Effect of cycloheximide and actinomycin D on germinating conidi

Cochrane, V. W.

Hitchcock, S. E.

English

Kansas State University

Fungal Genetics Reports Volume 15 Article 22 Effect of cycloheximide and actinomycin D on germinating conidia S. E. Hitchcock V. W. Cochrane 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 Hitchcock, S. E., and V.W. Cochrane (1969) "Effect of cycloheximide and actinomycin D on germinating conidia," Fungal Genetics Reports: Vol. 15, Article 22. https://doi.org/10.4148/1941-4765.1925 This Response of Neurospora to Various Antibiotics and other Toxic Chemicals 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. Effect of cycloheximide and actinomycin D on germinating conidia Abstract Effect of cycloheximide and actinomycin D on germinating conidia This response of neurospora to various antibiotics and other toxic chemicals is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol15/iss1/22 presence of inhibiting concentrations of thorazine were  characterized by a “rice-like” morphology. This pculior  morphologypersisted thrcoghout the growth of the culture, even after inhibition had been overcome.5 days in cultures containing thorazine in excess of 2 x lOAM.No growth occurred over o period ofAntimycin A: Inclusion of I pg/ml.  of ontimycin A in the culture medium her  been observed to lengthen the moss doublingtime of the mycelium during the exponential phase of growth from 3 to 7 hcurr. - - - Department of Biological  Sciences, Stan-ford University, Stanford, Colifornio  94305./Miicke, D. and  M. Popp. Effect of molochitegreen on growth  in presence of rurfoca octonh.The LD50  of malachite green was  determined to be 3 x IO-59 per100  ml, using N. crosso  3o6A, cultured for 5 days ot 27OC.  At 18OCthe woe  concGtr=of  molochitc  oreen  inhibits the m~celiol orcwthmore than 60% (control without malachite  green = 100%) and  at 3O’Cmore than 80%. The inhibitory effect of malachite green was  increased in the presence of the following surface octonts (Tween40 (10-4g/lW  ml>  Twecn  80 ( lO-4  g/lo0  ml ), Louryl pyri lnium  chloride ( low5  g/l00  ml ),d’ and  Dimethyl-benrylomino-acctic-dodccylomid (IO-  g/l00  ml ) ) ot 18’C and  at 36X  (except for LK ot 36X).  At 27’C  the inhibitory effect of the surfaceoctonh tested did not oppeor. The concentrations of surface octonts tested in these investigations did not influence the growthof mycelium if the medium was  free of malachite green. - - - Inrtitut  fijr  Physiolcgische  Chemic der Univerritiit  Rortock, 25Rortock 1, Lenimllae  70, DDR.Wilson, J.F. and  W.K.Boter.  Effects ofcycnovzxunide ot low  concentrations.We hove observed the following effects of cyclohsximidc  (Acti-dionc)  on the Cuk  Ridge and  Rockefeller-Lindegren wild type strainsof Ncurosporo  crmso:  I. Detectable inhibition of growth occurs at aconcentmtion  o~ug/ml.,  and  o concentration  of 0.5 w/ml causes. ,-.more than  75% inhibition of growth of rtonding cultures.2. Hyphcl tip of microcultures  (Wilson and Gornjcbrt  1966  Genetics 53:621  ) develop obnormol morphology after expc-sure  to concentrotionr  of 0.6, 0.7, or 0.8 pg/ml. This change is accompanied by pronounced cytoplormic  flow into the tips,with consequent swelling and dichotomous branching even in the presence of a hypertonic (14%) swr~ne  solution.3. Regenerotim  into punctured cells, typicolly  100% within 45 minutes ot 30X,  is totally inhibited in the presence ofI.  0 pg/ml. of a group of cells individually injected with cycloheximidc ot o conccntmtion  of 10.0 mg/ml.,  two-thirds sur-vived  and one-half of all injected cells retained the ability  to regenemte. Production, by obrorption,  of an introcellulor  levelof antibiotic comp-oroble  to that obtained by microinjection weld  require 100% vptoke  of the tot.1 cycloheximide content ofo microchamber  filled with o concentration  of I pg/ ml.A complete description of this study requires photographs of regenemtion  and  doto toc extensive for o brief summary. Thecomplete description will therefore be published elsewhere. We feel that,  even  on the bosir  of this brief description, coutionmust be exercised in the interpretation  of doto bored upon the use  of cycloheximide concentrations above  I.  0 pg/ml. In odd-ition, even though the cdxewotions  relating to regeneration ore consistent with mechanisms hosed  upon inhibition of proteinsynthesis, the tolerance of cells to the high injected concentration  suggests that the toxicity of cxternol cycldrcximide  moy re-sult  directly from effects upon the cell mcmbrone,  and only rccondorily  from inhibition of protein synthesis. - - - Departmentof Biology, University of North Carolina at Greensboro, Greensboro, North Corolino  27412.Hitchcock, S.E. end  V.W.Cochrone.  Effect of cyclo- In o study of the germination of conidio of wild type strainhsximids and  octinomycin  Don germinating conidio.Em 5297a  (ATCC*lO816),  the synthetic copacitier  of conidio incu-bated  in minimal medium with and  withwt  a carbon source were  in-vestigated. Conidio were grown  on Vogel’s medium N with ~1  % glu-cose and  2% agor. Conidio  were horverted  at 7 doyr, worhed,  and  incubated in Vogel’s liquid medium N with (colled  “germ-inoting”  ) or without (called “non-germinating” ) 2% glucose at o spare  concentration  of 10 ,mg/ml  wet weight.Cycloheximida  (Up+  Co. ) at concentrations of I, IO, and  100 pg./ml  (0. I,  1.0, 10.0 pg/mg wet weight) inhibits germ-inotion completely and  inhibits the incorporation of levcine-U-C14,  phcnylalonine-U-C14,  and proline-U-C14  into protein(hot TCA insoluble, hot NaOH so u e material ). Inhibition at I pg/ml was  usually greater  than 84%. ot IO  &ml  greaterI blthan  97%,  and  ot IM)  pg/ml greater  than  99%. The inhibitor hod complex effects on the amino  acid pools  which have notbeen analyzed.Figurer 1 and  2 show  the effect of cycloheximide  on RNA synthesis in “germinating” conidia.  When cycloheximide warodded  at the beginning of the incubation, germination was  inhibited and  RNA synthesis approximated that  in “non-germinating”conidio (Figure 1 ). When the inhibitor was  added after germination hod begun, RNA synthesis continued ot the control rotefor o short  period and  then leveled off and  never ottoined the level of the control (Figure 2). Addition of cyclohcximide ot30 minutes inhibited germination completely, while if odded  ot 80 minutes, it halted germimtion  after 20 minutes. It may beconcluded that, while some RNA synthesis occurs in the obrence  of protein synthesis, continued protein synthesis is requiredfor RNA synthesis ot the rate found in germinating conidio.Figure I.  The effect of cycloheximide  on RNA synthesis. Figure 2. The effect of cycioheximide  on RNA synthesisIncorporation of urocil-2-Cl4  (5 &flask,  0.075 mM) when added after the beginning of incubation. Cyclohex-into TCA insoluble material. V’germinotitg”;  U”‘non- imide was odded  to germinating conidio at 30 or  80 min-germinating”; CYCLO=cycloheximide,  lC@g/ml;  %G= uter.  Experimental conditions and symbols are os describedpercent germination of “germinating” conidia. for Figure 1.Table I.  Effect of octinomycin  D cm  RNA synthesis rend  germination.Actinomycin  D (pg/ml) 0 I I O 2 5 loo 250% germination ot 2.5 hrr. 40 40 3 8 40 -- 2 3CPM in TCA insolublematerial at  0.5 hrs. 2,529 2,417 2,155 2,182 2,208 1,613CPM in TCA insolublenwterial ot 1.5 hrs. 32,092 32,056 32,498 28,053 28,549 1 6 , 6 3 0Table I shows pelimilwry  results of the effect of actinomycin  D (gift of Merck, Sharpe  and D&me)  on RNA synthesis andgermination.  Concentrations of up to 100  &ml  had a small,  if any, effect on  RNA synthesis and  germination. An extremelyhigh level, 250 &ml,  inhibited incorporation of uracil-2-Cl4  into TCA insoluble material only by 48.2% and  germinationby 57.5%. It was not determined if the actincmycin  D was  taken up by conidia.This research was supported by NSF gmnt GE 1401.  - - - Department of Biology, Wesleyon University, Middletown,Connecticut 06457. Present address of SEH: Department of Biology, Case Western Reserve  University, Cleveland, Ohio44106.

Effect of cycloheximide and actinomycin D on germinating conidia

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Effect of cycloheximide and actinomycin D on germinating conidia

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