Bruenn, J.

Mortimer, R.K.

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Lawrence Berkeley National LaboratoryRecent WorkTitleTHE ISOLATION OF MONOSOMCS IN YEASTPermalinkhttps://escholarship.org/uc/item/5598d8q8AuthorsBruenn, J.Mortimer, R.K.Publication Date1970-04-01eScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaSubmitted to Journal of Bacteriology RtCE1VE O L&W RENa RAD%A1OH LABOikATORY  mm 5 1970 LIBRARY AND DOCUMF-N-TS  SECTION THE ISOLATION OF MONOSOMICS IN YEAST IJCRL-19759 Pre print &2 J. Bruenn and R. K. Mortimer April 1970 AEC Contract No, W.-7405-eng-48 TWO-WEEK LOAN COPY This is a library CirculatIng Copy which may be borrowed for two weeks. For a personal retention copy, call Tech. Info. Division, Ext. 5545 LAWRENCE RADIATION LABORATOR) P L () UNIVERSITY of CALIFORNIA BERKELE DISCLAIMER This document was prepared as an account of work sponsored by the United States Government. While this document is believed to contain correct information, neither the United States Government nor any agency thereof, nor the Regents of the University of California, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by its trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof, or the Regents of the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof or the Regents of the University of California. • THE ISOLATION OF MONQSOMICS IN YEAST J. Bruenn and R. K. Mortimer Division of Medical Physics, University of California, Berkeley -1 ABSTRACT Tetraplcid cultures of Saccharomyces cerevisiae triplex (A/A/A/a) • 	ab several loci were sporulated. All the diploid spores are expected to be homozygous (A/A) or heterozygous (A/a) and hence to have the domi- • nant phenotype. Cells lacking one of the chromosomes (monosomics) may show the recessive phenotype (a/-). So spores that grew on complete medium but failed to grw on the relevant synthetic single omission media were presumed to be monosomic. These isolates were further charac- • • 	terized by sporulation and several stable monosomics established. 0 • 	 • 	 • 	 . - 	- 	 S 	 -- 	 - - -2 • Nonosomics are cells diploid for all chromosomes but one, which • 	is present in the haploid condition. They have been identified as • 	occasional products of meiosis in tetraploid strains of yeast (5). • 	The four ascospores resulting from a single meiosis in a tetraploid are expected to be diploid, but a nondisjunctional event may result in the production of an ascus that contains one trisomic, one monosomic, and to diploid spores (5). In a tetraploid of the genotype A/A/A/a at a given locua normal meiosis would result in two spores of the genotype A/A and two of the genotype A/a.. All spores would have the phenotype conferred by the dominant allele. The appearance of spores with the recessive (a) phenotype can occur by mutation, gene conversion, or nondisjunction. Hence the recovery of phenotypically recessive spores from triplex (A/A/A/a) tetraploids constitutes a procedure for the iso-lation of specific monosomics. The monosomic condition may be confirmed by sporulation or further crosses. Extensive genetic mapping has been done on Saccharomyces cerevisiae using tetrad analysis (). This approach, however, is laborious when the genes to be located are not centromere linked. Part of the motiva-tion for designing a method of obtaining monosomics was the hope that • 		they might be useful for mapping. When a monosomic is sporulated, half the resulting spores are nullisotnic: they are haploid cells that lack the • 	chromosome that was monosomic in the parent. - The other spores are normal haploids. If the nullisomic spores can be induced to mate with haploids, • - 	then the resulting monosomics will have the same phenotype as the haploids • 	for any markers located on the rnonosomic chromosome. To mapa recessive gene, a haploid carrying that recessive gene would be mated with the spores -3 hybrids would be selected. 	Only if the unlocated marker is on the mono- somic chromosome would any of the hybrids have the recessive phenotype. Inhat case, half the zygote clones, or all the hybrids resulting from the mating of the haploid with nulliomic (N-l) 	spores, would have the recssive phenotype. l4onosomics are quite interesting for other reasons. 	Since the missing chromosome will segregate as a centromere linked lethal, cen- tromere linked genes present in heterozygous condition (B/b) in an a/a monosomic will segregate mostly 2B or 2b in the two viable spores. 	Con- sequently, a monosomic for any chromosome may be used to determine in one cross whether any segregating gene is centromere linked. 	Entire chromosomes may be mapped by the isolation of mutants in monosomic strains. 	Most mutations are recessive and if isolated in: the monosomic must be an the monosomic chromosome. 	Recessive geneS affecting meioti 0 recombinatibn are best isolated in ronosomics, where their effects can be assayed directly by sporulation : MATERIALS AND METHODS Media and techniques. Media and techniques are as described in Hawthorne and Mortimer (2). I..' Yeast Strains. Two tetraploid strains of Saccharomyces cerevisiae were used. The first (xB105) was constructed from two diploids, X299 • and Y0287-10, by a forced mating #). X2939 has the following genotype: 	a• 	adel-1 gall 	trpl-1 ,.ura3 	his2-1 0leul-1 	arg! a + 	+ + 	ura) + 	+ metl4 	asp5 	gal2 ,..lys7. Y02587 has the genotype 	a 	0 trpl-L. ural. 0 + . + . + . 	+ 	 . 	 a trpl-1 ural The resulting tetraploid was triplex(A/A/A/a) at the loci adel (chromo-some I), gall (chromosome II), his2 (chromqsome VI), leul (chromosome vii), arg (chromosome viii), met1 (cromosome xi) asp and 12l2 (chroosome and jjs7 (chromosome xiii).. The matins mixture was incubated for 25 hours on glucoe nutrient agar (c-cA) and then streaked onto complete-uracil for the selective growth of tetraploid zygotes. One of the tetra-p1oidsfro this cross was sporulated and disseced. 	.. 	 .. The second .tetraploid culture used Xl22la) has the folloiing geno type: , a 	,adel 	ga1l. 	trpl ura3 .. 	thr3 	his2 leul. . 	adé5 a + + + 	+ 	+ his2 ± 	+ 0arg4 thri Cu 	gal2 	1YS7 ade2= ade 3 hisS mel 	ma 	X 	meti. + 	+ 	cu + + 	+ 	+ 	his8 Me 1 ma a 	meti X1221a is tripie:at the loci. add (chromosome ). gall (chromosome 21), trpl (chromosome IV), ura and thr (chromosomeV), ..leul and ade (chromosome VII), arg4 and thri (cbromosome viii), gal2 (chromosome Xli), lvs7 (chromosome and ada2 and ade-9 (fragmenci) . This cetraploid was sporulaed and the asci were treated with snail enzyme. The asci were disrupted • 1 -5 - parent cells plated out to a density of about lO per Petri plate on yeast- 	- extract. +. peptone + dextose (YEPD). Cononies that failed, to grow after replica-plating on minimal+methionine+histidine or on  yeast extract+ galactose medLum were presumed to be monosomics They were checked for their ability to use galactose as a carbon source and for growth on a series .of synthetic single-omission media lacking uracil, • leucine, arginine, threonine, 	tryptophan, or lysine. -u -. 	 . 	 -. 	•.''' 	6.' RESjTS •$ixten asci from a sorulated citure of the tetraploid XBl. • 	were dissected, and their spores scored an omission media (Table 1). • 	Tha results are consistent with the genotype above, and one of the spores • 	(XB105-16C) appears to be rnonosomic for chromosome VI, since it has the coflferred by his2-1, which is located on that chromo recessive 'phenotype  some. XBIO5-16C was found to spo'rulate, andit in turn.was dissected. The results - t shown it Table .2. The monosomiC sgregateS as a recessive • 	lethal. As expected, centronere linkage is also.demonstrated, since • 	both viable spores in each ascus have the same genotype for gall (as • 	determined by com.plementatiofl) in 10 of 11 two-spored asci. This is • 	' consistent with he previously reported l5.5 second division segregaion of (5).  Atotal of 61+  suspected monosomicS were then isolated from 1000 colonies of the sporulated X1221a tetraploid as described above. Only 29 of the 6L cultureS sporulated. . the remainder pres.abiy were hoo zygous for one or the other of the natig t:oe alleles. Suseo:e monos'omicS for chromosomes I, II, V, VII, VIII and for fraent 1 were sporulated and dissected. One of these darned thr 7 and ura3 together on the (prestmiably) monosomiC chromosome V, and all the viable spores • 	' 	fom the sporulated isolate carried both thr and ura as expected. Many of the 29 isolates that spdrulated gav segregations of non-viable to viable spores that indicated that the isolate was a mixtre • • 	 ells, it was poible to isolate clones from of diploid and monosomic c  • 	these isolates which save four viable snorCS ErQm each ascus and were so jud, 	to e dtlo. Other clones onigindlly m300sonic apparenzl; ;'• 	 all 	•.e 	:h 	fastr some 4:O segregations for viable: non-viable spores. 	The diploid revertants were always honozygous for the marker that was used to iidicate monosomy in the original isolate. 	For example, when the presumed monosomic for a chroosome V carrying both.thr5 and ura3 diploidized, 	the diploid was -homozygous for both thr3 and ura5. 	The number of asci withthree or tour viable spores may not accurately represent the proportion of diploids. ) in a population of diploid and monosomic cells, since the monosoruc cells may sporulate with lower efficier.c 	In general, isolates presr'ed to be monos.ornic for the larger chromosomes or fragments grew poorly, 	sporu- lated poorly, and reverted more rapidly tb the diploid condition. 	The most slowly growIng isolate carried leul and ade 	in homo- or hemizygous condition 	Botn tnese nar1ers are located o- cnro-ioso'e VU, 	the logest 4 chromosome. Two stable monosomics were isolated fro-t among the 29 of 64 that would 	porulate. 	These two, Xl22la-7C and Xl2la-C, 	are both red cn YEPD and thus carry adel or ade2 in noio- or 	ie-azgous ccndto 	IN All the spores from X122'la-C and XlLila-7C 	aiied to comoleme: when crossed to adel 	testers of oo: - 	mativg types 	as 	t'aica:ed lr :aole Again, 	the 	nissi'g c1'romosoe segregates as a ceritrorlere linked lethal the centronere narkers leul, arg- 	trpl, ura 	and a give oearly the expected second division segregation frequerces (Table '-) 	These two strais ar 	LLeefo: 	onoso-'_c for cro-oso 	i'_c- 	oa::es 	:e aaei marker. 	They remaihmonosomic.aftr several transfers. The stable moosorucs so far isullated f.roin tetraoloid 	eioic pro- d.jccs in the nanner described are riorosorric for chromosorres I and VI :Strøtnrw.es has obtained similar results using DL-.parafluorophnyLalanine -9 . Discussion The recovery of spores showing the recessive phenotype at loci tri- M. plex inthe parent tetraploid is a convenient and relatively unambiguous • method of isolating monosomics. Those isolates which show a 2:2 segre-gationof viable to inviable spores must either be monosomic or hetero-zygous for a centromere linked lethal and homozygous for the recessive marker used to identify them. Since the latter possibility. reçuires two coincidental events whose individual spontaneous probabilities are low, itiS a much less likely explanation. When chromosomes are multiply marked (as in the case of the monosomic for chromosome V cited above) any explanation other than that of monosomy involves pbstulacing three - or more independent spontaneous events. The para_fluorophenylalafline treatment of diploids results in a constellation of effects at high frequency whose interpretation is difficult, so that it is a less use- - ful tethod for the isolation of rnonosomics () Work is now in progress on the isolation of recombina:ionl-ess mu-tantsin monosomics and on the mapping of unlocated genes by mating hap-• bids to monosomic spores. • -10 • - • 	Tab1 	I. 	TetradaflalYsis of the tetraploid XBIO5 Locus • Number of asci s egregating 3:1 2:2 1:3 	O: ]ul-1 7 	0 0 0 	0 adel-1 7 	0 0 0 	0 asp' 7 	0 0 0 	0 • 	.!.EL.1 0 7 0 	. 	 0 • urab 2 	.1 0 	0 his2-1 6 0 0 	• 	0 • 	 • colonies were scored for growth on synthetic complete media lacking the growth factors indicated. The ratioof prototrophiC to áuxotrophic spores. per ascus is givep. bBth ura3 and ural are in duplex condition in XBl. CThe one spore that failed to grow on compl 	e_his:dine 	:s designated XB1O5- 16C. 	Only the seven complete asci are shown.  0 	 - 	 - 41. I Table 2 Tetrad analysis of the monosouc XBl-lC Locus 	 Number of asci segregating 2 : 0a 	1:1 	0:2 ade-1-1 	 11 	0 	0 leul-1 	 11 	0 	0 asp5 	 11 	0 	0 b ura 	 1 his2-1 	 0 	 0 	11. ga liC  aprototroph s: auxotrophs bspores were not tested by cortp1ernentatiOn for ural and ura Spores were tested for complementation with haploid- cescers carry-ing gall in both mating types 	Since gal2 was also segregati'g some of the spores scored upos4tive ? above did no gro•: on gaiact3Se ed -None ofthe five-one-spored asci gave excepdoaal spores. The spore viability was -. -- 	 -- - : -12 Table 3. 	Tetrad analysis of the . onosOmiCs from X1221a - x1221a-7C x1221a-3C Number of asci segregating Number of asci segregating Locus 2:0 1:1 	0:2 2 : Oa 1:1 0:2 6 0 	6 9 0 0  leul ade ib 0 0 	12 0 0 9 6 2 4- 3 2 trpl 7 0 6 0 3 0 1. lys7' 12 0 	0. .9 0 0 3 5 	L. 4 '4 • 1 aproto t ro pls . : auxotrophs . 	 . bAll spores were adel: 	none carried the ade2 marker. Cj'jg type 	locus. 	a:c. - . 	 3. Table 4. Centrorlere linkage Monosomic %Second division %Second division Centromere segregation segregatifl marker exp&c ted • 	 leul x1221a-7c 0.0 .9 16.7 i68 .99 arg X1221a-3C 33.3 16.8 .17 0.0 o.94 .75 trpl X1221a-7C 0.0 .0.94 .76 trpl X1221a-3C ura3 X1221a-SC 11.1 10.2 .97 X1221a-7C 41.7 1.0 .99 a a X1221a-SC -l.0 .95 ll XB105-16C 9.1 13.3 .74 aThe percent  second diisiOfl segegatiOfl is c1culated on the assumption that the monosomiC segregates as a centronere linked lethal, so thatany 1:1 ascus represents a second division segregaciofl of a • 	 centrOmre linked narker. •bp ro i .Mortitner  and Hawthorne (5) Cp is the chi_square probability that the observed variation is • 	 • 	 due 	to chance. 	, - . -14 • Literature Cited 1. Chamberlain, N., 	Cutts, N.S., Rainbow, C. (1952). J. Gen. Microb. • I , 5 59 • lit • 	2 Hawthorne, D.C. 	& N9rtimer, R.K. 	(1960). Genetics 3. Mortimer, D.K. and Hawthorne, D.C. 	(1966). Genetics 	, 	165-115. i. Pomper, 	S., Daniels,K.M. and McKee, D.W. (1954). Genetics 59, - . - • 33-555. .. •  Roma, H., Phillips, M.M. and Sands, 	S.M. 1955). Genetics 	o, 	5556i  Sttcmnae, cY. 	(1968). 	Hereditas 59, 	191-220. - LEGAL NOTICE This report was prepared as an account of Government sponsored work. Neither the United States, nor the Commission, nor any person acting on behalf of the Commission: Makes any warranty or representation, expressed or implied, with respect to the accuracy, completeness, or usefulness of the informa-tion contained in this report, or that the use of any information, apparatus, method, or process disclosed in this report may not in-fringe privately owned rights; or Assumes any liabilities with respect to the use of, or for damages resulting from the use of any information, apparatus, method, or process disclosed in this report. As used in the above, "person acting on behalf of the Commission" includes any employee or contractor of the Commission, or employee of such contractor, to the extent that such employee or contractor of the Commission, or employee of such contractor prepares, disseminates, or pro-vides access to, any information pursuant to his employment or contract with the Commission, or his employment with such contractor. to  Z-;v ~u tri C) til C)  1-3 1.1~ 0 11 C) m 

THE ISOLATION OF MONOSOMCS IN YEAST

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