Reprinted from the Proceedings of the NarionaL ACADEMY OF SCIENCES, Vol. 43, No. 10, pp. 919-923. October, 1956. EXTRANUCLEAR TRANSMISSION IN YEAST HETEROKARYONS* By Roserr E,. Wricut anp Josaua LEDERBERGT DEPARTMENT OF GENETICS, UNIVERSITY OF WISCONSIN, MADISON, WISCONSIN Communicated August 15, 1957 Fowell' has given preliminary evidence for transient heterokaryosis, incidental to zygote formation, in a strain of Saccharomyces cerevisiae var. ellipsoideus. This paper constitutes (1) a verification of Fowell’s scheme and (2) its application to the demonstration of cytoplasmic transfer of factors associated with respiratory de- ficiency.” Stocks.—The cultures WY42 (a) and WY44 (a) were kindly supplied by R. R. Fowell as HMh*+ and HQ10c~, respectively. They grew dispersely, would not sporulate, and mated prolifically. Two auxotrophic mutants, WY91 and WY92, were obtained from 10‘ colonies of WY42 screened by replica plating after ultra- violet irradiation. WY91 showed normal respiration (A+) and had an absolute requirement for arginine and a partial requirement for leucine. Petite (R—) derivatives, 91-LP1 to LP9 (a@ Arg-R-), were obtained by acriflavine treatment.” WY92 required lysine and was also R~, as are many of the survivors after ultra- violet irradiation.? WY115 was obtained from 104 colonies of WY44 by similar methods. It re- sponded to the thiazole component of vitamin B; and was R~. An aThz~R+ segregant, WY110, was obtained from a hybrid of WY115 and WY42. An actidione-resistant mutant of WY110, 110-Ac3 (aThz~Ac'Rt), was selected on complete medium! containing 10 mg. actidione per liter. Ascus analysis of hybrids of WY92 and WY44 (H1, 2, 3) indicated that the former was a segregational petite’ (i.e., non-cytoplasmic) and was therefore unsuit- able for the projected experiments. 3:1 and 4:0 segregations for lysine were ob- tained. Non-mating, directly sporulating spore clones were recovered from these hybrids and from a hybrid of WY91 and 110-Ac3 (H4). Thirty asci from H4 were dissected, but only 26 spores from 18 asci were recovered.6 The only tetrad com- pletely recovered produced three prototrophic clones and one clone which required both arginine and thiazole. X-ray survival tests on WY42 and a hybrid (WY91 X WY44) indicated that both of the parental stocks were already diploid. Presumably they were homozy- gous for mating type. All the spores recovered from H4 showed some recombination of the parental markers, which are therefore not closely linked. H4 was actidione-resistant, though less so than 110-Ac3, and the segregants showed a range of levels of actidione resist~ ance. EXPERIMENTAL DESIGN AND PROCEDURE According to Fowell’s observations, many buds from mating pairs should have parental combinations of genes. In addition, because of the possibility of cyto- plasmic mixing in a fusion figure, some buds from R~ X Rt+ mating pairs should receive R* cytoplasm with the gene combination of the R~ parent. Their mani- 919 920 GENETICS: WRIGHT AND LEDERBERG Proc. N. A. 8. festation of the 2+ phenotype would indicate extranuclear transmission of the nor- mal respiratory factor. Conversely, extranuclear transmission of the suppressive factor’ would be indicated by the isolation of R~ buds containing the other markers of the R* parent. R- X &~ and kt X R* crosses were examined in control experiments. In the experiments to be reported, mass-mating mixtures were incubated for 4-8 hours before the isolation of mating pairs was begun.* An earlier series of ex- periments, in which 72 pairs were isolated 11-24 hours after setting up the mating mixtures, had not given any evidence of cytoplasmic transfer. Each mating pair was incubated until it had formed a progeny of 20-100 cells, which was then spread on a plate of complete medium. This was replica-plated to minimal* and complete media to detect auxotrophs among the progeny. Any auxotrophs were characterized for growth requirements by replica plating to a series of appropriately supplemented plates of minimal medium. Respiratory status was usually determined in tubes of acetate broth,!’ but in control experiments a plate of acetate medium was included in the series of replica plates. Sometimes buds were removed from the isolated mating pairs and cultured sepa- rately for characterization of growth requirements and respiratory status. Samples of auxotrophs from each plate were tested for mating type. Actidione resistance was tested in crosses involving 110-Ac3 or its R~ derivatives. ORIGIN OF THE PARENTAL-TYPE BUDS FROM MATING PAIRS The crosses involved WY91 or its R~ derivatives (a Arg~) and WY110 or its actidione-resistant or petite derivatives (aThz—Ac’ or Ac‘). Auxotrophs were found in the progeny of 91 out of 530 mating pairs which did not have buds present at the time of isolation or were stripped of existing buds. Thirty-three of these pairs yielded both types of auxotrophic progeny, and 58 gave one or the other type. The remaining 439 pairs gave exclusively prototrophic (hybrid) progeny. Proto- trophic progeny were also present on most of the plates containing auxotrophs. In 90 of the 91 sets of auxotrophic progeny, no recombination of the parental markers was observed. The remaining set, produced by a pair from the mating WY91 X 110-Ac3, contained Thz~ progeny, which, unlike the parental cultures, would not mate with either WY42 or WY44 and could be induced to sporulate, although poorly. They also showed an intermediate level of actidione resistance which, together with the weak sporulation, suggests that these auxotrophs were aneuploid hybrids. At best, this is a dubious case of recombination. Therefore, it can be concluded that the auxotrophs carry nuclei assorted from heterokaryons without intervening fusion, which would afford an opportunity for genic recombination. Thus any changes in respiratory status depend on extranu- clear transmission or on mutation of nuclear or cytoplasmic determinants of respi- ration. EXTRANUCLEAR TRANSMISSION R~ X &+ matings.—The suppressiveness of three of the 91-LP series of petites was determined.’ An #+ histidine-requiring culture,!! WY121, was used as the screening parent. 91-LP9 (a@Arg—R-) was found to be 76 per cent suppressive and was used in a series of crosses with WY110 (aThz-R+). The results of these and Vou. 43, 1957 GENETICS: WRIGHT AND LEDERBERG 921 all subsequent experiments are given in Table 1. One case of a change from aArg-R- to aArg-R+ was found among seven sets of progeny containing Arg- auxotrophs. In the same experiments, three of eight pairs which yielded Thz~ progeny gave some aThz-R-. TABLE 1 RESULTS OF EXPERIMENTS WITH Mating Pairs IsoLaTED AFTER 4-8 Hours IncuBATION oF Mating MrxtTuRES NUMBER OF Pairs GIvInG AUXOTROPHS NUMBER NUMBER OF Pairs or CHANGED OF GIVING AUXOTROPHIC RESPLRATORY PaIrs PROGENY Status Iso- Both Parent Parent Type or EXPERIMENT LATED -lrg~ Thz- Types a a R7~X Rt 91-LP9* X WY110 (aArg-R~ X a Thz-R*) 71 4 5 3 1 3 91-LP3t or 5¢ X WY110 or 110-Ac3 (aArg-R~ X a Thz-(Ac)Rt) 147 4 15 8 4 3 -¥% R- 91-LP3 or 5 X 110-P3, 4, or 5 (aArg-R- X aThz-R-) 268 7 8 iW 0 0 R+ xX Rt WY91 X WY1I0 or 110-Ac3 (@Arg-Rt X aThz-(Ac)R*) 135 7 12 14 0 0 * 76 per cent suppressive. + 27 per cent suppressive, ft 4 per cent suppressive. The next series of tests involved crosses of 91-LP3 (27 per cent suppressive) or 91-LP5 (4 per cent suppressive) with WY110 or 110-Ac3. Twelve pairs yielded Arg~ progeny, and 4 of these pairs gave some a Arg-R+ types, while only 3 of 23 pairs which gave Thz~ progeny showed a change from R*+ to R~. Thus the number and direction of the changes observed showed the expected correlation with the suppressiveness of the R- parent. R~ X R~ Control Matings —The occurrence of the mutation from R~ to Rt has never been definitely established, and the R- X R~ crosses so far reported have given exclusively R— progeny, unless one of the petites was segregational. However, a series of a Arg-R~ X aThz-R~ crosses (91-LP8 or 5 & 110-P3, 4- or 5-acriflavine &—- cultures from WY110) was made as a control for the changes from a Arg-R~ to a Arg-R*+ which were observed in the R~ X R* crosses. The progeny plates contained exclusively small-colony types, and these were all found to be R~ on examination of the acetate replica plates. Seven pairs gave a Agr-R~ progeny, 8 gave a7hz—R~, and 11 gave a mixture of these two types of auxotrophs. Thus 18 pairs gave a Arg—R~, and none gave a Arg~R*, in compari- son with the test series, where 5 of 19 pairs which yielded Arg~ progeny gave some a Arg-R*. Rt+ X R* Control Matings—Mutation from R+ to R~- is quite common during vegetative growth. Ephrussi et al.” calculated that the mutation rate is2 x 107* per cell generation. A+ X #*+ crosses were carried out to determine whether the R- parent (in R- X R*+ matings) had contributed to the ohserved frequency of changes from R+ to R~ in thiazole auxotrophs. Auxotrophic progeny were obtained from 33 of 135 pairs isolated from matings of WY91 and WY110 or 110-Ac3. All these were R+. One mating pair, which 922 GENETICS: WRIGHT AND LEDERBERG Proc, N. ALS. gave exclusively prototrophic progeny, contained 3 A~ among 100 A+ colonies. A bud removed from this pair also gave rise to an R~ clone. Seven pairs gave a Arg-R+ progeny, 12 gave a Thz-R*+, and 14 gave a mixture of the two types of auxotrophs. With the exception noted above, all the proto- trophic progeny were also R+. Thus 26 pairs gave aThz—R*+ progeny, and none gave aThz~R~, in comparison with the test series, where 6 of 31 pairs which yielded aThz— progeny gave some afhz~R-. Exact treatment!® of the 2 X 2 tables, 18/0:14/5 for Arg progeny and 26/0:25/6 for Thz- progeny, gives p = 0.03 and 0.02 that the observed changes in respiration are the same for R7~ X A+ versus R- X R- and for R~ X kt versus R+ &K Rt crosses, respectively. If mutation is independent of the other parent in the mating, it cannot account for the observed ahanges. The changes from R~ to R+ and from R+ to R- are therefore probably caused by cytoplasmic transmission of the factors responsible for normal respiration and the suppression of normal respiration in accord with the con- clusions of Ephrussi et al.” 7 The interested reader is referred to Wright!‘ for technical details and a discussion of remaining problems concerning these factors. SUMMARY Fowell’s interpretation that parental-type buds from mating pairs in a strain of S. cerevisiae var. ellipsoideus arise from transient heterokaryons has heen con- tirmed, In a series of experiments with doubly and triply marked stocks, carrying markers which were not. closely linked, no unequivocal case of reeombination was found. Progeny of isolated heterokaryons include clones of the parental genotype which show a change in respiratory phenotype. The latter therefore appears to be sub- ject to extranuclear transmission, in accord with the conclusion of Ephrussi ef al. * Paper No. 669. This work has been supported by grants from the National Cancer Institute (C-2157), Public Health Service, and from the Graduate School, University of Wisconsin, with funds allocated by the Wisconsin Alumni Research Foundation. { We are indebted to Drs. H. Roman, C. Raut, and B. Ephrussi for the provision of cultures related to this investigation, and to Dr. C. C. Lindegren for kindly bringing Fowell’s paper to our attention. 'R. R. Fowell, J. (ust. Brewing, 57, 180-195, 1951. 2? B. Ephrussi, Nucleo-cytoplasmic Relations in Micro-organisms (Loudon: Oxford University Press, 1953). 3C. Raut, J. Cellular Comp. Physiol., 44, 463-475, 1954. 4 Glucose, 10 gm.; tryptone (Difeo), 5 gm.; yeast extract (Difeo), 1 gm.; KH2PO,, 2 gm.; K»HPO,, 0.8 gm.; zinc (as sulfate), 400 ug.; iron (as ferrous ammonium sulfate), 150 ug.; copper (as sulfate), 25 wg.; water, 1 liter, 5 §. Y. Chen, B. Ephrussi, and H. Hottinguer, Heredity, 4, 337-51, 1950. 6 Four-spored asci were obtained from slants of Fowell’s sporulation medium (Nature, 170, 573, 1952). Asci were dissected in a moist chamber, with the aid of a DeFonbrune micromanipulator. Spore viability was low; only 27 per cent. of all spores isolated formed visible colonies. Many others formed small clones (4-50 cells) which did not develop further. In order to decrease the possibility of spore damage during ascus breakage, a wall-softening enzyme was obtained by growing Aerobacillus polymyzxa (strain C7, kindly supplied by Dr. Eliza- beth McCoy, Bacteriology Department, University of Wisconsin) for 4 days at 25° C. in the pres- ence of yeast asci suspended in nutrient broth containing I per cent NaCl. After centrifugation, solid ammonium sulfate, to give ca. three-fourths saturation, was added to 10 ml. of the supernate. The precipitate was dissolved in 1 ml. of sterile distilled water. After incubation of fresh asci for Vou, 43, 1957 GENETICS: WRIGHT AND LEDERBERG 928 3 hours in this preparation, many free spores were present, and intact asci broke after a minimum of manipulation. 7B. Ephrussi, H. Hottinguer, and H. Roman, these ProckEpines, 41, 1065-1071, 1955. § The oil-chamber technique of P. DeFonbrune, La Technique de micromanipulation (Paris: Masson & Cie, 1949), was used. * The synthetic medium of B. H. Olson and M. J. Johnson (J. Bacteriol., 69, 159-162, 1949) was used. Asparagine was omitted, and biotin and caleium pantothenate were the only vitamins wdded. This was supplemented as required with amino acids (10 mg/1) or 4-methyl-5-8-hydroxy- ethyl thiazole (0.1 mg/]). 0M. Ogur, G. Lindegren, and C. C. Lindegren, J. Bacteriol., 68, 391-392, 1954. This medium was modified for replica plating by omitting the indicator, reducing the glucose to 0.025 per cent, and adding agar. 11 Kindly supplied by Dr. S. Spiegelman as 33-E-26. 12 B, Ephrussi, P. L’Héritier, and H. Hottinguer, Ann. Inst. Pasteur, 77, 64-83, 1949. ‘SR. A. Fisher, Statistical Methods for Research Workers (Edinburgh: Oliver & Boyd, 1944). 4. EH. Wright, Hxrtranuclear Inheritance in Yeast Heterokaryons (doctoral dissertation, Uni- versity of Wisconsin, 1957) (University Microfilms, 313 N. First St., Ann Arbor, Michigan).