Grindle, M.

Woodward, D. O.

Fungal Genetics Reports

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M. Grindle

D. O. Woodward

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Variations among extrachromosomal mutants of N. crassa

Kansas State University

Fungal Genetics Reports Volume 12 Article 11 Variations among extrachromosomal mutants of N. crassa M. Grindle D. O. Woodward Follow this and additional works at: https://newprairiepress.org/fgr This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Recommended Citation Grindle, M., and D.O. Woodward (1967) "Variations among extrachromosomal mutants of N. crassa," Fungal Genetics Reports: Vol. 12, Article 11. https://doi.org/10.4148/1941-4765.1956 This Research Note is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact cads@k-state.edu. Variations among extrachromosomal mutants of N. crassa Abstract Variations among extrachromosomal mutants This research note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol12/iss1/11 Weston, N. J. and  A. G. De&k.  Amino acid Woodwardandh4mkres(l966  Pmt. N&l.  Acod. Sci.,U.S.  55:872)trompor+  in & (ml)  mutants of Neumsporo  cmuo. have shown on  amino acid substitution to occur in the mitochondrial  strut.- turd  protein (MSP) in certain gckr  mutants. It is clear that enzymes at-tacheb  to membranes containing an altered MSP may show  decreased affin-ity for wbrtmter, 0s  was  demonstrated in the case  of malate  dehydmgenase  (Mu&es  and Wwxhvwd  1966 Pmt. Notl.  Acod. Sci.U. S. 55: 1217). Furthermore, not  only ore membranes of mitochondrirr  altered in &mutants, but the mme  abnormal  stwcturolprotein is found in other membranes of the cell os  well (Woodward,  personal communication).\Yp have attempted to test the hypothesis that (I  permeate  system which is part  of or attached to a  membrane rmy  have an alter-ed activity when assacioted  with such an  amilx)  acid substitutea  structuml  protein. Table I comparer conidiol  phenylalamne  trans-port in two  wild type stmins  and ssverol  mi mutontr. Incubations were carried out  in the absence  of a carbon  source,  employingtechniques similar to those previously deabed  (De&k  and DeBusk  I%5 Biochim.  Biophyr. Act. 104: 139). Tronrport  by my-celiol pads  of wild type and mi-I  are also  compared, since mi-1 bils  to conidiote.Deriments  are for  more  difficult  do with precision.) The po2(Although rometimer  revealing, mycelial ex-strains failed to show a decreased transport  rate when comparedwith wild type stminr.  Surprisingly, in one  instance (mi-4 t ere  war a marked increme  in both the rate of tramper+  and capacityof conidia for phenylalanine.  However, segre(pnts  of=strain  show nomx~i  transport rates. The studier with phenylalanine  re-ported here and additional studier with other omim  acids indicate that the &phenotype has  little effect on  amino rrcid  tmns-port.Table  1. Phenylalanine  transport in fi strains.Time (min) 15 3 0 4 5 6 0 7 5Table  2. Phenylalanine  transport in wild type and  2 strains  in thep r e s e n c e  o f  m e t a b o l i c  u n c o u p l i n g  a g e n t s .Time (min) 15 3 0 4 5 6 0 7 5Strain74ASY7Am i - 2XTmi-4mmi-I*-280 473 513 650 680253 460 578 615 6 %342 617 758 833 092307 540 583 6 2 1 608455 978 1186 1 2 2 7 1 2 4 21 2 3 206 2 4 1 505 4608 7 1 9 8 3 5 1 383 454Valuer represent total phenylolanine  uptake  inStrain74A (control) 265 335 490 530 56074~ (N.N~) 80 140 220 215 1 6 0. ~,74A(Antimycin A) 7 9 1 0 0 180 185 24074A (DNP) 14.5 250 305 300 375mi-I  (contml) l&I 400 435 415 460mi-lz(Antimycin  (NUNS)6 0 85 110 1 0 0 a 5A) 4 5 5 0 5 4 5 5ml-1  (DNP) 100 1 1 0 i 9 7 80the absence of a c&on  source expressed asCPM/O.5 mg (dry weight) conidia; saturatingconcentmtions  of L-phenylolanine  were employed.Valuer represent total phenylalonine  uptake with mycelial discs in the ab-sews  of a carbon source expressed (II CPM,&  (dry weight) mycelia.Tiuierer et al. ( 1953 J. Biol.  them. 205: 423) have shown that the reqrimtion  of mi-I  (and  appm&a~ly  one-third that of wild type. Preliminary experiments have shownto both aride (0.5 mM) and  antimycin  A (0.025 ma/ml), the uncoupling agent DNP reduces relpimtion  by approximately 50%.Also, 0s  shown in Table II, uptake  of I4C p h enylalanine  by p&y  decrsared  when the cells were incubated with the above-men-tioned inhibitors. These data strongly suggest that the energy coupling rydem  for  active tmnsport  is M)+  dependent on the cyto-chrome terminal &dare  system.This work was supported in part  by a training Grant (TO1  GM01316)  fram the Natioml Institute of Health to Florida StateUniversity. - - - Genetics Labomtoriec,  Department of Biological Science, Florida Sbte University, Tollah-e,  Florida 32306.Grindle, M. and D. 0. Wwdward.  Variations The c mutants of N. cmsm  have been chamcterized  by their slowamong  extmchromolomol  mutants of N. crow growth, excess cytoc&me  c, absence of cytochmmes a + a3  and mater-- -* nal  inheritance of the factors  responsible for these phenotypic  tmitr(Mitchell and  Mitchell 1952 Pros.  N&l.  Acad. Sci. U.S. 38:205;Mitchell, Mitchell and Tirsiera 1953 Pmt. N&l.  Acad. Ssi. U.S. 39:606).  Some  of the mi strains analyrd  recently in thislaboratory,  however, differ in Lath  phenotype and breeding behavior from the strains dercribxin  the 1952 and 1953 reports.In view of this, the following data  should be useful to anyone  using mi strains  in research  pmgmmr  or for clan demonstmtionr.-We obtained cultures of mi-1 A, -1  a_ , SA  and  -3a from the Fungal  Genetics Stock Center in 1964 and additional cultures ofmi-I  A and -1  a from the Fm in 1945.in6.CulturGf  mi-2a, -4A, -5A, -6A, -7A and  -8a were obtained  from Mary MitchellEox  of the stmins  was subcultured  on VogeGiniGl  maumm)  & conidswere  plated on Vogel’s N agar  med-ium containing 1% sorbore  and 0.1% su~rosa  (MS) and on MS supplemented with 0.5% bacto-peptone  and 0.5% yeast extract(MSPY). Mxt of the strains were cmssed  both as  pmtoperithecial  and conidial  parents to the wild types STA4,  74-OR23-1Aand 74-ORB-la  and to auxotrophic  and morphological nuclear mutnnts  with the genetic background of these wild types. Asco-spores  from individual perithecia were plated on MS and MSPY (MSPY permitted growth of both pmtotmphic  and ouxotmphicprogeny and, since vegetative growth was  nwre  rapid on MSPY than  on MS, progeny could be scored earlier).Asexual (conidiol) progeny of the various  mi strains  could be classified into 4 main  types of colonies after  4 doyr ot 34’C  onMS medium; Type I “tiny” -- 0.5-1.0 mm. diometerwith sparse myceliol growth;Type  I1  “compact” -- oppmximately  2.0 mm.diameter  with considerable myceliol growth; Type III “large” -- opproximotely 4 mm. diameter with considerable myceliolgrowt$;  or Type IV “diffuse” -- opproximotely 6.0-8.0 mm. diameter with sparse myceliol growth. Progeny on MSPY mediumgrew more rapidly but the relative morphological  ond growth-rote differences were mointoined. Colonies of mi-I A and  -1 o( 1964 stocks) were  types I and IV, those of mi-1 A ond -1 o ( 1966  stocks)c!?d  also  those of the wild types we-type  III, &seof mi-io,  -4A,  -5A.  -6A,  -7A ond -go weretype  1, o7 those of mi-3A and -30  were types I ond II. In flasks  of liquid M,-the rote ofqowtrof  types  I&d  III Gre  similar and types 1 ond IV=  ot obzt 30-50%  the rote of the wild types. Althoughmycelio from flak  cultures of mi-2a,  -30, -4A,  -5A,  -&A, ZA ond -80  were deficient in cytochromes o + o3, cytochmmespectm of mi-I A, -lo and  -3Awere  &oscden&l  to those of the ad  types with appreciable  absorption for cytochromesa + o3 ot ST. Tytochmme  spectm of the 1964  mi- I stocks  were not determined.-Progeny from crosses of tiny, compact  or large mi strains x prototmphic  or ouxotrophic  normal  strains consisted of tiny, com-pact ond Iorge colonies (i.~.,  types I, 11 and IIIri;lus  those  with various  intermediate phenotypes (the 1964  stocks of mi-IAand -1 o were not tested ). That is, progeny of types 1, II ond III were recovered fmm 011  sexual  crosses involving 55sos pGperithecia1  parents irrespective of the phenotype of the mi parent.  Progeny from  the reciprocal crosses, nomml x *,were also  heterogeneous but no tiny colonies were recovered. G;bt  is, the foclvrs  responsible for the tiny phenotype were op-porently  not transmitted to sexuoi  progeny when the mi strains were used os the conidial  parents in crosws. The proportions oftiny progeny varied  considerably among  different mi~wxmol  cms~s  and  among different perithecio of the some cross, butmost perithecio gave  30-70%  tiny progeny. In cozmst,  tiny progeny were very rarely  recovered from crosses involving pairsof normal  stmins. Progeny with the tiny phenotype were deficient in cytochromes a + 03; those with the compact  or largephenotype were rwt  deficient in cytochromes a +a3.  Thus, regardless  of the phenotypes of ourfi  cultuures,  we  hove derivedstmins from them whose properties are similar to those of the original  Mitchell strain.Cultures of mi strains  that do not give phenotypicolly  heterogeneous progeny when cmssed  to normal strains  con be obtainedby selecting t’;y  fI progeny ondb.ckcrossing  these repeatedly  to o normal  strain such os 74-OR&lo. When tiny strains  ofmi-IA  derived in this manner  by Pahicio  St. Lowrencc  were crossed os protoperitheciol  parents to normal  strains with themR8-1  o genetic bockground, virtually 011  of the progeny hod the tiny phenotype.All of the mi stmins carry  some extrochromosomol  factor or factors that ore inherited only through the moternol porsnt ino sexual  crosc  The expression of these factors is influenced by mbny  nuclear genes such  that the colonial morphology, growth-rote ond cytochrome spectrum of on mi mutant con be modified by ossocioting  its extrochmmaomol  constituents with o varietyof genetic backgrounds. Our data  iacote  that,  when modifying nuclear genes hove been eliminated from the eight g strainsby witoble  bockcrossing  procedures, they hove very similoi  phenotypic ond genetic properties.Due to the slow growth of mi mutonts,  there must be strong selection in favor  of spontaneous  nuclear mutations (and also,perhops,  of extmchromoromarmutotions)  thot result in more  mpid growth ond conidiotion  and possibly also  some  risk of con-tamination by normal strains  during routine tmnsfer. It is not surprising, therefore, that mi stock, subjected to constant vege-tative transfer in various  institutions should  differ from the original mi stmins by MitcheGnd  Mitchell. It moy be advisableto derive new  mi strains  from the existing stock. by repeated bockcr&ng  to a standard wild type so thot 011  mi mutonts  wouldhove more sir&r  genetic backgrounds. - - - Stanford University, Department of Biological  Sciences, Stanfog,  California94305.

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Variations among extrachromosomal mutants of N. crassa

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