KARl heterokaryons was improved when the parental nuclei were diploid to an extent consistent with the ...... Further evidence for a link between the inviability of .... and chromosome size, and Walt Fangman, Paul Szauter, and. Connie Holm ...
MOLECULAR AND CELLULAR BIOLOGY, Mar. 1981, p. 245-253 0270-7306/81/030245-09$02.00/0
Vol. 1, No. 3
Internuclear Transfer of Genetic Information in karl-i/KARl Heterokaryons in Saccharomyces cerevisiae SUSAN K. DUTCHERt Department of Genetics, University of Washington, Seattle, Washington 98195
Heterokaryons of Saccharomyces cerevisiae have been constructed utilizing the karl-i mutation, which prevents nuclear fusion during conjugation (J. Conde and G. Fink, Proc. Natl. Acad. Sci. U.S.A. 73:3651-3655,1976). Each heterokaryon contained two haploid nuclei that were marked on several chromosomes. They segregated haploid progeny (cytoductants), most of which have the nuclear genotype of one or the other of the heterokaryon parents, but they occasionally segregated progeny having a recombinant genotype (exceptional cytoductants). Exceptional cytoductants receive the majority of their genome from one parent (the recipient) and a minority from the other (the donor). Transfer of two markers from the donor nucleus to the recipient is rarely coincident for markers located on different chromosomes but is nearly always coincident for those markers located on the same chromosome, suggesting that whole chromosomes are transferred from the donor nucleus to the recipient. In crosses of karl-i x KARl parents, either nucleus may act as a recipient or donor with equal probability. Recipient nuclei acquired 9 of the 10 chromosomes examined, with frequencies which were inversely correlated with the size of the chromosome. When a chromosome is acquired by the recipient nucleus, it either replaces its homolog or exists in a disomic condition. Haploid progeny emanating from karl x KARl crosses are frequently inviable. I tested whether this inviability might be the result of chromosome loss by donor nuclei. Viability of progeny from karl x KARl heterokaryons was improved when the parental nuclei were diploid to an extent consistent with the hypothesis, and diploid progeny which had become monosomic were recovered from these heterokaryons. The following sequence of events accounts for chromosome transfer in karl x KARl heterokaryons. After cell fusion, each nucleus in the heterokaryon has a probability of about 0.38 of losing one or more chromosomes. A nucleus sustaining such a loss can become a donor in a chromosome transfer event. If the other nucleus does not sustain a mortal chromosome loss, it can become a recipient in a transfer event. The chance of acquiring a chromosome lost by the donor is greater for smaller chromosomes than for larger ones and is about 0.05 for the average chromosome.
Conjugation of haploid yeast cells of opposite mating type normally produces diploid cells. Rare, transient heterokaryons are formed which produce haploid progeny that receive a cytoplasmic contribution from both of the parental cells. These haploid progeny presumably retain the nuclear genotype of one of the parents, differing from that parent only by the contribution of cytoplasm from the other parent. The mutant karl-I (3) disrupts nuclear fusion during conjugation, resulting in a high frequency of heterokaryotic zygotes which produce these unusual progeny. As is the case for most fungi, the nuclear membrane of yeast remains intact during mitosis so that the nuclear contents of the cell remain t Present address: The Rockefeller University, New York, NY 10021.
segregated from the cytoplasm (9). Yeast heterokaryons have been employed to study whether extrachromosomal genetic elements that show 4:0 segregation are acquired when only cytoplasm is transferred. In crosses of a parent with the extrachromosomal element and a parent without the extrachromosomal element, it was shown that mitochondria (20), 2,u deoxyribonucleic acid (DNA) (11), the 20S ribonucleic acid control element (6), URE3 (1), [psi] factor, and the killer factor (5) exhibited the properties expected for a cytoplasmic localization. It has been assumed in these studies that the nuclear genotypes of the haploid progeny remained unaltered, although Wright and Lederberg (20) reported one alteration in the nuclear genotype among the 90 zygotes that they examined. I have examined the progeny produced by heterokaryons and find internuclear transfer of 245
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DUTCHER
cytoductants, were selected in crosses of the following type: MATa KAR1 cyh2 x y M+ N+ [rho] x MATa karl-i X+ Y+ m n [rho'], where cyh2 is a recessive nuclear mutation conMATERIALS AND METHODS ferring resistance to cycloheximide, x, y, m, and Saccharomyces cerevisiae strains used are listed in n are nuclear mutations conferring auxotrophy Table 1. Strain 2401 was made homozygous at the for various nutrients, and [rho] and [rho'] sigmating type locus by inducing mitotic recombination nify the absence and presence of mitochondrial with a 1-min exposure to X-irradiation at a dose of 106 DNA, respectively. Cytoductants were selected rads/s. All heterokaryons were formed by growing cells to densities of 107 cells per ml in YM-1 medium by plating conjugation mixtures onto medium (7) and filtering 3 x 10e cells of each mating type onto containing cycloheximide with glycerol as the 25-mm-diameter Millipore filters to allow conjugation. carbon source. Neither of the two parents nor Haploid progeny (cytoductants) were selected on the true diploids arising from the cross grow on YEPG medium (7) containing 3 ,ug of cycloheximide this medium. Cytoductants in this cross that (Sigma Chemical Co., St. Louis, Mo.) per ml. Micro- have the genotype of the KARl parent bearing manipulation of zygotes and progeny was described the cyh2 allele and have received functional previously (submitted for publication). Buds that mitochondria from the cytoplasm of the karl-i formed at the poles of the zygotes (end buds) were examined exclusively because they have been shown parent will grow on this medium. The unselected to be mononucleate in greater than 95% of the cases nuclear markers x, y, m, and n were scored in examined, whereas medial buds were mainly binu- the cytoductants. Most cytoductants retained the nuclear genotype (x y M+ N+) of the KARl cleate (submitted for publication). Sporulation of MATa/MATa karl/karl cells was parent, but a small proportion of the cytoducachieved after conjugation with a MATa strain (8). tants had acquired the X+, Y+, m, or n markers These cells were allowed to conjugate for 3 h. Zygotes of the karl-i parent (Table 2). I shall term the were purified on a 20-ml 30 to 60% (wt/vol) sorbitol latter "exceptional cytoductants." gradient (modified from reference 18), washed three Of the 60,800 KARl cytoductants examined times with 0.3% potassium acetate with 0.1 mg of from the selective medium from four indepentryptophan per ml, and resuspended in 0.3% potassium acetate with 0.1 mg of tryptophan per ml at approxi- dent crosses, 349 exceptional cytoductants were mately 107 cells per ml. To avoid false tetrads during found. In cross I of Table 2, for example, 22,500 dissection, the spore walls of the sporulated zygotes cytoductants were examined, and 106 exceptional cytoductants were found. Forty-one bewere digested on the agar (4). came URA+ (chromosome XI), 44 cytoductants RESULTS became either MATa or nonmaters (chromoI have found that chromosomal genes are some III), and 21 cytoductants became prototransferred from one nucleus to another during trophic for the amino acids histidine, lysine, and abortive nuclear fusion in heterokaryons having tyrosine (chromosome II). The exceptional cytoductants cannot be acone parent with the karl-i mutation. Haploid
chromosomal genes among the progeny of karl1 x KARl heterokaryons but no transfer in the progeny of rare KARl x KARl heterokaryons.
progeny,
TABLE 1. S. cerevisiae strains used Strain
Source
Genotype
JCK525D A364A cyh2 500-19-1 500-19-1 cyh2 511-7-2 548-8-1
G. Fink L. Hartwell This study This study This study This study
MATa karl leul thrl MATa adel ade2 his7 lys2 tyrl ural cyh2 [rho] MATa karl leul thrl MATa karl leul thrl cyh2 [rho] MATa adel leu2 ural tyrl cyh2 [rho] MATa lys2 tyrl leu2 trpl hom3 ural asp5 ade2 cyh2
517-48-3 511-9-4 548-24-1 520-4-2 520-7-1 S2072A 2401
This study This study This study This study This study D. Hawthorne This study
MATa adel lys2 his4 trpl ural cyh2 [rho] MATa adel his4 ural asp5 MATa leu2 trpl ura3 his2 MATa ural met2 MATa met2 thrl
508-23-4 529-11-4
This study This study
[rho]
MATa/MATa arg4/arg4 leul/leul trpl/trpl MATa/MATa adel/ADEI ura3/URA3 thrl/THRJ trpl/trpl his7/his7 karl/karl cyh2/cyh2 MATa karl met2pha2 MATa cysl karl tyrl
S. CEREVISIAE karl-1/KARl HETEROKARYONS
VOL. 1, 1981
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TABLE 2. Exceptional cytoductants among KARl cytoductants produced from karl x KARI keterokaryons No. of exceptional cytoductants per chromosome:
Cross' I
II
II
IV
v
VI
vii
xi
xii
xvi
No. of double transfers CoinNo. of cytoductants cidence E examined Observed' peCtedd valuec
16.6 2 0.12 22,500 NT 21 44 NT NT NT 0 41 NT NT I Of 33.3 1 0.03 6,221 3 NT NT 8 NT NT NT 0 II 16 6.6 4 0.60 12,711 8 0 13 1 4 14 6 19 III NT 10 9.1 3 0.33 19,400 16 39 10 NT NT 0 39 NT NT IV 37 a Parents of crosses: cross I, JCK525D x A364A cyh2, cross II, 529-11-4 x 511-7-2; cross III, 500-19-1 x 548-81; cross IV, 500-19-1 x 517-48-3. b Observed/expected number of double transfers. 'The number of exceptional cytoductants that acquired two markers located on different chromosomes. d The expected number of double acquisitions was calculated by summing the probabilities of acquiring all pairwise combinations of two chromosomes and then multiplying by the number of cytoductants examined. This method assumes that each class is independent. 'NT, Not tested. f Both parents were tyrl, and no TYR' colonies were recovered.
counted for by mutation of the KARl cells. First, the frequency of exceptional cytoductants is 10'fold higher than the mutation frequency. Mutations of ural to URA+, mutations of MATa to MATa or to a nonmating phenotype, and simultaneous mutations of his7, lys2, and tyrl to prototrophy occurred with frequencies of 7 x 1o-7,
