t i c of e x t r a c e l l u l a r enzymes of microorganisms (EgamI and Nakamura, 1969)* The release of a c t i v i t y tnto minimal medium was more gradual and began e a r l i e r a f t e r entry of the culture Into the log phase of growth, In contrast to cultures of complete medium. at the two pH's ( f i g . 3).  (4.5  and 7.5)  The  Increases In rtbonuclease a c t i v i t y measured  p a r a l l e l each other during the period of release  This suggests that the period of synthesis and release are co-  Incidental for the enzymes Involved.  The total amount of RNase a c t i v i t y  greater In complete medium suggesting  that conditions in this medium were  was  more favourable for synthesis or release of RNase. (b) A stable mycelial statn grown in complete medium released  rtbonuclease  a c t t v l t y in a pattern s i m i l a r to a sportdtal culture In complete medium (Fig. 4).  Total RNase a c t i v i t y reached a level s i m i l a r to that of sporldta! c u l t u r e s .  The c o n s t i t u t i v e release of RNase a c t i v i t y . In both sportdtal and mycelial (possibly dtkaryotlc) cultures would suggest that such ts the case for at least a l l of the non-parasttic part of the l i f e cycle of U. horde!. (c) To determine whether the addition of RNA  to the culture medium had  inducible e f f e c t (I.e. Increase the amount of RNase release), the release of RNase a c t i v i t y from s t r a i n E^", grown In minimal medium plus 0.5% was measured ( F i g . 5).  yeast  RNA,  This figure shows, f i r s t l y , that the maximal level  an  Figure 2:  Growth (-o-) o f s t r a i n  and  cultured In e i t h e r minimal  or complete medium; and the release of RNase a c t i v i t y a t pH 7.5 ( — o — ) .  a) E ~/M; b) E^/C; 3  c) l ^ / M and  d) l ^ / C .  RNase  91  activity  GROWTH  (units  / ml)  (K.U.)  RNase  activity  GROWTH  (units/ml)  (K.  U.)  Figure 3:  Growth of s t r a i n E^  In minimal medium (a) and In complete  medium ( b ) . RNase a c t i v i t y was measured a t both pH 4.5 and pH 7.5.  RNase  activity  GRO./TH  (  (units/nil)  K.U.)  F i g u r e 4:  The growth c u r v e of  the s t a b l e m y c e l i a l  the r e l e a s e of pH 4.5  and pH 7.5  strain  (Myc/C) and  RNase a c t i v i t y .  RNase  activity  (units/in )  Figure 5- The release o f RNase a c t i v i t y by s t r a i n E^ medium supplemented with 0.5% yeast RNA.  grown on minimal  This graph a l s o  includes, f o r comparison, the growth of s t r a i n E^ release of RNase a c t i v i t y i n minimal medium.  and i t s  FIGURE  '©  »growth  E-j'/M  m  'growth  E^'/M+RNA  E  60=  .a.-7.5 a c t i v i t y  ©4.5 •A7.5  5  E^"/M|  activity  E-^'/M+RNA  activi ty  E^~/M+RNA  c  3  o  TO 0) TO  O  TIME  (Hours) U5  of RNase a c t i v i t y release by early stationary phase o f the E^ In RNA medium was noticeably less than that of a culture o f Eg medium.  Secondly, a s i g n i f i c a n t delay  culture In minimal  In the release of the RNase a c t i v i t y ,  r e l a t i v e to the start of log phase growth, was noted, as had s i m i l a r l y been noted with E^ (d) Considering  culture fn complete medium (e.g. Figs. 2 and 3). the release of RNase a c t i v i t y r e l a t i v e to the growth of  the c u l t u r e , It appeared (taking Into account the lag period between the inoculation o f the culture and f i r s t detecting RNase a c t i v i t y ) that the quantity of RNase a c t i v i t y was a function of c e l l density rather than of the age of the c u l t u r e .  A d i l u t i o n experiment in which culture £j  in completr  medium was grown to mid-log phase and then d i l u t e d to a o n e - f i f t h c e l l concentration was c a r r i e d out.  The d i l u t i o n was performed by taking one volume  of culture sample and adding i t to four volumes of either complete or minimal medium.  Growth and release of RNase a c t i v i t y was followed by the standard  methods, and these results are presented In f i g u r e 6.  The rates o f release  of a c t i v i t y ( i . e . the slope of the a c t i v i t y lines) o f the o r i g i n a l and the d i l u t e d cultures are s i m i l a r . The greatest rate of release In both cases occurred at the point of maximal c e l l  3.  density.  RNA AS A CARBON SOURCE FOR U. HORDEI  Sporldia of Ustilago horde! wore not able to grow on agar plates or in l i q u i d c u l t u r e which contained only yeast RNA (SI) and Vogel's s a l t s o l u t i o n , suggesting  that the RNA cannot serve the c e l l as a carbon source in the same  way as glucose.  Figure 6:  The d i l u t i o n experiment.  From a c u l t u r e of s t r a i n  In  complete medium (E^ /C), a sample was d i l u t e d Into four times i t s volume of minimal (E^ /C/M)  or complete medium  (E^ /C/C) a t 121 hr. Growth was followed f o r both the o r i g i n a l and the d i l u t e d c u l t u r e s . a c t i v i t y was measured at pH 7»5«  Release of RNase  FIGURE  TIME  6  (hours)  4.  CHEMICAL MUTAGENESIS  (a) Table I presents the assembled Information o f four d i f f e r e n t NG treatment t r i a l s .  The younger the c u l t u r e sampled f o r treatment, the  greater the k i l l i n g e f f e c t and probably the higher the mutation r a t e (though the data are Incomplete on t h i s p o i n t ) .  This i s In agreement with  the report that NG a c t s a t the r e p l i c a t i o n points (Cerda-Olmedo e£ aJL, 1968), and therefore an a c t i v e log phase c u l t u r e as In t r i a l NG IV i s predicted t o be a f f e c t e d by the treatment, more than an end-of-log o r e a r l y - l o g phase c u l t u r e such as that used In t r i a l NG I o r I I I . (b) Among approximately 14,000 c o l o n i e s from NG-treated c u l t u r e samples, no RNase-defIcient mutants were detected. (c) S i x t y - n i n e c o l o n i e s were picked from p l a t e s o f NG-treated c e l l s f o r t h e i r i n a b i l i t y t o grow on minimal medium, and one f o r having a p e c u l i a r colony morphology. Isolates.  Table II summarizes the preliminary screening o f these  F i f t y - f i v e o f the Isolates had sharp n u t r i t i o n a l d e f i c i e n c i e s  and were considered t o be auxotrophs. e x t r a c t and o f these: definitely);  A l l 55 responded s t r o n g l y t o yeast  seven were possjbly adenlne-defIcient (NG 48 and NG 64  seven were v l t a r a l n - d e f i c i e n t (probably n i a c i n o r r i b o f l a v i n ) ;  one responded weakly t o c y t o s l n e (NG 66), w h i l e another (NG 40) responded to a l l o f the bases except guanine, suggesting that t t Is a n u c l e i c a c i d mutant o f some k i n d .  NG 69, a morphological mutant, had the appearance o f  a length o f randomly c o i l e d rope.  5. CMC-P-TOLUENESULFONATE MODIFIED RNA  To measure the extent to which guanines i n the RNA had been blocked by the CMC a d d l t t o n r e a c t i o n , the RNase T. (10 mg/ml) h y d r o l y s i s o f CMC-RNA  Table I. HG treatments o f s t r a i n E^  of U. horde1.  Treatment C e l l Density Sampled Trial  Duration (minutes)  pH  KU  HG  I  J2,/  5.0  330  NG  i i  15  5.0  280  Viability count  During treatment (calc.)  1.1  .55 x 10  x 10  8  .8 x I O  8 8  .8  x 10  Posttreatment C e l l S u r v i v a l growth period Colony Percent (hrs.) count (calc.) 8.5 12.5  8  2.3 x 10 '1.5  * 10  Auxotrophs  7  18  5  7  5  t) 2 H) 15 c  NG i l l  15  5.7  350  2 x 16  IV  15  5.7  106  1.5 x 10  NG  8 7  1  x 10*  .75 x  10  8  9  3.3 x 10'  9.5  1.* x 10  6  12  33  Percent isolate auxotrophs numbers 1-6 7-H '•5 .43  0.7  a) Estimated; b) C a l c u l a t i o n based on the assumption that 10 hrs o f post-treatment growth represents 1.5 generations; c) A sample o f 1024 colonies from 'complete* plates with treated c e l l s .  15-31 32-70  Table 11. Preliminary screening of possible mutant Isolates.  Minimal medium plus supplement NG  Isolate  Minimal medium  1  -  2*3  -  k  1  5*6  3  -  9-11  I  7  Yeast extract •  •  •  -  -  12  1  13  1  \k  •  •  -  1  -  15 - 20  21  Vitamin solution  +  rz.  Amino, acids  -  -  B a s e s  Adenine  Description  1  Adenlne  t  leaky,yet low  1  Adenine  -  Adenine  Cytoslne  Uracil  thymine  Guanine  -  -  -  -  -  1  1  1  1  -  -  1  -  -  -  1  1  1  1  1  1  leaky  1  •  •  +  •  +  wild  I  1  1  t  1  1  leaky  •  •  •  •  wild  -  -  -  1  -  -  _  -  -  -  •-  -  -  -  -  1  0  c  Table II Continued  Minimal medium plus supplement B a s e s NG  isolate  Minimal medium  Yeast extract  Vitamin solution  Amino acids  Cytosine  Uracil  Thymine  Guanine  Adenine  Oescrlpti  •  -  -  -  -  Vitamin**  1  -  -  -  -  leaky  2k * 25  -  26  1  1  1  1  1  1  1  1  27  -  -  -  -• -  -  -  -  -  -  22 • 23  •  29  •-  -  1  -  30  1  1  -  1  1  1  1  1  1  -  -  -  -  -  1  1  1  1  1  1  wild  28  31 32  -  •  33 3^  1  35  •  36 37  +  -  •  Vitamin*  leaky  -  -  1  1  1  1  1  H  leaky  1  1  •  •  +  +  -  wild  1  -  -  -  -  •  •f  -  -  -  A  Vitamin Vitamin*  1  Table It Coatlimed  Ktntaat medium etas supplement  86  Mlfjfna! Isolate medium  38 39 40  Yeast extract  -  -  r  • *  41 - 47 %8 49-51  52 53 54  •  -  55  57 53  -  • •  • • • *  59 60 • 61  •  •m-  Amino acids  B a s e s — — • —— Cytoslne Uracil Thyaslne guanine  1  -  m.  1  mm  1  •  •  56  Vitamin solution  •  1  t  -  -  -  1 mm  «»  9*  mm  • •m  •  -  ^  •»  •» ^  mm  I  -  «•*'  *•»  «H  M  4tm  -mt  »  Adenine  1  Adenine  •  n.a»  •  M e n Ine  mm-  •m  «  •»  6  c  mm  mm  «•  Description  «M «•  1  Vitamin  1  Adenine  6  ro  Tab 1e I I Cont i nued  Minimal medkm p l u s  supplement B a s e s  NG isolate  Minimal medium  Yeast extract  62  1  +  63  -  bk  -  65  -  Amino acids  Cytokine  Uracil  Thymine  -  1  1  1  1  1  1  leaky  +  -  1  -  -  +  -  -  -  -  -  -  -  Adenine  +  +  -  -  -  -  -  66  -  +  -  -  1  -  -  -  67  -  +  -  1  -  -  68  1  +  -  1  I  1  69  +  +  +  1  +  +  70  -  +  -  1  -  .  Vitamin solution  .  -  '  Guanine  .  Adenine  -  1  -  -  -  b) c a s e i n h y d r o l y s a t e p l y s t r y p t o p h a n . definite.  d) p r o b a b l y n i a c i n or r i b o f l a v i n , s i n c e these a r e common to y e a s t e x t r a c t and v i t a m i n m i x t u r e . e) appearance l i k e a length o f randomly c o i l e d rope.  Vitamin  d  -  Cytosine  -  leaky  +  wild, morphological  1  a) + w i l d type g r o w t h ; 1 weak growth response; - no response. c) p r o b a b l e but not  Description  was compared with RNase hydrolysis of unmodified RNA.  Two preparations of  CMC-RNA had, respectively, Sh and 99 percent decrease In the formation of acid soluble products. CMC-p-toluenesulfonate  (The Inhibition o f RNase T^ a c t i v i t y , due to unreacted i n the CMC-RNA product, was not evaluated).  CMC-RNA  ts probably less acid-soluble than unmodified RNA f o r i t was found that pH 4.5 plates which contain RNA are c l e a r while containing CMC-RNA they formed a white p r e c i p i t a t e even before the addition of t r i c h l o r o a c e t i c acid.  RNase degradation of this white p r e c i p i t a t e was s t i l l possible f o r  cleared areas formed against the p r e c i p i t a t e wherever solution was spotted.  RNase-contatnlng  DISCUSSION  It was mentioned e a r l i e r that one consideration of t h i s study was to e s t a b l i s h whether e x t r a c e l l u l a r RNases were produced by U s t l l a g o hordei and, i f so, to determine whether they are i n any way s i m i l a r to those produced by U_. sphaerogena (Arlma et a K , 1968). The r e s u l t s i n d i c a t e :  I) that e x t r a c e l l u l a r ribonucleases are In f a c t  present a f t e r growth of U. horde1 s p o r l d i a and mycelta i n c u l t u r e media; and i i ) that the presence of ribonucleases i n the growth medium Is not e n t i r e l y due to release from ruptured c e l l s . The existence of three pH maxima ( F i g . 1 ) suggests that a t l e a s t three enzymes are present;  two o f these maxima ( I . e . those at pH 5.0 and pH 8.0)  compare with the pH optima shown by ribonucleases of U. sphaerogena.  Whether  each o f these optima corresponds to a s i n g l e RNase ( i n U_. sphaerogena, two were reported f o r each pH optimum; by biochemical methods.  Arima et^ aj_., 1 9 6 8 ) can only be e s t a b l i s h e d  Preliminary studies (author), using g e l f i l t r a t i o n  on Sephadex G - 7 5 (method of Arima et a l . , 1 9 6 8 ) suggested that there may be two RNases with pH maxima a t 4 . 5 (see Appendix B). Changes i n the c e l l u l a r environment resulted In n o t i c e a b l e e f f e c t s on the pattern of RNase s e c r e t i o n .  Release of RNase a c t i v i t y from s t r a i n E^" could  be delayed by the a d d i t i o n to minimal medium o f RNA or complete medium components. Several l i n e s of evidence would suggest that phosphate content i n the medium i s a p o s s i b l e c o n t r o l l i n g f a c t o r f o r the synthesis and s e c r e t i o n of e x t r a c e l l u l a r enzymes.  The fungus Asperg111us oryzae grown on phosphate-  f r e e medium,both synthesized and released I t s e x t r a c e l l u l a r amylase more r e a d i l y (Yurkevlch et_ aJL,  1967).  S i m i l a r l y , the production of e x t r a c e l l u l a r phytase by a number of  Aspergillus Isolates was strongly repressed by low levels ( 2 mg/100 ml) of Inorganic phosphorus;  It was, however, possible to overcome the phosphate-  Induced repression by Increasing the ratio of carbon to phosphorus in the growth medium. The authors (Shteh and Ware, 1968) concluded that since phytase in produced when concentrations of inorganic phosphate are limiting, the organism has the capacity to obtain Inorganic phosphate from organic phosphates when this becomes necessary. Arlma ct^ aj. (1968), who worked with U_. sphaerogena, replaced all phosphates In the culture medium with RNA and, following this, observed a significant Increase In RNase  , together with a smaller Increase in  and s t i l l smaller Increases In  and U^.  If these Increases were due to RNA  being a poor phosphate source (I.e. the enzymes being produced as a response to limiting concentrations of inorganic phosphate), It Is interesting to speculate on the reason for the differing levels of enzyme production.  In  future investigations of synthesis and release of extracellular RNases of tJ. horde! it would be of interest to Investigate the effects of limiting phosphate concentrations. The secretion of enzymes by microorganisms leads to speculation as to the function of such an activity.  In the case of U. sphaerogena, the secretion  of four extracellular RNases, possibly different from all different types of base specificities, can lead to the speculation that selection, at least In part, has been acting In the evolution of such a particular biological feature. However, to support such a speculation one must be able to attribute some role to the enzymes (I.e. some selective advantage associated with their presence). Extracellular enzymes are most often degradatlve enzymes and the view can be taken that they are "scavenger" enzymes, I.e. enzymes that make certain material In the cell's environment usable which would otherwise be wasted.  The  I n a b i l i t y of U. horde1 sportdta and mycella to grow on RNA  Indicates that the RNA used as carbon sources.  and  plus s a l t s  Its degradation products are apparently not being  Furthermore, If the RNases of U. horde1 did function  as scavenger enzymes to make use of any RNA  In the culture medium, they  would probably do so In conjunction with phosphomonoesterases (PMEs), for It ts generally considered that nucleotides (which represents the  final  products of RNase degradation) do not pass readily thocugh c e l l membranes; the phosphomonoesterases, acting at the c e l l surface to convert nucleotides to nucleosides, could thus mediate the movement of RNA the c e l l .  In this connection  degradation products  It w i l l be recalled that acid PME  activity  Into was  detected primarily on the c e l l surface rather than In the culture medium from which growing c e l l s had been removed. So f a r as the p a r a s i t i c phase of the l i f e cycle Is concerned, It Is difficult  to v i s u a l i z e any  Important function for the e x t r a c e l l u l a r RNases.  While It Is possible that e x t r a c e l l u l a r rlbonucleases may special way  contribute In some  to the success of U. horde1 during the p a r a s i t i c phase, the data do  not relate to the p a r a s i t i c phase.  The success o r f a i l u r e of  mutants would have provided useful Information concerning c e l l u l a r RNases during the pathogenic suggested a v i t a l  phase:  RNase-deficlent  the role of extra-  loss of pathogenicity would have  r o l e , whereas no loss of pathogenicity would have suggested  that their role during the p a r a s i t i c phase is dlspenslble. In considering the synthesis and translocation of e x t r a c e l l u l a r RNases, It Is interesting to speculate whether the many membrane-bound v e s i c l e s seen In electron micrographs of U_. horde! sporldla and mycella  (personal communication  Jane Robb and Carla Stein, Or. C. Person's laboratory) have a role In moving e x t r a c e l l u l a r RNases within the c e l l to the c e l l surface.  This could be  Investigated by histochemlcal methods using fluorescence-labelled antibodies against the p u r i f i e d e x t r a c e l l u l a r rlbonucleases.  If the v e s i c l e s were Involved  in the movement of e x t r a c e l l u l a r RNases the fluorescence-label led antibodies (see Shugar and Sierakowska, 1967 the v e s i c l e s .  for d e t a i l s ) should be concentrated  In  Likewise, vesicles which have been Isolated from c e l l s  (e.g. by the method of Matile, 1967)  should contain RNase a c t i v i t y .  It Is not possible to decide whether the f a i l u r e to obtain  rIbonuclease-  d e f i c i e n t mutants was due to the fact that no mutants were produced, or to the fact that the mutants having been produced went undetected.  In favour  of the f i r s t of these p o s s i b i l i t i e s Is the fact that the total number of mutations obtained was  small.  Under these conditions, the recovery of  s p e c i f i c mutants would be Influenced by the r e l a t i v e s t a b i l i t i e s of s p e c i f i c genetic l o c i .  If the change to rlbonuclease-defIclency occurs only rarely  It is possible that the total s i z e of the mutant sample was this type of mutant.  too small to Include  This point could be c l a r i f i e d In future work by  conditions in which mutagenicity  Is enhanced, for example by treating c e l l s  In mj.d-log phase when they are more susceptible to k i l l i n g by NG NG  IV).  (cf. t r i a l  Studies with bacteria have shown that NG-lnduced mutagenesis Is  effected at the region, or "point", of ONA 1968).  choosing  r e p l i c a t i o n (Cerda*-01roedo et^ al_.,  If this observation holds a l s o for jj. horde1, It should be possible to  find a stage, e i t h e r In the growth of a culture or In the c e l l cycle of synchronized  cultures, In which even higher y i e l d s of NG-lnduced mutations  can be obtained. In considering the second p o s s i b i l i t y (I.e. that rlbonuclease-defIcient mutants were In fact produced but not detected), It should be noted that If the excreted ribonucleases perform an Indlspenslble role within the c e l l before they are released, It Is not l i k e l y that RNase mutants (incapable of forming  the needed enzyme) could be recovered.  The screening procedures  were, however, based on the assumption that c e l l s d e f i c i e n t in e x t r a c e l l u l a r ribonucleases could nevertheless survive and reproduce.  The selection of  RNase-deficient mutants Is complicated by the fact that there are at least two and possibly more (as many as f i v e If the enzyme a c t i v i t y shown at pH 6.5 Is s i g n i f i c a n t ) enzymes.  The problem of detecting  loss of a c t i v i t y  of single enzymes would be less complicated If the production of two or more enzymes were controlled by a single genetic locus, and It would be a much simpler problem i f a l l e x t r a c e l l u l a r RNase a c t i v i t y were controlled by a single genetic locus.  However, since e x t r a c e l l u l a r RNase a c t i v i t y  seems not to represent a s t r i c t l y Inducible system, i t Is probable that the e x t r a c e l l u l a r RNases are not under coordinated control by a single  locus.  It was therefore necessary to employ a selection procedure In which i d e n t i f i c a t i o n of mutants was based on the loss of a c t i v i t y of single enzymes. The method used In this study was selective only on the basis of pH optima. RNA-contalnlng plates at pH 6.0  (or higher) were used to screen f o r loss of  enzymes with wide a l k a l i optima (see Materials and Methods).  But where there  Is overlapping of pH optima of d i f f e r e n t RNases, or where there is a c t i v i t y of nonspecific dtesterases,  the method becomes Ineffective, and It Is probable  that these two factors d i d interfere with the effectiveness of the screening. An a l t e r n a t i v e to this method would be to s h i f t the pH of the RNA-contalnlng plates to below 4.0, thus selecting for deficiency of enzymes with optima at pH 5.0 and, at the same time, minimizing the effects of enzymes with optima at pH 6.5 and higher. A second method of s e l e c t i o n , based on a l l optima and base s p e c i f i c i t y , had been considered but because of the i n a v a l l a b t 1 1 t y of the compound p-to1uenesu1fonate was not used.  CMC-  The rationale of this method, based on the  presence of the RNases reported by Arlma et al_. (1968) Is outlined  In Table  A disadvantage of this method, as with the f i r s t , Is that It does not Identify ribonuclease a c t i v i t y on the basis of Individual ctstrons and that d e f i n i t e and p o s i t i v e results are thus contingent on a multiple mutational event.  111.  Table HI.  S e l e c t i o n method f o r d e t e c t i n g e x t r a c e l l u l a r mutants u s i n g a CMC d e r i v a t i v e of  RNA.  Response to substrate Oeflelency In RNase activity  RNA — pN 4.5 pH 7.5  Total loss  1.  0  CMC-RNA pK 4.5  pH 7.5  -  2. pH 4.5 1o»» (U and Uj)  .  2  3. pH 7.5 loss  RNase d e f i c i e n t  *  d  •  -  <«•  -  <+  e  a) + enzyme activity present; - no enzyme activity; <* less activity than when using unmodified RNA substrate; «<+ very much less than when using unmodified RNA substrate* b) Uj and c)  present.  acting at cyttdlne and adenine.  d) Ug and U| present. e) U. and U„ acting at guanine.  3 The d i s a d v a n t a g e o f both these methods c o u l d p o s s i b l y be overcome by a a r a n g i n g t o s c r e e n f o r mutants whose s u r v i v a l capacity for rlbonuclease a c t i v i t y .  i s dependent on the  I t Is known t h a t the w i l d - t y p e  ( i . e . non-mutant) s t r a i n o f U_. h o r d e i cannot grow when p r o v i d e d w i t h as the o n l y s o u r c e o f c a r b o n .  RNA  But I f I t were p o s s i b l e t o s y n t h e s i z e a  s t r a i n which has a s p e c i f i c requirement f o r a preformed n u c l e o s i d e t h a t c o u l d be o b t a i n e d through d e g r a d a t i o n s o f RNA RNase and PME,  o r o f PDE  and PME  ( e i t h e r by a c o m b i n a t i o n o f  a c t i v i t y ) , such a s t r a i n s h o u l d be  c a p a b l e o f showing a growth response on RNA-supplemented medium.  If a  p o s i t i v e growth response were o b t a i n e d , i t s h o u l d then be p o s s i b l e t o s e l e c t f o r RNase mutants on the b a s i s o f f a i l u r e t o show the response. T h i s approach,  i f i t s h o u l d prove p r a c t i c a b l e , would thus s e l e c t f o r RNase  mutants on the b a s i s o f a u x o t r o p h y ;  the auxotrophs w o u l d , o f c o u r s e , r e q u i r e  further screening. The method J u s t o u t l i n e d c o u l d perhaps be m o d i f i e d so as t o e l i m i n a t e the p o s s i b l e involvement o f both PME  and PDE  activity.  The  modification  would r e q u i r e development o f a h y p o t h e t i c a l s t r a i n h a v i n g a requirement f o r s u b s t a n c e "X", which c o u l d be l i b e r a t e d , through a c t i o n o f i t s e x t r a c e l l u l a r RNase, from a n u c l e o s i d e 3 ( X ) phosphate. , _  e x i s t e n c e o f a system were a l l  i n which RNases  I f one were t o assume the  through t o  (Arima et_ a h ,  p r e s e n t , the base s p e c i f i c i t y o f endonucieases c o u l d then be used  t o some advantage  s i n c e i t would a l l o w f o r s e l e c t i o n , In sequence,  U^,  and f i n a l l y f o r the Uj d e f i c i e n c y  the U*2 and  Table IV). RNases),  1968)  A mutant t h a t Is d e f i c i e n t f o r  f o r the  (see o u t l i n e o f method,  (and t h e r e f o r e f o r a l l  four  i f one were o b t a i n e d , c o u l d be used as a p a r e n t a l s t r a i n In a t t e m p t i n g  t o d e v e l o p , through s e l e c t i o n o f back m u t a t i o n s , those c u l t u r e s t h a t d e f i c i e n t f o r s p e c i f i c RNases.  As a f i n a l m o d i f i c a t i o n , i t may  t o f i n d a compound w h i c h , a c t i n g as compound "X  11  remained  be p o s s i b l e  i n t h i s system, would be  toxic  Table IV.  Selection scheme f o r the detection o f three classes of e x t r a c e l l u l a r RNase d e f i c i e n t strains using mode) substrates.  Enzyme response  8  Model substrate 1. Uridine 3'-(X)P II  2. Adenosine 3'-(X)P it  3.  Genotype  2  S  n/a  n/a  U  wild  n/a  V  n/a  n/a  n/a  wild  n/a  •  •  U  °2* * 3* Guanos Ine 3'-(X)P wild U  b  n/a  m  .•  •  II  •  •  •  a) • ability to release compound X; - Inability to release compound X* b) not applicable since assuming absolute base specificity as suggested for each enzyme (Uj - U^ ) by Arlma at a l . (1968).  to the cells following Its release by RNase. This would automatically eliminate all cells excepting those that were entirely deficient in production of extracellular RNases and those which had become resistant to the toxic compound. The application of this general method using a model compound nucleoside 3'**(X)P is contingent on the following conditions: I) that the endonucleases are able to use the model compound as an efficient substrate, II) that the model compound Is stable and can be synthesized with reasonable ease and therefore in quantity, and ill) that the enzymes have absolute base spectflclty. The type of auxotrophs which were obtained in the NG study had been In many cases previously obtained by Hood (1966) using U.V. Irradiation. The only definite nucleic acid mutants (i.e. the adenine deficients) obtained here, were, In fact, the only nucleic acid mutant obtained by Hood. The mutants with wlvltamln requirement were suggested to be either for niacin or for riboflavin. study.  Of these two, Hood obtained only niacin mutants In his  If the NG vitamin mutants are found to be niacin requiring, they should  be considered In relationship to the hypothesis of Hood's that two metabolic pathways lead to synthesis of niacin. The majority of the mutants are s t i l l unidentified, though characteristically all responded to yeast extract; this observation was also made by Hood for his unclassified mutants. Attempts to produce rIbonucleasedefIclent strains of U. hordei should be continued for such mutants would be useful: 1.  to the biochemist Interested In RNases for sequence analysts, enzymology and evolutionary comparisons,  2.  to the biologist Interested In the role, the synthesis and the secretion of extracellular RNases,  38 3.  t o the g e n e t i c i s t I n t e r e s t e d i n f o r m a t i o n and Information,  k.  In the o r g a n i z a t i o n o f g e n e t i c  p o s s i b l e c o n t r o l s f o r the r e l e a s e o f such  and  t o the p l a n t p a t h o l o g i s t f o r the I n v e s t i g a t i o n o f a p o s s i b l e r o l e o f RNases In h o s t - p a r a s i t e , r e l a t i o n s h i p s .  SUMMARY AND  RJbonuclease a c t i v i t y was horde I had huerx grown. the d e t e c t e d Also,the  In c u l t u r e medium In which U s t l I ago  Some e v i d e n c e has been presented  not due  not due  t o suggest t h a t  t o r e l e a s e from r u p t u r e d  t o p h o s p h o d i e s t e r a s e s s i n c e these were not  surfaces.  r e l e a s e o f RNase (as measured by RNase a c t i v i t y ) was  the c e l l u l a r environment. w i t h y e a s t RNA  and pH 7»5  Influenced  by  With complete medium o r w i t h minimal medium e n r i c h e d  the r e l e a s e o f RNase was  delayed.  I t Is p o s t u l a t e d t h a t phosphate  s t a r v a t i o n w i l l encourage the e a r l y r e l e a s e o f RNase a c t i v i t y .  The  cells.  In the c e l l - f r e e medium and o n l y minor amounts were found t o be  associated with c e l l The  detected  r l b o n u c l e a s e a c t i v i t y was  a c t i v i t y was  detected  CONCLUSIONS  RNase a c t i v i t y appears t o be r e l e a s e d l e v e l o f RNase a c t i v i t y d e t e c t e d  The  h.S  pH  concurrently.  In the c u l t u r e medium was  o f the c e l l d e n s i t y , w h i l e the maximal l e v e l o f RNase a c t i v i t y was  a function In t u r n  determined by the r i c h n e s s of the c u l t u r e medium. The  d e t e c t i o n o f RNase a c t i v i t y cannot by I t s e l f be taken as an I n d i c a t i o n  t h a t RNases p l a y the r o l e o f " s c a v e n g e r " enzymas, s i n c e they c o u l d do o n l y In c o n j u n c t i o n w i t h phosphomonoesterases. phosphomonoesterase a c t i v i t y on the c e l l  W i t h the d e t e c t i o n o f a c i d  s u r f a c e , I t Is r e a s o n a b l e  a t t r i b u t e a " s c a v e n g e r " r o l e t o the e x t r a c e l l u l a r  to  ribonucleases.  Among h a p l o l d c e l l s o f U. horde1 t r e a t e d w i t h the chemical nitrosoguanldlne, f i f t y - f i v e  this  mutagen,  b i o c h e m i c a l l y d e f i c i e n t mutants were I s o l a t e d .  P r e l i m i n a r y s c r e e n i n g o f these mutants showed t h a t some were a d e n i n e and v i t a m i n mutants.  The  remaining  others  a u x o t r o p h s , e v e r y o n e o f w h i c h responded t o a  y e a s t e x t r a c t supplement, remain undetermined as t o t h e i r s p e c i f i c r e q u i r e m e n t .  The l e v e l o f mutagenesis was not as h i g h as t h a t r e p o r t e d w i t h irradiation  U.V.  In U_. horde 1 (Hood, 1966) so a more e x t e n s i v e s t u d y s h o u l d  made o f o p t i m a l c o n d i t i o n s f o r the use o f NG In t h i s o r g a n i s m .  Applying  the r e s u l t s o f such a s t u d y and u s i n g the s e l e c t i o n methods o u t l i n e d *  n e  P J s c u s s i p n , i t s h o u l d be p o s s i b l e t o produce and I d e n t i f y  strains  d e f i c i e n t In e x t r a c e l l u l a r r l b o n u c l e a s e s w i t h a s a t i s f a c t o r y l e v e l o f efficiency.  be  In  BIBLIOGRAPHY Adelberg, E.A., M. Handel, and G.C.C. Chen. 1965. Optimal conditions f o r mutagenesis by N-methyl-N'-nltro-N-nttrosoguanldlne In Escherichia col I K12. Biochem. Biophys. Res. Comm. Jjh 788-795. Arima, T., I. Uchida, and F. Egaml. 1968. I . Studies of e x t r a c e l l u l a r ribonucleases of U s t l l a g o sphaerogena; p u r i f i c a t i o n and p r o p e r t i e s . Biochem. J . 106: SoTToS. Arima, T., I. Uchida, and F. Egaml. 1968. I I . Characterization of substrate s p e c i f i c i t y with special reference to p u r l n e - s p e c i f t c ribonucleases. Biochem. J . 106: 609-613. Baker, R., and I. Tessman. 1968. D i f f e r e n t mutagenic s p e c i f i c i t i e s In phages S13 and T4: i n v i v o treatment with N-methy1-M -nltro-N-nitrosoguantdlne J . Mol. B i o l . 35*n»39-^8. 1  Beaton, J.D. I968. An e l e c t r o n microscope study of the mesosomes o f a pent" ci11inase-producing Staphylococcus. J . gen. M i c r o b i o l . 50: 37-42. Cerda-Olraedo, £., and P.C. Hanawalt. 1967. 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P e r s o n . 1969- G e n e t i c complementation h o r d e t . Can. J . Botany 47: 9-14. Egaml, F., and K. Nakamura. New Y o r k , I n c .  1969.  Cell Biology. 1n Us t i l a g o  Microbtal Ribonucleases.  Sprlnger-Verlag  E l s e n s t a r k , A . , R. E t s e n s t a r k , and R. van S i c k l e . 1365. M u t a t i o n o f S a l m o n e l l a typhtmurlurn by n1trosoguanIdIne. Mu t . Res. 2: 1-10. GItham, P.T. 1962. An a d d l t t o n r e a c t i o n s p e c i f i c f o r u r i d i n e and guanoslne n u c l e o t i d e s and I t s a p p l i c a t i o n t o t h e m o d i f i c a t i o n o f r i b o n u c l e a s e a c t i o n . J . Am. Chem. Soc. 84: 687-688.  42 G l i t z , O.G., and C A . Dekker. 1964. Studies on a rlbonuclease from Ustllago sphaerogena. I. P u r i f i c a t i o n and properties o f the enzyme. Biochemistry 3j 1391-1399. G l i t z , O.G., and C A . Dekker. 1964. Studies on a rlbonuclease from Ust11ago sphaerogena. 11. S p e c i f i c i t y of the enzyme. Biochemistry 3.: 1399-1*06, Ho, N.W.Y., and P.T. Gllham. 1967. The reversible chemical modification of u r a c i l , thymine and guanine nucleotides and the modification of the action of rlbonuclease on ribonucleic acid. Biochemistry 6_:  3632-3639. Holliday, R. 1961. The genetics of U. maydls.  Genet. Res. Comb. 2; 204-230.  Holllday, R., and R.E. H a l l t w e l l . 1968. An endonuclease-deftclent s t r a i n o f Ust11ago maydls. Genet. Res. Comb. V2: 95-98. Hood, C H . 1966. UV-lrradlatlon s e n s i t i v i t y and mutation production In the haploid sporldta o f Ust1lago hordeI. Ph.D. Thesis, University of Alberta, Edmonton. ™ " J e f f f i e s , C C , D.F. Holtman, and O.G. Guse. 1957. Rapid method f o r determining the a c t i v i t y of microorganisms on nucleic acids. J . Bact. 73: 590-591. Kushner, D.J., and H.R. Pollock. 1961. The location of cell-bound p e n i c i l l i n a s e In Bac111us subt111s. J . gen. Microbiol. 26: 255-265. Lampen, J.O. 1965. Secretion of enzymes by microorganisms. of Gen. Microbiologists 1j>: 115-133.  In Symp.  Lawley, P.D. I968. Methylatlon of DNA by N-methyl-N-nltrosourethane and N-methyl-N-nltroso-N'-nltroguanldlne. Nature 218: 580-581. Lederberg, J . , and E.M. Lederberg, 1952. Replica plating and Indirect selection of bacterial mutants. J . B ^ c t e r l o l . 63_: 399-406. Lee, J . C , N.W.Y. Ho, and P.T. Gllham. 1965, Preparation of r l b o t r l n u c l o tides containing terminal c y t l d l n e . Blochlm. Blophys. Acta 95:  503-504.  Loprieno, N., and C H , Clarke. 1965. Investigations on reversions to methionine independence induced by mutagens in Senl,20sacchart^ces_ pombe. Mutation Res. 1} 312-319. McCalla, D.R. 1968. Reaction of H-methy1~N *-nItro-H-n!trosoguanJdine and N-methyl-N-nltroso-p-toluenesulfonamlde with DNA J n vjjtro. Biochim. Blophys. Acta 155: 114-120. Mandell, J.D., and J . Greenberg. I960. A new chemical mutagen for bacteria, 1-methyl-3-n1tro-l-nltrosoguanId?ne. Blochem. Blophys. Res.  Comm. 3: 575~577-  MatHe, P. 1965. I n t r a c e l l u l a r ^ l o k a l l s a t i o n proteolytlscher enzyme von Neurospora crassa. I. Funktlon und subcellulare Vertellung proteolytlscher Enzyme. Z. Zeleforsch. 65*. 884-896.  Matlle, P., M. Jost, and H. Moor. 1965. Intracellulare Lokallsatlon proteolytlscher Enzyme von Neurospora crassa. 11. IdentlfIkatlon von proteasehaltlgen Z e l l s t r u k t u r e . Z. Z e l l f o r s c h . 6 8 : 2 0 5 - 2 1 6 . Matile. P., and A. Wlemken. 1967. The vacuole as the lysosome of the yeast c e l l . Archlv fur Mlkroblol. 56: 1 4 8 - 1 5 5 . Miiller, J . , and T. Glchner. 1964. Mutagenic a c t i v i t y of l-methyl-3-nltro1-nltrosoguanldlne on A^a^dhopjsls^. Nature 20U 1 1 4 9 - 1 1 5 0 . Naylor, R., N.W.Y. Ho, and P.T. Gllham. 1965. Selective chemical modifications of uridine and pseudourldlne In polynucleotides and their e f f e c t on the s p e c i f i c i t i e s of rlbonuclease and phosphodiesterases. J . Am. Chem. Soc. 87j 4209-4210. Nlshlmura, S., and M. Nomura. 1959. Rlbonuclease of Bac111us sub11 l i s . J . Blochem. (Tokyo) 4 6 : 161-167. Nlshlmura, S., and B i Maruo. 1960. I n t r a c e l l u l a r rlbonuclease from Bacl1lus s u b t I l l s . Biochlm. Blophys. Acta 4 0 : 355-357. Pollock, M.R. 1963. Exoenzymes. In The Bacteria 4: 121. Ed. by I.C. Gunsalus and R . Y , Stanier. N.Y. Academic Press. Razzell, W.E. 1 9 6 7 . Polynucleotidases In animal tissues.  321-325.  Experentia 2 3 :  Shleh, T . R . , and J.H. Ware. 1968. Survey of microorganisms f o r the production of e x t r a c e l l u l a r phytase. Appl. M i c r o b i o l . Jj6: 1348-1351. Shugar, 0 . , and H. Slerakowska. 1967. Mammalian n u c l e o l y t l c enzymes and their l o c a l i z a t i o n . Prog. N.A. and Mol. B i o l . Ed. J.N. Davidson 6 W.E. Cohn, 7_: 369-429. Stein, CW. 1970. An electron microscope study of a mycelial mutant of Ustllago horde I. M.Sc. Thesis, University of B r i t i s h Columbia. Subba Rao, P.V., K. Moore, and G.H.N. lowers. 1967. P u r i f i c a t i o n and properties of phenylalanine ammonia-lyase from Ustllago horde1. Can. J . Blochem. 4£: 1863-1872. Takahashl, K. I96I. Chromatographic p u r i f i c a t i o n and properties of rlbonuclease T p J . Blochem. (Tokyo), 4 9 : 1-8. Takai, N., T. Uchlda, and F. Egaml. 1967. Rlbonuclease, phosphodiesterases and phosphomonoesterases of Neurospora crassa In various culture conditions. J . Japan. Blochem. SocfTSelkagoku) 3 9 : 285-290. Velemlnsky, J . , T. Glchner, and V. Pojorny. 1967. The action of 1-alkyl3~nItro-1-n11rosoguanidine on the M. generation of barley and Arabidopsls thaliana (L.) Heynk. Biologia Plantarum (Praha),  9 : 249^2617  Vogel, H.J. 1956. A comment on growth medium for Neurospora crassa. Microbiol. Gen. B u l l . No. 13.  Von Tlgerstrom, R.G., and M . Smith. 1969. Preparation o f the 2,4dlnttrophenyl esters of thymidine 3'~ and thymidine 5*phosphate and t h e i r use as substrates f o r phosphodiesterases.  Biochemistry 8: 3067-3070.  Yanaglda, H., T. Uchida, and F. Egaml. 1964. Culture o f Ustllago zeae with RMA or poly U as phosphorus source. J. Agr. Chem. Soc.  Japan 38: 531-535. Yurkevlct), V.V., G.T. Kozureva, and M . I . Oergacheva. 1967. Secretion o f amylase by the fungus Aspergillus oryzac. Prlkladnaya Blokhlmlya I Mikroblologlya £ J 15FHF.  APPENDIX A.  1.  Vogel's (1956) s a l t s o l u t i o n contained:  123 9 sodium c i t r a t e ,  250 g monobasic potassium phosphate, 100 g ammonium n i t r a t e (anhyd.), 10 g magnesium sulphate, 5 g calcium c h l o r i d e , 5 ml trace element s o l u t i o n In 750 ml d i s t i l l e d water w i t h 2 ml chloroform. 2.  The trace element s o l u t i o n contained:  5 9 c i t r i c acid, 5 g  z i n c sulphate, 1 g ferrous ammonium sulphate, 0.25 9 copper sulphate, 0.05 g manganese sulphate, 0.05 9 b o r i c a c i d , 0.05 g sodium molybdate, 1 ml chloroform a l l In 95 ml d i s t i l l e d water.  Both these s o l u t i o n s were  stored a t room temperature. 3.  The vitamin s o l u t i o n contained:  100 mg thiamin, 50 mg r i b o f l a v i n ,  50 mg pyridoxtne, 200 mg calcium pantothenate, 50 mg para-amlno-benzotc a c i d , 200 mg n i c o t i n i c a c i d , 200 mg c h o l i n e c h l o r i d e , kOO rug I n o s i t o l and 50 mg f o l i c a c i d per one l i t e r d i s t i l l e d water.  APPEND IX B.  Flash-evaporation of standard minima) medium, from a stationary culture, to o n e - t h i r t i e t h the o r i g i n a l volume produced a syrupy which affected the column loading and e l u t l n g .  liquid  In future attempts to  establish the number o f RNases that are released by U_. horde 1, i t Is suggested  that cultures be grown on a glucose-1Imiting minimal medium.  This would allow f o r greater concentration of the medium and this provides a more d e f i n i t e RNase a c t i v i t y e l u t l o n p r o f i l e .  

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