Altered Chloroplast Ribosomal Proteins Associated withErythromycin- Resistant Mutants inTwoGenetic Systems ofChlamydomonas reinhardi

Thephenotype ofseveral erythromycin-re- sistant mutantsofChiamydomonasreinhardi wasfurther characterized intermsoftheelectrophoretic properties of theirchloroplast ribosomal proteins. Inmutantery-M2d asingle protein ofthelarge(52S)subunit hasaltered prop- erties, whichprobably result fromachangeinitsprimary sequence. Thismutationisinherited inaMendelian man- ner.Inmutantery-Ula, whichisinherited inauniparental manner,adifferent single protein ofthe52Ssubunitisal- tered. Thischangemightresult fromachangeineither the primarysequence oftheprotein orinsomeformofsecond- arymodification. Theseresults indicate thatthesetwo distinct genetic systemsmustcooperate intheproduction ofchloroplast ribosomes. Inprokaryotes, acomplete mixture oftheindividual com- ponents ofribosomes iscapable ofspecific invitro aggrega- tion into functional ribosomes (1-3). Thus,since allofthese components aresynthesized within a single compartment, thegenetic specification ofthefinal structure appears tobe complete oncethismixture issynthesized. Ineukaryotes, however, wheretheribosomes confined within organelles differ inoverall structure andmacromolecular composition fromthose contained inthecytoplasm, thegenetic specifica- tion ofribosomes mustadditionally determine thesubcellular localization ofthefinal product. We havebegunstudies of thegenetic control ofribosome synthesis intheunicellular greenalga, Chlamydomonas reinhardi, hoping thatwewill eventually identify somegeneral features ofthegenetic control ofsubcellular organization ineukaryotes. We haveshown(4)thatatleast threedifferent genetic lociinC.reinhardi canmutatetoproduce resistance to erythromycin, anantibiotic thatisknowntointeract with prokaryotic ribosomes, andthatinhibits growthinwild- typeC.reinhardi aswell. Twoofthese genesareinherited inanormal Mendelian manner; itseemslikely thattheyare located onnuclear chromosomes. Thethird isinherited ina uniparental manner. Sager haspresented strong arguments tosupport theviewthatuniparental genesarecarried on chloroplast DNA (5,6),buttheevidence isnotconclusive (7-9). Further characterization ofmutants showedthateach ofthese genesisinvolved indetermining theerythromycin- binding capacity ofthelarge (52S)subunit ofchloroplast ribosomes (4). Thus,theerythromycin-resistant phenotype identifies genesfromtwoseparate genetic systems that control thefinal structure ofribosomes confined tothechloro- plast. An elucidation oftheexactmolecular phenotype asso- ciated witheachmutantgeneisofprimary interest. From whatisknownofribosome structure todate, genetic control oferythromycin-binding capacity might involve coding for (a)theprimary structure ofindividual ribosomal proteins (10,11),(b)secondary modification ofribosomal proteins (12, 13),(c)secondary modification oftheribosomal RNA (14), and/or, possibly, (d)theprimary sequence oftheribo- somal RNA.Asafirst stepinthemolecular characterization oftheresistance mutations, inthis paperwepresent studies oftheelectrophoretic properties oftheproteins fromchloro- plast 52Sribosomal subunits fromwild-type anderythro- mycin-resistant mutantstrains.