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

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A STUDY OF E X T R A C E L L U L A R R I B O M J C L E A S E A C T I V I T Y IN U S T I L A G O HORDE I by N i l s T o r b e n B e c h - h i a n s e n B . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1968 A T H E S I S SUBMITTED IN P A R T I A L F U L F I L M E N T OF THE REUU I K tMbN I S H)K f HI- I1RGRFF OF MASTER OF S C I E N C E i n t h e D e p a r t m e n t o f B o t a n y We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE U N I V E R S I T Y OF B R I T I S H COLUMB IA J a n u a r y , 1970 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Co lumb ia , I a g ree tha t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s tudy . I f u r t h e r agree tha p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d tha t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Botany  The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada D a t e February 20, 1970 i f ABSTRACT E x t r a c e l l u l a r r ibonuclease a c t i v i t y was detected In cu l tu re media of s p o r l d i a l and mycel ia l cu l tures of Ust I lago horde!. The RNase a c t i v i t y was maximal at pH 5.0, 6.5 and 8.0. The secret ion of the RNase a c t i v i t y was a funct ion of the c e l l dens i ty . Release of pH k.S and pH 7.5 a c t i v i t y was co inc ident . Enrichment of the simple glucose and s a l t s medium delayed the I n i t i a l secre t ion of a c t i v i t y . The presence of RNA In the medium d id not enhance the amount of a c t i v i t y re leased. Furthermore, s ince the presence of RNA in the medium was not required for the re lease of the RNase a c t i v i t y Into the medium, i t is suggested the synthesis and secret ion is a c o n s t i t u t i v e func t i on . N-methyl -N ' -n i t ro-N-n l t rosoguanid lne was used to produce auxotrophic s t r a i n s . Se lec t ion methods for the detect ion of s t r a in s d e f i c i e n t in e x t r a -c e l l u l a r RNase a c t i v i t y are d i scussed. in TABLE OF CONTENTS Page INTRODUCTION 1 MATERIALS AND METHODS 8 1. B i o l o g i c a l M a t e r i a l 8 2. C u l t u r t n g 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 Mutagenesis, Detection and Screening o f Mutants. . . . 11 6. The Prep a r a t i o n of CMC-RNA 12 RESULTS 13 1. Evidence f o r E x t r a c e l l u l a r Rtbonucleases In U. horde I 13 2. Release o f Ribonuclease A c t i v i t y . 15 3. RNA as a Carbon Source f o r U_. hordei 20 k. Chemical Mutagenesis, Mutants Detected and Screened 22 5. CMC-p-Toluenesulfonate Modified RNA 22 DISCUSSION 29 SUMMARY AND CONCLUSIONS 39 BIBLIOGRAPHY *1 APPENDIX A . *»5 APPENDIX B *6 LIST OF TABLES Page Table I. NG treatments of strain E^ of U. horde! 23 Table II. Preliminary screening of possible mutant Isolates of U_. horde I 2k Table III. Selection method for detecting extracellular RNase-defIcient mutants using CMC modified RNA $k iv Table IV. Selection scheme for the detection of three classes of extracellular RNase-deficient mutants using model substrates 36 LIST OF FIGURES Page Figure 1. RNase activity of cell-free minimal medium from E« stationary culture, measured across pH range 3.0 to 9.2 14 Figure 2. Growth of strains Ej and 1^  cultured In either minimal or complete medium and the release of RHase activity (pH 7-5) 16 Figure 3. Growth of strain Ej In minimal and complete medium and the release of RNase activity (pH 4.5 and pH 7.5) 17 Figure 4. Growth of a stable mycelial strain in complete medium and the release of RNase activity (pH 4.5 and pH 7.5) 18 Figure 5. The release of RNase activity (pH 4.5 and pH 7.5) by strain E^~ grown on minimal medium plus RNA. 19 Figure 6. Dilution experiment 21 ACKNOWLEDGEMENTS The author wishes to extend his sincere appreciation to Professor Clayton Person for his suggestions and criticisms during the Investigation and especially In the preparation of this thesis; Gratitude is also expressed to Or. Michael Smith and Or. Michael Shaw, co-supervisors, for their encouragement and helpful discussions. A special word of thanks to Or. Smith who generously allowed me to use space and equipment In his lab, and who provided many useful criticisms during the preparation of this thesis. To Or. Peter Gilham, many thanks for Initiating my Interest in this study and offering helpful discussions. The discussions and constructive criticisms of my fellow-colleagues In the Botany and Biochemistry Departments during the research and preparation of this thesis are gratefully 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 occur 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 f u n g i . Sometimes t h e t r presence Is a r e s u l t o f the death 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 In other cases the enzymes are t r u l y e x t r a -c e l l u l a r (Lampen, 1965)* 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 which can be produced and released by the c e l l without any 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 greater than that compatible w i t h the c e l l s ' normal processes of growth and reproduction ( P o l l o c k , 1963)• Studies of 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 Davles (1963), by P o l l o c k (1963) and by Lampen (1965). Some general f e a t u r e s are revealed from a comparison of those e x t r a c e l l u l a r enzymes that have been stud i e d ( P o l l o c k , 1963): I) they are small In s i z e - l e s s than 80,000 M.W.; II) 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 primary s t r u c t u r e ; I I I ) they o f t e n need cal c i u m ions f o r a c t i v a t i o n and s t a b i l i z a t i o n ; and Iv) they occur most f r e q u e n t l y In gram-positive b a c t e r i a and f u n g i . Such g e n e r a l i z a t i o n s are based mainly 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 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 are g e n e r a l l y considered t o be synthesized by the 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. Studies of 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 Pol l o c h , 1961) showed that the l i b e r a t i o n of the enzyme requ i r e s membrane s y n t h e s i s . Beaton (1968), who s t u d i e d Staphylococcus aureus, suggested that r e l e a s e of p e n i c i l l i n a s e Involves a l t e r a t i o n of membranous s t r u c t u r e s . Lampen (1965) In h i s hypothesis v i s u a l i z e s the formation and s e c r e t i o n of p e n i c i l l i n a s e as being 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. This p r o p o s a l , based 2 on studies in prokaryotes, may not be t o t a l l y compatible with the phenomena of secretion In eukaryotes. In Neurospora crassa It has been suggested that e x t r a c e l l u l a r proteases located in membrane-bound vesicles are released e x t r a c e l l u l a r l y when they "cross the plasma membrane as intact p a r t i c l e s by means of Invaginations of the plasmalemma" (Nat l i e «Jt a L , 19^5). Electron micrographs have recently been obtained (Stein, 1970) which also support the Idea that I n t r a c e l l u l a r vesicles are Involved in 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 in higher organisms, for example wtth pancreatic c e l l s (production and release of zymogen). In this example zymogen is synthesized on the rIbosomes of the endoplasmic reticulum (E.R.), moves into the Interclsternal c a v i t i e s of the E.R. and then to vesicles of the Golgi complex by transient connections. These vesicles 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 Robertls, 1965)• In U. hordeI where there Is no Golgl complex (Stein, 1970), the production and release of e x t r a c e l l u l a r products cannot follow t h i s sequence of events. Although It Is apparent that there Is no single or standard mechanism of secretion used by a l l eukaryotlc c e l l s the process appears In a l l cases to involve movement, vi a v e s i c l e s , of proteins from their s i t e s of synthesis to the plasma membrane. Microbial ribonucleases, both i n t r a - and e x t r a - c e l l u l a r , have been reviewed in d e t a i l by Egaml and Nakamura (1969)* These authors noted that, In general, I n t r a c e l l u l a r RNases have no nucleic acid 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 heat-labile 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 (st 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 stationary phase and then continued to increase at a constant rate during the stationary phase. In a later 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 in such characteristics as optimum pH, heat s t a b i l i t y and ion requirement (Nlshlmura and Haruo, I960). This contrasts with the finding 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 affected by the conditions of culture; 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 for the RNase) was supplied as the sole 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) also found that an e x t r a c e l l u l a r guanyloribonuclease accumulated In the culture medium of U. sphaerogena when RNA was added as the sole carbon source. They therefore considered these to be Inducible enzymes. 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 base-specific. 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 in RNases and My following the addition of RNA as the sole source of phosphorus In the culture medium. In contrast with the results 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 in growing c e l l s i s discussed In d e t a i l by Egaml and Nakaraura (1969), who summarize their probable physiological roles as follows: 1) metabolism of RNAs; t l ) protection against penetration by phage RNA; i l l ) supply of nutrients 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 of DNA-speciflc endonuclease I by removing I n h i b i t o r y 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 of such RNases i t i s known that guanlne-rich n u c l e o t i d e s tend to form aggregates and i t Is t h e r e f o r e suggested that RNases s p e c i f i c f o r guanylfc a c i d phosphodtester bonds would y i e l d d i g e s t i o n products which may d i f f u s e through the c e l l membrane (Egami and Nakamura, 1969)* On the whole, the m i c r o b i a l e x t r a c e l l u l a r rtbonucleases, because of t h e i r ease of 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 great favour w i t h the biochemist w h i l e the general b i o l o g i c a l r o l e of such e x t r a c e l l u l a r products remains, f o r the most p a r t , unresolved. Species of the genus U s t l l a g o are u s u a l l y considered to be o b l i g a t e p a r a s i t e s because they are e n t i r e l y dependent on t h e i r host species during at l e a s t part of 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 species and t h e i r hosts are u s u a l l y h i g h l y s p e c i f i c and, although e x t r a c e l l u l a r enzymes o f several U s t l l a g o species have been s t u d i e d , there i s as yet no evidence to i n d i c a t e that the production of 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 to the success of these species during t h e i r pathogenic phase. One approach considered u s e f u l f o r e v a l u a t i n g the importance of e x t r a -c e l l u l a r enzymes of p a r a s i t i c fungt Involves the production of mutants d e f i c i e n t In the a b i l i t y to release a s p e c i f i c a c t i v i t y and the study of the a b t l i t y of such mutants to p a r a s i t i z e the host. Mutants tn U s t l l a g o hordet have t o date been produced only by UV I r r a d i a t i o n (Hood, 1966), but u s e f u l a p p l i c a t i o n of t h i s method of mutation required the treatment of synchronously growing c u l t u r e s . The chemical mutagen N-methyl-N'-nttro-N-nttrosoguantdtne (NG or nttrosoguanldine) was used tn the present study In attempts to produce RNase-defIcient mutants. H o l l l d a y and H a l l l w e l l (1968) have p r e v i o u s l y 5 used NG with Ustilago maydls to produce extracellular DNase-defIclent mutants. Mandell and Greenberg {1961) f i r s t reported the mutagenic action of NG In bacteria and this compound has subsequently been shown to be highly mutagenic. Adelberg et al_. ( 1965) studied the action of NG In fjchejjchta c o l i and found that a high yield of mutations was obtained in conditions which gave over 50 percent survival rate. Mutagenic action of NG has similarly been studied In Salmonella typhlmurium (Elsenstark et a l . , 1 9 6 5 ) , Schlzosaccharomyces pombe (Loprleno and Clarke, 1965) and Arabldopsls thai Iana (Muller and Gichner, 1 9 6 4 ) . Loprieno and Clarke ( 1965) in their study reported the following order for decreasing ratios of mutagenesis to lethality: NG > nitrosomethylurethane > ultraviolet light > nitrous acid. In v i t r o , NG acts on DNA and RNA to produce 7-methylguanine (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 is specific for the replicating region of the bacterial genome (Cerda"-01medb e£ a1_., 1 9 6 8 ) , but this has yet to be shown In fungi. In another in vivo study, Baker and Tessman ( 1968) found different mutagenic speci 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 In about equal frequences, whereas GC to AT transitions predominated In phage T 4 . The molecular environment of the replication region of the DNA (which should depend on the specific mechanism of replication, the nature of the DNA polymerase and the base composition of the DNA) Is suggested to be responsible for the differences. The authors point out that care should be taken in making generalizations about the specificity of a particular mutagen (e.g. beyond saying that NG Is an alkylating agent). The macromolecular action of NG In vivo is not restricted to DNA (Cerda-Olmedo and Hanawalt, 1 9 6 7 ) . Besides causing alteration of DNA which can be recognized and repaired by the dark repair mechanism, NG can Inactivate 6 proteins and Inhibit protein synthesis. To a lesser extent HG can also Inhibit RNA synthesis, resulting In a reduction In the rate of synthesis of proteins and the production of small amounts of non-functional protein. HG may also cause misreading of the genetic code, which can be suppressed by streptomycin. Whether HG or diazoraethane Is the reactive species tn vivo is s t i l l an unanswered question. The former view Is supported by Mandell and Greenberg (I960) and McCalla (1967) and the latter by Cerda-Olmedo and Hanawalt (1968), As well as having an efficient procedure for Inducing mutations, one must also have a selection method for detecting those mutants which are of specific Interest. For a detailed consideration of the problems Involved see the Discussion. In the present study, one approach that was considered for the selection of RNase-defIcient mutants Involved th® use of a derivative 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 specifically with the bases guanine, uracil and thymidine (Ko and Gilham, 1967). Pyrlmldine-speclfIc RNases are restricted to degradation at cytosine when the RNA substrate has previously been reacted with CMC-p-toluene-sulfonate. Similarly It should be feasible to limit the action of RNases of other base sp e c i f i c i t i e s (P.T. Gtlham, personal communication). The practical 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) for RNase on CMC-modified polynucleotides, and by Sanger et a l . (1968) tn the 7 restriction of RNase A on modified 5s RNA for partial hydrolyses to f a c i l i t a t e base sequence analysis. After establishing the presence of ribonuclease activity 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 for RNase activity. (Protease and amylase ac t i v i t i e s were also detected but these were not studied.) The study ts divided into two parts: one, the study of the RNase activity in the non-pathogenic phase of the l i f e cycle (nature of acti v i t y , secretion pattern, control of secretton); and two, chemical mutagenesis of U. hordet In attempts to produce mutants deficient In RNase activity which would be useful In considering the Importance of extracellular RNases In the host parasite relationship of U. hordeI and its host, cultivated barley (Hordeum vulgare L.). 8 MATERIALS AND METHODS 1. BIOLOGICAL MATERIAL The two standard monosporldlal (I.e. haplotd) lines of Ustllago hordeI (Pers.) Lagerh. used In this study were developed by Hood (1966) and designated by him as E^ and 1^ . Both these lines were used in enzyme studies while Ej only was used tn mutagenesis experiments. A mycelial culture derived from a complementation test (Dlnoor and Person, 1969) of two sporldlal lines derived from a single tellospore, Met Pan Arg A and Met Pan Arg a (Drs. Jean Mayo and CO. Person, personal communication), was studied for production of e x t r a c e l l u l a r RNase. 2. CULTURING Liquid cultures were grown In a New Brunswick psycrotherm incubator at 22° C on a shaking table (100 RPM). Agar plates were also incubated at 22° C. In order to measure c e l l density of cultures at different stages of growth, cultures were grown in l i q u i d culture in 125 ml Erlenmeyer flasks f i t t e d with Klett-Summerson color(metric tubes (side-arm f l a s k s ) . By turning the flasks sideways, the side-arm f i l l e d with culture medium whose t u r b i d i t y was then measured on a Klett-Summerson photoelectric colorimeter f i t t e d with a red f i l t e r . This allowed one to follow a culture from a c e l l density of 10^ cells/ml (0-5 K.U.) through to stationary phase which occurred at a c e l l 8 density near 2 x 10 cells/ml (*»00-/»50 K.U.). 9 3. CULTURE MEDIA AND SELECTION PLATES Culture media, minimal and complete, were prepared according to the procedures outltned by Hood (1966). Minimal medium contained 20 ml Vogel's s a l t solution (see Appendix A) and 10 g glucose per one l i t e r of d i s t i l l e d water. Complete medium was a minimal medium enriched with 5 g Dlfco yeast extract, 5 g s a l t - f r e e casein hydrolysate (N.B. Co.), 50 mg tryptophan and 10 ml vitamin solution (see Appendix A) per one l i t e r of 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 individual growth factors were added to minimal medium as required: amino acids, 100 mg; purines and pyrlmldlnes, 10 rag; and vitamins, 1 mg per l i t e r . I n i t i a l screening of auxotrophs was carried out on agar plates of minimal medium supplemented with yeast extract, vitamins, or casein hydrolysate. In amounts normally used for complete medium. Auxotrophs were stored on agar slants (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 solution to autoclaved regular minimal medium containing one percent agar. 4. ASSAYS (a) Rlbonuclease a c t i v i t y was determined quantitatively by measuring the absorbance of acid-soluble degradation products according to the assay for RNase T., described by Takahashi (1961) and modified by Arima et a l . (1968). 10 The reaction mixture contained 0.1 ml of enzyme solution, 0.25 ml of 0.2 H Trls buffer, pH 7.5, or 0.2 M sodium acetate buffer, pH k.$, 0.1 ral of -2 2 x 10 M E0TA, 0.3 ml of d i s t t l l e d water and 0.25 ml of yeast sodium ribonucleate (Schwarz), 10 mg/ml freshly prepared before use. The hydrolysis was allowed to proceed for 30 min at 37° C after the additton of RNA solution and was stopped with 0.25 ml of 0.75$ uranyl acetate In 254 perchloric acid. The reaction mixture was centrifuged and 0.2 ml of supernatant solution 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 ells of one centimeter light path. The amount of enzyme that under the standard assay conditions and thirty minutes of hydrolysis would produce an increase tn absorbance of one at 260 nm was defined as one enzyme unit (Takahashl, 1961). An alternative, qualitative assay for RNase activity similar to that used by Hoi Itday and Hall(well (1968) for ONase and earlier by Jeffries et aJL (1957) was employed In assaying for activity 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 carried 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 for about five days at 22° C or they were spotted with culture medium and the plates were floated tn a 37° C water bath for thirty minutes. To stop the hydrolysis, the plates were flooded with 10% trichloro-acetic acid (TCA). To test the validity of this assay, ten microliters of purified 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 precipitate 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 sensitive method for detecting actfvtty that may be due to RNases. 11 (b) Phosphodiesterase (PDE) I and II (both described by Razze l l , 1967) were assayed for by the method of Razzell (H. Smith, personal communication). For PDE I the stock solution 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 at 272 nm In 0.01 M HC1), pH 9.3, and 0.75 ml water. To 0.1 ml of stock warmed 2 minutes at 37° C about 20 m i c r o l i t e r of enzyme solution was added and incubated for one hour. The reaction was stopped with 0.25 R>1 0.3 H NaOH and the contents of the tube mixed by Inverting several times, made up to 1 ml with water and absorbance at 400 nm was determined. For phosphodiesterase II the stock solution contained 0.25 ml H ammonium acetate, pH 5*9, 0.05 ml 0.02 M sodium E0TA, 0.1 ml 2,4-dlnltrophenyl thymidine-^' phosphate, 10 mlcromoles per ml, and 0.55 ml water. The assay procedure was as for P0E I. The use of 2,4-dlnltrophenyl thymldlne-3* phosphate, a modification of Von TigerStrom and Smith (1969). allows direct quantitative 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 of p-nltrophenyl thyraidine-3' phosphate as a substrate, since p-nltrophenoxide has l i t t l e absorbance at pK 5.9. (c) Phosphomonoesterase, acid and a l k a l i n e , were measured by the method of Artma et a l . (1968). 5. CHEMICAL MUTAGENESIS, DETECTION AND SCREENING OF MUTANTS To Induce mutations, wi l d type, log phase sporidla ( E ^ ) grown In l i q u i d minimal medium were pelleted and resuspended at a concentration of about 10 c e l l s per ml In c i t r a t e buffer at pH 5.0 or 5.7 in 40 ml Nalgene tubes. S u f f i c i e n t NG stock solution was added to give a f i n a l concentration of 0.1 mg NG per ml of medium. Standard NG treatment lasted for f i f t e e n minutes at 2 2 - 2 4 C. Stock solution of 2 mg per ml of NG was made fresh with each 12 treatment. Treated cells were Immediately washed with citrate 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 for about ten hours before plating. Samples of cultures treated tn this way were diluted to give 50 to 100 colonies per plate when spread on complete medium. Colonies of these plates were replicated, using the method of Lederberg and Lederberg (1952), as follows; to minimal plates to determine auxotrophs, to RNA plates to detect RNase-deftctent colonies and, as a ftnal step, to complete plates to confirm that transfer of each colony had been made onto a l l previous plates. After five to ten days, each plate was examined and the auxotrophs were tentatively Identified; these were further defined by their abtltty to respond to casein hydrolysate, to vitamin solution or to yeast extract. Final classification of an auxotroph was based on a positive response to a stngl® compound added to minimal plates. Arlma et ej[, (1968) reported four extracellular RNases In Ustilago sphaerogena, two with sharp optima at pH 4.5 and two others with a broader optima at pH 7.5. These observations were taken as the basis for beginning thts study. RNA plates were made with a pH of 6.0 or greater In order to assay for deficiency of 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 for 26-30 hours. 13 RESULTS 1. EVIDENCE FOR EXTRACELLULAR RIBONUCLEASES IN U. HORDEI (a) Initial qualitative and quantitative assays for RNase activity in the medium of a stationary phase E^ culture gave positive results, both at pH 4.5 and 7*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 ) . in addition, a third maximum was obtained at pH 6.5; this maximum has not been reported previously. Some preliminary attempts at identifying the number of RNases released by U. horde1 were made (see Discussion). (b) Evidence that the RNase activity was due to the release of extra-cellular RNases came from observation of culture samples under the light microscope. In samples taken during the period of maximal Increase in RNase activity no rupture cells or c e l l fragments were seen. U. hordeI character-Istlcally does not lyse but rather, becomes mycelial towards the end of its log phase of growth. (c) A slight amount of activity (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 cel l s and assaying the cell-free culture medium was not detectable. Phospho-diesterase II activity was not detected on the c e l l surface or In the culture medium. (d) A ten day old liquid culture of strain Ej in minimal medium, that had been in the stationary phase of growth for five days, was assayed for phospho-monoesterase (PME) act i v i t y . Activity was detected only under acid conditions (pH 5.5) and primarily in ce 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 culture medium,In which s t r a i n had been grown to stationary phase, measured across the pH range 3*0 to 9.2. F I G U R E I pH UN ITS 2. RELEASE OF RlBONUCLEASE ACTIVITY (a) Figure 2 presents representative graphs to show the release of RNase activity for and 1^ * grown in complete and In minimal medium. A sharp increase In RNase activity occurred in late log phase and by early stationary phase maximal release was achieved. The release of extracellular enzymes during this phase of growth has been observed not only with RNases but also with many other extracellular enzymes, and may be regarded as a rather general characteristic of extracellular enzymes of microorganisms (EgamI and Nakamura, 1969)* The release of activity tnto minimal medium was more gradual and began earlier after entry of the culture Into the log phase of growth, In contrast to cultures of complete medium. The Increases In rtbonuclease activity measured at the two pH's (4.5 and 7.5) parallel each other during the period of release (f i g . 3). This suggests that the period of synthesis and release are co-Incidental for the enzymes Involved. The total amount of RNase activity was greater In complete medium suggesting that conditions in this medium were more favourable for synthesis or release of RNase. (b) A stable mycelial statn grown in complete medium released rtbonuclease acttvlty in a pattern similar to a sportdtal culture In complete medium (Fig. 4). Total RNase activity reached a level similar to that of sporldta! cultures. The constitutive release of RNase act 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 an inducible effect (I.e. Increase the amount of RNase release), the release of RNase act i v i t y from strain E^", grown In minimal medium plus 0.5% yeast RNA, was measured (Fig. 5). This figure shows, f i r s t l y , that the maximal level Figure 2: Growth (-o-) of s t r a i n and cultured In either minimal or complete medium; and the release of RNase a c t i v i t y at pH 7.5 ( — o — ) . a) E3~/M; b) E^/C; c) l^/M and d) l ^ / C . R N a s e a c t i v i t y ( u n i t s / m l ) R N a s e a c t i v i t y ( u n i t s / m l ) 91 GROWTH ( K . U . ) GROWTH (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 at both pH 4.5 and pH 7.5. R N a s e a c t i v i t y ( u n i t s / n i l ) GRO./TH ( K.U.) Figure 4: The growth curve of the s tab le mycel ia l s t r a i n (Myc/C) and the re lease of pH 4.5 and pH 7.5 RNase a c t i v i t y . R N a s e a c t i v i t y ( u n i t s / i n ) Figure 5- The release of RNase a c t i v i t y by s t r a i n E^ grown on minimal medium supplemented with 0.5% yeast RNA. This graph also includes, for comparison, the growth of s t r a i n E^ and i t s release of RNase a c t i v i t y in minimal medium. F IGURE 5 ' © » g r o w t h E - j ' /M m ' g r o w t h E^ '/M+RNA 60= E .a.-7.5 a c t i v i t y E^ " /M| © 4 . 5 a c t i v i t y E-^'/M+RNA • A7 . 5 a c t i v i t y E^~/M+RNA c 3 o TO 0) TO O T IME ( H o u r s ) U5 of RNase activity release by early stationary phase of the E^ culture In RNA medium was noticeably less than that of a culture of Eg In minimal medium. Secondly, a significant delay In the release of the RNase activity, relative to the start of log phase growth, was noted, as had similarly been noted with E^ culture fn complete medium (e.g. Figs. 2 and 3). (d) Considering the release of RNase activity relative to the growth of the culture, It appeared (taking Into account the lag period between the inoculation of the culture and f i r s t detecting RNase activity) that the quantity of RNase activity was a function of c e l l density rather than of the age of the culture. A dilution experiment in which culture £j in completr medium was grown to mid-log phase and then diluted to a one-fifth c e l l concentration was carried out. The dilution 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 activity was followed by the standard methods, and these results are presented In figure 6. The rates of release of activity (i.e. the slope of the activity lines) of the original and the diluted cultures are similar. The greatest rate of release In both cases occurred at the point of maximal c e l l density. 3. RNA AS A CARBON SOURCE FOR U. HORDEI Sporldia of Ustilago horde! wore not able to grow on agar plates or in liquid culture which contained only yeast RNA (SI) and Vogel's salt solution, 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 culture of s t r a i n In complete medium (E^ /C), a sample was diluted Into four times i t s volume of minimal (E^ /C/M) or complete medium (E^ /C/C) at 121 hr. Growth was followed for both the o r i g i n a l and the diluted cultures. Release of RNase a c t i v i t y was measured at pH 7»5« F I G U R E 6 T IME ( h o u r s ) 4. CHEMICAL MUTAGENESIS (a) Table I presents the assembled Information of four different NG treatment t r i a l s . The younger the culture sampled for treatment, the greater the k i l l i n g effect and probably the higher the mutation rate (though the data are Incomplete on th i s point). This i s In agreement with the report that NG acts at the re p l i c a t i o n points (Cerda-Olmedo e£ aJL, 1968), and therefore an active log phase culture as In t r i a l NG IV is predicted to be affected by the treatment, more than an end-of-log or early-log phase culture such as that used In t r i a l NG I or I I I . (b) Among approximately 14,000 colonies from NG-treated culture samples, no RNase-defIcient mutants were detected. (c) Sixty-nine colonies were picked from plates of NG-treated c e l l s for their i n a b i l i t y to grow on minimal medium, and one for having a peculiar colony morphology. Table II summarizes the preliminary screening of these Isolates. F i f t y - f i v e of the Isolates had sharp n u t r i t i o n a l deficiencies and were considered to be auxotrophs. A l l 55 responded strongly to yeast extract and of these: seven were possjbly adenlne-defIcient (NG 48 and NG 64 d e f i n i t e l y ) ; seven were vltaraln-deficient (probably niacin or r i b o f l a v i n ) ; one responded weakly to cytoslne (NG 66), while another (NG 40) responded to a l l of the bases except guanine, suggesting that tt Is a nucleic acid mutant of some kind. NG 69, a morphological mutant, had the appearance of a length of randomly coiled rope. 5. CMC-P-TOLUENESULFONATE MODIFIED RNA To measure the extent to which guanines in the RNA had been blocked by the CMC addltton reaction, the RNase T. (10 mg/ml) hydrolysis of CMC-RNA Table I. HG treatments of s t r a i n E^ of U. horde1. Treatment Cell Density Sampled Duration T r i a l (minutes) pH V i a b i l i t y KU count During treatment (calc.) Post-treatment Cell Survival growth period Colony Percent Auxotrophs Percent isolate (hrs.) count (calc.) auxotrophs numbers HG I J2,/ NG i i 15 5.0 330 1.1 x 108 .55 x 10 8.5 2.3 x 107 18 5.0 280 .8 x IO 8 8 .8 x 108 12.5 '1 .5 * 107 5 NG i l l NG IV 15 15 5.7 350 2 x 16 8 1 x 10* 5.7 106 1.5 x 107 .75 x 108 9 3.3 x 10' 12 9.5 1.* x 106 0.7 5 t) 2 H) c15 33 '•5 .43 1-6 7-H 15-31 32-70 a) Estimated; b) Calculation based on the assumption that 10 hrs of post-treatment growth represents 1.5 generations; c) A sample of 1024 colonies from 'complete* plates with treated c e l l s . Table 11. Preliminary screening of possible mutant Isolates. Minimal medium plus supplement NG Minimal Yeast Vitamin Amino, acids B a s e s Isolate medium extract solution Cytoslne Uracil thymine Guanine Adenine Description 1 - • - - - - - - 1 Adenlne c 2 * 3 - - - - - - - -k 1 • • 1 1 1 1 t leaky,yet low 5*6 - - - - - • - 1 Adenine 7 - - - - - - Adenine 0 3 - - 1 - - - - -9 - 1 1 I - 1 1 1 1 1 1 leaky 12 1 1 • • + • + wild 13 1 • - I 1 1 t 1 1 leaky \k • 1 - • • • • wild 15 - 20 - 1 - - - - -21 - + rz. - _ - - 1 Table II Continued Minimal medium plus supplement NG isolate Minimal medium Yeast extract Vitamin solution Amino acids B a s e s Cytosine Uracil Thymine Guanine Adenine Oescrlpti 22 • 23 - • - - - - - - Vitamin** 2k * 25 - • - 1 - - - -26 1 1 1 1 1 1 1 1 leaky 27 - - - - - - - -28 - - -• - - Vitamin* 29 • - - 1 - - - - -30 1 1 - 1 1 1 1 1 leaky 31 - - - - - - -32 - + - 1 - - - - -33 • - 1 1 1 1 1 1 wild 3^ 1 • - 1 1 1 1 1 H leaky 35 • 1 1 • • + + - wild 36 - • - 1 - - - - A Vitamin 37 - •f - - - - - Vitamin*1 Table It Coatlimed 86 Isolate Mlfjfna! medium Ktntaat medium etas supplement Description Yeast extract Vitamin solution B a s e s Amino — — • — — acids Cytoslne Uraci l Thyaslne guanine Adenine 38 - r 1 39 - • - - 1 Adenine 6 40 - * m. 1 • 1 t • n . a » c 41 - 47 mm 1 - -%8 • • • M e n Ine 49-51 1 -52 - • mm «» 9* » mm 53 • - mm mm-54 - • - •m « 55 • mm «• «M 56 • • - - - •» «• 57 • •m ^ •» «•*' *•» -53 - • • •» «H M Vitamin 1 59 * - ^ mm 4tm -mt 1 Adenine 6 60 • 61 •m- I -ro Tab 1e I I Cont i nued Minimal medkm plus supplement B a s e s NG Minimal Yeast V i tamin Amino i s o l a t e medium e x t r a c t s o l u t i o n ac ids Cytokine U r a c i l Thymine Guanine Adenine D e s c r i p t i o n 62 1 + - 1 1 1 1 1 1 leaky 63 - + - 1 - -bk - + - - - - - - - Adenine 65 - . + + - - - ' - - . - V i t a m i n d 66 - + - - 1 - - - - Cytos ine 67 - + - 1 - - 1 68 1 + - 1 I 1 1 - - leaky 69 + + + 1 + + + w i l d , morpholog ica l 70 - + - 1 - . - - -a) + w i l d type growth; 1 weak growth response; - no response. b) ca se in hyd ro l y sa te plys t ryptophan. c) probable but not d e f i n i t e . d) probably n i a c i n or r i b o f l a v i n , s i nce these are common to yeast e x t r a c t and v i t am in m ix tu re . e) appearance l i k e a length of randomly c o i l e d rope. 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. (The Inhibition of RNase T^ activity, due to unreacted CMC-p-toluenesulfonate in the CMC-RNA product, was not evaluated). CMC-RNA ts probably less acid-soluble than unmodified RNA for i t was found that pH 4.5 plates which contain RNA are clear while containing CMC-RNA they formed a white precipitate even before the addition of trichloroacetic acid. RNase degradation of this white precipitate was s t i l l possible for cleared areas formed against the precipitate wherever RNase-contatnlng solution was spotted. DISCUSSION It was mentioned e a r l i e r that one consideration of this study was to establish whether e x t r a c e l l u l a r RNases were produced by Ustllago hordei and, i f so, to determine whether they are in any way si m i l a r to those produced by U_. sphaerogena (Arlma et a K , 1968). The results indicate: I) that e x t r a c e l l u l a r ribonucleases are In fact present after growth of U. horde1 sporldia and mycelta in culture media; and i i ) that the presence of ribonucleases in 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 (Fig. 1 ) suggests that at least three enzymes are present; two of 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 of these optima corresponds to a single RNase (in U_. sphaerogena, two were reported for each pH optimum; Arima et^ aj_., 1 9 6 8 ) can only be established by biochemical methods. Preliminary studies (author), using gel f i l t r a t i o n on Sephadex G-75 (method of Arima et a l . , 1 9 6 8 ) suggested that there may be two RNases with pH maxima at 4 . 5 (see Appendix B). Changes in the c e l l u l a r environment resulted In noticeable effects on the pattern of RNase secretion. Release of RNase a c t i v i t y from s t r a i n E^" could be delayed by the addition to minimal medium of RNA or complete medium components. Several lines of evidence would suggest that phosphate content in the medium is a possible c o n t r o l l i n g factor for the synthesis and secretion of ex t r a c e l l u l a r enzymes. The fungus Asperg111us oryzae grown on phosphate-free medium,both synthesized and released Its e x t r a c e l l u l a r amylase more readily (Yurkevlch et_ aJL, 1 9 6 7 ) . 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 stil 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 Inability of U. horde1 sportdta and mycella to grow on RNA plus salts Indicates that the RNA and Its degradation products are apparently not being used as carbon sources. 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 ce l l surface to convert nucleotides to nucleosides, could thus mediate the movement of RNA degradation products Into the c e l l . In this connection It will be recalled that acid PME activity was detected primarily on the c e l l surface rather than In the culture medium from which growing cells had been removed. So far as the parasitic phase of the l i f e cycle Is concerned, It Is d i f f i c u l t to visualize any Important function for the extracellular RNases. While It Is possible that extracellular rlbonucleases may contribute In some special way to the success of U. horde1 during the parasitic phase, the data do not relate to the parasitic phase. The success or failure of RNase-deficlent mutants would have provided useful Information concerning the role of extra-cellular RNases during the pathogenic phase: loss of pathogenicity would have suggested a vital role, whereas no loss of pathogenicity would have suggested that their role during the parasitic phase is dlspenslble. In considering the synthesis and translocation of extracellular RNases, It Is interesting to speculate whether the many membrane-bound vesicles 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 extracellular 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 purified extracellular rlbonucleases. If the vesicles were Involved in the movement of extracellular RNases the fluorescence-label led antibodies (see Shugar and Sierakowska, 1967 for details) should be concentrated In the vesicles. Likewise, vesicles which have been Isolated from cells (e.g. by the method of Matile, 1967) should contain RNase activity. It Is not possible to decide whether the failure to obtain rIbonuclease-deficient 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 possibilities Is the fact that the total number of mutations obtained was small. Under these conditions, the recovery of specific mutants would be Influenced by the relative s t a b i l i t i e s of specific genetic l o c i . If the change to rlbonuclease-defIclency occurs only rarely It is possible that the total size of the mutant sample was too small to Include this type of mutant. This point could be c l a r i f i e d In future work by choosing conditions in which mutagenicity Is enhanced, for example by treating cells In mj.d-log phase when they are more susceptible to k i l l i n g by NG (cf. t r i a l NG IV). Studies with bacteria have shown that NG-lnduced mutagenesis Is effected at the region, or "point", of ONA replication (Cerda*-01roedo et^ al_., 1968). If this observation holds also for jj. horde1, It should be possible to find a stage, either In the growth of a culture or In the ce l l cycle of synchronized cultures, In which even higher yields of NG-lnduced mutations can be obtained. In considering the second possibility (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 likely that RNase mutants (incapable of forming the needed enzyme) could be recovered. The screening procedures were, however, based on the assumption that cells deficient in extracellular 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 five If the enzyme activity shown at pH 6.5 Is significant) enzymes. The problem of detecting loss of activity 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 extracellular RNase activity were controlled by a single genetic locus. However, since extracellular RNase activity seems not to represent a s t r i c t l y Inducible system, i t Is probable that the extracellular RNases are not under coordinated control by a single locus. It was therefore necessary to employ a selection procedure In which identification of mutants was based on the loss of activity 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 for loss of enzymes with wide alkali optima (see Materials and Methods). But where there Is overlapping of pH optima of different RNases, or where there is activity of nonspecific dtesterases, the method becomes Ineffective, and It Is probable that these two factors did interfere with the effectiveness of the screening. An alternative to this method would be to shift 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 selection, based on a l l optima and base specificity, had been considered but because of the inavallabt11ty of the compound CMC-p-to1uenesu1fonate was not used. The rationale of this method, based on the presence of the RNases reported by Arlma et al_. (1968) Is outlined In Table 1 1 1 . A disadvantage of this method, as with the f i r s t , Is that It does not Identify ribonuclease activity on the basis of Individual ctstrons and that definite and positive results are thus contingent on a multiple mutational event. Table HI. Se lec t ion method for detect ing e x t r a c e l l u l a r RNase d e f i c i e n t mutants using a CMC de r i va t i ve of RNA. Response to substrate0 RNA CMC-RNA Oeflelency In — RNase activity pN 4.5 pH 7.5 pK 4.5 pH 7.5 1. Total loss -2. pH 4.5 1o»» . (U2 and Uj) • - <«• 3. pH 7.5 loss * 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 present. c) acting at cyttdlne and adenine. d) Ug and U| present. e) U. and U„ acting at guanine. 3 The disadvantage of both these methods could p o s s i b l y be overcome by aaranging to screen f o r mutants whose s u r v i v a l i s dependent on the c a p a c i t y f o r rlbonuclease a c t i v i t y . I t Is known that the w i l d - t y p e ( i . e . non-mutant) s t r a i n of U_. hordei cannot grow when provided w i t h RNA as the only source of carbon. But If I t were p o s s i b l e to 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 nucleoside that could be obtained through degradations of RNA ( e i t h e r by a combination o f RNase and PME, or of PDE and PME a c t i v i t y ) , such a s t r a i n should be capable of showing a growth response on RNA-supplemented medium. I f a p o s i t i v e growth response were obtained, i t should then be p o s s i b l e to s e l e c t f o r RNase mutants on the basis of f a i l u r e to show the response. This approach, i f i t should 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 basis of auxotrophy; the auxotrophs would, of course, r e q u i r e f u r t h e r s creening. The method Ju s t o u t l i n e d could perhaps be modified so as to e l i m i n a t e the p o s s i b l e involvement of both PME and PDE a c t i v i t y . The m o d i f i c a t i o n would r e q u i r e development of a h y p o t h e t i c a l s t r a i n having a requirement f o r substance "X", which could 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 nucleoside 3 , _ ( X ) phosphate. If one were to assume the ex i s t e n c e o f a system i n which RNases through to (Arima et_ a h , 1968) were a l l present, the base s p e c i f i c i t y of endonucieases could then be used to 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, f o r the U^, the U*2 and and f i n a l l y f o r the Uj d e f i c i e n c y (see o u t l i n e of method, Table IV). A mutant that Is d e f i c i e n t f o r (and therefore f o r a l l f our RNases), i f one were obtained, could be used as a parental s t r a i n In attempting to develop, through s e l e c t i o n of back mutations, those c u l t u r e s that remained 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 be p o s s i b l e to f i n d a compound which, a c t i n g as compound "X11 i n t h i s system, would be t o x i c Table IV. Selection scheme for the detection of three classes of extracellular RNase deficient strains using mode) substrates. Enzyme response8 Model substrate Genotype U2 S 1. Uridine 3'-(X)P wild n/ab n/a n/a II UV n/a n/a n/a 2. Adenosine 3'-(X)P wild n/a • • it °2* * U3* n/a m • 3. Guanos Ine 3'-(X)P wild .• • 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 al. (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. Of these two, Hood obtained only niacin mutants In his study. 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 still unidentified, though character-istically all responded to yeast extract; this observation was also made by Hood for his unclassified mutants. Attempts to produce rIbonuclease-defIclent 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. to the g e n e t i c i s t Interested In the o r g a n i z a t i o n o f g e n e t i c information and p o s s i b l e c o n t r o l s f o r the release of such Information, and k. 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 of a p o s s i b l e r o l e of RNases In host-parasite, r e l a t i o n s h i p s . SUMMARY AND CONCLUSIONS RJbonuclease a c t i v i t y was detected In c u l t u r e medium In which U s t l I ago horde I had huerx grown. Some evidence has been presented to suggest that the detected rlbonuclease a c t i v i t y was not due to release from ruptured c e l l s . A l s o , t h e a c t i v i t y was not due to phosphodiesterases s i n c e these were not detected In the c e l l - f r e e medium and only minor amounts were found to be a s s o c i a t e d w i t h c e l l s u r f a c e s . The release of RNase (as measured by RNase a c t i v i t y ) was Influenced by the c e l l u l a r environment. With complete medium or wit h minimal medium enriched w i t h yeast RNA the r e l e a s e of RNase was delayed. It Is postulated that 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 of RNase a c t i v i t y . The pH h.S and pH 7»5 RNase a c t i v i t y appears to be released c o n c u r r e n t l y . The l e v e l of RNase a c t i v i t y detected In the c u l t u r e medium was a f u n c t i o n of the c e l l d e n s i t y , w h i l e the maximal l e v e l of RNase a c t i v i t y was In turn determined by the richness of the c u l t u r e medium. The d e t e c t i o n of 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 that RNases play the r o l e of "scavenger" enzymas, s i n c e they could do t h i s o n l y In c o n j u n c t i o n w i t h phosphomonoesterases. With the d e t e c t i o n of a c i d phosphomonoesterase a c t i v i t y on the c e l l s u r f a c e , I t Is reasonable to a t t r i b u t e a "scavenger" r o l e to the e x t r a c e l l u l a r r i b o n u c l e a s e s . Among ha p l o l d c e l l s o f U. horde1 tr e a t e d w i t h the chemical mutagen, n i t r o s o g u a n l d l n e , f i f t y - f i v e 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 screening of these mutants showed that some were adenine and others v i t a m i n mutants. The remaining auxotrophs,everyone of which responded to a yeast e x t r a c t supplement, remain undetermined as to t h e i r s p e c i f i c requirement. The l e v e l of mutagenesis was not as high as that reported w i t h U.V. i r r a d i a t i o n In U_. horde 1 (Hood, 1966) so a more extensive study should be made of optimal c o n d i t i o n s f o r the use of NG In t h i s organism. Applying the r e s u l t s of such a study and using the s e l e c t i o n methods o u t l i n e d In * n e PJscussipn, i t should be p o s s i b l e to produce and I d e n t i f y s t r a i n s d e f i c i e n t In e x t r a c e l l u l a r rlbonucleases w i t h a s a t i s f a c t o r y l e v e l of e f f i c i e n c y . BIBLIOGRAPHY Adelberg, E.A., M. Handel, and G.C.C. Chen. 1965. Optimal conditions for 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 ex t r a c e l l u l a r ribonucleases of Ustllago sphaerogena; p u r i f i c a t i o n and properties. 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 purlne-speciftc ribonucleases. Biochem. J . 106: 609-613. Baker, R., and I. Tessman. 1968. Different mutagenic s p e c i f i c i t i e s In phages S13 and T4: in vivo treatment with N-methy1-M 1-nltro-N-nitrosoguantdlne J . Mol. B i o l . 35*n»39-^8. Beaton, J.D. I968. An electron microscope study of the mesosomes of a pent" ci11inase-producing Staphylococcus. J . gen. Microbiol. 50: 37-42. Cerda-Olraedo, £., and P.C. Hanawalt. 1967. Macroraolecules action of n l t r o -soguanidtne In Escherichia c o l l . Blochlm. Biophys. Acta 142: 450-464. Cerda-Olmedo, E., P.C. Hanawalt, and N. Guerola. 1968. Mutagenesis of the rep l i c a t i o n point by nltrosoguantdlne: map and pattern of rep l i c a t i o n of the Escherichia colt chromosome. J . Mol. B i o l . 33: 705-719. " Craddock, V.M. 1968. The reaction of N-methyl-N'-nltro-nltrosoguantdtne with deoxyribonucleic acid. Biochem. J. 106: 921-922. Davies, R. 1963. M i c r o b t a l e x t r a c e l l u l a r enzymes, t h e i r uses and some f a c t o r s a f f e c t i n g t h e i r formation. In 81ochem1s: t r y o f Industrfa1 Microorganisms, Chap, 4. Ed. by C. Rainbow s A~H. Rose. London, Academic Press. DeRobertls, E.D.P., W.W. Nowlnki, and F.A. Saez. 1965. C e l l B i o l o g y . 4th ed. W.8. Saunders & Co. P h i l a d e l p h i a . Dtnoor, A., and C. Person. 1969- Genetic complementation 1n Us t i l a g o hordet. Can. J. Botany 47: 9-14. Egaml, F., and K. Nakamura. 1969. M i c r o b t a l Ribonucleases. Sprlnger-Verlag New York, Inc. E l s e n s t a r k , A . , R. Etsen s t a r k , and R. van S i c k l e . 1365. Mutation of Salmonella 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 to the m o d i f i c a t i o n of ribonuclease a c t i o n . J . Am. Chem. Soc. 84: 687-688. 42 G l i t z , O.G., and CA. Dekker. 1964. Studies on a rlbonuclease from Ustllago sphaerogena. I. Purification and properties of the enzyme. Biochemistry 3j 1391-1399. G l i t z , O.G., and CA. Dekker. 1964. Studies on a rlbonuclease from Ust11ago sphaerogena. 11. Specificity 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. Halltwell. 1968. An endonuclease-deftclent strain of Ust11ago maydls. Genet. Res. Comb. V2: 95-98. Hood, CH. 1966. UV-lrradlatlon sensitivity and mutation production In the haploid sporldta of Ust1lago hordeI. Ph.D. Thesis, University of Alberta, Edmonton. ™ " Jef f f i e s , C C , D.F. Holtman, and O.G. Guse. 1957. Rapid method for determining the activity of microorganisms on nucleic acids. J. Bact. 73: 590-591. Kushner, D.J., and H.R. Pollock. 1961. The location of cell-bound penicillinase In Bac111us subt111s. J. gen. Microbiol. 26: 255-265. Lampen, J.O. 1965. Secretion of enzymes by microorganisms. In Symp. of Gen. Microbiologists 1j>: 115-133. 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^cterlol. 63_: 399-406. Lee, J.C, N.W.Y. Ho, and P.T. Gllham. 1965, Preparation of rlbotrlnuclo-tides containing terminal cytldlne. Blochlm. Blophys. Acta 95: 503-504. Loprieno, N., and CH, 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 Jn 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. Intracellular^ lokallsation 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 Zellstrukture. Z. Zellforsch. 6 8 : 205 - 216 . Matile. P., and A. Wlemken. 1967. The vacuole as the lysosome of the yeast c e l l . Archlv fur Mlkroblol. 56: 1 48 -155 . Miiller, J . , and T. Glchner. 1964. Mutagenic activity of l-methyl-3-nltro-1-nltrosoguanldlne on A^a^dhopjsls^. Nature 20U 1149 -1150. Naylor, R., N.W.Y. Ho, and P.T. Gllham. 1965. Selective chemical modifications of uridine and pseudourldlne In polynucleotides and their effect on the specif 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) 46 : 161-167. Nlshlmura, S., and B i Maruo. 1960. Intracellular rlbonuclease from Bacl1lus subtIlls. 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. 1967. Polynucleotidases In animal tissues. Experentia 2 3 : 321-325. Shleh, T .R . , and J.H. Ware. 1968. Survey of microorganisms for the production of extracellular phytase. Appl. Microbiol. Jj6: 1348-1351. Shugar, 0 . , and H. Slerakowska. 1967. Mammalian nucleolytlc enzymes and their localization. Prog. N.A. and Mol. Biol. 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 British Columbia. Subba Rao, P.V., K. Moore, and G.H.N. lowers. 1967. Purification and properties of phenylalanine ammonia-lyase from Ustllago horde1. Can. J. Blochem. 4£: 1863-1872. Takahashl, K. I96I. Chromatographic purification and properties of rlbonuclease T p J. Blochem. (Tokyo), 49 : 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-alkyl-3~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. Bull. No. 13. Von Tlgerstrom, R.G., and M . Smith. 1969. Preparation of the 2,4-dlnttrophenyl esters of thymidine 3'~ and thymidine 5*-phosphate and their use as substrates for phosphodiesterases. Biochemistry 8: 3067-3070. Yanaglda, H., T. Uchida, and F. Egaml. 1964. Culture of 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 of amylase by the fungus Aspergillus oryzac. Prlkladnaya Blokhlmlya I Mikroblologlya £ J 15FHF. APPENDIX A. 1. Vogel's (1956) s a l t solution contained: 123 9 sodium c i t r a t e , 250 g monobasic potassium phosphate, 100 g ammonium ni t r a t e (anhyd.), 10 g magnesium sulphate, 5 g calcium chloride, 5 ml trace element solution In 750 ml d i s t i l l e d water with 2 ml chloroform. 2. The trace element solution contained: 5 9 c i t r i c a c i d , 5 g zinc sulphate, 1 g ferrous ammonium sulphate, 0.25 9 copper sulphate, 0.05 g manganese sulphate, 0.05 9 boric 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 solutions were stored at room temperature. 3. The vitamin solution 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 ac i d , 200 mg n i c o t i n i c a c i d , 200 mg choline chloride, kOO rug Inositol and 50 mg f o l i c acid 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 one-thirtieth the original volume produced a syrupy liquid which affected the column loading and elutlng. In future attempts to establish the number of 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 for greater concentration of the medium and this provides a more definite RNase activity elutlon profile. 

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