UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Supersuppression in Neurospora crassa Newcombe, Kenneth Donald 1973

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1973_A1 N49_3.pdf [ 3.4MB ]
Metadata
JSON: 831-1.0101162.json
JSON-LD: 831-1.0101162-ld.json
RDF/XML (Pretty): 831-1.0101162-rdf.xml
RDF/JSON: 831-1.0101162-rdf.json
Turtle: 831-1.0101162-turtle.txt
N-Triples: 831-1.0101162-rdf-ntriples.txt
Original Record: 831-1.0101162-source.json
Full Text
831-1.0101162-fulltext.txt
Citation
831-1.0101162.ris

Full Text

/ 7 - Z / 3 (Ll SOPEBSUPPBISSIOH IH HEOBOSPOBA CBASSA BY KENNETH DONALD MEWCOMBE, B.Sc, fl.Sc. A Thesis Submitted to the School of Graduate Studies in P a r t i a l Fulfilment of the fieguirements for the Degree ~> y Doctor of Philosophy in Genetics University of B r i t i s h Columbia December, 1972. We accept t h i s thesis as conforming to the required standard: July, 1973 In presenting t h i s thesis, I agree that the l i b r a r y s h a l l make i t f r e e l y available for reference and study. I further agree that the thesis may be copied extensively for scholarly purposes. However, i t i s understood that copying or publication of thi s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Kenneth Newcombe The Genetics Group of The University of B r i t i s h Columbia VANCOUVEB 8, B r i t i s h Columbia Canada ABSTRACT A population of 76 adenine-3E (ad-3B) heteroalleles (each produced by t r a n s i t i o n of a single base pair) has been tested for s u p p r e s s i b i l i t y bj 8 supersuppressors. . Suppression has been observed in ncnpolar and noncompleinenting str a i n s with AT or GC at the mutant s i t e , and in s t r a i n s with a polarized pattern of complementation and AT at the mutant s i t e . The r e s u l t s support e a r l i e r work indicating the existence of nonsense mutations and nonsense suppressors in Neurospora s t r a i n s . The results also suggest the existence of a novel species of tRNA which can recognise either a nonsense or a missense codon. I SUPERSUPPRESSION IH NEUROSPOBA CHASSA i i i I DOCTOR OF PHILOSOPHY UNIVERSITY OF BRITISH COLUMBIA (Genetics) Vancouver, B r i t i s h Columbia TITLE : Supersuppression i n Neurosrgora crassa AUTHOR : Kenneth Donald Newcombe, B.Sc. (McMaster University), M.Sc. (McMaster University) SUPERVISOR : Dr. A.J.F. G r i f f i t h s NUMBER OF PAGES : x i , 88 i .V;, ACKNOWLEDGEMENTS The study reported i n t h i s thesis could not have been completed without the generous help and advice of more people than I can l i s t here. Eut I am p a r t i c u l a r l y g r a t e f u l to both Miss. Laura Doliner and Miss. Judith Hale who have provided cheerful and accurate technical assistance throughout the project. I would l i k e tc thank Dr. I. B. Woods for his help with the graphs; Hr. G. White for his drawings of the Col l e c t i o n Gantry; and both Hrs. C. Hoods and my wife, Maybeth, for extensive help in preparing the text. I also acknowledge the kind assistance of Hr. Steve Eorden and Mrs. Andrea Berger i n u t i l i s i n g the UBC computing f a c i l i t i e s . I would l i k e to thank my supervisor Dr. A. J . F. G r i f f i t h s for his help at a l l stages of t h i s project. In every regard Dr. g r i f f i t h s has been a superior supervisor and I consider myself very fortunate to have had the p r i v i l e g e of working with him . v TABLE OF CONTESTS INTRODUCTION PAGE # THE SPECIFIC INDUCTION OF GC TO(AT TRANSITIONS BY HA 4 THE SPECIFIC ACTION OF HA IN NEUROSPORA 8 ADENINE BIOSYNTHESIS IN NEUROSPORA 10 NOMENCLATURE,AND TOPOGRAPHY OP THE AE-3 REGION 12 COMPLEMENTATION OF MUTANTS IN THE AD-3 REGION 15 SUPPRESSORS 18 MATERIALS AND METHODS NEUROSPORA STRAINS 27 CULTURE MEDIA 33 CROSSING NEUROSPORA STRAINS 34 ISOLATION OF PROGENY FROM CROSSES 35 GENOTYPE DETERMINATION 39 GROWTH RATE DETERMINATION 41 EXPERIMENTAL PROTOCOL 42 OBSERVATIONS THE SUPPRESSIBILITY OF AD-3B MUTANTS 47 SUPPRESSIBILITY OF ALLELES AT OTHER LOCI 48 THE GROWTH RATE OF OCTAD ISOLATES 48 v i TABLE OF CONTENTS JCONT.l DISCUSSION ANALYSIS OF DATA 57 NONSENSE-MISSENSE SUPPRESSION 59 THE PATTERN OF NONSENSE SUPPRESSION 65 APPENDIX SOMATIC VIGOR OF SUPERSUPPRESSED NEUROSPORA STRAINS 68 LITERATURE CITED 79 V i i 1 . DESCRIPTION OF AD-3B MUTANTS TESTED FOR SUPPRESSIBILITY 2 . DESCRIPTION OF ADDITIONAL STRAINS USED IN THIS STUDY 3. SUPPRESSIBILITY OF NONPOLARS WITH GC AT THE MUTANT SITE 4. SUPPRESSIBILITY OF NONPOLARS WITH AT AT THE MUTANT SITE 5 . SUPPRESSIBILITY OF POLARS WITH GC AT THE MUTANT SITE 6. SUPPRESSIBILITY OF POLARS WITH AT AT THE MUTANT SITE 7. SUPPRESSIBILITY OF NONCOMPLEMENTERS WITH GC AT THE MUTANT SITE 8. SUPPRESSIBILITY OF NONCOMPLEMENTERS WITH AT AT THE MUTANT SITE LIST OF TABLES JCONT..1 Page # 9. THE NUMBER OF SUPPBESSIBLE MUTANTS AS A FRACTION OF THE TOTAL NUMBER OF MUTANTS IN EACH CLASS 58 TABLE OF THE APPENDIX 1. PARTIAL GENOTYPES OF PROGENY IN FIGURES 1-8 OF THE APPENDIX 70 ix LIST OF FIGURES Page # 1. GENETIC BLOCKS OF ADENINE BIOSYNTHESIS IN NEUROSPORA 14 2. ALLELIC COMPLEMENTATION OF AD-JB MUTANTS 17 3. LINKAGE MAPS OF NEUROSPORA CRASSA 25 4. COLLECTION GANTRY 37 5. POSSIBLE TRANSITIONS FROM NONSENSE TO SENSE CODONS 60 6. CODONS 'RECOGNISED' BY ANTICODONS OF POSSIBLE NONSENSE-MISSENSE SUPPRESSING tRNA SPECIES 61 7. CODONS RELATED TO SPECIFIC SUPPR1SSIBLE HISSENSE CODONS 66 x FIGURES OF THE APPENDIX 1. GROWTH OF PARENTS AND SINGLE OCTAD PROGENY OF CROSS 1751 X 2*17-70 2. GROWTH OF PARENTS AND SINGLE OCTAD PROGENY OF CROSS 1750 X 2-17-28 3. GROWTH OF PARENTS AND SINGLE OCTAD PROGENY OF CROSS 1749 X 12-21-440 4. GROWTH OF PARENTS AND SINGLE OCTAD PROGENY OF CROSS 1751 X 2*17-28 5. GROWTH OF PARENTS AND SINGLE OCTAD PROGENY OF CROSS 1749 X 2-17-76 6. GROWTH OF PARENTS AND SINGLE OCTAD PROGENY OF CROSS 1749 X 2-17-22 7. GROWTH OF PARENTS AND SINGLE OCTAD PROGENY OF CROSS 1750 X 2-17-76 8. GROWTH OF PARENTS AND SINGLE OCTAD PROGENY OF CROSS 1687 X 2-17-149 xi INTRODUCTION 2 The work reported in t h i s thesis tests the s u p p r e s s i b i l i t y of t r a n s i t i o n mutants of the ad -3B locus by a number of supersuppressors. This appraisal i s of interest for two reasons. 1) I t provides new information for the assertion that the supersuppressors in Neurospora could be tRNA mediated suppressors; and that the suppressible ad-3B mutants are largely nonsense mutants of the sort described i n bacteria and yeast. 2) It provides the f i r s t quantitative assessment of the gross physiological consequences of supersuppression on the vigor of Neurospora str a i n s . (This work i s reported in the appendix). The jJ~3B locus i s uniquely suited to the methods of the present study. A large number of ad-3B a l l e l e s were available which had been characterised with respect to complementation pattern and r e v e r t i b i l i t y with the mutagen hydroxylamine (HA). These studies of the properties of ad-3B mutants have led to three assertions which w i l l be examined i n the remaining sections of the introduction . 1) In Heurospora, hydroxylamine (HA) i s presumed to cause primarily GC to AT t r a n s i t i o n s . 2) The pattern of intragenic complementation can be used to discriminate between missense and 3 nonsense mutations. 3 ) Supersuppressors are most l i k e l y to suppress nonsense mutations. As w i l l be shown these assertions can be combined to predict that only certain classes of ad -3B a l l e l e s should be suppressed by supersuppressors. The testing of t h i s prediction constitutes the main body of the thesis. 4 The s p e c i f i c induction of GC to AT tra n s i t i o n s bj HA The genetic alt e r a t i o n s in HA induced mutants have been characterised at the molecular l e v e l (Walling, 1971), and they are believed to be primarily GC to AT t r a n s i t i o n s . This apparent s p e c i f i c i y provides one c r i t e r i o n which might distinguish between missense mutants (which may have AT at the mutant site) and nonsense mutants (which must have AT at the mutant site) (see Mailing and deSerres, 1968a; 1968b; deSerres and Mailing,1969;) since nonsense mutants cannot arise 5® 2222 a s a r e s u l t of any AT to GC t r a n s i t i o n (see figure 5), the r e v e r t a b i l i t y of t r a n s i t i o n mutants after treatnent with HA may provide one basis f o r selecting those mutants which are most l i k e l y to be suppressible by nonsense suppressors. The v a l i d i t y of th i s approach depends upon a demonstration of the s p e c i f i c i t y of HA which i s inferr e d from the following four types of evidence. The question of whether t h i s evidence can be used to assert that HA i s s p e c i f i c in Neurospora i s examined in a subsequent section. 5 ! i P h y s i c a l evidence of HA s p e c i f i c i t y i n DNA-_ The UV a b s o r p t i o n of U and C i s decreased a f t e r r e a c t i o n with HA . The a b s o r p t i o n c h a r a c t e r i s t i c s of other bases are not changed i n d i c a t i n g t h a t HA r e a c t s s p e c i f i c a l l y with C i n DMA (Sbuster, 1961; Freese, 1963). 2. Evidence from i n v i t r o t r a n s c r i p t i o n ^ P o l y r i b o n u c l e o t i d e s have been found to a c t as templates i n r i b o n u c l e o s i d e t r i p h o s p h a t e i n c o r p o r a t i o n systems, and f o r more than a decade have been used to assess the molecular e f f e c t s of mutagenic agents ( B a s i l i s et a l , 1962; Fox et a l , 1963; P h i l l i p s et a l , 1965; 1966; Wilson et a l , 1966; and Singer et a l , 1970). When c y t o s i n e r e s i d u e s of a template are modified by HA, the a f f e c t e d r e s i d u e s e i t h e r cease to p a r t i c i p a t e i n RNA polymer formation or demonstrate ambiguous s p e c i f i c i t y ; sometimes the s p e c i f i c i t y of C and sometimes the s p e c i f i c i t y of T. i n an i n v i y g system an a f f e c t e d template r e s i d u e r e p l i c a t e d with the s p e c i f i c i t y of T would produce a GC to AT t r a n s i t i o n . In a d d i t i o n , where DNA r e p l i c a t i o n i s i n progress during HA treatment i t i s evident t h a t an a l t e r e d DNA p r e c u r s o r with ambiguous s p e c i f i c i t y c o u l d produce both GC to AT and AT/ to GC t r a n s i t i o n s . T h i s apparent r e d u c t i o n i n s p e c i f i c i t y would not be expected i n the treatment of pure phage suspensions or n o n r e p l i c a t i n g c e l l s (Budowsky et a l , 1972). Apparent r e d u c t i o n i n HA s p e c i f i c i t y seems u n l i k e l y to have been 6 a s i g n i f i c a n t factor in the c l a s s i f i c a t i o n of Neurospora revertants since the method of obtaining revertants involved HA treatment of conidia (Hailing, 1971). The only other base of EN A which can react with HA i s A, bat the i n v i t r o reaction rate i s two orders of magnitude lower than that of C under similar conditions. As yet no b i o l o g i c a l significance has been attached to t h i s finding (Budowsky et a l , 1971). 1*. Evidence from £hacje mutagenesis The following studies of phage mutagenesis are not conclusive; but the results are compatible with the physical and biochemical findings discussed above. It has been found that bacteriophage T U r l l mutants f a l l into two d i s t i n c t categories: those which are r e v e r t i b l e by HA; and those which are not (Freese et a l , 1961). Furthermore, bacteriohphage S13 host range mutants which are induced by HA, cannot be reverted by HA. Other host range mutants which are r e v e r t i b l e by HA, cannot be induced by i t (Tessman et a l , 1963) In themselves these experiments do not demonstrate an i n vivo s p e c i f i c i t y of HA; but they are strongly suggestive and completely compatible with the physical and biochemical evidence cited above. 7 H*. t.RNft Evidence for HA s p e c i f i c i t y : The production of GC to AT changes by HA has been d i r e c t l y demonstrated i n the anticodon region of a phage tRNA (Altman et a l , 1971). The A2 gene of phage $ 80 codes for a tyrosine tRNA which has A~u£* at the anticodon. The phage w i l l not normally suppress DAA mutations i n host bacteria. However, after HA treatment a group of phage str a i n s were selected which would suppress the host c e l l OAA l e s i o n . Phage s p e c i f i c tyrosine tRNA was isolated from suppressed host c e l l s and characterised by two dimensional f i n g e r p r i n t analysis following ribonuclease digestion. In each case the analysis demonstrated a single base change from C to U in the anticodon region. Such an a l t e r a t i o n could only have occurred as a consequence of a GC to AT a l t e r a t i o n i n the corresponding codon of the A2 gene i n each of the strains which were analysed. * For reasons of convenience and c l a r i t y the symbols for phosphoric acid residues have been omitted in describing codons (eg. ATC). Also, since codon-anticodon recogniticn i s most ea s i l y appreciated i f the anticodons are presented i n register with the codons, they appear throughout the text with a single arrow i n d i c a t i n g the position of the 5 prime terminus (eg. DAG). 8 The s p e c i f i c a c t i o n of HA i n Neutos_gora_. The evidence c i t e d so f a r suggests that HA s p e c i f i c a l l y induces GC to AT t r a n s i t i o n s ; a d d i t i o n a l evidence i s r e q u i r e d to demonstrate that t h i s mode of a c t i o n d e s c r i b e s the s i t u a t i o n i n Neurospora. F i r s t , HA r e v e r t s only part of a p o p u l a t i o n of mutants induced by n i t r o u s a c i d and e t h y l methanesulfonate; and i t r e v e r t s none of a p o p u l a t i o n induced by o-methylhydroxylamine ( M a i l i n g , 1966; 1971). N i t r o u s a c i d (for review see K r i e g , 1963a) and e t h y l methanesulphonate (Krieg, 1963b) probably both cause a l l types of t r a n s i t i o n events while O-methylhydroxylamine a c t s l i k e hydroxylamine ( M a i l i n g , 1971). In t h i s s o r t of experiment the c l a i m t h a t HA causes predominantly GC to AT t r a n s i t i o n s i s dependant upon the r e l i a b i l i t y with which the s p e c i f i c i t y of the other mutagens can be assessed. The a c t i o n of these mutagens has been reviewed and the a n a l y s i s i s i n agreement with other work which has a l r e a d y been presented ( M a i l i n g , 1971). Second, the spectrum of complementation p a t t e r n s (which w i l l be d i s c u s s e d l a t e r ) among HA induced ad~3,B mutants i s d i s t i n c t l y d i f f e r e n t from the spectrum found i n n i t r o u s a c i d induced mutants which are r e v e r t a b l e a f t e r treatment with HA. The HA induced group presumably has AT at the mutant s i t e and may i n c l u d e nonsense mutations; while the HA r e v e r t a b l e group presumably has GC at 9 the mutant s i t e and i s presumably comprised exclusively of missense mutants {Walling, 1971). As indicated e a r l i e r there are grounds for expecting differences i n the complementation patterns of nonsense and missense mutations. Before discussing the type of complementation referred to, the pathway of adenine biosynthesis and some general c h a r a c t e r i s t i c s of the ad-3 region w i l l be presented. 10 Adenine Biosynthesis in Neurgsgora * The process cf adenine biosynthesis i n Neurospora i s t y p i c a l of the process in microorganisms and i s e s s e n t i a l l y the same as the process i n avian l i v e r preparations where i t has been studied extensively. (Moyed et a l , 1957; for a review see Buchanan, 1960). The pathway, the genetic blocks, and the linkage location of genes af f e c t i n g adenine biosynthesis are i l l u s t r a t e d in figure 1. The figure has been revised from Buchanan to take i n more recent information including the most recent linkage analysis of the genes involved. The linkage group of each a l l e l e i s shown in parenthesis (see Buchanan, 1960; Bernstein, 1961; Fisher, 1967; and Radford, 1972). * The following abbreviations are used i n the description of adenine biosynthesis: AICAR 5-amino-1-ribosyl-4-imidazolecarboxamide-5•-phosphate AIR 5-amino-1-ribosylimidazole-5'-phosphate AMP adenosine monophosphate AMP-S 6-(succinylamino)-9-(ribofurancsyl 5'-phosphate) purine CAIR 5-amino-1-rribosyl-4-imidazolecarboxylic acid FGAM 2-formamino-fl-ribosylacetamide-5*-phosphate FGAR 2-formamido-N-ribosylacetamide-5•-phosphate GAR 2-amino-N-ribosylacetamide-5*-phosphate SAICAR H-(5-amino-1-ribosyl-4-imidazolecarbonyl)-Laspartic acid 11 The ad-3 region codes for the production of two enzymes: phosphoribosyl-amino-imidazole carboxylase, and phosphoribosyl-amino-imidazole-succinocarboxamide synthetase. Becent improvements in methods of enzyme preparation and assay have made i t possible to conclude that the ad-3A c i s t r o n i s responsible for synthetase a c t i v i t y , while the ad-3B cis t r o n i s responsible for the carboxylase (Fisher, 1969a; 1969b). Unfortunately the carboxylase has not yet been characterised in Neurospora. 12 Nomenclature and Topography of the ad-3 Region Designations of the genes af f e c t i n g adenine biosynthesis have evolved in the following manner. The ad mutants shown in figure 1 were formerly designated by l e t t e r s of the alphabet. For example, group A mutants were those which blocked the step from CAIR tc SAICAR; group B mutants blocked the step between AIR and CAIR; and so on (for example see Buchanan, 1960). At the time the present numerical convention was adopted i t was not known whether group A and group B mutants were a l l e l i c hence the designations ad-3A and ad-3B. The ad-3 region i s now known to consist of two genes, ad-3A and ad-3B separated by the x-region (deSerres, 1964; deSerres , 1969; G r i f f i t h s , 1970). Whether or not the two genes are transcribed on a single p o l y c i s t r o n i c message i s not known. However, polar interactions between a l l e l e s of both l o c i have not been described. Some of the genes discussed here and elsewhere i n the thesis have been mapped and are presented in figure 3. Figure 3 i s redrawn from a comprehensive study of linkage relationships done by Radford (1972); with additional information on the positions of supersuppressors from Seale (1971). Where precise information does not exist the approximate location of the gene i s denoted by two dotted l i n e s . The position of other genes i s described only as the l e f t or right arm of a p a r t i c u l a r linkage group. This information appears in figure 1 for the genes of the 13 adenine biosynthetic pathway and i s given for other genes l i s t e d i n table 1. Figure 1 Genetic Blocks of Adenine Biosynthesis in Neurospora (see text) GAR FGAR FGAM AIR CAIR SAICAR ~*-~ AICAR ad 9 ad 6 ad2 ad3B ad3A ad 4 (1R) (4R) (3R) (IR)- (1R) (3R) AICAR -H*- FAICAR IMP AMP ~S — ad 5 1 adV ad 4 (ID (6L) (3R) AMP 15 Complementation of Mutants i n the ad-3 Region Pairs of adenine reguiring mutants from d i f f e r e n t genes or sometimes from the same gene are known to complement and grow i n adenineless medium. Intergenic complementition between ad-3A and ad-3B mutants i s well known (for example see deserres, 1967). While intragenic complementation between a l l e l e s of the ad-3A locus i s unknown ( G r i f f i t h s , 1970) ; intragenic complementation between a l l e l e s of the ad-3B locus may take one of three d i s t i n c t patterns. 1) A mutant may f a i l to complement with any other a l l e l e s and i s known as a • noncomplementor•. ( 2) It may f a i l to complement with a l l mutants which l i e at the d i s t a l end of the c i s t r o n i n which case i t complements in •polar• fusion. Or 3) i t may complenent with mutants at the d i s t a l end of the ci s t r o n in which case i t complements i n *nonpolar* fashion (ie. a nonpdlar complementot must complement at the d i s t a l end and may complement elsewhere in the c i s t r o n ) . The s p e c i f i c complementation pattern of nitrous acid induced mutants used in this study are shown i n figure 2. The c i s t r o n i s divided into 17 complons, ccmplon #1 being the most d i s t a l of a l l . The complon coverage i s indicated by a series of bars extending across the complons i n which there are no complementating mutants (after deserres et a l , 1967). As 16 shown i n the figure the polar complementing group have been placed above the group of non-polar complementing mutants. Similar information for the HA induced mutants used in th i s study has not been published (see Hailing, 1971). While the molecular basis of a l l e l i c complementation i s not e n t i r e l y c l e a r ; the significance of polar, or noncomplementing patterns may be appreciated by considering the nature of chain terminating mutations. If only part of the polypeptide chain i s formed, then a l l the fragments have the proximal end in common (see Sarabhai et a l , 196H; Fowler et a l , 1966; and Nichols, 1970). If the fragment i s large enough to permit a l l e l i c complementation at a l l , complementation with mutations in positions corresponding to the missing d i s t a l section seems unl i k e l y . For t h i s reason i t has been assumed that nonsense mutants would show a polar or noncomplementing pattern in tests of intragenic complementation (Mailing et a l , 1968a). It follows that, of the mutants used in th i s study those which either f a i l to complement at a l l or which complement i n polar fashion are the most l i k e l y tc be nonsense mutants. The polar and noncomplementing mutants are therefore must l i k e l y to be suppressible by t-RNA mediated nonsense suppressors should they be found in Neurospora. The c h a r a c t e r i s t i c s of such suppressors are examined i n the remaining section of the introduction. Figure 2 Allelic Complementation of ad-3B Mutants ' ad-3B cistron allele #'s 2-17-153 93 163 16,46,131 200,264 76,128 74 58,150 51 129 37 114 130 17,22,79 68,106 34 30 47 1 2 3 4 5 6 7 8,9 10 11 12 131415 16 17 (see text) 18 Suppressors In the c l a s s i c a l sense a suppressor i s a mutation which can restore a normal (or partly normal) phenotype despite the persistance of some primary genetic lesion* The suppressor mutations may act • i n d i r e c t l y * or ' d i r e c t l y * (Gorini et a l , 1966). ! A suppressor which acts in an in d i r e c t fashion restores function by a l t e r i n g c e l l conditions in such a way that the o r i g i n a l mutation no longer affects c e l l phenotype (Cribbs, 1970). For example, the Jkyr-3 mutants of Neurospora are known to accumulate carbarmoyl phosphate. The £y.r-3 mutants act p l e i o t r o p i c a l l y to suppress the arginine requirement of ar<j-2 and arjg-3 mutants by supplying carbamoyl phosphate to overcome a deficiency i n the arginine pathway (Reissig, 1963a; 1963b). However, the action of i n d i r e c t suppressors i s generally not t h i s well defined (see Gajewski et a l , 1968; Stadler, 1967; and Hewmeyer, 1970). In contrast, the action of di r e c t suppressors has been elucidated in d e t a i l (for a review see Smith, 1972). The dir e c t or sc c a l l e d 'informational suppressors' (Gorini, 1970) act to a l t e r a primary genetic l e s i o n so that the corresponding protein product appears to be normal (or almost normal). Presumably an informational suppressor would not suppress a l l a l l e l e s of one gene, but some a l l e l e s of many genes (Cribbs, 1970). Such a 19 mode of action i s fundamentally d i f f e r e n t from that of the i n d i r e c t suppressors. Informational suppression { f i r s t entertained as a p o s s i b i l i t y by Xanofsky and St. Lawrence, 1960) was postulated to occur by at least three d i f f e r e n t mechanisms. In the f i r s t place d i r e c t suppression would be an expected consequence of a change i n some element of the RNA polymerase system re s u l t i n g in a l t e r a t i o n s to the f i d e l i t y of DNA dependant RNA t r a n s c r i p t i o n . Second, direct suppression could also occur i f changes in a ribosomal component produced se l e c t i v e a l t e r a t i o n s in the translation of an mRNA sequence. (Specific informational suppression i s not known to occur by either of these mechanisms.) F i n a l l y , informational suppression does occur as a res u l t of changes in the anticodon s p e c i f i c i t y of tRNA moieties which i n turn cause s e l e c t i v e a l t e r a t i o n s in the t r a n s l a t i o n a l process. Altered species of tRNA are known to cause the suppression of missense, nonsense, and frameshift mutations (Riddle, 1973; for a review of the above types of suppression see Smith, 1972). Nonsense suppressing tRNA 1s have been characterised in bacteria and yeast and w i l l be discussed following some general observations about the properties of nonsense suppressing tRNA's. There are three unique properties of tRNA mediated nonsense suppressors. F i r s t , they are characterised by an altered tRNA species. The altered tRNA i s known to recognise stop codons OAA, UAG and UGA because of a 20 s p e c i f i c a l t e r a t i o n i n the anticodon region (Smith, 1972). Second, suppressors are known to restore normal or near normal protein production in c e l l s with nonsense mutations. They act to ins e r t a s p e c i f i c amino acid at a point i n the polypeptide chain which corresponds to the position of the nonsense codon (Gorini, 1970). Such a substitution may be detected by amino acid sequence analysis (Gilmore et a l , 1968). F i n a l l y , since these suppressors function by 'mistranslating* s p e c i f i c t r i p l e t s they must be a l l e l e rather than gene s p e c i f i c (Smith, 1972). Two additional conseguences of a l t e r i n g a tRNA to recognise nonsense t r i p l e t s may be considered. F i r s t , the tRNA species should in s e r t an amino acid not only at a s i t e corresponding to a suppressible nonsense mutation, but everywhere the nonsense condon occurs. Second, the suppressing tRNA species no longer performs i t s usual function* Since most tRNA species have several isoaccepting forms any p a r t i c u l a r one may be dispensible. But whether t h i s i s i n fact the case depends on the possible reasons for the unexplained redundancy of tRNA genes. In t h i s regard i t i s s i g n i f i c a n t that haploid yeast st r a i n s producing two d i f f e r e n t species of nonsense suppressing tRNA suffer from the 'two suppressor e f f e c t ' . When i t i s not l e t h a l , t h i s two suppressor e f f e c t r e s u l t s i n morphologically aberrant c e l l s and retarded 21 growth (Gilmore, 1967). The physiological e f f e c t s of tieurospora supersuppressors are being investigated and a preliminary report appears i n the appendix. Supersuppressors i n Neurospora (which are not yet i d e n t i f i e d as tRNA mediated) are very similar to supersuppressors in yeast (which are known to be tRNA mediated) and the nonsense suppressors i n b a c t e r i a l systems. In bacteria nonsense mutations of phage can be suppressed by the host c e l l . Por example, i f T4 phage with a nonsense mutation i n the region corresponding to the coat protein i s used to i n f e c t a normal b a c t e r i a l c e l l , the b a c t e r i a l host produces only fragments of the coat protein (Sarabhai et a l , 1964). However , i f the b a c t e r i a l host produces a suppressing species of tRNA, a completed protein can be produced. At the point where they would otherwise terminate these coat proteins have a s p e c i f i c amino acid which i s inserted by the suppressing tRNA (weigett et a l , 1967).. The tRNA mediated suppressors were demonstrated i n yeast a f t e r i t became possible to i s o l a t e and characterise iso-1-cytrochrome (Sherman et a l , 1968; 1970). Nonsense mutants producing only the proximal portion of the cytochrome were is o l a t e d and the terminating t r i p l e t (UAA) was determined by amino acid analysis of cytochrome i n reverted s t r a i n s . This i n turn allowed the i d e n t i f i c a t i o n cf DAA 22 suppressing tRNA *s (Gilmore et a l , 1968; 1971). A l l e l e s responsible for the suppressing tRNA•s were known to cause simultaneous suppression of several b i o s y n t h e t i c a l l y unrelated mutants. For thi s reason the a l l e l l e s were c a l l e d supersuppressors by Hawthorne and Mortimer who f i r s t described them a decade ago (Hawthorne and Mortimer, 1963). Since then more than f i f t e e n d i s t i n c t supersuppressor genes have been described i n yeast and several have been subjected to extensive genetic analysis (Gilmore, 1967). Multiple suppression i s not r e s t r i c t e d to yeast. Besides i t s occurrence in Neurospora, which w i l l be described presently, i t i s known in Drosophila. Su2-HW suppresses Hairy Wing and diverse morphological t r a i t s at seven d i f f e r e n t l o c i (E.B. Lewis unpublished, cited by Wagner and M i t c h e l l , 1962). Unlike the supersuppressors in yeast, the supersuppressors in Neurospora are not d e f i n i t e l y known to be tRNA mediated nonsense suppressors; however, two types of evidence w i l l be presented which strongly suggests that they are. F i r s t , a suppressor of glutamate dehydrogenase deficiency (am) has recently been discovered (Seale, 1967). When suppressed, two noncomplementing stra i n s produce a glutamate dehydrogenase which d i f f e r s from wild type enzyme i n i t s s p e c i f i c a c t i v i t y and thermal s t a b i l i t y . Trjj£-2 mutants can also be suppressed and si m i l a r q u a l i t a t i v e 23 differences are observed between the normal and the multienzyme aggregate of suppressed tryp-2 mutants (Chalmers and Seale, 1971). In addition, the supersuppressors suppress seme of the genes of the ajom series as well as some t rXE~l a l l e l e s (Seale, 1970). In other work two supersuppressors; su-22, and su-33; have been shown to suppress seven di f f e r e n t noncomplementing arom mutants. Unsuppressed mutants normally lack a c t i v i t y for five.enzymes which catalyse part cf the aromatic synthetic pathway to chorismic acid (Case et a l , 1968) whereas i n suppressed s t r a i n s the a c t i v i t y of a l l f i v e i s restored. The above r e s u l t s are very si m i l a r to results in yeast in which i t was found that certain supersuppressible mutants produce broken fragments of the tryptophan synthetase enzyme (Manney, 1964). In yeast the r e s u l t s were c o r r e c t l y interpreted to indicate that the supersuppressors reversed the primary genetic l e s i c n at the time of t r a n s c r i p t i o n . Second, the observation that the Neurospora supersuppressors were a l l e l e rather than gene s p e c i f i c was v e r i f i e d by Seale, Case, and Barratt (1969) who crossed the eight a v a i l a l e supersuppressors with an assortment of mutants. (Since the suppressible mutants of that study provide a method of checking the i n t e g r i t y of supersuppressor stocks, the work i s considered again in the methods section of th i s report.) Two possible s i m i l a r i t i e s between yeast and Neurospora supersuppressors have not been 24 explored previously. In yeast i t has been observed that few of the mutants which part i c i p a t e i n a l l e l i c complementation are supersuppressible (Manney, 1964). Also, the mutants which are supersuppressible and which do complement • show a polarized pattern of intragenic complementation (Manney, 1964; fink, 1966). To summarize: i t has been shown that the supersuppressors of Neurospora clo s e l y resemble what are now known to be tBNA mediated nonsense suppressors i n yeast. It has also been shown that nonsense mutants should not be r e v e r t i b l e with HA and that they should complement in polar fashion i f they complement at a l l . Taken together these two approaches are predictive: the supersuppressors are expected to suppress polar or noncomplementing mutants with AT at the mutant s i t e . The ad-3B locus i s the only one i n which mutants have been extensively scored for both complementing pattern and s p e c i f i c r e v e r t i b i l i t y . Because of the very large number of well characterised mutants which are now available i t has been possible to test the above predictions by exclusive use of ad-3B a l l e l e s . 25 C o 9PP I t -9-II 11 1 1 * 1 I // A WW Cb 0 ) O ) Q J Qj ^ - O J ^ to 0 ) C Co Qj o 9 l\\ ^ • Q j Q J " O N J -fX 3' I CO O CO O 2 Q J CO CO Q J CO \ 3 CO CO c g n$s • i + Q J 3 of O J ~+-M A T E R I A L S AND METHODS 27 NEOROSPORA STRAINS Table I describes the adgnine-3B mutants used in t h i s study. Other stra i n s are described i n table I I . The use of the designations, * ssu-1? • and 'ssu-2?' presumably r e f l e c t s some uncertainty on the part cf previous workers regarding a l l e l e designations of stocks 1750 and 1719. Table 1 . Description of ad-3B mutants tested for s u p p r e s s i t i l i t y i s o l a t i o n # base pair complementation genotype ad-3B mutation of ad-3B at the c h a r a c t e r i s t i c s inducen by: mutants mutant s i t e 17 GC NP A/a, ad z3B NA - 22 i i i i II II - 30 t i II i t II - 34 i t II i i ii - 37 i t t i II i t - 47 i t i i II II - 51 u i i II I I - 58 t t i t II I t - 74 II i i II I t - 76 i t u II 11 - 79 II i i II il -106 i i i t i t I I -114 i t t i i t I I -128 i t i t n I I -130 i t I I i i I I -150 n II ii I I -164 II t i I I 68 AT NP A/a, ad-3D HA -129 II i i II I I 29 to 1 ro i rO tO i _v 1 t 1 *0 1 1 1 UJ 1 f 1 1 1 _> 1 1 t 1 t 1 CO 1 1 —1 VD CO tO tO _j •p •IT CO m Ul ON OJ XT 00 CT> O CT\ Ul O 00 vO CT. •Cr O 00 CD ro CT, I CO to I to I I I I OJ UJ CC CO CT Ul •c J w co 2 s rw a n >~3 S itJ '-3 H ru I* I ito io io if* \ so-lo !& I I |OJ : | S * \ 10) |0) I I ICO I CD 10) I to IO IO in-o o '73 n » C (!) ro 10.' ! » I I to ro 10) IS I ito IP | W I |cu lea IB) i IK Table 1 continued 5- 3- 12 GC NC A/a, ad-3B Hi - 1 7 7 - 45 -105 -166 -190 -212 II « II » 2-17- 14 GC NC A/a, acl-3B HA II ti it it it II ti H it it ii it II ti II it II ti it it II it II it ti II it it ti ti it H -228 " " ti . it 2-17-18 AT NC A/a, ad-3B Na - 26 11 " " " -126 -136 -142 -159 " 11 " " 12-21- 17 AT NC A, al-2, cot, 2^-2, ad-3B HA _ 22 " " " " - 28 " 11 " " _31 ti ii » »• _ 33 it it II » _ 35 »t •» '» " _ -7Q it II II i: — 76 II » » " — *J49 " " " " 31 i i i i i c c c 4= w CO NJ - » VD O CT\ CO CO CO \ I I I I co co co —» —» VO CO vO CO CO CT> vO O tT> HI tr o o rj rt c Q J Table 2. Description of additional s t r a i n s used i n t h i s study >train * - genotype a l l e l e linkage i s o l a t i o n genes #»s other than mt 987 A 74-OB23-1A 988 a 74-0B8-1a 1687 A; ssu-J, am WEN33r am 17 7B, 5R 1688 £> ss u-J, am WRN33, am17 7B, 5R 1750 a; s s u— 1 ? ¥319-44 7B 1689 A; ssu-2, am WBU35, am17 1B, 5R 1749 a; sju-2? ¥319-37 1B 1851 a; ssu-3, am WRU118, am17 1B, 5B 1852 *; ssu-4, am WRU18, am17 71, 5B 1751 a; ssu-5 ¥319-45 3 or 4 ? 1748 I ; ssu-6 ¥319-26 5B 1680 am am17 5B 1684 a; am am17 5B 1862 A; arom (p) Y306H54 2B 1854 a; arom jp) Y306H54 2B 1855 A; hist-3 ¥269M5, 43002 1B, B 1848 A; 15 3B 1850 trj£-2 10 6B 1846 tryp-2 TB31 6B 1845 I ; 41 6B 1024 I t ±Il£zl td140Ab 2B 33 CULTURE MEDIA The types of media used throughout th i s work have been described by Davis and deSerres (1970), however: 1) The advantages of modifying the Westergaard and Mitchell (1947) crossing medium are discussed below under the heading Isolation of Prccjejvy from Crosses . 2) Vegetative or sorbose media intended for n u t r i t i o n a l testing were made with washed agar to minimise background l e v e l s of possible supplements. One pound of Difco Bacto Agar was allowed to s e t t l e in 5 g a l . of water. The supernatant was removed by siphoning and the agar resuspended in fresh water. After 10 such washings the agar was recovered by pouring the s l u r r y into a fine meshed nylon bag which was pressed i n a wine press. The pressed agar was resuspended in 3 to 4 gal. of acetone and after 12 hours i t was again recovered in the nylon bag and spread on a clean surface to dry. F i n a l l y , a blender was used to give the dried agar a granular consistency. 34 CROSSING NEUROSPORA STRAINS Throughout t h i s study, cresses were made in 18 x 150 mm. culture tubes. A s t r i p of Whatman's #1 f i l t e r paper {approximately 20 x 120 mm.) was inserted into each tube which was then charged with 5 mis. of crossing medium. Crosses were i n i t i a t e d by the consecutive introduction of each parent as a drop or two of c o n i d i a l suspension. Subsequently the crosses were incubated at 25° C. Those intended for shot octad analysis were maintained i n the dark for 10-12 days, while those intended for random spore analysis were incubated for at least 14 days and were not protected from the l i g h t . 35 ISOLATION OF PROGENY FROM CROSSES Random spores and shot octads were isolated from crosses by the following techniques: I Random spore i s o l a t i o n : Spores discharged from the perithecia of a mature cross accumulate on the inner wall of the crossing tube. A loopful of s t e r i l e water was used to transfer the spores to a block cf H% agar. Tungsten needles were then used to i s o l a t e the spores into i n d i v i d u a l tubes of vegetative medium (eg. Threlkeld 1961, also Davis and de Serres 1970). Isolates were heat shocked for 40 minutes at 60° C. to induce germination. II Isolation of shot octads : The technique used d i f f e r s from that of Perkins who controls the amount of conidiation by using a morphological mutant, f l u f f y , in his crosses (Perkins 1966). Throughout t h i s work crossing medium contained 0.2% sucrose in contrast to the usual 2%; t h i s procedure greatly reduced conidiation but maintained high f e r t i l i t y , thereby making the use of f l u f f y unnecessary. The reduced conidiation and the use of f i l t e r paper permitted the removal of a l l perithecia from the cross tube. Perithecia, s t i l l mounted on the paper, were placed on s l i d e s , and held inverted over an agar c o l l e c t i o n block (see the description of adjustable platforms below). The l i g h t from a fluorescent desk lamp was then used to promote the discharge of ascospores. 36 As above, tungsten needles were used to i s o l a t e i n d i v i d u a l spores from groups cf eight. However, shot octad i s o l a t e s were incubated at 25° C for 10 days to obtain maximum germination after heat shocking. Two models of the platforms used for c o l l e c t i n g shot octads are described below: Model I (see figure 4) has been used for the c o l l e c t i o n of more than 2,000 octads., It consists of two tubing clamps (a) mounted on a p l a s t i c stand (b). The inverted s l i d e bearing the perithecia was placed on top of the clamps and a s l i d e bearing the agar c o l l e c t i o n block was placed across the two adjustable arms. As shown in the diagram, two such devices were mounted back to back on the same stand. Model II i s of more recent design. S l i d i n g p l a s t i c shelves (c) permit very close adjustment of the perithecia to the surface of the agar c o l l e c t i n g block. The shelves are held in contact with the p l a s t i c stand by means of an e l a s t i c band (e) and s i l i c o n e grease between the s l i d i n g surfaces (d) (Newcombe and G r i f f i t h s 1973). FIGURE 1 Diagram 1. A d j u s t a b l e p l a t f o r m s f o r the c o l l e c t i o n o f shot octads. ITT! L i i;iJilli l i f e " b Model 1. Side view. b Model 1. End view. 38 c L_ Model 2. Side view. 39 GENOTYPE DETERMINATION Random spores and shot octad i s o l a t e s were characterised by the following tests (as appropriate): 1 AJ-3B mutants^ accumulation of a c h a r a c t e r i s t i c purple pigment i n the mycelium and the medium allowed the v i s u a l i d e n t i f i c a t i o n of adenine auxotrophs (cf. Mailing 1966). Because of d i f f i c u l t y in scoring for adenine requirement in the presence of am , v e r i f i c a t i o n by n u t r i t i o n a l testing was done on slants of vegetative rather than plates of sorbose medium (cf. Case and Giles 1968). 2 Other n u t r i t i o n a l jutantsj. N u t r i t i o n a l c r i t e r i a (ie. the a b i l i t y or i n a b i l i t y to grow on appropriately supplemented sorbose media) were used to distinguish aromJjDp , hist-3 , _pan-2 , t r j p - J , tr_yj;-2 , and trvp-3 mutants from their wild type alternatives (Davis and de Serres 1970). Am mutants (which grow well on medium with 200 mg./l. alanine and 200 mg./l. monosodium glutamate), were distinguished by their poor growth on medium supplemented with 150 mg./I. of glycine. 1 Super suppressors,: In crosses between a suppressible mutant (m, ssu+) and a supersuppressing s t r a i n (m*, ssu), tetratype (TT) and ncnparental ditype (NPD) no a s c i contain one or two m, ssu spore pairs respectively. Such phenotypically wild type i s o l a t e s when mated to standard wild type strains (987 and 988) y i e l d recombinant progeny which display the o r i g i n a l mutant phenotype. Besides distinguishing between suppressed mutants and bona f i d e wild types, t h i s outcrossing procedure allows i d e n t i f i c a t i o n of a l l i s o l a t e s from NPD octads as ssu* or ssu. However, i n tetratype a s c i the d i s t i n c t i o n between m+, ssu+ and m+, ssu types could only be made by crossing such i s o l a t e s to a suppressible mutant. The result i n g octads would then be examined for evidence of suppression as described i n the experimental protocol. Octad i s o l a t e s were not characterised for supersuppressor a l l e l e alternatives where there was no evidence of suppression. H Other , Determinants: the a 1-2 a l l e l e produces an albino phenotype which i s e a s i l y distinguishable from the pink al t e r n a t i v e . Cot mutants grow normally at 25° C but at 37° C they form small dense colonies which are readi l y distinguishable from wild type cultures. 41 GROWTH RATE DETERMINATION A preliminary study of the vigor of suppressed and unsuppressed s t r a i n s was undertaken by measuring the rate of mycelial elongation in race tubes (Ryan et a l . . 1943; ci t e d by Davis and deSerres, 1970). Each s t e r i l e tube was charged with 25 ml. of vegetative medium and a st r a i n was subsequently inoculated at one end. The tube was incubated at room temperature and a daily record was made of the position of the advancing mycelial front. 42 EXPERIMENTAL PROTOCOL The ssu mutants were crossed to the aronj, am, h i s t and tryp mutants described in table 2. The supersuppressing character of the ssu mutants (already reported by Seale, Case, and Baratt 1969) was confirmed i n the course of th i s study by shot octad analysis of these crosses. However, the bulk of the experimental work involved an analysis of the s u p p r e s s i b i l i t y of the ad-3B mutants described in table 1. This analysis was accomplished by crossing available ad-3B strains to the ssu mutants. The genotype of such crosses f e l l into one of two categories: 1) A/a, ad-3B X A/a, am/+, ssu or 2) A, al-2, £an-2, cot, ad-3B X a, am/+, ssu where *A/a' or 'a!/*' indicate that either a l l e l e alternative was used depending upon the pa r t i c u l a r s t r a i n . Shot octads from these crosses were sorted and dealt with as follows: (1) Informative Octads were those which either d e f i n i t e l y demonstrated or f a i l e d to demonstrate the s u p p r e s s i b i l i t y of the ad-3B parent. Informative octads therefore contained: (a) 5 or more nonpuiple i s o l a t e s (b) 3 or 4 purple i s o l a t e s or (c) 2 nonidentical purple i s o l a t e s from crosses heterozygous for al^2 . 43 These i s o l a t e s were a l l tested for adenine requirement. Where 5 or more i s o l a t e s did not require adenine the ad-3B parent of the cross was tenta t i v e l y regarded as suppressible pending the i d e n t i f i c a t i o n of suppressed ad-3B types by the outcrossing procedure described in section 7. (2) S p i n fprmative Octads were those i n which: (a) the germination was i n s u f f i c i e n t for unambiguous analysis or (b) the octads were not s e l f consistent (see below). Except as described above for ad—JB, r a t i o s of segregating a l l e l e alternatives which were not sel f - c o n s i s t e n t (eg.5:3) were presumed to have resulted from the accidental i s o l a t i o n of spores from more than one ascus. Of the more than 2,000 octads is o l a t e d , 18 were not s e l f consistent and were discarded. The question of how many octads to i s o l a t e from each cross was approached i n the following manner. The i d e n t i f i c a t i o n of suppressible ad-3g mutants i s possible only i n NPD and TT octads since i t depends upon the attendant segregation of both the ad^3B and ssu a l l e l e s . Because ad-3B i s approximately 3 map units from the centromere on linkage group 1 (Badford 1972), the r e l a t i v e frequencies of PD, HPD and TT octads are e f f e c t i v e l y determined by the linkage 44 of the ssu genes to t h e i r centromeres. The probability of recovering only PD octads (and thereby f a i l i n g to i d e n t i f y a suppressible ad-3B mutant) was estimated for three t h e o r e t i c a l types of crosses described below: 1 Crosses involving ssu genes which segregate independantly from t h e i r centromeres were expected to produce PD octads at a frequency of 1/6. 2 Almost exclusive production of parental ditypes (PD*s) would occur i f an ssu gene were closely linked to the centromere of linkage group I. 3 Where an ssu gene i s linked to another centromere no more than 1/2 of the octads would be PD's. Ssu genes are only approximately located i n current linkage maps (the most recent information has been contributed by Seale 1972 ). However, the most unfavorable possible positions of ssu - J [, s s u - J ? , ssu-3, ssu-4 and ssu;6 would permit the detection of more than 1/2 of the suppressible ad^3B mutants by the i s o l a t i o n of even a single informative octad from each cross. In the case of ssu-2, ssu-2?, and ssu-5, the proportion of PD»s could not be determined a p r i o r i . OBSERVATIONS 46 THE SUPPRESSIBILITY OF AD-3B MUTANTS Tables 3 through 8 describe the s u p p r e s s i b i l i t y of six classes of ad~3B mutants as follows: I Crosses in which the ad-3B mutant was not suppressible are indicated by a followed in brackets by the number of informative octads i s o l a t e d from the cross; or by " r s " when the only available information was from random spore analysis. II Crosses in which the ad-3B parent was suppressible are indicated by a where suppression was v e r i f i e d by the •outcrossing procedure. Our experience with crosses of noncomplementing mutants having AT at the mutant s i t e has demonstrated the r e l i a b i l i t y of vi s u a l and n u t r i t i o n a l tests i n detecting suppression of ad73B mutants. Consequently, in the most recent crosses i t has not been necessary to v e r i f y suppression of the ad-3B parent by extensive backcrossing procedures. I t i s c r i t i c a l to the interpretation of the present experiments that each ad^3B stock was maintained i n s t r i c t homokaryotic condition. Ad-3B stocks were routinely tested for n u t r i t i o n a l requirements before being tested for su p ^ r s u p p r e s s i b i l i t y . The fact that tetratype (TT) asci were is o l a t e d from each suppressible s t r a i n i s additional evidence that even where progeny testing was not carried out, the r e s u l t s can not be due to adulteration of the ad;3B stocks. Crosses in which suppression has been inferred on the basis of vis u a l and n u t r i t i o n a l c r i t e r i a are indicated by a «*•". (This score has recently been v e r i f i e d in the course of futher investigations which are to be reported elsewhere). Blank spaces occur in the tables where crosses either have not been i n i t i a t e d or were too i n f e r t i l e to analyse. U8 SO£PfiJSSIBILITY OF ALLELES AT OTHER LOCI The s u p p r e s s i b i l i t y of aro|,a|, hiSi» a n ^ t r x p mutants has been reported e a r l i e r by Seale, Case and Barratt ( 1 9 6 9 ) . This work was repeated and confirmed to check stocks before they were used in the present programme. THE GROWTH RATE OF OCTAD ISOLATES The growth rates of octad i s o l a t e s are presented and discussed i n appendix 1. 49 Table 3 . Supp r e s s i b i l i t y cf ncnpclars with GC at the mutant s i t e . Supersuppressors ad^3B mutant 2-17 ssu- 1 16 87~ 1688 ssu; 1 ? ssu-2 17 50 1689 ssu;2? ssu;3 ssu-4 ssu-5 ssu-6 1749 ~ 1851~ "1852 175" 1748~ - 17 — ( 4) — ( 3) — ( 2) - 22 - I ") - ( »») - ( 1) - 30 - ( ") * |rs) - < 8) - ( 4) - ( «») - ( 2) - ( 6) " ( D - 3 4 * + - 37 - ( 3) - (rs) - ( *») - (rs) - ( 5) - ( D - ( 2) " ( 4) - 47 - (rs) - (rs) - ( 3) - ( 3) - ( D - 51 - ( 2) - ( 3) - t 2) - 58 - 1 4) - ( 4) - ( 1) - 74 - ( «») - (rs) - ( 2) - < 2) - ( 2) - ( 2) - < 3) - 76 -( 3) - (rs) - ( 3) - < 4) - ( «0 - ( 6) - 79 - ( *0 - ( «») - ( D -106 - ( »») - (rs) - ( 9) - < 2) - ( 4) - ( D - ( 2) -114 - ( <0 - (rs) - (10) - ( D - ( 5) - ( *») - ( 5) ~ ( D -128 - ( < » > - (rs) - ( 1) - ( *») - ( 5) - ( «) - ( D -130 *• ** * 4 -;150 - < 3) - ( *») -164 - ( 4) - ( «0 - ( «) 50 Table 4 Supp r e s s i b i l i t y cf ncnpclars with »T at the mutant s i t e . Supersuppressors ad-3B ssu-1 ssu-1 ? ssu-2 ssu-2? ssu-3 ssu-4 ssu-5 ssju—6 mutant 1687~ 1750 1689~ 1749 "l 8 51 1852~ ll5~\ 1748 1688 2-17 - 68 - ( 3) -(rs) -(10) -( 4) -( 4) -{ 4) - { 4) -( 4) -129 -( 6) - ( 2) -(12) - ( 5) -( 2) -( 4) -( 2) 12-21 - 1 - ( 3 ) - ( 1 ) - ( 2 ) - 58 - ( 5 ) - ( 1 ) - ( 2 ) - 62 -( 1) -( 1) -381 *• -( 1) - ( 4) -384 - ( 3) - ( 4) -385 - ( 5) - ( 5) -406 -( 4) -442 - ( 4) - ( 3) - ( 3) 51 Table 5 . Suppressibility cf pclars with GC at the mutant s i t e . Supersup pressors ad-3B ssu-J sgu-1? ssu-2 ssu-2? ssu-3 ssu-4 ssu-5 ssu-6 mutant "l687~ 1750 1689 1749 185~ 1852~ 175~1~ 1748 1688 2-17 - 87 -( 3) -{ }) - 98 - ( 3 ) - ( i s ) -13) - ( 3 ) - ( 3 ) - ( 2 ) - ( 2 ) -158 -( 3) -( 3) - ( 2) -163 - ( 2) -<ts) -( 5) -( 1) -( 3) -< 3) - { 3) -( 1) -200 - ( 3) -( 1) - ( 2) -264 - ( 6 ) -( 1) -387 -( 3) -( 3) -( 3) 52 Table 6 . Sup p r e s s i b i l i t y of polars with AT at the mutant s i t e . Supersuppressors ad;3B s s u - J sgu-1? ssu-2 ssu-2? ssu-3 ssu;4 ssu;5 ssu;6 mutant 1687 "1750 1689 1749 1851 1852~ 1751~ 1748~ 1688 2 - 1 7 - 16 - ( 2 ) -(rs) - ( 2 ) - ( 4 ) - ( 1 ) -{ 2) -( 1) - 46 -(rs) -(rs) - ( 4 ) - ( 2 ) - ( 2 ) - ( 2 ) - ( 2 ) -131 -( 2) -(rs) - ( 3) -( 4) - ( 1 ) -( 1) -( 1) 12-21 - 9 - ( 4) -{ 4) -( 4) -( 4) - 61 - ( 2) -157 * + -159 - ( 2 ) - ( 3 ) - ( 3) -388 -( 2) -{ 3) -( 2) 53 T a b l e 7 . S u p p r e s s i b i l i t y o f n c n c c m p l e m e n t e r s w i t h GC a t t h e m u t a n t s i t e . S u p e r s u p p r e s s o r s a g - 3 B EEU-J SE u - 1 ? s s u - 2 s s u - 2 ? s s u - 3 s s u - 4 s s u - 5 s s u - 6 m u t a n t 16 87 17 50 l 6 8 9 ~ 1 7 4 9 l 8 5 1 ~ 1 8 5 2 ~ " l 7 5 1 ~ 1 7 4 8 1 6 8 8 5 - 3 - 12 - | 4 ) - 1 7 7 - ( 3) - ( 2 ) 2 - 1 7 - 14 - ( 2) - ( D - 45 - ( 3) - ( 3) - ( 4) - ( 4) - ( 2 ) - 1 0 5 - ( 4) - ( r s ) - ( 2) " ( 2) - ( 1) - ( 3 ) - 1 6 6 - ( 2) -< 1) - 1 9 0 - ( D - ( r s ) - ( D - 2 1 2 - ( r s ) * + *• - 2 2 8 - ( r s ) -< D - ( r s ) - ( r s ) 54 Table 8 . Sup p r e s s i b i l i t y of nonccmplementers with AT at the mutant s i t e . Supersuppressors ad-jB ss u - J ssu-j? ssu-2 ssu-2? ssu-3 ssu-4 ssu-5 ssu-6 mutant 1687 1750 ~ 1689 1749 ~ 1"851 1852 ~175l "l748 1688 2-17 12-21 - 18 - (rs) -( 1) - ( 2) - ( «») - < 2) - 26 -( 3) - ( D - ( 2) - ( D -126 * + - ( 2) + - I 3) + -i 2) -136 - (rs) - ( 2) -142 - (rs) - ( 2) - ( 3) - ( 7) -( 2) -129 - (rs) - (rs) - ( 3) - ( - ( D - 17 - ( 3) - ( 5) -( 2) - ( D - 22 + * • - ( D + - 28 -( D • • - ( 2) - ( 3) - 31 -( 1) - 33 -( 3) - ( D - ( D - ( D - 36 - ( 2) - ( 2) - ( D - ( 2) - ( D - 70 -< 2) + - ( 3) - ( 2) - t 2) - 76 + + - < 2) - 1 2) -149 -( D • - ( 3) - ( 2) - ( 3) -186 -( 2) -< 3) - ( «*) - ( 2) -( 2) -( 1) I Table 8 (cent) . Su p p r e s s i b i l i t y of noncomplementers with AT at the mutant s i t e . Supersuppressors ad;3B ssu;1 ss u;J? ssu-2 ssu; 2 J mutant 1687~ 1750 1689 1749 1688 12-21 -190 -379 -386 -393 -398 -418 -428 -436 -440 " ( 2 ) - ( 3 ) -( 2) + - ( D -< D - ( D -i 2) - ( D - ( 2) -( 3) -( 2) - ( D -( 3) -( 2) - ( 2) -( 3) SSU;3 SSU;4 1851 1852~ 3) - ( 3) - ( D -( 2) - ( 1) - ( 3) S SU;5 SSU; 1751 174 8 "( 3) •» - < 2) -( 3) - ( D " ( 3) -( 1) DISCUSSION 57 ANALYSIS OF DATA The data presented in tables 3 through 8 of the observations are summarized i n table 9. In th i s table the number of suppressible ad-3B mutants i s expressed as a f r a c t i o n of the t o t a l number analysed for each of the 6 mutant classes. Of the 13 suppressible ad;3B mutants, 10 had AT at the mutant s i t e and only 3 had a nonpolarized pattern of complementation. Very few ad^3B mutants with a polarized pattern of complementation and with GC at the mutant s i t e were available for analysis. Of the 7 which were analysed, none proved to be suppressible. Table 9 shows that, as expected, the polar complementing and noncomplementing classes with AT at the mutant s i t e have the highest incidence of suppressible mutants , 9/33 (2755) compared with 4/43 (9%) for a l l ether classes combined. The difference i s s i g n i f i c a n t at the 5% l e v e l (chi-sguared='».24, df-1) and supports the assertion that the supersuppressors are nonsense suppressors, and that most of the nonsense mutants are in the polar complementing or noncomplementing populations with AT at the mutant s i t e . The discovery of suppressible mutants with GC at the mutant s i t e and suppressible mutants with a nonpolarized pattern of complementation i s sur p r i s i n g . These findings are discussed below. 58 Table 9 . The number of suppressible mutants as a f r a c t i o n of the t o t a l number of mutants in each cla s s . MUTANT COMPLEMENTATION PATTEBN Base Pair at the non- polarized non-Mutant S i t e polarized complementing GC AT 2/17 1/10 0/7 1/8 1/9 8/25 59 NOHSESSE-MISSENSE SUPPRESSION Ad-3B mutants with a nonpolarized pattern of complementation are most l i k e l y missense rather than nonsense mutants (de Serres et a l 1967). Also, i f as described e a r l i e r , the procedure used to select the ad-3E mutants r e a l l y i s s p e c i f i c enough to exclude transversion i mutants from the population tested, then i t fellows rigorously that those mutants with GC at the mutant s i t e must be missense mutants since a nonsense codon cannot aris e de novo by an AT to GC t r a n s i t i o n (see figure 5). If the ssu genes are nonsense suppressors, and i f the ad-3B mutants are a l l t r a n s i t i o n s , i t should not be possible to obtain suppressible mutants with GC at the mutant s i t e . Yet 3 of these were demonstrated. The suppression of presumed missense mutants i s not necessarily inconsistent with either the work suggesting that Neurospora supersuppressors are tRNA mediated supersuppressors, or the work suggesting that mutants can be r e l i a b l y c l a s s i f i e d as nonsense or missense on the basis of t h e i r complementation and reversion c h a r a c t e r i s t i c s . In f a c t , there are t h e o r e t i c a l grounds for predicting the suppression of p a r t i c u l a r missense mutants by seme nonsense suppressors. U, for example, at the wobble position of a tRNA anticodon can pair to adenine or guanine (Crick 1966). Thus any naturally occuring tRNA with anticodon ACU would be expected to recognise both nonsense (UGA) and missense (UGG) codons. 60 Figure 5, Possible t r a n s i t i o n s from nonsense to sense codons io RNA nonsense codons OA A UAG UGA codons related by AT to GC tra n s i t i o n s CAA (Glu N) OGA (nonsense) DAG (nonsense) CAG (GluN) OGG <Trp) CGA (Arg) OGG (Trp) codons related by GC to AT tran s i t i o n s none UAA (nonsense) DAA It follows rigorously from the above relationships that a nonsense codon cannot be generated by the AT to xGC t r a n s i t i o n of a single base pair of any sense codon . 61 figure 6. Codons •recognised* by anticodons of possible ncnsense-missense suppressing tRNA species anticodons codons 1. with no modified base in the anticodon Acti OGA (nonsense) UGG (Trp) 2. With inosjne i n the f i r s t or wobble pos i t i o n UAA (nonsense) . AUI OAU (Tyr) UAC (Tyr) OGA (nonsense) ACI 0G0 (Cys) UGC (Cys) 3- With pseudouridine i n the wobble position ^ DAA (nonsense) AUP DAG (nonsense) DAD (Tyr) DGA (nonsense) ACP UGG (Trp) DGD (Cys) 62 Inosine and pseudouridine are two of the minor tRNA nucleotides which can occur at the 5* end or the wobble position of the anticodon. In t h i s position inosine pairs with A, U and C while pseudouridine pairs with A, U and G allowing certain tRNA species to recognise both nonsense and missense codons. Figure 6 l i s t s a l l of the nonsense suppressing tRNA antidocons containing inosine (I) or pseudouridine (P) i n the wobble position and the s p e c i f i c nonsense and missense codons with which pairing can take place. Inosine and pseudouridine are the only minor components which are l i k e l y tc be found in Neurospora nonsense-missense suppressing tFNA species. Of the others which are described, uridine-5-oxyacetic acid and the unknown nucleotide "Qw pair with U, A or G and 0 or C respectively in the t h i r d position of the codon seguence. However, both appear to be unigue to E . c o l i tRNA (although a recent report indicates that •Q» i s c h a r a c t e r i s t i c of some Drosophila tRNA's; White et a l . , 1973). The 2-thiouridine derivatives: 2-thiouridine-5-acetic acid methylester, 5-methylaminomethyl-2-thiouridine and 5 methyl-2-thiouridine appear to prevent ambiguity or miscoding when they appear i n the wobble position. The function of minor nucleotides adjacent tc the anticodon i s unclear; however, they too may increase the precision of codon-anticodon pairing. A comprehensive review of minor components i n tRNA has recently been published by Nishimura (1972). 63 The suppression of a mutant with a nonpolarized pattern of complementation has been observed in yeast by Simarov, Mironova and Inge-Vechtomov who explain their results on the basis of the wobble c h a r a c t e r i s t i c s of inosine, pseudouridine and uridine-5-oxyacetic acid (Simarov et a l . 1971) . A similar explanation could account for the apparent suppression of missense mutants by nonsense suppressors which was observed i n t h i s study. However, i t i s necessary to emphasize that i t has not been established that the supersuppressor genes are in any way tBNA mediated; that they suppress nonsense, missense, or both types of mutants; nor that the nature of ad^3B lesions can be r e l i a b l y derived from consideration of s p e c i f i c r e v e r t a b i l i t y and complementation testing. But such an explanation i s predictive, and i t i s considered further because i t appears to be capable of reconciling a l l of the previous work i n t h i s area. Missense codons recognised by nonsense suppressing tBNA species may ari s e from s p e c i f i c "regular" codons by either GC to AT or AT to GC t r a n s i t i o n (see figure 7). For example, a suppressible missense mutant with GC at the mutant s i t e would only be expected where the t r a n s i t i o n a l event had led to the replacement of a tyrosine by a cysteine residue. Such mutants might occur infreguently i n view of the more numerous acceptable GC to AT t r a n s i t i o n s (shewn in the 61 rig h t column of figure 7). In fact, such an excess i s observed in Table 9, although the frequencies do not d i f f e r to any s i g n i f i c a n t extent. The discovery of what may be nonsense-missense suppression i n Neurospora and in yeast raises the p o s s i b i l i t y that some supersuppressors are genes regulating the post-transcriptional modification of tRNA nucleotides. A gene of t h i s sort has recently been described i n Drosophila (White et a l . , 1973). 65 THE PATTERN OF NONSENSE SUPPRESSION Each of the supersuppressors appears to suppress a unique group of ad-3B mutants. This finding can be explained on the basis of tRNA mediated suppression. The success of a tRNA species in suppressing nonsense codons would be expected to depend pa r t l y upon codon-anticodon recognition ( v a r i a b i l i t y might be expected from other sources such as a competing release factor of the sort found i n I i c o l i ). Because ssu genes could have arisen by a multistep process i t must be assumed that an anticodon recognising nonsense codons might occur i n any of the common tRNA species. It therefore seems unlikely that many of t n e ad-3B mutants ( i f they are being suppressed by a tRNA species) would produce an e n t i r e l y normal protein product. As indicated by Seale (1972), successful suppression of a nonsense mutant probably depends upon the location of the termination t r i p l e t in the mRNA sequence and the stringency of permissible amino acid substitutions at the corresponding residue. Figure 7. Codons related tc s p e c i f i c suppressible missense codons by AT to GC and GC to AT tr a n s i t i o n s codons recognised by possible nonsense missense suppressing tBNA species codons related by GC to AT t r a n s i t i o n codons related by AT to GC tr a n s i t i o n DAD (Tyr) UAC (Tyr) UGU (Cys) UGC (Cys) UGG (Trp) none D AU (Tyr) UAU (Tyr) UAC (Tyr) UGU (Cys) CAU (His) UGU (Cys) UAC (Tyr) CAC (His) UGC (Cys) CGU (Arg) UGC (Cys) CGC (Arg) UAG (nonsense) CGG (Arg) UGA (nonsense) 67 APPENDIX 68 SOMATIC VIGOR OF SUPERSUPPRESSEB JEUROSPOBA STBAIMS The vigor of Neurospora str a i n s can be assessed by measuring the daily mycelial elongation of str a i n s grown in the race tube apparatus described e a r l i e r . The growth of parents and progeny from eight of the ad-3E X ssu crosses has been investigated and i s displayed i n figures 1 to 8 of this appendix. In a l l cases growth determinations were made on media lacking adenine; under these conditions none cf the ad-3B parents showed any measurable growth and they have not been recorded in the figures. The ssu parent i s plotted with s o l i d rather than open c i r c l e s . Non-adenine reguiring progeny are conspicuously divided into f a s t and slow growers; the former being + , + or +, ssu types and the l a t t e r being exclusively ad-3B , ssu types (see table 1 of t h i s appendix). The r e l a t i v e l y slow growth of suppressed adenine mutants may r e f l e c t the r e s t r i c t i o n s on ef f e c t i v e amino acid substitution at the termination s i t e , or the a v a i l a b i l i t y of suppressor tRNA for competition-with a termination factor. The f a i l u r e to observe s i g n i f i c a n t differences in the growth rates of +, ssu and +,+ types implies that the supersuppressor a l l e l e s under study dc i 69 not i n t e r f e r e with the normal punctuation of t r a n s l a t i o n . Investigation of these phenomena and the phenomenon of stop-start growth in some of the suppressed adenine s t r a i n s i s being continued. i 70 Table 1 of the Appendix P a r t i a l Genotypes of Progeny i n F i g u r e s 1-8 of the Appendix Fig u r e 1 F i g u r e 2 F i g u r e 3 F i g u r e 4 F i g u r e 5 F i g u r e 6 F i g u r e 7 F i g u r e 8 12E2- 3 12E2- 4 9X1- 7 10C3- 1 10C3- 2 10H3- 1 AD-3B. SSU 8Z2- 3 8Z2- 4 8Z2- 5 8Z2- 7 9E2- 2 9E2- 3 9E2- 4 9E2- 5 12E2- 5 i OB ssu 9X1- 3 9X1- 4 10C3- 5 10G3- 6 10H3- 2 10H3- 3 10L2- 2 1012- 3 10S1- 1 10N1- 2 10H1- 3 10N1- 4 8Z2- 1 8Z2- 2 8Z2- 6 8Z2- 8 9E2- 1 9E2- 6 9E2- 7 12E2- 1 1212- 2 12E2- 6 1212- 7 9X1- 1 9X1- 2 9X1- 5 9X1- 6 10C3- 3 10C3- 4 10C3- 7 10C3- 8 10H3- 4 10H3- 5 10H3- 6 10H3- 7 10L2- 1 1012- 4 10L2- 5 10N1- 5 10H1- 6 10N1- 7 10N1- 8 -L L _ L L _ 50 100 150 200 TIME (hours) FIGURE 2. GROWTH OF PARENTS AND PROGENY OF CROSS 1750 x 2-17-28, ASCUS 9E1. 100 150 TIME (hours.) FIGURE 3. GROWTH OF PARENTS AND PROGENY OF CROSS 1749 x 12-21-440, ASCUS 12E2. 100 TIME (hours ) o—i— 200 50 S 30 O CD ^ 20 o Uj FIGURE 4 . G R O W T H OF PARENTS A N D P R O G E N Y OF CROSS 1751 x 2-17-28 , 9X7. ,5 74 2 0 0 TIME (hours) FIGURE 5. GROWTH OF PARENTS AND PROGENY OF CROSS 1749 x 2-17-76, ASCUS 10C3. TIME (hours) FIGURE 6. GROWTH OF PARENTS AND TIME (hours) FIGURE 7. GROWTH OF PARENTS AND TIME (hours ) 79 LITERATURE CITED ALTMAN, S., S. BRENNER and J. D. SMITH, (1971). I d e n t i f i c a t i o n of an ochre-suppressing anticodon. J. Moi. E i o l . 56 : 195-197. BASILIO, C , A.J. WAHBA, P.LENGYEL, J.P.SPEYER, and S. OCHOA, (1962). Synthetic polynucleotides and the amino acid code, V. P.N.A.S. D.S. 48 : 613-616. BERNSTEIN, H., (1961). Imidazole Compounds Accumulated by purine mutants of Neurosppra crassa. J . Gen. Microbiol. 25 : 41-46. BUCHANAN, J.M., (1960).„ The enzymatic synthesis of the purine nucleotides. In: Harvey Lectures (1958-1959) 54 : 104-130. BUDOWSKY, B.I. , E.D. SVERDLOV and G.S. MONASTYBSKAYA, (1971). Mechanism of the mutagenic action of Hydroxylamine IV. Biochim. Biophys. Acta 246 : 320-328. BUDOWSKY, E.I., E.D. SVERDLOV and T.N. SPASOKUKOTSKAYA, (1972). Mechanism of mutagenic action of Hydroxylamine VII. Biochim. Biophys. Acta 287 : 195-210. CASE, M.E. and N.H.GILES, (1968). Evidence for nonsense mutations i n the arom gene cluster of Neurosppra crassa . Genetics 60 : 49-58. CHALMERS, J.H. and T.W. SEALE, (1971)., Supersuppressible mutants i n Neurospora: Mutants at the trjjg^J and t r x E_2 l o c i a f f e c t i n g the structure of the multienzyme complex in the tryptophan pathway. Genetics 67: 353-363. 80 CRIBBS, R.M. (1970). Suppression of st r u c t u r a l gene mutations in bacteria. V i r g i n i a Journal of Science 2_1 : 6-13., CRICK, F.H.C., (1966). Codon-anticodon pairing: the wobble hypothesis. J. Hoi. B i o l . _19: 548. DAVIS, R.H. And F.J. deSERRES, (1970). Genetic and microbiological research techniques for Neurospora crass a . pp. 79-143. In: Methods in Enzjfmclojgjr Vol. XVII A. Edited by H. TABOR and C.' TABOR. Academic Press, N. ¥. deSERRES, F.J. (1964). Genetic analysis of the structure of the ad-3 region of Neurospora crassa by means of irreparable recessive l e t h a l mutations. Genetics 50 : 21-30. deSERRESr F. J,, (1967). Genetic analysis of the extent and type of functional i n a c t i v a t i o n i n irreparable recessive l e t h a l mutations in the ad-3 region of Neurospora crassa. Genetics 58 : 69-77. deSERRES, F.J., H.E. BROCKMAN, M.E. BARNETT and H.G. KOLMARK, (1967). A l l e l i c complementation among nitrous acid-induced ad-3B mutants of Neurospora crassa. Mutation Res. 4: 415-424. deSERRES, F.J. (1969). Comparison of the complementation and genetic maps of closely linked n o n a l l e l i c markers on linkage group 1 of Neurospora crassa . Mutation Res. 7 : 43-50. 81 DeSERRES, F.J., and H.V. MALLING, (1969). I d e n t i f i c a t i o n of the genetic a l t e r a t i o n s i n s p e c i f i c locus mutants at the molecular l e v e l . Japan. J. Genetics 44, Suppl. 1: 106-113. FBEESE, E., E.B. FBEESE, and E. BAOTZ, (1961). Chemical and Mutagenic S p e c i f i c i t y of Hydroxylamine. Proc. Natl. Acad.. S ci O.S. 47 : 845-855. FBEESE, E.B., (1963). Molecular Mechanism of Mutations. In: Molecular Genetics pt. 1, p. 207-269. Edited by H. TAYLOR. Academic Press, 8.Y. FINCHAM, J.B.S. (1970). Fungal Genetics. Ann. Bev. Genet., 4: 337-372. FINK, G.R. (1966). A clus t e r of genes c o n t r o l l i n g three enzymes in h i s t i d i n e biosynthesis i n Saccharofyces £®£e^isiae A Genetics 53 : 445-449. FISHER, C.R., (1967). Determination of the enzymatic functions controlled by the adzJA a n < j ad-3B l o c i in Neurospora crassa . Genetics 56 : 560. FISHER, C.B. (1969a). Phosphoribosyl-aminoimida2ole-succinocarboxamide synthetase from Neurospora crassa . Biochim. Biophys. Acta, J78 : 380-388. (1969b). Enzymology of the pigmented adenine-reguiring mutants of Sacharomycgs and Schizosaccharcm^ces Biochem. Biophys. Res. Commun. 34 : 306-310. FOWLER, A.V., and I. ZABIN, (1966). Colinearity of B-galactosidase with i t s gene by immunological detection of incomplete polypeptide chains. Science 54: 1027-1029. 82 FOX, C.F., W.S. ROBINSON, R. BASEL R. BASELKORN and S.B.WEISS, (1963). Enzymatic Synthesis of Ribonucleic Acid. J. B i o l . Chem. 239 : 186-195. GAJEWSKI, W. And J . LITWINSKA, (1968). Methionine l o c i and their suppressors in Aspergillus nidulans .. Molec. Gen. Genetics J02 : 210-220. GILMORE^ R. (1967). Super-suppresors in Saccharomyces cerevisiae. Genetics 56: 641-658. GILMORE, R.A., R.A. STEWARD, F. SHERMAN, (1968). Amino acid replacements resulting from super-supression of a nonsense mutant of yeast. Biochim. Biophys. Acta J6_1 : 270. GILMORE, R.A., J.W. STEWART, and F. SHERMAN, (1971). Amino acid replacements r e s u l t i n g from Super-suppression of nonsense mutants of iso-I-cytochrome c from yeast. J.Mol. B i o l . 6J : 157-173. GORINI, L. And J.R. BECKWITH, (1966). Suppression. Ann. Rev. Microb. 20 : 401-422. GORINI, L. (1970). Informational Suppression. Ann. Rev. Genet. Mi 107-134. GRIFFITHS, A.J.F. (1970). Topography of the ad-3 region of Neurgspora crassa . Can. J. Genet. Cytol. J2 : 420-424. HAWTHORNE, D.C, and R.K. MORTIMER (1963). Super-suppressors in yeast. Genetics 48: 617-620. 83 KRIEG, D.R., (1963a). S p e c i f i c i t y of chemical mutagenesis. Proc. Hucl. Acid Res. 2 : 125-168. , (1963b). Ethyl methanesulfonate-induced reversion of bacteriophage T4rII mutants. Genetics 48 : 561-580. MANNEY, T.R. (1964). Action of super-supressors in yeast i n re l a t i o n to a l l e l i c mapping and complimentation. Genetics 50: 109-121. MANNEY, T.R. (1968). Evidence for chain termination by supersuppressible mutants i n yeast. Genetics 60 : 719-733. MALLING, H.V., (1966). Hydroxylamine as a mutagenic agent for Neurgspgra crassa . Mutation Res. 3: 470-476. MALLING, H.V., and P.J. deSERRES, (1967). Relation between complementation patterns and genetic a l t e r a t i o n s in nitrous acid-induced ad-3B mutants of Neurospora crassa. Mutation Res. 4: 425-440. MALLING, H.V., and F.J. deSERRES, (1968a). Correlation between base-pair t r a n s i t i o n and complementation pattern i n nitrous acid-induced ad-3B mutants of Neurospora crassa. Mutation Res. 5: 359-371. , (1968b). I d e n t i f i c a t i o n of genetic a l t e r a t i o n s induced by ethyl methane sulfonate in Neurospora crassa. Mutation Res. 6: 181-193. MALLING, H.V., (1971). Hydroxylamine-induced purple mutants (ad-3) i n Neurospora crassa. II I d e n t i f i c a t i o n of genetic a l t e r a t i o n at the molecular l e v e l . Hereditas 68: 219-234. 84 MALLING, H.V. Ana F.J. deSERRES (1971). Hydroxylamine-induced purple adenine (ad-3) mutants i n Neurospora crassa . Mutation Res., J.2 : 35-46. MOYED, H.S., and B. MAGASANIK, (1957). Enzymes esse n t i a l for the biosynthesis of nucleic acid guanine; xanthosine 5'-phosphate aminase of Agrobacter aerogenes^ J. E i o l . Chem. 226 : 351-363. NEWCOMBE, K.D., and A.J.F. GRIFFITHS, (1973). Adjustable platforms for c o l l e c t i n g shot a s c i . Neurospora Newsletter 20. (In press). / NEWMEYER, D. (1970). A suppressor of the heterokaryon-incompatability associated with mating type in Neurospora crassa . Can. J. Genet. Cytol. J2 : 914-926. NICHOLS, J.L., (1970). Nucleotide sequence from the polypeptide chain termination region of the coat protein c i s t r o n i n bacteriophage R 17. Nature 225: 147. NISHIMURA, S., (1972). Minor components in transfer BNA: their characterization, l o c a t i o n , and function, pp. 49-85. In: Progress in nucleic acid research and molecular biology Vol. 12. Edited by J.N. DAVIDSON and W.E. CORN. Academic Press, N.Y. OSBOBN M., S. PERSON, S., PHILLIPS, F. FONK, (1967). A determination of mutagen s p e c i f i c i t y i n bacteria using nonsense mutants of Bacteriophage T4. J. Mcl. E i o l . 26 : 437-447. i 85 PERKINS, D.D. (1966). Details for c o l l e c t i o n of as c i as unordered groups of eight projected ascospores. Neurospora Newsletter 9: 11. PHILLIPS, J.H., D.M. BROWN, R. ADMAN, and L. GROSSMAN, (1965). The ef f e c t s of Hydroxylamine on Polynucleotide Templates for RNA Polymerase. J. Moi. B i o l . .12 : 816-828. PHILLIPS, J.H. , D.M.BROWN and L. GROSSMAN, (1966). The ef f i c i e n c y of induction of mutations by Hydroxylamine. J. Moi. B i o l . 18 : 405-419. RADFORD, A., (1972). Revised linkage maps for N., Crassa. Neurospora Newsletter _19: 25. REISSIG, J.L. (1963a). Induction of forward mutations in the £yr-3 region of Neurospora. J. Gen. Microbiol. 30 : 317-325. , (1963b). Spectrum of forward mutants in the £2Ezl region of Neurospora. J. Gen. Microbiol. 30 : 327-337. RIDDLE, D. L. And J. CARBON, (1973). Frameshift Suppression: nucleotide addition i n the anticodon of a glycine transfer RNA. Nature New B i o l . 242 : 230-234. SARABHAI, A.S., A.D. STRETTON, S. BRENNER and A. BOLLE, (1964). C o - l i n e a r i t y of the gene with the polypeptide chain. Nature 201: 13-17. 86 SEALE, T.W., (1967). Beversion of the am locus in Neurospora: evidence for nonsense suppressors. Genetics 58: 85-99. , (1972). , Supersuppressors i n Neurospora crassa I. Induction, genetic l o c a l i s a t i o n and relationship to a missense suppressor. Genetics 70: 385-396. SEALE, T.W., M. CASE and B.W. BABBATT, (1969). Supersuppressors in Neurospora c r a s s a . Neurospora Newsletter .15: 5. SEALE, T. M. (1970). Nonallelic super suppressors in Neurospora. (Abstr.) Genetics 64 : s57. SEALE, T.W., (1971). Super suppressors in Neurospora crassa I. Induction, genetic l o c a l i z a t i o n and relationship to a missense suppressor. Genetics 70 : 385-396. SHERMAN, F., J.W. STEWART, J.H. PARKER, E. INABER, N.A. SHIPMAN, G.J. PUTTERMAN, R.L. GARDISKY, E. HARGOLIASH, (1968). The mutational a l t e r a t i o n of the primary structure of yeast iso-I-cytochrome c. J. B i o l . Chem. 243 : 5446. SHERMAN, F., J.W. STEWART, J.H. PARKER, G.J. PUTTERMAN, B.B.L. AGRAWAL and B. MARGOLIASH, (1970). The relationship of gene structure and protein structure of iso-I-cytochrome c from yeast. Symp. Soc. Exp. B i o l . 24 : 85^107. SCHUSTER, H., (1961). The reaction of Tobacco Mosaic Virus Ribonucleic Acid with Hydroxylamine. J. Mol. B i o l . 3 : 447-457. 87 SIMAROV, B.V., L.N. MIRONOVA, and S.G. INGE-VECHTOHOV, (1-971). Nonsense-raissense suppression in yeast. Molec. Gen. Genetics 113: 302-307. SINGER, B. And H. FRAENKEL-CONRAT (1970). Messenger and Template A c t i v i t i e s of Chemically Modified Polynucleotides. Biochemistry 9 : 3694-3701. SMITH, J.D. (1972). The genetics of transfer tRNA. Ann. Rev. Genet. 6 : 235-256. STADLER, D.R. (1967). Suppressors of amino acid uptake mutants of Neurospora. Genetics 57 : 935-942. TESSMAN, I., R.K. PODDAR, S. KOMAR, (1964). I d e n t i f i c a t i o n of the altered bases in mutated single stranded DNA. J. Moi. B i o l . 9 : 352-363. TESSMAN, I., H. ISHIWA, S. KUMAR, (1965); Mutagenic e f f e c t s of Hydroxalamine i n vivo. Science .148 : 507-508. THRELKELD, S.F.H. . (1961). Seme studies of genetic recombination i n Neurospora. Ph.D. Dissertation, Cambridge. WESTERGAARD, M. and H.K. MITCHELL, (1947). Neurospora V. A synthetic medium favouring sexual reproduction. Am. J. Botany 34: 573-577. WHITE, B.N., G.M. TENER, J. HOLDEN and D.T.SOZUKI, (1973). A c t i v i t y of a transfer BNA modifying enzyme during the development of Drosophila and i t s relationship to the s u j s l locus. J. Moi. B i o l . 74: 635-651. 88 WILSON, R.G. And M.J. CALCUTS, (1966). The effects of hydroxylamine on the template properties of P o l y c y t i d y l i e acid. J. B i o l . Chem. 24J[ : 1725-1731. YANOFSKY, C. And P. ST. LAWRENCE, (1960). Gene Action. Ann. Rev. Microb. 14 : 311-340. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0101162/manifest

Comment

Related Items